Drugs containing active sulfur compounds as their main component

Abstract

Provided is a medical agent that exerts preventative and therapeutic effects against viral infections, by inhibiting proteases of viral origin on the basis of the effects of various physiological activities of an active sulfur compound present in vivo.

Description

Technical Field

[0001] The present invention relates to a drug containing an active sulfur compound as a main medicinal ingredient.

Background Art

[0002] The physiological importance of reactive sulfur molecular species / active sulfur molecular species (RSS) such as cysteine hydro-polysulfide (CysSSH) and glutathione polysulfide (GSSH) has been reported (Non-Patent Document 1).

[0003] Cysteine hydro-polysulfide (CysSSH) is produced in large amounts in various organisms, but little is known about its biosynthesis and physiological functions. The formation of extensive persulfides in cysteine-containing proteins of Escherichia coli and mammalian cells is clear, and this is thought to result from post-translational processes including chemical reactions related to sulfur metabolites in vivo.

[0004] There is an efficient CysSSH synthesis from the substrate L-cysteine, which is a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetase (CARS). Targeted disruption of the genes encoding mitochondrial CARS in mouse and human cells indicates that CARS plays an important role in endogenous CysSSH production, suggesting that these enzymes function as major cysteine persulfide synthases in vivo. CARS also catalyzes cotranslational cysteine persulfidation and is involved in mitochondrial biosynthesis and the regulation of bioenergy.

[0005] Therefore, elucidation of the production mechanism of CARS-dependent persulfide metabolites may help understand abnormal redox signaling under physiological and pathophysiological conditions and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction.

[0006] By establishing active sulfur metabolomics analysis using RSS metabolic profiling, the in vivo dynamics of significant amounts of RSS, which are produced endogenously and ubiquitously in both prokaryotes and eukaryotes, will be revealed. However, the chemical properties of polysulfides are not fully understood and remain unexplained due to their reactivity or complex redox activity characteristics.

[0007] Meanwhile, elucidating the pathogenesis of the novel coronavirus (SARS-CoV-2), which causes COVID-19, and establishing prevention and treatment methods are urgent priorities. SARS-CoV-2 protease inhibitors have been reported as therapeutic agents for COVID-19 (Non-Patent Literature 2, 3). Furthermore, WO / 2017 / 005768 discloses that the challenge of providing an intraocular irrigation solution that adequately protects intraocular tissue during ophthalmic surgery and is even safer can be solved by using an intraocular irrigation solution containing at least one type of active sulfur molecule, such as cysteine ​​persulfide or glutathione persulfide. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] WO / 2017 / 005768 publication [Non-patent literature]

[0009] [Non-Patent Document 1] NATURE COMMUNICATIONS|8:1177,Cysteinyl-tRNA synthetase governs cysteine ​​polysulfidation and mitochondrial bioenergetics,27 October 2017. [Non-Patent Document 2] Zhang L, et al. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α ketoamide inhibitors. Science 368:409-412 (2020). [Non-Patent Document 3] Jin Z, et al. Structure of Mpro from COVID-19 virus and discovery of its inhibitors. Nature 582:289-293 (2020). [Overview of the project] [Problems that the invention aims to solve]

[0010] The present invention aims to provide a drug that exerts preventive and therapeutic effects against viral infections by inhibiting virus-derived proteases based on the diverse physiological activity of active sulfur compounds present in living organisms. This is neither disclosed nor suggested in the aforementioned Patent Document 3. [Means for solving the problem]

[0011] To achieve the above objective, the first invention is characterized by being an antiviral drug containing an active sulfur compound as its main active ingredient. The active sulfur compound is, for example, R1S(S) n R2 (R1 and R2 are independently selected from amino acids and polypeptides having thiol groups, and represent the portion other than the thiol group. n is an integer of 2 or more, preferably 7 or less). The active sulfur compound is, for example, L-cysteine ​​(Cys), homocysteine ​​(Hcys), or glutathione (GSH) for R1 and R2, respectively. The active sulfur compound is, for example, glutathione polysulfide, and in particular GSSSG.

[0012] The virus is a coronavirus (CoV), particularly, the novel coronavirus (SARS-CoV-2). The antiviral drug is, for example, a drug for preventing and treating SARS-CoV-2 pneumonia.

[0013] The second invention is a drug for preventing and treating coronavirus disease 2019 (COVID-19) with glutathione poly(tri)sulfide (such as GSSSG) as the main active ingredient, the third invention is an immune regulator with glutathione poly(tri)sulfide as the main active ingredient, and the fourth invention is an anti-inflammatory agent with glutathione poly(tri)sulfide as the main active ingredient.

[0014] The fifth invention is a drug for preventing and treating pneumonia such as viral pneumonia with an active sulfur compound as the main component. The form of the active sulfur compound is as described above.

