Therapeutic Uses of Pleuromutilin

Abstract

Derivatives of pleuromutilin are provided for particular use in the treatment or prevention of non-bacterial mediated inflammatory diseases. The present invention provides a compound of formula (I), or a pharmaceutically acceptable salt, solvate, prodrug, or metabolite thereof: JPEG2026041886000019.jpg49166 (R is ethyl or vinyl, R 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, etc.

Description

[Technical Field]

[0001] The present invention relates to novel therapeutic uses of pleuromutilins. [Background technology]

[0002] Pleuromutilin is a compound of the formula: [ka] For example, natural antibiotics produced by the basidiomycete fungi Pleurotus mutilus and P. pasquerianus; see, for example, Merck Index, 12th Edition, item 7694.

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

[0004] Pharmaceutically active compounds (semisynthetic compounds) derived from pleuromutilins are inhibitors of ribosomal protein synthesis in bacteria. Representative examples of semisynthetic pleuromutilins for human use are retapamulin (approved as AltargetoP®, AltabaxP®), a topical agent approved for the short-term treatment of impetigo and infected small lacerations, abrasions, or sutured wounds, and lefamulin (approved as Xenleta®) for the treatment of community-acquired bacterial pneumonia (CABP) in adults. Tiamulin (Denagard®) and valnemulin (Econor®) are two other semisynthetic pleuromutilin derivatives that have been used systemically as antibiotics in veterinary medicine for many years. [ka]

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

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

[0007] In the case of an inappropriate response, the inflammatory response can be generalized, involving the entire body, such as sepsis, or it can primarily affect a specific organ. This can lead to the dysfunction of body organs, including the lungs. For example, acute lung injury (ALI) is defined as a syndrome of acute and persistent pulmonary inflammation accompanied by increased vascular permeability. It is characterized by inflammatory damage to the alveolar-capillary membrane and an excessive and uncontrolled inflammatory response within the lungs. Acute respiratory distress syndrome (ARDS), the most severe form of ALI, involves 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 multiple organ dysfunction syndrome, which plays a crucial role in the death of patients with 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 constant state of alert. Over time, chronic inflammation can have adverse effects on tissues and organs.

[0009] Valnemulin has been shown to reduce the concentrations of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β, in bronchoalveolar lavage fluid (BAL) and suppress the transcription of these cytokines in the lung (Chen, Zhang et al. 2010, Inflammation 33(5):306-14). This is consistent with in vitro studies (Zhang, Li et al. 2009, Int Immunopharmacol 9(7-8):810-816). Valnemulin may also effectively scavenge oxyradicals and inhibit epithelial permeability during the inflammatory response to lipopolysaccharide (LPS)-induced ALI, potentially contributing to its therapeutic effects. Furthermore, pre-administration of valnemulin significantly reduced the wet-to-dry ratio (W / D) in the lung and attenuated the development of pulmonary edema (Chen, Zhang et al. 2010, Inflammation 33(5):306-14).

[0010] We have published a preprint of a preliminary analysis of the anti-inflammatory activity of lefamulin in a lipopolysaccharide-induced pulmonary neutrophilia 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 lefamulin are set forth in the "Q1 2020 Nabriva Therapeutics PLC Earnings Call" dated May 11, 2020 (copies of which are available at https: / / www.yahoo.com / news / edited-transcript-nbrv-oq-earnings-144108621.html, downloaded on June 10, 2020, and in the May 11, 2020 press release (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 virus, certain adenoviruses, rhinoviruses, and coronaviruses, especially the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19), have a significant impact on public health.

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

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

[0015] WO 2009 / 106839 claims the use of tiamulin as an antiviral agent, and exemplifies the effects of tiamulin on influenza A virus and porcine reproductive and respiratory syndrome virus (PRRSV) types 1 and 2 in virus uptake assays 4 hours after inoculation with tiamulin at concentrations of 0.1 to 10 μg / mL compared with valnemulin, as well as the effect of tiamulin on endosomal pH. It states that valnemulin does not exhibit antiviral activity, and that other pleuromutilin antibiotics have not been found to be effective against viruses.

[0016] Alteration of endosomal or lysosomal pH by tiamulin and the consequent blockade of viral membrane fusion with endosomes and lysosomes, a prerequisite for viral entry, have been described as potential mechanisms of action.

[0017] CN Patent No. 103204787 and CN Patent No. 103242210 both disclose further pleuromutilin derivatives and generally mention their use in antiviral drugs, but do not disclose actual evidence of antiviral activity. Summary of the Invention [Means for solving the problem]

[0018] Surprisingly, it has now been found that the pleuromutilin derivatives disclosed in WO 2008 / 113089 exhibit immunomodulatory and anti-inflammatory effects independent of antibacterial activity.

[0019] Thus, in a first aspect, the present invention relates to a compound according to claims 1 to 6, in particular lefamulin, or any pharmaceutically acceptable salt, solvate, metabolite ester thereof, for particular use in the treatment or prevention of non-bacterially mediated inflammatory diseases.

[0020] In a further aspect, the present invention relates to a method for treating or preventing a non-bacterially mediated inflammatory disease, comprising administering a compound according to any one of claims 1 to 6, in particular lefamulin, or a pharmaceutically acceptable salt, solvate or ester of a metabolite thereof, to a subject in need of such treatment.

[0021] Furthermore, the compounds exhibit antiviral activity and are therefore also suitable for use in the treatment and prevention of diseases mediated by viruses, i.e. viral infections, and in methods for treating or preventing such diseases.

[0022] Thus, in another aspect, the present invention relates to a compound according to claims 1 to 6, in particular lefamulin, or a pharmaceutically acceptable salt, solvate, prodrug or metabolite thereof, for particular use in the treatment or prevention of viral infections and inflammatory conditions associated with or caused by viral infections, wherein 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.

