CN115957302A - Compound preparation LL-37-cGAMP and preparation method and application thereof - Google Patents

Abstract

The invention relates to a compound preparation (LL-37-cGAMP for short) consisting of a human-derived antibacterial peptide LL-37 and Cyclic dinucleotide Cyclic GMP-AMP (cGAMP), and a preparation method and application thereof. The sequence of the human antibacterial peptide LL-37 is shown as ID No. 1. The invention prepares LL-37-cGAMP compound preparation by combining human-derived antibacterial peptide LL-37 with cGAMP having cell membrane shuttle characteristic. On one hand, LL-37 can carry cGAMP molecules into cells efficiently, so that the immune activation capacity and the antiviral capacity of cGAMP are greatly improved (by 10-40 times); in addition, LL-37 has good antiviral property, and the defect that the antiviral effect of single cGAMP or LL-37 is not ideal is overcome by using LL-37 and cGAMP together.

Description

Compound preparation LL-37-cGAMP and preparation method and application thereof

Technical Field

The invention belongs to the field of biological preparations, and particularly relates to a novel compound preparation with immune regulation and antiviral functions, a preparation method and application thereof, in particular to a compound preparation for carrying biological micromolecule cGAMP by using humanized antibacterial peptide for combined immunotherapy and antiviral prevention and treatment, and a preparation method and application thereof.

Background

In recent years, the discovery of the cyclic GMP-AMP Synthase (cGAS) -cyclic GMP-AMP (cGAMP) -Stimulator of Interferon Genes (STING) signal pathway is a major breakthrough in the field of innate immunity. The cGAS-cGAMP-STING signal pathway plays an important role in the immune regulation of the body. There is a certain DNA damage response in body cells, and when this DNA is monitored and taken up by antigen presenting cells, it is recognized by cGAS and synthesizes the second messenger, cGAMP molecule. cGAMP binds and activates STING, and the activated STING recruits TBK1 and activates IRF3, so as to rapidly induce the expression of I-type interferon and downstream genes, and finally, powerfully inhibit and kill viruses. Thus, cGAMP, a good agonist for STING, is effective in killing viruses by activating innate immunity in the body. However, the content of natural cGAMP in human body is very limited, so that the exogenous cGAMP supplementation is promising for improving the body immunity and preventing and treating virus infection.

Although exogenous supplementation of cGAMP is an excellent strategy for regulating body immunity and resisting viruses, since cGAMP carries anions and has high hydrophilicity, cGAMP hardly penetrates cell membranes to enter cells to activate cGAS-STING innate immune pathways to play antiviral roles, which greatly limits the pharmaceutical potential and therapeutic effect of cGAMP. In addition, cGAMP alone has limited antiviral effects and is still difficult to achieve at a desirable level. In view of the above disadvantages, the development of safe and highly effective agents capable of enhancing cGAMP cell-entering efficiency and antiviral effect is of great significance for improving immune function and antiviral ability of human and animals.

Disclosure of Invention

Aiming at the problems that the current cGAMP is difficult to pass through cell membranes and the single antiviral effect is not ideal enough, the invention aims to provide a novel compound preparation LL-37-cGAMP with immune regulation and antiviral effects so as to improve the efficiency of the cGAMP entering cells and enhance the antiviral effect of the cGAMP, and a preparation method and application thereof.

In order to achieve the above objects, the present invention provides a novel immunomodulating and antiviral complex formulation.

The composition of the complex preparation comprises a cationic antibacterial peptide LL-37 and cGAMP or analogues thereof.

Wherein the amino acid sequence of the cationic antibacterial peptide LL-37 is shown as ID No.1, or the amino acid sequence with equivalent functions formed by replacing, deleting or adding one or more amino acids in the sequence.

The molecular structure of the cGAMP is shown as a formula I; the cGAMP analogue is other cyclic dinucleotide (such as 3,3-cGAMP, c-di-AMP, c-di-GMP and the like from bacteria) with similar functions or a small molecule with similar functions formed by micro-structural modification of cGAMP molecules.

Furthermore, the ratio of the cGAMP or the analogue thereof to the cationic antibacterial peptide LL-37 in the compound preparation is (0.1-300) mmol to (1-500) g.

Furthermore, the ratio of the cGAMP or the analogue thereof to the cationic antibacterial peptide LL-37 in the compound preparation is (1-3) mmol to (5-40) g.

The invention also provides a preparation method of the compound preparation.

