Application of harmine in preparation of antiviral drugs

Abstract

The invention discloses an application of harmine or a pharmaceutical salt thereof in preparation of an antiviral drug. The drug micromolecule harmine hydrochloride screened from a natural product library can significantly inhibit cracking and replication of KSHV, the application of harmine in KSHV virus resistance is reported for the first time, the effective drug concentration is low, high biosafety is achieved, and a new solution is provided for KSHV treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and in particular relates to an application of dehydrogenated harmine in the preparation of antiviral drugs. Background Art

[0002] Herpes virus is a type of virus with an envelope and a double-stranded DNA genome. Currently, there are 8 types of herpes virus that can infect humans, of which KSHV and EBV belong to γ-herpes virus. After the initial infection, the herpes virus enters a latent infection period and is a lifelong carrier. Under certain conditions, the virus is activated and enters a lytic replication period, which can cause tumors and other diseases.

[0003] Kaposi's sarcoma-associated herpesvirus (KSHV) is an important human tumor virus, which is closely related to the occurrence of malignant diseases such as Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). Among them, Kaposi's sarcoma is a common tumor and cause of death in AIDS patients. At present, there is a lack of highly effective antiviral drugs.

[0004] Dehydrogenated harmaline, also known as harmine, rutinine, molecular formula C 13 H 12 N2O, CAS: 442-51-3. Existing literature reports that its pharmacological effects mainly affect the central nervous system, cardiovascular system, muscles, and ion channels, such as radiation protection, anti-inflammatory, analgesic and antipruritic effects, anti-echinococcosis effects, and anti-psoriasis effects. Summary of the invention

[0005] The purpose of the present invention is to provide an application of dehydrogenated harmaline in the preparation of antiviral drugs.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a use of dehydrogenated harmine or a pharmaceutically acceptable salt thereof in the preparation of an antiviral drug.

[0008] In a preferred embodiment of the present invention, the virus is Kaposi's sarcoma-associated herpesvirus (KSHV).

[0009] The pharmaceutically acceptable salt is an acid addition salt formed by dehydrogenated echinopsine and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

[0010] In the application, dehydrogenated echinopsine or its pharmaceutically acceptable salt is used as a single active ingredient.

[0011] The second aspect of the present invention also provides a pharmaceutical preparation, which is prepared from dehydrogenated echinopsine or its pharmaceutically acceptable salt and medically acceptable excipients or other pharmaceutically acceptable ingredients.

[0012] The dosage form of the pharmaceutical preparation is selected from powders, tablets, granules, capsules, solutions, emulsions, and suspensions.

[0013] In a preferred embodiment of the present invention, the pharmaceutically acceptable salt of harmine used is an acid addition salt formed by harmine and hydrochloric acid, i.e., harmine hydrochloride, with a CAS number of 343-27-1 (Liu, H., Han, D., Liu, Y. et al. Harmine hydrochloride inhibits Akt phosphorylation and depletes the pool of cancer stem-like cells of glioblastoma. J Neurooncol 112, 39–48 (2013). https: / / doi.org / 10.1007 / s11060-012-1034-x), and the structure is shown below:

[0014]

[0015] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0016] The drug small molecule harmine hydrochloride screened from 902 natural product libraries in the present invention can significantly inhibit the lysis and replication of KSHV. It is the first report of the application of harmine in the antiviral treatment of KSHV. The drug cytotoxicity test found that 8 μM harmine treated iSLK219 cells still maintained 100% cell activity; the drug IC required for harmine hydrochloride to inhibit KSHV 50 Only 0.1135μM; not only is the effective drug concentration low, but it also has higher biosafety.

[0017] The IC value of ganciclovir, a clinically used anti-herpes virus, against KSHV in the BCBL1 cell model 50 is 5.1μM (Medveczky MM, Horvath E, Lund T, Medveczky PG. In vitro antiviral drugsensitivity of the Kaposi's sarcoma-associated herpesvirus.AIDS.1997Sep;11(11):1327-32.doi:10.1097 / 00002030-199711000-00006.PMID:9302441.).

[0018] The present invention provides a new solution for the treatment of KSHV. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the growth curve results of KSHV in the lytic replication phase in Example 1.

[0020] Figure 2 This is a schematic diagram of the fitting curve results of harmine hydrochloride inhibiting viral replication in Example 1. DETAILED DESCRIPTION

[0021] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0022] The acid addition salt formed by harmine and hydrochloric acid, namely harmine hydrochloride, is used in the embodiment of the present invention and has a CAS number of 343-27-1.

