JP2502659B2 - Pharmaceutical composition for antiviral agent containing hypericin or pseudohypericin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of hypericin and pseudohypericin as active substances for the manufacture of a pharmaceutical composition effective in inhibiting viral replication and destroying the virus. The pharmaceutical composition of the present invention is effective in systemic administration as well as local administration, and is essentially nontoxic. It is also effective against vesicular stomatitis virus, influenza virus, herpes simplex HSV-1 and HSV-2.

The active substance according to the invention is a perennial plant Hypericom triquetr.
It can be isolated from ifolium Turra (or Hypericum crispum L.).

Only a few compounds have hitherto been known as compounds having antiviral activity. One of the most preferred antiviral agents is interferon, which is currently used for human therapy. The production and purification of human interferon requires a very complicated process, and the amount available is limited. However, the mechanism of action of this antiviral protein is currently well understood, and it is known that interferon exerts its action by inducing an antiviral state in cells (Antivi
al Druqs and Interferon: Its activity and molecular basis, Ed.Y.
echiel Becker 1984, p.357, Martinus Nijhoff Publishi
ng Boston).

Hypericin and pseudohypericin are known and
Hypericum triquetrifolium Turr (or Hypericum
(known as crispum L.) and has been chemically identified as hypericin (1) having the following structure and pseudopipericin (2).
These compounds were isolated in pure form and structurally determined by NMR spectroscopy. These spectra have signals characteristic of polycyclic aromatic structures.

These two compounds are vesicular stomatitis virus (VS
V), influenza virus, herpes simplex HSV-1 and HSV-2, and the like, showing reliable and strong antiviral activity. The toxicity of these compounds was found to be low in humans and cultured tissues. The effects of these compounds on the viral uptake of the radioactive substance ³ H-uridine into RNA were tested, and it was found that these compounds suppress RNA synthesis and inhibit viral replication, resulting in virus destruction. Ivy.

In order to exert these effects, hypericin and pseudohypericin must be in direct contact with the virus.
Viral activity was assessed by pre-contacting the virus with low concentrations of these compounds and then examining how much the cells protected from cell destruction by the virus.

Given the effect of hypericin or pseudohypericin, topical administration for local infection is preferred.

These compounds are described in Brockmann et al. [H. Brockmann and W. Sanne, Discovery of pipericin and pseudopipericin, Chem. Ber. 90, 2480, (1957); H. Br.
ockmann, U. Frassen, D. Spitzner and H. Augustiniak, Isolation and structure determination of pseudohypericin, Tetrahedron Letter
s, 1991, (1974)], and NMR signals of these compounds have also been reported [H. Brockmann and D. Spitzner, Structure of pseudohypericin, Tetrahedron Letters 37, (1
975)]. The synthesis of hypericin has also been reported [H. Brockmann, F. Kluge and H. Muxfeldt, Total Synthesis of Hypericin, Chem. Ber. 90, 2302 (1957)].

It has been reported that a plant of the genus Hypericum (St. John's wort) exhibits antiviral activity [NADerbentseva
Et al., Effects of tannins of Hypericum perforatom against influenza virus, Mikrobiol.Zh. (Kiev) 34,768.
(1972) CA, 78,67532d; Mishenkova et al., ST. John's wor.
Antiviral activity of t and preparations obtained therefrom, Tr. S'ezda M
ikrobiol, Ukr.4th.222 (1975), CA, 85,197161Y),
However, it has never been reported that hypericin and pseudohypericin have antiviral activity.

Further, hypericin (1) has been reported to Merck Index that it has tonic and sedative activity on human tissues (Vol. 8, OG, Stecher Ed., 1968, p. 55).
8). It is also used as an antidepressant [Daniel
K., On the photodynamic substance hypericin, Hippokrates 20,
526 (1949)].