The preparation method of the compound preparation provided by the invention comprises the following steps: physically blending the human cationic antibacterial peptide LL-37 with cGAMP or an analogue thereof.

Further, the human-derived cationic antibacterial peptide LL-37 and cGAMP or analogues thereof are physically blended, dispersed in a solvent, and prepared into a suspension.

Preferably, the effective concentration of LL-37 in the suspension is 1-500. Mu.g/ml, further 5-40. Mu.g/ml, such as 5. Mu.g/ml, 20. Mu.g/ml, 40. Mu.g/ml or 300. Mu.g/ml, and the effective concentration of cGAMP is 0.1-300. Mu.M, further 1-3. Mu.M, such as 1. Mu.M, 2. Mu.M, 3. Mu.M or 222. Mu.M.

According to a particular embodiment of the invention, the effective concentration of LL-37 in the suspension is between 5 and 40. Mu.g/ml and the effective concentration of cGAMP is between 1. Mu.M and 2. Mu.M.

According to one embodiment of the invention, the effective concentration of LL-37 in the suspension is 20. Mu.g/ml and the effective concentration of cGAMP is 3. Mu.M.

According to one embodiment of the invention, the effective concentration of LL-37 in the suspension is 300. Mu.g/ml and the effective concentration of cGAMP is 222. Mu.M.

The solvent may be selected from water, dimethyl sulfoxide (DMSO), and the like.

The invention also provides application of the composite preparation.

The application of the composite preparation provided by the invention is (a 1) and/or (a 2) and/or (a 3) and/or (a 4) and/or (a 5) as follows:

(a1) The application of the compound preparation in preparing immunomodulators;

(a2) The application of the compound preparation in preparing virus inhibitors;

(a3) The application of the compound preparation in preparing products for preventing and/or treating diseases caused by virus infection;

(a4) The application of the compound preparation in immune regulation;

(a5) The application of the compound preparation in inhibiting viruses;

(a6) The application of the compound preparation in preventing and/or treating diseases caused by virus infection.

Illustratively, the product may be a pharmaceutical, nutraceutical, food or additive.

Said product may contain, in addition to said complex formulation, suitable carriers or excipients. The carrier material herein includes, but is not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl cellulose, etc.). Among these, water-soluble carrier materials are preferred. The materials can be prepared into various dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections and the like. Can be common preparation, sustained release preparation, controlled release preparation and various microparticle drug delivery systems. In order to prepare the unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets. For making the unit dosage form into pill form, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc. In order to prepare the unit dosage form into suppositories, various carriers known in the art can be widely used. As examples of the carrier, there may be mentioned, for example, polyethylene glycol, lecithin, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like. For preparing the unit dosage form into preparations for injection such as solution, emulsion, lyophilized powder and suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid ester, etc. In addition, for the preparation of isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol may be added to the preparation for injection, and in addition, a conventional cosolvent, a buffer, a pH adjuster, and the like may be added. In addition, colorants, preservatives, flavors, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation, if desired. The preparation can be used for administration by injection, including subcutaneous injection, intravenous injection, intramuscular injection, intracavity injection and the like; for luminal administration, such as rectally and vaginally; administration to the respiratory tract, e.g., nasally; administration to the mucosa.

The invention also provides a method for enhancing the immunological activity of an animal, comprising the steps of: the compound preparation of the present invention is administered to a recipient animal to enhance the immunological activity of the animal.

The present invention also provides a method of inhibiting viral infection in an animal comprising the steps of: the complex formulation of the present invention is administered to a recipient animal to inhibit viral infection of the animal.

The present invention also provides a method of treating a disease caused by a viral infection, comprising the steps of: the complex formulation of the present invention is administered to a recipient animal to treat a disease caused by a viral infection.

The immune regulation is specifically to enhance the immune activity, such as activating STING innate immune response and promoting the expression of cellular immune factors.

The virus of the invention may be a herpesvirus, such as HSV-1 virus or VSV virus; the virus may also be other than a herpesvirus, such as papilloma virus (HPV).

In the present invention, the inhibitory virus may also be referred to as an antiviral. The inhibiting of the virus may specifically be inhibiting replication of the virus.

In the present invention, the animal may be a mammal, such as a human; the animal may also be other animals than mammals, such as poultry and fish, infected with the virus.

