Use of a tlr4 inhibitor in the manufacture of a medicament for alleviating sorafenib-induced gastrointestinal toxicity

Abstract

The application discloses application of a TLR4 inhibitor in preparation of a medicine for relieving gastrointestinal toxicity caused by sorafenib, and relates to the field of biological medicines. The TLR4 inhibitor has a relieving effect on gastrointestinal toxicity induced by sorafenib, can readjust slow weight gain of mice caused by sorafenib, relieve intestinal mucosal injury of mice caused by sorafenib, and reduce intestinal oxidative stress caused by sorafenib.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the application of TLR4 inhibitor in the preparation of a drug for relieving gastrointestinal toxicity caused by sorafenib. BACKGROUND

[0002] Sorafenib is the first molecular targeted drug used for the treatment of hepatocellular carcinoma. A number of clinical studies have shown that sorafenib has good efficacy for patients with advanced hepatocellular carcinoma in different countries and regions and different liver disease backgrounds. However, sorafenib is often accompanied by a number of adverse reactions during use, and gastrointestinal toxicity is a very common adverse reaction of sorafenib, mainly in the form of diarrhea, nausea and vomiting, with an incidence rate of more than 40%, and some patients even develop gastrointestinal perforation. These adverse reactions often reduce the quality of life of patients, reduce the tolerance of sorafenib, lead to a reduction in the dosage or even discontinuation of clinical medication, and greatly affect the clinical efficacy.

[0003] Therefore, how to relieve the gastrointestinal side effects caused by sorafenib is a problem that needs to be solved in clinical practice at present. SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes the application of TLR4 inhibitor in the preparation of a drug for relieving gastrointestinal toxicity caused by sorafenib.

[0005] The application of TLR4 inhibitor according to the first aspect of the present application in the preparation of a drug for preventing and / or relieving gastrointestinal toxicity caused by sorafenib.

[0006] According to the application of the present application, at least the following beneficial effects are achieved:

[0007] The present application proves that TLR4 inhibitor has a relieving effect on gastrointestinal toxicity induced by sorafenib, can adjust the slow increase in body weight of mice caused by sorafenib, relieve intestinal mucosal injury of mice caused by sorafenib, and reduce intestinal oxidative stress caused by sorafenib. This not only widens the application of sorafenib in clinical treatment, but also provides evidence and basis for the research and development of drugs for inhibiting gastrointestinal toxicity of sorafenib, and has clinical practicability. The present application also expands the indications of TAK-242, and the reassuring safety also indicates its potential application in the future.

[0008] According to some embodiments of the present application, the drug can adjust the slow increase in body weight of mice caused by sorafenib.

[0009] According to some embodiments of the present application, the drug can prevent and / or relieve intestinal mucosal injury of mice caused by sorafenib.

[0010] According to some embodiments of the present application, the preventing and / or alleviating the intestinal mucosal damage in mice caused by sorafenib comprises at least one of A1) to A5);

[0011] A1) increasing the length of the colon;

[0012] A2) increasing the length of the small intestine;

[0013] A3) increasing the villus length of the ileum;

[0014] A4) increasing the ratio of villus length to crypt depth;

[0015] A5) improving at least one of the symptoms of ileal villus atrophy, loose structure, and defects.

[0016] According to some embodiments of the present application, the drug can reduce the intestinal oxidative stress caused by sorafenib.

[0017] According to some embodiments of the present application, the reducing the intestinal oxidative stress caused by sorafenib comprises at least one of B1) to B4);

[0018] B1) reducing the content of MDA in the intestine;

[0019] B2) increasing the activity of CAT;

[0020] B3) increasing the activity of SOD;

[0021] B4) increasing the total antioxidant capacity.

[0022] According to some embodiments of the present application, the TLR4 inhibitor can be a small molecule, a peptide, a protein, an antibody or an antigen-binding fragment thereof, an antibody mimetic, an aptamer, or a nucleic acid molecule.

[0023] According to some embodiments of the present application, the TLR4 inhibitor comprises at least one of TAK-242, E5531, E5564, (+) naloxone, (-) naloxone, (+) naltrexone, (-) naltrexone, T5342126, TP6034019, IAANS, auranofin, JTT705, curcumin, zangzhibenzoic acid A, berberine.

