Freeze-dried liposomes

Abstract

To provide a lyophilized liposome formulation containing two or more drugs that exhibits an excellent drug retention profile and also maintains its size distribution after lyophilization and reconstitution. [Solution] (a) A gel-phase liposome having a melting phase temperature (TC) of at least 37°C, wherein the liposome membrane of the liposome contains 20 mol% or less of cholesterol and at least 1 mol% of phosphatidylglycerol (PG) or phosphatidylinositol (PI), or both, wherein at least two anti-cancer drugs in a certain ratio are stably associated with the liposome; and (b) A cryoprotective substance present on the outside of the liposome, wherein the liposome substantially does not contain the inner cryoprotective substance, and the drug is substantially retained within the liposome, a pharmaceutical composition for the treatment of cancer.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Patent Application 61 / 550,047, filed on 21 October 2011, the contents of which are incorporated herein by reference in their entirety.

[0002] Technical field The present invention relates to a method and composition for producing lyophilized liposomes containing at least two therapeutic or diagnostic agents that can be stored for a long period of time. In one aspect, the present invention relates to low-cholesterol liposomes which optionally contain a cryoprotective substance in an external medium and have resistance to freeze / thaw and dehydration damage to the liposomes, and thus maintain the size and integrity of the liposomes. [Background technology]

[0003] Background of the present invention Liposomes are closed vesicles that have at least one lipid bilayer surrounding a water-soluble core. The space and lipid layer within liposomes can trap a variety of substances, including drugs, cosmetics, diagnostic agents, genetic material, and bioactive compounds. Because non-toxic lipids serve as the base of liposomes, they generally exhibit low toxicity. Low toxicity, along with the ability of liposomes to increase the lifespan of drugs in plasma circulation, makes liposomes a particularly useful vehicle for delivering pharmaceutical active agents. In many cases, liposome-delivered drugs yield superior clinical efficacy, along with the resulting reduced toxicity.

[0004] The practical application of liposomal formulations as drug delivery vehicles is limited by the chemical and physical stability of the formulation. Commercialization requires long-term stability at both chemical and physical levels. The use of freeze-dried (lyophilized) formulations, which avoid the degradation of unstable drugs and / or lipid components, offers some improvements in stability. However, during the lyophilization process, ice crystal formation can cause mechanical rupture, liposome aggregation and fusion (resulting in an increase in liposome size). Furthermore, when drug-containing liposomes are lyophilized and then reconstituted at room temperature, changes in the structure of the liposome bilayer sometimes occur, leading to accelerated drug leakage.

[0005] Conventional attempts in the preparation of lyophilized liposome compositions have relied on conventional liposomes, which are liquid at normal body temperature and whose lipid movement is fluid and uncontrollable. Such conventional liposomes can be classified into two categories. The first is gel-liquid crystal (T) C These are liposomes that are maintained in a liquid state because they contain lipids below body temperature (i.e., they can be in the liquid phase at body temperature). These liposomes are conventionally used in the field; however, a negative aspect of being fluid is poor drug retention for many encapsulating materials.

[0006] A second type of conventional liposome contains a large amount of membrane scaling agent, such as cholesterol (e.g., 30-45 mol%), so that liquid-gel transition never occurs. Cholesterol functions to increase bilayer thickness and fluidity while reducing liposome membrane permeability, protein interactions, and lipoprotein destabilization. These large amounts of cholesterol are most frequently used in liposome research and, while not sufficient to retain all drugs, have historically been taught to be necessary for adequate serum stability and drug retention in vivo. Some drugs exhibit superior drug retention both in vitro and in vivo in liposomes that contain virtually no cholesterol. See, for example, Non-Patent Literature 1.

[0007] On the other hand, gel-phase liposomes are more stable and exhibit improved drug retention. This invention relates to liposomes that are in the gel phase at body temperature (i.e., body temperature is the T temperature of the liposomes). C Liposomes with a lower cholesterol content are utilized. Gel-phase liposomes can be prepared using a variety of lipids; however, liposomes made from more saturated acyl side-chain phosphatidyl lipids (e.g., hydrogenated soybean PC, dipalmitoylphosphatidylcholine (DPPC), or distearoylphosphatidylcholine (DSPC)) are required to have less than 30% cholesterol to achieve a gel phase at body temperature. One example of a conventional liposome that does not exhibit a gel phase at body temperature is one made from egg phosphatidylcholine (EPC), which is remarkably leaky.

[0008] Conventional attempts in the preparation of lyophilized liposome compositions using conventional liposomes have involved either empty liposomes or liposomes containing only a single drug. These may utilize cryoprotective substances, generally sugars, both inside and outside the liposome, or a large osmotic gradient across the liposome membrane.

[0009] For example, cryoprotective substances have been used to protect "liquid" EPC liposomes encapsulating a single drug from freeze / thaw damage when sufficient amounts are present both inside and outside the liposome (ideally, these amounts are equal). See, for example, Patent Documents 1 and 2. The presence of 1% to 10% cryoprotective substance in the liposome fluid protects doxoluvcin-encapsulated lyophilized EPC liposome formulations, preferably with an internal molar osmotic concentration close to the physiological molar osmotic concentration. See, for example, Patent Document 3. It has been shown that the inability to include cryoprotective substance inside the liposome results in a loss of liposome integrity during reconstitution, particularly with respect to the retention of the encapsulated drug. See, for example, Non-Patent Document 2.

[0010] In one case, protection from vesicle aggregation and fusion, as well as loss of the captured agent, has also been reported for hydrogenated soybean PC:cholesterol:DSPE-mPEG (51:44:5 molar ratio) liposomes, in which the liposome formulation contains 44 mol% cholesterol, a cryoprotective agent, and a high concentration of salt in the extracellular solution. The presence of 44% cholesterol means that the liposomes can become liquid at or below body temperature. Furthermore, the protective effect is achieved only when there is a large osmotic gradient across the membrane such that the molar osmotic concentration outside the liposome is significantly higher than the molar osmotic concentration inside. See, for example, Patent Document 4.

[0011] Membrane-bound cryoprotective agents further improve the freeze-drying and freeze-drying resistance of these non-gel phase liposomes. In particular, sugars implanted on the liposome membrane surface via oligo(ethylene oxide) linkers consisting of 1 to 3 repeating units, in the form of EPC or EPC:cholesterol (1:1 molar ratio), have been reported to be cryoprotective agents for liposomes containing fluorescent probes. See, for example, Non-Patent Document 3; Non-Patent Document 4; and Patent Document 5. Baldeschwieler et al. reported that liposomes prepared using oligoethylene oxide linkers in the absence of terminal sugar groups could not be protected from fusion after freezing (Patent Document 5).

[0012] Trehalose in the extrasolution of PC liposome formulations encapsulating a single drug provides resistance to liposome aggregation and fusion (Patent Document 6). Another method for producing small liposomes stabilized against aggregation requires the formation of empty PC:cholesterol (1:1 molar ratio) liposomes, to which a solution of sugar and a single drug is added and then dried. During the drying process, a certain proportion of the reagent is captured within the liposomes. These liposomes are reported to be more stable during storage than in the absence of sugar. See, for example, Patent Document 7.

[0013] As mentioned earlier, most lyophilization techniques have focused on lyophilizing either empty liposomes or liposomes encapsulating a single drug. Lyophilization while maintaining integrity is more challenging when two or more drugs are encapsulated, especially when these drugs differ in their solubility. Encapsulating two or more drugs is sometimes useful because many life-threatening diseases, such as cancer, are influenced by multiple molecular mechanisms, and due to their complexity, the success of treatment achieved with a single drug is limited. Therefore, almost all cancer treatments involve combinations of more than one drug. This is also true for the treatment of other conditions, including infections and chronic diseases.

