Combination therapy of CTLA-4 inhibitors and nonsteroidal anti-inflammatory drugs for cancer treatment

Abstract

The present invention provides a combined anticancer treatment method of a CTLA-4 inhibitor and a non-steroidal anti-inflammatory drug. Specifically, the present invention provides a product combination, including: (i) a first pharmaceutical composition, the first pharmaceutical composition containing (a) a first active ingredient, the first active ingredient is a CTLA-4 inhibitor, and a pharmaceutically acceptable carrier; and (ii) a second pharmaceutical composition, the second pharmaceutical composition containing (b) a second active ingredient, the second active ingredient is a non-steroidal anti-inflammatory drug, and a pharmaceutically acceptable carrier; wherein the first pharmaceutical composition and the second pharmaceutical composition are different pharmaceutical compositions, or the same pharmaceutical composition. The product combination of the present invention can synergistically treat tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to a combined anti-cancer treatment method of a CTLA-4 inhibitor and a non-steroidal anti-inflammatory drug. Background Art

[0002] Recombinant anti-CTLA-4 fully human monoclonal antibody, which binds with high affinity to CTLA-4 on the T lymphocyte membrane, blocks the binding of CTLA-4 to B7-1 / B7-2, enables B7-1 / B7-2 to bind to the T cell co-stimulatory molecule signal molecule CD28, enhances and maintains the activation state of the anti-tumor lymphocyte co-stimulatory signal, promotes the activation, proliferation and infiltration of lymphocytes into tumor tissues, and ultimately kills tumor cells.

[0003] Although immune checkpoint inhibitors have made some breakthroughs in multiple cancer types, the efficacy of single-drug immunotherapy needs to be improved. Although the combination of CTLA4 inhibitors and PD1 inhibitors has significantly improved the remission rate and survival prognosis of patients, the toxicity of the combined drug has also increased significantly. At present, the clinical search is still underway for drugs that can assist immunotherapy and further improve the efficacy without increasing serious adverse reactions.

[0004] NSAIDs, represented by ibuprofen and aspirin, can reduce the production of prostaglandin E2 (PGE2) by inhibiting the cyclooxygenase (COX) pathway. It was previously believed that the COX2 pathway was related to tissue congestion, edema, and peripheral nerve pain.

[0005] There is an urgent need in this field to explore more effective and safer combination drugs that work in synergy with immunotherapy. Summary of the invention

[0006] The purpose of the present invention is to develop a more effective and safer combination of drugs that cooperates with immunotherapy. The purpose of the present invention is to provide a combination drug for treating tumors to improve the therapeutic effect of anti-CTLA-4 antibodies alone.

[0007] A first aspect of the present invention provides a product combination, comprising:

[0008] (i) a first pharmaceutical composition, the first pharmaceutical composition comprising (a) a first active ingredient, the first active ingredient being a CTLA-4 inhibitor, and a pharmaceutically acceptable carrier; and

[0009] (ii) a second pharmaceutical composition, the second pharmaceutical composition comprising (b) a second active ingredient, the second active ingredient being a nonsteroidal anti-inflammatory drug, and a pharmaceutically acceptable carrier;

[0010] Wherein, the first pharmaceutical composition and the second pharmaceutical composition are different pharmaceutical compositions, or the same pharmaceutical composition.

[0011] In another preferred embodiment, the inhibitor is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, or a combination thereof.

[0012] In another preferred embodiment, the antibodies include KD6001, Ipilimumab, Tremelimmab, Zalivrelimab, Quavonlimab, REGN4659, and YH001.

[0013] In another preferred embodiment, the antibody sequence of KD6001 is as shown in PCT / CN2015 / 095072.

[0014] In another preferred embodiment, the nonsteroidal anti-inflammatory drug is a nonsteroidal anti-inflammatory drug (NSAIDs).

[0015] In another preferred embodiment, the non-steroidal anti-inflammatory drug is selected from the following group: salicylic acid drugs, aniline drugs, acetic acid drugs, phenylacetic acid drugs, propionic acid drugs, oxicams drugs, coxibs drugs, or a combination thereof.

[0016] In another preferred embodiment, the salicylic acid drugs include aspirin and benolate.

[0017] In another preferred embodiment, the aniline drugs include acetaminophen and nimesulide.

[0018] In another preferred embodiment, the acetic acid drug includes indomethacin.

[0019] In another preferred embodiment, the phenylacetic acid drugs include diclofenac and sulindac.

[0020] In another preferred embodiment, the propionic acid drugs include ibuprofen, naproxen, and loxoprofen.

[0021] In another preferred embodiment, the oxicams include piroxicam and meloxicam.

[0022] In another preferred embodiment, the coxib drugs include celecoxib, valdecoxib, rofecoxib, etoricoxib, nimesulide, and parecoxib.

