New application of 4-hydroxyisoleucine

Abstract

The present invention belongs to the field of biomedicine technology, and specifically discloses a new use of 4-hydroxyisoleucine. The present invention first discovered through in vitro and in vivo experiments that 4-hydroxyisoleucine (4-HIL) can enhance the anti-tumor function of immune checkpoint inhibitors, and can reduce immune-related adverse events of the gastrointestinal tract, reduce the level of intestinal inflammation, and alleviate or treat gastrointestinal diseases (such as ulcerative colitis). Clinically, the treatment cost of ICB therapy is high, and 4-HIL combined with ICB therapy can effectively reduce the dosage of immune checkpoint inhibitors, reduce treatment costs, and enhance anti-tumor ability, alleviate immune-related adverse events such as gastrointestinal diseases, while reducing the patient's medication burden, and has broad clinical application prospects. The present invention provides a new candidate idea for the first-line treatment of immune checkpoint blocking therapy, and also provides a new candidate drug for the treatment of immune checkpoint inhibitor-related gastrointestinal diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a new use of 4-hydroxyisoleucine. Background Art

[0002] In recent years, cancer immunotherapy has been increasingly used in the first-line clinical treatment of cancer. Cancer immunotherapy is a new generation of tumor treatment methods that has developed rapidly after traditional treatments such as surgery, radiotherapy, and chemotherapy. Cancer immunotherapy has great clinical application prospects by restoring T cell activity, reversing the exhausted phenotype of T cells, and reactivating the immune system to enhance anti-tumor ability. Among them, tumor immunotherapy targeting immune checkpoint blockade (ICB) of CTLA-4, PD-1 and its ligand PD-L1 has been applied to the clinical treatment of many tumor types, such as melanoma, non-small cell lung cancer, and renal cancer.

[0003] At present, immune checkpoint inhibitors have become the core pillar of cancer treatment. In developed countries in North America, nearly half of metastatic cancer patients have received immune checkpoint blockade therapy. As of the end of 2021, there are 8 approved immune checkpoint inhibitors for the treatment of 17 different tumor types, including non-small cell lung cancer, lymphoma, melanoma, etc. Immune checkpoint blockade therapy is increasingly used in several new adjuvants and maintenance therapies, and is also often used in combination therapy, including other types of ICIs, cytotoxic chemotherapy, biological or targeted therapy. It is well known in the art that immune checkpoints are receptors expressed by immune cells that can dynamically regulate immune homeostasis and are related to T cell function. For example, PD-1 and its main ligand PD-L1 are expressed on T cells, tumor cells, and tumor-infiltrating myeloid cells, respectively. The interaction between these two proteins will lead to T cell exhaustion, reduced or absent immune cell function, lack of response to stimulation, and changes in transcriptional and epigenetic states. Immune checkpoints play a very important role in limiting autoimmune-related diseases, maintaining fetal tolerance during pregnancy, and preventing rejection of organ transplants. However, tumor cells use this interaction to maintain immune tolerance and establish an immunosuppressive microenvironment, thereby escaping immunity. Compared with PD-1, CTLA-4 plays a more important role in immune activation. Compared with the T cell co-stimulatory molecule CD28, CTLA-4 has a higher affinity for the dendritic cell ligand B7 (also known as CD80), thereby limiting the level of T cell activation in the startup phase. The expression of CTLA-4 can also enhance its function in the tumor microenvironment and promote the expansion of regulatory T (Treg) cells. Therefore, blocking the interaction between immune checkpoints and their ligands can inhibit the exhaustion of T cells, rejuvenate T cells or delay the aging of T cells, thereby exerting the anti-tumor ability of immune cells. Since 2011, the U.S. Food and Drug Administration (FDA) has approved a variety of immune checkpoint inhibitors targeting PD-1 and its ligands PD-L1 and CTLA-4, such as Pembrolizumab, Nivolumab (PD-1 monoclonal antibody), Atezolizumab (PD-L1 monoclonal antibody) and Ipilimumab (CTLA-4 monoclonal antibody) for clinical treatment, which have significantly improved the remission rate, overall survival rate and survival time of patients.

[0004] However, with the gradual application of immune checkpoint inhibitors (ICIs) in clinical practice, a key challenge has emerged, namely, the uncontrolled side effects on the immune system, which causes the overactivated immune system to produce a series of adverse reactions related to immune mechanisms, collectively referred to as immune-related adverse events (irAEs). The toxicity of ICIs is different from that of standard chemotherapy or other biological adjuvants. Most toxicities are caused by excessive immunity to normal organs. Compared with the negative effects of traditional tumor therapies, immune-related adverse events have a late onset, a long duration, and may involve any organ. If they cannot be treated in time, they will not only affect the patient's prognosis, but also force the interruption of tumor treatment, which can lead to death in severe cases. With the increase in the use of ICIs for cancer treatment, the annual cumulative number of irAEs has increased exponentially, with nearly 13,000 adverse events related to immune checkpoint inhibitors reported in 2018 alone. The incidence of ICB-induced irAEs of varying degrees can be as high as 70%-90%, and can affect almost every organ in the human body, including the skin, intestines, liver, kidneys, eyes, endocrine tissues, and even the central nervous system. Most immune-related adverse events occur early in the treatment process, but delayed events (defined as irAEs that occur after 1 year of treatment) also occur frequently. In a clinical report of a group of 118 patients, the incidence of high-grade delayed irAEs was 5.3%, and the most common irAEs were colitis, rash, and pneumonia. These adverse effects are usually low-grade, treatable or reversible. However, some adverse effects can be serious and can lead to permanent diseases.

