Sorbitol and PD-1 inhibitor combined pharmaceutical composition and application thereof

Abstract

The invention discloses a sorbitol and PD-1 inhibitor combined pharmaceutical composition and application thereof, and belongs to the technical field of biological medicine. The invention provides an anti-tumor combined pharmaceutical composition which comprises sorbitol and a PD-1 inhibitor, and meanwhile, the invention further relates to application of the pharmaceutical composition in preparation of anti-tumor products. It is found for the first time that sorbitol has indications for enhancing tumor immunotherapy, and sorbitol can be used in tumor immunotherapy to improve survival prognosis of tumor patients. The sorbitol is combined with the PD-1 inhibitor to effectively kill tumor cells, and compared with a PD-1 inhibitor single drug, the combined use of the sorbitol and the PD-1 inhibitor greatly improves the treatment effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a sorbitol and PD-1 inhibitor combined pharmaceutical composition and application thereof. Background Art

[0002] Recombinant anti-PD-1 fully human monoclonal antibody, which binds with high affinity to PD-1 on the T lymphocyte membrane, blocks the binding of PD-1 and PD-L1, thereby enhancing and maintaining the activation state of the co-stimulatory signal of anti-tumor lymphocytes, promoting the activation of the anti-tumor effect of lymphocytes, activation and infiltration of tumor tissues, and ultimately killing tumor cells.

[0003] At present, although immune checkpoint inhibitor therapy has made certain breakthroughs in multiple cancer types, the efficacy of single-agent immunotherapy needs to be improved, especially in some solid tumors, such as gastric cancer, intestinal cancer, and breast cancer. PD-1 inhibitors combined with chemotherapy drugs can improve the remission rate and survival prognosis of tumor patients to a certain extent, but some patients still cannot benefit from it. The KEYNOTE-585 study is the first global phase III clinical study on the application of immunotherapy combined with chemotherapy for gastric cancer to report survival follow-up data. In the main cohort, compared with the chemotherapy group alone, the addition of pembrolizumab can bring about an increase in the pCR rate in the short term (pCR rate: 12.9% vs. 2.0%), and the R0 resection rate reached 80%. Although there is a trend of benefit in long-term survival (EFS period: 44.4 months vs. 25.3 months), it is not statistically significant, and survival needs to be confirmed by longer follow-up.

[0004] The clinical indications of sorbitol are indigestion, abdominal distension, loss of appetite, constipation, stubborn abdominal distension and habitual constipation in the elderly. Its toxicity and side effects are minimal and it can be taken for a long time. At the same time, sorbitol oral solution is low in cost and is often used as an auxiliary drug to improve appetite in clinical practice.

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

[0006] In order to solve the above technical problems, the present invention discovered for the first time that sorbitol (powder, tablet, injection, etc.) can enhance the immunotherapy effect of PD-1 inhibitors and enhance anti-tumor immune function, and on this basis, developed a more effective combination drug combination that synergizes with tumor immunotherapy to enhance the efficacy of immunotherapy.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides an anti-tumor combined pharmaceutical composition, which comprises sorbitol and a PD-1 inhibitor.

[0009] Furthermore, the dosage form of the pharmaceutical composition is selected from injection, solution, emulsion, suspension, suppository, ointment, cream, spray, drop, powder, granule, granule, capsule, pill, tablet, patch or sustained-release preparation.

[0010] Furthermore, the sorbitol is administered once a week, and the PD-1 inhibitor is administered once every three days.

[0011] Furthermore, the PD-1 inhibitor is selected from nivolumab, toripalimab, pembrolizumab, sintilimab, carrelizumab or tislelizumab.

[0012] Furthermore, the pharmaceutical composition also contains one or more pharmaceutically acceptable carriers or excipients.

[0013] In addition, the present invention also provides the use of the pharmaceutical composition in the preparation of anti-tumor products.

[0014] Furthermore, the product is an anti-tumor drug.

