Application of axitinib in preparation of medicine for treating plexus neurofibroma

Abstract

The invention belongs to the field of medicines, and particularly relates to application of axitinib in preparation of a medicine for treating plexus neurofibroma. In the medicine, axitinib serves as the only effective component or one of the effective components. When axitinib in the medicine is used as one of the effective components, the effective components further comprise an MEK inhibitor. The MEK inhibitor is one selected from the group consisting of simetinib and trametinib. The invention proves that axitinib can play a role in inhibiting pNF by targeting an important gene OTUD3 for promoting the progress of pNF. Moreover, axitinib is a medicine already used for clinical treatment and is good in safety, and research results show that the neurofibroma inhibition effect can be improved by combined use of axitinib and the MEK inhibitor.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the use of axitinib in the preparation of a therapeutic drug for plexiform neurofibroma. Background Art

[0002] Plexiform neurofibroma (pNF) is one of the most common and challenging tumors in patients with type I neurofibromatosis (NF1). Currently, the treatment of pNF mainly relies on MEK inhibitors (MEKi), such as selumetinib. Selumetinib is an oral selective mitogen-activated protein kinase (MEK) inhibitor, which was approved by the US FDA in 2020 for the treatment of symptomatic and / or progressive, inoperable NF1-related pNF in patients aged 2 to 18 years. However, although selumetinib shows significant efficacy in some patients, such as partial remission (tumor volume reduction ≥ 20% compared to baseline and maintained for at least 4 weeks) observed in 70% of patients, the problem of treatment resistance still exists. Therefore, there is a need to find new therapeutic drugs for plexiform neurofibroma. Summary of the Invention

[0003] The purpose of the present invention is to provide the use of axitinib in the preparation of a therapeutic drug for plexiform neurofibroma to solve the problem of treatment resistance of selumetinib.

[0004] To achieve the above purpose, the present invention adopts the following technical solutions: In the first aspect, the present invention provides the use of axitinib in the preparation of a therapeutic drug for plexiform neurofibroma.

[0005] Plexiform fibroma is one of the typical clinical manifestations of type I neurofibromatosis, which can cause destructive harm to patients. At present, the main treatment method is surgical resection, and for patients who are difficult to surgically resect, oral selumetinib (MEK inhibitor) can be used. At the same time, selumetinib is also the only effective drug for the treatment of plexiform neurofibroma, and there are two major clinical dilemmas: treatment resistance and limited efficacy of single drug.

[0006] Axitinib is a VEGFR inhibitor, and pNF is a tumor with rich blood supply; the present invention has confirmed that axitinib can play an inhibitory role on pNF by targeting the important gene OTUD3 that promotes the progression of pNF. Moreover, axitinib is a drug that has been used in clinical treatment and has good safety. The research results of the present invention show that the combined use of axitinib and MEK inhibitor can improve the inhibitory effect on neurofibroma.

[0007] Furthermore, axitinib in the drug is used as the only active ingredient or one of the active ingredients.

[0008] Further, when axitinib is one of the active ingredients in the drug, the active ingredients further include a MEK inhibitor.

[0009] Further, the MEK inhibitor is selected from selumetinib and trametinib.

