Application of marine small molecule peptide and active variant thereof in preparation of medicines for treating skin diseases

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a marine small molecule peptide and an active variant thereof in preparation of a medicine for treating skin diseases, the marine small molecule peptide and the active variant thereof can effectively inhibit keratinocyte pan apoptosis induced by TNF-alpha and IFN-gamma, the formation of a PANoptosome complex is blocked through a multi-target synergistic effect, and the effect of treating the skin diseases is achieved. The oxidative stress is reduced, and mitochondria is stabilized, so that a skin inflammatory cell death network is inhibited from the source. The variant has the same high sequence as the peptide, retains the activity of inhibiting cell pan-apoptosis, and has enhanced stability, skin permeability, in vivo half-life or efficacy. Based on the action mechanism, the peptide and the variant thereof can be used for preparing medicines for preventing and treating intractable skin diseases such as epidermis necrolysis, psoriasis, atopic dermatitis, skin lupus erythematosus and lichen planus. The invention also provides a composition containing the peptide or the variant thereof, and dosage forms comprise an external preparation and an injection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a marine small molecule peptide and its active variants in the preparation of drugs for skin diseases. Background Technology

[0002] As the largest organ and the first line of immune barrier in the human body, the skin is a major medical challenge in clinical practice due to the protracted course, high recurrence rate, and treatment resistance of various chronic inflammatory and autoimmune skin diseases (such as epidermal necrolysis, psoriasis, atopic dermatitis, cutaneous lupus erythematosus, and lichen planus). Although these diseases present with diverse clinical manifestations, their core pathological features are highly consistent: the synergistic overexpression of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in the local skin lesions triggers abnormal keratinocyte death, forming a vicious cycle of "inflammation-cell death-inflammatory amplification," ultimately leading to skin tissue damage and functional impairment.

[0003] Existing clinical treatments (such as glucocorticoids, immunosuppressants, and single-cytokine targeted biologics) have significant limitations: either their targets are located upstream or parallel to the panapoptotic pathway, failing to directly intervene in the core process of abnormal keratinocyte death; or they suffer from limited response rates, significant long-term side effects (such as infection risk and skin atrophy), and a tendency to develop drug resistance. Therefore, developing novel drugs that can precisely target the core pathway of abnormal keratinocyte death, have high safety, and a clearly defined mechanism is an urgent need in the field of skin disease treatment.

[0004] Recent studies have revealed that panapoptosis, synergistically induced by TNF-α and IFN-γ, is a key form of abnormal keratinocyte death. This process is initiated by upstream sensors such as ZBP1, integrating pyroptosis, apoptosis, and necroptosis through the assembly of the PANoptosome complex, ultimately leading to cell disintegration and the release of inflammatory factors. Although existing research has clarified the core role of the ZBP1-mediated PANoptosome pathway in skin injury, no drugs have yet been found that can directly, efficiently, and safely inhibit this pathway. In particular, there is a lack of candidate molecules derived from natural marine organisms with well-defined structures and excellent drug-like properties, which presents a key technological gap for the development of this invention.

[0005] Marine biological resources are an important source of novel bioactive molecules. The applicant's prior patent (CN202410696181.X) discloses a marine small molecule peptide containing the amino acid sequence GGEGPPW, used for preventing and treating sunburn. Its mechanism of action is based on antioxidant activity that scavenges free radicals, targeting only physical skin damage caused by ultraviolet radiation. This is fundamentally different from the "TNF-α / IFN-γ-induced immune inflammatory skin disease" targeted by this invention in terms of pathological mechanism, therapeutic goal, and target. Although TNF-α / IFN-γ... The induced pan-apoptotic pathway exists in some inflammatory diseases, but the pathological microenvironment of skin diseases (such as psoriasis and epidermal necrolysis) differs fundamentally from that of sunburn: sunburn begins with UV-induced physical damage, with inflammatory factor release mainly consisting of IL-1β and TNF-α, and no autoimmune components involved; while diseases such as psoriasis and atopic dermatitis are centered on immune dysregulation, accompanied by activation of specific inflammatory axes such as IL-23 / IL-17, and the initiation threshold and signal amplification pathway of pan-apoptosis of keratinocytes are completely different from those of sunburn. Existing technologies do not disclose that "pan-apoptotic inhibitors can be applied across physical damage and immune diseases," nor do they suggest that the pan-apoptotic inhibitory activity of the marine small molecule peptide of this invention can cover the above-mentioned different pathological types of skin diseases. Applying this peptide and its variants to other diseases other than sunburn requires independent target validation, drug delivery system optimization, and pharmacodynamic evaluation.

[0006] In summary, given the technical limitations of existing therapies in precisely targeting the core pathway of pan-apoptosis in keratinocytes, and the lack of research and development of marine-derived pan-apoptotic inhibitors, this study combines novel bioactivity discoveries of known sequence peptides to develop a marine small molecule peptide and its active variants that can directly inhibit TNF-α / IFN-γ-induced pan-apoptosis in keratinocytes and is applicable to various immune-inflammatory skin diseases. This has significant clinical implications and application prospects. Summary of the Invention

[0007] The purpose of this invention is to provide an application of marine small molecule peptides and their active variants in the preparation of drugs for skin diseases, filling the gap in the clinical lack of drugs for precise intervention of the panapoptotic pathway of keratinocytes.

[0008] The objective of this invention is achieved through the following technical solution: This invention provides the use of a marine small molecule peptide, its active variant, or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of skin diseases, said marine small molecule peptide comprising an amino acid sequence as shown in SEQ ID NO.1.

[0009] Furthermore, the skin disease is selected from epidermal necrolysis, psoriasis, atopic dermatitis, cutaneous lupus erythematosus, and lichen planus.

[0010] Furthermore, the active variant has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.1 and retains the activity of inhibiting pan-apoptosis of keratinocytes.

[0011] Furthermore, the activity of retaining inhibition of pan-apoptosis of keratinocytes is such that, in a pan-apoptotic model of keratinocytes induced by the combined action of TNF-α and IFN-γ, the active variant, at an equimolar concentration, promotes cell survival by no less than 70% of the peptide shown in SEQ ID NO.1.

[0012] Furthermore, the active variant comprises one or more modifications selected from the following: (a) One or more amino acid residues are replaced by their corresponding D-type amino acid residues; (b) One or more amino acid residues are replaced by a conserved amino acid, wherein the conserved amino acid substitution is selected from glycine replaced by alanine or sarcosine, glutamic acid replaced by aspartic acid, proline replaced by an N-methylated amino acid, and tryptophan replaced by phenylalanine or tyrosine. (c) The N-terminus is acetylated, acylated, or linked to a cell-penetrating peptide sequence; (d) The C-terminus is amidated; (e) One or more amide bonds in the peptide backbone are N-methylated; (f) Formation of an intramolecular cyclization structure, wherein the cyclization is achieved through head-to-tail connection, disulfide bond between side chains, lactam bond or click chemical connection; (g) Covalently linked 1- or more polyethylene glycol molecules with molecular weights of 2kDa-20kDa.

[0013] Furthermore, the marine small molecule peptide or its active variants inhibit the panapoptotic pathway of keratinocytes through multi-target action.

[0014] Furthermore, the multi-target action includes one or more of the following: (a) Downregulate the expression or activity of one or more of the following proteins: ZBP1, AIM2, NLRP3, NLRP12, NLRC5, ASC, and FADD; (b) Inhibit phosphorylation activation of RIPK1 and / or RIPK3; (c) Reduce the level or activity of one or more of the following proteins: Cleaved Caspase-3, Cleaved Caspase-8, Bax, and Cytochrome C; (d) Inhibit phosphorylation of MLKL; (e) Reduce the level or activity of one or more of the following proteins: Cleaved Caspase-1, GSDMD-N, and GSDME-N.

[0015] Furthermore, the drug is a topical preparation or an injectable preparation; the topical preparation is selected from creams, gels, ointments, solutions or sprays; the injectable preparation is selected from lyophilized powder for injection or pre-filled injection solution.

[0016] Furthermore, in the topical or injectable formulation, the content of the marine small molecule peptide or its active variant is 0.3125ppm-20000ppm.

[0017] Furthermore, the lyophilized powder injection contains a lyophilization protectant selected from mannitol or sucrose.

[0018] The beneficial effects of this invention are as follows: (1) It pioneers entirely new therapeutic applications: For the first time, the marine small molecule peptide and its active variants have been discovered and confirmed to possess a novel biological activity of inhibiting TNF-α / IFN-γ-induced pan-apoptosis of keratinocytes. Based on this, it has been applied to the prevention and treatment of a series of refractory skin diseases characterized by abnormal keratinocyte death, such as psoriasis, atopic dermatitis, lichen planus, epidermal necrolysis, and cutaneous lupus erythematosus. This invention not only protects the core peptide itself but also extends to a family of variants that retain the core activity obtained through rational design, achieving a leap from a single molecule to a candidate drug system.

[0019] Furthermore, the aforementioned diseases have all been confirmed by existing technologies to possess the core pathological feature of "TNF-α / IFN-γ synergistic induction of pan-apoptosis in keratinocytes," and their pathological processes all depend on a vicious cycle of "inflammation-cell death-inflammatory amplification" (highly matching the target of this invention). This invention has validated the therapeutic effects of peptides and their variants in epidermal necrolysis (an acute severe model) and psoriasis (a chronic inflammation model) (Examples 11 and 12). The core pathological axis of the remaining diseases (atopic dermatitis, cutaneous lupus erythematosus, and lichen planus) is consistent with the aforementioned validated models, and no existing technology indicates that these diseases exhibit specific resistance to pan-apoptotic inhibitors.

[0020] (2) An innovative multi-target mechanism of action was revealed: For the first time, it was elucidated that this peptide and its active variants, in the treatment of skin diseases, synergistically inhibit upstream sensors such as ZBP1, key kinases such as RIPK1 / RIPK3, and multiple downstream execution pathways such as Caspase, MLKL, and GSDMD, thereby fundamentally blocking the formation and function of the PANoptosome death complex. This cutting-edge and complex multi-target mechanism has no logical connection with known simple antioxidant effects, constituting the core of the non-obvious technology.

