A polypeptide synergistic activation composition targeting the regulation of igf-1 synthesis and its use in tissue repair

Abstract

The application discloses a polypeptide synergistic activating composition for targeted regulation of IGF-1 synthesis and application thereof in tissue repair. The composition comprises at least twelve polypeptides, for example, basic regulation peptides for promoting IGF-1 transcription and secretion of liver cells, structural functional peptides derived from IGF-1 functional regions and optional MMP-cleavable IGF-1 regulation precursor polypeptides, which can release IGF-1 regulation fragments on demand in a high MMP microenvironment of injury, significantly up-regulate local IGF-1 expression, and promote wound healing and tissue regeneration.

Description

Technical Field

[0001] This invention relates to the field of peptide composition technology, and more particularly to a peptide synergistic activation composition that targets and regulates IGF-1 synthesis and its application in tissue repair. Background Technology

[0002] Insulin-like growth factor-1 (IGF-1) is one of the important growth factors in the body, mainly synthesized and secreted by hepatocytes, and plays a role in various tissues such as skin, skeletal muscle, and bone. IGF-1, by binding to its receptor, activates multiple downstream signaling pathways such as PI3K / Akt and MAPK, promoting cell proliferation, migration, and matrix synthesis, and inhibiting apoptosis. It plays a crucial role in wound repair, chronic ulcer healing, fracture healing, and tissue regeneration. Studies have shown that in refractory wounds such as diabetic foot ulcers, venous ulcers, and radiation-induced skin injuries, local IGF-1 levels and its signaling pathways are often in a state of persistently low activity, which is one of the important reasons for slow wound healing and poor repair quality.

[0003] Existing methods for increasing IGF-1 levels or activity mainly include: systemic application of recombinant human IGF-1 or its analogues, indirect increase of circulating IGF-1 through growth hormone or growth hormone secretagogues, and local injection of single growth factors or short peptides containing IGF-1-like activity. While these approaches can improve tissue repair to some extent, they generally suffer from problems such as short duration of action, the need for repeated administration, high preparation and storage costs, and the potential to cause hypoglycemia, abnormal proliferation, or even potential tumor-promoting risks. They also struggle to achieve precise and controllable regulation of IGF-1 signaling at the site of injury. Furthermore, single factors often act only on a specific step in the IGF-1 pathway, making it difficult to achieve synergistic regulation of IGF-1 gene transcription and local receptor activation in hepatocytes.

[0004] To improve safety and specificity, some studies have attempted to use short peptides or polypeptides to mimic key functional regions of growth factors, or to combine multiple functional peptides to promote angiogenesis, collagen deposition, and granulation tissue formation, thereby accelerating wound healing. However, these peptides typically focus on only one or a few biological effects, such as simply promoting fibroblast proliferation, angiogenesis, or antioxidation. They lack a systematic regulatory design targeting IGF-1 from synthesis and secretion to receptor activation, and their regulatory efficiency and controllability on the IGF-1 axis remain limited.

[0005] On the other hand, matrix metalloproteinases (MMPs) are widely present in wound and chronically inflamed tissues, especially with significantly elevated levels of MMP-2 and MMP-9. A high-MMP microenvironment accelerates the degradation of exogenous proteins and growth factors, weakening the effectiveness of traditional biologics. However, it also reflects the microenvironmental characteristics of the injury site and has the potential to serve as a "trigger signal" for selective drug release in the lesion area. Existing literature reports the introduction of MMP-sensitive peptides into drug delivery systems, utilizing MMP-mediated cleavage to achieve controlled release of small molecule drugs or some protein factors. However, research on the design of MMP-responsive precursors targeting short peptides with IGF-1 regulatory activity is limited, and a synergistic activation strategy oriented towards the IGF-1 axis and finely regulated by the high-MMP microenvironment of the wound has not yet been established.

[0006] In summary, there is currently a lack of peptide compositions capable of simultaneously and synergistically regulating multiple levels of IGF-1 pathways—including hepatocyte IGF-1 gene transcription, endogenous IGF-1 secretion, and IGF-1 receptor signaling activation—and utilizing the high MMP microenvironment at wound or inflammatory sites to achieve on-demand release of IGF-1 regulatory fragments. This limits the efficient, safe, and controllable activation of local IGF-1 pathways in scenarios such as refractory wounds and chronic tissue injuries. Therefore, developing a peptide composition that targets and regulates IGF-1 synthesis and signaling pathways for synergistic activation and promotes tissue repair has significant practical application value. Summary of the Invention

[0007] Given the shortcomings of existing technologies in regulating the IGF-1 axis, such as single action points, lack of synergistic design, and inability to fully utilize the high MMP microenvironment of trauma, the technical problem to be solved by this invention is to provide a polypeptide synergistic activation composition, its preparation method, and its application that can simultaneously and synergistically regulate multiple levels of IGF-1 gene transcription, endogenous IGF-1 secretion, and IGF-1 receptor signal activation in hepatocytes, and achieve on-demand release of IGF-1 regulatory fragments in the high MMP microenvironment of trauma, for improving the repair efficiency and quality of wounds and other tissue injuries.

[0008] To achieve the above objectives, the first aspect of the present invention provides a polypeptide co-activation composition for targeting and regulating IGF-1 synthesis, comprising a basic regulatory peptide and a structural-functional peptide.

[0009] The basic regulatory peptide is preferably a short peptide composed of 2 to 3 natural amino acid residues, and is an L-type linear peptide group, used to enhance the transcriptional activity of IGF-1 mRNA in hepatocytes and increase the secretion level of IGF-1. Preferably, the basic regulatory peptide is selected from one or more of Pro-Gly, Pro-Asp, Gly-Pro-Glu, and Gly-Pro. The basic regulatory peptide provides a stable source of IGF-1 by acting on hepatocytes or other IGF-1 synthesis-related cells.

[0010] The structural-functional peptide is preferably a short peptide composed of 4 to 8 natural amino acid residues, an L-type linear peptide, whose amino acid sequence is derived from the receptor-binding region or other functional segments of the IGF-1 protein, or a conserved site substitution variant thereof, which can maintain or enhance the activation ability of the IGF-1 receptor or stabilize the conformation of IGF-1. Preferably, the structural-functional peptide includes, but is not limited to, one or more of GPET, GPETLC, ETLCG, QFVCGD, CGDRGFY, FNKPTG, SSSRRAP, and GIVDECC, which can be used alone or in combination to enhance the cellular response to IGF-1 signaling and produce a synergistic effect with the basic regulatory peptide in promoting the overall activation of the IGF-1 axis.

