Use of compounds in the diagnosis and treatment of novel coronavirus infection

Abstract

The present application relates to the use of a compound in the manufacture of a medicament for preventing and / or treating a disease and / or a symptom caused by SARS-CoV-2 infection in a subject, or for reducing or inhibiting damage to a tissue caused by SARS-CoV-2 infection in a subject, or of a kit for diagnosing whether a subject is infected with SARS-CoV-2, or for distinguishing between a subject infected and not infected with SARS-CoV-2. Therefore, the compounds of the present application have great potential in the diagnosis and treatment of the novel coronavirus.

Description

Technical Field

[0001] This application relates to the field of biomedicine. Specifically, this application relates to the use of compounds in the preparation of pharmaceuticals or kits, wherein the pharmaceuticals are used to prevent and / or treat diseases and / or symptoms caused by SARS-CoV-2 infection in subjects, or to reduce or inhibit tissue damage caused by SARS-CoV-2 infection in subjects, and the kits are used to diagnose whether a subject is infected with SARS-CoV-2, or to distinguish between subjects infected and uninfected with SARS-CoV-2. Background Technology

[0002] The novel coronavirus (SARS-CoV-2) belongs to the coronavirus family, which is a large family of viruses. Currently known symptoms caused by coronavirus infections mainly include those caused by the novel coronavirus, as well as highly contagious and relatively severe diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS).

[0003] Currently, vaccines are a common means of preventing the novel coronavirus. Although vaccination slows the spread of the virus for a period of time, it cannot completely suppress its transmission. In addition, widely used antiviral drugs such as remdesivir, saquinavir, nelfinavir, amprenavir, ritonavir, and lopinavir mostly reduce viral load by inhibiting the activity of viral replication-related RNases, but do not directly protect the body from virus-induced tissue damage.

[0004] In summary, considering the common toxic side effects of antiviral drugs, there is an urgent need to find more comprehensive and safer treatment options and drugs for the novel coronavirus. Summary of the Invention

[0005] The applicant of this application obtained compounds (i.e., homocitrulline, trigonelline, and / or 4-guanidinobutyric acid) capable of determining whether a subject is infected with SARS-CoV-2 D614G by analyzing the metabolome of multiple tissues and serums from golden hamsters infected with the SARS-CoV-2 D614G variant (the first reported variant). Furthermore, the applicant found that homocitrulline, one of the three compounds, can treat SARS-CoV-2-infected golden hamster pneumonia, significantly reducing organ damage (e.g., lung injury), weight loss, and mortality caused by SARS-CoV-2 infection in golden hamsters.

[0006] Therefore, in a first aspect, this application provides the use of the following compounds in the preparation of pharmaceuticals or kits, wherein,

[0007] The drug is used in subjects to prevent and / or treat disease and / or symptoms caused by SARS-CoV-2 infection, or to reduce or inhibit tissue damage caused by SARS-CoV-2 infection in subjects;

[0008] The kit is used to diagnose whether a subject is infected with SARS-CoV-2, or to distinguish between subjects infected and uninfected with SARS-CoV-2; and the kit includes reagents or combinations of reagents for determining the level of the compound in the subject;

[0009] The compounds include any one, two, or three of the following: citrulline, trigonelline, and 4-guanidinobutyric acid.

[0010] Uses of drug preparation

[0011] This application provides the use of the following compounds in the preparation of a medicament, wherein the medicament is used in a subject to prevent and / or treat disease and / or symptoms caused by SARS-CoV-2 infection, or to reduce or inhibit tissue damage caused by SARS-CoV-2 infection in a subject.

[0012] In some embodiments, the compound comprises homocitrulline. In some embodiments, the compound comprises trigonelline. In some embodiments, the compound comprises 4-guanidinobutyric acid.

[0013] In some embodiments, the compound comprises homocitrulline and trigonelline. In some embodiments, the compound comprises homocitrulline and 4-guanidinobutyric acid. In some embodiments, the compound comprises trigonelline and 4-guanidinobutyric acid.

[0014] In some embodiments, the compound includes citrulline, trigonelline, and 4-guanidinobutyric acid.

[0015] In some embodiments, the CAS number for high-citrulline is 1190-49-4. In some embodiments, the CAS number for trigonelline is 535-83-1. In some embodiments, the CAS number for 4-guanidinobutyric acid is 463-00-3.

[0016] In some implementations, the disease caused by SARS-CoV-2 infection in the subjects is selected from novel coronavirus infection.

[0017] In some implementations, the symptoms of SARS-CoV-2 infection in the subjects are selected from weight loss, fever, cough, difficulty breathing, headache, chills, runny nose, muscle aches, loss of appetite, nausea, vomiting, abdominal pain, and diarrhea.

