CN116549428A - The New Application of 4-Hydroxyisoleucine - Google Patents

Abstract

The invention belongs to the technical field of biomedicine, and specifically discloses a new application of 4-hydroxyisoleucine. The present invention finds for the first time through in vivo and in vitro experiments that 4-hydroxyisoleucine (4-HIL) can enhance the anti-tumor function of immune checkpoint inhibitors, reduce immune-related adverse events in the gastrointestinal tract, and reduce the level of intestinal inflammation. Alleviate or treat gastrointestinal diseases (such as ulcerative colitis). Clinically, the treatment cost of ICB therapy is relatively high, but 4‑HIL combined with ICB therapy can effectively reduce the dosage of immune checkpoint inhibitors and reduce the cost of treatment. At the same time, it reduces the drug burden of patients and has broad clinical application prospects. The 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-related gastrointestinal diseases.

Description

The New Application of 4-Hydroxyisoleucine

technical field

The invention belongs to the technical field of biomedicine, and specifically relates to a new application of 4-hydroxyisoleucine.

Background technique

In recent years, cancer immunotherapy (Cancer immunotherapy) has been increasingly used in the first-line clinical treatment of cancer. Tumor immunotherapy is a new generation of tumor treatment that has developed rapidly following traditional treatment methods such as surgery, radiotherapy, and chemotherapy. Tumor immunotherapy has great clinical application prospects by restoring T cell viability, reversing T cell exhaustion phenotype, and reactivating the immune system to enhance anti-tumor ability. Among them, tumor immunotherapy targeting CTLA-4, PD-1 and its ligand PD-L1 (Immune checkpoint blocking, ICB) has been applied to a variety of tumor types, such as melanoma, non-small cell Clinical treatment of advanced lung cancer, kidney cancer, etc.

Currently, immune checkpoint inhibitors have become a central pillar of cancer treatment. In North America and other developed countries, 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 adjuvant and maintenance therapies, and is also commonly 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. Lack of response and alterations in transcriptional and epigenetic state. Immune checkpoints play an 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, establish an immunosuppressive microenvironment, and thus conduct immune escape. Compared with PD-1, CTLA-4 has a more important role in immune activation. CTLA-4 has a higher affinity for the dendritic cell ligand B7 (also known as CD80) than the co-stimulatory molecule CD28 of T cells, thereby limiting the level of T cell activation during the priming phase. Expression of CTLA-4 also enhances its function in the tumor microenvironment and promotes the expansion of regulatory T (Treg) cells. Therefore, blocking the interaction of immune checkpoints with their ligands can inhibit the exhaustion of T cells, rejuvenate T cells or delay the senescence 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) have entered clinical treatment and have greatly improved the remission rate, overall survival rate and survival period of patients.

However, with the gradual application of immune checkpoint inhibitors (ICIs) in clinical practice, a key challenge has emerged, that is, uncontrolled side effects on the immune system, resulting in a series of Adverse reactions related to immune mechanisms are collectively referred to as immune-related adverse events (Immune-related adverse events, irAEs). The toxicity of ICIs is different from that of standard chemotherapy or other biological adjuvants, and most of the toxicity is due to excessive immunity to normal organs. Compared with the negative effects of traditional tumor therapy, immune-related adverse events occur later, last longer, and may involve any organ. If they cannot be treated in time, they will not only affect the prognosis of patients, but also force the interruption of tumor treatment In severe cases, it can lead to death of the patient. 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 irAEs induced by ICB in varying degrees can reach up to 70%-90%, and can affect almost every organ of the human body, including skin, intestine, liver, kidney, eye, endocrine tissue, and even the central nervous system. Most immune-related adverse events occurred early in the course of treatment, but delayed events (defined as irAEs occurring after 1 year of treatment) also occurred frequently. In a clinical report of 118 patients, the incidence of high-grade delayed irAEs was 5.3%, and the most common irAEs were colitis, rash, and pneumonia, respectively. These adverse effects are usually low-grade and treatable or reversible. However, there are also some adverse effects that can be serious and can lead to permanent disease.

