Dendritic poly-L-lysine modified PACAP as well as preparation method and application thereof

Abstract

The invention particularly discloses dendritic poly-L-lysine modified PACAP as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The PACAP-DPL provided by the invention effectively overcomes the defect of insufficient in-vivo stability of natural PACAP on the premise of not weakening the biological function of PACAP, provides a new technical approach for the application of PACAP neuropeptides in Alzheimer's disease, and has good research value and potential application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a dendritic poly-L-lysine modified PACAP, its preparation method, and its application. Background Technology

[0002] Pituitary adenylate cyclase-activating polypeptide (PACAP) is an endogenous small neuropeptide, primarily existing in two natural forms: a full-length form consisting of 38 amino acids (PACAP-38) and a short-chain form consisting of 27 amino acids (PACAP-27). Current research indicates that PACAP has significant neurotrophic and neuroprotective effects in the central nervous system. PACAP-38 is the dominant form found in brain tissue. PACAP can promote intracellular cAMP production through the PAC1 receptor, thereby mediating related biological effects. Elevated cAMP levels can further activate downstream signaling pathways such as protein kinase A (PKA), regulating gene transcription, cell survival, and functional status.

[0003] Alzheimer's disease (AD) is the most common type of dementia, and currently, there are no effective disease-modifying treatments. Previous studies have shown that changes in PACAP levels in cerebrospinal fluid can serve as a potential biomarker for Alzheimer's disease, aiding in its diagnosis, and that decreased PACAP levels in brain tissue are correlated with AD pathology. Furthermore, animal experiments have demonstrated that exogenous administration of PACAP can improve cognitive function in AD model mice to some extent, suggesting that PACAP has certain research and application value in the intervention of Alzheimer's disease.

[0004] However, as a small molecule peptide, PACAP is easily degraded by various proteases in vivo, exhibiting poor stability, a short in vivo half-life, and a limited duration of efficacy, resulting in low bioavailability. Literature reports that the elimination half-life of PACAP-38 in the circulatory system is typically in the minute range, approximately 5–10 minutes in humans. Its rapid inactivation is mainly related to degradation processes mediated by in vivo proteases (such as dipeptidyl peptidase 4, DPP4). These physicochemical and pharmacokinetic characteristics limit the sustained efficacy of PACAP, thus restricting its long-term, stable application as a therapeutic drug in chronic neurological diseases such as Alzheimer's disease.

[0005] Therefore, how to effectively improve PACAP's in vivo stability, prolong its half-life, and enhance its sustained efficacy while maintaining its biological activity remains a pressing technical problem to be solved in this field. Summary of the Invention

[0006] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide a dendritic poly-L-lysine (DPL) modified PACAP (PACAP-DPL). Without weakening the biological function of PACAP, it effectively overcomes the deficiency of insufficient in vivo stability of natural PACAP, providing a new technical approach for the application of PACAP-like neuropeptides in Alzheimer's disease, and possesses good research value and potential application prospects.

[0007] (II) Technical Solution To achieve the above objectives, the present invention provides a PACAP-DPL having the structure shown in formula (1): (1); Where K represents lysine; R independently represents PACAP or .

[0008] In one embodiment, the PACAP is PACAP-38.

[0009] In one embodiment, it also includes pharmaceutically acceptable salts, esters, hydrates, solvates, metabolites, prodrugs, stereoisomers, tautomers, polymorphs, and / or isotope derivatives.

[0010] In one embodiment, R represents PACAP.

[0011] In one embodiment, the structure is as follows: .

[0012] In another aspect, the present invention provides a method for preparing the above-mentioned PACAP-DPL, comprising: S1: DPL-MAL is obtained by reacting EMCS (6-(maleimide)hexanoic acid succinimide ester) with DPL; S2: PACAP is reacted with DPL-MAL after a cysteine ​​residue is attached to the C-terminus of PACAP to obtain PACAP-DPL.

[0013] In another aspect, the present invention also provides a PACAP-DPL, which is obtained by the above preparation method.

