Application of futantinib in treatment of severe dengue liver injury

Abstract

The invention discloses an application of futantinib in treatment of severe dengue liver injury, belongs to the technical field of biology, and provides an application of futantinib or a pharmaceutically acceptable salt thereof in preparation of a pharmaceutical composition for preventing or treating the dengue liver injury and a pharmaceutical composition for regulating hepatocyte carbon metabolism of a dengue virus infected patient. The invention also provides a method for regulating the in-vitro dengue virus infected liver cell injury level and screening a medicine for assisting in treating and preventing dengue liver injury by futantinib or medicinal salt thereof.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the application of fotantinib in treating severe dengue fever liver damage. Background Art

[0002] Dengue fever is an acute infectious disease caused by dengue virus (DENV) transmitted by mosquitoes (Aedes aegypti and Aedes albopictus). According to the World Health Organization (WHO), dengue fever is prevalent in more than 100 countries in Africa, America and Southeast Asia. About 40% of the world's population is at risk of infection, with about 390 million cases of infection each year, of which 96 million are diagnosed. Dengue fever has become a serious global public health problem. Dengue virus infection causes flu-like symptoms such as high fever, headache, and muscle pain in patients, and can also develop into fatal severe dengue fever (SD), which is divided into dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Every year, there are about 50 million to 100 million symptomatic dengue patients, of which 3 million to 6 million symptomatic patients progress to fatal severe dengue fever. In 2023, dengue virus caused more than 5 million cases of dengue fever in 80 countries and more than 5,000 deaths from SD. In 2024, as of July, more than 10 million cases of dengue fever were reported in 176 countries, including more than 24,000 severe cases and 6,508 deaths. The DENV epidemic not only increases the economic burden of the country, but also seriously affects the lives and health of the people. However, there is currently no targeted therapeutic drug.

[0003] Liver damage is the most common complication in dengue patients, including abnormal blood biochemistry, hepatomegaly, acute liver failure (ALF), etc. Clinical studies have shown that abnormally elevated aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels can indicate severe disease symptoms and poor prognosis. Gross autopsy results showed that the livers of deceased patients showed pathological manifestations such as congestion, swelling, fatty degeneration and necrosis. In addition, deaths are usually associated with liver failure. Current studies have shown that hepatocellular damage is caused by multiple factors, including direct cell lesions caused by viral infection, indirect immune-mediated damage, and poor liver perfusion. Therefore, early diagnosis and treatment of liver damage may reduce the morbidity and mortality of patients with severe dengue, but there are no targeted drugs for SD liver damage. Summary of the invention

[0004] Severe dengue (SD) is the main cause of morbidity and mortality in patients infected with dengue virus. Liver damage is the most common complication of SD, including blood biochemical abnormalities, hepatomegaly, acute liver failure (ALF), etc. Liver damage is related to the severity of SD and may indicate a poor prognosis of the disease. Gross autopsy results showed that the livers of deceased patients showed pathological manifestations such as congestion, swelling, fatty degeneration and necrosis. However, at present, except for supportive therapy, there are no targeted drugs for the treatment of SD liver damage. Therefore, this patent aims to solve the problem of drug development for the prevention and treatment of SD liver damage.

[0005] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0006] The present invention provides the use of fotantinib or a pharmaceutically acceptable salt thereof in preparing a pharmaceutical composition for preventing or treating dengue fever liver damage.

[0007] Furthermore, the dengue fever liver injury includes severe dengue fever liver injury.

[0008] Furthermore, the severe dengue fever liver damage includes significantly elevated liver enzymes, severe liver function abnormalities, hepatomegaly, or symptoms of liver failure.

[0009] Furthermore, the pharmaceutical composition is administered orally, via suppository, around the lesion, on the lesion surface, topically, intravenously, parenterally, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally or subcutaneously.

[0010] Furthermore, the pharmaceutical composition includes pharmaceutically acceptable excipients.

[0011] Furthermore, the auxiliary materials include glidants, sweeteners, diluents, preservatives, colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, pH regulators and / or buffers, and emulsifiers.

[0012] In this context, the term "pharmaceutically acceptable" means that when these molecular entities and compositions are appropriately administered to animals or humans, they do not produce adverse, allergic or other adverse reactions. Specific examples of some substances that can be used as pharmaceutically acceptable excipients or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as wetting agents, such as sodium lauryl sulfate; colorants; flavorings; tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc. The composition of the present invention can be prepared into various dosage forms as required, and the physician can determine the dosage that is beneficial to the patient according to factors such as the patient type, age, weight, general disease condition, and administration method. The administration method can be, for example, injection or other treatment methods.

[0013] Furthermore, the pharmaceutically acceptable salt may be an acid addition salt or a base addition salt.

[0014] Further, the acid addition salt includes, but is not limited to, any one of the hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate and salicylate, malonate, adipate, caproate, arginine, fumarate, nicotinate, phthalate or oxalate of fotantinib, or a combination of at least two thereof.

[0015] Furthermore, the base addition salt includes but is not limited to lithium salt, sodium salt, potassium salt, barium salt, calcium salt, magnesium salt, aluminum salt, iron salt, ferrous salt, copper salt, zinc salt of fotantinib, or a salt of fotantinib with morpholine, diethylamine, triethylamine, isopropylamine, trimethylamine, lysine or histidine.

[0016] In this context, the "pharmaceutically acceptable salt" is non-toxic in the amount and concentration used. The preparation of such salts can facilitate pharmacological applications by changing the physical properties of the compound without preventing it from exerting its physiological effects.