[0024] According to some embodiments of the present application, the active ingredients of the drug comprise a TLR4 inhibitor and other ingredients that can be used to prevent and / or alleviate the gastrointestinal toxicity caused by sorafenib. The ingredients are preferably those that do not affect the effect of the TLR4 inhibitor. The ingredients include, but are not limited to, loperamide hydrochloride, montmorillonite powder, metoclopramide (metoclopramide), dexamethasone, diphenhydramine, chlorpromazine, ondansetron, or ridamer.

[0025] According to some embodiments of the present application, the medicine further comprises a pharmaceutically acceptable carrier or excipient.

[0026] According to some embodiments of the present application, the medicine is in the form of granules, hard capsules, soft capsules, tablets, emulsions or suspensions.

[0027] In the present application, the subject of the drug treatment is mammal, including human, mouse, etc. The person skilled in the art can convert the dosage of human to the dosage of animal per unit weight according to the dose conversion relationship between human and experimental animals. According to the conversion of the dosage per unit weight, the equivalent dose of mouse is about 0.75 times of human.

[0028] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent to those skilled in the art from the description, or can be learned by practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Technical roadmap for the alleviating effect of the TLR4 inhibitor of the present application on the gastrointestinal toxicity caused by sorafenib;

[0030] Figure 2 The weight change results of mice in each treatment group;

[0031] Figure 3 The colon (A) and small intestine (B) length detection results of mice in each treatment group;

[0032] Figure 4 The ileum histopathological changes of mice in each treatment group;

[0033] Figure 5 The ileum villus length (A), crypt depth (B) and V / C value (C) changes of mice in each treatment group;

[0034] Figure 6 The T-AOC (A), CAT (B), SOD activity (C) and MDA content (D) detection results of intestinal tissue of mice in each treatment group. DETAILED DESCRIPTION

[0035] The concept and technical effects of the present application will be described clearly and completely in combination with the embodiments below, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by the person skilled in the art without creative labor are within the protection scope of the present application.

[0036] Unless otherwise indicated, conventional methods and techniques of molecular biology, microbiology, and recombinant DNA techniques, as well as other conventional methods and techniques, can be used. Such methods and techniques are explained fully in the literature. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Techniques, 4th Ed., John Wiley & Sons, Inc., New York (1999); and the series Methods in Enzymology, Academic Press, San Diego, Calif. The disclosures of all of the references mentioned in this document are incorporated by reference.

[0037] When a numerical range is disclosed herein, the range is to be construed as having been recited continuously, and including every value from the lowest value to the highest value, and every value within such range. Further, when a range is stated to include integers, every integer within the range is included. In addition, when a plurality of ranges are provided to describe a characteristic or attribute, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood as including any and all sub-ranges subsumed therein.

[0038] In the embodiments of the present application, C57BL / 6J mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. and were raised in the SPF barrier environment of the Experimental Animal Center of Shenzhen Institute for Drug Control;

[0039] The mouse feed was provided by the Experimental Animal Center of Shenzhen Institute for Drug Control;

[0040] Sorafenib was purchased from MedChemExpress (MCE) with the item number HY-10201;

[0041] TLR4 inhibitor (TAK-242) was purchased from MedChemExpress (MCE) with the item number HY-11109;

[0042] Sodium carboxymethylcellulose (CMC) was purchased from Macklin with the item number C804625;

[0043] Dimethyl sulfoxide (DMSO) was purchased from Solarbio with the item number D8370;

[0044] Sterile normal saline was purchased from Guangzhou Yunsen Biotech Co., Ltd. with the item number GY-128;

[0045] Phosphate buffered saline (PBS) was purchased from Servicebio Biological Technology Co., Ltd. in Wuhan with the item number G4202;

[0046] Universal tissue fixative was purchased from Servicebio Biological Technology Co., Ltd. in Wuhan with the item number G1101;

[0047] Catalase (CAT) activity detection kit was purchased from Solarbio with the item number BC0205;

[0048] The total antioxidant capacity (T-AOC) detection kit was purchased from Solarbio, and the item number was BC1315;

[0049] The malondialdehyde (MDA) content detection kit was purchased from Solarbio, and the item number was BC0025;

[0050] The superoxide dismutase (SOD) activity detection kit was purchased from Solarbio, and the item number was BC0175.

