Application of polyethylene glycol modified interleukin 2, glucocorticoid and hyaluronic acid to treatment of atopic dermatitis

Abstract

The invention discloses a pharmaceutical composition for treating atopic dermatitis, which comprises polyethylene glycol modified interleukin 2, glucocorticoid and small molecular weight hyaluronic acid, and optionally comprises a pharmaceutically acceptable carrier, also provided are 10KD PEG modified IL2 and their use in the treatment of type I allergic diseases.

Description

PEGylated interleukin-2, glucocorticoids, and hyaluronic acid for the treatment of atopic dermatitis Technical Field

[0001] The present invention relates to polyethylene glycol-modified interleukin-2 (IL-2 / IL2), glucocorticoid and hyaluronic acid for treating type I hypersensitivity (allergic) reaction, in particular, to the treatment of atopic dermatitis. Background Art

[0002] IL-2 is a multi-directional cytokine that preferentially promotes Treg cell proliferation at low concentrations, but preferentially promotes Teff cell proliferation at high concentrations. Therefore, it is difficult to control the dose and the efficacy is unstable when used directly for immunomodulatory therapy [GRAβHOFF H, et al. Low-Dose IL-2 Therapy in Autoimmune and Rheumatic Diseases. Front Immunol, 2021 Apr 1; ​​12: 648408]. Previous studies have found that the use of PEG-modified IL-2 combined with glucocorticoid 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. "Effects of dexamethasone combined with IL-2 on the selective expansion of regulatory T cells in donor mice and the inhibition of acute graft-versus-host disease reaction." Chinese Journal of Hematology, 2009, 30(11): 726-30; MAJ, 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 this is because the spatial conformation of PEG-modified IL-2 changes, weakening its affinity for the IL-2 receptor β subunit, resulting in a significant reduction in the proliferation of CD4+CD25- T cells, while having little effect on the proliferation of CD4+CD25+ Tregs, ultimately leading to the dominant increase in Treg cells. At the same time, low-affinity PEG-IL-2, assisted by CD25 molecules, can still promote FoxO3a phosphorylation in Treg cells, enhancing resistance to glucocorticoid-induced immune cell apoptosis, while having no such protective effect on CD4+CD25- T cells.

[0003] Regulatory T cells (Tregs) are important immune sentinels that negatively regulate immunity by secreting anti-inflammatory cytokines (such as IL-10 and TGF-β), highly expressing PD-1 and CTLA-4, and releasing granzymes and perforins to cause apoptosis of effector T cells (Teff), thereby maintaining the body's immune balance and avoiding excessive immune activation. Immune imbalance caused by an increase in effector T cells or a decrease or functional defect in Treg cells is an important link in many allergic and autoimmune diseases, including allergic asthma, atopic dermatitis, allergic gastroenteritis, systemic lupus erythematosus, polyendocrinopathy enteropathy X-linked syndrome, etc. [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]. Regulatory T cells can be effectively used to treat such diseases.

[0004] The skin is the largest barrier tissue in the body and is exposed to various stimuli from the external environment for a long time. Therefore, the proportion of Treg cells in normal skin is higher than that in peripheral blood to maintain local immune homeostasis in the skin [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]. In inflammatory skin with damaged skin barrier, local immunity is overactivated, pro-inflammatory effector cells and cytokines increase significantly, and Treg cells increase compensatorily but are not enough 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 human population; 2) fatality, such as anaphylactic shock and asthma attacks; 3) prolonged, recurrent, and excruciating pain, such as allergic rhinitis and atopic dermatitis (eczema); 4) Type I allergic diseases share a common pathophysiological mechanism, consisting of three phases: sensitization, provocation, and effector phases, involving allergens, allergens (allergen-specific IgE), CD4+ Th2 helper cells, cytokines IL-4, IL-13, and IL-5, mast cells, and basophils. Type I allergic reactions include: 1) systemic anaphylaxis, including drug-induced anaphylactic shock and serum-induced anaphylactic shock; 2) respiratory allergic reactions, 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 cutaneous manifestation of type I allergy. AD affects 10%-20% of the population worldwide, and its prevalence is increasing year by year with the improvement of environmental hygiene levels in life. The chronic recurrence of AD has seriously affected the long-term quality of life of patients. Current clinical medications are aimed at relieving symptoms such as itching, redness, swelling, and exudation in the acute phase of dermatitis, and have achieved certain therapeutic effects, but the efficacy of the drugs is limited and short-lived, and the side effects of long-term medication cannot be ignored. Therefore, this field needs a safe, effective and long-term solution for the treatment and prevention of atopic dermatitis.

[0007] Summary of the Invention

[0008] In a first aspect, a pharmaceutical composition for treating atopic dermatitis is provided, comprising polyethylene glycol-modified interleukin-2 (PEG-IL2), a glucocorticoid, and low molecular weight hyaluronic acid (HA), and optionally a pharmaceutically acceptable carrier.

[0009] In a second aspect, a method for treating atopic dermatitis in a subject is provided, comprising administering to the subject a therapeutically effective amount of PEG-IL2, a glucocorticoid, and a low molecular weight HA.

[0010] In a third aspect, provided are uses of PEG-IL2, glucocorticoids, and HA in preparing a medicament for treating atopic dermatitis, and the use of PEG-IL2, glucocorticoids, and HA for treating atopic dermatitis, wherein the HA comprises low molecular weight HA.

[0011] In a fourth aspect, a kit is provided, comprising PEG-IL2, a glucocorticoid, and HA, wherein the HA comprises high molecular weight HA and / or low molecular weight HA.

[0012] In a fifth aspect, provided are the use of HA in preparing a medicament for increasing the efficacy of PEG-IL2 and / or glucocorticoids in treating atopic dermatitis and the use of HA for increasing the efficacy of PEG-IL2 and / or glucocorticoids in treating atopic dermatitis, wherein the HA comprises low molecular weight HA.

[0013] In a sixth aspect, provided is the use of 10KD PEG-modified IL2 in the preparation of a medicament for treating type I allergy.

[0014] In a seventh aspect, a method for treating type I allergy in a subject is provided, comprising administering 10 KD PEG-modified IL2 to the subject.

[0015] In an eighth aspect, a 10KD PEG-modified IL2 is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1: Preparation of polyethylene glycol (PEG)-modified interleukin-2. (a) SDS-PAGE electrophoresis of recombinant human interleukin-2 before (-) or after (+) PEG modification followed by silver staining. (b) Monitoring of purified PEG-modified IL-2. M, marker. (c) Activity assay of recombinant human interleukin-2 and PEG-modified recombinant human interleukin-2 of different molecular weights and structures. (d) Activity assay of PEG-modified recombinant human interleukin-2 (40 kD-PEG-IL-2) and recombinant human interleukin-2 (IL-2).

[0017] Figure 2: OVA-induced atopic skin inflammation (AD) mouse model. A: Photographs of normal and dermatitis mouse skin, showing signs of AD skin redness, scaling, and damage. B: H&E staining of the skin from both groups (x40 magnification), showing marked thickening of AD skin and infiltration of numerous inflammatory cells. C: Quantitative RT-PCR analysis of mRNA expression of inflammatory factors in skin. The vertical axis represents the 2^(-ΔΔCT) value of qPCR mRNA between experimental and normal mice. The blank control group was designated as "1." Expression of IL-4, IL-13, and IL-17 in AD skin was significantly upregulated, with statistically significant differences, while IFN-γ expression remained unchanged. D: ELISA analysis of serum IgE concentrations, showing a significant increase in serum IgE in AD mice. In the figures, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P = 0.00, and ns indicates P > 0.05.

[0018] Figure 3: A shows skin photographs of mice with dermatitis 3 days after treatment with buffer (PBS), 40kD PEG-IL-2, budesonide, and 40kD PEG-IL2 + budesonide. All groups showed relief of dermatitis. B shows H&E staining of the skin in each group, showing similar levels of inflammatory cell infiltration (magnification x20). C shows the dermatitis scores for each group, with only the PBS group and the budesonide-only group showing statistical differences. D shows flow cytometry analysis of Treg cell ratios in the skin of the blank control group, AD model group, and each treatment group. E shows Treg cell ratios, showing statistical differences in all groups compared to the blank group, but no statistical differences among the treatment groups. * indicates P < 0.05, *** indicates P < 0.001, and ns indicates P > 0.05.

[0019] Figure 4: A is a photo of the skin of dermatitis mice after administration of 40kdPEG-IL-2 + budesonide (Bude), HA alone, and 40kdPEG-IL-2 + budesonide + HA. The improvement of skin damage in the latter group was significantly better than that in the first two groups. B is a H&E staining of the skin pathology of each group. The degree of inflammatory cell infiltration in the skin of the 40kdPEG-IL-2 + budesonide + HA group was significantly lower than that in the other two groups (×20). C is the flow cytometry detection of skin CD4 + CD25 + FOXP3 + The proportion of Treg cells; D is a statistical graph showing skin dermatitis scores. The dermatitis score was significantly reduced in the 40kdPEG-IL-2 + budesonide plus HA group compared with the no HA group and the HA group alone; E is a statistical graph showing skin Treg cells. The HA group showed statistically significant differences compared with the no HA group and the HA group alone. In the figures, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates P > 0.05.

[0020] Figure 5: A shows skin photographs of mice with dermatitis treated with PBS, mice induced with dermatitis and then treated with drugs (HA+PEG-IL-2+budesonide) followed by OVA exposure, and mice sensitized with normal skin treated with drugs for three days followed by OVA exposure. The photos show that only three days of drug administration during dermatitis can prevent the development of dermatitis after seven consecutive days of OVA exposure. B shows H&E staining of the skin in the corresponding groups (magnification x20), showing a significant reduction in inflammatory cell infiltration in the skin of the groups treated with drugs after dermatitis. C shows the proportion of CD25+FOXP3+ Treg cells in skin CD4+ cells measured by flow cytometry after sensitized mice were treated with drugs and then exposed to OVA for three days. D shows the statistical analysis of skin dermatitis scores, showing statistically significant differences between the two drug groups. E shows the statistical analysis of skin Treg cells, showing statistically significant differences between the two drug groups. ***P<0.001, ns=P>0.05.

[0021] Figure 6: Under the condition of unchanged budesonide + HA, after treatment with 40kD PEG-IL-2, 20kD PEG-IL-2, 10kD branched PEG-IL-2, 10kD unbranched PEG-IL-2, and ordinary IL-2, A is the skin photos of mice in each group; B is the H&E staining of the corresponding skin pathology of each group, magnification x20; C is the flow cytometry detection of the proportion of Treg in the skin of each group; D is the statistical graph of the skin dermatitis score of each group. Compared with the 40kD-PEG-IL-2 group, there was no statistical difference in the 20kD-PEG-IL-2 group, the 10kD-PEG-IL-2 group (branched), and the 10kD-PEG-IL-2 group (unbranched), while there was no statistical difference between the ordinary IL-2 group and the budesonide alone group, but there was a statistical difference between these two groups compared with the 40kD-PEG-IL-2 group; E is the statistical graph of skin Treg, comparing the groups, the 10kD PEG-IL-2 significantly increased Tregs, with the difference reaching statistical significance. The PEG-IL-2 combined group had the lowest skin Treg ratio, which was not different from the budesonide alone group. In the figures, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates P > 0.05.

[0022] Figure 7: 3-day treatment with PEG-IL-2 modified with unbranched PEGylations of varying molecular weights in a physiological model mouse model revealed the percentages of FoxP3+ and PD-1+ cells in CD3+CD4+CD25+ cells. Splenic CD3+CD4+ lymphocytes were analyzed for the percentages of CD25+FoxP3+ cells and PD-1+ cells. Panel A shows the percentage of CD4+CD25+FoxP3+ cells, and Panel B shows the percentage of CD4+FoxP3+PD-1+ cells. Panel C shows the flow cytometry of CD4+CD25+FoxP3+ cells. Panel D shows the flow cytometry of CD4+FoxP3+PD-1+ cells. This study demonstrates that only 10kD PEG-IL-2 significantly upregulates regulatory T cells (Tregs) in this physiological mouse model and induces high expression of PD-1 in these Tregs (PD-1 enhances the suppressive function of Tregs). In the figure, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0023] Figure 8: A is a photograph of mouse skin with dermatitis and after treatment with 10kdPEG-IL-2 with or without HA. B is a H&E staining of the skin pathology of each group, magnification x20. C is flow cytometry analysis of the proportion of CD25+FOXP3+ Treg cells in skin CD4+ cells of mice treated with 10kdPEG-IL-2 with or without HA. D is a statistical graph of skin dermatitis scores, showing statistically significant differences between the two treatment groups and the control group, but no statistical differences between the two treatment groups. E is a statistical graph of skin Treg cells, showing statistically significant differences between the two treatment groups and the control group, but no statistical differences between the two treatment groups. * indicates P < 0.05, and ns indicates P > 0.05.

[0024] Figure 9: AHR analysis of allergic asthma mice treated with medication and PBS, as well as healthy mice, using changes in pulmonary (airway) resistance (RI) as an indicator of airway responsiveness. * indicates p < 0.05 compared to the blank control and PBS-treated mice. Topical application of 10kD-PEG-IL2 significantly reduced airway resistance.

