Use of raltitrexed in treating diseases caused by poxvirus infections

Abstract

Provided is a use of raltitrexed in relieving and / or treating diseases caused by poxvirus infections. In view of the observed strong inhibition effect of raltitrexed on poxviruses including monkeypox virus and vaccinia virus, raltitrexed is very likely to become a wonder drug for diseases caused by poxvirus infections including monkeypox virus and vaccinia virus. As a potential drug for treating diseases caused by poxvirus infections including monkeypox virus and vaccinia virus, raltitrexed has great medicinal value and is a promising candidate drug for treating diseases caused by poxvirus infections including monkeypox virus and vaccinia virus.

Description

Application of raltitrexed in the treatment of diseases caused by poxvirus infection

[0001] This application claims priority to an earlier application filed with the State Intellectual Property Office of China on September 4, 2023, with patent number 2023111319166, entitled “Use of raltitrexed in the treatment of diseases caused by poxvirus infection.” The entire text of that application is incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of medical technology and relates to a new use of the drug raltitrexed, and specifically relates to the use of raltitrexed in preventing and / or treating diseases caused by poxvirus infection. Background Art

[0003] On February 15, 2023, the World Health Organization announced that the monkeypox outbreak in multiple countries continued to constitute a "Public Health Emergency of International Concern." Currently, more than 30 countries (regions) continue to report cases of monkeypox. Some other countries (regions) may have insufficient testing and reporting of confirmed cases. After infection with the monkeypox virus (MPXV), patients often show symptoms of fever, drowsiness, muscle pain, headache, and a widespread characteristic rash, accompanied by swollen lymph nodes, which is also one of the prominent features of infection with the monkeypox virus. In addition, monkeypox virus infection can 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.

[0004] Monkeypox virus (MPXV) is classified in the genus Orthopoxvirus, family Poxviridae. The virus has two distinct evolutionary branches: Clade I, formerly known as the Central African (Congo Basin) branch; and Clade II, formerly known as the West African branch, which includes subclades IIa and IIb. Compared with Clade I, Clade II is associated with milder disease, fewer deaths, and limited human-to-human transmission. Genome sequencing results show that the variant strain prevalent in this monkeypox outbreak belongs to subclade IIb. Based on the characteristics of the virus, subclade IIb is further divided into lineages A.1, A.1.1, A.2, A.2.1, A.2.2, A.3, and B.1. The majority of patients in this outbreak were infected with strains belonging to the B.1 lineage, with a small number of patients infected with the A.2 lineage.

[0005] In addition to monkeypox virus, members of the Orthopoxvirus genus also include variola virus, vaccinia virus (VACV), cowpox virus, and molluscum contagiosum virus (MCV). Most of these viruses can cause systemic rashes. Phylogenetic analysis suggests that these viruses descended from a common ancestor and have evolved gradually over the course of evolution. Different species of the Orthopoxvirus genus can undergo recombination, and poxviruses within the same genus share similar antigenicity, leading to cross-reactivity. This means that the immune response induced by one virus can also protect against infection with another virus. For example, the smallpox vaccine cross-reacts with the immune response induced by monkeypox virus.

[0006] In 1926, Qi Changqing, head of the Smallpox Vaccine Section of the Third Department of the Tiantan Epidemic Prevention Office in Beijing, my country, isolated a smallpox virus from a patient at the Beijing Infectious Diseases Hospital. After passage through monkeys, rabbits, and cattle, he developed an attenuated strain—the "Tiantan strain" of vaccinia virus. The Tiantan strain has been used as a smallpox vaccine in my country, with hundreds of millions of people vaccinated. Its effectiveness and safety have played a significant role in my country's smallpox eradication efforts. Following smallpox eradication, this vaccine strain was used as a viral expression vector in the development of various recombinant vaccines. Given the effectiveness of smallpox vaccination against monkeypox and its favorable safety profile, the Tiantan strain of vaccinia virus represents a promising surrogate model for monkeypox virus and holds potential for use in the prevention of monkeypox.