[0023] In a further aspect, 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 according to any one of claims 1 to 6, in particular lefamulin, 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 lefamulin in its form as an acid addition salt with itaconic acid, particularly lefamulin itaconate. [Brief explanation of the drawings]

[0025] [Figure 1] Figure 1 demonstrates the effect of lefamulin in reducing total cell counts in bronchoalveolar lavage fluid (BALF) from mice challenged with lipopolysaccharide (LPS). Error bars indicate standard error of the mean. Diamonds above the bars indicate statistically significant reductions compared to untreated controls (p<0.05, Mann-Whitney test). [Figure 2] Figure 1 demonstrates the effect of lefamulin in reducing neutrophil counts in bronchoalveolar lavage fluid (BALF) from mice challenged with lipopolysaccharide (LPS). Error bars indicate standard error of the mean. Diamonds above the bars indicate statistically significant reductions compared to untreated controls (p<0.05, Mann-Whitney test). [Figure 3A] FIG. 1 demonstrates the effect of dexamethasone, lefamulin and valnemulin on the number of total cells in bronchoalveolar lavage fluid (BALF) of mice challenged with lipopolysaccharide (LPS). [Figure 3B] FIG. 1 demonstrates the effect of dexamethasone, lefamulin and valnemulin on the number of neutrophils in bronchoalveolar lavage fluid (BALF) of mice challenged with lipopolysaccharide (LPS). [Figure 3C] FIG. 1 demonstrates the effect of dexamethasone, lefamulin and valnemulin on the number of macrophages in bronchoalveolar lavage fluid (BALF) of mice challenged with lipopolysaccharide (LPS). [Figure 3D] Figure demonstrating the effect of dexamethasone, lefamulin, and valnemulin on the number of lymphocytes in bronchoalveolar lavage fluid (BALF) of mice challenged with lipopolysaccharide (LPS). Error bars indicate standard error of the mean. Diamonds above the bars indicate a statistically significant reduction compared to untreated controls (p<0.05, Mann-Whitney test). [Figure 4A] FIG. 10 demonstrates the effect of dexamethasone, lefamulin and valnemulin on the concentration of various markers, particularly TNF-α, in lung homogenates. [Figure 4B] FIG. 10 demonstrates the effect of dexamethasone, lefamulin and valnemulin on the concentration of various markers, particularly IL-6, in lung homogenates. [Figure 4C] FIG. 10 demonstrates the effect of dexamethasone, lefamulin and valnemulin on the concentration of various markers, particularly IL-1β, in lung homogenates. [Figure 4D] FIG. 10 demonstrates the effect of dexamethasone, lefamulin and valnemulin on the concentration of various markers, particularly GM-CSF, in lung homogenates. [Figure 4E] FIG. 10 demonstrates the effect of dexamethasone, lefamulin and valnemulin on the concentration of various markers, particularly CXCL1, in lung homogenates. [Figure 4F] FIG. 10 demonstrates the effect of dexamethasone, lefamulin and valnemulin on the concentration of various markers, particularly CXCL2, in lung homogenates. [Figure 4G] FIG. 10 demonstrates the effect of dexamethasone, lefamulin and valnemulin on the concentration of various markers, particularly CCL2, in lung homogenates. [Figure 4H] Figure 1 demonstrates the effect of dexamethasone, lefamulin, and valnemulin on the concentration of various markers, particularly MMP9, in lung homogenates. Error bars indicate the standard error of the mean. Diamonds above the bars indicate a statistically significant decrease (p<0.05, Mann-Whitney test) compared to untreated controls. [Figure 5]1A-1C demonstrate the effect of sodium itaconate and acetate and itaconate forms of lefamulin in reducing cell counts in bronchoalveolar lavage fluid (BALF) of mice challenged with lipopolysaccharide (LPS). Error bars indicate standard error of the mean. [Figure 6A] FIG. 1 demonstrates the effects of lefamulin and oseltamivir on clinical signs, particularly body weight, in a mouse model of influenza infection. [Figure 6B] FIG. 1 demonstrates the effects of lefamulin and oseltamivir on clinical signs, particularly clinical scores, in a mouse model of influenza infection. [Figure 6C] FIG. 1 demonstrates the effects of lefamulin and oseltamivir on clinical signs, particularly survival rate, in a mouse model of influenza infection. [Figure 6D] FIG. 10 demonstrates the effect of lefamulin and oseltamivir on clinical signs, particularly lung sample histopathology scoring, in a mouse model of influenza infection. [Figure 7] FIG. 1 demonstrates the effect of lefamulin and oseltamivir on lung viral titers in a mouse model of influenza infection determined as 50% tissue culture infectious dose (TCID50) in MDCK cells. [Figure 8A] FIG. 1 demonstrates the effect of lefamulin and oseltamivir on immune cell counts at days 3 and 6 post-infection in a mouse model of influenza infection. [Figure 8B] FIG. 1 demonstrates the effect of lefamulin and oseltamivir on bone marrow subsets in a mouse model of influenza infection at day 3 post-infection. [Figure 8C] FIG. 10 demonstrates the effect of lefamulin and oseltamivir on myeloid subsets (neutrophils, alveolar macrophages, macrophages) in an influenza-infected mouse model at day 6 post-infection. [Figure 8D] FIG. 1 demonstrates the effects of lefamulin and oseltamivir on lymphocyte subsets in a mouse model of influenza infection at day 3 post-infection. [Figure 8E] Figure 1 demonstrates the effects of lefamulin and oseltamivir on lymphocyte subsets in a mouse model of influenza infection at day 6 post-infection. A conventional one-way ANOVA, Dunnett's multiple comparisons against vehicle control treatment, was performed for 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] Figure demonstrating the effect of lefamulin against alphacoronavirus 229E (HCoV-229E) in MRC-5 cells at day 6 after infection with the virus. [Figure 9B] Figure demonstrating the effect of tiamulin against alphacoronavirus 229E (HCoV-229E) in MRC-5 cells on day 6 after infection with the virus. [Figure 9C] Figure demonstrating the effect of remdesivir against alphacoronavirus 229E (HCoV-229E) in MRC-5 cells at day 6 after infection with the virus. [Figure 10A] FIG. 10 demonstrates the effect of lefamulin against respiratory syncytial virus type A (RSVA2) in HEp2 cells at day 6 after infection with the virus. [Figure 10B] FIG. 10 demonstrates the effect of tiamulin on respiratory syncytial virus type A (RSVA2) in HEp2 cells at day 6 after infection with the virus. [Figure 10C] FIG. 10 demonstrates the effect of TMC353121 against respiratory syncytial virus type A (RSVA2) in HEp2 cells at day 6 after infection with the virus. DETAILED DESCRIPTION OF THE INVENTION

[0026] Lefamulin is an INN of the compound of general formula (I), more specifically, lefamulin is a compound of formula (VII): [ka] That is, 14-O-{[(1R,2R,4R)-4-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin (also known as "BC-3781").

[0027] Hereinafter, the term "lefamulin", when used generally without further explanation, is intended to encompass both the free base form of lefamulin and its salts and solvates.

[0028] Lefamulin is being developed for systemic use to treat serious 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 according to the present invention have been found to have immunomodulatory effects.

[0030] Experimental results indicate that in addition to antibacterial activity, lefamulin also has immunomodulatory and anti-inflammatory effects. Experiments using an in vivo model of lipopolysaccharide (LPS)-induced pulmonary inflammation in mice demonstrated that lefamulin dose-dependently reduced the number of total cells, particularly neutrophils, in bronchoalveolar lavage fluid (BALF), thereby inhibiting cellular infiltration. Furthermore, experiments also demonstrated a significant reduction in the concentration of pro-inflammatory mediators (e.g., cytokines, chemokines, and other factors, such as matrix metalloproteinase 9) at the inflammatory site at all doses tested.

