Use of sioroni in combination with immune checkpoint inhibitors for anti-tumor therapy

Abstract

The present application relates to the field of medicine, and in particular to the application of sirolimus combined with immune checkpoint inhibitors in anti-tumor therapy. The present application discloses the use of a combination of sirolimus or a derivative thereof and an immune checkpoint inhibitor in the preparation of a medicament for treating tumors, a pharmaceutical composition and a kit comprising the combination, and a method for treating tumors by administering a therapeutically effective amount of the pharmaceutical composition or the kit to a tumor patient in need thereof. The combination of the present application has a synergistic effect and is suitable for treating cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the application of sirolimus combined with immune checkpoint inhibitors in anti-tumor therapy. BACKGROUND

[0002] Tumor is a major disease that threatens human health, and the research on tumor treatment has always attracted widespread attention. Traditional tumor treatment methods include surgical treatment, radiotherapy, and chemical drug treatment. With the progress of medical science, new treatment methods such as targeted therapy and immunotherapy have emerged, providing new treatment options for cancer patients.

[0003] Targeted therapy is a method of designing corresponding targeted drugs for treatment at the cellular and molecular level, targeting the identified oncogenic sites. After the targeted drugs enter the human body, they will specifically select the oncogenic sites for binding and action, causing specific death of tumor cells, while not affecting the normal tissue cells around the tumor. Targeted therapy is the focus of the development of many anticancer drugs, and it is the cornerstone of precision medicine.

[0004] Sirolimus is a small molecule anti-tumor targeted drug targeting multiple protein kinases, which is an original compound independently developed by Shenzhen Microchip Biotechnology Co., Ltd. with complete intellectual property rights. Sirolimus has high selective inhibitory activity on VEGFR / PDGFR / c-Kit, Aurora B, and CSF-1R targets, and has three-pathway anti-tumor synergistic action mechanisms of anti-tumor angiogenesis, inhibition of tumor cell mitosis, and regulation of tumor inflammatory microenvironment, thereby playing a comprehensive anti-tumor role.

[0005] CN200910223861.5 discloses a naphthamide derivative, its preparation method and application. The compound has protein kinase inhibitory activity and histone deacetylase inhibitory activity, and can be used for treating diseases related to abnormal protein kinase activity or abnormal histone deacetylase activity, including inflammation, autoimmune diseases, cancer, nervous system diseases and neurodegenerative diseases, cardiovascular diseases, metabolic diseases, allergies, asthma, and hormone-related diseases.

[0006] CN201610856945.2 discloses non-solvated crystals A, B, and C of sirolimus, their preparation methods, and pharmaceutical compositions containing the crystals, as well as the application of the crystals in the preparation of drugs for treating diseases related to abnormal protein kinase activity or abnormal histone deacetylase activity.

[0007] CN201811550290.1 discloses a new use of sirolimus in the preparation of a drug for treating acute myeloid leukemia. The new use is the use of sirolimus or its pharmaceutically acceptable salt, ester, solvate in the preparation of a drug for preventing and / or treating acute myeloid leukemia. Sirolimus inhibits the growth of acute myeloid leukemia cells, induces apoptosis and colony formation through Src / Fyn / p38 and Erk / MEK signaling pathways.

[0008] Immunotherapy aims to activate the human immune system and rely on the body's immune function to kill cancer cells and tumor tissue. Immune checkpoint molecules such as PD-1 are mainly expressed on the surface of activated T lymphocytes. When its ligand PD-L1 or PD-L2 binds to PD-1, it transmits an immunosuppressive signal, so that T lymphocytes are in a state of immune tolerance or low immune function. When anti-PD-1 or PD-L1 antibodies are used to block the above signal pathway, the immunosuppressive signal of T lymphocytes is removed, thereby further activating T lymphocytes to exert anti-tumor activity such as killing and cytotoxicity. Therefore, immune checkpoint inhibitors such as PD-1 inhibitors or PD-L1 inhibitors have been approved for the clinical treatment of various tumors.

[0009] Targeted therapy drugs and immune checkpoint inhibitors can show certain efficacy in clinical tumor treatment, and research on the combination of the two is also increasing.

[0010] WO2015088847A1 discloses the use of pazopanib and PD-1 inhibitors in combination. The animal experiment data and phase I / II clinical trial results recorded in the specification show that the combination of the two has a significant effect on the treatment of cancer.

[0011] CN105960415A discloses the use of axitinib and PD-1 inhibitors in combination. The clinical trial scheme recorded in the specification shows that the combination of the two is expected to have a significant therapeutic effect on renal cell carcinoma.

[0012] WO2016141218A discloses the use of lenvatinib and PD-1 inhibitors in combination. The animal experiment data recorded in the specification show that the combination of the two has a significant effect on the treatment of cancer.

[0013] CN108601831B discloses the use of axitinib and PD-1 inhibitors in combination. The animal experiment data and phase I / II clinical trial results recorded in the specification show that the combination of the two has a significant effect on the treatment of cancer.

[0014] CN106963948A discloses the use of axitinib and PD-1 inhibitors in combination. The animal experiment data recorded in the specification show that the combination of the two has a significant effect on the treatment of colon cancer.

[0015] WO2019096194A1 discloses the use of apatinib and PD-1 inhibitor in combination, and the phase II clinical trial results recorded in the specification prove that the combination of the two has a significant effect on the treatment of small cell lung cancer.

[0016] CN109893654A discloses the use of apatinib and PD-1 inhibitor and IDO inhibitor in combination, and the animal experiment data recorded in the specification prove that the combination of the three has a significant effect on the treatment of colon cancer.