[0015] Furthermore, the anti-tumor drug also contains one or more pharmaceutically acceptable carriers or excipients.

[0016] Furthermore, the dosage form of the anti-tumor drug is selected from injection, solution, emulsion, suspension, suppository, ointment, cream, spray, drop, powder, granule, granule, capsule, pill, tablet, patch or sustained-release preparation.

[0017] Furthermore, the tumor includes lung cancer, gastric cancer, colorectal cancer, endometrial cancer, liver cancer, breast cancer, melanoma, leukemia, glioma, cervical cancer, esophageal cancer or sarcoma.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention discovered for the first time that sorbitol has an indication for enhancing tumor immunotherapy. Sorbitol can be used in tumor immunotherapy to improve the survival prognosis of tumor patients. Sorbitol combined with PD-1 inhibitors can effectively kill tumor cells, which greatly improves the therapeutic effect of PD-1 inhibitors alone.

[0020] In addition, the present invention also discovered for the first time that the concentration of sorbitol is upregulated in tumor patients who are sensitive to immunotherapy. It can be used as a tumor biomarker for evaluating the efficacy of immunotherapy, providing a theoretical basis for the subsequent clinical guidance of immunotherapy for tumor patients. At the same time, the application of sorbitol can also improve the situation in which immunotherapy is insensitive in patients with some tumor types. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a WB image of the expression of the key immune regulatory factor cGAS detected in Example 1;

[0022] Figure 2 This is the qPCR graph for detecting the transcription level of downstream immune factors in Example 1;

[0023] Figure 3 This is a WB image of the binding detection of metabolic enzyme AKR1B1 and cGAS key transcription factor SP1 in Example 2;

[0024] Figure 4 This is a diagram showing the binding energy distribution of sorbitol in the AKR1B1-SP1 complex in Example 2;

[0025] Figure 5 is a graph showing the change in binding energy between AKR1B1 and SP1 in Example 2;

[0026] Figure 6 is a graph showing changes in tumor volume and sorbitol concentration in vivo in Example 3;

[0027] Figure 7 This is a graph showing changes in tumor volume and mouse body weight in Example 3;

[0028] Figure 8 is a graph showing changes in tumor volume and sorbitol concentration in vivo in Example 3;

[0029] Fig. 9 This is a diagram of the treatment results of the patient in Example 4; DETAILED DESCRIPTION

[0030] In order to better illustrate the present invention, the following embodiments are listed. Obviously, the described embodiments are only a part of the present invention, not all embodiments. Based on the embodiments in the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0031] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0032] Through extensive and in-depth research, the inventors have discovered for the first time that the combination of sorbitol and PD-1 inhibitors can greatly enhance the immunotherapy effect of PD-1 inhibitors, effectively treat tumors, and have a synergistic effect. In addition, it can also effectively improve the efficacy of existing immunotherapy regimens in clinical practice. On this basis, the inventors have completed the present invention.

[0033] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are not conventional products that can be purchased through regular channels.

[0034] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0035] Example 1 Effect of adding sorbitol on the opening of tumor cell immune pathways

[0036] 1.1 Cell culture

[0037] Take human gastric cancer cell MKN45 and mouse colorectal cancer cell CT26 as examples.

[0038] Culture and subculture of human gastric cancer cell line MKN45 and mouse colorectal cancer cell line CT26: RPMI-1640 containing 10% fetal bovine serum was used as the culture medium, and the cells were kept in a saturated humidity, 37°C, and 5% CO. 2 Cultivate in a cell culture incubator under the following conditions. Observe the cell status every day. When the cells grow to 80-90%, discard the original culture medium, gently rinse once with PBS, and digest the cells with 0.25% trypsin (containing EDTA). According to the purpose of the experiment, subculture at a ratio of 1:2 or 1:3 every day. After 3 generations, take cells with good growth status, normal morphology and in the logarithmic growth phase for experiments.

