In Vitro Release Assay Method for Liposomal Aminoglycoside Formulations

Abstract

The present disclosure relates to in vitro release (IVR) methods for liposomal aminoglycoside formulations, particularly liposomal amikacin formulations.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 208,894, filed June 9, 2021, which is incorporated by reference in its entirety for all purposes. The present invention relates to an in vitro method for determining aminoglycoside release profiles from liposomal aminoglycoside formulations, particularly liposomal amikacin formulations. [Background technology]

[0002] Oral inhalation of liposomes encapsulating the antibiotic amikacin has been proposed to treat certain pulmonary infections. One such formulation, amikacin liposomal inhalation suspension (ALIS), was initially approved by the US Food and Drug Administration (FDA) in 2018 for oral inhalation by adults with limited or no alternative treatment options for the treatment of Mycobacterium avium complex (MAC) pulmonary disease as part of a combination antibiotic regimen in patients with non-negative sputum cultures after 6 months or more of background multidrug therapy. ALIS liposomes provide localized delivery of antibiotics at high concentrations at the site of infection while minimizing systemic exposure and therefore toxicity. Liposomes also provide long-term therapeutic effects and increase amikacin uptake into macrophages. Amikacin-containing liposome formulations, including certain ALIS formulations, are described, for example, in U.S. Pat. No. 6,233,633, ... each of which is incorporated herein by reference in its entirety.

[0003] However, inhalation delivery of liposomes is complicated by their sensitivity to shear-induced stress during nebulization, which can result in changes in physical characteristics. Under certain conditions that affect the integrity of the lipid bilayer membrane(s) of the liposome (e.g., addition of surfactants, proteins, ethanol, osmotic stress, and / or temperature increase), the contents inside the liposome can be induced to leak out suddenly and / or over a period of time. However, as long as the changes in properties are reproducible and meet acceptable criteria, they need not prohibit drug development.

[0004] According to the FDA's April 2018 industry guidance, Non-Patent Document 1, the in vitro release profile of the drug substance is a useful property for characterizing liposomal drug formulations, and a validated analytical procedure should be established. If the liposomal drug product is highly stable under physiological conditions, an in vitro quality control release test can be performed under non-physiological conditions to accelerate the release of the drug substance from the liposomes. However, because no liposomal formulations for inhalation have been approved by the FDA to date, no further guidance has been provided for developing validated analytical test methods. The present invention addresses this and other needs. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 7,544,369 [Patent Document 2] U.S. Patent No. 7,718,189 [Patent Document 3] U.S. Patent No. 8,226,975 [Patent Document 4] U.S. Patent No. 8,632,804 [Patent Document 5] U.S. Patent No. 8,642,075 [Patent Document 6] U.S. Patent No. 8,679,532 [Patent Document 7] U.S. Patent No. 8,802,137 [Patent Document 8] U.S. Patent No. 9,566,234 [Patent Document 9] U.S. Patent No. 9,827,317 [Patent Document 10] U.S. Patent No. 9,895,385 [Non-patent literature]

[0006] [Non-Patent Document 1] Liposome Drug Products: Chemistry, Manufacturing and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to an in vitro release (IVR) method for assessing aminoglycoside release profiles from liposomal aminoglycoside formulations, such as liposomal amikacin formulations suitable for inhalation. [Means for solving the problem]

[0008] One aspect of the IVR method for evaluating the aminoglycoside release profile from a liposomal aminoglycoside formulation is by conducting a dissolution test with the liposomal aminoglycoside formulation in one or more dialysis devices placed (or installed) in a dissolution medium (such as a buffer solution) that includes a stirring member (the stirring member is also referred to herein as a dissolution device). The liposomal aminoglycoside formulation includes an aminoglycoside encapsulated within a plurality of liposomes. The one or more dialysis devices each have a predetermined volume and each include a dialysis membrane with a predetermined molecular weight cut-off (MWCO). The dissolution medium includes a predetermined amount of a surfactant in a buffer solution. The dialysis membrane is permeable to the aminoglycoside, the dissolution medium, and the surfactant, such that bidirectional passage occurs across the dialysis membrane(s). Specifically, the surfactant diffuses into and through the one or more dialysis membranes over time, the surfactant increases the permeability of the liposomes to allow free aminoglycoside release from the liposomes, and the free aminoglycoside diffuses across the one or more dialysis membranes into the dissolution medium. During the dissolution test, the stirring member is operated. In one embodiment, the stirring member is a USP dissolution apparatus 2 (paddle). Preferably, the dialysis membrane is not permeable or is substantially impermeable to the aminoglycoside-encapsulating liposome. In a preferred embodiment, the dialysis membrane has a predetermined MWCO of about 20 kD to about 1500 kD. In a further embodiment, the dialysis membrane has a predetermined MWCO of about 1000 kD.

[0009] One embodiment of the IVR method comprises: (a) operating a dissolution device within a dissolution vessel, the dissolution vessel containing (i) a predetermined amount of a liposomal aminoglycoside formulation in one or more dialysis devices, each having a predetermined volume and a dialysis membrane with a predetermined molecular weight cut-off (MWCO), and (ii) a dissolution medium having a predetermined amount of a surfactant, each dialysis membrane being permeable to the aminoglycoside, the dissolution medium, and the surfactant, and the one or more dialysis devices being immersed in the dissolution medium; (b) removing an aliquot of the dissolution medium after a predetermined time interval; (c) analyzing the aliquot for free aminoglycoside content; (d) optionally repeating steps (b) and (c) after one or more additional time intervals. The aliquot removed from the dissolution medium may be subjected to solid phase extraction (such as with a cation exchange sorbent material) to remove the detergent prior to analysis of the aliquot for free aminoglycoside content.

[0010] In one embodiment, step (b) is performed a total of three, four, five, or six times. In yet another embodiment, step (b) is performed four times. In a further embodiment, the liposomal aminoglycoside formulation is an ALIS formulation. In one embodiment, the liposomal aminoglycoside formulation comprises an aminoglycoside or a pharma- ceutically acceptable salt thereof encapsulated in a plurality of liposomes. In one embodiment, the lipid component of the plurality of liposomes comprises an electrically neutral lipid. In a further embodiment, the electrically neutral lipid is comprised of dipalmitoylphosphatidylcholine (DPPC) and cholesterol. In one embodiment, analyzing the aliquot for free aminoglycoside content comprises performing high performance liquid chromatography (HPLC) on the aliquot. In a further embodiment, HPLC is performed using evaporative light scattering detection (ELSD) or charged aerosol detection (CAD) to determine the free aminoglycoside content. In a further embodiment, HPLC is used to determine the total aminoglycoside content of the aliquot. In one embodiment, step (a) of operating the dissolution apparatus is carried out for a time sufficient to (i) allow the surfactant to diffuse into and through the one or more dialysis membranes, and (ii) allow the free aminoglycoside to diffuse through the one or more dialysis membranes into the dissolution medium. In a further embodiment, the dissolution apparatus is a USP Apparatus 2 (paddle).

[0011] In another embodiment, step (a) of operating the dissolution device includes placing or positioning one or more dialysis devices (e.g., USP device 2 (paddles) or stir bars) in the dissolution medium within the dissolution vessel containing the dissolution device, and is carried out for a time sufficient to (i) allow the surfactant to diffuse into and through the one or more dialysis membranes, and (ii) allow the free aminoglycoside to diffuse through the one or more dialysis membranes and into the dissolution medium. In one embodiment, the one or more dialysis devices are immersed in the dissolution medium and are buoyant in the dissolution medium. The one or more dialysis devices can be fully or partially immersed in the dissolution medium and do not interfere with the dissolution device during operation. In another aspect of the IVR method for evaluating an aminoglycoside release profile from a liposomal aminoglycoside formulation, the method includes adding a predetermined amount of a surfactant (e.g., a surfactant and a buffer (e.g., a dissolution medium comprising a surfactant and a buffer)) to the liposomal aminoglycoside formulation at each predetermined time point to form a release solution at each predetermined time point, removing an aliquot of the release solution at a predetermined time interval (e.g., at 30 minutes) after each addition, and analyzing each aliquot for total and free aminoglycoside content. The method may further include removing an aliquot of the release solution at one or more predetermined time points after the final addition of surfactant and analyzing for total and free aminoglycoside content. In one embodiment, the analysis of total and free aminoglycoside content is performed by HPLC.

[0012] In one embodiment of this aspect, the IVR method comprises: (a) adding a predetermined amount of a dissolution medium comprising a surfactant and a buffer to a predetermined amount of a liposomal aminoglycoside formulation to obtain a release solution; (b) removing an aliquot of the release solution after a predetermined time interval and analyzing the aliquot for total and free aminoglycoside content; (c) increasing the ratio of surfactant to liposomal aminoglycoside formulation in the release solution (e.g., by adding a dissolution medium); (d) removing an aliquot of the release solution after a predetermined time interval and analyzing the aliquot for total and free aminoglycoside content; (e) optionally repeating steps (c) and (d) one or more times; (f) optionally repeating step (d) one or more times. In one embodiment, step (e) is performed a total of 3, 4, 5, or 6 times. In yet another embodiment, step (e) is performed 3 times and then step (f) is performed once. In a further embodiment, the liposomal aminoglycoside formulation is an ALIS formulation.

[0013] The IVR methods described herein are useful for characterizing liposomal aminoglycoside formulations and manufacturing conditions used to make liposomal aminoglycoside formulations, including liposomal amikacin formulations. The aminoglycoside can be in free base form or salt form, such as aminoglycoside sulfate (e.g., amikacin sulfate). The release profile generated by the IVR methods described herein reflects a product that actively leaks over a period of time (rather than instantaneously) and demonstrates the ability to completely release the encapsulated aminoglycoside. The IVR methods described herein can be completed within a normal workday, are distinguishable (i.e., they can distinguish batches of liposomal aminoglycoside formulations that have significantly different release rates from those that have sufficient quality, for example, due to differences in lipid to aminoglycoside weight ratios), and are adaptable to implementation in a quality control environment. Furthermore, the conditions and media used in the methods have physiological relevance.

