JPH0859481A - New anti-hiv agent - Google Patents

Abstract

PURPOSE: To provide an agent effective for the treatment of diseases caused by HIV (AIDS virus) such as AIDS and ARC. CONSTITUTION: This anti-HIV agent contains one or more compounds selected from phosphorothioester-bonded oligoribonucleotides expressed by either one of the following formulas (I) to (IV) as active components. (I) GsGsG; (II) GsGsGsG; (III) GsGsGsGsG; (IV) GsGsGsGsGsG (G is guanosine structure; s is phosphorothiophosphoric acid residue forming phosphorothioester bonds with adjacent G groups; each s positioned between two G groups forms an ester bond with the left side G through its 3' hydroxyl group and with the right side G through its 5' hydroxyl group).

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to HIV (Human).
The present invention relates to an anti-HIV agent containing, as an active ingredient, a phosphorothioester-bonded oligoribonucleotide that exhibits an excellent antiviral action for diseases caused by Immunodeficiency Virus).

[0002]

BACKGROUND ART As a conventional finding on a homo-oligomer of DNA, it has been reported by Agrawal et al. That a phosphorothioate conjugate of 15 bases and 20 bases has an inhibitory effect on HIV growth, and the effect is that a homologue having a length of 15 bases is used. It is reported that it is an oligomer and does not depend on the type of base. In addition, it is reported that its effectiveness was presumed to be based on the fact that it is a homo-oligomer having a long chain length and, therefore, acts effectively as a template for reverse transcriptase and competes with the nucleic acid of HIV. (Pro
c. Natl. Acad. Sci. USA, 85, 70
79, 1988).

In addition, it has been reported by Matsukura et al. That a 28-base deoxycytidine phosphorothioate conjugate has an HIV growth inhibitory action (Proc. Nat.
l. Acad. Sci. USA, 84, 7706, 19
87). Furthermore, as a conventional knowledge about oligoribonucleotides, Shibahara et al.
Methylinosine phosphorothioate conjugates have been shown to have HIV growth-inhibitory activity (Nucleic Aci
d Res. , 17, 239, 1989), and Crawford et al. Reported that polyadenine-polyuridine also has an HIV growth inhibitory effect (AIDS Re).
s. Human Retroviruses. 8, 28
5, 1992). However, since all of them are long-chain nucleotides having 14 to 28 bases or more, there is a problem in preparing them, and there are also problems in absorbability and stability when administered in vivo.

In order to solve such a problem, the present inventor has conducted various studies on the reduction of the molecular weight of an oligonucleotide compound having an HIV growth inhibitory activity, and the short-chain oligodeoxyguanylic acid of 4 to 8 bases was previously identified as HIV. It was found to have a growth inhibitory action.

The present inventor has conducted further research, and as a result,
6-base short-chain guanylate ribonucleotide has excellent HI
It was found that it has a V growth inhibitory action. The present invention is based on such findings.

The present invention is characterized by containing, as an active ingredient, one or more selected from phosphorothioester-bonded oligoribonucleotides represented by any of the following formulas (I) to (IV). The present invention provides an anti-HIV agent. GsGsG (I) GsGsGsG (II) GsGsGsGsG (III) GsGsGsGsGsG (IV) (In each formula, G represents a guanosine structure and s represents a phosphorothiophosphate residue forming a phosphorothioester bond with an adjacent G. , S located between the two G's are 3'to the G on the left side in the formula, respectively.
The hydroxyl group at the position and G on the right side respectively form an ester bond with the hydroxyl group at the 5'position).

The oligothioribonucleotide, which is an active ingredient of the anti-HIV agent of the present invention, can be synthesized by using a conventional method generally used for oligoribonucleotide synthesis. That is, for example, a support to which a guanosine monomer having a protected functional group is bound is prepared, to which a guanylic acid monomer having a protected functional group is bound, and then sulfurization of the phosphorus moiety is performed. After performing this operation sequentially, the protective group is removed and purification is performed. The phosphoamidite method is the preferred method (Scaringe e
t al, Nucleic Acids Res. 18
5433; Damha & Ogilvie, Met
hods in Mol. Biol. 20 81; Ec
kstein Ann. Rev. Biochem. 54
367).

In the case of solid phase synthesis by the phosphoramidite method, guanosine having a protected functional group bound to a support such as controlled pore glass (CPG) is prepared and the 5'hydroxyl group of this nucleoside is prepared. 4, which is a protecting group
4, remove the dimethoxytrityl group with trichloroacetic acid, etc.
An amidite reagent in which the amino group of a guanylate group is protected with an isobutyl group, (5'-o-dimethoxytrityl) -2'-o- (t-butyldimethylsilyl) ribonucleoside 3'-N, N-diisopropyl ( The cyanoethyl) phosphoamidite is condensed using tetrazole as the condensing agent. To prevent the synthesis of by-products,
The unreacted 5'hydroxyl group is acetylated with acetic anhydride, N-methylimidazole or the like. After that, the phosphorus portion is sulphidized using tetraethylthiuram disulfide to obtain a phosphorothiotriester. After repeating the above reaction until the target chain length is reached, by ammonia treatment, each internucleotide is converted from a triester to a diester, which is cleaved from the support and at the same time deprotection of the base portion is carried out. The 2'hydroxyl group is deprotected by N-butylammonium fluoride treatment to obtain the desired oligoribonucleotide.

