Combination therapy for treating nontuberculous mycobacterial lung disease

Abstract

Provided herein is a method of treating NTM pulmonary disease in a patient in need thereof. The method includes administering to the patient (i) a liposomal amikacin composition and (ii) a second active agent that is synergistic with amikacin for NTM during a dosing period. The liposomal amikacin composition includes amikacin or a pharma- ceutically acceptable salt thereof encapsulated in a plurality of liposomes, the lipid component of the liposomes being composed of one or more electrically neutral lipids. Administering the liposomal amikacin composition includes aerosolizing the composition to provide an aerosolized composition comprising a mixture of free amikacin and liposomally complexed amikacin, and administering the aerosolized composition to the patient's lungs via a nebulizer. The second active agent is administered to the patient orally, parenterally, or via inhalation, and can be administered at the same or different dosing interval as the liposomal amikacin composition.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 165,418, filed March 24, 2021, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] Nontuberculous mycobacterial (NTM) pulmonary infections in susceptible hosts can result in severe morbidity and ultimately death in some affected individuals. Due to rising infection rates, NTM pulmonary disease has become an emerging public health concern in the United States. NTM are ubiquitous in the environment. More than 80% of NTM pulmonary disease in the United States is due to Mycobacterium avium complex (MAC). In addition, M. kansasii, M. abscessus, and M. fortuitum are routinely isolated in subjects diagnosed with NTM pulmonary disease.

[0003] NTM lung disease, caused by Mycobacterium avium complex (MAC), is a potentially life-threatening progressive disease associated with symptoms of productive cough, fatigue, shortness of breath, fever, weight loss, decreased lung function, and hemoptysis. This NTM lung disease is often associated with other chronic debilitating underlying lung diseases such as bronchiectasis or COPD. When NTM lung disease occurs in patients without underlying pulmonary comorbidities, it is associated with progressive lung disease.

[0004] The prevalence of NTM lung disease in the United States has more than doubled in the last 15 years. The American Thoracic Society (ATS) / Infectious Disease Society of America (IDSA) reported a 2-year prevalence of pulmonary NTM infection of 8.6 / 100,000. The prevalence of pulmonary NTM infection increases with age to 20.4 per 100,000 in people at least 50 years of age, and is more prevalent in women (median age: 66 years, female: 59%).

[0005] Arikayce® (amikacin liposomal inhalation suspension or ALIS) is the first FDA-approved treatment for NTM lung disease. Specifically, ALIS is an aminoglycoside antibiotic indicated for the treatment of Mycobacterium avium complex (MAC) lung disease as part of a combination antibiotic regimen in adult patients with limited or no alternative treatment options and who have not obtained negative sputum cultures after at least 6 consecutive months of multidrug background therapy.

[0006] Although approved therapies exist for NTM lung disease, and other antibiotics have been used historically to treat NTM lung disease, available therapies can be poorly tolerated and can have significant adverse events. The present invention addresses this and other needs by providing a method of treating NTM lung disease in patients in need thereof. Summary of the Invention

[0007] Provided herein is a method of treating NTM pulmonary disease in a patient in need thereof. In one embodiment, the method includes administering to the patient (i) a liposomal amikacin composition and (ii) a second active agent that is synergistic with amikacin for NTM during an administration period. The liposomal amikacin composition includes amikacin or a pharma- ceutically acceptable salt thereof encapsulated in a plurality of liposomes, the lipid component of the liposomes being composed of one or more electrically neutral lipids. Administering the liposomal amikacin composition includes aerosolizing the composition to provide an aerosolized composition comprising a mixture of free amikacin and liposomally complexed amikacin, and administering the aerosolized composition to the patient's lungs via a nebulizer. The second active agent is administered to the patient orally, parenterally, or via inhalation, and can be administered at the same or different dosing interval as the liposomal amikacin composition.

[0008] In one embodiment, synergy is assessed by the Fractional Inhibitory Concentration Index (FICI) value.

[0009] In one embodiment, the second active agent is a carbapenem. In a further embodiment, the second active agent is imipenem, doripenem, biapenem, or tebipenem.

[0010] In yet another embodiment, the second active agent is rifabutin, rifampin, RV40, clofazimine, bedaquiline, or cefdinir.

[0011] In another embodiment, the second active agent is imipenem, rifabutin, rifampin, RV40, clofazimine, bedaquiline, cefdinir, doripenem, biapenem, or tebipenem.

[0012] The liposomal amikacin composition and the second active agent are administered to a patient in need of treatment during an administration period. The administration period is measured from the time (T1) when the patient is administered both the liposomal amikacin composition and the second active agent to the time (T2) when the patient is no longer administered both the liposomal amikacin composition and the second active agent. During the administration period, the liposomal amikacin composition and the second active agent do not have to be administered via the same route or via the same administration schedule for the same amount of time.

[0013] The second active agent, i.e., the active agent that is synergistic with amikacin for NTM, can be administered to the patient orally, parenterally, or locally via inhalation.For example, in one embodiment, the second active agent is administered orally.In another embodiment, the second active agent is administered parenterally.In a further embodiment, the second active agent is administered intravenously.In yet another embodiment, the second active agent is administered via inhalation, for example, via a nebulizer, a metered dose inhaler (MDI) or a dry powder inhaler (DPI).

[0014] The NTM lung disease, in one embodiment, is caused by Mycobacterium avium, Mycobacterium abscessus, or Mycobacterium avium complex (MAC) (Mycobacterium avium and Mycobacterium intracellulare) lung infection. In embodiments where the NTM lung disease is caused by Mycobacterium avium lung infection, the Mycobacterium avium can be Mycobacterium subsp. hominissuis (MAH). In one embodiment, the NTM lung disease is caused by Mycobacterium avium complex (MAC) (Mycobacterium avium and Mycobacterium intracellulare) lung infection. In one embodiment, the pulmonary NTM infection is a pulmonary recalcitrant NTM infection.

[0015] In one embodiment, the NTM pulmonary disease treated by the methods provided herein is caused by Mycobacterium abscessus or Mycobacterium avium complex. In one or more of the foregoing embodiments, the patient is a cystic fibrosis (CF) patient, a bronchiectasis patient, an asthma patient, or a COPD patient.

[0016] In one embodiment, the liposomal amikacin composition is a dispersion (i.e., a suspension) of liposomes. The liposomal portion of the composition includes a lipid component that includes one or more electrically neutral lipids. In a further embodiment, the electrically neutral lipids include phosphatidylcholine and a sterol (e.g., dipalmitoylphosphatidylcholine (DPPC) and cholesterol). In one embodiment, the amikacin in the liposomal amikacin composition is amikacin sulfate.

[0017] In one embodiment, a method of treating NTM disease includes, in part, aerosolizing a liposomal amikacin composition to obtain an aerosolized composition, and administering the aerosolized composition to the lungs of a patient in need of treatment, the aerosolized pharmaceutical composition comprising a mixture of free amikacin and liposomally complexed amikacin. In a further embodiment, the lipid component of the liposomal amikacin composition comprises phosphatidylcholine and a sterol (e.g., DPPC and cholesterol). In a further embodiment, the amikacin is present as a pharma- ceutically acceptable salt. In yet a further embodiment, the amikacin is amikacin sulfate.

[0018] The methods provided herein, in one embodiment, include achieving a negative NTM culture following administration of a liposomal amikacin composition and a second active agent. In one embodiment, the methods provided herein include achieving a negative conversion of NTM culture following a period of administration. A patient subject to the methods provided herein, in one embodiment, achieves a negative NTM sputum culture sooner than a patient administered either the liposomal amikacin composition or the second active agent alone. In one embodiment, a patient subject to the methods provided herein achieves a culture conversion, i.e., three consecutive negative NTM cultures, prior to a patient administered either the liposomal amikacin composition or the second active agent alone, with both patients having sputum cultures at the same time points.

[0019] In yet another embodiment of the invention provided herein, during or after the administration period, the patient exhibits an improvement in one or more respiratory symptoms as measured by QOL-B Respiratory domain score compared to the patient's one or more respiratory symptoms prior to the administration period. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 is a schematic diagram of one method for performing a checkerboard minimum inhibitory concentration (MIC) assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Nontuberculous mycobacteria are organisms found in soil and water that can cause severe lung disease in susceptible individuals, for which there are currently limited and no approved treatments. The prevalence of NTM disease is reported to be increasing and is believed to be higher than that of tuberculosis in the United States, according to a report from the American Thoracic Society. According to the National Center for Biotechnology Information, epidemiological studies indicate that NTM infections are increasing in developing countries, possibly due to the use of untreated water. Women with characteristic phenotypes are thought to be at higher risk for NTM infection, as are patients with defects in the cystic fibrosis transmembrane conductance regulator. In general, groups with NTM lung disease at higher risk for progressive morbidity and increased mortality are those with cavitary lesions, lower BMI, older age, and higher dependency index.

[0022] NTM lung disease is often a chronic condition that can lead to progressive inflammation and lung damage and is characterized by bronchiectasis and cavitary disease. NTM infections often require prolonged hospitalization for medical management. Treatment usually requires multi-drug regimens that may be poorly tolerated and of limited effectiveness, especially in severely ill patients or those who have failed prior treatment attempts. According to a company-sponsored patient chart review conducted by Clarity Pharma Research, approximately 50,000 patients with NTM lung disease were seen in clinics in the United States during 2011.

[0023] Management of NTM lung disease caused by nontuberculous mycobacterial infections involves long-term multidrug regimens that are often associated with drug toxicity and suboptimal outcomes. Achieving negative NTM cultures is one of the goals of treatment and represents the most clinically relevant microbiological endpoint in patients with NTM lung infections.

[0024] The invention described herein relates in part to a method of treating NTM pulmonary disease using a liposomal amikacin composition and a second active agent that is synergistic with amikacin.Without intending to be bound by theory, it is believed that the synergistic combination of anti-infective agents provides greater efficacy in certain NTM pulmonary disease treatment methods described herein compared to the use of liposomal amikacin compositions.Furthermore, without intending to be bound by theory, it is believed that the combination therapy approach retards antimicrobial resistance, thereby providing a more effective treatment option than the use of one anti-infective agent alone or a combination of anti-infective agents that is not synergistic.