[0014] A typical leakage profile of a liposomal aminoglycoside formulation under specific conditions can be established using the IVR method and then used to verify the consistency of quality of liposomal aminoglycoside formulations manufactured in different batches, different processes, or at different scales. Preferably, leakage at the beginning of the test is low, but leakage at the end of the test approaches about 100% or is complete leakage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Aspects of the invention relate to in vitro release (IVR) methods for assessing aminoglycoside release from liposomal aminoglycoside formulations in which the aminoglycoside is encapsulated within a plurality of liposomes. In embodiments described herein, the IVR method has one or more of the following characteristics: The IVR method does not represent the in vivo release profile of aminoglycosides from liposomal aminoglycoside formulations. The IVR profile obtained from this method reflects a liposomal product that actively releases aminoglycosides over time. · The IVR method demonstrates the ability to release all or substantially all of the aminoglycoside from the liposomal aminoglycoside formulation. · The conditions employed in IVR methods have some physiological references. · IVR methods are adaptable for implementation in a quality-controlled environment. · IVR procedures can be completed within a normal workday. IVR techniques do not induce an initial burst of aminoglycoside release from liposomal formulations, as this may mask evidence of "dose dumping" of the formulation.

[0016] The IVR method described herein is based in part on increasing the permeability of liposomes to aminoglycosides, such as amikacin, by surfactant binding and / or surfactant interaction with liposome membrane.Unlike other methods, aminoglycoside release by the IVR method described herein is not based on liposome membrane rupture.It has been found that the use of serum to dissolve liposome membrane is insufficient to induce nearly complete aminoglycoside release from net neutrally charged liposomes. Surprisingly, the high temperature and low osmolarity that served as the basis of previous IVR methods were insufficient to sufficiently increase the permeability of the lipid membrane of liposomes to affect complete aminoglycoside release from the liposomal aminoglycoside formulations described herein.As a result, the methods provided herein employ active liposomal membrane disruption to increase permeability.The active disruption technique employed herein increases membrane permeability without disrupting the membrane.In one embodiment, active disruption occurs by adding a surfactant to the liposomal aminoglycoside formulation.

[0017] Furthermore, there is biological relevance in accelerating the release of aminoglycosides from liposomal aminoglycoside formulations using surfactant disruptors. Although the exact molecular interactions between liposomes and biological agents have not been fully elucidated, it was found that the release of aminoglycosides from liposomal aminoglycoside formulations was significantly accelerated upon ex vivo incubation with liquefied Pseudomonas-infected sputum samples from cystic fibrosis patients (Meers et al. (2008). Journal of Antimicrobial Chemotherapy 61, pp. 859-868, the disclosure of which is incorporated herein by reference in its entirety for all purposes). A substantial portion of this activity was found to be associated with two small organic solvent-soluble molecules, (i) monorhamnolipids and (ii) dirhamnolipids.

[0018] In one aspect of the invention, an IVR method is provided. In one embodiment of this aspect, the liposomal aminoglycoside formulation is present in one or more dialysis devices, each having a predetermined volume and a dialysis membrane. In one embodiment, the predetermined volume of one dialysis device is 5 mL. In another embodiment, the predetermined volume of one dialysis device is 10 mL. The one or more dialysis devices are present in a container having a dissolution device and a dissolution medium disposed therein. The dissolution medium includes a surfactant. In an embodiment described herein, the one or more dialysis devices are immersed or substantially immersed in the dissolution medium while being buoyant in the dissolution medium. The dissolution device is operated, and the surfactant diffuses through the dialysis membrane(s) to trigger the release of the aminoglycoside from the liposomes. The free aminoglycoside then diffuses through the dialysis membrane into the external dissolution medium, from which aliquots are taken at predetermined time intervals and tested for free aminoglycoside content. Selection of an appropriate molecular weight cut-off (MWCO) dialysis membrane prevents diffusion of the liposomes into the external dissolution medium while allowing sampling of only free amikacin from the external dissolution medium. In one embodiment, analyzing the aliquot for free aminoglycoside content comprises performing high performance liquid chromatography (HPLC) on the aliquot. In a further embodiment, HPLC is performed using evaporative light scattering detection (ELSD) or charged aerosol detection (CAD) to determine the free aminoglycoside content. In a further embodiment, HPLC is performed to determine the total aminoglycoside content.

[0019] In another aspect, an IVR method is provided that is based in part on a stepwise approach in which additional surfactant is added to the liposomal aminoglycoside formulation over time. Without wishing to be bound by theory, it is believed that this approach simulates the continuous accumulation of proteins and other biological factors that interact with the liposomal membrane when the liposomal aminoglycoside formulation is administered to a patient, facilitating complete aminoglycoside release within a time frame appropriate for quality control analysis. In one embodiment of this aspect, the time frame appropriate for quality control analysis is about 2 hours to about 5 hours, or about 2.5 hours to about 5 hours, or about 3 hours to about 5 hours, or about 2.5 hours to about 4 hours. In a further embodiment, the time frame appropriate for quality control analysis is about 3 hours. In one embodiment, the time frame appropriate for quality control analysis is a time point that allows for subsequent immediate processing and testing of an aliquot of the resulting release solution within the same day to avoid storage and potential changes in the composition of the post-release aliquot. Additional aspects and embodiments of the invention are discussed in detail below.

[0020] definition Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The term "comprising" is open-ended and, in the context of a composition, refers to the elements listed. When used in the context of the compositions described herein, the term "comprising" can alternatively encompass compositions "consisting essentially of" or "consisting of" the recited components. As used herein, the term "liposome(s)" refers to a completely closed lipid bilayer membrane that contains an enclosed aqueous volume. Liposomes can be unilamellar vesicles (possessing a single 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) "tail" of the lipid monolayer faces toward the center of the bilayer, while the hydrophilic "head" faces toward the aqueous phase.

[0021] The term "aminoglycoside-encapsulated liposome" or "liposomal aminoglycoside" refers to a liposome encapsulating an aminoglycoside, which may be in any form, such as a salt form or a free base form. In one embodiment, the aminoglycoside is amikacin or amikacin sulfate. In embodiments described herein, the liposomal aminoglycoside formulation comprises an aminoglycoside encapsulated in multiple liposomes. The term "amikacin-encapsulating liposome" or "liposomal amikacin" refers to a liposome that encapsulates amikacin, which can be in any form, such as a salt form. In one embodiment, the liposome encapsulates amikacin sulfate. In the embodiments described herein, the liposomal amikacin formulation comprises amikacin encapsulated in multiple liposomes. The term "encapsulated" refers to an aminoglycoside that is associated with a liposome, either (i) as part of a complex with the liposome, (ii) within the aqueous phase of the liposome, (iii) within the hydrophobic liposome bilayer, (iv) at the interfacial headgroup region of the liposome bilayer, or (v) a combination thereof. The term "amikacin liposome inhalation suspension" or "ALIS" refers to an amikacin-encapsulated liposome formulation in which amikacin is present as amikacin sulfate and the lipid components of the liposomes consist of dipalmitoylphosphatidylcholine (DPPC) and cholesterol. ALIS contains amikacin at a concentration of about 70 mg / mL (amikacin base, e.g., 70 mg / mL ± 10%), about 40-56 mg / mL of total lipid (expressed as DPPC and cholesterol) (e.g., about 47 mg / mL of total lipid, and DPPC and cholesterol at a weight ratio of about 2:1 (DPPC:cholesterol), and a lipid to amikacin weight ratio of about 0.60 (lipid):1 (amikacin) to about 0.80 (lipid):1 (amikacin) (e.g., expressed as about 0.65 (lipid):1 (amikacin) to about 0.75 (lipid):1 (amikacin)).

[0022] As used herein, the term "dissolution medium" includes detergents and buffers, and in some instances is referred to herein as a "detergent / buffer mixture." In one embodiment, the detergent is mixed with the buffer to obtain the dissolution medium. In one embodiment, the dissolution medium is equilibrated prior to use. Described herein is a method for evaluating the aminoglycoside release profile from a liposomal aminoglycoside formulation. The liposomal aminoglycoside formulation subjected to the method described herein comprises an aminoglycoside or a pharma- ceutically acceptable salt thereof encapsulated in a plurality of liposomes. In one embodiment, the lipid component of the plurality of liposomes is composed of electrically neutral lipids. In one aspect of the IVR method described herein, one or more dialysis devices, dissolution devices, and dissolution media are used. The liposomal aminoglycoside formulation is present in one or more dialysis devices, each dialysis device having a predetermined volume and dialysis membrane. The liposomal aminoglycoside formulation is present in one or more dialysis devices, and the device(s) are placed in a container that includes (i) a detergent-enriched dissolution medium, and (ii) a dissolution device immersed in the dissolution medium. In one embodiment, the one or more dialysis devices are immersed in the dissolution medium and are buoyant and do not interfere with the dissolution device when it is operated. Without wishing to be bound by theory, bidirectional passage occurs across the one or more dialysis membranes during operation of the dissolution device, for example, detergent diffuses through the dialysis membrane(s) into the one or more dialysis devices over time, which permeabilizes the liposomal bilayer of the liposomes and induces free aminoglycoside release. The free aminoglycoside diffuses through the dialysis membrane(s) into the dissolution medium over time. The dissolution apparatus is operated and aliquots of the dissolution medium are removed at timed intervals for analysis of free aminoglycoside levels. The method may also employ a solid phase extraction (SPE) process to remove excess detergent from the aliquots prior to free aminoglycoside analysis.

[0023] In one embodiment, a dialyzer (eg, one dialyzer or two dialyzers) is inserted through the center of the buoyancy ring(s) to maintain buoyancy in the dissolution medium. In one embodiment of this aspect, the IVR method is carried out using a dissolution apparatus, e.g., United States Pharmacopeia (USP) Apparatus 2 (paddle) (see United States Pharmacopeia (USP) General Chapters Dissolution (2011), the disclosure of which is incorporated by reference in its entirety for all purposes), or stirring using a stir plate with a stir bar. In further embodiments, the IVR method is carried out at a controlled temperature, e.g., about 30°C to about 40°C, about 32°C to about 40°C, about 34°C to about 40°C, or about 36°C to about 40°C. In a preferred embodiment, the IVR method is carried out at 37°C ± 3°C. In one embodiment, the dissolution apparatus is run at about 75 to about 150 rpm (e.g., 150 rpm). In one embodiment, the dissolution apparatus is equipped with a paddle or stir bar and is operated at about 75 rpm to about 150 rpm (e.g., 150 rpm). In one embodiment, the surfactant is added at a concentration above its critical micelle concentration (CMC). In one embodiment, the predetermined volume of the dialysis device is 5 mL. In another embodiment, the predetermined volume of the dialysis device is 10 mL. In yet another embodiment, the predetermined volume of the dialysis device is about 4 mL to about 11 mL, or about 5 mL to about 10 mL.