[0017] Although the combination of targeted therapy drugs and immune checkpoint inhibitors has achieved remarkable curative effect, there are still two problems:

[0018] First, due to the complexity of the mechanism and process of tumor targeted therapy and tumor immunotherapy, the curative effect of the combination of targeted drugs and immune checkpoint inhibitors is difficult to predict, so extensive attempts are made to combine therapy to further improve the applicability and benefit rate of combined drugs for tumor patients, reduce the single drug dosage, improve the incidence and / or severity of adverse events (AE) in treatment, enhance the clinical control of tumor symptoms / progression, prolong the degree and duration of drug response in patients, which is of great significance, and it is also a big challenge in the field of tumor treatment.

[0019] Second, although the prior art discloses the combination of a variety of VEGFR inhibitors and PD-1 inhibitors in tumor treatment, however, ceritinib is not a traditional VEGFR class targeted inhibitor, compared with traditional VEGFR class targeted inhibitors such as sunitinib / sorafenib, ceritinib has unique inhibitory activity on aurora B, a key enzyme of mitosis, which has the potential to reduce genomic instability of tumor tissue and inhibit tumor cell metastasis. Ceritinib exerts anti-tumor activity through three complementary mechanisms of action: inhibition of tumor angiogenesis, cell mitosis and tumor inflammatory microenvironment, so it is worth further studying whether the combination of ceritinib and immune checkpoint inhibitors can produce a synergistic effect and thus improve the curative effect. SUMMARY

[0020] To solve the above technical problems, the present application provides the use of ceritinib or its derivative and immune checkpoint inhibitor in combination for preparing a drug for treating tumors.

[0021] The term "treatment" refers to any indication of successful or improved progress, severity and / or duration of a disease, pathology or condition, including any objective or subjective parameter, such as dose reduction, remission, alleviation of symptoms or making injury, pathology or condition more tolerable to the patient, slowing the rate of deterioration or decline, making the final point of deterioration less debilitating, or improving the patient's physical or mental well-being.

[0022] The term "inhibit" refers to a decrease in the activity, binding or expression of a polypeptide, or a reduction or amelioration of a disease, disorder or condition, or symptoms thereof. Inhibition as described herein can include partial or complete blockade of stimulation, reduction, prevention or delay of activation or binding, or inactivation, desensitization or down-regulation of protein or enzyme activity or binding.

[0023] The sirolimus or its derivatives and the immune checkpoint inhibitors as described herein employ a conventional effective amount.

[0024] The term "effective amount" refers to the amount of a drug that is sufficient to achieve a therapeutic goal or therapeutic effect in the course of therapy. An effective amount can be sufficient to reduce and / or ameliorate the progression, recurrence, severity and / or duration of a given disease, disorder or condition and / or symptoms related thereto, and / or to improve or enhance the prophylactic or therapeutic effect(s) of another therapy. An effective amount of one therapeutic drug in the combination as described herein can enhance the therapeutic efficacy of another therapeutic drug.

[0025] The sirolimus derivatives include pharmaceutically acceptable salts thereof and non-solvated crystals A, B and C thereof.

[0026] The non-solvated crystals A, B and C of sirolimus and the preparation method thereof are disclosed in CN201610856945.2, the contents of which are incorporated herein in their entirety.

[0027] In specific embodiments, the sirolimus or its derivatives are used in an amount of about 1-100 mg, preferably about 5-80 mg, more preferably about 10-50 mg, and most preferably about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg.

[0028] The immune checkpoint inhibitors include PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, BTLA inhibitors, VISTA inhibitors or LAG-3 inhibitors, preferably PD-1 inhibitors and PD-L1 inhibitors.

[0029] The term "PD-1 inhibitor" refers to a moiety, such as a compound, nucleic acid, polypeptide, antibody, including variants, isoforms, species homologs (e.g., mouse) of human PD-1, and analogs having at least one common epitope with PD-1, that decreases, inhibits, blocks, abrogates, or interferes with the activity or expression of PD-1. PD-1 inhibitors include small and large molecules, such as compounds, nucleic acids, polypeptides, antibodies, peptibodies, bifunctional antibodies, minibodies, single-chain variable fragments (ScFv), and fragments or variants thereof. Thus, a PD-1 inhibitor for use in the present application refers to any moiety that antagonizes the activity or expression of PD-1. PD-1 inhibitors include exemplary components such as Nivolumab, Pembrolizumab, Toripalimab, Sintilimab, SHR-1210, BGB-A317, Genolimzumab, Zimberelimab (AB122), AK101, AK104, AK105, GLS-010, BAT1306, CS1003, PDR001, Cemiplimab, and MEDI0680.

[0030] Nivolumab, also known as Opdivo, O drug, is developed by Medarex and Ono Pharmaceutical. In 2009, Bristol-Myers Squibb (BMS) acquired Nivolumab and obtained the patent of the drug. Nivolumab is the first approved PD-1 monoclonal antibody in the world, which can treat melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck cancer and other cancers. It is combined with anti-CTLA-4 ipilimumab (Yervoy) to form the first approved immunotherapy combination in the world, which can treat metastatic melanoma, and the dosage is 3 mg / kg.

[0031] Pembrolizumab, also known as Keytruda, K drug, is an antibody product of Merck & Co., which was listed in the United States in 2014, and its sales ranked second in the PD-1 antibody market after listing. It is a strong competitor of Nivolumab (nivolumab) of Bristol-Myers Squibb (BMS). The first approved indication of Pembrolizumab is melanoma, the second is combined with chemotherapy for non-small cell lung adenocarcinoma first-line treatment, and the third is single lung cancer (adenocarcinoma and squamous cell carcinoma) first-line treatment, and the dosage is 2 mg / kg.