[0014] Patent Document 8, incorporated herein by reference, reports that gel-phase liposomes with a transition temperature of 38°C or higher can be prepared using saturated phosphatidyl lipids (e.g., DPPC and DSPC) and small amounts (0-20%) of cholesterol, provided that the composition contains at least 1 mol% phosphoinositol (PI) or phosphatidylglycerol (PG). These liposomes have been shown to be stable to freezing at -20°C when containing an encapsulated irinotecan and phloxuridine (FUDR) combination. Simple freezing is generally less harsh and less destructive to the integrity of liposomes than lyophilization.

[0015] The use of liposomes as delivery vehicles for these combinations is advantageous if the liposomes contain drugs that are particularly non-antagonistic and have the ability to maintain their proportions. This general approach is described in detail in Patent Document 9, incorporated herein by reference. This patent teaches a method for determining non-antagonistic or synergistic proportions of various therapeutic agents, including antineoplastic agents that maintain such non-antagonistic or synergistic effects over a wide concentration range. This patent also teaches that it is necessary to deliver the drugs at the proportions in question and maintain those proportions by allowing the delivery vehicle to control the pharmacokinetics. The liposomes illustrated in this patent contain and maintain non-antagonistic or synergistic proportions of two or more therapeutic agents, including irinotecan and FUDR. Such combinations encapsulated in liposomes benefit from the advantage of being preserved in a lyophilized form, provided that the integrity of the liposomes and the concentrations and proportions of the drugs are maintained during reconstitution. A particularly useful combination of cytarabine and daunorubicin encapsulated in liposomes is described in Patent Document 10, which is also incorporated by reference in this disclosure.

[0016] The use of these in therapeutic procedures with remarkably good results is described in Patent Documents 11 and 12. Further formulations involving liposome encapsulation of desired drug delivery options are described in Patent Documents 13, 14, and 15. These formulations are merely good examples of useful compositions in which two or more therapeutic agents are contained within liposomes for delivery to the patient.

[0017] As described above, it has been difficult and unpredictable to prepare a stable lyophilized composition of liposomes that generally maintains the integrity of liposomes during reconstitution. For combinations of two or more agents, obtaining such a stable liposome composition is even more challenging. Therefore, the success of the method of the present invention in obtaining a lyophilized liposome containing two or more therapeutic or diagnostic agents and maintaining the integrity of liposomes during reconstitution is a remarkable achievement.

Prior Art Documents

Patent Documents

[0018]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Patent Document 10

Patent Document 11

Patent Document 12

Patent Document 13

Patent Document 14

[0019] [Non-Patent Document 1] Dos Santos, et al., Biochim. Biophs. Acta, (2002) 1561:188-201 [Non-Patent Document 2] “prevention of leakage requires the sugar be present both inside and outside the liposome” (Lowery, M. (June 2002) Drug Development and Delivery, Vol. 2, No. 4) [Non-Patent Document 3] Bendas, et al., Eur. J. Pharm. Sci. (1996) 4:211-222 [Non-Patent Document 4] Goodrich, et al., Biochem. (1991) 30:5313-5318 [Overview of the project] [Means for solving the problem]

[0020] Disclosure of the present invention It has been consistently reported that cryoprotective materials are required both inside and outside liposomes to maintain liposome integrity during reconstitution after lyophilization, particularly to ensure the retention of encapsulated drugs. We have identified stable liposomes that do not require an internal cryoprotective material, enabling successful lyophilization of liposomes encapsulating not just one, but two or more active drugs.

[0021] The present invention relates to a successful lyophilized gel-phase liposome formulation containing more than one therapeutic and / or diagnostic agent and without an internal cryoprotective substance. Thus, in one aspect, the present invention relates to a lyophilized liposome composition in which the liposomes stably associate with at least two therapeutic and / or diagnostic agents, and when the composition is reconstituted, the average diameter of the liposomes is maintained compared to the pre-lyophilized state, and the proportion of each agent remaining encapsulated within the liposomes is maintained at a satisfactory level. The integrity of the liposomes is therefore measured as the proportion of encapsulated agents retained after reconstitution of the liposomes. An additional parameter used as a criterion for satisfactory lyophilization is the minimum change in size distribution. A particularly important embodiment is one in which the agents are encapsulated within the liposomes in fixed proportions, and the proportions of these agents are maintained when the lyophilized form is reconstituted.

[0022] A typical condition for achieving this result is the gel-liquid crystal transition temperature (T C) includes the use of gel-phase liposomes that may reach or exceed human body temperature at least at room temperature. Body temperature is assumed to be approximately 37°C. The liposomes may be low-cholesterol liposomes stabilized with phosphatidylglycerol and / or phosphoinositol. The liposomes may contain substantially no internal cryoprotectant but may contain an external cryoprotectant on their surface and therefore can be freeze-dried in the presence of a cryoprotectant-containing liquid. The term “substantially no internal cryoprotectant” means liposomes that do not contain an internal cryoprotectant, as well as liposomes that contain an amount of cryoprotectant that does not affect the freezing and / or freeze-drying process of the liposomes (i.e., 125 mM or less of cryoprotectant, i.e., an “inactive” amount). Therefore, “substantially no internal cryoprotectant” stipulates that there is approximately 0–125 mM of cryoprotectant within the liposomes. It is important to note that preventing drug leakage after the freeze-drying process is significantly more difficult than maintaining liposome size. As mentioned above, drug retention after freeze-drying has historically been achieved through the use of cryoprotective substances both inside and outside the liposome.

[0023] Therefore, in one embodiment, the liposome has a membrane gel-liquid crystal transition temperature (T) higher than room temperature or 25°C or 37°C. C The lipid membrane is sufficiently gel-like at room temperature (e.g., 25°C) to aid in drug retention. The composition provides a composition suitable for use with extended shelf life, giving retention of encapsulated drugs, reduced aggregation and fusion during lyophilization and reconstitution. The enhanced protection from the lyophilization process is independent of the permeation potential. These liposomes maintain their size distribution profile and drug encapsulation profile over long periods under pharmaceutically relevant conditions.

[0024] A method for preparing a lyophilized liposome composition is, therefore, that the liposome membrane before freezing and lyophilization has a phase transition temperature TC The liposome may contain a cryoprotective substance at a selected concentration on its outer surface, which is less than 1°C. Preferably, the liposome contains a solution with a glass transition temperature (T°C) of both the solution containing the drug-encapsulated liposome and the extraliposome solution containing the cryoprotective substance. g It freezes at temperatures below )

