Cell membrane-permeable anti-human norovirus agent

Abstract

To provide an anti-human norovirus agent capable of suppressing infection and growth of human norovirus.SOLUTION: An anti-human norovirus agent contains, as an active ingredient, a fucose analog of the formula (V), for example.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an infectious disease therapeutic agent, and more specifically, an anti-human norovirus agent.

Background Art

[0002] Norovirus is one of the four virus genera in the family Caliciviridae, and currently, Norwalk virus is the only virus species.

[0003] Human norovirus mainly infects humans through food (seafood and water), and even a small number of virus particles, about 100 - 1000 in food, can cause infection, and it is highly infectious. Food poisoning caused by human norovirus accounts for more than 90% of viral food poisoning.

[0004] The main symptoms of human norovirus infection are nausea, vomiting, and diarrhea, and fever may also be observed. Vomiting and diarrhea can occur several times a day, up to more than 10 times in severe cases. The vomit may contain bile and intestinal contents, and the patient is stricken with severe pain.

[0005] According to data from North America, about 20 million people contract human norovirus infection annually, about 2 million people visit the hospital, 400,000 people are taken to the emergency department, 70,000 people are hospitalized, and 7,000 people die.

[0006] According to the estimates of the National Institute of Infectious Diseases, the number of cases of vomiting and diarrhea caused by human norovirus infection in Japan has exceeded 1 million to 3 million per year for decades, and this has had a serious social and economic impact. For these reasons, the demand from the Japanese people for antiviral drugs and vaccines against human norovirus is high, and administratively, the development priority has been placed fourth. In Japan, there are almost no death cases directly caused by human norovirus infection, but in the case of patients with other diseases and the elderly, there is a high risk of the condition becoming prolonged or severe. Also, even after the above symptoms subside, the excretion of highly infectious human norovirus in feces continues for 7 to 14 days, or up to two months or more in some cases, during which time patients cannot return to school or work. Furthermore, asymptomatic carriers of human norovirus are constantly detected at a rate of several percent of the total population. Since asymptomatic carriers of human norovirus are not aware of their own virus infection, for example, it has been reported that such asymptomatic carriers can cause large-scale food poisoning by working as food handlers.

[0007] Thus, human norovirus is a virus that has a high incidence of infection, causes food poisoning accompanied by patient suffering, has strong infectivity, and shows asymptomatic infection, but there is currently no specific drug. The basic approach is to prevent dehydration by oral or intravenous fluid replacement and conduct follow-up observations, and other symptomatic treatments such as administration of antiemetics and intestinal regulators are carried out. The use of antidiarrheal agents is discouraged, at least in the initial stage of the disease, because it can cause the virus to accumulate in the intestinal tract and delay recovery.

[0008] In 2002, a group in France reported that virus-like hollow particles (VLPs) of human norovirus bind to human histo-blood group antigens (HBGAs), and it was proposed that this molecule might be the receptor for human norovirus (Non-Patent Document 1).

[0009] Subsequently, verification by human volunteers progressed, and it was revealed that non-secretor individuals of HBGA are less susceptible to human norovirus infection, while secretor individuals of HBGA are more susceptible to human norovirus infection (Non-Patent Document 2).

[0010] However, currently, HBGA itself is not a receptor that mediates the entry of human norovirus into cells. This is based on the following reasons.

[0011] Secretor status, that is, whether HBGA is secreted into saliva or mucosa, is said to be related to the gene polymorphism of FUT2 (fucosyltransferase 2, the second out of 1 to 8). However, even if the FUT2 gene is introduced into cells where HBGA is not expressed on the cell surface to secrete HBGA and express it on the cell surface, infection of human norovirus into the cells does not occur. Therefore, HBGA itself cannot be said to be a receptor that mediates the entry of human norovirus into cells, and the causal relationship (direct or indirect relationship) between FUT2 gene polymorphism and human norovirus infection susceptibility has not yet been elucidated (Non-Patent Document 3).

[0012] Patent Document 1 is a prior art document that mainly discloses the use of fucose analogs, which are the active ingredients of the anti-norovirus agent of the present invention described later, for anti-cancer agent applications. Although Patent Document 1 has a general description regarding the use for treating infectious diseases, no examples are disclosed. Moreover, norovirus and its closely related diarrhea viruses are not disclosed among the viruses listed in Patent Document 1, and the mechanism of action in the infectious diseases described in Patent Document 1 is the enhancement of the immune response, which is completely different from the anti-norovirus agent of the present invention.

[0013] Patent Document 2 describes the 2D organoids used in the examples.

Prior Art Documents

Patent Documents

[0014]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Document

[0015]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0016] An object of the present invention is to provide an antiviral agent having a substantial therapeutic effect against human norovirus.

Means for Solving the Problems

[0017] The present inventors conducted studies to solve the above problems, and surprisingly found that administration of a fucose analog that inhibits sugar chain modification by fucosyltransferase (FUT) suppresses the infection of human norovirus in intestinal cells, which are infected cells of human norovirus, and further suppresses the growth of human norovirus in the cells, thereby completing the present invention.

[0018] That is, the present invention provides an anti-human norovirus agent (hereinafter also referred to as the anti-norovirus agent of the present invention) comprising a fucose analog that inhibits sugar chain modification by fucosyltransferase (FUT) or a salt thereof as an active ingredient. Here, it is preferable that the above fucosyltransferase contains FUT2.

[0019] More specifically, the present invention will be described as follows. The anti-norovirus agent of the present invention is an anti-human norovirus agent comprising a fucose analog of the following formula (I) or (II) or a salt thereof (hereinafter, the fucose analog includes its salt unless otherwise specified) as an active ingredient. In this specification, a functional group may be represented by an atomic symbol and "-". For example, "-OH" represents a hydroxyl group. Also, the number of carbon atoms may be shown as, for example, C1-C 10 (having 1 to 10 carbon atoms). The "salt" is pharmaceutically or biologically acceptable and may be any of a normal salt, an acidic salt, and a basic salt.

[0020] [Chemical formula] [In the formula, each of formula (I) or (II) is in the α or β anomer; R 1 , R 2 , R 3 , and R 4 are each independently a fluorine atom (F), a chlorine atom (Cl), -OH, -OC(O)H, -OC(O)C1-C 10 alkyl, -OC(O)C2-C 10 alkenyl, -OC(O)C2-C 10 alkynyl, -OC(O)aryl, -OC(O)heterocycle, -OC(O)C1-C 10 alkylene(aryl), -OC(O)C2-C 10 alkenylene(aryl), -OC(O)C2-C 10 alkynyl(aryl), -OC(O)C1-C 10 alkylene(heterocycle), -OC(O)C2-C 10 alkenylene(heterocycle), -OC(O)C2-C10 Alkynylene (heterocyclic ring), -OCH2OC(O)alkyl, -OCH2OC(O)Oalkyl -OCH2OC(O)aryl, -OCH2OC(O)Oaryl, -OC(O)CH2O(CH2CH2O) n CH3, -OC(O)CH2CH2O(CH2CH2O) n CH3, -O-tri-C1-C3alkylsilyl, and -OC1-C 10 selected from the group consisting of alkyl, and each n is an integer independently selected from 0 to 5; R 2a and R 3a each of which is independently selected from the group consisting of a hydrogen atom (H), F and Cl; R 5 is selected from the group consisting of -CH3, -CHF2, -CH=C=CH2, -C≡CH, -C≡CCH3, -CH2C≡CH, -C(O)OCH3, -CH(OAc)CH3, -CN, -CH2CN, -CH2X (X is F, a bromine atom (Br), Cl or an iodine atom (I).), and methoxylane; R 5 when it is other than -CH=C=CH2, -CH2F or -CHF2, R 1 , R 2 , R 3 , R 2a and R 3a at least one of is F or Cl.]

