JP5072264B2 - Sialic acid-containing sugar chain complex, method for producing the same, and anti-influenza virus agent containing the sialic acid-containing sugar chain complex - Google Patents

Description

  The present invention relates to a novel sialic acid-containing sugar chain complex, an efficient production method thereof, and an anti-influenza virus agent having the anti-influenza virus activity, which contains the sialic acid-containing sugar chain complex as an active ingredient About.

At present, the main methods for preventing or treating influenza include vaccination and administration of amantadine and Tamiflu which are approved for use as anti-influenza drugs.
However, it is already known that each of these prevention or treatment methods has advantages and disadvantages. For example, vaccines can prevent viral attachment to cells by secreting IgA produced by vaccination into the mucus of the respiratory system. It is known to be powerless. Amantadine inhibits the ion channel action of the M2 protein of influenza virus and can suppress the unhulling of influenza virus after invading the target cell, but the effect on B and C viruses without M2 protein is However, strong side effects and the development of resistant bacteria are regarded as problems. Tamiflu also inhibits the action of neuraminidase (NA), one of the spike proteins present in the envelope of influenza virus, and the replicated influenza virus is released from infected target cells and spreads to other cells. Although it is possible to suppress this, it is essential to take the drug at an early stage of infection for the manifestation of the effect, and in recent years, side effects on young people have become a problem.

  Research and development of new preventive or therapeutic methods for influenza that can overcome the problems of conventional vaccines and anti-influenza drugs as described above are widely performed. As a technique relating to such a method for preventing or treating influenza, for example, an anti-influenza virus agent containing a glycosphingolipid having a sugar chain containing a recognized site of a virus receptor as an active ingredient has been reported (see Patent Document 1). ). In Patent Document 1, among gangliosides that are glycosphingolipids containing sialic acid, GM4 (Neu5Acα2-3Gal-Cer), GM3 (Neu5Acα2-3Galβ1-4Glc-Cer), GD1a (Neu5Acα2-) extracted from natural products. 3Galβ1-3GalNAcβ1-4 (Neu5Acα2-3) Galβ1-4Glc-Cer) and SPG (sialylparagloboside: Neu5Acα2-3Galβ1-4GlcNAcβ1-4Galβ1-4Glc-Cer) were found to have infection-inhibiting activity against influenza virus. It is disclosed that the strength of infection prevention was in the order of SPG> GD1a> GM3> GM4.

However, the anti-influenza virus agent disclosed in Patent Document 1 has not yet been put into practical use, and gangliosides disclosed as the anti-influenza virus agent are derived from natural products such as minke whale brain and human erythrocytes. In view of the fact that a large-scale preparation of an active ingredient is desired for practical use as an influenza drug, it may be somewhat difficult because of being extracted.
In addition, the epidemic of influenza is a major problem, and existing drugs such as amantadine and Tamiflu, which are currently used, have drawbacks such as side effects and restrictions on the timing of administration, as described above. As for new influenza drugs having influenza virus activity and high safety, prompt research and development and practical application are desired at present.

JP 2001-233773 A

  An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the present invention provides a novel sialic acid-containing sugar chain complex having anti-influenza virus activity, an efficient production method thereof, and an excellent anti-influenza virus activity containing the sialic acid-containing sugar chain complex. It aims at providing the anti-influenza virus agent which has this.

In influenza viruses, hemagglutinin (HA), one of the spike proteins present in the envelope, recognizes sugar chains containing complex sialic acid present on the cell membrane surface of target cells and binds to the sugar chains. Thus, it is generally known to establish infection of target cells. The avian influenza virus specifically recognizes a sugar chain such as Neu5Ac (α2-3) Gal-, and the human influenza virus specifically recognizes a sugar chain such as Neu5Ac (α2-6) Gal-. It is thought to be a factor that creates specificity.
For viruses and bacteria that recognize the components of the target cell and bind to the target cell as described above, and establish infection, the same degree as the sugar chain of the target cell recognized by the virus or bacteria, or If a component having a stronger affinity is added from the outside, infection by the virus or bacteria is considered to be inhibited.

However, in spite of the sugar chain having the hemagglutinin (HA) recognition site as described above, 3′-sialyl lactose (3′-SL: Neu5Acα2-3Galβ1-4Glc) and 3′- which are sialyl oligosaccharides. Sialyl lactosamine (3′-SLN: Neu5Acα2-3Galβ1-4GlcNAc) did not show anti-influenza virus activity.
On the other hand, as a result of intensive studies, the present inventors bound compounds other than sugars to sialyl oligosaccharides that did not show anti-influenza virus activity as described above, and had a constant molecular weight. When a complex of a sialyl oligosaccharide and a compound other than a sugar was newly created so as to be within the range of the above, it was found that the resulting complex showed excellent anti-influenza virus activity. The present invention has been completed.

