Patent Application
20220062315
The present invention relates to a therapeutic agent for infections and, more particularly, to an agent against human norovirus (hereinafter may be referred to as an “anti-human norovirus agent”).
Norovirus is one of the four members belonging to the family Caliciviridae. Currently, norovirus is a single-species virus, which is Norwalk virus.
Human norovirus infection is caused mainly by the mediation of food (fish and shellfish, or water). The infection may occur even in the presence of a very small number (about 100 to 1,000) of virus particles, indicating considerably strong infection action of human norovirus. Food poisoning caused by human norovirus accounts for more than 90% of all cases of viral food poisoning.
The major symptoms of human norovirus infection include nausea, vomiting, diarrhea, and fever. Infected patients suffer vomiting and diarrhea several times a day, and more than 10 times a day in severe cases. Vomit may contain bile and intestinal substances, which can be extremely distressing.
According to some data of North America, annually, about 20 million persons suffer from human norovirus infection; about 2 million persons visit hospitals; 400,000 persons are conveyed by ambulance; 70,000 persons are hospitalized, and 7,000 persons die.
According to an estimation of the National Institute of Infection of Japan, cases of vomiting/diarrhea caused by human norovirus infection have been increasing to 1 to 3 million people per year over several decades, which has an serious impact on society and the economy. Under such circumstances, Japanese people highly seek an antiviral drug and vaccine against human norovirus, and development thereof has been placed at the fourth administrative position in order of priority. In Japan, there are very few cases where human norovirus infection is a direct cause of death, but a high risk of prolongation and severity is imposed in the case of co-presence of other diseases and in elderly people. Also, even if the aforementioned symptoms are mitigated, excretion of highly contagious human norovirus into feces lasts for 7 to 14 days, or for 2 months or longer in the case of a long period, making the patients unable to return to schools or workplaces during the period. Further, latent human norovirus infection cases are usually detected at a rate of several percent of the total population. Since such a latent human norovirus infection patient is not aware of viral infection by himself or herself, large-scale food poisoning has been reported to be caused by, for example, a latent infection patient working as a food service worker.
As described above, human norovirus is a food-toxic virus which results in high morbidity and which causes pain in patients. In addition, human norovirus is a highly contagious virus causing latent infection. However, no specific medicine against norovirus has hitherto been developed. Basically, oral water supply, drip infusion, or the like is performed for preventing dehydration, with follow-up observation. In addition, an antiemetic agent, an intestinal regulator, or the like is administered as symptomatic therapy. Use of an antidiarrheal is negatively guided at least in an initial onset stage, since norovirus remains in the intestinal tract, thereby retarding recovery.
In 2002, a French study group reported that the virus-like (hollow) particle (VLP) of human norovirus binds to human histo-blood group antigen (HBGA), and suggested that this molecule may be a possible receptor of human norovirus (Non-Patent Document 1).
Thereafter, through progress in verification by human volunteers, it has been elucidated that HBGA non-secretory individuals are less susceptible to human norovirus, and HBGA secretory individuals are more susceptible to human norovirus (Non-Patent Document 2).
However, at present, HBGA per se is not regarded as a receptor responsible for the invasion of human norovirus into cells, for the following reasons.
The status of a secretor (i.e., secretor status); that is, whether or not HBGA is secreted into saliva and mucous membrane, is thought to be related to the genetic polymorphism of FUT2 (fucosyltransferase 2 of fucosyltransferases 1-8). However, even if the FUT2 gene is introduced into a cell in which HBGA is not formed through gene expression on the cell surface so as to secrete HBGA and form HBGA through gene expression on the cell surface, infection of the cell with human norovirus is not established. Thus, HBGA itself is not regarded as a receptor responsible for invasion of human norovirus into cells, and the causal relationship (directly and indirectly) between the FUT2 gene polymorphism and human norovirus infection susceptibility has not been elucidated (Non-Patent Document 3).
Patent Document 1 is a prior art document which discloses use, as an anti-cancer agent, of the fucose analog serving as the active ingredient of the below-mentioned anti-norovirus agent of the present invention. Although Patent Document 1 generally discloses use of the fucose analog for the treatment of infections, no specific working examples thereof are disclosed. The viruses listed in Patent Document 1 do not include norovirus and related diarrhea viruses. Furthermore, the action mechanism against infections disclosed in Patent Document 1 is an enhanced immuno-response, which completely differs from that of the anti-norovirus agent of the present invention.
Patent Document 2 discloses a 2D organoid used in the Examples.
An object of the present invention is to provide an anti-norovirus agent having a substantial therapeutic effect to human norovirus.
The present inventors have conducted extensive studies for solving the aforementioned problems, and have surprisingly found that human norovirus infection of intestinal cells, which are susceptible to infection with human norovirus, and further, proliferation of the human norovirus in the intestinal cells, can be suppressed through administration of a fucose analog which inhibits glycosylation by fucosyltransferase (FUT). The present invention has been accomplished on the basis of this finding.
Accordingly, the present invention provides an anti-human norovirus agent comprising, as an active ingredient, a fucose analog which inhibits glycosylation by fucosyltransferase (FUT), or a salt thereof (hereinafter the agent may be referred to as “the anti-norovirus agent of the present invention”). Preferably, the fucosyltransferase includes FUT2.
The present invention will be described in more detail.
The anti-norovirus agent of the present invention is an anti-human norovirus agent comprising, as an active ingredient, a fucose analog represented by the below-provided formula (I) or (II) or a salt thereof (hereinafter, unless otherwise specified, the term “fucose analog” also encompasses a salt thereof). Notably, in the present description, a functional group may be denoted by an element symbol and “-”. For example, “—OH” denotes a hydroxyl group. The number of carbon atoms may be represented as, for example, C1-C10 (i.e., the number of carbon atoms being 1 to 10). The term “salt” refers to a pharmaceutically or biologically acceptable salt and may be any of a neutral salt, an acidic salt, and a basic salt.
