Application of raltitrexed in treating diseases caused by poxvirus infection

Abstract

The present invention relates to the use of raltitrexed in the alleviation and / or treatment of poxvirus infection. In view of the strong inhibition effect of raltitrexed on pox viruses including monkey pox viruses and vaccinia viruses, raltitrexed is very likely to become a specific medicine for diseases caused by infection of the pox viruses including the monkey pox viruses and the vaccinia viruses. The inventor considers that raltitrexed as a potential medicine for treating diseases caused by infection of poxviruses including monkey poxviruses and vaccinia viruses has great medicinal value, and is a promising candidate medicine for treating diseases caused by infection of poxviruses including monkey poxviruses and vaccinia viruses.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and relates to a new use of the drug raltitrexed, and specifically to the use of raltitrexed in preventing and / or treating diseases caused by poxvirus infection. Background Art

[0002] After infection with monkeypox virus (MPXV), patients often show fever, lethargy, muscle pain, headache and widespread characteristic rash, accompanied by swollen lymph nodes, which is also one of the significant characteristics of infection with MPXV. In addition, MPXV infection may also cause a series of complications, such as secondary infection, bronchopneumonia, sepsis, encephalitis, and keratitis that can cause visual impairment, and even endanger the patient's life.

[0003] Monkeypox virus (MPXV) is classified in the genus Orthopoxvirus of the family Poxviridae. The virus has two different evolutionary branches: Branch I, formerly known as the Central African (Congo Basin) branch; Branch II, formerly known as the West African branch, which contains subbranches IIa and IIb. Compared with branch I, branch II is associated with milder disease, fewer deaths, and limited human-to-human transmission. Genome sequencing results show that the variant strain of this monkeypox epidemic belongs to subbranch IIb. According to the characteristics of the virus, subbranch IIb is divided into A.1, A.1.1, A.2, A.2.1, A.2.2, A.3 and B.1 lineages. Most patients are infected with virus strains belonging to the B.1 lineage, and a small number of patients are still infected with the A.2 lineage.

[0004] In addition to monkeypox virus, members of the genus Orthopoxvirus also include variola virus, vaccinia virus (VACV), cowpox virus and molluscum contagiosum virus (MCV), most of which can cause systemic rashes. Phylogenetic analysis shows that these viruses originated from a common ancestor and gradually evolved during the evolution process. Recombination can occur between different species of the genus Orthopoxvirus. Poxviruses of the same genus have similar antigenicity and cross-reactions, that is, the immune response induced by one virus infection can also resist infection by other viruses. For example, the immune response induced by the smallpox vaccine and the monkeypox virus has cross-reactions.

[0005] In 1926, an attenuated strain, the "Vaccinia Virus Tiantan Strain", was isolated from a smallpox patient and passaged through monkeys, rabbits, cattle and other animals. The Vaccinia Virus Tiantan Strain has been used as a vaccine to prevent smallpox in my country. It has been vaccinated to hundreds of millions of people, with good efficacy and safety, and has played a huge role in the eradication of smallpox in my country. After smallpox was eradicated, the vaccine strain was used as a viral expression vector for the development of a variety of recombinant vaccines. In view of the effectiveness of smallpox vaccination in preventing monkeypox and its good safety, the Vaccinia Virus (Tiantan Strain) is a good alternative model for monkeypox virus and has the potential value for the immune prevention of monkeypox.

[0006] Sequencing analysis showed that the genetic similarity between the VACV reference sequence and the MPXV-2022 sequence was approximately 84%, the latter containing approximately 13% insertions / deletions and 3% single nucleotide polymorphisms. In addition, the derived T-cell epitopes of VACV were 71.6% similar to those of MPXV-2022, which were 89.2% similar to the T-cell epitopes of MPXV-CB (Congo Basin branch). The above results indicate that the two homologous sequences of MPXV, MPXV-2022 and MPXV-CB, have genetic similarities with the VACV reference sequence, which further confirms that vaccinia virus is a good alternative model for monkeypox virus.

