Screening methods for cytokine storm treatments and preventive agents and cytokine storm treatments and preventive agents

Abstract

To provide a superior "cytokine storm treatment / preventive agent screening method" and the "cytokine storm treatment / preventive agent".SOLUTION: A cytokine storm treatment / preventive agent screening method selects a test substance as an agent for treatment or prevention of a cytokine storm of a human. The test substance is co-administrated to a silkworm for inhibiting "the expansion or blackening of a silkworm front part, or the silkworm posterior contraction, caused when the fungus or the bacillus mort or a proteolytic enzyme to infect the silkworm is administered to the silkworm". A cytokine storm treatment / preventive agent screening method is provided for screening a proteolytic digestion enzyme inhibitor, a blood anticoagulant or an antithrombin medicine as the test substance. The cytokine storm treatment / preventive agent is the substance for inhibiting the action of proteolytic enzymes, the blood anticoagulant or the antithrombin medicine. Also, the cytokine storm treatment / preventive agent is a heparin and / or a nafamostat, a vetamethasone or soy meal.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for screening a cytokine storm treatment and preventive agent, and a cytokine storm treatment and preventive agent.

Background Art

[0002] Immunosuppressive agents are used for the treatment of various inflammatory diseases and organ transplantation for the purpose of suppressing excessive immune activation or immune response (for example, Non-Patent Document 1, etc.).

[0003] In the treatment of severe infectious diseases, in addition to eliminating pathogens using antibiotics, it is important to maintain the body fluid homeostasis of patients (Non-Patent Document 2). In sepsis and other diseases, many cases have been reported where excessive systemic immune activation (cytokine storm) accompanied by a large amount of cytokine release occurs when the disease worsens, but the prognosis is extremely poor (Non-Patent Document 3). Therefore, it is required to understand the pathological condition in which animals die due to excessive immune activation and to establish a treatment strategy based on it.

[0004] Currently used immunosuppressive agents can be classified into categories such as those that suppress the functions of immune cells, those that suppress the production of cytokines, etc., and those that exhibit cytotoxicity (Non-Patent Documents 1, 4), but further exploration of immunosuppressive agents, particularly cytokine storm treatment and preventive agents, is desired.

[0005] Under such circumstances, it is difficult to consider the pharmacokinetics and toxicity (ADMET) of candidate compounds in in vitro screening. Therefore, in the above-mentioned "understanding of the pathological condition" and "search for agents", the sacrifice of a large number of mammals is forced, which is a problem from an ethical point of view and also a problem in terms of cost. Substantially, screening that takes into account pharmacokinetics is impossible, at least in the initial stage of screening.

[0006] On the other hand, the present inventors have investigated a screening system using silkworms and have actually identified a novel antimicrobial compound that shows good therapeutic results in vertebrates through screening of antimicrobial compounds using a silkworm infection model (Patent Document 1).

[0007] Furthermore, the inventors have discovered that injecting the cell wall fraction of a fungus into the blood of a live silkworm causes the silkworm's muscles to contract, and have confirmed that this method of evaluating innate immune activity using the silkworm muscle contraction system can be used to quantitatively evaluate and screen for substances that activate the innate immune system in humans and other organisms (Patent Document 2, Non-Patent Document 5).

[0008] In the search for therapeutic and preventive agents (or potential candidates) for cytokine storms, there have been very few effective methods that take into account pharmacokinetics from the early stages of the search. Furthermore, as mentioned above, many patients suffer from inflammatory diseases caused by excessive immune response, and there has been a need for new cytokine storm treatments and preventative agents, as well as improved therapeutic effects with immunosuppressants. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2012-006917 [Patent Document 2] International Publication No. 2008 / 126905 [Non-patent literature]

[0010] [Non-Patent Document 1] Wiseman, AC (2016) Immunosuppressive Medications. Clin J Am Soc Nephrol 11, 332-343 [Non-Patent Document 2] Bochud, Pierre-Yves, Michel P. Glauser, and Thierry Calandra. "Antibiotics in sepsis." Intensive Care Med 27.Suppl 1 (2001): S33-S48. [Non-Patent Document 3] Tisoncik, Jennifer R., et al. "Into the eye of the cytokine storm." Microbiology and Molecular Biology Reviews 76.1 (2012): 16-3 [Non-Patent Document 4] Suthanthiran, M., Morris, RE, and Strom, TB (1996) Immunosuppressants: cellular and molecular mechanisms of action. Am J Kidney Dis 28, 159-172 [Non-Patent Document 5] Ishii K, Hamamoto H, Kamimura M. and Sekimizu K. (2008) Activation of the silkworm cytokines by bacterial and fungal cell wall components via a reactive oxygen species-mechanism, J. Biol. Chem. 25, 283(4), 2185-2191. [Overview of the project] [Problems that the invention aims to solve]

[0011] This invention has been made in view of the above-mentioned background art, and its objective is to provide an excellent "screening method for cytokine storm treatment and prevention agents" and a "cytokine storm treatment and prevention agent". [Means for solving the problem]

[0012] The inventors of the present invention conducted extensive research to solve the above problems and discovered that silkworms die accompanied by several specific changes in appearance due to live or dead bacteria. Furthermore, they discovered that there is a substance that, when administered to silkworms simultaneously with live or dead bacteria, suppresses the changes in the silkworms' appearance.

[0013] Furthermore, we found that the changes in the appearance of silkworms that occur when proteolytic enzymes are administered to them are identical to the "changes in the appearance of silkworms that occur when bacteria that infect silkworms or dead bacteria thereof are administered to them," as described above. Furthermore, it has been found that inhibiting the function of proteolytic enzymes in humans suppresses the "changes in the appearance of silkworms" described above when a substance known as a proteolytic enzyme inhibitor is simultaneously administered to silkworms, thus completing the present invention.

[0014] In other words, the present invention provides a screening method for cytokine storm treatments and preventive agents, characterized by selecting a test substance that suppresses the swelling or blackening of the anterior part of the silkworm, or the contraction of the posterior part of the silkworm, which occurs when a fungus that infects silkworms, dead bacteria of said fungus, or a product derived from said fungus is administered to the silkworm, by simultaneous administration to said silkworm, as an agent for treating or preventing cytokine storms in humans.

