Application of neratinib in preparation of inhibitor and medicine for inhibiting proliferation of monkey pox virus
Through the future application of natinib to inhibit the proliferation of monkeypox virus, the problem of lack of special treatment methods for monkeypox has been solved, and effective inhibition of the proliferation of monkeypox virus has been achieved, providing new ideas for the treatment of monkeypox.
Patent Information
- Application Number
- CN202510235869.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-28
- Publication Date
- 2025-04-25
AI Technical Summary
There is currently no effective treatment method to inhibit the proliferation of monkeypox virus, resulting in the lack of a special treatment plan for monkeypox.
In the future, Neratinib will be used to prepare inhibitors that inhibit monkeypox virus proliferation, and by binding to monkeypox virus, it will inhibit its proliferation.
It has achieved effective inhibition of monkeypox virus proliferation, expanded the application scenarios of lenatinib, and provided new ideas for the treatment of monkeypox.
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Figure CN119868358A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of new drug applications, and in particular to the application of neratinib in the preparation of inhibitors and drugs for inhibiting the proliferation of monkeypox virus. Background Art
[0002] Monkeypox is a rare disease caused by the monkeypox virus that usually affects rodents or non-human primates, but can also affect humans. The disease is mainly found in Central and West Africa. It was first discovered in monkeys during vaccine research in 1958 and has been detected in some buffalo and small mammals. Related studies have shown that long-term close contact, respiratory droplets, contaminated personal items, and direct contact with the rash area are the media of monkeypox transmission.
[0003] Monkeypox causes viral zoonoses, and the symptoms seen in humans are similar to those seen in patients with smallpox in the past. However, there is currently no specific treatment. The treatment of monkeypox is mainly supportive care, and smallpox-related vaccines and treatments may be considered. There is currently no good specific treatment drug. Summary of the invention
[0004] The main purpose of the present invention is to propose the use of neratinib in the preparation of inhibitors and drugs for inhibiting the proliferation of monkeypox virus, so as to broaden the application scenarios of neratinib and provide new ideas for the treatment of monkeypox.
[0005] To achieve the above objectives, the present invention proposes, on one hand, the use of the present invention in the preparation of an inhibitor for inhibiting the proliferation of monkeypox virus.
[0006] Optionally, the IC50 of neratinib for inhibition of monkeypox virus is between 40-200 nM.
[0007] Optionally, neratinib also includes pharmaceutically acceptable derivatives of neratinib.
[0008] Optionally, the dosage forms of neratinib and the pharmaceutically acceptable derivatives of neratinib include tablets, capsules or injections.
[0009] Another aspect of the present invention provides the use of neratinib in the preparation of a medicament for preventing and / or treating a disease, or reducing the risk of a disease; the disease is monkeypox.
[0010] The present invention applies neratinib to inhibit the proliferation of monkeypox virus, thereby achieving effective inhibition of the proliferation of monkeypox virus, broadening the application scenarios of neratinib and providing new ideas for the treatment of monkeypox. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.
[0012] Figure 1 This is a graph showing the results of an experiment to detect the inhibitory effect of neratinib on monkeypox virus in Examples 1-5 of the present invention;
[0013] Figure 2 It is a cell status diagram of the inhibition of monkeypox virus by Example 2 of the present invention and the blank control group.
[0014] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION
[0015] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.
[0016] Monkeypox is a viral zoonosis caused by infection with the monkeypox virus (MPXV), with symptoms in humans similar to those seen in patients with smallpox in the past. First discovered in monkeys during vaccine research in 1958, and detected in some buffalo and small mammals, the first human infection with the monkeypox virus was not discovered until 1970 in the Republic of Congo. Related studies have shown that long-term close contact, respiratory droplets, contaminated personal items, and direct contact with the rash area are the media of monkeypox transmission.
[0017] There is currently no good specific drug for the treatment of monkeypox, and there is no effective method for inhibiting the proliferation of the monkeypox virus.
[0018] In view of this, the present invention proposes, on one hand, the use of neratinib in the preparation of an inhibitor for inhibiting the proliferation of monkeypox virus.
[0019] Neratinib is currently mainly used for adjuvant treatment of breast cancer, especially for early HER2-positive breast cancer. Neratinib binds irreversibly to intracellular kinase domains such as epidermal growth factor receptor (EGFR), HER2 and HER4, thereby inhibiting receptor autophosphorylation and reducing the activation of downstream signaling pathways.
[0020] In the technical solution of the present invention, it is discovered for the first time that the application of neratinib in inhibiting the proliferation of monkeypox virus can effectively inhibit the proliferation of monkeypox virus, broaden the application scenarios of neratinib, and provide new ideas for the treatment of monkeypox.
