TMPRSS2 inhibitors

Abstract

To exhibit an antiviral action.SOLUTION: An antiviral composition contains extract from plants as an active ingredient. The antiviral composition can be expected to exhibit an antiviral action by inhibiting an activity of TMPRSS2.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention provides TMPRSS2 inhibitors Regarding. [Background technology]

[0002] As disclosed in Patent Document 1, compositions containing extracts from plants are known to be useful for treating viral diseases. For example, Patent Document 1 discloses a composition containing an extract from a plant of the genus Chamomile.

[0003] In recent years, research has been progressing on infectious diseases caused by SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2), a SARS-related coronavirus. Non-Patent Document 1 indicates that when SARS-CoV-2 invades cells, ACE2 (Angiotensin-converting enzyme 2) and TMPRSS2 (Transmembrane protease, serine 2), present on the cell membrane of the host cell, are involved. Specifically, it is described that after the spike protein of the virus binds to the receptor ACE2, the spike protein present on the surface of the virus is cleaved by the activity of TMPRSS2, a type II transmembrane serine protease. It is also described that cleavage of the spike protein promotes the entry of SARS-CoV-2 into host cells. It has also been shown that the entry of SARS-CoV-2 into host cells can be inhibited by camostat, a known serine protease inhibitor.

[0004] Non-Patent Document 2 suggests a relationship between SARS-CoV-2 infection and the oral cavity. Specifically, it has been shown that ACE2 and TMPRSS2 are expressed in the oral cavity, for example, in the tongue, taste buds, and gingival sulcus.

[0005] In addition, cleavage of spike proteins by TMPRSS2 is known to be involved in the infection of not only SARS-CoV-2 but also other coronaviruses. Furthermore, Non-Patent Document 3 indicates that TMPRSS2 is involved in the activation of influenza viruses.

[0006] TMPRSS2 activity may be involved in infection with various viruses, including SARS-CoV-2. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 2011-520867 [Non-patent literature]

[0008] [Non-Patent Document 1] “SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor”, Cell, April 16, 2020, volume181, p.271-280 [Non-patent document 2] "Existence of SARS-CoV-2 Entry Molecules in the Oral Cavity", International Journal of Molecular Sciences, 2020, 21(17), 6000 [Non-patent document 3] "Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium", Journal of Virology, Oct. 2006, Vol.80, No.19, p.9896-9898 Summary of the Invention [Problem to be solved by the invention]

[0009] There is a need for antiviral compositions that are effective in suppressing infection with viruses in which TMPRSS2 activity is involved in infection. [Means for solving the problem]

[0010] To solve the above problems TMPRSS2 inhibitors teeth, chamomile Contains an extract from the plant as an active ingredient 。 [Effects of the Invention]

[0011] Original Clearly According to By inhibiting the activity of TMPRSS2 It can exert antiviral effects. DETAILED DESCRIPTION OF THE INVENTION

[0012] One embodiment of the antiviral composition will be described below. The antiviral composition of this embodiment contains a plant extract as an active ingredient, and exerts its antiviral effect by inhibiting the activity of TMPRSS2.

[0013] <Extract> The extract from the plant will be described. In this embodiment, an effective extract is one that inhibits TMPRSS2 activity by 1% or more at a concentration of 0.01 w / v% during the reaction. The TMPRSS2 inhibition rate is preferably 50% or more, and more preferably 70% or more. The method for calculating the TMPRSS2 inhibition rate will be described later.

[0014] The plant extract is not particularly limited, but is preferably one extracted using a known extraction solvent. Examples of known extraction solvents include alcohols such as ethanol, glycerin, propylene glycol, and 1,3-butylene glycol, as well as water. The extraction solvent may also be a mixed solvent of the above alcohols and water.

[0015] The plant extract may be purified as necessary. The following extracts may be used singly or in combination of two or more. The plant used in the antiviral composition of this embodiment is not particularly limited, but examples thereof include plants of the Asteraceae family, the Onagraceae family, and the Fabaceae family.

[0016] As the plant of the Asteraceae family, specifically, the genus Deer Daisy can be mentioned, more specifically, chamomile (Matricaria chamomilla) can be mentioned. For example, the extract extracted from chamomile flowers can be used as the extract. Chamomile is also known as chamomile or kamyrure.