[0025] Figure 10: A is the skin photos after 10kd PEG-IL-2 + budesonide was added with large molecule HA, 2 / 3 large molecule HA + 1 / 3 small molecule HA, 1 / 2 large molecule HA + 1 / 2 small molecule HA, 1 / 3 large molecule HA + 2 / 3 small molecule HA, and small molecule HA respectively; B is the corresponding skin pathology H&E staining, magnification x20; C is the flow cytometry detection of the proportion of CD25+FOXP3+Treg cells in skin CD4+ cells; D is the statistical graph of skin dermatitis scores, showing the difference between the hyaluronic acid (HA) groups and budesonide alone. Compared with the budesonide group, the 1 / 3 large molecule HA + 2 / 3 small molecule HA group and the 2 / 3 large molecule HA + 1 / 3 small molecule HA group showed the most significant improvement in dermatitis, followed by the HA group, with statistical differences between the two groups. However, there was no statistical difference in dermatitis improvement between the large molecule HA group and the 1 / 2 large molecule HA + 1 / 2 small molecule HA group. E is a statistical chart of skin Tregs. Compared with the budesonide group alone, the combination of 1 / 3 large molecule HA + 2 / 3 small molecule HA showed the greatest increase in Tregs, with the highest statistical significance, but the difference was not statistically significant compared with the small molecule HA group (P>0.05). ns indicates P>0.05, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0026] Figure 11: A is the skin manifestation after 3 days of treatment with 10kDa PEG-IL-2 + dexamethasone (DXMS) or budesonide (Bude) + HA (large molecule: small molecule ratio is 1:2); B is the skin pathology H&E staining picture, magnification X20; C is the flow cytometry detection of the proportion of CD25+FOXP3+Treg cells in the skin CD4+ cells; D is the dermatitis score statistical chart, there is no statistical difference between the dexamethasone combination group and the budesonide combination group (P>0.05); E is the skin Treg proportion statistical chart, there is no statistical difference between the dexamethasone combination group and the budesonide combination group (P>0.05).

[0027] Figure 12: Figure 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 drug group determined by the aforementioned experiment, and clinical first-line budesonide treatment group. The IL-4, IL-13, and IL-17 in the combination drug group were downregulated more than those in the budesonide group, and the differences were statistically significant, while there was no difference in the expression of IFN-γ among the groups (P>0.05). Figure B shows the serum IgE concentration of each group detected by ELISA. The combination drug group reduced serum IgE more strongly than the budesonide group, and the differences were statistically significant. ns: P>0.05, *: P<0.05, **: P<0.01, ***: P<0.001.

[0028] Figure 13: 6-week OVA patch re-exposure images in the combination-treatment group and the AD control group. Panel A shows photographs of mice in the combination-treatment group and the AD control group 4 days after re-exposure to the OVA patch 6 weeks after OVA removal. Panel B shows H&E staining of the corresponding skin pathology, magnification x20. Panel C shows flow cytometry analysis of CD25+FOXP3+ Treg cells in the skin CD4+ cells of the two groups of mice. Panel D shows the dermatitis scores of the two groups, which showed statistically significant differences between the two groups (P < 0.05). Panel E shows the Treg cell ratio, which showed statistically significant differences between the two groups (P < 0.05). This study demonstrates that the efficacy of the combination-treatment group is maintained for at least 6 weeks. ns = P > 0.05, * = P < 0.05, and ** = P < 0.01.

[0029] Summary of the Invention

[0030] Unless otherwise indicated, all technical and scientific terms have the meanings commonly used by those skilled in the art. All patents, patent applications, publications, GenBank sequences, websites, and other public materials are incorporated herein by reference unless otherwise indicated. If a term in the present invention has multiple definitions, the definitions in this section shall prevail. When describing a URL or other identifier or address, it should be understood that the identifier may change and that specific information on the Internet is updated at any time. Relevant information can be found by searching the Internet. The disclosures thereof are provided as a basis for public gain.

[0031] As used herein, the terms "protein," "peptide," "polypeptide," and "amino acid sequence" are used interchangeably to refer to polymers of any length, for example, two or more amino acid residues. The term also includes amino acid polymers modified naturally or by human intervention; for example, by disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a tag or a biologically active component. Conventional single-letter or three-letter amino acid residue codes are used herein.

[0032] As used herein, the term "amino acid" or "aa" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the natural amino acids. Natural amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a natural amino acid (i.e., an α-carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group). Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid but that function in a manner similar to a natural amino acid.

[0033] Hyaluronic acid (HA) is a glycosaminoglycan that plays a role in a variety of physiological processes (Laurent TC et al (1992) FASEB J 6: 2397-2404). HA is a linear repeating polysaccharide, a straight-chain high-molecular-weight polysaccharide composed of (1-β-4) D-glucuronic acid and (1-β-3) N-acetyl-D-glucosamine disaccharide units repeatedly linked together. HA is present in the extracellular matrix of many cells, particularly in soft connective tissue. In connective tissue, bound water associated with hyaluronic acid creates spaces between tissues, thereby creating an environment suitable for cell movement and proliferation. Hyaluronic acid plays a role in biological phenomena related to cell motility, including rapid development, regeneration, repair, embryogenesis, embryological 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 adding the atomic weights of the repeating disaccharide units constituting the hyaluronic acid.

[0034] As used herein, high molecular weight HA refers to hyaluronic acid having a molecular weight of about 800 KD or greater.

[0035] As used herein, low molecular weight HA refers to hyaluronic acid having a molecular weight of about 300 KD or less.

[0036] Herein, the molecular weight of HA refers to the number average molecular weight of HA.

[0037] The hyaluronic acid used herein can be any suitable form of hyaluronic acid, including hyaluronate salts, such as, but not limited to, sodium hyaluronate, potassium hyaluronate, and magnesium hyaluronate. In one embodiment, the hyaluronic acid used herein comprises sodium hyaluronate.

[0038] The ability of large molecule HA to assist drug transdermal absorption and retention in the skin is relatively weak, but it has immunomodulatory ability, which can promote the expression of transcription factor FOXP3 by locally activated Treg and enhance the immunosuppressive function of Treg [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 an inflammatory effect and is not conducive to allergic dermatitis because it has agonist activity on TLR-2 and TLR-4, while high molecular weight HA reduces its ability to bind to receptors, forms a dense coating around cells and covers the receptor surface, reducing the proinflammatory effect of low molecular weight HA [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; 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].

[0039] Unless otherwise determined from the context, the ratio of high molecular weight HA to low molecular weight HA herein is a mass ratio.

[0040] Polyethylene glycol (PEG) is a non-toxic, water-soluble, neutral polymer with excellent biocompatibility and hemocompatibility. It has been used for topical, gastrointestinal, and intravenous administration in humans. Protein PEG modification involves activating one or both of the terminal groups of PEG to possess specific functional groups that are reactive toward at least one functional group in the target protein. PEG is then covalently bonded to the protein termini (N-terminus or C-terminus) or specific amino acids. PEG's binding sites are universal.

[0041] PEG is a polymer of ethylene glycol and ethylene oxide, also known as carbon wax, with the structural formula:

[0042] CH2(OH)-(CH2CH2O)n-CH2OH

[0043] PEG for drug modification may include branched and unbranched forms, ie, linear molecular forms (see, for example, EP0593868) and U / Y-shaped branched molecular forms (see, for example, EP0809996 and CN1243779C).

[0044] The U-branched PEG derivative (PEG2-NHS) has the following molecular formula:

[0045] In the formula, R and R' are each independently a low molecular weight alkyl group; n and n' are between 600-1500; and the average molecular weight of PEG is between 26KD-66KD.

[0046] The Y-type PEG derivative molecule is shown below:

[0047] Among them, P a and P b are the same or different polyethylene glycol molecules; j is an integer; R i is H, substituted or unsubstituted alkyl, substituted aryl, aralkyl or heteroalkyl, etc.; X1 and X2 are independently a linking group, for example, a group selected from the following: (CH2)n, (CH2) n OCO, (CH2) n NHCO, (CH2) n CO, and n is an integer of 1-10, and F is a terminal group, for example, selected from the following: hydroxyl, carboxyl, ester, acyl chloride, hydrazide, maleimide, pyridine disulfide, which can react with amino, hydroxyl or sulfhydryl groups on the therapeutic drug or the matrix to form a covalent bond.

[0048] As used herein, the molecular weight of PEG is determined by adding the atomic weights of the individual atoms that make up PEG.

[0049] Various methods are known in the art for determining the molecular weight of a molecule. For example, electrophoresis can be used to determine the molecular weight of a molecule by comparing it to a standard molecular weight marker. This is within the capabilities of those skilled in the art.

[0050] As used herein, "glucocorticoid" can be any synthetic or naturally occurring glucocorticoid. Glucocorticoids that can be used in the present invention are, for example, glucocorticoids that can be used to treat atopic dermatitis, examples of which 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, clobetasone, clobetasone propionate, clobetasone, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumoxonide, rofleponide, butixocort, and tipredane.

[0051] In the context of this specification sheets, unless otherwise indicated, any title for " glucocorticoid " includes all active salts, solvates or derivatives that can be generated from the glucocorticoid, for example, mentioning " dexamethasone " covers dexamethasone and its active salt, solvate or derivative with required activity. The example of the possible salt or derivative of glucocorticoid includes: sodium salt, sulfobenzoate, phosphate, isonicotinate, acetate, propionate, dihydrogen phosphate, palmitate, pivalate, fumarate and pharmaceutically acceptable ester (for example C1-C6 alkyl ester). Glucocorticoid and its active salt or derivative can also be their solvate form, for example hydrate form.

[0052] As used herein, "IL-2" refers to IL-2 of any origin, including mammalian origin, such as human, mouse, rat, primate, and porcine, and can be naturally occurring or obtained through recombinant or synthetic techniques, including recombinant IL-2 polypeptides produced by microbial hosts. IL-2 can be or comprise a naturally occurring polypeptide sequence, or can be an active variant of a naturally occurring IL-2 polypeptide. Preferably, the IL-2 polypeptide or active variant is derived from a human source, and includes recombinant human IL-2, particularly recombinant human IL-2 produced by a microbial host.

[0053] The IL2 that can be used in the present invention is any IL2 that can be used clinically to treat type I allergies, particularly atopic dermatitis. A person skilled in the art can obtain or determine which IL2 or its derivatives or variants can be used in the present invention.

[0054] As used herein, polyethylene glycol-modified IL-2 refers to IL-2 modified by PEG, such as unbranched PEG or branched PEG, by linking to any suitable site on IL-2.

[0055] As used herein, "treating" a subject with atopic dermatitis means that the subject's lesions or symptoms are partially or completely eliminated, or remain stable and do not progress after treatment. Treatment includes prevention, treatment, and / or cure. Prevention refers to preventing the underlying disease from occurring and / or preventing it from worsening or progressing. Prevention includes mitigating or eliminating one or more risk factors for the development of the disease. Because it is often impossible to determine whether a disease has never occurred, prevention also includes reducing the risk of developing or acquiring the disease.

[0056] As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to a dose that is at least sufficient to produce a therapeutic effect in a subject. For the active ingredients herein, a specific therapeutically effective amount can be preliminarily estimated using a variety of techniques known in the art. Dosage ranges suitable for human subjects can be determined, for example, using data obtained from cell culture experiments and other animal studies. Dosage levels and regimens can be determined based on known doses and regimens, extrapolated based on known properties if necessary, and / or empirically determined based on a variety of factors. Such factors include, for example, the subject's weight, overall health, age, the activity of the specific compound used, sex, diet, time of administration, drug combination, severity and course of the disease, as well as the patient's disease susceptibility and the judgment of the physician. After the patient's condition improves, a maintenance dose of the compound or composition can be administered, and if necessary, the dose, dosage form, and frequency of administration, or a combination thereof, can be changed. The exact dose and regimen should be based on the physician's judgment and the specific patient's circumstances.

[0057] The therapeutically effective amount depends largely on the nature of the drug, the condition of the patient and the nature and severity of the condition to be treated. The therapeutically effective amount may range from as low as 1 ng / kg, for example when using effective active substance in the treatment of local conditions such as atopic dermatitis, to as much as 10 mg / kg, more particularly in a dose range of 20 ng / kg to 1 mg / kg.

[0058] In this study, BALB / c mice were sensitized with three intraperitoneal injections of chicken ovalbumin (OVA). OVA challenge at different sites induced a typical type I hypersensitivity reaction, characterized by skin lesions and high airway reactivity, elevated serum IgE, pathological inflammatory cell infiltration in the skin, Th2 cell polarization, and elevated IL-4 and IL-3 expression. OVA-sensitized BALB / c mice are the most common experimental animal model for inducing type I hypersensitivity. Based on the OVA-induced mouse atopic dermatitis model, we first tried topical PEG-IL-2 combined with budesonide and compared their efficacy with the current first-line clinical treatment, topical glucocorticoids alone. Both treatments alleviated dermatitis symptoms in mice and increased the proportion of Treg cells in the skin to some extent. However, the efficacy of the combination therapy did not improve the efficacy compared with budesonide alone, and there was no statistically significant difference in the proportion of Treg cells in the skin. Given the presence of budesonide in the combination therapy, the alleviated dermatitis is likely due to the effects of the glucocorticoids.

[0059] The core of this study is to correct skin immune imbalance by increasing the proportion of Treg in the skin and enhancing its immunosuppressive ability through drugs to treat and prevent atopic dermatitis.

