KR20220068590A - A conjugate comprising sialic acid or a derivative thereof bonded on the surface of dendrimer core, and a phenylpropanoid-type compound, and use thereof - Google Patents

Abstract

The present invention relates to a conjugate and a use thereof, wherein the conjugate comprises: a core; sialic acid or a derivative thereof bonded to a surface thereof; and phenylpropanoid-based compounds. The conjugate provided by the present invention binds to hemagglutinin present on the surface of an influenza virus to inhibit the infection process of the influenza virus, thereby being capable of preventing or treating not only influenza virus infection, but also infections with influenza viruses exhibiting tolerance to anti-virus formulations. Thus, the conjugate can be widely used in developing formulations for preventing or treating influenza virus infections.

Description

A binder comprising sialic acid or a derivative thereof bound to the surface of a dendrimer core and a phenylpropanoid-based compound, and uses thereof AND USE THEREOF}

The present invention relates to a dendrimer core, a binder comprising sialic acid or a derivative thereof bound to a surface thereof, and a phenylpropanoid-based compound, and uses thereof.

Influenza virus (Influenza virus) is an RNA virus belonging to the family Orthomyxoviridae, and serotypes are divided into three types: type A, type B, and type C. Among them, type B and type C infection is confirmed only in humans, and type A infection has been confirmed in humans, horses, pigs, other mammals, and various types of poultry and wild birds. The serotypes of influenza A virus are classified according to the types of two proteins on the surface of the virus, hemagglutinin (HA) and neuraminidase (NA). 9 kinds of proteins) are known. HA serves to attach the virus to somatic cells, and NA allows the virus to penetrate into the cell. An influenza virus with a new antigenicity spreads every winter, and the virus spreads through livestock, birds, including humans, and monitoring of these viruses is very important. Accordingly, there is a demand for the development of primers and probes with high specificity and sensitivity.

이상으로 가열 조리하면 음식에 존재하는 바이러스가 사멸하는 것으로 알려져 있다.Influenza A (H1N1) virus, which has recently been attracting attention, is also called "H1N1 influenza" or "H1N1 influenza virus". was first discovered in May. Although the route of transmission of the virus has not yet been clearly elucidated, similar to the existing seasonal influenza virus, it is mainly through droplet infection, that is, coughing or sneezing of an infected person, from person to person, between infected and close contacts ( It is known to spread within about 2 m). Also, 70 for food

It is known that the virus present in food is killed by heating and cooking above.

The incubation period of the H1N1 influenza virus is estimated to be between 1 to 7 days, and symptoms include fever, chills, headache, cough, sore throat, runny nose, upper respiratory tract symptoms such as shortness of breath, muscle pain, joint pain, fatigue, vomiting, or It is known to cause diarrhea. It has been reported to be contagious from one day before symptoms onset to seven days after onset, although in children it can be as long as 10 days or more. In order to reduce the damage caused by the H1N1 influenza virus, research has been actively conducted to develop a method for early diagnosis of H1N1 influenza virus infection. For example, in Korea Patent Publication No. 2011-0064174, a monoclonal antibody specific only to novel influenza virus A/H1N1 excluding other influenza viruses, a fusion cell producing the same, a diagnostic kit including the monoclonal antibody, and a novel influenza virus A using the same A method for diagnosing /H1N1 is disclosed, and Korean Patent Publication No. 2011-0096940 discloses a novel influenza A virus using rapid immunochromatography using an antibody against the hemagglutinin (HA) antigen of the novel influenza virus. A diagnostic kit capable of diagnosing (Influenza A, H1N1) is disclosed, and Korea Patent Publication No. 2011-0127034 discloses an influenza A virus common PCR primer for detecting influenza A virus and a swine-derived influenza (swine flu, A method for rapidly and accurately detecting H1N1 influenza (A/Korea/01/2009(H1N1)) virus using a PCR primer specific to A/Korea/01/2009(H1N1)) virus is disclosed.

However, even if the virus infection is diagnosed, only oseltamivir (trade name Tamiflu) and Zanamivir (trade name Relenza) are the only agents that can treat the infected influenza virus, and even using these agents As a virus having resistance to it is generated, the development of a method for treating the virus is still insignificant.

