Use of gallocatechol in the preparation of a medicament for treating coronavirus or sepsis

Abstract

The present application provides the use of galloyl catechin gallate, a pharmaceutically acceptable salt, a prodrug or a hydrate thereof in the preparation of a medicament for preventing and / or treating a disease caused by a coronavirus or sepsis. The inventors found, and experimentally verified, that galloyl catechin gallate can significantly inhibit the coronavirus, and, in combination with another M pro inhibitor, Ebselen, can have a synergistic effect; in addition, it can also effectively inhibit sepsis, and thus is a very promising candidate drug for treating a disease caused by a coronavirus (particularly a novel coronavirus infection caused by SARS-CoV-2) and sepsis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the use of gallocatechin gallate, its pharmaceutically acceptable salts, prodrugs, or hydrates in the preparation of medicaments for the prevention and / or treatment of diseases or sepsis caused by coronaviruses. Background Technology

[0002] Novel coronavirus infection is a viral respiratory disease caused by SARS-CoV-2 infection. Currently, there are no specific drugs or effective treatments for novel coronavirus infection. Therefore, there is an urgent need to develop specific drugs against novel coronavirus infection.

[0003] SARS-CoV-2 is a single-stranded positive-sense RNA virus containing approximately 30,000 bases and 14 open reading frames, encoding a replicase, four structural proteins (S, E, M, N), 16 non-structural proteins, and nine accessory proteins. NSPs (non-structural proteins) play a crucial role in the viral replication and transcription cycles. NSP5, also known as the 3CLike protease, is the major protease of SARS-CoV-2 and is essential for the processing and maturation of viral polymers; therefore, it is considered an important potential drug target for combating the novel coronavirus.

[0004] Sepsis is a systemic inflammatory response syndrome (SIRS) caused by infection, clinically confirmed by the presence of bacteria or a highly suspected focus of infection. Sepsis is a significant clinical problem in critical care medicine, with its incidence steadily increasing. Millions of new sepsis patients are diagnosed globally each year, and more than a quarter of these patients die. It is one of the most common critical illnesses in intensive care units, characterized by rapid disease progression and a high clinical mortality rate, now surpassing myocardial infarction as the leading cause of death in intensive care units. In recent years, there have been increasing reports of the clinical use of traditional Chinese medicine preparations and integrated traditional Chinese and Western medicine in the treatment of sepsis, which has gradually become a research hotspot.

[0005] Gallatechin gallate (GCG) belongs to the catechin class of compounds, with CAS registry number 4233-96-9, and has the following structure: . Summary of the Invention

[0006] Purpose of the invention The object of this invention is to provide novel uses of gallocatechin gallate (GCG), its pharmaceutically acceptable salts, prodrugs or hydrates, namely, the use of the above compounds in the preparation of medicaments for the prevention and / or treatment of diseases or sepsis caused by coronaviruses.

[0007] Solution To achieve the objectives of this invention, the technical solution adopted is as follows: In a first aspect, the present invention provides the use of gallocatechin gallate, a pharmaceutically acceptable salt thereof, a prodrug or hydrate thereof in the preparation of a medicament for the prevention and / or treatment of disease caused by coronavirus in subjects.

[0008] In the specific implementation plan, the disease caused by the coronavirus is that caused by SARS. Cov 2. Infectious diseases or their complications caused by SARS, preferably those caused by SARS. Cov 2. Respiratory infections or their complications caused by [the virus / disease].

[0009] In the above-mentioned uses, the treatment or prevention is achieved by inhibiting SARS-CoV-2 M pro The enzyme activity of the protease is inhibited, thereby suppressing the replication of the novel coronavirus.

[0010] In a second aspect, the present invention provides the use of gallocatechin gallate, its pharmaceutically acceptable salt, prodrug or hydrate, in combination with ibuselenin in the preparation of a medicament for the prevention and / or treatment of diseases caused by coronaviruses.

[0011] In the above-mentioned applications, the ebselen used is a SARS-CoV-2 M... pro Another inhibitor of proteases.

[0012] In the above-mentioned uses, preferably, in the prepared drug, the molar ratio of gallocatechin gallate, its pharmaceutically acceptable salt, prodrug or hydrate to ibuselenin is (10~20):1, more preferably 16:1.

[0013] Furthermore, in the use described above in the first or second aspect, preferably, the prepared drug is an oral dosage form or an injectable dosage form; in a specific embodiment, the oral dosage form may be a tablet, capsule, granule, powder, solution, emulsion or suspension; in a specific embodiment, the injectable dosage form may be a solution or lyophilized powder for injection.

