A synergistic combination for treating chordoma and use thereof

Abstract

The application belongs to the field of medicine, and particularly relates to a synergistically treating chordoma combined drug and application thereof. The combined drug is Notch blocker PF03084014 and ELT or Notch blocker PF03084014 and DOX. Research proves that the Notch blocker PF0308401 targets and regulates the transcription expression of a key transcription regulatory factor Brachyury of chordoma, and inhibits tumor cell proliferation; and can obviously improve the drug resistance of chordoma to the chemotherapeutic drugs DOX and ELT. The application can provide an advantageous laboratory research basis and a transformation research direction for a chordoma drug treatment scheme.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a combination drug for the synergistic treatment of chordoma and its uses. Background Technology

[0002] Chordoma is a rare, malignant bone tumor originating from residual notochord tissue, commonly found in axial bone tissues such as the skull base and sacrococcygeal region. Although chordoma progresses slowly, patients often have a poor prognosis; the median survival is approximately 8-9 years, and the tumor recurrence rate is over 50%, with at least 20% of patients exhibiting aggressive tumor growth. Because chordoma is insensitive to traditional chemotherapy drugs and radiotherapy, its treatment primarily involves surgical resection. Currently, there are no FDA-approved standard chemotherapy drugs. Therefore, there is an urgent need to clarify and improve the chemical drug formulation for chordoma.

[0003] In chordomas, the transcription factor Brachyury is widely and highly expressed in chordoma tumor tissues and plays a crucial role in the occurrence and development of chordomas as a typical lineage-specific oncogene. Therefore, therapeutic strategies targeting the expression of the transcription factor Brachyury have great clinical application potential.

[0004] The Notch signaling pathway is a highly conserved cell signaling system that plays a crucial regulatory role in cell proliferation and differentiation, and in maintaining homeostasis. For example, during the development of vertebrate neural axons, activated Notch signaling can maintain the stemness of stem and progenitor cells and inhibit cell differentiation. Simultaneously, Notch signaling can also regulate intercellular signal transduction through cross-interactions with other signaling pathways. Abnormal Notch signaling is closely related to tumor development and progression. However, its specific role in chordoma remains unclear. This study found that the Notch blocker PF03084014 significantly inhibited chordoma tumor cell proliferation, promoted apoptosis, and targeted the inhibition of the chordoma marker transcription factor Brachyury. More importantly, we demonstrated through in vitro and in vivo experiments that the Notch blocker PF03084014 exhibits a synergistic effect with the chemotherapy drugs doxorubincin (DOX) and erlotinib (ELT), improving chemotherapy resistance to DOX and ELT. This provides favorable research direction support for the clinical translational research of chordoma. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a pharmaceutical use for the Notch blocker PF03084014 in combination with chemotherapy drugs to prepare anti-chordoma drugs.

[0006] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0007] A combination therapy for synergistic treatment of chordoma, the combination therapy comprising the Notch blocker PF03084014 and a chemotherapy drug, wherein the chemotherapy drug is doxorubicin or erlotinib.

[0008] Furthermore, the Notch blocker PF03084014 can improve the resistance of chordoma to the chemotherapy drugs doxorubicin or erlotinib.

[0009] Furthermore, in the combined drug, the mass ratio of PF03084014 to erlotinib is 2:1.

[0010] Furthermore, in the combined drug, the mass ratio of PF03084014 to doxorubicin is 5:2.

[0011] The present invention also provides the use of the combination drug in the preparation of products that inhibit the growth and / or metastasis of chordoma.

[0012] The present invention also provides a method for non-therapeutic inhibition of chordoma cell growth in vitro, the method comprising the step of: co-culturing chordoma cells with the drug combination.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] This invention discovers that the Notch blocker PF03084014 (PF for short) targets and regulates the transcriptional expression of Brachyury, a key transcriptional regulator in chordoma, thereby inhibiting tumor cell proliferation. Furthermore, it significantly improves the resistance of chordoma to chemotherapeutic drugs such as doxorubicin and erlotinib. This invention provides a favorable laboratory research foundation and translational research direction for chordoma drug treatment regimens. Attached Figure Description

[0015] Figure 1 The results of the combined effect index analysis of each treatment group in Example 1 are shown.

