A sensitizer for a PD-1 antibody drug and its application; an antitumor drug composition and its application.

Abstract

This invention discloses a sensitizer for PD-1 antibody drugs and its application, as well as an antitumor drug composition and its application. The sensitizer comprises dihydroartemisinin and niraparib, and the antitumor drug composition comprises dihydroartemisinin, niraparib, and a PD-1 antibody. This invention is the first to experimentally discover that dihydroartemisinin possesses a dual mechanism of action: "non-HRD-dependent PD-L1 upregulation" and "tumor-selective ferroptosis induction." This mechanism complements and synergizes with niraparib's "HRD-dependent PD-L1 upregulation + synthetic lethality" mechanism and the PD-1 antibody's "immune activation" mechanism. The antitumor drug composition formed by combining these three components can effectively reshape the tumor immune microenvironment, exerting a potent antitumor effect by upregulating PD-L1 expression in tumor cells, increasing the number of cytotoxic T cells, and inhibiting the proportion of regulatory T cells.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a sensitizer for PD-1 antibody drugs and its application, and an antitumor drug composition and its application. Background Technology

[0002] In the field of tumor immunotherapy, therapies targeting the immune checkpoints PD-1 / PD-L1 have attracted considerable attention. Programmed cell death protein 1 (PD-1) is an important immunosuppressive molecule, widely expressed in activated T cells and serving as a surface receptor for activated T cells. The PD-1 receptor, PD-L1, is almost undetectable in normal tissues, but is present in various human tumor tissues. Upon binding to PD-L1, PD-1 sends immunosuppressive signals to T cells, inhibiting T cell activation and preventing them from killing tumor cells, thus causing tumor immune escape. Blocking the PD-1 / PD-L1 interaction using drugs such as PD-1 antibodies can restore the functional effects of tumor-specific T cells.

[0003] However, the vast majority of cancer patients in clinical practice do not respond to PD-1 antibodies, and the treatment of such "PD-1 antibody-insensitive tumors" has become a core challenge in the industry. The treatment bottleneck of this type of tumor is mainly reflected in the "triple dilemma": (1) The expression level of PD-L1 in tumor cells is extremely low, and PD-1 antibodies lack effective targets, so they cannot block the PD-1 / PD-L1 signaling pathway to relieve immunosuppression; (2) The tumor microenvironment is in an "immune cold state", and the number of effector T cells infiltrating is insufficient. Even if the PD-1 pathway is partially blocked, there are still not enough immune effector cells to initiate anti-tumor killing; (3) The existing treatment regimens have a single anti-tumor mechanism, which is difficult to cover tumor cells with different biological backgrounds, especially the killing efficacy against non-DNA repair defective and drug-resistant tumor cells is low.

[0004] To improve the efficacy of PD-1 antibodies, existing technologies have explored the "niraparib + PD-1 antibody" dual-drug combination, which relies on the dual effects of niraparib: on the one hand, it provides targets for PD-1 antibodies by regulating the expression of PD-1 / PD-L1 pathway-related molecules; on the other hand, it directly kills tumor cells through a "synthetic lethal" effect. However, this dual-drug combination has significant technical limitations: the upregulation of PD-L1 by niraparib is strictly dependent on the tumor cell background, and it is only mildly effective in homologous recombination repair deficient (HRD) positive tumors, with no PD-L1 upregulation effect in HRD negative tumors; at the same time, the "synthetic lethal" mechanism only targets tumor cells with abnormal DNA repair function, and has no killing activity against HRD negative, non-DNA repair dependent tumor cells. As a result, the objective response rate (ORR) of the dual-drug combination for PD-1 antibody-unresponsive tumors remains at an extremely low level, failing to break through the treatment bottleneck of PD-1 antibody-unresponsive tumors.

[0005] Therefore, there is an urgent need in this field to find new drugs and treatment options to improve the treatment efficacy of PD-1 antibody-unresponsive tumors. Summary of the Invention

[0006] The purpose of this invention is to provide a sensitizer for PD-1 antibody drugs and its application, and an anti-tumor drug composition and its application, wherein the anti-tumor drug composition comprises dihydroartemisinin, niraparib and PD-1 antibody, for improving the therapeutic effect of PD-1 antibody-unresponsive tumors.

[0007] To address the aforementioned technical problems, the present invention first provides a sensitizer for PD-1 antibody drugs, wherein the sensitizer comprises: dihydroartemisinin and niraparib.

[0008] Optionally, the mass ratio of dihydroartemisinin to niraparib is 1:2 to 1:9.

[0009] In another aspect, the present invention provides the application of the aforementioned sensitizer in the preparation of a PD-1 antibody antitumor sensitizer, wherein the PD-1 antibody is applied to at least one of PD-L1 low-expressing tumors or immune desert tumors.

[0010] Optionally, the tumor includes at least one of the following: ovarian cancer, fallopian tube cancer, peritoneal cancer, prostate cancer, breast cancer, and pancreatic cancer.

[0011] In another aspect, the present invention provides an antitumor drug composition comprising: dihydroartemisinin, niraparib, and a PD-1 antibody.

[0012] Optionally, the mass ratio of dihydroartemisinin to niraparib is 1:2 to 1:9.

