Liposome compositions for delivery of compounds and methods thereof

Abstract

The present disclosure provides liposomal particles comprising one or more active pharmaceutical ingredients (APIs) comprising at least one poorly water-soluble API located in an aqueous phase compartment inside the particle, pharmaceutical compositions thereof, and methods of preparing the same, and methods of preparing the same. And the use of the drug-loaded liposome and the pharmaceutical composition thereof for treating patients (including various types of cancer patients) to achieve a synergistic treatment effect. Also disclosed are therapeutic kits, dosage forms, and methods of preparing the drug-loaded liposomes and pharmaceutical compositions thereof.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of U.S. Provisional Patent Application No. 63 / 416,483, filed on October 14, 2022, and the entire disclosure thereof is incorporated herein by reference in accordance with 35 USC § 119(e). Technical field

[0003] The present invention relates to liposome - based drug delivery systems and methods thereof, which are particularly suitable for delivering poorly water - soluble drug molecules, such as various anticancer drugs. Background art

[0004] In nanoparticle - based drug delivery systems, liposomes are a widely used drug carrier with many unique properties, including: (1) prolonged circulation half - life of the carrier - mediated drug; (2) reduced non - specific tissue uptake; (3) increased accumulation at solid tumor or inflammatory sites through the enhanced permeability and retention (EPR) effect; (4) improved delivery specificity and active targeting by surface modification with targeting agents; (5) mainly absorbed through endocytosis, potentially bypassing the multi - drug resistance mechanism; (6) a single delivery system carrying multiple drugs in the same carrier can lead to pharmacokinetic synchronization and control of each drug, thus enhancing the therapeutic effect through drug synergy; (7) the ability to adjust their relative proportions according to the pharmacological distribution of each compound; (8) improved drug solubility and bioavailability; (9) sustained drug release characteristics (Mamot, C., et al., Drug Resist. Updates, 2003, 6: 271 - 279).

[0005] To achieve an ideal high drug - to - lipid mass ratio and high drug encapsulation efficiency, remote loading or active loading techniques have been developed to encapsulate drug compounds in liposomes. For drug remote loading, a transmembrane ion gradient is first generated by encapsulating a trapping agent within the aqueous core of the liposome. During remote loading, amphiphilic drugs diffuse through the bilayer lipid membrane into the aqueous vesicle inner space. Once inside the liposome, the drug interacts with the pre - loaded trapping agent to form a precipitate, aggregate, or gelated complex that prevents membrane re - permeation, thus resulting in drug accumulation within the vesicle (Hood, R., et al., Lab Chip, 2014, 14: 3359 - 67).

[0006] Although remote loading can be used for efficient loading of small molecule drugs, the payload must have sufficient water solubility to achieve an ideal loading effect. Remote loading of compounds with poor water solubility has been a long-standing technical problem in this field. Hayes et al. explored the use of aprotic solvents (such as dimethyl sulfoxide (DMSO)) as solubilizing enhancers to increase the water solubility of compounds with poor water solubility for remote loading (Hayes, M., et al., US10004759B2). However, only a slight improvement in drug encapsulation efficiency can be achieved by this method. In addition, aprotic solvents such as DMSO may cause toxicity problems and also have an adverse effect on the physical stability of liposomes. Therefore, to date, it is still very challenging to efficiently load compounds with poor water solubility into liposomes through a remote loading process with a high drug-to-lipid mass ratio and high encapsulation efficiency. Summary of the Invention

[0007] The present invention provides a liposome composition encapsulating one, two or more drug compounds within its aqueous core, and wherein at least one compound is a drug with poor water solubility.

[0008] The present invention also provides a method for loading a compound with poor water solubility into liposomes at a high drug-to-lipid mass ratio and achieving a high drug encapsulation efficiency. The method generally comprises the following steps: First, a solubilizer is used to increase the solubility and concentration of the compound with poor water solubility in an external (i.e., outside the liposome) aqueous medium; then, a remote drug loading method is adopted to efficiently encapsulate the compound within the aqueous core of the liposome.

[0009] To remotely load poorly soluble weakly amphiphilic bases, a pH gradient needs to be established across the lipid membrane. Surprisingly, we observed that the pH value of the external medium of the liposome needs to be lower than that of the internal medium. This is completely opposite to the conventional pH gradient method reported previously for remote loading of weak bases (Madden, D., Lipid Chemistry and Physics, 1990, 53:37 - 46), in which the pH value of the external medium is higher than that of the internal medium.