[0018] In some embodiments, the tissue is tissue from the brain, heart, kidney, lung, pancreas, spleen, liver, testis, and / or ovary.

[0019] In some embodiments, the drug is used alone or in combination with other active ingredients (e.g., antiviral agents).

[0020] In some embodiments, the other active ingredients are selected from remdesivir, saquinavir, nelfinavir, amprenavir, ritonavir, and lopinavir.

[0021] In some embodiments, the drug further includes a pharmaceutically acceptable carrier and / or excipient.

[0022] In some implementations, the subject is a mammal.

[0023] In some implementations, the subject is a human being.

[0024] Applications of the preparation kit

[0025] This application provides the use of the following compounds in the preparation of a kit for diagnosing whether a subject is infected with SARS-CoV-2, or for distinguishing between subjects infected and uninfected with SARS-CoV-2; and the kit includes a reagent or combination of reagents for determining the level of the compound in the subject.

[0026] In some embodiments, the compound comprises homocitrulline. In some embodiments, the compound comprises trigonelline. In some embodiments, the compound comprises 4-guanidinobutyric acid.

[0027] In some embodiments, the compound comprises homocitrulline and trigonelline. In some embodiments, the compound comprises homocitrulline and 4-guanidinobutyric acid. In some embodiments, the compound comprises trigonelline and 4-guanidinobutyric acid.

[0028] In some embodiments, the compound includes citrulline, trigonelline, and 4-guanidinobutyric acid.

[0029] In some embodiments, the CAS number for high-citrulline is 1190-49-4. In some embodiments, the CAS number for trigonelline is 535-83-1. In some embodiments, the CAS number for 4-guanidinobutyric acid is 463-00-3.

[0030] In some implementations, a reduced expression level of the compound compared to the reference range for compounds (e.g., high-citrulline, trigonelline, and / or 4-guanidinobutyric acid) in uninfected healthy subjects indicates that the subject is infected with SARS-CoV-2.

[0031] Those skilled in the art can reasonably select methods and reagents for detecting the compound based on publicly available information in the prior art.

[0032] In some embodiments, the kit includes a first reagent or combination of reagents for determining the subject's high citrulline level, a second reagent or combination of reagents for determining the subject's trigonelline level, and a third reagent or combination of reagents for determining the subject's 4-guanidinobutyric acid level.

[0033] In some embodiments, the reagents (e.g., first, second, third reagents, or combinations thereof) determine the levels of compounds in the subject's biological sample by methods such as liquid chromatography-tandem mass spectrometry, chromatography and / or mass spectrometry, fluorescence assay, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy, radiochemical analysis, near-infrared spectroscopy, nuclear magnetic resonance spectroscopy, light scattering analysis, and turbidimetry.

[0034] In some embodiments, the reagent is used to determine the level of the compound in the subject's biological sample by liquid chromatography-tandem mass spectrometry.

[0035] In some embodiments, the kit may further include reagents and / or consumables for liquid chromatography-tandem mass spectrometry, reagents and / or consumables for mass spectrometry, or any combination thereof.

[0036] In some embodiments, the reagents and / or consumables for liquid chromatography-tandem mass spectrometry are selected from chromatographic columns, acetonitrile, ammonium acetate, ammonium formate, formic acid, glycylproline standards, isopropanol, methanol, or any combination thereof.

[0037] In some embodiments, the subject's biological sample is selected from whole blood (e.g., peripheral blood), plasma, serum, or any combination thereof; or the biological sample is obtained from the brain, heart, kidney, lung, pancreas, spleen, liver, testis, and / or ovary.

[0038] In some implementations, the subject is a mammal.

[0039] In some implementations, the subject is a human being.

[0040] Methods to test whether a treatment is effective

[0041] In a second aspect, this application provides a method for detecting the effectiveness of a therapy targeting disease and / or symptoms caused by SARS-CoV-2 infection or tissue damage caused by SARS-CoV-2 infection; the method includes:

[0042] (1) Detect the level of the target compound in the subject after receiving the therapy;

[0043] (2) Compare the above levels with the levels of the target compound in the subject before receiving the treatment; or compare the above levels with the levels of the target compound in a healthy subject.

[0044] In some implementations, the therapy is effective if the level of the target compound in the subject after receiving the therapy is higher than the level of the target compound in the subject before receiving the therapy, or if the level of the target compound in the subject after receiving the therapy is higher than the level of the target compound in a healthy subject.