Korean Patent Publication No. 2011-0096940

Under this background, as a result of intensive research efforts to develop a method for effectively treating influenza virus infection with low toxicity, the present inventors include sialic acid or its derivative and phenylpropanoid compound bound to the core and its surface. And, when using a conjugate characterized in that the sialic acid or its derivative and the phenylpropanoid compound are arranged at a predetermined interval, it is possible to treat influenza virus infection as well as to exhibit resistance to conventional antiviral agents It was confirmed that even infection caused by a virus can be treated, and the present invention was completed.

The present invention provides a conjugate comprising sialic acid or a derivative thereof bound to a core and a surface thereof and a phenylpropanoid-based compound, wherein the sialic acid or a derivative thereof is arranged at intervals of 1.0 to 6.0 nm, respectively will be.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating influenza virus infection comprising the conjugate.

Another object of the present invention is to provide a food composition for preventing or improving influenza virus infection comprising the conjugate.

Another object of the present invention is to provide a method for preventing or treating influenza virus infection, comprising administering the conjugate to an individual suspected of influenza virus infection or infected with influenza virus.

Another object of the present invention is to provide a method for inhibiting influenza virus infection, comprising the step of treating the conjugate to the isolated influenza virus.

The conjugate provided in the present invention binds to hemagglutinin present on the surface of the influenza virus and inhibits the influenza virus infection process, thereby preventing or treating influenza virus infection as well as exhibiting resistance to antiviral agents. Since it is possible to prevent or treat influenza virus infection, it can be widely used in the development of a formulation for preventing or treating influenza virus infection.

1 is a reaction scheme showing the reductive amination of the primary amino group of 6'-sialyllactose and PAMAM dendrimer (G4) using NaCNBH ₃ and the reductive amination of the resulting polymer and phenylpropanoid-based compound using NaCNBH ₃ .
2 is a spectrum showing the result of analyzing the properties of the conjugate by ¹ H-NMR method.
3 shows the structure of a PAMAM dendrimer (G4) conjugate with 10 molecules of trans-cinnamaldehyde, which is a representative 6'-sialyllactose or phenylpropanoid compound, among the polymers synthesized in the present invention.
Figure 4 shows the regularity of the distribution of 6'-sialyllactose and phenylpropanoid compounds and the size of the polymer in a PAMAM dendrimer conjugate having an ethylene diamine core (G4) with a 6'-sialyllactose and phenylpropanoid compound. will be.

As an embodiment for achieving the above object, the present invention includes a core and sialic acid or a derivative thereof bonded to the surface thereof and a phenylpropanoid-based compound, and the sialic acid or its derivative and phenylpropanoid The system compound provides a conjugate, characterized in that they are arranged at regular intervals from each other.

As used herein, the term "core" refers to a material that binds to sialic acid or a derivative thereof and serves as a support so that the sialic acid or derivative thereof and the phenylpropanoid-based compound can be arranged at regular intervals from each other. do.

In the present invention, the material constituting the core is not particularly limited thereto, as long as it includes a reactive group capable of binding to sialic acid or a derivative thereof and a phenylpropanoid-based compound, but as an example, polyamidoamine (PAMAM) ), polyhydroxybutyrate, polyhydroxyvalerate, polylysine, polylactic acid, polyglycolide, polycaprolactone, polypropylene fumarate, polydioxenone, polynucleotides, and copolymers thereof can be used alone or in combination. have.

In the present invention, a plurality of sialic acid or a derivative thereof and a phenylpropanoid-based compound are bonded to the surface of the core to be arranged at regular intervals from each other.

The shape of the core provided in the present invention is not particularly limited thereto, but as an example, it may be a spherical shape composed of a polymer body of the core component material, and as another example, a linear polymer body of the core component material has a plurality of shapes. It may be in the form of overlapping through folding, or as another example, it may be in the form of a dendrimer formed by using the core constituent material as a base compound.

As used herein, the term "dendrimer" refers to a macromolecule in which a base compound is overlapped with a tree branch-shaped molecular chain to form a spherical structure. Since it is easy and has a large number of reactive groups on the outermost surface, it exhibits unique chemical or physical properties, and it is possible to introduce various derivatives and reactive groups into the dense terminal groups on the surface. In particular, the dendrimer can be applied to various fields because it is easier to control and predict the structure compared to the linear polymer form.

