NOVEL THERAPEUTIC USE OF PLEUROMUTILINS

Abstract

A compound of formula (I)

##STR00001## wherein n is 0 to 4; m is 0 or 1 with the proviso that the sulphur atom and R.sub.3 are in vicinal position (if m=0 then R.sub.3 is in position 2′, and if m=1 then R.sub.3 is on position 1′); R is ethyl or vinyl; R.sub.1 is hydrogen or (C.sub.1-6)alkyl, R.sub.2 is hydrogen or (C.sub.3-6)cycloalkyl, or unsubstituted (C.sub.1-6)alkyl, or (C.sub.1-6)alkyl substituted by one or more of hydroxy; preferably one or two, methoxy, halogen, (C.sub.3-6)cycloalkyl, or R.sub.1 and R.sub.2 together with the nitrogen atom to which they are attached form a 5 to 7 membered heterocyclic ring containing at least 1 nitrogen atom or 1 nitrogen and 1 additional heteroatom e. g. selected from N or O, or R.sub.1 is hydroxy and R.sub.2 is formyl; R.sub.3 is OH, OR.sub.4, a halogen atom, or R.sub.3 is bound to 2′ and represents —O—(CH.sub.2).sub.p—O— with p is 2 or 3; R.sub.4 is unsubstituted (C.sub.1-6)alkyl or (C.sub.3-6)cycloalkyl, or a pharmaceutically acceptable salt, solvate, prodrug or metabolite thereof for the specific use in the treatment or prevention of a disease mediated by a virus.

Claims

1. A method for treating or preventing a disease mediated by a virus, comprising administering to a subject in need thereof a compound of formula (I) ##STR00005## wherein n is 0 to 4; m is 0 or 1 with the proviso that the sulphur atom and R.sub.3 are in vicinal position, if m=0 then R.sub.3 is in position 2′, and if m=1 then R.sub.3 is on position 1′; R is ethyl or vinyl; R.sub.1 is hydrogen or (C.sub.1-6)alkyl, R.sub.2 is hydrogen or (C.sub.3-6)cycloalkyl, or unsubstituted (C.sub.1-6)alkyl, or (C.sub.1-6)alkyl substituted by one or more of hydroxy, methoxy, halogen, or (C.sub.3-6)cycloalkyl, or R.sub.1 and R.sub.2 together with the nitrogen atom to which they are attached form a 5 to 7 membered heterocyclic ring containing at least 1 nitrogen atom or 1 nitrogen and 1 additional heteroatom selected from N or O, or R.sub.1 is hydroxy and R.sub.2 is formyl; R.sub.3 is OH, OR.sub.4, or a halogen atom, or R.sub.3 is bound to 2′ and R.sub.3 represents —O—(CH.sub.2).sub.p—O— with p being 2 or 3; and R.sub.4 is unsubstituted (C.sub.1-6)alkyl or (C.sub.3-6)cycloalkyl, or a pharmaceutically acceptable salt, solvate, prodrug or metabolite thereof.

2. The method according to claim 1, wherein the compound or the pharmaceutically acceptable salt, solvate, prodrug or metabolite thereof is selected from formulae (II), (III), (IV), (V), and (VI) ##STR00006## wherein in each formula, n, R.sub.1 and R.sub.2 are defined as in claim 1, and their pharmaceutically acceptable salts, solvates, prodrugs or metabolites.

3. The method according to claim 1, wherein the compound or the pharmaceutically acceptable salt, solvate, prodrug or metabolite thereof is selected from 14-O-{[(1R, 2R, 4R)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin; 14-O-{[(1S, 2S, 4S)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin; 14-O-{[(1R, 2R, 5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin; 14-O-{[(1S, 2S, 5R)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin; 14-O-{[(1R, 2R, 4S)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 4R) diastereomer thereof; 14-O-{[(1R, 2R, 5R)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin; 14-O-{[(1S, 2S, 5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin; 14-O-{[(1R, 2R, 3R)-3-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 3S) diastereomer thereof; 14-O-{[(1R, 2R, 4R)-4-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 4S) diastereomer thereof; 14-O-{[(1R, 2R, 4R)-4-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 4S) diastereomer thereof; 14-O-{[(1R, 2R, 5S)-5-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 5R) diastereomer thereof; 14-O-{[(1R, 2R, 5S)-5-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 5R) diastereomer thereof; 14-O-{[(1R, 2R, 4S)-4-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 4R) diastereomer thereof; 14-O-{[(1R, 2R, 5R)-5-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 5S) diastereomer thereof; 14-O-{[(1R, 2R, 3R)-3-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 3S) diastereomer thereof; 14-O-{[(1R, 2R, 3R)-3-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 3S) diastereomer thereof; 14-O-{[(1R, 2R, 4S)-4-(Formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 4R) diastereomer thereof; 14-O-{[(1R, 2R, 5S)-5-(Formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 5R) diastereomer thereof; 14-O-{[(1R, 2R, 3R/S)-3-(Formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 3R/S) diastereomer thereof; 14-O-{[(1R, 2R, 5S)-2-Hydroxy-5-methylamino-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 5R) diastereomer thereof; 14-O-{[(1R, 2R, 5S)-5-Allylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 5R) diastereomer thereof; 14-O-{[(1R, 2R, 5S)-2-Hydroxy-5-(2-methoxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 5R) diastereomer thereof; 14-O-{[(1R, 2R, 4R*)-2-Hydroxy-4-(2-hydroxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 4S*) diastereomer thereof; 14-O-{[(1R, 2R, 4R*)-4-Cyclohexylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 4S*) diastereomer thereof; 14-O-{[(1R, 2R, 4R*)-4-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 4S*) diastereomer thereof; 14-O-{[(1R, 2R, 5S*)-4-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 5R*) diastereomer thereof; 14-O-{[(1R, 2R, 4S*)-4-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 4R*) diastereomer thereof; 14-O-{[(1R, 2R, 5R*)-2-Hydroxy-5-morpholin-4-yl-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 5S*) diastereomer thereof; 14-O-{[(1R, 2R, 5S*)-2-Hydroxy-5-morpholin-4-yl-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S, 5R*) diastereomer thereof; 14-O-{[(1R, 2R, 5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin and the (1S, 2S, 5R) diastereomer thereof; 14-O-{[(1R, 2R, 5S)-5-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin and the (1S, 2S, 5R) diastereomer thereof; 14-O-{[(1R, 2R, 5R)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin and the (1S, 2S, 5S) diastereomer thereof; 14-O-{[(1R, 2R)-4-Aminomethyl-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S) diastereomers thereof; 14-O-{[5-Amino-2-chloro-cyclohexylsulfanyl]-acetyl}-mutilin; 14-O-{[4-Amino-2-chloro-cyclohexylsulfanyl]-acetyl}-mutilin; 14-O-[(4-Amino-1-hydroxy-cyclohexylmethylsulfanyl)-acetyl]-mutilin; 14-O-{[(1R, 2R)-2-Hydroxy-5-(3-methylamino-propyl)-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S) diastereomer thereof; 14-O-{[(1R, 2R)-2-Hydroxy-4-(3-methylamino-propyl)-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S) diastereomer thereof; 14-O-{[(1R, 2R)-5-(3-Amino-propyl)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S) diastereomer thereof; 14-O-{[(1R, 2R)-4-(3-Amino-propyl)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin and the (1S, 2S) diastereomer thereof; 14-O-{[(6R, 8R)-8-Amino-1,4-dioxa-spiro[4.5]dec-6-ylsulfanyl]-acetyl}-mutilin and the (6S, 8S) diastereomer thereof; 14-O-{[4-Amino-2-methoxy-cyclohexylsulfanyl]-acetyl}-mutilin; 14-O-{[5-Amino-2-methoxy-cyclohexylsulfanyl]-acetyl}-mutilin and their pharmaceutically acceptable salts, solvates, prodrugs or metabolites.