[0025] In one aspect, the invention provides a method of treating nontuberculous mycobacterial (NTM) pulmonary disease in a patient in need thereof. The method, in one embodiment, comprises administering to the patient during an administration period (i) a liposomal amikacin composition comprising amikacin or a pharma- ceutically acceptable salt thereof encapsulated in a plurality of liposomes, wherein the lipid component of the plurality of liposomes is comprised of one or more electrically neutral lipids, and (ii) a second active agent that is synergistic with amikacin.

[0026] In one embodiment, the one or more neutral lipids in the liposomal amikacin composition comprise a phospholipid and a sterol. In a further embodiment, the phospholipid is a phosphatidylcholine. In a further embodiment, the phosphatidylcholine is dipalmitoylphosphatidylcholine (DPPC). In a further embodiment, the sterol is cholesterol. In one embodiment, the second active agent that is synergistic with amikacin is imipenem, rifabutin, rifampin, RV40, clofazimine, levofloxacin, moxifloxacin, bedaquiline, cefdinir, doripenem, biapenem, tebipenem, ethambutol, or tetrandrine. In another embodiment, the second active agent that is synergistic with amikacin is imipenem, rifabutin, rifampin, RV40, clofazimine, bedaquiline, cefdinir, doripenem, biapenem, or tebipenem. In another embodiment, the second active agent that is synergistic with amikacin is rifabutin, rifampin, RV40, clofazimine, bedaquiline, cefdinir.

[0027] The NTM pulmonary disease treated by the methods provided herein, in one embodiment, is caused by Mycobacterium avium (e.g., Mycobacterium subgenus avium hominisuis (MAH), Mycobacterium abscessus, Mycobacterium chelonae, or Mycobacterium avium complex (MAC) (Mycobacterium avium and Mycobacterium intracellulare)). In one embodiment, the NTM pulmonary disease is caused by Mycobacterium avium complex (MAC) (Mycobacterium avium and Mycobacterium intracellulare). In one embodiment, the NTM pulmonary infection is a refractory nontuberculous mycobacterial pulmonary infection.

[0028] In one embodiment, the NTM lung disease is caused by Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium avium or Mycobacterium avium complex (MAC) lung infection. In one embodiment, a patient in need of treatment is administered the liposomal amikacin composition via inhalation and the second active agent is administered orally, intravenously or via inhalation. In one embodiment, the liposomal amikacin composition is administered once daily in a single administration session. In yet a further embodiment, the NTM lung disease is caused by Mycobacterium avium complex.

[0029] The NTM lung disease, in one embodiment, is associated with cavitary lesions. In one embodiment, the NTM lung disease is nodular NTM lung disease. In one embodiment, the NTM lung disease is nodular with cavitary lesions.

[0030] In one embodiment, the NTM lung disease is caused by Mycobacterium abscessus or Mycobacterium avium complex (MAC) lung infection. In one embodiment, the NTM lung disease is caused by a refractory nontuberculous mycobacterial lung infection.

[0031] In an embodiment of the method for treating NTM lung disease described herein, the liposomal amikacin composition and the second active agent are administered to a patient in need thereof during an administration period. The administration period is measured from the time (T1) when the patient is administered both the liposomal amikacin composition and the second active agent to the time (T2) when the patient is no longer administered both the liposomal amikacin composition and the second active agent. During the administration period, the liposomal amikacin composition and the second active agent do not need to be administered at the same time, at the same administration interval, or for the same duration.

[0032] During the administration period, the liposomal amikacin composition and the second active agent do not have to be administered via the same route or via the same administration schedule for the same time. For example, treatment with the liposomal amikacin composition and the second active agent can begin at the same time point (T1), and administration of the second active agent can be discontinued before administration of the liposomal amikacin is discontinued. In this scenario, the administration period is measured from T1 until administration of the liposomal amikacin composition is discontinued (T2). In another embodiment, administration of the liposomal amikacin composition and the second active agent begins at the same time point (T1) and ends at the same time point (T2).

[0033] In one embodiment, the administration period is at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, or at least 24 months. In another embodiment, the administration period is from about 6 months to about 24 months, or from about 6 months to about 18 months, or from about 6 months to about 12 months.

[0034] In one embodiment, the administration period is about 30 days to about 400 days, for example, about 45 days to about 300 days, or about 45 days to about 270 days, or about 80 days to about 200 days. In another embodiment, the administration period is about 80 days to about 400 days, for example, about 90 days to about 400 days, or about 100 days to about 400 days. In another embodiment, the administration period is about 100 days to about 500 days.

[0035] The term "treating" includes (1) preventing or delaying the appearance of clinical symptoms of a condition, disorder, or condition in a subject who may be afflicted or affected by the condition, disorder, or condition, but who has not yet experienced or exhibited clinical or subclinical symptoms of the condition, disorder, or condition; (2) inhibiting the condition, disorder, or condition (i.e., halting, reducing, or delaying the onset of the disease with respect to at least one of its clinical or subclinical symptoms, or, in the case of maintenance treatment, halting, reducing, or delaying its recurrence); and / or (3) alleviating the condition (i.e., causing a reduction in the condition, disorder, or condition, or at least one of its clinical or subclinical symptoms). The benefit to a subject being treated is statistically significant or at least perceptible to the subject or to the physician.

[0036] Treatment includes a therapeutic response that a user (e.g., a clinician) recognizes as an effective response to the combination therapy. In one embodiment, the therapeutic response is a reduction, inhibition, delay, or prevention of the growth or proliferation of one or more NTM, or the death of one or more NTM. In one embodiment, during or after the administration period, the patient achieves a negative NTM culture. The NTM culture, in one embodiment, is prepared from a sputum sample obtained from the patient. In another embodiment, the patient achieves a negative culture conversion of NTM during or after the administration period. As provided herein, converting a culture to negative refers to three consecutive negative NTM cultures. The three consecutive cultures can be prepared from patient samples obtained at spaced intervals, for example, at biweekly or monthly intervals.

[0037] By "effective amount" is meant an amount of the liposomal amikacin composition and second synergistically active agent used in the present invention sufficient to result in the desired therapeutic response.

[0038] As used herein, the term "about" refers to plus or minus 10% of the object that "about" modifies.

[0039] As described throughout, the methods and compositions described herein are used to treat NTM lung disease and include a combination of a liposomal amikacin composition and a second active agent. The second active agent, i.e., an active agent that is synergistic with amikacin for NTM, can be administered to the patient orally, parenterally, or locally via inhalation. Thus, the second active agent can be administered via the same route (inhalation) or a different route compared to the liposomal amikacin composition. When administered via inhalation, in one embodiment, the second active agent can be in the same composition as the liposomal amikacin composition, or a different composition. In one embodiment, the second active agent is administered orally. In another embodiment, the second active agent is administered parenterally. In a further embodiment, the second active agent is administered intravenously. In yet another embodiment, the second active agent is administered via inhalation, for example, via a nebulizer or dry powder inhaler (DPI).

[0040] In one embodiment, the second active agent is a carbapenem. In a further embodiment, the second active agent is imipenem, doripenem, biapenem, or tebipenem.

[0041] In yet another embodiment, the second active agent is rifabutin, rifampin, RV40, clofazimine, levofloxacin, moxifloxacin, bedaquiline, cefdinir, ethambutol, or tetrandrine. In a further embodiment, the second active agent is rifabutin, rifampin, RV40, clofazimine, bedaquiline, or cefdinir.

[0042] In another embodiment, the second active agent is imipenem, rifabutin, rifampin, RV40, clofazimine, levofloxacin, moxifloxacin, bedaquiline, cefdinir, doripenem, biapenem, tebipenem, ethambutol, or tetrandrine. In a further embodiment, the second active agent is imipenem, rifabutin, rifampin, RV40, clofazimine, bedaquiline, cefdinir, doripenem, biapenem, or tebipenem.

[0043] In one embodiment, amikacin is present in the liposomal amikacin composition as amikacin base, or a pharma- ceutically acceptable salt of amikacin, such as amikacin sulfate or diamikacin sulfate.

[0044] "Pharmaceutically acceptable salts" includes both acid and base addition salts. In one embodiment, pharma-ceutically acceptable salts are those acid addition salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable. Pharmaceutically acceptable acid addition salts may be formed with inorganic acids, such as, but not limited to, hydrochloric acid (HCl), hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or organic acids, such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutaric acid, glycer ... Examples of suitable carboxylic acids include nic acid, glutamic acid, 2-oxo-glutamic acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid (e.g., as the lactate salt), lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, acetic acid (e.g., as the acetate salt), tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid (TFA), and undecylenic acid. In one embodiment, the pharma- ceutically acceptable salt is HCl, TFA, lactate, or acetate, hi another embodiment, the pharma- ceutically acceptable salt is a sulfate.

[0045] The liposomal amikacin compositions provided herein include (i) amikacin or a pharma- ceutically acceptable salt thereof encapsulated in a plurality of liposomes, (ii) amikacin or a pharma- ceutically acceptable salt thereof complexed to the lipid bilayer or surface of a plurality of liposomes, or (iii) a combination thereof. "Liposome-complexed amikacin" includes embodiments in which (i) amikacin is encapsulated in a liposome, (ii) amikacin is associated with the liposome bilayer via covalent or non-covalent bonds, (iii) amikacin is present in the aqueous or hydrophobic bilayer phase or in the interfacial headgroup region of the liposome bilayer of the liposome, or (iv) any combination of the foregoing. In embodiments provided herein, substantially all of the amikacin in the liposomal amikacin composition is complexed to liposomes. For example, ≧95%, ≧96%, ≧97%, or ≧98% of the amikacin is complexed to liposomes prior to administration of the composition.

[0046] The methods provided herein include, in part, administering via inhalation to a patient in need thereof a composition comprising amikacin or a pharma- ceutically acceptable salt thereof encapsulated in a plurality of liposomes. In one embodiment, the lipid components of the plurality of liposomes comprise one or more electrically neutral lipids. In yet a further embodiment, the electrically neutral lipid comprises a sterol and a phospholipid. In yet a further embodiment, the sterol is cholesterol and the phospholipid is a net neutral phosphatidylcholine. In a further embodiment, the phosphatidylcholine is dipalmitoylphosphatidylcholine (DPPC).