[0023] In another preferred embodiment, the weight ratio of component (i) to component (ii) is 1:2-500, preferably, 1:2-200, more preferably, 1:2-100, more preferably, 1:2-50, more preferably, 1:2-10, more preferably, 1:2-6.

[0024] In another preferred embodiment, in the product combination, the content of the CTLA-4 inhibitor is 1%-99%, preferably, 1%-60%, and more preferably, 1%-30%.

[0025] In another preferred embodiment, in the product combination, the content of the non-steroidal anti-inflammatory drug is 1%-99%, preferably, 10%-90%, and more preferably, 50%-90%.

[0026] In another preferred embodiment, in the product combination, the component (i) and the component (ii) account for 0.01-99.99 wt %, preferably 0.1-90 wt %, and more preferably 1-80 wt % of the total weight of the product combination.

[0027] In another preferred embodiment, the dosage form of the pharmaceutical composition includes an injection form and an oral dosage form.

[0028] In another preferred embodiment, the oral dosage form includes tablets, capsules, films, and granules.

[0029] In another preferred embodiment, the dosage form of the pharmaceutical composition includes a sustained-release dosage form and a non-sustained-release dosage form.

[0030] A second aspect of the present invention provides a composition comprising:

[0031] (i) CTLA-4 inhibitors;

[0032] (ii) nonsteroidal anti-inflammatory drugs; and

[0033] (iii) a pharmaceutically acceptable carrier.

[0034] In another preferred embodiment, in the composition, the component (i) and the component (ii) account for 0.01-99.99 wt %, preferably 0.1-90 wt %, and more preferably 1-80 wt % of the total weight of the medicine kit.

[0035] In another preferred embodiment, the composition also includes other drugs for treating tumors.

[0036] In another preferred embodiment, the tumor comprises a solid tumor.

[0037] In another preferred embodiment, the tumor is selected from the following group: colorectal cancer, non-small cell lung cancer, small cell lung cancer, head and neck squamous cell carcinoma, liver cancer, cervical cancer, B-cell lymphoma, multiple myeloma, non-Hodgkin's lymphoma, melanoma, ovarian cancer, breast cancer, prostate cancer, pleural mesothelioma, renal cancer, urothelial carcinoma, nasopharyngeal carcinoma, endometrial cancer, glioma, gastric cancer, esophageal cancer, esophageal-gastric junction cancer, thyroid cancer, pancreatic cancer, bile duct cancer, primary brain cancer, or a combination thereof.

[0038] In another preferred embodiment, the other drugs for treating tumors are selected from the following group: immune checkpoint inhibitors, immune agonists, or a combination thereof.

[0039] In another preferred embodiment, the immune checkpoint inhibitor drugs include PD-1, 4-1BB, IDO (IDO2 and TDO), LAG-3, OX40, GITR, CD27, CD30, TIM-3, CD3, ICOS, CD73 / CD39, CD2 / SLAM family, PD-L1, CD47, TIGIT, CD73, CD33, CEACAM1 / 5 / 6, STING, WNT / Beta catenin, B7-H3, and VISITA.

[0040] In another preferred embodiment, the immune agonists include CD40, CSF1R, TLR family and its receptor IFN, IL factors and their receptors such as IL-2, IL-15, IL-21, GMCSF / GMCSFR, RIG-1, TNF family, CD25, chemokines CCR and CXC, NKG2D.

[0041] The third aspect of the present invention provides a medicine kit, comprising:

[0042] (a1) a first container, and a CTLA-4 inhibitor or a drug containing a CTLA-4 inhibitor in the first container;

[0043] (b1) a second container, and a nonsteroidal anti-inflammatory drug, or a medicament containing a nonsteroidal anti-inflammatory drug, in the second container.

[0044] In another preferred embodiment, the first container and the second container are the same or different containers.

[0045] In another preferred embodiment, the drug in the first container is a single-ingredient preparation containing a CTLA-4 inhibitor.

[0046] In another preferred embodiment, the medicine in the second container is a single-ingredient preparation containing non-steroidal anti-inflammatory drugs.

[0047] In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

[0048] In another preferred embodiment, the kit further contains instructions.

[0049] In another preferred embodiment, the description records the following description:

[0050] (a) A method for synergistically treating tumors by combining a CTLA-4 inhibitor and a nonsteroidal anti-inflammatory drug.

[0051] The fourth aspect of the present invention provides a use of a combination, wherein the combination includes a CTLA-4 inhibitor and a non-steroidal anti-inflammatory drug, for preparing a pharmaceutical composition or a kit, wherein the pharmaceutical composition or the kit is used to treat tumors.

