Methods for Treating Nontuberculous Mycobacterial Disease - Patent application

Abstract

The present disclosure provides bismuth-thiol (BT) compositions and methods for treating nontuberculous mycobacterial infections and associated conditions in subjects in need thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 298,124, filed January 10, 2022, which is incorporated by reference in its entirety herein. [Background technology]

[0002] background

[0002] The incidence of pulmonary infections due to nontuberculous mycobacteria (NTM), such as M. avium and M. abscessus, is increasing, and treatment options are limited. Approximately 80% of pulmonary NTM cases are associated with M. avium. These infections are common in patients with chronic pulmonary conditions, such as cystic fibrosis, and are associated with severe respiratory disease. Currently approved macrolide (clarithromycin) or azalide (azithromycin) therapies often result in resistance or are associated with side effects related to the duration of treatment, which can be 18-24 months and includes a minimum of three antibiotics. Treatment outcomes for M. abscessus infections are even more problematic, since the cure rate for patients with pulmonary infections is only 25-58%. As such, M. abscessus is referred to as an "incurable nightmare."

[0003]

[0003] Due to poor treatment outcomes and long duration of treatment combined with drug toxicity, there is an urgent medical need to develop more effective and safer regimens consisting of drugs with potent anti-NTM activity.

[0004]

[0004] The present disclosure addresses these and other problems related to the treatment of NTM diseases. Summary of the Invention [Means for solving the problem]

[0005] overview

[0005] In some embodiments, the present disclosure provides a method for treating an infection in a subject caused by nontuberculous mycobacteria (NTM), comprising administering to the subject an effective amount of a bismuth-thiol (BT) composition comprising a BT compound.

[0006]

[0006] In some embodiments, the present disclosure provides a method for reducing NTM intracellular bacterial load in a subject, the method comprising contacting infected cells of the subject with an effective amount of a bismuth-thiol (BT) composition comprising a BT compound.

[0007]

[0007] In some embodiments, the present disclosure provides a method for treating or providing prophylaxis against a nontuberculous mycobacterial (NTM) pulmonary infection in a subject in need of treatment or prophylaxis, the method comprising administering to the subject's lungs a BT composition comprising a BT compound for an administration period.

[0008]

[0008] In some embodiments, the present disclosure provides a method for treating a biofilm-associated nontuberculous mycobacterial (NTM) infection in the lungs of a subject in need thereof, comprising administering to the subject a BT composition comprising a BT compound.

[0009]

[0009] In some embodiments, the NTM infection is a pulmonary infection. In some embodiments, the NTM infection is an extrapulmonary infection. In some embodiments, the NTM infection is a chronic pulmonary infection.

[0010]

[0010] In some embodiments, the NTM infection is located in or on the lung mucosa, bronchi, alveoli, macrophages, and / or bronchioles. In some embodiments, the NTM infection is an NTM infection of the skin. In some embodiments, the NTM infection is an infection of the skin, bones, joints, lymphatic system, and / or soft tissue. In some embodiments, the NTM infection is located in a macrophage. In some embodiments, the NTM infection is located at least partially in a macrophage. In some embodiments, the macrophage cell is a THP-1 cell. In some embodiments, the NTM infection is located in a histiocyte.

[0011]

[0011] In some embodiments, the present disclosure provides a method for treating an NTM infection in a subject, comprising: (i) testing for the presence of bacterially infected macrophages in a biological sample from the subject; and (ii) if the sample tests positive for bacterially infected macrophages, administering to the subject an effective amount of a bismuth-thiol (BT) composition comprising a BT compound.

[0012]

[0012] In some embodiments, the present disclosure provides a method for treating an NTM infection in a subject having a macrophage infection, comprising administering to the subject an effective amount of a bismuth-thiol (BT) composition comprising a BT compound.

[0013]

[0013] In some embodiments, the method further includes testing for the presence of bacterially-infected macrophages in a biological sample from the subject, and if the sample tests positive for bacterially-infected macrophages, administering to the subject an effective amount of a bismuth-thiol (BT) composition.

[0014]

[0014] In some embodiments, the NTM infection is selected from the group consisting of M. avium, M. avium subsp. hominissuis (MAH), M. abscessus, M. chelonae, M. bolletii, M. kansasii, M. chimaera, M. ulcerans, M. avium complex (MAC) (M. avium and M. intracellulare), M. conspicuum, M. kansasii, M. peregrinum (M. peregrinum, M. immunogenum, M. xenopi, M. marinum, M. malmoense, M. massiliense, M. mucogenicum, M. nonchromogenicum, M. porcinum, M. scrofulaceum, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. terrae complex, M. haemophilum, M. genavense genavense, M. asiaticum, M. shimoidei, M. gordonae, M. nonchromogenicum, M. triplex, M. lentiflavum, M. celatum, M. fortuitum, M. fortuitum complex (M.In some embodiments, the NTM infection is caused by M. abscessus subsp. abscessus, M. abscessus subsp. bolletti, or M. abscessus subsp. massiliense. In some embodiments, the NTM infection is caused by M. avium or M. abscessus. In some embodiments, the NTM infection is caused by M. avium complex (MAC) (M. avium and M. intracellulare). In some embodiments, the NTM infection is caused by M. abscessus subsp. abscessus, M. abscessus subsp. bolletti, or M. abscessus subsp. massiliense.

[0015] In some embodiments, the NTM infection is resistant to standard of care antibiotic therapy. In some embodiments, the NTM infection is resistant to amikacin. In some embodiments, the NTM infection is resistant to macrolide or azalide therapy.

[0016] In some embodiments, the BT compound is selected from the group consisting of BisBAL, BisEDT, bis-dimercaprol, BisDTT, bis-2-mercaptoethanol, Bis-DTE, BisPyr, BisEry, BisTol, BisBDT, BisPDT, BisPyr / BAL, BisPyr / BDT, BisPyr / EDT, BisPyr / PDT, Bis-Pyr / Tol, BisPyr / Ery, bismuth-1-mercapto-2-propanol, BisEDT / CSTMN(1:1), BisPyr / CSTMN(1:1), BisBAL / CSTMN(1:1), BisTOL / CSTMN(1:1), and BisEDT / 2-hydroxy-1-propanethiol. In some embodiments, the BT compound is BisEDT or BisBAL. In some embodiments, the BT compound is BisEDT.

[0017]

[0017] In some embodiments, the BT composition is administered by inhalation. In some embodiments, the BT composition is administered to the lungs of a subject. In some embodiments, the BT composition is administered to the lungs of a subject by a nebulizer, dry powder inhalation, nanoparticle inhalation, metered dose inhalation, or any other method of inhaling a drug known in the art. In some embodiments, the dry powder is micronized. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the nanoparticles are dry nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the concentration of bismuth in the lungs after a single daily dose is about 0.03 μg / g lung tissue to about 3 μg / g lung tissue.

[0018] In some embodiments, the BT composition is administered once per month, twice per month, three times per month, four times per month, once every two weeks, once per week, twice per week, or three times per week. In some embodiments, the BT composition is administered once per week. In some embodiments, the BT composition is administered for a period of less than 24 months, less than 18 months, less than 12 months, less than 9 months, less than 6 months, less than 3 months, or less than 1 month. In some embodiments, the BT composition is administered for a period of 1 to 56 days.

[0019]

[0019] In some embodiments, the subject has a chronic pulmonary condition. In some embodiments, the chronic pulmonary condition is cystic fibrosis, chronic pneumonia, bronchiectasis, restrictive lung disease, interstitial lung disease, pulmonary hypertension, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), emphysema, or asthma. In some embodiments, the chronic pulmonary condition is cystic fibrosis, chronic bronchitis, emphysema, bronchiectasis, pulmonary fibrosis, asbestosis, pneumonitis, chronic obstructive pulmonary disease (COPD), or asthma. In some embodiments, the chronic pulmonary condition is cystic fibrosis. In some embodiments, the subject is immunocompromised.

[0020] In some embodiments, the subject is administered about 30 μg to about 3,000 μg of the BT compound per administration. In some embodiments, the subject is administered about 100 μg to about 1,000 μg of the BT compound per administration.

[0021] In some embodiments, the method further comprises administering an effective amount of an additional antibacterial agent. In some embodiments, the additional antibacterial agent is amikacin, clarithromycin, azithromycin, ethambutol, rifampicin, tigecycline, linezolid, imipenem, cefoxitin, or a combination thereof. In some embodiments, the additional antibacterial agent is amikacin or clarithromycin. In some embodiments, the additional antibacterial agent is amikacin. In some embodiments, administration of the BT composition and the additional antibacterial agent to a subject in need thereof results in a synergistic effect in the treatment of NTM infection.

[0022] In some embodiments, the BT composition and the additional antibacterial agent are administered simultaneously, separately, or sequentially. In some embodiments, the BT composition is administered in parallel with, prior to, or after the additional antibacterial agent. [Brief description of the drawings]

[0023] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]

[0023] Figure 1 shows mycobacterial survival versus controls in THP-1 macrophages infected with M. avium strain 104 (MAH 104), M. avium strain 3388 (MAH 3388), and M. avium cystic fibrosis patient strain DNA00703 after treatment with amikacin, BisEDT (soluble and insoluble forms), or a combination thereof. [Diagram 2]

[0024] Shown is mycobacterial survival versus control in THP-1 macrophages infected with M. abscessus strain 19977 (B), M. abscessus cystic fibrosis patient strain 01715 (C), and M. abscessus cystic fibrosis patient strain 00703 (D) following treatment with minimum inhibitory concentrations of amikacin, BisEDT (soluble and insoluble forms), or a combination thereof. [Figure 3A]

[0025] Transmission electron microscopy images (TEM) of uninfected THP-1 macrophages 72 hours after seeding are shown (control). [Figure 3B]

[0026] Transmission electron microscopy images of THP-1 macrophages 24 hours after infection with M. avium are shown (without BisEDT treatment). [Figure 3C]

[0027] Transmission electron microscopy images of THP-1 macrophages 48 hours after infection with M. avium are shown (without BisEDT treatment). [Figure 3D]

[0028] 1 shows transmission electron microscopy images of M. avium infected THP-1 macrophages 24 hours after treatment with BisEDT (BIZ). [Figure 3E]

[0029] Transmission electron microscopy images of THP-1 macrophages 24 hours after infection with M. abscessus are shown (without BisEDT treatment). [Figure 3F]

[0030] 1 shows transmission electron microscopy images of M. abscessus-infected THP-1 macrophages 24 hours after treatment with BisEDT (BIZ). [Figure 3G]

[0031] 1 shows transmission electron microscopy images of M. abscessus-infected THP-1 macrophages 48 hours after treatment with BisEDT (BIZ). [Figure 4]

[0032] FIG. 1 is a schematic showing the inoculation, exposure, and recovery steps of the MBEC assay used in Example 5 to measure the susceptibility of M. avium and M. abscessus biofilms to BisEDT. [Figure 5A]

[0033] FIG. 1 shows the biofilm biomass formed on the pegs after inoculation with M. avium for staining purposes with a crystal violet assay. [Figure 5B]

[0034] FIG. 1 shows the biofilm biomass formed on the pegs after inoculation with M. abscessus for staining purposes with a crystal violet assay. [Figure 6]

[0035] Normalized heatmap distribution of antibiotic resistance for Mycobacterium abscessus. [Figure 7]

[0036] A schematic diagram summarizing the study groups and dosing schedules for the in vivo chronic M. abscessus infection study in SCID mice is provided. Treatment groups consisted of baseline (to determine initial infection levels), vehicle, inhaled BisEDT (200 μg / kg / day), inhaled BisEDT (1000 μg / kg / day) or inhaled amikacin (100 mg / kg / day). Treatment continued for 28 days (6 days / week), after which lung and spleen samples were collected for CFU determination or histopathology. [Figure 8A]

[0037] 1 shows colony forming units (CFU) recovered from lung homogenates in an in vivo chronic M. abscessus infection study in SCID mice. [Figure 8B]

[0038] 1 shows colony forming units (CFU) recovered from spleen homogenates in an in vivo chronic M. abscessus infection study in SCID mice.

[0024] definition

[0039] Unless otherwise defined, all scientific and technical terms used herein have the meanings commonly understood by those skilled in the art in the field of this disclosure. The following references provide those skilled in the art with general definitions of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified.

[0025]

[0040] As used in the specification and claims, the word "comprise" and its conjugations are used in its open-ended sense to mean that the items following the word are included, but items not specifically mentioned are not excluded. The present disclosure may suitably "comprise", "consist of", or "consist essentially of" the steps, elements, and / or reagents recited in the claims.

[0026]

[0041] As used herein, the term "or" is understood to be inclusive unless specifically stated or clear from context. As used herein, the terms "a," "an," and "the" are understood to be singular or plural unless specifically stated or clear from context.

[0027]

[0042] Throughout this specification, the terms "about" and / or "approximately" may be used in conjunction with numerical values ​​and / or ranges. The term "about" is understood to mean a value close to the recited value. For example, "about 40 [units]" may mean within ±10% of 40 (e.g., 36-44), ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, within less than ±1%, or any other value or range therebetween. Additionally, the phrase "less than about [value]" or "greater than about [value]" should be understood in light of the definition of the term "about" provided herein. The terms "about" and "approximately" may be used interchangeably.

[0028]

[0043] The term "bismuth" refers to the 83rd element of the periodic table, or an atom or ion thereof. Bismuth can exist in either the metallic state or an ionized state, such as the III or V oxidation state. Bismuth ions can be complexed with anions to make bismuth salts, or form complex anions that can then be further complexed with one or more additional cations. Bismuth can also form covalent bonds to other atoms, such as sulfur.

[0029]

[0044] As disclosed herein, a "bismuth-thiol compound" or "BT compound" is a compound having a bismuth atom covalently bonded to one, two, or three other sulfur atoms present on one or more thiol compounds. The term "thiol" refers to a carbon-containing compound or fragment thereof that contains an -SH group and can be represented by the general formula R-SH. These thiol compounds include compounds having one, two, three, or more S atoms. The thiol compounds may have other functional groups such as alkyl, hydroxyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, amino, and other substituents. Thiol compounds having two or more S atoms can be chelated with a bismuth atom such that two S atoms of the same molecule are covalently bonded to the bismuth atom. Exemplary bismuth-thiol compounds are shown below: [ka] .

[0030]

[0045] The term "subject" to which administration is contemplated includes, but is not limited to, humans (i.e., male or female of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults)) and / or other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercially important mammals such as cows, pigs, horses, sheep, goats, cats, and / or dogs; and / or birds, including commercially important birds such as chickens, ducks, geese, quail, and / or turkeys. A preferred subject is a human.

[0031]

[0046] As used herein, the phrase "co-administration" refers to any form of administration of two or more different therapeutic compounds, where a second compound is administered while a previously administered therapeutic compound is still effective in the body (e.g., the two compounds are effective in the patient at the same time, which may involve a synergistic effect of the two compounds). For example, the different therapeutic compounds can be administered in the same formulation or in separate formulations, either concomitantly or sequentially. In certain embodiments, the different therapeutic compounds can be administered within 1 hour, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or within 1 week.

[0032]

[0047] "Co-administration" refers to any method of administration of two drugs such that the pharmacological actions of both drugs are evident in the patient at the same time. Thus, co-administration does not require that a single pharmaceutical composition, the same dosage form, or even the same route of administration be used to administer both drugs, nor that the two drugs be administered at exactly the same time. However, in some situations, co-administration will most conveniently be performed at substantially the same time, using the same dosage form and the same route of administration.

[0033]

[0048] As used herein, the term "in combination" or "in further combination" or "in further combination" refers to the use of additional prophylactic and / or therapeutic agents and the BT composition of the present disclosure. The use of the term "in combination" does not restrict the order in which prophylactic and / or therapeutic agents are administered to a subject. A first prophylactic or therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly, or following (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of a second prophylactic or therapeutic agent (different from the first prophylactic or therapeutic agent) to a subject.

[0034]

[0049] As used herein, a therapeutic agent that "prevents" a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in a treated sample relative to an untreated control sample, or delays the onset of or reduces the severity of one or more signs and symptoms of the disorder or condition relative to an untreated control sample.

[0035]

[0050] As used herein, the terms "prophylactic agent" and "prophylactic agents" refer to agents, such as the BT compositions of the present disclosure, that can be used to prevent, manage, or control one or more signs and symptoms of a disease or disorder, particularly a disease or disorder associated with microbial (e.g., bacterial and / or fungal) infection, such as diabetic foot infection.

[0036]

[0051] The term "treating" refers to one or more of alleviating, ameliorating, delaying, reducing, ameliorating, or managing at least one symptom of a condition in a subject. The term "treating" also refers to one or more of halting the occurrence or worsening of a condition, delaying its onset (i.e., the period before clinical signs of a condition), or reducing the risk thereof.

[0037]

[0052] The term "managing" includes therapeutic treatment as defined above. Managing includes achieving a steady state level of infection as determined by known methods in the art. Steady state may include one or more assessments of the severity of the infection, the size and location of the infection, the number of different microbial pathogens present in the infection, the level of antibiotic tolerant or resistant microbial pathogens, the extent of response to treatment such as with the BT compositions disclosed herein, the extent of biofilm formation and reduction, and side effects experienced by the subject. During the management of an infection, the infection may fluctuate in the amount or extent of infection, from increasing to decreasing in severity, the amount of side effects experienced by the subject, or other subject outcome indications. Over a period of time such as days, months, or years, the extent of infection management can be determined by assessing whether the clinical course of the infection has improved, is bacteriostatic, or worsened, by evaluating the above factors. In some embodiments, managing an infection includes successful treatment of microbial pathogens that are otherwise drug tolerant or resistant.

[0038]

[0053] The term "reducing the severity" of an infection refers to an improvement in the clinical course of the infection by any measurable criterion. Such criteria may include measurable indicators such as a reduction in the extent of the infection, whether the infection is considered acute, the number and identity of the microbial pathogen causing the infection, the extent / spread / amount of microbial (e.g., bacterial and / or fungal) biofilm, and side effects experienced by the subject. In some embodiments, reducing the severity of an infection is determined by measuring an improvement in the clinical signs and symptoms of the infection. In some embodiments, reducing the severity includes halting the steady decline in outcome to obtain a stabilized infection, allowing the subject to enter into successful management of the infection. In other embodiments, reducing the severity may result in substantial to complete treatment of the infection.

[0039]

[0054] In some embodiments, reducing the severity of infection and / or symptoms can be related to patient-reported outcomes ("PROs"). A PRO instrument is defined as any measure of a subject's health status that is elicited from a patient and determines how the patient "feels or functions in relation to his or her health status." PROs are particularly useful in reporting outcomes in the DFI and whether the severity of symptoms has been reduced or decreased. Such symptoms can be observable events, behaviors, or emotions (e.g., ability to walk fast, loss of appetite, expression of anger) or unobservable outcomes known only to the patient (e.g., perception of pain, depressed feelings). In some embodiments, reducing the severity of infection and / or symptoms can be determined by physician assessment as is commonly known in the art, for example, by an 8-item wound score.

[0040]

[0055] As used herein, "effective amount" refers to an amount that is sufficient to achieve a desired biological effect. As used herein, "therapeutically effective amount" refers to an amount that is sufficient to achieve a desired therapeutic effect. For example, a therapeutically effective amount can refer to an amount that is sufficient to improve at least one sign or symptom of an infection.

[0041]

[0056] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio.

[0042]

[0057] A "response" to a method of treatment may include, among other things, a reduction or amelioration of adverse signs and symptoms, a reduction in the progression of the infection or its symptoms, an increase in beneficial symptoms or clinical outcome, a reduction in side effects, stabilization of the infection, and partial or complete cure of the infection, partial or complete wound closure, a reduction in wound size, or complete or substantially complete re-epithelialization.

