HK1216010B - Engineering a control drug release profile via liposome composition in both aqueous and non-aqueous compartments - Google Patents

Description

Designing the composition of the aqueous and non-aqueous phases of liposomes to control drug release trends

Cross-reference to related applications

This application claims the benefit of U.S. Application No. 61/792,850, filed March 15, 2013, the entire disclosure of which is incorporated herein by reference.

Technical Field

The present invention relates to a pharmaceutical composition comprising at least one liposome, at least one multivalent counter ion donor or a pharmaceutically acceptable salt thereof, at least one monovalent counter ion donor or a pharmaceutically acceptable salt thereof, and a therapeutic agent. The present invention also relates to the use of the pharmaceutical composition in the manufacture of a pharmaceutical for inhibiting cancer cell growth.

Background Art

Liposomes are widely used as in vivo delivery vehicles for various therapeutic agents. Ideally, these liposomes should have high encapsulating efficiency and an extended retention profile (i.e., minimal drug release before reaching the target site).

The product is a liposomal formulation of vinorelbine, which utilizes liposomes to enhance the retention of vinorelbine before reaching its target site. While Phase 1 clinical trials demonstrated enhanced anti-cancer efficacy, the prolonged retention of vinorelbine in vivo also resulted in increased toxicity.

Therefore, there is a need to provide a liposome composition for delivering therapeutic agents with tunable retention properties that balances optimal anticancer efficacy with minimal side effects. The present invention addresses this need and other important needs.

Summary of the Invention

In one embodiment, the present invention is directed to a pharmaceutical composition comprising at least one liposome, at least one polyvalent counterion donor or a pharmaceutically acceptable salt thereof, at least one monovalent counterion donor or a pharmaceutically acceptable salt thereof, and a therapeutic agent, a derivative thereof, or a pharmaceutically acceptable salt thereof. Advantageously, the pharmaceutical composition provides tunable retention characteristics and tunable encapsulation percentage for the therapeutic agent.

In another embodiment, the present invention provides a pharmaceutical composition comprising at least one liposome having: a particle forming component selected from a phospholipid or a mixture of at least one phospholipid and cholesterol; 0.1 mM to 10 mM of a polyvalent counter ion donor or a pharmaceutically acceptable salt thereof; 150 mM to 450 mM of a monovalent counter ion donor or a pharmaceutically acceptable salt thereof; and a vinca alkaloid.

In a third embodiment, the present invention provides a pharmaceutical composition comprising at least one liposome having: a particle-forming component selected from a phospholipid or a mixture of at least one phospholipid and cholesterol; 1 milliequivalent (mEq) to 320 mEq per liter of a polyvalent counter-ion donor or a pharmaceutically acceptable salt thereof; 150 mM to 450 mM of a monovalent counter-ion donor or a pharmaceutically acceptable salt thereof; and an amphipathic therapeutic agent.

The present invention also relates to the use of the pharmaceutical composition described herein for the manufacture of a medicament for inhibiting cancer cell growth, comprising a therapeutically effective dose of the pharmaceutical composition. The present invention also relates to a method for inhibiting cancer cell growth in a subject in need thereof. The method comprises administering the pharmaceutical composition described herein, wherein the subject experiences reduced symptoms and signs of cancer. This method advantageously improves cancer cell inhibition while reducing toxicity.

It should be understood that statements containing such terms do not limit the subject matter described herein or limit the meaning or scope of the claims of the following patents. The embodiments of the invention covered by this patent are defined by the following claims, not by this Summary of the Invention. This Summary of the Invention is a high-level overview of various aspects of the invention and introduces some of the concepts further described in the following Implementation Section. This Summary of the Invention is not intended to identify the key or essential features of the claimed subject matter, nor is it intended to be used to isolate and determine the scope of the claimed subject matter. The subject matter will be understood by reference to the appropriate portions of the entire specification, any or all of the drawings, and each technical solution.

The present invention will be more easily understood with the help of the accompanying drawings and the following embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the precipitation of sodium polydextrose sulfate and vinorelbine in liposomes;

FIG2 shows the average tumor volume in the NanoVNB group, LV304 group, and normal saline (control) group;

FIG3 shows the mean survival time in the NanoVNB group, LV304 group, and normal saline (control) group;

FIG4 shows the skin toxicity scores in the NanoVNB group and the LV304 group.

DETAILED DESCRIPTION

definition

Unless otherwise indicated, the following terms when used above and in the present invention shall be understood to have the following meanings.

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, an "effective amount" includes a dosage of the pharmaceutical composition sufficient to reduce the symptoms and signs of cancer, including, but not limited to, weight loss, pain, and tumor mass, which can be measured clinically as palpable mass or radiologically via various imaging modalities.

As used herein, the term "treat" includes preventative (eg, prophylactic), palliative, and eradicative uses or results.

The terms "inhibit" and "suppress" include slowing down or stopping growth.