4. The method according to claim 1, wherein the compound or the pharmaceutically acceptable salt, solvate, prodrug or metabolite thereof is Lefamulin or its pharmaceutically acceptable salts, solvates, prodrugs or metabolites.

5. The method according to claim 1, wherein the compound or the pharmaceutically acceptable salt, solvate, prodrug or metabolite thereof is in form of a salt and/or a solvate.

6. The method according to claim 1, wherein the compound or the pharmaceutically acceptable salt, solvate, prodrug or metabolite thereof is Lefamulin in the form as Lefamulin acetate salt.

7. The method according to claim 1, wherein the disease is a respiratory disease.

8. The method according to claim 1, wherein the disease is an acute respiratory syndrome.

9. The method according to claim 1, wherein the virus is a positive- or negative-sense single-stranded RNA virus.

10. The method according to claim 1, wherein the disease is an airborne disease.

11.-15. (canceled)

16. The method according to claim 8, wherein the acute respiratory syndrome includes Influenza, Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) or COVID-19.

17. The method according to claim 9, wherein the virus is selected from Coronaviridae, Paramyxoviridae, Orthomyxoviridae, Flaviviridae, and Picornaviridae.

18. The method according to claim 17, wherein the Coranaviridae is human coronavirus.

19. The method according to claim 17, wherein the Paramyxoviridae is Paramyxovirinae or Pneumovirinae.

20. The method according to claim 19, wherein the Paramyxovirinae is Measles virus.

21. The method according to claim 19, wherein the Pneumovirinae is Respiratory Syncytial Virus.

22. The method according to claim 17, wherein the Orthomyxoviridae is Influenza virus.

23. The method according to claim 17, wherein the Flaviviridae is Dengue virus or Zika virus.

24. The method according to claim 17, wherein the Picornaviridae is Rhinovirus.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 demonstrates the effect of Lefamulin against alpha corona virus 229E (HCoV-229E) in MRC-5 cells 6 days post infections with the virus.

[0018] FIG. 2 demonstrates the effect of Tiamulin in the same assay.

[0019] FIG. 3 demonstrates the effect of Remdesivir in the same assay.

[0020] FIG. 4 demonstrates the effect of Lefamulin against respiratory syncytial virus type A in HEp2 cells 6 days post infections with the virus.

[0021] FIG. 5 demonstrates the effect of Tiamulin in the same assay.

[0022] FIG. 6 demonstrates the effect of TMC353121 in the same assay.

[0023] FIGS. 7A to 7D demonstrate the effect of Lefamulin and Oseltamivir on clinical signs in an Influenza infection mouse model, in particular on the body weight (A), clinical score (B), percentage survival (C), and lung sample histopathology scoring (D).

[0024] FIG. 8 demonstrates the effect of Lefamulin and Oseltamivir on lung viral titer in the Influenza infection mouse model determined as 50% tissue culture infective dose (TCID.sub.50) in MDCK cells.

DETAILED DESCRIPTION OF THE INVENTION

[0025] Lefamulin is the INN for a compound of generic formula (I), more particular, Lefamulin is a compound of formula (VII)

##STR00004##

[0026] i.e. 14-O-{[(1R, 2R, 4R)-4-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin (also known as “BC-3781”).

[0027] In the following, the term “Lefanulin”, when generally used without additional explanation, is intended to encompass both Lefamulin in free base form, as well as its salts and solvates.

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

[0029] The present invention refers to the treatment and prevention of a disease mediated by viruses, e.g. a viral disease or viral infection.

