A combination of trifluoperazine and a pd-1 inhibitor and uses thereof

Abstract

This invention relates to the pharmaceutical field, specifically to a combination drug of trifluoperazine and a PD-1 inhibitor and its uses. This invention, through the combined use of trifluoperazine and a PD-1 inhibitor in the treatment of oral squamous cell carcinoma, has achieved superior efficacy compared to monotherapy. The combination of trifluoperazine and a PD-1 inhibitor in this invention increases CD8... + T cell expression levels and CD3 + CD8 + CD25 + Regarding the proportion of T cell subsets, it has a synergistic effect, providing a new strategy for the treatment of oral squamous cell carcinoma and showing great application prospects.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a combination of trifluoperazine and a PD-1 inhibitor and its uses. Background Technology

[0002] Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors of the oral and maxillofacial region. Current treatment primarily involves comprehensive therapy with surgery, but the 5-year survival rate for advanced-stage patients remains low. In recent years, immune checkpoint inhibitors (ICIs) have shown heterogeneity in treatment responses in OSCC-related clinical trials. This suggests that the complex immunosuppressive network within the OSCC tumor microenvironment (TME) may limit the efficacy of ICIs. Therefore, exploring combination therapy strategies that reshape the TME and enhance anti-tumor immune responses has become a key direction for overcoming current treatment bottlenecks.

[0003] Drug repurposing, also known as drug repositioning, refers to the development of new indications for drugs that have already been approved or are in clinical trials. This concept has received considerable attention in the field of oncology treatment in recent years. Previous studies have shown that drugs such as metformin and simvastatin exhibit certain anti-tumor activity in OSCC. Non-anti-tumor drugs have shown great potential in overcoming drug resistance and in combination with immunotherapy, and have broad value for exploration in the comprehensive treatment of OSCC.

[0004] Trifluoperazine (TFP) is a classic phenothiazine antipsychotic that improves positive symptoms of schizophrenia by selectively antagonizing dopamine D2 receptors in the central nervous system. Epidemiological studies have shown that patients receiving antipsychotic treatment have a lower risk of developing various cancers. In recent years, the antitumor effects of TFP have gradually attracted attention: studies have found that TFP can induce cell cycle arrest and apoptosis in cancer cells, inhibiting the growth and metastasis of triple-negative breast cancer. TFP promotes ROS accumulation, damages mitochondria, and induces mitophagy by activating the AMPK / mTOR / ULK1 signaling pathway, inhibiting osteosarcoma cell growth without damaging normal cells. Studies have also indicated that TFP exerts its anti-triple-negative breast cancer and osteosarcoma growth effects without neurological side effects. However, the mechanism of action of TFP in the treatment of OSCC is not fully understood, and its therapeutic efficacy and safety require further in-depth research. Therefore, developing new combination therapies for the treatment of OSCC has significant research and application value. Summary of the Invention

[0005] To address the problems of existing technologies, this invention provides a combination drug of trifluoperazine and a PD-1 inhibitor and its uses.

[0006] A combination therapy for treating oral squamous cell carcinoma, wherein the combination therapy is a phenothiazine compound and an immune checkpoint inhibitor.

[0007] Preferably, the phenothiazine compound is selected from trifluoperazine, and the immune checkpoint inhibitor is selected from PD-1 inhibitors.

[0008] Preferably, the PD-1 inhibitor is Anti-hPD-1 agonist mAb HM266.

[0009] Preferably, the combined medication is trifluoperazine and a PD-1 inhibitor, administered separately or simultaneously.

[0010] Preferably, the weight ratio of trifluoperazine to PD-1 inhibitor is 5.4-6.6:1.

[0011] The above-mentioned combined drugs are used in the preparation of drugs for treating oral squamous cell carcinoma.

[0012] A pharmaceutical composition for treating oral squamous cell carcinoma, comprising a phenothiazine compound and an immune checkpoint inhibitor as active ingredients, and pharmaceutically acceptable excipients or auxiliary ingredients.

[0013] Preferably, the phenothiazine compound is selected from trifluoperazine, and the immune checkpoint inhibitor is selected from PD-1 inhibitors.

[0014] Preferably, the PD-1 inhibitor is Anti-hPD-1 agonist mAb HM266.

[0015] Preferably, the weight ratio of trifluoperazine to PD-1 inhibitor is 5.4-6.6:1.

