Combinations of pyridine thiolate oxide compounds and cystic fibrosis transmembrane conductance regulator modulators for use in the treatment of lung infections in subjects with cystic fibrosis

Abstract

The present invention relates to a combination therapy for treating infections caused by bacteria of the genus Mycobacterium and other microbial pathogens in subjects with cystic fibrosis comprising the sequential, simultaneous or separate administration of pyridine-2-thiol 1-oxides or their salts and cystic fibrosis transmembrane conductance regulator (CFTR) modulator compounds.

Description

[0001] COMBINATIONS OF PYRIDINE THIOLATE OXIDE COMPOUNDS AND CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR MODULATORS FOR USE IN THE TREATMENT OF LUNG INFECTIONS IN SUBJECTS WITH CYSTIC FIBROSIS

[0002] The present invention relates to a combination therapy for treating infections caused by bacteria of the genus Mycobacterium and other microbial pathogens in subjects with cystic fibrosis (CF) comprising the sequential, simultaneous or separate administration of pyridine-2-thiol 1 -oxides, or their salts, and cystic fibrosis transmembrane conductance regulator (CFTR) modulator compounds.

[0003] Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations of the gene encoding the CFTR that, mainly, affects people of Northern European descent. At present, there are new CFTR gene modulator compounds to treat subjects with CF. CFTR modulators are designed to correct the malfunctioning protein made by the CFTR mutated gene. Each of these modulators treat a different aspect of the defective CFTR protein, thus clinical symptoms can be improved by combining diverse modulators [1], Because different mutations cause different defects in the protein, the medications that have been developed so far are effective only in people with specific mutations.

[0004] New combinations such as Kalydeco®, Orkambi®, Symkevi® (Symdeko® in the US) or Kaftrio® (Trikafta® in the US) (from now on referred in here as “CFTR modulator therapies”) containing fixed dose combinations of medicaments ivacaftor, lumacaftor, tezacaftor and elexacaftor have been recently commercialized. Other potential CFTR modulators are currently under development [2, 3],

[0005] CF is often associated with pulmonary infections that complicate the management of people suffering from this disease. Among these pathogens, recalcitrant infections caused by Mycobacterium abscessus are of major concern but also those caused by Pseudomonas aeruginosa or Staphylococcus aureus. Identifying novel strategies to treat these types of infections has the potential to dramatically improve the life of subjects with CF.

[0006] CFTR modulator therapies are used in some CF populations with the capacity to correct the malfunctioning of the CFTR protein. When the CFTR protein is not working correctly, it is unable to help move chloride ions to the cell surface. Without the chloride to attract water to the cell surface, the mucus in various organs becomes thick and sticky. In the lungs, the mucus clogs the airways and traps microorganisms, like bacteria, leading to infections, inflammation, respiratory failure, and other complications. For this reason, avoiding the proliferation of pathogenic microorganisms is a top concern for subjects with CF. Recent studies have demonstrated the benefit of CFTR modulator therapies in the management of CF not only on their role as a CFTR modulators, but also due to their antimicrobial activity against several pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus and Mycobacterium abscessus ([4], [5], [6]), although the antimicrobial activity is mainly driven by the ivacaftor component of the CFTR modulator therapies.

[0007] Ivacaftor is a CFTR potentiator that directly acts on the ion channel, increasing its open-state (

[0001] , [4]). Bacteria also regulate their physiology through ion channels; so, ivacaftor could also increase the transport of the molecules across bacterial cell membranes [7], Moreover, the chemical structure of ivacaftor contains a quinolone ring, which may be the reason why it has these certain antibacterial properties ([4], [5])

[0008] The authors of the present invention have now discovered the synergistic effect of the combined use of pyridine-2 -thiol 1 -oxides and their water-soluble salts with at least one CFTR modulator, such as ivacaftor, tezacaftor, elexacaftor, or lumacaftor and multiple combinations thereof, against Mycobacterium abscessus and other bacterial pathogens in subjects with cystic fibrosis.

[0009] Therefore, it is an object of the present invention a pharmaceutical combination comprising:

[0010] (i) a compound of formula (I): wherein R is selected from the group consisting of COOR1, CONR22or CF3, wherein Ri is a linear C1-C3 alkyl group and R2 is selected between H and C1-C3 alkyl group; wherein X is selected from the group consisting of Na, Li, K, or Ca, or their crystalline forms; and ii) at least one CFTR modulator.

[0011] It is a further object of the present invention the above pharmaceutical combination according to the invention for use in medical field for the treatment of lung infections in subjects with cystic fibrosis.

[0012] In a preferred embodiment of the invention said combination therapy is of benefit to subjects with cystic fibrosis with infections of bacteria belonging to the genera Mycobacterium or Staphylococcus. In a more preferred embodiment of the invention, said bacteria are Mycobacterium abscessus or Staphylococcus aureus.

[0013] According to a preferred embodiment of the invention, said R substituent is selected between the group consisting of 5 -methoxy carbonyl, 5-ethoxycarbonyl and 5- [(dimethylamino)carbonyl] .

[0014] In a preferred embodiment of the present invention, X is Na.

[0015] In one of the most preferred embodiment of the invention the compound of formula (I) is sodium 5-(alkoxycarbonyl)pyridine-2 -thiolate 1 -oxide. In another preferred embodiment the compound is sodium 5 -(ethoxy carbonyl) pyridine-2-thiolate 1-oxide (in the following “VOMG” compound). According to a preferred embodiment of the present invention said cystic fibrosis transmembrane conductance regulator (CFTR) is selected from the group consisting of ivacaftor, tezacaftor, lumacaftor, and elexacaftor or combinations thereof.

[0016] In a preferred embodiment of the invention of the combination therapy for treating infections in subjects with cystic fibrosis the compound of formula (I) is sodium 5- (ethoxy carbonyl) pyridine-2 -thiolate 1 -oxide (in the following “VOMG” or compound 2) and said CFTR modulator is selected from the group consisting of ivacaftor, tezacaftor, lumacaftor, and elexacaftor or combinations thereof. Double combinations of CFTR modulators such as ivacaftor / tezacaftor, ivacaftor / elexacaftor, elexacaftor / tezacaftor or triple combinations of CFTR modulators such ivacaftor / tezacaftor / elexacaftor may be advantageously contemplated in the combination therapy according to the present invention.

[0017] In a further preferred embodiment, the CFTR modulators employed in combination with sodium 5-(ethoxy carbonyl) pyridine-2-thiolate 1 -oxide are ivacaftor, tezacaftor, lumacaftor and elexacaftor. According to another preferred embodiment sodium 5- (ethoxy carbonyl) pyridine-2 -thiolate 1 -oxide (VOMG) is used in combination with ivacaftor. In further preferred embodiment sodium 5-(ethoxycarbonyl) pyridine-2- thiolate 1 -oxide (VOMG) is used in combination with tezacaftor. In another preferred embodiment sodium 5-(ethoxy carbonyl) pyridine-2 -thiolate 1 -oxide is used in combination with ivacaftor.