[0060] In normal skin structure, substances with a molecular weight exceeding 4kD have difficulty penetrating the stratum corneum into the epidermis and dermis, especially when the substance is in a hydrophilic polar state. This study shows that combined use of drugs after allergen sensitization but before dermatitis (skin lesions) occurs cannot prevent the dermatitis induced by OVA allergen, indicating that HA cannot help drugs penetrate into skin with normal structure. This article shows that in an atopic dermatitis mouse model with damaged stratum corneum, the therapeutic effect of PEG-IL-2 combined with budesonide and low molecular weight hyaluronic acid was significantly improved, while Treg cells were upregulated, indicating that low molecular weight hyaluronic acid can help drugs with large molecular weight and hydrophilic polarity penetrate into the deep epidermis or dermis or remain in the deep epidermis or dermis (the deep epidermis or dermis is the immune response layer of atopic dermatitis). The smaller the molecular weight of the combined PEG-IL-2 (10kDa, 20kDa, and 40kDa PEG), the greater the increase in the proportion of Treg cells in the skin and the better the therapeutic effect on dermatitis. 10kD-IL-2 (molecular weight 25kDa) is more effective than 20kD-IL-2 (molecular weight 35kDa) and 40kD-IL-2 (molecular weight 55kDa). However, when ordinary interleukin-2 (molecular weight 15kDa) replaces PEG-IL-2 in the combination regimen, the molecular weight is reduced and the hydrophilic polarity of PEG is removed (reduced polarity facilitates drug penetration). The results showed that the increase in induced Treg cells and the improvement of skin inflammation were not as good as those of PEGylated interleukin-2, and the therapeutic effect was the same as that of budesonide alone. It seems that ordinary interleukin-2 cannot be penetrated by small molecule hyaluronic acid through damaged skin to the deep epidermis. However, Madeleine Witting et al. [WARD-HARTSTONGE KA, KEMP RA. 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] experiments showed that bovine serum albumin (BSA) with a molecular weight exceeding 60 kD can penetrate damaged skin and reach the epidermis.

[0061] In the lesional state, we also applied 10kD PEG-IL-2 (interleukin-2 modified with this PEG molecular weight has the advantage of preferentially inducing Treg proliferation) to the lesions alone, which also demonstrated therapeutic efficacy, although the efficacy was less pronounced than when combined with low-molecular-weight HA. This suggests that PEG-IL-2 can penetrate the skin even in the lesional state. Therefore, unPEG-modified interleukin-2 can also penetrate the damaged stratum corneum, but under the action of HA, it cannot reach or be retained in the deep epidermal and dermal layers where immune responses occur. While small-molecule HA restricts interleukin-2 to the superficial epidermis, PEG-modified interleukin-2 can penetrate the deep epidermal or dermal layers via low-molecular-weight hyaluronic acid to achieve therapeutic effects. We believe that the interaction between low-molecular-weight HA and PEG, which retains the drug in the deep active epidermal layers or dermis, is the primary reason for the superior efficacy. In the absence of PEG, low-molecular-weight HA retains the drug in the superficial epidermis, preventing it from being retained at sites of immune response.

[0062] Without wishing to be bound by any theory, the experiments presented herein demonstrate that the molecular weight of PEG-modified IL-2 combined with glucocorticoids varies within a certain range and does not significantly affect the therapeutic effects on the skin. It is possible that some form of binding between hyaluronic acid and PEG molecules helps PEG-IL-2 enter and / or be stably retained in the deep epidermis or dermis to exert its therapeutic effects, whereas ordinary IL-2 without PEG molecules cannot enter the deep epidermis or dermis with the help of HA, or can only stay in the epidermis for a short time, and the IL-2 is lost before it can effectively act. The specific binding method between PEG and HA molecules is still unclear, and there are currently no reports of interactions between the two.

[0063] There were no statistically significant differences in dermatitis manifestations, inflammatory cell infiltration, or Treg ratios in the skin between the 10kD branched PEG-IL-2 and 10kD unbranched PEG-IL-2 groups, indicating that conjugating PEG with IL-2 of varying structures had no significant effect on overall efficacy. This study demonstrates that variations in PEG molecular weight and structure have little impact on drug entry and retention in the deep epidermis or dermis.

[0064] Why does 10kD-IL-2 combined with glucocorticoids and low-molecular-weight HA induce better Tregs and therapeutic effects than 20kD or 40kD PEG-IL-2? Without wishing to be bound by any theory, it is likely that in addition to improving skin permeation through interaction with hyaluronic acid, the specific molecular weight of PEG modification may also alter the properties of interleukin-2. When BALB / c mice were injected with different molecular weights of 10kD, 20kD and 40kD non-branched PEG-modified interleukin-2 and ordinary IL-2 under physiological conditions, it was unexpectedly found that only the 10kD molecular weight PEG-modified interleukin-2 could preferentially induce Treg cell proliferation, and the induced Treg cells highly expressed PD-1 molecules. The high expression of PD-1 in Treg cells enhanced the inhibitory effect of Treg 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.].

[0065] Herein, the use of 10kD PEG IL-2 alone in combination with or without HA (not combined with glucocorticoids) can still improve the symptoms of dermatitis in mice, and the proportion of Treg cells in the skin of mice after treatment is increased to a certain extent compared with that in dermatitis model mice, and the difference is statistically significant, while the use of 40kD PEG-IL-2 alone has no therapeutic effect (see Figure 3). Therefore, the inventors believe that 10kD PEG-IL-2 itself is a new interleukin 2 that can preferentially induce Treg cell proliferation, while 20kD or 40kD PEG-modified interleukin 2 needs to be combined with glucocorticoids to preferentially induce Treg cell proliferation. Obviously, PEG modification of appropriate molecular weight will lead to changes in the biological activity of interleukin 2 that preferentially induces Treg.

[0066] In addition, the use of 10kD PEG-IL-2 in a mouse asthma model significantly reduced airway hyperresponsiveness in mice, but the addition of HA on this basis did not further improve airway hyperresponsiveness. Therefore, it is believed that 10kD PEG-IL-2 can reduce type I allergic reactions and alleviate the symptoms of various type I allergy-related diseases, once again proving that 10kD PEG-IL-2 is a new type of IL-2 with its own immune regulatory function.

[0067] Subsequently, this study selected 10kD PEG-IL-2 combined with budesonide and varied the HA molecular weight and combination ratio. It was found that the proportion of Tregs in the skin was highest in the large-molecule HA / small-molecule HA ratio of 1:2 group and the group using only small-molecule HA. It is believed that both large-molecule HA and small-molecule HA can enhance the efficacy of 10kD PEG-IL-2 combined with budesonide, but small-molecule HA has a greater auxiliary effect because it can more easily carry the combined drug into and retain it in the active layer of the skin. Therefore, as the amount of small-molecule HA increases, this auxiliary effect becomes stronger and the efficacy becomes better. However, when the amount of small-molecule HA increases to a level sufficient to carry most of the drug, the efficacy no longer increases.

[0068] Therefore, optimal efficacy is achieved when the ratio of high- and low-molecular-weight HA is optimal. Ultimately, a combination regimen of 10kDa PEG-IL-2 + budesonide + 1 / 3 high-molecular-weight HA + 2 / 3 low-molecular-weight HA was selected as the optimal regimen for this study. Further comparison with first-line clinical treatments demonstrated that this optimal regimen, identified in this study, more rapidly and effectively alleviated dermatitis symptoms, more rapidly reduced inflammatory factors and serum IgE concentrations, and significantly increased the proportion of skin Treg cells. Furthermore, even after six weeks of treatment, it prevented dermatitis recurrence and maintained a high proportion of skin Treg cells. This long-term efficacy may be related to the long-term, stable retention of HA-induced Treg cells in the skin. Therefore, HA may interact with Treg cells, anchoring them in the dermis. Long-term dermal Treg cells can autoregulate the skin's immune response, achieving long-term therapeutic effects. It also has the potential to prevent the onset of dermatitis following re-exposure to allergens in sensitized skin, potentially preventing seasonal allergies such as pollen.

[0069] In summary, a three-day, topical regimen of 10kDa PEG-IL-2 + budesonide (or dexamethasone) + 1 / 3 large molecule HA + 2 / 3 small molecule HA can rapidly and effectively relieve and prevent atopic dermatitis in mice, reduce local inflammatory cell infiltration, and increase the proportion of Treg cells in the skin, with effects lasting at least 6 weeks. This regimen, through the unexpected discovery of an interaction between PEG molecules and small molecular weight HA, addresses the difficulty of hydrophilic polar and large molecular weight protein drugs in penetrating and being retained in the deep epidermis or dermis of the skin. Furthermore, it was unexpectedly discovered that the 10kD molecular weight modified interleukin-2 alters the biological properties of interleukin-2, preferentially expanding Treg cells between Treg cells (which suppress allergic immunity) and effector cells (which cause allergic reactions), thereby alleviating type I hypersensitivity reactions. Therefore, 10kD PEG-IL-2 is a novel interleukin-2.

[0070] Without wishing to be bound by any theory, a possible mechanism is that 10kD PEG-IL-2 can advantageously amplify Tregs and promote their high expression of PD-1 to enhance immunosuppressive function, while preferentially protecting Treg cells against glucocorticoid-induced apoptosis, while small molecule HA can assist in drug absorption, and a certain form of combination with PEG molecules can help the drug to remain stably in the deep epidermis and dermis, and may also stabilize Treg cells in the deep epidermis and dermis of the skin, so as to achieve long-term stable local immunity and produce lasting therapeutic effects, while large molecule HA simultaneously enhances the immune regulation function of Tregs to inhibit the occurrence of allergic immune responses.

[0071] Therefore, in a first aspect, a pharmaceutical composition is provided, comprising polyethylene glycol-modified interleukin-2 (PEG-IL2), a glucocorticoid and low molecular weight hyaluronic acid (HA), and optionally a pharmaceutically acceptable carrier.

[0072] The PEG used to modify IL2 can be any suitable type or size of PEG, eg, linear or non-linear PEG, and / or any suitable size.

[0073] In one embodiment, the polyethylene glycol (PEG) used to modify IL-2 is a PEG having a molecular weight of about 100, 90, 80, 70, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 kD. In one embodiment, the PEG is a PEG having a molecular weight of about 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-10, 1-20, 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20, 3-100, 3-90, 3-80, 3-70, 3-60, 3-50, 3-40, 3-30, 3- or 10-20 kD PEG.

[0074] In one embodiment, the PEG is a PEG having a molecular weight of about 60 KD, 55 KD, 50 KD, 45 KD, 40 KD, 35 KD, 30 KD, 25 KD, 20 KD, 15 KD, 10 KD, or 5 KD.

[0075] In one embodiment, the PEG comprises branched or unbranched PEG. For example, the branched PEG can comprise branched PEG of about 10 KD and 20 KD; the unbranched PEG comprises unbranched PEG of about 10 KD, 20 KD, and 40 KD.

[0076] The PEG modification described herein can be at any suitable site in IL-2 for PEG modification, such as a lysine, serine, or threonine residue or the N-terminal α-amino group of IL-2. In one embodiment, the PEG modification is at the N-terminal amino acid residue of IL-2, such as a lysine, serine, or threonine residue at the N-terminus of IL-2. In one embodiment, the PEG modification is at the N-terminal α-amino group of IL-2. The PEG modification can be, for example, a single-site modification or a multi-site modification, preferably a single-site modification.

[0077] In one embodiment, the IL-2 is IL-2 from a human or non-human mammal, such as, for example, a cow, rat, mouse, dog, monkey, goat, sheep, cow, deer, horse, or cat.

[0078] In one embodiment, human IL-2 or an active variant thereof is used herein, preferably recombinantly produced. The nucleotide and amino acid sequences of human IL-2 are disclosed, for example, in Genbank ref 3558 or P60568, respectively. Unless otherwise specified, IL-2 is used in a substantially pure form, for example, 95% or greater purity, more preferably 96%, 97%, 98%, or 99% pure. IL-2 can be used as a monomeric or multimeric protein.

[0079] In one embodiment, the IL2 used herein comprises the amino acid sequence shown in SEQ ID NO:1.

[0080] The low molecular weight HA refers to HA having a molecular weight of about 300 KD or less, for example, about 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3 or 2 KD or less. In one embodiment, the low molecular weight HA refers to HA having a molecular weight of about 2-20, 2-15, 2-10, 2-5, 3-20, 3-15, 3-10 or 3-5 KD, for example, about 5 KD, 4 KD, 3 KD or 2 KD.

[0081] In one embodiment, the pharmaceutical composition further comprises high molecular weight HA, wherein the high molecular weight HA and the low molecular weight HA can be present in any ratio, provided that the mass ratio of the high molecular weight HA to the low molecular weight HA is about 2:1, or the mass of the high molecular weight HA accounts for less than 50% of the total mass of the high molecular weight HA and the low molecular weight HA, preferably less than 40%, 35%, 30%, 25%, 20%, 15%, 10% or less. Preferably, the pharmaceutical composition comprises high molecular weight HA and low molecular weight HA in a mass ratio of about 2:1 or about 1:2.

