WO2023164847A1

WO2023164847A1 - Combinational anti-cancer therapy with ctla-4 inhibitor and non-steroidal anti-inflammatory drug

Info

Publication number: WO2023164847A1
Application number: PCT/CN2022/078881
Authority: WO
Inventors: 蔡则玲; 陈羿; 张驰
Original assignee: 上海赛金生物医药有限公司
Priority date: 2022-03-02
Filing date: 2022-03-02
Publication date: 2023-09-07

Abstract

The present invention provides a combinational anti-cancer therapy with a CTLA-4 inhibitor and a non-steroidal anti-inflammatory drug. Specifically, the present invention provides a product combination comprising: (i) a first pharmaceutical composition, wherein the first pharmaceutical composition comprises (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, wherein the second pharmaceutical composition comprises (b) a second active ingredient, the second active ingredient being 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

Anti-cancer combination therapy of CTLA-4 inhibitors and nonsteroidal anti-inflammatory drugs

technical field

The invention relates to the field of biomedicine, in particular to an anti-cancer combined treatment method of a CTLA-4 inhibitor and a non-steroidal anti-inflammatory drug.

Background technique

Recombinant anti-CTLA-4 fully human monoclonal antibody, which blocks the binding of CTLA-4 to B7-1/B7-2 by binding to CTLA-4 with high affinity on the T lymphocyte membrane, making B7-1/B7- 2 It can combine with T cell co-stimulatory molecular signal molecule CD28, enhance and maintain the activation state of anti-tumor lymphocyte co-stimulatory signal, promote the activation, proliferation and infiltration of tumor tissue of lymphocytes, and finally kill tumor cells.

At present, although some breakthroughs have been made in the treatment of immune checkpoint inhibitors in multiple cancer types, the efficacy of single-drug immunotherapy needs to be improved. The combination of CTLA4 inhibitors combined with PD1 inhibitors has significantly improved the remission rate and survival prognosis of patients. , but the toxicity of the combined drug was also significantly increased. At present, we are still looking for drugs that can assist immunotherapy in clinical practice, so as to further improve the curative effect without increasing serious adverse reactions.

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

There is an urgent need in this field to explore more effective and safer drug combinations that work synergistically with immunotherapy.

Contents of the invention

The purpose of the present invention is to develop a more effective and safer combined drug combination synergized with immunotherapy. The purpose of the present invention is to provide a combined drug for treating tumors, so as to improve the therapeutic effect of anti-CTLA-4 antibody alone.

The first aspect of the present invention provides a product combination, including:

(i) a first pharmaceutical composition containing (a) a first active ingredient, which is a CTLA-4 inhibitor, and a pharmaceutically acceptable carrier; and

(ii) a second pharmaceutical composition, which contains (b) a second active ingredient, which 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.

In another preferred embodiment, the inhibitor is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, or combinations thereof.

In another preferred example, the antibodies include KD6001, Ipilimumab, Tremelimmab, Zalifrelimab, Quavonlimab, REGN4659, and YH001.

In another preferred example, the antibody sequence of KD6001 is as shown in PCT/CN2015/095072.

In another preferred example, the non-steroidal anti-inflammatory drugs are non-steroidal anti-inflammatory drugs (NSAIDs).

In another preferred example, the non-steroidal anti-inflammatory drugs are selected from the group consisting of salicylic acid drugs, aniline drugs, acetic acid drugs, phenylacetic acid drugs, propionic acid drugs, oxicam drugs, coxib drugs, or combinations thereof.

In another preferred example, the salicylic acid drugs include aspirin and benolate.

In another preferred example, the aniline drugs include acetaminophen and nimesulide.

In another preferred example, the acetic acid drug includes indomethacin.

In another preferred example, the phenylacetic acid drugs include diclofenac and sulindac.

In another preferred example, the propionic acid drugs include ibuprofen, naproxen and loxoprofen.

In another preferred example, the oxicam drugs include piroxicam and meloxicam.

In another preferred example, the coxibs include celecoxib, valdecoxib, rofecoxib, erecoxib, etoricoxib, nimesulide, and parecoxib.

In another preferred example, 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.

In another preferred example, in the product combination, the content of the CTLA-4 inhibitor is 1%-99%, preferably 1%-60%, more preferably 1%-30%.

