HK40013477A - Hyperstabilized liposomes increase targeting of mitotic cells - Google Patents

Description

Hyperstable liposomes increase targeting to mitotic cells

Background

The present invention relates to the field of cancer therapy. More particularly, the present invention relates to hyperstable liposomes useful for the treatment of cancer and methods of using the hyperstable liposomes for the treatment of cancer.

The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference and for convenience are individually grouped in the references.

Mitotic regulators should be good targets for anticancer drugs if the cancer is essentially a state of excessive cell division. Indeed, the success of Microtubule Targeting Agents (MTAs) as a class of drugs has prompted the search for more specific methods of inhibiting mitosis, thereby avoiding the MTA-associated peripheral neuropathy [1-3 ]. Enzyme regulators that play a key role in mitosis, such as Polo-like kinase (PLK) [4, 5], kinesin-spindle protein (KSP) [6, 7] and aurora kinase [8, 9], immediately become high priority drug targets. However, despite costing more than 100 billion dollars for developing 25 mitosis-specific drugs, their performance is frustrating with unreported clinical efficacy [10, 11 ].

However, mitotic regulatory enzymes may be intrinsically bad drug targets, since only a limited proportion of tumor cells are actually dividing at any one time. As briefly indicated by Komlodi-Pasztor et al [10], a "target must be present for targeted therapy to be effective. However, this argument suggests that mitotic regulatory enzymes may still be effective if tumor bioavailability is temporarily maintained. Preclinical testing of PLK inhibitor BI2536 showed tumor reduction only once every two weeks [4], a fact supporting the idea that sustained bioavailability increases the chance of capturing tumor cells in cell division behavior.

Liposomes are well known colloidal particles that have been used for drug delivery. It is well known that small molecules, including drugs, can be "remotely loaded" into liposomes by creating a physicochemical difference between the internal and external environment of the liposome [16-18 ]. Importantly, the drug should be membrane permeable in the external environment, but become electrically charged and therefore embedded upon diffusion into the internal environment (entrap). One way to make this difference, if the drug is weakly basic, is to encapsulate a buffering anion inside the liposome, so that a low pH is produced inside the liposome relative to the outside.

Liposomes are known to utilize fenestrations in tumor endothelium for access and persistence in tumor tissue [12, 13 ]]. This phenomenon, known as the Enhanced Permeability and Retention (EPR) effect, was first demonstrated with doxorubicin, which resulted in the liposomal cancer drug, Doxil^TM[14，15]. It demonstrated that the stability of liposome encapsulation is Doxil^TMA certified double-edged sword. In one aspect, drug exposure to healthy tissue is reduced. On the other hand, slow leakage of doxorubicin from liposomes can have a limiting effect on efficacy, since most cancer drugs require high tumor concentrations to be effective.

In contrast to doxorubicin, BI2536 is effective at 1000-fold the concentration of doxorubicin, which means that sustained exposure rather than maximal concentration should greatly enhance its efficacy.

It is desirable to develop a system that can maximize the time exposure of mitotic inhibitors to increase the fraction of dividing cancer cells that can be targeted by the mitotic inhibitor.

Disclosure of Invention

The present invention relates to the field of cancer therapy. More particularly, the present invention relates to hyperstable liposomes useful for the treatment of cancer and methods of using the same for the treatment of cancer.

Thus, in one aspect, the invention provides hyperstable liposomes encapsulating an antimitotic drug. In some embodiments, the anti-mitotic drug is BI2536, Ispinesib (SB 715992), MK 0457(VX 680), AZD1152, PHA 680632, PHA 739358, MLN8054, MLN8237, R763, AT9283, SNS 314, SU 6668, ENMD2076, BI 811283, CYC116, ENMD981693, MKC 1693, ON01910, GSK 461364, HMN214, BI6727, SB 743921, MK 0731, or ARRY 520. In some embodiments, the hyperstable liposomes can be prepared using any suitable liposome composition. In some embodiments, the hyperstable liposomes are sterically stabilized. In some embodiments, the hyperstable liposomes are prepared from a lipid mixture comprising HEPC: Chol: DSPE-PEG2000 (HEPC: hydrogenated egg L-a-phosphatidylcholine; cholesterol: cholesterol; DSPE-PEG-2000: 1, 2-distearoyl-sn-glycerol-3-phosphoethanoi-amine-N- [ methoxy (polyethylene glycol) -2000) in a molar ratio of 50: 45: 5 in some embodiments, the hyperstable liposomes contain an internal environment (lnermeilieu) having one or more anions, preferably two or more anions, that provide for slow release of the antimitotic agent from the hyperstable liposomes. The internal environment contains one or more cations. In some embodiments, the one or more cations may be as described herein. By using the techniques described herein, the optimal combination of anion and cation for a given antimitotic drug can be readily determined.

In some embodiments, pharmaceutical compositions are provided comprising the ultrastable liposomes described herein, with or without at least one pharmaceutically acceptable excipient and/or carrier. Suitable pharmaceutically acceptable excipients and carriers are well known in the art.

In a second aspect, the invention provides methods of treating cancer using the ultrastable liposomes described herein. According to the method, a therapeutically effective amount of the hyperstable liposomes is administered to a patient, e.g., a human, in need of treatment.

Brief Description of Drawings

Fig. 1 shows a procedure to study the ability to partition BI2536 into hexanol from multiple salt solutions.

FIG. 2 shows the relative fluorescence of BI2536 extracted from various monoanionic salt solutions into hexanol using the method described in FIG. 1. The partitioning of BI2536 into hexanol and the salt solution is influenced by the identity and concentration of the salt anion. Abbreviations used are citrate (C), acetate (a), phosphate (P), 2- (N-morpholino) ethanesulfonate (M) and hydrochloric acid (H). All salt solutions were 0.8M and adjusted to pH 3 with sodium as cation. Error bars indicate standard errors.

FIG. 3 shows the relative fluorescence of BI2536 extracted from various pairs of anion combinations into hexanol using the method described in FIG. 1. Adjusting the molar ratio of the paired anion combination can affect the partitioning of BI2536 into hexanol. All salts were 0.8M and adjusted to pH 3 with sodium as cation. The concentration of the monosalt was 0.8M. The numbers before the abbreviation indicate the concentration ratio of 0.8M to the total salt concentration.