[0018] The combination therapy according to the invention provides dual activity. First, it will maintain the gene modulating activity of ivacaftor, tezacaftor, lumacaftor and elexacaftor and, second, it will provide synergistic antibacterial activity being more potent than the sum of the independent drugs alone (elexacaftor, tezacaftor, and ivacaftor have little antibacterial activity by themselves and the combination of VOMG with CFTR modulators is more potent than just VOMG alone) thus, enhancing the antimicrobial activity and improving the potential beneficial outcome of the treatment.

[0019] The invention is further directed to a pharmaceutical combination of a compound of formula (I) with at least one CFTR modulator, or combinations thereof, for sequential, simultaneous or separate administration for use in the treatment of lung infections in subjects with cystic fibrosis.

[0020] Therefore, it is a further object of the present invention a kit of parts comprising: i) a compound of formula (I): wherein R is selected from the group consisting of COOR1, CONR22or CF3, wherein Ri is a linear C1-C3 alkyl group and R2 is selected between H and C1-C3 alkyl group; wherein X is selected from the group consisting of Na, Li, K, or Ca, or their crystalline forms; and ii) at least one CFTR modulator, or combinations thereof; for the simultaneous, separate and / or sequential administration to a subject in needs thereof. Preferably the subjects in needs thereof are subjects with cystic fibrosis and lung infections.

[0021] Therefore, it is an object of the present invention the kit of parts for use in medical field for the treatment of lung infections in subjects with cystic fibrosis.

[0022] According to preferred embodiments of the present inventions said lungs infections may be caused by pathogens belonging to Mycobacterium and Staphylococcus genera. Even more preferably these lung infections are caused by Mycobacterium abscessus or Staphylococcus aureus.

[0023] According to a preferred embodiment of the invention, said R substituent is selected between the group consisting of 5 -methoxy carbonyl, 5-ethoxycarbonyl and 5- [(dimethylamino)carbonyl] . In a preferred embodiment of the present invention, X is Na, preferably in combination with any of the above meaning of R.

[0024] In one of the most preferred embodiment of the invention the compound is sodium 5-(alkoxycarbonyl)pyridine-2-thiolate 1 -oxide. In another preferred embodiment the compound is sodium 5-(ethoxycarbonyl) pyridine-2-thiolate 1 -oxide (in the following “VOMG” compound).

[0025] According to a preferred embodiment of the present invention said CFTR modulator is selected from the group consisting of ivacaftor, tezacaftor, lumacaftor and elexacaftor or combinations thereof.

[0026] In a preferred embodiment of the invention of the combination therapy for treating lung infections in subjects with cystic fibrosis the compound of formula (I) is sodium 5-(ethoxycarbonyl) pyridine-2 -thiolate 1 -oxide (in the following “VOMG” or compound 2) and said CFTR modulator is selected from the group consisting of ivacaftor, tezacaftor, lumacaftor, and elexacaftor, or combinations thereof.

[0027] In a further preferred embodiment, the CFTR modulators employed in combination with sodium 5-(ethoxycarbonyl) pyridine-2-thiolate 1 -oxide (VOMG) are ivacaftor, tezacaftor, and elexacaftor. According to another preferred embodiment sodium 5- (ethoxy carbonyl) pyridine-2 -thiolate 1 -oxide is used in combination with tezacaftor. It is another object of the present invention a pharmaceutical composition comprising a compound of formula (I) according to anyone of the preceding claims as active principle together with pharmaceutically acceptable adjuvant and excipients for simultaneous, separate or sequential use in the treatment of lung infections in subjects with cystic fibrosis in combination with a pharmaceutical composition comprising at least one CFTR modulator as active principle or combinations thereof.

[0028] According to a preferred embodiment of the present invention said CFTR modulator is selected from the group consisting of ivacaftor, tezacaftor, lumacaftor, and elexacaftor, or combinations thereof.

[0029] In a preferred embodiment said pharmaceutical composition comprises ivacaftor, tezacaftor, lumacaftor, and elexacaftor as CTFR modulators. Even in a more preferably embodiment said pharmaceutical composition is the approved medicament Kalydeco®, Orkambi®, Symkevi® (Symdeko® in the US) or Kaftrio® (Trikafta® in the US).

[0030] According to a preferred embodiment the route of administration of the pharmaceutical compositions of the invention is systemic.

[0031] The compound of formula (I) of the present invention is water-soluble, thus it may be formulated in a wide variety of oral administration dosage forms and carriers. Oral administration can be in the form of tablets, coated tablets, dragee, hard and soft gelatin capsules, solutions, emulsions, syrups, or suspensions. Compounds of formula (I) are efficacious when administered by other routes of administration including continuous (intravenous drip) topical parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may include a penetration enhancement agent), buccal, nasal, nebulizer inhalation and suppository administration, among other routes of administration.

[0032] In another preferred embodiment the pharmaceutical formulation of the invention could be formulated for aerosol delivery, directly into the lungs, which is a local administration with reduced systemic exposure.

[0033] The compounds of the invention may be used in dosages from 0.01 - 1000 mg / kg body weight, preferably from 1-50 mg / kg body weight.

[0034] The invention further provides sodium 5-(alkoxycarbonyl)pyridine-2-thiolate 1- oxide for use in medical field for the treatment of lung infections in subjects with cystic fibrosis. In a preferred embodiment the invention relates to sodium 5- (ethoxy carbonyl) pyridine-2-thiolate 1 -oxide for use in medical field for the treatment of lung infections in subjects with cystic fibrosis. According to preferred embodiments said lungs infections may be caused by pathogens belonging to Mycobacterium and Staphylococcus genera. Even more preferably these lung infections are caused by Mycobacterium abscessus or Staphylococcus aureus.

[0035] The present invention will now be described for illustrative, but non-limiting purposes, according to a preferred embodiment with particular reference to the attached figures, in which: - Figure 1 shows combinatorial time-kill assays of VOMG in combination with commonly used antibiotics against Mycobacterium abscessus. Concentrations used are based on MIC values (i.e., 1 / lOxMIC, l / 4xMIC, IxMIC, 4xMIC or lOxMIC). Clarithromycin (CLA), MICCLA= 4 pg / mL; Amikacin (AMK), MICAMK= 32 pg / mL; Levofloxacin (LEVO), MICLEVO= 4 pg / mL; Imipenem (IMI), MICIMI= 4 pg / mL; Bedaquiline (BED A), MICBEDA= 0.5 pg / mL; Linezolid (LINE), MICLINE= 8 pg / mL; Rifabutin (RIF), MICRIF= 4 pg / mL; Clofazimine (CLOFA), MICCLOFA= 16 pg / mL; Tigecy cline (TIGE), MICTIGE= 4 pg / mL.