[0052] In another preferred embodiment, the tumor is selected from the following group: colorectal cancer, non-small cell lung cancer, small cell lung cancer, head and neck squamous cell carcinoma, liver cancer, cervical cancer, B-cell lymphoma, multiple myeloma, non-Hodgkin's lymphoma, melanoma, ovarian cancer, breast cancer, prostate cancer, pleural mesothelioma, renal cancer, urothelial carcinoma, nasopharyngeal carcinoma, endometrial cancer, glioma, gastric cancer, esophageal cancer, esophageal-gastric junction cancer, thyroid cancer, pancreatic cancer, bile duct cancer, primary brain cancer, or a combination thereof.

[0053] In another preferred embodiment, the effective concentration of the CTLA-4 inhibitor is 0.1-30 mg / kg, preferably, 0.2-20 mg / kg, preferably, 0.3-10 mg / kg.

[0054] In another preferred embodiment, the effective concentration of the non-steroidal anti-inflammatory drug is 0.1-100 mg / kg, preferably 0.3-30 mg / kg, preferably 1-15 mg / kg.

[0055] In another preferred embodiment, the pharmaceutical composition or kit comprises (a) a CTLA-4 inhibitor and a non-steroidal anti-inflammatory drug; and (b) a pharmaceutically acceptable carrier.

[0056] In another preferred embodiment, in the pharmaceutical composition or kit, the CTLA-4 inhibitor and non-steroidal anti-inflammatory drug account for 0.01-99.99wt%, preferably 0.1-90wt%, and more preferably 1-80wt% of the total weight of the pharmaceutical composition or kit.

[0057] In another preferred embodiment, the pharmaceutical composition or drug kit further comprises other drugs for treating tumors.

[0058] In another preferred embodiment, the other drugs for treating tumors are selected from the following group: immune checkpoint inhibitors, immune agonists, or a combination thereof.

[0059] The fifth invention of the present invention provides a method for treating tumors, comprising:

[0060] Administer a CTLA-4 inhibitor and a nonsteroidal anti-inflammatory drug, or the product combination of the first aspect of the present invention, the composition of the second aspect of the present invention, or the kit of parts of the third aspect of the present invention to a subject in need thereof.

[0061] In another preferred embodiment, the subject includes a human or non-human mammal suffering from a tumor.

[0062] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0063] In another preferred embodiment, the administration dosage of the CTLA-4 inhibitor is 0.1-30 mg / kg, preferably, 0.2-20 mg / kg, and more preferably, 0.3-10 mg / kg.

[0064] In another preferred embodiment, the dosage of the nonsteroidal anti-inflammatory drug is 0.1-100 mg / kg, preferably 0.3-30 mg / kg, more preferably 1-15 mg / kg.

[0065] In another preferred embodiment, the CTLA-4 inhibitor is administered once a week, once every two weeks, once every three weeks, once every 6 weeks, once every 12 weeks, or once every 24 weeks, preferably, once every 2-6 weeks.

[0066] In another preferred embodiment, the CTLA-4 inhibitor is administered for 1-4000 days, preferably 1-1000 days, and most preferably 1-365 days.

[0067] In another preferred embodiment, the nonsteroidal anti-inflammatory drug is administered 1-6 times a day, preferably 2-4 times a day.

[0068] In another preferred embodiment, the administration time of the nonsteroidal anti-inflammatory drug is 1-4000 days, preferably 1-1000 days, and most preferably 1-365 days.

[0069] In another preferred embodiment, the CTLA-4 inhibitor and the non-steroidal anti-inflammatory drug are administered simultaneously or sequentially.

[0070] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 The effect of KD6001 combined with ibuprofen on the body weight of MC38 tumor-bearing mice is shown. After colon cancer cells MC38 were implanted subcutaneously in mice, KD6001 or PBS was given by intraperitoneal injection 4 times on D8, D11, D15, and D19, and ibuprofen was given by gavage twice a day from D8 to D17. The mice were weighed twice a week. The weight values ​​of mice in each group are presented in the form of "arithmetic mean ± standard error". The individual weight data of mice at each time point were analyzed for statistical differences between groups, and the low, medium, and high dose groups were compared with the control group by one-way analysis of variance, and the P values ​​of each dose group were greater than 0.05.

[0072] Figure 2 The effect of KD6001 combined with ibuprofen on the tumor volume of MC38 tumor-bearing mice is shown. After colon cancer cells MC38 were implanted into mice subcutaneously, KD6001 or PBS was intraperitoneally injected 4 times on D8, D11, D15, and D19, and ibuprofen was gavaged twice a day from D8 to D17. The tumor volume was measured twice a week. The experiment ended on D26. A: Average tumor volume change curve of mice in each group; B: Tumor volume and weight at the end of the experiment; C: Mice were killed after the experiment, and the tumor was removed and photographed. The data of individual tumor volume of mice at the end of each experiment (D26) were analyzed for statistical differences between groups. Each drug-treated group was compared with the control group by T-test analysis. The tumor volume of mice in the KD6001 50μg + ibuprofen 200μg group and the KD6001 50μg + ibuprofen 60μg group was statistically significantly different from that of the KD6001 50μg monotherapy group. *p<0.05. DETAILED DESCRIPTION

[0073] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that the combination of the CTLA-4 inhibitor of the present invention and the non-steroidal anti-inflammatory drug can effectively treat tumors and has a synergistic effect. In addition, it can also effectively improve the anti-tumor effect of the anti-CTLA-4 antibody alone. On this basis, the inventors completed the present invention.

