JPH04264026A - Antiretrovirus agent - Google Patents

Abstract

PURPOSE:To provide an antiretroviral agent having extremely low toxicity and high safety, exhibiting retrovirus-proliferation inhibiting effect and effective for the treatment of various viral diseases caused by retrovirus. CONSTITUTION:The objective antiretroviral agent contains, as an active component, the extract of the flower of Rosa rugosa or tellimaglandin I and/or pedunculagin of formula I and formula II (R is H or OH) separable from the above extract. The antiretroviral agent suppresses the proliferation of retroviruses such as leukemia virus, breast carcinoma virus, Visna virus, Maedi virus and HIV (Human Immuno deficiency Virus) and HILV-T (adult T-cell leukemia virus) causing acquired immunodeficiency syndrome (AIDS) by inhibiting the RNA-dependent DNA synthetase (reverse transcriptase) necessary for the proliferation of the virus.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to an antiretroviral agent effective in treating various viral diseases caused by retroviruses.

0002

BACKGROUND OF THE INVENTION As research on viruses continues, treatments for viral diseases are gradually being established. In particular, acquired immunodeficiency syndrome (AI) has become a problem recently.
HIV (Human Immunoimmune Disease), which causes DS)
no deficiency virus), HT
LV-I (adult T-cell leukemia virus) and the like are known as retroviruses.

[Problem to be solved by the invention]

[0003] Retroviruses contain RN in their virus particles.
It is a virus that contains A-dependent DNA synthase (hereinafter referred to as reverse transcriptase) and propagates as follows.

[0004] After infecting a host cell, RNA is first transcribed into DNA by reverse transcriptase. ■This DNA is integrated into the host cell chromosome, and then mRNA is synthesized by the host cell's RNA synthetase. ■ Various viral 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.

[0005] The present inventors conducted research on the retrovirus growth inhibiting effect of various herbal medicines and found that Hanamasu flower extract, tellimagrandin I, and pedunclazine have a retrovirus growth inhibiting effect. .

[0006] The present invention is based on this knowledge, and includes an antiretroviral agent containing an extract of Hanamasu flower as an active ingredient, as well as an antiretroviral agent represented by the following formula II (wherein R represents a hydrogen atom or a hydroxyl group). It is an antiretroviral agent containing a compound, ie, telimagrandin I and/or a compound represented by the following formula III (wherein R represents a hydrogen atom or a hydroxyl group), ie, peduncladine, as an active ingredient.

[0007] The flower extract of Hanamasu is known to have antidiarrheal and hemostatic effects, and one of its components, tellimagrandin I, is known to have a herpes virus inhibitory effect. However, it was not previously known that it had an antiretroviral effect against HIV.

[0008] In addition, the Rosa rugosa used in the present invention may be Mica squid, which is used as a herbal medicine, or its original plant, Rosa rugosa.
Thunb. ), and its variety Shirobananasu (R
osa rugosa cv. Alba. ),
Rosa rugosa
cv. Flower parts such as Alboplena) may also be used.

[0009] Hereinafter, the extract of the flower of the Japanese lily will be referred to as the drug of the present invention, the compounds represented by formulas I and II will be referred to as the compounds of the formula, and these will be collectively referred to as the antiretroviral agent of the present invention.

The antiretroviral agent of the present invention can be obtained, for example, as follows.

[0011] That is, the flowers of Hermanus are ground into fine powder as necessary, and extracted with at least one solvent selected from hexane, diethyl ether, petroleum ether, ethyl acetate, chloroform, acetone, methanol, ethanol, and water. Inventive drugs can be obtained.

[0012] Although extraction may be carried out at room temperature, it is preferable to carry out the extraction at a temperature near the boiling point of the solvent used, taking into account extraction efficiency, extraction time, etc., and the extraction time is suitably 1 to 6 hours.

[0013] Further, the residue obtained by removing the solvent from the extract obtained as described above is dissolved in methanol, and the soluble portion is dissolved in hexane, diethyl ether, petroleum ether, ethyl acetate, chloroform, acetone, or acetonitrile. , methanol, ethanol, water, and sodium hydrogen phosphate as an elution solvent, porous polymers such as Diaion HP-20 and MCI gel CHP20P, Sephadex such as Sephadex LH-20, TSKgel 120T, etc. The compound of the formula can be obtained by subjecting it once or several times to column chromatography or high performance liquid chromatography using reverse phase silica gel, silica gel, polyamide, cellulose, etc. as a carrier. Further, desalting operation may be performed as necessary.

Next, a specific example of the production of the antiretroviral agent of the present invention will be shown.

Specific Example 1 Rosa rugosa Thun
b. ) 100g into fine powder and 60g using 800ml of water.
Digestion extraction was performed at ℃ for 2 hours, followed by filtration. By concentrating the filtrate to dryness, 27.3 g of water extract was obtained.