[0014] In another aspect, the present invention provides a pharmaceutical composition comprising: (1) The therapeutically effective amount of the above-mentioned PACAP-DPL; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0015] In another aspect, the present invention provides a pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0016] In one embodiment, the dosage form of the pharmaceutical preparation includes plasters, granules, lotions, liniments, powders, syrups, aerosols, sprays, extracts, elixirs, solutions, ointments, fluid extracts, emulsions, suspensions, decoctions, infusions, tablets, suppositories, injections, alcohol preparations, capsules, creams, lozenges, tinctures, pastes, pills, and / or capsules.

[0017] In one embodiment, the dosage form of the pharmaceutical preparation includes solutions and injections.

[0018] In another aspect, the present invention also provides a pharmaceutical product comprising the above-described pharmaceutical preparation.

[0019] In one embodiment, the pharmaceutical product is a vial or box.

[0020] In another aspect, the present invention also provides the use of the above-mentioned PACAP-DPL, pharmaceutical composition, pharmaceutical preparation and / or pharmaceutical product in the preparation of medicines for the prevention and / or treatment of Alzheimer's disease.

[0021] (III) Beneficial Effects This invention provides a dendritic poly-L-lysine-modified PACAP, its preparation method, and its applications. Compared with existing technologies, it has the following advantages: 1. Compared with unmodified PACAP, PACAP-DPL showed greater stability in the brains of wild-type mice and significantly prolonged its half-life in brain tissue.

[0022] 2. Cell experiments showed that PACAP-DPL can effectively activate the downstream cAMP signaling pathway, indicating that it achieves enhanced stability while maintaining the biological activity of PACAP.

[0023] 3. Animal experiments further confirmed that PACAP-DPL can significantly improve the cognitive function of 5xFAD Alzheimer's disease model mice. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a schematic diagram of the synthesis of PACAP-modified drugs; Figure 2 This is a graph showing the activation of cAMP by PACAP-modified drugs; Figure 3 This is a schematic diagram of PACAP-PLGA synthesis; Figure 4 This is a mass spectrometry characterization of PACAP38C; Figure 5 This is a PACAP-PLGA characterization diagram; Figure 6 This is a PACAP-PLGA brain metabolism analysis diagram; Figure 7 This is a schematic diagram of PACAP-DPL synthesis; Figure 8 This is the PACAP-DPL characterization diagram; Figure 9 This is a PACAP-DPL brain metabolism analysis diagram; Figure 10 This is a quantitative graph of PACAP-DPL brain metabolism analysis; Figure 11 This is a PACAP-DPL major organ metabolism analysis diagram; Figure 12 This is a quantitative chart of major organ metabolism analysis using PACAP-DPL; Figure 13 This is a graph showing the activation analysis of cAMP by PACAP-DPL; Figure 14 This is a graph showing the therapeutic effect of PACAP-DPL on AD. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Terms and Definitions As used herein, the term "pharmaceutical composition" refers to a composition comprising PACAP-DPL formulated with one or more pharmaceutically acceptable carriers.

[0028] The formulation of a pharmaceutical composition can be tailored to the application. In particular, pharmaceutical compositions can be formulated using methods known in the art to provide rapid, continuous, or delayed release of the active ingredient upon administration to mammals. For example, the formulation can be selected from any of the following: plasters, granules, lotions, liniments, lemonade, aromatic water, powders, syrups, eye ointments, liquids and solutions, aerosols, sprays, extracts, elixirs, ointments, fluid extracts, emulsions, suspensions, decoctions, infusions, eye drops, tablets, suppositories, injections, alcoholic preparations, capsules, creams, lozenges, tinctures, pastes, pills, and soft or hard gelatin capsules.

[0029] As used herein, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0030] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier used for the administration of therapeutic agents, encompassing a variety of excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0031] Pharmaceutically acceptable carriers in a composition include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption-delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, flow aids, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc., compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide complementary, additional, or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch, or talc, or liquid carriers, such as water, fatty oils, or liquid paraffin, are possible. Other examples of carriers include culture media, such as DMEM or RPMI; and cryogenic storage media containing components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates, and ion concentrations to balance intracellular states at low temperatures; and mixtures of organic solvents with water.

