JPH08245402A - Antiviral agent - Google Patents

Abstract

PURPOSE: To obtain the subject antiviral agent containing a chitin in which phosphoric acid group and sulfuric acid group are introduced or its salts as active ingredients, having low toxicity and high antiviral activity, retaining the action for a long time in vivo and effective against virus such as T lymphocyte-infectious virus. CONSTITUTION: This antiviral agent contains a chitin phosphated after being sulfated, in which phosphoric acid group and sulfuric acid group are introduced, e.g. in the range having 0.2-2 substituent degree per constituent monosaccharide of chitin as an active ingredient. Furthermore, the chitin is preferably obtained by phosphating chitin extracted by removal of protein and ash from crust of crab, etc., using orthophosphoric acid and then sulfating the phosphated chitin using sulfur trioxide-N,N-dimethylformamide complex containing N,N- dimethylformamide as a solvent. The dose of the chitin is preferably 0.1-100mg per kg weight.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antiviral agent which is a medicine useful for treating and preventing viral diseases.

[0002]

BACKGROUND OF THE INVENTION As is well known, viruses are parasites and are organisms that can grow only in living susceptible cells.
It is a cause of many infectious diseases, and causes viral inflammation, leukemia, malignant lymphoma, sarcoma, cancer, immunodeficiency, etc. in various animal species including humans. As a viral disease, recently acquired attention is recently acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV). This AIDS is the HI
V is a disease that infects T lymphocytes (CD4 positive cells) and destroys host cells, resulting in a decrease in cell-mediated immunity, which causes opportunistic infections and the development of malignant tumors.

Further, it is known that the proliferation of viruses takes the steps of attachment to host cells, entry into cells, transcription of genes, assembly of virus particles, release of virus particles by germination or cell lysis. , Antiviral agents have been used to inhibit any of the above processes. And
As an anti-AIDS agent, a reverse transcriptase inhibitor AZ
There are nucleoside derivatives such as T, ddC and ddI. Further, it is known that saccharides having a sulfate group such as heparin and sulfated alkyl oligosaccharides have a therapeutic and preventive effect on retroviral infections (Japanese Patent Laid-Open No. 63-4522).
No. 3, JP-A-5-139980).

[0004]

However, the conventional antiviral agents are not satisfactory in their therapeutic and prophylactic effects. For example, a nucleoside derivative has severe side effects such as headache, malaise, and sometimes psychosis such as stupor, in addition to causing extreme anemia by inhibiting the hematopoietic action of patients, and a resistant virus is generated. There is a problem.

[0005] Further, saccharides having a sulfate group such as heparin have a large anticoagulant action, and therefore, when administered in vivo, they block the blood coagulation system, which is important in biological defense, and rapidly decompose in vivo. However, there is a problem that it is metabolized and the blood concentration cannot be maintained.

Since the sulfated alkyl oligosaccharide is a substance having a low molecular weight, it has a problem that it is rapidly metabolized in vivo and its blood concentration cannot be maintained.

The present invention has been made in view of the above problems, and provides an antiviral agent having low toxicity, high antiviral activity, and long-term action in vivo. With the goal.

[0008]

That is, the antiviral agent according to claim 1 of the present invention comprises as an active ingredient chitin or a salt thereof having a phosphate group and a sulfate group introduced therein. In addition, the antiviral agent according to claim 2 has as an active ingredient chitin or a salt thereof which is phosphorylated and then sulfated. The antiviral agent according to claim 3 is effective against a T lymphocyte infectious viral disease in which the virus is a T lymphocyte infectious virus.

[0009]

The chitin having a phosphate group and a sulfate group used in the present invention has N-acetyl-D-glucosamine and D-glucosamine as constituent monosaccharides, and the proportion of D-glucosamine is 20%. The following, or N-acetyl-
It has a structure in which a phosphate group and a sulfate group are introduced into chitin, which is a polysaccharide consisting only of D-glucosamine. Examples of the method for producing this chitin into which a phosphate group and a sulfate group have been introduced include, for example, phosphorylation of extracted chitin by removing proteins from the shells of crabs, shrimps, etc., soft shells of squid, etc. And a method of sulfating. As for the order of phosphorylating and sulfating chitin, it is operationally efficient to perform sulfation after phosphorylation.

As the phosphorylation method, known methods such as a method using orthophosphoric acid and a method using diphosphorus pentoxide with methanesulfonic acid as a solvent (for example, "Last biomass chitin, chitosan", Chapter 2 , "2.3.
5 Sulfuric acid and phosphoric acid esterification reaction ", Chitin, Chitosan Kenkyukai, edited by Chitosan Kenkyukai, February 20, 1988, 1st edition, 1st edition, published by Gihodo Co., Ltd.). The degree of substitution of the phosphate group can be adjusted by appropriately adjusting the above.