[0017] In some embodiments, the acid addition salt is prepared using an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. In some embodiments, the acid addition salt is prepared using an organic acid, such as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, p-toluenesulfonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, dodecylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, etc.

[0018] In some embodiments, the pharmaceutical composition includes but is not limited to the following dosage forms, such as tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), pills, powders, granules, capsules (including soft capsules, microcapsules), lozenges, syrups, liquids, emulsions, suspensions, controlled release preparations (e.g., instant release preparations, sustained release preparations, sustained release microcapsules), aerosols, films (e.g., orally disintegrating films, oral mucosa-adhesive films) , injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), intravenous drips, transdermal absorption preparations, ointments, lotions, adhesive preparations, suppositories (e.g., rectal suppositories, vaginal suppositories), pills, nasal preparations, pulmonary preparations (inhalants), eye drops, etc., oral or parenteral preparations (e.g., intravenous, intramuscular, subcutaneous, intraorgan, intranasal, intradermal, instillation, intracerebral, intrarectal and other forms of administration, administration to the vicinity of the tumor and direct administration to the lesion).

[0019] The present invention provides a pharmaceutical composition for preventing or treating dengue fever liver damage, wherein the pharmaceutical composition comprises a preventive effective amount or a therapeutic effective amount of fotantinib or a pharmaceutically acceptable salt thereof.

[0020] Furthermore, the dengue fever liver injury includes severe dengue fever liver injury.

[0021] In some embodiments, the pharmaceutical composition further comprises other active ingredients that can be added, including but not limited to weight loss agents (appetite suppressants, slimming agents), antacids (anti-acidosis agents), anti-hypoxic agents, analgesics (anti-rheumatic drugs), anti-worm drugs, anti-allergic drugs, anti-anemic drugs, anti-arrhythmic agents, antibiotics (anti-infective drugs), anti-dementia drugs (nootropic drugs), anti-diabetic drugs, antidote, antiemetic drugs (anti-vertigo agents), anti-epileptic drugs, anti-hemorrhagic drugs (anti-fibrinolytic drugs and other hemostatic agents), anti-hypertensive drugs, anti-hypoglycemic drugs. Medications, antihypotensives, anticoagulants, antifungals, antiparasitics, anti-inflammatory drugs, cough suppressants (expectorants), antiatherosclerotics, bath and thermotherapy agents, beta-blockers, calcium channel blockers and inhibitors of the renin-angiotensin-aldosterone system, bronchial drugs (antiasthmatics), choleretics and biliary therapeutics, cholinergics, adrenocortical hormones, skin drugs, disinfectants (antibacterials), dietary agents (nutritionals), diagnostic agents and preparations for diagnosis, diuretics, blood circulation promoting agents, detoxification agents (drugs for the treatment of addiction), enzyme inhibitors, preparations for enzyme deficiencies agents, transporters, fibrinolytics, geriatric drugs, antigout preparations, cold and flu medicines and cough and sneezing medicines, gynecological drugs, hemorrhoid drugs (rectal drugs), liver drugs, hypnotics (sedatives), pituitary hormones, hypothalamic hormones and other regulatory peptides and their inhibitors, immunomodulators, infusions and standard injections, organ perfusions, cardiac drugs, caries prevention agents, periodontal degeneration drugs and other dental preparations, coronary preparations, laxatives, lipid-lowering drugs, local anesthetics (neurotherapeutics), gastrointestinal drugs, migraine drugs, mineral preparations, oral and throat drugs, muscle relaxants, anesthetics, neuropathy preparations and Other neurotropic agents, ophthalmic drugs, anti-osteoporotic agents (calcium- and bone metabolism regulators), otological drugs, anti-Parkinson drugs and other drugs for extrapyramidal disorders, psychiatric drugs, nasal drugs (sinus drugs), tonic drugs (tonic drugs), thyroid preparations, serums, immunoglobulins and vaccines, sex hormones and their inhibitors, antispasmodics (anticholinergic drugs), platelet aggregation inhibitors, tuberculosis drugs, mood-altering drugs, urological drugs, drugs for venous diseases, vitamins, wound and scar treatments, cytostatics and other antitumor drugs and protective agents, biomaterials, pharmaceutical synthetics.

[0022] As described herein, the term "prophylactically effective amount" is an amount sufficient to prevent a disease, disorder, or condition, or an amount sufficient to prevent one or more symptoms associated with a disease, disorder, or condition, or an amount to prevent the recurrence of a disease, disorder, or condition. A prophylactically effective amount of a drug refers to an amount of a therapeutic agent that provides a prophylactic benefit in the process of preventing a disease, disorder, or condition when used alone or in combination with other agents. The term "prophylactically effective amount" may include an amount that improves overall prevention, or an amount that enhances the prophylactic effect of other prophylactic agents.

[0023] As used herein, the term "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder, or condition, or to delay or minimize one or more symptoms associated with the disease, disorder, or condition. A therapeutically effective amount of a drug refers to an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a disease, disorder, or condition. The term "therapeutically effective amount" may include an amount that improves overall treatment, reduces or avoids symptoms or causes of a disease or condition, or enhances the therapeutic effect of other therapeutic agents.

[0024] In some embodiments, the effective amount can be determined according to various factors, including the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type of preparation, the age of the patient, the weight, general health, sex and diet of the patient, the time of administration, the route of administration, the secretion rate of the composition, the treatment period and concomitant medications.

