Multi-component pharmaceutical compositions and kits containing nitric oxide-releasing compounds, and methods of using the same

Abstract

This specification provides 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 results in a fractional inhibitory concentration index (FICI) of 1.0 or less for the combination of the first and second components. The first and second components can be formulated in a single combined composition or maintained as separate compositions. Also described herein are methods of treating microbial infections and methods of preventing, reducing, or removing biofilm formation caused by bacteria.

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Application No. 63 / 350,132, filed on June 8, 2022, the entire contents of which are incorporated herein by reference.

[0002] Field The present disclosure relates to multi - component pharmaceutical compositions and kits comprising two or more components such as nitric oxide - releasing compounds and antibacterial agents (e.g., antibiotics or antifungal agents). The present disclosure also relates to methods of using the contents of the multi - component pharmaceutical compositions and kits in the treatment of microbial infections and the inhibition of bacterial biofilm formation.

Background Art

[0003] Background Patients suffering from certain infectious diseases (e.g., chronic lung infections) are often prescribed multiple antibiotics simultaneously. Combinations of antibiotics are generally described as antagonistic (where the compounds inhibit each other), irrelevant (where one compound alone has the same effect as the combination), additive (where both compounds work equally well alone or in combination, so the combination is better than a single compound), or synergistic (where the compounds work better together than alone, so the effect is greater than the sum of the parts). Antagonistic combinations can have harmful effects on the patient and may, in some cases, cause morbidity in the patient. Therefore, it is important to evaluate whether antibiotics change each other's effectiveness.

Summary of the Invention

[0004] Summary Provided herein are multi-component pharmaceutical compositions and kits comprising at least two components that can act non-antagonistically to achieve a desired therapeutic effect (e.g., antibacterial effect). The multi-component pharmaceutical compositions and kits can comprise a first component comprising a nitric oxide (NO) releasing compound and a second component comprising a second therapeutic agent, where the second therapeutic agent comprises an antibiotic, an antifungal, or a combination thereof. In these pharmaceutical compositions and kits, the ratio of the first component to the second component results in a fractional inhibitory concentration index (FICI) of the combination of the first and second components of 1.0 or less (e.g., 0.5 or less or 0.3 or less). Details for calculating the FICI of the compositions are described in the Examples section below.

[0005] 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 alone (i.e., in the absence of the NO releasing compound) to exhibit an antibacterial effect against the microorganism. In some cases, the concentration of the second therapeutic agent in the second component is at least 10% lower, at least 20% lower, or at least 30% lower than the concentration of the second therapeutic agent required alone to exhibit an antibacterial effect against the microorganism. In some cases, the NO releasing compound in the first component is present in an amount effective to sensitize or resensitize the microorganism to the second therapeutic agent in the second component. Optionally, the first and second components are present in the pharmaceutical composition or kit as a single combined composition. Optionally, the first and second components are present in the pharmaceutical composition or kit as separate compositions. In some cases, the first component is suitable for nebulization and the second component is suitable for intravenous administration.

[0006] The NO releasing compound in the first component comprises at least two diazeniumdiolate groups present on one carbon atom, where each of the at least two diazeniumdiolate groups has a charge and each has an associated pharmaceutically acceptable cation to balance the charge on the diazeniumdiolate group, and the compound has a molecular weight of less than 500 g / mol excluding the associated pharmaceutically acceptable cation. Optionally, the compound has the following structure: [Chemical] In the formula, R is hydrogen, deuterium, C 1~12 alkyl, aryl, heteroaryl, alkylaryl, arylalkyl, or carbonyl, optionally substituted with one or more substituents, and the substituents are -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 alkyl), -NH-((CH2) d NH) e -H, and -NH-((CH2) d NH) e -(CH2) f H, independently selected from the group consisting of, 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, M+ is a pharmaceutically acceptable cation, and the ratio of the compound to the cation is such that the overall net charge of the compound is neutral.

[0007] In some examples, the cation is selected from the group consisting of sodium, potassium, lithium, calcium, magnesium, ammonium, and substituted ammonium. Optionally, the compound has the following structure: [Chem.]

[0008] 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 can be selected from the group consisting of aminoglycosides, monobactams, 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 can be selected from the group consisting of polyenes, azoles, allylamines, and echinocandins.

[0009] Optionally, the molar equivalent concentration ratio of the NO-releasing compound to the second therapeutic agent is from 0.1:1 to 10:1 (e.g., from 0.5:1 to 2:1). The multi-component pharmaceutical composition or kit and its individual components described herein can further comprise one or more additives (e.g., one or more preservatives, salts, chelating agents, viscosity modifiers, stabilizers, surfactants, antioxidants, buffers, or co-solvents, etc.).

[0010] Also described herein is a method of treating a microbial infection in a subject, the method comprising administering to the subject the components of the multi-component pharmaceutical composition or kit described herein. The method can comprise administering a first component comprising a nitric oxide (NO)-releasing compound described herein and administering a second component comprising a second therapeutic agent to the subject, 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 results in a fractional inhibitory concentration index (FICI) of the combination of the first and second components of 1.0 or less (e.g., 0.5 or less or 0.3 or less) in vitro.

[0011] Optionally, the first component comprising an NO-releasing compound and the second component comprising a second therapeutic agent are each independently administered to the subject orally, parenterally, intravenously, by inhalation, intraperitoneally, intracranially, intraspinally, intrathecally, intraventricularly, intramuscularly, subcutaneously, sublingually, buccally, intravaginally or transdermally. In some cases, the NO-releasing compound and the second therapeutic agent are administered using the same mode of administration. In other cases, the NO-releasing compound and the second therapeutic agent are administered using different modes of administration (e.g., the first component can be administered via a nebulizer and the second component can be administered intravenously). Optionally, the NO-releasing compound and the second therapeutic agent are administered simultaneously. In some cases, the first component comprising an NO-releasing compound and the second component comprising a second therapeutic agent are present in a single combined composition, and the single combined composition is administered to the subject. In other cases, the first component comprising an NO-releasing compound and the second component comprising a second therapeutic agent are maintained as separate compositions and are administered to the subject simultaneously (using the same or different modes of administration) or sequentially (using the same or different modes of administration). When administered sequentially, in some cases, the first component comprising an NO-releasing compound is administered prior to the second component comprising the second therapeutic agent.

[0012] The microbial infection can be a bacterial infection. The bacterial infection can be caused by Gram-positive bacteria, Gram-negative bacteria, or atypical bacteria. Optionally, the bacterial infection is caused by a Gram-positive bacterial species selected from the group consisting of Actinomyces spp., Bacillus spp., Clostridium spp., Corynebacterium spp., Enterococcus spp., Leuconostoc spp., Micrococcus spp., Nocardia spp., Propionibacterium spp., Staphylococcus spp., and Streptococcus spp.

[0013] 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, Haemophilus species, Helicobacter species, Klebsiella species, Raoultella species, Legionella species, Leptospira species, Listeria species, Moraxella species, Morganella species, Neisseria species, Orientia species, Pantoea species, Paracoccus species, Prevotella 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 includes Pseudomonas aeruginosa.

[0014] In some cases, the bacterial infection is caused by an atypical bacterial species selected from the group consisting of Mycobacterium species, Chlamydia / Chlamydophila species, and Mycoplasma species. Optionally, the bacterial infection is caused by an antibiotic-resistant bacterium.

[0015] Optionally, this method is carried out under aerobic conditions, anaerobic conditions, or microaerophilic conditions. In this 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 alone to exhibit an antibacterial effect against the microorganisms in the subject. In some cases, the concentration of the second therapeutic agent in the second component administered to the subject is at least 10% lower, at least 20% lower, or at least 30% lower than the concentration of the second therapeutic agent required alone to exhibit an antibacterial effect against the microorganisms in the subject. In some cases, the NO-releasing compound in the first component sensitizes or resensitizes the microorganisms to the second therapeutic agent.

[0016] Furthermore, provided herein is a method for preventing, reducing, or eliminating biofilm formation caused by bacteria, the method comprising contacting the bacteria with the contents of the multi-component pharmaceutical composition or kit described herein. The method can comprise administering a first component comprising a nitric oxide (NO) releasing compound described herein and a second component comprising a second therapeutic agent, where the second therapeutic agent comprises an antibiotic, an antifungal, or a combination thereof. In this method, the ratio of the first component to the second component results in an in vitro fractional inhibitory concentration index (FICI) of the combination of the first and second components of 1.0 or less. Further provided 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 the multi-component pharmaceutical composition or kit described herein. The method can comprise administering a first component comprising a nitric oxide (NO) releasing compound described herein and a second component comprising a second therapeutic agent, where the second therapeutic agent comprises an antibiotic, an antifungal, or a combination thereof. In this method, the ratio of the first component to the second component results in an in vitro fractional inhibitory concentration index (FICI) of the combination of the first and second components of 1.0 or less.

[0017] The details of one or more embodiments are set forth in the accompanying drawings and description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

Figure 1A

Figure 1B

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0019] This specification describes multi-component pharmaceutical compositions and multi-component kits containing two or more components. The first component in the pharmaceutical composition or multi-component kit contains a nitric oxide (NO)-releasing compound. The second component in the pharmaceutical composition or multi-component kit contains an antibacterial agent such as an antibiotic or an antifungal agent. This specification also describes methods of using the components of the pharmaceutical composition and / or multi-component kit in the treatment of microbial infections and the inhibition of bacterial biofilm formation.

[0020] In particular, the pharmaceutical composition and multi-component kit can be designed and adjusted with respect to the administration method so as to obtain the maximum therapeutic effect. As an example, as further described herein, the composition and multi-component kit composed of two or more components can be prepared by combining the components into a single composition before administration or storing them separately and administering them to the subject as separate compositions. When combined, the combined composition is administered by a single effective administration mode, as further described herein. When the components are separate (i.e., when the components are administered as separate compositions), the compositions can be administered simultaneously by the same administration mode or different administration modes, or can be administered sequentially using the same administration mode or different administration modes. Such administration methods are further described herein. The pharmaceutical compositions and kits described herein can have a multifaceted impact on the subject in a single treatment plan, so the flexibility of the administration method makes them more desirable in the treatment and prevention of microbial infections and the inhibition of bacterial biofilm formation. The components of the pharmaceutical composition and kit can be adapted to the desired type of delivery. In some cases, the first component is suitable for spraying and the second component is suitable for intravenous administration.

[0021] The pharmaceutical compositions and kits described herein can exhibit an enhanced and unexpected antibacterial effect due to the influence of the NO-releasing compound in the first component of the pharmaceutical composition and / or kit. First, since the NO-releasing compound itself releases nitric oxide, it contributes as an active pharmaceutical ingredient to the composition. The NO-releasing compound acts in conjunction with a second therapeutic agent, such as an antibacterial agent, to enhance the therapeutic effect of the treatment. In some cases, the NO-releasing compound and the second therapeutic agent can act additively, such that delivering a combination of the two agents is more effective than delivering one agent alone. In some cases, the NO-releasing compound and the second therapeutic agent can act synergistically, such that delivering a combination of the two agents has a greater effect than the sum of its parts. In other cases, the NO-releasing compound and the second therapeutic agent may act independently, such that only one compound is as effective as the combination.

