JP2018076280A - Pharmaceutical composition for treating zika virus infection - Google Patents

Abstract

The present invention provides an effective means for the treatment and / or prevention of Zika virus infection. A pharmaceutical composition for preventing and / or treating zika virus infection, comprising ribavirin. [Selection figure] None

Description

  Techniques relating to the treatment of Zika virus infection are disclosed.

  Currently, mosquito-borne zika viruses are prevalent in other countries. Along with this, the number of neonatal microcephaly patients is increasing. In recent years, it has become clear that Zika virus infection in pregnant women and microcephaly in newborns are involved. However, effective therapeutic agents and vaccines against Zika virus have not been developed yet. In order to reduce microcephaly patients, early treatment of Zika virus infected persons is required to prevent the spread of infection.

  On the other hand, 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) is a nucleic acid structural analog and is currently used for the treatment of hepatitis C in combination with interferon α-2α. (Patent Document 1).

JP2011-51898

  Under the present situation as described above, it is an object to provide an effective means for the treatment and / or prevention of Zika virus infection.

  As a result of intensive studies to solve the above problems, it has been found that ribavirin, which is known as a therapeutic agent for hepatitis C virus, is effective in suppressing the growth or infection of Zika virus. As a result of further research based on such knowledge, the following inventions are provided.

Item 1.
A pharmaceutical composition for preventing and / or treating zika virus infection, comprising ribavirin.
Item 2.
Item 2. The pharmaceutical composition according to Item 1, which is an oral dosage form.
Item 3.
A method for treating Zika virus infection, comprising administering ribavirin to a subject infected with Zika virus or suspected of being infected with Zika virus.
Item 4.
Use of ribavirin for producing a pharmaceutical composition for prevention and / or treatment of Zika virus infection.

  Effective treatment and / or prevention measures for Zika virus infection are provided. In one embodiment, a safe treatment and / or treatment for a Zika virus infection with reduced side effects is provided.

The results of evaluating the ability of ribavirin to inhibit Zika virus growth based on plaque formation are shown. (A) is the result when the virus titer is 0, 0.001, and 0.01, and (B) is the result when the virus titer is 0, 0.1, and 1. (C) is a result of relatively evaluating the number of plaques. The result of having evaluated the ability to suppress Zika virus growth by ganciclovir based on plaque formation is shown. (A) is the photograph which image | photographed formation of plaque, (B) is the result of having evaluated the number of plaques relatively. The result of having evaluated the concentration heterogeneity and safety | security of the Zika virus growth suppression ability by ribavirin is shown. The results of evaluating the ability of ribavirin to inhibit Zika virus growth using cells derived from mosquito larvae are shown. The results of evaluating the ability of ribavirin to inhibit Zika virus growth using cells derived from mosquito larvae are shown. The results of evaluating the ability of ribavirin to inhibit Zika virus growth using genetically modified mice highly sensitive to Zika virus infection are shown. The result of having evaluated the Zika virus growth inhibitory effect of ribavirin using the Vero cell is shown. The result of having evaluated the zika virus growth inhibitory effect of ribavirin using the cell of a human neuroblastoma is shown. The result of having evaluated the inhibitory effect of ribavirin with respect to apoptosis and cell death by Zika virus is shown. The result of having evaluated the inhibitory effect of ribavirin with respect to the expression of the viremia by Zika virus is shown. The result of having evaluated the influence of ribavirin on the survival rate of the mouse infected with Zika virus is shown.

  The pharmaceutical composition for treating and / or preventing zika virus infection (hereinafter also referred to as the pharmaceutical composition of the present invention) preferably contains ribavirin as an active ingredient. Ribavirin is a known compound, and as a method for obtaining it, a commercially available product may be used, or it may be produced based on a known method.

  The pharmaceutical composition of the present invention is administered to a subject infected with Zika virus or a subject who is likely to be infected. In general, infected patients exhibit symptoms such as fever, headache, conjunctival hyperemia, skin rash, myalgia, and joint pain. Therefore, in addition to ribavirin, the pharmaceutical composition of the present invention may contain a symptomatic treatment for these symptoms.

