JPH01216922A - Antiviral pharmocological composition containing hipericine and pseudohipericine - Google Patents

Abstract

PURPOSE: To obtain a pharmacological composition for an antiviral agent capable of controlling the reproduction of viruses and destroying them, containing hypericin or pseudohypericin. CONSTITUTION: This composition contains hypericin or pseudohypericin isolated from a perennial plant as an active ingredient. The composition is mixed with an excipient, an auxiliary, etc., and can be pharmaceutically manufactured. The composition can be prepared into a dosage form for oral administration such as tablet, sugar-coated tablet and capsule, rectal administration such as suppository, injection, solution for oral administration or aerosol. A dose is about 5 to 10mg/kg weight. The composition contains 1-5wt.% of hypericin or 2-5wt.% of pseudohypericin in terms of diseases of skin and mucosa. The composition is effective against viruses, especially against vesicular stomatitis virus, influenza virus, simple herpes virus, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of hyvericin and pseudohyperidine as active substances for the production of pharmaceutical compositions effective for inhibiting viral replication and destroying viruses. The pharmaceutical compositions of the present invention are effective for systemic as well as local administration and are essentially non-toxic. It is also effective against varicella stomatitis virus, influenza virus, herpes simplex +5v-i, H8V-2, etc.

The active substance according to the invention is a compound of the perennial plant Hypericomt.
11111 from riqljetrif01iul Turra (or Ilvpericulcrispgi L,).

Until now, only a few compounds have been known to have antiviral activity. One of the most preferred antiviral agents is interferon, currently used for human therapy. The production and purification of human interferon requires very complicated steps, and the availability of interferon is limited. However, the mechanism of action of this antiviral protein has now been largely elucidated, and it is known that interferon exerts its action by inducing an antiviral state in the am (^nti
Viral DrtlqS and rnterrer
on: Its activity and molecular basis, Ed, Yechiel
Becker 1984, 0.

357, Hartinus N1jhoff Pu
blishing Boston).

Piperidine and pseudopiperidine are known, and
hypericu+s triquetrifolium
Turr (or +1vperictil Cr
Piperidine (1), which can be isolated from 1SpUI L, and has the structure shown below, and pseudopipericine (chemically identified as δ). The structure was determined by NMR spectra.These spectra were
It has a signal specific to polycyclic aromatic 1M structure.

These two compounds are associated with vesicular stomatitis virus (VSV).
), influenza virus, herpes simplex H8V-
1 and H8V-2, etc., and shows strong antiviral activity. The toxicity of one of these compounds is
It was found to be low in humans and cultured tissues. Testing of the effects of these compounds on the incorporation of the radioactive substance Hl-uridine into RNA by viruses revealed that these compounds suppress RNA synthesis, inhibit viral replication, and thereby destroy the virus. It turned out that.

In order to exert these effects, piperidine and pseudopiperidine must come into direct contact with the virus. Viral activity was evaluated by exposing the virus to low concentrations of these compounds and then examining how well the cells were protected from cell destruction by the virus. Judging from their action, electrohypericin or pseudopiperidine is preferable for local administration against local infections.
.

These compounds are described in the article by Brockann et al. [It, Brockwann et al., 5anne, Discovery of Piperidine and Pseudopipericine, Chem, Ber, 90, 2480. (,1
957): H, Brockmann.

J, rranssen, o, sp t tlne
r and H, AuQuStirtiak, isolation and structure determination of pseudopiperidine, tetrahedron Let
ters, 1991. (1974)], and the NMR signals of these compounds have also been reported [
Il, Br0Ckllann and 0.5DitZnOr
, pseudohypericinno structure, Tetrahedron
Letters 37, (1975)]. The synthesis of hibericin has also been reported [H, Bro
ckIann, F, Kluge and H, Huxf.
eldt.

Total synthesis of Hibe IJ syn, Checker, 90,
2302 (1957)].

ttypertcum a plant (St, John's
wort) has been reported to exhibit antiviral activity [8,^.

) erbentseva et al., HleriCLII De
Effect of rfOratOl tannins on influenza virus, %1krobio1. Zh, (Ki
ev) 34.768 (1972):,^,,78.
67532d, formerly 5hOnkOVa et al., St. John
Antiviral activity of 's wort and preparations obtained therefrom, Tr, S'ezda Hikrobiol, Ukr.
4th, 222 (1975), C.A.
85.187161Y), however, it has not been reported at all that hypericin and pseudohypericin have antiviral activity.

