JPS6118526B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel therapeutic agent for DNA virus infections. A typical existing antiviral agent is 5-iodo-2'-deoxyuridine (hereinafter referred to as IDU).
), 9-β-D-arabinofuranosyl-adenine (hereinafter abbreviated as Ara-A)
It has been known. However, IDU has side effects such as teratogenicity and cannot be considered a safe antiviral agent. Furthermore, like IDU, Ara-A strongly suppresses the proliferation of animal cells, including humans, and there are concerns about its toxicity when used as a therapeutic drug. Furthermore, both IDU and Ara-A have extremely low solubility in water, making it difficult to obtain highly concentrated solutions when used as injections or eye drops. As a result of repeated research on the antiviral activity of 1-β-D-arabinofuranosyl-thymine-5'-monophosphate (hereinafter referred to as Ara-TMP), the present inventors found that Ara- Although TMP does not have the effect of directly inactivating viruses, it does show the effect of suppressing the production of progeny viruses by inhibiting the DNA synthesis of progeny viruses that occurs during virus replication. If these
Does not exhibit DNA synthesis inhibitory effect on DNA synthesis,
It was found that the proliferation of these animal cells was not inhibited at all. In addition, Ara-TMP is less toxic to normal cells than 1-β-D-arabinofuranosyl-thymine (hereinafter referred to as Ara-T), which is known to have low toxicity and strong antiviral effects. It was the same. In vivo, Ara-TMP becomes Ara-T by phosphatase present on the cell surface, and Ara-T
It is thought that it is taken up into cells as a. Ara−
T is phosphorylated by deoxypyrimidine kinase induced by virus infection and becomes Ara-
TMP (Antimicrobial Aging and Chemotherapy)
Agents and Chemotherapy) 12, pp243-254
(1977)), further phosphorylation occurs, and 1-β-
D-arabinofuranosyl-thymine-triphosphate and inhibits DNA replication (Cancer Research) 38, pp3076
~3079 (1978)). Ara-TMP is thought to express antiviral activity through this mechanism of action, and therefore, like Ara-T, it shows almost no toxicity to cells and only selectively inhibits viral proliferation. . The advantage of using Ara-TMP as a therapeutic agent for viral infections is that Ara-TMP has extremely high solubility in various solvents. In particular, Ara-T
The allowable use concentration in an aqueous solution is 0.5%, whereas the solubility of Ara-TMP in water is extremely high at over 50%. Therefore, Ara-TMP has the advantage that it can be used as a highly concentrated solution when prepared as a solution in water or syrup, and is particularly advantageous when used as an injection or eye drops, such as for eye diseases. . Ara-TMP exhibits antiviral activity against various viruses in in vivo and in vitro experiments.
In particular, Herpesviruses including Herpes simplex, Varicella Zoster in mammals, such as rabbits, herpetic keratitis in humans and herpetic keratitis in mice. It is effective against DNA virus infections. The drug according to the present invention has Ara-
It is prepared as a pharmaceutical composition consisting of TMP or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier selected depending on the method and form of administration of the drug. That is, the drug of the present invention is administered orally or parenterally depending on the subject to be treated for internal virus diseases or external tissue virus diseases, and is prepared into powders or granules with an appropriate drug carrier depending on the administration method. It can be prepared as a preparation such as a liquid for injection or internal use, tablets, suppositories, petals, ointments, creams, aerosols, and eye drops. Pharmaceutically acceptable salts of Ara-TMP include:
Examples include alkali metal salts such as lithium, sodium, and potassium, alkaline earth metal salts such as calcium and magnesium, and ammonium salts. For internal diseases in the body, Ara-TMP or a pharmaceutically acceptable salt thereof is orally or parenterally administered at a dose of about 10 to 1000 mg, preferably 50 to 500 mg per kg of body weight of the mammal. For oral administration, finely divided powders or granules which may contain diluents, dispersants and/or surfactants; solutions in water or syrup; or suspensions which may contain suspending agents; including binders and lubricants. Liquid tablets: Administered in dry form, in non-aqueous solutions or suspensions, and in capsules or capsules. Flavoring agents, preservatives, suspending agents, diluents, thickening agents, emulsifying agents, and excipients can also be contained in these pharmaceutical compositions as required.
Tablets and granules are preferred, especially for purposes of oral administration, and these may be coated with any coating agent. When administered parenterally such as injections or as eye drops for eye diseases, Ara-TMP
or about 0.1 to 50 pharmaceutically acceptable salts thereof
(W/V)%, preferably 0.5-20 (W/V)% as an aqueous solution. Preservatives, buffers, etc. can also be included in this solution. For diseases of tissues external to the body such as the eyes, mouth and skin, Ara-TMP or its pharmaceutical agents can be administered locally in the form of an ointment with a water-soluble ointment base or a cream with an oil-in-water cream base. Approximately 0.1-40 (W/V)% of acceptable salt, preferably
It can be administered at a concentration of 0.5-20 (W/V)%. Test Example 1 (Antiviral Activity Test in Tissue Culture) Human fetal lung-derived cells were cultured in Eagle's basic medium supplemented with 10% fetal bovine serum, and a few days later infected with _30TCID50 herpes virus types. After 30 minutes, the virus solution was removed, Eagle's basal medium supplemented with 2.5% fetal bovine serum containing the test drug diluted to 1/2 log10 was added, and cultured at 37°C for 3 days. Cell degeneration (CPE) caused by viral infection was observed under a microscope and divided into five CPE scores ranging from 40 to 4.
A score of 0 means that CPE is completely suppressed by the drug, and a score of 4 indicates that CPE is not suppressed at all and CPE occurs to the same degree as a control without drug. Scores 1 to 3 represent the degree of CPE between scores 0 and 4 in stages. The minimum inhibitory concentration (MIC) was defined as the lowest concentration at which the CPE score was 2 or less. Viral rating (VR) calculations were performed using the Sidwell and Huffman method (Applied Microbiology).
Microbiology) Vol.22.No.5.p797). The relationship between VR and antiviral activity was defined as + for VR 1 or higher, ++ for 2 or higher, +++ for 3 or higher, ± for 0.5 to 0.9, and - for 0.4 or lower. These results are shown in Table 1.