In vitro and in vivo experiments show that the compound preparation has remarkably enhanced immune activity and virus infection resistance compared with LL-37 or cGAMP alone. On a cell model, compared with the LL-37 or the cGAMP alone, the composition can not only more strongly activate the cGAS-STING innate immune signal pathway and promote the expression of immune factors such as IFN beta, but also more effectively resist the infection of herpes virus (HSV-1) and Vesicular Stomatitis Virus (VSV). Compared with the LL-37 or cGAMP alone, the compound preparation can also more strongly activate the cGAS-STING innate immune signal pathway, promote the expression of multiple immune factors such as IFN beta and the like, more effectively resist the infection of HSV-1 and VSV viruses and more remarkably relieve the weight loss of mice caused by virus infection in a mouse model.

The invention combines the human cationic antibacterial peptide LL-37 and the small molecular cGAMP into a compound preparation for the first time. Compared with the prior art, the invention has the beneficial effects that:

1. cGAMP is a small molecule with immunomodulatory activity. However, since cGAMP carries anions and has high hydrophilicity, cGAMP hardly penetrates cell membranes into cells to activate cGAS-STING innate immunity. LL-37 is a cationic antimicrobial peptide with amphiphilicity, and therefore, is very easy to pass through cell membranes. By combining LL-37 and cGAMP, LL-37 is utilized to combine and carry cGAMP to enter cells, so that the membrane penetrating efficiency of cGAMP is greatly improved, and the immune regulation capability and the antiviral capability of cGAMP are greatly improved (by 10-40 times);

2. the single cGAMP has limited antiviral effect and is difficult to achieve the ideal level. LL-37 has good antiviral activity, and the defect of weak antiviral effect of single cGAMP is overcome by using the two drugs in a compatible way.

The compound preparation can be used for preparing immune drugs (or health products, foods or additives and the like), antiviral drugs (or health products, foods or additives and the like) or drugs, health products, foods or additives and the like with immune regulation and antiviral functions.

Furthermore, the invention also provides a product (comprising medicines, health products, foods or additives and the like) containing the LL-37-cGAMP compound preparation. The product containing the LL-37-cGAMP compound preparation has excellent prevention and treatment effects on virus infection caused by HSV-1 and VSV; has excellent regulating and preventing effects on the reduction of immunity of the organism caused by virus infection.

The method of the invention can be adopted to prepare the compound medicine in large quantity and high efficiency, and the prepared compound preparation has excellent immunity enhancement and antiviral activity, has no obvious toxic or side effect, and has huge application value and potential.

The invention prepares the LL-37-cGAMP compound preparation by combining the human-derived antibacterial peptide LL-37 with the cell membrane shuttle characteristic and the cGAMP. On one hand, LL-37 can carry cGAMP molecules into cells efficiently, so that the immune activation capacity and the antiviral capacity of cGAMP are greatly improved (by 10-40 times); in addition, LL-37 has good antiviral property, and the defect that the antiviral effect of single cGAMP or LL-37 is not ideal is overcome by using LL-37 and cGAMP in a compatibility manner. In conclusion, the invention discloses a safe and efficient novel antiviral compound medicament which has good immunoregulation and antiviral effects on a cell model and an animal model. The research result provides a brand new thought and method for improving the immune function of the organism and preventing and treating the virus infection, has extremely high application value and development potential, and is beneficial to improving the health of human beings.

Drawings

FIG. 1 is a graph showing the activation of the STING-associated immune pathway by a LL-37-cGAMP complex formulation of example 1 of the present invention. (A) THP1-Lucia ISG cells were treated with a combination of cGAMP (1. Mu.M) and LL-37 at various concentrations (5,10,20,40. Mu.g/ml) to make LL-37-cGAMP complexes and the cellular ISRE luciferase activity was examined. ISRE luciferase activity of Lucia ISG cells represents the strength of activation of the STING pathway. (B) THP1 cells were treated with a cocktail of LL-37-cGAMP (2. Mu.M) and LL-37 at various concentrations (5,10,20,40. Mu.g/ml) and stimulation of STING signaling was assessed by immunoblotting (Western blotting). (C) RAW-Lucia ISG cells were treated and analyzed as in (A). (D) RAW264.7 cells were treated and analyzed as in (B). (E) BV2 cells were treated with LL-37-cGAMP complex formulation prepared by formulating cGAMP (2. Mu.M) with LL-37 (40. Mu.g/ml) and the activation of STING pathway was assessed by Western blotting.