Specific Example 2 27.3 g of the extract obtained in Specific Example 1 was mixed with 25 g of methanol.
Extraction was performed twice with 0 ml to obtain 6.5 g of methanol soluble portion. Of this, 3 g was used as an elution solvent for MCI
It was subjected to column chromatography using gel CHP20P (2.5φ×18.5cm).

[0017] 124 mg obtained by distilling off the solvent of the 15% methanol elution fraction was mixed with 20-30% acetonitrile (
T
It was subjected to reverse-phase silica gel column chromatography using SKgel 120T, and stepwise elution was carried out at 8 ml/min for 15 minutes.

Further, it was subjected to column chromatography using Sephadex LH-20, desalted with water, and then eluted with a mixture of water: ethanol: acetone (1:1:2) to obtain crude peduncladine. I got it.

Crude peduncladine was subjected to reversed phase silica gel column chromatography using TSKgel 120T using 15-30% acetonitrile (10mM sodium hydrogen phosphate) as an eluent, and 15 to 30% acetonitrile (10mM sodium hydrogen phosphate) was used as the eluent.
Stepwise elution for 5 minutes, then 10-25% acetonitrile (
Water) was used as the elution solvent and subjected to reverse phase silica gel column chromatography using TSKgel120T.
By stepwise elution for 15 min at ml/min, the literature [L
iebigs Annalen derchemi
c. , 1965, vol. 690, pp. 150-16
2, O. T. Schmidt, L. Wurtele
and H. 10.5 mg of peduncladine described in [Harreus] was obtained.

Specific Example 3 MCI gel CHP20P (2.5φ×18.
In column chromatography using
2 obtained by distilling off the solvent of the 30% methanol elution fraction
TSKgel1 was added to 72mg using 15-35% acetonitrile (10mM sodium hydrogen phosphate) as the elution solvent.
The mixture was subjected to reverse phase silica gel column chromatography using 20T, and stepwise elution was carried out at 10 ml/min for 30 minutes.

[0021] Further, it was subjected to column chromatography using Sephadex LH-20, and after desalting with water, it was eluted with a mixture of water: ethanol: acetone (1:1:2) to obtain crude Telima. 72 mg of Grandin I was obtained.

[0022] Crude telimagrandin was subjected to reverse phase silica gel column chromatography using TSKgel 120T using 20-35% acetonitrile (10mM sodium hydrogen phosphate) as an eluent, and the eluting rate was 8ml/min.
After stepwise elution for 5 minutes, the mixture was subjected to reverse phase silica gel column chromatography using TSKgel 120T again using 10-30% acetonitrile aqueous solution as the elution solvent.
By stepwise elution for 20 min at ml/min, the
hytochemistry, 1976, vol.
15, pp. 211-214, Cornelius
K. , Wilkins and Bruce A.
25.2 mg of telimagrandin I described in [Bohn] was obtained.

Next, the anti-retroviral effect of the anti-retroviral agent of the present invention will be explained with reference to experimental examples.

Experimental Example 1 Cells infected with murine leukemia virus (Rauscher strain) were cultured, and the method of Nakajima et al.
KEMIA RESEARCH 1973, LEU
KEMOGENESIS, ED. Y. ITO. AN
DR. M. DUTCHER, UNIV. O.F.T.
OKYO PRESS TOKYO/KARGER
, BASEL, PP. 603-605 (1975)], reverse transcriptase was isolated and purified. Next, a reaction mixture having the following composition was prepared.

・Reverse transcriptase
1 unit/ml ・Polyadenylic acid/oligothymidylic acid complex as template/primer complex [polyadenylic acid: oligothymidylic acid pdT12-18 (1:1)] manufactured by Pharmacia
5 μg/ml Tris-HCl (pH 8.0)
25mM potassium chloride
75mM Magnesium Chloride
8mM dithiothreitol
2mM・[3H]deoxythymidine triphosphate (1.55TBq/mmol) 0.2μM・Deoxythymidine triphosphate 9.8μM・Compound of the drug or formula of the present invention 0.05-1μg/
ml/purified water
Appropriate amount However, 1 unit means 37 units of reverse transcriptase.
dNTP (deoxynucleic acid triphosphate) 1n for 10 minutes at ℃
It is a unit of specific activity that consumes mol.

[0026] 50 μl of this reaction mixture was heated at 37°C for 30
The reaction product DNA was quantified using the ion exchange filter method, and the DNA of [3H]deoxythymidine triphosphate was
The incorporation of radioactivity into the compound was measured using a Beckman scintillation counter to determine the reverse transcriptase activity, and the inhibition rate at each concentration was calculated to determine the 50% inhibitory concentration (IC50) of the drug of the present invention or the compound of the formula.

As a result, the IC50 of the drug of the present invention was 0.
Among the compounds of the formula, the IC50 of telimagrandin I was 0.04 μg/ml, and the IC50 of peduncladine was 0.10 μg/ml.