[0023] Example 1

[0024] Cytotoxicity test of dehydrogenated echinopsine hydrochloride

[0025] iSLK.219 cells constructed by Dr. Don Ganem's Lab were used as a cell model for screening KSHV antiviral drugs (Myoung J, Ganem D. Generation of a doxycycline-inducible KSHV producer cellline of endothelial origin: maintenance of tight latency with efficient reactivation upon induction. J Virol Methods. 2011 Jun; 174(1-2): 12-21. doi: 10.1016 / j.jviromet.2011.03.012. Epub 2011 Mar 17. PMID: 21419799; PMCID: PMC3095772.). iSLK.219 cells carry recombinant KSHV.219 virus, and the viral genome contains a constitutive GFP reporter gene and a RFP reporter gene induced by doxycycline (Dox) to monitor the latent infection activity and lytic replication activity of the virus, respectively. Dox and NaB were added to induce iSLK.219 cells to enter the lytic replication state, and the supernatant was collected to detect the number of viral particle copies to detect the viral replication.

[0026] like Figure 1 As shown, Figure 1 This is a schematic diagram of the growth curve results of KSHV in the lytic replication phase in Example 1. As can be seen from the figure, iSLK.219 cells enter the KSHV virus lytic replication state after Dox and NaB induction, and the supernatant virus particles are collected and viral DNA is extracted. It is found that with the extension of the induction time, the replication of KSHV shows an upward trend.

[0027] DMSO was used to dilute dehydrogenated echinopsine hydrochloride to prepare 1000× different drug concentration stock solutions. The working concentrations of dehydrogenated echinopsine hydrochloride were 10μM, 8μM, 6μM, 5μM, 2μM, 1μM, 0.8μM, 0.6μM, 0.4μM, 0.2μM, 0.1μM, 0.01μM, 0.001μM, and 0μM (as control). The drug cytotoxicity test showed that compared with the control group, 8μM dehydrogenated echinopsine treated iSLK219 cells still maintained 100% cell activity.

[0028] Example 2

[0029] Antiviral activity test of dehydrogenated harmaline hydrochloride

[0030] The antiviral activity of dehydrogenated echinopsine against KSHV was further tested. iSLK.219 cells were seeded in 96-well cell plates, with approximately 4 × 10 4 Cells were cultured for 24 hours until the cell density reached 90%, and then replaced with a cell culture medium containing 1 μg / μL DOX and 1 mM NaB. At the same time, the prepared 1000× storage solution was added to the culture medium in proportion and diluted to the set drug working concentration. Culture was continued in a cell culture incubator for 48-72 hours. The 96-well cell culture plate was removed, the cell supernatant was collected, and viral DNA was extracted for qPCR detection. The inhibition ratio of dehydrogenated echinopsine hydrochloride to viral replication was calculated and a curve was drawn to obtain the drug IC 50 It is 0.1135μM.

[0031] The results are as follows Figure 2 As shown, Figure 2 The figure is a schematic diagram of the fitting curve results of the inhibition of virus replication by harmine hydrochloride. Figure 2 It can be seen that as the concentration of dehydrogenated echinopsine hydrochloride increases, the inhibitory effect on KSHV virus replication becomes greater, and the drug concentration required to inhibit the virus by 50% is 0.1135 μM.

[0032] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A use of dehydrogenated echinopsine or a pharmaceutically acceptable salt thereof in the preparation of an antiviral drug.

2. The use according to claim 1, characterized in that: The virus is Kaposi's sarcoma-associated herpes virus.

3. The use according to claim 1, characterized in that: The pharmaceutically acceptable salt is an acid addition salt formed by dehydrogenated echinopsine and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

4. The use according to any one of claims 1 to 3, characterized in that: In the application, dehydrogenated echinopsine or its pharmaceutically acceptable salt is used as a single active ingredient.

5. A pharmaceutical preparation, characterized in that The invention is prepared from dehydrogenated harmaline or its pharmaceutical salt and medically acceptable auxiliary materials or other pharmaceutically acceptable ingredients.

6. The pharmaceutical preparation according to claim 5, characterized in that The dosage form of the pharmaceutical preparation is selected from powders, tablets, granules, capsules, solutions, emulsions, and suspensions.

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