[0043]

[0058] "Antibiotic susceptibility or sensitivity" refers to whether a bacterium is successfully treated by a given antibiotic. Similarly, "antifungal susceptibility or sensitivity" refers to whether a fungus is successfully treated by a given antibiotic. Susceptibility testing can be performed by methods known in the art, such as the Kirby-Bauer method, the Stokes method, and the agar broth dilution method. The effectiveness of an antibiotic in killing bacteria or preventing bacterial growth can be observed as an area of ​​reduced or stable amounts of bacterial growth, respectively, on a medium, such as a wafer, agar, or broth culture.

[0044]

[0059] "Antibiotic tolerance" refers to the ability of microorganisms, such as bacteria or fungi, to naturally resist being killed by antibiotics. It is not caused by mutated microorganisms, but by microbial cells that exist in a transient, dormant, non-dividing state. Antibiotic or drug tolerance is caused by a small subpopulation of microbial cells called persisters. Persisters are not mutants, but dormant cells that can survive antimicrobial treatments that kill the majority of their genetically identical siblings. The persister cells have entered a non-growing or extremely slow-growing physiological state that makes them insensitive (refractory or tolerant) to the action of antimicrobial drugs. Similarly, "antibiotic tolerance" refers to the ability of bacteria to naturally resist being killed by antibiotics, and "antifungal tolerance" refers to the ability of fungi to naturally resist being killed by antibiotics.

[0045]

[0060] "Antimicrobial resistance" refers to the ability of a microorganism to resist the effects of medicines that once successfully treated that microorganism. Microorganisms that are resistant to multiple antimicrobial drugs are called multidrug resistant (MDR). Resistance occurs through one of three mechanisms: natural resistance in a particular type of bacteria, genetic mutations, or one species acquiring resistance from another. Mutations can result in drug inactivation, changes in drug binding sites, altered metabolic pathways, and reduced drug permeability.

[0046]

[0061] As used herein, the terms "antibacterial activity", "antifungal activity" and "antimicrobial activity" with respect to the BT compositions of the present disclosure refer to the ability to kill and / or inhibit the growth or reproduction of a particular microorganism. In certain embodiments, antibacterial or antimicrobial activity is assessed by culturing bacteria, such as gram-positive bacteria (e.g., S. aureus), gram-negative bacteria (e.g., A. baumannii, E. coli, and / or P. aeruginosa), or bacteria not classified as gram-positive or gram-negative, or fungi, according to standard techniques (e.g., in liquid culture or on an agar plate), contacting the culture with the BT compositions of the present disclosure, and monitoring cell growth after said contact. For example, in liquid culture, bacteria may be grown to an optical density ("OD") that represents the midpoint of exponential growth of the culture; the culture is exposed to one or more concentrations of the BT compounds of the present disclosure or variants thereof, and the OD is monitored against a control culture. A decrease in OD relative to a control culture represents antimicrobial activity (e.g., indicates lytic killing activity). Similarly, bacterial colonies can be allowed to form on agar plates, the plates exposed to the disclosed BT compositions or variants thereof, and the subsequent growth of colonies assessed relative to control plates. A decrease in colony size or total number of colonies indicates antimicrobial activity.

[0047]

[0062] "Biofilm" refers to any syntrophic consortium of microorganisms in which cells attach to each other and often to surfaces. These adherent cells are embedded within a slimy extracellular matrix composed of extracellular polymeric substances (EPS). Upon biofilm formation, the resistance of microorganisms to antibiotics is up to 1000 times greater than that of planktonic bacteria. Bacterial aggregates are clusters of laterally aligned cells that can initiate biofilm development, which has a more complex and dense 3D structure. In some embodiments, biofilms can include one or more species of bacteria (e.g., Pseudomonas aeruginosa and Staphylococcus aureus) and / or one or more different phyla (e.g., bacteria, viruses, and fungi).

[0048]

[0063] The term "infection" is used herein in its broadest sense and refers to any infection caused by a microbial bacterial infection, fungal infection or parasitic infection (e.g., protozoa, amoeba or helminths), such as a viral infection. Examples of such infections can be found in several well-known textbooks, such as "Medical Microbiology" (Greenwood, D., Slack, R., Peutherer, J., Churchill Livingstone Press, 2002); "Mims' Pathogenesis of Infectious Disease" (Mims, C., Nash, A., Stephen, J., Academic Press, 2000); "Fields" Virology. (Fields, BN, Knipe DM, Howley, PM, Lippincott Williams and Wilkins, 2001); and "The Sanford Guide To Antimicrobial Therapy," 26th Edition, JP Sanford et al. (Antimicrobial Therapy, Inc., 1996), which are incorporated herein by reference. For example, the presence of infection in a diabetic foot wound is defined by clinical signs and symptoms of infection or inflammation, not by culture of resident microorganisms. However, immediately following resolution of the clinical signs and symptoms of wound infection, most patients still have an underlying ulcer (e.g., a diabetic foot ulcer) that requires ongoing treatment to promote complete wound closure. However, it is noteworthy that many wound specialists believe that in addition to the clinically defined state of infection, there exists a less clinically evident pathological state known as "critical colonization." In this state, wounds may be delayed or halted in wound healing due to the subclinical presence of high levels of bacteria. This critical colonization, sometimes referred to as high "wound bioburden," is often polymicrobial and associated with biofilm-producing bacteria; it has been shown to induce or prolong the active inflammatory phase of repair, impeding the normal wound healing process.The bacterial cells that compose such biofilms are difficult to recognize because they often exist in a viable but non-culturable (VBNC) state (Pasquaroli 2013), but they are attached to surfaces and are typically more tolerant and resistant to antibiotics and antiseptics than their planktonic counterparts (Costerton 1999; Nguyen 2011). Thus, the term "infection" contemplates a clinically defined state of infection as well as "critical colonization."

[0049]

[0064] As used herein, "airway surface" and "pulmonary surface" include the surfaces of the airways of the lungs, such as the bronchi and bronchioles, the alveolar surfaces, and the nasal and sinus surfaces.

[0050]

[0065] As used herein, the term "volume median diameter" or "VMD" of an aerosol is a particle size diameter defined such that half of the population of aerosol particles are comprised of particles having a diameter larger than the VMD and half of the population of aerosol particles are comprised of particles having a diameter smaller than the VMD. VMD is typically measured by laser diffraction.

[0051]

[0066] "Mass Median Aerodynamic Diameter" or "MMAD" is a measure of the aerodynamic size of a dispersed aerosol particle. Aerodynamic diameter is used to describe an aerosolized particle in terms of its settling behavior and is generally the diameter of a unit density sphere in air that has the same settling velocity as the particle in question. Aerodynamic diameter encompasses the particle shape, density, and physical size of the particle. As used herein, MMAD refers to the midpoint or median of the aerodynamic particle size distribution of an aerosolized particle as determined by cascade impaction and / or laser time-of-flight and / or cascade impactor.

[0052]

[0067] "Mass Median Diameter" or "MMD" is a measure of the average particle size. Any of several commonly used techniques can be used to measure the average particle size.

[0053]

[0068] As used herein, "D90" refers to the 90th percentile of particle diameter (either fine particles or aerosolized particles). For example, if D90=1 μm, then 90% of the particles are smaller than 1 μm. Similarly, "D80" refers to the 80th percentile of the particle diameter (either fine particles or aerosolized particles), "D70" refers to the 70th percentile of the particle diameter (either fine particles or aerosolized particles), "D60" refers to the 60th percentile of the particle diameter (either fine particles or aerosolized particles), "D50" refers to the 50th percentile of the particle diameter (either fine particles or aerosolized particles), "D40" refers to the 40th percentile of the particle diameter (either fine particles or aerosolized particles), "D30" refers to the 30th percentile of the particle diameter (either fine particles or aerosolized particles), "D20" refers to the 20th percentile of the particle diameter (either fine particles or aerosolized particles), and "D10" refers to the 10th percentile of the particle diameter (either fine particles or aerosolized particles).

[0054]

[0069] As used herein, "monodisperse" refers to a population of particles (bulk or aerosol dispersion) that includes particles of substantially uniform MMD and / or MMAD and / or VMD.

[0055]

[0070] As used herein, the term "deposition efficiency" refers to the percentage of a delivered dose that is deposited in the area of ​​interest. Thus, the deposition efficiency of a method and / or system for delivering an aerosolized pharmaceutical to the lungs is the amount of aerosol by mass that is deposited in the lungs divided by the total amount of aerosol delivered to the nostrils by the system.

[0056]

[0071] As used herein, "substantially" or "substantially" refers to the complete or nearly complete extent or degree of an action, property, quality, state, structure, item, or result. For example, an object that is "substantially" surrounded would mean that the object is completely surrounded or nearly completely surrounded. The exact acceptable degree of deviation from absolute completeness may depend on the specific circumstances in some cases. In general, however, the nearness of completion would likely have the same overall result as if absolute and total completion had been obtained. The use of "substantially" applies equally when used in a negative sense to refer to the complete or nearly complete lack of an action, property, quality, state, structure, item, or result. For example, a composition that is "substantially free" of other active agents would be completely devoid of other active agents or nearly completely devoid of other active agents, so that it would have the same effect as if it were completely devoid of other active agents. In other words, a composition that is "substantially free" of a component or element or another active agent may still contain such an article as long as there is no measurable effect of them. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Detailed Description

[0072] Nontuberculous mycobacteria (NTM) are opportunistic pathogens that cause mostly TB-like lung disease in immunocompromised patients or those with pre-existing conditions such as cystic fibrosis (CF), chronic bronchitis, emphysema, bronchiectasis, pulmonary fibrosis, asbestosis, pneumonitis, chronic obstructive pulmonary disease (COPD), asthma, and other underlying lung diseases. The annual prevalence of NTM lung disease (NTM-PD) varies in different regions, ranging from 0.2 / 100000 to 9.8 / 1000001,2, with an overall alarming rate of increase3,4. The situation is even worse in vulnerable populations. Large epidemiological surveys in several countries and regions reported a high prevalence of 3.3-22.6% in CF patients, while COPD patients treated with inhaled corticosteroid therapy are associated with a 29-fold increased risk of NTM.

[0058]

[0073] Unfortunately, the development of effective drug regimens to treat NTM-PD has been a significant challenge, meaning that treatment options are often limited and patient outcomes are poor, in part due to the ability of NTM to form biofilms, lengthy treatment regimens, and drug-related side effects.

[0059]

[0074] To address these and other issues, the present disclosure provides bismuth-thiol compositions and methods for the treatment of NTM disease and related conditions. BT compositions have been established as highly effective antimicrobial agents for treating a variety of pathogens, including those involved in CF, wounds, and co-infections, as described in WO 2020 / 028558, WO 2020 / 028561, and WO 2021 / 195236, each of which is incorporated herein by reference.

[0060] How to use

[0075] In some embodiments, the present disclosure provides a method for treating an infection caused by a nontuberculous mycobacterium (NTM) in a subject, comprising administering to the subject an effective amount of a bismuth-thiol (BT) composition comprising a BT compound.

[0061]

[0076] In some embodiments, the BT compound is a BT compound disclosed herein. In some embodiments, the BT compound is selected from the group consisting of BisBAL, BisEDT, bis-dimercaprol, BisDTT, bis-2-mercaptoethanol, Bis-DTE, BisPyr, BisEry, BisTol, BisBDT, BisPDT, BisPyr / BAL, BisPyr / BDT, BisPyr / EDT, BisPyr / PDT, Bis-Pyr / Tol, BisPyr / Ery, bismuth-1-mercapto-2-propanol, BisEDT / CSTMN(1:1), BisPyr / CSTMN(1:1), BisBAL / CSTMN(1:1), BisTOL / CSTMN(1:1), and BisEDT / 2-hydroxy-1-propanethiol. In some embodiments, the BT compound is BisEDT or BisBAL. In some embodiments, the BT compound is BisEDT.

[0062]

[0077] In some embodiments, the NTM infection is a pulmonary infection. In some embodiments, the NTM infection is a chronic pulmonary infection. In some embodiments, the NTM infection is an extrapulmonary infection. In some embodiments, the NTM infection is a skin infection. In some embodiments, the extrapulmonary infection is an infection of the skin, bones, joints, lymphatic system, soft tissue, or a combination thereof.

[0063]

[0078] In some embodiments, the NTM infection is a biofilm-associated NTM infection. In some embodiments, the NTM infection comprises planktonic cells and a biofilm.

[0064]

[0079] In some embodiments, the NTM infection is caused by an antibiotic resistant strain of NTM. In some embodiments, the NTM infection is resistant to standard of care antimicrobials. In some embodiments, the NTM infection is resistant to macrolide or azalide therapy. In some embodiments, the NTM infection is resistant to aminoglycoside therapy. In some embodiments, the NTM infection is resistant to amikacin, clarithromycin, azithromycin, ethambutol, rifampicin, tigecycline, linezolid, imipenem, cefoxitin, or combinations thereof. In some embodiments, the NTM infection is resistant to amikacin. In some embodiments, the antimicrobial resistant NTM infection comprises a biofilm.

[0065]

[0080] In some embodiments, the NTM infection is selected from the group consisting of M. avium, M. avium subsp. hominissuis (MAH), M. abscessus, M. chelonae, M. bolletii, M. kansasii, M. ulcerans, M. avium complex (MAC) (M. avium and M. intracellulare), M. conspicuum, M. kansasii, M. peregrinum, M. immunogenum, M. xenopi (M. xenopi, M. marinum, M. malmoense, M. marinum, M. mucogenicum, M. nonchromogenicum, M. scrofulaceum, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. terrae complex, M. haemophilum, M. genavense, M. asiaticum, M. shimoidei, M. gordone, M. gordonae, M. nonchromogenicum, M. triplex, M. lentiflavum, M. celatum, M. fortuitum, M. fortuitum complex (M. fortuitum and M. chelonae), or combinations thereof.In some embodiments, the NTM infection is caused by M. abscessus, M. avium, M. intracellulare, M. fortuitum, M. gordonae, M. kansasii, M. avium complex (MAC), M. abscessus complex (MABSC) M. marinum, M. terrae, and M. cheloni. In some embodiments, the NTM infection is caused by M. abscessus, M. avium, or a combination thereof. In some embodiments, the NTM infection is caused by M. abscessus, M. avium complex (M. avium and M. intracellulare), or a combination thereof. In some embodiments, the NTM pulmonary infection is caused by M. avium complex (M. avium and M. intracellulare). In some embodiments, the M. avium is M. avium subsp. hominissuis. In some embodiments, the M. abscessus is M. abscessus subsp. abscessus, M. abscessus subsp. bolletti, or M. abscessus subsp. massiliense.

[0066]

[0081] In some embodiments, the NTM infection is located in or on the lung mucosa, bronchi, alveoli, macrophages, and / or bronchioles. In some embodiments, the NTM infection is an NTM infection of the skin. In some embodiments, the NTM infection is an infection of the skin, bones, joints, lymphatic system, and / or soft tissue. In some embodiments, the NTM infection is located in a macrophage. In some embodiments, the NTM infection is at least partially located in a macrophage. In some embodiments, the macrophage cell is a THP-1 cell. In some embodiments, the macrophage is an alveolar macrophage. In some embodiments, the macrophage is an M1 or M1-like macrophage. In some embodiments, the macrophage is infected with one or more strains of M. abscessus and / or M. avium. In some embodiments, the macrophages are infected with one or more strains of M. abscessus and / or M. avium complex. In some embodiments, the NTM infection is located in a histiocyte. In some embodiments, the infection is located in an osteoclast.

[0067]

[0082] In some embodiments, upon administration of the BT composition to a subject, the bacterial load in macrophages is reduced.

[0068]

[0083] In some embodiments, the subject in need of treatment has a chronic disease. In some embodiments, the subject has a chronic pulmonary condition. In some embodiments, the chronic pulmonary condition is cystic fibrosis, chronic pneumonia, bronchiectasis, restrictive lung disease, interstitial lung disease, pulmonary hypertension, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), emphysema, or asthma. In some embodiments, the chronic pulmonary condition is cystic fibrosis, chronic bronchitis, emphysema, bronchiectasis, pulmonary fibrosis, asbestosis, pneumonitis, chronic obstructive pulmonary disease (COPD), or asthma. In some embodiments, the chronic pulmonary condition is cystic fibrosis. In some embodiments, the subject is immunocompromised.

[0069]

[0084] In some embodiments, the subject with NTM disease also suffers from a co-morbid disease, such as malignancy and / or cardiovascular disease. In some embodiments, the co-morbid disease is selected from the group consisting of diabetes, mitral valve disorder, acute bronchitis, pulmonary hypertension, pneumonia, asthma, cystic fibrosis, pulmonary fibrosis, laryngeal malformation, tracheal malformation, bronchial malformation, aspergillosis, HIV, bronchiectasis, arrhythmia, cancer (including but not limited to tracheal cancer, bronchial cancer, prostate cancer, lung cancer, ovarian cancer, breast cancer, etc.), chronic heart failure, ischemic heart disease COPD, Crohn's disease, Sjogren's syndrome, rheumatoid arthritis, panbronchiolitis, systemic lupus erythematosus (SLE), systemic scleroderma, dyslipidemia, gastroesophageal reflux disease (GERD), or a combination thereof. In some embodiments, the co-morbidity is selected from the group consisting of diabetes, mitral valve disorders, acute bronchitis, pulmonary hypertension, pneumonia, asthma, tracheal carcinoma, bronchial carcinoma, lung cancer, cystic fibrosis, pulmonary fibrosis, laryngeal malformations, tracheal malformations, bronchial malformations, aspergillosis, HIV, bronchiectasis, or a combination thereof.

[0070]

[0085] In some embodiments, the BT composition is administered to a subject in need thereof by inhalation. In some embodiments, the BT composition is administered to a subject in need thereof by inhalation of an aerosol as described herein. In some embodiments, the BT composition is administered to the subject's lungs. In some embodiments, the BT composition is aerosolized and administered to the subject's lungs. In some embodiments, the BT composition is administered to the subject's lungs by nebulizer, dry powder inhalation, nanoparticle inhalation, metered dose inhalation, or any other method of inhaling a drug known in the art. In some embodiments, the dry powder is micronized. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the nanoparticles are dry nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the concentration of bismuth in the lungs after a single daily dose is about 0.03 μg / g lung tissue to about 3 μg / g lung tissue.

[0071]

[0086] In some embodiments, a subject in need thereof is administered about 30 μg to about 3,000 μg of the BT compound per administration, including all ranges and values ​​therebetween, such as about 30 μg, about 100 μg, about 200 μg, about 300 μg, about 400 μg, about 500 μg, about 600 μg, about 700 μg, about 800 μg, about 900 μg, about 1000 μg, about 1100 μg, About 1200μg, about 1300μg, about 1400μg, about 1500μg, about 1600μg, about 1700μg, about 1800μg, about 1900μg, about 2000μg, about 2100μg, about 2200μg, about 2300μg, about 2400μg, about 2500μg, about 2600μg, about 2700μg, about 2800μg, about 2900μg, or about 3000μg is administered. In some embodiments, the subject is administered about 100μg to about 2,000μg of the BT compound per administration. In some embodiments, the subject is administered about 100μg to about 3,000μg of the BT compound per administration. In some embodiments, the subject is administered about 1000 μg to about 3,000 μg of the BT compound per administration. In some embodiments, the subject is administered about 1000 μg to about 2,000 μg of the BT compound per administration. In some embodiments, the subject is administered about 2000 μg to about 3,000 μg of the BT compound per administration.