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

In another preference, in the product combination, the component (i) and component (ii) account for 0.01-99.99wt%, preferably 0.1-90wt%, more preferably Ground 1-80wt%.

In another preferred example, the dosage form of the pharmaceutical composition includes injection dosage form and oral dosage form.

In another preferred example, the oral dosage forms include tablets, capsules, films, and granules.

In another preferred example, the dosage forms of the pharmaceutical composition include sustained-release dosage forms and non-sustained-release dosage forms.

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

(i) CTLA-4 inhibitors;

(ii) NSAIDs; and

(iii) A pharmaceutically acceptable carrier.

In another preferred example, in the composition, the component (i) and component (ii) account for 0.01-99.99wt%, preferably 0.1-90wt%, more preferably Ground 1-80wt%.

In another preferred example, the composition further includes other drugs for treating tumors.

In another preferred example, the tumor includes a solid tumor.

In another preferred example, the tumor is selected from the group consisting of 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, Kidney Cancer, Urothelial Cancer, Nasopharyngeal Cancer, Endometrial Cancer, Glioma, Gastric Cancer, Esophageal Cancer , esophagogastric junction cancer, thyroid cancer, pancreatic cancer, cholangiocarcinoma, primary brain cancer, or a combination thereof.

In another preferred example, the other drugs for treating tumors are selected from the group consisting of immune checkpoint inhibitor drugs, immune stimulants, or combinations thereof.

In another preferred example, 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, VISITA.

In another preferred example, the immune stimulant includes CD40, CSF1R, TLR family and its receptor IFN, IL factor and its receptor such as IL-2, IL-15, IL-21, GMCSF/GMCSFR, RIG- 1. TNF family, CD25, chemokines CCR and CXC, NKG2D.

A third aspect of the present invention provides a kit, comprising:

(a1) a first container, and a CTLA-4 inhibitor located in said first container, or a medicine containing a CTLA-4 inhibitor;

(b1) A second container, and a non-steroidal anti-inflammatory drug, or a drug containing a non-steroidal anti-inflammatory drug, located in the second container.

In another preferred example, the first container and the second container are the same or different containers.

In another preferred example, the drug in the first container is a single preparation containing a CTLA-4 inhibitor.

In another preferred example, the medicine in the second container is a single preparation containing non-steroidal anti-inflammatory drugs.

In another preferred example, the dosage form of the drug is an oral dosage form or an injection dosage form.

In another preferred example, the kit also contains instructions.

In another preferred example, the description records the following instructions:

(a) A method for synergistically treating tumors by combining CTLA-4 inhibitors and non-steroidal anti-inflammatory drugs.

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

In another preferred example, the concentration of the CTLA-4 inhibitor is 0.1-30 mg/kg, preferably 0.2-20 mg/kg, preferably 0.3-10 mg/kg.

In another preferred example, the effect 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.

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

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

In another preferred example, the pharmaceutical composition or kit further includes other drugs for treating tumors.

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

Administer CTLA-4 inhibitors and non-steroidal anti-inflammatory drugs to subjects in need, or the product combination described in the first aspect of the present invention or the composition described in the second aspect of the present invention or the kit described in the third aspect of the present invention .

In another preferred example, the subject includes a human or a non-human mammal suffering from a tumor.

In another preferred example, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

In another preferred example, the dose of the CTLA-4 inhibitor is 0.1-30 mg/kg, preferably 0.2-20 mg/kg, more preferably 0.3-10 mg/kg.

In another preferred example, the dosage of the non-steroidal anti-inflammatory drug is 0.1-100 mg/kg, preferably 0.3-30 mg/kg, more preferably 1-15 mg/kg.

In another preferred example, the administration frequency of the CTLA-4 inhibitor is once a week or once every two weeks or once every three weeks or once every 6 weeks or once every 12 weeks or once every 24 weeks, preferably Yes, every 2-6 weeks.

In another preferred example, the administration time of the CTLA-4 inhibitor is 1-4000 days, preferably 1-1000 days, most preferably 1-365 days.

In another preferred example, the administration frequency of the non-steroidal anti-inflammatory drug is 1-6 times a day, preferably 2-4 times a day.

In another preferred example, the non-steroidal anti-inflammatory drug is administered for 1-4000 days, preferably 1-1000 days, most preferably 1-365 days.

