CN116211865B

CN116211865B - Application of compound Ibrutinib in preparation of antiviral drugs and pharmaceutical composition

Info

Publication number: CN116211865B
Application number: CN202310008765.9A
Authority: CN
Inventors: 彭辰; 牛康; 吴文学; 王玺茹
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2023-01-04
Filing date: 2023-01-04
Publication date: 2024-03-29
Anticipated expiration: 2043-01-04
Also published as: CN116211865A

Abstract

The invention provides an application of a compound Ibrutinib in preparing antiviral drugs and a pharmaceutical composition, and belongs to the technical field of biological medicines. The invention discovers that the kinase inhibitor Ibrutinib has broad-spectrum efficient antiviral activity for the first time, can efficiently inhibit 3 viruses (VACV-WR, VACV-MVA and LSDV) from infecting host cells and mice, obviously reduces the virus amount in the mice and relieves the infection symptoms. And the Ibrutinib has low cytotoxicity and no toxic or side effect on mice. The compound Ibrutinib can be used as a kinase inhibitor for effectively resisting various virus infections, and has wide application prospect in the fields of breeding industry and biomedicine.

Description

Application of compound Ibrutinib in preparation of antiviral drugs and pharmaceutical composition

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of a compound Ibrutinib in preparation of antiviral drugs and a pharmaceutical composition.

Background

In recent years, related researches on broad-spectrum antiviral preparations have been greatly advanced, the bottleneck and limitation that single host of the antiviral preparations and broad-spectrum preparations have relatively weak activity are broken through, and partial achievements are faced with application. Antiviral agent studies are based on two designs, one from the viral infection level and one from the host cell defense level. Antiviral agents are studied from the host cell defense level, i.e. by targeting proteins or molecules that are required for viral replication but are not important for the growth of the cell itself, to affect the life cycle of the virus, thereby achieving antiviral effects. The antiviral preparation for host cells can achieve the aim of broad-spectrum antiviral in a real sense, and can minimize the possibility of drug resistance generation, thus becoming an important research direction in the virology field. A number of anti-apoptotic signaling molecules and pathways associated with NF-. Kappa. B, PI3-K/AKT and STAT5 are regulated by BTK. The BTK-PI3K-AKT cell signal transduction pathway and the cell signal transduction pathway regulated by BTK such as NF-kappa B, STAT are important for virus replication. The virus such as VACV-WR, LSDV and the like has high infectivity, great harm to the breeding industry and human health, and no specific therapeutic medicine exists at present.

Disclosure of Invention

The invention aims to provide an application of a compound Ibrutinib in preparing antiviral drugs and a pharmaceutical composition, and discovers that a kinase inhibitor Ibrutinib has broad-spectrum efficient antiviral activity.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an application of a compound Ibrutinib in preparing antiviral drugs, wherein the compound Ibrutinib has a structure shown in a formula 1:

preferably, the antiviral drug is in the form of injection administration.

Preferably, the antiviral drug is in a cavity administration form.

Preferably, the virus comprises a poxviridae virus.

Preferably, the poxviridae include orthopoxvirus and capripoxvirus.

Preferably, the orthopoxvirus genus comprises vaccinia virus and the capripoxvirus genus comprises bovine nodular skin disease virus.

Preferably, the compound Ibrutinib is used in antiviral drugs at a concentration of 0.5-20 mu M.

The invention provides a pharmaceutical composition, the active ingredient of which comprises a compound Ibrutinib, wherein the compound Ibrutinib has a structure shown in a formula 1:

the invention provides an application of a compound Ibrutinib in preparing antiviral drugs, and discovers that the kinase inhibitor Ibrutinib has broad-spectrum efficient antiviral activity for the first time, can efficiently inhibit 3 viruses (VACV-WR, VACV-MVA and LSDV) of orthopoxvirus and capripoxvirus from infecting host cells and mice, obviously reduces the viral load in mice, relieves the infection symptoms, provides an alternative for developing safe and reliable antiviral drugs aiming at poxvirus infection, provides a basis and possibility for developing broad-spectrum efficient antiviral new drugs, and provides a novel safe and reliable technical means for guaranteeing sustainable development of agriculture and animal husbandry and human health. The results of the examples show that the compounds ibutinib can inhibit 3 viruses (VACV-WR, VACV-MVA, LSDV) of orthopoxvirus and capripoxvirus from infecting host cells (HeLa cells, MDBK cells) with high efficacy at a concentration of 0.5 to 20 μm, and can inhibit virus infection of mice with high efficacy at a dose of 2.5mg/kg body weight, significantly reducing or even eliminating symptoms in infected animals. When the concentration of the drug (compound Ibrutinib) is 10 mu M, the virus titer can be reduced by 484.2 times, and the Ibrutinib has lower cytotoxicity and no toxic or side effect on mice. The compound Ibrutinib can be used as a kinase inhibitor for effectively resisting various virus infections, and has wide application prospect in the fields of breeding industry and biomedicine.