Clinical data showed that nearly 60% of patients treated with the combination of PD-1 and CTLA-4 antibodies developed severe inflammatory reactions. The molecular mechanism of these immune-related adverse events is still unclear, and there is currently no optimal clinical treatment. Among them, the most common inflammatory response occurs at barrier sites, including the gastrointestinal mucosa, liver, skin and lungs. Among all immune-related adverse events, immune checkpoint inhibitor-induced gastrointestinal disease (such as colitis) is one of the most common serious irAEs and an important reason for discontinuation of tumor therapy, especially in the combination of PD-1 mAb In patients receiving CTLA-4 mAb. Among the adverse events induced by immune checkpoint inhibitors, the incidence rate of CTLA-4 inhibitors is about 35%, and the incidence rate of PD-1 inhibitors is about 20%. Combination therapy of CTLA-4 and PD-1 inhibitors The incidence rate is above 40%. Among them, the incidence of colitis was 12%, 1% and 14%, respectively. Gastrointestinal diseases are usually manifested clinically as diarrhea, less commonly as abdominal pain and blood in the stool, 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 was common among patients treated with ipilimumab, and diarrhea occurred in nearly half of patients receiving high doses or combination therapy. Severe colonic inflammation is less common, but still occurs in 10%-20%, and may 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 revealed a high proportion of lymphocytes and an increased number of apoptotic epithelial cells. As with most other serious irAEs, current treatment options consist only of high-dose corticosteroids or discontinuation of immune checkpoint blockade therapy.

Currently, the clinical impact of systemic corticosteroids on antitumor responses is unclear, but clinical retrospective data suggest that high doses of corticosteroids may reduce the efficacy of immunotherapy and reduce antitumor responses. Preliminary clinical data show that tumor necrosis factor α (TNF-α) blockers can effectively treat corticosteroid-resistant immune checkpoint inhibitor-associated enteritis, but there are still few types of related drugs, and the mechanism of action and side effects are also unclear. Not clear. Therefore, there is a need to develop a drug that can alleviate immune checkpoint inhibitor-associated gastrointestinal diseases without compromising anti-tumor immunity as a specific alternative treatment option for immune checkpoint inhibitor-associated enteritis .

Traditional Chinese medicine has a long history in Asian countries, dating back thousands of years. A commonly used class of traditional medicines includes medicinal mushrooms such as cnidium, cinnamon, and purple fungus, which contain various immunomodulatory and bioactive compounds. Fenugreek is a leguminous plant that has been used in traditional Chinese medicine to treat common ailments such as diabetes. Moreover, the extract of fenugreek seeds has shown anti-tumor activity in various tumor models. Although the current research has not clarified the anti-tumor active ingredients in the extract, the development of new anti-tumor preparations based on natural medicines still has broad clinical applications. Application prospects. 4-Hydroxyisoleucine (4-HIL) is one of the active components of fenugreek. 4-HIL is a non-protein amino acid and a branched-chain amino acid derivative, accounting for about the total content of free amino acids in fenugreek seeds 80% of. Studies have reported that the isoleucine in fenugreek can be converted into 4-HIL through a direct route, and has the activity of lowering blood sugar and promoting insulin secretion both in vivo and in vitro. Moreover, previous studies have found that 4-HIL can improve the disorders of glucose metabolism and lipid metabolism caused by obesity and reverse insulin resistance in the in vivo obesity model, but the research on its anti-tumor activity has not been reported yet.

Contents of the invention

The technical problem mainly solved by the present invention is to provide a new application of 4-hydroxyisoleucine for alleviating or treating gastrointestinal diseases related to immune checkpoint inhibitors.

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 immune checkpoint inhibitor-related gastrointestinal diseases.

Finally, the present invention also provides a use of 4-hydroxyisoleucine in the preparation of medicines for alleviating or treating gastrointestinal diseases.

In order to solve the problems of the technologies described above, the present invention provides the following technical solutions:

The application of 4-hydroxyisoleucine, the application is: the application of 4-hydroxyisoleucine in the preparation of drugs for alleviating or treating immune-related adverse events.

As a preferred embodiment of the present invention, the immune-related adverse events include but are not limited to immune-related adverse events in the gastrointestinal tract, that is, immune checkpoint inhibitor-related gastrointestinal diseases, due to the use of immune checkpoint inhibitors Gastrointestinal diseases caused by it.

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

As a preferred embodiment of the present invention, the immune checkpoint inhibitors include but are not limited to cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibitors, programmed cell death protein 1 (PD-1) inhibitors , programmed cell death-ligand 1 (PD-L1) inhibitor, programmed cell death-ligand 2 (PD-L2) inhibitor, lymphocyte activation gene 3 (LAG-3, or CD223) inhibitor, T One or more of cellular immunoglobulin mucin 3 (TIM-3) inhibitors and the like. In addition, inhibitors of the following immune checkpoints 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 immunoreceptor 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 antibody, double antibody, etc. The 4-hydroxyisoleucine has obvious synergistic effect on immune checkpoint inhibitors.