In addition, it has been reported by Merck Index that hyvericin (1) has tonic and sedative activities on human tissues (Vol. 8, o, e, 5techerEd.
,, 1968. p. 558). It is also used as an antidepressant [Daniel 1. Photodynamic substance Hiberisinnitsuite, formerly ppokrates 20, 52
6 (1949)].

Superintendent 1 blood! 11 Plants were harvested at flowering stage and their leaves were dried at 55°C, cut and milled, and then extracted with acetone. The raw material 1 was put into a Soxhlet extractor and extracted for several hours (5-10 hours) until the color of the extract disappeared. The solution showed red fluorescence. The solvent was distilled off under reduced pressure and the residue (959) was completely dried.

The resulting residue was fractionated using a chromatographic column (0.06 to 0.20 mm) packed with silica gel 60.

A drying process was used to carry out zero-magnetography. That is, 25 g of the remaining residue was dissolved in acetone, added to the same amount of silica gel 60, and the solvent was distilled off while swirling in a rotary evaporator until the solution was homogeneously dried. The resulting mixture was placed on top of the column and first eluted with CH2Cl2 until the solvent reached the bottom of the column. Then CH2Cl2-acetone-MeOH (75:15:
It was eluted with the mixed solvent of 10). When the red color becomes weak,
Reduce the noise of CHC12 and increase the ratio of mixed solvent to 55:1
It was set at 5:10. 250 d of each fraction was collected. Then, it was monitored by irradiating two main red fluorescent spots with ultraviolet light on a thin layer zero mudplate and determining the R value. The same solvent as described above was used as the developing solvent.

The chromatography was completed over approximately 2 days.

The two main components obtained were combined with silica gel 60 (0.04
Further separation and purification was carried out by flash chromatography using a slightly pressurized drying process using the same solvent as above (~0.06 mesh) and the same solvent as described above. The two main components are tohericin (R, value = 0.45. Yield - 0,
199) and pseudohypericin (R, value -0,35,
Yield - 0.73 g). The NMR spectra were previously reported in the literature (Brocksann (1
975) and 1bid).

Bioassay Antiviral activity was assessed by a radioactivity assay that measures the incorporation of the radioactive substance uridine into the viral RAN in infected cells. The cultured cells used in this assay were grown in Eagle's medium. These cells are human culture line 1111F, which was established in our laboratory.
S11 [JJeissenbach, 1
4. Zeevi, T., Landau and H. Revel.
, Identification of translation products of human Nbud interferon mRNA in reticulocyte lysates, Eur.

J. Bioches, 98.1-8 (1979)
The virus used in this assay is vesicular stomatitis virus grown in cultured cells. In this assay, antiviral activity was expressed as a decrease in the amount of labeled substance uptake, ie, inhibition of virus replication.

A simpler and faster assay method that takes into account the toxicity of the test compound to cells is to detect cytopathic effects (CPE) using a microscope.
). According to this method, the effects of antiviral active compounds on cells can be monitored over a long period of time.

The method was performed using a microplate with 96 wells (8X12). To determine the antiviral activity against toxicity of the test compound at the lowest concentration, a series of dilutions of the test compound were added to each well at low concentrations. Dilutions of the test compound were brought into contact with the virus for a short period of incubation (approximately 1 hour) before adding cells to the wells and then examined for cytopathic effects at regular intervals. In addition to the assay method described above, an assay method for measuring suppression of plaque formation by the virus in 41 m culture was also used for evaluation. Therefore, in this assay, the absence of plaque formation indicates that viral replication has been suppressed. This assay was used in the case of influenza virus and herpes simplex.

In the case of herpes simplex virus, the following method was used. That is, diluted solutions of hypericin and pseudohypericin at various concentrations were prepared using PBS (phosphate buffered saline). A fixed amount of virus was added to each dilution and the resulting mixture was incubated at 4°C for several hours. Then 60m
A monolayer of VerO cells in +dish was infected and assayed as follows.

1. Adsorption of virus (in mixture): 0.2 d/plate, 1 hour 1 m 37°C, 2. Overlay: 2% F in methylcellulose or hemlock hemlock
C8 (fetal bovine serum), incubation for 3 days in a humid CO2 incubator (after 1 hour adsorption treatment, virus mixture is not removed); 3. After staining, count the number of plaques.

Results Vesicular stomatitis assay Results of evaluation by CPE method, plant u, crispug+
The crude extract showed activity with Ill of 325 γ/d.