[0021] (3) Provides an effective clinical solution: Addressing the shortcomings of existing therapies in intervening in the panapoptotic pathway of the skin, this invention provides a well-structured, clearly defined, easily synthesized and optimized lead peptide, its active variants, and corresponding topical and injectable formulations. Experiments have demonstrated that this peptide and its variants can significantly inhibit panapoptosis of keratinocytes and improve skin conditions in cell and animal disease models.

[0022] (4) A creative recombination oriented towards new goals was completed: a completely new application purpose (treatment of skin diseases) and a completely new activity screening standard (i.e., inhibition of pan-apoptosis of keratinocytes) were given to the known peptide sequence (GGEGPPW) and conventional modification technology. This recombination and definition of existing substances and technologies oriented towards specific disease treatment goals has formed a complete and non-obvious technical solution.

[0023] (5) It demonstrates broad-spectrum therapeutic potential: Based on the understanding that multiple skin diseases share the core pathological axis of TNF-α / IFN-γ-keratinocyte panapoptosis, this single active peptide (and its variants) is expected to achieve intervention for multiple diseases such as psoriasis, atopic dermatitis, lichen planus, epidermal necrolysis (SJS / TEN) and cutaneous lupus erythematosus, and has the potential advantage of "one drug with multiple effects" and has broad application prospects.

[0024] (6) A clear foundation for sustainable development has been laid: The core peptide and its active variants have the characteristics of small molecular weight, well-defined structure, and ease of synthesis and rational modification. Through strategies such as D-type amino acid substitution, cyclization, and PEGylation, their stability, transdermal properties, pharmacokinetics, and efficacy can be systematically optimized, laying a solid material and design foundation for the development of novel skin-targeted drugs that combine efficacy and patient compliance.

[0025] Compared with existing clinical treatments, the advantages of this invention are further reflected in: 1) Compared to glucocorticoids (non-specific anti-inflammatory, long-term use leads to skin atrophy), the peptides of this invention precisely target the panapoptotic core pathway, and as demonstrated in Example 7, they are non-cytotoxic and do not damage the skin barrier; 2) Compared with single-cytokine targeted biological agents (such as anti-TNF-α antibodies with a response rate of about 40%-60%), the multi-target mechanism of the present invention (simultaneous inhibition of ZBP1, RIPK1 / RIPK3, Caspase, etc.) showed in the protein detection of Example 7 that it can comprehensively block the panapoptotic pathway. The disease remission rate in animal models (Examples 11 and 12) is significantly higher than the publicly available data of existing agents. 3) Compared with existing marine-derived peptides, this invention is the first to discover its pan-apoptotic inhibitory activity, realizing a leap from "chemical protection" to "biological regulation", and the mechanism of action is not logically related to antioxidation, constituting an independent technical contribution.

[0026] In summary, this invention is not merely a simple extension of known antioxidant activities, but rather represents a paradigm shift from chemical protection to biological regulation. It innovatively discovers and utilizes a previously unknown therapeutic efficacy that directly targets a core hub of immune regulation and cell death (the panapoptotic pathway). Through creative recombination, a novel and profound mechanism of action has been discovered and elucidated, thereby opening up entirely new therapeutic applications and providing a complete solution encompassing the core compound, its optimized variants, and formulations. This invention constructs a complete innovative chain from material basis and mechanism of action to therapeutic applications, providing technical support for addressing clinical challenges. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is the reversed-phase high-performance liquid chromatography (RP-HPLC) chromatogram of the marine small molecule peptide GGEGPPW in Example 1 of the present invention; Figure 2 The image shows the electrospray ionization mass spectrometry (ESI-MS) analysis of the marine small molecule peptide GGEGPPW in Example 1 of this invention. Figure 3 The image shows the results of HaCaT cell panapoptosis model construction and cell viability detection in Example 4 of this invention (**p<0.01). Figure 4 This is a graph showing the effect of simultaneous treatment with marine small molecule peptides and cytokines on the viability of HaCaT cells in Example 5 of the present invention (different letters indicate statistical differences). Figure 5 This is a graph showing the effect of marine small molecule peptides on the viability of HaCaT cells after cytokine induction treatment in Example 6 of the present invention (different letters indicate statistical differences). Figure 6 The image shows the fluorescence pattern of cell death morphology observed by Calcein-AM / PI fluorescence double staining in Example 7 of this invention. Green fluorescence represents live cells and red fluorescence represents dead cells, which visually demonstrates the protective effect of peptide treatment on cell death. Figure 7 This is a bar chart based on quantitative statistical analysis of cell mortality rate in Example 7 of the present invention (different letters indicate statistical differences). Figure 8This is a scatter plot of apoptosis detected by Annexin V-FITC / PI double staining in Example 7 of the present invention. Figure 9 This is a bar chart showing the quantitative statistical analysis of cell apoptosis rate in Example 7 of the present invention (different letters indicate statistical differences). Figure 10 This is an image of the cell ultrastructure observed by transmission electron microscopy (TEM) in Example 7 of the present invention; Figure 11 This is a fluorescence micrograph of the intracellular reactive oxygen species (ROS) level detected by the DCFH-DA fluorescent probe in Example 7 of the present invention; Figure 12 Based on Embodiment 7 of the present invention Figure 11 Quantitative statistical bar chart of intracellular ROS levels based on fluorescence intensity (different letters indicate statistical differences). Figure 13 This is a fluorescence micrograph of mitochondrial membrane potential detected by the JC-1 fluorescent probe in Example 7 of the present invention. In the figure, red fluorescence represents JC-1 polymer (normal membrane potential) and green fluorescence represents JC-1 monomer (decreased membrane potential). Figure 14 This is a bar chart showing the statistical ratio of JC-1 monomer / polymer in Example 7 of the present invention (different letters indicate statistical differences). Figure 15 This is a scatter plot of differentially expressed genes from transcriptome sequencing in Example 7 of the present invention. Figure 16 This is a bubble chart showing the KEGG pathway enrichment analysis of differentially expressed genes between the model group and the blank control group in Example 7 of this invention. Figure 17 This is a bubble chart showing the KEGG pathway enrichment analysis of differentially expressed genes between the peptide-treated group and the model group in Example 7 of this invention. Figure 18 The image shows the results of Western blotting detection of ZBP1-RIPK1 / RIPK3 signal axis protein expression in Example 7 of this invention (*p<0.05, **p<0.01). Figure 19 This is a graph showing the results of Western blotting detection of the expression of multiple pattern recognition receptor proteins in Example 7; including protein bands and quantitative bar graphs of AIM2, NLRP3, NLRP12, and NLRC5, demonstrating the multi-target inhibitory effect of peptides on multiple inflammatory sensors (**p<0.01). Figure 20The image shows the results of detecting the expression of key executive proteins of apoptosis and necroptosis using Western blotting in Example 7 of this invention (*p<0.05, **p<0.01). Figure 21 The image shows the results of detecting pyroptosis-related protein expression by Western blotting in Example 7 of this invention (*p<0.05, **p<0.01). Figure 22 The image shows the clinical score of skin damage in the mouse model of epidermal necrosis and lysis in Example 11 of this invention (**p<0.01). Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] Based on the novel applications of the marine small molecule peptide (containing the amino acid sequence GGEGPPW, SEQ ID NO: 1) discovered in this invention in inhibiting TNF-α / IFN-γ-induced pan-apoptosis of keratinocytes and treating related skin diseases, this invention not only protects the core sequence peptide itself but also explicitly covers the active variants obtained through rational design. These active variants, while maintaining a high degree of homology with the core sequence, are designed through specific structural modifications to systematically address the pharmaceutical challenges (drug stability, skin permeability, in vivo half-life, efficacy, etc.) faced when administered topically or systemically as a treatment for skin diseases, ultimately achieving superior preventative and / or therapeutic effects.

[0035] The screening and evaluation of the active variants in this invention are always based on their novel therapeutic mechanism-related activity of inhibiting TNF-α / IFN-γ-induced pan-apoptosis in keratinocytes. This standard is fundamentally different from the modification and screening based on chemical antioxidant activity that scavenges free radicals described in the background art.

[0036] To obtain candidate molecules suitable for different clinical scenarios (such as topical transdermal or injectable administration), one or more of the following strategies can be used to optimize the structure of the peptide: (1) Improve metabolic stability and resistance to proteolytic activity D-amino acid substitution: Replacing L-amino acid residues involved in peptide bonds in the sequence that are susceptible to cleavage by skin or in vivo proteases with their D-enantiomers (e.g., [D-Glu]). 3 [] or [D-Trp] 7 [Replacement]. This modification effectively resists hydrolysis by proteases and significantly prolongs the peptide's retention time at the site of action and its half-life in vivo.

[0037] N-methylation modification: Introducing a methyl group (-CO-N(CH3)-) onto the nitrogen atom of one or more amide bonds (-CO-NH-) in the peptide backbone. This modification can stereotactically hinder the recognition and cleavage of peptide bonds by proteases, and is a classic strategy to improve the metabolic stability of peptide compounds, while also moderately increasing membrane permeability.

[0038] Terminal modification: Acetylation or acylation of the N-terminus of a peptide, or amidation of the C-terminus, can eliminate terminal charge, resist exopeptidase degradation, and improve its physicochemical properties (such as lipid solubility).

[0039] (2) Enhance membrane permeability and intracellular delivery Linking cell-penetrating peptides (CPPs): Covalently linking a known cell-penetrating peptide sequence (such as Tat, Penetratin, etc.) to the N- or C-terminus of a peptide can significantly enhance its ability to cross the keratinocyte membrane and ensure that it acts on intracellular targets (such as ZBP1).

[0040] Linking a permeation-enhancing group: Linking a short-chain fatty acid (such as an octanoyl group) or other molecules known to promote skin penetration to the N-terminus can enhance the transdermal absorption efficiency of peptides when applied topically.

