Cyclic peptide and use thereof

Abstract

A cyclic peptide containing HMGB1 as a main target. The cyclic peptide significantly prevents or treats atopic dermatitis by means of inhibiting an inflammatory response, thereby relieving rash and pruritus phenotypes.

Description

A cyclic peptide and its use

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to Chinese patent application No. 202310770256.X, filed with the Patent Office of China on June 28, 2023, entitled “A cyclic peptide and its use,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to the fields of biomedicine and cosmetics, and in particular to a cyclic peptide and its use. Background Art

[0004] Atopic dermatitis is one of the most common dermatological diseases, characterized by an eczematous rash, including papules, blisters, dryness, and scaling, often accompanied by exudative crusting and intense, persistent pruritus. According to the Global Burden of Disease Study, the prevalence of atopic dermatitis in high-income countries is approximately 20% among children and as high as 10% among adults. Atopic dermatitis ranks 15th among all non-fatal diseases and carries the highest burden among skin diseases, measured in disability-adjusted life years (DALYs). Approximately tens of millions of people suffer from atopic dermatitis in my country. The pathogenesis of atopic dermatitis is driven by an interplay of genetic and environmental factors. Patients often have a genetic predisposition, along with multiple factors such as immune dysregulation, skin barrier dysfunction, and dysbiosis, resulting in a complex pathogenesis. Currently available topical medications and skincare products are less than ideal for controlling the disease, posing significant challenges to anti-inflammatory and skin barrier repair efforts.

[0005] High-mobility group protein B1 (HMGB1) is a highly conserved nuclear protein widely distributed in mammalian cells. With the discovery of its proinflammatory role, HMGB1 has become a hot topic in critical care medicine research in recent years. A review published by Cui Lixia et al. revealed the important role of HMGB1 in numerous skin diseases. For example, in atopic dermatitis, HMGB1 expression levels in serum and lesions are elevated compared with controls and positively correlated with dermatitis severity. In a mouse model of psoriasis vulgaris, HMGB1 expression levels are significantly elevated. In addition to its important role in skin diseases, related studies have shown that HMGB1 is also involved in physiological processes such as pigment regulation, inflammation suppression, immune regulation, and aging improvement. Currently known HMGB1 antagonists include, but are not limited to, cisplatin, quercetin, ethyl pyruvate, glycyrrhizin, and triptolide. However, most of the above-mentioned antagonists are chemical drugs or traditional Chinese medicines, which have limited effects, unclear mechanisms, and certain side effects. In summary, HMGB1, as an important target, provides researchers with a research object for the treatment of various diseases.

[0006] Based on the above-mentioned defects, the inventors of this application combined computer-assisted molecular simulation technology to screen a cyclic peptide that is effective in treating or alleviating atopic dermatitis. By constructing a mouse atopic dermatitis model, they verified the important role of the cyclic peptide in effectively alleviating dermatitis and reducing related inflammatory factors, and achieved significant technical effects.

[0007] Summary of the Invention

[0008] The present disclosure relates to a therapeutic agent effective in treating dermatitis;

[0009] In a first aspect, the present disclosure relates to a cyclic peptide having an amino acid sequence of CIPRFYLFYC.

[0010] In some embodiments, the cyclic peptide is formed by forming a ring with a disulfide bond between the first and last cysteines.

[0011] In some embodiments, modifications such as hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation can be performed on the amino acid side chain groups, amino terminus or carboxyl terminus of the cyclic peptide to obtain derivatives of the cyclic peptide without affecting its biological activity.

[0012] In a second aspect, the present disclosure relates to a pharmaceutical composition comprising the cyclic peptide and a pharmaceutically acceptable carrier.

[0013] In some embodiments, the pharmaceutically acceptable carrier is selected from isotonic saline, ethanol, phosphate-buffered saline, balanced salt solutions, and dimethyl sulfoxide (DMSO).

[0014] In other embodiments, the composition may be administered orally, by inhalation, rectally, vaginally, topically, nasally, ophthalmically, or parenterally.

[0015] In other embodiments, the composition is in a dosage form selected from the group consisting of an oral dosage form, a parenteral dosage form, a buccal dosage form, a sublingual dosage form, a nasal dosage form, an inhaler, a nebulizer, a topical dosage form, a transdermal dosage form, and a suppository.

[0016] In a third aspect, the present disclosure relates to use of the above-mentioned cyclic peptide or pharmaceutical composition in the preparation of a medicament for treating HMGB1-mediated diseases.