[0039] 1.2 Cell plating: Take cells in logarithmic growth phase, digest them with 0.25% trypsin, blow the cells evenly with RPMI-1640 cell culture medium containing 10% fetal bovine serum to disperse them into single cell suspension, and adjust the cell suspension concentration to 2-3×10 after counting. 5 / mL, 100 μL of cell suspension was evenly plated into 6-well plates and placed at 37°C with 5% CO 2 Culture in a saturated humidity incubator.

[0040] 1.3 Dosing: After 24-48 hours, when the cells grow to a suitable density (80-90%), set up three replicate wells according to experimental needs, add sorbitol at concentrations of 0mM, 1mM, 2mM, 4mM, 8mM, and 16mM, and continue to place the cell plate at 37°C and 5% CO 2 Incubate in a saturated humidity incubator for 72 h.

[0041] 1.4 Protein immunoblotting

[0042] Protein extraction, protein concentration determination (BCA method) and SDS polyacrylamide gel electrophoresis were performed respectively.

[0043] 1.5qPCR experiments

[0044] Reagents and materials: template DNA or cDNA; forward and reverse primers; qPCR premix (including DNA polymerase, dNTPs, buffer, etc.); SYBR Green or TaqMan probe (according to experimental requirements); sterile water; qPCR reaction plate or tube; sealing film.

[0045] Instruments: qPCR instrument; centrifuge; pipette and sterile pipette tip; ice box.

[0046] Primer design: Use primer design software (such as Primer3, NCBI Primer-BLAST) to design specific primers. The primer length is usually 18-22bp, the GC content is 40-60%, and the Tm value is 55-65°C. The final primer sequence is shown in Table 1:

[0047] Table 1 Primer sequence list

[0048]

[0049] Reaction system configuration: reaction system (taking 20 μL as an example), qPCR premix 10 μL, forward primer (10 μM) 0.8 μL, reverse primer (10 μM) 0.8 μL, DNA / cDNA template 2 μL, sterile water 6.4 μL.

[0050] Reaction program settings: preliminary denaturation: 95°C, 5 minutes.

[0051] Amplification cycles (40 cycles): denaturation: 95°C, 15 seconds, annealing: 55-60°C, 30 seconds (adjusted according to the primer Tm value), extension: 72°C, 30 seconds.

[0052] Melting curve analysis (SYBR Green assay): 95°C, 15 sec, 60°C, 1 min, 95°C, 15 sec.

[0053] 1.6 Experimental Results

[0054] like Figure 1 As described above, adding different concentrations of sorbitol significantly increased the protein of cGAS, a key immune regulatory factor in human gastric cancer cell line MKN45 and mouse colorectal cancer cell line CT26. Figure 2 As mentioned above, qPCR experiments suggest that sorbitol can significantly increase the transcription levels of downstream immune factors such as CXCL8, CXCL9, IFNA1, IFNA2 and IFNG in tumor cells and open up immune pathways.

[0055] This experiment successfully proved at the cellular level that sorbitol can open the immune pathway of tumor cells through cGAS. Sorbitol can improve the level of tumor cell immunosuppression.

[0056] Example 2 Study on the mechanism of sorbitol regulating immune pathways in tumor cells in vivo

[0057] 2.1 Experimental Materials

[0058] Drug: Sorbitol; Cell line: Human gastric cancer cell MKN45 cell line.

[0059] 2.2 Experimental methods

[0060] 2.2.1 Protein interaction assay (Co-IP)

[0061] (1) Sample preparation: First, collect cell or tissue samples, wash them three times with pre-cooled PBS buffer, and then add IP lysis buffer for lysis. Protease inhibitors need to be added during the lysis process to prevent protein degradation. After lysis, centrifuge the sample at 4°C and collect the supernatant for later use.

