JP2007070311A - Infectious disease preventive agent and infectious disease therapeutic agent comprising α-galactosylceramide - Google Patents

Abstract

Disclosed is a novel infectious disease therapeutic agent or infectious disease preventive agent that is not a vaccine.
An active ingredient is α-galactosylceramide and / or a pharmaceutically acceptable salt thereof.
[Effect] Administration of α-galactosylceramide and / or its pharmaceutically acceptable salt increases the number of immunocompetent cells and enhances the resistance to infectious diseases in the living body. Listeria, Salmonella, Escherichia coli, green It becomes a therapeutic or preventive agent for infectious diseases caused by concentrated bacteria or Staphylococcus aureus.
[Selection] Figure 1

Description

  The present invention relates to a preventive agent and a therapeutic agent for pathogenic microbial infections such as bacteria, fungi, viruses and parasites using α-galactosylceramide.

Until now, vaccines have been widely used as preventive measures against various infectious diseases. Vaccine uses the long-lasting specific immune response against the target pathogenic microorganism, but the duration of the effect varies greatly depending on the target pathogenic microorganism and may cause individual differences Therefore, it is not necessarily an effective preventive measure. In addition, in live vaccines, the attenuated strain used may be mutated, which is problematic in terms of safety.
In general, antibiotics such as antibacterial drugs, antifungal drugs, and antiviral drugs are used as therapeutic drugs in the case of suffering from infectious diseases. Antibiotics are administered to reduce (or kill) microorganisms in the body without destroying human cells, and to kill and eliminate pathogenic microorganisms remaining by the immune response of the living body. However, in recent years, multidrug-resistant bacteria have emerged due to heavy use of antibiotics, which has become a social problem such as nosocomial infections and opportunistic infections, so urgent measures are required. In this way, in addition to the limitations of existing preventive and therapeutic methods for infectious diseases, the number of affected people with re-emerging infectious diseases and emerging infectious diseases is increasing worldwide, so it can be provided safely, reliably and easily. There is an urgent need to develop new preventive and therapeutic methods.

In Patent Document 1, the use of glycosylceramide as an adjuvant for vaccines against infectious diseases and cancer was reported. However, it has not been known that α-galactosylceramide alone is effective in treating or preventing infection.
JP 2005-500336 JP

  An object of the present invention is to provide a safe and inexpensive infectious disease preventive agent or infectious disease therapeutic agent.

  The present inventor has intensively studied to solve the above problems. As a result, it has been found that administration of α-galactosylceramide alone increases the white blood cell count and remarkably enhances infection resistance against Listeria. From this, it was found that this compound is useful alone as a therapeutic or preventive agent for infectious diseases, and the present invention was completed.

That is, the present invention is as follows.
(1) An infectious disease preventive agent or an infectious disease therapeutic agent comprising α-galactosylceramide and / or a pharmaceutically acceptable salt thereof.
(2) The infectious disease preventive agent or infectious disease therapeutic agent according to (1), wherein the infectious disease is an infectious disease caused by bacteria.
(3) The infectious disease preventive agent or infectious disease therapeutic agent according to (2), wherein the infectious disease caused by bacteria is an infectious disease caused by Listeria monocytogenes, Salmonella, Escherichia coli, green bacterium, or Staphylococcus aureus.

Administration of α-galactosylceramide alone increases the number of immunocompetent cells, thereby enhancing resistance to infectious diseases in the living body. Therefore, α-galactosylceramide can be used alone as an infectious disease preventive or therapeutic agent.
Since α-galactosylceramide is a natural glycolipid derived from marine sponge and the like, it has few side effects on the living body and can be produced in large quantities and at low cost. Promising.

α−ガラクトシルセラミドの薬学的に許容される塩としては、無機酸付加塩（例えば塩酸塩、臭化水素酸塩、硫酸塩、リン酸塩等）、有機カルボン酸・スルホン酸付加塩（例えばギ酸塩、酢酸塩、トリフルオロ酢酸塩、マレイン酸塩、酒石酸塩、フマル酸塩、クエン酸塩、乳酸塩、メタンスルホン酸塩、ベンゼンスルホン酸塩、トルエンスルホン酸塩等）、あるいは、ナトリウム、カリウム、マグネシウム、カルシウム等の金属塩が挙げられる。なお、α−ガラクトシルセラミドは水和物であってもよい。 The present invention is described in detail below.
The agent for preventing or treating infection according to the present invention contains α-galactosylceramide and / or a pharmaceutically acceptable salt thereof as an active ingredient.
α-Galactosylceramide is a glycolipid composed of ceramide to which galactose is bound, represented by the following chemical formula. This is synthesized, for example, by the enzyme ceramide galactosyltransferase that binds the two parts together. Moreover, what was refine | purified from the commercially available thing or sponge etc. can also be used.