[0030] The results of the experiments show that besides its antibacterial activity, Lefamulin is also actively reducing the cytopathic effect mediated by different viruses. This antiviral effect was particularly shown for such viruses that are characterized in that they are positive- or negative sense single-stranded RNA viruses. Antiviral activity was shown for both enveloped and non-enveloped viruses, in particular several enveloped positive- or negative sense single-stranded RNA viruses (such as Coronaviridae, Paramyxoviridae, Orthomyxoviridae, and Flaviviridae). Moreover, some of the investigated viruses, including measles virus are known for a transmission involving the respiratory route, in particular airborne transmission. Corona virus and Respiratory Syncytial Virus also cause infections of the respiratory tract in humans.

[0031] In a preferred embodiment of the present invention, the virus is a positive- or negative-sense single-stranded RNA virus,

[0032] preferably the virus is selected from the group consisting of [0033] Coronaviridae including in particular human coronavirus, [0034] Paramyxoviridae including in particular Paramyxovirinae, such as Measles virus, and Pneumovirinae, such as Respiratory Syncytial Virus, [0035] Orthomyxoviridae including in particular Influenza virus, [0036] Flaviviridae including in particular Dengue virus and Zika virus, and [0037] Picomaviridae including in particular Rhinovirus.

[0038] In an other embodiment, the disease is an airborne disease. An airborne disease is mediated by a virus transmitted by the air.

[0039] Viral infections can affect various organs. In a preferred embodiment of the present invention, the disease is a respiratory disease, including upper and lower respiratory infections, in particular lower respiratory infections.

[0040] In particular, the disease is an acute respiratory syndrome, such as Influenza, Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) or COVID-19.

[0041] In a further embodiment of the present invention the disease is mediated by a virus selected from the group consisting of viruses of the virus families Coronaviridae, in particular a corona virus such as SARS-CoV, SARS-CoV2, MERS-CoV or HCoV-229E, Orthomyxoviridae, in particular an Influenza virus such as Influenza A and B viruses, Paramyxoviridae in particular Respiratory Syncytial Virus and Adenoviridae, in particular Adenovirus.

[0042] In an embodiment, the virus is a corona virus, in particular selected from the group consisting of SARS-CoV, SARS-CoV2, MERS-CoV, and HCoV-229E as well as mutations thereof. Such corona viruses are known to cause (severe) acute respiratory syndromes, such as SARS, MERS or COVID-19.

[0043] Treating, treatment or to treat as understood herein includes on one hand the complete curing, curation or to cure a condition (the disease mediated by a virus) such that it comes to its end and on the other hand also ameliorating, amelioration or to ameliorate a condition such that its symptoms are reduced at least partially or individually.

[0044] Treatment typically includes administering a compound as used according to the present invention to a subject in need thereof, i.e. a subject being diagnosed to have a disease mediated by a virus.

[0045] Preventing, prevention, or to prevent includes administering a compound before a condition is diagnosed or before onset of (all) disease symptoms of the condition.

[0046] Prevention of diseases mediated by viruses includes administering the compounds before onset of disease symptoms. Prevention may be considered after a subject has been infected with a virus but has not shown any symptoms, or wherein a subject has been exposed and/or is prone to exposition to a virus.

[0047] The appropriate dosage of the compound to be administered according to the present invention, in particular Lefamulin, will, of course, vary depending upon, for example, the individual host, the mode of administration and the nature and severity of the conditions being treated. However, in general, for satisfactory results in larger mammals, for example humans, an indicated daily dosage is in the range from about 0.5 mg to 3 g of a compound used according to the present invention conveniently administered, for example, in divided doses up to four times a day.

[0048] The compound used according to the present invention may be administered by any conventional route, for example enterally, e.g. including nasal, buccal, rectal, oral administration; parenterally, e.g. including intravenous, intramuscular, subcutaneous administration; or topically, e.g. including pulmonary, epicutaneous, intranasal, intratracheal administration, e.g. in form of coated or uncoated tablets, capsules, injectable solutions or suspensions, e.g. in the form of ampoules, vials, in the form of ointments, creams, gels, pastes, inhaler powder, foams, tinctures, lip sticks, drops, sprays, or in the form of suppositories, e.g. in analogous manner to the antibiotic agent tobramycin or macrolides, such as erythromycins, e.g. clarithromycin or azithromycin.

[0049] Preferably, the compound used according to the present invention is administered via inhalation, via intravenous or subcutaneous injection, or orally.

[0050] Preferred pharmaceutical compositions of Lefamulin for injection are disclosed in WO 2016/202788 A1 the contents of which are incorporated herein by reference.

[0051] The compound used according to the present invention, in particular Lefamulin, may be administered in the form of a pharmaceutically acceptable salt, e.g. an acid addition salt, or in free form, optionally in the form of a solvate.

[0052] In one embodiment, the compound is in the form of a salt and/or a solvate.

[0053] A salt of a compound used according to the present invention includes an acid addition salt. Pharmaceutically acceptable acid addition salts include salts of a compound used according to the present invention with an acid, e.g. hydrogen fumaric acid, fumaric acid, tartaric acid, ethane-1,2-disulphonic acid, maleic acid, naphthalin-1,5-sulphonic acid, acetic acid, malic acid, lactic acid, i.e. L-lactic acid, succinic acid, salicylic acid, azelaic acid, 2-[(2,6-dichlorophenyl)amino]benzene acetic acid, hydrochloric acid, deuterochloric acid, preferably hydrochloric acid, acetic acid, L-lactic acid, and maleic acid.

[0054] Of these, in the case of Lefamulin, the acetate salt of Lefamulin is especially preferred.

[0055] Preferred crystalline forms of Lefamulin as well as crystalline salt forms of Lefamulin are disclosed in WO 2011/146954 A1, the contents of which are incorporated herein by reference. Of these, the acetate salt of Lefamulin in crystalline Form B as disclosed in WO 2011/146954 A1 is especially preferred.