[0047] The lipid component of the plurality of liposomes may comprise one or more synthetic, semi-synthetic, or naturally derived lipids, including phospholipids, tocopherols, sterols, fatty acids, or combinations thereof. In one embodiment, the lipid component of the plurality of liposomes is comprised of electrically neutral lipids. In a further embodiment, the lipid component comprises DPPC and cholesterol.

[0048] In one embodiment, at least one phospholipid is present in the lipid components of the plurality of liposomes. In one embodiment, the phospholipid is electrically neutral. In one embodiment, the phospholipid is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidic acid (PA), one of the soybean counterparts described above, or one of the hydrogenated egg and soybean counterparts described above (e.g., hydrogenated egg PC, hydrogenated egg PC).

[0049] In one embodiment, the lipid components of the plurality of liposomes include dipalmitoylphosphatidylcholine (DPPC), a major constituent of natural pulmonary surfactant. In one embodiment, the lipid components of the plurality of liposomes include, or consist essentially of, DPPC and cholesterol, or consist of DPPC and cholesterol. In a further embodiment, the DPPC and cholesterol have a molar ratio ranging from about 19:1 (DPPC:cholesterol) to about 1:1 (DPPC:cholesterol), or from about 9:1 (DPPC:cholesterol) to about 1:1 (DPPC:cholesterol), or from about 4:1 (DPPC:cholesterol) to about 1:1 (DPPC:cholesterol), or from about 2:1 (DPPC:cholesterol) to about 1:1 (DPPC:cholesterol). In yet a further embodiment, the DPPC and cholesterol have a molar ratio of about 2:1 (DPPC:cholesterol), about 1.5:1 (DPPC:cholesterol), or about 1:1 (DPPC:cholesterol). In yet a further embodiment, the DPPC and cholesterol have a molar ratio of about 2:1 (DPPC:cholesterol).

[0050] In still further embodiments, the DPPC and cholesterol have a weight ratio of about 2:1 (DPPC:cholesterol), about 1.5:1 (DPPC:cholesterol), or about 1:1 (DPPC:cholesterol). In yet further embodiments, the DPPC and cholesterol have a weight ratio of about 2:1 (DPPC:cholesterol).

[0051] Other examples of lipids for use with the methods and compositions described herein include, but are not limited to, dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidecholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), disteroylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidyl-ethanolamine (DOPE), mixed phospholipids such as palmitoylstearoylphosphatidylcholine (PSPC), single acylated phospholipids such as mono-oleoyl-phosphatidylethanolamine (MOPE).

[0052] In one embodiment, the lipid component of the plurality of liposomes comprises a sterol. In a further embodiment, at least one lipid component comprises, or consists essentially of, a sterol and a phospholipid, or consists of a sterol and a phospholipid (e.g., a neutral phosphatidylcholine such as DPPC). Sterols for use in the present invention include, but are not limited to, cholesterol, esters of cholesterol, including cholesterol hemisuccinate, salts of cholesterol, including cholesterol hydrogen sulfate and cholesterol sulfate, ergosterol, esters of ergosterol, including ergosterol hemisuccinate, salts of ergosterol, including ergosterol hydrogen sulfate and ergosterol sulfate, lanosterol, esters of lanosterol, including lanosterol hemisuccinate, salts of lanosterol, including lanosterol hydrogen sulfate, lanosterol sulfate, and tocopherol. Tocopherols may include tocopherol, esters of tocopherol (including tocopherol hemisuccinate), salts of tocopherol (including tocopherol hydrogen sulfate and tocopherol sulfate). The term "sterol compound" includes sterols, tocopherols, and the like.

[0053] In one embodiment, at least one cationic lipid is provided in the lipid component of a plurality of liposomes of liposome-complexed amikacin. Cationic lipids that may be modified for use in the present invention include, but are not limited to, ammonium salts of fatty acids, phospholipids, and glycerides. Fatty acids include fatty acids with carbon chain lengths of 12 to 26 carbon atoms, either saturated or unsaturated. Some specific examples include, but are not limited to, myristylamine, palmitylamine, laurylamine, and stearylamine, dilauroylethylphosphocholine (DLEP), dimyristoylethylphosphocholine (DMEP), dipalmitoylethylphosphocholine (DPEP), and distearoylethylphosphocholine (DSEP), N-(2,3-di-(9-(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP), and combinations thereof.

[0054] In one embodiment, the lipid components of the liposomes present in the liposomal amikacin composition described herein are provided with at least one anionic lipid (negatively charged lipid) for use in a method of treating NTM pulmonary infection in a patient in need thereof. Negatively charged lipids that may be used include phosphatidyl-glycerol (PG), phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylserine (PS). Examples include, but are not limited to, DMPG, DPPG, DSPG, DMPA, DPPA, DSPA, DMPI, DPPI, DSPI, DMPS, DPPS, DSPS, and combinations thereof.

[0055] Without intending to be bound by theory, phosphatidylcholine such as DPPC aids in the uptake of amikacin by lung cells (e.g., alveolar macrophages) and helps to maintain amikacin in the lung. Negatively charged lipids such as PG, PA, PS and PI are believed to play a role in the sustained activity properties of inhaled compositions, as well as in the transport of compositions across the lung for systemic uptake (transcytosis), in addition to reducing particle aggregation. Without intending to be bound by theory, sterol compounds are believed to affect the release properties of compositions.

[0056] Liposomes are completely sealed lipid bilayers that contain an enclosed aqueous volume. Liposomes may be unilamellar vesicles (possessing one membrane bilayer) or multilamellar vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer), or a combination thereof. The bilayer is composed of two lipid monolayers with hydrophobic "tail" regions and hydrophilic "head" regions. The structure of the membrane bilayer is such that the hydrophobic (non-polar) "tails" of the lipid monolayers point towards the centre of the bilayer, while the hydrophilic "heads" point towards the aqueous phase.

[0057] In one embodiment, the weight ratio of lipid components to amikacin by weight in the liposomal amikacin composition (the weight ratio is also referred to herein as "lipid:amikacin" or "lipid to amikacin weight ratio") is 3:1 or less, 2.5:1.0 or less, 2:1 or less, 1.5:1 or less, 1:1 or less, or 0.75:1 or less. In one embodiment, the weight ratio of lipid to amikacin in the liposomal amikacin compositions provided herein is 0.7:1.0 or about 0.7:1.0 by weight. In another embodiment, the weight ratio of lipid to amikacin in the liposomal amikacin compositions provided herein is 0.75:1 (lipid:amikacin) or less (by weight). In one embodiment, the weight ratio of lipid to amikacin is about 0.10:1.0 to about 1.25:1.0, about 0.25:1.0 to about 1.25:1.0, about 0.50:1.0 to about 1.25:1.0, or about 0.6:1 to about 1.25:1.0. In another embodiment, the weight ratio of lipid to amikacin is about 0.60:1.0 (lipid:amikacin) to about 0.79:1.0 (lipid:amikacin).

[0058] In another embodiment, the lipid to amikacin weight ratio in the liposomal amikacin compositions provided herein is less than 3:1 (lipid:amikacin), less than 2.5:1.0 (lipid:amikacin), less than 2.0:1.0 (lipid:amikacin), less than 1.5:1.0 (lipid:amikacin), or less than 1.0:1.0 (lipid:amikacin). In yet another embodiment, the lipid to amikacin weight ratio is about 0.6:1.0 (lipid:amikacin) to about 0.8:1.0 (lipid:amikacin).

[0059] In one embodiment, the liposomal amikacin composition is amikacin liposomal inhalation suspension (ALIS), available under the trade name Arikayce®.

[0060] In one embodiment, the weight ratio of lipid to amikacin in the liposomal amikacin compositions provided herein is 0.7:1.0 (lipid:amikacin), about 0.7:1.0 (lipid:amikacin), about 0.5:1.0 (lipid:amikacin) to about 0.8:1.0 (lipid:amikacin), or about 0.6:1.0 (lipid:amikacin) to about 0.8:1.0 (lipid:amikacin). In a further embodiment, the liposomes provided herein are small enough to effectively penetrate bacterial biofilms. In one embodiment, the average diameter of a plurality of liposomes is about 150 nm to about 350 nm, or about 200 nm to about 400 nm, or about 250 nm to about 400 nm, or about 250 nm to about 300 nm, or about 200 nm to about 300 nm, as measured by light scattering. In still further embodiments, the average diameter of the plurality of liposomes is from about 260 nm to about 280 nm as measured by light scattering.

[0061] In one embodiment, the liposomal compositions described herein are produced by one of the methods described in U.S. Patent Application Publication No. 2013 / 0330400, or U.S. Patent No. 7,718,189, each of which is incorporated by reference in its entirety for all purposes. In another embodiment, the liposomal amikacin compositions are produced by one of the methods described in WO / 2019 / 213398, which is incorporated by reference in its entirety. In yet another embodiment, the liposomal amikacin compositions are produced by one of the methods described in WO / 2019 / 191627, which is incorporated by reference in its entirety. Other methods of producing liposomes are known in the art and can be used to produce the liposomal amikacin compositions herein. In one embodiment, one or more of the methods described in U.S. Patent Application Publication No. 2008 / 0089927, which is incorporated by reference in its entirety, are used herein to produce the liposomal amikacin compositions described herein.

[0062] In one embodiment, liposomes are produced by dissolving one or more lipids in an organic solvent to form a lipid solution, and amikacin coacervates are formed from mixing the lipid solution with an aqueous solution of amikacin. In a further embodiment, the organic solvent is ethanol. In yet a further embodiment, the lipid solution comprises a phospholipid and a sterol, e.g., DPPC and cholesterol.

[0063] In one embodiment, liposomes are made by sonication, extrusion, homogenization, swelling, electroforming, inverted emulsion, or reverse evaporation. The method of Bangham (J. Mol. Biol. (1965)) produces general multilamellar vesicles (MLVs). Lenk et al. (U.S. Pat. Nos. 4,522,803, 5,030,453, and 5,169,637), Fountain et al. (U.S. Pat. No. 4,588,578), and Cullis et al. (U.S. Pat. No. 4,975,282) disclose methods for producing multilamellar liposomes, which have substantially equal intermembrane solute distributions within each aqueous compartment. Paphadjopoulos et al. (U.S. Pat. No. 4,235,871) discloses the preparation of oligolamellar liposomes by reverse phase evaporation. Each method can be adapted for use with the present invention.