[0017] Specific examples of plants of the Onagraceae family include the genus Oenothera, and more specifically, Oenothera biennis. For example, an extract extracted from the seeds of Oenothera biennis can be used as the extract.

[0018] Specific examples of legume plants include the genus Sophora, more specifically Sophora flavescens. For example, an extract extracted from the roots of Sophora flavescens can be used as the extract. The plant used in the antiviral composition of the present embodiment is preferably an ingredient that can be incorporated into quasi-drugs, which can suppress side effects and enable safer use.

[0019] <Application form> The application form of the antiviral composition is not particularly limited, and it can be used as, for example, food, cosmetics, pharmaceuticals, or quasi-drugs.

[0020] When the antiviral composition is used as an oral composition such as a food, it can be applied to, for example, candy, troches, tablets, gum, granules, powder, jelly, syrup, beverages, etc.

[0021] When the antiviral composition is used as a topical composition such as a cosmetic, it can be applied, for example, to face packs, pastes, ointments, creams, gels, lotions, emulsions, beauty serums, and skin lotions. Furthermore, the antiviral composition is not limited to products used on the human body, and can also be applied as a liquid to be sprayed or applied to products that come into contact with the skin, such as bedding, clothing, furniture, fixtures, and other articles. Examples of materials for the target products include natural fibers, synthetic fibers, natural leather, artificial leather, synthetic leather, wood, synthetic resins, metals, and painted surfaces.

[0022] When the antiviral composition is used as an oral cavity composition or throat composition such as a quasi-drug, it can be applied, for example, to sprays, toothpastes, liquid toothpastes, mouthwashes, rinses, etc. Examples of sprays include nasal sprays, throat sprays, and oral sprays.

[0023] The antiviral composition can be used to cleanse the skin or the mouth, and to maintain healthy skin or the mouth. The antiviral composition can be used to suppress or alleviate sore throat, swollen throat, throat discomfort, sore throat, or hoarseness caused by throat inflammation. The antiviral composition can be used to sterilize, disinfect, or cleanse the skin, hands, the oral cavity, or the throat (pharynx). The antiviral composition can also be used for virus barrier, virus blocking, virus shutting, antivirus, and virus elimination.

[0024] <Action and Effects> The operation of this embodiment will be described. In this embodiment, a plant extract is used as an active ingredient. A composition containing the extract as an active ingredient has the effect of inhibiting the activity of TMPRSS2. In other words, the composition containing the extract as an active ingredient acts as a TMPRSS2 inhibitor.

[0025] TMPRSS2 is a type II transmembrane serine protease expressed on the cell membrane of host cells. For example, TMPRSS2, together with ACE2, which is also expressed on the cell membrane of host cells, is involved in the entry of SARS-CoV-2 into host cells. Specifically, after the viral spike protein binds to the ACE2 receptor, it is thought that the spike protein present on the surface of the virus is cleaved by TMPRSS2 during the virus's entry into the host cell. The virus, activated by spike protein cleavage, induces fusion of the viral outer membrane with the cell membrane, making it easier for the virus to enter the host cell.

[0026] According to the antiviral composition of this embodiment, the cleavage of the spike protein can be suppressed by inhibiting the activity of TMPRSS2. By suppressing viral activation, the invasion of the virus into host cells can be suppressed. Thus, the composition is expected to exert an antiviral effect.

[0027] The effects of this embodiment will be described. (1) The antiviral composition contains an extract from a plant as an active ingredient and exerts an antiviral effect.

[0028] The higher the rate of inhibition of TMPRSS2 activity, the more effectively spike protein cleavage can be suppressed. This effectively inhibits viruses activated by spike protein cleavage, such as SARS-CoV-2, from entering host cells. In other words, the higher the rate of TMPRSS2 inhibition, the more effective the antiviral effect that can be expected to suppress viral infection.

[0029] (2) The antiviral composition is useful not only against SARS-CoV-2 but also against viruses whose spike proteins can be activated by cleavage by TMPRSS2. For example, it is expected to exert antiviral effects against other coronaviruses, such as MERS-CoV, SARS-CoV, HCoV-229E, HCoV-OC43, and HCoV-NL63.