[0056] The present invention also provides the Lefamulin in its form as acid addition salt with itaconic acid, in particular Lefamulin itaconate. Lefamulin itaconate is disclosed herein as a new compound (Example 12). Itaconic acid can be deprotonated to the anions hydrogen itaconate and itaconate. The acid addition salt comprising Lefamulin as cation and an anion derived from itaconic acid is expected to be useful as antiviral agent.

[0057] The compound used according to the present invention, in particular Lefamulin, may be used for the pharmaceutical treatment contemplated herein alone or in combination with one or more other pharmaceutically active agents. Such other pharmaceutically active agents include e.g. other antiviral agents. Such other antiviral agents may preferably be selected from the group consisting of nucleoside and nucleotide analogues and RNA polymerase inhibitors, e.g.

[0058] 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, interferons (interferon alfa and/or beta), or other broad-spectrum antiviral agents.

[0059] 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 instruction for co-administration; and free combinations in which the pharmaceutically active agents are packaged separately, but instruction for simultaneous or sequential administration are given.

[0060] A pharmaceutical composition comprising a compound used according to the present invention, in particular Lefamulin may in addition comprise at least one pharmaceutically acceptable excipient, e.g. carrier or diluent, e.g. including fillers, binders, disintegrators, flow conditioners, lubricants, sugars and sweeteners, fragrances, preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating osmotic pressure and/or buffers.

[0061] Such pharmaceutical compositions may be manufactured according, e.g. analogously, to a method as conventional, e.g. by mixing, granulating, coating, dissolving, spray drying, or lyophilizing processes. Unit dosage form may contain, for example, from about 0.5 mg to about 3000 mg, such as 10 mg to about 600 mg.

[0062] A subject in need of a treatment as contemplated by the present invention may be any living subject suffering from a disease mediated by a virus.

[0063] Especially, the subject may be a human or an animal.

EXAMPLES

[0064] Herein, including the examples, the following abbreviations are used:

[0065] .sup.1H-NMR proton nuclear magnetic resonance spectroscopy

[0066] ° C. degrees Celsius

[0067] μM micromolar concentration

[0068] BALB/c laboratory-bred mouse strain

[0069] BC-3781 lefamulin

[0070] CC Cell Control

[0071] CoV corona virus

[0072] CPE cytopathic effects, in particular virus-induced

[0073] DMEM Dulbecco's modified Eagle's medium

[0074] DMF N,N-dimethylformamide

[0075] DMSOdimethylsulfoxide

[0076] EC.sub.50 Half maximal (fifty-percent) effective concentration

[0077] eq equivalents

[0078] FBS Fetal bovine serum

[0079] HeLa immortal human epithelial cell line

[0080] HEp2 human epithelial cell line

[0081] Huh7 human liver cell line

[0082] MDCK Madin-Darby Canine Kidney cells

[0083] MOI Multiplicity of infection

[0084] MRC-5 Medical Research Council cell strain 5

[0085] M molarity

[0086] MS mass spectrometry

[0087] m/z mass/charge ratio

[0088] MTBE methyl-tert-butylether

[0089] nm nanometer

[0090] TC.sub.50 Half maximal (fifty-percent) toxic concentration

[0091] TCID.sub.50 Fifty-percent (half maximal) tissue culture infective dose

[0092] VC reduction in viral CPE

[0093] XTT 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide

Example 1

[0094] Objective: The assay measured the inhibition of virus-induced cytopathic effects (CPE) and cell viability following alpha coronavirus 229E (HCoV-229E or CoV.sub.229E) in MRC-5 cells 6 days post infections with the virus by various concentrations of Lefamulin (BC-3781).

[0095] Methodology: MRC-5 cells were seeded in 96-well flat-bottom tissue culture plates (at a density of 3×10.sup.3 cells per well) and allowed to adhere overnight. Thereafter, diluted test compounds (Lefamulin as acetate, Tiamulin as fumarate) dissolved in DMSO were added to the plate and incubated for 4 hours prior to addition of the virus. The virus was added diluted to a pre-determined titer to yield 85-95% cell killing at 6 days post-infection (MOI of 0.001).

[0096] Following incubation at 37° C. and at 5% CO.sub.2 for 6 days, cell viability was measured by XTT tetrazolium dye staining. The optical density of the cell culture plate was determined spectrophotometrically at 450 and 650 rn. Percent reduction of the virus-infected cells and the percent cell viability of uninfected drug control wells were calculated to determine the effective concentration at which 50% of cytopathic effect was inhibited (EC.sub.50) and the cytotoxic concentration (TC.sub.50) using four parameter curve fit analysis. The antiviral compound Remdesivir served as positive control.

[0097] Results:

[0098] Surprisingly, Lefamulin reduced the viral CPE by 91.82% at a concentration of 10 M, which is a concentration that had no cytotoxic effect on the viability of the cell control. The calculated EC.sub.50 was 3.87 μM, at which 50% of the viral cytopathic effect was inhibited. At the Lefamulin concentration of 50 μM, Lefamulin displayed a cytotoxic effect; the calculated TC.sub.50 was 55.3 μM. The ratio of EC.sub.50 and TC.sub.50, known also as therapeutic index, was 14.3.

[0099] In contrast, Tiamulin at a concentration of 10 μM reduced the viral CPE only by 10.53% and no cytotoxic effect was observed. At the next higher test concentration of 50 μM the CPE was reduced by 81.68% and a cytotoxic effect was observed. The calculated EC.sub.50 was 24.4 μM and the calculated TC.sub.50 was 62.9 μM. The therapeutic index of Tiamulin was 2.58 and surprisingly much lower than that of Lefamulin.