[0025] In other aspects, the present invention also relates to a method for preparing lyophilized liposomes containing two or more therapeutic and / or diagnostic agents according to the embodiments described above, a method for reconstituting the lyophilized composition, a method for administering the reconstituted liposomes to an animal, and a method for treating an animal that is afflicted with, susceptible to, or suspected of being afflicted with a disease (e.g., cancer). The present invention also provides, for example, the following items: (Item 1) A freeze-dried gel-phase liposome composition, wherein the gel-phase liposomes have a melting phase temperature of at least 25°C (T C ) demonstrates stable association with at least two therapeutic and / or diagnostic agents, substantially does not contain an internal cryoprotective substance, and, Herein, the gel-phase liposome composition, when reconstituted, maintains the average diameter of the liposomes compared to the composition before lyophilization, and retains the drug within the liposomes. (Item 2) The composition according to item 1, wherein the proportion of the encapsulated agent is fixed, and when the composition is reconstituted, the proportion of the agent changes by 25% or less compared to the composition before freeze-drying. (Item 3) The liposome has a phase transition temperature (T) of at least 37°C. C The composition described in item 1, having ) (Item 4) The composition according to item 1, wherein the average diameter of the liposomes increases by 25% or less after lyophilization and during reconstitution of the liposomes, compared to the value measured before lyophilization. (Item 5) The composition according to item 4, wherein the average diameter of the liposomes increases by 15% or less after lyophilization and during reconstitution of the liposomes, compared to the value measured before lyophilization. (Item 6) The composition according to item 5, wherein the average diameter of the liposomes increases by 10% or less after lyophilization and during reconstitution of the liposomes, compared to the value measured before lyophilization. (Item 7) The composition according to item 1, wherein at least 75% of each drug is retained during the reconstitution of the liposomes. (Item 8) The composition according to item 7, wherein at least 85% of each drug is retained during the reconstitution of the liposomes. (Item 9) The composition according to item 8, wherein at least 90% of each drug is retained during the reconstitution of the liposomes. (Item 10) The composition according to item 1, wherein the size distribution of the liposomes changes by 25% or less after freeze-drying and reconstitution of the liposomes. (Item 11) The composition according to item 10, wherein the size distribution of the liposomes changes by 15% or less after lyophilization and reconstitution of the liposomes. (Item 12) The composition according to item 11, wherein the size distribution of the liposomes changes by 10% or less after freeze-drying and reconstitution of the liposomes. (Item 13) The composition according to any one of items 1 to 12, wherein the liposome contains 20 mol% or less of cholesterol and at least 1 mol% of phosphatidylglycerol (PG) and / or phosphoinositol (PI). (Item 14) The composition according to any one of items 1 to 12, wherein the cryoprotective substance is present on the outside of the liposome. (Item 15) The composition according to item 14, wherein the cryoprotective substance is sucrose. (Item 16) The composition according to any one of items 1 to 12, wherein the agent has log partition coefficients that differ by at least 1.0. (Item 17) The composition according to any one of items 1 to 12, wherein the agent is an anti-cancer drug. (Item 18) At least one of the anti-cancer drugs is a nucleoside analog, or The composition according to item 17, wherein at least one of the anti-cancer drugs is an anthracycline. (Item 19) The composition according to item 18, wherein the anti-cancer drugs are daunorubicin and cytarabine. (Item 20) The composition according to item 19, wherein the ratio of daunorubicin:cytarabine is 1:5 on a molar basis. (Item 21) The composition according to item 18, wherein the anti-cancer drugs are irinotecan and floxuridine. (Item 22) The composition according to item 21, wherein the molar ratio of irinotecan and floxuridine is 1:1 on a molar basis. (Item 23) A method for preparing a composition according to any one of items 1 to 12, comprising subjecting an aqueous medium containing gel-phase liposomes, which exhibit a melting-phase temperature (T C ) of at least 25°C, stably associate with at least two therapeutic and / or diagnostic agents, and substantially contain no inner cryoprotective substances, to lyophilization. (Item 24) The method according to item 23, wherein the medium containing gel-phase liposomes is frozen at a temperature below the glass transition temperature (T g ) of the medium. (Item 25) A method for preparing a pharmaceutical composition for administering a therapeutic and / or diagnostic agent to a subject, comprising reconstituting the liposome composition according to any one of items 1 to 12 in a pharmaceutical carrier to obtain a reconstituted composition. (Item 26) A method for administering a therapeutic and / or diagnostic agent to an animal subject, comprising administering a reconstituted composition described in item 25 to the subject. (Item 27) The method according to item 26, wherein the administration is parenteral. (Item 28) The method according to item 26 or 27, wherein the subject is human. [Brief explanation of the drawing]

[0026] [Figure 1] Figure 1 shows the particle size profile of CPX-1 liposomes before freezing.

[0027] [Figure 2] Figure 2 shows the particle size profiles of reconfigured CPX-1 liposomes immediately after freezing, lyophilization, and reconstitution.

[0028] [Figure 3] Figure 3 shows the particle size profile of reconstituted CPX-1 liposomes after one month of storage.

[0029] [Figure 4A] Figures 4A-4C show the particle size profiles of reconstituted CPX-1 liposomes after 6 months of storage. [Figure 4B] Figures 4A-4C show the particle size profiles of reconstituted CPX-1 liposomes after 6 months of storage. [Figure 4C] Figures 4A-4C show the particle size profiles of reconstituted CPX-1 liposomes after 6 months of storage. [Modes for carrying out the invention]

[0030] The present invention provides, for the first time, a lyophilized gel-phase liposome composition containing two or more therapeutic and / or diagnostic agents, such that the characteristics and properties of the reconstituted lyophilized composition are essentially consistent with those of the composition before lyophilization. These characteristics may include the average diameter, size distribution, and content of the liposomes. The content of the liposomes refers to the retention of the drug; in some embodiments, similarly, to the proportion of the drug that is retained.

[0031] The liposomes contain therapeutic and / or diagnostic agents, but in this application, “drug” is sometimes used as an abbreviation to specify these.

[0032] The gel-phase liposomes contain one or more lipid bilayers surrounding an inner compartment. These liposomes can be bilayer or monolayer vesicles. Monolayer liposomes (also known as monolayer vesicles or "ULVs") surround a single inner water-soluble compartment and are classified as either small monolayer vesicles (SUVs) or large monolayer vesicles (LUVs). LUVs and SUVs range in size from approximately 50–500 nm and 20–50 nm, respectively. Bilayer liposomes have two lipid membranes: an inner membrane surrounds a single inner water-soluble compartment, and a second, larger outer membrane surrounds the inner membrane, thus creating a second inner water-soluble compartment.

[0033] The maintenance of the size distribution of the gel-phase liposomes can be experimentally evaluated by obtaining particle size profiles, as shown in Figures 1-4 of this application. The size distribution determined by quasi-elastic light scattering is typically represented as a histogram showing the average diameter of the liposomes. The most commonly used important size distribution measures in this field are D10, D90, D99, or standard deviation or polydispersion index. A "D99" value means that 99% of the liposomes are either less than or greater than the referenced size. This is particularly useful when it is important to exclude either larger or smaller sizes, for example. For example, in certain embodiments, it is desirable to ensure that no liposomes with an average diameter greater than 200 nm are present.

[0034] A specific example with a D99 value of 178 nm is used for illustration. A D99 value measured at 178 nm (as seen in Table 1 of Example 2) ensures that at least 99% of the liposome aggregates are less than 178 nm. D10 and D90 values ​​for average diameter are also commonly used, where less than 10% of the aggregates are smaller than the minimum reference size (i.e., D10), and for D90, 90% of the aggregates are at or below the upper limit of the reference size. For example, as seen in batches 1 and 2, the D10 value is 68 nm, meaning less than 10% of the liposome aggregates are less than 68 nm. The D90 value for batches 1 and 2 indicates that 90% of the aggregates are 135 nm or 137 nm or less, respectively. Maintaining the size distribution of liposomes after lyophilization and reconstitution is defined herein as being demonstrated by showing that the reference value of the selected D value changes by no more than 50%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, or 5% during reconstitution compared to the D value before freezing and / or lyophilization. The selected D value may be an integer between 99, 98, 94, and 90, or D10.

[0035] One characteristic of lyophilized liposomes relates to the average diameter of the liposomes in the composition. Based on the diameter before freezing, the average diameter of the liposome composition is maintained in reconstruction if, after lyophilization and reconstruction, the average diameter of the liposomes does not increase by more than 50%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, or 5% on a volume-weighted basis. Concomitant values, such as a 10% increase in the average diameter of the liposomes and a 10% increase in the reference for D90 (or other D values ​​as listed above), are one measure to ensure that the size distribution of the particles (e.g., liposomes) has not changed. The overall properties of the dispersion can also be preferably evaluated on a volume-weighted basis, as shown in Figures 1-4.

[0036] More specifically, liposome compositions contain a size range that typically follows a Gaussian curve. The average diameter of liposomes may increase by only about 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, or 5% or less from their initial size during reconstitution after freezing, or during lyophilization and reconstitution. For example, a sample of liposomes with an average diameter of 90 nm is considered resistant to the effects of freezing and / or lyophilization if the average diameter is only 30% or less larger during reconstitution (i.e., about 117 nm or less). This size increase is more indicative of aggregation and fusion of the liposomes than would suggest. Sufficiently sensitive measurement techniques can be used to measure changes in size distribution or average diameter, and as a result, changes of less than 10% may be measured.

