RU2780489C2 - Lyophilized liposomes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for the treatment of cancer with a lyophilized liposomal composition with two incapsulated drugs is disclosed, where incapsulated drugs are antitumor agents daunorubicin and cytarabine.

EFFECT: these compositions show excellent profiles of drug retention, as well as preserve size distribution after lyophilization and reproduction.

11 cl, 3 ex, 6 tbl, 6 dwg

Description

Related Applications

This application claims priority to US Patent Application No. 61/550047, filed October 21, 2011, the contents of which are incorporated herein by reference in their entirety.

Technical field

The invention relates to compositions and methods for the production of lyophilized liposomes that contain at least two therapeutic or diagnostic agents that can be stored for prolonged periods of time. In one aspect, the invention relates to low cholesterol liposomes, optionally containing a cryoprotectant in the environment having resistance to freeze/thaw and dehydration degradation of the liposomes, thus maintaining their size and integrity.

State of the art prior to the invention

Liposomes are closed vesicles having at least one lipid bilayer surrounding an aqueous core. The intraliposomal space and lipid layer(s) can hold a wide variety of substances, including drugs, cosmetics, diagnostic reagents, genetic material, and bioactive compounds. Since non-toxic lipids serve as the basis for liposomes, they generally show low toxicity. The low toxicity, together with the ability of liposomes to increase the duration of plasma circulation of agents, makes liposomes suitable as carriers, especially for the delivery of pharmaceutically active agents. In many cases, liposome-delivered drugs result in superior clinical efficacy along with reduced toxicity.

The practical use of liposomal preparations as drug delivery vehicles is limited by the chemical and physical stability of the drug. Industrial release requires stability over a long period of time, both chemically and physically. The use of frozen or freeze-dried (lyophilized) formulations to avoid degradation of the labile drug and/or lipid components provides some improvement in stability. However, during the lyophilization process, the formation of ice crystals can lead to mechanical rupture, aggregation of liposomes and fusion (resulting in increased liposome size). Moreover, when drug-containing liposomes are lyophilized and then reconstituted at room temperature, changes in the structure of their bilayer(s) often occur, resulting in accelerated drug leakage.

Previous attempts to prepare lyophilized liposomal formulations have been based on standard liposomes, which are generally in a liquid phase at body temperature, the movement of lipids is fluid and uncontrolled. Such conventional liposomes fall into two categories. The former remain in a liquid state due to the fact that they contain lipids where the gel-to-liquid crystal transition temperature (T _c ) is below body temperature (ie, they will be in the liquid phase at body temperature). These liposomes are regularly used in the art, however, the downside of fluidity is that they have poor drug retention for many encapsulated agents.

The second type of conventional liposomes never undergo liquid-to-gel conversion because they contain high amounts of membrane stiffening agents such as cholesterol (eg 30-45 mol %). Cholesterol acts to increase bilayer thickness and fluidity while decreasing membrane permeability, protein interactions, and lipoprotein destabilization of the liposome. Such high amounts of cholesterol are most commonly used in liposomal studies and have previously been considered necessary for sufficient serum stability and drug retention in vivo , although not all drugs may be sufficiently retained. Some drugs exhibit better drug retention in both in vitro and in vivo substantially cholesterol-free liposomes. See, for example, Dos Santos, et al., Biochim. Biophs. Acta, (2002) 1561:188-201.

On the other hand, gel phase liposomes are more stable and exhibit improved drug retention. The invention takes advantage of liposomes that are in a gel phase at body temperature (ie, body temperature below the T _c of liposomes). Liposomes in the gel phase can be prepared from a range of lipids; however, liposomes formulated with a more saturated acyl side chain of phosphatidyl lipids, such as hydrogenated soy PC, dipalmitoyl phosphatidylcholine (DPPC), or distearoyl phosphatidylcholine (DSPC), require at least 30% cholesterol in order to form gel phases at body temperature. An example of conventional liposomes that are not in gel phases at body temperature are those prepared from egg phosphatidylcholine (EPC), which are highly leaky.

Previous attempts to prepare lyophilized liposomal compositions using conventional liposomes have involved either empty liposomes or liposomes containing only a single agent. They may use a cryoprotectant, typically a saccharide, both inside and outside the liposomes, or a large osmotic gradient across the liposomal membrane.

For example, cryoprotectants have been used to protect against freeze/thaw degradation of 'liquid' EPC liposomes encapsulating a single agent when present in sufficient amounts both inside and outside the liposomes, ideally when the amounts are equal. See, for example, US Pat. See, for example, US Pat. No. 4,927,571. Disruption of cryoprotectant incorporation within the interior of the liposome has been shown to result in loss of liposome integrity upon replication, especially in terms of retaining the encapsulated agent. As described, "the presence of a sugar both inside and outside the liposome is required to prevent leakage" (Lowery, M. (June 2002) Drug Development and Delivery, Vol. 2, No. 4).

In one case, protection against vesicle aggregation and fusion, as well as loss of retained drug, was also reported for hydrogenated soy PC:cholesterol:DSPE-mPEG liposomes (molar ratio 51:44:5), where the liposome preparation contains 44 mole % cholesterol, as well as a cryoprotectant and a high concentration of salt in the external environment. The presence of 44% cholesterol means that the liposomes will be in the liquid phase at or below body temperature. Moreover, the protective effect is realized only if there is a large osmotic gradient across the membrane, such that the osmotic concentration outside the liposome is significantly higher than the internal osmotic concentration. See, for example, WO01/05372.

Membrane-bound cryoprotectants also further improve the freeze and lyophilization resistance of these liposomes in the non-gel phase. In particular, sugars transplanted onto the surface of EPC or EPC:cholesterol liposomal membranes (1:1 molar ratio) via oligo(ethylene oxide) linkers consisting of one to three repeat units are reported to be cryoprotective for liposomes containing a fluorescent probe. See, for example, Bendas, et al., Eur. J Pharm. sci. (1996) 4:211-222; Goodrich, et al., Biochem. (1991) 30:5313-5318; US patent No. 4915951. Baldeschwieler, et al. reported that in the absence of a terminating sugar group, liposomes prepared with an oligoethylene oxide linker were not themselves able to provide fusion protection after freezing. US Patent No. 4915951.

Trehalose in the external environment of the PC liposomal formulation encapsulating a single agent provides resistance to liposomal aggregation and fusion. US Patent No. 6319517. Other methods for the production of small liposomes stabilized against aggregation require the formation of empty PC:cholesterol liposomes (1:1 molar ratio) to which a sugar solution and a single reagent are added and which are then dried. During the drying process, a percentage of the reagent is retained within the liposome. These liposomes are reported to be more storage stable than in the absence of sugar. See, for example, WO99/65465.

