Use of an inhibitor targeting c-rel in the manufacture of a medicament for treating glioblastoma

Abstract

The application discloses an application of an inhibitor targeting c-Rel in preparation of a medicine for treating glioblastoma, relates to the technical field of biological medicine, and discloses an application of a c-Rel protein inhibitor in preparation of a medicine for treating glioblastoma, wherein the medicine comprises: a) the c-Rel protein inhibitor; b) temozolomide; c) a PD-1 / PD-L1 pathway inhibitor; and the medicine is arranged to be used in combination with radiotherapy. The application discloses, for the first time, a key role of the c-Rel protein in formation of an immunosuppressive tumor microenvironment of glioblastoma, and establishes the c-Rel as a new target for treating glioblastoma. Furthermore, the application proposes a quadruple scheme of the c-Rel inhibitor combined with temozolomide, radiotherapy and the PD-1 / PD-L1 pathway inhibitor, realizes complete regression of a tumor in a mouse subcutaneous transplantation tumor model, and achieves 100% long-term (>80 days) tumor-free survival in an intracranial model, which is significantly superior to existing treatment schemes.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of an inhibitor targeting c-Rel in the preparation of drugs for treating glioblastoma. Background Technology

[0002] Glioblastoma (GBM) is a grade IV glioma, the most malignant type according to the World Health Organization (WHO) classification, and is the most common primary malignant tumor of the adult central nervous system. It is characterized by high invasiveness, cellular heterogeneity, and an immunosuppressive tumor microenvironment (TME). The current standard treatment is the Stupp regimen, which involves surgical resection followed by concurrent chemoradiotherapy with temozolomide (TMZ) and subsequent adjuvant TMZ chemotherapy. However, this regimen only provides limited prolongation of patient survival, with a median overall survival of approximately 14-16 months and a five-year survival rate of less than 10%. Almost all patients experience recurrence.

[0003] In recent years, immune checkpoint blockade therapies, represented by inhibitors of programmed death receptor-1 (PD-1) and its ligand (PD-L1), have achieved breakthroughs in various solid tumors. However, in large-scale phase III clinical trials targeting GBM (such as CheckMate 143 and KEYNOTE-028), they have failed to significantly improve overall patient survival, highlighting the inherent resistance of GBM to immunotherapy. Studies have shown that the TME of GBM is enriched with a large number of immunosuppressive cells, such as regulatory T cells (Tregs), tumor-associated macrophages / microglia (TAMs), and myeloid-derived suppressor cells (MDSCs), and contains various immunosuppressive factors, collectively forming a strong immune barrier. Therefore, developing novel treatment strategies that can effectively reverse or reshape the immunosuppressive TME of GBM has become crucial to improving the efficacy of existing therapies (including radiotherapy, chemotherapy, and immunotherapy).

[0004] The nuclear factor-κB (NF-κB) signaling pathway is a core pathway regulating inflammation, immune response, cell proliferation, and survival. c-Rel, an important member of the NF-κB / Rel protein family, is primarily expressed in immune cells and is crucial for the activation, proliferation, and functional differentiation of lymphocytes (especially T cells and B cells). Although the role of the NF-κB pathway in tumorigenesis is widely recognized, the specific function of the c-Rel subunit in GBM progression, particularly in regulating the immunosuppressive tumor microenvironment (TME) of GBM, remains unclear. Currently, there is a lack of GBM treatments targeting c-Rel specifically to synergistically enhance the efficacy of TMZ, radiotherapy, and PD-1 / PD-L1 inhibitors by modulating the TME. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an application of a c-Rel-targeting inhibitor in the preparation of a drug for treating glioblastoma, so as to overcome the immune resistance of GBM and improve the treatment response rate and survival benefit.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows.

[0007] The use of a c-Rel protein inhibitor in the preparation of a medicament for treating glioblastoma, the medicament comprising: a) the c-Rel protein inhibitor; b) temozolomide; c) a PD-1 / PD-L1 pathway inhibitor; and the medicament being configured for use in combination with radiotherapy.

[0008] More preferably, the c-Rel protein inhibitor is pentoxifylline.

[0009] More preferably, the PD-1 / PD-L1 pathway inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0010] More preferably, the c-Rel protein inhibitor, temozolomide, and PD-1 / PD-L1 pathway inhibitor are administered to the subject simultaneously, separately, or sequentially with the radiotherapy.

[0011] More preferably, the treatment is performed after surgical resection of the glioblastoma.

[0012] A pharmaceutical composition for treating glioblastoma comprises: a) a therapeutically effective amount of a c-Rel protein inhibitor; b) a therapeutically effective amount of temozolomide; and c) a therapeutically effective amount of a PD-1 / PD-L1 pathway inhibitor; wherein the pharmaceutical composition is configured to be administered in combination with radiotherapy.