Depending on the level of overlap of the base compounds forming the dendrimer, the generation of dendrimers can be specified and the first generation dendrimer (G1), the second generation dendrimer (G2), the third generation dendrimer (G3), the fourth generation dendrimer (G4) and the fifth generation. It can be expressed as a dendrimer (G5). That is, as one additional base compound is combined, the number of generations increases by one generation.

In the present invention, the dendrimer may use the material constituting the above-mentioned core as a base compound, wherein a plurality of sialic acid or a derivative thereof and a phenylpropanoid compound are bonded to the surface of the base compound, and are spaced apart from each other at regular intervals. It is not particularly limited as long as it can form a core that can be arranged, but as an example, it may be polyamidoamine, polylysine, polynucleotide, and the like, and as another example, it may be polyamidoamine.

As used herein, the term sialic acid, also called sialic acid or neuramic acid, has a chemical structure in which neuramic acid, which is an aldol condensate of pyruvic acid and mannosamine, is n-acetylated, n-glycolized or o-acetylated It means a kind of amino sugar with

In the present invention, the sialic acid may be used as a ligand that binds to hemagglutinin (HA) of the virus and inhibits its function. As the ligand, not only sialic acid but also derivatives thereof may be used.

As used herein, the term “derivative” refers to a similar compound obtained by chemically changing a part of a target compound, and may be a compound in which a hydrogen atom or a specific atomic group in the target compound is generally substituted by another atom or group.

In the present invention, the derivative can be interpreted as a derivative of sialic acid, as long as the derivative of sialic acid can bind to HA of the virus and inhibit its function, but is not particularly limited thereto. It may be a sialyl oligosaccharide in which a sugar is bound to an acid, and as another example, 3'-sialyllactose, 6'-Sialyllactose, sialyllactose-N- It may be tetraose (sialyl lacto-N-tetraose), disialyl lacto-N-tetraose (disialyl lacto-N-tetraose), or the like.

In the present invention, monosaccharides and disaccharides are reducing sugars, preferably monosaccharides rhamnose, glucose, and galactose, and disaccharides include lactose and maltose. However, the present invention is not limited thereto, and may also include trisaccharides.

As used herein, the term "conjugate" refers to a material in which a plurality of sialic acids or derivatives thereof and a phenylpropanoid-based compound are bonded to one core. Specifically, the conjugate may be a form in which sialic acid or a derivative thereof and a phenylpropanoid-based compound are bonded to the core at regular intervals. In addition, in the conjugate of the present invention, sialic acid or a derivative thereof and a phenylpropanoid compound are bonded to the core through a reactive group, or a plurality of sialic acid or a derivative thereof and a phenylpropanoid compound are bonded to the core through a linker. can be In this case, the reactive group of the core is not particularly limited as long as sialic acid or a derivative thereof and a phenylpropanoid-based compound can be bound to the core, and a reactive group known in the art may be used.

As described above, since the core is not limited in terms of its constituent material or form as long as sialic acid or a derivative thereof and a phenylpropanoid-based compound can be bound, the combination includes the core, sialic acid or a derivative thereof, and phenyl. By various combinations of propanoid-based compounds, it may be formed in various forms.

In the present invention, since a plurality of sialic acid, sialyllactose, or derivatives thereof are bound to the surface of the core and arranged at the same distance from each other, the core preferably has a diameter of at least a certain level. The diameter of the core is not particularly limited as long as a plurality of sialic acid, sialyllactose or derivatives thereof are bound to the surface thereof and arranged at the same distance from each other, but as an example, it may be 1.0 to 6.0 nm. may be, as another example, it may be 3.5 to 5.5 nm, as another example, it may be 4.0 to 6.0 nm, and as another example, it may be 4.5 to 5.4 nm.

According to an embodiment of the present invention, first, the aldehyde group of the reducing sugar of 6SL was reductively aminationed with the amino group of the PAMAM dendrimer having an ethylenediamine core (G4), and at this time, the resulting polymer was reductively ainated with a phenylpropanoid-based compound. Thus, various types of 6'-sialyllactose and phenylpropanoid-based compounds and a PAMAM dendrimer having an ethylene diamine core (G4) were prepared (FIG. 1). The number of 6SLs included therein was determined (Table 1).