[Table] Abbreviation for Tocin.
Test Example 2 (Treatment experiment for mouse inflammation caused by herpes virus) ICR mouse strain (male, 4 weeks old, weight 18-22 g)
I was infected with 32LD ₅₀ herpes virus type. Each dose of the test drug was intraperitoneally administered to mice nine times every 12 hours starting 4 hours after infection.
Phosphate buffered saline was administered as a control. The number of surviving mice 21 days after infection and the average survival days of mice that died within 21 days were as shown in the table below. In the Ara-TMP-2Na administration group, the mortality rate was significantly reduced and the survival period was prolonged, and IDU, Ara-C
No decrease in mortality rate or prolongation of survival days was observed in the treated group. In addition, mice in the Ara-C 100 mg/Kg administration group died due to toxicity.

[Table] Test Example 3 (Irritation test on rabbit ocular mucosa) 0.1 ml of a 2-40% aqueous solution of Ara-TMP-2Na was placed in one eye of a Japanese native rabbit (male, weighing around 4 kg).
After instillation, the eye was observed for hyperemia, floater formation, and corneal epithelial villan formation. Physiological saline was instilled into the other eye as a control. In observation from 0 to 24 hours, no hyperemia, edema, or corneal epithelium was observed in all three animals in one group at any concentration from 2 to 40%, indicating that Ara-TMP is almost irritating to the ocular mucosa. did not show. Test Example 4 (Treatment test for recurrent herpetic keratitis) Regarding two patients with recurrent herpetic keratitis,
When the eye was treated with 0.7% Ara-TMP-2Na aqueous solution five times a day, the ulcer shrank a few days after the start of the eye drop, and the ulcer disappeared and healed 10 to 12 days later. Test Example 5 (Acute toxicity test in mice) ICR mouse strain (female, 6 weeks old, weight 23-25 g)
2500 mg/Kg of Ara-TMP-2Na was administered by intravenous injection. During the one-week observation period, none of the 10 animals died, and their weight increased. That is, the LD ₅₀ of intravenous administration in mice was 2500 mg/Kg or more. In addition, similarly obtained mice were administered intraperitoneally.
LD ₅₀ is more than 10000 mg/Kg, and the toxicity of Ara-TMP is extremely low. Formulation example 1 (oil-in-water cream base) Ara-TMP-2Na 5.0g White petrolatum 25.0g Stearyl alcohol 25.0g Propylene glycol 12.0g Sodium lauryl sulfate 1.0g Ethyl paraoxybenzoate 0.025g Propyl paraoxybenzoate 0.015g Purified water Total amount 100 Formulation example 2 (aqueous ointment base) Ara-TMP-2NH ₄ 10.0g Polyethylene glycol 4000 50.0g Polyethylene glycol 400 50.0g Formulation example 3 (tablet) Ara-TMP-Ca 250mg Lactose 191mg Starch 50mg Polyvinylpyrodrine 5mg Magnesium stearate 4mg Total weight 500mg Formulation example 4 (eye drops) Ara-TMP-2Na 4g Benzalkonium chloride 1:50000 Purified water Total volume 100ml Formulation example 5 (injection) Ara-TMP-2K 20g Sodium chloride 4.5g Purified water Total amount 1000ml

Claims (1)

[Claims] 1 1-β-D-arabinofuranosyl-thymine-
A drug for treating DNA virus infections containing 5'-monophosphate or a pharmaceutically acceptable salt thereof as a main ingredient. 2. The therapeutic agent for DNA virus infection according to claim 1, which is in the form of an aqueous solution. 3. The therapeutic agent for DNA virus infections according to claim 2, wherein the aqueous solution is selected from the group consisting of internal solutions, injection solutions, and eye drops. 4. The therapeutic agent for DNA virus infection according to claim 1, which is in the form of eye drops. 5 1-β-D-arabinofuranosyl-thymine-
The DNA according to claim 2 or 4, wherein the dissolved concentration of 5'-monophosphate or a pharmaceutically acceptable salt thereof is 0.5 to 20 (W/V)%.
A drug for treating viral infections.