Therapeutic use of pleuromuciline

Abstract

2. A compound of formula (I) for particular use in the treatment or prevention of non-bacterially mediated inflammatory diseases, wherein n is 0 to 4, m is 0 or 1, with the proviso that the sulfur atom and R3 are in vicinal positions (when m=0, R3 is in position 2', and when m=1, R3 is in position 1'), R is ethyl or vinyl, and R1 is hydrogen or (C 1~6 ) alkyl, and R2 is hydrogen or (C 3~6 ) cycloalkyl, or unsubstituted (C 1~6 ) alkyl, or hydroxy, preferably substituted with one or more of one or two hydroxy (C 1~6 ) alkyl, methoxy, halogen, (C 3~6 )cycloalkyl, or R1 and R2 together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocycle containing at least one nitrogen atom, or one nitrogen and one further heteroatom, such as one selected from N or O, or R1 is hydroxy, R2 is formyl, R3 is OH, OR4, a halogen atom, or R3 is attached to 2' and represents -O-(CH2)pO- where p is 2 or 3, and R4 is unsubstituted (C 1~6 ) alkyl or (C 3~6 )cycloalkyl, or a pharmaceutically acceptable salt, solvate, prodrug, or metabolite thereof.

Description

Technical Field

[0001] The present invention relates to a novel therapeutic use of pleuromutilin.

Background Art

[0002] Pleuromutilin, a compound of the following formula,

Chemical Formula

[0003] Some further pleuromutilins having the main ring structure of pleuromutilin and substituted with a primary hydroxy group have been developed, for example, as antibacterial agents. Due to their remarkable antibacterial activity, a group of pleuromutilin derivatives, amino-hydroxy-substituted cyclohexylsulfanylacetylmutilin, as disclosed in WO 2008 / 113089, have been found to be particularly interesting. As described in WO 2008 / 113089, 14-O-{[(4-amino-2-hydroxy-cyclohexyl)-sulfanyl]-acetyl}-mutilin is a particularly useful compound due to its activity against Gram-positive and Gram-negative bacteria.

[0004] Pharmaceutically active compounds (semi-synthetic compounds) derived from pleuromucilins are inhibitors of ribosomal protein synthesis in bacteria. Representative examples of semi-synthetic pleuromucilins for human use are letapamulin (approved as AltargetoP®, AltabaxP®), a topical agent approved for the short-term treatment of impetigo and infectious fissures, abrasions, or sutures, and refamulin (approved as Xenleta®), for the treatment of community-acquired bacterial pneumonia (CABP) in adults. Thiamulin (Denagard®) and barnemurin (Econor®) are two other semi-synthetic pleuromucilin derivatives that have been used systemically as antibiotics in veterinary medicine for many years. [ka]

[0005] Approved semi-synthetic compounds derived from pleuromucilin have shown excellent activity against bacterial organisms, particularly Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus (including MRSA), Moraxella catarrhalis, Legionella pneumophila, Chlamydophila pneumoniae, and Mycoplasma pneumoniae.

[0006] Inflammatory diseases are caused by an inappropriate immune response. Inflammation can be associated with a variety of causes, including, for example, cell damage, ischemia, trauma, exposure to allergens or pathogens, and when the immune system attacks the body's own tissues (autoimmune diseases). An uncontrolled, for example, overexcited immune response and / or a persistent, i.e., chronic condition is considered an inflammatory disease.

[0007] In cases of inappropriate response, inflammatory responses can become systemic, involving the entire body and potentially leading to sepsis, or they can primarily affect specific organs. This can result in dysfunction of organs of the body, including, for example, the lungs. For instance, acute lung injury (ALI) is defined as a syndrome of acute and persistent pneumonia accompanied by increased vascular permeability. It is characterized by inflammatory damage to the alveolar-capillary membranes and an excessive, uncontrolled inflammatory response within the lungs. Acute respiratory distress syndrome (ARDS) is the most severe form of ALI, involving defective oxidation and inflammation, upregulation of adhesion molecules, increased production of cytokines and chemokines, and excessive lung cell apoptosis. These syndromes are associated with the development of multi-organ dysfunction syndrome, which plays a crucial role in patient mortality, often involving multiple transfusions, shock, sepsis, and ischemia-reperfusion, and remains refractory to treatment.

[0008] Chronic inflammation occurs when the immune response persists, keeping the body in a state of constant alert. Over time, chronic inflammation can have adverse effects on tissues and organs.

[0009] Barnemurin has been shown to reduce the concentrations of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β in bronchoalveolar lavage fluid and suppress the transcription of these cytokines in the lungs (Chen, Zhang et al. 2010, Inflammation 33(5):306-14), which is consistent with in vitro studies (Zhang, Li et al. 2009, Int Immunopharmacol 9(7-8):810-816). This may contribute to the therapeutic effect of barnemurin by suppressing the effective capture of oxyradicals and inhibition of epithelial permeability during the inflammatory response to lipopolysaccharide (LPS)-induced ALI. Furthermore, pre-administration of barnemurin may significantly reduce the wet-dry ratio (W / D) in the lungs and reduce the incidence of pulmonary edema (Chen, Zhang et al. 2010, Inflammation 33(5):306-14).

[0010] We published a preprint of a preliminary analysis of the anti-inflammatory activity of refamulin in a lipopolysaccharide-induced pulmonary neutrophilosis model (Hafner, Paukner, et al. 2020, bioRxiv 2020.06.23.168393, doi:https: / / doi.org / 10.1101 / 2020.06.23.168393).

[0011] Specific statements regarding the potential antiviral and anti-inflammatory effects of refamlin are included in the “Q1 2020 Nabriva Therapeutics PLC Earnings Call” dated May 11, 2020 (a copy thereof is available under https: / / www.yahoo.com / news / edited-transcript-nbrv-oq-earnings-144108621.html, downloaded on June 10, 2020, and in the press release dated May 11, 2020 (https: / / investors.nabriva.com / news-releases / news-release-details / nabriva-therapeutics-reports-first-quarter-2020-financial), downloaded on May 28, 2020).

[0012] Viral diseases are one of the leading causes of morbidity and mortality worldwide. Respiratory viruses such as influenza, respiratory syncytial viruses, certain adenoviruses, rhinoviruses, and coronaviruses, particularly the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19), have a significant impact on public health.

[0013] In Asheshov, Igor N. et al., *Antibiotics & Chemotherapy* 4 / 4 (1954), 380-394, the antiviral activity of pleuromucilin was first described, along with the antiviral activity of pleuromucilin itself against influenza A virus strain (PR8) at a concentration of 2 mg / mL. In contrast, pleuromucilin did not show antiviral activity against poliovirus in this study.

[0014] Furthermore, Alacorn, Balbino et al., Antiviral Research, 4(1984), 231-243 describes the antiviral activity of pleuromucilin against both DNA and RNA viruses, particularly herpes simplex virus type 1 (HSV-1), and its activity against varicella stomatitis virus (VSV), at a test compound concentration of 40 μM (15 μg / mL) that confers 50% protection (CPE50) against HSV-1-induced cytotoxic activity.

[0015] International Publication No. 2009 / 106839 advocates for the use of thiamlin as an antiviral agent, illustrating the effects of thiamlin on influenza A virus, porcine reproductive and respiratory syndrome virus (PRRSV) types 1 and 2 in a viral uptake assay 4 hours after inoculation with thiamlin concentrations of 0.1–10 μg / mL compared to valnemurin, as well as the effect of thiamlin on endosomal pH. It was stated that valnemurin did not show antiviral activity, and that other pleuromucilin antibiotics have not been found to be effective against viruses.

[0016] The alteration of endosomal or lysosome pH by thiamrin, and the subsequent inhibition of the fusion of the viral membrane with endosomes and lysosomes—a prerequisite for viral entry—were described as potential mechanisms of action.

[0017] Chinese Patent No. 103204787 and Chinese Patent No. 103242210 both disclose further pleuromucilin derivatives and generally refer to their use in antiviral drugs, but do not disclose actual evidence of antiviral activity. [Overview of the Initiative] [Means for solving the problem]

[0018] Surprisingly, it was found that the pleuromucilin derivative disclosed in International Publication No. 2008 / 113089 exhibits immunomodulatory and anti-inflammatory effects, regardless of its antibacterial activity.

[0019] Accordingly, in a first aspect, the present invention relates to the compounds described in claims 1 to 6, in particular refamulin, or any pharmaceutically acceptable salts, solvates, or metabolites thereof, for specific use in the treatment or prevention of non-bacterial inflammatory diseases.

[0020] In a further embodiment, the present invention relates to a method for treating or preventing inflammatory diseases not mediated by bacteria, and includes administering a compound according to any one of claims 1 to 6, in particular refamulin, or a pharmaceutically acceptable salt, solvate, or ester of a metabolite thereof, to a subject in need of such treatment.

[0021] Furthermore, the compound exhibits antiviral activity. Therefore, the compound is also suitable for use in the treatment and prevention of virus-borne diseases, i.e., viral infections, as well as in methods for treating or preventing such diseases.

[0022] Accordingly, in another aspect, the present invention relates to the compounds described in claims 1 to 6, in particular refamulin, or a pharmaceutically acceptable salt, solvate, prodrug or metabolite thereof, for specific use in the treatment or prevention of viral infections and inflammatory conditions associated with or caused by viral infections, wherein the compounds of formula I are administered both to treat and / or prevent viral infections themselves and to treat and / or prevent inflammatory conditions.

[0023] In a further embodiment, the present invention relates to a method for treating or preventing viral infections and inflammatory conditions associated with or caused by viral infections, comprising administering a compound, particularly refamulin, or a pharmaceutically acceptable salt, solvate, prodrug, or metabolite thereof, wherein both viral infections and inflammatory conditions are treated and / or prevented.