Chemical Preparation Method The plants were taken in the flowering stage, the leaves were dried at 55 ° C., cut and milled and then extracted with acetone. Raw material 1Kg
Was placed in a Soxhlet extractor and extracted for several hours (5-10 hours) until the color of the extract liquid disappeared. The solution showed red fluorescence. The solvent was distilled off under reduced pressure, and the residue (95 g) was completely dried. The obtained residue was fractionated by a chromatography column (0.06 to 0.20 mm) packed with silica gel 60. Chromatography was performed by a drying process. Immediately, 25 g of the residue was dissolved in acetone, the same amount of silica gel 60 was added, and the solvent was distilled off while vortexing with a rotary evaporator until it was uniformly dried. The resulting mixture was placed on top of the column, eluting first with CH ₂ Cl ₂ until the solvent reached the bottom of the column. Then CH ₂ Cl ₂ - acetone -MeOH (75:15:10)
It eluted with the mixed solvent of. When the red color weakens, CH ₂ Cl ₂
Was reduced to a mixed solvent ratio of 55:15:10. 250 ml of each fraction was collected. Then, the two main red fluorescent spots were irradiated with ultraviolet rays on a thin-layer chromatographic plate to monitor the R _f value by monitoring. The same solvent as described above was used as the developing solvent. Chromatography was completed in about 2 days. The two main components obtained were subjected to flash chromatography using silica gel 60 (0.04 to 0.06 mesh) and the same solvent as described above under a slight pressure and a drying process for further separation and purification. The two main components are hypericin (R _f value = 0.4
5, yield = 0.19 g) and pseudohypericin (R _f value = 0.35,
Yield = 0.73 g). The NMR spectrum was the same as the one already reported (Brockmann (1975), ibid).

Bioassay method antiviral activity, have been conducted under evaluation in radioactive Atsusei method of measuring radioactive substances ³ H- uridine incorporation into virus RAN in infected cells. The cultured cells used in this assay were grown in Eagle medium. This cell is derived from our laboratory-established human cultured fibroblast FS11 [T. Weissenbach, M. Zeevi, T. Landau and M. Re.
vel, human fibroblast interferon in reticulocyte lysates.
Identification of translation product of mRNA, Eur. J. Biochem., 98, 1-8 (1
979)]. The virus used in this assay is vesicular stomatitis virus grown in cultured cells. In this assay, antiviral activity was represented by a decrease in the amount of labeled substance incorporated, that is, inhibition of virus replication. A simpler and faster assay, which takes into account the toxicity of the test compound to cells, is to monitor the cytopathic effect (CPE) under a microscope. According to this method, the effect of the antivirally active compound on cells can be traced over a long period of time. This 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, serial dilutions of the test compound were added to each well at low concentration. After contacting the diluted solution of the test compound with the virus, the cells were incubated for a short time (about 1 hour) before adding cells to the wells, and then the cytopathic effect was examined at regular intervals. In addition to the above-mentioned assay method, evaluation was also performed using the assay method which measures plaque control inhibition by virus in monolayer culture. Therefore, this assay shows that viral replication was suppressed when plaque formation did not occur. This assay was used in the case of influenza virus and herpes simplex.

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

1. Adsorption of virus (in mixture); 0.2 ml / plate, 1 hour at 37 ° C 2. Overlay; 2% FCS in methylcellulose or agarose
(Fetal bovine serum), incubated in a humidified CO ₂ incubator for 3 days (after 1 hour adsorption treatment, the virus mixture was not removed), 3. After staining, count the number of plaques.

Results Evaluation of vesicular stomatitis virus by Atssei CPE method showed that crude extract of plant H. crispum
It exhibited activity at a concentration of 325γ / ml. 85γ by first purification
The activity was shown at / ml. At this stage, toxicity to cells in culture was significantly reduced. By the series of purification as described above, two compounds, hypericin and pseudohypericin, which are active at a concentration of 5γ / ml, were obtained (FIGS. 1 and 2).
See figure). FIG. 1 shows the delay in destruction of VSV-infected cells when the concentration of hypericin was several gamma per ml. At the optimal concentration of 9γ / ml, the virus remained viable for up to 140 hours. FIG. 2 shows that when the compound was contacted with the cells at the same concentration as in FIG. 1 (no virus), the cells continued to survive during the same incubation period.

