JP4961144B2 - Antiviral agent - Google Patents

Description

  The present invention relates to a novel antiviral agent comprising a sulfated polysaccharide in which a hydroxyl group of a raw material polysaccharide is partially sulfated or a compound having the sulfated polysaccharide as a partial structure.

Conventionally, with regard to the antiviral effect of sulfated polysaccharides, many sulfated polysaccharides including heparin have been studied (for example, see Non-patent Document 1 and Patent Document 1). However, when these are used as drugs, various activities possessed by sulfated polysaccharides have adverse effects on the contrary (for example, see Non-patent Document 1).
JP-A-64-52723 Trends in Glycoscience and Glycotechnology Vol.15, No.81, pp29-46 (2003)

  Therefore, development of a safe and inexpensive antiviral agent has been desired. In view of such circumstances, an object of the present invention is to provide an antiviral agent having low cytotoxicity at a low cost.

  The present inventor has intensively studied to solve the above problems. As a result, the present inventors have found that sulfated gellan in which the hydroxyl group of gellan is sulfated by 10% or more has a cytotoxicity-inhibiting action against HIV infection, has extremely low cytotoxicity, and has an inhibitory action on giant cell formation. Thus, the sulfated polysaccharide obtained by sulfating an inexpensive polysaccharide by an existing method was found to have an antiviral action with low cytotoxicity, and the present invention was completed.

（式中、Rはそれぞれ独立に選ばれるOHまたはOSO₃Hである。）
［４］前記硫酸化多糖が硫酸化ジェランであることを特徴とする前記［３］項記載の抗ウイルス剤。
［５］ヒトレトロウイルスに効果があることを特徴とする前記［１］〜［４］のいずれか１項記載の抗ウイルス剤。
［６］前記ヒトレトロウイルスがＨＩＶであることを特徴とする前記［５］記載の抗ウイルス剤。
［７］前記硫酸化多糖の平均分子量が５〜５００ｋＤａであることを特徴とする前記［１］〜［６］のいずれか１項記載の抗ウイルス剤。
［８］前記水酸基の１０％以上が硫酸化されていることを特徴とする前記［１］〜［７］のいずれか１項記載の抗ウイルス剤。 The present invention has the following configuration.
[1] An antiviral agent comprising a sulfated polysaccharide consisting of glucose, glucuronic acid, and rhamnose, wherein a part or all of hydroxyl groups constituting the sugar is sulfated as an active ingredient.
[2] The antiviral agent according to [1], wherein the sulfated polysaccharide is composed of repeating structural units composed of two molecules of glucose, one molecule of glucuronic acid, and one molecule of rhamnose.
[3] The antiviral agent according to [1], wherein the structural unit is a unit represented by the following formula (1).

(In the formula, each R is independently selected OH or OSO ₃ H.)
[4] The antiviral agent according to [3], wherein the sulfated polysaccharide is sulfated gellan.
[5] The antiviral agent according to any one of [1] to [4], which is effective for human retroviruses.
[6] The antiviral agent according to [5], wherein the human retrovirus is HIV.
[7] The antiviral agent according to any one of [1] to [6], wherein the sulfated polysaccharide has an average molecular weight of 5 to 500 kDa.
[8] The antiviral agent according to any one of [1] to [7], wherein 10% or more of the hydroxyl group is sulfated.

  According to the present invention, an antiviral agent having low cytotoxicity can be provided at low cost.

  Hereinafter, the present invention will be described in detail. In the present specification, sulfate esterification may be referred to as sulfation, and a polysaccharide having a hydroxyl group partially sulfated may be referred to as a sulfated polysaccharide.

  The antiviral agent according to the present invention is an antiviral agent comprising, as an active ingredient, a sulfated polysaccharide that is composed of glucose, glucuronic acid, and rhamnose and in which part or all of the hydroxyl groups constituting the sugar are sulfated. In particular, in the raw material polysaccharide, it is preferable that the abundance ratio of glucose, glucuronic acid, and rhamnose is constituted by repeating structural units of 2: 1: 1. The binding order of monosaccharides, the binding mode between monosaccharides (for example, α bond, β bond), and the binding position between monosaccharides are not particularly limited. In addition, in this invention, a sulfation rate means the value which represented the ratio by which sulfate esterification was carried out in percentage among the hydroxyl groups which raw material polysaccharide has. The sulfation rate of the sulfated polysaccharide used in the present invention is 10% or more, preferably 10 to 80%, and more preferably 20 to 50%. The average molecular weight of the sulfated polysaccharide is preferably 5 to 500 kDa, more preferably 5 to 30 kDa.