[0032] Toripalimab, also known as Tislelizumab, is the first listed domestic PD-1 inhibitor developed by Junshi Biosciences. It was approved for listing in 2018 and used for the treatment of locally advanced or metastatic melanoma after failure of standard treatment. Junshi Biosciences has prepared to submit the listing application of Tislelizumab for nasopharyngeal carcinoma and urothelial carcinoma indications.

[0033] Sintilimab, also known as signdyli single antibody, is a PD-1 monoclonal antibody jointly developed by Sinopharm and Eli Lilly in China. It can bind to the surface of T cell PD-1, block the binding between it and ligand PD-L1, so that T cells and autoimmune reactions can function normally, and then eliminate tumor cells. Sintilimab was approved in China at the end of 2018, and was included in the 2019 edition of CSCO lymphoma diagnosis and treatment guidelines, and was recommended for the treatment of relapsed / refractory classical Hodgkin's lymphoma (R / R-cHL).

[0034] SHR-1210, also known as cari Li single antibody, is a PD-1 inhibitor independently developed by Jiangsu Hengrui Medicine Co., Ltd. Carereli single antibody completed preclinical study in January 2015, and in January 2019, the U.S. Food and Drug Administration (FDA) approved the international multicenter phase III clinical trial of carereli single antibody combined with apatinib mesylate for the first-line treatment of liver cancer in the United States, Europe and China. On May 29, 2019, the National Drug Administration officially approved the listing of Aireka.

[0035] BGB-A317, also known as tirali single antibody, is a human monoclonal antibody developed by Baijishen. Tirali single antibody may restore the ability of CTL to kill cancer cells by binding to PD-1, and does not activate the receptor, thereby preventing the binding of PD-L1 to PD-1. On December 27, 2019, the marketing application (CXSS1800019) of tirali single antibody injection was approved by the State Drug Administration, which is used for the treatment of relapsed / refractory classical Hodgkin's lymphoma (r / r cHL).

[0036] Other PD-1 inhibitors include AK101, AK104 and AK105 of Lepe Medical, Geno single antibody of Jiahe Biological, GLS-010 of Yuheng Pharmaceutical, BAT1306 of Baotaite, CS1003 of Genscript Pharmaceutical, PDR001 of Novartis, Cemiplimab of Regeneron / Sanofi, MEDI0680 of AstraZeneca, Zimberelimab (AB122) of Arcus, etc.

[0037] The term“PD-L1 inhibitor” refers to a moiety, e.g., a compound, nucleic acid, polypeptide, antibody, including variants, isoforms, species homologs of human PD-L1 (e.g., mouse) and analogs having at least one common epitope with PD-L1, that decreases, inhibits, blocks, abrogates, or interferes with the activity, binding of PD-L1 to its receptor PD-1 or expression of PD-L1. PD-L1 inhibitors include small and large molecules, e.g., small molecule compounds, nucleic acids, polypeptides, antibodies, peptibodies, bifunctional antibodies, minibodies, single-chain variable fragments (ScFv), and fragments or variants thereof. Thus, a PD-L1 inhibitor for use in the present application refers to any moiety that antagonizes the activity of PD-L1, its binding to PD-1 or its expression. Exemplary components of PD-L1 inhibitors include Tecentriq (Atezolizumab), Imfinzi (Durvalumab), Bavencio (Avelumab), CS1001, TQB2450, SHR1316, lazertinib, bintrafusp alfa, Envafolimab (KN035), CA-170, CX-072, BGB-A333, BMS-936559, GEN-1046, KL-A167, and IO-103.

[0038] Tecentriq, Atezolizumab, T for short, is a PD-L1 immunosuppressant developed by Roche Pharmaceutical, which can bind to PD-L1 on tumor cells and tumor-infiltrating immune cells, block its action with PD-1 and B7-1 receptors, and thus restart T cells to kill cancer cells. In May 2020, Tecentriq (atezolizumab) was approved by the US FDA for a new indication as a first-line (initial) monotherapy for the treatment of metastatic non-small cell lung cancer (NSCLC) in adult patients, specifically: The FDA-approved detection method determines that the tumor has high expression of PD-L1 (PD-L1 staining of ≥ 50% of tumor cells [TC≥50%] or PD-L1 staining of tumor-infiltrating [IC] covering ≥ 10% of tumor area [IC≥10%]), non-squamous and squamous non-small cell lung cancer (NSCLC) in adult patients with advanced non-squamous and squamous non-small cell lung cancer (NSCLC) without EGFR or ALK mutations.

[0039] Durvalumab, also known as Imfinzi, is a human monoclonal antibody that directly targets PD-L1. It can block the interaction of PD-L1 with PD-1 and CD80 on T cells, resist the immune escape of tumors, and induce immune response. In 2020, AstraZeneca announced the detailed results of the latest analysis of the CASPIAN trial, a phase III trial of Imfinzi combined with standard of care (SoC) platinum-based chemotherapy (etoposide + cisplatin or carboplatin) for first-line treatment of extensive-stage small cell lung cancer (ES-SCLC) in adult patients. The results showed that the combination of anti-PD-L1 therapy Imfinzi and standard of care (SoC) platinum-based chemotherapy (etoposide + cisplatin or carboplatin) for first-line treatment of extensive-stage small cell lung cancer (ES-SCLC) in adult patients had a sustained clinical benefit in overall survival (OS).

[0040] Avelumab, also known as Bavencio, is a PD-L1 antibody drug jointly developed by Pfizer and Merck. In May 2020, Pfizer and Merck announced positive results of a phase III clinical trial of Bavencio (avelumab) maintenance therapy combined with best supportive care (BSC) in patients with advanced urothelial carcinoma (UC) after platinum-based chemotherapy. The median follow-up time for patients treated with avelumab + BSC and BSC alone was 19.6 months and 19.2 months, respectively. Among all enrolled patients, 358 cases were detected for tumor biomarker PD-L1 positive. The results showed that avelumab + BSC significantly prolonged the OS of patients compared with BSC alone (hazard ratio [HR] 0.69; 95% CI 0.56, 0.86; 1-sided p = 0.0005); the median OS of avelumab + BSC and BSC alone was 21.4 and 14.3 months, respectively, indicating that the OS of patients was prolonged by more than 7 months.