[0017] In some embodiments, the disease is dermatitis.

[0018] In other embodiments, the disease is dermatitis caused by bacterial, fungal or viral infection.

[0019] In other embodiments, the dermatitis is atopic dermatitis.

[0020] In other embodiments, the disease is dermatitis not caused by bacterial, fungal or viral infection.

[0021] In a fourth aspect, the present disclosure relates to use of the cyclic peptide or the pharmaceutical composition in preparing skin care products.

[0022] In a fifth aspect, the present disclosure relates to use of the cyclic peptide or the pharmaceutical composition in preparing cosmetics.

[0023] In a sixth aspect, the present disclosure relates to use of the cyclic peptide or the pharmaceutical composition in preparing a drug for treating dermatitis.

[0024] In a sixth aspect, the present disclosure relates to the use of the cyclic peptide and an antiviral agent or an antibacterial agent in the combined preparation of a drug for treating dermatitis.

[0025] The present inventors used computer-assisted molecular modeling to simulate and predict the structures of the HMGB1 protein and its associated binding proteins. They then annotated and analyzed the key interaction domains of HMGB1 and screened for potential binding sequences. Molecular dynamics simulations were used to further analyze and optimize the peptide structure. The key design principles were to analyze the secondary structure of the peptide sequence in its lower energy states and, through strategies such as amino acid substitution and cyclization, theoretically enhance its stability in solution. Specific peptides with potential HMGB1 binding activity and good stability, obtained through these methods, were synthesized and experimentally tested. Experimental validation confirmed that these sequences exhibited excellent therapeutic efficacy in treating or alleviating dermatitis, and they are expected to become monomeric active ingredients with specific anti-inflammatory activity in cosmetic applications.

[0026] In a seventh aspect, the present disclosure relates to a method for treating dermatitis, comprising administering the above-mentioned cyclic peptide or pharmaceutical composition to a subject.

[0027] The present disclosure relates to the dosage of the cyclic peptide. In some embodiments, the relevant dosage includes: 0.0001 μM-100 μM, preferably 1-20 μM, more preferably 5-12 μM, and most preferably 10 μM. In other embodiments, the relevant dosage is preferably 1-10 nM, more preferably 2-8 nM. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1: A) Predicted binding pattern of the cIY-8 peptide (CIPRFYLFYC) to HMGB1. B) Schematic diagram of the two-dimensional molecular structure of the cIY-8 peptide. C) Schematic diagram of the tertiary structure of the cIY-8 peptide. D) Sequence and structural homology comparison of the human and mouse HMGB1 proteins (human: Homo sapiens shown in blue; mouse: Mus musculus shown in green)

[0029] Figure 2: Results of atopic dermatitis experiments in mice.

[0030] Note: The upper part of Figure 2 is the MC903 group, which only used MC903; the lower part of Figure 2, in addition to the MC903 group, also used the cyclic peptide therapeutic agent cIY8 of the present application.

[0031] Figure 3: Results of atopic dermatitis itch experiment in mice.

[0032] Figure 4: Cytokine experimental results in a mouse atopic dermatitis model.

[0033] Figure 5A: Laser confocal microscopy results.

[0034] FIG5B : Competitive binding assay results of cIY8 and HMGB1. DETAILED DESCRIPTION

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terminology in this field, professionals are specifically referred to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0036] Although the numerical ranges and parameter approximations shown in the broad scope of this application, the numerical values ​​shown in the specific examples are recorded as accurately as possible. However, any numerical value is necessarily contained in a certain error, which is caused by the standard deviation present in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, a range of "1 to 10" should be considered to include any and all sub-ranges between a minimum of 1 and a maximum of 10 (including endpoints); that is, all sub-ranges starting with a minimum of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0037] Example 1: Screening and Acquisition of cIY8 Cyclic Peptide

[0038] Extracellular HMGB1 can bind to TLR4 and induce the expression and production of various cytokines and chemokines. HMGB1 transmits downstream inflammatory signals through TLR4 and TLR2. HMGB1 is composed of two structurally similar box structures, the A-box and B-box domains. Related studies have revealed the contributions of the HMGB1 A-box and B-box domains alone in TLR4 signaling activation. For example, the antibody mAb 2G7 can block the HMGB1 A-box and downstream signaling pathways.