[0010] On the one hand, the present invention provides drug-loaded liposome particles comprising an inner core and an outer lipid bilayer membrane, wherein the lipid bilayer membrane comprises an inner layer having an inner surface surrounding the inner core and an outer layer forming the outer surface of the liposome particle; and the inner core contains an aqueous liquid medium and one or more active pharmaceutical ingredients encapsulated by the bilayer membrane, wherein at least one active pharmaceutical ingredient has poor water solubility.

[0011] On the other hand, the present invention provides a pharmaceutical composition comprising the drug-loaded liposome particles of any embodiment of the present invention and a liposome dispersion liquid medium.

[0012] In another aspect, the present invention provides a method of treating a subject in need of treatment with a therapeutic agent, the method comprising administering to the subject a therapeutically effective amount of a drug-loaded liposomal particle or a pharmaceutical composition thereof according to any of the embodiments disclosed herein.

[0013] In another aspect, the present invention provides a method for preparing liposomes loaded with one or more active pharmaceutical ingredients, said active pharmaceutical ingredients comprising at least one active pharmaceutical ingredient having poor water solubility.

[0014] In another aspect, the present invention provides a therapeutic kit comprising a drug-loaded liposomal particle according to any of the embodiments disclosed herein, or a pharmaceutical composition comprising a drug-loaded liposomal particle according to any of the embodiments disclosed herein.

[0015] In an exemplary embodiment for the active loading of a poorly soluble weak amphiphilic base, the pH of the external medium is lower than the pH of the internal medium by 8.0, 7.0, 6.0, 5.0, 4.0, 3.0, 2.0, 1.0, 0.5, 0.25 or 0.1 units. In one embodiment, the pH of the internal medium is between 5.0 and 10.0, and the pH of the external medium is between 2.0 and 5.0.

[0016] In an exemplary embodiment, the present invention provides a pharmaceutical formulation containing liposomes having a membrane encapsulating an aqueous compartment. Encapsulated within the aqueous compartment are a remote loading trap agent and one, two or more drug compounds, and wherein at least one of the compounds has poor water solubility. In various embodiments, about 60%, about 70%, about 90%, about 95% or about 99% of each compound is encapsulated within the aqueous compartment of the liposome.

[0017] In another exemplary embodiment, the present invention provides a liposomal composition containing two or more therapeutic agents, including liposomes stably bound to those encapsulated compounds, and a drug-to-drug molar ratio of those encapsulated compounds that exhibits a non-antagonistic effect on the relevant cells or tumor homogenates.

[0018] In an exemplary embodiment, drug-loaded liposomes can be prepared according to the following steps:

[0019] a) forming a lipid dispersion in a solution containing a trap agent and optionally a buffer;

[0020] b) reducing the liposome particle size at an elevated temperature;

[0021] c) substantially removing the trap agent outside the liposomes to obtain drug-free liposomes;

[0022] d) Dissolve the active pharmaceutical ingredient in an aqueous solution in the presence of a cosolvent. When loading two or more drugs, at least one drug compound has poor water solubility;

[0023] e) Load the drug to form drug-loaded liposomes;

[0024] f) Optionally, remove the unloaded drug and solubilizer outside the liposomes; and

[0025] g) Optionally, form a dry form of the liposome product by lyophilization.

[0026] The technology proposed in the present invention content expands the range of functional drug compounds that can be encapsulated in liposomes. Other aspects and advantages will be better understood in combination with the drawings, detailed description, examples and claims. Brief Description of the Drawings

[0027] Figure 1 Shows the particle size distribution of drug-loaded liposomes characterized by dynamic light scattering. Using TEA-SOS as the scavenger and sodium salt of SBE-β-CD as the solubilizer for poorly soluble drugs. (A) Carfilzomib liposomes (B) Ceritinib liposomes (C) Dasatinib liposomes (D) Afatinib / dasatinib co-loaded liposomes (E) Doxorubicin / carfilzomib co-loaded liposomes and (F) Dasatinib / ceritinib co-loaded liposomes.