[0074] As used herein, Quavonlimab was developed by Merck & Co., and the sequence information is disclosed by PCT publication WO2020185722.

[0075] As used herein, REGN4659 was developed by Regeneron Pharmaceuticals and the sequence information is disclosed by PCT publication WO2019023482.

[0076] As used herein, YH001 was developed by Youhe Pharmaceuticals, and the sequence information was disclosed by Chinese application CN111406069.

[0077] CTLA-4 inhibitors

[0078] CTLA-4 is an immune checkpoint receptor on T lymphocytes, which inhibits the over-activation of T cells by binding to B7-1 (CD80) and B7-2 (CD86) on antigen-presenting cells. CTLA-4 inhibitors, such as CTLA-4 antibodies, can specifically bind to CTLA-4 on T lymphocytes, block the binding of CTLA-4 to B7 molecules, antagonize the inhibitory effect of CTLA-4 on T cell activation, thereby allowing the T lymphocyte co-stimulatory factor CD28 to bind to the B7 molecules on antigen-presenting cells, continuously activating T lymphocytes, and thus exerting their anti-tumor effects.

[0079] In the present invention, the CTLA-4 inhibitor is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, or a combination thereof.

[0080] In some embodiments, the CTLA-4 inhibitor is a CTLA-4 antibody, including KD6001, Ipilimumab, Tremelimmab, Zalivrelimab, Quavonlimab, REGN4659, YH001, preferably KD6001, whose antibody sequence is as shown in PCT / CN2015 / 095072 and Table 1 of the embodiments of the present invention.

[0081] Nonsteroidal anti-inflammatory drugs

[0082] Non-steroidal anti-inflammatory drugs, also known as non-steroidal anti-inflammatory drugs (NSAIDs), are a class of drugs with antipyretic, analgesic, and most of them also have anti-inflammatory and anti-rheumatic effects. They are the first choice for some rheumatic diseases, such as early rheumatoid arthritis, senile arthritis, and early ankylosing spondylitis. Studies have shown that cyclooxygenase (COX) plays an important role in the occurrence, development, and prognosis of tumors. Non-steroidal anti-inflammatory drugs can promote cancer cell apoptosis and inhibit tumor angiogenesis by inhibiting the synthesis of COX. Therefore, it can not only be used for chemotherapy of tumors, but also very likely to be used in clinical treatment.

[0083] In a preferred embodiment, the non-steroidal anti-inflammatory drug is selected from the group consisting of salicylates, anilines, acetics, phenylacetic acids, propionic acids, oxicams, coxibs, or combinations thereof.

[0084] Among them, salicylic acid drugs include: aspirin, benolate; aniline drugs include: acetaminophen, nimesulide; acetic acid drugs include: indomethacin; phenylacetic acid drugs include: diclofenac, sulindac;

[0085] Propionic acid drugs include: ibuprofen, naproxen, and loxoprofen; oxicams include: piroxicam and meloxicam.

[0086] Coxib drugs include: celecoxib, valdecoxib, rofecoxib, etoricoxib, nimesulide, and parecoxib.

[0087] The research of the present invention shows that the combined use of the non-steroidal anti-inflammatory drug of the present invention and CTLA-4 inhibitors, such as CTLA-4 antibodies, in the treatment of tumors can play a synergistic role and effectively improve the anti-tumor effect of anti-CTLA-4 antibodies alone. The combination of non-steroidal anti-inflammatory drugs and CTLA-4 inhibitors, such as CTLA-4 antibodies, has excellent therapeutic effect in the treatment of tumors and good prospects for clinical application.

[0088] Compositions and methods of administration

[0089] As used herein, the term "composition" includes (a1) a first active ingredient, which is a CTLA-4 inhibitor; (a2) a second active ingredient, which is a non-steroidal anti-inflammatory drug; and (b) a pharmaceutically acceptable carrier. In addition, the composition includes a pharmaceutical composition.

