Application of betrixaban or pharmaceutically acceptable salt thereof in preparation of antiviral drugs

Abstract

The invention discloses application of betrixaban or pharmaceutically acceptable salt thereof in preparation of antiviral drugs. Drug screening and in-vitro drug experiments find that betrixaban or pharmaceutically acceptable salts thereof can inhibit virus replication and infection ability, and can block virus-induced syncytial bodies, so that cell death caused by the syncytial bodies is reduced. In addition, the betrixaban or the pharmaceutically acceptable salt thereof provided by the invention has broad-spectrum antiviral activity, and also has the characteristics of safety and high efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to the use of betrixaban or a pharmaceutically acceptable salt thereof in the preparation of an antiviral drug. Background Art

[0002] In recent years, due to changes in the natural environment and the expansion of human activities, viral diseases have occurred frequently, posing a great threat to human public health and safety. The spread of viral diseases is a major public safety and health issue that threatens human health. Therefore, there is an urgent need to develop a safe, broad-spectrum and highly effective antiviral drug. Summary of the invention

[0003] In order to solve at least some of the technical problems in the above-mentioned prior art, the present invention provides the use of betrixaban or a pharmaceutically acceptable salt thereof in the preparation of an antiviral drug. Specifically, the present invention includes the following contents.

[0004] The first aspect of the present invention provides use of betrixaban or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, isomer, analog, derivative or metabolite thereof in the preparation of an antiviral drug.

[0005] In certain embodiments, according to the use of the present invention, the virus comprises a DNA virus and / or an RNA virus.

[0006] In certain embodiments, according to the use of the present invention, the DNA virus includes at least one of herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, cytomegalovirus, smallpox virus, monkeypox virus, adenovirus, human papillomavirus, parvovirus, bocavirus and hepatitis B virus.

[0007] In certain embodiments, according to the use of the present invention, the RNA virus includes at least one of vesicular stomatitis virus, encephalomyocarditis virus, mouse hepatitis virus, influenza A virus, influenza B virus, influenza C virus, dengue virus, Zika virus, Ebola virus, Marburg virus, Nipah virus, coronavirus, hepatitis A virus, hepatitis C virus, rotavirus, measles virus, human immunodeficiency virus, respiratory syncytial virus and rabies virus.

[0008] In certain embodiments, according to the use of the present invention, the antiviral comprises at least one of the following: (1) Inhibit virus invasion into host cells; (2) inhibiting viral genome replication; (3) inhibiting viral protein processing or assembly; (4) Inhibition of viral release; (5) Reducing the amount or activity of the virus.

[0009] In certain embodiments, according to the use of the present invention, the antiviral effect is achieved by administering a therapeutically effective amount of the drug to the subject.

[0010] In certain embodiments, according to the use of the present invention, the subject comprises a mammal.

[0011] In certain embodiments, according to the use of the present invention, the mammal comprises a human.

[0012] In certain embodiments, according to the use of the present invention, the therapeutically effective amount is 0.01-1000 mg / Kg.

[0013] The second aspect of the present invention provides the use of betrixaban or its pharmaceutically acceptable salt, solvate, hydrate, prodrug, isomer, analog, derivative or metabolite and other drugs in the preparation of a combined antiviral drug.

[0014] The present invention finds through drug screening and in vitro drug experiments that betrixaban or its pharmaceutically acceptable salt, solvate, hydrate, prodrug, isomer, analog, derivative or metabolite can inhibit viral replication and infection ability, and can block virus-induced syncytium, thereby reducing cell death caused by syncytium. In addition, betrixaban or its pharmaceutically acceptable salt, solvate, hydrate, prodrug, isomer, analog, derivative or metabolite of the present invention has a broad spectrum of antiviral activity, and has the characteristics of safety and high efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shown is the inhibitory effect of betrixaban on vesicular stomatitis virus (VSV) virus in human fibrosarcoma cells (2fTGH cells).

[0016] Figure 2 Inhibitory effects of betrixaban on transcription of other viruses and on syncytium formation are shown.

[0017] Figure 3 The inhibitory effect of betrixaban on VSV virus in other cells is shown.