[0041] (3) Conformational constraints and affinity optimization Intramolecular cyclization: This involves forming cyclic structures in peptide chains through head-to-tail cyclization (forming head-to-tail amide bonds), side-chain cyclization (such as introducing a pair of cysteine ​​residues to form a disulfide bond, or using Lys to form a lactam bond with the Glu / Asp side chain), or modern click chemistry methods (such as alkyne-azide cycloaddition). Cyclocyclization significantly stabilizes the active conformation, reduces conformational entropy, thereby increasing binding affinity and selectivity to target proteins, and enhancing their resistance to enzymatic degradation.

[0042] Conservative amino acid substitution: This involves rationally designing the substitution of one or more amino acids to fine-tune the physicochemical properties of a peptide while maintaining the core pharmacophore. Exemplary substitutions include: glycine (Gly) replaced by alanine (Ala) or sarcosine (Sar, N-methylglycine); glutamic acid (Glu) replaced by aspartic acid (Asp); proline (Pro) replaced by N-methylated amino acids or other cyclic amino acids; and tryptophan (Trp) replaced by phenylalanine (Phe) or tyrosine (Tyr).

[0043] (4) Improve pharmacokinetic properties (especially for injection administration) PEGylation: One or more polyethylene glycol (PEG) chains are covalently linked to the N-terminus, C-terminus, or via an introduced lysine (Lys) side chain of the peptide. Linear or branched PEG with a molecular weight of 2 kDa to 20 kDa is preferred. PEGylation significantly increases the apparent molecular weight of the peptide, effectively reduces renal clearance, substantially prolongs its plasma half-life, and reduces immunogenicity.

[0044] (5) Combinatorial optimization Those skilled in the art can combine the above-mentioned multiple modification strategies to design and synthesize variants with multiple optimized features (e.g., variants containing D-type amino acids, N-methylation, and terminal PEG modification) to systematically address multiple challenges such as stability, delivery, and pharmacokinetics, thereby obtaining candidate molecules that are more suitable for drug development.

[0045] The active variants obtained through the above rational design are an integral part of this invention. Together with the original sequence peptide, they constitute an innovative drug system that protects skin cells, inhibits pathological pan-apoptosis, and is used to treat specific skin diseases.

[0046] The inventors also emphasize that the above modification strategies are all designed based on the sequence characteristics of the core peptide (SEQ ID NO.1) of this invention (7 amino acid residues, including easily hydrolyzed peptide bonds and polar amino acids), and their scientific validity has been verified through representative variants (D-type amino acid substitution, cyclization, and terminal modification). 1) D-type amino acid substitution targets the easily hydrolyzed sites corresponding to Glu residues in the peptide chain. Example 2 confirms that this modification can retain core activity and improve stability. Similarly, D-type substitution of other amino acids that are easily recognized by proteases (such as Pro and Trp) can achieve stability optimization based on the same technical principle. 2) Cyclization modification enhances target affinity by fixing the active conformation. Example 3 confirms that its activity is superior to that of the original peptide. Other cyclization methods (such as lactam bonds and click chemical linkages) are conventional methods for optimizing peptide drugs and do not change the structure of the core pharmacophore. 3) The mechanism of action of modifications such as PEGylation and N-methylation (prolonging half-life and enhancing transdermal properties) has been clearly defined in the field of peptide drugs (refer to common knowledge in the field). Combined with the small molecule characteristics of the peptides of this invention, such modifications can be applied directly and their retention of core activity can be predicted without additional verification.

[0047] The following examples will specifically illustrate the preparation, activity verification, mechanism of action, formulation development, and efficacy evaluation of the peptides and their active variants.

[0048] Example 1: Solid-phase synthesis and structural confirmation of marine small molecule peptides The purpose of this embodiment is to provide a reproducible preparation method for the core active ingredient of this invention—the marine small molecule peptide GGEGPPW (Gly-Gly-Glu-Gly-Pro-Pro-Trp, SEQ ID NO.1), and to rigorously confirm its chemical purity and molecular structure, thus providing a material basis and quality assurance for this invention. Specifically, the method is as follows: (1) Synthesis by Fmoc solid-state synthesis method The Fmoc solid-phase synthesis strategy was employed, using 2-chlorotriphenylmethyl chloro resin (degree of substitution 1.03 mmol / g) as a support to sequentially link amino acids from the C-terminus to the N-terminus. The specific steps included: ① Resin swelling and initial amino acid loading: The resin was swollen with dichloromethane (DCM, 15 mL / g resin) for 30 minutes. After filtration, 3 molar excess of Fmoc-Trp (Boc), appropriate amount of N,N-dimethylformamide (DMF) and 10 molar excess of N,N-diisopropylethylamine (DIEA) were added. The mixture was shaken at room temperature for 60 minutes. The residual sites were blocked with methanol.

[0049] ② Deprotection-washing-detection cycle: Remove the liquid, add 20% piperidine DMF solution (15 mL / g resin), shake for 5 minutes, then remove the solution. Add an equal volume of fresh piperidine DMF solution again, and continue shaking for 15 minutes. Take a small amount of resin particles, wash with ethanol, add ninhydrin reagent, and heat at 105-110℃ for 5 minutes. A deep blue color indicates complete deprotection. Wash the resin twice each with DMF, DCM, and DMF, with each wash using 10 mL / g resin.

[0050] ③ Condensation-washing-detection cycle: Add 3 molar excess of the next Fmoc protected amino acid and an equimolar amount of O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, both dissolved in the minimum volume of DMF), immediately add 10 molar excess of DIEA, and react with shaking at room temperature for 30 minutes. Take a small amount of resin for ninhydrin detection; colorless indicates complete condensation. Wash the resin once with DMF, then twice with DCM, and finally twice with DMF, each time using 10 mL / g of resin as solvent.

[0051] ④ Sequence extension: Repeat steps ② to ③, connecting Fmoc-Pro, Fmoc-Pro, Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Gly, and Fmoc-Gly in order from C to N to complete the full sequence assembly.

[0052] ⑤ Final treatment and cutting: Wash the resin twice each with DMF, methanol, DMF and DCM in sequence, remove the solvent and then vacuum dry for 10 minutes. Add cutting solution (V / V: 95% trifluoroacetic acid, 1% water, 2% 1,2-ethylenedithiol, 2% triisopropylsilane, at 10 mL / g), and shake at room temperature for 120 minutes.

[0053] ⑥ Precipitation, purification, and lyophilization: Transfer the lysis buffer to a centrifuge tube, purge with nitrogen until viscous, add pre-cooled anhydrous diethyl ether to precipitate the small molecule peptide, centrifuge, discard the supernatant, wash the precipitate with diethyl ether a total of 6 times, and evaporate to dryness at room temperature. The crude product is purified by high performance liquid chromatography, the target fraction is collected, and after lyophilization, the target small molecule peptide is obtained as a white powder.

[0054] Using the aforementioned Fmoc solid-phase synthesis strategy, a marine small molecule peptide with the target sequence GGEGPPW was successfully prepared. After purification, its chemical purity and molecular structure were systematically confirmed using two complementary techniques: high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS).

[0055] (2) Purity analysis by high performance liquid chromatography Approximately 1.0 mg of the prepared marine small molecule peptide lyophilized powder was dissolved in an aqueous solution containing 0.1% trifluoroacetic acid and diluted to 1 mL. The solution was then filtered through a 0.22 μm filter membrane to obtain the test solution. High-performance liquid chromatography (HPLC) was used with a Kromasil 100-5C18 column (4.6 mm × 250 mm, 5 μm) at 25 ℃. Mobile phase A consisted of acetonitrile containing 0.1% TFA, and mobile phase B consisted of water containing 0.1% TFA. The flow rate was 1.0 mL / min, the detection wavelength was 220 nm, and the injection volume was 10 μL. Gradient elution was employed: from 0 to 20 min, mobile phase A linearly increased from 22% to 47%, while mobile phase B correspondingly decreased from 78% to 53%. At 20.1 min, mobile phase A was switched to 100% and held for a certain period for column washing and regeneration.

[0056] RP-HPLC chromatograms show ( Figure 1 The retention time of the main peak of the synthesized product was 6.808 min, and the peak shape was sharp and symmetrical. Calculations using the area normalization method showed that the area percentage of this main peak was as high as 98.28%, indicating that the chemical purity of the obtained peptide was higher than 98%, meeting the quality requirements for use as a pharmaceutical active ingredient.

[0057] (3) Structural confirmation by electrospray ionization mass spectrometry Solutions of marine small molecule peptides were analyzed using electrospray ionization mass spectrometry (ESI-MS) in positive ion mode. Key mass spectrometry parameters: capillary voltage 2500-3500 V, cone voltage 15-30 V, desolventizing temperature 450 ℃.

[0058] ESI-MS spectrum ( Figure 2 The results showed that, in positive ion mode, significant single-charge ion peaks [M+H] were observed at m / z 699.56, 350.36, and 1398.58, respectively. + Double-charged ion peak [M+2H]² + and the dimer ion peak [2M+H] + The above measured values ​​are respectively compared with the corresponding theoretical ion peaks ([M+H) of the target small molecule peptide GGEGPPW (molecular weight 698.74 Da). + m / z 699.74, [M+2H]² +m / z 350.37, [2M+H] + The molecular weight (m / z 1398.48) is highly consistent with the target sequence, with deviations all less than 0.2 m / z. This result confirms that the molecular weight of the synthesized product is consistent with the target sequence.

[0059] This embodiment establishes an efficient solid-phase synthesis and purification process for the marine small molecule peptide GGEGPPW (SEQ ID NO.1). The high chemical purity (>98%) and correct target molecular structure of the obtained product were confirmed by complementary analytical techniques, HPLC and ESI-MS. This provides a reliable and high-quality raw material guarantee for subsequent bioactivity evaluation, mechanism of action studies, and pharmacodynamic verification, laying the material foundation for this invention.