[0007] Currently, there is no licensed treatment for monkeypox in humans. The oral smallpox drugs brincidofovir and tecovirima have shown good results in different animal models of orthopoxvirus and monkeypox virus. The combination of brincidofovir and tecovirima further improves the effectiveness of the drug. Tecovirima is very effective against the mortality of primate monkeypox virus, and further studies should be implemented to verify the effectiveness of the drug. Modified vaccinia (MVA) is a smallpox vaccine with an efficacy of 85% in preventing monkeypox, but the protection rate of MVA against monkeypox under new and persistent mutations needs to be further confirmed. At the same time, there is still another concern that too many injections of vaccines such as influenza during pandemics may cause potential adverse problems. Safe and effective therapeutic drugs remain the focus of monkeypox virus epidemic prevention and control. Therefore, it is of great significance to develop safe, effective and broad-spectrum antiviral drugs for monkeypox virus infection and diseases caused by orthopoxvirus infection that may appear in the future. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides the use of raltitrexed in the preparation of a drug for preventing, alleviating and / or treating diseases caused by poxvirus infection.

[0009] According to an embodiment of the present invention, the poxvirus comprises an orthopoxvirus.

[0010] According to an embodiment of the present invention, the orthopoxvirus includes: monkeypox virus, vaccinia virus, smallpox virus, cowpox virus and molluscum contagiosum virus.

[0011] According to an embodiment of the present invention, the monkeypox virus includes the Central African (Congo Basin) branch and the West African branch.

[0012] According to an embodiment of the present invention, the West African clade comprises subclades IIa and IIb.

[0013] According to an embodiment of the present invention, the subclade IIb includes A.1, A.1.1, A.2, A.2.1, A.2.2, A.3 and B.1 lineages.

[0014] According to an embodiment of the present invention, the raltitrexed prevents poxviruses such as monkeypox virus and vaccinia virus, or treats diseases caused by poxviruses such as monkeypox virus and vaccinia virus, or alleviates or improves diseases caused by infection with poxviruses such as monkeypox virus and vaccinia virus.

[0015] According to an embodiment of the present invention, the inhibition of poxviruses such as monkeypox virus and vaccinia virus may be at the organism level or the cell level.

[0016] According to an embodiment of the present invention, the symptoms caused by infection with poxviruses such as monkeypox virus and vaccinia virus may be at least one of the following symptoms: fever, drowsiness, severe headache, muscle pain, swollen lymph nodes, ulcers in the mouth about 1-3 days after the fever, rashes in other parts of the body, etc.

[0017] According to an embodiment of the present invention, the raltitrexed can inhibit poxviruses at two stages, before entering the host cell (i.e., before endocytosis) and after entering the host cell (i.e., after endocytosis). Therefore, the raltitrexed is suitable for preventing, alleviating and / or treating diseases caused by poxviruses such as monkeypox virus and vaccinia virus infection.

[0018] The present invention also provides a pharmaceutical composition for alleviating and / or treating diseases caused by poxvirus infection, which comprises: raltitrexed and other drugs for treating diseases caused by poxvirus infection, such as brincidofovir and / or tecovirima.

[0019] According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0020] According to an embodiment of the present invention, the pharmaceutically acceptable excipient includes one, two or more of a physiologically or pharmaceutically acceptable carrier, diluent, vehicle and / or excipient.

[0021] According to an embodiment of the present invention, the pharmaceutical composition is in the form of a preparation selected from granules, tablets, capsules, solutions, suspensions and semisolid preparations.

[0022] The present invention also provides a method for preventing, alleviating and / or treating diseases caused by poxvirus infection, comprising: administering raltitrexed to an individual in need thereof.

[0023] According to an embodiment of the present invention, the method further comprises administering other drugs for treating diseases caused by poxvirus infection, such as brincidofovir and / or tecovirima, in combination with raltitrexed.

[0024] Beneficial Effects

[0025] The inventors have found that raltitrexed has good inhibitory activity against poxviruses, such as orthopoxviruses including monkeypox virus and vaccinia virus. Therefore, raltitrexed of the present invention has important clinical application value for the treatment of diseases caused by poxviruses, such as orthopoxviruses including monkeypox virus and vaccinia virus.

