JPS5936623A - Inducing agent for interferon - Google Patents

Abstract

PURPOSE:The titled pharmaceutical, containing a 9,19-cyclolanostane derivative isolated as a component of Astragali Radix as an active constituent, capable of stimulating a host to promote the production of interferon, and useful as an antiviral and anticancer agent. CONSTITUTION:An inducing agent for interferon containing a compound expressed by formula I (R1 is OH or formula II; R2 is a group expressed by formula II or IV) as an active constituent. The compound expressed by formula I is obtained by extracting Astragali Radix with methanol, etc. under warming, distributing and transferring the resultant extract to another solvent i.e. benzene, etc. to remove impurities, distributing and transferring the resultant substance to another solvent i.e. n-butanol, and subjecting the resultant extract to the silica gel chromatogaphy (chloroform, methanol and water). The compound expressed by formula I has the ability to induce the interferon and can be administered orally or parenterally as a remedy for viral diseases, e.g. hepatitis or herpes, and cancers. The LD50 is 3,000mg/kg or more in mice for the compound expressed by formula I (R1 is formula II; R2 is formula III) by the intraperitoneal or oral route, and the toxicity is very low.

Description

[Detailed description of the invention] The present invention relates to a novel interferon-inducing agent having the following properties.

- (Interferon (hereinafter abbreviated as IFN) is a viral growth inhibitory factor produced by animal cells in response to virus invasion or other stimuli. Numerous studies conducted since its discovery in 1957 have shown that it has antiviral effects and anti-lΦΦ
It is attracting a lot of attention as a substance that has cancer effects.

Although IFN is known to be produced by cells such as leukocytes, cultured fibroblasts, kidney cells, and HeLa cells, there are still problems in mass production from an industrial production standpoint.

In addition, IFN stimulates the host to promote IFN production.
-Research on inducers has been conducted, and several substances have been reported to be effective against viral diseases such as influenza, herpes, hepatitis, and cancer in clinical trials, but problems remain in terms of side effects. The current situation is that

In view of this situation, the present inventors have conducted extensive research on methanol extracts of traditional Chinese herbal medicines and their components, and as a result, have found IFN-inducing activity in the compound represented by general formula (1), and have completed the present invention.

・1'1 This compound was isolated and confirmed by the present inventors as a component of Aspergillus orientalis, which is frequently used in many prescriptions as a key ingredient for energization, tonicity, stasis, and diuresis. 11 27th Annual Meeting of the Pharmaceutical Society of Japan (1980), 11101st Annual Meeting of the Pharmaceutical Society of Japan (1980)
1981), and its biological/
+Tir1 has not been confirmed until now.

Hereinafter, 1.1 will explain in detail the method for producing the interferon-inducing agent of the present invention and the interferon-inducing effect.

However, in the test described in 1., two representative compounds (compound A and compound B) among the compounds of general formula (1) were used.
was used.

20(R),24(S)-Epoxy-16β,25-dihydroxy-3/j-0-[JI-n-(2-0-acetyl)-xylopyranosyl]-6α-〇-β-D-glucopyranosyl- 9,19-cyclola 20(R), 24(
S)-epoxy-6α,16β,25-trihydroxy-3β-0-(β-D-glucopyranosyl(l→2)-
β-D-xylopyranosyl)-9,19-cyclolanostane Compound A and Compound B are obtained by heating and extracting aspergillus with methanol, ethanol (both may contain water), water-containing acetone, water, etc. After removing impurities by dissolving and dissolving with hexane, benzene, carbon tetrachloride, dichloromethane, chloroform, ethyl acetate, ether, etc., the extract obtained by dissolving and dissolving with n-butanol was mixed with chloroform, methanol, and water. It can be easily obtained by subjecting it to silica gel column chromatography using a mixture of n-butanol, ethyl acetate, water, etc. as a developing solvent.

1F) l-M superactivity is determined by the precious wood method [Japan, J, Mjcrob
iol.

18(6), 449-456 (1974)) 1
(2) - The incorporation of uridine was determined by measuring with a scintillation counter.

7r) Su (ddY line, male, 10M age, body ff133±
2 g, 5 animals per group) were used as experimental animals, and each of Compound A and Compound B produced in the Production Example described later was added to 1 illll I! of physiological saline. 8 hours, 24 hours and 96 hours after intraperitoneal administration of 30 mg/kg, blood was collected by cutting the mouse tail, and this blood was injected at 300 rpm for 1 hour.
After 6 minutes for 0 minutes, the IFN titer in the collected blood was measured.

