Use of axitinib for the preparation of a drug for the treatment of plexiform neurofibroma

Abstract

The application belongs to the field of medicine, and particularly relates to application of axitinib in preparation of a medicine for treating plexiform neurofibroma. In the medicine, axitinib serves as the only effective component or one of the effective components. When axitinib serves as one of the effective components in the medicine, the effective components further include a MEK inhibitor. The MEK inhibitor is selected from one of selumetinib and trametinib. The application proves that axitinib can play a role in inhibiting plexiform neurofibroma (pNF) by targeting OTUD3, an important gene for promoting pNF progression. Moreover, axitinib is a drug that has been used for clinical treatment, and has good safety. The research result of the application shows that the combination use of axitinib and the MEK inhibitor can improve the inhibitory effect on neurofibroma.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the application of axitinib in the preparation of drugs for the treatment of neurofibromatosis plexus. Background Technology

[0002] Plexiform neurofibroma (pNF) is one of the most common and challenging tumors in patients with neurofibromatosis type 1 (NF1). Currently, treatment for pNF primarily relies on MEK inhibitors (MEKi), such as selumetinib. Selumetinib is an oral selective mitogen-activated protein kinase (MEK) inhibitor that was approved by the US FDA in 2020 for the treatment of symptomatic and / or progressive, unresectable NF1-related pNF in patients aged 2–18 years. However, despite the significant efficacy of selumetinib in some patients, such as partial remission observed in 70% of patients (tumor volume reduction ≥20% from baseline, maintained for at least 4 weeks), treatment resistance remains a concern. Therefore, the search for new therapeutic agents for plexiform neurofibroma is necessary. Summary of the Invention

[0003] The purpose of this invention is to provide the application of axitinib in the preparation of plexiform neurofibroma treatment drugs to solve the problem of selmetinib treatment resistance.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] In a first aspect, the present invention provides the use of axitinib in the preparation of a therapeutic drug for plexiform neurofibroma.

[0006] Pleistofibrillar fibroma is a typical clinical manifestation of neurofibromatosis type I, which can cause devastating damage to patients. The main treatment currently is surgical resection, and for patients who cannot undergo surgical resection, oral selumetinib (a MEK inhibitor) can be used. Selumetinib is also the only effective drug for treating plexus neurofibroma, but it faces two major clinical challenges: treatment resistance and limited efficacy as a single agent.

[0007] Axitinib is a VEGFR inhibitor, and pNF is a hypervascular tumor. This invention demonstrates that axitinib can inhibit pNF by targeting OTUD3, a key gene promoting pNF progression. Furthermore, axitinib is a drug already used in clinical treatment with a good safety profile. The results of this invention show that the combined use of axitinib and a MEK inhibitor can improve the inhibitory effect on neurofibromatosis.

[0008] Furthermore, axitinib is the sole active ingredient or one of the active ingredients in the drug.

[0009] Furthermore, when axitinib is one of the active ingredients in the drug, the active ingredients also include MEK inhibitors.

[0010] Furthermore, the MEK inhibitor is selected from selmetinib and trametinib.

[0011] Furthermore, the drug also includes pharmaceutically acceptable excipients. These pharmaceutically acceptable excipients include at least one conventional diluent (such as water for injection, microcrystalline cellulose, etc.), fillers (such as mannitol, sucrose, lactose, polyethylene glycol, Tween 80, sorbitol, menthol, liquid paraffin, petrolatum, stearic acid, glyceryl monostearate, lanolin, mineral oil, DMSO). At least one of the following: adhesives (such as carbomer, gum arabic, starch, cellulose, gelatin, polyvinylpyrrolidone, polyacrylamide, etc.), disintegrants (such as sodium carboxymethyl starch, croscarmellose sodium, hydroxypropyl methylcellulose, low-substituted hydroxypropyl cellulose, etc.), lubricants (such as talc, magnesium stearate, calcium stearate, solid polyethylene glycol, lecithin, silica, micronized silica, etc.), humectants (such as propylene glycol, glycerin, ethanol, etc.), stabilizers (such as disodium ethylenediaminetetraacetate, sodium thiosulfate, sodium metabisulfite, sodium sulfite, sodium bisulfite, ethanolamine, sodium bicarbonate, sodium acetate, nicotinamide, vitamin C, etc.), osmotic pressure regulators (such as sodium chloride, glucose, etc.), pH regulators (such as triethanolamine, sodium hydroxide, sodium citrate, etc.), and preservatives (such as chlorobutanol, parabens, ethylparaben, benzalkonium bromide, etc.). The excipients mentioned above can be in commonly used doses and mixed with axitinib and MEK inhibitors in commonly used ratios. Once the dosage of axitinib and MEK inhibitors is determined, the ratio between each pharmaceutical excipient can be adjusted as needed.