[0060] Example 2: Preparation and Characterization of D-Type Amino Acid Substitution Variants The purpose of this embodiment is to provide a method for preparing a marine small molecule peptide variant with enhanced metabolic stability. This variant replaces the L-glutamic acid (L-Glu) at the third position in the original peptide (SEQ ID NO.1) with its D-enantiomer (D-Glu), yielding a peptide with the sequence GG(DE)GPPW. By introducing the D-amino acid, the aim is to improve the peptide's resistance to protease hydrolysis, providing a candidate molecule for developing long-acting formulations for topical or systemic drug delivery. The specific steps are as follows: (1) Synthesis process The same Fmoc solid-phase synthesis strategy, resin support, and general operating procedures as in Example 1 were used. The key difference lies in the selection of specific amino acid raw materials: Resin loading and sequence extension: Using 2-chlorotriphenylmethylchloro resin as a carrier, amino acids were sequentially linked from the C-terminus to the N-terminus. The specific steps were the same as in Example 1, with only one change: when linking the third amino acid (corresponding to the Glu site of the prototype peptide), Fmoc-D-Glu (OtBu) was used as the synthetic starting material instead of Fmoc-L-Glu (OtBu) used in Example 1. The linking order, reaction conditions (deprotection, condensation, washing), cleavage, and precipitation steps of the remaining amino acids—Fmoc-Trp (Boc), Fmoc-Pro (two), and Fmoc-Gly (three)—were exactly the same as in Example 1. Purification: The obtained crude peptide was purified using preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The target fraction was collected and freeze-dried to obtain a white powder of the target variant peptide.

[0061] (2) Purity and structural characterization High-performance liquid chromatography (HPLC) purity analysis: The same analytical conditions as in Example 1 were used. The results showed that the retention time of the main peak of the synthesized product differed slightly from that of the prototype peptide (GGEGPPW), a difference consistent with the minor changes in chromatographic behavior caused by the introduction of D-amino acids. The area percentage of the main peak, calculated using the area normalization method, was 97.5%, indicating that the product has high chemical purity and meets the requirements for subsequent biological evaluation.

[0062] Electrospray ionization mass spectrometry (ESI-MS) confirmed the structure: Mass spectrometry analysis was performed in positive ion mode. The main observed peak was a single-charged ion [M+H]. + m / z 699.55. This measured value corresponds to the theoretical molecular weight (698.74 Da) of the target variant peptide GG(DE)GPPW, with a [M+H] value. + The theoretical value (m / z 699.74) is in high agreement, with the deviation within the allowable range of instrument error (<0.3 Da). This result confirms that the molecular weight of the synthesized product is consistent with the designed sequence, indicating that the D-glutamic acid substituted variant has been successfully synthesized.

[0063] This embodiment successfully established a chemical synthesis and purification method for the D-type amino acid substitution variant [D-Glu³]-GGEGPPW (GG(DE)GPPW). Characterization by HPLC and ESI-MS confirmed that the obtained product had the correct structure and high purity. This variant, as a representative example of modification to improve peptide metabolic stability, demonstrated its core biological activity of inhibiting pan-apoptosis of keratinocytes in Example 8, indicating that it fully retains the therapeutic function of the original peptide while possessing superior drug-like potential.

[0064] Example 3: Preparation and Characterization of Cyclic Variants This embodiment aims to provide a method for preparing and characterizing a conformationally restricted cyclized marine small molecule peptide variant. This variant has a cysteine ​​residue (Cys) added to both the N-terminus and C-terminus of the original sequence (SEQ ID NO.1), forming the linear sequence CGHEGPPWC. Cyclation via intramolecular disulfide bonds is then used to improve its biostability and potential target affinity, as detailed below: (1) Synthesis process The Fmoc solid-phase synthesis strategy was adopted, using 2-chlorotriphenylmethyl chloride resin (degree of substitution 1.03 mmol / g) as a carrier to sequentially link amino acids from the C-terminus to the N-terminus.

[0065] The key steps are as follows: ① Initial amino acid loading: After swelling the resin with dichloromethane (DCM), it is reacted with 3 molar excess of Fmoc-Cys(Trt)-OH in the presence of N,N-diisopropylethylamine (DIEA) to load the first cysteine.

[0066] ② Sequence extension: Fmoc-Trp(Boc), Fmoc-Pro, Fmoc-Pro, Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Gly, and Fmoc-Gly are sequentially linked, and finally the second Fmoc-Cys(Trt)-OH is linked to complete the assembly of the linear sequence "Cys-Gly-Gly-Glu-Gly-Pro-Pro-Trp-Cys". Ninhydrin detection was performed after each condensation reaction to ensure complete ligation.

[0067] ③Cut-off and deprotection of side chains: The peptide was cut off from the resin using a cutting solution (trifluoroacetic acid:water:1,2-ethylenedithiol:triisopropylsilane=94:2.5:2.5:1, V / V), and all side chain protecting groups (including the Trt protecting group of Cys) were removed simultaneously to obtain the crude linear peptide CGGEGPPWC.

[0068] ④ Oxidative cyclization: The obtained crude linear peptide was dissolved in 0.1 M, pH 8.0 phosphate buffer, with a peptide concentration of approximately 0.1 mg / mL. The solution was placed in an open container and gently stirred magnetically at room temperature to induce disulfide bonds (-SS-) between the thiol groups (-SH) of the two cysteine ​​residues within the molecule through air oxidation. The reaction was carried out for 24 hours.

[0069] ⑤ Purification: After the reaction was completed, the cyclized product was purified using preparative reversed-phase high-performance liquid chromatography (RP-HPLC). Column: Kromasil 100-5C18 (10 mm × 250 mm, 5 μm); Mobile phase: A was acetonitrile containing 0.1% TFA, and B was water containing 0.1% TFA; gradient elution. The main fraction was collected, lyophilized, and a white powder of the cyclized target peptide was obtained.

[0070] (2) Purity and structural characterization High-performance liquid chromatography (HPLC) analysis: Cyclic peptides typically retain earlier than their linear precursors. Analysis showed a single main peak with an area percentage of 96.8%. Electrospray ionization mass spectrometry (ESI-MS) analysis: Under non-reducing conditions, a single-charged ion peak [M+H] was observed. + The m / z value of 934.08 is consistent with the theoretical molecular weight (933.06 Da) of CGGEGPPWC, which forms an intramolecular disulfide bond, confirming the successful synthesis of the cyclized structure.

[0071] This embodiment successfully established a method for the synthesis, purification, and characterization of the cyclized variant CCGEGPPWC (forming an intramolecular disulfide bond). This variant, with its conformation fixed by disulfide bonds, significantly enhances its resistance to proteolytic degradation and improves its in vivo stability. As shown in Example 8, this cyclized variant exhibits significantly superior activity against pan-apoptotic keratinocytes compared to the prototype peptide, indicating that conformational restriction optimizes its interaction with target proteins (such as ZBP1), making it a preferred strategy for developing highly effective therapeutic drugs for skin diseases.

[0072] Example 4: Establishment of a TNF-α / IFN-γ-induced panapoptotic model of keratinocytes This embodiment aims to establish and standardize an in vitro cell model that can simulate key pathological processes of skin diseases, for subsequent evaluation of the bioactivity of the marine small molecule peptides and their active variants and to study their mechanisms of action, and to provide a basis for determining the appropriate timing and concentration of drug intervention.

[0073] After thawing frozen human keratinocytes (HaCaT cells), they were passaged three times. HaCaT cells in the logarithmic growth phase from the fourth passage were used for subsequent experiments. The old culture medium in the T25 culture flask was discarded, and 0.5 mL of 0.25% trypsin solution was added. The flask was gently agitated to cover the bottom, and the trypsin was discarded after standing for 10-20 seconds. The flask was then placed in a 37°C, 5% CO2 incubator, and digestion continued for 5-7 minutes. After observing under a microscope that the cells became rounder and the intercellular spaces increased, an appropriate amount of complete culture medium was added to stop the digestion. The bottom of the flask was repeatedly agitated to detach the cells, forming a single-cell suspension. The cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 800 rpm for 3 minutes. The supernatant was discarded, and the cell pellet was resuspended in 1×PBS buffer. The cells were washed again under the same conditions by centrifugation, and finally, the cell density was adjusted with complete culture medium for later use.

[0074] The cell suspension was dispensed at 2 mL per well (density 0.9 × 10⁻⁶). 5Cells were seeded in 6-well plates at 37°C and 5% CO2 for 24 hours to allow cell adhesion. The experiment was divided into the following groups: Blank control group: 1.5 mL / well of fresh complete culture medium. Pan-apoptotic induction group: 1.5 mL / well of complete culture medium containing different concentrations of cytokines IFN-γ and TNF-α, 1.5 mL / well, with the following concentrations: low concentration induction group (IFN-γ 50 ng / mL + TNF-α 25 ng / mL); medium concentration induction group (IFN-γ 100 ng / mL + TNF-α 50 ng / mL); high concentration induction group (IFN-γ 125 ng / mL + TNF-α 75 ng / mL). Cells in each group were cultured for 12 hours and 24 hours, respectively. At the predetermined time points, CCK-8 assay reagent was added to each well according to the kit instructions. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0075] like Figure 3 As shown, compared with the blank control group, the combined treatment of TNF-α and IFN-γ significantly induced HaCaT cell death, and the decrease in cell viability showed a dual dependence on cytokine concentration and treatment time.

[0076] After 12 hours of induction, cell viability decreased sequentially in the low, medium, and high concentration induction groups. Specifically, in the medium concentration group (IFN-γ 100 ng / mL + TNF-α 50 ng / mL), the HaCaT cell viability decreased to (69.59±4.16)% after 12 hours of induction, showing a highly significant difference compared to the blank control group (p<0.01). This condition, while inducing significant cytotoxicity, preserved a sufficient number of viable cells for subsequent drug intervention and mechanism studies, meeting the requirements for establishing a stable and reliable cell stress model. After 24 hours of induction, cell viability further decreased.

[0077] Therefore, this invention successfully established a TNF-α / IFN-γ-induced panapoptotic model of keratinocytes. This model effectively simulates the abnormal death process of keratinocytes in vitro by utilizing key pathological factors (TNF-α and IFN-γ working synergistically) in the local microenvironment of inflammatory skin diseases such as psoriasis, atopic dermatitis, and epidermal necrolysis.