[0013] In another aspect, the present invention provides the use of the aforementioned antitumor pharmaceutical composition in the preparation of antitumor drugs.

[0014] Optionally, the tumor is at least one of a PD-L1 low-expression tumor or an immune desert tumor.

[0015] Optionally, the pharmaceutical composition can effectively remodel the tumor immune microenvironment, and exert a synergistic and potent anti-tumor effect by upregulating PD-L1 expression in tumor cells, increasing the number of cytotoxic T cells, and inhibiting the proportion of regulatory T cells.

[0016] In another aspect, the present invention provides the application of dihydroartemisinin in the preparation of PD-L1 expression agonists in tumor cells, wherein the tumor is a PD-L1 low-expression tumor, and the PD-L1 low expression includes: PD-L1 expression level of TPS<1% or CPS<1.

[0017] Optionally, the tumor cells are pancreatic cancer cell line PANC02 or triple-negative breast cancer cell line 4T1.

[0018] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0019] Through a series of experiments, this invention has for the first time discovered and confirmed that dihydroartemisinin can upregulate the expression of PD-L1 in tumor cells. Moreover, this PD-L1 upregulation mechanism is independent of the HRD state of tumor cells and can be stably effective in both HRD-positive and HRD-negative PD-1 antibody-unresponsive tumors, thus providing a sufficient and universal target for PD-1 antibodies. In addition, dihydroartemisinin can also induce ferroptosis by regulating the iron metabolism balance and antioxidant system function of tumor cells. Furthermore, this ferroptosis induction mechanism is independent of the DNA repair function of tumor cells and has low cytotoxicity to normal cells.

[0020] Furthermore, dihydroartemisinin is combined with niraparib and PD-1 antibody to form the antitumor drug composition provided by the present invention. Animal experiments have demonstrated that the drug composition can effectively remodel the tumor immune microenvironment, and exert a synergistic and potent antitumor effect by upregulating PD-L1 expression in tumor cells, increasing the number of cytotoxic T cells and inhibiting the proportion of regulatory T cells. It is particularly suitable for treating malignant tumors with low PD-L1 expression, unresponsive to PD-1 antibody monotherapy, and / or insensitive to or resistant to niraparib. Attached Figure Description

[0021] Figure 1 The expression of PD-L1 in different types of tumor cells after treatment with different concentrations of DHA was compared.

[0022] A represents the PD-L1 expression results of the pancreatic cancer cell line PANC02;

[0023] B represents the PD-L1 expression result of the triple-negative breast cancer cell line 4T1;

[0024] *p<0.05, **p<0.01, ***p<0.001.

[0025] Figure 2 This is a comparison of the antitumor effects of different drug combinations on breast cancer mice treated in Example 2, wherein:

[0026] A shows photographs of tumor tissue from each group of mice;

[0027] B represents the growth curve of tumor volume in each group of mice;

[0028] C represents the tumor weight of each group of mice;

[0029] D represents the weight growth curves of mice in each group.

[0030] Figure 3 The image shows the expression of PD-L1 in the tumors of breast cancer mice after treatment with different drug combinations in Example 2.

[0031] Figure 4 The expression of GZMB in the tumors of breast cancer mice after treatment with different drug combinations in Example 2 is shown.

[0032] Figure 5 The image shows the expression of Foxp3 in the tumors of breast cancer mice after treatment with different drug combinations in Example 2.

[0033] Figure 6 This is a comparison of the antitumor effects of different drug combinations on breast cancer mice treated in Example 3, wherein:

[0034] A shows photographs of tumor tissue from each group of mice;

[0035] B represents the growth curve of tumor volume in each group of mice;

[0036] C represents the tumor weight of each group of mice;

[0037] D represents the weight growth curves of mice in each group.

[0038] Figure 7 This is a comparison of the antitumor effects of different drug combinations on pancreatic cancer mice treated in Example 4, wherein:

[0039] A shows photographs of tumor tissue from each group of mice;

[0040] B represents the growth curve of tumor volume in each group of mice;

[0041] C represents the tumor weight of each group of mice;

[0042] D represents the weight growth curves of mice in each group.

[0043] Figure 8 The image shows the expression of PD-L1 in the tumors of pancreatic cancer mice after treatment with different drug combinations in Example 4.

[0044] Figure 9 The image shows the expression of GZMB in the tumors of pancreatic cancer mice after treatment with different drug combinations in Example 4.

[0045] Figure 10 The image shows the expression of Foxp3 in the tumors of pancreatic cancer mice after treatment with different drug combinations in Example 4.

[0046] Figure 11 Example 5 compares the antitumor effects of different drug combinations on breast cancer mice after treatment, wherein:

[0047] A shows photographs of tumor tissue from each group of mice;

[0048] B represents the growth curve of tumor volume in each group of mice;

[0049] C represents the tumor weight of each group of mice;

[0050] D represents the weight growth curves of mice in each group. Detailed Implementation

[0051] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0052] Terminology Explanation

[0053] "TPS<1% or CPS<1": These are indicators used to describe low PD-L1 expression levels. The most commonly used PD-L1 indicators in clinical practice are TPS (Tumor Proportion Score) and CPS (Combined Positive Score). TPS<1% means that PD-L1-positive tumor cells account for less than 1% of the tested tumor tissue, indicating "low PD-L1 expression." CPS<1 means that the combined percentage of PD-L1-positive cells in tumor cells and immune cells is less than 1%, also indicating "low PD-L1 expression."