[0022] In addition to the combined effects (in some cases, additive, synergistic, or independent) of these agents, the NO-releasing compound can cause an additional effect of enhancing the impact that the second therapeutic agent has on the microorganism. For example, NO released from the NO-releasing compound can damage the bacterial resistance mechanism, thereby increasing antibacterial susceptibility and decreasing resistance over time. Such a combined effect, namely the increase in antibacterial susceptibility and the imparting of an antibacterial effect, results in a supra-additive effect (i.e., a synergistic effect) in the treatment of bacterial infections. Furthermore, NO released from the NO-releasing compound can restore the breakpoint susceptibility of the microorganism, such that it can be effective even against organisms that are multi-drug resistant and insensitive to antibiotics. Such a combined effect is a synergistic effect.

[0023] The released NO also directly affects the antimicrobial biofilm. Biofilm formation causes serious complications across a wide range of problems, not limited to the treatment of bacterial infections. For example, Pseudomonas aeruginosa and nontuberculous mycobacteria (among other pathogen hosts) have the ability to form biofilms in various locations, including the lungs of CF patients and on the surfaces of medical devices. Such biofilms are difficult to control or destroy once formed and are one of the main causes of nosocomial infections. Many antibiotics are ineffective in treating infections caused by bacteria growing within biofilms or require more drugs to treat biofilm-related bacteria compared to planktonic bacteria. Surprisingly, NO from the NO-releasing compound in the composition enables the co-administered (simultaneously or sequentially) antibiotic to be effective against the bacteria. In some cases, NO loosens the biofilm matrix, affects the redox state of the biofilm, activates stationary cells, and promotes active metabolism.

[0024] In particular, using a certain amount of NO can reduce the amount of the second therapeutic agent administered, yet still have the same effect as the second therapeutic agent (e.g., an antibacterial agent) administered alone. Since NO is effective against the microbial mechanism, the amount of antibacterial agent required to produce an antibacterial effect is at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, or at least 40% less than the amount of antibacterial agent administered alone, yet still has an equivalent or greater effect. Such multi-component kits showing these enhanced antibacterial effects are further described herein.

[0025] I. Multi-Component Pharmaceutical Compositions and Kits The multi-component pharmaceutical composition or multi-component kit described herein comprises at least two compositions that act non-antagonistically to achieve a desired therapeutic effect (e.g., an antibacterial effect). In some examples, the components within the multi-component pharmaceutical composition or kit act synergistically with respect to conferring an antibacterial effect. In some examples, the components within the multi-component pharmaceutical composition or kit act additively with respect to conferring an antibacterial effect. In other examples, the components within the multi-component pharmaceutical composition or kit act independently with respect to conferring an antibacterial effect.

[0026] The multi-component pharmaceutical composition or kit described herein comprises a first component that includes a nitric oxide (NO)-releasing compound, as will be described in further detail below. The first component can exhibit antibacterial properties, including antiviral, antibacterial, and antifungal properties. The first component described herein can further have anti-inflammatory properties and other beneficial therapeutic properties. The multi-component pharmaceutical composition or kit described herein further comprises a second component that includes a second therapeutic agent. The second therapeutic agent can be an antibacterial agent such as an antibiotic, an antifungal agent, or a combination thereof. The components of the pharmaceutical composition and kit are further described below.

[0027] The components of the multi-component pharmaceutical composition or kit can be packaged and administered in a variety of ways and manners. Thus, the components of the multi-component pharmaceutical composition or kit described herein can be adjusted to achieve the maximum antibacterial effect that provides a benefit to the subject. By way of example, the first and second components of the multi-component pharmaceutical composition or kit can be prepared such that the two compositions are included within a single combined composition. The single combined composition can then be administered to the subject via a desired mode of administration (e.g., oral, parenteral, intravenous, inhalation, intraperitoneal, intracranial, intraspinal, intrathecal, intraventricular, intramuscular, subcutaneous, sublingual, buccal, intracavernous, or transdermal).

[0028] In other examples, the first and second components of a multi-component pharmaceutical composition or kit can be prepared such that the two compositions are maintained as separate components (i.e., not otherwise mixed prior to administration to a subject). In some cases, the first and second components are contained in separate containers and may be exposed to different storage conditions prior to administration, depending on the requirements of the components of the individual compositions and as determined by one of ordinary skill in the art based on the disclosure herein. Optionally, the first and second components can be administered to a subject separately and simultaneously. Such administration can be via the same mode of administration or via different modes of administration. For example, the first component can be administered to the subject by inhalation and the second component can be administered to the subject orally. In other examples, the first component can be administered via a nebulizer and the second component can be administered intravenously. Optionally, the first and second components can be administered to the subject continuously. Such continuous administration can be via the same mode of administration (i.e., the first and second components are continuously administered but the same mode of administration is used) or via different modes of administration (i.e., the first and second components are continuously administered and different methods of administration are used). In some examples, the first component is administered prior to the second component. In other examples, the second component is administered prior to the first component.

[0029] a. Nitric oxide donor component The pharmaceutical active ingredient delivered via 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 nitric oxide-releasing prodrug, or a compound required for the promotion of the endogenous production of nitric oxide). As described herein, the first component in the form of a compound that releases NO can exhibit antibacterial properties including antiviral, antibacterial, and antifungal properties. The first component described herein can further have anti-inflammatory and other beneficial therapeutic properties.

[0030] The release of NO can be initiated thermally or via any of the decomposition strategies for N-diazinium dioates, nitrosamines, hydroxyl nitrosamines, nitrosothiols, hydroxylamines, hydroxyureas, metal complexes, labile moieties of organic nitrites, and organic nitrates. See Wang, P.G., et al., Nitric Oxide Donors. For Pharmaceutical and Biological Applications; Wliey-VCH. Weinheim, Germany, 2005; and Wang. P.G., et al., Chem. Rev., 102, 1091-1134 (2002). In some embodiments, the NO donor is an N-diazinium dioate (i.e., 1-amino-substituted diazen-1-ium-1,2-dioate). N-diazinium dioates are particularly attractive as NO donors because of their ability to spontaneously generate NO under biological conditions. See Hrabie, J.A. and Keefer, L.K. Chem. Rev., 102, 1135-1154 (2002); and Napoli, C. and Ianarro, L.J., Annu. Rev. Pharmacol. Toxicol., 43, 97-123 (2003). Several N-diazinium dioate compounds have been synthesized using a series of nucleophilic residues including primary and secondary amines, polyamines, and secondary amino acids. See Hrabie, J.A. and Keefer L.K. Chem. 102, 1135-1154 (2002). In the formation of N-diazinium dioalate, 1 equivalent of an amine reacts with 2 equivalents of nitrogen monoxide under high pressure. A base (e.g., an alkoxide such as methoxide) removes a proton from the amine nitrogen to generate an anionic and stable N(O)NO group. N-diazinium dioates are stable under ambient conditions but decompose spontaneously in an aqueous medium to generate NO at a rate depending on the pH, temperature, and / or the structure of the amine moiety.

[0031] In some cases, the first component can include at least one nitric oxide-releasing compound having at least two diazeniumdiolate groups on one carbon atom, and the two diazeniumdiolate groups each have a charge and each have an associated pharmaceutically acceptable cation to balance the charge on the diazeniumdiolate group, and the compound has a molecular weight of less than 500 g / mol excluding the associated pharmaceutically acceptable cation.

[0032] As described above, the compounds described herein can include at least one nitric oxide-releasing functional group. Various NO donors (e.g., diazeniumdiolates, S-nitrosothiols, metal nitrosyls, organic nitrates) are known to provide controlled exogenous NO release, but the diazeniumdiolate functional group (NONOate) in the compounds disclosed herein is particularly attractive due to its good stability and ease of storage, and also because it undergoes spontaneous proton-induced dissociation under physiological conditions to regenerate nitric oxide. Certain compounds include two diazeniumdiolate groups present on one carbon atom, and the two diazeniumdiolate groups each have a charge and each have a pharmaceutically acceptable cation to balance the charge on the diazeniumdiolate group. These compounds are small molecules (with a molecular weight of 500 g / mol or less excluding the cation as further described below) that release nitric oxide (NO) and exhibit antiviral, antibacterial, antifungal (including antibacterial properties), anti-inflammatory, and other beneficial therapeutic properties.

[0033] The compound optionally has the following structure represented by Formula I:

Chemical formula

[0034] In Formula I, R is hydrogen, deuterium, C 1~12It is alkyl, aryl, heteroaryl, alkylaryl, arylalkyl, or carbonyl. Optionally, R is substituted with one or more substituents, and the substituents are -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 alkyl), -NH-((CH2) d NH) e -H, and -NH-((CH2) d NH) e -(CH2) f H, and are independently selected from the group consisting of, and a, b, c, d, e, and f are independently selected from the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively.

[0035] Furthermore, in Formula I, M+ is a cation. For example, M+ can be a pharmaceutically acceptable cation. Optionally, the cation is selected from the group consisting of sodium, potassium, lithium, calcium, magnesium, and quaternary ammonium salts (e.g., ammonium or ammonium substituents).

[0036] In these compositions, the ratio of the compound to the cation is such that the overall net charge of the compound is neutral. When M+ is a cation other than monovalent, the ratio of the compound to the cation is such that the total positive charge equals the total negative charge. As an example, if the total charge of the compound is -3 and the total charge of the cation is +1, there will be three cations present for one compound.

[0037] For example, the compound can be represented by Structure I-A shown below:

Chemical Formula

[0038] As shown in Structure I-A above, the total charge of this compound is -3. Therefore, three cations (i.e., 3M+) are present to balance the charge of the compound (i.e., the total positive charge equals the total negative charge).

[0039] Examples of Structure I-A include the following compounds:

Chemical Formula

[0040] The compound can have a molecular weight of less than 500 g / mol, excluding the relevant cation (e.g., the relevant pharmaceutically acceptable cation). For example, the compound can have a molecular weight of 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, the molecular weight of the compound excluding the relevant cation can be from 100 g / mol to less than 500 g / mol, from 120 g / mol to 450 g / mol, from 150 g / mol to 400 g / mol, or from 175 g / mol to 350 g / mol.

[0041] Additional details regarding the mechanism of action of the compounds described herein, including nitric oxide delivery characteristics and advantageous properties of the compounds (e.g., storage stability), are described in 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". These documents are hereby incorporated by reference in their entireties.

