CN112294834B

CN112294834B - Application of decitabine in preparing medicament for treating herpes simplex virus

Info

Publication number: CN112294834B
Application number: CN202011353558.XA
Authority: CN
Inventors: 任哲; 郑楷; 王一飞; 肖吉
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2020-11-26
Filing date: 2020-11-26
Publication date: 2023-05-12
Anticipated expiration: 2040-11-26
Also published as: CN112294834A

Abstract

The invention provides application of decitabine in preparing a medicament for treating herpes simplex virus, relates to the technical field of medicaments, and is proved by a large number of experiments that the decitabine can realize the effect of preventing and/or treating the herpes simplex virus by regulating and controlling the expression of interferon, in particular up-regulating cGAS, IFI16, ifnb and ISG 15. Based on the above, the invention expands the medical field application of decitabine, not only can treat various malignant hematopathy, but also can inhibit herpes simplex virus, and is used for preparing medicaments for preventing and/or treating the herpes simplex virus.

Description

Application of decitabine in preparing medicament for treating herpes simplex virus

Technical Field

The invention relates to the technical field of medicines, in particular to application of decitabine in preparation of a medicament for treating herpes simplex virus.

Background

Herpes simplex virus is a common virus that severely threatens human health and is associated with a variety of diseases such as encephalitis, gingivitis, keratitis, and the like. Herpes viruses are susceptible to epithelial tissue, can remain in the human body for a long period of time after primary infection, even remain latent in the human body for a long period of time, resulting in a lifelong infection, and form a "dominant infection-latent infection-reactivation" cycle. In 2015, world Health Organization (WHO) first estimated infection with herpes simplex virus type 1 (HSV-1) worldwide, and more than 37.09 million people worldwide (aged 0-49 years) had been infected with HSV-1. Herpes Simplex Encephalitis (HSE) is the most common viral infectious disease of the central nervous system, with HSE morbidity accounting for approximately 20% of encephalitis morbidity, 90% of HSE being caused by HSV-1, with mortality rates as high as 60-70% without effective therapeutic intervention.

At present, the clinical medicines for treating herpes simplex virus are mainly nucleoside medicines and analogues thereof, such as Acyclovir (ACV), ganciclovir, valacyclovir and the like, and the action mechanism of the medicines is to influence the replication of viral genome by competing with normal deoxyribonucleoside triphosphate. However, since therapeutic agents are single and most of such agents have mutagenicity, ACV-resistant virus strains have appeared and become more serious clinically since the beginning of the use of ACV in 1960, and finding an effective and drug is a problem to be solved urgently by researchers in the field.

Decistabine (Decistabine) is a 2' -deoxycytidine analog that, upon phosphorylation by deoxycytidine kinase, is metabolically converted to the active nucleotide and incorporated into DNA. Decistabine forms a covalent complex with DNA methyltransferase Dnmt1, thereby depleting the enzymatic activity of the cell, inhibiting the function of DNA methyltransferase, and reducing the degree of methylation of DNA. In vitro studies have shown that Decistabine, while inhibiting DNA methylation, does not affect DNA synthesis and that non-proliferating cells are also insensitive to Decistabine. Decistabine has obvious curative effects on various malignant hematopathy including MDS, AML and chronic granulocytic leukemia. In 4 and 5 2006, european EMEA and U.S. FDA approved Decistabine are marketed for the treatment of MDS. In 2009, decistabine was approved in China for the treatment of MDS,2012, and the European Union approves of Decistabine for the treatment of primary or secondary AML patients diagnosed for the first time, aged 65 years and older.

However, the correlation between decitabine and herpes simplex virus infection has not been reported.

In view of this, the present invention has been made.

Disclosure of Invention

The main purpose of the invention is to provide the application of the decitabine in preparing medicaments for preventing and/or treating herpes simplex virus so as to expand the medical field application of the decitabine.

The invention provides application of decitabine in preparing a medicament for preventing and/or treating herpes simplex virus.

Further, the herpes simplex virus includes a type I herpes simplex virus or a type II herpes simplex virus.

Further, the herpes simplex virus comprises acyclovir resistant virus strains.

Further, the acyclovir resistant virus strain comprises a herpes simplex virus acyclovir resistant strain of type I.

Further, the decitabine plays a role in preventing and/or treating herpes simplex virus by regulating the expression of interferon.

Further, the medicine also comprises pharmaceutically acceptable auxiliary materials.

Further, the dosage form of the medicine comprises an oral preparation or an injection preparation.