In another preferred example, the CTLA-4 inhibitor and non-steroidal anti-inflammatory drugs are administered simultaneously or sequentially.

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 in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows the effect of KD6001 combined with ibuprofen on body weight of tumor-bearing MC38 mice. Colon cancer cells MC38 were subcutaneously implanted into mice and administered KD6001 or PBS by intraperitoneal injection four times on D8, D11, D15, and D19, and intragastrically administered ibuprofen 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 the mice at each time point were statistically analyzed for differences between groups. The low, middle 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 all greater than 0.05.

Figure 2 shows the effect of KD6001 combined with ibuprofen on tumor volume in tumor-bearing MC38 mice. Colon cancer cells MC38 were subcutaneously implanted into mice and administered KD6001 or PBS by intraperitoneal injection four times on D8, D11, D15, and D19, and intragastrically administered ibuprofen twice a day from D8 to D17. Tumor volume was measured twice a week. The experiment was ended on D26. A: The average tumor volume change curve of the mice in each group; B: The tumor volume and weight at the end of the experiment; C: The mice were killed after the experiment, and the tumors were stripped and photographed. At the end of each experiment (D26) the data of the individual tumor volume of the mice were statistically analyzed for differences between the groups. The T-test analysis was performed between each administration group and the control group respectively. KD6001 50 μg + ibuprofen 200 μg group, KD6001 50 μg + ibuprofen 60 μg Compared with the KD6001 50μg monotherapy group, there was a statistically significant difference in the tumor volume of the mice in the group. *p<0.05.

Detailed ways

Through extensive and in-depth research, the inventors discovered for the first time that the combined use of the CTLA-4 inhibitor of the present invention and non-steroidal anti-inflammatory drugs can effectively treat tumors and have a synergistic effect. In addition, it can also effectively improve the anti-CTLA Antitumor effect of -4 antibody single drug. On this basis, the present inventors have completed the present invention.

As used herein, Quavonlimab was developed by Merck, and its sequence information is disclosed in PCT publication WO2020185722.

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

As used herein, YH001 was developed by Ewell Pharmaceuticals, and the sequence information was disclosed by Chinese application CN111406069.

CTLA-4 inhibitors

CTLA-4 is an immune checkpoint receptor on T lymphocytes, which inhibits the excessive 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 combination of CTLA-4 and B7 molecules, and antagonize CTLA-4 to inhibit the activation of T cells, thereby making T lymphocytes Cell co-stimulatory factor CD28 binds to B7 molecules on antigen-presenting cells, continuously activates T lymphocytes, and thus exerts its anti-tumor effect.

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

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 antibody, including KD6001, Ipilimumab, Tremelimmab, Zalifrelimab, Quavonlimab, REGN4659, YH001, preferably KD6001, the antibody sequences thereof are shown in PCT/CN2015/095072 and Table 1 of the Examples of the present invention.

NSAIDs

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

In a preferred embodiment, non-steroid anti-inflammatory drugs are selected from the group consisting of salicylic acid drugs, aniline drugs, acetic acid drugs, phenylacetic acid drugs, propionic acid drugs, oxicam drugs, coxib drugs, or a combination thereof.

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

Propionic acid drugs include: ibuprofen, naproxen, loxoprofen; oxicam drugs include: piroxicam, meloxicam.

Coxibs include: celecoxib, valdecoxib, rofecoxib, erecoxib, etoricoxib, nimesulide, parecoxib.

The research of the present invention shows that the combination of non-steroidal anti-inflammatory drugs of the present invention and CTLA-4 inhibitors, such as CTLA-4 antibodies, can play a synergistic effect in the treatment of tumors, effectively improving the anti-CTLA-4 antibody single drug anti-tumor effect. For tumor effects, the combination of non-steroidal anti-inflammatory drugs and CTLA-4 inhibitors, such as CTLA-4 antibodies, has a good curative effect on tumors and has a good clinical application prospect.

Composition and method of application

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

In general, 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.