[0016] Figure 2 The images show the results of the cell clone sphere formation experiment in each treatment group of Example 1.

[0017] Figure 3 This is an analysis of the number of cell clones in each treatment group in Example 1.

[0018] Figure 4 The images show the tumor images of each treatment group at the end of the experiment in Example 1.

[0019] Figure 5 Tumor growth curves for different treatment groups in Example 1.

[0020] Figure 6 This is a comparison of tumor quality among the groups at the experimental endpoint of Example 1.

[0021] Figure 7 Example 1: Immunohistochemical staining was used to detect the expression of chordoma-related proteins in different treatment groups.

[0022] Figure 8 Example 1: Western blot analysis was used to detect the expression of chordoma-related proteins in different treatment groups.

[0023] Figure 9 The results of the combined effect index analysis of each treatment group in Example 2 are shown.

[0024] Figure 10 The images show the results of the cell clone sphere formation experiment in each treatment group of Example 2.

[0025] Figure 11 This is an analysis of the number of cell clones in each treatment group in Example 2.

[0026] Figure 12 The images show the tumor images of each treatment group at the end of the experiment in Example 2.

[0027] Figure 13 The tumor growth curves for different treatment groups in Example 2 are shown.

[0028] Figure 14 This is a comparison of tumor quality among the groups at the experimental endpoint of Example 2.

[0029] Figure 15 Example 2: Immunohistochemical staining was used to detect the expression of chordoma-related proteins in different treatment groups.

[0030] Figure 16 Example 2: Western blot analysis of the expression of chordoma-related proteins in different treatment groups. 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] Example 1

[0033] Notch blocker PF03084014 and ELT are used to treat chordoma.

[0034] I. Experimental Materials

[0035] 1. Chordoma U-CH1 cell line (Shanghai Zeye Biotechnology)

[0036] 2. Chordoma U-CH1 cell line tumor-bearing mouse model: 4-8 weeks old, female BALB / c nude mice (subcutaneously inoculated with tumor tissue, experiments started 1 month after inoculation).

[0037] II. Experimental Methods

[0038] 1. In vitro experiments

[0039] (1) CCK8 cell proliferation experiment

[0040] The single-cell suspension was prepared at a ratio of 5 × 10 3 Cells were evenly seeded into 96-well plates at a uniform density and incubated overnight at 37°C. Different concentrations of PF (0, 1.25, 2.5, 5 μM), ELT (0, 1.25, 2.5, 5 μM), and PF combined with ELT (Combo group) were added to the drug treatment groups, and incubation continued at 37°C for 72 hours. Subsequently, CCK8 reaction solution was added directly to each well at a 1:9 ratio. After culturing at 37°C for 30 minutes, the absorbance (optical density, OD) was read using a microplate reader at a wavelength of 450 nm. Furthermore, the synergistic antitumor effect was analyzed using CompuSyn software, and the combination index (CI) was recorded. CI < 1 indicates synergistic antitumor effect; CI = 1 indicates additive effect; CI > 1 indicates interactive effect. The results are shown below. Figure 1 As shown.

[0041] (2) Analysis of cell clone spheroid formation ability:

[0042] The collected single-cell suspensions were divided into 10... 3 Cells were evenly seeded into 6-well plates at a uniform density and incubated overnight at 37°C. Different concentrations of PF, ELT, or a combination of PF and ELT were then added. After 72 hours of incubation at 37°C, the culture medium was aspirated; the cells were washed twice with PBS, and then fresh complete culture medium was added. Incubation continued at 37°C for 3 weeks. Subsequently, the culture medium was aspirated, and 0.05% crystal blue staining solution was added. The plates were then incubated on a shaker at room temperature. After 30 minutes, the plates were rapidly shaken and rinsed with deionized water for 15 minutes. After the cell culture plates dried, photographs were taken, the number of colony-forming cells in the plates was counted, and statistical analysis was performed. The results are shown below. Figure 2-3 .