Further, the oral preparation comprises a tablet, a capsule, a granule, a pill, a syrup, an oral solution, an oral suspension or an oral emulsion.

Further, the injection preparation comprises injection or powder injection.

Further, the drug was administered at a dose of 10. Mu.M.

Compared with the prior art, the invention has the following beneficial effects:

a large number of experiments prove that the decitabine can realize the effect of preventing and/or treating the herpes simplex virus by regulating and controlling the expression of interferon, in particular up-regulating cGAS, IFI16, ifnb and ISG 15. Based on the above, the invention expands the medical field application of decitabine, not only can treat various malignant hematopathy, but also can inhibit herpes simplex virus, and is used for preparing medicaments for preventing and/or treating the herpes simplex virus.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1A is a graph showing the results of plaque size for the different treatment groups of cells provided in example 1 of the present invention;

FIG. 1B is a graph showing the statistics of the relative plaque areas of the cells of the different treatment groups provided in example 1 of the present invention;

FIG. 1C is a graph showing the statistics of the relative plaque numbers of cells of different treatment groups provided in example 1 of the present invention;

FIG. 1D is a graph showing the results of the number of plaques in cells from different treatment groups provided in example 1 of the present invention;

FIG. 2A is a graph showing the results of inhibiting the formation of viral plaques by adding 10. Mu.M Decistabine at various time points after infection with a virus according to example 2 of the present invention;

FIG. 2B is a graph showing the inhibition of virus by 10. Mu.M Decistabine added at various time points after infection with the virus according to example 2 of the present invention;

FIG. 3A is a graph showing the effect of different treatment groups on virus titer provided in example 3 of the present invention;

FIG. 3B is a graph showing the effect of different treatment groups on viral genomic DNA copy number provided in example 3 of the present invention;

FIG. 4 is a graph showing the effect of Western Blot detection of different treatment groups on viral protein content provided in example 4 of the present invention;

FIG. 5A is a graph showing the effect of different concentrations of Decistabine treatment groups on the plaque formation of ACV drug-resistant strain Blue strain provided in example 5 of the present invention;

FIG. 5B is a statistical graph showing the effect of different concentration Decistabine treatment groups on the plaque formation of ACV drug-resistant strain Blue strain provided in example 5 of the present invention;

FIG. 5C is a graph showing the effect of treatment groups of Decistabine at different concentrations on the plaque formation in ACV-resistant strain 153 strain provided in example 5 of the present invention;

FIG. 5D is a statistical graph showing the effect of different concentration Decistabine treatment groups on the plaque formation of ACV drug-resistant strain 153 strain virus provided in example 5 of the present invention;

FIG. 6 is a graph showing the effect of Decistabine on the expression of interferons cGAS, IFI16, ifnb and ISG15 provided in example 6 of the present invention.

Detailed Description

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms should be clear, however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extraneous definition. In the present application, the use of the term "including" and other forms is non-limiting unless otherwise specified.

Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally well known in the art and are performed according to conventional methods as described in various general and more specific references cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. Nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques therefor, are those well known and commonly employed in the art.

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

According to one aspect of the present invention there is provided the use of decitabine for the manufacture of a medicament for the prophylaxis and/or treatment of herpes simplex virus.

In the present invention, "preventing and/or treating" means that decitabine can be used for preventing and treating herpes simplex virus, or can be used for preventing and treating herpes simplex virus.

The invention does not limit the specific type of the herpes simplex virus, and all strains belonging to the herpes simplex virus are within the protection scope of the invention. Preferably, decitabine has a better antiviral effect when the subject is a type I herpes simplex virus.

Based on a large number of acyclovir resistant strains caused by the treatment of herpes simplex viruses by clinically using acyclovir at present, the invention discovers that decitabine has good inhibition effect on acyclovir resistant strains and can greatly improve the treatment effect of herpes simplex viruses.

Furthermore, in some embodiments of the invention, a novel mechanism of action of decitabine to inhibit herpes virus infection is also found: the effect of inhibiting virus proliferation is exerted by regulating the expression of interferon. The new action mechanism is completely different from the action mechanism of the traditional nucleoside analogues and decitabine in treating MDS, and provides a new strategy for developing antiviral drugs.

In some preferred embodiments, the medicament further comprises a pharmaceutically acceptable excipient.

