Use of rapamycin combined with PD-1 monoclonal antibody in preparation of drugs for treating colorectal cancer

Abstract

Provided are a use of rapamycin combined with a PD-1 monoclonal antibody in preparation of drugs for treating colorectal cancer, and a corresponding pharmaceutical composition. The tumor immune microenvironment is remodeled by means of a rapamycin formulation targeting tumor tissue, the infiltration of T lymphocytes in the tumor tissue is increased, and M2-related TAMs are inhibited, thereby enhancing the anti-tumor, especially anti-microsatellite stable colorectal cancer effect of the PD-1 monoclonal antibody.

Description

Application of rapamycin combined with PD-1 monoclonal antibody in the preparation of colorectal cancer therapeutic drugs

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application filed with the China Patent Office on September 1, 2023, with application number 202311128066.4 and invention name “Application of rapamycin combined with PD-1 monoclonal antibody to prepare drugs for the treatment of colorectal cancer”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application belongs to the field of tumor treatment. Specifically, the present application provides the use of rapamycin combined with PD-1 monoclonal antibody to prepare a drug for treating colorectal cancer and a corresponding pharmaceutical composition. Background Art

[0004] Colorectal cancer (CRC) has become one of the three most common malignancies in my country. With the continuous advancement of medical diagnosis and treatment, malignant tumor treatment has entered the era of immunotherapy. Currently, PD-1 inhibitors are used in the treatment of CRC in two populations. Microsatellite instability (MSI) or mismatch repair gene status (MMR) is the best predictor of efficacy, dividing CRC patients into two groups: the "dominant population"—MSI-H / dMMR CRC (MSI-H CRC for short) and the "ineffective population"—MSS / pMMR CRC (MSS CRC for short).

[0005] For patients with MSI-H advanced CRC, the NCCN and CSCO guidelines strongly recommend PD-1 monoclonal antibodies (pembrolizumab K and nivolumab O) as first-line treatment options. However, studies on immunotherapy for patients with MSS advanced CRC have not yielded satisfactory results. This type of CRC patient accounts for over 95% of all CRC patients. The urgent and strong need for treatment and the relatively scarce treatment options create a stark contradiction, creating a treatment dilemma that urgently needs to be overcome.

[0006] At present, the key to transforming MSS-type colorectal cancer, a "cold tumor" that is resistant to immunotherapy, into a "hot tumor" that is effective for immunotherapy is combination therapy. This can transform patients who were originally not suitable for PD-1 inhibitor treatment into those who can benefit from it and improve the treatment effect. Currently, the mainstream combinations are as follows: (1) Combination with another immunotherapy drug: PD-1 inhibitors combined with CTLA-4 antibodies have been approved for malignant melanoma. (2) Combination with chemotherapy: PD-1 inhibitors combined with chemotherapy have been approved for the first-line treatment of advanced non-squamous non-small cell lung cancer. Similar regimens have also shown good preliminary data for gastric cancer, colorectal cancer, triple-negative breast cancer, etc. (3) Combination with radiotherapy: PD-1 inhibitors combined with radiotherapy are used in tumors such as lung cancer and malignant melanoma. (4) Combination with targeted drugs: PD-1 inhibitors combined with anti-angiogenic targeted drugs or combined with EGFR inhibitors, but care should be taken as serious side effects may occur.

[0007] The mammalian target of rapamycin (mTOR) signaling pathway is a major regulator of cell growth and metabolism, occurring within two functionally distinct complexes: mTORC1 and mTORC2. The mTORC1 (mTOR complex 1) signaling pathway is a crucial pathway for cell growth and proliferation, involved in regulating autophagy, ribosome biogenesis, mRNA translation, and metabolism. Aberrant activation of the mTORC1 signaling pathway is highly common in malignant tumors. Therefore, drugs targeting mTORC1 are promising anticancer therapies.

[0008] The targeted mTOR inhibitor rapamycin holds great promise for cancer treatment. Previous studies have shown that the combination of rapamycin and 5-fluorouracil has a synergistic effect in the treatment of colorectal cancer. Therefore, we considered combining Rapa LPs with immunosuppressants to see if they could improve the tumor microenvironment and thereby exert a synergistic or stimulatory effect in the treatment of colorectal cancer.

[0009] Summary of the Invention

[0010] In order to further develop such combination schemes, on the one hand, the present application provides the use of rapamycin combined with PD-1 monoclonal antibody to prepare a drug for treating colorectal cancer.

[0011] Optionally, rapamycin and PD-1 monoclonal antibody are prepared in a single pharmaceutical composition, or rapamycin and PD-1 monoclonal antibody are used in combination.

[0012] Optionally, the rapamycin in the colorectal cancer therapeutic drug exists in the form of liposomes, micelles and self-microemulsification solutions.