[0072]

[0087] In some embodiments, the concentration of bismuth in the lungs after a single daily dose is about 0.03 μg / g lung tissue to about 3 μg / g lung tissue, e.g., about 0.03 μg / g lung tissue, about 0.1 μg / g lung tissue, about 0.25 μg / g lung tissue, about 0.5 μg / g lung tissue, about 0.75 μg / g lung tissue, about 1 μg / g lung tissue, about 1.25 μg / g lung tissue, about 1.5 μg / g lung tissue, about 1.75 μg / g lung tissue, about 2 μg / g lung tissue, about 2.25 μg / g lung tissue, about 2.5 μg / g lung tissue, about 2.75 μg / g lung tissue, or about 3 μg / g lung tissue, including all ranges and values ​​therebetween. In some embodiments, the concentration of bismuth in the lungs after a single daily dose is about 0.1 μg / g lung tissue to about 3 μg / g lung tissue. In some embodiments, the concentration of bismuth in the lungs after a single daily dose is about 1 μg / g lung tissue to about 3 μg / g lung tissue, in some embodiments, the concentration of bismuth in the lungs after 28 daily doses is about 0.3 μg / g lung tissue to about 60 μg / g lung tissue.

[0073]

[0088] In some embodiments, the BT composition is administered to a subject in need thereof three times per day, twice per day, once per day, every other day, once every third day, once per week, once per every other week, once per month, or once per every other month. In some embodiments, the BT composition is administered once per month, twice per month, three times per month, four times per month, once every two weeks, once per week, twice per week, or three times per week. In some embodiments, the BT composition is administered once per month, twice per month, three times per month, or four times per month. In some embodiments, the BT composition is administered once every two weeks, once per week, twice per week, or three times per week. In some embodiments, the BT composition is administered once per week. In some embodiments, the BT composition is administered once per day. In some embodiments, the BT composition is administered once per week. In some embodiments, the BT composition is administered once per month. In some embodiments, the BT composition is administered twice a month. In some embodiments, the BT composition is administered three times a month. In some embodiments, the BT composition is administered four times a month. In certain embodiments, the BT composition is administered once every other week. In some embodiments, the BT composition is administered once every three weeks. In some embodiments, the BT composition is administered chronically on a 4 week on / 4 week off dosing schedule. In some embodiments, the BT composition is administered chronically, for example as part of a basal therapy. In some embodiments, the BT composition is administered as needed.

[0074]

[0089] In some embodiments, the BT composition is administered for a period of less than 24 months, less than 18 months, less than 12 months, less than 9 months, less than 6 months, less than 3 months, or less than 1 month. In some embodiments, the BT composition is administered for a period of 1 month to 24 months. In some embodiments, the BT composition is administered for a period of 1 month to 18 months. In some embodiments, the BT composition is administered for a period of 1 month to 12 months. In some embodiments, the BT composition is administered for a period of 1 month to 6 months. In some embodiments, the BT composition is administered for a period of 1 month to 3 months. In some embodiments, the BT composition is administered for a period of 3 months to 24 months. In some embodiments, the BT composition is administered for a period of 3 months to 18 months. In some embodiments, the BT composition is administered for a period of 3 months to 12 months. In some embodiments, the BT composition is administered for a period of 3 months to 6 months. In some embodiments, the BT composition is administered for a period of 1 to 56 days. In some embodiments, the BT composition is administered for a period of 14 to 28 days. In some embodiments, the BT composition is administered once per month for the treatment period. In some embodiments, the BT composition is administered once every two weeks for a treatment period. In some embodiments, the BT composition is administered once per week for a treatment period. In some embodiments, the BT composition is administered twice per week for a treatment period. In some embodiments, the BT composition is administered three times per week for a treatment period.

[0075]

[0090] In some embodiments, the disclosure provides a method for reducing NTM intracellular bacterial load in a subject, comprising contacting infected cells of the subject with an effective amount of a bismuth-thiol (BT) composition comprising a BT compound.

[0076]

[0091] In some embodiments, contacting the cells with the BT composition reduces the intracellular bacterial load by about 10-fold to about 1000-fold. In some embodiments, the intracellular bacterial load reduces by about 10-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, or about 1000-fold. In some embodiments, contacting the cells with the BT composition prevents the intracellular bacterial load from increasing, i.e., the BT composition provides a bacteriostatic effect. In some embodiments, contacting the cells with the BT composition results in intracellular accumulation of the BT compound. In some embodiments, contacting the cells with the BT composition results in phagocytosis of the BT compound.

[0077]

[0092] In some embodiments, the infected cell is a phagocyte. In some embodiments, the infected cell is a macrophage cell. In some embodiments, the macrophage cell is a THP-1 cell. In some embodiments, the macrophage cell is an alveolar macrophage. In some embodiments, the macrophage cell is an M1 or M1-like macrophage. In some embodiments, the macrophage cell is infected with one or more strains of M. abscessus and / or M. avium. In some embodiments, the macrophage cell is infected with one or more strains of M. abscessus and / or M. avium complex. In some embodiments, the macrophage cell is a histiocyte. In some embodiments, the macrophage cell is an osteoclast.

[0078]

[0093] In some embodiments, the present disclosure provides a method of treating or providing prophylaxis against a nontuberculous mycobacterial (NTM) pulmonary infection in a subject in need of such treatment or prophylaxis, comprising administering to the subject's lungs a BT composition of the present disclosure comprising a BT compound described herein for an administration period.

[0079]

[0094] In some embodiments, administering to the lungs of the patient includes aerosolizing the BT composition to provide an aerosolized BT composition, and administering the aerosolized BT composition to the lungs of the subject. In some embodiments, administering the aerosolized BT composition to the lungs of the subject is by nebulizer, dry powder inhalation, nanoparticle inhalation, metered dose inhalation, or any other method of inhaling drugs known in the art. In some embodiments, the dry powder is micronized. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the nanoparticles are dry nanoparticles. In some embodiments, administering the aerosolized BT composition to the lungs of the subject is by nebulizer.

[0080]

[0095] In some embodiments, the aerosolized BT composition is administered for a dosing period once a day in a single dosing session. In some embodiments, the aerosolized BT composition is administered for a dosing period three times per week in a single dosing session. In some embodiments, the aerosolized BT composition is administered for a dosing period two times per week in a single dosing session. In some embodiments, the aerosolized BT composition is administered for a dosing period once per week in a single dosing session. In some embodiments, the aerosolized BT composition is administered for a dosing period once every two weeks in a single dosing session.

[0081]

[0096] In some embodiments, during a single dosing session, the aerosolized BT composition is administered over less than about 75 minutes, less than about 60 minutes, less than about 30 minutes, less than about 15 minutes, or less than about 5 minutes. In some embodiments, during a single dosing session, the aerosolized BT composition is administered over about 60 to about 75 minutes, about 45 to about 60 minutes, about 30 to about 45 minutes, about 20 to about 30 minutes, or about 15 to about 20 minutes.

[0082]

[0097] In some embodiments, the aerosolized BT composition is administered for a 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 18 months, or 24 months treatment period. In some embodiments, the aerosolized BT composition is administered for a 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months treatment period.

[0083]

[0098] In some embodiments, the present disclosure provides a method for treating a biofilm-associated nontuberculous mycobacterial (NTM) infection in the lungs of a subject in need thereof, comprising administering to the subject a BT composition of the present disclosure comprising a BT compound described herein.

[0084]

[0099] In some embodiments, the present disclosure provides a method of treating an NTM infection in a subject having a macrophage infection, comprising administering to the subject an effective amount of a bismuth-thiol (BT) composition of the present disclosure that includes a BT compound described herein.

[0085]

[0100] In some embodiments, the present disclosure provides a method of treating an NTM infection in a subject, comprising: (i) testing for the presence of bacterially infected macrophages in a biological sample from the subject; and (ii) if the sample tests positive for bacterially infected macrophages, administering to the subject an effective amount of a bismuth-thiol (BT) composition of the present disclosure, comprising a BT compound as described herein.

[0086]

[0101] In some embodiments, macrophages are tested for the presence of one or more pathogenic species of NTM. In some embodiments, macrophages are tested for the presence of M. abscessus and / or M. avium. In some embodiments, macrophages are tested for the presence of M. abscessus. In some embodiments, macrophages are tested for the presence of M. avium.

[0087]

[0102] In some embodiments, the methods disclosed herein further comprise administering an effective amount of an additional antibacterial agent. In some embodiments, the additional antibacterial agent is amikacin, clarithromycin, azithromycin, ethambutol, rifampicin, tigecycline, linezolid, imipenem, cefoxitin, or a combination thereof. In some embodiments, the additional antibacterial agent is amikacin or clarithromycin. In some embodiments, the additional antibacterial agent is amikacin. In some embodiments, the administration of the BT composition and the additional antibacterial agent results in a synergistic effect in the treatment of NTM infection. In some embodiments, the administration of the BT composition and amikacin results in a synergistic effect in the treatment of NTM infection. Thus, in some embodiments, an effective amount of the additional antibacterial agent is an amount that is ineffective in the treatment of NTM infection when administered without the BT composition, and an effective amount of the BT composition is an amount that is ineffective in the treatment of NTM infection when administered without the additional antibacterial agent.

[0088]

[0103] In some embodiments, the BT composition and the additional antibacterial agent are administered simultaneously, separately, or sequentially. In some embodiments, the BT composition is administered in parallel with, prior to, or after the additional antibacterial agent. In some embodiments, the BT composition and the additional antibacterial agent are combined and administered to a subject in need thereof. In some embodiments, the BT composition and the additional antibacterial agent are co-administered to a subject in need thereof. In some embodiments, the BT composition and the additional antibacterial agent are administered together to a subject in need thereof.

[0089] Bismuth-thiol composition

[0104] In some embodiments, the BT composition comprises a BT compound, which in some embodiments is selected from the group consisting of BisBAL, BisEDT, bis-dimercaprol, BisDTT, bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr / Bal, Bis-Pyr / BDT, BisPyr / EDT, Bis-Pyr / PDT, Bis-Pyr / Tol, Bis-Pyr / Ery, bismuth-1-mercapto-2-propanol, BisEDT / CSTMN(1:1), BisPYR / CSTMN(1:1), BisBAL / CSTMN(1:1), BisTOL / CSTMN(1:1), and BisEDT / 2-hydroxy-1-propanethiol.

[0090]

[0105] In some embodiments, the BT composition comprises a BT compound selected from the group consisting of:

[0091] [Table 1]

[0092]

[0106] In some embodiments, the BT compound is selected from the group consisting of BisEDT, Bis-Bal, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, or BisEDT / 2-hydroxy-1-propanethiol.

[0093]

[0107] In some embodiments, the BT compound is BisEDT or BisBAL. In some embodiments, the BT compound is BisEDT. In some embodiments, BisEDT has the structure: [ka] It is a compound having the formula:

[0094]

[0108] In some embodiments, the BT compounds of the present disclosure exhibit bactericidal effects. In some embodiments, the BT compounds exhibit bacteriostatic effects.

[0095]

[0109] When administered to a subject, such as a human, the composition or compound is preferably administered as a pharmaceutical composition, for example, comprising a compound of the present disclosure and a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art, and include, for example, aqueous solutions such as water, physiologically buffered saline, phosphate buffer, or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or organic esters for injection. In some embodiments, when such pharmaceutical compositions are for human administration, the aqueous solutions are pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to cause delayed release of the drug or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition can be in unit dose form, such as a lyophilisate, powder, liquid, syrup, injection, etc. for reconstitution. The composition can also be in a liquid suitable for topical administration.

[0096]

[0110] A pharma- ceutically acceptable carrier may contain a physiologically acceptable agent that acts, for example, to stabilize, increase the solubility, or increase the absorption of a compound, such as the compounds of the present disclosure. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose, or dextran; antioxidants, such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; salts; or other stabilizers or excipients. The choice of a pharma- ceutical acceptable carrier that includes a physiologically acceptable agent depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) may also be a liposome or other polymer matrix, into which, for example, the compounds of the present disclosure may be incorporated. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, metabolizable carriers that are relatively simple to prepare and administer.

[0097]

[0111] Other examples of materials which can act as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol. (11) polyols and sugar alcohols, such as glycerin, sorbitol, mannitol, xylitol, erythritol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other nontoxic, compatible substances, including salts, such as sodium chloride, utilized in pharmaceutical formulations.

[0098]

[0112] Preparations can be conveniently presented in unit dosage form and can be prepared by any method known in the field of pharmacy.The amount of active ingredient that can be combined with carrier material to produce single dosage form will vary depending on the subject being treated, specific mode of administration.The amount of active ingredient that can be combined with carrier material to produce single dosage form will generally be the amount of compound that produces therapeutic effect.

[0099]

[0113] In some embodiments, the BT composition further comprises one or more carriers selected from animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, polymers, talc, and zinc oxide. In some embodiments, the carrier is methylcellulose. In some embodiments, the carrier is poly(methyl methacrylate).

[0100]

[0114] The compositions can also be formulated to give slow or controlled release of the active ingredient therein, for example, using hydroxypropylmethylcellulose (HPMC), other polymer matrices, liposomes and / or microspheres in various proportions that give the desired release profile. They can be sterilized, for example, by filtration through a bacteria-retaining filter, by ionizing radiation (e.g., gamma photons), by autoclaving, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water or any other sterile injectable medium immediately prior to use.

[0101]

[0115] Suspensions may contain, in addition to the active compound, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0102]

[0116] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharma- ceutically acceptable carrier, and with any preservatives or buffers which may be required.

[0103]

[0117] In addition to the active compound, the BT composition includes one or more excipients or carriers, such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, polymers, salts, and zinc oxide, or mixtures thereof. In some embodiments, the BT composition is in the form of an aqueous solution. In some embodiments, the excipient includes a salt selected from sodium chloride or potassium chloride. In some embodiments, the excipient includes sodium chloride.

[0104]

[0118] In some embodiments, the BT composition is a powder, spray, ointment, paste, cream, lotion, liquid, patch, suspension, or gel. In some embodiments, the BT composition is a liquid. In some embodiments, the BT composition is an aerosol.

[0105]

[0119] The BT composition may contain any suitable concentration of the bismuth-thiol compound. In some embodiments, the BT composition may contain from about 0.25 mg / mL to about 15 mg / mL, from about 0.4 mg / mL to about 15 mg / mL, from about 0.6 mg / mL to about 15 mg / mL, from about 0.6 mg / mL to about 100 mg / mL, from about 5 mg / mL to about 100 mg / mL, from about 10 mg / mL to about 100 mg / mL, from about 25 mg / mL to about 100 mg / mL, from about 50 mg / mL to about 100 mg / mL, / mL, approximately 0.8 mg / mL to approximately 15 mg / mL, approximately 1 mg / mL to approximately 10 mg / mL, 2.5 mg / mL to approximately 10 mg / mL, approximately 4 mg / mL to approximately 10 mg / mL, approximately 5 mg / mL to approximately 1 0mg / mL, approximately 6mg / mL to approximately 10mg / mL, 0.6mg / mL to approximately 6mg / mL, approximately 4mg / mL to approximately 15mg / mL, approximately 6mg / mL to approximately 15mg / mL, approximately 50μg / mL to approximately and / or the total amount of the BT composition administered to the lung is about 0.25 mg to about 15 mg, about 0.4 mg to about 15 mg, about 0.6 mg to about 15 mg, about 0.8 mg to about 15 mg, about 0.9 mg to about 15 mg, about 10 mg to about 15 mg, about 15 mg to about 25 mg, about 25 mg to about 25 mg, about 30 mg to about 35 mg, about 40 mg to about 45 mg, about 50 mg to about 50 mg, about 60 mg to about 65 mg, about 70 mg to about 750 μg / mL, about 75 μg / mL to about 500 μg / mL, about 100 μg / mL to about 250 μg / mL, about 100 μg / mL to about 150 μg / mL, or about 75 μg / mL to about 150 μg / mL; and / or the total amount of the BT composition administered to the lung is about 0.25 mg to about 15 mg, about 0.4 mg to about 15 mg, about 0.6 mg to about 15 mg, about 0.8 mg to about 15 mg, about 0.9 ... ~ about 15 mg, about 1 mg to about 10 mg, 2.5 mg to about 10 mg, about 4 mg to about 10 mg, about 5 mg to about 10 mg, about 6 mg to about 10 mg, 0.6 mg to about 6 mg, about 4 mg to about 15 mg, about 6 mg to about 15 mg, about 50 μg to about 750 μg, about 75 μg to about 500 μg, about 100 μg to about 250 μg, about 100 μg to about 150 μg, or about 75 μg to about 150 μg. In a specific embodiment, the BT composition is administered as a dose of about 0.6 mg / mL to about 6 mg / mL.

[0106]

[0120] In some embodiments, the concentration of bismuth in the lungs after a single daily dose is about 0.03 μg / g lung tissue to about 3 μg / g lung tissue, for example, about 0.03 μg / g lung tissue, about 0.1 μg / g lung tissue, about 0.25 μg / g lung tissue, about 0.5 μg / g lung tissue, about 0.75 μg / g lung tissue, about 1 μg / g lung tissue, about 1.25 μg / g lung tissue, about 1.5 μg / g lung tissue, about 1.75 μg / g lung tissue, about 2 μg / g lung tissue, about 2.25 μg / g lung tissue, about 2.5 μg / g lung tissue, about 2.75 μg / g lung tissue, or about 3 μg / g lung tissue, including all ranges and values ​​therebetween. In some embodiments, the concentration of bismuth in the lungs after a single daily dose is about 0.1 μg / g lung tissue to about 3 μg / g lung tissue. In some embodiments, the concentration of bismuth in the lungs after a single daily dose is about 1 μg / g lung tissue to about 3 μg / g lung tissue, in some embodiments, the concentration of bismuth in the lungs after 28 daily doses is about 0.3 μg / g lung tissue to about 60 μg / g lung tissue.

[0107]

[0121] In some embodiments, the BT composition is administered three times per day, twice per day, once per day, every other day, once every third day, once per week, once per every other week, once per month, or once per every other month. In some embodiments, the BT composition is administered once per month, twice per month, three times per month, four times per month, once every two weeks, once per week, twice per week, or three times per week. In some embodiments, the BT composition is administered once per month, twice per month, three times per month, or four times per month. In some embodiments, the BT composition is administered once every two weeks, once per week, twice per week, or three times per week. In some embodiments, the BT composition is administered once per week. In some embodiments, the BT composition is administered once per day. In certain embodiments, the BT composition is administered once per week. In certain embodiments, the BT composition is administered once every other week. In some embodiments, the BT composition is administered chronically, on a 4 week on / 4 week off dosing schedule. In some embodiments, the BT composition is administered chronically, for example as part of a basal therapy. In some embodiments, the BT composition is administered as needed. As will be appreciated by those skilled in the art, the frequency of administration may depend on several factors, including the dosage and the route of administration. For example, if the BT composition is administered by aerosol administration, a low dosage, such as 100-1000 μg / mL, may be administered once or twice a day; however, a high dosage, such as 2.5-10 mg / mL, may be administered, for example, once or twice a week.

[0108]

[0122] In some embodiments of the present disclosure, the BT composition is a suspension of the BT compound in a polysorbate (e.g., polysorbate 80) and / or in a buffer (e.g., sodium phosphate buffer). For example, in some embodiments, the BT composition is a suspension of the BT compound in about 0.1% polysorbate 80 to about 1.0% polysorbate 80, including all ranges therebetween. For example, the BT composition is a suspension of the BT compound in about 0.1% polysorbate 80, about 0.2% polysorbate 80, about 0.3% polysorbate 80, about 0.4% polysorbate 80, about 0.5% polysorbate 80, about 0.6% polysorbate 80, about 0.7% polysorbate 80, about 0.8% polysorbate 80, about 0.9% polysorbate 80, or about 1% polysorbate 80. In some embodiments, the BT composition is a suspension of the BT compound in about 0.5% polysorbate 80.