[0005] Clinical data show that nearly 60% of patients treated with a combination of PD-1 and CTLA-4 antibodies experience severe inflammatory reactions. The molecular mechanisms of these immune-related adverse events are unclear, and there is currently no optimal clinical treatment. Among them, the most common inflammatory reactions occur at barrier sites, including the gastrointestinal mucosa, liver, skin, and lungs. Among all immune-related adverse events, immune checkpoint inhibitor-induced gastrointestinal diseases (such as colitis) are one of the most common severe irAEs and an important cause of interruption in tumor treatment, especially in patients with combined use of PD-1 monoclonal antibodies and CTLA-4 monoclonal antibodies. Among the adverse events induced by immune checkpoint inhibitors, the incidence of CTLA-4 inhibitors is about 35%, the incidence of PD-1 inhibitors is about 20%, and the incidence of combined treatment of CTLA-4 and PD-1 inhibitors is above 40%. Among them, the incidence of colitis is 12%, 1%, and 14%, respectively. Gastrointestinal diseases are usually manifested clinically as diarrhea, less often as abdominal pain and bloody stools, and in severe cases, patients may experience intestinal perforation, which is life-threatening. Studies have shown that CTLA-4 plays a more important role in intestinal homeostasis than PD-1 and its ligand PD-L1. For example, mild colitis is more common in patients treated with ipilimumab, and nearly half of patients receiving high-dose or combination therapy experience diarrhea. Severe colon inflammation is less common, but still has an incidence of 10%-20%, which can be life-threatening in severe cases. Studies have reported that immune checkpoint inhibitor-associated enteritis is usually characterized by persistent inflammation from the rectum to the cecum, and histopathological analysis has found a high proportion of lymphocytes and an increased number of apoptotic epithelial cells. Like most other severe irAEs, current treatments only include the use of high-dose corticosteroids or discontinuation of immune checkpoint blockade therapy.

[0006] At present, the effect of systemic corticosteroids on anti-tumor response is still unclear in clinical practice, but clinical retrospective data show that high-dose corticosteroids may reduce the efficacy of immunotherapy and reduce anti-tumor response. Preliminary clinical data show that tumor necrosis factor α (TNF-α) blockers can effectively treat immune checkpoint inhibitor-associated enteritis that is resistant to corticosteroids, but there are still few types of related drugs, and the mechanism of action and toxic side effects are not clear. Therefore, it is necessary to develop a drug that can be used to alleviate immune checkpoint inhibitor-associated gastrointestinal diseases without compromising anti-tumor immunity, as a specific alternative treatment for immune checkpoint inhibitor-associated enteritis.

[0007] Traditional Chinese medicine has a long history in Asian countries, dating back thousands of years. A common class of traditional medicines includes medicinal mushrooms, such as Cnidium monnieri, Cinnamon bark and Auricularia auricula, which contain a variety of immunomodulatory and bioactive compounds. Fenugreek is a plant of the Leguminosae family and has been used as a traditional Chinese medicine to treat common diseases such as diabetes. In addition, fenugreek seed extracts have shown antitumor activity in a variety of tumor models. Although current studies have not identified the antitumor active ingredients in the extracts, the development of new antitumor preparations based on natural medicines still has broad clinical application prospects. 4-Hydroxyisoleucine (4-HIL) is one of the active ingredients of fenugreek. 4-HIL is a non-protein amino acid and a branched-chain amino acid derivative, accounting for about 80% of the total free amino acid content in fenugreek seeds. Studies have reported that isoleucine in fenugreek can be converted to 4-HIL through a direct pathway, which has the activity of lowering blood sugar and promoting insulin secretion both in vivo and in vitro. In addition, previous studies have found that 4-HIL can improve glucose metabolism and lipid metabolism disorders caused by obesity in an in vivo obesity model and reverse insulin resistance, but research on its anti-tumor activity has not been reported. Summary of the invention

[0008] The main technical problem solved by the present invention is to provide a new use of 4-hydroxyisoleucine for alleviating or treating gastrointestinal diseases associated with immune checkpoint inhibitors.

[0009] Secondly, the present invention also provides a drug of 4-hydroxyisoleucine combined with ICB therapy, which can enhance the anti-tumor ability of ICB therapy while alleviating or treating gastrointestinal diseases associated with immune checkpoint inhibitors.

[0010] Finally, the present invention also provides a use of 4-hydroxyisoleucine in preparing a medicine for alleviating or treating gastrointestinal diseases.

[0011] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0012] The application of 4-hydroxyisoleucine is: the application of 4-hydroxyisoleucine in preparing a drug for alleviating or treating immune-related adverse events.

[0013] As a preferred embodiment of the present invention, the immune-related adverse events include but are not limited to gastrointestinal immune-related adverse events, i.e., immune checkpoint inhibitor-related gastrointestinal diseases, and gastrointestinal diseases caused by the use of immune checkpoint inhibitors.

[0014] As a preferred embodiment of the present invention, the immune checkpoint inhibitor-related gastrointestinal diseases include but are not limited to gastritis, enteritis (such as colitis, proctitis), etc.

