JPS6120525B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an injectable antiviral agent. The present invention was achieved from research carried out at or under the direction of the Institut Pasteur, and its object is a complex heteropolyanion compound containing a tungsten atom combined with an antimony atom. In particular, the present invention relates to such compounds and their use as medicaments for the treatment and prophylaxis of various viral infections. The compound according to the present invention is characterized in that the central atom is an antimony atom, and the group of ligands surrounding the central atom contains a heteropolyanion, which is a tungsten ion that is bonded to each other using oxygen atoms as bridges. For a review of the prior art in the field of heteropolyanions, see P. Souchay. “Ions
“mineraux condenses” (Masson et Cie, Edit)
Paris (1969)) and “Polyanions et
Gauthier―Villars, Edit―Paris
(1963)). However, these books do not contain any description of a method for producing a heteropolyanion in which the central atom is an antimony atom. The chemical definition of 2-antimonio-5-tungstate () and its production method are published in CRAcademic des Sciences, Paris, No. 27.
Volume (January 17, 1972 issue), pages 209-212. However, very recently, infrared and Raman spectra have shown that the above compound has a formula different from the one assigned to it, and the heteropolyanion has the empirical formula: [NaSb ₉ W ₂₁ O ₈₆ ]. It was confirmed that The heteropolyanion compound according to the present invention is both a complex compound and an acid salt. Such compounds can be present in neat form or in the form of ammonium salts or salts of metals, especially alkali metals and alkaline earth metals. As mentioned above, the central atom, the antimony atom,
It is an inorganic ion obtained by condensing tungsten atoms W around Sb. Therefore, the heteropolyanion compound according to the present invention is antimonio-dungsthosodate.
-tungsho- sodate). There are many hydrated forms of such heteropolyanions. All hydrated forms of antimoniotungstate are included within the scope of this invention. The present invention also relates to antiviral agents containing the active agent HPA23 compound in combination with a pharmaceutically acceptable carrier. According to a special form of provision, the antiviral agent of the present invention is injectable, eg, injected into humans or animals as an aqueous solution (saline solution) with a pH around neutrality. In another aspect of the invention, the invention relates to an antiviral agent containing a therapeutically effective amount of HPA in combination with interferon. It should be made clear that previous studies have shown that HPA23 itself does not act as an interferon inducer. In the present invention, it was confirmed that there is a synergistic effect between HPA23 and interferon. This synergy was demonstrated by administering 50 mg/Kgi.p. of HPA23 and 75,000 units of interferon ip or iv to a single mouse 1 and 6 hours, respectively, before subcutaneous inoculation with ₁₀₀ DL 50 encephalomyocarditis (EMC) virus. It became clear from the results. Especially showing band phenomenon
It was found that the effect of HPA against the VR129 strain of EMC virus was even greater. Therefore HPA23
can alter the production of interferons by the action induced by certain viruses, such as the EMC virus. Heteropolyanions are characterized by an affiliation series determined by the ratio ν=W/Sb. In the "11 series", the ratio ν=11. The production method and physicochemical properties of 11-tungstoantimoniate () and 11-tungsto bismusate () are described in P.
Souchay, Vol. 271, pp. 1337-1340 (November 30, 1970). In particular, the present invention has nine antimony atoms Sb in the center.
The purpose is to utilize 9-antimonio-21-tungsto-sodate, which is a heteropolyanion compound in which 21 tungsten atoms are located around these atoms and the characteristic ratio ν is equal to about 2.4. The present invention also includes all possible isomers of the above heteropolyanions. The present invention also includes the above-mentioned heteropolyanion compound and a mixture obtained by varying the pH and converting the above-mentioned antimoniotungstate. The novel heteropolyanion compound according to the present invention, that is, 9-antimonio-21-tungsto-sodate, is stable at a pH of 6.5 to 7, that is, near neutral pH. Such properties are advantageous when the above compound is used as a drug as described below. It is of interest for the purposes of the present invention that the heteropolyanion compounds are present in the form of pharmaceutically acceptable metal salts. Such salts are preferably selected from salts formed with alkali or alkaline earth metals and ammonium salts.
As such salts, sodium, potassium and ammonium salts are used. Heteropolyanion compounds can be used as such or in acid form, if desired. 9-antimonio in the form of ammonium salt
To obtain 21-Tungst-Sodate, Sb〓
The ion-containing aqueous solution is heated and reacted with 1M sodium tungstate solution. The reaction medium is maintained approximately neutral by adding a sufficient amount of concentrated ammonium hydroxide to render the reaction medium colorless. This results in the formation of the required ammonium salt precipitate. This is recovered by filtration and then processed in a conventional manner. The reaction temperature is below the boiling temperature of the reaction medium, for example about 80°C. Sb〓Ion-containing aqueous solution is SbCl ₃
Advantageously, it is prepared by dissolving it in a saturated solution of NH ₄ Cl. The final compound is ammonium 9-antimonio-
-21-Tungst-Sodate. Its pH in aqueous solution is approximately 6.7. This salt exists in the form of a hydrate of the following formula: [NaSb ₉ W ₂₁ O ₈₆ ](NH ₄ ) _{18.8H 2} O. In aqueous solution this salt exists as a quartet. This last compound is designated by the symbol TA or HPA23. Antimoniotungstosodate according to the invention is active in the prophylactic and therapeutic treatment of viral diseases in humans and animals. French patents 7045843 and 7143444 describe the use of complex compounds of tungsten-containing heteropolyanions as antiviral agents. However, although the compounds described in these two French patents are useful drugs against diseases caused by certain viruses, it is clear that the activity of these compounds against leukemia and sarcoma viruses in vivo is insufficient. is being used. A particularly effective known heteropolyanion is sodium tungstosilicate. This compound exhibits a direct virucidal effect, immobilizing the virus and solubilizing the viral material. Sodium tungstosilicate therefore acts both by pre-treating the cells and during virus adsorption. Sodium tungstosilicate acts on cell membranes. In the present invention, quite surprisingly, 9-antimonio--21