[0010] Further still, the drug further includes pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients include conventional diluents (such as at least one of water for injection, microcrystalline cellulose, etc.), fillers (such as at least one of mannitol, sucrose, lactose, polyethylene glycol, Tween 80, sorbitol, menthol, liquid paraffin, petrolatum, stearic acid, glyceryl monostearate, lanolin, mineral oil, DMSO, etc.), binders (such as at least one of carbomer, gum arabic, starch, cellulose, gelatin, polyvinylpyrrolidone, polyacrylamide, etc.), disintegrants (such as at least one of sodium carboxymethyl starch, cross-linked carboxymethyl cellulose sodium, hypromellose, low-substituted hydroxypropyl cellulose, etc.), lubricants (such as at least one of talc, magnesium stearate, calcium stearate, solid polyethylene glycol, lecithin, silicon dioxide, colloidal silica, etc.), wetting agents (such as at least one of propylene glycol, glycerol, ethanol, etc.), stabilizers (such as at least one of disodium edetate, sodium thiosulfate, sodium metabisulfite, sodium sulfite, sodium bisulfite, ethanolamine, sodium bicarbonate, sodium acetate, nicotinamide, vitamin C, etc.), osmotic pressure regulators (such as at least one of sodium chloride, glucose, etc.), pH regulators (such as at least one of triethanolamine, sodium hydroxide, sodium citrate, etc.), and preservatives (such as at least one of chlorobutanol, parabens, ethylparaben, benzalkonium bromide, etc.). The above excipients can be used in common dosages and mixed with axitinib and the MEK inhibitor in common ratios. After the dosages of axitinib and the MEK inhibitor are determined, the ratios between the various pharmaceutical excipients can be appropriately adjusted as needed.

[0011] Further still, the dosage form of the drug is a granule, tablet, capsule, pill, dripping pill, oral liquid preparation, gavage preparation, or injection dosage form.

[0012] In a second aspect, the present invention provides a drug for treating plexiform neurofibroma, and the drug uses the axitinib and the MEK inhibitor as active ingredients.

[0013] Further, the MEK inhibitor is selected from selumetinib and trametinib.

[0014] Further still, the drug further includes pharmaceutically acceptable excipients.

[0015] Further still, the dosage form of the drug is a granule, tablet, capsule, pill, dripping pill, oral liquid preparation, gavage preparation, or injection dosage form.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. Provide new treatment options: The application of axitinib in the treatment of plexiform neurofibroma (pNF) provides new treatment options for patients, especially for those who are resistant or intolerant to existing MEK inhibitor treatments.

[0017] 2. Potential for combination therapy: When axitinib is used in combination with MEK inhibitors (such as selumetinib, trametinib), the anti-tumor effect can be enhanced through multi-target synergistic effects, overcoming the limitations of single-drug therapy.

[0018] 3. Overcome treatment resistance: The combination therapy regimen can overcome the drug resistance problems that occur in existing treatments, improving the durability and effectiveness of treatment.

[0019] 4. Broad clinical application prospects: The drugs of the present invention have broad prospects in clinical applications and can improve the treatment effect and quality of life of pNF patients.

[0020] In summary, through the application of axitinib in the treatment of pNF and its combination therapy regimen, the present invention provides a new strategy for overcoming the deficiencies of existing treatments and has significant clinical value and application prospects. Brief Description of the Drawings

[0021] Figure 1 To detect the efficiency of knocking down and overexpressing OTUD3 by Western Blotting, A shows the detection results of the efficiency of knocking down OTUD3, and B shows the detection results of the efficiency of overexpressing OTUD3.

[0022] Figure 2 For the colony formation ability of plexiform neurofibroma cell lines after knocking down and overexpressing OTUD3, A shows the distribution of ipNF95.6 cells after knocking down OTUD3, B is the quantitative statistical chart of A, C shows the distribution of ipNF95.11C cells after overexpressing OTUD3, and D is the quantitative statistical chart of C.

[0023] Figure 3 For the effects of knocking down and overexpressing OTUD3 on MEK, p-MEK, ERK, p-ERK, and p-FOS in neurofibroma, A shows the expression levels of MEK, p-MEK, ERK, p-ERK, and p-FOS after knocking down OTUD3 detected by Western Blotting, and the relative intensity is normalized to Tubulin. B shows the expression levels of MEK, p-MEK, ERK, p-ERK, and p-FOS after overexpressing OTUD3 detected by Western Blotting, and the relative intensity is normalized to Tubulin.

[0024] Figure 4This is the docking site of axitinib and OTUD3 molecule.