[0024] In yet another aspect, the present invention relates to refamulin in the form of an acid addition salt with itaconic acid, and more particularly to itaconic acid refamulin. [Brief explanation of the drawing]

[0025] [Figure 1] This figure demonstrates the effect of refamulin on reducing the total cell count in bronchoalveolar lavage fluid (BALF) of mice loaded with lipopolysaccharide (LPS). Error bars indicate the standard error of the mean. Diamond marks above the bars indicate a statistically significant reduction (p<0.05, Mann-Whitney test) compared to the untreated control. [Figure 2] This figure demonstrates the effect of refamulin on the reduction of neutrophil count in bronchoalveolar lavage (BALF) fluid from mice loaded with lipopolysaccharide (LPS). Error bars indicate the standard error of the mean. Diamond marks above the bars indicate a statistically significant reduction (p<0.05, Mann-Whitney test) compared to the untreated control. [Figure 3A] This figure demonstrates the effects of dexamethasone, refamulin, and barnemurin on the total number of cells in bronchoalveolar lavage fluid (BALF) from mice loaded with lipopolysaccharide (LPS). [Figure 3B] This figure demonstrates the effects of dexamethasone, refamulin, and barnemurin on the number of neutrophils in bronchoalveolar lavage fluid (BALF) from mice loaded with lipopolysaccharide (LPS). [Figure 3C] This figure demonstrates the effects of dexamethasone, refamulin, and barnemurin on the number of macrophages in bronchoalveolar lavage (BALF) from mice loaded with lipopolysaccharide (LPS). [Figure 3D] This figure demonstrates the effects of dexamethasone, refamulin, and barnemurin on the number of lymphocytes in bronchoalveolar lavage fluid (BALF) from mice loaded with lipopolysaccharide (LPS). Error bars indicate the standard error of the mean. Diamond marks above the bars indicate a statistically significant decrease (p<0.05, Mann-Whitney test) compared to the untreated control. [Figure 4A] This figure demonstrates the effects of dexamethasone, refamulin, and barnemurin on the concentration of various markers in lung homogenates, particularly TNF-α. [Figure 4B] This figure demonstrates the effects of dexamethasone, refamulin, and barnemurin on various markers in lung homogenates, particularly the concentration of IL-6. [Figure 4C] This figure demonstrates the effects of dexamethasone, refamulin, and barnemurin on the concentration of various markers in lung homogenates, particularly IL-1β. [Figure 4D] This figure demonstrates the effects of dexamethasone, refamulin, and barnemurin on various markers in lung homogenates, particularly the concentration of GM-CSF. [Figure 4E] This figure demonstrates the effects of dexamethasone, refamulin, and barnemurin on the concentration of various markers in lung homogenates, particularly CXCL1. [Figure 4F] This figure demonstrates the effects of dexamethasone, refamulin, and barnemurin on the concentration of various markers in lung homogenates, particularly CXCL2. [Figure 4G] This figure demonstrates the effects of dexamethasone, refamulin, and barnemurin on the concentration of various markers in lung homogenates, particularly CCL2. [Figure 4H] This figure demonstrates the effects of dexamethasone, refamulin, and barnemurin on the concentration of various markers in lung homogenates, particularly MMP9. Error bars indicate the standard error of the mean. Diamond marks above the bars indicate a statistically significant reduction (p<0.05, Mann-Whitney test) compared to the untreated control. [Figure 5]This figure demonstrates the effects of refamulin in the forms of sodium itaconate, acetate, and itaconate on reducing cell counts in bronchoalveolar lavage fluid (BALF) from mice loaded with lipopolysaccharide (LPS). Error bars indicate the standard error of the mean. [Figure 6A] This figure illustrates the effects of refamulin and oseltamivir on clinical signs, particularly body weight, in a mouse model of influenza infection. [Figure 6B] This figure demonstrates the effects of refamulin and oseltamivir on clinical signs, particularly clinical scores, in a mouse model of influenza infection. [Figure 6C] This figure demonstrates the effects of refamulin and oseltamivir on clinical signs, particularly survival rate, in a mouse model of influenza infection. [Figure 6D] This figure demonstrates the effects of refamulin and oseltamivir on clinical signs, particularly on lung tissue pathology scoring, in a mouse model of influenza infection. [Figure 7] This figure demonstrates the effects of refamulin and oseltamivir on lung virus titers in an influenza-infected mouse model, as determined by the 50% tissue culture infectious dose (TCID50) in MDCK cells. [Figure 8A] This figure demonstrates the effects of refamulin and oseltamivir on the number of immune cells on days 3 and 6 post-infection in an influenza-infected mouse model. [Figure 8B] This figure demonstrates the effects of refamulin and oseltamivir on a bone marrow subset three days post-infection in an influenza-infected mouse model. [Figure 8C] This figure demonstrates the effects of refamulin and oseltamivir on bone marrow subsets (neutrophils, alveolar macrophages, and macrophages) 6 days post-infection in an influenza-infected mouse model. [Figure 8D] This figure demonstrates the effects of refamulin and oseltamivir on lymphocyte subsets 3 days post-infection in an influenza-infected mouse model. [Figure 8E] This figure demonstrates the effects of refamulin and oseltamivir on lymphocyte subsets 6 days post-infection in an influenza-infected mouse model. Standard one-way ANOVA and Dunnett's multiple comparisons against solvent-controlled treatments were performed at each time point. *, **, and *** represent significance levels of p<0.05, p<0.01, and p<0.001, respectively. Individual data points were excluded as outliers. [Figure 9A] This figure demonstrates the effect of refamulin on alpha-coronavirus 229E (HCoV-229E) in MRC-5 cells 6 days after viral infection. [Figure 9B] This figure demonstrates the effect of thiamrin on alpha-coronavirus 229E (HCoV-229E) in MRC-5 cells 6 days after viral infection. [Figure 9C] This figure demonstrates the effect of remdesivir on alpha-coronavirus 229E (HCoV-229E) in MRC-5 cells 6 days after viral infection. [Figure 10A] This figure demonstrates the effect of refamulin on respiratory syncytial virus type A (RSVA2) in HEp2 cells 6 days after viral infection. [Figure 10B] This figure demonstrates the effect of thiamrin on respiratory syncytial virus type A (RSVA2) in HEp2 cells 6 days after viral infection. [Figure 10C] This figure demonstrates the effect of TMC353121 on respiratory syncytial virus type A (RSVA2) in HEp2 cells 6 days after viral infection. [Modes for carrying out the invention]

[0026] Refamulin is the INN of the compound of general formula (I), and more specifically, refamulin is the compound of formula (VII), [ka] That is, 14-O-{[(1R,2R,4R)-4-amino-2-hydroxycyclohexylsulfanyl]-acetyl}mutilin (also known as "BC-3781").

[0027] In the following, the term "refamulin," when used without further explanation, is intended to encompass both refamulin in its free base form and its salts and solvates.

[0028] Refamlin was developed for systemic use to treat severe bacterial infections in humans and was approved in the United States in 2019 for medical use to treat adults with community-acquired bacterial pneumonia (CABP).

[0029] The compounds used in accordance with the present invention have been found to have immunomodulatory effects.

[0030] The experimental results indicate that, in addition to antibacterial activity, refamulin is also immunomodulatory and possesses anti-inflammatory effects. As evident from experiments using an in vivo model of lipopolysaccharide (LPS)-induced pneumonia in mice, refamulin demonstrated a dose-dependent reduction in total cells, particularly neutrophils, in bronchoalveolar lavage fluid (BALF), i.e., inhibiting cell infiltration. Furthermore, the experiments also showed a significant decrease in the concentration of pro-inflammatory mediators (e.g., cytokines, chemokines, and other factors, e.g., matrix metalloproteinase 9) at the site of inflammation at all doses tested.

[0031] Immune cell (e.g., neutrophil) infiltration is a multi-step immunological process involving the release of pro-inflammatory mediators (e.g., cytokines, chemokines, and matrix metalloproteinases). These cytokines upregulate the expression of cell adhesion molecules on capillary endothelium and subsequently mediate the migration of neutrophils into the alveolar space based on a chemotactic gradient. Similar mechanisms are expected for inflammatory processes in other sites of disease. Alterations in immune function (e.g., inhibition of pro-inflammatory mediator secretion) can treat or prevent inflammatory diseases based on inappropriate (over-excited or deregulated) or chronic inflammatory responses.

[0032] Accordingly, the present invention relates to the use of the compounds described in claims 1 to 6, particularly refamulin, in the treatment of inflammatory diseases, which are based on an inappropriate (over-excited or deregulated) or chronic inflammatory response. The diseases are not mediated by bacteria.

[0033] In bacterial-borne diseases, anti-inflammatory treatment may be desirable. This invention encompasses the treatment of inflammatory diseases not mediated by bacteria.

[0034] According to one embodiment, the inflammatory disease may be an inflammatory disease not mediated by microorganisms.

[0035] For the purposes of this invention, the term "microorganism" does not include viruses.

[0036] In preferred embodiments of the present invention, inflammatory diseases are the result of inappropriate (over-excited or deregulated) or chronic inflammatory responses, and in particular, diseases include hyperactive immune responses, neutrophil-dominant inflammatory diseases, autoimmune diseases, allergies, or dermatological inflammatory diseases.

[0037] In preferred embodiments, inflammatory diseases are hyperactive immune response states, including, for example, acute lung injury (ALI), including acute respiratory distress syndrome (ARDS), sepsis, and cytokine release syndromes, including cytokine storms. In these states, the immune response is deregulated and typically considered to be overexcited.

[0038] In particular, when not transmitted by bacteria, such an overreaction of the immune response may be a non-infectious state (i.e., not transmitted by any pathogen, including microorganisms and viruses) or a viral state.

[0039] In one embodiment, an inflammatory disease is a state of overreaction of the immune response mediated by a virus. In this embodiment, the state of overreaction of the immune response (e.g., acute lung injury (ALI), including acute respiratory distress syndrome (ARDS), sepsis, and cytokine release syndrome, including cytokine storm) may be associated with a viral infection.

[0040] In certain embodiments, the overreaction state of the immune response is mediated by a virus, particularly viral sepsis, or an acute respiratory disease associated with a viral infection such as influenza, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), or COVID-19.

[0041] In another preferred embodiment, the inflammatory disease is a neutrophil-dominant inflammatory disease, including, for example, chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis including diffuse panbronchiolitis (DPB), bronchiolitis obstructive, and noneosinophilic asthma.

[0042] In these diseases, the immune response is predominantly deregulated by neutrophil granulocyte activity. The medical need for treating neutrophil-dominant inflammatory diseases is clear, as classical anti-inflammatory therapies, such as corticosteroids, are ineffective, and nonsteroidal anti-inflammatory drugs either do not affect neutrophil function or may adversely prolong neutrophil lifespan. The results in Examples 1 and 2 demonstrate the specific effect of refamlin on neutrophil cell count.

[0043] According to one embodiment of the present invention, inflammatory diseases are autoimmune diseases. According to the present invention, examples of autoimmune diseases include multiple sclerosis, inflammatory bowel disease (Crohn's disease, colitis), rheumatoid arthritis, type 1 diabetes, and psoriasis. These diseases are based on a disregulated immune response, in which the immune response is directed towards the body's own tissues. Furthermore, these diseases are typically associated with chronic inflammation.

[0044] According to another embodiment of the present invention, inflammatory diseases are allergies. In allergies, the immune response is considered to be deregulated because the reaction to the allergen is not physiological.

[0045] In another embodiment, the inflammatory disease may be a respiratory inflammatory disease or a dermatological inflammatory disease, and preferably a respiratory inflammatory disease. Examples of respiratory inflammatory diseases include ALI (including ARDS), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), COVID-19, chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis including diffuse panbronchiolitis (DPB), obstructive bronchiolitis syndrome, or noneosinophilic asthma. Examples of dermatological inflammatory diseases include psoriasis, acne, or rosacea.