[0090] Typically, the active ingredients of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable carrier medium. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

[0091] The present invention also provides a pharmaceutical composition containing a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram-10 milligrams per kilogram of body weight per day. Preferably, the dosage of the EP4 receptor antagonist can be: 0.1 to 2000 mg per day for adults, preferably 1 to 300 mg / day. The dosage of the PD-1 inhibitor can be: 0.1 to 2000 mg every two weeks for adults, preferably 1 to 300 mg / two weeks. As a drug for synergistic tumor therapy, oral and non-oral preparations can be prepared. Oral administration can be made into common dosage forms such as tablets, powders, granules, capsules, etc. The excipients used can be one or more of starch, lactose, sucrose, mannose, hydroxymethyl cellulose, etc. The disintegrant can be one or more of potato starch, hydroxymethyl cellulose, etc. The binder can be one or more of gum arabic, corn starch, gelatin, dextrin, etc. In addition to the above dosage forms, oral preparations can also be made into emulsions, syrups, etc.

[0092] Non-oral preparations can be made into injections, which can be made into injections with water for injection, physiological saline, glucose water, or by adding a certain proportion of ethanol, propanol, ethylene glycol, etc. In addition, they can also be made into common dosage forms such as nasal drops, sprays, rectal suppositories, and rectal retention enema solutions.

[0093] In addition, the active ingredients of the present invention are particularly suitable for use in combination with other drugs for treating tumors (such as immune checkpoint inhibitors, immune agonists), so as to more effectively inhibit tumors.

[0094] Pill Box

[0095] The present invention also provides a medicine box, which contains:

[0096] (a1) a first container, and a CTLA-4 inhibitor or a drug containing a CTLA-4 inhibitor in the first container;

[0097] (b1) a second container, and a nonsteroidal anti-inflammatory drug, or a medicament containing a nonsteroidal anti-inflammatory drug, in the second container.

[0098] In a preferred embodiment, the first container and the second container are the same or different containers.

[0099] The formulation containing the CTLA-4 inhibitor may be a unit dosage form containing the CTLA-4 inhibitor, and the formulation containing the nonsteroidal anti-inflammatory drug may be a unit dosage form containing the nonsteroidal anti-inflammatory drug.

[0100] As used herein, the term "unit dosage form" means that for the convenience of taking, the composition is prepared into a dosage form required for a single dose, including but not limited to various solid doses (such as tablets), liquid doses, capsules, and sustained-release preparations.

[0101] In another preferred embodiment, the instructions record the following method of use:

[0102] (I) administering to a subject in need thereof a formulation comprising a CTLA-4 inhibitor and a formulation comprising a nonsteroidal anti-inflammatory drug simultaneously; and optionally (II) repeating steps (I)-(II).

[0103] The preparation of the present invention can be taken three times a day to once every twenty days, or once every ten days in a sustained release manner. The preferred mode is to take it once a day, because it is convenient for patients to adhere to it, thereby significantly improving the compliance of patients to take the medicine.

[0104] When taking the drug, the total daily dose in most cases should be lower than (or equal to or slightly higher than) the daily dose of each single drug in a few cases. Of course, the effective dose of the active ingredient used may vary depending on the mode of administration and the severity of the disease to be treated.

[0105] The main advantages of the present invention include:

[0106] (1) The present invention first discovered that the combination of the CTLA-4 inhibitor of the present invention and a non-steroidal anti-inflammatory drug can effectively treat tumors and has a synergistic effect. In addition, it can also effectively improve the anti-tumor effect of the anti-CTLA-4 antibody alone.

[0107] (2) The present invention discovers for the first time that the combined use of the nonsteroidal anti-inflammatory drugs (such as ibuprofen) of the present invention and CTLA-4 inhibitors (such as anti-CTLA-4 monoclonal antibodies) in the treatment of tumors can play a synergistic role and effectively improve the anti-tumor effect of the anti-CTLA-4 antibody alone. The combination of nonsteroidal anti-inflammatory drugs (such as ibuprofen) and CTLA-4 inhibitors (such as anti-CTLA-4 monoclonal antibodies) has excellent therapeutic effects and good prospects for clinical application.

[0108] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0109] Unless otherwise specified, the materials and reagents in the specification of the present invention are commercially available products.

[0110] Example 1 In vivo pharmacodynamic study of combined therapy (anti-CTLA-4 antibody and non-steroidal anti-inflammatory drug ibuprofen) on mouse solid cancer (taking colon cancer cell MC38 as an example)

[0111] 1. Materials:

[0112] Drug: Anti-CTLA-4 antibody: KD6001 injection, Shanghai Saijin Biopharmaceutical Co., Ltd., batch number 20180201; its sequence is shown in PCT / CN2015 / 095072. The detailed information of the sequence is shown in Table 1.

[0113] Table 1 Sequence information of KD6001

[0114]

[0115] Ibuprofen suspension: Shanghai Johnson & Johnson Pharmaceutical Co., Ltd., specification 100ml: 2g / bottle, batch number 190121046

[0116] Cell line: Colon cancer cell MC38, Shanghai Model Organisms Technology Co., Ltd.

