Nitric oxide-releasing antimicrobial compounds, formulations, and related methods

Abstract

Some embodiments of NO-releasing compounds are disclosed. In some embodiments, the structures are covalently modified to store and release nitric oxide. Some embodiments relate to the methods of making and using these structures. The compounds may be tailored to release nitric oxide in a controlled manner, and may be useful, for example, for treating or preventing microbial infections or reducing the microbial burden of microbial infections.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 971,624, filed February 7, 2020, and entitled "NITRIC OXIDE-RELEASING ANTIBACTERIAL COMPOUNDS, FORMULATIONS, AND METHODS PERTAINING THERETO," the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates generally to nitric oxide-releasing compounds, their synthesis, and their use as antibacterial compounds. Antibacterial compositions containing these compounds and methods of their use are also disclosed. [Background technology]

[0003] Respiratory viruses, including rhinoviruses and enteroviruses (Picornaviridae), influenza virus (Orthomyxoviridae), parainfluenza, metapneumoviruses, and respiratory syncytial viruses (Paramyxoviridae), coronaviruses (Coronaviridae), and some adenoviruses, also pose significant challenges to human health. Furthermore, exposure to respiratory viruses often leads to secondary bacterial infections.

[0004] Bacterial infections pose a major challenge to human health in community and hospital settings, and antimicrobial resistance has led to the development of multidrug-resistant bacteria, making them increasingly difficult to treat.

[0005] Biofilms are cooperative communities of bacteria encapsulated by an exopolysaccharide (EPS) matrix that protects them from the host immune response and antibiotics. Bacteria growing in biofilms are typically more resistant to antibiotics and disinfectants than planktonic cells, and this resistance increases with the age of the biofilm. Bacterial biofilms also exhibit increased physical resistance to desiccation, extreme temperatures, or light, and traditional antibiotic treatments are often ineffective in treating biofilms.

[0006] Conventional treatments for microbial infections typically involve the systemic administration of antimicrobial agents (antibiotics or antivirals), which can lead to drug resistance and many adverse effects, from hearing loss to gastrointestinal disorders. It would be advantageous to have alternative treatments for microbial infections that use orthogonal mechanisms of action to treat microbial infections.

[0007] Nitric oxide is known to have such an orthogonal antibacterial mechanism of action. See, for example, U.S. Patent Application Publication No. 2019 / 0322770. The exact mechanism by which nitric oxide (NO) terminates or inhibits the replication of various intracellular pathogens is not fully understood, but it likely involves reactivity against iron centers involved in cellular metabolism, imposition of nitrosative stress, and activation of host immunity. Nitric oxide is also understood to target cysteine ​​proteases (Saura et al., Immunity, Volume 10, Issue 1, January 1, 1999, Pages 21-28). NO nitrosylates cysteine ​​residues in the active sites of certain viral proteases, inhibiting protease activity and disrupting the viral life cycle. Because cysteine ​​proteases are important for the pathogenesis or replication of many viruses, bacteria, and parasites, NO can be used to treat microbial infections. Although long seen as a potentially beneficial therapeutic agent, administration of NO gas via inhalation is difficult and time-consuming, and antimicrobial levels are close to therapeutic, leaving little safety interval.

[0008] NO-releasing compounds (i.e., NO donors) have been proposed as therapeutic agents, often in the form of polymers bearing side chains containing nitric oxide-releasing moieties, such as nitrosothiols and diazeniumdiolates. Nitric oxide-releasing polymers have been underutilized as therapeutic agents to date, due at least in part to limited NO payloads, faster-than-desirable NO release rates, and a lack of targeted NO delivery.

[0009] It would be advantageous to have pharmaceutical compositions containing NO donors to deliver antibacterial concentrations of NO to patients. It would also be advantageous to have additional compounds, compositions, and methods for treating microbial infections, particularly compounds and methods effective in treating drug-resistant microorganisms and biofilms. The present invention provides such compounds, compositions, and methods. Summary of the Invention

[0010] Nitric oxide is a diatomic free radical that is endogenously produced and is involved in many biological processes.Exogenous NO delivery can be an effective strategy for treating or preventing microbial infections.Disclosed are nitric oxide-releasing compounds (also referred to as nitric oxide donors or NO donors), compositions containing such compounds, and methods for treating microbial infections using the compounds and compositions.

[0011] In one embodiment, the compounds disclosed herein have the formula: [ka] During the ceremony, X is selected from the group consisting of H, D, R, and RC(O)—; R is a C optionally substituted with one or more substituents as defined herein. 1~12 alkyl, aryl, heteroaryl, alkylaryl, or arylalkyl; M + is a pharmaceutically acceptable cation.

[0012] In some embodiments, M + For example, +2 or +3 where the ratio of compounds of Formula I to cations is such that the total positive charge equals the total negative charge. Thus, for a compound with a total charge of -3 and a cation with a total charge of +2, there are two compounds and three cations.

[0013] Representative positively charged cations include sodium, potassium, lithium, calcium, magnesium, and quaternary ammonium salts.

[0014] Methods for making these compounds are also disclosed. In one embodiment, compounds having R(CO)- moieties that do not contain acidic α CH (i.e., α to a carbonyl), such as aryl, heteroaryl, and branched alkyl groups, such as t-butyl groups, can be prepared by reacting all acidic α CH on the methyl groups of a compound having the formula R(CO)CH3 with nitric oxide in basic methanol to give a trisdiazeniumdiolate. A representative reaction is shown below. [ka]

[0015] As an example, the reaction product of acetophenone with nitric oxide in KOH / methanol is: [ka] is.

[0016] In another embodiment, compounds where X is H or D can be prepared by reacting nitric oxide with acetone in basic or deuterated methanol to give the tris-diazeniumdiolate.

[0017] In another embodiment, the NO-releasing compound has the structure of Formula II: [ka] In the formula, M + is as defined above with respect to Formula I. In some embodiments, the cation is sodium, lithium, potassium, or a quaternary ammonium salt.

[0018] Compounds of Formula II can be prepared, for example, by reacting acetone, acetonitrile, or ethanol with NO, optionally in the presence of a base (e.g., a methoxide / methanol solution), at elevated pressure (i.e., above atmospheric pressure, ideally above about 2 atmospheres, preferably above about 10 atmospheres) to form one or more diazeniumdiolate-containing species. In some embodiments, highly pure compounds (greater than about 80%, greater than about 90%, greater than about 95%, or greater than about 98%) can be produced according to the methods disclosed herein.

[0019] In yet another embodiment, the NO-releasing compound has the structure of Formula III. [ka]

[0020] In some embodiments, the compound of any of Formula I, II, or III has a purity of greater than 96%, greater than 97%, greater than 98%, greater than 99%, or greater than 99.5%. The present disclosure also relates to compounds having this purity level.

[0021] In one embodiment, the nitric oxide-releasing compound has an NO release half-life of 0.1 to 24 hours at normal physiological temperature and pH. In another embodiment, the NO release half-life is at least 15 minutes. In some embodiments, the compound has a total releasable NO pool in the range of 2 to 10 μmol of NO per mg of NO donor compound. In some embodiments, the compound has a total NO release duration in the range of 1 to 60 hours. In some embodiments, the total NO release after 4 hours is in the range of 0.1 to 1.0 μmol of NO per mg of compound.

[0022] The compounds can be formulated in a variety of pharmaceutical compositions for delivery by intravenous, inhalation, nebulization, intranasal administration, oral administration, injection, rectal or vaginal administration, and topical administration.

[0023] In one embodiment, the pharmaceutical composition comprises one or more nitric oxide-releasing compounds described herein and an aqueous solution. In one aspect of this embodiment, the nitric oxide-releasing compound has a water solubility of at least about 25 mg / ml in aqueous solution at a physiologically compatible pH.

[0024] The pharmaceutical composition may further contain one or more additional active agents depending on the type of microbial infection to be treated. For example, if the infection is a bacterial, viral, or fungal infection, one or more antibacterial, antiviral, or antifungal compounds may be present. Anti-inflammatory compounds may also be present.

[0025] In some embodiments, the composition may further comprise one or more of a chelating agent, a mucoadhesive agent, or a low molecular weight polyethylene glycol.

[0026] In one embodiment, the composition further comprises a gallium salt.

[0027] In certain embodiments, the small molecule nitric oxide donor is provided in a dilute solution (e.g., for nebulization, vaporization, or inhalation), while in other embodiments it is provided in the form of a gel or viscous liquid, e.g., for topical administration.

[0028] Also disclosed are methods for treating microbial infections. In some embodiments, the methods include delivering nitric oxide to a subject in need of antimicrobial treatment by delivering a compound of any of Formulas I, II, or III, and allowing the compound to decompose upon exposure to physiological pH and temperature to release nitric oxide. In some embodiments, the amount of compound administered is an effective amount of the compound or a composition containing the compound to produce the desired antimicrobial effect, i.e., treatment, prevention, or reduction of microbial burden.

[0029] Representative microorganisms include viruses, gram-positive bacteria, gram-negative bacteria, drug-resistant bacteria, molds, yeasts, fungi, and combinations thereof. In some embodiments, the microorganisms include one, two, or more of gram-positive bacteria, gram-negative bacteria, drug-resistant bacteria, fungi, yeasts, and viruses.

[0030] Without wishing to be bound by any particular theory, it is believed that the compounds generate nitric oxide and induce oxidative and / or nitrosative damage to microbial DNA and membrane structures, thereby treating microbial infections, preventing microbial infections, reducing microbial burden by reducing the number of viable microorganisms, and / or preventing microbial colonization or infection. In some embodiments, the NO donor of the compound or composition generates NO and induces damage to microbial membranes and / or DNA.

[0031] Representative microbial infections include bacterial, fungal, and viral infections, particularly those that result in gastrointestinal disorders, respiratory disorders, and sexually transmitted diseases.

[0032] Representative viral infections that can be treated include those associated with one or more of human immunodeficiency virus, herpes simplex virus, papillomavirus, parainfluenza virus, influenza, hepatitis, coxsackievirus, shingles, measles, mumps, rubella, rabies, pneumonia, hemorrhagic viral fever, H1N1, SARS, MERS, and SARS-CoV2. Representative fungal infections that can be treated include those associated with molds such as black mold, Candida albicans, and Aspergillus niger.

[0033] Exemplary bacterial infections that can be treated include Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, Group A streptococci, S. pneumoniae, Mycobacterium tuberculosis, Campylobacter jejuni, Salmonella, Shigella, carbapenem-resistant Enterobacteriaceae, methicillin-resistant Staphylococcus aureus, and Burkholderia cepacia. In some embodiments, the microbial burden comprises methicillin-resistant Staphylococcus aureus. In some embodiments, the microbial burden comprises carbapenem-resistant Enterobacteriaceae. In some embodiments, the microbial burden comprises Staphylococcus aureus. In some embodiments, the microbial burden comprises Pseudomonas aeruginosa. In some embodiments, the microbial burden comprises Burkholderia cepacia.

[0034] In some embodiments, the microorganism is a parasite.

[0035] In some embodiments, the microbial infection to be treated is present on an organic surface, such as human or animal skin, including epithelial tissue, or on the surface of a wound. In some embodiments, application does not cause skin irritation. The skin surface may be, for example, in the mouth or surrounding tissues (e.g., lips, nostrils, teeth, gums, etc.), oral mucosa, any part of the digestive tract, or the lungs, or any other part of the respiratory tract.

[0036] In some embodiments, an effective amount of the compound or composition is administered as a solution by nebulization. In some embodiments, the subject is a patient suffering from a pulmonary infection, and the compound is administered to treat, and ideally, eliminate, the infection.

[0037] In some embodiments, the subject has an infection of the gastrointestinal tract or another tissue or organ, and the composition of the present disclosure is administered as an anti-infective. Some chronic infections, such as those associated with implanted devices, chronic wounds, and cystic fibrosis, are frequently caused by biofilm-forming pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. Biofilms are cooperative communities of bacteria encapsulated by an exopolysaccharide (EPS) matrix that protects them from the host immune response and antibiotics. It has been reported that eradicating biofilms can require antibiotic concentrations up to 1,000 times higher than those required for planktonic bacteria. Resistant respiratory infections are particularly difficult to treat because they form protective biofilms in airway mucus and can survive for decades. Due to the resistance of biofilms to conventional antibacterial agents, there is a need in the art for new antibacterial compositions.

[0038] Gallium has been reported to have both antibacterial and immunosuppressive properties. Its antiviral activity has been demonstrated (Narayanasamy, et al., “Prolonged-acting, Multi-targeting Gallium Nanoparticles Potently Inhibit Growth of Both HIV and Mycobacteria in Co-Infected Human Macrophages,” Sci Rep 5, 8824 (2015)). Similarly, gallium damages key iron-dependent enzymes in bacteria (see Goss, et al., “Gallium Disrupts Bacterial Iron Metabolism and Has Therapeutic Effects in Mice and Humans with Lung Infections,” Sci Transl Med. 2018;10(460):eaat7520). This has led to the use of gallium citrate as a pharmaceutical (AR-501 by Aridis). Gallium has been studied against planktonic biofilms and in vivo PAs (see Kaneko, et al., J Clin Invest., 2007;117(4):877-888). When tested against planktonic, macrophage-grown nontuberculous mycobacteria (NTM), gallium showed some promise for treating chronic infections (see Abdalla et al., Antimicrob Agents Chemother. 2015;59(8):4826-4834).

[0039] Combining gallium with one or more of the nitric oxide donor compounds described herein can produce a synergistic antimicrobial effect against, for example, biofilms that are particularly difficult to treat with conventional antibacterial compositions. Such combinations can further include a siderophore. Compositions comprising gallium, at least one nitric oxide donor compound described herein, and optionally at least one siderophore, also exhibit synergistic effects against planktonic bacteria and biofilms.

[0040] The compositions and related methods described in more detail below describe specific actions taken by a physician. However, it should be understood that they may also include the direction of those actions by another party. Thus, an action such as "administering an NO-donating compound" includes "directing the administration of an NO-donating compound."

[0041] The above-discussed embodiments will be better understood by reference to the following detailed description. [Brief explanation of the drawings]

[0042] [Figure 1] 1 shows the FTIR spectrum of the compound of formula III. [Figure 2] An ion-exchange chromatogram of a composition containing the compound of Formula III is shown, with a retention time of 11 minutes (λ=252 nm). The UV absorbance spectrum of the analyte held for approximately 6 minutes, the UV absorbance spectrum of the analyte held for approximately 10 minutes, and the UV absorbance spectrum of the analyte held for approximately 11 minutes are shown in the inset. [Figure 3] 1 shows the 1H NMR spectrum of the compound of formula III in DO. [Figure 4] 1 shows the 13C NMR spectrum of the compound of formula III. [Figure 5] 2D NMR of the compound of formula III is shown. [Figure 6] A) HPLC chromatogram (IEX-UV) with the upper chromatogram showing the separation of components of the compound of formula III (designated MD3) before acid degradation, the middle chromatogram showing the separation of components after 5 hours of acid degradation, and the lower chromatogram showing the separation of components after 24 hours of acid degradation; B) H NMR spectra of the acid degradation components; C) Table of NOA sum for the compound of formula III before and after acid degradation. [Figure 7] 1 is a 1H NMR spectrum of the reaction product when the compound of formula III is neutralized (pH 7) at room temperature. [Figure 8]1 is a 13C NMR spectrum of the reaction product when the compound of formula III is neutralized (pH 7) at room temperature. [Figure 9] 1 shows a graphical representation of the nitric oxide assay (NOA) release profile for compound of formula III at pH 7.4, as measured by chemiluminescence, showing 6.7 μmol NO / mg of material released with a T of approximately 3.75 hours, suggesting that only a single diazeniumdiolate group is released, with a theoretical loading of 7.6 μmol NO / mg. [Figure 10] To compare the MBC results of 21 strains of P. aeruginosa when grown under aerobic and anaerobic conditions. [Figures 11A-11B] 1 is a graph showing the dose-dependent time kill results of P. aeruginosa as (CFU / ml) versus time (hours) after exposure to compounds of Formula III. [Figure 12] 1 shows the effect of pH on time-kill results of P. aeruginosa after exposure to a compound of formula III (0.125 mg / ml). [Figure 13] 1 shows the effect of pH on time-kill results of P. aeruginosa after exposure to a compound of formula III (0.0625 mg / ml). [Figure 14] 1 shows the effect of pH on time-kill results of P. aeruginosa after exposure to a compound of formula III (0.03125 mg / ml). [Figure 15] Chromatograms (impurity profiles) of different lots of compound of formula III prepared with the starting reactants acetonitrile (top), ethanol (middle), and acetone (bottom) are compared. DETAILED DESCRIPTION OF THE INVENTION

[0043] Although nitric oxide (NO) is antimicrobial, its role as a therapeutic agent has been underutilized to date, due at least in part to the limited NO payload of therapeutic compositions, faster-than-desirable NO release rates, and lack of targeted NO delivery.

[0044] Disclosed are NO-releasing compounds, compositions comprising such compounds, methods for producing such compounds and compositions, and methods for treating or preventing microbial infections or reducing microbial burden. In some embodiments, the compounds are present in pharmaceutical compositions that have desirable physical properties, such as viscosity and gelation.

[0045] The compounds are small molecules, i.e., free of associated cations, and have a molecular weight of less than about 500 g / mol, and in some embodiments, about 200 g / mol. One advantage of using molecules smaller than polymers is that the compounds can be prepared with relatively lower impurity levels than polymeric compounds. Furthermore, as described herein, the NO loading can be higher compared to polymeric compounds because the NO to backbone percentage composition can be maximized.

[0046] Small molecule precursor compounds that can be converted to the NO-releasing compounds described herein can be selected with relatively few reactive groups, e.g., alpha hydrogens adjacent to carbonyl groups, reducing the likelihood that many different species will result from the nitration reaction. As a result, nitrosylation of NO precursors can proceed with few or no partial reaction products, offering the potential for relatively pure products.

[0047] By knowing the structure of the small molecule, release kinetics can be more predictable than those obtained with polymers. Additionally, if desired, the cation associated with the negatively charged diazenium ion can be selected to also have desirable properties. For example, quaternary ammonium salts also have antibacterial properties.

[0048] Before the present disclosure is further described, it is to be understood that the present disclosure is not limited to the specific embodiments described below, since variations thereto may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology used is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present disclosure will be established by the appended claims.

[0049] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0050] The invention will be better understood with reference to the following definitions.

[0051] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter belongs. The terminology used in the description of the subject matter herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the subject matter.

[0052] The term "effective amount" as used herein broadly refers to an amount of the recited compound effective to treat, prevent, or reduce the microbial burden in a subject suffering from a microbial infection, including improving the subject's condition (e.g., in one or more symptoms), slowing or reducing the progression of the infection, preventing or delaying the onset of the infection, and / or modifying clinical parameters, diseases, or conditions, as is well known in the art.

[0053] For example, an effective amount can refer to the amount of a composition, compound, or agent that improves a condition in a subject by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0054] In some embodiments, the improvement in the condition may be a reduction in infection. In some embodiments, the improvement may be a reduction in bacterial load (e.g., bioburden) on a surface or in a subject. The actual dosage level of the active ingredient in the active composition of the presently disclosed subject matter may be varied to administer an amount of the active compound that is effective to achieve the desired response for a particular subject. The selected dosage level will depend on various factors, including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimum dose is administered, and in the absence of dose-limiting toxicity, the dose is titrated to the minimum effective amount. Determination and adjustment of effective doses, as well as evaluation of when and how to make such adjustments, are contemplated herein.

[0055] "Treating" or "treating" or "treatment" broadly refers to any type of action that imparts a desired antimicrobial effect, including treating or preventing a microbial infection, reducing microbial load, improving a subject's condition (e.g., in one or more symptoms), slowing or reducing the progression of an infection, and / or altering one or more clinical parameters.

[0056] The terms "disrupting" and "eradicating" broadly refer to the ability of the disclosed structures to combat biofilms. A biofilm may be partially eradicated or destroyed, meaning that the cells are no longer bound to each other or to a surface. A biofilm may be completely eradicated, meaning that the biofilm is no longer substantially an interconnected, cohesive, or continuous network of cells.

[0057] The term "nitric oxide donor" or "NO donor" broadly refers to species and / or molecules that donate, release, and / or directly or indirectly transfer nitric oxide species, and / or stimulate the endogenous production of nitric oxide species in vivo and / or increase endogenous levels of nitric oxide in vivo, such that the biological activity of the nitric oxide species is expressed at an intended site of action.

[0058] The terms "nitric oxide releasing" or "nitric oxide donating" refer to species and / or methods that donate, release, and / or directly or indirectly transfer any one (or more) of the three redox forms of nitric oxide (NO, NO, NO (e.g., NO)). In some embodiments, nitric oxide release is achieved such that the biological activity of the nitric oxide species is expressed at the intended site of action.

[0059] As used herein, the term "microbial infection" broadly refers to bacterial infections, fungal infections, viral infections, yeast infections, and other microorganisms, and combinations thereof.

[0060] The term "respiratory tract" includes not only the lungs but also the mouth, nasal passages, throat, esophagus, larynx, pharynx, and trachea. Within the lungs, therapy can target one or more of the bronchioles, bronchi, upper airways, and lower airways. The therapeutic approaches described herein can be used to treat or prevent, slow the progression of, or reverse the damage associated with many different types of respiratory disorders. Certain respiratory disorders are associated with microbial infections, and compounds used to treat the disorders can produce concentrations of nitric oxide effective to kill microorganisms. Certain respiratory disorders, such as COPD, emphysema, and both acute and chronic bronchitis, are associated with poor angiogenesis and / or can benefit from increased angiogenesis. Exogenous nitric oxide, released upon degradation of the compounds described herein, can increase angiogenesis and therefore may be particularly effective in treating such disorders.