[In the above formulas, each of formulas (I) and (II) represents an α-anomer or a β-anomer;
each of R1, R2, R3, and R4 is selected from the group consisting of a fluorine atom (F), a chlorine atom (Cl), —OH, —OC(O)H, —OC(O)C1-C10 alkyl, —OC(O)C2-C10 alkenyl, —OC(O)C2-C10 alkynyl, —OC(O) aryl, —OC(O) heterocycle, —OC(O)C1-C10 alkylene(aryl), —OC(O) C2-C10 alkenylene(aryl), —OC(O) C2-C10 alkynylene(aryl), —OC(O)C1-C10 alkylene(heterocycle), —OC(O) C2-C10 alkenylene(heterocycle), —OC(O) C2-C10 alkynylene(heterocycle), —OCH2OC(O)alkyl, —OCH2OC(O)O-alkyl-OCH2OC(O)aryl, —OCH2OC(O)O-aryl, —OC(O)CH2O(CH2CH2O)nCH3, —OC(O)CH2CH2O(CH2CH2O)nCH3, —O-tri-C1-C3 alkylsilyl, and —OC1-C10 alkyl. Each n is an integer of 0 to 5;
each of R2a and R3a is selected from the group consisting of a hydrogen atom (H), F, and Cl;
R5 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 represents F, a bromine atom (Br), Cl, or an iodine atom (I)), and methoxysilane; and
when R5 is a group other than —CH═C═CH2, —CH2F, or —CHF2, at least one of R1, R2, R3, R2a, and R3a is F or Cl.]
Among the above-mentioned members, the fucose analog or a salt thereof, which serves as an active ingredient of the anti-norovirus agent of the present invention, is preferably a fucose analog represented by formula (I) or a salt thereof. In addition to the salt form, a form of solvate is also acceptable.
The fucose analog represented by formula (I) or (II) is known to exhibit a fucosylation reducing effect of at least 10%, with respect to fucosylation of a protein in the case where no fucose analog is administered (Patent Document 1). In the present invention, “inhibition of glycosylation by fucosyltransferase (FUT)” means a fucosylation reduction effect of 10% or more.
The norovirus targeted by the anti-norovirus agent of the present invention is human norovirus. The anti-norovirus agent of the present invention can suppress human norovirus infection of intestinal cells, which are susceptible to infection with human norovirus, and proliferation of the human norovirus in the intestinal cells.
The fucose analog, which serves as an active ingredient of the anti-norovirus agent of the present invention, preferably has cell membrane permeability to human cells, at least to human intestinal cells, for exerting the anti-norovirus effect thereof. So long as the FUT inhibition effect of the fucose analog is not impaired, it is preferable that one or more groups selected from R1, R2, R3, R2a, and R3a are “—OC(O)C1-C10 alkyl,” preferably O-acetyl(OAc); i.e., “—OC(O)CH3” (Patent Document 3).
The fucose analog, which serves as an active ingredient of the anti-norovirus agent of the present invention, inhibits glycosylation by fucosyltransferase (FUT) in intestinal cells. Particularly, inhibition of glycosylation by FUT2 is observed along with the anti-norovirus effect.
The fucose analog represented by the aforementioned formula (I) or (II) is preferably a fucose analog represented by the below-provided formula (III) or (IV) or a salt thereof.
[In the above formulas, each of formulas (III) and (IV) represents an α-anomer or a β-anomer;
each of R1, R3, and R4 is —OH or -OAc;
R2 is a halogen atom which is F (fluorine atom) or Cl (chlorine atom), —OH, or -OAc; and
R5 is —CH3, —C≡CH, —C≡CCH3, or —CH2CCH.]
(1) In the above formula (III) or (IV), when R2 is a halogen atom which is F (fluorine atom) or Cl (chlorine atom), at least one of R1, R3, and R4 is preferably -OAc. More preferably, two or more groups of R1, R3, and R4 are -OAc. Most preferably, all of R1, R3, and R4 are -OAc.
In the above formula (III) or (IV), when R2 is a halogen atom which is F (fluorine atom) or Cl (chlorine atom), R5 is preferably —CH3, and the halogen atom is preferably F (fluorine atom).
(2) In the above formula (III) or (IV), when R5 is —C≡CH, —C≡CCH3, or —CH2C≡CH, R2 is preferably —OH or -OAc. At least one group of R4, R2, R3, and R4 is preferably -OAc. More preferably, two or more groups of R4, R2, R3, and R4 are -OAc. Most preferably, all of R4, R2, R3, and R4 are -OAc. In addition, in a preferred embodiment, the alkynyl group is —C≡CH.
(3) In (1) and (2) above, the fucose analog or a salt thereof, which serves as an active ingredient of the anti-norovirus agent of the present invention, is preferably a fucose analog represented by formula (III) or a salt thereof. In addition to the salt form, a form of solvate is also acceptable.
Conceivably, when the fucose analog represented by formula (I) or (II), or formula (III) or (IV), or a salt or solvate thereof, which serves as an active ingredient of the anti-norovirus agent of the present invention, is incorporated into intestinal cells, the fucose analog replaces the fucose moiety of GDP-fucose (i.e., a metabolic intermediate), to thereby inhibit normal glycosylation and, via this action, provide an anti-norovirus effect of interest. However, no precise mechanism thereof has been elucidated. More specifically, (a) invasion of human norovirus into intestinal cells (i.e., infection of the intestinal cells with human norovirus) is inhibited via the aforementioned action. However, as described above, there has not been found a receptor responsible for the invasion of human norovirus present on the cell surface into the cells. As has been recognized in relation to HIV, HCV, and HBV, a plurality of receptor molecules might be concertedly involved with infection of human norovirus. Thus, characterization of the receptor is currently in a chaotic state. In contrast, surprisingly, the fucose analog serving as an active ingredient of anti-norovirus agent of the present invention acts on an unknown receptor molecule or receptor molecule groups (one or more species thereof), whereby infection of intestinal cells with human norovirus is inhibited. More surprisingly, (b) with the above action, proliferation of human norovirus itself in intestinal cells can be inhibited.