[0036] - Figure 2 shows double, triple and quadruple combinatorial time-kill assays of Kaftrio® / Trikafta® components (TRI) with VOMG against Mycobacterium abscessus. ELX, elexacaftor; IVA, ivacaftor and TEZ, tezacaftor were tested at a fixed 16 pg / mL sub-MIC concentration. VOMG, sodium pyridine-2 -thiol 1- oxides. VOMG was tested at the lx MIC value.

[0037] - Figure 3 shows pairwise combinations of VOMG with different CFTR modulators against Mycobacterium abscessus. VOMG was tested at lx (left column) and 4x (right column) MIC values in combination with different CFTR modulators at a fixed sub-MIC concentration of 16 pg / mL. IVA, ivacaftor; TEZ, tezacaftor; ELX, elexacaftor.

[0038] - Figure 4 shows the triple combinations of VOMG with different CFTR modulators against Mycobacterium abscessus. Panel A: VOMG was tested at lx (left column) and 4x (right column) MIC values in combination with different CFTR modulators at a fixed sub-MIC concentration of 16 pg / mL. Panel B: Pairwise combinations of the CFTR modulators without VOMG. IVA, ivacaftor; TEZ, tezacaftor; ELX, elexacaftor.

[0039] - Figure 5 shows double, triple and quadruple combinatorial of the different Kaftrio® / Trikafta® components (TRI) with VOMG, clarithromycin and amikacin at (Panel A) lx MIC values and (Panel B) 4x MIC values against Mycobacterium abscessus. TRI, elexacaftor-ivacaftor-tezacaftor at a fixed 16 pg / mL concentration. VOMG, sodium pyridine-2-thiol 1 -oxides; CLA, clarithromycin and AMK, amikacin were tested based on their MIC values at IxMIC (panel A) and 4xMIC (panel B). - Figure 6 shows dose-response time-kill assays of ivacaftor and VOMG against Staphylococcus aureus and Pseudomonas aeruginosa. IVA, ivacaftor. Concentrations are in pg / mL.

[0040] - Figure 7 shows double, triple and quadruple combinatorial time-kill assays of Kaftrio® / Trikafta® components (TRI) with VOMG against Staphylococcus aureus. ELX, elexacaftor; IVA, ivacaftor; TEZ, tezacaftor; TRI, elexacaftor- ivacaftor-tezacaftor. Elexacaftor and tezacaftor were tested at sub-MIC values at a fixed 16 pg / mL concentration. Ivacaftor was tested at 16 pg / mL concentration, which is an over-MIC value with antimicrobial activity by itself. VOMG at 1 pg / mL or 4 pg / mL.

[0041] - Figure 8 shows pairwise combinations of VOMG tested at l / 4x (left column) and lx (right column) MIC values in combination with different CFTR modulators against Staphylococcus aureus. Ivacaftor was tested at 1 pg / mL (l / 4x MIC). Elexacaftor and tezacaftor at sub-MIC values at a fixed 16 pg / mL concentration. IVA, ivacaftor; TEZ, tezacaftor; ELX, elexacaftor.

[0042] - Figure 9 shows triple combinations of VOMG with different CFTR modulators against Staphylococcus aureus. Panel A: VOMG was tested at l / 4x (left column) and lx (right column) MIC values in combination with CFTR modulator compounds. Panel B: Pairwise combinations of the CFTR modulators without VOMG. Ivacaftor was tested at 1 pg / mL (l / 4x MIC). Elexacaftor and tezacaftor at sub-MIC values at a fixed 16 pg / mL concentration. IVA, ivacaftor; TEZ, tezacaftor; ELX, elexacaftor.

[0043] - Figure 10 shows quadruple combinations of VOMG with the Kaftrio® / Trikafta® components (TRI) against Staphylococcus aureus. Panel A: VOMG was tested at l / 4x (left) and lx (right) MIC values in combination with Kaftrio® / Trikafta® components (TRI). Panel B: Kaftrio® / Trikafta® components (TRI) without VOMG. Ivacaftor was tested at 1 pg / mL (l / 4x MIC). Elexacaftor and tezacaftor at sub-MIC values at a fixed 16 pg / mL concentration. IVA, ivacaftor; TEZ, tezacaftor; ELX, elexacaftor.

[0044] - Figure 11 shows double, triple and quadruple combinatorial time-kill assays of Kaftrio® / Trikafta® components (TRI) with VOMG against Mycobacterium smegmatis. ELX, elexacaftor; IVA, ivacaftor; TEZ, tezacaftor; TRI, elexacaftor- ivacaftor-tezacaftor. Compounds tested at sub-MIC values at a fixed 16 mg / L concentration. VOMG at 1 pg / mL or 4 pg / mL.

[0045] - Figure 12 shows double, triple and quadruple combinatorial time-kill assays of Kaftrio® / / Trikafta® components (TRI) with VOMG against Pseudomonas aeruginosa. ELX, elexacaftor; IVA, ivacaftor; TEZ, tezacaftor; TRI, elexacaftor- ivacaftor-tezacaftor. Compounds tested at sub-MIC values at a fixed 16 pg / mL concentration. VOMG at 1 pg / mL or 4 pg / mL.

[0046] The following non-limiting examples are now provided for a better illustration of the invention, in which different combination therapies of compound of formula (I), CFTR modulators and antimicrobial agents were tested and compared.

[0047] EXAMPLE 1 Synthetic reaction scheme of the compounds of formula (I)

[0048] Examples of representative compounds if formula (I) encompassed by the present invention and within the scope of the invention are provided in the following examples A-L.

[0049] All compounds were synthesized according below Scheme 1 wherein corresponding 2-chloropyridine was oxidized by freshly prepared solution of H2O2 / OC(NH2)2 in chloroform with formation pyridine N-oxide, which can be transferred to aim 2- thiopyridine by two methods:

[0050] A) by the reaction of 2-chloropyridine N-oxide with large excess of sodium hydrosulfide;

[0051] B) by the synthesis of pyridin-2-yl imidothiocarbamate as intermediate with follow its treatment by sodium hydrogen carbonate, pyridine-2 -thiol 1 -oxide derivatives easy form corresponding salts with metal hydroxides and these final compounds are solid, white and water-soluble compounds.

[0052] Scheme 1 a) H2O2 / OC(NH2)2, CHC13; b) NaHS, EtOH, water; c) SC(NH2)2, EtOH; d) NaHCCh, EtOH, water; e) XOH, MeOH, aceton.

[0053] The step of the synthesis according to Scheme 1 are herein explained in more detail: A solution of corresponding pyridine (1 mmol) in chloroform was treated by dry UHP (H2O2 / OC(NH2)2 (2 mmol) at 0°C. Trifluoroacertic acid anhydride was then slowly added to the reaction mixture (the reaction is exotermic). The reaction was followed by TLC until completion at room temperature. The reaction mixture was dissolved by water, formed precipitate was filtered off and washed by water. The desired pyridine N-oxide was recrystallized preferably from ethanol.