[0042] As used herein, the terms alkyl, alkenyl, and alkynyl include straight-chain and branched-chain monovalent substituents. Examples include methyl, ethyl, isobutyl, 3-butynyl, and the like. The scope 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 scopes of these groups useful in the compounds and methods described herein include C1-C 12 alkyl, C2-C 12 alkenyl, C2-C 12 alkynyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C4 alkyl, C2-C4 alkenyl, and C2-C4 alkynyl.

[0043] Heteroalkyl, heteroalkenyl, and heteroalkynyl are defined in the same manner as alkyl, alkenyl, and alkynyl, but can contain heteroatoms of O, S, or N, or combinations thereof, within the backbone. The scope of these groups useful in the compounds and methods described herein includes C1-C 20 heteroalkyl, C2-C 20 heteroalkenyl, and C2-C 20 heteroalkynyl. Additional scopes of these groups useful in the compounds and methods described herein include C1-C 12 heteroalkyl, C2-C 12 heteroalkenyl, C2-C 12 heteroalkynyl, C1-C6 heteroalkyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, C1-C4 heteroalkyl, C2-C4 heteroalkenyl, and C2-C4 heteroalkynyl.

[0044] The terms cycloalkyl, cycloalkenyl, and cycloalkynyl include cyclic alkyl groups having a single cyclic ring or multiple fused rings. Examples include cyclohexyl, cyclopentylethyl, and adamantanyl. The scope of these groups useful in the compounds and methods described herein includes C3-C 20 cycloalkyl, C3-C 20 cycloalkenyl, and C3-C 20 cycloalkynyl. Additional scopes of these groups useful 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.

[0045] The terms heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl are defined in the same manner as cycloalkyl, cycloalkenyl, and cycloalkynyl, but can include heteroatoms of O, S, or N, or combinations thereof, within the cyclic backbone. The scope 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 scope of these groups useful in the compounds and methods described herein includes C5-C 12 heterocycloalkyl, C5-C 12 heterocycloalkenyl, C5-C 12 heterocycloalkynyl, C5-C6 heterocycloalkyl, C5-C6 heterocycloalkenyl, and C5-C6 heterocycloalkynyl.

[0046] Aryl molecules include cyclic hydrocarbons incorporating one or more planar sets of usually six carbon atoms connected by the same number of delocalized electrons, such that they are, for example, composed of alternating single and double bonds arranged in a row. An example of an aryl molecule is benzene. Heteroaryl molecules include substitution along the main ring chain of atoms such as O, N, or S. When a heteroatom is introduced, an aromatic system can be formed by a set of five atoms, for example, four carbons and one heteroatom. Examples of heteroaryl molecules include furan, pyrrole, thiophene, imidazole, oxazole, pyridine, and pyrazine. Aryl and heteroaryl molecules can also include additional fused rings such as benzofuran, indole, benzothiophene, naphthalene, anthracene, and quinoline. Aryl and heteroaryl molecules can be attached at any position on the ring, unless otherwise specified.

[0047] As used herein, the term "alkoxy" is an alkyl group bonded through a single terminal ether linkage. As used herein, the term "aryloxy" is an aryl group bonded through a single terminal ether linkage. Similarly, as used herein, the terms "alkenyloxy", "alkynyloxy", "heteroalkyloxy", "heteroalkenyloxy", "heteroalkynyloxy", "heteroaryloxy", "cycloalkyloxy", and "heterocycloalkyloxy" are alkenyloxy, alkynyloxy, heteroalkyloxy, heteroalkenyloxy, heteroalkynyloxy, heteroaryloxy, cycloalkyloxy, and heterocycloalkyloxy groups, respectively, bonded through a single terminal ether linkage.

[0048] As used herein, the term "hydroxy" is represented by the formula -OH.

[0049] As used herein, the term "amine" or "amino" is represented by the formula -NZ 1 Z 2 wherein Z 1 and Z 2 are each independently a substituent described herein such as hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group.

[0050] The 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" means adding an alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl group at a position bonded to the main chain of alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl, for example, including substitution of hydrogen by one of these molecules. Examples of substituents include, but are not limited to, hydroxy, halogen (e.g., F, Br, Cl, or I), and carboxyl groups. Conversely, as used herein, the term "unsubstituted" means that alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl has hydrogen, i.e., has a complete complement of hydrogen corresponding to its saturation level and has no substitution, for example, linear decane (-(CH2)9-CH3).

[0051] The C-diazeniumdiolate described herein is a pH-responsive NO-releasing donor (also referred to herein as an NO-releasing compound or NO-releasing agent). When reacted with a proton under physiological conditions (e.g., 37 °C, pH 7.4), 2 moles of NO and 2 - 3 moles of nitroxyl compounds are produced from 1 mole of Compound 1 (MD3).

[0052] In some embodiments, the NO releasing compound is stable at various temperatures from freezing to room temperature of 25 °C (e.g., -20 °C, 0 °C, 5 °C, 20 °C, etc.) and is stable over long storage periods (e.g., 10 hours, 20 hours, 22 hours, 25 hours, 30 hours, etc., number of days such as 1 day, 3 days, 5 days, 6 days, 7 days, 15 days, 30 days, 45 days, etc., number of weeks such as 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, etc., number of months such as 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, etc., and even several years (1 year, 2 years, or more)).

[0053] In some cases, the compound has a total releasable NO storage amount such that the amount of NO per 1 mg of the compound ranges from 0.1 μmol to 23.0 μmol (for example, 0.1 μmol to 15 μmol per 1 mg of the compound, 0.5 μmol to 7.5 μmol per 1 mg of the compound, 1 μmol to 7.0 μmol per 1 mg of the compound, 1.5 μmol to 6.5 μmol per 1 mg of the compound, 2.0 μmol to 6.0 μmol per 1 mg of the compound, 2.5 μmol to 5.5 μmol per 1 mg of the compound, or 3.0 μmol to 5.0 μmol per 1 mg of the compound). For example, the total releasable NO storage amount of the compound used in the composition is 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, 2.2 μmol, 2.3 μmol, 2.4 μmol, 2.5 μmol, 2.6 μmol, 2.7 μmol, 2.8 μmol, 2.9 μmol, 3.0 μmol, 3.1 μmol, 3.2 μmol, 3.3 μmol, 3.4 μmol, 3.5 μmol, 3.6 μmol, 3.7 μmol, 3.8 μmol, 3.9 μmol, 4.0 μmol, 4.1 μmol, 4.2 μmol, 4.3 μmol, 4.4 μmol, 4.5 μmol, 4.6 μmol, 4.7 μmol, 4.8 μmol, 4.9 μmol, 5.0 μmol, 5.1 μmol, 5.2 μmol, 5.3 μmol, 5.4 μmol, 5.5 μmol, 5.6 μmol, 5.7 μmol, 5.8 μmol, 5.9 μmol, 6.0 μmol, 6.1 μmol, 6.2 μmol, 6.3 μmol, 6.4 μmol, 6.5 μmol, 6.6 μmol, 6.7 μmol, 6.8 μmol, 6.9 μmol, 7.0 μmol, 7.1 μmol, 7.2 μmol, 7.3 μmol, 7.4 μmol, 7.5 μmol, 7.6 μmol, 7.7 μmol, 7.8 μmol, 7.9 μmol, 8.0 μmol, 8.1 μmol, 8.2 μmol, 8.3 μmol, 8.4 μmol, 8.5 μmol, 8.6 μmol, 8.7 μmol, 8.8 μmol, 8.9 μmol, 9.0 μmol, 9.1 μmol, 9.2 μmol, 9.3 μmol, 9.4 μmol, 9.5 μmol, 9.6 μmol, 9.7 μmol, 9.8 μmol, 9.9 μmol, 10.0 μmol, 10.1 μmol, 10.2 μmol, 10.3 μmol, 10.4 μmol, 10.5 μmol, 10.6 μmol, 10.7 μmol, 10.8 μmol, 10.9 μmol, 11.0 μmol, 11.1 μmol, 11.2 μmol, 11.3 μmol, 11.4 μmol, 11.5 μmol, 11.6 μmol, 11.7 μmol, 11.8 μmol, 11.9 μmol, 12.0 μmol, 12.1 μmol, 12.2 μmol, 12.3 μmol, 12.4 μmol, 12.5 μmol, 12.6 μmol, 12.7 μmol, 12.8 μmol, 12.9 μmol, 13.0 μmol, 13.1 μmol, 13.2 μmol, 13.3 μmol, 13.4 μmol, 13.5 μmol, 13.6 μmol, 13.7 μmol, 13.8 μmol, 13.9 μmol, 14.0 μmol, 14.1 μmol, 14.2 μmol, 14.3 μmol, 14.4 μmol, 14.5 μmol, 14.6 μmol, 14.7 μmol, 14.8 μmol, 14.9 μmol, 15.0 μmol, 15.1 μmol, 15.2 μmol, 15.3 μmol, 15.4 μmol, 15.5 μmol, 15.6 μmol, 15.7 μmol, 15.8 μmol, 15.9 μmol, 16.0 μmol, 16.1 μmol, 16.2 μmol, 16.3 μmol, 16.4 μmol, 16.5 μmol, 16.6 μmol, 16.7 μmol, 16.8 μmol, 16.9 μmol, 17.0 μmol, 17.1 μmol, 17.2 μmol, 17.3 μmol, 17.4 μmol, 17.5 μmol, 17.6 μmol, 17.7 μmol, 17.8 μmol, 17.9 μmol, 18.0 μmol, 18.1 μmol, 18.2 μmol, 18.3 μmol, 18.4 μmol, 18.5 μmol, 18.6 μmol, 18.7 μmol, 18.8 μmol, 18.9 μmol, 19.0 μmol, 19.1 μmol, 19.2 μmol, 19.3 μmol, 19.4 μmol, 19.5 μmol, 19.6 μmol, 19.7 μmol, 19.8 μmol, 19.9 μmol, 20.0 μmol, 20.1 μmol, 20.It can be 2 μmol, 20.3 μmol, 20.4 μmol, 20.5 μmol, 20.6 μmol, 20.7 μmol, 20.8 μmol, 20.9 μmol, 21.0 μmol, 21.1 μmol, 21.2 μmol, 21.3 μmol, 21.4 μmol, 21.5 μmol, 21.6 μmol, 21.7 μmol, 21.8 μmol, 21.9 μmol, 22.0 μmol, 22.1 μmol, 22.2 μmol, 22.3 μmol, 22.4 μmol, 22.5 μmol, 22.6 μmol, 22.7 μmol, 22.8 μmol, 22.9 μmol, or 23.0 μmol.