[0021] In the above, the fucose analog or its salt which is the active ingredient of the anti-norovirus agent of the present invention is preferably the fucose analog or its salt of formula (I). Also, in addition to the salt form, the solvate form is also acceptable.

[0022] The fucose analog of the above formula (I) or (II) is known to have at least a 10% fucosylation reduction effect on the fucosylation of proteins when the administration of the fucose analog is not performed (Patent Document 1). The "inhibition of sugar chain modification by fucosyltransferase (FUT)" in the present invention means this fucosylation reduction effect of 10% or more.

[0023] The norovirus to which the anti-norovirus agent of the present invention is applicable is a human norovirus. The anti-norovirus agent of the present invention can suppress the infection of the virus to intestinal cells, which are infected cells of human norovirus, and further suppress the growth of human norovirus in the cells.

[0024] It is preferable that the fucose analog, which is an active ingredient of the anti-norovirus agent of the present invention, has cell membrane permeability to human cells, at least to intestinal cells, in order to exert its anti-norovirus effect. For this purpose, within the limit that does not impair the FUT inhibitory effect as a fucose analog, R 1 , R 2 , R 3 , R 2a and R 3a One or more of them are preferably “-OC(O)C1-C 10 alkyl”, preferably O-acetyl (OAc), that is, “-OC(O)CH3” (Patent Document 3).

[0025] The fucose analog, which is an active ingredient of the anti-norovirus agent of the present invention, inhibits the sugar chain modification by fucose transferase (FUT) in intestinal cells. In particular, the inhibition of sugar chain modification by FUT2 is recognized together with the anti-norovirus effect.

[0026] The fucose analog of the above formula (I) or (II) is preferably a fucose analog of the following formula (III) or (IV) or a salt thereof.

[0027] [Chemical formula] [In the formula, each of formula (III) or (IV) is an α or β anomer, R 1 , R 3 and R 4 each is -OH or -OAc, R 2is a halogen atom which is F (fluorine atom) or Cl (chlorine atom), -OH, or -OAc, R 5 is -CH3, -C≡CH, -C≡CCH3, or -CH2C≡CH.]

[0028] (1) In the above formula (III) or (IV), when R 2 is a halogen atom which is F (fluorine atom) or Cl (chlorine atom), R 1 , R 3 and R 4 at least one of them being -OAc is preferred, and it is more preferred that two or more of R 1 , R 3 and R 4 are -OAc, and it is most preferred that all of R 1 , R 3 and R 4 are -OAc.

[0029] Also, in the above formula (III) or (IV), when R 2 is a halogen atom which is F (fluorine atom) or Cl (chlorine atom), R 5 is preferably -CH3, and the halogen atom is preferably F (fluorine atom).

[0030] (2) In the above formula (III) or (IV), when R 5 is -C≡CH, -C≡CCH3, or -CH2C≡CH, R 2 is preferably -OH or -OAc. Also, at least one of R 1 , R 2 , R 3 and R 4 being -OAc is preferred, and it is more preferred that two or more of R 1 , R 2 , R 3 and R 4 are -OAc, and it is most preferred that R 1 , R 2 , R 3 and R 4That all are -OAc is one of the most preferred embodiments. Further, that the alkynyl group is -C≡CH is one of the preferred embodiments.

[0031] (3) In the above (1) and (2), the fucose analog or a salt thereof which is the active ingredient of the anti-norovirus agent of the present invention is preferably the fucose analog of formula (III) or a salt thereof. In addition to the salt form, a solvate form is also acceptable.

[0032] The fucose analog or a salt or solvate thereof of the above formula (I) or (II), or (III) or (IV), which is the active ingredient of the anti-norovirus agent of the present invention, is introduced into the interior of intestinal cells, and as the replacement of the fucose moiety of GDP-fucose, which is a metabolic intermediate, proceeds, the formation of normal sugar chains is inhibited, and it is considered that the desired anti-norovirus effect is exerted by this action, but the details of the causal relationship are unknown. Specifically, (a) the entry (infection) of human norovirus into intestinal cells is blocked by the above action. However, as described above, the receptor responsible for the entry of human norovirus into cells on the cell surface of human norovirus is not yet known. As observed in HIV, HCV, and HBV, there is even a possibility that the infection of human norovirus is achieved by the cooperative action of multiple receptor molecules in human norovirus, and the specific situation regarding the above receptor is confusing. The fucose analog, which is the active ingredient of the anti-norovirus agent of the present invention, surprisingly can act on this unknown receptor molecule or a group of molecules (any one or more thereof) to inhibit the infection of human norovirus to intestinal cells. Even more surprisingly, (b) the growth of human norovirus itself in intestinal cells can be inhibited along with the above action.

Advantages of the Invention

[0033] According to the present invention, there is provided an anti-norovirus agent capable of suppressing the infection and growth of human norovirus in intestinal cells and preventing and treating human norovirus infection.

Brief Description of the Drawings

[0034]

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Figure 8

Figure 9

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Figure 12

Figure 13

Figure 14

Mode for Carrying Out the Invention

[0035] [1] Fucose analog The active ingredient of the anti-norovirus agent of the present invention is a fucose analog (I) or (II) [including (III) or (IV)] as described above, or a salt thereof, and in some cases, a solvate.

[0036] R 1 、R 2 、R 3 、and R 4 In the atoms or functional groups that each of can independently take, the "alkyl group" is a saturated straight-chain hydrocarbon group or a saturated branched hydrocarbon group having a defined number of carbon atoms, which may or may not be substituted.

[0037] Examples of the unsubstituted alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, n-hexyl group, 2-hexyl group, 3-hexyl group, n-heptyl group, n-octyl group, n-nonyl group, n-decyl group, 1-methylbenzyl group, 2-methylbenzyl group, 3-methylbenzyl group, 4-methylbenzyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2,2-dimethylbutyl group, 2,3-dimethylbutyl group, 3,3-dimethylbutyl group, 1-ethylbutyl group, 2-ethylbutyl group, 1,1,2-trimethylpropyl group, 1,2,2-trimethylpropyl group, 1-ethyl-1-methylpropyl group, 1-ethyl-2-methylpropyl group, and the like.

[0038] The substituted alkyl group can be substituted by a halogen atom, and further can be substituted by one or more groups, preferably 1-3 groups, selected from -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), aryl group, -C(O)R′, -OC(O)R′, -C(O)OR′, -C(O)NH2, -C(O)NHR′, -C(O)N(R′)2, -NHC(O)R′, -SR′, -SO3R′, -S(O)2R′, -S(O)R′, -OH, =O, -NH2, -NH(R′), -N(R′)2, and -CN. Here, each R′ is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, and aryl group.

[0039] Examples of the alkenyl group that R′ can take include, but are not limited to, ethylene or vinyl group, allyl group, -1-butenyl, -2-butenyl, -isobutenylenyl, -1-pentenyl, -2-pentenyl, 3-methyl-1-butenyl, -2-methyl-2-butenyl, and -2,3-dimethyl-2-butenyl.