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 wait. In order to prepare a specific pharmaceutical dosage form, the drug also includes pharmaceutically acceptable carriers, including but not limited to excipients, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, buffers one or more of these.

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

As a preferred embodiment of the present invention, the dose of the drug is a pharmaceutically acceptable dose.

A drug for 4-hydroxyisoleucine combined with ICB therapy, the drug comprising 4-hydroxyisoleucine and an immune checkpoint inhibitor in a pharmacodynamic amount.

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 (that is, the content of immune checkpoint inhibitors is lower than that of the same type only Drugs that use immune checkpoint inhibitors as active ingredients), thereby reducing treatment costs and reducing the burden of medication for patients.

As a preferred embodiment of the present invention, the immune checkpoint inhibitors include but are not limited to cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibitors, programmed cell death protein 1 (PD-1) inhibitors , programmed cell death-ligand 1 (PD-L1) inhibitor, programmed cell death-ligand 2 (PD-L2) inhibitor, lymphocyte activation gene 3 (LAG-3, or CD223) inhibitor, T One or more of cellular immunoglobulin mucin 3 (TIM-3) inhibitors and the like. In addition, inhibitors of the following immune checkpoints 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 immunoreceptor 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 antibody, double antibody, etc.

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 wait. In order to be prepared into a specific pharmaceutical dosage form, in addition to the 4-hydroxyisoleucine and the immune checkpoint lowering inhibitor, the drug also includes a pharmaceutically acceptable carrier, including but not limited to excipients , preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, buffers and the like.

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

As a preferred embodiment of the present invention, the dose of the drug is a pharmaceutically acceptable dose.

The application of 4-hydroxyisoleucine, the application is: the application of 4-hydroxyisoleucine in the preparation of medicines for alleviating or treating gastrointestinal diseases.

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

As a preferred embodiment of the present invention, the content of 4-hydroxyisoleucine in the drug is a drug effect amount.

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 wait. In order to prepare a specific pharmaceutical dosage form, the drug also includes pharmaceutically acceptable carriers, including but not limited to excipients, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, buffers one or more of these.

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

As a preferred embodiment of the present invention, the dose of the drug is a pharmaceutically acceptable dose.

Beneficial effects of the present invention:

The present invention uses dextran sulfate sodium salt (DSS) to establish an animal model of ulcerative colitis, monitors its body weight, intestinal pathology and inflammatory cytokines, thereby judging the degree of intestinal inflammation in mice. The experimental results showed that after induction of DSS, the mice had symptoms of enteritis, significantly decreased body weight, decreased survival rate, increased infiltration of intestinal epithelial inflammatory cells, and significantly increased levels of inflammatory factors, indicating that the animal model of ulcerative colitis was successfully established. Using 4-hydroxy After treatment with isoleucine (4-HIL), the body weight loss of mice slowed down, the survival rate increased, the infiltration of intestinal epithelial inflammatory cells decreased, the levels of inflammatory factors decreased significantly, and the levels of anti-inflammatory factors increased, indicating that 4-HIL can relieve inflammation in vivo. Ulcerative colitis can be used to prepare medicines for alleviating or treating gastrointestinal diseases.

The present invention also uses DSS and immune checkpoint inhibitors to establish an ICB animal model, and monitors its tumor volume, tumor weight, body weight and intestinal pathology, thereby judging the anti-tumor effect and the degree of intestinal inflammation in mice. The experimental results showed that after DSS induction and ICIs administration, the tumor volume and tumor weight decreased, the body weight of the mice decreased significantly, the survival rate decreased, the intestinal epithelial inflammatory cell infiltration increased significantly, and the level of inflammatory factors increased significantly, while 4-HIL combined with ICIs treatment Later, it was found that the tumor volume and weight were significantly reduced, the weight of the mice was not significantly reduced, the survival rate was increased, the infiltration of intestinal epithelial inflammatory cells was reduced, the levels of inflammatory factors were reduced, and the levels of anti-inflammatory factors were increased. During the treatment period, the drug had no obvious side effects. It shows that 4-HIL combined with ICB therapy can relieve immune checkpoint inhibitor-related enteritis while enhancing the 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, enhance anti-tumor ability, relieve gastrointestinal diseases and other immune-related adverse events while reducing treatment costs , reduce the drug burden of patients, and has broad clinical application prospects.