Figure 4 shows that the release rate is related to EC50 of liposomal BI2536, but not to liposomal doxorubicin. The following anions, in single and paired combination, were used to generate a continuous drug release rate for gradient loading: citrate (C), acetate (A), phosphate (P), 2- (N-morpholino) ethanesulfonate (M) and hydrochloric acid (H). The double letter abbreviations represent pairs of anion combinations, each combination being half of the total concentration. Cytotoxicity (EC) of BI2536 (upper panel) and doxorubicin (lower panel) is shown for both hypotonic (water) and hypertonic conditions (sucrose)₅₀) Scatter plot against release rate. Bar chart shows EC for same data₅₀Relative to the formulation ordered by release rate. The dashed lines on all graphs indicate the EC for unencapsulated drug₅₀. The release rate is based on the amount of drug released after 12 hours of incubation. Spearman grade correlation (rs) and associated p-value are reported.

Fig. 5A and 5B show the control of citrate by adjusting citrate: phosphate ratio to modulate the potency of liposomal BI 2536. FIG. 5A: shows the EC measured at day 3 and day 8 for various citrate to phosphate (C: P) ratios₅₀vs. scatter plot of release rate. FIG. 5B: mice xenografted with HCT116 colorectal cancer cells were treated with single dose liposomes BI2536 formulated at various C: P ratios. 3 mice were used per experimental group. Tumor volume and weight are reported. Error bars indicate standard errors.

Fig. 6A and 6B show in vivo efficacy and toxicity of liposomal BI2536 on HCT116 xenograft mice. Tumor volume and weight and Kaplan-Meier survival curves are shown for treatment with a single dose on day 0 (fig. 6A) or double doses on days 0 and 7 (fig. 6B). All treatments with liposome BI2536 were formulated with the various citrate: phosphate (C: P) ratios described and administered at 340mg/kg after considering encapsulation efficiency. Free BI2536 was administered at a maximum tolerated dose of 100 mg/kg. Error bars indicate standard errors. Tumor volumes significantly different (P < 0.05) between the ultrastable liposomes (C: P ═ 1: 3) and the other groups from day 9 for single dose and from day 14 for double dose treatment. Mice treated with C: P (1: 3) survived significantly longer than the other groups (P < 0.05). The Kaplan-Meier curve shows the percent survival over time. The tick marks represent death events. The difference between BI-L2C6P and all other treatments was significant, reporting Mantel-Cox Log scale P-values for survival curves, showing that mice treated with C: P (1: 3) survived significantly longer for both single dose (P0.0164) and double dose (P0.0349).

FIGS. 7A-7D show the pharmacokinetic distribution and bioavailability of BI2536 after treatment with ultrastable liposomes BI 2536. (FIG. 7A): mice bearing HCT116 xenografts were treated with BI2536, which was encapsulated using various citrate: phosphate ratios. Each data point included 3 mice. BI2536 was extracted from tissue at multiple time points quantified by fluorimetry. Data points and error bars represent mean and standard error, respectively. The significant difference between the ultrastable liposomes (C: P ═ 1: 3) and the other groups (P < 0.05) is indicated by an asterisk. (FIG. 7B): tissue exposed to BI2536 as measured by area under the curve is shown. (FIG. 7C): the percentage of mitotically arrested cells at 1.5 and 5.5 days post-treatment is shown. Each bar is from 6 independent fields of 2 non-adjacent H & E stained sections. Error bars indicate standard errors. (FIG. 7D): typical H & E images of various processes are shown. Arrows refer to examples of mitotically arrested cells. Scale bar, 10 μm.

Fig. 8A and 8B show in vivo efficacy of liposomal BI2536 on HCT116 xenograft mice. Tumor volumes were shown on day 0 with single dose treatment, where the single dose treatment refers (fig. 8A) to liposomes formulated with various ratios of citrate: acetate and (fig. 8B) with various ratios of citrate: acetate salt but ammonium instead of sodium cation. All formulations were administered at 340mg/kg of BI 2536. Error bars represent standard error.

Detailed Description

The present invention relates to the field of cancer therapy. More particularly, the present invention relates to hyperstable liposomes useful for the treatment of cancer and methods of using the hyperstable liposomes for the treatment of cancer. It has been found that the extreme prolongation of the release of mitotic suppressive drugs from ultrastable liposomes improves the efficacy of cancer treatment by increasing the proportion of cancer cells that can be targeted. The slow release of mitotic inhibitory drugs from ultrastable liposomes is associated with cancer cell killing in vitro and in vivo. In one example, xenograft mice treated with a single dose of ultrastable liposome BI2536 experienced a sustained reduction in tumor volume for 12 days and had a 20% complete response in the treated mice. Treatment with two doses every other week increased the response rate in 75% of treated mice.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "about" or "approximately" means within a statistically significant range of a value. Such a range may be within an order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, more preferably within 10%, even more preferably within 5%. The allowable variations encompassed by the terms "about" or "approximately" depend on the particular system under study and can be readily understood by one of ordinary skill in the art.

As used herein, "cancer" refers to a group of diseases involving abnormal cell growth that may invade or spread to other parts of the body. Cancers include cancers such as glioma, head and neck cancer, kidney cancer, lung cancer, medulloblastoma, melanoma, merck cell carcinoma, mesothelioma, neuroblastoma, esophageal cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, testicular cancer, thyroid cancer; leukemias, such as acute myelogenous leukemia, chronic myelogenous leukemia, hairy cell leukemia, lymphocytic T cell leukemia, B cell leukemia; lymphomas such as anaplastic large cell lymphoma, B cell lymphoma, burkitt's lymphoma, hodgkin's lymphoma; and myeloma.

The term "mitotic inhibitory drug" refers to drugs that target mitotic regulatory enzymes, such as microtubule regulatory enzymes, Polo-like kinases (PLKs), kinesin-spindle proteins (KSPs), aurora kinases, and the like. The terms "anti-mitotic drug (anti-mitotic drug)" or "anti-mitosis drug (anti-mitosis drug)" are used interchangeably with "mitosis inhibitory drug".

As used herein, "ultrastable liposomes" refers to liposome-encapsulated drugs that are slowly released due in part to the anions and cations present in the internal environment of the liposome. The slowest release rate of the hyperstable liposomes is highly correlated with cancer cell killing.

The term "slow release of drug" means that when the liposome is suspended in 600mM sucrose, the drug quantitatively released from the liposome-encapsulated drug is less than 0.6% in 12 hours or less than 5% in 8 days.

In one aspect, the invention provides a hyperstable liposome encapsulating an antimitotic drug. In some embodiments, the antimitotic drug is a polo-like kinase inhibitor, such as BI2536, ON01910, GSK 461364, HMN214, or BI 6727. In other embodiments, the anti-mitotic drug is a kinesin spindle inhibitor, e.g., Ispinesib (SB 715992), SB 743921, MK 0731, or ARRY 520. In some embodiments, the antimitotic agent is an aurora kinase inhibitor, e.g., MK 0457(VX 680), AZD1152, PHA 680632, PHA 739358, MLN8054, MLN8237, R763, AT9283, SNS 314, SU 6668, ENMD2076, BI 811283, CYC116, ENMD981693, or MKC 1693. In some embodiments, the anti-mitotic agent is BI2536 or Ispinesib.

