A pharmaceutical composition and its use in the preparation of an antitumor drug

Abstract

The application discloses a kind of pharmaceutical composition and its application in preparation antitumor drug.The pharmaceutical composition of the application includes cyclic peptide SHAP and PARP1 inhibitor, and cyclic peptide SHAP can inhibit the homologous recombination repair after tumor cell DNA damage;And, one or more of the combination of small molecule cyclic peptide SHAP and PARP1 inhibitor, can reduce the tolerance of tumor to PARP1 inhibitor.The application discloses the extensive application potential of cyclic peptide SHAP and PARP1 inhibitor in tumor drug resistance, and has important clinical research value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a pharmaceutical composition and its application in the preparation of antitumor drugs. Background Technology

[0002] Genomic DNA is susceptible to various forms of DNA damage caused by endogenous replication and oxidative stress, or by exogenous factors such as ultraviolet radiation and ionizing radiation. On the one hand, when DNA damage occurs, cells can initiate response mechanisms and recruit DNA repair proteins to repair the damaged sites and maintain genomic stability. On the other hand, disruption of DNA damage response mechanisms can lead to genomic instability, inducing a range of human diseases, including tumors. A deeper understanding of DNA damage response mechanisms can not only help us elucidate the molecular mechanisms of tumor development and progression, but also provide new targeted therapy strategies for clinical practice.

[0003] Currently, radiotherapy and chemotherapy, widely used in clinical practice, work by directly or indirectly causing DNA damage in tumor cells, inducing apoptosis, and ultimately killing tumor cells. However, most chemotherapeutic drugs that cause DNA damage lack a clear target and have a killing effect on both malignant tumor cells and normal cells, producing strong toxic side effects on the body. Therefore, discovering the key signaling pathways and molecules that specifically depend on the tumor cell DNA damage response mechanism, and then developing targeted drugs accordingly, has significant practical implications and broad application prospects in clinical practice.

[0004] Poly(ADP-ribose) polymerase 1 (PARP1) was the first clinically applied molecular target related to DNA damage response mechanisms. The PARP1-mediated DNA damage response pathway is crucial for the repair of single / double-strand breaks in DNA. Normal cells can repair DNA breaks through other repair pathways, such as homologous recombination (HR). However, due to defective HR repair function in some tumor cells, they become overly reliant on the PARP1-mediated DNA damage repair pathway. Under these conditions, PARP1 inhibitors can induce tumor cell death through synthetic lethal effects without affecting normal cells.

[0005] Currently, several PARP1 inhibitors, including olaparib, niraparib, rucaparib, and taprazole, have been approved by the FDA for the clinical treatment of breast cancer, ovarian cancer, pancreatic cancer, and prostate cancer. However, PARP1 inhibitors only produce a synthetic lethal effect in individuals with specific DNA repair deficiencies, thus inhibiting tumor growth. Subsequent researchers have attempted to combine PARP1 inhibitors with other targeted inhibitors such as immune checkpoint inhibitors and MEK inhibitors to expand the applicability of these drugs. Therefore, identifying key signaling molecules contributing to PARP1 inhibitor resistance in tumors, improving PARP1 inhibitor resistance, and developing novel anti-tumor drugs are of paramount importance for clinical cancer treatment. Summary of the Invention

[0006] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a pharmaceutical composition comprising a cyclic peptide SHAP and a PARP1 inhibitor, which can be used in the preparation of antitumor drugs. The cyclic peptide SHAP can inhibit homologous recombination repair after DNA damage, and when used in combination with a PARP1 inhibitor, it can significantly enhance the antitumor effect and improve PARP1 inhibitor resistance.

[0007] According to a first aspect of the present invention, a pharmaceutical composition is provided comprising a cyclic peptide SHAP and a PARP1 inhibitor; the amino acid sequence of the cyclic peptide SHAP is shown in SEQ ID No. 1. Specifically, the amino acid sequence is LVRRCK-Nle-LCY.