[0007] Sequencing analysis showed that the genetic similarity between the VACV reference sequence and the MPXV-2022 sequence was approximately 84%, with the latter containing approximately 13% insertions / deletions and 3% single nucleotide polymorphisms. In addition, the VACV-derived T cell epitope was 71.6% similar to the MPXV-2022-derived T cell epitope, which was 89.2% similar to the T cell epitope of MPXV-CB (Congo Basin branch). The above results indicate that the two homologous sequences of MPXV, MPXV-2022 and MPXV-CB, have genetic similarity with the VACV reference sequence, which further confirms that vaccinia virus is a good alternative model for monkeypox virus.

[0008] Currently, there are no licensed treatments for monkeypox in humans. The oral smallpox drugs brincidofovir and tecovirima have shown promising results in various animal models of orthopoxvirus and monkeypox virus. Combining brincidofovir with tecovirima further enhances the drug's effectiveness. Tecovirima is highly effective against monkeypox virus mortality in primates, and further studies are warranted to validate its efficacy. Modified vaccinia (MVA), a smallpox vaccine, is 85% effective in preventing monkeypox, but its protection against new and persistent mutations requires further confirmation. Furthermore, concerns remain regarding the potential adverse effects of overuse of vaccines such as influenza and the novel coronavirus during pandemics. Safe and effective therapeutics remain a priority for monkeypox prevention and control. Therefore, developing safe, effective, and broad-spectrum antiviral drugs is crucial for the prevention and control of monkeypox outbreaks and future orthopoxvirus-induced diseases.

[0009] Summary of the Invention

[0010] To solve the above technical problems, the present invention provides the use of raltitrexed in the preparation of a medicament for preventing, alleviating and / or treating diseases caused by poxvirus infection.

[0011] According to an embodiment of the present invention, the poxvirus comprises an orthopoxvirus.

[0012] According to an embodiment of the present invention, the orthopoxvirus includes: monkeypox virus, vaccinia virus, smallpox virus, cowpox virus and molluscum contagiosum virus.

[0013] According to an embodiment of the present invention, the monkeypox virus includes the Central African (Congo Basin) branch and the West African branch.

[0014] According to an embodiment of the present invention, the West African clade comprises subclades IIa and IIb.

[0015] According to an embodiment of the present invention, the subclade lib includes A.1, A.1.1, A.2, A.2.1, A.2.2, A.3 and B.1 lineages.

[0016] 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.

[0017] According to an embodiment of the present invention, the inhibition of poxviruses such as monkeypox virus and vaccinia virus can be at the organism level or the cellular level.

[0018] According to an embodiment of the present invention, the symptoms caused by infection with poxviruses such as monkeypox virus and vaccinia virus can 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 fever, rashes in other parts of the body, etc.

[0019] According to an embodiment of the present invention, raltitrexed can inhibit poxviruses at two stages: before entering the host cell (i.e., pre-endocytosis) and after entering the host cell (i.e., post-endocytosis). Therefore, raltitrexed is suitable for preventing, alleviating and / or treating diseases caused by poxviruses, such as monkeypox virus and vaccinia virus infection.

[0020] The present invention also provides a pharmaceutical composition that can alleviate and / or treat diseases caused by poxvirus infection, which comprises: raltitrexed and other drugs for treating diseases caused by poxvirus infection, such as brincidofovir and / or tecovirima.

[0021] According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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. Beneficial effects

[0026] The inventors have discovered that raltitrexed has excellent inhibitory activity against poxviruses, such as orthopoxviruses, including monkeypox virus and vaccinia virus. Therefore, raltitrexed of the present invention has important clinical application value in the treatment of diseases caused by poxviruses, such as monkeypox virus and vaccinia virus, including orthopoxvirus infections.

[0027] The inventors found that raltitrexed has good inhibitory activity against vaccinia virus (VACV). 50 =0.003μM, CC 50 >100 μM, SI>10000. The above results indicate that raltitrexed has great potential clinical application value in preventing and / or treating diseases caused by poxviruses, such as monkeypox virus and vaccinia virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 is a one-step growth curve of the Tiantan strain of vaccinia virus.