[0046] In one embodiment of the present invention, the disease is not bacterial pneumonia and / or is not related to bacterial pneumonia.

[0047] The compounds used in accordance with the present invention have been found to possess both antiviral and immunomodulatory effects. Therefore, the compounds are useful in situations where the inflammatory state is associated with a viral infection, or where the inflammatory state is caused by a viral infection. In such situations, a dual effect is desirable.

[0048] In one embodiment, the inflammatory state is an overreaction of the immune response associated with or caused by a viral infection.

[0049] In particular, the compound is intended for the treatment of patients who require treatment for both viral infections and inflammatory conditions.

[0050] In a preferred embodiment, the inflammatory state is the result of an inappropriate (over-excited or deregulated) or chronic inflammatory response associated with or caused by a viral infection.

[0051] In further embodiments, specific uses of compounds administered according to the present invention, particularly refamulin, are provided for the treatment of patients who require treatment for both viral infections and inflammatory conditions.

[0052] In preferred embodiments, the inflammatory state is an overreaction of the immune response associated with or caused by a viral infection, including, for example, acute lung injury (ALI), including acute respiratory distress syndrome (ARDS), sepsis, and cytokine release syndrome, including cytokine storm. In these states, the immune response is deregulated and typically considered to be overexcited.

[0053] In certain embodiments, specific uses of compounds administered according to the present invention, particularly refamulin, are provided for treating patients suffering from acute respiratory syndromes associated with viral infections such as influenza, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), or COVID-19.

[0054] The experimental results (Examples 6 to 15) show that, in addition to its antibacterial activity, refamulin also actively reduces the cytotoxic activity mediated by various viruses. This antiviral effect was particularly demonstrated for viruses characterized by being single-stranded positive- or negative-strand RNA viruses. Antiviral activity was demonstrated for both enveloped and non-enveloped viruses, particularly for several enveloped single-stranded positive- or negative-strand RNA viruses (such as Coronaviridae, Paramyxoviridae, Orthomyxoviridae, and Flaviviridae). Furthermore, some of the viruses investigated, including the measles virus, are known to be transmitted through respiratory routes, particularly airborne transmission. Coronaviruses and respiratory syncytial viruses also cause respiratory tract infections in humans.

[0055] In preferred embodiments of the present invention, viral infections are transmitted by single-stranded positive- or negative-strand RNA viruses. Preferably, the virus is - In particular, the Coronaviridae family, which includes human coronaviruses. - Paramyxoviruses, especially measles virus sub Family (Paramyxoviri) n ae) , and Pneumovirinae subfamily, such as respiratory syncytial viruses. Paramyxoviridae (including) )、 - In particular, the Orthomyxoviridae family, which includes influenza viruses. - In particular, the Flaviviridae family, including dengue virus and Zika virus, and - In particular, the Picornaviridae family, which includes rhinoviruses. It is selected from the group consisting of the following.

[0056] In other embodiments, viral infections are airborne diseases. Airborne diseases are transmitted by viruses that are transmitted through the air.

[0057] Viral infections can affect various organs. In preferred embodiments of the present invention, the disease is a respiratory disease including upper and lower respiratory tract infections, particularly lower respiratory tract infections.

[0058] As understood herein, treating, treating, or to treat includes, on the one hand, complete curing, curation, or to cure a condition (inflammatory disease) so that it comes to an end, and on the other hand, ameliorating, improving, or to ameliorate a condition so that its symptoms are reduced at least partially or individually.

[0059] Treatment typically involves administering a compound used according to the present invention to a subject in need, for example, in one embodiment, a subject diagnosed with both a viral infection and an inflammatory condition associated with or caused by the viral infection. The subject may have a medical history including early symptoms of a viral infection and late symptoms of an inflammatory condition, i.e., the viral infection is diagnosed early and the inflammatory condition is diagnosed later (simultaneously). Therefore, in a preferred embodiment, the compound for use according to the present invention is administered to a subject exhibiting or diagnosed with both symptoms of a viral infection and an inflammatory condition associated with or caused by the viral infection.

[0060] Preventing, prevention, or to prevent includes administering a compound before the condition is diagnosed or before all disease symptoms of the condition develop.

[0061] For example, prevention according to the present invention may be considered when a subject is infected with a virus but does not show symptoms of viral infection (asymptomatic carrier), when a subject is exposed to and / or susceptible to viral exposure, or when a subject is diagnosed with a viral infection but is not yet in an inflammatory state. Thus, in one embodiment, the compound of formula I is administered to treat the viral infection itself and to prevent an inflammatory state associated with or caused by the virus.

[0062] The appropriate dosage of the compounds administered according to the present invention, particularly refamulin, will of course vary depending on, for example, the individual host, the mode of administration, and the nature and severity of the symptoms being treated. However, generally, for satisfactory results in larger mammals, such as humans, the indicated daily dosage is in the range of about 0.5 mg to 3 g of the compounds used according to the present invention, which are conveniently administered, for example, in divided doses up to four times a day.

[0063] The compounds used in accordance with the present invention can be administered by any conventional route, for example enterally, including nasal, buccal, rectal, and oral administration; parenterally, including intravenous, intramuscular, and subcutaneous administration; or topically, including pulmonary, epidermal, intranasal, and intratracheal administration, in the form of coated or uncoated tablets, capsules, injectable solutions or suspensions, for example in the form of ampoules, vials, ointments, creams, gels, pastes, inhalation powders, foams, tinctures, lipsticks, drops, sprays, or suppositories, in a manner similar to that of the antibiotic tobramycin or macrolides, for example erythromycin, for example clarithromycin or azithromycin.

[0064] Preferably, the compounds used in accordance with the present invention are administered by inhalation, intravenous or subcutaneous injection, or orally.

[0065] Preferred pharmaceutical compositions of refamlin for injection are disclosed in International Publication No. 2016 / 202788, which is incorporated herein by reference.

[0066] The compounds used in accordance with the present invention, in particular refamulin, can be administered in the form of pharmaceutically acceptable salts, such as acid addition salts, or in free form, and optionally in the form of solvates.

[0067] In one embodiment, the compound is in the form of a salt and / or a solvate.

[0068] Salts of the compounds used in accordance with the present invention include acid addition salts. Examples of pharmaceutically acceptable acid addition salts include salts of the compounds used in accordance with the present invention with acids, such as hydrogen fumarate, fumaric acid, tartaric acid, ethane-1,2-disulfonic acid, maleic acid, naphthalene-1,5-sulfonic acid, acetic acid, malic acid, lactic acid, i.e., L-lactic acid, succinic acid, salicylic acid, azelaic acid, 2-[(2,6-dichlorophenyl)amino]benzeneacetic acid, hydrochloric acid, deuterated hydrochloric acid, preferably hydrochloric acid, acetic acid, L-lactic acid, and maleic acid.

[0069] Among these, in the case of refamulin, the acetate salt of refamulin is particularly preferred.

[0070] Preferred crystalline forms of refamulin and crystalline salt forms of refamulin are disclosed in International Publication No. 2011 / 146954, which is incorporated herein by reference. Of these, the acetate of refamulin in crystalline form B disclosed in International Publication No. 2011 / 146954 is particularly preferred.

[0071] The present invention also provides refamulin in its form as an acid addition salt with itaconic acid, particularly itaconic acid refamulin. Compounds of formula VII in the form of itaconic acid refamulin, i.e., itaconic acid salts, are disclosed herein as novel compounds (Example 3). Itaconic acid can be deprotonated to the anions hydrogen itaconate and itaconate ions. Acid addition salts containing refamulin as a cation and anions derived from itaconic acid combine the anti-inflammatory effects of refamulin and itaconic acid (Example 4).

[0072] In preferred embodiments, the compound used according to the present invention is refamulin in the form of refamulin acetate or refamulin itaconate.

[0073] Compounds used in accordance with the present invention, particularly refamulin, can be used alone or in combination with one or more other pharmaceutically active agents in the pharmaceutical treatments envisioned herein. Such other pharmaceutically active agents include other immunomodulators, such as glucocorticoids, cytokines, interferons, or antiviral agents. Such antiviral agents can preferably be selected from the group consisting of nucleoside analogs and nucleotide analogs, RNA polymerase inhibitors, such as remdesivir or ribavirin, viral protease inhibitors, such as lopinavir or ritonavir, viral neuraminidase inhibitors, such as oseltamivir, and other agents used in antiviral therapy, such as hydroxychloroquine and interferons (interferon alpha and / or beta).

[0074] Combinations include fixed combinations in which two or more pharmaceutically active drugs are contained in the same formulation, kits in which two or more pharmaceutically active drugs from separate formulations are sold in the same package, for example, with instructions for simultaneous administration, and free combinations in which pharmaceutically active drugs are packaged separately but instructions for simultaneous or sequential administration are provided.

[0075] In another embodiment, the compounds used according to the present invention, particularly refamulin, are the sole active agent administered to patients diagnosed with viral infections and inflammatory conditions associated with or caused by viral infections. That is, the patient is treated with only one active agent to treat and / or prevent both the viral infection and the inflammatory condition.

[0076] A pharmaceutical composition containing compounds used in accordance with the present invention, particularly refamulin, may further include at least one pharmaceutically acceptable excipient, such as a carrier or diluent, such as a filler, binder, disintegrant, flow modifier, lubricant, sugar and sweetener, flavoring, preservative, stabilizer, wetting agent and / or emulsifier, solubilizer, salt and / or buffer for adjusting osmotic pressure.

[0077] Such pharmaceutical compositions can be manufactured, for example, by conventional methods, such as mixing, granulation, coating, dissolution, spray drying, or freeze-drying processes. A unit dosage form may contain, for example, about 0.5 mg to about 3000 mg, or, for example, 10 mg to about 600 mg.

[0078] The subjects requiring treatment as envisioned by the present invention may be any living subjects suffering from an inflammatory disease not transmitted by bacteria. The subjects may be humans or animals.

