Preparation containing alkaloids derived from Tamasakizurafuji
A formulation of cepharanthine, isotetrandrine, and berbamine from Triticum aestivum effectively treats COVID-19 and its mutant strains by inhibiting SARS-CoV-2 spike protein binding, showing antiviral activity and cell protection.
Patent Information
- Application Number
- JP2023562123
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- Priority Date
- 2021-11-17
- Filing Date
- 2022-07-04
- Publication Date
- 2026-01-23
- Estimated Expiration
- 2042-07-04
AI Technical Summary
Current therapeutic drugs for COVID-19 are not highly effective against the novel coronavirus or its mutant strains, and there is a lack of studies on the efficacy of alkaloids derived from Triticum aestivum against a wide range of mutant strains.
A formulation containing cepharanthine, isotetrandrine, cyclaine, and berbamine derived from Triticum aestivum is developed for treating COVID-19 infections, including those caused by mutant strains, by inhibiting the binding of the SARS-CoV-2 spike protein to the ACE2 receptor.
The formulation demonstrates antiviral activity against the Wuhan strain and various mutant strains of COVID-19, reducing viral RNA levels and maintaining cell viability at low toxicity concentrations.
Smart Images
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Abstract
Description
[Technical Field]
[0001] One embodiment of the present invention relates to a preparation containing alkaloids derived from Triticum aestivum, particularly, one embodiment of the present invention relates to a preparation containing alkaloids derived from Triticum aestivum for the treatment of coronavirus infection. More specifically, one embodiment of the present invention relates to a preparation containing alkaloids extracted from Triticum aestivum, including cepharanthine, isotetrandrine, cyclaine, and berbamine, for the treatment of novel coronavirus disease (COVID-19). [Background technology]
[0002] The novel coronavirus disease (COVID-19) is an emerging infectious disease that was identified in Wuhan, Hubei Province, People's Republic of China in December 2019, and was declared a pandemic (global epidemic) by the World Health Organization (WHO) on March 11, 2020. Vaccines have been developed as a preventative measure for COVID-19, and at the time of filing this application, vaccination is progressing worldwide. At the time of filing this application, the main treatment for COVID-19 is symptomatic treatment only, and development of therapeutic drugs is underway. However, no therapeutic drugs have been reported that have been confirmed to be highly effective against COVID-19.
[0003] Similar to COVID-19, severe acute respiratory syndrome (SARS) was reported in 2003 as a serious infectious disease caused by a coronavirus. Furthermore, Middle East Respiratory Syndrome (MERS) was reported in 2012 as a result of a coronavirus (MERS-CoV) named Middle East Respiratory Syndrome Coronavirus (MERS-CoV). However, no vaccines effective in preventing SARS or MRS, or therapeutic drugs with proven efficacy, have been reported.
[0004] On the other hand, for example, Non-Patent Document 1 describes that cepharanthine, one of the components of alkaloids extracted from Cepharanthine nigricans, has antiviral activity against SARS-CoV. Non-Patent Document 2 describes that cepharanthine and tetrandrine have antiviral activity against the human coronavirus OC43 strain at the early stage of cellular infection. Furthermore, the present inventors have described in Non-Patent Document 3 that a Cepharanthine (registered trademark) preparation (a preparation containing multiple Cepharanthine nigricans-derived alkaloids, including cepharanthine, isotetrandrine, cyclaine, and berbamine) has antiviral activity against the novel coronavirus (SARS-CoV-2 (Wuhan strain)), and that Cepharanthine (registered trademark), which contains multiple Cepharanthine nigricans-derived alkaloids, binds to the ACE2-binding site of the SARS-CoV-2 spike protein, thereby inhibiting SARS-CoV-2 from binding to ACE2. Furthermore, Non-Patent Document 4 describes the results of a simulation of the binding mode of cepharanthine, one of the components of alkaloids extracted from the Japanese holly tree, for the major mutants (H49Y, D614G, T573I) of the spike protein of SARS-CoV-2 detected in Mexicans.