[0032] The active substance in the product disclosed in this invention accounts for 0.001-99.9 wt% of the total weight of the composition. In one embodiment, the active ingredient is dissolved in a solvent at a concentration of 0.01-1 μg / μL; or 0.05-0.5 μg / μL; or 0.06-0.4 μg / μL; or 0.07-0.3 μg / μL; or 0.08-0.2 μg / μL, with the balance being a pharmaceutically acceptable carrier and other additives.

[0033] The dosage forms of the pharmaceutical compositions disclosed in this invention can be granules, tablets, lyophilized powders, suppositories, capsules, sublingual tablets, liquid solutions, nasal drops, sprays, and metered-dose sprays.

[0034] The pharmaceutical compositions of the present invention can be administered using any known method. One of a variety of methods known to those skilled in the art can be used to administer the substance, compound, or agent to a subject using the terms "give" or "apply".

[0035] For example, compounds or agents can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). Compounds or agents can also be suitably introduced via rechargeable or biodegradable polymeric devices or other devices (e.g., patches and pumps or formulations) that provide prolonged, slowed, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more prolonged periods.

[0036] As used herein, the term “therapeutic effective dose” refers to a dose that is adequately suited for medical treatment with a reasonable benefit / risk ratio for treating the disease, and the effective dose level includes subject type and severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant medications, and other factors known in the medical field.

[0037] As used herein, the term “treatment” for a symptom or patient refers to steps taken to achieve a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, symptom, or condition; substantially improving or alleviating the clinical or aesthetic symptoms of a symptom; substantially preventing the clinical or aesthetic symptoms of a disease, symptom, or condition; and avoiding harmful or unpleasant symptoms. Treatment also refers to accomplishing one or more of the following: (a) reducing the severity of the symptom; (b) limiting the development of characteristic symptoms of the symptom being treated; (c) limiting the exacerbation of characteristic symptoms of the symptom being treated; (d) limiting the recurrence of the symptom in patients who previously had the symptom; and / or (e) limiting the onset of symptoms in patients who previously did not have symptoms of the symptom.

[0038] The dendritic poly-L-lysine-modified PACAP described in this invention can also be used for medical aesthetic indications or cosmetic medical applications.

[0039] As used in this article, the terms "cosmetic indications" or "cosmetic medical uses" refer to cosmetic improvements that are not for therapeutic or disease treatment purposes, including but not limited to: improving skin appearance, reducing wrinkles, improving skin laxity, improving rough skin, improving dull skin, improving abnormal skin pigmentation, improving the appearance of skin aging, and improving skin contours.

[0040] As used in this article, the term "prevention" refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms.

[0041] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0043] Example 1: Synthesis of PACAP-modified drugs: A 39-peptide molecule (PACAP38C or PACAP39) with a cysteine ​​residue added to the C-terminus of the PACAP38 peptide sequence was synthesized using solid-phase synthesis. The ESI-MS results of the obtained product PACAP38C are shown below. Figure 4 As shown, and as Figure 1 The diagram shows the construction of three PACAP chemically modified delivery systems: polyethylene glycol (PEG) modification (… Figure 1In the B), poly(lactic-co-glycolic acid) copolymer (PLGA) modification ( Figure 1 A) Dendritic poly-L-lysine (DPL) modification ( Figure 1 (C) in order to develop new AD treatment drugs.

[0044] PEG and PLGA modification are drug chemical modification methods approved by the U.S. Food and Drug Administration for use in humans. DPL has good biocompatibility and is a common type of dendritic macromolecule used for biomodification. Some DPL-modified therapeutics have also entered the clinical trial stage.

[0045] The terminal thiol group of the synthesized PACAP38C was reacted with the terminal maleimide group of PEG, PLGA and DPL molecules to link the peptide molecules with the polymer molecules.