[0024] In one embodiment, the IVR method comprises: (a) operating a dissolution device within a dissolution vessel, the dissolution vessel containing (i) a predetermined amount of a liposomal aminoglycoside formulation in one or more dialysis devices, and (ii) a predetermined amount of a detergent (e.g., 5% Triton X-100 in DPBS), each dialysis membrane being permeable to an aminoglycoside, a dissolution medium, and the detergent and dissolution device being immersed in the dissolution medium within the dissolution vessel; (b) removing an aliquot of the dissolution medium after a predetermined time interval; (c) analyzing the aliquot for free aminoglycoside content; (d) optionally repeating steps (b) and (c) after one or more additional time intervals.

[0025] In one embodiment, operating the dissolution apparatus includes stirring the dissolution medium. In a further embodiment, stirring is performed using a dissolution apparatus that includes a paddle (e.g., USP Apparatus 2). In another embodiment, stirring is performed using a dissolution apparatus that includes a stir bar. In a further embodiment, the dissolution vessel is placed on a stir plate and prior to operating the stir bar. In one embodiment, step (a) of operating the dissolution apparatus is carried out for a time sufficient to (i) allow the surfactant to diffuse into and through the one or more dialysis membranes, and (ii) allow the free aminoglycoside to diffuse through the one or more dialysis membranes into the dissolution medium. In another embodiment, step (a) of operating the dissolution device comprises placing or placing one or more dialysis devices in the dissolution medium in a dissolution vessel containing a dissolution device (e.g., USP Apparatus 2 (paddle) or stir bar), and the dissolution device is operated for a time sufficient to (i) allow the surfactant to diffuse into and through the one or more dialysis membranes, and (ii) allow the free aminoglycoside to diffuse through the one or more dialysis membranes into the dissolution medium. It should be understood that when a stir bar is used as the dissolution device, the vessel is placed on a stir plate to operate the dissolution device.

[0026] Before operating the dissolution apparatus, perform the following steps: (i) adding a predetermined amount of a liposomal aminoglycoside formulation to one or more dialysis devices; and (ii) placing one or more dialysis membranes in a dissolution vessel having a dissolution medium containing a predetermined amount of surfactant may be performed. In one embodiment, the surfactant is equilibrated in the dissolution vessel prior to step (a). Equilibration can occur with or without stirring. In a further embodiment, the initial equilibration is carried out at about 30°C to about 40°C, for example, about 35°C to about 40°C, or about 37°C.

[0027] In one embodiment, the liposomal aminoglycoside formulation is equilibrated to ambient temperature for at least about 45 minutes, e.g., before adding the formulation to one or more dialysis devices or operating the dissolution device. In a further embodiment, prior to step (a), the liposomal aminoglycoside formulation is shaken and / or vortexed until it appears homogeneous and well mixed by visual inspection. In a further embodiment, the liposomal aminoglycoside formulation is transferred to a separate dissolution vessel to provide a predetermined amount of the liposomal aminoglycoside formulation used in step (a) and equilibrated with agitation (e.g., a USP dissolution apparatus with paddles or a stir plate with a stir bar). In one embodiment, equilibration is performed for about 30, about 60, about 90, about 120, or about 150 minutes. In a further embodiment, equilibration with agitation is performed at about 30°C to about 40°C, e.g., about 35°C to about 40°C, or about 37°C.

[0028] In one embodiment, prior to step (a), the liposomal aminoglycoside formulation and / or dissolution medium (comprising a surfactant in a buffer) are equilibrated in a separate dissolution vessel, e.g., for at least 30 minutes. In one embodiment, the liposomal aminoglycoside formulation is equilibrated to ambient temperature (e.g., for at least 45 minutes). In one embodiment, the dissolution medium is equilibrated to about 37° C. with agitation at about 75 rpm to about 150 rpm using a paddle or stir bar (e.g., for at least 30 minutes). In a further embodiment, an evaporation cover is placed over the vessel containing the dissolution medium during equilibration. In one embodiment, the dialysis membrane is composed of cellulose ester. In one embodiment, the one or more dialysis devices are one or more Float-A-Lyzer dialysis devices (e.g., available from Repligen Corp., Rancho Dominguez, California, USA). In one embodiment, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the free aminoglycoside (such as amikacin) diffuses across the membrane within about 12 or 24 hours during the IVR procedure. In one embodiment, the dialysis membrane has a molecular weight cut-off (MWCO) of about 20 kD to about 1500 kD, such as about 100 kD to about 1000 kD, such as about 20 kD, about 100 kD, about 300 kD or about 1000 kD. In a preferred embodiment, the dialysis membrane has a MWCO of about 1000 kD. Preferably, the dialysis membrane has a MWCO that allows free aminoglycosides, surfactants, and dissolution media to move freely across the dialysis membrane while preventing or substantially preventing liposomes from passing through the membrane.

[0029] In one embodiment, a single dialysis device is used in the IVR method. In a further embodiment, the dialysis device comprises a cellulose ester membrane having a MWCO of about 1000 kD. The cellulose ester membrane can comprise a single ester type or multiple ester types. In one embodiment, the IVR method includes (a) adding a predetermined amount of the liposomal aminoglycoside formulation to two dialysis devices, such as two Float-A-Lyzer dialysis devices (e.g., each having a MWCO of about 1000 kD). In a further embodiment, each dialysis device has a predetermined volume of about 5 mL or about 10 mL. In another embodiment, each dialysis device has a predetermined volume of about 5 mL to about 10 mL.

[0030] In one embodiment using two dialysis devices, each device has a predetermined volume of about 5 mL and a cellulose ester membrane. In a further embodiment, the volume of the liposomal aminoglycoside formulation is about 8 mL to about 10 mL and is divided approximately equally between each device. In a further embodiment, the volume of the liposomal aminoglycoside formulation is about 8 mL to about 9 mL, for example, about 8.4 mL. When two dialysis devices are used, the liposomal aminoglycoside formulation subjected to the IVR method can be divided equally or substantially equally between the two devices. In one embodiment, a single bundle float ring is used to hold both dissolution device floatants in the dissolution medium while the dissolution devices are operated.

[0031] The aliquots can be removed at various times, such as 1, 2, 6, and 24 hours after the start of the dissolution test. In another embodiment, the aliquots are removed 1, 2, 3, 6, and 24 hours after the start of the dissolution test. In yet another embodiment, the aliquots are removed 1, 2, 4, 6, and 24 hours after the start of the dissolution test. In yet another embodiment, the aliquots are removed 1, 2, 3, 4, 6, and 24 hours after the start of the dissolution test. In yet another embodiment, the aliquots are removed 1, 3, 4, 6, and 24 hours after the start of the dissolution test. In yet another embodiment, the aliquots are removed 1, 2, 3, 4, and 24 hours after the start of the dissolution test. In yet another embodiment, the aliquots are removed 1, 2, 3, 4, and 24 hours after the start of the dissolution test. In one embodiment, the aliquots removed from the dissolution medium in step (b) are subjected to solid phase extraction (SPE) before analyzing the aliquots for free aminoglycoside content. SPE can be used to remove excess detergent before analysis of the aliquots. SPE can be performed using a cation exchange sorbent material.

[0032] In one embodiment, the SPE filter (e.g., SPE cartridge) comprises a strong cation exchange material, such as, for example, Oasis MCX (1 cc / 30 mg) (available from Waters of Milford, Mass.). Without wishing to be bound by theory, the inventors theorize that the protonated aminoglycoside (such as amikacin) is retained on the SPE column while the detergent (e.g., Triton X-100, e.g., 5% Triton X-100 in PBS) may be washed away. The aminoglycoside (such as amikacin) is then eluted with a basic elution solution to obtain an aminoglycoside (such as amikacin) aliquot that may then be analyzed. A general SPE process is shown in the diagram below.

[0033] [Table 1]

[0034] After SPE, the amount of free aminoglycoside (eg, amikacin) can be determined by HPLC analysis. Some of the preferred parameters for this IVR method are: (i) dissolution apparatus: USP Apparatus 2 (paddle) operated at 150 RPM; (ii) dissolution media: 5% octylphenol ethoxylate (Triton X-100) in DPBS 1x maintained at about 37°C; (iii) media volume is about 900 mL in the dissolution vessel at a pH of about 7.1; (iv) two Float-A Lyzer dialysis devices with 1000 kD MWCO membrane (cellulose ester membrane) are used per vessel to allow testing of the entire amount of one vial of ALIS drug product (i.e., total dose of 8.4 mL); and (v) SPE is performed on sample pulls (aliquots) to remove octylphenol ethoxylate (Triton X-100) from the aliquots prior to free aminoglycoside analysis by HPLC.

[0035] In another preferred embodiment, the IVR method comprises: (a) adding a predetermined amount of a liposomal aminoglycoside (e.g., amikacin) formulation to a dialysis device containing a cellulose ester membrane; (b) placing the dialyzer in a dissolution vessel containing a dissolution medium comprising a predetermined amount of a surfactant (e.g., octylphenol ethoxylate) in a buffer solution (e.g., phosphate buffered saline or Dulbecco's phosphate buffered saline); (c) allowing the surfactant to diffuse through the dialysis membrane into the liposomal aminoglycoside (e.g., amikacin) formulation, thereby facilitating release of the aminoglycoside from the liposomes; (d) allowing free amikacin to diffuse through the cellulose ester membrane into a dissolution medium; (e) removing one or more aliquots of the dissolution medium at predetermined time(s) (e.g., four (4) or more, 1, 2, 3, 4, 6, 12, and 24 hours); (f) analyzing the aliquot for free aminoglycoside (e.g., amikacin) content. Prior to analysis for free aminoglycoside content, an aliquot can be subjected to SPE to remove detergents such as cation exchange sorbent materials. In one embodiment, the vessel includes a dissolution device that is operated once the dialysis device is placed in the dissolution vessel.

[0036] One preferred embodiment is a method for measuring the release profile of an aminoglycoside from a liposomal aminoglycoside formulation comprising a plurality of liposome-encapsulated aminoglycosides, the method comprising: (a) adding a predetermined amount of the liposomal aminoglycoside formulation to one dialysis machine or dividing it among two or more dialysis machines, each dialysis machine having a predetermined volume and a dialysis membrane made of a cellulose ester or a combination of cellulose esters; (b) placing one or more dialysis devices in a dissolution vessel containing a predetermined amount of octylphenol ethoxylate in Dulbecco's phosphate buffered saline and a dissolution device; (c) operating a dissolution apparatus within the dissolution vessel, the dissolution apparatus being a USP Apparatus 2 (paddle) operated at approximately 150 rpm and 37° C.; (d) removing an aliquot of the dissolution medium after a predetermined time interval; (e) analyzing the aliquot for free aminoglycoside content; (f) optionally repeating steps (d) and (e) after one or more additional time intervals. In another embodiment of the invention, the IVR method uses a stepwise approach in which a surfactant is initially added to a predetermined amount of liposomal aminoglycoside formulation to obtain a release solution, and additional surfactant is added to the release solution at one or more subsequent time points to increase the surfactant to lipid ratio.