Further, as the above-mentioned sulfation method, N, N
-Sulfur trioxide-N, using dimethylformamide as a solvent,
Known methods such as a method using an N-dimethylformamide complex, a method using chlorosulfonic acid and pyridine, a method using chlorosulfonic acid and dichloroethane, and a method using concentrated sulfuric acid (for example, "final biomass chitin, chitosan", second Chapter, “2.3.5 Sulfuric acid and phosphoric acid esterification reaction”, Chitin, Chitosan Kenkyukai, published February 20, 1988, 1st edition, 1st edition, Gihodo Publishing Co., Ltd.), and a sulfating agent can be added. By appropriately adjusting the amount, reaction time, reaction temperature and the like, the degree of substitution of the sulfate group can also be adjusted.

Here, the substitution degree of the phosphate group and the sulfate group per constituent monosaccharide of chitin having the phosphate group and the sulfate group introduced is preferably in the range of 0.1 to 1.9. , And the sum of both substitution degrees is preferably in the range of 0.2 to 2 per constituent monosaccharide. When the sum of both substitution degrees per constituent monosaccharide is less than 0.2, it is difficult to handle because it is difficult to dissolve in water, and the effect as an antiviral agent is low.

On the other hand, the salts of chitin having a phosphate group and a sulfate group used in the present invention include salts with inorganic bases such as sodium salt, potassium salt and ammonium salt, and diethanolamine salt, cyclohexylamine salt, Medically acceptable salts such as salts with organic bases such as amino acid salts are included.

The dose of chitin or a salt thereof having a phosphate group and a sulfate group introduced therein is an amount sufficient to produce the desired therapeutic effect in vivo, and generally 1 kg of body weight.
0.01 to 200 mg, preferably 0.1 to 10
The dose is 0 mg, and sufficient activity can be sustained by administration at long-term intervals such as weekly intervals.

The method of administration may be intravenous drip or injection. At the time of administration, chitin having a phosphate group and a sulfate group introduced thereinto may be combined with a pharmaceutical carrier such as a liquid excipient. It can be mixed and administered. Also,
Additives such as adjuvants, stabilizers, and pH adjusters can be added if necessary.

The effect of the antiviral agent containing chitin or a salt thereof having a phosphate group and a sulfate group introduced therein has not been clarified yet at present, but the phosphate group and the sulfate group are The introduced chitin or its salt is T
It is considered to prevent infection by inhibiting attachment of viruses such as lymphocyte infectious virus to host cells.

[0017]

The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 10 kg of soft-shelled squid was ground with a feather mill (passing through a 5 mm screen), and the ground product was put into a 1N aqueous sodium hydroxide solution (30 liters) and the temperature was adjusted to 1 at 90 ° C.
After being kept for a time and washed with water, it was immersed in a 0.1 N hydrochloric acid aqueous solution for 1 hour. Then, after washing with water, it is kept in a 1N aqueous sodium hydroxide solution (90 ° C.) for 1 hour, washed with water, and then dried in an oven (50 ° C.) for 5 hours to obtain purified squid chitin 1.2.
I got kg. The purified squid chitin was pulverized with a centrifugal fluidizing mill and pulverized into fine powder of 100 mesh or less.

And 150 ml of dimethylformamide
To the solution obtained by adding 150 g of urea and heating to 100 ° C. and dissolving, 10 g of the fine powdery chitin was added and dispersed, and then 26 ml of orthophosphoric acid was added and reacted at 150 ° C. for 3 hours to react chitin. Phosphorylation was performed. After adding 1000 ml of deionized water to this reaction solution, the pH was adjusted to 9 with a concentrated sodium hydroxide solution, the insoluble portion was removed by centrifugation, and dialysis was performed for 3 days. Further, the solution after dialysis was concentrated and freeze-dried to obtain about 12 g of sodium salt of phosphorylated chitin.

50 ml of pyridine was added to 5 g of the sodium salt of phosphorylated chitin prepared as described above, and the mixture was immersed overnight. Then, the insoluble portion was recovered by centrifugation, newly suspended in 100 ml of pyridine, 12.16 g of a complex of sulfur trioxide and pyridine was added, and the mixture was added at 60 ° C. in a nitrogen atmosphere at 60 ° C.
Let react for minutes. Then, the reaction solution was cooled to room temperature, 400 ml of ethanol was added to the reaction solution, the formed precipitate was filtered off, dissolved in 50 ml of deionized water, and the pH was adjusted to 9 with 5N sodium hydroxide. After adjustment, the insoluble portion was filtered and dialyzed for 4 days. Further, the solution after dialysis was concentrated and freeze-dried to obtain 4.4 g of chitin into which a phosphate group and a sulfate group were introduced. From the results of elemental analysis, the degree of substitution of the obtained chitin into which a phosphate group and a sulfate group had been introduced was found to be phosphorylation degree 1.18, sulfation degree 0.15
And the molecular weight is H using pullulan as a standard substance.
The result of PLC analysis was about 110,000.