Advantages of the Invention

[0015] According to the present invention, based on the diverse physiological activities of the active sulfur compounds present in the living body, for example, by inhibiting virus-derived proteases, etc., a drug can be provided that exhibits a preventive and / or therapeutic effect against viral infections.

Brief Description of the Drawings

[0016] [Figure 1] FIG. 1 is a block diagram showing the antiviral effect of a glutathione peroxidation preparation. [Figure 2] FIG. 2 is a diagram showing various COVID-19 treatment effects of a glutathione peroxidation preparation. [Figure 3] FIG. 3 is a graph showing the anti-influenza effect of GSSSG. [Figure 4] FIG. 4 is a graph showing the treatment effect of GSSSG on elastase-induced chronic obstructive pulmonary disease (COPD). [Figure 5] FIG. 5 is a graph showing the inhibitory effect of GSSSG on SARS-CoV-2 protease. [Figure 6]Figure 6 is a graph showing the antiviral effect of GSSSG.

Mode for Carrying Out the Invention

[0017] SARS-CoV-2 expresses two different cysteine (thiol) proteases based on its viral genome. These are papain-like protease (PLpro) and 3C-like protease (also called 3CLpro or main protease, Mpro), and these proteases are essential for viral replication in host cells. The crystal structures of these proteases have been elucidated.

[0018] The inventors established an invention that glutathione trisulfide (GSSSG, etc., G: glutathione), which is a donor of active sulfur molecules and one of the active sulfur compounds, specifically covalently binds to the thiol groups at the active centers of these two proteases, PLpro and 3CLpro, thereby strongly inhibiting their enzyme activities. Already, it has been reported that ebselen, which shows a similar inhibition mode, consequently exhibits a remarkable growth inhibitory effect against the novel coronavirus (SARS-CoV-2) (Non-Patent Documents 2 and 3 mentioned above). Since the nucleophilicity of the sulfur in the center of the oxidized polysulfide (GSSSG) is stronger than that of the reduced polysulfide (GSSSH), the oxidized polysulfide is more useful.

[0019] The inventors of this application have invented a novel anti-SARS-CoV-2 drug by utilizing the fact that GSSSG, as glutathione polysulfide, exerts a direct antiviral effect by inhibiting the protease activity of SARS-CoV-2 and its variants (Figure 1). Furthermore, by utilizing the potent inflammatory and immunosuppressive activity (inflammation and immunosuppression) of GSSSG (Figure 2), they have invented an integrated anti-COVID-19 treatment agent that goes beyond simple antiviral therapy by preventing the occurrence of cytokine storms, which are considered a pathological condition of severe (fulminant) COVID-19 pneumonia, and intractable diseases such as interstitial pneumonia and pulmonary fibrosis, which are considered important complications or sequelae (complications) (Figure 2). Glutathione polysulfide exerts an inhibitory effect on inflammation resulting from viral infections such as SARS-CoV-2.

[0020] In Figure 2, glutathione trisulfide achieves its antiviral effect by inhibiting two cysteine ​​proteases. Glutathione trisulfide suppresses interleukin-6 production induced by the immune response to SARS-CoV-2 infection, thereby achieving immunomodulatory, anti-inflammatory, and anti-cytokine effects. Furthermore, glutathione trisulfide reacts with and binds to nitric oxide (NO), enhancing NO-mediated vasodilation and platelet aggregation inhibition, thereby exerting a potent vascular protective effect against thrombosis, vasculitis, and vascular damage, which are known complications of COVID-19.

[0021] In the development of antiviral drugs, there has been active research into viral protease inhibitors. However, many compounds are specialized to inhibit only one protease, and in fact, they only exhibit inhibitory activity limited to each protease. There is no precedent for a drug like glutathione polysulfide (GSSSG) that simultaneously inhibits the two cysteine ​​proteases of SARS-CoV-2 and exerts potent therapeutic effects.

[0022] GSSSG exerts therapeutic effects against viral pneumonia through its potent anti-inflammatory action. In fact, it has been found that intranasal administration of GSSSG to mice exhibits preventive and therapeutic effects against influenza A virus pneumonia (Figure 3). Figure 3 shows the results of an example demonstrating the anti-influenza effect of GSSSG. Multiple B6 mice (10 mice per group, 8 weeks old) were infected with 2000 pfu / ml (PBS) of influenza virus PR8 strain (A / PR / 8 / 34 (H1N1), Akaike T. et al, Journal of Infectious Diseases, 170, 1023-1026, 1994). This administration was performed by intratracheal administration of 40 μL of PBS virus solution under anesthesia. Furthermore, GSSSG (50 μL, 1 mM) was administered intranasally under anesthesia 6 hours before viral inoculation, and the survival rate, lung injury (14 days after infection), and the number of various inflammatory cells (3 days after infection) of mice were measured and analyzed over time. The survival rate, presence or absence of lung injury, and the number of various inflammatory cells were compared between the GSSSG-administered group and the control group (PBS-administered group / GSSG-unadministered group). For lung injury, the pneumonia pattern was evaluated by HE staining. To measure the number of inflammatory cells, bronchoalveolar lavage (BAL) was performed using PBS, and the number of cells and cytokines in the BAL were analyzed.

