Measurement of entrapment capacity of liposomes

Abstract

The present invention relates to a method for determining the entrapped volume and entrapped water volume of a plurality of liposomes in a liposomal composition, which can be used to calculate the concentration of an active pharmaceutical ingredient entrapped within the liposomes and ensure the quality of a preformed liposomal composition.

Description

[Technical field]

[0001] The present invention relates to a method for determining the entrapment capacity of liposomes and its use in characterizing liposomes. [Background technology]

[0002] The trapped volume (Vt) and trapped water volume (Vtaq) of liposomes are important parameters in liposome characterization, especially since they define the volume available for encapsulation of the active pharmaceutical ingredient (API). Thus, the determination of Vt and Vtaq allows the calculation of the concentration of API entrapped within the liposomes in the liposome composition. In compositions containing empty liposomes, the trapped volume and trapped water volume are important features of the liposome structure and its properties.

[0003] The entrapment capacity of liposomes can be determined by various methods, most of which involve measuring the marker entrapped within the liposomes after removal of the unentrapped marker. This approach may be suitable for all types of liposomes, as long as the marker does not leak out of the liposomes and there is good separation between the liposomes and the extraliposomal medium. However, this approach requires that the marker be encapsulated during the liposome preparation. Therefore, it cannot be used to characterize batches designated for clinical use.

[0004] Oku et al. (Biochimica et Biophysica Acta (BBA)-Biomembranes, 691(2), 1982, 332-340) report a method to measure the volume of the aqueous compartment of liposomes using the fluorescent dye calcein. The percentage of total volume within the liposome is given as the percentage of fluorescence remaining after addition of cobalt(II) ions, which quench the fluorescence when chelated by calcein.

[0005] Perkins et al. (Chemistry and Physics of Lipids, 64, 1993, 197-217) report several methods for quantifying liposome entrapment capacity. The first method is 3 H2O and 14 C-glucose was used to mark the total water volume and the external volume. 14 Compared to C-glucose 3 The method relied on differences in the kinetics of membrane permeability of HO. The method was performed at 4°C to prevent glucose binding and permeation. 3 It reduced the risk of H2O vapor inhalation.The second method, called ViVo, was performed by adding the ESR spin probe 4-trimethylammonium TEMPO (CAT1) to a sample in a total volume of 1 mL and then using a standard curve to compare the probe concentration in the supernatant above the centrifuged pellet with the probe concentration in the total volume. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Oku et al., Biochimica et Biophysica Acta (BBA)-Biomembranes, 691(2), 1982, 332-340. [Non-Patent Document 2] Perkins et al., Chemistry and Physics of Lipids, 64, 1993, 197-217. Summary of the Invention [Problem to be solved by the invention]

[0007] There is an unmet need for accurate post-production measurements of liposome entrapment capacity that can be used to characterize liposomal compositions suitable for clinical use. [Means for solving the problem]

[0008] The present invention discloses a post-manufacturing method for determining the entrapped volume and entrapped water volume of liposomes and its use in characterizing liposomes for clinical use and in the quality control of batch production thereof.

[0009] The present invention is based in part on the discovery of a simple, accurate and reproducible method for determining the entrapped volume and entrapped water volume of liposomes. The method uses a marker with a molecular weight of 1 kDa or more that does not penetrate the liposomes, is not integrated or bound to the lipid membrane, and is present only in the external aqueous phase. When the liposomes are separated from the suspension (e.g., using centrifugation), an increase in the concentration of the marker in the liquid is measured, since both the lipid membrane and the intraliposomal aqueous medium occupy part of the dispersion volume but do not take up the marker. This increase in concentration indicates the entrapped volume. The calculation of the entrapped volume is made based on the volume of the external aqueous medium estimated from the ratio of the signal induced by the marker in the suspension after separation of the liposomes to the total signal before separation. In some embodiments, the calculation may be further made based on the measurement of the signal induced by the marker in the suspension after separation and in the precipitate containing the liposomes, compared to the total signal induced by the marker before separation.

[0010] Contrary to previously known methods that use probes that are not suitable as post-production methods or that permeate the lipid bilayer and rely on the zeta potential and surface charge of the liposomes, the method of the present invention is a post-production method that is sensitive, reproducible and less dependent on marker concentration, turbidity, quenching, etc. It is also relatively easy to perform and more accessible.

[0011] According to a first aspect, there is provided a method for determining the trapped volume (Vt) and trapped water volume (Vtaq) of a plurality of liposomes in a liposome composition, comprising the steps of: (i) obtaining a liposome composition comprising a plurality of liposomes at a predetermined lipid concentration suspended or dispersed in a fluid medium; (ii) adding a marker to the liposome composition, the marker being liposome-impermeable and having a molecular weight of 1 kDa or greater; (iii) determining the total volume of the liposome composition by measuring a signal induced by the marker in the liposome composition; (iv) separating the fluid medium from the plurality of liposomes to obtain a precipitate comprising the fluid medium and the plurality of liposomes and interstitial medium between the liposomes; (v) determining the volume of the fluid medium and the interstitial medium by measuring the signal induced by the marker in the separated fluid medium of step (iv); (vi) optionally determining the interstitial volume between liposomes in said precipitate by measuring a signal induced by said marker in said precipitate comprising a plurality of liposomes of step (iv) and interstitial medium between a plurality of liposomes; and (vii) calculating the trapped volume (Vt) and the trapped water volume (Vtaq) of the plurality of liposomes based on the volumes measured in steps (iii) and (v), or the volumes measured in steps (iii), (v), and (vi) and the predetermined concentration in step (i). A method is provided that includes:

[0012] It is to be understood that when step (vi) is performed, step (vii) includes calculating the trapped volume (Vt) and the trapped water volume (Vtaq) of the plurality of liposomes based on the volumes measured in steps (iii), (v), and (vi) and the predetermined concentration in step (i).

[0013] According to another aspect, there is provided a method for determining the trapped volume (Vt) and the trapped water volume (Vtaq) of a plurality of liposomes in a liposome composition, comprising the steps of: (i) obtaining a liposome composition comprising a plurality of liposomes at a predetermined lipid concentration suspended or dispersed in a fluid medium; (ii) adding a marker to the liposome composition, the marker being liposome-impermeable and having a molecular weight of 1 kDa or greater; (iii) determining the total volume of the liposome composition by measuring a signal induced by the marker in the liposome composition; (iv) separating the fluid medium from the plurality of liposomes to obtain a precipitate comprising the fluid medium and the plurality of liposomes and interstitial medium between the liposomes; (v) determining the volume of the fluid medium and the interstitial medium by measuring the signal induced by the marker in the separated fluid medium of step (iv); (vi) calculating the trapped volume (Vt) and the trapped water volume (Vtaq) of the plurality of liposomes based on the volumes measured in steps (iii) and (v) and the predetermined concentration of step (i). A method is provided that includes:

[0014] It should be understood that calculating the trapped volume (Vt) and trapped water volume (Vtaq) of the plurality of liposomes based on the volumes measured in steps (iii) and (v) includes calculating the ratio of the signal of the marker in the separated fluid medium after step (iv) to its signal in the liposome composition before step (iv).