As stated previously, most prior lyophilization technologies have been directed towards lyophilizing either empty liposomes or liposomes encapsulating a single agent. Integrity lyophilization, when two or more agents are encapsulated, is more difficult, especially if the reagents differ in their solubility characteristics. Encapsulation of two or more agents is often advantageous since many life-threatening diseases such as cancer are caused by multiple molecular mechanisms and, due to their complexity, treatment with a single agent has had limited success. Therefore, almost all cancer treatments involve combinations of more than one therapeutic agent. This is also true for the treatment of other conditions, including infections and chronic diseases.

PCT publication WO03/041681, incorporated herein by reference, discloses that gel-phase liposomes with transformation temperatures of 38° C. or more can be prepared using saturated phosphatidyl lipids such as DPPC and DSPC and reduced amounts (0 -20%) cholesterol if at least 1 mole % of phosphoinositol (PI) or phosphatidylglycerol (PG) is included in the compositions. It was demonstrated that these liposomes when containing a combination of encapsulated irinotecan and floxuridine (FUDR) were stable when frozen at -20°C. Simple freezing is generally less harsh and less damaging to the integrity of the liposome than lyophilization.

The use of liposomes as carriers for the delivery of these combinations is advantageous, especially if the liposomes include and are capable of maintaining a ratio of agents that are non-antagonistic. This general approach is described in detail in US Pat. No. 7,850,990, incorporated herein by reference. This patent describes how to determine non-antagonistic or synergistic ratios of various therapeutic agents, including antineoplastic antitumor agents, that retain non-antagonism or synergy over a wide range of concentrations. This patent describes what is essential to deliver drugs at an administered ratio and maintain that ratio while allowing delivery vehicles to control pharmacokinetics. This patent illustrates liposomes that contain, while maintaining ratios, non-antagonistic or synergistic ratios of two or more therapeutic agents, including irinotecan and FUDR. Such combinations encapsulated in liposomes will be advantageous when stored in lyophilized form if the integrity of the liposomes and the concentrations of agents and their ratios are maintained during reproduction. A particularly useful combination of cytarabine and daunorubicin encapsulated in liposomes is described in US Pat. No. 8,022,279, also incorporated herein by reference.

The use of these combinations in therapeutic protocols with surprisingly good results is described in PCT publication WO2007/050784 and PCT publication WO2008/101214. Additional formulations with liposomal encapsulated drug delivery methods are described in WO2009/097011 and WO2009/070761, as well as WO2010/043050. These formulations are a simple example of suitable formulations in which two or more therapeutic agents are contained in liposomes for delivery to a patient.

As described above, in general, the preparation of stable lyophilized liposome formulations that retain their integrity during reconstitution has been difficult and unpredictable. The preparation of such stable liposome formulations for the combination of two or more agents is even more difficult. Thus, the success of the method of the invention in obtaining lyophilized liposomes, where the liposomes contain two or more therapeutic or diagnostic agents and where they retain their integrity when reproduced, is an exceptional achievement.

Disclosure of invention

Accordingly, it has been reported that a cryoprotectant is required both inside and outside of liposomes in order to maintain the integrity of the liposome during replication after lyophilization, especially in order to ensure retention of the encapsulated agent. The authors of this discovery have identified stable liposomes that do not require any internal cryoprotectant for successful lyophilization of liposomes encapsulating not only one but two or more active agents.

The invention relates to successfully lyophilized liposomal preparations in the gel phase, which contain more than one therapeutic and/or diagnostic agent and do not contain an internal cryoprotectant. Thus, in one aspect, the invention is directed to a lyophilized liposome composition, wherein said liposomes are stably associated with at least two therapeutic and/or diagnostic agents, and where, when said composition is reconstituted, the average liposome diameter is maintained compared to the pre-lyophilization state, and the percentage of each of the agents that remain encapsulated in liposomes is maintained at a satisfactory level. Liposomal integrity is thus measured as the percentage of encapsulated agents retained after liposome reproduction. An additional parameter used as a criterion for satisfactory lyophilization is the minimum change in the size distribution. A particularly important embodiment is one in which the agents are encapsulated within liposomes in a specific ratio, and where the ratio of these agents is maintained when the lyophilized forms are reconstituted.

Typical conditions to achieve this result include the use of liposomes in a gel phase with gel-to-liquid crystal temperatures (T _c ) that are at least equal to room temperature and can be equal to or higher than human body temperature. The body temperature is considered to be approximately 37°C. The liposomes may be low cholesterol liposomes that are stabilized with phosphatidylglycerol and/or phosphoinositol. Liposomes essentially do not contain an internal cryoprotectant, but may contain an external cryoprotectant on their surface and thus can be lyophilized in the presence of a medium containing a cryoprotectant. The term "substantially no intrinsic cryoprotectant" is intended to include liposomes that do not contain an intrinsic cryoprotectant, as well as liposomes that contain an amount of cryoprotectant that does not interfere with the freezing and/or lyophilization of said liposomes (i.e., 125 mM cryoprotectant or less, which is the "inactive" quantity). Therefore, "substantially no intrinsic cryoprotectant" is defined as being approximately 0-125 mM of cryoprotectant within the liposomes. It is important to note that preventing drug leakage after the lyophilization process is significantly more difficult than maintaining the size of the liposomes. As mentioned above, drug retention after lyophilization has previously been achieved by applying a cryoprotectant both inside and outside the liposomes.

Thus, in one embodiment, the liposomes have membrane gel-to-liquid-crystal temperatures (T _c ) greater than room temperature, or greater than 25°C or 37°C in order to at least at room temperature, for example 25° C, the lipid membrane was sufficiently gel-like to aid drug retention. The compositions allow retention of encapsulated agents and reduced aggregation and fusion upon lyophilization and reconstitution, thereby providing usable compositions with extended shelf life. Improved protection against the lyophilization process is independent of osmotic potential. These liposomes retain their size distribution and drug encapsulation profiles for long periods of time under pharmaceutically relevant conditions.

Methods for preparing a composition of lyophilized liposomes may thus contain a cryoprotectant external to liposomes at a selected concentration, where the temperature of the liposomal membrane prior to freezing and lyophilization is below its phase transition temperature T _c . Preferably, the liposomes are frozen below the glass transition temperature (T _g ) of the solution that contains the drug-encapsulated liposomes, as well as the extraliposomal medium that contains the cryoprotectant.

The invention is also directed in other aspects to methods for preparing lyophilized liposomes containing two or more therapeutic and/or diagnostic agents according to the embodiments set forth above, methods for reproducing said lyophilized compositions, methods for administering replicated liposomes to patients, and methods for treating patients affected, susceptible disease or suspected lesion (eg, cancer).