[0054] Step b). A solution of pyridine N-oxide derivative (1.0 mmol) in ethanol was slowly treated by drop by solution of sodium hydrosulfide (5-7 mmol) at room temperature and was stored 2-3 hours at this temperature. The reaction mixture was cooled, diluted by water and formed solid was filtered off. The thiopyridine derivative was recrystallized preferably from ethyl acetate or ethanol. solution of pyridine N-oxide derivative (1.0 mmol) in aceton was treated by thiourea (1 / 3 mmol) and refluxed for 2-3 hours. The reaction mixture was evaporated in vacuum till 14 of the volume and cooled at -18°C for 6 hours. The formed precipitate was filtered off and residue was dissolved in small volume of water. This solution was treated by water solution of sodium hydrogen carbonate and formed sediment of pyridin-2-yl imidothiocarbamate derivative was filtered off. The compound was recrystallized preferably from ethyl acetate or ethanol. solution of pyridin- ■2-yl imidothiocarbamate derivative (1 mmol) in 20 ml of ethanol was treated by water solution of sodium hydrogencarbonate (2 mmol) at room temperature. The reaction mixture was stored for 3 hours at 60°C, cooled and treated by 0.1N hydrochloric acid water solution until pH~2. Formed residue was collected, dried and pyridine-2 -thiol 1 -oxide derivative was recrystallized from ethanol.

[0055] Step e). A solution of pyridine-2-thiol 1 -oxide derivative (1 mmol) in ethanol was treated by drop by alkali hydroxide (1 mmol) solved in minimal volume of water. For med sediment was collected and recrystallized from methanol or ethanol to give aim corresponding alkali salt of 5-(R)-pyridine-2-thiolate 1 -oxide as an off-white solid.

[0056] All compounds were obtained at a purity of not less than 98% in amounts sufficient to carry out all planned. The confirmation of the structure and investigation of the physicochemical properties of all synthesized compounds was performed by various modem methods described in the following section related to “Examples A-L”.

[0057] Examples A-L

[0058] A. Ethyl 6-mercaptonicotinate 1-oxide 11226084 (compound 1)

[0059] Yield 76%. Mass (El), m / z (Irelat.(%)): 199.2298 [M] (73). Anal. Calcd. for C8H9NO3S: (%) C, 48.23; H, 4.55; N, 7.03. Found (%) C, 48.07; H, 4.36; N, 7.18. 'H NMR (DMSO-d6): 8.74 (s, 1H, CH), 7.68 (d, 1H, J= 9.8 Hz, CH), 7.59 (d, 1H, J = 9.8 Hz, CH), 4.28 (q, 2H, J = 7.2 Hz, CH2), 1.28 (t, 3H, J= 6.9 Hz, CH3) ppm.13C NMR (50 MHz, DMSO) 5 164.47, 162.64, 137.28, 134.16, 125.91, 121.08, 61.07, 14.30 ppm. B. Sodium 5-(ethoxycarbonyl)pyridine-2 -thiolate 1-oxide 11326030 (compound 2, VOMG)

[0060] Yield 64%. Mass (El), m / z (Irelat.(%)): 198.2201 [M] (79). Anal. Calcd. for C8H8NNaO3S: (%) C, 43.44; H, 3.65; N, 6.33. Found (%) C, 43.35; H, 3.67; N, 6.28. 'HNMR (DMSO-de): 8.82 (s, 1H, CH), 7.62 (d, 1H, J= 9.8 Hz, CH), 7.51 (d, 1H, J = 9.8 Hz, CH), 4.23 (q, 2H, J= 7.2 Hz, CH2), 1.17 (t, 3H, J= 6.9 Hz, CH3)ppm.13C NMR (50 MHz, DMSO) 5 169.71, 164.58, 137.76, 136.33, 132.17, 125.71, 61.07, 14.31 ppm.

[0061] C. Methyl 6-mercaptonicotinate 1-oxide 11226067 (compound 3)

[0062] Yield 73%. Mass (El), m / z (Irelat.(%)): 185.2014 [M] (57). Anal. Calcd. for C7H7NO3S: (%) C, 45.40; H, 3.81; N, 7.56. Found (%) C, 45.17; H, 4.01; N, 7.59. 1H NMR (DMSO-d6): 8.74 (s, 1H, CH), 7.69 (d, 1H, J = 9.8 Hz, CH), 7.57 (d, 1H, J = 9.8 Hz, CH), 3.77 (s, 3H, OCH3) ppm. 13C NMR (50 MHz, DMSO) 5 164.61, 162.58, 137.79, 133.83, 125.87, 121.92, 52.23 ppm. D. Sodium 5-(methoxycarbonyl)pyridine-2-thiolate 1-oxide (compound 4)

[0063] Yield 86%. Mass (El), m / z (Irelat.(%)): 184.1935 [M] (65). Anal. Calcd. for C7H6NNaO3S: (%) C, 40.58; H, 2.92; N, 6.76. Found (%) C, 40.49; H, 3.03; N, 6.79. 'HNMR (DMSO-d6): 8.70 (s, 1H, CH), 7.68 (d, 1H, J= 9.8 Hz, CH), 7.51 (d, 1H, J = 9.8 Hz, CH), 3.75 (s, 3H, OCH3) ppm.13C NMR (50 MHz, DMSO) 5 172.82, 164.81, 138.10, 136.67, 132.17, 125.61, 52.29 ppm.

[0064] E. Sodium 5-(trifluoromethyl)pyridine-2 -thiolate 1-oxide 11326031 (compound 5)

[0065] Yield 80%. Mass (El), m / z (Irelat.(%)): 194.1554 [M] (79). Anal. Calcd. for C6H3F3NNaOS: (%) C, 37.12; H, 1.56; N, 7.21. Found (%) C, 37.20; H, 1.49; N, 7.27.XH NMR (DMSO-d6): 8.41 (s, 1H, CH), 7.97 (d, 1H, J= 9.7 Hz, CH), 7.67 (d, 1H, J= 9.7 Hz, CH) ppm.13C NMR (50 MHz, DMSO) 5 171.51, 134.12, 132.57, 132.34, 131.26, 131.08, 127.41, 123.34(q), 52.29 ppm.

[0066] F. Potassium 5-(aminocarbonyl)pyridine-2-thiolate 1-oxide (compound 6)

[0067] Yield 93%. Mass (El), m / z (Irelat.(%)): 169.1822 [M] (64). Anal. Calcd. for C6H5KN2O2S: (%) C, 34.60; H, 2.42; N, 13.45. Found (%) C, 34.82; H, 2.56; N, 13.51.1H NMR (DMSO-d6): 8.85 (s, 1H, CH), 8.04 (d, 1H, = 9.6 Hz, CH), 7.73 (d, 1H, J= 9.6 Hz, CH), 5.67 (br s, 2H, NH2) ppm.13C NMR (50 MHz, DMSO) 5 172.53, 170.60, 140.98, 134.61, 133.53, 129.44 ppm.