Initial purification showed activity at 85γ/d. At this stage, toxicity to the cells in culture was significantly reduced. A series of purifications as described above yielded two compounds, hibericin and pseudohyperidine, which exhibited activity at a concentration of 5.gamma./- (see FIGS. 1 and 2). FIG. 1 shows the degree of delay in destruction of ■Sv-infected It cells when hyvericin IIm was several gamma per 1. At the optimal 1 degree of 9γ/d, the virus remained viable for up to 140 hours. FIG. 2 shows that cells remained viable in the same incubation medium when the compounds were contacted with the cells (no virus) at the same concentrations as in FIG. 1, respectively.

To perform the radioactivity bioassay, the test compound pseudohypericin was prepared by contacting the virus with various concentrations of pseudohypericin and then adding the resulting mixture to 111 cells of culture medium. The results are shown in Figures 3 and 4. A decrease in the amount of uridine uptake from day 3 indicating inhibition of RNA synthesis was observed not only in vSv-infected cells but also in uninfected cells used as controls. A clear dose-dependent response was seen for different dilution concentrations. Also, the two virus dilutions used for virus infection were 50%
X10'l), f, v, /TSIA/, 100 x 106p
, f, v,/well (p, f, v, -77-') forming unit).

Effects on RNA are key to a compound's activity against viruses and explain its action on cells.

Assay Using Influenza Virus For influenza virus, the plaque bioassay method described above was performed. Regarding the mixture consisting of hypericin and pseudohypericin, influenza virus A/
Activity against Port-Cha 1sers/73 was tested. The results are summarized in Table 1. 0.15μ
96% inhibition υ1 was obtained at low f/d III, and 100% inhibition was obtained at all higher concentrations. Even at high concentrations, cells remained normal throughout the experimental period. In control experiments, the same mixture at the same concentration was contacted with tissue culture cells (non-sensitized). The cells grew normally and formed a monolayer.

Table 1 N (μ9/d) 1.5 0.75 0.
37 0.19 0.09 Number of control plaques 0 0 0 8
42 200 Suppression rate (X) 100
In the 100 10G 9679 test,
The concentration of the stock solution was determined from a concentration calibration curve prepared using 280 n- ultraviolet light. Saturated solutions of hibericin and busonoid hibericin were each filtered, and the transmittance was plotted on a curve to determine the concentration. This allowed us to obtain extremely accurate values.

The results obtained for each dilution are shown in Table 2. Second
As is clear from the table, hibericin has a concentration of 2.5 na/d.
(10-9or), pseudohypericin is 472n
It showed activity at a/at.

Table 2: Number of plaques per plate (%) 25 nQ/ll 0 10G2.5
ng/d O1000, 25no/a
te Final concentration of 11γ69 pseudohibericin 472 nO/d 1 10
G47.2no/d 14gg
(-15)4.72 no/d
1G000 As is clear from the above, the pharmaceutical composition of the present invention exhibits efficacy even when containing a small amount of the active ingredient hypericin or pseudohypericin. In general, fII of about 5 to about 10111/K1 body intoxication! Administered within the surrounding area.

Various dosage forms can be used in the present invention. For diseases of the skin and mucous membranes, hibericin or pseudohyveridine can be advantageously administered in topical forms such as lotions and ointments using polyethylene glycol.

These forms contain 1-5% by weight of pipericin and 2-5% by weight of pseudopericin. The active ingredients hypericin and pseudohypericin can also be administered together in any proportion.

In addition to local administration, systemic administration can also be carried out.

The dose is sufficient to be effective against the virus when it comes into contact with the virus anywhere in the system. Although some experimentation is required to determine the appropriate dosage depending on the route of administration (oral, parenteral, aerosol, etc.) and the type of virus, one skilled in the art is knowledgeable about the activity of the active ingredients of the present invention. undue experimentation will not be necessary to determine dosage. Determination of an effective amount is easy for those skilled in the art.

In addition to hypericin or pseudohypericin, the pharmaceutical compositions of the present invention may contain suitable pharmaceutically acceptable carriers such as excipients, adjuvants, etc., which facilitate the formulation of the active ingredient. . Oral preparations such as tablets, sugar-coated tablets, and capsules; preparations for rectal administration such as for cattle; solutions for injection or oral administration; preparations for administration by aerosol, etc.
It contains 1 to about 99% active ingredient, preferably about 25 to 85%, as well as excipients.