  The raw material polysaccharide of the sulfated polysaccharide constituting the antiviral agent of the present invention may be a chemically synthesized product, a fermented product of a naturally occurring microorganism, a seaweed extract, or the like, and its origin is particularly limited. Is not to be done. In addition, they can be used in the sulfation reaction after the molecular weight has been reduced in advance by hydrolysis with hydrochloric acid, sulfuric acid, trifluoroacetic acid or other acid and an alkali compound such as sodium hydroxide.

Specific examples of the raw material polysaccharide include a polysaccharide having a structural unit of the formula (2).

  More specifically, the raw material polysaccharide is gellan (gellan CAS 71010-52-1) obtained by deacylating and purifying a polysaccharide comprising the structural unit of formula (2), that is, a polysaccharide produced by Pseudomonas elodea. ). Gellan is a polysaccharide mainly composed of glucose, glucuronic acid, and rhamnose, and can be preferably used in the present invention because it can be obtained in large quantities at low cost.

The method for sulfating polysaccharides is not particularly limited, and generally known methods can be used. For example, a method of reacting chlorosulfonic acid in dimethylformamide (DMF) (International Journal of Biological Macromolecules 28, 381 (2001)) or DMF / SO ₃ in DMF. These include a method of causing a complex to act (artificial organ 26, 1 (1997)) and a method of causing a sulfur trioxide complex to act in dehydrated pyridine (Journal of the Chemical Society 81 (1959)). In addition, a method in which a sulfuric anhydride complex such as a dioxane-SO ₃ complex, a trimethylamine-SO ₃ complex, or a pyridine-SO ₃ complex is allowed to act can be used. By changing the molecular weight of the raw material polysaccharide and the reaction conditions, a sulfated polysaccharide having an arbitrary molecular weight and degree of sulfation can be obtained.

  The above-mentioned sulfated polysaccharides are preferably used for the antiviral agent of the present invention, but compounds having these sulfated polysaccharides as partial structures can also be used. Examples of the substrate into which the sulfated polysaccharide is introduced include a polysaccharide having an amino group, a polyamino acid having an amino group, and a polysaccharide having an amino group introduced therein. A specific example of a polysaccharide having an amino group is chitosan. A specific example of the polyamino acid having an amino group is polylysine. A specific example of the polysaccharide into which an amino group has been introduced is aminated cellulose. In addition, there is no problem even if it is combined with any compound as long as it does not lose the antiviral activity of the sulfated polysaccharide of the present invention.

  The method for introducing the sulfated polysaccharide into the substrate is not particularly limited as long as the antiviral activity of the antiviral agent of the present invention is not lost, but the carboxyl group of the sulfated polysaccharide is obtained using water-soluble carbodiimide (WSC) as a catalyst. A method of binding the amino group of the substrate with the reducing terminal aldehyde group of the sulfated polysaccharide and the amino group of the substrate are reacted under a weak alkaline condition and then treated with a reducing agent (sodium tetrahydroborate, dimethylamine borane, etc.) The method etc. can be mentioned.

  The prepared antiviral agent can be used for various viral diseases. In particular, it is administered before infection for viral diseases caused by infections such as human retroviruses such as HIV (Human Immunodeficiency virus) diseases. It can be used for prevention and can be used for treatment by administration after infection. In addition, the administration method of these antiviral agents needs to be appropriately performed depending on the disease state, and the main methods are drugs such as subcutaneous administration by injection, intravenous administration, oral administration in tablet form, intestinal administration as suppository, etc. As long as the administration method according to the form is selected as an appropriate form, there is no problem with any administration method or pharmaceutical form.

  Hereinafter, although the present invention is explained in detail using an example and a comparative example, the present invention is not limited to these examples. Definitions of terms and measurement methods used in Examples and Comparative Examples are as follows.