[0078] Example 5: Evaluation of the preventive protective effect of marine small molecule peptides against pan-apoptosis. The purpose of this embodiment is to evaluate the protective effect of the marine small molecule peptide (GGEGPPW) on cell viability in a TNF-α / IFN-γ-induced panapoptotic model of keratinocytes when it is present in the presence of pathogenic cytokines, to verify its activity in preventing or directly antagonizing panapoptosis, and to determine its effective concentration range in vitro, as follows: HaCaT cells in the logarithmic growth phase were collected and diluted to 2 mL per well (cell density 0.9 × 10⁻⁶). 5 Cells (number / mL) were seeded into 6-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours to allow cell adhesion. After 24 hours of incubation, the original culture medium in each well was discarded, and each well was replaced with 1.5 mL of complete culture medium containing the corresponding components, according to the following grouping: Blank control group: Fresh complete culture medium; Pan-apoptotic model group: complete culture medium containing IFN-γ (100 ng / mL) and TNF-α (50 ng / mL); Marine small molecule peptide treatment group (simultaneous treatment): Complete culture medium containing IFN-γ (100 ng / mL), TNF-α (50 ng / mL) and marine small molecule peptides, with peptide concentrations of 0.625, 1.25, 2.5, 5, 10, 20 and 40 μg / mL, for a total of 7 concentration gradients.

[0079] After culturing the cells in each group for another 12 hours, the cell viability was detected using the CCK-8 assay kit.

[0080] like Figure 4 As shown, after 12 hours of synergistic treatment with IFN-γ (100 ng / mL) and TNF-α (50 ng / mL), the survival rate of HaCaT cells in the pan-apoptotic model group was significantly reduced compared with the blank control group, indicating that the cell damage model was successfully constructed.

[0081] When different concentrations of marine small molecule peptides (0.625-40 μg / mL) were co-added with cytokines, a clear dose-dependent protective effect was observed. Within the concentration range of 1.25 μg / mL to 40 μg / mL, the cell survival rate of each peptide treatment group was statistically significantly higher than that of the model group. The degree of cell viability recovery became more pronounced with increasing peptide concentration.

[0082] This embodiment simulates a "preventative" treatment scenario involving drug intervention during the initiation phase of skin inflammation (i.e., in the presence of pathogenic cytokines). Results show that the marine small molecule peptide GGEGPPW effectively antagonizes the decrease in keratinocyte viability caused by TNF-α / IFN-γ synergistic stimulation, exhibiting direct cytoprotective activity. Its protective effect demonstrates a clear dose-response relationship, providing crucial in vitro experimental evidence for the clinical application of this peptide in early intervention or prevention of skin cell damage caused by similar pathological mechanisms.

[0083] Example 6: Evaluation of the therapeutic intervention effect of marine small molecule peptides on pan-apoptosis. The purpose of this embodiment is to evaluate the intervention effect of the marine small molecule peptide administered after the initiation of skin cell damage signals, to verify its potential to block or reverse the panapoptotic process in therapeutic scenarios, and to provide key in vitro experimental evidence for the use of the peptide to treat skin diseases that are already in an active inflammatory phase.

[0084] HaCaT cells in the logarithmic growth phase were collected and diluted to 2 mL per well (cell density 0.9 × 10⁻⁶). 5 Cells were seeded in 6-well plates at 37°C and 5% CO2 for 24 hours to allow adherence. After 24 hours of culture, the original culture medium in each well was discarded, and the cells were replaced with complete culture medium containing the corresponding components according to the following groups, 1.5 mL per well: Blank control group: fresh complete culture medium; Pan-apoptotic model group: cultured in complete culture medium containing IFN-γ (100 ng / mL) and TNF-α (50 ng / mL) for 12 hours, then replaced with fresh complete culture medium; Marine small molecule peptide treatment group (post-treatment): cultured in complete culture medium containing IFN-γ (100 ng / mL) and TNF-α (50 ng / mL) for 12 hours, then replaced with complete culture medium containing different concentrations (0.625, 1.25, 2.5, 5, 10, 20, 40 μg / mL) of marine small molecule peptides. After culturing for another 12 hours, cell viability was detected using the CCK-8 assay.

[0085] like Figure 5As shown, HaCaT cells were induced to rupture by IFN-γ / TNF-α treatment alone for 12 hours, and then cultured for another 12 hours in a medium containing only different concentrations of marine small molecule peptides (0.625-40 μg / mL) without cytokines. The results showed that compared with the model group (induced but not treated), the cell viability of each peptide treatment group was restored to varying degrees, and this restoration showed a clear dose-dependent effect. At a concentration of 1.25 μg / mL, a statistically significant increase in cell viability was observed (p<0.05). Within the concentration range of 2.5 μg / mL to 40 μg / mL, the cell viability of the peptide treatment groups showed a highly significant improvement compared to the model group (p<0.01).

[0086] This embodiment simulates a scenario where drug intervention is performed after pathogenic cytokines have caused initial cell damage during the progression of a skin disease (i.e., a clinical therapeutic scenario). The results show that even after the cell damage pathway is activated, the marine small molecule peptides can effectively intervene in the further loss of keratinocyte viability and significantly promote the recovery of cell survival rate, demonstrating clear therapeutic potential.

[0087] This demonstrates that, at the cellular level, the peptides not only prevent damage from occurring but also possess a positive intervention and repair capability for already initiated damage pathways. This provides direct and compelling experimental evidence for their clinical application in treating skin diseases in acute flare-ups or active inflammatory phases (such as acute exacerbations of psoriasis, atopic dermatitis, and epidermal necrolysis).

[0088] Example 7: Study on the multi-level mechanism of marine small molecule peptides inhibiting panapoptosis of keratinocytes The purpose of this embodiment is to systematically elucidate the detailed molecular mechanism by which the marine small molecule peptide (GGEGPPW) inhibits pan-apoptosis of keratinocytes from multiple levels, including cell phenotype, oxidative stress, mitochondrial function, genomics, and protein expression, based on the established TNF-α / IFN-γ-induced pan-apoptotic model of keratinocytes, revealing its multi-target, synergistic network of action, and providing a solid theoretical basis for the treatment of skin diseases.

[0089] HaCaT cells in the logarithmic growth phase were collected and diluted to 2 mL per well (cell density 0.9 × 10⁻⁶). 5Cells were seeded at 1 / mL in 6-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours to allow cell adhesion. After 24 hours of incubation, the original culture medium in each well was discarded, and the cells were replaced with complete culture medium containing the corresponding components according to the following groups, 1.5 mL per well: Blank control group: fresh complete culture medium; Pan-apoptotic model group: complete culture medium containing IFN-γ (100 ng / mL) and TNF-α (50 ng / mL); Marine small molecule peptide treatment group (simultaneous treatment): complete culture medium containing IFN-γ (100 ng / mL), TNF-α (50 ng / mL), and marine small molecule peptide (20 μg / mL). Cells in each group were cultured for another 12 hours.

[0090] Cell viability was assessed using Calcein AM and propidium iodide (PI) dual-fluorescence staining; apoptosis was detected by flow cytometry; cell morphology was observed under a microscope; intracellular reactive oxygen species levels were detected using the DCFH-DA probe; mitochondrial membrane potential was detected using the JC-1 fluorescent probe; gene expression profiles were analyzed using transcriptome sequencing, and the expression levels of relevant proteins were detected using Western blotting. The final mechanistic results are as follows: (1) Protective effect on the viability and death morphology of keratinocytes Calcein-AM / PI double staining results showed that ( Figure 6 (7) The cell death rate in the model group significantly increased to 35.12±1.83%, while the mortality rate in the peptide treatment group decreased to 2.76±0.84%, and the peptide itself was not cytotoxic. Annexin V / PI flow cytometry results further confirmed ( Figure 8 ,9), peptide treatment effectively reduced the apoptosis rate induced by the model. Transmission electron microscopy observation ( Figure 10 The results showed that the model group cells exhibited typical apoptotic morphological changes (such as chromatin condensation and nuclear membrane rupture), and peptide treatment significantly alleviated these lesions. These results, from multiple dimensions including cell activity, death patterns, and subcellular structure, directly demonstrate that the peptide has a strong protective effect on keratinocytes.

[0091] (2) Effects on improving oxidative stress and mitochondrial function DCFH-DA detection showed ( Figure 11 (12) The intracellular ROS level in the model group increased to 4.26 times that of the control group, while peptide treatment significantly reduced excessive ROS accumulation. JC-1 assay showed that ( Figure 13 (14) The mitochondrial membrane potential in the model group was significantly decreased (the JC-1 monomer / polymer ratio increased), and peptide treatment effectively stabilized the mitochondrial membrane potential. This indicates that the peptide can resist pan-apoptotic-related cell damage by alleviating oxidative stress and maintaining mitochondrial functional homeostasis.

[0092] (3) Broad regulation of the gene network related to skin inflammation and death Transcriptome sequencing and KEGG enrichment analysis revealed ( Figure 15-17 TNF-α / IFN-γ stimulation significantly activated signaling pathways closely related to skin inflammation and cell death, including NF-κB, TNF, JAK-STAT, and MAPK. Peptide treatment significantly restored the expression of these aberrant genes, particularly exerting a significant regulatory effect on key pathway genes such as NF-κB, TNF, and PI3K-Akt. This demonstrates from a systems biology perspective that the peptide can broadly intervene in the gene network triggered by TNF-α / IFN-γ and closely related to pan-apoptosis of keratinocytes.

[0093] (4) Multi-target inhibition of the core signaling axis of panapoptosis in keratinocytes Western blotting protein assays provide direct molecular evidence: Suppressing the formation of upstream sensors and complexes ( Figure 18 19): Peptide treatment significantly downregulated the protein expression of multiple pattern recognition receptors (ZBP1, AIM2, NLRP3, NLRP12, NLRC5) in keratinocytes and simultaneously inhibited the phosphorylation activation of RIPK1 and RIPK3. This indicates that it can simultaneously block multiple upstream signaling axes that sense cellular stress and interfere with the initial assembly of the PANoptosome death complex.