[0025] The present invention provides a method for regulating the damage level of dengue virus infected hepatocytes in vitro, the method comprising adding fotantinib or a pharmaceutically acceptable salt thereof to dengue virus infected hepatocytes in vitro.

[0026] Furthermore, the method is non-therapeutic.

[0027] The present invention provides the use of fotantinib or a pharmaceutically acceptable salt thereof in preparing a pharmaceutical composition for regulating carbon metabolism of liver cells of patients infected with dengue virus.

[0028] In an embodiment of the present invention, the hepatocytes are derived from any animal, including but not limited to humans, non-human primates, rodents, etc. Similarly, in an embodiment of the present invention, the "patient" refers to an animal subject, particularly a vertebrate subject, more particularly a mammalian subject. Suitable vertebrates falling within the scope of the present invention include but are not limited to any member of the subphylum Chordata, including primates, rodents (e.g., mice, rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), birds (e.g., chickens; ducks; geese; companion birds, such as canaries, budgies, etc.), marine mammals (e.g., dolphins, whales), reptiles (e.g., snakes, frogs, lizards, etc.) and fish. Preferred subjects are primates (e.g., humans, apes, monkeys, chimpanzees).

[0029] In this article, the "fostatinib" refers to fostamatinib, which is an oral spleen tyrosine kinase (SYK) inhibitor mainly used to treat chronic immune thrombocytopenia (ITP).

[0030] The present invention provides a method for non-therapeutic screening of drugs for assisting fotantinib or its pharmaceutically acceptable salts in treating and preventing dengue fever liver damage, the method comprising the step of adding candidate drugs to in vitro dengue virus-infected hepatocytes treated with fotantinib or its pharmaceutically acceptable salts.

[0031] As used herein, the terms "screening", "screening" refer to any method, technique, process or task intended to generate diagnostic and / or prognostic information. Thus, one skilled in the art will understand that the term screening encompasses determining whether an individual has, may have or develop, or is at risk of having or developing a disease, disorder or condition.

[0032] Furthermore, the method detects the levels of liver enzymes and liver function indicators in cells after administration of the candidate drug.

[0033] In some embodiments, the liver function index includes aspartate aminotransferase, total bilirubin value, serum bilirubin value, plasma albumin value, prothrombin time extension value, prothrombin ratio, ascites value and hepatic encephalopathy value.

[0034] The present invention provides a method for preparing dengue fever hepatocytes with high carbon metabolism, wherein the method comprises treating the dengue fever hepatocytes with fotantinib or a pharmaceutically acceptable salt thereof.

[0035] Compositions or methods described herein as "comprising" one or more named elements or steps are open ended, meaning that the named elements or steps are required, but other elements or steps may be added within the scope of the composition and method. To avoid redundancy, it is also understood that any composition or method described as "comprising" one or more named elements or steps also describes a corresponding more limited composition or method "consisting essentially of the same named elements or steps," meaning that the composition or method includes the named required elements or steps, and may also include other elements or steps that do not materially affect the basic and novel characteristics of the composition or method.

[0036] The term "drug candidate" as used herein generally refers to substances that may be used as drugs and can be evaluated by appropriate methods to obtain information on their biological activity, pharmacological effects and efficacy.

[0037] Advantages and beneficial effects of the present invention:

[0038] Previous studies used dengue virus type II N10 to infect Ifnar- / - mice and construct a SD mouse model with significant liver damage. We found that the liver mononuclear phagocytic system (HMPS), which includes Kupffer cells (macrophages in the liver), monocytes, and peripheral macrophages, promotes the activation of inflammation-related pathways. In addition, HMPS disorders have been shown to promote a variety of liver diseases. For example, infiltration of activated monocytes and monocyte-derived macrophages in fatty liver hepatitis can promote liver fibrosis. Therefore, HMPS is crucial for the stability of the liver microenvironment and is of great value for the treatment of SD patients.

[0039] Furthermore, through multi-omics and single-cell RNA sequencing, we found that hepatocyte carbon metabolism dysfunction and changes in HMPS cell components are important features of SD. By analyzing the disrupted kinase expression patterns in the liver of SD mice, we found and demonstrated that fostamatinib, which has been approved for the treatment of thrombocytopenia, can inhibit the progression of liver damage in SD mice by alleviating carbon metabolism dysfunction, thereby improving the survival of SD mice, which may be used in future clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a graph showing the establishment result of a mouse model of severe dengue fever with liver injury, wherein a is the construction process of the mouse model of severe dengue fever with liver injury, bd are graphs showing the disease severity of mice with severe dengue fever with liver injury, eg is the dengue virus load in serum, liver, and intestine, h is a graph showing liver lesions in mice infected with NGC, i is a graph showing liver injury results in mice with severe dengue fever, and j is a graph showing the ALT and AST test results of mice in the severe group and the NGC group.

[0041] Figure 2 This is a gross anatomy of the liver tissue of mice injected with inactivated dengue virus.

[0042] Figure 3 It is the main characteristic analysis diagram of liver lesions during SD, among which A is KEGG enrichment analysis, BC is PPI network analysis of the regulatory relationship between genes and receptors and ligands, DE is IFA co-localization analysis, F is the grayscale overlap result of IDH1 and S100A4, and G is the grayscale overlap result of IDH1 and LY6G.