[0012] Furthermore, the dosage form of the drug is granules, tablets, capsules, pills, drop pills, oral liquid preparations, oral enemas, or injectable dosage forms.

[0013] Secondly, the present invention provides a medicament for treating plexiform neurofibroma, wherein the medicament comprises axitinib and a MEK inhibitor as active ingredients.

[0014] Furthermore, the MEK inhibitor is selected from selmetinib and trametinib.

[0015] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0016] Furthermore, the dosage form of the drug is granules, tablets, capsules, pills, drop pills, oral liquid preparations, oral enemas, or injectable dosage forms.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. Providing new treatment options: The use of axitinib in the treatment of plexiform neurofibroma (pNF) provides patients with new treatment options, especially for those who are resistant to or intolerant of existing MEK inhibitor therapy.

[0019] 2. Potential for combination therapy: When axitinib is used in combination with MEK inhibitors (such as selmetinib and trametinib), the anti-tumor effect can be enhanced through multi-target synergistic effects, overcoming the limitations of monotherapy.

[0020] 3. Overcoming treatment resistance: Combination therapy can overcome drug resistance problems that have emerged in existing treatments, and improve the durability and effectiveness of treatment.

[0021] 4. Broad clinical application prospects: The drug of this invention has broad prospects in clinical application and can improve the treatment effect and quality of life of pNF patients.

[0022] In summary, this invention provides a new strategy to overcome the shortcomings of existing treatments through the application of axitinib in the treatment of pNF and its combination therapy, which has significant clinical value and application prospects. Attached Figure Description

[0023] Figure 1 To test the efficiency of OTUD3 knockdown and overexpression using Western blotting, A shows the efficiency test results for OTUD3 knockdown, and B shows the efficiency test results for OTUD3 overexpression.

[0024] Figure 2 To assess the clonogenic ability of plexiform neurofibroma cell lines after OTUD3 knockdown and overexpression, A shows the distribution of ipNF95.6 cells after OTUD3 knockdown, B is a quantitative statistical graph of A, C shows the distribution of ipNF95.11C cells after OTUD3 overexpression, and D is a quantitative statistical graph of C.

[0025] Figure 3 To investigate the effects of OTUD3 knockdown and overexpression on MEK, p-MEK, ERK, p-ERK, and p-FOS expression in neurofibromatosis, A shows Western blotting to detect the expression of MEK, p-MEK, ERK, p-ERK, and p-FOS after OTUD3 knockdown, with relative intensities normalized to Tubulin. B shows Western blotting to detect the expression of MEK, p-MEK, ERK, p-ERK, and p-FOS after OTUD3 overexpression, with relative intensities normalized to Tubulin.

[0026] Figure 4 This is the docking site between axitinib and the OTUD3 molecule.

[0027] Figure 5 To investigate 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 of ipNF95.6 cells detected by MTT assay; and C shows the effect of axitinib on the proliferation of ipNF95.11C cells detected by MTT assay.

[0028] Figure 6 To investigate the effect of axitinib on the proliferative capacity of neurofibroma cells using a clonogenic assay, A shows the distribution of neurofibroma cells after treatment with 0 μM or 10 μM axitinib, B shows the clonal count of ipNF95.6 cells after treatment with 0 μM or 10 μM axitinib, and C shows the clonal count of ipNF95.11C cells after treatment with 0 μM or 10 μM axitinib.

[0029] Figure 7 To illustrate the therapeutic effect of axitinib combined with trametinib on neurofibromatosis, 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 and ipNF95.11C cells treated with axitinib combined with trametinib, D shows the clonal count of ipNF95.6 cells treated with axitinib combined with trametinib, and E shows the clonal count of ipNF95.11C cells treated with axitinib combined with trametinib.

[0030] Figure 8 This study compares the inhibitory effects of axitinib and lapatinib on the cell activity of different plexiform neurofibroma cell lines. In this study, A represents ipNF95.6 cells and B represents ipNF95.11C cells. Detailed Implementation

[0031] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0032] The plexiform neurofibroma cell lines ipNF95.6 and ipNF95.11C involved in this invention were both donated by Professor Wang Zhichao of Shanghai Jiao Tong University and purchased by Professor Wang Zhichao from ATCC, the American Type Culture Collection. ipNF95.11C is disclosed 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 (CAS No.: 319460-85-0), Selumetinib (CAS No.: 606143-52-6), Trametinib (CAS No.: 871700-17-3), and Lapatinib (CAS No.: 231277-92-2) were all purchased from MedChemExpress (MCE).