[0011] Furthermore, the peptide co-activation composition targeting and regulating IGF-1 synthesis may further include an MMP-cleavable IGF-1 regulatory precursor peptide. The MMP-cleavable IGF-1 regulatory precursor peptide contains an MMP substrate sequence and an IGF-1 regulatory functional fragment, and is specifically cleaved through the high-MMP microenvironment at the site of injury or inflammation, thereby releasing the IGF-1 regulatory fragment on demand.

[0012] The MMP-cleavable IGF-1 regulatory precursor peptide comprises a substrate sequence that can be recognized and cleaved by matrix metalloproteinases (MMPs), and at least one structural functional fragment with IGF-1 regulatory activity, connected by flexible linker peptides at both ends. Preferably, the precursor peptide is an L-type linear peptide, and its N-terminus and C-terminus may contain one or more glycine residues as flexible linker arms to improve solubility and spatial conformation. The substrate sequence is preferably a peptide sequence that can be recognized by MMP-2, such as GPQGIWGQ, PLGLAG, etc., and the IGF-1 regulatory functional fragment is preferably a peptide containing the SSSRRAP sequence. Through the above structural design, the precursor peptide remains relatively stable at physiological basal MMP levels and is cleaved in the high MMP microenvironment of wounds or chronic inflammation sites, directionally releasing the fragment with IGF-1 regulatory activity, achieving local on-demand activation.

[0013] In a preferred embodiment, the peptide co-activation composition targeting and regulating IGF-1 synthesis comprises, by weight, at least two of three classes of components: a basic regulatory peptide, a structural-functional peptide, and an MMP-cleavable IGF-1 regulatory precursor peptide. The total mass of the basic regulatory peptide, the structural-functional peptide, and the MMP-cleavable precursor peptide constitutes at least 80% of the total peptide content in the composition. The preferred mass ratio of the basic regulatory peptide to the structural-functional peptide is 1-10:1-10, more preferably approximately 1-3:1-3.

[0014] For a peptide synergistic activation composition that targets and regulates IGF-1 synthesis by containing an MMP-cleavable IGF-1 regulatory precursor peptide, the mass fraction of the MMP-cleavable IGF-1 regulatory precursor peptide in the total peptide is preferably 5-40%, more preferably 10-30%. By adjusting the proportions of the three types of peptides, personalized synergistic regulation can be achieved for the degree of IGF-1 deficiency and MMP levels in different disease states.

[0015] The second aspect of this invention provides a method for preparing the above-mentioned peptide synergistic activation composition for targeting and regulating IGF-1 synthesis, comprising the following steps: First, an isotonic regulator, a buffer salt, and a lyophilization protectant are dissolved in water for injection to obtain a clear buffer excipient solution; then, the basic regulatory peptide, the structural functional peptide, and the MMP-cleavable IGF-1 regulatory precursor peptide are dissolved separately in water for injection, and added sequentially to the buffer excipient solution under stirring conditions. After mixing, the pH is adjusted to weakly acidic or neutral, and the volume is brought to a final volume with water for injection to obtain a peptide mixed solution; subsequently, the peptide mixed solution is pre-filtered, sterilized, dispensed, pre-frozen, and vacuum freeze-dried. After drying, an inert gas is introduced and the container is sealed to obtain the final product. The isotonic regulator is sodium chloride; the buffer salt is phosphate; and the lyophilization protectant is mannitol.

[0016] The preparation method is mild and can effectively maintain the bioactivity of peptides. The resulting formulation has good resolubility and storage stability, making it convenient for clinical use and transportation.

[0017] A third aspect of this invention provides the use of the above-described peptide synergistic activating composition in the preparation of a medicament for promoting tissue repair, wherein the tissue repair includes, but is not limited to, acute trauma, full-thickness skin defects, surgical incisions, chronic refractory ulcers, radiation or chemical skin injuries, soft tissue contusions and lacerations, and other tissue injuries accompanied by a decrease in local IGF-1 levels or an increase in MMP levels. The medicament can be prepared as a lyophilized powder for injection, a solution for injection, a local irrigation solution, a topical gel, a spray, or a sustained-release formulation combined with dressings or scaffold materials, and administered via local injection, topical application, or in combination with wound dressings.

[0018] Beneficial effects of the present invention

[0019] Compared with existing technologies, the combination of basic regulatory peptides and structural functional peptides in this invention can increase the IGF-1 mRNA and secretion level of hepatocytes by more than 2 times in vitro, which is significantly better than the composition containing only basic regulatory peptides. Moreover, the cell survival rate is ≥98%, indicating that it has good safety while increasing IGF-1 expression.

[0020] This invention introduces MMP-cleavable precursor peptides containing GPQGIWGQ or PLGLAG substrate sequences. In the presence of MMP-2, it can significantly increase the levels of IGF-1 mRNA and protein without increasing the total amount of peptide, while the inert precursor group remains essentially unchanged. This indicates that the invention can selectively enhance local effects by utilizing a high MMP microenvironment to achieve on-demand release.

[0021] In a mouse full-thickness skin trauma model, Examples 1-3 of this invention significantly improved the wound healing rate on days 7 and 10 and upregulated the expression of local IGF-1 and Ki67. Among them, Example 2, which contains Pep-MMP-IGF, can shorten the complete healing time to about 9 days, which is better than the control composition without precursor or containing only inert precursor. Detailed Implementation

[0022] Unless otherwise stated, the raw materials and reagents used in the embodiments of the present invention are all conventional commodities in the art, or can be prepared by methods known in the art; the percentages used are mass percentages; and the conditions not specified in the steps are conventional conditions for those skilled in the art.

[0023] Introduction to some raw material parameters and sources

[0024] All peptides used in the embodiments of this invention are L-type linear peptides with HPLC purity of not less than 95%, dried and stored at -20℃, and custom-obtained by solid-phase synthesis from Huatuo Biotechnology Co., Ltd.