  Examples of such symptomatic treatment drugs include: aspirin, aspirin aluminum, sazapyrine, etenzamide, salicylamide, ibuprofen, acetaminophen, isopropylantipyrine and the like antipyretic analgesics; caffeine, anhydrous caffeine Central nervous stimulants such as sodium benzoate caffeine; sedatives such as bromvalerylurea and allylisopropylacetylurea, chlorpheniramine maleate, diphenhydramine, diphenhydramine salicylate, clemastine fumarate, carbinoxamine maleate, mequitazine, alimemazine tartrate, hydrochloric acid Antihistamines such as diphenylpyraline and triprolidine hydrochloride; lysozyme chloride, bromelain, serrapeptase, semi-alkaline proteinase, glycyrrhizin , Anti-inflammatory drugs such as dipotassium glycyrrhizinate, ammonium glycyrrhizinate, glycyrrhetinic acid, sodium azulene sulfonate; belladonna (total) alkaloids, belladonna extract, isopropamide iodide, scopolamine hydrobromide, methylbenactidium bromide, timepidium bromide Anti-acetylcholine agents such as pirenzepine; sterilization of cetylpyridinium, cetylpyridinium chloride, popidone iodine, chlorhexidine gluconate, benzalkonium chloride, decalinium chloride, thymol, iodine / potassium iodide, phenol, chlorhexidine hydrochloride, creosote, benzethonium chloride Disinfectants; local anesthetics such as dibucaine hydrochloride, ethyl aminobenzoate, lidocaine, lidocaine hydrochloride, oxesasein; vitamin A, liver oil, vitamin B1, Vitamins such as Tamine B2, Vitamin B6, Vitamin B12, Vitamin C, Calcium Ascorbate, Vitamin D, Vitamin E, Tocophenol Calcium Succinate; Pantothenic Acid, Panthenol, Sodium Pantothenate, Calcium Pantothenate, Panthetin, Nicotinic Acid , Nicotinamide, glucuronic acid, glucuronolactone, aminoethyl sulfonic acid, biotin, γ-oryzanol, and other metabolic components; ground yellow, caihi, leopard, ginger, maoh, licorice, ginger, hange, shazenso, psycho, bukkuri , Herbal medicines such as Shinyi and their extracts (extracts, tinctures, etc.). As the symptomatic therapeutic agent, a single component may be blended in the pharmaceutical composition of the present invention, or two or more kinds may be blended in combination.

  The pharmaceutical composition of the present invention can be administered orally or parenterally. Examples of the preparation to be administered orally include tablets, capsules, powders, fine granules, granules, liquids, troches, and jelly. In addition, preparations for parenteral administration include injections, plasters, spirits, extracts, suppositories, suspensions, tinctures, ointments, poultices, nasal drops, inhalants, liniments, A lotion agent, an aerosol agent, etc. can be mentioned. You may mix | blend the pharmaceutical composition of this invention with a mouthwash, a throat spray, and a mouthwash. In one embodiment, the pharmaceutical composition of the present invention is preferably an orally administered preparation, more preferably a solid preparation such as a tablet, capsule, powder, fine granule, granule, troche, and jelly.

  These preparations can be produced by conventional production methods known to those skilled in the art, and additives can be blended as desired to prepare pharmaceutical preparations of such various dosage forms. Examples of the additive include an excipient, a binder, a disintegrant, a fluidizer, an emulsifier, and a stabilizer. These additives may be used alone or in combination of two or more.

  The dosage of the pharmaceutical composition of the present invention to a patient is appropriately determined in consideration of the patient's sex, age, weight, symptoms, administration method, frequency of administration, administration timing, and the like. In one embodiment, the pharmaceutical composition of the present invention is preferably administered orally to a patient, and the dose of ribavirin at that time is, for example, 1 to 30 mg / kg / day, preferably 5 to 20 mg / kg / day. Can be a day. The frequency of administration is not particularly limited, and can be, for example, once a day, twice a day, three times a day, or four times a day. The administration period is not particularly limited, and can be continued until symptoms are alleviated or eliminated, for example, 3 months, 2 months, 1 month, 3 weeks, 2 weeks, 1 week, 5 days, 4 days, 3 days, One day or two days. The dose may be changed during the administration period. For example, the initial dose (ribavirin) is set high (for example, 15 to 50 mg / kg, preferably 20 to 40 mg / kg), and the dose for the second transfer is stepwise (for example, every day, every 2 days, 3 days, Daily, every 4 days, every 5 days, every 6 days, every 1 week, every 2 weeks, every 3 weeks, or every month. In one embodiment, the pharmaceutical composition of the present invention is preferably administered to a patient by intravenous injection. The dose, frequency of administration, and administration period for intravenous administration may be the same as or different from those for oral administration exemplified above.