[0037] Another criterion for preserving integrity is the retention of the encapsulated drug. Unlike the average diameter, size distribution, and drug percentage, which are evaluated in comparison to values ​​before lyophilization, drug retention is evaluated after reconstitution as a relative value to the total amount of drug itself (i.e., based on the total drug in the lyophilized composition). The percentage of drug encapsulated within the liposome, or the percentage of drug in the extraliposome solution ("unencapsulated" %), is relative to the total amount of drug in the composition. In one embodiment, at least about 75% of the encapsulated drug is retained encapsulated after lyophilization and reconstitution. At least about 85% or at least 90% or 95% of each may be retained encapsulated. This can also be measured by the amount of unencapsulated drug in the surrounding solution and should not exceed 25%, 20%, 15%, 10%, or 5% of the initial encapsulated amount when reconstituting the lyophilized liposomes.

[0038] The proportion of the encapsulated therapeutic and / or diagnostic agent is maintained in the reconstitution if the proportion does not deviate by more than 20%, 10%, 9%, 8%, 7%, 6%, or 5% from the proportion in the composition itself before lyophilization. The proportion is expressed as a molar ratio.

[0039] In one embodiment, the average diameter of the liposomes after lyophilization and reconstitution may increase by 25% or less compared to the measured value before lyophilization. In another embodiment, the average diameter of the liposomes after lyophilization and reconstitution may change by 15% or less compared to the measured value before lyophilization. In yet another embodiment, the average diameter of the liposomes after lyophilization and reconstitution may change by 10%, 9%, 8%, 7%, 6%, or 5% or less compared to the measured value before lyophilization.

[0040] In some embodiments, the percentage of unencapsulated drug is 25% or less of the drug originally encapsulated during the reconfiguration of the liposomes. In other embodiments, the percentage of unencapsulated drug is 15% or less of the drug originally encapsulated during the reconfiguration of the liposomes. In other embodiments, the percentage of unencapsulated drug is 10% or less, or 9%, 8%, 7%, 6%, or 5% or less of the drug originally encapsulated during the reconfiguration of the liposomes.

[0041] In other words, in some embodiments, the percentage of retained encapsulated drug is 75% or more upon reconstitution of the liposomes. In other embodiments, the percentage of each encapsulated drug is 85%, 90%, or 95% or more upon reconstitution of the liposomes.

[0042] Combinations of these parameters are also included. For example, the average diameter may increase by no more than 25% and the percentage of encapsulated drug may be at least 90%, or the average diameter may increase by no more than 10% and the percentage of encapsulated drug may be at least 90%.

[0043] In some embodiments, the size distribution of the liposomes changes by 25% or less after lyophilization and reconstitution compared to before lyophilization. In other embodiments, the size distribution of the liposomes changes by 15%, 10%, 9%, 8%, 7%, 6%, or 5% or less after lyophilization and reconstitution compared to before lyophilization.

[0044] As described above, various combinations of these parameters, or criteria for determining whether the liposomes have been successfully lyophilized and reconstituted, are considered. For example, the encapsulated drug content being at least 85% may be combined with an increase in average diameter of 15% or less, and optionally with a change in size distribution of 5% or less. Each possible combination of these parameters is within the scope of the present invention.

[0045] Gel-phase liposomes can be produced using conventional techniques. For example, injection method (Deamer, et al., Acad. Sci. (1978) 308:250), surfactant method (Brunner, et al., Biochim. Biophys. Acta (1976) 455:322), freeze-thaw method (Pick, et al., Arch. Biochim. Biophys. (1981) 212:186), reverse-phase evaporation method (Szoka, et al., Biochim. Biophys. Acta. (1980) 601:559-571), sonication method (Huang, et al., Biochemistry (1969) 8:344), ethanol injection method (Kremer, et al., Biochemistry (1977) 16:3932), extrusion method (Hope, et al., Biochim. Biophys. Acta (1985) 812:55-65), and either the French press method (Barenholz, et al., FEBS Lett. (1979) 99:210) are mentioned.

[0046] These processes can be used in combination. Small monolayer vesicles (SUVs) can be prepared in particular by sonication, ethanol injection, and French press. Large monolayer vesicles (LUVs) can be prepared by injection, surfactant, freeze-thaw, reverse-phase evaporation, French press, or extrusion. Preferably, LUVs are prepared according to the extrusion method.

[0047] Lyophilization and reconstitution are carried out under conditions in which the liposomes are in the gel phase. The gel-liquid phase transition temperature of the liposomes must therefore be higher than room temperature (i.e., about 20-30°C), and more preferably higher than body temperature or above. Room temperature can vary considerably, but it is important that the lyophilization process begins under conditions in which the liposomes are in a gel state. In some embodiments, T CThe temperature is at least the same as body temperature (i.e., about 37°C). In some embodiments, the liposomes are prepared at a temperature below the phase transition temperature to maintain gel-like properties. Any suitable internal solution can be used. Typically, the internal solution is an aqueous medium. The internal solution is substantially free of cryoprotective substances (i.e., less than 125 mM of cryoprotective substance). The internal solution may contain less than 100 mM or less than 50 mM of cryoprotective substance, or it may not contain any cryoprotective substance at all.

[0048] Suitable T C Liposome formulations with a certain value may be "low-cholesterol" liposomes, that is, they are prepared and contain an amount of cholesterol (i.e., typically 20 mol% or less) that is insufficient to significantly alter the characteristics of the liposome's phase transition. Cholesterol higher than 20 mol% broadens the temperature range in which the phase transition occurs, and at higher cholesterol levels, the phase transition does not occur. Liposomes with low cholesterol may have less than 20 mol% or less than 15 mol%, or 10 mol%, 5 mol%, or less cholesterol, or may not have any cholesterol at all. Such liposomes may require, if necessary, at least 1 mol% of a stabilizer such as PG or PI.

[0049] In methods in which a cryoprotective substance is used, the cryoprotective substance is preferably present only in the external solution of the formulation. Typically, the cryoprotective substance is a disaccharide such as sucrose, maltose, trehalose, and lactose. The cryoprotective substance may be a disaccharide such as sucrose having a concentration of about 100 mM to 500 mM or about 250 to 400 mM or higher than 300 mM. The external solution may contain about 100 mM to 500 mM of cryoprotective substance and the internal solution may contain less than 125 mM, or the external solution may contain about 250 mM to 400 mM of cryoprotective substance and the internal solution may contain less than 100 mM, or the external solution may contain about 250 mM to 400 mM of cryoprotective substance and the internal solution may not contain any cryoprotective substance, or the external solution may contain about 250 mM to 400 mM of cryoprotective substance and the internal solution may not contain any cryoprotective substance. The cryoprotective substance may be a sugar such as sucrose.

[0050] Gel-phase liposome formulations can be lyophilized (or freeze-dried) using any suitable protocol. The initial temperature of the freeze-drying chamber is preferably the glass transition temperature (T) of the solution containing the external liquid, as well as the solution containing the liposomes encapsulating the drug. g ) is less than. For example, liposomes can be frozen at temperatures below about -5°C, about -10°C, about -20°C, about -30°C, or about -40°C. In some embodiments, when sucrose is used as the solution of the cryoprotective substance, the starting temperature of the freeze-drying chamber is the T of the sucrose solution. g It is below -32℃. g This includes the "glass transition temperature" and "glass phase transition temperature," which are the approximate midpoint temperatures at which a non-frozen solution undergoes a transition from a soft, viscous gel to a hard, relatively brittle form.

[0051] Lyophilized liposomes can be stored at or below room temperature. Some illustrated embodiments have liposomes stored at or below 5°C. Some other illustrated embodiments have liposomes stored at 25°C. Lyophilized products remain stable for at least about 6 months, at least 9 months, at least 1 year, or at least 24 to 36 months (e.g., maintaining relative particle size and preserving encapsulated drugs).