[0025] Basic regulatory peptides:

[0026] Pro-Gly, amino acid sequence: Pro-Gly, molecular formula: C7H 12 N₂O₃, relative molecular mass: 172.18; Pro-Asp, amino acid sequence: Pro-Asp, molecular formula: C₈H₂O 12 N2O5, relative molecular mass: 216.19; Gly-Pro-Glu, amino acid sequence: Gly-Pro-Glu; Gly-Pro, amino acid sequence: Gly-Pro. These basic regulatory peptides promote the transcription and secretory expression of the IGF-1 gene in hepatocytes.

[0027] Structural-functional peptides (short peptide fragments derived from key functional regions of IGF-1):

[0028] GPET: Gly-Pro-Glu-Thr; GPETLC: Gly-Pro-Glu-Thr-Leu-Cys; ETLCG: Glu-Thr-Leu-Cys-Gly; QFVCGD: Gln-Phe-Val-Cys-Gly-Asp; CGDRGFY: Cys-Gly-Asp-Arg-Gly-Phe-Tyr; FNKPTG: Phe-Asn-Lys-Pro-Thr-Gly; SSSRRAP: Ser-Ser-Ser-Arg-Arg-Ala-Pro; GIVDECC: Gly-Ile-Val-Asp-Glu-Cys-Cys. These structurally functional peptides mimic or originate from functional regions of the IGF-1 protein and can participate in IGF-1 receptor binding regulation, signaling pathway activation, and endogenous IGF-1 conformational stability.

[0029] MMP-cleavable IGF-1 regulatory precursor peptide (Pep-MMP-IGF), amino acid sequence: H-Gly-Gly-GPQGIWGQ-SSSRRAP-Gly-Gly-OH; "GPQGIWGQ" is a typical substrate sequence of MMP-2, which can be specifically cleaved in the high MMP microenvironment of trauma / inflammation; "SSSRRAP" is a structural functional peptide fragment with IGF-1 regulatory activity confirmed in this invention; the two Gly-Gly segments serve as flexible linkers, improving the conformation and solubility of the precursor peptide. Form: L-type linear peptide, HPLC purity ≥95%.

[0030] MMP-cleavable IGF-1 regulatory precursor peptide (Pep-PLGL-IGF), amino acid sequence, for example: H-Gly-Gly-Pro-Leu-Gly-Leu-Ala-Gly-SSSRRAP-Gly-Gly-OH. "Pro-Leu-Gly-Leu-Ala-Gly (PLGLAG)" is a substrate sequence recognized by multiple MMPs, exhibiting different affinity and cleavage efficiency for MMP-2 / 9 compared to GPQGIWGQ. The flanking Gly-Gly segments act as flexible linkers, improving the precursor peptide's conformation and solubility. Form: L-type linear peptide, HPLC purity ≥95%.

[0031] Pep-inert-IGF, an inert IGF-1 precursor peptide, has the amino acid sequence: H-Gly-Gly-GPQGIAGQ-SSSRRAP-Gly-Gly-OH. It is highly similar to the Pep-MMP-IGF sequence, except that "WG" in "GPQGIWGQ" is replaced with "AG". Experiments have shown that this site is no longer effectively cleaved by MMP-2. It also contains the SSSRRAP fragment, but under physiological conditions, it is not released via the MMP-mediated "on-demand release" mechanism. It serves as a control precursor peptide to demonstrate the contribution of MMP-cleavability to the overall effect. Form: L-type linear peptide, HPLC purity ≥95%.

[0032] PBS buffer, pH 7.4, concentration 0.01 mol / L.

[0033] Recombinant human MMP-2 is derived from Sigma-Aldrich.

[0034] MMP reaction buffer: 50 mmol / L Tris-HCl, 150 mmol / L NaCl, 5 mmol / L CaCl2, 0.05% Brij-35, pH 7.5.

[0035] Example 1

[0036] A peptide synergistic activation composition that targets and regulates IGF-1 synthesis, in the form of a lyophilized powder for injection.

[0037] The preparation method in this embodiment includes the following steps:

[0038] S1. Add 1.8L of water for injection to a sterilized stainless steel mixing tank, start stirring at 200rpm, and add 4.5g NaCl, 5.6g Na2HPO4•12H2O, 1.0g NaH2PO4•2H2O and 50.0g mannitol in sequence, stirring until completely dissolved to obtain a clear buffer excipient solution.

[0039] S2. Add 3.0g Pro-Gly, 3.0g Pro-Asp, 3.0g Gly-Pro-Glu, 3.0g Gly-Pro, 1.0g GPET, 1.0g GPETLC, 1.0g ETLCG, 1.0g QFVCGD, 1.0g CGDRGFY, 1.0g FNKPTG, 1.0g SSSRRAP, and 1.0g GIVDECC to 10mL of water for injection, and dissolve by shaking at room temperature. Then, slowly add each peptide solution to the clear buffer excipient solution sequentially at 50rpm. After the addition is complete, stir for 15min. Adjust the pH to 6.8, and then bring the volume to 2L with water for injection. Shake well to obtain the mixture.

[0040] S3. The mixed solution is pre-filtered through a 0.45μm filter membrane to remove particulates, and then filtered through a 0.22μm sterile filter under aseptic conditions into a sterilized storage tank. The temperature is controlled at 15℃ during the filtration process. The filtered solution is filled into 10mL glass vials at a rate of 2.0mL / bottle, with the filling volume error controlled within ±2%. The vials are then partially stoppered with rubber stoppers, placed on the shelf of a freeze dryer, and freeze-dried under vacuum. The rubber stoppers are then tightened under nitrogen protection, and the vials are sealed with an aluminum-plastic composite cap to obtain the peptide synergistic activation composition for targeting and regulating IGF-1 synthesis in this embodiment.

[0041] Freeze-drying process can:

[0042] Pre-freezing: Lower the shelf temperature to -40°C and maintain for 3 hours to ensure complete freezing;

[0043] First drying: The shelf temperature is raised to -25℃, the vacuum is about 100mTorr, and the drying time is 12 hours;

[0044] Secondary drying: The shelf temperature is gradually increased to 25℃, and the vacuum is maintained at approximately 50 mTorr for 6 hours;

[0045] After drying, sterile nitrogen gas is introduced into the vacuum until atmospheric pressure is reached.

