JPH037224A - Anti-retrovirus agent - Google Patents

Abstract

PURPOSE:To obtain an anti-retrovirus agent effective for various kinds of viral diseases caused by retrovirus, comprising a compound such as luteolin, morin or quercitrin as an active ingredient. CONSTITUTION:A compound shown by the formula (R1 is H, OH, -O-rhamnoside or -O-lutinoside; R2 to R7 are H or OH; with the proviso that compounds wherein R1 and R4 to R7 are H and R2 and R3 are OH and compounds wherein R4 and R7 are H, R1 to R3, R5 and R6 are OH are omitted) is contained as an active ingredient and pharmaceutically manufactured by a conventional procedure as it is or blended with an ordinarily used preparation additive and pharmaceutically manufactured by a conventional procedure to give the aimed substance. The substance may be prepared into an oral agent such as tablet, capsule, granule, fine granule or powder or parenteral drug such as injection or suppository. In the case of oral administration, a dose is 100mg to 6g per adult daily and administered dividedly several times. Digallic acid extracted and isolated from Scutellaria baicalensis has also inhibitory effects on multiplication of retrovirus.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an antiretroviral agent effective in treating various viral diseases caused by retroviruses.

[Prior Art and Problems] As research on viruses continues, treatments for viral diseases are gradually being established.

In particular, acquired immunodeficiency syndrome (AI) has become a problem recently.
DS), causing Hr V (Iiuman 1m
mun.

delicacy Virus), HTLV-1 (
Adult T-cell leukemia virus) and the like are known as retroviruses.

Retroviruses contain RNA-dependent DNA within their virus particles.
It is a virus that contains a synthetic enzyme (hereinafter referred to as reverse transcriptase) and propagates as follows.

■After infecting host cells, RNA is first converted to D by reverse transcriptase.
Transferred to NA.

■This DNA is integrated into the host cell chromosome, and then mRNA is synthesized by the host cell's RNA synthetase.

■Various virus proteins are produced by this mRNA.

There has been a desire to develop a drug that has an epoch-making therapeutic effect on human diseases caused by this retrovirus.

"Means for Solving the Problems" The present inventors conducted research on the retrovirus growth inhibitory effect of various herbal medicines, and found that baicalein and picarin were extracted and isolated from the herbal medicine Huanglium, and quercetin was extracted and isolated from Caica. They also discovered that hesperetin extracted and isolated from Chinpi has a retrovirus growth inhibiting effect, and filed a patent application (Patent Application No. 321 of 1988).
516, Patent Application No. 321517 of 1988, and Patent Application No. 321518 of 1988).

By continuing the research this time, we found that the following formula I (wherein R1 represents a hydrogen atom, a hydroxyl group, -0-rhamnoside, or -0-rutinoside, R7, R5, R5,
Rfl and R7 represent a hydrogen atom or a hydroxyl group.

However, compounds in which R, R,, R6, R11 and R7 are hydrogen atoms, R7 and R3 are hydroxyl groups, and R4 and R7 are hydrogen atoms, RRl, R1, R6
and compounds in which R6 is a hydroxyl group are excluded. The present inventors have discovered that the compound represented by the following formula and digalic acid have an inhibitory effect on retrovirus proliferation, and have completed the present invention.

That is, in the present invention, specific examples of compounds of the following formula r are as follows, and commercially available compounds can be used (wherein R1 is a hydrogen atom, a hydroxyl group, -〇-rhamnocyte, or -〇-rutinosodo, R1, R3, R4, R6, R8 and R
7 represents a hydrogen atom or a hydroxyl group.

However, compounds in which R1, R4, R6, R6, and R7 are hydrogen atoms, R, and R3 are hydroxyl groups, and R4 and R7 are hydrogen atoms, RI, R,, R3,
Excludes compounds in which R6 and R6 are hydroxyl groups. ) and digallic acid (hereinafter, the compound represented by formula I and digalic anod are collectively referred to as the compound of formula).