[0052] The captured drugs are therapeutic or diagnostic agents, and sometimes anticancer agents. Surprisingly, the contents and integrity of the gel-phase liposome composition are maintained even if the drugs have different solubility properties with respect to aqueous and non-aqueous solvents. Using the approach of the present invention, drugs with a difference of only 1.0 in the log partition coefficient (LogP) can be successfully retained. Differences in the log partition coefficient of 1.5, 2.0, or 3.0 can be similarly acceptable. One drug may be amphiphilic and the other may be water-soluble, or one may be hydrophobic and the other may be water-soluble. The LogP value is based on the dispersion coefficient between octanol and water, i.e., the base of the ratio of the amount of octanol to the amount of water when the compound undergoes phase separation is the logarithm of 10.

[0053] Examples of anticancer agents include anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, or idarubicin). These drugs are amphiphilic. Examples of such anticancer agents include nucleoside analogs (e.g., hydrophilic cytosine arabinoside, 5-FU, or FUDR). Other anticancer agents include amphiphilic camptothecin or camptothecin derivatives such as irinotecan. In some cases, both anthracyclines and nucleoside analogs are encapsulated, or both camptothecin or a camptothecin derivative and a nucleoside analog are encapsulated. Encapsulation and / or loading of drugs into liposomes can be performed using any suitable loading technique, including passive and active loading. Important embodiments include those described in U.S. Patents 7,850,990 and 8,022,279 cited above (i.e., combinations of irinotecan:floxuridine (FUDR) in a molar ratio of 1:1 and daunorubicin:cytarabine (AraC) in a molar ratio of 1:5). Specific formulations of these drugs designate CPX-1 and CPX-351, respectively.

[0054] The drug is incorporated into one or more water-soluble compartments within a liposome by either a passive or active loading procedure, or a combination thereof. In passive loading, the bioactive agent may simply be included in the preparation in which the liposome is formed, or the active agent may be added to the outside of the pre-formed liposome and passively loaded into the liposome according to a decreasing concentration gradient. If necessary, non-encapsulated material may be removed from the preparation by any suitable procedure. Alternatively, active loading procedures may be used, such as metal-based loading methods based on ion gradients, ion permeability pores, pH gradients, and metal complexes. One commonly used embodiment for suitable drugs is loading by a metal-based method.

[0055] Liposomes are approximately 80–500 nm in size. In one embodiment, liposomes have a diameter of less than 300 nm, sometimes less than 200 nm. In one example, the typical size of these liposomes is approximately 100 nm. In some embodiments, the liposome membrane consists of distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and cholesterol (CHOL). In some embodiments, the liposome membrane consists of 50–80% DSPC, 1–20% DSPG, and 1–20% CHOL. In other embodiments, the liposome membrane consists of 50–80% DSPC or DPPC, 1–20% DSPG or distearoyl phosphatidylinositol (DSPI), and 1–20% CHOL, and the liposome contains less than 125 mM of cryoprotective material in the liposome fluid. In some illustrated embodiments, the liposome membrane is composed of 50–80% DSPC or DPPC, 1–20% DSPG or DSPI, and 1–20% CHOL, and the liposome contains less than 50 mM cryoprotective material in the liposome fluid. In other illustrated embodiments, the liposome membrane is composed of DSPC, DSPG, and CHOL in a molar ratio of approximately 7:2:1, and does not contain cryoprotective material in the liposome fluid. In one example, the liposomes are prepared by water in oil derived from the liposome method, and the extruded liposomes are suspended in phosphate-buffered sucrose at pH 7.0. In another example, the extruded liposomes are suspended in sucrose. In one embodiment, the extruded liposomes are suspended in 250–400 mM sucrose.

[0056] Any suitable method for encapsulating the drug combination within liposomes may be used. In a specific embodiment, irinotecan and phloxuridine are simultaneously loaded into DSPC / DSPG / CHOL(7 / 2 / 1) pre-formation liposomes, thereby, using copper sulfate or copper gluconate in the internal solution, at 50°C, phloxuridine is passively loaded into the liposomes and irinotecan is actively loaded. See both U.S. Patents 7,850,990 and 7,238,367, co-applications, incorporated by reference. In another specific embodiment, cytarabine and daunorubicin are encapsulated within liposomes, thereby, using a copper gluconate / triethanolamine-based loading procedure, cytarabine is passively encapsulated into pre-formation liposomes with high trapping efficiency, and daunorubicin is actively accumulated within the liposomes. See, for example, both International Publication 05 / 102359 and International Publication 07 / 076117 A2 of the concurrently pending and jointly filed PCT applications, which are referred to in their entirety.

[0057] The freeze-dried compositions of the present invention offer convenience in terms of ease of storage, preservation, and transportation. These freeze-dried compositions maintain their characteristics over long periods of time.

[0058] For use, the compositions of the present invention are reconstituted in a suitable pharmaceutical carrier or medium.

[0059] These preparations for use are prepared according to standard reconstitution techniques using pharmaceutically acceptable carriers. Generally, ordinary physiological saline is used as a pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, dextrose, 0.4% sodium chloride, 0.3% glycine, and glycoproteins such as albumin, lipoproteins, and globulins for enhanced stability. These compositions can be sterilized by common and well-known sterilization techniques. The resulting aqueous solutions are packaged for use, or filtered under sterile conditions and lyophilized. The lyophilized preparations can be combined with a sterile aqueous solution before administration. The compositions may contain pharmaceutically acceptable adjuncts (such as pH adjusters and buffers, osmotic pressure adjusters, etc., e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride) as required for approximate physiological conditions. Furthermore, liposome suspensions may contain lipid protectants to protect lipids from damage by free radicals and lipid peroxidation during storage. Lipophilic free radical quenchers such as α-tocopherol and water-soluble iron-specific chelating agents (e.g., ferrioxamine) are suitable.

[0060] The reconstituted formulations may be administered to animals, including humans or other mammals (e.g., non-human primates, dogs, cats, cattle, horses, sheep, etc.), and used to treat a variety of diseases. Examples of the medical use of the compositions of the present invention include, but are not limited to, the treatment of cancer, cardiovascular diseases such as hypertension, arrhythmias, and restenosis, the treatment of bacterial, fungal, or parasitic infections, the treatment and / or prevention of diseases through the use of the compositions of the present invention as vaccines, the treatment of inflammation, or the treatment of autoimmune diseases. For the treatment of human diseases, a qualified physician will use established protocols to determine how the compositions of the present invention should be used in terms of dosage, schedule, and route of administration. In such applications, dose escalation may also be utilized to determine whether the bioactive agents encapsulated in the liposomes and lipid carriers of the present invention exhibit reduced toxicity to the healthy tissues of the subject.

[0061] The pharmaceutical composition is typically administered parenterally (e.g., intravenously), but other routes may be used. The dosage of the liposomal formulation depends on the patient's age, weight, and condition, the ratio of drug to lipids, and the opinion of the administering physician.

[0062] Overall, one process useful in the present invention comprises lyophilizing a liposome composition, wherein the liposomes comprise 20 mol% or less of cholesterol and two or more active agents, and the liposome membrane is below the phase transition temperature when in an external solution containing a cryoprotective substance at room temperature; storing the lyophilized liposomes; and reconstituting the lyophilized liposomes in a predetermined volume of water solubility. The liposomes may be lyophilized at a temperature below about -5°C, or below about -10°C and -20°C, or even below about -30°C or -40°C, and stored at room temperature (about 23-25°C) or below.

[0063] In one embodiment, the liposome composition is composed of any intermediate value of cholesterol, such as 2-20% cholesterol or 10% cholesterol.

[0064] In one embodiment, the lyophilized composition comprises about 10% cholesterol, liposomes composed of disaccharides at a selected concentration in an external solution, and reconstitution carried out at room temperature. C The cryoprotective substance is less than [amount missing] and exists unbound only on the outside of the liposome.

[0065] In another embodiment, upon reconstitution using a predetermined volume of aqueous medium, a lyophilized liposome composition containing two or more encapsulated drugs yields a liposome composition comprising: (a) liposomes containing less than 20 mol% cholesterol; (b) liposome sizes predominantly between approximately 80 and 500 nm; (c) one or more drugs captured in liposomes with an encapsulation percentage of approximately 95%, 90%, 85%, 80%, or 75% or more; and (d) a cryoprotective substance in the extraliposome solution between approximately 100 mM and 500 mM. In some embodiments, the cryoprotective substance is present in the extraliposome solution between 250 and 400 mM. In some embodiments, the cryoprotective substance is present in the extraliposome solution between approximately 9.5 and 10%.