Brief description of the drawings

1 shows the particle size profile of CPX-1 liposomes before freezing.

2 shows the particle size profile of reconstituted CPX-1 liposomes immediately after freezing, lyophilization and reconstitution.

3 shows the particle size profile of reconstituted CPX-1 liposomes after 1 month of storage.

4A-4C show the particle size profiles of reconstituted CPX-1 liposomes after 6 months of storage.

Methods for carrying out the invention

The invention provides for the first time lyophilized liposome compositions in the gel phase which contain two or more therapeutic and/or diagnostic agents such that the characteristics and properties of the reproduced lyophilized composition essentially correspond to the characteristics and properties of the composition before lyophilization. These characteristics may include the average diameter, size distribution and capacity of the liposomes. The capacity of liposomes refers to the retention of agents; in some embodiments, the ratios of agents are also stored.

Although liposomes contain therapeutic and/or diagnostic agents, in this application the term "drugs" is sometimes used as a shorthand for this.

Liposomes in the gel phase contain one or more lipid bilayers surrounding an internal compartment. These liposomes may be bilayer or single layer vesicles. Unilamellar liposomes (also known as unilamellar vesicles or "ULVs") surround a single internal aqueous compartment and are classified as either small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs). LUV and SUV range in size from about 50 to 500 nm and 20 to 50 nm, respectively. Bilayer liposomes have two lipid membranes where an inner membrane surrounds a single inner water compartment and a second large outer membrane surrounds the inner membrane, thus creating a second inner water compartment.

The maintenance of the size distribution of liposomes in the gel phase can be assessed experimentally by obtaining particle size profiles such as those shown in Figures 1-4 herein. The size distribution determined by quasi-elastic light scattering is typically presented as a histogram showing the average liposome diameter. Significant size distribution measurements most commonly used in the art are D10, D90, D99 or standard deviation or polydispersity index. "D99" values mean that 99% of the liposomes are smaller than the reference size or larger than the reference size. This is particularly useful if, for example, it is important to exclude either the top or bottom dimension. For example, in some embodiments, it is desirable to ensure that liposomes with an average diameter greater than 200 nm are not present.

A specific example in which a D99 value of 178 nm is present is used for illustration. The D99 value for measuring 178 nm (as seen in Table 1 of Example 2) ensures that at least 99% of the liposomal population is less than 178 nm. The D10 and D90 values for mean diameters, also commonly used, are values for which no more than 10% of the population is less than the minimum reference size (i.e. D10), and for D90 where 90% of the population is less than the upper limit reference size. For example, as seen in groups 1 and 2, the D10 value is 68 nm, so that no more than 10% of the population of liposomes is less than 68 nm. The D90 value demonstrates that 90% of the population is less than 135 or 137 nm for groups 1 and 2, respectively. Maintaining the size distribution of liposomes after lyophilization and reconstitution is defined herein as demonstrating that the reference value of the selected D values changes by no more than 50%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6% or 5% at reproduction compared to the value before freezing and/or lyophilization. Selected D values can be 99, 98, 94 and intermediate integer values for 90 or D10.

One characteristic of lyophilized liposomes relates to the average diameter of the liposomes in the composition. The average diameter of the liposomal composition is maintained at play when the average diameter of the liposomes does not increase by more than 50%, 25%, based on a bulk density of 20%, 15%, 10%, 9%, 8%, 7%, 6%, or 5% after lyophilization and when reproduced based on the diameter before freezing. A concomitant value such as a 10% increase in mean liposomal diameter along with a 10% increase for the reference for D90 (or other D value such as above) is one measure to ensure that the particle size distribution (e.g., liposomal) is not has changed. The overall distribution pattern can also be estimated, preferably on the basis of bulk density, as shown in FIGS. 1-4.

In more detail, the composition of liposomes contains a range of sizes, typically following a Gaussian curve. The average liposome diameter may increase by no more than about 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, or 5% of its original size when reproduced after freezing or lyophilization and playback. For example, a sample of liposomes having an average diameter of 90 nm would be considered resistant to freezing and/or lyophilization effects if the average diameter is no more than 30% larger in reproduction, i.e. is approximately 117 nm. If the size increases more than shown, this suggests that aggregation and fusion of liposomes has occurred. Sufficiently sensitive measurement technology can be applied to measure the change in size distribution or average diameter such that changes of less than 10% can be measured.

Another criterion for maintaining integrity is the retention of encapsulated agents. Unlike mean size distribution diameter and drug ratio, which are measured relative to pre-lyophilization values, drug retention is measured relative to total drug content per se after reconstitution, i.e. based on the total drug content of the lyophilized composition. The percentage of the drug encapsulated within the liposomes or the percentage of the drug in the external environment outside the liposomes (% "unencapsulated") is related to the total amount of the drug in the composition. In one embodiment, at least about 75% of the encapsulated agents are retained in their encapsulated form after lyophilization and upon reconstitution. At least about 85% of each may be retained encapsulated and/or at least about 90% or 95%. This can similarly be measured by the amount of unencapsulated drug in the environment, which should not be more than 25%, 20%, 15%, 10%, or 5% of the original encapsulated amounts when replicating lyophilized liposomes.

The ratios of the encapsulated therapeutic and/or diagnostic agents are maintained at play unless the ratios vary by more than 20%, 10%, 9%, 8%, 7%, 6%, or 5% of the ratio in the pre-lyophilized composition itself. Ratios are expressed as molar ratios.

In one embodiment, the average diameter of the liposomes after lyophilization and during reproduction of these liposomes will be increased by no more than 25% compared to the specified value measured before lyophilization. In another embodiment, the average diameter of the liposomes after lyophilization and during reproduction of these liposomes changes by no more than 15% compared to the specified value measured before lyophilization. In other embodiments, the average liposome diameter after lyophilization and upon reconstitution of said liposomes changes by no more than 10%, 9%, 8%, 7%, 6%, or 5% compared to the indicated value measured before lyophilization.

In some embodiments, the percentage of unencapsulated drug is no more than 25% of that originally encapsulated when replicating said liposomes. In other embodiments, the percentage of non-encapsulated drug is no more than 15% of that originally encapsulated when replicating said liposomes. In other embodiments, the percentage of unencapsulated drug is no more than 10%, or no more than 9%, 8%, 7%, 6%, or 5% of the originally encapsulated drug when replicating said liposomes.

In other words, in some embodiments, the implementation of the percentage of retained encapsulated drugs is not less than 75% when playing these liposomes. In other embodiments, the implementation of the percentage of each encapsulated drug is not less than 85% or 90% or 95% when playing these liposomes.