The present invention also provides a pharmaceutical composition, which contains 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, dextrose, 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 injection, for example, by conventional methods using physiological saline or 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 to 10 mg/kg body weight per day, preferably, the dosage of the EP4 receptor antagonist can be: 0.1-2000 mg per day for adults, preferably 1-300 mg/day . The dosage of the PD-1 inhibitor can be: 0.1-2000 mg every two weeks for adults, preferably 1-300 mg/two weeks. As a medicine for tumor co-treatment, it can be made into oral and non-oral preparations. Oral administration can be made into common dosage forms such as tablets, powders, granules, and capsules, and the used excipients can be one or more of starch, lactose, sucrose, mannose, and hydroxymethylcellulose. The disintegrant can be one or more of potato starch, hydroxymethyl cellulose and the like. The binder can be one or more of gum arabic, cornstarch, gelatin, dextrin and the like. In addition to the above dosage forms, oral preparations can also be made into emulsions, syrups and the like.

Non-oral preparations can be made into injections, which can be made into injections with water for injection, physiological saline, and glucose water, or a certain proportion of ethanol, propanol, ethylene glycol, etc. can be added thereto. In addition, it can also be made into common dosage forms such as nasal drops, sprays, rectal suppositories, and rectal retention enemas.

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 inhibitor drugs, immune stimulants), so as to inhibit tumors more effectively.

pill box

The present invention also provides a kit, which contains:

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

The preparation containing a CTLA-4 inhibitor may be a unit dosage form containing a CTLA-4 inhibitor, and the preparation containing a non-steroid anti-inflammatory drug may be a unit dosage form containing a non-steroid anti-inflammatory drug.

As used herein, the term "unit dosage form" refers to the preparation of a composition into a dosage form required for single administration for the convenience of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, sustained-release dosage forms, etc. agent.

In another preferred example, the instructions describe the following methods of use:

(I) simultaneously administering to a subject in need a formulation comprising a CTLA-4 inhibitor and a formulation comprising a non-steroidal anti-inflammatory drug; and optionally

(II) Repeat steps (I)-(II).

The formulation 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 form. The preferred way is to take it once a day, because it is easy for patients to adhere to it, thereby significantly improving the patient's compliance with medication.

When taking it, the total daily dose in most cases should be lower than (or in a small number of cases, equal to or slightly greater than) the usual daily dose of each single drug. Of course, the effective dose of the active ingredient used can vary with the mode of administration and the dose to be treated. Depending on the severity of the disease being treated, etc.

The main advantages of the present invention include:

(1) The present invention finds for the first time that the combined use of the CTLA-4 inhibitor of the present invention and non-steroidal anti-inflammatory drugs can effectively treat tumors and have a synergistic effect. In addition, it can also effectively improve the anti-CTLA-4 antibody monotherapy Antitumor effect.

(2) The present invention finds for the first time that the combined use of non-steroidal anti-inflammatory drugs (such as ibuprofen) and CTLA-4 inhibitors (such as anti-CTLA-4 monoclonal antibodies) of the present invention can play a synergistic effect in the treatment of tumors , effectively improve the anti-tumor effect of anti-CTLA-4 antibody single drug, and the curative effect of combining non-steroidal anti-inflammatory drugs (such as ibuprofen) and CTLA-4 inhibitors (such as anti-CTLA-4 monoclonal antibodies) Excellent, good prospects for clinical application.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are usually followed. Percentages and parts are by weight unless otherwise indicated.

Unless otherwise specified, the materials and reagents in the description of the present invention are all commercially available products.

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

1. Materials:

Drugs: anti-CTLA-4 antibody: KD6001 injection, Shanghai Saijin Biomedical Co., Ltd., batch number 20180201; its sequence is shown in PCT/CN2015/095072. The specific information of the sequence is shown in Table 1.

Table 1 Sequence information of KD6001

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

Cell line: colon cancer cell MC38, Shanghai Southern Model Biotechnology Co., Ltd.

Animals: human CTLA-4 gene knock-in (knock in) mice

2. Experimental method

2.1 Drug preparation preparation:

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

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

2.2 MC38 cell culture:

Mouse colon cancer cells MC38 were cultured in DMEM medium containing 10% FBS or DMEM/F12 medium containing 10% FBS, and passaged twice a week. On the day of inoculating the mice, treat the cells with trypsin, collect them in a 50ml centrifuge tube, centrifuge to pellet the cells, discard the supernatant, wash the cells twice with PBS, and finally suspend the cells in PBS at a cell density of 5×10 ⁶ cells /ml.