[0013] Optionally, the method for preparing the self-microemulsifying solution comprises:

[0014] (1) mixing two or more components selected from anhydrous ethanol, propylene glycol, polyethylene glycol 300, and polyethylene glycol 400, adding rapamycin, and optionally one or more antioxidants selected from 2,6-tert-butyl-4-methylphenol, bis(3,5-tert-butyl-4-hydroxyphenyl)sulfide, tetrakis[β-(3,5-tert-butyl-4-hydroxyphenyl)propionic acid], pentaerythritol ester, BHT, α-tocopherol, thioglycerol, and BHA to prepare a mother solution;

[0015] (2) using a surfactant selected from polyethylene glycol stearate, such as one or more of PEG-10 hydroxystearate, PEG-12 hydroxystearate, PEG-18 stearate, PEG-10 stearate, and PEG-15 stearate, anhydrous ethanol, water, and optionally one or more of a metal chelating agent selected from ethylenediaminetetraacetic acid, aminotriacetic acid, diethylenetriaminepentaacetic acid and its salts, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid, polyacrylic acid, polymethacrylic acid, hydrolyzed polymaleic anhydride, fumaric acid (fumaric acid)-propylene sulfonic acid copolymer, dihydroxyethylglycine, and sodium calcium edetate to prepare a diluent;

[0016] (3) Mixing the drug stock solution and the diluent to obtain the microemulsion solution.

[0017] Optionally, the method for preparing the self-microemulsifying solution comprises:

[0018] (1) Anhydrous ethanol and polyethylene glycol 300 solution are mixed to obtain a mixed solution, rapamycin is added to the mixed solution, and then an antioxidant and anhydrous citric acid are added to obtain a drug stock solution;

[0019] (2) Prepare diluent: measure polyethylene glycol-10 hydroxystearate, anhydrous ethanol, and water for injection, mix well, add metal chelating agent, and mix well to obtain diluent;

[0020] (3) Diluting the drug stock solution to the desired concentration with a diluent to obtain rapamycin self-microemulsion injection.

[0021] Optionally, the method for preparing the self-microemulsifying solution comprises:

[0022] (1) 0.5 ml of anhydrous ethanol and 0.5 ml of polyethylene glycol 300 solution were mixed to obtain a mixture, rapamycin was added to the mixture, and then 0.5 μg of antioxidant and 2.5 μg of anhydrous citric acid were added to obtain a 20 mg / ml drug stock solution;

[0023] (2) Prepare diluent: Measure 0.8 ml of polyethylene glycol-10 hydroxystearate, 0.2 ml of anhydrous ethanol, and 1.0 ml of water for injection, mix well, add 0.004 g of metal chelating agent, and mix well to obtain diluent.

[0024] (3) The drug mother solution and diluent are sterilized by filtration and then sealed and stored at 4°C. When used, the drug mother solution is diluted with the diluent to the desired concentration to obtain the rapamycin self-microemulsion injection.

[0025] Optionally, the PD-1 monoclonal antibody is selected from BioXcell, inVivoMab's anti-mouse PD-1CD279 monoclonal antibody, pembrolizumab, nivolumab, sintilimab, toripalimab, tislelizumab, and carrelizumab.

[0026] Optionally, the colorectal cancer is microsatellite stable colorectal cancer.

[0027] Optionally, the drug contains rapamycin combined with PD-1 monoclonal antibody.

[0028] Optionally, the weight ratio of rapamycin to PD-1 monoclonal antibody in the drug is 1:1-4:1.

[0029] Optionally, the weight ratio of rapamycin to PD-1 monoclonal antibody in the drug is 1:1-2:1.

[0030] Optionally, the weight ratio of rapamycin to PD-1 monoclonal antibody in the drug is 1:1-1:20.

[0031] On the other hand, the present application provides a drug for treating colorectal cancer, comprising rapamycin and PD-1 monoclonal antibody; rapamycin and PD-1 monoclonal antibody are prepared in a single pharmaceutical composition, or rapamycin and PD-1 monoclonal antibody are provided separately.

[0032] When used in combination, the dosage of rapamycin is 0.5-4 mg / kg, and the dosage of PD-1 monoclonal antibody is 0.5-80 mg / kg.

[0033] The present application also provides a method for treating colorectal cancer, comprising administering rapamycin and PD-1 monoclonal antibody in combination to a subject in need.

[0034] Optionally, rapamycin and PD-1 monoclonal antibody are prepared in a single pharmaceutical composition, or rapamycin and PD-1 monoclonal antibody are used in combination.

[0035] Optionally, the PD-1 monoclonal antibody is selected from BioXcell, inVivoMab's anti-mouse PD-1CD279 monoclonal antibody, pembrolizumab, nivolumab, sintilimab, toripalimab, tislelizumab, and carrelizumab.

[0036] Optionally, the rapamycin in the colorectal cancer therapeutic drug exists in the form of liposomes, micelles and self-microemulsification solutions.