Description of drawings

Fig. 1 is the experimental result of the acute colitis induced by 4-HIL in Experimental Example 1 in mice induced by DSS;

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

Figure 2 is the experimental results of 4-HIL alleviating immune checkpoint inhibitor-related enteritis and enhancing anti-tumor ability in Experimental Example 2;

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

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the drawings obtained in the experimental examples are briefly introduced above. 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 protection scope of the claims. For those skilled in the art, other related drawings can also be obtained based on these drawings without creative work.

Detailed ways

The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments and experimental examples. However, those skilled in the art should understand that the examples are only used to illustrate the technical solution of the present invention, and should not be regarded as limiting the protection scope of the present invention. Based on the following examples, all other implementations obtained by persons of ordinary skill in the art without creative efforts, such as implementations obtained after modification, deformation or simple replacement, shall fall within the protection scope of the present invention.

If no special instructions, the experimental methods used in the following examples and experimental examples are conventional methods; used raw materials (including biological materials), reagents, culture media, instruments, etc., if no special instructions, are commonly used in this field. Items that are available to the public or can be obtained through commercial channels; the terms and abbreviations involved are conventional meanings in the field, such as PBS is phosphate buffer saline.

Example 1

This embodiment provides an application of 4-hydroxyisoleucine, specifically: the application of 4-hydroxyisoleucine in the preparation of drugs 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.

Example 2

This embodiment provides an application of 4-hydroxyisoleucine, specifically: the application of 4-hydroxyisoleucine in the preparation of drugs for alleviating or treating immune-related adverse events in the gastrointestinal tract. The immune-related adverse events of 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).

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

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

Example 3

This embodiment provides an application of 4-hydroxyisoleucine, specifically: the application of 4-hydroxyisoleucine in the preparation of drugs for alleviating or treating gastrointestinal diseases. The gastrointestinal disease is specifically enteritis (including colitis).

This embodiment also provides a medicament for alleviating or treating gastrointestinal diseases, the medicament comprising a pharmacodynamic amount of 4-hydroxyisoleucine. The dosage form of the medicine is an injection; in addition to the above-mentioned effective ingredients, the medicine also includes pharmaceutical auxiliary materials such as preservatives and stabilizers. The administration route of the medicine is intravenous injection, and the dose is a clinical medicinal dose.

In other embodiments of the present invention, the medicament for relieving or treating symptoms can be prepared in any medicinal form, which can be prepared by using conventional technical means and common excipients based on the common knowledge in the field.

Experimental example 1 4-HIL alleviates acute colitis induced by DSS in mice

1. Experimental materials

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 an SPF-level animal experiment center, and the feeding conditions strictly followed the SPF-level standards. They were fed with sterilized drinking water and sterile feed. The ambient humidity was about 60%, the temperature was about 25°C, and light and dark were alternated for 12 hours. The mice acclimatized to the environment for a week, and the experiment began after no abnormalities were observed.

Experimental reagents: tissue cell lysate, purchased from Beijing Suo Laibao Technology Co., Ltd.; dextran sulfate sodium salt (DSS), purchased from Dalian Meilun Biotechnology Co., Ltd.; 4-HIL, purchased from Henan Julong Bioengineering Co., Ltd. Co., Ltd.; LightCycler 480SYBR Green I Master, purchased from Roche; reverse transcription reagents, purchased from Nanjing Nuoweizan Biotechnology Co., Ltd.

2. Experimental method

1. Establishment of acute colitis model in mice

Establishment of experimental animal model of ulcerative colitis induced by dextran sulfate sodium salt (Dextran Sulfate Sodium Salt, DSS): 21 female mice of C57/6N strain at 6-8 weeks were randomly divided into 3 groups according to body weight, each group 7 rats were normal control group, normal saline solvent group and 100mg/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 normal saline and 100 mg/kg 4-HIL for 9 consecutive days. The model group was given 3% sodium dextran sulfate dissolved in drinking water for 7 days, and then given normal drinking water for two days. The normal control group was given normal drinking water for 9 consecutive days. On the 10th day, they were killed by neck dislocation.

