Spermidine-polypeptide coupling compound as well as preparation method and application thereof

Abstract

The invention relates to a spermidine-polypeptide coupling compound as well as a preparation method and application thereof. The structural formula of the spermidine-polypeptide coupling compound is shown in the specification. The novel PDC compound is synthesized by a simple and efficient method, and the compound can reduce the protein expression quantity of senescence-related protein gamma H2A. X in vascular endothelial cells and visceral organ tissues, the content of senescence-related galactosidase (SA-beta-gal), the mRNA expression quantity and protein expression quantity of markers p16 and p21 and the ROS level; the PDC compound can reverse low proliferation of senescent cells and improve the conditions of vascular wall thickness and collagen deposition, and has a remarkable anti-aging effect. And the PDC compound has good druggability, has no obvious toxicity to main organs (lung, liver, kidney and spleen), has good safety, and can be used for preparing drugs for treating aging and related diseases thereof.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicines, and relates to an anti-aging medicine, in particular to a spermidine-polypeptide conjugate compound, a preparation method thereof and application thereof in anti-aging. BACKGROUND

[0002] With the intensification of global population aging, vascular diseases and organ function decline associated with aging have become a major public health problem. Vascular aging is a key link of the body's aging, and its characteristics include endothelial cell dysfunction, increased oxidative stress, chronic low-grade inflammation, and decreased mitochondrial function, etc., which is a key risk factor for leading to a variety of age-related diseases such as atherosclerosis, heart failure, cognitive decline, etc. At the same time, the accumulation of senescent cells in solid organs such as liver, lung, kidney, etc. will drive tissue fibrosis, loss of function and increase the risk of tumor occurrence, which directly affects the quality of life and life expectancy of the elderly.

[0003] At present, the intervention strategies for aging mainly include small molecule compounds, polypeptide drugs and natural products, etc., but all have obvious limitations. Small molecule compounds, such as spermidine, have the problems of poor targeting and high cytotoxicity. When administered systemically, it is difficult to enrich in specific senescent tissues or cells, and a high dose is often needed to take effect, thereby further increasing the risk of its toxic side effects. Polypeptide drugs have become a new focus of anti-aging research due to their high activity, good targeting and relatively low toxicity. For example, platelet factor 4 (PF4) is a large protein molecule with anti-aging activity, which is a protein composed of 70 amino acids, released by activated platelet alpha granules, and is a tetramer of polypeptide. However, its large molecular weight makes it inconvenient to be chemically synthesized, which limits its industrialization progress. Low molecular weight active peptides extracted from natural products (such as quinoa bran, marine organisms) also show anti-aging potential. For example, quinoa bran peptides with a molecular weight of less than 3 kDa can down-regulate the expression of senescence-related markers by inhibiting the p53 signaling pathway and improve oxidative stress. Marine active peptides usually have small molecular weight and have the characteristics of antioxidant and antihypertensive. However, the activity of these natural peptides is limited, and the separation and purification process is complex, which is difficult to meet the needs of drug development.

[0004] Therefore, it is necessary to develop a new anti-aging drug with good safety, high activity and simple synthesis for treating aging-related diseases. SUMMARY

[0005] Based on this, the purpose of the present application is to provide a new anti-aging drug.

[0006] The technical solutions for achieving the above-mentioned purposes include the following.

[0007] In a first aspect, the present application provides a spermidine-polypeptide conjugate compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, the structure of the spermidine-polypeptide conjugate compound being

[0008] .

[0009] In a second aspect, the present application provides use of the spermidine-polypeptide conjugate compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof in the preparation of a drug for inhibiting aging.

[0010] In a third aspect, the present application provides use of the spermidine-polypeptide conjugate compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof in the preparation of a drug for preventing and / or treating a disease related to aging.

[0011] In a fourth aspect, the present application provides a preparation method of the spermidine-polypeptide conjugate compound, comprising the following steps:

[0012] (1) synthesizing a linear protective polypeptide with a sequence of CPLYKKIIKKLLESC-Val-Cit;

[0013] (2) reacting the linear protective polypeptide with N1,N4-bis-BOC-spermidine to obtain a linear protective polypeptide conjugated with spermidine;

[0014] (3) deprotecting the linear protective polypeptide conjugated with spermidine, and then performing a ring closing reaction on cysteine residues in the linear protective polypeptide to form disulfide bonds, thereby obtaining the spermidine-polypeptide conjugate compound.

[0015] In a fifth aspect, the present application provides a drug for anti-aging or preventing and / or treating a disease related to aging, which is prepared from an active ingredient and a pharmaceutically acceptable adjuvant, and the active ingredient comprises the spermidine-polypeptide conjugate compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to the present application.