The pharmaceutical formulation of the present invention can be formed by methods known per se, such as mixing methods, granulation methods, sugar-coating methods, and the like. Oral preparations are prepared by mixing the active ingredient with a solid excipient, preferably with the addition of suitable auxiliaries, and then crushing the mixture, then granulating it and, if necessary, forming the core part of a tablet or sugar-coated tablet. You can get it by doing

Preferred excipients are sugars such as lactose and sucrose, fillers such as mannitol, sorbitol, cellulose II, tricalcium phosphate, calcium hydrogen phosphate, or corn starch, wheat starch, rice starch, potato. Binding agents include starch, gelatin, tragacanth, methylcellulose, human oxypropyropylmethylcellulose, carboxymethylcellulose, polyvinylvinylcellulose, and the like. If necessary, starch as mentioned above,
Carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, alginic acid, salts such as sodium alginate, etc. may be added as disintegrants. Auxiliary agents include lubricants and flow control agents, such as silica, talc, stearic acid, magnesium stearate, calcium stearate, polyethylene glycol, and the like. The core of the dragee is optionally provided with a coating that is resistant to gastric juices. For this purpose, concentrated sugar solutions can be used, preferably containing gum arabic, talc, polyvinylpyrrolidone, pyriethylene glycol, titanium dioxide, lacquer solutions, suitable organic solvents, solvent mixtures and the like. To form a film that is resistant to gastric juices, solutions of Cellulose Im products with acetyl cellulose phthalate, hydroxypropyl methyl cellulose phthalate, etc. may be used. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification of the preparation or to characterize the active ingredient.

Other formulations for oral administration include Bushfit capsules consisting of gelatin and a plasticizer such as glycerol or sorbitol. Bushfit capsules contain the active ingredient in the form of granules, such granules containing fillers such as lactose, binders such as starches, lubricants such as talc, magnesium stearate, and preferably stabilizers. It is a mixture. In soft capsules, the active ingredients are dissolved or suspended in liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. A stabilizer may be added to these.

Formulations for rectal administration include, for example, bovine formulations, which consist of the active ingredient and a bovine base. Preferred cattle bases include, for example, synthetic or natural triglycerides, paraffin hydrocarbons, polyethylene glycols, higher alkanols, and the like. In particular, gelatin capsules for rectal administration consisting of the active ingredient and a carrier may be used. Examples of bases include liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.

Preparations for parenteral administration include, for example, aqueous solutions of the active ingredient, such as water-soluble salts. Additionally, suspensions of the active ingredients may be administered as oily injection suspensions.

Suitable organic solvents or carriers include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyloleate triglyceride, and the like. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, and the like. Preferably, the suspending agent contains stabilizers.

Preferred forms of administration are those by aerosol spray, for systemic administration or for mucosal, mucosal,
It is used for local administration such as in the lungs. All conventional aerosol excipients and propellants can be used for this purpose. For aerosol administration, the active ingredients of the invention can be packaged in a pressurized container equipped with a suitable pulp system and actuator. Preferably, a meter pulp is used with a pulp chamber into which the active ingredient is recharged after each dose. All of these are known to those skilled in the art.

The Mizuyo M book described human treatment, but the present invention
This includes the treatment of all animals in which viral infections occur. Among these, humans are most preferred, but the present invention is not limited thereto.

All animal treatments that benefit from the invention include:
It is within the scope of the present invention.

[Brief explanation of the drawing]

FIG. 1 shows the degree of delay in destruction of vSv-infected cells in the presence of various concentrations of hypericin. FIG. 2 shows the degree of delay in destruction of uninfected m-vesicles in the presence of various concentrations of hyvericin. Figure 3 shows the amount of uridine taken up in 3 days during RNA synthesis, where -he is vSv (50λ/ae).
infected cells, -low is uninfected [I! Ii! The measurements were carried out using different concentrations of pseudohypericin. Figure 4 shows the amount of uridine taken up in 3 days during RNA synthesis, -Δ- is vSv (100λ/d)
Infected cells, -mouth- are uninfected mlI! The measurements were carried out using different concentrations of pseudohypericin.

Claims (4)

[Claims] (1) A pharmaceutical composition for antiviral agents containing an effective amount of hypericin or pseudohypericin as an active ingredient. 2. The pharmaceutical composition of claim 1, comprising about 5 to about 10 mg of hypericin or pseudohypericin and a pharmaceutically acceptable excipient per unit dosage form. (3) A pharmaceutical composition containing about 1% to about 5% by weight of hypericin or about 2% to about 5% by weight of pseudohypericin as an active ingredient, which is effective against viral diseases of the skin, mucous membranes or tissues. A polyethylene glycol pharmaceutical composition for external use against. (4) Claims for use against vesicular stomatitis virus, influenza virus or herpes simplex virus (
The pharmaceutical composition according to any one of 1) to (3).