[0043] Figure 4 It is a kinase prediction and kinase activity prediction diagram based on the proteome and phosphorylated proteome, where AB is the activation of kinases in the liver of SD mice at 4dpi, C is the difference in kinase expression levels between the uninfected group and SD mice, and D is the overlapping result of differentially activated kinases between different groups.

[0044] Figure 5This is an analysis diagram of the kinase expression patterns of hepatocytes and HMPS cell components during SD, wherein A is the co-localization result of kinase BTK with hepatocytes, BC is the co-localization result of BTK with cell components of the mononuclear phagocytic system, D is the change in the gray value level of BTK, E is the change in the gray value level of S100A4, F is the change in the gray value level of LY6G, G is the co-localization result of BTK with ALB, H is the co-localization result of BTK with S100A4, and I is the co-localization result of BTK with LY6G.

[0045] Figure 6 It is a relationship diagram between carbon metabolism-related genes and disordered expression kinases, where A is the result of screening differentially expressed genes between SD mice and control mice, B is the result of PPI network analysis, C is the result of Cytohubba screening, and D is the result of drug target screening.

[0046] Figure 7 This is a graph showing the effect of Fostamatinib on the survival status of SD mice, where A is the statistical result of body weight, B is the statistical result of death time, C is the statistical result of AST expression, and D is the statistical result of ALT expression.

[0047] Figure 8 The following are the results of the effect of Fostamatinib on liver injury during SD, where A is the local lesion image, B is the H&E staining result, C is the ImageJ quantification result of the lesion area, and D is the Mfuzz result.

[0048] Fig. 9 The figure shows the effect of fostamatinib on the carbon metabolism function of the liver of SD mice. AC is the IFA statistical result of Kupffer cells / monocytes / macrophages (S100A4+) and neutrophils (LY6G+), and DF is the GSEA analysis of the gene expression of carbon metabolism pathway in the liver of mice after fostamatinib treatment. DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0050] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the reagents, biological materials, etc. used in the following examples are all commercially available unless otherwise specified.

[0051] Example 1

[0052] 1. Experimental Materials

[0053] 1) Animals: Type I interferon receptor (Ifnar- / -) knockout mice on a C57BL / 6 background were purchased from Cyagen (Suzhou) Biotechnology Co., Ltd. All mice were housed in ABSL-2 at the Institute of Animal Science, Chinese Academy of Medical Sciences.

[0054] 2) Virus: Dengue virus (DENV): Type 2 New Guinea strain was purchased from ATCC, and the N10 adapted strain was obtained by subculturing the New Guinea strain in mice in our laboratory and stored at -80°C. All experiments involving dengue virus were performed in an animal biosafety level 2 (ABSL-2) or biosafety level 2 (BSL-2) laboratory authorized by the Institute of Laboratory Animals, Chinese Academy of Medical Sciences.

[0055] 3) RNA extraction and QPCR kits: QIAamp virus RNA Mini Kit (Qiagen, 52906) and TaqMan quantitative polymerase chain reaction (Qiagen, 204443).

[0056] 4) Biochemical kits.

[0057] 2. Experimental methods

[0058] 2.1 Establishment of a severe dengue fever mouse model with liver damage

[0059] 1) Experimental animal groups. For details, see Table 1.

[0060] Table 1

[0061]

[0062] 2) Viral RNA quantification

[0063] a) Total RNA extraction: QIAamp virus RNA Mini Kit (Qiagen, 52906) was used to extract total RNA from viruses or cells, that is, total RNA from virus solution was extracted by separation column method. Add 560 μL of Mixture and 140 μL of virus solution sample to a 1.5 mL EP tube, mix well, and incubate at room temperature for 10 min to lyse the virus and release nucleic acid. Then add 560 μL of anhydrous ethanol, mix well, and transfer 630 μL of the mixture to the collection column, centrifuge at 6000 g for 1 min, replace with a new collection tube, and absorb the remaining 630 μL of the mixed solution, and repeat the previous step. After the waste liquid in the collection column overflows, replace with a new collection tube, add 500 μL of AW1 solution, centrifuge at 6000 g for 1 min, and replace with a new collection tube. Add 500 μL of AW2 solution, centrifuge at 20000 g for 3 min, and replace with a new collection tube. Centrifuge at 20000 g for 1 min. Place the collection column in a sterile 1.5 mL EP tube, add 60 μL AVE solution, centrifuge at 6000 g for 1 min, and discard the collection column. The extracted total RNA can be directly used for subsequent qPCR experiments or stored at -80°C.

[0064] b) Preparation of standards and analysis of standard results: The above-mentioned Total RNA was reverse transcribed to obtain cDNA, and ordinary PCR was performed using the following upstream primer DENV-forward primer 1 and downstream primer DENV-reverse primer 2. The obtained DNA product was divided into two parts, and 5 μL was taken for agarose gel electrophoresis. If the target fragment band size is about 67 bp, the remaining part is used for the construction of the standard. The concentration of the constructed plasmid standard was measured using a NanoDrop spectrophotometer and marked as the original standard. Then, DEPC water was used for 10-fold continuous gradient dilution, and a preliminary experiment was performed. Finally, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 The diluted plasmid was used as the standard. A standard curve was established based on the relationship between the concentration of the plasmid standard and the Ct value, and a fitting formula was used to estimate the virus copy number of other samples.

[0065] 3) Real-time fluorescence quantitative PCR detection of DENV: TaqMan quantitative polymerase chain reaction (Qiagen, 204443) was then used to quantify viral RNA.

