Application of berberine sulfate in the preparation of drugs for the prevention and / or treatment of viral enteritis

Abstract

This invention relates to the use of berberine sulfate in the preparation of drugs for the prevention and / or treatment of viral enteritis. This invention is the first to propose the application of berberine sulfate in the treatment of viral intestinal inflammation, expanding the scope of application for this class of drugs. This invention has found that berberine sulfate can alleviate intestinal inflammation caused by papain-like proteins in animal models and may play a role in the treatment of intestinal inflammation caused by the novel coronavirus SARS-CoV-2.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the use of berberine sulfate in the preparation of drugs for the prevention and / or treatment of viral enteritis. Background Technology

[0002] The novel coronavirus infection (COVID-19) is a global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 RNA genome is approximately 30,000 bp in length, with the replicase gene occupying two-thirds of the 5' end of the viral genome. The replicase gene encodes two long overlapping polyproteins, pp1a and pp1ab. In the SARS-CoV-2 life cycle, the processing of pp1a and the conversion of pp1ab into 16 non-structural proteins (Nsps) are crucial steps required for RNA transcription and genome replication. Papain-like protein (PLpro) and major protease (Mpro) function as proteases, thereby participating in the viral replication and translation process. Simultaneously, SARS-CoV-2 PLpro has the function of removing ubiquitin and the ubiquitin-like interferon-stimulated gene 15 (ISG15) post-translational modification, inhibiting the host's antiviral interferon signaling pathway and playing an important role in the innate immune response during viral infection.

[0003] The human upper respiratory tract is a site of high ACE2 receptor expression in host cells mediating SARS-CoV-2 infection; therefore, the clinical symptoms of COVID-19 patients mainly manifest as upper respiratory tract reactions. In addition to common respiratory symptoms, gastrointestinal infection and digestive symptoms of SARS-CoV-2 have received attention since the beginning of the outbreak. Early studies indicated that approximately 10%-50% of COVID-19 patients in China reported gastrointestinal reactions during infection. Furthermore, a US study of 318 COVID-19 patients showed widespread gastrointestinal symptoms. COVID-19-related gastrointestinal symptoms mainly included diarrhea (24.2%), anorexia (17.9%), and nausea (17.9%), and some COVID-19 patients also experienced acute abdomen. Most importantly, some clinical studies have shown a close correlation between gastrointestinal symptoms and the severity of COVID-19, such as severe, critical, and respiratory distress. In fact, SARS-CoV-2 has a high replication efficiency in intestinal epithelial cells, which may be one of the reasons for the gastrointestinal symptoms.

[0004] Treatment options for viral gastrointestinal inflammation are currently limited. Some antiviral drugs, such as remdesivir and favipiravir, have been used to treat COVID-19, but they are primarily used to treat respiratory infections and are not effective for gastrointestinal symptoms. Therefore, finding new drugs to treat gastrointestinal symptoms is essential. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing berberine sulfate as a drug for the prevention and / or treatment of viral enteritis.

[0006] The molecular formula of berberine sulfate described in this invention is C0 19 H 15 NO8S, with a molecular weight of 417.39, has the following structural formula:

[0007]

[0008] In one aspect, the present invention provides the use of berberine sulfate or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and / or treatment of viral enteritis, wherein the virus is a coronavirus.

[0009] The coronaviruses include HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus), SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), and MERS-CoV (Middle East Respiratory Syndrome Coronavirus). Preferably, the coronavirus is SARS-CoV-2.

[0010] Another aspect of the present invention provides the use of berberine sulfate or a pharmaceutically acceptable salt thereof in the preparation of an enteritis drug induced by papain-like protein PLpro.

[0011] Preferably, the papain-like protein is the papain-like protein of SARS-CoV-2.

[0012] The present invention also provides a drug for treating intestinal inflammation caused by the novel coronavirus, the active ingredient of which includes berberine sulfate.

[0013] This invention also provides the use of berberine sulfate or a pharmaceutically acceptable salt thereof in the preparation of a drug that targets the binding interface between the coronavirus PLpro and ISG15 protein. The targeting effect is an inhibitory effect.

[0014] Further, the coronavirus includes HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus), SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), and MERS-CoV (Middle East Respiratory Syndrome Coronavirus). Preferably, the coronavirus is SARS-CoV-2.

