Multi-component pharmaceutical compositions and kits containing nitric oxide releasing compounds and methods of use thereof

Abstract

Provided herein are multi-component pharmaceutical compositions and kits comprising a first component comprising a nitric oxide (NO)-releasing compound, and a second component comprising a second therapeutic agent wherein the second therapeutic agent comprises an antibiotic, an antifungal agent, or a combination thereof. In these compositions, the ratio of the first component to the second component is such that the in vitro graded inhibitory concentration index (FICI) of the combination of the first component and the second component is 1.0 or less. The first component and the second component may be formulated into a single combined composition, or may be retained as a separate composition. Also described herein are methods of treating microbial infections, and methods of preventing, reducing or eliminating biofilm formation caused by bacteria.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 350,132, filed on June 8, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to multi-component pharmaceutical compositions and kits, which include two or more components, such as a compound that releases nitric oxide and an antimicrobial agent (e.g., an antibiotic or an antifungal agent). The present disclosure also relates to methods for treating microbial infections and inhibiting bacterial biofilm formation using the contents of the multi-component pharmaceutical compositions and kits. Background Art

[0004] Patients with certain infections (e.g., chronic lung infections) are often given multiple antibiotics at the same time. Combinations of antibiotics are often described as antagonistic (the compounds inhibit each other), unrelated (one compound is just as effective alone as the combination), additive (the two compounds are equally effective when used alone and in combination, so the combination is better than either compound alone), or synergistic (the compounds work better together than either one alone, so the effect is greater than the sum of their parts). Antagonistic combinations can have deleterious effects on patients and in some cases lead to morbidity. Therefore, it is important to assess whether antibiotics alter the efficacy of each other. Summary of the invention

[0005] Provided herein are multi-component pharmaceutical compositions and kits, which include at least two components that can play a role in a non-antagonistic manner to achieve a desired therapeutic effect (e.g., antimicrobial effect). Multi-component pharmaceutical compositions and kits may include a first component and a second component, wherein the first component includes a compound that releases nitric oxide (NO), and the second component includes a second therapeutic agent, wherein the second therapeutic agent includes an antibiotic, an antifungal agent, or a combination thereof. In these pharmaceutical compositions and kits, the ratio of the first component to the second component is such that the in vitro fractional inhibition concentration index (FICI) of the combination of the first component and the second component is 1.0 or lower (e.g., 0.5 or lower or 0.3 or lower). The details of calculating the FICI of the composition are provided in the Examples section below.

[0006] In some cases, the concentration of the second therapeutic agent in the second component is lower than the concentration of the second therapeutic agent required for the antimicrobial effect on the microorganism alone (i.e., in the absence of the compound releasing NO). In some cases, the concentration of the second therapeutic agent in the second component is at least 10%, at least 20%, or at least 30% lower than the concentration of the second therapeutic agent required for the antimicrobial effect on the microorganism alone. In some cases, the compound releasing NO in the first component is present in an amount that can effectively sensitize or re-sensitize the microorganism to the second therapeutic agent in the second component. Optionally, the first component and the second component are present in a pharmaceutical composition or a kit as a single combined composition. Optionally, the first component and the second component are present in a pharmaceutical composition or a kit as a separate composition. In some cases, the first component is suitable for atomization, and the second component is suitable for intravenous administration.

[0007] The NO releasing compound in the first component may comprise at least two diazeniumdiolate groups located on one carbon atom, each of the at least two diazeniumdiolate groups having a charge and each having an associated pharmaceutically acceptable cation to balance the charge on the diazeniumdiolate group, the molecular weight of the compound being less than 500 g / mol, excluding the associated pharmaceutically acceptable cation. Optionally, the compound has the following structure:

[0008] in

[0009] R is hydrogen, deuterium, C 1-12 Alkyl, aryl, heteroaryl, alkaryl, aralkyl or carbonyl, wherein the substituents are independently selected from the group consisting of: -OH, -NH2, -OCH3, -C(O)OH, -CH2OH, -CH2OCH3, -CH2OCH2CH2OH, -OCH2C(O)OH, -CH2OCH2C(O)OH, -CH2C(O)OH, -NHC(O)-CH3, -C(O)O((CH2) a O) b -H、-C(O)O((CH2) a O) b -(CH2) c H、-C(O)O(C 1-5 alkyl), -C(O)-NH-((CH2) d NH) e -H、-C(O)-NH-((CH2) d NH) e -(CH2) fH、-O-((CH2) a O) b -H、-O-((CH2) a O) b -(CH2) c H, -O-(C 1-5 Alkyl), -NH-((CH2) d NH) e -H and -NH-((CH2) d NH) e -(CH2) f H; a, b, c, d, e and f are each independently selected from integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and M + is a pharmaceutically acceptable cation, wherein the ratio of the compound to the cation is such that the overall net charge of the compound is neutral.

[0010] In some examples, the cation is selected from the group consisting of sodium ions, potassium ions, lithium ions, calcium ions, magnesium ions, ammonium ions, and substituted ammonium ions. Optionally, the compound has the following structure:

[0011]

[0012] Optionally, the second therapeutic agent in the second component of the multi-component pharmaceutical composition or kit comprises an antibiotic. In some cases, the antibiotic may be selected from the group consisting of aminoglycosides, monolactams, cephalosporins, quinolones, macrolides, polymyxins, and carbapenems. Optionally, the second therapeutic agent in the second component of the multi-component pharmaceutical composition or kit comprises an antifungal agent. The antifungal agent may be selected from the group consisting of polyenes, azoles, allylamines, and echinocandins.

[0013] Optionally, the molar equivalent concentration ratio of the NO-releasing compound to the second therapeutic agent is 0.1:1 to 10:1 (e.g., 0.5:1 to 2:1). The multi-component pharmaceutical composition or kit as described herein and its individual components may further comprise one or more additives (e.g., one or more preservatives, salts, chelating agents, viscosity modifiers, stabilizers, surfactants, antioxidants, buffers or cosolvents).

[0014] Also described herein is a method for treating a microbial infection in a subject, the method comprising administering to the subject a component of a multi-component pharmaceutical composition or a kit as described herein. The method may comprise administering a first component comprising a compound that releases nitric oxide (NO) as described herein, and administering to the subject a second component comprising a second therapeutic agent, wherein the second therapeutic agent comprises an antibiotic, an antifungal agent, or a combination thereof. In these methods, the ratio of the first component to the second component is such that the in vitro fractional inhibition concentration index (FICI) of the combination of the first component and the second component is 1.0 or lower (e.g., 0.5 or lower or 0.3 or lower).

[0015] Optionally, the first component including the compound releasing NO and the second component including the second therapeutic agent are independently administered to the subject orally, parenterally, intravenously, by inhalation, intraperitoneally, intracranial, intraspinal, intrathecal, intraventricular, intramuscularly, subcutaneously, sublingually, buccally, intracavitary or transdermally. In some cases, the compound releasing NO and the second therapeutic agent are administered using the same mode of administration. In other cases, the compound releasing NO and the second therapeutic agent are administered using different modes of administration (for example, the first component can be administered by a nebulizer, and the second component can be administered intravenously). Optionally, the compound releasing NO and the second therapeutic agent are administered simultaneously. In some cases, the first component comprising the compound releasing NO and the second component comprising the second therapeutic agent are present in a single combination composition, and the single combination composition is administered to the subject. In other cases, the first component comprising the compound releasing NO and the second component comprising the second therapeutic agent remain as separate compositions, and are administered to the subject as separate compositions simultaneously (using the same or different modes of administration) or in sequence (using the same or different modes of administration). When administered sequentially, in some cases, the first component comprising the NO-releasing compound is administered prior to the second component comprising the second therapeutic agent.

[0016] Microbial infection may be a bacterial infection. Bacterial infection may be caused by Gram-positive bacteria, Gram-negative bacteria or atypical bacteria. Optionally, bacterial infection is caused by Gram-positive bacterial species, and the Gram-positive bacterial species is selected from the group consisting of: Actinomyces species; Bacillus species; Clostridium species; Corynebacterium species; Enterococcus species; Leuconostoc species; Micrococcus species; Nocardia species; Propionibacterium species; Staphylococcus species; and Streptococcus species.

[0017] In some cases, the bacterial infection is caused by a Gram-negative bacterial species selected from the group consisting of: Acinetobacter species; Aeromonas species; Alcaligenes / Achromobacter species; Bacteroides species; Bartonella species; Bordetella species; Borrelia species; Brevundimonas species; Brucella species; Burkholderia species; Campylobacter species; Citrobacter species; Coxiella species; Ehrlichia species; Enterobacter species; Escherichia species; Francisella species; species); Haemophilus species; Helicobacter species; Klebsiella species; Leclercia species; Legionella species; Leptospira species; Listeria species; Moraxella species; Morganella species; Neisseria species; Orientia species; Pantoea species; Paracoccus species; Prevotellas species; Proteus species; Providencia species; Pseudomonas species; Ralstonia species; Rickettsia species; Roseomonas species;Salmonella species; Serratia species; Shigella species; Sphingomonas species; Stenotrophomonas species; Treponema species; Ureaplasma species; Vibrio species; and Yersinia species. Optionally, the Gram-negative bacterial species comprises Pseudomonas aeruginosa. ;

[0018] In some cases, the bacterial infection is caused by an atypical bacterial species selected from the group consisting of: Mycobacteria species; Chlamydia / Chlamidophila species; and Mycoplasma species. Optionally, the bacterial infection is caused by an antibiotic-resistant bacterium.

[0019] Optionally, the method is performed under aerobic conditions, anaerobic conditions, or microaerobic conditions. In the method, the concentration of the second therapeutic agent in the second component administered to the subject may be lower than the concentration of the second therapeutic agent required to exhibit an antimicrobial effect on microorganisms in the subject alone. In some cases, the concentration of the second therapeutic agent in the second component administered to the subject is at least 10%, at least 20%, or at least 30% lower than the concentration of the second therapeutic agent required to exhibit an antimicrobial effect on microorganisms in the subject alone. In some cases, the NO-releasing compound in the first component sensitizes or re-sensitizes the microorganism to the second therapeutic agent.

[0020] Further described herein is a method for preventing, reducing or eliminating biofilm formation caused by bacteria, the method comprising contacting the bacteria with the contents of a multi-component pharmaceutical composition or a kit as described herein. The method may include applying a first component comprising a compound releasing nitric oxide (NO) as described herein and a second component comprising a second therapeutic agent, wherein the second therapeutic agent comprises an antibiotic, an antifungal agent, or a combination thereof. In the method, the ratio of the first component to the second component is such that the in vitro graded inhibition concentration index (FICI) of the combination of the first component and the second component is 1.0 or lower. In addition, described herein is a method for treating a surface to prevent, reduce or eliminate biofilm formation caused by bacteria, the method comprising contacting the surface with the contents of a multi-component pharmaceutical composition or a kit as described herein. The method may include applying a first component comprising a compound releasing nitric oxide (NO) as described herein and a second component comprising a second therapeutic agent, wherein the second therapeutic agent comprises an antibiotic, an antifungal agent, or a combination thereof. In the method, the ratio of the first component to the second component is such that the in vitro graded inhibition concentration index (FICI) of the combination of the first component and the second component is 1.0 or lower.

[0021] The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1A and 1B The results show that MD3 combined with a second antibiotic has an effect on Figure 1A ) or anaerobic ( Figure 1B ) Graph showing the efficacy of PAK against Pseudomonas aeruginosa strains grown under 4% paraformaldehyde (PAGE) conditions. Abbreviations: ABX, antibiotic; TOB, tobramycin; AZT, aztreonam; CIP, ciprofloxacin; CST, colistin sulfate; CAZ, ceftazidime; MER, meropenem; GaCi, gallium citrate; GEN, gentamicin.

[0023] Figure 2 is a graph showing the efficacy of MD3 in combination with a second antibiotic against NTM grown under aerobic conditions. Abbreviations: ABX, antibiotic; AMK, amikacin; CLR, clarithromycin; GaCi, gallium citrate. NT, not tested.

[0024] Figure 3 is a graph showing the efficacy of MD3 in combination with a second antibiotic against S. aureus grown under aerobic conditions. Abbreviations: TOB, tobramycin; GaCi, gallium citrate.

[0025] Figure 4It is a diagram showing the number of Pseudomonas aeruginosa remaining in each mL sample after treatment with different concentrations of MD3. The measurement results of biofilm-related bacteria are shown in the left bar group (bars 1-6 from the left), while the measurement results of planktonic bacteria are shown in the right bar group (bars 7-12 from the left). Before treatment with MD3, the initial colony forming unit (CFU / mL) per mL was 9.50E+05, and is represented by a dotted line.

[0026] Figure 5 It is a diagram showing that MD3 eradicates Pseudomonas aeruginosa biofilm. Aerobic and anaerobic Pseudomonas aeruginosa biofilms were processed with MD3 (left side bar group) or tobramycin (right side bar group) to determine the minimum biofilm eradication concentration (MBEC), which is defined as a reduction of 3 logarithms in biofilm-related CFU. In each bar group, the left side bar represents aerobic conditions, while the right side bar represents anaerobic conditions. The detection limit is represented by a dotted line. DETAILED DESCRIPTION

[0027] Multi-component pharmaceutical compositions and multi-component kits comprising two or more components are described herein. The first component in the pharmaceutical composition or multi-component kit comprises a compound that releases nitric oxide (NO). The second component in the pharmaceutical composition multi-component kit comprises an antimicrobial agent, such as an antibiotic or an antifungal agent. Methods for treating microbial infections and inhibiting bacterial biofilm formation using the components of the pharmaceutical composition and / or multi-component kit are also described herein.

[0028] It is noteworthy that pharmaceutical compositions and multi-component kits can be designed and customized according to the mode of administration, so as to achieve maximum therapeutic effect. For example, and as further described herein, the composition and multi-component kit consisting of two or more components can be prepared so that the components are combined into a single composition before application, or kept separate and applied to the subject as a separate composition. When combined, the composition is applied by a single effective mode of administration as further described herein. When each component is separated (that is, when each component is applied as a separate composition), the composition can be applied simultaneously by the same mode of administration or different modes of administration, or the same mode of administration or different modes of administration can be used to apply in sequence. Such methods of administration are further described herein. The flexibility of the mode of administration increases the desirability of pharmaceutical compositions and kits described herein in the treatment and prevention of microbial infection and the inhibition of bacterial biofilm formation, because pharmaceutical compositions and kit components can produce many-sided effects on subjects in a single treatment regimen. Pharmaceutical compositions and kit components can be suitable for the desired delivery type. In some cases, the first component is suitable for atomization, and the second component is suitable for intravenous administration.

