Application of fotantinib in the treatment of severe dengue fever liver injury

Abstract

The present invention discloses the use of fotantinib in treating severe dengue fever liver damage, belonging to the field of biotechnology. 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, and in preparing a pharmaceutical composition for regulating the carbon metabolism of hepatocytes in patients infected with dengue virus. The present invention also provides a method for regulating the level of in vitro dengue virus-infected hepatocyte damage, and for screening a pharmaceutical method for assisting fotantinib or a pharmaceutically acceptable salt thereof in treating and preventing dengue fever liver damage.

Description

Technical Field

[0001] The present 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 the dengue virus (DENV) and transmitted by mosquitoes (Aedes aegypti and Aedes albopictus). According to the World Health Organization (WHO), dengue fever is endemic in over 100 countries in Africa, the Americas, and Southeast Asia. Approximately 40% of the world's population is at risk of infection, with approximately 390 million cases reported annually, of which 96 million develop the disease. Dengue fever has become a serious global public health problem. Dengue virus infection causes flu-like symptoms such as high fever, headache, and muscle aches, but can also progress to severe dengue fever (SD), which is fatal and can be divided into dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Approximately 50 to 100 million patients develop symptomatic dengue fever annually, of which 3 to 6 million progress to severe, fatal dengue fever. In 2023, dengue virus (DENV) caused over 5 million cases of dengue fever and over 5,000 deaths from dengue fever (SD) in 80 countries. By July 2024, over 10 million cases of dengue fever had been reported in 176 countries, including over 24,000 severe cases and 6,508 deaths. DENV epidemics not only impose a heavy economic burden on countries but also severely impact public health. However, no targeted therapeutics are currently available.

[0003] Liver damage is the most common complication in dengue patients, including abnormal blood biochemistry, hepatomegaly, and acute liver failure (ALF). 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 show that the livers of deceased patients exhibit pathological manifestations such as congestion, swelling, fatty degeneration, and necrosis. In addition, deaths are often associated with liver failure. Current studies have shown that liver cell damage is caused by multiple factors, including direct cytopathic effects of 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 fever, 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 the 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 show that the livers of deceased patients showed pathological manifestations such as congestion, swelling, fatty degeneration and necrosis. However, at present, in addition to 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 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, perilesional administration, lesion surface administration, topical application, intravenous administration, parenteral administration, intraperitoneal administration, intramuscular administration, intralesional administration, intrathecal administration, intranasal administration or subcutaneous administration.

[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] As used herein, the term "pharmaceutically acceptable" means that these molecular entities and compositions do not produce adverse, allergic or other untoward reactions when appropriately administered to animals or humans. 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; tableting agents, 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 needed, and a physician can determine the dosage that is beneficial to the patient based on factors such as the patient's 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] Furthermore, the acid addition salt includes, but is not limited to, any one or a combination of at least two of the hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate and salicylate, malonate, adipate, hexanoate, arginine, fumarate, nicotinate, phthalate or oxalate of fotantinib.