[0037] In one embodiment of this aspect, the IVR method comprises: (a) adding a predetermined amount of a dissolution medium comprising a surfactant and a buffer to a predetermined amount of a liposomal aminoglycoside formulation to obtain a release solution; (b) removing an aliquot of the release solution after a predetermined time interval and analyzing the aliquot for total and free aminoglycoside content; (c) increasing the ratio of surfactant to liposomal aminoglycoside formulation in the release solution (e.g., by adding surfactant, e.g., to the dissolution medium); (d) removing an aliquot of the release solution after a predetermined time interval and analyzing the aliquot for total and free aminoglycoside content; (e) optionally repeating steps (c) and (d) one or more times; (f) optionally repeating step (d) one or more times. Each addition of surfactant may be performed, for example, by adding a dissolution medium containing the surfactant in a buffer solution. In one embodiment, each addition of surfactant has the same dissolution medium, but the amount (volume) of dissolution medium may vary for each addition. In another embodiment, each addition of surfactant is performed with a mixture containing the same surfactant and the same buffer solution, but (i) the amount of dissolution medium and / or (ii) the amount of surfactant in the dissolution medium may vary for each addition.

[0038] In one embodiment, the method is carried out while operating a dissolution apparatus within a vessel. In one embodiment, the amount of liposomal aminoglycoside formulation is an amount pooled from two or more vials of formulation derived from the same manufacturing batch. Without wishing to be bound by theory, it is believed that because the liposomal aminoglycoside formulation is a homogenous suspension, the composite of the multiple vials will be equally reflective of each batch. In a further embodiment, step (e) is performed 1, 2, 3, or 4 times. In a further embodiment, step (e) is performed 3 times. In one embodiment, the liposomal aminoglycoside formulation is an ALIS formulation. The stepwise increase in the ratio of surfactant to liposomal aminoglycoside formulation in the release solution is designed in one embodiment to induce a generally logarithmic curve of the surfactant to lipid ratio with successive additions.Thus, in one embodiment, the volume of surfactant added is increased each time step (c) is performed.In one embodiment, the increase in surfactant volume is added to provide a sufficient and / or necessary surfactant concentration to increase liposome membrane permeability.

[0039] In another embodiment, the IVR method comprises: (a) adding a predetermined amount of a dissolution medium comprising a surfactant in a buffer to a predetermined amount of a liposomal aminoglycoside formulation to obtain a release solution; (b) removing an aliquot of the release solution after a predetermined time interval and analyzing the aliquot for total and free aminoglycoside content; (c) adding a predetermined amount of dissolution medium to the release solution; (d) removing an aliquot of the release solution after a predetermined time interval and analyzing the aliquot for total and free aminoglycoside content; (e) optionally repeating steps (c) and (d) one or more times; (f) optionally repeating step (d) one or more times.

[0040] In another embodiment, the IVR method comprises: (a) adding 0.5 mL of dissolution medium to 30 mL of the liposomal aminoglycoside formulation in a vessel equipped with a dissolution device to form a release solution; (b) 30 minutes after step (a) [time=0.5 hours], (i) removing 3 mL of the release solution and analyzing it for total and free aminoglycoside content, (ii) adding 1.7 mL of dissolution medium to the remaining portion of the release solution; (c) 30 minutes after step (b) [time=1 hour], (i) removing 3 mL of the release solution and analyzing it for total and free aminoglycoside content, (ii) adding 4.0 mL of dissolution medium to the remaining portion of the release solution; (d) 30 minutes after step (c) [time=1.5 hours], (i) removing 3 mL of the release solution and analyzing it for total and free aminoglycoside content, (ii) adding 16 mL of dissolution medium to the remaining portion of the release solution; (e) 30 minutes after step (d) [time=2 hours], (i) removing 20 mL of the release solution and analyzing it for total and free aminoglycoside content; and (ii) adding 100 mL of dissolution medium to the remaining portion of the release solution; (f) 30 minutes after step (e) [time=2.5 hours], removing 20 mL of the release solution and analyzing it for total aminoglycoside content and free aminoglycoside content; (g) 30 minutes after step (f) [time=3 hours], removing 20 mL of the release solution and analyzing it for total aminoglycoside content and free aminoglycoside content; The dissolution medium comprises octylphenol ethoxylate (e.g., as Triton™ X-100) in phosphate buffered saline (PBS) (such as Dulbecco's phosphate buffered saline), and a dissolution apparatus is operated during the method. In yet a further embodiment, the aminoglycoside is amikacin or a pharma- ceutically acceptable salt thereof (e.g., amikacin sulfate). In a further embodiment, the dissolution apparatus is a USP Apparatus 2 (paddle), and the method is carried out at about 37°C (e.g., 37°C±3°C) at 150 (±20) rpm. In one embodiment, the octylphenol ethoxylate in PBS is 200 ppm octyl mononol ethoxylate in PBS pH 7.2 or 7.4. In a further embodiment, the PBS pH is about 7.2.

[0041] In one embodiment, the dissolution medium is equilibrated in the dissolution vessel prior to step (a), with or without stirring, hi a further embodiment, the initial equilibration is at about 30°C to about 40°C, e.g., about 35°C to about 40°C, or about 37°C. In one embodiment of the IVR method provided herein, the liposomal aminoglycoside formulation is equilibrated to ambient temperature, for example, for at least about 45 minutes prior to step (a). In a further embodiment, prior to step (a), the liposomal aminoglycoside formulation is shaken and / or vortexed until it appears homogenous and is well mixed. In a further embodiment, the liposomal aminoglycoside formulation is transferred to a separate dissolution vessel to provide a predetermined amount of the liposomal aminoglycoside formulation used in step (a) and equilibrated with agitation using a stirring member (e.g., a USP dissolution apparatus with a paddle or a stir plate with a stir bar). In one embodiment, the equilibration is performed for about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, or about 150 minutes. In a further embodiment, the equilibration with agitation is performed at about 30°C to about 40°C, for example, about 35°C to about 40°C, or about 37°C.

[0042] In one further embodiment of the method described herein, prior to step (a), the liposomal aminoglycoside formulation and / or dissolution medium (including surfactant in a buffer) are equilibrated in a separate dissolution vessel, e.g., for at least 30 minutes. In one embodiment, the liposomal aminoglycoside formulation is equilibrated to ambient temperature (e.g., for at least 45 minutes). In one embodiment, the dissolution medium is equilibrated to about 37° C. with stirring at about 75 RPM to about 150 RPM using a stirring member such as a paddle or stir bar (e.g., for at least 30 minutes). In a further embodiment, an evaporation cover is placed on the vessel during equilibration. In the methods described herein, the surfactant present in the dissolution medium can be any type of surfactant, such as an anionic surfactant or a nonionic surfactant. In one embodiment, the surfactant is nonionic. In a further embodiment, the surfactant comprises a hydrophilic polyethylene oxide chain. In a further embodiment, the surfactant comprising a hydrophilic polyethylene oxide chain further comprises an aromatic hydrocarbon group that is lipophilic or hydrophobic. In one embodiment, the surfactant comprises octylphenoxypolyethoxyethanol (sold under the trade name Nonidet P-40 or IGEPAL CA). In one embodiment, the surfactant is a nonionic surfactant. In a further embodiment, the nonionic surfactant is octylphenol ethoxylate (available as Triton™ X-100 from Sigma Aldrich, St. Louis, MO). Suitable anionic surfactants for use in the dissolution medium used herein include rhamnolipids. In one embodiment, the surfactant has a critical micelle concentration (CMC) of less than about 5 mM, less than about 2 mM, or less than about 1 mM. For example, the non-ionic surfactant can have a CMC of about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, or 0.5 mM, or a CMC in the range of about 0.1 to about 1 mM, such as about 0.1 to about 0.5 mM.

[0043] The dissolution medium contains a surfactant and a buffer. In one embodiment, the surfactant is dissolved in a buffer to provide the dissolution medium. Suitable buffers include, but are not limited to, phosphate buffered saline, such as Dulbecco's phosphate buffered saline (DPBS). In a preferred embodiment, the dissolution medium is octylphenol ethoxylate in DPBS. The dissolution medium may be free or substantially free of alcohol, such as ethanol (e.g., containing less than about 5%, e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.1%, less than about 0.05%, or less than about 0.01% v / v). In one embodiment, the dissolution medium used in the method involving the use of a dialysis membrane comprises about 0.5 to about 10% v / v, about 1% to about 5% or 6% (v / v) Triton X-100 (octylphenol ethoxylate) in phosphate buffered saline (PBS) (such as Dulbecco's phosphate buffered saline, DPBS). In one embodiment, the dissolution medium comprises about 5% (v / v) octylphenol ethoxylate in phosphate buffered saline (PBS) (such as Dulbecco's phosphate buffered saline). In a preferred embodiment, the dissolution medium is 5% (v / v) octylphenol ethoxylate in phosphate buffered saline (PBS) (such as Dulbecco's phosphate buffered saline DPBS 1×) without magnesium and calcium (e.g., phosphate buffered saline (PBS) (such as Dulbecco's phosphate buffered saline).

[0044] The method is preferably carried out with a stirring member (e.g., paddle, stir bar) in the release solution or dissolution medium. In a preferred embodiment, the method is carried out with USP Apparatus 2 (paddle) at about 37° C. operated at about 150 rpm (i.e., the release solution / dissolution medium is in a vessel containing USP Apparatus 2 (paddle) operated at 150 rpm±20 rpm at 37° C.). In one embodiment, the IVR method is carried out in a 150 mL vessel. In one embodiment, the dissolution medium has a pH of about 7.1 or 7.2 to about 7.4. In one embodiment, the dissolution medium has a pH of about 7.2. In another embodiment, the dissolution medium has a pH of about 7.4.