[0028] Figure 2A 、 2B And Figure 2C shows the in vitro evaluation of the synergistic effect of combined drug combinations on different types of cancer cells. The representative figure is the relationship between the combination index (CI) value and the cell growth inhibition rate (represented by Fa) at different drug molar ratios, where CI values less than 1, approximately equal to 1, and greater than 1 indicate synergistic effect, additive effect, and antagonistic effect, respectively. Figure 2A : Effect of the combination of afatinib (AFA) and dasatinib (DAS) on the HCC-827 non-small cell lung cancer cell line. Figure 2B : Effect of the combination of dasatinib (DAS) and ceritinib (CER) on the HCC-827 non-small cell lung cancer cell line. Figure 2C : Use of carfilzomib (CAR) and doxorubicin (DOX) in combination for the H929 myeloma cell line.

[0029] Figure 3A And 3B Shows the morphological characterization of carfilzomib-loaded liposomes using cryogenic transmission electron microscopy (cryo-TEM). Figure 3A : Low magnification image; Figure 3B : High magnification image.

[0030] Figure 4Shows the pharmacokinetic (PK) curves of free carfilzomib solution and carfilzomib-loaded liposomes in BALB / c mice. Specific embodiments

[0031] When using liposomes to deliver a compound for treatment, it is generally desirable to load a high concentration of drug molecules in the liposomes to obtain a high drug-to-lipid mass ratio. Because this can reduce the amount of liposomes required for each treatment, thereby achieving the desired therapeutic effect of the compound. In addition, since some lipids used to prepare liposomes can produce adverse toxicities when the dose reaches a certain threshold level, using a higher drug-to-lipid mass ratio can avoid such adverse reactions. In addition, it is also highly desirable for liposomal drug products to have a high drug encapsulation efficiency, because it is well known that liposomal formulations can significantly change the pharmacokinetic characteristics of compounds, thereby enhancing the therapeutic effect, reducing drug-related toxic effects and increasing the overall therapeutic index.

[0032] First, solubilizers can be used to increase the concentration of poorly soluble compounds in the external aqueous medium of liposomes, and then a remote drug loading method can be used to effectively encapsulate the compounds in the aqueous core of liposomes, thereby increasing the drug-to-lipid mass ratio and drug encapsulation efficiency of poorly soluble compounds in liposomal formulations.

[0033] The present disclosure provides liposomes that encapsulate one, two or more drug compounds in their aqueous core, and wherein at least one compound is a poorly soluble drug. The present disclosure provides methods for preparing such liposomes, formulations containing such liposomes, and methods for preparing the liposomal formulations of the present disclosure.

[0034] In one aspect, the present invention provides drug-loaded liposomal particles that include an inner core and an outer lipid bilayer membrane, wherein the lipid bilayer membrane includes an inner layer having an inner surface surrounding the inner core and an outer layer forming the outer surface of the liposomal particle; and the inner core contains an aqueous liquid medium and one or more active pharmaceutical ingredients encapsulated by the bilayer membrane, wherein at least one active pharmaceutical ingredient has poor water solubility.

[0035] In some embodiments, the drug-loaded liposomal particles comprise an inner core and an outer lipid bilayer membrane, wherein the lipid bilayer membrane comprises an inner layer having an inner surface surrounding the inner core and an outer layer forming the outer surface of the liposomal particle; wherein the inner core contains an aqueous liquid medium and one or more active pharmaceutical ingredients encapsulated by the bilayer membrane, wherein at least one of the active pharmaceutical ingredients has poor water solubility; wherein the aqueous liquid medium of the inner core contains a trapping agent and optionally a buffer; and wherein the average particle size of the drug-loaded liposomal particles is between 10 nm and 450 nm, optionally between 25 nm and 300 nm or between 50 nm and 200 nm.

[0036] In some embodiments, in the drug-loaded liposome particles, the lipid bilayer membrane comprises:

[0037] a) Phospholipids selected from phosphatidylcholine (e.g., HSPC, DSPC, DPPC, and DMPC), phosphatidylglycerol (e.g., DSPG, DPPG, and DMPG), phosphatidylinositol, glyceroglycolipids, glycosphingolipids (e.g., sphingomyelin), and combinations thereof, wherein the amount of phospholipids is at least 10 mol% of the total lipids present in the liposome particles;

[0038] b) Cholesterol or its derivatives, the content of which is 5 mol% to 50 mol% of the total lipids present in the liposome particles;

[0039] And

[0040] c) Conjugated lipids that inhibit liposome aggregation, the amount of which is 0 mol% to 10

[0041] mol%, sometimes preferably 0.1 mol% to 10 mol%, sometimes preferably 1 mol% to 10 mol%, sometimes preferably 2 mol% to 8 mol%, sometimes preferably 3 mol% to 6 mol% of the total lipids present in the liposome particles.