[0026] The inventors found that raltitrexed has good inhibitory activity against vaccinia virus (VACV). 50 =0.003μM, CC 50 >100μM, SI>10000. The above results indicate that raltitrexed has great potential clinical application value in the prevention and treatment of diseases caused by poxviruses, such as monkeypox virus and vaccinia virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a one-step growth curve of the Tiantan strain of vaccinia virus.

[0028] Figure 2 EC of raltitrexed against vaccinia virus Tiantan strain in Vero E6 cells 50 Experimental results.

[0029] Figure 3 This is a graph showing the test results of different action stages of raltitrexed on Vero E6 cells infected with the Tiantan strain of vaccinia virus.

[0030] Figure 4 This is a graph showing the test results of different action stages of different concentrations of raltitrexed on Vero E6 cells infected with the Tiantan strain of vaccinia virus.

[0031] Figure 5 This is a graph showing the test results of the adsorption / internalization phase of raltitrexed on Vero E6 cells infected with the Tiantan strain of vaccinia virus. DETAILED DESCRIPTION

[0032] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0033] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0034] Example 1 Test of the anti-vaccinia virus activity of raltitrexed

[0035] 1. Raltitrexed inhibits vaccinia virus (VACV)

[0036] 1) Cell virus culture

[0037] Vero E6, an African green monkey kidney cell line, was obtained from the American Type Culture Collection (ATCC, No. 1586) and cultured in a 37°C, 5% CO2 incubator using DMEM medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco Invitrogen).

[0038] 2) Raltitrexed inhibits the EC of the Tiantan strain of vaccinia virus 50 and CC 50 Determination

[0039] EC 50 Detection: 2.5×10 4Vero E6 cells were inoculated into 96-well plates and cultured in an incubator at 37°C and 5% CO2 for 24 h. Then, raltitrexed solutions with final concentrations of 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.312 μM, 0.156 μM, 0.078 μM, 0.039 μM, 0.019 μM, 0.01 μM, 0.005 μM, 0.0025 μM, 0.00125 μM, 0.000625 μM, 0.0003125 μM, 0.000156 μM, 0.000078 μM, 0.000039 μM, 0.000019 μM, and 0 μM were added to the cell culture wells, respectively. The cells were infected with vaccinia virus, washed three times with PBS 2 hours after infection, and the medium containing only the drug was added. The final concentrations were 10μM, 5μM, 2.5μM, 1.25μM, 0.625μM, 0.312μM, 0.156μM, 0.078μM, 0.039μM, 0.019μM, 0.01μM, 0.005μM, 0.0025μM, 0.00125μM, 0.000625μM, 0.0003125μM, 0.000156μM, 0.000078μM, 0.000039μM, 0.000019μM, 0μM, and cultured in an incubator at 37°C, 5% CO2. The cytopathic effect was observed under a microscope 48 hours after infection with the Tiantan strain of vaccinia virus, and the expression of viral RNA and the reference gene GAPDH in the cells was quantitatively detected by qRT-PCR. GraphPad-Prism 8 software was used for data analysis and calculation of EC 50 .

[0040] CC 50 Detection: CC using CellTiter-Blue method 50 Vero E6 cells were seeded into 96-well cell culture plates, and the test was carried out when the cell density reached 60%-80%. After the Vero E6 cells were replaced with diluted drugs, the final concentrations were 10μM, 5μM, 2.5μM, 1.25μM, 0.625μM, 0.312μM, 0.156μM, 0.078μM, 0.039μM, 0.019μM, 0.01μM, 0.005μM, 0.0025μM, 0.00125μM, 0.000625μM, 0.0003125μM, 0.000156μM, 0.000078μM, 0.000039μM, 0.000019μM, 0μM. The cells were cultured at 37°C with 5% CO2 for 48 h. The luminescence intensity at 593 nm was detected using CellTiter-Blue reagent. The data were analyzed and CC was calculated using GraphPad-Prism 8 software.50 .