[0100] The antiviral compound Remdesivir was developed as a treatment for Ebola virus, and also is known to have antiviral activity against corona viruses (clinical investigation is ongoing). Thus, Remdesivir served as positive control herein. Remdesivir showed an EC.sub.50 of 0.11 M, a TC.sub.50 of >5 and a therapeutic index of >45.5.

TABLE-US-00001 MRC-5 cells infected with CoV.sub.229E Compound EC.sub.50 (μM) TC.sub.50 (μM) Therapeutic Index Lefamulin 3.87 55.3 14.3 Tiamulin 24.4 62.9 2.58 Remdesivir 0.11 >5.00 >45.5

[0101] The results are graphically displayed in FIGS. 1 (Lefamulin), 2 (Tiamulin) and 3 (Remdesivir) (VC . . . reduction in viral CPE, CC . . . Cell Control).

Example 2

[0102] Objective: The assay measured the inhibition of virus-induced cytopathic effects (CPE) and cell viability following alpha coronavirus 229E (HCoV-229E or CoV.sub.229E) in MRC-5 cells for Lefamulin at various treatment conditions.

[0103] Method: The assay was performed in analogy to Example 1 above with the following differences regarding the test candidate. Lefamulin (as acetate) was evaluated using different treatment conditions of 4 h, 1 h or 0 h incubation prior to virus addition and addition 1 h after infection For this particular set of experiments, coronavirus was diluted 1:200 in assay medium and added at 100 μL/well to achieve approximately 90% cell killing in the untreated virus control wells (MOI of 0.001).

[0104] Results:

[0105] The antiviral efficacy and cellular toxicity data are summarized in the table below. Lefamulin showed a time-dependent effect on the inhibition of the virus-induced cytopathic effects (CPE). In the treatment setting after viral exposure (1 h post-infection) a dose-dependent effect was observed. At a concentration of 50 μM the viral CPE was reduced by 86.83% (data not shown in detail).

TABLE-US-00002 Treatment condition MRC-5 cells infected with CoV.sub.229E Lefamulin EC.sub.50 (μM) TC.sub.50 (μM) Therapeutic Index 4 h pretreatment 2.18 >50.0 >22.9 1 h pretreatment 2.16 >50.0 >23.1 0 h pretreatment 3.08 >50.0 >16.2  1 h post-infection 15.1 >50.0 >3.31

Example 3

[0106] Objective: The assay measured the inhibition of virus-induced cytopathic effects (CPE) and cell viability following human respiratory syncytial virus (strain RSV.sub.A2) replication in HEp2 cells 6 days post infections with the virus by various concentrations of Lefamulin (BC-3781).

[0107] Methodology: HEp2 cells were seeded in 96-well flat-bottom tissue culture plates (at a density of 5×10.sup.3 cells per well) and allowed to adhere overnight. Thereafter, diluted test compounds (Lefamulin as acetate, Tiamulin as fumarate) in DMSO were added to the plate and incubated for 4 hours prior to addition of the virus. The virus was added diluted to a pre-determined titer to yield 85-95% cell killing at 6 days post-infection (MOI of 0.001).

[0108] Following incubation at 37° C. and at 5% CO.sub.2 for 6 days, cell viability was measured by XTT tetrazolium dye staining. The optical density of the cell culture plate was determined spectrophotometrically at 450 and 650 nm. Percent reduction of the virus-infected cells and the percent cell viability of uninfected drug control wells were calculated to determine the effective concentration at which 50% of cytopathic effect was inhibited (EC.sub.50) and the cytotoxic concentration (TC.sub.50) using four parameter curve fit analysis. The antiviral compound TMC353121 (RSV fusion inhibitor) served as positive control.

[0109] Results:

[0110] Surprisingly, Lefamulin reduced the viral cytopathic effect (CPE) by 92.17% and 100% at concentrations of 10 μM and 50 μM, respectively, which are concentrations that had no cytotoxic effect on the viability of the cell control. The calculated EC.sub.50 was 5.34 μM, at which 50% of the viral CPE was inhibited. At the Lefamulin concentration of 100 μM, Lefamulin displayed a cytotoxic effect; the calculated TC.sub.50 was 70.7 μM. The ratio of EC.sub.50 and TC.sub.50, known also as therapeutic index, was 13.2.

[0111] In contrast, Tiamulin at a concentration of 10 μM reduced the viral CPE only by 16.76% and a cytotoxic effect (84% viability) was observed at this concentration. At the next higher test concentration of 50 μM the viral CPE was reduced by 43.28% and at the cytotoxic effect was more pronounced (70.0% viability). The calculated EC.sub.50 was with >67.9 μM above the calculated TC.sub.50 of 67.9 μM. The therapeutic index of Tiamulin therefore could not be calculated. Surprisingly, the antiviral activity and the therapeutic index was much higher for Lefamulin than for Tiamulin.

[0112] The antiviral compound TMC353121 was developed as a specific respiratory syncytial virus fusion inhibitor (clinical investigation is ongoing). Thus, TMC353121 served as positive control herein. TMC353121 showed an EC.sub.50 of 0.006 M, a TC.sub.50 of >0.1 μM and a therapeutic index of >167.

TABLE-US-00003 HEp2 cells infected with human respiratory syncytial virus (RSVS.sub.A2) Compound EC.sub.50 (μM) TC.sub.50 (μM) Therapeutic Index Lefamulin 5.34 70.7 13.2 Tiamulin >67.9 67.9 — TMC353121 0.0006 >0.1 >167

[0113] The results are graphically displayed in FIGS. 4 (Lefanulin), 5 (Tiamulin) and 6 (TMC353121) (VC . . . Reduction in viral CPE, CC . . . Cell Control).

