阅读说明：本技术 伊文思蓝在制备药物中的用途及药物组合物 (Application of Evans blue in preparation of medicine and medicine composition ) 是由 陈绪林 肖宇 于 2019-10-23 设计创作，主要内容包括：本发明提出了伊文思蓝在制备治疗HBV病毒感染药物中的应用及一种用于治疗或者预防乙型肝炎病毒感染的药物组合物。所述药物或药物组合物可有效抑制乙型肝炎病毒的进入和病毒颗粒的组装,可有效用于治疗或者预防乙型肝炎病毒感染引起的肝炎。(The invention provides an application of Evans blue in preparing a medicine for treating HBV infection and a medicine composition for treating or preventing hepatitis B virus infection. The medicine or the pharmaceutical composition can effectively inhibit the entry of hepatitis B virus and the assembly of virus particles, and can be effectively used for treating or preventing hepatitis caused by hepatitis B virus infection.)

1. Use of Evans blue in preparing medicine for treating or preventing hepatitis B virus infection is provided.

2. A pharmaceutical composition for treating or preventing hepatitis b virus infection, comprising:

evans blue as active ingredient; and

a second agent that is different from evans blue and that is used to treat or prevent hepatitis b virus infection.

3. The pharmaceutical composition of claim 2, further comprising a pharmaceutically acceptable carrier.

4. The pharmaceutical composition of claim 2, wherein the second agent comprises at least one selected from the group consisting of: interferon, polyethylene glycol interferon, lamivudine, adefovir, entecavir, telbivudine, tenofovir.

5. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is in the form of a tablet, injection, powder, elixir, capsule, suspension, syrup, pill, sustained release formulation, controlled release formulation, or nano-formulation.

Technical Field

The invention relates to the field of biomedicine, in particular to application of Evans blue in preparation of a medicine and a pharmaceutical composition, and more particularly relates to application of Evans blue in preparation of a medicine and a pharmaceutical composition for treating or preventing hepatitis B virus infection.

Background

Hepatitis B Virus (HBV) infection is the most prevalent chronic viral infection in the world. HBV is transmitted mainly by blood transmission, sexual behavior transmission, mother-infant transmission and the like. Hepatitis b virus infection is a serious health problem worldwide, with about 2.2 billion HBV carriers worldwide, causing 768000 deaths each year, which is the tenth leading cause of death worldwide.

However, the current drugs for treating hepatitis B virus infection are only applied to clinic, and are divided into two forms of Interferon (IFN) and polyethylene glycol interferon (PEG-IFN), which are approved by FDA in 1990 and 2005; and five nucleoside analogue viral polymerase inhibitor drugs, namely Lamivudine (Lamivudine,3TC or LMV), Adefovir (Adefovir, ADV), Entecavir (Entecavir, ETV), Telbivudine (Telbivudine, LdT) and Tenofovir (Tenofovir disoproxil fumarate, TDF). However, interferon has human tolerance and has large side effect; polymerase inhibitors, however, do not clear cccDNA, and viral replication rebounds rapidly after drug withdrawal, and long-term administration also causes viral mutation and drug resistance.

Therefore, the screening of new anti-HBV infection drugs is very urgent and important.

Evans blue (Evans blue) has been a common biological dye commonly used for tissue section staining, Blood Brain Barrier (BBB) permeability detection and anti-hypersensitivity mediator assay. At the same time, evans blue has been reported in the literature to inhibit HIV infection.

However, the use of evans blue has yet to be further developed.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art.

The present application is based on the discovery and recognition by the inventors of the following facts:

evans blue can significantly inhibit HBV infection, and simultaneously, Evans blue can significantly inhibit the assembly of HBV virus particles. This suggests that evans blue has a dual anti-HBV effect of inhibiting virus entry into cells and inhibiting viral particle assembly.

Therefore, the invention provides a new application of Evans blue in preparing a medicine which targets HBV virus invasion and assembly of virus particles and is used for resisting hepatitis B virus infection.