[0054] "Immune desert tumors" refer to tumors in which there are almost no immune cells (such as CD8+) inside or around the tumor parenchyma. +The infiltration of key anti-tumor immune cells (such as T cells and NK cells) renders the entire tumor microenvironment like a "barren desert," devoid of effective anti-tumor immune responses. Because these tumors lack immune cells as targets for immunotherapy, therapies such as immune checkpoint inhibitors are often ineffective. Radiotherapy, combined with chemotherapy, and other methods are often necessary to first induce immune cell infiltration, creating conditions for subsequent immunotherapy.

[0055] As described in the background section, although PD-1 / PD-L1 immune checkpoint inhibitors represent a significant breakthrough in cancer treatment, the vast majority of cancer patients in clinical practice are PD-1 antibody non-responsive, making the treatment of such patients a core challenge in the field. Existing studies have combined niraparib with PD-1 inhibitors in an attempt to improve the treatment efficacy for PD-1 antibody non-responsive tumors, but the results have been unsatisfactory. This is mainly attributed to the fact that both the upregulation of PD-L1 by niraparib and its synthetic lethal effect on tumor cells are dependent on a strictly defined tumor cell background.

[0056] Therefore, this invention attempts to find drugs that can positively regulate PD-L1 expression in tumor cells with more diverse biological backgrounds, aiming to enhance the responsiveness of PD-1 antibody-unresponsive tumors to PD-1 antibodies and improve the treatment sensitivity and efficacy of such tumors. After extensive experimental screening and validation, this invention discovers for the first time that dihydroartemisinin (DHA) possesses a dual mechanism of action: "HRD-independent PD-L1 upregulation" and "tumor-selective ferroptosis induction," as detailed below:

[0057] (1) Non-HRD-dependent PD-L1 upregulation

[0058] This invention, through experiments, found that when DHA was co-cultured with the triple-negative breast cancer cell line 4T1 (HRD-positive PD-1 antibody-unresponsive tumor cells) and the pancreatic cancer cell line PANC02 (HRD-negative PD-1 antibody-unresponsive tumor cells), PD-L1 expression was significantly increased in both cell types. This indicates that DHA may provide more PD-1 antibody targets for PD-1 antibody-unresponsive tumors with different biological backgrounds, thereby broadening the applicable population for PD-1 antibodies and improving anti-tumor efficacy. Furthermore, experiments also showed that the upregulation of PD-L1 protein expression by DHA was significantly higher than that by niraparib.

[0059] Further investigation into the mechanism revealed that DHA can be activated by Fe... 2+The PD-L1-mediated oxidative stress signaling pathway activates the NF-κB and STAT3 signaling pathways, thereby promoting phosphorylation of the NF-κB / p65 subunit and STAT3 at the transcriptional level. Phosphorylated NF-κB / p65 forms a complex with STAT3, synergistically binding to the PD-L1 gene promoter region, ultimately significantly increasing the mRNA transcription level of the PD-L1 gene. Furthermore, at the protein level, DHA downregulates the ubiquitination efficiency of E3 ubiquitin ligase on PD-L1 protein, reducing proteasomal degradation of PD-L1 protein and thus prolonging its half-life on the tumor cell membrane. These two levels of action synergistically regulate a significant upregulation of PD-L1 expression in tumor cells. Moreover, this PD-L1 upregulation mechanism is independent of the HRD state of tumor cells, showing stable efficacy in both HRD-positive and HRD-negative PD-1 antibody-unresponsive tumors, thus providing a sufficient and universal target for PD-1 antibodies.

[0060] (2) Tumor-selective ferroptosis induction

[0061] DHA can induce ferroptosis by regulating the iron metabolism balance and antioxidant system function of tumor cells: on the one hand, DHA inhibits the activity of iron efflux proteins on the surface of tumor cell membranes and promotes the expression of iron influx proteins, leading to the release of free Fe in tumor cells. 2+ On the one hand, DHA accumulates in concentration; on the other hand, it downregulates the expression level of glutathione peroxidase 4 (GPX4) in tumor cells, promotes the activity of lipoxygenase (LOX), and accelerates the generation and accumulation of lipid peroxides (LPO) in tumor cells. These two aspects work together to synergistically induce ferroptosis in tumor cells.

[0062] This ferroptosis-inducing mechanism does not depend on the DNA repair function of tumor cells. It can efficiently kill HRD-negative, non-DNA repair-dependent tumor cells that are ineffective against the niraparib "synthetic lethality" mechanism, and has low cytotoxicity to normal cells—the expression level of GPX4 in normal cells is significantly higher than that in tumor cells, which can effectively clear lipid peroxides and avoid ferroptosis damage.