Example 4

[0114] Objective: The assay measured the inhibition of virus-induced cytopathic effects (CPE) and cell viability following human respiratory syncytial virus (strain RSV.sub.A2) replication in HEp2 cells changing the multiplicity of infection (MOI).

[0115] Methodology: The assay was performed in analogy to Example 3 above with the following differences regarding the test. The virus was added diluted to a pre-determined titer to yield 85-95% cell killing at 6 days post-infection, and the added amount was adopted to obtain MOIs of 0.003, 0.001, 0.0008, and 0.0004, respectively. Lefamulin (as acetate) was investigated in this study as well as TMC353121 for positive control.

[0116] Results:

[0117] The antiviral efficacy and cellular toxicity data are summarized in the tables below. The EC.sub.50 value in the low μM range for Lefamulin was reproduced at an MOI of 0.0004. In contrast, a higher MOI, thus higher viral load with respect to the investigated cells, reduced the antiviral effect of Lefamulin. This effect is less pronounced in the highly effective control substance TMC353121.

TABLE-US-00004 MOI/Compound HEp2 cells infected with human respiratory syncytial virus (RSV.sub.A2) Lefamulin EC.sub.50 (μM) TC.sub.50 (μM) Therapeutic Index 0.003 >63.2 63.2 — 0.001 32.3 65.9 2.04 0.0008 28.7 66.60 2.32 0.0004 2.34 67.4 28.8

TABLE-US-00005 MOI/Compound HEp2 cells infected with human respiratory syncytial virus (RSV.sub.A2) TMC353121 EC.sub.50 (μM) TC.sub.50 (μM) Therapeutic Index 0.003 0.0002 >0.1 500 0.001 0.0002 >0.1 >500 0.0008 0.0003 >0.1 >333 0.0004 0.00004 >0.1 >2500

Example 5

[0118] Objective: The assay measured the inhibition of virus-induced cytopathic effects (CPE) and cell viability following replication of the two different respiratory syncytial virus strains RSV A.sub.LONG and RSV B.sub.18537 in HEp2 cells.

[0119] Methodology: The assay was performed in analogy to Example 3 above with the difference that cells seeded with a density of 5×10.sup.3 cells per well were incubated with the virus strains RSV A.sub.LONG or RSV B.sub.13537, respectively, following a 4 hour cell pretreatment with the test compound at different concentrations. Virus was diluted and added in an amount yielding MOIs of 0.01 and 0.001 for RSV A.sub.LONG and RSV B.sub.18537, respectively.

[0120] Results:

[0121] The antiviral efficacy and cellular toxicity data are summarized in the tables below. The control compound TMC353121 was evaluated in parallel to Lefamulin and yielded an EC.sub.50 value of 0.01 nM against the investigated strains of RSV A and RSV B. Lefamulin yielded an EC.sub.50 value of 17.7 μM against the RSV B.sub.18537. Activity against RSV A.sub.LONG could not be determined due to the cytotoxicity to HEp2 cells with TC.sub.50 values of 71.1 μM in the assay.

TABLE-US-00006 HEp2 cells infected with respiratory syncytial virus (RSV A.sub.LONG) Compound EC.sub.50 (μM) TC.sub.50 (μM) Therapeutic Index Lefamulin >62.9 62.9 — TMC353121 0.00001 >1.00 >100000

TABLE-US-00007 HEp2 cells infected with respiratory syncytial virus (RSV B.sub.18537) Compound EC.sub.50 (μM) TC.sub.50 (μM) Therapeutic Index Lefamulin 17.7 71.1 4.02 TMC353121 0.00001 >1.00 >100000

Example 6

[0122] Objective: The assay measured the inhibition of virus-induced cytopathic effects (CPE) and cell viability during replication of Measles virus strain Edmonston in HeLa cells.

[0123] Method: HeLa cells were seeded in 96-well flat-bottom tissue culture plates (at a density of 5×10.sup.3 cells per well) and allowed to adhere overnight. Thereafter, diluted test compounds (Lefamulin as acetate, Ribavirin for control) were added to the plate and incubated for 4 hours prior to addition of the virus. Virus was added diluted to a pre-determined titer to yield 85-95% cell killing at 6 days post-infection (1:50 dilution, MOI of 0.008).

[0124] Cell viability determination and calculation of EC.sub.50 and TC.sub.50 were performed as described in Examples 1 and 3.

[0125] Results:

[0126] The antiviral efficacy and cellular toxicity data are summarized in the Table below. Ribavirin was evaluated as control compound in parallel to Lefamulin and yielded an EC.sub.50 value of 1.88 μg/mL. Surprisingly, Lefamulin yielded an even lower EC.sub.50 value of 0.89 μM with a high calculated TI of 81.7.

TABLE-US-00008 HeLa Cells infected with Measles virus (strain Edmonston) Compound EC.sub.50 (μM) TC.sub.50 (μM) Therapeutic Index Lefamulin 0.89 72.7 81.7 Ribavirin (μg/mL) 1.88 21.6 11.5

Example 7

[0127] Objective: The assay measured the inhibition of virus-induced cytopathic effects (CPE) and cell viability during replication of Dengue virus strain DENV2.sub.New Guinea in Huh7 cells.

[0128] Method: Huh7 cells were seeded in 96-well flat-bottom tissue culture plates (at a density of 5×10.sup.3 cells per well) and allowed to adhere overnight. Thereafter, diluted test compounds (Lefamulin as acetate, Ribavirin for control) were added to the plate and incubated for 4 hours prior to addition of the virus. The Dengue virus strain DENV2.sub.New Guinea was obtained from ATCC (VR-1584) and was grown in Rhesus monkey kidney cells for the production of stock virus pools. Virus was added diluted to a pre-determined titer to yield 85-95% cell killing at 6 days post-infection (MOI of 0.001).

