Use of at9283 in the preparation of a medicine for combating monkeypox virus

Abstract

The application provides application of AT9283 in preparation of an anti-monkeypox virus drug. Through in-vitro experiments, it is verified that the multi-target kinase inhibitor AT9283 can effectively inhibit replication of the monkeypox virus at a low-toxicity concentration, and a new use of AT9283 as an anti-monkeypox virus drug is disclosed, which provides an important basis and a candidate compound for development of a new, safe and effective monkeypox treatment drug.

Description

Technical Field

[0001] This invention relates to the field of antiviral drug development technology, specifically the application of AT9283 in the preparation of anti-monkeypox virus drugs. Background Technology

[0002] Monkeypox is a zoonotic infectious disease caused by monkeypox virus (MPXV). MPXV is a double-stranded DNA virus belonging to the genus Orthopoxvirus of the family Poxviridae, and is related to smallpox virus (Variola virus) and vaccinia virus (Vaccinia virus). Monkeypox virus is mainly transmitted through contact with the rash, bodily fluids, respiratory droplets, and contaminated items (such as clothing and bedding) of infected individuals. Clinical symptoms typically include fever, severe headache, swollen lymph nodes, back pain, myalgia, and fatigue, followed by a characteristic rash that spreads from the face to other parts of the body. In recent years, monkeypox virus has continued to evolve, mutating rapidly, and its strains have become increasingly complex, increasing its transmissibility and the potential risk of immune evasion, posing new challenges to prevention and control efforts.

[0003] Currently, there are no approved specific treatments for monkeypox virus. Clinical treatment primarily focuses on symptomatic and supportive care and management of complications, aiming to alleviate symptoms and prevent and manage secondary infections. Although antiviral drugs used against smallpox, such as tecovirimat, are available for treating monkeypox under emergency use authorization, accessibility is limited, and there is a potential risk of viral resistance. Therefore, developing safe, effective, and readily available novel anti-monkeypox drugs is a critical and urgent issue that needs to be addressed.

[0004] Drug repurposing (drug repurposing) strategies have become a rapid and effective way to deal with the threat of emerging infectious diseases because they can make full use of known drug pharmacological, efficacy, safety, and pharmacokinetic data, significantly reducing R&D risks, shortening the R&D cycle and costs.

[0005] AT9283 (CAS: 896466-04-9, Molecular Formula: C 19 H 23 N7O2 is a multi-target kinase inhibitor. Studies have shown that AT9283 can effectively inhibit the activity of multiple kinases, including Aurora A / B, JAK2 / 3, Abl (T315I), and Flt3 (IC50). 50The effective value ranges from 1 to 30 nM, and it can inhibit the growth and survival of various solid tumors both in vitro and in vivo. Currently, AT9283 is mainly studied for its anti-tumor therapeutic applications. Given that AT9283 targets multiple cellular kinases, and viral replication is highly dependent on host cell mechanisms, theoretically, AT9283 may affect the viral replication cycle by interfering with host cell signaling pathways. However, to date, there have been no reports, either domestically or internationally, of AT9283 exhibiting activity against monkeypoxvirus or other orthopoxviruses. Summary of the Invention

[0006] This invention proposes the application of AT9283 in the preparation of anti-monkeypox virus drugs, improving the accessibility of anti-monkeypox virus drugs and providing more options for the development of safe and effective anti-monkeypox virus drugs.

[0007] The technical solution of this invention is implemented as follows: This invention proposes the use of AT9283 or a pharmaceutically acceptable salt thereof in the preparation of anti-monkeypox virus drugs.

[0008] Furthermore, the application includes at least one of the following: (1) Inhibit viral invasion of host cells; (2) Inhibit viral genome replication; (3) Inhibit viral protein processing or assembly; (4) Reduce the amount or activity of the virus; (5) Inhibit the release of the virus.

[0009] Furthermore, the application is to achieve anti-monkeypox virus by administering an effective dose of AT9283 or a pharmaceutically acceptable salt thereof to the subject.

[0010] Preferably, the subject includes a mammal; the mammal includes a human.

[0011] Furthermore, the drug includes pharmaceutically acceptable excipients.

[0012] Preferably, the pharmaceutically acceptable excipients include at least one of the following: diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, cosolvents, solubilizers, preservatives, pH adjusters, osmotic pressure adjusters, surfactants, coating materials, antioxidants, antibacterial agents, or buffers.

[0013] Furthermore, the dosage form of the drug is tablets, capsules, granules, powders, or liquid preparations.

[0014] Furthermore, the drug administration route includes at least one of the following: intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, nasal administration, sublingual administration, nebulized administration, or transdermal administration.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention demonstrates that the multi-target kinase inhibitor AT9283 can effectively inhibit the replication of monkeypox virus at low toxic concentrations, revealing a new use of AT9283 as an anti-monkeypox virus drug, and providing important evidence and candidate compounds for the development of novel, safe and effective monkeypox treatment drugs. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The results of the cytotoxicity assay of AT9283 on A549 cells in Example 1 of this invention are shown.

