WO2011158388A1 - Sialic acid-containing sugar chain complex and method for producing same, anti-influenza virus agent and filter - Google Patents

Sialic acid-containing sugar chain complex and method for producing same, anti-influenza virus agent and filter Download PDF

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WO2011158388A1
WO2011158388A1 PCT/JP2010/065985 JP2010065985W WO2011158388A1 WO 2011158388 A1 WO2011158388 A1 WO 2011158388A1 JP 2010065985 W JP2010065985 W JP 2010065985W WO 2011158388 A1 WO2011158388 A1 WO 2011158388A1
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sialic acid
sugar chain
containing sugar
gal
influenza virus
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PCT/JP2010/065985
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French (fr)
Japanese (ja)
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隆志 寺林
稔 森田
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東光薬品工業株式会社
学校法人北里研究所
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Priority to JP2012520240A priority Critical patent/JP5736371B2/en
Publication of WO2011158388A1 publication Critical patent/WO2011158388A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
    • C07H5/06—Aminosugars

Definitions

  • the present invention provides a novel sialic acid-containing sugar chain complex, an efficient production method thereof, an anti-influenza virus agent containing the sialic acid-containing sugar chain complex as an active ingredient, and the anti-influenza virus agent Related to the filter.
  • hemagglutinin In influenza virus, hemagglutinin (HA), one of the spike proteins in the envelope, recognizes sugar chains containing sialic acids of complex carbohydrates 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 ( ⁇ 2-3) Gal-, and the human influenza virus specifically recognizes a sugar chain such as Neu5Ac ( ⁇ 2-6) Gal-. It is thought to be a factor that creates the specificity of
  • the main methods for preventing or treating influenza include vaccination and administration of amantadine (Symmetrel (registered trademark)) and oseltamivir (Tamiflu (registered trademark)) that have been approved for use as anti-influenza drugs.
  • amantadine Symmetrel (registered trademark)
  • oseltamivir Teamiflu (registered trademark)
  • a vaccine can prevent the attachment of virus to cells by secreting IgA produced by vaccination into the mucus of the respiratory system, but against different types of new viruses Is known to be powerless.
  • Amantadine inhibits the ion channel action of the M2 protein of influenza virus and can suppress the unhulling of the influenza virus after entering the target cell, but the effect on B and C viruses without M2 protein is
  • Tamiflu also inhibits the action of neuraminidase (NA), one of the spike proteins in the influenza virus envelope, and the replicated influenza virus is released from infected target cells and spreads to other cells.
  • NA neuraminidase
  • an anti-influenza virus agent containing a glycosphingolipid having a sugar chain containing a recognition site of a virus receptor as an active ingredient has been reported (see Patent Document 1).
  • gangliosides that are glycosphingolipids containing sialic acid GM4 (Neu5Ac ( ⁇ 2-3) Gal-Cer) and GM3 (Neu5Ac ( ⁇ 2-3) Gal ( ⁇ 1-4) extracted from natural products.
  • Neu5Ac ( ⁇ 2-3) Gal ( ⁇ 1-4) GlcNAc ( ⁇ 1-4) Gal ( ⁇ 1-4) Glc-Cer) has an anti-infection activity against influenza virus, and its infection-preventing power is strong Is disclosed in the order of SPG> GD1a> GM3> GM4.
  • 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.
  • gangliosides disclosed as the anti-influenza virus agent are derived from natural products such as minke whale brain and human erythrocytes.
  • an active ingredient is desired for practical use as an anti-influenza drug, it may be somewhat difficult because of being extracted.
  • the epidemic of influenza is a major problem, and existing drugs such as amantadine and Tamiflu, which have been used in the past, have drawbacks such as side effects, restrictions on the timing of administration, and the risk of the emergence of resistant strains. ing.
  • sialic acid-containing sugar chain complexes have anti-influenza virus activity (see Patent Document 2).
  • Patent Document 2 only the sialic acid-containing sugar chain complex having 12 carbon atoms of the fatty acid amide is disclosed, and the sialic acid-containing sugar chain complex having 12 carbon atoms of the fatty acid amide is disclosed.
  • Anti-influenza virus activity is insufficient, and further improvement of anti-influenza virus activity has been desired.
  • 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 is an object of the present invention to provide a highly safe anti-influenza virus agent that has no side effects and has no side effects and has high side effects, and a filter carrying the anti-influenza virus agent.
  • the sugar chain of the target cell recognized by the virus or bacteria is equal to or higher than that. If a component having a strong affinity is added from the outside, infection by the virus or bacteria is considered to be inhibited.
  • HA hemagglutinin
  • 3'-sialyl lactose 3'-SL: 3'-Neu5Ac ( ⁇ 2-3) Gal ( ⁇ 1- 4) Glc
  • 3′-sialyllactosamine 3′-SLN: 3′-Neu5Ac ( ⁇ 2-3) Gal ( ⁇ 1-4) GlcNAc did not show anti-influenza virus activity.
  • the present inventors have made extensive studies and obtained the following knowledge. That is, when a sialyl oligosaccharide that did not exhibit anti-influenza virus activity and has a certain molecular weight and a fatty acid having a certain number of carbon atoms bound thereto, a complex of the sialyl oligosaccharide and the fatty acid was newly prepared. The obtained complex was found to show excellent anti-influenza virus activity, and the present invention was completed.
  • a sialic acid-containing sugar chain containing sialic acid at one end has an amide bond with a fatty acid having 14 to 18 carbon atoms excluding sphingolipid, and a molecular weight of 400 to 1 except for the sphingolipid.
  • 400 a sialic acid-containing sugar chain complex.
  • sialic acid-containing sugar chain complex according to any one of ⁇ 1> to ⁇ 2>, wherein the sialic acid-containing sugar chain is composed of 1 to 5 monosaccharides.
  • sialic acid-containing sugar chain includes a sialylgalactose chain (Neu5Ac-Gal) composed of N-acetylneuraminic acid (Neu5Ac) and galactose (Gal).
  • the sialic acid-containing sugar chain is a 3′-sialylgalactose chain (3′-Neu5Ac ( ⁇ 2-3) Gal) or a 6′-sialylgalactose chain (6′-Neu5Ac ( ⁇ 2-6) Gal).
  • Sialic acid-containing sugar chains are 3′-sialyl lactose chains (3′-Neu5Ac ( ⁇ 2-3) Gal ( ⁇ 1-4) Glc), 6′-sialyl lactose chains (6′-Neu5Ac ( ⁇ 2-6) ) Gal ( ⁇ 1-4) Glc), 3′-Sialyllactosamine chain (3′-Neu5Ac ( ⁇ 2-3) Gal ( ⁇ 1-4) GlcNAc), 6′-Sialyllactosamine chain (6′-Neu5Ac ( ⁇ 2) -6) Gal ( ⁇ 1-4) GlcNAc), 3′-sialylamide lactose chain (3′-Neu5Ac1NH 2 ( ⁇ 2-3) Gal ( ⁇ 1-4) Glc), 6′-sialylamide lactose chain (6′- Neu5Ac1NH 2 ( ⁇ 2-6) Gal ( ⁇ 1-4) Glc), 3′-sialylamidolactosamine chain (3′-Neu5Ac1NH 2 ( ⁇ 2-6) Gal (
  • ⁇ 7> The sialic acid-containing sugar chain complex according to any one of ⁇ 1> to ⁇ 6>, wherein the fatty acid has 16 to 18 carbon atoms.
  • ⁇ 8> The sialic acid-containing sugar chain complex according to any one of ⁇ 1> to ⁇ 7>, wherein the molecular weight is 750 to 950.
  • ⁇ 9> The method for producing a sialic acid-containing sugar chain complex according to any one of ⁇ 1> to ⁇ 8>, wherein the sialic acid-containing sugar chain contains sialic acid at one end other than the sialic acid terminal
  • a method for producing a sialic acid-containing sugar chain complex wherein a fatty acid having 14 to 18 carbon atoms excluding sphingolipid is bonded to the end of the saccharide by a chemical reaction.
  • ⁇ 10> The method for producing a sialic acid-containing sugar chain complex according to ⁇ 9>, wherein a terminal other than the sialic acid terminal of the sialic acid-containing sugar chain is acylamidated.
  • ⁇ 11> The method for producing a sialic acid-containing sugar chain complex according to any one of ⁇ 9> to ⁇ 10>, wherein the carboxyl group of the sialic acid of the sialic acid-containing sugar chain is acylamidated.
  • ⁇ 12> The sialic acid-containing sugar chain and ammonium hydrogen carbonate are reacted, and then the obtained reaction product and a fatty acid are reacted in the presence of a condensing agent.
  • This is a method for producing a sialic acid-containing sugar chain complex.
  • An anti-influenza virus agent comprising the sialic acid-containing sugar chain complex according to any one of ⁇ 1> to ⁇ 8> as an active ingredient.
  • the anti-influenza virus agent according to ⁇ 13> wherein the sialic acid-containing sugar chain constituting the sialic acid-containing sugar chain complex has a hemagglutinin recognition site for influenza virus.
  • the hemagglutinin recognition site of influenza virus has 3′-sialylgalactose chain (3′-Neu5Ac ( ⁇ 2-3) Gal) and 6′-sialylgalactose chain (6′-Neu5Ac ( ⁇ 2-6) Gal)
  • a filter comprising the anti-influenza virus agent according to any one of ⁇ 13> to ⁇ 15>.
  • the above-described conventional problems can be solved, the object can be achieved, and a novel sialic acid-containing sugar chain complex having anti-influenza virus activity, an efficient production method thereof, and
  • a filter carrying an anti-influenza virus agent can be provided.
  • FIG. 1 is a diagram showing TLC results of a sialic acid-containing sugar chain complex (3′-SL—N—C12) produced by the conventional production method and the production method of the present invention.
  • FIG. 2 is a diagram showing TLC results of the sialic acid-containing sugar chain complex (3′-SL-N—C14) produced in Production Example 1.
  • FIG. 3A is a view showing a TLC result of a sialic acid-containing sugar chain complex (3′-SL—N—C12) produced by the production method of the present invention in Test Example 1.
  • “M” indicates a whale brain ganglioside mixed solution used as a marker.
  • FIG. 1 is a diagram showing TLC results of a sialic acid-containing sugar chain complex (3′-SL—N—C12) produced by the conventional production method and the production method of the present invention.
  • FIG. 2 is a diagram showing TLC results of the sialic acid-containing sugar chain complex (3′-SL-N—C14) produced in Production Example 1.
  • FIG. 3B shows the sialic acid-containing sugar chain complex (3′-SL-N-C12) produced in Test Example 1 and the sialic acid-containing sugar chain complex (3′-SL-N—C16) produced in Production Example 2. It is a figure which shows the result of TLC of).
  • FIG. 3C shows the sialic acid-containing sugar chain complex (3′-SL-N—C14) produced in Production Example 1 and the sialic acid-containing sugar chain complex (3′-SL—N—C18) produced in Production Example 3. It is a figure which shows the result of TLC of).
  • “M” indicates a whale brain ganglioside mixed solution used as a marker.
  • FIG. 4 shows the sialic acid-containing sugar chain complex produced in Production Example 1 (3′-SL-N-C14) and the sialic acid-containing sugar chain complex produced in Production Example 5 (6′-SLN-N-C14). And 6′-S (1NH 2 ) LN—N—C14) and TLC results of the sialic acid-containing sugar chain complex (6′-SL—N—C14) produced in Production Example 6. “M” indicates a whale brain ganglioside mixed solution used as a marker.
  • FIG. 5 is a proton NMR spectrum of sialyl oligosaccharide (3′-SL) used as a raw material for the sialic acid-containing sugar chain complex produced in Production Examples 1 to 3.
  • FIG. 5 is a proton NMR spectrum of sialyl oligosaccharide (3′-SL) used as a raw material for the sialic acid-containing sugar chain complex produced in Production Examples 1 to 3.
  • FIG. 5 is a proton NMR spectrum of sialyl oligo
  • FIG. 6 is a proton NMR spectrum of the sialic acid-containing sugar chain complex (3′-SL—N—C18) produced in Production Example 3.
  • FIG. 7 is a proton NMR spectrum of the sialic acid-containing sugar chain complex (3′-SLN—N—C14) produced in Production Example 4.
  • FIG. 8A is a diagram showing an LC / MS spectrum of the sialic acid-containing sugar chain complex (6′-SLN—N—C14) produced in Production Example 5.
  • FIG. 8B is a diagram showing an LC / MS spectrum of the sialic acid-containing sugar chain complex (6′-S (1NH 2 ) LN—N—C14) produced in Production Example 5.
  • FIG. 8C is a diagram showing a proton NMR spectrum of the sialic acid-containing sugar chain complex (6′-SLN—N—C14) produced in Production Example 5.
  • FIG. 8D is a diagram showing a proton NMR spectrum of the sialic acid-containing sugar chain complex (6′-S (1NH 2 ) LN—N—C14) produced in Production Example 5.
  • FIG. 9 is a proton NMR spectrum of sialyl oligosaccharide (6′-SLN) used as a raw material for the sialic acid-containing sugar chain complex produced in Production Example 5 and Production Example 8.
  • FIG. 10A is a proton NMR spectrum of the sialic acid-containing sugar chain complex (6′-SL—N—C14) produced in Production Example 6.
  • FIG. 10A is a proton NMR spectrum of the sialic acid-containing sugar chain complex (6′-SL—N—C14) produced in Production Example 6.
  • FIG. 10B is a diagram showing a proton NMR spectrum of the sialic acid-containing sugar chain complex (6′-S (1NH 2 ) LN—C14) produced in Production Example 6.
  • FIG. 11 is a proton NMR spectrum of sialyl oligosaccharide (6′-SL) used as a raw material for the sialic acid-containing sugar chain complex produced in Production Examples 6 and 7.
  • FIG. 12 is a proton NMR spectrum of the sialic acid-containing sugar chain complex (6′-SL—N—C18) produced in Production Example 7.
  • FIG. 13A is a diagram showing a proton NMR spectrum of the sialic acid-containing sugar chain complex (6′-SLN—N—C18) produced in Production Example 8.
  • FIG. 11 is a proton NMR spectrum of sialyl oligosaccharide (6′-SL) used as a raw material for the sialic acid-containing sugar chain complex produced in Production Examples 6 and 7.
  • FIG. 12 is a proton NMR spectrum of the
  • FIG. 14A is a diagram showing a change in body weight of a mouse in Test Example 5.
  • “ ⁇ ” is a control group
  • “*” is a 3′-SL-N-C14 oral administration control group
  • “ ⁇ ” is a 3′-SL-N-C14 oral administration group
  • “ ⁇ ” is 3′-SL-N -C14 nasal administration group and " ⁇ " indicate virus administration group.
  • Vertical axis body weight (g)
  • horizontal axis days after influenza virus administration.
  • FIG. 14B is a diagram showing the survival rate of mice in Test Example 5.
  • FIG. 14C is a graph showing IL-6 concentration in mouse serum in Test Example 5.
  • Vertical axis IL-6 concentration (pg / mL). “*” Indicates p ⁇ 0.05 (t test).
  • FIG. 14D is a graph showing the INF- ⁇ concentration in the serum of mice in Test Example 5.
  • Vertical axis INF- ⁇ concentration (pg / mL). “*” Indicates p ⁇ 0.05 (t test).
  • FIG. 14E is a diagram showing virus titers in mouse lung tissue in Test Example 5.
  • Vertical axis virus titer (PFU / mL). “*” Indicates p ⁇ 0.05 (t test).
  • FIG. 15A is a diagram showing changes in the survival rate of mice in Test Example 5.
  • FIG. Vertical axis survival rate (%), horizontal axis: days after influenza virus administration.
  • FIG. 15B is a graph showing IL-6 concentration in mouse serum in Test Example 5.
  • Vertical axis IL-6 concentration (pg / mL).
  • FIG. 15C is a diagram showing virus titers in mouse lung tissue in Test Example 5.
  • Vertical axis virus titer (PFU / mL).
  • FIG. 16 is a diagram for confirming sialidase resistance when the carboxyl group of sialic acid in the sialic acid-containing sugar chain complex is amidated in Test Example 10.
  • 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 fatty acid.
  • the molecular weight of the sialic acid-containing sugar chain complex is 400 to 1,400, preferably 430 to 950, more preferably 590 to 950, still more preferably 750 to 950, and particularly preferably 800 to 950.
  • a desired degree of anti-influenza virus activity may not be exhibited.
  • 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.
  • the molecular weight is in the range of 750 to 950, it is advantageous in that a micelle structure advantageous for anti-influenza virus activity is easily formed in an aqueous solution.
  • the fatty acid is a fatty acid having 14 to 18 carbon atoms, but a fatty acid having 16 to 18 carbon atoms is preferred from the viewpoint of strong anti-influenza activity.
  • sphingolipids are excluded from the fatty acids. Specific examples of sphingolipids excluded from the fatty acid include ceramide.
  • the fatty acid is preferably hydrophobic.
  • the sialic acid-containing sugar chain complex is considered to be amphiphilic as a whole and take a micelle structure in an aqueous solution.
  • a sugar chain having a recognition site for an influenza virus hemagglutinin (HA) protein is preferably located on the 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
  • the fatty acid is not particularly limited as long as it has 14 to 18 carbon atoms, and can be appropriately selected according to the purpose. Examples thereof include linear fatty acids, unsaturated fatty acids, branched fatty acids and the like. Among these, the fatty acid is preferably a straight-chain fatty acid from the viewpoint of easily forming a micelle structure advantageous for anti-influenza virus activity in an aqueous solution and stability.
  • 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, Use methods such as use in combination with an antioxidant are preferred.
  • 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 a binding site for influenza virus 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 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.
  • avian influenza virus has a sugar chain such as Neu5Ac ( ⁇ 2-3) Gal- in which sialic acid is bonded to position 3 of galactose, and human influenza virus has Neu5Ac ( ⁇ 2) in which sialic acid is bonded to position 6 of galactose. -6) It is known to specifically recognize each sugar chain such as Gal-.
  • 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.
  • the sialic acid-containing sugar chain includes a 3′-sialylgalactose chain (3′-Neu5Ac ( ⁇ 2-3) Gal) and a 6′-sialylgalactose chain (6′-Neu5Ac ( ⁇ 2-6) Gal).
  • 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 more preferably 3 Particularly preferred. That is, the sialic acid-containing sugar chain may be composed only of sialic acid, but is preferably composed of sialic acid and monosaccharide or disaccharide, and is preferably composed of sialic acid and disaccharide (total trisaccharide). Is particularly preferred.
  • the sugar chain part that binds to hemagglutinin (HA) of influenza virus is the leading disaccharide part of the sialic acid-containing sugar chain.
  • the avian influenza virus specifically recognizes a sugar chain such as Neu5Ac ( ⁇ 2-3) Gal
  • the human influenza virus specifically recognizes a sugar chain such as Neu5Ac ( ⁇ 2-6) Gal-.
  • the sialic acid-containing sugar chain complex of the present invention has a total of three sugars, if the third sugar is N-acetylated, N-acetylated when an aggregate (such as a micelle) is formed in an aqueous solution.
  • the monosaccharide adjacent to the sialic acid is not particularly limited and may be appropriately selected depending on the purpose. (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 may be appropriately selected depending on the purpose, but is preferably selected from the group consisting of galactose (Gal), glucose (Glc), galactosamine (GalNAc), and glucosamine (GlcNAc). .
  • galactosamine (GalNAc) and glucosamine (GlcNAc) are more preferable, and galactosamine (GalNAc) 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.
  • the sialic acid constituting the sialic acid-containing sugar chain is further amidated with a carboxyl group from the viewpoint of superior anti-influenza virus activity. Even when the carboxyl group of sialic acid is amidated, the molecular weight of the sialic acid-containing sugar chain complex is hardly affected. Therefore, it is preferable in that a large three-dimensional structural change does not occur at the hemagglutinin (HA) recognition site and binding to influenza virus is not inhibited. In addition, when the carboxyl group of sialic acid is amidated, the negative charge in the carboxyl group is eliminated, so that the sialic acid-containing sugar chain complex can be easily analyzed after MALDI-TOF MS analysis. This is advantageous.
  • sialidase activity occurs when the carboxyl group of sialic acid is amidated.
  • HA hemagglutinin
  • sialic acid-containing sugar chain of the host cell receptor Inhibiting sialidase activity is advantageous in that the cycle of influenza virus infection and proliferation can be suppressed.
  • the bond between the sialic acid-containing sugar chain and the fatty acid is not particularly limited as long as the fatty acid is amide-bonded at a terminal other than the sialic acid terminal of the sialic acid-containing sugar chain. You can choose.
  • the fatty acid is amide-bonded to a terminal other than the sialic acid terminal of the sialic acid-containing sugar chain means that the sugar of the sialic acid-containing sugar chain and the carboxyl group of the fatty acid are —NH—.
  • it is represented by the following general formula (I).
  • A represents sialic acid
  • B represents a monosaccharide
  • C represents a fatty acid
  • N represents 0 or an integer of 1 or more, preferably 0 to 4, more preferably 1 to 2, and particularly preferably 2.
  • the coupling method is not particularly limited and may be appropriately selected depending on the intended purpose.
  • Examples thereof include a method for introducing an acyl group corresponding to the fatty acid into the sialic acid-containing sugar chain.
  • the method for introducing the acyl group into the sialic acid-containing sugar chain is not particularly limited and may be appropriately selected depending on the intended purpose.
  • An introduction method by glycosidation is preferred, and an introduction method by N-glycosidation (acylamidation, amide bond) is particularly preferred from the viewpoint of stability.
  • the sialic acid-containing sugar chain complex is formed by binding the sialic acid-containing sugar chain and the fatty acid in any combination.
  • a preferred specific example of such a sialic acid-containing sugar chain complex is, for example, 3′-sialyllactosylmyristate amide (3′-Neu5Ac ( ⁇ 2-3) Gal ( ⁇ 1-4) Glc-N— C14; hereinafter sometimes referred to as “3′-SL-N—C14”.) 3′-Sialyllactosyl palmitic acid amide (3′-Neu5Ac ( ⁇ 2-3) Gal ( ⁇ 1-4) Glc-N -C16; hereinafter sometimes referred to as "3'-SL-N-C16") 3'-sialyl lactosyl stearamide (3'-Neu5Ac ( ⁇ 2-3) Gal ( ⁇ 1-4) Glc- N-C18; hereinafter referred to as “3′-SL-N-C18”), 6′-sialyl lac
  • 6′-S (1NH 2) L -N-C14 6'-sialyl amide lactosyl palmitic acid amide
  • 6′-S (1NH 2 ) L—N—C16 6′-sialylamide Lactosyl stearamide
  • 6′-Neu5Ac1NH 2 ( ⁇ 2-6) Gal ( ⁇ 1-4) Glc-N—C18 6′-sialylamide Lactosyl stearamide
  • 6′-Neu5Ac1NH 2 ( ⁇ 2-6) Gal ( ⁇ 1-4) Glc-N—C18 hereinafter sometimes referred to as “6′-S (1NH 2 ) LN—C18”
  • 3′-Sialylamide lactosaminyl myristic acid amide (3′-Neu5Ac1NH 2 ( ⁇ 2-3) Gal ( ⁇ 1-4) GlcNAc-N—C14; hereinafter, “3′-S (1NH 2 ) LN— N-C14 " 3′-Sialyl
  • 6′-sialylamidolactosaminyl palmitate (6′-Neu5Ac1NH 2 ( ⁇ 2-6) Gal ( ⁇ 1-4) GlcNAc-N—C16; hereinafter, “6′-S (1NH 2 ) LN—N -C16 "), 6'-sialylamidolactosaminyl stearamide (6'-Neu5Ac1NH 2 ( ⁇ 2-6) Gal ( ⁇ 1-4) GlcNAc-N-C18; hereinafter referred to as" 6 ' —S (1NH 2 ) LN—N—C18 ”))) 3′-Sialylgalactosylgalactosylmyristate amide (3′-Neu5Ac ( ⁇ 2-3) Gal ( ⁇ 1-4) Gal-N— C14), 3′-sialylgalactosylgalactosylpalmitate (3′-Neu5Ac ( ⁇ 2-3) Gal ( ⁇ 1-4) Gal-N-C16), 3′- Allo
  • the sialic acid-containing sugar chain complex has excellent anti-influenza virus activity, it can be suitably used as an active ingredient of the anti-influenza virus agent of the present invention described later.
  • a fatty acid 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. It is the method of making it manufacture.
  • the bonding method is preferably a method in which the terminal other than the terminal sialic acid of the sialic acid-containing sugar chain is acylamidated.
  • the sialic acid-containing sugar chain includes the 3′-sialyllactose chain (hereinafter sometimes referred to as “3′-SL”), the 6′-sialyllactose chain (hereinafter referred to as “6′-SL”).
  • 3′-sialyllactosamine chain hereinafter sometimes referred to as “3′-SLN”
  • 6′-sialyllactosamine chain hereinafter referred to as “6′-”.
  • the fatty acid is a fatty acid having 14 to 18 carbon atoms excluding sphingolipid, and an amide at the terminal other than the sialic acid terminal of the sialic acid-containing sugar chain.
  • An example of a method for producing such a sialic acid-containing sugar chain complex will be described with reference to an example of binding.
  • the aforementioned 3′-SL, 6′-SL, 3′-SLN, and 6′-SLN may be collectively referred to as “sialyl oligosaccharide”.
  • 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 such as 3′-SL, 6′-SL, 3′-SLN, 6′-SLN is not particularly limited and may be appropriately selected depending on the intended purpose.
  • human, bovine, etc. Sialyl oligosaccharides derived from mammals can be used.
  • 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. Since the sialic acid-containing sugar chain complex is a substance constructed from natural materials, it is advantageous in terms of high safety when used for an anti-influenza virus agent described later.
  • step (I) the sialyl oligosaccharide and ammonium hydrogen carbonate are reacted.
  • step (II) the sialyl oligosaccharide and ammonium hydrogen carbonate are reacted. More specifically, for example, it can be performed as follows.
  • step (I) is a step of reacting sialyl oligosaccharide with ammonium hydrogen carbonate.
  • the method for the reaction is not particularly limited and may be appropriately selected depending on the intended purpose. The method of making it, etc. are mentioned.
  • room temperature means 10 ° C. to 40 ° C., preferably 20 ° C. to 25 ° C.
  • the progress of the reaction between the sialyl oligosaccharide and ammonium hydrogen carbonate in the step (I) can be confirmed, for example, by thin layer chromatography (TLC).
  • the reaction solution obtained by the reaction may be subjected to the following step (II) after removing ammonium hydrogen carbonate together with moisture using, for example, a rotary evaporator. From the viewpoint of good reaction efficiency.
  • the step (II) is a step of reacting the reaction product obtained in the step (I) with a fatty acid.
  • the reaction method is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the reaction product obtained in the step (I) and a chlorinated fatty acid are subjected to a 0 ° C. condition in the presence of sodium carbonate.
  • the method of reacting the reaction product obtained in the step (I) with a fatty acid in the presence of a condensing agent under room temperature conditions is a sialyl oligosaccharide fatty acid in a short reaction time. Amides can be obtained, which is preferable in terms of good reaction efficiency.
  • the amount of the chlorinated fatty acid is not particularly limited, and is appropriately determined depending on the purpose. For example, it can be used in an amount such that the molar ratio of sialyl oligosaccharide is 5: 1 with respect to the amount of sialyl oligosaccharide used.
  • the condensing agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include carbodiimides such as WSC and DCC; and triazine-based condensations such as DMT-MM. These may be used alone or in combination of two or more. Among these, the condensing agent is preferable because DMT-MM can obtain a sialyl oligosaccharide fatty acid amide in a short reaction time and has good reaction efficiency.
  • Amides can be obtained.
  • Sialyl oligosaccharide fatty acid amides in which the carboxyl group of sialic acid is amidated and sialyl oligosaccharide fatty acid amides in which the carboxyl group of sialic acid is not amidated are known by anion exchange chromatography, silica gel column chromatography, etc. It can be separated by this method.
  • the sialyl oligosaccharide fatty acid amide is preferably further purified.
  • the method for purifying the sialyl oligosaccharide fatty acid amide is not particularly limited and may be appropriately selected depending on the intended purpose.
  • Examples thereof include Sephadex LH-20 (GE Healthcare Biosciences), Sephadex G-10 ( GE Healthcare Biosciences Co., Ltd., BioGel P-2 (Nippon Bio-Rad Laboratories Co., Ltd.), silica gel (for example, Iatrobeads 6RS 8060 (Mitsubishi Chemical Yatron Co., Ltd.)), C18 reverse phase column (Preparative C18 Bulk Packing Materials ( And a method of performing various column chromatography using Waters)) as a carrier.
  • the solvent in the reaction solution obtained in the step (II) is removed, and an equal amount of hexane is added to and mixed with the reaction solution, followed by centrifugation and removal of the supernatant. It is preferable to perform the washing in that the unreacted fatty acid can be removed.
  • the number of times of washing is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 2 times or more, and more preferably 2 to 3 times.
  • the method for confirming the sialic acid-containing sugar chain complex obtained by the above production method is not particularly limited and may be appropriately selected depending on the intended purpose.
  • thin layer chromatography, high performance liquid chromatography, mass Analytical methods such as analyzer, proton NMR, LC / MS (liquid chromatography / mass spectrometry) and the like can be mentioned.
  • the said manufacturing method can manufacture suitably the said sialic acid containing sugar_chain
  • the anti-influenza virus agent of the present invention contains at least the sialic acid-containing sugar chain complex of the present invention, and further contains other components as necessary.
  • 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 present in the envelope of influenza virus, and recognizes and binds to a sugar chain containing sialic acid of a complex carbohydrate present on the surface of a target cell membrane 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.
  • hemagglutinin recognition site of the sialic acid-containing sugar chain examples include 3′-sialylgalactose chain (3′-Neu5Ac ( ⁇ 2-3) Gal) and 6′-sialylgalactose chain (6′- Any of Neu5Ac ( ⁇ 2-6) Gal) is preferred.
  • 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 mass% to 70 mass% is preferable.
  • the anti-influenza virus agent may be the sialic acid-containing sugar chain complex itself.
  • 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 may be appropriately selected depending on the purpose within a range not impairing the effects of the present invention.
  • examples thereof include pharmaceutically acceptable carriers.
  • the pharmaceutically acceptable carrier is not particularly limited and may be appropriately selected depending on the dosage form of the anti-influenza virus agent.
  • 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.
  • 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.
  • oral solid agent, oral solution, injection, point Examples include nasal sprays, sprays, and inhaled powders.
  • oral solid preparation examples include tablets, coated tablets, granules, powders, capsules and the like.
  • filler and various additives as needed are added to the said sialic acid containing sugar_chain
  • 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.
  • a binder 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.
  • 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.
  • the oral liquid preparation examples include internal liquid preparations, syrups, and elixirs.
  • a manufacturing method of the said oral liquid agent A normal method can be used, For example, it can manufacture by adding an additive to the said sialic acid containing sugar_chain
  • 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
  • the injection examples include a solution, a suspension, and a solid agent for dissolving for use.
  • the method for producing the injection is not particularly limited, and a conventional method can be used. It can be produced by adding a local anesthetic or the like.
  • 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.
  • 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.
  • nasal drops include liquids, sprays, ointments and the like.
  • a normal method can be used, For example, it can manufacture by adding an additive to the said sialic acid containing sugar_chain
  • the additive is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the said anti-influenza virus agent may be used individually by 1 type, may be used together with the chemical
  • medical agent Although it can select suitably according to the objective, Oseltamivir (Tamiflu (trademark)), Zanamivir (relenza (trademark)), Amantadine (Symmetrel (trademark)) Existing anti-influenza virus drugs such as Tamiflu are preferred.
  • the anti-influenza virus agent preferably has anti-influenza virus activity both at the time of influenza virus infection and after infection, but the drug has anti-influenza virus activity mainly in the early stage of infection, and the anti-influenza virus Since the mechanism of action is different from that of a viral agent, the combined use of these agents is advantageous in that it has a synergistic effect.
  • administering There is no restriction
  • 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.
  • 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.
  • influenza virus to which the anti-influenza virus agent is applied is not particularly limited.
  • the anti-influenza virus agent can be expected to suppress infection of all influenza viruses of types A, B, and C, and all types of influenza viruses (H1N1- With regard to the influenza virus H15N9), an infection suppression effect can be expected.
  • the anti-influenza virus agent has excellent anti-influenza virus activity, has no restrictions on the administration time and age of administration, has no side effects, and is highly safe. Therefore, it is suitable for the prevention or treatment of influenza viruses. Is possible.
  • the anti-influenza virus agent can be supported on a substrate, or can be used by supporting it on a filter of the present invention described later.
  • the filter of the present invention carries the anti-influenza virus agent, and further has other configurations as necessary.
  • the amount of the anti-influenza virus agent carried in the filter is not particularly limited and can be appropriately selected depending on the purpose.
  • the method for supporting the anti-influenza virus agent on the filter is not particularly limited and can be appropriately selected depending on the purpose.
  • the anti-influenza virus agent is diluted as it is or with water or alcohol.
  • Examples of the anti-influenza virus agent in a state include a spraying method, a coating method, and a dipping method.
  • the filter for masks the filter for air conditioners, the filter for air cleaners, the filter for vacuum cleaners, the filter for care, the filter for bed mats And wall or ceiling filters.
  • the material used for the filter is not particularly limited and may be appropriately selected depending on the purpose. Examples thereof include polyethylene, polyester, polyamide, polyacryl, polypropylene, nylon, polycarbonate, rayon, and bis-maleimide triazine. Examples include plastic materials; natural fibers such as paper, cotton, hemp, and silk; and inorganic materials such as glass fibers.
  • the form of the filter is not particularly limited and may be appropriately selected according to the purpose. However, when the filter is a mask filter, gauze, non-woven fabric, and cloth are preferable.