[0068] G. Potassium 5-(piperidin-l-ylcarbonyl)pyridine-2-thiolate 1-oxide (compound 7)

[0069] Yield 87%. Mass (El), m / z (Irelat.(%)): 237.2992 [M] (60). Anal. Calcd. for C11H13KN2O2S: (%) C, 47.80; H, 4.74; N, 10.14. Found (%) C, 47.61; H, 4.61; N, 10.23. 'H NMR (DMSO-de): 8.67(s, 1H, CH), 7.99 (d, 1H, J= 9.7 Hz, CH), 7.69 (d, 1H, J= 9.7 Hz, CH), 3.35 (m, 4H, N(CH2)2), 1.93 (m, 4H, 2CH2), 1.37 (m, 2H, CH2) ppm.13C NMR (50 MHz, DMSO) 5 170.03, 167.55, 141.99, 133.92, 132.79, 132.18, 46.70, 26.15, 24.85 ppm. H. 6-Mercapto-N,N-dimethylnicotinamide 1 -oxide (compound 8)

[0070] Yield 65%. Mass (El), m / z (Irelat.(%)): 198.2433 [M] (34). Anal. Calcd. for C8H10N2O2S: (%) C, 48.47; H, 5.08; N, 14.13. Found (%) C, 48.53; H, 5.14; N, 14.22. 'HNMR (DMSO-d6): 8.67 (s, 1H, CH), 8.03 (d, 1H, J= 9.7 Hz, CH), 7.67 (d, 1H, J = 9.7 Hz, CH), 3.12 (s, 6H, N(CH3)2) ppm.13C NMR (50 MHz, DMSO) 5 167.87, 163.13, 135.71, 132.59, 131.06, 122.74, 35.62, 34.20 ppm.

[0071] I. Sodium 5 -[(dimethylamino)carbonyl]pyridine-2 -thiolate 1 -oxide (compound 9)

[0072] Yield 79%. Mass (El), m / z (Irelat.(%)): 197.2353 [M] (47). Anal. Calcd. for C8H9N2NaO2S: (%) C, 43.63; H, 4.12; N, 12.72. Found (%) C, 43.57; H, 4.11; N, 12.68.1H NMR (DMSO-d6): 8.73 (s, 1H, CH), 8.00 (d, 1H, = 9.7 Hz, CH), 7.71 (d, 1H, J = 9.7 Hz, CH), 3.12 (s, 6H, N(CH3)2) ppm.13C NMR (50 MHz, DMSO) 5 172.41, 167.57, 142.69, 133.92, 133.45, 132.39, 37.60, 34.27 ppm. L. Sodium 5-(isopropoxycarbonyl)pyridine-2-thiolate 1-oxide (compound 10)

[0073] Yield 63%. Mass (El), m / z (Irelat.(%)): 212.2466 [M] (53). Anal. Calcd. for C9HioNNa03S: (%) C, 45.95; H, 4.28; N, 5.95. Found (%) C, 45.87; H, 4.33; N, 6.04. 'H NMR (DMSO-de): 8.69 (s, 1H, CH), 8.16 (d, 1H, J= 9.7 Hz, CH), 7.49 (d, 1H, J = 9.7 Hz, CH), 4.93 (m, 1H, CH), 1.14 (d, 6H, J = 4.2 Hz, 2CH3) ppm.13C NMR (50 MHz, DMSO) 5 172.82, 164.50, 137.42, 135.99, 132.17, 125.75, 69.65, 17.98 ppm.

[0074] EXAMPLE 2: In vitro interaction profile of compound 2 (VOMG) in combination with different antibiotics against mycobacteria or other bacteria

[0075] The in vitro interaction profile of VOMG in combination with commonly used antibiotics clarithromycin, amikacin, levofloxacin, imipenem, bedaquiline, linezolid, rifabutin, clofazimine, tigecycline, against Mycobacterium abscessus has been determined.

[0076] Then the combination of compound 2 (VOMG) with either ivacaftor, elexacaftor and tezacaftor (active ingredients of Kaftrio® / Trikafta®) in either pairwise or higher degree combinations against Mycobacterium abscessus (and other bacterial pathogens) has been evaluated.

[0077] Additionally, the activity of compound 2 (VOMG) and ivacaftor against Staphylococcus aureus and Pseudomonas aeruginosa was assessed. Finally, the combination of the ivacaftor, elexacaftor and tezacaftor with compound 2 (VOMG) against Staphylococcus aureus, Mycobacterium smegmatis, and Pseudomonas aeruginosa was tested.

[0078] MATERIALS AND METHODS

[0079] Antibiotics, media and strains

[0080] Concentrations of the antibiotics used in time-kill combination assays were based on MIC values of the single compounds, as follows:

[0081] Clarithromycin (CLA), MICCLA= 4 pg / mL; Amikacin (AMK), MICAMK= 32 pg / mL; Levofloxacin (LEVO), MICLEVO= 4 pg / mL; Imipenem (IMI), MICIMI= 4 pg / mL; Bedaquiline (BEDA), MICBEDA= 0.5 pg / mL; Linezolid (LINE), MICLINE= 8 pg / mL; Rifabutin (RIF), MICRIF= 4 pg / mL; Clofazimine (CLOFA), MICCLOFA= 16 pg / mL; Tigecycline (TIGE), MICTIGE= 4 pg / mL. The MIC values of VOMG and the CFTR modulators against the different bacteria used in this study are shown in Table 1. Compounds were dissolved in the recommended solvent (DMSO or water).

[0082] The mycobacterial species were grown in Middlebrook 7H9 broth (BD Difco) supplemented with 10% (vol / vol) albumin, dextrose, and catalase (ADC).

[0083] The non-mycobacterial species were grown in cation-adjusted Mueller Hinton broth (MHB). Mycobacterium abscessus ATCC 19977, Mycobacterium smegmatis mc2155, Staphylococcus aureus CECT 794 and Pseudomonas aeruginosa ATCC 27853 were used in this study. All strains were grown at 37 °C.

[0084] Drug Susceptibility Testing

[0085] MIC determinations were performed by broth microdilution assays in a 96-well plate format by serial 2-fold dilutions of test compounds.

[0086] Briefly, bacterial cells were added to a final cell density of 5^ 105 cells / mL. Positive and negative growth controls were included in every plate for each strain.

[0087] Plates were incubated at 37°C for two days for Mycobacterium abscessus and Mycobacterium smegmatis and overnight for Staphylococcus aureus and Pseudomonas aeruginosa.

[0088] After the incubation period, the redox indicator MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide) was added to the wells and incubated overnight for Mycobacterium abscessus and Mycobacterium smegmatis and for 3 hours for Staphylococcus aureus and Pseudomonas aeruginosa.

[0089] Then, optical density was read at 580 nm to measure the MTT to formazan conversion, an indicator of bacterial growth.

[0090] The lowest drug concentration that inhibited conversion by 90% compared to the internal negative control was used to define the MIC value.

[0091] Experiments were done in duplicate at least three times.

[0092] Time-kill assays

[0093] Mycobacterium abscessus, Mycobacterium smegmatis, Staphylococcus aureus and Pseudomonas aeruginosa cultures were prepared at a final cell density of 105cells / mL.