[0043] 2. In vivo experiments

[0044] (1) Chordoma U-CH1 cell line tumor-bearing mouse models were randomly divided into groups and administered the drug orally once a day for 7 days, 7 days off, 7 days on, and 7 days off (a total of 28 days) in the following doses:

[0045] A: Blank control group (denoted as Ctrl group): No medication was administered;

[0046] B: Group PF03084014 (denoted as PF group): PF03084014 (25mg / kg);

[0047] C: ELT group (denoted as ELT group): ELT (25mg / kg);

[0048] D: PF combined with ELT (referred to as Combo group): PF03084014 (25mg / kg) and ELT (25mg / kg).

[0049] The diameter of the tumor was measured regularly using calipers, and a tumor growth curve was plotted. The weight of the nude mice was measured using an electronic balance. The mice were euthanized by carbon dioxide inhalation combined with cervical dislocation the day after the last administration. The tumors were removed, weighed, and their volume was calculated using the following formula:

[0050] V = 1 / 2(LW) 2 ); where L is the longest diameter of the tumor and W is the shortest diameter of the tumor;

[0051] The above experimental results are shown in Figure 4-6 .

[0052] (2) Immunohistochemical staining was used to detect the expression levels of chordoma-related proteins in different treatment groups. The results are shown in […]. Figure 7 .

[0053] (3) Western blot was used to detect the expression levels of chordoma-related proteins in different treatment groups. The results are shown in […]. Figure 8 .

[0054] In summary, in vitro experiments in this embodiment revealed a synergistic effect between PF and ELT. Compared with the control group and the PF and ELT groups, the Combo group significantly inhibited the formation of clonal spheroids in chordoma U-CH1 and UCL / JHH cells. Furthermore, in vivo experiments showed that, compared with the control group, the Combo group inhibited tumor cell proliferation and downregulated the protein expression levels of the chordoma tumor marker Brachyury and the cell proliferation marker Ki67. After PF and ELT acted synergistically on chordoma cells, they synergistically downregulated the protein expression levels of proliferation-related proteins PCNA and P-stat3 / stat3, and upregulated the protein expression levels of apoptosis proteins Cleaved-caspase3 and Cleaved-PARP.

[0055] Example 2

[0056] Notch blocker PF03084014 and DOX are used to treat chordoma.

[0057] I. Experimental Materials

[0058] Same as Example 1.

[0059] II. Experimental Methods

[0060] 1. In vitro experiments

[0061] (1) CCK8 cell proliferation experiment

[0062] The collected single-cell suspension was processed at a ratio of 5 × 10⁻⁶. 3 Cells were evenly seeded at a uniform density in 96-well plates and incubated overnight at 37°C. Different concentrations of PF (0, 1.25, 2.5, 5 μM), DOX (0, 1.25, 2.5, 5 μM), and PF combined with DOX (Combo group) were added to the drug treatment groups, and incubation continued at 37°C for 72 hours. Subsequently, CCK8 reaction solution was added directly to each well at a 1:9 ratio. After incubation at 37°C for 30 minutes, the absorbance (optical density, OD) was read using a microplate reader at a wavelength of 450 nm. Furthermore, the synergistic antitumor effect was analyzed using CompuSyn software, and the combination index (CI) was recorded. CI < 1 indicates synergistic antitumor effect; CI = 1 indicates additive effect; CI > 1 indicates interactive effect. The results are shown below. Figure 9 As shown.