  • the filter may have a single layer structure or a multilayer structure.
  • the anti-influenza virus agent only needs to be carried in at least one layer.
  • the filter of the present invention carries the anti-influenza virus agent, it has excellent anti-influenza virus activity, is highly safe, and can be manufactured at a low cost, so that inhalation of influenza virus floating in the air can be achieved. Can be efficiently prevented.
  • 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.
  • 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 fatty acid having a specified carbon number within a specified molecular weight range, It is possible to provide various types of complexes, and therefore, it is expected to provide anti-influenza virus agents 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 the animal species, they are clinically applied as new anti-influenza drugs after amantadine and Tamiflu, which are currently approved for use as anti-influenza drugs. Potential is expected.
  • influenza viruses have the ability to escape the body defense function of antibodies by slightly mutating proteins on the membrane surface.
  • the site of influenza virus that recognizes and binds to the receptor is well conserved, and the high specificity for the receptor is well maintained.
  • 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.
  • 3′-sialyllactose (derived from bovine colostrum, 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 confirmed 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.
  • TLC thin layer chromatography
  • chlorinated lauric acid was used in an amount such 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 intervals of about 30 minutes from the start and confirmed by TLC. 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.
  • TLC was developed with tetrahydrofuran / acetonitrile / 1-propanol / 0.6 M aqueous ammonium acetate / 28% by volume aqueous ammonia (5: 10: 50: 35: 0.3 (volume ratio)).
  • the amount of sialic acid in the obtained 3′-SL—N—C12 and the starting material 3′-SL was quantified with the resorcinol-hydrochloric acid method and after coloration with a densitometer (manufactured by Shimadzu Corporation). However, the yield of 3′-SL—N—C12 was 10% by mass or less.
  • the results of TLC 24 hours after the start of the reaction are shown in FIG.
  • 3′-SL-N-C12 was produced in the same manner as in the conventional method except that the reaction was carried out at room temperature by changing the reaction temperature to 0 ° C. using -4-methylmorpholine chloride (manufactured by Kokusan Chemical Co., Ltd.). The progress of the reaction was sampled at intervals of about 30 minutes from the start and confirmed by thin layer chromatography (TLC).
  • TLC thin layer chromatography
  • the reaction was terminated when the progress of the reaction reached saturation. After removing the solvent from the reaction solution, an equal amount of hexane was added to the reaction solution, and after sufficient stirring, the supernatant was removed by centrifugation. This operation was repeated twice to remove unreacted lauric acid. The reaction was then dissolved in water and loaded onto a C18 reverse phase column (Preparative C18 Bulk Packing Materials (Waters)), methanol: water (5: 5 (volume ratio)), then methanol: water (8 : 2 (volume ratio)), and fractions containing the desired product were collected.
  • TLC was developed with tetrahydrofuran / acetonitrile / 1-propanol / 0.6 M aqueous ammonium acetate / 28% by volume aqueous ammonia (5: 10: 50: 35: 0.3 (volume ratio)).
  • the yield of 3′-SL—N—C12 was determined by the same method as described above, and was 50% by mass. Further, TLC results after 30 minutes and 24 hours from the start of the reaction are shown in FIG. 1, and TLC results 30 minutes after the start of the reaction are shown in FIGS. 3A and 3B.
  • myristic acid (C14) was used in such an amount that the molar ratio was five times that of 3′-SL.
  • DMT-MM was used in an amount that was equivalent in molar ratio to the amount of myristic acid (C14) used.
  • the progress of the reaction was sampled at intervals of about 30 minutes from the start, and TLC (developing solvent: tetrahydrofuran / acetonitrile / 1-propanol / 0.6 M aqueous ammonium acetate solution / 28% by volume aqueous ammonia (5:10:50: 35: 0.3 (volume ratio)), and the reaction was terminated when the progress of the reaction reached saturation, and the result confirmed by TLC is shown in Fig. 2. A high yield was obtained in 30 minutes from the start of the reaction. Obtained.
  • 3′-sialyl lactosyl myristate amide (3′-SL-N—C14: 3′-Neu5Ac ( ⁇ 2-3) Gal ( ⁇ 1-4) Glc-N—C14 represented by the following structural formula, Molecular weight: 842) was obtained.
  • Ac represents an acetyl group.
  • FIG. 3C and FIG. 4 show the results of confirming 3′-SL-N—C14 after purification by TLC in the same manner as described above.
  • “M” indicates a whale brain ganglioside mixed solution (GM4, GM3, GM2, GM1, GD3, GD1a, GD1b, and GT1b) used as a marker.
  • the whale brain ganglioside mixed solution was prepared by the following method. Extracted by adding 5 times volume of chloroform / methanol mixed solution (2: 1 (volume ratio)) to the brain of minke whale (assigned from Japan Whale Research Institute) was further extracted by adding a 5-fold volume chloroform / methanol mixed solution (1: 2 (volume ratio)) to the brain tissue weight to obtain a total lipid fraction. The obtained total lipid fraction was prepared in a chloroform / methanol / water mixed solution (about 30: 60: 8 (volume ratio)), and an anion exchange (DEAE-Sephadex A-25 (GE Healthcare)) column. It was made to adsorb to.
  • the mixture was neutralized by adding an acetic acid / methanol mixed solution so that the amount of acetic acid added was equal to that of the NaOH, and concentrated by evaporating methanol under a nitrogen stream.
  • the dried product was dissolved in a small amount of water, desalted with a Sephadex G-10 column (manufactured by GE Healthcare), and the ganglioside fraction was freeze-dried.
  • This lyophilizate was dissolved in a chloroform / methanol solution (2: 1 (volume ratio)) and used as a standard sample for TLC.
  • the proton NMR spectrum of 3'-SL used as a raw material for producing 3'-SL-N-C14 was measured as proton nuclear magnetic resonance spectrum using acetone as an internal standard in heavy water (D 2 O) at 400 MHz. Is as shown in FIG. As peaks observed in the sialic acid-containing substance, a C3ax peak of sialic acid (Neu5Ac) was observed near 1.8 ppm, and a C3eq peak was observed near 2.75.
  • the anomeric proton peak of C1 of galactose (Gal) is divided around 4.5 ppm, and the anomeric proton peak of glucose (Glc) at the reducing end of the sugar chain is divided into ⁇ and ⁇ , which are around 4.65 ppm, and It was recognized near 5.2 ppm. Further, a C3 proton peak of galactose (Gal) was observed in the vicinity of 4.1 ppm. The peak near 4.7 ppm is a signal derived from water, and the peak near 2.225 ppm is a signal derived from acetone.
  • Production Example 1 a reaction solution containing 3′-SL-N—C16 was used in the same manner as in Production Example 1, except that a reaction solution containing 3′-SL—N—C16 was used instead of the reaction solution containing 3′-SL—N—C14. '-SL-N-C16 was purified. Further, 3′-SL—N—C16 was confirmed by TLC in the same manner as in Production Example 1. The results are shown in FIG. 3B.
  • Production Example 3 Production of 3'-SL-N-C18
  • 3′-sialyllactosyl stearamide (3 ′) represented by the following structural formula was used in the same manner as in Production Example 1 except that stearic acid (C18) was used instead of lauric acid.
  • -SL-N-C18 3'-Neu5Ac ( ⁇ 2-3) Gal ( ⁇ 1-4) Glc-N-C18, molecular weight: 898).
  • “Ac” represents an acetyl group.
  • a peak of methyl proton was observed around 0.9 ppm due to the introduction of stearic acid. Further, a methylene proton peak was observed near 1.3 ppm, an ⁇ -carbon proton peak was observed near 2.1 ppm, and a ⁇ -carbon proton peak was observed near a low magnetic field shift near 1.5 ppm. From the sum of the signals of the methylene protons and the signal of the methyl protons, it was confirmed to be 3′-SL—N—C18. The peak around 3.4 ppm is a signal derived from heavy water (DHO).
  • Production Example 4 Production of 3'-SLN-N-C14
  • 3′-sialyl-N-acetyllactosamine (3′-SLN) (manufactured by Sigma-Aldrich, molecular weight: 674) was used instead of 3′-sialyl lactose (3′-SL).
  • 3′-sialyllactosaminyl myristic acid amide (3′-SLN—N—C14: 3′-Neu5Ac ( ⁇ 2-3) Gal ( ⁇ 1 -4) GlcNAc-N-C14, molecular weight: 883) was obtained.
  • “Ac” represents an acetyl group.
  • Production Example 5 Production of 6′-SLN—N—C14 and 6′-S (1NH 2 ) LN—N—C14
  • 6′-sialyl-N-acetyllactosamine (6′-SLN) (manufactured by Sigma-Aldrich, molecular weight: 674) was used instead of 3′-sialyl lactose (3′-SL).
  • 6′-sialyllactosaminyl myristic acid amide (6′-SLN—N—C14: 6′-Neu5Ac ( ⁇ 2-6) Gal ( ⁇ 1) represented by the following structural formula.
  • 6′-SLN—N—C14 and 6′-S (1NH 2 ) LN—N—C14 were purified in the same manner as in Production Example 1. Further, 6′-SLN—N—C14 and 6′-S (1NH 2 ) LN—N—C14 were confirmed by TLC in the same manner as in Production Example 1. The results are shown in FIG. As shown in FIG. 4, when the carboxyl group of sialic acid is amidated, the polarity is lowered, and thus the mobility is increased. Further, LC / MS measurement was performed on 6′-SLN—N—C14 and 6′-S (1NH 2 ) LN—N—C14 under the following conditions. The spectrum of 6′-SLN-N—C14 is shown in FIG.
  • the proton nuclear magnetic resonance spectrum was measured in deuterated dimethyl sulfoxide (DMSO-d6) containing 2 % by mass heavy water (D 2 O) at 600 MHz with TMS as an internal standard.
  • DMSO-d6 deuterated dimethyl sulfoxide
  • D 2 O mass heavy water
  • the proton NMR spectrum of 6′-SLN—N—C14 is as shown in FIG. 8C
  • the proton NMR spectrum of 6′-S (1NH 2 ) LN—N—C14 is as shown in FIG. 8D.
  • FIG. 9 shows a proton NMR spectrum measured in heavy water (D 2 O).
  • Production Example 6 Production of 6′-SL—N—C14 and 6′-S (1NH 2 ) LN—C14
  • 6′-sialyl lactose (6′-SL) (derived from bovine colostrum, Sigma-Aldrich, molecular weight: 655) was used instead of 3′-sialyl lactose (3′-SL).
  • 6′-sialyl lactosyl myristate amide (6′-SL—N—C14: 6′-Neu5Ac ( ⁇ 2-6) Gal ( ⁇ 1- 4) Glc-N-C14, molecular weight: 842), and 6'-sialylamidolactosylmyristic acid amide (6'-S (1NH 2 ) LN-C14: 6'-Neu5Ac1NH 2 ( ⁇ 2-6) Gal ( ⁇ 1-4) Glc-N-C14, molecular weight: 841) was obtained.
  • Ac represents an acetyl group.
  • Production Example 1 a reaction solution containing 6′-SL—N—C14 and 6′-S (1NH 2 ) L—N—C14 was used in place of the reaction solution containing 3′-SL—N—C14. Except for this, 6′-SL—N—C14 and 6′-S (1NH 2 ) L—N—C14 were purified in the same manner as in Production Example 1. 6′-SL—N—C14 was confirmed by TLC in the same manner as in Production Example 1. The results are shown in FIG. LC / MS analysis was performed on 6′-SL—N—C14 and 6′-S (1NH 2 ) LN—C14 under the same conditions as in Production Example 5.
  • FIG. 11 shows the proton NMR spectrum measured in heavy water (D 2 O).
  • Production Example 7 Production of 6′-SL—N—C18 and 6′-S (1NH 2 ) LN—C18
  • 6′-sialyl lactose (6′-SL) (derived from bovine colostrum, Sigma-Aldrich, molecular weight: 655) was used instead of 3′-sialyl lactose (3′-SL).
  • 6′-sialyllactosyl stearic acid amide (6′-SL—N—C18: 6′-Neu5Ac ( ⁇ 2-6) Gal ( ⁇ 1- 4) Glc-N-C18, molecular weight: 898), and 6′-sialylamidolactosyl stearamide (6′-S (1NH 2 ) LN-C18: 6′-Neu5Ac1NH 2 ( ⁇ 2-6) Gal ( ⁇ 1-4) Glc-N-C18, molecular weight: 897) was obtained.
  • “Ac” represents an acetyl group.
  • Production Example 8 Production of 6'-SLN-N-C18 and 6'-S (1NH 2 ) LN-N-C18
  • 6′-sialyl-N-acetyllactosamine (6′-SLN) (manufactured by Sigma-Aldrich, molecular weight: 674) was used instead of 3′-sialyl lactose (3′-SL).
  • 6′-sialyllactosaminyl stearic acid amide (6′-SLN—N—C18: 6′-Neu5Ac ( ⁇ 2-6) Gal ( ⁇ 1) represented by the following structural formula.
  • 6′-SLN—N—C18 and 6′-S (1NH 2 ) LN—N—C18 were purified in the same manner as in Production Example 1.
  • the proton nuclear magnetic resonance spectrum was measured in deuterated dimethyl sulfoxide (DMSO-d6) containing 2 % by mass heavy water (D 2 O) at 600 MHz with TMS as an internal standard.
  • the proton NMR spectrum of 6′-SLN—N—C18 is as shown in FIG. 13A
  • the proton NMR spectrum of 6′-S (1NH 2 ) LN—N—C18 is as shown in FIG. 13B. From FIG.
  • sialic acid-containing sugar chain complexes synthesized in Production Examples 1 to 8 and Comparative Production Example 1 are shown below.
  • Test Example 2 Examination of anti-influenza virus activity by sialic acid-containing sugar chain complex (in vitro)) ⁇ Action after infection of sialic acid-containing sugar chain complex>
  • the anti-influenza virus activity of each sialic acid-containing sugar chain complex shown in Table 2 was evaluated by plaque measurement (PFU Assay) as follows. MDCK cells (NBL-2 cells, canine kidney cells) (manufactured by Dainippon Sumitomo Pharma Co., Ltd.) were added to E-MEM medium (EIB-Minimum Essential Medium) (GIBCO; Invitrogen Corporation) containing 10% by volume fetal bovine serum (FBS). In the microplate to which the product was added at 37 ° C. and 5% CO 2 .
  • E-MEM medium EIB-Minimum Essential Medium
  • FBS fetal bovine serum
  • Influenza virus A / PR / 8/34 strain (ATCC VR-95) (H1N1 type) was added to the monolayer MDCK cells at 100 PFU / mL and incubated at 37 ° C. for 1 hour for infection. .
  • the culture solution containing the influenza virus used for the infection was removed, and each sialic acid-containing sugar chain complex shown in Table 2 and oseltamivir (Tamiflu (registered trademark), manufactured by Roche), zanamivir as existing drugs (Relenza (registered trademark), manufactured by GlaxoSmithKline) and amantadine hydrochloride (manufactured by Sigma-Aldrich) in a range of 0.1 ⁇ g / mL to 500 ⁇ g / mL, respectively, are added to the microplate.
  • influenza virus growth inhibition rate (%) 100 ⁇ (number of plaques at the time of addition of sialic acid-containing sugar chain complex / number of plaques for control) ⁇ 100
  • the anti-influenza virus activity of the sialic acid-containing sugar chain complex increases as the carbon number increases.
  • the IC 50 value of the sialic acid-containing glycan complex is anti-influenza virus activity at 50 to 100-fold concentration, which is equivalent to that of amantadine hydrochloride. It was.
  • MDCK cells were prepared in a monolayer by the same method as described above.
  • influenza virus A / PR / 8/34 strain (ATCC VR-95) (H1N1 type) was added so as to be 100 PFU / mL, and each sialic acid-containing sugar chain complex shown in Table 3 LN-C14 obtained in Comparative Production Example 1 was added in the range of 0.1 ⁇ g / mL to 500 ⁇ g / mL as a control for existing drugs and sialic acid-containing sugar chain complexes, respectively, and incubated at 37 ° C. for 1 hour. I was infected.
  • sialic acid-containing sugar chain complex is a substance constructed from natural materials, it was suggested that the sialic acid-containing sugar chain complex is highly safe even at high concentrations.
  • ⁇ Repeated dose test> ICR mice (male and female, 6-week-old SPF, n 6, Nippon Charles River Co., Ltd.) were treated with a sialic acid-containing sugar chain complex (3′-SL-N-C14) at a dose of 1.5 mg / kg. Repeated intravenous administration once a day for 7 days, the animals were raised for a total of 7 days. An animal administered with PBS instead of the sialic acid-containing sugar chain complex was used as a control. During the breeding period, general symptoms were observed by measuring body weight, hematology, blood chemistry, and the like. As a result, there were no deaths, no effects on the general symptoms were observed compared to control animals, and no toxicity was observed.
  • oral administration group and virus administration group of sialic acid-containing sugar chain complex measurement of inflammatory cytokine in serum and measurement of virus titer in lung tissue by the following methods Went.
  • the glycoconjugate was found not to be toxic.
  • the survival rate results are shown in FIG. 14B.
  • the oral administration group of the sialic acid-containing sugar chain complex (3′-SL-N-C14 oral administration group) was found to have a significant life-prolonging effect with respect to the virus administration group.
  • the nasal administration group (3′-SL-N-C14 nasal administration group) of the sialic acid-containing sugar chain complex, suppression of weight loss was not observed, but the survival rate was compared with the virus administration group. As a result, a life-prolonging effect was recognized.
  • IL-6 and INF- ⁇ were measured by the following method. On the third day after inoculation with the influenza virus, blood of each animal was collected, and serum was obtained by centrifugation at 3000 rpm for 10 minutes at 4 ° C. Using this serum, the concentration of IL-6 was measured by ELISA (Mouse IL-6 Immunoassay: manufactured by Quantikine R & D SYSTEMS).
  • the serum was used to measure the concentration of INF- ⁇ using ELISA (Mouse IFN- ⁇ Immunoassay: manufactured by Quantikine R & D SYSTEMS).
  • the measurement result of IL-6 is shown in FIG. 14C
  • the measurement result of INF- ⁇ is shown in FIG. 14D.
  • suppression of inflammatory cytokine production induced by influenza virus infection was recognized by the sialic acid-containing sugar chain complex.
  • the inhibitory action on inflammatory cytokine production was significant in the 3′-SL-N—C14 oral administration group.
  • the statistical analysis was performed by t test (Student's t-test) (p ⁇ 0.05).
  • the influenza virus in the lung tissue was extracted, and the virus titer was measured by plaque measurement (PFU Assay). That is, MDCK cells prepared by the same method as in Test Example 2 were inoculated with a diluted series of influenza viruses in the lung tissue of each group and incubated at 37 ° C. for 1 hour to be infected.
  • the virus titer in the lung tissue was clearly decreased on the second day of infection in both the 3′-SL-N-C14 oral administration group and the 3′-SL-N-C14 nasal administration group. It was.
  • the statistical analysis was performed by t test (Student's t-test) (p ⁇ 0.05).
  • IL-6 As an inflammatory cytokine, the concentration of IL-6 was measured by the same method as described above. The measurement result of IL-6 is shown in FIG. 15B. From this result, suppression of IL-6 production was particularly strongly observed in 3′-SL-N—C14 and 6′-S (1NH 2 ) LN—C14, which are sialic acids with strong antiviral activity. It was found to be a sugar chain complex containing.
  • influenza virus infection process that is, each sialic acid-containing sugar chain complex was allowed to act during or after influenza virus infection.
  • the virus infection suppression effect was evaluated by plaque measurement (PFU Assay).
  • Test Example 2 the sialic acid-containing sugar chain complex was replaced with each sialic acid-containing sugar chain complex shown in Table 4, and the addition time of each sialic acid-containing sugar chain complex was according to the time shown in Table 4 Except for the above, IC 50 values were calculated in the same manner as in Test Example 2. The results are also shown in Table 4. Table 4 shows the average values obtained by performing the test 1 to 3 times.
  • Sialic acid-containing glycan complexes are affected at the time of infection or after infection because any sialic acid-containing glycan complex acts on hemagglutinin of influenza virus, especially at both the time of infection and after infection. It was found that the anti-influenza virus activity was enhanced by the action. Moreover, the sialic acid-containing sugar chain complex in which the carboxyl group was amidated tended to increase the anti-influenza virus activity by amidation.
  • anti-influenza virus activity was observed in any of the isolates, both at the time of influenza virus infection and after infection, as in A / PR / 8/34.
  • MDCK cells were cultured in the same manner as in Test Example 2, and the MDCK cells in a monolayer were infected with influenza virus A / PR / 8/34 strain in the same manner as in Test Example 2. After incubation at 37 ° C. for 1 hour, the culture solution containing influenza virus was removed, and after each time (1 to 28 hours) shown in Table 6 below, 3′-SL-N-C16 (50 ⁇ g / mL) or Tamiflu The agarose solution to which (1 ⁇ g / mL) was added was layered on the microplate and completely coagulated, followed by culturing at 37 ° C. under 5% CO 2 for 3 days. From the number of plaques formed in MDCK cells, the amount of free virus (PFU / mL) at each time was calculated. The results are shown in Table 6 below.
  • the sialic acid-containing sugar chain complex shows strong anti-influenza virus activity when added both at the time of infection and after infection, and in particular, when added after 16 hours, a tendency that the effect is clearly strengthened was confirmed. The effect was stronger than the existing drug Tamiflu. It has been reported that mature influenza virus appears after 6 hours after influenza virus infects cells and is almost completed by about 15 hours (Tamiflu (Pharmaceutical Interview Form: Chugai Pharmaceutical Co., Ltd.), Relenza (Pharmaceuticals) Interview form: GlaxoSmithKline Co., Ltd.)))) suggests that the sialic acid-containing sugar chain complex has the action of suppressing the growth of mature influenza virus and preventing reinfection.
  • Tamiflu and Relenza are selective neuraminidase inhibitors, it is thought that mature influenza virus is released from infected cells and acts on the final process of proliferation (Tamiflu (Pharmaceutical Interview Form: Chugai Pharmaceutical Co., Ltd.)) Relenza (see pharmaceutical interview form: GlaxoSmithKline, Inc.). Therefore, the sialic acid-containing sugar chain complex is advantageous in that it can suppress influenza virus activity at a stage prior to Tamiflu and Relenza.
  • Test Example 8 Anti-influenza virus effect of nonwoven fabric impregnated with sialic acid-containing sugar chain complex
  • Six general nonwoven fabrics used in the masks shown in Table 7 below were impregnated with 50 ⁇ g / cm 2 of each of the sialic acid-containing sugar chain complexes shown in Table 7 below and dried, and then the nonwoven fabrics were treated with influenza.
  • Virus (A / PR / 8/34) was allowed to act at 1.0 ⁇ 10 6 PFU / mL. After leaving still in the safety cabinet for 1 hour at room temperature, PBS was added to each nonwoven fabric, and influenza viruses were collect
  • MDCK cells prepared by the same method as in Test Example 2 were inoculated with a dilution series of the virus solution and incubated at 37 ° C. for 1 hour to be infected. After culturing, the agarose solution was overlaid and completely coagulated, and then cultured at 37 ° C. under 5% CO 2 for 3 days, and the influenza virus growth inhibition rate of each impregnated nonwoven fabric was measured. As a control, various nonwoven fabrics that were not impregnated with the sialic acid-containing sugar chain complex were used. The results are shown in Table 7.
  • Influenza virus (A / PR / 8/34) and the sialic acid-containing sugar chain complexes shown in Table 8 below were mixed according to the volumes shown in Table 8 and allowed to stand at room temperature for 10 minutes or 60 minutes.
  • the proliferation of these influenza viruses was measured by the plaque method (PFU Assay). That is, 100 PFU / mL of the influenza virus was added to MDCK cells prepared in the same manner as in Test Example 2, and the mixture was incubated at 37 ° C. for 1 hour for infection. After culturing, the agarose solution was overlaid and completely coagulated, then cultured at 37 ° C. under 5% CO 2 for 3 days, and the growth inhibition rate of influenza virus was measured respectively. The results are shown in Table 8.
  • PAEA-3′-SL was synthesized by performing a PAEA-forming reaction below 3′-sialyl lactose by the following method.
  • 3 ⁇ -sialyllactose (3′-SL) Sigma Aldrich 15 mg (24 ⁇ mol)
  • PAEA Wi- Pure Chemical Industries, Ltd.
  • 600 ⁇ L ethanol / 0.32 M hydroxylated Sodium (9: 1 (volume ratio) was added, and 8.3 mg (26 ⁇ mol) of DMT-MM (manufactured by Kokusan Chemical Co., Ltd.) (12.90% moisture) was added, and then left at room temperature for 3 hours.
  • a 3′-SL derivative (PAEA-3′-SL) was obtained.
  • “ ⁇ ” indicates that sialidase treatment has not been performed, and “+” indicates that sialidase treatment has been performed.
  • “M” indicates a whale brain ganglioside mixed solution (GM4, GM3, GM2, GM1, GD3, GD1a, GD1b, and GT1b) used as a marker. From the results shown in FIG. 16, 6′-S (1NH 2 ) LN—N—C18, in which the carboxyl group of sialic acid is amidated, has no change in mobility depending on the presence or absence of sialidase treatment, and exhibits sialidase resistance. all right.
  • PAEA-3′-SL in which the carboxyl group of sialic acid was modified by an amidation reaction by the above method, was similarly resistant to sialidase.
  • 6'-SLN-N-C18 in which the carboxyl group of sialic acid was not amidated was decomposed by sialidase treatment, and bands were detected in the vicinity of GT1b and at a slightly higher mobility than GM4.
  • the band near GT1b is free Neu5Ac cleaved from 6'-SLN-N-C18, and the band slightly more mobile than GM4 is lactosaminyl stearamide (LN-N-C18).
  • the sialic acid-containing sugar chain complex of the present invention has excellent anti-influenza virus activity, it can be suitably used as an anti-influenza virus agent. Moreover, since the method for producing a sialic acid-containing sugar chain complex of the present invention has a short reaction time and good reaction efficiency, the sialic acid-containing sugar chain complex can be efficiently produced. Furthermore, since the anti-influenza virus agent of the present invention contains the sialic acid-containing sugar chain complex, it has excellent anti-influenza virus activity, there is no restriction on the administration time, the age of the administration subject, etc., and there are no side effects. It can be suitably used for prevention or treatment of influenza, which is highly safe.

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Abstract

A sialic acid-containing sugar chain complex characterized by comprising a C14-18 fatty acid, excluding a sphingolipid, that is bonded, via an amide bond, to an end other than the sialic acid end of a sialic acid-containing sugar chain, which contains sialic acid at one end, and having a molecular weight of 400-1,400; an anti-influenza virus agent which comprises said complex as the active ingredient; and a filter which carries said anti-influenza virus agent.

Description

シアル酸含有糖鎖複合体及びその製造方法、並びに、抗インフルエンザウイルス剤、及びフィルターSialic acid-containing sugar chain complex, method for producing the same, anti-influenza virus agent, and filter
　本発明は、新規なシアル酸含有糖鎖複合体及びその効率的な製造方法、並びに、該シアル酸含有糖鎖複合体を有効成分として含有する抗インフルエンザウイルス剤、及び該抗インフルエンザウイルス剤を担持したフィルターに関する。 The present invention provides a novel sialic acid-containing sugar chain complex, an efficient production method thereof, an anti-influenza virus agent containing the sialic acid-containing sugar chain complex as an active ingredient, and the anti-influenza virus agent Related to the filter.
　インフルエンザウイルスは、そのエンベロープに存在するスパイクタンパク質の１つであるヘマグルチニン（ＨＡ）が、標的細胞の細胞膜表面に存在する複合糖質のシアル酸を含む糖鎖を認識し、該糖鎖に結合して、標的細胞への感染を成立させることが一般に知られている。
　また、鳥インフルエンザウイルスは、Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ－といった糖鎖を、ヒトインフルエンザウイルスは、Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ－といった糖鎖をそれぞれ特異的に認識し、このことがインフルエンザウイルス感染の特異性を作り出す要因であると考えられている。 In influenza virus, hemagglutinin (HA), one of the spike proteins in the envelope, recognizes sugar chains containing sialic acids of complex carbohydrates 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 the specificity of
　現在、インフルエンザの予防又は治療方法としては、ワクチンの予防接種や、抗インフルエンザ薬として使用認可されているアマンタジン（シンメトレル（登録商標））やオセルタミビル（タミフル（登録商標））の投与が主である。
　しかし、これらの予防又は治療方法には、それぞれ一長一短があることが既に知られている。例えば、ワクチンは、該ワクチンの接種により産生されたＩｇＡが、呼吸器系の粘液中に分泌されることにより、ウイルスの細胞への付着を防ぐことができるが、タイプの異なる新型ウイルスに対しては無力であることが知られている。また、アマンタジンは、インフルエンザウイルスのＭ２タンパク質のイオンチャンネル作用を阻害し、標的細胞に侵入した後のインフルエンザウイルスの脱殻を抑制することができるが、Ｍ２タンパク質のないＢ型、Ｃ型ウイルスに対する効果はなく、強い副作用や耐性菌の発現が問題とされている。また、タミフルは、インフルエンザウイルスのエンベロープに存在するスパイクタンパク質の１つであるノイラミニダーゼ（ＮＡ）の作用を阻害し、複製されたインフルエンザウイルスが感染した標的細胞から遊離して他の細胞に感染を広げることを抑制することができるが、その効果の発現のためには、感染初期（４８時間以内）の服用が必須とされ、また、近年、若年者に対する副作用が問題となっている。 At present, the main methods for preventing or treating influenza include vaccination and administration of amantadine (Symmetrel (registered trademark)) and oseltamivir (Tamiflu (registered trademark)) that have been 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, a vaccine can prevent the attachment of virus to cells by secreting IgA produced by vaccination into the mucus of the respiratory system, but against different types of new viruses Is known to be powerless. Amantadine inhibits the ion channel action of the M2 protein of influenza virus and can suppress the unhulling of the influenza virus after entering 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 in the influenza virus envelope, and the replicated influenza virus is released from infected target cells and spreads to other cells. Although it can be suppressed, in order to manifest its effects, it is essential to take it at the beginning of infection (within 48 hours), and in recent years, side effects on young people have become a problem.
　前記従来のワクチンや抗インフルエンザ薬の問題点を克服できるような、新たなインフルエンザの予防又は治療方法の研究開発は、広く行われている。例えば、ウイルス受容体の被認識部位を含む糖鎖を持つスフィンゴ糖脂質を有効成分とする抗インフルエンザウイルス剤が報告されている（特許文献１参照）。具体的には、シアル酸を含むスフィンゴ糖脂質であるガングリオシド類のうち、天然物から抽出したＧＭ４（Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ－Ｃｅｒ）、ＧＭ３（Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇｌｃ－Ｃｅｒ）、ＧＤ１ａ（Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－３）ＧａｌＮＡｃ（β１－４）（Ｎｅｕ５Ａｃ（α２－３））Ｇａｌ（β１－４）Ｇｌｃ－Ｃｅｒ）、及びＳＰＧ（シアリルパラグロボシド：Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）ＧｌｃＮＡｃ（β１－４）Ｇａｌ（β１－４）Ｇｌｃ－Ｃｅｒ）に、インフルエンザウイルスに対する感染阻止活性が認められ、その感染阻止力の強さは、ＳＰＧ＞ＧＤ１ａ＞ＧＭ３＞ＧＭ４の順であったことが開示されている。 Research and development of new methods for preventing or treating influenza that can overcome the problems of the conventional vaccines and anti-influenza drugs have been widely conducted. For example, an anti-influenza virus agent containing a glycosphingolipid having a sugar chain containing a recognition site of a virus receptor as an active ingredient has been reported (see Patent Document 1). Specifically, among gangliosides that are glycosphingolipids containing sialic acid, GM4 (Neu5Ac (α2-3) Gal-Cer) and GM3 (Neu5Ac (α2-3) Gal (β1-4) extracted from natural products. ) Glc-Cer), GD1a (Neu5Ac (α2-3) Gal (β1-3) GalNAc (β1-4) (Neu5Ac (α2-3)) Gal (β1-4) Glc-Cer), and SPG (sialylpara) Globoside: Neu5Ac (α2-3) Gal (β1-4) GlcNAc (β1-4) Gal (β1-4) Glc-Cer) has an anti-infection activity against influenza virus, and its infection-preventing power is strong Is disclosed in the order of SPG> GD1a> GM3> GM4.
　しかし、前記特許文献１に開示された抗インフルエンザウイルス剤は、未だ実用化には至っておらず、また、前記抗インフルエンザウイルス剤として開示されたガングリオシド類はミンククジラの脳やヒト赤血球といった天然物から抽出されたものであることから、抗インフルエンザ薬としての実用化には有効成分の大量調製が望まれる点を鑑みると、やや困難があるのではないかと考えられる。
　また、インフルエンザの流行は大きな問題であり、現在使用されているアマンタジンやタミフルなどの既存薬も、前記したようにそれぞれ副作用や服用時期の制限、耐性株の出現の危険性などといった欠点を有している。 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 amount of an active ingredient is desired for practical use as an anti-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 have been used in the past, have drawbacks such as side effects, restrictions on the timing of administration, and the risk of the emergence of resistant strains. ing.
　また、本出願人らは、以前にシアル酸含有糖鎖複合体が抗インフルエンザウイルス活性を有することを見出した（特許文献２参照）。
　しかし、この報告における実施例では脂肪酸アミドの脂肪酸の炭素数が１２のシアル酸含有糖鎖複合体しか開示されておらず、脂肪酸アミドの脂肪酸の炭素数が１２のシアル酸含有糖鎖複合体では抗インフルエンザウイルス活性が不十分であり、更なる抗インフルエンザウイルス活性の向上が望まれていた。 In addition, the present applicants have previously found that sialic acid-containing sugar chain complexes have anti-influenza virus activity (see Patent Document 2).