[0054] The compound can have a total NO release duration ranging from 0.1 to 60 hours after the start of activation. In some cases, the NO release can occur over about 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, 36 hours, 48 hours, or 60 hours. In some embodiments, within 2 hours after being added to phosphate buffered saline (PBS) buffer, the compound releases about 25%, 50%, 75%, 85%, 90%, 95%, 100% or more, or a range including and / or spanning the aforementioned values, i.e., the total weight percentage of bound NO. Optionally, the compound has a total NO release amount such that the amount of NO per 1 mg of the compound 4 hours after the start of NO release (also referred to as "activation") is 0.1 to 8.0 μmol.

[0055] In some embodiments, when using chemiluminescence-based nitric oxide detection, the compound has a release rate such that the release rate per hour is about 0.2%, 0.5%, 1.0%, 1.5%, 2.5%, 5.0%, 10% or less, or a range including and / or spanning the aforementioned values.

[0056] Optionally, the compounds used in the compositions described herein have an NO release half-life in the range of 0.01 to 24 hours. In some embodiments, the NO release half-life is equal to, or at least on the order of, 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 aforementioned values. In some embodiments, NO release occurs in the range of 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 less, or a range including and / or spanning the aforementioned values.

[0057] Optionally, the first component of the multi-component pharmaceutical composition is, or can include, the composition described in PCT / US2023 / 019807 entitled "Buffering Agent-Containing Compositions and Methods of Using Same", which is hereby incorporated by reference in its entirety.

[0058] b. The second therapeutic component As described above, the second component described herein includes a second therapeutic agent. The second therapeutic agent can, in some cases, be an antibiotic. Suitable antibiotics can include any antibiotic effective in the treatment of bacterial infections and / or the inhibition or disruption of biofilms, such as, 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), glycylcyclines (e.g., tigecycline), beta-lactams (e.g., penams), lipopeptides (e.g., daptomycin), oxazolidinones (e.g., linezolid), trimethoprim, and monobactams.

[0059] For example, the second component described in this specification includes antibiotics such as acetadapsone, sodium acetosulfonate, alamethicin, alexidine, amdinocillin, amdinocillin pivoxyl, amicycline, amifloxacin, amifloxacin mesylate, amikacin, amikacin sulfate, aminosalicylic acid, sodium aminosalicylic acid, amoxicillin, amphomycin, ampicillin, sodium ampicillin, sodium apramycin, aplasmomycin, aspartocin, astromicin sulfate, avilamycin, avoparcin, azithromycin, azocin, sodium azocin, aztreonam, bacampicillin hydrochloride, bacitracin, bacitracin methylene disalicylate, zinc bacitracin, bambermycin, benzoyl pascalcium, verithromycin, betamethycin sulfate, biapenem, vinylamycin, bifenamine hydrochloride, bispyrithione magnesium sulfate, butikacin, butirosin sulfate, capreomycin sulfate, carbadox, carbenicillin disodium, carbenicillin indanyl sodium, carbenicillin phenyl sodium, carbenicillin potassium, carmonam sodium, cefaclor, cefadroxil, cefamandole, cefamandole nafate, sodium cefamandole, cefaparole, cefatriaxone, sodium cefazafuril, cefazolin, sodium cefazolin, cefbuperazone, cefdinir, cefepime, cefepime hydrochloride, cefetamet, cefixime, cefmenoxime hydrochloride, cefmetazole, sodium cefmetazole, cefonicid monosodium, cefonicid sodium, sodium cefoperazone, ceforanide, cefotaxime sodium, cefotetan, cefotetan disodium, cefotiam hydrochloride, cefoxitin, sodium cefoxitin, cefpimizole, sodium cefpimizole, cefpiramide, sodium cefpiramide, cefpirome sulfate, cefpodoxime proxetil, cefprozil, cefroxadine, sodium cefurosime, ceftazidime, cefibutene, cefixime sodium, ceftriaxone sodium, cefuroxime, cefuroxime axetil, cefuroxime pivoxetil, sodium cefuroxime, cefacetrile sodium, cephalexin, cephalexin hydrochloride,Cefaloglycin, cefaloridine, cefalotin sodium, cefapirin sodium, cephradine, cetocycline hydrochloride, cetophenicol, chloramphenicol, chloramphenicol palmitate, chloramphenicol pantothenate complex, chloramphenicol sodium succinate, chlorhexidine phosphanilate, chloroxylenol, chlortetracycline bisulfate, chlortetracycline hydrochloride, cinoxacin, ciprofloxacin, ciprofloxacin hydrochloride, siromycin, clarithromycin, clinafloxacin hydrochloride, clindamycin, clindamycin hydrochloride, clindamycin hydrochloride palmitate, clindamycin phosphate, clofazimine, cloxacillin benzathine, cloxacillin sodium, cloxyquine, colistimethate sodium, colistin, colistin sulfate, kumermycin, kumermycin sodium, cyclacillin, cycloserine, dalfopristin, dapsone, daptomycin, demeclocycline, demeclocycline hydrochloride, demesacycline, denofungin, diaberidine, dicloxacillin, dicloxacillin sodium, dihydrostreptomycin sulfate, dipyrithione, dirithromycin, doxycycline, doxycycline calcium, doxycycline phosphatex, doxycycline hydrate, droxacycline sodium, enoxacin, episilin, epitetracycline hydrochloride, erythromycin, erythromycin acetate, erythromycin ethylsuccinate, erythromycin ethylsuccinate ester, glucosepotassium erythromycin, erythromycin lactobionate, erythromycin propionate, erythromycin stearate, ethambutol hydrochloride, ethionamide, fleroxacin, floxacin, fludarabine, flumequine, fosfomycin, fosfomycin trometamol, fumoxycillin, furazolidone chloride, furazolidone tartrate, fusidic acid sodium, fusidic acid, gentamicin sulfate, glomoxanam, gramicidin, haloprogin, hetacillin, hetacillin potassium, hexedine, ibafloxacin, imipenem, isoconazole, isepamicin, isoniazid, josamycin, kanamycin sulfate, kitasamycin, levoflutadone, levopropylcillin potassium, lexicomycin,Lincomycin, Lincomycin Hydrochloride, Lomefloxacin, Lomefloxacin Hydrochloride, Lomefloxacin Mesylate, Loracarbef, Mafenide, Meclocycline, Meclocycline Sulfosalicylate, Megalomycin Potassium Phosphate, Methacycline, Methacycline Hydrochloride, Methenamine, Methenamine Hippurate, Methenamine Mandelate, Methicillin Sodium, Metioprim, Metronidazole Hydrochloride, Metronidazole Phosphate, Mezlocillin, Mezlocillin Sodium, Minocycline, Minocycline Hydrochloride, Milinkamycin Hydrochloride, Monensin, Monensin Sodium, Nafcillin Sodium, Nalidixic Acid Sodium, Nalidixic Acid, Natamycin, Nebramycin, Neomycin Palmitate, Neomycin Sulfate, Neomycin Undecylenate, Netilmicin Sulfate, Neutramycin, Nifradene, Nifraldazone, Nifrate, Nitratone, Niflumadil, Nifrimide, Nifopirinol, Niflumizole, Nifurthiazole, Nitrosocycline, Nitrofurantoin, Nitromide, Norfloxacin, Novobiocin Sodium, Ofloxacin, Ornetoprim, Oxacillin, Oxacillin Sodium, Oxymonam, Oxymonam Sodium, Oxolinic Acid, Oxytetracycline, Oxytetracycline Calcium, Oxytetracycline Hydrochloride, Pardimicin, Parachlorophenol, Paulomycin, Perfloxacin, Perfloxacin Mesylate, Penamesillin, Penicillin G Benzathine, Penicillin G Potassium, Penicillin G Procaine, Penicillin G Sodium, Penicillin V, Penicillin V Benzathine, Penicillin V Hydrabamine, Penicillin V Potassium, Pentidone Sodium, Phenyl Aminosalicylate, Piperacillin Sodium, Pyrubenicillin Sodium, Pyridicillin Sodium, Pirrimycin Hydrochloride, Pivampicillin Hydrochloride, Pivampicillin Pamoate, Pivampicillin Probeneate, Polymyxin B Sulfate, Porfiromycin, Propicain, Pyrazinamide, Zinc Pyrithione, Decamine Acetate, Quinupristin, Racefennicol, Ramoplanin, Ranimicin, Relomycin, Repromycin, Rifabutin, Rifametane, Rifamexil, Rifamide, Rifampin, Rifapentine, Rifaximin,It can contain loritetracycline, loritetracycline nitrate, rosaramicin, rosaramicin butyrate, rosaramicin propionate, rosaramicin sodium phosphate, rosaramicin stearate, roxithromycin, roxarsone, roxithromycin, sancycline, sanfetrinem sodium, salmoxicillin, salpicillin, scopafungin, sisomicin, sisomicin sulfate, sparfloxacin, spectinomycin hydrochloride, spiramycin, starymicin hydrochloride, stefimycin, streptomycin sulfate, streptonicid, sulfabenz, sulfabenzamide, sulfacetamide, sulfacetamide sodium, sulfachitin, sulfadiazine, sulfadiazine sodium, sulfadoxine, sulfalen, sulfamerazine, sulfamethazine, sulfamethizole, sulfamethoxazole, sulfamonomethoxine, sulfamoxole, zinc sulfanilate, sulfanitran, sulfasalazine, sulfisoxazole, sulfisoxazole acetyl, sulfisoxazole diolamine, sulfomixine, slopenem, sultamicillin, sansirine sodium, talampicillin hydrochloride, teicoplanin, temafloxacin hydrochloride, temocillin, tetracycline, tetracycline hydrochloride, tetracycline phosphate complex, tetroxoprim, thiamphenicol, tifensillin potassium, ticarcillin cloxacillin sodium, ticarcillin disodium, ticarcillin monosodium, ticlopenton, thiodonium chloride, tobramycin, tobramycin sulfate, tosufloxacin, trimethoprim, trimethoprim sulfate, trisulfapyrimidine, troandromycin, trospectomycin sulfate, tyrothricin, vancomycin, vancomycin hydrochloride, virginiamycin, or zolbamycin.

[0060] In some cases, the second therapeutic agent may be an antifungal agent. Optionally, the antifungal agent can be selected from the group consisting of polyenes, azoles, allylamines, and echinocandins. Exemplary antifungal agents include, for example, amphotericin B, nystatin, flucytosine, natamycin, ketoconazole, econazole, miconazole, itraconazole, fluconazole, clotrimazole, griseofulvin, oxiconazole, terconazole, tioconazole, clotrimazole, silver sulfadiazine, ciclopirox olamine, and terbinafine.

[0061] Optionally, the second component of the multi-component pharmaceutical composition can be or can include the composition described in PCT / US2023 / 019807 entitled "Buffering Agent-Containing Compositions and Methods of Using Same", which is hereby incorporated by reference in its entirety.