The term "subject" may refer to a vertebrate suffering from cancer or a vertebrate deemed in need of cancer treatment. Subjects include warm-blooded animals, such as mammals, such as primates, and more preferably humans. Non-human primates are also subjects. The term "subject" includes domesticated animals, such as cats and dogs; livestock (e.g., cattle, horses, pigs, sheep, goats); and experimental animals (e.g., mice, rabbits, rats, gerbils, guinea pigs). Thus, veterinary uses and pharmaceutical dosage forms are contemplated herein.

The term "counterion donor" includes counterion donors that can form a salt with a therapeutic agent without reducing the activity of the therapeutic agent. In one embodiment, the therapeutic agent is an amphipathic acid with a net negative charge, and the counterion donor is a cation or an entity covalently linked to one or more cationic functional groups. In another embodiment, the therapeutic agent is an amphipathic base with a net positive charge, and the counterion donor is an anion or an entity covalently linked to one or more anionic functional groups. The counterion donor has high solubility in the agent-carrying component of the liposome, but has low liposome membrane (bilayer) permeability. Therefore, the counterion donor remains in the agent-carrying component during loading of the therapeutic agent and during storage.

All numbers herein may be understood as modified by the word "about".

As used herein, the term "alkyl" refers to a straight or branched chain saturated aliphatic group having 1 to about 10 carbon atoms. Alkyl groups may include multiple carbon atoms, such as _C1-2 , _C1-3 , C1-4, _C1-5 , _C1-6 , _C1-7 , _C1-8 , _C1-9 , _C1-10 , _C2-3 , C2-4, _{C2-5, C2-6, C3-4, C3-5 , C3-6 , C4-5 , C4-6 , and C5-6} . For example, _C1-6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, and the like. Alkyl may also refer to alkyl groups having up to 20 carbon atoms, such as, but not limited to, heptyl, octyl, nonyl, and decyl.

As used herein, the term "aryl" refers to an aromatic ring system having any suitable number of ring atoms and any suitable number of rings. An aryl group can include any suitable number of ring atoms, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring atoms and 6 to 10, 6 to 12, or 6 to 14 ring members. An aryl group can be a 15-membered monocyclic ring, fused to form a bicyclic or tricyclic group, or a biaryl group connected by a bond. Representative aryl groups include phenyl, naphthyl, and biphenyl. Other aryl groups include benzyl groups with a methylene linkage. Some aryl groups have 6 to 12 ring members, such as phenyl, naphthyl, or biphenyl. Other aryl groups have 6 to 10 ring members, such as phenyl or naphthyl. Some other aryl groups have 6 to 20 ring members, such as phenyl. The "substituted aryl" may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano and alkoxy.

The "pharmaceutically acceptable salts" of the amphoteric acids of the present invention are salts formed with bases, i.e., cationic salts such as alkali metal and alkaline earth metal salts, such as sodium, lithium, potassium, calcium, magnesium, and the four ammonium salts, such as ammonium, trimethylammonium, diethylammonium and tris-(hydroxymethyl)-methyl-ammonium salts.

Similar acid addition salts, such as those of mineral acids, organic carboxylic acids, and organic sulfonic acids (eg, hydrochloric acid, methanesulfonic acid, maleic acid), are also useful for basic therapeutic agents having moieties such as pyridyl as part of the structure.

liposomes

As used herein, the term "liposome" refers to multivesicular liposomes (MVLs), multilamellar vesicles (MLVs), or small or large unilamellar vesicles (ULVs). Liposomes are nanosized and comprise a particle-forming component and an agent-carrying component. The particle-forming component forms a closed lipid barrier, and the agent-carrying component comprises a medium enclosed by the particle-forming component.

The particle-forming component may be prepared from a phospholipid or a mixture of at least one phospholipid and cholesterol. Examples of phospholipids used in the present invention include, but are not limited to, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidic acid (EPA), egg phosphatidylinositol (EPI), soy ... phosphatidylcholine, SPC), soy phosphatidylglycerol (SPG), soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidic acid (SPA), soy phosphatidylinositol (SPI), soy phosphatidylcholine (S ...yl phosphatidylinositol (SPI), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-glycero-3-phosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol (DOPG), dimyristoylphosphatidylglycerol (DMPG), hexadecylphosphocholine (HEPC), hydrogenated soy phosphatidylcholine (HPC), dioctylphosphatidylcholine (DPC), dioctylphosphatidylglycerol (DPG), dioctylphosphatidylglycerol (DMPG), hexadecylphosphocholine (HEPC), hydrogenated soy phosphatidylcholine (DPC), dioctylphosphatidylcholine (DPG ...DMPG), dioctylphosphatidylcholine (DPG), dioctylphosphatidylglycerol (DMPG), dioctylphosphatidylcholine (DPG), dioctylphosphatidylglycerol (DMPG), dioctylphosphatidylcholine (DPG), dioctylphosphatidylglycerol (DMPG), dioctylphosphatidylcholine (DPG), dioctylphosphatidylcholine (DPG), dioctylphosphatidylcholine (DPG), dioctylphosphatidylcholine (DPG), dioctylphosphatidylcholine (DPG), dioctylphosphatidylcholine (DPG), di phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylstearoylphosphatidylcholine (PSPC), palmitoylstearoylphosphatidylglycerol (PSPG), monooleoylphosphatidylethanolamine (MOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (1-palmitoyl-2-oleoyl-sn-glycero-3 -phosphatidylcholine, POPC; distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidyl acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid acid, DOPA), dimyristoylphosphatidic acid (DMPA); distearoylphosphatidic acid (DSPA), dimyristoylphosphatidylinositol (DMPI), distearoylphosphatidylinositol (DSPI) and mixtures thereof.