However, in the examples in this report, only the sialic acid-containing sugar chain complex having 12 carbon atoms of the fatty acid amide is disclosed, and the sialic acid-containing sugar chain complex having 12 carbon atoms of the fatty acid amide is disclosed. Anti-influenza virus activity is insufficient, and further improvement of anti-influenza virus activity has been desired.
　したがって、優れた抗インフルエンザウイルス活性を有し、投与時期や投与対象の年齢などに制限がなく、かつ副作用がなく安全性の高い、新たな抗インフルエンザウイルス剤についての早急な研究開発及び実用化が望まれているのが現状である。 Therefore, rapid research and development and practical application of a new anti-influenza virus agent that has excellent anti-influenza virus activity, has no restrictions on the administration time and age of administration, has no side effects, and is highly safe. What is desired is the current situation.
特開２００１－２３３７７３号公報JP 2001-233773 A 特開２００７－３０８４４４号公報JP 2007-308444 A
　本発明は、従来における前記諸問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、抗インフルエンザウイルス活性を有する、新規なシアル酸含有糖鎖複合体及びその効率的な製造方法、並びに、該シアル酸含有糖鎖複合体を含有し、優れた抗インフルエンザウイルス活性を有し、投与時期や投与対象の年齢などに制限がなく、かつ副作用がなく安全性の高い抗インフルエンザウイルス剤、及び前記抗インフルエンザウイルス剤を担持したフィルターを提供することを目的とする。 This invention makes it a subject to solve the said various problems in the past and to achieve the following objectives. 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 is an object of the present invention to provide a highly safe anti-influenza virus agent that has no side effects and has no side effects and has high side effects, and a filter carrying the anti-influenza virus agent.
　標的細胞が有する成分を認識して標的細胞に結合し、感染を成立させるウイルスや細菌に対しては、前記ウイルスや細菌に認識される標的細胞の糖鎖と同程度の、又は、それ以上に強い親和性を有する成分を外部から添加すれば、前記ウイルスや細菌による感染が阻害されると考えられる。
　ところが、前記ヘマグルチニン（ＨＡ）認識部位を有する糖鎖であるにもかかわらず、シアリルオリゴ糖である、３’－シアリルラクトース（３’－ＳＬ：３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇｌｃ）及び３’－シアリルラクトサミン（３’－ＳＬＮ：３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）ＧｌｃＮＡｃ）は、抗インフルエンザウイルス活性を示さなかった。 For viruses and bacteria that recognize the components of the target cell, bind to the target cell, and establish infection, the sugar chain of the target cell recognized by the virus or bacteria is equal to or higher than that. If a component having a strong 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, 3'-sialyl lactose (3'-SL: 3'-Neu5Ac (α2-3) Gal (β1- 4) Glc) and 3′-sialyllactosamine (3′-SLN: 3′-Neu5Ac (α2-3) Gal (β1-4) GlcNAc) did not show anti-influenza virus activity.
　前記課題を解決するため、本発明者らは鋭意検討した結果、以下のような知見を得た。即ち、抗インフルエンザウイルス活性を示さなかったシアリルオリゴ糖に対して、一定の分子量を有し、一定の炭素数の脂肪酸を結合させた、シアリルオリゴ糖と脂肪酸との複合体を新たに作製したところ、得られた複合体は、優れた抗インフルエンザウイルス活性を示すようになったことを見出し、本発明の完成に至った。 In order to solve the above-mentioned problems, the present inventors have made extensive studies and obtained the following knowledge. That is, when a sialyl oligosaccharide that did not exhibit anti-influenza virus activity and has a certain molecular weight and a fatty acid having a certain number of carbon atoms bound thereto, a complex of the sialyl oligosaccharide and the fatty acid was newly prepared. The obtained complex was found to show excellent anti-influenza virus activity, and the present invention was completed.
　本発明は、本発明者らによる前記知見に基づくものであり、前記課題を解決するための手段としては、以下の通りである。即ち、
　＜１＞　一方の末端にシアル酸を含有するシアル酸含有糖鎖のシアル酸末端以外の末端に、スフィンゴ脂質を除く炭素数１４～１８の脂肪酸がアミド結合してなり、かつ分子量が４００～１，４００であることを特徴とするシアル酸含有糖鎖複合体である。
　＜２＞　シアル酸のカルボキシル基がアミド化された前記＜１＞に記載のシアル酸含有糖鎖複合体である。
　＜３＞　シアル酸含有糖鎖が、１個～５個の単糖で構成される前記＜１＞から＜２＞のいずれかに記載のシアル酸含有糖鎖複合体である。
　＜４＞　シアル酸含有糖鎖が、Ｎ－アセチルノイラミン酸（Ｎｅｕ５Ａｃ）と、ガラクトース（Ｇａｌ）とからなるシアリルガラクトース鎖（Ｎｅｕ５Ａｃ－Ｇａｌ）を含む前記＜１＞から＜３＞のいずれかに記載のシアル酸含有糖鎖複合体である。
　＜５＞　シアル酸含有糖鎖が、３’－シアリルガラクトース鎖（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ）、及び、６’－シアリルガラクトース鎖（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ）のいずれかを有する前記＜１＞から＜４＞のいずれかに記載のシアル酸含有糖鎖複合体である。
　＜６＞　シアル酸含有糖鎖が、３’－シアリルラクトース鎖（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇｌｃ）、６’－シアリルラクトース鎖（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）Ｇｌｃ）、３’－シアリルラクトサミン鎖（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）ＧｌｃＮＡｃ）、６’－シアリルラクトサミン鎖（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）ＧｌｃＮＡｃ）、３’－シアリルアミドラクトース鎖（３’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－３）Ｇａｌ（β１－４）Ｇｌｃ）、６’－シアリルアミドラクトース鎖（６’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－６）Ｇａｌ（β１－４）Ｇｌｃ）、３’－シアリルアミドラクトサミン鎖（３’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－３）Ｇａｌ（β１－４）ＧｌｃＮＡｃ）、６’－シアリルアミドラクトサミン鎖（６’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－６）Ｇａｌ（β１－４）ＧｌｃＮＡｃ）、３’－シアリルガラクトシルガラクトース鎖（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇａｌ）、６’－シアリルガラクトシルガラクトース鎖（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）Ｇａｌ）、３’－シアリルガラクトシルガラクトサミン鎖（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）ＧａｌＮＡｃ）、及び６’－シアリルガラクトシルガラクトサミン鎖（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）ＧａｌＮＡｃ）からなる群より選択される前記＜１＞から＜５＞のいずれかに記載のシアル酸含有糖鎖複合体である。
　＜７＞　脂肪酸の炭素数が、１６～１８である前記＜１＞から＜６＞のいずれかに記載のシアル酸含有糖鎖複合体である。
　＜８＞　分子量が、７５０～９５０である前記＜１＞から＜７＞のいずれかに記載のシアル酸含有糖鎖複合体である。
　＜９＞　前記＜１＞から＜８＞のいずれかに記載のシアル酸含有糖鎖複合体の製造方法であって、一方の末端にシアル酸を含有するシアル酸含有糖鎖のシアル酸末端以外の末端に、スフィンゴ脂質を除く炭素数１４～１８の脂肪酸を、化学反応により結合させることを特徴とするシアル酸含有糖鎖複合体の製造方法である。
　＜１０＞　シアル酸含有糖鎖のシアル酸末端以外の末端を、アシルアミド化する前記＜９＞に記載のシアル酸含有糖鎖複合体の製造方法である。
　＜１１＞　シアル酸含有糖鎖のシアル酸のカルボキシル基を、アシルアミド化する前記＜９＞から＜１０＞のいずれかに記載のシアル酸含有糖鎖複合体の製造方法である。
　＜１２＞　シアル酸含有糖鎖と炭酸水素アンモニウムとを反応させ、次いで、得られた反応物と脂肪酸とを縮合剤の存在下で反応させる前記＜９＞から＜１１＞のいずれかに記載のシアル酸含有糖鎖複合体の製造方法である。
　＜１３＞　前記＜１＞から＜８＞のいずれかに記載のシアル酸含有糖鎖複合体を有効成分として含有することを特徴とする抗インフルエンザウイルス剤である。
　＜１４＞　シアル酸含有糖鎖複合体を構成するシアル酸含有糖鎖が、インフルエンザウイルスのヘマグルチニン認識部位を有する前記＜１３＞に記載の抗インフルエンザウイルス剤である。
　＜１５＞　インフルエンザウイルスのヘマグルチニン認識部位が、３’－シアリルガラクトース鎖（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ）、及び、６’－シアリルガラクトース鎖（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ）のいずれかである前記＜１３＞から＜１４＞のいずれかに記載の抗インフルエンザウイルス剤である。
　＜１６＞　前記＜１３＞から＜１５＞のいずれかに記載の抗インフルエンザウイルス剤を担持したことを特徴とするフィルターである。 The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> A sialic acid-containing sugar chain containing sialic acid at one end has an amide bond with a fatty acid having 14 to 18 carbon atoms excluding sphingolipid, and a molecular weight of 400 to 1 except for the sphingolipid. , 400, a sialic acid-containing sugar chain complex.
<2> The sialic acid-containing sugar chain complex according to <1>, wherein a carboxyl group of sialic acid is amidated.
<3> The sialic acid-containing sugar chain complex according to any one of <1> to <2>, wherein the sialic acid-containing sugar chain is composed of 1 to 5 monosaccharides.
<4> Any one of <1> to <3>, wherein the sialic acid-containing sugar chain includes a sialylgalactose chain (Neu5Ac-Gal) composed of N-acetylneuraminic acid (Neu5Ac) and galactose (Gal). The sialic acid-containing sugar chain complex described.
<5> The sialic acid-containing sugar chain is a 3′-sialylgalactose chain (3′-Neu5Ac (α2-3) Gal) or a 6′-sialylgalactose chain (6′-Neu5Ac (α2-6) Gal). The sialic acid-containing sugar chain complex according to any one of <1> to <4>, having any of them.
<6> Sialic acid-containing sugar chains are 3′-sialyl lactose chains (3′-Neu5Ac (α2-3) Gal (β1-4) Glc), 6′-sialyl lactose chains (6′-Neu5Ac (α2-6) ) Gal (β1-4) Glc), 3′-Sialyllactosamine chain (3′-Neu5Ac (α2-3) Gal (β1-4) GlcNAc), 6′-Sialyllactosamine chain (6′-Neu5Ac (α2) -6) Gal (β1-4) GlcNAc), 3′-sialylamide lactose chain (3′-Neu5Ac1NH 2 (α2-3) Gal (β1-4) Glc), 6′-sialylamide lactose chain (6′- Neu5Ac1NH 2 (α2-6) Gal (β1-4) Glc), 3′-sialylamidolactosamine chain (3′-Neu5Ac1NH 2 (α2-3) Gal (β1-4) GlcNAc ), 6′-sialylamidolactosamine chain (6′-Neu5Ac1NH 2 (α2-6) Gal (β1-4) GlcNAc), 3′-sialylgalactosylgalactose chain (3′-Neu5Ac (α2-3) Gal (β1 -4) Gal), 6'-sialylgalactosylgalactose chain (6'-Neu5Ac (α2-6) Gal (β1-4) Gal), 3'-sialylgalactosylgalactosamine chain (3'-Neu5Ac (α2-3) Gal (Β1-4) GalNAc) and a 6′-sialylgalactosylgalactosamine chain (6′-Neu5Ac (α2-6) Gal (β1-4) GalNAc) selected from the group <1> to <5> The sialic acid-containing sugar chain complex according to any one of the above.
<7> The sialic acid-containing sugar chain complex according to any one of <1> to <6>, wherein the fatty acid has 16 to 18 carbon atoms.
<8> The sialic acid-containing sugar chain complex according to any one of <1> to <7>, wherein the molecular weight is 750 to 950.
<9> The method for producing a sialic acid-containing sugar chain complex according to any one of <1> to <8>, wherein the sialic acid-containing sugar chain contains sialic acid at one end other than the sialic acid terminal Is a method for producing a sialic acid-containing sugar chain complex, wherein a fatty acid having 14 to 18 carbon atoms excluding sphingolipid is bonded to the end of the saccharide by a chemical reaction.
<10> The method for producing a sialic acid-containing sugar chain complex according to <9>, wherein a terminal other than the sialic acid terminal of the sialic acid-containing sugar chain is acylamidated.
<11> The method for producing a sialic acid-containing sugar chain complex according to any one of <9> to <10>, wherein the carboxyl group of the sialic acid of the sialic acid-containing sugar chain is acylamidated.
<12> The sialic acid-containing sugar chain and ammonium hydrogen carbonate are reacted, and then the obtained reaction product and a fatty acid are reacted in the presence of a condensing agent. This is a method for producing a sialic acid-containing sugar chain complex.
<13> An anti-influenza virus agent comprising the sialic acid-containing sugar chain complex according to any one of <1> to <8> as an active ingredient.
<14> The anti-influenza virus agent according to <13>, wherein the sialic acid-containing sugar chain constituting the sialic acid-containing sugar chain complex has a hemagglutinin recognition site for influenza virus.
<15> The hemagglutinin recognition site of influenza virus has 3′-sialylgalactose chain (3′-Neu5Ac (α2-3) Gal) and 6′-sialylgalactose chain (6′-Neu5Ac (α2-6) Gal) The anti-influenza virus agent according to any one of <13> to <14>.
<16> A filter comprising the anti-influenza virus agent according to any one of <13> to <15>.
　本発明によれば、従来における前記諸問題を解決し、前記目的を達成することができ、抗インフルエンザウイルス活性を有する、新規なシアル酸含有糖鎖複合体及びその効率的な製造方法、並びに、該シアル酸含有糖鎖複合体を含有し、優れた抗インフルエンザウイルス活性を有し、投与時期や投与対象の年齢などに制限がなく、かつ副作用がなく安全性の高い抗インフルエンザウイルス剤、及び前記抗インフルエンザウイルス剤を担持したフィルターを提供することができる。 According to the present invention, the above-described conventional problems can be solved, the object can be achieved, and a novel sialic acid-containing sugar chain complex having anti-influenza virus activity, an efficient production method thereof, and The anti-influenza virus agent containing the sialic acid-containing sugar chain complex, having excellent anti-influenza virus activity, having no restrictions on the administration time or age of the administration target, and having no side effects and high safety, and A filter carrying an anti-influenza virus agent can be provided.
図１は、従来の製造方法及び本発明の製造方法で製造したシアル酸含有糖鎖複合体（３’－ＳＬ－Ｎ－Ｃ１２）のＴＬＣの結果を示す図である。FIG. 1 is a diagram showing TLC results of a sialic acid-containing sugar chain complex (3′-SL—N—C12) produced by the conventional production method and the production method of the present invention. 図２は、製造例１で製造したシアル酸含有糖鎖複合体（３’－ＳＬ－Ｎ－Ｃ１４）のＴＬＣの結果を示す図である。FIG. 2 is a diagram showing TLC results of the sialic acid-containing sugar chain complex (3′-SL-N—C14) produced in Production Example 1. 図３Ａは、試験例１において本発明の製造方法で製造したシアル酸含有糖鎖複合体（３’－ＳＬ－Ｎ－Ｃ１２）のＴＬＣの結果を示す図である。「Ｍ」は、マーカーとして使用したクジラ脳ガングリオシド混合液を示す。FIG. 3A is a view showing a TLC result of a sialic acid-containing sugar chain complex (3′-SL—N—C12) produced by the production method of the present invention in Test Example 1. “M” indicates a whale brain ganglioside mixed solution used as a marker. 図３Ｂは、試験例１で製造したシアル酸含有糖鎖複合体（３’－ＳＬ－Ｎ－Ｃ１２）及び製造例２で製造したシアル酸含有糖鎖複合体（３’－ＳＬ－Ｎ－Ｃ１６）のＴＬＣの結果を示す図である。FIG. 3B shows the sialic acid-containing sugar chain complex (3′-SL-N-C12) produced in Test Example 1 and the sialic acid-containing sugar chain complex (3′-SL-N—C16) produced in Production Example 2. It is a figure which shows the result of TLC of). 図３Ｃは、製造例１で製造したシアル酸含有糖鎖複合体（３’－ＳＬ－Ｎ－Ｃ１４）及び製造例３で製造したシアル酸含有糖鎖複合体（３’－ＳＬ－Ｎ－Ｃ１８）のＴＬＣの結果を示す図である。「Ｍ」は、マーカーとして使用したクジラ脳ガングリオシド混合液を示す。FIG. 3C shows the sialic acid-containing sugar chain complex (3′-SL-N—C14) produced in Production Example 1 and the sialic acid-containing sugar chain complex (3′-SL—N—C18) produced in Production Example 3. It is a figure which shows the result of TLC of). “M” indicates a whale brain ganglioside mixed solution used as a marker. 図４は、製造例１で製造したシアル酸含有糖鎖複合体（３’－ＳＬ－Ｎ－Ｃ１４）、製造例５で製造したシアル酸含有糖鎖複合体（６’－ＳＬＮ－Ｎ－Ｃ１４及び６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１４）、及び製造例６で製造したシアル酸含有糖鎖複合体（６’－ＳＬ－Ｎ－Ｃ１４）のＴＬＣの結果を示す図である。「Ｍ」は、マーカーとして使用したクジラ脳ガングリオシド混合液を示す。FIG. 4 shows the sialic acid-containing sugar chain complex produced in Production Example 1 (3′-SL-N-C14) and the sialic acid-containing sugar chain complex produced in Production Example 5 (6′-SLN-N-C14). And 6′-S (1NH 2 ) LN—N—C14) and TLC results of the sialic acid-containing sugar chain complex (6′-SL—N—C14) produced in Production Example 6. “M” indicates a whale brain ganglioside mixed solution used as a marker. 図５は、製造例１～３で製造したシアル酸含有糖鎖複合体の原料として使用したシアリルオリゴ糖（３’－ＳＬ）のプロトンＮＭＲのスペクトルを示す図である。FIG. 5 is a proton NMR spectrum of sialyl oligosaccharide (3′-SL) used as a raw material for the sialic acid-containing sugar chain complex produced in Production Examples 1 to 3. 図６は、製造例３で製造したシアル酸含有糖鎖複合体（３’－ＳＬ－Ｎ－Ｃ１８）のプロトンＮＭＲのスペクトルを示す図である。FIG. 6 is a proton NMR spectrum of the sialic acid-containing sugar chain complex (3′-SL—N—C18) produced in Production Example 3. 図７は、製造例４で製造したシアル酸含有糖鎖複合体（３’－ＳＬＮ－Ｎ－Ｃ１４）のプロトンＮＭＲのスペクトルを示す図である。FIG. 7 is a proton NMR spectrum of the sialic acid-containing sugar chain complex (3′-SLN—N—C14) produced in Production Example 4. 図８Ａは、製造例５で製造したシアル酸含有糖鎖複合体（６’－ＳＬＮ－Ｎ－Ｃ１４）のＬＣ／ＭＳのスペクトルを示す図である。FIG. 8A is a diagram showing an LC / MS spectrum of the sialic acid-containing sugar chain complex (6′-SLN—N—C14) produced in Production Example 5. 図８Ｂは、製造例５で製造したシアル酸含有糖鎖複合体（６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１４）のＬＣ／ＭＳのスペクトルを示す図である。FIG. 8B is a diagram showing an LC / MS spectrum of the sialic acid-containing sugar chain complex (6′-S (1NH 2 ) LN—N—C14) produced in Production Example 5. 図８Ｃは、製造例５で製造したシアル酸含有糖鎖複合体（６’－ＳＬＮ－Ｎ－Ｃ１４）のプロトンＮＭＲのスペクトルを示す図である。FIG. 8C is a diagram showing a proton NMR spectrum of the sialic acid-containing sugar chain complex (6′-SLN—N—C14) produced in Production Example 5. 図８Ｄは、製造例５で製造したシアル酸含有糖鎖複合体（６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１４）のプロトンＮＭＲのスペクトルを示す図である。FIG. 8D is a diagram showing a proton NMR spectrum of the sialic acid-containing sugar chain complex (6′-S (1NH 2 ) LN—N—C14) produced in Production Example 5. 図９は、製造例５及び製造例８で製造したシアル酸含有糖鎖複合体の原料として使用したシアリルオリゴ糖（６’－ＳＬＮ）のプロトンＮＭＲのスペクトルを示す図である。FIG. 9 is a proton NMR spectrum of sialyl oligosaccharide (6′-SLN) used as a raw material for the sialic acid-containing sugar chain complex produced in Production Example 5 and Production Example 8. 図１０Ａは、製造例６で製造したシアル酸含有糖鎖複合体（６’－ＳＬ－Ｎ－Ｃ１４）のプロトンＮＭＲのスペクトルを示す図である。FIG. 10A is a proton NMR spectrum of the sialic acid-containing sugar chain complex (6′-SL—N—C14) produced in Production Example 6. 図１０Ｂは、製造例６で製造したシアル酸含有糖鎖複合体（６’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１４）のプロトンＮＭＲのスペクトルを示す図である。FIG. 10B is a diagram showing a proton NMR spectrum of the sialic acid-containing sugar chain complex (6′-S (1NH 2 ) LN—C14) produced in Production Example 6. 図１１は、製造例６及び７で製造したシアル酸含有糖鎖複合体の原料として使用したシアリルオリゴ糖（６’－ＳＬ）のプロトンＮＭＲのスペクトルを示す図である。FIG. 11 is a proton NMR spectrum of sialyl oligosaccharide (6′-SL) used as a raw material for the sialic acid-containing sugar chain complex produced in Production Examples 6 and 7. 図１２は、製造例７で製造したシアル酸含有糖鎖複合体（６’－ＳＬ－Ｎ－Ｃ１８）のプロトンＮＭＲのスペクトルを示す図である。FIG. 12 is a proton NMR spectrum of the sialic acid-containing sugar chain complex (6′-SL—N—C18) produced in Production Example 7. 図１３Ａは、製造例８で製造したシアル酸含有糖鎖複合体（６’－ＳＬＮ－Ｎ－Ｃ１８）のプロトンＮＭＲのスペクトルを示す図である。FIG. 13A is a diagram showing a proton NMR spectrum of the sialic acid-containing sugar chain complex (6′-SLN—N—C18) produced in Production Example 8. 図１３Ｂは、製造例８で製造したシアル酸含有糖鎖複合体（６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１８）のプロトンＮＭＲのスペクトルを示す図である。FIG. 13B is a diagram showing a proton NMR spectrum of the sialic acid-containing sugar chain complex (6′-S (1NH 2 ) LN—N—C18) produced in Production Example 8. 図１４Ａは、試験例５におけるマウスの体重変動を示す図である。「○」は対照群、「＊」は３’－ＳＬ－Ｎ－Ｃ１４経口投与対照群、「■」は３’－ＳＬ－Ｎ－Ｃ１４経口投与群、「▲」は３’－ＳＬ－Ｎ－Ｃ１４経鼻投与群、及び「◆」はウイルス投与群を示す。縦軸：体重（ｇ）、横軸：インフルエンザウイルス投与後日数。FIG. 14A is a diagram showing a change in body weight of a mouse in Test Example 5. “○” is a control group, “*” is a 3′-SL-N-C14 oral administration control group, “■” is a 3′-SL-N-C14 oral administration group, and “▲” is 3′-SL-N -C14 nasal administration group and "♦" indicate virus administration group. Vertical axis: body weight (g), horizontal axis: days after influenza virus administration. 図１４Ｂは、試験例５におけるマウスの生存率を示す図である。縦軸：生存率（％）、横軸：インフルエンザウイルス投与後日数。FIG. 14B is a diagram showing the survival rate of mice in Test Example 5. Vertical axis: survival rate (%), horizontal axis: days after influenza virus administration. 図１４Ｃは、試験例５におけるマウスの血清中のＩＬ－６濃度を示す図である。縦軸：ＩＬ－６濃度（ｐｇ／ｍＬ）。「＊」は、ｐ＜０．０５（ｔ検定）を示す。FIG. 14C is a graph showing IL-6 concentration in mouse serum in Test Example 5. Vertical axis: IL-6 concentration (pg / mL). “*” Indicates p <0.05 (t test). 図１４Ｄは、試験例５におけるマウスの血清中のＩＮＦ－γ濃度を示す図である。縦軸：ＩＮＦ－γ濃度（ｐｇ／ｍＬ）。「＊」は、ｐ＜０．０５（ｔ検定）を示す。FIG. 14D is a graph showing the INF-γ concentration in the serum of mice in Test Example 5. Vertical axis: INF-γ concentration (pg / mL). “*” Indicates p <0.05 (t test). 図１４Ｅは、試験例５におけるマウスの肺組織中のウイルス力価を示す図である。縦軸：ウイルス力価（ＰＦＵ／ｍＬ）。「＊」は、ｐ＜０．０５（ｔ検定）を示す。FIG. 14E is a diagram showing virus titers in mouse lung tissue in Test Example 5. Vertical axis: virus titer (PFU / mL). “*” Indicates p <0.05 (t test). 図１５Ａは、試験例５におけるマウスの生存率の変化を示す図である。縦軸：生存率（％）、横軸：インフルエンザウイルス投与後日数。FIG. 15A is a diagram showing changes in the survival rate of mice in Test Example 5. FIG. Vertical axis: survival rate (%), horizontal axis: days after influenza virus administration. 図１５Ｂは、試験例５におけるマウスの血清中のＩＬ－６濃度を示す図である。縦軸：ＩＬ－６濃度（ｐｇ／ｍＬ）。FIG. 15B is a graph showing IL-6 concentration in mouse serum in Test Example 5. Vertical axis: IL-6 concentration (pg / mL). 図１５Ｃは、試験例５におけるマウスの肺組織中のウイルス力価を示す図である。縦軸：ウイルス力価（ＰＦＵ／ｍＬ）。FIG. 15C is a diagram showing virus titers in mouse lung tissue in Test Example 5. Vertical axis: virus titer (PFU / mL). 図１６は、試験例１０において、シアル酸含有糖鎖複合体におけるシアル酸のカルボキシル基がアミド化した場合のシアリダーゼ抵抗性を確認した図である。FIG. 16 is a diagram for confirming sialidase resistance when the carboxyl group of sialic acid in the sialic acid-containing sugar chain complex is amidated in Test Example 10.
（シアル酸含有糖鎖複合体）
　本発明のシアル酸含有糖鎖複合体は、シアル酸含有糖鎖と脂肪酸とが結合してなる。 (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 fatty acid.
＜分子量＞
　前記シアル酸含有糖鎖複合体の分子量としては、４００～１，４００であり、４３０～９５０が好ましく、５９０～９５０がより好ましく、７５０～９５０が更に好ましく、８００～９５０が特に好ましい。
　前記分子量が、４００未満であると、所望の程度の抗インフルエンザウイルス活性を示さないことがあり、１，４００を超えると、水溶液中で抗インフルエンザウイルス活性に有利なミセル構造を形成しないことがあり、そのため、所望の程度の抗インフルエンザウイルス活性を示さないことがある。一方、前記分子量が、７５０～９５０の範囲内であると、水溶液中で抗インフルエンザウイルス活性に有利なミセル構造を形成し易い点で、有利である。 <Molecular weight>
The molecular weight of the sialic acid-containing sugar chain complex is 400 to 1,400, preferably 430 to 950, more preferably 590 to 950, still more preferably 750 to 950, and particularly preferably 800 to 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 750 to 950, it is advantageous in that a micelle structure advantageous for anti-influenza virus activity is easily formed in an aqueous solution.
＜脂肪酸＞
　前記脂肪酸としては、炭素数１４～１８の脂肪酸であるが、炭素数１６～１８の脂肪酸が、抗インフルエンザ活性が強い点で好ましい。ただし、スフィンゴ脂質は、前記脂肪酸から除外されるものとする。前記脂肪酸から除外されるスフィンゴ脂質の具体例としては、セラミドが挙げられる。 <Fatty acid>
The fatty acid is a fatty acid having 14 to 18 carbon atoms, but a fatty acid having 16 to 18 carbon atoms is preferred from the viewpoint of strong anti-influenza activity. However, sphingolipids are excluded from the fatty acids. Specific examples of sphingolipids excluded from the fatty acid include ceramide.
　前記脂肪酸は、疎水性であることが好ましい。前記脂肪酸が疎水性であると、前記シアル酸含有糖鎖複合体は全体として両親媒性となり、水溶液中でミセル構造をとるようになると考えられる。前記シアル酸含有糖鎖複合体が水溶液中でミセル構造をとると、好ましくはインフルエンザウイルスのヘマグルチニン（ＨＡ）タンパク質の認識部位を有する糖鎖が、外側に位置するようになる。そのため、該糖鎖は、よりヘマグルチニンタンパク質に認識され易くなり、インフルエンザウイルスの該糖鎖への結合が促進されて、結果として、インフルエンザウイルスの本来の標的細胞への結合を、より効率よく阻害することができるようになると考えられる。 The fatty acid is preferably hydrophobic. When the fatty acid is hydrophobic, 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 (HA) protein is preferably located on the 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
　前記脂肪酸としては、炭素数１４～１８であれば、特に制限はなく、目的に応じて適宜選択することができ、例えば、直鎖脂肪酸、不飽和脂肪酸、分岐脂肪酸などが挙げられる。これらの中でも、前記脂肪酸は、水溶液中で抗インフルエンザウイルス活性に有利なミセル構造を形成し易い点や、安定性の点で、直鎖脂肪酸が好ましい。
　なお、前記不飽和脂肪酸は二重結合が酸化されやすく、抗インフルエンザウイルス剤中で不安定要素になり得るという欠点があることから、前記脂肪酸として前記不飽和脂肪酸を使用する場合には、例えば、酸化防止剤と併用するなどの使用方法が好ましい。 The fatty acid is not particularly limited as long as it has 14 to 18 carbon atoms, and can be appropriately selected according to the purpose. Examples thereof include linear fatty acids, unsaturated fatty acids, branched fatty acids and the like. Among these, the fatty acid is preferably a straight-chain fatty acid 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, Use methods such as use in combination with an antioxidant are preferred.
＜シアル酸含有糖鎖＞
　前記シアル酸含有糖鎖としては、一方の末端にインフルエンザウイルスの結合部位となるシアル酸を有する糖鎖であれば特に制限はなく、目的に応じて適宜選択することができるが、好ましい抗インフルエンザウイルス活性を有するという点で、インフルエンザウイルスのヘマグルチニン認識部位に該当する糖鎖部位を有していることが好ましい。
　インフルエンザウイルスのヘマグルチニンが糖鎖を認識するには、前記糖鎖がシアル酸を有していることのみならず、前記シアル酸と、前記シアル酸と結合しているガラクトースとの結合様式が重要であることが知られている。なお、鳥インフルエンザウイルスは、シアル酸がガラクトースの３位に結合した、Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ－といった糖鎖を、ヒトインフルエンザウイルスは、シアル酸がガラクトースの６位に結合した、Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ－といった糖鎖を、それぞれ特異的に認識することが知られている。 <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 a binding site for influenza virus 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 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. Note that 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 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-.
　したがって、前記シアル酸含有糖鎖は、一方の末端にインフルエンザウイルスの結合部位となるシアル酸を有することが必要であり、また、シアル酸とガラクトースが結合したシアリルガラクトース鎖を有することが好ましく、これらの中でも、前記シアル酸含有糖鎖は、３’－シアリルガラクトース鎖（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ）、及び、６’－シアリルガラクトース鎖（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ）のいずれかを有することがより好ましく、３’－シアリルラクトース鎖（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇｌｃ）、６’－シアリルラクトース鎖（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）Ｇｌｃ）、３’－シアリルラクトサミン鎖（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）ＧｌｃＮＡｃ）、６’－シアリルラクトサミン鎖（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）ＧｌｃＮＡｃ）、３’－シアリルアミドラクトース鎖（３’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－３）Ｇａｌ（β１－４）Ｇｌｃ）、６’－シアリルアミドラクトース鎖（６’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－６）Ｇａｌ（β１－４）Ｇｌｃ）、３’－シアリルアミドラクトサミン鎖（３’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－３）Ｇａｌ（β１－４）ＧｌｃＮＡｃ）、６’－シアリルアミドラクトサミン鎖（６’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－６）Ｇａｌ（β１－４）ＧｌｃＮＡｃ）、３’－シアリルガラクトシルガラクトース鎖（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇａｌ）、６’－シアリルガラクトシルガラクトース鎖（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）Ｇａｌ）、３’－シアリルガラクトシルガラクトサミン鎖（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）ＧａｌＮＡｃ）、及び、６’－シアリルガラクトシルガラクトサミン鎖（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）ＧａｌＮＡｃ）からなる群より選択されることが、特に好ましい。 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. Among them, the sialic acid-containing sugar chain includes a 3′-sialylgalactose chain (3′-Neu5Ac (α2-3) Gal) and a 6′-sialylgalactose chain (6′-Neu5Ac (α2-6) Gal). It is more preferable to have a 3′-sialyllactose chain (3′-Neu5Ac (α2-3) Gal (β1-4) Glc), 6′-sialyllactose chain (6′-Neu5Ac (α2-6) ) Gal (β1-4) Glc), 3′-Sialyllactosamine chain (3′-Neu5Ac (α2-3) Gal (β1-4) GlcN c), 6'-sialyllactosamine chain (6'-Neu5Ac (α2-6) Gal (β1-4) GlcNAc), 3'- sialyl amide lactose chain (3'-Neu5Ac1NH 2 (α2-3) Gal (β1 -4) Glc), 6'-Sialylamide lactose chain (6'-Neu5Ac1NH 2 (α2-6) Gal (β1-4) Glc), 3'-Sialylamide lactosamine chain (3'-Neu5Ac1NH 2 (α2- 3) Gal (β1-4) GlcNAc), 6′-Sialylamide lactosamine chain (6′-Neu5Ac1NH 2 (α2-6) Gal (β1-4) GlcNAc), 3′-Sialylgalactosylgalactose chain (3′- Neu5Ac (α2-3) Gal (β1-4) Gal), 6′-sialylgalactosylgalactose chain (6′-Neu5Ac (α2-6) Gal (β1-4) Gal), 3′-sialylgalactosylgalactosamine chain (3′-Neu5Ac (α2-3) Gal (β1-4) GalNAc), and 6′-sialylgalactosylgalactosamine chain (6′-Neu5Ac ( It is particularly preferred that it is selected from the group consisting of (α2-6) Gal (β1-4) GalNAc).