- Confirmed that tungst-sodate (TA) was able to protect mice from leukemia induced by Friend's virus and the plasmavariant described by CHIRIGOS. When this compound is injected on the day of virus inoculation, a complete protective effect with no observed leukemia or A partial protective effect can be achieved. Mice can be made immune to Friend's leukemia virus by co-injecting the virus and TA. When treatment starts 307 days after virus inoculation, 4
In two of the experiments, the mean weight of the spleen was significantly reduced after treatment with TA, and the increase in survival time was also reduced. TA can also delay the appearance of tumors in neonatal mice inoculated with M-MSV virus. TA does not induce interfaerons and has no effect on graft leukemia. The action of TA does not require direct interaction between the virus and this compound. The details of the pharmacological test are explained below. The unique properties of TA were completely unexpected even by those skilled in the art. Experiments in cell culture Ammonium antimonio tungsten sodate has no effect either in the pretreatment or during the virus adsorption period. Antimonio tungst sodate, as seen by electron microscopy (the tungsten part of the heteropolyanion does not conduct electrons), or labeled antimonio tungsten sodate
When using tungstate, it permeates the cell, as seen when recovered in some cellular parts, particularly the nucleus. This compound is effective for the first 24 hours after virus adsorption.
It works for hours, but also later, between hours 48 and 72. More rigorous testing revealed that antimonio tungstosodate's effects occur even between 69 and 72 hours. Antimonio Tungst Sodate is a desoxyribonucleic acid-polymerase (RNA) derived from the ribonucleic acid of sarcomatogenic and leukemogenic viruses (oncorna virus).
It significantly suppresses the enzymatic activity of dependent DNA-polymerase. The 50% inhibitory dose of antimonio tungst sodate is 1.25 μg/ml of the reaction mixture. The enzymatic reaction is stopped immediately upon addition of antimonio tungstosodate. The inhibitory effect of 9-antimonio-21-tungst-sodate is of the competitive type. This suppressive effect can be overcome by adding hybrid rA-oligo dT, but poly-rA
It cannot be overcome by oligo-dT alone or by the addition of viral enzymes. Antimonio tungst sodate is not immobilized in the rAdT form, nor in proteins or nucleic acids. In Vivo - Animal Studies - 9-antimonio-21-tungst-sodate reduces the increase in splenomegaly induced by Friend's virus and Chirigos'"plasmavariant". It also delays the appearance of Moloney virus-induced tumors in neonatal mice. Its activity is the same or superior to interfaeron, interferon inducers, or other antiviral compounds used under comparable experimental conditions. Experiments showed that this compound was not an inducer of interferon. This compound is active against three diseases: erythroblastic leukemia, undifferentiated leukemia, and sarcoma, and its effects are not specific to target cells. Its activity does not require direct contact with the virus, and the possibility of inhibition of virus transport from the peritoneal cavity to the blood is eliminated by the inhibitory effect observed when the virus is inoculated intravenously. The present invention also provides a method for treating or preventing a viral infection in a mammal, which comprises administering by injection a clinically effective amount of a sterile solution of 9-antimonio-21-tungsto-sodate. The invention will be illustrated with reference to the following examples. Example [Manufacture of NaSb ₉ W ₂₁ O ₈₆ (NH ₄ ) ₁₈ Sb ion-containing aqueous solution obtained by dissolving 11.4 g of SbCl ₃ in 50 ml of saturated NH ₄ Cl solution
Added to 125 ml of 1M aqueous sodium tungstate solution heated to °C. Just before the addition of the Sb ion-containing solution was complete, a sufficient amount of concentrated ammonium hydroxide to render the reaction medium colorless was added. The precipitated ammonium salt of the heteropolyanion was separated off, washed with dilute NH ₄ Cl solution and finally crystallized from distilled water. The compound produced was pure ammonium 9-antimonio-21-tungsto-sodate (hereinafter referred to as HPA23). This compound existed in the form of a tetramer in aqueous solution. This aqueous solution had a pH of around 6.7. This compound is
It was stable in an aqueous solution with a pH of around 7. In its natural state, this compound existed in the form of a white powder that was easily soluble in water. The crystallized product remained stable at room temperature. It was dissolved in physiological saline (0.9% NaCl aqueous solution) before use for pharmacological testing. FIG. 1 is an infrared spectrum of the product in potassium chloride solution. Figure 2 is the Raman spectrum of the product.
At PH 7.5 in a buffer containing 0.5 M Tris and 0.5 M NaCl, the product exhibits two striking reductive boularograms at potentials of -1.11 volts and -1.22 volts with respect to the saturated calomel electrode. Indicated. To obtain ammonium 9-antimonio-21-tungst-sodate labeled with antimony 125, the following operations were performed. : 2ml labeled antimony chloride (0.53mCi/
ml) containing saturated ammonium chloride ( _NH4Cl )
The solution was added to 3 ml of sodium tungstate solution (0.33 g/ml) at an elevated temperature of about 60°C. Labeled 9 - Antimonio - 21 - Tungst -
The ammonium salt of sodate precipitated during the manufacturing process and continued to precipitate during the cooling process. Separate the precipitate,
Washed with NH ₄ Cl solution and then recrystallized with water. It was then vacuum dried over phosphoric anhydride. Effect of the heteropolyanion HPA23 on Friend's leukemia We conducted tests on mice infected with Friend's leukemia. They injected a defined amount of the virus that causes the disease, and then recorded the death of all mice, usually between 30 and 40 days. In contrast, per mouse on day 2 from the day of virus injection.
0.5 mg of product HPA23 was injected. The results are shown in the table below.