FIG. 2 is a graph showing the effect of the LL-37-cGAMP complex preparation of example 2 on the expression level of various immune factors in mouse RAW264.7 cells.

FIG. 3 is a graph showing the effect of LL-37-cGAMP complex formulation on viral replication on THP1 cells in example 3 of the present invention. Wherein A is the effect of the LL-37-cGAMP complex formulation on HSV-1-GFP virus replication and B is the effect of the LL-37-cGAMP complex formulation on VSV-GFP virus replication.

FIG. 4 is a graph showing the remitting effect of the LL-37-cGAMP complex formulation of example 4 on the reduction in body weight of mice caused by HSV-1 infection.

FIG. 5 is a graph showing the inhibition of replication of HSV-1 in mice by the LL-37-cGAMP complex formulation of example 5 of the present invention. Where A was LL-37-cGAMP pre-treated for 1 hour, followed by infection with virus 24 hours later, HSV-1 virus titers in mouse livers were determined by plaque assay. B is LL-37-cGAMP, after 1 hour of pretreatment, and then 24 hours of infection with the virus, the expression level of the virus gene in the spleen of the mouse is detected by an RT-qPCR method.

FIG. 6 is a graph showing the effect of LL-37-cGAMP combinations on immunocytokine expression in mice in example 6 of the present invention. (A) Mice were treated with cGAMP, LL-37 or LL-37-cGAMP complex formulations for 4 and 8 hours, and spleens were collected and homogenized for RT-qPCR assay. (B) Mice were treated with cGAMP, LL-37 or LL-37-cGAMP complex formulations for 4 hours and 8 hours before collecting mouse sera for ELISA assays. Data are mean ± standard deviation. NS: p >0.05, p.ltoreq.0.05, p.ltoreq.0.01, p.ltoreq.0.001, and p.ltoreq.0.0001.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and improvements may be made by those skilled in the art without departing from the spirit and scope of the invention.

The amino acid sequence of LL-37 used in the examples below is shown as sequence 1 in the sequence listing.

LL-37 and cGAMP used in the examples described below were both commercially available from the open world.

The complex formulation of cGAMP and LL-37 in the following examples was prepared using water as the solvent.

Example 1 LL-37-cGAMP Complex formulations highly active STING innate immune response

THP1-Lucia ISG and RAW-Lucia ISG cells were constructed by stable expression of luciferase reporter gene under control of Interferon Stimulated Response Element (ISRE) in human THP1 and mouse RAW264.7 cells. ISRE luciferase activity of Lucia ISG cells represents the activation intensity for STING pathway.

THP1-Lucia ISG and RAW-Lucia ISG cells were treated with cGAMP (1,2. Mu.M) and LL-37 at various concentrations (5,10,20,40. Mu.g/ml) in a cocktail formulation and their effects on innate immunity were studied by examining cellular ISRE luciferase activity and STING pathway protein activation.

The results show that the LL-37-cGAMP compound drug can efficiently activate ISRE luciferase signals of two cells, can efficiently promote the expression of STING pathway proteins such as p-TBK1, p-STING and p-IRF3, and has a far stronger activating effect on STING innate immunity than LL-37 or cGAMP alone (10-40 times) (FIGS. 1A-1D).

Notably, the transmembrane delivery efficiency of LL-37 for cGAMP was even higher than that of the specialized perfringolysin O (PFO; 0.05. Mu.g/ml) (FIGS. 1A-1D).

In addition, cGAMP (2. Mu.M) was combined with LL-37 (40. Mu.g/ml) to prepare LL-37-cGAMP combined preparation. The LL-37-cGAMP complex formulation activated the STING innate immune response significantly more strongly on mouse BV2 cells than either LL-37 or cGAMP alone (FIG. 1E).

Example 2 LL-37-cGAMP Complex formulations efficiently induce the expression of cellular immune factors

The complex preparation of LL-37-cGAMP was prepared by formulating cGAMP (1. Mu.M) and LL-37 (40. Mu.g/ml), and RAW264.7 cells were treated with the complex preparation. RT-qPCR assays showed that the LL-37-cGAMP complex formulations induced significantly more potent expression of immunomodulatory cytokines downstream of the STING pathway, including TNF-. Alpha., ISG15, IFIT3, IL-6, and the chemokines CXCL10 and CCL5, etc., than LL-37 or cGAMP alone (FIG. 2).