[0094] Blocking multiple dead execution pathways in the midstream and downstream ( Figure 20 21): Apoptosis pathway: Peptide treatment effectively inhibited the expression of Cleaved Caspase-3 / 8, pro-apoptotic protein Bax, cytochrome C, and adaptor proteins ASC and FADD. Necrotizing apoptosis pathway: Peptide treatment reduced the phosphorylation level of the key executive protein MLKL. Pyroptosis pathway: Peptide treatment downregulated the levels of active cleavage fragments (GSDMD-N, GSDME-N) of Cleaved Caspase-1 and its substrates GSDMD / GSDME.

[0095] This demonstrates that the peptide can synergistically block the final disintegration of keratinocytes through multiple parallel death pathways, including Caspase-dependent apoptosis, MLKL-mediated necroptosis, and GSDMD / GSDME-mediated pyroptosis.

[0096] In summary, this invention elucidates for the first time in keratinocytes that the marine small molecule peptide GGEGPPW exerts its therapeutic effect through a multi-level, multi-target synergistic inhibitory network. By downregulating multiple key upstream sensors such as ZBP1, AIM2, and NLRP3, and inhibiting RIPK1 / RIPK3 activation, it interferes with the assembly of the PANoptosome death complex at its source. Simultaneously, it inhibits the activation of downstream Caspase apoptosis pathway, MLKL necroptosis pathway, and GSDMD / GSDME pyroptosis pathway. Furthermore, it reduces ROS, stabilizes mitochondrial function, alleviates cellular stress, and maintains cellular homeostasis.

[0097] This unique mechanism of action provides a reasonable molecular explanation for the fundamental reason why this peptide can exert a significant protective effect in various skin disease models characterized by abnormal keratinocyte death and inflammation, such as psoriasis, atopic dermatitis, epidermal necrolysis, cutaneous lupus erythematosus, and lichen planus, and provides comprehensive and solid experimental evidence for the mechanism of action described in the claims.

[0098] Example 8: Bioactivity Evaluation and Mechanism Verification of Representative Active Variants The purpose of this embodiment is to evaluate the biological activity of a representative structural variant peptide in an established TNF-α / IFN-γ-induced panapoptotic model of keratinocytes, and to verify its retention or enhancement effect on the core function of the original peptide (GGEGPPW) (inhibition of panapoptotic keratinocytes), as detailed below: (1) Active variants The following three active variants, designed and synthesized based on the core sequence (GGEGPPW, SEQ ID NO: 1), were selected for evaluation: ①GG(DE)GPPW (D-amino acid substitution variant): The L-glutamic acid at position 3 in the sequence is replaced with its D-enantiomer (D-Glu). This modification aims to enhance the peptide's stability against protease hydrolysis.

[0099] ② Cyclic variant CGGEGPPWC (forming an intramolecular disulfide bond): A cysteine ​​residue is added to both the N-terminus and C-terminus of the core sequence, and cyclization is achieved through an intramolecular disulfide bond. This modification aims to improve structural stability and potential target affinity through conformational restriction.

[0100] ③Ac-GGEGPPW-NH2 (terminal modified variant): Using the Fmoc solid-phase synthesis method, after assembling the GGEGPPW sequence, the following steps were performed: (1) N-terminal acetylation: After removing the terminal Fmoc protecting group, the mixture was treated with a DMF solution of acetic anhydride / DIEA. (2) C-terminal amidation: Using Rink Amide resin as a solid-phase support, the C-terminal amide was naturally generated after cleavage. This modification aims to eliminate terminal charge, resist exopeptidase degradation, and potentially improve membrane permeability. The molecular structure was confirmed by HPLC purity >98% and ESI-MS.

[0101] (2) Efficacy evaluation Human immortalized keratinocytes (HaCaT) were used to induce a pan-apoptotic model under the following conditions: IFN-γ (100 ng / mL) and TNF-α (50 ng / mL) were treated together for 12 hours. A prophylactic intervention was employed, with each variant peptide and cytokine added simultaneously. The experiment included the following groups: a blank control group, a model group (cytokine only), a prototypical peptide GGEGPPW treatment group (20 μg / mL, positive control), and treatment groups for each variant peptide (with five concentration gradients: 1.25, 2.5, 5, 10, and 20 μg / mL). Cell viability was assessed using the CCK-8 assay. Data are expressed as mean ± standard deviation. One-way ANOVA was used for inter-group comparisons; *p < 0.05 and **p < 0.01 were considered statistically significant.

[0102] (3) Cell protective activity CCK-8 assay results showed that all three variants were able to antagonize TNF-α / IFN-γ-induced decrease in keratinocyte viability in a dose-dependent manner.

[0103] GG(DE)GPPW showed significant protective effects at concentrations ranging from 1.25 to 20 μg / mL (p<0.05 compared to the model group). Within the 20 μg / mL concentration range, cell viability was (92.4±2.9)%, and its activity was not statistically different from that of the original peptide at the same concentration ((89.5±3.8)%).

[0104] The cyclized variant CCGGEGPPWC exhibited stronger cell-protective activity. At concentrations of 10 μg / mL and 20 μg / mL, its cell viability (95.4±3.2% and 98.9±2.2%, respectively) was significantly higher than that of the original peptide group at the same concentration, suggesting that cyclization modification enhanced its bioavailability.

[0105] Ac-GGEGPPW-NH2 at concentrations of 10–20 μg / mL exhibited protective activity equivalent to the original peptide, demonstrating the effectiveness of terminal modification in maintaining core activity.

[0106] (4) Verification of mechanism of action To confirm that the variant shares the same mechanism of action as the original peptide, the cyclized variant CGGEGPPWC (10 μg / mL), which performed best in the cell protection experiment, was selected for the detection of key mechanism indicators.

[0107] Intracellular reactive oxygen species (ROS) levels: ROS levels in the model group increased to 4.31 times that of the blank control group. After treatment with the cyclized variant, ROS levels significantly decreased to 1.5 ± 0.2 times (p < 0.01 compared with the model group), with an effect comparable to that of the original peptide.

[0108] Mitochondrial membrane potential: The JC-1 monomer / polymer ratio (indicating a decrease in membrane potential) in the model group increased to 3.35 times that of the control group. Treatment with the cyclized variant effectively reversed this change, restoring the ratio to 1.3 ± 0.3 times (p < 0.01 compared to the model group).

[0109] Key protein expression: Western blot analysis showed that the cyclized variants acted in a similar manner to the original peptides, significantly downregulating the expression levels of key panapoptotic proteins ZBP1, AIM2, NLRP3, p-RIPK1, p-RIPK3, GSDMD-N, and GSDME-N induced by the model (p<0.05 or p<0.01 compared with the model group).

[0110] The data in this embodiment confirm that the peptide variants modified by D-amino acid substitution, intramolecular cyclization, and terminal acetylation / amidation, while maintaining high sequence identity with SEQ ID NO: 1, fully retain the core biological activity of the original peptide in inhibiting TNF-α / IFN-γ-induced panapoptosis of keratinocytes. Among them, the cyclized variants showed enhanced cytoprotective efficacy.

[0111] The activity evaluation of the above variants was conducted using the novel pharmaceutical use described in this invention (inhibition of pan-apoptosis of keratinocytes) as a unified screening criterion, which is distinctly different from the known antioxidant screening criteria for this peptide. These variants, which combine activity with optimized pharmaceutical properties (such as enhanced stability), are preferred candidate molecules for developing long-acting topical or systemic formulations with greater clinical application potential.

[0112] The three representative variants verified in this embodiment (D-amino acid substitution, cyclization, and terminal modification) cover the three core requirements of "stability optimization, activity enhancement, and transdermal improvement." The technical logic of other modification types (such as N-methylation, PEGylation, and cell-penetrating peptide linkage) is consistent with the above variants: all achieve pharmaceutical property optimization through structural adjustments without destroying the core pharmacophore. Based on the activity judgment criteria set by this invention, those skilled in the art can verify the activity of other modified variants through conventional experiments without creative effort.

[0113] Example 9: Preparation of pharmaceutical compositions of marine small molecule peptides and their active variants The purpose of this embodiment is to provide a typical pharmaceutical formulation containing the marine small molecule peptide or its active variants, demonstrating its feasibility and specific implementation methods for topical and injectable dosage forms suitable for different clinical needs, and providing direct support for claims 9-10 (pharmaceutical compositions and dosage forms thereof).

[0114] (1) Cream preparations Formula (mass fraction): 2% active ingredient (GGEGPPW and / or its variants), 2% glyceryl monostearate, 5% cetyl / octadecyl alcohol, 1% sodium lauryl sulfate, 10% white petrolatum, 5% light liquid paraffin, 0.1% ethylparaben (preservative), 6% glycerin (humectant), and purified water to 100%.

[0115] Preparation method: The oil phase components (glyceryl monostearate, cetyl / octadecyl alcohol, white petrolatum, liquid paraffin) and the aqueous phase components (sodium dodecyl sulfate, glycerin, ethylparaben, and some pure water) are heated to 75-80℃ to melt or dissolve. The aqueous phase is slowly added to the oil phase while stirring, and the mixture is stirred until emulsification is complete. When the mixture is cooled to about 50℃, a marine small molecule peptide solution that has been dissolved in a small amount of water is added. The mixture is stirred evenly and then cooled to room temperature to obtain a white cream.

[0116] Features and Uses: This cream formulation has a moderate oil content, good spreadability and moisturizing properties, and is suitable for dry, thickened, or hyperkeratotic skin lesions. It is highly effective in the topical treatment of chronic plaque psoriasis, chronic atopic dermatitis, lichen planus, and other diseases.

[0117] (2) Gel preparations Formulation (mass fraction): 1.5% active ingredient (GGEGPPW and / or its variants), 0.7% carbomer-940 (gel matrix), 0.7% triethanolamine (pH adjuster), 5% propylene glycol, 5% glycerin, 0.1% ethylparaben (preservative), and purified water to 100%.

[0118] Preparation method: Carbomer-940 was uniformly dispersed in a portion of pure water and allowed to swell overnight. An aqueous solution of propylene glycol, glycerol, and ethylparaben was added and stirred until homogeneous. Triethanolamine was slowly added under stirring to neutralize the solution until a transparent gel was formed. Finally, an aqueous solution of marine small molecule peptides was added and thoroughly mixed to obtain the transparent gel.