Table 1 The results shown in Table 1 show that a single injection of the virus post-injection product HPA23 significantly reduces splenomegaly. This indicates regression of leukemia. Toxicity test of the product HPA23 Toxicity tests of the product "HPA23" were carried out on the following four animals: tricolor guinea pig, white New Zealand rabbit, beagle, dog, mouse. The product HPA23 was administered in the form of a physiological saline solution. In the case of guinea pigs, the experiment lasted for 42 days, during which time the product HPA23
was administered subcutaneously and intravenously in increasing doses between 2 and 15 mg/Kg/day. In the case of rabbits, the experiment continued for 42 days. The product HPA23 was injected intravenously at increasing doses between 2 and 32 mg/Kg/day. In Dogs The product HPA23 was similarly administered intravenously for 21 days in increasing doses between 20 and 100 mg/dog/day. Under the experimental conditions described above, the product "HPA23" was well tolerated by laboratory animals: guinea pigs, rabbits and dogs. However, in the case of guinea pigs, the product HPA23
It should be noted that when administered subcutaneously, such injections were followed by adverse phenomena in the form of local necrosis phenomena. In the case of guinea pigs, intravenous administration was extremely effective. In the case of rabbits, a similar phenomenon of necrosis occurred near the veins. In these three animals, the product HPA23
Overall tolerability was excellent when injected as follows: 24 consecutive days in guinea pigs and rabbits, 21 days in dogs At the end of the experiment, all animals were sacrificed and histological and Organs were examined from an anatomical point of view. No abnormalities were observed, so it could be concluded that the product HPA23 was well tolerated. Changes in body weight and body temperature were tracked for each animal. For dogs, kidney and liver balance sheets were prepared daily. All these factors were normal. Antiviral Potency Testing of Product HPA23 The following tests were carried out: (a) In vitro: (a ₁ ) Inhibition of the formation of infectious foci or plaques by Moloney's sarcomatogenic virus in fetal mouse cilioblasts; (a ₂ ) Inhibition of leukemia-inducing virus response (Tests X and C in Virology, 1970, 42, 1136), (a ₃ ) Inhibition of polymerase polymerization reaction by DNA adjusted by DNA [Nature, 1970,
225, 1209), (b) In vivo: -About Friend's leukemia; -About leukemia caused by inoculation with Kyrigos'"plasmavariant"; -About tumor formation by Moloney's sarcoma virus in newborn mice; Leukemogenicity and disease development were investigated using two different methods: - The spleens of the animals were removed 21 days after virus inoculation;
Methods for comparing spleen weights of treated and untreated animals; - Methods for determining survival time of treated animals compared to untreated animals; In vitro inhibitory properties of the product HPA23 Foci of transformation or Inhibition of spot formation: The average dose (ID ₅₀ ) showing a 50% inhibition effect calculated by the method of Reed and Muench was 15 μg/ml. a. No direct virus eradication effect was observed against the virus. In fact, when increasing doses of virus and antimoniotungstate (TA) were contacted for 1 hour at 0° C., no decrease in titer was observed, but activity decreased (see Table 2).