[0062] (2) Antibody-protein binding: The treated magnetic beads are mixed with the antibody and incubated on a flip mixer. After incubation, the magnetic beads are collected by magnetic separation and washed several times with binding / washing buffer to remove non-specifically bound proteins. Antigen-antibody binding: The target protein is mixed with the magnetic beads, and after further incubation, the magnetic beads are magnetically separated and washed to remove unbound proteins.

[0063] (3) Protein separation and detection: The magnetic beads and protein sample mixture are separated by SDS-PAGE electrophoresis, and then the presence of the target protein is detected by Western blot. If both the bait protein and the target protein can be detected in the precipitate, it indicates that there is an interaction between the two proteins.

[0064] (4) Experimental principle: Co-IP experiment is a classic method for studying protein interactions based on the specific interaction between antibodies and antigens. Proteins bound to a specific target protein are captured indirectly using target protein-specific antibodies to form an antibody-target protein complex. The complex is then fixed and precipitated using a beaded support that can bind to the antibody, and finally analyzed by SDS-PAGE and Western blot.

[0065] 2.2.2 GST-Pulldown Experiment

[0066] (1) Protein extraction

[0067] 1) Washing: Wash the sample with 1 mL of pre-cooled PBS (about 2 × 10 7 cells) twice, and try to dry the PBS in the last time;

[0068] 2) Lysis: Add 990mL Lysis buffer and 10μL Protease inhibitor (100X) according to the amount of cells, and fully lyse on ice for 20-30min, mixing by inverting every 5min;

[0069] 3) Ultrasound: ultrasonic cell disruptor was used for 5 min, power was 20%, ultrasonication was performed for 3 s, interval was 3 s, and ultrasonication was performed in an ice bath;

[0070] 4) Centrifugation: 4°C, 12000 rpm, 10 min, collect the supernatant.

[0071] (2) Preparation of magnetic beads

[0072] 1) Take out the Glutathione MagBeads from the 4°C refrigerator, mix them upside down several times to mix the magnetic beads and the solution evenly, and take 30 μL into two clean 1.5 mL Eppendorf tubes, respectively, as the control group and the experimental group;

[0073] 2) Add 0.5 mL of pre-cooled washing buffer to resuspend the magnetic beads, place on a magnetic stand for 1 min to separate the magnetic beads and solution, and carefully aspirate and discard the supernatant with a pipette;

[0074] 3) Repeat step 2) 2 times, washing 3 times in total;

[0075] (3) Magnetic beads bind to GST-X protein

[0076] 1) Add 200ug GST-X recombinant protein to the experimental group, and add no protein or 200ug GST recombinant protein to the control tube; make up the volume to 1mL with Incubation buffer, and incubate at room temperature for 2h without mixing;

[0077] 2) Place the two tubes on a magnetic stand and let stand for 1 min to separate the magnetic beads and solution, and carefully aspirate and discard the supernatant with a pipette;

[0078] 3) Add 0.5 mL of pre-cooled washing buffer, place on a magnetic stand for 1 min to separate the magnetic beads and solution, and carefully discard the supernatant with a pipette;

[0079] 4) Repeat step 3) 2 times, washing 3 times in total.

[0080] (4) Bait protein binding interaction protein Y (other recombinant proteins or total proteins except GST tag)

[0081] 1) Add 200ug Y recombinant protein or 500-1000ug total protein to each of the two tubes, add Incubation buffer to make the volume 1mL, and incubate at 4℃ with silent mixing overnight (about 16h);

[0082] 2) Place the two tubes on a magnetic stand and let stand for 1 min to separate the magnetic beads and solution, and carefully aspirate and discard the supernatant with a pipette;

[0083] 3) Add 0.5 mL of pre-cooled washing buffer, place on a magnetic stand for 1 min to separate the magnetic beads and solution, and carefully discard the supernatant with a pipette;

[0084] 4) Repeat step 3) 2 times, washing 3 times in total.