[0031] Immune cell (e.g., neutrophil) infiltration is a multistep 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, which then mediate the migration of neutrophils into the alveolar space based on a chemotactic gradient. Similar mechanisms are expected for inflammatory processes at other disease sites. Altering immune function (e.g., inhibiting pro-inflammatory mediator secretion) can treat or prevent inflammatory diseases based on inappropriate (overexcited or deregulated) or chronic inflammatory responses.

[0032] The present invention therefore relates to the use of compounds according to claims 1 to 6, in particular lefamulin, in the treatment of inflammatory diseases, which are based on an inappropriate (overexcited or deregulated) or chronic inflammatory response, and which are not bacterially mediated.

[0033] In bacterially mediated diseases, anti-inflammatory treatment may be desirable. The present invention encompasses the treatment of inflammatory diseases that are not bacterially mediated.

[0034] According to one embodiment, the inflammatory disease may be a non-microbially mediated inflammatory disease.

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

[0036] In a preferred embodiment of the invention, the inflammatory disease is the result of an inappropriate (overly excited or deregulated) or chronic inflammatory response, in particular the disease is an overactive state of the immune response, a neutrophil-dominated inflammatory disease, an autoimmune disease, an allergy, or a dermatological inflammatory disease.

[0037] In a preferred embodiment, the inflammatory disease is an overactive immune response, including, for example, acute lung injury (ALI), including acute respiratory distress syndrome (ARDS), sepsis, and cytokine release syndrome, including cytokine storm, in which the immune response is considered deregulated and typically over-excited.

[0038] In particular, if not mediated by bacteria, such an immune hyperresponsive state may be a non-infectious state (i.e., not mediated by any pathogen, including microorganisms and viruses) or a virally mediated state.

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

[0040] In certain embodiments, the hyperactive state of immune response is mediated by a virus, in particular viral sepsis or 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-predominant inflammatory disease, including, for example, chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis including diffuse panbronchiolitis (DPB), bronchiolitis obliterans syndrome, and non-eosinophilic asthma.

[0042] In these diseases, the immune response is predominantly deregulated by the activity of neutrophil granulocytes. There is a clear medical need for the treatment of neutrophil-dominated inflammatory diseases, as none of the classical anti-inflammatory therapies, such as corticosteroids, are effective, and nonsteroidal anti-inflammatory drugs either do not affect neutrophil function or may adversely extend neutrophil lifespan. The results of Examples 1 and 2 demonstrate the specific effect of lefamulin on neutrophil cell counts.

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

[0044] According to another embodiment of the invention, the inflammatory disease is allergy, in which the immune response is thought to be deregulated, as 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, preferably a respiratory inflammatory disease. Respiratory inflammatory diseases include, for example, 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), bronchiolitis obliterans syndrome, or non-eosinophilic asthma. Dermatological inflammatory diseases include, for example, psoriasis, acne, or rosacea.

[0046] In one embodiment of the invention, the disease is not and / or is not associated with bacterial pneumonia.

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

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

[0049] In particular, the compounds are for the treatment of patients in need of both treatment for a viral infection and treatment for an inflammatory condition.

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

[0051] In a further embodiment, there is provided the particular use of compounds administered in accordance with the present invention, particularly lefamulin, for the treatment of a patient in need of both treatment for a viral infection and treatment for an inflammatory condition.

[0052] In a preferred embodiment, the inflammatory condition is an overactive 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 conditions, the immune response is considered deregulated and typically over-excited.

[0053] In particular embodiments, there is provided a particular use of the compounds administered in accordance with the present invention, in particular lefamulin, 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, lefamulin also actively reduces cytotoxic activity mediated by various viruses. This antiviral effect was particularly demonstrated against viruses characterized as single-stranded positive- or negative-strand RNA viruses. Antiviral activity was demonstrated against both enveloped and non-enveloped viruses, particularly against several enveloped single-stranded positive- or negative-strand RNA viruses (e.g., Coronaviridae, Paramyxoviridae, Orthomyxoviridae, Flaviviridae, etc.). Furthermore, several of the viruses investigated, including the measles virus, are known to be transmitted via the respiratory route, particularly airborne transmission. Coronaviruses and respiratory syncytial viruses also cause respiratory tract infections in humans.

[0055] In a preferred embodiment of the invention, the viral infection is mediated by a single-stranded positive or negative strand RNA virus, Preferably, the virus is - Coronaviridae, which includes in particular human coronaviruses, - the family Paramyxoviridae, which includes in particular the subfamily Paramyxovirinae, such as the measles virus, and the subfamily Pneumovirinae, such as the respiratory syncytial virus; - Orthomyxoviridae, which includes in particular influenza viruses, Flaviviridae, including in particular the dengue and zika viruses, and - Picornaviridae, which includes especially the rhinoviruses is selected from the group consisting of:

[0056] In another embodiment, the viral infection is an airborne disease. Airborne diseases are transmitted by viruses that are transmitted through the air.

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

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

[0059] Treatment typically involves administering a compound used in accordance with the present invention to a subject in need thereof, for example, in one embodiment, a subject who has been 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 that includes early symptoms of the viral infection and late symptoms of the inflammatory condition, i.e., the viral infection is diagnosed early and the inflammatory condition is diagnosed later (simultaneously). Thus, in a preferred embodiment, a compound for use in accordance with the present invention is administered to a subject who exhibits or has been diagnosed with both 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 (any) disease symptoms of the condition occur.

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

[0062] Suitable dosages of the compounds administered according to the present invention, particularly lefamulin, will, of course, vary depending, for example, on the individual host, the mode of administration, and the nature and severity of the condition being treated. Generally, however, for satisfactory results in larger mammals, such as humans, indicated daily dosages will be in the range of about 0.5 mg to 3 g of the compounds used according to the present invention, conveniently administered, for example, in divided doses up to four times daily.

[0063] The compounds used according to the invention may be administered by any conventional route, for example enterally, including, for example, nasal, buccal, rectal, oral administration; parenterally, for example, including intravenous, intramuscular, subcutaneous administration; or topically, for example, including pulmonary, epidermal, intranasal, intratracheal administration, for example 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, inhalant powders, foams, tinctures, lipsticks, drops, sprays, or in the form of suppositories, in a manner similar to, for example, the antibiotic tobramycin or a macrolide, for example erythromycin, for example clarithromycin or azithromycin.

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

[0065] A preferred pharmaceutical composition of lefamulin for injection is disclosed in WO 2016 / 202788, the contents of which are incorporated herein by reference.