[0061] As disclosed herein, the treated "patient" or "subject" is, in some embodiments, a human patient; however, it should be understood that the principles of the presently disclosed subject matter indicate that the presently disclosed subject matter is effective against all vertebrate species, including mammals, which are intended to be encompassed by the terms "subject" and "patient." Suitable subjects are generally mammalian subjects. The subject matter described herein finds use in research as well as veterinary and medical applications. As used herein, the term "mammal" includes, but is not limited to, humans, non-human primates, cattle, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g., rats or mice), monkeys, and the like. Human subjects include neonates, infants, juveniles, adults, and geriatric subjects. A subject "in need" of the methods disclosed herein can be a subject experiencing and / or expected to experience a disease state, and the methods and compositions of the present invention are used for therapeutic and / or prophylactic treatment.

[0062] For the general chemical formulas provided herein, if no substituent is indicated, those skilled in the art will understand that the substituent is hydrogen. A bond not connected to an atom but shown indicates that the position of such a substituent is variable. A jagged line, a wavy line, or two wavy lines drawn through or at the end of a bond indicates that some additional structure is attached at that position. For many additional monomers disclosed herein but not explicitly shown in the structures, those skilled in the art of polymers will understand that these monomers can be added to modify the physical properties of the resulting polymeric material, even if elemental analysis does not indicate that such a distinction can be expected. Such physical properties include solubility, charge, stability, crosslinking, secondary and tertiary structure, etc. Furthermore, if stereochemistry is not indicated for compounds with one or more chiral centers, all enantiomers and diastereomers are included. Similarly, for any listed aliphatic or alkyl groups, all structural isomers thereof are also included. Unless otherwise noted, in the general formulas provided herein, A1-A n and referred to herein as alkyl groups are independently selected from alkyl or aliphatic groups, particularly alkyls having 20 or fewer carbon atoms, and even more typically lower alkyls having 10 or fewer atoms (e.g., methyl, ethyl, propyl, isopropyl, and butyl). The alkyl may be optionally substituted (e.g., substituted or unsubstituted as disclosed elsewhere herein). The alkyl may be a substituted alkyl group, such as an alkyl halide (e.g., —CX, where X is a halide and combinations thereof (either in-chain or attached thereto)), an alcohol (e.g., an aliphatic or alkyl hydroxyl, particularly a lower alkyl hydroxyl), or other similarly substituted moieties (e.g., amino-, amino acid-, aryl-, alkylaryl-, alkyl ester-, ether-, keto-, nitro-, sulfhydryl-, sulfonyl-, sulfoxide-modified-alkyl group).

[0063] The terms "amino" and "amine" refer to nitrogen-containing groups such as NR, NH, NHR, and NHR, where R can be as described elsewhere herein. Thus, as used herein, "amino" can refer to a primary amine, a secondary amine, or a tertiary amine. In some embodiments, one R of the amino group can be a diazeniumdiolate (e.g., NONO).

[0064] When a group is described as "optionally substituted," the group can be unsubstituted or substituted with one or more of the listed substituents. Similarly, when a group is described as being "unsubstituted or substituted" (or "substituted or unsubstituted"), if substituted, the substituents may be selected from one or more of the listed substituents. If no substituents are indicated, it means that the indicated "optionally substituted" or "substituted" group may be substituted with one or more groups individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, aryl(alkyl), cycloalkyl(alkyl), heteroaryl(alkyl), heterocyclyl(alkyl), hydroxy, alkoxy, acyl, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, nitro, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, amino, monosubstituted amine group, disubstituted amine group, monosubstituted amine(alkyl), disubstituted amine(alkyl), diamino group, polyamino group, diether group, and polyether group.

[0065] As used herein, "C" is a set of integers where "a" and "b" are integers. a ~C b" refers to the number of carbon atoms in the group. The designated group can contain from "a" to "b" (inclusive) carbon atoms. Thus, for example, a "C1 to C4 alkyl" or a "C1-C4 alkyl" group broadly refers to all alkyl groups having from 1 to 4 carbons, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)-, and (CH3)3C-. When "a" and "b" are not specified, the broadest ranges described by these definitions are assumed.

[0066] As used herein, the term "alkyl" broadly refers to a fully saturated aliphatic hydrocarbon group. The alkyl moiety may be branched or straight-chain. Examples of branched-chain alkyl groups include, but are not limited to, isopropyl, sec-butyl, t-butyl, and the like. Examples of straight-chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and the like. An alkyl group may have 1 to 30 carbon atoms (as it appears herein, numerical ranges such as "1 to 30" refer broadly to each integer in the given range; for example, "1 to 30 carbon atoms" means that the alkyl group may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms, although this definition also includes the occurrence of the term "alkyl" without a specified numerical range). An "alkyl" group may also be a medium-sized alkyl having 1 to 12 carbon atoms. An "alkyl" group may also be a lower alkyl having 1 to 6 carbon atoms. An alkyl group may be substituted or unsubstituted. By way of example only, "C1-C5 alkyl" indicates that there are 1 to 5 carbon atoms in the alkyl chain, e.g., the alkyl chain is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl (branched and straight chain), etc. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, and hexyl.

[0067] As used herein, the term "alkylene" broadly refers to a divalent, fully saturated, straight-chain aliphatic hydrocarbon group. Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, and octylene. An alkylene group is: [ka] , followed by the number of carbon atoms, followed by "*". For example, [ka] is intended to represent ethylene. An alkylene group may have 1 to 30 carbon atoms (as it appears herein, a numerical range such as "1 to 30" broadly refers to each integer in the given range; for example, "1 to 30 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 30 carbon atoms, but this definition also includes the occurrence of the term "alkylene" without a specified numerical range). An "alkylene" group may also be a medium-sized alkyl having 1 to 12 carbon atoms. An "alkylene" group may also be a lower alkyl having 1 to 6 carbon atoms. An alkylene group may be substituted or unsubstituted. For example, a lower alkylene group may be formed by replacing one or more hydrogens of the lower alkylene group and / or by replacing both hydrogens on the same carbon with C 3~6 It can be substituted by substituting with a monocyclic cycloalkyl group. (for example, [ka] ).

[0068] The term "alkenyl," as used herein, broadly refers to a monovalent straight or branched chain radical of 2 to 20 carbon atoms containing a carbon double bond, including, but not limited to, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, etc. Alkenyl groups can be unsubstituted or substituted.

[0069] The term "alkynyl" as used herein broadly refers to a monovalent straight or branched chain radical of 2 to 20 carbon atoms containing a carbon triple bond, including, but not limited to, 1-propynyl, 1-butynyl, 2-butynyl, etc. Alkynyl groups can be unsubstituted or substituted.

[0070] As used herein, "cycloalkyl" broadly refers to a fully saturated (no double or triple bonds) monocyclic or polycyclic (e.g., bicyclic) hydrocarbon ring system. When composed of two or more rings, the rings may be joined together in a fused, bridged, or spiro fashion. As used herein, the term "fused" broadly refers to two rings that share two atoms and one bond in common. As used herein, the term "bridged cycloalkyl" broadly refers to a compound in which a cycloalkyl contains a linkage of one or more atoms connecting non-adjacent atoms. As used herein, the term "spiro" broadly refers to two rings that share one atom in common, where the two rings are not connected by a bridge. Cycloalkyl groups can contain 3 to 30 atoms in the rings, 3 to 20 atoms in the rings, 3 to 10 atoms in the rings, 3 to 8 atoms in the rings, or 3 to 6 atoms in the rings. Cycloalkyl groups can be unsubstituted or substituted. Examples of mono-cycloalkyl groups include, but are in no way limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of fused cycloalkyl groups are decahydronaphthalenyl, dodecahydro-1H-phenalenyl, and tetradecahydroanthracenyl. Examples of bridged cycloalkyl groups are bicyclo[1.1.1]pentyl, adamantanyl, and norbornanyl. Examples of spirocycloalkyl groups are spiro[3.3]heptane and spiro[4.5]decane.

[0071] As used herein, "cycloalkenyl" broadly refers to a monocyclic or polycyclic (e.g., bicyclic) hydrocarbon ring system containing one or more double bonds in at least one ring, provided that if more than one is present, the double bonds cannot form a completely delocalized π-electron system throughout all rings (otherwise, the group is an "aryl" as defined herein). Cycloalkenyl groups can contain 3 to 10 atoms in the rings, 3 to 8 atoms in the rings, or 3 to 6 atoms in the rings. When composed of more than one ring, the rings may be connected together in a fused, bridged, or spiro fashion. Cycloalkenyl groups can be unsubstituted or substituted.

[0072] As used herein, "aryl" broadly refers to a carbocyclic (all carbon) monocyclic or polycyclic (such as bicyclic) aromatic ring system (including fused ring systems in which two carbon rings share a chemical bond) having a completely delocalized pi-electron system throughout all rings. The number of carbon atoms in an aryl group can vary. For example, aryl groups range from C6 to C 14 Aryl groups, C6-C 10The heteroaryl group may be an aryl group or a C6 aryl group. Examples of aryl groups include, but are not limited to, benzene, naphthalene, and azulene. The aryl group may be substituted or unsubstituted. As used herein, "heteroaryl" refers to a monocyclic or polycyclic (such as bicyclic) aromatic ring system (a ring system with a completely delocalized π-electron system) containing one or more heteroatoms (e.g., 1, 2, or 3 heteroatoms), i.e., elements other than carbon, including, but not limited to, nitrogen, oxygen, and sulfur. The number of atoms in the ring of a heteroaryl group may vary. For example, a heteroaryl group can contain 4 to 14 atoms in the ring, 5 to 10 atoms in the ring, or 5 to 6 atoms in the ring, e.g., 9 carbon atoms and 1 heteroatom, 8 carbon atoms and 2 heteroatoms, 7 carbon atoms and 3 heteroatoms, 8 carbon atoms and 1 heteroatom, 7 carbon atoms and 2 heteroatoms, 6 carbon atoms and 3 heteroatoms, 5 carbon atoms and 4 heteroatoms, 5 carbon atoms and 1 heteroatom, 4 carbon atoms and 2 heteroatoms, 3 carbon atoms and 3 heteroatoms, 4 carbon atoms and 1 heteroatom, 3 carbon atoms and 2 heteroatoms, or 2 carbon atoms and 3 heteroatoms. Furthermore, the term "heteroaryl" includes fused ring systems in which two rings share at least one chemical bond, such as at least one aryl ring and at least one heteroaryl ring, or at least two heteroaryl rings.Examples of heteroaryl rings include, but are not limited to, furan, furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, benzothiazole, imidazole, benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole, benzisoxazole, isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, purine, pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, and triazine. Heteroaryl groups can be substituted or unsubstituted.

[0073] As used herein, "heterocyclyl" or "heteroalicyclyl" broadly refers to 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, and up to 18-membered monocyclic, bicyclic, and tricyclic ring systems in which carbon atoms and one to five heteroatoms together comprise said ring system. However, heterocycles may optionally contain one or more unsaturated bonds positioned in such a way that a completely delocalized π-electron system does not occur throughout all rings. Heteroatoms are elements other than carbon, including, but not limited to, oxygen, sulfur, and nitrogen. Heterocycles may further contain one or more carbonyl or thiocarbonyl functional groups and, as defined, include oxo and thio systems such as lactams, lactones, cyclic imides, cyclic thioimides, and cyclic carbamates. When composed of two or more rings, the rings may be fused, bridged, or joined together in a spiro fashion. As used herein, the term "fused" refers to two rings that share two atoms and one bond. As used herein, the term "bridged heterocyclyl" or "bridged heteroalicyclyl" refers to a compound in which a heterocyclyl or heteroalicyclyl contains a linkage of one or more atoms connecting non-adjacent atoms. As used herein, the term "spiro" refers to two rings that share one atom in common, and the two rings are not connected by a bridge. A heterocyclyl or heteroalicyclyl group can contain 3 to 30 atoms in the rings, 3 to 20 atoms in the rings, 3 to 10 atoms in the rings, 3 to 8 atoms in the rings, or 3 to 6 atoms in the rings. For example, 5 carbon atoms and 1 heteroatom, 4 carbon atoms and 2 heteroatoms, 3 carbon atoms and 3 heteroatoms, 4 carbon atoms and 1 heteroatom, 3 carbon atoms and 2 heteroatoms, 2 carbon atoms and 3 heteroatoms, 1 carbon atom and 4 heteroatoms, 3 carbon atoms and 1 heteroatom, or 2 carbon atoms and 1 heteroatom. Additionally, any nitrogen in the heteroalicyclic may be quaternized. The heterocyclyl or heteroalicyclic group may be unsubstituted or substituted.Examples of such "heterocyclyl" or "heteroalicyclyl" groups include 1,3-dioxine, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3-dioxolane, 1,4-dioxolane, 1,3-oxathiane, 1,4-oxathiin, 1,3-oxathiolane, 1,3-dithiol, 1,3-dithiolane, 1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline, imidazolidine, isothiophene, tetrahydro-1,4-thiazine, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, tetrahydro-1,4-thi ... Examples of suitable amines include, but are not limited to, xazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholine, oxirane, piperidine N-oxide, piperidine, piperazine, pyrrolidine, azepane, pyrrolidone, pyrrolidione, 4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiopyran, thiamorpholine, thiamorpholine sulfoxide, thiamorpholine sulfone, and benzo-fused analogs thereof (e.g., benzimidazolidinone, tetrahydroquinoline, and / or 3,4-methylenedioxyphenyl). Examples of spiroheterocyclyl groups include 2-azaspiro[3.3]heptane, 2-oxaspiro[3.3]heptane, 2-oxa-6-azaspiro[3.3]heptane, 2,6-diazaspiro[3.3]heptane, 2-oxaspiro[3.4]octane, and 2-azaspiro[3.4]octane.

[0074] As used herein, "aralkyl" and "aryl(alkyl)" broadly refer to an aryl group connected as a substituent via a lower alkylene group. The lower alkylene and aryl groups of an aralkyl may be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.

[0075] As used herein, "cycloalkyl(alkyl)" broadly refers to a cycloalkyl group connected as a substituent via a lower alkylene group. The lower alkylene and cycloalkyl groups of the cycloalkyl(alkyl) may be substituted or unsubstituted.

[0076] As used herein, "heteroaralkyl" and "heteroaryl(alkyl)" broadly refer to a heteroaryl group connected as a substituent via a lower alkylene group. The lower alkylene and heteroaryl groups of a heteroaralkyl may be substituted or unsubstituted. Examples include, but are not limited to, 2-thienylalkyl, 3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl, as well as benzo-fused analogs thereof.

[0077] "Heteroalicyclyl(alkyl)" and "heterocyclyl(alkyl)" broadly refer to heterocyclic or heteroalicyclic groups connected as substituents via a lower alkylene group. The lower alkylene and heterocyclyl of (heteroalicyclyl)alkyl may be substituted or unsubstituted. Examples include, but are not limited to, tetrahydro-2H-pyran-4-yl(methyl), piperidin-4-yl(ethyl), piperidin-4-yl(propyl), tetrahydro-2H-thiopyran-4-yl(methyl), and 1,3-thiazinan-4-yl(methyl).

[0078] As used herein, the term "hydroxy" broadly refers to an --OH group.

[0079] As used herein, "alkoxy" broadly refers to the formula -OR, where R is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl), as defined herein. A non-limiting list of alkoxy is methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, phenoxy, and benzooxy. Alkoxy may be substituted or unsubstituted.

[0080] As used herein, "acyl" broadly refers to hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, aryl(alkyl), heteroaryl(alkyl), and heterocyclyl(alkyl) connected as a substituent through a carbonyl group. Examples include formyl, acetyl, propanoyl, benzoyl, and acryl. Acyl may be substituted or unsubstituted.

[0081] As used herein, a "cyano" group refers broadly to a "-CN" group.

[0082] As used herein, the term "halogen atom" or "halogen" means any one of the radioactive stable atoms in column 7 of the periodic table of the elements, such as fluorine, chlorine, bromine, and iodine.

[0083] A "thiocarbonyl" group broadly refers to a "-C(=S)R" group, where R can be the same as defined for O-carboxy. The thiocarbonyl can be substituted or unsubstituted. An "O-carbamyl" group refers to a "-OC(=O)N(R A R B ) group, where R A and R Bcan be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). O-carbamyl can be substituted or unsubstituted.

[0084] The "N-carbamyl" group is broadly defined as "ROC(=O)N(R A )-" group, where R and R A can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). N-carbamyl can be substituted or unsubstituted.

[0085] The "O-thiocarbamyl" group is broadly defined as "-OC(=S)-N(R A R B ) group, where R A and R B can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). O-thiocarbamyl can be substituted or unsubstituted.

[0086] The "N-thiocarbamyl" group is broadly defined as "ROC(=S)N(R A )-" group, where R and R A can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). N-thiocarbamyl can be substituted or unsubstituted.

[0087] A "C-amide" group is broadly defined as "-C(=O)N(R A R B ) group, where R A and R B can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). C-amides can be substituted or unsubstituted.

[0088] The "N-amide" group is broadly defined as "RC(=O)N(R A )-" group, where R and R A can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). N-amides can be substituted or unsubstituted.

[0089] The "S-sulfonamide" group is broadly defined as "-SO2N(R A R B ) group, where R A and R B can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). S-sulfonamides can be substituted or unsubstituted.

[0090] The "N-sulfonamide" group is broadly defined as "RSO2N(R A )-" group, where R and R Acan be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). N-sulfonamides can be substituted or unsubstituted.

[0091] An "O-carboxy" group refers broadly to the group "RC(=O)O-," where R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl), as defined herein. The O-carboxy can be substituted or unsubstituted.

[0092] The terms "ester" and "C-carboxy" broadly refer to the group "-C(=O)OR", where R can be the same as defined for O-carboxy. Esters and C-carboxy can be substituted or unsubstituted.

[0093] A "nitro" group broadly refers to a "-NO2" group.

[0094] A "sulfenyl" group refers broadly to the group "-SR," where R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). The sulfenyl can be substituted or unsubstituted.

[0095] A "sulfinyl" group refers broadly to the group "-S(=O)-R," where R can be the same as defined for sulfenyl. The sulfinyl can be substituted or unsubstituted.

[0096] A "sulfonyl" group refers broadly to a "SO2R" group, where R can be the same as defined for sulfenyl. The sulfonyl can be substituted or unsubstituted.

[0097] As used herein, "haloalkyl" broadly refers to an alkyl group in which one or more hydrogen atoms have been replaced with halogen (e.g., monohaloalkyl, dihaloalkyl, trihaloalkyl, and polyhaloalkyl). Such groups include, but are not limited to, chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1-chloro-2-fluoromethyl, 2-fluoroisobutyl, and pentafluoroethyl. Haloalkyl may be substituted or unsubstituted.

[0098] As used herein, "haloalkoxy" broadly refers to an alkoxy group in which one or more hydrogen atoms have been replaced by halogen (e.g., mono-haloalkoxy, di-haloalkoxy, and tri-haloalkoxy). Such groups include, but are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, 1-chloro-2-fluoromethoxy, and 2-fluoroisobutoxy. Haloalkoxy may be substituted or unsubstituted.

[0099] As used herein, the terms "amino" and "unsubstituted amino" refer broadly to the group --NH.sub.2.

[0100] A "monosubstituted amine" group is broadly defined as "-NHR A " group, where R A R can be alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl), as defined herein. AThe monosubstituted amine group may be, for example, a mono-alkylamine group, a mono-C1-C6 alkylamine group, a mono-arylamine group, a mono-C6-C 10 arylamine groups, etc. Examples of monosubstituted amine groups include, but are not limited to, -NH(methyl), -NH(phenyl), etc.

[0101] A "disubstituted amine" group can be broadly defined as "-NR A R B " group, where R A and R B R can independently be alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl), as defined herein. A and R B can be independently substituted or unsubstituted. Disubstituted amine groups include, for example, di-alkylamine groups, di-C1-C6 alkylamine groups, di-arylamine groups, di-C6-C 10 The disubstituted amine group may include, but is not limited to, -N(methyl), -N(phenyl)(methyl), -N(ethyl)(methyl), and the like. As used herein, a "monosubstituted amine(alkyl)" group broadly refers to a monosubstituted amine provided herein connected as a substituent via a lower alkylene group. The monosubstituted amine(alkyl) may be substituted or unsubstituted. Examples of the monosubstituted amine(alkyl) group include, for example, a mono-alkylamine(alkyl) group, a mono-C1-C6 alkylamine(C1-C6 alkyl) group, a mono-arylamine(alkyl) group, a mono-C6-C6 alkylamine(C1-C6 alkyl) group, a mono-arylamine(alkyl) group, a mono-C6-C6 alkylamine(C6-C6 alkyl) group, a mono-C6-C6 alkylamine(C6-C6 alkyl) group, a mono-arylamine(alkyl ... 10 Examples of monosubstituted amine (alkyl) groups include, but are not limited to, -CHNH(methyl), -CHNH(phenyl), -CHCHNH(methyl), -CHCHNH(phenyl), and the like.

[0102] As used herein, the term "disubstituted amine(alkyl)" broadly refers to the disubstituted amines provided herein that are connected as substituents via a lower alkylene group. The disubstituted amine(alkyl) may be substituted or unsubstituted. Examples of disubstituted amine(alkyl) groups include dialkylamine(alkyl) groups, di-C1-C6 alkylamine(C1-C6 alkyl) groups, di-arylamine(alkyl) groups, di-C6-C6 alkyl ... 10 Examples of disubstituted amine(alkyl) groups include, but are not limited to, -CHN(methyl), -CHN(phenyl)(methyl), -CHN(ethyl)(methyl), -CHCHN(methyl), -CHCHN(phenyl)(methyl), -NCHCH(ethyl)(methyl), and the like.