[0063] Based on the above research, it is evident that the "HRD-independent PD-L1 upregulation + ferroptosis induction" mechanism of DHA, the "HRD-independent PD-L1 upregulation + synthetic lethality" mechanism of niraparib, and the "immune activation" mechanism of PD-1 antibodies form a functional complement and synergistic effect. Therefore, this invention designs a combination of DHA, niraparib, and PD-1 antibodies to construct a synergistic closed-loop pathway of "target upregulation - immune activation - dual killing," achieving a synergistic anti-tumor effect of "1+1>2" against PD-1 antibody-unresponsive tumors. The specific synergistic logic is as follows:

[0064] a. Target Synergy: Niraparib's PD-L1 upregulation is HRD-dependent, covering only HRD-positive tumors; DHA's PD-L1 upregulation is HRD-independent, covering both HRD-positive and HRD-negative tumors. The synergistic effect of both can achieve high-amplitude PD-L1 upregulation in PD-1 antibody-unresponsive tumors across all HRD states, completely resolving the "insufficient target" problem of PD-1 antibodies and enabling PD-1 antibodies to effectively bind to targets in tumors with diverse biological backgrounds.

[0065] b. Immune Synergy: Niraparib activates the cGAS-STING signaling pathway, promoting the secretion of type I interferon and chemokines, and recruiting effector T cells to infiltrate the tumor microenvironment. When DHA induces ferroptosis in tumor cells, it releases damage-associated molecular patterns (DAMPs), further enhancing the activation efficiency and antigen-presenting capacity of dendritic cells. The synergistic effect of these two factors significantly increases the number and activity of effector T cells in the tumor microenvironment, transforming the "immunely cold" microenvironment into an "immunely hot" one, providing a sufficient effector cell basis for the immune activation effect of PD-1 antibodies.

[0066] c. Synergistic Killing Effect: Niraparib kills HRD-positive tumor cells with abnormal DNA repair function through a "synthetic lethality" mechanism; DHA kills HRD-negative tumor cells that are not DNA repair dependent through a "ferroptosis" mechanism. The two work synergistically to form a dual killing system of "DNA repair dependent + DNA repair dependent," covering tumor cells with different biological backgrounds in PD-1 antibody-unresponsive tumors. Simultaneously, effector T cells activated by the PD-1 antibody can specifically recognize antigens on the surface of tumor cells, further amplifying the dual killing effect and achieving an anti-tumor effect of "1+1>2."

[0067] Therefore, the present invention first provides a sensitizer for PD-1 antibody drugs, the sensitizer comprising: dihydroartemisinin and niraparib.

[0068] In some embodiments, the mass ratio of dihydroartemisinin to niraparib is 1:2 to 1:9.

[0069] In another aspect, the present invention provides the application of the aforementioned sensitizer in the preparation of a PD-1 antibody antitumor sensitizer, wherein the PD-1 antibody is applied to at least one of PD-L1 low-expressing tumors or immune desert tumors.

[0070] In some embodiments, the tumor includes at least one of ovarian cancer, fallopian tube cancer, peritoneal cancer, prostate cancer, breast cancer, and pancreatic cancer.

[0071] In another aspect, the present invention provides an antitumor drug composition comprising: dihydroartemisinin, niraparib, and a PD-1 antibody.

[0072] In some embodiments, the mass ratio of dihydroartemisinin to niraparib is 1:2 to 1:9.

[0073] In some embodiments, the pharmaceutical composition further includes at least one of a pharmaceutically acceptable carrier, excipient, wetting agent, emulsifier, and pH buffer. Suitable pharmaceutically acceptable carriers are well known to those skilled in the art. A thorough description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences. Pharmaceutically acceptable carriers in the composition may contain liquids such as water, phosphate buffer, Ringer's solution, physiological saline, balanced salt solution, glycerol, or sorbitol. Additionally, these carriers may contain auxiliary substances such as lubricants, flow aids, wetting agents or emulsifiers, pH buffers, and stabilizers such as albumin. In use, a safe and effective amount of the antitumor drug of the present invention is administered to mammals (such as humans). Of course, the specific dosage should also consider factors such as the route of administration and the patient's health condition, which are within the scope of a skilled physician's expertise. The precise effective amount for a given subject depends on the subject's body size and health condition, the nature and severity of the disease, and the chosen therapeutic agent and / or combination of therapeutic agents. For a given condition, the effective amount can be determined using routine experiments, and clinicians are capable of making that judgment.

[0074] In another aspect, the present invention provides the application of the aforementioned antitumor pharmaceutical composition in the preparation of antitumor drugs. The pharmaceutical composition can effectively remodel the tumor immune microenvironment, exerting a synergistic and potent antitumor effect by upregulating PD-L1 expression in tumor cells, increasing the number of cytotoxic T cells, and inhibiting the proportion of regulatory T cells.

[0075] In some embodiments, the tumor is at least one of PD-L1 low-expression (e.g., TPS<1% or CPS<1) tumors or immune desert tumors.

[0076] In some embodiments, the tumor includes at least one of ovarian cancer, fallopian tube cancer, peritoneal cancer, prostate cancer, breast cancer, and pancreatic cancer.

[0077] In another aspect, the present invention provides the application of dihydroartemisinin in the preparation of PD-L1 expression agonists in tumor cells, wherein the tumor is a PD-L1 low-expressing tumor.