Pharmaceutically acceptable excipients refer to excipients and additives used in the manufacture of medicaments and formulation prescriptions, and refer to substances which, apart from the active ingredient, have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations. The same medicinal auxiliary material can be used for medicinal preparations with different administration routes, and has different effects and uses. The pharmaceutically acceptable auxiliary materials added into the medicine provided by the invention can play roles in shaping, serving as a carrier or improving stability, and further have important functions of solubilization, dissolution assistance or sustained and controlled release and the like.

Typical, but non-limiting, pharmaceutically acceptable excipients include: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, antioxidants, chelating agents, permeation enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, or release retarders.

In some preferred embodiments, the dosage form of the medicament comprises an oral formulation or an injectable formulation.

When administered orally, the medicament may be in any orally acceptable formulation, such as, but not limited to, tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions or oral emulsions.

Wherein the carrier used for the tablet generally comprises lactose and corn starch, and optionally a lubricant such as magnesium stearate. Diluents used in capsules generally include lactose and dried corn starch. Oral suspensions are typically prepared by mixing the active ingredient with suitable emulsifying and suspending agents.

Optionally, some sweetener, flavoring agent or coloring agent can be added into the oral preparation.

When administered in an injectable form, the above-described drugs may be formulated into any injectable acceptable formulation, for example, but not limited to, injectable solutions or powder injections.

Among the carriers and solvents that can be used are water, ringer's solution and isotonic sodium chloride solution. In addition, the sterilized fixed oils may also be used as solvents or suspending media, such as mono-or diglycerides.

Preferably, the drug is administered at a dose of 10. Mu.M.

The technical solution and advantageous effects of the present invention are further described below in connection with preferred embodiments.

Unless otherwise specified, "virus" in examples 1-4, 6 of the present invention refers to HSV-1.

EXAMPLE 1 in vitro anti-inhibitory viral plaque size and plaque number of decitabine

Will be 2X 10 ⁵ Vero (purchased from ATCC) was inoculated into 12-well plates and the plates were placed in 5% CO ₂ The cells were cultured overnight at 37℃in a cell culture incubator. Cells were divided into virus control groups, decistabine (purchased from Selleck corporation, dissolved in DMSO to prepare 50mM, and the virus groups (10. Mu.M and 50. Mu.M) were treated with DMEM basic (1X) medium (Gibco corporation) diluted to working fluid concentration),after the Decistabine and the virus (HSV-1/F) are treated for 2 hours, the virus covering liquid containing the corresponding Decistabine concentration is changed for continuous culture for 72 hours, the size of the plaques is observed, the virus covering liquid is discarded, after the bacteria covering liquid is fixed by 4% paraformaldehyde, crystal violet is dyed, and the size of the plaques and the number of the plaques of the viruses are observed and counted.

The results are shown in FIGS. 1A and 1B, in which the Decistabine treated virus group (10. Mu.M and 50. Mu.M) significantly inhibited the size of viral plaques, and in which the Decistabine treated virus group (10. Mu.M and 50. Mu.M) significantly inhibited the number of viral plaques, as compared to the virus control group, as shown in FIGS. 1C and 1D, indicating that Decistabine is capable of inhibiting viral replication.

Example 2 Decistabine inhibits viral plaque formation at various time points after viral infection

Will be 1.6X10 ⁵ Vero was inoculated into 24-well plates and the plates were placed in 5% CO ₂ The cells were cultured overnight at 37℃in a cell culture incubator. Dividing cells into virus control group, treating the virus group with Decistabine, and changing into virus covering solution containing Decistabine concentration of corresponding 10 μm at 0h,2h,4h,6h,8h after virus infection, and placing into 5% CO ₂ The cell culture is continued for 72 hours at 37 ℃, the size of the plaques is observed, the virus covering liquid is discarded, after the virus covering liquid is fixed by 4% paraformaldehyde, crystal violet is dyed, and the size of the plaques and the number of the plaques are observed and counted.

As shown in fig. 2A and 2B, treatment of the viroset with 10 μm Decistabine significantly inhibited the number of viral plaques formed compared to the viroset.

Example 3 Decistabine inhibits replication of HSV-1

Will be 2X 10 ⁵ Vero was inoculated in 6-well plates and the plates were placed in 5% CO ₂ The cells were cultured overnight at 37℃in a cell culture incubator. The virus control group and Decistabine-treated virus group at concentrations of 2. Mu.M, 10. Mu.M and 50. Mu.M, respectively, were set, and after 24 hours of virus infection, freeze thawing was repeated 3 times, and then virus TICD50 was detected, respectively.