Therefore, this invention is based on the said knowledge by the present inventors, and as means for solving the said subject, it is as follows. That is,
<1> Terminals other than sialic acid terminals of sialic acid-containing sugar chains containing either 3′-sialylgalactose chain (Neu5Acα2-3Gal) or 6′-sialylgalactose chain (Neu5Acα2-6Gal) at one terminal And a sialic acid-containing sugar chain complex comprising a straight chain fatty acid having 8 to 18 carbon atoms (excluding sphingolipids) bonded with an amide bond and having a molecular weight of 400 to 1,400 It is.
< 2 > Sialic acid-containing sugar chains are 3′-sialyl lactose chains (Neu5Acα2-3Galβ1-4Glc), 6′-sialyl lactose chains (Neu5Acα2-6Galβ1-4Glc), 3′-sialyllactosamine chains (Neu5Acα2-3Galβ1- 4GlcNAc), 6′-sialyllactosamine chain (Neu5Acα2-6Galβ1-4GlcNAc), 3′-sialylgalactosylgalactose chain (Neu5Acα2-3Galβ1-4Gal), 6′-sialylgalactosylgalactose chain (Neu5Acα2-6Galβ1G) -Sialylgalactosylgalactosamine chain (Neu5Acα2-3Galβ1-4GalNAc) and 6′-sialylgalactosylgalactosamine chain (Neu5Acα2-6Galβ1-4Gal) NAc), and the sialic acid-containing sugar chain complex according to <1 > selected from the group consisting of:
< 3 > The method for producing a sialic acid-containing sugar chain complex according to any one of <1> to < 2 >, wherein a 3′-sialylgalactose chain (Neu5Acα2-3Gal) and 6 at one end The sialic acid-containing sugar chain containing any one of the '-sialylgalactose chain (Neu5Acα2-6Gal) is acylamidated at a terminal other than the sialic acid terminal to bond a linear fatty acid having 8 to 18 carbon atoms. This is a method for producing a sialic acid-containing sugar chain complex.
< 4 > The method for producing a sialic acid-containing sugar chain complex according to <3>, wherein the sialic acid-containing sugar chain is reacted with ammonium hydrogen carbonate, and then the resulting reaction product is reacted with a chlorinated fatty acid. .
< 5 > An anti-influenza virus agent comprising the sialic acid-containing sugar chain complex according to any one of <1> to < 2 > as an active ingredient.

  According to the present invention, the above-mentioned conventional problems can be solved, the object can be achieved, and a novel sialic acid-containing sugar chain complex having anti-influenza virus activity, its efficient production method, and An anti-influenza virus agent containing the sialic acid-containing sugar chain complex and having excellent anti-influenza virus activity can be provided.

(Sialic acid-containing sugar chain complex)
The sialic acid-containing sugar chain complex of the present invention is formed by binding a sialic acid-containing sugar chain and a compound other than a sugar and has a specific molecular weight. The sialic acid-containing sugar chain complex has an excellent anti-influenza virus activity and can be preferably used as an active ingredient of the anti-influenza virus agent of the present invention described later.

<Molecular weight>
The molecular weight of the sialic acid-containing sugar chain complex is in the range of 100 to 40,000, preferably 400 to 1,400, more preferably 430 to 950, still more preferably 590 to 950, and more preferably 750 to 950. Even more preferred is 800-950.
When the molecular weight is less than 400, a desired degree of anti-influenza virus activity may not be exhibited. When the molecular weight exceeds 1,400, a micelle structure advantageous for anti-influenza virus activity may not be formed in an aqueous solution. Therefore, the desired degree of anti-influenza virus activity may not be exhibited. On the other hand, when the molecular weight is in the range of 800 to 950, it is advantageous in that a micelle structure advantageous for anti-influenza virus activity can be easily formed in an aqueous solution.

<Compounds other than sugar>
The compound other than sugar is not particularly limited as long as it is a compound other than sugar, and can be appropriately selected depending on the purpose. However, sphingolipids are excluded from compounds other than the sugar. Specific examples of sphingolipids excluded from compounds other than the sugar include ceramide.

The compound other than the sugar is preferably a hydrophobic compound. If the compound other than the sugar is a hydrophobic compound, the sialic acid-containing sugar chain complex is considered to be amphiphilic as a whole and take a micelle structure in an aqueous solution. When the sialic acid-containing sugar chain complex has a micelle structure in an aqueous solution, a sugar chain having a recognition site for an influenza virus hemagglutinin protein (HA) is preferably located outside. Therefore, the sugar chain is more easily recognized by the hemagglutinin protein, and the binding of influenza virus to the sugar chain is promoted. As a result, the binding of influenza virus to the original target cell is more efficiently inhibited. It will be possible to
There is no restriction | limiting in particular as compounds other than the said sugar which is a hydrophobic compound, According to the objective, it can select suitably, For example, a fatty acid, an aromatic compound, etc. are mentioned. Among these, fatty acids are preferred because they are rich in molecular species and can be easily used for synthesis reactions.

There is no restriction | limiting in particular as said fatty acid, According to the objective, it can select suitably, For example, a linear fatty acid, unsaturated fatty acid, branched fatty acid etc. are mentioned. Among these, straight chain fatty acids are preferable from the viewpoint of easily forming a micelle structure advantageous for anti-influenza virus activity in an aqueous solution and stability. In addition, since the unsaturated fatty acid has a disadvantage that double bonds are easily oxidized and can be an unstable element in an anti-influenza virus agent, when using the unsaturated fatty acid as the fatty acid, for example, It is considered that a method of use such as using in combination with an antioxidant is preferable.
Among the linear fatty acids, linear fatty acids having 8 to 18 carbon atoms are preferable, and linear fatty acids having 12 to 14 carbon atoms are preferable in that a micelle structure advantageous for anti-influenza virus activity can be easily formed in an aqueous solution. More preferred.