[0085] (5) Elution

[0086] 1) Add 100uL of elution buffer to both tubes, boil in water for 10min, centrifuge at 12000rpm for 5min, take the supernatant, add 20uL 6X Loading buffer, boil in water for 8-10min, and record as control group and experimental group;

[0087] 2) Take two clean 1.5mL EP tubes, record them as Input control group and Input experimental group respectively, add 50ug of GST recombinant protein and Y recombinant protein to the Input control group, add 50ug of GST-X protein and Y recombinant protein to the Input experimental group (if it is total protein, add 100uL), add 1 / 5 volume of 6X Loading buffer, and boil in water bath for 8-10min.

[0088] 3) The control group, experimental group, Input control, and Input experiment were stored at -20℃ for future use.

[0089] (6) Western Blot: Take 30 μL of each of the control, experimental, Input control, and Input experimental samples and perform SDS-PAGE and Western blot detection.

[0090] 2.3 Experimental Results

[0091] like Figure 3 As mentioned above, SP1 protein is known to be a transcription factor of cGAS. Through Co-IP experiments and GST-Pulldown experiments, we found that the addition of sorbitol can enhance the binding of metabolic enzyme AKR1B1 to SP1 and increase the transcription level of cGAS. Figure 4 As shown in Figure 2, sorbitol can bind to the AKR1B1-SP1 complex and form stable hydrogen bonds, enhancing its stability. Figure 5 As shown, we further found that sorbitol can reduce the binding energy between AKR1B1 and SP1, making it more likely to bind.

[0092] It can be seen that the addition of sorbitol can enhance the binding ability of AKR1B1 and transcription factor SP1, and further enhance the cGAS transcription level, thereby opening the immune pathway.

[0093] Example 3 In vivo pharmacodynamic study of combined therapy on solid cancer in mice

[0094] 3.1 Experimental Materials

[0095] Drug: Anti-PD-1 antibody, Product information: Anti-Mouse CD279 (PD-1) (Clone RMP1-14)-Purified in vivo GOLD Functional Grade (USA); Brand: Leinco

[0096] Sorbitol; cell lines: mouse breast cancer cell 4T1, mouse colorectal cancer cell CT26; animals: Bal / B mice.

[0097] 3.2 Experimental methods

[0098] 3.2.1 Drug delivery preparation

[0099] Sorbitol was added to the daily diet of mice at a concentration of 5% (kg / L).

[0100] 3.2.2 Culture of mouse breast cancer cell 4T1:

[0101] Mouse breast cancer 4T1 cells were cultured in 1640 medium containing 10% FBS or DMEM / F12 medium containing 10% FBS and passaged twice a week. On the day of inoculation, cells were treated with trypsin and collected in a 50 ml centrifuge tube. 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.

[0102] 3.2.3 Culture of mouse colorectal cancer cells CT26:

[0103] Mouse colon cancer cell CT26 was cultured in 1640 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, collected in a 50 ml centrifuge tube, centrifuged and precipitated, the supernatant was discarded, the cells were washed twice with PBS, and finally suspended in PBS at a cell density of 5×10 6 cells / ml.

[0104] 3.2.4 Subcutaneous transplantation of mouse breast cancer cells 4T1

[0105] 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.

[0106] 3.2.5 Subcutaneous transplantation of mouse colorectal cancer cells CT26

[0107] 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.

[0108] 3.2.6 Drug administration to mice

[0109] The mice were administered by intraperitoneal injection, with a volume of 0.2 ml per mouse. AntiPD-1 was administered 4 times on D8, D12, D15, and D19, once a day. The weight of the mice and the tumor volume were measured once every 2-3 days starting from D5 (the weight and tumor volume were measured before administration during the administration period). (Tumor volume V (mm 3 ) = 1 / 2 × tumor long diameter a (mm) × tumor short diameter b 2 The specific experimental groups are shown in Table 2.

[0110] Table 2 Dosage groups for each group

[0111]

[0112]

[0113] 3.2.7 Statistics and Analysis

[0114] 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.