[0117] Animals: Human CTLA-4 gene knock-in mice

[0118] 2. Experimental Methods

[0119] 2.1 Preparation of drug delivery preparations:

[0120] Prepare 0.25 mg / ml CTLA-4 antibody (KD6001) injection solution with sterile PBS

[0121] Prepare 0.1 mg / ml, 0.3 mg / ml and 1 mg / ml ibuprofen suspensions with sterile water for injection

[0122] 2.2 MC38 cell culture:

[0123] Mouse colon cancer cell MC38 was cultured in DMEM medium containing 10% FBS or DMEM / F12 medium containing 10% FBS and passaged twice a week. On the day of inoculation, the cells were treated with trypsin and collected in a 50 ml centrifuge tube. The cells were centrifuged and precipitated, the supernatant was discarded, and the cells were washed twice with PBS. Finally, the cells were suspended in PBS at a cell density of 5×10 6 cells / ml.

[0124] 2.3 Subcutaneous transplantation of mouse colon cancer cells MC38:

[0125] The collected cells were placed on ice and brought to the mouse breeding facility. Each mouse was subcutaneously injected with 0.1 ml of cell suspension containing 5×10 5 cells.

[0126] 2.4 Drug administration to mice

[0127] The mice were administered by intraperitoneal injection, and the administration volume for each mouse was 0.2 ml. The drug was administered 4 times in total at D8, D12, D15, and D19. From D8 to D19, ibuprofen was administered by gavage, and the administration volume was 0.2 ml, twice a day. The weight of the mice and the tumor volume were measured twice a week (the weight and tumor volume were measured before administration during the administration period), (tumor volume V (mm3) = 1 / 2 × tumor long diameter a (mm) × tumor short diameter b2 (mm)). The specific experimental groups are shown in Table 2.

[0128] Table 2 Dosage grouping table

[0129]

[0130]

[0131] 2.5 Statistical analysis

[0132] The mouse body weight data, tumor volume, and tumor weight data of the experimental group and the control group were expressed in the form of (arithmetic mean ± standard error). T-test analysis was performed on the data between the groups to determine whether there were significant differences between the data between the groups.

[0133] 3. Experimental results

[0134] 3.1 Mouse weight monitoring results

[0135] The average weight changes of mice in each group are shown in Figure 1. The individual body weight data of mice at each time point were analyzed for statistical differences among the groups, and each group was compared with the control group by one-way analysis of variance, and the P value of each dose group was greater than 0.05. Under the conditions of this experiment, the weight of MC38 tumor-bearing mice was not affected by KD6001 2.5mg / kg + ibuprofen 10mg / kg, KD6001 2.5mg / kg + ibuprofen 3mg / kg, and KD6001 2.5mg / kg + ibuprofen 1mg / kg in the high, medium and low dose groups of the drug combination. The experimental results show that under the conditions of this experiment, the combination of KD6001 and ibuprofen has no obvious toxicity to mice.

[0136] 3.2 Results of mouse tumor volume monitoring

[0137] Figure 2 A shows the changes in the average tumor volume of each group of mice during the experiment. Starting from D15 (before the third administration), the average tumor volume of mice in the three combined administration groups began to show a significant decrease compared to the average tumor volume of mice in other groups, especially significantly decrease compared to the control group. At D26 (end of the experiment), the average tumor volume of mice in the control group was about 1908.6 mm 3 The average tumor volumes of mice in the KD6001 2.5 mg / kg, ibuprofen 10 mg / kg, ibuprofen 3 mg / kg, and ibuprofen 1 mg / kg groups were 1763.3 mm 3 、1189.6mm 3 、1975.8mm 3 、1344.2mm 3 The average tumor volumes of mice in the combined drug treatment groups of KD6001 2.5 mg / kg + ibuprofen 10 mg / kg, KD6001 2.5 mg / kg + ibuprofen 3 mg / kg, and KD6001 2.5 mg / kg + ibuprofen 1 mg / kg were 419.6 mm 3 、704.2mm 3 、872.8mm 3 ( Figure 2 B left). The weight of the tumors of mice in each group at the end of the experiment (D26) is shown in Figure 2 B (right). Compared with the tumor inhibition effect of KD6001 alone, the tumor inhibition effect of KD6001 combined with 10 mg / kg and combined with 3 mg / kg ibuprofen was significantly different (p values ​​were less than 0.01 and 0.05, respectively) ( Figure 2 B). Figure 2 C shows the photos of the tumors removed from the mice in each group.

[0138] The combination of different doses of ibuprofen and a fixed dose of KD6001 (KD6001 2.5 mg / kg) showed an additive effect in anti-tumor effects, and the additive effect increased with the increase in the dose of ibuprofen. The average tumor volume of the high, medium and low doses of ibuprofen combined with KD6001 was significantly smaller than that of the KD6001 monotherapy group.