<Sialic acid-containing sugar chain>
The sialic acid-containing sugar chain is not particularly limited as long as it is a sugar chain having a sialic acid that serves as an influenza virus binding site at one end, and can be appropriately selected according to the purpose. From the viewpoint of activity, it preferably has a sugar chain site corresponding to the hemagglutinin (HA) recognition site of influenza virus.
In order for influenza virus hemagglutinin to recognize sugar chains, not only does the sugar chain have sialic acid, but also the mode of binding between the sialic acid and galactose bound to the sialic acid is important. It is known that there is. The avian influenza virus has a sugar chain such as Neu5Ac (α2-3) Gal- in which sialic acid is bonded to position 3 of galactose, and the human influenza virus has Neu5Ac (α2) in which sialic acid is bonded to position 6 of galactose. -6) It is known to specifically recognize each sugar chain such as Gal-.

Therefore, the sialic acid-containing sugar chain needs to have a sialic acid that serves as a binding site for influenza virus at one end, and preferably has a sialylgalactose chain in which sialic acid and galactose are bonded. It is more preferable to have either a 3′-sialylgalactose chain (Neu5Acα2-3Gal) or a 6′-sialylgalactose chain (Neu5Acα2-6Gal).
Among them, the sialic acid-containing sugar chain includes 3′-sialyl lactose chain (Neu5Acα2-3Galβ1-4Glc), 6′-sialyllactose chain (Neu5Acα2-6Galβ1-4Glc), 3′-sialyllactosamine chain (Neu5Acα2-3Galβ1- 4GlcNAc), 6′-sialyllactosamine chain (Neu5Acα2-6Galβ1-4GlcNAc), 3′-sialylgalactosylgalactose chain (Neu5Acα2-3Galβ1-4Gal), 6′-sialylgalactosylgalactose chain (Neu5Acα2-6Galβ1G) -Sialylgalactosylgalactosamine chain (Neu5Acα2-3Galβ1-4GalNAc) and 6′-sialylgalactosylgalactosamine chain (Neu5Acα2-6Galβ1-4G) It is particularly preferred that it is selected from the group consisting of alNAc).

Further, the number of monosaccharides constituting the sialic acid-containing sugar chain can be appropriately selected according to the purpose, but is preferably 1 to 5, more preferably 2 to 3, and particularly preferably 3. . That is, the sialic acid-containing sugar chain may be composed only of sialic acid, but is more preferably composed of sialic acid and monosaccharide or disaccharide, and is composed of sialic acid and disaccharide (total trisaccharide). Is particularly preferred.
When the number of monosaccharides constituting the sialic acid-containing sugar chain is 2 or more, the monosaccharide adjacent to the sialic acid is not particularly limited and can be appropriately selected according to the purpose. Galactose (Gal) is preferred. Further, when the number of monosaccharides constituting the sialic acid-containing sugar chain is 3 or more, the monosaccharide adjacent to the sialic acid is further adjacent to the monosaccharide (the monosaccharide adjacent to the sialic acid). Is not particularly limited and can be appropriately selected according to the purpose, and among them, selected from the group consisting of galactose (Gal), glucose (Glc), galactosamine (GalNAc), and glucosamine (GlcNAc). Is preferred. Among these, galactosamine (GalNAc) and glucosamine (GlcNAc) are more preferable, and glucosamine (GlcNAc) is more preferable in that the binding property of the sialic acid-containing sugar chain to influenza virus is high and anti-influenza virus activity is expected to be excellent. Is particularly preferred.

<Bonding>
The bond between the sialic acid-containing sugar chain and the compound other than the sugar is not particularly limited as long as a compound other than the sugar is bound to a terminal other than the sialic acid terminal of the sialic acid-containing sugar chain. It can be selected as appropriate according to the conditions. For example, examples of the binding method when the compound other than the sugar is the fatty acid include a method for introducing an acyl group corresponding to the fatty acid into the sialic acid-containing sugar chain. As a method for introducing the acyl group into the sialic acid-containing sugar chain, for example, an introduction method by O-, N-, S-, C-glycosidation, etc. are preferable. -Introduction by glycosidation (acylamidation, amide bond) is particularly preferred.