[0103] As used herein, the term “diamino-” refers to “—N(R A )R B -N(R C )(R D ) group, wherein R A , R C , and R D may independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl), as defined herein; R B connects two "N" groups and (R A , R C , and R D and (independently of) may be a substituted or unsubstituted alkylene group. A、 R B , R C , and R D can further independently be substituted or unsubstituted.

[0104] As used herein, the term "polyamino" refers to a group consisting of "-(N(R A )R B-) n -N(R C )(R D For illustrative purposes, the term polyamino refers to -N(R A ) alkyl-N(R A ) alkyl-N(R A ) alkyl-N(R A ) alkyl-H. In some embodiments, the alkyl of the polyamino is disclosed elsewhere herein. While this example has only four repeating units, the term "polyamino" may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units. R A , R C , and R D may independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl), as defined herein, where R B connects two "N" groups and (R A , R C , and R D and (independently of) may be a substituted or unsubstituted alkylene group. A、 R C , and R D may further independently be substituted or unsubstituted. As described herein, polyamino comprises amine groups with intervening alkyl groups, where alkyl is as defined elsewhere herein.

[0105] As used herein, the term "diether-" refers to "-OR B OR A " group, wherein R A may be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl), as defined herein; R BR connects two "O" groups and can be a substituted or unsubstituted alkylene group. A can further independently be substituted or unsubstituted.

[0106] As used herein, the term "polyether" refers to a repeating -(OR B -) n OR A For purposes of illustration, the term polyether refers to an -Oalkyl-Oalkyl-Oalkyl-Oalkyl-OR group. A In some embodiments, the alkyl of the polyether is as disclosed elsewhere herein. While this example has only four repeating units, the term "polyether" may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units. R A R may be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl), as defined herein. B R can be a substituted or unsubstituted alkylene group. A may further independently be substituted or unsubstituted. As described herein, polyethers comprise ether groups having intervening alkyl groups, where alkyl is as defined elsewhere herein and may be optionally substituted.

[0107] Where the number of substituents is not specified (e.g., haloalkyl), one or more substituents may be present. For example, "haloalkyl" may include one or more of the same or different halogens. As another example, "C1-C3 alkoxyphenyl" may include one or more of the same or different alkoxy groups containing one, two, or three atoms. As used herein, a radical refers to a species with a single unpaired electron such that the species containing the radical can be covalently bonded to another species. Thus, in this context, a radical is not necessarily a free radical. Rather, a radical refers to a specific portion of a larger molecule. The term "radical" may be used interchangeably with the term "group."

[0108] When a range of integers is given, the range includes any number within the range and the numbers defining the endpoints of the range. For example, if the term "integers from 1 to 20" is used, the integers included in the range are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., up to and including 20.

[0109] When an amount, concentration, or other value or parameter is given as either a range, a preferred range, or a list of preferred upper and lower limits, this is understood to specifically disclose all ranges formed from any pairing of any upper or preferred value with any lower or preferred value, regardless of whether the ranges are individually disclosed. When a range of numerical values ​​is recited herein, unless otherwise stated, the range is intended to include its endpoints, and all integers and fractions within the range. It is not intended that the scope of the invention be limited to the specific values ​​recited when defining the range.

[0110] Ranges disclosed herein also encompass any and all overlaps, subranges, and combinations thereof. Words such as "up to," "at least," "greater than," "less than," and "between" include the recited numbers. Numbers preceded by terms such as "about" or "approximately" include the recited numbers. For example, "about 10 millipascal-seconds" includes "10 millipascal-seconds."

[0111] Also, as used herein, "and / or" broadly refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted as alternatives ("or").

[0112] Furthermore, as used herein, the term "about," when referring to a measurable value, such as an amount, dosage, time, temperature, etc., of a compound or agent of the invention, is meant to encompass a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount. The term "consisting essentially of" (and grammatical variations) shall be given its ordinary meaning and shall also mean that the referenced composition or method can contain additional components, so long as the additional components do not substantially alter the composition or method. The term "consisting of" (and grammatical variations) shall be given its ordinary meaning and shall also mean that the referenced composition or method is closed to the additional components. The term "comprising" (and grammatical variations) shall be given its ordinary meaning and shall also mean that the referenced composition or method is closed to the additional components.

[0113] The subject matter of the present disclosure will be described in more detail below. However, many modifications and other embodiments of the subject matter of the present disclosure described herein will occur to those skilled in the art to which the subject matter of the present disclosure relates, having the benefit of the teachings presented in the foregoing description. Therefore, it should be understood that the subject matter of the present disclosure is not limited to the specific embodiments disclosed, and modifications and other embodiments are intended to fall within the scope of the appended claims. In other words, the subject matter described herein includes all alternatives, modifications, and equivalents. In the event that one or more of the incorporated documents, patents, and similar materials differ or contradict this application, including, but not limited to, defined terms, term usage, described techniques, etc., this application shall prevail. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0114] I. Compound As disclosed elsewhere herein, some embodiments disclosed herein relate to small molecules capable of delivering NO to achieve antimicrobial activity. In some embodiments, the cation present in the small molecule possesses antimicrobial or other desirable physiological properties. In some embodiments, the compound is water-soluble.

[0115] In one embodiment, provided herein are NO-releasing compounds that exhibit potent antibacterial characteristics, comprising the structure of Formula I: [ka] During the ceremony, X is selected from the group consisting of H, D, R, and RC(O)—; R is C optionally substituted with one or more substituents. 1~12 alkyl, aryl, heteroaryl, alkylaryl, or arylalkyl; The substituents are independently -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, each instance of a, b, c, d, e, f, g, h, i, j, k, and l is independently selected from the integer 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; M + is a pharmaceutically acceptable cation.

[0116] In some embodiments, M + For example, +2 or +3 where the ratio of compounds of Formula I to cations is such that the total positive charge equals the total negative charge. Thus, for a compound with a total charge of -3 and a cation with a total charge of +2, there are two compounds and three cations.

[0117] Representative positively charged cations include sodium, potassium, lithium, calcium, magnesium, and quaternary ammonium salts.

[0118] In another embodiment, the compound has the structure: [ka] In the formula, M + refers to a pharmaceutically acceptable cation. The cation can be any pharmaceutically acceptable, non-toxic cation known to those skilled in the art, including, but not limited to, sodium, potassium, lithium, calcium, magnesium, ammonium, or substituted ammonium. Those skilled in the art will understand that when the cation (M) has a valence greater than 1, the ratio of negative charges in the methyltrisdiazeniumdiolate moiety to positive charges in the cation will be balanced. For example, when the cation (M) has a charge of +2, the ratio of negative charges in the methyltrisdiazeniumdiolate moiety to positive charges in the cation will be balanced. +2 The ratio of cation to methyltrisdiazeniumdiolate is 2 / 3, and if the cation (M) has a +3 charge, the ratio of cation to methyltrisdiazeniumdiolate is 1 / 1.

[0119] One representative compound is shown below. [ka]

[0120] Formula II is also described as methanetrisdiazeniumdiolate (MTDD) and Formula III is described as methanetrisdiazeniumdiolate sodium salt.

[0121] Although various NO donors (e.g., diazeniumdiolates, S-nitrosothiols, metal nitrosyls, organic nitrates) are known to provide controlled exogenous NO delivery, the diazeniumdiolate moieties in the compounds disclosed herein are attractive because of their good stability and easy storage, and because they spontaneously undergo proton-induced dissociation under physiological conditions to regenerate nitric oxide containing NO radicals.

[0122] The C-diazeniumdiolates described herein are pH-triggered NO release donors. Upon reaction with protons under physiological conditions (e.g., 37°C, pH 7.4), one mole of Formula III generates two moles of NO radicals and two to three moles of nitroxyl compound.

[0123] Some embodiments disclosed herein have one or more of the following advantages: efficient and unique synthetic routes and resulting small molecule chemical compositions.

[0124] In some embodiments, the NO-releasing compound is stable at various temperatures up to 20°C (e.g., 40°C, 45°C, 55°C, 60°C, 80°C, etc.) and over an extended storage period (e.g., 10 hours, 20 hours, 22 hours, 25 hours, 30 hours, etc., 1 day, 3 days, 5 days, 6 days, 7 days, 15 days, 30 days, 45 days, etc., 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, etc., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, etc., or even 1 year (or more)).

[0125] In some embodiments, the compound has an NO storage capacity (μmol of NO per mg of compound) of about 0.25, 0.4, 0.5, 1.0, 1.5, 2.0, 3.0, or greater than or equal to the ranges inclusive and / or ranging from the aforementioned values. In some embodiments, within 2 hours of addition to PBS buffer, the compound releases 25%, 50%, 75%, 85%, 90%, 95%, 100%, or greater than or equal to the ranges inclusive and / or ranging from the aforementioned values ​​of the total weight percent bound NO. In some embodiments, NO release when used to reduce or eliminate biofilms occurs in similar amounts, e.g., about 20-25%, about 30-50%, about 60-75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than or equal to the ranges inclusive and / or ranging from the aforementioned values ​​of the total weight percent bound NO.

[0126] In some embodiments, NO release can occur over a period of about 0.01 hours, 0.1 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or a range of values ​​inclusive and / or ranging from the aforementioned values. In some embodiments, the NO release half-life is about 0.01 hours, 0.1 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or a range of values ​​inclusive and / or ranging from the aforementioned values. In some embodiments, NO release is about 0.01 hours, 0.1 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, or a range of values ​​inclusive and / or ranging from the aforementioned values.

[0127] In some embodiments, the compound has a degradation rate / time in an amylase enzyme exposure assay of less than or equal to about 0.2%, 0.5%, 1.0%, 1.5%, 2.5%, 5.0%, 10%, or a range including and / or spanning the aforementioned values.

[0128] In some embodiments, the compounds have antibacterial activity. In some embodiments, the compounds can effectively eradicate or reduce the viability of microorganisms (e.g., prokaryotic cells, bacteria, viruses, protozoa, fungi, algae, amoeba, slime mold, etc.) that have low toxicity to native tissues and patient cells (e.g., eukaryotic cells, mammalian cells, human cells, etc.).

[0129] In some embodiments, the compounds provide a 90% or greater bacterial reduction in a bacterial viability assay performed under static conditions over a 2-hour period against one or more of P. aeruginosa, S. aureus, P. gingivalis, A. actinomycetemcomitans, A. viscosus, and / or S. mutans at concentrations of about 4 mg / ml, 2 mg / ml, 1 mg / ml, 0.5 mg / ml, 0.1 mg / ml, 0.05 mg / ml, or a range inclusive of and / or spanning the foregoing values. In some embodiments, the disclosed functionalized NO-releasing compounds provide a 99% or greater bacterial reduction and / or a 2-3 log reduction in a bacterial viability assay performed under static conditions over a 2-hour period against Gram-positive bacteria at concentrations of about 4 mg / ml, 2 mg / ml, 1 mg / ml, 0.5 mg / ml, 0.1 mg / ml, 0.05 mg / ml, or a range inclusive of and / or spanning the foregoing values. In some embodiments, the disclosed functionalized NO-releasing polymers provide a bacterial reduction of 99% or greater and / or a 2-3 log reduction in Gram-negative bacteria at concentrations of about 4 mg / ml, 2 mg / ml, 1 mg / ml, 0.5 mg / ml, 0.1 mg / ml, 0.05 mg / ml, or a range including and / or spanning the aforementioned values, in a bacterial viability assay performed under static conditions over a 2-hour period. In some embodiments, the bacterial reduction is greater than 95%, greater than 98%, or greater than 99%.

[0130] II. Compound Synthesis Some embodiments disclosed herein provide for the synthesis and characterization of the diazeniumdiolate NO donor-modified compounds described herein. The synthesis of compounds capable of controlled NO storage and release is important for exploiting the role of NO in physiology and developing NO-based therapeutics.

[0131] Some embodiments disclosed herein have one or more of the following advantages: efficient and unique synthetic routes and the chemical compositions of the resulting constructs. While certain compounds described herein have been previously disclosed, this disclosure describes their synthesis and utility in pharmaceutical compositions, and methods for treating or preventing microbial infections or reducing microbial burden.

[0132] There are several methods for making compounds of Formula I. In one embodiment, compounds having R(CO)- moieties that do not contain an acidic α CH (i.e., α to a carbonyl), such as aryl, heteroaryl, and branched alkyl groups, such as t-butyl groups, can be prepared by reacting all acidic α CH on the methyl groups of a compound having the formula R(CO)CH3 with nitric oxide in basic methanol to give a trisdiazeniumdiolate. A representative reaction is shown below. [ka]

[0133] As an example, the reaction product of acetophenone with nitric oxide in KOH / methanol is: [ka]

[0134] In another embodiment, compounds where X is H or D can be prepared by reacting nitric oxide with acetone in basic or deuterated methanol to give the tris-diazeniumdiolate.

[0135] Formula II can be prepared by many different approaches, including the reaction of ethanol, acetonitrile, or acetone with nitric oxide gas in the presence of a basic methanol solution. Ideally, the nitric oxide gas is present at a pressure greater than atmospheric pressure, more ideally greater than 2 atmospheres, and preferably greater than 10 atmospheres. Higher pressures help ensure complete reaction. If the reaction is incomplete, one of the by-products of the reaction is methane bis-diazeniumdiolate. [ka]

[0136] Methane bis-diazeniumdiolate does not release NO or NHO under physiological conditions.

[0137] Reacting acetone with nitric oxide in the presence of a basic methanol solution, such as sodium hydroxide or potassium hydroxide in methanol, at relatively high pressure tends to yield the purest compounds. 13 The C NMR is shown in Figure 8 and as can be seen in this figure, there is one product peak.

[0138] Using sodium hydroxide in methanol, M + Na + The proposed reaction mechanism behind this reaction is shown below, which provides a compound which is [ka]

[0139] The following proposed mechanism involves the decomposition of this compound to form nitric oxide. [ka]

[0140] Decomposition products 1 H and 13 The C NMR spectra are shown in Figures 7 and 8, respectively. Although not explicitly shown, it is expected that NO is not produced directly, but rather is formed when two moles of nitroxyl (HNO) are released, which then dimerize and react to form NO and HO as shown. Nitroxyl groups are reactive nitrogen molecules and may themselves play a role in enhancing the antibacterial activity of compounds of Formula III.

[0141] The decomposition pathway is particularly interesting because it clarifies how NO is produced from carbon-bonded diazeniumdiolates. Typically, carbon-bonded diazeniumdiolates do not produce NO. Instead, they produce HNO, which dimerizes to form nitrous oxide, NO. In this case, the initial decay of Formula III follows the expected HNO pathway, followed by the formation of an intermediate alcohol. Upon reconstitution of the alcohol intermediate, two moles of NO gas can be released. This is consistent with experimental results showing that one mole of Formula III compound releases approximately two moles of NO. NMR of the decomposed Formula III byproduct (shown in Figures 9 and 10) also agrees with the proposed structure for the fully decomposed molecule.

[0142] III. Pharmaceutical Compositions Also disclosed are pharmaceutical compositions comprising one or more compounds of Formula I, II, or III together with a suitable pharmaceutically acceptable carrier or excipient.

[0143] According to some embodiments, the compounds described herein can be present in aqueous solutions containing concentrations of about 100 μg / mL or more, such as about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 40 mg / mL or more. The amount of the second compound in the aqueous composition can be at least about 10% by weight based on the weight of the first compound, and can also be higher, for example, at least about 20% by weight, at least about 30% by weight, or at least about 50% by weight, on the same basis. The compounds in the aqueous composition are selected so that the compounds are mutually miscible.

[0144] In some embodiments, the compositions disclosed herein provide a NO-releasing compound discussed herein, which has an NO storage capacity (μmol of NO per mg of powder) of about 2.0, 4.0, 6.0, 8.0, or 10.0, or a range including and / or ranging from the aforementioned values, or greater. In some embodiments, within 2 hours of addition to the PBS buffer described in the Examples, the NO-releasing compound releases about 25%, 50%, 75%, 85%, 90%, 95%, 100%, or greater of the total weight percent bound NO, or a range including and / or ranging from the aforementioned values, or greater.

[0145] In some embodiments, the composition is in the form of a liquid, dry powder, gel, or aerosol. The composition may be provided in the form of a formulation loaded into a delivery device such as an inhaler.

[0146] In some embodiments, the compositions comprise a compound described herein at a concentration of about 1 mg / ml, 10 mg / ml, 20 mg / ml, 50 mg / ml, 100 mg / ml, 250 mg / ml, or a range up to and including and / or spanning the aforementioned values.

[0147] Pulmonary administration formulations In some embodiments, the compound is administered to the lungs (i.e., by pulmonary administration).In one particular embodiment, pulmonary administration typically involves inhaling the compound in the form of particles or droplets, for example, by nasal inhalation, oral inhalation, or both.The formulation can be developed to be aerosolized by a metered dose inhaler, dry powder inhaler, liquid spray, or nebulizer.Nebulization can be achieved by compressed air, ultrasonic energy, or vibrating mesh to form multiple droplets or solid particles containing the NO-releasing compound.

[0148] In one aspect of this embodiment, the particles may be formulated as an aerosol (i.e., droplets of a stable dispersion or suspension of particles comprising one or more of the compounds described herein in a gaseous medium). Particles delivered by aerosol may be deposited in the respiratory tract by gravitational settling, inertial impaction, and / or diffusion.

[0149] Whether administered by inhalation or nebulization, the particles or droplets can be administered in two or more separate administrations (doses).

[0150] In one embodiment, the composition is administered by inhalation to treat bacterial infections associated with cystic fibrosis, including, but not limited to, stenotrophomonis, mybacterium avium intracellulaire and m. abcessus, burkhoderia cepacia, and Pseudomonas aeruginosa (P. aeruginosa) infections.

[0151] Biodegradable particles can be used for controlled release and delivery of the compounds described herein. Aerosols have been developed to deliver therapeutic agents to the respiratory tract. Adjei, A. and Garren, J. Pharm Res. 7, 565-569 (1990), and Zanen, P. and Lamm, J.-WJ Int. J. Pharm. 114, 111-115 (1995).

[0152] porous particles The respiratory tract includes the upper airways, including the oropharynx and larynx, followed by the lower airways, including the trachea, followed by bifurcation into the bronchi and bronchioles. The upper and lower airways are called the conducting airways. The terminal bronchioles then divide into respiratory bronchioles, which lead to the final respiratory zone, the alveoli, or deep lung. Gonda, I., "Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract," in Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313, 1990. The deep lung, or alveoli, is the primary target of inhaled therapeutic aerosols for systemic drug delivery.

[0153] Therefore, it may be important to deliver antiviral particles to the deep lung (i.e., the alveolar region of the lung). Relatively large particles tend to become trapped in the oropharyngeal cavity, which can lead to excessive loss of inhaled drug. Relatively small particles can be delivered to the deep lung but may be phagocytosed. One method for delivering relatively large particles (sized to avoid phagocytosis) that are light enough to avoid excessive trapping in the oropharyngeal cavity is to use porous particles.

[0154] In one embodiment, particles for delivering the compounds described herein to the alveolar region of the lung are porous "aerodynamically light" particles, as described in U.S. Patent No. 6,977,087. Aerodynamically light particles can be made from biodegradable materials and typically have a density of 0.4 g / cm. 3The particles have a tap density of less than 100 μm and a mass average diameter of 5 μm to 30 μm. The particles may be formed from a biodegradable material, such as a biodegradable polymer. For example, the particles may be formed from a functionalized polyester graft copolymer consisting of a linear alpha hydroxy acid polyester backbone having at least one amino acid group incorporated herein and at least one poly(amino acid) side chain extending from the amino acid group in the polyester backbone. In one embodiment, aerodynamically light particles with a large average diameter, e.g., greater than 5 μm, can be used to enhance delivery of one or more of the compounds described herein to the alveolar region of the lung.

[0155] aqueous solution In some embodiments, the compounds disclosed herein are administered and delivered as an aqueous solution, for example, topically, intranasally, intravenously, by injection, and by spray. In some embodiments, the solution contains one or more salts and is isotonic.

[0156] Oral Drug Delivery Vehicles In some embodiments, the composition can be in the form of, for example, tablets, pills or capsules, which are prepared by conventional techniques using pharmaceutically acceptable excipients.Representative excipients include binders (for example, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose), fillers (for example, lactose, microcrystalline cellulose or calcium hydrogen phosphate), lubricants (for example, magnesium stearate, talc or silica), disintegrants (for example, potato starch or sodium starch glycolate), wetting agents (for example, sodium lauryl sulfate), suspending agents, solubilizers, and their mixtures.

[0157] Tablets can be coated by methods known in the art. For example, the therapeutic agent can be combined with hydrochlorothiazide and formulated as a pH-stabilized core with an enteric or delayed-release coating that protects the therapeutic agent until it reaches the target organ.

[0158] Liquid preparations for oral or topical administration can be in the form of, for example, solution, syrup or suspension, or they can be presented as a dry product that is to be reconstituted with water or another suitable vehicle before use.Such liquid preparations can be prepared by conventional techniques using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils), and preservatives (e.g., methyl or propyl p-hydroxybenzoate or sorbic acid).Preparations can also contain buffer salts, flavoring agents, coloring agents and sweeteners as needed.Preparations for oral administration can be suitably formulated to achieve controlled release of active compounds.For buccal administration, the composition can be in the form of tablets or lozenges formulated in a conventional manner.

[0159] Nanoparticle Composition The compounds described herein can also be administered in the form of nanoparticle compositions. In one embodiment, the controlled-release nanoparticle formulation comprises a nanoparticle active agent and a rate-controlling polymer that functions to extend the release of the agent after administration. In this embodiment, the composition can release the active agent for a period ranging from about 2 to about 24 hours, or up to 30 days or more, after administration. Exemplary controlled-release formulations containing nanoparticle forms of the active agent are described, for example, in U.S. Patent No. 8,293,277.