[0016] The present application has the following beneficial effects:

[0017] This invention synthesizes a novel PDC compound using a simple and efficient method. This compound exerts its anti-aging effect by regulating the DNA repair protein FBL. It reduces the protein expression of aging-related protein γH2A.X, the content of aging-related galactosidase (SA-β-gal), the mRNA and protein expression levels of markers p16 and p21, and ROS levels in vascular endothelial cells and organ tissues. The PDC compound reverses the low proliferative capacity of senescent cells, improves vascular wall thickness and collagen deposition, and exhibits significant anti-aging effects. Furthermore, the PDC compound has good drug-like properties, low cytotoxicity, no significant toxicity to major organs (lung, liver, kidney, spleen), and good safety profile, making it suitable for preparing drugs to treat aging and related diseases.

[0018] The PDC compound of the present invention has a small molecule polypeptide moiety, which has a relatively simple structure, is relatively easy to synthesize, and is easy to store and transport, and is expected to become a promising conjugate drug for the treatment of aging and related diseases. Attached Figure Description

[0019] Figure 1 This is an HPLC chromatogram of PDC compounds.

[0020] Figure 2 This is a schematic diagram of the mass spectrometry used to characterize the structure of PDC compounds.

[0021] Figure 3 Results of cytotoxicity assays for PDC compounds and spermidine (SPD). *** indicates P < 0.001 PDC vs SPD.

[0022] Figure 4 This diagram illustrates the staining of aging galactosidase in RAECs and HPMECs, where Control represents the control group and Model represents the angiotensin II (Ang II)-induced aging group. ### indicates P<0.001 vs Control, ** indicates P<0.01 vs Model, and *** indicates P<0.001 vs Model.

[0023] Figure 5 The effect of PDC compounds on FBL protein expression in the aging model HMEC-1 cells.

[0024] Figure 6 The results show the protein expression levels of γH2A.X and P21. **** indicates P < 0.0001, and ns indicates no significant difference.

[0025] Figure 7Results of detection of PDC inhibition of aging genes P16 and P21 in human microvascular endothelial cells and human lung microvascular endothelial cells. ** indicates P<0.01, **** indicates P<0.0001, and ns indicates no significant difference.

[0026] Figure 8 The results show the expression of ROS in a PDC-inhibited Ang II-induced human lung microvascular endothelial cell senescence model. **** indicates P < 0.0001.

[0027] Figure 9 To improve the extent of Edu proliferation in an Ang II-induced human lung microvascular endothelial cell senescence model using PDC. ### indicates P<0.001 vs Control, ** indicates P<0.01 vs Model, *** indicates P<0.001 vs Model.

[0028] Figure 10 To improve the degree of cell cycle arrest in an Ang II-induced human lung microvascular endothelial cell senescence model using PDC. ### indicates P<0.001 vs Control, ** indicates P<0.01 vs Model, *** indicates P<0.001 vs Model.

[0029] Figure 11 Results of PDC reducing the expression of creatine kinase isoenzymes, alanine aminotransferase, and lipofuscin in a mouse model of D-galactosidase-induced aging. ### indicates P<0.001 vs Control, ** indicates P<0.01 vs Model, *** indicates P<0.001 vs Model.

[0030] Figure 12 PDC effectively reduced the expression of the aging marker protein P21 in the heart tissue of a mouse aging model induced by D-galactosidase, and its performance in reducing P21 was superior to that of the positive control drugs NMN and CoQ10.

[0031] Figure 13 Results of hematoxylin-eosin (H&E) staining and Masson staining were shown.

[0032] Figure 14 Hematoxylin and eosin (H&E) staining results showed that PDC did not exhibit toxicity in the key organs (lung, liver, kidney, and spleen) of mice fed with PDC for six months. ### indicates P<0.001 vs Control, ** indicates P<0.01 vs Model, *** indicates P<0.001 vs Model.

[0033] Figure 15 To investigate the effect of PDC on the levels of aging-related biomarkers galactosidase in a D-galactose-induced aging mouse model by aging galactosidase staining (SA-β-gal), the results confirmed that PDC can reverse D-galactosidase-induced aging. Detailed Implementation

[0034] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0035] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0036] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0037] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0038] Some embodiments of the present invention relate to a spermidine-peptide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, wherein the structural formula of the spermidine-peptide conjugate is as follows:

[0039] .

[0040] The spermidine-peptide conjugate of the present invention can be prepared using conventional solid-phase synthesis methods. First, a linear polypeptide CPLYKKIIKKLLESC-Val-Cit is synthesized, then N1,N4-bis-BOC-spermine is conjugated to it, followed by disulfide cyclization with hydrogen peroxide to obtain the final product.