[0040] Examples of the alkynyl group that R′ can take include, but are not limited to, ethynyl group, propynyl group, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1-pentynyl, and the like.

[0041] R 1 、R 2 、R 3 、and R 4 In the atoms or functional groups that each of R 、R 、R 、and R can independently take, the “alkenyl group” and the “alkynyl group” are unsaturated straight-chain hydrocarbon groups or unsaturated branched hydrocarbon groups with the defined number of carbon atoms, which may or may not be substituted.

[0042] The alkenyl chain has at least one double bond in the chain, and the alkynyl chain has at least one triple bond in the chain.

[0043] Examples of the unsubstituted alkenyl group include, but are not limited to, the same alkenyl groups that R′ can take, such as ethylene or vinyl group, allyl group, -1-butenyl, -2-butenyl, -isobutenylenyl, -1-pentenyl, -2-pentenyl, 3-methyl-1-butenyl, -2-methyl-2-butenyl, and -2,3-dimethyl-2-butenyl.

[0044] Examples of the unsubstituted alkynyl group include, but are not limited to, the same alkynyl groups that R′ can take, such as ethynyl group, propynyl group, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1-pentynyl, and the like.

[0045] When substituted, the alkenyl and alkynyl groups may be substituted with halogen atoms, and may further be substituted with one or more groups, preferably 1 to 3 groups, selected from -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), aryl groups, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S3R', -S(O)2R', -S(O)R', -OH, =O, -NH2, -NH(R'), -N(R')2, and -CN, etc. Here, each R' is the same as the "substituted R'" above.

[0046] R 1 , R 2 , R 3 , and R 4 In the atoms or functional groups each of which may be independently selected, "alkylene (group)" refers to a substituted or unsubstituted saturated branched or saturated straight-chain hydrocarbon group having two monovalent radical centers having a number of carbon atoms in the defined range. Representative unsubstituted alkylenes include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decalene, 1,4-cyclohexylene, and the like.

[0047] The substituted alkylene group may be substituted with a halogen atom, and may further be substituted with one or more groups, preferably 1 to 3 groups, selected from -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), an aryl group, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -SOR', -S(O)2R', -S(O)R', -OH, =O, -NH2, -NH(R'), -N(R')2, and -CN. Here, each R' is the same as the "substituted R'" above.

[0048] R 1, R 2 , R 3 , and R 4 In each of the atoms or functional groups that R

[0049] “Alkenylene (group)” is an alkenyl group (as described above) that is an unsaturated, branched, straight-chain or cyclic hydrocarbon group having two monovalent group centers derived by elimination of two hydrogen atoms from the same or two different carbon atoms of the parent alkene. The “alkenylene” group may be unsubstituted or substituted as described above for alkenyl groups. In some embodiments, the “alkenylene” group is unsubstituted.

[0050] R 1 , R 2 , R 3 , and R 4 In each of the atoms or functional groups that R

[0051] The aryl group being replaced is capable of being substituted by a halogen atom and further by one or more groups, preferably 1 - 3 groups, selected from -O-(C1 - C8 alkyl), -O-(C2 - C8 alkenyl), -O-(C2 - C8 alkynyl), aryl group, -C(O)R′, -OC(O)R′, -C(O)OR′, -C(O)NH2, -C(O)NHR′, -C(O)N(R′)2, -NHC(O)R′, -SR′, -SO3R′, -S(O)2R′, -S(O)R′, -OH, =O, -NH2, -NH(R′), -N(R′)2, and -CN etc. Here, each R′ is the same as the above-mentioned "substituted R′".

[0052] R 1 、R 2 、R 3 、and R 4 In the atoms or functional groups that each of R

[0053] can independently assume, "heterocyclic ring" means a substituted or unsubstituted monocyclic ring system having 3 - 7 or 3 - 10 ring atoms, wherein at least one ring atom is a heteroatom selected from N, O, P or S. One or more of the N, C or S atoms in the heterocyclic ring may be oxidized. The monocyclic heterocyclic ring preferably has 3 to 7 ring members (e.g., 2 - 6 carbon atoms and 1 - 3 heteroatoms independently selected from N, O, P, and S). The ring containing heteroatoms can be aromatic or non-aromatic. Unless otherwise specified, the heterocyclic ring is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure.Heterocyclic rings are described in Paquette, "Principles of Heterocyclic Chemistry" (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7 and 9; "The Chemistry of Heterocyclic Compounds, A Series of Monographs" (John Wiley & Sons, New York, since 1950), particularly Chapters 13, 14, 16, 19 and 28; and J. Am. Chem. Soc., 82:5566 (1960). Examples of unsubstituted "heterocyclic" groups include, but are not limited to, pyridyl group, dihydropyridyl group, tetrahydropyridyl group (piperidyl group), thiazolyl group, pyrimidinyl group, furanyl group, thienyl group, pyrrolyl group, pyrazolyl group, imidazolyl group, tetrazolyl group, fucosyl group, aziridinyl group, azetidinyl group, oxiranyl group, oxetanyl group, tetrahydrofuranyl group, etc.

[0054] Substituted heterocyclic groups can be substituted by halogen atoms and further by one or more groups, preferably 1 - 3 groups, selected from -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), aryl group, -C(O)R′, -OC(O)R′, -C(O)OR′, -C(O)NH2, -C(O)NHR′, -C(O)N(R′)2, -NHC(O)R′, -SR′, -SO3R′, -S(O)2R′, -S(O)R′, -OH, =O, -NH2, -NH(R′), -N(R′)2, and -CN, etc. Here each R′ is the same as the above-mentioned "substituted R′".

[0055] As more detailed examples of heterocyclic groups, carbon-bonded heterocyclic groups are at the following positions: at the 2-, 3-, 4-, 5- or 6-position of pyridine; at the 3-, 4-, 5- or 6-position of pyridazine; at the 2-, 4-, 5- or 6-position of pyrimidine; at the 2-, 3-, 5- or 6-position of pyrazine; at the 2-, 3-, 4- or 5-position of furan, tetrahydrofuran, thiophene, thienyl, pyrrole or tetrahydropyrrole; at the 2-, 4- or 5-position of oxazole, imidazole or thiazole; at the 3-, 4- or 5-position of isoxazole, pyrazole or isothiazole; at the 2- or 3-position of aziridine; at the 2-, 3- or 4-position of azetidine; and can be bonded.

[0056] Examples of such a carbon-bonded heterocyclic group include a 2-pyridyl group, 3-pyridyl group, 4-pyridyl group, 5-pyridyl group, 6-pyridyl group, 3-pyridazinyl group, 4-pyridazinyl group, 5-pyridazinyl group, 6-pyridazinyl group, 2-pyrimidinyl group, 4-pyrimidinyl group, 5-pyrimidinyl group, 6-pyrimidinyl group, 2-pyrazinyl group, 3-pyrazinyl group, 5-pyrazinyl group, 6-pyrazinyl group, 2-thiazolyl group, 4-thiazolyl or 5-thiazolyl, and the like.

[0057] The nitrogen-bonded heterocyclic group can be bonded at the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline or 1H-indazole; at the 2-position of isoindole or isoindoline; and at the 4-position of morpholine. Representative nitrogen-bonded heterocyclic groups include a 1-aziridyl group, 1-azetidyl group, 1-pyrrolyl group, 1-imidazolyl group, 1-pyrazolyl group, 1-piperidinyl group and the like.