[0015] According to yet another aspect, there is provided a method for determining the trapped volume (Vt) and the trapped water volume (Vtaq) of a plurality of liposomes in a liposome composition, comprising the steps of: (i) obtaining a liposome composition comprising a plurality of liposomes at a predetermined lipid concentration suspended or dispersed in a fluid medium; (ii) adding a marker to the liposome composition, the marker being liposome-impermeable and having a molecular weight of 1 kDa or greater; (iii) determining the total volume of the liposome composition by measuring a signal induced by the marker in the liposome composition; (iv) separating the fluid medium from the plurality of liposomes to obtain a precipitate comprising the fluid medium and the plurality of liposomes and interstitial medium between the liposomes; (v) determining the volume of the fluid medium and the interstitial medium by measuring the signal induced by the marker in the separated fluid medium of step (iv); (vi) determining the interstitial volume between liposomes in the precipitate by measuring a signal induced by the marker in the precipitate comprising a plurality of liposomes of step (iv) and interstitial medium between a plurality of liposomes; and (vii) calculating the trapped volume (Vt) and the trapped water volume (Vtaq) of the plurality of liposomes based on the volumes measured in steps (iii), (v), and (vi) and the predetermined concentration of step (i). A method is provided that includes:

[0016] In one embodiment, determining the trapped volume (Vt) and the trapped water volume (Vtaq) comprises determining a specific trapped volume and a specific trapped water volume, respectively.

[0017] According to some embodiments, the marker is water soluble. According to other embodiments, the marker has a molecular weight of at least about 2 kDa. In certain embodiments, the marker is a radioactive marker. In certain embodiments, the marker is inulin. In other specific embodiments, the marker is 14 C-carboxyinulin.

[0018] In further embodiments, the signal induced by the marker is measured using at least one technique selected from radiation detection, calorimetry, fluorescence, high performance liquid chromatography (HPLC), liquid chromatography coupled with mass spectrometry (LC / MS), gas chromatography (GC), GC-coupled mass spectrometry (GC-MS), infrared spectroscopy, Raman spectroscopy, NMR, colorimetry, UV-VIS spectroscopy, and combinations thereof. Each possibility represents a separate embodiment.

[0019] In various embodiments, the liposome composition comprises a plurality of liposomes selected from the group consisting of small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs), oligolamellar vesicles (OLVs), multilamellar vesicles (MLVs), multivesicular liposomes (MVLs), and mixtures or combinations thereof, with each possibility representing a separate embodiment.

[0020] In certain embodiments, the liposome composition comprises a plurality of liposomes having a size ranging from about 0.3 μm to about 50 μm, including each value within the specified range, while in other embodiments, the liposome composition comprises a plurality of liposomes having a size ranging from about 1 μm to about 40 μm, including each value within the specified range.

[0021] In various embodiments, the liposome composition includes a plurality of liposomes having a lipid concentration of about 50 mM to about 200 mM, including values ​​within the specified ranges, hi certain embodiments, the liposome composition includes a plurality of liposomes having a lipid concentration of about 100 mM to about 150 mM, including values ​​within the specified ranges.

[0022] In some embodiments, the liposome composition comprises a plurality of empty liposomes essentially free of an active pharmaceutical ingredient. In other embodiments, the liposome composition comprises a plurality of liposomes that encapsulate an active pharmaceutical ingredient. In accordance with the latter embodiment, the method of the present invention further comprises determining the concentration of the active pharmaceutical ingredient encapsulated by the plurality of liposomes.

[0023] In various embodiments, separation of the fluid medium from the plurality of liposomes is accomplished by centrifugation, filtration, or decantation. Each possibility represents a separate embodiment. In one embodiment, separation of the fluid medium from the plurality of liposomes provides a fluid medium that is substantially free of liposomes.

[0024] In a currently preferred embodiment, separation of the fluid medium from the plurality of liposomes is accomplished by centrifugation.

[0025] In some embodiments, centrifugation of the liposome composition is carried out at a relative centrifugal force of about 10,000 to about 30,000 (×g), including each value within the specified range.

[0026] In further embodiments, the centrifugation of the liposome composition is carried out at a temperature of about 2° C. to about 25° C., including each value within the specified range.

[0027] In additional embodiments, centrifugation of the liposome composition is carried out for about 10 minutes to about 100 minutes, including each value within the specified range.

[0028] In various embodiments, the methods disclosed herein are useful for quality control of liposomal compositions, and according to these embodiments, the methods further comprise comparing the measured trapped volume (Vt) and trapped water volume (Vtaq) to reference trapped volume (Vt) and trapped water volume (Vtaq) values, and rejecting the liposomal composition if the measured trapped volume (Vt) and trapped water volume (Vtaq) differ from the reference values ​​by more than ±10%.

[0029] In other embodiments, a method disclosed herein is useful for controlling the quality of a liposomal composition comprising an active pharmaceutical ingredient (API) encapsulated in a plurality of liposomes, the method comprising: determining a concentration of the API encapsulated in a plurality of liposomes in the liposomal composition from the trapped volume (Vt) and trapped water volume (Vtaq) disclosed herein, comparing the determined API concentration to a reference API concentration, and rejecting the liposomal composition if the determined API concentration differs from the reference API concentration by more than ±10%.

[0030] According to some embodiments, there is provided a liposome composition formulated for intra-articular administration comprising: a polyol tonicity agent; and a plurality of liposomes consisting essentially of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a ratio of about 45:55, wherein the composition is essentially free of a pharma- ceutical active agent, and the plurality of liposomes are characterized by a particular trapped water capacity in the range of about 1 to about 4 μl / mg lipid (inclusive of each value within the specified range), as determined by any one of the methods disclosed herein.

[0031] According to another aspect, there is provided a liposomal composition formulated for intra-articular administration comprising a plurality of liposomes consisting essentially of a fluid medium; a polyol tonicity agent; and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a ratio of about 45:55, the composition being essentially free of a pharma- ceutical active agent, the plurality of liposomes comprising: (i) adding a marker to the liposome composition, the marker being liposome-impermeable and having a molecular weight of 1 kDa or greater; (ii) determining the total volume of the liposome composition by measuring a signal induced by the marker in the liposome composition; (iii) separating the fluid medium from the plurality of liposomes and determining, after separation, the volume of the fluid medium and the interstitial medium between the liposomes by measuring a signal induced by the marker in the fluid medium; and (iv) calculating the trapped volume (Vt) and trapped water volume (Vtaq) of said plurality of liposomes based on the volumes determined in steps (ii) and (iii) and the concentrations of lipids forming said liposomes. The present invention is characterized by a specific captured water capacity in the range of about 1 to about 4 μL / mg lipid (inclusive of each value within the specified range), as measured by a method comprising:

[0032] According to various embodiments, step (iii) of determining the volume of the fluid medium and the interstitial medium between the liposomes further comprises determining the interstitial volume between the liposomes in the precipitate by measuring a signal induced by a marker in the precipitate after separation from the fluid medium. In one embodiment, the separation is performed by centrifugation.

[0033] According to some embodiments, the fluid medium comprises a histidine buffer. According to other embodiments, the polyol is a linear polyol. According to still other embodiments, the polyol is selected from the group consisting of mannitol, dextrose, lactose, trehalose, and combinations thereof. Each possibility represents a separate embodiment. According to certain embodiments, the polyol is mannitol. According to various embodiments, the weight ratio of the plurality of liposomes to the polyol tonicity agent ranges from about 6:1 to about 2:1, including all values ​​of ratio within the specified range.

[0034] According to further embodiments, the plurality of liposomes consists essentially of a combination of DMPC and DPPC, wherein DMPC is present in a weight percentage ranging from about 1% (by weight) to about 10% (by weight), and DPPC is present in a weight percentage ranging from about 2% (by weight) to about 12% (by weight), including values ​​within the specified range. According to certain embodiments, the liposome composition has a pH ranging from about 5 to about 8, including values ​​within the specified range. According to other embodiments, the plurality of liposomes is characterized by a specific trapped water capacity of about 1.5 to about 3.5 μL / mg lipid, including values ​​within the specified range. According to still other embodiments, the plurality of liposomes is characterized by a specific trapped water capacity of about 2 to about 3 μL / mg lipid, including values ​​within the specified range. According to further embodiments, the plurality of liposomes is characterized by a trapped water capacity of about 10% to about 35%, including values ​​within the specified range. According to additional embodiments, the plurality of liposomes are characterized by a trapped water capacity of about 20% to about 30%, including each value within the specified range.