[0051] Mitigation effect of TLR4 inhibitor on gastrointestinal toxicity caused by sorafenib

[0052] After 30 C57BL / 6J mice (20g±2g, male, five weeks old) were adaptively fed for one week, they were randomly divided into four groups, namely the normal control group (Control, n=7), the sorafenib group (Sorafenib, n=7), the TLR4 inhibitor group (TAK-242-C, n=8), and the sorafenib+TLR4 inhibitor combination group (TAK-242-S, n=8). The total administration treatment lasted for 21 days, and the body weight of the mice was recorded every day. The mice were sacrificed 24h after the last administration, the intestinal tissues of the mice were collected, and the colon length and small intestine length were recorded; at the same time, the ileum end tissue with a length of 1-1.5cm was cut, washed with 4℃ pre-cooled PBS solution, and then placed in a general tissue fixative for 24h, and sent to Wuhan Sivier Biological Technology Co., Ltd. for hematoxylin-eosin (H&E) staining for histopathological analysis (image acquisition was performed using Aperio GT 450 automatic large-capacity digital pathology slide scanner, and histopathological evaluation was performed; three samples were randomly selected from each group of samples, and ileum villus length and crypt depth were measured using Aperio ImageScope pathology slide viewing software (n=3)); the rest of the intestinal tissues were quickly frozen in liquid nitrogen and stored in an ultra-low temperature freezer for subsequent biochemical index (CAT activity, T-AOC, MDA content, SOD activity) analysis (n=6, six samples were randomly selected from each group of samples), and the steps were performed according to the instructions of the corresponding kit.

[0053] The technical roadmap of the experiment is shown in Figure 1 The administration of the mice in each group is as follows:

[0054] The normal control group (Control): carbowax 0.5% sodium carboxymethylcellulose solution was orally administered at a dose of 0.1mL / kg once a day;

[0055] Sorafenib group (Sorafenib): Sorafenib suspension solution was orally administered at a dose of 120 mg / kg, with a volume of 0.1 mL / kg, once a day;

[0056] TLR4 inhibitor group (TAK-242-C): TAK-242 solution was intraperitoneally injected at a dose of 3 mg / kg, with a volume of 0.1 mL / kg, once every 2 days;

[0057] Sorafenib + TLR4 inhibitor combination group (TAK-242-S): Sorafenib suspension solution was orally administered at a dose of 120 mg / kg, with a volume of 0.1 mL / kg, once a day; TAK-242 solution was intraperitoneally injected at a dose of 3 mg / kg, with a volume of 0.1 mL / kg, once every 2 days.

[0058] The preparation method of the sorafenib suspension solution is as follows: 0.5 g of sodium carboxymethyl cellulose is accurately weighed, and 100 mL of distilled water is taken in a glass beaker; the beaker is placed on a heating type magnetic stirrer, and the weighed sodium carboxymethyl cellulose (CMC) is scattered on the water surface in small amounts for multiple times, while stirring, until the solution is clear and transparent, to obtain a 0.5% sodium carboxymethyl cellulose solution (prepared and used immediately); sorafenib is added to the 0.5% sodium carboxymethyl cellulose solution and ultrasonically dissolved, to obtain the sorafenib suspension solution.

[0059] The preparation method of the TAK-242 solution is as follows: TAK-242 is dissolved in DMSO, and then diluted in sterile normal saline to a final DMSO concentration of 1% (prepared and used immediately).

[0060] Statistical analysis was performed using GraphPad Prism 8. Unless otherwise stated, all values are expressed as mean ± standard error (Mean ± SEM). Differences between groups were analyzed using one-way ANOVA or two-way ANOVA. In all cases, p<0.05 was considered statistically significant, where * indicates a significant difference (p<0.05), ** indicates a highly significant difference (p<0.01), and *** indicates a highly significant difference (p<0.001).