[0064] Unilamellar vesicles can be made from MLVs by a number of techniques, such as the extrusion methods of U.S. Patent Nos. 5,008,050 and 5,059,421. Sonication and homogenization can be used to produce smaller unilamellar liposomes from larger ones (see, e.g., Chapman et al., "Physical studies of phospholipids. X. The effect of sonication of aqueous dispersions of egg yolk lecithin," Biochim Biophys Acta. 163(2):255-61 (1968), incorporated herein by reference in its entirety for all purposes.

[0065] The liposome preparation of Bangham et al. (J. Mol. Biol. 13, 1965, pp. 238-252) involves suspending phospholipids in an organic solvent, which is then evaporated to dryness, leaving a phospholipid film on the reaction tube. An appropriate amount of aqueous phase is then added and mixed to "swell" to obtain liposomes. The resulting liposomes consist of multilamellar vesicles (MLVs), which are dispersed by mechanical means. This preparation provides the basis for the development of small sonicated unilamellar vesicles and large unilamellar vesicles described in Papahadjopoulos et al. (Biochim. Biophys. Acta. 135, 1967, pp. 624-638, the entire contents of which are incorporated by reference).

[0066] Techniques for making large unilamellar vesicles (LUVs), such as reverse phase evaporation, injection, and detergent dilution, can be used to prepare liposomes for use in the pharmaceutical compositions presented herein. A review of these and other methods for preparing liposomes can be found in the text Liposomes, Marc Ostro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1, which is incorporated herein by reference. Also see Szoka, Jr. et al. (Ann. Rev. Biophys. Bioeng. 9, 1980, p. 467), which is also incorporated herein by reference in its entirety for all purposes.

[0067] Other methods for preparing liposomes include forming reverse-phase evaporation vesicles (REV) (U.S. Pat. No. 4,235,871). Other types of liposomes that have been characterized as having substantially the same lamellar solute distribution can also be used. This type of liposome is termed stable plurilamellar vesicles (SPLV) and includes the single-phase vesicles defined in U.S. Pat. No. 4,522,803 and described in U.S. Pat. No. 4,588,578, and the frozen and thawed multilamellar vesicles (FATMLV) described above.

[0068] Liposomes are formed using various sterols and their water-soluble derivatives, such as cholesterol hemisuccinate; see, for example, U.S. Patent No. 4,721,612. Mayhew et al., PCT Publication No. WO 1985 / 00968, described a method for reducing the toxicity of drugs by encapsulating the drugs in liposomes containing alpha-tocopherol and certain derivatives thereof. Also, various tocopherols and their water-soluble derivatives can be used to form liposomes and to prepare liposomal amikacin as described herein. For example, the method disclosed in PCT Publication No. 1987 / 02219, the entirety of which is incorporated herein by reference, can be used.

[0069] The liposomal amikacin composition, in one embodiment prior to nebulization, comprises liposomes having an average diameter in the range of about 150 nm to about 400 nm, e.g., about 150 nm to about 350 nm, as measured by light scattering. In one embodiment, the average diameter of the liposomes in the composition is about 150 nm to about 300 nm, about 200 nm to about 300 nm, about 210 nm to about 290 nm, about 220 nm to about 280 nm, about 230 nm to about 280 nm, about 240 nm to about 280 nm, about 250 nm to about 280 nm, or about 260 nm to about 280 nm.

[0070] In the methods provided herein, a patient in need of NTM pulmonary disease treatment is co-administered (i) a liposomal amikacin composition via inhalation, e.g., nebulizer, and (ii) a second active agent that is synergistic with amikacin during the administration period. In one embodiment, the patient is administered about 450 mg, about 500 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, or about 610 mg of amikacin once daily during the administration period. In another embodiment, the amount of amikacin provided in the composition administered once daily to the patient in need of treatment is about 500 mg to about 600 mg, or about 500 mg to about 650 mg, or about 525 mg to about 625 mg, or about 550 mg to about 600 mg. In one embodiment, the amount of amikacin administered to the subject is about 560 mg and is provided in 8 mL of the composition. In one embodiment, the amount of amikacin administered to a subject is about 590 mg and is provided in 8 mL to 10 mL of a composition, e.g., 8 mL to 9 mL of a liposomal suspension in 1.5% NaCl. In one embodiment, the amount of amikacin in a liposomal amikacin composition administered to a patient once daily during an administration period is about 590 mg and is provided in 8 mL to 9 mL of a composition. The amount of amikacin provided in the composition is about 450 mg, about 500 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, or about 610 mg. In another embodiment, the amount of amikacin provided in the composition is about 500 mg to about 650 mg, or about 525 mg to about 625 mg, or about 550 mg to about 600 mg. In one embodiment, the amount of amikacin administered to a patient once per day during the administration period is about 590 mg and is provided in 8 mL to 9 mL of a composition for nebulization. In one embodiment, the amount of amikacin administered to a patient once per day during the administration period is about 590 mg and is provided in 8.2 mL to 8.6 mL of a composition for nebulization. In one embodiment, the liposomal amikacin composition is a 8.3 mL to 8.5 mL composition.

[0071] In one embodiment, the liposomal amikacin compositions provided herein comprise about 60 mg / mL to about 80 mg / mL amikacin, e.g., about 65 mg / mL to about 80 mg / mL amikacin, about 65 mg / mL to about 75 mg / mL amikacin. In one embodiment, the liposomal amikacin compositions provided herein comprise about 60 mg / mL amikacin, about 65 mg / mL amikacin, about 70 mg / mL amikacin, about 75 mg / mL amikacin, about 80 mg / mL amikacin, about 85 mg / mL amikacin, or about 90 mg / mL amikacin. In a further embodiment, the amikacin is amikacin sulfate.

[0072] The liposomal amikacin composition is administered as an aerosol via nebulization to a patient in need of treatment. In one embodiment, the liposomal amikacin composition is administered once daily in a single administration session for the administration period. In another embodiment, the method comprises administering the liposomal amikacin composition to a patient in need thereof every other day or every third day for the administration period. In yet another embodiment, the method comprises administering the liposomal amikacin composition to a patient in need thereof twice daily for the administration period.

[0073] During the administration period, in one embodiment, a patient in need of NTM pulmonary disease treatment is administered a liposomal amikacin composition via nebulization, where about 500 mg to about 1000 mg of amikacin is administered daily in a single administration session, e.g., about 500 mg of amikacin to about 700 mg of amikacin (e.g., about 590 mg of amikacin) is administered daily in a single administration session during the administration period.

[0074] In one embodiment, the liposomal amikacin composition is provided in a suspension of about 8 mL. In one embodiment, the density of the liposomal amikacin composition is about 1.05 grams / mL, and in one embodiment, about 8.4 grams of the liposomal amikacin composition is present in the composition of the present invention per dose. In a further embodiment, the entire amount of the composition is administered to a subject in need thereof.

[0075] In one embodiment, the liposomal amikacin composition provided herein comprises amikacin and a lipid component comprising at least one phospholipid and cholesterol. In a further embodiment, the liposomal amikacin composition comprises amikacin sulfate, DPPC and cholesterol. In one embodiment, the liposomal amikacin composition is a composition provided in Table 1 or Table 2 below. [Table 1] [Table 2]

[0076] In embodiments in which the liposomal amikacin and / or second active agent are delivered via inhalation, inhalation can be accomplished via a nebulizer, which provides an aerosol mist of the composition for delivery to the patient's lungs.

[0077] As used herein, an "aerosol" is a gas suspension of liquid particles. The aerosols provided herein include particles of liposomal dispersions.

[0078] A "nebulizer" or "aerosol generator" is a device that converts a liquid into an aerosol of a size that can be inhaled into the respiratory tract. Pulmonary ultrasonic electronic nebulizers (e.g., passive electronic mesh nebulizers, active electronic mesh nebulizers, and vibrating mesh nebulizers, etc.) are applicable for use in the present invention, provided that the particular nebulizer emits an aerosol with the desired characteristics at the desired exit rate.

[0079] The process of converting bulk liquid into small droplets by air pressure is called atomization. The operation of a pneumatic nebulizer requires a supply of pressurized gas as the driving force required for liquid atomization. Ultrasonic nebulizers use electricity introduced by a piezoelectric element in a liquid reservoir to convert liquid into respirable droplets. Various types of nebulizers are described in Respiratory Care, Vol. 45, No. 6, pp. 609-622 (2000), the disclosure of which is incorporated herein by reference in its entirety. The terms "nebulizer" and "aerosol generator" are used interchangeably throughout this specification. "Inhalation device", "inhalation system", and "atomizer" are also used interchangeably in the literature with the terms "nebulizer" and "aerosol generator".

[0080] "Mass median diameter" or "MMD" is the average particle diameter by mass as determined by laser diffraction or impactor measurements.

[0081] "Mass median aerodynamic diameter" or "MMAD" is a calculated impactor measurement, such as an Anderson Cascade Impactor (ACI) or Next Generation Impactor (NGI), normalized to the aerodynamic separation of aqueous aerosol droplets. Gas flow rates are, in one embodiment, 28 liters / min for the Anderson Cascade Impactor (ACI) and 15 liters / min for the Next Generation Impactor (NGI). "Geometric standard deviation" or "GSD" is a measure of the spread of the aerodynamic particle size distribution.

[0082] In one embodiment, the liposomal amikacin composition and / or the second active agent are delivered to a patient in need of treatment via a nebulizer selected from the group consisting of an electronic mesh nebulizer, a pneumonic (jet) nebulizer, an ultrasonic nebulizer, an exhalation-enhanced nebulizer, and an exhalation-actuated nebulizer. In a further embodiment, the nebulizer is portable.

[0083] In one embodiment, the liposomal amikacin composition is delivered to a patient in need of treatment via a nebulizer once daily in a single administration session. In yet a further embodiment, the nebulizer is one of the nebulizers described in U.S. Patent Application Publication No. 2013 / 0330400, which is incorporated herein by reference in its entirety for all purposes.