[0013] More preferably, the c-Rel protein inhibitor is pentoxifylline, and the PD-1 / PD-L1 pathway inhibitor is an anti-PD-1 antibody.

[0014] More preferably, the pharmaceutical composition is an injection, an oral preparation, or a topical preparation.

[0015] More preferably, the c-Rel protein inhibitor is a small molecule compound, antibody, antisense oligonucleotide, or siRNA that has the function of inhibiting the activity or expression of c-Rel protein.

[0016] The beneficial effects of adopting the above technical solution are as follows: This study is the first to clearly reveal the key role of c-Rel in the immunosuppressive TME of GBM, establishing it as a potential new target for treating GBM, especially for enhancing the efficacy of immunotherapy, and providing a new direction for drug development.

[0017] The TRPP quadruple therapy proposed in this invention, with c-Rel inhibitors as the core, has shown significantly better anti-tumor activity than the TMZ+RT+αPD-1 triple therapy in various preclinical GBM models, and can achieve complete tumor regression, providing a practical new strategy for solving GBM treatment resistance.

[0018] This approach is not simply a combination of drugs. By regulating immunosuppressive cells in the TME, c-Rel inhibitors effectively reverse the "immune cold" state of GBM, creating conditions for T cells to recognize antigens released from immunogenic cell death induced by radiotherapy and chemotherapy, and for PD-1 inhibitors to relieve T cell exhaustion, thus producing a synergistic effect.

[0019] All components of the protocol (TMZ, RT, PTXF, and αPD-1 antibody) are drugs or mature treatments that have been approved for clinical use. Their safety as monotherapy is known, which greatly reduces the development risk and cycle of combination therapy and has the potential to achieve rapid clinical translation. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the difference in c-Rel mRNA expression between glioblastoma and normal brain tissue, based on analysis of a public database, in Example 1 of this invention.

[0021] Figure 2 This is a schematic diagram illustrating the verification of c-Rel protein expression levels in paired adjacent normal tissue and tumor tissue from a clinical glioblastoma patient in Example 1 of the present invention; wherein, Figure 2 'a' represents a statistical bar chart. Figure 2 b is a representative Western blotting image.

[0022] Figure 3 This is a scatter plot showing the correlation between c-Rel protein expression and PD-1 protein expression in clinical glioblastoma samples in Example 1 of this invention.

[0023] Figure 4 This is a schematic diagram illustrating the anti-tumor effect of the c-Rel inhibitor PTXF combined with TMZ, RT and αPD-1 in a mouse GL261 glioma subcutaneous transplantation model in Example 2 of the present invention, showing the tumor growth curve.

[0024] Figure 5 This is a schematic diagram illustrating the antitumor effect and survival benefit of the c-Rel inhibitor PTXF combined with TMZ, RT, and αPD-1 in a mouse intracranial GL261 glioma model in Example 2 of the present invention; wherein, Figure 5 a shows the survival curves of mice in different treatment groups. Figure 5 b shows a schematic diagram of fluorescence in a representative mouse brain tumor.

[0025] Figure 6 This is a schematic diagram illustrating the antitumor effect and survival benefit of the c-Rel inhibitor PTXF combined with TMZ, RT, and αPD-1 in a mouse intracranial CT2A glioma model in Example 2 of the present invention; wherein, Figure 6 a shows the survival curves of mice in different treatment groups. Figure 6 b shows a schematic diagram of fluorescence in a representative mouse brain tumor.

[0026] Figure 7 The figures show the tumor growth curves for each group in Example 2.

[0027] Figure 8 The survival curves of mice in each group in Example 2 are shown. Detailed Implementation

[0028] The following embodiments illustrate the present invention in detail. All raw materials and equipment used in the present invention are commercially available products and can be directly obtained through market purchase. The following embodiments are intended to facilitate a better understanding of the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent companies. Unless otherwise specified, all percentages in the following embodiments refer to mass percentages.

[0029] It should be understood that, when used in this specification and the appended claims, the term "comprising" indicates the presence of a described feature, integral, step, operation, element, and / or component, but does not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components, and / or a collection thereof. It should also be understood that the term "and / or" as used in this specification and the appended claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes such combinations. As used in this specification and the appended claims, the term "if" may be interpreted, depending on the context, as "when," "once," "in response to determination," or "in response to detection." Similarly, the phrase "if determined" or "if detected [the described condition or event]" may be interpreted, depending on the context, as meaning "once determined," "in response to determination," "once detected [the described condition or event]," or "in response to detection [the described condition or event]."

[0030] Furthermore, in the description of this application and the appended claims, the terms "first," "second," "third," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance. References to "one embodiment" or "some embodiments" in this application mean that one or more embodiments of this application include a specific feature, structure, or characteristic described in connection with that embodiment. Therefore, the phrases "in one embodiment," "in some embodiments," "in other embodiments," "in still other embodiments," etc., appearing in different parts of this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. The terms "comprising," "including," "having," and variations thereof mean "including but not limited to," unless otherwise specifically emphasized.