[0037] Optionally, the method for preparing the self-microemulsifying solution comprises:

[0038] (1) mixing two or more components selected from anhydrous ethanol, propylene glycol, polyethylene glycol 300, and polyethylene glycol 400, adding rapamycin, and optionally one or more antioxidants selected from 2,6-tert-butyl-4-methylphenol, bis(3,5-tert-butyl-4-hydroxyphenyl)sulfide, tetrakis[β-(3,5-tert-butyl-4-hydroxyphenyl)propionic acid], pentaerythritol ester, BHT, α-tocopherol, thioglycerol, and BHA to prepare a mother solution;

[0039] (2) using a surfactant selected from polyethylene glycol stearate, such as one or more of PEG-10 hydroxystearate, PEG-12 hydroxystearate, PEG-18 stearate, PEG-10 stearate, and PEG-15 stearate, anhydrous ethanol, water, and optionally one or more of a metal chelating agent selected from ethylenediaminetetraacetic acid, aminotriacetic acid, diethylenetriaminepentaacetic acid and its salts, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid, polyacrylic acid, polymethacrylic acid, hydrolyzed polymaleic anhydride, fumaric acid (fumaric acid)-propylene sulfonic acid copolymer, dihydroxyethylglycine, and sodium calcium edetate to prepare a diluent;

[0040] (3) Mixing the drug stock solution and the diluent to obtain the microemulsion solution.

[0041] Optionally, the method for preparing the self-microemulsifying solution comprises:

[0042] (1) Anhydrous ethanol and polyethylene glycol 300 solution are mixed to obtain a mixed solution, rapamycin is added to the mixed solution, and then an antioxidant and anhydrous citric acid are added to obtain a drug stock solution;

[0043] (2) Prepare diluent: measure polyethylene glycol-10 hydroxystearate, anhydrous ethanol, and water for injection, mix well, add metal chelating agent, and mix well to obtain diluent;

[0044] (3) Diluting the drug stock solution to the desired concentration with a diluent to obtain rapamycin self-microemulsion injection.

[0045] Optionally, the method for preparing the self-microemulsifying solution comprises:

[0046] (1) 0.5 ml of anhydrous ethanol and 0.5 ml of polyethylene glycol 300 solution were mixed to obtain a mixture, rapamycin was added to the mixture, and then 0.5 μg of antioxidant and 2.5 μg of anhydrous citric acid were added to obtain a 20 mg / ml drug stock solution;

[0047] (2) Prepare diluent: Measure 0.8 ml of polyethylene glycol-10 hydroxystearate, 0.2 ml of anhydrous ethanol, and 1.0 ml of water for injection, mix well, add 0.004 g of metal chelating agent, and mix well to obtain diluent;

[0048] (3) The drug mother solution and diluent are sterilized by filtration and then sealed and stored at 4°C. When used, the drug mother solution is diluted with the diluent to the desired concentration to obtain the rapamycin self-microemulsion injection.

[0049] Optionally, the colorectal cancer is microsatellite stable colorectal cancer.

[0050] Optionally, the weight ratio of rapamycin to PD-1 monoclonal antibody in the drug is 1:1-4:1.

[0051] Optionally, the weight ratio of rapamycin to PD-1 monoclonal antibody in the drug is 1:1-2:1.

[0052] Optionally, the weight ratio of rapamycin to PD-1 monoclonal antibody in the drug is 1:1-1:20.

[0053] PD-1 monoclonal antibodies currently on the market or under development can be used in the technical solutions of this application after appropriate experiments and selection.

[0054] The rapamycin preparations in this application can be prepared using various methods known in the art, not limited to the methods in the examples. For example, reference may be made to the preparation methods in Chinese patent applications No. 201910918643.7, No. 202011065631.3, and No. 202210106827.5.

[0055] The components of the drug of this application may be in any clinically acceptable dosage form, including various oral and parenteral dosage forms. For oral administration, the drug may be in the form of tablets, capsules, soft capsules, oral liquids, syrups, granules, dripping pills, orally disintegrating tablets, sustained-release tablets, sustained-release capsules, controlled-release tablets, controlled-release capsules, etc.; for parenteral administration, the drug may be in the form of aqueous injections, lyophilized powder injections, sterile powder injections, infusions, etc.

[0056] Those skilled in the art can use various pharmaceutically acceptable carriers or excipients to construct the above-mentioned dosage forms, such as solvents, co-solvents, pH regulators, osmotic pressure regulators, stabilizers, antioxidants, fillers, binders, lubricants, disintegrants, coating agents, sustained-release agents, controlled-release agents, adsorption carriers, etc.