[0084] The working principle of pulmonary nebulizers is generally known to those skilled in the art and is described, for example, in Respiratory Care, Vol. 45, No. 6, pp. 609-622 (2000), which is incorporated herein by reference in its entirety. Briefly, pneumatic nebulizers use a supply of pressurized gas as the driving force required for liquid atomization. Compressed gas is delivered, which creates a region of negative pressure. The solution to be aerosolized is then delivered into the gas stream and sheared into a liquid film. This film is unstable and breaks into droplets due to surface tension. Smaller particles, i.e., particles with MMAD and FPF properties, can then be formed by placing a baffle in the aerosol stream. In one embodiment of a pulmonary nebulizer, the gas and solution are mixed before exiting the exit port (nozzle) and contacting the baffle. In another embodiment, mixing does not occur until the liquid and gas exit the exit port (nozzle). In one embodiment, the gas is air, O2 and / or CO2.

[0085] In one embodiment, the droplet size and discharge rate can be adjusted in the pulmonary nebulizer that can be used in the methods provided herein. However, consideration should be given to the composition to be nebulized and whether the composition's properties (e.g., percentage (%) of amikacin) change due to changes in the nebulizer. For example, in one embodiment, the gas velocity and / or the velocity of the pharmaceutical composition are changed to achieve the discharge rate and droplet size of the present invention. Additionally or alternatively, the flow rate of the gas and / or the solution can be adjusted to achieve the droplet size and discharge rate of the present invention. For example, when the gas velocity is increased, in one embodiment, the droplet size is decreased. In one embodiment, the ratio of the pharmaceutical composition flow to the gas flow is adjusted to achieve the droplet size and discharge rate of the present invention. In one embodiment, when the ratio of the liquid flow to the gas flow is increased, the particle size is increased.

[0086] In one embodiment, the discharge rate of the pulmonary nebulizer is increased by increasing the filling volume in the liquid reservoir. Without wishing to be bound by theory, the increase in discharge rate may be due to the reduction of the dead volume of the nebulizer. In one embodiment, the nebulization time is shortened by increasing the flow to power the nebulizer. See, for example, Clay et al. (1983). Lancet 2, pp. 592-594 and Hess et al. (1996). Chest 110, pp. 498-505.

[0087] In one embodiment, a storage bag is used to capture aerosol during the nebulization process, and then the aerosol is delivered to the subject by inhalation.In another embodiment, the nebulizer provided herein comprises a valved open-vent design.In this embodiment, when the patient inhales through the nebulizer, the output of the nebulizer increases.During the exhalation phase, the one-way valve redirects the flow from the patient away from the nebulizer chamber.

[0088] In one embodiment, the nebulizer provided herein is a continuous nebulizer.In other words, there is no need to refill the nebulizer with pharmaceutical composition during administration of a single dose.Rather, the nebulizer has a capacity of at least 8mL, at least 8.4mL, at least 8.6mL, at least 8.8mL, at least 9mL, at least 9.4mL, at least 9.6mL, at least 9.8mL, or at least 10mL.

[0089] In one embodiment, the nebulizer provided herein does not use an air compressor and therefore does not generate airflow. In one embodiment, the aerosol is generated by an aerosol head inserted into the mixing chamber of the device. When the patient inhales, air enters the mixing chamber through a one-way inhalation valve at the back of the mixing chamber and carries the aerosol to the patient through the mouthpiece. When exhaling, the patient's breath flows through a one-way exhalation valve in the mouthpiece of the device. In one embodiment, the nebulizer continues to generate aerosol in the mixing chamber, which the subject inhales during the next breath, and this cycle continues until the drug reservoir of the nebulizer is empty.

[0090] In one embodiment, the nebulization time of the liposomal amikacin compositions provided herein, e.g., ALIS, during an administration session is less than 20 minutes, less than 18 minutes, less than 16 minutes, or less than 15 minutes. In one embodiment, the nebulization time of an effective amount of the liposomal amikacin composition provided herein is less than 15 minutes, or less than 13 minutes. In one embodiment, the nebulization time of an effective amount of the liposomal amikacin composition provided herein is about 13 minutes. In another embodiment, the nebulization time of the liposomal amikacin compositions provided herein during an administration session is about 13 minutes to about 17 minutes, or about 13 minutes to about 16 minutes, or about 13 minutes to about 15 minutes.

[0091] In one embodiment, the liposomal amikacin compositions described herein are administered once daily to a patient in need thereof.

[0092] In one embodiment, the liposomal amikacin composition comprises about 550 mg to about 600 mg of amikacin, DPPC and cholesterol, and the lipid to amikacin weight ratio of the composition is 0.75:1.0 (lipid:amikacin) or less, for example, about 0.7:1.0 (lipid:amikacin) or about 0.5:1.0 (lipid:amikacin) to about 0.8:1.0 (lipid component:amikacin).

[0093] In one embodiment, prior to spraying of the liposomal amikacin composition, about 95% to about 100% of the amikacin present in the composition is liposomally complexed. In a further embodiment, the amikacin is amikacin sulfate. In another embodiment, prior to spraying, about 95% to about 99% or about 96% to about 99% of the amikacin present in the composition is liposomally complexed. In a further embodiment, the amikacin is amikacin sulfate. In one embodiment, about 97% or more of the amikacin present in the liposomal amikacin composition is liposomally complexed prior to spraying. In a further embodiment, the amikacin is amikacin sulfate.

[0094] Upon nebulization of the liposomal amikacin compositions described herein, i.e., for administration to a patient in need of treatment for NTM pulmonary disease, an aerosolized composition is formed, in one embodiment, the aerosolized composition has a mass median aerodynamic diameter (MMAD) of about 1.0 micrometer to about 4.2 micrometers as measured by Anderson Cascade Impactor (ACI). In one embodiment, the aerosolized composition has a MMAD of about 3.2 μm to about 4.2 μm as measured by ACI. In one embodiment, the aerosolized composition has a MMAD of about 1.0 μm to about 4.9 μm as measured by Next Generation Impactor (NGI). In one embodiment, the aerosolized composition has a MMAD of about 4.4 μm to about 4.9 μm as measured by NGI.

[0095] The fine particle fraction (FPF) of the aerosolized composition, in one embodiment, is greater than or equal to about 64% when measured by an Anderson Cascade Impactor (ACI), or greater than or equal to about 51% when measured by a Next Generation Impactor (NGI). In one embodiment, the FPF of the aerosolized composition is greater than or equal to about 70% when measured by an ACI, greater than or equal to about 51% when measured by an NGI, or greater than or equal to about 60% when measured by an NGI.

[0096] Upon nebulization, the liposomes in the liposomal amikacin composition leak amikacin from the liposomes, as described above. In one embodiment, upon nebulization, about 20% to about 45% of the liposomal complexed amikacin is released from the liposomes, thereby providing an aerosol comprising a mixture of free and liposomal complexed amikacin. In a further embodiment, the amikacin is amikacin sulfate. In a further embodiment, about 25% to about 45%, or about 30% to about 40% of the liposomal complexed amikacin is released from the liposomes, thereby providing an aerosol comprising a mixture of free and liposomal complexed amikacin. In a further embodiment, the amikacin is amikacin sulfate.

[0097] In another embodiment, the amount of liposome-complexed amikacin after nebulization is about 55% to about 80%, e.g., about 55% to about 75%, or about 55% to about 70%, or about 60% to about 70%. These percentages are also referred to herein as "percentage of associated amikacin after nebulization." In one embodiment, the percentage of associated amikacin after nebulization is about 55% to about 75%, or e.g., about 60% to about 70%. In a further embodiment, the amikacin is amikacin sulfate.

[0098] In one embodiment, the percentage of associated amikacin after nebulization is measured by recovering the aerosol from the air by condensation in a cold trap, followed by quantification of free and associated amikacin.

[0099] As provided throughout, the methods described herein include, in part, administering to a patient in need of NTM pulmonary disease treatment during an administration period, (i) an effective amount of a liposomal amikacin composition via inhalation, and (ii) a second active agent that is synergistic with amikacin. The second active agent can be delivered together with the liposomal amikacin, i.e., in the same composition, or in a separate composition. When present in a different composition, the second active agent can be delivered to a patient in need of treatment orally, parenterally, or topically via inhalation during an administration period.

[0100] Synergy can be assessed according to one skilled in the art, for example, via in vitro MIC assays and by calculating the Fractional Inhibitory Concentration Index (FICI) value. FICI is determined by the following equation: Table 3 provides a means for interpreting FICI values ​​for amikacin and a second active agent.

number

[0101] In one embodiment, the second active agent is a carbapenem. In a further embodiment, the second active agent is imipenem, doripenem, biapenem, or tebipenem.

[0102] In yet another embodiment, the second active agent is rifabutin, rifampin, RV40, clofazimine, levofloxacin, moxifloxacin, bedaquiline, cefdinir, ethambutol, or tetrandrine. In a further embodiment, the second active agent is rifabutin, rifampin, RV40, clofazimine, bedaquiline, or cefdinir.

[0103] In another embodiment, the second active agent is imipenem, rifabutin, rifampin, RV40, clofazimine, levofloxacin, moxifloxacin, bedaquiline, cefdinir, doripenem, biapenem, tebipenem, ethambutol, or tetrandrine. In a further embodiment, the second active agent is imipenem, rifabutin, rifampin, RV40, clofazimine, bedaquiline, cefdinir, doripenem, biapenem, or tebipenem.

[0104] Each of the aforementioned active agents has been shown to be synergistic with amikacin against NTM, as detailed in the Examples section herein.

[0105] The second active agent compound is administered via inhalation in one embodiment. In a further embodiment, the second active agent is present in a liposomal amikacin composition. For example, the second active agent is provided as a free agent in the composition. Alternatively, the second active agent is administered via inhalation in a separate composition. The second active agent is administered via inhalation in one embodiment as a "free" anti-infective. In other words, in this embodiment, the second active agent is not liposomally complexed. However, in another embodiment, the second active agent is liposomally complexed and administered via inhalation.

[0106] In one embodiment, the second active agent is administered orally to a patient in need of NTM pulmonary disease treatment.

[0107] In one embodiment, the second active agent is administered parenterally to a patient in need of NTM pulmonary disease treatment. In a further embodiment, the second active agent is administered intravenously to a patient in need of NTM pulmonary disease treatment.