[0030] (3) TMPRSS2 is known to be involved in infection with viruses other than coronaviruses, and the antiviral composition is useful against viruses whose infection may be affected by the protease activity of TMPRSS2, such as influenza virus and human metapneumovirus (hMPV).

[0031] (4) The antiviral composition inhibits the activity of TMPRSS2 present in host cells. Therefore, even if a virus mutates, if the mutated virus is still activated by the activity of TMPRSS2, it is expected to exert an antiviral effect by inhibiting the activity of TMPRSS2.

[0032] (5) It is possible to provide an oral composition, an external composition, an oral cavity composition, and a throat composition that exhibit antiviral activity. (6) When used as an oral composition, the antiviral composition is expected to act in the oral cavity, where TMPRSS2 and ACE2 are co-expressed. In other words, when the co-expression of TMPRSS2 and ACE2 is involved in the entry of viruses into host cells, such as SARS-CoV-2, the antiviral composition is expected to be effective in suppressing infection of the virus through the oral cavity.

[0033] Similarly, when the antiviral composition is used as a throat composition, it can be expected that the antiviral composition will also act on the oral cavity. Furthermore, when the antiviral composition is used as an oral composition, it can be expected that the antiviral composition will act on the oral cavity. In particular, a form that is likely to remain in the oral cavity, such as candy, is useful.

[0034] (7) The antiviral composition is expected to be effective in preventing viral infections, treating viral infections, inhibiting viral proliferation, suppressing viral infection, inhibiting viral infection promotion, inhibiting viral infection-related enzymes, and inhibiting viral infection-related enzymes.

[0035] (Example of change) This embodiment can be modified as follows: This embodiment and the following modifications can be combined and implemented within the scope of technical compatibility.

[0036] The extract as an active ingredient is not limited to an extract from a plant. For example, an antiviral composition may contain an extract from seaweed as an active ingredient. 〈Technical thought〉 The technical ideas that can be understood from the above-described embodiment and modified examples will be described.

[0037] (A) An antiviral composition containing an extract from seaweed as an active ingredient. (B) An antiviral composition containing an extract from a plant or seaweed as an active ingredient. (C) The plant includes at least one selected from chamomile, sophora flavescens, and evening primrose. [Example]

[0038] The antiviral composition will be described in more detail based on the following examples. Note that the antiviral composition is not limited to the configurations described in the examples. It should be noted that the following Examples 2 to 6 should be replaced with Reference Examples 2 to 6, respectively. This study was based on the method described in "An Enzymatic TMPRSS2 Assay for Assessment of Clinical Candidates and Discovery of Inhibitors as Potential Treatment of COVID-19," ACS Pharmacology & Translational Science, September 7, 2020, 3(5), pp. 997-1007.

[0039] <<Calculation principle of TMPRSS2 inhibition rate>> The principle of the method for calculating the TMPRSS2 inhibition rate is explained below. TMPRSS2 and a peptide substrate degraded by TMPRSS2 activity were used to calculate the TMPRSS2 inhibition rate. TMPRSS2 was used as "transmembrane protease, serine 2, LS-G57269-100" from LifeSpan Biosciences, Inc. The peptide substrate used was "Boc-Gln-Ala-Arg-MCA, 3135-v" from Peptide Institute, Inc.

[0040] When the peptide substrate "Boc-Gln-Ala-Arg-MCA" is decomposed by TMPRSS2, the fluorescent substance AMC (7-Amino-4-methylcoumarin) is released. AMC can be detected by fluorescence measurement at an excitation wavelength of 340 nm and an emission wavelength of 440 nm. TMPRSS2 activity can be measured based on the fluorescence intensity of the generated AMC.

[0041] When a TMPRSS2 inhibitor is added to the system in addition to the peptide substrate and TMPRSS2, TMPRSS2 activity is inhibited according to the inhibitory potency of the added TMPRSS2 inhibitor. In other words, the amount of AMC produced decreases according to the inhibitory potency of the TMPRSS2 inhibitor. Therefore, the inhibitory potency of each TMPRSS2 inhibitor against TMPRSS2 can be calculated based on the fluorescence intensity. Specifically, the TMPRSS2 inhibition rate can be calculated using the following formula (Formula 1):

[0042]

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[0043] <<Evaluation Test>> The TMPRSS2 inhibition rate was evaluated using the extracts of Examples 1 to 6 shown in Table 1 as TMPRSS2 inhibitors. The concentration of each TMPRSS2 inhibitor during the reaction was 0.01 w / v%. Details of the TMPRSS2 inhibitor in each Example are as follows.