In one embodiment, the particle-forming component is free of fatty acids or cationic lipids (ie, lipids with a net positive charge at physiological pH).

In another embodiment, the particle forming component includes a hydrophilic polymer having a long chain of highly hydrated, flexible neutral polymer attached to a phospholipid molecule. Without being bound by any theory, the hydrophilic polymer can stabilize the liposome and prolong its circulation time in vivo. Examples of hydrophilic polymers include, but are not limited to, polyethylene glycol (PEG) having a molecular weight of about 2,000 to about 5,000 daltons, methoxy PEG (mPEG), gangsen GM ₁ , polysialic acid, polylactic acid (also known as polylactide), polyglycolic acid (also known as polyglycolide), polylactic acid polyglycolic acid, polyvinyl alcohol, polyvinyl pyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polyasparagine, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyvinyl methyl ether, polyhydroxyethyl acrylate, derivatized cellulose (such as hydroxymethyl cellulose or hydroxyethyl cellulose), and synthetic polymers.

In one set of embodiments, the phospholipid is selected from DSPC and DSPE-PEG, wherein the molecular weight of PEG is about 2,000 Daltons (hereinafter DSPE-PEG ₂₀₀₀ ).

In another group of embodiments, the molar ratio of DSPC, cholesterol, and DSPE-PEG ₂₀₀₀ is about 3:2:0.45.

The particle composition may further comprise a lipid-conjugate of an antibody or peptide as a targeting moiety, thereby enabling the liposome to specifically bind to target cells carrying a target molecule. Examples of target molecules include, but are not limited to, epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), carcinoembryonic antigen (CEA), and erbB-2/neu (HER2).

The liposome preferably has an average particle size of about 30 nm to about 200 nm, more preferably about 50 nm to about 150 nm.

The liposomes prepared in the present invention can be produced by conventional techniques for preparing vesicles. Such techniques include the ether injection method (Deamer et al., Acad. Sci. (1978) 308:250), the surfactant method (Brunner et al., Biochim. Biophys. Acta (1976) 455:322), the freeze-thaw method (Pick et al., Arch. Biochim. Biophys. (1981) 212:186), the reverse phase evaporation method (Szoka et al., Biochim. Biophys. Acta. (1980) 601:559), and the like. 71), ultrasonic treatment (Huang et al., Biochemistry (1969) 8:344), ethanol injection method (Kremer et al., Biochemistry (1977) 16:3932), extrusion method (Hope et al., Biochim. Biophys. Acta (1985) 812:5565), French compression method (Barenholz et al., FEBS Lett. (1979) 99:210), and the method described in detail in Szoka, K, Jr. et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980). All of the above processes are basic techniques for forming liposome vesicles and are incorporated herein by reference.

therapeutic agents

The therapeutic agent can be any suitable therapeutic agent. In one embodiment, the therapeutic agent is an anticancer agent. Non-limiting examples of anticancer agents include vinca alkaloids, topoisomerase inhibitors, taxane compounds, derivatives thereof, or pharmaceutically acceptable salts thereof.

Examples of vinca alkaloids include, but are not limited to, vinorelbine, vincristine, vinblastine, and vindesine.

Examples of topoisomerase inhibitors include, but are not limited to, topotecan, camptothecin, irinotecan, etoposide, and doxorubicin.

Examples of taxane compounds include, but are not limited to, paclitaxel.

Monovalent relative ion donor

In one embodiment, the therapeutic agent is a zwitterionic base with a net positive charge, and the monovalent counterion donor within the liposome can be selected from an anion or an entity covalently linked to an anionic functional group. The anion or anionic functional group has a valence of -1, -2, or -3.

Non-limiting examples of monovalent counter ion donors include benzenesulfonic acid and 4-hydroxybenzenesulfonic acid as illustrated below:

In another embodiment, a pharmaceutically acceptable salt of a monovalent counter-ion donor comprises a) an anion or an entity covalently linked to an anionic functional group; and b) one or more cations, wherein the anion or the anionic functional group forms an ion pair with the cations.

The anion or anionic functional group can be selected from one or more of the following: citrate, sulfate, sulfonate, phosphate, pyrophosphate, tartrate, succinate, maleate, borate, carboxylate, glucuronate, chloride, hydroxide, nitrate, cyanate or bromide. In one embodiment, the anion and anionic functional group are selected from one or more of the following: citrate, sulfate, sulfonate, phosphate, pyrophosphate or carboxylate.