[0129] Cell viability determination as well as EC.sub.50 and TC.sub.50 calculations were performed as described in Examples 1 and 3.

[0130] Results:

[0131] The antiviral efficacy and cellular toxicity data are summarized in the Table below. Ribavirin was evaluated as control compound in parallel to Lefamulin and yielded an EC.sub.50 value of 4.73 μg/mL. Lefamulin yielded an EC.sub.50 value of 6.79 μM. Ribavirin and Lefamulin both showed a certain cytotoxicity for this specific cell line at concentrations of 48.5 μg/mL and 23.3 μM, respectively.

TABLE-US-00009 Huh7 Cells infected with Dengue virus (strain DENV2.sub.New Guinea) Compound EC.sub.50 (μM) TC.sub.50 (μM) Therapeutic Index Lefamulin 6.79 23.3 3.43 Ribavirin (μg/mL) 4.73 48.5 10.3

Example 8

[0132] Objective: The assay measured the inhibition of virus-induced cytopathic effects (CPE) and cell viability during replication of Zika virus strain ZIKV.sub.PRVABC59 in Huh7 cells following a 4 hour cell pretreatment.

[0133] Method: Huh7 cells were seeded in 96-well flat-bottom tissue culture plates (at a density of 5×10.sup.3 cells per well) and allowed to adhere overnight. Thereafter, diluted test compounds (Lefamulin as acetate, Sofosbuvir for control) were added to the plate and incubated for 4 hours prior to addition of the virus. The Zika virus strain PRVABC59 obtained from ATCC (catalog VR-1843) was obtained from ATCC (VR-1584) and was grown in Rhesus monkey kidney cells for the production of stock virus pools. Virus was added diluted to a pre-determined titer to yield 85-95% cell killing at 6 days post-infection (MOI of 0.001).

[0134] Cell viability determination as well as EC.sub.50 and TC.sub.50 calculations were performed as described in Examples 1 and 3.

[0135] Results:

[0136] The antiviral efficacy and cellular toxicity data are summarized in the Table below. The control compound Sofosbuvir was evaluated in parallel to Lefamulin and yielded an EC.sub.50 value of 0.65 μg/mL. Lefamulin yielded an EC.sub.50 value of 2.78 μM with a calculated therapeutic index of 8.42.

TABLE-US-00010 Huh7 Cells infected with Zika virus (strain ZIKV.sub.PRVABC59) Compound EC.sub.50 (μM) TC.sub.50 (μM) Therapeutic Index Lefamulin 2.78 23.4 8.42 Sofosbuvir 0.65 >10 >15.4

Example 9

[0137] Objective: The assay measured the inhibition of virus-induced cytopathic effects (CPE) and cell viability during replication of human rhinovirus strain HRV16 strain 11757 in H1-HeLa cells following a 4 hour cell pretreatment.

[0138] Method: H1-HeLa cells were seeded in 96-well flat-bottom tissue culture plates (at a density of 5×10.sup.3 cells per well) and allowed to adhere overnight. Thereafter, diluted test compounds (Lefamulin as acetate, Rupintrivir for control) were added to the plate and incubated for 4 hours prior to addition of the virus. The virus HRV16.sub.11757 was added diluted to a pre-determined titer to yield 85-95% cell killing in the untreated virus control wells (MOI of 0.0005).

[0139] Cell viability determination as well as EC.sub.50 and TC.sub.50 calculations were performed as described in Examples 1 and 3.

[0140] Results:

[0141] Rupintrivir, a protease inhibitor developed for treatment of rhinoviruses, was evaluated in parallel and yielded an EC.sub.50 value of 4.90 nM. Lefamulin yielded an EC.sub.50 value of 9.34 μM with a calculated therapeutic index of 2.58.

TABLE-US-00011 MDCK Cells infected with rhinovirus HRV16.sub.11757 Compound EC.sub.50 (μM) TC.sub.50 (μM) Therapeutic Index Lefamulin 9.34 24.1 2.58 Rupintrivir 0.0049 >0.10 >20.4

Example 10

[0142] Objective: The assay measured the inhibition of virus-induced cytopathic effects (CPE) and cell viability during replication of influenza virus strain A/PR/8/34 in MDCK cells following a 4 hour cell pretreatment.

[0143] Method: MDCK cells were seeded in 96-well flat-bottom tissue culture plates (at a density of 5×10.sup.1 cells per well) and allowed to adhere overnight.

[0144] Thereafter, diluted test compounds (Lefamulin as acetate, Oseltamivir for control) were added to the plate and incubated for 4 hours prior to addition of the virus. The influenza virus strain A/PR/8/34 was added diluted to a pre-determined titer to yield 90% cell killing in the untreated virus control wells (MOI of 0.0004).

[0145] Cell viability determination as well as EC.sub.50 and TC.sub.50 calculations were performed as described in Examples 1 and 3.

[0146] Results:

[0147] Oseltamivir as established influenza drug was evaluated in parallel and yielded an EC.sub.50 value of 0.06 μM. Lefamulin was cytotoxic to MDCK cells at concentrations greater than 23 μM. A maximum inhibition of the influenza mediated CPE by 18.4% was measured at 5 μM Lefamulin. Therefore, and because cytotoxicity was observed at 50 μM, an EC.sub.50 could not be determined for Lefamulin. However, an in vivo activity was observed in an Influenza infection mouse model (see Example 11 below) using a related mouse adapted Influenza A strain (Influenza A/Puerto Rico/8/34 (H1N1)).

TABLE-US-00012 MDCK Cells infected with influenza virus (strain A/PR/8/34) Compound EC.sub.50 (μM) TC.sub.50 (μM) Therapeutic Index Lefamulin >23.8 23.8 — Oseltamivir 0.06 >200 >333

Example 11

[0148] Objective: Effects of Lefamulin were investigated in an in vivo Influenza virus infection model. In the Influenza virus model, mice were challenged with an influenza A (H1N1) strain adapted to mice.