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

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

[0017] In some embodiments, the acid addition salts are prepared using inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. In some embodiments, the acid addition salts are prepared using organic acids 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 injection, intravenous injection, intramuscular injection, intraperitoneal injection), 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, etc., 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, which comprises a preventive or therapeutically 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 (nociceptive 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, antihypertensives, anticoagulants, antifungals, antiparasitics, anti-inflammatory drugs, cough suppressants (expectorants), antiatherosclerotics, bath and thermotherapy agents, beta-blockers, calcium channel blockers and renin-angiotensin-aldosterone system inhibitors, bronchial drugs (antiasthmatics), choleretics and biliary therapeutics, cholinergics, adrenocortical hormones, skin medications, disinfectants (antibacterials), dietary supplements (nutritionals), diagnostics and preparations for diagnosis, diuretics, blood circulation promoting drugs, detoxification drugs (drugs for the treatment of addiction), enzyme inhibitors, preparations for enzyme deficiencies agents, transport proteins, fibrinolytics, geriatric drugs, antigout preparations, cold and flu medicines and cough and sneezing medicines, gynecological drugs, hemorrhoid medicines (rectal medicines), liver medicines, hypnotics (sedatives), pituitary hormones, hypothalamic hormones and other regulatory peptides and their inhibitors, immunomodulators, infusions and standard injections, organ perfusates, cardiac drugs, caries prevention drugs, periodontal degeneration drugs and other dental preparations, coronary preparations, laxatives, lipid-lowering drugs, local anesthetics (neurotherapeutic agents), gastrointestinal drugs, migraine drugs, mineral preparations, oral and throat medicines, muscle relaxants, anesthetics, neuropathy preparations and Other neurotropic agents, ophthalmic drugs, anti-osteoporotics (calcium- and bone metabolism regulators), otological drugs, anti-Parkinson drugs and other drugs for extrapyramidal disorders, psychiatric drugs, nasal drugs (sinus drugs), tonics (tonic drugs), thyroid preparations, serums, immunoglobulins and vaccines, sex hormones and their inhibitors, antispasmodics (anticholinergics), platelet aggregation inhibitors, tuberculosis drugs, mood-altering drugs, urological drugs, drugs for venous diseases, vitamins, wound and scar treatments, cytostatics and other anti-tumor drugs and protective agents, biomaterials, pharmaceutical synthetics.

[0022] As used 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 sufficient 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, alone or in combination with other agents, that provides a prophylactic benefit in preventing a disease, disorder, or condition. The term "prophylactically effective amount" can 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" can include an amount that improves overall treatment, reduces or avoids symptoms or causes of a disease or condition, or enhances the therapeutic effects of other therapeutic agents.

[0024] In some embodiments, the effective amount can be determined based on 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 level of damage to hepatocytes infected with dengue virus in vitro. The method comprises adding fotantinib or a pharmaceutically acceptable salt thereof to hepatocytes infected with dengue virus 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 in 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 "fostamatinib" refers to fostamatinib, 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 that have been treated with fotantinib or its pharmaceutically acceptable salts.

[0031] As used herein, the terms "screening" and "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, is likely to have, or is developing, 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 markers include aspartate aminotransferase, total bilirubin value, serum bilirubin value, plasma albumin value, prothrombin time prolongation 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, which 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 compositions and methods. To avoid redundancy, it should also be understood that any composition or method described as "comprising" one or more named elements or steps also describes the 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 further 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 establish a SD mouse model with significant liver damage. We found that the hepatic mononuclear phagocytic system (HMPS), composed of 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 has important value for the treatment of SD patients.

[0039] Furthermore, through multi-omics and single-cell RNA sequencing, we discovered that hepatocyte carbon metabolism dysfunction and altered HMPS cellular composition are key features of SD. By analyzing the expression patterns of disrupted kinases in the livers of SD mice, we discovered and demonstrated that fostamatinib, an approved thrombocytopenia treatment, can inhibit the progression of liver damage in SD mice by alleviating carbon metabolism dysfunction, thereby improving the survival of SD mice. This may be exploited in future clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a diagram showing the results of establishing a mouse model with severe dengue fever and liver damage, where a represents the process of establishing the mouse model with severe dengue fever and liver damage, bd represents the disease severity results of mice with severe dengue fever and liver damage, eg represents the dengue virus load in serum, liver, and intestine, h represents the liver lesion of mice infected with NGC, i represents the liver damage results of mice with severe dengue fever, and j represents the ALT and AST test results of mice in the severe group and NGC group.

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

[0042] Figure 3 This is the main feature analysis diagram of liver lesions during SD, where 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 overlap result of IDH1 grayscale and S100A4 grayscale, and G is the overlap result of IDH1 grayscale and LY6G grayscale.