[0029] Figure 2 shows the EC of raltitrexed against vaccinia virus Tiantan strain in Vero E6 cells 50 Experimental results.

[0030] FIG3 shows the test results of different action stages of raltitrexed on Vero E6 cells infected with the Tiantan strain of vaccinia virus.

[0031] FIG4 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.

[0032] FIG5 is a graph showing the test results of the adsorption / internalization stage of raltitrexed on Vero E6 cells infected with the Tiantan strain of vaccinia virus. DETAILED DESCRIPTION

[0033] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative 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 encompassed within the scope of protection that the present invention is intended to protect.

[0034] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0035] Example 1 Test of anti-vaccinia virus activity of raltitrexed

[0036] 1. Raltitrexed inhibits vaccinia virus (VACV)

[0037] 1) Cell virus culture

[0038] Vero E6, an African green monkey kidney cell line, was obtained from the American Type Culture Collection (ATCC, No. 1586) and cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco Invitrogen) at 37°C in a 5% CO2 incubator.

[0039] 2) Raltitrexed inhibits the EC of the Tiantan strain of vaccinia virus 50 and CC 50 Determination

[0040] EC 50 Detection: 2.5×10 4 Vero E6 cells were seeded into 96-well plates and cultured in a 37°C, 5% CO2 incubator 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 0.01% vaccinia virus and 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, and 0μM, and cultured in an incubator at 37°C with 5% CO2. Cytopathic effects were observed under a microscope 48 hours after infection with the Tiantan strain of vaccinia virus. 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 to analyze the data and calculate the EC 50 .

[0041] CC 50 Detection: CC was performed using the CellTiter-Blue method 50Vero E6 cells were seeded into 96-well cell culture plates, and the test was performed 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, and 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, and the data were analyzed and calculated using GraphPad-Prism 8 software. 50 .

[0042] 3) Raltitrexed dosing time experiment

[0043] 1×10 5 Vero E6 cells were seeded into 24-well plates and cultured in a 37°C, 5% CO₂ incubator for 24 hours. Raltitrexed was then added to the wells for both the entire time period and before cell entry (or serial dilutions to final concentrations of 0.15 μM, 0.03 μM, 0.06 μM, and 0.15 μM, respectively). All experimental groups and the virus control group were infected with virus at an MOI of 0.01 for 2 hours at 37°C, 5% CO₂. After 2 hours of infection, cells were washed three times with PBS and the medium was replaced. Drug-only medium was added to the wells for both the entire time period and after cell entry, while drug-free medium was added to the wells for both the pre-invasion and control groups. The final concentration of drug-containing medium was 0.15 μM (or serial dilutions to final concentrations of 0.03 μM, 0.06 μM, and 0.15 μM, respectively). The cells were cultured in a 37°C, 5% CO₂ incubator. Ten hours after viral infection, qRT-PCR was used to quantify the expression of viral RNA and the reference gene GAPDH in cells. Data were analyzed using GraphPad-Prism 8 software.

[0044] 4) Determination of the inhibitory effect of raltitrexed on viral adsorption / internalization

[0045] 1×10 5Vero E6 cells were seeded into 24-well plates and cultured in a 37°C, 5% CO2 incubator for 24 hours. Raltitrexed was added to a final concentration of 0.15 μM in the wells used to measure viral adsorption, and the cells were infected with virus at an MOI of 5. Raltitrexed was not added to the wells used to measure viral internalization, and the cells were directly infected with virus at an MOI of 5. The plates were then placed at 4°C for 2 hours to allow sufficient viral adsorption to the cell surface. The supernatant from the wells used for viral adsorption was then aspirated and discarded. The cells were gently rinsed three times with ice-cold PBS (previously stored in a 4°C refrigerator). Quantitative RT-PCR was then performed to quantify the expression of viral RNA on the cell surface and the internal reference gene GAPDH. Data were analyzed using GraphPad-Prism 8 software.