[0041] For example, some embodiments of the present invention relate to a method for preparing the spermidine-peptide coupling compound, comprising the following steps:

[0042] (1) A linear protective polypeptide with the synthetic sequence CPLYKKIIKKLLESC-Val-Cit;

[0043] (2) The linear protective polypeptide is reacted with N1,N4-bis-BOC-spermidine to obtain a linear protective polypeptide coupled with spermidine.

[0044] (3) After deprotecting the linear protected polypeptide coupled with spermidine, the cysteine ​​residues therein undergo a cyclization reaction to form a disulfide bond, thereby obtaining the spermidine-polypeptide coupled compound.

[0045] In some embodiments, the reaction in step (2) is carried out under the activation of 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide.

[0046] In some embodiments, the reaction in step (2) is carried out at a temperature of 20°C-30°C for 3-5 hours.

[0047] In some embodiments, the cyclization reaction described in step (3) is carried out in the presence of hydrogen peroxide at a mass concentration of 4%-6%.

[0048] In some embodiments, the cyclization reaction in step (3) is carried out at a temperature of 20°C-30°C for 0.8 hours to 2 hours.

[0049] This invention, through extensive experimental data, confirms that the spermidine-peptide conjugate or its pharmaceutically acceptable salt or stereoisomer described herein can exert anti-aging effects by regulating the DNA repair protein FBL; it can reduce the expression level of γH2A.X protein in vascular endothelial cells and organ tissues; it can reduce the content of aging-related galactosidase in vascular endothelial cells and organ tissues; it can reduce the mRNA expression of aging-related proteins p16 and p21 in vascular endothelial cells and organ tissues; it can reduce the ROS level in vascular endothelial cells and organ tissues; it can reverse the low proliferation of senescent cells; and it can reduce vascular thickening and collagen deposition in blood vessels.

[0050] Based on this, some embodiments of the present invention also relate to the use of the spermidine-peptide conjugate or its pharmaceutically acceptable salt or stereoisomer in the preparation of medicaments for inhibiting aging.

[0051] In some embodiments, the aging includes vascular aging and organ tissue aging, such as vascular endothelial cell aging, and aging of organs such as the liver, spleen, lungs, kidneys, blood vessels, rectum, testes, and heart. Among these, vascular endothelial cell aging is particularly characterized by pulmonary microvascular endothelial cell aging and aortic vascular endothelial cell aging.

[0052] Some embodiments of the present invention also relate to the use of the spermidine-peptide conjugate or its pharmaceutically acceptable salt or stereoisomer in the preparation of medicaments for the prevention and / or treatment of aging-related diseases.

[0053] Some embodiments of the present invention also relate to a medicament for anti-aging or prevention and / or treatment of aging-related diseases, prepared from an active ingredient and pharmaceutically acceptable excipients, said active ingredient comprising the spermidine-peptide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

[0054] The amount of active ingredients contained therein is within the safe and effective range. "Safe and effective range" means that the amount of active ingredients is sufficient to significantly improve the condition without causing serious side effects.

[0055] "Pharmaceutical acceptable excipients" refer to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity.

[0056] "Compatibility" here refers to the ability of the components in the composition or formulation to interact with and incorporate with the active ingredient of the present invention without significantly reducing the efficacy of the active ingredient.

[0057] Pharmaceutically acceptable examples of excipients (or carriers) include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween®), wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0058] The administration method of the active ingredient, pharmaceutical composition, or pharmaceutical preparation of the present invention is not particularly limited. Representative administration methods include, but are not limited to, oral, transdermal, rectal, and parenteral (intravenous, intramuscular, or subcutaneous) administration. That is, the dosage form of the pharmaceutical preparation includes, but is not limited to, capsules, granules, tablets, pills, powders, drops, ointments, patches, liniments, sprays, powders, suppositories, sustained-release preparations, and injections.

[0059] Solid dosage forms for oral administration include capsules, granules, tablets, pills, and powders. In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or excipient or carrier), such as sodium citrate or dicalcium phosphate, or with the following components:

[0060] (a) Fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol and silica;

[0061] (b) Adhesives, such as hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic;

[0062] (c) Moisturizers, such as glycerin;

[0063] (d) Disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginate, certain complex silicates, and sodium carbonate;

[0064] (e) Slow solvents, such as paraffin;

[0065] (f) Absorption accelerators, for example, quaternary ammonium compounds;

[0066] (g) Wetting agents, such as cetyl alcohol and glyceryl monostearate;

[0067] (h) Adsorbents, such as kaolin; and

[0068] (i) Lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain a buffer.

[0069] The solid dosage form can also be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active ingredient from this composition can be delayed in a portion of the digestive tract. Examples of suitable encapsulating components are polymers and waxes.