[0045] In some embodiments, the compound comprises homocitrulline. In some embodiments, the compound comprises trigonelline. In some embodiments, the compound comprises 4-guanidinobutyric acid.

[0046] In some embodiments, the compound comprises homocitrulline and trigonelline. In some embodiments, the compound comprises homocitrulline and 4-guanidinobutyric acid. In some embodiments, the compound comprises trigonelline and 4-guanidinobutyric acid.

[0047] In some embodiments, the compound includes citrulline, trigonelline, and 4-guanidinobutyric acid.

[0048] In some implementations, the subject is a mammal.

[0049] In some implementations, the subject is a human being.

[0050] Terminology Definition

[0051] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0052] As used herein, "severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)," also known as "novel coronavirus," belongs to the β-coronavirus genus and is an enveloped, single-stranded, positive-sense RNA virus. The genome sequence of SARS-CoV-2 is known to those skilled in the art and can be found, for example, in GenBank: MN908947. SARS-CoV-2 contains at least three membrane proteins, including a surface spike protein (S), an integrated membrane protein (M), and a membrane protein (E). Like SARS-CoV, the receptor for SARS-CoV-2 specifically binds to angiotensin-converting enzyme 2 (ACE2) on host cells via the receptor-binding domain (RBD) on the S protein, leading to viral membrane fusion and cellular entry. This receptor plays a crucial role in viral infection of cells.

[0053] As used in this article, the terms “novel coronavirus” and “SARS-CoV-2” have the same meaning and are used interchangeably.

[0054] As used in this article, the term "novel coronavirus infection" refers to pneumonia caused by infection with the novel coronavirus.

[0055] As used herein, the term "reference value" refers to a predetermined value for a compound, derived from the level of the compound in a control sample (e.g., a biological sample obtained from a healthy population). Reference values ​​can be used as thresholds to distinguish between subjects who may be at risk of a disease and those who are not. Reference values ​​can be relative values, numerical ranges with upper and lower limits, averages, medians, etc. Those skilled in the art can select appropriate control samples, determine, and obtain reference values ​​according to methods disclosed in the prior art.

[0056] In some implementations, a reduced expression level of the compound (high-citrulline, trigonelline, and / or 4-guanidinobutyric acid) compared to the reference range for the compound in uninfected healthy subjects indicates the level after the subject was infected with SARS-CoV-2.

[0057] As used in this article, the term "metabolomics" refers to the technique of using chromatography-mass spectrometry to detect metabolites with a molecular weight of less than 1,000 Daltons.

[0058] As used herein, the term "subject" refers to mammals, including but not limited to humans, rodents (mice, rats, guinea pigs), dogs, horses, cattle, cats, pigs, monkeys, chimpanzees, etc. In some embodiments, the subject is a human.

[0059] As used herein, the term "metabolite" or "metabolic product" refers to a substance produced during chemical or physical processes in the human body. It includes any chemical or biochemical product of metabolic processes, such as any compound produced through the processing, cleavage, or consumption of biomolecules. Examples of such molecules include, but are not limited to: acids and related compounds; mono, di, and tricarboxylic acids (saturated, unsaturated, aliphatic and cyclic, aryl, alkylaryl); aldehydes, keto acids; lactone forms; gibberellins; abscisic acid; alcohols, polyols, derivatives, and related compounds; ethanol, benzyl alcohol, methanol; propylene glycol, glycerol, phytol; inositol, furfuryl alcohol, menthol; aldehydes, ketones, quinones, derivatives, and related compounds; acetaldehyde, butyraldehyde, benzaldehyde, acrolein, furfural, glyoxal; acetone, butanone; anthraquinones; carbohydrates; monosaccharides, disaccharides, trisaccharides; alkaloids, amines, and other bases; pyridines (including nicotinic acid and nicotinamide); pyrimidines (including cytosine and thymine). Purines (including guanine, adenine, xanthine / hypoxanthine, kinetin); pyrrole; quinoline (including isoquinoline); morphinan, tropane, cinchonans, nucleotides, oligonucleotides, derivatives and related compounds; guanosine, cytosine, adenosine, thymidine, inosine; amino acids, oligopeptides, derivatives and related compounds; esters; phenols and related compounds; heterocyclic compounds and derivatives; pyrrole, tetrapyrrole; flavonoids; indole; lipids (including fatty acids and triglycerides), derivatives and related compounds; carotenoids, phytopenic oleoresin and sterols, isoprene-like compounds, including terpenes; and modified forms of the above molecules.

[0060] In some embodiments, the CAS number for high-citrulline is 1190-49-4. In some embodiments, the CAS number for trigonelline is 535-83-1. In some embodiments, the CAS number for 4-guanidinobutyric acid is 463-00-3.