Then, as a result of analyzing the influenza virus infection inhibitory ability of the conjugate in vitro, the infection inhibitory efficacy of the H1N1 virus was confirmed (Table 3).

Next, as a result of analyzing the binding affinity of the binder to HA, it was confirmed that all of them exhibit high binding affinity with the HA protein (Table 4).

In another embodiment, the present invention provides a pharmaceutical composition for preventing or treating influenza virus infection comprising the conjugate.

The content of the binder contained in the pharmaceutical composition provided in the present invention is not particularly limited thereto, as long as it can exhibit an effect of preventing or treating influenza virus infection, but as an example, it will be 0.0001 to 50% by weight relative to the weight of the total composition. and, as another example, may be 0.01 wt% to 10 wt%.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition, and the carrier may include a non-naturally occurring carrier. have.

The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. In the present invention, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient, for example, starch, calcium carbonate, sucrose or lactose. It is prepared by mixing (lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc., which are commonly used simple diluents such as water and liquid paraffin, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, etc. can be used.

In another embodiment, the present invention includes the step of administering the conjugate or a pharmaceutical composition comprising the conjugate to an individual suspected of having influenza virus infection or having an influenza virus infection. Prevention of influenza virus infection or a method of treatment.

The conjugate of the present invention or a pharmaceutical composition comprising the same may be administered in a pharmaceutically effective amount, and the term, “pharmaceutically effective amount” of the present invention, means treating a disease at a reasonable benefit/risk ratio applicable to medical treatment. It means an amount that is sufficient and does not cause side effects, and the effective dose level is the patient's sex, age, weight, health status, disease type, severity, drug activity, drug sensitivity, administration method, and administration time. , route of administration, and rate of excretion, duration of treatment, factors including drugs used in combination or concomitantly, and other factors well known in the medical arts can be readily determined by one of ordinary skill in the art.

As an example, the conjugate of the present invention is, as an example, 1×10 ⁷ to 1×10 ¹¹ , as another example, 1×10 ⁸ to 5×10 ¹⁰ , as another example, 5×10 ⁸ to 2×10 ¹⁰ may be administered, but is not limited thereto.

As another example, in the case of the pharmaceutical composition of the present invention as a standard, it may be administered at 0.0001 to 100 mg/kg of body weight per day based on the solid content, and more specifically, 0.001 to 100 mg/kg of body weight. For administration, the recommended dosage may be administered once a day, or divided into several administrations.

In the method for preventing or treating influenza virus infection of the present invention, the route and mode of administration for administering the pharmaceutical composition are not particularly limited, and any pharmaceutical composition comprising the conjugate can reach the desired site. It may depend on the route of administration and the mode of administration. Specifically, the pharmaceutical composition may be administered through a variety of oral or parenteral routes, and non-limiting examples of the route of administration include oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, and those administered intranasally or through inhalation.

As another embodiment, the present invention provides a food composition for preventing or improving influenza virus infection comprising the conjugate.

As used herein, the term “improvement” refers to any action that at least reduces a parameter related to a condition to be treated, for example, the severity of a symptom by administration of a composition comprising a conjugate of the present invention.

As used herein, the term “food” refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages , vitamin complexes, health functional foods, and health foods, and includes all foods in a conventional sense.

Since the food composition of the present invention can be consumed on a daily basis, an effect of improving influenza virus infection at a high level can be expected, and thus it can be very usefully used for health promotion purposes.

The functional food (functional food) is the same term as food for special health use (FoSHU), and in addition to nutritional supply, it is processed to efficiently exhibit bioregulatory functions and has high medical effects. means food. Here, 'function (sex)' refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body. The food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, the formulation of the food may be prepared without limitation as long as it is a formulation recognized as a food. The composition for food of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage of not having side effects that may occur during long-term administration of drugs using food as a raw material, and has excellent portability. The food of the invention can be ingested as an adjuvant to enhance the effect of improving influenza virus infection.

The health food means food having an active health maintenance or promotion effect compared to general food, and health supplement food means food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and dietary supplement are preferred.