　また、前記シアル酸含有糖鎖を構成する単糖の個数としては、目的に応じて適宜選択することができるが、１個～５個が好ましく、２個～３個がより好ましく、３個が特に好ましい。即ち、前記シアル酸含有糖鎖としては、シアル酸のみからなるものであってもよいが、シアル酸と一糖又は二糖からなるものがより好ましく、シアル酸と二糖（合計三糖）からなるものが特に好ましい。 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 more preferably 3 Particularly preferred. That is, the sialic acid-containing sugar chain may be composed only of sialic acid, but is preferably composed of sialic acid and monosaccharide or disaccharide, and is preferably composed of sialic acid and disaccharide (total trisaccharide). Is particularly preferred.
　一般に、インフルエンザウイルスのヘマグルチニン（ＨＡ）と結合する糖鎖部は、シアル酸含有糖鎖の先端二糖部分であることが知られている。上述のとおり、例えば、鳥インフルエンザウイルスは、Ｎｅｕ５Ａｃ（α２－３）Ｇａｌといった糖鎖を、ヒトインフルエンザウイルスは、Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ－といった糖鎖をそれぞれ特異的に認識する。
　本発明のシアル酸含有糖鎖複合体が合計三糖を有する場合、第三番目の糖がＮ－アセチル化されていると、水溶液中で集合体（ミセル等）を形成した際に、Ｎ－アセチル化されていない場合と比較して単分子専有面積が広がることでスペース的な余裕ができ、各分子の糖鎖提示性が高まるために、インフルエンザウイルスによりＮｅｕ５Ａｃ（α２－３）ＧａｌやＮｅｕ５Ａｃ（α２－６）Ｇａｌ等の構造がより認識され易くなり、また隣接分子による障害がなくなるために、インフルエンザウイルスの該糖鎖への結合が促進されて、結果として、インフルエンザウイルスの本来の標的細胞への結合を、より効率よく阻害することができるようになると考えられる。 In general, it is known that the sugar chain part that binds to hemagglutinin (HA) of influenza virus is the leading disaccharide part of the sialic acid-containing sugar chain. As described above, for example, 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-.
In the case where the sialic acid-containing sugar chain complex of the present invention has a total of three sugars, if the third sugar is N-acetylated, N-acetylated when an aggregate (such as a micelle) is formed in an aqueous solution. Compared to the case where the molecule is not acetylated, the space occupied by a single molecule is expanded, so that a space can be afforded and the sugar chain presentation of each molecule is enhanced. α2-6) The structure of Gal or the like is more easily recognized, and since there is no obstacle by neighboring molecules, the binding of influenza virus to the sugar chain is promoted, and as a result, the original target cell of influenza virus is restored. It is considered that the binding of can be inhibited more efficiently.
　前記シアル酸含有糖鎖を構成する単糖の個数が２個以上である場合、前記シアル酸に隣接する単糖としては、特に制限はなく、目的に応じて適宜選択することができるが、ガラクトース（Ｇａｌ）が好ましい。また、前記シアル酸含有糖鎖を構成する単糖の個数が３個以上である場合、前記シアル酸に隣接する単糖に、更に隣接する単糖（シアル酸の隣の隣の単糖）としては、特に制限はなく、目的に応じて適宜選択することができるが、ガラクトース（Ｇａｌ）、グルコース（Ｇｌｃ）、ガラクトサミン（ＧａｌＮＡｃ）、及び、グルコサミン（ＧｌｃＮＡｃ）からなる群より選択されることが好ましい。
　これらの中でも、インフルエンザウイルスに対する前記シアル酸含有糖鎖の結合性が高く、抗インフルエンザウイルス活性により優れることが期待されるという点で、ガラクトサミン（ＧａｌＮＡｃ）、グルコサミン（ＧｌｃＮＡｃ）がより好ましく、ガラクトサミン（ＧａｌＮＡｃ）が特に好ましい。 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 may be appropriately selected depending on the purpose. (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 may be appropriately selected depending on the purpose, but is preferably selected from the group consisting of galactose (Gal), glucose (Glc), galactosamine (GalNAc), and glucosamine (GlcNAc). .
Among these, galactosamine (GalNAc) and glucosamine (GlcNAc) are more preferable, and galactosamine (GalNAc) 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.
　また、前記シアル酸含有糖鎖を構成するシアル酸は、更にカルボキシル基がアミド化されていることが、抗インフルエンザウイルス活性により優れる点で好ましい。
　シアル酸のカルボキシル基がアミド化された場合であっても、シアル酸含有糖鎖複合体の分子量にはほとんど影響しない。したがって、ヘマグルチニン（ＨＡ）の認識部位に大きな立体構造の変化が生じることがなく、インフルエンザウイルスとの結合を阻害することがない点で好ましい。
　また、シアル酸のカルボキシル基がアミド化すると、カルボキシル基におけるマイナスチャージがなくなるため、合成後にシアル酸含有糖鎖複合体をＭＡＬＤＩ－ＴＯＦ　ＭＳ分析する際に分析を容易に行うことができるようになる点で有利である。
　更に、シアル酸のカルボキシル基がアミド化されると、シアリダーゼ活性に対する抵抗性が生じる。インフルエンザウイルスが感染細胞から娘ウイルスが放出される際には、娘ウイルスのヘマグルチニン（ＨＡ）と、宿主細胞のレセプターのシアル酸含有糖鎖との結合を切ることが必要となるため、インフルエンザウイルスのシアリダーゼ活性を阻害することで、インフルエンザウイルスの感染及び増殖のサイクルを抑制することができる点で有利である。 In addition, it is preferable that the sialic acid constituting the sialic acid-containing sugar chain is further amidated with a carboxyl group from the viewpoint of superior anti-influenza virus activity.
Even when the carboxyl group of sialic acid is amidated, the molecular weight of the sialic acid-containing sugar chain complex is hardly affected. Therefore, it is preferable in that a large three-dimensional structural change does not occur at the hemagglutinin (HA) recognition site and binding to influenza virus is not inhibited.
In addition, when the carboxyl group of sialic acid is amidated, the negative charge in the carboxyl group is eliminated, so that the sialic acid-containing sugar chain complex can be easily analyzed after MALDI-TOF MS analysis. This is advantageous.
Furthermore, resistance to sialidase activity occurs when the carboxyl group of sialic acid is amidated. When the influenza virus is released from the infected cell, it is necessary to break the bond between the daughter virus hemagglutinin (HA) and the sialic acid-containing sugar chain of the host cell receptor. Inhibiting sialidase activity is advantageous in that the cycle of influenza virus infection and proliferation can be suppressed.
＜結合＞
　前記シアル酸含有糖鎖と前記脂肪酸との結合としては、前記シアル酸含有糖鎖のシアル酸末端以外の末端に、前記脂肪酸がアミド結合されていれば、特に制限はなく、目的に応じて適宜選択することができる。
　ここで、「シアル酸含有糖鎖のシアル酸末端以外の末端に、前記脂肪酸がアミド結合している」とは、シアル酸含有糖鎖の糖と、脂肪酸のカルボキシル基とが、－ＮＨ－を介して結合していることをいい、例えば、下記一般式（Ｉ）で表される。
　Ａ－（Ｂ）ｎ－ＮＨ－Ｃ　・・・一般式（Ｉ）
　上記一般式（Ｉ）において、「Ａ」はシアル酸を表し、「Ｂ」は単糖を表し、「Ｃ」は脂肪酸を表す。また、「ｎ」は０又は１以上の整数を表し、０～４が好ましく、１～２がより好ましく、２が特に好ましい。 <Bonding>
The bond between the sialic acid-containing sugar chain and the fatty acid is not particularly limited as long as the fatty acid is amide-bonded at a terminal other than the sialic acid terminal of the sialic acid-containing sugar chain. You can choose.
Here, “the fatty acid is amide-bonded to a terminal other than the sialic acid terminal of the sialic acid-containing sugar chain” means that the sugar of the sialic acid-containing sugar chain and the carboxyl group of the fatty acid are —NH—. For example, it is represented by the following general formula (I).
A- (B) n —NH—C General formula (I)
In the above general formula (I), “A” represents sialic acid, “B” represents a monosaccharide, and “C” represents a fatty acid. “N” represents 0 or an integer of 1 or more, preferably 0 to 4, more preferably 1 to 2, and particularly preferably 2.
　前記結合方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記シアル酸含有糖鎖への、前記脂肪酸に対応したアシル基の導入法などが挙げられる。前記シアル酸含有糖鎖への前記アシル基の導入法としては、特に制限はなく、目的に応じて適宜選択することができるが、Ｏ－グリコシド化、Ｎ－グリコシド化、Ｓ－グリコシド化、Ｃ－グリコシド化による導入法などが好ましく、Ｎ－グリコシド化（アシルアミド化、アミド結合）による導入法が、安定性の点で特に好ましい。 The coupling method is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a method for introducing an acyl group corresponding to the fatty acid into the sialic acid-containing sugar chain. The method for introducing the acyl group into the sialic acid-containing sugar chain is not particularly limited and may be appropriately selected depending on the intended purpose. However, O-glycosidation, N-glycosidation, S-glycosidation, C An introduction method by glycosidation is preferred, and an introduction method by N-glycosidation (acylamidation, amide bond) is particularly preferred from the viewpoint of stability.
　前記シアル酸含有糖鎖複合体は、前記シアル酸含有糖鎖と前記脂肪酸とが、任意の組み合わせで結合してなる。
　このような、前記シアル酸含有糖鎖複合体の好ましい具体例としては、例えば、３’－シアリルラクトシルミリスチン酸アミド（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１４；以下、「３’－ＳＬ－Ｎ－Ｃ１４」と称することがある。）、３’－シアリルラクトシルパルミチン酸アミド（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１６；以下、「３’－ＳＬ－Ｎ－Ｃ１６」と称することがある。）、３’－シアリルラクトシルステアリン酸アミド（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１８；以下、「３’－ＳＬ－Ｎ－Ｃ１８」と称することがある。）、６’－シアリルラクトシルミリスチン酸アミド（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１４；以下、「６’－ＳＬ－Ｎ－Ｃ１４」と称することがある。）、６’－シアリルラクトシルパルミチン酸アミド（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１６；以下、「６’－ＳＬ－Ｎ－Ｃ１６」と称することがある。）、６’－シアリルラクトシルステアリン酸アミド（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１８；以下、「６’－ＳＬ－Ｎ－Ｃ１８」と称することがある。）、３’－シアリルラクトサミニルミリスチン酸アミド（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１４；以下、「３’－ＳＬＮ－Ｎ－Ｃ１４」と称することがある。）、３’－シアリルラクトサミニルパルミチン酸アミド（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１６；以下、「３’－ＳＬＮ－Ｎ－Ｃ１６」と称することがある。）、３’－シアリルラクトサミニルステアリン酸アミド（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１８；以下、「３’－ＳＬＮ－Ｎ－Ｃ１８」と称することがある。）、６’－シアリルラクトサミニルミリスチン酸アミド（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１４；以下、「６’－ＳＬＮ－Ｎ－Ｃ１４」と称することがある。）、６’－シアリルラクトサミニルパルミチン酸アミド（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１６；以下、「６’－ＳＬＮ－Ｎ－Ｃ１６」と称することがある。）、６’－シアリルラクトサミニルステアリン酸アミド（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１８；以下、「６’－ＳＬＮ－Ｎ－Ｃ１８」と称することがある。））、３’－シアリルアミドラクトシルミリスチン酸アミド（３’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－３）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１４；以下、「３’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１４」と称することがある。）、３’－シアリルアミドラクトシルパルミチン酸アミド（３’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－３）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１６；以下、「３’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１６」と称することがある。）、３’－シアリルアミドラクトシルステアリン酸アミド（３’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－３）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１８；以下、「３’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１８」と称することがある。）、６’－シアリルアミドラクトシルミリスチン酸アミド（６’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－６）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１４；以下、「６’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１４」と称することがある。）、６’－シアリルアミドラクトシルパルミチン酸アミド（６’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－６）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１６；以下、「６’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１６」と称することがある。）、６’－シアリルアミドラクトシルステアリン酸アミド（６’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－６）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１８；以下、「６’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１８」と称することがある。）、３’－シアリルアミドラクトサミニルミリスチン酸アミド（３’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－３）Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１４；以下、「３’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１４」と称することがある。）、３’－シアリルアミドラクトサミニルパルミチン酸アミド（３’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－３）Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１６；以下、「３’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１６」と称することがある。）、３’－シアリルアミドラクトサミニルステアリン酸アミド（３’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－３）Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１８；以下、「３’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１８」と称することがある。）、６’－シアリルアミドラクトサミニルミリスチン酸アミド（６’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－６）Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１４；以下、「６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１４」と称することがある。）、６’－シアリルアミドラクトサミニルパルミチン酸アミド（６’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－６）Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１６；以下、「６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１６」と称することがある。）、６’－シアリルアミドラクトサミニルステアリン酸アミド（６’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－６）Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１８；以下、「６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１８」と称することがある。））、３’－シアリルガラクトシルガラクトシルミリスチン酸アミド（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇａｌ－Ｎ－Ｃ１４）、３’－シアリルガラクトシルガラクトシルパルミチン酸アミド（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇａｌ－Ｎ－Ｃ１６）、３’－シアリルガラクトシルガラクトシルステアリン酸アミド（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇａｌ－Ｎ－Ｃ１８）、６’－シアリルガラクトシルガラクトシルミリスチン酸アミド（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）Ｇａｌ－Ｎ－Ｃ１４）、６’－シアリルガラクトシルガラクトシルパルミチン酸アミド（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）Ｇａｌ－Ｎ－Ｃ１４）、６’－シアリルガラクトシルガラクトシルステアリン酸アミド（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）Ｇａｌ－Ｎ－Ｃ１８）、３’－シアリルガラクトシルガラクトサミニルミリスチン酸アミド（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）ＧａｌＮＡｃ－Ｎ－Ｃ１４）、３’－シアリルガラクトシルガラクトサミニルパルミチン酸アミド（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）ＧａｌＮＡｃ－Ｎ－Ｃ１６）、３’－シアリルガラクトシルガラクトサミニルステアリン酸アミド（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）ＧａｌＮＡｃ－Ｎ－Ｃ１８）、６’－シアリルガラクトシルガラクトサミニルミリスチン酸アミド（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）ＧａｌＮＡｃ－Ｎ－Ｃ１４）、６’－シアリルガラクトシルガラクトサミニルパルミチン酸アミド（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）ＧａｌＮＡｃ－Ｎ－Ｃ１６）、６’－シアリルガラクトシルガラクトサミニルステアリン酸アミド（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）ＧａｌＮＡｃ－Ｎ－Ｃ１８）、これらのシアル酸のカルボキシル基がアミド化されたシアル酸含有糖鎖複合体などが挙げられる。 The sialic acid-containing sugar chain complex is formed by binding the sialic acid-containing sugar chain and the fatty acid in any combination.
A preferred specific example of such a sialic acid-containing sugar chain complex is, for example, 3′-sialyllactosylmyristate amide (3′-Neu5Ac (α2-3) Gal (β1-4) Glc-N— C14; hereinafter sometimes referred to as “3′-SL-N—C14”.) 3′-Sialyllactosyl palmitic acid amide (3′-Neu5Ac (α2-3) Gal (β1-4) Glc-N -C16; hereinafter sometimes referred to as "3'-SL-N-C16") 3'-sialyl lactosyl stearamide (3'-Neu5Ac (α2-3) Gal (β1-4) Glc- N-C18; hereinafter referred to as “3′-SL-N-C18”), 6′-sialyl lactosyl myristate amide (6′-Neu5Ac (α2-6) Gal (β1-4) Glc -N-C14 Hereinafter, it may be referred to as “6′-SL-N-C14”.), 6′-sialyllactosyl palmitate (6′-Neu5Ac (α2-6) Gal (β1-4) Glc-N-C16 Hereinafter referred to as “6′-SL-N—C16”), 6′-sialyl lactosyl stearamide (6′-Neu5Ac (α2-6) Gal (β1-4) Glc-N— C18; hereinafter may be referred to as “6′-SL-N—C18”.) 3′-Sialyllactosaminyl myristic acid amide (3′-Neu5Ac (α2-3) Gal (β1-4) GlcNAc- N-C14; hereinafter sometimes referred to as “3′-SLN-N-C14”.) 3′-Sialyllactosaminyl palmitic acid amide (3′-Neu5Ac (α2-3) Gal (β1-4) GlcNAc-N- 16; hereinafter sometimes referred to as “3′-SLN-N—C16”.) 3′-Sialyllactosaminyl stearamide (3′-Neu5Ac (α2-3) Gal (β1-4) GlcNAc- N-C18; hereinafter sometimes referred to as “3′-SLN-N-C18”), 6′-sialyllactosaminyl myristic acid amide (6′-Neu5Ac (α2-6) Gal (β1-4) GlcNAc-N-C14; hereinafter sometimes referred to as “6′-SLN-N-C14”), 6′-sialyllactosaminyl palmitate amide (6′-Neu5Ac (α2-6) Gal (β1- 4) GlcNAc-N-C16; hereinafter sometimes referred to as “6′-SLN-N-C16”), 6′-sialyllactosaminyl stearamide (6′-Neu5Ac (α2-6) Ga l (β1-4) GlcNAc-N—C18; hereinafter referred to as “6′-SLN-N—C18”. )) 3′-Sialylamide lactosylmyristate amide (3′-Neu5Ac1NH 2 (α2-3) Gal (β1-4) Glc-N—C14; hereinafter “3′-S (1NH 2 ) LN -C14 ")) 3'-Sialylamide lactosyl palmitic acid amide (3'-Neu5Ac1NH 2 (α2-3) Gal (β1-4) Glc-N-C16; hereinafter referred to as"3'- S (1NH 2 ) L—N—C16 ”)) 3′-Sialylamido lactosyl stearamide (3′-Neu5Ac1NH 2 (α2-3) Gal (β1-4) Glc-N— C18;. which hereinafter may be referred to as "3'-S (1NH 2) L -N-C18 "), 6'-sialyl amide lactosyl myristic acid amide (6'-Neu5Ac1NH 2 (α2-6) al (β1-4) Glc-N- C14;. which hereinafter may be referred to as "6'-S (1NH 2) L -N-C14 "), 6'-sialyl amide lactosyl palmitic acid amide (6 ' —Neu5Ac1NH 2 (α2-6) Gal (β1-4) Glc-N—C16; hereinafter referred to as “6′-S (1NH 2 ) L—N—C16”), 6′-sialylamide Lactosyl stearamide (6′-Neu5Ac1NH 2 (α2-6) Gal (β1-4) Glc-N—C18; hereinafter sometimes referred to as “6′-S (1NH 2 ) LN—C18” 3′-Sialylamide lactosaminyl myristic acid amide (3′-Neu5Ac1NH 2 (α2-3) Gal (β1-4) GlcNAc-N—C14; hereinafter, “3′-S (1NH 2 ) LN— N-C14 " 3′-Sialylamide lactosaminyl palmitic acid amide (3′-Neu5Ac1NH 2 (α2-3) Gal (β1-4) GlcNAc-N—C16; hereinafter referred to as “3′-S ( 1NH 2 ) LN—N—C16 ”)) 3′-Sialylamide lactosaminyl stearamide (3′-Neu5Ac1NH 2 (α2-3) Gal (β1-4) GlcNAc-N—C18 Hereinafter referred to as “3′-S (1NH 2 ) LN—N—C18”), 6′-sialylamidolactosaminyl myristic acid amide (6′-Neu5Ac1NH 2 (α2-6) Gal ( β1-4) GlcNAc-N—C14; hereinafter referred to as “6′-S (1NH 2 ) LN—N—C14”. ), 6′-sialylamidolactosaminyl palmitate (6′-Neu5Ac1NH 2 (α2-6) Gal (β1-4) GlcNAc-N—C16; hereinafter, “6′-S (1NH 2 ) LN—N -C16 "), 6'-sialylamidolactosaminyl stearamide (6'-Neu5Ac1NH 2 (α2-6) Gal (β1-4) GlcNAc-N-C18; hereinafter referred to as" 6 ' —S (1NH 2 ) LN—N—C18 ”))) 3′-Sialylgalactosylgalactosylmyristate amide (3′-Neu5Ac (α2-3) Gal (β1-4) Gal-N— C14), 3′-sialylgalactosylgalactosylpalmitate (3′-Neu5Ac (α2-3) Gal (β1-4) Gal-N-C16), 3′- Allylgalactosylgalactosyl stearamide (3′-Neu5Ac (α2-3) Gal (β1-4) Gal-N-C18), 6′-sialylgalactosylgalactosylmyristate amide (6′-Neu5Ac (α2-6) Gal ( β1-4) Gal-N-C14), 6′-sialylgalactosylgalactosylpalmitate amide (6′-Neu5Ac (α2-6) Gal (β1-4) Gal-N-C14), 6′-sialylgalactosylgalactosyl stearin Acid amide (6′-Neu5Ac (α2-6) Gal (β1-4) Gal-N-C18), 3′-sialylgalactosylgalactosaminylmyristic acid amide (3′-Neu5Ac (α2-3) Gal (β1- 4) GalNAc-N-C14), 3'-sialylgalactosylgalactosaminylpa Lumicinamide (3'-Neu5Ac (α2-3) Gal (β1-4) GalNAc-N-C16), 3'-Sialylgalactosylgalactosaminyl stearamide (3'-Neu5Ac (α2-3) Gal (β1 -4) GalNAc-N-C18), 6'-sialylgalactosylgalactosaminylmyristic acid amide (6'-Neu5Ac (α2-6) Gal (β1-4) GalNAc-N-C14), 6'-sialylgalactosylgalacto Saminyl palmitic acid amide (6′-Neu5Ac (α2-6) Gal (β1-4) GalNAc-N-C16), 6′-sialylgalactosylgalactosaminyl stearic acid amide (6′-Neu5Ac (α2-6) Gal (Β1-4) GalNAc-N-C18), the carboxyl group of these sialic acids is amino Etc. of sialic acid-containing sugar chain complex thereof.
＜用途＞
　前記シアル酸含有糖鎖複合体は、優れた抗インフルエンザウイルス活性を有しているため、後述する本発明の抗インフルエンザウイルス剤の有効成分として好適に利用可能である。 <Application>
Since the sialic acid-containing sugar chain complex has excellent anti-influenza virus activity, it can be suitably used as an active ingredient of the anti-influenza virus agent of the present invention described later.
（シアル酸含有糖鎖複合体の製造方法）
　本発明のシアル酸含有糖鎖複合体の製造方法は、一方の末端にシアル酸を含有するシアル酸含有糖鎖のシアル酸末端以外の末端に、脂肪酸を、化学反応により任意の結合方法で結合させて製造する方法である。前記結合方法は、前記シアル酸含有糖鎖のシアル酸末端以外の末端を、アシルアミド化する方法が好ましい。 (Method for producing sialic acid-containing sugar chain complex)
In the method for producing a sialic acid-containing sugar chain complex of the present invention, a fatty acid 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. It is the method of making it manufacture. The bonding method is preferably a method in which the terminal other than the terminal sialic acid of the sialic acid-containing sugar chain is acylamidated.
＜シアリルオリゴ糖脂肪酸アミドの製造方法＞
　以下に、前記シアル酸含有糖鎖が前記３’－シアリルラクトース鎖（以下、「３’－ＳＬ」と称することがある。）、前記６’－シアリルラクトース鎖（以下、「６’－ＳＬ」と称することがある。）、前記３’－シアリルラクトサミン鎖（以下、「３’－ＳＬＮ」と称することがある。）、及び、前記６’－シアリルラクトサミン鎖（以下、「６’－ＳＬＮ」と称することがある。）のいずれかであり、かつ、前記脂肪酸がスフィンゴ脂質を除く炭素数１４～１８の脂肪酸であって、前記シアル酸含有糖鎖のシアル酸末端以外の末端にアミド結合している場合を例に挙げ、このようなシアル酸含有糖鎖複合体の製造方法の一例を説明する。
　なお、以下、前記した３’－ＳＬ、６’－ＳＬ、３’－ＳＬＮ、６’－ＳＬＮを総称して、「シアリルオリゴ糖」と称することがある。
　また、以下、前記シアリルオリゴ糖と前記脂肪酸とがアミド結合してなるシアル酸含有糖鎖複合体を、「シアリルオリゴ糖脂肪酸アミド」と称することがある。前記シアリルオリゴ糖脂肪酸アミドは、前記シアル酸含有糖鎖複合体の好ましい態様の１つである。 <Method for producing sialyl oligosaccharide fatty acid amide>
Hereinafter, the sialic acid-containing sugar chain includes the 3′-sialyllactose chain (hereinafter sometimes referred to as “3′-SL”), the 6′-sialyllactose chain (hereinafter referred to as “6′-SL”). 3′-sialyllactosamine chain (hereinafter sometimes referred to as “3′-SLN”), and 6′-sialyllactosamine chain (hereinafter referred to as “6′-”). And the fatty acid is a fatty acid having 14 to 18 carbon atoms excluding sphingolipid, and an amide at the terminal other than the sialic acid terminal of the sialic acid-containing sugar chain. An example of a method for producing such a sialic acid-containing sugar chain complex will be described with reference to an example of binding.
Hereinafter, the aforementioned 3′-SL, 6′-SL, 3′-SLN, and 6′-SLN may be collectively referred to as “sialyl oligosaccharide”.
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.
－シアリルオリゴ糖－
　３’－ＳＬ、６’－ＳＬ、３’－ＳＬＮ、６’－ＳＬＮなどの前記シアリルオリゴ糖としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ヒト、ウシ等の哺乳動物由来のシアリルオリゴ糖などを使用することができる。また、前記シアリルオリゴ糖としては、市販品を使用することもできる。前記市販品は、例えば、シグマアルドリッチ社、生化学工業株式会社などから入手することができる。
　前記シアル酸含有糖鎖複合体は、天然の材料で構築した物質であることから、後述する抗インフルエンザウイルス剤に使用した場合、安全性が高い点で有利である。 -Sialyl oligosaccharide-
The sialyl oligosaccharide such as 3′-SL, 6′-SL, 3′-SLN, 6′-SLN is not particularly limited and may be appropriately selected depending on the intended purpose. For example, human, bovine, etc. Sialyl oligosaccharides derived from 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.
Since the sialic acid-containing sugar chain complex is a substance constructed from natural materials, it is advantageous in terms of high safety when used for an anti-influenza virus agent described later.
－シアリルオリゴ糖のアシルアミド化－
　前記シアリルオリゴ糖を、例えば、アシルアミド化することにより、前記脂肪酸が前記シアリルオリゴ糖にアミド結合したシアリルオリゴ糖脂肪酸アミドを得ることができる。
　前記シアリルオリゴ糖のアシルアミド化は、例えば、前記シアリルオリゴ糖と炭酸水素アンモニウムとを反応させ（以下、「工程（Ｉ）」と称することがある。）、次いで、得られた反応物と脂肪酸とを反応させる（以下、「工程（ＩＩ）」と称することがある。）ことにより行うことができる。より具体的には、例えば、以下のようにして行うことができる。 -Acylamidation of sialyl oligosaccharides-
For example, by acylating the sialyl oligosaccharide, a sialyl oligosaccharide fatty acid amide in which the fatty acid is amide-bonded to the sialyl oligosaccharide can be obtained.
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 and fatty acid are reacted. (Hereinafter sometimes referred to as “step (II)”). More specifically, for example, it can be performed as follows.
－工程（Ｉ）－
　前記工程（Ｉ）は、シアリルオリゴ糖と炭酸水素アンモニウムとを反応させる工程である。
　前記反応させる方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記シアリルオリゴ糖を、過飽和の炭酸水素アンモニウム水溶液に添加し、撹拌しながら、室温条件下で反応させる方法などが挙げられる。ここで、室温とは、１０℃～４０℃をいい、２０℃～２５℃が好ましい。 -Step (I)-
The step (I) is a step of reacting sialyl oligosaccharide with ammonium hydrogen carbonate.
The method for the reaction is not particularly limited and may be appropriately selected depending on the intended purpose. The method of making it, etc. are mentioned. Here, room temperature means 10 ° C. to 40 ° C., preferably 20 ° C. to 25 ° C.
　前記工程（Ｉ）におけるシアリルオリゴ糖と炭酸水素アンモニウムとの反応の進行具合は、例えば、薄層クロマトグラフィー（ＴＬＣ）により確認することができる。 The progress of the reaction between the sialyl oligosaccharide and ammonium hydrogen carbonate in the step (I) can be confirmed, for example, by thin layer chromatography (TLC).
　前記工程（Ｉ）の反応終了後、前記反応により得られた反応液は、例えば、ロータリーエバポレーターなどを用いて、水分と共に、炭酸水素アンモニウムを除去した後、以下の工程（ＩＩ）に供することが、反応効率がよい点で好ましい。 After completion of the reaction in the step (I), the reaction solution obtained by the reaction may be subjected to the following step (II) after removing ammonium hydrogen carbonate together with moisture using, for example, a rotary evaporator. From the viewpoint of good reaction efficiency.
－工程（ＩＩ）－
　前記工程（ＩＩ）は、前記工程（Ｉ）で得られた反応物と、脂肪酸とを反応させる工程である。
　前記反応方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記工程（Ｉ）で得られた反応物と塩化脂肪酸とを、炭酸ナトリウムの存在下、０℃条件下で反応させる方法、前記工程（Ｉ）で得られた反応物と脂肪酸とを、縮合剤の存在下、室温条件下で反応させる方法などが挙げられる。これらの中でも、前記工程（ＩＩ）は、前記工程（Ｉ）で得られた反応物と脂肪酸とを、縮合剤の存在下、室温条件下で反応させる方法が、短い反応時間でシアリルオリゴ糖脂肪酸アミドを得ることができ、反応効率がよい点で好ましい。 -Step (II)-
The step (II) is a step of reacting the reaction product obtained in the step (I) with a 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 the step (I) and a chlorinated fatty acid are subjected to a 0 ° C. condition in the presence of sodium carbonate. And a method of reacting the reaction product obtained in the step (I) with a fatty acid in the presence of a condensing agent at room temperature. Among these, in the step (II), the method of reacting the reaction product obtained in the step (I) with a fatty acid in the presence of a condensing agent under room temperature conditions is a sialyl oligosaccharide fatty acid in a short reaction time. Amides can be obtained, which is preferable in terms of good reaction efficiency.
　前記工程（ＩＩ）が、前記反応物と前記塩化脂肪酸とを、炭酸ナトリウムの存在下で反応させる方法で行われる場合、前記塩化脂肪酸の使用量としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記シアリルオリゴ糖の使用量に対して、モル比で、塩化脂肪酸：シアリルオリゴ糖＝５：１となるような量で使用することができる。
　また、前記炭酸ナトリウムの使用量としても、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記塩化脂肪酸の使用量に対して、モル比で、炭酸ナトリウム：塩化脂肪酸＝１：１となるような量で使用することができる。 When the step (II) is performed by a method in which the reactant and the chlorinated fatty acid are reacted in the presence of sodium carbonate, the amount of the chlorinated fatty acid is not particularly limited, and is appropriately determined depending on the purpose. For example, it can be used in an amount such that the molar ratio of sialyl oligosaccharide is 5: 1 with respect to the amount of sialyl oligosaccharide used.
Also, 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 chlorinated fatty acid used. : 1 can be used in such an amount.
　前記工程（ＩＩ）が、前記反応物と前記脂肪酸とを、縮合剤の存在下で反応させる方法で行われる場合、前記脂肪酸の使用量としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記シアリルオリゴ糖の使用量に対して、モル比で、脂肪酸：シアリルオリゴ糖＝５：１となるような量で使用することができる。
　また、前記縮合剤の使用量としても、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記脂肪酸の使用量に対して、モル比で、縮合剤：脂肪酸＝１：１となるような量で使用することができる。 When the step (II) is performed by a method of reacting the reactant and the fatty acid in the presence of a condensing agent, the amount of the fatty acid to be used is not particularly limited and is appropriately selected depending on the purpose. For example, it can be used in such an amount that the molar ratio of fatty acid: sialyl oligosaccharide = 5: 1 with respect to the amount of the sialyl oligosaccharide used.
Further, the amount of the condensing agent used is not particularly limited and may be appropriately selected depending on the purpose. For example, condensing agent: fatty acid = 1: 1 in molar ratio with respect to the amount of fatty acid used. Can be used in such amounts.
　前記縮合剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ＷＳＣ、ＤＣＣ等のカルボジイミド；ＤＭＴ－ＭＭ等のトリアジン系縮合などが挙げられる。これらは、１種単独で使用してもよく、２種以上を併用してもよい。これらの中でも、前記縮合剤は、ＤＭＴ－ＭＭが、短い反応時間でシアリルオリゴ糖脂肪酸アミドを得ることができ、反応効率がよい点で好ましい。 The condensing agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include carbodiimides such as WSC and DCC; and triazine-based condensations such as DMT-MM. These may be used alone or in combination of two or more. Among these, the condensing agent is preferable because DMT-MM can obtain a sialyl oligosaccharide fatty acid amide in a short reaction time and has good reaction efficiency.