In another embodiment, the entity to which the anionic functional group is attached can be a natural or synthetic organic or inorganic compound. Examples of entities include, but are not limited to, non-polymers such as benzene, oligonucleotides, and monosaccharides; or polymers such as polyvinyl, polyols (such as glycerol, sorbitol, and mannitol), polysaccharides, polypeptides, glycoproteins, and polynucleotides.

The cation of the pharmaceutically acceptable salt may be selected from one or more of the following: calcium ion, magnesium ion, sodium ion, potassium ion, manganese ion or NR4 ⁺ , wherein R is H or an organic group (such as an alkyl or aryl group) or a mixture thereof. In one embodiment, the cation is ammonium.

A second embodiment of the present invention provides an amphoteric acidic therapeutic agent, wherein the monovalent relative ion donor within the liposome can be selected from or include a cation or an entity covalently linked to a cationic functional group, wherein the cation or cationic functional group has a valence of +1, +2, or +3.

Pharmaceutically acceptable salts of monovalent counter-ion donors comprise a) an anion or an entity covalently linked to an anionic functional group; and b) one or more anions, wherein the cation or the cationic functional group forms an ion pair with the one or more anions.

In one embodiment, the monovalent counter ion donor is ammonium sulfate. In another embodiment, the concentration of the monovalent counter ion donor is from about 100 to about 500 mM, or any value or range therebetween in increments of 10 mM (e.g., 80 mM, 320 mM). In another embodiment, the concentration of the monovalent counter ion donor is from about 150 to about 450 mM. In another embodiment, the concentration of the monovalent counter ion donor is from about 200 mM to about 400 mM. In another embodiment, the concentration of the monovalent counter ion donor is about 300 mM.

Multivalent counter ions

In one embodiment, the therapeutic agent is a zwitterionic base and forms an insoluble salt with at least one multivalent counter-ion donor or a pharmaceutically acceptable salt thereof within the liposome.

In another embodiment, the multivalent counter-ion donor comprises an entity covalently linked to one or more anionic functional groups, wherein the anionic functional groups have a valence of -1, -2, or -3. A pharmaceutically acceptable salt of the multivalent counter-ion donor comprises a) an entity covalently linked to one or more anionic functional groups; and b) one or more cations, wherein the anionic functional groups form ion pairs with the cations.

The anion functional group of the multivalent counterion is selected from one or more of the following: citrate, sulfate, sulfonate, phosphate, pyrophosphate, tartrate, succinate, maleate, borate, carboxylate, glucuronide, chloride, hydroxide, nitrate, cyanate or bromide. In one embodiment, the anion functional group is selected from one or more of the following: citrate, sulfate, sulfonate, phosphate, pyrophosphate or carboxylate. The anion functional groups of the multivalent counterion donor can be different from each other. For example, chondroitin sulfate is a multivalent counterion donor with different anion functional groups on the same entity, as illustrated below:

The cation may be selected from one or more of calcium, magnesium, sodium, potassium, manganese, _NR4 ⁺ , wherein R is H or an organic group such as an alkyl or aryl group, and mixtures thereof. In one embodiment, the cation is ammonium.

Another embodiment of the present invention provides an amphoteric acidic therapeutic agent, wherein the polyvalent counter-ion donor within the liposome comprises an entity covalently linked to one or more cationic functional groups, wherein the cationic groups have a valence of +1, +2, or +3. The amphoteric acid forms an insoluble salt with the polyvalent counter-ion donor and is retained within the liposome.

Pharmaceutically acceptable salts of multivalent counter-ion donors comprise a) an entity covalently linked to one or more cationic functional groups; and b) one or more anions, wherein the cationic functional groups form ion pairs with the anions.

The entity of the multivalent counter-ion donor can be a natural or synthetic organic or inorganic compound. Non-limiting examples of entities include, but are not limited to: non-polymers such as benzene, oligonucleotides, and monosaccharides; or polymers such as polyvinyl, polyols (such as glycerol, sorbitol, and mannitol), polysaccharides (such as dextran and chitosan), polypeptides, glycoproteins, and polynucleotides.

In one embodiment, the multivalent counter-ion donor is selected from one or more of the following: sulfated heparin, carrageenan, mucin, sulfated hyaluronic acid, chondroitin sulfate, keratin sulfate, dermatan sulfate, or sulfated polysaccharides. Non-limiting examples of sulfated polysaccharides include polydextrose sulfate having a molecular weight of about 1,600 Daltons to about 8,000 Daltons.

In one embodiment, the pharmaceutically acceptable salt of polydextrose sulfate is selected from ammonium polydextrose sulfate or sodium polydextrose sulfate.

Pharmaceutical compositions

In one embodiment, the pharmaceutical composition of the present invention comprises a combination of: (a) at least one liposome having a particle-forming component selected from a phospholipid or a mixture of at least one phospholipid and cholesterol, (b) at least one multivalent counter ion donor or a pharmaceutically acceptable salt thereof; (c) at least one monovalent counter ion donor or a pharmaceutically acceptable salt thereof; and (d) an amphoteric therapeutic agent, a derivative thereof, or a pharmaceutically acceptable salt thereof.