[0041] 3) Raltitrexed dosing time experiment

[0042] 1×10 5 Vero E6 cells were inoculated into 24-well plates and cultured in an incubator at 37°C and 5% CO2 for 24 hours. Then, 0.15 μM raltitrexed (or 0.03 μM, 0.06 μM, 0.15 μM raltitrexed in gradient dilution) was added to the cell culture wells during the whole period and before cell entry. At the same time, the cells were infected with virus at MOI=0.01 in all experimental groups and the virus control group, and the cells were infected for 2 hours at 37°C and 5% CO2. After 2 hours of infection, the cells were washed with PBS for 3 times and the medium was replaced. The medium containing only the drug was added to the cell culture wells during the whole period and after cell entry. The medium without the drug was added to the cell culture wells before cell entry and the control group. The final concentration of the medium containing the drug was 0.15 μM (or the final concentration of the gradient dilution was 0.03 μM, 0.06 μM, 0.15 μM), and the cells were cultured in an incubator at 37°C and 5% CO2. Ten hours after virus infection, the expression of viral RNA and the reference gene GAPDH in cells was quantitatively detected by qRT-PCR, and data were analyzed using GraphPad-Prism 8 software.

[0043] 4) Determination of the inhibitory effect of raltitrexed on virus adsorption / internalization

[0044] 1×10 5 Vero E6 cells were inoculated into a 24-well plate, cultured in an incubator at 37°C and 5% CO2 for 24 hours, and then raltitrexed with a final concentration of 0.15 μM was added to the cell culture wells for determining virus adsorption, and the cells were infected with viruses at MOI=5. Raltitrexed was not added to the cell culture wells for determining virus internalization, and the cells were directly infected with viruses at MOI=5. The cell culture plate was then placed at 4°C for 2 hours to allow the virus to fully adsorb on the cell surface, and then the supernatant in the cell culture wells for determining virus adsorption was discarded, and the cells were gently rinsed with ice PBS (previously placed in a 4°C refrigerator) (washed 3 times), and then the expression of cell surface viral RNA and cell internal reference gene GAPDH was quantitatively detected by qRT-PCR. Data analysis was performed using GraphPad-Prism 8 software.

[0045] For the cell culture wells for measuring virus internalization, after gently rinsing with ice PBS three times, add raltitrexed with a final concentration of 0.15 μM to the wells, continue to culture in an incubator at 37°C and 5% CO2 for 1 hour, then gently rinse with ice PBS once, then add DMEM medium pre-dissolved with proteinase K, add 150 microliters (containing 0.5 microliters of proteinase K) to each well, then continue to place in an incubator at 37°C and 5% CO2, observe under a microscope every 1 minute, and after the cell morphology becomes round, gently aspirate the cell-containing medium, place it in a 1.5mL centrifuge tube, centrifuge at 500rpm for 3 minutes, discard the supernatant, resuspend with PBS, discard the supernatant, and quantitatively detect the expression of viral RNA and the cell reference gene GAPDH in the cell by qRT-PCR. Data analysis was performed using GraphPad-Prism 8 software.

[0046] 5) Virus sample processing and detection

[0047] RNA was extracted using the Axygen TM Multi-Purpose Total RNA Mini Kit (Axygene, Catalog No. AP-MN-MS-RNA-250G) according to the manufacturer's instructions. Reverse transcription was performed using the Hifair II 1st strand cDNA Synthesis Kit (Shanghai Yisheng Biotechnology, Catalog No.: 11121ES60) that can digest gDNA, and qRT-PCR was performed using Quantstudio Real-Time PCR Detection Reagent (Applied biosystems, Foster City, CA, USA). SYBR-Green qPCR amplification was performed: 95°C for 5 min, 40 cycles, 95°C for 10 s, 55°C for 20 s, and 72°C for 31 s. Normalization was achieved by detecting the GAPDH gene.

[0048] 2. Experimental results

[0049] The one-step growth curve of the Tiantan strain of vaccinia virus is shown in Figure 1 ,Depend on Figure 1 It can be seen that when cells were infected with MOI = 0.1, the viral load of the vaccinia virus Tiantan strain reached a peak at 24 hours after infection.