Specifically, the health functional food is a food prepared by adding the compound of the present invention to food materials such as beverages, teas, spices, gum, and confectionery, or encapsulating, powdering, suspension, etc., and when ingested, It means to bring an effect, but unlike general drugs, it has the advantage that there are no side effects that may occur when taking the drug for a long time using food as a raw material.

The food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used.

In addition, the food composition may include additional ingredients that are commonly used in food compositions to improve odor, taste, vision, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr); and amino acids such as lysine, tryptophan, cysteine, and valine.

In addition, the food composition includes a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), a disinfectant (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), an antioxidant (butylhydroxyanisole (BHA), butyl hydro Loxytoluene (BHT), etc.), coloring agents (tar pigments, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, etc.) , sodium, etc.), flavorings (vanillin, lactones, etc.), swelling agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (foaming agents), film agents, gum base agents, foam inhibitors, solvents, improving agents, etc. It may contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.

As an example of the food composition of the present invention, it may be used as a health drink composition, and in this case, it may contain various flavoring agents or natural carbohydrates as an additional component like a conventional drink. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as taumatine, stevia extract; A synthetic sweetener such as saccharin or aspartame may be used. The ratio of the natural carbohydrate is generally about 0.01 per 100 mL of the health beverage composition of the present invention? 0.04 g, specifically about 0.02 ? It can be 0.03 g.

In addition to the above, the health drink composition includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid, pectic acid salts, alginic acid, alginic acid salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol, a carbonation agent, or the like. In addition, it may contain the pulp for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These components may be used independently or in combination. Although the ratio of these additives is not very important, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

In another embodiment, the present invention provides a method for inhibiting infection of influenza virus, comprising the step of treating the conjugate or the conjugate to the influenza virus.

As described above, in the conjugate of the present invention, sialic acid or a derivative thereof contained therein binds with hemagglutinin present on the surface of the influenza virus to form a vesicle, thereby causing the virus to attach, enter the cell, and endo It inhibits the fusion/release process, participates in viral entry into cells, and can have the effect of lowering the expression level of NA, which is increased by virus infection.

Therefore, the conjugate of the present invention can be used to inhibit influenza virus infection in vitro, in vivo, or ex vivo.

In particular, when it is desired to inhibit influenza virus infection in vitro or ex vivo in order to conduct a study on the infectivity of influenza virus in a target cell, the conjugate is diluted in a buffer to an appropriate concentration. Then, by treating the virus solution or the virus-infected cells, it is possible to inhibit the infection of the influenza virus to the cells.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Synthesis of a conjugate of sialyllactose, phenylpropanoid-based compound and PAMAM dendrimer

First, the aldehyde group of the reducing sugar of 6SL was subjected to reductive amination with the amino group of the PAMAM dendrimer having an ethylene diamine core (G4), and at this time, the resulting polymer was subjected to reductive amination with a phenylpropanoid compound to form various types of 6'-sialyl. A conjugate of lactose and a phenylpropanoid-based compound and a PAMAM dendrimer having an ethylene diamine core (G4) was prepared (FIG. 1).

Roughly, in 0.1 M sodium borate buffer (pH 9.5) containing 50 mM NaCNBH ₃ , PAMAM dendrimer (G4) and different amounts of 6'-sialyllactose were mixed, and each of the resulting substances was mixed with 50 mM NaCNBH ₃ . Under the containing 0.1M sodium borate buffer (pH 9.5), various amounts of phenylpropanoid compounds were added and reacted (Table 1). In each of the first and second steps, the reaction solution was continuously reacted for 5 days in a dark place and at room temperature while slowly stirring the reaction solution. After the reaction is completed, all materials except the final polymer are removed by filtration using Amicon centrifugal filters (Millipore) of an appropriate size (MWCO 3 K).

, the solvent was replaced with deionized water. After filtration, the solution was freeze-dried, and a sample of the obtained white powder was dried and stored until use.