The sialic acid-containing sugar chain complex is formed by combining the sialic acid-containing sugar chain and a compound other than the sugar in any combination.
Such, preferred specific examples of the sialic acid-containing sugar chain complexes, for example, 3'-sialyl lactosyl lauric acid amide _{(Neu5Acα2-3Galβ1-4Glc- NHCO - (CH 2)} 10 CH 3), 6 ' - sialyl lactosyl lauric acid amide _{(Neu5Acα2-6Galβ1-4Glc- NHCO - (CH 2)} 10 CH 3), 3'- sialyl lactosamine Samini Lula Gaulin acid amide _{(Neu5Acα2-3Galβ1-4GlcNAc- NHCO - (CH 2)} 10 CH _3), 6'-sialyl lactosamine Samini Lula Gaulin acid amide _{(Neu5Acα2-6Galβ1-4GlcNAc- NHCO - (CH 2)} 10 CH 3), 3'- sialyl lactosyl myristic acid amide (Neu5Acα2-3Galβ1-4Glc- NHCO _{- (CH 2) 12 CH 3} ), 6'- sialyl lactosyl myristic acid amide _{(Neu5Acα2-6Galβ1-4Glc- NHCO - (CH 2)} 12 CH 3), 3'- sialyl lactosaminoglycans sulfonyl myristic acid amide (Neu5Acarufa2- _{3Galβ1-4GlcNAc- NHCO (CH 2) 12 CH} 3), 6'- sialyl lactosaminoglycans sulfonyl myristic acid amide _{(Neu5Acα2-6Galβ1-4GlcNAc- NHCO - (CH 2)} 12 CH 3), 3'- sialyl-galactosyl-galactosyl laurate amide _{(Neu5Acα2-3Galβ1-4Gal- NHCO - (CH 2)} 10 CH 3), 6'- sialyl-galactosyl-galactosyl lauric acid amide (Neu5Acα2-6Galβ1-4Gal- NHCO - (CH ₂ ) ₁₀ CH _3), 3'- sialyl-galactosyl galactosamine mini Lula Gaulin acid amide _{(Neu5Acα2-3Galβ1-4GalNAc- NHCO - (CH 2)} 10 CH 3), 6'- sialyl-galactosyl galactosamine mini Lula Gaulin acid amide (Neu5Acarufa2-6Galbeta1 _{-4GalNAc- NHCO - (CH 2) 10} CH 3), 3'- sialyl-galactosyl-galactosyl myristic acid amide _{(Neu5Acα2-3Galβ1-4Gal- NHCO - (CH 2)} 12 CH 3), 6'- sialyl-galactosyl-galactosyl myristic acid amide _{(Neu5Acα2-6Galβ1-4Gal- NHCO - (CH 2)} 12 CH 3), 3'- sialyl-galactosyl galactosamine mini Le myristic acid amide (Neu5Acarufa2-3Ga _{β1-4GalNAc- NHCO - (CH 2) 12} CH 3), 6'- sialyl-galactosyl galactosamine mini Le myristic acid amide _{(Neu5Acα2-6Galβ1-4GalNAc- NHCO - (CH 2)} 12 CH 3) , and the like.

(Method for producing sialic acid-containing sugar chain complex)
In the sialic acid-containing sugar chain complex, a compound other than a sugar is bonded to a terminal other than the sialic acid terminal of a sialic acid-containing sugar chain containing sialic acid at one terminal by a chemical reaction by an arbitrary bonding method. Can be manufactured. In particular, when the compound other than the saccharide constituting the sialic acid-containing sugar chain complex is a fatty acid, for example, the sialic acid-containing sugar chain may be converted to the sialic acid by acylamidating a terminal other than the sialic acid terminal. It is preferable to produce the containing sugar chain complex.

<Method for producing sialyl oligosaccharide fatty acid amide (one embodiment of sialic acid-containing sugar chain complex)>
Hereinafter, the sialic acid-containing sugar chain may be referred to as the 3′-sialyl lactose chain (hereinafter sometimes referred to as 3′-SL) or the 6′-sialyl lactose chain (hereinafter referred to as 6′-SL). ), The 3′-sialyl lactosamine chain (hereinafter sometimes referred to as 3′-SLN), and the 6′-sialyl lactosamine chain (hereinafter sometimes referred to as 6′-SLN). And the sialic acid-containing sugar chain complex is exemplified by a case where the compound other than the sugar is a fatty acid and is amide-bonded to a terminal other than the sialic acid terminal of the sialic acid-containing sugar chain. An example of a body manufacturing method will be described.
Hereinafter, the 3′-SL, 6′-SL, 3′-SLN, and 6′-SLN may be collectively referred to as sialyl oligosaccharides.
Hereinafter, the sialic acid-containing sugar chain complex formed by amide bond between the sialyl oligosaccharide and the fatty acid may be referred to as sialyl oligosaccharide fatty acid amide. The sialyl oligosaccharide fatty acid amide is one of preferred embodiments of the sialic acid-containing sugar chain complex.

-Sialyl oligosaccharide-
There is no restriction | limiting in particular as said sialyl oligosaccharides, such as 3'-SL, 6'-SL, 3'-SLN, 6'-SLN, It can select suitably according to the objective, For example, a human, a cow, etc. Sialyl oligosaccharides derived from these mammals can be used. Moreover, a commercial item can also be used as said sialyl oligosaccharide. The said commercial item can be obtained from Sigma-Aldrich, Seikagaku Corporation, etc., for example.

-Acylamidation of sialyl oligosaccharides-
A sialyl oligosaccharide fatty acid amide in which the fatty acid is amide-bonded to the sialyl oligosaccharide can be obtained by acylating the sialyl oligosaccharide, for example.
In the acylamidation of the sialyl oligosaccharide, for example, the sialyl oligosaccharide and ammonium hydrogen carbonate are reacted (hereinafter sometimes referred to as step (I)), and then the obtained reaction product is reacted with a chlorinated fatty acid. (Hereinafter sometimes referred to as step (II)). More specifically, for example, it can be performed as follows.