[0046] For the cell culture wells assaying viral internalization, after gently rinsing three times with ice-cold PBS, raltitrexed was added to the wells at a final concentration of 0.15 μM. The cells were incubated in a 37°C, 5% CO2 incubator for 1 hour, then gently rinsed once with ice-cold PBS. DMEM culture medium pre-dissolved with proteinase K was added, with 150 μL (containing 0.5 μL of proteinase K) added to each well. The cells were then incubated in a 37°C, 5% CO2 incubator and observed under a microscope every minute. When the cells became rounded, the culture medium containing the cells was gently aspirated and placed in a 1.5 mL centrifuge tube. The cells were centrifuged at 500 rpm for 3 minutes, the supernatant discarded, and the cells were resuspended in PBS. After discarding the supernatant, the expression of viral RNA and the cellular reference gene GAPDH in the cells was quantitatively detected by qRT-PCR. Data analysis was performed using GraphPad-Prism 8 software.

[0047] 5) Virus sample processing and testing

[0048] RNA was extracted using the Axygen™ Multipurpose 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) with gDNA digestion. Quantstudio Real-Time PCR Detection Reagents (Applied Biosystems, Foster City, CA, USA) were used for qPCR amplification using the SYBR Green method: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s, 55°C for 20 s, and 72°C for 31 s. Normalization was achieved by detecting the GAPDH gene.

[0049] 2. Experimental Results

[0050] The one-step growth curve of the vaccinia virus Tiantan strain is shown in FIG1 . As shown in FIG1 , when cells were infected with an MOI of 0.1, the viral load of the vaccinia virus Tiantan strain reached a peak at 24 hours after infection.

[0051] EC of raltitrexed against vaccinia virus Tiantan strain in Vero E6 cells 50 The experimental results are shown in Figure 2. As shown in Figure 2, different concentrations of raltitrexed have a good inhibitory effect on vaccinia virus and have a dose-dependent effect.

[0052] In addition, as shown in Figure 2, the EC of raltitrexed against vaccinia virus in Vero E6 cells 50 =0.003μM, CC 50 >100μM, SI>10000.

[0053] Figure 3 shows the test results of raltitrexed at different stages of action on vaccinia virus Tiantan strain infected Vero E6 cells. As shown in Figure 3, raltitrexed at a concentration of 0.15 μM can inhibit vaccinia virus infection in Vero E6 cells at both the pre- and post-cytosolic stages.

[0054] Figure 4 shows the test results of different concentrations of raltitrexed on different stages of vaccinia virus infection in Vero E6 cells. As shown in Figure 4, at a low concentration (0.03 μM), raltitrexed has a weaker effect on the pre-cytosolic phase of vaccinia virus infection in Vero E6 cells, but still effectively inhibits the post-cytosolic phase. The inhibitory effect of raltitrexed on vaccinia virus infection in Vero E6 cells is generally dose-dependent.

[0055] Figure 5 shows the results of testing the adsorption and internalization phases of raltitrexed in Vero E6 cells infected with the Tiantan strain of vaccinia virus. As shown in Figure 5, raltitrexed at a concentration of 0.15 μM inhibits both the adsorption and internalization phases of the pre-cytosolic process of vaccinia virus infection in Vero E6 cells, with a greater degree of inhibition of internalization.

[0056] In summary, raltitrexed demonstrated a dose-dependent antiviral effect and low cytotoxicity against vaccinia virus infection in Vero E6 cells. Currently, there are no specific drugs for monkeypox virus and vaccinia virus. Therefore, this invention holds important implications for the future clinical use of monkeypox virus and vaccinia virus.

[0057] Given its observed strong inhibitory effect against vaccinia virus, raltitrexed has great potential as a specific drug for diseases caused by orthopoxviruses, including monkeypox and vaccinia viruses. The inventors believe that raltitrexed has significant medicinal value as a potential treatment for orthopoxvirus infections, including monkeypox and vaccinia viruses, and is a promising candidate for the treatment of diseases caused by orthopoxviruses, including monkeypox and vaccinia viruses.

[0058] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection 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.

Images