The present invention enables provision of an anti-norovirus agent which inhibits infection of intestinal cells with human norovirus and proliferation of the human norovirus in the intestinal cells and which can prevent and treat human norovirus infections.
[1] Fucose Analogs
As mentioned above, the active ingredient of the anti-norovirus agent of the present invention is a fucose analog (I) or (II) [including (III) or (IV)], a salt thereof, or, in some cases, a solvate thereof.
In each of R1, R2, R3, and R4 which can independently represent an atom or a functional group, the “alkyl group” is a substituted or unsubstituted, saturated linear-chain or saturated branched hydrocarbyl group having a predetermined number of carbon atoms.
Examples of the unsubstituted alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, n-hexyl, 2-hexyl, 3-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, 1-methylbenzyl, 2-methylbenzyl, 3-methylbenzyl, 4-methylbenzyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, and 1-ethyl-2-methylpropyl.
The substituted alkyl group may be an alkyl group substituted by a halogen atom, or may be an alkyl group substituted by one or more groups, preferably 1 to 3 groups, selected from among —O—(C1-C8 alkyl), —O—(C2-C8 alkenyl), —O—(C2-C8 alkynyl), aryl, —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, —CN, etc. R′ is independently selected from —H, —C1-C8 alkyl, —C2-C8 alkenyl, —C2-C8 alkynyl, and aryl.
Examples of possible R′ of the alkenyl group include, but are not limited to, ethenyl, vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, and 2,3-dimethyl-2-butenyl.
Examples of possible R′ of the alkynyl group include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and 3-methyl-1-pentynyl.
In each of R1, R2, R3, and R4 which can independently represent an atom or a functional group, each of the “alkenyl group” and the “alkynyl group” is a substituted or unsubstituted, unsaturated linear-chain or unsaturated branched hydrocarbyl group having a predetermined number of carbon atoms.
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.
Similar to the examples of possible R′ of the alkenyl group, examples of the unsubstituted alkenyl group include, but are not limited to, ethenyl, vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, and 2,3-dimethyl-2-butenyl.
Similar to the examples of possible R′ of the alkynyl group, examples of the unsubstituted alkynyl group include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and 3-methyl-1-pentynyl.
When being substituted, the alkenyl group and the alkynyl group may be substituted by a halogen atom, or may be substituted by one or more groups, preferably 1 to 3 groups, selected from among —O—(C1-C8 alkyl), —O—(C2-C8 alkenyl), —O—(C2-C8 alkynyl), aryl, —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, —CN, etc. Definition of R′ is the same as mentioned above.
In each of R1, R2, R3, and R4 which can independently represent an atom or a functional group, the “alkylene (group)” denotes a substituted or unsubstituted, saturated branched or saturated linear-chain hydrocarbyl group including two monovalent core moieties and having a predetermined number of carbon atoms. Typical examples of the unsubstituted alkylene group include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, and 1,4-cyclohexylene.
The substituted alkylene group may be an alkylene group substituted by a halogen atom, or may be an alkylene group substituted by one or more groups, preferably 1 to 3 groups, selected from among —O—(C1-C8 alkyl), —O—(C2-C8 alkenyl), —O—(C2-C8 alkynyl), aryl, —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, —CN, etc. Definition of R′ is the same as mentioned above.
In each of R4, R2, R3, and R4 which can independently represent an atom or a functional group, the “alkenylene (group)” denotes an unsaturated branched, linear-chain, or cyclic hydrocarbyl group including two monovalent core moieties derived through elimination of two hydrogen atoms from a single or two different carbon atoms of the parent alkene of the aforementioned alkenyl group. The “alkenylene” group may be unsubstituted, or may be substituted as in the case of the alkenyl group. In some embodiments, the “alkenylene” group is unsubstituted.
The “alkynylene (group)” is a substituted or unsubstituted, unsaturated branched, unsaturated linear-chain, or unsaturated cyclic hydrocarbyl group including two monovalent core moieties derived through elimination of two hydrogen atoms from a single or two different carbon atoms of the parent alkyne of the aforementioned alkynyl group.
In each of R4, R2, R3, and R4 which can independently represent an atom or a functional group, the “aryl (group)” refers to a substituted or unsubstituted monovalent aromatic hydrocarbyl group having 6 to 20 carbon atoms, which group is derived through elimination of one hydrogen atom from one carbon atom of the parent aromatic ring system. Some aryl groups are represented by a symbol “Ar” in an exemplary structure. Typical examples of the unsubstituted aryl group include, but are not limited to, groups derived from benzene, substituted benzene, phenyl, naphthalene, anthracene, biphenyl, etc.
The substituted aryl group may be an aryl group substituted by a halogen atom, or may be an aryl group substituted by one or more groups, preferably 1 to 3 groups, selected from among —O—(C1-C8 alkyl), —O—(C2-C8 alkenyl), —O—(C2-C8 alkynyl), aryl, —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, —CN, etc. Definition of R′ is the same as mentioned above
In each of R4, R2, R3, and R4 which can independently represent an atom or a functional group, the “heterocycle” refers to a substituted or unsubstituted single cyclic system having 3 to 7 or 3 to 10 ring-forming atoms in which at least one of them is a hetero atom selected from N, O, P, and S.
One or more N, C, or S atoms in the heterocycle may be oxidized. A single heterocycle preferably has a 3- to 7-membered ring (e.g., 2 to 6 carbon atoms and 1 to 3 hetero atoms independently selected from N, O, P, and S). The hetero atom-containing ring may be an aromatic ring or a non-aromatic ring. Unless otherwise specified, the heterocycle is bound at any hetero atom or any carbon atom thereof that provides structural stability.
The definition of “heterocycle” is described in Paquette, “Principles of Heterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularly in Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A Series of Monographs” (John Wiley & Sons, New York, 1950 to), particularly in Chapters 13, 14, 16, 19, and 28; and J. Am. Chem. Soc., 82:5566 (1960). Examples of the unsubstituted “heterocycle” group include, but are not limited to, pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl), thiazolyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, fucosyl, aziridinyl, azetidinyl, oxyranyl, oxetanyl, and tetrahydrofuranyl.