In some embodiments, any suitable liposomal composition can be used to prepare the hyperstable liposomes. In some embodiments, the hyperstable liposomes are sterically stabilized. In some embodiments, the hyperstable liposomes are prepared from a lipid mixture comprising HEPC: Chol: DSPE-PEG2000 (HEPC: hydrogenated egg L- α -phosphatidylcholine; Chol: cholesterol; DSPE-PEG-2000: 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamin N- [ methoxy (polyethylene glycol) -2000]) in a molar ratio of 50: 45: 5.

In some embodiments, the hyperstable liposomes contain an internal environment with one or more anions, preferably an internal environment with two or more anions, which provides for slow release of the antimitotic agent from the hyperstable liposomes. In some embodiments, the one or more anions, or preferably two or more anions, can be citrate, acetate, phosphate, 2- (N-morpholino) ethanesulfonate, chloride, citrate and acetate, citrate and 2- (N-morpholino) ethanesulfonate, citrate and chloride, acetate and phosphate, acetate and 2- (N-morpholino) ethanesulfonate, acetate and chloride, phosphate and 2- (N-morpholino) ethanesulfonate, phosphate and chloride, 2- (N-morpholino) ethanesulfonate, and chloride. In some embodiments, the chloride ion is in the HCL form. In some embodiments, the ratio of the two anions may be from about 1: 7 to about 7: 1. In other embodiments, the ratio of the two anions may be from about 1: 3 to about 3: 1. In some embodiments, the anion is citrate: phosphate, in a ratio of from about 1: 3 to about 1: 7, preferably about 1: 3. In some embodiments, the anion is citrate to acetate, in a ratio of about 1: 3 to about 3: 1, preferably about 1: 3. In some embodiments, the internal environment contains one or more cations. In some embodiments, the one or more cations may be sodium, ammonium, triethylammonium, copper, magnesium, zinc, or iron. For a given anti-mitotic drug, the optimal combination and ratio of anions and cations can be readily determined experimentally in mice, for example by using the techniques described herein.

In some embodiments, the ultrastable liposomes of the invention may comprise one or more anions of the invention in any suitable form (e.g., in the form of an acid or salt comprising a polyanion and a cation, preferably in the form of a salt). The amount of anion can be stoichiometrically equal to the amount of cation or different from the amount of cation. In some embodiments, the hyperstable liposomes of the present invention contain one or more anionic salts of cations, wherein there is a concentration gradient or pH gradient of cations across the liposome membrane. In another embodiment, the ultrastable liposomes of the invention contain one or more salts of the ammonium anions of the invention. In another embodiment, the ultrastable liposomes of the invention contain anions within the ultrastable liposomes, and the anions in the medium containing the ultrastable liposomes are partially or substantially removed by any suitable method known to those skilled in the art, such as dilution, ion exchange chromatography, size exclusion chromatography, dialysis, ultrafiltration, absorption, precipitation, and the like. In some embodiments, the hyperstable liposomes with embedded anions also have a transmembrane gradient effective to retain the substance within the hyperstable liposome. Examples of such transmembrane gradients are pH gradients, electrochemical potential gradients, cationic ion gradients or solubility gradients. Methods for generating transmembrane gradients are conventional in the liposome art.

In some embodiments, the hyperstable liposomes enter the target cells by exploiting windowing (fencing) in the tumor endothelium. In other embodiments, the hyperstable liposomes of the invention may also be targeted liposomes, e.g., liposomes containing one or more targeting moieties or biodistribution modifiers on the surface of the liposome. The targeting moiety can be any agent capable of specifically binding to or interacting with the desired target. In some embodiments, the targeting moiety is a ligand. In some embodiments, the ligand preferentially binds to and/or internalizes into a cell (target cell) in which the liposome-embedded entity exerts its desired effect. The ligand is typically a member of a binding pair, wherein the second member is present on or in a target cell or in a tissue comprising said target cell. See, for example, U.S. patent No. 8,922,970, which is incorporated herein by reference.

Liposomes of the invention can be prepared by any suitable method known or later discovered by those skilled in the art. See, e.g., Gregoriadis [25], U.S. patent No. 8,992,970, and U.S. patent No. 9,023,384, each incorporated herein by reference. Liposomes are typically manufactured using a variety of methods in which water-soluble (hydrophilic) materials are entrapped by using an aqueous solution of these materials as the hydration fluid or by adding a drug/drug solution at some stage during liposome manufacture. Fat-soluble (lipophilic) substances are dissolved in organic solutions of the constitutive lipids and then evaporated to a dry drug containing a lipid film, followed by hydration. These methods involve loading the embedded reagents prior to or during the manufacturing process (passive loading). However, certain types of compounds with ionizable groups and compounds that exhibit lipid solubility and water solubility can be introduced into liposomes after formation of intact vesicles (remote or active loading).

When preparing liposomes with mixed lipid compositions, the lipids are first dissolved and mixed in an organic solvent to ensure that a homogeneous mixture of lipids is formed. In some embodiments, the organic solvent is chloroform or chloroform: a methanol mixture. Once the lipids are well mixed in the organic solvent, the solvent is removed to obtain a lipid film. In some embodiments, the organic solvent is removed by rotary evaporation under reduced pressure, resulting in a thin lipid film on the side of the round bottom flask. The lipid film is typically dried thoroughly overnight under high vacuum to remove residual organic solvent. Hydration of the dried lipid film is accomplished simply by adding the aqueous buffer solution to a container of dried lipid and stirring at a temperature above the lipid transition temperature. This method produces a population of Multilamellar Liposomes (MLVs) that are non-uniform in size and shape (e.g., 1-5 μm in diameter). Liposome size reduction techniques, such as sonication for Single Unilamellar Vesicle (SUV) formation or extrusion through polycarbonate filters that form Large Unilamellar Vesicles (LUVs). Additional details and other methods of preparing liposomes with encapsulated drugs can be found in Fritze et al [16], Dua et al [20], Laouini et al [21], U.S. Pat. No. 8,992,970 and U.S. Pat. No. 9,023,384, each of which is incorporated herein by reference.