[0079] example In this specification, the following abbreviations are used, including in examples. 1 H-NMR Proton Nuclear Magnetic Resonance Spectroscopy Celsius (℃) μM micromolar concentration ALI acute lung injury ARDS acute respiratory distress syndrome BALB / c experimental mouse strain BALF (Bronchoalveolar Lavage Solution) BC-3781 Refamlin CC cell control CCL2 chemokine (CC motif) ligand 2 CoV coronavirus CPE cytotoxic activity, especially viral-induced activity CXCL1 chemokine (CXC motif) ligand 1 CXCL2 chemokine (CXC motif) ligand 2 DMEM Dulbecco's Modified Eagle Medium DMF (N,N-dimethylformamide) DMSO (Dimethyl Sulfoxide) eq equivalent EC 50 Half (50%) effective concentration FBS Fetal Bovine Serum GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor) HeLa immortal human epithelial cell line HEp2 human epithelial cell line Huh7 human hepatocyte cell line IL-1β Interleukin-1 beta IL-6 Interleukin-6 (IL-6) IN nasal cavity IP intraperitoneal LPS (Lipopolysaccharide) MDCK (Maidin Darby Canine Kidney Cells) MMP9 Matrix Metallopeptidase 9 MOI Multiplicity of infection MRC-5, UK Medical Research Council cell line 5 M molar concentration MS mass spectrometry m / z mass / charge ratio MTBE methyl-tert-butyl ether nm (nanometer) PBS phosphate buffer PO oral SC subcutaneous TC 50 Half (50%) toxic concentration TCID 50 50% (half) tissue culture infectious dose TNF-α (tumor necrosis factor alpha) Decrease in VC virus CPE XTT 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide

[0080] Example 1 Objective: To investigate the anti-inflammatory effect of refamlin in a model of LPS-induced pulmonary neutrophilism by determining the total cell count and neutrophil count in BALF 4 hours after LPS administration.

[0081] Methods: Six groups of animals, each consisting of eight BALB / c mice, were tested. The compound refamulin was tested at subcutaneous doses of 35, 70, and 140 mg / kg, administered 30 minutes prior to intranasal LPS loading. Dexamethasone, a reference compound and known clinically used anti-inflammatory drug, was orally administered at a dose of 0.5 mg / kg 1 hour prior to LPS loading. All mice were anesthetized prior to LPS loading and intraperitoneally injected with a mixture of ketamine hydrochloride and xylazine hydrochloride. Healthy control mice were administered 50 μL of saline, and all other animals were intranasally administered 5 μg of E. coli-derived LPS / 50 μL of saline to induce pulmonary neutrophilia. Approximately 4 hours after LPS administration, the mice were euthanized by intraperitoneal administration of an overdose of anesthetic. Tracheostomy was performed, and a tracheal catheter was clamped into the trachea. The lungs were washed three times with cold PBS in a total volume of 1 mL (0.4, 0.3, and 0.3 mL). The collected BALF was placed in an Eppendorf test tube and centrifuged in a benchtop Eppendorf centrifuge (5 min / 3500 rpm / 4°C). The resulting cell plug was resuspended in 600 μL of PBS by vortex. The BALF was immediately analyzed for total cell count and differential cell count using an automated blood analyzer Sysmex XT-2000iV. Statistical analysis was performed using GraphPad Prism version 5.04 (GraphPad Software, Inc., La Jolla, California, USA). The Mann-Whitney test was used to determine the difference between groups, and a P < 0.05 was considered statistically significant.

[0082] result Figures 1-2 show the results of BALF analysis (total cell count and neutrophil count) 4 hours after intranasal LPS loading. LPS loading induced a statistically significant increase in total cell count and neutrophil count compared to the saline loading control. Compared to LPS-loaded animals, mice pre-treated with the reference substance dexamethasone PO 1 hour prior to loading showed a statistically significant decrease in both total cell count and neutrophil count in BALF. In a dose-dependent manner, all groups treated with refamulin at the prescribed dose also showed a statistically significant decrease in total cell count and neutrophil count (p<0.05 compared to lipopolysaccharide loading). Nearly complete impairment of cell influx was observed at the highest dose of 140 mg / kg. The refamulin-related decrease in BALF cell count was comparable to that observed after treatment with the reference substance dexamethasone, a known anti-inflammatory agent, at all refamulin doses. Notably, exposure to SC at a dose of 70 mg / kg of refamlin per day corresponds to the exposure achieved in humans when administered the approved clinical daily dose of 2 × 600 mg orally or 2 × 150 mg intravenously for the treatment of bacterial pneumonia.

[0083] In the early stages of ARDS, there is widespread neutrophilic alveolitis with destruction of alveolar epithelium and endothelial barrier, and inhibiting neutrophil pulmonary infiltration, which leads to the formation of protein-rich edema in the interstitium and alveolar space, may be beneficial (Matthay, Ware et al. 2012, Clin Invest 122(8):2731-2740).

[0084] Example 2 Objective: In a second study comparable to the above, the activity of refamulin at doses of 10, 30, and 100 mg / kg was further investigated and compared with barnemurin in a mouse LPS-induced pulmonary neutrophilosis model, including the determination of cell counts in BALF and the evaluation of cytokine and chemokine concentrations.

[0085] Methods: Mice were anesthetized intraperitoneally (IP) with a combination of ketamine and xylazine before intranasal (IN) LPS administration. For IN infusion, mice were held in a tilted supine position with their heads raised to 60-75 degrees above their feet during and after the infusion (approximately 1 minute). Loading was performed 30 minutes after treatment with 50 μL of saline / 5 μg of LPS in the mouse. Non-loaded control mice were given 50 μL of saline. The group size was 8 animals per group.

[0086] Thirty minutes before LPS loading, 1 mg / kg of the reference compound dexamethasone (positive control) was administered intraperitoneally (IP). Dexamethasone was dissolved in 0.5% methylcellulose in water. The dose was 10 mL / kg. 10, 30, or 100 mg / kg of refamulin or barnemurin were administered subcutaneously (SC) between the shoulders 30 minutes before LPS loading. Refamulin and barnemurin were dissolved in 0.9% physiological saline. 0.9% physiological saline was used as a solvent control. The dose was 10 mL / kg. Four hours after LPS loading, mice were sacrificed by overdose of ketamine (200 mg / kg) and xylazine (16 mg / kg), and BALF and lung tissue were subsequently sampled.

[0087] To collect bronchoalveolar lavage fluid (BALF), a cannula was inserted into the trachea, and the lung was then washed with three volumes of PBS (0.4, 0.3, and 0.3 mL, totaling 1 mL). The collected BALF was placed in an Eppendorf test tube and centrifuged in a benchtop Eppendorf centrifuge (5 min / 1303xg / 4°C). The cell plugs were resuspended in 600 μL of PBS by shaking the contents of the closed tube on a vortex. The resuspended BAL cells were immediately analyzed for total cell count and differential cell count using an automated blood analyzer, Sysmex XT-2000iV.

[0088] Lung tissue samples were taken to determine specific markers. The lung was exposed, the chest was gently opened, and the lung was resected by cutting both sides of the sternum and ribs and trimming the posterior portion. The lung was removed and placed in pre-weighed sterile Precellys. After sampling, the Precellys test tube was weighed again, rapidly frozen, and kept at -80°C until analysis. The tissue was homogenized.

[0089] The concentrations of CCL2, CXCL1, CXCL2, GM-CSF, IL-6, and TNF-α were determined using the Mouse Premixed Multi-Analyte Kit according to the manufacturer's protocol (R&D Systems). Standard dilution series and samples were prepared in parallel and incubated with the biotin antibody cocktail. Concentrations were determined using the respective streptavidin-phycoerythrin conjugates and read using a Luminex200 instrument. The concentrations of these markers were determined by interpolation from standard curves using XPONENT software (Applied Cytometry). The measured values ​​(MFI) were blank-corrected, and a standard curve-fitted 5-parameter logistic coefficient was used.

[0090] For the analysis of MMP-9 and IL-1β, whole mouse lungs were thawed from -80°C and homogenized in 1 mL of PBS supplemented with protease inhibitors in Precellys CK28 hard tissue tubes using Precellys instruments and programs. The mixture was shaken at 6800 rpm for 30 seconds with a 15-second pause, and this process was repeated three times. After homogenization, the samples were centrifuged at 18000 × g at 4°C for 10 minutes, and the supernatant was collected for analysis. The concentrations of MMP-9 and IL-1β were determined using the Mouse DuoSet ELISA kit according to the manufacturer's protocol (R&D Systems). Standard dilution series and samples were treated in parallel using plates prepared with MMP-9 or IL-1β capture antibodies. The concentrations were determined using the respective detection antibodies and readout reagents. Absorbance was measured at 450 nm using a SpectraMax i3 instrument. The concentrations of MMP9 and IL-1β in the samples were determined by interpolation from standard curves.

[0091] Statistical analysis was performed using GraphPad Prism software (version 8.1.1). Outliers were identified using the Grubbs test. Nonparametric statistics (Mann-Whitney test) were performed to evaluate total cell count, differential cell count, and marker concentrations. Differences between treatment groups relative to the solvent group were considered statistically significant at p<0.05.

[0092] result The results of BALF analysis (cell count) are shown in Figures 3A-3D. LPS loading induced a significant increase in total cell count and neutrophil count compared to the saline loading control. Subcutaneous pretreatment with 10, 30, or 100 mg / kg of refamulin dose-dependently reduced the total cell count in BALF (Figure 3A). The change was statistically significant at 100 mg / kg (p<0.05). As in previous studies, the effect was comparable to that of the anti-inflammatory drug dexamethasone (positive control). A dose-dependent and significant reduction in neutrophil cells was observed at all concentrations of refamulin examined (Figure 3B). In contrast, the cell counts of macrophages and lymphocytes were not significantly affected by refamulin or varnemurin (Figures 3C and 3D).

[0093] The pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) were increased in lung homogenates after LPS loading in a mouse model compared to saline control (Figures 4A-4C). Subcutaneous pretreatment with 10, 30, or 100 mg / kg of refamulin reduced TNF-α and IL-6 levels by more than 50% (Figures 4A and 4B), while barnemurin showed lower activity compared to the same dose. Subcutaneous pretreatment with 100 mg / kg of refamulin significantly reduced IL-1β levels in lung homogenates by 24% (Figure 4C).

[0094] Granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine known to affect cell migration, was increased in an LPS-induced mouse model (Figure 4D). Refamulin nearly completely inhibited GM-CSF in lung homogenates. Barnemurin also showed a decrease in GM-CSF levels, but its activity was lower than that of refamulin at all doses tested (Figure 4D).

[0095] The concentrations of chemokine (CXC motif) ligand 1 (CXCL1), (CXC motif) ligand 2 (CXCL2), and (CC motif) ligand 2 (CCL2) increased in lung homogenates after LPS loading in a mouse model compared to saline controls (Figures 4E-4G). Both CXCL1 and CXCL2 levels were significantly reduced by subcutaneous pretreatment with refamulin at 10, 30, or 100 mg / kg (Figures 4E and 4F). Lower doses of barnemurin (10 and 30 mg / kg) had a less significant effect on CXCL1 and CXCL2 compared to refamulin. LPS-induced CCL2 increase in lung homogenates was completely inhibited by all doses of refamulin examined, although barnemurin was less active, particularly at doses of 10 and 30 mg / kg (Figure 4G).