[0041] Envafolimab (KN035) is the first domestic nanobody targeting PD-L1, developed by Kangning Jierui. KN035 was authorized to Sidu Di in March 2016 and filed for clinical trials in May of the same year. KN035 is a PD-L1 nanobody-Fc fusion protein with IgG1 subtype, which has been modified by C221S, D265A, and P331G to remove ADCC and other Fc effector effects. KN035 is a room temperature stable, subcutaneous injection formulation, easy to store and use. KN035 is a PD-L1 single-domain antibody Fc fusion protein, based on this unique design, it has advantages in safety, convenience, and compliance, and can be used for patients who are not suitable for intravenous infusion, while having lower medical costs. Currently, clinical trials are being conducted in China, the United States, and Japan for multiple tumor indications, and some indications have entered phase III clinical trials.

[0042] CS1001, TQB2450 and SHR1316 are PD-L1 inhibitors developed by Simcere Pharmaceutical, Changsheng Pharmaceutical and Hengrui Pharmaceutical, respectively.

[0043] In specific embodiments, the immune checkpoint inhibitor is used in an amount of about 1-1000 mg, preferably about 10-800 mg, more preferably 20-500 mg, most preferably about 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 300 mg, 400 mg, 500 mg.

[0044] The tumor includes colon cancer, liver cancer, lung cancer, gastric cancer, intestinal cancer, breast cancer, cervical cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, fallopian tube tumor, peritoneal tumor, melanoma, glioma, glioblastoma, hepatocellular carcinoma, head and neck tumor, leukemia, lymphoma and myeloma.

[0045] The present application also provides a pharmaceutical composition comprising the aforementioned combination.

[0046] The pharmaceutical composition of the present application includes any pharmaceutical composition administered to a patient by any route, such as oral, transmucosal (nasal, inhalation, pulmonary, sublingual, vaginal or rectal), parenteral (subcutaneous, intravenous, bolus injection, intramuscular or intra-arterial), transdermal, etc.

[0047] The dosage form of the pharmaceutical composition of the present application includes tablets, granules, capsules and the like solid preparations, as well as liquid dosage forms suitable for parenteral administration to a patient and liquid dosage forms suitable for parenteral administration to a patient.

[0048] The pharmaceutical composition of the present application includes one or more excipients, and suitable excipients are well known to those skilled in the art of pharmacy.

[0049] The present application also provides a kit comprising the aforementioned combination.

[0050] The kit of the present application can include the aforementioned combination with the same or different formulations. Each component of the aforementioned combination in the kit can be provided in separate individual containers or provided in the same container.

[0051] In specific embodiments, the sotorasib or derivative thereof in the kit is formulated for oral administration, such as a solid formulation of a tablet, granule or capsule. The immune checkpoint inhibitor can be provided in the form of, for example, a lyophilized powder or a solution for parenteral administration.

[0052] In specific embodiments, the sotorasib or derivative thereof in the kit and the immune checkpoint inhibitor are unit formulations with the same or different specifications.

[0053] In specific embodiments, the sotorasib or derivative thereof and the immune checkpoint inhibitor are placed in the same container or in different containers, respectively.

[0054] In specific embodiments, the sotorasib or derivative thereof is a gastrointestinal administration dosage form, preferably an oral preparation; and the immune checkpoint inhibitor is a parenteral administration dosage form, preferably an injection preparation.

[0055] The kit further comprises instructions for use or other information.

[0056] The present application also provides a method for treating a tumor, which comprises administering to a tumor patient in need a therapeutically effective amount of the aforementioned pharmaceutical composition or kit. The present application provides a method for treating a tumor as a combination therapy.

[0057] The tumor includes colon cancer, liver cancer, lung cancer, stomach cancer, intestinal cancer, breast cancer, cervical cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, fallopian tube tumor, peritoneal tumor, melanoma, glioma, glioblastoma, hepatocellular carcinoma, head and neck tumor, leukemia, lymphoma and myeloma.

[0058] The term "combination therapy" refers to administration of two or more pharmaceutical components. The timing of the combination therapy is dependent, in part, on the combination administered. Conjoint administration is intended to include concurrent or sequential administration of the components. The components of the present application can be administered separately or in combination to the patient. The components described herein can be used in combination with each other and in combination with other active agents known to be useful in the treatment of cancer.

[0059] The term "administering" refers to the act of delivering the combination to a subject by a route such as oral, mucosal, topical, suppository, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration. Parenteral administration includes intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Administration typically occurs after the onset of the disease, disorder, or condition, or symptom thereof, but in some cases, can occur prior to the onset of the disease, disorder, or condition, or symptom thereof.

[0060] Preferably, the aforementioned pharmaceutical composition or kit is administered to the tumor patient as a first-line therapy.

[0061] The aforementioned pharmaceutical composition or kit can also be administered to the tumor patient as a second-line, third-line, fourth-line, fifth-line or sixth-line therapy.

[0062] Preferably, the tumor patient has not been treated or has been treated at least once with an anti-tumor therapy.

[0063] The anti-tumor therapy includes surgery, chemotherapy, radiotherapy, targeted therapy and immunotherapy or a combination thereof.

[0064] In the method, the sirolimus or derivative thereof and the immune checkpoint inhibitor are administered simultaneously, separately or sequentially.