[0018] Figure 2 This is a pharmacodynamic curve of AT9283 inhibiting monkeypox virus replication in Example 2 of the present invention. Detailed Implementation

[0019] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] This invention, through systematic in vitro experiments, is the first to demonstrate that the multi-target kinase inhibitor AT9283 can effectively inhibit monkeypox virus replication at low toxic concentrations, and its EC50... 50 The value was 1.92 μM. This discovery reveals a new use for AT9283 as an anti-monkeypox virus drug, providing important scientific evidence and candidate compounds for the development of novel, safe, and effective monkeypox treatments.

[0021] Based on this, the present invention provides the use of AT9283 or a pharmaceutically acceptable salt thereof in the preparation of anti-monkeypox virus drugs.

[0022] In this invention, the "pharmaceutically acceptable salt" includes, but is not limited to: pharmaceutically acceptable acid addition salts, such as: salts of inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid and sulfuric acid, and salts of organic acids such as acetic acid, ethanesulfonic acid, benzenesulfonic acid, benzoic acid, citric acid, fumaric acid, gluconic acid, glycolic acid, hydroxyethanesulfonic acid, lactic acid, lactobionic acid, maleic acid, malic acid, mesylic acid, succinic acid, p-toluenesulfonic acid and tartaric acid; and salts of pharmaceutically acceptable bases selected from ammonium salts, alkali metal salts (such as sodium salts, potassium salts) and alkaline earth metal salts (such as magnesium salts, calcium salts), as well as salts of glycerol (2-amino-2-hydroxymethyl-1,3-propanediol), diethanolamine, lysine or ethylenediamine.

[0023] In this invention, "antiviral" refers to improving a condition before or after the onset of a disease or functional disorder. This degree of remission or prevention, measured by any standard technique, is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to an untreated control group under equivalent conditions. Beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state, delay or slowing of disease progression, improvement or mitigation of the disease state, and reduction.

[0024] In this invention, the antiviral applications include, but are not limited to, the following: (1) Inhibit viral invasion of host cells; (2) Inhibit viral genome replication; (3) Inhibit viral protein processing or assembly; (4) Reduce the amount or activity of the virus; (5) Inhibit the release of the virus.

[0025] In this invention, anti-monkeypox virus is achieved by administering a therapeutically effective amount of the drug to a subject. Subjects include, but are not limited to, mammals, including groups that are susceptible to carrying and transmitting monkeypox virus, such as humans, rodents (squirrels, mice), primates (monkeys, apes), and other mammals.

[0026] The therapeutically effective dose described in this invention refers to a pharmaceutically acceptable dosage, i.e., an amount of AT9283 or its pharmaceutically acceptable salt sufficient to significantly improve the condition without causing serious side effects. The daily dosage is typically 0.01-1000 mg / kg, preferably 0.01-500 mg / kg, or 0.01-400 mg / kg, or 0.01-300 mg / kg, or 0.01-200 mg / kg, or 0.01-150 mg / kg, or 0.01-100 mg / kg, or 0.01-50 mg / kg, or 0.01-40 mg / kg, or 0.01-30 mg / kg, or 0.01-20 mg / kg. It can be administered as a single daily dose, divided into multiple daily doses, or at intervals.

[0027] In this invention, the drug further includes pharmaceutically acceptable excipients. Preferably, the pharmaceutically acceptable excipients include at least one of the following: diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, cosolvents, solubilizers, preservatives, pH adjusters, osmotic pressure adjusters, surfactants, coating materials, antioxidants, antibacterial agents, or buffers.

[0028] In this invention, the dosage form of the drug is tablet, capsule, granule, powder, or liquid preparation.

[0029] In this invention, the drug administration route includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, nebulized administration, or transdermal administration; more preferably, oral administration.

[0030] This invention also provides the use of AT9283 or a pharmaceutically acceptable salt thereof in combination with other drugs to prepare anti-monkeypox virus drugs. Other drugs include, but are not limited to, known ones such as tecovirimat.

[0031] Example 1: Detection of AT9283 cytotoxicity in A549 cells

[0032] This embodiment aims to evaluate the in vitro toxicity of AT9283 to host cells A549 in order to determine the safe concentration range for subsequent antiviral experiments.

[0033] Cell culture: Human non-small cell lung cancer cells A549 (purchased from the American Type Culture Collection, ATCC) were revived and cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase were used for experiments.

[0034] Experimental grouping and treatment: After digesting A549 cells in the logarithmic growth phase, they were subjected to 2×10⁻⁶... 4 Cells were seeded at a density of 200 μL per well in 48-well culture plates. After 16 hours of culture until cell confluence reached approximately 80%, the supernatant was discarded and replaced with DMEM maintenance medium containing 2% FBS. The experiment included an AT9283 treatment group, a Tecovirimat positive control group, a blank control group, and a cell control group. AT9283 and Tecovirimat were prepared at four concentration gradients: 0, 0.5 μM, 1 μM, and 5 μM, with three replicates for each concentration. The blank control group had three replicates. The control group (DMSO) had three replicates.