In another embodiment, the pharmaceutical composition of the present invention comprises the following combination: (a) at least one liposome having a particle-forming component selected from a mixture of one or more phospholipids and cholesterol, (b) at least one multivalent counter-ion donor or a pharmaceutically acceptable salt thereof at a concentration of about 0.1 mM to about less than 10 mM; (c) at least one monovalent counter-ion donor or a pharmaceutically acceptable salt thereof at a concentration of about 150 mM to about 450 mM; and (d) a vinca alkaloid. In another embodiment, the particle-forming component further comprises a hydrophilic polymer.

Advantageously, by combining a multivalent counter ion donor or a pharmaceutically acceptable salt thereof with a monovalent counter ion donor or a pharmaceutically acceptable salt thereof, the encapsulation efficiency and/or retention characteristics of the therapeutic agent can be modulated to maintain therapeutic efficacy but minimize toxicity.

In one set of embodiments, the total valence equivalents per liter of the anionic functional groups of the multivalent counter-ion donor, or a pharmaceutically acceptable salt thereof, is from about 1 to about 160 milliequivalents (mEq), from about 3 to 160 mEq, from about 1 to about 320 mEq, from about 1 to about 250 mEq, from about 3 to about 250 mEq, from about 160 to about 250 mEq, from about 160 to about 320 mEq, or any value or range of 1 mEq increments between 1 and 320 mEq (e.g., 23 mEq, 233 mEq). In another embodiment, the anionic functional groups of the multivalent counter-ion donor, or a pharmaceutically acceptable salt thereof, are sulfate.

In another set of embodiments, the concentration of the multivalent relative ion donor or a pharmaceutically acceptable salt thereof is from about 2 mM to less than 8 mM, from about 0.1 mM to less than 8 mM, from about 0.1 mM to less than about 10 mM, from about 2 mM to less than 10 mM, or any value or range in 0.1 mM increments between 0.1 mM and 10 mM (e.g., 1.5 mM, 8.3 mM).

The pharmaceutical compositions can be formulated for any suitable route of administration, including intracranial, intracerebral, intraventricular, intrathecal, intraspinal, oral, topical, rectal, transdermal, subcutaneous, intravenous, intramuscular, intranasal, intraperitoneal, intratumoral, and the like.

The dosage of the pharmaceutical compositions of the present invention can be determined by those skilled in the art based on these examples. Unit doses and multiple doses are contemplated, each offering advantages in certain clinical settings. According to the present invention, the actual amount of the pharmaceutical composition to be administered may vary depending on the age, weight, general condition, cancer type, toxicity, and other factors of the individual being treated, and is at the discretion of the medical professional.

In some embodiments, as demonstrated in FIG1 , at least a portion of the therapeutic agent (such as vinorelbine) forms a salt with a pharmaceutically acceptable salt of a multivalent counter-ion donor and precipitates in the intraliposomal aqueous solution core.

Methods to inhibit cancer cell growth

The present invention is directed to methods of inhibiting the growth of cancer cells in a subject, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition described herein, thereby reducing symptoms and signs of cancer and/or toxicity in the subject.

The pharmaceutical compositions can be administered alone or as an adjunct to surgery, e.g., before surgery to reduce tumor size and/or after surgery to reduce the likelihood of metastasis, e.g., by inhibiting the growth and migration of circulating tumor cells through the bloodstream.

The pharmaceutical composition can be administered before, after, or simultaneously with one or more anti-cancer treatments, including conventional chemotherapeutic agents, targeted cancer therapy, or radiation therapy.

Conventional chemotherapeutic agents include DNA synthesis inhibitors, alkylating agents, antifolates, metabolic inhibitors, or combinations thereof.

Targeted cancer therapies are drugs that inhibit cancer cell growth by interfering with specific target molecules required for carcinogenesis and cancer growth, rather than simply interfering with rapidly dividing cells (such as with conventional chemotherapeutic agents). Targeted cancer therapies include the administration of kinase inhibitors, angiogenesis inhibitors, epidermal growth factor receptor (EGFR) inhibitors, HER2/neu receptor inhibitors, or a combination thereof.

Radiation therapy uses high-energy radiation to shrink tumors and kill cancer cells. Examples of radiation therapy include the administration of X-rays, gamma rays, and charged particles.

The following examples further illustrate the present invention. These examples are intended only to illustrate the present invention and should not be construed as limiting the present invention.

Example 1: Preparation of liposomes

Liposomes were prepared by solvent injection. Lipids including DSPC, DSPE-PEG ₂₀₀₀ , and cholesterol were combined at a molar ratio of 3:0.045:2 and dissolved in 99.9% ethanol in a flask at approximately 60°C. Lipid solubilization was performed using a tabletop ultrasonic bath.

The dissolved lipid solution was added to a 1.0 mM sodium phosphate solution at 100 mL/min using a peristaltic pump and the two solutions were mixed. The lipid mixture was then passed through polycarbonate membranes with pore sizes of 0.2 μm and 0.1 μm, respectively, 6-10 times. Liposomes (or large multilamellar vesicles) were formed with an average vesicle diameter of approximately 100-120 nm (measured using a Malvern ZetaSizer Nano ZS-90).