Mixing ratio of PAMAM dendrimer (G4) and 6'-sialyllactose and mixing ratio of product and phenylpropanoid compound concrete
dendrimer (G4)
6'-sialyllactose
primary polymer
monosaccharides and disaccharides
20SL-G4
80
118.8
-
-
10tCA-20SL-G4
80
118.8
53.0
tCA 3.2
20tCA-20SL-G4
80
118.8
53.0
tCA 8.5
30tCA-20SL-G4
80
118.8
53.0
tCA 26.4
10HMCA-20SL-G4
80
118.8
53.0
HMCA 4.3
20HMCA-20SL-G4
80
118.8
53.0
HMCA 11.4
30HMCA-20SL-G4
80
118.8
53.0
HMCA 35.6
16SL-G4
80
94.4
-
-
20tCA-16SL-G4
80
94.4
48.1
tCA 8.5
20HMCA-16SL-G4
80
94.4
48.1
HMCA 11.4

Example 2: Characterization of 6'-sialyllactose and phenylpropanoid-based compound and PAMAM dendrimer (G4) conjugate

The properties of the 6'-sialyllactose and phenylpropanoid-based compound synthesized in Example 1 and the PAMAM dendrimer (G4) conjugate were analyzed using ¹ H-NMR and dynamic light scattering (DLS).

Example 2-1: ^One H-NMR analysis

The NMR spectrum for the conjugate of PAMAM (G4) dendrimer, 6'-sialyllactose and phenylpropanoid-based compound was measured in deuterium oxide (D ₂ O) using a Bruker AVANCE III 700 spectrometer (FIG. 2), and the data Through the analysis, average values of 6'-sialyllactose and phenylpropanoid-based compounds bound to PAMAM dendrimer (G4) were calculated (Table 2 and FIG. 2).

¹ Average value of PAMAM (G4) dendrimer, 6'-sialyllactose and phenylpropanoid compound measured through H-NMR analysis concrete
6'-sialyllactose
Number of glyceraldehyde derivatives
20SL-G4
20.1
-
10tCA-20SL-G4
20.1
10.3
20tCA-20SL-G4
20.1
20.0
30tCA-20SL-G4
20.1
27.5
10HMCA-20SL-G4
20.1
10.0
20HMCA-20SL-G4
20.1
19.8
30HMCA-20SL-G4
20.1
26.8
16SL-G4
16.0
-
20tCA-16SL-G4
16.0
20.3
20HMCA-16SL-G4
16.0
27.1

From the obtained results, the structure of the synthesized polymer could be predicted (FIG. 3).

Example 2-2: Dynamic light scattering (DLS) analysis

The regularity and size of the PAMAM dendrimer (G4), 6'-sialyllactose and phenylpropanoid compound complexes were determined by the hydrodynamic diameter (dynamic light scattering) obtained by DLS (dynamic light scattering) using Beckman Coulter DelsaMax PRO (Brea, California, USA) ( hydrodynamic diameter) and polydispersity were determined through data analysis (Table 3 and FIG. 4). Briefly, samples were dissolved in 10 mM PBS at pH 7.4 and set in 5 duplicates at 20 °C, and data were acquired using DelsaMax Analysis Software version 1.0.

Regularity and size of PAMAM dendrimer (G4), 6'-sialyllactose, and phenylpropanoid compound conjugates obtained by DLS analysis concrete
hydrodynamic diameter (nm)
Polydispersity (%Pd)
PAMAM G4
5.3 ± 0.51
19.9 ± 1.70
20SL-G4
5.9 ± 0.83
23.1 ± 2.30
10tCA-20SL-G4
7.0 ± 0.10
25.0 ± 5.50
20tCA-20SL-G4
6.3 ± 0.10
27.7 ± 2.00
30tCA-20SL-G4
13.3 ± 0.80
50.7 ± 4.9
10HMCA-20SL-G4
6.2 ± 0.10
27.3 ± 3.4
20HMCA-20SL-G4
6.5 ± 0.10
27.7 ± 4.2
30HMCA-20SL-G4
8.8 ± 0.10
28.5 ± 1.5
16SL-G4
5.9 ± 0.20
22.0 ± 0.25
20tCA-16SL-G4
11.4 ± 0.90
46.5 ± 2.50
20HMCA-16SL-G4
7.4 ± 0.20
29.7 ± 2.16

From the results obtained by DLS analysis, 6'-sialyllactose and phenylpropanoid compound conjugates of PAMAM dendrimer (G4) are relatively regular except for 30tCA-20SL-G4 and 20tCA-16SL-G4. was shown.