[0109]

[0123] In some embodiments, the present disclosure provides a bismuth-thiol (BT) composition comprising a BT compound (e.g., BisEDT) suspended therein, the BT composition comprising a plurality of particles. In some embodiments, the present disclosure provides a bismuth-thiol (BT) composition comprising a BT compound (e.g., BisEDT) suspended therein, the BT composition comprising a plurality of microparticles. In some embodiments, the D90 of the particles / microparticles is 4.5 μm, or 4.0 μm, or 3.5 μm, or 3.0 μm, or 2.5 μm, or 2.0 μm, or 1.9 μm, or 1.8 μm, or 1.7 μm, or 1.6 μm, or 1.5 μm or less, or any range therebetween. In some embodiments, the D90 of the particles / microparticles is 1.9 μm or less. In another embodiment, the D90 of the particles / microparticles is 1.6 μm or less. In another embodiment, the particle / particulate has a D50 of 2.5 μm, or 2.0 μm, or 1.5 μm, or 1.3 μm, or 1.2 μm, or 1.1 μm, or 1.0 μm, or 0.9 μm, or 0.87 μm, or 0.72 μm or less, or any range therebetween. In another embodiment, the particle / particulate has a D10 of 0.9 μm, or 0.8 μm, or 0.7 μm, or 0.6 μm, or 0.50 μm, or 0.40 μm, or 0.39 μm, or 0.38 μm, or 0.37 μm, or 0.36 μm, or 0.35 μm, or 0.34 μm, or 0.33 μm or less, or any range therebetween.

[0110]

[0124] In some embodiments, the bismuth-thiol (BT) composition of the present disclosure comprises a BT compound suspended therein and comprises a plurality of particles, wherein the D90 of the particles is about 1.6 μm or less. In some embodiments, the bismuth-thiol (BT) composition comprises a BT compound (e.g., BisEDT) suspended therein and comprises a plurality of microparticles, wherein the D90 of the microparticles is about 1.6 μm or less.

[0111]

[0125] In some embodiments, the BT composition comprises a BT compound (e.g., BisEDT) at a concentration of greater than about 0.1 mg / mL, about 0.05% to about 1.0% polysorbate 80 (Tween 80®), about 0.05 to 40 mM sodium chloride, and optionally about 2 to 20 mM sodium phosphate at about pH 7.4.

[0112]

[0126] A variety of buffers may be used in the context of the present disclosure, as will be readily apparent to one of skill in the art. For example, in some embodiments, suitable buffers include phosphate buffers such as sodium or potassium citrate, citric acid, sodium phosphate, boric acid, sodium bicarbonate, and various mixed phosphate buffers including combinations of Na2HPO4, NaH2PO4, and KH2PO4. In some embodiments, sodium phosphate buffers are used. In some embodiments, sodium citrate buffers are used. Without being bound to a particular theory, changes in airway surface liquid pH may be responsible for the defective host defense in cystic fibrosis shortly after birth. Changes in lung pH may affect the airway surface liquid environment, improving airway defense and altering the disease course. Thus, the formulation pH may vary from about 5 to about 10. In some embodiments, the formulation pH is about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10. In some embodiments, the formulation pH is about 7.4.

[0113]

[0127] In some embodiments, the BT composition is a suspension of the BT compound in about 0.5% polysorbate 80 in sodium phosphate buffer at a pH of about 7.4. In some embodiments, the BT compound is present in the composition at a concentration ranging from about 100 μg / mL to about 1000 mg / mL, including all integers and ranges therebetween. For example, in some embodiments, the BT compound is present in the composition at a concentration of from about 100 μg / mL to about 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL, 1000 μg / mL, 10 mg / mL, 25 mg / mL, 50 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL, 175 mg / mL, 200 mg / mL, 225 mg / mL, 250 mg / mL, 275 mg / mL, 300 mg / mL, 325 mg / mL, 350 mg / mL , 375mg / mL, 400mg / mL, 425mg / mL, 450mg / mL, 475mg / mL, 500mg / mL, 525mg / mL, 550mg / mL, 575mg / mL, 600mg / mL, 625mg / mL, 650mg / mL, 675mg / mL, 700mg / mL, 725mg / mL, 750mg / mL, 775mg / mL, 800mg / mL, 825mg / mL, 850mg / mL, 875mg / mL, 900mg / mL, 925mg / mL, 950mg / mL, 975mg / mL, about 1000mg / mL. In some embodiments, the BT compound is present in the composition at a concentration ranging from about 100μg / mL to about 1000μg / mL.

[0114]

[0128] In some embodiments, the composition osmolality is further adjusted with additives such as NaCl or TDAPS to obtain a desired osmolality. For example, in some embodiments, the composition osmolality is adjusted with sodium chloride to an osmolality ranging from about 100 mOsmol / kg to about 500 mOsmol / kg, including all integers and ranges therebetween. In some embodiments, the composition osmolality is from about 290 mOsmol / kg to about 310 mOsmol / kg. For example, in some embodiments, the osmolality of the composition is about 290 mOsmol / kg, 291 mOsmol / kg, 292 mOsmol / kg, 293 mOsmol / kg, 294 mOsmol / kg, 295 mOsmol / kg, 296 mOsmol / kg, 297 mOsmol / kg, 298 mOsmol / kg, 299 mOsmol / kg, 300 mOsmol / kg, 301 mOsmol / kg, 302 mOsmol / kg, 303 mOsmol / kg, 304 mOsmol / kg, 305 mOsmol / kg, 306 mOsmol / kg, 307 mOsmol / kg, 308 mOsmol / kg, 309 mOsmol / kg, or about 310 mOsmol / kg. In some embodiments, the osmolality is about 300 mOsmol / kg.

[0115]

[0129] In some embodiments, the BT composition is a suspension of BisEDT in a polysorbate (e.g., polysorbate 80) in a buffer (e.g., sodium phosphate buffer). In some embodiments, the BT composition is a suspension of BisEDT in about 0.5% polysorbate 80 in a sodium phosphate buffer at a pH of about 7.4. In some embodiments, the BT composition is a suspension of BisEDT in about 0.5% polysorbate 80 in a sodium phosphate buffer at a pH of about 7.4, the composition having an osmolality of about 300 mOsmol / kg (e.g., adjusted to 300 mOsmol / kg with sodium chloride). In some embodiments, BisEDT is present at a concentration of about 100 μg / mL, 250 μg / mL, 500 μg / mL, 750 μg / mL, 1000 μg / mL, 2.5 mg / mL, 10 mg / mL, 25 mg / mL, 50 mg / mL, 75 mg / mL, or about 100 mg / mL.

[0116]

[0130] In some embodiments, the BT composition is delivered to the lungs of a subject. In some embodiments, the BT composition is administered to the lungs of a subject by a nebulizer, dry powder inhalation, nanoparticle inhalation, metered dose inhalation, or any other method of inhaling a drug known in the art. In some embodiments, the dry powder is micronized. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the nanoparticles are not lipid nanoparticles. In some embodiments, the nanoparticles are dry nanoparticles. In some embodiments, the BT composition is a suspension formulation for pulmonary delivery. For example, the BT composition is a suspension formulation that is ultimately administered by inhalation either orally and / or intranasally.

[0117]

[0131] Thus, in some embodiments, the BT composition is in the form of an aerosol. In some embodiments, the BT composition is aerosolized by a device such as a nebulizer. In some embodiments, the aerosol comprises a plurality of dispersed droplets in a gas, the droplets comprising a BT composition comprising bismuth-1,2-ethanedithiol (BisEDT) suspended therein, the BT composition comprising a plurality of BisEDT particles / particulates having a D90 as disclosed herein, e.g., a D90 of less than about 2 μm; at least 70%, at least 80%, or at least 90% of the droplets have an MMAD as disclosed herein, e.g., an MMAD of about 0.4 μm to about 5 μm as measured by cascade impaction or laser time of flight.

[0118]

[0132] In some embodiments, the aerosol is administered by inhalation, orally or intranasally, using an aerosol device such as a nebulizer. Known nebulizers, such as PARI IC Plus, can administer the disclosed aerosol as an aqueous solution, optionally in buffered saline. The solution can be provided to the subject in the form of an ampoule for use in the nebulizer. The nebulizer can be reusable and includes a compressor that provides the formulation for a period of time, such as about 10-15 minutes or longer, for example, for a period of 45-60 minutes. Known compressors, such as APRI Vios Air and DeVilbiss Pulmo-aide, are suitable for administration. The nebulizer administers the formulation topically to pulmonary tissues, such as mucous membranes, bronchi and / or bronchioles, alveoli, and deep lung alveoli. The formulations can penetrate the lung mucosa and biofilms to reduce microbial (e.g., bacterial or fungal) biofilms, impair the growth of microbial (e.g., bacterial or fungal) biofilms, prevent the formation of microbial (e.g., bacterial or fungal) biofilms, reduce planktonic growth, and / or inhibit planktonic growth.

[0119]

[0133] In other embodiments, a nasal aerosol device may be used to administer the formulation.

[0120]

[0134] Powders and sprays can contain, in addition to the active compounds, excipients such as methylcellulose, sodium chloride, PMMA, lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, dipalmitoylphosphatidylcholine (DPPC), leucine, polyethylene glycol, or mixtures of these substances. Sprays can additionally contain conventional propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0121]

[0135] An exemplary BT composition formulation is a neutral pH, isotonic, buffered aqueous solution of BT compound particles / microparticles and a non-ionic surfactant. In certain embodiments, the buffer is a phosphate buffer to which NaCl has been added. In some embodiments, the microparticle size is about 1-5 μm D 50 The formulation can be delivered using a commercially available compressed air jet nebulizer. In some embodiments, the formulation concentration is about 0.1 μg / mL to about 100 mg / mL.

[0122]

[0136] In some embodiments, the disclosure provides an aerosol composition comprising a plurality of droplets dispersed in a gas, said droplets comprising a BT compound suspended therein, wherein at least 60%, 65%, 70%, 75%, 80%, 90%, or 95% of the droplets have a mass median aerodynamic diameter (MMAD) of about 0.4 μm to about 5 μm as measured by laser time-of-flight and / or cascade impactor. In some embodiments, at least 60%, 65%, 70%, 75%, 80%, 90%, or 95% of the droplets have an MMAD of about 0.4 μm to about 7 μm, or about 0.5 μm to about 5 μm, or about 0.7 μm to about 4 μm, or about 0.7 μm to about 3.5 μm, or about 0.8 μm to about 3.5 μm, or about 0.9 μm to about 3.5 μm, or about 0.9 μm to about 3 μm, or about 0.8 μm to about 1.8 μm, or about 0.8 μm to about 1.6 μm, or about 0.9 μm to about 1.4 μm, or about 1.0 μm to about 2.0 μm, or about 1.0 μm to about 1.8 μm, including all ranges therebetween. In some embodiments, at least 60%, 65%, 70%, 75%, 80%, 90%, or 95% of the droplets have an MMAD of about 0.8 μm to about 1.6 μm, or about 0.9 μm to about 3.5 μm, or about 0.9 μm to about 3 μm, or about 0.9 μm to about 1.4 μm, or about 1.0 μm to about 2.0 μm, or about 1.0 μm to about 1.8 μm, and all ranges therebetween.

[0123]

[0137] In some embodiments, the plurality of droplets has a D90 of less than about 10 μm. For example, in some embodiments, the plurality of droplets has a D90 of less than about 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, or about 1 μm. In some embodiments, the plurality of droplets has a D90 of less than about 3 μm. In some embodiments, the plurality of droplets has a D90 in the range of about 1 μm to about 5 μm, or about 2 μm to about 6 μm, or about 2 μm to about 4 μm, or about 2 μm to about 3 μm, or about 1 μm to about 4 μm, or about 1 μm to about 3 μm.

[0124]

[0138] In some embodiments, the plurality of droplets are dispersed in a continuous gas phase.

[0125]

[0139] In some embodiments, the BT compound (e.g., BisEDT) is suspended in the droplets. The BT compounds of the present disclosure may have little or no solubility in conventional solvents and aerosol carriers and thus exist substantially as a suspension of BT particles in the aerosol droplets. For example, in some embodiments, the BT compound (such as BisEDT) has less than 1% solubility in the aerosol carrier and thus exists primarily (>99%) as a solid.

[0126]

[0140] In some embodiments, the droplets further comprise polysorbate 80 (e.g., about 0.05% to about 1%) and optionally a buffer at a pH of about 7.4 (e.g., sodium phosphate or sodium citrate); and / or sodium chloride.

[0127]

[0141] The aerosols of the present disclosure have a very narrow MMAD distribution, which is beneficial due to the need to focus the particle population in a target size range and the need to minimize or eliminate the fraction of products outside the respirable range or "fines", i.e., particles typically less than 0.4 μm in diameter. The ability to create a narrow droplet size distribution in the appropriate size range provides control over the initial evaporation rate and allows for high deposition efficiency. The limiting factor at the lower end of the particle aerosol droplet size is the BT particle size (e.g., BisEDT particle size). Aerosolized droplets cannot be smaller than the BisEDT particulate size. Therefore, the BT particle size distribution, as well as the uniformity and consistent reproducibility of the BT particle size distribution, are important and beneficial properties to support the production of safe, effective and efficient aerosolized BisEDT pharmaceutical products for inhalation purposes. Thus, in some embodiments, the aerosols of the present disclosure achieve deposition efficiencies of greater than 3%, greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, and greater than 80%. In some embodiments, deposition efficiency refers to deposition in the deep lung regions of the lung, for example in the deep alveoli. In some embodiments, the aerosols of the present disclosure achieve deposition efficiencies upon aerosolization by a nebulizer. For example, the nebulizer is a jet nebulizer. In some embodiments, the jet nebulizer is a Pari LC Plus jet nebulizer or a Pari LC SPRINT jet nebulizer. In some embodiments, the nebulizer has an inlet pressure of about 10 to about 40 psig (e.g., 20 to 25 psig). In some embodiments, the inlet flow is about 3 L / min to about 8 L / min (e.g., 5.2 L / min). In some embodiments, the exhaust flow is about 3 L / min to about 8 L / min (e.g., 5 L / min).

[0128]

[0142] Since the alveolar region of the lung has a minimal thickness (0.5 μm to 2.5 μm) separating the bloodstream from the lumen, conventional pulmonary drugs that are deposited on the alveolar epithelium have extremely short pulmonary residence times for systemic absorption. Thus, conventional pulmonary treatments typically require frequent dosing to maintain adequate levels of drug at tissue levels. However, the aerosolized particles of the present disclosure have surprisingly been found to have exceptionally long residence times in the lung (measured as half-life) and reduced mucociliary clearance and macrophage uptake relative to conventional pulmonary treatments. Furthermore, the long residence times of the aerosols of the present disclosure minimize systemic effects and associated systemic side effects. Without being bound by a particular theory, it is believed that the aerosolized particles / microparticles dissolve slowly on the lung lumen, and thus systemic exposure is dissolution rate-limiting. Furthermore, the increased pulmonary residence time results in a significant reduction in microbial colonization due to the continuous presence of BT particles / microparticles.

[0129]

[0143] Thus, in some embodiments, when the aerosol is deposited in the lung (e.g., in the deep alveoli), the BT compound has an average half-life of at least 2 days. For example, the BT compound has an average half-life of about 2, 3, 4, or 5 days. In some embodiments, the BT compound is BisEDT. In specific embodiments, the lung tissue half-life of BisEDT is 30 hours or more, 40 hours or more, 50 hours or more, 60 hours or more, 70 hours or more, 80 hours or more, 90 hours or more, 100 hours or more, 110 hours or more, 125 hours or more, or 150 hours or more. In specific embodiments, the lung tissue half-life is after a single dose by inhalation. In another embodiment, the lung tissue is from a rat. In another embodiment, the lung tissue half-life of BisEDT is determined by using the protocol in Example 8 herein.

[0130]

[0144] In another embodiment, the lung tissue half-life of BisEDT is 80 hours or more when the formulations described herein are administered to rats using a Pari LC plus jet nebulizer to give the rats a single dose of 100 μg / kg lungs. In another embodiment, the lung tissue half-life of BisEDT is 90 hours or more. In another embodiment, the lung tissue half-life of BisEDT is 100 hours or more.

[0131]

[0145] In another embodiment, after delivery of the aerosolized composition to a subject, at least 60%, 65%, 70%, 75%, 80%, 90%, or 95% of the dose is deposited in the lungs, rather than in the oropharyngeal region and conducting airways. In a specific embodiment, at least 80% of the dose is deposited in the lungs, rather than in the oropharyngeal region and conducting airways. In another embodiment, at least 90% of the dose is deposited in the lungs, rather than in the oropharyngeal region and conducting airways.

[0132]

[0146] It has not been previously known that aerosolized pulmonary therapy has a narrow distribution of aerosol particles that achieves high deposition efficiency coupled with exceptionally long lung residence times for continuous therapy and little to no systemic absorption.

[0133]

[0147] In some embodiments, after delivery of the aerosolized composition to a subject, at least 60%, 65%, 70%, 75%, 80%, 90%, or 95% of the dose is deposited in the lungs, rather than in the oropharyngeal region and conducting airways. In some embodiments, at least 80% of the dose is deposited in the lungs, rather than in the oropharyngeal region and conducting airways. In another embodiment, at least 90% of the dose is deposited in the lungs, rather than in the oropharyngeal region and conducting airways. In another embodiment, percent deposition is determined by administering the formulations described herein to rats using a Pari LC plus jet nebulizer.

[0134]

[0148] In another embodiment, the lung tissue half-life of BisEDT is 80 hours or more when the formulations described herein are administered to rats using a Pari LC plus jet nebulizer to give the rats a single dose of 100 μg / kg lungs. In another embodiment, the lung tissue half-life of BisEDT is 90 hours or more. In another embodiment, the lung tissue half-life of BisEDT is 100 hours or more.

[0135]

[0149] In another embodiment, a method of the invention comprises treating, managing, or reducing the severity of a cystic fibrosis (CF) symptom associated with an NTM infection in a subject comprising administering to the subject an aerosol comprising a plurality of dispersed droplets, wherein the droplets comprise a bismuth-thiol (BT) composition comprising particles of a BT compound suspended therein, wherein the particles have a D90 (e.g., as measured by laser diffraction) of less than about 5 μm, and / or at least 70%, 80%, or 90% of the droplets have a mass median aerodynamic diameter (MMAD) of about 0.4 μm to about 5 μm (e.g., as measured by cascade impaction or laser time-of-flight).

[0136]

[0150] In another embodiment, a method of the invention comprises treating, managing, or reducing the severity of a cystic fibrosis (CF) symptom associated with an NTM infection in a subject comprising administering to the subject an aerosol comprising a plurality of dispersed droplets, wherein the droplets comprise a bismuth-thiol (BT) composition comprising microparticles of a BT compound suspended therein, the microparticles having a D90 (e.g., as measured by laser diffraction) of less than about 5 μm and / or at least 70%, 80%, or 90% of the droplets have a mass median aerodynamic diameter (MMAD) of about 0.4 μm to about 5 μm (e.g., as measured by cascade impaction or laser time-of-flight).

[0137]

[0151] In some embodiments, the composition is a suspension of particles / particulates having a volume mean diameter (VMD) of about 0.4 μm to about 5 μm. In some embodiments, at least 60%, 65%, 70%, 75%, 80%, 90%, or 95% of the particles / particulates have a VMD of about 0.4 μm to about 5 μm, or about 0.6 μm to about 2.5 μm, or about 0.7 μm to about 4 μm, or about 0.7 μm to about 3.5 μm, or about 0.7 μm to about 3.0 μm, or about 0.9 μm to about 3.5 μm, or about 0.9 μm to about 3 μm, or about 0.8 μm to about 1.8 μm, or about 0.8 μm to about 1.6 μm, or about 0.9 μm to about 1.4 μm, or about 1.0 μm to about 2.0 μm, or about 1.0 μm to about 1.8 μm and all ranges therebetween. In some embodiments, at least 60%, 65%, 70%, 75%, 80%, 90%, or 95% of the particles / particulates have a VMD of about 0.6 μm to about 2.5 μm, or about 0.8 μm to about 1.6 μm, or about 0.9 μm to about 3.5 μm, or about 0.9 μm to about 3 μm, or about 0.9 μm to about 1.4 μm, or about 1.0 μm to about 2.0 μm, or about 1.0 μm to about 1.8 μm, and all ranges therebetween. In some embodiments, the particles / particulates have a D90 of less than 5 μm, 4 μm, 3 μm, 2 μm, or about 1 μm. In some embodiments, the particles / particulates have a D90 of less than about 3 μm. In some embodiments, the particles / particulates have a D90 in the range of about 1 μm to about 5 μm, or about 2 μm to about 4 μm, or about 2 μm to about 3 μm, or about 1 μm to about 4 μm, or about 1 μm to about 3 μm, or about 1 μm to about 2 μm.