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

In some embodiments, the size distribution of liposomes changes by no more than 25% after lyophilization and when reproducing said liposomes compared to prior lyophilization. In other embodiments, the size distribution of the liposomes changes by no more than 15%, 10%, 9%, 8%, 7%, 6%, or 5% after lyophilization and when reconstituting said liposomes compared to prior to lyophilization.

As noted above, various combinations of these parameters or criteria are contemplated for successful complete lyophilization and reproduction of liposomes, e.g., at least 85% encapsulated drugs combined with no more than 15% increase in mean diameter, optionally combined with no more than 5% change size distribution. Each of the possible combinations of these parameters is covered by the scope of the invention.

Liposomes in the gel phase can be formed by conventional techniques, for example, the ether injection method (Deamer, et al., Acad. Sci. (1978) 308:250), the 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) , an extrusion method (Hope, et al., Biochim. Biophys. Acta (1985) 812:55-65) and a French press method (Barenholz, et al., FEBS Lett. (1979) 99:210).

Such processes may be applied in combination. Small unilamellar vesicles (SUVs) in particular can be prepared by the sonication method, the ethanol injection method, and the French press method. Large unilamellar vesicles (LUV) can be prepared by the ether injection method, the surfactant method, the freeze-thaw method, the reverse phase evaporation method, the French press, or the extrusion method. Preferably, the LUVs are prepared according to an extrusion process.

Lyophilization and reproduction are carried out under conditions in which the liposomes are in the gel phase. Therefore, the temperature of the transformation from gel to liquid of liposomes should be higher than room temperature, i.e. about 20-30°C, and more preferably equal to or above body temperature. Room temperatures can vary considerably, but it is important that the freeze-drying process be started under conditions where the liposomes are in a gel state. In some embodiments, T _c is at least equal to body temperature (ie, approximately 37°C). In some embodiments, the liposomes are prepared at a temperature below the phase transition temperature in order to maintain a gel-like state. Any suitable indoor environment may be used. Typically, the indoor environment is an aqueous environment. The internal environment essentially does not contain cryoprotectant (ie less than 125 mm cryoprotectant). The internal environment may contain less than 100 mm cryoprotectant, or less than 50 mm cryoprotectant, or no cryoprotectant.

Liposomal formulations that have suitable T _c values may be "low cholesterol" liposomes, ie. prepared when present and containing an amount of cholesterol that is not sufficient to significantly alter the phase transition characteristics of the liposome, ie, typically 20 mole % or less of cholesterol. Greater than 20 mole % of cholesterol widens the range of temperatures at which the phase transition occurs, with the phase transition disappearing at higher cholesterol levels. A liposome having a low cholesterol content will have less than 20 mole % or less than 15 mole % cholesterol, or 10 mole % or 5 mole % or less cholesterol, or no cholesterol. Such liposomes optionally require at least 1 mole % of a stabilizing agent such as PG or PI.

In these methods where a cryoprotectant is used, the cryoprotectant is preferably present only in the external environment of the formulation. Typically, cryoprotectants are disaccharides such as sucrose, maltose, trehalose and lactose. The cryoprotectant may be a disaccharide, such as sucrose, having a concentration that is about 100 mM to about 500 mM, or about 250-400 mM, or greater than 300 mM. The external environment may contain from about 100 mM to 500 mM of cryoprotectant, and the internal environment contains less than 125 mM of cryoprotectant, or the external environment contains from about 250 mM to 400 mM of cryoprotectant, and the internal environment contains less than 100 mM of cryoprotectant, or the external environment contains about 250 mM to 400 mM cryoprotectant and the internal environment contains less than 50 mM cryoprotectant, or the external environment contains approximately 250 mM to 400 mM cryoprotectant and the internal environment does not contain cryoprotectant. The cryoprotectant may be a saccharide such as sucrose.

The gel phase of the liposomal formulations can be lyophilized or freeze dried using any suitable protocol. The initial temperature of the lyophilization chamber is preferably below the glass transition temperature (T _g ) of the solution which contains the external medium also containing the drug-encapsulated liposomes. For example, liposomes can be frozen at a temperature lower than about -5°C, or lower than about -10°C, or lower than about -20°C, or lower than about -30°C, or lower, than approximately -40°C. In some embodiments, when sucrose is used as the cryoprotectant solution, the initial temperature of the lyophilization chamber is less than -32° C., which is the T _g of the sucrose solution. " _Tg " includes "glass transition temperature" and "glass transition temperature", which is an approximate average temperature at which an unfrozen solution undergoes a transformation from a soft, viscous gel to a hard and relatively brittle form.

Lyophilized liposomes may be stored at or below room temperature. In some exemplary embodiments, liposomes are used that are stored at or below 5°C. In some other exemplary embodiments, liposomes are used that are stored at 25°C. The lyophilized product remains stable (e.g., maintains its relative particle size and keeps the drug encapsulated) for at least about six months, or at least about nine months, or at least about one year, or at least about twenty four to thirty-six months.

Retained agents are therapeutic or diagnostic agents, often anti-cancer agents. Surprisingly, liposomal formulations in the gel phase retain content and integrity even when the agents differ in their solubility characteristics in water and non-aqueous solvents. Using the approach of the invention, agents that differ in log distribution coefficients (LogP) by up to 1.0 can be successfully retained. Differences in log distribution coefficients of 1.5 or 2.0 or 3.0 may also be tolerable. One of the agents may be amphipathic while the others are water soluble, or one may be hydrophobic while the others are water soluble. The LogP values are based on the separation factors between octanol and water, i.e. are the base 10 logarithm of the ratio of the amount in octanol to the amount in water when the compound undergoes phase separation.

Anticancer agents may contain an anthracycline (eg daunorubicin, doxorubicin, epirubicin or idarubicin). These agents are amphipathic. Anticancer agents may contain a nucleoside analogue, for example, cytosine arabinoside, 5-FU or FUDR, which are hydrophilic. Other anti-cancer agents include camptothecin or a camptothecin derivative such as irinotecan, which are amphipathic. Both the anthracycline and the nucleoside analog are encapsulated in some cases, or both the camptothecin or camptothecin derivative and the nucleoside analog are encapsulated. Encapsulation and/or loading of agents into liposomes can be accomplished using any suitable loading techniques, including passive and active loading. Important embodiments include the combinations of irinotecan:floxuridine (FUDR) in a 1:1 molar ratio and daunorubicin:cytarabine (AraC) in a 1:5 molar ratio as described in US Pat. No. 7,850,990 and US Pat. No. 8,022,279 above. The particular formulations of these drugs are designated CPX-1 and CPX-351, respectively.