In some embodiments, the ultrastable liposomes of the invention are formulated at the nanoscale using saturated phosphatidylcholine coupled to high cholesterol content to reduce membrane permeability. In some embodiments, the hyperstable liposomes are further formulated using pegylation or other conjugation for steric stabilization. In other embodiments, the saturated phosphatidylcholine can be replaced by other membrane-forming phospholipids. In some embodiments, the hyperstable liposomes are prepared using conventional techniques or those described herein. In some embodiments, the membrane-forming phospholipid is a saturated Phosphatidylcholine (PC), any synthetic Phosphatidylcholine (PC) with a saturated fatty acid tail, or a membrane-forming lipid. In some embodiments, the synthetic PC may be dimyristoyl-phosphatidylcholine, dipalmitoyl-phosphatidylcholine, or distearoyl-phosphatidylcholine. In some embodiments, the saturated Phosphatidylcholine (PC) is Hydrogenated Egg Phosphatidylcholine (HEPC). In other embodiments, the film-forming lipid can be saturated sphingomyelin, saturated phosphatidylethanolamine, saturated phosphatidylglycerol, saturated phosphatidylinositol, or saturated phosphatidylserine.

In some embodiments, the conjugate can be polyethylene glycol, polypropylene glycol, polybutylene glycol, or a copolymer of polyalkylene glycols (e.g., a block copolymer of polyethylene glycol and polypropylene glycol), dextran, pullulan, polysucrose, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, or carrageenan. In some embodiments, polyethylene glycol (PEG) is used as the conjugate (C-PEG). In some embodiments, the molecular weight of the PEG ranges from about 500 to about 10000, preferably from about 1000 to about 5000, more preferably about 2000.

In some embodiments, the PEG or other conjugate is conjugated to: distearoylphosphatidylethanolamine (DSPE), Dipalmitoylphosphatidylethanolamine (DPPE), Dimyristoylphosphatidylethanolamine (DMPE), Distearoylglycerol (DSG), Dimyristoylglycerol (DMG), a cholesterylated conjugate, an Stearoyl (STR) conjugate, a C8 ceramide conjugate, or a C16 ceramide conjugate. In some embodiments, the conjugate is PEG2000, and the conjugate is DSPE-PEG2000, DPPE-PEG2000, DMPE-PEG2000, DSG-PEG2000, DMG-PEG2000, cholesterylated-PEG 2000, STR-PEG2000, C8 ceramide-PEG 2000, or C16 ceramide-PEG 2000.

In some embodiments, the sterically stabilized liposomes are prepared from a preparative mixture of PC: cholesterol: C-PEG, wherein the molar ratio of PC: cholesterol is typically in the range of 2: 1 to 1: 1, wherein the C-PEG is typically present at 5% (mol/mol). In some embodiments, the preparative mixture of PC to cholesterol to C-PEG has a molar ratio of 50: 45: 5. In some embodiments, the preparative mixture is HEPC to cholesterol to DSPE-PEG 2000. In some embodiments, the preparative mixture of HEPC: cholesterol: DSPE-PEG2000 has a molar ratio of 50: 45: 5.

In some embodiments, the liposomes are prepared by dissolving the preparation mixture described herein in chloroform. The solution was dried to a film under rotary evaporation and then under vacuum overnight. The membrane is hydrated with a hydration buffer containing the desired salt solution (e.g., as described herein) as the internal environment of the liposome and submerged in a water bath sonicator. The liposome mixture is first sonicated and then extruded to form SUVs. In some embodiments, the SUV is dialyzed against sucrose to alter the external environment of the liposome

In some embodiments, mitotic-inhibiting drugs are actively loaded into liposomes by pH gradient methods well known in the art. In some embodiments, the mitosis-inhibiting drug is first coated as a thin film in a suitable container and then dried. In some embodiments, the liposomes are present in a 3: 1 lipid: drug concentration was loaded and diluted with water to the desired concentration. The mixture was then incubated in a high temperature water bath to facilitate loading and subsequently dialyzed against sucrose to remove unencapsulated drug.

In some embodiments, the ultrastable liposomes of the invention are prepared as follows. Lipid mixture of HEPC: Chol: DSPE-PEG2000 was dissolved in chloroform at a molar ratio of 50: 45: 5. The mixture was dried in a round bottom flask under rotary evaporation to a thin lipid film and further dried under high vacuum overnight before hydration with the desired salt solution as the internal environment of the liposomes. The resulting 100mM lipid suspension was sonicated with a bath sonicator for 1 hour and then extruded 10 times through a double-stacked 100nm nucleocore filter using a Lipex thermoearrel Extruder to form Single Unilamellar Vesicles (SUVs). These SUVs were dialyzed at 4 ℃ against 300mM sucrose, and the fresh sucrose solution was changed three times over 24 hours to exchange the external environment of the liposomes. Liposomes were stored in glass tubes at 4 ℃ until intended for use.

In one example, mitotic inhibitor BI2536 was actively loaded into liposomes by a pH gradient method. BI2536 was first coated as a thin film in a scintillation vial by dissolving in ethanol and then drying under rotary evaporation. The BI2536 films were further dried under vacuum for at least 24 hours. The liposomes are present in a 3: 1 lipid: drug concentration loading and dilution with water to a final concentration of about 50 to about 70mM lipid. The mixture was then incubated in a 70 ℃ water bath to facilitate loading and then dialyzed against 300mM sucrose for at least 36 hours to remove unencapsulated BI 2536. After dialysis, the liposomes were stored in glass tubes until use.

In some embodiments, the ultrastable liposomes of the invention are very stable during storage, e.g., as measured by the percentage of embedded entities released outside the ultrastable liposome or remaining inside the ultrastable liposome after a period of time from initial loading of the entities within the ultrastable liposome of the invention. For example, the ultrastable liposome composition of the invention is stable at 4 ℃ for at least 6 months.

The following are advantageous: the liposome-embedded antimitotic agent remains encapsulated in the liposome until the hyperstable liposome reaches the site of its intended action (e.g., a tumor in the case of administration of a liposomal antimitotic drug in a patient). The ultrastable liposomes of the invention exhibit surprising stability under in vivo conditions (e.g., in mammalian blood) to prevent release (leakage) of the embedded antimitotic drug. It is noteworthy that the inventive hyperstable liposomes, although having such a low in vivo drug release rate in the blood circulation, show significant in vitro antitumor activity. The ultrastable liposomes of the present invention provide an unexpected combination of high efficiency and low toxicity of the embedded antimitotic drug.

In some embodiments, provided are liposome compositions comprising the ultrastable liposomes described herein in an aqueous medium. In some embodiments, the hyperstable liposomes separate the internal aqueous space from the aqueous medium by a membrane. In some embodiments, the membrane comprises 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000], hydrogenated egg L- α -phosphatidylcholine, and cholesterol. In some embodiments, embedded within the hyperstable lipid is an antimitotic drug, an anion, and a cation, wherein the concentration of the embedded antimitotic drug within the hyperstable lipid exceeds the concentration of the antimitotic drug in the aqueous medium.