[0065] The beneficial effects of the present application are:

[0066] (1) Compared with the solvent control group, the proportion of CD4 and CD8 subgroups of spleen lymphocytes in the two groups of mice administered with sirolimus and PD-1 antibody alone was increased, from an average proportion of 5.8% (CD4) and 3.5% (CD8) in the solvent control group to 9.9%, 12% (CD4) and 6.1%, 6% (CD8), respectively; while the proportion of CD4 and CD8 subgroups of spleen lymphocytes in the group administered with sirolimus and PD-1 antibody in combination was significantly increased to 15.8% and 9.3%.

[0067] (2) Compared with the solvent control, the relative tumor inhibition rates of the two groups of mice administered with sirolimus and PD-1 antibody alone were 39% and 32%, respectively, while the relative tumor inhibition rate was significantly increased to 70% when the two drugs were used in combination. The above results show that the combination of sirolimus and PD-1 antibody has good immune-enhancing and synergistic anti-tumor activity in tumor-bearing mice.

[0068] (3) Compared with the solvent control, the relative tumor inhibition rates of the two groups of mice administered with sirolimus and PD-1 antibody alone were 26% and 39%, respectively, while the relative tumor inhibition rate was significantly increased to 51% when the two drugs were used in combination. The results show that the combination of sirolimus and immune checkpoint inhibitor anti-PD-1 antibody has good synergistic anti-tumor activity in the mouse tumor model.

[0069] (4) The combination of Xioroni (2.5 mg / kg), the reference PD-L1 antibody (1 mg / kg, 3 mg / kg), Xioroni (2.5 mg / kg) and the reference PD-L1 antibody (1 mg / kg), and Xioroni (2.5 mg / kg) and the reference PD-L1 antibody (3 mg / kg) had a significant inhibitory effect on the growth of MC38-hPD-L1 subcutaneously transplanted tumors at the tested dose, and did not produce toxic side effects on animals when they worked, and had good safety.

[0070] The above experimental data show that the combination of Xioroni or its derivatives and immune checkpoint inhibitors produces a synergistic effect, which is suitable for the treatment of cancer. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1 Figure 6 shows the effect of Xioroni and PD-1 antibody alone and in combination on the proportion of spleen lymphocyte subsets in tumor-bearing mice;

[0072] Figure 2 Figure 7 shows the effect of Xioroni and PD-1 antibody alone and in combination on the efficacy of colon cancer cell line CT-26 inoculated tumors in mice;

[0073] Figure 3 Figure 8 shows the growth curve of tumor volume of each group of mice showing the effect of Xioroni and PD-1 antibody alone and in combination on the efficacy of H22 liver cancer cell line inoculated tumors in mice; data is expressed as "mean ± standard error";

[0074] Figure 4 Figure 9 shows the tumor weight at the end of the experiment of each group of mice showing the effect of Xioroni and PD-1 antibody alone and in combination on the efficacy of H22 liver cancer cell line inoculated tumors in mice; data is expressed as "mean ± standard error";

[0075] Figure 5 Figure 10 shows the body weight change curve of each group of mice showing the effect of Xioroni and PD-1 antibody alone and in combination on the efficacy of H22 liver cancer cell line inoculated tumors in mice; data is expressed as "mean ± standard error";

[0076] Figure 6 Figure 11 shows the body weight change rate curve of each group of mice showing the effect of Xioroni and PD-1 antibody alone and in combination on the efficacy of H22 liver cancer cell line inoculated tumors in mice; data is expressed as "mean ± standard error". DETAILED DESCRIPTION

[0077] The application discloses application of Xioroni combined with an immune checkpoint inhibitor in antitumor treatment, and those skilled in the art can improve process parameters to realize the application according to the content of the present application. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are regarded as included in the application. The application and the pharmaceutical composition are described through preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the application and the pharmaceutical composition to realize and apply the application without departing from the content, spirit and scope of the application.

[0078] Example 1: Pharmacodynamic experiment of Balb / c mice inoculated with colon cancer cell line CT-26 to construct a tumor model

[0079] Experimental materials:

[0080] The mouse colon cancer cell line CT-26 is purchased from the Cell Resource Center of Shanghai Life Science Research Institute of Chinese Academy of Sciences, and is routinely cultured at 37 DEG C under the condition of 5% CO2, and the culture solution is DMEM (Gibco) containing 10% fetal bovine serum (Fetal bovine serum, FBS; Gibco) and 1% Penicillin-Streptomycin (HyClone); trypsin is purchased from Gibco. Chiauranib is synthesized by Shenzhen Weicai Biotechnology Co., Ltd., and the purity is greater than 99%. Anti-mouse PD-1 antibodies are purchased from Bio X cell company. Fluorescein-labeled anti-CD4 and CD8 antibodies for detecting mouse lymphocyte subgroups are purchased from eBiosciences company. Normal 6-8-week-old Balb / c mice are purchased from Guangdong Medical Experimental Animal Center.

[0081] Experimental method:

[0082] A large number of CT-26 cells are cultured and kept in a logarithmic growth state. After the cell number reaches the required number, the cells are collected by trypsin digestion, washed with a large amount of PBS for 2 times to remove trypsin and serum components, centrifuged at room temperature and 800 rpm for 10 min, and the supernatant is discarded. The cells are resuspended with FBS-free DMEM culture solution, and the cell concentration is adjusted to 5x10 6 / mL.

[0083] Under sterile conditions, the cell suspension is injected into the back of Balb / c mice subcutaneously at 100 μL / needle, and each mouse is injected with one needle. A 1 mL disposable medical syringe is used for injection, so that the injection site and direction of each mouse are basically consistent.