[0046] Comparative Example 1

[0047] A polypeptide composition containing only basic regulatory peptides, formulated as a lyophilized powder for injection.

[0048] The comparative preparation method includes the following steps:

[0049] S1. Add 1.8L of water for injection to a sterilized stainless steel mixing tank, start stirring at 200rpm, and add 4.5g NaCl, 5.6g Na2HPO4•12H2O, 1.0g NaH2PO4•2H2O and 50.0g mannitol in sequence, stirring until completely dissolved to obtain a clear buffer excipient solution.

[0050] S2. Add 5.0g Pro-Gly, 5.0g Pro-Asp, 5.0g Gly-Pro-Glu, and 5.0g Gly-Pro to 10mL of water for injection, and dissolve by shaking at room temperature. Then, slowly add each peptide solution to the clear buffer excipient solution sequentially at 50rpm. After the addition is complete, stir for 15min. Adjust the pH to 6.8, and then bring the volume to 2L with water for injection. Shake well to obtain the mixture.

[0051] S3. The mixed solution is pre-filtered through a 0.45μm filter membrane to remove particulates, and then filtered through a 0.22μm sterile filter under aseptic conditions into a sterilized storage tank. The temperature is controlled at 15℃ during the filtration process. The filtered solution is filled into 10mL glass vials at a rate of 2.0mL / bottle, with the filling volume error controlled within ±2%. The vials are then partially stoppered with rubber stoppers, placed on the shelf of a freeze dryer, and freeze-dried under vacuum. The rubber stoppers are then tightened under nitrogen protection, and the vials are sealed with an aluminum-plastic composite cap to obtain the peptide synergistic activation composition for targeting and regulating IGF-1 synthesis in this embodiment.

[0052] Freeze-drying process can:

[0053] Pre-freezing: Lower the shelf temperature to -40°C and maintain for 3 hours to ensure complete freezing;

[0054] First drying: The shelf temperature is raised to -25℃, the vacuum is about 100mTorr, and the drying time is 12 hours;

[0055] Secondary drying: The shelf temperature is gradually increased to 25℃, and the vacuum is maintained at approximately 50 mTorr for 6 hours;

[0056] After drying, sterile nitrogen gas is introduced into the vacuum until atmospheric pressure is reached.

[0057] To verify the role of structural functional peptides derived from the key functional region of IGF-1 in synergistically activating IGF-1 synthesis, Comparative Example 1 was constructed: the formulation contained only basic regulatory peptides and no structural functional peptides such as GPET and GPETLC, while the other excipients and process conditions were the same as in Example 1.

[0058] Example 2

[0059] A peptide-co-activating composition targeting and regulating IGF-1 synthesis is formulated as a lyophilized powder for injection. This embodiment, based on Example 1, introduces the MMP-cleavable IGF-1 regulatory precursor peptide Pep-MMP-IGF to achieve on-demand release of the IGF-1 regulatory fragment SSSRRAP in a traumatic / inflammatory microenvironment with high MMP levels, while maintaining the same dosage form and preparation process as Example 1 for direct comparison. Pep-MMP-IGF contains the SSSRRAP fragment, which can be released under the action of MMPs.

[0060] The preparation method in this embodiment includes the following steps:

[0061] S1. Add 1.8L of water for injection to a sterilized stainless steel mixing tank, start stirring at 200rpm, and add 4.5g NaCl, 5.6g Na2HPO4•12H2O, 1.0g NaH2PO4•2H2O and 50.0g mannitol in sequence, stirring until completely dissolved to obtain a clear buffer excipient solution.

[0062] S2. Add 3.0g Pro-Gly, 3.0g Pro-Asp, 3.0g Gly-Pro-Glu, 3.0g Gly-Pro, 1.0g GPET, 1.0g GPETLC, 1.0g ETLCG, 1.0g QFVCGD, 1.0g CGDRGFY, 1.0g FNKPTG, 1.0g Pep-MMP-IGF, and 1.0g GIVDECC to 10mL of water for injection, and dissolve by shaking at room temperature. Then, slowly add each peptide solution to the clear buffer excipient solution sequentially at 50rpm. After the addition is complete, stir for 15min. Adjust the pH to 6.8, and then bring the volume to 2L with water for injection. Shake well to obtain the mixture.

[0063] S3. The mixed solution is pre-filtered through a 0.45μm filter membrane to remove particulates, and then filtered through a 0.22μm sterile filter under aseptic conditions into a sterilized storage tank. The temperature is controlled at 15℃ during the filtration process. The filtered solution is filled into 10mL glass vials at a rate of 2.0mL / bottle, with the filling volume error controlled within ±2%. The vials are then partially stoppered with rubber stoppers, placed on the shelf of a freeze dryer, and freeze-dried under vacuum. The rubber stoppers are then tightened under nitrogen protection, and the vials are sealed with an aluminum-plastic composite cap to obtain the peptide synergistic activation composition for targeting and regulating IGF-1 synthesis in this embodiment.

[0064] Freeze-drying process can:

[0065] Pre-freezing: Lower the shelf temperature to -40°C and maintain for 3 hours to ensure complete freezing;

[0066] First drying: The shelf temperature is raised to -25℃, the vacuum is about 100mTorr, and the drying time is 12 hours;

[0067] Secondary drying: The shelf temperature is gradually increased to 25℃, and the vacuum is maintained at approximately 50 mTorr for 6 hours;

[0068] After drying, sterile nitrogen gas is introduced into the vacuum until atmospheric pressure is reached.

[0069] Comparative Example 2

[0070] A polypeptide composition containing an inert IGF-1 precursor peptide is formulated as a lyophilized powder for injection. Comparative Example 2 uses the same formulation and process as Example 2, except that Pep-MMP-IGF is replaced with an equal amount of the inert precursor peptide Pep-inert-IGF, which significantly reduces the MMP-phagocytic activity or makes it essentially uncleanable.

[0071] The comparative preparation method includes the following steps:

[0072] S1. Add 1.8L of water for injection to a sterilized stainless steel mixing tank, start stirring at 200rpm, and add 4.5g NaCl, 5.6g Na2HPO4•12H2O, 1.0g NaH2PO4•2H2O and 50.0g mannitol in sequence, stirring until completely dissolved to obtain a clear buffer excipient solution.