-Step (I)-
In step (I), a sialyl oligosaccharide is reacted with ammonium hydrogen carbonate. There is no restriction | limiting in particular as this reaction method, According to the objective, it can select suitably, For example, the said sialyl oligosaccharide is added to supersaturated ammonium hydrogencarbonate aqueous solution, room temperature (25 degreeC) conditions, stirring. The method of making it react under is mentioned.

  Moreover, the progress of the reaction between the sialyl oligosaccharide and ammonium hydrogen carbonate in the step (I) can be confirmed by, for example, thin layer chromatography (TLC).

  After completion of the reaction in step (I), ammonium hydrogen carbonate is removed together with moisture using, for example, a rotary evaporator. The reaction product obtained after removal is then subjected to the following step (II).

-Step (II)-
In step (II), the reaction product obtained in step (I) is reacted with chlorinated fatty acid. The reaction method is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the reaction product obtained in step (I) and a chlorinated fatty acid are reacted at 0 ° C. in the presence of sodium carbonate. The method of making it react with is mentioned.

  There is no restriction | limiting in particular as the usage-amount of the said chlorinated fatty acid, According to the objective, it can select suitably, For example, a molar ratio with respect to the usage-amount of the said sialyl oligosaccharide, a chlorinated fatty acid: sialyl oligosaccharide = 5 : 1 can be used in such an amount. The amount of sodium carbonate used is not particularly limited and may be appropriately selected depending on the purpose. For example, sodium carbonate: fatty acid chloride = 1 in molar ratio with respect to the amount of the chlorinated fatty acid used. : 1 can be used in such an amount.

  A sialyl oligosaccharide fatty acid amide can be produced by the steps (I) and (II). Examples of the sialyl oligosaccharide fatty acid amide include Sephadex LH-20 (GE Healthcare Biosciences), Sephadex G-10 (GE Healthcare Biosciences), BioGel P-2 (Nippon Bio-Rad Laboratories, Inc.). ), Silica gel (for example, Iatrobeads 6RS 8060 (Mitsubishi Chemical Yatron Co., Ltd.)) and the like can be purified by various column chromatography.

<Method for confirming manufactured sialic acid-containing sugar chain complex>
The sialic acid-containing sugar chain complex obtained by the above production method can be confirmed by, for example, thin layer chromatography, high performance liquid chromatography, mass spectrometer, proton NMR and the like.

  The sialic acid-containing sugar chain complex obtained by the production method has excellent anti-influenza virus activity, and can be preferably used as an active ingredient of the anti-influenza virus agent of the present invention described later.

(Anti-influenza virus agent)
The anti-influenza virus agent of the present invention contains at least the sialic acid-containing sugar chain complex, and further contains other components as necessary. The anti-influenza virus agent may be the sialic acid-containing sugar chain complex itself.

<Sialic acid-containing sugar chain complex>
The sialic acid-containing sugar chain complex contained in the anti-influenza virus agent preferably has an influenza virus hemagglutinin (HA) recognition site in the sialic acid-containing sugar chain part. The hemagglutinin is a kind of spike protein that exists in the envelope of influenza virus, and recognizes and binds to sugar chains containing sialic acid of complex carbohydrates present on the membrane surface of target cells to establish infection. It has been known. Since the sialic acid-containing sugar chain complex has this hemagglutinin recognition site, it can bind to influenza virus, and therefore, it can inhibit binding of influenza virus to the original target cell. It is done.
Specifically, the hemagglutinin recognition site of the sialic acid-containing sugar chain is preferably any of 3′-sialylgalactose chain (Neu5Acα2-3Gal) and 6′-sialylgalactose chain (Neu5Acα2-6Gal). It is done.

  The content of the sialic acid-containing sugar chain complex (active ingredient) in the anti-influenza virus agent is not particularly limited and can be appropriately selected according to the dosage form of the anti-influenza virus agent, 60-70 mass% is preferable.

<Other ingredients>
The other components other than the sialic acid-containing sugar chain complex that can be contained in the anti-influenza virus agent are not particularly limited and are appropriately selected depending on the purpose within a range not impairing the effects of the present invention. For example, a pharmaceutically acceptable carrier and the like can be mentioned. The pharmaceutically acceptable carrier is not particularly limited and may be appropriately selected depending on the dosage form of the anti-influenza virus agent.
In addition, the content of the other components is not particularly limited, and for example, the content of the sialic acid-containing sugar chain complex (active ingredient) in the anti-influenza virus agent is within a desired range. It can be appropriately selected according to the purpose.

<Dosage form>
The dosage form of the anti-influenza virus agent is not particularly limited, and can be appropriately selected according to, for example, the administration method of the anti-influenza virus agent. For example, oral solid agent, oral solution, injection, inhalation Examples include powders.