Example 3 LL-37-cGAMP Complex formulations efficiently inhibit viral replication on cells

The cGAMP (3 mu M) and LL-37 (20 mu g/ml) are combined to prepare the LL-37-cGAMP compound preparation. THP1 cells were infected with HSV-1-GFP (MOI = 1) or VSV-GFP (MOI = 1), and virus-infected cells were then treated with the above-described combination drug, and virus replication was examined after drug treatment.

The results showed that both LL-37 and cGAMP alone showed a weak antiviral effect (FIG. 3). In contrast, LL-37-cGAMP complex drugs showed significantly greater antiviral effects and almost completely prevented the proliferation and replication of HSV-1-GFP (FIG. 3A) and VSV-GFP (FIG. 3B) on THP1 cells.

Example 4 LL-37-cGAMP Complex formulation is effective in inhibiting weight loss in mice caused by viral infection

Mice were divided into 5 groups: the wild-type (WT) group, the GAS knock-out (cGAS-/-) group, the cGAS-/- + cGAMP group, the cGAS-/- + LL-37 group, and the cGAS-/- + LL-37 cGAMP group, each group containing 5 mice. Mouse endogenous cGAMP is synthesized by cGAS, and in order to avoid interference of endogenous cGAMP with the results of the experiment, mice of both WT and cGAS-/-genes were studied simultaneously in this experiment. The cGAMP (222. Mu.M) and LL-37 (300. Mu.g/ml) are combined to prepare LL-37-cGAMP compound preparation. Mice were treated for 1 hour by intraperitoneal injection of 200. Mu.L of physiological saline, LL-37 solution, cGAMP solution, or LL-37-cGAMP complex formulation solution. Then, do notThe mice of the same group were injected intravenously with HSV-1 (1X 10 per mouse) ⁷ pfu). The body weight of the mice was recorded daily until the mice were euthanized. The results show that the LL-37-cGAMP complex formulation protects the host better and reduces the weight loss of mice caused by viral infection compared to cGAMP alone or LL-37 treatment (FIG. 4).

Example 5 LL-37-cGAMP Complex formulations efficiently inhibit viral replication in mice

Mice were divided into 5 groups: group of wide-type (WT), group of GAS gene knock-out (cGAS-/-), group of cGAS-/- + cGAMP, group of cGAS-/- + LL-37+ cGAMP, and 5 individuals per group. Mouse endogenous cGAMP is synthesized by cGAS, and in order to avoid interference of endogenous cGAMP with the results of the experiment, mice of both WT and cGAS-/-genes were studied simultaneously in this experiment. The cGAMP (222. Mu.M) and LL-37 (300. Mu.g/ml) are combined to prepare LL-37-cGAMP compound preparation. Mice were treated for 1 hour by intraperitoneal injection of 200. Mu.L of physiological saline, LL-37 solution, cGAMP solution, or LL-37-cGAMP complex solution. Then, different groups of mice were injected intravenously with HSV-1 (1X 10 per mouse) ⁷ pfu)。

Mouse spleens were harvested 24 hours after virus infection and homogenized, and HSV-1 virus titers in the mouse spleens were determined by plaque assay. Briefly, vero cells were incubated with appropriate dilutions of spleen homogenate samples for 1 hour, then covered with DMEM containing 1% fbs and 1.5% methylcellulose for 72 hours. Cells were then fixed with methanol and stained with 0.1% crystal violet, and plaques were counted to calculate virus titers. To quantify HSV-1 specific genes in infected tissues, DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen). The expression of specific genes of HSV-1, such as pol, gD, gB, etc., was measured by qPCR.

The results showed that LL-37 or cGAMP alone showed a weak effect of inhibiting viral replication (FIG. 5). The LL-37-cGAMP complex formulation showed significantly stronger antiviral effects compared to LL-37 or cGAMP alone, and almost completely prevented HSV-1 from multiplying and replicating in the mouse spleen (FIG. 5).

Example 6 LL-37-cGAMP Complex formulations efficiently induce immune factor expression in mice

Mouse endogenous cGAMP is synthesized by cGAS, and in order to avoid interference of endogenous cGAMP with the results of the assay, studies were performed using cGAS-/-mice in this assay. Mice were divided into 7 groups: control group, cGAMP treated 4 hours group, LL-37+ cGAMP treated 4 hours group, cGAMP treated 8 hours group, LL-37+ cGAMP treated 8 hours group. Each group had 3 mice. The cGAMP (222. Mu.M) and LL-37 (300. Mu.g/ml) are combined to prepare LL-37-cGAMP compound preparation. 200. Mu.L of physiological saline, LL-37 solution, cGAMP solution or LL-37-cGAMP complex solution was intraperitoneally injected into mice. Mouse spleen homogenates were collected for RT-qPCR assays after 4 or 8 hours of treatment and mouse sera were collected for ELISA assays.