[0149] Methodology: Adult female BALB/c mice were randomly allocated to three experimental groups of 15 animals and allowed to acclimatize for one week. Treatments were administered subcutaneously starting at Day −1. The negative control group received a vehicle administered twice per day. Lefamulin was investigated at different dosing. In the low dose regime 35 mg/kg of Lefamulin were administered twice daily (corresponding to a dose of 70 mg/kg/day) from Day −1 to Day 6. In the high dose regime, Lefamulin was administered with a dose of 105/mg/kg/day in three injections until day 3 and was altered from Day 3 on due to administration problems at the injection site. The following doses were 70 mg/kg administered twice daily (corresponding to 140 mg/kg/day). On Day 0, all groups were challenged with influenza A/Puerto Rico/8/34 (H1N1).

[0150] During the study, animals were scored daily for clinical signs of influenza virus infection to include abnormal coat condition (piloerection), abnormal posture (hunched), abnormal breathing (rapid and/or irregular breathing rate), reduced mobility, ocular discharged, eye closure and/or survival. The signs of severity of disease were added for the scoring system yielding a maximum possible score of 5. When clinical signs were judged as severe, individual animals were taken out of the study prior to the scheduled end of the study.

[0151] At Day 6, lung tissue was dissected, assessed for gross pathology, preserved in fixative and stored for histopathology.

[0152] Lung consolidation was scored as follows after macroscopic evaluation: >50% (across all lobes) of field occupied by intra-alveolar edema/haemorrhage; extensive vascular degeneration. Lungs removed and fixated on Day 6 were evaluated microscopically in the histopathology. Four main readouts were evaluated (Bronchial/Bronchiolar Degeneration/Hyperplasia, Broncho-interstitial Inflammation, Alveolar Inflammation/Degeneration, Alveolar Edema/Haemorrhage) and scored yielding a maximum total histopathology score of 16, wherein a lower number indicates less signs of histopathological anomalies.

[0153] Lung samples were also processed and stored for Day 3 and Day 6 viral titre. On Day 3 and 6, lungs were collected, homogenised and clarified to determine viral load by TCID.sub.50 assay on Madin-Darby Canine Kidney (MDCK) cells.

[0154] Results:

[0155] The results of the clinical monitoring are shown in FIGS. 7A to 7D. The positive control treatment with Oseltamivir worked as expected. A reduction was observed in clinical scores and bodyweight loss for Oseltamivir. Lefamulin did not have a significant effect on bodyweight at the investigated doses (FIG. 7A). At high dose, Lefamulin resulted in an increase in clinical scores and decrease in survival compared to vehicle, which might relate to the local tolerability (SC) issues of the investigated dosage, concentration and formulation (FIGS. 7B and C). With the lower dose of Lefamulin, 90% survival was achieved, whereas only 20% of the vehicle group survived until day 6 (FIG. 7C).

[0156] Moreover, a significant improvement in the macroscopically evaluated lung consolidation was observed for Lefamulin at the low dose. In histopathology, a spectrum of overall individual animal scores (4-13 range) were evident within the specimens examined (FIG. 7D). Lesions were similar to those described within the literature. Increasing severities/distribution area of alveolar pathology were correlated with increases in bronchiolar degeneration/proliferation, broncho-interstitial inflammation and alveola oedema/haemorrhage. Treatment with the high dose of Lefamulin resulted in a significant reduction in bronchial degeneration and alveolar inflammation resulting in an overall significant reduction in histopathology score in this group compared to the vehicle treated control and comparable to Oseltamivir.

[0157] Lung viral titre decreased in all groups between Day 3 and Day 6 (FIG. 8). Lefamulin at both doses and Oseltamivir resulted in reduced lung viral titres when compared with the vehicle treated control.

[0158] The clinical readout and the reductive effect on the lung viral titer further supports the potential of Lefamulin in the treatment of viral diseases.

Example 12

[0159] Objective: The example aims at the synthesis of Lefamulin itaconate as a potential new active pharmaceutical ingredient comprising Lefamulin in protonated form as cation and itaconate as an anion derived from the dicarboxylic itaconic acid.

[0160] Methodology and Result:

[0161] To a solution of lefamulin as free base (1 g, 1 eq) in DMF (2 mL) itaconic acid was added (0.5 eq) and stirred at room temperature overnight. The resulting reaction mixture was added dropwise to MTBE. The obtained precipitate was filtered, washed with MTBE and dried under reduced pressure to receive Lefamulin itaconate (1.20 g) in the form of a colorless solid.

[0162] .sup.1H-NMR (400 MHz, DMSO-d.sub.6, δ, ppm, characteristic signals, mutilin numbering system described in Bemer, H.; Schulz, G.; Schneider H. Tetrahedron 1980, 36, 1807-1811): 6.15 (dd, 1H, H-19, J=17.6, 11.2 Hz), 5.63 (s, 0.5H, itaconate), 5.55 (d, 1H, H-14, J=8.0 Hz), 5.25-5.00 (m, 2.5H, H-20, itaconate), 3.55 and 3.29 (AB, 2H, H-22, J=15.2 Hz), 3.43 (d, 1H, H-11, J=5.6 Hz), 3.05 (s, 1H, itaconate), 1.37 (s, 3H, CH.sub.3-15), 1.06 (s, 3H, CH.sub.3-18), 0.82 (d, 3H, CH.sub.3-17, J=7.2 Hz), 0.63 (d, 3H, CH.sub.3-16, J=6.8 Hz).

[0163] MS m/z: 508 [M+H.sup.+], 552 [M+HCOO.sup.−].