In some embodiments, pharmaceutical compositions are provided comprising the ultrastable liposomes described herein, with or without at least one pharmaceutically acceptable excipient and/or carrier. In some embodiments, the pharmaceutically acceptable carrier is physiological saline, isotonic dextrose, isotonic sucrose, Ringer's (Ringer) solution, and Hank's (Hank) solution. Buffering substances may be added to provide a pH optimal for storage stability. For example, a pH between about 6.0 and about 7.5, more preferably a pH of about 6.5, is optimal for liposome membrane lipid stability and provides excellent retention of the embedded entity. The following are exemplary buffer substances: histidine, hydroxyethylpiperazine-ethylsulphonate (HEPES), morpholine-ethylsulphonate (MES), succinate, tartrate and citrate, typically at concentrations of 2-20 mM. Other suitable carriers include, for example, water, aqueous buffered solutions, 0.4% NaCl, 0.3% glycine, and the like. Protein, carbohydrate or polymer stabilizers and tonicity adjusting agents, such as gelatin, albumin, dextran or polyvinylpyrrolidone, may be added. The tonicity of the composition may be adjusted to a physiological level of 0.25-0.35mol/kg with dextrose or a more inert compound such as lactose, sucrose, mannitol or dextrin. These compositions may be sterilized by conventional well-known sterilization techniques, such as by filtration. The resulting aqueous solution may be packaged for use or filtered under sterile conditions and lyophilized, the lyophilized formulation being combined with a sterile aqueous medium prior to administration.

In some embodiments, pharmaceutically acceptable excipients may be used as needed 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 and the like. In addition, the hyperstable liposomal suspension may include lipid protectants that protect the storage of lipids from free radicals and lipid peroxidation damage. Lipophilic radical quenchers are suitable, such as alpha-tocopherol and water soluble iron specific chelators (e.g. ferric amine).

The concentration of the ultrastable liposomes of the invention in the pharmaceutical composition can vary widely, i.e. typically less than about 0.05% by weight or at least about 2-10% by weight-up to 30 to 50% by weight, and is selected primarily by liquid volume, viscosity, etc., depending on the particular mode of administration selected. For example, the concentration may be increased to reduce the fluid load associated with the treatment. This may be particularly desirable for patients with atherosclerosis-associated congestive heart failure or severe hypertension. Alternatively, the pharmaceutical composition consisting of the irritating lipid may be diluted to a low concentration to reduce inflammation at the site of administration.

In a second aspect, the invention provides methods of treating cancer using the ultrastable liposomes described herein. In some embodiments, the amount of the hyperstable liposomal pharmaceutical composition administered will depend on the particular antimitotic drug entrapped within the hyperstable liposomes, the cancer being treated, the type of hyperstable liposomes used, and the judgment of the clinician. Generally, the amount of the hyperstable liposomal pharmaceutical composition administered will be sufficient to deliver a therapeutically effective dose of the particular antimitotic drug.

The amount of the hyperstable liposomal pharmaceutical composition necessary to deliver a therapeutically effective dose can be determined by conventional in vitro and in vivo methods common in the art of pharmaceutical testing. See, for example, Budman et al [22 ]. Therapeutically effective dosages of various antimitotic drugs are well known to those skilled in the art; and according to the invention, the antimitotic drug delivered by the pharmaceutical composition of the invention provides at least the same, or 2-fold, 4-fold or 10-fold higher activity than that obtained by administering the same amount of antimitotic drug in its conventional non-liposomal formulation. Typically, the dosage of the hyperstable liposomal pharmaceutical composition of the present invention ranges from about 0.005 to about 500mg therapeutic entity per kilogram body weight, most typically from about 0.1 to about 100mg therapeutic entity per kg body weight.

Typically, the pharmaceutical compositions of the present invention are prepared as topical or injectable liquid solutions or suspensions. However, solid forms suitable for dissolution or suspension in a liquid carrier prior to injection can also be prepared. The compositions may also be formulated into enteric-coated tablets or gel capsules according to methods known in the art.

The hyperstable liposome composition of the present invention may be administered in any manner that is medically acceptable, which may depend on the cancer being treated. Possible routes of administration include injection, by parenteral routes, e.g., intramuscular, subcutaneous, intravenous, intraarterial, intraperitoneal, intraarticular, intradural, intrathecal, or otherwise, and oral, nasal, ocular, rectal, vaginal, topical, or pulmonary, e.g., by inhalation. For the delivery of liposomal antimitotic drugs formulated according to the present invention to tumors of the central nervous system, slow, sustained intracranial infusion of liposomes directly into the tumor (convection enhanced delivery or CED) is particularly advantageous. See Saito et al [23] and Mamot et al [24 ]. The composition may also be applied directly to the tissue surface. Also specifically included in the present invention are sustained release, pH dependent release or other specific chemical or environmental condition mediated release applications, such as by depot injection or erodible implants.

As shown in the examples below, methods of identifying sustained release ultrastable liposome formulations using combined anion diversity are described. Although the examples focus on citrate: phosphate anion pairs, but other hyperstable anion pairs found in the screen (fig. 4) will also produce similar results to BI 2536. When the citrate radical: phosphate pair is represented by citrate: acetate replacement, mice treated with a single dose of this replacement liposome form showed a ratio of citrate to citrate in the ratio of 1: 3: the maximal tumor reduction by acetate was similar in potency (fig. 8A). A natural way to add more diversity is to change the cationic species. For example, the replacement of sodium cations with ammonium for citrate: acetate significantly increased the rate of tumor regression (fig. 8B).

Ultrastable liposomes solve two conceptual problems. Prolonged time availability allows the anti-mitotic drug to capture more tumor cells during replication. Furthermore, the low sustained drug concentrations achieved by ultrastable liposomes are unlikely to trigger mitotic slippage (slippage) [19 ]. This means that there should be few tumor cells that escape the intended effect of the drug. This approach can be applied to any antimitotic drug, not just BI 2536. Therefore, ultrastable encapsulation has the potential to restore clinical utility to 24 drugs belonging to this class. One advantage of ultrastable liposomes is extended bioavailability over a single dose time scale of two weeks, which would enable clinicians to achieve higher efficacy at lower frequency doses. Another advantage is the lack of irreversible neuropathy after treatment with ultrastable liposomes compared to other drug classes, which is an attractive feature of mitotic inhibitors from a toxicity point of view. The description of the general method for reactivating failed mitotic inhibitors opens the door for many possibilities and demonstrates that the idea of not inhibiting mitosis is deficient; but in fact the delivery is important.

Examples

The invention is described by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention in any way. Standard techniques well known in the art or those specifically described below are used.