[0043] Figure 4 It is a kinase prediction and kinase activity prediction diagram based on the proteome and phosphorylated proteome. Among them, AB is the kinase activation status of SD mouse liver at 4 dpi, 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 5Figure 3 is an analysis of the kinase expression patterns of hepatocytes and HMPS cell components during SD, where A shows the colocalization result of kinase BTK with hepatocytes, BC shows the colocalization result of BTK with cellular components of the mononuclear phagocyte system, D shows the change in the gray value level of BTK, E shows the change in the gray value level of S100A4, F shows the change in the gray value level of LY6G, G shows the colocalization result of BTK with ALB, H shows the colocalization result of BTK with S100A4, and I shows the colocalization result of BTK with LY6G.

[0045] Figure 6 This is a relationship diagram between carbon metabolism-related genes and disordered expression kinases. 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] Figure 9 The following is a graph showing the effect of fostamatinib on the carbon metabolism function of the liver in 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 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 Zoology, Chinese Academy of Medical Sciences.

[0054] 2) Viruses: Dengue virus (DENV) type 2 New Guinea strain was purchased from ATCC. The N10-adapted strain was obtained in our laboratory by subculturing the New Guinea strain in mice and stored at −80°C. All experiments involving dengue virus were conducted in an animal biosafety level 2 (ABSL-2) or biosafety level 2 (BSL-2) laboratory authorized by the Institute of Laboratory Animal Science, 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. See Table 1 for details.

[0060] Table 1

[0061]

[0062] 2) Viral RNA quantification

[0063] a) Total RNA Extraction: Total RNA from viruses or cells was extracted using the QIAamp Virus RNA Mini Kit (Qiagen, 52906). This involves extracting total RNA from viral fluid using a separation column method. To a 1.5 mL EP tube, add 560 μL of the mixture and 140 μL of the viral fluid sample, mix thoroughly, and incubate at room temperature for 10 minutes to lyse the virus and release the nucleic acid. Then, add 560 μL of anhydrous ethanol and mix thoroughly. Transfer 630 μL of this mixture to a collection column and centrifuge at 6000 g for 1 minute. Replace the collection tube, aspirate the remaining 630 μL of the mixture, and repeat the previous step. Once the collection column overflows, replace the collection tube, add 500 μL of AW1 solution, centrifuge at 6000 g for 1 minute, and replace the collection tube. Add 500 μL of AW2 solution, centrifuge at 20,000 g for 3 minutes, and replace the collection tube. Centrifuge at 20,000 g for 1 minute. Place the collection column in a sterile 1.5 mL EP tube, add 60 μL of AVE solution, centrifuge at 6000 g for 1 minute, 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 was about 67 bp, the remaining part was 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 viral 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 utilized hematoxylin-eosin staining (H&E), which stains nucleic acids within the cytoplasm and nucleus purplish-blue and extracellular matrix and cytoplasmic components red, thereby distinguishing cellular structures. The staining process is briefly described below. Pathological sections were immersed in xylene I, II, and III for 10 minutes each. They were then immersed in anhydrous ethanol I and II for 5 minutes each, and in 95%, 80%, 70%, and 50% ethanol for 5 minutes each. The sections were then placed in hematoxylin stain for 15 minutes and slowly rinsed with tap water. The sections were then differentiated in 1% hydrochloric acid-ethanol for 5-20 seconds, followed by a slow rinse in tap water for 1-3 minutes. The sections were then dehydrated with 2% ammonia solution for 30 seconds to 1 minute, followed by 2-minute debluing. The sections were then dehydrated with 70% and 80% ethanol for 2 minutes each. Then place it in eosin staining for 30s to 2min, dehydrate 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 biochemistry test

[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 twice, and the serum was separated and directly used for subsequent analysis or stored at -80°C.

[0080] b) On-device testing: Use a fully automated biochemical analyzer. First, turn on the instrument and perform a self-test according to the program. Then, clean the instrument twice, empty the waste container, and replace the reaction cuvette. 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 sample according to the standard curve.