[0066] The compounds used according to the present invention, in particular lefamulin, may be administered in the form of a pharmaceutically acceptable salt, for example an acid addition salt, or in free form, optionally in the form of a solvate.

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

[0068] Salts of the compounds used according to the present invention include acid addition salts. Pharmaceutically acceptable acid addition salts include salts of the compounds used according to 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, and bihydrochloric acid, preferably hydrochloric acid, acetic acid, L-lactic acid, and maleic acid.

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

[0070] Preferred crystalline forms of lefamulin and crystalline salt forms of lefamulin are disclosed in WO 2011 / 146954, the contents of which are incorporated herein by reference. Of these, crystalline form B of lefamulin acetate, disclosed in WO 2011 / 146954, is particularly preferred.

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

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

[0073] The compounds used according to the present invention, particularly lefamulin, can be used alone or in combination with one or more other pharmaceutically active agents in the pharmaceutical treatments contemplated 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 drugs 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 agents are in the same formulation; kits, in which two or more pharmaceutically active agents in separate formulations are sold in the same package, e.g., with instructions for simultaneous administration; and free combinations, in which the pharmaceutically active agents are packaged separately but with instructions for simultaneous or sequential administration.

[0075] In another embodiment, the compounds used according to the invention, particularly lefamulin, are the only active agents administered to a patient diagnosed with a viral infection and an inflammatory condition associated with or caused by the viral infection, i.e., the patient is treated with only one active agent to treat and / or prevent both the viral infection and the inflammatory condition.

[0076] Pharmaceutical compositions containing the compounds used according to the present invention, in particular lefamulin, may further comprise at least one pharmaceutically acceptable excipient, such as a carrier or diluent, such as a filler, binder, disintegrant, flow regulator, lubricant, sugar and sweetener, flavoring, preservative, stabilizer, wetting agent and / or emulsifier, solubilizer, salt for regulating osmotic pressure and / or buffer.

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

[0078] The subject in need of treatment contemplated by the present invention may be any living subject suffering from a non-bacterially mediated inflammatory disease. The subject may be a human or an animal.

[0079] example The following abbreviations are used herein, including examples: 1 H-NMR proton nuclear magnetic resonance spectroscopy ℃ Celsius μM micromolar ALI acute lung injury ARDS acute respiratory distress syndrome BALB / c laboratory mouse strain BALF bronchoalveolar lavage fluid BC-3781 Lefamulin CC Cell control CCL2 chemokine (CC motif) ligand 2 CoV coronavirus CPE Cytotoxic activity, especially virus-induced CXCL1 chemokine (CXC motif) ligand 1 CXCL2 chemokine (CXC motif) ligand 2 DMEM Dulbecco's Modified Eagle's 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 intranasal IP intraperitoneal LPS lipopolysaccharide MDCK Madin-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 solution PO Oral SC subcutaneous TC 50 Half (50%) toxic concentration TCID 50 50% (half) tissue culture infectious dose TNF-α tumor necrosis factor alpha Reduction of VC viral CPE XTT 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide

[0080] Example 1 Objective: We investigated the anti-inflammatory effect of lefamulin in a model of LPS-induced pulmonary neutrophilia by determining the total cell and neutrophil counts in BALF 4 h after LPS administration.

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

[0082] result The results of BALF analysis (total cell counts and neutrophil cell counts) 4 hours after intranasal LPS challenge are shown in Figures 1 and 2. LPS challenge induced a statistically significant increase in total cell counts and neutrophil counts compared with saline-challenged controls. Compared with LPS-challenged animals, mice pretreated with the reference substance dexamethasone PO 1 hour before challenge showed a statistically significant decrease in both total cell counts and neutrophil cell counts in BALF. There was also a statistically significant decrease in total cell counts and neutrophil cell counts in all groups treated with lefamulin at the indicated doses in a dose-dependent manner (p < 0.05 vs. lipopolysaccharide challenge). A nearly complete impairment of cell influx was observed at the highest dose of 140 mg / kg. The lefamulin-associated decrease in BALF cell counts was comparable to that seen after treatment with the reference substance dexamethasone, a known anti-inflammatory drug, at all lefamulin doses. Of note, the SC exposure of 70 mg / kg lefamulin per day corresponds to the exposure achieved in humans when administered the approved clinical daily doses 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 the alveolar epithelial and endothelial barriers, which leads to the formation of protein-rich edema in the interstitium and alveolar spaces. Inhibition of neutrophil pulmonary infiltration 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, we further investigated the activity of lefamulin at doses of 10, 30, and 100 mg / kg in a mouse model of LPS-induced pulmonary neutrophilia, including the determination of cell numbers in BALF as well as the assessment of cytokine and chemokine concentrations, and compared it with valnemulin.

[0085] Methods: Prior to intranasal (IN) LPS administration, mice were anesthetized intraperitoneally (IP) using a combination of ketamine and xylazine. For IN instillation, mice were held in a tilted supine position with their head elevated 60-75 degrees above their feet during and after the instillation period (approximately 1 min). Challenge occurred 30 min after treatment with 5 μg of LPS in 50 μL of saline per mouse. Non-challenged control mice received 50 μL of saline. Group size was 8 animals per group.

[0086] 1 mg / kg of the reference compound dexamethasone (positive control) was administered intraperitoneally (IP) 30 min before LPS challenge. Dexamethasone was dissolved in 0.5% methylcellulose in water. The dose volume was 10 mL / kg. 10, 30, or 100 mg / kg of lefamulin or valnemulin was administered subcutaneously (SC) between the shoulders 30 min before LPS challenge. Lefamulin and valnemulin were dissolved in 0.9% saline. 0.9% saline was used as a vehicle control. The dose volume was 10 mL / kg. Four hours after LPS challenge, mice were sacrificed by an overdose of ketamine (200 mg / kg) and xylazine (16 mg / kg), after which BALF and lung tissue samples were taken.

[0087] To collect bronchoalveolar lavage fluid (BALF), a cannula was inserted into the trachea, and the lungs were then lavaged with three volumes of PBS (0.4, 0.3, and 0.3 mL, totaling 1 mL). The collected BALF was placed in an Eppendorf tube and centrifuged in a tabletop Eppendorf centrifuge (5 min at 1303 x g at 4°C). The cell plug was resuspended in 600 μL of PBS by shaking the closed tube contents on a vortex mixer. The resuspended BAL cells were immediately analyzed for total and differential cell counts using an automated hematology analyzer, the Sysmex XT-2000iV.

[0088] Lung tissue samples were collected to determine specific markers. The lungs were exposed and excised by gently opening the chest and cutting both sides of the sternum and ribs, trimming the back. The lungs were removed and placed in pre-weighed sterile Precellys tubes. After sampling, the Precellys tubes were weighed again, flash-frozen, and placed at -80°C until analysis. The tissue was homogenized.