[Table] b Using antimoniotungstate 125 _sb in the virus, contact the virus with the labeled antimoniotungstate,
When this mixture was centrifuged through a density gradient (5-55% sucrose), the virus reached equilibrium (1.14-1.15 g/ml) and did not become significantly radioactive. Therefore, no fixation of the antimoniotung state occurred. c Ammonium antimoniotungstate was not toxic to cells at doses showing an inhibitory effect: cell growth was unaffected by the presence of antimoniotungstate: the same number of cells from treated flasks as from untreated flasks. Cells were collected. As far as the contamination of tritiated thymidine (for DNA synthesis), tritiated thymidine (for RNA synthesis), and seleno-75-methionine (for protein synthesis) was concerned, the distribution was the same. d Antimoniotungstate had no effect during pretreatment or virus absorption. The best protection was obtained when the product HPA23 was added after virus infection. Inhibition of syncytia formation (test
XC) The average dose showing a 50% inhibitory effect was 16μg/
ml (see experimental examples in Table 3). Suppression of polymerase polymerization reaction by RNA adjusted by DNA (Nature, 1970, 225,
1209) - Experimental results (protocol) by D.Baltimore; - Exogenous reaction: primer oligo
dT, template rA; - ID ₅₀ : 1.4 μg/ml relative to the reaction medium; - Immediate termination of the polymerization reaction from the addition of antimoniotungstate; the inhibitory effect was irreversible: 50% in the enzyme solution
The fact that the reaction rate does not change when cultured with the addition of an amount of heteropolyanion exhibiting an inhibitory effect and when diluted to 1/10 indicates that the inhibitory effect is reversible. Antimoniotungstate: -inhibits polymerases; -does not react with ribonucleic acids and desoxynucleic acids; -does not act on proteases (pronase, pepsin, trypsin); -does not immobilize on proteins (albumin) and is present in murine oncorna DNA from viral RNA
inhibits the polymerase of