[0115] 3.2.8 Results of mouse tumor volume monitoring

[0116] Figure 6The changes in the average volume of colorectal cancer tumors in each group of mice during the experiment are shown. It can be clearly seen that the tumors of the control group, the group only adding sorbitol, and the mice treated with antiPD-1 alone increased significantly after Day 12, while the tumors of the mice treated with sorbitol and antiPD-1 were significantly inhibited from growing, with statistically significant differences (P<0.05). The T-test analysis of the individual tumor volume data of the mice in the combined drug group and the antiPD-1 monotherapy group showed that there was also a statistically significant difference between the combined drug group and the monotherapy group (P<0.05). At the same time, we also performed sorbitol testing on the peeled mouse tumors, and the results showed that the sorbitol concentration in the tumor tissue of the mice fed with sorbitol increased significantly, with statistical significance (P<0.05).

[0117] Figure 7 The photos and weight comparison of the colorectal cancer tumors peeled off from each group of mice are shown. The tumor volume of the mice in the combined drug group was statistically significantly different from that in the PBS group (P<0.05). At the end of the D20 experiment, the average tumor volume of the mice in each group is shown in Table 3 below:

[0118] Table 3 Average volume of colorectal cancer tumors in mice in each group

[0119]

[0120] To further explore the role of sorbitol in other tumors, we also conducted validation in mouse breast cancer tumor models, such as Figure 8 As shown, compared with the antiPD-1 monotherapy group, the tumor size of mice in the combination treatment group was significantly inhibited, and the tumor even disappeared in one mouse.

[0121] In this experiment, 5% sorbitol was fed to the mice with drinking water and antiPD-1 was injected intraperitoneally. The mice were given the drug once every three days for a total of 5 times. At the same time, the antiPD-1 and 5% sorbitol monotherapy groups and the PBS negative control group were set up to investigate whether the combination of antiPD-1 and sorbitol has a synergistic effect in the anti-tumor effect of mouse colon cancer CT26 and breast cancer 4T1. Single-agent antiPD-1 (intraperitoneal injection) had a certain effect on tumor growth, but there was no significant statistical difference. The application of sorbitol alone had no significant effect on tumor growth. After the combined application of sorbitol and antiPD-1, the mouse tumor showed that sorbitol and antiPD-1 had a synergistic effect in inhibiting tumor growth.

[0122] Example 4 Preliminary application of a clinical treatment plan for enhancing the efficacy of PD-1 inhibitors with sorbitol (this study has passed the ethics review of this unit)

[0123] Overall design: This is an exploratory study based on the concept of tumor metabolism and immunity, using oral sorbitol to enhance the SOX chemotherapy regimen combined with tislelizumab (PD-1 inhibitor) for the neoadjuvant treatment of advanced gastric / gastroesophageal junction adenocarcinoma. The study was divided into three phases: screening phase, treatment phase (neoadjuvant therapy, surgery and adjuvant therapy phase), and follow-up phase. Forty subjects were enrolled in this study and randomly divided into two groups. Group A: oral sorbitol + PD1 inhibitor + SOX 3 cycles (1 cycle = every 3 weeks [Q3W]); Group B: placebo + PD1 inhibitor + SOX 3 cycles (1 cycle = every 3 weeks [Q3W]).

[0124] In the above clinical protocols, all patients signed informed consent and passed the unit ethics.

[0125] During this experiment, nivolumab, toripalimab, pembrolizumab, sintilimab, carrelizumab or tislelizumab can be selected as PD-1 inhibitors.