2. Observe and record the diseases of experimental animals

Since the establishment of the experimental animal model of ulcerative colitis, the living conditions of the mice were continuously observed and recorded every day, and the body weight was measured until the end of the modeling. After the modeling, the experimental animals in each group were killed, and the colon tissue of the experimental mice was collected to observe the lesions of the colon tissue, and measure and record the length of the colon.

3. Paraffin sections of colon tissue and hematoxylin-eosin (Hematoxylin&Eosin, H&E) staining

Take the mouse large intestine tissue fixed in 10% formalin solution, wash it three times with PBS, and place the sample in the following solutions for ethanol dehydration and transparency. The steps are: 75% ethanol solution for 20 minutes; 80% ethanol solution for 20 minutes minutes; 95% ethanol solution I for 20 minutes; 95% ethanol solution II for 20 minutes; absolute ethanol I for 20 minutes; absolute ethanol II for 20 minutes; xylene for 10 minutes to be transparent. The dehydrated and transparent samples were embedded in paraffin, sectioned with a microtome (10 μm), and fixed on glass slides for H&E staining. Hydration steps: dewax the slices in a 60°C incubator for 30 minutes; dewax with xylene I for 3 minutes; dewax with xylene II for 3 minutes; dewax with xylene III for 3 minutes; dewax with ethanol I for 3 minutes; II 3 minutes; 95% ethanol solution I 3 minutes; 95% ethanol solution II 3 minutes; 75% ethanol solution 3 minutes; deionized water 3 minutes. H&E staining steps for sample slices: hematoxylin staining for 10 minutes; deionized water wash 2 times; 1% hydrochloric acid differentiation for 30 seconds; deionized water wash 1 time, 1% ammonia water 30 seconds to turn blue; deionized water wash 1 time; 75% ethanol solution for 3 minutes; 95% ethanol solution for 3 minutes; staining with eosin in 95% ethanol solution for 10 minutes; 95% ethanol solution for 2 washes; absolute ethanol for 1 minute; xylene I for 3 minutes; xylene II for 3 minutes; Toluene III 3 min. The slices were placed in a fume hood to dry naturally, sealed with neutral gum, and photographed under a microscope.

4. Real-time fluorescence quantitative PCR

4.1. Extraction of RNA

(1) After the tissue is ground in liquid nitrogen, add 1mL of tissue cell lysate, pipette until no agglomerated tissue pieces are visible, and the whole solution is clear but not viscous.

(2) Use the commercialized kit Total RNAIsolation Hanbook, and follow the instructions to extract total RNA.

(3) Take 2 μL of the extracted total RNA, and use NanoDrop to detect the RNA concentration.

4.2. Reverse transcription of total RNA into cDNA

(1) Reverse transcription reagents were stored at -20°C. After melting, the components were centrifuged slightly and placed on ice.

(2) Put the sterile nuclease-free PCR tube on ice in advance, firstly carry out the synthesis of the first strand, and add the following components in order: 4×gDNAwiper mix 4 μL, 1 μg RNA (volume=1 μg/RNA concentration), DEPC H ₂ O to make up to 16 μL.

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

(4) Put the PCR tube into the PCR instrument, set the program: 42°C, 2min.

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

(6) Set the program of the PCR instrument as follows: 37°C for 15 minutes; 85°C for 5s; terminate the reaction, and the obtained product is cDNA, which is stored at -80°C for later use.

4.3. Fluorescence quantitative PCR

(1) Using β-actin as an internal reference gene, the primers in Table 1 (as shown in SEQ ID NO: 1-10) were synthesized by BGI.

Table 1 Fluorescent quantitative PCR primers

(2) The reaction system was 12 μL, and the components in Table 2 were added sequentially on ice in the dark.

Table 2 Fluorescent quantitative PCR reaction system

(3) After all the solutions are added, mix the solutions evenly with a row gun, paste the sealing film, and place it in a centrifuge for 5 minutes at 3500 rpm.

(4) After centrifugation, put the fluorescent quantitative PCR reaction plate into the fluorescent quantitative PCR instrument, and set the reaction program in Table 3.

Table 3 Fluorescent quantitative PCR reaction program

5. Statistics

Statistical analysis of differences between groups was performed using Unpaired Student's T-test. Data are presented as Means ± SEM unless otherwise stated. When the P value is less than 0.05, the difference is considered to be statistically significant, * is the difference compared with the normal control group, *P<0.05, **P<0.01, ***P<0.001, # is LPS+IFN- The difference between the γ group and the control group, #P<0.05, ##P<0.01, ###P<0.001. The result is shown in Figure 1.