[0021] Optionally, the IC50 of neratinib for inhibition of monkeypox virus is between 40-200 nM.
[0022] Optionally, neratinib also includes pharmaceutically acceptable derivatives of neratinib.
[0023] Optionally, the dosage forms of neratinib and the pharmaceutically acceptable derivatives of neratinib include tablets, capsules or injections.
[0024] Another aspect of the present invention provides the use of neratinib in the preparation of a medicament for preventing and / or treating a disease, or reducing the risk of a disease; the disease is monkeypox.
[0025] In the present invention, the term "prevention and / or treatment" not only includes preventing and / or treating diseases, but also generally includes reducing or inhibiting the number of pathogens associated with the disease, preventing the onset of the disease, slowing down or reversing the progression of the disease, preventing or slowing down the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptom associated with it and / or preventing the disease and / or any symptom associated with it from further increasing in severity, preventing, reducing or reversing any physiological damage caused by the disease, and any pharmacological effect that is generally beneficial to the patient being treated. The drugs of the present application do not need to achieve a complete cure or eradication of any symptoms or manifestations of the disease to form a viable therapeutic agent. As recognized in the relevant art, drugs used as therapeutic agents can reduce the severity of a given disease state, but do not need to eliminate every manifestation of the disease to be considered a useful therapeutic agent. Similarly, prophylactically administered treatments constitute viable preventive agents that do not need to completely and effectively prevent the onset of the disease. Simply reducing the effects of the disease in a subject (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood that the disease will occur or worsen, is sufficient.
[0026] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.
[0027] Example 1
[0028] Inhibition of monkeypox virus by neratinib
[0029] Vero-E6 cells were plated into 24-well plates at a cell density of 6 × 10 4 The cells were placed in a cell culture incubator and allowed to stand for 12 hours.
[0030] The monkeypox virus was diluted with DMEM medium containing 2% fetal bovine serum (FBS) to a final concentration of 2×10 5 Pieces / mL.
[0031] Neratinib was diluted with DMEM medium containing 2% FBS so that the final concentration of neratinib was 40 nM.
[0032] The culture medium in the Vero-E6 cell culture plate was removed, leaving the Vero-E6 cells, 500 μL of diluted culture medium containing monkeypox virus was added to each well, and the plate was incubated in a cell culture incubator for 1 hour.
[0033] The Vero-E6 cells were taken out of the incubator, the medium containing the virus in the Vero-E6 cell culture plate was removed, the Vero-E6 cells were left, washed with 500 μL PBS, and a DMEM medium containing 2% FBS with a final concentration of 40 nM neratinib was added, and the cells were placed in a cell incubator for 48 hours.
[0034] The cultured Vero-E6 cells were taken out of the cell culture incubator, placed in a -80°C refrigerator, and incubated for 20 minutes. Then, the cells were taken out of the -80°C refrigerator, placed in a 37°C cell culture incubator, and incubated for 20 minutes. This process was repeated twice.
[0035] Place the cells that have been frozen and thawed twice on the cell operation table, mix them by pipetting, and take 200 μL of the culture medium containing the cells for nucleic acid extraction.
[0036] The nucleic acid in the samples was extracted using the Daan Gene Nucleic Acid Extraction Kit (#DA0620).
[0037] Take 10 μL of the extracted nucleic acid solution and dilute it to 50 μL with double distilled water to use as a template for qPCR detection.
[0038] Design monkeypox virus primers, mix the upstream primer and the downstream primer together, and the final concentration is 1 μM. The sequence of the upstream primer is F3L-F: cttccgtcaatgtctacacaggc. The sequence of the downstream primer is F3L-R: cgttggtctacgacaatggatgc.
[0039] Use Novozyme SYBR Green Mixture (#Q712) to detect the content of target nucleic acid fragments in the sample. Prepare the reaction system according to Table 1, and run the qPCR program according to Table 2.
[0040] Table 1 qPCR reaction system
[0041] Components volume SYBR Green mix 10μL Upstream primer + downstream primer mixture (1 μM) 5μL template 5μL
[0042] Table 2qPCR reaction procedure
[0043] Reaction temperature Reaction time Number of cycles 95℃ 5min 1 95℃ 10s 40 60℃ 20s 40 72℃ 20s 40
[0044] See also Figure 1 By analyzing the content of monkeypox virus treated with neratinib, the inhibition rate of neratinib on monkeypox virus was calculated. The inhibition rate of 40nM neratinib on monkeypox virus after 48h was 15.07%.