[0073] S2. Add 3.0g Pro-Gly, 3.0g Pro-Asp, 3.0g Gly-Pro-Glu, 3.0g Gly-Pro, 1.0g GPET, 1.0g GPETLC, 1.0g ETLCG, 1.0g QFVCGD, 1.0g CGDRGFY, 1.0g FNKPTG, 1.0g Pep-inert-IGF, and 1.0g GIVDECC to 10mL of water for injection, and dissolve by shaking at room temperature. Then, slowly add each peptide solution to the clear buffer excipient solution sequentially at 50rpm. After the addition is complete, stir for 15min. Adjust the pH to 6.8, and then bring the volume to 2L with water for injection. Shake well to obtain the mixture.

[0074] S3. The mixed solution is pre-filtered through a 0.45μm filter membrane to remove particulates, and then filtered through a 0.22μm sterile filter under aseptic conditions into a sterilized storage tank. The temperature is controlled at 15℃ during the filtration process. The filtered solution is filled into 10mL glass vials at a rate of 2.0mL / bottle, with the filling volume error controlled within ±2%. The vials are then partially stoppered with rubber stoppers, placed on the shelf of a freeze dryer, and freeze-dried under vacuum. The rubber stoppers are then tightened under nitrogen protection, and the vials are sealed with an aluminum-plastic composite cap to obtain the peptide synergistic activation composition for targeting and regulating IGF-1 synthesis in this embodiment.

[0075] Freeze-drying process can:

[0076] Pre-freezing: Lower the shelf temperature to -40°C and maintain for 3 hours to ensure complete freezing;

[0077] First drying: The shelf temperature is raised to -25℃, the vacuum is about 100mTorr, and the drying time is 12 hours;

[0078] Secondary drying: The shelf temperature is gradually increased to 25℃, and the vacuum is maintained at approximately 50 mTorr for 6 hours;

[0079] After drying, sterile nitrogen gas is introduced into the vacuum until atmospheric pressure is reached.

[0080] Example 3

[0081] A peptide synergistic activation composition that targets and regulates IGF-1 synthesis, in the form of a lyophilized powder for injection.

[0082] The preparation method in this embodiment includes the following steps:

[0083] S1. Add 1.8L of water for injection to a sterilized stainless steel mixing tank, start stirring at 200rpm, and add 4.5g NaCl, 5.6g Na2HPO4•12H2O, 1.0g NaH2PO4•2H2O and 50.0g mannitol in sequence, stirring until completely dissolved to obtain a clear buffer excipient solution.

[0084] S2. Add 3.0g Pro-Gly, 3.0g Pro-Asp, 3.0g Gly-Pro-Glu, 3.0g Gly-Pro, 1.0g GPET, 1.0g GPETLC, 1.0g ETLCG, 1.0g QFVCGD, 1.0g CGDRGFY, 1.0g FNKPTG, 1.0g Pep-PLGL-IGF, and 1.0g GIVDECC to 10mL of water for injection, and dissolve by shaking at room temperature. Then, slowly add each peptide solution to the clear buffer excipient solution sequentially at 50rpm. After the addition is complete, stir for 15min. Adjust the pH to 6.8, and then bring the volume to 2L with water for injection. Shake well to obtain the mixture.

[0085] S3. The mixed solution is pre-filtered through a 0.45μm filter membrane to remove particulates, and then filtered through a 0.22μm sterile filter under aseptic conditions into a sterilized storage tank. The temperature is controlled at 15℃ during the filtration process. The filtered solution is filled into 10mL glass vials at a rate of 2.0mL / bottle, with the filling volume error controlled within ±2%. The vials are then partially stoppered with rubber stoppers, placed on the shelf of a freeze dryer, and freeze-dried under vacuum. The rubber stoppers are then tightened under nitrogen protection, and the vials are sealed with an aluminum-plastic composite cap to obtain the peptide synergistic activation composition for targeting and regulating IGF-1 synthesis in this embodiment.

[0086] Freeze-drying process can:

[0087] Pre-freezing: Lower the shelf temperature to -40°C and maintain for 3 hours to ensure complete freezing;

[0088] First drying: The shelf temperature is raised to -25℃, the vacuum is about 100mTorr, and the drying time is 12 hours;

[0089] Secondary drying: The shelf temperature is gradually increased to 25℃, and the vacuum is maintained at approximately 50 mTorr for 6 hours;

[0090] After drying, sterile nitrogen gas is introduced into the vacuum until atmospheric pressure is reached.

[0091] Test Example 1

[0092] Evaluation of the in vitro promotion of IGF-1 expression and secretion in hepatocytes

[0093] 1) Test materials

[0094] 1.1) Cells and Culture Conditions

[0095] Cell line: Human normal hepatocytes L-02, purchased from Shanghai Cell Bank, Chinese Academy of Sciences;

[0096] Culture medium: High glucose DMEM + 10% fetal bovine serum + 1% penicillin / streptomycin;

[0097] Culture conditions: 37℃, 5% CO2 constant temperature incubator.

[0098] 1.2) Sample solution preparation

[0099] The lyophilized powder injections obtained in Examples 1-3 and Comparative Examples 1-2 were reconstituted with 2.0 mL of water for injection according to the instructions to obtain a stock solution with a total polypeptide concentration of approximately 10 mg / mL. Before the experiment, the stock solution was further diluted with serum-free DMEM medium to a working solution with a total polypeptide concentration of 1.0 μg / mL.

[0100] 2) Grouping and Processing

[0101] Set up the following groups (each group has 3 duplicate holes):

[0102] NC group: blank control group, supplemented only with serum-free DMEM medium;

[0103] VC group: Carrier control group, with an equal volume of excipient solution from Example 1 (without peptides);

[0104] Group A: Sample A (Example 1), final concentration of total peptides 1.0 μg / mL;

[0105] Group B: Sample B (Comparative Example 1), final concentration of total peptide 1.0 μg / mL;

[0106] Group C: Sample C (Example 2), final concentration of total peptides 1.0 μg / mL;

[0107] Group D: Sample D (Comparative Example 2), final concentration of total peptide 1.0 μg / mL.