  The pharmaceutical composition of the present invention may be produced as a single preparation containing a plurality of components according to the present invention and administered, or each component according to the present invention may be divided into separate preparations. A kit preparation that allows simultaneous or sequential administration of preparations may be used.

  In one embodiment, Zika virus infection is prevented by administering the pharmaceutical composition of the present invention to a subject suspected of being infected with Zika virus. A subject suspected of being infected with Zika virus is, for example, a human who has visited an area where Zika virus infection is prevalent, or a person who has developed such a Zika virus infection or a Zika virus infection. Mention may be made of humans in contact. Administration of the pharmaceutical composition of the present invention for the purpose of prevention is performed within a certain period of time from when a human visits an area where Zika virus infection is prevalent or when a human contacts a human who develops Zika virus. It is preferable to carry out. The fixed period can be, for example, 2 months, 1 month, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these.

Test Example 1 Ribavirin's ability to inhibit Zika virus growth (Vero cells)
Vero cells are cell lines derived from the kidney epithelial cells of African green monkeys, and are highly sensitive to viral infections because they lack the cluster of type I interferon genes that play an important role in suppressing viral growth. Is a stock. The plaque assay is an effective technique for measuring virus titer. Vero cells adhering to the dish are infected with the virus, and methylcellulose is overlaid thereon. Thereby, it is possible to prevent the virus released from the infected cells from infecting distant cells. A virus that infects one cell infects only adjacent cells, and the infected cell spreads concentrically from the initial infection cell. This part of the Vero cell is killed and peeled off to form a plaque. By counting the number of plaques, a plaque forming unit (PFU) can be calculated to determine the virus titer.

Vero cells were seeded at 2 × 10 ^{5 /} well on a 12-well plate. The next day, the Zika virus was infected with five levels of virus titers of multiplicity of infection (MOI) 0, 0.001, 0.01, 0.1, and 1 (MOI determines the number of viruses per cell). Represent). One hour after infection, 2% methylcellulose was overlaid, and ribavirin was added in four concentrations, 0, 10, 100, and 1000 μg / ml. Four days after the infection, plaque forming unit (PFU) was calculated by counting the number of plaques, the virus titer was determined, and the viral growth inhibitory effect of ribavirin was evaluated.

  In Zika virus-infected Vero cells not treated with ribavirin, more plaques were observed as the MOI increased (FIGS. 1 (A) and (B)). On the other hand, when ribavirin was allowed to act, plaque formation was suppressed depending on the concentration of ribavirin (FIGS. 1 (A) and (B)). FIG. 1C is a graph showing the ratio of the number of plaques in the ribavirin-added group to the number of plaques in the ribavirin-unadded group at each MOI. In the group to which ribavirin was added, plaque formation was suppressed depending on its concentration, and when 1000 μg / ml ribavirin was allowed to act, it was found that almost no plaque was formed even if the MOI value was high. From the above results, it has been clarified that riribavirin suppresses the growth of Zika virus in Vero cells, which are mammalian cells, and its effect increases in a concentration-dependent manner.

Test Example 2: Ganciclovi's ability to inhibit Zika virus growth Tests similar to those of Test Example 1 were conducted except that ribavirin in Test Example 1 was changed to ganciclovir. However, Zika virus was infected with three levels of virus titers of MOI 0, 0.1, and 1, and ganciclovir was added at four levels of 0, 0.5, 1, and 2 μM. Ganciclovir is used as a therapeutic agent for cytomegalovirus and herpes simplex virus infections and has a DNA polymerase inhibitory action.

  In each MOI, almost the same number of plaques was observed regardless of the concentration of ganciclovir (FIG. 2 (A)). In addition, no inhibitory effect on plaque formation was observed in any concentration of ganciclovir. From the above results, it was clarified that ganciclovir does not have the ability to suppress the growth of Zika virus in Vero cells.

Test Example 3: Concentration heterogeneity and safety evaluation Vero cells were seeded at a rate of 2 × 10 ^{5 /} well on a 12-well plate. The next day, Vero cells were infected with Zika virus (ZIKV) at a viral titer of MOI 1. One hour after infection, ribavirin was added at 12 concentrations, 0, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 750, 1000, 1500, and 2000 μg / ml. Four days after the infection, each well was photographed under an inverted microscope to evaluate the survival of Vero cells.