[0066] 3.1) Primer and probe sequences are shown in Table 2.

[0067] Table 2

[0068]

[0069] 3.2) Prepare the qPCR reaction system as shown in Table 3.

[0070] Table 3

[0071]

[0072]

[0073] 3.3) Set up the qPCR reaction program as shown in Table 4.

[0074] Table 4

[0075]

[0076] 4) Mouse histopathological analysis (H&E staining)

[0077] This study used hematoxylin-eosin staining (H&E staining), which can make the nucleic acid in the cytoplasm and the nucleus purple-blue, and the components in the extracellular matrix and cytoplasm red, thereby distinguishing the cell structure. The following is a brief description of the staining process. The pathological sections were immersed in xylene I, II, and III for 10 minutes each. Then they were immersed in anhydrous ethanol I and II for 5 minutes each, and 95%, 80%, 70%, and 50% ethanol for 5 minutes each. They were placed in hematoxylin dye for 15 minutes and slowly rinsed with tap water. Differentiate with 1% hydrochloric acid ethanol for 5 to 20 seconds, and then slowly rinse with tap water for 1 to 3 minutes. 2% ammonia water turned blue for 30 seconds to 1 minute, and 70% and 80% ethanol were dehydrated for 2 minutes respectively. Then place it in eosin staining for 30s to 2min, dehydrate it in 95% ethanol for 1min, place it in anhydrous ethanol I and II for 2min each, place it in xylene I and II for 5min, and finally use neutral gum to seal the slide.

[0078] 5) Blood biochemical testing

[0079] a) Blood collection: On days 0, 2, 4, and 6 after DENV infection, mice were anesthetized with tribromoethanol, and blood was collected from the submandibular venous plexus. The blood was centrifuged at 300 g for 10 min × 2 times to separate the serum, which was directly used for subsequent measurements or stored in a -80°C refrigerator.

[0080] b) On-machine testing: Use a fully automatic biochemical analyzer for testing. First, turn on the instrument and perform a self-test according to the program. Then clean the instrument twice, empty the waste liquid bucket, and replace the reaction cup. Select the test items: ALT and AST. Then place negative and positive controls, standards, and experimental samples for testing. Finally, record and export the test results.

[0081] c) Calculation: Calculate the samples according to the standard curve.

[0082] 3. Experimental results

[0083] To study the differences in disease progression between severe dengue and dengue fever, and to explore the impact of organ failure on severe dengue, we established a severe dengue mouse model. To further evaluate whether the severe dengue mouse model had liver damage, we performed a gross autopsy on the mice and collected liver tissue samples on days 0, 2, 4, 6, and 8 after infection ( Figure 1 a). The results showed that on the 6th day after infection, the liver surface of mice infected with N10 had a large number of focal lesions, and a small number of mice also had subcapsular hemorrhage with multiple petechiae, which is consistent with the disease manifestations of coagulopathy. However, the liver of mice infected with NGC had only a small number of focal lesions ( Figure 1 h). The results showed that the liver of mice with severe dengue fever was severely damaged. In order to determine the extent of tissue damage and the duration of damage, we used H&E staining to simultaneously observe the pathological characteristics of the liver. The liver of mice with severe dengue fever (N10) showed hepatocellular necrosis and a large number of inflammatory cell infiltration ( Figure 1 i). During the progression of severe dengue fever, clinical patients developed varying degrees of liver damage. Therefore, in order to further verify whether liver damage occurred in mice with severe dengue fever, we used blood biochemistry experiments to detect the changes in transaminase levels in mild and severe groups over time. The results showed that the ALT of mice infected with N10 in the severe group increased significantly from the second day after infection (about 4 times higher than that in the NGC group, P<0.05). On the sixth day after infection, the ALT and AST of mice in the N10 group were significantly higher than those in the NGC group (ALT: 2 times, P<0.01; AST: 2 times, P<0.05) ( Figure 1 j). Increased ALT and AST indicated liver damage in mice with severe dengue, suggesting liver cell damage. Increased viral load in the severe dengue mouse model ( Figure 1 eg), increased disease severity ( Figure 1 bd), organ damage, and abnormally elevated transaminases characterize the disease symptoms in clinically critically ill patients.

[0084] Based on the above results, in order to prove that DENV replication caused liver damage in mice and to exclude the possibility that DENV structural proteins caused liver damage in mice, we inactivated the above two dengue viruses and injected them into mice through the tail vein. The animals were dissected and observed on the 6th day after injection. The results showed that the inactivated dengue viruses failed to cause liver surface lesions in mice ( Figure 2 ), which suggests that hepatocyte necrosis during DENV infection may be caused by viral replication.

[0085] Example 2

[0086] 1. Experimental methods

[0087] 1) RNA-Sequencing

[0088] RNA-seq for this study was performed by Beijing Novogene Co., Ltd. First, RNA was extracted from the liver, and the RNA integrity and total amount were detected using an Agilent 2100 bioanalyzer, and then the library was constructed and QC was performed using a Qubit instrument and real-time fluorescence quantitative PCR. Only samples that passed QC were further analyzed. Subsequently, different libraries were combined according to the effective concentration and the required downstream data volume, and then Illumina sequencing was performed. After quality control evaluation, different libraries were combined according to the effective concentration and target data volume and sequenced with Illumina.