[0062] c. Optional additives The multi-component pharmaceutical compositions and kits described herein, and / or one or more individual components / compositions within the multi-component pharmaceutical compositions and kits, can further include one or more additives. The one or more additives can include, for example, one or more preservatives, salts, chelating agents, stabilizers, surfactants, antioxidants (e.g., N-acetylcysteine or glutathione), buffering agents and / or co-solvents. The multi-component pharmaceutical compositions and kits, and / or the individual compositions within the kits, can also include, if necessary, wetting or emulsifying agents, lubricants, flow promoters, skin softeners, moisturizers, thickeners, and / or flavoring agents.

[0063] In some cases, one or more additives can include viscosity reducing agents, natural and synthetic antibiofilm agents (e.g., chitosan), biofilm dispersants, natural and synthetic anti-quorum sensing agents (e.g., autoinducer-2 or N-acyl homoserine lactone), siderophores, iron chelating agents, iron mimetics (e.g., gallium-containing compounds such as gallium (Ga) and gallium azole (Ga-azole)), anti-persister cell agents (e.g., 4-(4,7-dimethyl-1,2,3,4-tetrahydro-naphthalen-1-yl) pentanoic acid (DMNP)), antimicrobial peptides (AMP) (e.g., LL-37 or lactoferricin), efflux pump inhibitors, and / or bacteriophage therapy.

[0064] Optionally, the additive can be present in an amount of less than 1 wt% in the multi-component pharmaceutical composition or kit. For example, the amount of the additive can 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%. Optionally, the amount of the additive can be 0.1 - 0.9 wt%, 0.2 - 0.8 wt%, or 0.3 - 0.7 wt%.

[0065] The viscosity modifier can optionally be included in the multi-component pharmaceutical compositions and kits described herein, and / or in one or more of the individual compositions within the multi-component pharmaceutical compositions and kits. Optionally, the viscosity modifier can be included in the multi-component pharmaceutical composition or kit in an amount of up to 5 wt% (e.g., 0.1 wt% - 5 wt%, 0.5 wt% - 4.5 wt%, 1.0 wt% - 4.0 wt%, 1.5 wt% - 3.5 wt%, or 2.0 wt% - 3.0 wt%). For example, the viscosity modifier can 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%.

[0066] d. Synergistic, additive, unrelated compositions As outlined above, in some embodiments, the multi-component pharmaceutical compositions or kit components described herein can be synergistic, additive, or unrelated with respect to the relationship between the first and second components. Specifically, the ratio of the first component to the second component results in a fractional inhibitory concentration index (FICI) of the combination of the first and second components of 1.0 or less (e.g., 0.5 or less, or 0.3 or less) in vitro. In some cases, an FICI value of 0.5 or less is considered synergistic, a value of 0.51 - 1 is considered additive, and a value greater than 1 and less than 4 is considered unrelated.

[0067] The nature and amounts of the first and second components of each composition can be selected such that the desired relationship (synergistic, additive, or unrelated) is achieved. For example, the molar equivalent concentration ratio of the NO-releasing compound to the second therapeutic agent can be selected such that the desired relationship is achieved. Optionally, the molar equivalent concentration ratio of the NO-releasing compound to the second therapeutic agent is from 0.1:1 to 10:1 (e.g., 0.5:1 to 2:1). In some cases, the molar equivalent concentration ratio of the NO-releasing compound 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.

[0068] The concentration of the second therapeutic agent in the second component may be lower than the concentration of the second therapeutic agent required alone to exhibit an antibacterial effect against 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 alone to exhibit an antibacterial effect against 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 alone to exhibit an antibacterial effect against 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 alone to exhibit an antibacterial effect against the microorganism. Optionally, the NO-releasing compound is present in an amount effective to sensitize or resensitize the microorganism to the second therapeutic agent.

[0069] II. Methods of Making Compounds The compounds described in this specification can be prepared in various 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 in this specification can be prepared from readily available starting materials. The optimal reaction conditions can vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art.

[0070] Variations of the compounds of Formula I and those described in this specification include the addition, deletion, or rearrangement of the various components described for each compound. Similarly, when one or more chiral centers are present within the molecule, all possible chiral variants are included. Further, the synthesis of the compounds may involve the protection and deprotection of various chemical groups. The use of protection and deprotection, as well as the selection of appropriate protecting groups, can be determined by those skilled in the art. The chemical properties of the protecting groups can be found, for example, in Wuts, Greene’s Protective Groups in Organic Synthesis, 5th. Ed., Wiley & Sons, 2014, the entire content of which is incorporated herein by reference.

[0071] The reactions for generating the compounds described in this specification can be carried out in a solvent that can be selected by those skilled 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 reaction is carried out, i.e., temperature and pressure. The reaction can be carried out in one solvent or a mixture of two or more solvents. The formation of the product or intermediate can be monitored according to any suitable method known in the art. For example, the formation of the product can be monitored by spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H-NMR or 13 C-NMR), infrared spectroscopy (IR), spectrophotometry (e.g., UV-visible light), or mass spectrometry (MS), or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).

[0072] Optionally, the compounds described herein can be prepared according to the synthetic methods described in 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", the entire contents of each of which are hereby incorporated by reference.

[0073] III. Pharmaceutical Compositions In some cases, a multi-component composition that includes 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 can include a buffering agent. A buffering agent can be included to control the pH of the composition. In some examples, the buffering agent is included to maintain the pH of the composition between 5.5 and 8.5. For example, the buffering agent can be included to maintain the pH of the composition between 6.0 and 8.0, between 6.7 and 7.5, or between 7.0 and 7.5 (e.g., 7.4).

[0074] The buffer can have a buffering strength of 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 can have a buffering strength of 0.1 molar equivalent, 0.2 molar equivalent, 0.3 molar equivalent, 0.4 molar equivalent, 0.5 molar equivalent, 0.6 molar equivalent, 0.7 molar equivalent, 0.8 molar equivalent, 0.9 molar equivalent, 1.0 molar equivalent, 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.

[0075] Generally, the buffer can be any buffer that is generally regarded as safe for use as an inert 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, phosphate buffers in the range of 0.01 to 1 M. 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 in the evaluation of the analytical properties of the composition.

[0076] Specifically, certain examples of the compounds described herein, such as MD3 (further described below), include sodium cations as counter ions. When measuring the amount of the compound in a specific formulation such as an aerosol formulation, the amount of sodium cation is calculated. In such situations, the presence of sodium in the buffer (e.g., sodium phosphate buffer) can make the calculation of the compound unclear. In other examples, the presence of potassium in the buffer can affect the proton pump when administered to humans, and as a result, can affect the pH of the epithelial lining fluid. Potassium does not increase the alkalinity of the composition, but an increase in alkalinity can prevent NO release from the nitric oxide-releasing compound, which is a beneficial property.

[0077] One or more buffers including acetate buffer, benzoate buffer, citrate buffer, lactate buffer, maleate buffer, and tartrate buffer can be included in the composition. Optionally, the one or more buffers include HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer.

[0078] In some examples, the composition substantially does not contain a carbonate buffer. In some examples, the composition substantially does not contain hydrochloric acid, sulfuric acid, or citric acid. As used herein, the term “substantially does not contain” the indicated component (e.g., carbonate buffer, hydrochloric acid, sulfuric acid, and / or citric acid) means that the pharmaceutical composition may contain 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) based on the weight of the pharmaceutical composition.

[0079] In the case of a pharmaceutical composition and in some cases where the multi-component kit composition is a pharmaceutical composition, the composition can include a pharmaceutically acceptable carrier. As used herein, the term “carrier” encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizing agent, lipid, stabilizer, or other material well known in the art used in therapeutic formulations. The selection of the carrier for use in the composition will depend on the intended mode of administration of the composition.

[0080] Suitable liquid carriers can be aqueous or non-aqueous carriers. Examples of suitable non-aqueous carriers include propylene glycol, polyethylene glycol, and oils including those of animal, vegetable, or synthetic origin such as petroleum, peanut oil, soybean oil, mineral oil, sesame oil, and olive oil. 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 physiological saline and buffer media. When the composition is a pharmaceutical composition for intravenous administration, water or an aqueous carrier is preferred. Physiological saline solutions as well as aqueous dextrose solutions and aqueous glycerol solutions can also be used as liquid carriers, particularly for injection solutions. The composition can also optionally contain wetting or emulsifying agents, lubricants, flow promoters, skin softeners, humectants, thickeners, flavoring agents, preservatives, and pH buffers. A pH buffer can be included to control the pH of the composition. In some examples, the buffer is included to maintain the pH of the composition at 5-8.5, thereby being able to determine the release rate of nitric oxide (NO). For example, the buffer can be included to maintain the pH of the composition at 5.2-8.3, 5.5-8.0, 6.0-8.0, 6.8-8.0, or 7.0-7.8 (e.g., 7.4). Examples of suitable buffers include phosphate buffers such as phosphate buffered saline (PBS), e.g., 0.01-0.1 M phosphate buffer, acetate buffer, benzoate buffer, citrate buffer, lactate buffer, maleate buffer, and tartrate buffer. Buffered carriers such as Hank's solution, Ringer's solution, dextrose solution, 5% human serum albumin, Ringer dextrose, dextrose and sodium chloride, lactated Ringer's solution and fixed oils, polyethylene glycol, polyvinylpyrrolidone, or lecithin can be used. Monoethanolamine, diethanolamine, tromethamine, and glycine solutions can also be used as suitable buffers. Non-aqueous vehicles such as liposomes and fixed oils can also be used. The formulation must be compatible with the mode of administration. Additional carriers for use in the composition are described in the section on pharmaceutical compositions herein.

[0081] Furthermore, one or more of the compounds, compositions, and formulations described herein can be combined with other agents, including for the treatment of diseases and disorders associated with the lung, digestive tract, liver, and biliary tract. For example, in the case of cystic fibrosis, the compositions and formulations described herein can be combined with mucolytics (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), agents that target nonsense mutations (e.g., PTC124). Further examples of additional agents that can be combined with the compounds described herein include additional antibiotics (e.g., aminoglycosides, antipseudomonal penicillins, and cephalosporins), additional antibacterial agents (e.g., rifabutin), ethambutol, clarithromycin, clofazimine, aztreonam, steroidal and nonsteroidal anti-inflammatory agents (e.g., ibuprofen and prednisone), pentoxifylline, dornase alfa, or ursodeoxycholic acid.

[0082] One or more of the compounds and compositions described herein can be provided in the form of an inhaler or nebulizer for inhaled therapy, with or without additional agents. As used herein, inhaled therapy refers to the delivery of therapeutic agents, such as the compounds and compositions described herein, to the airways in aerosol form (i.e., lung delivery). As used herein, the term aerosol refers to very fine liquid or solid particles that are carried to the site of therapeutic application by a pressurized propellant gas. When a pharmaceutical aerosol is used, the aerosol contains one or more of the compounds and compositions described herein that can be dissolved, suspended, or emulsified in a mixture of a fluid carrier and / or propellant. The aerosol can be in the form of a solution, suspension, emulsion, powder, or semisolid formulation. In the case of a powder, if the device is a dry powder inhaler that is actuated by respiration, a propellant gas is not required. The aerosol used is intended to be administered via the patient's airways as very fine solid particles or as a liquid mist.