[0008] In this invention, the cyclic peptide SHAP is a key signaling molecule that is specifically dependent on the DNA damage response mechanism of tumor cells. The cyclic peptide SHAP can significantly inhibit the homologous recombination (HR) repair efficiency of tumor cells and inhibit the DNA double-strand break (DSB) damage response of tumor cells, and this process has a dose-dependent effect. As a key signaling molecule dependent on drug resistance, the cyclic peptide SHAP can improve the sensitivity of PARP1 inhibitors and can be used in combination with PARP1 inhibitors to treat tumor-related diseases, especially for the treatment of drug-resistant tumors.

[0009] In some embodiments of the present invention, the structure of the cyclic peptide SHAP is: Ac-LVRRCK-Nle-LCY-NH2, wherein the cyclic peptide SHAP forms a cyclic peptide structure by cyclizing two cysteine ​​residues in the amino acid sequence with m-dibenzyl halide.

[0010] In some embodiments of the present invention, the PARP1 inhibitor includes at least one of olaparib, rucaparib, and niraparib. Specifically, one or more PARP1 inhibitors can be used in combination with the cyclic peptide SHAP, and corresponding pharmaceutical compositions can be prepared for antitumor therapy.

[0011] In some embodiments of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable salt and / or carrier.

[0012] In some embodiments of the present invention, the concentration of the cyclic peptide SHAP is 0.5–20 μM; and / or the concentration of the PARP1 inhibitor is 0.01–100 μM.

[0013] Preferably, the concentration of the cyclic peptide SHAP is 0.5–10 μM; and / or the concentration of the PARP1 inhibitor is 1–100 μM. More preferably, the concentration of the cyclic peptide SHAP is 0.5–2 μM; and / or the concentration of the PARP1 inhibitor is 1–10 μM. In some embodiments, the dosage concentration of the cyclic peptide SHAP is 10 mg / kg, and the dosage concentration of the PARP1 inhibitor is 10 mg / kg.

[0014] According to another aspect of the present invention, the use of the above-described pharmaceutical composition in the preparation of a therapeutic antitumor drug is proposed.

[0015] In this invention, the combined use of the cyclic peptide SHAP and a PARP1 inhibitor can efficiently kill tumor cells in vivo and in vitro through synthetic lethal effects, significantly increasing the sensitivity of tumor cells to PARP1 inhibitors. Therefore, this invention, by combining the cyclic peptide SHAP and a PARP1 inhibitor, can greatly broaden the clinical application scope of PARP1 inhibitors and provides a new solution to the drug resistance problem currently faced in the clinical treatment of PARP1 inhibitors, possessing extremely high clinical application value and broad market transformation potential.

[0016] In some embodiments of the present invention, the antitumor drug is an anti-gastrointestinal tumor drug.

[0017] In some embodiments of the present invention, the antitumor drug is a drug for treating tumor diseases resistant to PARP inhibitors. Preferably, the combination of the cyclic peptide SHAP and a PARP1 inhibitor can enhance the sensitivity of tumor cells to the PARP1 inhibitor.

[0018] In some embodiments of the present invention, the cyclic peptide SHAP can inhibit homologous recombination repair after DNA damage in tumor cells.

[0019] Preferably, the cyclic peptide SHAP can significantly reduce the ability of ZMYND8 to recruit to DNA double-strand break sites.

[0020] Preferably, the cyclic peptide SHAP can significantly inhibit the protein degradation rate of γ-H2AX and suppress the DNA double-strand break damage response in tumor cells.

[0021] Preferably, the combination of the cyclic peptide SHAP and the PARP1 inhibitor can inhibit the repair of DNA double-strand breaks in tumor cells and enhance the killing effect of tumor cells.

[0022] In some embodiments of the present invention, the combination of the cyclic peptide SHAP and the PARP1 inhibitor can inhibit tumor growth through a synthetic lethal effect.

[0023] Preferably, the combined use of the cyclic peptide SHAP and a PARP1 inhibitor can inhibit tumor growth through a synthetic lethal effect, even when DNA damage repair is normal.