[0029] Due to the impact of the compound of the release NO in the first component of the pharmaceutical composition and / or the test kit, the pharmaceutical composition as herein described and the test kit can show enhanced, unexpected antimicrobial effect.First, because the compound of the release NO releases nitric oxide, it itself is the contributing factor to the active pharmaceutical ingredient of the composition.The compound of the release NO and the second therapeutic agent (such as antimicrobial agent) act in a collaborative manner to enhance the therapeutic effect of treatment.In some cases, the compound of the release NO and the second therapeutic agent can act additively so that the combination of the two medicaments of delivery is better than a kind of medicament effect of delivery.In some cases, the compound of the release NO and the second therapeutic agent can act synergistically so that the combined effect of the two medicaments of delivery is greater than the sum of its parts.In other cases, the compound of the release NO and the second therapeutic agent can act irrelevantly so that a kind of compound is used separately and is equally effective as the combination.

[0030] In addition to this collaborative effect of medicament (additive, synergistic or even irrelevant in some cases), the compound that releases NO may also produce another effect, so that the second therapeutic agent can enhance its effect on microorganisms. For example, the NO released from the compound that releases NO can destroy the drug resistance mechanism of bacteria, thereby increasing antimicrobial sensitivity and reducing drug resistance over time. The result produced by this combined effect (that is, increasing antimicrobial sensitivity and imparting antimicrobial effect) in treating bacterial infections is greater than the additive result (in other words, synergistic effect). In addition, the NO released from the compound that releases NO can restore the breakpoint sensitivity of microorganisms, making organisms with multidrug resistance and insensitive to antibiotics become effective. This combined effect is a synergistic effect.

[0031] The released NO also has a direct impact on antimicrobial biofilms. Biofilm formation causes serious complications in a wide range of problems (including but not limited to the treatment of bacterial infections). For example, Pseudomonas aeruginosa and non-tuberculous mycobacteria (as well as many other pathogens) have the ability to establish biofilms in multiple locations, including in the lungs of CF patients and on the surfaces of medical devices. Once formed, such biofilms are difficult to control and destroy, and are one of the main causes of nosocomial infections. Many antibiotics are ineffective for treating infections caused by bacteria growing in biofilms, or more drugs are required to treat biofilm-associated bacteria compared to planktonic bacteria. Surprisingly, the NO from the NO-releasing compound in the composition enables (simultaneously or sequentially) co-administered antibiotics to be effective against bacteria. In some cases, NO loosens the biofilm matrix, affects the redox state of the biofilm, and activates quiescent cells, thereby promoting active metabolism.

[0032] It is noteworthy that the use of a certain amount of NO can reduce the amount of the second therapeutic agent to be used, and the effect is still the same as the second therapeutic agent (e.g., antimicrobial) used alone. Due to the effectiveness of NO to the microbial body, the amount of the antimicrobial agent required for the antimicrobial effect is less than at least 10%, less than at least 15%, less than at least 20%, less than at least 25%, less than at least 30%, less than at least 35% or less than at least 40% of the amount of the antimicrobial agent used alone, while still having the same or greater impact. This paper further describes such a multi-component kit that demonstrates these enhanced antimicrobial effects.

[0033] I. Multi-component pharmaceutical compositions and kits

[0034] As described herein, multi-component pharmaceutical compositions or multi-component kits include at least two compositions that play a role in a non-antagonistic manner to achieve the desired therapeutic effect (e.g., antimicrobial effect). In some instances, the components in the multi-component pharmaceutical compositions or kits play a role in a synergistic manner to impart antimicrobial effect. In some instances, the components in the multi-component pharmaceutical compositions or kits play a role in an additive manner to impart antimicrobial effect. In other instances, the components in the multi-component pharmaceutical compositions or kits play a role in an unrelated manner to impart antimicrobial effect.

[0035] A multi-component pharmaceutical composition or kit as described herein comprises a first component comprising a compound that releases nitric oxide (NO), as further described below. The first component may exhibit antimicrobial properties, including antiviral, antibacterial and antifungal properties. The solid components described herein may further have anti-inflammatory properties and other beneficial therapeutic properties. A multi-component pharmaceutical composition or kit as described herein further comprises a second component comprising a second therapeutic agent. The second therapeutic agent may be an antimicrobial agent, such as an antibiotic, an antifungal agent or a combination thereof. The components of the pharmaceutical composition and kit are further described below.

[0036] The multi-component pharmaceutical composition or kit components can be packaged and applied in various ways and modes, respectively. Therefore, the multi-component pharmaceutical composition or kit components described herein can be customized to achieve maximum antimicrobial impact, so that the subject benefits. For example, the first component and the second component of the multi-component pharmaceutical composition or kit can be prepared so that the two compositions are in a single combined composition. The single combined composition can then be applied to the subject by a desired mode of administration (e.g., oral, parenteral, intravenous, by inhalation, intraperitoneal, intracranial, intraspinal, intrathecal, intraventricular, intramuscular, subcutaneous, sublingual, buccal, intracavitary or transdermal).

[0037] In other examples, the first component and the second component of a multi-component pharmaceutical composition or a kit can be prepared so that the two compositions remain as separate components (i.e., not otherwise mixed before being applied to a subject). In some cases, the first component and the second component are contained in separate containers and may be subjected to different storage conditions before application, as required by the individual composition components and as determined by a person of ordinary skill in the art based on the disclosure provided herein. Optionally, the first component and the second component can be applied to a subject simultaneously, respectively. Such application can be carried out by the same mode of administration or by different modes of administration. For example, the first component can be applied to a subject by inhalation, and the second component is applied to a subject orally. In other examples, the first component can be applied by a nebulizer, and the second component is applied intravenously. Optionally, the first component and the second component can be applied to a subject in sequence. Such sequential application can be carried out by the same mode of administration (i.e., applying the first and second components in sequence, but using the same mode of administration) or by different modes of administration (i.e., applying the first and second components in sequence, but using different modes of administration). In some examples, the first component is applied before the second component. In other examples, the second component is applied before the first component.

[0038] a. Nitric Oxide Donor Component

[0039] The active pharmaceutical ingredient delivered by the first component described herein is nitric oxide and can be included in the first component in the form of a compound that releases nitric oxide (NO) (e.g., a nitric oxide donor, a prodrug that releases nitric oxide, or a compound required to promote endogenous production of nitric oxide). As described herein, the first component in the form of a compound that releases NO can exhibit antimicrobial properties, including antiviral, antibacterial and antifungal properties. The stabilizing component described herein can further have anti-inflammatory properties and other beneficial therapeutic properties.

[0040] NO release can be initiated by thermal initiation or by any degradation strategy of unstable portions of N-diazenium diolates, nitrosamines, hydroxynitrosamines, nitrosothiols, hydroxylamines, hydroxyureas, metal complexes, organic nitrites, and organic nitrates. See Wang, PG et al., Nitric Oxide Donors. For Pharmaceutical and Biological Applications; Wliey-VCH. Weinheim, Germany, 2005; and Wang. PG et al., Chem. Rev., 102, 1091-1134 (2002). In some embodiments, the NO donor is an N-diazenium diolate (i.e., 1-amino substituted ethyl nitrene-1-cation-1,2-diolate). N-diazenium diolates are particularly attractive as NO donors because they can spontaneously generate NO under biological conditions. See Hrabie, JA and Keefer, LK, Review of Chemistry, 102, 1135-1154 (2002); and Napoli, C. and Ianarro, LJ, Annu. Rev. Pharmacol. Toxicol., 43, 97-123 (2003). Several N-diazenium diolates have been synthesized using a range of nucleophilic residues covering primary amines, secondary amines, polyamines and secondary amino acids, see Hrabie, JA and Keefer LK, Review of Chemistry, 102, 1135-1154 (2002). In the formation of N-diazenium diolates, one equivalent of amine reacts with two equivalents of nitric oxide under high pressure. A base (e.g., an alkoxide such as methoxide) removes a proton from the amine nitrogen to produce a stable anionic N(O)NO group. Although stable under ambient conditions, N-diazenium diolates spontaneously decompose in aqueous media to generate NO at a rate that depends on pH, temperature, and / or the structure of the amine moiety.

[0041] In some cases, the first component may include at least one nitric oxide releasing compound, wherein the nitric oxide releasing compound has at least two diazonium diolate groups located on one carbon atom, each of the at least two diazonium diolate groups having a charge and each having an associated pharmaceutically acceptable cation to balance the charge on the diazonium diolate group, the compound having a molecular weight of less than 500 g / mol and containing no associated pharmaceutically acceptable cation.

[0042] As described above, the compounds described herein may include at least one functional group that releases nitric oxide. Although various NO donors (e.g., diazonium diolates, S-nitrosothiols, metal nitroso, organic nitrates) are known to provide controlled exogenous NO release, the diazonium diolate functional group (NONOate) in the compounds disclosed herein is attractive for its good stability and easy storage and spontaneous proton-triggered dissociation under physiological conditions to regenerate nitric oxide. Certain compounds include two diazonium diolate groups located on one carbon atom, each of which has a charge and each has a related pharmaceutically acceptable cation to balance the charge on the diazonium diolate group. The compound is a small molecule (molecular weight of 500 g / mol or less, free of cations, as further described below) that releases nitric oxide (NO) and exhibits antimicrobial properties (including antiviral, antibacterial and antifungal properties), anti-inflammatory properties and other beneficial therapeutic properties.

[0043] Optionally, the compound has the following structure, as represented by Formula I:

[0044]

[0045] In Formula I, R is hydrogen, deuterium, C 1-12 alkyl, aryl, heteroaryl, alkaryl, aralkyl or carbonyl. Optionally, R is substituted with one or more substituents, wherein the substituents are independently selected from the group consisting of: -OH, -NH2, -OCH3, -C(O)OH, -CH2OH, -CH2OCH3, -CH2OCH2CH2OH, -OCH2C(O)OH, -CH2OCH2C(O)OH, -CH2C(O)OH, -NHC(O)-CH3, -C(O)O((CH2) a O) b -H、-C(O)O((CH2) a O) b -(CH2) c H、-C(O)O(C 1-5 alkyl), -C(O)-NH-((CH2) d NH) e -H、-C(O)-NH-((CH2) d NH) e -(CH2) f H、-O-((CH2) a O) b -H、-O-((CH2) a O) b -(CH2) cH、-O-(C 1-5 Alkyl), -NH-((CH2) d NH) e -H and -NH-((CH2) d NH) e -(CH2) f H, wherein a, b, c, d, e and f are each independently selected from the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0046] In addition, in Formula I, M + is a cation. For example, M + It may be a pharmaceutically acceptable cation. Optionally, the cation is selected from the group consisting of sodium ions, potassium ions, lithium ions, calcium ions, magnesium ions, and quaternary ammonium salts (eg, ammonium ions or substituted ammonium ions).

[0047] In these compositions, the ratio of compound to cation is such that the overall net charge of the compound is neutral. + In the case of a cation with a valence other than one, the ratio of compound to cation is such that the total positive charge equals the total negative charge. For example, for a compound with a total charge of negative three and a cation with a total charge of positive one, there will be one compound and three cations.

[0048] For example, a compound may be represented by structure IA as follows:

[0049]

[0050] As shown in the structure IA above, the overall charge of the compound is negative three. Therefore, there are three cations (i.e., 3 M + ) to balance the charge of the compound (i.e., the total positive charge equals the total negative charge).

[0051] Examples of structure IA include the following compounds:

[0052]

[0053] The molecular weight of the compound can be less than 500 g / mol, excluding related cations (e.g., related pharmaceutically acceptable cations). For example, the molecular weight of the compound can be 450 g / mol or less, 400 g / mol or less, 350 g / mol or less, 300 g / mol or less, 250 g / mol or less, or 200 g / mol or less. Optionally, removing related cations, the molecular weight of the compound can be 100 g / mol to less than 500 g / mol, 120 g / mol to 450 g / mol, 150 g / mol to 400 g / mol, or 175 g / mol to 350 g / mol.

[0054] Additional details regarding the mechanism of action of the compounds described herein, including their nitric oxide delivery properties and the advantageous properties of the compounds (e.g., storage stability), are described in the following documents: PCT / US2021 / 016841, entitled "Nitric Oxide-Releasing Antibacterial Compounds, Formulations, and Methods Pertaining Thereto"; PCT / US2021 / 016854, entitled "Nitric Oxide-Releasing Antibacterial Compounds, Formulations, and Methods Pertaining Thereto"; and / or PCT / US2021 / 016869, entitled "Nitric Oxide-Releasing Antibacterial Compounds, Formulations, and Methods Pertaining Thereto", each of which is incorporated herein by reference in its entirety.

[0055] As used herein, the terms alkyl, alkenyl, and alkynyl include straight and branched monovalent substituents. Examples include methyl, ethyl, isobutyl, 3-butynyl, and the like. The range of these groups useful in the compounds and methods described herein includes C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Alkynyl. Additional ranges of these groups that can be used in the compounds and methods described herein include C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C4 alkyl, C2-C4 alkenyl and C2-C4 alkynyl.

[0056] Heteroalkyl, heteroalkenyl, and heteroalkynyl groups are defined similarly to alkyl, alkenyl, and alkynyl groups, but may contain O, S, or N heteroatoms, or combinations thereof, within the backbone. The range of these groups useful in the compounds and methods described herein includes C1-C 20 Heteroalkyl, C2-C 20 Heteroalkenyl and C2-C 20 Additional ranges of these groups that can be used in the compounds and methods described herein include C1-C 12 Heteroalkyl, C2-C 12 Heteroalkenyl, C2-C 12 C1-C6 heteroalkyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, C1-C4 heteroalkyl, C2-C4 heteroalkenyl and C2-C4 heteroalkynyl.

[0057] The terms cycloalkyl, cycloalkenyl, and cycloalkynyl include cyclic alkyl groups having a single cyclic ring or multiple condensed rings. Examples include cyclohexyl, cyclopentylethyl, and adamantyl. The range of these groups that can be used in the compounds and methods described herein includes C3-C 20 Cycloalkyl, C3-C 20 Cycloalkenyl and C3-C 20 Additional ranges of these groups that can be used in the compounds and methods described herein include C5-C 12 Cycloalkyl, C5-C 12 Cycloalkenyl, C5-C 12 cycloalkynyl, C5-C6 cycloalkyl, C5-C6 cycloalkenyl and C5-C6 cycloalkynyl.