[0035] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. These exemplary methods and / or materials are not necessarily intended to be limiting. [Brief description of the drawings]

[0036] [Figure 1] FIG. 1 is a schematic diagram of an Eppendorf vial after centrifugation showing the different volumes used to calculate the captured volume according to certain embodiments of the present invention. [Diagram 2] 1 is a calibration curve of the optical density of ferricyanide in water and HMB at a wavelength of 420 nm. [Diagram 3] Figure 1 shows the upper phase capture capacity and turbidity measured using ferricyanide dye for various samples: A - 3 mM lipid; B - 52 mM lipid; C - 122 mM lipid; D - 160 mM lipid; E - 206 mM lipid; F - 305 mM lipid; G - 10 mM NaCl; H - 100 mM NaCl; I - 150 mM NaCl; J - 3000 mM NaCl; K-MLVs; L-LMVs; and M-MLVs. [Figure 4] FIG. 13 shows the specific trapped water volume (light grey) and specific trapped volume (dark grey) determined using supernatant and total volume (left), and supernatant and pellet (right). [Diagram 5] Figure 1 shows the % trapped water capacity and % RSD at different lipid concentrations. The results are based on the radioactivity of the supernatant (n=3). [Figure 6] 1 is a graph showing specific trapped water capacity (μL trapped volume / mg lipid) versus lipid concentration. The average specific trapped water capacity was 2±0.1 μL / mg lipid (n=15). [Figure 7] 1 is a graph showing the specific trapped water capacity at different 14C-carboxyinulin concentrations (CPM normalized to the total sample weight). [Figure 8] FIG. 1 shows the fold change in specific trapped water capacity of liposomal compositions containing HSPCs and cholesterol, or HSPCs alone (n=2). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present invention relates to a method for the non-destructive determination of the trapped volume (Vt) and trapped water volume (Vtaq) of a liposomal composition, which can be used to control the quality of the production of liposomal compositions with improved accuracy and reproducibility.

[0038] While currently used methods for measuring the entrapment capacity of liposomes require the inclusion of a marker within the liposome, the present invention defines a different approach, in which a radioactive marker 14 A marker with a molecular weight of ≧1 kDa that is liposome-impermeable, such as C-carboxyinulin, is used. When a marker that cannot cross the lipid membrane is used, the marker distributes only in the external aqueous phase of the liposomes. Separation of the fluid medium from the liposomes (e.g., using centrifugation) results in sedimentation of the liposomes such that substantially no liposomes remain in the supernatant. The concentration of the marker in the supernatant can be measured to calculate the volume of the external aqueous phase. The volume of the external aqueous phase, including the interstitial volume between the liposomes, and the calculated lipid volume are subtracted from the total volume of the liposome composition to represent the liposome-entrapped volume.

[0039] This method overcomes the limitations of the previous approaches, since it is a post-manufacturing method that can be used on a small portion of already formed liposomes and does not involve the inclusion of a marker in the liposomes that would make them unsuitable for clinical use. Also, when using an entrapped marker to measure the entrapped volume, it is necessary that the marker is distributed uniformly in all aqueous compartments. However, it is known that in multilamellar vesicles (MLVs), the entrapped solute is not always uniformly distributed due to differences between solute and water permeability. The method disclosed herein provides accurate measurement of the entrapped volume of various liposomes, including MLVs, and can be performed without modifying the liposome structure. The method uses a high molecular weight polar marker that does not permeate the lipid bilayer, allowing for accurate and reproducible measurement of entrapped volume and entrapped water volume. The method of the present invention can be used for quality control and physical evaluation of batch production of empty liposomes and API-loaded liposomes.

[0040] Some embodiments of the present invention are described herein with reference to the accompanying drawings. The description, together with the drawings, will make apparent to those skilled in the art how some embodiments can be practiced. The drawings are for illustrative purposes, and no attempt is made to show the structure of the embodiments in more detail than is necessary for a fundamental understanding of the present invention. For clarity, some objects depicted in the drawings are not drawn to scale.

[0041] The present invention provides a method for determining the trapped volume (Vt) and trapped water volume (Vtaq) of a plurality of liposomes in a liposome composition, comprising several steps, some of which can be performed sequentially or simultaneously in any order, with each possibility representing a separate embodiment. As used herein, the term "trapped volume" refers to the sum of the volume occupied by the lipid membrane constituting the liposome and the volume of the intraliposomal aqueous medium. In some aspects and embodiments, the method disclosed herein comprises measuring the trapped water volume. As used herein, the term "trapped water volume" refers to the volume of the intraliposomal aqueous medium. In further aspects and embodiments, the method of the present invention provides for measuring the specific trapped volume and the specific trapped water volume, which are the trapped volume and the trapped water volume, respectively, normalized by the weight of lipid.

[0042] As used herein, the term "liposome" refers to a vesicle characterized by an internal aqueous core surrounded by a lipid membrane that typically comprises a phospholipid bilayer. Exemplary phospholipids include, but are not limited to, phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerol, or any derivative or combination thereof. Each possibility represents a separate embodiment. Suitable phosphatidylcholines within the scope of the present invention include, but are not limited to, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and mixtures or combinations thereof. Each possibility represents a separate embodiment. Suitable phosphatidylethanolamines include 1,2-dilauroyl-L-phosphatidyl-ethanolamine (DLPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DphPE), 1,3-dipalmitoyl-sn-glycero-2-phosphoethanolamine (1,3-DPPE), 1-palmitoyl-3-o ... These include, but are not limited to, leioyl-sn-glycero-2-phosphoethanolamine (1,3-POPE), biotin-phosphatidylethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and mixtures or combinations thereof. Each possibility represents a separate embodiment.Suitable phosphatidylglycerols include, but are not limited to, 1,2-dimyristoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (DMPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-distearoylphosphatidylglycerol (DSPG), and mixtures or combinations thereof. Each possibility represents a separate embodiment.

[0043] In accordance with the principles of the present invention, the methods disclosed herein can be used to determine the entrapment capacity of liposomes having structures including small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs), oligolamellar vesicles (OLVs), multilamellar vesicles (MLVs), multivesicular liposomes (MVLs), and mixtures or combinations thereof. Each possibility represents a separate embodiment.

[0044] The method of the present invention can be performed on empty liposomes or on liposomes encapsulating an active pharmaceutical ingredient (API). Each possibility represents a separate embodiment. Since the marker according to the principles of the present invention is not incorporated into the liposomes, the presence of the active pharmaceutical ingredient encapsulated within the liposomes does not interfere with the determination of the entrapped volume. A correlation between the entrapped volume and the concentration of the active pharmaceutical ingredient encapsulated within the liposomes can provide a measure of the API concentration. Thus, by determining the entrapped volume of a liposome, the concentration of the active pharmaceutical ingredient encapsulated therein can be evaluated. Thus, the present invention further provides a method for determining the concentration of the active pharmaceutical ingredient encapsulated within a plurality of liposomes in a liposomal composition, comprising determining the entrapped volume and / or the entrapped water volume of a plurality of liposomes encapsulating the active pharmaceutical ingredient according to the methods disclosed herein.