[0089] 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 a biotin-antibody cocktail. Concentrations were determined using the respective streptavidin-phycoerythrin conjugates and read on a Luminex 200 instrument. Concentrations of these markers were determined by interpolation from the standard curve using XPONENT software (Applied Cytometry). Measurements (MFI) were blank-corrected and a 5-parameter logistic fit of the standard curve was used.

[0090] For MMP-9 and IL-1β analysis, whole mouse lungs were thawed from -80°C and homogenized in 1 mL of PBS supplemented with protease inhibitors in a Precellys CK28 hard tissue tube using the Precellys instrument and program. The homogenization was repeated three times, shaking at 6,800 rpm for 30 seconds with a 15-second pause. After homogenization, the samples were centrifuged at 18,000 x g for 10 minutes at 4°C, and the supernatant was collected for analysis. MMP-9 and IL-1β concentrations were determined using the Mouse DuoSet ELISA kit according to the manufacturer's protocol (R&D Systems). Standard dilution series and samples were run in parallel using plates prepared with MMP-9 or IL-1β capture antibodies. 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 MMP-9 and IL-1β in the samples were determined by interpolation from the standard curve.

[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 used to evaluate total and differential cell counts and marker concentrations. Differences between treatment groups relative to the vehicle group were considered statistically significant at p < 0.05.

[0092] result The results of BALF analysis (cell counts) are shown in Figures 3A–3D. LPS challenge induced a significant increase in total cell and neutrophil counts compared with saline-challenged controls. Subcutaneous pretreatment with 10, 30, or 100 mg / kg lefamulin dose-dependently reduced total cell counts 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 counts was observed at all concentrations of lefamulin tested (Figure 3B). In contrast, macrophage and lymphocyte counts were not significantly affected by lefamulin or lefamulin (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) are increased in lung homogenates after LPS challenge in mouse models compared with saline controls (Figures 4A-C). Subcutaneous pretreatment with 10, 30, or 100 mg / kg lefamulin reduced TNF-α and IL-6 levels by more than 50% (Figures 4A and 4B), whereas valnemulin was less active compared with the same doses. Subcutaneous pretreatment with 100 mg / kg lefamulin 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 the LPS-induced mouse model (Figure 4D). Lefamulin almost completely inhibited GM-CSF in lung homogenates. Valnemulin also showed a reduction in GM-CSF levels, but was less active than lefamulin at all doses tested (Figure 4D).

[0095] Concentrations of the chemokines (C-X-C motif) ligand 1 (CXCL1), (C-X-C motif) ligand 2 (CXCL2), and (C-C motif) ligand 2 (CCL2) were increased in lung homogenates after LPS challenge in a mouse model compared with saline controls (Figures 4E-G). Both CXCL1 and CXCL2 levels were significantly reduced by subcutaneous pretreatment with lefamulin at 10, 30, or 100 mg / kg (Figures 4E and 4F). Lower doses of valnemulin, 10 and 30 mg / kg, were less effective against CXCL1 and CXCL2 compared with lefamulin. LPS-induced increases in CCL2 in lung homogenates were completely inhibited by lefamulin at all doses tested, whereas valnemulin was less active, especially at doses of 10 and 30 mg / kg (Figure 4G).

[0096] Matrix metallopeptidase 9 (MMP9), a regulator of neutrophil migration, was increased in the LPS-induced mouse model (Figure 4H). Subcutaneous pretreatment with 30 or 100 mg / kg lefamulin reduced MMP9 levels in lung homogenates by 26 or 53%, respectively, whereas 10 mg / kg did not alter MMP9 concentrations. Valnemulin 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 lefamulin in a murine LPS-induced pulmonary neutrophilia model by inhibiting neutrophil infiltration into the lung and reducing (pro)inflammatory factors such as cytokine and chemokine levels in the lung.

[0098] The inhibitory potential of lefamulin in this model, at clinically relevant doses, is comparable in magnitude to the established anti-inflammatory drug dexamethasone (positive control) for most of the parameters investigated. Surprisingly, when compared with valnemulin at the same dose, lefamulin was shown to be a more potent inhibitor than valnemulin for most of the (pro-)inflammatory markers.

[0099] LPS administration can induce pathological and biological changes similar to those seen in ARDS or ALI, including increased neutrophil infiltration and pulmonary cytokines, which have been widely studied 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 lung may contribute to tissue damage, particularly in chronic disease or ARDS (Giacalone, Vincent D et al. 2020, Int J Mol Sci 21(3):851).

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

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

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

number

[0103] Example 4 Objective: In a modified model of LPS-induced pulmonary neutrophilia similar to that in Example 1, the anti-inflammatory effects of various lefamulin salts were investigated with a readout 24 hours after LPS challenge.

[0104] Methods: Aqueous solutions of test compounds were administered as a single subcutaneous dose (5 μg / animal) 30 minutes before intranasal LPS challenge. Immediately before LPS challenge, groups of mice (n = 6) were anesthetized by intraperitoneal (IP) injection of ketamine (2 mg / mouse) and xylazine (0.08 mg / mouse). To induce pulmonary neutrophilia, control mice received 50 μL of saline, while all other animals received 5 μg of LPS in 50 μL of saline intranasally. As a control, saline (0.86% NaCl) was administered intranasally as a negative control for LPS challenge. Lefamulin was investigated in its acetate form (Lef.Ac) and its itaconate form (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 a subcutaneous injection of saline (0.86% NaCl).

[0105] The total cell count and granulocyte cell count in the BALF were determined 24 hours after intranasal LPS challenge as in Example 1.

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

[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, an ester derivative of itaconic acid (4-octyl itaconate) showed protective effects in an LPS-induced sepsis model (Liao, Shan-Ting et al. 2019 Nat Comm 10(1);5091).

[0108] The itaconate salt of lefamulin showed the most pronounced effect on cell counts compared to lefamulin acetate or sodium itaconate alone. In this study, lefamulin itaconate treatment resulted in the lowest total and granulocyte cell counts, thus suggesting an interesting synergistic or additive effect of the itaconate form of lefamulin.

[0109] Example 5 Objective: We investigated the effects of lefamulin in an in vivo influenza virus model, in which mice were challenged with a mouse-adapted influenza A (H1N1) strain. The mouse immune system is particularly suitable for testing effects on virus-induced immune responses in a murine influenza virus infection model.