[0061] As used herein, the term “pharmaceutical acceptable” means something recognized in the pharmaceutical industry as suitable for use in animals, and particularly in humans. As used herein, the term “pharmaceutical acceptable carrier and / or excipient” means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters (including, but not limited to, phosphate buffers), surfactants (including, but not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80), adjuvants, ionic strength enhancers (including, but not limited to, sodium chloride), diluents, excipients, media for containing or administering therapeutic agents, and any combination thereof.

[0062] As used herein, pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including petroleum-derived, animal-, plant-based, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when administering pharmaceutical compositions intravenously. Saline solutions, as well as aqueous solutions of dextran and glycerol, can also be used as liquid carriers, particularly for injectable solutions.

[0063] Pharmaceutically acceptable excipients, as used herein, may include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc. If desired, the pharmaceutical composition may also include a wetting agent, or an emulsifier such as sodium hyaluronate, or a pH buffer. The pharmaceutical composition may be in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, etc.

[0064] Beneficial effects of the invention

[0065] This application, through analysis of the metabolomes of multiple tissues from subjects infected with SARS-CoV-2 and uninfected healthy subjects, obtained compounds (i.e., homocitrulline, trigonelline, and / or 4-guanidinobutyric acid) capable of determining whether a subject is infected with SARS-CoV-2. Furthermore, the applicant of this application found that the compounds, particularly homocitrulline, can treat subjects infected with SARS-CoV-2, significantly reducing organ damage (e.g., lung injury), weight loss, and mortality caused by SARS-CoV-2 infection. Therefore, the compounds of this application have significant potential in the diagnosis and treatment of novel coronavirus.

[0066] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0067] Figure 1 This study showcases hallmark metabolites of SARS-CoV-2 D614G virus infection in hamsters and their association with disease. Among them, Figure 1 Figure A shows an overview of the metabolic changes in various organs (brain, heart, kidney, lung, pancreas, spleen, liver, testis, and serum) on the fifth day after D614G infection of hamsters; Figure 1 The statistical analysis of the number of organs with significant changes in metabolites in hamsters B after SARS-CoV-2 infection at 1 dpi, 3 dpi, 5 dpi, 7 dpi, and 11 dpi compared with hamsters not infected with SARS-CoV-2 (0 dpi) (vertical axis represents metabolite name, and horizontal axis represents the number of organs with significant changes in metabolites at that time point). Figure 1 C in the figure shows the changing trends of homocitrulline, 4-guanidinobutanoic acid, and trigonelline in lung tissue; Figure 1 In the figure, D represents the ROC curve analysis of metabolite changes over time.

[0068] Figure 2 The results showed that high-citrulline content inhibited AAV-N pseudovirus-induced lung injury in C57BL / 6 mice, where... Figure 2 In the figure, A represents a volcano plot of metabolomics analysis of lung tissue from AAV-N-infected mice; Figure 2 B and C in the table represent HE staining of lung tissue and its scoring.

[0069] Figure 3 The study demonstrated the protective and therapeutic effects of high-citrulline levels on hamsters infected with SARS-CoV-2 D614G and Beta strains; among which, Figure 3 In this context, A represents the high-citrulline replenishment experimental protocol; Figure 3 B in the figure shows the changes in body weight of hamsters infected with D614G or Beta before and after treatment; Figure 3 C in the figure shows the survival rate of hamsters infected with the Beta strain and those in the high-citrulline treatment group; Figure 3 D in the figure shows the cytokine expression in hamsters after treatment; Figure 3E in the figure demonstrates the inhibitory effect of high citrulline on virus-mediated lung injury. Detailed Implementation

[0070] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0071] Unless otherwise specified, the experiments and methods described in the embodiments are performed in accordance with conventional methods well known in the art and described in various references. For example, conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in this invention can be found in Sambrook, Fritsch, and Maniatis, *Molecular Cloning: A Laboratory Manual*, 2nd edition (1989); *Current Protocols in Molecular Biology* (edited by FM. Ausubel et al., (1987)); the *Methods in Enzymology* series (academic publishing company): *PCR 2: A PRACTICAL APPROACH* (edited by MJ. MacPherson, BD. Hames, and GR. Taylor, (1995)); and *Animal Cell Culture*. CELLCULTURE (edited by R.R. Freshney (1987)).

[0072] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0073] Example 1. Obtaining metabolomic specimens of whole organ systems from SARS-CoV-2-infected hamsters.