[0015] The present invention also provides a drug that targets and inhibits the binding of the SARS-CoV-2 virus PLpro to the ISG15 protein, the active ingredient of which includes berberine sulfate.

[0016] The beneficial effects of this invention include:

[0017] This invention marks the first discovery of the application of berberine sulfate and its pharmaceutically acceptable salts in the preparation of drugs for the prevention and / or treatment of viral enteritis, thereby expanding their scope of use. Specifically, papain-like protein (PLpro) is involved in the replication and translation process of SARS-CoV-2 virus. SARS-CoV-2 PLpro has the function of removing ubiquitin and ubiquitin-like interferon-stimulated gene 15 (ISG15) post-translational modifications, inhibiting the host antiviral interferon signaling pathway. Examples of this invention demonstrate that the papain-like protein of the novel coronavirus SARS-CoV-2 can induce colonic inflammation in mice. Furthermore, in vitro experiments show that berberine sulfate can effectively inhibit the activity of papain-like protein PLpro binding to ISG15, with an IC50 value of [value missing]. 50 The concentration was 4.48 μM. Animal experiments showed that berberine sulfate could effectively relieve colon inflammation caused by SARS-CoV-2 PLpro, indicating that berberine sulfate has a positive effect in the treatment of viral intestinal inflammation. Attached Figure Description

[0018] Figure 1 This is the result of establishing a mouse colonic inflammation model using papain-like protein, an important immunomodulatory protein of the novel coronavirus SARS-CoV-2, in Example 1 of this invention. Figure A shows an H&E stained section, with the red arrow indicating obvious inflammatory cell infiltration; Figure B shows the real-time quantitative PCR detection results of the mRNA levels of inflammatory factors TNFα, IL-1α, and ISG15 in the colon of the papain-like protein treatment group and the blank control.

[0019] Figure 2This is the result of determining the half-maximal inhibitory concentration (IC50) of berberine sulfate in inhibiting the interaction between PLpro and ISG15 using the fluorescence polarization method in Example 2 of this invention. The horizontal axis represents the concentration of berberine sulfate, and the vertical axis represents the inhibition rate of berberine sulfate on the binding of papain-like protein to ISG15. The half-maximal effective concentration (IC50) of berberine sulfate in inhibiting the binding of PLpro and ISG15 was calculated based on the inhibition rate. 50 value.

[0020] Figure 3 This figure shows the results of using berberine sulfate in Example 3 of the present invention to induce a mouse colonic inflammation model induced by papain-like protein, an important immunomodulatory protein of SARS-CoV-2. Figure A shows an H&E stained section, indicating that berberine sulfate can inhibit inflammatory cell infiltration; Figure B shows the real-time quantitative PCR results of a significant decrease in the levels of inflammatory factors TNFα, IL-1α, and ISG15 after treatment with berberine sulfate. Detailed Implementation

[0021] To better understand the content of this invention, the following description, in conjunction with the accompanying drawings and specific implementation methods, will further illustrate the content of this invention. However, the scope of protection of this invention is not limited to the following embodiments.

[0022] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in this specification is for the purpose of describing particular embodiments and is not intended to limit the scope of the invention.

[0023] The following examples primarily utilize papain-like protein, an important immunomodulatory protein of SARS-CoV-2, to establish a mouse model of colitis. Based on the function of papain-like protein in removing post-translational modifications of interferon-ubiquitin-stimulated gene 15 (ISG15), a method for screening inhibitors targeting the papain-like protein-ISG15 interface was established. In this example, the activity of berberine sulfate in blocking the interaction interface between papain-like protein and ISG15 was evaluated using fluorescence polarization. Simultaneously, berberine sulfate was applied to a mouse model of colitis induced by papain-like protein stimulation to observe its anti-inflammatory effect.

[0024] The C57BL / 6J mice used in this embodiment of the invention were purchased from the Animal Experiment Center of Southern Medical University.

[0025] In this embodiment of the invention, berberine sulfate was purchased from Shanghai Taosu Biochemical Technology Co., Ltd., with a purity >99%.

[0026] The ISG15-FITC used in this embodiment of the invention was purified and prepared in our laboratory.

[0027] The papain-like protein used in the embodiments of the present invention was recombinantly expressed and purified in Escherichia coli in our laboratory.

[0028] The acetic acid solution, chloroform, anhydrous ethanol, and isopropanol used in the embodiments of this invention were purchased from Guangzhou Chemical Reagent Company.