[0024] Preferably, the combined use of the cyclic peptide SHAP and a PARP1 inhibitor can enhance the tumor's response to PARP1 inhibitors in PARP1 inhibitor-resistant tumor cells through a synthetic lethal effect.

[0025] The present invention has at least the following beneficial effects: (1) The cyclic peptide SHAP can effectively inhibit the repair of DNA double-strand breaks in tumor cells, thereby playing a role in tumor suppression; (2) The combination of cyclic peptide SHAP and one or more PARP1 inhibitors can reduce the drug resistance of tumor cells to PARP1 inhibitors, providing a solution to the drug resistance problem in clinical treatment; (3) The cyclic peptide SHAP has good stability and is not easily degraded, and has broad application prospects for anti-tumor clinical treatment.

[0026] General definitions and terms

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of any conflict, the definitions provided herein shall prevail.

[0028] When used with a value variable, the terms "about" or "approximately" usually mean that the value of the variable and all values ​​of the variable are within the experimental error (e.g., within a 95% confidence interval for the mean) or within ±10% of the specified value, or a wider range.

[0029] The terms “comprising,” “including,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps. Those skilled in the art will understand that the foregoing terms such as “comprising” encompass the meaning of “consisting of.” The expression “consisting of” excludes any unspecified elements, steps, or ingredients. The expression “substantially constitutes” limits the scope to the specified elements, steps, or ingredients, plus optional elements, steps, or ingredients that do not materially affect the essential and novel features of the claimed subject matter. It should be understood that the expression “comprising” encompasses both the expressions “substantially constitutes” and “consisting of.”

[0030] When describing numerical or range endpoints in this document, it should be understood that the disclosure includes the specific values ​​or endpoints referenced.

[0031] As used herein, the terms “one or more” or “at least one” refer to one, two, three, four, five, six, seven, eight, nine or more.

[0032] The term "pharmaceutically acceptable" refers to a substance that, within the bounds of normal medical judgment, is suitable for contact with a patient's tissues without undue toxicity, irritation, allergic reactions, etc., has a reasonable benefit-risk ratio, and is effective for its intended use. The term "pharmaceutically acceptable salt" refers to a salt made from a pharmaceutically acceptable, non-toxic base or acid, including inorganic or organic bases and inorganic or organic acids.

[0033] The term "amino acid" refers to the basic building blocks of proteins, giving proteins their specific molecular structure and enabling them to have biochemical activity. Attached Figure Description

[0034] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0035] Figure 1 This is a schematic diagram of the structure of the small molecule cyclic peptide SHAP in an embodiment of the present invention;

[0036] Figure 2 Figure 1 shows the experimental results of the effect of cyclic peptide SHAP on DNA damage repair in Example 1 of the present invention. In this figure, A is the effect of the reporter system HR detection of the MST1 / 2 activator SHAP on the DSB damage response; B is the DNA laser experiment evaluating the effect of cyclic peptide SHAP on the recruitment of ZMYND8 to the DSB damage site; C is the statistical analysis of the fluorescence intensity in B using ImageJ software; and D is the Western blot results of cell proteins at different time points after the control group and the cyclic peptide SHAP group were incubated with the DNA damage inducer etoposide for 2 hours.

[0037] Figure 3 The figures shown are the cell activity detection results of cyclic peptide SHAP combined with PARP1 inhibitor rucapranib in Example 2 of the present invention. In this figure, A is the cell activity result of cyclic peptide SHAP combined with rucapranib in AZ-521 cells, and B is the cell activity result of cyclic peptide SHAP combined with rucapranib in AGS cells.

[0038] Figure 4 The figures shown are the cell activity detection results of cyclic peptide SHAP combined with PARP1 inhibitor olaparib in Example 3 of the present invention. In this figure, A is the cell activity result of cyclic peptide SHAP combined with olaparib in AZ-521 cells, and B is the cell activity result of cyclic peptide SHAP combined with olaparib in AGS cells.