[0046] Synthesis of PACAP-PLGA: One equivalent of PLGA-COOH and 100 equivalents of EDC / NHS were dissolved in DMF solution and mixed and stirred for 24 h. After activation by EDC / NHS, the terminal carboxyl group of the PLGA-COOH molecule reacted with the amino group of 200 equivalents of the small molecule linking unit MAL-NH2 for 2 h to generate PLGA-MAL molecules. After dialysis to remove unreacted small molecule impurities, it was further reacted with 0.67 equivalents of PACAP38C for 24 h. The maleimide group and thiol group linked to form a PACAP-PLGA amphiphilic chain. When dispersed in water, it can form micelles with a particle size of approximately 100 nm, achieving brain delivery of PACAP across the BBB, thereby improving drug accumulation and efficacy in the brain. Figure 3 ).

[0047] The synthesis of PACAP-PEG involves mixing 1 equivalent of PACAP with 1.5 equivalents of mPEG. 5K -MAL molecules were dissolved in PBS solution with a pH of 6.5, mixed and stirred for 24 h, and then obtained by lyophilization after dialysis to remove salt.

[0048] Synthesis of PACAP-DPL: Using solid-phase synthesis, a second-generation DPL molecule with four lysine residues at the end (containing eight amino groups) and a third-generation DPL molecule with eight lysine residues at the end (containing sixteen amino groups) were constructed. Take 0.17 mmol of 1 mmol / g 2-chlorotriphenylmethyl chloride resin, swell it in anhydrous DMF for 60 minutes, and drain it under nitrogen. Then, dissolve 2 equivalents of Fmoc-Lys(Fmoc)-OH relative to the chlorine groups of the resin in 2 mL of anhydrous DMF, add 4 equivalents of DIPEA, and stir under nitrogen for 5 minutes. Add this solution to the resin and react at room temperature under nitrogen for 2 hours. After the reaction, add a methanol / DIPEA / DMF (2:1:7, v / v) mixture and block unreacted sites under nitrogen for 30 minutes. Wash twice with 2 mL of DMF, then treat twice with 20% piperidine / DMF solution (10 minutes each time) to remove the Fmoc protecting group, exposing the two primary amino groups to obtain O-generation polylysine. Wash 5 times with DMF and store under nitrogen.

[0049] Two equivalents of each of Fmoc-Lys(Fmoc)-OH, HBTU, and HOBT were dissolved in 4 mL of anhydrous DMF. Four equivalents of DIPEA were added, and the mixture was activated in nitrogen for 2 minutes before being added to the resin. The reaction was carried out at room temperature under nitrogen for 2 hours. After washing twice with 2 mL of DMF, the mixture was treated twice with 20% piperidine / DMF solution (10 minutes each time) to remove the Fmoc protecting group and expose the four primary amino groups, yielding first-generation polylysine.

[0050] Four equivalents of each of Fmoc-Lys(Fmoc)-OH, HBTU, and HOBT were dissolved in 6 mL of anhydrous DMF. Eight equivalents of DIPEA were added, and the mixture was activated in nitrogen for 2 minutes before being added to the resin. The reaction was carried out at room temperature under nitrogen for 2 hours. After washing twice with 2 mL of DMF, the mixture was treated twice with 20% piperidine / DMF solution (10 minutes each time) to remove the Fmoc protecting group and expose eight primary amino groups, yielding second-generation polylysine.

[0051] Eight equivalents of each of Fmoc-Lys(Fmoc)-OH, HBTU, and HOBT were dissolved in 8 mL of anhydrous DMF. Sixteen equivalents of DIPEA were added, and the mixture was activated in nitrogen for 2 minutes before being added to the resin. The reaction was carried out at room temperature under nitrogen for 2 hours. After washing twice with 2 mL of DMF, the mixture was treated twice with 20% piperidine / DMF solution (10 minutes each time) to remove the Fmoc protecting group, exposing sixteen primary amino groups to obtain third-generation polylysine.