[0015] As a preferred embodiment of the present invention, the immune checkpoint inhibitor includes but is not limited to one or more of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibitors, programmed cell death protein 1 (PD-1) inhibitors, programmed cell death-ligand 1 (PD-L1) inhibitors, programmed cell death-ligand 2 (PD-L2) inhibitors, lymphocyte activation gene 3 (LAG-3, or CD223) inhibitors, T cell immunoglobulin mucin 3 (TIM-3) inhibitors, etc. In addition, the following immune checkpoint inhibitors can also be used: galectin 3 (GAL3), galectin 9 (GAL9), B and T lymphocyte weakening factor (BTLA), B7-H1, B7-H3, B7-H4, T cell immune receptor TIGIT / Vstm3 / WUCAM / VSIG9 with Ig and ITIM domains, V domain Ig inhibitor of T cell activation (VISTA), etc. The immune checkpoint inhibitor can be in the form of monoclonal antibodies, double antibodies, etc. The 4-hydroxyisoleucine has a significant synergistic effect on immune checkpoint inhibitors.

[0016] As a preferred embodiment of the present invention, the dosage form of the drug is a pharmaceutically acceptable dosage form, including but not limited to (lyophilized) powder injection, injection, tablet, pill, capsule, spray, dispersion, etc. In order to prepare a specific pharmaceutical dosage form, the drug also includes a pharmaceutically acceptable carrier, including but not limited to one or more of excipients, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, buffers, etc.

[0017] As a preferred embodiment of the present invention, the administration route of the drug is a pharmaceutically acceptable route, including but not limited to oral, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, topical administration, and transdermal, intranasal or oral inhalation.

[0018] As a preferred embodiment of the present invention, the dosage of the drug is a pharmaceutically acceptable dosage.

[0019] A drug for 4-hydroxyisoleucine combined with ICB therapy, the drug comprising a pharmaceutically effective amount of 4-hydroxyisoleucine and an immune checkpoint inhibitor.

[0020] As a preferred embodiment of the present invention, the content of 4-hydroxyisoleucine in the drug can be appropriately increased to reduce the content of immune checkpoint inhibitors (i.e., the content of immune checkpoint inhibitors is lower than that of drugs of the same type that only use immune checkpoint inhibitors as active ingredients), thereby reducing treatment costs and alleviating the medication burden on patients.

[0021] As a preferred embodiment of the present invention, the immune checkpoint inhibitor includes but is not limited to one or more of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibitors, programmed cell death protein 1 (PD-1) inhibitors, programmed cell death-ligand 1 (PD-L1) inhibitors, programmed cell death-ligand 2 (PD-L2) inhibitors, lymphocyte activation gene 3 (LAG-3, or CD223) inhibitors, T cell immunoglobulin mucin 3 (TIM-3) inhibitors, etc. In addition, the following immune checkpoint inhibitors can also be used: galectin 3 (GAL3), galectin 9 (GAL9), B and T lymphocyte weakening factor (BTLA), B7-H1, B7-H3, B7-H4, T cell immune receptor TIGIT / Vstm3 / WUCAM / VSIG9 with Ig and ITIM domains, V domain Ig inhibitor of T cell activation (VISTA), etc. The immune checkpoint inhibitor can be in the form of monoclonal antibodies, double antibodies, etc.

[0022] As a preferred embodiment of the present invention, the dosage form of the drug is a pharmaceutically acceptable dosage form, including but not limited to (lyophilized) powder injection, injection, tablet, pill, capsule, spray, dispersion, etc. In order to prepare a specific pharmaceutical dosage form, the drug includes a pharmaceutically acceptable carrier in addition to a pharmacologically effective amount of 4-hydroxyisoleucine and an immune checkpoint inhibitor, including but not limited to one or more of an excipient, a preservative, a stabilizer, a wetting agent, an emulsifier, a salt for adjusting osmotic pressure, a buffer, etc.

[0023] As a preferred embodiment of the present invention, the administration route of the drug is a pharmaceutically acceptable route, including but not limited to oral, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, topical administration, and transdermal, intranasal or oral inhalation.

[0024] As a preferred embodiment of the present invention, the dosage of the drug is a pharmaceutically acceptable dosage.

[0025] The application of 4-hydroxyisoleucine is: the application of 4-hydroxyisoleucine in preparing medicines for alleviating or treating gastrointestinal diseases.

[0026] As a preferred embodiment of the present invention, the gastrointestinal diseases include but are not limited to gastritis, enteritis (such as colitis, proctitis), peptic ulcer and the like.

[0027] As a preferred embodiment of the present invention, the content of 4-hydroxyisoleucine in the drug is an effective amount.

[0028] As a preferred embodiment of the present invention, the dosage form of the drug is a pharmaceutically acceptable dosage form, including but not limited to (lyophilized) powder injection, injection, tablet, pill, capsule, spray, dispersion, etc. In order to prepare a specific pharmaceutical dosage form, the drug also includes a pharmaceutically acceptable carrier, including but not limited to one or more of excipients, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, buffers, etc.

[0029] As a preferred embodiment of the present invention, the administration route of the drug is a pharmaceutically acceptable route, including but not limited to oral, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, topical administration, and transdermal, intranasal or oral inhalation.