[0045] In a preferred embodiment of any of the methods described herein, the aminoglycoside is amikacin or a pharma- ceutically acceptable salt thereof, such as amikacin sulfate. In a preferred embodiment of any of the methods described herein, the dissolution medium comprises octylphenol ethoxylate in PBS, such as DPBS, at a pH of, for example, about 7.1, about 7.2, or about 7.4. In a further embodiment of any of the methods described herein, the dissolution medium comprises about 20 to about 600 ppm of octylphenol ethoxylate in DPBS, such as about 100, 150, 200, 250, 300, 350, or 400 ppm of octylphenol ethoxylate. The octylphenol ethoxylate may have an ethoxylate molar content of about 4 to about 16, such as about 8 to about 12. In a preferred embodiment, the octylphenol ethoxylate has an ethoxylate molar content of about 9.5. In another preferred embodiment, the dissolution medium comprises about 200 ppm of octylphenol ethoxylate with an ethoxylate molar content of about 9.5 in DPBS. In one embodiment, the dissolution medium comprises (by volume) 5% surfactant in a buffer, for example 5% octylphenol ethoxylate in PBS.

[0046] In further embodiments of any of the methods described herein, aliquots are stored at about 2-8 °C prior to further analysis, e.g., analysis of free and / or total aminoglycoside (e.g., amikacin) content. In further embodiments of any of the methods described herein, removed aliquots of the release solution are tested by HPLC analysis to determine free amikacin content and, optionally, total amikacin content within one week of their collection. In one embodiment of any of the methods described herein, a removed aliquot of the dissolution medium or release solution is stored at about 2-8° C. (until tested) and tested by HPLC analysis to determine free amikacin content and optionally total amikacin content within one week of taking the aliquot.

[0047] During manufacturing, individual batches of liposomal aminoglycoside formulations can be tested using the in vitro methods described herein. For example, one embodiment is a method for preparing a liposomal aminoglycoside formulation that includes aminoglycoside-encapsulated liposomes. The method includes: (a) obtaining a manufacturing batch of a liposomal aminoglycoside formulation comprising an aminoglycoside-encapsulated liposome; (b) determining the release profile of a sample of the formulation within the batch by any of the IVR methods described herein; (c) optionally, if the release profile meets predetermined criteria, incorporating some or all of the remaining batch of the liposomal aminoglycoside formulation into one or more dosage forms (e.g., vials).

[0048] Liposomal aminoglycoside preparations The liposomal aminoglycoside formulation comprises an aminoglycoside or a pharma- ceutically acceptable salt thereof encapsulated in a plurality of liposomes, and, optionally, an aminoglycoside in free form (i.e., unencapsulated). In a preferred embodiment, the aminoglycoside is amikacin or a pharma- ceutically acceptable salt thereof (e.g., amikacin sulfate). The percentage of liposomal aminoglycoside refers to the percentage of aminoglycoside present in the liposomal aminoglycoside formulation that is encapsulated in liposomes (based on 100% total content of aminoglycosides in any form in the formulation). The total and free aminoglycoside content in the liposomal aminoglycoside formulation (such as before or after nebulization) can be measured by high performance liquid chromatography (HPLC). In one embodiment, HPLC is performed using evaporative light scattering detection (ELSD) or charged aerosol detection (CAD) to determine the total and free aminoglycoside content.

[0049] In one embodiment, the percentage of liposomally associated aminoglycoside in the formulation subjected to one of the IVR methods described herein ranges from about 70% to about 100%. In another embodiment, the percentage of liposomally associated aminoglycoside in the formulation subjected to one of the IVR methods described herein ranges from about 80% to about 100%. In yet another embodiment, the percentage of liposomally associated aminoglycoside in the liposomal aminoglycoside formulation (prior to performing the IVR method) ranges from about 80% to about 100%, about 80% to about 99%, about 90% to about 100%, about 90% to about 99%, or about 95% to about 99%. In one embodiment, at least about 95% of the aminoglycoside is liposomally associated prior to being subjected to one of the IVR methods described herein. In a further embodiment, at least about 97% of the aminoglycoside is liposomally associated prior to being subjected to one of the IVR methods described herein. The percentage of liposome-associated aminoglycoside in the formulation will vary when performing one of the IVR methods described herein, depending on the starting materials and the conditions used to perform a particular IVR method. In one embodiment, the liposomal aminoglycoside formulation has one of the following release profiles according to the IVR method described in Example 1. The release profiles provided below can be an average of multiple samples from a single batch or from a single sample from a batch. In one embodiment, samples can be pooled from multiple vials (such as 6 or 12 vials) from the same liposomal aminoglycoside batch.

[0050] [Table 2]

[0051] In one embodiment, the liposomal aminoglycoside formulation (e.g., ALIS) releases (i) about 20% or less at 0.5 hours, (ii) about 35% or less at 1.0 hours, (iii) about 50% or less at 1.5 hours, (iv) about 65% or less at 2.0 hours, (v) about 75% or less at 3.0 hours, (vi) about 83% or less at 4.0 hours, (vii) about 84% or less at 6.0 hours, (viii) about 97% or less at 12.0 hours, (ix) about 70% or less at 24 hours, or (x) any combination of any of the foregoing, where percent aminoglycoside released = [free aminoglycoside (mg / mL) / total aminoglycoside (mg / mL)] x 100%, according to the IVR method described in Example 2. The release profile provided can be an average of multiple samples from a single batch or from a single sample from a batch. In one embodiment, samples can be pooled from multiple vials from the same liposomal aminoglycoside batch. In alternative embodiments, the liposomal aminoglycoside formulation has one of the following release profiles according to the IVR method described in Example 2. The release profiles provided below can be an average of multiple samples from a single batch or from a single sample from a batch. In one embodiment, samples can be pooled from multiple vials derived from the same liposomal aminoglycoside batch.

[0052] [Table 3]

[0053] In one embodiment, the liposomal aminoglycoside formulation (e.g., ALIS) releases (i) 20% or less of the aminoglycoside at 0.5 hours, (ii) 50% or less of the aminoglycoside at 1.5 hours, (iii) 80% or more of the aminoglycoside at 3.0 hours, or (iv) any combination of any of the foregoing, according to the IVR method described in Example 2, where percent aminoglycoside released = [free aminoglycoside (mg / mL) / total aminoglycoside (mg / mL)] x 100%. The release profile provided can be an average of multiple samples from a single batch or from a single sample from a batch. In one embodiment, samples can be pooled from multiple vials from the same liposomal aminoglycoside batch.

[0054] The liposomal aminoglycoside formulation may include one or more pharma- ceutically acceptable excipients for inhalation formulations, such as a solvent (preferably water), an isotonicity agent (such as sodium chloride), and a pH adjuster (such as sodium hydroxide). The liposomal aminoglycoside formulation may be contained in a vial. One embodiment is a kit comprising a plurality of vials, each containing a liposomal aminoglycoside formulation comprising aminoglycoside-encapsulated liposomes. The liposomal aminoglycoside formulation in each vial may be from a manufacturing batch of the liposomal aminoglycoside formulation, the batch being verified to have a predetermined in vitro release profile by the in vitro method described herein. In one embodiment, the kit comprises 28 vials. In another embodiment, the kit comprises 7, 14, 21, 35, 42, 49, or 56 vials. The kit may further comprise a nebulizer.

[0055] Aminoglycosides In further embodiments, the aminoglycoside in the liposomal aminoglycoside formulation is amikacin, apramycin, arbekacin, astromycin, capreomycin, dibekacin, framycetin, gentamicin, hygromycin B, isepamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodestreptomycin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin or verdamycin, or a pharma- ceutically acceptable salt of one of the foregoing, or any combination of any of the foregoing. In further embodiments, the aminoglycoside is AC4437, dibekacin, K-4619, sisomicin, amikacin, dactimicin, isepamicin, rhodestreptomycin, arbekacin, etimicin, KA-5685, sorbistin, apramycin, framycetin, kanamycin, spectinomycin, astromycin, gentamicin, neomycin, sporaricin, bekanamycin, H107, netilmicin, streptomycin, boformycin, hygromycin, paromomycin, tobramycin, bourramycin, hygromycin B, plazomycin, verdamycin, capreomycin, inosamycin, ribostamycin, vertilmycin, a pharmaceutically acceptable salt thereof, or any combination of any of the above. The aminoglycoside may be in free base or salt form. When an aminoglycoside has one or more chiral centers, each unique stereoisomer and any combination mixtures thereof (including racemic mixtures) are included unless otherwise specified. When an aminoglycoside has one or more unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are included herein. When an aminoglycoside exists in tautomeric forms (such as keto-enol tautomers), each tautomeric form is contemplated as being included herein.

[0056] In one preferred embodiment, the aminoglycoside is amikacin or a pharma- ceutically acceptable salt thereof (e.g., amikacin sulfate). Amikacin sulfate refers to the disulfate salt of amikacin, i.e., D-streptamine, O-3-amino-3-deoxy-α-D-glucopyranosyl-(1→6)-O-[6-amino-6-deoxy-α-D-glucopyranosyl-(1→4)]-N1-(4-amino-2-hydroxy-1-oxobutyl)-2-deoxy-, (S)-, sulfate (1:2), having a chemical formula of C22H43N5O13·2H2SO4 and a molecular weight of 781.76. The liposomes described herein may contain one or more aminoglycosides, such as two aminoglycosides. In a preferred embodiment, the liposomes contain only one aminoglycoside, such as amikacin or a pharma- ceutically acceptable version thereof (e.g., amikacin sulfate).

[0057] Liposomes The liposomes described herein contain a lipid component, including one or more lipids. The lipids can be synthetic, semi-synthetic or natural lipids, including phospholipids, tocopherols, sterols, fatty acids, negatively charged lipids, and cationic lipids. In one embodiment, the aminoglycoside is impermeable to the bilayer of the liposome. In one embodiment of any of the methods described herein, the lipid component of the liposome comprises or consists of one or more net neutral lipids. In one embodiment, the lipid components of the liposome comprise or consist of net neutral phospholipids and cholesterol. In a further embodiment, the net neutral lipid is phosphatidylcholine. In one embodiment, the phosphatidylcholine is dipalmitoylphosphatidylcholine (DPPC). In one embodiment, the lipid components of the plurality of liposomes include phospholipids, such as dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (EPC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidic acid (PA), the soybean counterparts of the above, such as soybean phosphatidylcholine (SPC), SPG, SPI SPS, SPE, and SPA, hydrogenated egg and soybean counterparts (e.g., HEPC and HSPC), phospholipids consisting of ester bonds of fatty acids at the 2- and 3-positions of the glycerol position containing chains of 12-26 carbon atoms, and different head groups at the 1-position of the glycerol including choline, glycerol, inositol, serine, or ethanolamine, and the corresponding phosphatidic acid. The chains on these fatty acids may be saturated or unsaturated, and phospholipids may be composed of fatty acids of different chain lengths and different degrees of unsaturation.