[0052] After synthesis, the second- and third-generation polylysine were washed three times each with DMF, isopropanol, and cyclohexane. The resin was then treated with a dichloromethane solution (v / v) containing 1% trifluoroacetic acid for 2 hours, and the filtrate was collected. The filtrate was concentrated by purging with nitrogen, precipitated with cold diethyl ether, and the white solid was collected by centrifugation and dried under vacuum to obtain the second-generation or third-generation DPL.

[0053] Next, 10 mg of second-generation DPL and 108 mg, or 32 equivalents, of the small molecule linker 6-(maleimide)hexanoic acid succinimide ester (EMCS) were dissolved in 5 mL of N,N-dimethylformamide (DMF) and stirred in the dark for 24 h. Maleimide group modification was carried out by the click reaction between the terminal amino group of DPL and the succinimide group (NHS) of EMCS to obtain the dendritic molecule DPL-MAL with thiol reactivity. Similarly, 10 mg of third-generation DPL and 216 mg, or 64 equivalents, of solid EMCS were dissolved in 10 mL of DMF and stirred in the dark for 24 h to obtain third-generation DPL-MAL.

[0054] Further, 0.34 mg of second-generation DPL-MAL and 20 mg (32 equivalents) of PACAP38C were mixed in 5 mL of DMF and stirred in the dark for 24 h to obtain second-generation PACAP-DPL; 0.34 mg of third-generation DPL-MAL and 20 mg (64 equivalents) of PACAP38C were mixed in 5 mL of DMF and stirred in the dark for 24 h to obtain third-generation PACAP-DPL. Figure 7 ).

[0055] Example 2: Screening of PACAP-modified drugs: A stable, high-expression HEK293T cell line of PAC1R was constructed using cell transfection technology. First, HEK293T cells were seeded in culture dishes. When the cell confluence reached 70%–80%, the expression plasmid carrying the PAC1R gene was transfected into the cells using PEI transfection reagent. An empty vector plasmid transfection group served as a negative control. After 6–8 hours of transfection, the medium was replaced with fresh complete medium, and the cells were cultured for another 48 hours. Puromycin was added for drug selection, and the culture was continued with regular changes of the drug-containing medium until all untransfected cells died. The surviving cells were further subjected to single-clone picking and amplification culture. Finally, the mRNA and protein expression levels of PAC1R were detected by qPCR and immunofluorescence to verify and establish a stable, high-expression cell line.

[0056] The experiment was divided into two groups: a control group received no treatment, while the drug intervention group received PACAP38, PACAP39 prepared in Example 1, PACAP-PLGA, or PACAP-DPL (final concentration 100 nM). Both groups of cells were incubated at 37°C for 20 minutes, then washed with PBS and lysed with cell lysis buffer. Subsequent procedures were performed according to the ELISA kit (RD KGE002B) instructions.

[0057] Immunofluorescence results showed that, compared with untransfected HEK293T cells, the expression level of PAC1R on the cell membrane surface of transfected cells was significantly increased, indicating that the localization and expression of PAC1R on the cell membrane were as expected. Figure 2 (A in the middle).

[0058] PACAP-PLGA significantly promoted intracellular cAMP production, with activation levels comparable to unmodified PACAP, suggesting that PLGA modification does not affect PACAP's biological activity. In contrast, PACAP-PEG showed a significantly reduced ability to activate cAMP, inducing approximately 57.9% less cAMP levels than unmodified PACAP, suggesting that PEG linkage may adversely affect PACAP's binding to PAC1 or the receptor activation process. Figure 2 (B in the middle).

[0059] Based on the above results, and considering that PEG modification significantly reduced the bioactivity of PACAP, this strategy was not adopted further.

[0060] Example 3: Characterization of PACAP-PLGA: PLGA is a biodegradable, low-toxicity medical functional polymer with strong hydrophobicity, while PACAP38 peptide has strong hydrophilicity. The amphiphilic chain formed by the connection of the two can self-assemble into stable micelles in aqueous solution through hydrophobic interactions. This can improve the stability of PACAP in vivo and provide a basis for subsequent ligand functionalization for brain-targeted delivery of PACAP.