[0018] Figure 4 The results of a study on the antiviral mechanism of betrixaban are shown. DETAILED DESCRIPTION

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0022] use One aspect of the present invention provides use of betrixaban or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, isomer, analog, derivative or metabolite thereof in the preparation of an antiviral drug.

[0023] The term "pharmaceutically acceptable salt" as used herein refers to organic and inorganic salts of the compound betrixaban of the present invention. Pharmaceutically acceptable salts are well known in the art, and examples thereof include, but are not limited to: inorganic acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, perchlorates; organic acid salts, such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates; or other methods described in books and literature, such as ion exchange methods, to obtain these salts. Other pharmaceutically acceptable salts include adipate, malate, 2-hydroxypropionate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate and the like.

[0024] The term "solvate" as used herein refers to an association or complex formed by one or more solvent molecules and the compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, aminoethanol, and the like.

[0025] The term "hydrate" as used herein refers to an association or complex formed with the compound of the present invention when the solvent molecule is water.

[0026] The term "isomer" as used herein includes enantiomeric, diastereomeric, and geometric (or conformational) isomeric forms of a given structure. For example, the present invention includes R and S configurations, Z and E double bond isomers, Z and E conformational isomers, single stereochemical isomers, and enantiomeric, diastereomeric, and geometric (or conformational) isomer mixtures for each asymmetric center. Unless otherwise indicated, the present invention includes all tautomeric forms of the disclosed structures of betrixaban.

[0027] The term "prodrug" as used herein refers to a compound that is converted into a compound represented by the following formula (I) or formula (II) in vivo: Formula (I); Formula (II).

[0028] The term "prodrug" refers to that the parent drug molecule is a pharmacologically inactive derivative, which requires spontaneous or enzymatic biotransformation in vivo to release active drugs. Prodrugs are variants or derivatives of the compounds of the present invention, which have a group that can be cleaved under metabolic conditions. When prodrugs undergo solvent degradation or undergo enzymatic degradation under physiological conditions, they become compounds of the present invention with pharmaceutical activity in vivo. Prodrug forms generally provide solubility, tissue compatibility or delayed release advantages in mammalian organisms (see Bundgard, Design of Prodrugs (prodrug design), pp.7-9, 21-24, Elsevier, Amsterdam 1985 and Silverman, The Organic Chemistry of Drug Designand Drug Action (organic chemistry and drug action of drug design), pp.352-401, Academic Press, San Diego, CA, 1992). Prodrugs known in the art include acid derivatives well known to those skilled in the art, such as esters prepared by reacting the parent acid with a suitable alcohol, or amides prepared by reacting the parent acid compound with an amine, or with a basic group which reacts to form an acylated base derivative.

[0029] As used herein, the term "metabolite" refers to a product obtained by metabolism of a specific compound or its salt in vivo. The metabolite of a compound can be identified by techniques known in the art, and its activity can be characterized by methods known in the art. Such a product can be obtained by administering a compound through oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic cleavage, etc. Accordingly, the present invention includes metabolites of compounds of formula (I) or (II), including metabolites produced by contacting a compound of formula (I) or (II) of the present invention with a mammal or a cell line derived therefrom for a period of time.

[0030] In the present invention, the term "derivative" refers to a product derived from the substitution of atoms or functional groups in the compound of formula (I) or (II) by other atoms or functional groups. The derivative is not specifically limited and includes but is not limited to esters, acylates or metal complexes of the compound of formula (I) or (II).

[0031] In the present invention, the virus includes DNA virus and / or RNA virus. Wherein, examples of the DNA virus include but are not limited to herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, cytomegalovirus, smallpox virus, monkeypox virus, adenovirus, human papillomavirus, parvovirus, bocavirus, hepatitis B virus, etc.; examples of the RNA virus include but are not limited to vesicular stomatitis virus, encephalomyocarditis virus, mouse hepatitis virus, influenza A virus, influenza B virus, influenza C virus, dengue virus, Zika virus, Ebola virus, Marburg virus, Nipah virus, coronavirus, hepatitis A virus, hepatitis C virus, rotavirus, measles virus, human immunodeficiency virus, respiratory syncytial virus, rabies virus, etc. In a preferred embodiment, the DNA virus is herpes simplex virus type 1. In another preferred embodiment, the RNA virus is vesicular stomatitis virus. In another preferred embodiment, the RNA virus is encephalomyocarditis virus. In another preferred embodiment, the RNA virus is mouse hepatitis virus. In yet another preferred embodiment, the RNA virus is influenza A virus.