[0110] Methods: Adult female BALB / c mice were randomly assigned to three experimental groups of 15 animals and allowed to acclimate for 1 week. Treatment began on day -1 via subcutaneous administration. The negative control group received vehicle administered twice daily. Various doses of lefamulin were investigated. In the low-dose regimen, 35 mg / kg lefamulin was administered twice daily from day -1 to day 6 (equivalent to a dose of 70 mg / kg / day). In the high-dose regimen, lefamulin was administered at a dose of 105 mg / kg / day for three injections until day 3, when it was changed due to administration issues at the injection site. The following doses were administered: 70 mg / kg twice daily (equivalent to 140 mg / kg / day). On day 0, all groups were challenged with influenza A / Puerto Rico / 8 / 34 (H1N1).

[0111] During the study, animals were scored daily for clinical signs of influenza virus infection, including abnormal coat condition (piloerection), abnormal posture (crouching), abnormal breathing (tachypnea and / or irregular respiratory rate), decreased mobility, eye discharge, eye closure, and / or survival. Signs of disease severity were added to a scoring system, resulting in a maximum possible score of 5. If clinical signs were deemed severe, individual animals were removed from the study before the scheduled end of the study.

[0112] On day 6, lung tissue was dissected, assessed for gross lesions, preserved in fixative, and archived for histopathology.

[0113] After macroscopic evaluation, lung consolidation was assessed as follows: intra-alveolar edema / hemorrhage occupying more than 50% of the field (across all lobes), and extensive vascular degeneration. Lungs removed and fixed on day 6 were evaluated microscopically for histopathology. Four main readouts were assessed (bronchial / bronchial degeneration / hyperplasia, bronchointerstitial inflammation, alveolar epithelial inflammation / degeneration, and alveolar epithelial / hemorrhage) and scored to obtain a maximum total histopathology score of 16, with lower numbers indicating fewer signs of histopathological abnormalities.

[0114] Lung samples were also processed and stored for virus titration on days 3 and 6. On days 3 and 6, lungs were harvested, homogenized, clarified, and assayed for TCID using Madin-Darby canine kidney (MDCK) cells. 50 Viral load was determined by assay.

[0115] On days 3 and 6, bronchoalveolar lavage fluid (BALF) samples were collected, and cells were processed for flow cytometry analysis of immune cell components using the antibody panel detailed below. Absolute cell numbers were counted using flow cytometry counting beads. Cells were gated on viable single events. The effects of test treatments on immune cell subsets were 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 a procedural error.

[0116] result The results of clinical monitoring are shown in Figures 6A-6D. The positive control treatment with oseltamivir performed as expected. Decreases in clinical scores and weight loss were observed with oseltamivir. Lefamulin did not significantly affect body weight at the doses studied (Figure 6A). At higher doses, lefamulin resulted in increased clinical scores and decreased survival compared to vehicle, which may be related to local tolerability (SC) issues at the dose, concentration, and formulation studied (Figures 6B and 6C). At lower doses of lefamulin, 90% survival was achieved, but only 20% of the vehicle group survived to day 6 (Figure 6C).

[0117] Furthermore, a significant improvement in macroscopically assessed lung consolidation was observed with low-dose lefamulin. Histopathology revealed a range of overall individual animal scores (ranging from 4 to 13) within the specimens examined (Figure 6D). The lesions were similar to those described in the literature. Increased extent / area of ​​distribution of alveolar pathology correlated with increased bronchiolar degeneration / proliferation, bronchointerstitial inflammation, and alveolar edema / hemorrhage. Treatment with high-dose lefamulin resulted in a significant reduction in bronchial degeneration and alveolar inflammation, resulting in an overall significant reduction in histopathology scores in this group compared with vehicle-treated controls, comparable to oseltamivir.

[0118] Lung viral titers decreased in all groups between days 3 and 6 (Figure 7). Both doses of lefamulin and oseltamivir reduced lung viral titers compared to vehicle-treated controls.

[0119] The results of BALF flow cytometry are shown in Figures 8A-8E. Total immune cell counts in BALF increased from days 3 to 6 in all test groups (Figure 8A), confirming the expected immune cell infiltration induced by progressive viral infection, which causes lung tissue inflammation and cytopathies. Lefamulin significantly reduced total immune cell infiltration in the lung by day 6 at both doses tested (p<0.05). Figures 8B-8E show the cell counts of specific immune cell subsets on days 3 and 6. Lefamulin significantly reduced neutrophil infiltration at both doses tested by day 6 (Figure 8C). When administered at higher doses, lefamulin significantly reduced inflammatory monocyte infiltration in the lung on day 6 after H1N1 challenge (Figure 8C). NK, CD4, and CD8 cell infiltration in the lung was reduced on day 6 at both doses tested (Figure 8E). The reduction in B cells was significant only at the highest dose on day 6 (Fig. 8E).

[0120] The BALF results also confirmed the immunomodulatory effects of lefamulin in the context of a viral disease model, particularly on neutrophil infiltration into the lung. The reducing effects on clinical readouts and pulmonary viral titers further support the potential of lefamulin in the treatment of viral diseases.

[0121] Example 6 Objective: The assay was designed to evaluate the efficacy of various concentrations of lefamulin (BC-3781) in the treatment of alphacoronavirus 229E (HCoV-229E or CoV 229E ) Virus-induced cytotoxic activity (CPE) and inhibition of cell viability were measured in MRC-5 cells 6 days after infection.

[0122] Methods: MRC-5 cells were seeded into 96-well flat-bottom tissue culture plates (3 × 10 per well). 3 Cells were plated at a density of 1000 x 1000 cells and allowed to adhere overnight. Diluted test compounds (lefamulin as the acetate salt and tiamulin as the fumarate salt) dissolved in DMSO were then added to the plates and incubated for 4 hours before adding virus. The virus was diluted to a predetermined titer and added, resulting in 85-95% cell death 6 days post-infection (MOI of 0.001).

[0123] After 6 days of incubation at 37°C and 5% CO2, 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 reduction of virus-infected cells and percent cell viability of uninfected drug control wells were calculated, and the effective concentration at which 50% of the cytotoxic activity was inhibited (EC2) was determined using a four-parameter curve-fit analysis. 50 ) and cytotoxic concentration (TC 50 ) was determined. The antiviral compound remdesivir served as a positive control.

[0124] result Remarkably, lefamulin reduced viral CPE by 91.82% at a concentration of 10 μM, a concentration that had no cytotoxic effect on the viability of cell controls. The calculated EC 50 The TC was 3.87 μM, which inhibited 50% of the viral cytotoxicity. At a lefamulin concentration of 50 μM, lefamulin showed a cytotoxic effect, with a calculated TC 50 The EC2, also known as the therapeutic index, was 55.3 μM. 50 and T.C. 50The ratio was 14.3.