[0126] Study objective: To investigate the safety and efficacy (major pathological response rate, MPR) of oral sorbitol in enhancing the efficacy of neoadjuvant chemotherapy combined with tislelizumab (PD-1 inhibitor) in patients with locally advanced gastric cancer;

[0127] Study subjects: A total of 40 patients diagnosed with advanced gastric / gastroesophageal junction adenocarcinoma were included in this study;

[0128] Specific research methods:

[0129] (1) Screening period: Subjects must undergo screening and evaluation within 14 days before the first medication to determine whether they meet the study conditions. Specific conditions are: histologically confirmed, untreated HER2-negative gastric cancer or gastroesophageal junction (GEJ) cancer, clinical stage cT3~4N+M0, and histological examination confirms that it is mainly adenocarcinoma. Only Siewert type III and Siewert type II subjects who do not require combined thoracotomy are allowed to enroll in the gastroesophageal junction (GEJ) cancer group. For patients who meet the inclusion and exclusion criteria, routine blood, imaging-related examinations and Helicobacter pylori tests will be completed, and further molecular level tests will be performed, including the MSI status of the biopsy pathological tissue before treatment, PD-1 expression CPS score, EBER detection, etc.

[0130] (2) Neoadjuvant therapy (pre-surgery) period: 3 cycles

[0131] The enrolled subjects were randomly divided into two groups.

[0132] Group A (sorbitol supplementary diet group): PD1 inhibitor + SOX 3 cycles (1 cycle = every 3 weeks [Q3W]); during the neoadjuvant treatment week, oral sorbitol 2-4g / time, three times a day (taken with meals).

[0133] Group B (placebo supplementary diet group): PD1 inhibitor + SOX 3 cycles (1 cycle = every 3 weeks [Q3W]); during the neoadjuvant treatment week, oral placebo 2-4g / time, three times a day (taken with meals). The placebo was selected as glucose solution.

[0134] (3) Surgery: After evaluation of the efficacy of neoadjuvant therapy

[0135] After neoadjuvant therapy, subjects who are determined to be eligible for surgery by research should undergo surgery within 2-6 weeks after the last dose of neoadjuvant therapy (including oral medication). For subjects who are unable to undergo surgery, the researchers will make a comprehensive judgment based on the clinical situation to determine the subsequent treatment plan. A small amount of pathological tissue should be taken during the operation, and the AKR1B1 / cGAS target and sorbitol content should be tested.

[0136] (4) Adjuvant therapy (post-surgery) period: 5 cycles

[0137] Adjuvant therapy phase: After surgery, the blind was unmasked and adjuvant therapy could be started 3 to 12 weeks after surgery (after 12 weeks, the investigator could decide whether to continue using the trial drug). Subjects in groups A and B were divided into groups according to TRG grading. Subjects with a TRG score of 0-1 continued to receive the original treatment regimen, and subjects with a TRG score greater than 2 only received SOX chemotherapy for a maximum of 5 cycles, or until the investigator determined that the subject lost clinical benefit, died, could not tolerate toxicity, withdrew informed consent, or for other reasons specified in the protocol (whichever occurred first).

[0138] (5) Follow-up period: including safety follow-up and survival follow-up. After the end of treatment, all subjects will be followed up. For subjects who discontinue treatment for reasons other than PD, they will be followed up according to the original planned frequency until PD occurs, informed consent is withdrawn, or they are lost to follow-up (whichever occurs first). After the end of treatment and safety follow-up, all subjects will be followed up for survival (OS data will be collected every 3 months ± 14 days) until death, withdrawal of informed consent, loss to follow-up, or termination of the study (whichever occurs first).

[0139] The preliminary results of the study are shown in Table 4 below:

[0140] Table 4 Preliminary study results of clinical treatment programs

[0141]

[0142] Fig. 9The treatment results of two gastric cancer patients are given: the two gastric cancer patients had no obvious adverse reactions during the treatment, and both achieved MPR (patient 1, TRG=1; patient 2, TRG=0).