[0009]

EXPERIMENTAL EXAMPLES Next, the present invention will be described in more detail with reference to experimental examples, but the gist of the present invention is not limited by these experimental examples. <Anti-AIDS virus (HIV) activity test using MT-4 cells (T cell line)> 6 MT-4 cells were placed in a 96-well microplate containing various phosphorothioester-bonded oligoribonucleotides that had been serially diluted in advance. × 1
Scatter in an amount of 0 ⁴ cells / hole, and further AIDS virus [HI
V-1 (IIIB; about ₅₀ TCID ₅₀ (50% culture cell infection concentration) / well) was infected and cultured at 37 ° C. under 5% carbon dioxide for 5 days. Was measured by the MTT method, and the results are shown in Table 1. In the table, in each oligoribonucleotide, the meaning of s in the formula is
It has the same meaning as defined above and G represents guanosine.

[0010]

[Table 1]

As shown in Table 1, the anti-H of the present invention
It was confirmed that the oligoribonucleotide, which is an active ingredient of the IV agent, has an extremely short length of 3 to 6 bases and exhibits an excellent anti-HIV action. No cytotoxicity was shown at a concentration of 100 μg / ml.

The safety of oligoribonucleotide, which is an active ingredient of the anti-HIV agent of the present invention, was confirmed by using a 4-base-bonded one by intravenously administering 20 mg / kg to mice. In another test, no death was observed even after oral administration of the substance at 100 mg / kg.

The novel anti-HIV agent according to the present invention is AID
It is particularly useful as a therapeutic agent for retrovirus infectious diseases such as S, and is formulated using a pharmaceutical carrier to be orally administered, such as tablets, capsules, granules, fine granules, and powders, and injections, It may be a parenteral preparation such as a drip infusion or a suppository. The dosage form is not specified, and an appropriate dosage form is selected and used if necessary, and examples thereof include oral administration agents such as tablets, capsules, granules, fine granules, and powders, Parenteral administration agents such as injections and suppositories are mentioned.
The dose required as an oral preparation varies depending on the age, body weight, and degree of illness of the patient, but the usual adult dose is 0.1 g.
Take ~ 6g in divided doses several times daily.

To prepare the above-mentioned oral administration preparations such as tablets, capsules and granules, for example, starch, lactose, sucrose, mannitol, carboxymethylcellulose and inorganic salts can be used, and they are produced by a conventional method. It In this type of formulation, a binder, a disintegrant, a surfactant, a lubricant, a fluidity enhancer, a flavoring agent, a colorant, a fragrance, etc. are appropriately selected and used in addition to the above-mentioned excipients. You can Specific examples of each are as follows.

[Binder] Starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch,
Methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, crystalline cellulose, ethyl cellulose, polyvinyl cellulose, macrogol.

[Disintegrant] Starch, hydroxypropyl starch, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, carboxymethyl cellulose, low-substituted hydroxypropyl cellulose.

[Surfactant] sodium lauryl sulfate,
Soy lecithin, sucrose fatty acid ester, polysorbate 80. [Lubricant] Talc, wax, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, polyethylene glycol. [Fluidity promoter] Light anhydrous silicic acid, dry aluminum hydride gel, synthetic aluminum silicate, magnesium silicate.

The oligoribonucleotides represented by the above formulas I to IV can also be administered as suspensions, emulsions, syrups and elixirs, and these various dosage forms have flavoring agents, coloring agents, and coloring agents. Agents can be included. The required amount as a parenteral agent varies depending on the age, weight, and degree of disease of the patient, but is usually about 1 to 100 mg per day of the above-mentioned oligoribonucleotide in adults, intravenous injection, intravenous infusion, subcutaneous injection. It is administered by injection or intramuscular injection. This parenteral preparation can be manufactured by a conventional method. In that case, generally, distilled water for injection, physiological saline, glucose aqueous solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol and the like can be used as the diluent. In addition, a bactericidal agent, a preservative, and a stabilizer can be added if necessary. From the viewpoint of stability, this parenteral preparation is frozen in a vial after filling, and then water is removed by an ordinary freeze-drying technique. The solution can be reconstituted. When formulating, if necessary,
Tonicity agents, stabilizers, preservatives, soothing agents and the like can be added as appropriate. Examples of other parenteral agents include liquid preparations for external use, coating agents such as ointments, suppositories for rectal administration, and the like, all of which can be manufactured by a conventional method.

[0019]

The present invention will be described in detail below with reference to examples, but the present invention is not limited to the examples. Example 1 According to the above prescription, a part of and is mixed until homogeneous, compression-molded, then crushed, and the remaining amount and is added and mixed, and compression-molded with a tableting machine to give one tablet. 200 mg tablets were obtained. This one tablet contains GsGsGs
Contains 20 mg of G. This tablet is taken in 4 to 8 tablets per day for adults in several divided doses.

Example 2 According to the above prescription, and and are mixed until homogeneous, kneaded by a conventional method, and dried by an extrusion granulator.
After crushing, this was mixed with and and the mixture was compression-molded with a tablet machine to obtain 200 mg tablets. This one tablet contains 20 mg of GsGsGsG. This tablet is taken in 4 to 8 tablets per day for adults in several divided doses.

Example 3 On the other hand, after dissolving and, the mixture was poured into a 5 ml ampoule, and heat sterilized at 121 ° C. for 15 minutes to obtain an injection.

[0022]

The novel anti-HIV agent according to the present invention is
Diseases caused by (AIDS virus), AIDS, AR
It is effective in treating C.

Claims (1)

[Claims] 1. An active ingredient comprising one or more selected from phosphorothioester-linked oligoribonucleotides represented by any of the following formulas (I) to (IV). Anti-HIV agent. GsGsG (I) GsGsGsG (II) GsGsGsGsG (III) GsGsGsGsGsG (IV) (In each formula, G represents a guanosine structure and s represents a phosphorothiophosphate residue forming a phosphorothioester bond with an adjacent G. , S located between the two G's are 3'to the G on the left side in the formula, respectively.
The hydroxyl group at the position and G on the right side respectively form an ester bond with the hydroxyl group at the 5'position).