[0015] Furthermore, the present invention provides a screening method for cytokine storm treatments and preventive agents, characterized by selecting a test substance that suppresses the swelling or blackening of the anterior part of the silkworm, or the contraction of the posterior part of the silkworm, which occurs when a proteolytic enzyme is administered to the silkworm, by simultaneous administration to the silkworm, as an agent for treating or preventing cytokine storms in humans.

[0016] Furthermore, the present invention provides a screening method for cytokine storm treatments and preventive agents, which uses silkworms as the test substance to screen for substances that treat or prevent human cytokine storms, using a substance that inhibits the function of proteases (protease inhibitor).

[0017] The present invention also provides a method for screening a cytokine storm therapeutic or prophylactic agent, which comprises screening a substance for treating or preventing a human cytokine storm using a protease inhibitor as a test substance.

[0018] The present invention also provides the above-described method for screening a cytokine storm therapeutic or prophylactic agent, which comprises screening a substance for treating or preventing a human cytokine storm using a blood anticoagulant or an antithrombin agent as the test substance with the use of silkworms.

[0019] The present invention also provides a method for screening a cytokine storm therapeutic or prophylactic agent, which comprises screening a candidate substance for treating or preventing a human cytokine storm using a blood anticoagulant or an antithrombin agent as a test substance.

[0020] The present invention also provides a candidate substance for treating or preventing a human cytokine storm, which is screened using the above-described method for screening a cytokine storm therapeutic or prophylactic agent.

[0021] The present invention also provides a cytokine storm therapeutic or prophylactic agent, which is a substance (protease inhibitor) that inhibits the action of a protease.

[0022] The present invention also provides the above-described cytokine storm therapeutic or prophylactic agent, in which the substance (protease inhibitor) that inhibits the action of the protease is nafamostat. That is, the present invention provides a cytokine storm therapeutic or prophylactic agent that is nafamostat.

[0023] Furthermore, the present invention provides a cytokine storm treatment and prevention agent characterized by being a blood anticoagulant or an antithrombin agent.

[0024] Furthermore, the present invention provides a cytokine storm treatment and prevention agent in which the blood anticoagulant or antithrombin agent is heparin or nafamostat. In other words, the present invention provides a cytokine storm treatment and prevention agent in which heparin is present.

[0025] Furthermore, the present invention provides a cytokine storm treatment and preventive agent characterized by being a mixture of heparin and nafamostat.

[0026] Furthermore, the present invention provides a cytokine storm treatment and preventive agent characterized by being betamethasone.

[0027] Furthermore, the present invention provides a cytokine storm treatment and preventive agent characterized by being obtained by soybeans as they are, dried, fermented, crushed, or a combination thereof. [Effects of the Invention]

[0028] According to the present invention, agents used to treat or prevent human cytokine storms can be suitably screened using silkworms, which pose fewer ethical challenges. Moreover, as will be described later, evaluation that takes into account metabolism in the body can be performed from the early stages of screening using silkworms, thus suppressing and mitigating the elimination of candidate substances in the later stages of screening.

[0029] This invention reveals that the specific external changes in silkworms that occur when proteolytic enzymes are administered to them are similar to the external changes in silkworms that occur when "bacteria that infect silkworms, dead bacteria of said bacteria, or products derived from said bacteria" are administered to them, i.e., the external changes in silkworms that occur due to a "cytokine storm in silkworms." This suggests that substances that inhibit the activity of protease enzymes (protease inhibitors) may have the effect of treating or preventing cytokine storms in silkworms.

[0030] Furthermore, protein degradation was actually observed in the body fluids of silkworms that experienced a cytokine storm. In addition, a large number of aggregates were formed in the blood of the silkworms at this time (Example 2), suggesting that an abnormality in the blood coagulation system was occurring.

[0031] In this invention, it has been found that a substance that inhibits the action of protease enzymes (protease inhibitor) has the effect of treating or preventing "cytokine storms in silkworms" (see Examples). Therefore, it is considered that this substance also has the effect of treating or preventing cytokine storms in humans. Therefore, substances that suppress the specific external changes in silkworms described above (i.e., "cytokine storms in silkworms") could potentially become therapeutic or prophylactic agents for cytokine storms in humans (agents used to treat or prevent cytokine storms in humans). At the very least, they could be candidates for therapeutic or prophylactic agents for cytokine storms in humans.

[0032] In other words, if a test substance that suppresses "the above-mentioned specific appearance changes," namely "swelling or blackening of the anterior part of the silkworm, or contraction of the posterior part of the silkworm, which occurs when a proteolytic enzyme is administered to silkworms," ​​is screened using silkworms, this screening method could be an excellent screening method for human cytokine storm treatments and preventives.

[0033] The inventors have already reported that the "immune activity of a certain substance against silkworms" correlates with the "immune activity of the same substance in mammals such as humans." Furthermore, the inventors have already reported that antibacterial activity in mammals such as humans can be evaluated using a silkworm infection model, and that the in vivo dynamics of silkworms and mammals such as humans are similar. This invention is a screening method for cytokine storm treatments and preventive agents using silkworms, and is based on the results of studies conducted using silkworms. Based on the above facts, it is a valid screening method for "cytokine storm treatments and preventive agents in mammals such as humans."

[0034] In fact, when nafamostat, a serine protease inhibitor used in human clinical practice, was administered (injected) to silkworms simultaneously, neither the "specific appearance change" caused by the administration of trypsin, a protease, nor the "specific appearance change" caused by the administration of heat-killed Pseudomonas aeruginosa bacteria (Figure 2(a)) were observed (Figure 1(b) of Example 5 and Figure 2(b) of Example 6).

[0035] Furthermore, when silkworms were simultaneously administered (injected) betamethasone (a steroid), heparin (an anticoagulant), and soybean powder instead of nafamostat, the same "suppression of the above-mentioned specific appearance changes" as when nafamostat was administered simultaneously was observed (Examples 8 and 9).