2.3 Mouse colon cancer cell MC38 subcutaneously transplanted into mice:

Put the collected cells on ice and bring them to the place where the mice were raised, and subcutaneously inject 0.1ml of cell suspension in the back of each mouse, containing 5×10 ⁵ cells.

2.4 Administration of mice

The way of administration to mice is intraperitoneal injection, and the administration volume of each mouse is 0.2ml. A total of 4 administrations were administered on D8, D12, D15, and D19. D8-D19 ibuprofen was administered by intragastric administration, the administration volume was 0.2ml, twice a day. The body weight and tumor volume of the mice were measured twice a week (the body 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.

Table 2 Dosage grouping table

2.5 Statistical Analysis

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

3. Experimental results

3.1 Mouse body weight monitoring results

The average body weight changes of the mice in each group are shown in Figure 1. Statistical difference analysis between groups was performed on the individual body weight data of mice at each time point, and each group was compared with the control group by one-way analysis of variance, and the P values of each dose group were all greater than 0.05. Under the conditions of this experiment, the mice in the high, middle and low dose groups were given KD6001 2.5mg/kg+Ibuprofen 10mg/kg, KD6001 2.5mg/kg+Ibuprofen 3mg/kg, KD6001 2.5mg/kg+Ibuprofen 1mg /kg had no effect on the body weight of tumor-bearing MC38 mice. The experimental results showed that under the experimental conditions, the combination of KD6001 and ibuprofen had no obvious toxicity to mice.

3.2 Tumor volume monitoring results in mice

Figure 2A shows the changes in the average tumor volume of mice in each group during the experiment. From D15 (before the third administration), the average tumor volume of mice in the three combined administration groups began to show significantly smaller than that of other groups, especially significantly smaller than that of the control group. D26 (the end of the experiment), the average tumor volume of the mice in the control group was about 1908.6mm ³ The average tumor volumes of mice in the kg group were 1763.3mm ³ , 1189.6mm ³ , 1975.8mm ³ , and 1344.2mm ³ ; KD6001 2.5mg/kg + ibuprofen 10mg/kg, KD6001 2.5mg/kg + ibuprofen The mean tumor volumes of the mice in the fen 3 mg/kg, KD6001 2.5 mg/kg+ibuprofen 1 mg/kg groups were 419.6 mm ³ , 704.2 mm ³ , and 872.8 mm ^{3 ,} respectively (Fig. 2B left). The weights of the tumors of mice in each group at the end of the experiment (D26) are shown in Fig. 2B (right). Compared with the tumor suppressive effect of single drug KD6001, the tumor suppressive effects of KD6001 combined with 10 mg/kg and combined with 3 mg/kg ibuprofen were significantly different (p values were less than 0.01 and 0.05, respectively) (Fig. 2B). Figure 2C shows the photos of the tumors removed from mice in each group.

Different doses of ibuprofen combined with a fixed dose of KD6001 (KD6001 2.5mg/kg) all showed an additive effect of anti-tumor effect, and the additive effect increased with the increase of the combined dose of ibuprofen. The average volume of tumors in the dose combined with KD6001 group was significantly smaller than that in the KD6001 monotherapy group.

Statistical analysis of the tumor volume between the dose group and the control group, each dose group and the control group were carried out T-test analysis of the individual tumor volume data of the mice, the results showed that the tumor volume of the mice in the combined drug group was significantly higher than that in the PBS group There was a statistically significant difference (p<0.05). The combined administration group and the KD6001 monotherapy group performed T-test analysis on the individual tumor volume data of the mice, and the results showed that there was also a statistically significant difference between the combined administration group and the monotherapy group (p<0.05) .

Table 3 shows the relative tumor volume (RTV) and relative tumor proliferation rate (T/C%) results of the dose groups in this experiment. From the perspective of relative tumor volume (RTV) and relative tumor proliferation rate (T/C%), the tumors of mice in the KD6001 2.5mg/kg group and ibuprofen 10mg/kg group treated with single drug at the end of the test (D26) were relatively Inhibition rates (T/C) were 112.3% and 68.1% respectively; KD6001 2.5mg/kg+ibuprofen 10mg/kg, KD6001 2.5mg/kg+ibuprofen 3mg/kg, KD6001 2.5mg/kg+buprofen The T/C of the mice in the profen 1mg/kg group were 31.7%, 43.0%, and 54.6% on D26, respectively. The anti-tumor effects of three different doses of combination therapy were better than those of the monotherapy group, showing that the combination of KD6001 and ibuprofen has a synergistic effect, and the best effect is KD6001 2.5mg/kg+ ibuprofen 10mg/kg, The weight ratio of KD6001 and ibuprofen is 1:4.