[0061] The obtained product was analyzed by infrared spectroscopy (IR). Figure 5 A) DLS ( Figure 5 C), Zeta potential measurement ( Figure 5 (B in the middle) and transmission electron microscopy ( Figure 5 Characterization was performed using methods such as (D) to indicate that the target drug had been successfully synthesized. Figure 5 ).

[0062] Example 4: Metabolic analysis of PACAP-PLGA: A cy5 fluorescent molecule was covalently linked to the lysine residue at position 36 of PACAP38C, and cy5-PACAP-PLGA micelles were synthesized using the preparation method described in Example 1. Wild-type C57B6J 8-9 week old male mice were used, and cy5-PACAP or cy5-PACAP-PLGA was injected intraperitoneally once (PACAP concentration 0.5ug / ul, total 200ul).

[0063] After intervention in mice, at 4h, 8h, 24h, 72h, and 5 days post-drug administration, the heart was perfused with 1xPBS for 4min (to remove residual fluorescence interference in the blood), and brain tissue was dissected. The fluorescence intensity of the brain tissue in each group was detected using the cy5 near-infrared channel on a multifunctional molecular imaging system to compare drug entry into the brain. It was found that cy5-PACAP-PLGA did not increase the drug's half-life in the brain compared to unmodified cy5-PACAP. Figure 6 A, fluorescence plot; B, quantization plot; n=3, ● and ▲ represent raw data points).

[0064] Example 5: PACAP-DPL characterization: The above experimental results indicate that the PACAP-PEG conjugate did not exhibit significant biological functions; although PACAP-PLGA has certain biological activity, in vivo experiments showed that it failed to effectively improve the stability of the drug in brain tissue, nor did it significantly prolong its in vivo half-life.

[0065] Based on the above results, further research was conducted on PACAP-DPL. Second-generation and third-generation DPL were used to conjugate PACAP, and the conjugation products were analyzed by SDS-PAGE electrophoresis to evaluate their conjugation efficiency. Simultaneously, Western blot was used to verify the conjugation bands displayed in the SDS-PAGE gel to confirm whether they were PACAP-related proteins.

[0066] The obtained product was determined by zeta potential measurement ( Figure 8 A) SDS-PAGE Figure 8 B, F), Western blot ( Figure 8 E in the middle) and MALDI-TOF MS ( Figure 8 C in PACAP n -DPL represents the connection of n PACAPs (n is 1~8) and other methods were used for characterization. The results showed that the synthesized second-generation PACAP-DPL had a significantly higher zeta potential than the free peptide PACAP, proving that PACAP was successfully modified.

[0067] SDS-PAGE showed that the obtained product had a high molecular weight, and the linkage product of second-generation DPL and PACAP had a higher linkage efficiency than that of third-generation DPL and PACAP.

[0068] The results of MALDI-TOF mass spectrometry also confirmed the increase in the molecular weight of the product. Further Western blot analysis confirmed the presence of PACAP molecules in these products with higher molecular weights, indicating that PACAP was successfully linked to the DPL molecule.

[0069] Example 6: Metabolic analysis of PACAP-DPL: A cy5 fluorescent molecule was covalently linked to the lysine residue at position 36 of PACAP38C, and then linked to the DPL-MAL molecule according to the preparation method in Example 1 to obtain a fluorescent cy5-PACAP-DPL molecule.

[0070] BALB / c nude mice were intraperitoneally injected with equal amounts of cy5-PACAP and cy5-PACAP-DPL (200 μL, 0.5 ug / ul cy5-PACAP), and small animal fluorescence imaging was performed on the nude mice at different time points after injection. The results showed that both drugs could enter the mouse brain.

[0071] Analysis of the fluorescence intensity in the brain demonstrated that the modified cy5-PACAP-DPL molecule has a longer half-life in the brain than cy5-PACAP. Figures 9-10 It has significant advantages over PACAP-PLGA.

[0072] Figure 11 and Figure 12 The metabolic process of cy5-PACAP-DPL in major organs other than the brain was shown within 15 min to 2 h after administration.