Example 1

Materials and methods

Liposome preparation: 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (DSPE-PEG2000) and hydrogenated egg L- α -phosphatidylcholine (HEPC) were purchased from Lipoid and cholesterol (Chol) from Sigma-Aldrich. Lipid mixture of HEPC: Chol: DSPE-PEG2000 was dissolved in chloroform (Takara) at a molar ratio of 50: 45: 5. The mixture was dried to a thin lipid film under rotary evaporation (eye NVC-2200/N-1100) in a round bottom flask and further dried under high vacuum overnight before hydration with the desired salt solution as the internal environment of the liposomes. The resulting 100mM lipid suspension was sonicated for 1 hour with a bath sonicator (S30H Elmasonic) and then extruded 10 times through a bilayer-stacked 100nm nucleocore filter (Whatman) using a Lipex Thermobarrel Extruder (Northern Lipids) to form Single Unilamellar Vesicles (SUV). These SUVs were dialyzed at 4 ℃ against 300mM sucrose (Sigma) and the fresh sucrose solution was changed three times over 24 hours to exchange the external environment of the liposomes. Liposomes were stored in glass tubes at 4 ℃ until intended for use.

Loading BI2536 into liposomes: BI2536 (Axon) was actively loaded into liposomes by a pH gradient method. The necessary BI2536 was first coated as a thin film in a scintillation vial by dissolving in ethanol and subsequently drying under rotary evaporation. The BI2536 films were further dried under vacuum for at least 24 hours. Liposomes were loaded at a lipid: drug concentration of 3: 1 and diluted with water to a final concentration of 50 to 75mM lipid. The mixture was then incubated in a 70 ℃ water bath to facilitate loading and subsequently dialyzed in 300mM sucrose for at least 36 hours to remove unencapsulated BI 2536. After dialysis, the liposomes were stored in glass tubes until use and a portion of the sucrose dialysate was stored at 4 ℃ for determination of downstream encapsulation efficiency.

Determination of BI2536 encapsulation: the amount of BI2536 loaded into the liposomes was determined by direct calculation (in vitro studies) or reverse calculation (for animal studies). For direct calculation, 1 μ l of liposomes was diluted with 20 μ l ethanol and read by fluorescence measurement using 360nm excitation and 470nm emission (Tecan Infinite M200). The amount of BI2536 was determined by comparison to a standard curve. For the reverse calculation, unencapsulated BI2536 was extracted from 1.5ml of dialysate by vortexing for 1 hour using 100. mu.l of 1-nonanol (Merck). The nonanol and sucrose phases were separated by brief centrifugation and the fluorescence intensity of the 20ul nonanol layer was measured using 330nm excitation and 370nm emission (Tecan Infinite M200). The concentration of BI2536 in the dialysate was determined by comparison to a standard curve and then the encapsulation efficiency was calculated by this formula.

Wherein a ═ BI2536 in dialysate]_{Without drug loading}And B ═ BI2536 in dialysate]_{Sample (I)}

Cell culture: HCT116(CCL-247, human colorectal cancer) was purchased from American Type Culture Collection (ATCC) and cultured using McCoy's 5A medium (life technology) supplemented with 10% fetal bovine serum (Thermo Scientific). Cells were incubated at 37 ℃ with 5% CO₂Incubate and passage every 2 to 3 days when the confluency (confluency) reaches-80%.

Determination of EC₅₀: will be about 7X 10³Individual HCT116 cells were seeded into 96-well plates and reached-50% confluence after overnight incubation. The medium in the wells was replaced with fresh medium supplemented with free BI2536 or liposomal BI 2536. The BI2536 concentration used was generated by serial dilution of 1 μ M BI 2536. Wells containing medium only served as blanks. For each formulation, at least 3 replicates were performed. SYBR Green I (Life Technologies) was used to quantify DNA as a measure of cell survival. This was done by first incubating the cells with 50 μ l of 0.2% sodium dodecyl sulfate for 2 hours at 37 ℃ to lyse them. Then 150. mu.L of SYBRGreen solution (diluted 1: 750 in water) was added to each well and the fluorescence intensity was measured using a Tecan plate reader (Ex: 497 nm/Em: 520 nm). The fluorescence intensity values were input into GraphPad Prism V5. By setting the maximum fluorescence valueLogistic regression (logistic regression) curves and ECs were determined for 100% survival and with the lowest fluorescence value set to 0% survival₅₀。

Animal studies: all animal experiments were approved by the institutional animal care and use committee of the life sciences laboratory of thaumatin and National University of Singapore (NUS). Female NCr nude mice (5-8 weeks old) were purchased from Singapore/InVivos and subcutaneously xenografted with HCT116 cells. At 600cm as described above²HCT116 cells were cultured in petri dishes (Corning), and when the fusion degree reached-80%, 5 mice were transplanted using each petri dish.

Efficacy studies: free BI2536 (dissolved in 0.1N HCl, saline) or the indicated liposomal BI2536 formulation was administered by slow tail intravenous injection 7 days after transplantation with HCT 116. The tumor volume is at least 150mm³And using length x width²X 0.5. All measurements were made using a vernier caliper, and mice were weighed every other day. Mice were hydrated subcutaneously with 1ml hartmann's solution daily for 5 days after treatment to ensure complete hydration of the mice.

Pharmacokinetic studies: mice bearing HCT116 xenografts were treated with the indicated free BI2536 or liposomal BI2536 preparations and euthanized at the indicated time points post-treatment to collect heart, tumor, muscle, kidney, liver and spleen. Organs were weighed and stored at-80 ℃ prior to tissue treatment. The tissue was treated by immersion in chaotropic agent 8M urea (Vivantis) and homogenized in a Bertin homogenizer using 0.5mm diameter zirconia beads (Biospec). Homogenized tissue was spun at top speed for 1 hour on a bench top centrifuge and 800 μ l of supernatant was collected for BI2536 extraction using 100 μ l nonanol and gentle spinning for 1 hour. The nonanol and sucrose phases were separated by brief centrifugation and 20ul of the nonanol layer was read by fluorescence measurement (Ex: 330 nm/Em: 370nm) using a Tecan plate reader. BI2536 was quantified by comparison to a standard curve and then normalized to the weight of the tissue.

Histology: mice were sacrificed on the indicated days post-treatment for tumor tissue collection. Tumor tissues were frozen in OCT medium (Sakura Finetek) and stored at-80 ℃ prior to sectioning. 10 μm tumor tissue sections were obtained using a CM3050S cryostat (Leica). The sectioned tissue was fixed in methanol and immediately stained with hematoxylin and eosin (H & E). For H & E staining, tumor sections were first stained with filtered Harris solution (Sigma), washed with running tap water, immersed in acid-alcohol (1% hydrochloric acid, 70% ethanol) and further washed with tap water. The tissue sections were then immersed in 0.2% ammonia (Sigma) until blue. After washing in tap water for 10 minutes, the tissue sections were stained with eosin-flame red (Sigma and Merck, respectively) and immersed in 95% ethanol to wash away excess dye. Tissue sections were dried overnight and then fixed with permount (fisher). All H & E stained sections were observed and bright field images were obtained using an Axioplan 2 microscope (Carl Zeiss, Inc) in combination with a DXM 1200F camera (Nikon) and a 63X objective.