[0125] In contrast, tiamulin at a concentration of 10 μM reduced viral CPE by only 10.53%, and no cytotoxic effect was observed. At the next highest concentration tested, 50 μM, CPE was reduced by 81.68%, and a cytotoxic effect was observed. The calculated EC 50 is 24.4 μM, and the calculated TC 50 The therapeutic index of tiamulin was 2.58, which was surprisingly much lower than that of lefamulin.

[0126] The antiviral compound remdesivir was developed as a treatment for Ebola virus and is known to have antiviral activity against coronaviruses (currently in clinical trials). Therefore, remdesivir served as a positive control herein. Remdesivir had an EC of 0.11 μM. 50 , more than 5 TC 50 and a therapeutic index of >45.5. [Table 1]

[0127] The results are shown graphically in Figures 9A (lefamulin), 9B (tiamulin), and 9C (remdesivir) (VC is reduction in viral CPE, CC is cell control).

[0128] Example 7 Objective: The assay evaluates the efficacy of alphacoronavirus 229E (HCoV-229E or CoV) against lefamulin under various treatment conditions. 229E ) Inhibition of virus-induced cytotoxic activity (CPE) and cell viability was measured in MRC-5 cells after infection.

[0129] Methods: Assays were performed similarly to Example 6 above, with the following differences for test candidates. Lefamulin (as the acetate salt) was evaluated using different treatment conditions: 4, 1, or 0 hours of incubation before virus addition and addition 1 hour post-infection. In this particular set of experiments, coronavirus was diluted 1:200 in assay medium and added at 100 μL / well to achieve approximately 90% cell kill in untreated virus control wells (MOI of 0.001).

[0130] result The antiviral effect and cytotoxicity data are summarized in the table below. Lefamulin showed a time-dependent effect on inhibiting virus-induced cytotoxic activity (CPE). In the treatment setting after virus exposure (1 hour post-infection), a dose-dependent effect was observed. 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 evaluates the efficacy of various concentrations of lefamulin (BC-3781) in the prevention and treatment of human respiratory syncytial virus (RSV) infection. A2 Virus-induced cytotoxic activity (CPE) and inhibition of cell viability were measured in HEp2 cells 6 days after replicative infection.

[0132] Methods: HEp2 cells were seeded into 96-well flat-bottom tissue culture plates (5 × 10 per well). 3 Cells were plated at a density of 1000 x 1000 cells per well and allowed to adhere overnight. Test compounds (lefamulin as the acetate salt and tiamulin as the fumarate salt) diluted in DMSO were then added to the plates and incubated for 4 hours before virus addition. Virus was diluted to a predetermined titer and added, resulting in 85-95% cell death 6 days post-infection (MOI of 0.001).

[0133] After 6 days of incubation at 37°C and 5% CO2, 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 reduction of virus-infected cells and percent cell viability of uninfected drug control wells were calculated, and the effective concentration at which 50% of the cytotoxic activity was inhibited (EC2) was determined using a four-parameter curve-fit analysis. 50 ) and cytotoxic concentration (TC 50 ) was determined. The antiviral compound TMC353121 (RSV fusion inhibitor) served as a positive control.

[0134] result Surprisingly, lefamulin reduced the viral cytotoxic activity (CPE) by 92.17% and 100% at concentrations of 10 μM and 50 μM, respectively, concentrations that had no cytotoxic effect on the viability of cell controls. 50 The TC was 5.34 μM, which inhibited 50% of viral CPE. At a lefamulin concentration of 100 μM, lefamulin showed cytotoxic effects, with a calculated TC 50 The EC2, also known as the therapeutic index, was 70.7 μM. 50 and T.C. 50 The ratio was 13.2.

[0135] In contrast, tiamulin at a concentration of 10 μM reduced viral CPE by only 16.76%, a concentration at which a cytotoxic effect (84% viability) was observed. At the next highest concentration tested, 50 μM, viral CPE was reduced by 43.28%, and the cytotoxic effect was more pronounced (70.0% viability). The calculated EC 50 calculated TC of 67.9 μM 50 The antiviral activity and therapeutic index of lefamulin were significantly higher than those of tiamulin, exceeding 67.9 μM. Therefore, the therapeutic index of tiamulin could not be calculated. Surprisingly, the antiviral activity and therapeutic index of lefamulin were much higher than those of tiamulin.

[0136] The antiviral compound TMC353121 was developed as a specific respiratory syncytial virus fusion inhibitor (currently in clinical trials). Therefore, TMC353121 served as a positive control herein. TMC353121 had an EC of 0.006 μM. 50 , TC above 0.1 μM 50 and a therapeutic index of >167. [Table 3]

[0137] The results are shown graphically in Figures 10A (lefamulin), 10B (tiamulin), and 10C (TMC353121) (VC is reduction in viral CPE, CC is cell control).

[0138] Example 9 Objective: This assay involves the detection of human respiratory syncytial virus (RSV) at varying multiplicities of infection (MOI). A2 Virus-induced cytotoxic activity (CPE) and inhibition of cell viability were measured in HEp2 cells after replication.

[0139] Methods: Assays were performed similarly to Example 8 above, with the following test differences. Virus was diluted to the desired titer to achieve 85-95% cell death at day 6 post-infection, with doses used to achieve MOIs of 0.003, 0.001, 0.0008, and 0.0004, respectively. Lefamulin (as the acetate salt) was tested in this study, and TMC353121 was tested as a positive control.

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

[0141] Example 10 Objective: This assay compares two different respiratory syncytial virus strains, RSV A and B. LONG and RSV B 18537 Inhibition of virus-induced cytotoxic activity (CPE) and cell viability were measured in HEp2 cells after replication of the virus.

[0142] Methods: After pre-treating cells with different concentrations of test compounds for 4 hours, 5 × 10 cells were cultured per well. 3 Cells were seeded at a density of 100 μg / ml with the virus strain RSV A. LONG or RSV B 18537 The assay was performed as in Example 8 above, with the difference that the virus was incubated with RSV A. LONG and RSV B 18537 were added in amounts resulting in 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 lefamulin and had an EC of 0.01 nM against the investigated strains of RSV A and RSV B. 50 Lefamulin was shown to be effective against RSV B 18537 EC of 17.7 μM 50 RSV A LONG The activity against TC in the assay was cytotoxic to HEp2 cells. 50 The value was 71.1 μM and therefore could not be determined. [Table 6] [Table 7]

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

[0145] Methods: HeLa cells were seeded into 96-well flat-bottom tissue culture plates (5 × 10 per well). 3 Cells were allowed to adhere overnight. Dilutions of test compounds (lefamulin as acetate, ribavirin as control) were then added to the plates and incubated for 4 hours before adding virus. Virus was diluted to the desired titer and added, resulting in 85–95% cell death 6 days post-infection (1:50 dilution, MOI 0.008).