[0014] Thirdly, the present invention provides a pharmaceutical composition for the prevention and / or treatment of diseases caused by coronaviruses, comprising gallocatechin gallate, a pharmaceutically acceptable salt thereof, a prodrug or hydrate thereof, and ibuselenium, and optionally pharmaceutically acceptable excipients.

[0015] In the above pharmaceutical composition, preferably, the molar ratio of the gallocatechin gallate, its pharmaceutically acceptable salt, prodrug or hydrate to ibuselenium is (10~20):1, more preferably 16:1.

[0016] In the above-described pharmaceutical composition, preferably, the pharmaceutically acceptable excipients include one or more of the following: carriers, excipients, and diluents; some examples suitable as carriers, excipients, and diluents include: lactose, dextrose, sucrose, sorbitol, mannitol, starch, resin, gum arabic, calcium phosphate, alginate, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, methylcellulose, methylparaben and propylparaben, talc, magnesium stearate, and liquid paraffin, one or more of the following.

[0017] In one possible implementation, pharmaceutically acceptable excipients also include lubricants, wetting agents, emulsifiers and suspending agents, preservatives, sweeteners or flavoring agents, etc.

[0018] The above-mentioned pharmaceutical composition may be in the form of an oral formulation or an injectable formulation; Preferably, the oral preparation is a tablet, capsule, granule, powder, solution, emulsion, or suspension; Preferably, the injectable formulation is a solution or a lyophilized powder for injection.

[0019] Fourthly, the present invention provides the use of gallocatechin gallate, its pharmaceutically acceptable salt, prodrug, or hydrate in the preparation of medicaments for the prevention and / or treatment of sepsis.

[0020] In the above uses, the treatment of sepsis includes one or more of the following: (1) improving the survival rate of sepsis patients; (2) reducing inflammation of various organs; (3) reducing the degree of damage to various organs.

[0021] In the above-mentioned uses, preferably, the subject is a human being; and when the subject is a human being, the effective dose of gallocatechin gallate, its pharmaceutically acceptable salt, prodrug or hydrate in the prepared drug is 5 to 20 mg / kg / day.

[0022] In the above-mentioned uses, preferably, the prepared drug is an oral dosage form or an injectable dosage form; in specific embodiments, the oral dosage form can be a tablet, capsule, granule, powder, solution, emulsion or suspension; in specific embodiments, the injectable dosage form can be a solution or lyophilized powder for injection.

[0023] Fifthly, the present invention also provides a method for preventing and / or treating diseases caused by coronaviruses, comprising the steps of administering to a subject in need a preventive and / or therapeutically effective amount of gallocatechin gallate, a pharmaceutically acceptable salt thereof, a prodrug or hydrate thereof, or a pharmaceutical composition as described in the third aspect.

[0024] In the specific implementation plan of the above-mentioned methods for preventing and / or treating diseases caused by coronaviruses: Preferably, the disease caused by the coronavirus is caused by SARS. Cov 2. Infectious diseases or their complications caused by SARS, preferably those caused by SARS. Cov 2. Respiratory infectious diseases or their complications caused by SARS-CoV-2; preferably, the treatment or prevention is achieved by inhibiting SARS-CoV-2 M. pro The enzyme activity of the protease is inhibited, thereby suppressing the replication of the novel coronavirus.

[0025] In a sixth aspect, the present invention also provides a method for preventing and / or treating sepsis, comprising the steps of administering to a subject in need a preventive and / or therapeutically effective amount of gallocatechin gallate, a pharmaceutically acceptable salt thereof, a prodrug, or a hydrate thereof.

[0026] In the specific implementation of the above-described use or method for preventing and / or treating diseases or sepsis caused by coronaviruses, preferably, the subject is selected from mammals; alternatively, the subject is selected from humans, mice, rats, dogs, rabbits, pigs, and monkeys.

[0027] As used in this invention, the term "effective amount" refers to the quantity or dose of an active ingredient administered to a patient, whether once or multiple times, to provide the intended effect to the diagnosed or treated patient. The effective amount can be determined by the participating diagnostic physician as a person skilled in the art, using known techniques and observations obtained in similar circumstances. In determining the effective amount or dose of the administered active ingredient, the participating diagnostic physician should consider a variety of factors, including but not limited to: the species of mammal; size, age, and general health; the specific disease involved; the extent or severity of the disease; the individual patient's response; the specific compound administered; the administration method; the bioavailability properties of the administered formulation; the chosen dosing regimen; the use of concomitant drug therapies; and other relevant circumstances.

