Application of Linsitinib as ADV7 virus inhibitor

Abstract

The invention discloses an application of Linsitinib as an ADV7 (adenosine diphosphate 7) virus inhibitor. According to an anti-ADV7 activity research experiment of the Linsitinib, the compound Linsitinib inhibits the cytopathic effect (CPE) generated by ADV7 on a host cell Hela, the cell survival rate is enhanced, the progeny virus yield is reduced, and the compound Linsitinib has the potential to be applied to preparation of anti-ADV7 virus drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of antiviral drugs, and in particular to the application of Linsitinib in the preparation of drugs against ADV7 virus. Background Art

[0002] Human adenovirus 7 (ADV7) is a widely distributed non-enveloped double-stranded DNA virus with virus particles having a diameter of 90-100 nm. The capsid is icosahedral, and its replication does not depend on the division of host cells. Human adenovirus (ADV) infection is very common and can cause various diseases in humans, mainly respiratory tract infections. The adenoviruses related to respiratory diseases are mainly three types, namely ADV-3, ADV-5, and ADV-7. It is known that the risk factors for adenoviruses to cause serious diseases are relatively small. It can commonly cause acute respiratory tract fever, but it has a great impact on children. In severe cases, it can lead to pediatric pneumonia. ADV7 has been confirmed to be an important cause of pediatric pneumonia. ADV7 can cause severe or fatal respiratory tract infections in children under 7 years old and is prone to cause outbreaks, endangering the health of infants and young children. Currently, there is also a lack of drugs with strong specificity and small side effects for ADV7 infection in clinical practice, and symptomatic treatment is mainly adopted at present. Therefore, it is also imperative to develop specific drugs against ADV-7 infection.

[0003] Linsitinib (OSI-906) is an effective and novel small molecule inhibitor that inhibits insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF-1R) kinases, with corresponding IC50 values of 75 nM and 35 nM. 3T3 / hulGF-1R (LISN) cell assays showed that Linsitinib inhibits ligand-dependent autophosphorylation of IGF-1R and its downstream signaling pathways, including phosphorylation of pERK1 / 2, pAKT, and p-p70S6K. Linsitinib has an anti-proliferative effect on a variety of cancer cell lines, including colorectal cancer cells (SW620), breast cancer cells (DY4475), and mouse fibroblasts (3T3 / hulGF-1R), with corresponding EC50 values of 21 nM, 86 nM, and 78 nM.

[0004] We evaluated the inhibitory activity of Linsitinib against ADV7 virus. Summary of the Invention

[0005] The purpose of the present invention is to aim at the above-mentioned situation and provide Linsitinib as an effective inhibitor against ADV7 virus.

[0006] Linsitinib has the following chemical structural formula: 。

[0007] The implementation of the object of the present invention is that Linsitinib is tested for its activity by standard viral activity test methods.

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows: Through a large number of biological experiments, the present invention finds that Linsitinib has the activity against ADV7 virus. Specifically, it can inhibit the cytopathic effect caused by ADV7 virus, enhance the survival rate of infected cells, inhibit the replication and proliferation of ADV7 virus in cells, and reduce the yield of progeny virus. This indicates that this compound has the potential to prepare specific therapeutic drugs against ADV7 infection and has great clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 It is the effect of Linsitinib on the survival rate of Hela cells against ADV7.

[0010] Figure 2 It is the inhibitory effect of Linsitinib on the CPE of Hela cells caused by ADV7.

[0011] Figure 3 It is the inhibitory effect of Linsitinib on the yield of progeny virus of ADV7. DETAILED DESCRIPTION OF THE INVENTION

[0012] The following is a further description of the technical solution of the present invention through examples. The Linsitinib used in the present invention is purchased from a reagent company.

[0013] The present invention relates to the application of Linsitinib in the preparation of drugs against ADV7 virus.

[0014] The said application means that Linsitinib is added with pharmaceutically acceptable excipients and carriers for the preparation of preparations against ADV7 virus.

[0015] The said preparations are granules, tablets, pills, capsules, injections or dispersants.

[0016] The Linsitinib of the present invention has the following chemical structural formula: .

[0017] Regarding the application of Linsitinib of the present invention as an anti-ADV7 virus, it is found that the anti-ADV7 virus effect of Linsitinib is obvious. This indicates that this compound has the potential to prepare specific therapeutic drugs against ADV7 infection and has great clinical application prospects.

[0018] The present invention conducted an experiment on the anti-ADV7 activity of the above-mentioned compound, and the experimental situation is as follows: In the following text, unless otherwise specified, the materials and operation methods used in the present invention are well-known in the art.

[0019] 1. Test content: Analysis of the anti-ADV7 activity of the compound: The present invention will evaluate the anti-ADV7 activity of Linsitinib by combining the analysis of cytopathic effect and the detection method of MTT for cell viability.