[Table] Inhibitory effect of antimoniotungstate on various viruses of the oncornavirus group a. Rabies virus The following results were obtained from the experiment: - ID ₅₀ : 4 μg/ml; - Does not act directly on the virus; - Dosage used It is non-toxic to cells. b. Other Viruses The product HPA23 is active in vitro against other viruses such as vesicular stomatitis virus (VSV), vexing myocarditis virus, among others. ``In vivo'' inhibitory properties We have already shown that ammonium antimoniotungstate has extremely strong antiviral activity ``in vitro.'' Several tests were conducted to clarify the antiviral activity "in vivo." For this, experimental leukemia in mice,
In Friend's leukemia, leukemia induced by Chirigos's "plasma variant", leukemia was measured from the following points: (1) degree of splenomegaly on day 21 (2) whether antimoniotungstate was administered or not. Table 4 shows the antiviral activity of antimoniotungstate against two types of leukemia. product
As a result of HPA23 injected 30 minutes before virus inoculation,
A marked reduction in splenomegaly was observed. This indicates that the treated mice produced less virus in the spleen or plasma, since significantly less virus was obtained compared to control mice (Table 5). Table 6 reports the results of experiments in which the dosage of antimoniotungstate was varied. From Table 6 μ
It can be seen that a single injection of less than g/mouse provides excellent protection. Table 7 lists the viruses and products to obtain the effect.
This shows that it is not necessary to inject HPA23 in the same way. Only oral administration was ineffective (Experiment 5). Table 8 shows that the protected animals become immune to the virus. That is, even if treated and protected mice are injected with the virus anew, the animals do not become ill. When examining the survival rate of the animals, it was revealed that all the control animals had died on the 30th day. Most treated animals survived until day 85. Remove plasma and spleen from living animals;
After inoculating normal mice, these mice were found to have never had leukemia. The treated animals were therefore cured of the disease. Similarly, the treatment was found to be effective when administered after the onset of illness. Good when the product HPA23 is injected on days 2, 4, 6, and 8, or once again on day 10, or several times over days 14, 16, 18, and 20. protection was provided. Other experiments showed a delay in splenomegaly induced by Moloney's sarcoma in neonatal mice. Finally, the antimoniotung state is 100
% lethal dose of EMC (encephalomyocarditis) virus. Conclusion Ammonium 9-antimonio-21-tungsto-sodate has a broad antiviral spectrum, is active "in vivo" and is well tolerated. The above detailed results clearly demonstrate the effectiveness of tungsten-containing heteropolyanions combined with antimony in the treatment of viral infections. Therefore, TA or HPA23 is a useful antiviral agent and is also active against encephalomyocarditis virus (vesicular stomatitis virus) in vivo. Administration is by injection. A suitable dosage form is a unit dosage form in sterile ampoules containing medicinal quantities. The active compound can be combined with a vehicle suitable for injection. Aqueous solutions or mineral oils can be used as carriers in the veterinary field. For humans, a 0.9% NaCl aqueous solution is preferred as it is suitable for administration at approximately neutral pH. Preferably, the active compound is completely dissolved in such a solution.