[0028] Beneficial effects This invention provides the use of gallocatechin gallate (GCG), its pharmaceutically acceptable salts, prodrugs, or hydrates in the preparation of medicaments for the prevention and / or treatment of diseases or sepsis caused by coronaviruses. The inventors have discovered, and experimentally demonstrated, that gallocatechin gallate can bind to SARS-CoV-2 M. pro It also inhibits the in vitro enzyme activity of the novel coronavirus, specifically targeting the main protease M of the SARS-CoV-2 virus. pro IC 50 The concentration was 5.774 ± 0.805 μM, thus significantly inhibiting the SARS-CoV-2 virus; furthermore, when combined with another M... pro When used in combination with the inhibitor ebselen, it exhibits a significant synergistic effect. The research of this invention also demonstrates that gallocatechin gallate can effectively inhibit sepsis. Therefore, gallocatechin gallate (GCG), its pharmaceutically acceptable salts, prodrugs, or hydrates are promising candidates for treating coronavirus-related diseases (particularly novel coronavirus infection caused by SARS-CoV-2) and sepsis. Attached Figure Description

[0029] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0030] Figure 1 shows the gallocatechin gallate (Gallocatechin gallate) Figure 1A ) and epigallocatechin gallate ( Figure 1B ) against SARS-CoV-2 M pro IC50 of the inhibitory effect on in vitro enzyme activity 50 ; Figure 2 shows Ebselen ( Figure 2A The combination of gallocatechin gallate (Ebselen) and ethyl gallate (Ebselen) Figure 2B ) against SARS-CoV-2 M pro IC50 of the inhibitory effect on in vitro enzyme activity 50 ; Figure 3 This study demonstrates the effect of gallocatechin gallate on the survival of septic mice; Figure 4 The effect of gallocatechin gallate on sepsis scores in septic mice was shown. Figure 5This study demonstrates the effect of gallocatechin gallate on the expression of the inflammatory cytokine TNF-α in septic mice; Figure 6 This study demonstrates the effect of gallocatechin gallate on the expression of the inflammatory cytokine IL-4 in septic mice; Figure 7 HE staining of tissue sections showing the effects of gallocatechin gallate on various organs of septic mice. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0032] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0033] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0034] In the following embodiments, the test materials used and their sources include: 1. Experimental drugs and reagents Gallic catechin gallate: powder form, provided by Shanghai Taosu Biochemical Technology Co., Ltd.; Epigallocatechin gallate: 10 mM solution, provided by Shanghai Taosu Biochemical Technology Co., Ltd.; Cefpirome sulfate: powder, Shanghai Yuanye Biotechnology Co., Ltd.; SARS-CoV-2 Mpro Enzyme Activity Detection Kit: Beyotime; Ebselen: Azure Sky; ELISA kit: Shanghai Enzyme-Linked Biotechnology Co., Ltd. Chloral hydrate: Product No.: Sigma-23100, Shanghai Beinuo Biotechnology Co., Ltd.; Formaldehyde solution: Product No.: 3A6A16163, Ailan (Shanghai) Chemical Technology Co., Ltd.; Hematoxylin dye solution: Product No.: ZLI-9610, Tianjin Junyao Trading Co., Ltd.; Water-soluble eosin dye solution: Product No.: ZLI-9613, Tianjin Junyao Trading Co., Ltd.

[0035] 2. Main Instruments Multifunctional microplate reader: Manufacturer: Thermo Fisher Electronic balance: Shanghai Ohaus Instruments Co., Ltd., Model: AR2202CN; Benchtop Refrigerated Centrifuge: Thermo Fisher Scientific, Model: ST16R; 4℃ Refrigerator: Qingdao Haier Co., Ltd., Model: BCD-192KJ; Vortex mixer: Haimen Qilin Bell Instrument Manufacturing Co., Ltd., Model: QZ-861.

[0036] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0037] Example 1: The effect of gallocatechin gallate ((-)-GCG) or epigallocatechin gallate (EGCG) on SARS-CoV-2 M pro Inhibition of in vitro enzyme activity Detection method: The assay was performed in a 96-well plate, with a reaction volume of 100 μL. pro The final concentration of the protease used was 0.3 μM, and the final concentration of the drug used was a serial dilution gradient from 0 to 100 μM. The reaction buffer consisted of: 50 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.3, and the final concentration of the substrate was 20 μM. The experimental groups were as follows: blank control group, each well containing 93 μL of reaction buffer, 5 μL of DMSO, and 2 μL of substrate; 100% enzyme activity control group, each well containing 92 μL of reaction buffer, 1 μL of DMSO, and 1 mM EDTA. pro 5 μL DMSO and 2 μL substrate; for the drug assay group, add 92 μL reaction buffer and 1 μL M to each well. pro 5 μL of different concentrations of gallocatechin gallate or epigallocatechin gallate and 2 μL of substrate were added to a 96-well plate according to the above grouping. pro After adding the protease and different concentrations of the drug, the mixture was incubated at 37°C for 5 min, followed by the addition of the substrate and incubation at 37°C in the dark for 10 min. Fluorescence values ​​were then measured at excitation and emission wavelengths of 340 nm and 490 nm, respectively. The effects of different concentration gradients of gallocatechin gallate or epigallocatechin gallate on M were calculated. proThe inhibition rate of in vitro protease activity was calculated as follows: Inhibition rate (%) = (RFU 100% enzyme activity control - RFU sample) / (RFU 100% enzyme activity control - RFU blank control) × 100%. Finally, the IC50 was plotted and fitted using Graphpad Prism 7.0. 50 .