[0084] After 8 days of inoculation, the tumor grows to an average volume of about 100 mm 3Around 1000 mice bearing tumors were randomly divided into four groups (n=9 per group): a solvent control group, a cioroni group (20 mg / kg, gavage, once daily), a PD-1 antibody group (10 mg / kg, intraperitoneal injection, twice weekly), and a cioroni and PD-1 antibody combination therapy group. After marking, the mice were housed separately. Medication was administered daily according to the group, and tumor formation was observed. Every two days, the longest diameter and the widest diameter perpendicular to it of the tumor were measured using calipers. The tumor volume was calculated using the formula TS = length × (width)² / 2 and recorded. Each mouse was administered the drug once daily via gavage. After the last administration on day 14, the mice were sacrificed, and the spleen was harvested and prepared as a single-cell suspension. 1 × 10⁻⁶ cells were collected. 6 Splenic cells were stained with fluorescein-labeled anti-CD4 and CD8 antibodies (1:200) at 4°C for 30 min. After washing, they were resuspended in PBS and analyzed using a flow cytometer (FACSCanto II). The relative tumor inhibition rate of each drug treatment group was calculated as: (mean tumor volume in the solvent control group - mean tumor volume in the drug treatment group) / mean tumor volume in the solvent control group × 100%.

[0085] Experimental results:

[0086] like Figure 1 As shown, compared with the solvent control group, the two groups treated alone, namely cioronide and PD-1 antibody, showed a certain increase in the proportion of CD4 and CD8 lymphocyte subsets in the spleen of mice, from an average of 5.8% (CD4) and 3.5% (CD8) in the solvent control group to 9.9% and 12% (CD4) and 6.1% and 6% (CD8), respectively; while the combination of cioronide and PD-1 antibody significantly increased the proportion of CD4 and CD8 subsets in the spleen to 15.8% and 9.3%, respectively.

[0087] like Figure 2 As shown, compared with the solvent control, the relative tumor inhibition rates of cioronide and PD-1 antibody alone were 39% and 32%, respectively, while the combined use of the two drugs significantly improved the relative tumor inhibition rate to 70%. These results indicate that the combined use of cioronide and PD-1 antibody has good immune-enhancing and synergistic anti-tumor activity in tumor-bearing mice.

[0088] Example 2: Pharmacological experiment on the establishment of a tumor model by subcutaneous transplantation of mouse hepatocellular carcinoma H22 cell line into female BALB / c mice.

[0089] Laboratory animals:

[0090] BALB / c mice, female, 7-9 weeks old (the age of mice at the time of tumor cell inoculation), weighing about 22 g, purchased from Shanghai Lingchuang Biotechnology Co., Ltd., production license number: SCXK (Shanghai) 2018-0003, animal qualification certificate number: 20180003015834. Raising environment: SPF level.

[0091] The experimental animals were raised in a constant temperature and humidity independent ventilation box, the temperature of the feeding room was 20-26℃, the humidity was 40-70%, the air exchange was 10-20 times / hour, the day and night light and dark alternating time was 12h / 12h; continuously supply of cobalt 60 radiation sterilization of mouse full price granular feed, unlimited free intake, drink tap water (high pressure steam sterilization after use), drinking water bottle uninterrupted water supply, free intake. The mouse box is a polysulfone mouse box, which is used after high pressure sterilization, with a size of 325mm×210mm×180mm; the bedding is high pressure sterilized corn cob, 5 animals per box; the experimental animals are marked by ear tags.

[0092] Experimental materials:

[0093] Xioroni was provided by Shenzhen Microchip Biotechnology Co., Ltd., batch number: 20101008, white powder, sealed at room temperature. Mouse PD-1 antibody: batch number: 0920L765, package specification: 30mg, 10.1mg / mL, provided by China-US Crown Biotechnology (Taicang) Co., Ltd., colorless solution, 4℃ sealed storage. CMC-Na; supplier: SIGMA-ALDRICH; batch number: SLBK436V. Tween 80; supplier: SIGMA-ALDRICH; batch number: WXBC7734V. PBS; supplier: Hyclone; batch number: AF29477519.

[0094] Experimental method:

[0095] Mouse hepatoma H22 cells (purchased from CCTCC; cell number TC-00011) were cultured in RPMI-1640 culture medium (purchased from American Gibco Company) containing 10% fetal bovine serum. The H22 cells in the exponential growth phase were collected, resuspended with PBS to an appropriate concentration, and then used for subcutaneous tumor inoculation in mice. Female mice were subcutaneously inoculated with 1×10 6 H22 cells. When the average volume of the tumor was 104mm 3 According to the tumor size, the mice were randomly divided into groups (see Table 1), and the drug was administered on the same day. The day of tumor cell inoculation was defined as day 0.

[0096] Table 1. Drug administration scheme

[0097]

[0098] Note: 1. The administration volume was 10 μL / g; 2. Xtoro was administered first, followed by the administration of the mouse PD-1 antibody, without a specific administration interval. QD refers to once a day, and BIW refers to twice a week.

[0099] Experimental results:

[0100] On the 19th day of the experiment (15th day of administration), the average tumor volume of the solvent control group was 2060.99 mm 3 . The experiment was terminated, the tumor-bearing mice were photographed, the tumors were removed, and the tumor weights were measured. The tumor volume calculation formula was: tumor volume (mm 3 ) = 1 / 2 × (a × b 2 ) (where a represents the long diameter, and b represents the short diameter)

[0101] On the 19th day of the experiment (15th day of administration), the average tumor volume of the solvent control group was 2060.99 mm 3 . At the end of the experiment, the average tumor volume of the mouse PD-1 antibody 5 mg / kg treatment group was 1266.93 mm 3 , which was statistically significantly different from the control group (p = 0.0242), and the TGI was 39%; the average tumor volume of the test drug Xtoro 5 mg / kg treatment group was 1527.40 mm 3 , which was not statistically significantly different from the control group (p = 0.173), and the TGI was 26%; the average tumor volume of the test drug Xtoro 5 mg / kg combined with mouse PD-1 antibody 5 mg / kg treatment group was 1013.80 mm 3 , which was statistically significantly different from the control group (p = 0.00126), and the TGI was 51%. Tumor samples were collected at the end of the experiment and the tumor weights were measured, and the results were consistent with the tumor volume measurements. The tumor growth of each treatment group and the control group is shown in Table 2, Figure 3 and Figure 4 .