3. Experimental results

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

No animal in each group died. The mice in the normal group had no weight loss, soft or bloody stools, decreased activity, and poor hair. The model groups induced by dextran sodium sulfate (DSS) all had the following symptoms of varying degrees: listlessness, decreased activity, messy hair, decreased food intake, weight loss, diarrhea, soft stools or bloody stools, and colonic tissue. Decreased colon length.

4-HIL affects the colon length of dextran sodium sulfate-induced ulcerative colitis in mice, as shown in Figure 1A and Figure 1B, the mice in the normal group had no hematochezia, and the intestinal contents were good; the colon of mice in the model group The length of the colon was significantly reduced, blood in the stool appeared in the intestine, and the intestinal content was sticky; the 4-HIL drug group could inhibit the decrease in the length of the colon induced by DSS, and there was no obvious blood in the intestine. The experimental results showed that 4-HIL had a therapeutic effect on DSS-induced ulcerative colitis mice.

4-HIL affects the body weight change results of dextran sodium sulfate-induced ulcerative colitis in mice, as shown in Figure 1C, the weight of mice in the experimental control group given normal drinking water gradually increased; DSS-induced ulcerative colitis The body weight of the mice in the model group decreased significantly, and began to decrease on the 5th day of DSS induction, and reached the lowest level on the 9th day, and the 4-HIL drug group could inhibit the weight loss of the mice induced by DSS. The experimental results showed that 4-HIL had a therapeutic effect on DSS-induced ulcerative colitis mice.

4-HIL affects the histopathological HE staining results of dextran sodium sulfate-induced ulcerative colitis in mice. As shown in Figure 1D, the colon tissue structure of mice in the experimental control group given normal drinking water is intact, and the crypts are clearly visible; DSS-induced ulcerative colitis model group mouse colon pathological section HE staining showed: intestinal wall edema, thickening, inflammatory cells infiltrating the mucosa and submucosa, crypt structure destruction, goblet cell destruction and disappearance; 4-HIL The colon tissue morphology of the mice in the drug group was improved, in which the goblet cells and their crypts were in good shape, the mucosal damage was reduced, and the infiltration of inflammatory cells was reduced. The experimental results showed that 4-HIL had a therapeutic effect on DSS-induced ulcerative colitis lesions in mice.

2. 4-HIL inhibits the mRNA levels of inflammatory factors in the colon tissue of mice with ulcerative colitis

Select the same part of mouse colon tissue, extract total RNA, and detect the expression levels of related cytokine mRNAs. The experimental results are shown in Figure 1E. DSS-induced mRNA levels of inflammatory factors TNF-α and IL-1β in mouse colon tissue Compared with the normal control group, the mRNA levels of inflammatory factors TNF-α and IL-1β in the 4-HIL drug group decreased significantly compared with the DSS-induced group, and the anti-inflammatory factor TGF-β and IL-10 mRNA levels increased. The experimental results showed that DSS induced an increase in the levels of inflammatory factors in mouse colon tissue, and 4-HIL had a therapeutic effect on DSS-induced ulcerative colitis mice.

Experimental example 2 4-HIL relieves immune checkpoint inhibitor-associated enteritis

1. Experimental materials

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 an SPF-level animal experiment center, and the feeding conditions strictly followed the SPF-level standards. They were fed with sterilized drinking water and sterile feed. The ambient humidity was about 60%, the temperature was about 25°C, and light and dark were alternated for 12 hours. The mice acclimatized to the environment for a week, and the experiment began after no abnormalities were observed.

2. Experimental method

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

A total of 28 female mice of C57/6N strain at 6-8 weeks were randomly divided into 4 groups, 7 mice in each group, including normal control group, dextran sulfate sodium salt group, immune checkpoint blockade treatment group and immune checkpoint treatment group. Point blocking combined with 150mg/kg 4-HIL drug group. 4-HIL was suspended in 0.9% saline. The model group was given 3% dextran sulfate sodium salt dissolved in drinking water for 5 days, and then given 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. 200 μL of cell suspension was subcutaneously injected into the right side of C57 mice with a 1 mL syringe, and the total number of cells injected in each mouse was 1×10 ⁵ . When the tumor volume reached 30-60 mm ³ , the mouse was weighed with an electronic balance and the mouse tumor was measured with a digital display caliper the next day, and the mouse tumor volume change was calculated and recorded according to the following formula: V=1/2×a(long )×b(width)×c(height). On the 7th and 14th days after tumor bearing, tumor immune checkpoint inhibitors (anti-CTLA-4 antibody 100 μg/mouse/time, anti-PD-1 antibody 100 μg/mouse/time) were injected intraperitoneally. Fourteen days after administration, the mice were sacrificed by cervical dislocation.