[0030] As a preferred embodiment of the present invention, the dosage of the drug is a pharmaceutically acceptable dosage.

[0031] Beneficial effects of the present invention:

[0032] The present invention uses dextran sulfate sodium salt (DSS) to establish an ulcerative colitis animal model, monitors its body weight, intestinal pathology and inflammatory cytokines, and thus determines the degree of intestinal inflammation in mice. Experimental results show that after DSS induction, mice develop enteritis symptoms, significantly reduce body weight, reduce survival rate, increase in intestinal epithelial inflammatory cell infiltration, and significantly increase the level of inflammatory factors, indicating that the ulcerative colitis animal model is successfully established, and after treatment with 4-hydroxyisoleucine (4-HIL), the weight loss of mice slows down, the survival rate increases, the infiltration of intestinal epithelial inflammatory cells decreases, the level of inflammatory factors significantly decreases, and the level of anti-inflammatory factors increases, indicating that 4-HIL can relieve ulcerative colitis in vivo, and can be used to prepare drugs for relieving or treating gastrointestinal diseases.

[0033] The present invention also uses DSS and immune checkpoint inhibitors to establish an ICB animal model, monitors its tumor volume, tumor weight, body weight and intestinal pathology, so as to judge the anti-tumor effect and the degree of intestinal inflammation in mice. The experimental results show that after DSS induction and ICIs administration, the tumor volume and tumor weight are reduced, the weight of mice is significantly reduced, the survival rate is reduced, the infiltration of intestinal epithelial inflammatory cells is significantly increased, and the level of inflammatory factors is significantly increased. After 4-HIL combined with ICIs treatment, it was found that the tumor volume and tumor weight were significantly reduced, the weight of mice was not significantly reduced, the survival rate was increased, the infiltration of intestinal epithelial inflammatory cells was reduced, the level of inflammatory factors was reduced, the level of anti-inflammatory factors was increased, and the drug had no obvious toxic and side effects during treatment, indicating that 4-HIL combined with ICB therapy can relieve immune checkpoint inhibitor-related enteritis while enhancing anti-tumor ability, and can be used to prepare anti-tumor drugs combined with ICB therapy. Due to the high cost of immune checkpoint inhibitors, 4-HIL combined with ICB therapy can effectively reduce the dosage of immune checkpoint inhibitors, while enhancing anti-tumor ability and alleviating immune-related adverse events such as gastrointestinal diseases, reducing treatment costs, and reducing the burden of medication for patients, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The experimental results of 4-HIL in Experimental Example 1 for alleviating acute colitis induced by DSS in mice;

[0035] In the figure, A, B: mouse colon length and statistical results; C: mouse body weight; D: H&E staining of mouse colon tissue; E: real-time fluorescence quantitative PCR detection of mRNA levels of related cytokines in colon tissue.

[0036] Figure 2 The experimental results of Experimental Example 2 show that 4-HIL alleviates immune checkpoint inhibitor-associated enteritis and enhances anti-tumor ability;

[0037] In the figure, A: mouse body weight; B: mouse tumor volume; C: mouse tumor weight; D: H&E staining of mouse colon tissue; E, F: mouse colon length and statistical results.

[0038] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the above drawings obtained in the experimental examples are briefly introduced. It should be understood that the above drawings only show some experimental examples of the present invention and should not be regarded as any limitation on the scope of protection of the claims. For ordinary technicians in this field, other related drawings can also be obtained based on these drawings without creative work. DETAILED DESCRIPTION

[0039] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments and experimental examples. However, it should be understood by those skilled in the art that the embodiments are only used to illustrate the technical solution of the present invention and should not be regarded as limiting the scope of protection of the present invention. Based on the following embodiments, all other implementation schemes obtained by ordinary technicians in the field without making creative work, such as modification, deformation or simple replacement, should belong to the scope of protection of the present invention.

[0040] Unless otherwise specified, the experimental methods used in the following embodiments and experimental examples are all conventional methods; the raw materials (including biological materials), reagents, culture media, instruments, etc. used are all commonly used in the art, available to the public or commercially available unless otherwise specified; the terms and abbreviations involved have the conventional meanings in the art, such as PBS is phosphate buffered saline.

[0041] Example 1

[0042] This embodiment provides an application of 4-hydroxyisoleucine, specifically: application of 4-hydroxyisoleucine in the preparation of a drug for alleviating or treating immune-related adverse events. The adult dosage of 4-hydroxyisoleucine is 5-50 mg / kg body weight, preferably 10-20 mg / kg body weight.

[0043] Example 2

[0044] This embodiment provides an application of 4-hydroxyisoleucine, specifically: the application of 4-hydroxyisoleucine in the preparation of a drug for alleviating or treating immune-related adverse events in the gastrointestinal tract. The immune-related adverse events in the gastrointestinal tract are specifically immune checkpoint inhibitor-related gastrointestinal diseases, that is, gastrointestinal diseases caused by the use of immune checkpoint inhibitors, such as enteritis (including colitis).

[0045] This embodiment also provides a drug for 4-hydroxyisoleucine combined with ICB therapy, the drug includes a pharmacological amount of 4-hydroxyisoleucine and an immune checkpoint inhibitor, the immune checkpoint inhibitor is a CTLA-4 and / or PD-1 monoclonal antibody, and its content is lower than that of the same type of drug that only uses immune checkpoint inhibitors as an effective ingredient. The dosage form of the drug is an injection; in addition to the above-mentioned effective ingredients, the drug also includes pharmaceutical excipients such as preservatives and stabilizers. The route of administration of the drug is intravenous injection, and the dosage is a clinical pharmaceutical dose.