[0016] The combination of trifluoperazine and a PD-1 inhibitor of this invention demonstrates superior efficacy in inhibiting OSCC tumor growth compared to the single-drug combination. The combination therapy of this invention significantly increases CD8 expression levels, exhibiting a synergistic effect compared to the single-drug combination. The CD3 expression level in the combination therapy group... + CD8 + CD25 + The proportion of T cell subsets was significantly higher in the group treated with the drug alone than in the group treated with the drug alone, which also indicates that the combination drugs of the present invention have a synergistic effect and have a good application prospect in the treatment of OSCC.

[0017] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0018] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0019] Figure 1 A heatmap showing the expression of cGAS-STING pathway-related genes in TFP and control group samples.

[0020] Figure 2 Gross images of orthotopic xenografts in the control group, aPD-1 group, TFP group, and combination therapy group.

[0021] Figure 3 Tumor growth curves for the control group, aPD-1 group, TFP group, and combination therapy group ( P<0.001, which is the result of comparing the aPD-1, TFP, and TFP+PD-1 groups with the control group.

[0022] Figure 4 The tumor volume change ratios were compared among the control group, aPD-1 group, TFP group, and combination therapy group.

[0023] Figure 5 The results of CD8 immunohistochemistry of head and neck lymph node tissues from the control group, aPD-1 group, TFP group, and combination therapy group are shown.

[0024] Figure 6 CD3+ ions from head and neck lymph node tissues of the control group, aPD-1 group, TFP group, and combination therapy group. + CD8 + CD25 + T cell subset flow cytometry analysis and statistical results ( (P<0.01). Detailed Implementation

[0025] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0026] Example 1: Combination therapy for oral squamous cell carcinoma This embodiment provides a combination drug for treating oral squamous cell carcinoma, which involves administering trifluoperazine and the PD-1 inhibitor Anti-hPD-1 agonist mAb HM266 (this PD-1 inhibitor recognizes the same antigenic epitope as Anti-mouse PD-1 (CD279)-InVivo (clone number: RMP1-14), recognizing the proximal membrane region of amino acids 38-48) separately. TFP administration: TFP was prepared with corn oil to a concentration of 20 mg / mL. Each mouse received 20 mg / kg of the drug, or 20 g of mouse. 20 µL was administered intraperitoneally once daily. PD-1 inhibitor administration: Mice were directly injected with a stock solution containing 200 µg of anti-PD-1 antibody (PBS buffer, pH 7.4, PD-1 antibody concentration of 6.55 mg / mL), administered intraperitoneally every two days. During the experiment, the weight ratio of TFP to PD-1 inhibitor used in each mouse was 6:1. In other embodiments, the synergistic effect was still observed even when the weight ratio of TFP to PD-1 inhibitor fluctuated, for example, when the weight ratio of TFP to PD-1 inhibitor was 5.4:1 and 6.6:1.

[0027] The technical solution of the present invention will be further illustrated by the following experiments.

[0028] Experiment Example 1: The role of TFP in the cGAS-STING signaling path Sample preparation: HSC-3 cells in the logarithmic growth phase were collected. The TFP group was treated with a concentration of 20 μM, while the control group was cultured routinely. After 4 hours, the cells were digested with trypsin, washed twice with PBS buffer, and then centrifuged to collect the cells. Each group was configured with 3 replicates.

[0029] RNA extraction and library construction: Total RNA was extracted from cells using TRIzol reagent, strictly following the manufacturer's instructions. RNA purity was assessed and quantified using a NanoDrop 2000 spectrophotometer (Thermo Fischer), and RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies). Transcriptome libraries were constructed using the VAHTS Universal V5 RNA-seq Library Prep kit, following the kit's instructions.

[0030] Sequencing: The constructed library was sequenced using the Llumina Novaseq 6000 sequencing platform, generating 150bp paired-end reads during the sequencing process. The raw reads in FASTQ format were processed using FASTP software to remove low-quality reads and obtain high-quality clean reads for subsequent data analysis. The clean reads were aligned to a reference genome using HISAT2 software to calculate gene expression levels (FPKM), and read counts for each gene were obtained using HTSeq-count. Principal component analysis (PCA) was performed on the gene count data using R software (version 3.2.0). Differentially expressed genes were analyzed using DESeq2 software, defining genes that met the q-value < 0.05 and fold change > 2 or fold change < 0.5 as differentially expressed genes (DEGs). Using the transcriptome sequencing data, a cGAS-STING pathway heatmap was generated through gene expression pattern analysis.