[0078] In some embodiments, the tumor cells are pancreatic cancer cell line PANC02 or triple-negative breast cancer cell line 4T1.

[0079] The experimental process and results of the present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0080] Unless otherwise specified, the experimental methods used in the following examples were performed under conventional or manufacturer-recommended conditions. Unless otherwise specified, the materials and reagents used in the following examples were commercially available. Niraparib was purchased from Zai Lab (Approval No.: H20190035), dihydroartemisinin was purchased from Fuyuan Pharmaceutical (Approval No.: H20067334), anti-PD-1 (PD-1 antibody) was purchased from Bioxcell (Ultra-LEAF™ Purified anti-mouse CD279 (PD-1), BE0146); and corn oil was purchased from Jintaiyang Grain and Oil Company.

[0081] Tumor cell lines: Triple-negative breast cancer cell line 4T1 and pancreatic cancer cell line PANC02 were selected, both purchased from the China Center for Type Culture Collection (CCTCC).

[0082] Example 1: Effect of dihydroartemisinin (DHA) on PD-L1 expression in tumor cells

[0083] (I) Experimental Procedure

[0084] Reagent preparation: Dihydroartemisinin (DHA) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution, which was then diluted to the required concentration with complete culture medium (DMEM medium) during the experiment.

[0085] Triple-negative breast cancer cell line 4T1 and pancreatic cancer cell line PANC02, both in logarithmic growth phase, were seeded into six-well plates at a density of 4 × 10⁻⁶ cells / well. 5 Cells were cultured at a concentration of 100 cells / well, and then passaged. After cell expansion, each well was treated with different concentrations (0 μM, 1 μM, 5 μM, 10 μM) of DHA for 24 h. After treatment, cells were collected, and the expression of PD-L1 in each well was detected by flow cytometry.

[0086] (II) Experimental Results

[0087] like Figure 1 As shown in Figures AB, flow cytometry results showed that, compared with the control group (0 μM), DHA treatment significantly increased the expression level of PD-L1 in triple-negative breast cancer cell line 4T1 and pancreatic cancer cell line PANC02.

[0088] The above results indicate that DHA can effectively upregulate the PD-L1 expression level in tumor cell lines with low PD-L1 expression, and the upregulation effect is independent of the HRD status of tumor cells. It can be stably effective in both HRD-positive PD-1 antibody-unresponsive tumors (triple-negative breast cancer cell line 4T1) and HRD-negative PD-1 antibody-unresponsive tumors (pancreatic cancer cell line PANC02).

[0089] Example 2: Evaluation of the in vivo therapeutic effect of the combination of dihydroartemisinin, niraparib, and PD-1 antibody on breast cancer.

[0090] (I) Experimental Procedure

[0091] 4T1 breast cancer cells were inoculated into 6-week-old female BALB / c mice (4 × 10⁻⁶ cells / year). 5 A subpapular orthotopic tumor model was established in vivo (1 cell / animal). The tumor volume reached 50 mm. 3 Afterwards, the mice were grouped and administered drugs according to the following combinations: (1) control group (no drug), (2) anti-PD-1 (100 μg / each dose) group, (3) dihydroartemisinin (10 mg / kg) group, (4) niraparib (40 mg / kg) group, (5) dihydroartemisinin (10 mg / kg) + niraparib (40 mg / kg) group, (6) dihydroartemisinin (10 mg / kg) + anti-PD-1 (100 μg / each dose) group, (7) niraparib (40 mg / kg) + anti-PD-1 (100 μg / each dose) group, (8) dihydroartemisinin (10 mg / kg) + niraparib (40 mg / kg) + anti-PD-1 (100 μg / each dose) group. The administration method for niraparib and / or DHA is as follows: after dissolving in a solvent, the niraparib or a combination of the two is administered by gavage. Specifically, DHA, niraparib, or a combination of the two are first dissolved in 10% DMSO, and then corn oil is added to make up to the required volume. 100 μL / mouse is administered by gavage once a day. The control group mice are administered the same volume of the same solvent by gavage. The administration method for PD-1 antibody is as follows: intraperitoneal injection, once every 5 days.

[0092] Tumor weight, volume, and body weight of mice in each group were monitored every two days. Tumor volume was calculated using the formula: Tumor volume (mm²) 3 = 0.5 × (length) × (width) 2 Fourteen days after administration, the mice were sacrificed, the tumors were dissected, and the expression level of PD-L1 and related indicators of tumor-infiltrating T cells in each group of tumor cells were detected by flow cytometry.

[0093] (II) Experimental Results

[0094] 1. The combined use of dihydroartemisinin, niraparib, and PD-1 antibody exerted a synergistic anti-tumor effect in breast cancer mice.

[0095] like Figure 2According to data from the Chinese A / D test and Table 1, compared with the control group, the tumor inhibition rates of PD-1 antibody, dihydroartemisinin, and niraparib alone were 8.32%, 7.97%, and 8.43%, respectively. None of the three drugs showed significant anti-tumor effects when used alone. Furthermore, the tumor inhibition rate of the combination of dihydroartemisinin and niraparib was 8.54%, which also did not show significant anti-tumor effects.