[0042] In some embodiments, in the drug-loaded liposome particles, the conjugated lipids that inhibit liposome aggregation include polyethylene glycol (PEG)-lipid conjugates.

[0043] In some embodiments, in the drug-loaded liposome particles, the average molecular weight of PEG is in the range of about 1,500 daltons to about 2,500 daltons.

[0044] In some embodiments, in the drug-loaded liposome particles, the average molecular weight of PEG is about 2,000 daltons.

[0045] In some embodiments, in the drug-loaded liposome particles, the PEG-lipid conjugate is mPEG2000-DSPE or PEG2000-DMG.

[0046] In some embodiments, in the drug-loaded liposome particles, the liquid medium in the core contains a trapping agent and no buffer.

[0047] In some embodiments, in the drug-loaded liposome particles, the liquid medium in the core contains a trapping agent and a buffer.

[0048] In some embodiments, in the drug-loaded liposome particles, the buffer is selected from acetic acid, citric acid, histidine, HEPES, lactic acid, succinic acid, phosphoric acid, tris(hydroxymethyl)aminomethane (Tris), and their salts.

[0049] In some embodiments, in the drug-loaded liposome particles, the trapping agent is selected from ammonium sulfate; ammonium salts or substituted ammonium salts of polyanionic sulfobutylether cyclodextrins; ammonium salts or substituted ammonium salts of polyanionic sulfated carbohydrates; ammonium salts or substituted ammonium salts of polyphosphoric acid; metal salts; and combinations thereof.

[0050] The ammonium salts of polyanionic sulfobutylether cyclodextrins are selected from TEA-SBE-α-cyclodextrin, TEA-SBE-β-cyclodextrin, TEA-SBE-γ-cyclodextrin, Tris-SBE-α-cyclodextrin, Tris-SBE-β-cyclodextrin, Tris-SBE-γ-cyclodextrin; the ammonium salts of polyanionic sulfated carbohydrates are selected from TEA-SOS, Tris-SOS; the ammonium salts of polyphosphoric acid are selected from triethylammonium inositol hexaphosphate, tris(hydroxymethyl)aminomethane inositol hexaphosphate; the metal salts are selected from acetates, carbonates, citrates, halides, sulfates, gluconates of calcium, copper, zinc, magnesium, manganese, nickel, cobalt.

[0051] In some embodiments, the average particle size of the drug-loaded liposome particles is between 10 nm and 450 nm, sometimes preferably between 25 nm and 300 nm, and sometimes preferably between 50 nm and 200 nm.

[0052] In some embodiments, in the drug-loaded liposome particles, the active pharmaceutical ingredient is selected from afatinib, abemaciclib, abiraterone, acalabrutinib, alectinib, amitinib, alpelisib, anlotinib, apatinib, avapritinib, axitinib, baricitinib, belinostat, binimetinib, bortezomib, bosutinib, brigatinib, bupivacaine, cabozantinib, capecitabine, carfilzomib, crizotinib, ceritinib, cobimetinib, copanlisib, crizotinib, dabrafenib, dacomitinib, dasatinib, delanzomib, docetaxel, doxorubicin, duvelisib, enasidenib, encorafenib, entrectinib, erdafitinib, erlotinib, everolimus, fedratinib, fostamatinib, fruquintinib, gefitinib, gemcitabine, gilteritinib, glasdegib, icotinib, ibrutinib, idarubicin, idelalisib, imatinib,ivosidenib, isatuximab, ixazomib, lapatinib, larotrectinib, lenalidomide, lenvatinib, lorlatinib, marizomib, mitotane, mitoxantrone, neratinib, nidasudil, nilotinib, nintedanib, niraparib, olaparib, oprozomib, osimertinib, paclitaxel, palbociclib, panobinostat, pazopanib, pemetrexed, pemigatinib, pexidartinib, ponatinib, pralsetinib, quizartinib, radotinib, regorafenib, ribociclib, ripretinib, rivastigmine, romidepsin, rucaparib, ruxolitinib, selpercatinib, selumetinib, sirolimus, sonidegib, sorafenib, sunitinib, talazoparib, tazemetostat, temsirolimus, tepotinib, tivozanib, tofacitinib, topotecan, trametinib, tucatinib, tucidinostat, upadacitinib, vandetanib, vemurafenib, venetoclax, vinorelbine, vismodegib, vorinostat, zanubrutinib and their free bases, medicinal salts, derivatives and mixtures.