Preparation of BI2536 in buffer: BI2536 was first dissolved in ethanol and then coated by spin drying onto 1.5ml microcentrifuge tubes. The BI 2636 coated tubes were then further dried under high vacuum overnight and then resuspended in the indicated salt solution to reach a final concentration of 500 μ M. To ensure that the coated BI2536 was completely dissolved, the tube was briefly vortexed and bath sonicated for 1 minute before being used for characterization.

Extracting hexanol: 1ml of the indicated buffer was extracted at BI2536 with 100. mu.l of 1-hexanol (Merck) by brief shaking for 1 hour. The hexanol and buffer layers were separated by brief centrifugation and 20ul of the hexanol layer was analyzed for fluorescence intensity using a Tecan plate reader (Ex: 330nm, Em: 370 nm).

And (3) determining the stability of the liposome: fluorescence dequencing using leaky BI2536 was used as a measure of liposome instability. All fluorescence readings were performed using a Tecan plate reader (Ex: 280nm, Em: 385 nm). Triton-X100(sigma) was added to reach a final concentration of 0.2% to completely release BI2536 from the liposomes and fluorescence readings were taken again. To calculate the fraction of BI2536 released, the fluorescence reading before triton addition was divided by the fluorescence reading after triton addition. For stability measurements, the liposome formulations were diluted 50-fold with water or 600mM sucrose solution and fluorescence was measured initially using a Tecan plate reader and after 12 hours of incubation at 37 ℃ using a Tecan plate reader. To determine long-term stability, liposomes were diluted in water or 600mM sucrose and stored in a 37 ℃ incubator and fluorescence readings were taken on the indicated days in a similar manner.

Example 2

The release rate of liposome BI2536 was inversely related to tumor cell killing

The extent to which the anionic species and concentration will affect the physicochemical properties of BI2536 was studied by using the following solutions adjusted to pH 3: sodium citrate (C), sodium acetate (A), sodium phosphate (P), 2- (N-morpholino) ethanesulfonic acid (M) and hydrochloric acid (H). The tendency of partitioning BI2536 to hexanol from these solutions was measured at various concentrations (fig. 1). It was observed that the anionic species did influence the efficiency of hexanol extraction, and that the efficiency increased (P, a) or decreased (M, C, H) in an anion concentration-dependent manner (fig. 2). It was further observed that by using pairwise combinations of these anions, the diversity of these hexanol extraction efficiency curves could be further improved (fig. 3). It was concluded from these results that these anions can be used in combination to generate a library of liposome preparations with different release rates.

To identify the ultrastable sustained release form of liposome BI2536, liposome encapsulated forms of all 15 monoanions and dianions were prepared and BI2536 was remotely loaded into their interiors. BI2536 fluorescence was quenched when encapsulated at high concentrations in liposomes. Thus, the release of BI2536 from liposomes can be measured by the increase in fluorescence due to dequenching. Using this method, leakage of BI2536 for each formulation was measured versus time under both hypertonic (600mM sucrose) and hypotonic (pure water) conditions (fig. 4). As expected, a range of release rates from fast (a, H, AH) to slow (all combinations with citrate) was observed. The rank order of these release rates did not differ significantly between the hypertonic and hypotonic environments, indicating that the liposome internal environment determines the drug release rate of liposome BI2536, rather than the external osmotic pressure. To examine whether the ultrastable sustained-release liposomes were associated with cancer cell killing, HCT116 colorectal cancerCells were incubated with serial dilutions of various liposome formulations and their EC's were added₅₀Values were calculated as a measure of efficacy. Consistent with the hypothesis associated with hyperstability and cytotoxicity, release rates were found with EC₅₀There is a high correlation between them. In contrast, no similar correlation was found when the same experiment was performed with doxorubicin (fig. 4). This finding is consistent with the following view: i.e. although mitotic inhibitors may benefit from hyperstability, for other types of drugs the situation is just the opposite, i.e. sustained release would not be advantageous for said other types of drugs.

Example 3

Anion ratio-modulated release rate and in vivo efficacy

Since the two anions with the slowest release rates were citrate alone and a combination of citrate and phosphate (fig. 4), studies were conducted to alter citrate: phosphate ratio to determine if it would have a substantial effect on efficacy. At a release rate and EC comparable to those of the previous pair₅₀Formulations in the same manner were tested covering ratios of 0: 1, 1: 3, 1: 1, 3: 1 and 1: 0. Whether these variables are measured on day 3 or day 8 of the cytotoxicity assay of the cells or not, rates of release and EC are consistently observed₅₀The same trend (fig. 5A). Interestingly, it was noted that citrate: the slowest release was achieved at a phosphate ratio of 1: 3, indicating that the combined ratio is synergistic and not merely an average of the results for citrate alone and phosphate alone. The results further indicate that the optimal citrate to maximize in vivo efficacy in actual solid tumors was identified: the phosphate ratio is important. To identify this ratio, the ratio of citrate: liposomes with phosphate ratios of 1: 7, 1: 4, 1: 3, 1: 2 and 1: 0.5 treated mice with established human colorectal cancer xenografts. Consistent with the previously observed 1: 3 ratio, the ratio between citrate: optimal in vivo efficacy was observed at phosphate ratios of 1: 3 and 1: 4 (FIG. 5B). Importantly, the reduction in tumor volume for these ratios lasted for more than two weeks, an observation consistent with the expected slow release of these hyperstable liposomes. Although 1: 4 ratioThe antitumor effect produced in this case was greater than 1: 3, but it also resulted in more weight loss. Therefore, 1: 3 was used as the optimal ratio for the subsequent experiments. BI2536 encapsulated in this manner is referred to as "ultrastable".

Example 4

Ultrastable liposomes BI2536 to generate complete responses in mice

Nude mice bearing HCT116 xenograft tumors were treated with a single intravenous injection of either ultrastable liposomal BI2536 or liposomal BI2536 using only citrate or phosphate as anions. Free unencapsulated BI2536 was used as a control. The xenografts treated with ultrastable liposomes decreased in volume within 12 days, reproducing the prolonged therapeutic effect we observed previously in animal experiments (figure 6A). In contrast, liposomes using only citrate or phosphate alone were indistinguishable from the free drug, indicating that the combination of anions produced a synergistic effect, whereas the anions alone did not. Mice treated with ultrastable BI2536 tended to have heavier post-treatment weights, a trend consistent with the expected lower toxicity when drug release was prolonged. Importantly, ultrastable BI2536 significantly improved mouse survival, with complete responses observed in two-tenths of the mice (table 1). No complete reaction was observed in the other experimental groups.