[0058] The above R 1 、R 3 and R 4 each independently can be taken as “-OC(O)C1-C 10 alkyl” of “C1-C 10"Alkyl" refers to an alkyl group having 1 to 10 carbon atoms, which is a saturated straight-chain group or a branched hydrocarbon group with or without substitution of the carbon atoms. Examples of the unsubstituted alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, n-hexyl group, 2-hexyl group, 3-hexyl group, n-heptyl group, n-octyl group, n-nonyl group, n-decyl group, 1-methylbenzyl group, 2-methylbenzyl group, 3-methylbenzyl group, 4-methylbenzyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2,2-dimethylbutyl group, 2,3-dimethylbutyl group, 3,3-dimethylbutyl group, 1-ethylbutyl group, 2-ethylbutyl group, 1,1,2-trimethylpropyl group, 1,2,2-trimethylpropyl group, 1-ethyl-1-methylpropyl group, 1-ethyl-2-methylpropyl group, etc. When the unsubstituted alkyl group is a methyl group, "-OC(O)C1-C 10 alkyl" is O-acetyl (-OAc), that is, "-OC(O)CH3", and is exemplified as a preferred form. Also in this case, one or more of R 1 R 3 and R 4 are preferably -OAc, more preferably two or more are -OAc, and all three being -OAc is one of the most preferred embodiments.

[0059] The further substituted alkyl group can be substituted by a halogen atom, and further, it can be substituted by one or more groups, preferably 1-3 groups selected from -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), aryl group, -C(O)R′, -OC(O)R′, -C(O)OR′, -C(O)NH2, -C(O)NHR′, -C(O)N(R′)2, -NHC(O)R′, -SR′, -SO3R′, -S(O)2R′, -S(O)R′, -OH, =O, -NH2, -NH(R′), -N(R′)2, and -CN, etc. Here, each R′ is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, and aryl group.

[0060] Examples of the alkenyl group that R′ can take include, but are not limited to, ethylene or vinyl group, allyl group, -1-butenyl, -2-butenyl, -isobutenylenyl, -1-pentenyl, -2-pentenyl, 3-methyl-1-butenyl, -2-methyl-2-butenyl, and -2,3-dimethyl-2-butenyl, etc.

[0061] Examples of the alkynyl group that R′ can take include, but are not limited to, ethynyl group, propynyl group, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1-pentynyl, etc.

[0062] The fucose analog (I) or (II) [including (III) or (IV)], or their salts or solvates can be produced by known methods. For example, as disclosed in Patent Document 3, using L-galactose as a starting material, protecting the hydroxyl groups with isopropylidene, applying known means to make the side chain other than the protected part into a desired substituent (for example, adding an alkynyl group to the carbon at the 5th position), and then deprotecting the above isopropylidene part, and optionally performing a known acetylation treatment on the deprotected hydroxyl group.

[0063] Also, it is possible to use commercially available products, including outsourcing the synthesis.

[0064] [2] Pharmaceutical composition The fucose analog (I) or (II) [(including (III) or (IV))], which is the active ingredient of the anti-norovirus agent of the present invention, or a salt or solvate thereof (hereinafter, in the unit of this pharmaceutical composition, also referred to as fucose analog) can be used directly for anti-norovirus applications, but it is also possible to make it into a pharmaceutical composition (hereinafter, also referred to as composition) with an appropriate formulation. The composition contains a therapeutically effective amount of fucose analog and typically contains one or more carriers (for example, sterilized liquids such as water and oil, including those derived from petroleum, animals, plants or synthesis, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.). Water is a typical carrier when the composition is administered intravenously. Furthermore, physiological saline, dextrose and glycerol aqueous solutions can also be used as liquid carriers for injection solutions. Suitable excipients include, for example, amino acids, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The composition can also contain, if desired and as necessary, wetting agents, emulsifiers, pH buffers, disintegrants, sweeteners, preservatives, dyes / colorants, etc. These compositions can take forms such as solutions, syrups, elixirs, suspensions, enteric solvents, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc.

[0065] When the composition is in the form of capsules such as enteric capsules and gelatin capsules, in addition to the above materials, it may also contain liquid carriers such as polyethylene glycol, cyclodextrin, and fatty oils.

[0066] The content of the fucose analog in the composition can be freely selected in the range of 0.01 - 99% by mass with respect to the composition, depending on the dosage form, administration form, etc.

[0067] The dosage of the fucose analog by the anti-norovirus agent of the present invention administered to humans is 0.01 - 500 mg / kg body weight, more preferably 0.1 - 200 mg / kg body weight.

[0068] The administration form of the anti-norovirus agent of the present invention can be selected according to the purpose.

[0069] For example, during an outbreak of gastroenteritis caused by human norovirus, before eating foodstuffs (such as shellfish) that may be contaminated with human norovirus or before traveling to an area where human norovirus is prevalent, when administering the norovirus agent of the present invention for the purpose of preventing human norovirus infection, administration should be carried out on the day of the act - 4 days or more before, preferably 3 - 4 days before. The dosing interval should be set so that the concentration of the fucose analog in the body does not decrease extremely over time during the dosing period. Although it varies depending on the dosage form and the dose, it is standard to administer at least once a day.

[0070] For example, when suffering from gastroenteritis caused by human norovirus, administer as early as possible. The dosing interval should be set so that the concentration of the fucose analog in the body does not decrease extremely over time, as described above. Although it varies depending on the dosage form and the dose, it is standard to administer at least once a day. The dosing period is preferably until the human norovirus RNA disappears from the feces.

[0071] As described later, the anti-norovirus agent of the present invention prevents the infection of human norovirus in intestinal cells. After the administration is stopped, the fucose derivative is removed from the target intestinal cells, and no cytotoxicity is observed. It is characterized by being gentler to the body and being easy for long-term administration. Because the anti-norovirus agent of the present invention has such characteristics, it is easy to administer to people with weak resistance, people in an immunocompromised state, and the elderly. Also, because long-term administration is possible, secondary infections can be prevented by administration after the disappearance of the poisoning symptoms.

Examples

[0072] Hereinafter, examples of the present invention will be disclosed. The scope of the present invention is not limited to this disclosure. In this example section, the active ingredient of the anti-norovirus agent of the present invention is also referred to as "fucose analog".

[0073] [Materials] (1) Culture of 2D organoids derived from intestinal stem cells and human norovirus The 2D organoids derived from intestinal stem cells are those disclosed in Patent Document 2, "Step 1 of three-dimensionally culturing human intestinal epithelial stem cells, human intestinal epithelial cells, or a tissue containing at least any of these cells on an extracellular matrix to obtain 3D organoids, dispersing the 3D organoids obtained in the above step 1 to prepare single cells, and monolayer-culturing the single cells on an extracellular matrix to obtain 2D organoids in which the epithelial cells constituting the human intestinal lumen containing differentiated villus cells and goblet cells have a monolayer structure. The 2D organoids for culturing the infection and growth of human diarrhea viruses (including norovirus).". With the 2D organoids, it has become possible to culture human norovirus, which was impossible in the past.

[0074] In this example, 2D organoids derived from norovirus-sensitive human intestinal stem cells were prepared by the method disclosed in

[0130] -

[0139] of Patent Document 2 and used in each test.

[0075] Specific steps and the like are as follows. The media and drugs used in these steps can all be selected from commercially available products, but it is also possible to manufacture them in-house and use them as needed.