[0044] Example 1 Maruzen Pharmaceutical Co., Ltd.'s "Chamomile Extract BG-J" was used as the extract extracted from chamomile flowers.

[0045] Example 2 The extract used was Falcorex Clara B from Ichimaru Falcos Co., Ltd., extracted from Sophora flavescens roots.

[0046] Example 3 The extract used was "Luna White B" from Ichimaru Falcos Co., Ltd., extracted from evening primrose seeds.

[0047] Example 4 The extract extracted from the flower heads of Artemisia capillaris was "Falcorex Artemisia capillaris B" by Ichimaru Falcos Co., Ltd. Artemisia capillaris is a plant of the Asteraceae family, Artemisia genus.

[0048] Example 5 The extract extracted from Calendula officinalis flowers was "Calendula officinalis Extract BG-J" by Maruzen Pharmaceutical Co., Ltd. Calendula officinalis is a plant of the Asteraceae family, Calendula genus.

[0049] Example 6 The extract extracted from eucalyptus leaves used was "Eucalyptus Extract BG" by Maruzen Pharmaceutical Co., Ltd. Eucalyptus is a plant of the genus Eucalyptus in the family Myrtaceae.

[0050] Preparation of Reagents (1x Assay Buffer) A buffer containing 150 mM NaCl and 50 mM Tris-HCl, pH 8.0, was prepared.

[0051] (peptide substrate) "Boc-Gln-Ala-Arg-MCA" was diluted with 1x Assay Buffer to prepare a 10 µM substrate solution. (TMPRSS2 inhibitor) The extract was diluted with distilled water so that the concentration of the TMPRSS2 inhibitor extract was 0.04 w / v%.

[0052] (TMPRSS2 solution) TMPRSS2 was diluted with 1× Assay Buffer to prepare a 0.2 μM TMPRSS2 solution.

[0053] Test Method A black 96-well plate was used, and 25 μL of the prepared TMPRSS2 inhibitor was added to each well. 25 μL of 1× Assay Buffer was added instead of the TMPRSS2 inhibitor to the wells for measuring BG2 and RFUc.

[0054] 25 μL of the prepared TMPRSS2 solution was added to each well. 25 μL of 1× Assay Buffer was added instead of the TMPRSS2 solution to the wells for measuring BG1 and BG2.

[0055] 50 μL of the prepared 10 μM substrate solution was added to each well. The plate was covered and incubated at 37°C for 60 minutes. The plate was measured using a plate reader (Cytation5, BioTek Instruments, Inc.) at an excitation wavelength of 340 nm and a fluorescence wavelength of 440 nm. Based on the measurement results, the TMPRSS2 inhibition rate for each example was calculated. Furthermore, evaluation was performed according to the following criteria. The results are shown in Table 1.

[0056] Evaluation criteria for antiviral activity ○○○(Excellent): TMPRSS2 inhibition rate is 70% or higher. ○○ (Good): TMPRSS2 inhibition rate is 50% or more but less than 70%. ○ (Acceptable): TMPRSS2 inhibition rate is 1% or more but less than 50%.

[0057] <Test Results> As shown in Table 1, it was confirmed that TMPRSS2 activity was inhibited in all Examples. In Examples 1 to 3, the TMPRSS2 inhibition rate was 50% or more. That is, in Examples 1 to 3, the antiviral activity was evaluated as good. In particular, in Examples 1 and 2, the TMPRSS2 inhibition rate was 70% or more, and even better antiviral activity can be expected.

[0058] As positive controls, tests were conducted using camostat mesylate (FUJIFILM Wako Pure Chemical Corporation) and nafamostat mesylate (FUJIFILM Wako Pure Chemical Corporation). Tests using camostat mesylate at a reaction concentration of 10 nM confirmed a 90% TMPRSS2 inhibition rate. Tests using nafamostat mesylate at a reaction concentration of 1 nM confirmed a 102% TMPRSS2 inhibition rate.

[0059] Table 1

Claims

[Claim 1] A TMPRSS2 inhibitor containing an extract from chamomile as an active ingredient.

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