[0046] In other embodiments of the present invention, the immune checkpoint inhibitors include but are not limited to one or more of CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, D-L2 inhibitors, LAG-3 inhibitors, TIM-3 inhibitors, etc., and the immune checkpoint inhibitors can be in the form of monoclonal antibodies or dual antibodies.

[0047] Example 3

[0048] This embodiment provides an application of 4-hydroxyisoleucine, specifically: application of 4-hydroxyisoleucine in the preparation of a drug for alleviating or treating a gastrointestinal disease, wherein the gastrointestinal disease is specifically enteritis (including colitis).

[0049] This embodiment also provides a drug for alleviating or treating gastrointestinal diseases, the drug comprising a pharmacologically effective amount of 4-hydroxyisoleucine. The drug is in the form of an injection; in addition to the above-mentioned pharmacologically effective ingredients, the drug also comprises pharmaceutical excipients such as preservatives and stabilizers. The drug is administered by intravenous injection, and the dosage is a clinical pharmaceutical dosage.

[0050] In other embodiments of the present invention, the drug for alleviating or treating a disease can be prepared into any medicinal acute drug using conventional technical means and commonly used excipients based on the common knowledge in the art.

[0051] Experimental Example 1 4-HIL alleviates DSS-induced acute colitis in mice

[0052] 1. Experimental Materials

[0053] Experimental animals: Female C57 / 6N mice, 6-8 weeks old, SPF grade, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The mice were raised in the SPF animal experimental center. The feeding conditions strictly followed the SPF standard. They were fed with sterilized drinking water and sterile feed. The ambient humidity was about 60%, the temperature was about 25°C, and the light and dark cycle was 12 hours. The mice were adapted to the environment for one week, and the experiment was started after no abnormalities were observed.

[0054] Experimental reagents: tissue cell lysis buffer, purchased from Beijing Solebow Technology Co., Ltd.; dextran sulfate sodium salt (DSS), purchased from Dalian Meilun Biotechnology Co., Ltd.; 4-HIL, purchased from Henan Julong Bioengineering Co., Ltd.; LightCycler 480SYBR Green I Master, purchased from Roche; reverse transcription reagent, purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0055] 2. Experimental Methods

[0056] 1. Establishment of acute colitis model in mice

[0057] Establishment of experimental animal model of ulcerative colitis induced by dextran sulfate sodium salt (DSS): A total of 21 female mice of C57 / 6N strain of 6-8 weeks old were randomly divided into 3 groups according to body weight, with 7 mice in each group, namely normal control group, saline solvent group and 100 mg / kg 4-HIL drug group. 4-HIL was suspended in 0.9% saline. On the first day of the modeling experiment, the model group was given 200 μL of saline and 100 mg / kg 4-HIL for gavage for 9 consecutive days. After the model group was given 3% dextran sulfate sodium salt dissolved in drinking water for 7 days, it was given normal drinking water for two days. The normal control group was given normal drinking water for 9 consecutive days. The mice were killed by cervical dislocation on the 10th day.

[0058] 2. Observe and record the disease conditions of experimental animals

[0059] Since the establishment of the ulcerative colitis experimental animal model, the survival status of the mice was continuously observed and recorded every day, and the body weight was measured until the model was completed. After the model was completed, the experimental animals in each group were killed, and the colon tissues of the experimental mice were taken to observe the lesions of the colon tissues, and the colon length was measured and recorded.

[0060] 3. Paraffin sections and hematoxylin-eosin (H&E) staining of colon tissue

[0061] Mouse colon tissue fixed in 10% formalin solution was taken, washed 3 times with PBS, and placed in the following solutions for ethanol dehydration and transparency, the steps are: 75% ethanol solution for 20 minutes; 80% ethanol solution for 20 minutes; 95% ethanol solution I for 20 minutes; 95% ethanol solution II for 20 minutes; anhydrous ethanol I for 20 minutes; anhydrous ethanol II for 20 minutes; xylene for 10 minutes for transparency. The dehydrated and transparent samples were embedded in paraffin, sliced ​​(10μm) by a microtome, and fixed on a slide for H&E staining. Hydration steps: the slices were placed in a 60℃ incubator for 30 minutes for dewaxing; xylene I for 3 minutes for dewaxing; xylene II for 3 minutes for dewaxing; xylene III for 3 minutes; anhydrous ethanol I for 3 minutes; anhydrous ethanol II for 3 minutes; 95% ethanol solution I for 3 minutes; 95% ethanol solution II for 3 minutes; 75% ethanol solution for 3 minutes; deionized water for 3 minutes. The sample sections were stained with H&E as follows: hematoxylin staining for 10 minutes; washed twice with deionized water; differentiated with 1% hydrochloric acid for 30 seconds; washed once with deionized water, and blued with 1% ammonia for 30 seconds; washed once with deionized water; 75% ethanol solution for 3 minutes; 95% ethanol solution for 3 minutes; 95% ethanol solution with eosin for 10 minutes; washed twice with 95% ethanol solution; anhydrous ethanol for 1 minute; xylene I for 3 minutes; xylene II for 3 minutes; xylene III for 3 minutes. The sections were placed in a fume hood to dry naturally, sealed with neutral gum, and photographed under a microscope.