[0108] Rifabutin, in one embodiment, is the second active agent used in one of the methods described herein. In one embodiment, rifabutin is administered orally or intravenously to a patient in need of treatment. In one embodiment, rifabutin is administered once a day to a patient during the administration period. In another embodiment, rifabutin is administered twice a day to a patient during the administration period. Rifabutin, in one embodiment, is administered daily at a dose of 150 mg or 300 mg during the administration period.

[0109] Rifampin, a semisynthetic antibiotic derivative of rifamycin SV, in one embodiment is the second active agent used in one of the methods described herein. In one embodiment, rifabutin is administered orally or intravenously to a patient in need of treatment. In one embodiment, the dose of rifampin is about 300 mg to about 600 mg per administration. In a further embodiment, the dose of rifampin is 300 mg or 600 mg per administration. In a further embodiment, rifabutin is administered once a day to a patient for the administration period.

[0110] RV40, a vancomycin derivative, is used in one embodiment as a second active agent in one of the methods described herein. Methods for making RV40 are disclosed in PCT Publication No. WO2018 / 217800, the entirety of which is incorporated herein by reference. In one embodiment, RV40 is administered intravenously to a patient in need of treatment. In another embodiment, RV40 is administered via inhalation, for example, via a DPI or nebulizer.

[0111] In another embodiment, the second active agent is clofazimine. In one embodiment, clofazimine is administered orally. In another embodiment, clofazimine is administered via inhalation, via a nebulizer or DPI. Various inhalation formulations of clofazimine have been described and are suitable for use in the present methods. For example, the formulations described in PCT Publication WO2019 / 110099, which is incorporated herein by reference in its entirety, can be used in the methods provided herein. In another embodiment, clofazimine dry powder formulations for administration via DPI can be used. See, for example, Brunaugh et al. (2017). Mol. Pharmaceutics 14, pp. 4019-4031, which is incorporated herein by reference in its entirety. Administration can be performed once a day or twice a day during the administration period.

[0112] The fluoroquinolone levofloxacin, in one embodiment, is used as the second active agent in one of the methods of treating NTM lung disease provided herein. In one embodiment, levofloxacin is administered orally or intravenously to a patient in need of treatment. For example, levofloxacin can be administered orally or intravenously as described in the prescribing information for Levaquin® (levofloxacin).

[0113] In one embodiment, the fluoroquinolone moxifloxacin (marketed under the trade name Avelox®) is used as the second active agent in one of the methods of treating NTM pulmonary disease provided herein. In one embodiment, moxifloxacin is administered orally or intravenously to a patient in need of treatment. In one embodiment, administration is performed once daily for the administration period. In a further embodiment, the patient is administered moxifloxacin orally, intravenously, or sequentially (intravenously followed by orally). In a further embodiment, the patient is administered 400 mg once daily for the administration period.

[0114] Bedaquiline, in one embodiment, is the second active agent used in one of the methods of treating NTM pulmonary disease provided herein. In one embodiment, bedaquiline is administered orally to a patient in need of treatment. In a further embodiment, bedaquiline is administered to a patient once daily at about 100 mg to about 400 mg for the administration period. In another embodiment, a patient is administered 400 mg once daily for two weeks, followed by 200 mg three times weekly for 22 or more weeks.

[0115] In another embodiment, bedaquiline is administered via inhalation.Various inhalation formulations of bedaquiline have been described and are suitable for use in the present method.For example, the formulations described in PCT Publication Nos. WO2020 / 123336 and WO2019 / 193609 can be used in the methods provided herein, which are incorporated herein by reference in their entirety.In another embodiment, bedaquiline dry powder formulations for administration via DPI can be used.See, for example, Momin et al. (2019).Pharmaceutics11,502,doi:10.3390 / Pharmaceutics11100502, which is incorporated herein by reference in its entirety.

[0116] The cephalosporin antibiotic cefdinir can be used as a second active agent in one of the methods described herein. Cefdinir is sold under the trade name Omnicef®. In one embodiment, cefdinir is administered to a patient once or twice daily. In one embodiment, cefdinir is administered orally. In a further embodiment, the dose of cefdinir is 300 mg or 600 mg per day.

[0117] In one embodiment, the second active agent is a carbapenem. In one embodiment, the carbapenem is imipenem, doripenem, biapenem, or tebipenem. In a further embodiment, imipenem is used as the second active agent in one of the methods described herein. Imipenem is a beta-lactam antibiotic that has been shown to be synergistic with amikacin against certain NTM strains and can therefore be used in the methods described herein. In one embodiment, imipenem is administered intravenously to a patient in need of treatment. In one embodiment, imipenem is administered with cilastatin to prevent its inactivation by the renal enzyme dehydropeptidase 1.

[0118] In one embodiment, doripenem, a beta-lactam antibiotic in the carbapenem class, is used as the second active agent in one of the methods of treating NTM pulmonary disease provided herein. In one embodiment, doripenem is administered intravenously to a patient in need of NTM pulmonary disease treatment. In a further embodiment, 500 mg of doripenem is administered once a day, twice a day, or three times a day during the administration period. When doripenem is administered twice a day or three times a day, the administrations are spaced 8 hours apart.

[0119] Biapenem is another carbapenem that can be used in the methods described herein. In one embodiment, biapenem is administered intravenously to patients in need of NTM lung disease treatment.

[0120] Tebipenem is another carbapenem that can be used in the methods described herein. In one embodiment, tebipenem is orally administered to a patient who needs NTM lung disease treatment. In one embodiment, tebipenem is administered as ester tebipenem pivoxil due to its improved absorption and bioavailability compared to the non-ester form.

[0121] Ethambutol, in one embodiment, is the second active agent used in the methods provided herein. In a further embodiment, the ethambutol is administered orally to the patient.

[0122] In one embodiment, the calcium channel blocker tetraindoline is used in the methods provided herein as the second active agent.In a further embodiment, the tetraindoline is administered orally.In a further embodiment, the tetraindoline is administered once a day.For example, 60 mg of tetraindoline can be administered once a day in the methods provided herein during the administration period.

[0123] The NTM lung disease treated by the methods provided herein, in one embodiment, is caused by one of the following NTM species: Mycobacterium avium complex, Mycobacterium kansasii, Mycobacterium abscessus, or Mycobacterium fortuitum. In a further embodiment, the NTM lung disease is caused by Mycobacterium avium complex. In another embodiment, the NTM lung disease is caused by Mycobacterium abscessus lung infection.

[0124] In one embodiment, the NTM lung disease treated by the methods provided herein is newly diagnosed and the treatment regimens described in the methods provided herein represent first line therapy.

[0125] As used herein, the term "newly diagnosed" means (i) untreated, initially diagnosed NTM lung disease based on a positive sputum culture for an NTM strain; or (ii) untreated NTM lung disease diagnosed on the basis of a newly positive sputum culture for NTM followed by a previously diagnosed NTM lung infection on the basis of a previously positive sputum culture for NTM, where the previously diagnosed NTM lung disease is treated, treatment for the previously diagnosed NTM lung disease is discontinued once a negative sputum culture is achieved, and the negative sputum culture for NTM returns to a newly positive sputum culture at least 6 months after cessation of treatment for the previously diagnosed NTM lung disease.

[0126] In one embodiment, the patient subjected to one of the treatment methods provided herein is a patient who has previously been non-responsive to a different NTM treatment, in other words, in one embodiment, the patient subjected to one of the treatment methods described herein is refractory to a previous treatment.

[0127] In another embodiment, the methods provided herein are practiced for the treatment or prevention of one or more NTM pulmonary infections in a CF patient. In a further embodiment, the liposomal amikacin composition administered to a patient in need of treatment is one of the compositions set forth in Table 1 or Table 2 above.

[0128] In one embodiment, the patient is a bronchiectasis patient. In one embodiment, the bronchiectasis is non-cystic fibrosis bronchiectasis (NCFBE). In another embodiment, the bronchiectasis is associated with CF.

[0129] The patient undergoing the methods described herein, in one embodiment, has a co-morbid condition. For example, in one embodiment, the patient in need of treatment with one of the methods described herein has, in addition to a pulmonary NTM infection, diabetes, mitral valve disorder (e.g., mitral valve prolapse), acute bronchitis, pulmonary hypertension, pneumonia, asthma, tracheal carcinoma, bronchial carcinoma, lung cancer, cystic fibrosis, pulmonary fibrosis, laryngeal abnormalities, tracheal abnormalities, bronchial abnormalities, aspergillosis, HIV, or bronchiectasis. In one embodiment, the patient in need of treatment of an NTM pulmonary disease with one of the methods provided herein has been diagnosed with chronic obstructive pulmonary disease (COPD). In yet another embodiment, the patient in need of treatment of an NTM pulmonary infection is an asthma patient. In a further embodiment, the composition administered to the patient in need of treatment is one of the compositions described in Table 1 or Table 2 above.

[0130] In one embodiment, the patient in need of treatment with one of the methods described herein is a CF patient, a bronchiectasis patient, a ciliary dyskinesia patient, a chronic smoker, a chronic obstructive pulmonary disease (COPD) patient, or a patient who has previously been non-responsive to treatment. In a further embodiment, the CF patient is treated for an NTM lung infection with one of the methods provided herein. In yet another embodiment, the patient is a bronchiectasis patient, a COPD patient, or an asthma patient.

[0131] In one embodiment, the treatment includes a patient achieving a negative NTM sputum culture. The negative sputum culture may be achieved during or after the administration period. In one embodiment, a patient's sputum sample is obtained from the patient at regular time intervals during and / or immediately after the administration period and then used to prepare NTM cultures.

[0132] In another embodiment, the treatment includes a patient who achieves conversion of NTM sputum culture to negative during or after the administration period. As provided herein, conversion of sputum culture to negative refers to three consecutive negative NTM cultures. Samples can be taken during the administration period or during the administration period and combinations thereafter. Three consecutive cultures can be prepared from patient samples taken at spaced intervals, for example, at biweekly or monthly intervals. The time to conversion is, in one embodiment, about 90 days, or about 100 days or about 110 days. In another embodiment, the time to conversion is from about 90 days to about 200 days, from about 90 days to about 190 days, from about 90 days to about 180 days, from about 90 days to about 160 days, from about 90 days to about 150 days, from about 90 days to about 140 days, from about 90 days to about 130 days, from about 90 days to about 120 days, from about 90 days to about 110 days, from about 90 days to about 110 days, or from about 90 days to about 100 days.