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

V8 represents the average tumor volume on day 8 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 PBS group

4. Experimental conclusion

In this experiment, through the combined administration of intraperitoneal injection of KD6001 and intragastric administration of ibuprofen, KD6001 was given to colon cancer-bearing MC38 mice twice a week for a total of 4 times; ibuprofen was administered twice a day for a total of 4 times. After 12 days of drug administration, KD6001, ibuprofen single-drug administration group and PBS negative control group were set up at the same time to investigate whether the combination of KD6001 and ibuprofen had a synergistic effect on the anti-tumor effect of the mouse colon cancer MC38 model.

Single drug KD6001 has no effect on tumor growth (RTV is 112.3%) at a dose of 2.5 mg/kg (intraperitoneal injection), and the inhibitory effect is 0. The RTV of ibuprofen alone at a dose of 10mg/kg is 68.1%, which has a partial inhibitory effect (about 30% inhibitory effect) on the growth of tumors, while KD6001 (dose is 2.5mg/kg) and 10mg/kg ibuprofen When fen is used in combination, its RTV is 31.7%, and the inhibitory effect reaches about 70%, showing that KD6001 and ibuprofen have a synergistic effect in inhibiting tumor growth. When KD6001 (dose is 2.5mg/kg) is combined with medium and low dose (for example, the dose is 1mg/kg or 3mg/kg) ibuprofen, the effect of inhibiting tumor growth is also due to the application of ibuprofen or KD6001 alone, and also Both showed the synergistic effect of KD6001 and ibuprofen in inhibiting tumor growth.

In summary, under the conditions of this experiment, ibuprofen combined with KD6001 has a synergistic antitumor effect.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content 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 appended claims of the present application.

Claims

A product combination, characterized in that it includes:

(i) a first pharmaceutical composition containing (a) a first active ingredient, which is a CTLA-4 inhibitor, and a pharmaceutically acceptable carrier; and

(ii) a second pharmaceutical composition, which contains (b) a second active ingredient, which 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 according to claim 1, wherein the inhibitor is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, or combinations thereof.
The product combination according to claim 2, wherein the antibodies include KD6001, Ipilimumab, Tremelimmab, Zalifrelimab, Quavonlimab, REGN4659 , YH001.
The product combination according to claim 1, wherein the non-steroidal anti-inflammatory drugs are non-steroidal anti-inflammatory drugs (NSAIDs).
The product combination according to claim 1, wherein the weight ratio of the component (i) to the 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.
A composition, characterized in that the composition comprises:

(i) CTLA-4 inhibitors;

(ii) NSAIDs; and

(iii) A pharmaceutically acceptable carrier.
The composition according to claim 6, further comprising other drugs for treating tumors.
A medicine box, is characterized in that, comprises:

(a1) a first container, and a CTLA-4 inhibitor located in said first container, or a medicine containing a CTLA-4 inhibitor;

(b1) A second container, and a non-steroidal anti-inflammatory drug, or a drug containing a non-steroidal anti-inflammatory drug, located in the second container.
A use of a combination, characterized in that the combination includes a CTLA-4 inhibitor and a non-steroidal anti-inflammatory drug for preparing a pharmaceutical composition or kit, and the pharmaceutical composition or kit is used for treating tumors.
The use according to claim 9, wherein the tumor is selected from the group consisting of colorectal cancer, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma of the head and neck, liver cancer, cervical cancer, B-cell lymphoid tumor, multiple myeloma, non-Hodgkin's lymphoma, melanoma, ovarian cancer, breast cancer, prostate cancer, pleural mesothelioma, renal cancer, urothelial carcinoma, nasopharyngeal carcinoma, endometrial carcinoma, colloid tumor, gastric cancer, esophageal cancer, esophagogastric junction cancer, thyroid cancer, pancreatic cancer, cholangiocarcinoma, primary brain cancer, or a combination thereof.