[0096] After introducing PD-1 antibody for combination therapy, the experimental results of the comparison groups (1), (2), (3), (4), (6), and (7) showed that, compared with the use of PD-1 antibody, niraparib, or dihydroartemisinin alone, although the combination regimen of PD-1 antibody with niraparib or dihydroartemisinin could improve the tumor inhibition rate, the anti-tumor effect was still weak and did not achieve the ideal therapeutic effect. Among them, the tumor inhibition rate of dihydroartemisinin combined with PD-1 antibody was 28.64%, and the tumor inhibition rate of niraparib combined with PD-1 antibody was 24.55%.

[0097] When dihydroartemisinin, niraparib, and PD-1 antibody are used in combination, the tumor inhibition rate can reach 86.19%, which is significantly better than any single or dual-drug regimen of PD-1 antibody, dihydroartemisinin, and niraparib. The synergistic effect is calculated using the formula: Q = Ea / Ee, where Ea is the actual combined tumor inhibition rate, and Ee = 1 - (1 - Ea / Ee). A (1-E) B (1-E) C E A E B E C The tumor inhibition rate of each single drug is shown. Calculations show that the synergistic effect Q value of the antitumor drug composition of this invention reaches 3.79, exhibiting a strong synergistic effect far exceeding "1+1>2" (Q value determination criteria: Q value > 1.15, synergistic effect; Q value = 0.85~1.15, simple addition; Q value < 0.85, antagonistic effect).

[0098] Furthermore, the monitoring results of weight changes in experimental animals showed that, compared with the control group, the weight of each experimental mouse did not fluctuate significantly, indicating that the anti-tumor drug composition provided by the present invention, while exerting a significant anti-tumor effect, did not show obvious toxicity and had good safety.

[0099] Table 1. Comparison of tumor inhibition rates of various drugs and compositions in breast cancer.

[0100]

[0101] 2. Exploration of the anti-tumor mechanism

[0102] To further explore the mechanism of action of the three drugs in combination, this invention further examined the expression level of PD-L1 in tumor cells and related indicators of tumor-infiltrating T cells.

[0103] The results are as follows Figure 3 As shown, compared with the control group, the PD-L1 expression level of tumor cells in the three-drug combination group (group 8) was significantly increased, from 57.9% in the control group to 82.1%, indicating that the anti-tumor drug composition provided by the present invention can effectively upregulate the expression level of PD-L1 in tumor cells, creating abundant targets for the PD-1 antibody, and thus providing an important prerequisite for the PD-1 antibody to exert its anti-tumor effect efficiently.

[0104] In addition, regarding tumor-infiltrating T cells, compared with the control group, the expression level of the cytotoxic T cell-related factor globular mycotoxin B (GZMB) in the three-drug combination group (group 8) was significantly increased from 9.36% to 40.3%. Figure 4 The expression level of the regulatory T cell (Treg) marker Foxp3 decreased significantly from 40% to 16.2%. Figure 5 ).

[0105] The above results demonstrate that the combination of dihydroartemisinin, niraparib, and PD-1 antibody in the pharmaceutical composition provided by this invention can effectively reshape the tumor immune microenvironment, and exert a synergistic and potent anti-tumor effect by upregulating PD-L1 expression in breast cancer tumor cells, increasing the number of cytotoxic T cells, and inhibiting the proportion of regulatory T cells.

[0106] Example 3: Evaluation of the in vivo therapeutic effect of the combination of dihydroartemisinin, palbociclib, and PD-1 antibody on breast cancer.

[0107] The present invention also explored the use of other chemotherapy drugs that may enhance PD-L1 expression (palbociclib in this embodiment) in combination with PD-1 antibody and dihydroartemisinin, and evaluated the anti-tumor effect.

[0108] (I) Experimental Procedure

[0109] 4T1 breast cancer cells were inoculated into 6-week-old female BALB / c mice (4 × 10⁻⁶ cells / year). 5 A subpapular orthotopic tumor model was established in vivo (1 cell / animal). The tumor volume reached 50 mm. 3Afterwards, the mice were grouped and administered drugs according to the following combinations: (1) control group (no drug), (2) anti-PD-1 (100 μg / each dose) group, (3) dihydroartemisinin (10 mg / kg) group, (4) palbociclib (100 mg / kg) group, (5) dihydroartemisinin (10 mg / kg) + palbociclib (100 mg / kg) group, (6) dihydroartemisinin (10 mg / kg) + anti-PD-1 (100 μg / each dose) group, (7) palbociclib (100 mg / kg) + anti-PD-1 (100 μg / each dose) group, (8) dihydroartemisinin (10 mg / kg) + palbociclib (100 mg / kg) + anti-PD-1 (100 μg / each dose) group. The drug composition was dissolved as follows: 10% DMSO + corn oil, 100 μL / mouse was administered by gavage, and the control group mice were administered the same volume of solvent by gavage.

[0110] Tumor weight and volume, as well as mouse body weight, were monitored every two days. Tumor volume was calculated using the formula: Tumor volume (mm²) 3 = 0.5 × (length) × (width) 2 Fourteen days after administration, the mice were sacrificed and the tumors were dissected.