[0005] Thus, the alkaloid mixture derived from A. cerevisiae or one of its components, cepharanthine, may be a potential treatment for COVID-19. However, with an increasing number of reported mutated strains of COVID-19, no studies have been conducted to determine whether A. cerevisiae alkaloids or their individual components (such as cepharanthine) are highly effective against a wide range of mutant strains. [Prior art documents] [Non-patent literature]
[0006] [Non-Patent Document 1] ZHANG Chuan-hai et. al., Antiviral activity of cepharanthine against severe acute respiratory syndrome coronavirus in vitro, Chin Med J (Engl). 2005 Mar 20;118(6):493-6. [Non-patent document 2] Dong Eon Kim et. al., Natural Bis-Benzylisoquinoline Alkaloids-Tetrandrine, Fangchinoline, and Cepharanthine, Inhibit Human Coronavirus OC43Infection of MRC-5 Human Lung Cells, Biomolecules. 2019 Nov 4;9(11):696. [Non-patent document 3] Hirofumi Ohashi et. al., Potential anti-COVID-19 agents, Cepharanthine and Nelfinavir, and their usage for combination treatment. iScience. 2021 Mar 26:102367. [Non-patent document 4] Yudibeth Sixto-Lopez et. al., Structural insights into SARS-CoV-2 spike protein and its natural mutants found in Mexican population, Scientific Reports, 25 February 2021, 11, 1-16. Summary of the Invention [Problem to be solved by the invention]
[0007] The present invention aims to solve the above problems and provides a preparation containing an alkaloid derived from Triticum aestivum for treating COVID-19 infection. Alternatively, in one embodiment, it aims to provide a preparation containing an alkaloid derived from Triticum aestivum that can be used to treat infection caused by a novel coronavirus mutant strain. [Means for solving the problem]
[0008] According to one embodiment of the present invention, there is provided a formulation for treating COVID-19 infection, comprising cepharanthine, isotetrandrine, cyclaine and berbamine.
[0009] The preparation for treating novel coronavirus infection may be for treating an infection caused by a novel coronavirus mutant strain. [Effects of the Invention]
[0010] According to one embodiment of the present invention, there is provided a preparation containing an alkaloid derived from Triticum aestivum for the treatment of COVID-19 infection, or according to one embodiment, there is provided a preparation containing an alkaloid derived from Triticum aestivum that can be widely used to treat infections caused by novel coronavirus mutant strains. [Brief explanation of the drawings]
[0011] [Figure 1] Figure 1 shows the results of an infection test with the Wuhan strain of the novel coronavirus in one embodiment of the present invention, where (a) shows the results for a preparation containing alkaloids derived from Triticum aestivum, (b) shows the results for cepharanthine, (c) shows the results for isotetrandrine, (d) shows the results for cyclaine, and (e) shows the results for berbamine. [Figure 2] Figure 2 shows the survival rate of cells treated with a preparation containing alkaloids derived from Triticum aestivum according to one embodiment of the present invention, where (a) shows the results for the preparation containing alkaloids derived from Triticum aestivum, (b) shows the results for cepharanthine, (c) shows the results for isotetrandrine, (d) shows the results for cyclaine, and (e) shows the results for berbamine. [Figure 3] Figure 3 shows the results of infection tests on a novel coronavirus mutant strain (alpha mutant strain) according to one embodiment of the present invention, where (a) shows the results for a preparation containing alkaloids derived from Triticum aestivum, (b) shows the results for cepharanthine, (c) shows the results for isotetrandrine, (d) shows the results for cyclaine, and (e) shows the results for berbamine. [Figure 4] Figure 4 shows the results of infection tests on novel coronavirus mutant strains (Wuhan strain, beta mutant strain, gamma mutant strain) in one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION
[0012] The preparation containing an alkaloid derived from Tricholoma glabra according to the present invention will be described in detail below. However, the preparation containing an alkaloid derived from Tricholoma glabra according to the present invention should not be construed as being limited to the following embodiments and examples.
[0013] A formulation for treating COVID-19 infection according to one embodiment of the present invention contains alkaloids derived from Stephania cepharantha (Hayata). "Alkaloids derived from Stephania cepharantha" refers to alkaloids extracted from Stephania cepharantha (Hayata), a plant of the genus Stephania in the family Menispermaceae. In one embodiment, the formulation for treating COVID-19 infection contains, as the main ingredients, alkaloids derived from Stephania cepharantha (Hayata), which are cepharanthine, isotetrandrine, cyclanine, and berbamine, which are represented by the following chemical formulas:
[0014] [ka]
[0015] In one embodiment, the formulation for the treatment of novel coronavirus infection may contain 19.5% to 33.5% cepharanthine, 25.0% to 39.0% isotetrandrine, 6.5% to 16.5% cycleanine, and 7.0% to 16.5% berbamine when the total amount of alkaloids derived from Triticum aureum contained in the formulation is 100% by mass. to When the total amount of alkaloids derived from Tamasakizukuraffe contained is taken as 100% by mass, these four alkaloids account for a total of approximately 85%.