It has not been previously known that the compound of the formula has antiviral effects, particularly antiretroviral effects.

Digaric acid can be isolated from plants, for example, as follows.

That is, digalic acid-containing plants are extracted using one or more mixed solvents selected from water, methanol, ethanol, acetone, and ethyl acetate, and the resulting extract is extracted as is or concentrated, and then extracted with Sephadex. Sephadex such as LH-20, MCr-gel
It can be obtained by subjecting it to column chromatography at least 61 times using a porous polymer gel such as Cf (P 20 P) as a carrier.

It should be noted that thugaric acid exists in two isomers, methacrylic acid and balazacric acid, and these are interconverted in a solution.

Digaric acid according to the invention includes meta-tugalic acid, para-digalic acid and mixtures of both these compounds in any proportion.

A specific example of the production of tsugaric acid is shown below.

Specific example: 5 kg of Chinese chinensis (Sudajii) leaves were mixed with 40ρ of water-acetone (
]・4) Extracted three times with the mixed solution, concentrated the extract (distilled off acetone), extracted the aqueous layer with 1 g of ethyl acetate, concentrated and dried the ethyl acetate extract, and obtained an extract of 2009. This extract was subjected to column chromatography using Sephadex L) (-20) and eluted by sequentially changing the mixing ratio of water and methanol.

The eluate containing water and methanol at a mixing ratio of III to 3 was concentrated and dried to obtain a crude fraction. This crude fraction was mixed with ethanol,
The mixture was subjected to Sephadex using water-methanol (1:4) as an eluent and subjected to column chromatography using H-20. Next, the eluate was subjected to column chromatography using M (1gelc HP20P) using a solvent in which the water-methanol mixing ratio was successively changed, and the eluate was concentrated at a water-methanol mixing ratio of 4:1 to 73. By drying, m-digallic acid (m-digalli acid)
c acid) 460η was obtained.

The physicochemical properties of metartugaric acid obtained as described above were consistent with the literature values shown below.

[M, NiN15hiza, T, Yamagishi,
G, Nonaka and 1. N15hioka, J
,Chem,Soc,Perkin Trans,,1
．． 2963.1982] Next, the anti-retroviral effect of the compound of the formula will be explained with reference to experimental examples.

Experimental Example: Cells infected with murine leukemia virus (Rauscher strain) were cultured, and a middle and high school method [C0NPARATIVE LEUK
EMIARESEARCll 1973. LEUKEM
OGENESIS, ED, Y, ITO, ^ND RM,
DUTCHER, UNIV, OP TOKYO PRES
S TOKYO/KARGERBASEL, PP, 6
03-605 (1975)], reverse transcriptase was isolated and purified.

Next, a reaction mixture having the following composition was prepared.

0 Reverse transcriptase I unit/-〇 Polyadenylic acid/oligothymidylic acid complex as template/brimer complex [Polyadenylic acid (manufactured by Methylmacia)]
: Oligothymidylic acid (manufactured by Methylmacia) = 4:I]
21/mlO Tris-HCl (pHs, o) 50+MO dithiothreitol 51M0 Potassium chloride 50 0 Manganese chloride 02 0['f(]deoxythymidine triphosphate 0.
0IffM (hereinafter abbreviated as [3H]dTTP)
(400 cpm/psi) 0 grise
Roll 15 (v/v)%
0 Purified water Appropriate amount of water 1 unit means that reverse transcriptase converts dNTPs in 1 hour at 37°C.
(deoxynucleic acid triphosphate) is a specific activity unit that consumes In-.

The compound of the formula and purified water were added to 20 sides of this reaction mixture to make 507J, and the incorporation of radioactivity into the acid-insoluble fraction of ['H]dTTP was measured using a Beckman anti-enrichment counter. The inhibition rate at each concentration was calculated based on the enzyme activity.

The results are shown in Table 1.