[0023] Furthermore, the therapeutic effect of GSSG was confirmed in bronchial asthma, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis models (Figure 4). Idiopathic interstitial pneumonia and pulmonary fibrosis have been reported as sequelae of SARS in the past, and there are concerns that similar intractable lung diseases may occur with COVID-19. GSSG provides broad preventive and therapeutic effects on all pathological conditions, including the entire course of SARS-CoV-2 infection from the early to late stages, as well as prognosis and complications.

[0024] Figure 4 shows an example demonstrating the therapeutic effect of GSSSG on elastase-induced COPD, using Cars2-deficient B6 mice. *1In a porcine pancreatic elastase (PPE)-induced emphysema model, GSSSG (50 μL, 1 mM) was administered via the airway three times: 6 hours prior, 2 days later, and 4 days later. The number of inflammatory cells in bronchial alveolar lavage fluid (BALF) was measured 5 days after PPE administration, and emphysema lesions were analyzed by CT. The number of inflammatory cells was measured in the same manner as in the example shown in Figure 3. In the GSSSG-administered group (GSSSG(+)), the number of inflammatory cells was lower compared to the GSSSG-free group (GSSSG(-) / control group), and as can be seen from the images obtained from CT, emphysema was confirmed to be in remission compared to the GSSSG-free group. *1:NATURE COMMUNICATIONS|8:1177,Cysteinyl-tRNA synthetase governs cysteine ​​polysulfidation and mitochondrial bioenergetics,27 October 2017.

[0025] While various active sulfur molecules exist in living organisms, the main one is GSSSG, which is glutathione with an excess of sulfur molecules attached. Because it is relatively stable and can be synthesized in large quantities with high purity, the inventor has been investigating its medicinal effects. On the other hand, it has been found that such active sulfur is also formed on the thiol group of cysteine ​​in proteins. For example, it has been revealed that the activity of the cysteine ​​thiol in the active site of thiol proteases such as papain is suppressed by this sulfur addition reaction (persulfidation).

[0026] Furthermore, the inventors have found that GSSSG not only improves severe pneumonia in a mouse model of influenza pneumonia, but also reduces airway and lung damage and pulmonary fibrosis in mouse models of bronchial asthma, COPD, and pulmonary fibrosis by suppressing the production of inflammatory cytokines. Generally, it is known that COVID-19 becomes more severe in patients with such underlying diseases. In the aforementioned CARS2-deficient mice, cytokines such as IL-6 are elevated, confirming the anti-cytokine activity of active sulfur molecules (RSS) such as GSSSG.

[0027] A major advantage of GSSSG in drug administration is that it can be administered locally to the target organs of the virus not only by injection and intravenous fluid, but also as an inhaled drug (dry powder) and eye drops. A similar approach, nitric oxide (NO) inhalation therapy, is being tested, but NO is toxic at high concentrations, difficult to set and adjust the dosage, and NO itself is a mediator of inflammation and oxidative stress, raising concerns that it may actually worsen the pathogenesis of COVID-19 infection. In particular, it may worsen the condition in patients with underlying lung disease.

[0028] Glutathione polysulfide possesses significant advantages from the perspective of its potent anti-inflammatory and immunomodulatory functions. Specifically, it is expected to have therapeutic effects in suppressing the occurrence of various complications such as cytokine storms and post-infection pulmonary fibrosis. Furthermore, because its site of action precisely targets the thiol group at the active site of the viral protease, it is expected to be highly effective in combination therapy with other drugs. Moreover, since GSSG can target not only viruses but also a wide range of host proteases, it can be applied as a preventive and therapeutic agent against various viruses, including coronaviruses other than SARS-CoV-2 and influenza.

[0029] Polysulfides, which are suitable as active sulfur compounds, include reduced and oxidized forms, and R-(S) n -SH(n=1,2,····) is the reduced form, and R-(S) n-R(n=2,3,····) is the oxidized form.

[0030] Figure 5 shows the results of the SARS-CoV-2 protease inhibition test by GSSSG. GSSSG is described in paragraph

[0025] of Japanese Patent Application No. 2017-543669.