[0061] The results are shown in Figures 2 to 6

[0062] During the experiment, the body weight of the normal control group mice remained stable and increased. Compared with the normal control group, after administration, the body weight of the sorafenib group mice decreased significantly in the early experimental period, and began to recover slowly in the later experimental period, but still showed a slow trend of weight gain. The combination of TAK-242 can adjust the slow weight gain of mice caused by sorafenib. As Figure 2 shown.​

[0063] The length of each intestinal segment is one of the important parameters for evaluating intestinal inflammation. Compared with the normal control group, the colon and small intestine length of the sorafenib group mice was significantly shortened, while the combination of TAK-242 reversed the shortening of the colon and small intestine length caused by sorafenib. As shown in Figure 3 .

[0064] The villi and crypt of the ileum of the normal control group mice were arranged regularly and had complete morphology. The ileum villi of the sorafenib group mice were atrophic, the structure was loose, and even had symptoms such as defects. After the combination of TAK-242, the atrophy of the ileum villi, the loose structure, and even the defects were lighter. As shown in Figure 4 .

[0065] Villi length, crypt depth, and the ratio of villi length to crypt depth (V / C) are common indicators for evaluating the morphological structure of the intestinal tract, which can be used to characterize the health status of the intestinal tract; the shortening of the villi and the deeper crypts will reduce the nutrient absorption of the intestinal surface area, and the decrease of the V / C ratio indicates that the mucosa is damaged and the digestive and absorptive capacity is decreased. Compared with the normal control group, the villi length of the ileum of the sorafenib group mice was significantly reduced, the crypt depth changed little, and the V / C value was significantly reduced; compared with the sorafenib group, the villi length of the ileum of the sorafenib + TLR4 inhibitor combination group mice was significantly increased, and the V / C value was significantly increased. As shown in Figure 5 .

[0066] The MDA level and the activity of antioxidant enzymes can be used to evaluate the oxidative stress state of the intestinal tissue. Compared with the normal control group, the total antioxidant capacity of the intestinal tissue of the sorafenib group mice was reduced, the CAT activity and SOD activity were significantly reduced, and the MDA content was increased (indicating that the body was in a state of lipid peroxidation); while the combination of TAK-242 could significantly alleviate the decrease of CAT activity and SOD activity and the increase of MDA content of the intestinal tissue caused by sorafenib.

[0067] In summary, TAK-242 can slow down the increase of body weight caused by sorafenib, alleviate the intestinal mucosal damage caused by sorafenib, and reduce the intestinal oxidative stress caused by sorafenib.

[0068] The above describes the embodiments of the present application in detail in combination with the drawings, but the present application is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the purpose of the present application.

Claims

1. The use of a TLR4 inhibitor in the preparation of a drug for preventing and / or alleviating gastrointestinal toxicity caused by sorafenib; wherein the TLR4 inhibitor is TAK-242.

2. The application according to claim 1, characterized in that, The drug can reverse the slow weight gain in mice caused by sorafenib.

3. The application according to claim 1, characterized in that, The drug can prevent and / or alleviate sorafenib-induced intestinal mucosal damage in mice.

4. The application according to claim 3, characterized in that, The prevention and / or relief of sorafenib-induced intestinal mucosal damage in mice includes at least one of A1) to A5); A1) Increase colon length; A2) Increase the length of the small intestine; A3) Increase the length of the villi in the ileum; A4) Increase the ratio of villus length to crypt depth; A5) Improves at least one of the following symptoms: ileal villus atrophy, loose structure, and defects.

5. The application according to claim 1, characterized in that, The drug can reduce intestinal oxidative stress caused by sorafenib.

6. The application according to claim 5, characterized in that, The reduction of sorafenib-induced intestinal oxidative stress includes at least one of B1 to B4; B1) Reduces intestinal MDA levels; B2) Increases CAT activity; B3) Increases SOD activity; B4) Improves overall antioxidant capacity.

7. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

8. The application according to claim 1, characterized in that, The dosage form of the drug is granules, hard capsules, soft capsules, tablets, emulsions, or suspensions.

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