Drawings

FIG. 1 is a graph showing the results of 10. Mu.M Ibrutinib inhibiting VACV-WR virus titres on THP1 cells;

FIG. 2 is a graph showing the results of Ibrutinib inhibiting VACV-WR viral titers on THP1 cells;

FIG. 3 is a graph of cytotoxicity results of different doses of Ibrutinib (1, 5, 10, 20. Mu.M) against THP 1;

FIG. 4 is a graph showing the results of 10. Mu.M Ibrutinib inhibiting VACV-WR virus titer on A549 cells;

FIG. 5 is a graph showing the results of ibutinib inhibiting VACV-WR viral titers on A549 cells;

FIG. 6 is a graph of the results of different doses of Ibrutinib (1, 5, 10, 20. Mu.M) on A549 cytotoxicity;

FIG. 7 is a graph showing the result of inhibiting transcription of the intermediate gene D13LmRNA of VACV-WR by Ibrutinib;

FIG. 8 is a graph showing the results of Ibrutinib inhibiting transcription of the A3LmRNA of the VACV-WR late gene;

FIG. 9 is a graph showing the results of Ibrutinib interference VACV-WR DNA synthesis;

FIG. 10 is a graph showing the results of a mouse weight curve;

FIG. 11 is a graph showing the results of a mouse survival curve;

FIG. 12 is a graph of the results of Ibrutinib inhibition of lung toxic load in mice;

FIG. 13 is a graph showing the results of Ibrutinib inhibition of spleen viral load in mice;

FIG. 14 is a graph showing the results of Ibrutinib inhibition of renal viral load in mice;

FIG. 15 is a graph of results of Ibrutinib inhibition of brain viral load in mice;

FIG. 16 is a graph showing the results of dose-dependent inhibition of VACV-MVA viral titers by Ibrutinib;

figure 17 is a graph of the results of dose dependent inhibition of LSDV viral titers by ibutinib.

Detailed Description

in the present invention, the compound ibutinib is preferably derived from a small molecule library of compounds from selectk corporation.

In the present invention, the antiviral drug is preferably in the form of an injection administration type or a cavity administration type.

In the present invention, the virus preferably includes a poxviridae virus, preferably including orthopoxvirus genus and capripoxvirus genus; the orthopoxvirus genus preferably comprises vaccinia virus (VACV-WR, VACV-MVA); the capripoxvirus preferably comprises bovine nodular skin disease virus (LSDV). In an embodiment of the invention, the virus is in particular a VACV-WR, VACV-MVA or LSDV.

In the present invention, the compound ibutinib is preferably used at a concentration of 0.5 to 20 μm, more preferably 10 μm in an antiviral drug.

Ibutinib is an inhibitor that is effective in targeting BTK, i.e., by targeting BTK to affect its activity. BTK is involved in transcriptional regulation, and BTK and NF- κB form an autoregulation network, wherein BTK regulates not only its own transcription but also NF- κB transcription. The invention discovers that the knocking down of BTK by using siRNA technology can limit the replication capacity of poxvirus, and over-expression of BTK promotes poxvirus replication, further research shows that after poxvirus infects cells, the 223 rd tyrosine phosphorylation level of BTK increases along with the increase of the infection time, and BTK has a nuclear entering phenomenon during poxvirus infection. These phenomena indicate that BTK has the ability to positively regulate poxvirus replication. The present invention for the first time found that ibutinib can limit poxvirus replication by effectively targeting BTK.

A number of anti-apoptotic signaling molecules and pathways associated with NF-. Kappa. B, PI3-K/AKT and STAT5 are regulated by BTK. BTK is involved in activating the Ras/Raf/Mek/Erk signaling cascade, a mitogen-activated protein kinase (MAPK) pathway that can be widely activated and which transmits extracellular signals into the nucleus; when Raf activity is activated in the cell, the level of Erk phosphorylation downstream is increased, which in turn phosphorylates eIF4E by the downstream key regulator Mnk kinase, making the latter more prone to initiate cap structure-dependent translation. The present invention discovers for the first time that ibutinib can inhibit the invasion and replication of a variety of viruses by affecting the pathways described above.

the technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Routine experiments in the examples:

1. preparation of cells

HeLa cells were passaged using DMEM medium containing 10% fetal bovine serum and 1% green streptomycin, and the maintenance medium was DMEM medium containing 2.5% fetal bovine serum and 1% green streptomycin.

