Use of jidalisib in the preparation of a medicament for combating monkeypox virus

Abstract

The application provides application of Gidalizie in preparation of an anti-monkeypox virus drug, and proves that the multi-target inhibitor Gidalizie can effectively inhibit replication of the monkeypox virus at a low-toxicity concentration through in-vitro experiments, reveals a new use of Gidalizie as an anti-monkeypox virus drug, and 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 gidalisylse 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 in the family Poxviridae, and is related to viruses such as Variola virus and Vaccinia virus.

[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] Gedatolisib (CAS No.: 1197160-78-3, molecular formula: C) 32 H 41Gidaricare (N9O4), also known as PKI-587 or PF-05212384, is a novel, potent, and reversible pan-PI3K / mTOR dual inhibitor that simultaneously targets all type I PI3K subtypes (α / β / γ / δ), mTORC1, and mTORC2. It exhibits highly selective multi-target inhibition, overcoming drug resistance by blocking multiple nodes to prevent compensatory activation. Compared to single-isomer selective PI3K inhibitors or single-target mTOR inhibitors, this drug effectively delays the development of drug resistance by blocking the multi-node compensatory mechanism of the PAM pathway. Gidaricare was initially developed by Pfizer and its clinical development was further advanced by Celcuity. Clinical studies have shown that in a phase III clinical trial for the treatment of HR+ / HER2- advanced breast cancer resistant to PI3KCA wild-type and CDK4 / 6 inhibitors, it achieved the clinically meaningful improvement in progression-free survival (PFS), setting a new record for the best median progression-free survival (mPFS) in this indication.

[0006] The above studies suggest that gidalithide may be more effective in treating monkeypox-infected individuals or as an interventional treatment for high-exposure populations. However, there are currently no studies on gidalithide as an anti-monkeypox drug. Summary of the Invention

[0007] This invention proposes the application of gidalisole 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.

[0008] The technical solution of this invention is implemented as follows: This invention proposes the use of gidalisyl or a pharmaceutically acceptable salt thereof in the preparation of anti-monkeypox virus drugs.

[0009] 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.

[0010] Furthermore, the application is to achieve anti-monkeypox virus by administering an effective dose of gidaliside or a pharmaceutically acceptable salt thereof to the subject.

[0011] Preferably, the subject includes a mammal; the mammal includes a human.

[0012] Furthermore, the drug includes pharmaceutically acceptable excipients.

[0013] 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.

[0014] Furthermore, the dosage form of the drug is tablets, capsules, granules, powders, or liquid preparations.

[0015] 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.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention demonstrates that the multi-target inhibitor gidalithide can effectively inhibit the replication of monkeypox virus at low toxic concentrations, revealing a new use of gidalithide 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

[0017] 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.

[0018] Figure 1 The results of the cytotoxicity assay of Gidarisoprostol on A549 cells in Example 1 of this invention are shown.

[0019] Figure 2 This is a pharmacodynamic curve of gidalisol inhibiting monkeypox virus replication in Example 2 of the present invention. Detailed Implementation

[0020] 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.

[0021] This invention, through systematic in vitro experiments, is the first to demonstrate that the multi-target inhibitor gedatolisib can effectively inhibit monkeypox virus replication at low toxic concentrations, and its EC50... 50The value was 3.03 μM. This discovery reveals a new use for gidalisole as an anti-monkeypox virus drug, providing important scientific evidence and candidate compounds for the development of novel, safe, and effective monkeypox treatments.

[0022] Based on this, the present invention provides the use of gidalisyl or a pharmaceutically acceptable salt thereof in the preparation of anti-monkeypox virus drugs.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] The therapeutically effective dose described in this invention refers to a pharmaceutically considered effective dosage, i.e., an amount of gidalisyl 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, or divided into multiple daily doses, or at intervals.

[0028] 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.

[0029] In this invention, the dosage form of the drug is tablet, capsule, granule, powder, or liquid preparation.

[0030] 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.

[0031] This invention also provides the use of gidarisothi 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.

[0032] Example 1: Cytotoxicity assay of Gidarisoprost in A549 cells

[0033] This embodiment aims to evaluate the in vitro toxicity of gidalisol to host cells A549 in order to determine the safe concentration range for subsequent antiviral experiments.

[0034] 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.

[0035] 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 a gidalisol treatment group, a tecovirimat positive control group, a blank control group, and a cell control group. Gidalisol and tecovirimat were administered 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.

[0036] 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.

[0037] The results of the cytotoxicity assay of Gidarisoprostol on A549 cells are shown in the figure below. Figure 1 As shown. Figure 1 The horizontal axis represents the concentration of gidalisol (μM), and the vertical axis represents the percentage of cytotoxicity (%). Figure 1 The green curve represents the gidaritase treatment group, and the gray curve represents the positive control drug tecovirimat treatment group. The results show that gidaritase at a concentration of 5 μM inhibited A549 cells by approximately 12.73%, indicating low cytotoxicity and good safety within the tested concentration range.

[0038] Example 2: Efficacy evaluation of gidalisylpropionate in inhibiting monkeypox virus replication in vitro

[0039] This embodiment aims to evaluate the inhibitory effect of guidalise on monkeypox virus replication in an A549 cell model.

[0040] 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.

[0041] 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%, 1%, and 5 μM in 48-well plates and incubated overnight. The culture medium was discarded, and 2% FBS DMEM maintenance medium containing different concentrations of tecorimex (0, 0.5, 1, and 5 μM) was added, 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.

[0042] 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.

[0043] 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): 12.5 μL 2×One Step RT-PCR Buffer III, 0.5 μL Takara Ex TaqHS, 0.5 μL PrimeScript RT Enzyme Mix II, 1.5 μL forward primer (10 μM), 1.5 μL reverse primer (10 μM), 0.5 μL probe (10 μM), 2 μL RNA template, and RNase-free water to a final volume of 25 μL. 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), for 40 cycles.

[0044] The efficacy test results of gidarisoprost in inhibiting monkeypox virus replication are as follows: Figure 2 As shown, Figure 2In the graph shown, the horizontal axis represents the concentration of gidaridine (μM), and the vertical axis represents the viral replication inhibition rate (%). The green curve represents the gidaridine-treated group, and the gray curve represents the positive control group treated with tecovirimat. The results show that the inhibitory effect of gidaridine on monkeypox virus is dose-dependent, with an EC50% for monkeypox virus at an MOI of 0.1. 50 It is 3.03 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 gidaliside 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 gidaliside or a pharmaceutically acceptable salt thereof to the subject.

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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