[0089] RNA-Sequencing Raw Data Processing

[0090] First, the raw reads were processed using fastp software to remove reads containing splice sites, poly-N sequences, and low-quality reads to obtain clean reads. Then the Q20, Q30, and GC content values ​​of the clean data were calculated. The index of the reference genome was constructed using HISAT2 v2.0.5, and the opposite-end clean reads were aligned to the reference genome using HISAT2v2.0.5. Featurecots (1.5.0-p3) was then used to calculate the number of reads for each gene. Genes were then calculated based on their length, and the FPKM of each gene was calculated. The DESeq2 R software package (1.20.0) was used to calculate feature values ​​and perform differential expression analysis of the two conditions / groups. P value <0.05 and |fold change (FC)|>2 were defined as deg.

[0091] 2) scRNA-seq preparation

[0092] In the ABSL-2 laboratory, fresh liver tissue was first rinsed with PBS and then stored in sCelLiveTM tissue preservation solution. In the ABSL-2 laboratory, fresh liver tissue was washed three times with Hanks balanced salt solution (HBSS), cut into small pieces by mechanical dissociation, and then washed with 3 mL of sCelLive TM Tissue dissociation solution (Singleron) was used at 37°C using Singleron PythoN TM The cell suspension was collected and filtered through a 40 μm sterile filter. Red blood cell lysis buffer (RCLB, Singleron), incubate at room temperature for 5-8 minutes to remove red blood cells. Centrifuge at 300×g for 5 minutes at 4℃, remove the supernatant, and resuspend in PBS at a concentration of 1000 cells / μL. The single cell processing system was used to prepare a single cell suspension (2×10 5 cells / mL) onto a microwell chip. Subsequently, the barcoded beads are collected from the microwell chip. Next, the mRNA captured by the barcoded beads is reverse transcribed and then used to obtain cDNA. Finally, PCR amplification is performed. The amplified cDNA is then fragmented and ligated to sequencing adapters. scRNA-seq libraries were constructed using the protocol outlined in the Single Cell RNA Library Kit (Singleron).1 Individual libraries were diluted to 4 nM, pooled, and sequenced on an Illumina NovaSeq 6000 with paired-end reads of 150 bp.

[0093] scRNA-seq raw data processing: The raw reads of scRNA sequencing were processed using the CeleScope v1.10.0 pipeline (https: / / github.com / singleron-RD / CeleScope) to generate a gene expression matrix.

[0094] 3) Protein preparation and LC-MS / MS analysis

[0095] Lysis buffer (containing 1% SDS, 1% protease inhibitors, and 1% phosphatase inhibitors) was added to 100 mg of liver tissue, and the mixture was heated to 100°C for 30 minutes to inactivate. After homogenization, the supernatant was centrifuged at 12,000 g for 10 minutes at 4°C, and the protein concentration was determined by the BCA method. The protein sample was then added to pre-cooled acetone, vortexed, and then added to pre-cooled acetone. The mixture was precipitated at -20°C for 2 hours and washed 2-3 times with pre-cooled acetone after precipitation. The protein sample was then redissolved in 200 mm TEAB and dispersed by ultrasound. The ratio of trypsin to protein was 1:50, and the initial digestion was performed overnight. The sample was reduced with 5 mM dithiothreitol at 37°C for 60 minutes and then alkylated with 11 mM iodoacetamide at room temperature in the absence of light for 45 minutes. Subsequently, the peptides were desalted using a StrataX solid phase extraction column.

[0096] The peptides required for phosphorylated proteomics analysis were dissolved in enrichment buffer (50% acetonitrile / 0.5% acetic acid), and the resulting solution was transferred to the pre-washed IMAC material and incubated with gentle agitation on a rotary shaker. Subsequently, the material was washed three times with a buffer solution consisting of 50% acetonitrile / 0.5% acetic acid and 30% acetonitrile / 0.1% trifluoroacetic acid. Subsequently, the phosphopeptides were eluted with 10% ammonia water, and the eluate was collected and freeze-dried under vacuum. After extraction, the material was desalted according to the instructions provided with the C18 ZipTips and freeze-dried under vacuum in preparation for liquid mass spectrometry analysis.

[0097] The peptides were dissolved in mobile phase A of the LC system and separated using a Vanquish Neo ultra-high performance liquid chromatography (UHPLC) system. The peptides were then injected into the NSI ion source for ionization and subsequently analyzed by Orbitrap star mass spectrometry. The ion source voltage was set to 1900 V. The parent ions of the peptides were detected and analyzed using the Orbitrap detector, while the secondary fragment ions were detected and analyzed using the Astral detector.

[0098] 4) Immunofluorescence staining (IFA)

[0099] Liver sections (5 μm thickness) were immersed in xylene and alcohol and incubated with the following primary antibodies overnight at 4°C, including rabbit anti-albumin (ALB) monoclonal antibody (1:100, Abcam, ab207327), rabbit anti-IDH1 monoclonal antibody (1:100, Abcam, ab230949), rabbit anti-ly6g antibody (1:100, CST, 87048S), mouse anti-s100a4 antibody (1:500, proteintech, 66489-1), and rabbit anti-BTK monoclonal antibody (1:100, Abcam, ab208937). After washing with PBS, the cells were incubated with the following secondary antibodies at room temperature for 1 hour, including donkey anti-mouse IgG (1:1000, Alexa Fluor R 488, ab150105, Life technologies), donkey anti-rabbit IgG (1:1000, Alexa Fluor R 594, ab150064, Life technologies) and donkey anti-rabbit IgG (1:1000, Alexa Fluor R 488, ab150073, Life technologies). The average gray value of the positive staining area was analyzed using ImageJ.