[0139] The statistical difference analysis of tumor volume between the dose group and the control group was performed. The individual tumor volume data of mice in each dose group were analyzed by T-test. The results showed that the tumor volume of mice in the combination drug group was statistically significantly different from that in the PBS group (p<0.05). The individual tumor volume data of mice in the combination drug group and the KD6001 monotherapy group were analyzed by T-test. The results showed that there was also a statistically significant difference between the combination drug group and the monotherapy group (p<0.05).

[0140] Table 3 shows the results of relative tumor volume (RTV) and relative tumor proliferation rate (T / C%) of the experimental dose groups. From the perspective of relative tumor volume (RTV) and relative tumor proliferation rate (T / C%), at the end of the experiment (D26), the relative tumor inhibition rates (T / C) of mice in the KD6001 2.5 mg / kg group and the ibuprofen 10 mg / kg group treated with single drug were 112.3% and 68.1%, respectively; the T / C of mice in the KD6001 2.5 mg / kg + ibuprofen 10 mg / kg, KD6001 2.5 mg / kg + ibuprofen 3 mg / kg, and KD6001 2.5 mg / kg + ibuprofen 1 mg / kg groups treated with combined drug administration were 31.7%, 43.0%, and 54.6%, respectively, at D26. The anti-tumor effects of the three different doses of combined treatment were better than those of the single-drug treatment group, indicating that the combination of KD6001 and ibuprofen has a synergistic effect. The best effect was KD6001 2.5 mg / kg + ibuprofen 10 mg / kg, and the weight ratio of KD6001 to ibuprofen was 1:4.

[0141] Table 3 Relative tumor volume (RTV) and relative tumor proliferation rate (T / C)

[0142]

[0143]

[0144] V8 represents the average tumor volume on the 8th day after MC38 cells were inoculated into mice (D8, before the first administration), and Vt represents the average tumor volume at the indicated time. Ibuprofen: ibuprofen; RTV: relative tumor volume; T / C: tumor proliferation rate relative to the PBS group

[0145] 4. Experimental Conclusion

[0146] In this experiment, KD6001 was given to MC38 mice bearing colon cancer twice a week for a total of 4 times by combined intraperitoneal injection of KD6001 and oral administration of ibuprofen. Ibuprofen was administered twice a day for a total of 12 days. At the same time, a KD6001 and ibuprofen single-drug group and a PBS negative control group were set up to investigate whether the combination of KD6001 and ibuprofen has a synergistic effect in the anti-tumor effect of the drug combination in the mouse colon cancer MC38 model.

[0147] KD6001 alone at a dose of 2.5 mg / kg (intraperitoneal injection) had no effect on tumor growth (RTV was 112.3%), and the inhibitory effect was 0. The RTV of ibuprofen alone at a dose of 10 mg / kg was 68.1%, which had a partial inhibitory effect on tumor growth (about 30% inhibitory effect), while when KD6001 (at a dose of 2.5 mg / kg) and 10 mg / kg ibuprofen were used in combination, its RTV was 31.7%, and the inhibitory effect reached about 70%, indicating that KD6001 and ibuprofen had a synergistic effect in inhibiting tumor growth. When KD6001 (at a dose of 2.5 mg / kg) was combined with medium and low doses (for example, a dose of 1 mg / kg or 3 mg / kg) of ibuprofen, the inhibitory effect on tumor growth was also due to the use of ibuprofen alone or KD6001 alone, which also showed the synergistic effect of KD6001 and ibuprofen in inhibiting tumor growth.

[0148] In summary, under the experimental conditions, the combination of ibuprofen and KD6001 has a synergistic anti-tumor effect.

[0149] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Shanghai Saijin Biopharmaceutical Co., Ltd. <120> Combination therapy of CTLA-4 inhibitors and nonsteroidal anti-inflammatory drugs for cancer treatment <130> P2021-3269 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <400> 1 Ser Tyr Gly Met His 1 5 <210> 2 <211> 14 <212> PRT <213> Artificial sequence <400> 2 Val Ile Trp Tyr Asp Gly Ser Arg Gln Tyr Tyr Ala Asp Ser 1 5 10 <210> 3 <211> 9 <212> PRT <213> Artificial sequence <400> 3 Gly Gly Phe Trp Gly Ala Phe Asp Ile 1 5 <210> 4 <211> 12 <212> PRT <213> Artificial sequence <400> 4 Arg Ala Ser Gln His Val Ile Ser Ser Tyr Leu Ala 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <400> 5 Gly Ala Ser Ser Arg Asp Thr 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial sequence <400> 6 Gln Gln Tyr Gly Thr Ser Pro Trp Thr Phe 1 5 10 <210> 7 <211> 447 <212> PRT <213> artificial sequence <400> 7 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Arg Gln Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Phe Trp Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr 100 105 110 Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 8 <211> 215 <212> PRT <213> artificial sequence <400> 8 Asp Val Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Gly Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ile Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Val Tyr Gly Ala Ser Ser Arg Asp Thr Gly Val Ser Asp Arg Phe Thr 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Ser Ala Val Tyr Phe Cys Gln Gln Tyr Gly Thr Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215