[0053] In some embodiments, in the drug-loaded liposome particles, the active pharmaceutical ingredient is selected from:

[0054] a) Carfilzomib encapsulated alone;

[0055] b) Dasatinib encapsulated alone;

[0056] c) Ceritinib encapsulated alone;

[0057] d) Carfilzomib and doxorubicin encapsulated together;

[0058] e) Dasatinib and ceritinib encapsulated together;

[0059] f) Afatinib and dasatinib encapsulated together;

[0060] g) Carfilzomib and doxorubicin in a molar ratio of about 1:50 to about 1:1000;

[0061] h) Dasatinib and ceritinib in a molar ratio of from about 30:1 to about 1:30; and

[0062] i) Afatinib and dasatinib in a molar ratio of from about 30:1 to about 1:30.

[0063] In some embodiments, the core of the drug-loaded liposome particles co-encapsulates two or more (sometimes preferably two or three, and sometimes more preferably two) active pharmaceutical ingredients. When two or more (such as two or three) active pharmaceutical ingredients are encapsulated, at least one (sometimes preferably two or three) of the pharmaceutical ingredients has poor water solubility.

[0064] On the other hand, the present invention provides a pharmaceutical composition comprising the drug-loaded liposome particles of any embodiment of the present invention and a liposome dispersion liquid medium.

[0065] In some embodiments, in the pharmaceutical composition, the liposome dispersion liquid medium comprises water, a buffer, and an osmotic pressure regulator.

[0066] In some embodiments, in the pharmaceutical composition, two or more active pharmaceutical ingredients are co-encapsulated in the core of the liposome particles and can be released to act synergistically, thereby exerting efficacy.

[0067] In some embodiments, in the pharmaceutical composition, the synergistic manner includes that after the pharmaceutical composition is administered to a subject, the active pharmaceutical ingredients can maintain a synergistic molar ratio in the blood for at least one hour.

[0068] In some embodiments, in the pharmaceutical composition, the synergistic molar ratio is such a molar ratio that when this ratio is provided to cancer-related cancer cells in an in vitro assay, within a drug concentration range where the cell growth inhibition ranges from about 0.20 to about 0.80 (i.e., the fraction of affected cells ranges from about 20% to about 80%), it exhibits at least a 20% synergistic effect within the cell growth inhibition range.

[0069] In some embodiments, in the pharmaceutical composition, the two active pharmaceutical ingredients are in a molar ratio ranging from about 1000:1 to about 1:1000, sometimes preferably from 500:1 to 1:500, sometimes more preferably from 100:1 to 1:100, sometimes more preferably from 50:1 to 1:50, sometimes more preferably from 10:1 to 1:10, sometimes more preferably from 5:1 to 1:5, sometimes more preferably from 2:1 to 1:2.

[0070] In some embodiments, in a pharmaceutical composition, the buffer in the liposome dispersion liquid medium is selected from acetic acid, citric acid, histidine, HEPES, lactic acid, succinic acid, phosphate, tromethamine (Tris), and their salts; the osmotic pressure regulator in the liposome dispersion liquid medium is selected from sucrose, glucose, mannitol, trehalose, and sodium chloride.

[0071] In some embodiments, in a pharmaceutical composition, the pH of the liposome dispersion liquid medium ranges from 5.0 to 10.0, sometimes preferably from 6.0 to 8.0, sometimes more preferably from 6.5 to 7.5, sometimes more preferably from 6.8 to 7.2, and sometimes even more preferably 7.0.

[0072] In another aspect, the present invention provides a method for treating a subject in need of treatment with a therapeutic agent, the method comprising administering to the subject a therapeutically effective amount of a drug-loaded liposome particle or a pharmaceutical composition thereof according to any of the embodiments disclosed herein.

[0073] In some embodiments, in a treatment method, the subject is a cancer patient in need of treatment with two or more cancer drugs in a synergistic manner.