[Table]

【table】

[Table] Inoculated intraperitoneally.

【table】

【table】

【table】

Table: In an advantageous method of administration, insoluble calcium phosphate is added to the aqueous solution of the active compound, for example at 5 mg/solution per solution.
Add in the amount of ml. Tests in mice have shown that slow-acting drugs can be produced by such a method. Product HPA23* (radioactive form) 0.9%, NaCl
It was administered intraperitoneally to mice together with calcium phosphate in an aqueous solution. One mouse was sacrificed every day, and the localization of the product HPA23* was investigated. Product HPA23*
The largest portion of was found in the liver (hepatic localization), a relatively significant portion was found in the kidneys, and a relatively minor portion was found in the lungs. Under such conditions, the product
HPA23* remained in the animal's organs for longer than 15 days. In the absence of calcium phosphate, the product HPA23* actually disappeared in 5 hours. In contrast, when calcium phosphate was added, the product HPA23* was found to pass through the organ very gradually, and the amount removed with urine increased over time. The invention is not limited to the above detailed description. The salts of 9-antimonio-21-tungsto-sodate can be utilized as active agents in pharmaceuticals that can be used against a wide range of viral infections for protective or therapeutic treatment. Such drugs can be used in combination with other active agents having antiviral activity. Ammonium 21 as mentioned above
-Dangst-9-antimonyate (AmTA) was found to be active against murine leukemia and sarcoma viruses in vitro and in vivo. AmTA also exhibits broad antiviral activity in vitro and protects mice against murine encephalomyocarditis virus infection. The in vivo activity of AmTA against the cytotoxic effects induced by various RNA viruses was investigated in the culture of monkey kidney and human WI-38 cells grown in BME using 10% calf serum. In the media of monkey kidney and human WI-38 cells, the highest non-cytotoxic AmTA concentration was 125 when added to a confluent monolayer.
When the compound was added to the culture medium at a concentration of 30 μg/ml at the time of inoculation, no inhibition of cell growth was observed. When the culture medium was treated with AmTA concentrations of 15-125 μg/ml approximately 1 hour before virus inoculation, the cytotoxic effects (CPE) of several DNA and RNA viruses could be significantly suppressed. These test results are shown in Table 9 for "virus evaluation" and the dose that shows 50% inhibition effect.
Shown in (4). The coefficient VR indicates the degree of inhibition of the cytotoxic effect of the virus when the test compound is used at various doses starting from the highest non-toxic dose. In the case of human rhinovirus type 1A, CPE
It was found that in parallel with the suppression of the virus, the production of new viral progeny was also suppressed. The kinetics of inhibitory activity was studied for rhinovirus 1A and Herpes simplex virus (HF): a. When 30 μg/ml of the compound was added 1 hour before virus inoculation and the compound remained in the medium thereafter. is extremely active and the reduction in virus titer in 24 hours is 3.5 log ₁₀
However, when the medium was mixed with 125 μg/ml
No inhibitory activity was observed when the medium was treated with AmTA for 2 hours and washed immediately before virus inoculation:c Antiviral activity was observed when the medium was washed free of compounds 2 hours before virus inoculation. A significant decrease in: VR when unwashed
= 1.8 and VR = 0.7: d For both viruses, there was still a large suppression of CPE when AmTA was added to the medium 6 hours after virus inoculation at low multiplicity of infection (>0.01 TCID ₅₀ /cell). Although an effect (VR=1.0) was observed, when this compound was added 24 hours after virus inoculation, the activity was just at the lower limit (VR=0.2). Virus suspension 2.5mg/ml of AmTA
AmTA had no direct inactivating effect on the infectivity of rhinovirus 1A and influenza A2 viruses when incubated with PBS solution for 1 hour at 37°C. Encephalomyocarditis virus (EMC) strains showed similar results.
Also obtained in the case of V77. LB57 cells were grown in Eagle's minimal essential medium containing 10% fetal serum. using speckle technique
The action time of AmTA was investigated. 40 μg/ml AmTA
Table 10 shows the results of a representative experiment using As with rhinovirus 1A and herpis simplex virus, pretreatment and treatment during absorption were inactive. AmTA was active at later stages of the viral response. The in vivo antiviral activity of AmTA was investigated in several lethal infections of Charles River CD-1 adult mice. Murine encephalomyocarditis virus (EMC) strain ATCC
Table 11 shows the results of a series of comparative experiments conducted using VR129. Animals are significantly protected when the compound is administered intraperitoneally (ip) or subcutaneously (se) 1 hour before inoculation of 6-10 TCID ₅₀ /mouse, but oral (pc) treatment has no effect. I understood. The most effective dose is 50mg/Kg
(ip), the acute LD 50 of the compound was 1/15 of the acute LD ₅₀ of the compound in mice using the same method _.
(treated daily for 5 consecutive days). The lowest dose causing weight loss was 5 times higher (250mg/
Kg, ip), but a slight temporary sedative effect was observed even at 60mg/Kgi.p. When the compound was administered to mice 4 hours after virus inoculation, 100 mg/Kg
(ip) and a single treatment at 50 mg/Kg (ip) (Table 11) significantly protected against EMC. The timing of compound administration was relatively late considering that untreated animals in this experimental system died within 4-5 days after virus inoculation. However, when mice were infected with EMC strain V77, protection by AmTA was still significant but significantly reduced under similar experimental conditions. AmTA, in doses found to be effective against EMC viruses, is effective against the following viruses: Herpis simplex (Virtue strain, i.
p., iv or ic inoculation], Semliki Forest arbovirus (se), West Nile arbovirus (ip), mouse foot and mouth disease (mouse-adapted
foot-and-mouth disease) type C virus (sc
or ip), A2/ANN Arbor/
It did not show any protective effect on mice infected with 2/62 virus (aerosol). The compound is also inactive against influenza viruses when co-administered (iv or ic) with the influenza virus, and when administered intranasally at 100 mg/Kg repeatedly before and after virus inoculation. It was inactive against infection. In summary, AmTA exhibits a broad spectrum of antiviral activity in cell culture media. Its mechanism of action is currently unknown, but under the experimental conditions used
AmTA did not exhibit antitoxicity against these viruses and did not inhibit virus absorption and penetration. In vivo, AmTA was repeatedly found to be active only against murine leukemia virus and EMC infections.