[0063] (2) Analysis of cell clone spheroid formation ability:

[0064] The collected single-cell suspensions were divided into 10... 3 Cells were evenly seeded into 6-well plates at a uniform density and incubated overnight at 37°C. Different concentrations of PF, DOX, or DOX combined with ELT (Combo) were then added. After 72 hours of incubation at 37°C, the culture medium was aspirated; the cells were washed twice with PBS, and fresh complete culture medium was added. Incubation continued at 37°C for 3 weeks. Subsequently, the culture medium was aspirated, and 0.05% crystal blue staining solution was added. The plates were then incubated on a shaker at room temperature. After 30 minutes, the plates were rapidly shaken and rinsed with deionized water for 15 minutes. After the cell culture plates dried, photographs were taken, the number of colony-forming cells in the plates was counted, and statistical analysis was performed. The results are shown below. Figure 10-11 .

[0065] 2. In vivo experiments

[0066] (1) Chordoma U-CH1 cell line tumor-bearing mouse models and UCL / JHH PDX models were randomly divided into groups and administered the drug orally once a day for 7 days, 7 days off, 7 days on, and 7 days off (a total of 28 days) in the following doses:

[0067] A: Blank control group (denoted as Ctrl group): No medication was administered;

[0068] B: Group PF03084014 (denoted as PF group): PF03084014 (25mg / kg);

[0069] C: DOX group (denoted as DOX group): DOX (5mg / kg);

[0070] D: PF combined with DOX (referred to as Combo group): PF03084014 (25mg / kg) and ELT (5mg / kg).

[0071] The diameter of the tumor was measured regularly using calipers, and a tumor growth curve was plotted. The weight of the nude mice was measured using an electronic balance. The mice were euthanized by carbon dioxide inhalation combined with cervical dislocation the day after the last administration. The tumors were removed, weighed, and their volume was calculated using the following formula:

[0072] V = 1 / 2(LW) 2 ); where L is the longest diameter of the tumor and W is the shortest diameter of the tumor;

[0073] The above experimental results are shown in Figure 12-14 .

[0074] (2) Immunohistochemical staining was used to detect the expression of chordoma-related proteins in different treatment groups. The results are shown in […]. Figure 15 .

[0075] (3) Western blot analysis was performed to detect the expression of chordoma-related proteins in different treatment groups. The results are shown in […]. Figure 16 .

[0076] In summary, in vitro experiments in this embodiment revealed a synergistic effect between PF and ELT. Compared with the control group, the PF group, and the DOX group, the Combo group significantly inhibited the formation of clonal spheroids in chordoma U-CH1 and UCL / JHH cells. Furthermore, in vivo experiments showed that, compared with the control group, the Combo group inhibited tumor cell proliferation and downregulated the protein expression levels of the chordoma tumor marker Brachyury and the cell proliferation marker Ki67. After PF and DOX synergistically acted on chordoma cells, they synergistically downregulated the protein expression levels of proliferation-related proteins PCNA and c-myc, and upregulated the protein expression levels of apoptosis proteins Cleaved-caspase 3 and Cleaved-PARP. Moreover, PF and DOX also synergistically inhibited the protein expression level of Hes1, a downstream protein of the Notch signaling pathway, and suppressed the protein expression level of the chordoma tumor marker Brachyury.

[0077] 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.

[0078] 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.

[0079] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of a composition for the manufacture of a medicament for the treatment of chordoma, characterized in that, The composition is composed of Notch blocker PF03084014 and a chemotherapy drug, wherein the chemotherapy drug is doxorubicin or erlotinib, the Notch blocker PF03084014 can improve the drug resistance of chordoma to the chemotherapy drug doxorubicin or erlotinib, and the composition can inhibit the growth and / or metastasis of chordoma.

2. Use according to claim 1, characterized in that, The mass ratio of PF03084014 to erlotinib is 2:

1.

3. Use according to claim 1, characterized in that, The mass ratio of PF03084014 to doxorubicin is 5:

2.

4. A method for in vitro non-therapeutic inhibition of growth of medullosarcoma cells using the composition of claim 1, characterized in that, The method comprises culturing chordoma cells and the composition.

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