[0031] Example 1: Expression characteristics and clinical relevance of c-Rel in glioblastoma 1.1 Bioinformatics Data Analysis: RNA-seq data (n=473) from glioblastoma (GBM) patients were downloaded from the Chinese Glioma Genome Atlas (CGGA) database, and RNA-seq data (n=1152) from normal brain tissue were downloaded from the Genotype-Tissue Expression (GTEx) project. After batch effect correction using R language and the limma package, the expression levels of the c-Rel gene (REL) were compared. The results showed that c-Rel mRNA expression was significantly upregulated in GBM tissue compared to normal brain tissue (p < 0.001), see [link to relevant documentation]. Figure 1 .

[0032] 1.2 Validation of Protein Levels in Clinical Samples: Tumor and adjacent non-tumor tissues were obtained during surgery from 45 patients with histologically confirmed GBM who visited the Department of Neurosurgery at the Second Hospital of Hebei Medical University. This protocol was approved by the Research Ethics Committee of the Second Hospital of Hebei Medical University. Specimens were flash-frozen in liquid nitrogen, and c-Rel protein expression was detected by Western blotting. ImageJ software was used to quantify the grayscale values ​​of the bands, and normalization was performed based on the expression level in adjacent normal tissues. Statistical results showed that c-Rel protein expression was higher in over 80% of GBM tumor tissues than in their paired adjacent normal tissues (paired t-test, p < 0.01), see [link to relevant documentation]. Figure 2 a and 2b.

[0033] 1.3 Correlation analysis between c-Rel and immune checkpoint molecules: Western blot was used to detect the expression levels of c-Rel and PD-1 proteins in tumor tissues of the 45 GBM patients. The results showed a significant positive correlation between c-Rel protein expression scores and PD-1 protein expression scores (r=0.52, p < 0.001). Figure 3 This suggests that GBM with high c-Rel expression may be accompanied by a more active PD-1 / PD-L1 immune checkpoint pathway.

[0034] Example 2: Validation of the efficacy of combination therapy targeting c-Rel in preclinical animal models 2.1 Cells and Animals: The mouse glioma cell lines GL261 and CT2A were provided by the Academy of Military Medical Sciences and the Shenzhen Hospital of Southern Medical University, respectively. Both cell lines were identified by STR analysis. SPF-grade adult female mice (C57BL / 6 mice, 20-25g, 6-8 weeks old) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (catalog number #11001). Mice were maintained under SPF conditions in a monitored environment (22℃ ± 3℃, 60% ± 5% humidity, 12:12h light). All mice received a standard diet with unlimited access to food and water. After acclimatization, mice were randomly assigned to control and experimental groups for subsequent experiments. All animal experimental protocols were approved by the Animal Care and Use Committee of the Second Hospital of Hebei Medical University and conducted in accordance with the National Institutes of Health's Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised in 1996).

[0035] 2.2 Efficacy assessment: Subcutaneous model: GL261 (2×10) 6(200 μl of phosphate-buffered saline (PBS) was injected into the right or left flank of C57BL / 6 mice. Tumors were measured twice weekly using calipers, and tumor volume was calculated using the formula (width² × length) / 2. When the tumor reached approximately 150 mm³ (11–13 days post-inoculation), mice were stratified randomized to experimental groups to maintain a balance in initial tumor size. Mice were euthanized when tumors exceeded 2000 mm³ or the longest tumor reached 20 mm. Tumor volume was measured twice weekly using vernier calipers. Results showed that tumors grew rapidly in the control group; while the TMZ+RT+αPD-1 group slowed growth but did not control tumor progression; and the TRPP quadruple therapy group induced complete tumor regression in all mice with no recurrence during the observation period. Figure 4 .

[0036] Intracranial or in situ model: GL261-luc cells or CT2A-luc cells (2×10⁻⁶) were injected using a stereotactic instrument and a microinjector. 5 Each mouse (cells dissolved in 5 μl PBS) was stereotactically injected into the right or left striatum (coordinates: 2 mm posterior to the anterior fontanelle, 1.5 mm lateral, 3 mm depth). Tumor growth was monitored by bioluminescence imaging. Once significant tumor growth was detected (5–7 days post-inoculation), mice were stratified and randomized to experimental groups to maintain balance in initial tumor size. Mice with a weight loss >20% were euthanized. Neurological symptoms were observed daily, and survival was recorded. Figure 5 As shown in figure a, the median survival of mice in the TRPP treatment group was significantly longer than that in other groups, and 100% of the mice were still alive at the end of the observation period (>80 days), achieving long-term tumor-free survival. Figure 6 As shown in a, the median survival of CT2A mice in the TRPP treatment group was significantly longer than that in the control group, and 80% of the mice were still alive at the end of the observation period (>80 days), achieving long-term tumor-free survival.