[0029] The reverse transcription kit and qPCR SYBR Green Master Mix used in the embodiments of this invention were purchased from Shanghai Yisheng Biotechnology.

[0030] The saline solution used in this embodiment of the invention was purchased from Sewell Biotechnology.

[0031] The PEG300 and Tween 80 used in the embodiments of this invention were purchased from Maclean Chemical Reagents.

[0032] The DMSO used in this embodiment of the invention was purchased from Beyotime Biotechnology.

[0033] Example 1: Establishment of a mouse model of colonic inflammation induced by papain-like protein stimulation

[0034] 1. Establishment of an intestinal inflammation model

[0035] Eight-week-old C57BL / 6J mice were allowed free access to food and water in a constant temperature environment of 26℃ with a 14 / 10-hour periodic light and dark cycle. The mice were pre-divided into a blank control group, an acetic acid control group, and a model group, with five mice in each group.

[0036] 1) The animals were fasted for 24 hours before modeling.

[0037] 2) Prepare 1% (v / v) acetic acid with physiological saline as a reagent to promote intestinal inflammation.

[0038] 3) Anesthetize mice by intraperitoneal injection of 40 mg / kg of 1% sodium pentobarbital. Approximately 5 minutes later, smear the surface of the tubing with glycerin and insert it into the mouse's anus to a depth of 4 cm. Attach a syringe to the other end of the tubing and inject 500 μL of 1% acetic acid solution; the control group received an equal volume of physiological saline. Invert the mice for 1 minute and irrigate twice with 500 μL of PBS. After the procedure, return the mice to their cages and provide food.

[0039] 4) Sixteen hours later, the protein group mice were injected intraperitoneally with SARS-CoV-2 papain-like protein at a dose of 2.5 mg / kg, prepared with PBS as the solvent to the required concentration. The blank group and acetic acid group were injected with the same volume of PBS.

[0040] 5) After 10 hours, observe the diarrhea status of the mice. Anesthetize the mice by intraperitoneal injection of 40 mg / kg 1% sodium pentobarbital, open the thoracic and abdominal cavities, and collect liver, kidney, lung, and intestinal tissues.

[0041] 2. Tissue RNA extraction

[0042] 1) Remove the tissue from the mouse tissue stored at -80°C and thaw it on ice. Subsequent procedures were performed in a clean bench to avoid interference from RNases.

[0043] 2) Transfer the intestinal tissue from each group to homogenization tubes, and add grinding beads and 200 μL of Trizol. Place the samples in a biosample homogenizer and run at 60 Hz for 60 seconds to ensure thorough homogenization, then let stand for 5 minutes.

[0044] 3) Add 40 μL of chloroform, mix quickly and evenly, and let stand for 2 minutes.

[0045] 4) Place the sample in a centrifuge at 4°C and centrifuge at 11,000 rpm for 15 minutes. After centrifugation, the sample will clearly separate into three phases, from top to bottom: aqueous phase, protein phase, and organic phase. Carefully transfer the aqueous phase containing tissue RNA to a new centrifuge tube.

[0046] 5) Add 0.5 mL of isopropanol and shake well immediately, then let stand for 10 minutes.

[0047] 6) Centrifuge at 11,000 rpm at 4°C for 10 minutes. A white precipitate, which is RNA, will be observed at the bottom of the centrifuge tube. Discard the supernatant, add 1 mL of 75% anhydrous ethanol to wash the precipitate, centrifuge at 6,700 rpm at 4°C for 5 minutes, and repeat this step three times. Finally, retain the precipitate.

[0048] 7) After drying the precipitate at room temperature for 5-10 minutes, add an appropriate amount of enzyme-free water at 55℃ to dissolve the precipitate and obtain a tissue RNA solution.

[0049] 3. Reverse transcription and real-time quantitative PCR

[0050] According to the instructions of the reverse transcription kit, the extracted tissue RNA was quantitatively reverse transcribed to obtain cDNA.

[0051] Real-time quantitative PCR (qRT-PCR) was performed according to the instructions for the qPCR SYBR Green Master Mix. Each sample was tested in triplicate. β-actin was used as an internal control. Relative RNA expression levels were calculated using the 2-ΔΔCt method.

[0052] 4. Results

[0053] like Figure 1As shown, H&E staining indicated that 500 μg of PLpro could induce a significant intestinal inflammatory response. qRT-PCR results showed that, compared with the control group, the expression levels of TNF-α, IL-1α mRNA, and ISG15 mRNA in the PLpro protein-treated group were significantly increased, indicating an intestinal inflammatory response.