The substituted heterocycle group may be a heterocycle group substituted by a halogen atom, or may be a heterocycle group substituted by one or more groups, preferably 1 to 3 groups, selected from among —O—(C1-C8 alkyl), —O—(C2-C8 alkenyl), —O—(C2-C8 alkynyl), aryl, —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, —CN, etc. Definition of R′ is the same as mentioned above
More specific examples of the heterocycle group include a carbon-bound heterocycle group. The positions of the heterocycle available for bonding at a carbon atom are as follows: 2-, 3-, 4-, 5-, or 6-position of pyridine; 3-, 4-, 5-, or 6-position of pyridazine; 2-, 4-, 5-, or 6-position of pyrimidine; 2-, 3-, 5-, or 6-position of pyrazine; 2-, 3-, 4-, or 5-position of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole; 2-, 4-, or 5-position of oxazole, imidazole, or thiazole; 3-, 4-, or 5-position of isoxazole, pyrazole, or isothiazole; 2- or 3-position of aziridene; and 2-, 3-, or 4-position of azetidine.
Examples of the carbon-bound heterocycle group include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyridazinyl, 3-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.
The nitrogen-bound heterocycle group may be bound at 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; 2-position of isoindole or isoindoline; and 4-position of morpholine. Typical examples of the nitrogen-bound heterocycle group include 1-aziridyl, 1-azetidyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.
The “C1-C10 alkyl” of “—OC(O)C1-C10 alkyl,” which R1, R3, and R4 can independently represent, is an alkyl group having 1 to 10 carbon atoms and a substituted or unsubstituted, saturated linear-chain or saturated branched hydrocarbyl group having the corresponding number of carbon atoms. Examples of the unsubstituted alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, n-hexyl, 2-hexyl, 3-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, 1-methylbenzyl, 2-methylbenzyl, 3-methylbenzyl, 4-methylbenzyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, and 1-ethyl-2-methylpropyl. When the unsubstituted alkyl group is a methyl group, the “—OC(O)C1-C10 alkyl” is —O-acetyl(-OAc); i.e., “—OC(O)CH3,” which is exemplified as a preferable embodiment of “—OC(O)C1-C10 alkyl.” In this case, in one preferred embodiment, one or more groups of R1, R3, and R4 are -OAc. In one more preferred embodiment, two or more groups of R1, R3, and R4 are -OAc. In one most preferred embodiment, all three groups of R1, R3, and R4 are -OAc.
In addition, the substituted alkyl group may be an alkyl group substituted by a halogen atom, or may be an alkyl group substituted by one or more groups, preferably 1 to 3 groups, selected from among —O—(C1-C8 alkyl), —O—(C2-C8 alkenyl), —O—(C2-C8 alkynyl), aryl, —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, —CN, etc. R′ is independently selected from among —H, —C1-C8 alkyl, —C2-C8 alkenyl, —C2-C8 alkynyl, and aryl.
Examples of possible R′ of the alkenyl group include, but are not limited to, ethenyl, vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, and 2,3-dimethyl-2-butenyl.
Examples of possible R′ of the alkynyl group include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and 3-methyl-1-pentynyl.
The fucose analog (I) or (II) [including (III) or (IV)], a salt thereof, or a solvate thereof may be produced through a known method. For example, as disclosed in Patent Document 3, L-galactose is used as a starting material, and the corresponding hydroxyl groups thereof are protected via formation of an isopropylidene moiety. The side chains other than the protected moiety are transformed into a substituent of interest (e.g., addition of an alkynyl group to 5-position of carbon atom) by known means. The isopropylidene moiety is removed for deprotection, and the deprotected hydroxyl groups may optionally be subjected to a known acetylation treatment.
The fucose analog used in the invention may be a commercial product (including outsourcing synthesis).
[2] Pharmaceutical Composition
The fucose analog (I) or (II) [including (III) or (IV)], a salt thereof, or a solvate thereof (in the section of “Pharmaceutical composition,” collectively also referred to as a “fucose analog”) is an active ingredient of the anti-norovirus agent of the present invention. The fucose analog as is can be used as an anti-norovirus agent. Alternatively, the fucose analog may be appropriately formulated, to thereby provide a pharmaceutical composition (hereinafter may also be referred to simply as a “composition”). The composition contains a therapeutically effective amount of the fucose analog and, typically, one or more carriers (e.g., sterilized liquids such as water or oil, including an oily ingredient synthesized or derived from petroleum, animal, or plant, for example, peanut oil, soybean oil, mineral oil, or sesame oil). Water is a typical carrier of the composition for intravenous administration. Further, physiological saline and, water solution of dextrose and glycerol may also be used as a liquid carrier for injection. Examples of appropriate excipients include amino acid, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dry skim milk, glycerol, propylene glycol, water, and ethanol. If needed, the composition may optionally contain a hygro-spreader, an emulsifier, a pH buffer, a disintegrant, a sweetener, a preservative, a dye/colorant, etc. The thus-provided compositions may be in the form of solution, syrup, elixir, suspension, enteric agent, emulsion, tablet, pill, capsule, powder, sustained release preparation, or the like.
When the composition has a capsule form such as an enteric capsule or gelatin capsule, the composition may further contain a liquid carrier such as polyethylene glycol, cyclodextrin, or fatty oil, in addition to the aforementioned materials.
The composition may have a fucose analog content falling within a range of 0.01 to 99 mass %, depending on dosage form, mode of administration, etc.
In administration of the anti-norovirus agent of the present invention to human subjects, the dosage of the fucose analog is 0.01 to 500 mg/kg-body weight, more preferably 0.1 to 200 mg/kg-body weight.
The mode of administering the anti-norovirus agent of the present invention may be chosen according to the purpose.