[0146] Determination of cell viability and EC 50 and T.C. 50 The calculations were carried out as described 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 alongside lefamulin and had an EC 50 Surprisingly, lefamulin had an even lower EC value of 0.89 μM. 50 values ​​and had a high calculated TI of 81.7. [Table 8]

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

[0149] Methods: Huh7 cells were seeded into 96-well flat-bottom tissue culture plates (5 × 10 per well). 3The cells were allowed to adhere overnight. Diluted test compounds (lefamulin as acetate salt, ribavirin as control) were then added to the plates and incubated for 4 hours before adding the virus. New Guinea The virus was obtained from ATCC (VR-1584) and propagated in rhesus monkey kidney cells to produce a stock virus pool. The virus was diluted to a predetermined titer and added to the cells, resulting in 85-95% cell death at 6 days post-infection (MOI of 0.001).

[0150] Determination of cell viability and EC 50 and T.C. 50 The calculations were carried out as described 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 alongside lefamulin and had an EC 50 Lefamulin had an EC value of 6.79 μM. 50 Both ribavirin and lefamulin showed specific cytotoxicity towards this particular cell line at concentrations of 48.5 μg / mL and 23.3 μM, respectively. [Table 9]

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

[0153] Methods: Huh7 cells were seeded into 96-well flat-bottom tissue culture plates (5 × 10 per well). 3The cells were allowed to adhere overnight. Diluted test compounds (lefamulin as the acetate salt, sofosbuvir as a control) were then 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 (catalog VR-1584) and propagated in rhesus monkey kidney cells for the production of stock virus pools. The virus was diluted to a predetermined titer and added, resulting in 85-95% cell death 6 days post-infection (MOI of 0.001).

[0154] Determination of cell viability and EC 50 and T.C. 50 The calculations were carried out as described in Examples 6 and 8.

[0155] result The antiviral efficacy and cytotoxicity data are summarized in the table below. The reference compound sofosbuvir was evaluated in parallel with lefamulin and had an EC 50 Lefamulin produced an EC50 value of 2.78 μM with a calculated therapeutic index of 8.42. [Table 10]

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

[0157] Methods: H1-HeLa cells were seeded into 96-well flat-bottom tissue culture plates (5 × 10 per well). 3 The cells were allowed to adhere overnight. Diluted test compounds (lefamulin as the acetate salt, rupintrivir as a control) were then added to the plates and incubated for 4 hours before adding the virus. 11757When diluted to a predetermined titer and added, 85–95% cell killing was achieved in untreated virus control wells (MOI of 0.0005).

[0158] Determination of cell viability and EC 50 and T.C. 50 The calculations were carried out as described in Examples 6 and 8.

[0159] result Rupintrivir, a protease inhibitor developed for the treatment of rhinovirus, was evaluated in parallel and had an EC of 4.90 nM. 50 Lefamulin had an EC value of 9.34 μM. 50 The calculated therapeutic index was 2.58. [Table 11]

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

[0161] Methods: MDCK cells were seeded into 96-well flat-bottom tissue culture plates (5 × 10 per well). 3 Cells were allowed to adhere overnight. Dilutions of test compounds (lefamulin as the acetate salt, oseltamivir as a control) were then added to the plates and incubated for 4 hours before virus addition. Influenza virus strain A / PR / 8 / 34 was added at a predetermined titer, resulting in 90% cell death in untreated virus control wells (MOI of 0.0004).

[0162] Determination of cell viability and EC 50 and T.C. 50 The calculations were carried out as described in Examples 6 and 8.

[0163] result Oseltamivir, an established influenza drug, was evaluated in parallel and had an EC of 0.06 μM 50 Lefamulin was cytotoxic to MDCK cells at concentrations above 23 μM. A maximum inhibition of influenza-mediated CPE of 18.4% was measured at 5 μM lefamulin. Therefore, EC 50 could not be determined for lefamulin. Of note, in vivo activity was observed in an influenza infection mouse model (see Example 5 above) using a related mouse-adapted influenza A strain (Influenza A / Puerto Rico / 8 / 34(H1N1)). [Table 12]

Claims

1. A pharmaceutical preparation for oral administration comprising about 600 mg of refamulin, or about 600 mg of refamulin in the form of a pharmaceutically acceptable salt, solvate, or prodrug thereof.

2. The pharmaceutical preparation according to claim 1, wherein the preparation is in the form of a tablet or a capsule.

3. A pharmaceutical formulation comprising refamulin, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, for use in a method for treating or preventing an inflammatory disease characterized by neutrophilic inflammation.

4. The pharmaceutical formulation according to claim 3, wherein the method reduces the level of a pro-inflammatory cytokine, chemokine, or metalloproteinase, preferably selected from TNF-α, IL-6, IL-1β, GM-CSF, CXCL1, CXCL2, CCL2, or MMP-9, in a subject.

5. The pharmaceutical preparation according to claim 3 or 4, wherein the disease is COPD, cystic fibrosis, bronchiectasis, diffuse panbronchiolitis, obstructive bronchiolitis syndrome, or noneosinophilic asthma.

6. The pharmaceutical preparation according to any one of claims 3 to 5, wherein refamlin, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, is administered by inhalation, injection, or orally.

7. The pharmaceutical formulation according to any one of claims 3 to 6, wherein refamulin, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, is administered as an orally administered pharmaceutical formulation comprising about 600 mg of refamulin, or about 600 mg of refamulin in the form of a pharmaceutically acceptable salt, solvate, or prodrug thereof.

8. The pharmaceutical preparation according to claim 7, wherein the preparation is in the form of a tablet or a capsule.

9. A pharmaceutical formulation comprising refamulin, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, for use in a method for treating or preventing neutrophilic inflammation.

10. The pharmaceutical formulation according to claim 9, wherein the method reduces the level of a pro-inflammatory cytokine, chemokine, or metalloproteinase, preferably selected from TNF-α, IL-6, IL-1β, GM-CSF, CXCL1, CXCL2, CCL2, or MMP-9, in a subject.

11. The pharmaceutical preparation according to claim 9 or 10, wherein the disease is COPD, cystic fibrosis, bronchiectasis, diffuse panbronchiolitis, obstructive bronchiolitis syndrome, or noneosinophilic asthma.

12. The pharmaceutical preparation according to any one of claims 9 to 11, wherein refamlin, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, is administered by inhalation, injection, or orally.

13. The pharmaceutical formulation according to any one of claims 9 to 12, wherein refamulin, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, is administered as an orally administered pharmaceutical formulation comprising about 600 mg of refamulin, or about 600 mg of refamulin in the form of a pharmaceutically acceptable salt, solvate, or prodrug thereof.

14. The pharmaceutical preparation according to claim 13, wherein the preparation is in the form of a tablet or a capsule.

Images