　なお、前記工程（ＩＩ）において、前記工程（Ｉ）で得られた反応物と、脂肪酸とを反応させた際に、副生成物として、シアル酸のカルボキシル基がアミド化されたシアリルオリゴ糖脂肪酸アミドを得ることができる。
　シアル酸のカルボキシル基がアミド化されたシアリルオリゴ糖脂肪酸アミドと、シアル酸のカルボキシル基がアミド化さていないシアリルオリゴ糖脂肪酸アミドとは、陰イオン交換クロマトグラフィーや、シリカゲルカラムクロマトグラフィーなどで、公知の方法により分離することができる。 In the step (II), a sialyloligosaccharide fatty acid in which the carboxyl group of sialic acid is amidated as a by-product when the reaction product obtained in step (I) is reacted with a fatty acid. Amides can be obtained.
Sialyl oligosaccharide fatty acid amides in which the carboxyl group of sialic acid is amidated and sialyl oligosaccharide fatty acid amides in which the carboxyl group of sialic acid is not amidated are known by anion exchange chromatography, silica gel column chromatography, etc. It can be separated by this method.
－精製工程－
　前記シアリルオリゴ糖脂肪酸アミドは、更に精製されることが好ましい。前記シアリルオリゴ糖脂肪酸アミドを精製する方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、Ｓｅｐｈａｄｅｘ　ＬＨ－２０（ＧＥ　ヘルスケア　バイオサイエンス株式会社）、Ｓｅｐｈａｄｅｘ　Ｇ－１０（ＧＥ　ヘルスケア　バイオサイエンス株式会社）、ＢｉｏＧｅｌ　Ｐ－２（日本バイオ・ラッド　ラボラトリーズ株式会社）、シリカゲル（例えば、Ｉａｔｒｏｂｅａｄｓ　６ＲＳ　８０６０（株式会社　三菱化学ヤトロン））、Ｃ１８逆相カラム（Ｐｒｅｐａｒａｔｉｖｅ　Ｃ１８　Ｂｕｌｋ　Ｐａｃｋｉｎｇ　Ｍａｔｅｒｉａｌｓ（ウォーターズ社製））等を担体として用いた、各種カラムクロマトグラフィーを行う方法などが挙げられる。
　なお、前記精製を行う前に、前記工程（ＩＩ）で得られた反応液中の溶媒を除去し、前記反応液と等量のヘキサンを加えて混合した後、遠心分離し、上清を除去する洗浄を行うことが、未反応の脂肪酸を除去できる点で好ましい。
　前記洗浄回数としては、特に制限はなく、目的に応じて適宜選択することができるが、２回以上が好ましく、２回～３回がより好ましい。 -Purification process-
The sialyl oligosaccharide fatty acid amide is preferably further purified. The method for purifying the sialyl oligosaccharide fatty acid amide is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include Sephadex LH-20 (GE Healthcare Biosciences), Sephadex G-10 ( GE Healthcare Biosciences Co., Ltd., BioGel P-2 (Nippon Bio-Rad Laboratories Co., Ltd.), silica gel (for example, Iatrobeads 6RS 8060 (Mitsubishi Chemical Yatron Co., Ltd.)), C18 reverse phase column (Preparative C18 Bulk Packing Materials ( And a method of performing various column chromatography using Waters)) as a carrier.
Before carrying out the purification, the solvent in the reaction solution obtained in the step (II) is removed, and an equal amount of hexane is added to and mixed with the reaction solution, followed by centrifugation and removal of the supernatant. It is preferable to perform the washing in that the unreacted fatty acid can be removed.
The number of times of washing is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 2 times or more, and more preferably 2 to 3 times.
＜製造されたシアル酸含有糖鎖複合体の確認方法＞
　前記製造方法により得られたシアル酸含有糖鎖複合体を確認する方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、薄層クロマトグラフィー、高速液体クロマトグラフィー、質量分析計、プロトンＮＭＲ、ＬＣ／ＭＳ（液体クロマトグラフィー／質量分析）等の分析方法などが挙げられる。 <Method for confirming manufactured sialic acid-containing sugar chain complex>
The method for confirming the sialic acid-containing sugar chain complex obtained by the above production method is not particularly limited and may be appropriately selected depending on the intended purpose. For example, thin layer chromatography, high performance liquid chromatography, mass Analytical methods such as analyzer, proton NMR, LC / MS (liquid chromatography / mass spectrometry) and the like can be mentioned.
＜用途＞
　前記製造方法は、本発明の前記シアル酸含有糖鎖複合体を好適に製造できるため、後述する本発明の抗インフルエンザウイルス剤の有効成分の製造方法として、好適に利用可能である。 <Application>
Since the said manufacturing method can manufacture suitably the said sialic acid containing sugar_chain | carbohydrate complex of this invention, it can utilize suitably as a manufacturing method of the active ingredient of the anti-influenza virus agent of this invention mentioned later.
（抗インフルエンザウイルス剤）
　本発明の抗インフルエンザウイルス剤は、本発明の前記シアル酸含有糖鎖複合体を少なくとも含有し、必要に応じて、更にその他の成分を含有する。 (Anti-influenza virus agent)
The anti-influenza virus agent of the present invention contains at least the sialic acid-containing sugar chain complex of the present invention, and further contains other components as necessary.
＜シアル酸含有糖鎖複合体＞
　前記抗インフルエンザウイルス剤に含有される前記シアル酸含有糖鎖複合体は、好ましくは、そのシアル酸含有糖鎖部分に、インフルエンザウイルスのヘマグルチニン（ＨＡ）認識部位を有する。前記ヘマグルチニンは、インフルエンザウイルスのエンベロープに存在するスパイクタンパク質の１種であり、標的細胞の膜表面に存在する複合糖質のシアル酸を含む糖鎖を認識し、結合して、感染を成立させることが知られている。前記シアル酸含有糖鎖複合体は、このヘマグルチニン認識部位を有していることから、インフルエンザウイルスと結合可能であり、そのため、インフルエンザウイルスの本来の標的細胞への結合を阻害することができると考えられる。
　前記シアル酸含有糖鎖の前記ヘマグルチニン認識部位としては、具体的には、３’－シアリルガラクトース鎖（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ）、及び、６’－シアリルガラクトース鎖（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ）のいずれかが好ましく挙げられる。 <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 present in the envelope of influenza virus, and recognizes and binds to a sugar chain containing sialic acid of a complex carbohydrate present on the surface of a target cell membrane 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.
Specific examples of the hemagglutinin recognition site of the sialic acid-containing sugar chain include 3′-sialylgalactose chain (3′-Neu5Ac (α2-3) Gal) and 6′-sialylgalactose chain (6′- Any of Neu5Ac (α2-6) Gal) is preferred.
　前記抗インフルエンザウイルス剤における前記シアル酸含有糖鎖複合体（有効成分）の含有量としては、特に制限はなく、前記抗インフルエンザウイルス剤の剤型などに応じて、適宜選択することができるが、６０質量％～７０質量％が好ましい。また、前記抗インフルエンザウイルス剤は、前記シアル酸含有糖鎖複合体そのものであってもよい。 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 mass% to 70 mass% is preferable. The anti-influenza virus agent may be the sialic acid-containing sugar chain complex itself.
＜その他の成分＞
　前記抗インフルエンザウイルス剤に含有され得る、前記シアル酸含有糖鎖複合体以外のその他の成分としては、特に制限はなく、本発明の効果を損なわない範囲内で、目的に応じて適宜選択することができ、例えば、薬学的に許容され得る担体などが挙げられる。
　前記薬学的に許容され得る担体としても、特に制限はなく、前記抗インフルエンザウイルス剤の剤型などに応じて、適宜選択することができる。
　また、前記その他の成分の含有量としても、特に制限はなく、例えば、前記抗インフルエンザウイルス剤における前記シアル酸含有糖鎖複合体（有効成分）の含有量が所望の範囲内となるように、目的に応じて適宜選択することができる。 <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 may be appropriately selected depending on the purpose within a range not impairing the effects of the present invention. Examples thereof include pharmaceutically acceptable carriers.
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.
＜剤型＞
　前記抗インフルエンザウイルス剤の剤型としては、特に制限はなく、例えば、前記抗インフルエンザウイルス剤の投与方法などに応じて適宜選択することができ、例えば、経口固形剤、経口液剤、注射剤、点鼻剤、スプレー剤、吸入散剤などが挙げられる。 <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, point Examples include nasal sprays, sprays, and inhaled powders.
－経口固形剤－
　前記経口固形剤としては、例えば、錠剤、被覆錠剤、顆粒剤、散剤、カプセル剤などが挙げられる。
　前記経口固形剤の製造方法としては、特に制限はなく、常法を使用することができ、例えば、前記シアル酸含有糖鎖複合体に、賦形剤、及び必要に応じて各種添加剤を加えることにより、製造することができる。ここで、前記賦形剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、乳糖、白糖、塩化ナトリウム、ブドウ糖、デンプン、炭酸カルシウム、カオリン、微結晶セルロース、珪酸などが挙げられる。また、前記添加剤としても、特に制限はなく、目的に応じて適宜選択することができ、例えば、結合剤、崩壊剤、滑沢剤、着色剤、矯味／矯臭剤などが挙げられる。 -Oral solid preparation-
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.
　前記結合剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、水、エタノール、プロパノール、単シロップ、ブドウ糖液、デンプン液、ゼラチン液、カルボキシメチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルスターチ、メチルセルロース、エチルセルロース、シェラック、リン酸カルシウム、ポリビニルピロリドンなどが挙げられる。
　前記崩壊剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、乾燥デンプン、アルギン酸ナトリウム、カンテン末、炭酸水素ナトリウム、炭酸カルシウム、ラウリル硫酸ナトリウム、ステアリン酸モノグリセリド、乳糖などが挙げられる。
　前記滑沢剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、精製タルク、ステアリン酸塩、ホウ砂、ポリエチレングリコールなどが挙げられる。
　前記着色剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、酸化チタン、酸化鉄などが挙げられる。
　前記矯味／矯臭剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、白糖、橙皮、クエン酸、酒石酸などが挙げられる。 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.
－経口液剤－
　前記経口液剤としては、例えば、内服液剤、シロップ剤、エリキシル剤などが挙げられる。
　前記経口液剤の製造方法としては、特に制限はなく、常法を使用することができ、例えば、前記シアル酸含有糖鎖複合体に添加剤を加えることにより、製造することができる。ここで、前記添加剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、矯味／矯臭剤、緩衝剤、安定化剤などが挙げられる。 -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 liquid agent, 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.
　前記矯味／矯臭剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、白糖、橙皮、クエン酸、酒石酸などが挙げられる。
　前記緩衝剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、クエン酸ナトリウムなどが挙げられる。
　前記安定化剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、トラガント、アラビアゴム、ゼラチンなどが挙げられる。 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.
－注射剤－
　前記注射剤としては、例えば、溶液、懸濁液、用事溶解用固形剤などが挙げられる。
　前記注射剤の製造方法としては、特に制限はなく、常法を使用することができ、例えば、前記シアル酸含有糖鎖複合体に、ｐＨ調節剤、緩衝剤、安定化剤、等張化剤、局所麻酔剤などを添加することにより、製造することができる。ここで、前記ｐＨ調節剤及び前記緩衝剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、クエン酸ナトリウム、酢酸ナトリウム、リン酸ナトリウムなどが挙げられる。また、前記安定化剤としても、特に制限はなく、目的に応じて適宜選択することができ、例えば、ピロ亜硫酸ナトリウム、ＥＤＴＡ、チオグリコール酸、チオ乳酸などが挙げられる。前記等張化剤としても、特に制限はなく、目的に応じて適宜選択することができ、例えば、塩化ナトリウム、ブドウ糖などが挙げられる。前記局所麻酔剤としても、特に制限はなく、目的に応じて適宜選択することができ、例えば、塩酸プロカイン、塩酸リドカインなどが挙げられる。 -Injection-
Examples of the injection include a solution, a suspension, and a solid agent for dissolving for use.
The method for producing the injection is not particularly limited, and a conventional method can be used. 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.
－点鼻剤－
　前記点鼻剤としては、例えば、液剤、スプレー剤、軟膏剤などが挙げられる。
　前記点鼻剤の製造方法としては、特に制限はなく、常法を使用することができ、例えば、前記シアル酸含有糖鎖複合体に添加剤を加えることにより、製造することができる。ここで、前記添加剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、塩化ベンザルコニウム、クエン酸、Ｄ－ソルビトール、グリセリン、エデト酸ナトリウム、結晶セルロース、ポリソルベート８０、ポリビニルアルコール、フェニルエチルアルコール、ｐＨ調節剤などが挙げられる。 -Nasal agent-
Examples of the nasal drops include liquids, sprays, ointments and the like.
There is no restriction | limiting in particular as a manufacturing method of the said nasal drop, 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, the additive is not particularly limited and may be appropriately selected depending on the intended purpose. For example, benzalkonium chloride, citric acid, D-sorbitol, glycerin, sodium edetate, crystalline cellulose, polysorbate 80 , Polyvinyl alcohol, phenylethyl alcohol, pH adjuster and the like.
－スプレー剤－
　前記スプレー剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、エアゾール剤、粉剤、乳剤、液剤、水和剤などが挙げられる。
　スプレー剤の製造方法としては、特に制限はなく、常法を使用することができ、例えば、米国特許第２，８６８，６９１号明細書及び米国特許第３，０９５，３５５号明細書に記載の方法などを使用して製造することができる。 -Spray agent-
There is no restriction | limiting in particular as said spray agent, According to the objective, it can select suitably, For example, an aerosol agent, a powder agent, an emulsion, a liquid agent, a wettable powder etc. are mentioned.
There is no restriction | limiting in particular as a manufacturing method of a spray agent, A normal method can be used, for example, as described in US Patent 2,868,691 and US Patent 3,095,355. It can be manufactured using a method or the like.
＜使用＞
　前記抗インフルエンザウイルス剤は、１種単独で使用されてもよく、他の成分を有効性とする薬剤と併用してもよく、また他の成分を有効性とする薬剤に配合されてもよい。
　前記他の薬剤としては、特に制限はなく、目的に応じて適宜選択することができるが、オセルタミビル（タミフル（登録商標））、ザナミビル（リレンザ（登録商標））、アマンタジン（シンメトレル（登録商標））等の既存の抗インフルエンザウイルス薬が好ましく、タミフルがより好ましい。
　前記抗インフルエンザウイルス剤は、インフルエンザウイルス感染時及び感染後のいずれの時期においても好適に抗インフルエンザウイルス活性を有するが、前記薬剤は、主に感染初期に抗インフルエンザウイルス活性を有し、前記抗インフルエンザウイルス剤とは作用機序が異なることから、これらを併用することにより、相乗効果を有する点で有利である。また、例えば、併用する薬剤の有効濃度を単独使用の約１／１０レベルまで低下させても十分な抗インフルエンザウイルス活性を得ることができ、使用に起因する副作用を軽減できる安全性の高いインフルエンザの感染予防又は治療薬となる。 <Use>
The said anti-influenza virus agent may be used individually by 1 type, may be used together with the chemical | medical agent which makes another component effective, and may be mix | blended with the chemical | medical agent which makes another component effective.
There is no restriction | limiting in particular as said other chemical | medical agent, Although it can select suitably according to the objective, Oseltamivir (Tamiflu (trademark)), Zanamivir (relenza (trademark)), Amantadine (Symmetrel (trademark)) Existing anti-influenza virus drugs such as Tamiflu are preferred.
The anti-influenza virus agent preferably has anti-influenza virus activity both at the time of influenza virus infection and after infection, but the drug has anti-influenza virus activity mainly in the early stage of infection, and the anti-influenza virus Since the mechanism of action is different from that of a viral agent, the combined use of these agents is advantageous in that it has a synergistic effect. In addition, for example, it is possible to obtain sufficient anti-influenza virus activity even if the effective concentration of the drug used in combination is reduced to about 1/10 level of single use, and the highly safe influenza that can reduce side effects caused by use. Infectious prevention or treatment.
＜投与＞
　前記抗インフルエンザウイルス剤の投与方法としては、特に制限はなく、例えば、前記抗インフルエンザウイルス剤の剤型などに応じて適宜選択することができ、経口又は非経口で投与することができる。 <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.
　前記抗インフルエンザウイルス剤の投与量としても、特に制限はなく、投与対象個体の年齢、体重、体質、症状、他の薬剤の投与有無など、様々な要因を考慮して適宜選択することができる。 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.
　また、個体への投与時期にも、特に制限はなく、目的に応じて適宜選択することができる。例えば、インフルエンザウイルスの感染前に投与してもよく、感染後に投与してもよい。前記抗インフルエンザウイルス剤は、投与時期に制限がないため、既存の薬剤と比べて有利である。 Moreover, there is no restriction | limiting in particular also in the time of administration to an individual | organism, It can select suitably according to the objective. For example, it may be administered before or after infection with influenza virus. The anti-influenza virus agent is advantageous compared to existing drugs because there is no restriction on the administration time.
＜対象＞
　前記抗インフルエンザウイルス剤の投与対象となる動物種としては、インフルエンザウイルスに感染する可能性のある動物種であれば特に制限はなく、目的に応じて適宜選択することができ、例えば、ヒト、トリ、サル、ブタ、ウシ、ヒツジ、ヤギ、イヌ、ネコ、マウス、ラットなどが挙げられる。 <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.
　また、前記抗インフルエンザウイルス剤の適用対象となるインフルエンザウイルスの種類としても、特に制限されるものではない。前記抗インフルエンザウイルス剤は、Ａ型、Ｂ型、Ｃ型全てのインフルエンザウイルスについて、感染抑制効果が期待でき、また、Ａ型インフルエンザウイルスについては、その由来を問わず、全ての亜型（Ｈ１Ｎ１～Ｈ１５Ｎ９）のインフルエンザウイルスについて、感染抑制効果が期待できる。 Also, 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 suppress infection of all influenza viruses of types A, B, and C, and all types of influenza viruses (H1N1- With regard to the influenza virus H15N9), an infection suppression effect can be expected.
＜用途＞
　前記抗インフルエンザウイルス剤は、優れた抗インフルエンザウイルス活性を有し、投与時期や投与対象の年齢などに制限がなく、かつ副作用がなく安全性が高いため、インフルエンザウイルスの予防又は治療に好適に利用可能である。
　また、前記抗インフルエンザウイルス剤は、基材に担持することもでき、後述する本発明のフィルターなどに担持させて利用することもできる。 <Application>
The anti-influenza virus agent has excellent anti-influenza virus activity, has no restrictions on the administration time and age of administration, has no side effects, and is highly safe. Therefore, it is suitable for the prevention or treatment of influenza viruses. Is possible.
In addition, the anti-influenza virus agent can be supported on a substrate, or can be used by supporting it on a filter of the present invention described later.
（フィルター）
　本発明のフィルターは、前記抗インフルエンザウイルス剤を担持し、必要に応じて、更にその他の構成を有する。 (filter)
The filter of the present invention carries the anti-influenza virus agent, and further has other configurations as necessary.
　前記フィルターにおいて、前記抗インフルエンザウイルス剤を担持させる量としては、特に制限はなく、目的に応じて適宜選択することができる。
　前記フィルターに、前記抗インフルエンザウイルス剤を担持させる方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記抗インフルエンザウイルス剤をそのまま、あるいは水やアルコール等で希釈した状態の抗インフルエンザウイルス剤を、噴霧する方法、被覆する方法、浸漬する方法などが挙げられる。 The amount of the anti-influenza virus agent carried in the filter is not particularly limited and can be appropriately selected depending on the purpose.
The method for supporting the anti-influenza virus agent on the filter is not particularly limited and can be appropriately selected depending on the purpose. For example, the anti-influenza virus agent is diluted as it is or with water or alcohol. Examples of the anti-influenza virus agent in a state include a spraying method, a coating method, and a dipping method.
　前記フィルターとしては、特に制限はなく、目的に応じて適宜選択することができ、例えば、マスク用フィルター、エアコン用フィルター、空気清浄機用フィルター、掃除機用フィルター、介護用フィルター、ベットマット用フィルター、壁乃至天井用フィルターなどが挙げられる。 There is no restriction | limiting in particular as said filter, According to the objective, it can select suitably, For example, the filter for masks, the filter for air conditioners, the filter for air cleaners, the filter for vacuum cleaners, the filter for care, the filter for bed mats And wall or ceiling filters.
　前記フィルターに用いられる材質としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ポリエチレン、ポリエステル、ポリアミド、ポリアクリル、ポリプロピレン、ナイロン、ポリカーボネート、レーヨン、ビスマイレイミドトリアジン等のプラスチック材；紙、綿、麻、絹等の天然繊維；ガラス繊維等の無機材料などが挙げられる。 The material used for the filter is not particularly limited and may be appropriately selected depending on the purpose. Examples thereof include polyethylene, polyester, polyamide, polyacryl, polypropylene, nylon, polycarbonate, rayon, and bis-maleimide triazine. Examples include plastic materials; natural fibers such as paper, cotton, hemp, and silk; and inorganic materials such as glass fibers.
　前記フィルターの形態としては、特に制限はなく、目的に応じて適宜選択することができるが、前記フィルターが、マスク用フィルターである場合、ガーゼ、不織布、布が好ましい。 The form of the filter is not particularly limited and may be appropriately selected according to the purpose. However, when the filter is a mask filter, gauze, non-woven fabric, and cloth are preferable.
　前記フィルターの目の粗さや厚みとしては、特に制限はなく、目的に応じて適宜選択することができる。
　また、前記フィルターは、単層構造及び多層構造のいずれの構造であってもよい。多層構造の場合、前記抗インフルエンザウイルス剤は、少なくとも１層に担持されていればよい。 There is no restriction | limiting in particular as the coarseness and thickness of the said filter, According to the objective, it can select suitably.
The filter may have a single layer structure or a multilayer structure. In the case of a multilayer structure, the anti-influenza virus agent only needs to be carried in at least one layer.
　本発明のフィルターは、前記抗インフルエンザウイルス剤を担持しているため、優れた抗インフルエンザウイルス活性を有し、安全性が高く、安価に製造可能であるため、空気中に浮遊するインフルエンザウイルスの吸入を効率よく防ぐことができる。 Since the filter of the present invention carries the anti-influenza virus agent, it has excellent anti-influenza virus activity, is highly safe, and can be manufactured at a low cost, so that inhalation of influenza virus floating in the air can be achieved. Can be efficiently prevented.
（効果）
　本発明のシアル酸含有糖鎖複合体は、抗インフルエンザウイルス活性を有しており、本発明の抗インフルエンザウイルス剤の有効成分として、好ましく利用可能である。また、前記シアル酸含有糖鎖複合体は、本発明のシアル酸含有糖鎖複合体の製造方法により効率的に得ることができる。
　更に、本発明のシアル酸含有糖鎖複合体は、規定の分子量の範囲内で、シアル酸含有糖鎖と規定の炭素数を有する脂肪酸とを任意に組み合わせて設計及び製造することができるため、様々な型の複合体を提供することが可能であり、したがって、様々な効果を有する抗インフルエンザウイルス剤の提供が期待される。 (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.
Furthermore, 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 fatty acid having a specified carbon number within a specified molecular weight range, It is possible to provide various types of complexes, and therefore, it is expected to provide anti-influenza virus agents having various effects.
　本発明の抗インフルエンザウイルス剤は、好ましい抗インフルエンザウイルス活性を有し、Ａ型、Ｂ型、Ｃ型を問わず、全てのインフルエンザウイルスに対して感染抑制効果が期待でき、また、Ａ型インフルエンザウイルスについては、全ての亜型について、その動物種由来を問わず、感染抑制効果が期待できることから、現在抗インフルエンザ薬として使用認可されているアマンタジンやタミフルに次ぐ新たな抗インフルエンザ薬として、臨床応用の可能性が期待される。 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 the animal species, they are clinically applied as new anti-influenza drugs after amantadine and Tamiflu, which are currently approved for use as anti-influenza drugs. Potential is expected.
　また、インフルエンザウイルスは、膜表面のタンパク質を僅かに変異させることで、抗体という生体防御機能を免れる能力を持つ。それに比べ、インフルエンザウイルスの、レセプターを認識し結合する部位はよく保存されており、レセプターに対する高い特異性はよく保持されている。本発明の抗インフルエンザウイルス剤は、このウイルス－レセプター結合の選択的特異性による感染の、競争的阻害効果を利用することに着目したものである。したがって、本発明の抗インフルエンザウイルス剤は、単独で使用する場合だけでなく、インフルエンザワクチンや、作用機構の異なる既存薬との併用においても、インフルエンザに対する感染予防と治療に対する大きな効果が期待される。 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 the high specificity for the receptor is well maintained. 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.
　以下に本発明の実施例を挙げて本発明を具体的に説明するが、本発明はこれらの実施例に何ら限定されるものではない。 Hereinafter, the present invention will be specifically described with reference to examples of the present invention, but the present invention is not limited to these examples.
（試験例１：製造方法の検討）
＜従来法による３’－ＳＬ－Ｎ－Ｃ１２の製造＞
　特開２００７－３０８４４４号公報に開示された方法で、下記構造式で表される３’－シアリルラクトシルラウリン酸アミド（３’－ＳＬ－Ｎ－Ｃ１２：３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１２、分子量：８１４）を、以下のようにして製造した。下記構造式において「Ａｃ」は、アセチル基を表す。
Figure JPOXMLDOC01-appb-C000001
 (Test Example 1: Examination of production method)
<Production of 3′-SL-N-C12 by Conventional Method>
3′-sialyl lactosyl lauric acid amide (3′-SL—N—C12: 3′-Neu5Ac (α2-3) Gal represented by the following structural formula is disclosed by the method disclosed in JP-A-2007-308444. (Β1-4) Glc-N-C12, molecular weight: 814) was prepared as follows. In the following structural formula, “Ac” represents an acetyl group.
Figure JPOXMLDOC01-appb-C000001
　３’－シアリルラクトース（３’－ＳＬ）（ウシ初乳由来、シグマアルドリッチ社製、分子量：６３３）を、過飽和炭酸水素アンモニウム水溶液に溶解し、室温で４日間、撹拌を行った。途中、反応の進行度合いを、薄層クロマトグラフィー（ＴＬＣ）で確認した。反応終了後、ロータリーエバポレーターを用いて、水分と共に炭酸水素アンモニウムを、反応物から除去した。
　次いで、得られた反応物を、０℃条件下、炭酸ナトリウム存在下で、塩化ラウリン酸と反応させた。ここで、塩化ラウリン酸は、３’－ＳＬの使用量に対し、モル比で５倍となるような量で使用した。また、炭酸ナトリウムは、塩化ラウリン酸の使用量に対し、モル比で等量となるような量で使用した。
　反応の進行状況は、開始から約３０分間間隔で、サンプリングを行い、ＴＬＣにより確認し、反応の進行が飽和に達した時点で反応を終了した。反応液に等量のクロロホルムを加え、未反応の塩化ラウリン酸を抽出し除去した。ＴＬＣは、テトラヒドロフラン／アセトニトリル／１－プロパノール／０．６Ｍ酢酸アンモニウム水溶液／２８体積％アンモニア水（５：１０：５０：３５：０．３（体積比））で展開した。
　得られた３’－ＳＬ－Ｎ－Ｃ１２、及び、出発物質の３’－ＳＬのシアル酸量を、レゾシノール－塩酸法、及び、呈色後にデンシトメーター（株式会社島津製作所製）で定量したところ、３’－ＳＬ－Ｎ－Ｃ１２の収率は、１０質量％以下であった。また、反応開始２４時間後のＴＬＣの結果を図１に示す。 3′-sialyllactose (3′-SL) (derived from bovine colostrum, 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 confirmed 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 an amount such 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 intervals of about 30 minutes from the start and confirmed by TLC. 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. TLC was developed with tetrahydrofuran / acetonitrile / 1-propanol / 0.6 M aqueous ammonium acetate / 28% by volume aqueous ammonia (5: 10: 50: 35: 0.3 (volume ratio)).
The amount of sialic acid in the obtained 3′-SL—N—C12 and the starting material 3′-SL was quantified with the resorcinol-hydrochloric acid method and after coloration with a densitometer (manufactured by Shimadzu Corporation). However, the yield of 3′-SL—N—C12 was 10% by mass or less. The results of TLC 24 hours after the start of the reaction are shown in FIG.
＜本発明の方法による３’－ＳＬ－Ｎ－Ｃ１２の製造＞
　前記従来法において、塩化ラウリン酸に代えてラウリン酸を用い、また炭酸ナトリウムに代えて縮合剤（ＤＭＴ－ＭＭ：４－（４，６－ｄｉｍｅｔｈｏｘｙ－１，３，５－ｔｒｉａｚｉｎ－２－ｙｌ）－４－ｍｅｔｈｙｌｍｏｒｐｈｏｌｉｎｉｕｍｃｈｌｏｒｉｄｅ、国産化学株式会社製）を用い、反応を０℃に変えて室温で行ったこと以外は、従来法と同様の方法で３’－ＳＬ－Ｎ－Ｃ１２を製造した。
　反応の進行状況は、開始から約３０分間間隔で、サンプリングを行い、薄層クロマトグラフィー（ＴＬＣ）により確認し、反応の進行が飽和に達した時点で反応を終了した。
　反応液の溶媒を除去した後、前記反応液と等量のヘキサンを加え、十分に攪拌した後、遠心分離して上清を除去した。この操作を、更に２度行うことで、未反応のラウリン酸を除去した。次いで、反応物を水で溶解し、Ｃ１８逆相カラム（Ｐｒｅｐａｒａｔｉｖｅ　Ｃ１８　Ｂｕｌｋ　Ｐａｃｋｉｎｇ　Ｍａｔｅｒｉａｌｓ（ウォーターズ社製））に負荷し、メタノール：水（５：５（体積比））、次いで、メタノール：水（８：２（体積比））の順で溶出し、目的物を含む画分を回収した。ＴＬＣは、テトラヒドロフラン／アセトニトリル／１－プロパノール／０．６Ｍ酢酸アンモニウム水溶液／２８体積％アンモニア水（５：１０：５０：３５：０．３（体積比））で展開した。
　３’－ＳＬ－Ｎ－Ｃ１２の収率を前記同様の方法で求めたところ、５０質量％であった。また、反応開始３０分間後と、２４時間後のＴＬＣの結果を図１に、反応開始３０分間後のＴＬＣの結果を図３Ａ及び３Ｂに示す。 <Production of 3'-SL-N-C12 by the method of the present invention>
In the above conventional method, lauric acid is used in place of chlorinated lauric acid, and a condensing agent (DMT-MM: 4- (4,6-dimoxy-1,3,5-triazin-2-yl) is used in place of sodium carbonate. 3′-SL-N—C12 was produced in the same manner as in the conventional method except that the reaction was carried out at room temperature by changing the reaction temperature to 0 ° C. using -4-methylmorpholine chloride (manufactured by Kokusan Chemical Co., Ltd.).
The progress of the reaction was sampled at intervals of about 30 minutes from the start and confirmed by thin layer chromatography (TLC). The reaction was terminated when the progress of the reaction reached saturation.
After removing the solvent from the reaction solution, an equal amount of hexane was added to the reaction solution, and after sufficient stirring, the supernatant was removed by centrifugation. This operation was repeated twice to remove unreacted lauric acid. The reaction was then dissolved in water and loaded onto a C18 reverse phase column (Preparative C18 Bulk Packing Materials (Waters)), methanol: water (5: 5 (volume ratio)), then methanol: water (8 : 2 (volume ratio)), and fractions containing the desired product were collected. TLC was developed with tetrahydrofuran / acetonitrile / 1-propanol / 0.6 M aqueous ammonium acetate / 28% by volume aqueous ammonia (5: 10: 50: 35: 0.3 (volume ratio)).
The yield of 3′-SL—N—C12 was determined by the same method as described above, and was 50% by mass. Further, TLC results after 30 minutes and 24 hours from the start of the reaction are shown in FIG. 1, and TLC results 30 minutes after the start of the reaction are shown in FIGS. 3A and 3B.
　試験例１の結果、本発明の製造方法は、従来法と比較して、短時間でシアル酸含有糖鎖複合体を得ることができ、更に収率が高いことがわかった。 As a result of Test Example 1, it was found that the production method of the present invention was able to obtain a sialic acid-containing sugar chain complex in a short time as compared with the conventional method, and the yield was higher.
（製造例１：３’－ＳＬ－Ｎ－Ｃ１４の製造）
　３’－シアリルラクトース（３’－ＳＬ）（ウシ初乳由来、シグマアルドリッチ社製、分子量：６３３）を、過飽和炭酸水素アンモニウム水溶液に溶解し、室温で４日間、撹拌を行った。途中、反応の進行度合いを、薄層クロマトグラフィー（ＴＬＣ）で確認した。反応終了後、ロータリーエバポレーターを用いて、水分と共に炭酸水素アンモニウムを、反応物から除去した。
　次いで、得られた反応物を、室温条件下、ＤＭＴ－ＭＭ存在下で、ミリスチン酸（Ｃ１４）と反応させた。ここで、ミリスチン酸（Ｃ１４）は、３’－ＳＬの使用量に対し、モル比で５倍となるような量で使用した。また、ＤＭＴ－ＭＭは、ミリスチン酸（Ｃ１４）の使用量に対し、モル比で等量となるような量で使用した。
　反応の進行状況は、開始から約３０分間間隔で、サンプリングを行い、ＴＬＣ（展開溶媒：テトラヒドロフラン／アセトニトリル／１－プロパノール／０．６Ｍ酢酸アンモニウム水溶液／２８体積％アンモニア水（５：１０：５０：３５：０．３（体積比））により確認し、反応の進行が飽和に達した時点で反応を終了した。ＴＬＣで確認した結果を図２に示す。反応開始３０分間で、高い収率が得られた。 (Production Example 1: Production of 3'-SL-N-C14)
3′-sialyllactose (3′-SL) (derived from bovine colostrum, 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 confirmed 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 myristic acid (C14) in the presence of DMT-MM at room temperature. Here, myristic acid (C14) was used in such an amount that the molar ratio was five times that of 3′-SL. In addition, DMT-MM was used in an amount that was equivalent in molar ratio to the amount of myristic acid (C14) used.