[0076] (a) Cell culture medium First, add human recombinant R-spondin 1 (e.g., manufactured by R&D systems) to Advanced DMEM / F-12 medium (e.g., manufactured by Thermo Ficher SCIENTIFIC) to a final concentration of 1 μg / mL, add noggin (e.g., manufactured by Peprotech) to a final concentration of 100 ng / mL, and add A83-01 (e.g., manufactured by Tocris) to a final concentration of 500 nM (hereinafter referred to as NRA medium).

[0077] Furthermore, prepare media by adding each of Wnt3a at a final concentration of 300 ng / mL, IGF1 at a final concentration of 500 ng / mL (e.g., manufactured by Biolegend), FGF2 at a final concentration of 50 ng / mL (e.g., manufactured by peprotech), and EGF at a final concentration of 50 ng / mL (e.g., manufactured by Thermo Ficher SCIENTIFIC) in the following combinations. ·WNRA + IGF1 + FGF2 medium ·ENRA medium

[0078] The meanings of the abbreviations used in these media are as follows. W: Wnt3A E: EGF N: Noggin R: Rspondin1 A: A-83-01

[0079] (b) Preparation of 2D organoids For example, based on an ethical research plan approved by the Ethics Committee of Keio University School of Medicine, normal mucosa is collected as a part at least 5 cm or more away from the gastrointestinal tumor from healthy individuals and gastrointestinal tumor patients who have given explanations and consent. The collected tissue is used to extract epithelial cells with EDTA or liberase TH and embed them in Matrigel (registered trademark).

[0080] Seed Matrigel (registered trademark) containing epithelial cells (hereinafter also referred to as intestinal stem cells) into a 48-well plate and culture it. Specifically, it is as follows.

[0081] The cultured intestinal stem cells were seeded into a 48-well plate together with 25 μL of Matrigel® (manufactured by BD Bioscience). 100 μL of the WNRA+IGF1+FGF2 medium prepared in (a) was added to each well, and the cells were cultured at 37°C. The medium was changed every two days during the culture, and the cells were cultured for 7 days to obtain 3D organoids derived from intestinal stem cells.

[0082] On the other hand, 50 μL / well of 2.5% Matrigel® diluted with PBS(-) was added to a 96-well plate and incubated at 37°C for 1 hour or more. Then, each well was washed three times with PBS(-) to prepare a well plate for 2D organoid preparation.

[0083] The above 3D organoids were dissociated using, for example, TrypLE™ Express (manufactured by Thermo Fisher SCIENTIFIC) to obtain single cells. After washing these single cells with ENRA medium, they were suspended in ENRA medium containing Y-27632 (a Rock inhibitor: manufactured by Wako Pure Chemical Industries, Ltd., for example) at a final concentration of 10 μM. This cell suspension was 5 seeded at 1×10

[0084] (c) Cultivation of human norovirus The feces of a human norovirus-infected patient contain a large amount of infectious human norovirus. A suspension of the feces of this human norovirus-infected patient in distilled water or the like to a final concentration of about 10% is designated as a "10% fecal suspension emulsion". This 10% fecal suspension emulsion is diluted about 10-fold with ENRA medium and added to the 2D organoids in each well above at 5 μL / well, followed by incubation for about 3 hours, whereby human norovirus can be grown and cultured. As a result of verification, it has been confirmed that the human norovirus grows about 100,000 times the initial amount in the culture system using these 2D organoids.

[0085] In this example, among the noroviruses of genetic group 2 (GII) that account for 80 - 90% of infected patients, which were obtained from the feces of past human norovirus-infected patients in the above-described manner and stocked in advance, as lineages (Lineage) included in GII, three isolates of GII.6 classified into "Lineage 1", GII.3 classified into "Lineage 2", and GII.4 classified into "Lineage 3" (GII.6: K2017 strain, GII.3: TCH2004 strain, GII.4: K2017 strain) were used as test subjects.

[0086] (2) Virus-like hollow particles (VLP) VLP is a structure obtained by incorporating the structural protein region of the human norovirus genome into baculovirus and expressing it in insect cells, which closely resembles human norovirus particles. VLP has a structure identical to that of human norovirus itself and has antigenicity equivalent to that of virus particles, but it does not have genomic RNA inside, is hollow, and is non-infectious.

[0087] In this example, a sequence encoding the structural protein region in norovirus GII.4 (AB447456) was incorporated into baculovirus and expressed in insect cells (SF9 cells), and the resulting product was used.

[0088] (3) Drugs As one of the fucose analogs used in this example, (3S,4R,5R,6S)-3-fluoro-6-methyltetrahydro-2H-pyran-2,4,5-triyl triacetate (manufactured by Merck & Co., Inc.; hereinafter also referred to as 2fp fucose) was used. 2fp fucose is a form in the fucose analog of formula (III) where R 1 , R 3 and R 4 are -OAc and R 2 is F. Specifically, it is a fucose analog represented by the following formula (V).

[0089] [Chemical formula]

[0090] As another one of the fucose analogs used in this example, 6-alkynyl fucose (1,2,3,4-tetra-O-acetyl-6,7-dideoxy-L-galacto-hept-6-ynopyranose) (manufactured by Peptide Institute, Inc.; hereinafter also referred to as 6a fucose) was used. 6a fucose is a fucose analog of formula (III) in which R 1 , R 2 , R 3 and R 4 are -OAc and R 5 is -C≡CH. Specifically, it is a fucose analog represented by the following formula (VI).

[0091] [Chemical formula]

[0092] As commercially available antiviral agents for comparison, sofosbuvir, Lamivudine, Ribavirin, and Mizoribine were obtained.

[0093] Growth inhibition test of human norovirus using 2fp fucose (1) Cytotoxicity of 2fp fucose against 2D organoids Continuous treatment with 2fp fucose for 7 days was performed on 2D organoids at 3.0 - 1000 μM, and the number of dead cells after treatment was determined by trypan blue staining. This test was performed independently 3 times. The results are shown in Figure 2. The error bars in the figure indicate SEM.

[0094] As shown in Figure 2, even 7 days after treatment, no significant difference was observed in the cell viability at all 2fp fucose treatment amounts compared to the non-treated case (0 μM).​​

[0095] This result indicates the safety against 2fp-fucose-treated cells.

[0096] (2) Influence of 2fp-fucose treatment on HBGA expression on the surface of 2D organoids Continuous 2fp-fucose treatment (0, 8, 40, 200, 1000 μM) for 4 days was performed on 2D organoids, and whether FITC (fluorophore)-labeled UEA-1 (Ulex europaeus agglutinin-1) binds to the decrease in fucose-modified proteins containing HBGA on the cell surface was examined. Note that UEA-1 reacts with fucose-modified proteins.

[0097] First, it was revealed that the expression level of α-linked fucose containing HBGA on the surface of 2D organoids decreased in a concentration-dependent manner with respect to 2fp-fucose by 2fp-fucose treatment, and this tendency became clear especially when exceeding 200 μM (Figure 3(a)). Figure 3(a) is a black-and-white photographic image.