[0039] Based on the above information, the inventors of the present application identified a new polypeptide inhibitor molecule cIY8 targeting HMGB1 through a polypeptide-protein complex prediction method and a molecular force field-based simulation method.

[0040] 1. Download the three-dimensional structures of HMGB1 and related proteins from the RCSB Protein Data Bank (RCSB PDB) (https: / / www.RCSB.org) and the AlphaFold Protein Structure Database, which provides over 200 million predicted protein structures (https: / / AlphaFold.ebi.ac.uk / ). AlphaFold2Multimer generated predicted binding sites for peptide-protein complexes (Figure 1A).

[0041] 2. ALPHAPICKLE software was used to analyze the predicted alignment errors and predicted local distance difference tests. Chimera and PyMol software were used to simulate the molecular interactions of the complex structure. Molecular dynamics simulations of peptides and peptide-protein complexes were performed using GROMACS software. Dynamics simulations were performed for 3.0 ns in simulated aqueous solution at 300 K.

[0042] 3. Chimera and PyMol software were used to analyze the peptide and peptide-protein complex structures. Chemical Sketch was used to generate a two-dimensional schematic diagram and a three-dimensional simulation of the peptide's chemical structure (Figures 1B and 1C). Based on the similarity of the amino acid sequences of specific regions of mouse and human HMGB1 (Figure 1D), the biological effects of the cIY8 cyclic peptide were confirmed through mouse functional experiments.

[0043] Example 2: Construction of atopic dermatitis model and drug efficacy verification

[0044] 2.1 Mouse pruritus experiment

[0045] To verify the relevant drug efficacy, an atopic dermatitis model was constructed and the efficacy was verified. Studies have shown that topical application of MC903 can induce atopic dermatitis syndrome in mice (Wang Can et al., Establishment of a calcipotriol-induced atopic dermatitis mouse model, 2016); therefore, MC903 (calcipotriol), commonly used in the field, was used to model the atopic dermatitis phenotype. The cyclic peptide of the present application was named: cIY8 in this experiment. In addition, to better avoid the influence of individual factors of mice, each mouse was treated with MC903 alone in the left ear and MC903 + cIY8 in the right ear.

[0046] The specific steps are as follows:

[0047] 1. Eight-week-old C57 / 6J mice were used for the experiment, with 3 mice in each group.

[0048] 2. Use a pipette to apply 20ul / mouse of MC903 (2nM) to the left ear of each mouse; for the right ear: apply 20ul / mouse of a mixture of MC903 (2nM) + HMGB1 inhibitor cIY8 (100μM) for 7 consecutive days, once a day.

[0049] 3. On the 8th day, animal behavior analysis was performed. The number of itching on both sides of the mice within 1 hour was recorded using a Nikon camera. A positive reaction was defined as the mouse lifting its hind legs and scratching the back of its neck. The number of itching on both sides was counted and the mice were photographed at the same time.

[0050] 4. Based on gross skin lesion analysis (see Figure 2), the treated mice showed significantly less ear edema, thickening, and erythema than the untreated group. Furthermore, the treated mice scratched significantly less frequently than the untreated mice (see Figure 3), a statistically significant difference.

[0051] The experimental results show that cIY8 can effectively reduce skin lesions and itching symptoms caused by dermatitis.

[0052] 2.2 Cytokine detection experiment

[0053] The expression of cytokines in mice after drug administration was detected. The specific steps are as follows:

[0054] Total RNA from lesional skin was extracted using TRIzol (Invitrogen) and then The DNA was reverse transcribed into cDNA using the One-Step gDNA Removal and cDNA Synthesis SuperMix Kit. qRT-PCR was then performed. The total volume was 20 μL, including 10 μL PowerUp SYBR Green (Applied Biosystems), 1 μL forward substrate, 1 μL reverse primer, and 2 μL cDNA. Cycling was performed at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 58-65°C for 34 seconds. GAPDH was used as an internal control. MC903 served as the modeling group, MC903 + cIY8 served as the drug-treated group, and the untreated group served as the healthy control. For relevant primers, please refer to Table 1.

[0055] Table 1: Primers used in this experiment

[0056] The experimental results showed (Figure 4): Compared with the modeling group, cIY8 can significantly inhibit the expression of various inflammatory cytokines (IL-1α, IL-4, IL-6, IL-13, IL-31) in the lesions of atopic dermatitis model mice, which explains the mechanistic reason why cIY8 can relieve dermatitis and reduce itching.