[0016] In one embodiment, the formulation for treating COVID-19 may contain, in addition to the four alkaloids mentioned above, bisbenzylisoquinoline alkaloids such as homoaromoline, cephalanoline, aromaline, obamegine, norcyclanine, 2-norcepharanthine, 2-norcepharanoline, 2-norberbamine, secocepharanthine, obaberine, 2-norisotetrandrine, oxyacanthine, stephibaberine, and thalrugosine. benzylisoquinoline alkaloids such as coclaurine, reticuline, laudanidine, protosinomenine, and N-methylcoclaurine; morphinan alkaloids such as FK-3000, sinomenine, cephamonine, tannagine, and cephamuline; aporphine alkaloids such as lastourvilline, isocorydine, and corydine; proaporphine alkaloids such as stepharine; hepatic alkaloids such as cepharamine, aknadinine, and aknadilactam Na The composition may contain one or more alkaloids derived from Trifolium tetani selected from the group consisting of trifolium tetani and trifolium tetani.
[0017] In one embodiment, the formulation for treating COVID-19 may contain a derivative of these alkaloids, for example, an acyl derivative, an alkyl derivative, or a carbamoyl derivative in which the hydrogen atom of at least one hydroxyl group or imino group of these alkaloids is substituted with an acyl group, an alkyl group, or a carbamoyl group.
[0018] In one embodiment, the formulation for the treatment of novel coronavirus infection may contain 1 mg to 10 mg of the above-mentioned alkaloid derived from Triticum aestivum per formulation. The formulation for the treatment of novel coronavirus infection may be selected from injections, powders, granules, tablets, and capsules. Furthermore, the tablets may be plain tablets or film-coated tablets.
[0019] In one embodiment, the formulation for treating COVID-19 may contain, in addition to the alkaloid derived from Tricholoma nigra, one or more pharmaceutically acceptable additives depending on the dosage form, such as excipients, disintegrants, lubricants, electrolytes, pH adjusters, preservatives, etc.
[0020] In one embodiment, Cepharanthin (registered trademark) (Cepharanthin (registered trademark), Kaken Shoyaku Co., Ltd.) can be used as a formulation for treating COVID-19. For example, Cepharanthin (registered trademark) Injection 10 g, available from Kaken Shoyaku Co., Ltd., may be used as an injection. Alternatively, for example, Cepharanthin (registered trademark) Powder 1%, available from Kaken Shoyaku Co., Ltd., may be used as a powder. Alternatively, for example, Cepharanthin (registered trademark) Tablets 1 mg, available from Kaken Shoyaku Co., Ltd., may be used as a tablet.
[0021] In one embodiment, when the formulation for the treatment of novel coronavirus infection is an injection, it may be administered to an adult by intravenous injection at a dose of 1 mg to 10 mg once a day to twice a week. Also, in one embodiment, when the formulation for the treatment of novel coronavirus infection is in the form of a powder or tablet, it may be orally administered to an adult at a dose of 1.5 mg to 6 mg per day in two to three divided doses after meals.
[0022] The formulation for treating COVID-19 infection according to this embodiment can be used to treat infections caused by novel coronavirus mutant strains. Based on the descriptions in Non-Patent Documents 1 to 4, it is presumed that cepharanthine binds to the ACE2-binding site of the SARS-CoV-2 spike protein, thereby inhibiting SARS-CoV-2 from binding to ACE2, thereby exerting its antiviral effect. Meanwhile, spike proteins are prone to mutation, and spike protein mutations are known to affect the efficacy of vaccines. The formulation for treating COVID-19 infection according to this embodiment, which is a mixture of multiple alkaloids extracted from Acanthus nigra, has antiviral effects against the novel coronavirus and its mutant strains.