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[Table] Treatment time 0 indicates the time of virus inoculation; 40 μg/ml AmTA was added to the culture medium of the treated group.

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[Table] Table 13 Ammonium 21 in vitro and in vivo
- Tungst - 9 - Preliminary test results on virus susceptibility in rabbits when using antimonyate sodate 1 reduction in lesions in vitro This tungst antimonyate
Suppressed the formation of foci in BHK ₂₁ C ₁₃ S cells. Ammonium 21 2 hours after virus inoculation
When tungst-9-antimonyate sodium was added, it was found that the dose that showed a 50% inhibitory effect was 4 μg/ml. It was not as effective when added after 48 hours, and an increase in the number of spots was observed when added after 72 hours. Preincubation also suppressed the appearance of foci. 2 Response cycle (table) [Mechanism in which genetic matenial becomes 2 times] When 20γ/ml ammonium 21-tungst-9-antimonyate sodate is used in one cycle, By counting the spots, the reduction was found to be about 10 ¹ after 22-30 hours. At 50 γ/ml, the difference from the control was about 10 ² . In vivo, toxic doses were determined for mice using various inoculation methods. Inoculum and ammonium 21-
Protection could be achieved by premixing tungst-9-antimonyate with sodate. 100% effective dose is 2.5mg/15 in mice
It was hot at g. Conclusion The susceptibility of rabbit viruses to the action of ammonium 21-tungst-9-antimonyate sodate was confirmed in vitro, even though there was no direct effect of tungst-antimonyate on viruses at effective doses in vivo. The results were clear and promising.

[Table] Kg day rat

Claims (1)

[Claims] 1 Heteropolyanion 9-antimonio-
-21-An injectable antiviral agent characterized by containing a pharmaceutically acceptable non-toxic salt of tungst-sodate and a drug carrier under sterile conditions and in a single dosage form. 2. The antiviral agent according to claim 1, wherein the cation is selected from the group consisting of ammonium, alkali, and alkaline earth cations. 3. The antiviral agent according to claim 1, which contains calcium phosphate. 4 Heteropolyanion 9-antimonio-
-21-An injectable antiviral agent comprising a pharmaceutically acceptable non-toxic salt of tungst-sodate, interferon, and a drug carrier under sterile conditions and in a single dosage form.