[0058] The terms heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl are defined similarly to cycloalkyl, cycloalkenyl, and cycloalkynyl, but may contain O, S, or N heteroatoms, or combinations thereof, within the cyclic backbone. The range of these groups useful in the compounds and methods described herein includes C3-C 20 Heterocycloalkyl, C3-C 20 Heterocycloalkenyl and C3-C 20 Heterocycloalkynyl. Additional ranges of these groups that can be used in the compounds and methods described herein include C5-C 12 Heterocycloalkyl, C5-C 12 Heterocycloalkenyl, C5-C 12 C5-C6 heterocycloalkynyl, C5-C6 heterocycloalkyl, C5-C6 heterocycloalkenyl and C5-C6 heterocycloalkynyl.

[0059] For example, aryl molecules include cyclic hydrocarbons that incorporate one or more planar carbon atom groups, typically six carbon atoms, which are connected by delocalized electrons in the same number as when they are composed of alternating single and double covalent bonds. An example of an aryl molecule is benzene. Heteroaryl molecules include substitutions along their cyclic atom backbones (such as O, N or S). When heteroatoms are introduced, a group of five atoms (for example, four carbon atoms and one heteroatom) can produce an aromatic system. Examples of heteroaryl molecules include furans, pyrroles, thiophene, imidazoles, oxazoles, pyridines and pyrazines. Aryl and heteroaryl molecules can also include additional condensed rings, such as benzofuran, indole, benzothiophene, naphthalene, anthracene and quinoline. Unless otherwise indicated, aryl and heteroaryl molecules can be connected at any position on the ring.

[0060] The term alkoxy as used herein is an alkyl group bonded by a single terminal ether bond. The term aryloxy as used herein is an aryl group bonded by a single terminal ether bond. Similarly, the terms alkenyloxy, alkynyloxy, heteroalkoxy, heteroalkenyloxy, heteroalkynyloxy, heteroaryloxy, cycloalkoxy and heterocycloalkoxy as used herein are alkenyloxy, alkynyloxy, heteroalkoxy, heteroalkenyloxy, heteroalkynyloxy, heteroaryloxy, cycloalkoxy and heterocycloalkoxy groups bonded by a single terminal ether bond, respectively.

[0061] The term hydroxyl group as used herein is represented by the formula—OH.

[0062] As used herein, the term amine or amino group is represented by the formula: NZ 1 Z 2 Indicates that Z 1 and Z 2 Each may be a substituent group as described herein, such as a hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group.

[0063] Alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl or heterocycloalkyl molecules used herein may be substituted or unsubstituted. As used herein, the term "substituted" includes adding an alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl or heterocycloalkyl group to a position connected to the backbone of an alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl or heterocycloalkyl group, for example, replacing a hydrogen with one of these molecules. Examples of substituted groups include, but are not limited to, hydroxyl, halogen (e.g., F, Br, Cl or I) and carboxyl. In contrast, as used herein, the term "unsubstituted" indicates that the alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl or heterocycloalkyl group has a full complement of hydrogen, i.e., no substitution commensurate with its degree of saturation, such as linear decane (-(CH2)9-CH3).

[0064] The C-diazenium diolates described herein are pH-triggered NO release donors (also referred to herein as NO release compounds or NO release agents). When reacting with protons under physiological conditions (e.g., 37° C., pH 7.4), 1 mole of compound 1 (MD3) generates two moles of NO and 2 to 3 moles of nitroxyl compounds.

[0065] In several embodiments, the NO-releasing compound is stable at various temperatures from frozen to room temperature 25°C (e.g., -20°C, 0°C, 5°C, 20°C, etc.), and is stable over long-term storage time periods (e.g., 10 hours, 20 hours, 22 hours, 25 hours, 30 hours, etc., several days such as 1 day, 3 days, 5 days, 6 days, 7 days, 15 days, 30 days, 45 days, etc., several weeks such as 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, etc., several months such as 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, etc., or even several years (1 year, 2 years or more)).

[0066] In some cases, the total releasable NO storage of the compound is in the range of 0.1 μmol to 23.0 μmol per mg of compound (e.g., 0.1 μmol to 15 μmol per mg of compound, 0.5 μmol to 7.5 μmol per mg of compound, 1 μmol to 7.0 μmol per mg of compound, 1.5 μmol to 6.5 μmol per mg of compound, 2.0 μmol to 6.0 μmol per mg of compound, 2.5 μmol to 5.5 μmol per mg of compound, or 3.0 μmol to 5.0 μmol per mg of compound). For example, the total releasable NO storage of the compound used in the composition can be 0.1 μmol, 0.2 μmol, 0.3 μmol, 0.4 μmol, 0.5 μmol, 0.6 μmol, 0.7 μmol, 0.8 μmol, 0.9 μmol, 1.0 μmol, 1.1 μmol, 1.2 μmol, 1.3 μmol, 1.4 μmol, 1.5 μmol, 1.6 μmol, 1.7 μmol, 1.8 μmol, 1.9 μmol, 2.0 μmol, 2.1 μmol, or 3.5 μmol per mg of the compound. ol, 2.2μmol, 2.3μmol, 2.4μmol, 2.5μmol, 2.6μmol, 2.7μmol, 2.8μmol, 2.9μmol, 3.0μmol, 3.1μmol, 3.2μmol, 3.3μmol, 3.4μmol, 3.5μmol, 3.6μmol, 3.7μmol, 3.8μmol, 3.9μmol, 4.0μmol, 4.1μmol, 4.2μmol, 4.3μmol, 4.4μmol, 4.5μmol, 4.6μmol mol, 4.7μmol, 4.8μmol, 4.9μmol, 5.0μmol, 5.1μmol, 5.2μmol, 5.3μmol, 5.4μmol, 5.5μmol, 5.6μmol, 5.7μmol, 5.8μmol ,5.9μmol, 6.0μmol, 6.1μmol, 6.2μmol, 6.3μmol, 6.4μmol, 6.5μmol, 6.6μmol, 6.7μmol, 6.8μmol, 6.9μmol, 7.0μmol, 7.1 μmol, 7.2μmol, 7.3μmol, 7.4μmol, 7.5μmol, 7.6μmol, 7.7μmol, 7.8μmol, 7.9μmol, 8.0μmol, 8.1μmol, 8.2μmol, 8.3μmo l, 8.4μmol, 8.5μmol, 8.6μmol, 8.7μmol, 8.8μmol, 8.9μmol, 9.0μmol, 9.1μmol, 9.2μmol, 9.3μmol, 9.4μmol, 9.5μmol, 9.6μmol、9.7μmol、9.8μmol、9.9μmol、10.0μmol、10.1μmol、10.2μmol、10.3μmol、10.4μmol、10.5μmol、10.6μmol、10.7μmol、10.8μmol、10.9μmol、11.0μmol、11.1μmol、11.2μmol、11.3μmol、11.4μmol、11.5μmol、11.6μmol、11.7μmol、11.8μmol、11.9μmol、12.0μmol、12.1μmol、12.2μmol、12.3μmol、12.4μmol、12.5μmol、12.6μmol、12.7μmol、12.8μmol、12.9μmol、13.0μmol、13.1μmol、13.2μmol、13.3μmol、13.4μmol、13.5μmol、13.6μmol、13.7μmol、13.8μmol、13.9μmol、14.0μmol、14.1μmol、14.2μmol、14.3μmol、14.4μmol、14.5μmol、14.6μmol、14.7μmol、14.8μmol、14.9μmol、15.0μmol、15.1μmol、15.2μmol、15.3μmol、15.4μmol、15.5μmol、15.6μmol、15.7μmol、15.8μmol、15.9μmol、16.0μmol、16.1μmol、16.2μmol、16.3μmol、16.4μmol、16.5μmol、16.6μmol、16.7μmol、16.8μmol、16.9μmol、17.0μmol、17.1μmol、17.2μmol、17.3μmol、17.4μmol、17.5μmol、17.6μmol、17.7μmol、17.8μmol、17.9μmol、18.0μmol、18.1μmol、18.2μmol、18.3μmol、18.4μmol、18.5μmol、18.6μmol、18.7μmol、18.8μmol、18.9μmol、19.0μmol、19.1μmol、19.2μmol、19.3μmol、19.4μmol、19.5μmol、19.6μmol、19.7μmol、19.8μmol、19.9μmol、20.0μmol、20.1μmol、20.2μmol、20.3μmol、20.4μmol、20.5μmol、20.6μmol、20.7μmol, 20.8μmol, 20.9μmol, 21.0μmol, 21.1μmol, 21.2μmol, 21.3μmol, 21.4μmol, 21.5μmol, 21.6μmol, 21.7μmol, 21.8μmol, 2 1.9μmol, 22.0μmol, 22.1μmol, 22.2μmol, 22.3μmol, 22.4μmol, 22.5μmol, 22.6μmol, 22.7μmol, 22.8μmol, 22.9μmol or 23.0μmol. .

[0067] The total duration of NO release after activation of the compound can be in the range of 0.1 to 60 hours. In some cases, NO release can occur within the following time period: about 0.1 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours or 60 hours. In some embodiments, within 2 hours of addition to a phosphate buffered saline (PBS) buffer solution, the compound releases greater than or equal to about 25%, 50%, 75%, 85%, 90%, 95%, 100% or a range including and / or spanning the above values ​​according to the total wt% of NO bound thereto. Optionally, the total amount of NO released by the compound 4 hours after the start of NO release (also referred to as "activation") is 0.1-8.0 μmol NO per mg of the compound.

[0068] In some embodiments, using chemiluminescent nitric oxide detection, the hourly release rate of the compound is less than or equal to about: 0.2%, 0.5%, 1.0%, 1.5%, 2.5%, 5.0%, 10% or a range including and / or spanning the above values.

[0069] Optionally, the NO release half-life of the compounds used in the compositions described herein is in the range of 0.01-24 hours. In several embodiments, the NO release half-life is equal to or at least about: 0.01 hour, 0.1 hour, 0.25 hour, 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or a range including and / or spanning the above values. In some embodiments, the time period over which NO release occurs is less than or equal to about: 0.01 hour, 0.1 hour, 0.25 hour, 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, or a range including and / or spanning the above values.

[0070] Optionally, the first component of the multi-component pharmaceutical composition can be or include a composition as described in PCT / US2023 / 019807, entitled “Buffering Agent-Containing Compositions and Methods of Using Same,” which is incorporated herein by reference in its entirety.

[0071] b. Second Therapeutic Agent Component

[0072] As described above, the second component described herein includes a second therapeutic agent. In some cases, the second therapeutic agent can be an antibiotic. Suitable antibiotics can include any antibiotics that are effective for treating bacterial infections and / or inhibiting or destroying biofilms, and include, for example, tetracyclines (e.g., minocycline), quinolones (e.g., ciprofloxacin, levofloxacin and nalidixic acid), aminoglycosides (e.g., amikacin, gentamicin, kanamycin and tobramycin), carbapenems (e.g., meropenem), cephalosporins (e.g., ceftriaxone and ceftazidime), macrolides (e.g., erythromycin and clarithromycin), polypeptides (e.g., colistin and polymyxin B), sulfonamides (e.g., sulfamethoxazole), glycine (e.g., tigecycline), beta lactams (e.g., penicillin), lipopeptides (e.g., daptomycin), oxazolidinones (e.g., linezolid), trimethoprim and monolactams.