In order to carry out a radioactive bioassay, the test compound pseudohypericin was pre-contacted with the virus at various concentrations of pseudohypericin and then the resulting mixture was added to the cultured cells. The results are shown in FIGS. 3 and 4. A decrease in the amount of ³ H-uridine uptake indicating RNA synthesis inhibition is observed not only in VSV-infected cells but also in non-infected cells used as a control. A clear dose-dependent response was seen for different dilution concentrations. The two virus dilutions used to carry out the virus infection were 50 x 10 ⁶ p.
Reproducible results were obtained for fv / well, 100 × 10 ⁶ pfv / well (pfv = plaque forming units).
The effect on RNA is key to the activity of the compound against viruses and explains its effect on cells.

Atsushi using influenza virus For the influenza virus, the plaque bioassay method described above was carried out. A mixture consisting of hypericin and pseudohypericin was tested for activity against influenza virus A / Port-Chalmers / 73. The results are summarized in Table 1. 96% inhibition was obtained at a low concentration of 0.15 μg / ml, 100
% Inhibition was obtained. Even at high concentrations, the cells remained normal throughout the experiment. In control experiments, the same mixture at the same concentration was contacted with tissue culture cells (non-sensitized). The cells grew normally and formed monolayer cells.

Assay using herpes simplex virus strain KOS (HSV-1) In this test, the concentration of the stock solution was determined from the concentration calibration curve prepared using ultraviolet rays of 280 nm. Saturated solutions of hypericin and pseudonoid hypericin were filtered, respectively, and the transmittance was plotted on a curve to determine the concentration.
With this, an extremely accurate value was obtained.

The results obtained for each dilution are shown in Table 2. As is clear from Table 2, hypericin was found to have a concentration of 2.5 ng / ml (10
^-9 gr) and pseudohypericin showed activity at 472 ng / ml.

As is apparent from the above, the pharmaceutical composition of the present invention is effective even when it contains a small amount of the active ingredient hypericin or pseudohypericin. Generally, about 5 to about 10 mg / K
It is administered in the range of g body weight.

Various dosage forms can be utilized in the present invention. For skin and mucous membrane diseases, hypericin or pseudohypericin can be advantageously administered in a topical administration form such as a lotion using polyethylene glycol or an ointment. In the case of these forms, 1-5% by weight of hypericin and 2-5% by weight of pseudohypericin are contained. The active ingredients hypericin and pseudohypericin can also be administered together in any proportion.

In addition to local administration, systemic administration can also be performed. The dose is sufficient to effectively act against the virus in contact with the virus in any of the strains. Although some experimentation is required to determine an appropriate dose depending on the administration route (oral, parenteral, aerosol, etc.) and the type of virus, those skilled in the art who are knowledgeable about the activity of the active ingredient of the present invention Will not require undue experimentation to determine dosage. Determination of an effective amount is straightforward to those of ordinary skill in the art.

In addition to hypericin or pseudohypericin, the pharmaceutical compositions of the present invention may include suitable pharmaceutically acceptable carriers such as excipients, adjuvants and the like, which facilitate the formulation of the active ingredient. . Oral preparations such as tablets, dragees and capsules; rectal preparations such as suppositories; injection or oral solution preparations; aerosol dosage preparations, etc.
Excipients are included with 1 to about 99%, preferably about 25 to 85% of the active ingredient.