(1) Average molecular weight (kDa): The synthesized sulfated gellan was dissolved in 0.2 mol / l-NaCl aqueous solution (prepared with ion-exchanged water) at a concentration of 1.0 mg / ml, and the same NaCl aqueous solution was used as the eluent. Gel filtration by HPLC. Here, Shodex (registered trademark) Ionpak KS-804 (Showa Denko) and KS-G (Showa Denko) were used for the column, and the eluate was detected by a differential refractive index detector. Pre-run pullulan (Shodex STANDARD P-82 (Showa Denko)) measured under the same conditions is gel filtered to create a calibration curve of elution time and molecular weight, and the elution time of synthesized sulfated gellan is Was applied to determine the average molecular weight of the sulfated gellan.

(2) Sulfation rate (%): The ratio of sulfate esterification of the hydroxyl groups of the raw material polysaccharide was expressed as a percentage. The total amount of S in the synthesized sulfated polysaccharide was measured by elemental analysis using an ICP emission spectrometer, and the amount of free S released from the sulfated polysaccharide main body was measured by ion chromatography. The sulfation rate was calculated from the amount of bound S obtained by subtracting the amount of free S from the total amount of S. The conditions for ion chromatography are as follows.
Separation column: ICS-A23 (Yokogawa)
Column temperature: 40 ° C
Eluent: 3mmol / l Sodium carbonate aqueous solution Flow rate 1ml / min
Remover: 15mmol / l sulfuric acid aqueous solution, flow rate 1ml / min

(3) Cytotoxicity inhibition test and cytotoxicity test using Molt4 cells: Two types of plates for measuring cytotoxicity and anti-HIV activity of anti-HIV substances are prepared. Both plates used 96-well flat-bottom culture plates, and 200 μl of a sample solution diluted to a predetermined concentration with 10% FCS-containing RPMI 1640 medium was added to the leftmost 8 wells. Place 100 μl of medium in the remaining holes and transfer the solution from the leftmost hole to the next hole 100 μl at a time to perform 2-fold serial dilution up to 11 holes. In the 12th hole, the drug concentration is 0 and cell growth and HIV infection The control was. Two rows of cytotoxicity and anti-HIV activity measurement plates were used for each type of test drug. Molt4 cells in the logarithmic growth phase of 2 × 10 ⁶ per plate were collected by centrifugation on a plate diluted with a test agent, suspended in 10 ml of medium on a plate for cytotoxicity measurement, and 100 μl was added to all wells. On the other hand, a stock solution of HIV (HTLV-IIIB) was added to 2 × 10 ⁶ Molt4 cells collected by centrifugation on a plate for anti-HIV measurement so as to be 100 TCID _50, and the plate was infected at 37 ° C. for 1 hour. The cells precipitated by centrifugation were resuspended in 10 ml of medium, and 100 μl was added to all holes of the anti-HIV activity measurement plate.
On the fifth day of the culture, the cytotoxic effect (CPE) and cytotoxicity due to HIV were observed with a microscope. If there was cell damage or cytotoxicity, cell degeneration occurred, and the cells were rounded and squeezed. Under the microscope, the cells looked dark.

(4) Giant cell formation inhibition test: A sample with confirmed effectiveness was subjected to a giant cell formation inhibition test using Molt 4 cells. Add 500 μl of a sample solution diluted to a predetermined concentration to a flat bottom 24-well plate. Persistent infection Molt4 / HIV cell suspension suspended in the medium 1 × 10 ⁶ cells / ml to 250 [mu] l / Anakuwae further uninfected Molt4 cell suspension of 1 × 10 ⁶ cells / ml to 250 [mu] l / Anakuwae, stirring, CO ₂ and mixed culture at 37 ° C. Giant cell formation is observed several hours after the mixed culture, and becomes significant after 20 hours. Determine the giant cell formation inhibitory effect of the sample by microscopy.

<Example 1>
2 g of gellan (manufactured by Wako Pure Chemical Industries, Ltd.) was added in advance to 200 ml of 0.5 mol / l-trifluoroacetic acid aqueous solution and reacted at 80 ° C. for 30 minutes for hydrolysis. The obtained low molecular weight gellan was added to 5 g of DMF under a nitrogen gas seal and stirred for 10 hours at room temperature to swell. Thereafter, the temperature was raised to 40 ° C., 14 g of DMF / SO ₃ complex (SO ₃ 18% by mass) was added, and the mixture was reacted for 6 hours. After completion of the reaction, the reaction mixture was ice-cooled, 0.3 g of water was added to decompose the unreacted DMF / SO ₃ complex, and the reaction was stopped. Subsequently, twice the volume of ethanol was added to precipitate the reaction product and collected by filtration. The recovered precipitate was dissolved in 20 ml of ion-exchanged water, neutralized with 1 mol / l-NaOH, and precipitated again with 2 volumes of ethanol. Thereafter, the recovered precipitate was added to pure water and purified with a double volume of ethanol to perform purification and washing three times in total, followed by drying at 50 ° C. under reduced pressure for one day, and 1.7 g of sulfated gellan powder ( The yield was 61% by mass. It was 5.6 kDa when the average molecular weight of sulfated gellan was measured by the above-mentioned method. The hydroxyl group sulfation rate was 24.4%.