[0073] For example, the second component described herein can include an antibiotic such as acetaminophensulfone; sodium sulfadiphenylsulfone; aletomycin; arixidine; amdenocillin; amdenocillin diester; amicycline; amlofloxacin; amlofloxacin mesylate; amikacin; amikacin sulfate; para-aminosalicylic acid; sodium para-aminosalicylate; amoxicillin; amphomycin; ampicillin; ampicillin sodium; apacillin sodium; apramycin; aspartocin; astamicin sulfate; avilamycin; avoparcin; azithromycin; azlocillin; azlocillin sodium; aztreonam; bacampicillin hydrochloride; bacitracin; bacitracin methylenedisalicylate; bacitracin zinc; bambemycin; calcium benzylsalicylates; erythromycin B; betamycin sulfate; biapenem; biniramycin; benzylsalicylamide hydrochloride ester; magnesium sulfate pyrithione; butikacin; butyryl sulfate; capreomycin sulfate; carbadox; carbenicillin disodium; indole penicillin sodium; carbenicillin sodium; carbenicillin potassium; carumonam sodium; cefaclor; cefoperazone; cefuroxil; cefmandole; cefmandole sodium; cefoperazone; ceftriaxone; cefazolin; cefazolin; cefoperazone Sodium cefuroxime; cefuroxime; cefdinir; cefepime; cefepime hydrochloride; cefotecol; cefixime; cefmenoxime hydrochloride; cefmetazole; cefmetazole sodium; cefaclor monosodium; cefaclor sodium; cefoperazone sodium; cefoperazone sodium; cefoperazone; ceftriaxone sodium; cefotetan; cefotetan disodium; cefotiam hydrochloride; cefoxitin; cefoxitin sodium; ceftriaxone; ceftriaxone sodium; cefoxitin Cefpiramide; Cefpiramide sodium; Cefpiramide sulfate; Cefpodoxime proxetil; Cefuroxime; Cefuroxime sodium; Ceftazidime; Ceftibuten; Cefuroxime sodium; Ceftriaxone sodium; Cefuroxime; Cefuroxime axetil; Cefuroxime pixetoxime; Cefuroxime sodium; Ceftriaxone sodium; Cefalexin; Cefalexin hydrochloride; Cefuroxime; Ceftriaxone; Cephalothin; Cephalothin sodium; Cefpirin sodium; Cefuroxime; Tetracycline hydrochloride; Chloramphenicol acetyl; Chloramphenicol; Chloramphenicol palmitate; Chloramphenicol pantothenate complex; Chloramphenicol sodium succinate; Chlorhexidine aminophenylphosphate; Chloroxylenol; Chlortetracycline hydrogen sulfate; Chlortetracycline hydrochloride; Cinofloxacin; Ciprofloxacin; Ciprofloxacin hydrochloride; Ciromycin; Clarithromycin; Clindamycin hydrochloride; Clindamycin; Clindamycin hydrochloride; Hydrochloride Clindamycin palmitate; Clindamycin phosphate; Clofazimine; Cloxacillin benzathine; Cloxacillin sodium; Cloquinol; Colistin sodium methane sulfonate; Colistin; Colistin sulfate; Coumarin; Coumarin sodium; Cyclohexillin; Cycloserine; Dalfopristin; Dapsone; Daptomycin; Demeclocycline; Demeclocycline hydrochloride; Demeclocycline; Diaminoveratridine; Dicloxacillin; Dicloxacillin sodium; Dihydrostreptomycin sulfate; Dithiothiopyridine; Dirithromycin; Doxycycline; Doxycycline calcium; Doxycycline phosphate complex; Doxycycline hydrochloride; Oxyfuroxine sodium; Enoxacin; Epicillin; Epitetracycline hydrochloride; Erythromycin; Erythromycin acetate stearate; Erythromycin estolate; Erythromycin ethylsuccinate; Erythromycin gluconate; Erythromycin lactobionate; Erythromycin propionate;Erythromycin stearate; ethambutol hydrochloride; ethionamide; fleroxacin; flucloxacillin; fludeuterated alanine; flumequine; fosfomycin; fosfomycin tromethamine; fumocillin; furazolidinium chloride; furthimazole tartrate; sodium fusidate; fusidic acid; gentamicin sulfate; glomonam; gramicidin; haloprogin; hetacillin; hetacillin potassium; hexedine; ibaflopax; imipenem; isoconazole; isopamicin; isoniazid; josamycin; kanamycin sulfate; kitasamycin; levofuratazone; levophenoxypropyl penicillin potassium; lexisomycin; lincomycin; lincomycin hydrochloride; lomefloxacin; lomefloxacin hydrochloride; lomefloxacin mesylate; loracarbef; sulfamethoxazole; meclocycline; meclocycline sulfosalicylate; meclocycline potassium dihydrogen phosphate; meclocycline Quindox; Meropenem; Methoxythiazine Hydrochloride; Hexamethylenetetracycline; Hexamethylenetetracycline Hippurate; Hexamethylenetetracycline Mandelate; Methicillin Sodium; Metipriline; Metronidazole Hydrochloride; Metronidazole Phosphate; Mezlocillin; Mezlocillin Sodium; Minocycline; Minocycline Hydrochloride; Milindoxine Hydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium; Nalidixic Acid; Natamycin; Darkomycin; Neomycin Palmitate; Neomycin Sulfate; Neomycin Undecylenate; Netilmicin Sulfate; Neutramycin; Nifuradine; Nifuradine; Nifuroxone; Nifuratel; Nifuron; Nifurdazid; Nifuramide; Nifuranolol; Nifuroxil; Nifurthiazole; Nitrocycline; Nitrofurantoin; Nitamide; Norfloxacin; Neomycin Sodium; Ofloxacin; Omeprim; Oxacillin; Oxacillin Sodium; Oxamo oximemonam sodium; oxolinic acid; oxytetracycline; oxytetracycline calcium; oxytetracycline hydrochloride; padramycin; parachlorophenol; paolomycin; pefloxacin; pefloxacin mesylate; penacillin; benzathine penicillin G; penicillin G potassium; procaine penicillin G; penicillin G sodium; penicillin V; benzathine penicillin V; hydrilamine penicillin V; penicillin V potassium; penicillin sodium; p-aminosalicylic acid phenyl ester; piperacillin sodium; pirbenicillin sodium; pirdicillin sodium; pirlimycin hydrochloride; pirimicin hydrochloride; pirimicin pamoate; pirimicin phenylpropionate; polymyxin B sulfate; methyl mitomycin; prupicacin; pyrazinamide; pyrithione zinc; quindecyl acetate; quinupristin; racemic thiamphenicol; ramoplanin; ranimycin; ralomycin; repulamicin; li Fubutin; riformestane; rifaximin; rifaamide; rifampicin; rifapentine; rifaximin; hydropyridine; pyridine nitrate; rosomycin; rosomycin butyrate; rosomycin propionate; rosomycin sodium phosphate; rosomycin stearate; rosofloxacin; roxarsone; roxithromycin; sanfepenem sodium; samoxicillin; sapicillin; hydromycin; sisomicin; sisomicin sulfate; sparfloxacin; spectinomycin hydrochloride; spiramycin; distamycin hydrochloride; streptomycin; streptomycin sulfate; streptomycin nicotinamide; sulfadiazine; sulfadiazine; sulfadiazine; sulfadiazine sodium; sulfadoxine; sulfamethoxazole; sulfadiaz ...Sulfadimethoxazole; Sulfadimethoxine; Sulfadimethoxazole; Sulfadimethoxine; Sulfadimethoxine; Sulfadimethoxazole ... Tetracycline complex; tetracycline; chloramphenicol sulfone; phenthiacillin potassium; ticarcillin sodium toluene; ticarcillin disodium; ticarcillin monosodium; ticarcillinone; chlorbenzimidone chloride; tobramycin; tobramycin sulfate; tosufloxacin; trimethoprim; trimethoprim sulfate; triple sulfadiazine; troleandomycin; spectinomycin sulfate; gramicidin; vancomycin; vancomycin hydrochloride; vilgimycin; or zolbomycin. ;

[0074] In some cases, the second therapeutic agent can be an antifungal agent. Optionally, the antifungal agent can be selected from the group consisting of polyenes, azoles, allylamines and echinocandins. For example, exemplary antifungal agents include amphotericin B, nystatin, flucytosine, natamycin, ketoconazole, econazole, miconazole, itraconazole, fluconazole, clotrimazole, griseofulvin, oxiconazole, terconazole, tioconazole, clotrimazole, silver sulfadiazine, ciclopirox olamine and terbinafine.

[0075] Optionally, the second component of the multi-component pharmaceutical composition can be or include a composition as described in PCT / US2023 / 019807, entitled “Compositions Containing Buffer and Methods of Use Thereof,” which is incorporated herein by reference in its entirety.

[0076] c. Optional Additives

[0077] The multi-component pharmaceutical compositions and kits described herein and / or one or more individual components / compositions in the multi-component pharmaceutical compositions and kits may further include one or more additives. The one or more additives may include, for example, one or more preservatives, salts, chelating agents, stabilizers, surfactants, antioxidants (e.g., N-acetylcysteine ​​or glutathione), buffers and / or cosolvents. If desired, the multi-component pharmaceutical compositions and kits and / or the individual compositions in the kits may also contain wetting agents or emulsifiers, lubricants, glidants, emollients, humectants, thickeners and / or flavoring agents.

[0078] In some cases, the one or more additives can include viscosity reducers, natural and synthetic anti-biofilm agents (e.g., chitosan), biofilm dispersants, natural and synthetic anti-quorum sensing agents (e.g., autoinducer-2 or N-acylhomoserine lactone), iron carriers, iron chelators, iron mimetics (e.g., gallium (Ga) and gallium-containing compounds such as gallium azoles (Ga-azole)), anti-persistent cell agents (e.g., 4-(4,7-di-methyl-1,2,3,4-tetrahydro-naphthalen-1-yl) pentanoic acid (DMNP)), antimicrobial peptides (AMPs) (e.g., LL-37 or lactoferrin peptide), efflux pump inhibitors and / or phage therapy.

[0079] Optionally, the amount of additives present in the multi-component pharmaceutical composition or kit may be less than 1 wt.%. For example, the amount of additives may be less than 0.9 wt.%, less than 0.8 wt.%, less than 0.7 wt.%, less than 0.6 wt.%, less than 0.5 wt.%, less than 0.4 wt.%, less than 0.3 wt.%, less than 0.2 wt.%, or less than 0.1 wt.%. The amount of additives may optionally be 0.1 to 0.9 wt.%, 0.2 to 0.8 wt.%, or 0.3 to 0.7 wt.%.

[0080] The viscosity modifier may be optionally included in the multi-component pharmaceutical composition and kit as described herein and / or in one or more separate compositions within the multi-component pharmaceutical composition and kit. Optionally, the amount of viscosity modifier included in the multi-component pharmaceutical composition or kit may be up to 5 wt.% (e.g., 0.1 wt.% to 5 wt.%, 0.5 wt.% to 4.5 wt.%, 1.0 wt.% to 4.0 wt.%, 1.5 wt.% to 3.5 wt.%, or 2.0 wt.% to 3.0 wt.%). For example, the viscosity modifier may be 0.1 wt.%, 0.5 wt.%, 1.0 wt.%, 1.5 wt.%, 2.0 wt.%, 2.5 wt.%, 3.0 wt.%, 3.5 wt.%, 4.0 wt.%, 4.5 wt.%, or 5.0 wt.%.

[0081] d. Synergistic, additive and unrelated combinations

[0082] As described above, in some embodiments, the multi-component pharmaceutical compositions or kit components described herein may be synergistic, additive, or unrelated in the relationship between the first component and the second component. Specifically, the ratio of the first component to the second component is such that the in vitro fractional inhibition concentration index (FICI) of the combination of the first component and the second component is 1.0 or less (e.g., 0.5 or less or 0.3 or less). In some cases, FICI values ​​of 0.5 or less are considered synergistic, values ​​of 0.51 to 1 are considered additive, and values ​​greater than 1 to 4 are considered unrelated.

[0083] The properties and amounts of the first and second components of each composition can be selected to achieve a desired relationship (synergistic, additive, or unrelated). For example, the molar equivalent concentration ratio of the compound that releases NO to the second therapeutic agent can be selected to achieve a desired relationship. Optionally, the molar equivalent concentration ratio of the compound that releases NO to the second therapeutic agent is 0.1:1 to 10:1 (e.g., 0.5:1 to 2:1). In some cases, the molar equivalent concentration ratio of the compound that releases NO to the second therapeutic agent is 0.1:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0084] The concentration of the second therapeutic agent in the second component can be lower than the concentration of the second therapeutic agent required for the antimicrobial effect of the microorganism. In some cases, the concentration of the second therapeutic agent in the second component is at least 10% lower than the concentration of the second therapeutic agent required for the antimicrobial effect of the microorganism. In other cases, the concentration of the second therapeutic agent in the second component is at least 20% lower than the concentration of the second therapeutic agent required for the antimicrobial effect of the microorganism. In still other cases, the concentration of the second therapeutic agent in the second component is at least 30% lower than the concentration of the second therapeutic agent required for the antimicrobial effect of the microorganism. Optionally, the compound releasing NO exists in an amount that can effectively make the microorganism sensitive or re-sensitive to the second therapeutic agent.

[0085] II. Methods of preparing the compounds

[0086] The compounds described herein can be prepared in a variety of ways. The compounds can be synthesized, for example, using various synthetic methods. At least some of these methods are known in the field of synthetic organic chemistry. The compounds described herein can be prepared from readily available starting materials. Optimal reaction conditions may vary with the specific reactants or solvents used, but such conditions can be determined by those skilled in the art.

[0087] The changes of Formula I and the compounds described herein include the addition, reduction or movement of various components as described for each compound. Similarly, when there are one or more chiral centers in the molecule, all possible chiral variants are included. In addition, compound synthesis can involve the protection and deprotection of various chemical groups. Those skilled in the art can determine the use of protection and deprotection and the selection of appropriate protecting groups. For example, the chemical substances of protecting groups can be found in the following documents: Wuts, Greene "Protective Groups in Organic Synthesis", 5th edition, Wiley & Sons, 2014, which is incorporated herein by reference in its entirety.

[0088] The reactions for producing the compounds described herein can be carried out in a solvent, which can be selected by a person of ordinary skill in the art of organic synthesis. The solvent can be substantially non-reactive with the starting materials (reactants), intermediates or products under the conditions under which the reactions are carried out, i.e., temperature and pressure. The reactions can be carried out in one solvent or in a mixture of more than one solvent. The formation of products or intermediates can be monitored according to any suitable method known in the art. For example, the formation of products or intermediates can be monitored by methods such as nuclear magnetic resonance spectroscopy (e.g., 1 H-NMR or 13 Product formation can be monitored by spectroscopic means such as C-NMR), infrared spectroscopy (IR), spectrophotometry (e.g., UV-visible), or mass spectrometry (MS), or by chromatographic methods such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).

[0089] Optionally, the compounds described herein can be prepared according to the synthetic methods described in the following documents: PCT / US2021 / 016841, entitled "Antibacterial compounds, formulations and related methods that release nitric oxide"; PCT / US2021 / 016854, entitled "Antibacterial compounds, formulations and related methods that release nitric oxide"; and / or PCT / US2021 / 016869, entitled "Antibacterial compounds, formulations and related methods that release nitric oxide", each of which is incorporated herein by reference in its entirety.

[0090] III. Pharmaceutical Compositions

[0091] In some cases, the multi-component composition (including a multi-component kit) is a pharmaceutical composition. In the case of a pharmaceutical composition, and in some cases where the multi-component kit composition is a pharmaceutical composition, the composition may include a buffer. A buffer may be included to control the pH of the composition. In some instances, a buffer is included to maintain the pH of the composition between 5.5 and 8.5. For example, a buffer may be included to maintain the pH of the composition between 6.0 and 8.0, 6.7 and 7.5, or 7.0 and 7.5 (e.g., 7.4).

[0092] The buffer strength of the buffer can be 0.1 to 2.0 molar equivalents (e.g., 0.1 to 1.5 molar equivalents, 0.2 to 1.25 molar equivalents, or 0.3 to 1.0 molar equivalents). For example, the buffer strength of the buffer can be 0.1 molar equivalents, 0.2 molar equivalents, 0.3 molar equivalents, 0.4 molar equivalents, 0.5 molar equivalents, 0.6 molar equivalents, 0.7 molar equivalents, 0.8 molar equivalents, 0.9 molar equivalents, 1.0 molar equivalents, 1.1 molar equivalents, 1.2 molar equivalents, 1.3 molar equivalents, 1.4 molar equivalents, 1.5 molar equivalents, 1.6 molar equivalents, 1.7 molar equivalents, 1.8 molar equivalents, 1.9 molar equivalents, or 2.0 molar equivalents.

[0093] In general, the buffer can be any buffer that is recognized as safe for use as an inactive ingredient suitable for inhalation. In some embodiments, the buffer used in the compositions described herein includes a phosphate buffer. Examples of suitable phosphate buffers include, for example, 0.01-1M phosphate buffer. Optionally, the phosphate buffer is a potassium phosphate buffer. The counter cation of the buffer used in the composition can be selected to enhance the biological activity of the composition, or to minimize the complexity of the analytical characterization of the composition.

[0094] Specifically, certain examples of compounds described herein, such as MD3 (as further described below), include sodium cations as counterions. When measuring the amount of a compound in a certain formulation (such as an aerosol formulation), the amount of sodium cations is calculated. In these cases, the presence of sodium in a buffer (e.g., a sodium phosphate buffer) can confuse compound calculations. In other examples, when administered to humans, the presence of potassium in a buffer may have an effect on the proton pump and the pH of the resulting epithelial lining fluid. Potassium does not increase the alkalinity of the composition, which is beneficial because an increase in alkalinity can interfere with the release of NO by a nitric oxide-releasing compound.

[0095] One or more buffers may be included in the composition, including acetate buffer, benzoate buffer, citrate buffer, lactate buffer, maleate buffer and tartrate buffer. Optionally, one or more buffers include HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer.