[0045] Example 2
[0046] Inhibition of monkeypox virus by neratinib
[0047] Vero-E6 cells were plated into 24-well plates at a cell density of 6 × 10 4 The cells were placed in a cell culture incubator and allowed to stand for 12 hours.
[0048] The monkeypox virus was diluted with DMEM medium containing 2% fetal bovine serum (FBS) to a final concentration of 2×10 5 Pieces / mL.
[0049] Neratinib was diluted with DMEM medium containing 2% FBS so that the final concentration of neratinib was 200 nM.
[0050] The culture medium in the Vero-E6 cell culture plate was removed, leaving the Vero-E6 cells, 500 μL of diluted culture medium containing monkeypox virus was added to each well, and the plate was incubated in a cell culture incubator for 1 hour.
[0051] The Vero-E6 cells were taken out of the incubator, the medium containing the virus in the Vero-E6 cell culture plate was removed, the Vero-E6 cells were left, washed with 500 μL PBS, and a DMEM medium containing 2% FBS with a final concentration of 200 nM neratinib was added, and the cells were placed in a cell incubator for 48 hours.
[0052] The cultured Vero-E6 cells were taken out of the cell culture incubator, placed in a -80°C refrigerator, and incubated for 20 minutes. Then, the cells were taken out of the -80°C refrigerator, placed in a 37°C cell culture incubator, and incubated for 20 minutes. This process was repeated twice.
[0053] Place the cells that have been frozen and thawed twice on the cell operation table, mix them by pipetting, and take 200 μL of the culture medium containing the cells for nucleic acid extraction.
[0054] The nucleic acid in the samples was extracted using the Daan Gene Nucleic Acid Extraction Kit (#DA0620).
[0055] Take 10 μL of the extracted nucleic acid solution and dilute it to 50 μL with double distilled water to use as a template for qPCR detection.
[0056] Design monkeypox virus primers, mix the upstream primer and the downstream primer together, and the final concentration is 1 μM. The sequence of the upstream primer is F3L-F: cttccgtcaatgtctacacaggc. The sequence of the downstream primer is F3L-R: cgttggtctacgacaatggatgc.
[0057] Use Novozyme SYBR Green Mixture (#Q712) to detect the content of target nucleic acid fragments in the sample. Prepare the reaction system according to Table 1, and run the qPCR program according to Table 2.
[0058] Table 1 qPCR reaction system
[0059] Components volume SYBR Green mix 10μL Upstream primer + downstream primer mixture (1 μM) 5μL template 5μL
[0060] Table 2qPCR reaction procedure
[0061] Reaction temperature Reaction time Number of cycles 95℃ 5min 1 95℃ 10s 40 60℃ 20s 40 72℃ 20s 40
[0062] See also Figure 1 By analyzing the content of monkeypox virus treated with neratinib, the inhibition rate of neratinib on monkeypox virus was calculated. The inhibition rate of 200nM neratinib on monkeypox virus after 48h was 53.04%.
[0063] Example 3
[0064] Inhibition of monkeypox virus by neratinib
[0065] Vero-E6 cells were plated into 24-well plates at a cell density of 6 × 10 4 The cells were placed in a cell culture incubator and allowed to stand for 12 hours.
[0066] The monkeypox virus was diluted with DMEM medium containing 2% fetal bovine serum (FBS) to a final concentration of 2×10 5 Pieces / mL.
[0067] Neratinib was diluted with DMEM medium containing 2% FBS so that the final concentration of neratinib was 1 μM.
[0068] The culture medium in the Vero-E6 cell culture plate was removed, leaving the Vero-E6 cells, 500 μL of diluted culture medium containing monkeypox virus was added to each well, and the plate was incubated in a cell culture incubator for 1 hour.
[0069] The Vero-E6 cells were taken out of the incubator, the medium containing the virus in the Vero-E6 cell culture plate was removed, the Vero-E6 cells were left, washed with 500 μL PBS, and a DMEM medium containing 2% FBS with a final concentration of 1 μM neratinib was added, and the cells were placed in a cell incubator for 48 hours.
[0070] The cultured Vero-E6 cells were taken out of the cell culture incubator, placed in a -80°C refrigerator, and incubated for 20 minutes. Then, the cells were taken out of the -80°C refrigerator, placed in a 37°C cell culture incubator, and incubated for 20 minutes. This process was repeated twice.
[0071] Place the cells that have been frozen and thawed twice on the cell operation table, mix them by pipetting, and take 200 μL of the culture medium containing the cells for nucleic acid extraction.