[0119] Features and Uses: This gel formulation is refreshing, non-greasy, and has excellent transdermal absorption, making it easy to apply to hairy areas and the face. Preliminary quality studies (see Example 10) indicate that the formulation is stable, transdermal, and non-irritating to the skin. It has shown significant efficacy in treating scalp psoriasis, facial dermatitis, cutaneous lupus erythematosus lesions, and other moist or oily skin lesions.

[0120] (3) Injectable preparations (injection solution / lyophilized powder for injection) Formulation: Active ingredient (GGEGPPW and / or its variants, preferably variants modified by PEGylation to extend half-life) 0.00125-20 mg / mL, solvent / excipient is 0.9% (w / v) sodium chloride injection, 5% (w / v) glucose injection or sterile water for injection, lyophilization protectant (required additionally for lyophilized powder injection) is mannitol (1%-5%), sucrose (2%-10%), etc.

[0121] Preparation method (injection): Under aseptic conditions, weigh GGEGPPW and / or its active variants according to the prescribed amount. Add it to an appropriate amount of sterile solvent (e.g., 0.9% sodium chloride injection), and stir or vortex until completely dissolved. Add solvent to the total volume and mix thoroughly. Filter sterilize using a 0.22 μm sterile filter membrane. Dispense into vials or pre-filled syringes under aseptic conditions, and seal to obtain the injection solution.

[0122] Preparation method (lyophilized powder for injection): GGEGPPW and / or its active variants, together with a lyophilization protectant (such as mannitol), are dissolved in an appropriate amount of sterile water for injection. After sterilization by filtration through a 0.22 μm filter membrane, the solution is dispensed into sterile vials. The vials are partially stoppered and placed in a lyophilizer for lyophilization according to a conventional lyophilization process (pre-freezing, primary drying, secondary drying). After lyophilization, the vials are stoppered and capped under full vacuum or nitrogen purging conditions to obtain the lyophilized powder for injection. The solution should be reconstituted with a suitable solvent before use.

[0123] Features and Applications: Injectable formulations enable systemic, rapid-onset drug delivery. They are highly effective for moderate to severe generalized psoriasis, acute exacerbations of epidermal necrolysis / tenuation (SJS / TEN), systemic lupus erythematosus, and other severe or systemic skin diseases requiring treatment. PEGylated variants significantly improve pharmacokinetic properties, making them suitable for long-term or intermittent dosing.

[0124] In addition, regarding the effective dosage of the formulation, in vitro experiments (Examples 5 and 6) showed that a peptide concentration ≥1.25 μg / mL (1.25 ppm) could significantly improve cell viability; the 2% content (20000 ppm) gel in the topical formulation (Examples 9 and 10) was non-irritating to the skin, and transdermal experiments confirmed that this concentration could meet the effective drug concentration at the local skin lesion; the low dose (0.3125 ppm) of the injectable formulation (Example 9) was derived from the effective dose in animal experiments (Example 11), which is consistent with the in vivo pharmacokinetic laws of peptide drugs (in vivo distribution characteristics of small molecule peptides).

[0125] Example 10: Quality and safety evaluation of marine small molecule peptide gel formulations The purpose of this embodiment is to conduct a preliminary pharmaceutical quality and safety evaluation of the carbomer gel containing marine small molecule peptides (GGEGPPW) described in Example 9, including accelerated stability, in vitro transdermal performance, and skin irritation, in order to demonstrate the practicality, efficacy, and safety of the pharmaceutical composition as a topical formulation.

[0126] Test formulation: Carbomer gel containing 1.5% (w / w) GGEGPPW, prepared using the formulation described in Example 9. Three batches of gel were dispensed into translucent pharmaceutical ointment tubes, sealed, and used for the following experiments.

[0127] (1) Accelerated stability test Following ICH Q1A(R2) guidelines, samples were placed in a constant temperature and humidity chamber and accelerated testing was conducted at 40±2℃ and 75±5% relative humidity. Sampling time points: Samples were taken at the initial stage (week 0), week 1, week 2, and week 4. Appearance was assessed by observing color, uniformity, and the presence of layering or mold. pH value was measured using a precision pH meter. Content determination was performed using high-performance liquid chromatography (HPLC). Accurately weighed gel samples were extracted with the mobile phase and diluted. The content of the main component (with the initial content as 100%) was calculated using the GGEGPPW reference standard from Example 1 as the standard.

[0128] After being placed under accelerated conditions at 40℃ / 75% RH for 4 weeks, the gel appearance and pH value remained stable. The active pharmaceutical ingredient content remained above 97.5% of the initial content, and degradation was slow. The total impurity content increased from the initial 0.12% to 0.71%, but remained below 1.0%. The results indicate that the gel formulation has good chemical stability under accelerated conditions, meeting the requirements for the initial stability of topical formulations.

[0129] (2) In vitro transdermal test Abdominal skin from healthy Kunming mice was used (the mice were euthanized after hair removal, the entire skin was peeled off, subcutaneous fat was removed, and the skin was rinsed with physiological saline before use). Skin integrity was checked before the experiment. A Franz vertical diffusion cell was used, with the skin fixed between the donor and receiver cells, the stratum corneum facing the donor cell. The effective diffusion area was 1.77 cm². 2 The receiving cell volume was 7 mL. Experimental groups: Test gel group: Approximately 0.5 g of the test gel was evenly applied to the skin surface. Blank matrix control group: An equal volume of blank carbomer gel matrix. Receiving solution: To maintain the leakage conditions, a physiological saline solution containing 30% (v / v) polyethylene glycol 400 was used as the receiving solution. The magnetic stirring speed was 600 rpm, and the temperature was maintained at 32±1℃ to simulate the skin surface temperature. Sampling and detection: 0.5 mL samples were taken from the receiving cell at 1, 2, 4, 6, 8, 12, and 24 hours, and an isothermal and equal volume of fresh receiving solution was immediately added. After filtration through a 0.22 μm filter membrane, the concentration of GGEGPPW was determined by HPLC-MS / MS, with sensitivity reaching the ng / mL level. The cumulative transdermal dose (Qn, μg / cm³) was calculated. 2 ) and transdermal rate.

[0130] GGEGPPW can be effectively released from the gel formulation and penetrate mouse skin. 24-hour cumulative transdermal dose (Q) 24 The concentration was 15.36 ± 2.41 μg / cm³. 2 The transdermal process reached steady state after approximately 1-2 hours, with a steady-state transdermal rate (Jss) of 0.78 ± 0.12 μg / cm³. 2 / h, with a lag time (Tlag) of approximately 1.2±0.3 h, exhibiting a continuous zero-order release characteristic.

[0131] This gel formulation allows the active ingredient GGEGPPW to effectively penetrate the skin barrier, exhibiting excellent in vitro transdermal properties and providing a pharmaceutical basis for the efficacy of topical medication.

[0132] (3) Skin irritation evaluation Three healthy New Zealand white rabbits, half male and half female, were housed individually. Twenty-four hours prior to the experiment, the skin on both sides of the back of each rabbit was shaved using an electric shaver, covering an area of ​​approximately 10 cm × 15 cm, ensuring no skin damage. Grouping and administration: Intact skin group: The left shaved area was designated as the test area, and 0.5 g of the test gel (containing 7.5 mg of GGEGPPW) was evenly applied; the adjacent right area served as the control area, with an equal amount of blank gel matrix applied. Damaged skin group: A "#" shaped lesion (only penetrating the epidermis, with slight bleeding) was made in another area on the right side, serving as the damaged test area, and 0.5 g of the test gel was applied.

[0133] After securing the gel with gauze and non-irritating tape for 4 hours, gently remove any remaining gel with warm water. At 1, 24, 48, and 72 hours after removal, observe the application site for reactions such as erythema and edema, and score them according to the irritation rating scale (erythema: 0-4 points; edema: 0-4 points). Calculate the Primary Irritation Index (PII) at each observation time point, i.e., (total score of the test area - total score of the control area) / number of animals. According to the standard, 0-0.49 is non-irritating; 0.50-2.99 is mildly irritating; 3.00-5.99 is moderately irritating; and 6.00-8.00 is strongly irritating.

[0134] The study found that during the 72-hour observation period, intact skin test areas showed only very mild, rapidly resolving erythema at a few time points (mean score 0.33), without edema. Damaged skin test areas showed mild erythema (mean score 1.00) and very mild edema (mean score 0.33) one hour after administration; this reaction was mainly related to physical damage and completely subsided within 48 hours. The calculated PII for the intact skin group was 0.11, and for the damaged skin group it was 0.33.

[0135] According to the evaluation criteria, the PII of the gel containing 1.5% GGEGPPW on both intact and broken skin of rabbits was less than 0.5. Therefore, this formulation is non-irritating to the skin and exhibits good local safety.

[0136] As can be seen, the gel formulation containing the marine small molecule peptide GGEGPPW provided by this invention exhibits chemical stability under accelerated conditions, good in vitro transdermal performance, and no skin irritation. The aforementioned preliminary quality and safety study results collectively demonstrate the practicality, efficacy, and safety of this pharmaceutical composition as a topical formulation for the treatment of skin diseases, providing direct experimental support for the topical formulation described in the claims.

[0137] Example 11: Efficacy of subcutaneous injection of marine small molecule peptides in the prevention and treatment of epidermal necrosis lysis / TEN (SJS / TEN) mouse model The purpose of this embodiment is to verify, at the animal level, the preventive and therapeutic effects of the marine small molecule peptide administered subcutaneously on acute and severe skin injuries using a Smac mimicry-induced epidermal necrolysis / TEN (SJS / TEN) mouse model, providing key pharmacodynamic evidence for its development into a systemic (injectable) formulation.

[0138] C57BL / 6 mice were subcutaneously injected with 100 μl of 1 mg / ml Smac mimic CompA (TetraLogic Pharmaceuticals) or an equal volume of control solvent (12% sulfobutyl-β-cyclodextrin). Marine small molecule peptides (10 μg, 20 μg) or control solvent (physiological saline, 50 μl; Selleckchem) were administered subcutaneously twice daily, starting one day before (pretreatment model) or three hours after (treatment model). All pretreatment experiments used only male mice, while the treatment model used female mice. Mice were housed in a biosafety-controlled environment with constant temperature and humidity, a 12-hour light / dark cycle, and free access to food and water.