[0094] Then, 2.5 mL of these inocula were transferred to each well of a 24-well plate. Different concentrations and combinations of compounds were added to each well and plates were incubated at 37°C.

[0095] At every time point, 20 pL of each sample were taken and serially diluted in 180 pL of phosphate-buffered saline solution (PBS) with 0.1% tyloxapol, then 2.5 pL of each dilution were plated out on agar plates containing 0.4% of activated charcoal in order to remove potential compound carryover. Moreover, 10 pL of each sample without dilution were also plated, in order to lower the limit of detection to 2xLogio CFU / mL. Agar plates were incubated for 4 days (Mycobacterium abscessus and Mycobacterium smegmatis) and overnight (Staphylococcus aureus and Pseudomonas aeruginosa) at 37°C, and CFU were determined by counting the colonies. Experiments were performed in duplicate at least three times.

[0096] Time-kill assays against nine commonly used antibiotics in the treatment of mycobacterial infections were carried out in the presence or absence of VOMG at different concentrations against Mycobacterium abscessus (Figure 1).

[0097] Subsequent double and triple combinatorial time-kill assays of ivacaftor, elexacaftor and tezacaftor (active ingredients of Kaftrio® / Trikafta®) with or without VOMG against Mycobacterium abscessus ATCC 19977 were carried out as well (Figure 2). Elexacaftor, ivacaftor and tezacaftor were tested at 16 pg / mL. VOMG was tested at different MIC value ratios, i.e., lx MIC, 4x MIC. Pairwise combinations of VOMG with different CFTR modulators against Mycobacterium abscessus ATCC 19977 were assessed (Figure 3). VOMG was tested at lx and 4x MIC values against different CFTR modulators at a fixed sub-MIC concentration of 16 gg / mL. MIC of CFTR modulators against Mycobacterium abscessus is equal or higher than 64 gg / mL (see Table 1).

[0098] Also, triple combinations of VOMG with different CFTR modulators against Mycobacterium abscessus ATCC 19977 were assessed (Figure 4, Panel A). The activity of the pairwise combinations of CFTR modulator (in the absence of VOMG) was also tested (Figure 4, Panel B). VOMG was tested at lx and 4x MIC values against different CFTR modulators at a fixed sub-MIC concentration of 16 gg / mL. MIC of CFTR modulators against Mycobacterium abscessus is equal or higher than 64 gg / mL (see Table 1).

[0099] Double, triple and quadruple combinatorial time-kill assays testing combinations of the triad elexacaftor-ivacaftor-tezacaftor (TRI) with either compound 2 (VOMG), amikacin, clarithromycin or a combination of all of them were performed (Figure 5). TRI, elexacaftor-ivacaftor-tezacaftor at a fixed 16 gg / mL concentration.

[0100] Compounds were tested at different ratios of their MIC values, i.e., Sodium pyridine- 2-thiol 1 -oxides (VOMG) (1 gg / mL), clarithromycin (4 gg / mL) and amikacin (32 gg / mL).

[0101] Dose-response time-kill assays of ivacaftor and VOMG against Staphylococcus aureus and Pseudomonas aeruginosa were also carried out. Concentrations are expressed in gg / mL (Figure 6).

[0102] Accordingly, double, triple and quadruple combinatorial time-kill assays of the Kaftrio® / Trikafta® individual components with VOMG were performed against Staphylococcus aureus CECT 794. Elexacaftor and tezacaftor were tested at fixed sub-MIC values (16 gg / mL). Ivacaftor was tested at 16 gg / mL concentration, which is an over-MIC value with antimicrobial activity by itself. VOMG was tested at 1 gg / mL or 4 gg / mL (Figure 7).

[0103] Pairwise combinations of VOMG with different CFTR modulators were performed against Staphylococcus aureus. VOMG was tested at l / 4x and lx MIC values (see Table 1 for reference). Ivacaftor was tested at 1 gg / mL (l / 4x MIC). The MIC of tezacaftor and elexacaftor against Staphylococcus aureus is higher than 64 gg / mL. Elexacaftor and tezacaftor were tested at 16 gg / mL (see Figure 8).

[0104] Additionally, triple combinations of VOMG with different CFTR modulators against Staphylococcus aureus were tested. VOMG was tested at l / 4x and lx MIC values (see Table 1 for reference). Ivacaftor was tested at 1 gg / mL (l / 4x MIC). The MIC of tezacaftor and elexacaftor against Staphylococcus aureus is higher than 64 gg / mL. Elexacaftor and tezacaftor were tested at 16 gg / mL (see Figure 9, Panel A). The activity of the pairwise combinations of CFTR modulator (in the absence of VOMG) was also tested (Figure 9, Panel B).

[0105] Moreover, quadruple combinations of VOMG with different CFTR modulators were performed against Staphylococcus aureus (see Figure 10, Panel A).

[0106] The activity of the triple combination of CFTR modulator (in the absence of VOMG) was also tested (see Figure 10, Panel B).

[0107] VOMG was tested at l / 4x and lx MIC values. Ivacaftor was tested at 1 gg / mL. The MIC of tezacaftor and elexacaftor against Staphylococcus aureus is higher than 64 gg / mL. Elexacaftor and tezacaftor were tested at 16 gg / mL.

[0108] Double, triple and quadruple combinatorial time-kill assays of the Kaftrio® / Trikafta® individual components with VOMG were performed against Mycobacterium smegmatis (Figure 11) and Pseudomonas aeruginosa (Figure 12) as well. Elexacaftor, ivacaftor, tezacaftor were tested at fixed sub-MIC values (16 gg / mL). VOMG was tested at 1 gg / mL or 4 gg / mL.

[0109] RESULTS

[0110] The MIC values of VOMG and the CFTR modulators used in this study are shown in Table 1.

[0111] Tezacaftor and elexacaftor were not active (MIC >64 gg / mL) against any of the strains tested. Ivacaftor was inactive against Pseudomonas aeruginosa, showed moderate activity against Staphylococcus aureus, with low activity against both mycobacterial species (Mycobacterium abscessus and Mycobacterium smegmatis). Table 1. Antibacterial activity of Kaftrio® / / Trikafta® active ingredients against different bacteria. Minimal Inhibitory Concentrations (MIC) are reported.

[0112] The results of the time-kill assays against the nine antibiotics used in combination with compound 2 of the invention (VOMG) did not identify any synergistic interaction (see Figure 1). The results depicted in Figure 1 clearly show that VOMG does not interact with other commonly used antibiotics, thus, the synergy with CFTR modulators is not evident.