[0032] In the present invention, "antiviral" refers to improving the condition before or after the disease or dysfunction occurs. Compared with an untreated reference group under the same conditions, the degree of relief or prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% as measured by any standard technique. Beneficial or desired clinical results include, but are not limited to, the following, whether detectable or undetectable, including relief of symptoms, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and reduction (whether partial or complete). Antiviral situations include, but are not limited to (1) inhibiting virus invasion of host cells; (2) inhibiting viral genome replication; (3) inhibiting viral protein processing or assembly; (4) inhibiting viral release; (5) reducing the amount or activity of the virus.

[0033] In the present invention, antiviral treatment is achieved by administering a therapeutically effective amount of the drug to a subject. The subject includes, but is not limited to, mammals, and examples of mammals include, but are not limited to, humans, mice, rabbits, cats, dogs, cattle, sheep, pigs, and the like.

[0034] In the present invention, the drug contains a therapeutically effective amount of betrixaban or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, isomer, analog, derivative or metabolite thereof, in particular betrixaban maleate, and optionally, a pharmaceutically acceptable excipient or carrier. The term "pharmaceutically acceptable excipient or carrier" as used herein refers to a pharmaceutically acceptable material, composition or medium, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, and such pharmaceutically acceptable material, composition or medium participates in carrying or transporting the agent from one organ or part of the body to another organ or another part of the body. Each excipient or carrier is "acceptable" in the sense that it is compatible with the other ingredients of the formulation and does not harm the patient.

[0035] In the present invention, the administration mode of the drug is not particularly limited, and representative administration modes include, but are not limited to, oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and topical administration. Accordingly, the drug of the present invention can be prepared into various clinically acceptable dosage forms, including oral dosage forms, injection dosage forms, topical dosage forms or external dosage forms, etc.

[0036] The therapeutically effective amount described in the present invention refers to the effective dosage considered pharmaceutically, that is, the amount of the active compound (i.e., betrixaban or its pharmaceutically acceptable salt, solvate, hydrate, prodrug, isomer, analog, derivative or metabolite, especially betrixaban maleate) is sufficient to significantly improve the condition without causing serious side effects. The daily dosage of betrixaban or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, isomer, analog, derivative or metabolite thereof is generally 0.01-1000 mg / Kg, preferably 0.01-500 mg / Kg, or 0.01-400 mg / Kg, or 0.01-300 mg / Kg, or 0.01-200 mg / Kg, or 0.01-150 mg / Kg, or 0.01-100 mg / Kg, or 0.01-50 mg / Kg, or 0.01-40 mg / Kg, or 0.01-30 mg / Kg, most preferably 0.01-20 mg / Kg. Exemplary effective dosages are, for example, 0.01 mg / Kg, 0.05 mg / Kg, 0.1 mg / Kg, 0.2 mg / Kg, 0.3 mg / Kg, 0.4 mg / Kg, 0.5 mg / Kg, 0.75 mg / Kg, 0.95 mg / Kg, 1 mg / Kg, 1.25 mg / Kg, 1.5 mg / Kg, 1.75 mg / Kg, 2 mg / Kg, 2.5 mg / Kg, 2.75 mg / Kg, 3 mg / Kg, 3.25 mg / Kg, 3.5 mg / Kg, 3.75 mg / Kg, 4 mg / Kg, 4.25 mg / Kg, 4.5 mg / Kg, 4.75 mg / Kg, 5 mg / Kg, 5.25 mg / Kg, 5.5 mg / Kg, 5.75 mg / Kg, 6 18 mg / Kg, 19 mg / Kg, 20 mg / Kg, 21 mg / Kg, 22 mg / Kg, 23 mg / Kg, 24 mg / Kg, 25 mg / Kg, 26 mg / Kg, 27 mg / Kg, 28 mg / Kg, 29 mg / Kg, 30 mg / Kg, 31 mg / Kg, 32 mg / Kg, 33 mg / Kg, 34 mg / Kg, 35 mg / Kg, 36 mg / Kg, 37 mg / Kg, 38 mg / Kg, 39 mg / Kg, 40 mg / Kg, 41 mg / Kg, 42 mg / Kg, 43 mg / Kg, 44 mg / Kg, 45 mg / Kg, 46 mg / Kg, 47 mg / Kg, 48 mg / Kg, 49 mg / Kg, 50 mg / Kg, 51 mg / Kg, 52 mg / Kg, 53 mg / Kg It can be administered as a single dose once a day, divided into multiple doses per day, or administered at intervals.