2. Culture of viruses

Culture of VACV-WR, VACV-MVA, LSDV cytotoxicity: about 10 flasks of HeLa, DF-1, BHK cells of T175 were cultured, virus stock was inoculated at 0.05MOI, the cells were scraped off 48 hours after infection, and transferred to a 50mL centrifuge tube, centrifuged at 2500rpm for 10min, and the supernatant was discarded. Resuspended with 1.5mL of 10mM Tris-Cl/each T175 flask and placed on ice. Cell suspension was lysed by homogenization 40 times in a glass homogenizer with a tight pestle, transferred to a 50mL tube and centrifuged at 300g for 5 minutes at 4 ℃ and the supernatant was saved; sonicating the combined supernatants (lysates) and sonicating on ice for 90 seconds; the sonicate was layered onto a 36% sucrose pad in a sterile centrifuge tube with a maximum tube volume of 38.5mL and the tube had to be filled to the top 2-3 mm. Make up volume with 10mM Tris-Cl (pH 9.0); the supernatant was aspirated and discarded by centrifugation at 33000g for 80 min, and the virions were resuspended with 1mL of 1mM Tris-Cl, pH9.0, and handled on ice.

3. Plaque assay for measuring viral titre

1) Trypsin digests the fused monolayer of BS-C-1 cells;

2) 5X105 BS-C-1 cells were plated per well in a 6-well plate, at 37℃overnight in a 5% carbon dioxide incubator,

3) 8 10-fold diluted viruses were prepared in DMEM medium (2% fbs,1% ps);

4) Removing the culture medium from the six-well plate, infecting cells in the wells with 1mL of 10-6, 10-7 and 10-8 virus dilutions (two replicates for each dilution), and after 1 to 2 hours in a carbon dioxide incubator, discarding the virus inoculum;

5) Cells were covered with 2mL DMEM (2% fbs,1% ps) containing 0.5% methylcellulose and placed in a carbon dioxide incubator for 2 days;

6) The medium was removed, 0.5mL of 0.1% crystal violet was added to each well and incubated at room temperature for 20min;

7) The crystal violet was blown in, the plate was rinsed with water, the wells were allowed to dry, and the titer was determined by counting plaques in the wells and multiplying by the dilution factor.

8) PFU/mL = number of plaques x dilution of virus ≡inoculation volume (mL).

4. Detection of cytotoxicity of drugs (MTT method)

1) The log phase cells were collected, the cell suspension concentration was adjusted, and the cells were plated in 96-well plates at 180. Mu.L per well and 3000-10000 cells/well.

2) Placing at 37deg.C 5% CO ₂ Culturing in an incubator to adhere cells to the wall, and culturing for 24 hours;

3) Test compounds were added at appropriate concentrations (5, 10, 20 μm) and incubation continued for 24h;

4) The supernatant was carefully aspirated, 90. Mu.L of fresh medium was added, and 10. Mu.L of MTT solution was added, and the culture was continued for 4 hours.

5) Then, the supernatant was aspirated, 110. Mu.L of Formazan solution was added to each well, and the mixture was placed on a shaker and shaken at a low speed for 10 minutes to sufficiently dissolve the crystals, and the absorbance of each well was measured at 490nm in an ELISA.

6) At the same time, zeroing wells (medium, MTT, formazan solution) and control wells (cells, drug-dissolving medium of the same concentration, medium, MTT, formazan solution) were set, and 3 multiplex wells were set for each group.

Example 1

Effect of the Compound Ibrutinib on VACV-WR Virus replication

1) THP1 cells were infected with VACV-WR strain at 3MOI, different doses of ibutinib (0,0.5,1,5, 10, 20 μm) were added simultaneously at the time of virus infection (prepared with DMSO, the same applies below), DMSO control was set simultaneously, after incubation for 2h, the supernatant was discarded, washed 2 times with PBS, cell maintenance solution was added, cell culture was incubated in an incubator for 24h, samples were collected for titration on BSC cells, and cytopathy was observed. The results showed that 10. Mu.M Ibrutinib was able to inhibit VACV-WR replication with reduced cytopathic effect compared to the non-dosed group (FIGS. 1, 10) ² ，10 ³ ，10 ⁴ Represents the virus dilution); the drug-loaded groups inhibited viral titers in comparison to the non-drug-loaded groups, which were dose-dependent (FIG. 2), and different doses of drug Ibutinib (1, 5, 10, 20 μm) was almost non-toxic to THP1 cells (fig. 3).

2) A549 cells were infected with 3MOI VACV-WR strain, different doses of Ibutinib (0,0.5,1,5, 10, 20. Mu.M) (prepared with DMSO, the same applies below) were added simultaneously at the time of virus infection, DMSO control was applied simultaneously, after incubation for 2 hours, the supernatant was discarded, washed 2 times with PBS, cell maintenance solution was added, cell incubator was cultured for 24 hours, samples were collected for titration on BSC cells, and cytopathy was observed. The results showed that 10. Mu.M Ibrutinib was able to inhibit VACV-WR replication with reduced cytopathic effect compared to the non-dosed group (FIGS. 4, 10) ² ，10 ³ ，10 ⁴ Represents the virus dilution); the dosing group inhibited viral titers and was dose dependent (fig. 5), and different doses of drug (1, 5, 10, 20 μm) were non-toxic to a549 cells (fig. 6) compared to the non-dosing group.