[0031] The results showed that, compared with the control group, most cGAS-STING signaling-related genes were upregulated in the TFP-treated group, suggesting that TFP treatment may play a regulatory role in the OSCC cGAS-STING pathway. Figure 1 ).

[0032] Experimental Example 2: Efficacy of TFP combined with PD-1 monoclonal antibody in the treatment of OSCC Male C57BL / 6 mice aged 4-5 weeks were randomly divided into four groups: IGG group, aPD-1 group, TFP group, and TFP+aPD-1 group (TFP and aPD-1 levels were consistent with those in the TFP and aPD-1 groups). Grouping information is shown in Table 1.

[0033] Table 1 The drugs were administered via intraperitoneal injection. TFP was administered daily, while aPD-1 and the isotype control antibody were administered every two days. The side length of the tongue tumor in mice was measured at the time of administration.

[0034] The results showed that the tumor volume in the IGG group was relatively large; the tumor volume in the aPD-1 group and the TFP group was smaller than that in the IGG group; the tumor volume in the TFP+aPD-1 group was significantly smaller than that in the other three groups, which clearly demonstrated that the combined treatment had a stronger inhibitory effect on tumor growth. Figure 2Tumor growth curves showed that, over time, the tumor volume in the IGG group increased rapidly, while the tumor growth rate in the aPD-1 and TFP groups was slower than that in the IGG group. The tumor volume growth in the TFP+aPD-1 group was extremely slow, and the tumor volume at each time point was significantly smaller than that in the other groups. Figure 3 The statistical results of the average tumor volume growth rate showed that the average tumor volume growth rate was 23.5% in the IGG group, 11.98% in the PD1 group, and 12.3% in the TFP group, while the growth rate in the combined treatment group was only 3.10%. Figure 4 The results indicate that the combination of PD1 and TFP significantly reduced the average growth rate of tumor volume. These results demonstrate that TFP combined with PD-1 monoclonal antibody can significantly enhance the inhibitory effect on OSCC growth, and compared with TFP or PD-1 monoclonal antibody alone and the control group, the combination therapy shows superior efficacy in inhibiting tumor growth.

[0035] Experimental Example 3: TFP combined with PD-1 monoclonal antibody against CD8 + Effects of T cell activation At the endpoint of the above mouse experiments, tumor tissues were collected from the four groups (IGG, aPD-1, TFP, and TFP+aPD-1) and investigated using IHC assays to explore the effect of TFP combined with PD-1 monoclonal antibody on CD8. + The effect of T cell count.

[0036] The results showed that CD8 expression was enhanced after treatment with TFP or PD-1 monoclonal antibody, and CD8 expression was further enhanced after TFP was combined with PD-1 monoclonal antibody. Figure 5 The average expression levels of the IGG group (isotype control group), TFP group, PD-1 monoclonal antibody group, and TFP and PD-1 monoclonal antibody combination group were 0.2, 0.6, 1.0, and 2.4, respectively. Based on the expression level of the IGG group, the increase in expression level in the TFP and PD-1 monoclonal antibody combination group (2.2) was greater than the sum of the increase in expression level in the single-drug groups (1.2). Therefore, this confirms that the combination of TFP and PD-1 monoclonal antibody in this invention has a synergistic effect.