[0033] Example 1: Deubiquitinating enzyme OTUD3 promotes neurofibroma progression via the MEK / ERK signaling pathway

[0034] 1. Experimental Methods

[0035] In the plexiform neurofibroma cell line ipNF95.6, shOTUD3#1 (sequence shown in SEQ ID NO.1) and shOTUD3#2 (sequence shown in SEQ ID NO.1) specifically knocked down the OTUD3 gene (Gene ID: 23252) were transfected. Tubulin was used as an internal control to detect whether OTUD3 was successfully knocked down. In ipNF95.11C cells, a plasmid carrying the OTUD3 gene was transfected, and Tubulin was used as an internal control to detect whether OTUD3 was overexpressed. The effects of OTUD3 knockdown and overexpression on the proliferative activity of the plexiform neurofibroma cell line were detected using clonogenic assays and CCK8 assays. The activity of the MEK / ERK signaling pathway was detected in OTUD3-knockdown ipNF95.6 cells and OTUD3-overexpressing ipNF95.11C cells.

[0036] SEQ ID NO. 1: 5'-TGGAAATCAGGGCTTAAAT-3'.

[0037] SEQ ID NO. 2: 5'-GAGTTACACATCGCATATC-3'.

[0038] 2. Experimental Results

[0039] To determine the functional importance of OTUD3 in the development of neurofibroma, we established an OTUD3 knockdown ipNF95.6 cell line using shOTUD3-targeting lentivirus, such as... Figure 1 As shown in A; simultaneously, we stably overexpressed OTUD3 in the ipNF95.11C cell line using an OEOTUD3 lentivirus carrying the HA tag, as shown in Figure A; Figure 1 As shown in B. To further verify whether OTUD3 intervention affects the progression of neurofibroma, we assessed the proliferation capacity of individual cells using a colony formation assay and the proliferation and activity of tumor cells using an MTT assay. After 2 weeks of culture, the colony formation and proliferation capacity of the OTUD3 knockdown groups (shOTUD3#1 and shOTUD3#2) were lower than those of the OTUD3 knockdown negative control group (NC) (e.g., ...). Figure 2 As shown in A and B), the OTUD3 overexpression group (HA-OTUD3) showed stronger colony formation and proliferation abilities than the OTUD3 overexpression negative control group (EV). Figure 2 (As shown in C and D). Therefore, we used Western blotting to detect the expression levels of the MEK / ERK signaling pathway in human NF1-deficient Schwann cell lines before and after OTUD3 intervention. The results are as follows... Figure 3 As shown in A and B, knocking down OTUD3 in ipNF95.6 leads to a decrease in the total protein levels of MEK and ERK, as well as a decrease in the phosphorylation levels of MEK (p-MEK), ERK (p-ERK), and Fos (p-Fos). In contrast, overexpression of OTUD3 in ipNF95.11C upregulates the total protein levels of MEK and ERK, as well as the levels of p-MEK, p-ERK, and p-Fos.

[0040] The above results indicate that OTUD3 plays a pro-cancer role in neurofibromas and exerts its effects through the classic MEK / ERK signaling pathway. Therefore, targeting and inhibiting OTUD3 to suppress neurofibromas is a question that needs further discussion.

[0041] Example 2: Axitinib can bind to the deubiquitinase OTUD3, thereby inhibiting the progression of neurofibroma.

[0042] 1. Experimental Methods

[0043] Neurofibromatosis cell lines ipNF95.6 and ipNF95.11C were treated with axitinib (5 μM) in DMEM high-glucose medium. Cell proteins were extracted 36 hours after treatment, and the MEK / ER signaling pathway activity was detected by Western blotting. Cell viability at different time points was detected using CCK8 assay after treatment with axitinib (5 μM); changes in cell viability were also detected using a colony formation assay.