The above results confirmed the excellent antiretroviral effect of the compound of formula.

As mentioned above, the compound of the formula has the effect of suppressing the proliferation of retroviruses by inhibiting the reverse transcriptase activity necessary for their proliferation, so it can be applied to any retrovirus. I can do it.

Specific examples of retroviruses include leukemia virus, sarcoma virus, breast cancer virus, Visna virus, Maedei virus, HIVSHTLVI, and the like.

Next, an acute toxicity test for oral administration of a compound of formula <I
d Y male rats and Wistar
When carried out using a strain of male rats, the compound of the formula I'
There were no deaths even after oral administration of d/ni9.

Thus, the compound of the formula has extremely low toxicity and high safety.

Next, the dosage and formulation of compounds of formula will be described.

The compounds of the formula can be administered to animals and humans neat or with conventional pharmaceutical carriers.

The dosage form is not particularly limited and may be selected and used as required, including tablets, capsules, granules, fine granules,
Examples include oral preparations such as powders, parenteral preparations such as injections, and suppositories.

In order to achieve the desired effect as an oral agent, it is usually necessary for an adult to administer IOOINK~69 by weight of the compound of the formula several times a day, although this will vary depending on the age, weight, and severity of the disease of the patient. It seems appropriate to take it.

In the present invention, oral preparations such as tablets, capsules, and granules are manufactured according to conventional methods using, for example, starch, lactose, sucrose, mannitol, carboxymethyl cellulose, cornstarch, inorganic salts, and the like.

In this type of preparation, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, colorants, fragrances, etc. can be used in the excipients as appropriate. . Specific examples of each are shown below.

[Binder] Starch, dextrin, gum arabic powder, gelatin,
Hydroxypropyl starch, methylcellulose, sodium carboxylmethylcellulose, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, macrogol.

[Disintegrant] Starch, hydroquine propyl starch, sodium carboxyl methyl cellulose, calcium carboxome dyl cellulose, carboxymethyl cellulose, low substituted hydroxypropyl cellulose.

[Surfactant Sodium colauryl sulfate, soybean lentil, sucrose fatty acid ester, polysorbate 80° [Lubricant] Talc, waxes, hydrogenated vegetable oil, sucrose fatty acid ester, magnenoum stearate, carnoum stearate, aluminum stearate, Polyethylene glycol.

"Fluidity promoter" Light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate.

Furthermore, the antiretroviral agent of the present invention can be administered as a suspension, emulsion, syrup, or elixir, and these various forms may contain flavorings and coloring agents. good.

In order to exert the desired effect as a parenteral agent, it is usually necessary for adults to administer 1 to 100 M9 of the compound of the formula per day by intravenous injection or infusion, although this will vary depending on the age, weight, and severity of the disease of the patient. Intravenous, subcutaneous, and intramuscular injections are considered appropriate.

This parenteral preparation is manufactured according to a conventional method, and generally uses distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol, etc. as a diluent. I can do it. Furthermore, a bactericide, a preservative, and a stabilizer may be added as necessary. In addition, from the viewpoint of stability, this parenteral preparation may be filled into a vial or the like, then frozen, water removed by ordinary freeze-drying techniques, and a liquid preparation prepared from the freeze-dried product immediately before use. Furthermore, isotonizing agents, stabilizers, preservatives, soothing agents, etc. may be added as appropriate.

Other parenteral preparations include liquid preparations for external use, liniments such as ointments, suppositories for rectal administration, and the like, which are manufactured according to conventional methods.

Example 1 ■Corn starch 449 ■Crystalline cellulose 40g ■Carboxymethyl cellulose calcium 59 ■Light silicic anhydride 0.59 ■Magnesium stearate 0.59 ■Luteolin
10g total 1009 According to the above recipe, ① to ② were mixed uniformly and compressed using a tablet machine to obtain tablets of 200 pieces.