3) HeLa cells were infected with 3MOI VACV-WR for 8h, and DMSO, araC (40 ug/mL) or Ibrutinib (10. Mu.M) were added, respectively. After 2h incubation, the supernatant was discarded, washed 2 times with PBS, and cell maintenance fluid was added. After 8h of culture in a cell culture box, the supernatant is discarded, cells are collected, RNA is sampled and extracted at a designated time point, quantitative is 1ug, qPCR is performed by reverse transcription of cDNA, and real-time fluorescent quantitative PCR experiments are performed to detect mRNA transcription levels of the mid-stage gene D13L and the late-stage gene A3L of the virus. The results indicate that Ibrutinib inhibits transcription of the VACV-WR midterm gene D13L mRNA (FIG. 7), and that Ibrutinib inhibits transcription of the VACV-WR midterm gene A3L mRNA (FIG. 8).

4) HeLa cells VACV-WR were infected at 3MOI for 8h, 12h, and DMSO, araC (40 ug/mL) or Ibrutinib (10. Mu.M) were added, respectively. DNA was sampled at the indicated time points and qPCR was performed to detect virus E11L, and the results indicated that Ibrutinib interfered with VACV DNA synthesis (FIG. 9).

5) 16 Balb/c mice were equally divided into 4 groups, with no infection, infection + high dose (2.5 mg/kg), infection + medium dose (1.25 mg/kg), infection + low dose (0.625 mg/kg). Mice were anesthetized with isoflurane and then inoculated intranasally with WR at a lethal dose (10 ⁶ pfu). Mice began to receive treatment 1, 3, 5 days post infection. Ibrutinib (2.5 mg/kg,1.25mg/kg,0.625 mg/kg) is added daily and the mice are observed daily for each index. For survival studies, mice were weighed daily until death. At the whole processDuring the course of the experiment, mice weights were recorded daily for up to 14 days, and weight loss was calculated, indicating that ibutinib treatment group reduced virus-induced weight loss compared to the Vehicle-free group (Vehicle) (fig. 10). Survival experimental results showed that the ibutinib treated group prolonged survival of mice compared to the non-drug group (fig. 11). The viral load of each tissue organ (lung, spleen, kidney, brain) was titrated using plaque assay, and the results indicated that the ibutinib-treated group significantly reduced the viral load of lung, spleen, kidney, brain compared to the non-drug group, and had a dose dependence (fig. 12-15).

Example 2

Effect of the Compound Ibrutinib on VACV-MVA Virus replication

DF1 cells were infected with 3MOI VACV-MVA strain, different doses of Ibutinib (0,0.5,1,5, 10, 20. Mu.M) (prepared with DMSO) were added simultaneously during virus infection, DMSO control was added simultaneously, after incubation for 2 hours, the supernatant was discarded, washed 2 times with PBS, cell maintenance solution was added, the cells were cultured in a cell culture incubator for 24 hours, and samples were collected for titration on DF1 cells, and the results were shown in FIG. 16. The results in fig. 16 demonstrate that the dosing group inhibited viral titers in comparison to the non-dosing group, and was dose dependent.

Example 3

Effect of the Compound Ibrutinib on LSDV Virus replication

MDBK cells were infected with LSDV at 3MOI, different doses of Ibrutinib (0,0.5,1,5, 10, 20. Mu.M) (in DMSO) were added simultaneously at the time of virus infection, DMSO controls were simultaneously provided, after 2h incubation, the supernatant was discarded, washed 2 times with PBS, cell maintenance solution was added, the cells were incubated in a cell incubator for 72h, and samples were collected for titration on MDBK cells, and the results are shown in FIG. 17. The results in fig. 17 demonstrate that the dosing group inhibited viral titers in comparison to the non-dosing group, and was dose dependent.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The application of a compound ibutinib in preparing antiviral drugs is characterized in that the compound ibutinib has a structure shown in a formula 1:

the virus is one or more of vaccinia virus VACV-WR, vaccinia virus VACV-MVA and bovine nodular skin disease virus LSDV.

2. The use according to claim 1, wherein the antiviral medicament is in the form of an injectable dosage form.

3. The use according to claim 1, wherein the antiviral medicament is in a luminal form.

4. Use according to any one of claims 1 to 3, wherein the compound ibutinib is used in an antiviral drug at a concentration of 0.5 to 20 μm.

5. The use according to claim 4, wherein the compound ibutinib is used in an antiviral drug at a concentration of 10 μm.