[0036] Therefore, by using substances that inhibit the function of proteases (protease inhibitors) or anticoagulants such as antithrombin drugs (substances that control coagulation) as the screening population according to the present invention, it is possible to efficiently screen and find human cytokine storm treatments and preventive agents.

[0037] Screening for cytokine storm treatments and preventives using silkworms has a significant advantage over in vitro screening systems because it allows for consideration of the in vivo pharmacokinetics and toxicity (ADMET) of the test substance.

[0038] Furthermore, since silkworms pose fewer ethical issues, the in vivo dynamics and toxicity (ADMET) of the test substance can be considered in the early stages of a large-scale search (screening) of test substances. Therefore, this invention makes it possible to search for new cytokine storm treatments and preventive agents. Conventional screening methods would have missed certain substances (substances that could not be discovered or invented as new cytokine storm treatments or preventive agents). For example, substances that "don't have extremely high cytokine storm prevention effects but have good pharmacokinetics" can now be selected (and not missed) as "cytokine storm treatments or preventive agents."

[0039] According to the present invention, since it utilizes the "actions and mechanisms within the body of a living silkworm," screening that also takes into account the in vivo pharmacokinetics of the test substance is possible. Therefore, even from the initial screening stage when there are many test substances, it is possible to screen for cytokine storm treatments and preventive agents that also take into account "good in vivo pharmacokinetics." For example, it becomes possible to screen for drugs that treat "symptoms resulting from excessive immune function" from an early stage. This can reduce the probability of a drug failing in the final stages of human clinical trials.

[0040] This invention opens the way for screening new cytokine storm treatments and preventive agents, and enables the construction of a cytokine storm suppression model using silkworms. The screening method of the present invention can be applied to screening a small number of "substances that will ultimately (definitely) be established as immunoactivators," but it is particularly preferable to apply it to the initial stages of narrowing down cytokine storm treatment and prevention agents, as this makes it easier to exert the effects of the present invention. Therefore, the present invention is also a screening method for "substances that can be candidates for cytokine storm treatment and prevention agents."

[0041] Furthermore, because silkworms are used, there are fewer ethical issues regarding experimental animals. In other words, because silkworms are used, ethical problems are less likely to arise even in the early screening stage where many test substances are sacrificed, making it useful. [Brief explanation of the drawing]

[0042] [Figure 1] These are photographs of silkworms that died after being injected with trypsin (Figure 1(a)) and silkworms that did not die after being simultaneously injected with nafamostat, a serine protease inhibitor (Figure 1(b)) (Examples 1 and 5). [Figure 2] These are photographs of silkworms that died after being injected with heat-killed Pseudomonas aeruginosa bacteria (ACPA) (Figure 2(a)) and silkworms that did not die after being simultaneously injected with nafamostat, a serine protease inhibitor (Figure 2(b)) (Examples 2 and 6). [Figure 3] This figure shows the analysis results of silkworms that died after being injected with heat-killed Pseudomonas aeruginosa suspended in DMSO (DMSO / ACPA). (a) A graph showing the increase in the amount of protease-encoding gene mRNA in the blood of the dead silkworms (Example 2). (b) A photograph of the protein electrophoresis results showing that protein degradation was observed in the blood of the dead silkworms (Measurement Example 1, Example 2). [Figure 4] This figure shows the results of evaluating the therapeutic effect of a mixture of nafamostat and heparin using silkworms in Example 9. [Modes for carrying out the invention]

[0043] The present invention will be described below, but it is not limited to the following specific embodiments and can be modified as appropriate within the scope of the technical idea. This specification discloses at least the following inventions: [1] A screening method for cytokine storm treatments and preventive agents, characterized by selecting a test substance that suppresses the swelling or blackening of the anterior part of the silkworm, or the contraction of the posterior part of the silkworm, which occurs when a fungus that infects silkworms, dead bacteria of said fungus, or a product derived from said fungus is administered to silkworms, by simultaneous administration to said silkworms, as an agent for treating or preventing cytokine storms in humans. [2] A screening method for cytokine storm treatments and preventive agents, characterized by selecting a test substance that suppresses the swelling or blackening of the anterior part of the silkworm, or the contraction of the posterior part of the silkworm, which occurs when a proteolytic enzyme is administered to the silkworm, by simultaneous administration to the silkworm, as an agent for treating or preventing cytokine storms in humans. [3] A method for screening cytokine storm treatments and preventive agents according to [1] or [2], wherein the swelling of the anterior part of the silkworm is induced by the activation of paralytic peptides within the silkworm body. [4] The method for screening cytokine storm treatments and preventive agents according to [1] or [2], wherein the blackening of the anterior part of the silkworm is due to melaninization of the silkworm blood by activation of phenol oxidase in the silkworm body. [5] A method for screening cytokine storm treatments and preventive agents according to [1] or [2], wherein the contraction of the posterior part of the silkworm is induced by the activation of the silkworm's innate immune system. [6] A method for screening cytokine storm treatments and preventive agents according to any one of [1] to [5], wherein a substance that inhibits the function of proteases (protease inhibitor) is used as the test substance, and silkworms are used to screen for substances that treat or prevent human cytokine storms. [7] A screening method for cytokine storm treatments and preventive agents, characterized by screening for candidate substances that can treat or prevent human cytokine storms using a substance that inhibits the function of protease (protease inhibitor) as the test substance. [8] A method for screening cytokine storm treatments and preventive agents according to any one of [1] to [5], wherein a blood anticoagulant or antithrombin agent is used as the test substance, and silkworms are used to screen for substances that treat or prevent human cytokine storms. [9] A screening method for cytokine storm treatments and preventive agents, characterized by screening for candidate substances that can treat or prevent human cytokine storms using blood anticoagulants or antithrombin drugs as test substances.

[10] A candidate substance for treating or preventing a human cytokine storm, characterized by being screened using the screening method for cytokine storm treatments and preventive agents described in any of [1] to [9].

[11] A cytokine storm treatment and preventative agent characterized by being a substance that inhibits the function of protease enzymes (protease inhibitor).

[12] The cytokine storm treatment and prevention agent described above, wherein the substance that inhibits the action of the aforementioned protease (protease inhibitor) is nafamostat

[11] .