[0074] In some embodiments, the subject is a cancer patient in need of treatment with two or more cancer drugs in a synergistic manner; wherein the cancer is bladder cancer (including accelerated and metastatic bladder cancer), breast cancer (e.g., estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer; HER-2 positive breast cancer; HER-2 negative breast cancer, progesterone receptor-positive breast cancer, progesterone receptor-negative breast cancer; estrogen receptor-negative, HER-2 negative, and progesterone receptor-negative breast cancer (i.e., triple-negative breast cancer); inflammatory breast cancer), colon cancer (including colorectal cancer), kidney cancer (e.g., transitional cell carcinoma), liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer, lung adenocarcinoma and squamous cell carcinoma). Genitourinary tract, such as ovarian cancer (including fallopian tube cancer and peritoneal cancer), cervical cancer, prostate cancer, testicular cancer, kidney cancer and ureteral cancer, lymphatic system cancer, rectal cancer, laryngeal cancer, pancreatic cancer (including pancreatic exocrine cancer), esophageal cancer, gastric cancer, gallbladder cancer, thyroid cancer, skin cancer (including squamous cell carcinoma), brain cancer (including glioblastoma multiforme), head and neck cancer (e.g., occult primary cancer), and soft tissue cancer (e.g., Kaposi's sarcoma (e.g., AIDS-related Kaposi's sarcoma), leiomyosarcoma, angiosarcoma, and histiocytoma).

[0075] In some embodiments, the cancer is selected from multiple myeloma, chronic myeloid leukemia, lung cancer (including small cell lung cancer and non-small cell lung cancer), lung adenocarcinoma, and squamous cell carcinoma.

[0076] In another aspect, the present invention provides a method for preparing liposomes loaded with one or more active pharmaceutical ingredients, comprising the following steps:

[0077] a) Prepare a lipid dispersion in a solution containing a trapping agent and optionally a buffer to form a suspension containing liposome particles;

[0078] b) Reduce the liposome particle size by heating the suspension to a high temperature (50 °C or above);

[0079] c) Remove the trapping agent in the suspension substantially outside the liposomes to obtain drug-free liposomes;

[0080] d) Dissolve one or more active pharmaceutical ingredients (APIs) in an aqueous solution in the presence of a solubilizer to obtain an API solution;

[0081] e) Incubate the drug-free liposomes in step c) with the API solution including the solubilizer in step d) at an elevated temperature (50 °C or above), thereby forming liposome particles, which liposome particles include an aqueous core loaded with one or more APIs, and the API is wrapped by a lipid bilayer membrane, wherein the drug-loaded liposome particles are suspended in an external liquid medium;

[0082] f) Optionally, further remove the unloaded drug molecules and solubilizer outside the liposome particles obtained in step e) by dialysis, ultracentrifugation or / and size exclusion chromatography; and

[0083] g) Optionally, form dry liposome particles loaded with one or more APIs by lyophilizing the liposome particles obtained in step e) or step f).

[0084] In some embodiments, in step a), the solution includes a trapping agent and does not include a buffer, such that in the prepared drug-loaded liposome particles, the liquid medium in the core includes the trapping agent and does not include a buffer.

[0085] In some embodiments, in step a), the solution contains both a trapping agent and a buffer, such that in the prepared drug-loaded liposome particles, the liquid medium in the core contains both the trapping agent and the buffer.

[0086] In some embodiments, in the method for preparing liposomes loaded with one or more active pharmaceutical ingredients, the solubilizer is selected from cyclodextrin and its derivatives, polyvinylpyrrolidone, polyethylene glycol and its derivatives, sorbitol, nonionic surfactants and combinations thereof.

[0087] In some embodiments, in the method for preparing liposomes loaded with one or more active pharmaceutical ingredients, the solubilizer is sulfobutyl ether-β-cyclodextrin or hydroxypropyl-β-cyclodextrin, or a salt thereof.

[0088] In some embodiments, in a method for preparing liposomes loaded with one or more active pharmaceutical ingredients, step e) of incubating the unloaded liposomes with a drug solution results in at least 50% of the total API being encapsulated within the aqueous core of the liposome particles and less than 50% of the total API molecules being present in the external liquid medium.

[0089] In some embodiments, in a method for preparing liposomes loaded with one or more active pharmaceutical ingredients, at the start of drug loading step e), the pH of the liquid medium of the liposome core is in the range of about 5.0 to about 10.0, and the pH of the external medium outside the liposome particles is in the range of about 2.0 to about 5.0.