In a first aspect of the invention, the invention proposes the use of evans blue for the preparation of a medicament. According to an embodiment of the present invention, the medicament is for inhibiting entry of hepatitis b virus into a cell; or treating or preventing hepatitis B virus infection. According to the embodiment of the present invention, evans blue can also significantly inhibit nucleocapsid assembly of HBV and HBV viral capsid total DNA within liver cells, and thus, the use of evans blue proposed by the present invention for preparing a medicament which can significantly inhibit hepatitis b virus particle assembly; or significant treatment or prevention of hepatitis B virus infection.

According to an embodiment of the present invention, the use of evans blue for the preparation of a medicament may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the medicament is for treating or preventing hepatitis b virus infection in a human. In some embodiments of the present invention, the inventors found that evans blue can significantly inhibit HBV against human hepatoma cell line Huh7D^hNTCPInfection of human liver primary cells (PHH) and porcine primary hepatocytes (hNTCP-PPH) stably expressing human NTCP protein. Meanwhile, evans blue can also obviously inhibit the assembly of HBV nucleocapsid and the level of nucleocapsid DNA in human hepatoma cell line HepAD38 cells. Therefore, the use of Evans blue proposed by the present invention in the preparation of a medicament for the treatment or prevention of hepatitis B virus infection in humans can be further improved.

According to an embodiment of the present invention, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. According to the embodiment of the invention, the pharmaceutically acceptable carrier can improve the stability of the pharmaceutical composition and the availability of the effective ingredients, thereby further improving the treatment or prevention of the hepatitis B virus infection by the pharmaceutical composition.

According to an embodiment of the invention, the pharmaceutical composition further comprises a second agent, which is different from evans blue and is used for treating or preventing hepatitis b virus infection. According to an embodiment of the present invention, the second agent in combination with evans blue further improves the treatment or prevention of hepatitis b virus infection by the pharmaceutical composition.

According to an embodiment of the invention, the second agent comprises at least one selected from the group consisting of: interferon (IFN), interferon-polyethylene glycol (PEG-IFN), Lamivudine (Lamivudine,3TC or LMV), Adefovir (Adefovir, ADV), Entecavir (Entecavir, ETV), Telbivudine (Telbivudine, LdT), Tenofovir (Tenofovir disoproxil fumarate, TDF). Lamivudine, adefovir, entecavir, telbivudine and tenofovir are HBV virus polymerase inhibitor drugs. The target point of Evans blue for inhibiting HBV virus infection is different from the second medicament, and Evans blue takes HBV invasion and nucleocapsid assembly as the target point, so that the risk of drug-resistant mutation of the virus does not exist, and the aim of continuously and effectively eliminating the hepatitis B virus can be fulfilled. According to an embodiment of the present invention, at least one of the above second agents is combined with evans blue, and the therapeutic or prophylactic effect of the pharmaceutical composition on hepatitis b virus infection is more significant.

According to an embodiment of the present invention, the pharmaceutical composition is in the form of a tablet, injection, powder, elixir, capsule, suspension, syrup, pill, sustained-release preparation, controlled-release preparation or nano-preparation. The pharmaceutical compositions in various dosage forms are administered in a predictable administration manner to inhibit HBV invasion and viral particle assembly, thereby further effectively treating or preventing hepatitis B virus infection.

According to some embodiments of the present invention, the pharmaceutical composition having evans blue as an active ingredient may include a pharmaceutically acceptable carrier, and the dosage form and administration manner of the pharmaceutical composition are not particularly limited. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants and flavoring agents. For injectable formulations, pharmaceutically acceptable carriers may include buffers, preservatives, analgesics, solubilizers, isotonic agents (isotonics agents) and stabilizers. For formulations for topical administration, pharmaceutically acceptable carriers may include bases, excipients, lubricants and preservatives. The pharmaceutical composition of the present invention may be prepared in various dosage forms in combination with the above pharmaceutically acceptable carrier. For example, for oral administration, the pharmaceutical compositions may be prepared as tablets, capsules, elixirs, suspensions or syrups. For injectable preparations, the pharmaceutical compositions may be prepared in ampoules, e.g. in single dose dosage form, or in unit dosage forms, e.g. in multidose containers. The pharmaceutical composition can also be prepared into solution, suspension, tablet, pill, capsule, long-acting preparation, sustained-release preparation, controlled-release preparation or nano-preparation.