[0070] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0071] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0072] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0073] The present invention will be further described in detail below with reference to specific embodiments.

[0074] Example 1: Synthesis of spermidine-peptide conjugates

[0075]

[0076] First, a linear polypeptide CPLYKKIIKKLLESC-Val-Cit was synthesized using a solid-phase synthesis method and coupled with N1,N4-bis-BOC-spermine. Then, 5% hydrogen peroxide was added to perform disulfide bond cyclization. All reactions were carried out at room temperature, and the specific steps are as follows:

[0077] 1.1 Select a reactor (20*250 mm, No. 1 sand core) and write the peptide name on the reactor as a label. This process adopts peptide solid-phase chemical synthesis (Fmoc solid-phase chemical synthesis). In the following process, AA represents amino acid.

[0078] 1.2 Weigh 1 g of 2-chlorotriphenylmethyl chloride resin and 2-Cl CTC resin (2-Cl resin) using an electronic balance, place them in a reactor, and add 119.226 mg of dichloromethane (DCM) to soak for 30 min.

[0079] 1.3 Linking the first C-terminal amino acid: Weigh 0.3 mmol of Fmoc-Cit-OH (the first C-terminal amino acid) into a centrifuge tube, dissolve it in 5 mL of DCM, then add 0.3 mmol of N,N-diisopropylethylamine (DIEA) and shake well. Use a disposable pipette to add the solution to the reactor from the previous step, bubble the reaction under nitrogen for 90 min, and after the reaction is complete, add a mixture of 2 mL methanol and 4 mL DCM and react for another 20 min.

[0080] 1.4 Washing: After the liquid in the reactor is evacuated by a circulating water vacuum pump, industrial-grade dimethylformamide (DMF) is added to the reactor through a wash bottle. The volume of the reagent is about 3 times the volume of the resin, so that the resin is completely immersed in the solution. Nitrogen gas is bubbled and washed for 30 seconds. Then, the liquid in the reactor is evacuated by a circulating water vacuum pump for about 30 seconds. This operation is repeated 4 times.

[0081] 1.5 De-Fmoc: Add 20% piperidine / DMF solution to the reactor using a wash bottle. The reagent volume should be approximately 3 times the resin volume to ensure the resin is completely immersed in the solution. Bubble the reaction with nitrogen for 20 min.

[0082] 1.6 Washing: Refer to 1.4, and replace the industrial-grade DMF with analytical-grade DMF during the fifth wash.

[0083] 1.7 Resin Detection: Using a long-necked pipette, place 10-20 resin particles from the reactor at the bottom of the test tube. Then, using a dropper, add two drops each of reagent A (5g ninhydrin-100ml anhydrous ethanol) and reagent B (analytical grade pyridine) to the test tube, ensuring sufficient contact between the resin and the reagents. Heat the test tube at 100℃ for 2 minutes and observe the resin color. If the resin shows color, it indicates successful Fmoc removal in step 1.5. If no color develops, repeat steps 1.5-1.7.

[0084] 1.8 Coupling: Weigh 1 mmol Fmoc-Val-OH (C-terminal second position) and 1 mmol HOBT (1-hydroxybenzotriazole) into a centrifuge tube, dissolve them thoroughly in 20 mL DMF, then add 1 mmol DIC (N,N'-diisopropylcarbodiimide), mix for 1 min, and add to the dried resin. React under nitrogen bubbling for 1 h. Do not add DIC directly to the reactor. Do not add the crystals generated during pre-activation of AA to the reactor. If the solution in the reactor cannot be shaken evenly to ensure uniform resin mixing during the reaction, a small amount of DMF can be added.

[0085] 1.9 Resin test: Refer to 1.7 and observe the color of the resin. If there is no color, it indicates that the connection is complete and proceed to step 1.10; if there is color, repeat step 1.8.

[0086] 1.10 Washing, same as 1.6.

[0087] 1.11 Repeat steps 1.5-1.10 until peptide inoculation is complete. Finally, remove Fmoc (see 1.5), then wash (see 1.4), add a mixture of 1 mmol Boc anhydride and 5 mL DCM, then add 1 mmol DIEA to block the reaction for 20 min, then wash (see 1.4), and finally wash the resin 3 times with DCM and dry it.

[0088] 1.12 Modification of C-terminal small molecule (N1,N4-bis-BOC-spermidine): The dried resin was added to a 50 mL centrifuge tube, followed by 30 mL of a 20% TFE (2,2,2-trifluoroethanol) / DCM mixture. The mixture was shaken for 2 h, filtered, and then lyophilized by rotary evaporation to obtain the fully protected peptide. The fully protected peptide was dissolved in 20 mL of DCM, and a mixed solution containing 1 mmol HOBT and 1.2 mmol N1,N4-bis-BOC-spermidine dissolved in 2 mL of DMF was added. Then, 1 mmol DIC was added, and the mixture was shaken for 4 h before being lyophilized by rotary evaporation.