[0057] The present application provides a new method for treating microsatellite-stable colorectal cancer, which is the combined application of rapamycin and immunotherapy for colorectal cancer, specifically the combination of PD-1 monoclonal antibody and rapamycin preparation. By targeting the rapamycin preparation in tumor tissue, the tumor immune microenvironment is reshaped, the infiltration of T lymphocytes in tumor tissue is increased, and M2-related TAMs are inhibited, thereby enhancing the anti-tumor effect of PD-1 monoclonal antibody, especially the anti-microsatellite-stable colorectal cancer effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 shows the results of an in vitro MTT assay of cells.

[0059] FIG2 shows the pharmacodynamic experimental results of different concentrations of RL on CT-26 tumor-bearing mice.

[0060] FIG3 is a graph showing the growth curve of transplanted tumors.

[0061] Figure 4 is a comparison of tumor sizes for each tumor treatment regimen.

[0062] FIG5 is a statistical diagram of tumor weight for each tumor treatment regimen.

[0063] Figure 6 is a Western blot image of PD-L1 expression in each group after administration and an IHC image of SMA.

[0064] Figure 7 shows the effects of rapamycin solution combined with anti-PD-1 antibodies on immune cells (CD8+T, CD4+T, M1, M2 macrophages, and regulatory T cells).

[0065] FIG8 is a graph showing the ELISA results of rapamycin solution combined with anti-PD-1 antibody (IFN-γ, TNF-α). DETAILED DESCRIPTION

[0066] Example 1 Preparation of rapamycin liposomes

[0067] Rapamycin liposomes were prepared using an anhydrous ethanol solution injection method. Particle size, potential, drug loading, and encapsulation efficiency were measured, and the structure was characterized by TEM. The specific methods are as follows:

[0068] 100 mg of rapamycin, 900 mg of phospholipid, and 100 mg of cholesterol (stabilizer) are dissolved in 7 ml of dichloromethane (organic phase solvent) to obtain an organic phase mixture; the organic phase mixture is added to PBS (aqueous phase solvent) at a rate of 1-10 drops / min, and stirred at a temperature of ≤40°C and a stirring speed of 600 rpm for 60 minutes to obtain a colostrum solution; the colostrum solution is homogenized six times in a homogenizer at a homogenization pressure of 900 bar, and then 5% trehalose (lyoprotectant) is added and mixed, and the mixture is sterilized by filtration through a microporous filter membrane with a pore size of 0.22 μm, and freeze-dried to obtain a liposome lyophilized powder of rapamycin preparation.

[0069] Example 2 Preparation of Rapamycin Micelles

[0070] Rapamycin micelles were prepared using an organic solvent injection method. Particle size, potential, drug loading, and encapsulation efficiency were determined. TEM was used to characterize the structure of the rapamycin liposomes. The specific methods are as follows:

[0071] 100 mg of rapamycin API and 900 mg of mPEG-PLA block polymer were added to 7 mL of acetone to form an organic phase. The organic phase was aspirated into a syringe and added dropwise at a rate of 5 drops per minute to 100 mL of PBS buffer solution stirred at 500 rpm, followed by stirring at 800 rpm for 120 minutes at room temperature. The organic solvent was recovered under reduced pressure at 40°C. The mixture was centrifuged at 4000 rpm for 30 minutes, the supernatant was collected, and sterilized by filtration through a 0.22 μm pore microporous membrane to obtain a micellar solution. Lactose was added to the micellar solution, sterile-filtered through a 0.22 μm pore microporous membrane, and freeze-dried to obtain a rapamycin micelle preparation.

[0072] Example 3 Preparation of Rapamycin Self-Microemulsifying Solution Injection (LPMSY)

[0073] (1) 0.5 ml of anhydrous ethanol and 0.5 ml of polyethylene glycol 300 solution were mixed to obtain a mixture, rapamycin was added to the mixture, and then 0.5 μg of antioxidant and 2.5 μg of anhydrous citric acid were added to obtain a 20 mg / ml drug stock solution;

[0074] (2) Prepare diluent: Measure 0.8 ml of polyethylene glycol-10 hydroxystearate, 0.2 ml of anhydrous ethanol, and 1.0 ml of water for injection, mix well, add 0.004 g of metal chelating agent, and mix well to obtain diluent.

[0075] (3) The drug mother solution and diluent are sterilized by filtration and then sealed and stored at 4°C. When used, the drug mother solution is diluted with the diluent to the desired concentration to obtain the rapamycin self-microemulsion injection.

[0076] Example 4 In vitro cytotoxicity assay

[0077] Mouse colorectal cancer cells (CT-26) were selected and the in vitro cytotoxicity of rapamycin injection was detected by the tetrazolium reduction method (MTT method), and the IC50 was calculated.