For example, the anti-norovirus agent of the present invention is administered to a subject in order to prevent infection of the subject with human norovirus, before eating a food (e.g., shellfish) potentially and latently infected with human norovirus in the period of prevalence of human noroviral gastroenteritis, or before traveling to an area where human noroviral infection is prevalent. In such a case, administration of the anti-norovirus agent is started at least four days before the day of eating or traveling, preferably 3 to 4 days before the day. The administration interval should be determined so that the fucose analog concentration of the body does not considerably lower over time during the administration period. The interval varies depending on the form and amount of dosage, but the agent is normally administered at least once a day.
In the case where a subject is affected by human noroviral gastroenteritis, the agent is administered as soon as possible. As described above, the administration interval should be determined so that the fucose analog concentration of the body does not considerably lower over time during the administration period. The interval varies depending on the form and amount of dosage, but the agent is normally administered at least once a day. The administration is preferably continued until the human noroviral RNA disappears in the feces of the subject.
As mentioned hereinbelow, the anti-norovirus agent of the present invention prevents human norovirus infection in intestinal cells. After termination of the administration, the fucose derivative is exhausted from the intestinal cells, providing no cytotoxicity. Thus, the agent of the present invention is milder to the human body. By virtue of the above characteristics, the agent of the present invention can be easily administered for a long period of time. As a result, the anti-norovirus agent of the present invention can be positively administered to a human subject having a reduced tolerance or in immunodeficiency, or to elderly person. In addition, since the anti-norovirus agent of the present invention can be administered for a long period, secondary infections can be avoided by the administration thereof after disappearance of the symptoms of poisoning.
A working example of the present invention will next be described. However, the example should not be construed as limiting the scope of the present invention thereto. In the section of “Examples,” the active ingredient of the anti-norovirus agent of the present invention may also be referred to as a “fucose analog.”
[Materials]
(1) 2D Organoid Derived from Intestinal Stem Cells and Culturing of Human Norovirus
The 2D organoid derived from intestinal stem cells refers to a 2D organoid for infection and culture of human diarrhea virus (including norovirus) disclosed in Patent Document 2. According to Patent Document 2, the 2D organoid is “obtained by Step 1 in which human intestinal epithelial stem cells, human intestinal epithelial cells, or a tissue including at least one of these cells is subjected to 3-dimensional culturing on the extracellular matrix, to thereby obtain a 3D organoid; and
by Step 2 in which the above-obtained 3D organoid is dispersed to prepare single cells; the single cells are subjected to monolayer culturing on an extracellular matrix; and there is obtained a 2D organoid having a single-layer structure of the epithelial cells for forming the human intestinal cavity which include differentiated trophoblastic cells and caliciform cells.” By virtue of the 2D organoid, culturing human norovirus, which was not previously achieved, can be successfully performed.
In the Example, a 2D organoid derived from norovirus-susceptible human intestinal stem cells was prepared through a technique disclosed in paragraphs [0130] to [0139] of Patent Document 2, and used in the tests carried out in the Example.
Specific steps thereof will next be described. The culture media and agents employed in these steps may be selected from corresponding commercial products, or may be produced in the laboratory in accordance with needs.
(a) Cell Culture Media
To an Advanced DMEM/F-12 medium (product of, for example, Thermo Fisher SCIENTIFIC), human recombinant R-Spondin 1 (product of, for example, R&D systems) is added to a final concentration of 1 μg/mL; Noggin (product of, for example, Peprotech) is added to a final concentration of 100 ng/mL; and A83-01 (product of, for example, Tocris) is added to a final concentration of 500 nM. The thus-prepared medium is referred to as an “NRA medium.”
Subsequently, any of Wnt3a (final concentration: 300 ng/mL), IGF1 (product of, for example, Biolegend) (final concentration: 500 ng/mL), FGF2 (product of, for example, Peprotech) (final concentration: 50 ng/mL), and EGF (product of, for example, Thermo Fisher SCIENTIFIC) (final concentration: 50 ng/mL), are added thereto, to thereby prepare the following combination media.
Definitions of the abbreviations of the media are as follows.
(b) Preparation of 2D Organoid
In one procedure, according to the research plan approved by Keio University School of Medicine, the Ethics Committee, normal mucous membrane samples (a portion 5 cm away from the tumor) are collected from healthy subjects and gastrointestinal tract tumor patients from which informed consent has been obtained. Each collected tissue sample is treated with EDTA or Liberase TH, to thereby extract epithelial cells. The cells are embedded in Matrigel (registered trademark).
Epithelial cells (hereinafter may be referred to as intestinal stem cells) embedded in Matrigel (registered trademark) are disseminated onto a 48-well plate and cultured. The specific procedure conducted is as follows.
Cultured intestinal stem cells were disseminated with 25 μL of Matrigel (registered trademark) (product of BD Bioscience) to a 48-well plate. The WNRA+IGF1+FGF2 medium prepared in (a) was added to each well (100 μL/well), and the cells were cultured at 37° C. From the start of culturing, the medium was exchanged every two days. The culturing was continued for 7 days, to thereby yield a 3D organoid derived from the intestinal stem cells.
Separately, 2.5% Matrigel (registered trademark) diluted with PBS(−) was added to a 96-well plate (50 μL/well), and the plate was subjected to incubation at 37° C. for 1 hour or longer. Subsequently, the wells were washed thrice with PBS(−), to thereby produce a well plate for preparing a 2D organoid.
The aforementioned 3D organoid is degraded by use of, for example, TrypLE (trademark) Express (product of Thermo Fisher SCIENTIFIC), to thereby yield single cells. The thus-obtained single cells are washed with an ENRA medium and suspended in an ENRA medium containing Y-27632 (Rock inhibitor, product of, for example, Wako Pure Chemical) (final concentration: 10 μM). The cell suspension is disseminated onto the wells of the aforementioned well plate at 1×105 cells/well, and allowed to stand for 3 hours or longer while the cells remain in contact with the plate, to thereby produce a 2D organoid derived from the intestinal stem cells. Unless otherwise specified, the “2D organoid derived from the intestinal stem cells” will be described simply as a “2D organoid.”