[0089] 1.13 Prepare the cutting reagent (taking 100 mL of cutting reagent as an example). The formula is: 95 mL TFA (trifluoroacetic acid) + 1 mL water + 2 mL EDT (1,2-ethylenedithiol) + 2 mL Tis (triisopropylsilane). Set aside. The preparation amount is generally 1 g resin plus 10 mL cutting reagent. 1 mL of cutting reagent is washed with 10 mL of diethyl ether.

[0090] 1.14 Cutting, Deprotection, and Sedimentation: Anhydrous diethyl ether was placed at -20°C for pre-cooling (pre-cooling time ≥ 2 h). The lyophilized product obtained in 1.12 was placed into a centrifuge tube, and 1 mL of cutting reagent was added. The tube was shaken at room temperature for 2 h to separate the peptide from the resin and remove the protecting groups on the peptide side chains. After 2 h, the cutting filtrate was filtered into 10 mL of ice-cold diethyl ether. After centrifugation, the supernatant was removed to obtain the crude peptide.

[0091] 1.15 Peptide Purification

[0092] Column: 20*250 mm Daisogel, 8 microns

[0093] Mobile phase: A: 0.1% TFA + Water; B: 0.1% TFA + Acetonitrile

[0094] Flow rate: 10 mL / min

[0095] The sample was loaded via pump A, and then a 10% acetonitrile solution was run for 5 minutes to begin gradient elution.

[0096] Time (min) B.conc

[0097] 0 10%

[0098] 5 15%

[0099] 45 55%

[0100] Sample peaks were collected and detected to obtain a linear polypeptide CPLYKKIIKKLLESC-Val-Cit coupled with spermidine, with an analytical purity of 97%.

[0101] 1.16 Cyclation of the peptide: The linear peptide CPLYKKIIKKLLESC-Val-Cit coupled with spermidine was prepared into a 0.10 mg / mL peptide solution using 50 mM NH4HCO3 aqueous solution as solvent. Then, 5% hydrogen peroxide was added and reacted for 1 h (200 mg peptide, total dissolved volume 2000 mL, hydrogen peroxide content 100 mL) to form disulfide bonds on the cysteine ​​residues. The sample was loaded via pump A and purified using the same method described in 1.15 above. The sample peaks were collected and detected.

[0102] 1.17 Sample Lyophilization: Transfer the sample to a lyophilization dish and lyophilize in a freeze dryer for 24 h to obtain a pure spermidine-peptide conjugate (PDC) with an analytical purity greater than 95% (e.g., Figure 1 (As shown). Its mass spectrum is as follows. Figure 2 As shown.

[0103] Example 2: CCK-8 cell viability assay

[0104] Methods: HMEC-1 cells in logarithmic growth phase were seeded at an appropriate density in 96-well plates and pre-cultured at 37°C in a 5% CO2 incubator to allow cell adhesion. The old culture medium was discarded, and the experimental groups were treated with fresh culture medium containing different concentrations of the drug (PDC and SPD treatment groups: 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM, respectively), while the control group used drug-free medium. After the predetermined treatment time (24 h), 10 μL of CCK-8 solution was added directly to each well, and the 96-well plate was returned to the incubator for 2 hours in the dark. The absorbance (OD value) of each well was measured using a microplate reader at 450 nm.

[0105] like Figure 3 As shown, the survival rate of HMEC-1 cells in the PDC compound group was significantly higher than that in the spermidine-treated group; spermidine (SPD) has significant cytotoxicity, while the PDC compound of this invention has virtually no cytotoxicity, minimal toxic side effects, and good safety.

[0106] Example 3: Effects of PDC on the levels of the aging biomarker β-galactosidase (SA-β-gal) and FBL protein expression in an aging model.

[0107] Modeling and drug administration: Twelve hours before modeling, HPMECs and RAECs in each group (including control group, model group, and drug administration group) were starved in serum-free culture medium. Subsequently, HPMECs and RAECs in the model group and drug administration group were stimulated with angiotensin II at a final concentration of 2 μmol / L for at least 48 hours. Fresh serum-free culture medium was used every 24 hours. The concentrations of PDC in the drug administration group were 1 μM, 2.5 μM, and 5 μM, respectively. 100 μM β-nicotinamide mononucleotide (NMN) and 500 nM spermidine (SPD, spermidine has high cytotoxicity and the concentration should not be too high) were used as positive control drugs.