[0083] The propellant of an aerosol package containing one or more of the compositions described herein is capable of generating pressure within the container to release the compound when the valve of the aerosol package is opened. Various types of propellants can be utilized, such as fluorinated hydrocarbons (e.g., trichloromonofluoromethane, dichlorodifluoromethane, and dichlorotetrafluoroethane, etc.) and compressed gases (e.g., nitrogen, carbon dioxide, nitrous oxide, or Freon). The vapor pressure of the aerosol package is determined by the propellant used. By varying the ratio of each component propellant, any desired vapor pressure within the limits of the vapor pressure of the individual propellants can be obtained.

[0084] As described above, one or more of the compositions described herein can be provided with a nebulizer, which is a device that generates very fine liquid particles of substantially uniform size in a gas. The liquid containing one or more of the compounds and / or compositions described herein can be dispersed in the form of a mist as droplets having a diameter of about 5 mm or less. The small droplets can be carried by a flow of air or oxygen through the outlet tube of the nebulizer. The resulting mist can penetrate the patient's airway.

[0085] Additional inhalers useful for the delivery of 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 hereby incorporated by reference in its entirety for at least the compositions taught therein). For example, a powder composition containing one or more of the compounds described herein can be administered to a patient with or without a lubricant, carrier, or propellant. Delivery of one or more compounds in powder form can be carried out using conventional devices for administering powder pharmaceutical compositions by inhalation.

[0086] Depending on the intended mode of administration, the pharmaceutical composition can be in a solid, semi-solid or liquid dosage form, such as, for example, tablets, suppositories, pills, capsules, powders, solutions, or suspensions, and preferably can be in a unit dosage form suitable for single administration of an exact dosage. The composition comprises a therapeutically effective amount of the compound or its derivative described herein in combination with a pharmaceutically acceptable carrier, and may further contain other agents, pharmaceuticals, carriers, or diluents. Pharmaceutically acceptable means a substance that is not biologically or otherwise undesirable, and can be administered to humans in combination with the selected compound without causing unacceptable biological effects or interacting in a harmful manner with other components of the pharmaceutical composition in which it is contained.

[0087] For the preparation of pharmaceutical preparations containing pharmaceutically acceptable carriers and these materials, see, for example, Remington: The Science and Practice of Pharmacy, edited by Adeboye Adejare, 23rd Edition, Academic Press (2021). Examples of physiologically acceptable carriers include buffers such as phosphate buffer, citrate buffer, and buffers with other organic acids, antioxidants containing 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, lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and / or nonionic surfactants such as TWEEN® (ICI, Inc., Bridgewater, NJ), polyethylene glycol (PEG), and PLURONICS® (BASF, Florham Park, NJ).

[0088] Compositions containing a compound or a derivative thereof described herein suitable for parenteral injection may 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 (such as propylene glycol, polyethylene glycol, glycerol, etc.), suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Suitable fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0089] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, dispersing agents, etc. Prevention of the activity of microorganisms can be enhanced by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. Also, isotonic agents such as sugars, sodium chloride, etc. may be included. Prolonged absorption of injectable pharmaceutical dosage forms can be brought about by the use of agents that delay absorption, such as aluminum monostearate and gelatin.

[0090] Solid dosage forms for oral administration of the compounds and compositions or derivatives thereof described herein include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds or derivatives thereof described herein are combined with at least one inert conventional excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, such as glycerol, (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) dissolution retardants, such as paraffin, (f) absorption promoters, such as quaternary ammonium compounds, (g) wetting agents, such as cetyl alcohol and glycerol 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 include buffering agents.

[0091] Solid compositions of the same type can also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycol.

[0092] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be formulated using coatings and shells such as enteric coatings and others known in the art. These can include opacifying agents and can also be formulated in compositions that delay the release of the active compound in specific parts of the intestinal tract. Examples of implantable compositions that can be used are polymeric substances and waxes. The active compound can also, if appropriate, be in microencapsulated form together with one or more of the excipients described above.

[0093] Liquid dosage forms for oral administration of the compounds and compositions or derivatives thereof described herein include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid dosage forms may include inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, especially 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.

[0094] In addition to such inert diluents, the compositions may also contain additional agents such as wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, or perfuming agents.

[0095] Suspensions may contain, in addition to the active compound, additional agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth, or mixtures of these substances.

[0096] Compositions for rectal administration of the compounds or derivatives thereof described herein can optionally be provided as suppositories by mixing the compound with a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol, or suppository wax, which is solid at room temperature but liquid at body temperature and thus melts in the rectum or vaginal cavity to release the active ingredient.

[0097] Dosage forms for topical administration of the compounds and compositions or derivatives thereof described herein include ointments, powders, sprays, inhalants, gels, pastes, creams, and lotions. The compounds or derivatives thereof described herein are mixed under sterile conditions with a physiologically acceptable carrier and optionally any preservative, buffer, or propellant. Ophthalmic formulations, ointments, powders, and solutions are also considered to be within the scope of application of the compositions.

[0098] As described above, the composition can comprise one or more of the compounds described herein or pharmaceutically acceptable salts thereof. As used herein, the term pharmaceutically acceptable salts refers to salts of the compounds described herein or derivatives thereof that are suitable for use in contact with the tissues of a subject within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, etc., are commensurate with a reasonable benefit / risk ratio, and are effective for their intended use, and zwitterionic forms of the compounds described herein if possible. The term salts refers to relatively non-toxic inorganic and organic acid addition salts of the compounds described herein. These salts can be prepared in situ during the isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt so formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, methanesulfonate, and laurylsulfonate. These can include cations based on alkali metals and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium, and non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. (See S.M. Barge et al., J. Pharm. Sci. (1977) 66, 1, the entire contents of which are incorporated herein by reference at least for the compositions taught therein).

[0099] Administration of the compounds and compositions described herein, or pharmaceutically acceptable salts thereof, can be carried out for a period of time effective to treat a disorder using a therapeutically effective amount of the compounds and compositions described herein, or pharmaceutically acceptable salts thereof. The effective amount of the compounds and compositions described herein, or pharmaceutically acceptable salts thereof described herein, can be determined by one of ordinary skill in the art and includes exemplary dosages for mammals, i.e., from about 0.01 to about 200 mg / kg body weight of the active compound per day, which can be administered in a single dose or in the form of individual divided doses such as 1 to 4 times a day. Alternatively, the dosage can be from about 0.05 to about 190 mg / kg body weight of the active compound per day, from about 0.1 to about 180 mg / kg body weight of the active compound per day, from about 0.25 to about 175 mg / kg body weight of the active compound per day, from about 0.5 to about 150 mg / kg body weight of the active compound per day, from about 0.5 to 100 mg / kg body weight of the active compound per day, from about 0.5 to about 75 mg / kg body weight of the active compound per day, from about 0.5 to about 50 mg / kg body weight of the active compound per day, from about 0.5 to about 25 mg / kg body weight of the active compound per day, from about 1 to about 20 mg / kg body weight of the active compound per day, from about 1 to about 10 mg / kg body weight of the active compound per day, about 20 mg / kg body weight of the active compound per day, about 10 mg / kg body weight of the active compound per day, or about 5 mg / kg body weight of the active compound per day. One of ordinary skill in the art will understand that the specific dosage level and frequency of administration for any particular subject can vary and depend on various factors including the activity of the specific compound used, the metabolic stability and duration of action of that compound, the species, age, weight, general health, sex and diet of the subject, the method and time of administration, the rate of excretion, drug combinations, and the severity of the particular condition.

[0100] IV. Method of Use A method for treating a microbial infection in a subject is provided herein. The method includes administering to the subject a multi-component pharmaceutical composition or a composition of a kit described herein. The method can include administering a first component comprising a nitric oxide (NO) releasing compound (and optionally having antibacterial properties, anti-inflammatory properties, and / or other beneficial therapeutic properties including antiviral properties, antibacterial properties, and antifungal properties), and administering to the subject a second component comprising a second therapeutic agent, where the second therapeutic agent includes an antibiotic, an antifungal agent, or a combination thereof.

[0101] In some cases of these methods, the concentration of the second therapeutic agent in the second component administered to the subject can be lower than the concentration of the second therapeutic agent required alone to exhibit an antibacterial effect against the microorganisms within the subject. In some cases, the concentration of the second therapeutic agent administered to the subject is at least 10% lower, at least 20% lower, or at least 30% lower than the concentration of the second therapeutic agent required alone to exhibit an antibacterial effect against the microorganisms within the subject. Furthermore, the compositions described herein are as effective as, or more effective than, the second therapeutic agent administered alone. In some cases, the NO releasing compound sensitizes or resensitizes the microorganisms to the second therapeutic agent such that the second therapeutic agent exhibits a new or greater antibacterial effect.

[0102] The NO-releasing compound and the second therapeutic agent are administered to the subject by any suitable method including, as further described herein, oral, parenteral, intravenous, inhalation, intraperitoneal, intracranial, intraspinal, intrathecal, intraventricular, intramuscular, subcutaneous, sublingual, buccal, intracavernous or transdermal. The NO-releasing compound and the second therapeutic agent can be administered using the same mode of administration or via different modes of administration. In some examples, the NO-releasing compound and the second therapeutic agent are administered simultaneously, while in other examples, the NO-releasing compound and the second therapeutic agent are administered sequentially. Optionally, the NO-releasing compound is administered before the second therapeutic agent. In some cases, the NO-releasing compound is administered within 24 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour of administering the second therapeutic agent to the subject.

[0103] Optionally, the microbial infection treated by the compositions described herein can be a bacterial infection. The bacterial infection can be caused by Gram-positive bacteria, Gram-negative bacteria, or atypical bacteria. Optionally, the bacterial infection is caused by Gram-positive bacterial species such as Actinomyces spp., Bacillus spp., Clostridium spp., Corynebacterium spp., Enterococcus spp., Leuconostoc spp., Micrococcus spp., Nocardia spp., Propionibacterium spp., Staphylococcus spp., or Streptococcus spp.