[0096] Matrix metallopeptidase 9 (MMP9), a regulator of neutrophil migration, was increased in an LPS-induced mouse model (Figure 4H). Subcutaneous pretreatment with 30 or 100 mg / kg of refamulin reduced MMP9 levels in lung homogenates by 26% or 53%, respectively, but did not alter MMP9 concentrations at 10 mg / kg. Barnemurin reduced MMP9 concentrations by 50% only at the highest dose investigated, 100 mg / kg (Figure 4H).

[0097] In conclusion, this study demonstrated the anti-inflammatory properties of refamlin in a mouse LPS-induced pulmonary neutrophilism model by inhibiting neutrophil infiltration into the lungs and reducing inflammatory (pro-inflammatory) factors such as cytokine and chemokine levels in the lungs.

[0098] In this model, the inhibitory activity of refamulin, at clinically appropriate doses, was comparable to that of the established anti-inflammatory drug dexamethasone (positive control) for most of the parameters investigated. Surprisingly, compared to valnemurin at the same dose, refamulin was shown to be a more potent inhibitor than valnemurin for most inflammatory (pro-inflammatory) markers.

[0099] LPS administration can induce pathological and biological changes similar to those seen in ARDS or ALI, including neutrophil infiltration and increased pulmonary cytokines, which have been widely tested in experimental models of acute lung injury (Matute-Bello Gustavo et al. 2008, Am J Physiol Lung Cell Mol Physiol 295:L379-399). Furthermore, studies suggest that increased neutrophil recruitment to the lungs may contribute to tissue damage, particularly in chronic diseases or ARDS (Giacalone, Vincent D et al. 2020, Int J Mol Sci 21(3):851).

[0100] The robust anti-inflammatory effects already observed with low doses of refamulin (e.g., 10–30 mg / kg) in the described mouse models of neutrophilic inflammation suggest that refamulin either inhibits LPS-induced pro-inflammatory signaling to reduce neutrophil accumulation or inhibits LPS-induced neutrophil infiltration into the lungs to reduce cytokine and chemokine levels. Both potential mechanisms are consistent with the dose-dependent anti-inflammatory effects observed in vivo after refamulin pretreatment.

[0101] Example 3 Objective: This example aims to synthesize itaconic acid refamulin as a potential novel pharmaceutical active ingredient, containing the protonated form of refamulin as a cation and the itaconic acid ion as an anion derived from dicarboxyitaconic acid.

[0102] Methods and Results To a solution of refamulin (1 g, 1 equivalent) as a free base in DMF (2 mL), itaconic acid (0.5 equivalents) was added and stirred overnight at room temperature. The resulting reaction mixture was added dropwise to MTBE. The resulting precipitate was filtered, washed with MTBE, and dried under reduced pressure to obtain refamulin itaconic acid (1.20 g) in the form of a colorless solid.

number

[0103] Example 4 Objective: Similar to Example 1, the anti-inflammatory effects of various refamulin salts were investigated in another modified model of LPS-induced pulmonary neutrophilism, read out 24 hours after LPS loading.

[0104] Methods: An aqueous solution of the test compound was administered as a single subcutaneous dose (5 μg / animal) 30 minutes prior to intranasal LPS loading. Immediately before LPS loading, the mouse group (n=6) was anesthetized by intraperitoneal (IP) injection of ketamine (2 mg / mouse) and xylazine (0.08 mg / mouse). To induce pulmonary neutrophilia, 50 μL of saline was administered to the control group of mice, and 5 μg of LPS / 50 μL of saline was administered intranasally to all other animals. As a control, saline (0.86% NaCl) was administered intranasally as a negative control for LPS loading. Lefamlin was investigated in the form of its acetate (Lef.Ac) and its itaconate (Lef.Itacon, synthesized as in Example 3 above), both administered at a dose of 70 mg / kg. For comparison, sodium itaconate (Na.Itacon) was investigated at a dose of 55 mg / kg. Untreated controls received subcutaneous injections of physiological saline (0.86% NaCl).

[0105] The total cell count and granulocyte cell count in BALF were determined 24 hours after intranasal LPS loading, similar to Example 1.

[0106] result The results of the BALF analysis (cell count) are shown in Figure 5. Refamulin acetate showed a reduction in cell count comparable to the effects shown in previous studies (Examples 1 and 2). Sodium itaconate itself had already shown some reduction in BALF cell count.

[0107] The properties of itaconic acid and its derivatives as immunomodulatory agents have been previously described (Yu Xia-Hua et al. 2019, Immunology & Cell Biology 97:134-141). For example, the ester derivative of itaconic acid (itaconic acid 4-octyl) showed protective effects in an LPS-induced sepsis model (Liao, Shan-Ting et al. 2019 Nat Comm 10(1);5091).

[0108] Refamlin itaconate showed the most significant effect on cell count compared to refamlin acetate or sodium itaconate alone. In this study, treatment with refamlin itaconate resulted in the lowest total cell count and granulocyte cell count, thus suggesting an interesting synergistic or additive effect of the itaconate form of refammlin.

[0109] Example 5 Objective: To investigate the effects of refamulin in an in vivo influenza virus model. In the influenza virus model, mice were loaded with an influenza A(H1N1) strain adapted for mice. The mouse immune system is particularly suitable for testing the effects of virus-induced immune responses in a mouse model of influenza virus infection.

[0110] Methods: Adult female BALB / c mice were randomly assigned to three experimental groups of 15 animals each and acclimatized for one week. Treatment was initiated with subcutaneous administration on day -1. The negative control group received a solvent administered twice daily. Refamulin was investigated at various doses. In the low-dose therapy, 35 mg / kg of refamulin was administered twice daily from day -1 to day 6 (equivalent to a dose of 70 mg / kg / day). In the high-dose therapy, refamulin was administered at a dose of 105 mg / kg / day in three injections until day 3, after which it was changed due to administration problems at the injection site. The following dose was 70 mg / kg twice daily (equivalent to 140 mg / kg / day). On day 0, all groups were administered influenza A / Puerto Rico / 8 / 34(H1N1).

[0111] During the study, animals were scored daily for clinical signs of influenza virus infection, including abnormal hair condition (erect hair), abnormal posture (crouching), abnormal respiration (tachypnea and / or irregular respiratory rate), decreased mobility, eye discharge, closed eyes, and / or survival. Signs of disease severity were added to the scoring system, with a maximum possible score of 5. If clinical signs were judged to be severe, individual animals were withdrawn from the study before the scheduled end of the study.

[0112] On the sixth day, lung tissue was dissected, macroscopic lesions were evaluated, and the tissue was preserved in fixative solution for histopathological examination.

[0113] After macroscopic evaluation, pulmonary sclerosis was assessed as widespread vascular degeneration, with alveolar edema / hemorrhage occupying more than 50% of the field of view (across all lobes). On day 6, the lungs were removed and fixed, and then microscopically evaluated histopathologically. Four main readouts were evaluated (bronchial / bronchial degeneration / hyperplasia, bronchial interstitial inflammation, alveolar epitheliitis / degeneration, alveolar epithelium / hemorrhage) and scored to obtain a maximum total histopathological score of 16, where a lower number indicates fewer signs of histopathological abnormalities.

[0114] Lung samples were also treated and stored for viral titers on days 3 and 6. On days 3 and 6, lungs were collected, homogenized, and clarified, and TCID was performed using Maidin Darby canine kidney (MDCK) cells. 50 The viral load was determined by assay.

[0115] On days 3 and 6, bronchoalveolar lavage (BALF) samples were collected, and cells were treated for flow cytometry analysis of immune cell components using the antibody panel detailed below. Absolute cell counts were performed using flow cytometry counting beads. Cells were gated from viable single events. The effect of the test treatment on immune cell subsets was analyzed using the following markers: CD45, TCRβ, CD3, CD4, CD8, CD19, Ly6C, Ly6G, MHCII, CD11b, CD11c, CD49b, Siglec-F, CD64, and viability dye. Flow cytometry data for the oseltamivir-treated group on day 3 were not collected due to treatment errors.

[0116] result The results of clinical monitoring are shown in Figures 6A to 6D. Positive control treatment with oseltamivir functioned as expected. Oseltamivir resulted in a reduction in clinical scores and weight loss. Refamulin did not have a significant effect on body weight at the doses investigated (Figure 6A). At high doses, refamulin resulted in increased clinical scores and decreased survival compared to the solvent, which may be related to local tolerance (SC) issues of the doses, concentrations, and formulations investigated (Figures 6B and 6C). 90% survival was achieved with lower doses of refamulin, while only 20% of the solvent group survived to day 6 (Figure 6C).

[0117] Furthermore, a significant improvement in macroscopically assessed pulmonary sclerosis was observed with low-dose refamulin. Histopathologically, a range of overall individual animal scores (4–13) was revealed within the examined specimens (Figure 6D). The lesions were similar to those described in the literature. Increased degree / distribution of alveolar pathology correlated with increased bronchiolar degeneration / proliferation, bronchointerstitial inflammation, and alveolar edema / hemorrhage. Treatment with high-dose refamulin resulted in a significant reduction in bronchial degeneration and alveolar inflammation, and consequently, a significant overall reduction in histopathological scores in this group compared to the solvent-treated control, comparable to oseltamivir.

[0118] Lung viral titers decreased in all groups between day 3 and day 6 (Figure 7). Both refamulin and oseltamivir doses resulted in reduced lung viral titers compared to solvent-treated controls.

[0119] The flow cytometry results for BALF are shown in Figures 8A to 8E. The total number of immune cells in BALF increased from day 3 to day 6 in all test groups (Figure 8A), confirming the expected immune cell infiltration induced by a progressive viral infection causing inflammation and cytopathogenesis in lung tissue. Refamlin significantly reduced total immune cell infiltration in the lungs by day 6 at both tested doses (p<0.05). Figures 8B to 8E show the cell counts of specific immune cell subsets at days 3 and 6. Refamlin significantly reduced neutrophil infiltration by day 6 at both tested doses (Figure 8C). At higher doses, refamlin significantly reduced inflammatory monocyte infiltration in the lungs at day 6 after H1N1 loading (Figure 8C). NK, CD4, and CD8 cell infiltration in the lungs decreased at day 6 at both tested doses (Figure 8E). The reduction in B cells was significant only at the highest dose on day 6 (Figure 8E).

[0120] The BALF results confirmed the immunomodulatory effects of refamulin, particularly on neutrophil infiltration in the lungs, in association with viral disease models. The reduction in clinical readout and lung viral titers further supports the potential of refamulin in the treatment of viral diseases.

[0121] Example 6 Objective: In the assay, the inhibition of virus-induced cytotoxic activity (CPE) and cell viability was measured in MRC-5 cells 6 days after infection with various concentrations of lefamulin (BC-3781) against alpha coronavirus 229E (HCoV-229E or CoV 229E ).