-Oral solid agent-
Examples of the oral solid preparation include tablets, coated tablets, granules, powders, capsules and the like.
There is no restriction | limiting in particular as a manufacturing method of the said oral solid preparation, A normal method can be used, For example, an excipient | filler and various additives as needed are added to the said sialic acid containing sugar_chain | carbohydrate complex. Can be manufactured. Here, the excipient is not particularly limited and may be appropriately selected depending on the intended purpose. For example, lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid, etc. Is mentioned. Moreover, there is no restriction | limiting in particular also as said additive, According to the objective, it can select suitably, For example, a binder, a disintegrating agent, a lubricant, a coloring agent, a flavoring / flavoring agent etc. are mentioned.

The binder is not particularly limited and may be appropriately selected depending on the intended purpose. For example, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxy Examples include propyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, and polyvinyl pyrrolidone.
The disintegrant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include dry starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose. Is mentioned.
The lubricant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include purified talc, stearate, borax, and polyethylene glycol.
The colorant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include titanium oxide and iron oxide.
The flavoring / flavoring agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include sucrose, orange peel, citric acid, and tartaric acid.

-Oral solution-
Examples of the oral liquid preparation include internal liquid preparations, syrups, and elixirs.
There is no restriction | limiting in particular as a manufacturing method of the said oral solution, A normal method can be used, For example, it can manufacture by adding an additive to the said sialic acid containing sugar_chain | carbohydrate complex. Here, there is no restriction | limiting in particular as said additive, According to the objective, it can select suitably, For example, a flavoring / flavoring agent, a buffering agent, a stabilizer, etc. are mentioned.

The flavoring / flavoring agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include sucrose, orange peel, citric acid, and tartaric acid.
There is no restriction | limiting in particular as said buffering agent, According to the objective, it can select suitably, For example, sodium citrate etc. are mentioned.
There is no restriction | limiting in particular as said stabilizer, According to the objective, it can select suitably, For example, tragacanth, gum arabic, gelatin, etc. are mentioned.

-Injection-
Examples of the injection include a solution, a suspension, and a solid agent for dissolving for use.
There is no restriction | limiting in particular as a manufacturing method of the said injection, For example, a pH regulator, a buffer, a stabilizer, an isotonic agent can be used for the said sialic acid containing sugar_chain | carbohydrate complex. It can be produced by adding a local anesthetic or the like. Here, there is no restriction | limiting in particular as said pH regulator and said buffer, According to the objective, it can select suitably, For example, sodium citrate, sodium acetate, sodium phosphate etc. are mentioned. Moreover, there is no restriction | limiting in particular as said stabilizer, According to the objective, it can select suitably, For example, sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid etc. are mentioned. The tonicity agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include sodium chloride and glucose. The local anesthetic agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include procaine hydrochloride and lidocaine hydrochloride.

<Administration>
There is no restriction | limiting in particular as the administration method of the said anti-influenza virus agent, For example, it can select suitably according to the dosage form etc. of the said anti-influenza virus agent, and can administer orally or parenterally.

  The dose of the anti-influenza virus agent is not particularly limited, and can be appropriately selected in consideration of various factors such as the age, weight, constitution, symptom, and presence / absence of administration of other drugs.

  Moreover, there is no restriction | limiting in particular also in the time of administration to an individual | organism, According to the objective, it can select suitably. For example, it may be administered before or after infection with influenza virus.

<Target>
The animal species to be administered with the anti-influenza virus agent is not particularly limited as long as it is an animal species that can be infected with influenza virus, and can be appropriately selected according to the purpose. , Monkeys, pigs, cows, sheep, goats, dogs, cats, mice, rats and the like.

  In addition, the type of influenza virus to which the anti-influenza virus agent is applied is not particularly limited. The anti-influenza virus agent can be expected to have an infection-suppressing effect for all influenza viruses of types A, B and C, and for influenza A viruses, all subtypes (H1N1 to H1N1- With regard to the influenza virus H15N9), an infection suppression effect can be expected.

(effect)
The sialic acid-containing sugar chain complex of the present invention has anti-influenza virus activity and can be preferably used as an active ingredient of the anti-influenza virus agent of the present invention. The sialic acid-containing sugar chain complex can be efficiently obtained by the method for producing a sialic acid-containing sugar chain complex of the present invention.
In addition, since the sialic acid-containing sugar chain complex of the present invention can be designed and manufactured by arbitrarily combining a sialic acid-containing sugar chain and a compound other than a sugar within a specified molecular weight range, It is possible to provide a complex of a type, and therefore, it is expected to provide an anti-influenza virus agent having various effects.

The anti-influenza virus agent of the present invention has preferable anti-influenza virus activity, and can be expected to have an infection-inhibiting effect on all influenza viruses, regardless of type A, B, or C. Since all subtypes can be expected to have an infection-inhibiting effect regardless of their animal species, they can be clinically applied as new influenza drugs after amantadine and Tamiflu, which are currently approved for use as influenza drugs. There is expected.
In addition, influenza viruses have the ability to escape the body defense function of antibodies by slightly mutating proteins on the membrane surface. In comparison, the site of influenza virus that recognizes and binds to the receptor is well conserved and retains a high specificity for the receptor. The anti-influenza virus agent of the present invention focuses on utilizing the competitive inhibitory effect of infection due to the selective specificity of the virus-receptor binding. Therefore, the anti-influenza virus agent of the present invention is expected to have a great effect on prevention and treatment of influenza infection not only when used alone but also in combination with an influenza vaccine or an existing drug having a different mechanism of action.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

(Example 1: 3'-sialyl lactosyl lauric acid amide)
<Manufacturing>
Sialic acid-containing sugar chain complex of the present invention the 3'-sialyl lactosyl lauric acid amide (3'-SL-N-C12 : Neu5Acα2-3Galβ1-4Glc- NHCO - (CH 2) 10 CH 3) ( molecular weight: 814) was prepared as follows.