[0045] In some aspects and embodiments, the methods of the invention use a liposome composition comprising a fluid medium and a plurality of liposomes at a predetermined concentration of lipid, typically expressed in weight units. As used herein, the term "liposome composition" refers to a composition comprising a plurality of liposomes suspended or dispersed in a suitable liquid medium, e.g., an aqueous medium such as a buffer. When referring to a liposome composition, it should be understood that the reference includes a reference to a portion of said composition sampled to provide information regarding the entrapment volume of the entire composition. Typically, the concentration of lipid in the liposome composition ranges from about 50 mM to about 200 mM, including each value within the range specified. Exemplary concentration ranges included within the scope of the invention are about 50 mM to about 150 mM, about 50 mM to about 100 mM, and about 100 mM to about 200 mM, including each value within the range specified. In accordance with the principles of the invention, the concentration of lipid in the liposome composition allows for the volume occupied by the liposomal lipid membrane in the composition to be determined. The volume of lipid can be calculated by dividing the weight of lipid by its density. Typically, the density of the lipids forming the liposome membrane can be measured using a suitable density meter (e.g., Anton Paar density meter). Alternatively, the density can be obtained from a reference sample or calculated according to various models.

[0046] In certain aspects and embodiments, a marker with a molecular weight of 1 kDa or more is added to the composition. According to the principles of the present invention, the marker is a compound that is not internalized by the liposome and does not interact with the liposome membrane, thereby making it liposome impermeable. According to some embodiments, the marker is a polar molecule that is preferably water-soluble. Markers within the scope of the present invention include compounds that include a detectable moiety that induces a signal that can be measured and quantified by known techniques, such as spectrometry. Suitable markers include, but are not limited to, radioactive agents, chromophores, fluorescent compounds, phosphorescent compounds, magnetic compounds, and heavy metal clusters. Each possibility represents a separate embodiment. An exemplary marker is a radioactive marker that includes a radioisotope that emits radiation as a measurable signal. An additional exemplary marker is a fluorescent compound that emits light of a specific wavelength upon exposure to radiation from an external source, and the emitted light can be used as a measurable signal. The signal induced by the marker can be measured according to methods known in the art, for example, using various detectors and monitors. Techniques that can be used to measure the signal include, but are not limited to, radiation detection, calorimetry, fluorescence, high performance liquid chromatography (HPLC), liquid chromatography coupled with mass spectrometry (LC / MS), gas chromatography (GC), GC-mass spectrometry (GC-MS), infrared spectroscopy, Raman spectroscopy, NMR, colorimetry, UV-VIS spectroscopy, and combinations thereof. Each possibility represents a separate embodiment.

[0047] In accordance with the principles of the present invention, the markers include compounds having a molecular weight of at least 1 kDa, preferably at least 2 kDa. Typically, the molecular weight of the markers ranges from 1 kDa to 1,000 kDa, including each value within the specified range. Exemplary molecular weights of the markers include 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, Examples include, but are not limited to, 95 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, or 1,000 kDa. Each possibility represents a separate embodiment.

[0048] In some aspects and embodiments, the markers include polysaccharides, such as, but not limited to, inulin, FITC-inulin, dextran, FITC-dextran, and derivatives thereof. Each possibility represents a separate embodiment. In other aspects and embodiments, the markers include carbohydrates, polypeptides, or synthetic polymers, such as, but not limited to, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, horseradish peroxidase (HRP). Each possibility represents a separate embodiment. Markers within the scope of the present invention typically have a molecular weight of at least 1 kDa, although it should be understood that markers having a molecular weight of less than 1 kDa that do not penetrate liposomes and are not integrated or bound to lipid membranes are also expected to provide accurate measurements of captured volume in accordance with the principles of the present invention. Such markers include, but are not limited to, disaccharides, such as sucrose, trisaccharides, and oligosaccharides.

[0049] In some currently preferred aspects and embodiments, the methods of the invention use radioactive markers. Exemplary radioactive markers within the scope of the invention include: 14 C-carboxyl-inulin, 125 Examples of suitable radioactive materials include, but are not limited to, I-polyvinylpyrrolidone, and radioactive carbohydrates. Each possibility represents a separate embodiment. 14 Radioactive markers such as C-carboxyl-inulin do not interact with liposomes and can be added at concentrations of about 0.1 microcuries (μci) / mL to about 10 μci / mL, including values ​​within the ranges specified. Exemplary concentration ranges included within the scope of the invention are about 0.1 μci / mL to about 5 μci / mL, about 0.5 μci / mL to about 5 μci / mL, and about 0.5 μci / mL to about 3 μci / mL, including values ​​within the ranges specified.

[0050] According to the principles provided herein, the marker is not available to the liposomal content. Therefore, its concentration in the extraliposomal medium increases when it is mixed with the liposomal composition. In some aspects and embodiments, the method of the present invention comprises measuring the signal induced by the marker in the liposomal composition. For example, when a radioactive marker is used, the method comprises measuring the radioactivity / weight (disintegrations per minute (DPM) / gr) of the total liposomal composition. Since the density of the phospholipids used is very close to 1 (less than 1.05), for such liposomes, the radioactivity / weight is substantially interchangeable with the radioactivity / volume (DPM / mL). (DPM / mL) of the total liposomal dispersion is (DPM / ml) total The volume of the liposome composition is determined by measuring the volume of the liposome composition.

[0051] Measurement of radioactivity can be carried out as known in the art using a suitable liquid scintillation counter, eg, a Packard 1900 TR scintillation counter, a Packard Tri-Carb liquid scintillation counter, etc.

[0052] After measuring the volume occupied by the entire liposome composition, the fluid medium is separated from the plurality of liposomes. Separation of the fluid medium from the plurality of liposomes can be performed, for example, by centrifugation, filtration, or decantation, as known in the art. Each possibility represents a separate embodiment. Typically, the liposome composition supplemented with a marker is centrifuged to precipitate the plurality of liposomes. In accordance with the principles of the present invention, the centrifugation is performed such that the supernatant solution is substantially free of liposomes. Typically, liposomes with sizes in the range of about 0.3 μm to about 50 μm, for example, in the range of about 1 μm to about 40 μm, can be precipitated with sufficient centrifugation. In some embodiments where the composition includes small liposomes, ultracentrifugation should be used to achieve complete precipitation. In some aspects and embodiments, centrifugation of the liposome composition is performed at a relative centrifugal force of about 10,000 to about 30,000 (×g), including each value within the specified range. Exemplary relative centrifugal forces include, but are not limited to, about 10,000, about 11,000, about 12,000, about 13,000, about 14,000, about 15,000, about 16,000, about 17,000, about 18,000, about 19,000, about 20,000, about 21,000, about 22,000, about 23,000, about 24,000, about 25,000, about 26,000, about 27,000, about 28,000, about 29,000, or about 30,000 (×g), with each possibility representing a separate embodiment. Centrifugation may be performed at room temperature, although centrifugation at lower temperatures is also contemplated within the scope of the present invention. Typically, centrifugation is performed at a temperature range of about 2° C. to about 25° C., including each value within the specified range. Exemplary temperatures include, but are not limited to, about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about 10° C., about 11° C., about 12° C., about 13° C., about 14° C., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., or about 25° C., with each possibility representing a separate embodiment. Typically, centrifugation at the above relative centrifugal forces for about 10 minutes to about 100 minutes is sufficient to obtain a supernatant that is substantially free of liposomes.Exemplary durations of centrifugation include, but are not limited to, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 minutes, with each possibility representing a separate embodiment.