[0138]

[0152] In some embodiments, the dispersed droplets have an MMAD of about 0.4 μm to about 5 μm. In some embodiments, at least 60%, 65%, 70%, 75%, 80%, 90%, or 95% of the droplets have an MMAD of about 0.4 μm to about 7 μm, or about 0.5 μm to about 5 μm, or about 0.7 μm to about 4 μm, or about 0.7 μm to about 3.5 μm, or about 0.8 μm to about 3.5 μm, or about 0.9 μm to about 3.5 μm, or about 0.9 μm to about 3 μm, or about 0.8 μm to about 1.8 μm, or about 0.8 μm to about 1.6 μm, or about 0.9 μm to about 1.4 μm, or about 1.0 μm to about 2.0 μm, or about 1.0 μm to about 1.8 μm, and all ranges therebetween. In some embodiments, at least 60%, 65%, 70%, 75%, 80%, 90%, or 95% of the droplets have an MMAD of about 0.8 μm to about 1.6 μm, or about 0.9 μm to about 3.5 μm, or about 0.9 μm to about 3 μm, or about 0.9 μm to about 1.4 μm, or about 1.0 μm to about 2.0 μm, or about 1.0 μm to about 1.8 μm, and all ranges therebetween. In some embodiments, the droplets have a D90 of less than about 10 μm. For example, in some embodiments, the droplets have a D90 of less than about 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, or about 1 μm. In some embodiments, the droplets have a D90 of less than about 3 μm. In some embodiments, the plurality of droplets have a D90 in the range of about 1 μm to about 5 μm, or about 2 μm to about 6 μm, or about 2 μm to about 4 μm, or about 2 μm to about 3 μm, or about 1 μm to about 4 μm, or about 1 μm to about 3 μm.

[0139]

[0153] In some embodiments, the plurality of droplets are dispersed in a continuous gas phase.

[0140]

[0154] In some embodiments, the aerosol comprises a BT compound disclosed herein.

[0141]

[0155] In some embodiments of the presently disclosed compositions, at least 60%, 65%, 70%, 75%, 80%, 90%, or 95% of the particles / particulates have a volume mean diameter of about 0.6 μm to about 2.5 μm. In some embodiments, substantially all of the particles / particulates have a VMD of about 0.6 μm to about 2.5 μm. In some embodiments, at least 70% of the dispersed droplets have a MMAD of about 0.9 μm to about 3 μm. In some embodiments, the composition is a suspension of particles / particulates having a volume mean diameter (VMD) of about 0.6 μm to about 2.5 μm and / or a mass median aerodynamic diameter (MMAD) of about 0.9 μm to about 3 μm. In some embodiments, the bismuth-thiol (BT) composition comprises a plurality of particles / particulates comprising a BT compound, substantially all of the particles / particulates having a volume mean diameter of about 0.4 μm to about 5 μm, and the BT compound is BisEDT or BisBAL. In some embodiments, the BT compound is BisEDT.

[0142]

[0156] In some embodiments, the compositions disclosed herein are aerosolized by a nebulizer. For example, the nebulizer is a jet nebulizer or a vibrating mesh nebulizer. In some embodiments, the jet nebulizer is a Pari LC Plus jet nebulizer or a Pari LC SPRINT jet nebulizer. In some embodiments, the nebulizer has an inlet pressure of about 10 to about 40 psig (e.g., 20 to 25 psig). In some embodiments, the inlet flow is about 3 L / min to about 8 L / min (e.g., 5.2 L / min). In some embodiments, the exhaust flow is about 3 L / min to about 8 L / min (e.g., 5 L / min).

[0143]

[0157] The actual dosage level of the active ingredient in the pharmaceutical composition can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0144]

[0158] The selected dose level will depend on a variety of factors, including the activity of the particular compound or combination of compounds or their esters, salts, or amides being employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular compound being employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and similar factors well known in the medical arts.

[0145]

[0159] A physician or veterinarian of ordinary skill in the art can easily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can begin administration of the pharmaceutical composition or compound at a lower level than required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. A "therapeutically effective amount" refers to the concentration of the compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound varies according to the subject's weight, sex, age, and medical history. Other factors that affect the effective amount may include, but are not limited to, the severity of the subject's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered together with the compound of the present disclosure. A larger total dosage can be delivered by repeated administration of the drug. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference).

[0146]

[0160] Generally, a suitable dosage of an active compound used in the compositions and methods of the present disclosure will be that amount of the compound that is the lowest dosage effective to produce a therapeutic effect. Such an effective dosage will generally depend upon the factors described above.