[0108] Detection of aging marker β-galactosidase levels: After modeling and drug administration, cells were fixed with 4% polymethanol and then stained with a cell senescence β-galactosidase staining kit (Beyotime, product number: C0602). After staining overnight, the number of positive cells was observed under a Leica microscope and the data were statistically analyzed using Prism 10 software.

[0109] FBL protein expression detection: After Ang II modeling and drug administration (PDC drugs: 1μM, 2.5 μM and 5μM, positive control only 100μM NMN) in HMEC-1 cells, cells were collected, total protein was extracted, and FBL protein expression was detected by Western blot experiment.

[0110] The results of the analysis of β-galactosidase levels are as follows: Figure 4 As shown, PDC compounds significantly reduced the levels of β-galactosidase, a biomarker associated with aging.

[0111] The results of FBL protein expression are as follows: Figure 5 As shown, the expression of fibrillarin (FBL) protein, which is related to DNA repair, was decreased in the model, while the administration of PDC compound significantly increased the expression of FBL protein.

[0112] Example 4: Effects of PDC on the levels of aging biomarkers γH2A.X and P21 in an in vitro aging model

[0113] After inducing and treating the cell senescence model as described in Example 3, cells were lysed in ice-cold RIPA buffer containing 1 mM benzyl sulfonyl fluoride. Proteins were obtained by centrifugation at 12,000 rpm for 15 minutes at 4°C. An equal volume of 10 µg of protein was loaded onto a 10–15% SDS-PAGE unit and transferred to a nitrocellulose NC membrane via electroblotting. The NC membrane was then blocked with 5% skim milk powder, stained with a 1:1000 diluted primary antibody, and incubated overnight at 4°C. Cell membrane analysis was performed using a peroxidase-conjugated secondary antibody at a 1:10,000 dilution. Antigen-antibody complexes were detected using enhanced chemiluminescence assays, visualized using an AMERSHAmersham Image Quant 800 system, and analyzed using ImageJ software.

[0114] Analysis results as follows Figure 6 As shown, PDC compounds can effectively reduce the expression levels of senescence proteins γH2A.X and P21 in HPMECs cells.

[0115] Example 5: Effect of PDC on mRNA levels in an in vitro aging model

[0116] Methods: Twelve hours before modeling, human microvascular endothelial cells (HMEC-1) and human lung microvascular endothelial cells (HPMECs) in all groups (including control, model, and drug-treated groups) were starved in serum-free culture medium. Subsequently, the model and drug-treated groups were stimulated with angiotensin II at a final concentration of 2 μmol / L for at least 48 hours, with fresh serum-free culture medium every 24 hours. The PDC concentrations in the drug-treated groups were 1 μM, 2.5 μM, and 5 μM, respectively. 100 μM β-nicotinamide mononucleotide (NMN) and 500 nM spermidine (SPD) were used as positive controls. After stimulation and drug treatment, total RNA was isolated using TRIzol reagent. Approximately 1 μg of total RNA from each sample was reverse transcribed into cDNA using a transgene kit, and then polymerase chain reaction (PCR) was performed using UltraSYBR buffer to detect in vitro aging biomarkers and inflammatory markers.

[0117] like Figure 7 As shown, PDC can significantly reduce the mRNA expression of aging-related proteins P16 and P21.

[0118] Example 6 Detection of Intracellular Reactive Oxygen Species (ROS)

[0119] Methods: ROS detection kits were purchased from Beyotime. HPMECs cells were cultured at 5 × 10⁶ cells / year. 4 / wells were seeded at a density of 96 plates. One day after seeding, the culture wells were treated with Ang II (2 μM) and different doses of PDC solution for 24 hours. The PDC concentrations for the treatment groups were 1 μM, 2.5 μM, and 5 μM, respectively. 100 μM β-nicotinamide mononucleotide (NMN) and 500 nM spermidine (SPD) were used as positive controls. To obtain dissociated endothelial cells for ROS assay, the culture medium was first removed and the cells were washed three times with PBS. DCFH-DA was diluted to a final concentration of 10 μM with serum-free medium and added to the culture. The cells were incubated at 37°C for 20 minutes. Fluorescence was read at 488 nm using an IN Cell Analyzer 6000 plate reader (Life, USA). Fluorescence intensity indicated intracellular ROS.

[0120] like Figure 8 As shown, PDC compounds can significantly reduce the expression level of ROS in HPMECs cells.