The progress of the reaction was sampled at intervals of about 30 minutes from the start, and TLC (developing solvent: tetrahydrofuran / acetonitrile / 1-propanol / 0.6 M aqueous ammonium acetate solution / 28% by volume aqueous ammonia (5:10:50: 35: 0.3 (volume ratio)), and the reaction was terminated when the progress of the reaction reached saturation, and the result confirmed by TLC is shown in Fig. 2. A high yield was obtained in 30 minutes from the start of the reaction. Obtained.
　３０分間反応させた反応液の溶媒を除去した後、前記反応液と等量のヘキサンを加え、十分に攪拌した後、遠心分離して上清を除去した。この操作を、更に２度行うことで、未反応のミリスチン酸を除去した。次いで、反応物を水で溶解し、Ｃ１８逆相カラム（Ｐｒｅｐａｒａｔｉｖｅ　Ｃ１８　Ｂｕｌｋ　Ｐａｃｋｉｎｇ　Ｍａｔｅｒｉａｌｓ（ウォーターズ社製））に負荷し、メタノール：水（５：５（体積比））、次いで、メタノール：水（８：２（体積比））の順で溶出し、目的物を含む画分を回収した。
　これにより、下記構造式で表される３’－シアリルラクトシルミリスチン酸アミド（３’－ＳＬ－Ｎ－Ｃ１４：３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１４、分子量：８４２）を得た。下記構造式において「Ａｃ」は、アセチル基を表す。
　精製後の３’－ＳＬ－Ｎ－Ｃ１４について、前記同様の方法でＴＬＣにより確認した結果を図３Ｃ及び図４に示す。なお、図３Ａ～Ｃ及び図４において、「Ｍ」は、マーカーとして使用したクジラ脳ガングリオシド混合液（ＧＭ４、ＧＭ３、ＧＭ２、ＧＭ１、ＧＤ３、ＧＤ１ａ、ＧＤ１ｂ、及びＧＴ１ｂ）を示す。
Figure JPOXMLDOC01-appb-C000002
 After removing the solvent from the reaction solution reacted for 30 minutes, hexane was added in an amount equal to that of the reaction solution, sufficiently stirred, and then centrifuged to remove the supernatant. This operation was further performed twice to remove unreacted myristic acid. The reaction was then dissolved in water and loaded onto a C18 reverse phase column (Preparative C18 Bulk Packing Materials (Waters)), methanol: water (5: 5 (volume ratio)), then methanol: water (8 : 2 (volume ratio)), and fractions containing the desired product were collected.
Thus, 3′-sialyl lactosyl myristate amide (3′-SL-N—C14: 3′-Neu5Ac (α2-3) Gal (β1-4) Glc-N—C14 represented by the following structural formula, Molecular weight: 842) was obtained. In the following structural formula, “Ac” represents an acetyl group.
FIG. 3C and FIG. 4 show the results of confirming 3′-SL-N—C14 after purification by TLC in the same manner as described above. 3A to 3C and FIG. 4, “M” indicates a whale brain ganglioside mixed solution (GM4, GM3, GM2, GM1, GD3, GD1a, GD1b, and GT1b) used as a marker.
Figure JPOXMLDOC01-appb-C000002
　なお、前記クジラ脳ガングリオシド混合液は、以下の方法で調製した。
　ミンククジラの脳（日本鯨類研究所より譲渡）に、脳組織重量に対して５倍容量のクロロホルム／メタノール混合溶液（２：１（体積比））を添加して抽出した後、この抽出物を更に前記脳組織重量に対して５倍容量のクロロホルム／メタノール混合溶液（１：２（体積比））を添加して抽出し、総脂質画分を得た。得られた総脂質画分をクロロホルム／メタノール／水混合溶液（約３０：６０：８（体積比））に調製し、陰イオン交換（ＤＥＡＥ－Ｓｅｐｈａｄｅｘ　Ａ－２５（ＧＥヘルスケア社製））カラムに吸着させた。前記総脂質画分の５倍容量のクロロホルム／メタノール／水混合溶液（３０：６０：８（体積比））、次いでメタノールで中性成分及び塩基性成分を洗浄後、前記総脂質画分の５倍容量の０．１Ｍ酢酸ナトリウム／メタノール混合溶液にて吸着成分を溶出した。溶出された溶液に１Ｍ　ＮａＯＨ／メタノール混合溶液を必要量加え、０．０５Ｍ濃度とし、室温にて３０分間放置し、エステル結合を含むリン脂質類を分解した（以下、「弱アルカリ処理」と称することがある。）。弱アルカリ処理後、酢酸の添加量が、前記ＮａＯＨと等量になるように酢酸／メタノール混合溶液を添加して中和し、窒素気流下でメタノールを蒸発させて濃縮した。次いで、乾燥物を少量の水に溶解し、Ｓｅｐｈａｄｅｘ　Ｇ－１０　カラム（ＧＥヘルスケア社製）で脱塩を行い、ガングリオシド画分を凍結乾燥した。この凍結乾燥物を、クロロホルム／メタノール溶液（２：１（体積比））で溶解し、ＴＬＣの標準試料として使用した。 The whale brain ganglioside mixed solution was prepared by the following method.
Extracted by adding 5 times volume of chloroform / methanol mixed solution (2: 1 (volume ratio)) to the brain of minke whale (assigned from Japan Whale Research Institute) Was further extracted by adding a 5-fold volume chloroform / methanol mixed solution (1: 2 (volume ratio)) to the brain tissue weight to obtain a total lipid fraction. The obtained total lipid fraction was prepared in a chloroform / methanol / water mixed solution (about 30: 60: 8 (volume ratio)), and an anion exchange (DEAE-Sephadex A-25 (GE Healthcare)) column. It was made to adsorb to. After washing the neutral component and the basic component with a 5-fold volume chloroform / methanol / water mixed solution (30: 60: 8 (volume ratio)) of the total lipid fraction and then methanol, the total lipid fraction 5 The adsorbed components were eluted with a double volume of 0.1 M sodium acetate / methanol mixed solution. Necessary amount of 1M NaOH / methanol mixed solution was added to the eluted solution to adjust the concentration to 0.05M and left at room temperature for 30 minutes to decompose phospholipids containing ester bonds (hereinafter referred to as “weak alkali treatment”). Sometimes.). After the weak alkali treatment, the mixture was neutralized by adding an acetic acid / methanol mixed solution so that the amount of acetic acid added was equal to that of the NaOH, and concentrated by evaporating methanol under a nitrogen stream. Next, the dried product was dissolved in a small amount of water, desalted with a Sephadex G-10 column (manufactured by GE Healthcare), and the ganglioside fraction was freeze-dried. This lyophilizate was dissolved in a chloroform / methanol solution (2: 1 (volume ratio)) and used as a standard sample for TLC.
　なお、３’－ＳＬ－Ｎ－Ｃ１４の製造原料として使用した３’－ＳＬについて、プロトン核磁気共鳴スペクトルとして、アセトンを内部標準とし、４００ＭＨｚにおいて重水（Ｄ２Ｏ）中で測定したプロトンＮＭＲスペクトルは、図５に示す通りである。シアル酸含有物質に認められるピークとして、シアル酸（Ｎｅｕ５Ａｃ）のＣ３ａｘのピークが１．８ｐｐｍ付近に、Ｃ３ｅｑのピークが２．７５付近に認められた。また、ガラクトース（Ｇａｌ）のＣ１のアノマープロトンのピークが４．５ｐｐｍ付近に、糖鎖還元末端のグルコース（Ｇｌｃ）のアノマープロトンのピークが、αとβとに分かれ、それぞれ４．６５ｐｐｍ付近、及び５．２ｐｐｍ付近に認められた。更に、ガラクトース（Ｇａｌ）のＣ３のプロトンのピークが４．１ｐｐｍ付近に認められた。なお、４．７ｐｐｍ付近のピークは水由来のシグナルであり、２．２２５ｐｐｍ付近のピークはアセトン由来のシグナルである。 The proton NMR spectrum of 3'-SL used as a raw material for producing 3'-SL-N-C14 was measured as proton nuclear magnetic resonance spectrum using acetone as an internal standard in heavy water (D 2 O) at 400 MHz. Is as shown in FIG. As peaks observed in the sialic acid-containing substance, a C3ax peak of sialic acid (Neu5Ac) was observed near 1.8 ppm, and a C3eq peak was observed near 2.75. In addition, the anomeric proton peak of C1 of galactose (Gal) is divided around 4.5 ppm, and the anomeric proton peak of glucose (Glc) at the reducing end of the sugar chain is divided into α and β, which are around 4.65 ppm, and It was recognized near 5.2 ppm. Further, a C3 proton peak of galactose (Gal) was observed in the vicinity of 4.1 ppm. The peak near 4.7 ppm is a signal derived from water, and the peak near 2.225 ppm is a signal derived from acetone.
（製造例２：３’－ＳＬ－Ｎ－Ｃ１６の製造）
　製造例１において、ラウリン酸に代えて、パルミチン酸（Ｃ１６）を用いたこと以外は製造例１と同様の方法で、下記構造式で表される３’－シアリルラクトシルパルミチン酸アミド（３’－ＳＬ－Ｎ－Ｃ１６：３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１６、分子量：８７０）を得た。下記構造式において「Ａｃ」は、アセチル基を表す。
　製造例１において、３’－ＳＬ－Ｎ－Ｃ１４を含む反応液に代えて、３’－ＳＬ－Ｎ－Ｃ１６を含む反応液を用いたこと以外は、製造例１と同様の方法で、３’－ＳＬ－Ｎ－Ｃ１６を精製した。更に、３’－ＳＬ－Ｎ－Ｃ１６を製造例１と同様の方法でＴＬＣにより確認を行った。結果を図３Ｂに示す。
Figure JPOXMLDOC01-appb-C000003
 (Production Example 2: Production of 3'-SL-N-C16)
In Production Example 1, 3′-sialyllactosyl palmitic acid amide (3 ′) represented by the following structural formula was prepared in the same manner as in Production Example 1 except that palmitic acid (C16) was used instead of lauric acid. -SL-N-C16: 3'-Neu5Ac (α2-3) Gal (β1-4) Glc-N-C16, molecular weight: 870) was obtained. In the following structural formula, “Ac” represents an acetyl group.
In Production Example 1, a reaction solution containing 3′-SL-N—C16 was used in the same manner as in Production Example 1, except that a reaction solution containing 3′-SL—N—C16 was used instead of the reaction solution containing 3′-SL—N—C14. '-SL-N-C16 was purified. Further, 3′-SL—N—C16 was confirmed by TLC in the same manner as in Production Example 1. The results are shown in FIG. 3B.
Figure JPOXMLDOC01-appb-C000003
（製造例３：３’－ＳＬ－Ｎ－Ｃ１８の製造）
　製造例１において、ラウリン酸に代えて、ステアリン酸（Ｃ１８）を用いたこと以外は製造例１と同様の方法で、下記構造式で表される３’－シアリルラクトシルステアリン酸アミド（３’－ＳＬ－Ｎ－Ｃ１８：３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１８、分子量：８９８）を得た。下記構造式において「Ａｃ」は、アセチル基を表す。
　製造例１において、３’－ＳＬ－Ｎ－Ｃ１４を含む反応液に代えて、３’－ＳＬ－Ｎ－Ｃ１８を含む反応液を用いたこと以外は、製造例１と同様の方法で、３’－ＳＬ－Ｎ－Ｃ１８を精製した。
　更に、３’－ＳＬ－Ｎ－Ｃ１８を製造例１と同様の方法でＴＬＣにより確認を行った。結果を図３Ｃに示す。また、プロトン核磁気共鳴スペクトルとして、ＴＭＳ（テトラメチルシラン）を内部標準とし、６００ＭＨｚにおいて２質量％重水（Ｄ２Ｏ）を含有する重ジメチルスルホキシド（ＤＭＳＯ－ｄ６）中で測定したプロトンＮＭＲスペクトルは、図６に示す通りである。３’－ＳＬと比較して、ステアリン酸が導入されたことにより、メチルプロトンのピークが０．９ｐｐｍ付近に認められた。また、メチレンプロトンのピークが１．３ｐｐｍ付近に認められ、更にα炭素のプロトンのピークが２．１ｐｐｍ付近に、β炭素のプロトンのピークが１．５ｐｐｍ付近に低磁場シフトして認められた。このメチレンプロトンのシグナルの総和と、メチルプロトンのシグナルとから、３’－ＳＬ－Ｎ－Ｃ１８であることが確認された。なお、３．４ｐｐｍ付近のピークは重水（ＤＨＯ）由来のシグナルである。
Figure JPOXMLDOC01-appb-C000004
 (Production Example 3: Production of 3'-SL-N-C18)
In Production Example 1, 3′-sialyllactosyl stearamide (3 ′) represented by the following structural formula was used in the same manner as in Production Example 1 except that stearic acid (C18) was used instead of lauric acid. -SL-N-C18: 3'-Neu5Ac (α2-3) Gal (β1-4) Glc-N-C18, molecular weight: 898). In the following structural formula, “Ac” represents an acetyl group.
In Production Example 1, a reaction solution containing 3′-SL—N—C18 was used instead of the reaction solution containing 3′-SL—N—C14, except that a reaction solution containing 3′-SL—N—C18 was used. '-SL-N-C18 was purified.
Further, 3′-SL—N—C18 was confirmed by TLC in the same manner as in Production Example 1. The results are shown in FIG. 3C. In addition, as a proton nuclear magnetic resonance spectrum, a proton NMR spectrum measured in deuterated dimethyl sulfoxide (DMSO-d6) containing 2 mass% heavy water (D 2 O) at 600 MHz with TMS (tetramethylsilane) as an internal standard is This is as shown in FIG. Compared with 3′-SL, a peak of methyl proton was observed around 0.9 ppm due to the introduction of stearic acid. Further, a methylene proton peak was observed near 1.3 ppm, an α-carbon proton peak was observed near 2.1 ppm, and a β-carbon proton peak was observed near a low magnetic field shift near 1.5 ppm. From the sum of the signals of the methylene protons and the signal of the methyl protons, it was confirmed to be 3′-SL—N—C18. The peak around 3.4 ppm is a signal derived from heavy water (DHO).
Figure JPOXMLDOC01-appb-C000004
（製造例４：３’－ＳＬＮ－Ｎ－Ｃ１４の製造）
　製造例１において、３’－シアリルラクトース（３’－ＳＬ）に代えて、３’－シアリル－Ｎ－アセチルラクトサミン（３’－ＳＬＮ）（シグマアルドリッチ社製、分子量：６７４）を用いたこと以外は製造例１と同様の方法で、下記構造式で表される３’－シアリルラクトサミニルミリスチン酸アミド（３’－ＳＬＮ－Ｎ－Ｃ１４：３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１４、分子量：８８３）を得た。下記構造式において「Ａｃ」は、アセチル基を表す。
　製造例１において、３’－ＳＬ－Ｎ－Ｃ１４を含む反応液に代えて、３’－ＳＬＮ－Ｎ－Ｃ１４を含む反応液を用いたこと以外は、製造例１と同様の方法で、３’－ＳＬＮ－Ｎ－Ｃ１４を精製した。
　プロトン核磁気共鳴スペクトルとして、ＴＭＳを内部標準とし、６００ＭＨｚにおいて２質量％重水（Ｄ２Ｏ）を含有する重ジメチルスルホキシド（ＤＭＳＯ－ｄ６）中で測定した。３’－ＳＬＮ－Ｎ－Ｃ１４のプロトンＮＭＲスペクトルは図７に示す通りである。
Figure JPOXMLDOC01-appb-C000005
 (Production Example 4: Production of 3'-SLN-N-C14)
In Production Example 1, 3′-sialyl-N-acetyllactosamine (3′-SLN) (manufactured by Sigma-Aldrich, molecular weight: 674) was used instead of 3′-sialyl lactose (3′-SL). 3′-sialyllactosaminyl myristic acid amide (3′-SLN—N—C14: 3′-Neu5Ac (α2-3) Gal (β1 -4) GlcNAc-N-C14, molecular weight: 883) was obtained. In the following structural formula, “Ac” represents an acetyl group.
In Production Example 1, a reaction solution containing 3′-SLN—N—C14 was used instead of the reaction solution containing 3′-SL—N—C14, except that a reaction solution containing 3′-SLN—N—C14 was used. '-SLN-N-C14 was purified.
The proton nuclear magnetic resonance spectrum was measured in deuterated dimethyl sulfoxide (DMSO-d6) containing 2 % by mass heavy water (D 2 O) at 600 MHz with TMS as an internal standard. The proton NMR spectrum of 3′-SLN—N—C14 is as shown in FIG.
Figure JPOXMLDOC01-appb-C000005
（製造例５：６’－ＳＬＮ－Ｎ－Ｃ１４及び６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１４の製造）
　製造例１において、３’－シアリルラクトース（３’－ＳＬ）に代えて、６’－シアリル－Ｎ－アセチルラクトサミン（６’－ＳＬＮ）（シグマアルドリッチ社製、分子量：６７４）を用いたこと以外は製造例１と同様の方法で、下記構造式で表される６’－シアリルラクトサミニルミリスチン酸アミド（６’－ＳＬＮ－Ｎ－Ｃ１４：６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１４、分子量：８８３）、及び６’－シアリルアミドラクトサミニルミリスチン酸アミド（６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１４：６’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－６）Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１４、分子量：８８２）を得た。下記構造式において「Ａｃ」は、アセチル基を表す。
　製造例１において、３’－ＳＬ－Ｎ－Ｃ１４を含む反応液に代えて、６’－ＳＬＮ－Ｎ－Ｃ１４及び６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１４を含む反応液を用いたこと以外は、製造例１と同様の方法で、６’－ＳＬＮ－Ｎ－Ｃ１４及び６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１４を精製した。更に、６’－ＳＬＮ－Ｎ－Ｃ１４及び６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１４を製造例１と同様の方法でＴＬＣにより確認を行った。結果を図４に示す。図４より、シアル酸のカルボキシル基がアミド化されると、極性が低くなるため、移動度が大きくなった。
　また、６’－ＳＬＮ－Ｎ－Ｃ１４及び６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１４について、下記条件にてＬＣ／ＭＳの測定を行った。６’－ＳＬＮ－Ｎ－Ｃ１４のスペクトルは図８Ａに、６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１４のスペクトルは図８Ｂに示す。ＬＣ／ＭＳの分析により、シアル酸のカルボキシル基がアミド化されると、分子量が１小さくなることが確認された。また、ＬＣ／ＭＳの分析（逆相クロマトグラフィー）では、前記ＴＬＣ（順相クロマトグラフィー）の結果と、移動度が逆転した。
［ＬＣ／ＭＳ分析条件］
　カラム：１．０ｍｍ×１０ｍｍ　Ｃ１８　ｃｏｌｕｍｎ（Ｃａｄｅｎｚａ　ＣＤ－Ｃ１８、Ｉｍｔａｋｔ社製）
　溶媒：Ａ液：０．０５体積％　ギ酸水
　　　　Ｂ液：アセトニトリル／ギ酸水（９０／１０（体積比））（含０．０５体積％　ギ酸）
　　　　（０分間～５分間：０体積％Ｂ液（一定）、５分間～１０分間：０体積％Ｂ液～１００体積％Ｂ液（濃度勾配）、１０分間～２０分間：１００体積％Ｂ液（一定））
　流速：０．２　ｍＬ／分間
　質量分析範囲：ｍ／ｚ　３４０～２，０００
Figure JPOXMLDOC01-appb-C000006
Figure JPOXMLDOC01-appb-C000007
 (Production Example 5: Production of 6′-SLN—N—C14 and 6′-S (1NH 2 ) LN—N—C14)
In Production Example 1, 6′-sialyl-N-acetyllactosamine (6′-SLN) (manufactured by Sigma-Aldrich, molecular weight: 674) was used instead of 3′-sialyl lactose (3′-SL). Except for 6′-sialyllactosaminyl myristic acid amide (6′-SLN—N—C14: 6′-Neu5Ac (α2-6) Gal (β1) represented by the following structural formula. -4) GlcNAc-N-C14, molecular weight: 883), and 6'-sialylamidolactosaminyl myristic acid amide (6'-S (1NH 2 ) LN-N-C14: 6'-Neu5Ac1NH 2 (α2-6 ) Gal (β1-4) GlcNAc-N—C14, molecular weight: 882). In the following structural formula, “Ac” represents an acetyl group.
In Production Example 1, a reaction solution containing 6′-SLN-N—C14 and 6′-S (1NH 2 ) LN—N—C14 was used in place of the reaction solution containing 3′-SL—N—C14. Except for this, 6′-SLN—N—C14 and 6′-S (1NH 2 ) LN—N—C14 were purified in the same manner as in Production Example 1. Further, 6′-SLN—N—C14 and 6′-S (1NH 2 ) LN—N—C14 were confirmed by TLC in the same manner as in Production Example 1. The results are shown in FIG. As shown in FIG. 4, when the carboxyl group of sialic acid is amidated, the polarity is lowered, and thus the mobility is increased.
Further, LC / MS measurement was performed on 6′-SLN—N—C14 and 6′-S (1NH 2 ) LN—N—C14 under the following conditions. The spectrum of 6′-SLN-N—C14 is shown in FIG. 8A, and the spectrum of 6′-S (1NH 2 ) LN—N—C14 is shown in FIG. 8B. LC / MS analysis confirmed that the molecular weight was reduced by 1 when the carboxyl group of sialic acid was amidated. In the LC / MS analysis (reverse phase chromatography), the mobility was reversed from the result of the TLC (normal phase chromatography).
[LC / MS analysis conditions]
Column: 1.0 mm × 10 mm C18 column (Cadenza CD-C18, manufactured by Imtakt)
Solvent: Liquid A: 0.05% by volume aqueous formic acid Liquid B: acetonitrile / water formic acid (90/10 (volume ratio)) (including 0.05% by volume formic acid)
(0 minute to 5 minutes: 0 volume% B liquid (constant), 5 minutes to 10 minutes: 0 volume% B liquid to 100 volume% B liquid (concentration gradient), 10 minutes to 20 minutes: 100 volume% B liquid ( Constant))
Flow rate: 0.2 mL / min Mass spectrometric range: m / z 340 to 2,000
Figure JPOXMLDOC01-appb-C000006
Figure JPOXMLDOC01-appb-C000007
　また、プロトン核磁気共鳴スペクトルとして、ＴＭＳを内部標準とし、６００ＭＨｚにおいて２質量％重水（Ｄ２Ｏ）を含有する重ジメチルスルホキシド（ＤＭＳＯ－ｄ６）中で測定した。６’－ＳＬＮ－Ｎ－Ｃ１４のプロトンＮＭＲスペクトルは図８Ｃに、６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１４のプロトンＮＭＲスペクトルは図８Ｄに示す通りである。 The proton nuclear magnetic resonance spectrum was measured in deuterated dimethyl sulfoxide (DMSO-d6) containing 2 % by mass heavy water (D 2 O) at 600 MHz with TMS as an internal standard. The proton NMR spectrum of 6′-SLN—N—C14 is as shown in FIG. 8C, and the proton NMR spectrum of 6′-S (1NH 2 ) LN—N—C14 is as shown in FIG. 8D.
　なお、６’－ＳＬＮ－Ｎ－Ｃ１４及び６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１４の製造原料として使用した６’－ＳＬＮについて、プロトン核磁気共鳴スペクトルとして、アセトンを内部標準とし、４００ＭＨｚにおいて重水（Ｄ２Ｏ）中で測定したプロトンＮＭＲスペクトルは、図９に示す通りである。 For 6′-SLN used as a raw material for producing 6′-SLN—N—C14 and 6′-S (1NH 2 ) LN—N—C14, as a proton nuclear magnetic resonance spectrum, acetone was used as an internal standard, and 400 MHz. FIG. 9 shows a proton NMR spectrum measured in heavy water (D 2 O).
（製造例６：６’－ＳＬ－Ｎ－Ｃ１４及び６’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１４の製造）
　製造例１において、３’－シアリルラクトース（３’－ＳＬ）に代えて、６’－シアリルラクトース（６’－ＳＬ）（ウシ初乳由来、シグマアルドリッチ社製、分子量：６５５）を用いたこと以外は製造例１と同様の方法で、下記構造式で表される６’－シアリルラクトシルミリスチン酸アミド（６’－ＳＬ－Ｎ－Ｃ１４：６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１４、分子量：８４２）、及び６’－シアリルアミドラクトシルミリスチン酸アミド（６’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１４：６’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－６）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１４、分子量：８４１）を得た。下記構造式において「Ａｃ」は、アセチル基を表す。
　製造例１において、３’－ＳＬ－Ｎ－Ｃ１４を含む反応液に代えて、６’－ＳＬ－Ｎ－Ｃ１４及び６’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１４を含む反応液を用いたこと以外は、製造例１と同様の方法で、６’－ＳＬ－Ｎ－Ｃ１４及び６’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１４を精製した。
　６’－ＳＬ－Ｎ－Ｃ１４を、製造例１と同様の方法でＴＬＣにより確認を行った。結果を図４に示す。また、６’－ＳＬ－Ｎ－Ｃ１４及び６’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１４について製造例５と同じ条件でＬＣ／ＭＳ分析を行ったところ、製造例５と同様に、シアル酸のカルボキシル基がアミド化されると、分子量が１小さくなることが確認された。
　更に、プロトン核磁気共鳴スペクトルとして、ＴＭＳを内部標準とし、６００ＭＨｚにおいて２質量％重水（Ｄ２Ｏ）を含有する重ジメチルスルホキシド（ＤＭＳＯ－ｄ６）中で測定した。６’－ＳＬ－Ｎ－Ｃ１４のプロトンＮＭＲスペクトルは図１０Ａに、６’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１４のプロトンＮＭＲスペクトルは図１０Ｂに示す通りである。
Figure JPOXMLDOC01-appb-C000008
Figure JPOXMLDOC01-appb-C000009
 (Production Example 6: Production of 6′-SL—N—C14 and 6′-S (1NH 2 ) LN—C14)
In Production Example 1, 6′-sialyl lactose (6′-SL) (derived from bovine colostrum, Sigma-Aldrich, molecular weight: 655) was used instead of 3′-sialyl lactose (3′-SL). Except for 6′-sialyl lactosyl myristate amide (6′-SL—N—C14: 6′-Neu5Ac (α2-6) Gal (β1- 4) Glc-N-C14, molecular weight: 842), and 6'-sialylamidolactosylmyristic acid amide (6'-S (1NH 2 ) LN-C14: 6'-Neu5Ac1NH 2 (α2-6) Gal (Β1-4) Glc-N-C14, molecular weight: 841) was obtained. In the following structural formula, “Ac” represents an acetyl group.
In Production Example 1, a reaction solution containing 6′-SL—N—C14 and 6′-S (1NH 2 ) L—N—C14 was used in place of the reaction solution containing 3′-SL—N—C14. Except for this, 6′-SL—N—C14 and 6′-S (1NH 2 ) L—N—C14 were purified in the same manner as in Production Example 1.
6′-SL—N—C14 was confirmed by TLC in the same manner as in Production Example 1. The results are shown in FIG. LC / MS analysis was performed on 6′-SL—N—C14 and 6′-S (1NH 2 ) LN—C14 under the same conditions as in Production Example 5. As in Production Example 5, sialic acid It was confirmed that the molecular weight was reduced by 1 when the carboxyl group of was amidated.
Further, proton nuclear magnetic resonance spectra were measured in deuterated dimethyl sulfoxide (DMSO-d6) containing 2 % by mass heavy water (D 2 O) at 600 MHz with TMS as an internal standard. The proton NMR spectrum of 6′-SL—N—C14 is as shown in FIG. 10A, and the proton NMR spectrum of 6′-S (1NH2) L—N—C14 is as shown in FIG. 10B.
Figure JPOXMLDOC01-appb-C000008
Figure JPOXMLDOC01-appb-C000009
　なお、６’－ＳＬ－Ｎ－Ｃ１４及び６’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１４の製造原料として使用した６’－ＳＬについて、プロトン核磁気共鳴スペクトルとして、アセトンを内部標準とし、４００ＭＨｚにおいて重水（Ｄ２Ｏ）中で測定したプロトンＮＭＲスペクトルは、図１１に示す通りである。 For 6′-SL used as a raw material for producing 6′-SL—N—C14 and 6′-S (1NH 2 ) LN—C14, acetone was used as an internal standard and 400 MHz as a proton nuclear magnetic resonance spectrum. FIG. 11 shows the proton NMR spectrum measured in heavy water (D 2 O).
（製造例７：６’－ＳＬ－Ｎ－Ｃ１８及び６’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１８の製造）
　製造例３において、３’－シアリルラクトース（３’－ＳＬ）に代えて、６’－シアリルラクトース（６’－ＳＬ）（ウシ初乳由来、シグマアルドリッチ社製、分子量：６５５）を用いたこと以外は製造例３と同様の方法で、下記構造式で表される６’－シアリルラクトシルステアリン酸アミド（６’－ＳＬ－Ｎ－Ｃ１８：６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１８、分子量：８９８）、及び６’－シアリルアミドラクトシルステアリン酸アミド（６’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１８：６’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－６）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１８、分子量：８９７）を得た。下記構造式において「Ａｃ」は、アセチル基を表す。
　製造例１において、３’－ＳＬ－Ｎ－Ｃ１４を含む反応液に代えて、６’－ＳＬ－Ｎ－Ｃ１８及び６’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１８を含む反応液を用いたこと以外は、製造例１と同様の方法で、６’－ＳＬ－Ｎ－Ｃ１８及び６’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１８を精製した。
　プロトン核磁気共鳴スペクトルとして、ＴＭＳを内部標準とし、６００ＭＨｚにおいて２質量％重水（Ｄ２Ｏ）を含有する重ジメチルスルホキシド（ＤＭＳＯ－ｄ６）中で測定した。６’－ＳＬ－Ｎ－Ｃ１８のプロトンＮＭＲスペクトルは図１２に示す通りである。
　また、６’－ＳＬ－Ｎ－Ｃ１８及び６’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１８について製造例５と同じ条件でＬＣ／ＭＳ分析を行ったところ、製造例５と同様に、シアル酸のカルボキシル基がアミド化されると、分子量が１小さくなることが確認された。
Figure JPOXMLDOC01-appb-C000010
Figure JPOXMLDOC01-appb-C000011
 (Production Example 7: Production of 6′-SL—N—C18 and 6′-S (1NH 2 ) LN—C18)
In Production Example 3, 6′-sialyl lactose (6′-SL) (derived from bovine colostrum, Sigma-Aldrich, molecular weight: 655) was used instead of 3′-sialyl lactose (3′-SL). Except for 6′-sialyllactosyl stearic acid amide (6′-SL—N—C18: 6′-Neu5Ac (α2-6) Gal (β1- 4) Glc-N-C18, molecular weight: 898), and 6′-sialylamidolactosyl stearamide (6′-S (1NH 2 ) LN-C18: 6′-Neu5Ac1NH 2 (α2-6) Gal (Β1-4) Glc-N-C18, molecular weight: 897) was obtained. In the following structural formula, “Ac” represents an acetyl group.
In Production Example 1, a reaction solution containing 6′-SL—N—C18 and 6′-S (1NH 2 ) L—N—C18 was used instead of the reaction solution containing 3′-SL—N—C14. Except for this, 6′-SL—N—C18 and 6′-S (1NH 2 ) LN—C18 were purified in the same manner as in Production Example 1.
The proton nuclear magnetic resonance spectrum was measured in deuterated dimethyl sulfoxide (DMSO-d6) containing 2 % by mass heavy water (D 2 O) at 600 MHz with TMS as an internal standard. The proton NMR spectrum of 6′-SL—N—C18 is as shown in FIG.
LC / MS analysis of 6′-SL—N—C18 and 6′-S (1NH 2 ) L—N—C18 was conducted under the same conditions as in Production Example 5. As in Production Example 5, sialic acid It was confirmed that when the carboxyl group of was amidated, the molecular weight was reduced by one.
Figure JPOXMLDOC01-appb-C000010
Figure JPOXMLDOC01-appb-C000011
（製造例８：６’－ＳＬＮ－Ｎ－Ｃ１８及び６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１８の製造）
　製造例３において、３’－シアリルラクトース（３’－ＳＬ）に代えて、６’－シアリル－Ｎ－アセチルラクトサミン（６’－ＳＬＮ）（シグマアルドリッチ社製、分子量：６７４）を用いたこと以外は製造例３と同様の方法で、下記構造式で表される６’－シアリルラクトサミニルステアリン酸アミド（６’－ＳＬＮ－Ｎ－Ｃ１８：６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１８、分子量：９３９）及び６’－シアリルアミドラクトサミニルステアリン酸アミド（６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１８：６’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－６）Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１８）、分子量：９３８）を得た。下記構造式において「Ａｃ」は、アセチル基を表す。
　製造例１において、３’－ＳＬ－Ｎ－Ｃ１４を含む反応液に代えて、６’－ＳＬＮ－Ｎ－Ｃ１８及び６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１８を含む反応液を用いたこと以外は、製造例１と同様の方法で、６’－ＳＬＮ－Ｎ－Ｃ１８及び６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１８を精製した。
　プロトン核磁気共鳴スペクトルとして、ＴＭＳを内部標準とし、６００ＭＨｚにおいて２質量％重水（Ｄ２Ｏ）を含有する重ジメチルスルホキシド（ＤＭＳＯ－ｄ６）中で測定した。６’－ＳＬＮ－Ｎ－Ｃ１８のプロトンＮＭＲスペクトルは図１３Ａに、６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１８のプロトンＮＭＲスペクトルは図１３Ｂに示す通りである。図１３Ｂより、８ｐｐｍ付近（約７．０ｐｐｍ～８．１ｐｐｍ）に糖鎖還元末端のアミン化によるアミドプロトンが確認された。
　また、６’－ＳＬＮ－Ｎ－Ｃ１８及び６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１８について製造例５と同じ条件でＬＣ／ＭＳ分析を行ったところ、製造例５と同様に、シアル酸のカルボキシル基がアミド化されると、分子量が１小さくなることが確認された。
Figure JPOXMLDOC01-appb-C000012
Figure JPOXMLDOC01-appb-C000013
 (Production Example 8: Production of 6'-SLN-N-C18 and 6'-S (1NH 2 ) LN-N-C18)
In Production Example 3, 6′-sialyl-N-acetyllactosamine (6′-SLN) (manufactured by Sigma-Aldrich, molecular weight: 674) was used instead of 3′-sialyl lactose (3′-SL). Except for 6′-sialyllactosaminyl stearic acid amide (6′-SLN—N—C18: 6′-Neu5Ac (α2-6) Gal (β1) represented by the following structural formula. -4) GlcNAc-N-C18, molecular weight: 939) and 6'-sialylamidolactosaminyl stearamide (6'-S (1NH 2 ) LN-N-C18: 6'-Neu5Ac1NH 2 (α2-6) Gal (β1-4) GlcNAc-N—C18), molecular weight: 938). In the following structural formula, “Ac” represents an acetyl group.