[0147] Numbered embodiments 1. A method for treating an infection in a subject caused by nontuberculous mycobacteria (NTM), comprising administering to the subject an effective amount of a bismuth-thiol (BT) composition comprising a BT compound. 2. The method of embodiment 1, wherein the infection is a pulmonary infection. 3. The method of embodiment 1, wherein the infection is an extrapulmonary infection. 4. The method of any one of embodiments 1-3, wherein the NTM infection is caused by an antibiotic-resistant strain of NTM. 5. NTM infections include M. avium, M. avium subsp. hominissuis (MAH), M. abscessus, M. chelonae, M. bolletii, M. kansasii, M. ulcerans, M. avium complex (MAC) (M. avium and M. intracellulare), M. conspicuum, M. kansasii, M. peregrinum, M. immunogenum, M. xenopi (M. xenopi, M. marinum, M. malmoense, M. marinum, M. mucogenicum, M. nonchromogenicum, M. scrofulaceum, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. terrae complex, M. haemophilum, M. genavense, M. asiaticum, M. shimoidei, M. gordone, M. The method of any one of embodiments 1-4, wherein the bacterial strain is caused by M. gordonae, M. nonchromogenicum, M. triplex, M. lentiflavum, M. celatum, M. fortuitum, M. fortuitum complex (M. fortuitum and M. chelonae), or a combination thereof. 6. The method of any one of embodiments 1-4, wherein the NTM infection is caused by M. abscessus, M. avium, or a combination thereof. 7. The method of any one of embodiments 1-4, wherein the NTM pulmonary infection is caused by the M. avium complex (M. avium and M. intracellulare). 8. The method of any one of embodiments 5-7, wherein the M. avium is M. avium subsp. hominissuis. 9. The method of any one of embodiments 1-8, wherein the NTM infection is a biofilm-associated NTM infection. 10. The method of any one of embodiments 1, 2, and 4-9, wherein the NTM infection is a chronic pulmonary infection. 11. The method of any one of embodiments 1-10, wherein the NTM infection is located in or on the lung mucosa, bronchi, alveoli, macrophages, and / or bronchioles. 12. The method of any one of embodiments 1-11, wherein the NTM infection is located at least in part in macrophages. 13. The method of embodiment 11 or 12, wherein the macrophages are THP-1 macrophages. 14. The method of any one of embodiments 11-13, wherein upon administration of the BT composition to a subject, the bacterial load in macrophages is reduced. 15. The method of any one of embodiments 11-14, wherein the macrophages are infected with one or more strains of M. abscessus and / or M. avium. 16. The method of any one of embodiments 1-15, wherein the NTM infection is resistant to treatment with amikacin. 17. The method of any one of embodiments 1-16, wherein the NTM infection is resistant to macrolide or azalide therapy. 18. The method of any one of embodiments 1-17, wherein the subject has a chronic pulmonary condition. 19. The method of embodiment 18, wherein the chronic pulmonary condition is cystic fibrosis, chronic bronchitis, emphysema, bronchiectasis, pulmonary fibrosis, asbestosis, pneumonitis, chronic obstructive pulmonary disease (COPD), or asthma. 20. The method of embodiment 18 or 19, wherein the chronic pulmonary condition is cystic fibrosis. 21. The method of any one of embodiments 1-20, wherein the subject is administered about 30 μg to about 3,000 μg of the BT compound per administration. 22. The method of embodiment 21, wherein the subject is administered about 100 μg to about 1,000 μg of the BT compound per administration. 23. The method of any one of embodiments 1-22, wherein the BT composition is administered once per month, twice per month, three times per month, four times per month, once every two weeks, once per week, twice per week, or three times per week. 24. The method of any one of embodiments 1-23, wherein the BT composition is administered once or twice daily. 25. The method of any one of embodiments 1-24, wherein the BT compound is administered to the lungs of the subject. 26. The method of any one of embodiments 1-25, wherein the BT compound is administered by inhalation. 27. The method of any one of embodiments 1-26, wherein the BT composition is administered for a period of less than 24 months, less than 18 months, less than 12 months, less than 9 months, less than 6 months, less than 3 months, or less than 1 month. 28. The method of any one of embodiments 1-27, wherein the BT composition is administered for a period of 1 to 56 days. 29. The method of any one of embodiments 1 to 27, wherein the BT composition is administered for a period of 14 to 28 days. 30. The method of any one of embodiments 25-29, wherein the concentration of bismuth in the lungs after a single daily dose is from about 0.03 μg / g lung tissue to about 3 μg / g lung tissue. 31. The method of any one of embodiments 25-29, wherein the concentration of bismuth in the lungs after 28 daily doses is from about 0.3 μg / g lung tissue to about 60 μg / g lung tissue. 32. The method of any one of the preceding embodiments, wherein the BT compound is selected from BisBAL, BisEDT, bis-dimercaprol, BisDTT, bis-2-mercaptoethanol, BisDTE, BisPyr, BisEry, BisTol, BisBDT, BisPDT, BisPyr / BAL, BisPyr / BDT, BisPyr / EDT, BisPyr / PDT, BisPyr / Tol, BisPyr / Ery, bismuth-1-mercapto-2-propanol, BisEDT / CSTMN(1:1), BisPyr / CSTMN(1:1), BisBAL / CSTMN(1:1), BisTOL / CSTMN(1:1), and BisEDT / 2-hydroxy-1-propanethiol. 33. The method of embodiment 32, wherein the BT compound is selected from BisEDT, BisBAL, BisPyr, BisEry, BisTol, BisBDT, or BisEDT / 2-hydroxy-1-propanethiol. 34. The method of embodiment 32, wherein the BT compound is BisEDT or BisBAL. 35. The method of embodiment 32, wherein the BT compound is BisEDT. 36. The method of any one of embodiments 1-35, further comprising administering to a subject in need thereof an effective amount of amikacin, clarithromycin, azithromycin, ethambutol, rifampicin, tigecycline, linezolid, imipenem, cefoxitin, or a combination thereof. 37. A method for treating an NTM infection in a subject having a macrophage infection, comprising administering to the subject an effective amount of a bismuth-thiol (BT) composition comprising a BT compound. 38. The method of embodiment 37, further comprising testing for the presence of bacteria-infected macrophages in a biological sample from the subject, and if the sample tests positive for bacteria-infected macrophages, administering to the subject an effective amount of a bismuth-thiol (BT) composition. 39. The method of embodiment 37 or 38, wherein the macrophage cells are THP-1 cells. 40. NTM infections include M. avium, M. avium subsp. hominissuis (MAH), M. abscessus, M. chelonae, M. bolletii, M. kansasii, M. ulcerans, M. avium complex (MAC) (M. avium and M. intracellulare), M. conspicuum, M. kansasii, M. peregrinum, M. immunogenum, M. xenopi (M. xenopi, M. marinum, M. malmoense, M. marinum, M. mucogenicum, M. nonchromogenicum, M. scrofulaceum, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. terrae complex, M. haemophilum, M. genavense, M. asiaticum, M. shimoidei, M. gordone, M. gordonae, M. nonchromogenicum, M. triplex, M. lentiflavum, M. celatum, M. fortuitum, M. fortuitum complex (M. fortuitum and M. chelonae).chelonae), or a combination thereof. 41. The method of any one of embodiments 37-40, wherein the NTM infection is caused by M. abscessus, M. avium, M. intracellulare, M. fortuitum, M. gordonae, M. kansasii, M. avium complex, M. marinum, M. terrae, M. cheloni, or combinations thereof. 42. The method of any one of embodiments 37-40, wherein the NTM infection is caused by M. abscessus, M. avium, or a combination thereof. 43. The method of any one of embodiments 37-40, wherein the NTM pulmonary infection is caused by the M. avium complex (M. avium and M. intracellulare). 44. The method of any one of embodiments 40-43, wherein the M. avium is M. avium subsp. hominissuis. 45. The method of any one of embodiments 37-44, wherein the NTM infection is a biofilm-associated NTM infection. 46. ​​The method of any one of embodiments 37-45, wherein the BT compound is selected from BisBAL, BisEDT, bis-dimercaprol, BisDTT, bis-2-mercaptoethanol, BisDTE, BisPyr, BisEry, BisTol, BisBDT, BisPDT, BisPyr / BAL, BisPyr / BDT, BisPyr / EDT, BisPyr / PDT, BisPyr / Tol, BisPyr / Ery, bismuth-1-mercapto-2-propanol, BisEDT / CSTMN(1:1), BisPyr / CSTMN(1:1), BisBAL / CSTMN(1:1), BisTOL / CSTMN(1:1), and BisEDT / 2-hydroxy-1-propanethiol. 47. The method of any one of embodiments 37-45, wherein the BT compound is BisEDT or BisBAL. 48. The method of any one of embodiments 37-45, wherein the BT compound is BisEDT. 49. The method of any one of embodiments 37-48, wherein the BT composition is administered once per month, twice per month, three times per month, four times per month, once every two weeks, once per week, twice per week, or three times per week. 50. The method of any one of embodiments 37-48, wherein the BT composition is administered once or twice daily. 51. The method of any one of embodiments 37-50, wherein the BT composition is administered for a period of less than 24 months, less than 18 months, less than 12 months, less than 9 months, less than 6 months, less than 3 months, or less than 1 month. 52. The method of any one of embodiments 37-51, wherein the BT compound is administered for a period of 1 to 56 days. 53. The method of any one of embodiments 37-52, wherein the BT compound is administered for a period of 14 to 28 days. 54. The method of any one of embodiments 37-52, further comprising administering to a subject in need thereof an effective amount of amikacin, clarithromycin, azithromycin, ethambutol, rifampicin, tigecycline, linezolid, imipenem, cefoxitin, or a combination thereof. 55. The method of any one of embodiments 37-53, further comprising administering amikacin to a subject in need thereof. 56. The method of embodiment 55, wherein the effective amount of amikacin, clarithromycin, azithromycin, ethambutol, rifampicin, tigecycline, linezolid, imipenem, cefoxitin, or a combination thereof is an amount that is ineffective in treating an NTM infection when administered without the BT composition. 57. The method of embodiment 55, wherein administration of an effective amount of the BT composition and an effective amount of amikacin results in a synergistic effect in the treatment of NTM infection. 58. The method of any one of embodiments 37-57, wherein the subject has a chronic pulmonary condition. 59. The method of embodiment 58, wherein the chronic pulmonary condition is cystic fibrosis, chronic bronchitis, emphysema, bronchiectasis, pulmonary fibrosis, asbestosis, pneumonitis, chronic obstructive pulmonary disease (COPD), or asthma. 60. The method of any one of embodiments 58 or 59, wherein the chronic pulmonary condition is cystic fibrosis. 61. The method of any one of embodiments 37-60, wherein the subject is administered about 30 μg to about 3,000 μg of the BT compound per day. 62. The method of any one of embodiments 37-60, wherein the subject is administered about 100 μg to about 1,000 μg of the BT compound per day. 63. The method of any one of embodiments 37-62, wherein the BT composition is administered to the lungs of the subject. 64. The method of any one of embodiments 37-63, wherein the BT composition is administered by inhalation. 65. The method of embodiment 63 or 64, wherein the concentration of bismuth in the lungs after a single daily dose is from about 0.03 μg / g lung tissue to about 3 μg / g lung tissue. 66. The method of any one of embodiments 63-65, wherein the concentration of bismuth in the lungs after 28 daily doses is from about 0.3 μg / g lung tissue to about 60 μg / g lung tissue. 67. The method of any one of embodiments 37-66, wherein the subject in need of treatment has previously been non-responsive to NTM therapy. 68. The method of any one of embodiments 37-67, wherein the subject in need of treatment has previously been non-responsive to amikacin. 69. A method for reducing NTM intracellular bacterial load in a subject, comprising contacting infected cells of the subject with an effective amount of a bismuth-thiol (BT) composition comprising a BT compound. 70. The cells are selected from the group consisting of M. avium, M. avium subsp. hominissuis (MAH), M. abscessus, M. chelonae, M. bolletii, M. kansasii, M. ulcerans, M. avium complex (MAC) (M. avium and M. intracellulare), M. conspicuum, M. kansasii, M. peregrinum, M. immunogenum, M. xenopi (M. xenopi, M. marinum, M. malmoense, M. marinum, M. mucogenicum, M. nonchromogenicum, M. scrofulaceum, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. terrae complex, M. haemophilum, M. genavense, M. asiaticum, M. shimoidei, M. gordone, M. 70. The method of embodiment 69, wherein the host is infected with M. gordonae, M. nonchromogenicum, M. triplex, M. lentiflavum, M. celatum, M. fortuitum, M. fortuitum complex (M. fortuitum and M. chelonae), or combinations thereof. 71. The method of embodiment 69 or 70, wherein the cell is infected with M. abscessus, M. avium, M. intracellulare, M. fortuitum, M. gordonae, M. kansasii, M. avium complex, M. marinum, M. terrae, M. cheloni, or a combination thereof. 72. The method of any one of embodiments 69-71, wherein the cells are infected with M. abscessus, M. avium, or a combination thereof. 73. The method of any one of embodiments 69-71, wherein the cells are infected with the M. avium complex (M. avium and M. intracellulare). 74. The method of any one of embodiments 70-73, wherein the M. avium infection is an M. avium subsp. hominissuis infection. 75. The method of any one of embodiments 69-74, wherein the infected cell is a macrophage. 76. The method of embodiment 75, wherein the macrophages are THP-1 macrophages. 77. The method of any one of embodiments 69-76, wherein contacting the cell with the BT composition results in phagocytosis of the BT compound. 78. The method of embodiment 77, wherein the intracellular bacterial load is reduced by about 10-fold to about 1000-fold. 79. The method of embodiment 77 or 78, wherein the intracellular bacterial load is reduced by about 10-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, or about 1000-fold. 80. The method of any one of embodiments 69-76, wherein contacting the cells with the BT composition prevents the intracellular bacterial load from increasing. 81. The method of any one of embodiments 75-80, wherein the macrophages are infected with one or more strains of M. abscessus. 82. The method of any one of embodiments 75-80, wherein the macrophages are infected with one or more strains of M. avium. 83. The method of any one of embodiments 69-82, wherein the BT composition comprises a BT compound selected from the group consisting of BisBAL, BisEDT, bis-dimercaprol, BisDTT, bis-2-mercaptoethanol, Bis-DTE, BisPyr, BisEry, BisTol, BisBDT, BisPDT, BisPyr / BAL, BisPyr / BDT, BisPyr / EDT, BisPyr / PDT, BisPyr / Tol, BisPyr / Ery, bismuth-1-mercapto-2-propanol, BisEDT / CSTMN(1:1), BisPyr / CSTMN(1:1), BisBAL / CSTMN(1:1), BisTOL / CSTMN(1:1), and BisEDT / 2-hydroxy-1-propanethiol. 84. The method of any one of embodiments 69-82, wherein the BT compound is selected from the group consisting of BisEDT, BisBAL, BisPyr, BisEry, BisTol, BisBDT, or BisEDT / 2-hydroxy-1-propanethiol. 85. The method of any one of embodiments 69-82, wherein the BT compound is BisEDT or BisBAL. 86. The method of any one of embodiments 69-82, wherein the BT compound is BisEDT. 87. The method of any one of embodiments 69-86, wherein the BT compound exhibits a bacteriostatic effect. 88. The method of any one of embodiments 69-86, wherein the BT compound exhibits a bactericidal effect. 89. The method of any one of embodiments 69-88, wherein the subject has a pulmonary infection. 90. The method of embodiment 89, wherein the pulmonary infection is a chronic pulmonary condition. 91. The method of embodiment 90, wherein the chronic pulmonary condition is cystic fibrosis, chronic bronchitis, emphysema, bronchiectasis, pulmonary fibrosis, asbestosis, pneumonitis, chronic obstructive pulmonary disease (COPD), or asthma. 92. The method of embodiment 90, wherein the chronic pulmonary condition is cystic fibrosis. 93. The method of any one of embodiments 69-92, wherein the subject is administered about 30 μg to about 3,000 μg of the BT compound per day. 94. The method of any one of embodiments 69-92, wherein the subject is administered about 100 μg to about 1,000 μg of the BT compound per day. 95. The method of any one of embodiments 69-94, wherein the BT composition is administered once per month, twice per month, three times per month, four times per month, once every two weeks, once per week, twice per week, or three times per week. 96. The method of any one of embodiments 69-95, wherein the BT composition is administered once or twice daily. 97. The method of any one of embodiments 69-96, wherein the BT composition is administered to the lungs of the subject. 98. The method of any one of embodiments 69-97, wherein the BT composition is administered by inhalation. 99. The method of any one of embodiments 69-98, wherein the BT composition is administered for a period of less than 24 months, less than 18 months, less than 12 months, less than 9 months, less than 6 months, less than 3 months, or less than 1 month. 100. The method of any one of embodiments 69-98, wherein the BT composition is administered for a period of 1 to 56 days. 101. The method of any one of embodiments 69-98, wherein the BT composition is administered for a period of 14 to 28 days. 102. The method of any one of embodiments 97-101, wherein the concentration bismuth in the lungs after a single daily dose is from about 0.03 μg / g lung tissue to about 3 μg / g lung tissue. The method of any one of embodiments 97-101, wherein the concentration of bismuth in the lungs after a daily dose of 103.28 is from about 0.3 μg / g lung tissue to about 60 μg / g lung tissue. 104. The method of any one of embodiments 69-103, further comprising contacting infected cells of the subject with amikacin. 105. The method of embodiment 104, wherein contacting the infected cells with a combination of the BT composition and amikacin exhibits a synergistic effect in reducing intracellular bacterial load. 106. A method of treating or providing prophylaxis against a nontuberculous mycobacterial (NTM) pulmonary infection in a subject in need of such treatment or prophylaxis, comprising: A method comprising administering to the lungs of a subject a BT composition comprising a BT compound for an administration period. 107. The method of embodiment 106, wherein administering to the patient's lungs comprises aerosolizing the BT composition to provide an aerosolized BT composition and administering the aerosolized BT composition to the subject's lungs. 108. The method of embodiment 107, wherein administering the aerosolized BT composition to the subject's lungs is by nebulizer, dry powder inhalation, nanoparticle inhalation, or metered dose inhalation. 109. The method of any one of embodiments 106-108, wherein the aerosolized BT composition is administered once per day in a single dosing session for the dosing period. 110. The method of embodiment 109, wherein during a single dosing session, the aerosolized BT composition is administered in less than about 75 minutes, less than about 60 minutes, less than about 30 minutes, less than about 15 minutes, or less than about 5 minutes. 111. The method of embodiment 106, wherein during a single dosing session, the aerosolized BT composition is administered over about 60 to about 75 minutes, about 45 to about 60 minutes, about 30 to about 45 minutes, about 20 to about 30 minutes, or about 15 to about 20 minutes. 112. The method of any one of embodiments 106-111, wherein the aerosolized BT composition is administered for a treatment period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. 113. The method of any one of embodiments 106-111, wherein the aerosolized BT composition is administered for a 6-month treatment period. 114. The method of any one of embodiments 106-111, wherein the subject in need of treatment of prophylaxis has cystic fibrosis, bronchiectasis, chronic obstructive pulmonary disease (COPD), or asthma. 115. The method of any one of embodiments 106-111, wherein the subject in need of treatment or prevention has cystic fibrosis. 116. The method of any one of embodiments 106-115, wherein the subject in need of treatment or prevention has previously been non-responsive to NTM therapy. 117. The method of any one of embodiments 106-116, wherein the subject in need of treatment or prevention has previously been non-responsive to amikacin. 118. The method of any one of embodiments 106-117, wherein the subject in need of treatment or prevention has a concomitant condition selected from the group consisting of diabetes, mitral valve disorder, acute bronchitis, pulmonary hypertension, pneumonia, asthma, tracheal carcinoma, bronchial carcinoma, lung cancer, cystic fibrosis, pulmonary fibrosis, laryngeal malformation, tracheal malformation, bronchial malformation, aspergillosis, HIV, and bronchiectasis. 119. NTM lung infections have been reported with M. avium, M. avium subsp. hominissuis (MAH), M. abscessus, M. chelonae, M. bolletii, M. kansasii, M. ulcerans, M. avium, M. avium complex (MAC) (M. avium and M. intracellulare), M. conspicuum, M. kansasii, M. peregrinum, M. immunogenum (M. immunogenum, M. xenopi, M. marinum, M. malmoense, M. marinum, M. mucogenicum, M. nonchromogenicum, M. scrofulaceum, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. terrae complex, M. haemophilum, M. genavense, M. asiaticum, M. schimoidei, M. shimoidei, M. gordonae, M. nonchromogenicum, M. triplex, M. lentiflavum, M. celatum, M. fortuitum, M. fortuitum complex (M. fortuitum and M. chelonae).chelonae), or a combination thereof. 120. The method of any one of embodiments 106-117, wherein the NTM lung infection is an M. avium infection. 121. The method of embodiment 120, wherein the M. avium infection is an M. avium subsp. hominissuis infection. 122. The method of any one of embodiments 106-117, wherein the NTM pulmonary infection is M. avium complex (M. avium and M. intracellulare). 123. The method of any one of embodiments 106-117, wherein the NTM lung infection is an M. abscessus infection. 124. The method of any one of embodiments 106-123, wherein the NTM pulmonary infection is a macrolide-resistant NTM pulmonary infection. 125. The method of any one of embodiments 106 to 124, wherein the NTM lung infection is a biofilm-associated infection. 126. The method of any one of embodiments 106-124, wherein the subject exhibits NTM culture conversion to negative during or after the administration period. 127. The method of embodiment 126, wherein the time to NTM culture conversion to negative is about 10 days, about 20 days, about 30 days, about 40 days, about 50 days, about 60 days, about 70 days, about 80 days, about 90 days, about 100 days or about 110 days. 128. The method of embodiment 126, wherein the time to NTM culture conversion to negative is about 20 days to about 200 days, about 20 days to about 190 days, about 20 days to about 180 days, about 20 days to about 160 days, about 20 days to about 150 days, about 20 days to about 140 days, about 20 days to about 130 days, about 20 days to about 120 days, about 20 days to about 110 days, about 30 days to about 110 days, or about 30 days to about 100 days. 129. The method of any one of embodiments 106-128, wherein the subject experiences an improvement in FEV1 for at least 15 days after the administration period has ended, compared to the subject's FEV1 before the administration period. 130. The method of any one of embodiments 106-129, wherein the subject experiences an improvement in blood oxygen saturation for at least 15 days after the administration period has ended, compared to the subject's blood oxygen saturation prior to the administration period. 131. The method of embodiment 129, wherein the subject's FEV1 is increased by at least 5% compared to the subject's FEV1 prior to the administration period. 132. The method of embodiment 129, wherein the subject's FEV1 is increased by at least 10% compared to the subject's FEV1 prior to the administration period. 133. The method of embodiment 129, wherein the subject's FEV1 is increased by at least 15% compared to the subject's FEV1 prior to the administration period. 134. The method of embodiment 129, wherein the subject's FEV1 is increased by 5% to 50% compared to the FEV1 prior to the administration period. 135. The method of any one of embodiments 106-134, wherein the subject exhibits an increase in the number of meters walked in a 6-minute walk test (6MWT) compared to the number of meters walked by the subject before receiving the treatment. 136. The method of embodiment 135, wherein the increase in the number of meters walked in the 6MWT is at least about 5 meters. 137. The method of embodiment 135, wherein the increase in the number of meters walked in the 6MWT is at least about 10 meters. 138. The method of embodiment 135, wherein the increase in the number of meters walked in the 6MWT is between about 5 meters and about 50 meters. 139. The method of embodiment 135, wherein the increase in the number of meters walked in the 6MWT is between about 15 meters and about 50 meters. 140. The method of any one of embodiments 106-139, wherein the BT composition comprises a BT compound selected from the group consisting of BisBAL, BisEDT, bis-dimercaprol, BisDTT, bis-2-mercaptoethanol, Bis-DTE, BisPyr, BisEry, BisTol, BisBDT, BisPDT, BisPyr / BAL, BisPyr / BDT, BisPyr / EDT, BisPyr / PDT, Bis-Pyr / Tol, BisPyr / Ery, bismuth-1-mercapto-2-propanol, BisEDT / CSTMN(1:1), BisPyr / CSTMN(1:1), BisBAL / CSTMN(1:1), BisTOL / CSTMN(1:1), and BisEDT / 2-hydroxy-1-propanethiol. 141. The method of any one of embodiments 106-139, wherein the BT compound is selected from the group consisting of BisEDT, BisBAL, BisPyr, BisEry, BisTol, BisBDT, or BisEDT / 2-hydroxy-1-propanethiol. 142. The method of any one of embodiments 106-139, wherein the BT compound is BisEDT or BisBAL. 143. The method of any one of embodiments 106-139, wherein the BT compound is BisEDT. 144. The method of any one of embodiments 106-143, wherein the concentration of bismuth in the lungs after a single daily dose is from about 0.03 μg / g lung tissue to about 3 μg / g lung tissue. 145. A method for treating a biofilm-associated nontuberculous mycobacteria (NTM) infection in the lungs of a subject in need thereof, comprising administering to the subject a BT composition comprising a BT compound. 146. The method of embodiment 145, wherein the subject has a chronic pulmonary condition. 147. The method of embodiment 146, wherein the chronic pulmonary condition is cystic fibrosis, chronic bronchitis, emphysema, bronchiectasis, pulmonary fibrosis, asbestosis, pneumonitis, chronic obstructive pulmonary disease (COPD), or asthma. 148. The method of embodiment 145 or 146, wherein the chronic pulmonary condition is cystic fibrosis. 149. The method of any one of embodiments 145 to 148, wherein the biofilm is caused by an antibiotic-resistant strain of a microorganism. 150.NTM pulmonary infections have been reported with M. avium, M. avium subsp. hominissuis (MAH), M. abscessus, M. chelonae, M. bolletii, M. kansasii, M. ulcerans, M. avium, M. avium complex (MAC) (M. avium and M. intracellulare), M. conspicuum, M. kansasii, M. peregrinum, M. immunogenum (M. immunogenum, M. xenopi, M. marinum, M. malmoense, M. marinum, M. mucogenicum, M. nonchromogenicum, M. scrofulaceum, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. terrae complex, M. haemophilum, M. genavense, M. asiaticum, M. schimoidei, M. shimoidei, M. gordonae, M. nonchromogenicum, M. triplex, M. lentiflavum, M. celatum, M. fortuitum, M. fortuitum complex (M. fortuitum and M. chelonae).chelonae), or a combination thereof. 151. The method of any one of embodiments 145-149, wherein the NTM lung infection is an M. avium infection. 152. The method of any one of embodiments 145-149, wherein the NTM pulmonary infection is M. avium complex (M. avium and M. intracellulare). 153. The method of any one of embodiments 150-152, wherein the M. avium infection is an M. avium subsp. hominissuis infection. 154. The method of any one of embodiments 145-149, wherein the NTM lung infection is an M. abscessus infection. 155. The method of any one of embodiments 145-154, wherein the BT composition comprises a BT compound selected from the group consisting of BisBAL, BisEDT, Bis-dimercaprol, BisDTT, Bis-2-mercaptoethanol, Bis-DTE, BisPyr, BisEry, BisTol, BisBDT, BisPDT, BisPyr / BAL, BisPyr / BDT, BisPyr / EDT, BisPyr / PDT, Bis-Pyr / Tol, BisPyr / Ery, bismuth-1-mercapto-2-propanol, BisEDT / CSTMN(1:1), BisPyr / CSTMN(1:1), BisBAL / CSTMN(1:1), BisTOL / CSTMN(1:1), and BisEDT / 2-hydroxy-1-propanethiol. 156. The method of any one of embodiments 145-154, wherein the BT compound is selected from the group consisting of BisEDT, BisBAL, BisPyr, BisEry, BisTol, BisBDT, or BisEDT / 2-hydroxy-1-propanethiol. 157. The method of any one of embodiments 145-154, wherein the BT compound is BisEDT or BisBAL. 158. The method of any one of embodiments 145-154, wherein the BT compound is BisEDT. 159. The method of any one of embodiments 145-158, wherein the subject is administered about 30 μg to about 3,000 μg of the BT compound per day. 160. The method of any one of embodiments 145-158, wherein the subject is administered about 100 μg to about 1,000 μg of the BT compound per day. 161. The method of any one of embodiments 145-160, wherein the BT composition is administered once per month, twice per month, three times per month, four times per month, once every two weeks, once per week, twice per week, or three times per week. 162. The method of any one of embodiments 145-160, wherein the BT composition is administered once or twice a day. 163. The method of any one of embodiments 145-162, wherein the BT compound is administered to the lungs of the subject. 164. The method of any one of embodiments 145-163, wherein the BT compound is administered by inhalation. 165. The method of any one of embodiments 145-164, wherein the BT composition is administered for a period of less than 24 months, less than 18 months, less than 12 months, less than 9 months, less than 6 months, less than 3 months, or less than 1 month. 165. The method of any one of embodiments 145-164, wherein the BT composition is administered for a period of 1 to 56 days. 166. The method of any one of embodiments 145-164, wherein the BT composition is administered for a period of 14 to 28 days. 167. The method of any one of embodiments 145-166, wherein the concentration of bismuth in the lungs after a single daily dose is from about 0.03 μg / g lung tissue to about 3 μg / g lung tissue. 168. The method of any one of embodiments 145-166, wherein the concentration of bismuth in the lungs after 28 daily doses is from about 0.3 μg / g lung tissue to about 60 μg / g lung tissue. 169. A method of treating an NTM infection in a subject, comprising: (i) testing for the presence of bacteria-infected macrophages in a biological sample from a subject; and (ii) if the sample tests positive for bacterially infected macrophages, administering to the subject an effective amount of a bismuth-thiol (BT) composition comprising a BT compound. The method includes: 170. The method of embodiment 169, wherein the NTM infection is a pulmonary infection. 171. Macrophages are responsible for the proliferation and proliferation of M. avium, M. avium subsp. hominissuis (MAH), M. abscessus, M. chelonae, M. bolletii, M. kansasii, M. ulcerans, M. avium complex (MAC) (M. avium and M. intracellulare), M. chimaera, M. conspicuum, M. peregrinum, M. immunogenum, M. xenopi (M. xenopi, M. marinum, M. malmoense, M. mucogenicum, M. nonchromogenicum, M. scrofulaceum, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. terrae complex, M. haemophilum, M. genavense, M. gordonae, M. fortuitum, M. fortuitum complex 171. The method of embodiment 169 or 170, wherein the bacteria is tested for the presence of M. fortuitum and M. chelonae complex (M. fortuitum and M. chelonae), or a combination thereof. 172. The method of any one of embodiments 169-171, wherein the macrophages are tested for the presence of M. abscessus, M. avium, M. intracellulare, M. fortuitum, M. gordonae, M. kansasii, M. avium complex, M. marinum, M. terrae, M. cheloni, or combinations thereof. 173. The method of any one of embodiments 169-171, wherein the macrophages are tested for the presence of M. abscessus, M. avium, or a combination thereof. 174. The method of any one of embodiments 169-171, wherein the macrophages are tested for the presence of the M. avium complex (M. avium and M. intracellulare). 175. The method of any one of embodiments 171-174, wherein the M. avium infection is an M. avium subsp. hominissuis infection. 176. The method of any one of embodiments 169-171, wherein macrophages are tested for the presence of M. abscessus. 177. The method of any one of embodiments 169-176, wherein the subject is administered about 30 μg to about 3,000 μg of the BT compound per day. 178. The method of any one of embodiments 169-176, wherein the subject is administered about 100 μg to about 1,000 μg of the BT compound per day. 179. The method of any one of embodiments 169-178, wherein the BT composition is administered once per month, twice per month, three times per month, four times per month, once every two weeks, once per week, twice per week, or three times per week. 180. The method of any one of embodiments 169-179, wherein the BT composition is administered once or twice daily. 181. The method of any one of embodiments 169-180, wherein the BT composition is administered to the lungs of the subject. 182. The method of any one of embodiments 169-181, wherein the BT compound is administered by inhalation. 183. The method of any one of embodiments 169-182, wherein the BT composition is administered for a period of less than 24 months, less than 18 months, less than 12 months, less than 9 months, less than 6 months, less than 3 months, or less than 1 month. 184. The method of any one of embodiments 169-182, wherein the BT composition is administered for a period of 1 to 56 days. 185. The method of any one of embodiments 169-182, wherein the BT composition is administered for a period of 14 to 28 days. 186. The method of any one of embodiments 169-185, wherein the concentration of bismuth in the lungs after a single daily dose is from about 0.03 μg / g lung tissue to about 3 μg / g lung tissue. 187. The method of any one of embodiments 169-186, wherein the concentration of bismuth in the lungs after 28 daily doses is from about 0.3 μg / g lung tissue to about 60 μg / g lung tissue. 188. The method of any one of embodiments 169-187, wherein the BT compound is selected from the group consisting of BisBAL, BisEDT, bis-dimercaprol, BisDTT, bis-2-mercaptoethanol, Bis-DTE, BisPyr, BisEry, BisTol, BisBDT, BisPDT, BisPyr / BAL, BisPyr / BDT, BisPyr / EDT, BisPyr / PDT, Bis-Pyr / Tol, BisPyr / Ery, bismuth-1-mercapto-2-propanol, BisEDT / CSTMN(1:1), BisPyr / CSTMN(1:1), BisBAL / CSTMN(1:1), BisTOL / CSTMN(1:1), and BisEDT / 2-hydroxy-1-propanethiol. 189. The method of any one of embodiments 169-187, wherein the BT compound is selected from the group consisting of BisEDT, BisBAL, BisPyr, BisEry, BisTol, BisBDT, or BisEDT / 2-hydroxy-1-propanethiol. 190. The method of any one of embodiments 169-187, wherein the BT compound is BisEDT or BisBAL. 191. The method of any one of embodiments 169-187, wherein the BT compound is BisEDT. 192. The method of any one of embodiments 1-191, wherein the pharmaceutical composition comprises the BT compound suspended therein and one or more pharma- ceutically acceptable excipients, and the composition is formulated for topically administering the BT compound to the lungs of a subject. 193. The method of embodiment 192, wherein the composition comprises a plurality of particles comprising the BT compound. 194. The method of embodiment 193, wherein the BT composition comprises a BT compound at a concentration of greater than about 0.1 mg / mL, about 0.05% to about 1.0% polysorbate 80, about 0.05 mM to 40 mM sodium chloride, and optionally about 2 mM to 20 mM sodium phosphate at about pH 7.4. 195. The method of embodiment 194, wherein the BT compound is BisEDT. 196. The method of any one of embodiments 192 to 195, wherein local administration to the lungs of the subject comprises aerosolizing the BT composition to provide an aerosolized BT composition, and administering the aerosolized BT composition to the lungs of the subject. 197. The method of embodiment 196, wherein administering the aerosolized BT composition to the subject's lungs is by a nebulizer. 198. The method of embodiment 196 or 197, wherein the aerosolized BT composition is administered for the dosing period once per day in a single dosing session. 199. The method of embodiment 198, wherein during a single dosing session, the aerosolized BT composition is administered in less than about 75 minutes, less than about 60 minutes, less than about 30 minutes, less than about 15 minutes, or less than about 5 minutes. 200. The method of embodiment 198, wherein during a single dosing session, the aerosolized BT composition is administered over about 60 to about 75 minutes, about 45 to about 60 minutes, about 30 to about 45 minutes, about 20 to about 30 minutes, or about 15 to about 20 minutes. EXAMPLES

[0148] Working Example

[0161] The following examples are provided to illustrate the present disclosure and should not be construed as limiting thereof.