Example 3: Influenza virus infection inhibition analysis of sialyllactose, phenylpropanoid-based compound and PAMAM dendrimer (G4) conjugate in vitro

First, a half-log serial dilution of the conjugate to be tested was prepared using PBS. 50 μl of the prepared dilution was mixed with an equal volume of 1 × 10 ³ TCID50 influenza H1N1 strain (A/PR/8/34)). The mixture was incubated at 37° C. for 1 hour, then 100 μl of MDCK cells at 1.5 × 10 ⁵ cells/ml were added thereto, and then further cultured at 5% CO 2 and 37° C. for 18 hours. After the culture was completed, a fixative (acetone:PBS=4:1, v/v) was added to the cultured MDCK cells and fixed for 10 to 15 minutes. Then, by performing ELISA analysis using anti-NP mouse antibody (Millipore) and anti-mouse goat antibody-HRP conjugate (Jackson Immunoresearch), influenza nuclear protein (NP) contained in each MDCK cell was detected. At this time, the infection rate for each MDCK cell was calculated using the absorbance for the negative control (uninfected cells, infection rate 0%) and positive control (cells infected without inhibitor, infection rate 100%), and statistical analysis of the calculated infection rate was performed using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA) (Table 4).

As shown in Table 4, in vitro conditions, the sialyllactose, phenylpropanoid compound, and PAMAM dendrimer (G4) conjugate showed a strong overall inhibitory effect on influenza virus infection, and the strongest in 16SL-G4 and 20tCA-16SL-G4 showed inhibitory effect.

Effects of sialyllactose, phenylpropanoid-based compounds and PAMAM dendrimer (G4) conjugates in inhibiting influenza H1N1 virus infection in vitro concrete
IC ₅₀ (μM)
CC ₅₀ (μM)
20SL-G4
3.3
352.3
10tCA-20SL-G4
3.7
506.2
20tCA-20SL-G4
10.5
783.0
30tCA-20SL-G4
5.5
661.5
10HMCA-20SL-G4
2.9
288.8
20HMCA-20SL-G4
5.4
401.6
30HMCA-20SL-G4
18.0
520.3
16SL-G4
0.5
359.8
20tCA-16SL-G4
1.0
705.5
20HMCA-16SL-G4
1.3
582.0

Claims (12)

As a binder comprising a core and sialic acid or a derivative thereof bound to a surface thereof and a phenylpropanoid-based compound,
The conjugate, characterized in that the sialic acid or its derivative is arranged at an interval of 1.0 to 6.0 nm, respectively.
According to claim 1,
The core includes polyamidoamine, polyhydroxybutyrate, polyhydroxyvalerate, polylysine, polylactic acid, polyglycolide, polycaprolactone, polypropylene fumarate, polydioxenone, polynucleotides, copolymers thereof and these Which is composed of a material selected from the group consisting of a combination of, the conjugate.
According to claim 1,
The core is a dendrimer, the conjugate.
According to claim 1,
The core will have a diameter of 3.0 to 6.0 nm, the binder.
According to claim 1,
The sialic acid, sialyllactose or derivatives thereof are 3'-sialyllactose (3'-Sialyllactose), 6'-sialyllactose (6'-Sialyllactose), sialyllacto-N-tetraose (sialyl lacto-) N-tetraose), disialyl lacto-N-tetraose (disialyl lacto-N-tetraose), and a compound selected from the group consisting of combinations thereof, the conjugate.
According to claim 1,
The sialic acid or a derivative thereof is bonded to a reactive group of the core or is bonded to the core through a linker, the conjugate.
According to claim 1,
The sialic acid or derivatives thereof are arranged at intervals of 1.6 to 3.5 nm, the conjugate.
A pharmaceutical composition for preventing or treating influenza virus infection, comprising the conjugate of any one of claims 1 to 7.
A food composition for preventing or improving influenza virus infection, comprising the conjugate of any one of claims 1 to 7.
A method for preventing or treating influenza virus infection, comprising administering the conjugate of any one of claims 1 to 7 to an individual suspected of or infected with influenza virus.
11. The method of claim 10,
The method of claim 1, wherein the influenza virus is an influenza A virus or an influenza B virus.
A method for inhibiting infection of an influenza virus, comprising the step of treating the conjugate of any one of claims 1 to 7 to the influenza virus.

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