This tablet contains Luteolin 20 and Shoulder 9, and adults should take 5 to 7 tablets a day in several doses.

Example 2 ■Crystalline cellulose 84.59 ■Magnesium stearate 059 ■Carboxymethylcellulose calcium 5g ■Morin IOg meter
1009 According to the above recipe, mix ■, ■, and part of ■ uniformly, compression mold, crush, add remaining amounts of ■ and Part 200 m9
tablets were obtained.

This tablet contains <20 mgh of morin, and adults should take 5 to 7 tablets a day in several doses.

Example 3 ■Crystalline cellulose 34. ．． 59 ■ 10% hydroquine propyl cellulose ethanol solution 509 ■ Carboxymethylcellulose calcium 5 g ■ Magnenium stearate 0.59 ■ Coerntrin 10 g total 100 g According to the above recipe, mix ■, ■, and ■ uniformly, and make a paste by a conventional method. After granulating with an extrusion granulator, drying and crushing, (1) and (2) were mixed and compression molded with a tablet machine to obtain some 20019 tablets.

This tablet contains 20m9 of quercitrin, and adults should take 5 to 7 tablets a day in several doses.

Example 4 ■Corn starch 849■Magnesium stearate 0 59■Carboxymethyl cellulose calcium 5g ■Light silicic anhydride 0.5g■Rutin
Lo! ? Total: 1009 According to the above recipe, ① to ② were uniformly mixed, compression molded using a compression molding machine, crushed using a crusher, and sieved to obtain granules.

This granule IS+ contains rutin l OO119, and adults should take 1 to 2.5 g per day in several doses.

Example 5 ■ Crystalline cellulose 40 g ■ 10% hydroxypropyl cellulose ethanol solution 509 ■ Quercetagetin 109 Total 100 g According to the above recipe, ■ to ■ were mixed uniformly and made into a suspension. After going into an extrusion granulator and granulating, the mixture was dried and sieved to obtain granules.

1 g of this granule contains quercetagetin 100119, and an adult should take 1 to 2.59 doses per day in several doses.

Example 6 ■Corn starch 89.59■Light silicic acid anhydride 0.5g■Myricetin
101i1 total 1009 Mix ■～■ uniformly according to the above recipe, 200 M
9 was filled into a No. 2 capsule.

One capsule of this preparation contains 20 m9 of myricetin, and adults should take 5 to 7 capsules a day in several doses.

Example 7 ■Corn starch 449 ■Crystalline cellulose 409 ■Carboxymethylcellulose calcium 5g ■Light anhydrous silicic acid 0.5g■Magnesium stearate 0.59■Digital acid
109 Total 1009 According to the above recipe, ① to ② were mixed uniformly and compressed using a tablet machine to obtain a portion of 200 ff9 tablets.

These tablets contain 20 Mg of digalic acid, and adults should take 5 to 7 tablets a day in several doses.

Example 8 ■ Distilled water for injection Appropriate amount ■ Glucose 2003!9 ■ 6,7-
Dihydroxyflavone + 019 total amount 1
577 After dissolving ■ and ■ in distilled water for injection, they were injected into a 5d ampoule and autoclaved at +21'C for 15 minutes to obtain an injection.

Claims (2)

[Claims] (1) The following formula I ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R_1 is a hydrogen atom, a hydroxyl group, -O-rhamnoside,
or -O-rutinoside, R_2, R_3, R_
4, R_5, R_6 and R_7 represent a hydrogen atom or a hydroxyl group. However, compounds in which R_1, R_4, R_5, R_6, and R_7 are hydrogen atoms, R_2 and R_3 are hydroxyl groups, and R_4 and R_7 are hydrogen atoms, and R_1, R_2, R_3, R_5, and R_
Excludes compounds where 6 is a hydroxyl group. ) An antiretroviral agent whose active ingredient is a compound represented by (2) Antiretroviral agent containing digalic acid as an active ingredient.