TABLE 1

Table of complete reactions to various treatments

When the same experiment was repeated with two therapeutic doses separated by 7 days instead of a single dose of ultrastable liposomes, the therapeutic effect was extended even to a longer duration (fig. 6B) and resulted in a complete response in 75% of the mice (table 1). In contrast, no complete reaction was observed in the other experimental groups. The ultrastable liposomes were not only more effective but well tolerated, while all other experimental groups showed post-treatment toxicity.

Example 5

Ultrastable liposomes prolong the tumor presence and efficacy of BI2536

A reasonable explanation for the improved efficacy observed with ultrastable liposomes is that the drug half-life is improved compared to conventional pegylated liposomes. Nude mice treated with a single dose of ultrastable liposomes showed significantly higher tumor concentrations of BI2536 compared to control liposomes containing citrate or phosphate alone (fig. 7A; table 2). This trend persists throughout the 9.5 day measurement period, after which the reduction in tumor volume renders tissue treatment impractical. Tumors exposed to the hyperstable liposome BI2536 (measured by area under the curve) were 5 times higher than exposed to citrate liposomes and 3 times higher than exposed to phospholiposomes. Drug concentrations in healthy tissues (spleen, muscle, kidney, heart and liver) were similarly elevated for the hyperstable liposomes, although this trend was not statistically significant after 10 hours (fig. 7A; table 2). Despite the higher tissue concentrations, the toxicity of the hyperstable liposomes was lower than the control liposomes, indicating that most of the BI2536 in the hyperstable liposomes were still safely encapsulated while circulating through healthy tissues. In combination with the general increase in area under the curve, this data indicates that ultrastable encapsulation increases the circulatory half-life of BI2536, thereby enhancing the filling and retention of BI2536 within the tumor lumen (fig. 7B). BI2536 (or any anti-mitotic chemotherapy) is marked by its ability to inhibit mitosis in tumors. To examine whether the higher bioavailability of BI2536 could explain the problem of differences between the ultrastable liposomes and the control, histological analysis of tumor samples was performed on xenografts 1.5 and 5.5 days after single dose treatment (fig. 7C and 7D). On day 1.5, all liposome formulations of BI2536 and encapsulated free BI2536 correlated with mitotic patterns observed in approximately 25% of tumor nuclei in tissue sections. However, by day 5.5, the ultrastable liposomal BI2536 was associated with a significantly higher proportion of mitotically hindered cells compared to the control liposomes and free drug. This extended time bioavailability is believed to be responsible for improving the efficacy of the ultrastable encapsulated BI 2536.

TABLE 2

Comparison of tissue p-values (two-tailed inequality variance t-test)

BI2536 concentration resulting from treatment with ultrastable liposomes (C: P ═ 1: 3) compared to other treatments (C: P ═ 1: 0 and C: P ═ 0: 1)

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

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Claims (17)

1. A hyperstable liposome comprising an internal environment separated from an external environment by a membrane, said internal environment comprising an antimitotic drug, one or more anions and one or more cations embedded in said internal environment, wherein said embedded antimitotic drug is released at a slow rate of less than 0.6% over 12 hours or at a slow rate of less than 5% over 8 days when said liposome is suspended in 600mM sucrose.

2. The ultrastable liposome of claim 1, wherein the one or more anions are citrate, acetate, phosphate, 2- (N-morpholino) ethanesulfonate, chloride, citrate and acetate, citrate and 2- (N-morpholino) ethanesulfonate, citrate and chloride, acetate and phosphate, acetate and 2- (N-morpholino) ethanesulfonate, acetate and chloride, phosphate and 2- (N-morpholino) ethanesulfonate, phosphate and chloride, 2- (N-morpholino) ethanesulfonate and chloride.

3. The hyperstable liposome of claim 1 or 2, wherein the two anions are present in a ratio of about 1: 7 to about 7: 1.

4. The ultrastable liposome of claim 3, wherein the one or more anions are citrate and phosphate or citrate and acetate or acetate and phosphate.

5. The ultrastable liposome of any one of claims 1-4, wherein the one or more cations are sodium, ammonium, triethylammonium, copper, magnesium, zinc, or iron.

6. The hyperstable liposome of any of claims 1-5, wherein the antimitotic drug is a polo-like kinase inhibitor, such as BI2536, ON01910, GSK 461364, HMN214, or BI 6727; kinesin spindle inhibitors, such as Ispinesib (SB 715992), SB 743921, MK 0731 or ARRY 520; or an aurora kinase inhibitor, such as MK 0457(VX 680), AZD1152, PHA 680632, PHA 739358, MLN8054, MLN8237, R763, AT9283, SNS 314, SU 6668, ENMD2076, BI 811283, CYC116, ENMD981693, or MKC 1693.

7. The hyperstable liposome of claim 6, wherein the antimitotic drug is BI2536 or Ispinesib.

8. The hyperstable liposome of any of claims 1-7, wherein the membrane comprises 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (DSPE-PEG2000), hydrogenated egg L-a-phosphatidylcholine (HEPC), and cholesterol (Chol).

9. The ultrastable liposome of claim 8, wherein the membrane comprises HEPC: Chol: DSPE-PEG2000 in a molar ratio of 50: 45: 5.

10. A liposome composition comprising the hyperstable liposome of any one of claims 1-9 in an aqueous medium.

11. A pharmaceutical composition comprising a hyperstable liposome according to any one of claims 1-9.

12. The pharmaceutical composition of claim 11, wherein the composition further comprises at least one pharmaceutically acceptable excipient and/or carrier.

13. A method of treating cancer in a subject comprising administering to a subject in need thereof a therapeutically effective amount of the hyperstable liposome of any one of claims 1-9 or the liposome composition of claim 10 or the pharmaceutical composition of claim 11 or 12.

14. Use of the hyperstable liposome of any of claims 1-9 in the manufacture of a medicament for treating cancer in a subject.

15. Use of a hyperstable liposome according to any one of claims 1 to 9 or a liposome composition according to claim 10 or a pharmaceutical composition according to claim 11 or 12 for the treatment of cancer in a subject.

16. The ultrastable liposome of any one of claims 1-9 for use in the preparation of a medicament for treating cancer in a subject.

17. The ultrastable liposome of any one of claims 1-9 or the liposome composition of claim 10 or the pharmaceutical composition of claim 11 or 12 for use in treating cancer in a subject.