[0082] The high molecular weight HA refers to HA having a molecular weight greater than or equal to about 800 KD, for example, greater than or equal to about 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 KD or more. In one embodiment, the high molecular weight HA refers to HA with a molecular weight of about 800-2000, 800-1900, 800-1800, 800-1700, 800-1600, 800-1500, 900-2000, 900-1900, 900-1800, 900-1700, 900-1600, 900-1500, 1000-2000, 1000-1900, 1000-1800, 1000-1700, 1000-1600 or 1000-1500 KD, in particular, HA with a molecular weight of about 800-1500 KD.

[0083] In one embodiment, the glucocorticoid is selected from dexamethasone, budesonide, beclomethasone dipropionate, ciclesonide, hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, clobetasone butyrate, triamcinolone acetonide, fluocinolone, mometasone furoate, clobetasone, clobetasone propionate, clobetasone, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumonide, roflenide, buticort, tipredan, and derivatives thereof. In one embodiment, the glucocorticoid comprises dexamethasone, budesonide, and derivatives thereof.

[0084] In one embodiment, the pharmaceutical composition is used to treat atopic dermatitis. Preferably, atopic dermatitis is atopic dermatitis with skin lesions. As used herein, atopic dermatitis with skin lesions refers to atopic dermatitis in which the structure of the stratum corneum of the skin is damaged, such as the cornified capsule and lipid membrane, and the structure of the granular layer, the last line of defense of the skin barrier, is damaged, manifesting as chronic recurrent severe itching, erythema, edema, blisters, serous exudation, etc.

[0085] In one embodiment, provided herein is a pharmaceutical composition comprising:

[0086] -10KD PEG-modified IL2,

[0087] -dexamethasone or budesonide, and

[0088] - HA, comprising: (i) low molecular weight HA, or (ii) 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.

[0089] As used herein, "10KD PEG-modified IL-2" is IL-2 modified with a PEG having a molecular weight of about 10KD, wherein the PEG can be any suitable type of PEG, such as a linear or non-linear PEG. "About 10KD" means that the sum of the atomic weights of the atoms comprising the PEG is 10KD ± 10%, 10KD ± 5%, 10KD ± 4%, 10KD ± 3%, 10KD ± 2%, 10KD ± 1%, 10KD ± 0.5%, or even 10KD ± 0.1%. In a preferred embodiment, the 10KD PEG-modified IL-2 is IL-2 whose N-terminus is modified with a PEG having a molecular weight of about 10KD (e.g., SEQ ID NO: 1).

[0090] The pharmaceutical composition can also include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. The use of such media and agents is well known in the art. Active ingredients used herein, such as glucocorticoids and polyethylene glycol-modified interleukin-2, can be mixed with one or more pharmaceutically acceptable additives, diluents or carriers. Examples of pharmaceutically acceptable carriers include, for example, but 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 can also include additives such as fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsifiers and other solvents or solubilizers or materials that achieve a storage effect.

[0091] The pharmaceutical composition can be formulated into any suitable form for topical administration (e.g., locally or topically), such as powder, spray, foam, solution, ointment, cream, etc. The pharmaceutical composition can be formulated into solid, liquid, gel or other forms.

[0092] The pharmaceutical compositions can be provided in bulk and unit dose form, as well as in the form of openable or perforable implants, capsules, blister packs, or cartridges as known in the art. Kits are also provided, comprising a delivery device, the pharmaceutical compositions herein, and optionally other suitable additives, such as other therapeutic compounds, excipients, surfactants (for use as therapeutic agents and formulation components), antioxidants, flavorings and coloring agents, fillers, volatile oils, buffers, dispersants, surfactants, antioxidants, flavorings, bulking agents, propellants, and preservatives, in separate containers, and instructions for use of the kit components.

[0093] In a second aspect, a method for treating atopic dermatitis in a subject is provided, comprising administering to the subject a therapeutically effective amount of PEG-IL2, a glucocorticoid, and a low molecular weight HA.

[0094] The PEG-IL2 administered to a subject can be any suitable type or size of PEG, for example, linear or non-linear PEG and / or PEG-modified IL-2 of any suitable size.

[0095] In one embodiment, the PEG-IL2 administered to a subject is modified with a PEG having a molecular weight of about 100, 90, 80, 70, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 kD. In one embodiment, the PEG is modified with a PEG having a molecular weight of about 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-10, 1-20, 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20, 3-100, 3-90, 3-80, 3-70, 3-60, 3-50, 3-40, 3-30, 3- or 10-20 kD PEG.

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

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

[0098] In one embodiment, the PEG modification is at the N-terminal amino acid residue of IL-2, such as a lysine, serine, or threonine residue at the N-terminus of IL-2. In one embodiment, the PEG modification is at the N-terminal α-amino group of IL-2. The PEG modification can be, for example, a single-site modification or a multi-site modification, preferably a single-site modification.

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

[0100] In one embodiment, the IL2 used herein comprises the amino acid sequence shown in SEQ ID NO:1.

[0101] The low molecular weight HA refers to HA having a molecular weight of about 300 KD or less, for example, about 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3 or 2 KD. In one embodiment, the low molecular weight HA refers to HA having a molecular weight of about 2-20, 2-15, 2-10, 2-5, 3-20, 3-15, 3-10 or 3-5 KD, for example, about 5 KD, 4 KD, 3 KD or 2 KD. In one embodiment, the low molecular weight HA refers to HA having a molecular weight of about 4 KD.

[0102] In one embodiment, the method comprises administering a low molecular weight HA having a molecular weight of about 5 KD or less, such as about 4 KD, 3 KD, or 2 KD. In one embodiment, the low molecular weight HA refers to a HA having a molecular weight of about 4 KD.

[0103] In one embodiment, the method further comprises administering to the subject a high molecular weight HA, wherein the high molecular weight HA and the low molecular weight HA can be administered in any ratio, provided that the mass ratio of the administered high molecular weight HA to the low molecular weight HA is about 2:1, or the mass of the administered high molecular weight HA accounts for less than 50% of the total mass of the administered high molecular weight HA and the low molecular weight HA, preferably less than 40%, 35%, 30%, 25%, 20%, 15%, 10% or less. Preferably, the HA administered to the subject comprises high molecular weight HA and low molecular weight HA in a mass ratio of about 1:2.

[0104] The high molecular weight HA refers to HA having a molecular weight greater than or equal to about 800 KD, for example, greater than or equal to about 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 KD or more. In one embodiment, the high molecular weight HA refers to HA with a molecular weight of about 800-2000, 800-1900, 800-1800, 800-1700, 800-1600, 800-1500, 900-2000, 900-1900, 900-1800, 900-1700, 900-1600, 900-1500, 1000-2000, 1000-1900, 1000-1800, 1000-1700, 1000-1600 or 1000-1500 KD, in particular, HA with a molecular weight of about 800-1500 KD.

[0105] 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, clobetasone, clobetasone propionate, clobetasone, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumonide, roflenide, buticort, tipredan, and derivatives thereof. In one embodiment, the glucocorticoid comprises dexamethasone, budesonide, or a derivative thereof.

[0106] In one embodiment, PEG-IL2, glucocorticoid, and HA may be administered to the diseased area simultaneously or sequentially in any order.

[0107] In one embodiment, the method comprises administering to a subject a therapeutically effective amount of the pharmaceutical composition of the first aspect.

[0108] As used herein, the subject is a human or non-human subject suffering from atopic dermatitis, non-limiting examples of which include humans, other mammals such as cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, horses, cats, and other non-mammals. In some embodiments, the subject is a human.

[0109] In one embodiment, the subject's atopic dermatitis is atopic dermatitis with skin lesions.

[0110] In one embodiment, the method comprises administering to a subject:

[0111] -10KD PEG-modified IL2,

[0112] -dexamethasone or budesonide, and

[0113] - HA, comprising: (i) low molecular weight HA, or (ii) 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.

[0114] In one embodiment, the method comprises administering to a subject a pharmaceutical composition comprising:

[0115] -10KD PEG-modified IL2,

[0116] -dexamethasone or budesonide, and

[0117] - HA, comprising: (i) low molecular weight HA, or (ii) 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.

[0118] As used herein, administration refers to the topical application (e.g., local or topical application) of the active substance or pharmaceutical composition to the affected area of ​​atopic dermatitis. Administration can be carried out by any method and auxiliary means known in the art, such as direct application, spraying, instillation, patch, etc.

[0119] In a third aspect, provided are the use of PEG-IL2, glucocorticoids and low molecular weight HA in the preparation of a drug (particularly, the drug is a topical or external drug) or a kit for treating atopic dermatitis; and the use of PEG-IL2, glucocorticoids and low molecular weight HA for treating atopic dermatitis.

[0120] The low molecular weight HA refers to HA having a molecular weight of about 300 KD or less, for example, about 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3 or 2 KD. In one embodiment, the low molecular weight HA refers to HA having a molecular weight of about 2-20, 2-15, 2-10, 2-5, 3-20, 3-15, 3-10 or 3-5 KD, for example, about 5 KD, 4 KD, 3 KD or 2 KD. In one embodiment, the low molecular weight HA refers to HA having a molecular weight of about 4 KD.

[0121] In one embodiment, PEG-IL2 is a PEG having a molecular weight of about 100, 90, 80, 70, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 kD. In one embodiment, the PEG is a PEG having a molecular weight of about 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-10, 1-20, 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20, 3-100, 3-90, 3-80, 3-70, 3-60, 3-50, 3-40, 3-30, 3- or 10-20 kD PEG.

[0122] In one embodiment, the PEG is a PEG having a molecular weight of about 60 KD, 55 KD, 50 KD, 45 KD, 40 KD, 35 KD, 30 KD, 25 KD, 20 KD, 15 KD, 10 KD, or 5 KD.

[0123] In one embodiment, the PEG comprises branched or unbranched PEG. For example, the branched PEG can comprise branched PEG of about 10 KD and 20 KD; the unbranched PEG comprises unbranched PEG of about 10 KD, 20 KD, and 40 KD.

[0124] In one embodiment, the PEG modification is at the N-terminal amino acid residue of IL-2, such as a lysine, serine, or threonine residue at the N-terminus of IL-2. In one embodiment, the PEG modification is at the N-terminal α-amino group of IL-2. The PEG modification can be, for example, a single-site modification or a multi-site modification, preferably a single-site modification.

[0125] In one embodiment, the IL-2 is human or non-human mammalian IL-2, such as cow, rat, mouse, dog, monkey, goat, sheep, cow, deer, horse, cat, etc.

[0126] In one embodiment, the IL2 used herein comprises the amino acid sequence shown in SEQ ID NO:1.

[0127] In one embodiment, further provided is the use of PEG-IL2, glucocorticoids, high molecular weight HA and low molecular weight HA in the preparation of a medicament or kit for treating atopic dermatitis; and PEG-IL2, glucocorticoids, high molecular weight HA and low molecular weight HA for treating atopic dermatitis, wherein the high molecular weight HA and the low molecular weight HA can be in any ratio, provided that the mass ratio of the high molecular weight HA to the low molecular weight HA is about 2:1, or the mass of the high molecular weight HA accounts for less than 50% of the total mass of the high molecular weight HA and the low molecular weight HA, preferably less than 40%, 35%, 30%, 25%, 20%, 15%, 10% or less. Preferably, the mass ratio of the high molecular weight HA to the low molecular weight HA is about 1:2.

[0128] The high molecular weight HA refers to HA having a molecular weight greater than or equal to about 800 KD, for example, greater than or equal to about 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 KD or more. In one embodiment, the high molecular weight HA refers to HA with a molecular weight of about 800-2000, 800-1900, 800-1800, 800-1700, 800-1600, 800-1500, 900-2000, 900-1900, 900-1800, 900-1700, 900-1600, 900-1500, 1000-2000, 1000-1900, 1000-1800, 1000-1700, 1000-1600 or 1000-1500 KD, in particular, HA with a molecular weight of about 800-1500 KD.

[0129] In one embodiment, the glucocorticoid is selected from dexamethasone, budesonide, beclomethasone dipropionate, ciclesonide, hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, clobetasone butyrate, triamcinolone acetonide, fluocinolone, mometasone furoate, clobetasone, clobetasone propionate, clobetasone, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumonide, roflenide, buticort, tipredan, and derivatives thereof. In one embodiment, the glucocorticoid comprises dexamethasone, budesonide, or a derivative thereof.

[0130] In one embodiment, the atopic dermatitis is atopic dermatitis with skin lesions.

[0131] In a fourth aspect, a kit is provided, comprising PEG-IL2, a glucocorticoid, and low molecular weight HA.

[0132] The kit may further comprise other reagents (eg, solvents, buffers, etc.) required for formulating the composition comprising PEG-IL2, glucocorticoid and HA into a suitable pharmaceutical agent, and optionally instructions.

[0133] In one embodiment, the PEG-IL2 used is a PEG-modified IL2 having a molecular weight of about 100, 90, 80, 70, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 kD. In one embodiment, the PEG is a PEG-modified IL2 having a molecular weight of about 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-10, 1-20, 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20, 3-100, 3-90, 3-80, 3-70, 3-60, 3-50, 3-40, 3-30, 3- or 10-20 kD PEG.

[0134] In one embodiment, the PEG is a PEG having a molecular weight of about 60 KD, 55 KD, 50 KD, 45 KD, 40 KD, 35 KD, 30 KD, 25 KD, 20 KD, 15 KD, 10 KD, or 5 KD.