[0121] Example 7 Edu cell proliferation detection

[0122] Methods: HPMECs in logarithmic growth phase were seeded at an appropriate density in culture plates. Senescence model induction and drug treatment were performed as in Example 3 (using 100 μM β-nicotinamide mononucleotide (NMN) and 500 nM spermidine (SPD) as positive controls). After cell modeling and drug treatment, an appropriate amount of Edu working solution (20 μM) was added to the culture medium, and the cells were incubated at 37 ℃ in a 5% CO2 incubator for 2-4 hours. The culture medium was discarded, and the cells were washed with PBS. The cells were fixed with 4% paraformaldehyde for 15-30 minutes, followed by permeabilization with 0.5% Triton X-100 for 10-20 minutes. Azide and Cu, labeled with fluorescent dyes, were then added. 2+ The reaction solution was incubated at room temperature in the dark for 30 minutes to specifically label Edu that had incorporated DNA. All cell nuclei were counterstained with DAPI. Finally, the cells were washed with PBS and observed and counted under a fluorescence microscope. The ratio of Edu-positive (proliferating cells) to the total number of cells is the cell proliferation rate.

[0123] like Figure 9 As shown, PDC compounds can reverse the low proliferation of senescent cells and significantly promote the proliferation of senescent cells.

[0124] Example 8: Flow Cytometry Cell Cycle Detection

[0125] Methods: HPMECs in logarithmic growth phase were seeded at an appropriate density in culture plates. Senescence model induction and drug treatment were performed as in Example 3 (using 100 μM β-nicotinamide mononucleotide (NMN) and 500 nM spermidine (SPD) as positive controls). After cell modeling and drug treatment, adherent cells were digested with trypsin (without EDTA), and digestion was terminated with the previously collected supernatant. After centrifugation (1000 g, 5 min), cells were gently resuspended in pre-chilled PBS and washed 1-2 times to thoroughly remove residual culture medium and trypsin. The cell suspension was slowly added dropwise to pre-chilled 70% ethanol (do not add ethanol directly to the cell pellet), while vortexing to mix, and fixed at 4°C for at least 30 min. Ethanol was removed by centrifugation, and cells were washed once with PBS to remove the fixative. PBS containing RNase A (working concentration typically 50-100 μg / mL) was added, and the cells were incubated at 37°C for 30 min to degrade RNA and avoid interference with DNA quantification. Add PI staining solution (propidium iodide, final concentration generally 50 μg / mL) and incubate in the dark for 15-30 minutes. Analyze the cells using flow cytometry. FlowJo et al. fitted the PI fluorescence histogram and calculated the percentage of cells in G0 / G1, S, and G2 / M phases.

[0126] like Figure 10 As shown, PDC compounds can restore the cell cycle of senescent cells.

[0127] Example 9: D-galactosidase-induced aging in mice and detection of blood parameters

[0128] Methods: Six-week-old, healthy C57BL / 6J mice weighing 22-26g were randomly selected and divided into a control group and a model group (model group, administered D-galactose intraperitoneally at a dose of 500 mg / kg daily). The model group received different concentrations of PDC compounds via intraperitoneal injection (1.25 mg / kg / 3 days, 2.5 mg / kg / 3 days, 5 mg / kg / 3 days, 10 mg / kg / 3 days). Positive controls included NMN and coenzyme Q10, both administered via gavage at 100 mg / kg / day. Each group contained at least 10 mice. After 180 days (6 months), mice were anesthetized with isoflurane, and blood was collected by enucleation. Serum was then separated by centrifugation at 3000 rpm for 10 minutes, and the expression of creatine kinase isoenzymes, alanine aminotransferase, and lipofuscin was detected.

[0129] like Figure 11 As shown, PDC compounds can significantly inhibit the overexpression of creatine kinase isoenzyme, alanine aminotransferase and lipofuscin in a D-galactose-induced aging mouse model.

[0130] Example 10: Therapeutic effect of PDC on a D-galactose-induced aging mouse model

[0131] Mice were induced to age using the method described in Example 9 and then drugged. After 180 days (6 months), heart tissue was taken, fixed overnight with 4% paraformaldehyde, and then embedded in paraffin for immunofluorescence staining. In this example, immunofluorescence double staining experiments were performed on cell nuclei and p21 protein. It was found that p21 expression was extremely high in the model group, while p21 expression in tissues injected with different concentrations of PDC was significantly lower than in the model group, and its ability to reduce p21 was higher than that of NMN and Coenzyme Q10. Simultaneously, coronary artery section staining of the heart revealed that the coronary arteries in the model group showed increased wall thickness and aggravated collagen deposition. Administration of different concentrations of PDC reduced the additional vascular thickening and collagen deposition within the blood vessels. NMN and Coenzyme Q10 did not have this effect.

[0132] like Figure 12 As shown, the PDC compound can significantly reduce the expression of P21 senescence protein.

[0133] like Figure 13 As shown, PDC compounds can effectively reduce the thickening and collagen deposition of coronary artery tissue in a mouse aging model induced by D-galactosidase, and their therapeutic effect is superior to that of the positive control drugs NMN and CoQ10.