[0078] CT-26 cells were seeded in 96-well plates at 3.5 × 10 cells per well. 3 Six replicate wells were seeded at each concentration. After the cells adhered, serum-free medium was replaced with medium and allowed to stand overnight. Then, drug-containing medium was switched to rapamycin injection at concentrations of 60, 40, 30, 20, 10, 5, 2.5, 1.25, and 0 μg mL⁻¹. After 48 hours of incubation, the medium was removed and replaced with serum-free medium containing 0.5 mg mL⁻¹ MTT. Incubation continued for another 4 hours. The medium was then removed, 100 μl of DMSO was added, and the mixture was mixed. The absorbance was measured on a microplate reader at 490 nm. Cell viability and IC₅₀ values ​​were calculated using GraphPrism. Figure 1 shows that the median lethal dose of rapamycin injection (LPMSY-01) for CT-26 cells was 7.764 μg / ml.

[0079] Example 5 Animal Experiment

[0080] 1. Preparation of Transplanted Tumor Mouse Model

[0081] CT-26 cells (MSS mouse colorectal cancer cells) in the logarithmic growth phase were collected, counted, and resuspended in serum-free medium 1640 to adjust the cell concentration to 5.0×10 6 cells / mL, the cells were placed in an ice box, the mixture was drawn up with a 1 mL syringe, and injected into the subcutaneous tissue of the right axilla of BALB / c mice. Each animal was inoculated with 200 μL (1×10 6 Cells / mouse) were used to establish a CT-26 transplant tumor model. The animal status was regularly observed, and the tumor diameter was measured using an electronic vernier caliper to calculate the tumor volume and monitor the tumor growth. 3 The experiment was divided into 4 groups, with 6 mice in each group (see Table 1 for specific grouping and dosing schedule). After the start of the experiment, the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal body weight was weighed and recorded.

[0082] The formula for calculating tumor volume (TV) is as follows: TV (mm 3 )=l×w2 / 2

[0083] Wherein, l represents the long diameter of the tumor (mm); w represents the short diameter of the tumor (mm).

[0084] 2. Preparation of Colorectal Cancer Liver Metastasis Mouse Model

[0085] CT-26 cells (MSS mouse colorectal cancer cells) in the logarithmic growth phase were collected, counted, and resuspended in serum-free medium 1640 to adjust the cell concentration to 2.0×10 7 cells / mL, placed the cells in an ice box, aspirated the cell suspension with an insulin needle, and inoculated each animal with 50 μL (1×10 6 A colorectal cancer liver metastasis model was established by splenic injection. The animals were regularly observed to monitor tumor metastasis. Dosing began the day after model establishment. The mice were divided into four groups, each consisting of six mice (see Table 1 for specific groupings and dosing schedules). Tumor diameters were measured and tumor volumes calculated twice weekly after the start of the experiment. Animal body weights were also recorded.

[0086] 3. Experimental Grouping and Dosage Regimen

[0087] First, we investigated the therapeutic effects of different concentrations of rapamycin liposomes on CT26 (MSS-type CRC) tumor-bearing mice. Compared with the saline group, rapamycin liposomes significantly inhibited tumor growth, with significant differences. The inhibition rates of 1, 2, and 4 mg / kg rapamycin liposomes were 47.77%, 50.82%, and 52.98%, respectively.

[0088] A preliminary study investigated the therapeutic efficacy of rapamycin injection (LPMSY-01, prepared as in Example 3) combined with anti-PD-1 monoclonal antibody in mice bearing microsatellite-stable colorectal cancer. Previous studies have shown that 2 mg / kg of rapamycin administered via the tail vein has a significant anti-colorectal cancer effect, and this dose was used in this study.

[0089] Table 1. Dosage regimen for efficacy experiments in nude mouse xenograft tumor model and liver metastasis model

[0090] 4. Preparation of drug delivery preparations

[0091] ① Preparation of blank solvent

[0092] 0.9% normal saline was used as the solvent control group.

[0093] ②Preparation of Rapamycin Injection (LPMSY-01)

[0094] Take 1 bottle of LPMSY-01 injection (about 20 mg / mL), dilute it with the first step diluent to obtain a solution with a concentration of 4.00 mg·mL-1, and then further dilute it with normal saline to a 0.2 mg·mL-1 dosing preparation, which is ready for use.

[0095] ③ Preparation of PD-1 monoclonal antibody (BioXcell, inVivoMab anti-mouse PD-1 (CD279))

[0096] The concentration of the PD-1 single antigen solution was 9.78 mg·mL-1. After the first dilution with normal saline, a solution with a concentration of 1.00 mg·mL-1 was obtained. Each mouse was given 200 μL (i.e., 200 ug / mouse) and it was prepared and used immediately.

[0097] 5. Evaluation Criteria

[0098] For transplanted tumor models, the relative tumor proliferation rate (T / C%) is recommended as the experimental evaluation indicator. In principle, the evaluation criteria are: T / C (%) > 40% is considered ineffective; T / C (%) ≤ 40%, and P < 0.05 after statistical analysis is considered effective.

[0099] The results showed that the combined administration had a significant inhibitory effect on the transplanted tumor, with a T / C (%) of 31.21%, indicating that the combined administration was effective in inhibiting the tumor, while the single administration was ineffective. The data are summarized in Table 2.