The liposome mixture was dialyzed and concentrated by a tangential flow dialysis system against 0.9% (w/w) sodium chloride and 9% (w/w) sucrose solution using a Millipore Pellicon 2 mini ultrafiltration module Biomax-100C (0.1 m ² ), and then sterilized by filtration using a 0.2 μm sterile filter membrane.

Example 2: Effect of Monovalent Relative Ion Donors on Encapsulation Efficiency and Retention Characteristics

The pharmaceutical composition was prepared by mixing the liposomes described in Example 1 with ammonium sulfate (a monovalent counter ion donor). 300 mM and 600 mM ammonium sulfate were used to create a gradient across the lipid bilayer of the liposomes, enabling remote loading of vinorelbine. The encapsulation efficiency and retention characteristics of liposomal vinorelbine were evaluated using an in vitro plasma release assay, and the results are summarized in Table 1.

Results: The data showed that ammonium sulfate could effectively load or encapsulate vinorelbine into liposomes. However, ammonium sulfate was not effective in retaining vinorelbine in the liposomes, with less than 30% of vinorelbine remaining encapsulated in the liposomes after 24 hours of plasma incubation.

Table 1. Characteristics of pharmaceutical compositions with monovalent counter ion donors

Example 3: Effect of multivalent counterion donors on encapsulation efficiency and retention characteristics

Sodium polyglucose sulfate (8,000 (8K) Daltons) was converted to ammonium polyglucose sulfate (a pharmaceutically acceptable salt of polyglucose sulfate) using a DOWEX ion exchange column. Two pharmaceutical compositions were prepared by mixing the liposomes described in Example 1 with 4 mM and 8 mM ammonium polyglucose sulfate, respectively, then actively loading approximately 2 mg of vinorelbine and incubating at approximately 60°C.

The encapsulation efficiency and retention characteristics of liposomal vinorelbine in these two pharmaceutical compositions were evaluated using an in vitro plasma release assay, and the results are summarized in Table 2.

Results: 8 mM ammonium polyglucose sulfate produced an encapsulation efficiency of 93%, while 4 mM ammonium polyglucose sulfate produced an encapsulation efficiency of less than 90%. Similarly, the LV009 formulation in Table 4 included only a multivalent counterion donor and had an encapsulation efficiency of less than 90% after 24 hours of plasma incubation, while retention was 98.74%.

Table 2. Characteristics of pharmaceutical compositions with multivalent counter ion donors

Example 4: Effect of Combinations of Monovalent and Multivalent Counter Ion Donors

In vitro studies were performed to evaluate the combination of monovalent and polyvalent counter-ion donors with respect to the retention characteristics of liposomal vinorelbine.

Liposomes prepared according to Example 1 were mixed with 300 mM ammonium sulfate (monovalent counter ion donor) and various concentrations of sodium polydextrose sulfate (multivalent counter ion donor salt).

The encapsulation efficiency and retention characteristics of liposomal vinorelbine were evaluated using a 24-hour in vitro plasma release assay, and the results are summarized in Table 3.

Results: The data showed that various combinations of monovalent and polyvalent counterion donors maintained vinorelbine encapsulation efficiency while maintaining a liposome size of approximately 100 nm. Furthermore, the retention characteristics of liposomal vinorelbine depended on the concentration of the polyvalent counterion donor. 8 mM sodium polydextrose sulfate was associated with a higher percentage of vinorelbine retention at 24 hours (78.9%) compared to 2 mM sodium polydextrose sulfate (51.8%).

Table 3. Characteristics of liposomal vinorelbine with combinations of monovalent and multivalent counterion donors

Example 5: Effects of various multivalent phases on ion-donating salts

In vitro studies were performed to evaluate the effects of different polyvalent counterion donor salts on the retention characteristics of liposomal vinorelbine.

Liposomes prepared according to Example 1 were mixed with 300 mM ammonium sulfate (AS) and two different polyvalent counterion donor salts: polydextrose sulfate (DS) sodium salt and DS ammonium salt.

The encapsulation efficiency and retention characteristics of liposomal vinorelbine were evaluated using a 24-hour in vitro plasma release assay, and the results are summarized in Table 4.

Results: The data show that the sodium and ammonium salts of polydextrose sulfate were equally effective in retaining vinorelbine in liposomes after 24 hours of plasma incubation. Furthermore, when the concentration of the polyvalent counterion donor or its salt was 10 mM, the retention characteristics of the monovalent and polyvalent counterion donor combinations (100% and 94.2% vinorelbine retained in liposomes after 24 hours) were similar to those of the polyvalent counterion donor combination (98.7% vinorelbine retained in liposomes after 24 hours). This compares to the data in Table 3, which show that the retention characteristics of liposomal vinorelbine depended on the concentration of the polyvalent counterion donor when the concentration of the polyvalent counterion donor was less than 10 mM.

Table 4. Characteristics of liposomal vinorelbine with combinations of monovalent and multivalent counter-ion donors

Example 6: Effects of various molecular weights of multivalent phase ion donors

Effect of the Molecular Weight of the Multivalent Counterion Donor on the Retention Characteristics of Liposomal Vinorelbine Liposomes prepared according to Example 1 were mixed with ammonium sulfate and with 5K and 8K polydextrose sulfate, respectively.