Claims

1. A product combination for treating colorectal cancer, characterized in that: include: (i) a first pharmaceutical composition, the first pharmaceutical composition comprising (a) a first active ingredient, the first active ingredient being a CTLA-4 inhibitor, and a pharmaceutically acceptable carrier; and (ii) a second pharmaceutical composition, the second pharmaceutical composition comprising (b) a second active ingredient, the second active ingredient being a nonsteroidal anti-inflammatory drug, and a pharmaceutically acceptable carrier; Wherein, the first pharmaceutical composition and the second pharmaceutical composition are different pharmaceutical compositions, or the same pharmaceutical composition; The CTLA-4 inhibitor is a CTLA-4 antibody, the non-steroidal anti-inflammatory drug is a propionic acid drug, the propionic acid drug is ibuprofen, the antibody is KD6001, the amino acid sequence of VH CDR1 in the KD6001 antibody is shown in SEQ ID NO.1, the amino acid sequence of VH CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO.3; and the amino acid sequence of VL CDR1 is shown in SEQ ID NO.4, the amino acid sequence of VL CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of VL CDR3 is shown in SEQ ID NO.

6.

2. The product combination according to claim 1, characterized in that: The weight ratio of the component (i) to the component (ii) is 1:2-500.

3. The product combination according to claim 2, characterized in that: The weight ratio of the component (i) to the component (ii) is 1:2-200.

4. The product combination according to claim 3, characterized in that: The weight ratio of the component (i) to the component (ii) is 1:2-100.

5. The product combination according to claim 4, characterized in that: The weight ratio of the component (i) to the component (ii) is 1:2-50.

6. A composition for treating colorectal cancer, characterized in that: The composition comprises: (i) CTLA-4 inhibitors; (ii) nonsteroidal anti-inflammatory drugs; and (iii) a pharmaceutically acceptable carrier; The CTLA-4 inhibitor is a CTLA-4 antibody, the non-steroidal anti-inflammatory drug is a propionic acid drug, the propionic acid drug is ibuprofen, the antibody is KD6001, the amino acid sequence of VH CDR1 in the KD6001 antibody is shown in SEQ ID NO.1, the amino acid sequence of VH CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO.3; and the amino acid sequence of VL CDR1 is shown in SEQ ID NO.4, the amino acid sequence of VL CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of VL CDR3 is shown in SEQ ID NO.

6.

7. The composition according to claim 6, characterized in that The composition also includes other drugs for treating tumors.

8. The composition according to claim 7, characterized in that The other drugs for treating tumors are selected from the following group: immune checkpoint inhibitors, immune agonists, or a combination thereof.

9. A medicine kit for treating colorectal cancer, characterized in that: include: (a1) a first container, and a CTLA-4 inhibitor or a drug containing a CTLA-4 inhibitor in the first container; (b1) a second container, and a nonsteroidal anti-inflammatory drug, or a medicament containing a nonsteroidal anti-inflammatory drug, located in the second container; The CTLA-4 inhibitor is a CTLA-4 antibody, the non-steroidal anti-inflammatory drug is a propionic acid drug, the propionic acid drug is ibuprofen, the antibody is KD6001, the amino acid sequence of VH CDR1 in the KD6001 antibody is shown in SEQ ID NO.1, the amino acid sequence of VH CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO.3; and the amino acid sequence of VL CDR1 is shown in SEQ ID NO.4, the amino acid sequence of VL CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of VL CDR3 is shown in SEQ ID NO.

6.

10. A use of a combination, characterized in that, The combination comprises a CTLA-4 inhibitor and a non-steroidal anti-inflammatory drug, and is used to prepare a pharmaceutical composition or a drug kit, wherein the pharmaceutical composition or the drug kit is used to treat a tumor; The tumor is selected from the following group: colorectal cancer, the CTLA-4 inhibitor is a CTLA-4 antibody, the non-steroidal anti-inflammatory drug is a propionic acid drug, the propionic acid drug is ibuprofen, the antibody is KD6001, the amino acid sequence of VHCDR1 in the KD6001 antibody is shown in SEQ ID NO.1, the amino acid sequence of VH CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of VHCDR3 is shown in SEQ ID NO.3; and the amino acid sequence of VL CDR1 is shown in SEQ ID NO.4, the amino acid sequence of VLCDR2 is shown in SEQ ID NO.5, and the amino acid sequence of VL CDR3 is shown in SEQ ID NO.

6.

11. The use according to claim 10, characterized in that The effective concentration of the CTLA-4 inhibitor is 0.1-30 mg / kg.

12. The use according to claim 10, characterized in that The non-steroidal anti-inflammatory drug has an effective concentration of 0.1-100 mg / kg.

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