[0098] On the other hand, AF488 (Alexa Fluor 488; green fluorophore) was labeled on the VLP of norovirus GII.4, and the purified fluorescent-labeled VLP was added to observe the degree of binding of the VLP to the surface of 2fp-fucose-treated 2D organoids. The 2fp-fucose treatment concentration was 0, 8, 40, 200, 1000 μM (4-day treatment) as in the case of the above UEA-1. As a result, the binding amount of the VLP decreased in a concentration-dependent manner with respect to 2fp-fucose, and a clear decrease in the adsorption of the VLP on the surface of 2D organoids was observed when the 2fp-fucose concentration exceeded 40 μM (Figure 3(b)). Figure 3(b) is a black-and-white photographic image.

[0099] The 2fp fucose concentration at which the decrease in the adsorption of the above-mentioned VLP (GII.4) to the 2D organoid surface becomes clear is "40 μM", which is a low concentration compared to "200 μM or more" (described later) at which the growth inhibitory effect in the GII.4 isolate of norovirus shown in Fig. 4 becomes clear. On the other hand, the clear decrease in fluorescence intensity of the above-mentioned UEA-1 to the 2D organoid occurs at "more than 200 μM of 2fp fucose", overlapping with the 2fp fucose concentration at which the growth inhibitory effect in the human norovirus isolate shown in Fig. 4 above becomes clear. This suggests that when the 2fp fucose concentration is 40 μM, although the function of the unknown human norovirus receptor (or receptor group) on the 2D organoid surface is decreasing, it is not completely impaired. This is because although the VLP adsorbs to the structure on the cell surface of the 2D organoid that adsorbs human norovirus particles and appears as fluorescence, a considerable number of VLPs that do not are considered to be taken up into the 2D organoid. The VLP taken up into the 2D organoid is rapidly decomposed and the presence of the fluorescent dye is no longer recognized, and is recognized as a decrease in fluorescence.

[0100] On the other hand, UEA-1 has the property of binding to proteins widely modified with fucose. When the 2fp fucose concentration exceeds 40 μM and reaches about 200 μM, 2fp fucose spreads to the proteins inside and on the cell surface that should originally be modified with fucose, which is considered to appear as an obvious UEA-1 binding inhibition phenomenon on the cell surface. There are also many fucose-modified proteins inside the cell, which should be involved in the reaction after the uptake of human norovirus particles. The function of these intracellular fucose-modified proteins is hindered by the inhibition of their production by 2fp fucose that has reached about 200 μM, and as a result, the growth of human norovirus inside the cell is considered to be inhibited.

[0101] Thus, it is strongly suggested that the fucose analog, which is the active ingredient of the anti-norovirus agent of the present invention, has the function of not only inhibiting the uptake of human norovirus into cells but also inhibiting its growth inside the cells.

[0102] (3) Effect of 2fp fucose treatment on human norovirus growth To examine the effect of 2fp fucose treatment on human norovirus growth for each norovirus lineage, after 3 days of 2fp fucose treatment, the above-mentioned isolates of norovirus GII.6, GII.3, and GII.4 were infected into the above-mentioned 2D organoids, and the virus growth amounts on the 3rd and 6th days after infection were measured (Figure 4). As a result, as shown in Figure 4, for the GII.3 isolate, complete inhibition of virus growth was observed at 40 μM or higher. For the GII.6 isolate, a complete growth inhibition effect was observed at 200 μM or higher. For the GII.4 isolate, a slight growth inhibition effect was observed from 40 μM, but a complete growth inhibition effect was observed at 200 μM or higher. From these results, at a concentration of 200 μM or higher, a complete growth inhibition effect was observed in human noroviruses of these lineages.

[0103] Next, to examine the effect of the number of days of prior 2fp fucose treatment on the anti-human norovirus effect of the fucose analog, the above-mentioned GII.4 isolate was treated with 2fp fucose at several concentrations over 1 - 4 days. The results are shown in Figure 5. As shown in Figure 5, in the 1-day treatment and 2-day treatment, the human norovirus grew more than 100-fold on the 3rd day after inoculation, and neither a growth inhibitory effect nor a concentration dependence was observed with 2fp fucose. However, in the 3-day treatment group, variability was observed in the effect where a growth inhibitory effect appeared with 200 μM treatment. In the 4-day treatment group, an effect appeared from 8 μM treatment, and the growth inhibition effect increased concentration-dependently up to 200 μM treatment. On the other hand, some growth of human norovirus was observed with 1000 μM treatment. Therefore, in this 2D organoid system, the highest growth inhibition effect was observed with 3 - 4 days of 200 μM treatment. This test was conducted independently 3 times. Error bars indicate SEM.

[0104] (4) Comparison of the effects of 2fp fucose and other virus growth inhibitors For 2D organoids, the following treatments were carried out for 3 days: (a) 200 μM of 2fp fucose, (b) 1 μM of sofosbuvir, (c) 1 μM of Lamivudine, (d) 400 μM of Ribavirin, (e) 100 μM of Mizoribine. Then, they were infected with human norovirus (the above-mentioned isolate of norovirus GII.4), and the growth inhibitory effect was measured (Figure 6). The test was independently conducted 3 times. Error bars indicate SEM. Only for 2fp fucose, almost no increase in viral RNA titer was observed from day 0 to day 7, and a growth inhibitory effect on human norovirus was recognized. For the other inhibitors, the viral RNA titer increased to the same level as the group without drug addition (No drug), and no growth inhibitory effect on human norovirus was shown at all.

[0105] Investigation of the mechanism of human norovirus growth inhibition by 2fp fucose (1) FUT gene expression profile and cytotoxicity by 2fp fucose After being taken up by cells, 2fp fucose is incorporated into α-fucose modification by fucose transferase (FUT). However, since some side chains are different from fucose, the next sugar chain cannot be extended from 2fp fucose. 2fp fucose is an analog of fucose that inhibits complete sugar chain modification by such a mechanism. 2fp fucose inhibits sugar modification by all fucose transferases found in mammalian cells, from FUT1 and FUT2 that perform α1-2 fucose transfer to FUT3-11 that perform α1-3 fucose transfer.

[0106] Here, the degree of intracellular expression of FUT from FUT1 to FUT11 in 2D organoids sensitive to human norovirus was examined by RT-PCR. The results are shown in Figure 7.

[0107] ​​As shown in Fig. 7, among FUT1 and 2 responsible for α1-2 fucose transfer, FUT2 was well expressed, but the expression of FUT1 was about 1 / 200 of that of FUT2. Among FUT3-11 responsible for α1-3 fucose transfer, the expressions of FUT3, 4, 6, 8, 10, and 11 were observed, but the expressions of FUT5, 7, and 9 were at background levels. Even when the functions of FUT2, 3, 4, 6, 8, 10, and 11 that should originally be expressed in 2D organoids were inhibited by 2fp fucose, no effect on cell viability was observed.

[0108] (2) Relationship between human norovirus growth and HBGA using FUT2 gene knockout cells To examine the relationship between the secretor status of HBGA and FUT2 gene expression in 2D organoids, the FUT2 gene of 2D organoids was knocked out, and the expression of the FUT2 gene in the knockout 2D organoids was confirmed by Western blotting (Fig. 8). In Fig. 8, "WT" indicates the wild 2D organoids as a control, and "KO" indicates the knockout 2D organoids. The marker used was "Bio-Rad Precision Plus Protein TM Dual Color Standard 1610374". The left electrophoresis diagram shows the results of examining FUT2, and the right electrophoresis diagram shows the results of examining β-actin as a control. In the left electrophoresis diagram, the band present only in WT between the 5th and 6th markers from the top is FUT2, and in the right electrophoresis diagram, the band present in both WT and KO between the 5th from the top is β-actin. That is, the expression of FUT2 in the knockout 2D organoids had decreased to a level where it could not be detected by Western blotting. Since the amount of β-actin in the non-knocked-out 2D organoids (wild type) and the knocked-out 2D organoids was the same, it was confirmed that the amount of cells subjected to electrophoresis and Western blotting was equal.