Drugs are incorporated into the aqueous internal compartment(s) of liposomes by either passive or active loading procedures, or some combination thereof. In passive loading, the biologically active agent may be readily incorporated into the formulation from which the liposomes are formed, or alternatively, the active agent may be added outside of the pre-prepared liposomes and loaded passively along its concentration gradient into the liposomes. Optionally, the unencapsulated material may be removed from the formulation by any suitable procedure. Alternatively, active loading procedures such as ion gradients, ionophores, pH gradients, and metal based loading procedures based on metal complexation can be applied. One embodiment commonly used for suitable drugs is loading via metal-based procedures.

Liposomes are approximately 80-500 nm in size. In one embodiment, the liposomes are less than 300 nm in diameter, sometimes less than 200 nm. In one example, the nominal size of these liposomes is approximately 100 nm. In some embodiments, the liposomal membrane is composed of distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), and cholesterol (CHOL). In some embodiments, the implementation of the liposome membrane is composed of 50-80% DSPC, 1-20% DSPG and 1-20% CHOL. In other embodiments, the liposomal membrane is composed of 50-80% DSPC or DPPC, 1-20% DSPG or distearoylphosphatidylinositol (DSPI), 1-20% CHOL, and the liposomes contain less than 125 mM cryoprotectant in the intraliposomal medium. In some exemplary embodiments, the liposomal membrane is composed of 50-80% DSPC or DPPC, 1-20% DSPG or DSPI, 1-20% CHOL, and the liposomes contain less than 50 mM cryoprotectant in the intraliposomal medium. In other exemplary embodiments, the liposomal membrane is composed of DSPC, DSPG and CHOL in a molar ratio of approximately 7:2:1 and does not contain a cryoprotectant in the internal liposomal medium. In one case, liposomes are prepared by a water-in-oil liposome preparation process and the extruded liposomes are suspended in phosphate buffered sucrose at pH 7.0. Alternatively, the extruded liposomes are suspended in sucrose. In one embodiment, the extruded liposomes are suspended in 250-400 mM sucrose.

Any suitable means for encapsulating the drug combination in liposomes may be used. In a specific embodiment, irinotecan and floxuridine are co-loaded into preformed DSPC/DSPG/CHOL (7/2/1) liposomes, whereby floxuridine is passively loaded into liposomes and irinotecan is actively loaded at 50°C using copper sulfate or copper gluconate during internal environment. See US Pat. Nos. 7,850,990 and 7,238,367 to the same applicants, both of which are incorporated herein by reference. In another specific embodiment, cytarabine and daunorubicin are liposome encapsulated, whereby cytarabine is passively encapsulated in preformed liposomes and daunorubicin actively accumulates within the liposomes with high uptake efficiencies using a copper gluconate/triethanolmamine loading procedure. See, for example, co-pending PCT applications WO05/102359 and WO07/076117A2 of the same applicants, both of which are also incorporated by reference in their entirety.

The lyophilized compositions of the invention provide ease of storage, storage and ease of transport. These lyophilized compositions retain their characteristics for extended periods of time.

For use, the compositions of the invention are formulated in a suitable pharmaceutical carrier or medium.

These formulations for use are prepared according to standard reproduction techniques using a pharmaceutically acceptable carrier. Basically, an isotonic solution will be used as a pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, dextrose, 0.4% sodium chloride, 0.3% glycine and the like, including glycoproteins for improved stability such as albumin, lipoprotein, globulin, etc. These compositions may be sterilized by conventional well known sterilization techniques. The resulting aqueous solutions may be packaged for use or aseptically filtered and lyophilized, wherein the lyophilized preparation is combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable excipients, as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. .d. Additionally, the liposome suspension may contain lipid protecting agents that protect lipids from free radical and lipid peroxidation damage during storage. Lipophilic free radical quenchers such as alpha-tocopherol and water-soluble iron-specific chelators such as ferrioxamine are suitable.

Generic formulations may be administered to subjects including humans or other mammalian species such as non-human primates, dogs, cats, cattle, horses, sheep, and the like, and may be used to treat a variety of diseases. Examples of medical applications of the compositions of this invention include, but are not limited to, the treatment of cancer, the treatment of cardiovascular diseases such as hypertension, cardiac arrhythmia and restenosis, the treatment of bacterial, fungal or parasitic infections, the treatment and/or prevention of disease through the use of compositions according to of this invention as a vaccine, treatment of inflammation or treatment of autoimmune diseases. For the treatment of human diseases, the skilled physician will determine how the compositions of this invention are to be used in relation to dose, schedule, and route of administration using established protocols. Dosage escalation may also be used in such applications if the liposome-encapsulated bioactive agents and lipid carriers of this invention exhibit reduced toxicity to healthy patient tissues.

Pharmaceutical compositions are typically administered parenterally, eg intravenously, but other routes may be used. Dosage for liposomal formulations will depend on the ratio between the drug and lipids and the opinion of the leading physician, based on the age, weight and condition of the patient.

In general, one process useful in the invention comprises lyophilizing a composition of liposomes, wherein said liposomes contain 20 mole % or less of cholesterol and two or more active agents, and wherein the liposomal membrane is at a temperature below its phase transition at room temperature and in an external environment. , which contains a cryoprotectant; storage of lyophilized liposomes; and reproducing the lyophilized liposomes in a predetermined aqueous volume. The liposomes are lyophilized at a temperature lower than about -5°C, or lower than about -10°C, or lower than about -20°C, or even lower than about -30°C or -40°C, and may be stored at or below room temperature (approximately 23-25°C).

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

In one embodiment, the lyophilized composition contains liposomes composed of about 10% cholesterol, a disaccharide at a selected concentration in an environment where reproduction is performed at room temperature below T _c and where the cryoprotectant is unbound and present only on the outside of the liposomes.

In another embodiment, a lyophilized liposome composition containing two or more encapsulated drugs, when reconstituted with a predetermined volume of aqueous medium, results in a liposome composition comprising: (a) liposomes containing 20 mole % or less cholesterol, (b) liposome size preferably about 80-500 nanometers, (c) retained(s) liposome agent(s), where the percentage of encapsulation of the specified(s) agent(s) is not less than about 95%, 90%, 85%, 80% or 75 %; and (d) between about 100-500 mM cryoprotectant in the external liposome medium. In some embodiments, between 250-400 mM cryoprotectant is present in an external liposome medium. In some embodiments, approximately 9.5-10% cryoprotectant is present in the external liposome medium.

In one embodiment, single-layer or bilayer gel-phase liposomes containing 20 mole % or less cholesterol, at least two drugs, and at least about 300 mM sucrose on the outside of the liposomes are lyophilized, and when reconstituted, at least about 90% of each encapsulated drugs are encapsulated and the average liposome diameter changes by less than about 25%.