[0053] Following centrifugation and near complete sedimentation of the liposomes, the supernatant is collected and the volume of the extraliposomal medium (the interstitial volume present between the supernatant or separated fluid medium and the sedimented liposomes) can be determined by measuring the signal induced by the marker. For example, if a radioactive marker is used, the radioactivity (DPM / mL) of the supernatant can be determined. sup According to the principles of the present invention, (DPM / mL) sup represents the radioactivity / mL of extraliposomal medium, i.e., the volume of the supernatant and the volume of the interstitial extraliposomal medium (trapped between the liposomes) of the precipitate. Thus, if a radioactive tracer is added to the liposomal dispersion after the liposomes are formed, (DPM / mL) total and (DPM / mL) sup The ratio of the total water volume outside the liposomes (V free , Equation 1) is calculated.

[0054] The volume trapped in the liposomes (Vt) is then calculated by subtracting the volume outside the liposomes from the total volume of the dispersion (Equation 2). The trapped water volume (Vtaq) is calculated by subtracting the volume of liposomal lipids (Vtaq) from the liposome trapped volume (Vtaq). lipids ) is calculated by subtracting V lipids is determined from a known liposomal lipid weight and a known lipid density. The ratio of Vt or Vtaq to lipid used to prepare the liposomes represents the specific Vt and specific Vtaq, respectively. These are the most relevant descriptors of the liposomes in the liposomal dispersion. A diagram of an Eppendorf tube with the definition of the different volumes occupied by each component (lipid, free (non-liposomal volume including interstitial volume), and intraliposomal aqueous phase (Vtaq)) is shown in Figure 1.

[0055]

number

[0056] Optionally, the signal induced by the marker in a precipitate containing a plurality of liposomes and the interstitial medium between the liposomes can be further measured and used to determine the interstitial volume between the liposomes in the precipitate.

[0057] It is within the scope of the present invention to use the determined Vt and Vtaq as well as the specific Vt and specific Vtaq as descriptors of the quality of the liposome-containing dispersion. According to these embodiments, the method of the present invention further comprises comparing the determined entrapped volume to a reference entrapped volume value and rejecting the liposome composition if the determined entrapped volume differs from the reference entrapped volume value by more than plus or minus 10%. When comparing the determined entrapped volume to a reference entrapped volume value, it can be compared to a standard entrapped volume known to be acceptable for clinical use. The standard entrapped volume can be an exact numerical value or it can be a range. For example, it can be an absolute amount and an acceptable percentage deviation, i.e. ±10%, ±5%, etc. The calculated entrapped volume is compared to the standard entrapped volume range and the liposome composition is rejected if it is outside the range by more than ±10%. Typically, the reference entrapped volume value corresponds to a range of values ​​acceptable according to the specifications of the liposome composition, and a failure occurs if the difference corresponds to more than 10% lower than the lower limit of the range or more than 10% higher than the upper limit of the range. A representative standard entrapment capacity of a liposome composition can be, for example, about 0.1 to about 10 μL / mg lipid, about 0.3 to about 5 μL / mg lipid, or about 1 to about 4 μL / mg lipid, including each value within the specified range. The standard entrapment capacity can be obtained from a control sample that can be measured before, during, or after measuring the entrapment capacity of a liposome composition for which quality control is desired. As a further alternative, the standard entrapment capacity can be an absolute value based on a previous analysis. If the measured entrapment capacity differs significantly (more than ±10%) from the standard reference value, this difference indicates a manufacturing defect.

[0058] Additionally or alternatively, the entrapped volume can be used to calculate the concentration of API encapsulated within the liposomes. The concentration of API can then be used to control the quality of the liposome composition by comparing it to a reference concentration and rejecting the liposome composition if the measured API concentration differs from the reference concentration by more than ±10%. The reference API concentration can be an amount known to be acceptable to exert a therapeutic effect on a patient treated with the API. The reference API concentration can be an exact numerical value or a range. For example, it can be an absolute amount with an acceptable percentage deviation or it can be a standard API concentration range. If the API concentration of the liposome composition determined by the method of the present invention is outside the range of the reference concentration by more than ±10%, the liposome composition is rejected.

[0059] As mentioned above, the method of the present invention advantageously provides a post-production measurement of the capture volume. Thus, only a small portion of the sample can be collected for use in the method of the present invention, and the remaining composition can be used for clinical and other applications. Typically, multiple samples are collected for the measurement of capture volume to minimize the effects of experimental deviation, non-uniform collection, and / or non-representative collection of samples. Thus, comparison of the average value with a reference value is within the scope of the present invention.

[0060] Also within the scope of the present invention is a liposomal composition formulated for intra-articular administration, comprising a plurality of liposomes consisting essentially of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a ratio of about 45:55, essentially free of pharmaceutical actives. As used herein, the term "substantially free of pharmaceutical actives" refers to a liposomal composition that, in some embodiments, contains less than a therapeutically effective amount of a pharmaceutical active known for use in joint lubrication, treating joint dysfunction, reducing joint pain, irritation and / or wear, or any combination thereof. In some embodiments, the liposomal composition is free of lubricants, such as, inter alia, glycosaminoglycans, or pharma- ceutically acceptable salts, esters, or derivatives thereof, such as hyaluronic acid, or hyaluronic acid-containing salts or esters. A plurality of liposomes in the liposome composition are characterized by a particular trapped water capacity in the range of about 1 to about 4 μL / mg lipid as measured by any method disclosed herein, including each value within the specified range.

[0061] According to some aspects and embodiments, the fluid medium comprises a histidine buffer. According to other aspects and embodiments, the polyol is a linear polyol. According to additional aspects and embodiments, the polyol is a sugar or sugar alcohol having at least five hydroxyl groups. Exemplary polyols include, but are not limited to, mannitol, dextrose, lactose, trehalose, and combinations thereof. Each possibility represents a separate embodiment. Currently, it is preferred to use mannitol as the polyol. According to various embodiments, the weight ratio of the plurality of liposomes to polyol ranges from about 6:1 to about 2:1, including all iterations of ratios within the specified ranges.

[0062] The present invention encompasses liposome compositions having a specific trapped water capacity in the range of about 1 to about 4 μL / mg lipid as measured by any of the methods disclosed herein, the liposome compositions comprising a plurality of liposomes consisting essentially of a fluid medium, a polyol, and a combination of DMPC and DPPC, DMPC being present in a weight percentage in the range of about 1% (by weight) to about 10% (by weight) and DPPC being present in a weight percentage in the range of about 2% (by weight) to about 12% (by weight), each value including each value within the range specified. Typically, the liposome compositions have a pH in the range of about 5 to about 8, each value including each value within the range specified.

[0063] In certain aspects and embodiments, the plurality of liposomes in the liposome composition are characterized by a particular trapped water volume in the range of about 1.5 to about 3.5 μL / mg lipid, or about 2 to about 3 μL / mg lipid, inclusive. In other aspects and embodiments, the plurality of liposomes in the liposome composition are characterized by a trapped water volume in the range of about 10% to about 35%, or about 20% to about 30%, inclusive.

[0064] As used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise. Thus, for example, a reference to a "lipid" includes a plurality of such lipids. It should be noted that the terms "and" or "or" are generally used in the sense of including "and / or" unless the context clearly dictates otherwise. As used herein, the term "about" is meant to encompass a variation of ±10%.

[0065] The following examples are provided to more fully illustrate certain embodiments of the present invention, but should not be construed as limiting the broad scope of the invention. Those skilled in the art can readily devise numerous variations and modifications of the principles disclosed herein without departing from the scope of the present invention. EXAMPLES

[0066] Comparative Example: Measurement of Entrapped Aqueous Phase of Liposomes Using Potassium Ferricyanide Dye The trapped water capacity (Vtaq) of liposome compositions containing empty liposomes formed from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a ratio of approximately 45:55 was evaluated by hydrating the phospholipid-containing mixture with a solution of 4% mannitol containing 10 mM histidine buffer, pH 6.5.