[0104] Optionally, a bacterial infection may be caused by a gram-negative bacterial species such as Acinetobacter spp., Aeromonas spp., Alcaligenes / Achromobacter spp., Bacteroides spp., Bartonella spp., Bordetella spp., Borrelia spp., Brevundimonas spp., Brucella spp., Burkholderia spp., Campylobacter spp., Citrobacter spp., Coxiella spp., Ehrlichia spp., Enterobacter spp., Escherichia spp., Francisella spp., Haemophilus spp., Helicobacter spp., Klebsiella spp., Raoultella spp., Legionella spp., Leptospira spp., Listeria spp., Moraxella spp., Morganella spp., Neisseria spp., Orientia spp., Pantoea spp., Paracoccus spp., Prevotella spp., Proteus spp., Providencia spp., Pseudomonas spp. (e.g., Pseudomonas aeruginosa), Ralstonia spp., Rickettsia spp., Roseomonas spp., Salmonella spp., Serratia spp., Shigella spp., Sphingomonas spp., Stenotrophomonas spp., Treponema spp., Ureaplasma spp., Vibrio spp., or Yersinia spp.

[0105] Optionally, a bacterial infection may be caused by an atypical bacterial species such as Mycobacterium spp., Chlamydia / Chlamydophila spp., or Mycoplasma spp. In some cases, a bacterial infection may be caused by or develop into an antibiotic-resistant bacterium such as antibiotic-resistant Burkholderia cepacia, carbapenem-resistant Enterobacteriaceae (CRE) enterobacteria, drug-resistant Campylobacter, drug-resistant nontyphoidal 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, erythromycin-resistant group A Streptococcus, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), and vancomycin-resistant Enterococcus (VRE).

[0106] Also provided herein is a method for preventing, reducing, or removing biofilm formation caused by bacteria. The method includes contacting the bacteria with an effective amount of the multi-component pharmaceutical composition or compositions of the kit described herein. The effective amount of the composition(s) can be an amount that prevents, reduces, and / or removes bacterial biofilm formation and includes that the concentration of a second therapeutic agent administered to a subject is lower than the concentration of the second therapeutic agent required alone to exhibit an antibacterial effect against the bacteria.

[0107] The compositions described herein are useful for preventing, reducing, or removing biofilm formation caused by bacteria in humans (e.g., pediatric and geriatric populations), animals (e.g., for veterinary use), and on surfaces (e.g., the surface of medical devices).

[0108] The methods described herein can further include selecting a subject infected with, or at risk of infection with, bacteria. Optionally, the method can further include selecting a subject infected with, or at risk of infection with, bacteria that can develop resistance to antibiotics. Subjects at risk of infection with the aforementioned bacteria include infants, the elderly, subjects with a weakened immune system, hospitalized subjects, subjects living in facilities (e.g., nursing homes), subjects having invasive medical devices (e.g., urinary catheters), subjects with open wounds, and subjects who have contacted other people infected with bacteria.

[0109] The methods of treatment or prevention described herein can further include treatment with one or more additional agents (e.g., a second biofilm inhibitor). As noted above, the one or more additional agents, compounds, and compositions or pharmaceutically acceptable salts thereof described herein can be administered in any order, including simultaneous administration and sequential administration with time intervals of up to several days. These methods can also include multiple administrations of the one or more additional agents, compounds, and compositions or pharmaceutically acceptable salts thereof described herein. The administration of the one or more additional agents, compounds, and compositions or pharmaceutically acceptable salts thereof described herein can be carried out in the same manner or in different manners. When treating with one or more additional agents, the compounds and compositions or pharmaceutically acceptable salts thereof described herein can be combined with a pharmaceutical composition comprising the one or more additional agents.

[0110] The methods and compounds described herein are useful for both prophylactic and therapeutic treatment. As used herein, the terms "treating" or "treatment" include prevention, delay of onset, reduction of symptoms or signs after onset, eradication, or delay of exacerbation, and prevention of recurrence. In the case of prophylactic use, a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof described herein is administered to a subject before onset (e.g., before obvious signs of bacterial biofilm formation), during early onset (e.g., when initial signs and symptoms of bacterial biofilm formation appear), or after bacterial biofilm formation has been established. Prophylactic administration can be carried out from several hours to several years before the symptoms of an infectious disease appear. Prophylactic administration can be used, for example, for the prophylactic treatment of subjects or surfaces exposed to Pseudomonas aeruginosa. Therapeutic treatment includes contacting bacteria with a therapeutically effective amount of the compositions described herein after bacterial biofilm formation has been observed.

[0111] Optionally, the contacting can be carried out under aerobic conditions (also referred to as an aerobic environment). As used herein, the term aerobic conditions refers to conditions characterized by the presence of free oxygen (O2). Optionally, the contacting can be carried out under anaerobic conditions (also referred to as an anaerobic environment). As used herein, the term anaerobic conditions refers to conditions lacking free oxygen (O2). Optionally, the contacting can be carried out under microaerobic conditions (also referred to as a microaerobic environment). As used herein, the term microaerobic conditions refers to conditions having a low level of free oxygen (O2), being below normal atmospheric oxygen levels and being between aerobic and anaerobic conditions.

[0112] Also provided herein is a method of treating a surface to prevent, reduce, or remove biofilm formation caused by bacteria. The method of treating a surface to prevent, reduce, or remove bacterial biofilm formation includes contacting the surface with an effective amount of the multi-component pharmaceutical composition or compositions of the kit described herein. The effective amount of the composition can be an amount that prevents, reduces, and / or removes bacterial biofilm formation on the surface and includes a concentration of a second therapeutic agent in a second component that is lower than the concentration of the second therapeutic agent required alone to exhibit an antibacterial effect against the bacteria. Optionally, the surface is a surface of the human body such as a mucosal surface. Optionally, the mucosal surface is a mucosal surface of the lung or upper respiratory tract.

[0113] V. Kits for the Treatment of Microbial Infections and the Prevention, Reduction, and Removal of Biofilm Formation Also provided herein are kits for treating microbial infections in a subject, kits for preventing, reducing, or removing biofilm formation caused by bacteria, and kits for treating or pretreating a surface to prevent, reduce, or remove biofilm formation caused by bacteria. The kits can include any of the compositions described herein. For example, the kit can include a NO releasing agent (e.g., a compound of Formula I) and a second therapeutic agent. Optionally, the kit can further include a carrier (e.g., a pharmaceutically acceptable carrier).

[0114] The kit can include a delivery means by inhalation (e.g., an inhaler or nebulizer) or an oral formulation of any of the compositions described herein. The kit can further include instructions for use of the kit (e.g., instructions for treating a subject or contacting a surface), one or more containers (for the compound(s), composition(s), or second biofilm inhibitor(s)), means for administering the compound or composition, and / or a carrier.

[0115] Optionally, the multi-component kit can include one or more containers. The kit can include a first container containing a first component that includes a nitric oxide-releasing compound. Optionally, the kit can include a second container containing a second therapeutic agent. Optionally, the kit can include a third container for combining the components of the first and second containers for optional use when the first and second components are administered as a single combined composition.

[0116] As used herein, "treatment", "treat", or "treating" refers to a method of reducing one or more signs of a disease or condition. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of one or more signs of a disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms or signs of the disease in a subject as compared to a control. As used herein, a control refers to an untreated state. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% as compared to the natural or control level. It is understood that treatment does not necessarily refer to a cure or complete disappearance of the disease, condition, or signs of the disease or condition.

[0117] As used herein, the terms "prevent", "preventing", or "prevention" of a disease or disorder refer to an act, such as administration of a composition or therapeutic agent, that suppresses or delays the onset or severity of one or more symptoms of the disease or disorder, which occurs before or approximately contemporaneously with the subject beginning to exhibit one or more symptoms of the disease or disorder.

[0118] As used herein, references to a decrease, reduction, or inhibition include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control level. The meaning of such terms may include, but does not necessarily include, complete elimination.

[0119] As used herein, the term "subject" means both mammals and non-mammals. Mammals include, for example, humans, non-human primates such as apes and monkeys, cows, horses, sheep, rats, mice, pigs, and goats. Non-mammals include, for example, fish and birds.

[0120] Throughout this application, various publications are referenced. The disclosures of these publications are hereby incorporated by reference in their entirety into this application.

Examples

[0121] The following examples are described below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to encompass all aspects of the subject matter disclosed herein, but rather 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 will be apparent to those skilled in the art.

[0122] Example 1: Efficacy of an NO-Releasing Compound in Combination with Other Antibiotics for Treating Pseudomonas aeruginosa Lung Infection Patients suffering from certain infectious diseases such as chronic pulmonary infections are often prescribed multiple antibiotics simultaneously. Therefore, it is important to evaluate whether NO-releasing agents change the effectiveness of commonly used antibiotics and whether commonly used antibiotics affect the effectiveness of NO-releasing agents. In this study, MD3 is used as a representative NO-releasing agent.

[0123] To quantify the combined effect of MD3 and secondary antibiotics, a checkerboard assay was used, which is a two-dimensional two-drug serial dilution method for quantifying the effects of each drug alone and in combination. In patients with underlying lung diseases such as bronchiectasis, a large amount of mucus in the lungs mainly causes microaerophilic (low oxygen) or anaerobic (no oxygen) bacterial infections. Therefore, combinations of MD3 and six secondary antibiotics were evaluated against the representative microorganism Pseudomonas aeruginosa under both aerobic and anaerobic conditions.

[0124] The antibiotics tested in this study were selected to represent the diverse mechanisms of action of the antibiotics used in the treatment of pulmonary Pseudomonas aeruginosa infections shown in Table 1.

Table 1

[0125] Methods Species and Strains The strains used in these studies and their sources are shown in Table 2.

Table 2

[0126] Checkerboard Assay The checkerboard method was performed as follows. MD3 and the second antibiotic were tested at 4 times (4X) their minimum inhibitory concentration (MIC) alone (see Table 3). Stock solutions of MD3 and secondary antibiotics were prepared at a final concentration of 4X (i.e., 16X the MIC, called a 4X stock).

[0127] MD3 and the secondary antibiotic were tested alone at 4X their minimum inhibitory concentration (MIC) alone. For example, the MIC MD3 against the aerobically grown Pseudomonas aeruginosa PAK strain TOB was 31.25 μg / ml, and the MIC against TOB

[0128] was 3.125 μg / ml, and thus the highest concentrations tested in the checkerboard assay were 125 μg / ml for MD3 and 12.5 μg / ml for TOB. MD3 and the antibiotic were tested over a 7-point range of 2-fold concentrations as detailed in Table 3. 6 5 6 Bacterial cultures were prepared by streaking from frozen glycerol stocks stored at -80 °C onto agar plates (see Table 4). After suspending several well-separated colonies in 1X PBS, they were diluted to 1 × 10

[0129] To prepare the checkerboard assay plate, phosphate-buffered saline (PBS) was added to wells B1 - H8 of a 96-well plate. Next, 200 μl of 4X second antibiotic stock was added to the empty wells A1 - A8 of the 96-well plate, and then serial dilutions were made 1:1 in a downward direction of the plate using a multi-channel pipette and stopped at row G. The last 100 μl in row G was discarded. Row H did not contain the secondary antibiotic. This measures the MIC of MD3 only. At this step, all wells contain the target concentration of 4X second antibiotic. Next, 100 μl of 4X MD3 stock solution was added to wells A1 - H1, and serial dilutions were made 1:1 from across the plate up to column 7 using a multi-channel pipette. The last 100 μl in column 7 was discarded. Column 8 did not contain any MD3. This measures the MIC of the secondary antibiotic only. After this step, all wells contain 2X of the target concentration of both MD3 and the second antibiotic. The wells in column 9 contain media only and function as a media control. The wells in column 10 contain media and bacteria and function as a growth control. Finally, 100 μl of a bacterial culture at 5×10 5 CFU / ml was added to each well. The checkerboard assay plate was incubated at 37 °C until growth was observed in the growth control wells (see Table 4).

