JPH0834736A - New antihiv agent - Google Patents

Abstract

PURPOSE:To obtain a new antiHIV agent effective in treating diseases caused by HIV (AIDS virus), AIDS and AIDS related complex (ARC). CONSTITUTION:This antiHIV agent contains one or more selected from phosphorothioester-binding type oligoribonucleotides represented by any of the following formulas (I) to (IV): pGsGsG (I), pGsGsGsG (II), pGsGsGsGsG (III) and pGsGsGsGsGsG (IV) [G denotes a guanosine structure; (p) denotes a phosphoric acid residue bound through a monoester bond to hydroxyl group at the 5'-position of the terminal G at the 5'-position; each (s) located between two G's respectively denotes a phosphorothiophosphoric acid residue forming the phosphorothio ester bond with the adjacent G and forms ester bonds with hydroxyl group at the 3'-position of G on the left-hand side in each formula and hydroxyl group at the 5'-position of G on the right-hand side therein].

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 deoxytidine phosphorothioate conjugate has an HIV growth inhibitory action (Proc. Natl,
Acad, Sci. USA, 84, 7706, 198.
7). Furthermore, as a conventional finding regarding oligoribonucleotides, Shibahara et al. Showed that a 20-base 2'-o-methylinosine phosphorothioate conjugate has an HIV growth inhibitory action (Nucleic Acid).
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. As a result of further studies, the present inventor has found that a short-chain guanylate ribonucleotide having 2 to 5 bases has an excellent HIV 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. pGsGsG (I) pGsGsGsG (II) pGsGsGsGsG (III) pGsGsGsGsGsG (IV) (In each formula, G represents a guanosine structure, and s represents a phosphorothiophosphate residue forming a phosphorothioester bond with an adjacent G. , S is an ester bond with G on the left side in the formula and the hydroxyl group at the 3'position and G on the right side with respect to the 5'position hydroxyl group, and p is 5 It represents a phosphoric acid residue that is monoester-bonded to the 5'-position hydroxyl group at the'-terminal G).

The oligothioribonucleotide, which is the active ingredient of the anti-HIV agent of the present invention, can be synthesized by a conventional method using the solid phase phosphoramidite method generally used for oligoribonucleotide synthesis. For example, the example is as follows.

A commercially available support having a functional group-protected nucleic acid monomer bound thereto is used as a starting material, and if necessary, a nucleic acid monomer protected with an amino group and an acidic hydroxyl group are sequentially bound thereto to give a crude oligoribonucleotide. It is prepared by a method of obtaining a nucleotide, removing the protecting group, and purifying. A suitable method is arbitrarily selected depending on the kind of desired oligothioribonucleotide.

In the case of solid phase synthesis by the phosphoamidite method, a 3'-terminal nucleoside corresponding to the RNA of interest is bound to a support such as a commercially available control pore glass (CPG), and this nucleoside is used. 4,4'-dimethoxytrityl group, which is a protective group for the 5'hydroxyl group, is removed with trichloroacetic acid or the like, and tetrazole is used as a condensing agent.
Amidite reagent (5'-o-dimethoxytrityl-) in which the amino group of a nucleobase as a commercially available monomer corresponding to the sequence from the 3'end side is protected with an isobutyl group.
2'-o- (t-butyldimethylsilyl) ribonucleoside 3'-N, N-diisopropyl (cyanoethyl)
A phosphoramidite) is condensed. The unreacted 5'hydroxyl group is acetylated with acetic anhydride, N-methylimidazole or the like to prevent the synthesis of by-products. After this,
Sulfidation of the phosphoric acid moiety is carried out using tetraethylthiuram disulfide, leading to phosphorothiotriesters.
After repeating the above reaction until the target chain length is reached,
By treatment with thiophenol, each internucleotide was converted from a triester to a diester, and a phosphorylating reagent (2- [2-4,4'dimethoxytrityloxy] ethylsulfonyl (2-cyanoethyl)-
(N, N-diisopropyl) -phosphoamidite) is condensed. Then, by treating with ammonia, the base portion is deprotected at the same time as being cut out from the support, and the tetra-N
The 2'-hydroxyl group is deprotected by treatment with -butylammonium fluoride to obtain the desired 5'-terminal phosphorylated phosphorothioate 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)> MT-4 cells were placed in a 96-well microplate containing various phosphorothioate-binding oligoribonucleotides, which had been serially diluted in advance, to prepare 6x10 MT-4 cells.
⁴ cells / hole, and then AIDS virus [HIV
-1 (IIIB; about ₅₀ TCID ₅₀ (50% culture cell infectious concentration) / well), and incubated at 37 ° C. under 5% carbon dioxide for 5 days. It was determined by measuring living cells by the MTT method, and the results are shown in Table 1. In the table, in each oligoribonucleotide shown, the meanings of p and s have the same meanings 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 5'-terminal phosphorylated phosphorothioate-linked oligoguanylic acid having 3 to 6 bases, which is an active ingredient of the IV agent, exhibits an excellent anti-HIV effect. 100
There was no cytotoxicity at the concentration of μg / ml.

The safety of ribonucleotide, which is an active ingredient of the anti-HIV agent of the present invention, was confirmed by using a 4-base-bond ribonucleotide, which was intravenously administered to mice at 20 mg / kg. In another test, 100 of the same substance
No deaths were observed even after oral administration of 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,
A flavoring agent and a coloring agent can be contained in these various dosage forms.

The required amount as a parenteral agent depends on the age of the patient,
Although it varies depending on the body weight and the degree of disease, the amount of the above-mentioned oligoribonucleotide is usually 1 to 100 mg / day for an adult and is administered by intravenous injection, intravenous infusion, subcutaneous 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, if necessary, a bactericidal agent, a preservative,
Stabilizers can be added. 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. At the time of formulation, an isotonic agent, a stabilizer, a preservative, a soothing agent and the like may be added as necessary. 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.

[0020]

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 pGsGsG
It contains 20 mg of sG. This tablet is for adults 1
Take 4 to 8 tablets daily 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 single tablet contains 20 mg of pGsGsGsG. 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.

[0023]

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. pGsGsG (I) pGsGsGsG (II) pGsGsGsGsG (III) pGsGsGsGsGsG (IV) (In each formula, G represents a guanosine structure, and p represents a phosphate that is monoester-bonded to the 5'-hydroxyl group at the 5'-terminal G. Each s, which represents a residue and is located between the two G's, is
Represents a phosphorothiophosphoric acid residue forming a phosphorothioester bond with an adjacent G, wherein G on the left side in the formula is its 3'-hydroxyl group, and G on the right side is its 5'-hydroxyl group. What is forming an ester bond).