[0067] A common method used to determine trapped Vt and Vtaq is based on the use of dyes that do not interact with and penetrate liposome membranes, such as potassium ferricyanide. The use of potassium ferricyanide for the measurement of Vt was performed as described below.

[0068] A standard solution of potassium ferricyanide (0.2 mg / mL) was prepared and mixed with a pre-weighed sample of the liposome composition (including empty liposomes without drug). To remove small (submicron) liposomes, the sample was centrifuged at 14,000 rpm for 10-30 min. The supernatant was then removed and weighed. The standard solution of potassium ferricyanide was added to the liposome-containing precipitate in a 1:1 weight ratio, followed by mixing using vortexing and centrifugation at 14,000 rpm for 30-60 min. Of note, mixing was difficult, so prolonged vortexing, heating, and sonication were used. The supernatant solution was then collected. The absorbance of the ferricyanide dye was measured using a UV-VIS spectrophotometer at 420 nm, and the concentration of ferricyanide was calculated based on a calibration curve generated under the same conditions (Figure 2).

[0069] An alternative procedure was also adopted. Standard solutions of potassium ferricyanide (1.25-50 mg / mL) were prepared and added to approximately 1 gram of weighed samples of liposomal dispersions of empty (drug-free) liposomes, followed by mixing using a vortex and centrifugation at 14,000 rpm for 1-3 hours. The supernatant solution was collected and the absorbance of the ferricyanide concentrate was measured using a UV-VIS spectrophotometer at 420 nm and 600 nm. Since ferricyanide does not absorb light at 600 nm, the absorbance at this wavelength is likely due to the turbidity contribution of small liposomes in the supernatant (Barenholz et al., Liposome Technology 1, 1993, 527-616). Because the turbidity was so high, it was not possible to measure the absorbance of the entire liposomal dispersion, so to calculate Vt, the absorbance of the supernatant was read at 420 nm against the same volume of a reference dispersion without liposomes. The turbidity of the supernatant of the liposome dispersion did not give consistent absorbance values ​​at 420 nm, and therefore no reliable values ​​of Vt and Vtaq could be determined using this method (OD of the supernatant (without ferricyanide) 420nm was 0.065 compared to 0.049 for the histidine mannitol buffer alone).

[0070] The results (Figure 3) show the effect of the turbidity of the supernatant on the absorbance at 420 nm. Notably, even after 2 h of centrifugation the turbidity was relatively high, i.e. the optical density values ​​at 600 nm of the liposomes in the upper phase were 0.055-0.063. The absorbance at 600 nm shows that there is a much greater turbidity at 420 nm. Therefore, the values ​​of Vt obtained with this method are spurious. Therefore, this approach does not allow the calculation of the Vt of MLV compositions containing DMPC / DPPC phospholipids.

[0071] Example 1: 14 Determination of entrapped volume and entrapped water volume of liposomes using C-carboxyl-inulin Therefore, an alternative approach was developed that does not rely on absorbance, but does not interact with and / or penetrate liposomes. 14C-carboxylinulin was selected as the tracer / marker.

[0072] The trapped water volume (Vtaq) of liposome compositions containing empty liposomes formed from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a ratio of about 45:55 by hydrating the phospholipid-containing mixture with a solution in 4% mannitol containing 10 mM histidine buffer, pH 6.5, was measured as a radioactive marker. 14 C-carboxyl-inulin was used to evaluate Vt and Vtaq of the liposomal compositions and the specific Vt and Vtaq were measured using two approaches based on the supernatant and the pellet (Figure 4).

[0073] 14 The C-carboxyl-inulin solution (aqueous) was added to pre-weighed samples containing the liposome composition. A portion of the sample (100-200 mg) was transferred to a pre-weighed 20 mL scintillation vial and weighed. Another portion (250-350 mg) was transferred to an Eppendorf tube and centrifuged at 20817 relative centrifugal force (rcf) for 30 min at 4 °C (Centrifuge 5810R, Eppendorf, Germany). The aqueous supernatant was collected, transferred to a pre-weighed Eppendorf tube and weighed again. A portion of the supernatant in the range of approximately 100-200 mg was transferred to a pre-weighed scintillation vial and weighed. A solvable was added and the solution was mixed at 50 °C for approximately 1 h to dissolve the liposomes. Liquid scintillation cocktail (Opti-Fluor) was added to each vial and the vials were stored overnight at 5-8 °C protected from light. Radioactive counts as disintegrations per minute (DPM) were measured in a liquid scintillation beta counter (Packard 1900 TR scintillation counter).

[0074] The trapped water volume (Vtaq) was calculated in two ways (Figure 4). In the first method, the total trapped volume (Vt) occupied by the liposomes (Vt including lipid volume and trapped water volume Vtaq) was calculated from the ratio of DPM / mL of the supernatant to DPM / mL of the total liposome dispersion (before liposome precipitation). This ratio allows the calculation of the % of the total volume occupied by the liposomes from the total volume of the liposome dispersion. The above ratio can be converted to volume. Vtaq was then calculated by subtracting the lipid volume from Vt. Vtaq was calculated according to the above formulas 1-3, where V free is the sum of the supernatant and interstitial fluid volumes; V interstitial is the free (non-liposomal) water volume between the sedimented liposomes after centrifugation, and V lipids is the volume of the lipid calculated by dividing the lipid mass by the density. V free (V interstitial was calculated according to Eq. 1.

[0075] The relative standard deviation (%RSD) was calculated for this measurement (n=5). The specific trapped water volume (Vtaq) calculated based on the radioactivity of the upper phase (supernatant) was 1.9±0.06 μL / mg lipid. The specific trapped volume (Vt) calculated based on the radioactivity of the upper phase was 2.9±0.06 μL / mg lipid, with a %RSD of 2.9% (Figure 4).

[0076] Example 2: Determination of entrapment capacity at different lipid concentrations The liposome samples were diluted with 10 mM iso-osmotic histidine buffer pH 6.5 in 4% mannitol. Based on the ratio of DPM / mL of the supernatant to DPM / mL of the total liposome dispersion, the specific trapped water capacity was measured according to the method described in Example 1. A linear correlation was observed between the total lipid concentration and the % trapped water capacity. The relative standard deviation increased exponentially as the lipid concentration decreased (Figure 5). The specific Vtaq of the liposome samples was calculated by taking the average of the specific trapped water capacity (n=15) (trapped volume μL / mg lipid) normalized by lipid weight. The specific Vtaq obtained was 2.0±0.3 μL / mg lipid (Figure 6). The results clearly show that the specific Vtaq (μL / mg lipid) remains the same regardless of the total lipid concentration in the same liposome composition. Therefore, the method of measuring the trapped capacity is consistent and reliable. The fact that the specific trapped capacity is independent of the lipid (liposome) concentration indicates that 14 This clearly demonstrates the absence of interaction between C-carboxyinulin and liposomes.

[0077] Example 3: Different 14 Determination of entrapment dose at C-carboxyl-inulin concentration Measurement of trapped water capacity (Vtaq) 14 To verify that C-carboxyl-inulin is not affected by its interaction with liposomes, different 14 The trapped water capacity at C-carboxyl-inulin concentration was measured based on the radioactivity of the supernatant according to the method described in Example 1. Figure 7 shows that the mean (n=6) of the specific trapped water capacity was 2.2% ± 0.1 with an RSD of 4.3%, which is consistent with the results shown in Table 1. 14 C-carboxyl-inulin, when added to preformed liposome dispersions, has been shown not to interact with the liposome membrane and not to penetrate the liposomes, as confirmed by Schafer et al., American Journal of Physiology-Legacy Content 206, 1964, 985-991.