[0111] (II) Experimental Results

[0112] like Figure 6 As shown in the AD diagram, the experimental results of groups (1) to (8) show that, compared with the control group, none of the experimental groups using dihydroartemisinin, palbociclib, or PD-1 antibody alone or in any combination showed significant anti-tumor effects. This indicates that not all chemotherapy drugs can exhibit synergistic anti-tumor effects when used in combination with PD-1 antibody and dihydroartemisinin.

[0113] Example 4: Evaluation of the in vivo therapeutic effect of the combined use of dihydroartemisinin, niraparib, and PD-1 antibody on pancreatic cancer.

[0114] (I) Experimental Procedure

[0115] Pancreatic cancer cells PAN02 were inoculated into 6-week-old female C57BL / 6 mice (5 × 10⁻⁶). 5 A subcutaneous tumor model was established in vivo using 1 cell / animal. The tumor volume reached 50 mm. 3Subsequently, the mice were grouped and administered the drugs according to the following combinations: (1) control group (no drug administration), (2) anti-PD-1 (100 μg / dose) group, (3) dihydroartemisinin (10 mg / kg) group, (4) niraparib (40 mg / kg) group, (5) dihydroartemisinin (10 mg / kg) + niraparib (40 mg / kg) group, (6) dihydroartemisinin (10 mg / kg) + anti-PD-1 (100 μg / dose) group, (7) niraparib (40 mg / kg) + anti-PD-1 (100 μg / dose) group, and (8) dihydroartemisinin (10 mg / kg) + niraparib (40 mg / kg) + anti-PD-1 (100 μg / dose) group. The drug administration method was the same as in Example 2, which can be found in the corresponding content in Example 2 and will not be repeated here.

[0116] Tumor weight, volume, and body weight of mice in each group were monitored every two days. Tumor volume was calculated using the formula: Tumor volume (mm²) 3 = 0.5 × (length) × (width) 2 Fourteen days after administration, the mice were sacrificed, the tumors were dissected, and the expression level of PD-L1 and related indicators of tumor-infiltrating T cells in each group of tumor cells were detected by flow cytometry.

[0117] (II) Experimental Results

[0118] 1. The combined use of dihydroartemisinin, niraparib, and PD-1 antibody exerted a synergistic anti-tumor effect in mice with pancreatic cancer.

[0119] like Figure 7 According to the data in Table 2, compared with the control group, the tumor inhibition rates of PD-1 antibody, dihydroartemisinin, niraparib alone and the combination of dihydroartemisinin and niraparib were 19.64%, 18.33%, 19.54% and 18.15%, respectively, all showing relatively similar weak anti-tumor effects.

[0120] After introducing PD-1 antibody for combination therapy, the experimental results of the comparison groups (1), (2), (3), (4), (6), and (7) showed that, compared with the use of PD-1 antibody, niraparib, or dihydroartemisinin alone, although the combination regimen of PD-1 antibody with niraparib or dihydroartemisinin could improve the tumor inhibition rate, it did not show a significant synergistic effect, and the anti-tumor effect still did not reach the ideal therapeutic effect. Among them, the tumor inhibition rate of dihydroartemisinin combined with PD-1 antibody was 37.26%, and the tumor inhibition rate of niraparib combined with PD-1 antibody was 37.72%.

[0121] When dihydroartemisinin, niraparib and PD-1 antibody are used in combination, the tumor inhibition rate can reach 75.49%, which is significantly better than any single or dual-drug combination of PD-1 antibody, dihydroartemisinin and niraparib. It shows a strong synergistic effect far greater than "1+1>2", with a synergistic effect Q value of 1.6.

[0122] Table 2 Comparison of tumor inhibition rates of various drugs and compositions in pancreatic cancer

[0123]

[0124] 2. Exploration of the anti-tumor mechanism

[0125] Further investigation was conducted to examine the expression level of PD-L1 in tumor cells and related indicators of tumor-infiltrating T cells.

[0126] The results are as follows Figure 8 As shown, compared with the control group, the PD-L1 expression level of tumor cells in the three-drug combination group (group 8) was significantly increased, from 59.3% in the control group to 87.3%.

[0127] In addition, regarding tumor-infiltrating T cells, compared to the control group, the expression level of the cytotoxic T cell-related factor globular mycotoxin B (GZMB) in the three-drug combination group (group 8) was significantly increased from 13.5% to 34.5%. Figure 9 The expression level of the regulatory T cell (Treg) marker Foxp3 decreased significantly from 50.9% to 21.2%. Figure 10 ).

[0128] The above results demonstrate that the combination of dihydroartemisinin, niraparib, and PD-1 antibody in the pharmaceutical composition provided by this invention can produce a synergistic effect, significantly enhancing the therapeutic effect on pancreatic cancer, and has good safety, providing an effective treatment option for PD-1 non-responsive tumors.

[0129] Example 5: In vivo antitumor effects of different drug dosage compositions

[0130] This embodiment further utilizes a mouse breast cancer model to investigate the effects of different drug dosages on tumor suppression. The specific design involved fixing the PD-1 dosage (the clinically standard dosage) and evaluating the antitumor effects of different drug dosage combinations by adjusting the dosage changes of dihydroartemisinin (DHA) and niraparib within their respective clinically appropriate dosage ranges.