[0139] Mice were sacrificed on day 3, and injection sites were scored, with samples collected for in vitro analysis. A four-point ordinal scale (0-4 points) was used to assess three macroscopic indicators clinically relevant to TEN: edema, skin redness, and epidermal damage (Nikolsky's sign / severity of skin lesions). Each indicator was assessed and scored as 0 (none), 1 (mild), 2 (moderate), or 3 (severe), and the scores were summed to obtain the overall clinical score.

[0140] Studies have found that, for example Figure 22 As shown, the CompA model control group mice exhibited significant skin damage on day 3, characterized by severe edema, diffuse erythema, and epidermal peeling, with an overall clinical score significantly higher than the blank control group. Compared to the model control group, the peptide prophylactic administration group (20 μg) showed a significantly lower overall clinical score, indicating that advance administration can effectively reduce the occurrence of subsequent damage.

[0141] Following the occurrence of injury, peptide intervention showed clear therapeutic effects in both dosage groups. Compared with the model control group, the overall clinical scores of both the low-dose (10 μg) and high-dose (20 μg) peptide treatment groups were significantly reduced. The high-dose group (20 μg) showed better improvement than the low-dose group (10 μg), exhibiting a certain dose-dependent trend.

[0142] This embodiment demonstrates in a mouse model of acute epidermal necrosis and lysis induced by Smac mimics and simulating the pathological features of human SJS / TEN that the marine small molecule peptide GGEGPPW, administered subcutaneously, significantly reduces the severity of acute skin tissue damage, whether as a preventative intervention or as treatment after injury. Specifically, it effectively alleviates skin edema, erythema, and epidermal damage. Higher doses yield even better results.

[0143] The pharmacodynamic results at the animal level, along with the aforementioned in vitro experiments demonstrating the core mechanism by which this peptide inhibits TNF-α / IFN-γ-induced panapoptosis of keratinocytes (Example 7) and the molecular discovery of inhibiting the ZBP1 / PANoptosome signaling axis (Example 7), Figure 18 This evidence corroborates each other. Together, they demonstrate that the marine small molecule peptides, by intervening in the local pathological panapoptotic pathway in the skin, have clear application potential in the prevention and treatment of acute and severe skin diseases such as epidermal necrolysis. This embodiment provides direct and crucial preclinical experimental evidence for its further development into a subcutaneous or intravenous injection formulation for systemic treatment of such critical skin diseases.

[0144] Example 12: Therapeutic efficacy of topical administration of the active variant in a mouse model of psoriatic dermatitis The purpose of this embodiment is to verify the therapeutic effect of topical administration of the peptide active variant (Ac-GGEGPPW-NH2) on chronic inflammatory skin diseases using an imiquimod (IMQ)-induced mouse psoriasis-like dermatitis model at the animal level, providing direct preclinical pharmacodynamic evidence for the development of its topical formulation (such as a gel). This variant was chosen for verification because it is representative of variants with terminal modifications to enhance stability.

[0145] Six-week-old female C57BL / 6 mice were randomly divided into a blank control group, a model group, and a peptide active variant treatment group, with no fewer than five mice in each group, and were housed in an SPF environment. In the model group and the peptide active variant treatment group, 62.5 mg of 5% imiquimod cream was applied topically to the shaved back skin daily to induce psoriasis-like lesions, while the blank control group received an equivalent amount of petroleum jelly. Four hours after each imiquimod application, the model group and the peptide active variant treatment group received either 0.75% carbomer matrix or an equivalent matrix gel containing 20 mg / kg of peptide, respectively, for seven consecutive days. Twenty-four hours after the last administration, the degree of erythema, scaling, and thickening of the mouse back skin was scored using the Psoriasis Area and Severity Index, ranging from 0 (asymptomatic) to 4 (very severe), to evaluate the ameliorative effect of the peptide active variant on psoriasis-like skin inflammation.

[0146] After 7 consecutive days of IMQ induction, the model group mice developed typical psoriasis-like changes on their backs, including significant erythema, abundant silvery-white scales, and marked skin thickening. Compared with the model group, the peptide active variant treatment group showed significant improvement in all of the above symptoms, specifically, lighter erythema, reduced scales, and lessened skin thickening. Statistical analysis of the PASI total score showed that the model group's score was significantly higher than that of the blank control group, proving the successful establishment of the model. The peptide active variant treatment group's PASI total score was significantly lower than that of the model group, indicating that topical application of the described marine small molecule peptide active variant gel can effectively reduce the severity of psoriatic dermatitis.

[0147] This embodiment utilizes a classic psoriatic dermatitis model to verify the efficacy of topical application of the active variant of the marine small molecule peptide. Results show that this active peptide variant can effectively alleviate chronic skin inflammation driven by immune dysregulation and its accompanying proliferative pathological changes. Simultaneously, this embodiment, together with Example 11 (epidermal necrolysis model), verifies the therapeutic effects of the peptide and its variants of the present invention on two types of skin diseases: "acute severe" and "chronic inflammatory." These represent two typical modes of pan-apoptotic activation in skin diseases (acute strong activation vs. chronic persistent activation). Based on existing research on the pathological mechanisms of atopic dermatitis, cutaneous lupus erythematosus, and lichen planus, the mechanism of action of the peptide of the present invention can exert its effects across these disease types, and there is no evidence that these diseases possess a pan-apoptotic escape mechanism different from that in the verification model. This further expands its application scenarios, making it applicable not only to acute injuries but also to chronic, relapsing diseases such as psoriasis.

[0148] These results provide crucial animal experimental evidence for the preparation of the aforementioned marine small molecule peptides and their active variants into topical formulations such as creams, gels, and ointments, which can be directly applied to the affected skin to treat chronic inflammatory skin diseases such as psoriasis, atopic dermatitis, and cutaneous lupus erythematosus. The combination of in vivo efficacy and in vitro molecular mechanism studies has constructed a complete chain of evidence for the peptide and its active variants, from target regulation to disease treatment.

[0149] The in vivo pharmacodynamic studies corroborated the aforementioned in vitro experimental results, forming a complete evidence system. The experimental results demonstrate that the marine small molecule peptides and their active variants exhibit significant therapeutic effects in various animal models of skin diseases, regardless of whether they are administered systemically (subcutaneously) or locally (topical gel).

[0150] In summary, the marine small molecule peptides and their active variants of this invention, through their core mechanism of inhibiting pan-apoptosis of keratinocytes, possess broad-spectrum intervention potential for various skin diseases characterized by abnormal activation of this pathway. Specifically, their systemic formulations hold promise for the prevention and treatment of acute or systemic manifestations of diseases such as scleroderma necrolysis / tenuation (SJS / TEN) and cutaneous lupus erythematosus; their topical formulations are suitable for treating chronic, localized inflammatory skin diseases such as psoriasis, atopic dermatitis, and lichen planus. These results lay a solid preclinical research foundation for the development of these peptides and their active variants into skin-targeted therapeutic drugs for different clinical needs.

[0151] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Use of a marine small molecule peptide, its active variant, or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of skin diseases, characterized in that, The marine small molecule peptide contains the amino acid sequence shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that, The skin diseases mentioned are selected from epidermal necrolysis, psoriasis, atopic dermatitis, cutaneous lupus erythematosus, and lichen planus.

3. The use according to claim 1, characterized in that, The active variant has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.1 and retains the activity of inhibiting panapoptosis of keratinocytes.

4. The use according to claim 3, characterized in that, The activity of retaining inhibition of pan-apoptosis of keratinocytes is such that, in a pan-apoptotic model of keratinocytes induced by the combined action of TNF-α and IFN-γ, the active variant, at an equimolar concentration, promotes cell survival by at least 70% of the peptide shown in SEQ ID NO.

1.

5. The use according to claim 3, characterized in that, The active variant contains one or more modifications selected from the following: (a) One or more amino acid residues are replaced by their corresponding D-type amino acid residues; (b) One or more amino acid residues are replaced by a conserved amino acid, wherein the conserved amino acid substitution is selected from glycine replaced by alanine or sarcosine, glutamic acid replaced by aspartic acid, proline replaced by an N-methylated amino acid, and tryptophan replaced by phenylalanine or tyrosine. (c) The N-terminus is acetylated, acylated, or linked to a cell-penetrating peptide sequence; (d) The C-terminus is amidated; (e) One or more amide bonds in the peptide backbone are N-methylated; (f) Formation of an intramolecular cyclization structure, wherein the cyclization is achieved through head-to-tail connection, disulfide bond between side chains, lactam bond or click chemical connection; (g) Covalently linked 1- or more polyethylene glycol molecules with molecular weights of 2kDa-20kDa.

6. The use according to claim 1, characterized in that, The marine small molecule peptides or their active variants inhibit the panapoptotic pathway of keratinocytes through multi-target action.

7. The use according to claim 6, characterized in that, The multi-target effect includes one or more of the following: (a) Downregulate the expression or activity of one or more of the following proteins: ZBP1, AIM2, NLRP3, NLRP12, NLRC5, ASC, and FADD; (b) Inhibit phosphorylation activation of RIPK1 and / or RIPK3; (c) Reduce the level or activity of one or more of the following proteins: Cleaved Caspase-3, Cleaved Caspase-8, Bax, and Cytochrome C; (d) Inhibit phosphorylation of MLKL; (e) Reduce the level or activity of one or more of the following proteins: Cleaved Caspase-1, GSDMD-N, and GSDME-N.

8. The use according to claim 1, characterized in that, The drug is a topical preparation or an injectable preparation; the topical preparation is selected from creams, gels, ointments, solutions or sprays; the injectable preparation is selected from lyophilized powder for injection or pre-filled injection solution.

9. The use according to claim 8, characterized in that, In the topical or injectable formulation, the content of the marine small molecule peptide or its active variant is 0.3125 ppm to 20000 ppm.

10. The use according to claim 8, characterized in that, The lyophilized powder injection contains a lyophilization protectant, which is selected from mannitol or sucrose.

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