Pharmaceutically acceptable salts of α-galactosylceramide include inorganic acid addition salts (eg, hydrochloride, hydrobromide, sulfate, phosphate, etc.), organic carboxylic acid / sulfonic acid addition salts (eg, formic acid). Salt, acetate, trifluoroacetate, maleate, tartrate, fumarate, citrate, lactate, methanesulfonate, benzenesulfonate, toluenesulfonate, etc.), or sodium, potassium And metal salts such as magnesium and calcium. The α-galactosylceramide may be a hydrate.

α-Galactosylceramide activates immunocompetent cells such as neutrophils, NK cells, γδ type T cells, CD8 ⁺ T cells and increases the number of these cells, thereby causing bacteria, fungi, viruses, or parasites Increases infection resistance against insects. Therefore, it can be effectively used as a therapeutic agent for infectious diseases caused by bacteria, fungi, viruses, parasites and the like. Moreover, since it also has the effect of increasing the number of leukocytes in the living body to enhance immunity in advance, it can be effectively used as a preventive agent for infectious diseases as described above. For example, by administering α-galactosylceramide before surgery, postoperative infection can be prevented in advance.
Of these, it is particularly preferably used as a prophylactic or therapeutic agent for bacterial infections. Examples of infections caused by bacteria include infections caused by Listeria monocytogenes, Salmonella, (pathogenic) Escherichia coli, green bacteria, Staphylococcus aureus, and the like, but infections caused by Listeria or Salmonella are also included. preferable.

The infectious disease therapeutic agent and infectious disease preventive agent comprising α-galactosylceramide is prepared from α-galactosylceramide or a pharmaceutically acceptable salt thereof according to known means generally used in the production of pharmaceutical preparations. It can be safely administered as it is or mixed with a pharmacologically acceptable carrier, for example, as pharmaceutical preparations such as tablets, powders, granules, capsules, solutions, injections, suppositories, sustained-release agents, etc. it can.
The content of α-galactosylceramide in the preparation may be 1 to 100% by weight of the whole preparation, but is preferably 10 to 100% and more preferably 50 to 100%.
The dose of α-galactosylceramide varies depending on the administration subject, target organ, symptom, administration method and the like and is not particularly limited, but is generally about 0.1 to 100 mg per day per patient (with a body weight of 60 kg). , Preferably about 1.0-50 mg, more preferably about 1.0-20 mg. The administration method of α-galactosylceramide is appropriately selected and is not particularly limited, but is preferably intravenous injection.

  Examples of pharmacologically acceptable carriers include excipients, lubricants, binders and disintegrants in solid formulations, or solvents, solubilizers, suspending agents, isotonic agents, buffers in liquid formulations. And soothing agents. If necessary, additives such as conventional preservatives, antioxidants, colorants, sweeteners, adsorbents, wetting agents and the like can be used in appropriate amounts. Examples of the excipient include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid and the like. Examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like. Examples of the binder include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, sodium carboxymethylcellulose and the like. Examples of the disintegrant include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, carboxymethyl starch sodium, L-hydroxypropyl cellulose, and the like. Examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil and the like. Examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like. Examples of the suspending agent include surfactants such as polysorbate, stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; Examples thereof include hydrophilic polymers such as polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose. Examples of isotonic agents include glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol and the like. Examples of the buffer include buffer solutions of phosphate, acetate, carbonate, citrate and the like. Examples of soothing agents include benzyl alcohol. Examples of the preservative include parahydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like. Examples of the antioxidant include sulfite, ascorbic acid, α-tocopherol and the like.

The infectious disease preventive agent or therapeutic agent comprising α-galactosylceramide of the present invention is a drug used for the prevention or treatment of infectious diseases based on the fact that α-galactosylceramide itself has a therapeutic or preventive effect on infectious diseases. It does not mean a drug containing α-galactosylceramide together with other active ingredients as an adjuvant or the like.