(c) Culturing of Human Norovirus
Feces of a human norovirus-infected patent contains a large amount of infectious human norovirus. Thus, the feces of a human norovirus-infected patient is suspended in distilled water or the like to a final concentration of about 10%, to thereby prepare a “10% feces suspension.” The 10% feces suspension is about 10-fold diluted with an ENRA medium. The diluted suspension is added to each of the aforementioned wells having the 2D organoid (5 μL/well) and subjected to incubation for about 3 hours, to thereby proliferate human norovirus. As a result of verification, it has been confirmed that human norovirus proliferates to about 100,000 times the initial count through the culture system employing the 2D organoid.
In the Example, there were tested strains of human norovirus obtained from feces of human norovirus infection patients through the aforementioned procedure and stocked before the test. Specifically, three strains belonging to the genetic group 2 (GII) which accounts for 80 to 90% of the infected patients were selected as test targets. The isolated strain lineages included in GII (
(2) Virus-Like Particles (VLPs)
A VLP (virus-like particle) is a particle extremely similar to a human norovirus and obtained by incorporating a structural protein domain of the human norovirus genome into a baculovirus and expressing the genetic domain in insect cells. Since the structure of VLP is completely the same as that of a particle of human norovirus, VLP has the same immunogenicity as that of the virus particle. However, VLP having no genomic RNA therein (i.e., conceptually hollow) is not infectious.
In the Example, a nucleotide sequence (AB447456) encoding a structural protein domain of norovirus GII.4 was incorporated into a baculovirus, and the sequence was expressed in insect cells (SF9 cells). The expression product was used in the Example.
(3) Agents
In the Example, (3S,4R,5R,6S)-3-fluoro-6-methyltetrahydro-2H-pyran-2,4,5-triyl triacetate (product of Merck, and hereinafter may be referred to as 2fp-fucose) was used as a fucose analog. 2fp-Fucose is one embodiment of the fucose analog represented by formula (III), wherein each of R4, R3, and R4 is -OAc, and R2 is F, and more specifically, a fucose analog represented by the following formula (V).
In the Example, 1,2,3,4-tetra-O-acetyl-6,7-dideoxy-L-galacto-hept-6-ynopyranose) (product of Peptide Institute, and hereinafter may be referred to as 6a-fucose) was used as another fucose analog. 6a-Fucose is one embodiment of the fucose analog represented by formula (III), wherein each of R4, R2, R3, and R4 is -OAc, and R5 is —CCH, and more specifically, a fucose analog represented by the following formula (VI).
As comparative compounds, commercial antiviral agents; i.e., Sofosbuvir, Lamivudine, Ribavirin, and Mizoribine were provided.
[Human Norovirus Proliferation Inhibition Test by Use of 2fp-Fucose]
(1) Cytotoxicity of 2fp-fucose to 2D organoid
The 2D organoid was treated with 2fp-fucose (3.0 to 1,000 μM) for 7 continuous days. In each case, the number of dead cells was determined through Trypan blue staining. The test was repeatedly performed three times.
As shown in
Thus, the safety of 2fp-fucose to cells has been confirmed by the above results.
(2) Effect of 2fp-Fucose Treatment on HBGA Expression on the Surface of 2D Organoid
The 2D organoid was subjected to a 2fp-fucose treatment (0, 8, 40, 200, and 1,000 μM) for 4 continuous days. Reduction of fucose-modified proteins including HBGA formed on the cell surfaces was investigated by checking binding of FITC (phosphor)-labeled UEA-1 (Ulex europaeus agglutinin-1). Notably, UEA-1 reacts with a fucose-modified protein.
Through the 2fp-fucose treatment, the amount of α-link fucose including HBGA formed via gene expression on the surface of the 2D organoid was found to decrease in a concentration-dependent manner with respect to 2fp-fucose. The tendency was found to be more obvious, particularly when the concentration exceeded 200 μM (
Separately, VLPs of norovirus GII.4 were labeled with AF488 (Alexa Fluor 488; green phosphor) and purified. The thus-prepared labeled VLPs were added to the 2fp-fucose-treated 2D organoid, and a state of binding of VLPs to the surface of the 2fp-fucose-treated 2D organoid was observed. Similar to the case of UEA-1, the 2fp-fucose concentration in the treatment was 0, 8, 40, 200, and 1,000 μM (for 4 treatment days). As a result, the VLP binding amount was found to decrease in a concentration-dependent manner with respect to 2fp-fucose. When the 2fp-fucose concentration exceeded 40 μM, definite reduction in VLP adsorption on the surface of 2D organoid was observed (
The 2fp-fucose concentration at which obvious reduction in adsorption of the VLP (GII.4) onto the surface of the 2D organoid was observed was “40 μM.” The concentration is lower than “200 μM or higher” (mentioned hereinbelow) at which the effect of inhibiting proliferation of isolated norovirus GII.4 was clearly confirmed as shown in
Meanwhile, UEA-1 can bind to a wide variety of fucose-modified proteins. When the 2fp-fucose concentration exceeds 40 μM and reaches about 200 μM, 2fp-fucose is widely spread over the proteins to be modified with fucose, the proteins being present in the cells and on the surfaces of the cells. The presence of 2fp-fucose in such a state is thought to achieve a substantial level of inhibition of binding UEA-1 on the cell surfaces. There are a variety of fucose-modified proteins in the cells, and the modified proteins should be involved in reaction after incorporation of human norovirus particles. The actions of fucose-modified proteins in the cells are impeded via inhibition of formation of the proteins by 2fp-fucose at a concentration elevated to about 200 μM. As a result, intracellular proliferation of human norovirus is conceivably inhibited.
As described above, highly provably, the fucose analog serving as the active ingredient of the anti-norovirus agent of the present invention prevents incorporation of human norovirus into cells and inhibits proliferation thereof in the cells.
(3) Effect of 2fp-Fucose Treatment on Proliferation of Human Norovirus
The effect of 2fp-fucose treatment on proliferation of human norovirus was investigated for each lineage of norovirus. Specifically, after the 2fp-fucose treatment for 3 days, the 2D organoid was infected with each of the aforementioned norovirus strains GII.6, GII.3, and GII.4, and the viral proliferation amount was determined on days 3 and 6 after infection (
Next, there was investigated the effect of the period of time (the number of days) of the 2fp-fucose treatment performed in advance on the anti-human norovirus action of the fucose analog. Specifically, the strain GII.4 was treated with 2fp-fucose (several concentrations) for 1 to 4 days.