[0160] The nanoparticle composition comprises particles of the active agent described herein, with a non-crosslinked surface stabilizer adsorbed on or associated with their surface. The average particle size of the nanoparticles is typically less than about 800 nm, more typically less than about 600 nm, and even more typically less than about 400 nm, less than about 300 nm, less than about 250 nm, less than about 100 nm, or less than about 50 nm. In one aspect of this embodiment, at least 50% of the particles of the active agent have an average particle size of less than about 800, 600, 400, 300, 250, 100, or 50 nm, respectively, as measured by light scattering techniques.

[0161] Various surface stabilizers are typically used with nanoparticle compositions to prevent particle agglomeration or aggregation.Representative surface stabilizers include gelatin, lecithin, dextran, acacia gum, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glycerol monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycol, polyoxyethylene stearate, colloidal silicon dioxide, phosphate, sodium dodecyl sulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, amorphous cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, polyvinylpyrrolidone. , tyloxapol, poloxamer, poloxamine, poloxamine 908, dialkyl ester of sodium sulfosuccinate, sodium lauryl sulfate, alkylaryl polyether sulfonate, mixture of sucrose stearate and sucrose distearate, p-isononylphenoxypoly-(glycidol), SA9OHCO, decanoyl-N-methylglucamide, n-decyl-D-glucopyranoside, n-decyl-D-maltopyranoside, n- The stabilizer is selected from the group consisting of dodecyl-D-glucopyranoside, n-dodecyl-D-maltoside, heptanoyl-N-methylglucamide, n-heptyl-D-glucopyranoside, n-heptyl-D-thioglucoside, n-hexyl-D-glucopyranoside, nonanoyl-N-methylglucamide, n-nonyl-D-glucopyranoside, octanoyl-N-methylglucamide, n-octyl-D-glucopyranoside, and octyl-D-thioglucopyranoside. Lysozyme can also be used as a surface stabilizer for nanoparticle compositions.

[0162] Representative rate-controlling polymers into which the nanoparticles can be formulated include chitosan, polyethylene oxide (PEO), polyvinyl acetate phthalate, gum arabic, agar, guar gum, cereal gum, and the like. gums), dextran, casein, gelatin, pectin, carrageenan, wax, shellac, hydrogenated vegetable oils, polyvinylpyrrolidone, hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), hydroxypropyl methylcellulose (HPMC), sodium carboxymethylcellulose (CMC), poly(ethylene) oxide, alkyl celluloses, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, hydrophilic cellulose derivatives, polyethylene glycol, polyvinylpyrrolidone, cellulose acetate, cellulose acetate butyrate, cellulose acetate phthalate, cellulose acetate trimellitate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinylacetyl diethylaminoacetate, poly(alkyl methacrylates), poly(vinyl acetate), polymers derived from acrylic or methacrylic acid and their respective esters, and copolymers derived from acrylic or methacrylic acid and their respective esters.

[0163] Methods for making nanoparticle compositions are described, for example, in U.S. Pat. Nos. 5,518,187 and 5,862,999 (both for "Method of Grinding Pharmaceutical Substances"), U.S. Pat. No. 5,718,388 (for "Continuous Method of Grinding Pharmaceutical Substances"), and U.S. Pat. No. 5,510,118 (for "Process of Preparing Therapeutic Compositions Containing Nanoparticles").

[0164] Controlled-release formulations In a preferred embodiment, the active compound is prepared with a carrier that protects the compound from rapid excretion from the body, for example, controlled-release formulations, including but not limited to implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used.For example, enteric-coated compounds can be used to protect against digestion by stomach acid.The method for preparing such formulations will be clear to those skilled in the art.Suitable materials can also be obtained commercially.

[0165] Liposomal suspensions (including but not limited to liposomes targeted to infected cells using monoclonal antibodies against viral antigens) are also preferred as pharmaceutically acceptable carriers.They can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811 (incorporated by reference).For example, liposomal preparations can be prepared by dissolving suitable lipids (such as stearoyl phosphatidylethanolamine, stearoyl phosphatidylcholine, arachadoyl phosphatidylcholine, and cholesterol) in an inorganic solvent that is then evaporated, leaving a thin film of dried lipid on the surface of the container.Then, an aqueous solution of active compound is introduced into the container.The container is then swirled by hand to release lipid material from the side of the container and disperse lipid aggregates, thereby forming a liposomal suspension.

[0166] Mucoadhesive Mucoadhesion is currently defined as adhesion between two materials, at least one of which is a mucosal surface. Compounds such as NO donors are often delivered locally because their half-lives are often shorter than the time required for systemic distribution. The mucoadhesives described herein enable formulations suitable for mucoadhesive drug delivery systems (buccal, nasal, ocular, gastric, vaginal, and rectal). Mucoadhesive-containing topical and local systems have been shown to exhibit enhanced bioavailability. For example, they typically utilize mucosal tissues, which have a high surface area and high blood flow, enhancing absorption (compared to non-mucoadhesive formulations).

[0167] In some embodiments, the mucoadhesive is a mucoadhesive polymer. In some embodiments, the mucoadhesive has multiple hydrophilic groups, such as hydroxyl, carboxyl, amide, and sulfate. These groups allow binding to mucus or cell membranes through physical and chemical interactions, such as hydrogen bonding, hydrophobic interactions, electrostatic interactions, or conformational interactions. Hydrophilicity increases by attracting water for greater hydration, and when in a gelatinous state, the mucoadhesive physically swells. Aspects to consider when selecting an appropriate mucoadhesive include: 1. Toxicity of the mucoadhesive and any potential degradation products, NO donors, and nitrosating environments, taking into account absorption and mucosal surface irritation; 2. Formulation compatibility with NO donors, 3. Biochemical mucoadhesive properties with mucus and epithelial cell surfaces (adhesion, kinetics, specificity, effects on pH and osmolality, stability, etc.), and 4. Commercial considerations (regulatory status, cost, availability, etc.).

[0168] In one embodiment, mucoadhesives adhere to mucosal surfaces through nonspecific, noncovalent interactions that are primarily electrostatic in nature. In another embodiment, mucoadhesives adhere to mucosal surfaces through hydrophilic functional groups that hydrogen bond with similar groups on biological substrates. In another embodiment, mucoadhesives adhere to mucosal surfaces through specific receptor sites on cells or mucus surfaces. For example, lectins and thiolated polymers adhere to mucosal surfaces through specific receptor sites on cells or mucus surfaces. As used herein, lectins are proteins or glycoprotein complexes of non-immune origin that can selectively bind sugars in a noncovalent manner. Lectins have been proposed to bind to carbohydrates on mucus or epithelial cell surfaces. Thiolated polymers or thiomers have pendant thiols that impart hydrophilicity to, for example, polyacrylate or cellulosic polymer backbones. The thiol groups can form stable covalent bonds with mucus glycoproteins, resulting in increased persistence and improved bioavailability.

[0169] Many such mucoadhesives are known in the art. Useful mucoadhesive polymers include, but are not limited to, carbopol, N-isopropylacrylamide, polyvinyl alcohol / polyvinylpyrrolidone, dextran, hydroxyethyl methacrylate / methacrylic acid, polyvinyl alcohol, polyacrylamide, polyethylene glycol / polylactic acid, carboxymethyl chitosan, and collagen. Mucoadhesives may also include polycarbophil and other acrylate / methacrylate polymers, anionic polymers based on methacrylic acid esters, which form pH-selectively dissolvable hydrogels that dissolve within a physiologically specific pH range, generally from about pH 5.5 to about pH 7.5 (allowing physiological conditions to interact with NO and further initiate NO release). Such formulations that dissolve in a pH range from about 5.5 to about 6.0 are useful for targeting the duodenum. Dissolution at higher pHs generally targets the lower intestine. For example, a dissolution pH of about 6.5 to about 7.0 may be useful for targeting the colon.

[0170] In some embodiments, the mucoadhesive comprises a water-soluble polymer. In particular, the water-soluble polymer may or may not form a hydrogel to some extent when hydrated, but is capable of forming a fluid aqueous solution. Mucoadhesives of this type include, but are not limited to, polyols and polysaccharides, and hydroxylated celluloses (hydroxypropylmethylcellulose and hydroxymethylcellulose).

[0171] In certain embodiments, the mucoadhesive extends the resonance time of the nitric oxide donor at the target site, for example, the airways.

[0172] In certain other embodiments, the mucoadhesive may also have adhesion specificity to biofilms containing pathogenic species, for example, some alginate oligomers are known to interact with Pseudomonas aeruginosa biofilms.

[0173] chelating agents In exemplary embodiments, the compositions disclosed herein may include one or more chelating agents. According to one aspect, a chelating agent is included to capture trace metals to prevent potential adverse effects on the NO donor compound. Exemplary chelating agents are known in the art and include, for example, ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA), as well as other compounds described in Baldari et al., "Current Biomedical Use of Copper Chelation Therapy," Int J Mol Sci. 2020;21(3):1069. (2020).

[0174] gallium One aspect of the present invention is a pharmaceutical composition that utilizes the synergistic antibacterial properties of NO and gallium. Synergy is a concept known in the art, and methods for determining synergy are provided below. A mini-review of antibacterial synergy measurements was prepared by Christopher Dern of Virginia Commonwealth University and can be found at jcm.asm.org / content / jcm / 52 / 12 / 4124.full.pdf (incorporated herein by reference in its entirety).

[0175] In an exemplary embodiment, a pharmaceutical composition is disclosed that includes a nitric oxide-releasing compound and an aqueous solution containing gallium. In one embodiment, the nitric oxide-releasing compound is a diazeniumdiolate or a nitrosothiol. In one embodiment, the gallium is at least partially complexed in the aqueous solution with a citrate moiety. The aqueous solution may also include chloride ions. These chloride ions remain, for example, when gallium chloride is dissolved in the solution. The aqueous solution may also include nitrate ions. These nitrate ions remain, for example, when gallium nitrate is dissolved in the solution. Citrates or other chelating agents described herein may be included in the formulation to stabilize gallium against the formation of gallium hydroxide, which is not highly soluble in water and can precipitate. In one embodiment, the form of gallium is gallium nitrate.

[0176] Further provided herein are antimicrobial compositions comprising gallium and a nitric oxide-releasing cyclodextrin and their use in reducing microbial load. The inventors of the present application have surprisingly discovered that not only are compositions comprising a nitric oxide donor and gallium effective against planktonic bacteria and biofilms, but that these compositions also exhibit a synergistic effect against them.

[0177] Provided herein are compositions comprising gallium and one or more of the compounds described herein, as well as methods for treating various pathophysiologies using such compositions that utilize the synergistic effects of gallium in combination with an NO-donating compound, taking advantage of the full potential of NO-releasing pharmacological compounds and compositions to utilize enhanced NO-releasing characteristics and beneficial physical properties. In some embodiments, provided herein are compositions that are highly effective as antibacterial agents. In some embodiments, provided herein are compositions with beneficial antibacterial properties. In some embodiments, the polymers and / or scaffolds disclosed herein have advantageous activity as mucoadhesives or chelators described herein.

[0178] The gallium can be gallium(III), gallium nitrate (Ga(NO3)3), gallium chloride, a pharmaceutically acceptable salt, a pharmaceutically acceptable complex, or a combination thereof.

[0179] The composition may contain about 0.001 mg to 100 mg of gallium. For example, the composition may contain about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, or 0.01 mg of gallium. The composition may contain about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 mg of gallium. The composition may contain about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg of gallium. The composition may contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg of gallium. The composition may include about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg of gallium.

[0180] The composition may comprise gallium dosed at about 0.001 mg to 100 mg of gallium per kg of patient. The composition may comprise gallium dosed at about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg of gallium per kg of patient.

[0181] Gallium may be in the form of a pharmaceutically acceptable salt or a pharmaceutically acceptable complex. In one embodiment, the pharmaceutically acceptable salt is an anion selected from the group consisting of nitrate, citrate, chloride, acetate, isocitrate, tartrate, and mixtures thereof. In another embodiment, the anion is a compound described herein, i.e., M + is gallium. When the anion is not a compound described herein, gallium is present with a different anion and the compound is present with a different cation, but some metathesis may occur, resulting in the formation of a gallium salt of a compound described herein at equilibrium.

[0182] Pharmaceutically acceptable complexing agents, also referred to herein as chelating agents, may be mannitol, maltolate or derivatives, protoporphyrin IX or derivatives, lactoferrin, transferrin, ferritin, bacterial siderophores belonging to the catecholate, hydroxamate and hydroxycarboxylate groups, bacterial hemophores, and any iron chelator.

[0183] Siderophore Siderophores have been used in drugs for iron and aluminum overload therapy and antibiotics for improved targeting. For example, iron-chelating microbial siderophores can be conjugated to antibiotics or antibacterial agents to enhance uptake and antibacterial activity. Some embodiments disclosed herein relate to pharmaceutical formulations comprising at least one nitric oxide-releasing moiety, optionally in combination with at least one siderophore. In some embodiments, at least one siderophore is functionalized with an NO-donating group, and the composition further comprises at least one pharmaceutically acceptable excipient.

[0184] transplant In some embodiments, the disclosed compositions can also be formulated as preparations for implantation.Thus, for example, the compositions can be formulated with suitable polymers or hydrophobic materials (for example, as emulsions in acceptable oils) or ion exchange resins, or as sparingly soluble derivatives (for example, as sparingly soluble salts).The compositions can also be formulated as rectal compositions (for example, suppositories or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides), creams or lotions, or transdermal patches.

[0185] Composition characteristics In some embodiments, the combination of all the various components of the pharmaceutical composition, including the molecular weight, concentration, or other chemical characteristics of the compound, and the other components in the composition, contributes to the tunability of the properties of the compositions disclosed herein. In some embodiments, by varying one or more of these characteristics, one or more properties of the composition can be adjusted according to the preferred properties described herein. In some embodiments, NO release rate, antibacterial effect, water solubility, degradation rate, viscosity, gel hardness (if the formulation forms a gel), viscoelasticity, elastic modulus, etc. can be adjusted.

[0186] In embodiments in which a polymer is present in a formulation with a small molecule NO-releasing compound described herein, the properties of the composition can be adjusted by adjusting the molecular weight of the particular polymer used in the formulation. In some embodiments, the weight average molecular weight (M w ) (kDa) is 2.5, 5.0, 7.0, 10, 15, 30, 50, 100, 200, 500, 750, 1,000, 2,000, 10,000, or a range including and / or spanning the foregoing values, or greater. In some embodiments, the number average molecular weight (M n ) (kDa) is 2.5, 5.0, 7.0, 10, 15, 30, 50, 90, 100, 200, 500, 700, 1,000, 2,000, 10,000, or a range inclusive and / or spanning the aforementioned values, or greater. In some embodiments, the polymers disclosed herein can have n repeating units. In some embodiments, n is 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, or a range inclusive and / or spanning the aforementioned values, or greater. In some embodiments, size exclusion chromatography (SEC) can be used to measure the molecular weight of the backbone structures disclosed herein. In some embodiments, a multi-angle light scattering (SEC-MALS) detector can be used. In some embodiments, the backbone structures can be characterized using their polydispersity index. The polydispersity index (PDI) is a measure of the distribution of molecular weights in a given polymer sample. The PDI can be calculated by dividing the weight average molecular weight and the number average molecular weight. In some embodiments, the backbone structure has a PDI of about 1.05, 1.1, 1.2, 1.3, 1.5, 1.7, 1.8, 1.9, 2.0, or a range including and / or spanning the aforementioned values.

[0187] Representative polymers include those disclosed in U.S. Patent Application No. 62 / 441,742, U.S. Patent Application No. 62 / 483,505, International Application No. PCT / IB2018 / 050051, U.S. Patent Application No. 62 / 447,564, International Application No. PCT / IB2018 / 052144, U.S. Patent Application No. 14 / 421525, U.S. Patent Application No. 62 / 639,119, and U.S. Patent Application No. 62 / 737,603, each of which is incorporated by reference in its entirety for all purposes.

[0188] In some embodiments, the composition (including all components) may be water-soluble and / or miscible with one another. In some embodiments, the composition is soluble in water (at about 20° C.) at a concentration of about 1 mg / ml, 10 mg / ml, 20 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, 500 mg / ml, or a range of values ​​inclusive and / or spanning the aforementioned values ​​or greater.

[0189] According to some embodiments, the NO donor can be formulated in a pharmaceutical formulation at a concentration of 100 μg / mL or greater, e.g., higher, e.g., about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, 25 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, or about 200 mg / mL or greater. In exemplary embodiments, the polymer species can be formulated in a pharmaceutical formulation at a concentration of 100 μg / mL or greater, e.g., higher, e.g., about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, 25 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, or about 200 mg / mL or greater. The amount of polymer in the aqueous composition can be at least about 10% by weight, based on the weight of the NO donor, and may also be higher, for example, at least about 20%, at least about 30%, or at least about 50% by weight, on the same basis. Any combination of NO donor and polymer in the aqueous composition is selected to be miscible with each other. As described above, an NO donor and a polymer having antibacterial activity are considered to be miscible with each other if, upon visual inspection, at least about 90% of the polymer components remain soluble in each other after 24 hours of mixing in water at a concentration of 1 mg / ml of each polymer and maintaining at room temperature. Surprisingly, such mutual miscibility of the water-soluble polymer with the NO donor can be achieved despite the expected phase separation at the concentration and molecular weight of 1 mg / ml described herein. The aqueous compositions described herein can be prepared by mixing the individual formulation components with water, for example, at room temperature with stirring.

[0190] In some embodiments, the compositions disclosed herein have properties characteristic of a viscous fluid and / or gel. In some embodiments, the gel point of the composition is at room temperature (in water or PBS) at concentrations (w / w%) of about 0.5%, 1%, 2.5%, 5%, 10%, or a range of values ​​inclusive and / or inclusive of the foregoing. In some embodiments, the composition may have a gel point in water. In some embodiments, the composition gels in water (at about 20°C) at concentrations of about 0.5 mg / ml, 1 mg / ml, 10 mg / ml, 20 mg / ml, 50 mg / ml, 100 mg / ml, 250 mg / ml, or a range of values ​​inclusive and / or inclusive of the foregoing. In some embodiments, at a concentration of a 5% w / w solution, the viscosity (at 20°C) of the polymer is about 10, 50, 100, 1,000, 2,000, 5,000, 10,000, or a range of values ​​inclusive and / or inclusive of the foregoing. In some embodiments, the intrinsic viscosity of the polymer is about 0.5 m 3 / kg, 1.0m 3 / kg, 2.0m 3 / kg, 4.0m 3 / kg, 8.0m 3 / kg or greater, or a range including and / or spanning the aforementioned values.

[0191] In some embodiments, at a concentration of a 5% w / w solution, the composition has a hardness of 1.0 mN, 2.5 mN, 5 mN, 10 mN, 15 mN, 20 mN, 30 mN, 50 mN, or a range inclusive and / or ranging therefrom, or greater than the foregoing. In some embodiments, at a concentration of a 5% w / w solution, the formulation has a work of adhesion (mN*mm) of about 1.0, 2.5, 5, 10, 15, 20, 30, 50, 100, or a range inclusive and / or ranging therefrom, or greater than the foregoing. In some embodiments, at a concentration of a 5% w / w solution, the composition has a storage modulus (G') of about 250, 500, 1,000, 2,000, 4,000, 5,000, 10,000, or a range inclusive and / or ranging therefrom, or greater than the foregoing. In some embodiments, at a concentration of a 5% w / w solution, the composition has a modulus of elasticity (G") of 25, 50, 100, 200, 400, 500, 1000, 2000, 5000, 10000, or a range including and / or spanning the foregoing values, or greater. In some embodiments, the aqueous composition is characterized by a barrier activity, as measured by a reduction in the diffusion rate of an anionic dye, of greater than 2 log at a total backbone concentration of 40 mg / ml or less.

[0192] In some embodiments, the formulation is a gel, which is stable at various temperatures up to 20°C (e.g., 40°C, 45°C, 55°C, 60°C, 80°C, etc.) and over an extended storage period (e.g., 10 hours, 20 hours, 22 hours, 25 hours, 30 hours, etc., 1 day, 3 days, 5 days, 6 days, 7 days, 15 days, 30 days, 45 days, etc., 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, etc., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, etc., or even 1 year (or more)).

[0193] In some embodiments, the viscosity of the composition increases with increasing temperature or decreases with decreasing temperature. For example, the composition has a relatively high viscosity (e.g., in the form of a gel) when above the gelation temperature, and its viscosity decreases (e.g., in the form of a liquid) when cooled below the gelation temperature. In some embodiments, the polymers disclosed herein may therefore be reversible polymers (e.g., thermoreversible polymers), in which the transition from liquid to gel can be reversed upon exposure to appropriate conditions. For example, as described above, the compositions of the present disclosure include thermoreversible polymers, in which the viscosity of the composition can change depending on the temperature of the composition. In some embodiments, the tunability of the viscosity allows for tailoring of the composition profile upon delivery (e.g., more liquid at the delivery temperature and more viscous at, for example, body temperature).

[0194] In some embodiments, the compositions are characterized by their degree of swelling when exposed to water. In some embodiments, the percent swelling of the compositions disclosed herein is less than or equal to about 100, 250, 500, 1000, 2000, 5000, or a range including and / or spanning the aforementioned values. In other words, the compositions may swell or otherwise expand 2x, 4x, 5x, 10x, 20x, 50x, 100x, or more.