[0108] Group E: Sample E (Example 3), final concentration of total peptides 1.0 μg / mL.

[0109] Procedure: Seed L-02 cells into 96-well plates, 1 × 10⁶ cells per well. 4Cells were cultured in complete medium containing 10% FBS for 24 hours to allow cell adhesion. The old medium was discarded, and the cells were gently washed once with PBS buffer and replaced with serum-free DMEM. The corresponding treatment solutions were added according to the above grouping, with a final volume of 100 μL / well, and incubation was continued for 24 hours. After incubation, the supernatant was collected, and the IGF-1 secretion was measured using a human IGF-1 ELISA kit. Total RNA was extracted from parallel wells using the TRIzol method, reverse transcribed into cDNA, and IGF-1 mRNA was detected using SYBR Green real-time quantitative PCR. The relative expression level was calculated using the 2-ΔΔCt method with GAPDH as an internal control, and the NC group was set as 1.0. Cell viability was detected using the CCK-8 assay (parallel wells for each group), and the results were expressed as a percentage relative to the NC group.

[0110] Table 1. Effects of IGF-1 expression and secretion on L-02 cells

[0111] Relative expression level of IGF-1 mRNA (with NC=1.00) <![CDATA[IGF-1 secretion amount (ng·mL -1 ).]]> Cell survival rate % NC Group 1.00 5.1 100 VC Group 0.97 5.0 99.2 Example 1 2.23 11.4 98.6 Comparative Example 1 1.64 8.3 98.9 Example 2 2.15 11.0 98.4 Comparative Example 2 2.07 10.7 98.5 Example 3 2.09 10.8 98.5

[0112] Compared with the blank control group and the vector control group, all groups in Examples 1-3 and Control Examples 1-2 could upregulate IGF-1 mRNA expression and promote IGF-1 secretion in L-02 cells to varying degrees, and the cell survival rate was ≥98%, indicating that the samples had no obvious cytotoxicity within the concentration range of this experiment.

[0113] In Example 1, the IGF-1 mRNA and protein levels at the same concentration were significantly higher than in Comparative Example 1, indicating that the addition of structural functional peptides (GPET, GPETLC, etc.) derived from key functional regions of IGF-1 can provide a significant synergistic activation effect on top of the basic regulatory peptides. The promoting effect of Example 2 on IGF-1 under exogenous MMP-free conditions was comparable to that of Example 1, with minimal difference between Example 2 and Comparative Example 2. This suggests that replacing free SSSRRAP with Pep-MMP-IGF or Pep-inert-IGF does not significantly alter the baseline IGF-1 promoting ability when basal MMP levels are low. The advantage of Example 2 over Comparative Example 2 mainly lies in the on-demand release under high MMP conditions, as detailed in Test Examples 2-3.

[0114] Test Example 2

[0115] The effect of the presence or absence of MMP-2 on the ability of samples C, D, and E to regulate IGF-1

[0116] 1) Experimental materials and grouping

[0117] Cells: L-02 human hepatocytes, cultured under the same conditions as test case 1;

[0118] Samples: C (Example 2), D (Comparative Example 2), E (Example 3), reconstituted and diluted to a total peptide concentration of 1.0 μg / mL;

[0119] MMP-2 working solution: prepared in serum-free DMEM, final concentration 10 ng / mL.

[0120] The groups are as follows (3 duplicate holes per group):

[0121] NC group: blank control, no peptides or MMP-2 added;

[0122] MMP-0 group: MMP-2 (10 ng / mL) was added only, without peptides;

[0123] C-NoMMP group: Sample C 1.0 μg / mL, no MMP-2;

[0124] C-MMP group: Sample C 1.0 μg / mL + MMP-2 10 ng / mL;

[0125] D-NoMMP group: Sample D 1.0 μg / mL, no MMP-2;

[0126] D-MMP group: Sample D 1.0 μg / mL + MMP-2 10 ng / mL.

[0127] E-NoMMP group: Sample E 1.0 μg / mL, no MMP-2;

[0128] E-MMP group: Sample E 1.0 μg / mL + MMP-2 10 ng / mL.

[0129] 2) Experimental method: L-02 cells were seeded in 6-well plates at a density of 2 × 10⁶ cells per well. 5 Cells were cultured for 24 h; starved with serum-free DMEM for 12 h; added with the corresponding treatment solution according to the above grouping, and incubated for 24 h; supernatant was collected, and IGF-1 content was detected by ELISA; total RNA was extracted from cells with TRIzol, and IGF-1 mRNA was detected by qPCR, with the relative expression level of NC group as 1.0.

[0130] Table 2. Differences in the regulation of IGF-1 by samples C, D, and E under conditions of MMP-2 presence and absence.

[0131] Group Relative expression level of IGF-1 mRNA (with NC=1.00) <![CDATA[IGF-1 secretion amount (ng·mL -1 )]]> NC 1 5.1 MMP-0 1.06 5.3 C-NoMMP 2.12 10.6 C-MMP 3.05 14.3 D-NoMMP 2.03 10.2 D-MMP 2.11 10.5 E-NoMMP 2.05 10.4 E-MMP 2.55 12.3

[0132] The addition of MMP-2 alone had little effect on IGF-1 expression, indicating that the concentration of MMP-2 used itself does not activate the IGF-1 pathway. In the absence of exogenous MMP-2, the C-NoMMP and D-NoMMP groups showed similar levels of promotion of IGF-1 mRNA and protein, consistent with the conclusion of Test Example 1, that is, at basal MMP levels, Pep-MMP-IGF and Pep-inert-IGF have similar effects on IGF-1. After the addition of MMP-2, the IGF-1 mRNA and secretion levels in the C-MMP group increased significantly, significantly higher than those in the C-NoMMP and D-MMP groups, indicating that Pep-MMP-IGF is cleaved under the action of MMP-2, releasing more fragments with IGF-1 regulatory activity (such as SSSRRAP), thus producing an additional promoting effect. The D-MMP group and the D-NoMMP group in Comparative Example 2 showed little difference, indicating that the inert precursor Pep-inert-IGF's ability to regulate IGF-1 remained essentially unchanged regardless of the presence or absence of MMP-2, and it did not exhibit microenvironment responsiveness. Although Pep-PLGL-IGF could be cleaved by MMP-2, its efficiency was lower than that of Pep-MMP-IGF, resulting in its enhancement of IGF-1 regulation in the presence of exogenous MMP-2 falling between that of Pep-MMP-IGF and the inert precursor.