[0057] 2.3 HMGB1 targeted inhibition mechanism experiments

[0058] 1. Human fibroblasts (HFF-1P27) were seeded in confocal microplates and allowed to adhere for 24 hours.

[0059] 2. HMGB1 (500 ng / ml) and cIY8 (concentration gradient, specifically: 10 μM, 1 μM, 500 nM, 100 nM) were co-incubated at room temperature for 30 minutes and then added to the cells. The cells were cultured in a 37°C incubator for 1 hour and then the samples were collected.

[0060] 3. Follow the instructions of the Biyuntian NFκB p65 Activation Detection Kit (Cat. No. SN368).

[0061] 4. After staining, take photos using a Leica DMI8 laser confocal microscope, and select three fields of view for each sample.

[0062] 5. Use Image J image processing software to perform data statistics and analysis. The images are displayed at a 20X image field of view.

[0063] The experimental results showed ( FIG5A - FIG5B ) that cIY8 could competitively bind to HMGB1, and this competitive binding was concentration-dependent.

[0064] The above experimental results show that the cyclic peptide obtained in this application can effectively and competitively bind to HMGB1, inhibit its downstream signaling pathway, relieve inflammatory response, and effectively treat or relieve the itching symptoms caused by atopic dermatitis, achieving unexpected technical effects.

[0065] The above describes the embodiments of the present disclosure. However, the present disclosure is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the scope of protection of the present disclosure. Industrial Applicability

[0066] The present disclosure provides a cyclic peptide and its use. The cyclic peptide has excellent therapeutic efficacy in treating or alleviating dermatitis and is expected to become a monomeric functional ingredient with specific good anti-inflammatory activity in cosmetic applications.

Claims

1. A cyclic peptide, whose amino acid sequence is CIPRFYLFYC.

2. The cyclic peptide according to claim 1, wherein the cyclic peptide is formed by a disulfide bond between the first and last cysteines.

3. A pharmaceutical composition comprising the cyclic peptide according to any one of claims 1 or 2 and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition of claim 3, wherein the pharmaceutically acceptable carrier is selected from isotonic saline, ethanol, phosphate buffered saline, balanced salt solution and dimethyl sulfoxide (DMSO).

5. The pharmaceutical composition of any one of claims 3-4, which is in a dosage form selected from the group consisting of oral dosage forms, parenteral dosage forms, buccal dosage forms, sublingual dosage forms, nasal dosage forms, inhalers, nebulizers, topical dosage forms, transdermal dosage forms and suppositories.

6. Use of the cyclic peptide according to any one of claims 1 to 2 or the pharmaceutical composition according to any one of claims 3 to 5 in the preparation of a medicament for treating HMGB1-mediated diseases.

7. The use according to claim 6, wherein the disease is dermatitis.

8. The use according to claim 6 or 7, wherein the disease is atopic dermatitis.

9. The use according to any one of claims 6 to 8, wherein the disease is dermatitis caused by non-bacterial or non-viral infection.

10. Use of the cyclic peptide according to any one of claims 1 to 2 or the pharmaceutical composition according to any one of claims 3 to 5 in the preparation of skin care products.

11. Use of the cyclic peptide according to any one of claims 1 to 2 or the pharmaceutical composition according to any one of claims 3 to 5 in the preparation of cosmetics.

12. Use of the cyclic peptide according to any one of claims 1 to 2 and an antiviral agent or an antibacterial agent in the combined preparation of a drug for treating dermatitis.

13. The use according to claim 12, wherein the dermatitis is atopic dermatitis.

14. The use according to claim 12 or 13, wherein the dermatitis is caused by non-bacterial or non-viral infection.

15. A method for treating dermatitis, characterized in that: The method comprises administering the cyclic peptide according to any one of claims 1 to 2 or the pharmaceutical composition according to any one of claims 3 to 5 to a subject.

16. The method according to claim 15, characterized in that The dosage of the cyclic peptide is 0.0001 μM-100 μM.

17. The method according to claim 15 or 16, characterized in that The dosage of the cyclic peptide is 1-20 μM.

18. The method according to any one of claims 15 to 17, characterized in that: The dosage of the cyclic peptide is 5-12 μM.

19. The method according to any one of claims 15 to 17, characterized in that: The dosage of the cyclic peptide is 1-10 nM.

20. The method according to claim 19, characterized in that The dosage of the cyclic peptide is 2-8 nM.

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