In Production Example 1, instead of the reaction solution containing 3′-SL-N—C14, a reaction solution containing 6′-SLN—N—C18 and 6′-S (1NH 2 ) LN—N—C18 was used. Except for this, 6′-SLN—N—C18 and 6′-S (1NH 2 ) LN—N—C18 were purified in the same manner as in Production Example 1.
The proton nuclear magnetic resonance spectrum was measured in deuterated dimethyl sulfoxide (DMSO-d6) containing 2 % by mass heavy water (D 2 O) at 600 MHz with TMS as an internal standard. The proton NMR spectrum of 6′-SLN—N—C18 is as shown in FIG. 13A, and the proton NMR spectrum of 6′-S (1NH 2 ) LN—N—C18 is as shown in FIG. 13B. From FIG. 13B, amide protons due to amination of the reducing end of the sugar chain were confirmed in the vicinity of 8 ppm (about 7.0 ppm to 8.1 ppm).
Further, when LC / MS analysis was performed on 6′-SLN—N—C18 and 6′-S (1NH 2 ) LN—N—C18 under the same conditions as in Production Example 5, as in Production Example 5, sialic acid It was confirmed that the molecular weight was reduced by 1 when the carboxyl group of was amidated.
Figure JPOXMLDOC01-appb-C000012
Figure JPOXMLDOC01-appb-C000013
（比較製造例１：ＬＮ－Ｃ１４の製造）
　製造例１において、３’－シアリルラクトース（３’－ＳＬ）に代えて、ラクトサミン（ＬＮ）（ナカライテスク株式会社製、分子量：３８４）を用いたこと以外は製造例１と同様の方法で、下記構造式で表されるラクトサミニルミリスチン酸アミド（ＬＮ－Ｃ１４：Ｇａｌ（β１－４）ＧｌｃＮＡｃ－Ｎ－Ｃ１４、分子量：５９１）を得た。下記構造式において「Ａｃ」は、アセチル基を表す。
　製造例１において、３’－ＳＬ－Ｎ－Ｃ１４を含む反応液に代えて、ＬＮ－Ｃ１４を含む反応液を用いたこと以外は、製造例１と同様の方法で、ＬＮ－Ｃ１４を精製した。
Figure JPOXMLDOC01-appb-C000014
 (Comparative Production Example 1: Production of LN-C14)
In Production Example 1, instead of 3′-sialyllactose (3′-SL), lactosamine (LN) (manufactured by Nacalai Tesque, Inc., molecular weight: 384) was used in the same manner as in Production Example 1, A lactosaminyl myristic acid amide (LN-C14: Gal (β1-4) GlcNAc-N-C14, molecular weight: 591) represented by the following structural formula was obtained. In the following structural formula, “Ac” represents an acetyl group.
In Production Example 1, LN-C14 was purified by the same method as in Production Example 1 except that a reaction solution containing LN-C14 was used instead of the reaction solution containing 3′-SL-N—C14. .
Figure JPOXMLDOC01-appb-C000014
　下記に、製造例１～８及び比較製造例１で合成したシアル酸含有糖鎖複合体を、まとめて示す。 The sialic acid-containing sugar chain complexes synthesized in Production Examples 1 to 8 and Comparative Production Example 1 are shown below.
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000015
　なお、製造例１～４において、本実施例においては、プロトン核磁気共鳴スペクトル、ＬＣ／ＭＳ分析、ＴＬＣ等で確認を行っていないが、シアル酸のカルボキシル基がアミド化されたシアル酸含有アミド糖鎖複合体は、シアリルオリゴ糖をアシルアミド化の際に副生成物として合成されるため、製造例１においては、３’－シアリルアミドラクトシルミリスチン酸アミド（３’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－３）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１４）、製造例２においては、３’－シアリルアミドラクトシルパルミチン酸アミド（３’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－３）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１６）、製造例３においては、３’－シアリルアミドラクトシルステアリン酸アミド（３’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－３）Ｇａｌ（β１－４）Ｇｌｃ－Ｎ－Ｃ１８）、製造例４においては、３’－シアリルアミドラクトサミニルミリスチン酸アミド（３’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－３）Ｇａｌ（β１－４）ＧｌｃＮａｃ－Ｎ－Ｃ１４）が合成されているものと推察される。 In Production Examples 1 to 4, in this example, although confirmation was not performed by proton nuclear magnetic resonance spectrum, LC / MS analysis, TLC, etc., a sialic acid-containing amide in which the carboxyl group of sialic acid was amidated Since the sugar chain complex is synthesized as a by-product during acylamidation of sialyl oligosaccharide, in Production Example 1, 3′-sialylamidolactosylmyristate amide (3′-Neu5Ac1NH 2 (α2-3 ) Gal (β1-4) Glc-N—C14), and in Production Example 2, 3′-sialylamidolactosyl palmitic acid amide (3′-Neu5Ac1NH 2 (α2-3) Gal (β1-4) Glc-N -C16), in Production Example 3, 3'-sialylamidolactosyl stearamide (3'-Neu5Ac1NH 2 (α2-3) Ga l (β1-4) Glc-N-C18), and in Production Example 4, 3′-sialylamidolactosaminylmyristic acid amide (3′-Neu5Ac1NH 2 (α2-3) Gal (β1-4) GlcNac-N -C14) is assumed to be synthesized.
（試験例２：シアル酸含有糖鎖複合体による抗インフルエンザウイルス活性の検討（ｉｎ　ｖｉｔｒｏ））
＜シアル酸含有糖鎖複合体の感染後の作用＞
　表２に示す各シアル酸含有糖鎖複合体の抗インフルエンザウイルス活性を、以下のようにしてプラック測定（ＰＦＵ　Ａｓｓａｙ）により評価した。
　ＭＤＣＫ細胞（ＮＢＬ－２細胞、イヌ腎細胞）（大日本住友製薬株式会社製）を、１０体積％ウシ胎仔血清（ＦＢＳ）含有Ｅ－ＭＥＭ培地（Ｅａｇｌｅ－Ｍｉｎｉｍｕｍ　Ｅｓｓｅｎｔｉａｌ　Ｍｅｄｉｕｍ）（ＧＩＢＣＯ；インビトロジェン株式会社製）を添加したマイクロプレートで、３７℃、５％ＣＯ２条件下で培養した。モノレーヤーになったＭＤＣＫ細胞に、１００ＰＦＵ／ｍＬとなるようにインフルエンザウイルスＡ／ＰＲ／８／３４株（ＡＴＣＣ　ＶＲ－９５）（Ｈ１Ｎ１型）を加え、３７℃にて１時間インキュベートして感染させた。感染後、感染に使用したインフルエンザウイルスを含む培養液を除去し、表２に示す各シアル酸含有糖鎖複合体、並びに、既存薬として、オセルタミビル（タミフル（登録商標）、ロッシュ社製）、ザナミビル（リレンザ（登録商標）、グラクソ・スミス・クライン社製）、及びアマンタジン塩酸塩（シグマアルドリッチ社製）をそれぞれ０．１μｇ／ｍＬ～５００μｇ／ｍＬの範囲で加えたアガロース液を前記マイクロプレートに重層し、完全に凝固した後、３７℃、５％ＣＯ２の条件下で３日間培養した。
　各シアル酸含有糖鎖複合体を添加していない細胞を対照とし、次式により、各シアル酸含有糖鎖複合体を添加した場合のインフルエンザウイルス増殖抑制率を算出し、ＩＣ５０値（対照の５０％までインフルエンザウイルスの増殖を抑制する濃度）を算出した。結果を下記表２に示す。なお、表２は、前記試験を１回～３回行った平均値を示す。
　　インフルエンザウイルス増殖抑制率（％）＝１００－（シアル酸含有糖鎖複合体添加時のプラック数／対照のプラック数）×１００ (Test Example 2: Examination of anti-influenza virus activity by sialic acid-containing sugar chain complex (in vitro))
<Action after infection of sialic acid-containing sugar chain complex>
The anti-influenza virus activity of each sialic acid-containing sugar chain complex shown in Table 2 was evaluated by plaque measurement (PFU Assay) as follows.
MDCK cells (NBL-2 cells, canine kidney cells) (manufactured by Dainippon Sumitomo Pharma Co., Ltd.) were added to E-MEM medium (EIB-Minimum Essential Medium) (GIBCO; Invitrogen Corporation) containing 10% by volume fetal bovine serum (FBS). In the microplate to which the product was added at 37 ° C. and 5% CO 2 . Influenza virus A / PR / 8/34 strain (ATCC VR-95) (H1N1 type) was added to the monolayer MDCK cells at 100 PFU / mL and incubated at 37 ° C. for 1 hour for infection. . After infection, the culture solution containing the influenza virus used for the infection was removed, and each sialic acid-containing sugar chain complex shown in Table 2 and oseltamivir (Tamiflu (registered trademark), manufactured by Roche), zanamivir as existing drugs (Relenza (registered trademark), manufactured by GlaxoSmithKline) and amantadine hydrochloride (manufactured by Sigma-Aldrich) in a range of 0.1 μg / mL to 500 μg / mL, respectively, are added to the microplate. After complete coagulation, the cells were cultured for 3 days under conditions of 37 ° C. and 5% CO 2 .
The cells without the addition of the sialic acid-containing sugar chain complexes as a control, the following equation to calculate the influenza virus growth inhibition rate when adding each sialic acid-containing sugar chain complex, IC 50 values (control of The concentration at which the growth of influenza virus was suppressed to 50% was calculated. The results are shown in Table 2 below. Table 2 shows the average values obtained by performing the test 1 to 3 times.
Influenza virus growth inhibition rate (%) = 100− (number of plaques at the time of addition of sialic acid-containing sugar chain complex / number of plaques for control) × 100
Figure JPOXMLDOC01-appb-T000016
Figure JPOXMLDOC01-appb-T000016
　表２より、シアル酸含有糖鎖複合体の抗インフルエンザウイルス活性は、炭素数が多くなる程強くなることがわかった。
　既存薬であるオセルタミフル及びリレンザに比べると、シアル酸含有糖鎖複合体のＩＣ５０値は、５０倍～１００倍濃度での抗インフルエンザウイルス活性であり、アマンタジン塩酸塩とは同等の濃度であった。 From Table 2, it was found that the anti-influenza virus activity of the sialic acid-containing sugar chain complex increases as the carbon number increases.
Compared with oseltamiflu and Relenza, which are existing drugs, the IC 50 value of the sialic acid-containing glycan complex is anti-influenza virus activity at 50 to 100-fold concentration, which is equivalent to that of amantadine hydrochloride. It was.
＜シアル酸含有糖鎖複合体の感染時及び感染後の作用＞
　ＭＤＣＫ細胞は、前記同様の方法でモノレーヤーに調製した。このＭＤＣＫ細胞に、１００ＰＦＵ／ｍＬとなるようにインフルエンザウイルスＡ／ＰＲ／８／３４株（ＡＴＣＣ　ＶＲ－９５）（Ｈ１Ｎ１型）を加え、同時に、表３に示す各シアル酸含有糖鎖複合体及び既存薬、並びに、シアル酸含有糖鎖複合体の対照として比較製造例１で得られたＬＮ－Ｃ１４をそれぞれ０．１μｇ／ｍＬ～５００μｇ／ｍＬの範囲で加え、３７℃にて１時間インキュベートして感染させた。感染後、感染に使用したインフルエンザウイルスを含む培養液を除去し、表３に示す各シアル酸含有糖鎖複合体及び既存薬を、前記感染時と同様の濃度で加えたアガロース液を前記マイクロプレートに重層し、完全に凝固した後、３７℃、５％ＣＯ２の条件下で３日間培養した。培養後、前記同様の方法で、ＩＣ５０値を算出した。結果を下記表３に示す。なお、表３は、前記試験を１回～３回行った平均値を示す。 <Effects of sialic acid-containing sugar chain complex during and after infection>
MDCK cells were prepared in a monolayer by the same method as described above. To this MDCK cell, influenza virus A / PR / 8/34 strain (ATCC VR-95) (H1N1 type) was added so as to be 100 PFU / mL, and each sialic acid-containing sugar chain complex shown in Table 3 LN-C14 obtained in Comparative Production Example 1 was added in the range of 0.1 μg / mL to 500 μg / mL as a control for existing drugs and sialic acid-containing sugar chain complexes, respectively, and incubated at 37 ° C. for 1 hour. I was infected. After infection, the culture solution containing the influenza virus used for the infection was removed, and the agarose solution added with each sialic acid-containing sugar chain complex and existing drugs shown in Table 3 at the same concentration as in the infection was added to the microplate. After complete coagulation, the cells were cultured at 37 ° C. under 5% CO 2 for 3 days. After culturing, IC 50 values were calculated by the same method as described above. The results are shown in Table 3 below. Table 3 shows the average values obtained by performing the test 1 to 3 times.
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-T000017
　表３の結果より、カルボキシル基をアミド化したシアル酸含有糖鎖複合体は、カルボキシル基がアミド化されていないシアル酸含有糖鎖複合体と同等の強い抗インフルエンザウイルス活性が認められた。シアル酸を有さないラクトースにミリスチン酸を導入したオリゴ糖誘導体（ＬＮ－Ｃ１４）では抗インフルエンザウイルス活性が全く確認されなかった。したがって、これらのシアル酸含有糖鎖複合体の抗インフルエンザウイルス活性は、界面活性作用ではなく、シアル酸を介した特異的な抗インフルエンザウイルス活性であることが示唆された。 From the results in Table 3, it was confirmed that the sialic acid-containing sugar chain complex in which the carboxyl group was amidated showed strong anti-influenza virus activity equivalent to that of the sialic acid-containing sugar chain complex in which the carboxyl group was not amidated. Anti-influenza virus activity was not confirmed at all in the oligosaccharide derivative (LN-C14) in which myristic acid was introduced into lactose having no sialic acid. Therefore, it was suggested that the anti-influenza virus activity of these sialic acid-containing sugar chain complexes is not a surfactant activity but a specific anti-influenza virus activity via sialic acid.
（試験例３：シアル酸含有糖鎖複合体の保存安定性の検討）
　前記表３に示す各シアル酸含有糖鎖複合体を４℃にて１．５年間保存した。長期保存後の各シアル酸含有糖鎖複合体について、試験例２と同様にしてプラック測定（ＰＦＵ　Ａｓｓａｙ）により抗インフルエンザウイルス活性を評価することで、その安定性について試験した。その結果、１．５年間保存後においても抗インフルエンザウイルス活性は安定に維持されていることが確認された。 (Test Example 3: Examination of storage stability of sialic acid-containing sugar chain complex)
Each sialic acid-containing sugar chain complex shown in Table 3 was stored at 4 ° C. for 1.5 years. Each sialic acid-containing sugar chain complex after long-term storage was tested for its stability by evaluating anti-influenza virus activity by plaque measurement (PFU Assay) in the same manner as in Test Example 2. As a result, it was confirmed that the anti-influenza virus activity was stably maintained even after storage for 1.5 years.
（試験例４：シアル酸含有糖鎖複合体の毒性試験）
＜単回投与試験＞
　ＩＣＲマウス（雌雄、６週齢ＳＰＦ、ｎ＝６、日本チャールス・リバー株式会社）に、シアル酸含有糖鎖複合体（３’－ＳＬ－Ｎ－Ｃ１４）を１．５ｍｇ／ｋｇの用量で単回静脈内投与し、７日間飼育した。シアル酸含有糖鎖複合体に代えて、ＰＢＳを投与した動物を対照とした。飼育期間中、体重測定、血液学的検査、血液化学的検査等により一般症状の観察を行った。
　その結果、死亡例はなく、対照動物と比較して前記一般症状への影響は認められず、毒性は認められなかった。シアル酸含有糖鎖複合体は、天然の材料で構築した物質であるため、高濃度でも安全性が高いことが示唆された。 (Test Example 4: Toxicity test of sialic acid-containing sugar chain complex)
<Single dose study>
ICR mice (male and female, 6-week-old SPF, n = 6, Nippon Charles River Co., Ltd.) were treated with a sialic acid-containing sugar chain complex (3′-SL-N-C14) at a dose of 1.5 mg / kg. The iv vein was administered and reared for 7 days. An animal administered with PBS instead of the sialic acid-containing sugar chain complex was used as a control. During the breeding period, general symptoms were observed by measuring body weight, hematology, blood chemistry, and the like.
As a result, there were no deaths, no effects on the general symptoms were observed compared to control animals, and no toxicity was observed. Since the sialic acid-containing sugar chain complex is a substance constructed from natural materials, it was suggested that the sialic acid-containing sugar chain complex is highly safe even at high concentrations.
＜反復投与試験＞
　ＩＣＲマウス（雌雄、６週齢ＳＰＦ、ｎ＝６、日本チャールス・リバー株式会社）に、シアル酸含有糖鎖複合体（３’－ＳＬ－Ｎ－Ｃ１４）を１．５ｍｇ／ｋｇの用量で１日１回、７日間反復静脈内投与し、合計７日間飼育した。シアル酸含有糖鎖複合体に代えて、ＰＢＳを投与した動物を対照とした。飼育期間中、体重測定、血液学的検査、血液化学的検査等により一般症状の観察を行った。
　その結果、死亡例はなく、対照動物と比較して前記一般症状への影響は認められず、毒性は認められなかった。 <Repeated dose test>
ICR mice (male and female, 6-week-old SPF, n = 6, Nippon Charles River Co., Ltd.) were treated with a sialic acid-containing sugar chain complex (3′-SL-N-C14) at a dose of 1.5 mg / kg. Repeated intravenous administration once a day for 7 days, the animals were raised for a total of 7 days. An animal administered with PBS instead of the sialic acid-containing sugar chain complex was used as a control. During the breeding period, general symptoms were observed by measuring body weight, hematology, blood chemistry, and the like.
As a result, there were no deaths, no effects on the general symptoms were observed compared to control animals, and no toxicity was observed.
（試験例５：シアル酸含有糖鎖複合体の抗インフルエンザウイルス活性の検討（ｉｎ　ｖｉｖｏ））
＜投与方法の検討＞
　ＢＡＬＢ／ｃマウス（雌、６週齢、日本クレア株式会社）に、製造例１で得られたシアル酸含有糖鎖複合体（３’－ＳＬ－Ｎ－Ｃ１４）を１ｍｇ／ｋｇの用量で、１日２回（計３日間）、経鼻投与（３’－ＳＬ－Ｎ－Ｃ１４経鼻投与群、ｎ＝５）又は経口投与（３’－ＳＬ－Ｎ－Ｃ１４経口投与群、ｎ＝５）した。
　最初にシアル酸含有糖鎖複合体を投与してから４時間後にインフルエンザウイルスＡ／ＰＲ／８／３４株（ＴＣＩＤ５０／ｍＬ＝１０－６／ｍＬ）を１０μＬ（致死量７５％～１００％）経鼻接種した。
　なお、シアル酸含有糖鎖複合体及びインフルエンザウイルスを投与しなかった動物を対照群（ｎ＝５）、シアル酸含有糖鎖複合体を投与せず、前記同様の方法でインフルエンザウイルスのみを経鼻接種させた動物をウイルス投与群（ｎ＝５）とした。また、シアル酸含有糖鎖複合体を経口投与し、インフルエンザウイルスを投与しなかった動物を経口投与対照群（ｎ＝５）とした。
　前記各群について、以下の方法で、体重変動及び生存率の観察を行った。また、シアル酸含有糖鎖複合体の経鼻投与群及び経口投与群、並びに、ウイルス投与群について、以下の方法で、血清中の炎症性サイトカインの測定、及び肺組織中のウイルス力価の測定を行った。 (Test Example 5: Examination of anti-influenza virus activity of sialic acid-containing sugar chain complex (in vivo))
<Examination of administration method>
In a BALB / c mouse (female, 6 weeks old, CLEA Japan, Inc.), the sialic acid-containing sugar chain complex (3′-SL-N-C14) obtained in Production Example 1 was administered at a dose of 1 mg / kg. Twice a day (total 3 days), nasal administration (3′-SL-N-C14 nasal administration group, n = 5) or oral administration (3′-SL-N-C14 oral administration group, n = 5 )did.
Four hours after the first administration of the sialic acid-containing sugar chain complex, 10 μL of influenza virus A / PR / 8/34 strain (TCID 50 / mL = 10 −6 / mL) (lethal dose 75% to 100%) Nasal inoculation.
It should be noted that the animals that were not administered with the sialic acid-containing glycan complex and influenza virus were the control group (n = 5), and the sialic acid-containing glycan complex was not administered, and only the influenza virus was transnasally treated in the same manner as described above. The inoculated animal was used as a virus administration group (n = 5). In addition, an animal that was orally administered with a sialic acid-containing sugar chain complex and was not administered with influenza virus was used as an orally administered control group (n = 5).
About each said group, the weight fluctuation and the survival rate were observed with the following method. For nasal administration group, oral administration group and virus administration group of sialic acid-containing sugar chain complex, measurement of inflammatory cytokine in serum and measurement of virus titer in lung tissue by the following methods Went.
－体重変動、生存率－
　インフルエンザウイルスを接種してから、１日１回、７日間、生存の確認及び体重測定を行った。なお、体重変動を観察した動物は、各群ｎ＝５である。
　体重測定の結果を図１４Ａに示す。この結果より、ウイルス投与群と比較して、シアル酸含有糖鎖複合体の経口投与群（３’－ＳＬ－Ｎ－Ｃ１４経口投与群）において有意な体重減少抑制が認められた。また、シアル酸含有糖鎖複合体のみを経口投与した対照群（３’－ＳＬ－Ｎ－Ｃ１４経口投与対照群）は、何も投与しなかった対象群と同様体重変動がなく、シアル酸含有糖鎖複合体は毒性がないことがわかった。
　生存率の結果を図１４Ｂに示す。この結果、シアル酸含有糖鎖複合体の経口投与群（３’－ＳＬ－Ｎ－Ｃ１４経口投与群）は、ウイルス投与群に対して有意な延命効果が認められた。また、シアル酸含有糖鎖複合体の経鼻投与群（３’－ＳＬ－Ｎ－Ｃ１４経鼻投与群）は、体重減少の抑制は認められなかったものの、生存率は、ウイルス投与群と比較して、延命効果が認められた。 -Body weight fluctuation, survival rate-
After inoculation with influenza virus, survival was confirmed and body weight was measured once a day for 7 days. In addition, the animal which observed the weight fluctuation is n = 5 for each group.
The result of the body weight measurement is shown in FIG. 14A. From these results, a significant suppression of weight loss was observed in the oral administration group (3′-SL-N-C14 oral administration group) of the sialic acid-containing sugar chain complex as compared with the virus administration group. In addition, the control group (3′-SL-N-C14 oral administration control group) to which only the sialic acid-containing glycan complex was orally administered had no change in body weight and contained sialic acid in the same manner as the subject group to which nothing was administered. The glycoconjugate was found not to be toxic.
The survival rate results are shown in FIG. 14B. As a result, the oral administration group of the sialic acid-containing sugar chain complex (3′-SL-N-C14 oral administration group) was found to have a significant life-prolonging effect with respect to the virus administration group. In the nasal administration group (3′-SL-N-C14 nasal administration group) of the sialic acid-containing sugar chain complex, suppression of weight loss was not observed, but the survival rate was compared with the virus administration group. As a result, a life-prolonging effect was recognized.
－炎症性サイトカインの測定－
　ウイルス投与群（ｎ＝５）、３’－ＳＬ－Ｎ－Ｃ１４経鼻投与群（ｎ＝５）、及び３’－ＳＬ－Ｎ－Ｃ１４経口投与群（ｎ＝５）について、炎症性サイトカインとして、ＩＬ－６及びＩＮＦ－γの濃度を以下の方法で測定した。
　インフルエンザウイルスを接種してから３日目に各動物の血液を採取し、４℃にて３０００ｒｐｍで１０分間遠心分離することにより血清を得た。この血清を用い、ＥＬＩＳＡ法（Ｍｏｕｓｅ　ＩＬ－６　Ｉｍｍｕｎｏａｓｓａｙ：Ｑｕａｎｔｉｋｉｎｅ　Ｒ＆Ｄ　ＳＹＳＴＥＭＳ社製）を用いてＩＬ－６の濃度を測定した。また、前記血清を用い、ＥＬＩＳＡ法（Ｍｏｕｓｅ　ＩＦＮ－γ　Ｉｍｍｕｎｏａｓｓａｙ：Ｑｕａｎｔｉｋｉｎｅ　Ｒ＆Ｄ　ＳＹＳＴＥＭＳ社製）を用いてＩＮＦ－γの濃度を測定した。ＩＬ－６の測定結果を図１４Ｃに、ＩＮＦ－γの測定結果を図１４Ｄに示す。この結果より、インフルエンザウイルス感染によって誘導される炎症性サイトカイン産生の、シアル酸含有糖鎖複合体による抑制が認められた。特に、炎症性サイトカイン産生の抑制作用は、３’－ＳＬ－Ｎ－Ｃ１４経口投与群において有意であった。なお、統計解析は、ｔ検定（Ｓｔｕｄｅｎｔ’ｓ　ｔ－ｔｅｓｔ）で行った（ｐ＜０．０５）。 -Measurement of inflammatory cytokines-
Inflammatory cytokines for virus administration group (n = 5), 3′-SL-N-C14 nasal administration group (n = 5), and 3′-SL-N-C14 oral administration group (n = 5) The concentrations of IL-6 and INF-γ were measured by the following method.
On the third day after inoculation with the influenza virus, blood of each animal was collected, and serum was obtained by centrifugation at 3000 rpm for 10 minutes at 4 ° C. Using this serum, the concentration of IL-6 was measured by ELISA (Mouse IL-6 Immunoassay: manufactured by Quantikine R & D SYSTEMS). In addition, the serum was used to measure the concentration of INF-γ using ELISA (Mouse IFN-γ Immunoassay: manufactured by Quantikine R & D SYSTEMS). The measurement result of IL-6 is shown in FIG. 14C, and the measurement result of INF-γ is shown in FIG. 14D. From this result, suppression of inflammatory cytokine production induced by influenza virus infection was recognized by the sialic acid-containing sugar chain complex. In particular, the inhibitory action on inflammatory cytokine production was significant in the 3′-SL-N—C14 oral administration group. The statistical analysis was performed by t test (Student's t-test) (p <0.05).
－肺組織中のウイルス力価の測定－
　ウイルス投与群、３’－ＳＬ－Ｎ－Ｃ１４経鼻投与群、及び３’－ＳＬ－Ｎ－Ｃ１４経口投与群について、インフルエンザウイルスを接種してから２日目（ｎ＝５／群）及び３日目（ｎ＝５／群）に解剖を行い、肺を摘出した。各群の肺組織をホモジナイズすることにより、肺組織中のインフルエンザウイルスを抽出し、プラック測定（ＰＦＵ　Ａｓｓａｙ）によりウイルス力価を測定した。
　即ち、試験例２と同様の方法で調製したＭＤＣＫ細胞に、各群の肺組織中のインフルエンザウイルスの希釈系列液を接種し、３７℃にて１時間インキュベートして感染させた。培養後、アガロース液を重層し、完全に凝固した後、３７℃、５％ＣＯ２の条件下で３日間培養し、各群のウイルス力価を測定した。
　結果を図１４Ｅに示す。この結果より、肺組織中のウイルス力価は、３’－ＳＬ－Ｎ－Ｃ１４経口投与群及び３’－ＳＬ－Ｎ－Ｃ１４経鼻投与群共に、感染２日目で明らかな減少が認められた。なお、統計解析は、ｔ検定（Ｓｔｕｄｅｎｔ’ｓ　ｔ－ｔｅｓｔ）で行った（ｐ＜０．０５）。 -Measurement of virus titer in lung tissue-
The virus administration group, 3′-SL-N-C14 nasal administration group, and 3′-SL-N-C14 oral administration group, the second day after inoculation with influenza virus (n = 5 / group) and 3 On the day (n = 5 / group), dissection was performed and the lungs were removed. By homogenizing the lung tissue of each group, the influenza virus in the lung tissue was extracted, and the virus titer was measured by plaque measurement (PFU Assay).
That is, MDCK cells prepared by the same method as in Test Example 2 were inoculated with a diluted series of influenza viruses in the lung tissue of each group and incubated at 37 ° C. for 1 hour to be infected. After culturing, the agarose solution was overlaid, and after complete coagulation, the cells were cultured at 37 ° C. under 5% CO 2 for 3 days, and the virus titer of each group was measured.
The result is shown in FIG. 14E. From these results, the virus titer in the lung tissue was clearly decreased on the second day of infection in both the 3′-SL-N-C14 oral administration group and the 3′-SL-N-C14 nasal administration group. It was. The statistical analysis was performed by t test (Student's t-test) (p <0.05).
＜既存薬との比較検討＞
　製造例１で得られた３’－ＳＬ－Ｎ－Ｃ１４、製造例３で得られた３’－ＳＬ－Ｎ－Ｃ１８、製造例４で得られた３’－ＳＬＮ－Ｎ－Ｃ１４、製造例５で得られた６’－ＳＬＮ－Ｎ－Ｃ１４及び６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１４、製造例６で得られた６’－ＳＬ－Ｎ－Ｃ１４と、既存薬剤タミフルとの比較検討を以下の方法で行った。
　ＢＡＬＢ／ｃマウス（雌、５週齢、日本クレア株式会社）に、前記各シアル酸含有糖鎖複合体を１ｍｇ／ｋｇ、及びタミフルを１ｍｇ／ｋｇの用量で経口投与し（ｎ＝５／群）、これを１日２回、３日間行った。
　最初にシアル酸含有糖鎖複合体を投与してから４時間後にインフルエンザウイルスＡ／ＰＲ／８／３４株を１００ＰＦＵ／マウスを経口投与した。
　シアル酸含有糖鎖複合体及びタミフルに代えて、ＰＢＳを投与した動物を対照群（ｎ＝５）とした。また、インフルエンザウイルスのみを投与した群をウイルス投与群（ｎ＝５）とした。 <Comparison with existing drugs>
3'-SL-N-C14 obtained in Production Example 1, 3'-SL-N-C18 obtained in Production Example 3, 3'-SLN-N-C14 obtained in Production Example 4, Production Example 6′-SLN—N—C14 and 6′-S (1NH 2 ) LN—N—C14 obtained in Step 5 , 6′-SL-N—C14 obtained in Production Example 6, and the existing drug Tamiflu A comparative study was conducted by the following method.
BALB / c mice (female, 5 weeks old, CLEA Japan, Inc.) were orally administered with each sialic acid-containing sugar chain complex at a dose of 1 mg / kg and Tamiflu at a dose of 1 mg / kg (n = 5 / group). This was done twice a day for 3 days.
Four hours after the first administration of the sialic acid-containing sugar chain complex, 100 PFU / mouse of the influenza virus A / PR / 8/34 strain was orally administered.
An animal administered with PBS instead of the sialic acid-containing sugar chain complex and Tamiflu was used as a control group (n = 5). Moreover, the group which administered only influenza virus was made into the virus administration group (n = 5).
－生存率の評価－
　シアル酸含有糖鎖複合体の投与終了後、更に６日間飼育し、合計９日間生存を観察し、生存率を算出した。結果を図１５Ａに示す。この結果より、３’－ＳＬ－Ｎ－Ｃ１４がタミフルと同様に１００％の生存率であった。 -Assessment of survival rate-
After the administration of the sialic acid-containing sugar chain complex, the animals were reared for another 6 days, the survival was observed for a total of 9 days, and the survival rate was calculated. The results are shown in FIG. 15A. From this result, 3'-SL-N-C14 had a survival rate of 100%, similar to Tamiflu.
－炎症性サイトカインの測定－
　炎症性サイトカインとして、ＩＬ－６の濃度を前記同様の方法で測定した。ＩＬ－６の測定結果を図１５Ｂに示す。この結果より、ＩＬ－６の産生の抑制は、特に３’－ＳＬ－Ｎ－Ｃ１４、６’－Ｓ（１ＮＨ２）Ｌ－Ｎ－Ｃ１４に強く認められ、これらは抗ウイルス活性が強いシアル酸含有糖鎖複合体であることがわかった。 -Measurement of inflammatory cytokines-
As an inflammatory cytokine, the concentration of IL-6 was measured by the same method as described above. The measurement result of IL-6 is shown in FIG. 15B. From this result, suppression of IL-6 production was particularly strongly observed in 3′-SL-N—C14 and 6′-S (1NH 2 ) LN—C14, which are sialic acids with strong antiviral activity. It was found to be a sugar chain complex containing.