[0113] Subsequent time-kill assays showed that combinations of compound 2 of the invention (VOMG) with either ivacaftor, elexacaftor and tezacaftor in either pair- wise or higher degree combinations had different levels of synergistic interactions against Mycobacterium abscessus and Staphylococcus aureus (Figures 2-5, and Figures 7-10, respectively). This makes the combinations more effective than the activity of the single compounds alone. The fact that no interaction was observed between compound 2 (VOMG) and commonly used antibiotics (Figure 1) but an interaction was found with the Kaftrio® / / Trikafta® active ingredients indicates that these novel interactions are not obvious or predictable for a skilled artisan in the field. The time-dependent interaction profile of the different Kaftrio® / / Trikafta® components and VOMG against Mycobacterium abscessus is shown in Figure 2.

[0114] First, neither ivacaftor nor elexacaftor or tezacaftor were active alone, in pair-wise or triple combinations (Figure 2, upper left and right panels). However, when compound 2 (VOMG) was added to the combination in either two drug combinations (Figure 2, lower left panel) or in three or even four drug combinations (Figure 2, lower right panel), the combinations were more potent than the drugs alone, as seen by a more pronounced decline in the bacterial load (logic CFU / mL) and control of the regrowth at the latter time point (day 15).

[0115] The interaction between VOMG and each of the different CFTR modulators was dose-dependent against Mycobacterium abscessus (see Figure 3). While VOMG displayed slight interaction with ivacaftor and elexacaftor (as previously reported in Figure 2), a clear interaction was observed with tezacaftor, being this more potent when the concentration of VOGM increased.

[0116] The interaction between VOMG and pairwise combination of CFTR modulator compounds was dose-dependent against Mycobacterium abscessus as well (see Figure 4). Similar to data shown in Figure 3, the interaction was stronger when in the presence of tezacaftor. Combination of VOMG plus tezacaftor and either ivacaftor or elexacaftor show a potent dose dependent interaction, being the later (VOMG+TEZ+ELX) the most active. The combination without tezacaftor (VOMG+IVA+ELX) showed a modest improvement of activity at shorter time points but without sterilizing capacity. Importantly, none of the pairwise combinations of the CFTR modulator compounds had activity against Mycobacterium abscessus, proving the critical role of VOMG in the synergistic activity of the combinations of the invention.

[0117] Next, the added value of these combinations was evaluated in comparison with the interaction that the two main antibiotics used in the treatment of Mycobacterium abscessus infections could have with the Kaftrio® / / Trikafta® components. To this aim, similar time-kill assays were performed testing combinations of elexacaftor- ivacaftor-tezacaftor with either VOMG, amikacin, clarithromycin or a combination of all of them, and the results are depicted in Figure 5. Here, a strong interaction between compound 2 (VOMG) and the Kaftrio® / / Trikafta® active ingredients (elexacaftor-ivacaftor-tezacaftor) was observed as evidenced by the strong killing effect of the combination (Figure 5A, upper left panel). This interaction was not observed when the triad elexacaftor- ivacaftor-tezacaftor was tested in combination with either clarithromycin, amikacin (Figure 5A, upper right panel). Importantly, compound 2 (VOMG) enhanced the activity of the triad plus clarithromycin and amikacin combination (Figure 5 A, lower panel). Similar results were observed when the concentrations of CLA, AMK and VOMG were increased (4xMIC). At these concentrations, VOMG was strongly bactericidal alone and in combination with the triad elexacaftor-ivacaftor-tezacaftor (Figure 5B, upper left panel). CLA and AMK showed minor interactions with the triad elexacaftor-ivacaftor-tezacaftor (Figure 5B, upper right panel), which not even in dual combination (CLA+AMK+TRI) had rapid bactericidal activity. The addition of VOMG greatly enhanced this activity (Figure 5B, lower panel).

[0118] The activity of compound 2 (VOMG) or ivacaftor is not only limited to Mycobacterium abscessus. Similarly antibacterial activity was previously observed for other pathogens such as Mycobacterium smegmatis and Staphylococcus aureus ([3], [4]) as shown in Table 1. Values >64 pg / mL are considered as no antibacterial activity.

[0119] In order to verify this fact, the activity of ivacaftor and VOMG by time-kill assays against Staphylococcus aureus and Pseudomonas aeruginosa was tested. Activity of ivacaftor was observed against Staphylococcus aureus at concentrations of 4-16 pg / mL, but at the tested concentrations no activity was identified against Pseudomonas aeruginosa (MIC >64 pg / mL) (Figure 6).

[0120] Staphylococcus aureus and Pseudomonas aeruginosa are common pathogens found in pulmonary infections in subjects with cystic fibrosis. In order to assess the potential value that a VOMG-TRI (elexacaftor-ivacaftor-tezacaftor) combination could have in the treatment of these type of infections, it was evaluated whether the strong synergistic interaction found against Mycobacterium abscessus could be also identified against Staphylococcus aureus and Pseudomonas aeruginosa. The non- pathogenic Mycobacterium smegmatis (a model organisms for the pathogenic Mycobacterium tuberculosis used in drug discovery for anti-tuberculosis drugs) was also included to assess whether this combination could also be widespread against other mycobacterial species.

[0121] The strong synergistic interaction of VOMG plus TRI (elexacaftor-ivacaftor- tezacaftor) identified against Mycobacterium abscessus was also maintained against Staphylococcus aureus (Figure 10) and Mycobacterium smegmatis (Figure 11), but not against Pseudomonas aeruginosa (Figure 12). This may be explained to the fact that VOMG was not active against Pseudomonas aeruginosa (MIC > 64 pg / mL) (see Table 1) and that bacteria like Pseudomonas aeruginosa has a low outer membrane permeability and an efficient system of detoxification from the antibiotics involving expression of efflux pumps that expel drugs out of the cell, for which VOMG could be a substrate since its MIC value in the presence of the efflux pump inhibitor phenylalanine-arginine beta-naphthyl ami de (PApN) is reduced to 32 pg / mL.

[0122] In addition, other important interactions were also identified such as ivacaftor plus VOMG, ivacaftor-tezacaftor plus VOMG or elexacaftor-ivacaftor plus VOMG, among others, against Staphylococcus aureus (Figure 7).

[0123] Figure 7 shows dose response activity of VOMG and different CFTR modulators alone against Staphylococcus aureus.

[0124] Looking at the results obtained considering the compounds alone in comparison with the results wherein pairwise and higher order combinations of VOMG with different CFTR modulators against Staphylococcus aureus CECT 794 were tested (Figures 8, 9 and 10), it can be inferred the following.

[0125] First, the interaction between VOMG and the different CFTR modulators against Staphylococcus aureus was dose-dependent. While VOMG displayed no interaction at 1 / 4 x MIC values, pairwise interactions were clearly seen when the dose of VOMG was increased to IxMIC for all three compounds, i.e. ivacaftor, elexacaftor and tezacaftor.

[0126] The alleged lack of interaction previously observed in Figure 7 (wherein Ivacaftor has an MIC of 4 pg / mL against Staphylococcus aureus) was due to the fact that, in that experiment, ivacaftor was used a concentration of 16 pg / mL of CFTR modulator, which has activity by itself, thus masking any potential interaction with VOMG. Although, it is worth noting that in Figure 7 ivacaftor by itself showed an initial killing with a re-growth after 24 hours, while in combination with VOMG this activity was maintained over the length of the experiment.