[0062] 4. Real-time fluorescence quantitative PCR

[0063] 4.1 RNA extraction

[0064] (1) After the tissue is ground in liquid nitrogen, add 1 mL of tissue cell lysis buffer and pipette until no clumping tissue is visible and the solution is clear and non-viscous.

[0065] (2) Use the commercial kit Total RNA Isolation Hanbook to extract total RNA according to the instructions.

[0066] (3) Take 2 μL of the extracted total RNA and detect the RNA concentration using NanoDrop.

[0067] 4.2. Reverse transcription of total RNA into cDNA

[0068] (1) The reverse transcription reagents were stored at -20°C. After thawing, each component was gently centrifuged and placed on ice.

[0069] (2) Sterile nuclease-free PCR tubes were placed on ice in advance. First, the first-strand synthesis was performed by adding the following components in order: 4 μL of 4×g DNA wiper mix, 1 μg of RNA (volume = 1 μg / RNA concentration), and DEPC H2O to make up to 16 μL.

[0070] (3) If the RNA template contains secondary structures or has a high GC content, gently mix the template and primers and centrifuge briefly.

[0071] (4) Place the PCR tube into the PCR instrument and set the program: 42°C, 2 min.

[0072] (5) Place the PCR tube on ice, add 4 μL 5×HiscriptⅢRT supermix to the first-strand synthesis system, mix gently, and centrifuge briefly.

[0073] (6) Set the PCR instrument program as follows: 37°C for 15 min; 85°C for 5 s; terminate the reaction and store the resulting product, cDNA, at -80°C for later use.

[0074] 4.3 Fluorescence quantitative PCR

[0075] (1) β-actin was used as the internal reference gene, and the primers in Table 1 (as shown in SEQ ID NOs: 1-10) were commissioned to be synthesized by BGI.

[0076] Table 1 Fluorescence quantitative PCR primers

[0077]

[0078] (2) The reaction system is 12 μL. Add the components listed in Table 2 in sequence on ice and away from light.

[0079] Table 2 Fluorescence quantitative PCR reaction system

[0080]

[0081] (3) After all the solutions are added, mix the solutions evenly with a spray gun and cover with sealing film, then place in a centrifuge and centrifuge at 3500 rpm for 5 minutes.

[0082] (4) After centrifugation, place the fluorescence quantitative PCR reaction plate into the fluorescence quantitative PCR instrument and set the reaction program in Table 3.

[0083] Table 3 Fluorescence quantitative PCR reaction procedure

[0084]

[0085] 5. Statistics

[0086] Unpaired Student's T-test was used for statistical analysis of intergroup differences. Unless otherwise specified, data are expressed as Mean ± SEM. When the P value was less than 0.05, the difference was considered statistically significant. * is the difference compared with the normal control group, *P < 0.05, **P < 0.01, ***P < 0.001, # is the difference between the LPS + IFN-γ group and the control group, #P < 0.05, ##P < 0.01, ###P < 0.001. Results are shown in Figure 1 shown.

[0087] 3. Experimental Results

[0088] 1. 4-HIL alleviates ulcerative colitis in C57 / 6N mice

[0089] No death occurred in any group of experimental animals. The mice in the normal group did not lose weight, have soft or bloody stools, decreased activity, or hair deterioration. The model groups induced by dextran sulfate sodium (DSS) all showed the following symptoms to varying degrees: mental depression, decreased activity, messy hair, decreased food intake, weight loss, diarrhea, soft or bloody stools, and decreased colon length in colon tissue.

[0090] Effects of 4-HIL on colon length in dextran sulfate sodium-induced ulcerative colitis in mice Figure 1 A. Figure 1As shown in B, the normal group mice had no blood in their stools and the intestinal contents were good; the model group mice had significantly reduced colon length, blood in their stools, and viscous intestinal contents; the 4-HIL drug group could inhibit the reduction in colon length induced by DSS, and there was no obvious blood in the intestine. The experimental results show that 4-HIL has a therapeutic effect on DSS-induced ulcerative colitis mice.

[0091] 4-HIL affects the body weight changes in mice with ulcerative colitis induced by dextran sulfate sodium. Figure 1 As shown in C, the weight of mice in the experimental control group given normal drinking water gradually increased; the weight of mice in the DSS-induced ulcerative colitis model group decreased significantly, starting from the 5th day of DSS induction and reaching the lowest level on the 9th day. The 4-HIL drug group could inhibit the weight loss of mice induced by DSS. The experimental results show that 4-HIL has a therapeutic effect on DSS-induced ulcerative colitis mice.

[0092] 4-HIL affects the histopathological HE staining results of dextran sulfate sodium-induced ulcerative colitis in mice, such as Figure 1 As shown in D, the colon tissue structure of the mice in the experimental control group given normal drinking water was intact, and the crypts were clearly visible; HE staining of the colon tissue pathological sections of the mice in the DSS-induced ulcerative colitis model group showed: edema and thickening of the intestinal wall, infiltration of inflammatory cells in the mucosal layer and submucosal layer, destruction of the crypt structure, destruction and disappearance of goblet cells; the colon tissue morphology of the mice in the 4-HIL drug group was improved, among which the goblet cells and their crypts were relatively intact, the mucosal damage was reduced, and the infiltration of inflammatory cells was reduced. The experimental results show that 4-HIL has a therapeutic effect on mice with DSS-induced ulcerative colitis.