[0066] In one embodiment, monolayer or bilayer gel-phase liposomes containing 20 mol% or less of cholesterol, at least two drugs, and at least about 300 mM of sucrose outside the liposomes are lyophilized, and upon reconstitution, at least about 90% of each encapsulated drug is encapsulated, and the average liposome diameter changes by less than about 25%.

[0067] As used herein, “a” or “an” means “at least one” or “one or more” unless the context makes it clear that only a single referent is intended.

[0068] The following examples are provided for illustrative purposes only and are not intended to limit the invention. [Examples]

[0069] Example 1 Freeze-drying of CPX-1 Irinotecan and floxuridine were encapsulated together in DSPC / DSPG / cholesterol (7:2:1 molar ratio) liposomes and designated CPX-1. The lyophilized CPX-1 resulted in stable drug-loaded liposomes, with minimal leakage of the active pharmaceutical ingredient from the reconstituted dosage form. Irinotecan hydrochloride used in CPX-1 has a predicted Log partition coefficient (LogP) of 3.94. Furoxuridine has a predicted LogP of -1.14.

[0070] Thermal analysis was performed on various lots of the CPX-1 liposomal drug product to determine the glass transition temperature (T g Information was provided regarding changes in heat capacity and other exothermic events. The decay temperature of the CPX-1 liposomal drug product was determined for two lots by lyophilization microscopy. These results were used in determining the final lyophilization cycle.

[0071] The sample consisted of a blue-green bulk aqueous suspension formulated in liposomes containing two active pharmaceutical ingredients, irinotecan hydrochloride and phloxuridine, in a 1:1 ratio. The sample was stored at ambient relative humidity at -20°C (or possibly -80°C), thawed overnight in a refrigerator, and thoroughly mixed before filling and freeze-drying.

[0072] Using a Cycle 1:20 mL Class A pipette, 20 cc of CPX-1 was filled into 60 cc glass molded vials. Twenty-four vials were loaded into a LyoStar II Tray Dryer, thermocouple probes were attached to two product vials to record the product temperature, and the vials were freeze-dried for four and a half days. After backfilling the vials with nitrogen gas to a chamber pressure of approximately 720,000 mTorr, the vials were stoppered, removed from the Tray Dryer, and labeled Lot TP-CPX1-001-032405. Some of the freeze-dried vials were then placed to accelerated stability at 25°C and 40°C, while the remaining vials were stored at -20°C.

[0073] Cycle 2: Approximately 21 mL of CPX-1 was filled into a 60 cc glass-forming vial and a 50 cc glass tube vial, respectively. These vials were LyoStarII TM The vials were loaded into a tray dryer, with one thermocouple probe attached to one product vial in the upper row and one to one product vial in the lower row. Upon completion of the freeze-drying cycle, the vials were backfilled with nitrogen gas to a chamber pressure of approximately 720,000 mTorr, plugged, removed from the tray dryer, and labeled as Lot TP-CPX1-002-041305T. Some of the freeze-dried vials were then placed to accelerated stability at 25°C and 40°C, while the remaining vials were stored at -20°C.

[0074] Cycle 3: The lyophilized samples were prepared in the same manner as in Cycle 2, except that they were filled into 50cc glass vials. The sealed vials were labeled CPX-1 formulation, Lot TP-CPXl-003-051105T. Some of the lyophilized vials were then placed to accelerated stability at 25°C and 40°C, while the remaining vials were stored at -20°C.

[0075] Cycle 4: Lyophilized samples were prepared in the same manner as in Cycle 2, except that CPX-1 was filled into 50cc glass vials. The sealed, lyophilized vials were labeled CPX-1 formulation, Lot TP-CPXl-004-051805T, and stored at -20°C, 5°C, 25°C, and 40°C for stability testing.

[0076] Cycle 5: Lyophilized samples were prepared in the same manner as in Cycle 2, filled into 50cc glass vials. The sealed and lyophilized vials were labeled CPX-1 formulation TP-CPXl-005-062705T-300. The CPX-1 liposome formulation vials were stored in a stability chamber at -20°C, 5°C, and 25°C.

[0077] The first lyophilization cycle was performed. The primary objective of the first lyophilization cycle (Cycle 1) was to determine whether the formulated bulk CPX-1 liposome formulation (CPX-1) could be successfully lyophilized gently in a two-step primary drying phase. The success of this lyophilization cycle was evaluated by analyzing the temperature and pressure profiles of the formulation, as well as by visually inspecting the appearance of the lyophilized cake.

[0078] The freeze-dried product profile for Cycle 1 showed that bulk ice was removed during the primary drying stage at -10°C. This was evident in the fact that the product temperature was slightly above the shelf temperature. Furthermore, the thermocouple pressures measuring true pressure and partial pressure of water vapor decreased toward the pressure of the capacitive pressure gauge or that of the true pressure. In addition, the freeze-dried formulation vial appeared dry with little to no evidence of cake collapse. However, some analytes were observed to be concentrated or layered. To optimize this cycle, the heat treatment phases and primary drying stage were modified for the second run.

[0079] The second freeze-drying cycle was performed. The second freeze-drying cycle (Cycle 2) was carried out using the same gentle primary and secondary drying phases as in Cycle 1. To maximize the ice load in the freeze-dryer, vials filled with deionized water were loaded into the available shelf space. The success of this freeze-drying cycle was also evaluated by measuring the temperature and pressure profiles, as well as by visually inspecting the freeze-dried cake.

[0080] Even though the product temperature and pressure profiles over a 4.5-day cycle were similar for both 50cc glass tube vials and 60cc molded glass vials, the formulation in the 50cc tube vials appeared to freeze-dry in a more uniform manner. After reaching the secondary drying shelf temperature, the product temperature exceeded the ice barrier (i.e., the product temperature rose above 0°C) for approximately 8.5 hours, indicating the completion of bulk ice sublimation. However, these vials were not sufficiently dried. The product temperature fell below the shelf temperature at the end of the primary drying phase, and the difference between the thermocouple pressure and the capacitive pressure gauge pressure remained unchanged from the beginning of the run throughout the secondary drying phase, indicating the presence of substantial bulk ice in the vials.

[0081] The shelf temperature used in the primary drying phase did not provide enough energy to drive and complete the sublimation rate of the product. Therefore, a third freeze-drying cycle was performed, using shelf temperatures and chamber pressures that increased the sublimation of ice (while ensuring that the decay temperature was not exceeded) to drive and complete the primary drying phase.

[0082] The third freeze-drying cycle was performed. Based on the results of cycle 2 and thermal analysis, the shelf temperature and chamber pressure for cycle 3 were adjusted to promote primary drying, while maintaining the product temperature below the estimated collapse temperature of -20°C. To maximize the ice load, the cycle was performed under fully loaded run conditions.

[0083] The profile plot obtained in Cycle 3 showed that the initial primary drying shelf temperature of -20°C at a pressure of 100 mTorr did not sufficiently drive the sublimation of bulk ice over 40 hours under fully loaded run conditions. Furthermore, the thermocouple pressure trace did not significantly decrease toward the capacitive pressure gauge until the end of the primary drying phase at -10°C, due to the limited duration of this phase. However, the profile illustrated that a second primary drying shelf temperature and duration of -10°C could maintain a product temperature below the -20°C collapse temperature until all bulk ice sublimated, as evidenced by the sharp rise in product temperature that ultimately exceeded the shelf temperature at the end of this phase.

[0084] The fourth freeze-drying cycle was executed. To finally determine the shelf temperature for the primary drying phase, the fourth freeze-drying cycle (cycle 4) used a shelf temperature of -10°C for a longer duration, along with a 6-hour initial primary drying phase at -20°C under fully loaded run conditions.