(4) Comparison in Effect Between 2fp-Fucose and Other Viral Proliferation Inhibitors
The 2D organoid was treated with an agent for 3 days. The tested agents were (a) 2fp-fucose (200 μM), (b) Sofosbuvir (1 μM), (c) Lamivudine (1 μM), (d) Ribavirin (400 μM), and (e) Mizoribine (100 μM). Subsequently, the thus-treated 2D organoid was infected with human norovirus (the norovirus strain GII.4), and the proliferation inhibition effect was determined (
[Studies on Mechanism of Human Norovirus Proliferation Inhibition by 2fp-Fucose]
(1) FUT gene expression profile and cytotoxicity by 2fp-fucose
After incorporation into cells, 2fp-fucose is involved in α-fucose modification by the mediation of fucosyltransferase (fucose transferase) (FUT). However, since 2fp-fucose differs from fucose in terms of a part of side chains, further sugar chain extension from 2fp-fucose is inhibited. Therefore, 2fp-fucose is a fucose analog which can inhibit complete glycosylation by virtue of the mechanism. 2fp-Fucose inhibits glycosylation by all types of fucosyltransferase which have ever been found in mammalian cells, specifically including FUT1 and FUT2 involved in al-2 fucosylation and FUT3-11 involved in al-3 fucosylation.
In this experiment, the level of formation of FUT (including FUT1 to FUT11) via gene expression in the human norovirus-susceptible 2D organoid was investigated through RT-PCR.
As shown in
(2) Studies on relationship between proliferation of human norovirus and HBGA by use of FUT2 gene-knocked-out cells
The relationship between HBGA secretor status and expression of an FUT2 gene in the 2D organoid was investigated. Specifically, the FUT2 gene of the 2D organoid was knocked out, and expression of the FUT2 gene in the knocked-out 2D organoid was confirmed through western blotting (
Next, green phosphor AF488-labelled VLPs of GII.4 were brought into contact with a human norovirus-knocked-out 2D organoid or the wild-type 2D organoid, and binding of VLPs to the surface of each 2D organoid was confirmed (
Next, the FUT2 gene was returned to the above FUT2 gene-knocked-out 2D organoid (i.e., a rescue operation) for trying to form FUT2 again via gene expression. The 2D organoid infected with human norovirus was red-stained with an anti-human norovirus VP1 antibody. As a result, only a limited amount of stained cells were observed among the cells spread in the vision field. The presence of the stained cells indicated the presence of a small amount of human norovirus-infected 2D organoid onto which VLPs were attached (bright spots in the monochrome photo image in
Finally, each of the wild-type 2D organoid (WT), the FUT2 gene-knocked-out 2D organoid (FUT2KO), and the FUT2 gene-returned 2D organoid (FUT2Res) was infected with human norovirus (the aforementioned norovirus GII.4 strain). Percent 2D organoid proliferation was determined in each case, and the results were compared (
As shown in
FUT2Res cells are prepared by incorporating the FUT2 gene to FUT2KO cells. Thus, FUT2 gRNA and Cas9, working for knocking out FUT2, are continuously expressed in the cells. As a result, a newly incorporated FUT2 gene is gradually knocked out. Conceivably, even when the FUT2 gene is newly incorporated into FUT2KO cells, the human norovirus proliferation performance cannot be revived to such a level as attained in the case of the wild type, and thus the percent proliferation is inferior to the case of WT.
The organoid gains human norovirus infection susceptibility in the process of differentiation to intestinal cells. The above test results match the hypothesis that FUT2 works in the differentiation process and directly or indirectly affects maturation of a human norovirus infection receptor that takes human norovirus into cells at the cell surfaces. However, as described above, there has been elucidated no precise mechanism including a course of formation of FUT2 via gene expression to generation of infection-susceptible cells.
[Human norovirus proliferation inhibition test by use of 6-alkynylfucose]
To a medium containing the 2D organoid, 6a-fucose was added so as to adjust a final concentration of 0 μM, 1.6 μM, 8 μM, 40 μM, and 200 μM, and culturing for differentiation induction was performed for 4 days. The culturing was performed by means of a 96-well plate in which 3 wells were dedicated for each 6a-fucose addition system, and one test plate was used for one human norovirus tested. The human norovirus tested included the GII.4 strain and GII.6 strain. The culturing was performed in a CO2 gas incubator controlled at 37° C.
On day 4 of culturing, the medium was removed, and the plate was washed with a new medium. Subsequently, each human norovirus was added at 105 RNA copies to the plate for infection, and static culturing was performed for 3 hours. After completion of the infection treatment, human norovirus remaining in the medium was removed together with the medium, and the plate was washed thrice with a new medium.
After washing, 6a-fucose was added again to the new medium so as to adjust a final concentration to 0 μM, 1.6 μM, 8 μM, 40 μM, and 200 μM, and culturing was performed for 6 days.
A portion (10 μL) of the culture medium was collected from each well at three timings: (a) immediately after the addition to the new medium, (b) on day 3, and (c) on day 6. The sample was centrifuged at 10,000 g for 10 minutes, and the cell precipitate was removed. The human norovirus genomic RNA contained in the supernatant (1 μL) was quantitated through real-time RT-PCR.
Notably, cultured cells (2D organoid) in the wells other than the wells in which proliferation was observed in culturing were still alive in the form of mono-layer sheet. Thus, no cytotoxicity attributed to addition of 6a-fucose was observed in any of the tested concentrations.
[Studies on the effect of treatment with 2fp-fucose or 6a-fucose after viral infection]
After differentiation induction, about 4 days are required for complete differentiation from the organoid to the small intestine epithelial cells. Then, cell division is substantially terminated, and, if no medium replacement is conducted, cell death starts on day 7 to day 9. As is also clear from the test results, the effect of a fucose analog emerges on day 2 of addition, and the effect peaks on day 4. Meanwhile, human norovirus increases in the culture medium (i.e., release of new virus to the culture medium begins) on the day following the infection, and proliferation can be observed. The proliferation lasts until day 6.