[0025] Figure 5 This is the mechanism by which axitinib inhibits the progression of neurofibroma. A shows the effect of axitinib on the expression level of the MEK / ERK signaling pathway detected by Western Blotting. B shows the effect of axitinib on the proliferation ability of ipNF95.6 cells detected by MTT assay. C shows the effect of axitinib on the proliferation ability of ipNF95.11C cells detected by MTT assay.

[0026] Figure 6 This is the effect of axitinib on the proliferation ability of neurofibroma detected by colony formation assay. A shows the distribution of neurofibroma cells after treatment with 0 μM or 10 μM axitinib. B shows the statistical chart of the number of colonies of ipNF95.6 cells after treatment with 0 μM or 10 μM axitinib. C shows the statistical chart of the number of colonies of ipNF95.11C cells after treatment with 0 μM or 10 μM axitinib.

[0027] Figure 7 This is the therapeutic effect of axitinib combined with trametinib on neurofibroma. A shows the growth curve of ipNF95.6 cells treated with axitinib combined with trametinib. B shows the growth curve of ipNF95.11C cells treated with axitinib combined with trametinib. C shows the distribution of ipNF95.6 cells and ipNF95.11C cells treated with axitinib combined with trametinib. D shows the statistical chart of the number of colonies of ipNF95.6 cells treated with axitinib combined with trametinib. E shows the statistical chart of the number of colonies of ipNF95.11C cells treated with axitinib combined with trametinib.

[0028] Figure 8 This is the comparison of the inhibitory effects of axitinib and lapatinib on the cell viability of different plexiform neurofibroma cell lines. Among them, A is ipNF95.6 cells and B is ipNF95.11C cells. Detailed implementation mode

[0029] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0030] The plexiform neurofibroma cell lines ipNF95.6 and ipNF95.11C involved in the present invention were both donated by Professor Zhichao Wang of Shanghai Jiao Tong University. Professor Zhichao Wang purchased them from ATCC, the American Type Culture Collection. ipNF95.11C was published in the literature "Li Y, Liu J, Huang J, et al. Reduced PTPRS expression promotes epithelial-mesenchymal transition of Schwann cells in NF1-related plexiform neurofibromas. [J]. Cancer letters, 2024, 599217151." Axitinib (English name: Axitinib; English abbreviation: Axi; CAS No.: 319460-85-0), Selumetinib (English name: Selumetinib; English abbreviation: Sel, CAS No.: 606143-52-6), Trametinib (English name: Trametinib; English abbreviation: Tra, CAS No.: 871700-17-3), Lapatinib (English name: Lapatinib; English abbreviation Lap, CAS No.: 231277-92-2) were all purchased from MedChemExpress (MCE).

[0031] Example 1: Deubiquitinase OTUD3 promotes the progression of neurofibroma through the MEK / ERK signaling pathway 1. Experimental method In the plexiform neurofibroma cell line ipNF95.6, shOTUD3#1 (the sequence is shown in SEQ ID NO.1) and shOTUD3#2 (the sequence is shown in SEQ ID NO.1) that specifically knock down the OTUD3 gene (Gene ID: 23252) were transfected. Using Tubulin as an internal reference, it was detected whether OTUD3 was successfully knocked down. In ipNF95.11C cells, a plasmid carrying the OTUD3 gene was transfected, and using Tubulin as an internal reference, it was detected whether OTUD3 was overexpressed. The clone formation assay and CCK8 assay were used to detect the effects of knocking down and overexpressing OTUD3 on the proliferation activity of the plexiform neurofibroma cell line. The activity of the MEK / ERK signaling pathway was detected in ipNF95.6 cells with knocked-down OTUD3 and ipNF95.11C cells with overexpressed OTUD3.

[0032] SEQ ID NO.1: 5′-TGGAAATCAGGGCTTAAAT-3′。

[0033] SEQ ID NO.2: 5'-GAGTTACACATCGCATATC-3'.