[0096] In some instances, the composition is substantially free of carbonate buffer. In some instances, the composition is substantially free of hydrochloric acid, sulfuric acid or citric acid. As used herein, the term "substantially free" from indicated components (e.g., carbonate buffer, hydrochloric acid, sulfuric acid and / or citric acid) means that based on the weight of the pharmaceutical composition, the pharmaceutical composition may include less than 1%, less than 0.1%, less than 0.01%, less than 0.001% or less than 0.0001% of the component (e.g., carbonate buffer, hydrochloric acid, sulfuric acid and / or citric acid).

[0097] In the case of a pharmaceutical composition, and in some cases where the multi-component kit composition is a pharmaceutical composition, the composition may include a pharmaceutically acceptable carrier. As used herein, the term carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material used in pharmaceutical formulations as known in the art. The choice of carrier for use in the composition will depend on one or more intended modes of administration of the composition.

[0098] Suitable liquid carriers can be aqueous carriers or non-aqueous carriers. Examples of suitable non-aqueous carriers include propylene glycol, polyethylene glycol and oils, including those oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, olive oil, etc. Organic esters such as ethyl oleate are also suitable non-aqueous carriers. Aqueous carriers include water, ethanol, glycerol, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. When the composition is a pharmaceutical composition administered intravenously, water or aqueous carriers are preferred. Saline solutions and dextran and glycerol aqueous solutions can also be used as liquid carriers, particularly injectable solutions. If desired, the composition can also contain a wetting agent or emulsifier, a lubricant, a glidant, an emollient, a humectant, a thickener, a flavoring agent, a preservative or a pH buffer. A pH buffer can be included to control the pH of the composition. In some instances, the buffer is included to maintain the pH of the composition between 5 and 8.5, which can determine the release rate of nitric oxide (NO). For example, a buffer may be included to maintain the pH of the composition between 5.2 and 8.3, 5.5 and 8.0, 6.0 and 8.0, 6.8 and 8.0, or 7.0 and 7.8 (e.g., 7.4). Examples of suitable buffers include phosphate buffers, such as phosphate buffered saline (PBS), for example, 0.01-0.1M phosphate buffer, acetate buffer, benzoate buffer, citrate buffer, lactate buffer, maleate buffer, and tartrate buffer. Buffer carriers may be used, such as Hanks's solution, Ringer's solution, glucose solution, 5% human serum albumin, Ringer's dextrose, glucose and sodium chloride, lactated Ringer's oil and fixed oil, polyethylene glycol, polyvinyl pyrrolidone, or lecithin. Monoethanolamine, diethanolamine, triethanolamine, and glycine solutions may also be used as suitable buffers. Liposomes and non-aqueous vehicles such as fixed oils may also be used as carriers. The formulation should be suitable for the mode of administration. Additional carriers for use in the compositions are described in the Pharmaceutical Compositions section herein.

[0099] In addition, one or more compounds, compositions and formulations described herein can be combined with other agents, including therapeutic agents for diseases and conditions associated with the lungs, digestion, liver and bile ducts. For example, in the case of cystic fibrosis, the compositions and formulations described herein can be combined with mucus thinning drugs (e.g., dornase alfa, N-acetylcysteine ​​and hypertonic saline), bronchodilators (e.g., metaproterenol sulfate, pirbuterol acetate, salmeterol, albuterol and terbutaline sulfate), P2Y2-receptor agonists (e.g., denufosol) and agents targeting nonsense mutations (e.g., PTC124). Further examples of additional pharmaceutical agents that can be combined with the compounds described herein include additional antibiotics (e.g., aminoglycosides, antipseudomonal penicillins, and cephalosporins), additional antimicrobial drugs (e.g., rifabutin), ethambutol, clarithromycin, clofazimine, aztreonam, steroidal and nonsteroidal anti-inflammatory drugs (e.g., ibuprofen and prednisone), pentoxifylline, dornase alfa, or ursodeoxycholic acid.

[0100] One or more compounds and compositions described herein (with or without additional medicaments) can be provided in the form of an inhaler or nebulizer for inhalation therapy. As used herein, inhalation therapy refers to the delivery of therapeutic agents (compounds and compositions as described herein) to the respiratory tract (i.e., pulmonary delivery) in the form of an aerosol. As used herein, the term aerosol refers to an extremely fine liquid or solid particle carried to the treatment application site by a propellant gas under pressure. When a pharmaceutical aerosol is used, the aerosol contains one or more compounds and compositions described herein, and the compound and composition can be dissolved, suspended or emulsified in a mixture of a fluid carrier and / or a propellant. The aerosol can be in the form of a solution, a suspension, an emulsion, a powder or a semisolid preparation. In the case of a powder, when the device is a breath-activated dry powder inhaler, no propellant gas is required. The aerosol used is intended to be applied as fine solid particles or a liquid mist through the patient's respiratory tract.

[0101] When the valve on the aerosol package is opened, the propellant of the aerosol package containing one or more compositions as described herein can form pressure in the container to discharge the compound. Various types of propellants can be utilized, such as fluorinated hydrocarbons (for example, trichloromonofluoromethane, dichlorodifluoromethane and dichlorotetrafluoroethane) and compressed gases (for example, nitrogen, carbon dioxide, nitrous oxide or freon). The vapor pressure of the aerosol package can be determined by one or more propellants adopted. By changing the ratio of each component propellant, any desired vapor pressure can be obtained within the limit of the vapor pressure of each propellant.

[0102] As mentioned above, one or more compositions as described herein can be provided with a nebulizer, which is an instrument for generating substantially uniform fine liquid particles of size in a gas. The liquid containing one or more compounds as described herein and / or compositions can be dispersed into droplets with a diameter of about 5 mm or less in the form of mist. The droplets can be carried by air or oxygen streams through the outlet pipe of the nebulizer. The mist thus produced can penetrate into the respiratory tract of the patient.

[0103] Additional inhalants for delivering the compositions described herein include oral sprays, mists, metered dose inhalers, and dry powder generators (see Gonda, J. Pharm. Sci. 89:940-945, 2000, which is incorporated herein by reference in its entirety at least for the inhalation delivery methods taught therein). For example, a powder composition containing one or more compounds as described herein (with or without a lubricant, carrier, or propellant) can be administered to a patient. One or more compounds in powder form can be delivered using conventional devices for administering powder pharmaceutical compositions by inhalation.

[0104] According to one or more expected modes of administration, the pharmaceutical composition can be in solid, semisolid or liquid dosage forms, such as tablets, suppositories, pills, capsules, powders, liquids or suspensions, preferably in a unit dosage form suitable for single administration of precise doses. The composition will include a therapeutically effective amount of a compound as described herein or a derivative thereof and a pharmaceutically acceptable carrier, and may additionally include other pharmaceutical agents, medicaments, carriers or diluents. Pharmaceutically acceptable means not being a biologically or otherwise undesirable material that can be administered to an individual with the selected compound without causing unacceptable biological effects or interacting in a harmful manner with other components of the pharmaceutical composition containing the material.

[0105] The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described, for example, in Remington: The Science and Practice of Pharmacy, ed. Adeboye Adejare, 23rd ed., Academic Press (2021). Examples of physiologically acceptable carriers include buffers such as phosphate buffers, citrate buffers, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as (ICI Inc.; Bridgewater, NJ), polyethylene glycol (PEG), and PLURONICS TM (BASF; Florham Park, NJ).

[0106] Compositions containing compounds described herein or derivatives thereof suitable for parenteral injection can include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injection solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, etc.), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters, such as ethyl oleate. For example, coatings such as lecithin can be used, in the case of dispersions, by maintaining the desired particle size and by using a surfactant to maintain appropriate fluidity.

[0107] These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersants. Prevention of microbial action can be promoted by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). Isotonic agents such as sugar, sodium chloride, etc. can also be included. Prolonged absorption of injectable drug forms can be achieved by using agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0108] Solid dosage forms for oral administration of the compounds and compositions described herein, or derivatives thereof, include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compound or its derivative described herein is mixed with at least one inert conventional excipient (or carrier) such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and acacia, (c) humectants such as glycerol, (d) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate, (e) solution retarders such as paraffin, (f) absorption promoters such as quaternary ammonium compounds, (g) wetting agents such as cetyl alcohol and glyceryl monostearate, (h) adsorbents such as kaolin and bentonite, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate or mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0109] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols.

[0110] Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other coatings known in the art. The dosage form may contain an opacifying agent and may also be such a composition that releases one or more active compounds in a delayed manner in a specific part of the intestinal tract. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compound may also be in microencapsulated form, with one or more of the above-mentioned excipients where appropriate.

[0111] Liquid dosage forms for oral administration of the compounds and compositions described herein or their derivatives include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (specifically cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, or mixtures of these substances, etc.

[0112] Besides such inert diluents, the composition may also include additional agents such as wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, or perfuming agents.

[0113] In addition to the active compounds, the suspensions may contain additional agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances and the like.

[0114] Compositions of the compounds described herein or their derivatives for rectal administration are optionally suppositories which can be prepared by mixing the compound with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ordinary temperatures but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredients.

[0115] Dosage forms for topical administration of the compounds and compositions described herein or derivatives thereof include ointments, powders, sprays, inhalants, gels, pastes, creams and lotions. The compounds described herein or derivatives thereof are mixed with a physiologically acceptable carrier and any preservatives, buffers or propellants that may be required under aseptic conditions. Ophthalmic formulations, ointments, powders and solutions are also considered to be within the scope of the composition.

[0116] As described above, the composition may include one or more of the compounds described herein or a pharmaceutically acceptable salt thereof. As used herein, the term pharmaceutically acceptable salt refers to a salt of a compound described herein or a derivative thereof, which is suitable for contact with the tissue of a subject within the scope of reasonable medical judgment without excessive toxicity, irritation, allergic reaction, etc., commensurate with a reasonable benefit / risk ratio, and effective for its intended use and, where possible, the zwitterionic form of the compound described herein. The term salt refers to relatively nontoxic inorganic and organic acid addition salts of the compounds described herein. These salts can be prepared in situ during the separation and purification of the compound, or by reacting the purified compound in the form of a free base with a suitable organic or inorganic acid alone and separating the salt thus formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, methanesulfonate, glucoheptonate, lactobionate, methanesulfonate and laurylsulfonate, etc. These salts may include cations based on alkali metals and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. (See SM Barge et al., J. Pharm. Sci. (1977) 66, 1, which is incorporated herein by reference in its entirety at least with respect to the compositions taught therein).

[0117] The administration of the compounds and compositions described herein or their pharmaceutically acceptable salts can be carried out using a therapeutically effective amount of the compounds and compositions described herein or their pharmaceutically acceptable salts as described herein for a period of time effective to treat the condition. The effective amount of the compounds and compositions described herein or their pharmaceutically acceptable salts as described herein can be determined by one of ordinary skill in the art and includes an exemplary dose of the active compound of about 0.01 to about 200 mg / kg body weight per day for a mammal, which can be administered in a single dose or in the form of separate divided doses, such as 1 to 4 times per day. Alternatively, the dosage can be from about 0.05 to about 190 mg / kg body weight of active compound per day, from about 0.1 to about 180 mg / kg body weight of active compound per day, from about 0.25 to about 175 mg / kg body weight of active compound per day, from about 0.5 to about 150 mg / kg body weight of active compound per day, from about 0.5 to about 100 mg / kg body weight of active compound per day, from about 0.5 to about 75 mg / kg body weight of active compound per day, from about 0.5 to about 50 mg / kg body weight of active compound per day, from about 0.5 to about 25 mg / kg body weight of active compound per day, from about 1 to about 20 mg / kg body weight of active compound per day, from about 1 to about 10 mg / kg body weight of active compound per day, about 20 mg / kg body weight of active compound per day, about 10 mg / kg body weight of active compound per day, or about 5 mg / kg body weight of active compound per day. Those skilled in the art will appreciate that specific dosage levels and dosage frequencies for any particular subject may vary and will depend on a variety of factors, including the activity of the specific compound employed, the metabolic stability and length of action of the compound, the species, age, weight, general health, sex and diet of the subject, mode and time of administration, rate of excretion, drug combination and the severity of the particular condition.

[0118] IV. How to use

[0119] Provided herein are methods for treating microbial infections in subjects. The method includes administering to the subject a composition in a multi-component pharmaceutical composition or a kit as described herein. The method may include administering a first component as described herein comprising a compound that releases nitric oxide (NO) (and optionally having antimicrobial properties (including antiviral, antibacterial and antifungal properties), anti-inflammatory properties and / or other beneficial therapeutic properties), and administering to the subject a second component comprising a second therapeutic agent, wherein the second therapeutic agent comprises an antibiotic, an antifungal agent, or a combination thereof.

[0120] In some cases of these methods, the concentration of the second therapeutic agent in the second component applied to the subject may be lower than the concentration of the second therapeutic agent required for the antimicrobial effect of the microorganism in the subject's body alone. In some cases, the concentration of the second therapeutic agent applied to the subject is at least 10%, at least 20%, or at least 30% lower than the concentration of the second therapeutic agent required for the antimicrobial effect of the microorganism in the subject's body alone. Even so, compositions as described herein are equally effective compared to the second therapeutic agent used alone, or even more effective. In some cases, the compound releasing NO makes the microorganism sensitive or re-sensitive to the second therapeutic agent, so that the second therapeutic agent shows a new or larger antimicrobial effect.

[0121] The compound that releases NO and the second therapeutic agent can be administered to the subject by any suitable method, including (as further described herein) orally, parenterally, intravenously, by inhalation, intraperitoneally, intracranial, intraspinal, intrathecal, intraventricular, intramuscularly, subcutaneously, sublingually, buccally, intracavitary or transdermally. The compound that releases NO and the second therapeutic agent can be administered using the same mode of administration or by different modes of administration. In some cases, the compound that releases NO and the second therapeutic agent are administered simultaneously, while in other cases, the compound that releases NO and the second therapeutic agent are administered sequentially. Optionally, the compound that releases NO can be administered before the second therapeutic agent is administered. In some cases, the compound that releases NO is administered 24 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less before the second therapeutic agent is administered to the subject.

[0122] Optionally, the microbial infection treated by the compositions described herein can be a bacterial infection. The bacterial infection may be caused by gram-positive bacteria, gram-negative bacteria or atypical bacteria. In some cases, the bacterial infection is caused by gram-positive bacterial species, such as actinomycetes, bacillus, clostridium, corynebacterium, enterococcus, leuconostoc, micrococcus, nocardia, propionibacterium, staphylococcus or streptococcus.