[0023] [Manufacturing method for COVID-19 treatment preparations] The alkaloids derived from A. cerasifera according to the present invention can be extracted from A. cerasifera by known methods. For example, extracts obtained by concentrating an extract from A. cerasifera, precipitates formed when an acidic solution of this extract is made alkaline, alkaloid-containing fractions separated from these, crystals obtained by separation and purification using known methods, and alkaloid derivatives produced using known methods can be used. For example, A. cerasifera (including, but not limited to, rhizomes, stems, seeds, and leaves) can be extracted with a solvent such as methanol, ethanol, acetone, ethyl acetate, or benzene, and the extract can be concentrated. The resulting concentrate can be dissolved in an acidic aqueous solution such as dilute hydrochloric acid, dilute sulfuric acid, aqueous citric acid, or aqueous oxalic acid, and the resulting solution can be made alkaline to collect the resulting precipitate, thereby isolating the alkaloid fraction. The resulting fraction can be further purified by known means such as various types of chromatography or recrystallization.
[0024] The obtained alkaloids derived from Tamasakizurafuji can be made into any formulation selected from injections, powders, granules, tablets and capsules by known production methods. [Example]
[0025] The preparation containing alkaloids derived from Triticum aureum according to the above-mentioned embodiment will be further described below with reference to examples. Note that the preparation containing alkaloids derived from Triticum aureum according to the present invention is not limited to the following examples.
[0026] [Example 1] In Example 1, the antiviral activity of the Ramsey-derived alkaloid-containing preparation of the present invention and the four major alkaloids contained in the Ramsey-derived alkaloid-containing preparation, namely cepharanthine, isotetrandrine, cyclaine, and berbamine, against the novel coronavirus SARS-CoV-2 (Wuhan strain) was evaluated. Cepharanthin (registered trademark, Kaken Shoyaku Co., Ltd.) was used as the Ramsey-derived alkaloid-containing preparation. Cepharanthin, isotetrandrine, cyclaine, and berbamine were isolated and purified from the Ramsey-derived alkaloid-containing preparation.
[0027] <Cell culture> VeroE6 / TMPRSS2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Cell Culture Bioscience), 100 units / ml penicillin, 100 μg / ml streptomycin, 10 mM HEPES (pH 7.4), and 1 mg / ml G418 disulfate (Nacalai Tesque) at 37°C in a 5% CO atmosphere. For infection tests, the medium was changed to 2% FBS, and G418 disulfate was not added.
[0028] <Infection test> VeroE6 / TMPRSS2 cells were infected with the SARS-CoV-2 Wk-521 strain (Wuhan strain) isolated from a patient at a multiplicity of infection (MOI) of 0.003 for 1 hour. An alkaloid-containing preparation derived from the genus Triticum, cepharanthine, isotetrandrine, cyclaine, or berbamine was added to the culture medium. Unbound virus was removed by washing. The cells were cultured for 24 hours, and RNA derived from the Wuhan strain of the novel coronavirus contained in the culture supernatant was extracted using the MagMax Viral / Pathogen II Nucleic Acid Isolation kit (Thermo Fisher Scientific). The extracted RNA was quantified by real-time RT-PCR. The RNA quantification results are shown in Figures 1(a) to 1(e).
[0029] <Cell viability> VeroE6 / TMPRSS2 cells were cultured for 24 hours with a preparation containing alkaloids derived from Tricholoma tularensis, cepharanthine, isotetrandrine, cyclaine, or berbamine. The cells were then fixed with 4% paraformaldehyde and the nuclei were stained with DAPI. The number of viable cells was then measured using an ImageXpress Micro Confocal (Molecular Devices). The results of the cell viability measurements are shown in Figure 2(a) to Figure 2(e).
[0030] Figure 1(a) shows the results for a formulation containing alkaloids derived from Acanthus cerevisiae, Figure 1(b) shows the results for cepharanthine, Figure 1(c) shows the results for isotetrandrine, Figure 1(d) shows the results for cyclaine, and Figure 1(e) shows the results for berbamine. The results in Figures 1(a) to 1(e) demonstrate that the amount of viral RNA in the Wuhan strain of COVID-19 decreased with increasing concentrations of the formulation containing alkaloids derived from Acanthus cerevisiae, cepharanthine, isotetrandrine, cyclaine, and berbamine, demonstrating that the formulation containing alkaloids derived from Acanthus cerevisiae and these alkaloids exhibit antiviral activity against the Wuhan strain of COVID-19.