[13] A cytokine storm treatment and preventive agent characterized by being a blood anticoagulant or antithrombin agent.

[14] The cytokine storm treatment and prophylactic agent according to the claim, wherein the blood anticoagulant or antithrombin agent is heparin or nafamostat

[13] .

[15] A cytokine storm treatment and preventive agent characterized by being a mixture of heparin and nafamostat.

[16] A cytokine storm treatment and preventive agent characterized by being betamethasone.

[17] A cytokine storm treatment and preventive agent characterized by being obtained from soybeans in their natural state, dried, fermented, ground, or a combination thereof.

[0044] The present invention provides a screening method for cytokine storm treatment and prevention agents, characterized by selecting a test substance that suppresses swelling or blackening of the anterior part of the silkworm, or contraction of the posterior part of the silkworm, which occurs when a fungus that infects silkworms, dead bacteria of said fungus, or a product derived from said fungus is administered to the silkworm, by simultaneous administration to said silkworm, as an agent for treating or preventing human cytokine storms.

[0045] Furthermore, the present invention's screening method for cytokine storm treatment and prevention agents is characterized by selecting a test substance that suppresses the swelling or blackening of the anterior part of the silkworm, or the contraction of the posterior part of the silkworm, which occurs when a proteolytic enzyme is administered to the silkworm, by simultaneous administration to the silkworm, as an agent for treating or preventing cytokine storms in humans.

[0046] Here, the above-mentioned bacteria administered to silkworms are not particularly limited as long as they can infect silkworms and kill the individual silkworms. However, in terms of safety for experimenters, versatility, and ease of handling, examples include bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli; and fungi such as Candida. Among these, Staphylococcus aureus and Pseudomonas aeruginosa are particularly preferred for the reasons mentioned above.

[0047] The substances administered to silkworms are fungi that infect silkworms, dead fungi, or products derived from such fungi. The fungi can be live or dead, and include, for example, live fungi, heat-treated fungi, moist fungi, and dried fungi. "Products derived from fungi" include parts of fungi (components of fungi), fungal products, and processed fungal cells obtained by treating fungi in some way. Specific examples of bacterial-derived products include, for example, cultures such as bacterial culture supernatant; suspensions; dilutions; concentrates; pastes; dried products such as spray-dried, freeze-dried, vacuum-dried, and drum-dried products; liquefied products; dilutions; crushed products; sterilized products such as heat-sterilized and radiation-sterilized products; extracts from the cultures; enzyme-treated products; and so on. Hereafter, the terms "live bacteria, dead bacteria, and products derived from the bacteria" may be abbreviated simply as "dead bacteria, etc."

[0048] Regarding the "method of administering the fungus to silkworms," ​​it is sufficient to introduce the above-mentioned "dead bacteria, etc." into the silkworm's body, and there are no particular limitations, but injection into the silkworm's blood or intestinal tract, or mixing into the feed (dropping or adding dead bacteria, etc. to the feed) are preferred. In the case of injection, it is preferable to use a solution in which the "dead bacteria, etc." are dispersed in a solvent or dispersion medium. Here, the "solvent or dispersion medium" is not particularly limited as long as it does not itself cause lethality to experimental animals (silkworms), but dimethyl sulfoxide (DMSO) and the like are preferred in terms of safety, cost, and track record of use.

[0049] The single dose (volume) of the above solution administered to silkworms is not particularly limited, but is preferably 1 to 200 μL / g per gram of silkworm, more preferably 5 to 150 μL / g, and particularly preferably 20 to 100 μL / g. The dosage of "dead bacteria, etc." itself is not particularly limited and depends on the type of bacteria, but it is preferably 50 to 500 μL per gram of silkworm, more preferably 100 to 400 μL, and especially preferably 150 to 300 μL, as the equivalent amount of overnight culture medium.

[0050] The silkworms used can be from any stage from the 1st to the 5th instar, but the 4th or 5th instar is preferred. While there are no particular limitations on the rearing temperature of silkworms during the implementation of this invention, it is preferably constant, preferably 7 to 45°C, more preferably 10 to 40°C, and particularly preferably 20 to 35°C. It is preferable that no further feeding (in terms of quantity) is provided to the silkworms after administering the "dead bacteria, etc."

[0051] Preferably, the dosage and other settings should be set so that the time from the administration of "dead bacteria, etc." to the confirmation of "specific changes in the appearance of silkworms" or "death of silkworms" is 15 minutes to 3 days, more preferably 30 minutes to 1 day, and particularly preferably 1 hour to 5 hours. Within the above-mentioned elapsed time (number of days), if a single point in time elapses (number of days), external changes will occur when they are expected to occur (silkworms that are expected to die will die), and external changes will not occur when they are not expected to occur (silkworms that are expected to survive will survive). As a result, the outcome saturates with respect to the passage of time, and the evaluation becomes stable. In addition, time is not wasted by unnecessarily long elapsed times (number of days).

[0052] Specific external changes in silkworms that occur when dead bacteria or other substances are administered to them include "swelling or blackening of the front of the silkworm, or contraction of the rear of the silkworm." Hereafter, the above terms in parentheses may be simply abbreviated as "specific external changes in silkworms."

[0053] The "swelling of the silkworm's anterior region" is induced by the activation of paralysis peptides, which are insect cytokines, within the silkworm's body. The "blackening of the silkworm's anterior region" is due to the melanization of the silkworm's blood caused by the activation of phenol oxidase within the silkworm's body. The contraction of the silkworm's posterior region is induced by the activation of the silkworm's innate immune system.

[0054] These "specific external changes in silkworms" that occur when "dead bacteria, etc." are administered to silkworms closely resemble paralysis caused by an excessive immune response in mammals, or shock death caused by the action of proteolytic enzymes in mammals. It was found that these phenomena also occur when silkworms are administered "dead bacteria, etc." This suggests that a lethal immune response (cytokine storm) can be induced in silkworms as well.

[0055] Here, the above-mentioned test substances are not particularly limited as long as they have even a slight possibility of being candidates for cytokine storm treatment or prevention (substances that can be screened). They may be living organisms such as bacteria, viruses, or inanimate objects, single compounds or mixtures, and liquids or solids.