[0130] In the experiment to grow Pseudomonas aeruginosa under anaerobic conditions, 90 mM KNO3 was added to CAMHB medium, and the checkerboard assay plate was incubated in an anaerobic box containing an anaerobic sachet. Anaerobicity was confirmed by co-incubated anaerobic test strips.

Table 4

[0131] Interpretation of Checkerboard Assay Results The effect of the antibiotic combination is classified according to the fractional inhibitory concentration index (FICI) as described by Chou and Talay (1984) using Equation 1, where MIC A and MICB are the values determined for drugs A and B when tested separately, MIC AB and MIC BA are the concentrations of drugs A and B in the most effective combination in the checkerboard assay.

Number

[0132] The FICI was calculated for each well along the growth / no-growth boundary of the checkerboard assay plate. If the FICI of any well was greater than 4, antagonism was reported; otherwise, the lowest FICI value was reported. An FICI value ≤ 0.5 is considered synergistic, a value of 0.51 - 1 is additive, and a value greater than 1 and less than 4 is irrelevant. The rationale for this analysis method is that if any of the ratios of antibiotics has a synergistic effect, there may be a synergistic effect in the combination even if the other ratios are not synergistic. The classification of synergistic and antagonistic effects was reported only when observed in at least two biological replicates. If the experiment was repeated twice and the same classification was observed (e.g., synergistic effect observed in both replicates), the average FICI for the drug combination was reported. If the results of the two replicates did not match in the classification, a third experiment had to be performed and the average FICI from the three replicate experiments used for the final classification.

[0133] Validity of the assay The checkerboard assay is considered valid if all of the following statements are true: - The starting inoculum is confirmed to be between 1×10 5 ~9.0×10 6 CFU / ml, - The starting inoculum produces pure single colonies on agar, - Growth is observed in the growth control wells within a time suitable for the species, as described in the "Bacterial Cultures" section, - At least 7 out of 8 growth control wells are turbid, - At least 7 out of 8 medium control wells were clear, - The MIC for each individual agent was within ±2 wells of the previously established MIC of that agent against that strain.

[0134] Results The results of checkerboard assays of MD3 in combination with secondary antibiotics tested against Pseudomonas aeruginosa are shown in Figures 1A and 1B. Under aerobic conditions, MD3 showed synergistic effects with aztreonam, ciprofloxacin, colistin, ceftazidime, and gallium citrate, was additive with tobramycin and meropenem, and was unrelated to gentamicin (Figure 1A). Under anaerobic conditions, MD3 was additive with all 6 antibiotics tested, tobramycin, aztreonam, ciprofloxacin, colistin, ceftazidime, and meropenem. Importantly, no antagonistic effects were observed in any of the MD3 / antibiotic combinations tested, either under aerobic or anaerobic conditions. See Table 5.

Table 5

[0135] The results of checkerboard assays of MD3 in combination with secondary antibiotics tested against NTM species are shown in Figure 2. MD3 was additive with amikacin when used against Mycobacterium abscessus strains N0010 and N0046, Mycobacterium chimaera, and Mycobacterium massiliense. MD3 and clarithromycin were additive when used against Mycobacterium chimaera and Mycobacterium massiliense. MD3 and gallium citrate were additive when used against Mycobacterium abscessus strains N0010 and N0046. Importantly, no antagonistic effects were observed in any of the combinations tested. See Table 6.

Table 6

[0136] Finally, the results of the checkerboard assay of MD3 in combination with secondary antibiotics tested against Staphylococcus aureus are shown in Figure 3. When used against Staphylococcus aureus strain N0040, MD3 was independent of tobramycin and had a synergistic effect with gallium citrate. Also, no antagonistic effect was observed for any of the combinations tested. See Table 7.

Table 7

[0137] In summary, the efficacy of MD3 in combination with other antibiotics commonly used in the treatment of lung infections was evaluated to assess potential drug-drug interactions. The studies were mainly conducted on Pseudomonas aeruginosa, the most common and most harmful pathogen for patients with underlying lung diseases such as bronchiectasis in cystic fibrosis. In the studies using Pseudomonas aeruginosa, antibiotics across six classes were evaluated under both aerobic and anaerobic conditions. The MD3 / antibiotic combinations did not show antagonistic effects, suggesting that MD3 is a good candidate for combination therapy. Indeed, synergistic effects were observed when MD3 was combined with aztreonam, ciprofloxacin, colistin, ceftazidime, and gallium citrate, and additive effects were observed when MD3 was combined with tobramycin and meropenem. These results support the possibility of combining MD3 with any of these conventional antibiotics in the management of Pseudomonas aeruginosa infections.

[0138] Additional studies were conducted to evaluate the combination of MD3 and antibiotics commonly used in the treatment of NTM infections, which are usually treated with at least three antibiotics for at least six months at a time. The results of these studies indicate that MD3 is additive with amikacin and clarithromycin, two of the most commonly prescribed antibiotics for NTM lung infections. These results support the possibility of combining MD3 with amikacin or MD3 with clarithromycin in the treatment of NTM infections.

[0139] Additional studies were conducted to evaluate the drug interactions of Staphylococcus aureus, a Gram-positive bacterium. In this species, the combination of MD3 and tobramycin was found to be irrelevant, suggesting that the combination of MD3 and tobramycin should not reduce the efficacy of either drug.

[0140] Example 2: Efficacy of NO-Releasing Compounds in Combination with Other Antifungal Drugs against Aspergillus fumigatus and Candida auris

[0141] As detailed in Example 1 for antibiotics, it is important to evaluate whether NO-releasing agents change the efficacy of commonly used antifungal drugs and whether commonly used antifungal drugs affect the efficacy of NO-releasing agents. In this study, MD3 is used as a representative NO-releasing agent.

[0142] To quantify the combined effect of MD3 and secondary antifungal drugs, the checkerboard assay was used as described above in Example 1. The antibiotics tested in this study included voriconazole, a triazole antifungal drug that inhibits the growth of fungi causing infections, itraconazole, an azole antifungal drug that inhibits fungal growth, and amphotericin B, a polyene antifungal drug that binds to ergosterol in the fungal cell membrane, creating holes in the membrane and causing leakage of cell components and cell death.

[0143] The fungal species used in this study included Aspergillus fumigatus (strain N0219) and Candida auris (strain N0220). The checkerboard method was performed as described above in Example 1. The effects of the antifungal drug combinations were classified according to the fractional inhibitory concentration index (FICI) at 24 and 48 hours as described above in Example 1. The results of the checkerboard assay of MD3 in combination with secondary antifungal drugs are shown in Table 8.

Table 8

[0144] In summary, MD3 is at least additive to voriconazole against Aspergillus fumigatus and Candida auris. Furthermore, MD3 is at least additive to itraconazole against Aspergillus fumigatus and Candida auris. The combinations of MD3 and the antifungal drugs tested were superior to the single compounds because both compounds were equally effective alone or in combination. Importantly, no antagonism was observed for any of the MD3 / antifungal drug combinations tested. See Table 8.

[0145] Example 3: Inhibition of Pseudomonas aeruginosa biofilm formation A 96-well plate containing serial dilutions of MD3 or medium only (untreated control) was prepared. The test concentrations of MD3 included 0.00391 mg / mL, 0.00781 mg / mL, 0.0156 mg / mL, 0.3125 mg / mL, and 0.0625 mg / mL. Pseudomonas aeruginosa was added to the wells at a concentration of 10 6 colony-forming units (CFU) / mL. A peg lid was inserted into the plate, and the biofilm was grown on the peg for 18 - 24 hours. Next, the peg was rinsed with phosphate-buffered saline (PBS) to remove planktonic bacteria. The biofilm was disrupted and plated to count the colony-forming units associated with the biofilm.

[0146] Figure 4 shows the number of biofilm-associated bacteria and planktonic Pseudomonas aeruginosa remaining per 1 mL of sample after treatment with various concentrations of MD3. The measured values for biofilm-associated bacteria are shown in the left bar group (starting from the left, bars 1 - 6), and the measured values for planktonic bacteria are shown in the right bar group (starting from the left, bars 7 - 12). The starting CFU / mL before treatment with MD3 was 9.50E+05 and is indicated by the dotted line. As shown in Figure 4, MD3 effectively prevented Pseudomonas aeruginosa from forming a biofilm on the peg at a concentration of 0.0625 mg / mL. Surprisingly, the biofilm prevention concentration of MD3 for Pseudomonas aeruginosa was approximately 2-fold lower than the minimum inhibitory concentration (MIC) of MD3 for planktonic Pseudomonas aeruginosa, which is 0.125 mg / mL. See Table 9.

Table 9

[0147] Example 4: Eradication of Pseudomonas aeruginosa biofilm The minimum biofilm eradication concentration (MBEC) of MD3 was determined for Pseudomonas aeruginosa biofilms grown under aerobic and anaerobic conditions. MBEC is defined as a 3-log reduction in biofilm-related CFU. Biofilms were generated in cation-adjusted Mueller-Hinton broth (CAMHB) at 37 °C for 24 h using an MBEC Assay™ growth apparatus (Innovotech, Edmonton, Alberta, Canada), and further treated with MD3 or tobramycin for 18 - 24 h. The remaining biofilm was disrupted by sonication and plated to measure the surviving CFU / mL after one treatment.

[0148] As shown in Figure 5, MD3 eradicated Pseudomonas aeruginosa biofilms at similar concentrations under both aerobic and anaerobic conditions. Tobramycin, used as a comparative example, was effective only against aerobic biofilms. The potent and broad antibacterial activity of MD3 indicates that this compound is effective in the treatment of Pseudomonas aeruginosa infections.

[0149] The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as examples of some aspects of the claims, and all compositions and methods functionally equivalent are within the scope of this disclosure. Various modifications of the compositions and methods in addition to those shown and described herein are intended to be included within the scope of the appended claims. Further, although only specific representative compositions and embodiments of these compositions and methods are specifically described herein, other compounds and methods are intended to be included within the scope of the appended claims. Accordingly, steps, elements, components, or combinations of components, although they may be specifically recited herein, are not excluded from other combinations of steps, elements, components, and components, whether or not explicitly described.

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