[0133] In some embodiments, the therapeutic response resulting from the methods provided herein is measured by one or more Patient Reported Outcomes (PROs) generated from PRO methods, such as Quality of Life Questionnaire-Bronchiectasis (QOL-B), Patient Global Impression of Severity (PGIS)-Respiratory, PROMIS Fatigue Short Form 7a (PROMIS F-SF 7a), and Patient Global Impression of Severity (PGIS)-Fatigue.

[0134] The QOL-B is a validated, self-administered, reported outcome questionnaire used to assess symptoms, function, and health-related quality of life in adults with non-CF bronchiectasis. See Quittner AL, Abbott J, Georgiopoulos AM, et al. Quality of Life Questionnaire-Bronchiectasis: final psychometric analyses and determination of minimal important differences scores. Thorax. 2016;71(1):26-34, incorporated herein by reference in its entirety. It measures outcomes over a 1-week recall period. The questionnaire includes 37 items across 8 scales (physical, role, vitality, emotional, social, treatment burden, health perception, and respiratory). Each of the 37 items is scored 1-4, and each of the 8 scores is standardized on a 0-100 scale, with higher scores indicating fewer symptoms or better function or quality of life. Scores are calculated for seven domains: physical, role, vitality, emotional, social, treatment burden, health perception, and respiratory.

[0135] In one embodiment of the methods disclosed herein, the treatment comprises an improvement in one or more respiratory symptoms of the patient during or after the administration period, as measured by QOL-B Respiratory Domain Score, compared to one or more of the patient's respiratory symptoms prior to treatment, or compared to one or more of the respiratory symptoms of a patient administered either a liposomal amikacin composition or a second active agent during the same administration period. In one embodiment, the patient's one or more respiratory symptoms are improved about three months to about six months after the administration period has ended. In another embodiment, the patient's one or more respiratory symptoms are improved about three months to about twelve months after the administration period has ended.

[0136] In one embodiment, the treatment comprises an improvement in one or more QOL-B non-respiratory domains for the patient during or after the administration period, compared to the patient's score in one or more QOL-B non-respiratory domains prior to treatment, or compared to the score in one or more QOL-B non-respiratory domains for the patient administered either the liposomal amikacin composition or the second active agent during the same administration period. In a further embodiment, the patient's score in one or more QOL-B non-respiratory domains is improved during the administration period. In yet a further embodiment, the patient's score in one or more QOL-B non-respiratory domains is improved about one month after the administration period has ended. In yet a further embodiment, the patient's score in one or more QOL-B non-respiratory domains is improved at least about one month after the administration period has ended. In yet a further embodiment, the patient's score in one or more QOL-B non-respiratory domains is improved about one month to about three months after the administration period has ended. In yet a further embodiment, the patient's score in one or more QOL-B non-respiratory domains is improved about three months after the administration period has ended. In yet further embodiments, the patient's score in one or more QOL-B non-respiratory domains is improved at least about three months after the administration period has ended. In yet further embodiments, the patient's score in one or more QOL-B non-respiratory domains is improved about three months to about six months after the administration period has ended. In yet further embodiments, the patient's score in one or more QOL-B non-respiratory domains is improved about three months to about twelve months after the administration period has ended.

[0137] The PGIS Respiratory Score is a simple categorical assessment of the severity of symptoms. The scale is 0=not at all to 5=very severe. In one embodiment of the methods disclosed herein, the treatment comprises an improvement in one or more respiratory symptoms for the patient during or after the administration period, as measured by the PGIS Respiratory Domain Score, compared to one or more of the patient's respiratory symptoms before treatment, or compared to a score of the PGIS Respiratory Domain Score for the patient administered either the liposomal amikacin composition or the second active agent during the same administration period. In a further embodiment, the patient's one or more respiratory symptoms are improved during the administration period. In an even further embodiment, the patient's one or more respiratory symptoms are improved after the administration period. In an even further embodiment, the patient's one or more respiratory symptoms are improved about one month after the administration period. In an even further embodiment, the patient's one or more respiratory symptoms are improved at least about one month after the administration period. In another embodiment, the patient's one or more respiratory symptoms are improved about one month to about three months after the administration period ends. In another embodiment, the patient's one or more respiratory symptoms are improved about three months after the administration period has ended. In yet another embodiment, the patient's one or more respiratory symptoms are improved at least about three months after the administration period has ended. In another embodiment, the patient's one or more respiratory symptoms are improved about three months to about six months after the administration period has ended. In another embodiment, the patient's one or more respiratory symptoms are improved about three months to about twelve months after the administration period has ended.

[0138] PROMIS F-SF 7a is a self-administered questionnaire that assesses a range of self-reported symptoms over the past 7 days, from mild subjective fatigue to overwhelming, debilitating, and persistent fatigue that is likely to reduce one's ability to function normally in family or social roles. See Ameringer S, Elswick RK, Jr., Menzies V, et al. Psychometric Evaluation of the Patient- Reported Outcomes Measurement Information System Fatigue-Short Form Across Diverse Populations. Nurs Res. 2016;65(4):279-289, incorporated herein by reference in its entirety. Fatigue is divided into the experience of fatigue (frequency, duration, intensity) and its impact on physical, mental, and social activities across 7 items. Response options are a 5-point Likert scale ranging from 1=never to 5=always. PROMIS F-SF 7a is not disease-specific, but universal.

[0139] In one embodiment of the methods disclosed herein, the treatment comprises improving one or more fatigue symptoms of the patient during or after the administration period as measured by a PROMIS F-SF 7a score, compared to the one or more fatigue symptoms of the patient prior to treatment, or compared to the one or more fatigue symptoms of the patient administered either the liposomal amikacin composition or the second active agent during the same administration period. In one embodiment, the one or more fatigue symptoms of the patient are improved during the administration period. In one embodiment, the one or more fatigue symptoms of the patient are improved after the administration period. In another embodiment, the one or more fatigue symptoms of the patient are improved about one month after the administration period ends. In yet a further embodiment, the one or more fatigue symptoms of the patient are improved at least about one month after the administration period. In yet a further embodiment, the one or more fatigue symptoms of the patient are improved about one month to about three months after the administration period. In yet a further embodiment, the one or more fatigue symptoms of the patient are improved about three months after the administration period. In yet a further embodiment, the one or more fatigue symptoms of the patient are improved at least about three months after the administration period. In yet further embodiments, the patient's one or more fatigue symptoms improve about three months to about six months after the administration period. In yet further embodiments, the patient's one or more fatigue symptoms improve about three months to about twelve months after the administration period.

[0140] The PGIS fatigue score is a simple categorical assessment of symptom severity, on a scale from 0 = not at all to 5 = very severe.

[0141] In one embodiment of the methods disclosed herein, the treatment comprises improving one or more fatigue symptoms of the patient during or after the administration period as measured by a PGIS fatigue score, compared to the one or more fatigue symptoms of the patient prior to treatment, or compared to one or more fatigue symptoms of the patient administered either the liposomal amikacin composition or the second active agent during the same administration period. In a further embodiment, the one or more fatigue symptoms of the patient are improved during the administration period. In an even further embodiment, the one or more fatigue symptoms of the patient are improved after the administration period. In an even further embodiment, the one or more fatigue symptoms of the patient are improved about one month after the administration period. In an even further embodiment, the one or more fatigue symptoms of the patient are improved at least about one month after the administration period. In an even further embodiment, the one or more fatigue symptoms of the patient are improved about one month to about three months after the administration period. In an even further embodiment, the one or more fatigue symptoms of the patient are improved about three months after the administration period. In an even further embodiment, the one or more fatigue symptoms of the patient are improved at least about three months after the administration period. In yet further embodiments, the patient's one or more fatigue symptoms improve about three months to about six months after the administration period. In yet further embodiments, the patient's one or more fatigue symptoms improve about three months to about twelve months after the administration period.

[0142] In some embodiments, the patient experiences improved pulmonary function for at least 15 days after the administration period has ended, compared to the patient's pulmonary function prior to treatment. For example, the patient experiences an increase in FEV1, an increase in blood oxygen saturation, or both. In some embodiments, the patient has an FEV1 (after an administration period or treatment cycle) that is increased by at least 5% relative to the FEV1 prior to the administration period. In other embodiments, the FEV1 is increased by 5%-50% relative to the FEV1 prior to the administration period. In other embodiments, the FEV1 is increased by 25 mL-500 mL relative to the FEV1 prior to the administration period. In some embodiments, the blood oxygen saturation is increased by at least 1% relative to the oxygen saturation prior to the administration period.

[0143] In one embodiment, the 6-minute walk test (6MWT) is used to evaluate the effectiveness of the therapeutic methods provided herein. The 6MWT is a practical and simple test used to objectively evaluate functional exercise capacity and measures the distance a patient can walk in a 6-minute period (see American Thoracic Society. (2002). Am J Respir Crit Care Med. 166, pp. 111-117, which is incorporated herein by reference in its entirety for all purposes).

[0144] In one embodiment, a patient undergoing one of the NTM methods described herein experiences an increase in the number of meters walked in the 6MWT compared to before undergoing the treatment. The increased number of meters walked in the 6MWT is, in one embodiment, about 5 meters, about 10 meters, about 15 meters, about 20 meters, about 25 meters, about 30 meters, about 35 meters, about 40 meters, about 45 meters, or about 50 meters. In another embodiment, the increased number of meters walked in the 6MWT is at least about 5 meters, at least about 10 meters, at least about 15 meters, at least about 20 meters, at least about 25 meters, at least about 30 meters, at least about 35 meters, at least about 40 meters, at least about 45 meters, or at least about 50 meters. In yet another embodiment, the increased number of meters walked in the 6MWT is about 5 meters to about 50 meters, or about 5 meters to about 40 meters, or about 5 meters to about 30 meters, or about 5 meters to about 25 meters.