[0056] The term "simultaneous administration" as described above is not limited to administering the "dead bacteria, etc." and the "test substance" completely at the same time (for example, by simultaneous injection). The timing of administration may vary as long as the dead bacteria, etc. and the test substance coexist within the silkworm's body.

[0057] The test substance may be administered to silkworms and allowed to circulate for a period of time before administering the dead bacteria, etc., to the silkworms, or they may be administered completely simultaneously. Alternatively, the dead bacteria, etc. may be administered to the silkworms and allowed to circulate for a period of time before administering the test substance. However, since the death of silkworms administered with the dead bacteria, etc. occurs in a short period of time, it is preferable to administer the test substance to the silkworms and allowed to circulate for a period of time before administering the dead bacteria, etc., to the silkworms, or to administer them completely simultaneously.

[0058] It is preferable to evaluate silkworm groups administered with a positive or negative control substance in parallel with the administration of the test substance, but in this case, it is preferable that the rearing conditions, elapsed time, etc., be substantially the same (compared under identical conditions).

[0059] As described above, in this invention, it was found that substances that inhibit the function of protease enzymes (protease inhibitors) suppress the "specific changes in the appearance of silkworms" and "death of silkworms" that occur when dead bacteria, etc., are administered to silkworms. As a result, the "screening method for cytokine storm treatment and prevention agents" of the present invention was discovered (the present invention was completed).

[0060] The present invention is a screening method for cytokine storm treatment and prevention agents, characterized by selecting a test substance that suppresses the aforementioned "specific appearance changes in silkworms" that occur when proteases are administered to silkworms, by being administered to the silkworms simultaneously.

[0061] Regarding the administration of "protein-degrading enzymes," all aspects, including preferred ranges, are the same as those (content) for the administration of "dead bacteria, etc." described above, including the method of administration to silkworms, the age of the silkworms used, the test substance, "specific external changes in silkworms," ​​the time from the time of administration to the observation of "specific external changes in silkworms," ​​and the "definition of simultaneous administration."

[0062] The proteolytic enzyme used in the above-described "Screening Method for Cytokine Storm Therapy and Prevention Agents" of the present invention is not particularly limited in terms of the peptide chain cleavage site, the molecular weight of the easily degraded protein (protease, peptiase, etc.), catalytic mechanism, etc., but examples include trypsin, chymotrypsin, papain, and other innate immune activators.

[0063] Based on the aforementioned scientific content, and considering that a "substance that inhibits the function of proteases (e.g., nafamostat)" as a test substance suppressed and prevented "specific changes in the appearance of silkworms" when "dead bacteria, etc." were administered to silkworms, it is preferable to select a "substance that inhibits the function of proteases" as the test substance, i.e., as the screening population, or to select a "substance that can act as a 'protease inhibitor' in humans or an agent known as a 'protease inhibitor'."

[0064] In other words, the present invention's screening method for cytokine storm treatment and prevention agents is preferably a method that uses silkworms as the population of test substances for screening, with protease inhibitors being substances that inhibit the function of proteases, in order to screen for substances that treat or prevent human cytokine storms.

[0065] The above screening method uses silkworms, but it is not limited to silkworm screening. Generally, when screening for substances that treat or prevent cytokine storms, protease inhibitors can be selected as the test substance, and screening can be conducted using this as the population (starting from there). Examples of experimental animals in this case include mammals, including humans.

[0066] It is not necessarily true that all substances screened by the screening method of this invention will be manufactured and sold as cytokine storm treatments or preventive agents. Therefore, the present invention is also a screening method for cytokine storm treatments and preventive agents, characterized by screening for substances that are candidates for treating or preventing human cytokine storms. According to the present invention, screening can be performed using silkworms, taking into account the pharmacokinetics in the human body. By using the screening method of the present invention in the initial stages of screening, the probability of discovering substances that will be manufactured and sold as cytokine storm treatments and preventive agents can be increased (the number of substances in the population can be reduced).

[0067] In fact, as shown in Example 7, the screening method described above was confirmed to be feasible and useful when using mice as experimental animals. In other words, mice administered with proteases exhibited shock symptoms and died. This result indicates that the innate immune system in mammals, such as mice, also underwent lethal hyperactivation (cytokine storm) by proteases (see Example 7).

[0068] Furthermore, nafamostat, already known as a protease inhibitor, suppressed the shock death of the mice (see Example 7). In other words, nafamostat, a protease inhibitor, showed therapeutic effects against shock death in mammals (mice) caused by protease.

[0069] Accordingly, the present invention is also a screening method for cytokine storm treatments and preventive agents, characterized by screening for substances that treat or prevent human cytokine storms using a substance that inhibits the function of proteases (protease inhibitor) as the test substance (preferably using experimental animals such as mammals).

[0070] This invention has revealed that substances that inhibit proteolytic enzymes can be used to treat and prevent cytokine storms. This indicates that proteolytic enzyme inhibitors can be candidates for use as "cytokine storm treatments and preventive agents" with limited applications.

[0071] This invention has shown that substances that inhibit the function of proteases in general (protease inhibitors), as well as agents already known as protease inhibitors, can be used as therapeutic and preventive agents for cytokine storms. Therefore, the present invention is characterized by being a substance that inhibits the function of protease enzymes (protease inhibitor), and is also a substance that can be used as a treatment or preventive agent for cytokine storms, or a candidate for such a substance.

[0072] The present invention is also a cytokine storm treatment and preventive agent in which nafamostat is the substance that inhibits the function of the above-mentioned protease enzyme (protease inhibitor). In other words, the present invention is also a cytokine storm treatment and preventive agent consisting of existing protease inhibitors such as nafamostat.

[0073] Similar effects were observed when using steroid drugs or anticoagulants such as antithrombin drugs instead of substances that inhibit the action of proteases (protease inhibitors) (Examples 8 and 9).

[0074] Since "antithrombin drugs and other blood anticoagulants" (e.g., nafamostat, heparin) and steroid drugs (e.g., betamethasone) suppressed and prevented "specific changes in the appearance of silkworms" as test substances, it is preferable in the screening method of the present invention to use blood anticoagulants, antithrombin drugs, or steroid drugs as test substances, or to use "agents that are known to exhibit the above-mentioned effects in humans."