[0054] Example 2: Detection of interfacial activity of berberine sulfate in blocking the binding of papain-like protein to ISG15

[0055] 1. Drug inhibitory activity experiment

[0056] 1) Prepare the required buffer solution for the experiment: 25mM NaH2PO4, 150mM NaCl, 0.1mg / ml BSA and 7.5μM ZnCl2.

[0057] 2) Take papain-like protein PLpro, with a molecular weight of 36.8kD, and dilute PLpro with buffer to a concentration of 2μM.

[0058] 3) Take ISG15-FITC protein with a molecular weight of 9.5kD, and dilute ISG15-FITC with buffer to a concentration of 200nM.

[0059] 4) In this experiment, the initial drug concentration was 1 μM (solvent was dimethyl sulfoxide DMSO). For the half-diluted drug, a total of 8 concentration gradients were set, and 3 replicates were set for each concentration.

[0060] 5) Take 50 μL of diluted PLpro and place it in a 96-well blackboard.

[0061] 6) Add 1 μL of berberine sulfate to the well.

[0062] 7) Use 1 μL LDMSO instead of berberine sulfate in the control well.

[0063] 8) Mix well and incubate for 10 minutes.

[0064] 9) Take 50 μL of diluted ISG15-FITC into the same well, so that the final concentrations of PLpro and ISG15-FITC are 1 μM and 100 nM, respectively.

[0065] 10) Incubate at room temperature for 1 hour.

[0066] 11) Place the sample in an ELISA reader to read the fluorescence value. The excitation wavelength is 483 nm and the emission wavelength is 535 nm.

[0067] 2. Results

[0068] like Figure 2As shown, using the copy number of the DMSO group as a reference, and based on the inhibition rates of different drug concentrations, GraphPad Prism 8.4.2 software was used to fit the drug inhibition rate curves and calculate the half-maximal inhibitory concentration (IC50) of berberine sulfate on the binding of SARS-CoV-2 PLpro to ISG15. 50 The value is 4.48 μM.

[0069] Example 3: The effect of berberine sulfate on relieving colonic inflammation caused by SARS-CoV-2 papain-like protein stimulation in animals.

[0070] 1. Experimental Design

[0071] Eight-week-old C57BL / 6J mice were housed in an environment with a constant temperature of 26°C and a 14 / 10-hour light / dark cycle, with free access to food and water. Dosages of 25 mg / kg and 100 mg / kg were administered, with each dose serving as a dosing group and the drug solvent serving as a control group. Five mice were in each group. The compound was dissolved in 10% DMSO, 40% PEG300, 5% Tween 80, and 45% physiological saline. In this experiment, a pre-treatment approach was used, with administration via gavage for three consecutive days prior to model establishment. After administration, the animal model was established according to the method described in Example 1.

[0072] 2. Experimental Methods

[0073] 2.1 Modeling Method

[0074] Perform according to the method in Example 1.

[0075] 2.2 RNA extraction and reverse transcription

[0076] Perform according to the method in Example 1.

[0077] 2.3 Real-time quantitative PCR

[0078] Perform according to the method in Example 1.

[0079] 3. Results

[0080] like Figure 3 As shown, treatment with berberine sulfate significantly reduced the expression levels of TNFα, IL-1α mRNA and ISG15 mRNA in the mouse intestine, and significantly alleviated intestinal inflammation in mice.

[0081] The above description is merely a specific embodiment of the present invention and not all embodiments. Any equivalent modifications made to the technical solutions of the present invention by those skilled in the art upon reading this specification are within the scope of the claims of the present invention.

Claims

1. The use of berberine sulfate or its pharmaceutically acceptable salt in the preparation of medicaments for the prevention and / or treatment of viral enteritis, characterized in that, The virus is a coronavirus, specifically SARS-CoV-2; the molecular formula of the berberine sulfate is C2. 19 H 15 NO8S, with a molecular weight of 417.39, has the following structural formula:

2. The application according to claim 1, characterized in that, The viral enteritis mentioned is enteritis induced by the papain-like protein of SARS-CoV-2.

3. The application according to claim 1, characterized in that, The berberine sulfate or its pharmaceutical formulations contain a papain-like protein that can accept salts to inhibit coronaviruses, which binds to ISG15.

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