[0082] 3. Experimental results

[0083] To investigate the differences in disease progression between severe dengue fever and dengue fever, and to explore the impact of organ failure on severe dengue fever, we established a severe dengue fever mouse model. To further assess whether liver damage occurs in the severe dengue fever mouse model, we performed gross autopsies 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 day 6 after infection, mice infected with N10 had a large number of focal lesions on the surface of the liver, and a small number of mice also had subcapsular hemorrhage with multiple petechiae, which is consistent with the disease manifestations of coagulopathy. However, the livers 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 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 the 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). Elevated ALT and AST levels indicate liver damage in mice with severe dengue fever, suggesting liver cell damage. The viral load in the severe dengue fever mouse model is increased ( Figure 1 eg), increased disease severity ( Figure 1 bd), organ damage, and abnormally elevated transaminases characterize the disease symptoms of clinically severe 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 hepatocellular 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 tested for integrity and total amount using an Agilent 2100 Bioanalyzer. Libraries were then constructed and quality-controlled using a Qubit instrument and real-time quantitative PCR. Only samples that passed QC were further analyzed. Subsequently, libraries were combined based on their effective concentration and the desired downstream data volume, and then sequenced using Illumina. After quality control assessment, the libraries were combined based on their effective concentration and target data volume, and sequenced using 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. The Q20, Q30, and GC content values of the clean data were then calculated. The index of the reference genome was constructed using HISAT2 v2.0.5, and the paired clean reads were aligned with the reference genome using HISAT2v2.0.5. Featurecots (1.5.0-p3) was then used to calculate the number of reads for each gene. The genes were then counted based on their length, and the FPKM of each gene was calculated. The DESeq2 R software package (1.20.0) was used to calculate the feature values, and differential expression analysis between the two conditions / groups was performed. 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 medium. 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. Incubate at room temperature for 5-8 minutes in erythrocyte lysis buffer (RCLB, Singleron) to remove erythrocytes. Centrifuge at 300×g for 5 minutes at 4°C, 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) are loaded 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 with 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 CeleScope v1.10.0 pipeline (https: / / github.com / singleron-RD / CeleScope) was used to process the raw reads of scRNA sequencing and 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 the enzymes. After homogenization, the supernatant was centrifuged at 12,000 g for 10 minutes at 4°C, and protein concentration was determined using the BCA assay. The protein sample was then added to pre-chilled acetone, vortexed, and then added to pre-chilled acetone. The mixture was precipitated at -20°C for 2 hours and washed two to three times with pre-chilled acetone. The protein sample was then redissolved in 200 mM TEAB and dispersed by sonication. An initial digestion was performed overnight using a 1:50 ratio of trypsin to protein. 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 phosphoproteomic 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, 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 chromatography-mass spectrometry analysis.

[0097] The peptides were dissolved in mobile phase A of the liquid chromatography system and separated using a Vanquish Neo ultra-high-performance liquid chromatography (UHPLC) system. Subsequently, the peptides were injected into the NSI ion source for ionization and subsequently analyzed by Orbitrap 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 membranes were incubated with the following secondary antibodies at room temperature for 1 hour: 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 mean grayscale value of the positively stained areas was analyzed using ImageJ.