[0075] In other words, the present invention's screening method for cytokine storm treatment and prevention agents is preferably a method for screening substances that treat or prevent human cytokine storms using silkworms, with blood anticoagulants or antithrombin drugs as the test substances.

[0076] The above screening method uses silkworms, but it is not limited to silkworm screening. Generally, when screening for substances that treat or prevent cytokine storms, anticoagulants such as antithrombin drugs can be selected as the test substance, and screening can be conducted using this as the population (starting from there). Examples of experimental animals in this case include mammals, including humans.

[0077] Accordingly, the present invention is also a screening method for cytokine storm treatments and preventive agents, characterized by screening for candidate substances that can treat or prevent human cytokine storms, using a blood anticoagulant or antithrombin drug as the test substance (preferably using experimental animals such as mammals).

[0078] This invention has shown that anticoagulants such as antithrombin drugs and steroid drugs can be used as therapeutic and preventive agents for cytokine storms. Therefore, the present invention is a cytokine storm treatment / preventive agent characterized by being a blood anticoagulant or an antithrombin agent, or a substance that could be a candidate for a cytokine storm treatment / preventive agent.

[0079] Specifically, the present invention is also a cytokine storm treatment and preventive agent in which the above-mentioned blood anticoagulant or antithrombin agent is heparin or nafamostat. In other words, the present invention is also a cytokine storm treatment and preventive agent in which heparin or nafamostat is used.

[0080] When the therapeutic and preventive effects of a mixture of heparin and nafamostat on cytokine storms were evaluated, a synergistic effect was observed when the two were mixed (Example 9). Therefore, the present invention is also a cytokine storm treatment and preventive agent characterized by being a mixture of heparin and nafamostat. Here, "and" means mixed or used in combination.

[0081] The mass mixing ratio of heparin to nafamostat is preferably 95:5 to 60:40, more preferably 92:8 to 70:30, and particularly preferably 90:10 to 80:20. Too much heparin can counteract the effects of nafamostat due to its toxicity, and too much nafamostat can counteract the effects of heparin due to its toxicity.

[0082] Furthermore, the steroid drug betamethasone and soy flour were also found to have therapeutic and preventive effects against cytokine storms. Therefore, the present invention is both a cytokine storm treatment and preventive agent characterized by being betamethasone, and a cytokine storm treatment and preventive agent characterized by being soy flour.

[0083] The above-mentioned soy flour is preferably obtained by using soybeans as they are, or by drying, fermenting, grinding, or a combination of these processes on soybeans. In other words, the present invention is also a cytokine storm treatment and preventive agent characterized by being obtained by soybeans as they are, dried, fermented, crushed, or a combination thereof. [Examples]

[0084] The present invention will be described in detail below based on examples, but the present invention is not limited to the specific scope of the following examples. Hereinafter, when "%" is used, it means "mass%" when it relates to mass.

[0085] Preparation Example 1 <Preparation of silkworms used in the experiment> We used silkworm eggs (sold by Ehime Silkworm Seed Co., Ltd.) that were purchased and raised in the laboratory until they reached the 5th instar larva. The silkworms were reared at 27°C, and silkworms that were fed artificial feed for the first two days of the 5th instar (5th instar, 3rd day) were used in the experiment. Each silkworm used in the experiment weighed approximately 2g.

[0086] Measurement Example 1 <Measurement of protein degradation in silkworm blood (protein electrophoresis)> Protein electrophoresis (SDS-polyacrylamide gel electrophoresis) is performed using TGX FastCast. The procedure was performed using Acrylamide Kit, 12% (Bio-Rad). Samples were prepared using SDS sample buffer (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), to which 2-mercaptoethanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added. 1 μL of silkworm blood was added to each well, and electrophoresis was performed under constant voltage conditions of 200V (see Figure 3(b)).

[0087] Example 1 <Induction of a cytokine storm in silkworms by trypsin> 50 μL of a trypsin solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (concentration 50 mg / mL, solvent: 10% dimethyl sulfoxide (DMSO) / (PBS)(-))) was injected into the blood of the silkworms described above, and these silkworms were reared at 27°C without being fed. Trypsin is a type of digestive enzyme found in pancreatic juice, and is a type of protease that hydrolyzes specific peptide bonds.

[0088] Two hours after injection, it was determined whether the silkworms had died, accompanied by swelling or blackening (melanization) of the anterior part of the silkworm and contraction of the posterior part.

[0089] <<Results>> Silkworms injected with trypsin died with swelling and blackening of the anterior part and contraction of the posterior part, similar to when administered with heat-killed Pseudomonas aeruginosa bacteria (Example 2) (Figure 1(a)). These results indicate that the innate immune system functioning in the silkworm's blood underwent lethal hyperactivation due to the action of proteases. In other words, silkworms injected with trypsin died from a cytokine storm (Figure 1(a)).

[0090] Example 2 <Induction of cytokine storm in silkworms by heat-killed Pseudomonas aeruginosa bacteria> A solution (DMSO / ACPA) was prepared by suspending heat-killed Pseudomonas aeruginosa strain PAO1 (autoclaved) in 80% DMSO / water, and 50 μL of this solution was injected into the blood of silkworms. Subsequently, these silkworms were reared at 27°C without being fed. If a silkworm died two hours after injection, accompanied by swelling or blackening (melaninization) of the anterior part of the silkworm and contraction of the posterior part, it was determined to be a death due to a "cytokine storm in silkworms."

[0091] <<Results>> Silkworms injected with heat-killed Pseudomonas aeruginosa suspended in DMSO died with swelling and blackening of the anterior part and contraction of the posterior part, similar to when trypsin was administered (Example 1) (Figure 2(a)).

[0092] At this time, the mRNA level of clip4, a cytokine-related gene that encodes a protease in the blood, increased (Figure 3(a)), and degradation of blood proteins was observed (Figure 3(b)).