As shown in fig. 3A, the viral titers of the 2 μm, 10 μm and 50 μm Decistabine treated virosomes were significantly reduced compared to the virosomes, indicating that such drugs have an anti-HSV-1 replicating effect.

Will be 2X 10 ⁵ Vero was inoculated in 6-well plates and the plates were placed in 5% CO ₂ The cells were cultured overnight at 37℃in a cell culture incubator. The virus control group and Decistabine-treated virus group at concentrations of 2. Mu.M, 10. Mu.M and 50. Mu.M, respectively, were set, and after 24 hours of virus infection, DNA was extracted and the viral DNA copy number was measured.

As shown in fig. 3B, 2 μΜ,10 μΜ, and 50 μΜ of Decitabine treated virosomes significantly reduced viral copy number compared to virosomes, indicating that such drugs have anti-HSV-1 genomic replication effects.

Example 4 Decistabine is capable of inhibiting expression of HSV-1 viral proteins

Will be 2X 10 ⁵ Vero was inoculated in 6-well plates and the plates were placed in 5% CO ₂ The cells were cultured overnight at 37℃in a cell culture incubator. Setting a cell control group, treating the virus group with the virus control group and Decistabine with the concentration of 0.16 mu M, 0.8 mu M, 4 mu M, 20 mu M and 100 mu M respectively, collecting cells after virus infection for 24 hours, extracting total proteins, and detecting the change of the content of the viral proteins by Western Blot.

As shown in FIG. 4, decistabine treatment of the virosomes at concentrations of 0.16 μM, 0.8 μM, 4 μM, 20 μM and 100 μM, respectively, reduced the viral protein gB and VP5 levels in a concentration-dependent manner compared to the viral control, demonstrating that Decitabine is capable of inhibiting HSV-1 infection.

Example 5 Decistabine is capable of inhibiting infection with acyclovir-resistant Virus strains

Will be 2X 10 ⁵ Vero was inoculated into 12-well plates and the plates were placed in 5% CO ₂ The cells were cultured overnight at 37℃in a cell culture incubator. Dividing cells into an ACV drug-resistant virus Blue virus strain control group and an ACV drug-resistant virus 153 virus strain control group, treating the ACV drug-resistant virus Blue virus strain and the ACV drug-resistant virus 153 virus strain group (2 mu M, 10 mu M and 50 mu M), after the combination of the Decistabine and the virus is treated for 2 hours, changing into a virus covering solution containing the corresponding Decistabine concentration, continuously culturing for 72 hours, observing the size of plaques, discarding the virus covering solution, fixing with 4% paraformaldehyde, staining with crystal violet, and observing and counting the number of the plaques of the viruses.

The results are shown in FIGS. 5A, 5B, 5C and 5D, where Decistabine treatment of the virosomes (2 μM, 10 μM and 50 μM) significantly inhibited the number of viral plaques, indicating that Decitabine is able to inhibit infection of acyclovir-resistant strains.

EXAMPLE 6 Decistabine upregulation of interferon-related Gene expression

Will be 2X 10 ⁵ Mouse macrophages Raw264.7 were seeded in 6-well plates and the plates were placed in 5% CO ₂ The cells were cultured overnight at 37℃in a cell culture incubator. Dividing the cells into a virus control group and a 10 mu M Decistabine treatment virus group, changing into a virus maintenance solution containing 10 mu M Decistabine concentration after the Decistabine and the virus are treated for 2 hours, continuously culturing for 4 hours, extracting RNA, and detecting the expression of interferon related pathway genes cGAS, IFI16, ifnb and ISG15 by qPCR.

The results are shown in FIG. 6, where 10. Mu.M Decistabine treated virus group, the expression of cGAS, IFI16, ifnb and ISG15 genes was significantly up-regulated compared to the virus control group, probably as a regulatory mechanism for Decistabine antiviral.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. Use of decitabine for the preparation of a medicament for the prevention and/or treatment of herpes simplex virus; the herpes simplex virus is a type I herpes simplex virus acyclovir drug resistant strain.

2. The use according to claim 1, wherein decitabine acts as a prophylactic and/or therapeutic agent for herpes simplex virus by modulating the expression of interferon.

3. The use according to claim 1, wherein the medicament further comprises pharmaceutically acceptable excipients.

4. The use according to claim 1, wherein the dosage form of the medicament comprises an oral formulation or an injectable formulation.

5. The use according to claim 4, wherein the oral formulation is selected from the group consisting of tablets, capsules, granules, pills and syrups.

6. The use according to claim 4, wherein the injectable formulation comprises an injectable solution or a powder injection.