[0122] Methods: MRC-5 cells were seeded in 96-well flat-bottom tissue culture plates (at a cell density of 3×10 3 cells per well) and allowed to adhere overnight. Then, diluted test compounds dissolved in DMSO (lefamulin as acetate, tiamulin as fumarate) were added to the plates and incubated for 4 hours before adding the virus. When the virus was diluted to a predetermined titer and added, 85-95% cell death was obtained 6 days after infection (MOI was 0.001).

[0123] After incubation at 37°C and 5% CO2 for 6 days, cell viability was measured by XTT tetrazolium dye staining. The optical density of the cell culture plates was determined spectrophotometrically at 450 nm and 650 nm. The percent decrease in virus-infected cells and the percent cell survival in non-infected drug control wells were calculated, and the effective concentration (EC 50 ) at which 50% of the cytotoxic activity was inhibited and the cytotoxic concentration (TC 50 ) were determined using four-parameter curve fit analysis. The antiviral compound remdesivir served as a positive control.

[0124] Results Surprisingly, lefamulin decreased virus CPE by 91.82% at a concentration of 10 μM, which is a concentration that does not have a cytotoxic effect on the viability of cell controls. The calculated EC 50 was 3.87 μM, and 50% of the virus cytotoxic activity was inhibited. At a lefamulin concentration of 50 μM, lefamulin showed a cytotoxic effect, and the calculated TC 50 was 55.3 μM. The EC 50 , also known as the therapeutic index, and TC 50The ratio was 14.3.

[0125] In contrast, thiamrin at a concentration of 10 μM reduced viral CPE by only 10.53%, and no cytotoxic effect was observed. At the next highest test concentration of 50 μM, CPE decreased by 81.68%, and a cytotoxic effect was observed. Calculated EC 50 It is 24.4 μM, and the calculated TC 50 The concentration was 62.9 μM. The therapeutic index for thiamlin was 2.58, which was surprisingly much lower than the therapeutic index for refamlin.

[0126] The antiviral compound remdesivir was developed as a treatment for the Ebola virus and is also known to have antiviral activity against coronaviruses (in clinical trials). Therefore, remdesivir served as a positive control in this specification. Remdesivir was administered in a 0.11 μM EC25 solution. 50 , TC exceeding 5 50 It also showed a treatment index of over 45.5. [Table 1]

[0127] The results are shown graphically in Figures 9A (Refamlin), 9B (Thiamlin), and 9C (Remdesivir) (VC represents the reduction in viral CPE, and CC represents the cell control).

[0128] Example 7 Objective: The assay aims to evaluate alpha-coronavirus 229E (HCoV-229E or CoV) against refamulin under various treatment conditions. 229E In MRC-5 cells after infection, we measured the inhibition of virus-induced cytotoxic activity (CPE) and cell viability.

[0129] Methods: The assay was performed in the same manner as in Example 6 above, with the following differences for the test candidates. Refamulin (as acetate) was evaluated using different treatment conditions: 4-hour, 1-hour, or 0-hour incubation before virus addition and addition 1 hour after infection. In this particular series of experiments, coronavirus was diluted 1:200 in assay medium and added at 100 μL / well to achieve approximately 90% cell death in the untreated virus control well (MOI of 0.001).

[0130] result The antiviral efficacy and cytotoxicity data are summarized in the table below. Refamlin showed a time-dependent effect in inhibiting virus-induced cytotoxicity (CPE). A dose-dependent effect was observed when the treatment was administered post-viral exposure (1 hour post-infection). At a concentration of 50 μM, viral CPE was reduced by 86.83% (data not shown in detail). [Table 2]

[0131] Example 8 Objective: This assay aims to evaluate the efficacy of human respiratory syncytial virus (RSV) at various concentrations of refamulin (BC-3781). A2 In HEp2 cells 6 days after replication infection, we measured the inhibition of virus-induced cytotoxicity (CPE) and cell viability.

[0132] Method: HEp2 cells were seeded in a 96-well flat-bottom tissue culture plate (5 × 10 per well). 3 The cells were adhered overnight (at their respective densities). Then, diluted test compounds (refamulin as acetate, thiamulin as fumarate) in DMSO were added to the plates and incubated for 4 hours before adding the virus. When the virus was added after being diluted to a predetermined titer, 85–95% cell death was achieved 6 days after infection (MOI of 0.001).

[0133] After incubation at 37°C and 5% CO2 for 6 days, cell viability was measured by XTT tetrazolium staining. The optical density of the cell culture plates was spectrophotometrically determined at 450 nm and 650 nm. The percentage reduction in virus-infected cells and the percentage of cell viability in uninfected drug control wells were calculated, and the effective concentration (EC2) at which 50% of cytotoxic activity is inhibited was determined using 4-parameter curve-fit analysis. 50 ) and cytotoxic concentration (TC 50 The following was determined: The antiviral compound TMC353121 (RSV fusion inhibitor) was used as a positive control.

[0134] result Remarkably, refamulin reduced the cytotoxic activity (CPE) of the virus by 92.17% and 100% at concentrations of 10 μM and 50 μM, respectively, which are concentrations that have no cytotoxic effect on the viability of cell controls. Calculated EC 50 At a concentration of 5.34 μM, 50% of viral CPE was inhibited. At a refamulin concentration of 100 μM, refamulin showed cytotoxic effects, and the calculated TC 50 The concentration was 70.7 μM. EC is also known as the therapeutic index. 50 and TC 50 The ratio was 13.2.

[0135] In contrast, thiamrin at a concentration of 10 μM reduced viral CPE by only 16.76%, and a cytotoxic effect (84% survival rate) was observed at this concentration. At the next highest test concentration of 50 μM, viral CPE was reduced by 43.28%, and the cytotoxic effect was more pronounced (70.0% survival rate). Calculated EC 50 This is the calculated TC of 67.9 μM. 50 It was higher than that, exceeding 67.9 μM. Therefore, the therapeutic index of thiamlin could not be calculated. Surprisingly, the antiviral activity and therapeutic index of refamlin were much higher than that of thiamlin.

[0136] The antiviral compound TMC353121 was developed as a specific respiratory syncytial virus fusion inhibitor (in clinical trials). Therefore, TMC353121 served as the positive control in this specification. TMC353121 was tested at 0.006 μM EC2. 50 , TC greater than 0.1 μM 50 It also showed a treatment index of over 167. [Table 3]

[0137] The results are shown graphically in Figures 10A (Refamulin), 10B (Thiamulin), and 10C (TMC353121) (VC represents the reduction of viral CPE, and CC represents the cell control).

[0138] Example 9 Objective: This assay aims to alter the multiplicity of infection (MOI) of human respiratory syncytial virus (RSV) A2 In HEp2 cells after replication, we measured the inhibition of virus-induced cytotoxicity (CPE) and cell viability.

[0139] Methods: The assay was performed in the same manner as in Example 8 above, with the following differences in the test. The virus was diluted to a predetermined titer and added so that 85-95% cell death was obtained on day 6 post-infection, and the amount added was selected to obtain MOIs of 0.003, 0.001, 0.0008, and 0.0004, respectively. In this test, refamulin (as acetate) was investigated, and TMC353121 was investigated as a positive control.

[0140] result The antiviral efficacy and cytotoxicity data are summarized in the table below. EC levels of refamlin in the low μM range. 50 The values ​​were reproduced at an MOI of 0.0004. In contrast, higher MOIs, and therefore higher viral loads relative to the cells examined, reduced the antiviral effect of refamulin. This effect was less pronounced with the highly effective control substance TMC353121. [Table 4] [Table 5]

[0141] Example 10 Objective: This assay uses two different respiratory syncytial virus strains, RSV A LONG and RSV B 18537 In replicated HEp2 cells, we measured the inhibition of virus-induced cytotoxic activity (CPE) and cell viability.

[0142] Method: After pre-treating cells with different concentrations of the test compound for 4 hours, 5 × 10⁶ cells were collected per well. 3 Cells seeded at cell density with the virus strain RSV A LONG Or RSV B 18537 The assay was performed in the same manner as in Example 8 above, with the difference being that each was incubated separately. The virus was diluted and RSV A LONG and RSV B 18537 Each was added in amounts that produced MOIs of 0.01 and 0.001, respectively.

[0143] result The antiviral efficacy and cytotoxicity data are summarized in the table below. The control compound TMC353121 was evaluated in parallel with refamulin, and showed an EC of 0.01 nM against the RSV A and RSV B strains being investigated. 50 The value was obtained. Refamlin is RSV B 18537 For comparison, 17.7 μM EC 50 The value was obtained. RSV A LONG The activity against HEp2 cells is cytotoxic, and in the assay, TC 50 The value was 71.1 μM, so a determination could not be made. [Table 6] [Table 7]

[0144] Example 11 Objective: The assay measured the inhibition of virus-induced cytotoxic activity (CPE) and cell viability during replication by the measles virus strain Edmonston in HeLa cells.

[0145] Method: HeLa cells were seeded into a 96-well flat-bottom tissue culture plate (5 × 10 per well). 3 The cells were adhered overnight (at their respective densities). Then, diluted test compounds (refamulin as acetate, ribavirin as control) were added to the plates and incubated for 4 hours before adding the virus. When the virus was added at a predetermined titer, 85–95% cell death was achieved 6 days after infection (1:50 dilution, MOI 0.008).

[0146] Determination of cell viability and EC 50 and TC 50 The calculation was performed as shown in Examples 6 and 8.

[0147] result The antiviral efficacy and cytotoxicity data are summarized in the table below. Ribavirin was evaluated as a control compound in parallel with refamulin at 1.88 μg / mL EC18. 50 A value was obtained. Surprisingly, refamrin had an even lower EC of 0.89 μM. 50 It yielded a value and had a high calculated TI of 81.7. [Table 8]

[0148] Example 12 Objective: The assay aims to evaluate the dengue virus strain DENV2 in Huh7 cells. New Guinea We measured the inhibition of virus-induced cytotoxic activity (CPE) and cell viability during replication.

[0149] Method: Huh7 cells were seeded in a 96-well flat-bottom tissue culture plate (5 × 10 per well). 3The cells were adhered overnight (at their respective dengue density). Then, diluted test compounds (refamulin as acetate, ribavirin as control) were added to the plates and incubated for 4 hours before adding the virus. (Dengue virus strain DENV2) New Guinea The virus was obtained from ATCC (VR-1584) and grown in rhesus monkey kidney cells to produce a stock virus pool. When the virus was diluted to a predetermined titer and added, 85-95% cell death was achieved 6 days after infection (MOI was 0.001).

[0150] Determination of cell viability and EC 50 and TC 50 The calculation was performed as shown in Examples 6 and 8.