[0078] Example 4: Determination of the entrapment capacity of multilamellar vesicles (MLVs) and large multivesicular vesicles (LMVs) To determine Vt and Vtaq as reliable descriptors of the quality of liposomal dispersions 14 To demonstrate the utility of the C-inulin approach, a comparison was made between Vt and Vtaq of liposomal compositions containing multilamellar vesicles (MLVs) and large multivesicular vesicles (LMVs).LMVV liposomal compositions were prepared from MLVs by repeated cycles of freezing and thawing, similar to the method described in Cohen et al., Journal of Controlled Release 160, 2012, 346-352.

[0079] Liposomal compositions consisting of MLVs were prepared as follows. MLVs of hydrogenated soy phosphatidylcholine (HSPC) and cholesterol or HSPC alone in a 2:1 molar ratio were prepared in saline by mixing phosphatidylcholine (with or without cholesterol) in ethanol at 65-70°C and then injecting into an aqueous solution (water or saline). The liposomal compositions contained a phosphatidylcholine concentration of approximately 75 mM. A portion of the composition was then subjected to 10 cycles of freezing and thawing to induce conversion of MLVs to LMVs. It is known that repeated freezing and thawing of liposomes dramatically increases their trapped water capacity (Sriwongsitanont et al., The Open Colloid Science Journal, 4, 2010, 1-6; Elorza et al., Journal of microencapsulation, 10(2), 1993, 237-248; Mayer et al., Biochimica et Biophysica Acta (BBA)-Biomembranes, 817(1), 1985, 193-196). Upon freezing, the liposome bilayer is disrupted and destabilized (MacDonald et al., Liposome Technology, 1, 1993, 209-228), but upon thawing, the exposed hydrophobic core fuses to form new vesicles with a reduced number of lamellae (Pick, Archives of biochemistry and biophysics, 212(1), 1981, 186-194).

[0080] Liposomes were characterized by size distribution and phosphatidylcholine concentration. Both MLV and LMV containing samples (same lipid composition) had the same lipid concentration and similar mean diameter (albeit with slightly different size distributions), but visual inspection of the vials after centrifugation revealed a significantly increased pellet volume for LMV compared to MLV. The trapped volume (Vt) and even trapped water volume Vtaq of LMV were found to be significantly higher than MLV.

[0081] The results are summarized in Table 1 and shown in FIG. Table 1. [Table 1]

[0082] Thus, the methods of the present invention provide accurate measurements of Vt, Vtaq, specific Vt, and specific Vtaq, and demonstrate a significant difference in the entrapment capacity of MLVs compared to LMVs, particularly the entrapment capacity of LMVs was significantly higher than that of MLVs.

[0083] Example 5: Comparison of capture capacity measured using the method of the present invention and a method using calcium ions as a capture marker 14For further validation of the Vtaq determination method based on C-inulin, a method for direct determination of the entrapment volume by encapsulating a marker in the aqueous phase of liposomes upon lipid hydration was applied and compared with the method of the present invention. In particular, calcium ions were selected as a water-soluble membrane-impermeable marker to directly determine the Vtaq of MLVs composed of DMPC / DPPC at a 45:55 molar ratio. Liposomes were prepared by injecting an ethanolic mixture of lipids into an aqueous medium containing 20 mM calcium ions (as calcium acetate) in 4% mannitol (histidine mannitol buffer, HMB). Exchange of the buffer and removal of ethanol and unencapsulated calcium ions were performed by repeated cycles of precipitation (by centrifugation) and repeated washing of the liposomes with the same HMB without calcium acetate. These repeated cycles of precipitation and washing of the MLVs were performed to almost completely remove the calcium-containing external medium and replace it with isoosmolar HMB. The pH and osmolality of the washing medium and the hydration medium containing calcium acetate were adjusted to 6.5, 272, and 279 mOsmo / kg, respectively. The external medium containing calcium ions was replaced by HMB medium by five successive centrifugation and washing steps. The lipid concentration and osmolality of the final liposome dispersion were 148 mM total PC (105 mg / mL) and 279 mOsmo / kg, respectively. The concentration of calcium ions trapped within the liposomes was measured using inductively coupled plasma (IPC)-optical emission spectroscopy (ICP-OES). To correct for the void volume and subtract its contribution from the amount of intraliposomal calcium, calcium ions were added to the liposome dispersion after liposome preparation. 14 C-inulin was used to measure the interstitial volume (by measuring the radioactivity of the precipitate containing the liposomes and the interstitial medium between the liposomes, as described above). For comparison, the trapped water capacity of the same liposome composition was also measured, as described in Example 1. 14 C-inulin using external marker, (DPM / mL) sup and (DPM / mL) total It was measured from the ratio of

[0084] Calcium concentrations were measured using ICP-OES before and after buffer exchange and removal of ethanol and calcium ions from the extraliposomal medium by repeated cycles of centrifugation and washing. Calcium concentrations remaining in the extraliposomal medium after centrifugation were also measured in the upper phase of the liposomes. Due to the high organic load, liposomal samples were treated with acid digestion at 200°C for 4 hours, which is not expected to affect calcium measurements. Total lipid concentrations were measured using a modified Bartlett method (Shmeeda H. et al., Methods in enzymology, 367, 2003, 272).

[0085] The trapped water capacity (Vtaq) was calculated according to the following formula:

number

[0086] Table 2. [Table 2]

[0087] The trapped water capacity (Vtaq) measured by the trapped calcium marker was 24.42% (%Vt aq= ((6.034-0.109) / 148 x 202 / 19.27) x 100%-7.1%-10.45% = 24.42%), the specific trapped water capacity (Vtaq) was 2.3 μL / mg lipid, and the specific trapped capacity (Vt) was 4 μL / mg lipid. The external capacity was also calculated using the external calcium concentration before buffer exchange and was determined to be 58%. Both results were 14 The method was compared with the method of the present invention using C-inulin marker as follows. The trapped water capacity of liposomes was measured using two different approaches: 1-based on the ratio of supernatant radioactivity to total radioactivity and 2-based on pellet radioactivity (interstitial fluid radioactivity). Similar results were obtained using both approaches. The relative standard deviation (%RSD) was calculated for both approaches (n=5). The calculated specific trapped water capacity (Vtaq) and specific trapped volume (Vt) results are summarized in Table 3.

[0088] Table 3. [Table 3]

[0089] Thus, the results of the trapped water volume (Vtaq) of liposomes obtained using trapped calcium ions (inside the liposomes) as a marker were 14 The results are almost identical to those obtained with the C-inulin external marker. Therefore, to determine the Vt and Vtaq of the liposome dispersion according to certain embodiments of the present invention, 14The use of C-inulin external liposomal medium marker results in an easy, simple, sensitive, reliable, accurate and reproducible method. The method can be applied to preformed liposome dispersions and does not require the inclusion of a liposomal internal marker. The method has also been shown to be independent of the turbidity of the supernatant after liposome precipitation, thus overcoming the problems often encountered when measuring the extraliposomal medium using low molecular weight chromophores as markers. The method allows the determination of both Vt and Vtaq, which are important descriptors of liposomal properties and can be used for quality assurance of preformed liposomes.

[0090] While particular embodiments of the present invention have been illustrated and described, it will be apparent that the present invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention, as set forth in the following claims.