[0039] Figure 5 The figure shows the experimental results of the synthetic lethal effect of the cyclic peptide SHAP combined with the PARP1 inhibitor rucapranib on mouse tumor cells in Example 4 of the present invention. In the figure, A is a flowchart of the mouse experiment and a graph of tumor volume change, B is an anatomical photograph of the mouse tumor, and C is a graph of tumor weight change in the mouse. Detailed Implementation

[0040] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0041] The basic experimental procedures used in the embodiments of the present invention, such as cell culture, immunoblotting, and cell viability experiments, are described below, but are not limited to this method.

[0042] Basic experimental methods:

[0043] (1) Cell culture:

[0044] Cell lines AGS, AZ-521, HGC-27, and 293A were all obtained from the Cell Bank of the Chinese Academy of Sciences. AGS, AZ-521, and HGC-27 were cultured in RPMI 1640 medium (Invitrogen), while 293A was cultured in DMEM medium (Invitrogen). 10% serum, 100 μg / ml penicillin, and 100 μg / ml streptomycin were added to the culture medium. Cells were cultured at 37°C in an incubator with 5% carbon dioxide.

[0045] (2) Immunoblotting:

[0046] Prepare protein samples according to experimental requirements. In a 95℃ metal bath for 10 minutes, centrifuge at 12000 rpm for 2 minutes. Add an equal volume of the supernatant to the wells of an SDS-PAGE gel of appropriate concentration. Electrophoresis is performed at 80V for 20 minutes on a stacking gel and at 120V for 60 minutes on a separating gel. After electrophoresis, remove the gel and assemble the transfer apparatus in the following order: filter paper, SDS-PAGE gel, PVDF membrane, filter paper. Transfer the membrane at a constant current of 300mA for 60-120 minutes in a 4℃ cold storage. After transfer, remove the PVDF membrane and place it in 5% skim milk prepared with PBST buffer. Incubate on a shaker at room temperature for 1 hour. Wash the PVDF membrane three times with PBST buffer, 5-10 minutes each time.

[0047] Add the primary antibody diluted with 3% BSA solution at the specified ratio and incubate overnight on a shaker at 4°C. Wash the membrane three times with PBST buffer, 10 minutes each time. Add the secondary antibody diluted with 5% skim milk at the specified ratio and incubate on a shaker at room temperature for 1-2 hours. Wash the PVDF membrane three times with PBST buffer, 10 minutes each time. Place the PVDF membrane in ECL chromogenic solution and develop at room temperature for 1 minute, then image using a chemiluminescence imaging system.

[0048] (3) Cell viability assay:

[0049] Cell proliferation was assessed using the ATP cell viability assay kit (CellTiter-Lum Plus chemiluminescence cell viability assay kit, Beyotime). Cells were seeded at the same density in 96-well plates, with 100 μL of culture medium per well and 500-2000 cells per well. After overnight incubation to allow cell attachment, different concentrations of cyclic peptide SHAP and PARP1 inhibitors were added, either alone or together. Wells containing only culture medium and no cells served as blank control wells. After several hours of incubation according to different experiments, the plates were removed from the incubator and equilibrated to room temperature. 100 μL of CellTiter-Lum Plus reagent, equal in volume to the cell culture medium, was added to each well. The plates were shaken at room temperature for 2 minutes, followed by incubation at room temperature for 10 minutes. Chemiluminescence signals were detected using a multi-mode microplate reader.

[0050] STRIPAK (Striatin interacting phosphatase and kinase) is a class of multi-component supramolecular complexes containing both kinases and phosphatases, identified in recent years. Highly conserved evolutionarily, STRIPAK supramolecular complexes are widely involved in regulating biological processes such as cell growth, proliferation, and apoptosis, and are closely related to the development of human diseases such as tumors. The cyclic peptide SHAP (STRN3-derived Hippo-activating Peptide) is a peptide inhibitor that can restore Hippo kinase. SHAP can efficiently and specifically block the binding of STRN3 and PP2A in the core component of the STRIPAK supramolecular complex, inhibiting the dephosphorylation of MST1 / 2 and reactivating the antitumor activity of MST1 / 2 kinases.