[0034] 2. Experimental results To determine the functional importance of OTUD3 in the development of neurofibroma, we established an OTUD3 knockdown ipNF95.6 cell line using shOTUD3-targeted lentivirus, as shown in A of Figure 1 ; meanwhile, we stably overexpressed OTUD3 in the ipNF95.11C cell line using OEOTUD3 lentivirus carrying an HA tag, as shown in B of Figure 1 . To further verify whether the intervention of OTUD3 affects the progression of neurofibroma, we evaluated the proliferation ability of single cells through a colony formation assay and the proliferation and viability of tumor cells through an MTT assay. After 2 weeks of culture, the colony formation ability and proliferation ability of the OTUD3 knockdown group (shOTUD3#1 and shOTUD3#2) were lower than those of the OTUD3 knockdown negative control group (NC) (as shown in A and B of Figure 2 ), while the colony formation ability and proliferation ability of the OTUD3 overexpression group (HA-OTUD3) were stronger than those of the OTUD3 overexpression negative control group (EV) (as shown in C and D of Figure 2 ). Therefore, we detected the expression levels of the MEK / ERK signaling pathway in human NF1-deficient Schwann cell lines before and after OTUD3 intervention by Western Blotting technology. The results are shown in A and B of Figure 3 . Knockdown of OTUD3 in ipNF95.6 resulted in a decrease in the total protein levels of MEK and ERK, as well as the phosphorylation levels of MEK (p-MEK), ERK (p-ERK), and Fos (p-Fos); while overexpression of OTUD3 in ipNF95.11C upregulated the total protein levels of MEK and ERK and the levels of p-MEK, p-ERK, and p-Fos.

[0035] The above results indicate that OTUD3 plays a pro-cancer role in neurofibroma and acts through the classical MEK / ERK signaling pathway. Therefore, targeted inhibition of OTUD3 to inhibit neurofibroma is an issue that we need to further discuss.

[0036] Example 2: Axitinib can bind to the deubiquitinase OTUD3 and thereby inhibit the progression of neurofibroma 1. Experimental methods Treat plexiform neurofibroma cell lines ipNF95.6 and ipNF95.11C with axitinib (5 μM) (the culture medium is high-glucose DMEM medium). Extract cell proteins after 36 hours of treatment, and detect the activity of the MEK / ERK signaling pathway by Western blotting. Treat plexiform neurofibroma cell lines ipNF95.6 and ipNF95.11C with axitinib (5 μM), and detect the cell viability at different time points by CCK8; at the same time, detect the change in cell viability by colony formation assay.

[0037] 2. Experimental results To find drug molecules targeting OTUD3, we predicted OTUD3-related interacting proteins in advance and found a certain degree of association between axitinib and OTUD3 through analysis. Since the association between axitinib and OTUD3 has not been reported, we confirmed the binding interaction between axitinib and OTUD3 by cellular thermal shift assay and surface plasmon resonance technology (as Figure 4 ). To verify that axitinib can inhibit the effect of pNF by targeting the interaction with OTUD3, we detected the expression level of the MEK / ERK signaling pathway after axitinib intervention (5 μM) by Western Blotting technology. The results are as Figure 5 shown in A of Figure 6 . In human NF1-deficient Schwann cell lines ipNF95.6 and ipNF95.11C, the total protein levels of MEK and ERK decreased, and the phosphorylation levels of MEK (p-MEK), ERK (p-ERK), and Fos (p-Fos) decreased. To further verify whether axitinib has an inhibitory effect on the progression of neurofibroma, we evaluated the proliferation ability of single cells by colony formation assay and evaluated the proliferation and viability of tumor cells by MTT assay. The results are as

[0038] shown in A, B, and C of

[0039] . After intervention with 5 μM axitinib, the cell colony formation ability and proliferation ability were weakened compared with the NC group. 1. Experimental methods Treat plexiform neurofibroma cell lines ipNF95.6 and ipNF95.11C with the MEK inhibitor (trametinib, 20 μM) and axitinib (5 μM) separately and in combination (Trametinib, 20 μM; Axitinib, 5 μM). After 2 weeks, detect the cell viability at different time points using CCK8; simultaneously, detect the change in cell viability using colony formation assay.