[0037] Combined use One aspect of the present invention provides the use of betrixaban or its pharmaceutically acceptable salt, solvate, hydrate, prodrug, isomer, analog, derivative or metabolite and other drugs in the preparation of a combined antiviral drug.

[0038] In the present invention, other drugs include but are not limited to remdesivir, ribavirin, favipiravir, acyclovir, valacyclovir, famciclovir, ganciclovir, brivudine, interferon, oseltamivir phosphate, peramivir, zanamivir, mabaloxavir, favipiravir, arbidol, etc.

[0039] Example The inhibitory effect of betrixaban on viruses is exemplified below.

[0040] 1. Inhibitory effect of betrixaban on VSV in 2fTGH cells 1.1 Experimental methods 2fTGH cells were treated with betrixaban (DMSO as solvent) and VSV at concentrations of 10, 40, 60, and 80 μM, respectively. DMSO+VSV was used as the control group. After 12 h and 24 h of treatment, the expression levels of VSV gene and protein were detected by qPCR and Western Blot, respectively, and the content of viral particles in the cell supernatant was detected by plaque assay.

[0041] 1.2 Experimental Results The results are as follows Figure 1 Specifically, the qPCR results showed that as the concentration of betrixaban increased, the relative amount of VSV viral gene expression decreased, and the effect of the drug on inhibiting viral transcription was not enhanced by the drug action time ( Figure 1 A). Western Blot results showed that compared with the internal reference protein, the expression of VSV virus protein decreased with the increase of betrixaban drug concentration ( Figure 1 B), Figure 1 The concentrations of betrixaban in B are 20, 40, and 80 μM from left to right. The results of the virus plaque assay showed that the virus titer decreased significantly with the increase of betrixaban concentration. Compared with the DMSO group, when the concentration of betrixaban was 60 μM, the virus load was almost 0, indicating that the replication and infection ability of the virus was negatively correlated with the concentration of betrixaban ( Figure 1 C).

[0042] In summary, betrixaban can reduce VSV viral transcription and expression, and inhibit viral replication and infection ability.

[0043] 2. Inhibitory effects of betrixaban on other viruses 2.1 Experimental Methods 2fTGH cells were treated with betrixaban and herpes simplex virus type 1 (HSV-1) at concentrations of 20, 40, and 80 μM, respectively, and DMSO+HSV-1 was used as the control group for 12 h; 2fTGH cells were treated with betrixaban and encephalomyocarditis virus (EMCV) at concentrations of 20, 40, and 80 μM, respectively, and DMSO+EMCV was used as the control group for 12 h; 2fTGH cells were treated with betrixaban and mouse hepatitis virus (MHV) at concentrations of 20, 40, and 80 μM, respectively, and DMSO+MHV was used as the control group for 12 h; 2fTGH cells were treated with betrixaban and influenza A virus (IAV) at concentrations of 20, 40, and 80 μM, respectively, and DMSO+IAV was used as the control group for 12 h, and the expression changes of viral genes were quantitatively detected by qPCR technology. In addition, the virus group and DMSO group were used as controls to observe the effect of betrixaban on the syncytium formation induced by HSV-1 and MHV in 2fTGH and J774.1 cells.

[0044] 2.2 Experimental Results The results are as follows Figure 2 Specifically, the qPCR relative quantitative results showed that betrixaban inhibited the transcription of HSV-1, EMCV, MHV and IAV viral genes, and as the drug concentration increased, the relative expression of viral genes decreased ( Figure 2 A). The cell syncytium experiment showed that the cell syncytium formation in the betrixaban-treated group was significantly reduced compared with the virus group and DMSO group, indicating that betrixaban can effectively inhibit the cell syncytium formation caused by MHV and HSV infection ( Figure 2 B).