[0135] In one embodiment, the PEG comprises branched or unbranched PEG. For example, the branched PEG can comprise branched PEG of about 10 KD and 20 KD; the unbranched PEG comprises unbranched PEG of about 10 KD, 20 KD, and 40 KD.

[0136] The PEG modification described herein can be at any suitable site in IL-2 for PEG modification, such as a lysine, serine, or threonine residue or the N-terminal α-amino group of IL-2. In one embodiment, the PEG modification is at the N-terminal amino acid residue of IL-2, such as a lysine, serine, or threonine residue at the N-terminus of IL-2. In one embodiment, the PEG modification is at the N-terminal α-amino group of IL-2. The PEG modification can be, for example, a single-site modification or a multi-site modification, preferably a single-site modification.

[0137] In one embodiment, the IL-2 is human or non-human mammalian IL-2, such as cow, rat, mouse, dog, monkey, goat, sheep, cow, deer, horse, cat, etc.

[0138] In one embodiment, human IL-2 or an active variant thereof is used herein, preferably recombinantly produced. The nucleotide and amino acid sequences of human IL-2 are disclosed, for example, in Genbank ref 3558 or P60568, respectively. Unless otherwise specified, IL-2 is used in a substantially pure form, for example, 95% or greater purity, more preferably 96%, 97%, 98%, or 99% pure. IL-2 can be used as a monomeric or multimeric protein.

[0139] In one embodiment, the IL2 used herein comprises the amino acid sequence shown in SEQ ID NO:1.

[0140] The low molecular weight HA refers to HA having a molecular weight of about 300 KD or less, for example, about 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3 or 2 KD. In one embodiment, the low molecular weight HA refers to HA having a molecular weight of about 2-20, 2-15, 2-10, 2-5, 3-20, 3-15, 3-10 or 3-5 KD, for example, about 5 KD, 4 KD, 3 KD or 2 KD. In one embodiment, the low molecular weight HA refers to HA having a molecular weight of about 4 KD.

[0141] In one embodiment, the kit further comprises high molecular weight HA, wherein the high molecular weight HA and the low molecular weight HA can be present in any ratio, provided that the mass ratio of the high molecular weight HA to the low molecular weight HA is about 2:1, or the mass of the high molecular weight HA accounts for less than 50% of the total mass of the high molecular weight HA and the low molecular weight HA, preferably less than 40%, 35%, 30%, 25%, 20%, 15%, 10% or less. Preferably, the kit comprises high molecular weight HA and low molecular weight HA in a mass ratio of about 1:2.

[0142] The high molecular weight HA refers to HA having a molecular weight greater than or equal to about 800 KD, for example, greater than or equal to about 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 KD or more. In one embodiment, the high molecular weight HA refers to HA with a molecular weight of about 800-2000, 800-1900, 800-1800, 800-1700, 800-1600, 800-1500, 900-2000, 900-1900, 900-1800, 900-1700, 900-1600, 900-1500, 1000-2000, 1000-1900, 1000-1800, 1000-1700, 1000-1600 or 1000-1500 KD, in particular, HA with a molecular weight of about 800-1500 KD.

[0143] In one embodiment, the kit comprises a glucocorticoid 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, clobetasone, clobetasone propionate, clobetasone, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumonide, roflenide, buticort, tipredan, and derivatives thereof. In one embodiment, the glucocorticoid comprises dexamethasone, budesonide, or a derivative thereof.

[0144] In a fifth aspect, provided are the use of low molecular weight HA in the preparation of a medicament for increasing the efficacy of PEG-IL2 and / or glucocorticoids in treating atopic dermatitis; and the use of low molecular weight HA for increasing the efficacy of PEG-IL2 and / or glucocorticoids in treating atopic dermatitis.

[0145] The low molecular weight HA refers to HA having a molecular weight of about 300 KD or less, for example, about 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3 or 2 KD. In one embodiment, the low molecular weight HA refers to HA having a molecular weight of about 2-20, 2-15, 2-10, 2-5, 3-20, 3-15, 3-10 or 3-5 KD, for example, about 5 KD, 4 KD, 3 KD or 2 KD. In one embodiment, the low molecular weight HA refers to HA having a molecular weight of about 4 KD.

[0146] In one embodiment, provided is the use of high molecular weight HA and low molecular weight HA in the preparation of a medicament for increasing the efficacy of PEG-IL2 and / or glucocorticoids in treating atopic dermatitis; and the use of high molecular weight HA and low molecular weight HA for increasing the efficacy of PEG-IL2 and / or glucocorticoids in treating atopic dermatitis, wherein the high molecular weight HA and low molecular weight HA can be in any proportion, provided that the mass ratio of high molecular weight HA to low molecular weight HA is about 2:1, or the mass of high molecular weight HA accounts for less than 50% of the total mass of high molecular weight HA and low molecular weight HA, preferably less than 40%, 35%, 30%, 25%, 20%, 15%, 10% or less, preferably, the mass ratio of high molecular weight HA to low molecular weight HA is about 1:2.

[0147] The high molecular weight HA refers to HA having a molecular weight greater than or equal to about 800 KD, for example, greater than or equal to about 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 KD or more. In one embodiment, the high molecular weight HA refers to HA with a molecular weight of about 800-2000, 800-1900, 800-1800, 800-1700, 800-1600, 800-1500, 900-2000, 900-1900, 900-1800, 900-1700, 900-1600, 900-1500, 1000-2000, 1000-1900, 1000-1800, 1000-1700, 1000-1600 or 1000-1500 KD, in particular, HA with a molecular weight of about 800-1500 KD.

[0148] In one embodiment, the atopic dermatitis is atopic dermatitis with skin lesions. In particular, the agent for treating atopic dermatitis is a topical or external agent.

[0149] In a sixth aspect, there is provided a use of 10KD PEG-modified IL2 in the preparation of a medicament for treating type I allergy, particularly, the medicament for treating type I allergy is a topical or external agent.

[0150] In one embodiment, the modified IL2 is branched or unbranched PEG-modified. In one embodiment, the modified IL2 is branched PEG-modified. In one embodiment, the modified IL2 is unbranched PEG-modified.

[0151] In one embodiment, the PEG modification is at the N-terminal amino acid residue of IL-2, such as a lysine, serine, or threonine residue at the N-terminus of IL-2. In one embodiment, the PEG modification is at the N-terminal α-amino group of IL-2. The PEG modification can be, for example, a single-site modification or a multi-site modification, preferably a single-site modification.

[0152] In one embodiment, the IL-2 is human or non-human mammalian IL-2, such as cow, rat, mouse, dog, monkey, goat, sheep, cow, deer, horse, or cat.

[0153] In one embodiment, IL2 comprises the amino acid sequence shown in SEQ ID NO:1.

[0154] In one embodiment, type I allergic reactions include 1) systemic allergic reactions, including drug anaphylactic shock and serum anaphylactic shock, 2) respiratory allergic reactions such as allergic rhinitis and allergic asthma, 3) digestive tract allergic reactions such as allergic gastroenteritis, 4) skin allergic reactions such as urticaria, atopic dermatitis (eczema) and angioedema.

[0155] In a seventh aspect, a method for treating type I allergy in a subject is provided, comprising administering 10 KD PEG-modified IL2 to the subject.

[0156] In one embodiment, the 10KD PEG-modified IL-2 administered by the method is modified with a branched or unbranched PEG of about 10KD. In one embodiment, the IL-2 is modified with a branched PEG of 10KD. In one embodiment, the IL-2 is modified with an unbranched PEG of 10KD.

[0157] In one embodiment, the PEG modification is at the N-terminal amino acid residue of IL-2, such as a lysine, serine, or threonine residue at the N-terminus of IL-2. In one embodiment, the PEG modification is at the N-terminal α-amino group of IL-2. The PEG modification can be, for example, a single-site modification or a multi-site modification, preferably a single-site modification.

[0158] In one embodiment, the IL-2 is human or non-human mammalian IL-2, such as cow, rat, mouse, dog, monkey, goat, sheep, cow, deer, horse, cat, etc.

[0159] In one embodiment, the administered IL2 comprises the amino acid sequence shown in SEQ ID NO:1.

[0160] In one embodiment, type I allergic reactions include: 1) systemic allergic reactions, including drug anaphylactic shock and serum anaphylactic shock; 2) respiratory allergic reactions such as allergic rhinitis and allergic asthma; 3) digestive tract allergic reactions such as allergic gastroenteritis; 4) skin allergic reactions such as urticaria, atopic dermatitis (eczema) and angioedema.

[0161] In one embodiment, the subject is a human or non-human subject suffering from atopic dermatitis, non-limiting examples of which include humans, other mammals such as cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, horses, cats, and other non-mammals. In some embodiments, the subject is a human.

[0162] The administration may be by any route suitable for treating the allergic reaction, such as intravenous, local, topical, subcutaneous, intraperitoneal, etc. In one embodiment, the administration is local administration, such as topical application.

[0163] The medicament may be in any suitable dosage form, such as powder, spray, foam, solution, ointment, emulsion, etc., and / or may be in solid, liquid, gel or other forms. The medicament may also contain auxiliary substances such as the pharmaceutically acceptable carriers mentioned above.

[0164] In an eighth aspect, a 10KD PEG-modified IL2 is provided.

[0165] In one embodiment, the IL-2 is IL-2 from humans or non-human mammals, such as cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, horses, cats, etc. In particular, the IL-2 comprises the amino acid sequence of SEQ ID NO: 1, and preferably, the IL-2 is modified at its N-terminus with the PEG single site.

[0166] The PEG can be any suitable type of PEG, for example, a linear or non-linear PEG. The "about 10 kD" means that the sum of the atomic weights 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 even 10 kD ± 0.1%.

[0167] As used herein, the word "or" is intended to include "and" unless the context indicates otherwise.

[0168] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, an optionally included step means that the step exists or does not exist; an optionally present pharmaceutically acceptable carrier means that the pharmaceutically acceptable carrier is included or not included.

[0169] As used herein, the term "about" refers to a numerical range that includes the specific value and that one skilled in the art would reasonably consider to be similar to the specific value. In certain embodiments, the term "about" refers to within the standard error of measurement using commonly accepted methods of measurement in the art. For example, in certain embodiments, about refers to ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, or even ±0.5% of the specific value.

[0170] As used herein, when a specific value or ratio is listed for a feature in the specification, a range consisting of any two of the values ​​or ratios is also included. For example, when the values ​​1, 2, 3, and 4 are listed, 1-2, 1-3, 1-4, 2-3, 2-4, and 3-4 are also included. Example

[0171] The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or embodiment of the present invention. The scope of the present invention is defined by the appended claims. In combination with this specification and common knowledge in the art, a person of ordinary skill in the art can clearly understand the scope defined by the claims. Without departing from the spirit and scope of the present invention, those skilled in the art can make any modifications or changes to the technical solution of the present invention, and such modifications and changes are also included in the scope of the present invention. The methods used in the following examples are all conventional methods unless otherwise specified.

[0172] Materials and Methods

[0173] 1. Experimental Materials

[0174] (1) Experimental instruments and consumables

[0175] (2) Experimental materials and reagents

[0176] 1. Reagent Kit

[0177] 2. Flow cytometry antibodies

[0178] 3. Other Reagents

[0179] 4. Experimental Animals

[0180] Female BALB / c mice, 6 weeks old, SPF grade, were purchased from Zhejiang Weitonglihua Laboratory Animal Co., Ltd. and maintained at the Laboratory Animal Department of Fudan University. Animal husbandry and handling procedures throughout this experiment complied with international standards for the use of laboratory animals. All animals underwent quarantine and acclimatization to the experimental environment for 1 week before the experiment began.

[0181] Example 1: Preparation of polyethylene glycol (PEG)-modified interleukin-2

[0182] PEG with a molecular weight of 10 kDa and 20 kDa has two structures: branched (Y-AALD-10 kD, Y-AALD-20 kD) and unbranched (M-ALD-10 kD, M-ALD-20 kD), and PEG with a molecular weight of 40 kDa is unbranched (M-ALD-40 kD) (all purchased from Beijing Jiankai Technology Co., Ltd.).

[0183] IL-2 (sequence shown in SEQ ID NO: 1) was replaced with sodium acetate buffer (pH 4-6) and mixed with M-ALD-20kD (Beijing Jiankai Technology Co., Ltd.) unbranched PEG in a proportion of 1:2 to 1:6 (mass ratio). The mixture was reduced with sodium cyanoborohydride and reacted at 2-10°C for 3-18 hours to obtain crude IL-2 (PEG). The crude IL-2 (PEG) product was subjected to cation exchange chromatography and preliminary separation under an acetic acid sodium acetate buffer system. The target protein peak was collected for reverse phase chromatography and gradient elution was performed with an acetonitrile trifluoroacetic acid system. The target protein peak was collected for cation exchange chromatography and concentrated under an acetic acid sodium acetate buffer system. The target protein peak was collected and finally replaced with an acetic acid sodium acetate buffer by ultrafiltration. 10kDa, 20kD, and 40kDa unbranched PEG-modified IL-2 and 10kDa and 20kD branched PEG-modified IL-2 were prepared separately. The process flow was consistent with that for the preparation of 10kDa and 20kDa unbranched PEG-modified IL-2 (PEG).