[0035] Toxicity assay: After 72 hours of drug treatment, add 25 μL of CCK-8 solution to each well, mix gently, and incubate for 1-4 hours at 37°C in a 5% CO2 incubator. After sufficient color development, measure the absorbance (OD value) at 450 nm using a microplate reader.

[0036] The results of the cytotoxicity assay of AT9283 on A549 cells are shown in the figure below. Figure 1 As shown. Figure 1 The horizontal axis represents the concentration of AT9283 drug (μM), and the vertical axis represents the percentage of cytotoxicity (%). Figure 1 The yellow curve represents the AT9283 treatment group, and the gray curve represents the positive control group treated with tecovirimat. The results showed that AT9283 at a concentration of 5 μM inhibited A549 cells by approximately 29.3%, indicating low cytotoxicity and good safety within the tested concentration range.

[0037] Example 2: Efficacy evaluation of AT9283 in inhibiting monkeypox virus replication in vitro

[0038] This embodiment aims to evaluate the inhibitory effect of AT9283 on monkeypox virus replication in an A549 cell model.

[0039] Virus and Cells: Monkeypox virus (MPXV, West African branch strain, deposited at the Wuhan Institute of Virology, Chinese Academy of Sciences) was amplified in A549 cells, and the viral titer was determined using a plaque formation assay. A549 cell culture was performed as in Example 1.

[0040] Drug treatment and viral infection: A549 cells were treated at a rate of 2 × 10⁻⁶. 4Cells were seeded at a density of 0.5 μM / well in 48-well plates and cultured overnight. The culture medium was discarded, and 2% FBS DMEM maintenance medium containing different concentrations of AT9283 (0, 0.5, 1, 5 μM) was added to each well, with three replicates for each concentration. A positive control group (tecovirimat at the same concentration) and a virus control group (no drug) were also included. After 1 hour of drug pretreatment, the drug-containing culture medium was discarded and replaced with fresh maintenance medium containing the corresponding drug concentration. Monkeypox virus was inoculated at a multiplicity of infection (MOI) of 0.1, and the plates were incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0041] Viral load detection: RNA extraction: 48 hours after infection, cell culture supernatant and cell lysate were collected, and total viral RNA was extracted according to the instructions of the Qiagen ViralRNA Mini Kit.

[0042] qRT-PCR detection: The viral gene copy number was quantitatively detected using a one-step qRT-PCR method with TaqMan probes. The primers and probe sequences used targeted conserved genes of MPXV. Forward primer: 5'-AGTGGATTAACACCGGAACAA-3' Reverse primer: 5'-CGACCGCGCTAGAATTACAA-3' Probe: 5'-FAM-CGTACCAGCTATGTTTACTGCTGCGT-TAMRA-3' Reaction system (25 μL): 2×One Step RT-PCR Buffer III 12.5 μL, Takara Ex TaqHS 0.5 μL, PrimeScript RT Enzyme Mix II 0.5 μL, forward primer (10 μM) 1.5 μL, reverse primer (10 μM) 1.5 μL, probe (10 μM) 0.5 μL, RNA template 2 μL, and RNase-free water to 25 μL.

[0043] Reaction conditions: 42℃ for 5 min (reverse transcription); 95℃ for 10 s (pre-denaturation); then 95℃ for 5 s, 60℃ for 30 s (collect fluorescence signal), 40 cycles.

[0044] The efficacy test results of AT9283 in inhibiting monkeypox virus replication are as follows: Figure 2 As shown, Figure 2In the graph shown, the horizontal axis represents the AT9283 drug concentration (μM), and the vertical axis represents the viral replication inhibition rate (%). The yellow curve represents the AT9283 treatment group, and the gray curve represents the positive control group treated with tecovirimat. The results show that the inhibitory effect of AT9283 on monkeypox virus is dose-dependent, with an EC50% for monkeypox virus at an MOI of 0.1. 50 The value is 1.92 μM.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of AT9283 or a pharmaceutically acceptable salt thereof in the preparation of antimonoplasmosis drugs.

2. The application according to claim 1, characterized in that, Anti-monkeypox virus was achieved by administering an effective dose of AT9283 or a pharmaceutically acceptable salt thereof to the subjects.

3. The application according to claim 2, characterized in that, The subjects were mammals.

4. The application according to claim 3, characterized in that, The mammal in question is a human.

5. The application according to claim 1, characterized in that, The drug includes pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that, The pharmaceutically acceptable excipients include at least one of the following: diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, cosolvents, solubilizers, preservatives, pH adjusters, osmotic pressure adjusters, surfactants, coating materials, antioxidants, antibacterial agents, or buffers.

7. The application according to any one of claims 1-5, characterized in that, The dosage form of the drug is tablets, capsules, granules, powders, or liquid preparations.

8. The application according to any one of claims 1-5, characterized in that, The drug administration route includes at least one of the following: intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, nasal administration, sublingual administration, nebulized administration, or transdermal administration.

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