[0038] The results are shown in Figure 1. Figure 1 shows that gallocatechin gallate inhibits M pro IC50 of proteases 50 It is 5.774 ± 0.805 μM (see Figure 1A Epigallocatechin gallate for M pro IC50 of the inhibitory effect of protease on in vitro enzyme activity 50 It was 27.49 ± 5.355 μM (see...) Figure 1B This indicates that the ratio of gallocatechin gallate to gallocatechin gallate has a positive effect on SARS-CoV-2 M. pro The inhibitory effect of protease on in vitro enzyme activity is stronger, with the former having an inhibitory effect nearly five times that of the latter, making it a more promising candidate for a drug to inhibit the novel coronavirus.

[0039] Example 2: Ebselen alone or in combination with gallocatechin gallate ((-)GCG) to inhibit SARS-CoV-2 M pro Inhibition of in vitro enzyme activity Detection method: The assay was performed in a 96-well plate, with a reaction volume of 100 μL. pro The final concentration of the protease used was 0.3 μM, and the final concentration of the drug used was a serial dilution gradient from 0 to 100 μM. The reaction buffer consisted of: 50 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.3, and the final concentration of the substrate was 20 μM. The experimental groups were as follows: blank control group, each well containing 93 μL of reaction buffer, 5 μL of DMSO, and 2 μL of substrate; 100% enzyme activity control group, each well containing 92 μL of reaction buffer, 1 μL of DMSO, and 1 mM EDTA. pro 5 μL DMSO and 2 μL substrate; for the drug assay group, add 92 μL reaction buffer and 1 μL M to each well. pro 5 μL of ebselen at different concentrations or ebselen at different concentrations, plus 5 μM (-) GCG and 2 μL of substrate. Following the above grouping, add M to a 96-well plate. proAfter adding the protease and different concentrations of the drug, the mixture was incubated at 37°C for 5 min, followed by the addition of the substrate and incubation at 37°C in the dark for 10 min. Fluorescence values ​​were then measured at excitation and emission wavelengths of 340 nm and 490 nm, respectively. The effects of different concentration gradients of ebselen or different concentrations of ebselen combined with (-) GCG on M were calculated. pro The inhibition rate of in vitro protease activity was calculated as follows: Inhibition rate (%) = (RFU 100% enzyme activity control - RFU sample) / (RFU 100% enzyme activity control - RFU blank control) × 100%. Finally, the IC50 was plotted and fitted using Graphpad Prism 7.0. 50 .

[0040] The results are shown in Figure 2. Figure 2 shows that Ebselen inhibits M pro IC50 of proteases 50 It is 0.1222 μM (see Figure 2A ), while Ebselen combined with (-) GCG for M pro IC50 of the inhibitory effect of protease on in vitro enzyme activity 50 It is 0.04535 μM (see Figure 2B This indicates that the combination of ebselen and (-) GCG is more effective against SARS-CoV-2 M than ebselen alone. pro The inhibitory effect of the protease on in vitro enzyme activity is stronger, with the former's inhibitory effect being nearly three times that of the latter, indicating that the combination of the two has a good synergistic effect.

[0041] Example 3: Inhibitory effect of gallocatechin gallate ((-)-GCG) on sepsis 1. Model establishment and drug administration C57BL / 6J mice aged 6-8 weeks and weighing between 22-24 g were selected for the experiments in this embodiment. These mice were randomly divided into five groups: control group (blank control, no modeling); model group (negative control, given physiological saline); positive control group (cefpirome sulfate treatment group); gallocatechin gallate 50 mg / kg treatment group; and gallocatechin gallate 100 mg / kg treatment group.