[0151] result The antiviral efficacy and cytotoxicity data are summarized in the table below. Ribavirin was evaluated as a control compound in parallel with refamulin at 4.73 μg / mL EC18. 50 The value was obtained. Refamrin was 6.79 μM EC 50 The results showed that both ribavirin and refamulin exhibited specific cytotoxicity against this particular cell line at concentrations of 48.5 μg / mL and 23.3 μM, respectively. [Table 9]

[0152] Example 13 Objective: The assay aims to test Zika virus strain ZIKV in Huh7 cells after 4 hours of cell pretreatment. PRVABC59 We measured the inhibition of virus-induced cytotoxic activity (CPE) and cell viability during replication.

[0153] Method: Huh7 cells were seeded in a 96-well flat-bottom tissue culture plate (5 × 10 per well). 3The cells were adhered overnight (at a given cell density). Then, diluted test compounds (refamulin as acetate, sofosbuvir as control) were added to the plates and incubated for 4 hours before adding the virus. Zika virus strain PRVABC59, obtained from ATCC (catalog VR-1843), was obtained from ATCC (VR-1584) and grown in rhesus monkey kidney cells to produce a stock virus pool. When the virus was added at a predetermined titer, 85-95% cell death was achieved 6 days after infection (MOI of 0.001).

[0154] Determination of cell viability and EC 50 and TC 50 The calculation was performed as shown in Examples 6 and 8.

[0155] result The antiviral efficacy and cytotoxicity data are summarized in the table below. The control compound sofosbuvir was evaluated in parallel with refamulin, and the EC2 was 0.65 μg / mL. 50 A value was obtained. Refamlin resulted in an EC50 value of 2.78 μM, and the calculated therapeutic index was 8.42. [Table 10]

[0156] Example 14 Objective: The assay measured the inhibition of virus-induced cytotoxic activity (CPE) and cell viability during replication by human rhinovirus strain HRV16 11757 in H1-HeLa cells after 4 hours of cell pretreatment.

[0157] Method: H1-HeLa cells were seeded in a 96-well flat-bottom tissue culture plate (5 × 10⁶ cells per well). 3 The cells were adhered overnight (at the specified cell density). Then, diluted test compounds (refamulin as acetate, lupintrivir as control) were added to the plates and incubated for 4 hours before adding the virus. (Virus HRV16) 11757When diluted to a predetermined titer and added, 85-95% cell death was achieved in the untreated virus control well (MOI was 0.0005).

[0158] Determination of cell viability and EC 50 and TC 50 The calculation was performed as shown in Examples 6 and 8.

[0159] result Lupintrivir, a protease inhibitor developed for the treatment of rhinovirus, was evaluated in parallel, and the EC2 was 4.90 nM. 50 The value was obtained. Refamrin is 9.34 μM EC 50 The resulting value and calculated treatment index was 2.58. [Table 11]

[0160] Example 15 Objective: The assay measured the inhibition of virus-induced cytotoxic activity (CPE) and cell viability during replication by influenza virus strain A / PR / 8 / 34 in MDCK cells after 4 hours of cell pretreatment.

[0161] Method: MDCK cells were seeded into a 96-well flat-bottom tissue culture plate (5 × 10 per well). 3 Cells were adhered overnight (at cell density). Then, diluted test compounds (refamulin as acetate, oseltamivir as control) were added to the plates and incubated for 4 hours before adding the virus. When influenza virus strain A / PR / 8 / 34 was added at the specified titer, 90% cell death was achieved in the untreated virus control well (MOI 0.0004).

[0162] Determination of cell viability and EC 50 and TC 50 The calculation was performed as shown in Examples 6 and 8.

[0163] result Oseltamivir, an established influenza drug, was evaluated in parallel, and 0.06 μM EC 50 Values ​​were obtained. Refamulin was cytotoxic to MDCK cells at concentrations above 23 μM. The maximum inhibition rate of influenza-mediated CPE was measured at 5 μM refamulin at 18.4%. Therefore, since cytotoxicity was observed at 50 μM, EC 50 We were unable to determine the efficacy of refamrin. Notably, in vivo activity was observed in an influenza-infected mouse model using the relevant mouse-compatible influenza A strain (influenza A / puerto rico / 8 / 34(H1N1)) (see Example 5 above). [Table 12] Preferred embodiments of the present invention include the following. (1) For specific uses in the treatment or prevention of inflammatory diseases not transmitted by bacteria, A compound of formula (I), [ka] During the ceremony, n is between 0 and 4. m is 0 or 1, where sulfur atoms and R 3 They are in close proximity (when m=0, R 3 It is at position 2', and if m=1, R 3 (is at position 1'), R is ethyl or vinyl. R 1 is hydrogen or (C 1~6 ) is alkyl, R 2 is hydrogen, or -(C 3~6 ) Cycloalkyl, or -Unsubstituted (C 1~6 ) alkyl, or -Hydroxy, preferably one or two hydroxyl groups Replaced with one or more of the following (C 1~6 ) alkyl, -Methoxy, -halogen, -(C 3~6 )It is a cycloalkyl, and is R 1 and R 2 They, together with the nitrogen atoms to which they are bonded, form a 5- to 7-membered heterocycle containing at least one nitrogen atom, or one nitrogen and one further heteroatom selected from, for example, N or O, or R 1 is hydroxyl, R 2 It is formyl, R 3 OH, OR 4 , halogen atom, or R 3 It is bonded to 2', and p is 2 or 3 -O-(CH 2 ) p -O- represents, R 4 is non-substitution (C 1~6 ) Alkyl or (C 3~6 ) A compound that is cycloalkyl, or its pharmaceutically acceptable salts, solvates, prodrugs, or metabolites. (2) The compound is of formula (II), (III), (IV), (V) and (VI) [Chemical formula] (In the formula, n, R in each case) 1 and R 2 (As defined in claim 1) A compound for use according to claim 1, characterized by being selected from the group consisting of the following compounds. (3) The aforementioned compound, 14-O-{[(1R,2R,4R)-4-amino-2-hydroxycyclohexylsulfanyl]-acetyl}mutiline 14-O-{[(1S,2S,4S)-4-amino-2-hydroxycyclohexylsulfanyl]-acetyl}mutilin 14-O-{[(1R,2R,5S)-5-amino-2-hydroxycyclohexylsulfanyl]-acetyl}mutiline 14-O-{[(1S,2S,5R)-5-amino-2-hydroxycyclohexylsulfanyl]-acetyl}mutilin 14-O-{[(1R,2R,4S)-4-amino-2-hydroxycyclohexylsulfanyl]-acetyl}mutilin and its (1S,2S,4R) diastereomers 14-O-{[(1R,2R,5R)-5-amino-2-hydroxycyclohexylsulfanyl]-acetyl}mutiline 14-O-{[(1S,2S,5S)-5-amino-2-hydroxycyclohexylsulfanyl]-acetyl}mutilin <008]] 14-O-{[(1R,2R,5R)-5-diethylamino-2-hydroxycyclohexylsulfanyl]-acetyl}mutilin and its (1S,2S,5S) diastereomers ​​​​​​​​​​​ 14-O-{[(1R,2R,3R)-3-ethylamino-2-hydroxycyclohexylsulfanyl]-acetyl}mutilin and its (1S,2S,3S) diastereomers 14-O-{[(1R,2R,3R)-3-diethylamino-2-hydroxycyclohexylsulfanyl]-acetyl}mutilin and its (1S,2S,3S) diastereomers 14-O-{[(1R,2R,4S)-4-(formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}mutilin and its (1S,2S,4R) diastereomers 14-O-{[(1R,2R,5S)-5-(formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}mutilin and its (1S,2S,5R) diastereomers 14-O-{[(1R,2R,3R / S)-3-(formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}mutilin and its (1S,2S,3R / S) diastereomers 14-O-{[(1R,2R,5S)-2-hydroxy-5-methylamino-cyclohexylsulfanyl]-acetyl}mutilin and its (1S,2S,5R) diastereomers 14-O-{[(1R,2R,5S)-5-allylamino-2-hydroxycyclohexylsulfanyl]-acetyl}mutilin and its (1S,2S,5R) diastereomers 14-O-{[(1R,2R,5S)-2-hydroxy-5-(2-methoxy-ethylamino)-cyclohexylsulfanyl]-acetyl}mutilin and its (1S,2S,5R) diastereomers 14-O-{[(1R,2R,4R * )-2-hydroxy-4-(2-hydroxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilin and its (1S,2S,4S * ) diastereomer 14-O-{[(1R,2R,4R * )-4-cyclohexylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and its (1S,2S,4S * ) diastereomer 14-O-{[(1R,2R,4R * )-4-cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and its (1S,2S,4S * ) diastereomer 14-O-{[(1R,2R,5S * )-4-cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and its (1S,2S,5R * ) diastereomer 14-O-{[(1R,2R,4S * )-4-cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and its (1S,2S,4R * ) diastereomer 14-O-{[(1R,2R,5R * )-2-hydroxy-5-morpholine-4-yl-cyclohexylsulfanyl]-acetyl}-mutilin and its (1S,2S,5S * ) diastereomer 14-O-{[(1R,2R,5S * )-2-hydroxy-5-morpholine-4-yl-cyclohexylsulfanyl]-acetyl}-mutilin and its (1S,2S,5R * ) diastereomer 14-O-{[(1R,2R,5S)-5-amino-2-hydroxycyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin and its (1S,2S,5R) diastereomers 14-O-{[(1R,2R,5S)-5-ethylamino-2-hydroxycyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin and its (1S,2S,5R) diastereomers 14-O-{[(1R,2R,5R)-5-amino-2-hydroxycyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin and its (1S,2S,5S) diastereomers 14-O-{[(1R,2R)-4-aminomethyl-2-hydroxycyclohexylsulfanyl]-acetyl}mutilin and its (1S,2S) diastereomers 14-O-{[5-amino-2-chlorocyclohexylsulfanyl]-acetyl}-mutilin 14-O-{[4-amino-2-chlorocyclohexylsulfanyl]-acetyl}-mutilin 14-O-[(4-amino-1-hydroxy-cyclohexylmethylsulfanyl)-acetyl]-mutilin 14-O-{[(1R,2R)-2-hydroxy-5-(3-methylaminopropyl)-cyclohexylsulfanyl]-acetyl}mutilin and its (1S,2S) diastereomers 14-O-{[(1R,2R)-2-hydroxy-4-(3-methylaminopropyl)-cyclohexylsulfanyl]-acetyl}mutilin and its (1S,2S) diastereomers 14-O-{[(1R,2R)-5-(3-amino-propyl)-2-hydroxycyclohexylsulfanyl]-acetyl}mutilin and its (1S,2S) diastereomers 14-O-{[(1R,2R)-4-(3-amino-propyl)-2-hydroxycyclohexylsulfanyl]-acetyl}mutilin and its (1S,2S) diastereomers 14-O-{[(6R,8R)-8-amino-1,4-dioxa-spiro[4.5]deca-6-ylsulfanyl]-acetyl}mutilin and its (6S,8S) diastereomers 14-O-{[4-amino-2-methoxy-cyclohexylsulfanyl]-acetyl}-mutilin and 14-O-{[5-amino-2-methoxycyclohexylsulfanyl]-acetyl}mutilin A compound for use according to claim 1 or 2, characterized by being selected from the group consisting of the following. (4) The compound for use according to any one of claims 1 to 3, characterized in that the compound is refamulin. (5) The compound for use according to any one of claims 1 to 4, characterized in that the compound is in the form of a salt and / or a solvate. (6) The compound for use according to any one of claims 1 to 5, characterized in that the compound is refamulin in the form of refamulin acetate or refamulin itaconate. (7) The compound for use according to any one of claims 1 to 6, characterized in that the inflammatory disease is the result of an inappropriate or chronic inflammatory response. (8) The compound for use according to any one of claims 1 to 7, characterized in that the inflammatory disease is a state of excessive immune response, a neutrophil-dominant inflammatory disease, an autoimmune disease, an allergy, or a dermatological inflammatory disease, particularly a state of excessive immune response or a neutrophil-dominant inflammatory disease. (9) The compound for use according to claim 8, wherein the inflammatory disease is an overreaction of the immune response selected from the group consisting of acute lung injury (ALI), including acute respiratory distress syndrome (ARDS), sepsis, and cytokine release syndrome, including cytokine storm. (10) The compound for use according to claim 9, wherein the inflammatory disease is a neutrophil-dominant inflammatory disease selected from the group consisting of chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis including diffuse panbronchiolitis (DPB), obstructive bronchiolitis syndrome, and noneosinophilic asthma. (11) The compound for use according to claim 8 or 9, wherein the inflammatory disease is a state of overreaction of the immune response mediated by a virus, in particular viral sepsis, or an acute respiratory disease associated with a viral infection such as influenza, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), or COVID-19. (12) The compound for use according to any one of claims 1 to 11, characterized in that the inflammatory disease is an inflammatory disease not mediated by microorganisms. (13) The compound for use according to any one of claims 1 to 12, characterized in that the inflammatory disease is a condition associated with or caused by a viral infection, and the compound of formula I is administered both to treat and / or prevent the viral infection itself and to treat and / or prevent the inflammatory condition. (14) The compound for use according to claim 13, characterized in that the inflammatory state is the result of an inappropriate or chronic inflammatory response related to or caused by the viral infection. (15) The compound for use according to claim 13, characterized in that the inflammatory state is an overreaction of the immune response related to or caused by the viral infection. (16) A compound for use according to any one of claims 13 to 15, for the treatment of a patient who requires both treatment for the viral infection and treatment for the inflammatory condition. (17) The viral infection is transmitted by a single-stranded positive-strand or negative-strand RNA virus, Preferably, the virus is - In particular, the Coronaviridae family, which includes human coronaviruses. - In particular, the Paramyxoviridae family, which includes the Paramyxovirinae subfamily such as the measles virus, and the Pneumovirinae subfamily such as the respiratory syncytial virus, - In particular, the Orthomyxoviridae family, which includes influenza viruses. - In particular, the Flaviviridae family, including dengue virus and Zika virus, and - In particular, the Picornaviridae family, which includes rhinoviruses. A compound for use according to any one of claims 13 to 16, characterized by being selected from the group consisting of the following. (18) The compound for use according to any one of claims 13 to 17, characterized in that the viral infection is an airborne disease. (19) The compound for use according to any one of claims 1 to 18, characterized in that the viral infection is a respiratory disease. (20) A method for treating or preventing an inflammatory disease not mediated by bacteria, comprising administering a compound according to any one of claims 1 to 6, in particular refamulin, or a pharmaceutically acceptable salt, solvate, or ester of a metabolite thereof, to a subject in need of such treatment. (21) The method according to claim 20, wherein the inflammatory disease is the result of an inappropriate or chronic inflammatory response. (22) The method according to claim 20 or 21, wherein the inflammatory disease is a state of excessive immune response, a neutrophil-dominant inflammatory disease, an autoimmune disease, an allergy, or a dermatological inflammatory disease, particularly a state of excessive immune response or a neutrophil-dominant inflammatory disease. (23) The method according to claim 22, wherein the inflammatory disease is an overreaction of the immune response selected from the group consisting of acute lung injury (ALI), including acute respiratory distress syndrome (ARDS), sepsis, and cytokine release syndromes, including cytokine storms. (24) The method according to claim 22, wherein the inflammatory disease is a neutrophil-dominant inflammatory disease selected from the group consisting of chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis including diffuse panbronchiolitis (DPB), bronchiolitis obstructive, and noneosinophilic asthma. (25) The method according to claim 22 or 23, wherein the state of excessive immune response is mediated by a virus, particularly viral sepsis, or an acute respiratory disease associated with a viral infection such as influenza, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), or COVID-19. (26) The method according to any one of claims 20 to 25, wherein the inflammatory disease is an inflammatory disease not mediated by microorganisms. (27) The method of treatment or prevention according to any one of claims 20 to 26, wherein the inflammatory disease is an inflammatory condition associated with or caused by a viral infection, and is administered both to treat and / or prevent the viral infection itself, and to treat and / or prevent the inflammatory condition. (28) The method according to claim 27, wherein the inflammatory condition is the result of an inappropriate or chronic inflammatory response related to or caused by the viral infection. (29) The method according to claim 27, characterized in that the inflammatory state is an overreaction of the immune response related to or caused by the viral infection. (30) The method according to any one of claims 27 to 29 for the treatment of a patient who requires both treatment for the viral infection and treatment for the inflammatory condition. (31) The viral infection is transmitted by a single-stranded positive-strand or negative-strand RNA virus, preferably the virus is - In particular, the Coronaviridae family, which includes human coronaviruses. - In particular, the Paramyxoviridae family, which includes the Paramyxovirinae subfamily such as the measles virus, and the Pneumovirinae subfamily such as the respiratory syncytial virus, - In particular, the Orthomyxoviridae family, which includes influenza viruses. - In particular, the Flaviviridae family, including dengue virus and Zika virus, and - In particular, the Picornaviridae family, which includes rhinoviruses. A treatment method according to any one of claims 27 to 30, selected from the group consisting of the following. (32) The treatment method according to any one of claims 27 to 31, wherein the viral infection is an airborne disease. (33) The treatment method according to any one of claims 27 to 32, wherein the viral disease is a respiratory disease. (34) Refamulin in the form of an acid addition salt with itaconic acid, in particular itaconic acid refamulin.