[0058] Other examples of lipids 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). In a further embodiment, the lipid component comprises a sterol. In a further embodiment, the lipid component comprises a sterol and a phospholipid, or consists essentially of a sterol and a phospholipid, or consists of a sterol and a phospholipid. 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 and tocopherols.

[0059] In one embodiment, the lipid component can include dipalmitoylphosphatidylcholine (DPPC), a major constituent of natural pulmonary surfactant. In one embodiment, the lipid component includes, or consists essentially of, DPPC and cholesterol, or consists of DPPC and cholesterol. In a further embodiment, the DPPC and cholesterol have a molar ratio ranging from about 19:1 to about 1:1, or from about 9:1 to about 1:1, or from about 4:1 to about 1:1, or from about 2:1 to about 1:1, or from about 1.86:1 to about 1:1. In yet a further embodiment, the DPPC and cholesterol have a molar ratio of about 2:1 or about 1:1. In a further embodiment, the lipid component comprises DPPC and cholesterol.For example, the liposome may comprise amikacin sulfate and may comprise DPPC and cholesterol as lipid components.In a further embodiment, the aminoglycoside is amikacin, for example amikacin sulfate.In one embodiment, the aminoglycoside (for example, amikacin) is impermeable to the bilayer of the liposome.

[0060] Suitable liposomal aminoglycoside formulations are described in U.S. Pat. Nos. 7,718,189, 8,226,975, 9,566,234, and 9,895,385, each of which is incorporated by reference herein in its entirety for all purposes. To minimize dose volume and reduce patient administration time, in one embodiment, liposomal encapsulation of an aminoglycoside (e.g., the aminoglycoside amikacin) is highly efficient, and then the lipid to aminoglycoside weight ratio is as low as possible and / or practical, while keeping the liposomes small enough to penetrate the patient's mucus and biofilms. In one embodiment, the weight ratio of lipid to aminoglycoside (sometimes expressed as lipid:aminoglycoside) in the liposomal aminoglycoside formulation can be about 0.7 (lipid):1.0 (aminoglycoside), about 0.5 (lipid):1.0 (aminoglycoside) to about 0.8 (lipid):1.0 (aminoglycoside), about 0.55 (lipid):1.0 (aminoglycoside) to about 0.8 (lipid):1.0 (aminoglycoside), about 0.55 (lipid):1.0 (aminoglycoside) to about 0.75 (lipid):1.0 (aminoglycoside), or about 0.6 (lipid):1.0 (aminoglycoside) to about 0.8 (lipid):1.0. In further embodiments, the liposomes provided herein are small enough to effectively penetrate bacterial biofilms. The sustained activity profile of the liposomal product can be modulated by the properties of the lipid membrane and by the inclusion of other excipients in the composition.

[0061] The liposomal aminoglycoside formulation, in one embodiment before spraying, comprises liposomes having an average diameter in the range of about 0.01 microns to about 3.0 microns, for example, about 0.2 to about 1.0 microns, as measured by light scattering. In one embodiment, the average diameter of the liposomes in the composition is about 125 nm to about 360 nm, about 125 nm to about 350 nm, about 150 nm to about 350 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. In a further embodiment, the average diameter (before spraying) of the plurality of liposomes, as measured by light scattering, is 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. In yet a further embodiment, the average diameter of the plurality of liposomes is from about 260 nm to about 280 nm as measured by light scattering.

[0062] In one embodiment, the pH of the liposomal aminoglycoside formulation ranges from about 6.1 to about 7.1, such as from about 6.1 to about 6.8. In one embodiment, the liposomal compositions described herein are produced by one of the methods described in U.S. Patent Application Publication Nos. 2013 / 0330400 and 2008 / 0089927, and U.S. Patent No. 7,718,189, each of which is incorporated by reference in its entirety for all purposes. Certain methods for making liposomal aminoglycoside formulations are described in U.S. Patent Application Publication No. 2021 / 0015750 and PCT Publication No. WO2019 / 213398, each of which is incorporated by reference in its entirety for all purposes. In one embodiment, liposomes may be formed by dissolving one or more lipids forming a lipid solution in an organic solvent (e.g., ethanol) and mixing an aqueous solution of an aminoglycoside with the lipid solution to form an aminoglycoside coacervate. In a preferred embodiment, the lipid solution comprises a phospholipid and a sterol, such as DPPC and cholesterol.

[0063] Liposomes can be produced by ultrasonication, extrusion, homogenization, swelling, electroforming, inverted emulsion or reverse evaporation. Bangham's method (J. Mol. Biol. 13:238-252, 1965) produces general multilamellar vesicles (MLVs). Lenk et al. (U.S. Pat. Nos. 4,522,803, 5,030,453 and 5,169,637, each of which is incorporated herein by reference in its entirety), Fountain et al. (U.S. Pat. No. 4,588,578, each of which is incorporated herein by reference in its entirety) and Cullis et al. (U.S. Pat. No. 4,975,282, each of which is incorporated herein by reference in its entirety) disclose methods for producing multilamellar liposomes, which have substantially equal intermembrane solute distribution in each aqueous compartment. U.S. Patent No. 4,235,871 (incorporated herein by reference in its entirety) discloses the preparation of oligomembranous liposomes by reverse phase evaporation. Each of the present methods is suitable for preparing liposomal aminoglycoside formulations for use in the present invention.

[0064] Unilamellar vesicles can be made from MLVs by a number of techniques, such as the extrusion method of U.S. Patent Nos. 5,008,050 and 5,059,421, the disclosures of each of which are incorporated herein by reference in their entirety. Sonication and homogenization can be used as such to generate smaller unilamellar liposomes from larger ones. The liposome formulation 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 vessel. An appropriate amount of aqueous phase is then added, and the mixture is "swelled" to obtain liposomes. The resulting liposomes consist of multilamellar vesicles (MLVs), which are dispersed by mechanical means. This formulation 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 disclosures of each of the aforementioned publications, including patents, are incorporated herein by reference in their entirety.

[0065] The techniques for making large unilamellar vesicles (LUV), such as reverse phase evaporation, injection, and detergent dilution, can be used to prepare liposomes for use in the liposomal aminoglycoside formulations presented herein.Reviews 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.See also 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. Other methods of making liposomes include forming reverse-phase evaporation vesicles (REV), see U.S. Patent 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 designated stable plurilamellar vesicles (SPLV), as defined in U.S. Patent No. 4,522,803, and includes the single-phase vesicles described in U.S. Patent No. 4,588,578, and the frozen and thawed multilamellar vesicles (FATMLV), discussed above. The disclosures of each of the foregoing patents are incorporated herein by reference in their entirety.

[0066] Liposomes have been 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 85 / 00968, described a method for reducing the toxicity of drugs by encapsulating the drugs in liposomes containing alpha-tocopherol and certain derivatives thereof.Also, liposomes have been formed using various tocopherols and their water-soluble derivatives.See, PCT Publication No. WO 87 / 02219.The disclosures of each of the aforementioned patents and patent application publications are incorporated herein by reference in their entirety.

[0067] treatment The liposomal aminoglycoside formulations prepared can be administered to treat various pulmonary infections, such as mycobacterial infections (e.g., pulmonary infections caused by nontubercular mycobacteria, also referred to herein as nontuberculous mycobacteria (NTM) infections), as described in U.S. Patent Nos. 7,544,369, 7,718,189, 8,226,975, 8,632,804, 8,642,075, 8,679,532, 8,802,137, 9,566,234, 9,827,317 and 9,895,385, each of which is incorporated herein by reference in its entirety. In one embodiment, the liposomal aminoglycoside formulation is administered by inhalation. For example, liposomal aminoglycoside formulations can be aerosolized, such as with a nebulizer, and administered by inhalation. Suitable methods for administering liposomal aminoglycoside formulations using a nebulizer are described in U.S. Patent No. 9,566,234, which is incorporated herein by reference. The liposomal aminoglycoside formulation may be administered by nebulization. For example, the liposomal aminoglycoside formulation may be administered using one or more of the methods disclosed in U.S. Pat. No. 9,566,234 or U.S. Pat. No. 9,895,385 (the disclosures of each of which are incorporated herein by reference in their entirety). In another embodiment, the percentage of liposomal aminoglycoside after nebulization is about 50% to about 80%, for example, about 50% to about 75%, about 50% to about 70%, about 55% to about 75%, or about 60% to about 70%. In another embodiment, the percentage of liposomal aminoglycoside after nebulization is about 65% to about 75%.

[0068] One embodiment is a method of treating a pulmonary infection in a patient, comprising administering to the patient a therapeutically effective amount of a liposomal aminoglycoside formulation comprising an aminoglycoside-encapsulated liposome, wherein the liposomal aminoglycoside formulation is derived from a manufacturing batch of the liposomal aminoglycoside formulation and has been verified to have a predetermined in vitro release profile, the release profile being determined by the in vitro methods described herein. Pulmonary infections (such as in patients with cystic fibrosis or bronchiectasis) that may be treated with the methods of the invention include Pseudomonas (e.g., P. aeruginosa, P. paucimobilis, P. putida, P. fluorescens, and P. acidovorans), staphylococcal, Methicillin-resistant Staphylococcus aureus (MRSA), streptococcal (including Streptococcus pneumoniae), Escherichia coli, Klebsiella, Enterobacter, Serratia, Haemophilus, Yersinia pesos, Burkholderia pseudomallei, B. cepacia, B. gladioli, B. multivorans, B. vietnarniensis, and Mycobacterium tuberculosis infections. Liposomal aminoglycoside formulations are also useful in treating, for example, pulmonary nontuberculous mycobacterial (NTM) infections, such as pulmonary M. avium, M. avium subsp. hominissuis (MAH), M. abscessus, M. chelonae, M. bolletii, M. kansasii, M. ulcerans, M. avium, M. avium complex (MAC) (M. avium and M. intracellulare), M. conspicuum, M. peregrinum, M. immunogenum, M. xenopi, M. marinum, M. malmoense, M. marinum, M. mucogenicum, M. nonchromogenicum, M. scrofulaceum, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. terrae complex, M. haemophilum, M. genavense, M. The composition may be administered to treat infections with M. gordonae, M. ulcerans, M. fortuitum, or the M. fortuitum complex (M. fortuitum and M. chelonae).