As used herein, the use of the term in the singular means "at least one" or "one or more", unless it is obvious from the context that only a single designated entity is meant.

The following examples are provided to illustrate and not limit the invention.

Example 1

Lyophilization CPX-1

Irinotecan and floxuridin 1:1 were co-encapsulated in DSPC/DSPG/cholesterol liposomes (7:2:1 molar ratio) and designated CPX-1. Lyophilized CPX-1 resulted in stable drug-loaded liposomes such that there was minimal leakage of active pharmaceutical ingredients from the generic dosage form. Irinotecan hydrochloride used in CPX-1 has a predicted log partition coefficient (LogP) of 3.94. Floxuridine has a predicted LogP of 1.14.

Conducted thermal analyzes for liposomal drug product CPX-1 using various kits to provide information on the glass transition temperature (T _g ), changes in heat capacity and other exothermic phenomena. The collapse temperature of the CPX-1 liposomal drug product was determined by freeze-drying microscope for two sets. These results were used to determine the final lyophilization cycle.

The kits consisted of a bluish-green bulk aqueous suspension formulated with liposomes containing the two active pharmaceutical ingredients, irinotecan hydrochloride and floxuridine, in a 1:1 ratio. Samples were stored at -20° C. (or in some cases -80° C.) with ambient relative humidity and thawed overnight in a refrigerator and thoroughly mixed before filling and lyophilization.

Cycle 1: Using a 20 ml class A pipette, 20 cm ³ CPX-1 was filled into 60 cm ³ molded glass vials. Twenty-four vials were loaded into a LyoStarII drying rack with two product vials equipped with thermoelectric probes to record the product temperature and freeze-dried for four and a half days. After filling the vials with nitrogen gas to a chamber pressure of approximately 720,000 mTorr, the vials were stoppered, removed from the rack dryer, and labeled with Lot TP-CPX1-001-032405. Several lyophilized vials were then placed in an accelerated stability test at 25°C and 40°C, while the remaining vials were stored at -20°C.

Cycle 2: Approximately 21 ml of CPX-1 was filled into 60 cm ³ molded glass vials and 50 cm ³ tubular glass vials, respectively. The vials were loaded into a LyoStarII™ Shelf Dryer with one thermoelectric probe in the product vial on the top shelf and one product vial on the bottom shelf. At the end of the lyophilization cycle, the vials were filled with nitrogen gas to reach a chamber pressure of approximately 720,000 mTorr, stoppered, removed from the rack dryer, and labeled with Lot TP-CPX1-002-041305T. Several lyophilized vials were then placed in an accelerated stability test at 25°C and 40°C, while the remaining vials were stored at -20°C.

Cycle 3: Lyophilization kits were prepared in the same manner as in Cycle 2, except for filling 50 ^cc tubular glass vials. The stoppered vials were labeled as drug product CPX-1, Lot TP-CPX1-003-051105T. Several lyophilized vials were then placed in an accelerated stability test at 25°C and 40°C, while the remaining vials were stored at -20°C.

Cycle 4: Lyophilization kits were prepared in the same manner as in Cycle 2, except that CPX-1 was filled into 50 ^cc tubular glass vials. The stoppered lyophilized vials were labeled as drug product CPX-1, Lot TP-CPX1-004-051805T and stored for stability studies at -20°C, 5°C, 25°C and 40°C.

Cycle 5: Lyophilization kits were prepared in the same manner as in Cycle 2 and filled into 50 ^cc tubular glass vials. The stoppered freeze-dried vials were labeled as drug product CPX-1, TP-CPX1-005 062705T-300. Vials with CPX-1 liposomal drug product were stored in climate chambers at -20°C, 5°C and 25°C.

First test of the lyophilization cycle. The primary goal for the first trial of the lyophilization cycle (cycle 1) was to determine if the formulated bulk CPX-1 liposomal drug product (CPX-1) could be successfully freeze-dried with a careful initial two step drying phase. The success of this lyophilization test was assessed by analyzing the drug product temperature and pressure profiles and visually monitoring the appearance of the lyophilized mass.

The lyophilization product profile for cycle 1 showed that most of the ice was removed during the initial drying step at -10°C. This was clearly due to the fact that the temperature of the product was slightly above the temperature of the shelf. Also, the thermocouple pressure, which is a measure of true pressure with the addition of the partial pressure of water vapor, was reduced relative to the capacitance gauge pressure or true pressure. In addition, the lyophilized drug product vials appeared to be dried with little or no evidence of lyophilized mass collapse. However, some analyte concentration or separation was observed. To optimize the cycle, the heat treatment phases and the initial drying steps were changed in the second test.

Second test of the lyophilization cycle. A second lyophilization test (cycle 2) was performed using similarly cautious initial and secondary drying phases as in cycle 1. To maximize ice loading into the lyophilizer, vials filled with deionized water were loaded into unoccupied shelf space. The success of the lyophilization test was also assessed by temperature and pressure profiles and by visual inspection of the lyophilized mass.

It appeared that the drug product in the 50 cm ³ tubular vials was freeze-dried in a more homogeneous manner, despite the fact that the product temperature and pressure profiles for the 50 cm ³ tubular glass vial and the 60 cm ³ molded glass vial were similar in a four and a half day cycle. Approximately eight and a half hours after reaching the secondary temperature of the drying shelf, the temperature of the product indicates the completion of the sublimation of the main volume of ice by passing the ice barrier (ie, the temperature of the product exceeds 0°C). However, these vials are not sufficiently dried. The product temperature below the shelf temperature at the end of the initial drying phase and the difference between the thermocouple pressure and the capacitance gauge pressure were unchanged from the start of the test until the end of the secondary drying phase, indicating the presence of a significant main volume of ice in the vials.

Because the shelf temperatures used in the initial drying phase did not provide enough energy to bring the degree of sublimation of the product to completion, a third lyophilization cycle was performed to bring the initial drying phase to completion using the shelf temperature and chamber pressure, which increases the sublimation of ice. without exceeding the collapse temperature.

Third test of the lyophilization cycle. Based on the results of cycle 2 and thermal analyzes, the shelf temperature and chamber pressure for cycle 3 were adjusted to facilitate initial drying while keeping product temperatures below the calculated collapse temperature of -20°C. To maximize the ice load, the cycle was run under full load conditions.