[0074] This application used liquid chromatography-tandem mass spectrometry (LC / MS) to investigate the metabolite levels in multiple tissues and organs of Egyptian golden hamsters infected with the SARS-CoV-2 D614G strain (which have been shown to express the ACE2 receptor and can well simulate the clinical indications of patients infected with the novel coronavirus in existing COVID-19 infection models, hereinafter referred to as "hamsters").

[0075] The hamsters used in this experiment were housed in a specific pathogen-free animal facility supported by the P3 laboratory of the Joint Institute of Virology, Shantou University and the University of Hong Kong. All infectious SARS-CoV-2 experiments were conducted in a Biosafety Level 3 (BSL-3) laboratory and an Animal Biosafety Level 3 (ABSL-3) laboratory. Virus infection experiments were carried out in the P3 laboratory. Hamsters were administered SARS-CoV-2 D614G strain via nasopharyngeal drip and sacrificed at 0 dpi (uninfected), 1 dpi, 3 dpi, 5 dpi, 7 dpi, and 11 dpi. Various tissues and organs were then collected and weighed (heart, liver, spleen, lung, kidney, pancreas, brain, and testes / ovaries). The collected tissues were placed in 1 ml of pre-cooled methanol, flash-frozen in liquid nitrogen, and stored at -80°C. After obtaining tissues at all time points, they were thawed, ground, and then thoroughly inactivated by water bath at 56°C for 1 hour. Metabolite extraction was performed in the P2 laboratory after the tissues were removed from the P3 laboratory.

[0076] Example 2. Metabolomics Experiments of Tissues and Organs

[0077] Extraction of metabolites from serum and organ samples:

[0078] Metabolites from hamster organs and serum obtained at 0 dpi (uninfected with virus), 1 dpi, 3 dpi, 5 dpi, 7 dpi, and 11 dpi in Example 1 were extracted separately—lipids and polar metabolites were extracted separately using the MTBE biphasic method. Specifically, 1 mL of tissue homogenate was first freeze-thawed in liquid nitrogen to fully disrupt the tissue cells, then transferred to a 5 mL centrifuge tube. The original tube was rinsed with 100 μL of methanol and transferred to another 5 mL centrifuge tube. Then, 2.75 mL of methyl tert-butyl ether (MTBE) was added, and the mixture was vortexed thoroughly (1200 rpm, 20 minutes, room temperature). After that, 1 mL of ultrapure water was added, and the mixture was vortexed again for 5 minutes. Finally, the mixture was centrifuged at 12000 g and 7°C for 15 minutes to separate the lipid and polar layers. Equal amounts of the lipid layer (upper layer) and the polar metabolite layer (lower layer) were collected, concentrated, evaporated to dryness, and then analyzed.

[0079] (1) Chromatographic and mass spectrometric acquisition conditions:

[0080] The chromatography used was ultra-high performance liquid chromatography (Shimpack UFLC SHIMADZU CBM30A). The main liquid phase conditions included: pre-column: Waters ACQUITY UPLC BEH C18 1.7μm, 2.1mm*5mm; column: Waters ACQUITY UPLC BEH C18 1.7μm, 2.1mm*100mm. Mobile phase: Phase A was a 100% ultrapure aqueous solution containing 0.3% (3 mL / L) NH3H2O ​​(>28%) and 15 mM (1.156 g / L) CH3COONH4; Phase B was acetonitrile / isopropanol (90:10, v / v). Elution gradient: 0 to 1.0 min, 95% B; 1 to 15.0 min, 95% to 45% B; 15.0 to 17 min, 45% B; 17 to 17.5 min, 45% to 95% B; 17.5 to 22 min, 95% B. Flow rate: 0.4 mL / min; column temperature: 40℃; injection volume: 5 μL. Tandem mass spectrometry was performed using Orbitrap ID-X. The main mass spectrometry conditions included an electrospray ionization source and desolvation temperatures of 150℃ and 550℃, respectively. Mass spectrometry voltage: 5500V (+), -4500V (-). Ion source gas I: 55 psi, gas II: 60 psi, curtain gas: 25 psi. Collision-induced ionization parameter: High. In the triple quadrupole, each ion pair was scanned and detected based on the optimized declustering voltage and collision energy.

[0081] (2) Quality Control (QC) Setup:

[0082] Take 10 µl from each sample and mix to prepare a quality control sample. When testing the samples, inject one quality control sample after every ten samples, following the sample order.

[0083] Example 3. Data Processing and Statistical Analysis

[0084] Mass spectrometry data processing used Compound Discoverer and Trace Finder software in conjunction with a self-built metabolomics library for qualitative and quantitative analysis of metabolites. Metabolites or lipids with less than 80% detection in a single mass spectrometry sample were deleted, and missing values ​​were filled using the median value in the MetaboAnalyst R package.