  As shown in FIG. 3, it was confirmed that Vero cells were killed and detached in a well having a ribavirin concentration of 125 μg / ml or less. On the other hand, Vero cells survived and adhered to the dish in wells having a ribavirin concentration of 250 μg / ml or more. Vero cells were alive even when the ribavirin concentration was 2000 μg / ml. From these results, it was clarified that ribavirin showed a virus growth inhibitory effect by plaque assay at 100 μg / ml, and could suppress cell death at 250 μg / ml. Moreover, even if the ribavirin concentration was increased, it did not adversely affect the survival of Vero cells, suggesting that the safety of ribavirin is high.

Test Example 4: Ribavirin's ability to suppress Zika virus growth (C6 / 36 cells: RT-PCR)
C6 / 36 cells are a cell line derived from the hatched larvae of Aedes albopictus, and are useful for mosquito-borne virus propagation experiments. C6 / 36 cells were seeded at 2 × 10 ⁵ / well on a 12-well plate. The next day, C6 / 36 cells were infected with Zika virus at a virus titer of MOI 0.001. One hour after infection, ribavirin was added in four concentrations, 0, 10, 100, and 1000 μg / ml. Four days after the infection, C6 / 36 cells were collected with TORIZOL and RNA was extracted. A reverse transcription reaction was performed on the extracted RNA to obtain cDNA. Real-time RT-PCR was performed on this cDNA using a primer that recognizes a base sequence specific to Zika virus. In addition, as an internal control, a primer for C6 / 36 cell 40S ribosomal protein S17 was used.

  As shown in FIG. 4, it was found that when ribavirin was allowed to act at a concentration of 10 μg / ml, the amount of zikavirus was reduced to about 1/8 compared with the case where ribavirin was not allowed to act. In addition, when it was allowed to act at a concentration of 100 μg / ml or 1000 μg / ml, almost no Dika virus was detected. From these results, ribavirin suppressed the growth of Zika virus in C6 / 36 cells. It became clear that the effect became high depending on the concentration. This indicates that ribavirin has the ability to suppress the growth of Zika virus even in the mosquito cells that are vectors.

Test Example 5: Ribavirin's ability to inhibit Zika virus growth (C6 / 36 cells: RT-PCR)
RT-PCR was performed on the RNA extracted from C6 / 36 cells in Test Example 4 using a primer that recognizes a base sequence specific to Zika virus. The sample after PCR was electrophoresed on an agarose gel, and the presence or absence of a band and the density were evaluated.

  As shown in FIG. 5, it was confirmed that the density of the band detected depending on the concentration of ribavirin was reduced. From this result, as in Test Example 4, it was shown that ribovirin has the ability to suppress the growth of Zika virus even in mosquito cells, which is a vector.

Test Example 6: Ribavirin's ability to protect against Zika virus infection (in vivo)
Mice have low susceptibility to Zika virus, and infection does not occur even when inoculated with a high titer virus. It has been reported (Lazear et al., Cell Host Microbe. 2016 May 11; 19 (5): 720-30). Therefore, a STAT1-deficient mouse essential for transmitting the signal downstream of the receptor for type I interferon was prepared, and it was confirmed that it was highly susceptible to Zika virus infection.

Six STAT1-deficient mice were administered 1 × 10 ⁴ PFU of Zika virus subcutaneously. On the day of infection, 3 animals were given 25 mg of ribavirin intraperitoneally per animal. On the other hand, the remaining 3 animals were intraperitoneally administered with an equal amount of PBS as a view. Two days after the infection, blood was collected, and the viral RNA in the serum was analyzed by the RT-PCR method.

  RT-PCR analysis was performed on a serum sample using a primer that specifically detects the base sequence of Zika virus, and the presence or absence of viremia was confirmed. As a result, in the ribavirin non-administered group, a band was detected in 3 out of 3 animals, but in the ribavirin-administered group, a band was not detected in 3 out of 3 animals (FIG. 6). From this result, it became clear that ribavirin suppresses viremia caused by Zika virus at the biological level.