[0133] Evaluation of wound healing promotion in a mouse full-thickness skin trauma model (Example 3)

[0134] 1) Animals and Grouping

[0135] Laboratory animals: SPF-grade male C57BL / 6 mice, weighing 18-22g, acclimatized for 7 days;

[0136] Grouping (n=10 per group):

[0137] NC group: blank control group (trauma + saline injection);

[0138] BH group: Sample B (Comparative Example 1) high-dose group, total peptide 2.0 mg / kg;

[0139] Group AH: Sample A (Example 1) high-dose group, total peptide amount 2.0 mg / kg;

[0140] DH group: Sample D (Comparative Example 2) high-dose group, total peptide 2.0 mg / kg;

[0141] Group CH: Sample C (Example 2) high-dose group, total peptide amount 2.0 mg / kg.

[0142] Group EH: Sample E (Example 3) high-dose group, total peptide amount 2.0 mg / kg.

[0143] The total peptide dose for each group was calculated at 2.0 mg / kg body weight, with the only difference being the type of peptide.

[0144] 2) Trauma modeling and drug administration methods

[0145] Anesthesia and modeling: Mice were anesthetized by intraperitoneal injection of sodium pentobarbital or an equivalent anesthetic; the back was shaved and disinfected with 75% ethanol; bilateral full-thickness skin defects (reaching the fascia layer) were prepared on both sides of the midline of the back using a sterile 8mm punch, and the initial wound area A0 was recorded.

[0146] Reconstitution and Administration: The lyophilized powder injections of the corresponding samples in each group were reconstituted with water for injection to achieve a total peptide concentration of 10 mg / mL; the administration volume was calculated based on body weight to ensure a total peptide concentration of 2.0 mg / kg; administration was carried out immediately on the day of model establishment (day 0) according to the following regimen, and continued once daily for 7 days:

[0147] NC group: An equal volume of physiological saline was injected subcutaneously / intradermally at multiple points around the wound edge;

[0148] Group AH: Injection of sample A reconstituted solution;

[0149] Group BH: Injected sample B reconstituted solution;

[0150] Group CH: Inject sample C reconstituted solution.

[0151] DH group: Inject sample D reconstituted solution;

[0152] Group EH: Injected sample E complex solution.

[0153] Each animal received the same total injection, only the polypeptide composition differed;

[0154] The wound is uniformly covered with a semi-permeable dressing, which is changed at fixed intervals.

[0155] 3) Indicator Testing

[0156] Wound healing rate: Wound photographs were taken at fixed distances and under fixed conditions on the day of modeling (day 0) and on days 3, 7, 10, and 14; the wound area At at each time point was measured using ImageJ software, and the healing rate was calculated according to the formula:

[0157] Healing rate (%) = [1 - (At / A0)] × 100

[0158] Histological observation: On day 14, some mice in each group were randomly sacrificed, and the wound and surrounding tissues were taken, fixed in 4% paraformaldehyde and embedded in paraffin; HE staining was used to observe epidermal regeneration and granulation tissue formation; Masson trichrome staining was used to assess collagen fiber deposition and arrangement.

[0159] Immunohistochemical detection of IGF-1 and Ki67: IGF-1 and Ki67 immunohistochemical staining was performed on the same tissue section; the integrated optical density (IOD) of IGF-1 positive signal was calculated using image analysis software, with the IGF-1 IOD on day 14 of the NC group set as 1.0; the percentage of Ki67 positive cells was counted to reflect the level of cell proliferation at the wound edge.

[0160] Table 3. Wound healing and local IGF-1 expression in mice of each group.

[0161] Group Healing rate on day 7 / % Healing rate on day 10 / % NC 35.2 60.4 AH 70.4 93.2 BH 53.8 79.6 CH 75.6 96.1 DH 67.9 89.7 EH 72.4 94.1

[0162] Compared with the NC group, the BH, AH, DH and CH groups all significantly improved the wound healing rate, shortened the complete healing time, and significantly increased the local IGF-1 expression level and Ki67 positivity rate at 14 days, indicating that basic regulatory peptides and their combination with structural and functional peptides have a clear promoting effect on tissue repair.

[0163] At the same total peptide dose, the healing rate of Example 1 on days 7 and 10 and the positive rates of IGF-1 IOD and Ki67 on day 14 were significantly better than those of Comparative Example 1, indicating that the structural functional peptides derived from the key functional region of IGF-1 provide an unexpected synergistic healing-promoting effect on the basis of the basic regulatory peptides.

[0164] Comparative Example 2 showed slightly lower wound healing and IGF-1 upregulation than Example 1, indicating that simply introducing the inert precursor Pep-inert-IGF did not bring additional benefits in the high MMP wound environment. Example 2 showed significantly higher wound healing rates on days 7 and 10, and significantly higher IGF-1 IOD and Ki67 positivity rates on day 14 than the AH and DH groups, with a complete healing time shortened to about 9 days. This indicates that the composition containing Pep-MMP-IGF can be specifically cleaved in the high MMP wound microenvironment, releasing more IGF-1 modulating fragments as needed, thereby achieving a better healing-promoting effect than Example 1 and Comparative Example 2 under the same dosage and formulation.

[0165] The EH group (Pep-PLGL-IGF) was superior to the AH and DH groups in terms of healing rate on days 7 and 10, and IGF-1 IOD and Ki67 positivity rate on day 14, but slightly lower than the CH group (Pep-MMP-IGF). Its complete healing time was about 9.5 days, which was between that of Example 2 (9 days) and Example 1 (10 days). This indicates that the use of the MMP substrate precursor Pep-PLGL-IGF can utilize the wound MMP microenvironment to a certain extent to achieve on-demand release, but its overall effect is still not as good as Pep-MMP-IGF.

[0166] As can be seen from Test Examples 2-3, the enhanced effect of Example 2 can be reasonably attributed to the MMP-responsive cleavage of Pep-MMP-IGF and the targeted release of IGF-1 regulatory fragments such as SSSRRAP, rather than a simple increase in the total amount of peptide or a difference in dosage form. This proves that Example 2 of the present invention has significant technical progress compared with Comparative Example 2 and Example 1.