[0134] Example 11 PDC Animal Toxicity Experiment

[0135] Eight groups of C57BL / 6J mice were used for model construction and drug administration as described in Example 9. The mice were administered the drug by injection and observed continuously for 180 days. Major organs were fixed overnight with 4% polymethyl methacrylate (PMMA) and then embedded in paraffin for H&E staining. Figure 14 As shown in the results, no mice died within 180 days after administration, and no abnormalities were observed in their food and water intake, spontaneous activity, etc. No obvious organic lesions were observed in the lungs, liver, kidneys, spleen, or other tissues.

[0136] Example 12: PDC reduces the levels of aging-related biomarkers and β-galactosidase.

[0137] Eight groups of C57BL / 6J mice were used for model construction and drug administration as described in Example 9. The mice were administered the drug by injection and observed continuously for 180 days. After anesthetizing the mice with isoflurane, the major organs were fixed overnight with 4% polymethanol, followed by SA-β-gal staining. Figure 15 As shown, PDC compounds can reduce the content of β-galactosidase, an aging-related marker, in the liver, spleen, lungs, kidneys, blood vessels, rectum, and testes, indicating that PDC can reverse D-galactosidase-induced aging, and the therapeutic effect on some organs (lungs, rectum, and testes) is better than that of the positive control drugs NMN and CoQ10.

[0138] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A spermidine-peptide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that, The structural formula of the spermidine-peptide conjugate is as follows: 。 2. The use of the spermidine-peptide conjugate of claim 1 or its pharmaceutically acceptable salt or stereoisomer in the preparation of an anti-aging medicament.

3. The use of the spermidine-peptide conjugate of claim 1 or its pharmaceutically acceptable salt or stereoisomer in the preparation of a medicament for anti-vascular aging.

4. The use of the spermidine-peptide conjugate of claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the preparation of a medicament for anti-vascular endothelial cell aging.

5. The use of the spermidine-peptide conjugate of claim 1 or its pharmaceutically acceptable salt or stereoisomer in the preparation of a medicament for inhibiting organ tissue aging.

6. The application according to claim 5, characterized in that, The organs mentioned are the liver, spleen, lungs, kidneys, blood vessels, rectum, testes, and heart.

7. The use of the spermidine-peptide conjugate of claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the preparation of a medicament for the prevention and / or treatment of aging-related diseases.

8. The application according to any one of claims 2-7, characterized in that, The spermidine-peptide conjugate or its pharmaceutically acceptable salt or stereoisomer can reduce the expression of γH2A.X protein in vascular endothelial cells and organ tissues; And / or, the spermidine-peptide conjugate or its pharmaceutically acceptable salt or stereoisomer can reduce the content of age-related galactosidase in vascular endothelial cells and organ tissues; And / or, the spermidine-peptide conjugate or its pharmaceutically acceptable salt or stereoisomer can exert anti-aging effects by regulating the DNA repair protein FBL; And / or, the spermidine-peptide conjugate or its pharmaceutically acceptable salt or stereoisomer can reduce the mRNA and protein expression levels of aging-related proteins p16 and p21 in vascular endothelial cells and organ tissues. And / or, the spermidine-peptide conjugate or its pharmaceutically acceptable salt or stereoisomer can reduce ROS levels in vascular endothelial cells and organ tissues; And / or, the spermidine-peptide conjugate or its pharmaceutically acceptable salt or stereoisomer can reverse the low proliferation of senescent cells; And / or, the spermidine-peptide conjugate or its pharmaceutically acceptable salt or stereoisomer can reduce vascular thickening and intravascular collagen deposition.

9. A method for preparing the spermidine-peptide conjugate according to claim 1, characterized in that, Includes the following steps: (1) A linear protective polypeptide with the synthetic sequence CPLYKKIIKKLLESC-Val-Cit; (2) The linear protective polypeptide is reacted with N1,N4-bis-BOC-spermidine to obtain a linear protective polypeptide coupled with spermidine. (3) After deprotecting the linear protected polypeptide coupled with spermidine, the cysteine ​​residues therein undergo a cyclization reaction to form a disulfide bond, thereby obtaining the spermidine-polypeptide coupled compound. Preferably, the reaction in step (2) is carried out under the activation of 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide; Preferably, the reaction in step (2) is carried out at a temperature of 20°C-30°C for 3-5 hours. Preferably, the cyclization reaction described in step (3) is carried out under the action of hydrogen peroxide with a mass concentration of 4%-6%; Preferably, the cyclization reaction in step (3) is carried out at a temperature of 20°C-30°C for 0.8 hours to 2 hours.

10. A drug for anti-aging or prevention and / or treatment of aging-related diseases, characterized in that, It is prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the spermidine-peptide conjugate of claim 1 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

Images