Test Example 7
Vero cells were infected with Zika virus at a virus titer of MOI 5 × 10 ⁻⁵ . One hour after infection, ribavirin was applied at concentrations of 0, 10, 20, 40, and 80 μg / ml. Four days later, quantitative RT-PCR analysis was performed, and the viral growth inhibitory effect of ribavirin was evaluated. The results are shown in FIG. The bar graph shows the average value of three independent experiments, and the error bar shows the standard deviation (*: P <0.05, **: P <0.01). As shown in FIG. 7, ribavirin significantly suppressed the growth of Zika virus in Vero cells at 20 μg / ml. Further, the growth of Zika virus was almost completely suppressed at 80 μg / ml.

Test Example 8
SH-SY5Y cells, a human neuroblastoma cell line, were infected with Zika virus at a viral titer of MOI 5 × 10 ⁻⁴ . One hour after infection, ribavirin was applied at concentrations of 0, 10, 20, 40, and 80 μg / ml. Four days later, quantitative RT-PCR analysis was performed to evaluate the effect of ribavirin on the growth inhibition of the virus. The results are shown in FIG. The bar graph shows the average value of three independent experiments, and the error bar shows the standard deviation (**: P <0.01). As shown in FIG. 8, ribavirin significantly suppressed the growth of Zika virus in SH-SY5Y cells at 10 μg / ml. Further, the growth was almost completely suppressed at 20 μg / ml.

Test Example 9
Vero cells were infected with Zika virus at a virus titer of MOI 5 × 10 ⁻³ . One hour after infection, ribavirin was applied at each concentration shown in FIG. Four days later, the ratio of apoptotic cells and dead cells was measured by FACS analysis (FIG. 9). Annexin V positive represents apoptotic cells and Zombie red positive represents dead cells. As shown in FIG. 9, ribavirin suppressed apoptosis and cell death by Zika virus in a concentration-dependent manner. Ribavirin at a concentration of 250 μg / ml or more almost completely suppressed cell death of Vero cells caused by Zika virus infection. Further, cytotoxicity was not confirmed even when a high concentration of ribavirin at 2000 μg / ml was allowed to act. The ratio of apoptotic cells was highest when 125 μg / ml ribavirin was applied, but the ratio decreased when a higher concentration of ribavirin was applied.

Test Example 10
STAT1-deficient mice were infected with 1.0 × 10 ⁴ PFU of Zika virus by subcutaneous administration. 15 mg of ribavirin was administered intraperitoneally (n = 5) for 3 consecutive days from the day of infection (0, 1, and 2 days after infection). A control group (n = 5) received the same amount of PBS. RNA was purified from serum and viremia was assessed by RT-PCR analysis. The results are shown in FIG. 10 (Ribavirin (−): Ribavirin non-administration group, Ribavirin (+): Ribavirin administration group, N: Negative control, P: Positive control). Here, the negative control was obtained by performing RT-PCR with Primase against ZIKV genome using RNase-free water as a sample, and the positive control was RT using Primer against ZIKV genome with RNA purified from virus dilution as a sample. -PCR was performed. The number of samples has decreased since day 6 after infection, which is due to the death of the individual. In the Ribavirin non-administered group, viremia was confirmed 2 days after infection, and strong viremia continued until 4 days after infection. Viremia disappears from day 5 after infection, but all individuals die by day 8 after infection. In the ribavirin-administered group, suppression of viremia was confirmed until the fourth day after infection. However, viremia began to be confirmed from day 5 after infection, and all died by day 8 after infection. From this result, it became clear that administration of ribavirin in the early stage of infection can suppress viremia.

Test Example 11
STAT1-deficient mice were infected with Zika virus by subcutaneous administration of 1.0 × 10 ⁴ PFU per mouse. From the day of infection, 10 mg of ribavirin was administered intraperitoneally every day (n = 5). A control group (n = 5) received the same amount of PBS. Thereafter, the survival rate was analyzed. The results are shown in FIG. 11 (rib (−): riribavirin non-administered group, rib (+): riribavirin-administered group, **: P <0.01). As shown in FIG. 11, in the ribavirin non-administered group, all individuals died by the seventh day after infection. On the other hand, in the ribavirin-administered group, although all individuals died by the ninth day after infection, it became clear that the survival period was significantly extended compared to the non-administered group.

  From the results of Test Examples 10 and 11, it is considered desirable not only in the early stage of infection with Zika virus, but also to continuously extend the life of subjects infected with Zika virus by administering a certain concentration of ribavirin continuously.

Claims (2)

A pharmaceutical composition for preventing and / or treating zika virus infection, comprising ribavirin. The pharmaceutical composition according to claim 1, which is in an oral dosage form or an intravenous injection form.