[0051] Studies have shown (Yang Tang, et al. Selective Inhibition of STRN3-Containing PP2A Hosphatase Restores Hippo Tumor-Suppressor Activity in Gastric Cancer. Cancer Cell, 2020, 38(1):115-128) that the small molecule cyclic peptide SHAP can efficiently and specifically block the binding of STRN3, the core component of the STRPAK supramolecular complex, to PP2A, thereby increasing MST1 / 2 kinase activity and exhibiting tumor-suppressive function. Without affecting other normal functions of PP2A, the cyclic peptide SHAP can specifically block the dephosphorylation of MST1 / 2 by targeting the STRN3-PP2A interaction interface. The structure of the small molecule cyclic peptide SHAP is as follows: Figure 1 As shown, its sequence is Ac-LVRRCK-Nle-LCY-NH2. In the sequence of the cyclic peptide SHAP, Nle is leucine. The cyclic peptide SHAP is obtained by acylation, amination, and cyclization of the peptide chain LVRRCK-Nle-LCY. The cyclization reaction is carried out by the reaction of the thiol groups of the two cysteine ​​residues (C) in the sequence with the meta-dibenzyl halide (NH2). X is a halogen (Br, Cl, etc.) that reacts to form a cyclic peptide structure.

[0052] In some embodiments of the present invention, a pharmaceutical composition of cyclic peptide SHAP and a PARP1 inhibitor is provided. Cyclic peptide SHAP can inhibit DNA homologous recombination damage repair, reduce the ability of ZYMND8 to recruit to DNA double-strand break damage sites, and inhibit the DNA double-strand break damage response of tumor cells. The combination of cyclic peptide SHAP and a PARP1 inhibitor can inhibit the growth of tumor cells through a synthetic lethal effect even when DNA damage repair is normal.

[0053] The following examples illustrate the pharmacological mechanism of action of the cyclic peptide SHAP alone or in combination with a PARP1 inhibitor.

[0054] Example 1: The effect of cyclic peptide SHAP on DNA damage repair

[0055] This embodiment studies the effect of the cyclic peptide SHAP on DNA damage repair, demonstrating that the cyclic peptide SHAP can effectively inhibit DNA homologous recombination damage repair. The specific experimental steps and methods are as follows:

[0056] a) HR-GFP reporter system detection experiment: Cells were electroporated using a NEPA GENE at 150V and 975 μF to transfect the I-SceI expression vector (pCBASce) and the GR-GFP reporter plasmid into 293A cells. After 48 hours of electroporation, the fluorescence intensity of the cells was analyzed using a BD FACS CantoII flow cytometer.

[0057] b) Laser microbeam irradiation experiment: HGC-27 cells were seeded in 35 mm confocal dishes and cultured overnight. They were then transfected with GFP-ZMYND8 plasmid and incubated with 10 mM 5-bromo-2'-deoxyuridine (B9285, Sigma) for 24 hours. Cells were stained with Hoechst 33342 (6249, Thermo Fisher) at room temperature for 15 minutes and washed three times with PBS. Subsequently, the cells were placed in the cell culture chamber (37°C, 5% CO2) of a live cell workstation (Eclipse Ti, Nikon). A 405 nm laser was used to irradiate the designated area 20 times using NIS-Elements software, with one image acquired every 30 seconds. The fluorescence intensity in the images was statistically analyzed using ImageJ software.

[0058] c) Western blot: The 293A cell culture method is as described in the basic experimental method (1). Etoposide and cyclic peptide SHAP were prepared at a concentration of 10 μM. Cells were stimulated with 10 μM etoposide for 1 hour. Cell samples were collected at different time points and Western blot experiments were performed. The experimental method is the same as the basic experimental method (2).