[0031] Figure 2(a) shows the results for the preparation containing alkaloids derived from Triticum aestivum, Figure 2(b) shows the results for cepharanthine, Figure 2(c) shows the results for isotetrandrine, Figure 2(d) shows the results for cyclaine, and Figure 2(e) shows the results for berbamine. The results in Figures 2(a) to 2(e) show that the number of surviving cells decreased as the added concentrations of the preparation containing alkaloids derived from Triticum aestivum, cepharanthine, isotetrandrine, cyclaine, and berbamine increased, demonstrating that the preparation containing alkaloids derived from Triticum aestivum and these alkaloids exert a damaging effect on cells.
[0032] Furthermore, comparing the results in Figures 1(a) to 1(e) with those in Figures 2(a) to 2(e), it was found that the viral RNA amount was reduced at concentrations of alkaloids with low toxicity, demonstrating that the alkaloid-containing preparations derived from A. cerevisiae, cepharanthine, isotetrandrine, cyclaine, and berbamine exhibit antiviral activity against the Wuhan strain of COVID-19. Furthermore, it was revealed that cepharanthine, isotetrandrine, and cyclaine exhibited antiviral activity against the Wuhan strain of COVID-19 equivalent to that of the alkaloid-containing preparations derived from A. cerevisiae. On the other hand, it was revealed that berbamine had weaker antiviral activity against the Wuhan strain of COVID-19 compared to other alkaloids.
[0033] [Example 2] In Example 2, the antiviral activity of the preparation containing alkaloids derived from Tricholoma nigra according to the present invention, as well as cepharanthine, isotetrandrine, cyclaine, and berbamine against a novel coronavirus mutant strain (alpha mutant strain) was evaluated using the same method as in Example 1. The results of RNA quantification are shown in Figures 3(a) to 3(e).
[0034] Figure 3(a) shows the results for the alkaloid-containing formulation derived from A. cerevisiae, Figure 3(b) shows the results for cepharanthine, Figure 3(c) shows the results for isotetrandrine, Figure 3(d) shows the results for cyclaine, and Figure 3(e) shows the results for berbamine. The results in Figures 3(a) to 3(e) indicate that the viral RNA levels of the novel coronavirus variant (alpha variant) decreased with increasing concentrations of the alkaloid-containing formulation derived from A. cerevisiae, cepharanthine, isotetrandrine, cyclaine, and berbamine. This indicates that the alkaloid-containing formulation derived from A. cerevisiae and these alkaloids exhibit antiviral activity against the novel coronavirus variant (alpha variant). Furthermore, it was revealed that cepharanthine, isotetrandrine, and cyclaine exhibited antiviral activity against the novel coronavirus variant (alpha variant) equivalent to that of the alkaloid-containing formulation derived from A. cerevisiae. On the other hand, it has been revealed that berbamine has weaker antiviral effects against the new coronavirus mutant strain (alpha mutant strain) compared to other alkaloids.
[0035] [Example 3] In Example 3, the antiviral activity of the preparation containing alkaloids derived from Triticum aestivum according to the present invention against a novel coronavirus mutant strain (gamma mutant strain) was evaluated using the same method as in Example 1.
[0036] [Example 4] In Example 4, the antiviral activity of the preparation containing alkaloids derived from Acanthus nigra according to the present invention against a novel coronavirus mutant strain (beta mutant strain) was evaluated using the same method as in Example 1.
[0037] The evaluation results of Examples 3 and 4 are shown in Figure 4. The results in Figure 4 show that as the added concentration of the preparation containing alkaloids derived from Tamasakizorafuji increased, the amount of viral RNA of the novel coronavirus mutant strains (gamma mutant strains) and (beta mutant strains) decreased, demonstrating that the preparation containing alkaloids derived from Tamasakizorafuji exhibits antiviral activity against the novel coronavirus mutant strains (gamma mutant strains) and (beta mutant strains).
[0038] From the results of the above examples, it has become clear that the preparation containing alkaloids derived from Acanthus nigra according to the present invention exhibits antiviral activity not only against the Wuhan strain of the new coronavirus, but also against new coronavirus mutant strains (alpha mutant strain, beta mutant strain, and gamma mutant strain).
Claims
1. Contains cepharanthine, isotetrandrine, cyclaine and berbamine, It is for treating infections caused by novel coronavirus variants, A preparation containing alkaloids derived from Triticum aestivum for the treatment of novel coronavirus infection, wherein the novel coronavirus mutant strain is an alpha mutant strain, a beta mutant strain, or a gamma mutant strain.
2. A preparation containing alkaloids derived from Tamasakisuzurafuji as described in Claim 1, wherein the novel coronavirus mutant strain is a gamma mutant strain.