3′-sialyl lactose (3′-SL) (derived from bovine colostrum, manufactured by Sigma-Aldrich) (molecular weight: 633) was dissolved in a supersaturated aqueous ammonium hydrogen carbonate solution and stirred at room temperature for 4 days. On the way, the progress of the reaction was checked by thin layer chromatography (TLC). After completion of the reaction, ammonium hydrogen carbonate along with moisture was removed from the reaction product using a rotary evaporator. Next, the obtained reaction product was reacted with chlorinated lauric acid in the presence of sodium carbonate under the condition of 0 ° C. Here, chlorinated lauric acid was used in such an amount that the molar ratio was 5 times the amount of 3′-SL used. Further, sodium carbonate was used in an amount equivalent to the molar amount of the lauric acid chloride used. The progress of the reaction was sampled at about one hour intervals from the start and confirmed by TLC, and the reaction was terminated when the progress of the reaction reached saturation. An equal amount of chloroform was added to the reaction solution, and unreacted chlorinated lauric acid was extracted and removed.
After chloroform extraction, the obtained 3'-sialyl lactosyl lauric acid amide (3'-SL-N-C12) was confirmed by thin layer chromatography (TLC). In addition, the obtained 3′-sialyl lactosyl lauric acid amide (3′-SL-N-C12) was converted into Sephadex LH-20 (GE Healthcare Biosciences), Sephadex G-10 (GE Healthcare Biosciences). Co., Ltd.), BioGel P-2 (Nippon Bio-Rad Laboratories Co., Ltd.), and silica gel (for example, Iatrobeads 6RS 8060 (Mitsubishi Chemical Yatron Co., Ltd.)) were used as carriers and purified.
After purification, the obtained 3′-sialyl lactosyl lauric acid amide (3′-SL-N-C12) was analyzed for molecular weight, purity, LC-MS (liquid chromatography-mass spectrometry), and proton NMR. And the structure was confirmed. These results are shown in FIG. 2, FIG. 3A, FIG. 3B, and Table 1.

The reaction scheme in Example 1 is shown in FIG. 1, and the thin layer chromatography image after the chloroform extraction is shown in FIG. In FIG. 2, lane 1 shows gangliosides (G _M4 , G _M1 , G _D1a , G _D1b , G _T1b ) extracted from minke whale brain as a control. Lane 2 shows a sample solution (including 3′-SL-N—C12 obtained) after chloroform extraction. Lane 3 shows the chloroform extract (including the unreacted chlorinated lauric acid removed) after the chloroform extraction operation.
The LC-MS results are shown in FIGS. 3A and 3B. FIG. 3A shows a chromatogram image of LC, and FIG. 3B shows the results of mass spectrometry of components contained in the peak of FIG. 3A. In FIG. 3B, the mass is expressed as a suspicious molecular ion (M + H) ⁺ quantity, so the mass shows about 815, which is 814 + 1.
Table 1 below shows proton NMR measurement results (chemical shifts of 3'-SL and 3'-SL-N-C12).

  From Table 1, it can be seen that by modifying 3'-SL, αH1 disappeared and all became βH1, and signals of amide proton, alkylmethyl, and methylene proton appeared.

[Evaluation]
<Evaluation of anti-influenza virus activity>
The anti-influenza virus activity of the obtained 3′-sialyl lactosyl lauric acid amide (3′-SL-N-C12) of Example 1 was evaluated as follows.
In this evaluation method, A / PR / 8/34 (ATCC VR-95) (H1N1 type) is used as an influenza virus, and MDCK cells (NBL-2 cells) are used as cells to be infected with influenza virus. , Dog kidney cells) (manufactured by Dainippon Sumitomo Pharma Co., Ltd.). Moreover, E-MEM culture medium (Eagle-Minimum Essential Medium) (GIBCO; manufactured by Invitrogen Corporation) was used as the culture solution.

MDCK cells were cultured in a microplate supplemented with 10% by volume fetal bovine serum (FBS) -containing E-MEM medium at 37 ° C. and 5% CO ₂ . The influenza virus A / PR / 8/34 strain serially diluted 10-fold was added to the MDCK cells that became monolayers and incubated at 37 ° C. for 1 hour for infection. The virus solution used for infection was removed, E-MEM medium was added, and the mixture was incubated at 37 ° C. for 4 days. During and after infection with influenza virus, 3′-sialyl lactosyl lauric acid amide (3′-SL-N-C12) of Example 1 was added to the medium at a concentration of 100 μM, and the cytopathic effect by the virus under a microscope. Was observed.