The phosphoric acid group- and sulfate group-containing chitin prepared as described above was dissolved in physiological saline at a concentration of 1 mg / ml, and 0.1N hydrochloric acid and 0.1N hydroxylated solution were prepared. The pH was adjusted to 7.2 with sodium. And dehydration, diarrhea, loss of appetite, loss of energy, and stomatitis and gingivitis coexist, and pet check FeLV / FIV, feline leukemia virus antigen / anti-cat immunodeficiency virus antibody simultaneous test kit (Dainippon Pharmaceutical Co., Ltd.) ) Was diagnosed as positive for T-lymphocyte infectious feline immunodeficiency virus infection (FIV),
It was used for treatment of 8-year-old hybrids and castrated cats weighing 3.3 kg.
No concomitant medication was used throughout the course of treatment.

As a result of administering 3 ml of the above-prepared phosphate and sulfate group-introduced chitin solution into the cephalic vein (0.9 mg / kg body weight) at 1-week intervals, 1 week after the first administration. With dehydration, diarrhea, loss of appetite,
Loss of energy, clinical symptoms such as stomatitis and gingivitis disappear,
No subsequent recurrence of these symptoms was seen thereafter.

Comparative Example 10 g of the squid chitin fine powder prepared in the above example was immersed in 45 ml of pyridine for 12 hours. Separately, pyridine 60m
1 l, and chlorosulfonic acid 15
After adding ml and sufficiently cooling with ice, the pyridine suspension of chitin prepared above was added and reacted at 90 ° C. for 60 minutes. Then, after cooling the reaction solution to room temperature, the reaction solution was dropped into 1 liter of ethanol, and the generated precipitate was collected. The precipitated portion was redissolved in 500 ml of water, neutralized with 5N sodium hydroxide, the insoluble portion was removed by filtration, and then dialyzed in deionized water for 10 days. Furthermore, the solution after dialysis is concentrated and freeze-dried to obtain 10 g.
To obtain sulfated chitin. The degree of substitution of the obtained sulfated chitin was found to be 1.4 by the elemental analysis.

1 mg of the sulfated chitin produced above
The solution was dissolved in physiological saline at a concentration of / ml, and the pH was adjusted to 7.0 with 0.1 N sodium hydroxide. And dehydration, diarrhea, loss of appetite, loss of energy, and stomatitis and gingivitis coexist, and pet check FeLV / FIV, feline leukemia virus antigen / anti-cat immunodeficiency virus antibody simultaneous test kit (Dainippon Pharmaceutical Co., Ltd.) ) Was diagnosed as positive for T-lymphocyte infectious feline immunodeficiency virus infection (FIV),
It was used for the treatment of males and cats weighing 2.3 kg and 3 years old. No concomitant medication was used throughout the course of treatment.

Solution 3 of sulfated chitin prepared as described above
ml was administered into the cephalic vein every 3 days (1.3 mg / kg body weight), and as a result, clinical symptoms such as dehydration, diarrhea, loss of appetite, loss of energy, and stomatitis and gingivitis improved slightly after 2 weeks. However, even if it was administered for 1 month, F
IV antibody did not turn negative.

[0026]

The antiviral agent containing chitin or a salt thereof having a phosphate group and a sulfate group introduced as described above as an active ingredient is used for treating a virus, particularly a T lymphocyte infectious virus infection. It has excellent preventive effects, long-term action in vivo, and low side effects. Further, since it is easy to manufacture, it can be supplied at a low cost, and if phosphorylated chitin and then sulfated, chitin having a phosphate group and a sulfate group introduced therein can be more efficiently produced.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Satoshi Miyazaki 5-133, Higashi 5-chome, Koyama-cho, Tottori City, Tottori Prefecture

Claims (3)

[Claims] 1. An antiviral agent containing chitin or a salt thereof having a phosphate group and a sulfate group introduced therein as an active ingredient. 2. The antiviral agent according to claim 1, wherein the chitin is phosphorylated and then sulfated. 3. The antiviral agent according to claim 1 or 2, wherein the virus is a T lymphocyte infectious virus.