[0149]

[0162] Example 1: Representative synthesis of BT compounds

[0163] Representative synthesis of BisEDT with 1.25 h addition of thiol via syringe pump at 20° C. and polypropylene cloth for filtration: BisEDT synthesis was carried out on a 10 g scale. A 1 L jacketed reactor was charged with USP water (480 mL, 48 vol.) followed by 70% HNO3 (34 mL, 3.4 vol.). A solution of bismuth subnitrate (10 g, 6.84 mmol) in water (43 mL, 4.3 vol.) and 70% HNO3 (14 mL, 1.4 vol.) was added at 20° C. The reaction mixture was cooled to 15° C. for the addition of 95% ethanol. Then, 95% ethanol (180 mL, 18 vol.) was added slowly (ethanol addition was exothermic and the temperature reached 22° C.). The temperature was then adjusted back to 20° C. Following this, 1,2 ethanedithiol (4.3 mL, 7.5 mmol in 94 mL 95% ethanol, 9.4 vol) was added dropwise over a period of 1.25 hours at a batch temperature of 20° C., during which time it turned to a yellow suspension. The reaction was stirred at 20° C. overnight. The reaction mixture was filtered through polypropylene cloth and washed with 95% ethanol (45 mL, 4.5 vol). The wet cake was placed back into the reactor and slurried in 95% ethanol (380 mL, 38 vol) for 2 hours at 20° C. The suspension was then filtered (same cloth) and washed with 95% ethanol (30 mL, 3 vol). The wet cake was reslurried in 95% EtOH (170 mL, 17 vol) at 20° C., filtered (same cloth) and washed with 95% ethanol (30 mL, 3 vol). The wet cake was then slurried in acetone (170 mL, 17 vol) at 20° C. overnight followed by filtration (same cloth) and acetone washing (20 mL, 2 vol). The acetone (170 ml, 17 vol) treatment was repeated on the solid and stirred for 2 h. The suspension was filtered (same cloth), washed with acetone (30 mL, 3 vol), died at 45° C. and dried at 45° C. (18 h) to give a bright yellow solid (10.81 g 91.0%).

[0150]

[0164] Representative synthesis of BisEDT by 1 hour addition of thiol via syringe pump at 15°C and polypropylene cloth for filtration: The synthesis BisEDT was carried out on a 10 g scale and the temperature profile was monitored by a data logger. Ethanedithiol was added by syringe pump over 1 hour at 15°C and filtration was carried out using a PP filter cloth. A 1 L jacketed reactor was charged with USP water (480 mL, 48 vol) and cooled to 15°C, followed by the addition of 70% HNO3 (34 mL, 3.4 vol). A solution of bismuth subnitrate (10 g, 6.84 mmol) in water (43 mL, 4.3 vol) and 70% HNO3 (14 mL, 1.4 vol) was added at the same temperature. Then 95% ethanol (180 mL, 18 vol) was added slowly (ethanol addition was exothermic and the temperature reached 22.5°C). It was then allowed to cool to 15°C. Subsequently, 1,2 ethanedithiol (4.3 mL, 7.5 mmol in 94 mL 95% ethanol, 9.4 vol) was added dropwise over 1 h at a batch temperature of 15° C. The reaction was stirred overnight at 15° C. The reaction mixture was filtered through polypropylene cloth and washed with 95% ethanol (45 mL, 4.5 vol). The wet cake was placed back into the reactor and slurried in 95% ethanol (380 mL, 38 vol) for 2 h at 20° C. The suspension was then filtered (same cloth) and washed with 95% ethanol (30 mL, 3 vol). The wet cake was reslurried in 95% EtOH (170 mL, 17 vol) at 20° C., filtered (same cloth) and washed with 95% ethanol (30 mL, 3 vol). The wet cake was then slurried in acetone (170 mL, 17 vol) at 20° C. overnight followed by filtration (same cloth) and acetone washing (20 mL, 2 vol). The acetone (170 ml, 17 vol) treatment was repeated on the solid and stirred for 2 h. The suspension was filtered (same cloth), washed with acetone (30 mL, 3 vol), killed at 45° C. and dried at 45° C. (18 h) to give a bright yellow solid (10.43 g 87.8%).

[0151]

[0165] Example 2: MIC activity of BisEDT and control drugs against M. avium and M. abscessus bacterial strains

[0166] Test Design

[0167] Mycobacterial strains were used to determine MICs for BisEDT and amikacin. Strains included cystic fibrosis patient isolates from the National Jewish Hospital. A range of high to low antibiotic doses were used to determine bacterial susceptibility. M. avium strain 104, M. avium strain 3388, and CF patient strain DNA00133 were grown to log phase (7 days) on 7H10 Middlebrook medium plates supplemented with 10% OADC (oleic acid, albumin, dextrose, catalase), while M. abscessus strain 19977, CF patient strain DNA00703, and CF patient strain DNA01715 achieved log phase (4 days). For amikacin, 128 μg / ml was added to 1 ml of 7H9 Middlebrook Broth supplemented with 10% OADC and then diluted 1:1 to reach a concentration of 1 ug / ml. For BisEDT, two compound physical states were tested. First, BisEDT was solubilized in DMSO (further denoted soluble BisEDT). Second, BisEDT was made into a suspension to mimic the in vivo test (further denoted insoluble BisEDT). For each type of BisEDT, 16 μg / ml was added to 1 ml of 7H9 Broth supplemented with OADC and then diluted 1:1 to reach a concentration of 0.125 μg / ml. Control tubes without antibiotic were also included for each antibiotic tested. 10% for each strain were added to DMSO (further denoted soluble BisEDT). Second, BisEDT was made into a suspension to mimic the in vivo test (further denoted insoluble BisEDT). For each type of BisEDT, 16 μg / ml was added to 1 ml of 7H9 Broth supplemented with OADC and then diluted 1:1 to reach a concentration of 0.125 μg / ml. Control tubes without antibiotic were also included for each antibiotic tested. 9 Bacterial inoculum was measured by optical density (OD) at 595 nm. 10 μl of these inoculum was added to each antibiotic tube and incubated at 37° C. and 200 rpm in a shaking incubator. Growth of slow growing strains was measured after 10 days of incubation, while fast growing strains were incubated for 5 days. After incubation, 100 μl was withdrawn from each tube and placed in a 96-well plate for OD readings. Susceptible, intermediate, or resistant bacteria were determined by turbidity.

[0152]

[0168] MIC activity of mycobacterial strains:

[0169] For M. avium strains, the amikacin MIC was determined to be 8 μg / ml, with the exception of the CF patient strain (DNA00133) that was resistant to 128 μg / ml. Soluble BisEDT inhibited all strains at 4 μg / ml, and insoluble BisEDT showed inhibition of the amikacin-resistant patient strain DNA0133 at 8 μg / ml (Table 1).

[0153] [Table 2]

[0154]

[0170] M. abscessus strains had variable inhibition by amikacin. Strain 19977 was susceptible at 32 μg / ml, patient-derived strain 00703 was susceptible at 64 μg / ml, and patient-derived strain 01715 was susceptible at 16 μg / ml. As shown by the data in Table 2, all M. abscessus strains were more susceptible to BisEDT formulations (1-4 μg / ml) than to amikacin.

[0155] [Table 3]

[0156]

[0171] Conclusions: BisEDT demonstrated improved in vitro activity, with MICs for the treatment of M. abscessus infections that were 16- to 32-fold lower than amikacin.

[0157]

[0172] Example 3: BisEDT treatment of THP-1 macrophages infected with M. avium or M. abscessus

[0173] the purpose

[0174] To investigate the efficacy of BisEDT in reducing M. avium and M. abscessus levels in vitro.

[0158]

[0175] Test Design

[0176] THP-1 macrophages were differentiated with 50ng / ml PMA for 24 hours and subsequently placed in medium for 24 hours prior to infection. Differentiated THP-1 macrophages were infected or not infected with M. avium or M. abscessus strains for 1 hour at a multiplicity of infection (MOI) of 5. Cells were washed twice followed by a 1 hour antibiotic treatment with 200μg / ml amikacin to remove extracellular bacteria. After the antibiotic step, cells were washed once more and BisEDT (BIZ) or amikacin was added once to selected wells (see in vitro doses below). Treatments included vehicle, amikacin, soluble BisEDT (S Biz), insoluble BisEDT (I Biz), and amikacin + BisEDT. The time points for sample preparation were 72 hours post-infection for M. abscessus strains and 4 and 7 days post-infection for M. avium strains. At the indicated time points, cell culture medium was removed and replaced with 400 ul of 0.1% triton x-100 in H2O for lysis. Wells were pipetted 25 times to disrupt cells, which were then diluted and plated for CFU counting.

[0159]

[0177] result

[0178] Using the MICs determined for each M. avium strain (see Table 3), M. avium survival was determined in THP-1 macrophages (Figure 1). Infectious CFU was determined 1 hour after infection and was designated as day 0 / pre-drug. As shown in Figure 1 (first panel), the CFU count of untreated M. avium strain 104 (vehicle) increased over a 4-day or 7-day period. Treatment with amikacin greatly reduced the bacterial load in each case. Treatment of this same strain with soluble BisEDT (S Biz; DMSO solution) or insoluble BisEDT (I Biz) provided a bacteriostatic effect. When BisEDT was combined with amikacin, the bacterial load was greatly reduced in both periods. A similar pattern was observed for M. avium strain 3388 (Figure 1, second panel). However, in the CF patient strain 00133, which is resistant to amikacin, a bacteriostatic effect was observed, as each of the BisEDT formulations was effective in maintaining CFU numbers at levels similar to those of the initial infection (Fig. 1 , third panel).

[0160] [Table 4]

[0161]

[0179] Using the MICs determined for each M. abscessus strain (see Table 4), M. abscessus survival was determined in THP-1 macrophages (Figure 2). Infectious CFU was determined 1 hour after infection and designated as day 0 / pre-drug. After 3 days, the bacterial load of untreated M. abscessus strain 19977 increased inside THP-1 cells. BisEDT reduced the bacterial load in THP-1 cells (Figure 2, first panel). Amikacin treatment somewhat reduced the CFU count from the initial infection rate, but the difference was non-significant. However, as the data show, combining BisEDT with amikacin produced a synergistic affect, as the bacterial CFU count in macrophages was reduced compared to when each drug was administered alone (Figure 2, first panel). In both CF patient strains, a similar pattern was obtained - amikacin treatment was bacteriostatic, whereas treatment with BisEDT greatly reduced the bacterial load in macrophages. A synergistic effect was also observed when BisEDT and amikacin were combined (Figure 2, second and third panels).

[0162] [Table 5]

[0163]

[0180] Conclusion – In Vitro Testing

[0181] BisEDT is bactericidal in macrophages infected with the three clinical isolates of M. avium tested, including an isolate of M. avium that is amikacin resistant.

[0164]

[0182] As shown in Figure 1, for MAH 104, statistically significant differences were found between the vehicle 4 and amikacin 4 data and between the vehicle 7 and amikacin 7 data (p values ​​of 0.0272 and 0.0017, respectively). For MAH 3388, statistically significant differences were found between the vehicle 4 data and the combination of amikacin and insoluble BisEDT 4, between the vehicle 7 data and the combination of amikacin and insoluble BisEDT 7, and between the vehicle 7 and amikacin 7 data (p values ​​of 0.0133, 0.0085, and 0.0220, respectively). All analyses used the Kruskal-Wallis test.

[0165]

[0183] Thus, the efficacy of BisEDT against M. avium strains may be increased in combination with amikacin. A synergistic effect was observed, as the effect of the combined treatment was greater than either compound used alone.

[0166]

[0184] BisEDT is also highly active against M. abscessus strains in vitro: THP-1 macrophages infected with clinically significant strains of M. abscessus and treated with BisEDT demonstrated a reduction in intracellular M. abscessus after 3 days of treatment.

[0167]

[0185] As shown in Figure 2, for MAB 19977, statistically significant differences were found between the vehicle and soluble BisEDT groups and between the vehicle and amikacin + soluble BisEDT groups (p values ​​of 0.0370 and 0.0029, respectively). For CF patient strain 1715, statistically significant differences were found between the vehicle and soluble BisEDT groups and between the vehicle and amikacin + soluble BisEDT groups (p values ​​of 0.0295 and 0.0029, respectively). For CF patient strain 007303, statistically significant differences were found between the vehicle and soluble BisEDT groups and between the vehicle and amikacin + soluble BisEDT groups (p values ​​of 0.0466 and 0.0029, respectively). All analyses used Kruskal-Wallis tests.

[0168]

[0186] Thus, in the macrophage system, BisEDT was shown to be highly active against M. abscessus, both alone and in combination with amikacin. BisEDT was bactericidal by itself (a 3 log reduction in M. abscessus levels was observed in CF patient isolates) and its efficacy against M. abscessus was increased when combined with amikacin. The data suggest that BisEDT is capable of killing M. abscessus intracellularly.

[0169]

[0187] Example 4: Mechanism of action of BisEDT on intracellular NTM

[0188] THP-1 macrophages were differentiated with 50ng / ml PMA for 24 hours and subsequently placed in medium for 24 hours prior to infection. Differentiated THP-1 macrophages were infected with M. avium or M. abscessus for 1 hour at a multiplicity of infection (MOI) of 10. Infected cells were washed twice followed by a 1 hour antibiotic treatment with 200μg / ml amikacin to remove extracellular bacteria. After the antibiotic step, cells were washed once more and 4μg / ml insoluble BisEDT (BIZ) was added to selected wells. Treatments included: (1) untreated, (2) untreated + MAH 104 infection, (3) untreated + M. abscessus infection, (4) BIZ treatment, (5) BIZ treatment + MAH 104 infection, and (6) BIZ treatment + M. abscessus infection. Sample preparation time points were 24 and 48 hours post-infection. Transmission Electron Microscopy Cells were not washed prior to TEM sample preparation to preserve any extracellular bacteria that may have been present. Cells were detached by treatment with 5 mM EDTA for 30 minutes, quenched with 1X HBSS, and suspended in fixation buffer with 2.5% glutaraldehyde, 1% formaldehyde, and 0.1 M sodium cacodylate for 24 hours before being submitted to the electron microscopy facility for processing. Samples were sectioned, dehydrated, and visualized with a FEIT Titan 80-200 TEM / STEM microscope.

[0170]

[0189] result

[0190] As shown in the TEM images in Figures 3A-3F, BisEDT appears to act (primarily or secondarily) on the mycobacterial cell wall, as observed by the loss of integrity of the polar region early on and the loss of integrity of the entire cell wall at more advanced stages. After 2 days of macrophage infection, no significant differences were observed between the two bacteria. M. abscessus appears to leave the macrophages faster than M. avium (3 days vs. 5 days). Analysis of the images suggests that the compound enters the eukaryotic cell by pinocytosis or by an energy-dependent transport mechanism.

[0171]

[0191] Example 5: Efficacy of BisEDT against biofilm-grown CF-associated pathogens

[0192] Background / Scope

[0193] The emergence of antibiotic-resistant bacterial strains has reduced the efficacy of antibiotics commonly used to treat chronic infections in CF patients. Furthermore, it has been shown that the biofilms formed by many of these bacterial strains also hinder the activity of these antibiotics. Identification and development of novel drugs that are effective against both drug-resistant bacterial strains and biofilms is urgently needed for the treatment of chronic infections in CF patients.

[0172]

[0194] The scope of this study is to evaluate the anti-biofilm performance of BisEDT against the performance of antibiotics currently used to treat infections in CF patients, using a minimum biofilm eradicating concentration (MBEC) assay.

[0173]

[0195] MBEC assay

[0196] In this study, the MBEC assay was followed as described in the Innovotech Procedural Manual, version 2.1 with minor modifications to evaluate the antibiofilm activity of the antimicrobial compounds under study. The MBEC value refers to the minimum biofilm eradicating concentration of each antimicrobial agent for each strain. The MBEC value is defined as the minimum antibiotic concentration at which the optical density (OD) value at recovery is less than 10% of the control OD value and no growth is observed by visual inspection of the wells.

[0174]

[0197] KK

[0198] Several strains of eight microbial species (Table 5A) were tested with different sets of antimicrobial agents (Table 5B) at various concentrations to determine the MBEC value for each strain. Fresh liquid cultures were grown from single colonies in Mueller-Hinton II cation-adjusted (CAMHB) broth to late logarithmic stage for storage. Strains were stored in 15% glycerol stocks at -80°C.

[0175] [Table 6]

[0176] [Table 7]

[0177]

[0199] procedure

[0200] Step 1: Inoculation. All strains were streaked from frozen stocks onto TSA plates and grown overnight at 37°C, except for mycobacteria, which were streaked onto 7H10 medium for activation (Table 6). From the plates, one colony was picked into organism specific medium (OSM) for inoculation and grown to mid-log phase (OD 0.2-0.8) at 37°C and 225 RPM in a shaker incubator. At mid-log phase, the culture was diluted to an OD of 0.05 in OSM medium (Table 6). 150 μL of it was aliquoted into each corresponding well in a 96-well MBEC plate (inoculation plate). The MBEC plate was then statically incubated at 37°C for the appropriate time for each species (Table 7). During incubation, biofilms formed on the pegs.

[0178]

[0201] Step 2: Exposure. After incubation, the peg-lids with biofilms were washed twice with PBS and placed on a second 96-well plate (exposure plate) with 200 μl of antibiotic dilution. The exposure plate was then incubated at 37° C. for 18 hours to expose the biofilms to antibiotics.

[0179]

[0202] Step 3: Recovery. In the final step, the peg-lids (with antibiotic-treated biofilms) were again washed twice with PBS and placed on a third 96-well plate (recovery plate) containing 200 μl growth medium per well to grow any bacterial cells resistant to any concentration of antibiotic. Recovery of resistant cells was assessed by end-point measurement of the optical density at 600 nm (OD600) of each well by a plate reader (FIG. 4).

[0180] [Table 8]

[0181] [Table 9]

[0182]

[0203] result

[0204] Antibiotic-tested M. abscessus and M. avium strains were evaluated for biofilm formation on pegs of MBEC plates. Biofilm formation was assessed by crystal violet assay. Briefly, pegs were washed twice with PBS, after which they were immersed in a 96-well plate with 0.1% crystal violet for 10 min. The pegs were then washed twice again with PBS, and biofilm formation was indicated by a purple appearance (see Figures 5A-5B).

[0183]

[0205] M. avium complex. Five strains of M. avium complex were tested, but none formed biofilms on the pegs (Figure 5A). Therefore, further evaluation of antimicrobial antibiofilm activity could not be performed for these strains.

[0184]

[0206] M. abscessus. Six clinical isolates of M. abscessus were tested: MABS1, MABS2, MABS4, MABS7, MABS9 and MABS11. All strains were resistant to high concentrations of imipenem, cefoxitin, amikacin and clarithromycin, except for strain MABS4, which was sensitive to amikacin at a concentration of 8 μg / mL and clarithromycin at a concentration of 4 μg / mL. Strains MABS1, MABS2, MABS9 and MABS11 were also resistant to BisEDT at a concentration of 32 μg / mL, whereas strains MABS4 and MABS7 were sensitive to BisEDT at concentrations of 0.5 μg / mL or less.