[0085] Statistical analysis of the data was primarily performed using R, utilizing packages such as "statTarget", "ggplot2", "ggrepel", and "ggpubr". Some statistical analyses and graphical representations were also performed using GraphpadPrism and Microsoft Office Excel. Polar metabolomics data underwent D5-phenylalanine internal standard correction, sample size correction, area normalization correction, and QC correction; nonpolar metabolomics data underwent sample size correction, area normalization correction, and QC correction. QC correction employed the QC-RFSC method from the statTarget package, with a key parameter Ntree of 500 and a CV of 30%. Data pre-screening followed the commonly used 80% rule in metabolomics, and blank values ​​were filled using the KNN algorithm. Differences in metabolites were analyzed using the FDR-corrected t-test and Log2 Fold change. Statistical significance was analyzed using the FDR-corrected t-test and two-way ANOVA. A p-value < 0.05 was considered statistically significant. A p-value ≥ 0.05 was considered insignificant and denoted as ns. A p-value |log2FC| ≥ 0.5 and p < 0.05 was considered significant. P-values ​​0.01 ≤ p < 0.05 were denoted as *, 0.001 ≤ p < 0.01 as **, 0.0001 ≤ p < 0.001 as ***, and p < 0.0001 as ****.

[0086] Experimental results

[0087] The experimental results of metabolomics are as follows Figure 1 As shown, where, Figure 1 Figure A shows the number of changes in the levels of polar and nonpolar (lipid) metabolites in different tissues and organs (brain, heart, kidney, lung, pancreas, spleen, liver, testis, and serum) on the fifth day after viral infection of hamsters. It can be seen that viral infection mediates metabolic changes in multiple organs, with the most significant changes in serum, lung, and liver metabolism. Figure 1 Figure B shows the statistical data on the number of organs with significant changes in differential metabolites in hamsters at 1 dpi, 3 dpi, 5 dpi, 7 dpi, and 11 dpi after SARS-CoV-2 infection, compared with uninfected hamsters (0 dpi). (The vertical axis represents the metabolite name, and the horizontal axis represents the number of organs with significant changes in metabolites at that time point). It can be seen that trigonelline, homocitrulline, and 4-guanidinobutyric acid all showed significant changes in 7 or more organs at 1 dpi, 3 dpi, 5 dpi, 7 dpi, and 11 dpi after infection. Figure 1C in the figure shows the changing trends of homocitrulline, 4-guanidinobutanoic acid, and trigonelline in lung tissue; Figure 1 Figure D shows the ROC curve analysis of the three high-frequency altered metabolites in serum and lung tissue. In serum, the AUC value (area under the curve) of high-citrulline was as high as 1.000, the AUC value of 4-guanidinobutyric acid was 0.800, the AUC value of trigonelline was 0.7680, and the AUC value of the combination of the three was 0.8764. In lung tissue, the AUC value of high-citrulline was as high as 1.000, the AUC value of 4-guanidinobutyric acid was 0.900, the AUC value of trigonelline was as high as 1.000, and the AUC value of the combination of the three was 0.9704.

[0088] In summary, this application used liquid chromatography-tandem mass spectrometry (LC / MS) to investigate the metabolite levels in multiple tissues and organs of SARS-CoV-2-infected hamsters, revealing metabolic changes in the brain, heart, kidneys, lungs, pancreas, spleen, liver, testes, and serum at different time points (1 dpi, 3 dpi, 5 dpi, 7 dpi, 11 dpi) after SARS-CoV-2 infection. Biomarkers of common metabolic changes in organs caused by viral infection were identified—high-citrulline, trigonelline, and 4-guanidinobutyric acid. ROC curve analysis showed that these three biomarkers, whether used alone or in combination, can effectively diagnose whether a subject is infected with SARS-CoV-2, or distinguish between infected and uninfected subjects.

[0089] Example 4. Experiment on AAV-N-induced lung injury in C57BL / 6 mice

[0090] In this embodiment, a pseudovirus-induced lung injury model was constructed by infecting C57BL / 6 mouse lung tissue with SARS-CoV-2 nucleocapsid protein pseudovirus (AAV-N) and its blank control virus (AAV-Control). This experiment was conducted in a P2 animal laboratory. Mice were divided into four groups, with six mice in each group. AAV-N and AAV-Control viruses were purchased from Shanghai Heyuan Biotechnology (catalog numbers H16499 and H14397, respectively).

[0091] First control group: 5*10e 11 AAV-Control was resuspended in 200 μL of physiological saline and injected into mice via the tail vein. The mice were then fed normally, and samples were collected 21 days after the control virus injection.