Claims

1. A pharmaceutical composition for use in the treatment or prevention of inflammatory diseases not transmitted by bacteria, The pharmaceutical composition comprising refamlin or a pharmaceutically acceptable salt or solvate thereof.

2. The pharmaceutical composition according to claim 1, characterized in that refamlin is in the form of a salt and / or a solvate.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that refamulin is refamulin in the form of refamulin acetate or refamulin itaconate.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that refamlin is refamlin in the form of refamlin itaconate.

5. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that the inflammatory disease is the result of an inappropriate or chronic inflammatory response.

6. The pharmaceutical composition according to any one of claims 1 to 5, characterized in that the inflammatory disease is a state of excessive immune response, a neutrophil-dominant inflammatory disease, an autoimmune disease, an allergy, or a dermatological inflammatory disease.

7. The pharmaceutical composition according to claim 6, wherein the inflammatory disease is an overreaction state of the immune response selected from the group consisting of acute lung injury (ALI), including acute respiratory distress syndrome (ARDS), sepsis, and cytokine release syndrome, including cytokine storm.

8. The pharmaceutical composition according to claim 6, wherein the inflammatory disease is a neutrophil-dominant inflammatory disease selected from the group consisting of chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis including diffuse panbronchiolitis (DPB), bronchiolitis obstructive, and noneosinophilic asthma.

9. The pharmaceutical composition according to claim 6 or 7, wherein the inflammatory disease is a state of excessive immune response mediated by a virus.

10. The pharmaceutical composition according to any one of claims 1 to 9, characterized in that the inflammatory disease is an inflammatory disease not mediated by microorganisms.

11. The pharmaceutical composition according to any one of claims 1 to 10, characterized in that the inflammatory disease is a condition associated with or caused by a viral infection, and refamlin is administered both to treat and / or prevent the viral infection itself, and to treat and / or prevent the inflammatory condition.

12. The pharmaceutical composition according to claim 11, characterized in that the inflammatory state is the result of an inappropriate or chronic inflammatory response related to or caused by the viral infection.

13. The pharmaceutical composition according to claim 11, characterized in that the inflammatory state is an overreaction of the immune response related to or caused by the viral infection.

14. A pharmaceutical composition according to any one of claims 11 to 13, for the treatment of a patient who requires both treatment for the viral infection and treatment for the inflammatory condition.

15. The pharmaceutical composition according to any one of claims 11 to 14, characterized in that the viral infection is transmitted by a single-stranded positive-strand or negative-strand RNA virus.

16. The pharmaceutical composition according to any one of claims 11 to 15, characterized in that the viral infection is an airborne disease.

17. The pharmaceutical composition according to any one of claims 11 to 16, characterized in that the viral infection is a respiratory disease.

18. Refamrin in the form of an acid addition salt with itaconic acid.

19. The pharmaceutical composition according to claim 9, wherein the aforementioned condition includes a viral sepsis or an acute respiratory disease related to a viral infection.

20. The pharmaceutical composition according to claim 19, wherein the viral infection is selected from influenza, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and COVID-19.

21. The pharmaceutical composition according to claim 15, wherein the virus is selected from the Coronaviridae family, Paramyxoviridae family, Orthomyxoviridae family, Flaviviridae family and Picornaviridae family.

22. The pharmaceutical composition according to claim 21, wherein the virus of the Coronavirus family is a human coronavirus.

23. The pharmaceutical composition according to claim 21, wherein the virus of the family Paramyxoviridae is a virus of the subfamily Paramyxovirinae or Pneumovirinae.

24. The pharmaceutical composition according to claim 23, wherein the virus of the Paramyxovirinae subfamily is the measles virus.

25. The pharmaceutical composition according to claim 23, wherein the virus of the subfamily Pneumovirinae is an esophageal syncytial virus.

26. The pharmaceutical composition according to claim 21, wherein the virus of the Orthomyxoviridae family is an influenza virus.

27. The pharmaceutical composition according to claim 21, wherein the virus of the Flaviviridae family is dengue virus or Zika virus.

28. The pharmaceutical composition according to claim 21, wherein the virus of the Picornaviridae family is a rhinovirus.

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