[0020] 2. Test method: 2.1.1 Toxicity of the compound to host Hela cells Seed Hela cells in a 96-well plate and culture them in a 37 °C, 5% CO2 incubator until they grow into a confluent monolayer. Then discard the cell culture medium and add cell maintenance medium containing different concentrations of the test compound to continue the culture. After 48 h, visually observe and record the cytotoxicity under a microscope, and determine the cell viability by the MTT method. Use SPSS 11.5 software to calculate the median cytotoxic concentration (CC50) of the drug on the cells. Cell viability = (average OD value of the drug group / average OD value of the cell control group) × 100%. 492 value / average OD value of the cell control group 492 value) × 100%.

[0021] 2.1.2 Inhibitory activity of the compound against ADV7 Seed Hela cells in a 96-well plate and culture them in a 37 °C, 5% CO2 incubator until they grow into a confluent monolayer. Then discard the culture medium and infect the cells with 100 TCID50 of ADV7 virus solution for 1.5 h. Add different concentrations of the test compound (ribavirin as the positive control drug) to incubate the cells. After continuing the culture for about 48 h, when about 90% of the CPE lesions appear in the virus control well, observe the cytopathic effect (CPE) under a microscope. Observation and recording method of CPE: No cytopathic effect is recorded as -, less than 25% cytopathic effect is recorded as +, 25%-50% cytopathic effect is recorded as ++, 50%-75% cytopathic effect is recorded as ++++, and more than 75% cytopathic effect is recorded as ++++.

[0022] After the CPE observation is completed, use the MTT method to detect the inhibition rate of the drug against ADV7. The specific steps are as follows: Add 50 μL of MTT (5 mg·mL -1 -1) to each well, incubate for 3-4 h, then remove the supernatant, and add an equal volume of DMSO to dissolve the precipitate. Read the corresponding absorbance (OD 492Value). The inhibition rate of the drug against ADV7 was calculated using the following formula. The half-maximal effective concentration (EC50) of the drug was calculated using SPSS 11.5 software.

[0023]

[0024] 2.1.3 Therapeutic index (TI) of the drug TI = CC50 / EC50. The higher the therapeutic index, the greater the antiviral potential.

[0025] 3. Experimental results: Table 1 Cytotoxicity of Linsitinib and its anti-ADV7 activity

[0026] The test results of the cytotoxicity of the compound and its anti-ADV7 activity are shown in Table 1. The concentration-dependent effect of the compound on the survival rate of Hela cells treated with Linsitinib is as Figure 1 shown. It was found that at 12.5 µM of Linsitinib, the maximum inhibition rate was approximately 95.3%. The inhibitory effect of the compound on the CPE of Hela cells caused by ADV7 is as Figure 2 shown. Under the action of the compound, the cytopathic effect of Hela cells caused by ADV7 was inhibited and the cell survival rate increased.

[0027] In this invention, an in-depth study on the anti-ADV7 activity of Linsitinib was carried out, and an experiment on the inhibitory effect of the compound on the production of ADV7 progeny virus was implemented. The experimental situation is as follows: 1. Test content To detect the inhibitory effect of the compound on the production of ADV7 progeny virus after ADV7 infects Hela cells.

[0028] 2. Test method Hela cells in the logarithmic growth phase were seeded in 24-well plates. After growing into a monolayer, 100 TCID 50 ADV7 was used to infect the cells. After incubation at 37 °C for 1.5 h, the virus solution was removed, and the cells were washed three times with PBS. Then, a cell maintenance solution containing a concentration of 12.5 µM was added. After 48 h, the cells and the supernatant culture solution were collected. After three cycles of freezing and thawing at -20 °C and 37 °C for lysis, the virus titer of ADV7 was measured by the TCID 50 method.

[0029] 3. Test results As Figure 3As shown, the virus titers of compound-treated Hela cells were significantly decreased compared to the virus control group, with a 3.9 log decrease compared to the virus control.

[0030] In summary, Linsitinib has strong inhibitory activity against ADV7, can inhibit the death of Hela cells caused by ADV7 virus, and can be prepared into a drug that is clinically effective against ADV7 infection.

[0031] It should be understood that the above embodiments are only used to illustrate the content of the present invention and not to limit the protection scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Application of linsitinib in anti-ADV7 virus drugs, characterized in that: The chemical structural formula of the compound Linsitinib with anti-EV71 activity is shown as follows: 。 2. Use of Linsitinib as an anti-ADV7 virus drug according to claim 1, characterized in that: The inhibition rate of Linsitinib against ADV7 is approximately 95.3% at 12.5 µM.

3. Use of Linsitinib as an ADV7 virus inhibitor according to claim 1, wherein: The application mentioned above refers to the use of this compound, added with pharmaceutically acceptable excipients and carriers, for preparing a preparation against ADV7 virus.

4. Use of Linsitinib as an ADV7 virus inhibitor according to claim 1, characterized in that: The preparation is a granule, tablet, pill, capsule, injection or dispersant.

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