The following examples illustrate the present invention more specifically. However, the present invention is not limited to the following examples.
α-Galactosylceramide (α-GalCer) was extracted from sponge, separated, purified, put into a glass vial and lyophilized. The lyophilized α-galactosylceramide was stored refrigerated at 2-8 ° C. At the time of use, it was redissolved in distilled water (preferably 0.9% aqueous sodium chloride solution in weight percent concentration) and then administered to inbred mice (C57BL / 6) by intravenous injection. The safety (cell proliferation ability and cell survival rate) for living bodies has been confirmed in safety tests.

1. α-GalCer enhances infection resistance against Listeria monocytogenes
To 8-week-old inbred mice (C57BL / 6), 100 μl of α-GalCer or control vehicle (Vehicle) (α-GalCer was adjusted to 50 μg / kg) was administered via the tail vein. Two hours after administration, Listeria monocytogenes 1500 cfu was infected from the tail vein, and the number of bacteria in organs (liver and spleen) 4 days later was compared (FIG. 1). Compared with the untreated group (Nil), the α-GalCer-treated group showed a significant (about 1/1000) decrease in the number of bacteria in the organ. A slight decrease in the number of bacteria in the organ was also observed in the vehicle-treated group, but the number of bacteria was significantly higher than that in the α-GalCer-treated group.

2. Effect of α-GalCer on pathological changes in various organs caused by Listeria infection Listeria is an intracellular parasitic bacterium, like tuberculosis, and is known to cause tissue damage. Therefore, the influence on tissue damage was examined using the above infection experiment (FIG. 2). As described above, mice infected with α-GalCer or Vehicle were infected with Listeria, and when the spleen and liver were removed on the 4th day of infection, the spleens of the untreated group and the Vehicle-treated group were salami-like after infection. On the other hand, no gross changes were observed in the α-GalCer-treated group (the same state as before infection).
In addition, after preparing a pathological specimen of the liver, the degree of tissue damage was microscopically examined by HE staining. In the livers of the untreated group and the vehicle-treated group, inflammatory cells infiltrated significantly after infection, and many granulomas were formed. Although a slight inflammatory cell infiltration was observed in the α-GalCer-treated group, no significant change was observed compared to that before infection (FIG. 3).

3. Dynamic analysis of NKT (α-GalCer / CD1d tetramer-reactive) cells using a flow cytometer After intravenous injection of α-GalCer (1 μg) into C57BL / 6 mice, NKT (α-GalCer / Results of analysis of CD1d tetramer-reactive cell dynamics with a flow cytometer (Uninfected) and α-GalCer (1 μg) intravenously injected into C57BL / 6 mice 2 hours before infection, then Listeria (1.5 x 10 ³ ) shows the result (Infected) when the presence or absence of NKT (α-GalCer / CD1d tetramer-reactive) cells in the liver on the fourth day after infection was analyzed with a flow cytometer (FIG. 4). NKT (α-GalCer / CD1d tetramer-reactive) cells were frequently observed in the livers of the untreated group and the vehicle-treated group. However, in the α-GalCer-treated group, NKT (α-GalCer / CD1d tetramer reactive) cells were no longer detectable. On the other hand, NKT (α-GalCer / CD1d tetramer-reactive) cells decreased even after infection.

4). Effect of α-GalCer on increasing white blood cell count When α-GalCer (1 μg) is intravenously injected into C57BL / 6 mice alone (Uninfected), the total number of white blood cells present in the liver and α-GalCer (1 μg 2 hours before infection) ) Was intravenously injected, and the total number of leukocytes present in the liver on the 4th day after infection with Listeria (1500 cfu) was measured (FIG. 5). Compared with the total number of leukocytes present in the livers of the untreated group and the vehicle-treated group, the α-GalCer-treated group showed a significant increase in the total number of leukocytes from the first day after treatment. In any group, an increase in the total number of white blood cells was observed due to infection, but the number was further increased by α-GalCer treatment.

5. Effect of α-GalCer on neutrophil count increase When α-GalCer (1 μg) is intravenously injected into C57BL / 6 mice alone (uninfected), the number of neutrophils present in the liver, and α- 2 hours before infection After intravenous injection of GalCer (1 μg), the number of neutrophils present in the liver on the 4th day after infection with Listeria (1500 cfu) was measured (FIG. 6). Compared with the number of neutrophils present in the liver of the untreated group and the vehicle-treated group, the α-GalCer-treated group showed a significant increase in the number of neutrophils from the first day after treatment. In any group, an increase in the number of neutrophils was observed due to infection, but a significant decrease was observed in the α-GalCer-treated group compared to the other groups.