  This sulfated gellan was found to have a cytotoxic effect at 3.8 μg / ml or more. Further, no cytotoxicity was observed at least at 1000 μg / ml. Furthermore, a giant cell formation inhibitory effect was observed at 200 μg / ml or more.

<Example 2>
Sulfated in the same manner as in Example 1 except that 2 g of normal gellan not reduced in molecular weight was used as a raw material, 3.6 g of chlorosulfonic acid was used as a sulfating agent, and the reaction temperature was 50 ° C. However, a sulfated gellan having a molecular weight of 23 kDa and a hydroxyl group sulfation rate of 36.6% was obtained.

  This sulfated gellan was found to have a cytotoxic effect at 1.9 μg / ml or more. Further, no cytotoxicity was observed at least at 1000 μg / ml. Furthermore, a giant cell formation inhibitory effect was observed at 200 μg / ml or more.

<Example 3>
Gellan was sulfated under the same conditions as in Example 2 except that gellan reduced in molecular weight by the method according to Example 1 was used as a raw material. As a result, the molecular weight was 13 kDa and the hydroxylation rate was 39.8%. Sulfated gellan was obtained.

  This sulfated gellan was found to have a cytotoxic effect at 1.9 μg / ml or more. Further, no cytotoxicity was observed at least at 1000 μg / ml. Furthermore, a giant cell formation inhibitory effect was observed at 40 μg / ml or more.

<Comparative Example 1>
When 2 g of normal gellan not reduced in molecular weight was used as a starting material and sulfated by the method according to Example 1, a sulfated gellan having a molecular weight of 4.2 kDa and a hydroxylation rate of 9.0% was obtained. It was.

  The cytotoxicity of this sulfated gellan was at least 1000 μg / ml or more, but the cytotoxic effect was as high as 500 μg / ml or more, and no giant cell formation inhibitory effect was observed.

<Conclusion>
Table 1 summarizes the results of the examples and comparative examples.

Thus, when sulfated gellan with a high average sulfation rate (about 20% or more) is used, a cytotoxic effect can be obtained at a low concentration, cytotoxicity cannot be detected, and a giant cell formation inhibitory effect can be obtained. It was. In contrast, when sulfated gellan with a low average sulfation rate (less than about 10%) was used (Comparative Example 1), cytotoxicity could not be detected, but the cytotoxic effect could be detected only at a higher concentration. No giant cell formation inhibitory effect was detected. This indicates that sulfated gellan having an average sulfation rate of 10% or more can be produced at low cost, has high antiviral activity, and low cytotoxicity.
In addition, since the cytotoxic effect of sulfated gellan is effective on cells infected with viruses, antiviral agents are effective for both prevention (administration before infection) and treatment (administration after infection). It was found that it can be used.

Claims (6)

An antiviral agent containing a sulfated polysaccharide as an active ingredient ,
The sulfated polysaccharide is constituted by a repeating structure unit represented by the following formula (1), antiviral agents in which a part or all of the hydroxyl groups contained in said sulfated polysaccharide is characterized in that it is sulfated .

(In the formula, each R is independently selected OH or OSO ₃ H.) Antiviral agent according to claim 1, wherein the sulfated polysaccharide is characterized by a sulfated gellan. The antiviral agent according to claim 1 or 2, which is effective against human retroviruses. 4. The antiviral agent according to claim 3, wherein the human retrovirus is HIV. The antiviral agent according to any one of claims 1 to 4 , wherein the sulfated polysaccharide has an average molecular weight of 5 to 500 kDa. Antiviral agent according to any one of claims 1 to 5 in which 10% or more of the hydroxyl groups are characterized by being sulfated.