[0145] In another embodiment, a patient undergoing one of the NTM methods described herein experiences an increase in the number of meters walked in the 6MWT compared to a patient undergoing treatment with either a liposomal amikacin composition or a second active agent for the same administration period. The increased number of meters walked in the 6MWT is, in one embodiment, about 5 meters, about 10 meters, about 15 meters, about 20 meters, about 25 meters, about 30 meters, about 35 meters, about 40 meters, about 45 meters, about 50 meters, about 60 meters, about 70 meters, or about 80 meters. In another embodiment, the increased number of meters walked in the 6MWT is at least about 5 meters, at least about 10 meters, at least about 15 meters, at least about 20 meters, at least about 25 meters, at least about 30 meters, at least about 35 meters, at least about 40 meters, at least about 45 meters, or at least about 50 meters. In yet another embodiment, the number of walking meter increments in the 6MWT is from about 5 meters to about 80 meters, or from about 5 meters to about 70 meters, or from about 5 meters to about 60 meters, or from about 5 meters to about 50 meters. EXAMPLES

[0146] The present invention will be further illustrated by reference to the following examples, however, it should be noted that these examples, like the above-mentioned embodiments, are merely illustrative and should not be construed as limiting the scope of the present invention in any way.

[0147] Example 1: Synergistic combinations of anti-infective drugs against nontuberculous mycobacteria To analyze synergistic combinations of active agents against nontuberculous mycobacteria (NTM), a checkerboard minimum inhibitory concentration (MIC) is performed to determine the effect on the efficacy of a particular combination of active agents compared to the individual MICs. The comparison is expressed as a Fractional Inhibitory Concentration Index (FICI) and takes into account the combination of active agents that produces the greatest change from the MIC of the individual active agents. In the examples provided herein, MICs are evaluated for the Mycobacterium abscessus ATCC 19977 Type strain. However, these methods are applicable to other NTM species described herein.

[0148] Assay setup and reagents The following protocol is performed for each pair of active agents (referred to in this section as Active Agent X and Active Agent Y).

[0149] Fresh cultures of slow-growing NTM strains (exemplified herein by Mycobacterium abscessus ATCC 19977) were prepared in 7H10 + 10% oleic acid albumin dextrose catalase (OADC) agar or 7H9 + 10% OADC broth.

[0150] Approximately 5×10 8 Bacterial inocula in CFU / mL were prepared in Hanks Balanced Salt Solution (HBSS) using standard protocols.

[0151] The inoculum suspension was diluted 1:10 in HBSS, then 1:100 (total dilution of 1:1,000 for 5 × 10 5 The cultures were diluted to achieve 100 colony forming units (CFU) / mL into the respective volumes of broth required, 15 mL per checkerboard plate.

[0152] Checkerboard plate wells were filled with 100 μL of the diluted inoculum suspension (100 μL per well). All plate wells in rows B through H were filled, while row A was initially left empty.

[0153] Active Agent-X was diluted in pre-inoculation broth such that the highest dose of Active Agent-X tested was present in 100 μL of broth. Dilutions were performed in 5 mL culture tubes.

[0154] Using a multipipette, a 100 μL aliquot of active drug-X dilution was added to each well in row A of the checkerboard plate.

[0155] 100 μL of Active Agent-X dilution (i.e., highest dose tested) was added to row B. Active Agent-X was mixed 10 times in row B via pipette.

[0156] The contents of row B were then serially diluted 1:1 by mixing 10 times in every row through row G. 100 μL in row G was discarded to obtain 100 μL in every well of the plate.

[0157] The wells in row H were left empty.

[0158] Active agent-Y was diluted in pre-inoculation broth such that 2x the highest dose of active agent-Y to be tested was present in 100 μL of broth. Dilutions were made in 5 mL culture tubes.

[0159] To each well in column 1, 100 μL of Active Drug-Y stock was added (8 wells total).

[0160] Using a multichannel pipette, the contents of each well in column 1 were mixed 10 times and 100 μL of the contents of column 1 were transferred to column 2.

[0161] The contents of column 2 were serially diluted 1:1 through column 11 by mixing 10 times across each column. 100 μL of column 11 was discarded to obtain 100 μL in all wells of the plate. No material was added to column 12 at this step.

[0162] At this stage, the checkerboard plate was completely filled with (i) active drug-X standalone MIC, which is column 12; (ii) active drug-Y standalone MIC, which is row H; (iii) negative (no drug) control, which is well H12; and a mixed drug matrix of concentrations in all other wells of the plate (see schematic in Figure 1).

[0163] Checkerboard plates were placed in zip-top bags and incubated at 37°C for 7 days unless otherwise stated.

[0164] The absorbance value of each well was measured at 595 nm after a 7-day incubation period using a plate reader (BioTek Synergy H1). The MIC value was determined based on the absorbance value. The FICI value was then calculated as follows:

[0165] FICI measurement FICI is determined by the following equation:

number

[0166] A and B are the MICs of the combination of active agent-X and active agent-Y (in a single well), and MICA and MICB are the MICs of each anti-infective individually. [Table 4]

[0167] Combinations are determined to be synergistic based on the values ​​set forth above in Table 4.

[0168] Table 5 provides the results for all active agent combinations tested. [Table 5] ********

[0169] All references, patents, patent applications, publications, product descriptions, and protocols cited throughout this application are hereby incorporated by reference in their entirety for all purposes.

[0170] The embodiments described and discussed herein are intended only to teach those skilled in the art the best way known to the inventor to make and use the invention. The above-described embodiments of the invention may be modified and varied without departing from the invention, as will be understood by those skilled in the art in light of the above teachings. It is therefore understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described. Therefore, the foregoing description and drawings are by way of example only, and the disclosure is described in detail by the following claims.

Claims

1. Use of (i) a liposomal amikacin composition and (ii) a second active agent in the manufacture of a medicament for the treatment of nontuberculous mycobacterial (NTM) pulmonary disease in a patient in need thereof, comprising: the second active agent is synergistic with amikacin against the NTM; The liposomal amikacin composition comprises amikacin or a pharma- ceutically acceptable salt thereof encapsulated in a plurality of liposomes, the lipid component of the plurality of liposomes being comprised of one or more electrically neutral lipids; The liposomal amikacin composition is administered to the patient once daily in a single administration session, and administering includes aerosolizing the liposomal amikacin composition to provide an aerosolized composition comprising a mixture of free amikacin and liposomally complexed amikacin, and administering the aerosolized pharmaceutical composition by inhalation to the patient's lungs via a nebulizer.

2. 2. The use of claim 1, wherein the amikacin or a pharma- ceutically acceptable salt thereof is amikacin or amikacin sulfate.

3. The use according to claim 1 or 2, wherein the one or more electrically neutral lipids comprise (i) an electrically neutral phospholipid or (ii) an electrically neutral phospholipid and a sterol.

4. The use according to any one of claims 1 to 3, wherein the one or more electrically neutral lipids consist of phosphatidylcholine and a sterol.

5. The use according to any one of claims 1 to 3, wherein the one or more electrically neutral lipids comprise dipalmitoylphosphatidylcholine (DPPC) and cholesterol.

6. The use of any one of claims 1 to 5, wherein the liposomal amikacin composition is a liposomal suspension having a volume of about 8 mL to about 10 mL.

7. 7. The use of any one of claims 1 to 6, wherein the liposomal amikacin composition comprises about 500 mg to about 650 mg of amikacin or a pharma- ceutically acceptable salt thereof, or about 550 mg to about 625 mg of amikacin or a pharma- ceutically acceptable salt thereof, or about 550 mg to about 600 mg of amikacin or a pharma- ceutically acceptable salt thereof.

8. The use according to any one of claims 1 to 7, wherein the second active agent is administered orally, parenterally, intravenously or via inhalation.

9. 9. The use of any one of claims 1 to 8, wherein the second active agent is an anti-infective, a carbapenem (e.g., imipenem, doripenem, biapenem, or tebipenem), rifabutin, rifampin, RV40, clofazimine, bedaquiline, or cefdinir.

10. The NTM pulmonary disease is selected from the group consisting of Mycobacterium avium (M. avium) pulmonary disease, Mycobacterium abscessus (M. abscessus) pulmonary disease, Mycobacterium chelonae (M. chelonae) pulmonary disease, Mycobacterium borretii (M. bolletii) pulmonary disease, Mycobacterium kansasii (M. kansasii) pulmonary disease, Mycobacterium ulcerans (M. ulcerans) pulmonary disease, Mycobacterium avium complex (MAC) (Mycobacterium avium and Mycobacterium pulmonary disease), ... Mycobacterium intracellulare pulmonary disease, Mycobacterium conspicuum pulmonary disease, Mycobacterium peregrinum pulmonary disease, Mycobacterium immunogenum pulmonary disease, Mycobacterium xenopi pulmonary disease, Mycobacterium malmoense pulmonary disease, Mycobacterium marinum pulmonary disease, M. mucogenicum pulmonary disease, M. nonchromogenicum pulmonary disease, M. scrofulaceum pulmonary disease, M. simiae pulmonary disease, M. smegmatis pulmonary disease, M. szulgai pulmonary disease, M. terrae pulmonary disease, Mycobacterium terrae complex pulmonary disease, M. haemophilum pulmonary disease, M. genavense pulmonary disease, M. asiaticum lung disease, M. shimoidei lung disease, M. gordonae lung disease, M. triplex lung disease, M. lentiflavum lung disease, M. celatum lung disease, M. fortuitum lung disease, M. fortuitum complex (M. fortuitum and M. chelonae) lung disease,or a combination thereof.

11. The use according to any one of claims 1 to 10, wherein the NTM lung disease is Mycobacterium abscessus lung disease.

12. The use according to any one of claims 1 to 10, wherein the NTM lung disease is Mycobacterium avium complex (Mycobacterium avium and Mycobacterium intracellulare) lung disease.

13. The use according to any one of claims 1 to 12, wherein the patient presents with negative NTM sputum cultures during or after the administration period.

14. The use according to any one of claims 1 to 12, wherein during or after the administration period, the patient exhibits a conversion of NTM sputum cultures to negative.

15. 15. The use of claim 14, wherein the NTM sputum culture conversion to negative comprises three consecutive NTM negative cultures.

16. 16. The use of any one of claims 1 to 15, wherein during or after said administration period, the patient shows improvement in said one or more respiratory symptoms as measured by QOL-B respiratory domain score compared to said one or more respiratory symptoms of the patient before receiving said treatment.

17. 16. The use according to any one of claims 1 to 15, wherein during or after the administration period, the patient experiences an increase in the number of metres walked in a 6 Minute Walk Test (6MWT) compared to the number of metres walked by the patient before receiving the treatment.

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