[0050] EC of raltitrexed against vaccinia virus Tiantan strain in Vero E6 cells 50 The experimental results are shown in Figure 2 ,Depend on Figure 2 It can be seen that different concentrations of raltitrexed have a good inhibitory effect on vaccinia virus and have a dose-dependent effect.

[0051] In addition, by Figure 2 It can be seen that the EC of raltitrexed against vaccinia virus in Vero E6 cells 50=0.003μM, CC 50 >100μM, SI>10000.

[0052] Figure 3 The results of the test of different action stages of raltitrexed on Vero E6 cells infected with the Tiantan strain of vaccinia virus. Figure 3 It can be seen that raltitrexed at a concentration of 0.15 μM can exert an inhibitory effect on vaccinia virus infection of Vero E6 cells both before and after entry into the cells.

[0053] Figure 4 The results of the test of different concentrations of raltitrexed on the Vero E6 cells infected with the Tiantan strain of vaccinia virus at different stages. Figure 4 It can be seen that at a low concentration (0.03 μM), the effect of raltitrexed on the pre-cytosolic stage of vaccinia virus infection of Vero E6 cells becomes weaker, but it can still effectively inhibit the post-cytosolic stage, and the inhibitory effect of raltitrexed on vaccinia virus infection of Vero E6 cells is generally dose-dependent.

[0054] Figure 5 This is a test result of the adsorption / internalization phase of raltitrexed on Vero E6 cells infected with the Tiantan strain of vaccinia virus. Figure 5 It can be seen that raltitrexed at a concentration of 0.15 μM can inhibit the adsorption and internalization stages of vaccinia virus infection of Vero E6 cells before entry into the cells, but the degree of inhibition on internalization is stronger.

[0055] In summary, raltitrexed has a good dose-dependent antiviral effect and low cytotoxicity against vaccinia virus infection in Vero E6 cells. However, there is currently no specific drug for monkeypox virus and vaccinia virus, so the present invention has important reference significance for the clinical use of monkeypox virus and vaccinia virus in the future.

[0056] Given the strong inhibitory effect of raltitrexed on vaccinia virus observed, raltitrexed is very likely to become a specific drug for diseases caused by orthopoxvirus infections including monkeypox virus and vaccinia virus. The inventor believes that as a potential drug for treating orthopoxvirus infections including monkeypox virus and vaccinia virus, raltitrexed has significant medicinal value and is a promising candidate drug for treating diseases caused by orthopoxvirus infections including monkeypox virus and vaccinia virus.

[0057] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of raltitrexed in the preparation of a drug for preventing, alleviating and / or treating diseases caused by poxvirus infection.

2. The use according to claim 1, characterized in that: The poxviruses include orthopoxviruses.

3. The use according to claim 1 or 2, characterized in that: The orthopoxviruses include: monkeypox virus, vaccinia virus, smallpox virus, cowpox virus and molluscum contagiosum virus.

4. The use according to any one of claims 1 to 3, characterized in that: The monkeypox virus includes the Central African branch and the West African branch.

5. The use according to claim 4, characterized in that: The Central African branch is the Congo Basin branch.

6. The use according to any one of claims 1 to 5, characterized in that: The West African clade includes subclades IIa and IIb.

7. The use according to any one of claims 1 to 6, characterized in that: The subclade IIb includes the A.1, A.1.1, A.2, A.2.1, A.2.2, A.3 and B.1 lineages.

8. A pharmaceutical composition for alleviating and / or treating diseases caused by poxvirus infection, characterized in that: Includes raltitrexed and other medicines that treat diseases caused by poxvirus infections.

9. The pharmaceutical composition according to claim 8, characterized in that The other drugs for treating diseases caused by poxvirus infection are brincidofovir and / or tecovir.

10. The pharmaceutical composition according to claim 8 or 9, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable excipient; The pharmaceutically acceptable excipients include one, two or more of physiologically or pharmaceutically acceptable carriers, diluents, vehicles and / or excipients.

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