[0093] 2.4-HIL inhibits the mRNA level of inflammatory factors in colon tissue of ulcerative colitis model mice

[0094] The same part of mouse colon tissue was selected to extract total RNA and detect the expression level of relevant cytokine mRNA. The experimental results are as follows Figure 1 As shown in E, the mRNA levels of inflammatory factors TNF-α and IL-1β in the colon tissue of DSS-induced mice were significantly increased compared with the normal control group, while the mRNA levels of inflammatory factors TNF-α and IL-1β in the colon tissue of mice in the 4-HIL drug group were significantly decreased compared with the DSS-induced group, and the mRNA levels of anti-inflammatory factors TGF-β and IL-10 increased. The experimental results show that DSS-induced an increase in the level of inflammatory factors in the colon tissue of mice, and 4-HIL has a therapeutic effect on DSS-induced ulcerative colitis mice.

[0095] Experimental Example 2 4-HIL relieves immune checkpoint inhibitor-associated enteritis

[0096] 1. Experimental Materials

[0097] Experimental animals: Female C57 / 6N mice, 6-8 weeks old, SPF grade, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The mice were raised in the SPF animal experimental center. The feeding conditions strictly followed the SPF standard. They were fed with sterilized drinking water and sterile feed. The ambient humidity was about 60%, the temperature was about 25°C, and the light and dark cycle was 12 hours. The mice were adapted to the environment for one week, and the experiment was started after no abnormalities were observed.

[0098] 2. Experimental Methods

[0099] 1. Establishment of tumor immune checkpoint inhibitor-associated enteritis model

[0100] A total of 28 6-8 week old female mice of C57 / 6N strain were randomly divided into 4 groups, with 7 mice in each group, namely normal control group, dextran sulfate sodium salt group, immune checkpoint blockade treatment group and immune checkpoint blockade combined with 150 mg / kg 4-HIL drug group. 4-HIL was suspended in 0.9% normal saline. The modeling group was given 3% dextran sulfate sodium salt dissolved in drinking water for 5 days, and then normal drinking water for 12 days. The normal control group was given normal drinking water for 14 consecutive days. MC38 cells were digested with trypsin, centrifuged, resuspended in PBS, counted under a microscope, and the cell concentration was adjusted with PBS. Use a 1mL syringe to take 200μL of cell suspension and inject it subcutaneously into the right side of C57 mice. The total number of cells injected per mouse was 1×10 5 When the tumor volume reaches 30-60mm 3 The mice were weighed with an electronic balance and the tumors were measured with a digital vernier caliper every other day. The changes in the tumor volume were calculated and recorded according to the following formula: V = 1 / 2 × a (length) × b (width) × c (height). On the 7th and 14th days after tumor loading, tumor immune checkpoint inhibitors (anti-CTLA-4 antibody 100 μg / mouse / time, anti-PD-1 antibody 100 μg / mouse / time) were intraperitoneally injected. After 14 days of administration, the mice were killed by cervical dislocation.

[0101] 2. Observe and record the disease conditions of experimental animals

[0102] Since the establishment of the tumor immune checkpoint inhibitor-associated enteritis model, the survival status of the mice was continuously observed and recorded every day, and the body weight and tumor volume were measured until the model was completed. After the model was completed, the experimental animals in each group were killed, and the colon and tumor tissues of the experimental mice were taken to observe the lesions of the colon tissues, and the colon length was measured and recorded.

[0103] 3. Paraffin sections and hematoxylin-eosin (H&E) staining of colon tissue

[0104] Mouse colon tissue fixed in 10% formalin solution was taken, washed 3 times with PBS, and placed in the following solutions for ethanol dehydration and transparency, the steps are: 75% ethanol solution for 20 minutes; 80% ethanol solution for 20 minutes; 95% ethanol solution I for 20 minutes; 95% ethanol solution II for 20 minutes; anhydrous ethanol I for 20 minutes; anhydrous ethanol II for 20 minutes; xylene for 10 minutes for transparency. The dehydrated and transparent samples were embedded in paraffin, sliced ​​(10μm) by a microtome, and fixed on a slide for H&E staining. Hydration steps: the slices were placed in a 60℃ incubator for 30 minutes for dewaxing; xylene I for 3 minutes for dewaxing; xylene II for 3 minutes for dewaxing; xylene III for 3 minutes; anhydrous ethanol I for 3 minutes; anhydrous ethanol II for 3 minutes; 95% ethanol solution I for 3 minutes; 95% ethanol solution II for 3 minutes; 75% ethanol solution for 3 minutes; deionized water for 3 minutes. The sample sections were stained with H&E as follows: hematoxylin staining for 10 minutes; washed twice with deionized water; differentiated with 1% hydrochloric acid for 30 seconds; washed once with deionized water, and blued with 1% ammonia for 30 seconds; washed once with deionized water; 75% ethanol solution for 3 minutes; 95% ethanol solution for 3 minutes; 95% ethanol solution with eosin for 10 minutes; washed twice with 95% ethanol solution; anhydrous ethanol for 1 minute; xylene I for 3 minutes; xylene II for 3 minutes; xylene III for 3 minutes. The sections were placed in a fume hood to dry naturally, sealed with neutral gum, and photographed under a microscope.