[0102] Compared with the solvent control, the relative tumor inhibition rates of the Xtoro and mouse PD-1 antibody single administration groups were 26% and 39%, respectively, while the combined use of the two drugs significantly increased the relative tumor inhibition rate to 51%. The results showed that the combination of Xtoro and immune checkpoint inhibitor anti-PD-1 antibody had good synergistic anti-tumor activity in vivo in the mouse tumor model.

[0103] Table 2. Pharmacodynamic analysis table of each group in the mouse H22 hepatocarcinoma subcutaneous transplantation model

[0104]

[0105]

[0106] Note:

[0107] a. Data are expressed as "mean ± standard error".

[0108] b. T / C% = T RTV / C RTV x 100%; TGI% = (1 - T / C) x 100%; (T RTV : average RTV of treatment group; C RTV : average RTV of control group; RTV = V t / V0, V0 is the tumor volume of the animal at grouping, V t is the tumor volume of the animal after treatment)

[0109] c. The significant difference of relative tumor volume between control group and each treatment group was compared by one-way ANOVA and Tukey HSD test. P value less than 0.05 was considered as significant difference.

[0110] Body weight was measured 3 times per week, and the body weight change after administration of the treatment group and the control group was shown in Figure 5 、 Figure 6 and Table 3. As can be seen from Figure 5 、 Figure 6 and Table 3, except that 1 mouse in the 5 mg / kg mouse PD-1 antibody treatment group died, the mice in the other groups did not have significant body weight loss during the experiment, and no obvious drug toxicity was observed, and the mice tolerated well during the treatment.

[0111] Table 3. Body weight change of each group at the end of the experiment in the mouse liver cancer H22 subcutaneous transplantation model

[0112]

[0113] Note: a. Data are expressed as "mean ± standard error".

[0114] Example Three Pharmacodynamic experiment based on B-hPD-1 / hPD-L1 humanized mouse MC38-hPD-L1 colon cancer animal model

[0115] Experimental materials:

[0116] Mouse colon cancer MC38 cells were purchased from Shunran Shanghai Biotechnology Co., Ltd. Cells were cultured at 37°C in a 5% CO2 incubator in Dulbecco's Modified Eagle's Medium containing 10% inactivated fetal bovine serum. Beijing Biocytogen Gene Biotechnology Co., Ltd. genetically modified MC38 cells to overexpress human PD-L1 while simultaneously knocking out mouse PD-L1; these cells were named MC38-hPD-L1 cells. The reference PD-L1 antibody used in the experiment was constructed and produced in the in-house based on the sequence of the marketed antibody drug MPDL3280A.

[0117] Experimental methods:

[0118] Cell Culture and Tumor Inoculation

[0119] After the cells reached a certain number, they were collected, and the MC38-hPD-L1 cells resuspended in PBS were cultured at 5 × 10⁻⁶ cells / mL. 5 A concentration of 0.1 mL / mole was administered subcutaneously to the right side of B-hPD-1 / hPD-L1 humanized mice. The tumor volume was 0.1 mL / mouse. When the average tumor volume reached approximately 104 mm², the inoculation was completed. 3 Mice were selected based on tumor volume and body weight and randomly assigned to 6 experimental groups, with 8 mice in each group. Drug administration began on the day of group assignment. Specific administration protocols are shown in Table 4 below.

[0120] Table 4. Dosing Regimen

[0121]

[0122] Note a: The administration volume is calculated based on the experimental animal's body weight at 10 μL / g.

[0123] b: QD means once a day, Q3D means once every three days.

[0124] Experimental results:

[0125] (1) Response and weight changes in experimental animals after drug administration

[0126] During the experiment, the experimental animals maintained good activity and appetite during the administration period, and their weight increased to a certain extent, indicating that the animals tolerated the test product well. The weight changes of all animals are shown in Table 5.

[0127] Table 5. Effects of the test product on the body weight of B-hPD-1 / hPD-L1 humanized mice transplanted with MC38-hPD-L1 colon cancer cells.

[0128]

[0129] Note: a: Mean ± standard error;

[0130] b: Statistical comparison of the body weight of the administration group and the control group on the 27th day of administration grouping, T-test analysis.

[0131] (2) Tumor volume inhibition results

[0132] The results were recorded and the tumor inhibition rate (TGITV) was calculated. Statistical analysis between groups was performed according to the tumor volume, and the results were entered into Table 6 and Table 7.

[0133] At the end of the experiment (i.e., on the 27th day of administration grouping), compared with the control group, the Xiaroni (2.5 mg / kg) group, the reference PD-L1 antibody (1 mg / kg, 3 mg / kg) group, the Xiaroni (2.5 mg / kg) combined with the reference PD-L1 antibody (1 mg / kg) group, and the Xiaroni (2.5 mg / kg) combined with the reference PD-L1 antibody (3 mg / kg) group had a significant inhibitory effect on the growth of MC38-hPD-L1 tumor subcutaneous transplanted tumors at the tested dose (P < 0.05).