[0100] 2. Experimental results

[0101] To clarify the changes in liver function in mice during SD, we performed KEGG enrichment analysis on the differentially expressed genes or proteins in each group. The results showed that the 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 the KEGG enrichment results of differentially expressed genes / proteins in multi-omics with the KEGG enrichment results of differentially expressed genes in cells from single-cell RNA sequencing and verified them. 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 liver cells, we combined hepatocytes, Kupffer cells (KCs) & monocytes / macrophages to analyze the KEGG enrichment results of differentially expressed genes / proteins in multi-omics and validated them. The interaction network was constructed by comparing the differentially expressed genes enriched in the carbon metabolism pathway revealed by CellChat in neutrophils with the receptor and ligand NaSTRING database in the activation pathway ( 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 peaks and valleys of the grayscale values of IDH1 are similar to those of S100A4 ( Figure 3 F) and LY6G( Figure 3 G) Gray value overlap. It indicates that hepatocytes, KCs, Alterations in intercellular communication with neutrophils contribute to 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 development of liver lesions. We then analyzed the activation and expression of kinases in the liver during SD based on proteomic and phosphoproteomic results ( 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 whose proteomic and phosphoproteomic activities were significantly altered in the livers of SD mice compared with uninfected controls, the expression levels of most kinases were upregulated ( Figure 4 C). The overlapping results of differentially activated kinases among the 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) colocalizes with the positive signals of kinases I and II, confirming the disruption of the kinase expression pattern and the activation of the mononuclear phagocytic system in SD livers.

[0104] Given that the cell types with perturbed kinase expression profiles coincide with those with dysfunctional carbon metabolism, we hypothesized that liver 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 primarily upregulated in hepatocytes and HMPS cell fractions. We then screened 14 overlapping differentially expressed genes from livers of SD and control mice, which 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 all 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 disrupted kinase expression patterns promote hepatic carbon metabolism dysfunction during SD.

[0105] In order to explore therapeutic drugs for SD-induced liver injury and discover new targets, we used kinases with altered activity as drug targets to screen potential anti-liver injury drugs ( Figure 6 D). The results showed that fostamatinib, an FDA-approved drug for the treatment of thrombocytopenia, inhibited most of the upregulated kinases in the liver of SD mice, including BTK. Combined with kinase expression levels revealed by single-cell RNA sequencing, we also identified HEPs and HMPs as the primary target cells of fostamatinib. Therefore, we subsequently treated SD mice with liver damage with fostamatinib.

[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), pathomorphological analysis and multi-omics analysis of liver tissues. All mouse experiments were approved by the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences, Institute of Laboratory Animals, and were performed in accordance with the established operating protocol (authorization number: WW22001). In order to study the effect of fotantinib on liver damage in SD mice, dengue virus (N10 strain) infected Ifnar- / - mice were used, and they were weighed every day after infection and monitored until the 6th day. Starting from the first day after infection (1dpi), 80mg / kg body weight of fotantinib or solvent alone was injected intraperitoneally 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.

[0112] 3. Experimental results

[0113] To investigate the effect of fostamatinib treatment on the survival of SD mice, Ifnar- / - mice were infected with DENV-2N10. From 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 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 damage 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 in 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 also evaluated the expression levels of common liver injury markers in serum, and the results showed that compared with SD mice, the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in mice treated with fostamatinib were significantly decreased ( 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 ( Figure 9 IFA showed that after fostamatinib treatment, the positive staining signals of Kupffer cells / monocytes / macrophages (S100A4+) and neutrophils (LY6G+) were reduced ( Figure 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 increased in the livers of mice treated with fostamatinib ( Figure 9 AC), while the positive staining of BTK decreased after fostamatinib treatment ( Figure 9 AC). In addition, with IFA ( Figure 9 AC) results and Mfuzz( Figure 8 D) The results were consistent. GSEA analysis demonstrated that most genes involved in carbon-rich metabolic pathways were upregulated in the liver of mice after fostamatinib treatment ( Figure 9 DF).

[0116] In summary, fostamatinib treatment not only alleviates SD-induced liver carbon metabolism dysfunction and kinase expression pattern disturbances, but also inhibits the abnormal activation and infiltration of HMPS cellular components, improving survival during SD. Our invention provides an effective drug strategy for treating 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, hepatomegaly, or symptoms of liver failure. The use according to claim 1 , wherein the pharmaceutical composition is administered orally, intravenously, intraperitoneally, intramuscularly or subcutaneously.

5. The use according to claim 1, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

6. The use according to claim 5, wherein the excipients include glidants, sweeteners, diluents, preservatives, colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, pH regulators and / or buffers, and emulsifiers.

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