[0092] Second experimental group: 5*10e 11The AAV-N pseudovirus was resuspended in 200 μL of pre-cooled saline and injected into mice via the tail vein. The mice were then fed normally, and lung tissue was obtained by euthanizing the mice 21 days after the virus injection for pathological and metabolomics experiments.

[0093] Group 3, low-dose treatment group: 5*10e 11 The AAV-N pseudovirus was resuspended in 200 μL of physiological saline and injected into mice via the tail vein. Seven days later, high-citrulline (20 mg / kg) was administered intraperitoneally. The administration was repeated every one day for 14 days, for a total of seven administrations. Twenty-one days after the virus injection, the mice were sacrificed to obtain lung tissue for pathological and metabolomics experiments.

[0094] Group 4, high-dose treatment group: 5*10e 11 The AAV-N pseudovirus was resuspended in 200 μL of physiological saline and injected into mice via the tail vein. Seven days later, high-citrulline (100 mg / kg) was administered intraperitoneally. The administration was repeated every one day for 14 days, for a total of seven administrations. Twenty-one days after the virus injection, the mice were sacrificed to obtain lung tissue for pathological and metabolomics experiments.

[0095] Experimental results are as follows Figure 2 As shown, Figure 2 The study showed that high citrulline content reduced AAV-N pseudovirus-induced lung injury in C57BL / 6 mice. Figure 2 In the figure, A is a volcano plot of metabolomics analysis of lung tissue from AAV-N infected mice. The results show that high citrulline is downregulated in AAV-N infected mice compared to AAV-Control (control group), indicating that virus-mediated N protein lung expression can partially mimic SARS-CoV-2 infection. Figure 2 B and C in the figure represent HE staining and scores of mouse lung tissue, indicating that high-citrulline supplementation can reduce lung damage caused by AAV-N infection.

[0096] Example 5. Hamster experiment using high-citrulline treatment for SARS-CoV-2 virus infection.

[0097] This study infected hamsters with different SARS-CoV-2 virus mutant strains and tested the therapeutic effect of high-citrulline treatment. The hamsters were divided into five groups of eight each. The hamsters used in this experiment were housed in a specific pathogen-free animal facility. The SARS-CoV-2 D614G mutant strain (GISAID No: EPI_ISL_2779639, hereinafter referred to as D614G strain) and the B.1.351 (Beta) mutant strain (GISAID No: EPI_ISL_2779638, hereinafter referred to as beta strain) used in this study were supported by the P3 laboratory of the Joint Institute of Virology, Shantou University and the University of Hong Kong. All infectious SARS-CoV-2 experiments were conducted in a Biosafety Level 3 (BSL-3) laboratory and an Animal Biosafety Level 3 (ABSL-3) laboratory. Viral infection experiments were carried out in the P3 laboratory.

[0098] Both D614G and Beta strains of the virus were passaged in Vero cells (#CCL-81 ATCC), and virus stock solutions were prepared. Vero cells were cultured in DMEM medium containing 2% fetal bovine serum (FBS), 5 μg / mL TPCK-trypsin, penicillin-streptomycin, and 30 mM magnesium chloride. After collection, the viruses were stored in an ultra-low temperature freezer, and viral titers were determined using the Vero cell plaque assay. Hamster challenge experiments—6-8 week old male hamsters were lightly anesthetized with isoflurane and challenged with 1×10⁻⁶ cells / mL of acetylcholine. 4 The PFU virus stock solution was diluted in 200 μL PBS and inoculated into hamsters via the nasal cavity.

[0099] The first group served as the control group, and the hamsters were not treated. They were collected from the hamsters in the control group at the same time point as the experimental group.

[0100] The second group was the D614G strain virus infection group, which used D614G strain virus to infect hamsters.

[0101] The third group was the Beta mutant virus infection group; hamsters were infected with the Beta mutant strain.

[0102] The fourth group was the D614G virus infection treatment group. Hamsters were infected with the D614G virus strain, and intraperitoneal administration (200 mg / kg high-citrulline) was started one day after infection, once a day, for 5 days.

[0103] The fifth group was the treatment group after infection with the Beta mutant strain. Hamsters were infected with the D614G Beta mutant strain and administered intraperitoneally (200 mg / kg high-citrulline) once a day for 5 days starting 1 day after infection.

[0104] The weight changes of the hamsters were recorded daily. On the seventh day, the hamsters in the experimental group were euthanized, and metabolomics (as described in Case 1 and 2) and pathological experiments were carried out respectively.