The results showed that the LL-37-cGAMP complex formulation significantly improved the promotion effect of the STING pathway-associated CXCL10, IFITM1, MX1, viperin, ISG15, IFIT3, etc. gene expression compared with cGAMP alone (FIG. 6A). In addition, LL-37-cGAMP complex significantly increased the expression levels of immune factors such as IFN beta and CXCL10 in mouse serum (FIG. 6B).

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

SEQUENCE LISTING

<110> Qinghua university

<120> composite preparation LL-37-cGAMP, and preparation method and application thereof

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 37

<212> PRT

<213> Artificial sequence

<400> 1

Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu

1 5 10 15

Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val

20 25 30

Pro Arg Thr Glu Ser

35

Claims (10)

1. A complex formulation, the composition of which comprises a cationic antibacterial peptide LL-37 and cGAMP or an analog thereof;

wherein, the amino acid sequence of the cationic antibacterial peptide LL-37 is shown as SEQ ID No.1, or the amino acid sequence with the same function is formed by replacing, deleting or adding one or more amino acids in the sequence;

the molecular structure of the cGAMP is shown as a formula I; the cGAMP analogue is other cyclic dinucleotide with similar functions or a small molecule with similar functions formed by the cGAMP molecule after micro-structural modification.

2. The complex formulation according to claim 1, characterized in that: the ratio of cGAMP or analogues thereof to the cationic antibacterial peptide LL-37 in the compound preparation is (0.1-300) mmol to (1-500) g.

3. A method for preparing the complex formulation of claim 1 or 2, comprising the steps of: and (3) physically blending the cationic antibacterial peptide LL-37 with cGAMP or an analogue thereof.

4. The production method according to claim 3, characterized in that: physically blending the cationic antibacterial peptide LL-37 and cGAMP or analogues thereof, and dispersing in a solvent to prepare a suspension;

preferably, the effective concentration of LL-37 in the suspension is 1-500. Mu.g/ml, further 5-40. Mu.g/ml; the effective concentration of cGAMP or the analogue thereof in the suspension is 0.1-300 mu M, and further 1-3 mu M;

preferably, the solvent is selected from water or dimethyl sulfoxide.

5. Use of a complex formulation according to claim 1 or 2 or produced by a method according to claim 3 or 4, wherein the use is (a 1) and/or (a 2) and/or (a 3) and/or (a 4) and/or (a 5) and/or (a 6):

(a1) The application of the compound preparation in preparing immunomodulators;

(a2) The application of the compound preparation in preparing a virus inhibitor;

(a3) The application of the compound preparation in preparing products for preventing and/or treating diseases caused by virus infection;

(a4) The use of the complex formulation in immunomodulation;

(a5) The application of the compound preparation in inhibiting viruses;

(a6) The application of the compound preparation in preventing and/or treating diseases caused by virus infection.

6. A product comprising the complex formulation of claim 1 or 2 or produced by the method of claim 3 or 4.

7. The product of claim 6, wherein: the product has the following application (b 1) and/or (b 2) and/or (b 3):

(b1) For use in immunomodulation;

(b2) Inhibiting viruses;

(b3) Preventing and/or treating diseases caused by virus infection.

8. The product according to claim 6 or 7, characterized in that: the product comprises medicine, health product, food or additive.

9. A method of inhibiting viral infection in an animal comprising the steps of: administering the complex formulation of claim 1 or 2 or the complex formulation prepared by the method of claim 3 or 4 or the product of any one of claims 6 to 8 to a recipient animal to inhibit viral infection of the animal;

or a method of treating a disease caused by a viral infection, comprising the steps of: administering the complex formulation of claim 1 or 2 or the complex formulation prepared by the method of claim 3 or 4 or the product of any one of claims 6 to 8 to a recipient animal to treat a disease caused by a viral infection.

10. A method of enhancing immunity in an animal comprising the steps of: administering the complex formulation of claim 1 or 2 or the complex formulation prepared by the method of claim 3 or 4 or the product of any one of claims 6 to 8 to a recipient animal to enhance the immunity of the animal.

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