[0100] 2. Experimental results

[0101] To clarify the changes in liver function of mice during SD, we performed KEGG enrichment analysis on differentially expressed genes or proteins in each group. The results showed that liver metabolic dysfunction in mice with severe dengue fever ( Figure 3 A). To further clarify the significant molecular features of liver metabolic dysfunction during SD, we combined and validated the KEGG enrichment results of differentially expressed genes / proteins in multi-omics with the KEGG enrichment results of differentially expressed genes in cells in single-cell RNA sequencing. Therefore, we demonstrated that carbon metabolism is the main metabolic pathway of liver dysfunction during SD. To further demonstrate the relationship between changes in intracellular communication and carbon metabolic dysfunction in SD hepatocytes, we combined hepatocytes, Kupffer cells (KCs) & monocytes / macrophages The interaction network was constructed by comparing the differentially expressed genes enriched in carbon metabolism pathways revealed by CellChat in neutrophils and the receptor and ligand nanoSTRING database in the activation pathways ( Figure 3 BC). The PPI network showed significant interactions between these genes and receptors and ligands ( Figure 3 B), some genes are regulated by these receptors and ligands ( Figure 3 C). In addition, IFA also suggested that the carbon metabolism pathway-related gene IDH1 was also mainly co-localized with Kupffer cells / monocytes / macrophages (S100A4+) and neutrophils (LY6G+) ( Figure 3 D and Figure 3 E). At 6 dpi, the grayscale value peaks and valleys of IDH1 and S100A4 ( Figure 3 F) and LY6G( Figure 3 G) Gray value overlap. It indicates that during SD, hepatocytes, KCs, Alterations in intercellular communication with neutrophils promote disturbances in liver carbon metabolism.

[0102] Kinases are involved in a variety of cellular processes, and abnormal activation of kinases has been reported to promote the occurrence of liver lesions. We then analyzed the activation and expression of kinases in the liver during SD based on the results of proteomic and phosphoproteomic analyses ( Figure 4 The results showed that most kinases in the liver of SD mice were activated at 4 dpi. Figure 4 A and Figure 4 B). More importantly, among the top 10 kinases with significant changes in proteome and phosphoproteome activities in liver of SD mice, the expression levels of most kinases were upregulated compared with those of uninfected controls ( Figure 4 C). Overlapping results of differentially activated kinases between groups also confirmed that most kinases were activated during SD ( Figure 4 D), thus indicating that the expression pattern of kinases in the liver is significantly disrupted during SD.

[0103] To verify the expression of abnormally expressed kinases in SD hepatocytes, immunofluorescence analysis (IFA) showed that the predicted upregulated kinase BTK was mainly co-localized with hepatocytes (ALB+)( Figure 5 A). BTK also partially colocalizes with cellular components of the mononuclear phagocytic system (Kupffer cells / monocytes / macrophages, S100A4+; neutrophils, LY6G+) ( Figure 5 B and Figure 5 C). BTK after infection ( Figure 5 D), S100A4( Figure 5 E) and LY6G( Figure 5 In addition, ImageJ also confirmed that the positive signal of BTK was significantly higher than that of ALB ( Figure 5 G)、S100A4( Figure 5 H) and LY6G( Figure 5 I), thus confirming the disruption of the kinase expression pattern and the activation of the mononuclear phagocytic system in SD livers.

[0104] Given that cell types with perturbed kinase expression profiles coincide with cell types with dysfunctional carbon metabolism, we hypothesized that hepatic carbon metabolism dysfunction during SD is associated with changes in kinase expression profiles. Single-cell RNA sequencing confirmed that carbon metabolism-related differentially expressed genes and perturbed kinases were mainly upregulated in hepatocytes and HMPS cell fractions. We then screened 14 overlapping differentially expressed genes from livers of SD mice and control mice that were enriched in carbon metabolism pathways ( Figure 6 A). The PPI network further revealed that these overlapping genes are regulated by fostamatinib target kinases ( Figure 6 B), the top 20 hub genes screened by Cytohubba overlap with the differentially expressed genes enriched in the carbon metabolism pathway between SD mouse liver HEPs and control mice ( Figure 6 C), indicating that disruption of kinase expression patterns promotes dysfunction of hepatic carbon metabolism during SD.

[0105] In order to explore therapeutic drugs for SD-induced liver injury and discover new targets, we used the kinases with altered activity as drug targets to screen potential anti-liver injury drugs ( Figure 6 D), and found that fostamatinib, which has been approved by the FDA for the treatment of thrombocytopenia, can inhibit most of the upregulated kinases in the liver of SD mice, including BTK. Combined with the expression levels of kinases in cells shown by single-cell RNA sequencing, we also found that HEPs and HMPS are the main target cells of fostamatinib. Therefore, we subsequently used fostamatinib to treat SD mice with liver damage.

[0106] Example 3

[0107] 1. Experimental Materials

[0108] Fostamatinib (R788) (MCE, HY-13038A) is an oral precursor of the active compound R406.