The profile plot obtained for cycle 3 showed that the initial initial temperature of the drying shelf, -20° C. at 100 mTorr, did not sufficiently sublimate the bulk of the ice for 40 hours under full load conditions. Also, the thermocouple pressure curve did not decrease significantly with respect to the capacitive pressure gauge pressure until the end of the initial drying phase at -10°C due to the limited duration of this phase. However, the profile showed that the second initial drying shelf temperature of -10°C and the duration were able to keep the product temperature below the collapse temperature of -20°C until the entire volume of ice was sublimated, which was evident from the rapid increase in product temperature, ultimately with an elevated shelf temperature at the end of the phase.

Fourth freeze-dry cycle test. For the final determination of the shelf temperature(s) for the initial drying phase in the fourth lyophilization cycle (cycle 4), a -10°C shelf temperature was used for a longer period of time with a 6 hour initial initial drying step at -20°C under full load conditions.

Based on the lyophilization cycle profile and visual observation, a shelf temperature of -20° C. for the initial drying phase appeared to provide a slight advantage in drying the vials. Product temperature samples exceeded shelf temperature at -10°C after a hold time of approximately 60 hours. The thermocouple pressure during the secondary drying phase indicated that the vials had a relatively low residual moisture since the product temperature profile closely matched the shelf temperature profile.

Drug encapsulation, liposomal particle size, and average residual moisture were calculated for vials of the lyophilized product. In the applied Karl Fischer method, an average residual moisture content of 3.1% was obtained, which was not an over-dried liposomal product. Also, in analyzes of particle size and percent encapsulation of irinotecan, the lyophilized product was found to be unchanged compared to the pre-lyophilized material. However, the percentage of unencapsulated floxuridine increased from 7.0% in the pre-lyophilized main volume to 8.6% in the lyophilized product when stored at -20° C. for 13 weeks. Also, after exposing the product for a week to 25°C, the percentage of non-encapsulated floxuridine increased to 11.8%, which exceeded the provisional specification by less than 10% of non-encapsulated floxuridine for this drug product.

Fifth freeze-dry cycle test. The goal of the fifth test of the lyophilization cycle (cycle 5) was to reduce the temperature of the shelf for the secondary drying phase from +20°C to +10°C in order to minimize leakage of floxuridine while achieving a suitable residual moisture under full load conditions. The moisture content for this material was evaluated during the secondary phase of drying, periodically measuring the pressure increase.

The drug product profile for cycle 5 showed that the bulk of the ice was largely sublimated after 72-84 hours of initial drying at -10°C. Moreover, it appeared that the material was dried sufficiently by applying a shelf temperature of +10° C. at 50 mTorr for 12 hours, based on pressure detector differences and pressure rise studies.

To evaluate the suitability of this lyophilized material, the playtime for the lyophilized drug product vials from cycles 4 and 5 was evaluated using 19 ml of water injected through the stoppers with a 18 gauge needle and a 30 cm ³ syringe. It was determined that the average playback time was 40 and 93 seconds for cycles 4 and 5, respectively. Moreover, the Karl Fischer results for cycle 5 showed an average residual moisture of 3.2%, which correlated well with the moisture content of the vials from cycle 4.

The encapsulation of drugs and the size of the liposomal particle were also determined. For the cycle 5 lyophilized drug product, the percentage of unencapsulated irinotecan was 0.4% at -20°C after 7 weeks of storage and 0.9% at 25°C after 4 weeks of storage. The particle size for the lyophilized drug product increased only slightly after 8 weeks of storage at 5°C compared to the drug product stored at -20°C, but increased by almost 10 nm after only 4 weeks of storage at 25°C. Not surprisingly, the percentage of unencapsulated floxuridine showed a similar trend in particle size changes. The percentage of unencapsulated floxuridine was 5.5% at -20°C after 7 weeks, 7.7% after 8 weeks at 5°C and 18.7% at 25°C after 4 weeks.

The fifth test of the lyophilization cycle, which applied a reduced shelf temperature during its secondary drying phase, produced an acceptable lyophilized CPX-1 liposomal drug product with increased retention of the encapsulated product.

Example 2

Particle size profile remains unchanged over time in lyophilized liposomes

Experiments were performed to test the effect of freezing, lyophilization and storage on the size distribution of the double-loaded CPX-1 and CPX-351 liposomes. CPX-351 is a formulation of daunorubicin and cytarabine in a 1:5 molar ratio in liposomes, which is distearoyl phosphocholine (DSPC):distearoyl phosphatidylglycerol (DSPG):cholesterol (CHOL) in a 7:2:1 molar ratio. Daunorubicin has a predicted LogP of 1.68. Cytarabine has a predicted LogP of 2.17.

The particle size distribution of the co-loaded liposomes was measured before and after freezing and lyophilization of the liposome, as well as after one and six months of storage of the lyophilized preparations.

CPX-1 liposomes were prepared with an external buffer of 300 mM sucrose, 20 mM phosphate, pH 7.0. Aliquots of 900 μl were added to 2 ml vials, placed in a metal tray (pre-cooled to -20°C) and stored at -20°C overnight. After freezing, the samples were transferred to a lyophilizer (pre-cooled to -20°C). A vacuum was applied and the shelf temperature was maintained at -20°C for 7 hours, and subsequently increased to -10°C for approximately 16 hours. Then, in the third temperature step, the temperature of the shelf was raised further to 4° C. over the next 3 hours and then completed with 3 hours of drying at room temperature. The dried samples were hydrated with 1 ml H ₂ O and the lyophilized mass was readily dissolved. The samples were then analyzed using dynamic light scattering (DLS).

Pre-frozen CPX-1 liposomes showed an average diameter size of 110 nm (FIG. 1). The size of the liposomes immediately after lyophilization and rehydration was observed to be 116 nm (FIG. 2). Two samples of lyophilized CPX-1 liposomes were stored at 5° C. for one month or six months and the size of the liposomes in the rehydrated formulations was measured. The average liposome size for each was 117 nm and 123 nm, respectively (FIGS. 3 and 4, respectively). 1-3 show the volume-weight distribution. Fig.4B shows a comparable volume-mass distribution. The results of other less preferred algorithms are shown in FIGS. 4A and 4C. Unless otherwise stated, the average diameter refers to the volume-weight distribution.

Experiments similar to those shown in Figures 1-4 were also performed for CPX-351 liposomes.

As noted above, CPX-351 is a liposomal fixed combination formulation of the anticancer drugs cytarabine and daunorubicin hydrochloride. Liposomes were prepared using DSPC, DSPG and CHOL at a molar ratio of 7:2:1 and buffered with copper gluconate-triethanolamine, pH 7.4. Raw liposomes are obtained by extrusion to achieve a size distribution of liposomal particles, where the average diameter of the liposomes should be between 80 nm and 110 nm with a D99 not greater than 200 nm (dynamic light scattering analysis). Cytarabine is encapsulated through a passive loading mechanism. Daunorubicin is encapsulated by an active copper-mediated mechanism to achieve a molar ratio of cytarabine:daunorubicin of 5:1. All non-encapsulated drug substances were removed and the bulk of the buffer was replaced by diafiltration. Multiple volumes of 300 mM sucrose were replaced to complete the CPX-351 liposomes, which then underwent lyophilization optimization. Dried samples of CPX-351 were reproduced with 19 ml of H ₂ O and they readily reconstituted the liposomal dispersion. The samples were then analyzed using dynamic light scattering (DLS).