[0123] Optionally, the bacterial infection may be caused by a Gram-positive bacterial species, such as Acinetobacter, Aeromonas, Alcaligenes / Achromobacter, Bacteroides, Bartonella, Bordetella, Borrelia, Brevundimonas, Brucella, Burkholderia, Campylobacter, Citrobacter, Coxsella, Ehrlichia, Enterobacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Luxembourg, Legionella, Leptospira, Listeria, Moraxella, Morganella, Neisseria, Orientia, Pantoea, Paracoccus, Prevotella, Proteus, Providencia, Pseudomonas (e.g., Pseudomonas aeruginosa), Ralstonia, Rickettsia, Roseomonas, Salmonella, Serratia, Shigella, Sphingomonas, Stenotrophomonas, Treponema, Ureaplasma, Vibrio, or Yersinia.

[0124] Optionally, the bacterial infection may be caused by an atypical bacterial species, such as mycobacteria, Chlamydia / Chlamydophila, or mycoplasma. In some cases, bacterial infections may be caused by or may develop into antibiotic-resistant bacteria, such as antibiotic-resistant Burkholderia cepacia, carbapenem-resistant Enterobacteriaceae (CRE) enterobacteria, drug-resistant Campylobacter, drug-resistant non-typhoidal Salmonella, drug-resistant Shigella, multidrug-resistant Acinetobacter, multidrug-resistant Escherichia coli, multidrug-resistant Klebsiella pneumoniae, multidrug-resistant Neisseria gonorrhoeae, multidrug-resistant Pseudomonas aeruginosa, antibiotic-resistant Clostridium difficile, drug-resistant Streptococcus pneumoniae, clindamycin-resistant Group B Streptococcus Streptococcus, erythromycin-resistant Group A Streptococcus, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), and vancomycin-resistant Enterococcus (VRE).

[0125] Also provided herein are methods for preventing, reducing or eliminating biofilm formation caused by bacteria. The method comprises contacting the bacteria with an effective amount of a multi-component pharmaceutical composition or a composition in a kit as described herein. The effective amount of the composition can be an amount that prevents, reduces and / or eliminates bacterial biofilm formation, and includes a concentration of the second therapeutic agent administered to the subject that is lower than the concentration of the second therapeutic agent required to exhibit an antimicrobial effect on the bacteria alone.

[0126] The compositions described herein can be used to prevent, reduce or eliminate biofilm formation caused by bacteria on humans (eg, children and elderly populations), animals (eg, veterinary applications), and surfaces (eg, medical device surfaces).

[0127] The methods described herein may further include selecting a subject infected or at risk of infection with bacteria. Optionally, the method may further include selecting a subject infected or at risk of infection with bacteria that can produce resistance to antibiotics. Subjects at risk of infection with bacteria as described above include infants, the elderly, immunocompromised subjects, hospitalized subjects, subjects living in institutions (e.g., nursing homes), subjects with invasive medical devices (e.g., catheters), subjects with open wounds, and subjects of other people who have been exposed to infected bacteria.

[0128] Treatment or prevention methods as described herein may further include treatment with one or more additional medicaments (e.g., a second biofilm inhibitor). As described above, one or more additional medicaments as described herein and compounds and compositions or their pharmaceutically acceptable salts may be administered in any order (including simultaneous administration) and at intervals up to several days in sequence. The method may also include more than one administration of one or more additional medicaments and / or compounds and compositions or their pharmaceutically acceptable salts as described herein. One or more additional medicaments as described herein and compounds and compositions or their pharmaceutically acceptable salts may be administered by the same or different modes. When treated with one or more additional medicaments, compounds and compositions or their pharmaceutically acceptable salts may be combined into pharmaceutical compositions comprising one or more additional medicaments as described herein.

[0129] The methods and compounds as described herein can be used for both preventive and therapeutic treatments. As used herein, the term treatment (treating or treatment) includes prevention; delayed onset; mitigation, elimination or delay of the aggravation of signs or symptoms after onset; and prevention of recurrence. For preventive use, a therapeutically effective amount of a compound as described herein and a composition or a pharmaceutically acceptable salt thereof is applied to a subject before the onset of a bacterial biofilm (e.g., before obvious signs of bacterial biofilm formation), during early onset (e.g., after initial signs and symptoms of bacterial biofilm formation), or after formation is determined. Prophylactic administration can be performed within a few hours to several years before symptoms of infection appear. For example, prophylactic administration can be used for prophylactic treatment of subjects or surfaces exposed to Pseudomonas aeruginosa. Therapeutic treatment involves contacting the bacteria with a therapeutically effective amount of a composition as described herein after observing bacterial biofilm formation.

[0130] Optionally, contact can be made under aerobic conditions (also referred to as aerobic environment). As used herein, the term aerobic conditions refers to conditions characterized by the presence of free oxygen (O2). Optionally, contact can be made under anaerobic conditions (also referred to as anaerobic environment). As used herein, the term anaerobic conditions refers to conditions lacking free oxygen (O2). Optionally, contact can be made under microaerobic conditions (also referred to as microaerobic environment). As used herein, the term microaerobic conditions refers to conditions with a lower free oxygen (O2) content, which means lower than normal atmospheric oxygen content and between aerobic and anaerobic conditions.

[0131] Also provided herein is a method for treating a surface to prevent, reduce or eliminate the formation of a biofilm caused by bacteria. The method for treating a surface to prevent, reduce or eliminate the formation of a bacterial biofilm includes contacting the surface with an effective amount of a multi-component pharmaceutical composition as described herein or a composition in a kit. The effective amount of the composition can be an amount that prevents, reduces and / or eliminates the formation of a bacterial biofilm on the surface, and includes a concentration of the second therapeutic agent in the second component that is lower than the concentration of the second therapeutic agent required for the antimicrobial effect on the bacteria alone. Optionally, the surface is a human body surface, such as a mucosal surface. Optionally, the mucosal surface is a mucosal surface of the lungs or upper respiratory tract.

[0132] V. Kits for treating microbial infections and preventing, reducing or eliminating biofilm formation

[0133] Also provided herein are kits for treating microbial infections in subjects; kits for preventing, reducing or eliminating biofilm formation caused by bacteria; and kits for treating or pretreating surfaces to prevent, reduce or eliminate biofilm formation caused by bacteria. The kit may include any of the compositions described herein. For example, the kit may include an NO releaser (e.g., a compound of formula I) and a second therapeutic agent. Optionally, the kit may further include a carrier (e.g., a pharmaceutically acceptable carrier).

[0134] The kit may include a device for delivery by inhalation (e.g., an inhaler or nebulizer) or an oral formulation of any composition described herein. In addition, the kit may include instructions for use of the kit (e.g., instructions for treating a subject or contacting a surface), one or more containers (for holding the compound, composition, or second biofilm inhibitor), a device for administering the compound or composition, and / or a carrier.

[0135] Optionally, the multi-component kit may include one or more containers. The kit may include a first container comprising a first component comprising a compound that releases nitric oxide. Optionally, the kit may include a second container comprising a second therapeutic agent. Optionally, the kit may include a third container for combining the components in the first and second containers for optional use when the first and second components are administered as a single combined composition.

[0136] As used herein, the term treatment (treatment, treat or treating) refers to a method for alleviating one or more symptoms of a disease or condition. Therefore, in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in severity of one or more symptoms of a disease or condition. For example, if one or more symptoms or signs of a subject's disease are reduced by 10% compared to a control, the method for treating the disease is considered to be a therapeutic method. As used herein, a control refers to an untreated situation. Therefore, compared to a natural or control level, reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any percentage reduction between 10% and 100%. It should be understood that treatment does not necessarily refer to curing or completely eliminating a disease, condition, or the symptoms of a disease or condition.

[0137] As used herein, the terms prevent, preventing, and prevention of a disease or condition refer to actions performed before or about the same time as a subject begins to develop one or more symptoms of the disease or condition, such as administration of a composition or therapeutic agent, thereby inhibiting or delaying the onset or severity of one or more symptoms of the disease or condition.

[0138] As used herein, reference to reduction, decrease or inhibition includes changes of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to control levels. Such terms may include (but do not necessarily include) complete elimination.

[0139] As used herein, subject means both mammals and non-mammals. Mammals include, for example, humans, non-human primates (e.g., apes and monkeys), cattle, horses, sheep, rats, mice, pigs and goats. Non-mammals include, for example, fish and birds.

[0140] Throughout this application, various publications are cited. The disclosures of these publications are hereby incorporated by reference into this application in their entirety.

[0141] Examples

[0142] The following examples are set forth below to illustrate methods and results according to the disclosed subject matter. These examples are not intended to include all aspects of the subject matter disclosed herein, but are intended to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the subject matter described herein that would be apparent to one skilled in the art.

[0143] Example 1: Efficacy of NO-releasing compounds in combination with other antibiotics in treating Pseudomonas aeruginosa lung infections

[0144] Patients with certain infections, such as chronic lung infections, often take multiple antibiotics at the same time. Therefore, it is important to evaluate whether NO-releasing agents alter the efficacy of commonly used antibiotics and vice versa. For the purposes of this study, MD3 was used as a representative NO-releasing agent.

[0145] To quantify the combined effects of MD3 and a second antibiotic, a checkerboard assay, a two-dimensional, two-agent serial dilution method that quantifies the effects of each drug alone and in combination, was used. In patients with underlying lung disease such as bronchiectasis, large amounts of mucus in the lungs can lead to bacterial infections that are primarily microaerophilic (very little oxygen) or anaerobic (no oxygen) infections. Therefore, combinations of MD3 and six second antibiotics were evaluated against the representative organism Pseudomonas aeruginosa under both aerobic and anaerobic conditions.

[0146] The antibiotics tested in this study were selected to represent the different mechanisms of action of antibiotics used to treat P. aeruginosa infections in the lungs, as shown in Table 1 .

[0147] Table 1. Antibiotics used in this study

[0148]

[0149]

[0150] method

[0151] Species and strains

[0152] The strains used in these studies and their sources are shown in Table 2 .

[0153] Table 2. Species and strains used in this study

[0154]

[0155] Abbreviations: UNC, University of North Carolina at Chapel Hill; BcRLR = Burkholderia cepacia Research Laboratory and Repository; CSU, Colorado State University; MDR, multidrug-resistant; MRSA, methicillin-resistant Staphylococcus aureus.

[0156] Checkerboard assay

[0157] The checkerboard method was performed as follows. MD3 and the second antibiotic were tested separately at four times (4X) their minimum inhibitory concentration (MIC) (see Table 3). Stock solutions of MD3 and the second antibiotic were prepared at 4X of the final concentration (i.e., 16X of the MIC, referred to as 4X stock solutions).

[0158] MD3 and a second antibiotic were tested alone at 4X their minimum inhibitory concentration (MIC). For example, the MIC against aerobic Pseudomonas aeruginosa strain PAK MD3 was 31.25 μg / ml, and the MIC TOB =3.125 μg / ml; therefore, the highest concentration tested in the checkerboard assay was 125 μg / ml MD3 and 12.5 μg / ml TOB. MD3 and antibiotics were tested over a 7-point range of 2-fold concentrations, as detailed in Table 3.

[0159] Table 3. Concentrations tested in the checkerboard assay plate, shown in μg / ml.

[0160]

[0161]

[0162] Abbreviations: TOB, tobramycin; ATM, aztreonam; CIP, ciprofloxacin; CST, colistin; CAZ, ceftazidime; MER, meropenem; AMK, amikacin; CLR, clarithromycin.

[0163] Prepare bacterial cultures by streaking on agar plates from frozen glycerol stocks stored at -80°C (see Table 4). Suspend several well-isolated colonies in 1X PBS and then dilute to 1×10 in liquid culture. 6 CFU / ml. Dilute the culture to 5×10 5CFU / ml and then added to the antibiotic combination in the checkerboard assay plate. Because colonies from strains N0010, N0046, and N0124 were difficult to suspend uniformly, they were first subcultured in 7H9 liquid medium containing Middlebrook ADC supplement and 0.05% Tween-80 and then incubated at 37°C with shaking for 3 days. This subculture was used to prepare 1×10 6 CFU / ml culture. Note: Tween-80 promotes homogenous growth; however, it is not included in the checkerboard assay medium because it has been shown to affect the susceptibility of mycobacteria to antimicrobial drugs (Van Boxtel, 1990).

[0164] To prepare the checkerboard assay plate, phosphate buffered saline (PBS) was added to wells B1 to H8 of a 96-well plate. Next, 200 μl of 4X stock solution of the second antibiotic was added to empty wells in A1 to A8 of the 96-well plate and serially diluted down the plate at a 1:1 ratio using a multichannel pipette, stopping at row G. The last 100 μl in row G was discarded. Row H contained no second antibiotic - it only measured the MIC of MD3. At this step, all wells contained 4X target concentration of the second antibiotic. Next, 100 μl of 4X stock solution of MD3 was added to wells A1 to H1 and serially diluted down the plate at a 1:1 ratio using a multichannel pipette to column 7. The last 100 μl in row 7 was discarded. Column 8 did not contain any MD3 - it only measured the MIC of the second antibiotic. After this step, all wells contained 2X target concentrations of MD3 and the second antibiotic. The wells in column 9 contained only medium as a medium control. The wells in column 10 contained medium and bacteria as a growth control. Finally, 100 μl of 5 × 10 5 CFU / ml of bacterial culture was added to each well. The checkerboard assay plates were incubated at 37°C until growth was observed in the growth control wells (see Table 4).

[0165] For experiments where P. aeruginosa was grown under anaerobic conditions, 90 mM KNO3 was added to the CAMHB medium and the checkerboard assay plates were incubated in an anaerobic chamber containing an anaerobic pouch. Anaerobiosis was confirmed by co-incubation of anaerobic test strips.

[0166] Table 4. Checkerboard assay conditions used.

[0167]

[0168] Abbreviations: MHA, Mueller-Hinton agar; CAMHB, cation-adjusted Mueller-Hinton broth; BHI, brain heart infusion; OADC, Middlebrook supplement containing oleic acid, bovine albumin, dextrose, and catalase; ADC, Middlebook supplement containing bovine albumin, dextrose, and catalase.

[0169] Interpretation of chessboard test results

[0170] The effects of antibiotic combinations were classified according to the Fractional Inhibitory Concentration Index (FICI) as described by Chou and Talay (1984) using Equation 1, where MIC A and MIC B are the values ​​determined for agents A and B when tested separately, and the MIC AB and MIC BA are the concentrations of agents A and B in the most effective combination in the checkerboard assay, respectively.