[0134] Table 6. Effect of the test product on the tumor volume of MC38-hPD-L1 colon cancer cell transplanted B-hPD-1 / hPD-L1 humanized mice

[0135]

[0136] Note: a: mean ± standard error;

[0137] b: Statistical comparison of the tumor volume of the administration group and the control group on the 27th day of administration grouping, T-test analysis, *P < 0.05, **P < 0.01.

[0138] Table 7. Comparison of the effect of different administration combinations on the tumor volume of MC38-hPD-L1 colon cancer cell transplanted B-hPD-1 / hPD-L1 humanized mice

[0139]

[0140] Note: Statistical comparison of the tumor volume of the administration group and the control group on the 27th day of administration grouping, T-test analysis, *P < 0.05, **P < 0.01.

[0141] (3) Tumor weight inhibition results

[0142] The experiment was ended on the 27th day of administration grouping. The tumor weight results of each group are summarized in Table 8.

[0143] After data analysis, at the end of the experiment, compared with the control group, Xi'elunai (2.5 mg / kg), reference PD-L1 antibody (1 mg / kg, 3 mg / kg), Xi'elunai (2.5 mg / kg) combined with reference PD-L1 antibody (1 mg / kg), Xi'elunai (2.5 mg / kg) combined with reference PD-L1 antibody (3 mg / kg) had significant inhibitory effect on the growth of MC38-hPD-L1 tumor subcutaneous transplanted tumor at the tested dose (P<0.05).

[0144] Table 8. Inhibitory effect of test products on tumor weight of MC38-hPD-L1 colon cancer cell transplanted B-hPD-1 / hPD-L1 humanized mice

[0145]

[0146] Note: a: mean ± standard error.

[0147] b: the tumor weight of the test drug group at the end of the experiment and the control group was statistically compared, T-test analysis, *P<0.05, **P<0.01.

[0148] Summary of the experiment:

[0149] In this experiment, Xi'elunai (2.5 mg / kg), reference PD-L1 antibody (1 mg / kg, 3 mg / kg), Xi'elunai (2.5 mg / kg) combined with reference PD-L1 antibody (1 mg / kg), Xi'elunai (2.5 mg / kg) combined with reference PD-L1 antibody (3 mg / kg) had significant inhibitory effect on the growth of MC38-hPD-L1 tumor subcutaneous transplanted tumor at the tested dose, and did not produce toxic side effects to animals when they worked, and had good safety.

[0150] The above provides a detailed introduction to the application of the combination of sirolimus and immune checkpoint inhibitors in anti-tumor treatment, the principles and implementation manners of the present application are described in this paper by using specific examples, the above example description is only used to help understand the method of the present application and its core idea, including the best mode, and also enables any person skilled in the art to practice the present application, including manufacturing and using any device or system, and implementing any combined method. It should be pointed out that for ordinary skilled in the art, without departing from the principles of the present application, some improvements and modifications can be made to the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application. The scope of the patent protection of the present application is limited by the claims, and can include other embodiments that can be thought by those skilled in the art. If these other embodiments have structural elements that are not different from the language expression of the claims, or if they include equivalent structural elements that are not substantially different from the language expression of the claims, then these other embodiments should also be included in the scope of the claims.

Claims

1. Use of siolimab or a pharmaceutically acceptable salt thereof in combination with a PD-1 inhibitor / PD-L1 inhibitor for the manufacture of a medicament for treating a tumor; the unit formulation dosage of siolimab or a pharmaceutically acceptable salt thereof is 5-80 mg; the unit formulation dosage of the PD-1 inhibitor / PD-L1 inhibitor is 1-1000 mg; the tumor is selected from colon cancer.

2. The use according to claim 1, wherein the unit formulation dosage of siolimab or a pharmaceutically acceptable salt thereof is 10-50 mg.

3. The use according to claim 1, wherein the PD-1 inhibitor is selected from Nivolumab, Pembrolizumab, Toripalimab, Sintilimab, SHR-1210, BGB-A317, Genolimzumab, Zimberelimab, AK101, AK104, AK105, GLS-010, BAT1306, CS1003, PDR001, Cemiplimab and MEDI0680.

4. The use according to claim 1, wherein the PD-L1 inhibitor is selected from Tecentriq, Imfinzi, Bavencio, CS1001, TQB2450, SHR1316, lazertinib, bintrafusp alfa, Envafolimab, CA-170, CX-072, BGB-A333, BMS-936559, GEN-1046, KL-A167 and IO-103.

5. The use according to claim 1, wherein the unit formulation dosage of the PD-1 inhibitor / PD-L1 inhibitor is 10-800 mg.

6. The use according to claim 5, wherein the unit formulation dosage of the PD-1 inhibitor / PD-L1 inhibitor is 20-500 mg.

7. A pharmaceutical composition for treating colon cancer, comprising a combination of siolimab or a pharmaceutically acceptable salt thereof and a PD-1 inhibitor / PD-L1 inhibitor as described in any one of claims 1-6.

8. A kit for treating colon cancer, comprising a combination of siolimab or a pharmaceutically acceptable salt thereof and a PD-1 inhibitor / PD-L1 inhibitor as described in any one of claims 1-6.

9. The kit according to claim 8, wherein the siolimab or a pharmaceutically acceptable salt thereof and the PD-1 inhibitor / PD-L1 inhibitor are unit formulations with the same or different specifications.

10. The kit according to any one of claims 8-9, wherein the siolimab or a pharmaceutically acceptable salt thereof and the PD-1 inhibitor / PD-L1 inhibitor are placed in the same container or in different containers, respectively.

11. The kit according to claim 10, wherein the siolimab or a pharmaceutically acceptable salt thereof is in a gastrointestinal administration dosage form; and the PD-1 inhibitor / PD-L1 inhibitor is in a parenteral administration dosage form.

Images