These results confirmed the excellent antiretroviral effects of the drug of the present invention and the compound of the formula.

As described above, the antiretroviral agent of the present invention has the effect of suppressing the proliferation of retroviruses by inhibiting the reverse transcriptase activity necessary for the proliferation of retroviruses. It can be applied to any virus.

[0030] Specific examples of retroviruses include leukemia virus, sarcoma virus, breast cancer virus, Visna virus, Maedi virus, HIV, HTLV-I, and the like.

Next, the antiretroviral agent of the present invention was subjected to an acute toxicity test by oral administration using male ddY mice and male Wistar rats. There were no deaths even after oral administration, and in the compound of the formula, 100 mg/k
There were no deaths even after oral administration of g.

[0032] Thus, the antiretroviral agent of the present invention has extremely low toxicity and high safety.

Next, the dosage and formulation of the antiretroviral agent of the present invention will be explained. The antiretroviral agent of the present invention can be administered to animals and humans as is or together with a conventional pharmaceutical carrier. The dosage form is not particularly limited and may be selected and used as appropriate,
Oral preparations such as tablets, capsules, granules, fine granules, powders,
Examples include parenteral preparations such as injections and suppositories.

[0034] In order to exert the desired effect as an oral preparation, the antiretroviral agent of the present invention usually requires a weight of 10% for an adult, although it varies depending on the age, weight, and severity of the disease of the patient.
It seems appropriate to take 0 to 6000 mg in divided doses several times a day.

[0035] 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.

[0036] In addition to the excipients mentioned above, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, fragrances, etc. may be used in this type of preparation as appropriate. Can be done. Specific examples of each are shown below.

[Binder] Starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch,
Methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, 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, waxes, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol.

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

[0042] The antiretroviral agent of the present invention can also be administered as a suspension, emulsion, syrup, or elixir, and these various dosage forms may contain flavoring agents and coloring agents. It's okay.

In order to exert the desired effect as a parenteral agent, it depends on the age, weight, and severity of the disease of the patient;
Normally for adults, the weight of the antiretroviral agent of the present invention is 1
Intravenous injections, intravenous drips, subcutaneous injections, and intramuscular injections of 1 to 100 mg per day are considered appropriate.

This parenteral preparation is manufactured according to a conventional method, and diluents generally include distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, and polyethylene glycol. etc. can be used. 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, as necessary, tonicity agents, stabilizers, preservatives,
A soothing agent or the like may be added.

Other parenteral preparations include liquid preparations for external use, liniments such as ointments, suppositories for intrarectal administration, etc.
Manufactured according to conventional methods.

[0046] Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto in any way.

Example 1 According to the above recipe, ① to ② were mixed uniformly and compressed using a tablet machine to obtain tablets each weighing 200 mg. One tablet contains 20 mg of the drug obtained in Example 1, and adults take 5 to 15 tablets a day in several doses.

Example 2 According to the above recipe, ■, ■, and part of ■ were uniformly mixed, compressed and molded, and then pulverized, the remaining amounts of ■ and ■ were added, mixed, and compressed using a tablet machine. Molded tablet 200mg
tablets were obtained. One tablet contains 20 mg of the compound obtained in Example 2, and adults should take 5 to 15 tablets a day in several doses.

Example 3 ■, ■, and ■ were uniformly mixed according to the above recipe, and the mixture was homogenized by a conventional method and granulated using an extrusion granulator.
After drying and crushing, (1) and (2) were mixed and compressed using a tablet machine to obtain tablets each weighing 200 mg. One tablet contains 20 mg of the compound obtained in Example 3, and adults should take 5 to 15 tablets a day in several doses.

Example 4 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. 1 g of this granule contains 100 mg of the drug obtained in Example 1, and adults should take 1 to 3 g in several doses per day.

Example 5 According to the above recipe, ① to ② were uniformly mixed and made into a paste. After granulation using an extrusion granulator, the mixture was dried and sieved to obtain granules. 1 g of this granule contains 100 mg of the compound obtained in Example 2, and adults should take 1 to 3 g in several doses per day.

Example 6 ■~■ were mixed uniformly according to the above recipe, and 200
mg was filled into No. 2 capsules. One capsule of this preparation contains 20 mg of the compound obtained in Example 3, and adults should take 5 to 15 capsules a day in several doses.

Example 7

Claims (3)

[Claims] [Claim 1] An antiretroviral agent containing an extract of flowers of Hermanus as an active ingredient. 2. An antiretroviral agent containing a compound represented by the following formula II (wherein R represents a hydrogen atom or a hydroxyl group) as an active ingredient. 3. An antiretroviral agent containing a compound represented by the following formula II (wherein R represents a hydrogen atom or a hydroxyl group) as an active ingredient.