[0171] Equation 1

[0172]

[0173] The FICI of each well is calculated along the growth / no growth boundary of the chessboard assay plate. If the FICI of any well is greater than 4, it is reported as antagonism; otherwise, the lowest FICI value is reported. FICI values ​​≤ 0.5 are considered synergistic, values ​​of 0.51 to 1 are additive, and values ​​greater than 1 to 4 are irrelevant. The basic principle of this analytical method is that if any ratio of MD3: antibiotic is synergistic, then this combination is likely to be synergistic even if other ratios are not synergistic. The classification is reported only when synergy and antagonism are observed in at least two biological replicates. If the experiment is repeated twice and the same classification is observed (for example, synergy is observed in both replicates), the average FICI of the drug combination is reported. If the classification results of the two replicates are inconsistent, a third experiment must be performed and the average FICI of the three replicates used for the final classification.

[0174] Determination of validity

[0175] A chessboard determination is considered valid if all of the following statements are true:

[0176] -Confirm that the starting inoculum is 1×10 5 -9.0×10 6 CFU / ml between;

[0177] - The starting inoculum produces pure single colonies on agar;

[0178] - Growth is observed in the growth control wells for a time appropriate to the species as described in the 'Bacterial cultures' section;

[0179] - At least 7 of the 8 growth control wells were turbid;

[0180] - At least 7 of the 8 media control wells are clear; and

[0181] -The MIC for each individual agent was ± 2 wells of the pre-determined MIC for that agent for that strain.

[0182] result

[0183] Figure 1A and 1B The results of the chessboard assay for MD3 in combination with a second antibiotic tested against Pseudomonas aeruginosa are shown in . Under aerobic conditions, MD3 was synergistic with aztreonam, ciprofloxacin, colistin, ceftazidime, and gallium citrate, additive with tobramycin and meropenem, but not with gentamicin ( Figure 1A ). Under anaerobic conditions, MD3 had an additive effect with all six antibiotics tested (tobramycin, aztreonam, ciprofloxacin, colistin, cefuroxime, and meropenem). Importantly, no antagonism was observed with any of the MD3 / antibiotic combinations tested under aerobic or anaerobic conditions. See Table 5.

[0184] Table 5. FICI values ​​obtained from the checkerboard assay for P. aeruginosa.

[0185]

[0186] Abbreviations: ATM, aztreonam; CAZ, ceftazidime; CIP, ciprofloxacin; CST, colistin sulfate; GaCi, gallium citrate; GEN, gentamicin; MER, meropenem; TOB, tobramycin. NT, not tested.

[0187] Figure 2 The results of the checkerboard assay for the use of MD3 in combination with a second antibiotic tested against NTM are shown in Table 6. MD3 had an additive effect with amikacin when used against M. abscessus strains N0010 and N0046, M. chimaericum, and M. massiliense. MD3 and clarithromycin had an additive effect when used against M. chimaericum and M. massiliense. MD3 and gallium citrate had an additive effect when used against M. abscessus strains N0010 and N0046. Importantly, no antagonism was observed with any of the combinations tested. See Table 6.

[0188] Table 6. FICI values ​​obtained from the checkerboard assays of NTM. These were performed under aerobic conditions.

[0189]

[0190]

[0191] Abbreviations: AMK, amikacin; GaCi, gallium citrate; CLR, clarithromycin. NT, not tested.

[0192] at last, Figure 3 The results of the checkerboard assay for MD3 in combination with a second antibiotic tested against S. aureus are shown in Table 7. When used against S. aureus strain N0040, MD3 was independent of tobramycin and had a synergistic effect with gallium citrate. Again, no antagonism was observed for any of the tested combinations. See Table 7.

[0193] Table 7. FICI values ​​obtained from the checkerboard assay for S. aureus. These were performed under aerobic conditions.

[0194]

[0195] Abbreviations: GaCi, gallium citrate; TOB, tobramycin. NT, not tested.

[0196] In summary, the efficacy of MD3 in combination with other antibiotics commonly used to treat lung infections was studied to evaluate any potential drug-drug interactions. Pseudomonas aeruginosa was studied primarily, which is the most common and harmful pathogen in patients with underlying lung diseases such as cystic fibrosis bronchiectasis. In the study of P. aeruginosa, antibiotics covering six classes of antibiotics were evaluated under aerobic and anaerobic conditions. The MD3 / antibiotic combination did not show antagonism, suggesting that MD3 is an ideal candidate for combination therapy. In fact, a synergistic effect was observed when MD3 was used in combination with aztreonam, ciprofloxacin, colistin, ceftazidime, and gallium citrate, and an additive effect was observed when MD3 was used in combination with tobramycin and meropenem. These results support the potential combination of MD3 with one of these traditional antibiotics for the management of P. aeruginosa infections.

[0197] Additional studies were conducted to evaluate the combination of MD3 with antibiotics commonly used to treat NTM infections, which are usually treated with at least three antibiotics for at least six months at a time. The results of these studies showed that MD3 has an additive effect with amikacin and clarithromycin, two of the most commonly used antibiotics for treating NTM lung infections. These results support the potential combination of MD3 and amikacin or MD3 and clarithromycin for the treatment of NTM infections.

[0198] Additional studies were performed to evaluate drug-drug interactions in the gram-positive organism Staphylococcus aureus. In this species, the combination of MD3 and tobramycin had no effect, suggesting that the combination of MD3 and tobramycin does not reduce the efficacy of either drug.

[0199] Example 2: Efficacy of NO-releasing compounds in combination with other antifungal drugs against Aspergillus fumigatus and Candida auris

[0200] As described in detail above with respect to antibiotics in Example 1, it is important to evaluate whether NO releasers would alter the efficacy of commonly used antifungal agents and vice versa. For the purposes of this study, MD3 was used as a representative NO releaser.

[0201] To quantify the combined effects of MD3 and a second antifungal agent, a checkerboard assay was used as described above in Example 1. The antibiotics tested in this study included voriconazole (a triazole antifungal that inhibits the growth of fungi causing infection), itraconazole (an azole antifungal that inhibits fungal growth), and amphotericin B (a polyene antifungal that binds to ergosterol on the fungal cell membrane, forming pores in the membrane and allowing cell components to leak out, leading to cell death).

[0202] The fungal species used in this study included Aspergillus fumigatus (strain N0219) and Candida auris (strain N0220). The checkerboard method was performed as described in Example 1 above. At the 24-hour and 48-hour time points, the effects of the antifungal combinations were classified according to the graded inhibitory concentration index (FICI) as described in Example 1 above. The checkerboard assay results for MD3 in combination with a second antifungal drug are shown in Table 8.

[0203] Table 8. FICI values ​​obtained from the checkerboard assay for fungal species.

[0204]

[0205] In conclusion, for Aspergillus fumigatus and Candida auris, MD3 Voriconazole At least In addition, for Aspergillus fumigatus and Candida auris, MD3 With itraconazole At least There is an additive effect. The combination of MD3 and the tested antifungal drugs (two compounds) is equally effective when used alone and in combination, so the combination is more effective than the single compound. Importantly, no antagonism was observed for any of the tested MD3 / antifungal combinations. See Table 8.

[0206] Example 3: Inhibition of Pseudomonas aeruginosa biofilm formation

[0207] Prepare 96-well plates containing serial dilutions of MD3 or medium alone (untreated control). Test concentrations of MD3 include 0.00391 mg / mL, 0.00781 mg / mL, 0.0156 mg / mL, 0.3125 mg / mL, and 0.0625 mg / mL. Add 10 6 Colony forming units (CFU) / mL of Pseudomonas aeruginosa. The nail cap is inserted into the plate and the biofilm is allowed to grow on the nail for 18 to 24 hours. The nail is then rinsed with phosphate buffered saline (PBS) to remove planktonic bacteria. The biofilm is disrupted and plated to count biofilm-associated colony forming units.

[0208] Figure 4 The number of biofilm-associated bacteria and planktonic Pseudomonas aeruginosa bacteria remaining in each mL of sample after treatment with different concentrations of MD3 is shown. The measurements of biofilm-associated bacteria are shown in the left group of bars (bars 1-6 from the left), while the measurements of planktonic bacteria are shown in the right group of bars (bars 7-12 from the left). Before treatment with MD3, the starting CFU / mL was 9.50E+05 and is represented by the dotted line. Figure 4 As shown, at a concentration of 0.0625 mg / mL, MD3 effectively prevented Pseudomonas aeruginosa from forming biofilm on nails. Surprisingly, the Pseudomonas aeruginosa biofilm prevention concentration of MD3 is about one-half of the minimum inhibitory concentration (MIC) of MD3 to planktonic Pseudomonas aeruginosa (which is 0.125 mg / mL). See Table 9.

[0209] Table 9

[0210] Compound MIC Biofilm Prevention Concentration MD3 0.125mg / mL 0.0625mg / mL

[0211] Example 4: Eradication of Pseudomonas aeruginosa biofilm

[0212] The minimum biofilm eradication concentration (MBEC) of MD3 was determined against P. aeruginosa biofilms grown under aerobic and anaerobic conditions. The MBEC was defined as a 3-log reduction in biofilm-associated CFU. TM Biofilms were grown in a growth apparatus (Innovotech; Edmonton, Alberta, Canada) at 37°C in cation-adjusted Miller-Hayden broth (CAMHB) for 24 hours and treated with MD3 or tobramycin for an additional 18-24 hours. The remaining biofilms were disrupted with ultrasound and plated to determine the CFU / mL surviving after a single treatment.

[0213] like Figure 5As shown, MD3 eradicates Pseudomonas aeruginosa biofilms at similar concentrations under aerobic and anaerobic conditions. Tobramycin was used as a comparative example, which is only effective against aerobic biofilms. The potent and broad-spectrum antibacterial activity of MD3 indicates that the compound can effectively treat Pseudomonas aeruginosa infections.

[0214] The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended to be illustrations of several aspects of the claims, and any compositions and methods that are functionally equivalent are within the scope of the present disclosure. In addition to the compositions and methods shown and described herein, various modifications to the methods are intended to fall within the scope of the appended claims. Further, although only certain representative compositions, methods, and various aspects of these compositions and methods are specifically described, other compounds and methods are also intended to fall within the scope of the appended claims. Therefore, the combination of steps, elements, components, or compositions may be explicitly mentioned herein, however, all other combinations of steps, elements, components, and compositions are included, even if not explicitly stated.

Claims

1. A pharmaceutical composition comprising: a first component comprising a nitric oxide (NO) releasing compound; and a second component, the second component comprising a second therapeutic agent, wherein the second therapeutic agent comprises an antibiotic, an antifungal agent, or a combination thereof, wherein the ratio of the first component to the second component is such that the in vitro fractional inhibition concentration index (FICI) of the combination of the first component and the second component is 1.0 or less.

2. The pharmaceutical composition of claim 1, wherein the concentration of the second therapeutic agent in the second component is lower than the concentration of the second therapeutic agent required to exhibit an antimicrobial effect against a microorganism alone.

3. The pharmaceutical composition of claim 2, wherein the concentration of the second therapeutic agent in the second component is at least 10% lower than the concentration of the second therapeutic agent required to exhibit an antimicrobial effect against a microorganism alone.

4. The pharmaceutical composition of claim 2 or 3, wherein the concentration of the second therapeutic agent in the second component is at least 20% lower than the concentration of the second therapeutic agent required to exhibit an antimicrobial effect against a microorganism alone.

5. The pharmaceutical composition according to any one of claims 2 to 4, wherein the concentration of the second therapeutic agent in the second component is at least 30% lower than the concentration of the second therapeutic agent required to exhibit an antimicrobial effect against a microorganism alone.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the NO-releasing compound in the first component is present in an amount effective to sensitize or re-sensitize a microorganism to the second therapeutic agent in the second component.

7. according to the pharmaceutical composition described in any one in claim 1 to 6, the compound of wherein said release nitric oxide (NO) comprises the compound with at least two diazonium cation dialkolate ion groups (diazeniumdiolate group) being positioned at a carbon atom, described at least two diazonium cation dialkolate ion groups have electric charge separately and have relevant pharmaceutically acceptable cation separately to balance the described electric charge on described diazonium cation dialkolate ion group, the molecular weight of described compound is lower than 500g / mol, does not comprise described relevant pharmaceutically acceptable cation.

8. The pharmaceutical composition according to claim 7, wherein the compound has the following structure: in R is hydrogen, deuterium, C 1-12 Alkyl, aryl, heteroaryl, alkaryl, aralkyl or carbonyl, wherein the substituents are independently selected from the group consisting of: -OH, -NH2, -OCH3, -C(O)OH, -CH2OH, -CH2OCH3, -CH2OCH2CH2OH, -OCH2C(O)OH, - CH2OCH2C(O)OH、-CH2C(O)OH、-NHC(O)-CH3、-C(O)O((CH2) a O) b -H、- C(O)O((CH2) a O) b -(CH2) c H、-C(O)O(C 1-5 alkyl), -C(O)-NH-((CH2) d NH) e -H、-C(O)- NH-((CH2) d NH) e -(CH2) f H, -O-((CH2) a O) b -H, -O-((CH2) a O) b -(CH2) c H, -O-(C 1-5 alkane ), -NH-((CH2) d NH) e -H and -NH-((CH2) d NH) e -(CH2) f H; a, b, c, d, e and f are each independently selected from the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and M + is a pharmaceutically acceptable cation, wherein the ratio of the compound to the cation is such that the overall net charge of the compound is neutral.

9. The pharmaceutical composition of claim 8, wherein the cation is selected from the group consisting of sodium ions, potassium ions, lithium ions, calcium ions, magnesium ions, ammonium ions, and substituted ammonium ions.

10. The pharmaceutical composition according to claim 8 or 9, wherein the compound has the following structure:

11. A pharmaceutical composition according to any one of claims 8 to 10, wherein the compound has the following structure:

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the second therapeutic agent in the second component comprises an antibiotic.

13. The pharmaceutical composition according to claim 12, wherein the antibiotic is selected from the group consisting of aminoglycosides, monolactams, cephalosporins, quinolones, macrolides, polymyxins and carbapenems.

14. The pharmaceutical composition of any one of claims 1 to 11, wherein the second therapeutic agent in the second component comprises an antifungal agent.

15. The pharmaceutical composition according to claim 14, wherein the antifungal agent is selected from the group consisting of polyenes, azoles, allylamines and echinocandins.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the in vitro FICI is 0.5 or lower.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the in vitro FICI is 0.3 or lower.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein the molar equivalent concentration ratio of the NO-releasing compound to the second therapeutic agent is 0.1:1 to 10:

1.

19. The pharmaceutical composition of claim 18, wherein the molar equivalent concentration ratio of the NO-releasing compound to the second therapeutic agent is 0.5:1 to 2:

1.