[0100] Table 2. Data statistical summary table

[0101] 6. Investigation of the mechanism of action

[0102] Tumor tissues from mice were paraffin-embedded and sectioned, and tissue proteins were extracted for ELISA analysis. Immunohistochemical staining was performed on tumor tissue sections to investigate changes in PD-L1 protein levels in tumor tissues of CT26 tumor-bearing mice after drug treatment. The levels of α-SMA+ cancer-associated fibroblasts, T cells, IFN-γ, and TNF-α in tumor tissues were also investigated. Western blot and immunohistochemical staining of tumor tissues revealed that anti-PD-1 antibodies had no significant effect on PD-L1 expression on tumor surfaces, whereas rapamycin solution alone or in combination with rapamycin downregulated PD-L1 expression on tumor surfaces. Furthermore, IHC staining revealed that rapamycin solution alone or in combination with rapamycin downregulated the activation of α-SMA+ cancer-associated fibroblasts in tumor tissues. Anti-PD-1 antibody treatment slightly increased the infiltration of CD8+ T cells in tumor tissues, while there was no significant change in CD4+ T cells. Rapa alone or in combination with rapamycin increased the infiltration of both CD8+ and CD4+ T cells. After treatment with anti-PD-1 antibodies, no significant changes were observed in the number of M1 and M2 macrophages and regulatory T cells in tumor tissue. Rapa alone or in combination with rapamycin increased M1 macrophage infiltration, while M2 macrophage and Treg infiltration decreased. Elisa results suggest that rapamycin solution alone or in combination with rapamycin increased tumor tissue secretion of IFN-γ and TNF-α.

Claims

1. Application of rapamycin combined with PD-1 monoclonal antibody in the preparation of drugs for the treatment of colorectal cancer.

2. The use according to claim 1, wherein rapamycin and PD-1 monoclonal antibody are prepared in a single pharmaceutical composition, or rapamycin and PD-1 monoclonal antibody are used in combination.

3. The use according to claim 2, wherein the rapamycin in the colorectal cancer treatment drug exists in the form of liposomes, micelles and self-microemulsification solutions.

4. The use according to claim 3, wherein the method for preparing the self-microemulsification solution comprises: (1) mixing two or more components selected from anhydrous ethanol, propylene glycol, polyethylene glycol 300, and polyethylene glycol 400, adding rapamycin, and optionally one or more antioxidants selected from 2,6-tert-butyl-4-methylphenol, bis(3,5-tert-butyl-4-hydroxyphenyl)sulfide, tetrakis[β-(3,5-tert-butyl-4-hydroxyphenyl)propionic acid], pentaerythritol ester, BHT, α-tocopherol, thioglycerol, and BHA to prepare a mother solution; (2) using a surfactant selected from polyethylene glycol stearate, such as PEG-10 hydroxystearate, PEG-12 hydroxystearate, PEG-18 stearate, PEG-10 stearate, PEG-15 stearate, anhydrous ethanol, water, and optionally a metal chelating agent selected from ethylenediaminetetraacetic acid, aminotriacetic acid, diethylenetriaminepentaacetic acid and its salts, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid, polyacrylic acid, polymethacrylic acid, hydrolyzed polymaleic anhydride, fumaric acid (fumaric acid)-propylene sulfonic acid copolymer, dihydroxyethylglycine, sodium calcium edetate, or one or more metal chelating agents to prepare a diluent; (3) Mixing the drug stock solution and the diluent to obtain the microemulsion solution.

5. The use according to claim 4, wherein the method for preparing the self-microemulsification solution comprises: (1) mixing anhydrous ethanol and polyethylene glycol 300 solution to obtain a mixed solution, adding rapamycin to the mixed solution, and then adding an antioxidant and anhydrous citric acid to obtain a drug mother solution; (2) preparing a diluent: measuring polyethylene glycol-10 hydroxystearate, anhydrous ethanol, and water for injection, mixing them well, adding a metal chelating agent, and mixing well to obtain a diluent; (3) Diluting the drug stock solution to a desired concentration with a diluent to obtain rapamycin self-microemulsion injection.

6. The use according to claim 5, wherein the method for preparing the self-microemulsification solution comprises: (1) 0.5 ml of anhydrous ethanol and 0.5 ml of polyethylene glycol 300 solution were mixed to obtain a mixed solution, rapamycin was added to the mixed solution, and then 0.5 μg of antioxidant and 2.5 μg of anhydrous citric acid were added to obtain a 20 mg / ml drug stock solution; (2) preparing a diluent: taking 0.8 ml of polyethylene glycol-10 hydroxystearate, 0.2 ml of anhydrous ethanol, and 1.0 ml of water for injection, mixing them well, adding 0.004 g of a metal chelator, and mixing well to obtain a diluent; (3) The drug mother solution and the diluent are sterilized by filtration and then sealed and stored at 4°C. When used, the drug mother solution is diluted with the diluent to a desired concentration to obtain the rapamycin self-microemulsion injection.