[0037] Tumor-bearing mice were treated with: temozolomide TMZ, 20 mg / kg, three days a week (Monday, Wednesday, and Friday) for up to 6 weeks; pentoxifylline PTXF, 60 mg / kg, five days a week (Wednesday to Sunday) for up to 6 weeks; αPD-1 (InVivoMab anti-mouse PD-1), 200 μg / mouse, administered every other Wednesday, Friday, and Sunday for up to 5 weeks; and / or RT, 10 Gy X-ray irradiation on the first Wednesday (day 3). Each drug was administered intraperitoneally to the mice in a volume of 100 μl. Radiation (10 Gy, 6 Gy / min) was administered to the mice under anesthesia via X-Rad 320 (Precision X-Ray Irradiation). The mice were placed on a small animal bed of the X-RAD SmART radiotherapy system. The irradiation field was 1 cm x 1 cm, and the rest of the body was shielded with a 5 mm thick lead plate.

[0038] 2.3 Statistical Analysis To verify the antitumor efficacy and synergistic effect of the TRPP quadruple therapy, the following control groups were set up in the GL261 subcutaneous xenograft model: CON group (blank control), RP group (RT+PTXF), PP group (PTXF+αPD-1), and TRP group (TMZ+RT+αPD-1), which were compared with the TRPP group (TMZ+RT+PTXF+αPD-1). Tumor growth curves for each group are shown below. Figure 7 As shown, the results indicated that the tumors in the CON group exhibited rapid progressive growth; while the RP and PP groups could delay tumor growth to some extent, they could not effectively control tumor progression; the TRP group could partially inhibit tumor growth, but the tumors still relapsed or progressed during the observation period; and the TRPP quadruple therapy group induced complete tumor regression in all mice, with no recurrence during the observation period.

[0039] Survival curves for each group of mice are as follows: Figure 8 As shown in Table 1, the median survival time and 80-day survival rate are as follows.

[0040] Table 1 Median survival time and long-term survival rate of mice in each group

[0041] The log-rank test (Mantel-Cox) was used for overall comparison and pairwise comparison, and the results are shown in Table 2.

[0042] Table 2. Log-rank test results of survival curves among groups.

[0043] Five groups of overall comparison: x 2=38.2, df=4, p<0.0001.

[0044] The above results indicate that the TRPP quadruple therapy regimen was statistically significantly different from each control group (p<0.01). Crucially, the comparison between the TRPP group and the TRP group (x<0.01) is particularly significant. 2 =7.8, p=0.005) This demonstrates that adding PTXF to the TRP triple regimen (TMZ+RT+αPD-1) to form the TRPP quadruple regimen extended the median survival of mice from 61 days to over 80 days, and increased the 80-day survival rate from 50.0% to 100%, indicating that the addition of PTXF produced a significant synergistic effect.

[0045] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0046] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. The use of a c-Rel protein inhibitor in the preparation of a medicament for treating glioblastoma, characterized in that, The drug comprises: a) the c-Rel protein inhibitor; b) temozolomide; c) a PD-1 / PD-L1 pathway inhibitor; and the drug is configured for use in combination with radiotherapy.

2. The application according to claim 1, characterized in that, The c-Rel protein inhibitor is pentoxifylline.

3. The application according to claim 1 or 2, characterized in that, The PD-1 / PD-L1 pathway inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

4. The application according to any one of claims 1-3, characterized in that, The c-Rel protein inhibitor, temozolomide, and PD-1 / PD-L1 pathway inhibitor were administered to the subject simultaneously, separately, or sequentially with the radiotherapy.

5. The application according to any one of claims 1-4, characterized in that, The treatment was performed after surgical removal of the glioblastoma.

6. A pharmaceutical composition for treating glioblastoma, characterized in that, The pharmaceutical composition comprises: a) a therapeutically effective amount of a c-Rel protein inhibitor; b) a therapeutically effective amount of temozolomide; and c) a therapeutically effective amount of a PD-1 / PD-L1 pathway inhibitor; wherein the pharmaceutical composition is configured to be administered in combination with radiotherapy.

7. The pharmaceutical composition according to claim 6, characterized in that, The c-Rel protein inhibitor is pentoxifylline, and the PD-1 / PD-L1 pathway inhibitor is an anti-PD-1 antibody.

8. The pharmaceutical composition according to claim 6 or 7, characterized in that, The pharmaceutical composition is an injection, oral preparation, or topical preparation.

9. The application according to any one of claims 1-5 or the pharmaceutical composition according to any one of claims 6-8, characterized in that, The c-Rel protein inhibitor is a small molecule compound, antibody, antisense oligonucleotide, or siRNA that has the function of inhibiting the activity or expression of c-Rel protein.

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