The encapsulation efficiency and retention characteristics of liposomal vinorelbine were evaluated using a 24-hour in vitro plasma release assay, and the results are summarized in Table 5.

Results: The total valency of the multivalent counterion donor affected the retention characteristics of liposomal vinorelbine. The data indicated that multivalent counterion donors with higher valency were associated with greater encapsulation of vinorelbine at 24 hours.

Table 5. Characteristics of liposomal vinorelbine with multivalent counterion donors of various molecular weights

Example 7: Tunable Retention Properties Using Combinations of Monovalent and Polyvalent Counter-Ion Donors

Various pharmaceutical compositions were prepared by mixing the liposomes of Example 1 with various concentrations of ammonium sulfate and polydextrose sulfate, followed by active loading of vinorelbine. The encapsulation efficiency and retention characteristics of liposomal vinorelbine were evaluated using a 24-hour in vitro plasma release assay, and the results are summarized in Tables 6 to 8.

Results in Table 6: At 72 hours, 72.2% of the encapsulated vinorelbine remained in the NanoVNB composition (the pharmaceutical composition contained only the polyvalent counterion donor, triethylammonium octasulfate). This high retention at 72 hours may contribute to toxicity, most notably skin toxicity. On the other hand, all of the encapsulated vinorelbine in the LV005 composition (the pharmaceutical composition contained only a monovalent counterion donor) was released at 72 hours, which is associated with low therapeutic efficacy. By combining monovalent and polyvalent counterion donors, a range of retention characteristics for liposomal vinorelbine was achieved. Notably, the total valence equivalent of the polyvalent counterion donor or its pharmaceutically acceptable salt was from about 1 to about 240 mEq.

Table 6. Characteristics of pharmaceutical compositions containing 100 mM and 300 mM monovalent counter ion donors and various concentrations of polyvalent counter ion donors.

Table 7: Characteristics of pharmaceutical compositions with various concentrations of monovalent counter ion donors and a fixed concentration (0.3 mM) of polyvalent counter ion donors

Results in Table 7: The encapsulation efficiency of vinorelbine was higher than 70% using 100 mM to 500 mM ammonium sulfate.

Table 8: Characteristics of pharmaceutical compositions with various concentrations of monovalent counter ion donors and a fixed concentration (0.3 mM) of polyvalent counter ion donors

Results in Table 8: Using 200 to -400 mM ammonium sulfate (monovalent relative ion donor), more than 30% of liposomal vinorelbine was retained after 24 hours of incubation.

Example 8: In vivo anti-cancer evaluation using HT-29 human colon cancer cells

In vivo anti-cancer evaluation of the LV304 pharmaceutical composition was performed using an orthotopic HT-29 human colon tumor model in mice.

Mice had free access to drinking water and food at all times during the experiment.

The study design included 3 study groups as follows:

NanoNVB group: 6 mice were administered vinorelbine in the form of NanoVNB at 25 mg/kg once daily on days 0, 3, 6, and 9 by intravenous injection.

LV304 group: 6 mice were administered vinorelbine in the form of LV304 pharmaceutical composition at 25 mg/kg by intravenous injection once a day on days 0, 3, 6, and 9.

Control group: Six mice were given intravenous injection of normal saline once a day on days 0, 3, 6, and 9.

During the study, the following outcomes were measured:

■Tumor growth percentage change (T/C%) was calculated by the following formula:

(Tumor weight _{day x} - tumor weight _{day 0} ) _treated /(tumor weight _{day x} - tumor weight _{day 0} ) _control x 100%.

■ Maximum weight change compared to day 0 weight.

■ Average tumor doubling time (TDT). It is widely used to quantify tumor growth rate and is calculated by the following formula:

(Day x - Day 0)

■ Day x is the time at which the tumor volume doubles compared to the stage size.

■ Skin toxicity score, evaluated and graded based on the parameters listed in Table 9.

Table 9. Skin toxicity scores

result:

Table 10 shows that the percentage change in tumor growth (T/C%) on day 8 was similar between the NanoVNB and LV304 groups (-41.0% for NanoVNB and -42.4% for LV304). The mean tumor doubling time (mean TDT) was >78 days in the NanoVNB group, 67.1 days in the LV304 group, and 7.6 days in the control group. In addition, mice receiving LV304 showed fewer side effects (less weight loss and lower skin toxicity scores) compared to mice receiving NanoVNB.

Figure 2 shows the mean tumor volumes in the NanoVNB, LV304, and saline (control) groups. The results indicate that the mean tumor volumes in the NanoVNB and LV304 groups were less than 200 mm ³ throughout the study, whereas the mean tumor volume in the control group exceeded 3000 mm ³ on day 40.

These results indicate that LV304 is an effective anti-cancer therapeutic agent and exhibits fewer side effects compared to NanoVNB.