[0131] (I) Experimental Procedure

[0132] 4T1 breast cancer cells were inoculated into 6-week-old female BALB / c mice (4 × 10⁻⁶ cells / year). 5A subpapular orthotopic tumor model was established in vivo (1 cell / animal). The tumor volume reached 50 mm. 3 Then, the mice were divided into groups and administered drugs according to the following combinations: (1) control group (no drug), (2) anti-PD-1 (100 μg / dose) group, (3) dihydroartemisinin (10 mg / kg) + niraparib (40 mg / kg) group, (4) dihydroartemisinin (10 mg / kg) + niraparib (40 mg / kg) + anti-PD-1 (100 μg / dose) group, (5) dihydroartemisinin (10 mg / kg) + niraparib (20 mg / kg) + anti-PD-1 (100 μg / dose) group, (6) dihydroartemisinin (10 mg / kg) + niraparib (50 mg / kg) + anti-PD-1 (100 μg / dose) group, (7) dihydroartemisinin (5 mg / kg) + niraparib (40 mg / kg) + anti-PD-1 (100 μg / dose) group. (8) Dihydroartemisinin (12.5 mg / kg) + niraparib (40 mg / kg) + anti-PD-1 (100 μg / time) group, (9) Dihydroartemisinin (15 mg / kg) + niraparib (35 mg / kg) + anti-PD-1 (100 μg / time) group, (10) Dihydroartemisinin (5 mg / kg) + niraparib (45 mg / kg) + anti-PD-1 (100 μg / time) group. Among them, the administration method of niraparib and / or DHA is: after dissolving in a solvent, it is administered by gavage. Specifically, DHA, niraparib or a combination of the two are first dissolved in 10% DMSO, and then corn oil is added to make up to the required volume. 100 μL / mouse is administered by gavage once a day. The control group mice are administered the same volume of the same solvent by gavage. The administration method of PD-1 antibody is: intraperitoneal injection, once every 5 days.

[0133] Tumor weight, volume, and body weight of mice in each group were monitored every two days. Tumor volume was calculated using the formula: Tumor volume (mm²) 3 = 0.5 × (length) × (width) 2 Fourteen days after administration, the mice were sacrificed and the tumors were dissected.

[0134] (II) Experimental Results

[0135] The results are as follows Figure 11 As shown in the AD diagram, compared with the use of PD-1 antibody alone, and dihydroartemisinin + niraparib, the combination of dihydroartemisinin, niraparib and PD-1 antibody showed a stronger anti-tumor effect. Moreover, within the experimental dosage range, different dose combinations of dihydroartemisinin + niraparib + PD-1 antibody all showed significant synergistic anti-tumor effects. Specific tumor inhibition rate data can be found in Table 3.

[0136] Table 3 Comparison of tumor inhibition rates of different drug compositions in breast cancer

[0137]

[0138] Based on the above experimental results, the effective dose of the antitumor drug composition provided by the present invention for mice is: DHA: 5 mg / kg~15 mg / kg; NIR: 20 mg / kg~50 mg / kg; PD-1 antibody (100 μg / time). According to the FDA's Center for Drug Evaluation and Research (CDER) guidance published in 2005, "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers" (see table on page 7 of the guidance), the effective dosage for a 60 kg person is as follows: DHA: 25 mg to 75 mg / day (usually administered orally); NIR: 100 mg to 250 mg / day (usually administered orally); PD-1 antibody (500 μg / dose, intravenously or subcutaneously). The dosing cycle of PD-1 antibody is not only related to the drug manufacturer and specific model, but also to factors such as the type of disease and the patient's physical condition. Currently, the dosing cycle of PD-1 antibodies on the market is usually once every 2 to 6 weeks. The actual dosing regimen needs to be individualized by the clinician based on the patient's specific condition, treatment response, and safety assessment. Furthermore, the physician can optimize the medication decision in a timely manner based on the dynamic monitoring results during the treatment process to ensure the effectiveness and safety of the treatment.

[0139] In summary, this invention provides a sensitizer for PD-1 antibody drugs and its application, as well as an antitumor drug composition and its application. The antitumor drug composition comprises dihydroartemisinin, niraparib, and a PD-1 antibody. Animal experiments have confirmed that the drug composition provided by this invention can exert a synergistic and potent antitumor effect by upregulating PD-L1 expression in tumor cells, increasing the number of cytotoxic T cells, and inhibiting the proportion of regulatory T cells. It is particularly suitable for treating malignant tumors with low PD-L1 expression, unresponsiveness to PD-1 antibody monotherapy, and / or insensitivity to or resistance to niraparib.

[0140] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A sensitizer for a PD-1 antibody drug, characterized in that, The sensitizer is composed of dihydroartemisinin and niraparib, wherein the mass ratio of dihydroartemisinin to niraparib is 1:2 to 1:

9.

2. The use of the sensitizer according to claim 1 in the preparation of PD-1 antibody antitumor sensitizer, characterized in that, The tumor is at least one of breast cancer or pancreatic cancer.

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