Claims

1. 1. A method for determining the trapped volume (Vt) and trapped water volume (Vtaq) of a plurality of liposomes in a liposome composition, comprising: (i) obtaining a liposome composition comprising a plurality of liposomes at a predetermined lipid concentration suspended or dispersed in a fluid medium; (ii) adding a marker to the liposome composition, wherein the marker is liposome-impermeable and has a molecular weight of 1 kDa or greater; (iii) determining the total volume of the liposome composition by measuring a signal induced by the marker in the liposome composition; (iv) separating the fluid medium from the plurality of liposomes to obtain a precipitate comprising the separated fluid medium and the plurality of liposomes and interstitial medium between the liposomes; (v) determining the volume of the separated fluid medium and the pore medium by measuring the signal induced by the marker in the separated fluid medium of step (iv); (vi) optionally, determining the interstitial volume between liposomes in the precipitate by measuring a signal induced by the marker in the precipitate comprising a plurality of liposomes of step (iv) and interstitial medium between the plurality of liposomes; and (vii) calculating the trapped volume (Vt) and the trapped water volume (Vtaq) of the plurality of liposomes based on the volumes measured in steps (iii) and (v), or the volumes measured in steps (iii), (v), and (vi) and the lipid volumes of the plurality of liposomes obtained from the predetermined concentrations of step (i).

2. 10. The method of claim 1, wherein determining the trapped volume (Vt) of a plurality of liposomes in the liposome composition comprises determining a specific trapped volume or a specific trapped water volume.

3. 2. The method of claim 1, wherein step (vi) is performed, and step (vii) comprises calculating the trapped volume (Vt) and the trapped water volume (Vtaq) of the plurality of liposomes based on the volumes determined in steps (iii), (v), and (vi) and the lipid volume of the plurality of liposomes resulting from the predetermined concentration in step (i).

4. 2. The method of claim 1, wherein step (vi) is not performed, and step (vii) comprises calculating the trapped volume (Vt) and the trapped water volume (Vtaq) of the plurality of liposomes based on the volumes determined in steps (iii), (v), and the lipid volumes of the plurality of liposomes obtained from the predetermined concentration in step (i), and preferably step (vii) comprises calculating the ratio of the signal induced by the marker in the separated fluid medium after step (iv) to the signal induced by the marker in the liposome composition before step (iv).

5. The method according to any one of claims 1 to 4, wherein the marker is water-soluble and / or has a molecular weight of at least about 2 kDa and / or is a radioactive marker, preferably wherein the marker is 14 C-carboxyinulin.

6. 5. The method of claim 1, wherein the liposome composition comprises a plurality of liposomes selected from the group consisting of small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs), oligolamellar vesicles (OLVs), multilamellar vesicles (MLVs), multivesicular liposomes (MVLs), and mixtures or combinations thereof; and / or the liposome composition comprises a plurality of liposomes with a size ranging from about 0.3 μm to about 50 μm; and / or the liposome composition comprises a plurality of liposomes having a lipid concentration of about 50 mM to about 300 mM; and / or the liposome composition comprises a plurality of empty liposomes essentially free of a pharmaceutically active agent; or the liposome composition comprises a plurality of liposomes encapsulating an active pharmaceutical ingredient, preferably further comprising determining the concentration of the active pharmaceutical ingredient encapsulated in the plurality of liposomes.

7. 5. The method of any one of claims 1 to 4, wherein step (iv) provides a fluid medium that is substantially free of liposomes and / or step (iv) is carried out by centrifugation, filtration or decantation.

8. 8. The method of claim 7, wherein step (iv) is carried out by centrifugation, preferably wherein step (iv) is carried out at a relative centrifugal force of about 10,000 to about 30,000 (×g), and / or wherein step (iv) is carried out at a temperature of about 2° C. to about 25° C., and / or wherein step (iv) is carried out for about 10 minutes to about 100 minutes.

9. 1. A method for quality control of a liposome composition, comprising: (i) determining the trapped volume (Vt) and the trapped water volume (Vtaq) of a plurality of liposomes in the liposome composition of any one of claims 1 to 4; (ii) comparing the determined captured volume (Vt) and captured water volume (Vtaq) with reference captured volume (Vt) and captured water volume (Vtaq) values; and (iii) rejecting the liposome composition if the determined trapped volume (Vt) and trapped water volume (Vtaq) differ from the reference values ​​by more than ±10%.

10. The liposome composition comprising a pharmaceutical active ingredient encapsulated within a plurality of liposomes, the method comprising: (i) determining the concentration of the active pharmaceutical ingredient encapsulated within a plurality of liposomes; (ii) comparing the determined active pharmaceutical ingredient concentration with a reference active pharmaceutical ingredient concentration; and (iii) rejecting the liposome composition if the determined active pharmaceutical ingredient concentration differs from the reference active pharmaceutical ingredient concentration by more than ±10%.

10. The method of claim 9, comprising:

11. 1. A liposome composition formulated for intra-articular administration, comprising a plurality of liposomes consisting essentially of a fluid medium; a polyol tonicity agent; and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a ratio of about 45:55, wherein the composition is essentially free of a pharmaceutically active agent, and the plurality of liposomes comprises: (i) adding a marker to the liposome composition, wherein the marker is liposome-impermeable and has a molecular weight of 1 kDa or greater; (ii) determining the total volume of the liposome composition by measuring a signal induced by the marker in the liposome composition; (iii) separating the fluid medium from the plurality of liposomes to obtain a separated fluid medium and a precipitate comprising a plurality of liposomes and interstitial medium between the liposomes, and measuring a signal induced by the marker in the separated fluid medium to determine the volume of the fluid medium and the interstitial medium between the liposomes; and (iv) calculating the trapped volume (Vt) and trapped water volume (Vtaq) of the plurality of liposomes based on the lipid volume of the plurality of liposomes obtained from the volumes measured in steps (ii) and (iii) and the concentrations of lipids forming the liposomes; A liposome composition characterized by having a specific entrapped water capacity in the range of about 1 to about 4 μL / mg lipid.

12. The liposome composition of claim 11, wherein step (iii) further comprises determining the interstitial volume between liposomes in the precipitate by measuring a signal induced by the marker in the precipitate after separation, and / or the marker has a molecular weight of at least about 2 kDa, and / or the marker is water-soluble, and / or the marker is a radioactive marker, preferably 14 C-carboxyinulin, and / or separation of the fluid medium from the plurality of liposomes is performed by centrifugation.

13. 13. The liposome composition of claim 11 or 12, wherein the plurality of liposomes is selected from the group consisting of small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs), oligolamellar vesicles (OLVs), multilamellar vesicles (MLVs), multivesicular liposomes (MVLs), and mixtures or combinations thereof, and preferably the plurality of liposomes are multilamellar vesicles (MLVs); and / or the plurality of liposomes have a size ranging from about 0.3 μm to about 50 μm; and / or the concentration of the lipids forming the liposomes ranges from about 50 mM to about 300 mM; and / or the composition has a pH of about 5 to about 8.

14. 13. The liposome composition according to claim 11 or 12, wherein the fluid medium is a histidine buffer, and / or the polyol is a linear polyol, and / or the polyol is selected from the group consisting of mannitol, dextrose, lactose, trehalose, and combinations thereof, preferably the polyol is mannitol, and / or DMPC is present in the composition in a weight percentage ranging from about 1% to about 10% by weight, and DPPC is present in the composition in an amount percentage ranging from about 2% to about 12% by weight.

15. 13. The liposome composition of claim 11 or 12, wherein the specific trapped water capacity is in the range of about 1.5 to about 3.5 μL / mg lipid, preferably about 2 to about 3 μL / mg lipid, and / or the trapped water capacity is in the range of about 10% to about 35%, preferably about 20% to about 30%.

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