[0085] Based on the freeze-drying cycle profile and visual observations, a shelf temperature of -20°C for the initial primary drying phase appeared to offer little benefit in drying the vials. The product temperature probe exceeded a shelf temperature of -10°C after approximately 60 hours of holding time. Since the product temperature profile closely matched the thermocouple pressure at the shelf temperature, the thermocouple pressure during the secondary drying phase indicated that the vials had relatively low residual moisture.

[0086] The encapsulation of the pharmaceutical substance, the particle size of the liposomes, and the average residual moisture content were evaluated for vials of the lyophilized product. The Karl Fischer method used recovered an average residual moisture content of 3.1%, indicating that the liposome product was not excessively dried. Analysis of particle size and irinotecan encapsulation percentage revealed that the lyophilized product was unchanged compared to the pre-lyophilized material. However, the percentage of unencapsulated fulocusuridine increased from 7.0% in the pre-lyophilized bulk to 8.6% in the lyophilized product after 13 weeks of storage at -20°C. Furthermore, after stressing the product at 25°C for 4 weeks, the percentage of unencapsulated fulocusuridine increased to 11.8%, exceeding the provisional standard of less than 10% for unencapsulated fulocusuridine in this formulation.

[0087] The fifth freeze-drying cycle was performed. The goal of the fifth freeze-drying cycle (cycle 5) was to determine the shelf temperature for the secondary drying phase from +20°C to +10°C in order to minimize phloxuridine leakage while achieving appropriate residual moisture under fully loaded run conditions. The moisture content of the material was assessed by periodically measuring the pressure rise during the secondary drying phase.

[0088] The formulation profile for Cycle 5 showed that the bulk ice had largely sublimated after 72–84 hours of primary drying at -10°C. Furthermore, based on differences in pressure detectors and pressure increase tests, the substance appears to dry sufficiently using a shelf temperature of +10°C at 50 mTorr for 12 hours.

[0089] To assess the suitability of this lyophilized material, the reconstitution time for lyophilized formulation vials from both cycles 4 and 5 was evaluated using an 18-gauge needle and a 30cc syringe, with 19mL of water injected through the stopper. The mean reconstitution times were determined to be 40 and 93 seconds for cycles 4 and 5, respectively. Furthermore, the Karl Fischer result for cycle 5 recovered an average residual moisture content of 3.2%, which was consistent with the average residual moisture content of vials from cycle 4.

[0090] The encapsulation of the pharmaceutical substance and the particle size of the liposomes were also evaluated. For the lyophilized formulations of Cycle 5, the percentage of unencapsulated irinotecan was 0.4% after 7 weeks of storage at -20°C and 0.9% after 4 weeks of storage at 25°C. The particle size of the lyophilized formulations increased only slightly after 8 weeks of storage at 5°C compared to the formulations stored at -20°C, but increased by almost 10 nm after only 4 weeks of storage at 25°C. Unsurprisingly, the percentage of unencapsulated fulocusuridine showed a similar trend to the change in particle size. The percentage of unencapsulated fulocusuridine was 5.5% after 7 weeks at -20°C, 7.7% after 8 weeks at 5°C, and 18.7% after 4 weeks at 25°C.

[0091] By performing a fifth freeze-drying cycle using a reduced shelf temperature during the secondary drying phase, we successfully produced an acceptable freeze-dried CPX-1 liposome formulation with increased inclusion retention.

[0092] Example 2 In freeze-dried liposomes, the particle size profile remains unchanged over time. Experiments were conducted to investigate the effects of freezing, lyophilization, and storage on the size distribution of double-loaded CPX-1 and CPX-351 liposomes. CPX-351 is a formulation containing daunorubicin and cytarabine in a molar ratio of 1:5 within liposomes where distearoylphosphocholine (DSPC):distearoylphosphatidylglycerol (DSPG):and cholesterol (CHOL) are in a molar ratio of 7:2:1. Daunorubicin has a predicted LogP of 1.68. Cytarabine has a predicted LogP of -2.17.

[0093] The particle size distribution of simultaneously loaded liposomes was measured not only before and after freezing and lyophilization of the liposomes, but also one month and six months after storage of the lyophilized formulation.

[0094] CPX-1 liposomes were prepared with 300 mM sucrose, 20 mM phosphate, and an external buffer at pH 7.0. A fixed volume of 900 μl was added to a 2 mL vial, placed in a metal dish (pre-cooled to -20°C), and stored overnight at -20°C. After freezing, the sample was transferred to a lyophilizer (pre-cooled to -20°C). The shelf temperature was maintained at -20°C for 7 hours under reduced pressure, and then raised to -10°C over approximately 16 hours. For the third temperature step, the shelf temperature was further increased to 4°C over the next 3 hours, and then dried at room temperature for 3 hours to complete the process. The dried sample was hydrated with 1 mL of H2O, and the lyophilized cake was immediately dissolved. The sample was then analyzed using dynamic light scattering (DLS).

[0095] Pre-frozen CPX-1 liposomes exhibited an average diameter of 110 nm (Figure 1). The liposome size immediately after lyophilization and rehydration was observed to be 116 nm (Figure 2). Two samples of lyophilized CPX-1 liposomes were stored at 5°C for 1 month or 6 months, and the liposome size of the rehydrated compositions was observed. The average liposome sizes were 117 nm and 123 nm, respectively (Figures 3 and 4). Figures 1-3 show the volume-weighted distribution. Figure 4B shows the comparative volume-weighted distribution. Results from other less favorable algorithms are shown in Figures 4A and 4C. Unless otherwise specified, average diameter refers to the volume-weighted distribution.

[0096] Similar experiments to those shown in Figures 1-4 were also performed on CPX-351 liposomes.

[0097] As described above, CPX-351 is a liposomal formulation of a fixed combination of the anti-cancer drugs cytarabine and daunorubicin hydrochloride. The liposomes are prepared using DSPC, DSPG, and CHOL in a molar ratio of 7:2:1 in copper gluconate-triethanolamine buffer, pH 7.4. Crude liposomes are extruded, resulting in a size distribution of liposome particles with an average liposome diameter between 80 nm and 110 nm and a D99 of less than 200 nm (analysis by dynamic light scattering). Cytarabine is encapsulated by a passive loading mechanism. Daunorubicin is encapsulated by an active copper-mediated mechanism, achieving a molar ratio of cytarabine:daunorubicin of 5:1. Any unencapsulated drugs are removed, and the bulk buffer is modified by diafiltration. Multiple volumes of 300 mM sucrose are exchanged to form the final CPX-351 liposome, which is then subjected to lyophilization optimization. The dried CPX-351 sample is reconstituted with 19 mL of H2O to immediately rearrange the liposome dispersion. The sample is then analyzed using dynamic light scattering (DSL).

[0098] Pre-freezed CPX-351 liposomes exhibited an average diameter of approximately 100 nm. For batch 1 (labeled "1C001" in Table 1 below), the liposome sizes immediately after freezing and subsequent freeze-drying / rehydration were observed to be 99 nm and 100 nm, respectively. For the second batch, 1D002, the CPX-351 liposome sizes immediately after freezing and subsequent freeze-drying / rehydration were observed to be 104 nm and 105 nm, respectively.

[0099] These results demonstrate that DSPC / DSPG low-cholesterol liposomes simultaneously loaded with either irinotecan + phloxuridine or cytarabine + daunorubicin effectively maintain their size distribution profile during freezing, lyophilization, and long-term storage. These results also indicate that these low-cholesterol gel-phase liposomes are resistant to aggregation and fusion, which commonly occur during freezing and lyophilization, particularly in the absence of high levels of cholesterol. [Table 1]

[0100] Example 3 In freeze-dried liposomes, the drug encapsulation percentage remains unchanged over time. Experiments were conducted to investigate the effects of freezing and / or lyophilization and storage on the degree of drug leakage from double-loaded CPX-1 or CPX-351 liposomes.