[0195] In certain embodiments, the compositions disclosed herein have a gelation temperature similar to a subject's normal body temperature, such as human body temperature, or 37°C. The gelation temperature refers to the intersection of a plot of elastic modulus and a plot of viscous modulus. In some cases, when a composition is below its gelation temperature, it has a relatively low viscosity, such as in a liquid form. In some instances, when a composition is above its gelation temperature, its viscosity increases (e.g., polymerizes) so that it is in a gel form. A composition that transitions from a liquid to a gel can facilitate administration of the composition to a subject, for example, by facilitating injection of a low-viscosity (e.g., liquid) composition at a temperature below its gelation temperature. After injection of the composition into a target treatment site, the temperature of the composition can increase due to heat absorption from surrounding body tissue, resulting in an increase in the viscosity of the composition (e.g., transition from a liquid to a gel, or polymerization), thereby providing structural and / or geometric support to the body tissue at the target treatment site. In some cases, gelation of the composition at the target treatment site can also facilitate retention of the composition at the treatment site by reducing diffusion and / or migration of the composition away from the treatment site. In certain embodiments, the composition has a gelation temperature of 30° C. to 40° C., for example, 32° C. to 40° C., including 35° C. to 40° C. In certain instances, the composition has a gelation temperature of 37° C.

[0196] Combination therapy The compounds described herein can be combined with conventional antibacterial compounds, for example, in addition to administering one or more of the compounds described herein, a patient can also be administered a conventional antibacterial agent.

[0197] Examples of conventional antibiotic agents include amikacin, tobramycin, gentamicin, piperacillin, mezlocillin, ticarcillin, imipenem, ciprofloxacin, ceftazidime, aztreonam, ticarcillin-clavulanate, dicloxacillin, amoxicillin, trimethoprim-sulfamethoxazole, cephalexin, piperacillin-tazobactam, linezolid, daptomycin, vancomycin, metronidazole, clindamycin, colistin, tetracycline, levofloxacin, amoxicillin and clavulanate (Augmentin®), cloclocillin, cyclosporin ... These include, but are not limited to, oxacillin, dicloxacillin, cefdinir, cefprozil, cefaclor, cefuroxime, erythromycin / sulfisoxazole, erythromycin, clarithromycin, azithromycin, doxycycline, minocycline, tigecycline, imipenem, meropenem, colitimethate / Colistin®, methicillin, oxacillin, nafcillin, carbenicillin, azlocillin, piperacillin and tazobactam (Zosyn®), cefepime, ethambutol, rifampin, and meropenem.

[0198] These antibiotics can also be combined with compounds that bind to or adsorb bacterial toxins, which can be particularly useful when bacterial toxins cause tissue damage. For example, Pseudomonas aeruginosa produces a variety of toxins that lead to cell lysis and tissue damage in the host. Type II toxins include exotoxin U (Exo U), which degrades the plasma membrane of eukaryotic cells, resulting in lysis; phospholipase C (PLC), which damages cellular phospholipids, causing tissue damage and stimulating inflammation; alkaline protease, which causes tissue damage; cytotoxins that damage the cell membranes of white blood cells and cause microvascular injury; elastase, which destroys elastin, a protein that is a component of lung tissue; and pyocyanin, a green-blue water-soluble pigment that catalyzes the formation of tissue-damaging toxic oxygen radicals, impairs ciliary function, and stimulates inflammation. Examples of compounds that bind these toxins include polyphenols and polyanionic polymers.

[0199] When the microorganism is a fungus, antifungal agent can be administered simultaneously.The typical antifungal agent that can be used includes fluconazole, posaconazole, viroconazole, itraconazole, echinocandin, amphotericin, and flucytosine.The selection of appropriate antifungal agent can be made by the treating physician.The following is a summary of fungal lung infections and their treatment.

[0200] Histoplasmosis is caused by the fungus Histoplasma capsulatum, and conventional treatments include itraconazole for mild and chronic lung disease, and a combination of itraconazole and amphotericin B (AmB) for moderate to severe histoplasmosis.

[0201] Blastomycosis is caused by Blastomyces dermatitis, and conventional treatments include itraconazole for mild to moderate disease and liposomal AmB (L-AmB) followed by itraconazole for life-threatening pulmonary infections.

[0202] Sporotrichosis is caused by Sporothrix schenckii, and conventional treatment for mild to moderate pulmonary disease involves itraconazole, while AmB followed by itraconazole is recommended for severe disease.

[0203] Coccidioidomycosis is caused by Coccidioides immitis and Coccidioides posadasii. Immunocompetent infected hosts may not require treatment, but immunocompromised patients are treated with fluconazole or itraconazole, and in severe cases, AmB followed by an azole.

[0204] Opportunistic fungal infections primarily occur in patients who are prone to immunodeficiency through congenital or acquired disease processes. Representative opportunistic infections are discussed below.

[0205] Aspergillosis is caused by Aspergillus, and related disorders include invasive pulmonary aspergillosis (IPA), chronic necrotizing aspergillosis, aspergilloma, and allergic bronchopulmonary aspergillosis. Conventional treatments for IPA include voriconazole, lipid-based AmB preparations, echinocandins, and posaconazole.

[0206] Cryptococcosis is an opportunistic infection seen in immunocompromised individuals, including those with HIV or AIDS and organ transplant recipients. Conventional treatment includes AmB (with or without flucytosine) followed by oral fluconazole. Fluconazole therapy is recommended for immunosuppressed or immunocompetent patients with mild to moderate symptoms.

[0207] Candidiasis occurs when Candida species colonize the pulmonary parenchyma. Many severely ill patients are treated empirically with broad-spectrum antibiotics. If these cases continue to clinically deteriorate and do not improve, empirical antifungal therapy is indicated. Triazole antifungals and echinocandins exhibit excellent lung penetration and can be used in addition to AmB preparations to treat pulmonary candidiasis.

[0208] Mucormycosis often develops in patients with diabetes mellitus, organ or hematopoietic stem cell transplants, neutropenia, or malignant tumors. Pulmonary mucormycosis is primarily observed in patients with neutropenia or a predisposing condition due to corticosteroid use. Conventional antifungal agents have difficulty penetrating lung tissue due to fungal adherence to and damage to endothelial cells, fungal vascular invasion, vascular thrombosis, and subsequent tissue necrosis. Therefore, conventional treatment involves debridement of necrotic tissue and antifungal therapy using AmB preparations, posaconazole, and iron chelation therapy.

[0209] Pneumocystis jirovecii pneumonia (PCP) occurs in patients with HIV / AIDS, hematologic and solid malignancies, organ transplants, and diseases requiring immunosuppressants. PCP is highly resistant to common antifungal therapies, including AmB and triazole antifungals, but can be treated with trimethoprim / sulfamethoxazole. Second-line treatments are primaquine plus clindamycin, atovaquone, IV pentamidine, or dapsone.

[0210] The antifungal agents identified herein can be co-administered with the therapeutic approaches described herein.

[0211] If a patient has a viral lung infection, they can be administered traditional antiviral agents used against such viruses. The choice of antiviral agent typically depends on the viral infection being treated. Influenza viruses are typically treated with oseltamivir (Tamiflu), zanamivir (Relenza), or peramivir (Rapivab), and RSV is treated with ribavirin (Virazol). Coronaviruses have also been treated with Tamiflu, ribavirin, certain anti-HIV compounds, and certain interferons, including Betaferon, Alferon, Multiferon, and Wellferon.

[0212] Combination therapy for specific use in the treatment of Covid-19 infection The compounds described herein can be combined with additional compounds useful for treating disease states that are also treated by the release of NO. Specifically, the compounds discussed below can be used in combination therapy to treat Covid-19 infection or other respiratory infections with similar pathology.

[0213] Various compounds that can be combined with the compounds described herein are discussed below.

[0214] In one aspect of this embodiment, the at least one other active agent is selected from the group consisting of fusion inhibitors, entry inhibitors, protease inhibitors, polymerase inhibitors, antiviral nucleosides such as remdesivir, GS-441524, N4-hydroxycytidine, and other compounds disclosed in U.S. Pat. No. 9,809,616 and their prodrugs, viral entry inhibitors, viral maturation inhibitors, JAK inhibitors, angiotensin-converting enzyme 2 (ACE2) inhibitors, SARS-CoV-specific human monoclonal antibodies, including CR3022, and agents of distinct or unknown mechanism.

[0215] Umifenovir (also known as Arbidol) is a representative fusion inhibitor.

[0216] Representative entry inhibitors include Camostat, luteolin, MDL28170, SSAA09E2, SSAA09E1 (which acts as a cathepsin L inhibitor), SSAA09E3, and tetra-O-galloyl-β-D-glucose (TGG). The chemical formulas of some of these compounds are provided below. [ka]

[0217] Other entry inhibitors include: [ka]

[0218] Remdesivir, sofosbuvir, ribavirin, IDX-184, and GS-441524 have the following formula: [ka]

[0219] Additionally, compounds that inhibit the cytokine storm, such as dexamethasone to combat blood clots, anticoagulants and / or platelet aggregation inhibitors, or compounds that chelate iron ions released from hemoglobin by viruses such as COVID-19, can be administered.

[0220] Representative ACE-2 inhibitors include sulfhydryl-containing agents such as alacepril, captopril (capoten), and zefnopril; dicarboxylate-containing agents such as enalapril (vasotec), ramipril (altace), quinapril (accupril), perindopril (coversyl), lisinopril (listril), benazepril (lotensin), imidapril (tanatril), trandolapril (mavik), and cilazapril (inhibace); and phosphonate-containing agents such as fosinopril (fositen / monopril).

[0221] For example, when used to treat or prevent infectious diseases, the active compound or its prodrug or pharmaceutically acceptable salt can be administered in combination with or alternately with another antiviral agent, including but not limited to those of the above formula.Generally, in combination therapy, effective dosages of two or more drugs are administered together, while during alternation therapy, effective dosages of each drug are administered consecutively.Dosage depends on the absorption, inactivation, and excretion rate of the drug, as well as other factors known to those skilled in the art.It should be noted that dosage values ​​will also vary depending on the severity of the condition to be alleviated.It should be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition.

[0222] Some agents for combination with the compounds described herein are disclosed in Ghosh et al., “Drug Development and Medicinal Chemistry Efforts Toward SARS-Coronavirus and Covid-19 Therapeutics,” ChemMedChem 10.1002 / cmdc.202000223.

[0223] Non-limiting examples of antiviral agents that may be used in combination with the compounds disclosed herein include those listed below.

[0224] Compounds for inhibiting cytokine storm Through its activation, the inflammatory response must be regulated to prevent damaging systemic inflammation, also known as a "cytokine storm." This is accomplished by numerous cytokines with anti-inflammatory properties, such as IL-10 and transforming growth factor β (TGF-β). Each cytokine acts on a different part of the inflammatory response. For example, products of a Th2 immune response suppress Th1 immune responses, and vice versa.

[0225] By resolving inflammation, collateral damage to surrounding cells can be minimized, with little long-term damage to the patient. Thus, in addition to using the compounds described herein to inhibit viral infection, one or more compounds that inhibit cytokine storm can be simultaneously administered.

[0226] Compounds that inhibit the cytokine storm include compounds that target fundamental immune pathways such as the chemokine network and the cholinergic anti-inflammatory pathway.

[0227] JAK inhibitors, such as JAK1 inhibitors and JAK2 inhibitors, can inhibit cytokine storm, and in some cases also have antiviral properties.Representative JAK inhibitors include those disclosed in U.S. Patent No. 10,022,378, such as Jakafi, tofacitinib and baricitinib, as well as LY3009104 / INCB28050, pacritinib / SB1518, VX-509, GLPG0634, INC424, R-348, CYT387, TG10138, AEG3482, and their pharmaceutically acceptable salts and prodrugs.

[0228] HMGB1 antibodies and COX-2 inhibitors can be used to downregulate cytokine storm. Examples of such compounds include Actemra (Roche). Celebrex (celecoxib), a COX-2 inhibitor, can be used. IL-8 (CXCL8) inhibitors can also be used.

[0229] Chemokine receptor CCR2 antagonists, such as PF-04178903, can reduce pulmonary immunopathology.

[0230] Selective α7Ach receptor agonists such as GTS-21 (DMXB-A) and CNI-1495 can be used. These compounds reduce TNF-α. HMGB1, a late mediator of sepsis, downregulates the IFN-γ pathway and prevents LPS-induced suppression of IL-10 and STAT3 mechanisms.

[0231] Compounds for treating or preventing blood clots Viruses that cause respiratory infections, including coronaviruses such as Covid-19, can be associated with blood clots in the lungs, and clots that can also damage the heart.

[0232] The compounds described herein can be co-administered with compounds that inhibit clot formation, e.g., blood thinners or compounds that break down existing clots, such as tissue plasminogen activator (TPA), Integrilin (eptifibatide), abciximab (ReoPro), or tirofiban (Aggrastat).

[0233] Blood thinners prevent blood clots from forming and existing clots from growing larger. There are two main types of blood thinners: anticoagulants, such as heparin or warfarin (also called Coumadin), which slow the biological process that creates clots, and antiplatelet agents, such as Plavix and aspirin, which prevent blood cells called platelets from clumping together to form clots.

[0234] As an example, Integrilin® is typically administered at a dosage of 180 mcg / kg as an intravenous bolus administered as soon as possible after diagnosis, with a continuous infusion of 2 mcg / kg / min (after the initial bolus) for up to 96 hours of therapy.

[0235] Representative platelet aggregation inhibitors include glycoprotein IIB / IIIA inhibitors, phosphodiesterase inhibitors, adenosine reuptake inhibitors, and adenosine diphosphate (ADP) receptor inhibitors, which can optionally be administered in combination with an anticoagulant.

[0236] Representative anticoagulants include coumarins (vitamin K antagonists), heparin and their derivatives, including unfractionated heparin (UFH), low molecular weight heparin (LMWH), and ultra-low molecular weight heparin (ULMWH); synthetic pentasaccharide inhibitors of factor Xa, including fondaparinux, idraparinux, and idrabiotaparinux; direct acting oral anticoagulants (DAOCs), such as dabigatran, rivaroxaban, apixaban, edoxaban, and betrixaban; and antithrombin protein therapeutics / thrombin inhibitors, such as the bivalent drugs hirudin, lepirudin, and bivalirudin and the monovalent argatroban.

[0237] Representative platelet aggregation inhibitors include pravastatin, Plavix (clopidogrel bisulfate), Pletal (cilostazol), Effient (prazgrel), Aggrenox (aspirin and dipyridamole), Brilinta (ticagrelor), caplacizumab, Kengreal (cangrelor), Persantine (dipyridamole), Ticlid (ticlopidine), and Yosprala (aspirin and omeprazole).

[0238] Additional compounds that can be used Additional compounds and compound classes that may be used in combination therapy include: monoclonal antibodies (mAbs), arbidol (umifenovir), Actemra (tocilizumab), APN01 (Aperion Biologics), ARMS-1 (containing cetylpyridinium chloride (CPC)), ASC09 (Ascletis Pharma), AT-001 (Applied Therapeutics Inc.) and other aldose reductase inhibitors (ARIs), ATYR1923 (aTyr Pharma, Inc.), Aviptadil (Relief Therapeutics), Azvudine, Bemcentinib, BLD-2660 (Blade Therapeutics), Bevacizumab, Brensocatib, Calquence (acalabrutinib), Camostat mesylate (TMPRSS2 inhibitor), Camrelizumab, CAP-1002 (Capricor) Therapeutics), CD24Fcm, Clevudine, (OncoImmune), CM4620-IE (CalciMedica Inc., a CRAC channel inhibitor), Colchicine, restorative plasma, CYNK-001 (Sorrento Therapeutics), DAS181 (Ansun Pharma), Desferal, Dipyridamole (Persantine), Dociparstat sodium (DSTAT), Duvelisib, Eculizumab, EIDD-2801 (Ridgeback Biotherapeutics), Emapalumab, Fadraciclib (CYC065), and seliciclib (roscovitine) (Cyclin-dependentkinase (CDK) inhibitors), Farxiga (dapagliflozin), Favilavir / Favipiravir / T-705 / Avigan, Galidesivir, Ganovo (danoprevir), Gilenya (fingolimod) (sphingosine 1-phosphate receptor modulator), Gimsilumab, IFX-1, Ilaris (canakinumab), intravenous immunoglobulin, Ivermectin (importin α / β inhibitor), Kaletra / Aluvia (lopinavir / ritonavir), Kevzara (sarilumab), Kineret (anakinra), LAU-7b (fenretinide), lenzilumab, leronlimab (PRO) 140), LY3127804 (anti-Ang2 antibody), Leukin (sargramostim, granulocyte-macrophage colony-stimulating factor), losartan, valsartan, telmisartan (angiotensin II receptor antagonist), meplasmab, Metabloc (LSALT peptide, DPEP1 inhibitor), methylprednisolone and other corticosteroids, MN-166 (ibudilast, macrophage migration inhibitory factor (MIF) inhibitor), MRx-4DP0004 (bifidobacterium breve strain, 4D Pharma), Nafamostat (serine protease inhibitor), neuraminidase inhibitors such as Tamiflu (oseltamivir), Nitazoxanide (nucleocapsid (N) protein inhibitor), Nivolumab, OT-101 (Mateon), Novaferon (artificial interferon), oseltamivir (yeliva) (sphingosine kinase-2 inhibitor), Otilimab, PD-1 blocking antibodies, pegylated interferons such as pegylated interferon lambda, Pepcid (famotidine), Piclidenoson (A3 adenosine receptor agonist), Prezcobix (darunavir), PUL-042 (Pulmotect, Inc., toll-like receptor (TLR) binder), Rebif (interferon beta-1a), RHB-107 (upamostat) (serine protease inhibitor, RedHill BiopharmaLtd.), Selinexor (a selective inhibitor of nuclear export (SINE)), SNG001 (Synairgen, inhaled interferon beta-1a), solnatide, stem cells including mesenchymal stem cells, MultiStem (Athersys), and PLX (Pluristem Therapeutics), Sylvant (siltuximab), Thymosin, TJM2 (TJ003234), Tradipitant (a neurokinin-1 receptor) antagonist), Truvada (emtricitabine and tenofovir), Ultomiris (ravulizumab-cwvz), Vazegepant (a CGRP receptor antagonist or blocker), and Xofluza (baloxavir marboxil).

[0239] Repurposed antiviral drugs Many pharmaceutical agents, including those active against other viruses, have been evaluated and found to be active against Covid-19. Any of these compounds can be combined with the compounds described herein. Representative compounds include lopinavir, ritonavir, niclosamide, promazine, PNU, UC2, cinanserin (SQ10,643), calmidazolium (C3930), tannic acid, 3-isotheaflavin-3-gallate, theaflavin-3,3'-digallate, glycyrrhizin, S-nitro-N-acetylpenicillamine, nelfinavir, niclosamide, chloroquine, hydroxychloroquine, 5-benzyloxygramin, ribavirin, interferons such as interferon (IFN)-α, IFN-β, and pegylated versions thereof, and combinations of these compounds with ribavirin, chlorpromazine hydrochloride, triflupromazine hydrochloride, gemcitabine, imatinib mesylate, dasatinib, and imatin.

[0240] IV. Types of microorganisms that can be treated The following are non-limiting examples of microorganisms, including bacteria, viruses, and fungi, that can be treated using the compounds described herein.

[0241] Types of bacterial infections that can be treated In one embodiment, the compounds described herein are used to treat bacterial infections of the respiratory tract. Examples of treatable pathogens include Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus lugdunensis, Staphylococcus epidermidis, Streptococcus milleri / anginous, Streptococcus pyogenes, non-tuberculous mycobacterium, Mycobacterium tuberculosis, Burkholderia spp., Achromobacter xylosoxidans, Pandoraeasputorum, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, Haemophilus pittmaniae, Serratia marcescens, Candida albicans, drug-resistant Candida albicans, Candida glabrata, Candida krusei, Candida guilliermondii, Candida auris, Candida tropicalis, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Aspergillus flavus, Morganella morganii, Inquilinus limosus, Ralstonia mannitolilytica, Pandoraea apista, Pandoraea pnomenusa, Pandoraea sputorum, Bdellovibrio bacteriovorus, Bordetella bronchiseptica, Vampirovibrio chlorellavorus, Actinobacter baumanni, Cupriadidus metallidurans, Cupriavidus pauculus, Cupriavidus respiraculi, Delftia acidivordans, Exophiliadermatitidis, Herbaspirillum frisingense, Herbaspirillum seropedicae, Klebsiella pneumoniae, Pandoraea norimbergensis, Pandoraea pulmonicola, Pseudomonasmendocina, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas stutzeri, Ralstonia insidiosa, Ralstonia pickettii, Neisseriagonorrhoeae, NDM-1 positive E. coli, Enterobacter cloaca, vancomycin-resistant E. faecium, vancomycin-resistant E. faecalis, E. faecium, E. faecalis, clindamycin-resistant S. agalactiae, S. agalactiae, Bacteroides fragilis, Clostridium difficile, Streptococcus pneumonia, Moraxella catarrhalis, Haemophilus haemolyticus, Haemophilus parainfluenzae, Chlamydophilia pneumoniae, Mycoplasma pneumoniae、Atopobium、Sphingomonas、Saccharibacteria、Leptotrichia、Capnocytophaga、Oribacterium、Aquabacterium、Lachnoanaerobaculum、Campylobacter、Acinetobacter、Agrobacterium; Bordetella; Brevundimonas; Chryseobacterium; Delftia; Enterobacter; Klebsiella; Pandoraea; Pseudomonas; Ralstonia、 and Prevotella.

[0242] Representative nontuberculous mycobacteria include Mycobacterium abscessus, Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium avium complex, Mycobacterium marinum, Mycobacterium terrae, and Mycobacterium cheloni.

[0243] Representative Burkholderia genera include Burkholderia cepacia, Burkholderia cepacia complex, Burkholderia multivorans, Burkholderia cenocepacia, Burkholderia stabilis, Burkholderia vietnamiensis, Burkholderia dolosa, Burkholderia ambifaria, Burkholderia anthina, Burkholderia pyrrocinia, Burkholderia gladioli, Burkholderia ubonensis, Burkholderia arboris, Burkholderia latens, Burkholderia lata, Burkholderia metallica, Burkholderia seminalis, Burkholderia contaminans, and Burkholderia diffusa.