－肺組織中のウイルス力価の測定－
　インフルエンザウイルスを接種してから２日目（ｎ＝５／群）に解剖を行い、肺を摘出し、肺組織中のウイルス力価を前記同様の方法で測定した。結果を図１５Ｃに示す。この結果より、シアル酸含有糖鎖複合体は、タミフルと同等のウイルス力価であり、明らかにインフルエンザウイルス感染を抑制していることが確認された。 -Measurement of virus titer in lung tissue-
On the second day (n = 5 / group) after inoculation with influenza virus, dissection was performed, the lung was removed, and the virus titer in the lung tissue was measured by the same method as described above. The result is shown in FIG. 15C. From this result, it was confirmed that the sialic acid-containing sugar chain complex has a virus titer equivalent to that of Tamiflu and clearly suppresses influenza virus infection.
（試験例６：インフルエンザウイルス感染の生活環での作用効果）
＜シアル酸含有糖鎖複合体添加時期の検討＞
　各シアル酸含有糖鎖複合体の抗ウイルス活性の作用機序を解析するために、インフルエンザウイルスの感染過程、即ち、インフルエンザウイルス感染時又は感染後に各シアル酸含有糖鎖複合体を作用させてインフルエンザウイルスの感染抑制効果についてプラック測定（ＰＦＵ　Ａｓｓａｙ）により評価した。 (Test Example 6: Effects of life cycle of influenza virus infection)
<Examination of the time of addition of sialic acid-containing sugar chain complex>
In order to analyze the mechanism of action of the antiviral activity of each sialic acid-containing sugar chain complex, influenza virus infection process, that is, each sialic acid-containing sugar chain complex was allowed to act during or after influenza virus infection. The virus infection suppression effect was evaluated by plaque measurement (PFU Assay).
　試験例２において、シアル酸含有糖鎖複合体を表４に示す各シアル酸含有糖鎖複合体に代え、この各シアル酸含有糖鎖複合体の添加時期を表４に示す時期に従ったこと以外は、試験例２と同様の方法でＩＣ５０値を算出した。結果を表４に併せて示す。なお、表４は、前記試験を１回～３回行った平均値を示す。 In Test Example 2, the sialic acid-containing sugar chain complex was replaced with each sialic acid-containing sugar chain complex shown in Table 4, and the addition time of each sialic acid-containing sugar chain complex was according to the time shown in Table 4 Except for the above, IC 50 values were calculated in the same manner as in Test Example 2. The results are also shown in Table 4. Table 4 shows the average values obtained by performing the test 1 to 3 times.
Figure JPOXMLDOC01-appb-T000018
Figure JPOXMLDOC01-appb-T000018
　シアル酸含有糖鎖複合体は、いずれのシアル酸含有糖鎖複合体も、インフルエンザウイルスのヘマグルチニンに作用することから感染時又は感染後に作用し、特にインフルエンザウイルス感染時及び感染後の両方の時期で作用させることにより抗インフルエンザウイルス活性が増強されることがわかった。また、カルボキシル基をアミド化したシアル酸含有糖鎖複合体は、アミド化することで抗インフルエンザウイルス活性が強まる傾向にあった。 Sialic acid-containing glycan complexes are affected at the time of infection or after infection because any sialic acid-containing glycan complex acts on hemagglutinin of influenza virus, especially at both the time of infection and after infection. It was found that the anti-influenza virus activity was enhanced by the action. Moreover, the sialic acid-containing sugar chain complex in which the carboxyl group was amidated tended to increase the anti-influenza virus activity by amidation.
＜インフルエンザウイルスの種類の検討＞
　シアル酸含有糖鎖複合体の、Ａ／ＰＲ／８／３４以外のインフルエンザウイルス株に対する効果について検討した。
　試験例２において、シアル酸含有糖鎖複合体を表５に示す各シアル酸含有糖鎖複合体に代え、この各シアル酸含有糖鎖複合体の添加時期を表５に示す時期に従い、インフルエンザウイルスをＡ／ＰＲ／８／３４に代えて、インフルエンザウイルスＡ型でありＨ１Ｎ１タイプのＡ／山形／３２／８９、Ａ／山形／１２０／８６、Ａ／北九州／１５９／９３、又はインフルエンザウイルスＢ型のＢ／三重／１／９３を用いたこと以外は、試験例２と同様の方法でＩＣ５０値を算出した。結果を表５に併せて示す。なお、表５は、前記試験を１回行った平均値を示す。 <Examination of influenza virus types>
The effect of the sialic acid-containing sugar chain complex on influenza virus strains other than A / PR / 8/34 was examined.
In Test Example 2, the sialic acid-containing sugar chain complex was replaced with each sialic acid-containing sugar chain complex shown in Table 5, and the addition time of each sialic acid-containing sugar chain complex was determined according to the time shown in Table 5 and the influenza virus Instead of A / PR / 8/34, it is influenza virus type A and H1N1 type A / Yamagata / 32/89, A / Yamagata / 120/86, A / Kitakyushu / 159/93, or influenza virus type B The IC 50 value was calculated in the same manner as in Test Example 2 except that B / Mie / 1/93 was used. The results are also shown in Table 5. Table 5 shows average values obtained by performing the test once.
Figure JPOXMLDOC01-appb-T000019
Figure JPOXMLDOC01-appb-T000019
　表５より、いずれの分離株もＡ／ＰＲ／８／３４と同様にインフルエンザウイルス感染時及び感染後のいずれであっても抗インフルエンザウイルス活性が認められた。 As shown in Table 5, anti-influenza virus activity was observed in any of the isolates, both at the time of influenza virus infection and after infection, as in A / PR / 8/34.
（試験例７：シアル酸含有糖鎖複合体添加時間と抗インフルエンザウイルス活性の検討）
　シアル酸含有糖鎖複合体（３’－ＳＬ－Ｎ－Ｃ１６）及びタミフルの作用時間による、ＭＤＣＫ細胞に接種したインフルエンザウイルスの遊離に対する影響を検討した。 (Test Example 7: Examination of sialic acid-containing sugar chain complex addition time and anti-influenza virus activity)
The effect of the sialic acid-containing sugar chain complex (3′-SL-N-C16) and Tamiflu on the release of influenza virus inoculated into MDCK cells was examined.
　試験例２と同様の方法でＭＤＣＫ細胞を培養し、モノレーヤーになったＭＤＣＫ細胞に、試験例２と同様の方法でインフルエンザウイルスＡ／ＰＲ／８／３４株に感染させた。３７℃にて１時間インキュベートした後、インフルエンザウイルスを含む培養液を除去し、下記表６に示す各時間（１時間から２８時間）後に３’－ＳＬ－Ｎ－Ｃ１６（５０μｇ／ｍＬ）又はタミフル（１μｇ／ｍＬ）をそれぞれ加えたアガロース液を前記マイクロプレートに重層し、完全に凝固した後、３７℃、５％ＣＯ２の条件下で３日間培養した。ＭＤＣＫ細胞に形成されたプラーク数から、各時間での遊離ウイルス量（ＰＦＵ／ｍＬ）を算出した。結果を下記表６に示す。 MDCK cells were cultured in the same manner as in Test Example 2, and the MDCK cells in a monolayer were infected with influenza virus A / PR / 8/34 strain in the same manner as in Test Example 2. After incubation at 37 ° C. for 1 hour, the culture solution containing influenza virus was removed, and after each time (1 to 28 hours) shown in Table 6 below, 3′-SL-N-C16 (50 μg / mL) or Tamiflu The agarose solution to which (1 μg / mL) was added was layered on the microplate and completely coagulated, followed by culturing at 37 ° C. under 5% CO 2 for 3 days. From the number of plaques formed in MDCK cells, the amount of free virus (PFU / mL) at each time was calculated. The results are shown in Table 6 below.
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-T000020
　表６より、シアル酸含有糖鎖複合体は、感染時及び感染後共に添加することで強い抗インフルエンザウイルス活性を示し、特に１６時間以降に添加すると、明らかに作用が強くなる傾向が確認され、その作用は既存薬のタミフルよりも強かった。
　インフルエンザウイルスが細胞に感染して６時間以降から成熟インフルエンザウイルスが出現し、約１５時間頃までにほぼ終了することが報告されている（タミフル（医薬品インタビューフォーム：中外製薬株式会社）、リレンザ（医薬品インタビューフォーム：グラクソ・スミスクライン株式会社）参照）ことから、シアル酸含有糖鎖複合体は、成熟インフルエンザウイルスの増殖を抑制し、再感染を防ぐ作用を有することが示唆される。
　タミフル及びリレンザは、選択的ノイラミニダーゼ阻害剤であることから、成熟インフルエンザウイルスが感染細胞から遊離し、増殖の最後の過程に作用すると考えられている（タミフル（医薬品インタビューフォーム：中外製薬株式会社）、リレンザ（医薬品インタビューフォーム：グラクソ・スミスクライン株式会社）参照）。したがって、シアル酸含有糖鎖複合体は、タミフル及びリレンザよりも前の段階でインフルエンザウイルス活性を抑制できる点で有利である。 From Table 6, the sialic acid-containing sugar chain complex shows strong anti-influenza virus activity when added both at the time of infection and after infection, and in particular, when added after 16 hours, a tendency that the effect is clearly strengthened was confirmed. The effect was stronger than the existing drug Tamiflu.
It has been reported that mature influenza virus appears after 6 hours after influenza virus infects cells and is almost completed by about 15 hours (Tamiflu (Pharmaceutical Interview Form: Chugai Pharmaceutical Co., Ltd.), Relenza (Pharmaceuticals) Interview form: GlaxoSmithKline Co., Ltd.))) suggests that the sialic acid-containing sugar chain complex has the action of suppressing the growth of mature influenza virus and preventing reinfection.
Since Tamiflu and Relenza are selective neuraminidase inhibitors, it is thought that mature influenza virus is released from infected cells and acts on the final process of proliferation (Tamiflu (Pharmaceutical Interview Form: Chugai Pharmaceutical Co., Ltd.)) Relenza (see pharmaceutical interview form: GlaxoSmithKline, Inc.). Therefore, the sialic acid-containing sugar chain complex is advantageous in that it can suppress influenza virus activity at a stage prior to Tamiflu and Relenza.
（試験例８：シアル酸含有糖鎖複合体を含浸させた不織布の抗インフルエンザウイルス効果）
　下記表７に示すマスクに使用されている一般的な６種類の不織布に、下記表７に示すシアル酸含有糖鎖複合体をそれぞれ５０μｇ／ｃｍ２含浸させ、乾燥させた後、該不織布にインフルエンザウイルス（Ａ／ＰＲ／８／３４）を１．０×１０６ＰＦＵ／ｍＬ作用させた。室温にて１時間、安全キャビネット内に静置した後、各不織布にＰＢＳを加えて該不織布からインフルエンザウイルスを回収した。このウイルス溶液を用い、不織布に残存したウイルス量を、プラック法（ＰＦＵ　Ａｓｓａｙ）により測定した。
　即ち、試験例２と同様の方法で調製したＭＤＣＫ細胞に、前記ウイルス溶液の希釈系列液を接種し、３７℃にて１時間インキュベートして感染させた。培養後、アガロース液を重層し、完全に凝固した後、３７℃、５％ＣＯ２の条件下で３日間培養し、各含浸不織布のインフルエンザウイルスの増殖抑制率を測定した。なお、対照としては、シアル酸含有糖鎖複合体を含浸させなかった各種不織布を用いた。結果を表７に示す。 (Test Example 8: Anti-influenza virus effect of nonwoven fabric impregnated with sialic acid-containing sugar chain complex)
Six general nonwoven fabrics used in the masks shown in Table 7 below were impregnated with 50 μg / cm 2 of each of the sialic acid-containing sugar chain complexes shown in Table 7 below and dried, and then the nonwoven fabrics were treated with influenza. Virus (A / PR / 8/34) was allowed to act at 1.0 × 10 6 PFU / mL. After leaving still in the safety cabinet for 1 hour at room temperature, PBS was added to each nonwoven fabric, and influenza viruses were collect | recovered from this nonwoven fabric. Using this virus solution, the amount of virus remaining on the nonwoven fabric was measured by the plaque method (PFU Assay).
That is, MDCK cells prepared by the same method as in Test Example 2 were inoculated with a dilution series of the virus solution and incubated at 37 ° C. for 1 hour to be infected. After culturing, the agarose solution was overlaid and completely coagulated, and then cultured at 37 ° C. under 5% CO 2 for 3 days, and the influenza virus growth inhibition rate of each impregnated nonwoven fabric was measured. As a control, various nonwoven fabrics that were not impregnated with the sialic acid-containing sugar chain complex were used. The results are shown in Table 7.
Figure JPOXMLDOC01-appb-T000021
 （＊１）ＬＰＯ　２５２Ｄ　ＰＰ　スパンボンド　２５ｇ／ｍ２（株式会社ツジトミ製）
（＊２）３２０１Ａ　スパンボンド　ＰＰＴ　１００％　２０ｇ／ｍ２（東洋紡績株式会社製）
（＊３）３３０１Ａ　スパンボンド　ＰＰＴ　１００％　３０ｇ／ｍ２（東洋紡績株式会社製）
（＊４）ＧＣ－２５　サーマルボンド　ＰＰ／ＰＥ　２５ｇ／ｍ２（三幸毛糸紡績株式会社製）
（＊５）ＧＣ－３０　サーマルボンド　ＰＰ／ＰＥ　３０ｇ／ｍ２（三幸毛糸紡績株式会社製）
（＊６）ＢＴ－０６０５Ｗ　３０ｇ／ｍ２（ユニセル株式会社製）
Figure JPOXMLDOC01-appb-T000021
 (* 1) LPO 252D PP Spunbond 25g / m 2 (Tsuji Tomi Co., Ltd.)
(* 2) 3201A Spunbond PPT 100% 20 g / m 2 (Toyobo Co., Ltd.)
(* 3) 3301A Spunbond PPT 100% 30 g / m 2 (Toyobo Co., Ltd.)
(* 4) GC-25 Thermal Bond PP / PE 25g / m 2 (Sanko Yarn Spinning Co., Ltd.)
(* 5) GC-30 Thermal Bond PP / PE 30g / m 2 (Sanko Yarn Spinning Co., Ltd.)
(* 6) BT-0605W 30g / m 2 (Unicel Corporation)
　表７の結果より、いずれの不織布もインフルエンザウイルスの増殖抑制率が８０％以上を示し、シアル酸含有糖鎖複合体を不織布に固定することが可能であることが確認された。 From the results shown in Table 7, it was confirmed that all of the nonwoven fabrics exhibited an influenza virus growth inhibition rate of 80% or more, and it was possible to fix the sialic acid-containing sugar chain complex to the nonwoven fabric.
（試験例９：シアル酸含有糖鎖複合体のインフルエンザウイルスへの作用の検討）
　インフルエンザウイルス（Ａ／ＰＲ／８／３４）と、下記表８に示すシアル酸含有糖鎖複合体とを、それぞれ表８の容量に従い混合し、室温にて１０分間又は６０分間静置した。これらのインフルエンザウイルスの増殖について、プラック法（ＰＦＵ　Ａｓｓａｙ）により測定した。
　即ち、試験例２と同様の方法で調製したＭＤＣＫ細胞に、前記インフルエンザウイルスを１００ＰＦＵ／ｍＬ添加し、３７℃にて１時間インキュベートして感染させた。培養後、アガロース液を重層し、完全に凝固した後、３７℃、５％ＣＯ２の条件下で３日間培養し、それぞれインフルエンザウイルスの増殖抑制率を測定した。結果を表８に示す。 (Test Example 9: Examination of action of sialic acid-containing sugar chain complex on influenza virus)
Influenza virus (A / PR / 8/34) and the sialic acid-containing sugar chain complexes shown in Table 8 below were mixed according to the volumes shown in Table 8 and allowed to stand at room temperature for 10 minutes or 60 minutes. The proliferation of these influenza viruses was measured by the plaque method (PFU Assay).
That is, 100 PFU / mL of the influenza virus was added to MDCK cells prepared in the same manner as in Test Example 2, and the mixture was incubated at 37 ° C. for 1 hour for infection. After culturing, the agarose solution was overlaid and completely coagulated, then cultured at 37 ° C. under 5% CO 2 for 3 days, and the growth inhibition rate of influenza virus was measured respectively. The results are shown in Table 8.
Figure JPOXMLDOC01-appb-T000022
Figure JPOXMLDOC01-appb-T000022
　表８の結果、感染前に、シアル酸含有糖鎖複合体とインフルエンザウイルスとを予め接触させることで、インフルエンザウイルスの細胞への感染能力が低下することがわかった。また、この作用は、１０分間接触させたインフルエンザウイルスより、６０分間作用させたインフルエンザウイルスの感染力が低下していた。これは、シアル酸含有糖鎖複合体が、インフルエンザウイルスのヘマグルチニンに吸着したことからウイルスの感染能力が低下したものと推察される。
　この結果から、シアル酸含有糖鎖複合体をフィルターなどに担持させることにより、インフルエンザウイルスへの感染の予防に好適にできることが示唆される。 As a result of Table 8, it was found that the ability of the influenza virus to infect cells was reduced by contacting the sialic acid-containing sugar chain complex and the influenza virus in advance before infection. Moreover, the infectivity of the influenza virus made to act for 60 minutes was falling rather than the influenza virus made to contact for 10 minutes for this effect | action. This is presumably because the infectious ability of the virus was reduced because the sialic acid-containing sugar chain complex was adsorbed on hemagglutinin of influenza virus.
This result suggests that carrying a sialic acid-containing sugar chain complex on a filter or the like can be suitably used for prevention of infection with influenza virus.
（試験例１０：シアル酸含有糖鎖複合体のシアリダーゼ抵抗性の検討）
　製造例８で得られた６’－ＳＬＮ－Ｎ－Ｃ１８及び６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１８、並びに、下記方法で合成した２－（２－ピリジルアミノ）エチルアミン・２塩酸－３’－シアリルラクトース（ＰＡＥＡ－３’－ＳＬ）、シアル酸（Ｎｅｕ５Ａｃ、シグマアルドリッチ社製）をそれぞれ０．０１ｍｇ用い、０．１Ｍ　酢酸緩衝液（ｐＨ５．０）に懸濁し、シアリダーゼ（別名ノイラミニダーゼ；ＥＣ３．２．１．１８、Ｃｌｏｓｔｒｉｄｉｕｍ　ｐｅｒｆｒｉｇｅｎｓ由来、シグマアルドリッチ社製）を１０ｍＵ／ｍＬとなるように添加し、３７℃で３０分間反応させた。 (Test Example 10: Examination of sialidase resistance of sialic acid-containing sugar chain complex)
6′-SLN—N—C18 and 6′-S (1NH 2 ) LN—N—C18 obtained in Production Example 8, and 2- (2-pyridylamino) ethylamine · dihydrochloride-3 synthesized by the following method Using 0.01 mg each of '-sialyl lactose (PAEA-3'-SL) and sialic acid (Neu5Ac, Sigma-Aldrich), suspended in 0.1 M acetate buffer (pH 5.0), sialidase (also known as neuraminidase; EC 3.2.1.18, derived from Clostridium perfrigens ( manufactured by Sigma-Aldrich)) was added so as to be 10 mU / mL, and reacted at 37 ° C. for 30 minutes.
　ＰＡＥＡ－３’－ＳＬは、以下の方法で３’－シアリルラクトース以下のＰＡＥＡ化反応を行うことにより合成した。
　３’－シアリルラクトース（３’－ＳＬ）（シグマアルドリッチ社製）１５ｍｇ（２４μｍｏｌ）と、ＰＡＥＡ（和光純薬工業株式会社製）５．５ｍｇ（２６μｍｏｌ）に、６００μＬのエタノール／０．３２Ｍ　水酸化ナトリウム（９：１（体積比））を加え、更にＤＭＴ－ＭＭ（国産化学株式会社製）（１２．９０％水分）８．３ｍｇ（２６μｍｏｌ）を加えた後、室温で３時間放置することにより、３’－ＳＬ誘導体（ＰＡＥＡ－３’－ＳＬ）を得た。 PAEA-3′-SL was synthesized by performing a PAEA-forming reaction below 3′-sialyl lactose by the following method.
3 μ-sialyllactose (3′-SL) (Sigma Aldrich) 15 mg (24 μmol), PAEA (Wako Pure Chemical Industries, Ltd.) 5.5 mg (26 μmol), 600 μL ethanol / 0.32 M hydroxylated Sodium (9: 1 (volume ratio)) was added, and 8.3 mg (26 μmol) of DMT-MM (manufactured by Kokusan Chemical Co., Ltd.) (12.90% moisture) was added, and then left at room temperature for 3 hours. A 3′-SL derivative (PAEA-3′-SL) was obtained.
　シアリダーゼ処理により得られた各反応物と、製造例１で用いたクジラ脳ガングリオシド混合液とを用い、ＴＬＣにより、クロロホルム／メタノール／０．２質量％塩化カルシウム水溶液（６０：４０：１０（体積比））で展開した。結果を図１６に示す。 Using each reaction product obtained by sialidase treatment and the whale brain ganglioside mixed solution used in Production Example 1, chloroform / methanol / 0.2 mass% calcium chloride aqueous solution (60:40:10 (volume ratio) was measured by TLC. )). The results are shown in FIG.
　図１６において、「－」は、シアリダーゼ処理を行っていないもの、「＋」は、シアリダーゼ処理を行ったものを示す。また、「Ｍ」は、マーカーとして使用したクジラ脳ガングリオシド混合液（ＧＭ４、ＧＭ３、ＧＭ２、ＧＭ１、ＧＤ３、ＧＤ１ａ、ＧＤ１ｂ、及びＧＴ１ｂ）を示す。
　図１６の結果より、シアル酸のカルボキシル基がアミド化された６’－Ｓ（１ＮＨ２）ＬＮ－Ｎ－Ｃ１８は、シアリダーゼ処理の有無により移動度に変化はなく、シアリダーゼ抵抗性を示すことがわかった。また、上記方法でシアル酸のカルボキシル基をアミド化反応で修飾したＰＡＥＡ－３’－ＳＬも同様にシアリダーゼに対する抵抗性が認められた。
　一方、シアル酸のカルボキシル基がアミド化されていない６’－ＳＬＮ－Ｎ－Ｃ１８は、シアリダーゼ処理により分解され、ＧＴ１ｂ付近と、ＧＭ４よりわずかに移動度が大きい位置にバンドが検出された。ＧＴ１ｂ付近のバンドは、６’－ＳＬＮ－Ｎ－Ｃ１８から切り離された遊離Ｎｅｕ５Ａｃであり、ＧＭ４よりわずかに移動度の大きいバンドは、ラクトサミニルステアリン酸アミド（ＬＮ－Ｎ－Ｃ１８）である。 In FIG. 16, “−” indicates that sialidase treatment has not been performed, and “+” indicates that sialidase treatment has been performed. “M” indicates a whale brain ganglioside mixed solution (GM4, GM3, GM2, GM1, GD3, GD1a, GD1b, and GT1b) used as a marker.
From the results shown in FIG. 16, 6′-S (1NH 2 ) LN—N—C18, in which the carboxyl group of sialic acid is amidated, has no change in mobility depending on the presence or absence of sialidase treatment, and exhibits sialidase resistance. all right. In addition, PAEA-3′-SL, in which the carboxyl group of sialic acid was modified by an amidation reaction by the above method, was similarly resistant to sialidase.
On the other hand, 6'-SLN-N-C18 in which the carboxyl group of sialic acid was not amidated was decomposed by sialidase treatment, and bands were detected in the vicinity of GT1b and at a slightly higher mobility than GM4. The band near GT1b is free Neu5Ac cleaved from 6'-SLN-N-C18, and the band slightly more mobile than GM4 is lactosaminyl stearamide (LN-N-C18).
　本発明のシアル酸含有糖鎖複合体は、優れた抗インフルエンザウイルス活性を有するため、抗インフルエンザウイルス剤に好適に利用可能である。
　また、本発明のシアル酸含有糖鎖複合体の製造方法は、短い反応時間で、かつ、反応効率がよいため、前記シアル酸含有糖鎖複合体を効率よく製造することができる。
　更に、本発明の抗インフルエンザウイルス剤は、前記シアル酸含有糖鎖複合体を含有するため、優れた抗インフルエンザウイルス活性を有し、投与時期や投与対象の年齢などに制限がなく、かつ副作用がなく安全性の高い、インフルエンザの予防又は治療に好適に利用可能である。 Since the sialic acid-containing sugar chain complex of the present invention has excellent anti-influenza virus activity, it can be suitably used as an anti-influenza virus agent.
Moreover, since the method for producing a sialic acid-containing sugar chain complex of the present invention has a short reaction time and good reaction efficiency, the sialic acid-containing sugar chain complex can be efficiently produced.
Furthermore, since the anti-influenza virus agent of the present invention contains the sialic acid-containing sugar chain complex, it has excellent anti-influenza virus activity, there is no restriction on the administration time, the age of the administration subject, etc., and there are no side effects. It can be suitably used for prevention or treatment of influenza, which is highly safe.

Claims (14)

  1. 　一方の末端にシアル酸を含有するシアル酸含有糖鎖のシアル酸末端以外の末端に、スフィンゴ脂質を除く炭素数１４～１８の脂肪酸がアミド結合してなり、かつ分子量が４００～１，４００であることを特徴とするシアル酸含有糖鎖複合体。 A sialic acid-containing sugar chain containing sialic acid at one end has an amide bond with a fatty acid having 14 to 18 carbon atoms excluding sphingolipids at a terminal other than the sialic acid terminal, and has a molecular weight of 400 to 1,400. A sialic acid-containing sugar chain complex characterized by being.
  2. 　シアル酸のカルボキシル基がアミド化された請求項１に記載のシアル酸含有糖鎖複合体。 The sialic acid-containing sugar chain complex according to claim 1, wherein the carboxyl group of sialic acid is amidated.
  3. 　シアル酸含有糖鎖が、１個～５個の単糖で構成される請求項１から２のいずれかに記載のシアル酸含有糖鎖複合体。 The sialic acid-containing sugar chain complex according to any one of claims 1 to 2, wherein the sialic acid-containing sugar chain is composed of 1 to 5 monosaccharides.
  4. 　シアル酸含有糖鎖が、Ｎ－アセチルノイラミン酸（Ｎｅｕ５Ａｃ）と、ガラクトース（Ｇａｌ）とからなるシアリルガラクトース鎖（Ｎｅｕ５Ａｃ－Ｇａｌ）を含む請求項１から３のいずれかに記載のシアル酸含有糖鎖複合体。 The sialic acid-containing sugar according to any one of claims 1 to 3, wherein the sialic acid-containing sugar chain includes a sialylgalactose chain (Neu5Ac-Gal) composed of N-acetylneuraminic acid (Neu5Ac) and galactose (Gal). Chain complex.
  5. 　シアル酸含有糖鎖が、３’－シアリルガラクトース鎖（Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ）、及び、６’－シアリルガラクトース鎖（Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ）のいずれかを有する請求項１から４のいずれかに記載のシアル酸含有糖鎖複合体。 The sialic acid-containing sugar chain has either a 3'-sialylgalactose chain (Neu5Ac (α2-3) Gal) or a 6'-sialylgalactose chain (Neu5Ac (α2-6) Gal). The sialic acid-containing sugar chain complex according to any one of the above.
  6. 　シアル酸含有糖鎖が、３’－シアリルラクトース鎖（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇｌｃ）、６’－シアリルラクトース鎖（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）Ｇｌｃ）、３’－シアリルラクトサミン鎖（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）ＧｌｃＮＡｃ）、６’－シアリルラクトサミン鎖（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）ＧｌｃＮＡｃ）、３’－シアリルアミドラクトース鎖（３’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－３）Ｇａｌ（β１－４）Ｇｌｃ）、６’－シアリルアミドラクトース鎖（６’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－６）Ｇａｌ（β１－４）Ｇｌｃ）、３’－シアリルアミドラクトサミン鎖（３’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－３）Ｇａｌ（β１－４）ＧｌｃＮＡｃ）、６’－シアリルアミドラクトサミン鎖（６’－Ｎｅｕ５Ａｃ１ＮＨ２（α２－６）Ｇａｌ（β１－４）ＧｌｃＮＡｃ）、３’－シアリルガラクトシルガラクトース鎖（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）Ｇａｌ）、６’－シアリルガラクトシルガラクトース鎖（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）Ｇａｌ）、３’－シアリルガラクトシルガラクトサミン鎖（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ（β１－４）ＧａｌＮＡｃ）、及び６’－シアリルガラクトシルガラクトサミン鎖（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ（β１－４）ＧａｌＮＡｃ）からなる群より選択される請求項１から５のいずれかに記載のシアル酸含有糖鎖複合体。 Sialic acid-containing sugar chains are 3′-sialyllactose chains (3′-Neu5Ac (α2-3) Gal (β1-4) Glc), 6′-sialyllactose chains (6′-Neu5Ac (α2-6) Gal ( β1-4) Glc), 3′-sialyllactosamine chain (3′-Neu5Ac (α2-3) Gal (β1-4) GlcNAc), 6′-sialyllactosamine chain (6′-Neu5Ac (α2-6) Gal (β1-4) GlcNAc), 3′-sialylamide lactose chain (3′-Neu5Ac1NH 2 (α2-3) Gal (β1-4) Glc), 6′-sialylamide lactose chain (6′-Neu5Ac1NH 2 ( α2-6) Gal (β1-4) Glc), 3′-sialylamidolactosamine chain (3′-Neu5Ac1NH 2 (α2-3) Gal (β1-4) GlcNAc), 6 ′ -Sialylamide lactosamine chain (6'-Neu5Ac1NH 2 (α2-6) Gal (β1-4) GlcNAc), 3'-sialylgalactosylgalactose chain (3'-Neu5Ac (α2-3) Gal (β1-4) Gal ), 6′-sialylgalactosylgalactose chain (6′-Neu5Ac (α2-6) Gal (β1-4) Gal), 3′-sialylgalactosylgalactosamine chain (3′-Neu5Ac (α2-3) Gal (β1-4) A sialic acid according to any one of claims 1 to 5, selected from the group consisting of :) GalNAc), and a 6'-sialylgalactosylgalactosamine chain (6'-Neu5Ac (α2-6) Gal (β1-4) GalNAc) Containing sugar chain complex.
  7. 　請求項１から６のいずれかに記載のシアル酸含有糖鎖複合体の製造方法であって、一方の末端にシアル酸を含有するシアル酸含有糖鎖のシアル酸末端以外の末端に、スフィンゴ脂質を除く炭素数１４～１８の脂肪酸を、化学反応により結合させることを特徴とするシアル酸含有糖鎖複合体の製造方法。 The method for producing a sialic acid-containing sugar chain complex according to any one of claims 1 to 6, wherein a sialic acid-containing sugar chain containing sialic acid at one end has a sphingolipid at a terminal other than the sialic acid terminal. A method for producing a sialic acid-containing sugar chain complex, wherein a fatty acid having 14 to 18 carbon atoms excluding is bonded by a chemical reaction.
  8. 　シアル酸含有糖鎖のシアル酸末端以外の末端を、アシルアミド化する請求項７に記載のシアル酸含有糖鎖複合体の製造方法。 The method for producing a sialic acid-containing sugar chain complex according to claim 7, wherein the terminal other than the sialic acid terminal of the sialic acid-containing sugar chain is acylamidated.
  9. 　シアル酸含有糖鎖のシアル酸のカルボキシル基を、アシルアミド化する請求項７から８のいずれかに記載のシアル酸含有糖鎖複合体の製造方法。 The method for producing a sialic acid-containing sugar chain complex according to any one of claims 7 to 8, wherein the carboxyl group of the sialic acid of the sialic acid-containing sugar chain is acylamidated.
  10. 　シアル酸含有糖鎖と炭酸水素アンモニウムとを反応させ、次いで、得られた反応物と脂肪酸とを縮合剤の存在下で反応させる請求項７から９のいずれかに記載のシアル酸含有糖鎖複合体の製造方法。 The sialic acid-containing sugar chain complex according to any one of claims 7 to 9, wherein a sialic acid-containing sugar chain is reacted with ammonium hydrogen carbonate, and then the obtained reaction product and a fatty acid are reacted in the presence of a condensing agent. Body manufacturing method.
  11. 　請求項１から６のいずれかに記載のシアル酸含有糖鎖複合体を有効成分として含有することを特徴とする抗インフルエンザウイルス剤。 An anti-influenza virus agent comprising the sialic acid-containing sugar chain complex according to any one of claims 1 to 6 as an active ingredient.
  12. 　シアル酸含有糖鎖複合体を構成するシアル酸含有糖鎖が、インフルエンザウイルスのヘマグルチニン認識部位を有する請求項１１に記載の抗インフルエンザウイルス剤。 The anti-influenza virus agent according to claim 11, wherein the sialic acid-containing sugar chain constituting the sialic acid-containing sugar chain complex has a hemagglutinin recognition site of influenza virus.
  13. 　インフルエンザウイルスのヘマグルチニン認識部位が、３’－シアリルガラクトース鎖（３’－Ｎｅｕ５Ａｃ（α２－３）Ｇａｌ）、及び、６’－シアリルガラクトース鎖（６’－Ｎｅｕ５Ａｃ（α２－６）Ｇａｌ）のいずれかである請求項１２に記載の抗インフルエンザウイルス剤。 The hemagglutinin recognition site of influenza virus is any of 3′-sialylgalactose chain (3′-Neu5Ac (α2-3) Gal) and 6′-sialylgalactose chain (6′-Neu5Ac (α2-6) Gal) The anti-influenza virus agent according to claim 12.
  14. 　請求項１１から１３のいずれかに記載の抗インフルエンザウイルス剤を担持したことを特徴とするフィルター。 A filter comprising the anti-influenza virus agent according to any one of claims 11 to 13.
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