7. The use according to any one of claims 1 to 6, wherein the PD-1 monoclonal antibody is selected from the group consisting of anti-mouse PD-1 CD279 monoclonal antibody, pembrolizumab, nivolumab, sintilimab, toripalizumab, tislelizumab, and carrelizumab from BioXcell, inVivoMab.

8. The use according to any one of claims 1-7, wherein the colorectal cancer is microsatellite stable colorectal cancer.

9. The use according to any one of claims 1 to 8, wherein the weight ratio of rapamycin to PD-1 monoclonal antibody in the drug is 1:1-4:

1.

10. The use according to claim 9, wherein the weight ratio of rapamycin to PD-1 monoclonal antibody in the drug is 1:1-2:

1.

11. The use according to any one of claims 1 to 9, wherein the weight ratio of rapamycin to PD-1 monoclonal antibody in the drug is 1:1-1:

20.

12. A drug for treating colorectal cancer, characterized in that: The drug includes rapamycin and PD-1 monoclonal antibody. 13 . The drug according to claim 12 , wherein rapamycin and PD-1 monoclonal antibody are prepared in a single pharmaceutical composition, or rapamycin and PD-1 monoclonal antibody are provided separately.

14. The drug according to claim 13, wherein rapamycin is present in the form of liposomes, micelles and self-microemulsification solutions.

15. The drug according to claim 14, wherein the method for preparing the self-microemulsification solution comprises: (1) mixing two or more components selected from anhydrous ethanol, propylene glycol, polyethylene glycol 300, and polyethylene glycol 400, adding rapamycin, and optionally one or more antioxidants selected from 2,6-tert-butyl-4-methylphenol, bis(3,5-tert-butyl-4-hydroxyphenyl)sulfide, tetrakis[β-(3,5-tert-butyl-4-hydroxyphenyl)propionic acid], pentaerythritol ester, BHT, α-tocopherol, thioglycerol, and BHA to prepare a mother solution; (2) using a surfactant selected from polyethylene glycol stearate, such as PEG-10 hydroxystearate, PEG-12 hydroxystearate, PEG-18 stearate, PEG-10 stearate, PEG-15 stearate, anhydrous ethanol, water, and optionally a metal chelating agent selected from ethylenediaminetetraacetic acid, aminotriacetic acid, diethylenetriaminepentaacetic acid and its salts, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid, polyacrylic acid, polymethacrylic acid, hydrolyzed polymaleic anhydride, fumaric acid (fumaric acid)-propylene sulfonic acid copolymer, dihydroxyethylglycine, sodium calcium edetate, or one or more metal chelating agents to prepare a diluent; (3) Mixing the drug stock solution and the diluent to obtain the microemulsion solution.

16. The drug according to claim 15, wherein the method for preparing the self-microemulsification solution comprises: (1) mixing anhydrous ethanol and polyethylene glycol 300 solution to obtain a mixed solution, adding rapamycin to the mixed solution, and then adding an antioxidant and anhydrous citric acid to obtain a drug mother solution; (2) preparing a diluent: measuring polyethylene glycol-10 hydroxystearate, anhydrous ethanol, and water for injection, mixing them well, adding a metal chelating agent, and mixing well to obtain a diluent; (3) Diluting the drug stock solution to a desired concentration with a diluent to obtain rapamycin self-microemulsion injection.

17. The drug according to claim 16, wherein the method for preparing the self-microemulsification solution comprises: (1) 0.5 ml of anhydrous ethanol and 0.5 ml of polyethylene glycol 300 solution were mixed to obtain a mixed solution, rapamycin was added to the mixed solution, and then 0.5 μg of antioxidant and 2.5 μg of anhydrous citric acid were added to obtain a 20 mg / ml drug stock solution; (2) preparing a diluent: taking 0.8 ml of polyethylene glycol-10 hydroxystearate, 0.2 ml of anhydrous ethanol, and 1.0 ml of water for injection, mixing them well, adding 0.004 g of a metal chelator, and mixing well to obtain a diluent; (3) The drug mother solution and the diluent are sterilized by filtration and then sealed and stored at 4°C. When used, the drug mother solution is diluted with the diluent to a desired concentration to obtain the rapamycin self-microemulsion injection.

18. The drug according to any one of claims 12 to 17, wherein the PD-1 monoclonal antibody is selected from the group consisting of anti-mouse PD-1 CD279 monoclonal antibody from BioXcell, inVivoMab, pembrolizumab, nivolumab, sintilimab, toripalizumab, tislelizumab, and carrelizumab.

19. The drug according to any one of claims 12 to 18, wherein the colorectal cancer is microsatellite stable colorectal cancer.