[0069] In one embodiment, the patient is a cystic fibrosis (CF) patient with an NTM infection, such as an NTM infection caused by MAC. In another embodiment, the patient is a non-CF patient with an NTM infection, such as an NTM infection caused by MAC. In one embodiment, the MAC patient is refractory to prior treatment. The prior treatment includes, for example, a combination of a macrolide antibiotic and ethambutol. In a further embodiment, the macrolide antibiotic is azithromycin, clarithromycin, erythromycin, carbomycin A, josamycin, kitamycin, midecamycin, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, or a combination thereof. In another embodiment, the prior treatment includes a combination of a macrolide antibiotic, ethambutol, and a rifamycin compound. In a further embodiment, the rifamycin compound is rifampin or rifabutin. In a further embodiment, the prior treatment comprises a combination of a macrolide antibiotic selected from azithromycin, clarithromycin, erythromycin, carbomycin A, josamycin, kitamycin, midecamycin, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, roxithromycin, or combinations thereof, ethambutol, and a rifamycin compound. In another embodiment, the patient treated with one of the methods presented herein is an NTM patient and is NTM treatment naive.

[0070] Working Example The present invention will now be further described by the following non-limiting examples. In applying the disclosure of these examples, it should be clearly noted that the examples are merely illustrative of the invention and should not be construed as limiting the scope of the invention in any way, inasmuch as numerous variations and equivalents encompassed by the invention will become apparent to those of skill in the art upon reading this disclosure.

[0071] Example 1 The method to determine the release of amikacin from the ALIS over time is performed using a USP dissolution apparatus II (paddle method) and a Float-A-Lyzer® dialysis device (available from Repligen Corp., Rancho Dominguez, CA, USA) using a detergent (5% Triton® X-100) in buffer (Dulbecco's phosphate buffered saline (DPBS)) solution as the medium. The ALIS drug product is loaded into the Float-A-Lyzer® dialysis device and placed in the medium. A constant temperature and mixing rate is achieved using the dissolution apparatus. Sample aliquots are removed from the container at designated time points up to 24 hours, filtered by solid phase extraction to remove detergent, and analyzed for amikacin content to determine amikacin release rate.

[0072] Instruments and Equipment Dialysis Device: 1000 kDa MWCO, 5-mL volume, Float-A-Lyzer G2 (cellulose ester dialysis membrane), Repligen, catalog no. G235062. Solid Phase Extraction (SPE) Cartridges: Oasis MCX (1cc / 30mg), Waters, Catalog No. 186001881

[0073] Preparation of solutions All volumes may be adjusted as long as the ratios remain the same. 5.0% Triton X-100 in DPBS 1X (culture medium) To prepare 6000 mL, slowly transfer 300-mL of Triton X-100 (BioXtra) into 5700 mL of DPBS 1× buffer while mixing with a magnetic stir bar and stir plate. 10% Perfluoropentanoic Acid (PFPA) in H2O To prepare 10 mL, transfer 1 mL of PFPA to a 10-mL volumetric flask. Dilute to volume with HPLC-grade HO and mix well. ·20%EtOH (H2O:EtOH, 80:20, v / v) To prepare 1000 mL, mix 200 mL of EtOH with 800 mL of deionized water. Cleaning solution (HO:MeOH:formic acid, 50:50:0.005, v / v) To prepare 1,000 mL, transfer 500 mL of HPLC grade HO containing 500 µL of methanol (MeOH) and 50 µL of formic acid into a suitable container and mix well. Elution solution (0.05N NaOH in 20% methanol) To prepare 500 mL, pipette 2.5 mL of 10 N NaOH into a suitable container containing 400 mL of HPLC grade HO and 100 mL of MeOH. Mix well. Diluent (H2O:n-PrOH:PFPA, 70:30:0.3, v / v) To prepare 1,000 mL of diluent, transfer 700 mL of deionized water, 300 mL of n-propanol (n-PrOH), and 3 mL of PFPA to a suitable container and mix well. Amikacin liposomal suspension for inhalation (ALIS)

[0074] Equipment Conditions The USP dissolution apparatus II is operated at 37° C. (±2° C.) and a paddle speed of 150 rpm (±10 rpm). The medium is 5.0% v / v Triton X-100 (±0.2%) in DPBS 1× (900 mL). 5 mL samples are taken at 1, 2, 6, and 24 hours.

[0075] Assay procedure Transfer 900 mL of medium into six different vessels. Place evaporation covers over the vessels and equilibrate to 37 °C with paddle speed agitation at 150 rpm. Pre-Treatment A Twelve Float-A-Lyzer dialysis device was pretreated as follows: The cap of the dialysis device was removed and the device was filled with 20% EtOH. The cap was replaced and the dialysis device was immersed in the same alcohol solution (20% EtOH) for 30 minutes. The device was removed from the alcohol solution and the 20% EtOH solution was emptied from the device. The interior and exterior of the device was thoroughly flushed with a continuous stream of deionized water, then the device was filled with deionized water, the cap was replaced and the dialysis device was immersed in water for 30 minutes. The device was removed from the deionized water and the deionized water was emptied from within the device. The interior and exterior of the device was then flushed with a continuous stream of deionized water. The device was loaded by shaking and / or tapping off any remaining drops of deionized water.

[0076] ALIS vials (6 or 12 vials) were equilibrated to ambient temperature (minimum 45 minutes). Vials were shaken or vortexed until samples appeared homogenous and well mixed. Each ALIS vial was assigned to a specific container and labeled accordingly. The contents of each ALIS vial were poured directly into the dialysis equipment (Float-A-Lyzers) by distributing evenly between two pairs of pre-conditioned Float-A-Lyzers, each with a predetermined volume of 5 mL. Once the dialysis devices were filled, the caps on the devices were replaced. Each pair of Float-A-Lyzers was assigned to a single dissolution vessel. This process was repeated for all vials to be tested. The loaded Float-A-Lyzer was attached to the cover / adapter assembly of the dissolution apparatus. The rotation of the dissolution apparatus paddles was stopped and each Float-A-Lyzer / cover / adapter was placed into the container. It was ensured that the Float-A-Lyzer would not accidentally crimp when placed into the container. The height of the adapter was adjusted as necessary to ensure that each Float-A-Lyzer did not contact the paddle while remaining submerged in the dissolution medium. Dissolution is initiated and samples (5 mL aliquots) are taken after 1, 2, 6, and 24 hours.

[0077] Sample solid phase extraction (SPE) pretreatment 62.5 μL of 10% PFPA was pipetted into a 5 mL sample pull (aliquot) and gently vortexed until well mixed. The SPE cartridge was loaded into the extraction manifold using the long needle valve. An empty collection tube was placed into the extraction manifold test tube rack. The extraction manifold vacuum pressure was adjusted to a set point of 15 inHg. One cartridge volume (approximately 1 mL) of HPLC grade HO was transferred to each cartridge. All long needle valves in use were opened and the vacuum pump was turned on. Once all the HO had passed through and all the long needle valves were closed, the pump was shut off. A 1-mL PFPA-pretreated sample was pipetted into each cartridge and allowed to sit in the cartridge for 5 min. All in-use long-needle valves were opened and the vacuum pump was turned on. Once all sample had passed, the pump was shut off. The cartridge was washed with six cartridge volumes of wash solution (approximately 6 mL). After washing, the pump was left on for 20 minutes to allow the cartridge to dry. Upon completion, the pump was stopped and all long needle valves were closed. The waste tube was replaced with a fresh and appropriately labeled collection tube. 0.5 mL of elution solution was pipetted into each cartridge and allowed to sit in each cartridge for 5 min. All long needle valves in use were opened and the vacuum pump was turned on. After the elution solution had passed through all cartridges, the pump was shut off. All long needle valves were closed and the elution procedure was repeated for a total of 1-mL elution. The tubes were vortexed to mix the eluted solution.

[0078] Sample preparation for HPLC analysis Final dilutions from the combined elution solution were made according to Table 1. Ensure that the final sample dilutions were mixed well by vortexing. [Table 4] All sample preparations are analyzed. Samples were rediluted or concentrated as necessary to fit within the calibration curve. Eluates were placed in vials and injected directly as necessary to get as close as possible to the calibration range.

[0079] calculation The release rate for each dissolution vessel is calculated as follows:

number

[0080] [Table 5]

[0081] Example 2 material Triton™ X-100 is a nonionic surfactant available from The Dow Chemical Company (Midland, MI). Triton X-100 refers to octylphenol ethoxylate (polyethylene glycol tert-octylphenyl ether (also known as CAS number 9002-93-1) with a cloud point of 66° C. (1 wt. % active aqueous solution), an HLB of 13.4, a molar ethoxylate (EO) of 9.5, a pH of 6 for a 5% aqueous solution, a pour point of 1 (° C.), a viscosity of 240 cP at 25° C., a density of 1.061 g / mL at 25° C., and a flash point (closed cup) of 251° C. by ASTM D93. Dulbecco's Phosphate Buffered Saline (DPBS, 1X, Corning®, calcium and magnesium free) is an aqueous salt solution containing potassium chloride (0.2 g / L), potassium dihydrogen phosphate (0.2 g / L), sodium chloride (8 g / L), and disodium phosphate (1.15 g / L) available from ThermoFischer Scientific (Waltham, Mass.).

[0082] device High-performance liquid chromatography (HPLC): equipped with an evaporative light scattering detector (ELSD) (Sedex 85, Sedere). HPLC column; 3 μm particle size, 4.6 mm ID x 150 mm length; Hypersil Gold, Thermo, catalog number 25003-154630 Filter: Centrisart I inverted centrifugal concentrator, 20,000 molecular weight cutoff, Sartorius, catalog number 13249E USP Dissolution Apparatus 2 (paddle) with small round-bottom dissolution vessel (150 mL)

[0083] Determination of in vitro release of liposomal amikacin for inhalation (ALIS) An assay of amikacin release rate over time from ALIS was performed using serial dilutions of ALIS in the presence of octylphenol ethoxylate surfactant (Triton™ X-100, referred to as “Triton” in this example for brevity) in buffer (Dulbecco’s phosphate buffered saline, pH 7.2). Amikacin leakage rate from liposomes is achieved utilizing a USP dissolution apparatus 2 (paddles) to maintain constant temperature and agitation. Samples are incubated and left agitated for up to 3 hours, and sample aliquots are drawn every 30 minutes to characterize the release rate over time. Collected samples are analyzed for total and free amikacin content by high performance liquid chromatography with evaporative light scattering detection (ELSD) to determine amikacin release rate.