2. Observe and record the diseases of experimental animals

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 end of the model. After the modeling, the experimental animals in each group were killed, and the colon and tumor tissues of the experimental mice were collected to observe the lesions of the colon tissue, and measure and record the length of the colon.

3. Paraffin sections of colon tissue and hematoxylin-eosin (Hematoxylin&Eosin, H&E) staining

Take the mouse large intestine tissue fixed in 10% formalin solution, wash it three times with PBS, and place the sample in the following solutions for ethanol dehydration and transparency. The steps are: 75% ethanol solution for 20 minutes; 80% ethanol solution for 20 minutes minutes; 95% ethanol solution I for 20 minutes; 95% ethanol solution II for 20 minutes; absolute ethanol I for 20 minutes; absolute ethanol II for 20 minutes; xylene for 10 minutes to be transparent. The dehydrated and transparent samples were embedded in paraffin, sectioned with a microtome (10 μm), and fixed on glass slides for H&E staining. Hydration steps: dewax the slices in a 60°C incubator for 30 minutes; dewax with xylene I for 3 minutes; dewax with xylene II for 3 minutes; dewax with xylene III for 3 minutes; dewax with ethanol I for 3 minutes; II 3 minutes; 95% ethanol solution I 3 minutes; 95% ethanol solution II 3 minutes; 75% ethanol solution 3 minutes; deionized water 3 minutes. H&E staining steps for sample slices: hematoxylin staining for 10 minutes; deionized water wash 2 times; 1% hydrochloric acid differentiation for 30 seconds; deionized water wash 1 time, 1% ammonia water 30 seconds to turn blue; deionized water wash 1 time; 75% ethanol solution for 3 minutes; 95% ethanol solution for 3 minutes; staining with eosin in 95% ethanol solution for 10 minutes; 95% ethanol solution for 2 washes; absolute ethanol for 1 minute; xylene I for 3 minutes; xylene II for 3 minutes; Toluene III 3 min. The slices were placed in a fume hood to dry naturally, sealed with neutral gum, and photographed under a microscope.

4. Statistics

The data statistics method is the same as that of Experimental Example 1. The result is shown in Figure 2.

3. Experimental results

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

Establish a mouse subcutaneous melanoma model, use anti-CTLA-4 antibody and anti-PD-1 antibody to simulate clinical ICB therapy, that is, treat tumor patients with combination therapy of PD-1 monoclonal antibody and CTLA-4 monoclonal antibody, and observe the effect of ICB in mice The anti-tumor effect in the model, the experimental results are shown in 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 of the ICB treatment group, that is, 4-HIL combined with ICB therapy can effectively inhibit the growth of melanoma in mice (Figure 2B) . Comparing the tumor weight of the mice on day 21, the tumor weight of the mice treated with 4-HIL combined with ICB was significantly smaller than that of the mice treated with ICB (Fig. 2C). The experimental results show that 4-HIL combined with ICB therapy can effectively enhance its anti-tumor ability.

2. 4-HIL relieves tumor immune checkpoint inhibitor-associated enteritis

No animal in each group died. The mice in the normal group had no weight loss, soft or bloody stools, decreased activity, and poor hair. The simple dextran sodium sulfate (DSS) group and the dextran sodium sulfate-induced ICB treatment group also experienced the following symptoms of varying degrees: listlessness, decreased activity, hair disorder, decreased food intake, decreased body weight, and diarrhea Symptoms such as soft stools or blood in the stool, and the colonic tissue shows the performance of decreased colon length.

The effect of 4-HIL on the length of the lesion colon in the immune checkpoint inhibitor-associated enteritis model mice, as shown in Figure 2E and Figure 2F, the mice in the normal group had no hematochezia, and the intestinal contents were good; dextran sulfate Compared with the dextran sodium sulfate (DSS) group, the mice in the sodium-induced ICB treatment group had significantly decreased colon length, blood in the stool, and viscous intestinal contents; the 4-HIL combined with ICB treatment group could inhibit the induction of DSS The length of the colon is reduced, and there is no obvious blood in the intestine. Experimental results show that 4-HIL can relieve tumor immune checkpoint inhibitor-associated colitis.