[0127] These results confirm the synergistic interaction of VOMG in combination with ivacaftor, tezacaftor and elexacaftor against Staphylococcus aureus. This is further confirmed by the results obtained with triple combinations of VOMG with different CFTR modulators against Staphylococcus aureus CECT 794 (see Figure 9). Again, the interaction of VOMG with pairwise combinations CFTR gene modulator compounds was dose-dependent, displaying a synergistic interaction at IxMIC values (see right column of Figure 9, Panel A).

[0128] This synergistic interaction was exclusively due to the presence of VOMG since the pairwise CFTR gene modulator compounds alone did not show any relevant synergistic interaction (Figure 9, Panel B).

[0129] Similar results were obtained with quadruple combinations of VOMG with different CFTR gene modulators against Staphylococcus aureus CECT 794 (see Figure 10). Again, the interaction of VOMG in combination with all CFTR gene modulator compounds was dose-dependent, displaying a synergistic interaction at l / 4xMIC values (Figure 10 Panel A left) but this being more potent at IxMIC (Figure 10 Panel A right). This synergistic interaction was due to the presence of VOMG since the triple CFTR gene modulator compounds combination was not able to clear the bacterial culture (Figure 10 Panel B), while this case the case when VOMG was added at IxMIC.

[0130] In summary, the above data demonstrate the synergistic interaction of VOMG in combination with at least one CFTR modulator against Mycobacterium abscessus or Staphylococcus aureus infections.

[0131] The above results demonstrate that the novel VOMG-CFTR modulator(s) combination can be effective not only against Mycobacterium abscessus infections, but also against infections caused by other mycobacteria, such as Mycobacterium smegmatis, or other gram-positive bacteria, such as Staphylococcus aureus. BIBLIOGRAPHY

[0132] [1] Gramegna et al., 2023. Respiratory Research. 24(316). https: / / doi.org / 10.1186 / sl2931-023-02612-l

[0133] [2] Zaher etal., 2021. Cureus. 3(7):el6144. doi: 10.7759 / cureus.16144 [3] Regard et al. 2023. Semin Respir Crit Care Med. 44(2):186-195. doi: 10.1055 / s- 0042-1758851

[0134] [4] Cigana c / a / ., 2023. Microbiol Spectr.l l(l) doi: 10.1128 / spectrum.04083-22.

[0135] [5] Reznikov et al., 2014. Journal of Cystic Fibrosis. 13 (5). doi: 10.1016 / j.jcf.2014.02.004 [6] Giron et al., Enferm Infecc Microbiol Clin. 41(3): 193-195. doi: 10.1016 / j.eimce.2022.05.018

[0136] [7] Millar et al. 2018. Journal of Clinical Pharmacy and Therapeutics, 43(6). doi: https: / / doi.org / 10. I l l 1 / jcpt.12722

Claims

CLAIMS1. A pharmaceutical combination comprising:(i) a compound of formula (I):wherein R is selected from the group consisting of COOR1, CONR22or CF3, wherein Ri is a linear C1-C3 alkyl group and R2 is selected between H and C1-C3 alkyl group; wherein X is selected from the group consisting of Na, Li, K, or Ca, or their crystalline forms; and ii) at least one cystic fibrosis transmembrane conductance regulator (CFTR) modulator.

2. A pharmaceutical combination according to claim 1, for use in medical field for the treatment of lung infections in subjects with cystic fibrosis.

3. A pharmaceutical combination for use according to claim 2, wherein R in formula (I) is selected from the group consisting of 5 -methoxy carbonyl, 5-ethoxycarbonyl and 5-[(dimethylamino)carbonyl],4. A pharmaceutical combination for use according to anyone of claim 2-3 wherein X is Na.

5. A pharmaceutical combination for use according to claim 4, wherein the compound of formula (I) is sodium 5-(alkoxycarbonyl)pyridine-2 -thiolate 1 -oxide.

6. A pharmaceutical combination for use according to claim 5, wherein the compound of formula (I) is sodium 5-(ethoxy carbonyl) pyridine-2-thiolate 1 -oxide.

7. A pharmaceutical combination for use according to anyone of claim 2-6, wherein said cystic fibrosis transmembrane conductance regulator (CFTR) modulator is selected from the group consisting of ivacaftor, tezacaftor, lumacaftor and elexacaftor or combinations thereof.

8. A pharmaceutical combination for use according to claim 7, wherein the compound of formula (I) is sodium 5 -(ethoxy carbonyl) pyridine-2-thiolate 1-oxide and said cystic fibrosis transmembrane conductance regulator (CFTR) is selected from the group consisting of ivacaftor, tezacaftor, lumacaftor and elexacaftor or combinations thereof.

9. A pharmaceutical combination for use according to claim 8, wherein the compound of formula (I) is sodium 5 -(ethoxy carbonyl) pyridine-2 -thiolate 1-oxide and said cystic fibrosis transmembrane conductance regulator (CFTR) modulator is a combination of ivacaftor, tezacaftor, and elexacaftor.

10. A pharmaceutical combination for use according to claim 8, wherein the compound of formula (I) is sodium 5 -(ethoxy carbonyl) pyridine-2-thiolate 1-oxide and said cystic fibrosis transmembrane conductance regulator (CFTR) modulator is tezacaftor or ivacaftor.

11. A pharmaceutical combination for use according to anyone of the preceding claims 2-10, wherein said lung infections are caused by bacteria belonging to Mycobacterium or Staphylococcus genera.

12. A kit of parts comprising: i) a compound of formula (I):wherein R is selected from the group consisting of COOR1, CONR22or CF3, wherein Ri is a linear C1-C3 alkyl group and R2 is selected between H and C1-C3 alkyl group; wherein X is selected from the group consisting of Na, Li, K, or Ca, or their crystalline forms; and ii) at least one cystic fibrosis transmembrane conductance regulator (CFTR) modulator; for the simultaneous, separate and / or sequential administration to a subject in needs thereof.

13. A pharmaceutical composition comprising a compound of formula (I) according to anyone of the preceding claims as active principle together with pharmaceutically acceptable adjuvant and excipients for simultaneous, separate or sequential use for the treatment of lung infections in subjects with cystic fibrosis in combination with a pharmaceutical composition comprising at least one cystic fibrosis transmembrane conductance regulator (CFTR) modulator as active principle.

14. A pharmaceutical composition for use according to claim 13, for systemic administration.

15. A pharmaceutical composition for use according to claim 13, for aerosol delivery.

16. Sodium 5-(alkoxycarbonyl)pyridine-2 -thiolate 1 -oxide for use in medical field for the treatment of lung infections in subjects with cystic fibrosis in combination with at least one CFTR modulator.

17. Sodium 5-(ethoxycarbonyl) pyridine-2 -thiolate 1-oxide for use in medical field for the treatment of lung infections in subjects with cystic fibrosis in combination with at least one CFTR modulator.

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