[0185] [Table 10]

[0186]

[0207] To facilitate comparison of BisEDT with antibiotics that have widely differing effective concentrations, we normalized the MBEC values ​​on a scale of 0 to 1, where 0 represents complete susceptibility and 1 represents complete resistance. To do this, the following formula was applied to each MBEC value along with the maximum and minimum concentrations of the associated antibiotic: ([MBEC]-[Ab] 最小 ) / ([Ab] 最大 -[Ab] 最小 )

[0187]

[0208] BisEDT was found to be effective against two strains of M. abscessus (MABS7 and MABS4) compared to control antibiotics, with the isolates evaluated showing little susceptibility to any of the control antibiotics (Figure 6).

[0188]

[0209] The M. abscessus data showed some variability between experimental replicates. This is likely due in part to the variable clumping of cells observed in the biofilm biomass on the pegs. The clumping of cells also affected the OD readings. Therefore, visual inspection of the plates was performed to address this challenge. For replicates with varying results, the final MBEC value was determined based on the higher MBEC concentration observed during the replicate. Although four of the six M. abscessus strains showed resistance to BisEDT at concentrations higher than 32 μg / mL, the same strains also showed that their recovery was significantly slower at concentrations higher than 0.125 μg / mL compared to their recovery at lower concentrations, indicating that BisEDT affects the growth of these strains at concentrations lower than their MBEC values.

[0189]

[0210] None of the five M. avium strains tested formed biofilms, therefore antibiotic testing of the biofilms was not possible.

[0190]

[0211] The efficacy of BisEDT against biofilms of multidrug-resistant (MDR) M. abscessus strains makes it a promising next-generation antibiotic candidate.

[0191]

[0212] Example 6: Efficacy of BisEDT against chronic NTM infection in mice

[0213] background

[0214] BisEDT (pravibismane) is a novel bioenergetic inhibitor antibiotic that affects energy flow in bacterial cell membranes preventing ATP production. This mechanism of action is becoming increasingly common for new antibiotics. Our aim is to evaluate the efficacy of inhaled BisEDT against pulmonary and M. abscessus infections in a mouse model. Since 2012, the collection of small molecule inhibitors of bioenergetics has expanded dramatically. Inhibitors are now a major component (>30%) of all new antimycobacterial drugs in clinical trials and are included in >65% of phase 3 trial regimens. Bioenergetic inhibitors have been only marginally explored for efficacy in NTM infections and can act as an alternative for resistant NTM infections. These formulations can improve patient clearance outcomes against previously resistant strains of NTM.

[0192]

[0215] Inhaled delivery of antimicrobial agents is becoming a preferred application for the treatment of pulmonary infections. The advantages of inhalation include site-specific drug targeting avoiding systematic administration, higher therapeutic concentrations at the site of infection, reducing off-target effects and any toxicity. Early studies in rats have shown that doses as high as 59ug / kg / day were well tolerated with no adverse effects on the animals for a period of 28 days. This study aims to identify new treatments for pulmonary NTM that will minimize discomfort and treatment costs for affected individuals and perhaps also shorten the duration of infection.

[0193]

[0216] Test Design

[0217] In vivo studies were performed in mice, a model of chronic M. abscessus infection. Animals were treated for 28 days and grouped according to dose: vehicle, low dose BisEDT, high dose BisEDT, and amikacin. At the end of treatment, blood, lungs, and spleens were collected for analysis. Since BisEDT showed strong efficacy against M. abscessus in vivo, further studies were performed to investigate the tendency of the bacteria to develop resistance, both naturally and from strains collected from mouse lungs. Histopathology studies were performed on lung samples from the in vivo studies.

[0194]

[0218] M. abscessus infection protocol

[0219] A total of 60 6-8 week old female SCID / Beige mice were used. Mice were placed with up to 4 animals per cage and allowed 1 week of acclimation prior to infection.

[0195]

[0220] The M. abscessus strain 19977 (MAB) inoculum used in this study was generated at low passage number from a highly virulent frozen stock. Bacteria were grown on 7H10 medium agar plates at 37°C until logarithmic phase after 4 days. Once confluent, as determined by optical density (OD), bacteria were added to 10 9 Inoculum was made by suspending in Hank's Balanced Salt Solution (HBSS) at 1000 x g for 24 h and quantified by serial dilution to determine the CFU / mL of the suspension. At this higher bacterial concentration, only 20 μL of inoculum was required to produce 10 8 It was necessary to place the bacteria.

[0196]

[0221] anesthesia

[0222] On the day of infection, mice were anesthetized with isoflurane by the drop jar method. The mouse was held by the neck in a standard one-handed grip, and the nose of the mouse was held of a tube containing a gauze containing a small amount of isoflurane. The mouse's breathing was monitored, and when the fast breathing first slowed, the mouse was removed from direct anesthesia and 20 μL of bacterial suspension was placed in the left nostril. The mouse then breathed slowly into the droplet. Each mouse was then held in the hand until full consciousness was obtained, and the mouse was then returned to its home cage. After the infection was initiated, 3 weeks were allowed to elapse for the bacteria to establish chronic lung lesions for final treatment.

[0197]

[0223] After each infection was established, 12 mice were euthanized the day before treatment began to determine baseline bacterial load. Mice were euthanized using CO2 according to the recommendations of the Guide for the Care and Use of Laboratory Animals (NRC 2011). Lungs and spleens were collected, homogenized, and then serially diluted for CFU enumeration.

[0198]

[0224] Drug Inhalation Protocol

[0225] A CH technologies nasal-only inhalation exposure chamber was used for this experiment. Mice were divided into four treatment groups: vehicle (buffer without BisEDT), amikacin (100 μg / kg / day), BisEDT "low" (200 μg / kg / day), and BisEDT "high" (1000 μg / kg / day). Each treatment group consisted of 12 mice. Ten of these mice were used for CFU determination and two mice were used for histology / pathology / pulmonary pharmacokinetics. Treatments were performed six days per week over a total course of 28 days. 10 mL of test article was made to create the correct level of deposition dose for each drug tested. Amikacin at 20 mg / mL in HBSS was made and stored at 4°C until required. BisEDT "high" at a concentration of 8 mg / mL was made once a week and stored at room temperature. BisEDT "low" was diluted from 8 mg / mL to 1.6 mg / mL on the day of treatment. Each treatment used a Pari LC star reusable nebulizer. The animal was restrained and placed on the inhalation apparatus. The breathing hose was connected to the inhalation apparatus and the compressor was connected to the nebulizer. The airflow rate from the compressor to the nebulizer remained at 8.5 L / min, which generated a misty fog that each mouse inhaled for 45 minutes.

[0199]

[0226] Sample and data collection

[0227] After 28 days of treatment with BisEDT, animals were euthanized by CO2 and secondary cardiac puncture, and whole blood was collected for quantification of BisEDT concentrations. Lungs and spleens from CFU mice were homogenized in 1:1 H2O:Dey-Engley neutralizing broth to inactivate BisEDT. CFU was determined after serial dilution. Mice and lung pairs for histology were separated, one in formalin and the other weighed and flash frozen in liquid N2 to determine tissue concentrations of BisEDT.

[0200]

[0228] The spleen was also placed in formalin. Samples for histological diagnosis were processed by the Veterinary Diagnostic Laboratory. Slides were stained with hematoxylin-eosin (HE stain) for general pathology as well as acid-fast stain to highlight mycobacterial infection in the lung tissue.

[0201]

[0229] The dose groups and study schedule for the M. abscessus in vivo study are summarized in Table 9 and FIG.

[0202] [Table 11]

[0203]

[0230] Results of the M. abscessus chronic pulmonary infection study

[0231] BisEDT administration was well tolerated by the mice, who responded and were within normal weight range at the end of treatment. Data for CFU recovery from lung and spleen homogenates are provided in Figures 8A and 8B, respectively. Using a Kruskal-Wallis test, lung CFU data showed a dose-dependent response to BisEDT as well as a statistically significant difference between the high dose group and vehicle control (p-value = 0.0163).

[0204]

[0232] As shown in FIG. 8A, administration of 200 μg / kg BisEDT (low) to mice infected with M. abscessus provided a bacteriostatic effect, as indicated by a reduction in CFU counts in lung homogenates. BisEDT is bactericidal at 1000 μg / kg (high) delivered to the lungs of mice infected with M. abscessus. In the macrophage system, it was also highly active (bactericidal) against all three clinical strains tested. Furthermore, activity against M. abscessus in this mouse model suggests that BisEDT is active against M. abscessus in the biofilm phenotype.

[0205]

[0233] histopathology

[0234] After 28 days of inhalation exposure to either vehicle or BisEDT aerosol atmosphere as described above, lungs and spleens were removed from vehicle- and antibiotic-treated mice. Mice for histology and lung tissue samples were prospectively selected. Each was split into their respective lung pairs. One half was fixed in formalin pending further histological processing, and the other half was weighed and frozen pending bioanalysis for quantification of BisEDT in lung tissue. Spleens were also fixed in formalin prior to further processing.

[0206]

[0235] Samples were processed for histological diagnosis: slides of tissue sections were prepared and stained with hematoxylin-eosin (HE stain) for general pathology, but in each case duplicate slides were prepared for acid-fast staining to highlight mycobacterial infection in the lung tissue.

[0207]

[0236] Example 7: Evaluation of resistance to BisEDT in mouse lung homogenates

[0237] Since BisEDT demonstrated in vivo efficacy against M. abscessus, studies were performed to investigate the propensity of M. abscessus to develop resistance to the drug. The first study attempted to show whether bacteria surviving the inhalation treatment developed resistance: bacteria from treated lung homogenates were exposed to MIC concentrations and growth was assessed. Some samples appeared to show growth at previously determined MIC levels, typically indicating the development of resistance after treatment. However, in this case, a confirmatory MIC study showed that the MIC of the bacteria had not changed significantly and that M. abscessus had not developed resistance during the course of treatment. The second study attempted to determine the concentration of bacteria required for natural resistance to emerge at a bactericidal concentration of BisEDT.

[0208]

[0238] Lung homogenates from M. abscessus mouse infections were thawed from freezing for 2 hours at 37°C. To reduce the effect of the Dey-Engley inactivation buffer, mouse homogenates were diluted 1:10 in water before samples were replated onto 7H10 agar plates containing 1ug / ml BisEDT (MIC level). Once plated, samples were incubated at 37°C for 3 days and plate colonies were counted to determine resistant CFU.

[0209]

[0239] Colony forming units were compared from initial plating without BisEDT in the agar plate to replating on agar plates containing 1 μg / mL BisEDT. The percent resistance determined is provided in Table 10.

[0210] [Table 12]

[0211]

[0240] Colony forming units were compared from initial plating without BisEDT in the agar plate to replating on agar plates containing 1 μg / mL BisEDT. The ratios are expressed as percentages in Table 10 above and appeared to indicate the development of resistance to BisEDT.

[0212]

[0241] To ensure that the observable colonies were truly resistant to BisEDT and not simply the result of artifacts of the distribution of BisEDT on the inactivated medium or agar plates still present in the lung homogenates, randomly selected isolates were grown on 7H10 from multiple mice to reach confluency, and then standard MIC analysis was performed as described above. As shown in Table 11, the MIC level from this study was determined to be 0.5 μg / mL. The previous MIC level of BisEDT was determined to be 1 μg / mL. Generally, a difference of at least 2 MIC levels must be observed to be considered a significant change in resistance. Therefore, this difference is not considered a change in resistance. The MBC levels of these selected isolates also did not indicate a change in resistance.

[0213]

[0242] Conclusion: There is little evidence of resistance development in a long-term M. abscessus study among mice treated with BisEDT for 28 days.

[0214] [Table 13]

[0215]

[0243] Example 8. Assessment of the emergence of resistance in M. abscessus to BisEDT treatment

[0244] The emergence of resistance is determined by using a bactericidal concentration of BisEDT and different amounts of M. abscessus bacteria. This procedure is used to determine the spontaneous emergence of resistance in bacterial suspensions.

[0216]

[0245] M. abscessus was grown to logarithmic phase on 7H10 agar at 37°C for 3 days. 11A bacterial suspension of 100 μL was made in PBS-T (phosphate buffered saline containing 0.05% tween 20). Ten tubes of 7H9 broth with 4ug / mL BisEDT or 7H9 broth alone (900 μL each) were made, and 100 μL of the high bacterial suspension was added to one tube and then serially diluted (1:10) into each of the other tubes. After adding the bacterial suspension and mixing thoroughly, the tubes were incubated at 37°C in a shaking incubator at 200 rpm for 3 days to allow the antibiotic to affect the bacteria. The level of bacterial growth was determined from the optical density by taking 100 mL of the bacterial suspension and placing it in a flat-bottom 96-well plate for reading on an Epoch spectrophotometer at 595 nm.

[0217] [Table 14]

[0218]

[0246] result

[0247] At the MBC or bactericidal concentration of BisEDT (4 μg / mL), MABs were 10 10 The bacteria were finally able to overcome the antibiotic (Table 12). This bacterial growth is comparable to the same amount of bacteria grown in 7H9 broth alone without antibiotics. MAB grew readily in 7H9 broth, as reflected by the optical density shown above.

[0219]

[0248] Conclusion: Incubation in the presence of a bactericidal concentration of BisEDT (4 μg / mL) abolishes natural resistance to the compound, even at bacterial inocula of 10 10 This means that M. abscessus requires high concentrations of the bacteria to become spontaneously resistant to this level of BisEDT.

[0220] Incorporation by Reference

[0249] All publications and patents mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0221] Equivalent

[0250] While specific embodiments of the subject disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the present disclosure will be apparent to those of skill in the art upon review of this specification and the following claims. The full scope of the present disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

1. A composition for use in treating an infection in a subject caused by nontuberculous mycobacteria (NTM), the composition comprising a therapeutically effective amount of a bismuth-thiol (BT) compound.

2. The composition for use according to claim 1, wherein the infection is a pulmonary infection.

3. The composition for use according to claim 1, wherein the infection is an extrapulmonary infection.

4. The composition for use according to any one of claims 1 to 3, wherein the NTM infection is caused by an antibiotic-resistant strain of NTM.

5. The NTM infection may be caused by any of the following: M. avium, M. avium subsp. hominissuis (MAH), M. abscessus, M. chelonae, M. bolletii, M. kansasii, M. ulcerans, M. avium complex (MAC) (M. avium and M. intracellulare), M. conspicuum, M. kansasii, M. peregrinum, M. immunogenum, M. xenopi, M. M. marinum, M. malmoense, M. marinum, M. mucogenicum, M. nonchromogenicum, M. scrofulaceum, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. terrae complex, M. haemophilum, M. genavense, M. asiaticum, M. shimoidei, M. gordonae, M.

2. The composition for use of claim 1, wherein the fungus is caused by M. nonchromogenicum, M. triplex, M. lentiflavum, M. celatum, M. fortuitum, M. fortuitum complex (M. fortuitum and M. chelonae), or a combination thereof.

6. 10. The composition for use of claim 1, wherein the NTM infection is caused by M. abscessus, M. avium, or a combination thereof.

7. The NTM lung infection is caused by the M. avium complex (M. avium and M. intracellulare).

2. The composition for use according to claim 1 .

8. 8. The composition for use according to any one of claims 5 to 7, wherein the M. avium is M. avium subsp. hominissuis.

9. The composition for use according to any one of claims 1 to 3, wherein the NTM infection is a biofilm-associated NTM infection.

10. 3. The composition for use according to claim 1 or 2, wherein the NTM infection is a chronic pulmonary infection.

11. 2. The composition for use of claim 1, wherein the NTM infection is located in or on the lung mucosa, bronchi, alveoli, macrophages, and / or bronchioles.

12. 2. The composition for use of claim 1, wherein the NTM infection is located at least in part in macrophages.

13. The composition for use according to claim 11, wherein the macrophages are THP-1 macrophages.

14. The composition for use according to any one of claims 11 to 13, wherein upon administration of the BT composition to the subject, the bacterial load in the macrophages is reduced.

15. 14. The composition for use according to any one of claims 11 to 13, wherein the macrophages are infected with one or more strains of M. abscessus and / or M. avium.

16. The composition for use according to any one of claims 1 to 3, wherein the NTM infection is resistant to treatment with amikacin.

17. The composition for use according to any one of claims 1 to 3, wherein the NTM infection is resistant to macrolide or azalide therapy.

18. The composition for use according to any one of claims 1 to 3, wherein the subject has a chronic pulmonary condition.

19. 19. The composition for use according to claim 18, wherein the chronic pulmonary condition is cystic fibrosis, chronic bronchitis, emphysema, bronchiectasis, pulmonary fibrosis, asbestosis, pneumonitis, chronic obstructive pulmonary disease (COPD), or asthma.

20. 19. The composition for use according to claim 18, wherein the chronic pulmonary condition is cystic fibrosis.

21. The composition for use according to any one of claims 1 to 3, wherein the subject is administered about 30 μg to about 3,000 μg of the BT compound per administration.

22. 22. The composition for use according to claim 21, wherein the subject is administered about 100 μg to about 1,000 μg of the BT compound per administration.

23. 4. The composition for use according to any one of claims 1 to 3, wherein the BT composition is administered to the subject once per month, twice per month, three times per month, four times per month, once every two weeks, once per week, twice per week, or three times per week.

24. The composition for use according to any one of claims 1 to 3, wherein the BT composition is administered to the subject once or twice a day.

25. The composition for use according to any one of claims 1 to 3, wherein the BT compound is administered to the lungs of the subject.

26. The composition for use according to any one of claims 1 to 3, wherein the BT compound is administered by inhalation.

27. 4. The composition for use according to claim 1, wherein the BT composition is administered to the subject for a period of less than 24 months, less than 18 months, less than 12 months, less than 9 months, less than 6 months, less than 3 months, or less than 1 month.

28. The composition for use according to any one of claims 1 to 3, wherein the BT composition is administered to the subject for a period of between 1 day and 56 days.

29. The composition for use according to any one of claims 1 to 3, wherein the BT composition is administered to the subject for a period of 14 to 28 days.

30. 26. The composition for use according to claim 25, wherein the concentration of bismuth in the lung after a single daily dose is from about 0.03 μg / g lung tissue to about 3 μg / g lung tissue.

31. 26. The composition for use according to claim 25, wherein the concentration of bismuth in the lung after 28 daily doses is from about 0.3 μg / g lung tissue to about 60 μg / g lung tissue.

32. The BT compound is BisBAL, BisEDT, Bis-dimercaprol, BisDTT, Bis-2-mercaptoethanol, BisDTE, BisPyr, BisEry, BisTol, BisBDT, BisPDT, BisPyr / BAL, BisPyr / BDT, BisPyr / EDT, BisPyr / PDT, BisPyr / Tol, BisPyr 4. The composition for use according to any one of claims 1 to 3, wherein the hydroxybenzoate is selected from BisEDT / CSTMN (1:1), BisPyr / CSTMN (1:1), BisBAL / CSTMN (1:1), BisTOL / CSTMN (1:1), and BisEDT / 2-hydroxy-1-propanethiol.

33. 33. The composition for use according to claim 32, wherein the BT compound is selected from BisEDT, BisBAL, BisPyr, BisEry, BisTol, BisBDT, or BisEDT / 2-hydroxy-1-propanethiol.

34. 33. The composition for use according to claim 32, wherein the BT compound is BisEDT or BisBAL.

35. 33. The composition for use according to claim 32, wherein the BT compound is BisEDT.

36. 33. The composition for use of claim 32, further comprising administering to the subject in need thereof an effective amount of amikacin, clarithromycin, azithromycin, ethambutol, rifampicin, tigecycline, linezolid, imipenem, cefoxitin, or a combination thereof.

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