The pharmaceutical preparation of the present invention can be formed by a method known per se, for example, a mixing method, a granulating method, a sugar-coated tableting method and the like. The oral preparation is preferably prepared by mixing the active ingredient with a solid excipient, preferably by adding a suitable auxiliary agent, pulverizing the mixture, and then granulating the mixture, if necessary, as a core portion of a tablet or a dragee. It can be obtained by

Preferred excipients are lactose, sugars such as sucrose, mannitol, sorbitol, cellulose preparations, fillers such as tricalcium phosphate, calcium hydrogen phosphate, or corn starch, congi starch,
Rice starch, potato starch, gelatin, tragacanth,
It is a binder such as methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, and polyvinylpyrrolidone. If desired, the above-mentioned starch, carboxymethyl starch, cross-linked polypinylpyrrolidone, agar, salts of alginic acid, sodium alginate and the like may be added as a disintegrant. As an auxiliary agent,
Lubricants and flow control agents such as silica, talc, stearic acid, magnesium stearate, calcium stearate, polyethylene glycol and the like. The core portion of the sugar-coated tablet is optionally provided with a film resistant to gastric juice. For this purpose, preferably concentrated sugar solutions containing gum arabic, talc, polyvinylpyrrolidone, pyriethylene glycol, titanium dioxide, Rucker solution, suitable organic solvents, solvent mixtures and the like can be used. A solution of a cellulose preparation such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate may be used to form a film that is resistant to gastric juice. Dyestuffs or pigments may be added to the tablets, dragee coatings for identification or to characterize the active ingredient.

Other formulations used for oral administration include push-fit capsules consisting of gelatin and a plasticizer such as glycerol and sorbitol. Pushfit capsules contain the active ingredient in the form of granules, such granules comprising fillers such as lactose, binders such as starch,
Lubricants such as talc and magnesium stearate, and more preferably a mixture of stabilizers and the like. In the case of soft capsules, the active ingredient is dissolved or suspended in a liquid such as fatty oil, liquid paraffin, liquid polyethylene glycol. Stabilizers may be added to these.

Formulations used for rectal administration include, for example, suppositories, which consist of the active ingredient and a suppository base. Preferred suppository bases include, for example, synthetic or natural triglycerides, paraffin hydrocarbons, polyethylene glycols and higher alkanols. In particular, a gelatin rectal capsule containing the active ingredient and a base can be used. Examples of the base include liquid triglyceride, polyethylene glycol, paraffin hydrocarbon and the like.

Formulations for parenteral administration include aqueous solutions of active ingredients such as water-soluble salts. In addition to these, suspensions of the active ingredient can be administered as oily injection suspensions. Suitable fat-soluble solvents or carriers include fatty oils such as sesame oil and synthetic fatty acid esters such as ethyl oleate triglyceride. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, and the like. Preferably, the suspension contains a stabilizer.

A preferred dosage form is an aerosol spray dosage form, which is used as a systemic administration of these or as a local administration of mucous membranes, lungs and the like. Both conventional aerosol excipients and propellants can be used for this purpose. For aerosol administration, the active ingredients of the invention may be packaged in a pressurized container equipped with a suitable valve system and actuator. It is preferable to use a meter valve with the valve chamber, in which the active ingredient is recharged after each administration. All of these are known to those skilled in the art.

Although the specification describes treatment of humans, the present invention includes treatment of all animals in which a viral infection occurs. Of these, humans are the most preferable, but the present invention is not limited thereto. All animal treatments which would benefit from the present invention are within the scope of the present invention.

[Brief description of drawings]

FIG. 1 shows the delay of destruction of VSV-infected cells in the presence of various concentrations of hypericin. FIG. 2 shows the delay of destruction of uninfected cells in the presence of various concentrations of hypericin. FIG. 3 shows the amount of ³ H-uridine incorporated during RNA synthesis, where −Δ− is VSV (50 λ / ml) infected cells, −
□ -indicates non-infected cells, which were measured using different concentrations of pseudohypericin. FIG. 4 shows the amount of ³ H-uridine incorporated during RNA synthesis, where −Δ− is VSV (100 λ / ml) infected cells,
-□ -indicates non-infected cells, which were measured using different concentrations of pseudohypericin.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Dahlia Rottman Rehoboth, Israel Alcalai Street 6 (72) Inventor Vincent Bande Verde Belgium, Brussels, Louis. Herebout, 27 (56) References Mikrobiol. Zh. Vol. 32 No. 4 (1970) p. 494-497

Claims (4)

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