[0045] It can be seen that betrixaban has a broad-spectrum antiviral activity and can block virus-induced syncytia, thereby reducing cell death caused by syncytia.

[0046] 3. Inhibitory effect of betrixaban on VSV in other cells 3.1 Experimental Methods A549, HeLa, HT29, Pan02, and Raw264.7 cells were treated with betrixaban (80 μM) and VSV, and the expression of viral genes was quantitatively analyzed by qPCR. In addition, the Mock group and DMSO group were used as controls, and HeLa cells were treated with 30 and 50 μM betrixaban and VSV-GFP (green fluorescent protein), respectively, and their fluorescence intensity was observed by fluorescence microscopy.

[0047] 3.2 Experimental Results The results are as follows Figure 3 Specifically, the qPCR results showed that the expression level of VSV virus mRNA in the experimental group (betrixaban + VSV) was significantly lower than that in the control group (DMSO + VSV). This shows that betrixaban can significantly inhibit the transcriptional expression of VSV in these cell lines ( Figure 3 A). The microscopic results showed that compared with the control group, the number of green fluorescent cells decreased significantly with the increase of betrixaban concentration, indicating that betrixaban can inhibit the expression of VSV-GFP ( Figure 3 B). In summary, betrixaban has significant antiviral activity and can inhibit viral replication in a variety of cell lines.

[0048] 4. Study on the antiviral mechanism of betrixaban 4.1 Experimental Methods cGAS knockout and wild-type 2fTGH cells were treated with 20, 40, and 80 μM betrixaban and VSV, respectively, and then qPCR was used to detect the expression levels of antiviral genes MX1 and MX2 and the inhibition of VSV viral transcription.

[0049] 4.2 Experimental Results The results are as follows Figure 4 As shown by Figure 4 A shows that compared with the betrixaban control group, the expression of MX1 and MX2 genes in the cGAS knockout group was significantly reduced, indicating that the cGAS pathway was inhibited. Figure 4 As shown in Figure 2, the results of VSV virus mRNA gene detection showed that in the wild-type 2fTGH model, compared with the DMSO control group, the expression of VSV mRNA in the betrixaban group was significantly decreased, and the expression level decreased with the increase of drug concentration; similarly, in the cGAS knockout model, the expression level of VSV mRNA in the betrixaban group decreased with the increase of drug concentration. This shows that betrixaban not only produces antiviral effects through the cGAS pathway, that is, cGAS is an important pathway among multiple antiviral pathways.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of betrixaban or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, isomer, analog, derivative or metabolite thereof in the preparation of an antiviral drug.

2. The use according to claim 1, characterized in that The viruses include DNA viruses and / or RNA viruses.

3. The use according to claim 2, characterized in that: The DNA virus includes at least one of herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, cytomegalovirus, smallpox virus, monkeypox virus, adenovirus, human papillomavirus, parvovirus, bocavirus and hepatitis B virus.

4. The use according to claim 2, characterized in that: The RNA virus includes at least one of vesicular stomatitis virus, encephalomyocarditis virus, mouse hepatitis virus, influenza A virus, influenza B virus, influenza C virus, dengue virus, Zika virus, Ebola virus, Marburg virus, Nipah virus, coronavirus, hepatitis A virus, hepatitis C virus, rotavirus, measles virus, human immunodeficiency virus, respiratory syncytial virus and rabies virus.

5. The use according to claim 1, characterized in that: The anti-virus includes at least one of the following situations: (1) Inhibit virus invasion into host cells; (2) inhibiting viral genome replication; (3) inhibiting viral protein processing or assembly; (4) Inhibition of viral release; (5) Reducing the amount or activity of the virus.

6. The use according to claim 1, characterized in that: The antiviral effect is achieved by administering a therapeutically effective amount of the drug to the subject.

7. The use according to claim 6, characterized in that The subject includes a mammal.

8. The use according to claim 7, characterized in that The mammals include humans.

9. The use according to claim 6, characterized in that The therapeutically effective amount is 0.01-1000 mg / Kg.

10. Use of betrixaban or its pharmaceutically acceptable salt, solvate, hydrate, prodrug, isomer, analog, derivative or metabolite and other drugs in the preparation of combined antiviral drugs.

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