[0101] The amounts of irinotecan and phloxuridine encapsulated in simultaneously loaded CPX-1 liposomes were measured not only immediately after lyophilization ("start") but also after 6 and 9 months of storage at 5°C. Stability tests demonstrated that the encapsulation percentage (%) of irinotecan was 99% immediately after lyophilization, 97% after 6 months of storage, and 97% after 9 months of storage (Table 2 below). Similarly, the encapsulation percentage of phloxuridine was 98% immediately after lyophilization and 95% after both 6 and 9 months of storage at 5°C (Table 3 below).

[0102] The effects of freezing and lyophilization on drug encapsulation percentage were also investigated for CPX-351 liposomes. As shown in Table 4 below, the amount of cytarabine encapsulated in simultaneously loaded CPX-351 liposomes was measured to be 100% immediately after freezing ("complete freezing") and 98% immediately after lyophilization ("complete lyophilization") in two separate batches (1C001 and 1D002). The encapsulation percentage of daunorubicin was 99% in both batches, both immediately after freezing and lyophilization. Drug encapsulation is stable even when CPX-351 is stored at 5°C or 25°C (Tables 5 and 6 below).

[0103] These results clearly demonstrate that both CPX-1 and CPX-351 gel-phase liposomes, which incorporate small amounts of cholesterol and cryoprotective substances in an external solution, can be efficiently frozen, dehydrated, and reconstituted with minimal leakage of both encapsulated drugs. [Table 2] [Table 3] [Table 4] [Table 5] [Table 6]

[0104] (1) A lyophilized gel-phase liposome composition, wherein the composition is (a) A molten phase temperature of at least 37°C (T C A gel-phase liposome that exhibits the following characteristics, and whose liposome membrane contains 20 mol% or less of cholesterol, and contains at least 1 mol% of phosphatidylglycerol (PG) or phosphatidylinositol (PI), or both thereof, Herein, a gel-phase liposome in which at least two therapeutic and / or diagnostic agents are stably associated with the liposome; and, (b) cryoprotective substance present on the outside of the liposome Includes, Here, the liposome substantially does not contain an inner cryoprotective substance, A composition wherein, when the lyophilized gel-phase liposome composition is reconstituted in a pharmaceutical carrier, the average diameter of the liposomes is substantially maintained compared to the composition before lyophilization, and the drug is substantially retained within the liposomes, A composition in which at least 75% of each drug is retained during the reconstitution of the liposomes, and the retention of said drugs is maintained for at least 6 months when stored at 5°C to 25°C.

[0105] (2) The composition according to (1), wherein the liposome membrane comprises distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), and cholesterol (CHOL).

[0106] (3) The composition according to (1), wherein the liposome membrane comprises 50 to 80 mol% distearoylphosphatidylcholine (DSPC) or dipalmitoylphosphatidylcholine (DPPC), 1 to 20 mol% distearoylphosphatidylglycerol (DSPG) or distearoylphosphatidylinositol (DSPI), and 1 to 20 mol% cholesterol (CHOL).

[0107] (4) The composition according to (3), wherein the liposome membrane comprises 50-80 mol% distearoylphosphatidylcholine (DSPC), 1-20 mol% distearoylphosphatidylglycerol (DSPG), and 1-20 mol% cholesterol (CHOL).

[0108] (5) The composition according to (4), wherein the liposome membrane contains the components DSPC:DSPG:CHOL in a molar ratio of 7:2:1.

[0109] (6) The composition according to any one of (1) to (5), wherein the drug is an anti-cancer drug.

[0110] (7) The composition according to (6), wherein the anti-cancer drug is daunorubicin and cytarabine, or irinotecan and phloxuridine.

[0111] (8) The composition according to any one of (1) to (7), wherein the encapsulated agent is in a fixed proportion, and when the composition is reconstituted, the proportion of the agent changes by 25% or less compared to the composition before freeze-drying.

[0112] (9) The composition according to any one of (1) to (8), wherein the average diameter of the liposomes increases by 25% or less after lyophilization and during reconstitution of the liposomes, compared to the value measured before lyophilization.

[0113] (10) The composition according to any one of (1) to (9), wherein the average diameter is maintained for at least 6 months when stored at 5°C to 25°C.

[0114] (11) The composition according to any one of (1) to (10), wherein the size distribution of the liposomes changes by 25% or less after lyophilization and reconstitution of the liposomes.

[0115] (12) A method for preparing a composition according to any one of (1) to (11), comprising subjecting an aqueous medium containing gel-phase liposomes to freeze-drying. Here, the liposome has a melting phase temperature of at least 37°C (T C The liposome exhibits the following characteristics, and the liposome membrane of the liposome contains 20 mol% or less of cholesterol and at least 1 mol% of phosphatidylglycerol (PG) or phosphatidylinositol (PI), or both; and the liposome stably associates with at least two therapeutic and / or diagnostic agents and substantially does not contain an inner cryoprotective substance. A method in which a protective substance is present on the outside.

[0116] (13) The aqueous medium containing gel-phase liposomes has a glass transition temperature (T g The method according to (12), which is frozen at a temperature below )

[0117] (14) A method for preparing a pharmaceutical composition for administering a therapeutic and / or diagnostic agent to a subject, comprising reconstituting a liposome composition described in any one of (1) to (11) in a pharmaceutical carrier to obtain the reconstituted composition.

[0118] (15) The reconstituted composition according to (14) for use in a method of administering therapeutic and / or diagnostic agents to animals.

Claims

1. A lyophilized gel-phase liposome composition, wherein the composition is (a) Gel-phase liposomes, wherein the membrane of the liposomes contains DSPC:DSPG:CHOL in a ratio of 7:2:1, and the liposomes are stably associated with daunorubicin and cytarabine in a certain ratio; and, (b) Cryoprotective substance present on the outside of the liposome Includes, Herein, a composition in which the liposomes substantially do not contain an inner cryoprotective substance.

2. The composition according to claim 1, wherein the ratio of daunorubicin to cytarabine is 1:

5.

3. The composition according to claim 1 or 2, wherein when the composition is reconstituted in a pharmaceutical carrier, the average diameter of the liposomes is maintained compared to the composition before lyophilization, and daunorubicin and cytarabine are substantially retained within the liposomes.

4. The composition according to claim 1 or 2, wherein when the composition is reconstituted, the ratio of daunorubicin to cytarabine changes by 25% or less compared to the composition before freeze-drying.

5. The composition according to claim 1 or 2, wherein the average diameter of the liposomes increases by 25% or less after lyophilization and during reconstitution of the liposome composition, compared to the value measured before lyophilization.

6. The composition according to claim 5, wherein the average diameter is maintained for at least six months when stored at 5°C to 25°C.

7. The composition according to claim 1 or claim 2, wherein at least 75% of each of Dow's rubicin and cytarabine are maintained when the liposome composition is reconstituted.

8. The composition according to claim 7, wherein the average diameter is maintained for at least 6 months when stored at 5°C to 25°C.

9. The composition according to claim 1 or claim 2, wherein the size distribution of the liposomes changes by 25% or less after freeze-drying and reconstitution of the liposome composition.

10. A method for preparing the composition according to claim 1, comprising freeze-drying an aqueous medium containing gel-phase liposomes in the presence of an external protective substance, Herein, the liposomes, the membrane of which the liposomes contain 7:2:1 DSPC:DSPG:CHOL as components; and a certain ratio of daunorubicin and cytarabine is stably associated with the liposomes; and the method substantially does not contain an inner cryoprotective substance.

11. The method according to claim 10, wherein the aqueous medium containing gel-phase liposomes is frozen at a temperature below the glass transition temperature (Tg) of the medium.

12. A method for preparing a pharmaceutical composition for administering daunorubicin and cytarabine to a subject, comprising reconstituting a liposome composition described in any one of claims 1 to 8 in a pharmaceutical carrier to obtain a reconstituted composition.

13. A composition prepared by the method of claim 12 for use in a method of administering daunorubicin and cytarabine to an animal.

14. A composition prepared by the method of claim 12 for use in a method for treating cancer in animals.

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