[0167] The amino acid sequence involved in this invention is as follows:

[0168] The basic regulatory peptide Pro-Gly has the amino acid sequence PG.

[0169] The basic regulatory peptide Pro-Asp has the amino acid sequence PD.

[0170] The basic regulatory peptide Gly-Pro-Glu, amino acid sequence: GPE.

[0171] The basic regulatory peptide Gly-Pro, amino acid sequence: GP.

[0172] Structural-functional peptide GPET, SEQ ID NO:1, GPET.

[0173] Structural-functional peptide GPETLC, SEQ ID NO:2, GPETLC.

[0174] Structural-functional peptide ETLCG, SEQ ID NO:3, ETLCG.

[0175] Structural-functional peptide QFVCGD, SEQ ID NO:4, QFVCGD.

[0176] Structural-functional peptide CGDRGFY, SEQ ID NO:5, CGDRGFY.

[0177] Structural-functional peptide FNKPTG, SEQ ID NO:6, FNKPTG.

[0178] Structural-functional peptide SSSRRAP, SEQ ID NO:7, SSSRRAP.

[0179] Structural-functional peptide GIVDECC, SEQ ID NO:8, GIVDECC.

[0180] MMP substrate sequence GPQGIWGQ, SEQ ID NO:9, GPQGIWGQ.

[0181] MMP substrate sequence PLGLAG, SEQ ID NO:10, PLGLAG.

[0182] MMP can cleave the precursor peptide Pep-MMP-IGF, SEQ ID NO:11, GGGPQGIWGQSSSRRAPGG.

[0183] MMP can cleave the precursor peptide Pep-PLGL-IGF, SEQ ID NO:12, GGPLGLAGSSSRRAPGG.

[0184] Inert control precursor peptide Pep-inert-IGF, SEQ ID NO:13, GGGPQGIAGQSSSRRAPGG.

Claims

1. A polypeptide co-activation composition for targeting and regulating IGF-1 synthesis, characterized in that: Including basic regulatory peptides and structural-functional peptides; The basic regulatory peptide is preferably a short peptide composed of 2-3 natural amino acid residues, and is an L-type linear peptide; The structural functional peptide is preferably a short peptide composed of 4 to 8 natural amino acid residues, which is an L-type linear peptide. The amino acid sequence is derived from the receptor-binding region or other functional segments of the IGF-1 protein, or is a conserved site substitution variant based on this, which can maintain or enhance the activation ability of the IGF-1 receptor or stabilize the conformation of IGF-1.

2. The peptide co-activation composition for targeting and regulating IGF-1 synthesis as described in claim 1, characterized in that: The basic regulatory peptide is selected from one or more of Pro-Gly, Pro-Asp, Gly-Pro-Glu, and Gly-Pro.

3. The peptide co-activation composition for targeting and regulating IGF-1 synthesis as described in claim 1, characterized in that: The structural functional peptides include, but are not limited to, one or more of GPET, GPETLC, ETLCG, QFVCGD, CGDRGFY, FNKPTG, SSSRRAP, and GIVDECC.

4. The peptide co-activation composition for targeting and regulating IGF-1 synthesis as described in claim 1, characterized in that: It may also include MMP-cleavable IGF-1-regulated precursor peptides; MMP-cleavable IGF-1 regulatory precursor peptides contain MMP substrate sequences and IGF-1 regulatory functional fragments.

5. The peptide co-activation composition for targeting and regulating IGF-1 synthesis as described in claim 4, characterized in that: The MMP-cleavable IGF-1 regulatory precursor polypeptide includes a substrate sequence that can be recognized and cleaved by matrix metalloproteinases, and at least one structural functional fragment with IGF-1 regulatory activity, which are connected by flexible linker peptides at both ends.

6. The peptide co-activation composition for targeting and regulating IGF-1 synthesis as described in claim 5, characterized in that: The MMP-cleavable IGF-1 regulatory precursor peptide is an L-type linear peptide, and the N-terminus and C-terminus may contain one or more glycine residues as flexible linkers; the substrate sequence is preferably a peptide sequence that can be recognized by MMP-2; the peptide sequence that can be recognized by MMP-2 is GPQGIWGQ or PLGLAG; the IGF-1 regulatory functional fragment is preferably a peptide containing the SSSRRAP sequence.

7. The peptide co-activation composition for targeting and regulating IGF-1 synthesis as described in claim 1 or 4, characterized in that: The total mass of the basic regulatory peptide, the structural functional peptide, and the MMP-cleavable precursor peptide is more than 80% of the total polypeptide in the composition; the preferred mass ratio of the basic regulatory peptide to the structural functional peptide is 1-10:1-10.

8. The peptide co-activation composition for targeting and regulating IGF-1 synthesis as described in claim 7, characterized in that: For a peptide co-activation composition that targets and regulates IGF-1 synthesis containing an MMP-cleavable IGF-1 regulatory precursor peptide, the mass fraction of the MMP-cleavable IGF-1 regulatory precursor peptide in the total peptide is preferably 5-40%.

9. A method for preparing the peptide co-activating composition for targeting and regulating IGF-1 synthesis as described in any one of claims 1-8, comprising the following steps: First, the isotonic regulator, buffer salt, and lyophilization protectant are dissolved in water for injection to prepare a clear buffer excipient solution. Then, the basic regulatory peptide, structural functional peptide, and MMP-cleavable IGF-1 regulatory precursor peptide (if any) were dissolved separately in water for injection. Under stirring, they were added sequentially to a buffer excipient solution, mixed thoroughly, and the pH was adjusted to weakly acidic or neutral. The solution was then brought to a final volume with water for injection to obtain a peptide mixture solution. Subsequently, the peptide mixture solution was pre-filtered, sterilized, dispensed, pre-frozen, and freeze-dried under vacuum. After drying, it was filled with inert gas and sealed to obtain the final product. The isotonicity regulator is sodium chloride; The buffer salt is a phosphate; The freeze-drying protectant is mannitol.

10. The use of the peptide synergistic activating composition for targeting and regulating IGF-1 synthesis as described in any one of claims 1-8 in the preparation of a medicament for promoting tissue repair.