4-HIL affects the body weight change results of immune checkpoint inhibitor-associated enteritis model mice, as shown in Figure 2A, the weight of mice in the experimental control group given normal drinking water gradually increased; dextran sodium sulfate (DSS) The body weight of the mice in the ICB treatment group induced by dextran sodium sulfate and dextran sodium sulfate decreased significantly, and the body weight of the mice in the model group began to decline on the 13th day until it reached the lowest level on the 21st day, and the 4-HIL administration treatment group could inhibit the induction of DSS mice lost weight. Experimental results show that 4-HIL can relieve tumor immune checkpoint inhibitor-associated colitis.

4-HIL affects the histopathological HE staining results of the colon of immune checkpoint inhibitor-associated enteritis model mice, as shown in Figure 2D, the colon tissue structure of the normal control group mice is intact, and the crypts are clearly visible; dextran sodium sulfate (DSS) group and dextran sodium sulfate-induced ICB treatment group mouse colon pathological sections HE staining showed: intestinal wall edema, thickening, inflammatory cells infiltrating the mucosa and submucosa, crypt structure destruction, goblet cell destruction and disappeared, and the inflammatory response induced by dextran sodium sulfate in the ICB treatment group was more serious, 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 them, the morphology of goblet cells and their crypts was relatively intact, the mucosal damage was reduced, and the infiltration of inflammatory cells was reduced. Experimental results show that 4-HIL can relieve tumor immune checkpoint inhibitor-associated colitis.

Through in vivo and in vitro experiments, the present invention found for the first time that 4-HIL can alleviate ulcerative colitis in vitro, and that 4-HIL combined with ICB therapy can not only enhance the anti-tumor level, but also reduce immune checkpoint inhibitor-related enteritis and reduce intestinal inflammation. Inflammation levels.

The 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-related enteritis.

Although the technical solution of the present invention has been described in detail with general description, specific implementation and experimental examples above, it should be noted that the embodiments and experimental examples are only used to illustrate the technical solutions and technical effects of the present invention. It should not be regarded as any limitation on the protection scope of the present invention. Simple deformations, modifications or improvements made based on the technical concept of the present invention all belong to the protection scope of the present invention.

Claims (10)

1. The application of 4-hydroxyisoleucine, characterized in that: the application of the 4-hydroxyisoleucine in the preparation of drugs for alleviating or treating immune-related adverse events. 2. The application according to claim 1, characterized in that: the immune-related adverse events include but not limited to immune checkpoint inhibitor-related gastrointestinal diseases. 3. The application according to claim 2, characterized in that: the immune checkpoint inhibitor-related gastrointestinal diseases include but not limited to gastritis, enteritis, and peptic ulcer. 4. The application according to claim 2, wherein the immune checkpoint inhibitors include but not limited to CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, One or more of LAG-3 preparations and TIM-3 inhibitors; The 4-hydroxyisoleucine has a synergistic effect on immune checkpoint inhibitors. 5. The application according to claim 1, characterized in that: the dosage form of the medicine is a pharmaceutically acceptable dosage form; And/or, the route of administration of the drug is a pharmaceutically acceptable route; And/or, the dose of the drug is a pharmaceutically acceptable dose. 6. The drug of 4-hydroxyisoleucine combined with ICB therapy, characterized in that: the drug includes a pharmacodynamic amount of 4-hydroxyisoleucine and an immune checkpoint inhibitor. 7. The medicine according to claim 6, characterized in that: the content of the immune checkpoint inhibitor in the medicine is lower than that of the same type of medicine that only uses the immune checkpoint inhibitor as the active ingredient. 8. The drug according to claim 6, wherein the immune checkpoint inhibitors include but not limited to CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, One or more of LAG-3 preparations and TIM-3 inhibitors. 9. The medicine according to claim 6, characterized in that: the dosage form of the medicine is a pharmaceutically acceptable dosage form; And/or, the route of administration of the drug is a pharmaceutically acceptable route; And/or, the dose of the drug is a pharmaceutically acceptable dose. 10. The application of 4-hydroxyisoleucine, characterized in that: the application of said 4-hydroxyisoleucine in the preparation of medicines for alleviating or treating gastrointestinal diseases.