Generally, in the human body, the primary infection of small intestine epithelial cells with human norovirus and viral proliferation in the cells cause the death of small intestine epithelial cells. When the dead cells exfoliate, stem cells present in the intestinal crypt undergo cell division, and normally differentiation-induced epithelial cells are supplied. Shortly, intestinal villus is regenerated. The thus-supplied epithelial cells are newly infected with human norovirus released from the small intestine epithelial cells to the intestinal tract through the primary infection/intracellular proliferation. When new human norovirus infection occurs, the second cycle of proliferation of human norovirus starts in the newly supplied cells. At this stage, viral proliferation control by immunoresponse of the host generally begins. Thus, conceivably, viral infection/proliferation is gradually suppressed.
In order to design an effective administration form of the anti-norovirus agent of the present invention adapted to the aforementioned human norovirus infection/proliferation cycle, how the fucose analog acts on the human norovirus infection/proliferation cycle is desirably monitored over time.
However, currently, no laboratory animal is available for monitoring the human norovirus infection/proliferation cycle. Thus, difficulty is encountered in integrally monitoring proliferation of human norovirus, cell regeneration, new cell supply, re-infection, and immunoresponse in vivo.
Under such circumstances, the time-over effect of administration of a fucose analog after infection with human norovirus was determined in vitro under the following conditions.
(a) The 2D organoid was disseminated to a 96-well plate. After completion of differentiation induction, two fucose analogs (i.e., 2fp-fucose and 6a-fucose) were each added on day 5 to a concentration of 200 μM, to thereby provide addition groups. The results are shown in
After the infection treatment, the human norovirus remaining in the medium was removed together with the medium, and the plate was washed thrice with a new medium. After washing, the aforementioned active ingredients were each added to the new medium to a concentration of 200 μM (addition groups), and no addition was performed with respect to the untreated (i.e., non-addition) group. Culturing was performed for 3 days. A portion (10 μL) of the medium was sampled from each well, and the samples were centrifuged at 10,000 g for 10 minutes, to thereby remove cell precipitates. The amount of human norovirus genomic RNA in the supernatant (1 μL) was determined through real-time RT-PCR.
At this stage, the untreated group exhibited a 10-fold rise in RNA titer with respect to the 2fp-fucose administration system and a 150-fold rise in RNA titer with respect to the 6a-fucose administration system. In contrast, no rise in RNA titer (i.e., proliferation of human norovirus) was observed in the two addition groups. The results are shown in
(b) In the untreated group, human norovirus infection-susceptible cells still remained. Thus, new infection of the remaining infection-susceptible cells with human norovirus may possibly occur. Accordingly, the effect of the anti-norovirus agent of the present invention on the remaining infection-susceptible cells was investigated through the following procedure.
In a manner similar to that of the (a) described above, the untreated group was subjected to culturing for 3 days, and then the medium of each culture was removed. The system was washed and infected again with human norovirus (i.e., reinfection: reinf.), and the virus was removed together with the medium. After completion of washing, a new medium was added, and culturing was performed for 3 days. In a manner similar to the above, the amount of human norovirus released to the supernatant was measured as an RNA titer. In the reinfection test, a 20-fold rise in RNA titer with respect to the initial infection observed in the aforementioned (a) was observed (see the bars of reinf in
(c) Next, the effect of the fucose analog on reinfection was investigated. Generally, the onset of norovirus symptoms in humans occurs about 24 to about 48 hours after infection with human norovirus. Therefore, there was assumed a model case of administration of an effective amount of the anti-norovirus agent of the present invention immediately after the onset. In this case, according to the aforementioned findings, the anti-viral effect was expected to be attained on the second day or later after the initial infection with human norovirus. Also, in the presence of the fucose analog, 3 days after the fucose analog treatment, cells derived through differentiation induction of the crypt stem cells after the initial infection were to be brought into contact with the virus.
In the test system, the effect of the anti-norovirus agent of the present invention provided after human norovirus infection was estimated on the basis of the following study. Specifically, among the fucose analog-treated 2D organoid cells, the living cells on growth day 3 (i.e., most of them were not infected with human norovirus, by virtue of the effect of the fucose analog) were subjected to a secondary infection operation (i.e., reinfection). The effect of the fucose analog on the reinfection was investigated.
In a specific procedure, the addition groups were prepared through the same procedure as employed in (a) above. The primarily infected cells were cultured in the presence of the fucose analog (200 μM) for 3 days, and the medium was removed from each culture system. After completion of washing, the cells were infected again with human norovirus, and then the virus was removed together with the medium. After completion of the next washing, a medium containing the fucose analog (200 μM) (represented by Reinf+2f-fuc or Reinf+6a-fuc), or a fucose analog-free medium (represented by Reinf-2f-fuc or Reinf-6a-fuc) was added thereto, and culturing was performed for further 3 days. Similar to the above case, the amount of human norovirus released to the supernatant was determined as an RNA titer. The results are shown in
As is clear from
The test results described above have revealed the following. That is, when a human subject is primarily infected with human norovirus, and the subject continuously takes an effective amount of the anti-norovirus agent of the present invention from the onset, continuous emergence of symptoms attributed to reinfection possibly occurring after day 2 or day 3 of the primary infection can be effectively suppressed, although it is difficult to avoid all the symptoms caused by primary infection. Thus, use of the anti-norovirus agent of the present invention is remarkably advantageous for mitigating the gravity and the period of pain of human norovirus infection patients. Furthermore, in the case where the anti-norovirus agent of the present invention is used to a human norovirus patient after onset of symptoms, the period of human norovirus excretion to feces, which is a problem as a cause of spread of human norovirus infection, is expected to be considerably shortened.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018-244213 | Dec 2018 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2019/051496 | 12/27/2019 | WO | 00 |