[0042] On Day 0, mice in both the model group and the treatment group were intraperitoneally injected with a 10% fecal dilution to establish a sepsis model. Two hours before modeling, the treatment group was intraperitoneally injected with 100 mg / kg cefpirome sulfate, 50 mg / kg gallocatechin gallate, and 100 mg / kg gallocatechin gallate, respectively. On Day 1, the drugs were administered again; on Day 2 (48 hours after modeling), the mice were euthanized.

[0043] 2. Evaluation indicators: Survival analysis: Survival curves were established based on the mortality rates of mice in each group 48 hours after modeling.

[0044] Mouse sepsis score: Based on the MSS mouse sepsis scoring principle, mice were scored on their appearance, level of consciousness, activity level, responsiveness to stimuli, eye condition, respiratory rate, and respiratory quality. The MSS score data are shown in Table 1 below. Figure 3 As shown.

[0045] Table 1

[0046] Inflammatory factor detection: Blood was collected from the eyes of all animals on the second day after modeling. Serum was collected by centrifugation at 3000 rpm for 10 minutes at room temperature. The serum was used for subsequent ELISA detection of cytokines IL-4 and TNF-α.

[0047] Pathological sections: After sampling on the second day, the heart, liver, spleen, lung, and kidney tissues of mice were placed in 5% formaldehyde fixative and incubated overnight at room temperature on a shaker. The tissues were then dehydrated, fixed, sectioned, stained, and photographed. Finally, the degree of inflammation in each organ was observed.

[0048] The results of the study on the effect of gallocatechin gallate on the survival of mice with fecal dilution-induced septicemia are as follows: Figure 3 As shown, Figure 3 The results showed that the mortality rate of septic mice in the model group was high after modeling, with a 24-hour mortality rate of 50%; while the survival of mice in the gallocatechin gallate treatment group, whether the treatment group was 50 mg / kg or 100 mg / kg (the survival curves of the two groups overlapped somewhat), was significantly better than that of the model group.

[0049] The results of the detection of the effects of gallocatechin gallate on the activity and mental state of fecal dilution-induced septic mice are as follows: Figure 4 As shown in Table 1, Figure 4 The results in Table 1 show that, as can be seen from the MSS scores, gallocatechin gallate significantly improved the activity and mental state of septic mice at both treatment concentrations of 50 mg / kg and 100 mg / kg.

[0050] Effects of gallocatechin gallate on the expression levels of inflammatory cytokines TNF-α and IL-4 in fecal dilution-induced septic mice. Figure 5-6 As shown, Figure 5-6The results showed that, regardless of the treatment concentration of 50 mg / kg or 100 mg / kg, gallocatechin gallate significantly downregulated the levels of inflammatory factors IL-4 and TNF-α in the serum of septic mice, and the degree was comparable to that of the positive control group of cefpirome sulfate.

[0051] Effects of gallocatechin gallate on the morphology of various organs and tissues in fecal dilution-induced septic mice, such as... Figure 7 As shown, Figure 7 The results showed that, regardless of the treatment concentration of 50 mg / kg or 100 mg / kg, gallocatechin gallate significantly improved the degree of inflammation and damage in various organs of septic mice.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A SARS-CoV-2 Mpro protease inhibitor composition, characterized in that, The SARS-CoV-2 Mpro protease inhibitor composition comprises gallocatechin gallate or a pharmaceutically acceptable salt thereof, and ibuselenium, and optionally a pharmaceutically acceptable excipient, wherein the molar ratio of the gallocatechin gallate or a pharmaceutically acceptable salt thereof to ibuselenium is (10–20):

1.

2. The SARS-CoV-2 Mpro protease inhibitor composition according to claim 1, wherein, The molar ratio of the gallocatechin gallate or its pharmaceutically acceptable salt to ibuselin is 16:

1.

3. The SARS-CoV-2 Mpro protease inhibitor composition according to claim 1 or 2, wherein it is in a formulation selected from the following: tablets, capsules, granules, powders, solutions, emulsions, suspensions or lyophilized powders.

4. The use of gallocatechin gallate or a pharmaceutically acceptable salt thereof in combination with ibuselenin in the preparation of SARS-CoV-2 Mpro protease inhibitors, wherein, In the SARS-CoV-2 Mpro protease inhibitor, the molar ratio of gallocatechin gallate or its pharmaceutically acceptable salt to ibuselenin is (10–20):

1.

5. The use according to claim 4, wherein, In the SARS-CoV-2 Mpro protease inhibitor, the molar ratio of gallocatechin gallate or its pharmaceutically acceptable salt to ibuselenin is 16:

1.

6. The use according to claim 4 or 5, wherein, The SARS-CoV-2 Mpro protease inhibitor is selected from the following formulations: tablets, capsules, granules, powders, solutions, emulsions, suspensions, or lyophilized powders.

Images