[0109] Figure 9 shows the results of examining the state of HBGA on the cell surface of knockout 2D organoids and wild-type 2D organoids, using the binding of FITC (fluorophore)-labeled UEA-1 to both as an indicator. In Figure 9, "UEA1" is a black-and-white photographic image showing the distribution and intensity of fluorescence when both are contacted with FITC-labeled UEA-1, and "Merge" is a composite of the said "UEA1" and an image of cell nucleus staining (blue) with Hoechst (registered trademark) 33342. As shown here, when the expression of FUT2 disappeared pinpointedly due to the knockout of the FUT gene, HBGA on the surface of 2D organoids was hardly observed.

[0110] Next, VLPs of GII.4 labeled with the green fluorophore AF488 were brought into contact with human norovirus knockout 2D organoids and wild-type 2D organoids to examine whether VLPs bind to the surfaces of both (Figure 10). As a result, almost no binding of VLPs was observed in the knockout 2D organoids. "Merge" in Figure 10, which is a black-and-white photographic image, is a composite of the image of the said "VLPs" and that of cell nucleus staining (blue) with Hoechst (registered trademark) 33342.

[0111] Next, the FUT2 gene was restored (rescued) to the above-mentioned FUT2 gene knockout 2D organoids to attempt to restore the expression of FUT2. When 2D organoids infected with human norovirus were stained red with an anti-human norovirus VP1 antibody, stained cells were observed with a low probability among the cell groups distributed on the whole surface, and it was confirmed that there were slightly some human norovirus-infected 2D organoids with VLPs attached to the surface (bright spots in Figure 11, which is a black-and-white photographic image). This suggests that HBGA was restored with low efficiency on the surface of the knockout 2D organoids.

[0112] Finally, wild-type 2D organoids (WT), FUT2 gene knockout 2D organoids (FUT2KO), and 2D organoids with the FUT2 gene restored (FUT2Res) were infected with human norovirus (the isolate of norovirus GII.4 described above), and the growth rates of the respective 2D organoids were compared and examined (Figure 12).

[0113] As shown in Figure 12, an increase in RNA titer was observed on the 3rd and 6th days in WT, and the growth of human norovirus was confirmed. However, no increase in RNA titer was observed in the FUT2 gene knockout 2D organoids, and no human norovirus growth occurred at all. And in the cells with the FUT2 gene restored in the knockout 2D organoids, although the increase rate was lower compared to the wild type (WT), an increase in RNA titer was observed, and it was confirmed that the human norovirus was in a state where it could grow.

[0114] Since FUT2Res cells are cells with the FUT2 gene introduced into FUT2KO cells, the FUT2 gRNA and Cas9 that had been working to knockout FUT2 in the cells continued to be constantly expressed. Therefore, the newly introduced FUT2 gene was also gradually knocked out. For this reason, even if the FUT2 gene is introduced into FUT2KO cells again, the human norovirus growth ability does not return to the same level as the wild type, and it is considered that the growth rate is also lower than that of WT.

[0115] Organoids become susceptible to human norovirus infection during the process of differentiating into intestinal cells. It is considered by this test that FUT2 acts during its maturation process and directly or indirectly affects the maturation of the human norovirus infection receptor that draws human norovirus into the cell on the cell surface. However, as described above, the mechanism including the process from FUT2 expression to becoming an infection-susceptible cell has not yet been elucidated.

[0116] Growth inhibition test of human norovirus using 6-alkynyl fucose ​​For 2D organoids, 6a-fucose was added to the medium at final concentrations of 0 μM, 1.6 μM, 8 μM, 40 μM, and 200 μM, and differentiation induction culture was performed for 4 days. The culture was carried out in 3 wells each on a 96-well plate, and each addition system of 6a-fucose was used as a test plate for each test human norovirus. As the test human noroviruses, two types, the above-mentioned GII.4 isolate and GII.6 isolate, were used. The culture was performed in a carbon dioxide incubator adjusted to 37°C.

[0117] On the 4th day after the start of the culture, the medium was removed, and after washing with a new medium, 10 5 RNA copies were added and left standing for 3 hours to infect. After the infection treatment, the human norovirus in the medium was removed together with the medium, and a washing operation with a new medium was performed 3 times.

[0118] After washing, 6a-fucose was added again to the new medium at final concentrations of 0 μM, 1.6 μM, 8 μM, 40 μM, and 200 μM, and the culture was performed for 6 days.

[0119] 10 μL of the medium was collected from each well immediately after adding the new medium (a), 3 days later (b), and 6 days later (c) as described above, centrifuged at 10,000 g for 10 minutes to remove cell sediment, and the amount of human norovirus genomic RNA contained in 1 μL of the supernatant was quantified by real-time RT-PCR.

[0120] The results are shown in Fig. 13. In Fig. 13, (a) shows the results using 6a-fucose for the GII.4 isolate of human norovirus, and (b) shows the results using 6a-fucose for the GII.6 isolate of human norovirus. The vertical axis in Fig. 13 is the copy number (RNA titer) of human norovirus genomic RNA, and the numbers below each group of bar graphs indicate the culture time at the start of the test, respectively.

[0121] The growth inhibitory effect on the GII.4 strain shown in Fig. 13(a) appeared at a 6a-fucose concentration of 1.6 μM. In the untreated group, an increase in the RNA titer more than 100-fold that at the start of culture was observed on the 6th day of culture, whereas all the 6a-fucose treatment groups at each concentration were suppressed to an RNA titer of 100-fold or less that at the start of culture. Specifically, at a 6a-fucose concentration of 8 μM, the increase in the RNA titer was further suppressed compared to the case of 1.6 μM, and was suppressed to an increase of about 10-fold that at the start of culture. At a 6a-fucose concentration of 40 μM, it slightly exceeded the measured value at the start of culture on the 6th day of culture, but almost no increase in the RNA titer was observed. At a 6a-fucose concentration of 200 μM, the increase in the RNA titer was completely inhibited.

[0122] The growth inhibitory effect on the GII.6 strain shown in Fig. 13(b) was not observed at a 6a-fucose concentration of 1.6 μM. However, at 8 μM, the growth of the human norovirus genome was strongly suppressed, and a slight growth exceeding the measured value on the 0th day was observed on the 3rd and 6th days of culture. At a 6a-fucose concentration of 40 μM or higher, the growth was completely inhibited.

[0123] In addition, the cultured cells (2D organoids) survived as a monolayer sheet except for the wells in which growth was observed by the above culture, and no cytotoxicity due to the addition of 6a-fucose was observed at all treatment concentrations.

[0124] Investigation of the treatment effect after virus infection by 2fp fucose and 6a fucose (1) Explanation of the test system It takes about 4 days for organoids to fully differentiate into small intestinal epithelial cells after induction of differentiation. Thereafter, cell division almost stops, and if the medium is not replaced, they start to die in 7 - 9 days. As can be seen from the above results, the effect of the fucose analog appears on the 2nd day after addition and reaches a plateau 4 days later. On the other hand, for human norovirus, the release of newly produced virus into the medium starts on the day after infection, growth can be observed, and the growth continues until the 6th day after infection.