20. The pharmaceutical composition according to any one of claims 1 to 19, further comprising one or more additives.

21. The pharmaceutical composition of claim 20, wherein the one or more additives comprise one or more preservatives, salts, chelating agents, viscosity modifiers, stabilizers, surfactants, antioxidants, buffers, or co-solvents.

22. A multi-component kit comprising: a first component comprising a nitric oxide (NO) releasing compound; and a second component, the second component comprising a second therapeutic agent, wherein the second therapeutic agent comprises an antibiotic, an antifungal agent, or a combination thereof, wherein the ratio of the first component to the second component is such that the in vitro fractional inhibition concentration index (FICI) of the combination of the first component and the second component is 1.0 or less.

23. The kit of parts according to claim 22, wherein the concentration of the second therapeutic agent in the second component is lower than the concentration of the second therapeutic agent required to exhibit an antimicrobial effect against a microorganism alone.

24. The kit of parts according to claim 23, wherein the concentration of the second therapeutic agent in the second component is at least 10% lower than the concentration of the second therapeutic agent required to exhibit an antimicrobial effect against a microorganism alone.

25. The kit of parts according to claim 23 or 24, wherein the concentration of the second therapeutic agent in the second component is at least 20% lower than the concentration of the second therapeutic agent required to exhibit an antimicrobial effect against a microorganism alone.

26. The kit of parts according to any one of claims 23 to 25, wherein the concentration of the second therapeutic agent in the second component is at least 30% lower than the concentration of the second therapeutic agent required to exhibit an antimicrobial effect against a microorganism alone.

27. The kit of parts according to any one of claims 22 to 26, wherein the NO-releasing compound in the first component is present in an amount effective to sensitize or re-sensitize a microorganism to the second therapeutic agent in the second component.

28. A kit of parts according to any one of claims 22 to 27, wherein the first component and the second component are present as a single combined composition.

29. The kit of parts according to any one of claims 22 to 27, wherein the first component and the second component are present as separate compositions.

30. The kit of parts according to claim 29, wherein the first component is suitable for nebulization and the second component is suitable for intravenous administration.

31. A multi-component kit according to any one of claims 22 to 30, wherein the compound that releases nitric oxide (NO) comprises a compound having at least two diazonium diolate groups located on one carbon atom, each of the at least two diazonium diolate groups having a charge and each having an associated pharmaceutically acceptable cation to balance the charge on the diazonium diolate group, the molecular weight of the compound being less than 500 g / mol, excluding the associated pharmaceutically acceptable cation.

32. The multi-component kit according to claim 31, wherein the compound has the following structure: in R is hydrogen, deuterium, C 1-12 Alkyl, aryl, heteroaryl, alkaryl, aralkyl or carbonyl, wherein the substituents are independently selected from the group consisting of: -OH, -NH2, -OCH3, -C(O)OH, -CH2OH, -CH2OCH3, -CH2OCH2CH2OH, -OCH2C(O)OH, - CH2OCH2C(O)OH、-CH2C(O)OH、-NHC(O)-CH3、-C(O)O((CH2) a O) b -H、- C(O)O((CH2) a O) b -(CH2) c H、-C(O)O(C 1-5 alkyl), -C(O)-NH-((CH2) d NH) e -H、-C(O)- NH-((CH2) d NH) e -(CH2) f H, -O-((CH2) a O) b -H, -O-((CH2) a O) b -(CH2) c H, -O-(C 1-5 alkane ), -NH-((CH2) d NH) e -H and -NH-((CH2) d NH) e -(CH2) f H; a, b, c, d, e and f are each independently selected from the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and M + is a pharmaceutically acceptable cation, wherein the ratio of the compound to the cation is such that the overall net charge of the compound is neutral.

33. The kit of parts according to claim 32, wherein the cation is selected from the group consisting of sodium ions, potassium ions, lithium ions, calcium ions, magnesium ions, ammonium ions, and substituted ammonium ions.

34. A multi-component kit according to claim 32 or 33, wherein the compound has the following structure:

35. A multi-component kit according to any one of claims 32 to 34, wherein the compound has the following structure:

36. The kit of parts according to any one of claims 22 to 35, wherein the second therapeutic agent in the second part comprises an antibiotic.

37. The kit of parts according to claim 36, wherein the antibiotic is selected from the group consisting of aminoglycosides, monolactams, cephalosporins, quinolones, macrolides, polymyxins and carbapenems.

38. The kit of parts according to any one of claims 22 to 35, wherein the second therapeutic agent in the second component comprises an antifungal agent.

39. The kit of parts according to claim 38, wherein the antifungal agent is selected from the group consisting of polyenes, azoles, allylamines and echinocandins.

40. The kit of parts according to any one of claims 22 to 39, wherein the in vitro FICI is 0.5 or less.

41. The kit of parts according to any one of claims 22 to 40, wherein the in vitro FICI is 0.3 or less.

42. The kit of parts according to any one of claims 22 to 41, wherein the molar equivalent concentration ratio of the NO-releasing compound to the second therapeutic agent is from 0.1:1 to 10:

1.

43. The kit of parts according to claim 42, wherein the molar equivalent concentration ratio of the NO-releasing compound to the second therapeutic agent is 0.5:1 to 2:

1.

44. The kit of parts according to any one of claims 22 to 43, wherein one or both of the first component and the second component further comprises one or more additives.

45. The multi-component kit of claim 44, wherein the one or more additives comprise one or more preservatives, salts, chelating agents, viscosity modifiers, stabilizers, surfactants, antioxidants or co-solvents.

46. ​​A method of treating a microbial infection in a subject, the method comprising administering to the subject a multi-component pharmaceutical composition or kit comprising: a first component comprising a nitric oxide (NO) releasing compound; and a second component, the second component comprising a second therapeutic agent, wherein the second therapeutic agent comprises an antibiotic, an antifungal agent, or a combination thereof, wherein the ratio of the first component to the second component is such that the in vitro fractional inhibition concentration index (FICI) of the combination of the first component and the second component is 1.0 or less.

47. The method of claim 46, wherein the first component and the second component are each independently administered to the subject orally, parenterally, intravenously, by inhalation, intraperitoneally, intracranialy, intraspinal, intrathecally, intracerebroventricularly, intramuscularly, subcutaneously, sublingually, buccally, intracavitary or transdermally.

48. The method of claim 46 or 47, wherein the first component and the second component are administered using the same mode of administration.

49. The method of claim 46 or 47, wherein the first component and the second component are administered using different modes of administration.

50. The method of claim 49, wherein the first component is administered by nebulizer and the second component is administered intravenously.

51. The method of any one of claims 46 to 50, wherein the first component and the second component are administered to the subject simultaneously.

52. The method of claim 51, wherein the first component and the second component are present in a single combined composition and the single combined composition is administered to the subject.

53. The method of claim 51, wherein the first component and the second component are administered to the subject as separate compositions.

54. The method of claim 53, wherein the first component and the second component are administered to the subject simultaneously as separate compositions.

55. The method of claim 54, wherein the first component and the second component are administered to the subject using the same mode of administration.

56. The method of claim 54, wherein the first component and the second component are administered to the subject using different modes of administration.

57. The method of claim 56, wherein the first component is administered by nebulizer and the second component is administered intravenously.

58. The method of any one of claims 46 to 50, wherein the first component and the second component are administered sequentially to the subject as separate compositions.

59. The method of claim 58, wherein the first component and the second component are administered to the subject using the same mode of administration.

60. The method of claim 58, wherein the first component and the second component are administered to the subject using different modes of administration.

61. The method of claim 60, wherein the first component is administered by nebulizer and the second component is administered intravenously.

62. The method of any one of claims 58 to 61, wherein the first component comprising the NO-releasing compound is administered prior to administration of the second component comprising the second therapeutic agent.

63. The method of any one of claims 46 to 62, wherein the microbial infection is a bacterial infection.

64. The method of claim 63, wherein the bacterial infection is caused by Gram-positive bacteria, Gram-negative bacteria, or atypical bacteria.

65. The method of claim 64, wherein the bacterial infection is caused by a Gram-positive bacterial species selected from the group consisting of: Actinomyces species; Bacillus species; Clostridium species; Corynebacterium species; Enterococcus species; Leuconostoc species; Micrococcus species; Nocardia species; Propionibacterium species; Staphylococcus species; and Streptococcus species.

66. The method of claim 64, wherein the bacterial infection is caused by a Gram-negative bacterial species selected from the group consisting of: Acinetobacter species; Aeromonas species; Alcaligenes / Achromobacter species; Bacteroides species; Bartonella species; Bordetella species; Borrelia species; Brevundimonas species; Brucella species; Burkholderia species; Campylobacter species; Citrobacter species; Coxiella species; Ehrlichia species; Enterobacter species; Escherichia species; Francisella species; species); Haemophilus species; Helicobacter species; Klebsiella species; Leclercia species; Legionella species; Leptospira species; Listeria species; Moraxella species; Morganella species; Neisseria species; Orientia species; Pantoea species; Paracoccus species; Prevotellas species; Proteus species; Providencia species; Pseudomonas species; Ralstonia species; Rickettsia species;Roseomonas species; Salmonella species; Serratia species; Shigella species; Sphingomonas species; Stenotrophomonas species; Treponema species; Ureaplasma species; Vibrio species; and Yersinia species.

67. The method of claim 66, wherein the Gram-negative bacterial species comprises Pseudomonas aeruginosa.

68. The method of claim 64, wherein the bacterial infection is caused by an atypical bacterial species selected from the group consisting of: Mycobacteria species; Chlamydia / Chlamidophila species; and Mycoplasma species.

69. The method of any one of claims 46 to 68, wherein the bacterial infection is caused by antibiotic-resistant bacteria.

70. The method of any one of claims 46 to 69, wherein the method is performed under aerobic conditions.

71. The method of any one of claims 46 to 69, wherein the method is performed under anaerobic conditions.

72. The method of any one of claims 46 to 69, wherein the method is performed under microaerobic conditions.

73. The method of any one of claims 46 to 72, wherein the concentration of the second therapeutic agent administered to the subject is lower than the concentration of the second therapeutic agent required to exhibit an antimicrobial effect against microorganisms in the subject alone.

74. The method of any one of claims 46 to 73, wherein the concentration of the second therapeutic agent in the second component administered to the subject is at least 10% lower than the concentration of the second therapeutic agent required alone to exhibit an antimicrobial effect against microorganisms in the subject.

75. The method of any one of claims 46 to 74, wherein the concentration of the second therapeutic agent in the second component administered to the subject is at least 20% lower than the concentration of the second therapeutic agent required to exhibit an antimicrobial effect against microorganisms in the subject alone.

76. The method of any one of claims 46 to 75, wherein the concentration of the second therapeutic agent in the second component administered to the subject is at least 30% lower than the concentration of the second therapeutic agent required to exhibit an antimicrobial effect against microorganisms in the subject alone.

77. The method of any one of claims 46 to 76, wherein the NO-releasing compound in the first component sensitizes or re-sensitizes a microorganism to the second therapeutic agent.

78. A method according to any one of claims 46 to 77, wherein the nitric oxide (NO) releasing compound comprises a compound having at least two diazonium diolate groups located on one carbon atom, each of the at least two diazonium diolate groups having a charge and each having an associated pharmaceutically acceptable cation to balance the charge on the diazonium diolate group, the molecular weight of the compound being less than 500 g / mol, excluding the associated pharmaceutically acceptable cation.

79. The method of claim 78, wherein the compound has the structure: in R is hydrogen, deuterium, C 1-12 Alkyl, aryl, heteroaryl, alkaryl, aralkyl or carbonyl, wherein the substituents are independently selected from the group consisting of: -OH, -NH2, -OCH3, -C(O)OH, -CH2OH, -CH2OCH3, -CH2OCH2CH2OH, -OCH2C(O)OH, - CH2OCH2C(O)OH、-CH2C(O)OH、-NHC(O)-CH3、-C(O)O((CH2) a O) b -H、- C(O)O((CH2) a O) b -(CH2) c H、-C(O)O(C 1-5 alkyl), -C(O)-NH-((CH2) d NH) e -H、-C(O)- NH-((CH2) d NH) e -(CH2) f H, -O-((CH2) a O) b -H, -O-((CH2) a O) b -(CH2) c H, -O-(C 1-5 alkane ), -NH-((CH2) d NH) e -H and -NH-((CH2) d NH) e -(CH2) f H; a, b, c, d, e and f are each independently selected from the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and M + is a pharmaceutically acceptable cation, wherein the ratio of the compound to the cation is such that the overall net charge of the compound is neutral.

80. The method of claim 79, wherein the cation is selected from the group consisting of sodium ions, potassium ions, lithium ions, calcium ions, magnesium ions, ammonium ions, and substituted ammonium ions.

81. The method of claim 79 or 80, wherein the compound has the structure:

82. The method of any one of claims 79 to 81, wherein the compound has the structure:

83. The method of any one of claims 46 to 82, wherein the second therapeutic agent in the second component comprises an antibiotic.

84. The method of claim 83, wherein the antibiotic is selected from the group consisting of aminoglycosides, monolactams, cephalosporins, quinolones, macrolides, polymyxins, and carbapenems.

85. The method of any one of claims 46 to 82, wherein the second therapeutic agent in the second component comprises an antifungal agent.

86. The method of claim 85, wherein the antifungal agent is selected from the group consisting of polyenes, azoles, allylamines, and echinocandins.

87. The method of any one of claims 46 to 86, wherein the molar equivalent concentration ratio of the NO-releasing compound to the second therapeutic agent is from 0.1:1 to 10:

1.

88. The method of claim 87, wherein the molar equivalent concentration ratio of the NO-releasing compound to the second therapeutic agent is 0.5:1 to 2:

1.

89. A method of preventing, reducing or eliminating biofilm formation caused by bacteria, the method comprising contacting the bacteria with a multi-component kit comprising: a first component comprising a nitric oxide (NO) releasing compound; and a second component, the second component comprising a second therapeutic agent, wherein the second therapeutic agent comprises an antibiotic, an antifungal agent, or a combination thereof, wherein the ratio of the first component to the second component is such that the in vitro fractional inhibition concentration index (FICI) of the combination of the first component and the second component is 1.0 or less.

90. A method of treating a surface to prevent, reduce or eliminate biofilm formation caused by bacteria, the method comprising contacting the surface with a multi-component kit comprising: a first component comprising a nitric oxide (NO) releasing compound; and a second component, the second component comprising a second therapeutic agent, wherein the second therapeutic agent comprises an antibiotic, an antifungal agent, or a combination thereof, wherein the ratio of the first component to the second component is such that the in vitro fractional inhibition concentration index (FICI) of the combination of the first component and the second component is 1.0 or less.

Images