20. The drug according to any one of claims 12 to 19, wherein the weight ratio of rapamycin to PD-1 monoclonal antibody in the drug is 1:1-4:

1. 21 . The drug according to claim 20 , wherein the weight ratio of rapamycin to PD-1 monoclonal antibody in the drug is 1:1-2:

1.

22. The drug according to any one of claims 12 to 19, wherein the weight ratio of rapamycin to PD-1 monoclonal antibody in the drug is 1:1-1:

20.

23. A method for treating colorectal cancer, comprising administering rapamycin and PD-1 monoclonal antibody in combination to a subject in need thereof.

24. The method according to claim 23, wherein rapamycin and PD-1 monoclonal antibody are prepared in a single pharmaceutical composition, or rapamycin and PD-1 monoclonal antibody are used in combination.

25. The method according to claim 23 or 24, wherein the PD-1 monoclonal antibody is selected from the group consisting of anti-mouse PD-1 CD279 monoclonal antibody, pembrolizumab, nivolumab, sintilimab, toripalizumab, tislelizumab, and carrelizumab from BioXcell, inVivoMab.

26. The method according to claim 23 or 24, wherein the rapamycin in the drug for treating colorectal cancer is in the form of liposomes, micelles and self-microemulsification solutions.

27. The method according to claim 26, wherein the method for preparing the self-microemulsification solution comprises: (1) mixing two or more components selected from anhydrous ethanol, propylene glycol, polyethylene glycol 300, and polyethylene glycol 400, adding rapamycin, and optionally one or more antioxidants selected from 2,6-tert-butyl-4-methylphenol, bis(3,5-tert-butyl-4-hydroxyphenyl)sulfide, tetrakis[β-(3,5-tert-butyl-4-hydroxyphenyl)propionic acid], pentaerythritol ester, BHT, α-tocopherol, thioglycerol, and BHA to prepare a mother solution; (2) using a surfactant selected from polyethylene glycol stearate, such as PEG-10 hydroxystearate, PEG-12 hydroxystearate, PEG-18 stearate, PEG-10 stearate, PEG-15 stearate, anhydrous ethanol, water, and optionally a metal chelating agent selected from ethylenediaminetetraacetic acid, aminotriacetic acid, diethylenetriaminepentaacetic acid and its salts, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid, polyacrylic acid, polymethacrylic acid, hydrolyzed polymaleic anhydride, fumaric acid (fumaric acid)-propylene sulfonic acid copolymer, dihydroxyethylglycine, sodium calcium edetate, or one or more metal chelating agents to prepare a diluent; (3) Mixing the drug stock solution and the diluent to obtain the microemulsion solution.

28. The method according to claim 27, wherein the method for preparing the self-microemulsification solution comprises: (1) mixing anhydrous ethanol and polyethylene glycol 300 solution to obtain a mixed solution, adding rapamycin to the mixed solution, and then adding an antioxidant and anhydrous citric acid to obtain a drug mother solution; (2) preparing a diluent: measuring polyethylene glycol-10 hydroxystearate, anhydrous ethanol, and water for injection, mixing them well, adding a metal chelating agent, and mixing well to obtain a diluent; (3) Diluting the drug stock solution to a desired concentration with a diluent to obtain rapamycin self-microemulsion injection.

29. The method according to claim 28, wherein the method for preparing the self-microemulsification solution comprises: (1) 0.5 ml of anhydrous ethanol and 0.5 ml of polyethylene glycol 300 solution were mixed to obtain a mixed solution, rapamycin was added to the mixed solution, and then 0.5 μg of antioxidant and 2.5 μg of anhydrous citric acid were added to obtain a 20 mg / ml drug stock solution; (2) preparing a diluent: taking 0.8 ml of polyethylene glycol-10 hydroxystearate, 0.2 ml of anhydrous ethanol, and 1.0 ml of water for injection, mixing them well, adding 0.004 g of a metal chelator, and mixing well to obtain a diluent; (3) The drug mother solution and the diluent are sterilized by filtration and then sealed and stored at 4°C. When used, the drug mother solution is diluted with the diluent to a desired concentration to obtain the rapamycin self-microemulsion injection.

30. The method according to any one of claims 23-29, wherein the colorectal cancer is microsatellite stable colorectal cancer.

31. The method according to any one of claims 23-30, wherein the weight ratio of rapamycin to PD-1 monoclonal antibody in the drug is 1:1-4:

1.

32. The method according to claim 31, wherein the weight ratio of rapamycin to PD-1 monoclonal antibody in the drug is 1:1-2:

1.

33. The method according to any one of claims 23-30, wherein the weight ratio of rapamycin to PD-1 monoclonal antibody in the drug is 1:1-1:

20.

34. The method according to any one of claims 23-30, wherein the dosage of rapamycin in the drug is 0.5-4 mg / kg, and the dosage of PD-1 monoclonal antibody is 0.5-80 mg / kg.

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