[0184] The obtained PEG-modified interleukin-2 with different molecular weights and structures are represented as follows: the unbranched ones include 10kD PEG-IL-2, 20kD PEG-IL-2 and 40kD PEG-IL-2; the branched ones include 20kD (branched)-IL-2, 10kD PEG (branched)-IL-2; and the electrophoresis patterns of other types are similar and not shown.

[0185] The selected PEG type primarily modified the N-terminus of the peptide chain. After modification with 20kD unbranched PEG, the molecular weight of recombinant human IL-2 (regular) increased from 15kD to 35kD, with high purity (Figures 1a and 1b). Activity assays using the CTLL-2 cell-based assay confirmed that the specific activity of the PEG-modified IL-2 was comparable to that of the IL-2 working standard (Figures 1c and 1d).

[0186] The sequence of human IL-2 used is shown in SEQ ID NO: 1 (5′-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT-3′).

[0187] Example 2: Construction of a mouse atopic (allergic) dermatitis model

[0188] The mouse atopic dermatitis model was established with slight modifications according to the method described in the literature [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 mouse. 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]. Chicken ovalbumin (OVA)-sensitized BALB / c mice are a commonly used mouse model for type I allergic diseases. 10 μg of OVA was mixed with 2 mg of aluminum hydroxide and dissolved in 200 μL of phosphate buffered saline (PBS) solution. The mice were sensitized by intraperitoneal injection. The injection was performed once every other week (on days 0, 7, and 14), with each mouse injected 3 times, 200 μL each time. On day 14, the mice were anesthetized using intraperitoneal injection of pentobarbital. During anesthesia, the hair on the back skin was removed with electric clippers and depilatory cream. After the hair was removed, medical tape was directly applied to the back skin for a few seconds and then torn off. This was repeated about 4 times to destroy the stratum corneum on the surface of the skin. Subsequently, the OVA patch was directly adhered to the mouse skin to induce atopic dermatitis in the mice. The OVA patch was made with a 1.5x1.5cm 2A sterile gauze was moistened with a 1 mg / mL OVA solution (dissolved in PBS) to prepare a solution (approximately 100 μL of OVA solution). A transparent dressing was applied to the depilated dorsal skin and changed daily for 3–7 days until dermatitis symptoms appeared. On the 4th–5th day of dermatitis induction (when dermatitis symptoms were most pronounced), the skin condition of the mice's dorsal skin was photographed and scored for skin inflammation. The mice were then euthanized, and skin tissue, spleen, and blood from the inflamed area were collected for further testing.

[0189] Example 3: Construction of mouse asthma model

[0190] The initial sensitization process for mice was similar to that used in the type I allergic disease mouse model: 10 μg of OVA and 2 mg of aluminum hydroxide were dissolved in 200 μL of phosphate-buffered saline (PBS) and injected intraperitoneally to sensitize the mice. Each mouse received three injections of 200 μL of PBS, administered once every week (on days 0, 7, and 14). On day 14, the mice were challenged intranasally with 20 μg of 2% OVA in sterile saline. Later, local airway administration of therapeutic drugs was performed via intratracheal spraying using the tip of a microneedle.

[0191] Example 4: Assessment of airway responsiveness in mice

[0192] Mice were anesthetized with sodium pentobarbital, tracheotomized, and mechanically ventilated. Changes in pulmonary resistance after aerosol inhalation of 3.125 mg / ml, 6.25 mg / ml, and 12.5 mg / ml methacholine were measured using the invasive Buxco small animal pulmonary function testing system.

[0193] 1. Grouping and drug administration

[0194] During the whole experiment, mice were randomly divided into 28 groups, with 6 mice in each group. The drug administration method for each treatment group of AD model mice was the aforementioned patch method, with a 1.5x1.5cm 2 Sterile gauze was wetted in drug solution (containing approximately 100 μL of drug solution and / or 50 μL of hyaluronic acid). The drugs were dissolved in PBS solution at the following concentrations: 100,000 IU / ml of regular IL-2 or PEG-IL-2, 20 μg / ml of budesonide, 100 μg / mL of dexamethasone, and 50 mg / mL of hyaluronic acid (HA). Drug administration began on the 2nd day of the experiment and continued for three consecutive days, with daily dressing changes. Aside from the different dosing regimens, all other conditions remained the same across groups. Three days later, mice were euthanized, and skin tissue, spleen, and blood samples were collected from the drug administration site for further analysis. The specific grouping scheme is as follows (except for 10kD PEG, which has two branched and unbranched structures. For groups not specified, both 40kD PEG and 20kD PEG are unbranched):

[0195] 1) To verify the effectiveness of drug treatment for dermatitis in mice, the mice were divided into the following groups: (1) blank control group; (2) atopic dermatitis (AD) model group; (3) PBS treatment group; (4) budesonide (Bude) 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 + small molecule HA treatment group; (8) small molecule HA treatment group;

[0196] 2) To investigate the effects of different molecular weights of PEG-IL-2 and different structural PEG-modified IL-2 on the therapeutic effect, the following groups were added: (1) 10kD (unbranched) PEG-IL-2 + budesonide + small molecule HA treatment group; (2) 10kD (branched) PEG-IL-2 + budesonide + small molecule HA treatment group; (3) 20kD PEG-IL-2 + budesonide + small molecule HA treatment group; (4) conventional IL-2 + budesonide + small molecule HA treatment group;

[0197] 3) To investigate the effect of different ratios of large and small molecular weight HA on the therapeutic effect, the following groups were added: (1) 10kD PEG-IL-2 + budesonide + HA (large molecule / small molecule = 1:2) treatment group; (2) 10kD PEG-IL-2 + budesonide + HA (large molecule / small molecule = 1:1) treatment group; (3) 10kD PEG-IL-2 + budesonide + HA (large molecule / small molecule = 2:1) treatment group; (4) 10kD PEG-IL-2 + budesonide + large molecular weight HA treatment group;

[0198] 4) To compare the effects of different forms of glucocorticoids on the therapeutic efficacy, an additional treatment group was established: ⒄10kD PEG-IL-2 + dexamethasone + HA (large molecule / small molecule = 1:2), with a dexamethasone concentration of 100μg / ml, 80-100μl (8-10μg) each time.

[0199] 5) To explore the long-term therapeutic effect of the drug: After the original atopic (allergic) dermatitis (AD) model mice were successfully established, the control group was induced with OVA patches to induce dermatitis without any treatment. The dermatitis subsided on its own after being separated from the antigen and then separated from the OVA environment for 6 weeks. After 6 weeks, dermatitis was induced with OVA patches again. The mice were euthanized, and the skin, spleen, and blood of the original administration site were taken for further testing; after dermatitis was induced with OVA in the experimental group, the 10kDa PEG-IL-2 + budesonide + HA (large molecule / small molecule = 1:2) regimen was used for treatment. After being separated from the OVA environment for 6 weeks, dermatitis was induced with OVA patches again after 6 weeks. The mice were euthanized, and the skin, spleen, and blood of the original administration site were taken for further testing.

[0200] 6) Other control groups: (18) Sensitized mice were first treated with 10 kd PEG-IL-2 + budesonide + small molecule HA for 3 consecutive days and then induced with OVA to induce dermatitis; (19) AD model mice were treated with 10 kd PEG-IL-2 + HA (small molecule);

[0201] 7) Asthma model mice: (20) PBS treatment group; (21) 10kDa PEG-IL-2 treatment group: each mouse was given 5000 IU 10kDa PEG-IL-2 once a day for three consecutive days; (22) combined treatment group: 10kDa PEG-IL-2 (5000 IU) + HA (1.25 mg) was given once a day for three consecutive days; (23) blank control group

[0202] 8) To understand the differences in the induction of regulatory T cells (Treg) by interleukin-2 modified with different molecular weights of PEG, five groups of physiological model mice were intraperitoneally injected with non-branched PEG-modified PEG-IL-2 and ordinary IL-2 of different molecular weights at a dose of 400,000 IU / time / week.

[0203] 2Skin inflammation status score

[0204] The skin inflammation status score assesses the severity of dermatitis from a clinical perspective. The total score ranges from 0 to 12 points, with individual scores ranging from 0 to 3 points (0, none; 1, mild; 2, moderate; 3, severe) based on edema, erythema, scaling, and erosion. The final score is the sum of the individual scores [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):1920-33].

[0205] 3. Histopathological staining

[0206] After euthanasia of mice, the back skin of the mice was collected and fixed with 4% paraformaldehyde at room temperature for more than 24 hours. The skin was then dehydrated with alcohol, embedded in paraffin, and sectioned longitudinally. After dewaxing, H&E staining was performed, and the skin tissue pathology was observed under an optical microscope.

[0207] 4. Flow cytometry detection of Treg cell proportion in skin and spleen

[0208] 1) Sample collection: After euthanasia of mice, spleen and skin of the experimental site on the back (about 1.5x1.5cm) were collected. 2 ), were immersed in PBS solution;

[0209] 2) Tissue processing: The spleen was ground and passed through a 70 μm pore size cell strainer to obtain a spleen single cell suspension; the skin was first cut into 1x1 mm pieces. 2 The cells were then digested at 37°C for approximately 50 minutes, with manual shaking every 5 minutes. After digestion, the cells were briefly centrifuged and the supernatant discarded. The cells were washed twice with PBS, ground, and filtered through a 70 μm filter to obtain a single-cell suspension.

[0210] 3) Lysis of red blood cells: After transferring the above single-cell suspension to a centrifuge tube, add 3-5 mL of red blood cell lysis buffer to the tube to resuspend the cells. After 2 minutes, centrifuge at 500g and 4°C for 5 minutes. Remove the supernatant and wash the cell pellet twice with PBS.

[0211] 4) Blocking: Count cells under a microscope and take about 5x10 6 For each cell, CD16 / 32 antibody was diluted 1:100, added to the cell suspension, incubated at 4°C for 15 minutes, and washed twice with PBS;

[0212] 5) Cell surface antigen staining: dilute CD3, CD4, and CD25 antibodies at a ratio of 1:100, add to the cell suspension, incubate at 4°C in the dark for 30 minutes, and wash twice with PBS;

[0213] 6) Fixation: Discard the supernatant and fix with the fixative in the transcription factor fixation / permeabilization kit (Biolegend, 424401) at 4°C in the dark for 1 hour;

[0214] 7) Permeabilization: Add an equal volume of permeabilization buffer to the fixative, centrifuge at 500 g, 4°C for 5 minutes, and wash twice with the permeabilization buffer.

[0215] 8) Intracellular antigen staining: Dilute FOXP3 antibody in permeabilization buffer at a ratio of 1:100, add to the cell suspension, incubate at room temperature in the dark for 1 hour, then wash once with permeabilization buffer and once with PBS;

[0216] 9) Flow cytometer: Resuspend the cells in PBS and analyze them using a flow cytometer and FlowJo software.

[0217] 5. Real-Time PCR Detection of Skin Cytokine Expression

[0218] 1) Sampling: Mouse dorsal skin was minced with ophthalmic scissors on ice, and 1 mL of Trizol reagent was added. The skin was then homogenized using a tissue homogenizer.

[0219] 2) RNA extraction: Add 200 μL of chloroform, shake vigorously for 15 seconds, let stand at room temperature for 15 minutes, centrifuge at 13,000 rpm at 4°C for 15 minutes, remove the supernatant to another RNase-free 1.5 mL centrifuge tube, add an equal amount of isopropanol, mix by inversion, let stand at room temperature for 15 minutes, centrifuge at 12,000 rpm at 4°C for 10 minutes, and discard the supernatant;

[0220] 3) Precipitation: Add 30 μL of pre-cooled 75% ethanol for washing, centrifuge and discard the supernatant;

[0221] 4) Dissolution: Add 30 μL of DEPC water to dissolve the RNA and measure the RNA concentration using a spectrophotometer.

[0222] 5) Reverse transcription: Based on the measured RNA concentration, strictly follow the instructions of the SYBR Green Reverse Transcription Kit (Tiangen, FP205-01) to obtain 1 μg of cDNA per tube;

[0223] 6) RT-PCR assay: A 20 μL reaction system was used, and the PCR kit (Takara, RR047A) was strictly followed. The PCR instrument was used and data were analyzed using Excel software.

[0224] 7) The primers included in the experiment are as follows:

[0225] Table 1: Primer sequences

[0226] 6. ELISA determination of mouse serum IgE levels

[0227] 1) Collection: After mice were anesthetized with pentobarbital, approximately 500 mL of blood was collected from the heart and allowed to stand at room temperature for 1 hour. The mice were then euthanized. Serum was obtained by centrifugation at 3500 rpm for 15 minutes at room temperature 1 hour later.

[0228] 2) Referring to the preliminary experimental results, serum was diluted 4-fold or 10-fold and the procedures of the mouse serum IgE ELISA kit (Xinbosheng Biotechnology, EMC117.48) were strictly followed. Standard samples and serum samples were tested simultaneously, with one replicate well set in each well. The color development results were measured using a microplate reader at a detection wavelength of 450 nm.

[0229] 3) Use Excel to draw a standard curve and curve equation, and calculate the serum IgE concentration.

[0230] 3. Data Analysis and Processing Methods

[0231] Flow cytometry data were analyzed using Flowjo software. Statistical analysis was performed using Graphpad Prism 7 or Excel. Results are presented as mean ± standard error. A P value < 0.05 (indicated by an asterisk) indicated significant differences between the data. The presence of more asterisks indicates a smaller P value and a more significant difference between the data.