[0244] In some embodiments, the bacteria are drug-resistant, and in some aspects of these embodiments, the bacteria are multi-drug resistant. For example, the bacteria can be resistant to antibiotics such as amikacin, aztreonam, methicillin, vancomycin, nafcillin, gentamicin, ampicillin, chloramphenicol, doxycycline, colistin, delamanid, presomanid, clofazimine, bedaquiline, and / or tobramycin.

[0245] Bacteria can develop resistance to these drugs, but cannot so easily develop resistance to the nitric oxide-based approaches described herein.

[0246] Treatable viral infections RNA and DNA viruses that can be treated are summarized below.

[0247] RNA viruses Currently, five orders and 47 families of RNA viruses are recognized, with many unassigned species and genera. Related to, but distinct from, the RNA viruses are viroids and RNA satellite viruses.

[0248] There are several major taxa: leviviruses and related viruses, picornaviruses, alphaviruses, flaviviruses, dsRNA viruses, and negative-strand viruses (Wolf et al., “Origins and Evolution of the Global RNA Virome,” mBio, 9(6) (November 2018)).

[0249] Positive-strand RNA viruses are the largest single group of RNA viruses, with 30 families. Of these, three groups are recognized: the picornavirata, which includes bymoviruses, comoviruses, nepoviruses, nodaviruses, picornaviruses, potyviruses, obemoviruses, and a subset of luteoviruses (Beet Western Yellows Virus and Potato Leaf Roll Virus); and the flavivirata, which includes carmoviruses, dianthoviruses, flaviviruses, pestiviruses, statoviruses, tombusviruses, single-stranded RNA bacteriophages, hepatitis C virus, and a subset of luteoviruses (Barley Yellow Dwarf Virus). The alpha-like group (Rubivirata) includes alphaviruses, caraviruses, furoviruses, hordeiviruses, potexviruses, rubiviruses, tobraviruses, tricornaviruses, tymoviruses, apple chlorotic leaf spot virus, beet yellows virus, and hepatitis E virus.

[0250] A division of the alpha-like (Sindbis-like) supergroup has been proposed into two groups: the "altovirus" group, which includes alphaviruses, furoviruses, hepatitis E virus, hordeiviruses, tobamoviruses, tobraviruses, tricornaviruses, and rubiviruses, and the "typovirus" group, which includes apple chlorotic leaf spot virus, curlaviruses, potexviruses, and tymoviruses.

[0251] There are five positive-strand RNA virus groups containing four, three, three, three, and one order, respectively. These 14 orders contain 31 virus families (including 17 plant virus families) and 48 genera (including 30 plant virus genera). Alphaviruses and flaviviruses can be divided into two families: Togaviridae and Flaviridae.

[0252] This analysis also suggests that the dsRNA viruses are not closely related to each other, but instead belong to four additional classes, Birnaviridae, Cystoviridae, Partitiviridae, and Reoviridae, as well as one additional order (Totiviridae) of the class of positive-stranded ssRNA viruses, in the same subphylum as the positive-stranded RNA viruses.

[0253] There are two major clades: the first includes the families Caliciviridae, Flaviviridae, and Picornaviridae, and the second includes the families Alphatetraviridae, Birnaviridae, Cystoviridae, Nodaviridae, and Permutotretraviridae.

[0254] Satellite viruses include the Sarthroviridae, which includes the genera Albetovirus, Aumaivirus, Papanivirus, Virtovirus, and Macronovirus.

[0255] Double-stranded RNA viruses (dsRNA viruses) comprise 12 families and several unassigned genera and species recognized within this group. These families include Amalgaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae, Megabirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae (including Rotavirus), Totiviridae, and Quadriviridae. Botybirnavirus is a single genus, with unassigned species including Botrytis porri RNA virus 1, Circulifer tenellus virus 1, Colletotrichum camelliae filamentous virus 1, Cucurbit yellows-associated virus, Sclerotinia sclerotiorum debilitation-associated virus, and Spisstilus festinus virus 1.

[0256] Positive-sense ssRNA viruses (positive-sense single-stranded RNA viruses) comprise 3 orders and 34 families, as well as several unclassified species and genera. The order Nidovirales includes the families Arteriviridae and Coronaviridae, which include coronaviruses such as SARS-CoV and SARS-CoV-2, Mesoniviridae, and Roniviridae. The order Picornavirales includes the families Dicistroviridae, Iflaviridae, and Marnaviridae, as well as the Picornaviridae, which includes poliovirus, rhinovirus (common cold virus), and hepatitis A virus; the family Secoviridae, which includes the subfamily Comovirinae; and the genus Bacillariornavirus and species of Kelp fly virus. The order Tymovirales includes the families Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, and Tymoviridae.Several families have not been assigned to any of these orders, including the families Alphatetraviridae, Alvernaviridae, Astroviridae, Barnaviridae, Benyviridae, Botourmiaviridae, Bromoviridae, Caliciviridae, which includes Norwalk virus (i.e., norovirus), Carmotetraviridae, Closteroviridae, and Flaviviridae, which includes yellow fever virus, West Nile virus, hepatitis C virus, dengue virus, and Zika virus. The families Fusariviridae, Hepeviridae, Hypoviridae, Leviviridae, Luteoviridae (including Barley yellow dwarf virus), Polycipiviridae, Narnaviridae, Nodaviridae, Permutotetraviridae, Potyviridae, Sarthroviridae, Statoviridae, Togaviridae (including Rubella virus, Ross River virus, Sindbis virus, and Chikungunya virus), Tombusviridae, and Virgaviridae. Unassigned genera include Blunervirus, Cilevirus, Higrevirus, Idaeovirus, Negevirus, Ourmiavirus, Polemovirus, Sinaivirus, and Sobemovirus.Unassigned species include Acyrthosiphon pisum virus, Bastrovirus, Blackford virus, Blueberry necrotic ring blotch virus, Cadicistrovirus, Chara australis virus, Extra small virus, Goji berry chlorosis virus, Harmonia axyridis virus 1, Hepelivirus, Jingmen tick virus, Le Blanc virus, Nedicistrovirus, Nesidiocoris tenuis virus 1, Niflavirus, Nylanderia fulva virus 1, Orsay virus, Osedax japonicus RNA virus 1, Picalivirus, planarian secretory cell nidovirus, Plasmopara halstedii virus, Rosellinia necatrix fusarivirus 1, Santeuil virus, Secalivirus, Solenopsis invicta virus 3, and Wuhan large pig roundworm virus.

[0257] Satellite viruses include the family Sarthroviridae and the genera Albetovirus, Aumaivirus, Papanivirus, Virtovirus, and chronic honeybee paralysis virus. This group currently includes six classes, seven orders, and 24 families. Several unassigned species and genera remain unclassified.

[0258] Negative-sense ssRNA viruses (negative-sense single-stranded RNA viruses), with the exception of hepatitis D virus, belong to a single phylum, Negarnaviricota, which includes two subphyla, Haploviricotina and Polyploviricotina, and four classes, Chunqiuviricetes, Milneviricetes, Monjiviricetes, and Yunchangviricetes. The Polyploviricotina subphylum includes two classes, Ellioviricetes and Insthoviricetes.

[0259] There are also several unassigned species and genera. The phylum Negarnaviricota includes the subphylum Haploviricotina, the class Chunqiuviricetes, the order Muvirales, and the family Qinviridae. The class Milneviricetes includes the order Serpentovirales and the family Aspiviridae. The class Monjiviricetes includes the order Jingchuvirales and the family Chuviridae.

[0260] The Mononegavirales include the families Bornaviridae (including Borna disease virus), Filoviridae (including Ebola and Marburg viruses), Mymonaviridae, Nyamiviridae, Paramyxoviridae (including measles, mumps, Nipah, Hendra, and NDV), Pneumoviridae (including RSV and metapneumovirus), Rhabdoviridae (including rabies), and Sunviridae, as well as the genera Anphevirus, Arlivirus, Chengtivirus, Crustavirus, and Wastrivirus. The Yunchangviricetes class includes the order Goujianvirales and the family Yueviridae.

[0261] The subphylum Polyploviricotina includes the class Ellioviricetes, the order Bunyavirales, and the families Arenaviridae, Cruliviridae, Feraviridae, Fimoviridae, Hantaviridae, Jonviridae, Nairoviridae, Peribunyaviridae, Phasmaviridae, Phenuiviridae, Tospoviridae, and the genus Tilapineviridae, which includes Lassa virus.

[0262] The class Insthoviricetes includes the order Articulavirales and the families Amnoonviridae, which includes the Taastrup virus, and Orthomyxoviridae, which includes the influenza virus.

[0263] The genus Deltavirus includes the hepatitis D virus.

[0264] Specific viruses include those associated with infection of mucosal surfaces of the respiratory tract, such as betacoronaviruses (SARS-COV-2 and MERS-COV), rhinoviruses, influenza viruses (including influenza A and B), and parainfluenza. In general, orthomyxoviruses and paramyxoviruses can be treated.

[0265] DNA viruses DNA viruses are viruses that use DNA as their genetic material and replicate using a DNA-dependent DNA polymerase. The nucleic acid is usually double-stranded DNA (dsDNA), but can also be single-stranded DNA (ssDNA). DNA viruses belong to either Group I or Group II of the Baltimore classification system of viruses. Single-stranded DNA usually expands to double-stranded DNA in infected cells. Group VII viruses, such as hepatitis B, contain DNA genomes, but are not considered DNA viruses according to the Baltimore classification. They replicate via an RNA intermediate, so are considered reverse-transcription viruses. Well-known diseases such as smallpox, herpes, and chickenpox are caused by such DNA viruses.

[0266] Some families have circular genomes (Baculoviridae, Papovaviridae, and Polydnaviridae), while others have linear genomes (Adenoviridae, Herpesviridae, and some phages). Some families have linear genomes that are circularly permuted (phage T4 and some Iridoviridae). Still others have linear genomes with covalently closed ends (Poxviridae and Phycodnaviridae).

[0267] Fifteen families are represented, including all three families of the order Herpesvirales and the following families: Ascoviridae, Ampullaviridae, Asfarviridae, Baculoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae, Lipothrixviridae, Nimaviridae, and Poxviridae.

[0268] Of these, species of the order Herpesvirales, which includes the families Alloherpesviridae, Herpesviridae (which includes human herpesviruses and varicella-zoster), and Adenoviridae (which includes viruses that cause human adenovirus infections), and Malacoherpesviridae, infect vertebrates.

[0269] The Asfarviridae, which includes African swine fever virus; the Iridoviridae; the Papillomaviridae; the Polyomaviridae, which includes Simian virus 40, JC virus, and BK virus; and the Poxviridae, which includes cowpox and smallpox, infect vertebrates. The Anelloviridae and Circoviridae also infect animals (mammals and birds, respectively).

[0270] The Smacoviridae family includes several single-stranded DNA viruses isolated from the feces of various mammals. This family includes 43 species, including six genera: Bovismacovirus, Cosmacovirus, Dragsmacovirus, Drosmacovirus, Huchismacovirus, and Porprismacovirus. Circo-like virus Brazil hs1 and hs2 have also been isolated from human feces. An unrelated group of ssDNA viruses includes the bovine stool-associated circular virus and chimpanzee stool-associated circular virus species.

[0271] Animal viruses include parvovirus-like viruses, which have a linear, single-stranded DNA genome but, unlike parvoviruses, have a bisected genome. This group includes hepatopancreatic parvo-like viruses and lymphoidal parvo-like viruses. Parvoviruses have frequently invaded the germ lineage of various animal species, including mammals.

[0272] Human respiratory-associated PSCV-5-like viruses have been isolated from the respiratory tract.

[0273] Representative viruses associated with pulmonary infections that can be treated using the methods described herein include coronaviruses, picornaviruses, influenza viruses (including influenza A and B), the common cold, respiratory syncytial virus (RSV), adenoviruses, parainfluenza, rhinoviruses, and SARS. In general, orthomyxoviruses and paramyxoviruses can be treated.

[0274] In addition to being associated with respiratory infections that cause bronchitis, sinusitis, and / or pneumonia, human papillomavirus (HPV) is associated with certain throat cancers.

[0275] Types of fungal infections that can be treated Exemplary fungal infections that can be treated include Candida albicans, drug-resistant Candida albicans, Candida glabrata, Candida krusei, Candida guilliermondii, Candida auris, Candida tropicalis, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, and / or Aspergillus flavus.

[0276] V. Treatment method The compounds disclosed herein can be used for anti-infective / antibacterial applications. Depending on the location of infection, the compounds and / or compositions discussed herein can be administered to infected tissue, for example, by inhalation, spraying, intranasal delivery, direct injection or application. Administration can also include parenteral administration (e.g., intravenous, intramuscular, or intraperitoneal injection), subcutaneous administration, administration into vascular space, and / or administration into joints (e.g., intra-articular injection).

[0277] The compounds may also be administered topically, vaginally, rectally, buccally, intrathecally, and intraarterially, or applied as a liquid or gel to the treatment site.

[0278] In some embodiments, the compounds allow for the efficient reduction in viability and / or the efficient eradication of microorganisms (e.g., prokaryotes, bacteria, protozoa, fungi, algae, amoeba, slime molds, etc.) In particular, such compounds are effective, alone or in combination with other known antibacterial or antiviral therapies, against such microorganisms that have developed at least some degree of drug resistance to conventional antibacterial or antiviral therapies.

[0279] Unlike conventional antibacterial treatments, NO, an endogenously produced free radical, eradicates bacteria using various mechanisms, including but not limited to lipid peroxidation, membrane protein nitrosation, and DNA damage via reactive oxygen / nitrogen species (e.g., peroxynitrite, dinitrogen trioxide). Due to its multiple killing mechanisms, NO can significantly reduce the risk of promoting microbial resistance. Furthermore, NO has an improved ability to actively degrade both the biofilm matrix and mucosal structures, thus enabling more efficient biocidal activity and mucociliary clearance in infections associated with biofilms and / or excessive mucosa.

[0280] NO is also a potent antibacterial agent that acts on bacteria through nitrosative and / or oxidative stress. NO is a broad-spectrum antibacterial agent, and in some embodiments, the compounds described herein deliver NO, thus eradicating both bacteria and biofilms, potentially through the formation of reactive NO byproducts (e.g., peroxynitrite and nitrogen trioxide) that cause oxidative and nitrosative damage to microbial DNA and / or membrane structure. Advantageously, the broad mechanism by which NO exerts its antibacterial effect reduces the risk of bacterial resistance developing. Thus, the NO-releasing compounds described herein are useful in combating bacterial infections. The antibacterial efficacy of NO-releasing materials depends on both the NO payload and the associated release kinetics. In some cases, a high total NO amount is an important parameter for effectively assessing the storage capacity of a suitable compound. However, excessively rapid NO release and high NO storage can result in undesirable toxicity to mammalian cells. Therefore, challenges exist in preparing biocompatible NO-releasing materials with high NO storage and low cytotoxicity, and these challenges, among others, are addressed according to some embodiments disclosed herein.

[0281] Real-time detection of NO can be performed using a chemiluminescence-based nitric oxide analyzer (NOA). The total NO storage and dissociation kinetics of water-soluble NO donors were measured under physiological conditions (pH 7.40, 37 °C). The resulting NO release parameters (e.g., total NO storage, NO release half-life, maximum flux, and conversion efficiency) were evaluated for their suitability as pharmaceuticals. In general, small molecule derivatives of many compounds can be engineered to exhibit large NO storage capacities with consistent NO release kinetics (e.g., species with consistent NO release half-lives between 0.5 h and 24 h).

[0282] Treating drug-resistant bacteria In some embodiments, the microbial load that is reduced and / or eliminated comprises drug-resistant bacteria. In some embodiments, the drug-resistant bacteria comprises carbapenem-resistant Enterobacteriaceae. In some embodiments, the drug-resistant bacteria comprises methicillin-resistant Staphylococcus aureus. In some embodiments, the microorganisms include human immunodeficiency virus, herpes simplex virus, papillomavirus, parainfluenza virus, influenza, hepatitis, coxsackievirus, shingles, measles, mumps, rubella, rabies, pneumonia, (hemorrhagic viral fever, H1N1, etc.), prions, parasites, fungi, molds, yeasts, and bacteria (both gram-positive and gram-negative), including Candida albicans, Aspergillus niger, Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and Staphylococcus aureus (S. aureus), Group A streptococci, S. pneumoniae, Mycobacterium tuberculosis, Campylobacter, among others. jejuni, Salmonella, Shigella, P. gingivalis, A. actinomycetemcomitans, A. viscosus, and / or S. mutans, as well as various drug-resistant bacteria. The terms microorganism and microbe are used interchangeably. Microorganisms can include wild-type, genetically engineered, or modified organisms.

[0283] Treating lung infections The method described herein can be used to treat, prevent, manage or reduce the severity of one or more lung disease or infectious disease-related symptoms and infectious diseases in subjects.This method involves administering one or more of the compounds described herein to subjects.Compound can be administered to mouth, nasal cavity, throat, esophagus, larynx, pharynx, trachea, bronchiole, bronchus, upper respiratory tract, lower respiratory tract, subcutaneously or via implant (for example, below ribs and into the thoracic cavity) and combinations thereof.

[0284] In some embodiments, the compound is nebulized, inhaled, or delivered intranasally. In certain embodiments, the method comprises inhaling particles comprising one or more of the compounds described herein aerosolized by nebulization. Nebulizers generally use compressed air or ultrasonic power to create inhalable aerosol droplets of the particles or a suspension thereof. In this embodiment, the aerosol droplets of the particles or a suspension thereof are delivered to the subject via pulmonary delivery by nebulization. In another embodiment, the method comprises inhaling aerosolized particles by a pMDI, wherein the particles or a suspension thereof are suspended in a suitable propellant system (including but not limited to hydrofluoroalkanes (HFAs)) containing at least one liquefied gas in a pressurized container sealed with a metering valve. Actuation of the valve delivers a metered dose of the aerosol spray of the particles or a suspension thereof.

[0285] In one embodiment, the compound is administered during pulmonary lavage, which can be whole lung lavage or bronchoalveolar lavage (BAL).In BAL, also known as bronchoalveolar lavage, a bronchoscope is passed through the mouth or nose into the lungs, and fluid is spurted into a small part of the lung, then collected and examined.The compound can move through the fluid and treat the entire fluid-coated part of the lung.

[0286] Bronchoalveolar lavage is commonly used to diagnose infections in people with immune system problems, pneumonia in people on mechanical ventilators, some types of lung cancer, and lung scarring (interstitial lung disease). It is the most common method used to sample epithelial lining fluid (ELF) and determine the protein composition of lung airways. It is often used in immunological studies as a means of sampling cells (e.g., T cells) or pathogen levels (e.g., influenza virus) in the lungs. Compounds described herein can be administered during this procedure. Whole lung lavage (WLL, or "lung lavage") is a treatment for pulmonary alveolar proteinosis. Compound therapy can also be administered while the lungs are being lavaged, allowing the fluid to contact the entire fluid-coated surface of the lungs.

[0287] In some embodiments, the compounds are used to treat or prevent microbial infections, including those caused by viruses such as coronaviruses, including SARS, MERS, and SARS-CoV2.

[0288] In other embodiments, the infection is caused by a spore-forming microorganism, such as certain bacteria and all fungi. Most medications are only active against bacteria or fungi when they are not in spore form, so treatment must be administered over an extended period of time to allow the spores to become active bacteria / fungi and be treated with an antimicrobial agent.

[0289] The compounds described herein are effective not only in killing active bacteria / fungi but also in killing spores. Therefore, the methods described herein can be used to shorten treatment duration. For example, treatment of infectious diseases such as tuberculosis or nontuberculous mycobacterial infections (NTM) requires approximately one year for effective treatment, primarily due to the continued presence of spores. Because drugs commonly used to treat HIV are incompatible with those used to treat tuberculosis, the duration of treatment often leads to poor patient compliance, especially for patients co-infected with HIV. A major cause of drug interactions in the management of tuberculosis / HIV co-infection is the effect of the antibacterial compound rifampicin, which induces the cytochrome P450 system, which affects the metabolism of many drugs used to treat HIV. The methods described herein can be used to minimize treatment duration, thus increasing patient compliance and minimizing or avoiding problems associated with drug interactions with drugs used to treat HIV.

[0290] In one embodiment, the lung disease or infection is a result of or associated with cystic fibrosis. Carbocysteine ​​is a mucolytic agent that can help break down mucus and can be co-administered with the compounds described herein.

[0291] In another embodiment, the subject has at least one pulmonary infection, and if there is more than one pulmonary infection, the infections are simultaneous or consecutive.

[0292] In some embodiments, the lung infection is in one lung, and in other embodiments, it is in both lungs, and can be in one or more of the three lobes of the right lung or one or both of the two lobes of the left lung.

[0293] Examples of lung infections that can be treated include bronchiectasis infections, pneumonia, valley fever, allergic bronchopulmonary aspergillosis (ABPA), ventilator-acquired pneumonia, hospital-acquired pneumonia, community-acquired pneumonia, ventilator-associated tracheobronchitis, lower respiratory tract infections, nontuberculous mycobacterial diseases, anthrax, Legionnaires' disease, whooping cough, bronchitis, bronchiolitis, COPD-related infections, and infections associated with post-lung transplantation.

[0294] Lung infections can be caused by one or more bacterial or fungal pathogens.

[0295] Where the pulmonary infection is a CF-associated pulmonary infection, the methods described herein can be used to treat, manage, or reduce the severity of the CF-associated pulmonary infection.

[0296] In some embodiments, the pulmonary infection is located in or on the pulmonary mucosa, bronchi and / or bronchioles.

[0297] In some embodiments, the pulmonary infection is located on or within a bacterial biofilm, an aggregated bacterium, a fungal biofilm, or an aggregated fungus.