[0037] Experiment Example 4: Effect of TFP combined with PD-1 monoclonal antibody on the proportion of T cells with specific phenotypes The proportion of T cells with specific phenotypes in different treatment groups was detected by flow cytometry. To identify the T cell status within the tumor microenvironment, this invention measured CD3... + CD8 + CD25 +T cell subsets were used to characterize activated T cells. At the endpoint of the mouse experiment in Example 2, head and neck lymph node tissues from each group of mice were collected and gently rinsed in pre-chilled petri dishes containing PBS buffer. The tissues were carefully cut into small pieces using ophthalmic scissors, gently ground with a grinder, and the cell suspension was collected and filtered through a 200-mesh cell sieve. The cells were centrifuged at 300g for 5 min, the supernatant was carefully discarded, and the cells were washed twice with PBS buffer, centrifuged again at 300g for 5 min, and the supernatant was carefully discarded. Three volumes of erythrocyte lysis buffer (Servicebio, G2015-500ML) were added to the cell pellet, and lysis was performed at room temperature for 5 min. After lysis, the cells were washed twice more with PBS buffer, centrifuged, and the cell pellet was collected for later use. Staining was then performed. The cells were first resuspended in 1000 μL of PBS buffer, and then 1 μL of Fixable Viability Stain 520 (BD Biosciences, 564407) was added to each tube of cell suspension and incubated at room temperature in the dark for 10 min. After incubation, centrifuge at 300g for 5 min, discard the supernatant, wash with PBS buffer, and centrifuge again at 300g for 5 min, discarding the supernatant. Resuspend the cells in 100 μL of PBS buffer, and add CD3 antibody (2.5 μL / tube), CD8 antibody (2.5 μL / tube), and CD25 antibody (2.5 μL / tube) to each tube of cell suspension sequentially. Incubate at room temperature in the dark for 30 min. After incubation, centrifuge the cell suspension at 300g for 5 min, discard the supernatant, wash with PBS buffer, centrifuge at 300g for 5 min, discard the supernatant, and then centrifuge at 350g for 5 min, discarding the supernatant. Finally, resuspend the cells in 300 μL of PBS buffer and perform flow cytometry analysis. Flow cytometry results were analyzed using CytExpert software (2.4).

[0038] The results showed that: in CD3 + CD8 + CD25 + Regarding the proportion of T cell subsets, the proportion of cells in the TFP+aPD-1 group was significantly higher than that in the control group, the aPD1 group, or the TFP group. Figure 6 Specifically, in CD3 + CD8 + CD25 + Regarding the proportion of T cell subsets, the TFP and PD-1 monoclonal antibody combination group was significantly higher than the sum of the groups treated with each drug alone: ​​control group, aPD1 group, TFP group, and TFP and aPD1 combination group. + CD8 + CD25 + The proportions of T cell subsets were 0.18%, 0.15%, 0.22%, and 0.32%, respectively. Compared with the control group, the CD3 percentage in the TFP and PD-1 monoclonal antibody combination group was significantly higher.+ CD8 + CD25 + The increase in the proportion of T cell subsets (0.14%) was greater than the sum of the increases in the single-drug groups (0.01%), confirming that the combination of TFP and PD-1 monoclonal antibody in this invention has a synergistic effect.

[0039] In summary, this invention reveals that TFP may play a regulatory role in the cGAS-STING pathway of OSCC. Combining TFP with a PD-1 monoclonal antibody for the treatment of OSCC achieved superior efficacy compared to monotherapy. The combined use of TFP and a PD-1 monoclonal antibody in this invention increases CD8 levels. + T cell expression levels and CD3 + CD8 + CD25 + Regarding the proportion of T cell subsets, it has a synergistic effect, and the combination drug of the present invention has a very good application prospect.

Claims

1. A combination drug for treating oral squamous cell carcinoma, characterized in that, The combined medications are phenothiazine compounds and immune checkpoint inhibitors.

2. The combined medicament according to claim 1, characterized in that, The phenothiazine compound is selected from trifluoperazine, and the immune checkpoint inhibitor is selected from PD-1 inhibitors.

3. The combined medicament according to claim 2, characterized in that, The PD-1 inhibitor is Anti-hPD-1agonist mAb HM266.

4. The combined medicament according to claim 2, characterized in that, The combined medication is trifluoperazine and a PD-1 inhibitor, administered separately or simultaneously.

5. The combined medicament according to claim 2, characterized in that, The weight ratio of trifluoperazine to PD-1 inhibitor is 5.4-6.6:

1.

6. Use of the combined medicament according to any one of claims 1-5 in the preparation of a medicament for treating oral squamous cell carcinoma.

7. A pharmaceutical composition for treating oral squamous cell carcinoma, characterized in that, It is a formulation made with phenothiazine compounds and immune checkpoint inhibitors as active ingredients, and pharmaceutically acceptable excipients or auxiliary ingredients.

8. The pharmaceutical composition according to claim 7, characterized in that, The phenothiazine compound is selected from trifluoperazine, and the immune checkpoint inhibitor is selected from PD-1 inhibitors.

9. The pharmaceutical composition according to claim 8, characterized in that, The PD-1 inhibitor is Anti-hPD-1agonist mAb HM266.

10. The pharmaceutical composition according to claim 8, characterized in that, The weight ratio of trifluoperazine to PD-1 inhibitor is 5.4-6.6:1.

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