[0084] 1) Solution Formulation All volumes may be adjusted as long as the ratios remain the same. 200 ppm Triton X-100 in DPBS buffer (Triton / PBS buffer) To prepare 500 mL, 100 μL of Triton X-100 was transferred to 500 mL of DPBS buffer using a positive displacement pipette and mixed well. H 2 1.5% Sodium Chloride (NaCl) in 2O (1.5% NaCl): To prepare 1 L of 1.5% NaCl, approximately 15.0 (±0.1) grams of NaCl was dissolved in 1000 mL of H2O and mixed well. Diluent: 3:7 (v / v) n-propyl alcohol (n-PrOH) / H2O containing 0.3% perfluoropentanoic acid (PFPa) 2 O To prepare 1 L of diluent, 700 mL of HO, 300 mL of n-PrOH, and 3 mL of PFPA were transferred to a suitable container and mixed well. Solvent A: 1:1 (v / v) containing 0.5% PFPA - PrOH:H 2 O To prepare 1 L of solvent A, 500 mL of n-PrOH, 500 mL of HO, and 5 mL of PFPA were transferred to a suitable container and mixed well. Mobile phase: 65:35 (v / v) MeOH:H containing 0.3% PFPA 2 O To prepare 1 L of mobile phase, 650 mL of MeOH, 350 mL of H2O, and 3 mL of PFPA were transferred to a suitable container and mixed well.

[0085] 2) Equipment conditions USP Dissolution Apparatus 2 (Paddle) Speed: 150(±20)RPM Temperature: 37℃ (±3℃)

[0086] 3) Assay procedure Sample preparation Triton / PBS buffer was equilibrated to 37° C. in the dissolution vessel (agitation was not required, but may be used optionally). Samples were allowed to equilibrate to ambient temperature (minimum 45 minutes). Samples were shaken or vortexed until they appeared homogenous and well mixed. 30 mL of ALIS was transferred to a separate dissolution vessel and equilibrated to 37° C. with stirring for approximately 1 hour. Time = 0 hours 0.5 mL of Triton / PBS buffer was added to 30 mL of equilibrated ALIS sample to form the "ALIS mixture" in the dissolution vessel. Time = 0.5 hours 3 mL of the ALIS mixture was removed from the container and transferred to an appropriately labeled container. This sample will be analyzed for total and free amikacin content. Each sample removed at t = 0.5-3 h can be stored at 2-8 °C for up to 1 week. 1.7 mL of Triton / PBS buffer was added to the remaining portion of the ALIS mixture in the vessel and incubated with stirring for 0.5 h. Time = 1.0 hour 3 mL of the ALIS mixture was removed from the container and transferred to an appropriately labeled container. This sample will be analyzed for total and free amikacin content. 4.0 mL of Triton / PBS buffer was added to the remaining portion of the ALIS mixture in the vessel and incubated with stirring for 0.5 h. Duration = 1.5 hours 3 mL of the ALIS mixture was removed from the container and transferred to an appropriately labeled container. This sample will be analyzed for total and free amikacin content. 16 mL of Triton / PBS buffer was added to the remaining portion of the ALIS mixture in the vessel and incubated with stirring for 0.5 h. Time = 2.0 hours 20 mL of the ALIS mixture was removed from the container and transferred to an appropriately labeled container. This sample will be analyzed for total and free amikacin content. 100 mL of Triton / PBS buffer was added to the remaining portion of the ALIS mixture in the vessel and incubated with stirring for 0.5 h. Duration = 2.5 hours 20 mL of the ALIS mixture was removed from the container and transferred to an appropriately labeled container. This sample will be analyzed for total and free amikacin content. The ALIS mixture was incubated and stirred for 0.5 h. Time = 3.0 hours 20 mL of the ALIS mixture was removed from the container and transferred to an appropriately labeled container. This sample will be analyzed for total and free amikacin content.

[0087] 4) Sample preparation for HPLC analysis Total Amikacin Content Using a positive displacement pipette, two-step dilutions were prepared, first in solvent A and then in diluent for samples taken from each time point as indicated in Table 3. Samples expire in 2 weeks when stored at 2-8 °C.

[0088] [Table 6]

[0089] Free Amikacin Content Dilution A: Dilutions of the ALIS mixture in 1.5% NaCl were prepared as described in Table 4.

[0090] [Table 7]

[0091] Dilution A was transferred to the filter. For Centrisart filter devices, the inner filter section was removed and approximately 2.5 mL of Dilution A was transferred to the outer Centrisart tube. The inner tube was reinserted and brought into contact with the sample surface. The filter was left in contact with the sample for at least 5 minutes. Samples were centrifuged at 2500 xg for 15 minutes at ambient conditions. The filtrate was collected from the inner Centrisart tube and stored in a suitable container. Dilutions B and C: Further dilutions of the filtrate collected for each time point were prepared as shown in Tables 5 and 6. The sample (Dilution C) will expire in 2 weeks when stored at 2-8°C.

[0092] [Table 8]

[0093] [Table 9]

[0094] All sample formulations were analyzed for total and free amikacin content by HPLC and ELSD as follows.

[0095] Preparation of standards Amikacin stock standard solution: 3.3 mg / mL amikacin solution in HO. Approximately 82.5 mg of amikacin standard (USP, catalog number 1019508) was weighed and quantitatively transferred into a 25-mL volumetric flask. Amikacin was dissolved in the appropriate amount of HO and mixed well. The actual amikacin concentration was calculated. Amikacin Working Standard Solution. Using a positive displacement pipette, a series of dilutions of the amikacin stock standard solution were prepared in diluent as outlined in Table 7 below.

[0096] [Table 10]

[0097] Amikacin Check Stock Standard Solution. 3.3 mg / mL amikacin in HO. Prepared by the same procedure as Amikacin Stock Standard Solution. Check standard working solution: 56 μg / mL amikacin in diluent. Prepare dilutions of amikacin check stock standard solution according to working standard 3 in Table 7. Kanamycin stock solution: 3.0 mg / mL in HO. 30 mg of Kanamycin Sulfate Reference Standard (USP, Catalog No. 1355006) was weighed and transferred to a 10-mL volumetric flask. The reference standard was dissolved in HO, diluted to volume, and mixed well. Lysis solution: 56 μg / mL amikacin and 60 μg / mL kanamycin in diluent. 0.85 mL of amikacin stock or check stock solution and 1.0 mL of kanamycin stock solution were pipetted into a 50-mL volumetric flask, diluted to volume with diluent, and mixed well.

[0098] HPLC settings Isocratic flow rate: 1.1mL / min (±0.1mL / min) Injection volume: 15μL Column temperature: 30℃ (±3℃) Run Time: 7.5 minutes HPLC injection scheme Diluent Blank: Inject as many times as necessary to obtain a clean baseline Injection accuracy (work standard 3): 6 injections Solution: Single injection Linearity standard (5 levels): 2 injections each from low to high Check working standard: 2 injections (only when a new stock standard is prepared) Samples: 2 injections each (maximum 6 samples or 12 injections) Linearity standard (5 levels): 2 injections each from low to high ELSD Settings These parameters are validated for Sedex 85. Parameters may need to be modified if a different ELSD model is utilized. Obtained: 10 Drift tube temperature: 55°(±5°)C Pressure: 3.0 bar (±0.3 bar) Filter: 5S

[0099] 5) calculation The % amikacin release is determined by: % released = [free amikacin (mg / ml) / total amikacin (mg / ml)] x 100% A graph of % release versus time was plotted to represent the % release of amikacin over time. * * * * * * *

[0100] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the appended claims. Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

Claim 1 A method for evaluating the aminoglycoside release profile of a liposomal aminoglycoside formulation comprising an aminoglycoside encapsulated within a plurality of liposomes, comprising: (a) operating a dissolution device within a dissolution vessel, said dissolution vessel comprising (i) a predetermined amount of said liposomal aminoglycoside formulation in one or more dialysis devices each having a dialysis membrane with a predetermined volume and a predetermined molecular weight cut-off (MWCO), and (ii) a dissolution medium having a predetermined amount of a surfactant, each dialysis membrane being permeable to said aminoglycoside, said dissolution medium, and said surfactant and substantially impermeable to liposomes, said dissolution device and said one or more dialysis devices being immersed in said dissolution medium; (b) removing an aliquot of said dissolution medium after a predetermined time interval; (c) analyzing said aliquot for free aminoglycoside content; (d) optionally repeating steps (b) and (c) after one or more additional time intervals. Claim 2 The method of claim 1, further comprising, prior to operating said dissolution device: (a) adding a predetermined amount of said liposomal aminoglycoside formulation to said one or more dialysis devices; (b) placing said one or more dialysis devices within said dissolution medium. Claim 3 The method of claim 1, wherein said one or more dialysis devices each comprise a dialysis membrane having a molecular weight cut-off (MWCO) of from about 20 kD to about 1500 kD. Claim 4 The method of claim 3, wherein said one or more dialysis devices each comprise a dialysis membrane having a molecular weight cut-off (MWCO) of about 1000 kD. Claim 5 The method of claim 1, wherein each dialysis membrane is composed of a cellulose ester. Claim 6 The method of claim 1, wherein said dissolution vessel comprises two dialysis devices, each containing a portion of said liposomal aminoglycoside formulation. Claim 7 The method of claim 1, wherein said surfactant is a nonionic surfactant. Claim 8 The method of claim 7, wherein said dissolution medium comprises from about 0.5 to about 10% v / v octylphenol ethoxylate in phosphate buffered saline. Claim 9 The method of claim 1, wherein operating said dissolution device comprises agitating said dissolution medium with a paddle. Claim 10 The method according to claim 9, wherein the paddle is operated at about 75 rpm to about 150 rpm.

11. The method according to claim 1, wherein the dissolving device is a USP device 2 (paddle) operated at about 150 rpm.

12. The method according to claim 1, wherein step (b) includes performing solid phase extraction on the aliquot to remove a surfactant before analyzing the aliquot for free aminoglycoside content.

13. The method according to claim 12, wherein the solid phase extraction is carried out using a cation exchange absorbent material.

14. The method according to claim 1, wherein aliquots of the dissolution medium are taken after 1, 2, 6, and 24 hours.

15. The method according to any one of claims 1 to 14, wherein the aminoglycoside is amikacin or a pharmaceutically acceptable salt thereof.

16. The method according to any one of claims 1 to 14, wherein the aminoglycoside is amikacin sulfate.

17. The method according to claim 1, wherein the lipid component of the plurality of liposomes comprises net neutral lipids.

18. The method according to claim 1, wherein the lipid component of the plurality of liposomes comprises net neutral phospholipids and sterols.

19. The method according to claim 18, wherein the sterol is cholesterol.

20. The method according to claim 18 or 19, wherein the net neutral phospholipid is net neutral phosphatidylcholine.