[0059] Experimental results and analysis:

[0060] Disruptions in the DNA damage response mechanism can lead to genomic instability and induce tumors. This study investigated the effect of the cyclic peptide SHAP on DNA damage repair, demonstrating that SHAP can effectively inhibit DNA homologous recombination damage repair. The HR reporter system was used to detect the effect of the MST1 / 2 activator SHAP on the DSB damage response. The results showed that compared with the control group, SHAP-treated cells exhibited a significantly decreased DSB repair efficiency, and a dose-dependent effect was observed. Figure 2 (A). ZMYND8, as a histone modification reader, can recruit NuRD complexes, exposing damaged DNA sites and thus promoting homologous recombination-mediated DSB damage repair. The effect of SHAP on its recruitment to DSB damage sites was evaluated using laser microbeam irradiation experiments. Results showed that, compared with the control group, GFP-ZMYND8 recruitment was significantly weakened in cells treated with the cyclic peptide SHAP, and at the same time points, the fluorescence intensity near the DSB damage sites (marked by the box lines) was lower. Figure 2 Figures B and C show that the cyclic peptide SHAP can inhibit the recruitment of ZYMND8 to DSB damage sites. Furthermore, we further validated its inhibitory function by detecting the protein degradation rate of γ-H2AX. As shown in the figure, we found that SHAP-treated cells showed a significant increase in p-MST1 / 2 expression, consistent with previous reports (Tang et al., 2020). SHAP can break the binding of PP2Aa to STRN3, thereby releasing MST1 / 2 kinases. Moreover, after etoposide treatment induced similar levels of DNA damage, at different time points, the protein degradation rate of γ-H2AX in the control group was significantly faster than that in the SHAP-treated group, indicating that the cyclic peptide SHAP inhibits the DSB damage response in tumor cells.

[0061] Example 2: Effect of cyclic peptide SHAP combined with PARP1 inhibitor rucapranib on cell viability

[0062] This embodiment examines the effect of the cyclic peptide SHAP combined with the PARP1 inhibitor rucapranib on cell viability. The specific experimental steps and methods are as follows:

[0063] Cell viability assay: AGS, AZ-521, and other cell culture methods are as described in basic experimental method (1). Different concentrations of lucappanil (10...) were prepared. -2 ~10 2 The cell viability of different cells was measured using the same experimental method as the basic experimental method (3) and cyclic peptide SHAP (0-2 μM).

[0064] Experimental results and analysis:

[0065] This embodiment used cell viability assays to detect the cell viability of the cyclic peptide SHAP combined with the PARP1 inhibitor rucapranib. In AZ-521 cells, the cell viability of the cyclic peptide SHAP combined with the PARP1 inhibitor rucapranib was significantly decreased. Figure 3 In AGS cells, the cellular activity of the cyclic peptide SHAP combined with the PARP1 inhibitor rucapranib showed the same trend. Figure 3 (B) The above results show that the combination of cyclic peptide SHAP and the PARP1 inhibitor rucapanib can exhibit good synthetic lethality against different tumor cells. The experimental results show that the lethality of rucapanib or cyclic peptide SHAP alone was poor, demonstrating that the antitumor activity was significantly enhanced when used in combination.

[0066] Example 3: Effect of cyclic peptide SHAP combined with PARP1 inhibitor oxapanediol on cell viability

[0067] This embodiment examines the effect of the cyclic peptide SHAP combined with the PARP1 inhibitor oxapranib on cell viability. The specific experimental steps and methods are as follows:

[0068] Cell viability assay: AGS, AZ-521, and other cell culture methods are as described in basic experimental method (1). Different concentrations of oxapane (10... -2 ~10 2 The cell viability of different cells was measured using the same experimental method as the basic experimental method (3) and cyclic peptide SHAP (0-2 μM).

[0069] Experimental results and analysis:

[0070] This embodiment used cell viability assays to detect the cell viability of the cyclic peptide SHAP combined with the PARP1 inhibitor oxapane. In AZ-521 cells, the cell viability of the cyclic peptide SHAP combined with the PARP1 inhibitor oxapane was significantly decreased. Figure 4 In AGS cells, the cellular activity of the cyclic peptide SHAP combined with the PARP1 inhibitor oxapaneri showed the same trend. Figure 4 (B) The results above show that the combination of cyclic peptide SHAP and the PARP1 inhibitor oxapanebide exhibits good synthetic lethality against different tumor cells. Meanwhile, the experimental results show that the lethality of oxapanebide or cyclic peptide SHAP alone was poor, demonstrating that the antitumor activity was significantly enhanced when used in combination.