[0093] Protein degradation was observed in the body fluids of silkworms that experienced a cytokine storm. Furthermore, a large number of aggregates were found in the silkworms' blood, suggesting an abnormality in the blood coagulation system.

[0094] Example 3 <Effects of protease inhibitor cocktails on silkworm external changes caused by trypsin> In Example 1, trypsin was injected into the silkworm bloodstream, and at the same time, a "protease inhibitor cocktail" (manufactured by Sigma-Aldrich) with the composition "104 mM AEBSF, 80 μM aprotinin, 4 mM bestatin, 1.4 mM E-64, 2 mM leupeptin, and 1.5 mM pepstatin A" was mixed with the trypsin and injected into the silkworm bloodstream. Two hours after injection, swelling or blackening (melaninization) of the anterior part of the silkworm and contraction of the posterior part were observed.

[0095] <<Results>> The protease inhibitor cocktail suppressed the swelling or blackening (melanization) of the anterior part of the silkworm and the contraction of the posterior part, both caused by trypsin. In other words, neither the "specific appearance changes of the silkworm" as in Example 1, nor silkworm death, caused by trypsin, was observed (not shown). No cytokine storm was observed in the silkworms.

[0096] Example 4 <Effects of a protease inhibitor cocktail on silkworm appearance changes caused by dead Pseudomonas aeruginosa bacteria> In Example 2, a suspension of heat-killed Pseudomonas aeruginosa strain PAO1 (autoclaved) in 80% DMSO / water (DMSO / ACPA) was injected into the silkworm blood. Simultaneously, the same protease inhibitor cocktail used in Example 3 was injected into the silkworm blood in the same manner as in Example 3. Two hours after injection, swelling or blackening (melaninization) of the anterior part of the silkworm and contraction of the posterior part were observed.

[0097] <<Results>> The protease inhibitor cocktail suppressed the swelling or blackening (melaninization) of the anterior part of silkworms and the contraction of the posterior part caused by a suspension of heat-killed (autoclaved) Pseudomonas aeruginosa strain PAO1 in 80% DMSO / water. In other words, the "cytokine storm" in silkworms caused by the dead Pseudomonas aeruginosa was not observed (not shown).

[0098] Example 5 <Effects of nafamostat on silkworm external changes caused by trypsin> In Example 1, trypsin was injected into the silkworm bloodstream, and at the same time, nafamostat (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), a serine protease inhibitor used in human clinical practice, was mixed with the trypsin and injected into the silkworm bloodstream. Two hours after injection, swelling or blackening (melaninization) of the anterior part of the silkworm and contraction of the posterior part were observed.

[0099] <<Results>> Nafamostat suppressed the swelling or blackening (melanization) of the anterior part of the silkworm and the contraction of the posterior part, both caused by trypsin. In other words, neither the "specific appearance changes of the silkworm" as in Example 1, nor silkworm death, caused by trypsin, were observed (Figure 1(b)). No cytokine storm was observed in the silkworms.

[0100] Example 6 <Effects of nafamostat on changes in silkworm appearance caused by dead Pseudomonas aeruginosa bacteria> In Example 2, dead Pseudomonas aeruginosa bacteria (DMSO / ACPA) were injected into the silkworm blood, and at the same time, nafamostat, the same as that used in Example 5, was injected into the silkworm blood in the same manner as in Example 5. Two hours after injection, swelling or blackening (melaninization) of the anterior part of the silkworm and contraction of the posterior part were observed.

[0101] <<Results>> Nafamostat suppressed swelling or blackening (melanization) of the anterior part of silkworms and contraction of the posterior part caused by dead Pseudomonas aeruginosa bacteria. In other words, no cytokine storm in silkworms caused by dead Pseudomonas aeruginosa bacteria was observed (Figure 2(b)).

[0102] Example 7 <Effect of nafamostat on trypsin-induced mouse death> 100 μL of a trypsin solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., concentration 500 mg / mL, solvent: physiological saline) was injected into the peritoneal cavity of mice, and these mice were then raised at 27°C without being fed. In addition, while trypsin was injected into the peritoneal cavity of mice, 1 mg of nafamostat (Fujifilm Wako Pure Chemical Corporation), a serine protease inhibitor used in human clinical practice, was mixed with the trypsin and injected into the peritoneal cavity of the mice simultaneously. Convulsions and cardiac arrest were observed sequentially for up to 1 day after injection. Trypsin is a type of digestive enzyme found in pancreatic juice, and is a type of protease that hydrolyzes specific peptide bonds.

[0103] <<Results>> Mice injected with trypsin exhibited shock symptoms and died within 10 minutes to 1 day. This result indicates that the innate immune system in mice underwent lethal hyperactivation due to the action of proteases. In other words, the mice injected with trypsin died due to a cytokine storm. Furthermore, nafamostat suppressed trypsin-induced shock death in mice. In other words, nafamostat showed a therapeutic effect against trypsin-induced shock death in mice.

[0104] <Summary of Examples 1-7> In the examples, it was shown that injecting trypsin, a proteolytic enzyme, into the blood of silkworms can induce a lethal immune response (cytokine storm) characterized by anterior swelling and blackening. Furthermore, the breakdown of body fluid proteins, which is the action of proteases, was also observed when heat-killed Pseudomonas aeruginosa bacteria were suspended in 80% DMSO and injected into the blood of silkworms. These silkworms died, accompanied by anterior swelling and blackening, similar to the case when trypsin was injected.

[0105] Furthermore, similar to the lethal immune response (cytokine storm) observed in silkworms administered with proteolytic enzymes, mice administered with proteolytic enzymes also exhibited shock symptoms and died.

[0106] These results suggest that shock death caused by the action of proteases, previously known only in higher animals, can also occur in insects such as silkworms, and that this shock death can also occur during lethal bacterial infections.

[0107] Furthermore, the examples demonstrated that protease inhibitors have a "therapeutic effect" against cytokine storms in silkworms, taking into account their favorable in vivo pharmacokinetics. In particular, the fact that nafamostat, a serine protease inhibitor used in human clinical practice, showed therapeutic efficacy demonstrates that cytokine storms can be treated with protease inhibitors, and that it is possible to "search for novel (human) cytokine storm treatments and preventive agents" using silkworms.