[0232] result

[0233] 1. OVA can induce skin inflammation in mice

[0234] This study used an OVA-induced atopic dermatitis (AD) mouse model, using the modeling protocol described previously. Compared with the blank control group, the AD model group showed significant edema, erythema, scaling, and erosion on the dorsal skin (Figure 2A), and the skin condition score increased from 0 to an average of 6.99 (Table 2). Pathological sections revealed thickened skin, an incomplete stratum corneum, and a large infiltration of inflammatory cells in the dermatitis mice (Figure 2B). RT-PCR was used to examine changes in inflammatory cytokine levels in the dorsal skin. Results showed increased expression of the Th2 cytokines IL-4 and IL-13, as well as the Th17 cytokine IL-17, in the skin of AD mice compared with the blank control group, while expression of the Th1 cytokine IFN-γ remained unchanged (Figure 2C). ELISA also significantly increased serum IgE levels in AD mice (Figure 2D). The OVA-induced BALB / c mouse model of allergic disease is a typical type I allergic disease model.

[0235] Table 2: Mouse dermatitis scoring table Six mice were scored for edema, erythema, scaling, and erosion in each group, and the total score ranged from 0 to 12. The PBS-treated group served as the control. * indicates P < 0.05, and ▲ indicates P < 0.01. ▲▲ indicates P > 0.05 between the standard IL-2 + budesonide + small molecule HA group and the budesonide group.

[0236] 2. PEG-IL-2 combined with budesonide has limited therapeutic effect on atopic dermatitis model mice

[0237] In previous studies, our team used a short-term nebulized inhalation regimen of 40kDa polyethylene glycol-modified IL-2 (PEG-IL-2) combined with dexamethasone to promote Treg cell proliferation and upregulate the Treg / Tcon cell ratio in the alveolar lavage fluid of asthma model mice. It can significantly reduce airway hypersensitivity and correct the abnormal polarization of immune Th2 cells [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 tried to use the same regimen for local skin administration to treat mouse dermatitis, with the model group and PBS treatment group as controls. After 3 days of administration, it was observed that compared with the AD model mice, the dermatitis symptoms of the mice in each treatment group were alleviated to varying degrees, and the dermatitis scores were all reduced. Compared with the PBS treatment group, the dermatitis score of the budesonide group alone was more reduced and statistically significant (P < 0.05), but the dermatitis scores of the 40kD PEG-IL-2 group alone and the 40kD PEG-IL-2 + budesonide group were not statistically different from those of the PBS group (Table 2, Figure 3C). Pathological sections showed that the 40kD There was no significant improvement in skin thickness or inflammatory cell infiltration in the PEG-IL-2 and PBS groups, and no significant change in the proportion of CD4+CD25+FOXP3+ regulatory T cells (Tregs) in skin CD4+ cells detected by flow cytometry. However, the skin thickness and inflammatory cell infiltration in mice treated with budesonide alone (currently the standard first-line treatment) and 40kD PEG-IL-2 combined with budesonide were relatively reduced, and the proportion of Treg cells in the skin increased, which was statistically significant compared with the physiological model control group (blank control). However, there was no statistical difference in the proportion of Treg cells between the treatment groups (Figure 3).

[0238] 3. HA can enhance the effect of PEG-IL-2 combined with budesonide in the treatment of atopic dermatitis in mice

[0239] These results indicate that the original combination therapy (40kD PEG-IL-2 combined with glucocorticoids) did not improve efficacy compared to the standard clinical regimen (glucocorticoids). We hypothesize that this is due to the drug's inability to penetrate the deeper layers of the epidermis and dermis to exert its effect. To enhance drug absorption, we administered a separate group of mice with dermatitis with a low-molecular-weight HA (40kD PEG-IL-2, budesonide, and low-molecular-weight HA) to the original combination therapy for three days. The results showed that compared with the group without HA, the group treated with HA showed significant and rapid relief of skin inflammation, with skin returning to normal and a statistically significant decrease in dermatitis scores (Table 2, Figure 4D). Pathological sections revealed a significant decrease in skin thickness and inflammatory cell infiltration, and flow cytometry revealed a statistically significant increase in the proportion of Tregs in the skin, reaching 19.8%. Furthermore, while the control group receiving low-molecular-weight HA alone alleviated dermatitis symptoms to some extent, it did not upregulate skin Tregs (Figure 4). This may be related to the moisturizing properties of HA molecules, confirming that low-molecular-weight HA has no immunomodulatory effects.

[0240] 4. HA combined with drug treatment can prevent the recurrence of dermatitis in dermatitis mice, while HA alone has no preventive effect on the normal skin of sensitized mice

[0241] As previously mentioned, after the initial OVA-induced dermatitis, we combined HA with PEG-IL-2 and budesonide for 3 days. We then attempted to induce dermatitis with topical OVA application, and used mice treated with PBS for 3 days as controls. The results showed that none of the mice in the drug-treated group developed obvious dermatitis during the 7-day topical application period, while mice in the control group developed dermatitis of comparable severity to the initial irritation. We also selected a separate group of mice that had been sensitized with intraperitoneal OVA injection but had not yet developed topical OVA-induced dermatitis. We first applied PEG-IL-2, budesonide, and small-molecule HA topical application for 3 consecutive days, followed by topical OVA application. Dermatitis was successfully induced within 2–4 days of OVA application, and the dermatitis score and skin Treg cell ratio were not statistically different from those in AD model mice (Figure 5). It is possible that HA may not facilitate drug penetration into the skin after allergen sensitization without the induction of dermatitis (lesions).

[0242] 5. Effects of PEG-modified IL-2 with different molecular weights and structures on combined drug treatment of AD mice

[0243] To explore whether the molecular weight of PEG-IL-2 and the modification mode of IL-2 by PEG play a key role in the efficacy of combined drug use, we set up four treatment groups: 20kD PEG-IL-2 + budesonide + small molecule HA, 10kD branched PEG-IL-2 + budesonide + small molecule HA, 10kD (unbranched) PEG-IL-2 + budesonide + small molecule HA, and ordinary IL-2 + budesonide + small molecule HA. The aforementioned 40kD PEG-IL-2 + budesonide + small molecule HA treatment group was used as a control (the molecular weight of the unindicated structure is 40kD PEG and 20kD PEG, both of which are unbranched). The results showed that the PEG-IL-2 treatment groups with different molecular weights and modification methods could significantly alleviate the dermatitis symptoms of mice, with a significant decrease in dermatitis scores and no statistically significant differences between the groups (see Table 2, 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 groups were statistically significant. Under the same 10kDa PEG molecule, there was no statistical difference in the Treg ratio between the unbranched PEG-IL-2 group and the branched PEG-IL-2 group. Interestingly, the molecular weight of ordinary IL-2 (15kDa) is lower than that of PEG-IL-2 (40 / 20 / 10+15kDa), but the efficacy of the ordinary IL-2+budesonide+small molecule HA treatment group was significantly weaker than that of the PEG-IL-2+budesonide+small molecule HA treatment groups. The skin still showed a little scaling. Compared with the 40kD PEG-IL-2+budesonide+small molecule HA treatment group, the dermatitis score was higher, averaging 2.33 points, with a statistically significant difference (P<0.05), and the proportion of Treg in the skin was also the lowest, with a statistically significant difference (P<0.05) (Figure 6). There was no difference in the dermatitis score between the ordinary IL-2+budesonide+small molecule HA group and the budesonide alone group (p>0.05). In terms of inducing skin FOXP3 + CD25 + There was no difference in T regulatory cells between the two groups (p>0.05), indicating that conventional interleukin-2 was ineffective (see Table 2, Figures 6D and E). Small HA molecules did not help conventional IL-2 (15 kDa) penetrate the epidermis and enter the deeper layers of the skin. However, they did help 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 greater than 15 kDa and have been modified with hydrophilic polarity, penetrate deeper into the skin and exert their therapeutic effects. This unexpected discovery allows us to discover that HA can interact with PEG to allow ultra-high molecular weight protein drugs to penetrate the stratum corneum and surface layers of the skin, or to be retained in the dermis (the immune response layer of atopic dermatitis).

[0244] 6.10kd-PEG-IL-2 promotes high expression of FoxP3 and PD-1 in regulatory T cells (Treg)

[0245] We injected PEG-IL-2 modified with non-branched PEG of different molecular weights into the peritoneal cavity of physiological model mice and detected the expression of FoxP3+ regulatory T cells and PD-1 in splenocytes on the 4th day. We unexpectedly found that 10kd-PEG-IL-2 could upregulate Treg cells, while ordinary IL-2, 20kd-PEG-IL-2, and 40kd-PEG-IL-2 could not upregulate Treg. The level of regulatory T cells in the 10kd-PEG-IL-2 group was higher than that in the ordinary IL-2 group, the 20kd-PEG-IL-2 group, and the 40kd-PEG-IL-2 group, and the difference was statistically significant (see Figure 7A).

[0246] In addition, we examined the expression of PD-1 on the surface of Treg cells and found that conventional IL-2 can promote the expression of PD-1 on regulatory T cells. The PD-1 expression level 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 expression level of PD-1 on the surface of Treg cells in the 10kd-PEG-IL-2 group was similar to that in the conventional IL-2 group, and the difference was also not statistically significant (Figure 7B). PD-1 expression on Treg cells enhances the inhibitory effect of Treg cells on effector T cells (Teff).

[0247] 7. Single use of 10kd PEG-IL-2 can increase the proportion of skin Treg cells and has a therapeutic effect on atopic dermatitis

[0248] We topically applied 10kd PEG-IL-2 and 10kd PEG-IL-2 + HA to treat dermatitis in AD mice. Three days later, we observed that skin redness, swelling, and erosion in both groups had improved compared to the previous period. However, the skin remained rough, and the rash had not completely subsided. The dermatitis score decreased, which was statistically significant compared to the original AD model mice. Compared to the original AD model mice, the mice treated with HA had smoother skin and lower dermatitis scores. Pathological sections also showed that skin thickening in both groups was alleviated and inflammatory cell infiltration was reduced. Skin flow cytometry results showed that compared with AD model mice, CD4+CD25+FoxP3+Treg cells were upregulated in the skin of both treatment groups, with statistically significant differences, but no statistical differences were found between the two groups (Figure 8).

[0249] 8.10kd PEG-IL-2 can alleviate airway hyperresponsiveness in asthmatic mice

[0250] We then established four additional groups of mice to further investigate the immunomodulatory effects of 10kd PEG-IL-2 in another classic allergic reaction model, the mouse asthma model. After successful establishment of the mouse asthma model, the mice were divided into three groups and administered PBS, 10kd PEG-IL-2, or 10kd PEG-IL-2 + HA intratracheally daily via a micronebulizer. Three days later, healthy mice were used as blank controls to assess airway responsiveness.

[0251] As shown in Figure 7, 10kd PEG-IL-2 effectively reduced airway resistance and airway hyperresponsiveness in asthmatic mice, with statistically significant differences compared to the blank control and PBS groups. However, HA did not further enhance the effect of 10kd PEG-IL-2 in alleviating airway hyperresponsiveness in the asthmatic mouse model.

[0252] 9. Effects of different ratios of large and small molecular weight HA on combined drug treatment of AD mice

[0253] As previously mentioned, small-molecule HA has a strong adjuvant drug absorption effect but lacks immunomodulatory function. Large-molecule HA has relatively poor skin permeability but can enhance the immunomodulatory capacity of activated Treg cells in the local environment. Therefore, after confirming the immunomodulatory effect of 10kDa PEG-IL-2, to further explore the effects of different molecular weight HAs and their combination ratios on the combination treatment of AD mice, we selected four groups of AD model mice and, without changing the 10kDa PEG-IL-2 + budesonide regimen, treated them with large-molecule HA, 2 / 3 large-molecule HA + 1 / 3 small-molecule HA, 1 / 2 large-molecule HA + 1 / 2 small-molecule HA, and 1 / 3 large-molecule HA + 2 / 3 small-molecule HA, respectively. The small-molecule HA combination groups served as controls. The results showed that all groups significantly and rapidly alleviated dermatitis symptoms, with reduced dermatitis scores. However, the 1 / 2 large-molecule HA + 1 / 2 small-molecule HA and large-molecule HA groups did not significantly improve inflammation compared with the budesonide alone group (Table 2, Figure 10D). Pathological sections revealed significantly thinner skin thickness and reduced inflammatory cell infiltration in all groups of mice, with the small-molecule HA group and the 1 / 3 large-molecule HA + 2 / 3 small-molecule HA group showing the best results, followed by the 1 / 3 small-molecule HA + 2 / 3 large-molecule HA group. Flow cytometry analysis of mouse skin revealed that, with the exception of the 1 / 2 large-molecule HA + 1 / 2 small-molecule HA and large-molecule HA groups, which showed no statistically significant difference compared to the budesonide alone group, all other treatments increased the proportion of skin Tregs, with the small-molecule HA group and the 1 / 3 large-molecule HA + 2 / 3 small-molecule HA group showing the greatest increases, significantly different from the budesonide group (Figures 10C, E). This suggests that both large-molecule and small-molecule HA can enhance the efficacy of PEG-IL-2 combined with budesonide in treating mouse dermatitis, but the effect of small-molecule HA is greater. Low-molecular-weight hyaluronic acid can help the PEGylated protein drug interleukin-2 penetrate and be retained in the skin's immune response layer. The interaction between low-molecular-weight hyaluronic acid and PEG is essential for the entry of large-molecule interleukin-2 proteins into the skin. Skin lesions can disrupt the structure of the skin's stratum corneum, such as the cornified capsule and lipid membrane, as well as the granular layer, the last line of defense of the skin barrier. Whether this is necessary for interleukin-2 to enter the skin and exert its immune effects remains uncertain. Considering the immunomodulatory function of large-molecule HA, we believe that a regimen of 10kDa PEG-IL-2 + budesonide + 1 / 3 large-molecule HA + 2 / 3 small-molecule HA can both increase the proportion of skin Tregs and enhance Treg function, making it the optimal dosing regimen at present.