[0071] Example 4: Synthetic lethal effect of cyclic peptide SHAP combined with PARP1 inhibitor on tumor cells in mice

[0072] This embodiment studies the synthetic lethal effect of the cyclic peptide SHAP combined with a PARP1 inhibitor on tumor cells in vivo in mice. The specific experimental steps and methods are as follows:

[0073] a) Establishment of a mouse tumor model: Healthy male nude mice (4 weeks old) were obtained from the Shanghai Laboratory Animal Center and kept under pathogen-free conditions according to the guidelines of the Institutional Animal Care and Use Committee of the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (SIBCB, Shanghai). Animal use license number No. SIBCB-SIBCB-NAF-14-004-S329-023 was issued by the SIBCB Animal Core Facility. In the tumor formation experiment, AZ-521 cells were used at a rate of 5 × 10⁻⁶ cells / mL. 6 A dose of one per mouse was subcutaneously injected into the axilla of mice to induce tumor formation.

[0074] b) Mouse administration: When the tumor volume reaches 100 mm² 3 Mice were intratumorally injected with the PARP1 inhibitor rucapanib (10 mg / kg) and / or the cyclic peptide SHAP (10 mg / kg) for 5 consecutive days. Tumor length and width were measured every 3 days, and tumor volume was calculated (tumor volume = width). 2 (×length×0.523). Two weeks later, the mice were euthanized, the tumors were dissected, and their weight was measured.

[0075] Experimental results and analysis:

[0076] This embodiment investigated the effect of the combination of cyclic peptide SHAP and a PARP1 inhibitor on tumor growth in a mouse tumor model. The combination of cyclic peptide SHAP and the PARP1 inhibitor rucapranib significantly reduced the volume and weight of mouse tumors. Figure 5 This indicates that the combination of cyclic peptide SHAP and a PARP1 inhibitor can produce a good synthetic lethal effect on mouse tumors. These results show that cyclic peptide SHAP can improve PARP1 inhibitor tolerance at the in vivo level and significantly enhance the killing ability against tumor cells.

[0077] This invention discovers that the cyclic peptide SHAP is a key signaling molecule specifically dependent on the DNA damage response mechanism of tumor cells. SHAP can significantly inhibit the HR repair efficiency of tumor cells and suppress the DSB damage response, and this process exhibits a dose-dependent effect. The combined use of SHAP and a PARP1 inhibitor can efficiently kill tumor cells in vivo and in vitro through synthetic lethal effects, significantly increasing the sensitivity of tumor cells to PARP1 inhibitors. Therefore, this invention, by combining SHAP and a PARP1 inhibitor, greatly expands the clinical application scope of PARP1 inhibitors and provides a technical solution to the drug resistance problem currently faced in clinical treatment with PARP1 inhibitors, possessing extremely high clinical application value and broad market transformation potential.

[0078] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified. sequence list <110> Shanghai Tenth People's Hospital <120> A pharmaceutical composition and its use in the preparation of antitumor drugs <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <400> 1 Leu Val Ala Ala Cys Leu Asn Leu Cys Thr 1 5 10

Claims

1. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a cyclic peptide SHAP and a PARP1 inhibitor; the cyclic peptide SHAP is formed by cyclizing the thiol groups of two cysteine ​​residues in the amino acid sequence Ac-LVRRCK-Nle-LCY-NH2 with a m-dibenzyl halide to form a cyclic peptide structure, and the structural formula of the m-dibenzyl halide is as follows: X is a halogen, Br or Cl; The PARP1 inhibitor is olaparib or rucaparib; The concentration of the cyclic peptide SHAP is 0.5~20 μM, and the concentration of the PARP1 inhibitor is 0.01~100 μM.

2. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition further includes pharmaceutically acceptable salts and / or carriers.

3. The use of the pharmaceutical composition as described in claim 1 in the preparation of a medicament for inhibiting the growth of tumor cells AGS or AZ-521.

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