[0044] 2. Experimental Results

[0045] To identify drug molecules targeting OTUD3, we pre-predicted OTUD3-related interacting proteins and discovered a certain degree of association between axitinib and OTUD3. Since the association between axitinib and OTUD3 had not been previously reported, we confirmed the binding interaction between axitinib and OTUD3 using cell thermal transfer experiments and surface plasmon resonance (SPR) technology. Figure 4 To verify that axitinib can inhibit pNF by targeting the interacting OTUD3, we used Western blotting to detect the expression levels of the MEK / ERK signaling pathway after axitinib intervention (5 μM). The results are as follows: Figure 5 As shown in Figure A, in the human NF1-deficient Schwann cell lines ipNF95.6 and ipNF95.11C, the total protein levels of MEK and ERK were decreased, as were the phosphorylation levels of MEK (p-MEK), ERK (p-ERK), and Fos (p-Fos). To further verify whether axitinib inhibits the progression of neurofibroma, we assessed the proliferation capacity of single cells using a clonogenic assay and evaluated tumor cell proliferation and activity using an MTT assay. The results are as follows: Figure 6 As shown in A, B, and C, cell colony formation and proliferation were weakened after intervention with 5 μM axitinib compared to the NC group.

[0046] These results indicate that axitinib can inhibit pNF by binding to OTUD3, a key protein in pNF progression, and targeting the MEK / ERK signaling pathway. This provides a theoretical basis for combining selumetinib (a MEK inhibitor) with axitinib for the treatment of pNF.

[0047] Example 3: Combination use of axitinib to improve the sensitivity of MEK inhibitors in the treatment of neurofibromatosis

[0048] 1. Experimental Methods

[0049] Neurofibromatosis cell lines ipNF95.6 and ipNF95.11C were treated separately and co-treated with MEK inhibitors (trametinib, 20 μM) and axitinib (5 μM) (trametinib, 20 μM; axitinib, 5 μM). Cell viability at different time points was detected by CCK8 assay after 2 weeks. Changes in cell viability were also detected by colony formation assay.

[0050] 2. Experimental Results

[0051] Since the results of Example 2 have demonstrated that axitinib can inhibit pNF by binding to OTUD3, a key protein in pNF progression, and targeting the MEK / ERK signaling pathway, we investigated whether axitinib could enhance the sensitivity of neurofibromatosis treated with MEK inhibitors, alleviate MEK inhibitor resistance, and improve efficacy. We evaluated the effect of combination therapy using clonogenic and MTT assays. The results are as follows: Figure 7 As shown in A, B, C, D, and E, in the human NF1-deficient Schwann cell lines ipNF95.6 and ipNF95.11C, MEK inhibitors alone can reduce the clonal and proliferative capacity of neurofibromas, while the combined use of Axi and MEK inhibitors can significantly enhance the efficacy of MEK inhibitors and further reduce the clonal and proliferative capacity of neurofibromas.

[0052] The above results indicate that axitinib can enhance the sensitivity of MEK inhibitors to neurofibromatosis, which provides a direction for addressing treatment resistance to MEK inhibitors in clinical practice.

[0053] Example 4: Study on the inhibitory activity of lapatinib on neurofibromatosis cells

[0054] 1. Experimental Methods

[0055] Neurofibromatosis cell lines ipNF95.6 and ipNF95.11C were treated with tyrosine kinases (lapatinib, 400 nM and 800 nM) and axitinib (5 μM), respectively, and cell viability at different time points was detected by CCK8 assay.

[0056] 2. Experimental Results

[0057] Axitinib is a tyrosine kinase. In this example, lapatinib was used to treat neurofibromatosis cells. MTT assay revealed that lapatinib did not effectively inhibit neurofibromatosis cell activity. Figure 8 As shown in Figures A and B. The results indicate that not all tyrosine kinases can effectively inhibit plexiform neurofibroma cell activity.

[0058] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0059] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. Use of axitinib for the preparation of a medicament for the treatment of plexiform neurofibroma, characterized in that, The effective components of the medicine are axitinib and a MEK inhibitor, and the MEK inhibitor is trametinib.

2. The use of axitinib according to claim 1 for the preparation of a medicament for the treatment of plexiform neurofibroma, characterized in that, The medicine further comprises pharmaceutically acceptable adjuvants.

3. The use of axitinib according to claim 1 for the preparation of a medicament for the treatment of plexiform neurofibroma, characterized in that, The dosage form of the medicine is granules, tablets, capsules, pills, oral liquid preparations or injection administration dosage forms.

4. A medicament for treating plexiform neurofibroma, characterized by, The effective components of the medicine are axitinib and a MEK inhibitor, and the MEK inhibitor is trametinib.

5. The medicament for treating plexiform neurofibroma according to claim 4, wherein The medicine further comprises pharmaceutically acceptable adjuvants.

6. The medicament for treating plexiform neurofibroma according to claim 5, wherein The dosage form of the medicine is granules, tablets, capsules, pills, oral liquid preparations or injection administration dosage forms.

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