[0040] 2. Experimental results Since the results of Example 2 have demonstrated that axitinib can bind to the important protein OTUD3 for pNF progression, target and inhibit the MEK / ERK signaling pathway, and thus play an inhibitory role on pNF, we explored whether axitinib can enhance the sensitivity of MEK inhibitor in treating neurofibroma, relieve the treatment resistance of MEK inhibitor, and enhance its efficacy. We evaluated the effect of combination therapy through colony formation and MTT assays. The results are as Figure 7 shown in A, B, C, D, and E of. In human NF1-deficient Schwann cell lines ipNF95.6 and ipNF95.11C, the use of MEK inhibitor alone can reduce the colony and proliferation ability of neurofibroma, while the combined use of Axi and MEK inhibitor can significantly enhance the efficacy of MEK inhibitor and more greatly reduce the colony and proliferation ability of neurofibroma.

[0041] The above results indicate that axitinib can enhance the sensitivity of MEK inhibitor in treating neurofibroma, which provides a direction for solving the treatment resistance of clinical MEK inhibitor.

[0042] Example 4: Study on the inhibition of plexiform neurofibroma cell activity by lapatinib 1. Experimental method Treat plexiform neurofibroma cell lines ipNF95.6 and ipNF95.11C with the tyrosine kinase (lapatinib, 400 nM and 800 nM) and axitinib (5 μM) separately, and detect the cell viability at different time points using CCK8.

[0043] 2. Experimental results Axitinib is a tyrosine kinase. In this example, treating plexiform neurofibroma cells with lapatinib, it was found by MTT detection that lapatinib cannot effectively inhibit the activity of plexiform neurofibroma cells, as Figure 8 shown in A and B of. The results indicate that not all tyrosine kinases can effectively inhibit the activity of plexiform neurofibroma cells.

[0044] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To prevent repetition, preferred embodiments of the present invention are described.

[0045] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made by those skilled in the art once they learn of the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

Claims

1. Application of axitinib in the preparation of drugs for the treatment of plexiform neurofibromas.

2. The use of axitinib according to claim 1 in the preparation of a drug for treating plexiform neurofibroma, characterized in that: In the drug, axitinib is used as the only active ingredient or one of the active ingredients.

3. The use of axitinib in the preparation of a drug for treating plexiform neurofibroma according to claim 2, characterized in that: When axitinib is used as one of the active ingredients in the drug, the active ingredients also include a MEK inhibitor.

4. The use of axitinib in the preparation of a drug for treating plexiform neurofibroma according to claim 3, characterized in that: The MEK inhibitor is selected from one of selumetinib and trametinib.

5. The use of axitinib according to any one of claims 1 to 4 in the preparation of a drug for treating plexiform neurofibroma, characterized in that: The drug also includes pharmaceutically acceptable excipients.

6. The use of axitinib according to any one of claims 1 to 4 in the preparation of a drug for treating plexiform neurofibroma, characterized in that: The dosage form of the medicine is granules, tablets, capsules, pills, dripping pills, oral liquid preparations, intragastric lavage or injection dosage form.

7. A drug for treating plexiform neurofibroma, characterized in that: The drug contains the axitinib and MEK inhibitor described in claim 3 as active ingredients.

8. A drug for treating plexiform neurofibroma according to claim 7, characterized in that: The MEK inhibitor is selected from one of selumetinib and trametinib.

9. A drug for treating plexiform neurofibroma according to claim 7, characterized in that: The drug also includes pharmaceutically acceptable excipients.

10. The drug for treating plexiform neurofibroma according to claim 7, characterized in that: The dosage form of the medicine is granules, tablets, capsules, pills, dripping pills, oral liquid preparations, intragastric lavage or injection dosage form.

Images