[0072] The nucleic acid in the samples was extracted using the Daan Gene Nucleic Acid Extraction Kit (#DA0620).
[0073] Take 10 μL of the extracted nucleic acid solution and dilute it to 50 μL with double distilled water to use as a template for qPCR detection.
[0074] Design monkeypox virus primers, mix the upstream primer and the downstream primer together, and the final concentration is 1 μM. The sequence of the upstream primer is F3L-F: cttccgtcaatgtctacacaggc. The sequence of the downstream primer is F3L-R: cgttggtctacgacaatggatgc.
[0075] Use Novozyme SYBR Green Mixture (#Q712) to detect the content of target nucleic acid fragments in the sample. Prepare the reaction system according to Table 1, and run the qPCR program according to Table 2.
[0076] Table 1 qPCR reaction system
[0077] Components volume SYBR Green mix 10μL Upstream primer + downstream primer mixture (1 μM) 5μL template 5μL
[0078] Table 2qPCR reaction procedure
[0079] Reaction temperature Reaction time Number of cycles 95℃ 5min 1 95℃ 10s 40 60℃ 20s 40 72℃ 20s 40
[0080] See also Figure 1 By analyzing the content of monkeypox virus treated with neratinib, the inhibition rate of neratinib on monkeypox virus was calculated. The inhibition rate of 1μM neratinib on monkeypox virus after 48h was 88.37%.
[0081] Example 4
[0082] Inhibition of monkeypox virus by neratinib
[0083] Vero-E6 cells were plated into 24-well plates at a cell density of 6 × 10 4 The cells were placed in a cell culture incubator and allowed to stand for 12 hours.
[0084] The monkeypox virus was diluted with DMEM medium containing 2% fetal bovine serum (FBS) to a final concentration of 2×10 5 Pieces / mL.
[0085] Neratinib was diluted with DMEM medium containing 2% FBS so that the final concentration of neratinib was 5 μM.
[0086] The culture medium in the Vero-E6 cell culture plate was removed, leaving the Vero-E6 cells, 500 μL of diluted culture medium containing monkeypox virus was added to each well, and the plate was incubated in a cell culture incubator for 1 hour.
[0087] The Vero-E6 cells were taken out of the incubator, the medium containing the virus in the Vero-E6 cell culture plate was removed, the Vero-E6 cells were left, washed with 500 μL PBS, and a DMEM medium containing 2% FBS with a final concentration of 5 μM neratinib was added, and the cells were placed in a cell incubator for 48 hours.
[0088] The cultured Vero-E6 cells were taken out of the cell culture incubator, placed in a -80°C refrigerator, and incubated for 20 minutes. Then, the cells were taken out of the -80°C refrigerator, placed in a 37°C cell culture incubator, and incubated for 20 minutes. This process was repeated twice.
[0089] Place the cells that have been frozen and thawed twice on the cell operation table, mix them by pipetting, and take 200 μL of the culture medium containing the cells for nucleic acid extraction.
[0090] The nucleic acid in the samples was extracted using the Daan Gene Nucleic Acid Extraction Kit (#DA0620).
[0091] Take 10 μL of the extracted nucleic acid solution and dilute it to 50 μL with double distilled water to use as a template for qPCR detection.
[0092] Design monkeypox virus primers, mix the upstream primer and the downstream primer together, and the final concentration is 1 μM. The sequence of the upstream primer is F3L-F: cttccgtcaatgtctacacaggc. The sequence of the downstream primer is F3L-R: cgttggtctacgacaatggatgc.
[0093] Use Novozyme SYBR Green Mixture (#Q712) to detect the content of target nucleic acid fragments in the sample. Prepare the reaction system according to Table 1, and run the qPCR program according to Table 2.
[0094] Table 1 qPCR reaction system
[0095] Components volume SYBR Green mix 10μL Upstream primer + downstream primer mixture (1 μM) 5μL template 5μL
[0096] Table 2qPCR reaction procedure
[0097] Reaction temperature Reaction time Number of cycles 95℃ 5min 1 95℃ 10s 40 60℃ 20s 40 72℃ 20s 40
[0098] See also Figure 1 By analyzing the content of monkeypox virus treated with neratinib, the inhibition rate of neratinib on monkeypox virus was calculated. The inhibition rate of 5μM neratinib on monkeypox virus after 48h was 95.23%.
[0099] Example 5
[0100] Inhibition of monkeypox virus by neratinib
[0101] Vero-E6 cells were plated into 24-well plates at a cell density of 6 × 10 4 The cells were placed in a cell culture incubator and allowed to stand for 12 hours.