[0298] In yet other embodiments, the pulmonary infection is located in the sputum.

[0299] The bacterial pathogen may be a gram-positive or gram-negative bacterium and may include one or more of a bacterial biofilm and a planktonic bacterium.

[0300] Because the compounds described herein can penetrate and disrupt biofilms, in embodiments where a bacterial biofilm is present, the methods can involve (i) reducing the bacterial biofilm, (ii) impairing the growth of the bacterial biofilm, and (iii) preventing the reformation of the bacterial biofilm.

[0301] In yet other embodiments, fungal pathogens are present, which can include planktonic and / or biofilm fungi.

[0302] The method can be used to treat, manage, or reduce the severity of pulmonary infections by one or both of preventing infection by bacterial or fungal pathogens, reducing bacterial or fungal pathogens, and / or reducing sputum viscosity.

[0303] The method can treat, manage, or reduce the severity of a pulmonary infection by preventing the production or secretion of exotoxins from bacterial or fungal pathogens, inhibiting cell viability or cell growth of planktonic cells of bacterial or fungal pathogens, inhibiting biofilm formation by bacterial or fungal pathogens, and inhibiting biofilm viability or growth.

[0304] By using the method described in this specification, it can be killed. For example, Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus lugdunensis, Staphylococcus epidermidis, Streptococcus milleri / anginous, Streptococcus pyogenes, Mycobacterium tuberculosis, Burkholderia, Achromobacter xylosoxidans, Pandoraeasputorum, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans、Haemophilus pittmaniae、Serratia marcescens、Candida albacans、Candida parapsilosis、Candida guilliermondii、Morganella morganii、Inquilinus limosus、Ralstonia mannitolilitica、Pandoraea apista、Pandoraea pnomenusa、Pandoraea sputorum、Bdellovibrio bacteriovorus、Bordetellabronchiseptica、Vampirovibrio chlorellavorus、Actinobacter baumanni、Cupriadidus metallidurans、Cupriavidus apulus、Cupriavidus respiraculi、Delftia acidivordans、Exophilia dermatitidis、Herbaspirillum frisingense、Herbaspirillum seropedicae、Klebsiella pneumoniae、Pandoraea norimbergensis、Pandoraea pulmonicola、Pseudomonas mendocina、PseudomonasPseudoalcaligenes, Pseudomonas putida, Pseudomonas stutzeri, Ralstonia insidiosa, Ralstonia pickettii, Neisseria gonorrhoeae, NDM-1 positive E. coli, Enterobacter cloaca, vancomycin-resistant E. faecium, vancomycin-resistant E. faecalis, E. faecium, E. faecalis, clindamycin-resistant S. agalactiae, S. agalactiae, Bacteroides fragilis, Clostridium difficile, Streptococcus pneumonia, Moraxella catarrhalis, Haemophilus haemolyticus, Haemophilus parainfluenzae, Chlamydophilia pneumoniae, Mycoplasma pneumoniae, Atopobium, Sphingomonas, Saccharibacteria, Leptotrichia, Capnocytophaga, Oribacterium, Aquabacterium, Lachnoanaerobaculum, Campylobacter, Acinetobacter; Agrobacterium; Bordetella; Brevundimonas; Chryseobacterium; Delftia; Enterobacter; Klebsiella; Pandoraea; Pseudomonas; Ralstonia, and Prevotella.

[0305] Common lung infections include inhalation anthrax, whooping cough (also known as pertussis, caused by Bordetella pertussis), streptococcus (pneumococcus, Streptococcus pneumoniae), mycobacteria, including mycobacteria tuberculosis and nontuberculous mycobacterial (NTM) lung disease (Mycobacterium avium complex (MAC), M abscessus, M kansasii, M malmoense, M szulgai, and M xenopi).

[0306] The methods described herein can reduce the severity of one or more of the following symptoms in a treated subject: cough, wheezing, dyspnea, bronchiectasis, nasal polyps, hemoptysis, respiratory failure, and pulmonary exacerbation.

[0307] Administration of the compounds described herein to treat or prevent coronavirus infection The compounds described herein can be delivered to one or more areas of a patient's respiratory tract using a nebulizer or nasal spray for a sufficient period of time to treat or prevent coronavirus infection, for example. The nitric oxide produced when the compounds decompose can be effective in treating or preventing coronavirus infection. Exposure to the compounds and the nitric oxide produced by these compounds does not significantly damage lung tissue, even over long periods of time.

[0308] The compound can be administered anywhere along the respiratory tract, depending on the patient's infection status. If the virus is not present in large numbers in the lungs and is primarily confined to the patient's mouth, nose, and throat, therapy can be limited to those areas of the respiratory tract.

[0309] This approach can also be used prophylactically in patients who are at risk of developing coronavirus infection due to previous or suspected exposure to an individual with coronavirus infection.

[0310] When the patient's lungs are infected, it is more desirable to administer the compound directly to the lungs.The compound can be administered, for example, via a nebulizer.In addition to treating viral infection, the compound can also be useful for preventing secondary infections, such as bronchitis or pneumonia, which are caused by bacteria and often follow viral infections.In some cases, minimizing the risk of secondary infections can be even more important than treating the underlying viral infection.

[0311] Tracking the progress of treatment is important, especially when patients have an active infection and will experience serious adverse consequences if treatment is not successful, or when they have a disease such as COPD that progresses over time, and it may be important to monitor disease progression.

[0312] Methods for tracking the progress of treatment include taking periodic pulse oximeter readings and taking periodic chest X-rays, ultrasounds, and CT scans. Residual microbial infections can also be checked, for example, using ELISA tests or other tests that look for antibodies specific to a particular microbial infection, as well as analyzing blood or sputum samples for residual infection. Patient temperature can also be tracked, particularly to track treatment for short-term microbial infections.

[0313] For pulmonary inflammatory disorders, it may be useful to track the progress of treatment using regular pulmonary function testing, which may include not only spirometry but also performance tests (such as the distance a patient can walk in a given period of time).

[0314] Treatment of cystic fibrosis Cystic fibrosis (CF) is a genetic disorder characterized by impaired mucociliary clearance and chronic bacterial infection. As demonstrated herein, in some embodiments, nitric oxide (NO) possesses broad-spectrum antibacterial activity against CF-associated bacteria, making it an attractive alternative to conventional antibiotics. Treatment with NO limits bacterial resistance due to its multiple killing mechanisms (e.g., induction of nitrosative and oxidative stress). It has surprisingly been found that storing NO in the compounds described herein improves bactericidal efficacy and reduces systemic cytotoxicity. Treatment is effective against planktonic and biofilm-based pathogens, and cytotoxicity assays on mammalian lung cells demonstrate minimal harm to treated subject cells. Surprisingly, the combination of gallium with an NO donor has been found to be synergistically effective.

[0315] CF is a debilitating disease characterized by chronic bacterial infection of the lungs, resulting in a reduced life expectancy of 20 years. Genetic defects in the CF transmembrane conductance regulator (CFTR) result in the loss of ions (e.g., Cl) to the airway surface liquid. -) This disrupts the normal transport of blood and inhibits water transport. Thus, the airway epithelium becomes dehydrated and produces thick mucus that cannot be efficiently removed via the mucociliary clearance mechanism. As goblet cells continually excrete mucin into the dehydrated airways, mucus accumulation accelerates until cilia become damaged or nonfunctional, preventing them from clearing mucus from the airways. Planktonic bacteria thrive in this quiescent environment, promoting the formation of complex communities of pathogenic bacteria known as biofilms. The exopolysaccharide matrix produced by these biofilms inhibits oxygen diffusion, creating pockets of anaerobic environments and altering bacterial metabolism. This combination of a thick mucus layer and a robust biofilm significantly reduces the antibacterial efficacy of common CF therapies.

[0316] Inflammatory disorders of the lungs There are several additional lung diseases with an inflammatory component. Representative disorders include asthma, COPD, chronic bronchitis, emphysema (and co-administration with retinoids (e.g., retinoid A) can help rebuild alveoli), acute bronchitis (viral or bacterial), lung diseases affecting the air sacs (alveoli) and / or interstitium, including pneumonia, tuberculosis (caused by Mycobacterium tuberculosis), emphysema, pulmonary edema (whether caused by COPD, heart failure, or direct injury to the lung), lung cancer, acute respiratory distress syndrome (ARDS), pneumonia, interstitial lung disease (ILD), sarcoidosis, idiopathic pulmonary fibrosis, and autoimmune diseases.

[0317] Disorders such as asthma, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis are the result of an ongoing inflammatory process. Asthma is a persistent disease of the bronchi in which the airways overreact to external factors such as smoke, air pollution, and allergens. The tissue lining the airways becomes inflamed, causing the bronchial tubes to narrow. This in turn causes symptoms of shortness of breath. Patients complain of shortness of breath and difficulty moving air in and out of their lungs.

[0318] COPD is another inflammatory disease affecting both the airways and lung tissue. It can manifest as a combination of chronic obstructive bronchitis and emphysema, the former resulting from chronic inflammation of the bronchial tubes and the latter resulting from destruction of the alveoli.

[0319] Pulmonary fibrosis is another chronic lung disease caused by scarring or thickening of the lungs, which affects oxygen exchange.

[0320] Symptoms of these lung diseases include difficulty breathing, shortness of breath, inability or decreased ability to exercise, coughing with or without blood or mucus, and painful breathing. In asthma, wheezing and chest tightness are common symptoms, along with coughing and shortness of breath. Patients with COPD typically present with a chronic cough accompanied by excessive mucus production, as well as symptoms similar to asthma. Pulmonary fibrosis can cause dry cough, fatigue, unexplained weight loss, and musculoskeletal pain.

[0321] Chest x-rays may reveal scar tissue, pulmonary hyperinflation, a flattened diaphragm, or thickened bronchial walls. Computed tomography scans, spirometry, arterial blood gases, and other tests may be appropriate depending on the clinical presentation and medical history.

[0322] Conventional therapies for pulmonary disease may be symptomatic but not curative. Treatment initially consists of corticosteroids, beta-agonists, leukotriene modifiers or receptor antagonists, or methylxanthines such as theophylline. In the early stages of pulmonary disease, these agents can be administered as monotherapy, but as the disease progresses, treatment is likely to consist of multiple agents, as well as supplemental oxygen and pulmonary rehabilitation.

[0323] Patients suffering from these disorders can benefit from treatment with the compounds described herein. Because patients suffering from inflammatory respiratory disorders often have underlying microbial infections, it can be useful to combine anti-inflammatory compounds with NO-producing compounds to treat both inflammatory lung diseases and respiratory infections.

[0324] Depending on the specific pulmonary disorder being treated, the progress of treatment can be tracked in different ways. Treatment of microbial infections can be tracked, for example, by tracking the severity of symptoms, the presence of fever, and the use of pulse oximetry. Treatment of pulmonary inflammatory diseases can be followed by X-rays, pulmonary function tests, and the like. A challenge test is a pulmonary function test used to confirm the diagnosis of asthma. The patient inhales a small amount of a substance known to cause symptoms in asthmatics, such as histamine or methacholine. After inhaling the substance, pulmonary function is assessed. After treatment, it can be determined whether the decline in pulmonary function after inhaling these substances is alleviated compared to before treatment began, indicating that the treatment is effective for such patients.

[0325] Treating dental erosion Dental caries (e.g., dental caries) is another important disease affecting 60%–70% of school-age children and the majority of adults in most industrialized countries. 11% of the total population suffers from severe periodontitis, which is responsible for tooth loss and systemic diseases such as coronary artery disease, cardiovascular disease, stroke, and adverse pregnancy outcomes. Among the >700 oral microorganisms, saprophytic bacteria (e.g., Streptococcus mutans, Actinomyces difficile) and periodontal pathogens (e.g., Porphyromonas gingivalis, Actinomyces difficile) play a major role in the initiation and progression of oral diseases. Oral diseases are one of the most common health problems faced by humans. Gram-positive oncogenic bacteria (e.g., Streptococcus mutans, Actinomyces difficile) and Gram-negative periodontal pathogenic bacteria (e.g., Porphyromonas gingivalis, Aggregatibacter actinomyces) represent major aggravating factors associated with the evolution and progression of dental caries and periodontal disease, respectively. Unfortunately, current treatments for combating these pathogens are associated with undesirable side effects. For example, the systemic use of antibiotics can result in gastrointestinal disorders and promote bacterial resistance. Chlorhexidine, a common oral antiseptic, can alter taste, stain teeth and tongue, and irritate the oral mucosa. Macromolecular NO delivery vehicles (e.g., silica nanoparticles, gold, etc.) kill Gram-negative periodontal pathogens. However, these materials have not been demonstrated to kill Gram-positive carrion-producing bacteria at safe concentrations (e.g., concentrations that are bactericidal but non-toxic to mammalian cells). Similar to nanomaterials, the lack of biodegradability and potential cytotoxicity of silica nanoparticles hinders their future for biomedical applications. Current research is also focusing on the use of nanomaterials, including silver, gold, zinc, and copper, as alternatives to traditional antibiotics, which have struggled to foster bacterial resistance. However, these nanomaterials can accumulate in the body, causing cumulative toxicity, limiting their future for certain applications. The development of oral therapeutics capable of killing these disease-causing bacteria is crucial for maintaining a healthy oral cavity. In some embodiments, the compositions disclosed herein (eg, comprising an NO scaffold) solve one or more of these problems, or other problems.

[0326] Providing wound care An unmet need in the fields of wound healing, general surgery, and orthopedics is an antimicrobial material that can release NO at the required rate and degrade within a desired time frame. This degradation rate can be tailored (through appropriate formulation) to the healing cycle of each specific condition and / or the time when the wound is at high risk of infection. Examples of these conditions include hernia repair, diabetic foot ulcer healing, and orthopedic tendon repair, to name a few. In some embodiments, the compounds and materials disclosed herein are directed to compositions with tunable degradation times.

[0327] Some embodiments provide a method for treating a tissue defect, comprising positioning any of the compounds described herein in the tissue defect. In some embodiments, the tissue defect is a wound. Some embodiments provide a method for treating a wound, performing tissue repair, and / or providing tissue and organ replenishment. In some embodiments, the first step in treating a tissue defect, wound, and / or tissue replenishment and replacement includes identifying a patient in need of an antimicrobial scaffold to assist in the repair and healing of the tissue defect, wound healing, or tissue replenishment.

[0328] A non-limiting list of patients in need of an antimicrobial compound includes patients suffering from tissue defects. In some embodiments, patients in need of an antimicrobial scaffold suffer from wounds, including burns, skin ulcers, lacerations, bullet holes, animal bites, and other wounds prone to infection. The antimicrobial compound can also be used to treat diabetic foot ulcers, venous leg ulcers, pressure ulcers, amputation sites, other skin trauma, or other wounds or diseases. Patients in need of an antimicrobial scaffold also include patients in need of tendon, ligament, fascia, and dura mater repair and replacement. The compound can also be used in tissue replacement procedures, including, but not limited to, rotator cuff repair, Achilles tendon repair, leg or arm tendon or ligament repair (e.g., torn ACL), vaginal prolapse repair, bladder slings for urinary incontinence, post-operative breast reconstruction, hernia repair, staple or suture reinforcement, bariatric surgery repair, pelvic floor reconstruction, dura mater repair, gum repair, bone grafting, and reconstruction. Furthermore, patients in need of an antimicrobial scaffold also include patients in need of tissue or organ replacement. In some embodiments, the compositions described herein can be used to replenish and / or replace tissue by acting as a filler and / or artificial extracellular matrix. In such applications, antimicrobial scaffolds can be used to support cell and tissue growth. Briefly, cells can be harvested from a patient or living host and seeded onto the antimicrobial scaffold either in vivo or ex vivo. Then, as the patient's natural tissue invades the material, the material degrades and adjusts to exclude only naturally occurring tissue and cells from bacterial infection.

[0329] In some embodiments, applications also include, inter alia, the delivery of therapeutic molecules to localized sites, use as adhesives or sealants, use as viscosity aids, and use in wound healing. The stabilized compositions can also be used as tissue fillers, dermal fillers, bone fillers, bulking agents, e.g., urethral or esophageal bulking agents, and embolization agents, as agents for repairing cartilage defects / injuries, and as agents for enhancing bone repair and / or growth. In some embodiments, compositions comprising the antimicrobial scaffold can be placed in or on a patient, for example, in a void to fill the space.

[0330] In some embodiments, the compound is used to repair damaged tissue. In some embodiments, the composition is formulated for administration to a target treatment site in a subject. For example, the composition can be formulated to facilitate administration to damaged or infected tissue in a subject.

[0331] In some embodiments, after administration of a composition (e.g., an antimicrobial scaffold containing gallium and NO donor), the composition may increase in temperature due to the absorption of heat from the subject's surrounding body tissue. In some embodiments, the subject's body temperature is sufficient to cause the composition to increase in viscosity (e.g., transition from a liquid to a gel). In some embodiments, the increase in viscosity (e.g., gelation) may result in a three-dimensional network sufficient to provide structural and / or geometric support to body tissue, such as cardiac tissue (e.g., cardiac tissue in an infarcted area). In some embodiments, the composition can be injected in vivo using a syringe or catheter. In some embodiments, the composition may be injected directly into the treatment site or may be partially preheated in a syringe to increase the viscosity of the composition prior to injection. In some embodiments, a preheated formulation may reduce the likelihood that a low-viscosity composition may diffuse and / or migrate from the intended tissue region after injection.

[0332] In some embodiments, after administration of a composition (eg, comprising an antimicrobial compound of Formula III), the composition may increase in temperature due to absorption of heat from the subject's surrounding body tissue.

[0333] The present invention is further defined in the following examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only. [Example]

[0334] Example 1: Synthesis and characterization of compounds of formula III This example relates to the synthesis and identification of one embodiment of a compound of Formula III, which has the following properties, advantages, and / or uses:

[0335] In some embodiments, the molecule possesses antibacterial properties, allowing the NO-releasing material to act as an antibacterial agent. The disclosed compounds have been found to be the product of specific high-pressure nitric oxide synthesis strategies. In this context, the compounds can be trace-formed to the main components of the reaction, depending on reaction conditions such as NO pressure, base content, temperature, and reactant content.

[0336] Methane trisdiazeniumdiolate, sodium salt was prepared according to the following procedure in Table 1. [Table 1]

[0337] The synthesized compounds were isolated and tested to confirm their identity by FTIR, HPLC, UV-Vis spectroscopy, 1 H NMR, and 13 C NMR analysis was used to support the conclusion that the synthesis yielded the compound of formula III.

[0338] Referring now to Figure 1, an FTIR spectrum of the compound of Formula III according to the present disclosure is shown. Referring now to Figure 2, an ion-exchange chromatogram of the compound of Formula III having a retention time of 11 minutes (λ = 252 nm) is shown. The UV absorbance spectrum of the analyte held for approximately 6 minutes, the UV absorbance spectrum of the analyte held for approximately 10 minutes, and the UV absorbance spectrum of the analyte held for approximately 11 minutes are shown in the inset.

[0339] FIG. 3 shows the structure of a compound of formula III. 1 The H NMR of the compound of formula III is shown. The peak at 7.5 ppm is assigned to a single proton on the compound of formula III. 13The C NMR of the compound of Formula III is shown. The peak at 100 ppm is assigned to a single carbon on the compound of Formula III. Figure 5 shows the 2D NMR of the compound of Formula III. Figure 6 shows A) an HPLC chromatogram (IEX-UV) with the top chromatogram (red) showing the separation of components before acid degradation of the compound of Formula III (designated MD3), the middle chromatogram (blue) showing the separation of components after 5 hours of acid degradation, and the bottom chromatogram (black) showing the separation of components after 24 hours of acid degradation. B) The separation of components after acid degradation. 1 C) A table of NOA sum for the compound of formula III before and after acid decomposition. 1 FIG. 8 is a H NMR spectrum of a composition containing the product of the compound of formula III after decomposition at room temperature and neutral pH. 13 C NMR spectrum.

[0340] The analytical results for the compound of formula III are summarized in Table 2. [Table 2]

[0341] Antibacterial activity The NO release of compound of formula III at pH 7.4 is shown in Figure 9. The release profile was measured by chemiluminescence and showed a T of approximately 3.75 hours. 1 / 2 showed a release of 6.7 μmol NO / mg of material. Consistent with the decomposition pathway described, 2 moles of NO are released per mole of Formula III, which translates to a theoretical yield of 7.6 μmol NO / mg.

[0342] The antibacterial efficacy of compounds of formula III according to the synthesis examples against various Pseudomonas strains is shown in Tables 3-5. The results of testing various amounts of compound mixed with a representative excipient (β-cyclodextrin) are shown in Table 6. [Table 3] [Table 4] [Table 5] [Table 6]

[0343] Example 2: In vitro antibacterial activity of compounds of formula III against P. aeruginosa Minimum inhibitory concentration / minimum bactericidal concentration (MIC / MBC) assays were performed using CLSI methods to evaluate the efficacy of compounds of formula III against several laboratory and clinical isolates of P. aeruginosa.

[0344] As used herein, the minimum bactericidal concentration (MBC) is defined as the lowest concentration of an antibiotic that kills 99.9% of bacteria, and the minimum inhibitory concentration (MIC) is defined as the lowest concentration of an antibacterial component or agent that is bacteriostatic (prevents visible bacterial growth).

[0345] MICs are used to evaluate the antimicrobial efficacy of various compounds by measuring the effectiveness of reducing concentrations of antibiotics / antiseptics over a defined period of time in terms of inhibiting microbial population growth. These evaluations can be very useful during the research and development phase of a product to determine the appropriate concentration needed in the final product.

[0346] Varying concentrations of the compound are inoculated into cultured bacteria and the results are measured using agar dilution or broth dilution (macro or micro) to determine at what level the MIC endpoint is established.