[0143] Final pathology report of patient 1: 1. (After neoadjuvant treatment for gastric malignant tumor biopsy in another hospital) (gastroesophageal junction) well-differentiated adenocarcinoma - tumor infiltrates the muscularis mucosa, mucus is visible in the submucosal layer, and no atypical epithelial cells are found - pathological stage: ypT1a - lymphatic vessel invasion (-) - neural invasion (-) - lymph nodes (0 / 29) grouping: pericardial lymph nodes (1), (1.3 group) lymph nodes (8), (2 group) lymph nodes (1), (4 group) lymph nodes (3), (7.8.9 group) lymph nodes (12), (10 group) lymph nodes (1), (811 group) lymph nodes (2) and (912 group) lymph nodes (1) No cancer metastasis was found on the sections. - Resection margin: esophageal lateral resection margin (-) gastric body lateral resection margin (-) esophageal (proximal) resection margin (-) greater omentum (-) Immunohistochemical staining showed tumor cells: P53 (partially positive, tested two wax blocks). TRG grade of tumor after neoadjuvant therapy: grade 1 (moderate reaction, graded according to 0-3) 2. Chronic cholecystitis with gallstones. (Another consultation specimen pathology number: CI24-09390)

[0144] Patient 2 was clinically diagnosed with leather gastritis, a type of gastric cancer that is extremely insensitive to immunotherapy. After the application of a new regimen of sorbitol combined with immunotherapy, complete remission was achieved. The final pathological results showed that after neoadjuvant chemotherapy for gastric cancer (gastric antrum and body), no clear cancer residue was found under the microscope in the original tumor bed, and multiple mucus lakes were formed, the deepest of which was located in the subserosa, and floating chronic inflammatory cells were seen in the mucus lakes. - No intravascular embolism and nerve invasion were found: - No cancer was found in the lateral resection margin of the anus, pylorus, esophagus and greater omentum: - Mucus lake formation was observed in (group 3) lymph nodes (1 / 5) and (group 4) lymph nodes (4 / 14), and no viable tumor cells were found. No metastasis was found in (group 1) lymph nodes (5), (groups 5 and 6) lymph nodes (3), (group 2) lymph nodes (5), (group 7, 8 and 9) lymph nodes (2), (group 11) lymph nodes (3) and (group 12) lymph nodes (4). Tumor TRG grade: 0 (no residual cancer cells, complete response). (Pathology number of the consultation specimen is CI24-09201)

[0145] This study showed that sorbitol combined with immunotherapy has excellent safety and efficacy in humans.

[0146] In summary, we found for the first time that sorbitol and anti-PD-1 immune agents have a synergistic anti-tumor effect. Sorbitol can be used to treat cancer patients, and combined with immunotherapy has good therapeutic prospects.

[0147] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An anti-tumor combined pharmaceutical composition, characterized in that: The pharmaceutical composition comprises sorbitol and a PD-1 inhibitor.

2. The pharmaceutical composition according to claim 1, characterized in that The dosage form of the pharmaceutical composition is selected from injection, solution, emulsion, suspension, suppository, ointment, cream, spray, drop, powder, granule, granule, capsule, pill, tablet, patch or sustained-release preparation.

3. The pharmaceutical composition according to claim 1, characterized in that The sorbitol is administered once a week, and the PD-1 inhibitor is administered once every three days.

4. The pharmaceutical composition according to claim 1, characterized in that The PD-1 inhibitor is selected from nivolumab, toripalimab, pembrolizumab, sintilimab, carrelizumab or tislelizumab.

5. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients.

6. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of anti-tumor products.

7. The use according to claim 6, characterized in that: The product is an anti-tumor drug.

8. The use according to claim 7, characterized in that: The anti-tumor drug also contains one or more pharmaceutically acceptable carriers or excipients.

9. The use according to claim 8, characterized in that: The dosage form of the anti-tumor drug is selected from injection, solution, emulsion, suspension, suppository, ointment, cream, spray, drop, powder, granule, granule, capsule, pill, tablet, patch or sustained-release preparation.

10. The use according to claim 9, characterized in that: The tumor includes lung cancer, gastric cancer, colorectal cancer, endometrial cancer, liver cancer, breast cancer, melanoma, leukemia, glioma, cervical cancer, esophageal cancer or sarcoma.

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