[0102] The monkeypox virus was diluted with DMEM medium containing 2% fetal bovine serum (FBS) to a final concentration of 2×10 5 Pieces / mL.
[0103] Neratinib was diluted with DMEM medium containing 2% FBS so that the final concentration of neratinib was 25 μM.
[0104] The culture medium in the Vero-E6 cell culture plate was removed, leaving the Vero-E6 cells, 500 μL of diluted culture medium containing monkeypox virus was added to each well, and the plate was incubated in a cell culture incubator for 1 hour.
[0105] The Vero-E6 cells were taken out of the incubator, the medium containing the virus in the Vero-E6 cell culture plate was removed, the Vero-E6 cells were left, washed with 500 μL PBS, and a DMEM medium containing 2% FBS with a final concentration of 25 μM neratinib was added, and the cells were placed in a cell incubator for 48 hours.
[0106] The cultured Vero-E6 cells were taken out of the cell culture incubator, placed in a -80°C refrigerator, and incubated for 20 minutes. Then, the cells were taken out of the -80°C refrigerator, placed in a 37°C cell culture incubator, and incubated for 20 minutes. This process was repeated twice.
[0107] Place the cells that have been frozen and thawed twice on the cell operation table, mix them by pipetting, and take 200 μL of the culture medium containing the cells for nucleic acid extraction.
[0108] The nucleic acid in the samples was extracted using the Daan Gene Nucleic Acid Extraction Kit (#DA0620).
[0109] Take 10 μL of the extracted nucleic acid solution and dilute it to 50 μL with double distilled water to use as a template for qPCR detection.
[0110] Design monkeypox virus primers, mix the upstream primer and the downstream primer together, and the final concentration is 1 μM. The sequence of the upstream primer is F3L-F: cttccgtcaatgtctacacaggc. The sequence of the downstream primer is F3L-R: cgttggtctacgacaatggatgc.
[0111] Use Novozyme SYBR Green Mixture (#Q712) to detect the content of target nucleic acid fragments in the sample. Prepare the reaction system according to Table 1, and run the qPCR program according to Table 2.
[0112] Table 1 qPCR reaction system
[0113] Components volume SYBR Green mix 10μL Upstream primer + downstream primer mixture (1 μM) 5μL template 5μL
[0114] Table 2qPCR reaction procedure
[0115] Reaction temperature Reaction time Number of cycles 95℃ 5min 1 95℃ 10s 40 60℃ 20s 40 72℃ 20s 40
[0116] See also Figure 1 By analyzing the content of monkeypox virus treated with neratinib, the inhibition rate of neratinib on monkeypox virus was calculated. The inhibition rate of 25μM neratinib on monkeypox virus after 48h was 97.99%.
[0117] The test results of Examples 1 to 5 are as follows Figure 1 As shown, Figure 1 “Relative virus activity” refers to the activity of monkeypox virus, “Ctrl” refers to the blank control group, and “**” represents that the sig value is less than 0.01, thus having a significant difference.
[0118] The blank control group refers to the culture medium without the addition of neratinib.
[0119] Depend on Figure 1 The results showed that compared with the blank control group, when the added amount of neratinib was 40nM, the inhibition rate of monkeypox virus was 15%. As the added amount of neratinib gradually increased, the inhibition rate of monkeypox virus gradually increased. When the added amount of neratinib was 25μM, the inhibition rate of monkeypox virus was close to 98%, and the monkeypox virus had almost no activity.
[0120] The cell status after the monkeypox virus was applied to Example 2 and the blank control group was tested. The test results are as follows: Figure 2 As shown, Figure 2 (Left) is the control group, which is a state diagram of Vero-E6 cells after monkeypox virus infection, and is not treated with lenatinib; Figure 2 (Right) is the experimental group, which is a diagram of the cell status after Vero-E6 cells were infected with monkeypox virus and treated with 200nM neratinib for 48 hours.
[0121] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.
Claims
1. The use of neratinib in the preparation of inhibitors for inhibiting the proliferation of monkeypox virus.
2. The use according to claim 1, characterized in that The IC50 of neratinib for inhibition of monkeypox virus is between 40-200nM.
3. The use according to claim 1, characterized in that The neratinib also includes pharmaceutically acceptable derivatives of neratinib.
4. The use according to claim 3, characterized in that The dosage forms of the neratinib and the pharmaceutically acceptable derivatives of neratinib include tablets, capsules or injections.
5. The use of neratinib in the preparation of a drug, characterized in that: The drug is used to prevent and / or treat a disease, or reduce the risk of a disease; the disease is monkeypox.