[0347] The minimum bactericidal concentration (MBC) can be determined from broth dilutions of MIC tests by replating onto agar plates without the test agent and incubating for 24 hours. The MBC is identified by determining the lowest concentration of an antimicrobial agent that reduces the viability of the initial bacterial inoculum by a predetermined percentage, such as ≥99.9%. The MBC is complementary to the MIC. While the MIC test indicates the lowest level of an antimicrobial agent that significantly inhibits growth, the MBC indicates the lowest level of an antimicrobial agent that results in microbial death. In other words, if the MIC indicates inhibition, plating bacteria onto agar may still result in organism growth because the antimicrobial agent did not cause death. An antimicrobial agent is typically considered bactericidal if its MBC is ≤4x the MIC.

[0348] The Clinical and Laboratory Standards Institute (CLSI) has established protocols and standards for establishing MICs and MBCs for products. The common methodology utilized for MICs is CLSI M07-A9, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. CLSI has also developed methods specific to yeast, filamentous fungi, and anaerobic bacteria. For MBC measurements, CLSI M26-A, Methods for Determining the Bactericidal Activity of Antimicrobial Agents, is the accepted industry standard.

[0349] As shown in Table 7, the compound of formula III (referred to in the table as MD3) was effective against all nine strains tested, resulting in a 3 log reduction of bacteria (MBC) at 0.125 mg / ml. [Table 7]

[0350] Thus, MD3 is effective against P. aeruginosa, the major pathogen affecting cystic fibrosis patients.

[0351] Example 3: In vitro efficacy of the compound of formula III against the nontuberculous mycobacterium, Mycobacterium abscessus MIC / MBC assays were performed using the CLSI method described above to evaluate the efficacy of the compound of Formula III against several laboratory and clinical isolates of Mycobacterium abscessus, a common NTM species. As shown in Table 8 below, the compound of Formula III was effective against all nine strains tested, resulting in a 3-log reduction in bacterial viability (MBC) at 1 mg / ml. [Table 8]

[0352] The data show that the compounds have in vitro efficacy over P. aeruginosa.

[0353] Example 4: Formula III is a broad spectrum antibacterial agent. To evaluate the efficacy of the compound of Formula III against additional clinical isolates of various pathogens, MIC / MBC assays were performed using CLSI methods. The results are shown in Table 9 below. The compound of Formula III is referred to in the table as MD3. [Table 9]

[0354] The data indicate that Formula III has broad spectrum antimicrobial activity.

[0355] Example 5: In vitro efficacy of cyclodextrin against P. aeruginosa biofilms P. aeruginosa biofilms grown on peg-lids in 96-well plates were exposed to the compound of Formula III for 18-24 hours, then the biofilms were isolated and surviving bacteria enumerated. Data showed that the compound of Formula III eradicated P. aeruginosa biofilms. A concentration of 0.391 mg / ml MD3 was sufficient to eradicate the bacterial biofilms (>3 log reduction).

[0356] Example 6: In vitro efficacy against P. aeruginosa phenotypes under aerobic and anaerobic growth conditions As an extension of the work performed in Example 2, the antimicrobial activity of the compound of Formula III was compared against 21 strains of P. aeruginosa under aerobic and anaerobic growth conditions. The results are presented in Figure 10. Of the 21 strains tested, the activity of the compound of Formula III was identical for 18 strains. Because these two bacterial strains could not be grown under anaerobic conditions, comparison of the two strains was not possible. Only one strain showed a difference in susceptibility, but the difference was minimal. Thus, the compound of Formula III exhibits highly consistent antimicrobial activity against P. aeruginosa under both aerobic and anaerobic growth conditions.

[0357] Example 7: In vitro efficacy - time kill assay The bactericidal activity of compounds of Formula III was evaluated over time against P. aeruginosa. P. aeruginosa cultures were grown at 37°C in either PBS or cation-adjusted Mueller-Hinton broth (CAMHB) containing various concentrations of compounds of Formula III, and bacteria were quantified at various time points during growth. In PBS, bacteria survive but lack the nutrients to replicate. In CAMHB, bacteria have the nutrients necessary for growth and replicate to high titers over time.

[0358] As shown in Figures 11A-B, in both PBS and CAMHB, the compound of Formula III (referred to as MD3 in the graphs) eradicated bacteria in a dose- and time-dependent manner. In the PBS-buffered system, bacterial titers decreased below the detection limit for all concentrations of MD3 used, but the time required to kill the bacteria increased with decreasing dose. A similar trend was observed in the CAMHB system; however, due to bacterial growth in CAMHB, the lowest two doses of MD3 evaluated were insufficient to kill all bacteria. Bacteria that survived for up to 8 hours rapidly reappeared to establish titers close to those of the untreated control at 24 hours. All doses above 0.125 mg / ml were effective enough to kill all bacteria within 8 hours. However, the lower the dose, the longer it took to completely kill the bacteria.

[0359] Example 8: Nitric oxide is key to the antibacterial activity of compounds of formula III. The activity of the compound of Formula III against P. aeruginosa and S. aureus was compared to that of its degradation product (referred to in the table as MD3NO-lib) using the CSLI method described above. The primary degradation product of the compound of Formula III is N-hydroxylformamide under physiological conditions. In P. aeruginosa, the MBC of the compound of Formula III was 64-fold lower than that of MD3NO-lib, and in S. aureus, the MBC of MD3 was 16-fold lower than that of MD3OG-lib. These data are shown in Table 10 below. The compound of Formula III is referred to in the table as MD3. [Table 10]

[0360] Because compounds of Formula III function by releasing NO, and NO-Lib does not, it is reasonable to conclude that NO is a significant driver of the bactericidal activity of Formula III in vitro.

[0361] To support the important role that NO plays in the antimicrobial activity of compounds of Formula III, three different pH conditions were tested: 6.4, 7.6, and 8.4. The rate of NO release is pH dependent. At lower pH, compounds of Formula III decompose faster, releasing NO. In turn, the more NO released, the faster Bacteria are killed. Figure 12 shows a graph of the time-kill assay results demonstrating the dependence of bacterial kill rates on pH. The raw results are the average of untreated samples at each pH, ​​indicating that differences in kill rates are due to the different amounts of NO released at each time point at the corresponding pH conditions, rather than due to the pH itself.

[0362] Example 9: Animal toxicity studies with compounds of formula III. Animal studies were conducted using severe combined immunodeficient (SCID) mice, using the study design outline in Table 11. No adverse effects were observed, even at the highest dose, Group 6 (100 mg / kg). All mice remained bright, alert, and responsive throughout the study period. [Table 11]

[0363] Example 12: Comparison of the efficacy of MD3 and MD2 In some cases, the synthesis of MD3 also produced an impurity, referred to herein as MD2, or methane bis-diazeniumdiolate, having the formula: [ka]

[0364] The activity of mixtures of these compounds was evaluated against P. aeruginosa (PAK) with the objective of determining how the ratio of the compound of formula III (referred to in the table as MD3) to MD2 in different samples affected the activity of the mixture against P. aeruginosa. [Table 12]

[0365] The results shown in Table 12 indicate that the higher the percentage of compound of Formula III in a sample relative to the percentage of MD2, the better the activity against PAK. It should be noted that when samples are decomposed under simulated physiological conditions, MD2 does not decompose to any significant extent. It also does not grow as a degradation product of compound of Formula III. Therefore, MD2 does not release NO or HNO when exposed to phytochemical temperatures and pH. In tables showing the percentage of MD3, the balance to 100% is primarily MD2, with minor amounts of other impurities.

[0366] Example 13: pH vs. efficacy of compounds of formula III The efficacy of compound of Formula III against PAK was evaluated at various pH values: 6.4, 7.6, and 8.4. All of these represent physiological pH values, albeit at different locations within the human body. For example, the oral pH of the proximal small intestine ranges from 5.5 to 7.0, gradually increasing to 6.5 to 7.5 in the distal ileum. There is a decrease in luminal pH from the terminal ileum to the cecum (range 5.5 to 7.5). The pH in the colon can range from 7.9 to 8.5. Normal blood pH is 7.40, which is approximately the same as the pH of the lungs. The pH of saliva is 6.5 to 7.5.

[0367] As shown in Figure 12 below, the compound of formula III at a dosage of 0.125 mg / ml at pH 6.4 reduced the concentration of PAK (CFU / ml) by 10 6 ~10 2 The compound of formula III at a dosage of 0.125 mg / ml at pH 8.4 was sufficient to reduce the concentration of PAK (CFU / ml) to 10 in 6 hours, and the concentration was maintained at this level for up to 25 hours. In contrast, the compound of formula III at a dosage of 0.125 mg / ml at pH 8.4 reduced the concentration of PAK (CFU / ml) to 10 in 6 hours. 6 ~10 5 The compound of formula III at a dosage of 0.125 mg / ml, pH 7.4, was sufficient to reduce the concentration of PAK (CFU / ml) to 10 in 4 hours, and the concentration was maintained at this level for up to 25 hours. 6 ~102 The PAK concentration in the untreated control was 10% throughout the experiment. 6 It remained as it was.

[0368] As shown in Figure 13 below, when the experiment was repeated, the compound of Formula III was used at a dosage of 0.0625 mg / ml at pH 6.4, resulting in a PAK (CFU / ml) concentration of 10 6 ~10 2 and concentrations were maintained at this level for up to 25 hours. In contrast, the compound of formula III at a dosage of 0.125 mg / ml, pH 8.4, reduced the concentration of PAK (CFU / ml) to 10 6 ~10 5 is sufficient to reduce the 6 The compound of formula III at a dosage of 0.125 mg / ml, pH 7.4, reduced the concentration of PAK (CFU / ml) to 10 in 6 hours. 6 ~10 2 The PAK concentration in the untreated control was sufficient to reduce the PAK concentration to 100% throughout the experiment, and the concentration remained at this level for up to 25 hours. 6 It remained as it was.

[0369] As shown in Figure 14 below, when the experiment was repeated, the compound of Formula III was used at a dosage of 0.03125 mg / ml at pH 6.4, reducing the concentration of PAK (CFU / ml) to 10 in 3 hours. 6 ~10 2 The compound of formula III at a dosage of 0.03125 mg / ml, pH 8.4 was insufficient to significantly reduce the concentration of PAK (CFU / ml), and the concentration remained at this level for up to 25 hours. In contrast, MD3 at 0.03125 mg / ml, pH 7.4 reduced the concentration of PAK (CFU / ml) by 10% in 8 hours. 6 From 10 2 The PAK concentration in the untreated control was 10% throughout the experiment. 6 It remained as it was.

[0370] Based on the data, it can be concluded that the efficacy of compounds of formula III is pH dependent due to the role of pH in influencing the rate of NO release from compounds of formula III.

[0371] Example 14: Comparison of various reactants with respect to purity and synthesis process optimization The compound of Formula III can be prepared using acetone, ethanol, or acetonitrile as starting materials, as well as other compounds with similar functional groups; however, when prepared from ethanol or acetonitrile (or any other compound), the impurity profile is unique. The following series of chromatograms show samples of the compound of Formula III prepared from acetone, ethanol, and acetonitrile. In each case, a different set of impurities was observed. The material prepared from ethanol was comparable in purity to that prepared from acetone (>90% area), while the material prepared from acetonitrile was significantly less pure (<40% area).

[0372] A process for synthesizing the compound of formula III, starting from acetone, was optimized. A series of experiments was performed to evaluate the effect of starting acetone concentration (14 vs. 30 mg / mL), base equivalents (4 vs. 6), NO pressure (2.5 vs. 20 bar), and temperature (10 vs. 20°C) on the yield and purity of the product produced. Table 13 shows the results of an experiment conducted over 4 days at an acetone concentration of 14 mg / mL and a temperature of 20°C. Each condition was performed in duplicate. [Table 13]

[0373] The best overall conditions identified from these experiments are listed here: acetone concentration of 14 mg / mL, NO pressure of 20 bar, and base equivalents of 4. Temperature had no effect under these conditions between 10°C and 20°C. Under these conditions, an average area purity of approximately 97% was achieved with a yield >95%.

[0374] Example 15: Comparison of the antibacterial activity of the compound of formula III with other NO-releasing compounds Other NO-donor compounds have been investigated for their potential antimicrobial activity. However, different NO donors are expected to have different ranges of antimicrobial activity due to their various chemical properties, such as loading capacity, release rate, water solubility, pKa, and molecular weight. Therefore, the antibacterial activity of compound of Formula III was compared with two other NO-donor compounds synthesized and characterized in our laboratory. The first NO-donor compound was a hepta-substituted ethanolamine β-cyclodextrin compound in which all seven secondary amines were functionalized with diazeniumdiolate groups, and the second NO-donor compound was 2,6-cis-dimethylpiperidine functionalized with diazeniumdiolate groups. The results of the antibacterial activity of these compounds are shown in Table 14. [Table 14]

[0375] From the compiled data, it is clear that the antibacterial activity of the compound of Formula III is greater than that of either of the other two NO donor molecules. Part of the increased activity can be attributed to its high NO loading capacity, which is not higher than cis-DMP / NO, yet is four times more effective at killing bacteria. This suggests that the extended NO half-life of the compound of Formula III compared to the other NO donor compounds plays a similarly important role. Upon decomposition at neutral pH, the compound of Formula III also releases nitroxyl (HNO) in addition to NO. HNO is another reactive nitrogen compound. Nitroxyl forms a dimer with itself, which rearranges to yield one mole of NO and one mole of water.

[0376] It is contemplated that various combinations or subcombinations of the specific attributes and aspects of the embodiments disclosed above may be made and still fall within one or more inventions. Furthermore, any specific attribute, aspect, method, property, characteristic, quality, attribute, element, etc. disclosed herein in connection with an embodiment may be used in all other embodiments described herein.

[0377] Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the disclosed invention. Accordingly, it is intended that the scope of the invention(s) disclosed herein not be limited by the specific disclosed embodiments described above.

[0378] Moreover, while the invention is susceptible to various modifications and alternative forms, specific examples thereof are shown in the drawings and described in detail herein. It should be understood, however, that the invention should not be limited to the particular forms or methods disclosed, but rather, the invention is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the various embodiments described and the appended claims. Any method disclosed herein need not be performed in the order recited.

[0379] The methods disclosed herein include specific actions taken by a practitioner, but they may also include any third-party instruction of those actions, either explicitly or implicitly. For example, an action such as "administering an NO-donating composition" includes "instructing administration of the NO-donating composition." Additionally, when features or aspects of the present disclosure are described in terms of a Markusch group, those skilled in the art will recognize that the present disclosure is also described in terms of individual members of the Markusch group, or any subgroup of members of the Markusch group.

[0380] The contents of all documents referenced herein are incorporated herein by reference for all purposes.

Claims

1. 1. An antibacterial composition for the treatment or prevention of pulmonary microbial infections comprising an NO-releasing compound of formula I and a pharmaceutically acceptable carrier or excipient, wherein formula I has the following structure: 【Chemical 1】 During the ceremony, X is selected from the group consisting of H and D; M ＋ is a pharmaceutically acceptable cation and M+ is a cation having a valency other than 1, the ratio of said compound of formula I to said cation is such that the total positive and negative charges are equal.

2. 10. The composition of claim 1, wherein the cation is selected from the group consisting of sodium, potassium, lithium, calcium, magnesium, ammonium, and substituted ammonium.

3. The compound has the following structure: 【Chemistry 2】 In the formula, M ＋ The composition of claim 1 , wherein refers to a pharmaceutically acceptable cation.

4. 2. The composition of claim 1, wherein the compound has the following structure: 【Chemistry 3】

5. 10. The composition of claim 1, further comprising a mixture of the compound of formula I and one or more thermal or acid decomposition products.

6. 10. The composition of claim 1, further comprising water such that the concentration of the compound of Formula I in the composition is about 0.01 to 98%.

7. The composition of any one of claims 1 to 6, in the form of a sprayable solution.

8. the compound has a total releasable NO store in the range of 0.1 to 8.0 μmol of NO per mg of the compound; the compound has an NO half-life in the range of 0.1 to 24 hours; the compound has a total NO release duration ranging from 1 to 60 hours; or 7. The composition of any one of claims 1 to 6, wherein the total NO release after 4 hours is in the range of 0.1 to 7.0 μmol NO / mg of compound.

9. 10. Use of a composition according to any one of claims 1 to 6 in the manufacture of a medicament for treating a pulmonary microbial infection, preventing a pulmonary microbial infection or reducing the pulmonary microbial burden in a patient.

10. 10. The use of claim 9, wherein the microorganisms associated with the microbial infection comprise two or more of gram-positive bacteria, gram-negative bacteria, fungi, yeasts, and viruses.

11. 10. The use according to claim 9, wherein the microbial infection is caused by a bacterium associated with cystic fibrosis.

12. 10. The use according to claim 9, wherein the microbial infection is an infection caused by a virus selected from the group consisting of coronavirus, picornavirus, orthomyxovirus, paramyxovirus, cytomegalovirus, and adenovirus.

13. The use of claim 12, wherein the virus is SARS, MERS, or SARS-CoV2.

14. 10. The use of claim 9, wherein the composition is formulated for inhalation, nebulization, intranasal delivery, or delivery by aerosol.

15. 10. The use of claim 9, wherein the medicament further comprises a second antibacterial agent, an anti-inflammatory agent, an anticoagulant, a platelet aggregation inhibitor, a chelating agent, an antibody, a gallium salt, or a siderophore.

16. The above microorganisms are Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus lugdunensis, Staphylococcus epidermidis, Streptococcus milleri / anginous, Streptococcus pyogenes, non-mycobacterial, Mycobacterium tuberculosis tuberculosis, Burkholderia genus, Achromobacter xylosoxidans, Pandoraeasputorum, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, Haemophilus pittmaniae, Serratia marcescens, Candida albicans, drug-resistant Candida albicans, Candida glabrata, Candida krusei, Candida Guilliermondii, Candida auris, Candida tropicalis, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Aspergillus flavus, Morganella morganii, Inquilinus limosus, Ralstonia mannitolytica, Pandoraa apista, Pandoraa pneumonusa, Pandoraa spitorum, Bdellovibrio bacterion mmetallidurans、Cupriavidus pauculus、Cupriavidus respiraculi、Del､tia acidivrdans、Exophilia dermatitisidis、Herbaspirillumm frorererereresseropedicae, Klebsiella pneumoniae, Pandoraea norimbergensis, Pandoraea pulmonicola, Pseudomonas mendocina, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas stutzeri, Ralstonia insidiosa, Ralstonia pickettii, Neisseria gonorrhoeae, NDM-1 positive E. coli, Enterobacter cloaca, vancomycin resistant E. faecium, vancomycin resistant E. faecalis, E. faecium, E. faecalis, clindamycin-resistant S. faecalis. agalactiae, S. agalactiae, Bacteroides fragilis, Clostridium difficile, Streptococcus pneumonia, Moraxella catarrhalis, Haemophilus haemolyticus, Haemophilus parainfluenzae, Chlamydophilia pneumoniae, Mycoplasma pneumoniae, Atopobium, Sphingomonas, Saccharibacteria, Leptotrichia, Capnocytophaga, Oribacterium, Aquabacterium, Lachnoanaerobaculum, Campylobacter, Acinetobacter, Agro 10. The use of claim 9, wherein the bacterium is selected from the group consisting of bacterium; Bordetella; Brevundimonas; Chryseobacterium; Delftia; Enterobacter; Klebsiella; Pandoraea; Pseudomonas; Ralstonia, and Prevotella.

17. 10. The use of claim 9, wherein the patient has an infection selected from the group consisting of bronchiectasis infection, pneumonia, valley fever, allergic bronchopulmonary aspergillosis (ABPA), ventilator-acquired pneumonia, hospital-acquired pneumonia, community-acquired pneumonia, ventilator-associated tracheobronchitis, lower respiratory tract infection, nontuberculous mycobacterial disease, anthrax, legionellosis, whooping cough, bronchitis, bronchiolitis, COPD-related infections, and infections associated after lung transplantation.

18. The pharmaceutical agent is selected from the group consisting of amikacin, tobramycin, gentamicin, piperacillin, mezlocillin, ticarcillin, imipenem, ciprofloxacin, ceftazidime, aztreonam, ticarcillin-clavulanate, dicloxacillin, amoxicillin, trimethoprim-sulfamethoxazole, cephalexin, piperacillin-tazobactam, linezolid, daptomycin, vancomycin, metronidazole, clindamycin, colistin, tetracycline, levofloxacin, amoxicillin and clavulanate (Augmentin®), cloxacillin, dicloxacillin, 10. The use of claim 9, further comprising a compound selected from the group consisting of rifampin, cefdinir, cefprozil, cefaclor, cefuroxime, erythromycin / sulfisoxazole, erythromycin, clarithromycin, azithromycin, doxycycline, minocycline, tigecycline, imipenem, meropenem, colitimethate / Colistin®, methicillin, oxacillin, nafcillin, carbenicillin, azlocillin, piperacillin and tazobactam (Zosyn®), cefepime, ethambutol, rifampin, and meropenem.

19. 10. The use according to claim 9, wherein the microorganism is a fungus selected from the group consisting of Histoplasma capsulatum, Blastomyces dermatitidis, Sporothrix schenckii, Coccidioides immitis, Coccidioides posadasii, Aspergilli, Cryptococcosis, Candida, Mucormycosis, and Pneumocystis jirovecii.

20. 19. The use of claim 18, wherein the medicament further comprises an antifungal compound selected from the group consisting of fluconazole, posaconazole, viroconazole, itraconazole, echinocandin, amphotericin, trimethoprim, sulfamethoxazole, primaquine, clindamycin, atovaquone, IV pentamidine, dapsone, and flucytosine.

21. Use of a composition described in any one of claims 1 to 6 in the manufacture of a medicament configured to deliver nitric oxide to a subject in need of treatment or prevention of a pulmonary microbial infection and / or reduction of the microbial load of a pulmonary microbial infection.

Images