[0031] It can be produced based on the description. Recombinant papain-like prosthesis (PLpro) and main protease (3CLpro) derived from SARS-CoV-2 were incubated with 0.3 mM GSSSG or ebselen (2-phenyl-1,2-benzoisoserenazole-3(2H)-one) and NO (nitric oxide) at 37°C for 15 minutes, and the protease activity was measured using an MCA fluorescent substrate and a fluorescence plate reader (excitation wavelength 380 nm, fluorescence wavelength 460 nm).

[0031] As shown in Figure 5, GSSSG exhibited similarly strong inhibitory activity against the two aforementioned proteases compared to the control (GSSSG(-)) with ebselen. The strong inhibitory activity of ebselen against the two proteases has been reported in the previously mentioned Non-Patent Documents 3 and 4. PLpro and 3CLpro were synthesized based on the publicly available SARS-CoV-2 genome.

[0032] For the cloning of the SARS-CoV-2 papain-like protease and main protease, artificial genes were synthesized from the SARS-CoV-2 genome (Accession number: NC_045512) for the papain-like protease (4946-5923) and main protease (10055-10972) regions. The nucleotide sequences were approximated to the codon usage frequency of E. coli. After cloning the papain-like protease into the pET53 vector, NiCo21 (DE3) was transformed, and after cloning the main protease into the pE SUMO vector, BL21 (DE3) was transformed. IPTG induction allowed the papain-like protease to be expressed as a His-tagged fusion protein and the main protease as a His-tagged SUMO-tagged fusion protein in E. coli. These recombinant proteins were purified with Ni-NTA agarose, and the main protease recombinant protein underwent further treatment with SUMO protease to remove the His tag and SUMO tag.

[0033] The papain-like protease and main protease sequences used for gene cloning are shown below. Papain-like protease sequence listing 1 Main protease sequence listing 2

[0034] The concentration of GSSSG in the preparation is usually 0.0003 μg / mL or higher, may be 0.003 μg / mL or higher, may be 0.01 μg / mL or higher, may be 0.03 μg / mL or higher, may be 0.1 μg / mL or higher, may be 0.3 μg / mL or higher, may be 1 μg / mL or higher, may be 3 μg / mL or higher, may be 10 μg / mL or higher, and may be 30 μg / mL or higher. On the other hand, the upper limit is usually 1000 μg / mL or lower, may be 300 μg / mL or lower, may be 100 μg / mL or lower, and may be 30 μg / mL or lower.

[0035] Next, a model was created by infecting African green monkey renal epithelial cells (VeroE6 / TMPRSS2 cells, obtained from the JCRB Cell Bank) overexpressing type II transmembrane serine protease (TMPRSS2) with the SARS-CoV-2 strain JPN / TY / WK-521 (obtained from the National Institute of Infectious Diseases) at an MOI of 0.01 and allowing them to grow. The anti-SARS-CoV-2 activity of GSSSG was confirmed by quantitative PCR using the 2019 novel coronavirus detection reagent kit (Shimadzu Corporation) and the CFX Connect Real-Time System (Bio-Rad Laboratories, Inc.). The model consisted of GSSSG-administered groups (10 μM, 100 μM, 300 μM) and a GSSSG-free group.

[0036] Figure 6 shows the test results for the anti-SARS-CoV-2 activity of GSSSG. As shown in Figure 6, increasing the dose of GSSSG resulted in a decrease in RNA viral load.

[0037] Glutathione trisulfide can be synthesized, for example, according to publication WO2018 / 117186. GS(S) n G(n=1-7) can also be synthesized based on 7606pp-7611pp / PNAS / May 27, 2014 / vol. 111 / no. 21 (www.pnas.org / cgi / doi / 10.1073 / pnas.1321232111). GSH(20mM) was reacted with 20mM NaHS at room temperature for 15 minutes in the presence of I2(20mM) in 20mM Tris-HCl buffer (pH 7.4). The reaction mixture was GS(S) n G was subjected to RP-HPLC for purification. HPLC was performed using a Waters e2695 series UV detector set to 210 nm. The sample was run on a TSKgel ODS-80Ts column (4.6 × 150 mm; Tosoh) at 40°C, with mobile phases A (H2O + 0.1% trifluoroacetic acid) and B (methanol) at a flow rate of 1 ml / min with a linear gradient from 2% to 70% B over 15 minutes. GS(S)n For each of G(n=3-7), the same effect as GSSSG can be observed.

Claims

1. It contains an active sulfur compound as its main active ingredient. The active sulfur compound is GSSSG, A pharmaceutical product (composition) for the treatment or prevention of chronic obstructive pulmonary disease (COPD).

2. An inhaled agent for the treatment or prevention of chronic obstructive pulmonary disease (COPD), comprising the pharmaceutical agent described in claim 1.

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