The IFN titer was determined using the diluted serum above and tested against water-vesting stomatitis virus (VSV: New Jersey strain) against mouse LY cells [a fibroblast cell line derived from mouse subcutaneous adipose tissue].
The RN^ synthesis accompanying the proliferation of ``H-uridine'' was measured by the amount of uptake of ``H-uridine,'' and the dilution factor of IFN that suppressed the uptake by 50% was calculated, and the titer was determined as 1Fhl6.The old H standard IFN was used as the standard. The results are shown in Table 1 below for country 119j#.

Table 1 Interferon (IFN) titer The IFN titers induced by Compound A or Compound B both showed high values 24 hours after administration.

Next, the relationship between the dosage of Compound A and Compound B and the IFN-induced activity will be explained.

Mouse CddY strain, female, 100 weeks old, weight 33±2 g.

Using 5 animals in each group as experimental animals, Compound A and Compound B produced in the production example described below were each adjusted in physiological saline at doses of 3 mg/kn, 30 mu/kg, and 300 mg.
24 hours after injecting into the IILI cavity, blood was collected by cutting the tail of the mouse, and the following procedure was performed in the same manner as above.
FN-induced activity was examined. The results are given in international units.
It is shown in Table 2 below.

Table 2: Interferon (IFN)-inducing activity The IFN-inducing activity of Compound A or Compound B was expressed by administration of 30 days/kg or more.

Furthermore, in the above test example, compound A or compound B
VSV virus infection induced in mouse serum by
! 11 It was confirmed that the inhibitory substance C was inactivated by trypsin treatment at 37° for 0.3 hours.

(6) Therefore, it is clear that IFN is induced by Compound A or Compound B.

In addition, the acute toxicity (LD%) of Compound A and Compound B was 3000 mg / kg or more, and the toxicity was extremely low.

The compound of general formula (1) can be administered orally or parenterally, and dosage forms for oral administration include tablets, coatings, powders, granules, capsules, syrups, etc. The dosage forms that can be used include injections and suppositories.

Adjustment of these dosage forms is carried out using pharmaceutically acceptable excipients, binders, lubricants, disintegrants, suspending agents, emulsifiers, preservatives, stabilizers, and dispersing agents as appropriate. . The dosage varies depending on symptoms, body weight, etc., but 1 for adults.
The daily dose is 20-2000 mg, preferably 100-5
00 mg can be administered in 1 to 4 divided doses.

□ As mentioned above, compounds of the general formula [1] represented by Compound A or Compound B (9) are useful as IF) inducers, and are widely used as therapeutic agents for viral diseases and cancer. They can be used in combination.

Production examples are listed below for specific explanation, and representative compositions of formulation examples are illustrated.

Production example: 1 kg of commercially available Ml (Australian japonica, produced in Korea), cut into small pieces, was mixed with 3Q
of methanol and extracted at 70'O for 3 hours. The extraction residue was extracted 4 times using the same method. The Vv liquid was combined and the solvent was distilled off under reduced pressure to obtain 150 g of an extract.

All 71 of this extract was dissolved in three 50% aqueous methanol, first 3Q of n-hexane, then 311 of chloroform.
1 was used to transfer the solution four times each, and the transferred material was removed. Methanol was distilled off from the remaining water-containing methanol liquid layer under reduced pressure, and 1.5 Q of butanol was added to the residue for distribution and dissolution, and the same operation was repeated four times. The tobutanol used for the dissolution was combined, the solvent was distilled off under reduced pressure, and the extraction was carried out (1
0) 50g of extract was obtained.

20g of this extract was added to a mixed solvent of chloroform, methanol, and water [chloroform:methanol:water=7:1:0-
After mixing at a ratio of 5 parts (volume ratio) and allowing it to stand for a day and night, the lower layer was subjected to silica gel column chromatography, and after the start of elution, both fractions 12 to 14Q were collected, and after drying, the lower layer was The solvent was distilled off to obtain compound A OJg. Further, after the start of elution, a 18-20M fraction was collected, dried, and then the solvent was distilled off under reduced pressure to obtain 4 g of compound BO. The melting point and infrared absorption spectrum of the obtained compound were as follows.

Compound A Melting point: 268-271°0 Infrared absorption spectrum (all-'') Y: a', : 3400 (by,), 1739
Compound B Melting point, 216-220"0 infrared absorption spectrum (φ-") KBr: 3350 (br-) 'mayo Formulation example (1) Tablet (11) Compound A 100mg mannitol 35mg microcrystalline cellulose
26mg10 Ital Silica
13+ng tarri
12mg polyvinylpyrrolidone 10mg stearin M magnesium
4 hours 0) Granule Compound B 100 mg starch 30 hours lactose sugar
32mg crystalline cellulose 42mg polyvinyl alcohol 6mg [3] Injection

Claims (1)

[Scope of Claims] 1) An interferon inducer containing a compound represented by general formula (1): as an active ingredient.