The pre-lyophilized CPX-351 liposomes exhibited an average diameter size of approximately 100 nm. The size of the liposomes immediately after freezing and then lyophilization/rehydration was determined to be 99 nm and 100 nm, respectively, for Lot 1 ("1C001" in Table 1 below). For the second batch of 1D002, the size of CPX-351 liposomes immediately after freezing and then lyophilization/rehydration was determined to be 104 nm and 105 nm, respectively.

These results demonstrate that low cholesterol DSPC/DSPG liposomes co-loaded with either irinotecan and floxuridine or cytarabine and daunorubicin effectively retain their size distribution profiles when frozen as well as lyophilized and for longer shelf life. These results also demonstrate that such gel phase liposomes formulated with low cholesterol are resistant to the aggregation and fusion that typically occurs as a result of freezing and lyophilization, especially in the absence of high cholesterol levels.

Table 1
Effects of Freezing and Lyophilization on the Size of CPX-351 Liposomes 1C001 (batch 1) 1D002 (batch 2) Liposomes CPX-351 Frozen Freeze-dried/rehydrated Frozen Freeze-dried/rehydrated Average diameter (nm) 99 100 104 105 D10 (nm) 68 68 74 71 D90 (nm) 135 137 137 142 D99 (nm) 178 182 178 191

Example 3

Percent drug encapsulation remains unchanged over time in lyophilized liposomes

Experiments were performed to test the effect of freezing and/or freeze-drying and storage on the degree of drug leakage from dual-loaded CPX-1 or CPX-351 liposomes.

The amount of encapsulated irinotecan and floxuridin in the co-loaded CPX-1 liposomes was measured immediately after lyophilization ("baseline"), as well as 6 and 9 months after storage at 5°C. Stability studies demonstrated that the percentage (%) of irinotecan encapsulation was 99% immediately after lyophilization, 97% after six months of storage, and 97% after nine months of storage (Table 2 below). Similarly, the percent encapsulation of floxuridine was 98% immediately after lyophilization and 95% after both six and nine months of storage at 5°C (table 3 below).

For CPX-351 liposomes, the effect of freezing and lyophilization effects on percent drug encapsulation was also investigated. As seen in Table 4 below, the amount of encapsulated cytarabine in co-loaded CPX-351 liposomes was measured to be 100% immediately after freezing ("frozen") and 98% after lyophilization ("lyophilized") in two separate lots (1C001 and 1D002). The percentage of encapsulation of daunorubicin was 99% both immediately after freezing and after lyophilization in both batches. Drug encapsulation was also stable when CPX-351 was stored at 5°C or 25°C (see tables 5 and 6).

These results clearly demonstrate that both CPX-1 and CPX-351 gel phase liposomes, incorporating low amounts of cholesterol and cryoprotectant in external solution, can be efficiently frozen, dehydrated and replicated with minimal leakage of both encapsulated drugs.

table 2
Percentage of irinotecan encapsulation in replicated CPX-1 liposomes Stability intervals Test Initial 6 months 9 months % encapsulation of irinotecan 99% 97% 97%

Table 3
Percentage of floxuridine encapsulation in replicated CPX-1 liposomes Stability intervals Test Initial 6 months 9 months % encapsulation of floxuridine 98% 95% 95%

Table 4
Percent Encapsulation of Cytarabine and Daunorubicin in Replicated CPX-351 Liposomes Liposomes CPX-351 1C001 1D002 frozen Lyophilized frozen Lyophilized % cytarabine encapsulation 100 98 100 98 % daunorubicin encapsulation 99 99 99 99

Table 5
CPX-351: Cytarabine Encapsulation Percentage Time after lyophilization Stored at 5°C Stored at 25°C Batch 1C001 Initial 98 98 3 months 98 98 6 months 98 98 9 months 98 - Batch 1D001 Initial 98 98 3 months 98 99 6 months 99 99 9 months 98 -

Table 6
CPX-351: Daunorubicin Encapsulation Percentage Time after lyophilization Stored at 5°C Stored at 25°C Batch 1C001 Initial 99 99 3 months 99 99 6 months 99 99 9 months 99 - Batch 1D001 Initial 99 99 3 months 99 99 6 months 99 99 9 months 99 -

Claims (11)

1. A method of treating a condition in a subject, wherein the condition is cancer, and wherein the method comprises administering to said subject a composition obtained by reproducing a lyophilized liposome composition in a gel phase, wherein said liposomes in the gel phase exhibit a melting phase temperature (T _c ) of at least 37°C and are stably associated with two therapeutic agents, and essentially do not contain an internal cryoprotectant, where the therapeutic agents are antitumor agents, where the antitumor agents are daunorubicin and cytarabine, where the ratio of daunorubicin: cytarabine is 1:5 on a molar basis, and where, when said liposomal composition in the gel phase is reconstituted, the average diameter of the liposomes is maintained compared to said composition prior to lyophilization, and said agents are retained in the liposomes. 2. The method of claim 1, wherein said encapsulated agents have fixed ratios, and wherein, upon reproducing said liposomal composition in the gel phase, said agent ratios change by no more than 25% compared to said composition prior to lyophilization. 3. The method of claim 1, wherein when said composition is reconstituted, the average liposome diameter is increased by no more than 25% compared to said value measured prior to lyophilization. 4. The method of claim 1, wherein at least 75% of each agent is retained in reproducing said liposomes. 5. The method of claim 4, wherein at least 85% of each agent is retained in reproducing said liposomes. 6. The method of claim 5, wherein at least 90% of each agent is retained in reproducing said liposomes. 7. The method of claim 1, wherein when said composition is reconstituted, the size distribution of the liposomes is changed by no more than 25%. 8. The method of claim 7, wherein when said composition is reconstituted, the size distribution of the liposomes is changed by no more than 10%. 9. The method according to any one of claims 1 to 8, wherein the liposomes contain no more than 20 mole % cholesterol and contain at least 1 mole % phosphatidylglycerol (PG) and/or phosphoinositol (PI). 10. The method according to any one of claims 1 to 9, wherein said administration is parenteral. 11. The method according to any one of claims 1-10, wherein the subject is a human.

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