[0109] 2. Experimental methods

[0110] Animal administration experiment: 6-8 week old Ifnar- / - mice were intraperitoneally injected with tribromoethanol (500 mg kg -1 ) After anesthesia, 1×10 8 PFU dengue virus (N10 strain), and pathomorphological analysis and multi-omics analysis were performed on liver tissues. All mouse experiments were approved by the Laboratory Animal Use and Care Committee (IACUC) of the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences, and were performed in accordance with established operating protocols (authorization number: WW22001). To study the effect of fotantinib on liver damage in SD mice, Ifnar- / - mice infected with dengue virus (N10 strain) were used, weighed every day after infection, and monitored until the 6th day. Starting from the first day after infection (1dpi), fotantinib or solvent alone was injected intraperitoneally at 80mg / kg body weight once a day until

[0111] Liver tissue samples were collected from euthanized mice at 2, 4, and 6 dpi. If infected animals lost 20% or 15% of their body weight for three consecutive days, they were euthanized directly.

[0112] 3. Experimental results

[0113] To investigate the effect of fostamatinib treatment on the survival status of SD mice, Ifnar- / - mice were infected with DENV-2N10. On the first day after infection, the mice were orally administered fostamatinib until the sixth day after infection. The results showed that the body weight of SD mice and mice treated with fostamatinib was significantly reduced compared with that of uninfected mice ( Figure 7 A). Notably, all SD mice died at 5 dpi, while fostamatinib-treated mice died at 6 dpi ( Figure 7 B) These results suggest that fostamatinib treatment may improve survival in SD patients compared with SD mice.

[0114] We further evaluated whether fostamatinib has a therapeutic effect on liver injury in mice. Compared with the uninfected control group, SD mice had increased vascular leakage in the liver and focal lesions in some areas ( Figure 8A), while the fostamatinib treatment group significantly reduced vascular leakage and no focal lesions were observed ( Figure 8 A). H&E staining showed that the livers of SD mice showed changes in hepatocyte structure, loss of cell integrity, vacuolation, hemorrhage, and increased inflammatory cell infiltration, while the liver damage of mice treated with fostamatinib was significantly milder ( Figure 8 B). The percentage of lesion area was also quantified using ImageJ ( Figure 8 C). We found that fostamatinib treatment significantly reduced the percentage of lesion area in SD livers ( Figure 8 C). In addition, we evaluated the expression levels of common liver injury markers in serum, and the results showed that the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in mice treated with fostamatinib were significantly decreased compared with those in SD mice ( Figure 7 CD). Therefore, fostamatinib treatment significantly alleviated SD-induced liver lesions.

[0115] Furthermore, we analyzed the infiltration of mononuclear phagocytes in the liver of SD mice after fostamatinib treatment ( Fig. 9 IFA showed that after fostamatinib treatment, the positive staining signals of Kupffer cells / monocytes / macrophages (S100A4+) and neutrophils (LY6G+) were reduced ( Fig. 9 AC), suggesting that fostamatinib can significantly alleviate the abnormal activation of the mononuclear phagocytic system during SD. We also found that the positive signals of ALB and IDH1 also increased in the livers of mice treated with fostamatinib ( Fig. 9 AC), while the positive staining of BTK decreased after fostamatinib treatment ( Fig. 9 AC). In addition, with IFA ( Fig. 9 AC) results and Mfuzz( Figure 8 D) The results are consistent. GSEA analysis demonstrated that after fostamatinib treatment, most genes in carbon-enriched metabolic pathways in mouse liver were upregulated ( Fig. 9 DF).

[0116] In summary, fostamatinib treatment can not only alleviate SD-induced liver carbon metabolism dysfunction and kinase expression pattern disorder, but also inhibit the abnormal activation and infiltration of HMPS cell components and improve the survival status during SD. Our invention provides an effective drug strategy for the treatment of liver lesions in SD patients.

Claims

1. Use of fotantinib or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for preventing or treating dengue fever liver damage.

2. The use according to claim 1, wherein the dengue fever liver injury comprises severe dengue fever liver injury.

3. The use according to claim 2, wherein the severe dengue fever liver damage includes significantly elevated liver enzymes, severe abnormal liver function, hepatomegaly, or symptoms of liver failure.

4. according to the use described in claim 1, described pharmaceutical composition is by oral administration, suppository administration, perilesional administration, lesion surface administration, local spot use, intravenous administration, parenteral administration, peritoneal administration, muscle administration, intralesional administration, intrathecal administration, intranasal administration or subcutaneous administration.

5. The use according to claim 1, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient; Preferably, the auxiliary materials include glidants, sweeteners, diluents, preservatives, colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, pH regulators and / or buffers, and emulsifiers.

6. A pharmaceutical composition for preventing or treating liver damage caused by dengue fever, comprising a preventive or therapeutically effective amount of fotantinib or a pharmaceutically acceptable salt thereof; Preferably, the dengue fever liver injury includes severe dengue fever liver injury.

7. A method for regulating the level of damage to dengue virus-infected hepatocytes in vitro, the method comprising adding fotantinib or a pharmaceutically acceptable salt thereof to dengue virus-infected hepatocytes in vitro.

8. Use of fotantinib or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for regulating carbon metabolism in hepatocytes of patients infected with dengue virus.

9. A method for screening drugs for non-therapeutic purposes to assist fotantinib or its pharmaceutically acceptable salts in treating and preventing dengue liver damage, the method comprising the step of adding candidate drugs to in vitro dengue virus-infected hepatocytes that have been treated with fotantinib or its pharmaceutically acceptable salts; Preferably, the method detects the levels of liver enzymes and liver function indicators in cells after administration of the candidate drug.

10. A method for preparing dengue virus-infected hepatocytes with high carbon metabolism, the method comprising treating dengue virus-infected hepatocytes with fotantinib or a pharmaceutically acceptable salt thereof.

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