[0105] 4. Statistics

[0106] The data statistics method is the same as that of Experimental Example 1. Figure 2 shown.

[0107] 3. Experimental Results

[0108] 1. 4-HIL combined with ICB therapy enhances its anti-tumor ability

[0109] A subcutaneous melanoma model was established in mice. Anti-CTLA-4 and anti-PD-1 antibodies were used to simulate clinical ICB therapy, i.e., the combined therapy of PD-1 and CTLA-4 monoclonal antibodies for the treatment of tumor patients. The anti-tumor effect of ICB in the mouse model was observed. The experimental results are as follows: Figure 2 Comparing the tumor volume and tumor growth rate, the tumor growth rate of mice in the 4-HIL combined with ICB treatment group was significantly slowed down, and the volume was significantly smaller than that in the ICB treatment group, that is, 4-HIL combined with ICB therapy can effectively inhibit the growth of mouse melanoma ( Figure 2 B). Comparing the tumor weight of mice on the 21st day, the tumor weight of mice treated with 4-HIL combined with ICB was significantly smaller than that of mice treated with ICB ( Figure 2 C). The experimental results showed that 4-HIL combined with ICB therapy can effectively enhance its anti-tumor ability.

[0110] 2. 4-HIL alleviates tumor immune checkpoint inhibitor-associated enteritis

[0111] There was no death in any of the experimental animals. The mice in the normal group did not lose weight, have soft or bloody stools, decreased activity, or poor hair. The simple dextran sulfate sodium (DSS) group and the dextran sulfate sodium-induced ICB treatment group all showed the following symptoms to varying degrees: mental depression, decreased activity, messy hair, decreased food intake, weight loss, diarrhea, soft or bloody stools, and reduced colon length in colon tissue.

[0112] Effect of 4-HIL on the length of lesion colon in mice with immune checkpoint inhibitor-associated enteritis model, such as Figure 2 E. Figure 2 As shown in F, the normal group mice had no blood in their stools and good intestinal contents; the mice in the ICB treatment group induced by dextran sulfate sodium had significantly reduced colon length, blood in the intestine, and viscous intestinal contents compared with the dextran sulfate sodium (DSS) group; the 4-HIL combined with ICB treatment group could inhibit the reduction of colon length induced by DSS, and there was no obvious blood in the intestine. The experimental results show that 4-HIL can alleviate tumor immune checkpoint inhibitor-related enteritis.

[0113] 4-HIL affects the weight changes of mice in the immune checkpoint inhibitor-associated enteritis model. Figure 2 As shown in A, the weight of mice in the experimental control group given normal drinking water gradually increased; the weight of mice in the dextran sulfate sodium (DSS) group and the dextran sulfate sodium-induced ICB treatment group decreased significantly, and the weight of mice in the model group began to decrease on the 13th day until it reached the lowest level on the 21st day. The 4-HIL administration treatment group could inhibit the DSS-induced weight loss of mice. The experimental results show that 4-HIL can alleviate tumor immune checkpoint inhibitor-associated enteritis.

[0114] 4-HIL affects the histopathological HE staining results of the colon in mice with immune checkpoint inhibitor-associated enteritis model, such as Figure 2As shown in D, the colon tissue structure of mice in the normal control group was intact, and the crypts were clearly visible; HE staining of the colon tissue pathological sections of mice in the dextran sulfate sodium (DSS) group and the dextran sulfate sodium-induced ICB treatment group showed: intestinal wall edema and thickening, inflammatory cells infiltrated the mucosal layer and submucosal layer, crypt structure destruction, goblet cell destruction and disappearance, and the inflammatory response in the dextran sulfate sodium-induced ICB treatment group was more severe, indicating that ICB treatment aggravated the inflammatory level of DSS-induced colitis; while the colon tissue morphology of mice in the 4-HIL combined with ICB treatment group was improved, among which the goblet cells and their crypts were relatively intact, mucosal damage was reduced, and inflammatory cell infiltration was reduced. The experimental results show that 4-HIL can alleviate tumor immune checkpoint inhibitor-related enteritis.

[0115] Through in vivo and in vitro experiments, the present invention discovered for the first time that 4-HIL can relieve ulcerative colitis in vitro, and that 4-HIL combined with ICB therapy can not only enhance the anti-tumor level, but also alleviate immune checkpoint inhibitor-associated enteritis and reduce the level of intestinal inflammation.

[0116] The present invention provides a new candidate idea for the first-line treatment of immune checkpoint blockade therapy, and also provides a new candidate drug for the treatment of immune checkpoint inhibitor-associated enteritis.

[0117] Although the technical solution of the present invention has been described in detail above with general descriptions, specific implementation methods and experimental examples, it should be noted that the embodiments and experimental examples are only used to illustrate the technical solution and technical effects of the present invention, and should not be regarded as any limitation on the protection scope of the present invention. Simple deformation, modification or improvement based on the technical concept of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. The application of 4-hydroxyisoleucine is characterized by: The invention relates to an application of the 4-hydroxyisoleucine in preparing a medicine for alleviating or treating ulcerative colitis.

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