[0125] ​​Generally, in the human body, when small intestinal epithelial cells die and exfoliate due to the first infection and intracellular proliferation by human norovirus, the stem cells present in the crypt part divide, and the replenishment of normally differentiation-induced epithelial cells is carried out, and eventually the regeneration of the villi progresses. In the newly supplied cells in this way, the nascent human norovirus released into the intestinal tract from the above small intestinal epithelial cells due to the first infection and intracellular proliferation newly infects, and the growth cycle of the second human norovirus begins to turn in the newly supplied cells. At this stage, usually, the control of virus growth by the host immune response starts, so it is considered that the infection and growth of the virus are gradually suppressed.

[0126] Regarding the anti-norovirus agent of the present invention, in order to design an effective dosage form applied to the above-mentioned infection and growth cycle of human norovirus, it is desirable to monitor how the fucose analog acts in the above-mentioned infection and growth cycle over time.

[0127] However, currently, there are no experimental animals that can monitor the infection and growth of human norovirus, and it is difficult to monitor human norovirus growth and cell regeneration, supply, reinfection, and immune response in vivo. Therefore, the time-dependent effect of the addition of the fucose analog after human norovirus infection was measured in vitro using the following conditions.

[0128] (2) Contents and results of the test system (Figure 14) (a) 2D organoids were seeded in 96-well plates, and after induction of differentiation, on the 5th day, groups were created in which the fucose analogs [2fp fucose (results shown in Figure 12(a)), 6a fucose (results shown in Figure 14(b))] were added at a concentration of 200 μM each (addition group) and a group without addition (untreated group). Then, on the 2nd day, the medium was removed, washed, and infected with the above two types of GII.4 and GII.6 isolates.

[0129] After the infection treatment, the human norovirus in the medium was removed together with the medium, and a washing operation was performed three times with a new medium. After washing, the above active ingredients were added again to the new medium, with the addition group being added to a concentration of 200 μM, and the untreated group was left without addition, and the cells were cultured for 3 days. 10 μL of the medium was collected from each well, centrifuged at 10,000 g for 10 minutes to remove the cell sediment, and the amount of human norovirus genomic RNA contained in 1 μL of the supernatant was quantified by real-time RT-PCR.

[0130] At this time, in the untreated group, an increase in the RNA titer of more than 10 times (2-fucose administration system) or more than 150 times (6a-fucose administration system) was observed, but in the addition group, no growth of human norovirus (increase in RNA titer) was observed in both administration systems. This result is shown in the two bars from the left end of Figures 14(a) and (b) (the bars for untreated and 2f-fuc or 6a-fuc respectively).

[0131] (b) In the above untreated group, human norovirus-infected susceptible cells still remained, indicating that new infection and growth occurred in the remaining susceptible cells due to additional infection with human norovirus. Therefore, in order to examine the effect of the anti-norovirus agent of the present invention on the remaining susceptible cells, the following operations were performed.

[0132] The medium was removed from each of the systems cultured for 3 days as the untreated group in the same manner as in (a) above. After the washing operation, the cells were reinfected with human norovirus, and then the virus was removed together with the medium. After the washing operation, a new medium was added, and the cells were further cultured for 3 days. The amount of human norovirus released into the supernatant was measured as the RNA titer in the same manner as above. In this reinfection test, an increase in the RNA titer of more than 20 times, which was higher than the first infection in (a) above, was observed (the bar for reinf in Figure 14(a)). That is, it was shown that the virus newly infected the cells by reinfection, and the virus concentration in the medium increased compared to the first infection.

[0133] (c) Next, examine the effect of the fucose analog on this reinfection. Since normal humans usually develop symptoms about 24 - 48 hours after norovirus infection, assume that an effective amount of the anti-norovirus agent of the present invention is administered immediately after the onset of symptoms. In this case, from the above findings, it is expected that the antiviral effect will appear 2 days or more after the initial norovirus infection. Also, the cells differentiated from the stem cells in the crypt part after the first infection will come into contact with the virus 3 days later in the presence of the fucose analog.

[0134] In this test system, among the 2D organoids that have passed 3 days after treatment with the above fucose analog, a second infection (reinfection) operation is performed on the surviving cells (most of which have not yet been infected with norovirus due to the effect of the fucose analog), and by examining the effect of the fucose analog on this reinfection operation, the effect brought about by the anti-norovirus agent of the present invention after norovirus infection is estimated.

[0135] Specifically, as in (a) above, as an addition group, the medium was removed from each of the systems cultured for 3 days in the presence of the fucose analog (200 μM) from the first infection, and after the washing operation, the cells were reinfected with norovirus. Then, the virus together with the medium was removed, and after the re-washing operation, a medium containing 200 μM of the fucose analog (Reinf+2f-fuc or Reinf+6a-fuc), or not containing it (Reinf-2f-fuc or Reinf-6a-fuc) was added, and the cells were further cultured for 3 days. At that time, the amount of norovirus released into the supernatant was measured as an RNA titer in the same manner as above. This result is shown in the two bars from the right end of FIGS. 14(a)(b) (the bars of Reinf+2f-fuc or Reinf+6a-fuc, or the bars of Reinf-2f-fuc or Reinf-6a-fuc).

[0136] As a result, it has been clarified that whether or not a fucose analog is added after the reinfection, cells that grew for 3 days in the presence of the active ingredient since the initial infection with norovirus do not accept reinfection with norovirus.

[0137] (3) Conclusion From the above results, when a human is infected with norovirus and continuously takes an effective amount of the anti-norovirus agent of the present invention at the time of onset, although it is difficult to avoid all the symptoms caused by the initial infection, it has been clarified that the continuous manifestation of symptoms caused by reinfection that occurs generally 2-3 days after infection can be effectively suppressed. This is very beneficial in terms of alleviating the pain of norovirus-infected patients qualitatively and in terms of duration. Furthermore, by using the anti-norovirus agent of the present invention after onset, it is expected that when a human is infected with norovirus, the excretion period of norovirus in feces, which has been a problem as a cause of the spread of norovirus infection, can be significantly shortened.

Claims

1. A cell membrane-permeable anti-human norovirus agent comprising, as an active ingredient, a fucose analogue represented by the following formula (III) or (IV) or a salt thereof: 【Chemistry 1】 wherein formula (III) or (IV) is an α or β anomer according to (1) or (2) below: (1) R １ , R ３ and R ４ Each of R is -OH or -OAc, and at least one is -OAc; ２ is a halogen atom which is F (fluorine atom) or Cl (chlorine atom), and R 5 is —CH 3 ; (2) Each of R 1 , R 2 , R 3 and R 4 is -OH or -OAc, and at least one is -OAc, and R 5 is -C≡CH.]

2. In the cell membrane-permeable anti-human norovirus agent, In the case of (1), R １ , R ３ and R ４ all of are -OAc; In the case of (2), R １ , R ２ , R ３ and R ４ All of are -OAc; The cell membrane-permeable anti-human norovirus agent according to claim 1.

3. The cell membrane-permeable anti-human norovirus agent according to claim 2, wherein the halogen atom of R 2 in (1) is F (fluorine atom).

4. The cell membrane-permeable anti-human norovirus agent according to any one of claims 1 to 3, wherein the fucose analog or a salt thereof as an active ingredient is a fucose analog of formula (III) or a salt thereof.

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