According to the observation result of the cytopathic effect by the virus, the anti-influenza virus activity of 3′-SL-N-C12 was evaluated. The evaluation results are shown in Table 2. In Table 2, “+” and “−” in the column of “antiviral activity” indicate the following criteria, respectively.
+: Compared with amantadine (Comparative Example 3, which will be described later) which is an approved drug, the virus suppression effect is comparable (having anti-influenza virus activity comparable to amantadine).
-: Compared with amantadine (Comparative Example 3, which will be described later) which is an approved drug, the virus suppression effect is weak (anti-influenza virus activity is weak compared with amantadine).

<Evaluation of cytotoxicity>
Moreover, the cytotoxicity of 3'-sialyl lactosyl lauric acid amide (3'-SL-N-C12) of Example 1 was determined by adding only 3'-SL-N-C12 without adding influenza virus to the MDCK cells. The state of cell degeneration upon addition of was evaluated by observing under a microscope. The evaluation results are shown in Table 2. In Table 2, “−” and “+” in the column of “cytotoxicity” indicate the following criteria, respectively.
-: No cell degeneration is observed.
+: Cell degeneration is observed (clearly different from virus-induced cell degeneration).

(Comparative Examples 1-3)
Moreover, instead of 3'-SL-N-C12 in Example 1, 3'-SL (3'-sialyl lactose: derived from bovine colostrum, manufactured by Sigma-Aldrich) (Comparative Example 1), 3'-SLN ( 3′-sialyllactosamine: derived from bovine colostrum, manufactured by Sigma-Aldrich) (Comparative Example 2) and amantadine (manufactured by Sigma-Aldrich) (Comparative Example 3), respectively, were used in the same manner as in Example 1. The anti-influenza virus activity and cytotoxicity at each concentration of 100 μM were evaluated. The results are shown in Table 2.

  From Table 2, the sialic acid-containing sugar chain complex of the present invention has an excellent anti-influenza activity comparable to that of amantadine as an existing drug, and is not cytotoxic in comparison with amantadine. It turns out that it is superior. Therefore, it was shown that the sialic acid-containing sugar chain complex of the present invention can be suitably used as an active ingredient of an anti-influenza virus agent.

  The anti-influenza virus agent of the present invention containing the sialic acid-containing sugar chain complex of the present invention as an active ingredient has preferable anti-influenza virus activity, and is expected to be clinically applicable as a new influenza drug. Is.

FIG. 1 is a diagram showing a reaction scheme in the production of 3'-sialyllactosyllauric acid amide, which is a preferred example of the sialic acid-containing sugar chain complex of the present invention. FIG. 2 is a thin-layer chromatographic image of 3'-sialyllactosyllauric acid amide (lane 2), which is a preferred example of the sialic acid-containing sugar chain complex of the present invention. FIG. 3A is an LC (liquid chromatography) image in LC-MS analysis of 3′-sialyl lactosyl lauric acid amide, which is a preferred example of the sialic acid-containing sugar chain complex of the present invention. FIG. 3B shows the results of mass spectrometry of the peak components of the LC image (FIG. 3A) in LC-MS analysis of 3′-sialyllactosyllauric acid amide, which is a preferred example of the sialic acid-containing sugar chain complex of the present invention. It is a figure.

Claims (5)

A sialic acid-containing sugar chain containing either a 3′-sialylgalactose chain (Neu5Acα2-3Gal) or a 6′-sialylgalactose chain (Neu5Acα2-6Gal) at one end has a carbon other than the sialic acid end. A sialic acid-containing sugar chain complex characterized in that a linear fatty acid of 8 to 18 (excluding sphingolipids) is an amide bond and has a molecular weight of 400 to 1,400. Sialic acid-containing sugar chains are 3′-sialyllactose chains (Neu5Acα2-3Galβ1-4Glc), 6′-sialyllactose chains (Neu5Acα2-6Galβ1-4Glc), 3′-sialyllactosamine chains (Neu5Acα2-3Galβ1-4GlcNAc), 6′-sialyllactosamine chain (Neu5Acα2-6Galβ1-4GlcNAc), 3′-sialylgalactosylgalactose chain (Neu5Acα2-3Galβ1-4Gal), 6′-sialylgalactosylgalactose chain (Neu5Acα2-6Galβ1-4Gal), 3′-sialyllacto Galactosamine chain (Neu5Acα2-3Galβ1-4GalNAc) and 6′-sialylgalactosylgalactosamine chain (Neu5Acα2-6Galβ1-4G) lNAc), sialic acid-containing sugar chain complex as claimed in claim 1 selected from the group consisting of. A method for producing a sialic acid-containing sugar chain complex according to claim 1, wherein a 3′-sialylgalactose chain (Neu5Acα2-3Gal) and a 6′-sialylgalactose chain ( A sialic acid-containing sugar chain complex characterized by acylating a terminal other than the sialic acid terminal of a sialic acid-containing sugar chain containing any of Neu5Acα2-6Gal) to bond a linear fatty acid having 8 to 18 carbon atoms Body manufacturing method. The method for producing a sialic acid-containing sugar chain complex according to claim 3, wherein the sialic acid-containing sugar chain is reacted with ammonium hydrogen carbonate, and then the obtained reaction product is reacted with a chlorinated fatty acid. An anti-influenza virus agent comprising the sialic acid-containing sugar chain complex according to claim 1 as an active ingredient.