Table 10. Anticancer evaluation of NanoVNB, LV304, and saline in the HT-29 human colon cancer model

*Number of days after day 0

Example 9: In vivo anti-cancer evaluation using the PC14PE6/AS2 human lung adenocarcinoma orthotopic model

In vivo anti-cancer evaluation of the LV304 pharmaceutical composition was performed using an orthotopic PC14PE6/AS2 lung tumor model in mice.

The study design included 3 study groups as follows:

NanoVNB group: 6 mice were given a single intravenous injection of 50% of the maximum tolerated dose (MTD) of NanoVNB on day 0 (1/2 MTD = 7.5 mg/kg vinorelbine).

LV304 group: 6 mice were given a single intravenous injection of LV304 pharmaceutical composition at 50% of the MTD on day 0 (1/2 MTD = 10 mg/kg vinorelbine).

Control group: Six mice were given a single intravenous injection of normal saline on day 0.

During the study, the following outcomes were measured:

■ Maximum weight change compared to day 0 weight.

■Average survival time.

Results: Referring to Table 11, the average survival time of mice after a single NanoVNB injection was 33.8 days, after a single LV304 injection was 34.2 days, and after a single saline injection was 21.4 days. Figure 3 shows that the survival time in the NanoVNB and LV304 groups was significantly longer than that in the saline (control) group (p < 0.01).

Table 11. Anticancer evaluation of NanoVNB, LV304, and saline in the PC14PE6/AS2 human lung adenocarcinoma orthotopic model

Treatment group Maximum BW change % (day)* Mean survival time ± SD (days) Normal saline -3.6(18) 21.4±2.0 NanoVNB -12.6(9) 33.8±6.6 LV304 -17.0(9) 34.2±4.8

*Days after drug administration on Day 0

Example 10: In vivo skin toxicity assessment using the SCID mouse model

In vivo dermal toxicity assessment of the LV304 pharmaceutical composition was conducted using BALB/c mice. Mice had free access to drinking water and food during the study and were randomly divided into three study groups as follows:

NanoVNB group: 6 mice received 7.5 mg/kg vinorelbine in the form of NanoVNB on days 0 and 9, and 5 mg/kg vinorelbine on days 3 and 6 via daily intravenous injection.

LV304 group: 6 mice received 7.5 mg/kg vinorelbine as LV304 on days 0 and 9 and 5 mg/kg vinorelbine on days 3 and 6 via daily intravenous injection.

Control group: 6 mice received intravenous injection of normal saline once a day on days 0, 3, 6, and 9.

During the study, skin toxicity was assessed and scored based on the grading system in Table 9.

Results: The skin toxicity scores in the NanoVNB and LV304 groups are shown in Figure 4. On day 60 of the trial, the skin toxicity in the LV304 group was significantly less than that in the NanoVNB group.

When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and subcombinations of specific embodiments of the range are contemplated.

The disclosures of each patent, patent application, and publication cited or described herein are incorporated by reference in their entirety.

Those skilled in the art will appreciate that many changes and modifications may be made to the preferred embodiments of the present invention without departing from the spirit of the present invention. Therefore, it is intended that the appended claims cover all such equivalent variations that fall within the true spirit and scope of the present invention.

Claims (10)

1. A pharmaceutical composition comprising: (a) at least one liposome, said liposome having a particle-forming component selected from (i) phospholipids and (ii) at least one mixture of phospholipids and cholesterol; (b) Polydextrose sulfate or its pharmaceutically acceptable salts at a concentration of 0.1 mM to 3 mM; (c) Ammonium sulfate with a concentration of 100 mM to 500 mM; and (d) Vinca alkaloid. 2. The pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable salt of the polydextrose sulfate is a group consisting of: (i) a polydextrose sulfate ion; and (ii) a cation, wherein the polydextrose sulfate ion and the cation form an ion pair. 3. The pharmaceutical composition of claim 2, wherein the cation comprises at least one cation selected from the group consisting of: calcium ions, magnesium ions, sodium ions, potassium ions, manganese ions, and NR ₄₊ and mixtures ^thereof , wherein R is H or an organic group. 4. The pharmaceutical composition of claim 3, wherein the cation is an ammonium ion. 5. The pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable salt of the polydextrose sulfate is sodium polydextrose sulfate or ammonium polydextrose sulfate. 6. The pharmaceutical composition of claim 1, wherein the polydextrose sulfate has a molecular weight of 1,600 Daltons to 8,000 Daltons. 7. The pharmaceutical composition of claim 1, wherein the concentration of the ammonium sulfate is from 150 mM to 450 mM. 8. A pharmaceutical composition comprising: (a) A liposome having a particle-forming component selected from phospholipids or a mixture of at least one phospholipid and cholesterol; (b) 0.1 mM to 3 mM polydextrose sulfate or a pharmaceutically acceptable salt thereof; (c) 150 mM to 450 mM ammonium sulfate; and (d) Vinca minor alkaloids. 9. The pharmaceutical composition of claim 8, wherein the liposome further comprises a hydrophilic polymer. 10. Use of the pharmaceutical composition according to any one of claims 1 to 9 in the preparation of a medicament for inhibiting cancer cell growth.