6). Effect of α-GalCer on increasing number of NK cells When α-GalCer (1 μg) was intravenously administered to C57BL / 6 mice alone (uninfected), the number of NK cells present in the liver, and α-GalCer (2 hours before infection) 1 μg) was intravenously injected, and the number of NK cells present in the liver on the 4th day after infection with Listeria (1500 cfu) was measured (FIG. 7). In the α-GalCer-treated group, a marked increase in the number of NK cells was observed on the 4th day after treatment, compared with the number of NK cells present in the liver of the untreated group and the vehicle-treated group. In any group, an increase in the number of NK cells was observed due to infection, but the α-GalCer-treated group showed a significantly higher value compared to the other groups. In addition, the expression of Mac-1 (activation marker) in NK cells was significantly increased as compared with other groups on the first day after α-GalCer treatment.

7). Effect of α-GalCer on increasing number of γδ-type T cells When α-GalCer (1 μg) was intravenously injected into C57BL / 6 mice alone (uninfected), the number of γδ-type T cells present in the liver and 2 hours before infection After intravenous injection of α-GalCer (1 μg), the number of γδ T cells present in the liver on the 4th day after infection with Listeria (1500 cfu) was measured (FIG. 8). In the α-GalCer-treated group, a significant increase in the number of γδ-type T cells was observed on the 4th day after the treatment, compared with the number of γδ-type T cells present in the livers of the untreated group and the vehicle-treated group. In any group, an increase in the number of γδ-type T cells was observed due to infection, but the α-GalCer-treated group showed a significantly higher value than the other groups.

8). Effect of α-GalCer on CD8 ⁺ T cell number increase When α-GalCer (1 μg) is intravenously injected into C57BL / 6 mice alone (Uninfected), the number of CD8 ⁺ T cells present in the liver and 2 hours before infection After intravenous injection of α-GalCer (1 μg), the number of CD8 ⁺ T cells present in the liver on the 4th day after infection with Listeria (1500 cfu) was analyzed (FIG. 9). Compared with the number of CD8 ⁺ T cells present in the liver of the untreated group and the vehicle treated group, the α-GalCer treated group showed a significant increase in the number of CD8 ⁺ T cells on the fourth day after the treatment. In any group, an increase in the number of CD8 ⁺ T cells was observed due to infection, but the α-GalCer-treated group showed a significantly higher value compared to the other groups.

The figure which shows the growth inhibitory effect with respect to the Listeria monocytogenes of (alpha) -GalCer. The figure (photograph) which shows the pathological image of the spleen by listeria infection in the mouse | mouth which did not administer (Nil) or vehicle and (alpha) -GalCer administration. The figure (photograph) which shows the result of the HE dyeing | staining of a liver in the Listeria infection mouse | mouth which administered non-administration (Nil) or Vehicle and (alpha) -GalCer. The figure which shows the result of the dynamic analysis of NKT ((alpha) -GalCer / CD1d tetramer reactivity) cell by a flow cytometer. The figure which shows the total white blood cell count when (alpha) -GalCer is administered to a Listeria infected or non-infected mouse | mouth (or the time of vehicle administration, or the time of non-administration). The figure which shows the number of neutrophils when (alpha) -GalCer is administered to a Listeria infected or non-infected mouse (or at the time of Vehicle administration, or at the time of non-administration). The figure which shows the number of NK cells when (alpha) -GalCer is administered to the Listeria infected or non-infected mouse (or at the time of vehicle administration, or at the time of non-administration). The figure which shows the number of (gamma) delta type T cells when (alpha) -GalCer is administered to a Listeria infection or a non-infected mouse | mouth (or the time of vehicle administration or the time of non-administration). The figure which shows the number of CD8 ⁺ T cells when α-GalCer is administered to Listeria-infected or non-infected mice (or when Vehicle is administered or not).

Claims (3)

An infectious disease preventive agent or an infectious disease therapeutic agent comprising α-galactosylceramide and / or a pharmaceutically acceptable salt thereof. The infectious disease preventive agent or infectious disease therapeutic agent according to claim 1, wherein the infectious disease is an infectious disease caused by bacteria. The infectious disease preventive agent or infectious disease therapeutic agent according to claim 2, wherein the infectious disease caused by bacteria is an infectious disease caused by Listeria monocytogenes, Salmonella, Escherichia coli, green-concentrated bacteria, or Staphylococcus aureus.

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