According to the embodiment of the present invention, the pharmaceutical composition of the present invention, which contains evans blue as an active ingredient, can significantly inhibit the invasion of hepatitis b virus and the assembly of viral particles at a non-cytotoxic concentration, and thus can be administered in the treatment or prevention of hepatitis b virus infection.

The term "administering" as used herein means introducing a predetermined amount of a substance into a patient by some suitable means. The drug or pharmaceutical composition of the present invention can be administered by any common route as long as it can reach the intended tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, cortical, oral, topical, nasal, pulmonary and rectal, but the invention is not limited to these exemplified modes of administration. However, because of oral administration, the active ingredients of orally administered compositions should be coated or formulated to prevent degradation in the stomach. Preferably, the composition of the present invention can be administered in an injectable formulation. In addition, the pharmaceutical compositions of the present invention may be administered using a specific device that delivers the active ingredient to the target cells.

The administration frequency and dose of the pharmaceutical composition of the present invention can be determined by a number of relevant factors, including the type of disease to be treated, the administration route, the age, sex, body weight and severity of the disease of the patient and the type of drug as an active ingredient. According to some embodiments of the invention, the daily dose may be divided into 1, 2 or more doses in a suitable form for administration 1, 2 or more times over the entire period, as long as a therapeutically effective amount is achieved.

The term "therapeutically effective amount" refers to an amount of a compound sufficient to significantly ameliorate some of the symptoms associated with a disease or condition, i.e., to provide a therapeutic effect for a given condition and administration regimen. For example, in the treatment of hepatitis b virus infection, a drug or compound that reduces, prevents, delays, inhibits or retards any symptoms of the disease or disorder should be therapeutically effective. A therapeutically effective amount of a pharmaceutical composition or compound need not cure a disease or condition, but will provide treatment for a disease or condition such that the onset of the disease or condition in an individual is delayed, prevented or prevented, or the symptoms of the disease or condition are alleviated, or the duration of the disease or condition is altered, or the disease or condition becomes less severe, or recovery is accelerated, for example.

The term "treatment" is used to refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects resulting from the disease. As used herein, "treatment" encompasses the treatment of disease (primarily hepatitis b virus infection) in mammals, particularly humans, including: (a) preventing the development of a disease (e.g., preventing hepatitis b virus infection) or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting a disease, e.g., arresting disease progression; or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administering to an individual in need thereof a pharmaceutical composition comprising evans blue as an active ingredient as described herein.

Drawings

FIG. 1 is a graph showing the result of measuring anti-HBV activity of Evans blue (Evans blue) according to example 1 of the present invention,

wherein FIG. 1A is a structural diagram of Evans blue,

FIG. 1B is Evans blue vs. Huh7D^hNTCPThe cytotoxicity of (a) and the inhibition rate of HBeAg in the supernatant,

FIG. 1C is a graph showing the real-time quantitative PCR results of total HBV DNA in suppressor cells in which Evans blue can be dose-dependently,

FIG. 1D is a dose-dependent inhibition of myr-preS1-FITC binding to Huh7D by Evans blue^hNTCPOn the surface of the cell, the cell surface,

FIG. 1E is a graph of the results of dose-dependent inhibition of HBV infection of human primary hepatocytes by Evans blue,

FIG. 1F is a graph of results of porcine primary hepatocytes stably expressing human NTCP protein by dose-dependent inhibition of HBV infection by Evans blue;

FIG. 2 is a graph showing the results of Evans blue affecting the assembly of HBV viral particles according to example 2 of the present invention;

wherein FIG. 2A shows cytotoxicity and antigen levels of HepAD38 cells treated with Evans blue for 4 days,

FIG. 2B shows the effect of Evans blue on intracellular HBV total DNA,

FIG. 2C shows the effect of Evans blue on intracellular HBV nucleocapsid (HBV capsid) and total DNA of nucleocapsid;

FIG. 2D shows the effect of Evans blue on the expression level of HBV core protein.

Detailed Description

The following examples of the present invention will be described in detail with reference to specific examples, which are provided for illustration of the present invention and should not be construed as limiting the scope of the present invention. The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruke et al, Huang Petang et al) or according to product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.