[0254] 10. The type of glucocorticoid has no significant effect on the efficacy of combined medication

[0255] To investigate the effects of different types of glucocorticoids combined with PEG-IL-2 and HA on the treatment of dermatitis in mice, we administered a separate group of mice with a combination of 10kDa PEG-IL-2 + dexamethasone (+ HA (large molecule: small molecule ratio of 1:2)) and 10kDa PEG-IL-2 + budesonide + HA (large molecule: small molecule ratio of 1:2) as a control. The results showed that both groups significantly and rapidly alleviated dermatitis symptoms, reduced inflammatory cell infiltration, and increased the proportion of skin Tregs, with no statistically significant differences between the two groups (see Table 2 and Figure 11).

[0256] Efficacy of a 10 kDa PEG-IL-2 combined with budesonide and 1 / 3 large molecule HA + 2 / 3 small molecule HA regimen

[0257] Significantly better than the first-line clinical solution (topical glucocorticoids)

[0258] As previously mentioned, we selected a 10kDa PEG-IL-2 combined with budesonide and a 1 / 3 large molecule HA + 2 / 3 small molecule HA dosing regimen as the optimal regimen. To further compare this with the current first-line clinical treatment regimen (topical glucocorticoids—i.e., budesonide alone), we used RT-PCR to examine changes in the levels of inflammatory cytokines IL-4, IL-13, IL-17, and IFN-γ in the skin of mice in both groups. We also measured serum IgE concentrations in both groups by ELISA, using a blank group and an AD model group as controls. The results showed that compared with the control group, skin expression of IL-4, IL-13, and IL-17 was downregulated in both treatment groups, with the combination group showing a more pronounced decrease, while IFN-γ expression remained unchanged (P>0.05). Serum IgE concentrations also decreased in both groups, with the combination group showing a greater decrease (see Figures 12A and 12B). The above data all prove that the combination therapy we selected (10kDa PEG-IL-2 combined with budesonide and 1 / 3 large molecule HA + 2 / 3 small molecule HA) is significantly more effective than the clinical first-line treatment regimen (budesonide alone group).

[0259] The efficacy of a 10 kDa PEG-IL-2 combined with budesonide and 1 / 3 large molecule HA + 2 / 3 small molecule HA regimen in treating atopic dermatitis in mice lasts for at least 6 weeks

[0260] To investigate the long-term efficacy of our established 10kDa PEG-IL-2 combined with budesonide and a 1 / 3 large HA + 2 / 3 small HA regimen, we randomly selected two groups of AD model mice. One group received a brief 3-day treatment with this regimen, induced by OVA sensitization followed by an OVA patch. The other group received no treatment. After 3 days, both groups remained untreated and housed under identical conditions. Six weeks later, an OVA patch was applied directly to the skin of both groups to induce atopic dermatitis. The untreated AD mice served as a control group.

[0261] The results showed that dermatitis could be re-induced in the control group AD mice 6 weeks later, and the severity of the dermatitis and the proportion of skin Tregs were the same as those of the first induced dermatitis, proving that the mice were still in an OVA-sensitized state after 6 weeks, excluding the possibility that the mice were no longer sensitive to OVA after 6 weeks. However, the mice in the combination group of 10kDa PEG-IL-2 combined with budesonide and 1 / 3 large molecule HA + 2 / 3 small molecule HA were unable to re-induce obvious atopic dermatitis after 6 weeks. The skin remained relatively normal with low dermatitis scores (see Table 2, Figure 13A). Pathological sections showed that the skin of the AD control group was thickened and infiltrated with a large number of inflammatory cells, while the combination group did not show significant thickening and only a small number of inflammatory cells infiltrated (Figure 13B). The proportion of skin Tregs remained as high as approximately 19.5% (vs 14.2% in the control group), a statistically significant difference (Figures 13C, 13D, and 13E). The above results prove that the administration regimen of 10kDa PEG-IL-2 combined with budesonide and 1 / 3 large molecule HA + 2 / 3 small molecule HA can not only effectively alleviate the symptoms of atopic dermatitis in mice, but also prevent the recurrence of dermatitis, and the effect can last for at least 6 weeks.

Claims

1. A pharmaceutical composition for treating atopic dermatitis, comprising polyethylene glycol-modified interleukin 2 (PEG-IL2), a glucocorticoid and a low molecular weight hyaluronic acid (HA), and optionally a pharmaceutically acceptable carrier, wherein the molecular weight of the low molecular weight HA is less than or equal to about 300KD, preferably about 2-20KD, and more preferably about 4KD.

2. The pharmaceutical composition of claim 1, wherein the PEG-IL2 is modified with polyethylene glycol (PEG) having a molecular weight of about 100, 90, 80, 70, 60, 50 or 40 KD or less, Preferably, the PEG is a PEG with a molecular weight of about 5-100, 5-90, 5-80, 5-70, 5-60, 5-50 or 10-40 KD, Preferably, the IL-2 comprises the amino acid sequence of SEQ ID NO: 1, and more preferably, the IL-2 is single-site modified with the PEG at its N-terminus.

3. The pharmaceutical composition of claim 1 or 2, wherein 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, clofluocinolone, clobetasone propionate, clofluocinolone, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumonide, roflenide, buticort, tipredan and derivatives thereof, preferably selected from the group consisting of dexamethasone, budesonide and derivatives thereof.

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

5. The pharmaceutical composition of any one of claims 1 to 4, comprising 10KD PEG-modified IL2, a glucocorticoid and HA, wherein the HA comprises (i) a low molecular weight HA or (ii) a high molecular weight HA and a low molecular weight HA in a mass ratio of about 1:2, and preferably the glucocorticoid is selected from dexamethasone, budesonide and derivatives thereof.

6. Use of PEG-IL2, glucocorticoids and HA in the preparation of a medicament or kit for treating atopic dermatitis, wherein the HA comprises low molecular weight HA, wherein the molecular weight of the low molecular weight HA is less than or equal to about 300KD, preferably about 2-20KD, and more preferably about 4KD.

7. The method of claim 6, wherein the PEG-IL2 is modified with polyethylene glycol (PEG) having a molecular weight of about 100, 90, 80, 70, 60, 50 or 40 KD or less, Preferably, the PEG is a PEG having a molecular weight of about 5-100, 5-90, 5-80, 5-70, 5-60, 5-50 or 10-40 KD, Preferably, the IL-2 comprises the amino acid sequence of SEQ ID NO: 1, and more preferably, the IL-2 is single-site modified with the PEG at its N-terminus.

8. The method of claim 6 or 7, wherein 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, clofluocinolone, clobetasone propionate, clofluocinolone, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumonide, roflenide, buticort, tipredan and derivatives thereof, preferably selected from the group consisting of 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, wherein the mass ratio of high molecular weight HA to low molecular weight HA is about 2:1, or the mass of high molecular weight HA accounts for less than 50% of the total mass of high molecular weight HA and low molecular weight HA, preferably less than 40%, 35%, 30%, 25%, 20%, 15%, 10% or less, more preferably, the mass ratio of high molecular weight HA to low molecular weight HA is less than or equal to about 1:2, preferably about 1:2, wherein the molecular weight of the high molecular weight HA is greater than or equal to about 800KD, for example, about 800-2000KD, preferably about 800-1500KD.

10. The use according to any one of claims 6 to 9, wherein PEG-IL2 is 10KD PEG-modified IL2, and 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, 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 a therapeutically effective amount of PEG-IL2, a glucocorticoid, and a low molecular weight HA, wherein the molecular weight of the low molecular weight HA is less than or equal to about 300 KD, preferably about 2-20 KD, and more preferably about 4 KD.

12. The method of claim 11, wherein the PEG-IL2 is modified with polyethylene glycol (PEG) having a molecular weight of about 100, 90, 80, 70, 60, 50 or 40 kD or less, Preferably, the PEG is a PEG with a molecular weight of about 5-100, 5-90, 5-80, 5-70, 5-60, 5-50 or 10-40 KD, Preferably, the IL-2 comprises the amino acid sequence of SEQ ID NO: 1, and more preferably, the IL-2 is single-site modified with the PEG at its N-terminus.

13. The method of claim 11 or 12, wherein 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, fluocorticoid, mometasone furoate, clofluocorticone, clobetasone propionate, clofluocorticone, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumonide, roflenide, buticort, tipredan and derivatives thereof, preferably selected from the group consisting of dexamethasone, budesonide and derivatives thereof.

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

15. The method of any one of claims 11 to 14, wherein The administered PEG-IL2 is 10KD PEG-modified IL2, and the administered 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. Preferably, the administered glucocorticoid is selected from dexamethasone, budesonide and derivatives thereof.

16. A kit, preferably for treating atopic dermatitis in a subject, comprising PEG-IL2, glucocorticoids and low molecular weight HA, wherein the molecular weight of the low molecular weight HA is less than or equal to about 300 KD, preferably about 2-20 KD, and more preferably about 4 KD.

17. The kit of claim 16, wherein the PEG-IL2 is modified with polyethylene glycol (PEG) having a molecular weight of about 100, 90, 80, 70, 60, 50 or 40 KD or less, Preferably, the PEG is a PEG with a molecular weight of about 5-100, 5-90, 5-80, 5-70, 5-60, 5-50 or 10-40 KD, Preferably, the IL-2 comprises the amino acid sequence of SEQ ID NO: 1, and more preferably, the IL-2 is single-site modified with the PEG at its N-terminus.

18. The kit of claim 16 or 17, wherein 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, clofluocinolone, clobetasone propionate, clofluocinolone, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumonide, roflenide, buticort, tipredan and derivatives thereof, preferably selected from the group consisting of dexamethasone, budesonide and derivatives thereof.

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

20. The kit of any one of claims 16-19, comprising 10KD PEG-modified IL2, a glucocorticoid and HA, wherein the HA comprises (i) a low molecular weight HA or (ii) a high molecular weight HA and a 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 preparation of a medicament for increasing the efficacy of PEG-IL2 and / or glucocorticoids in treating atopic dermatitis, wherein the molecular weight of the low molecular weight HA is less than or equal to about 300KD, for example, about 2-20KD, preferably about 4KD.

22. Use of high molecular weight HA and low molecular weight HA in the preparation of a medicament for increasing the efficacy of PEG-IL2 and / or glucocorticoids in treating atopic dermatitis, wherein the mass ratio of high molecular weight HA to low molecular weight HA is about 2:1, or the mass of high molecular weight HA accounts for less than 50% of the total mass of high molecular weight HA and low molecular weight HA, preferably less than 40%, 35%, 30%, 25%, 20%, 15%, 10% or less, more preferably, the mass ratio of high molecular weight HA to low molecular weight HA is less than or equal to about 1:2, preferably about 1:2, The molecular weight of the high molecular weight HA is greater than or equal to about 800 KD, for example, about 800-2000 KD, preferably about 800-1500 KD. Preferably, the molecular weight of the low molecular weight HA is less than or equal to about 300 KD, for example, about 2-20 KD, preferably about 4 KD.

23. Use of PEG-IL2 in the preparation of a medicament for treating a type I allergic disease in a subject, wherein the PEG-IL2 is 10KD PEG-modified IL2, preferably, the IL-2 comprises the amino acid sequence of SEQ ID NO: 1, more preferably, IL-2 is modified at its N-terminus by the PEG single site, preferably, the type I allergic disease is selected from systemic allergic reactions including drug anaphylactic shock and serum anaphylactic shock, respiratory allergic reactions such as allergic rhinitis and allergic asthma, digestive tract allergic reactions such as allergic gastroenteritis, skin allergic reactions such as urticaria, atopic dermatitis (eczema) and angioedema.

24. A method for treating type I allergy in a subject, comprising administering to the subject 10KD PEG-modified IL-2, preferably, the IL-2 comprises the amino acid sequence of SEQ ID NO: 1, more preferably, IL-2 is modified at its N-terminus by the PEG single site, preferably, the type I allergic disease is selected from systemic allergic reactions including drug anaphylactic shock and serum anaphylactic shock, respiratory allergic reactions such as allergic rhinitis and allergic asthma, digestive tract allergic reactions such as allergic gastroenteritis, skin allergic reactions such as urticaria, atopic dermatitis (eczema) and angioedema. 25.10KD PEG-modified IL-2, preferably, the IL-2 comprises the amino acid sequence of SEQ ID NO: 1, more preferably, IL-2 is modified with the PEG single site at its N-terminus.