[0073] Example 7: PACAP-DPL activation analysis of cAMP: The cell culture method was the same as in Example 2. The results showed that PACAP-DPL intervention in monoclonal HEK293T-PAC1R cells could significantly stimulate cAMP production, indicating that PACAP-DPL has functional activity comparable to PACAP at the cellular level, and DPL modification does not affect the function of PACAP. Figure 13 (n=3, ●, ■ and ▲ represent the original data points).

[0074] Example 8: Verification of the therapeutic effect of PACAP-DPL on AD: The classic Alzheimer's disease model, the 5xFAD transgenic mouse, was used as the research subject. The 5xFAD mouse is an APP / PS1 double transgenic mouse carrying mutations in five familial Alzheimer's disease-related genes. Among these, the APP-related mutations include K670N / M671L (Swedish), I716V (Florida), and V717I (London), while the presenilin-1 (PS1)-related mutations include M146L and L286V. This model mouse can exhibit typical Alzheimer's disease pathological features at an early stage, including β-amyloid plaque deposition, neuronal loss, glial cell proliferation, and learning and memory impairment.

[0075] Sex- and age-matched wild-type (WT) mice and 5xFAD mice were randomly assigned to four groups: WT+ddH2O, WT+PACAP-DPL, 5xFAD+ddH2O, and 5xFAD+PACAP-DPL. Intervention was initiated at 3 months of age and continued until 5 months of age, with administration via nasal inhalation. Behavioral assessments were performed at 5 months of age.

[0076] The dosage of PACAP-DPL was 0.6 μg / dose (concentration 0.1 μg / μL), dissolved in ddH2O, with a total administration volume of 6 μL. 3 μL was administered to each nostril, 5 days a week. The control group received an equal volume of ddH2O.

[0077] Cognitive behavioral assessment primarily employed the Barnes maze test to assess spatial learning and memory abilities in mice. Results showed that PACAP-DPL significantly improved learning in 5xFAD mice. Figure 14 A) and spatial memory function ( Figure 14 (B in the middle).

[0078] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0079] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dendritic poly-L-lysine-modified PACAP, characterized in that, It has the structure shown in equation (1): （1）； Where K represents lysine; R independently represents PACAP or .

2. The dendritic poly-L-lysine-modified PACAP according to claim 1, characterized in that, It also includes pharmaceutically acceptable salts, esters, hydrates, solvates, metabolites, prodrugs, stereoisomers, tautomers, polymorphs and / or isotope derivatives.

3. The dendritic poly-L-lysine-modified PACAP according to claim 1, characterized in that, R stands for PACAP.

4. A method for preparing dendritic poly-L-lysine-modified PACAP as described in any one of claims 1-3, characterized in that, include: S1: React EMCS with DPL to obtain DPL-MAL; S2: After attaching a cysteine ​​residue to the C-terminus of PACAP, it reacts with the DPL-MAL to obtain the dendritic poly-L-lysine-modified PACAP.

5. The preparation method according to claim 4, characterized in that, The PACAP in S2 is PACAP38.

6. A pharmaceutical composition, characterized in that, include: (1) A therapeutically effective amount of the dendritic poly-L-lysine modified PACAP according to any one of claims 1-3 or the dendritic poly-L-lysine modified PACAP obtained by the preparation method according to claim 4 or 5; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

7. A pharmaceutical preparation, characterized in that, Includes the pharmaceutical composition of claim 6.

8. The pharmaceutical preparation according to claim 7, characterized in that, The drug preparation is administered via the nasal cavity and / or abdominal cavity.

9. A pharmaceutical product, characterized in that, Includes the pharmaceutical formulation as described in claim 7 or 8.

10. The use of the dendritic poly-L-lysine modified PACAP according to any one of claims 1-3, or the dendritic poly-L-lysine modified PACAP obtained by the preparation method according to claim 4 or 5, the pharmaceutical composition according to claim 6, the pharmaceutical formulation according to claim 7 or 8, and / or the pharmaceutical product according to claim 9 in the preparation of a drug for the prevention and / or treatment of Alzheimer's disease.

Images