[0108] Example 8 Similar to Example 4, in addition to protease inhibitors, other drugs were screened for the "cytokine storm in silkworms" caused by dead Pseudomonas aeruginosa suspended in DMSO. Specifically, we investigated the therapeutic effects of over 1,000 types of drugs, including antibiotics and vitamins, as well as plant extracts, on cytokine storms in silkworms. In other words, similar to Example 4, we observed swelling or blackening (melanization) of the anterior part of the silkworm and contraction of the posterior part.

[0109] <<Results>> The above screening results showed that betamethasone (a steroid), heparin (an anticoagulant), and soybean powder all suppressed the swelling or blackening (melaninization) of the anterior part of silkworms and the contraction of the posterior part caused by a suspension of heat-killed (autoclaved) Pseudomonas aeruginosa strain PAO1 in 80% DMSO / water. In other words, silkworms administered these substances simultaneously did not exhibit the "cytokine storm" caused by the dead Pseudomonas aeruginosa (not shown).

[0110] Here, "soybean powder" refers to powder obtained by drying, fermenting, grinding, or a combination of these processes using soybeans as they are.

[0111] Example 9 In the same manner as in Example 4, the therapeutic effect of a mixture of nafamostat and heparin on a "cytokine storm in silkworms" caused by dead Pseudomonas aeruginosa suspended in DMSO was investigated. Both nafamostat and heparin are used in humans as medications affecting the blood coagulation system. Specifically, nafamostat is used as an antithrombin drug, and heparin is used as an anticoagulant.

[0112] <<Results>> As shown in Figure 4, a mixture of nafamostat and heparin showed a synergistic therapeutic effect against "cytokine storms in silkworms" caused by dead Pseudomonas aeruginosa suspended in DMSO. The numbers in Figure 4 represent the percentage of silkworms (5 in total) that escaped the "silkworm cytokine storm."

[0113] In other words, neither nafamostat at 50 μg / larva nor heparin at 250 μg / larva showed significant therapeutic effects when used alone, but a clear therapeutic effect was observed when both were used in combination (see Figure 4). Specifically, at least the "mixture of nafamostat at 50 μg / larva and heparin at 250 μg / larva" (circled in Figure 4) showed a clear therapeutic effect (see Figure 4). This indicates that mixing nafamostat and heparin produces a synergistic effect.

[0114] Furthermore, even when the dosage is standardized to the combined amount of both (300 μg / larva), Figure 4 shows that 300 μg / larva of heparin alone is less effective than the mixture of 50 μg / larva of nafamostat and 250 μg / larva of heparin. Furthermore, administering large doses of nafamostat alone, such as 300 μg / larva, is not practical because it can lead to toxicity.

[0115] <<Results>> The steroid betamethasone, the anticoagulant heparin, and soybean powder suppressed the swelling or blackening (melaninization) of the anterior part of silkworms and the contraction of the posterior part caused by a suspension of heat-killed (autoclaved) Pseudomonas aeruginosa strain PAO1 in 80% DMSO / water. In other words, the "cytokine storm" in silkworms caused by the dead Pseudomonas aeruginosa was not observed (not shown).

[0116] <Summary of Examples 8 and 9> Steroid drugs; drugs affecting the blood coagulation system such as antithrombin drugs and anticoagulants; and soybean powder have been shown to be substances with "therapeutic effects" against cytokine storms in silkworms, taking into account their favorable pharmacokinetics. In particular, the fact that betamethasone, heparin, and nafamostat, which are used in human clinical practice, have shown therapeutic effects indicates that cytokine storms in humans can be treated with steroids, anticoagulants, and antithrombin drugs.

[0117] Furthermore, this study demonstrates that by using already known steroid drugs, anticoagulants, and antithrombin drugs as test substances, or by using substances with such effects as test substances, it is possible to screen for substances that could be candidates for superior cytokine storm treatments or preventive agents, or substances that treat or prevent human cytokine storms.

[0118] A mixture of nafamostat and heparin has been found to be effective as a treatment and preventative agent for cytokine storms. [Industrial applicability]

[0119] According to the present invention, it is possible to screen for cytokine storm treatments and preventive agents that take into account their pharmacokinetics, and novel cytokine storm treatments and preventive agents can be provided. Therefore, the present invention can be widely used in fields such as the study, manufacture, sale, and use of pharmaceuticals, pharmaceutical raw materials, health foods, etc.

Claims

1. A screening method for cytokine storm treatment and prevention agents, characterized in that a test substance that suppresses swelling or blackening of the anterior part of a silkworm, or contraction of the posterior part of a silkworm, which occurs when Pseudomonas aeruginosa, dead Pseudomonas aeruginosa, or a Pseudomonas aeruginosa-derived product is administered to a silkworm, is selected as a candidate agent for treating or preventing a cytokine storm in humans.

2. A method for screening cytokine storm treatment and prevention agents according to Claim 1, comprising administering a solution of heat-killed Pseudomonas aeruginosa bacteria suspended in dimethyl sulfoxide to silkworms.

3. A method for screening cytokine storm treatments and preventive agents according to claim 1 or 2, wherein the swelling of the anterior part of the silkworm is induced by the activation of paralytic peptides within the silkworm body.

4. A method for screening cytokine storm treatments and preventive agents according to claim 1 or claim 2, wherein the blackening of the anterior part of the silkworm is due to melanization of the silkworm blood by activation of phenol oxidase in the silkworm body.

5. A method for screening cytokine storm treatments and preventive agents according to claim 1 or 2, wherein the contraction of the posterior part of the silkworm is induced by the activation of the silkworm's innate immune system.

6. A method for screening cytokine storm treatments and preventive agents according to any one of claims 1 to 5, wherein a substance that inhibits the function of proteases (protease inhibitor) is used as the test substance, and silkworms are used to screen for candidate substances that treat or prevent human cytokine storms.

7. A method for screening cytokine storm treatments and preventive agents according to any one of claims 1 to 5, wherein a blood anticoagulant or antithrombin agent is used as the test substance to screen for candidate substances for treating or preventing human cytokine storms using silkworms.

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