[0105] Lung tissue cytokine detection: ELISA kits were used to detect the corresponding cytokines—IL-4 (Catalog No.: CSB-EL011659HA, Cusabio), IL-6 (Catalog No.: CSB-E14304HA, Cusabio), IFN-γ (Catalog No.: CSB-EL011050HA, Cusabio), and IL-1β (Catalog No.: CSB-E14259HA, Cusabio). After treatment, tail lobes of hamster lung tissue were obtained. 1 ml of pre-chilled PBS buffer was added per 100 mg of tissue, and the homogenate was ground in an ice bath, followed by freeze-thaw and sonication. The resulting homogenate was centrifuged at 12000g, and the supernatant was used for ELISA detection. The ELISA assay was performed according to the instructions. In short, the standard and the sample to be tested (the supernatant extracted by the above method) were added to the assay plate in sequence, incubated at 37°C for 90 minutes, the supernatant was discarded, and the sample was gently tapped to remove excess liquid. Then, the biotinylated antibody was added, and the plate was incubated at 37°C for 60 minutes. The supernatant was discarded, and the plate was washed 5 times. After removing excess liquid, the substrate solution was added, and the plate was incubated at 37°C for 15 minutes. After adding the stop solution, the plate was measured at 450 nm using a microplate reader. The cytokine concentration in each sample was determined based on the standard curve.

[0106] Experimental results are as follows Figure 3 As shown, Figure 3 The study demonstrated the protective and therapeutic effects of high-citrulline levels on D614G and Beta strain-infected hamsters; among which, Figure 3 In this context, A represents the high-citrulline replenishment experimental protocol; Figure 3 Figure B shows the changes in hamster body weight at different infection times, which indicates that high citrulline intake effectively rescues weight loss caused by viral infection; Figure 3 C in the figure represents the survival rate of hamsters at different infection times. This result indicates that high citrulline significantly inhibits the mortality rate caused by viral infection. Figure 3 D in the figure shows the cytokine levels in lung tissue homogenate, which indicates that high citrulline significantly inhibits the levels of pro-inflammatory cytokines induced by viral infection. Figure 3 E in the image shows the HE staining results of lung tissue pathology before and after treatment. It is clearly seen that high-citrulline treatment inhibited pathological phenomena such as tissue damage area, inflammatory cell infiltration, alveolar hemorrhage, and bronchial inflammatory infiltration. This result shows that high-citrulline can reduce lung damage caused by D614G strain infection.

[0107] These experimental results indicate that high-citrulline levels decreased sharply after SARS-CoV-2 virus infection in hamsters, reaching their lowest level at 5 days post-infection (dpi). High-citrulline provided good protection against both SARS-CoV-2 nucleocapsid protein-induced lung injury and SARS-CoV-2 virus-induced pneumonia, particularly significantly reducing lung injury and protecting hamsters from death caused by SARS-CoV-2 Beta strain infection (hamsters treated with the high-citrulline of this application achieved a 100% survival rate). Therefore, the high-citrulline of this application has high potential for the prevention and / or treatment of diseases and / or symptoms caused by SARS-CoV-2 virus.

[0108] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. Use of homocitrulline for the preparation of a medicament, wherein, The drug is used in subjects to prevent and / or treat novel coronavirus infection caused by SARS-CoV-2 infection, or to reduce or inhibit lung damage to tissues caused by SARS-CoV-2 infection in subjects.

2. The use according to claim 1, wherein, The drug may be used alone or in combination with other active ingredients.

3. The use according to claim 2, wherein, The other active ingredients are selected from remdesivir, saquinavir, nelfinavir, amprenavir, ritonavir, and lopinavir.

4. The use according to claim 1, wherein, The drug also includes a pharmaceutically acceptable carrier.

5. The use of reagents or combinations of reagents for determining high citrulline levels in subjects in the preparation of kits for diagnosing SARS-CoV-2 infection.

6. The use according to claim 5, wherein, The reagent was used to determine the level of high-citrulline in the subject's biological sample by the following methods: chromatography and / or mass spectrometry, fluorescence assay, electrophoresis, immunoaffinity, ultraviolet spectroscopy, radiochemical analysis, near-infrared spectroscopy, nuclear magnetic resonance spectroscopy, light scattering analysis, and turbidimetry.

7. The use according to claim 5, wherein, The reagent was used to determine the level of high citrulline in the subject's biological sample by liquid chromatography-tandem mass spectrometry.

8. The use according to claim 6, wherein, The subject's biological sample was selected from whole blood, plasma, serum, or any combination thereof.

9. The use according to any one of claims 1-8, wherein the subject is a mammal.

10. The use according to any one of claims 1-8, wherein the subject is a human being.

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