[0090] In some embodiments, the method includes step f) of removing unloaded drug molecules and solubilizers outside the liposome particles obtained in step e) by dialysis, ultracentrifugation, or / and size exclusion chromatography; and step g) of forming dry liposome particles loaded with one or more APIs by lyophilizing the liposome particles obtained in step e) or step f). This process produces "dry" liposome particles loaded with APIs.

[0091] In some embodiments, the method only includes step f), that is, removing unloaded drug molecules and solubilizers outside the liposome particles obtained in step e) by dialysis, ultracentrifugation, or / and size exclusion chromatography, without including step g), that is, forming dry liposome particles loaded with one or more APIs by lyophilizing the liposome particles obtained in step e) or step f). This method can produce "wet" liposome particles loaded with APIs.

[0092] On the other hand, the present invention provides a therapeutic kit, including a first container containing a plurality of drug-loaded liposome particles according to any of the embodiments disclosed herein, and a second container containing a liposome dispersion liquid medium, wherein the drug-loaded liposome particles and the liposome dispersion liquid medium can be mixed in the first container or the second container to form a dispersion for administration to a subject in need of treatment; or, optionally, including a container containing a liposome pharmaceutical composition according to any of the embodiments disclosed herein for administration to a subject in need of treatment.

[0093] In some embodiments, sometimes preferably, the therapeutic kit includes a first container containing a plurality of drug-loaded liposome particles according to any of the embodiments disclosed herein, and a second container containing a liposome dispersion liquid medium, wherein the drug-loaded liposome particles and the liposome dispersion liquid medium can be mixed in the first container or the second container to form a liposome dispersion ready to be administered to a subject in need of treatment.

[0094] In some embodiments, and sometimes preferably, a therapeutic kit comprises a separate container that contains a liposomal pharmaceutical composition according to any of the embodiments disclosed herein and is ready for use in a subject in need of treatment. The liquid pharmaceutical composition can be prepared directly by a liposome drug-loading process according to any of the embodiments disclosed herein, or by mixing isolated drug-loaded liposome particles (wet or dry) with a liposome dispersion liquid medium.

[0095] In some embodiments, in the therapeutic kit, the liposome dispersion liquid medium comprises water, a buffer, and an osmotic pressure regulator.

[0096] In some embodiments, in the therapeutic kit, the buffer of the liposome dispersion liquid medium is selected from acetic acid, citric acid, histidine, HEPES, lactic acid, succinic acid, phosphates, tromethamine (Tris), and their salts; and the osmotic pressure regulator is selected from sucrose, glucose, mannitol, trehalose, and sodium chloride.

[0097] In some embodiments, in the therapeutic kit, the pH value of the liposome dispersion liquid medium is from 5.0 to 10.0, preferably from 6.0 to 8.0.

[0098] In some embodiments, the therapeutic kit further comprises a syringe and / or a needle suitable for administering the liposome dispersion to a subject.

[0099] In some embodiments, the therapeutic kit further contains instructions for mixing the drug-loaded liposome particles with the liposome dispersion liquid medium for administering the mixture to a subject in need of treatment.

[0100] Those skilled in the art should understand that the present invention encompasses any and all reasonable combinations of any two or more of the embodiments described in various aspects of the present invention.

[0101] In an exemplary embodiment, the present invention provides a pharmaceutical formulation comprising liposomes having a membrane encapsulating an aqueous compartment. The aqueous compartment encapsulates a remote loading trap, an optional buffer, and one, two, or more drug compounds, and wherein at least one of the compounds has poor water solubility. In various embodiments, about 50%, about 70%, about 90%, about 95%, or about 99% of each compound is encapsulated in the aqueous compartment of the liposome.

[0102] In an exemplary embodiment, the present invention provides a pharmaceutical composition wherein the drug loading is about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt% or higher. The drug loading in the final drug product is defined as follows:

[0103]

[0104] In an exemplary embodiment, the interaction between a poorly water-soluble compound and a solubilizer occurs through physical interactions (such as charge, hydrophobic interactions, or hydrogen bonding), and the compound is not covalently linked to the components of the solubilizer or liposome.

[0105] Preferably, a liposome composition containing two or more therapeutic agents provided herein will include liposomes stably bound to these compounds, and the encapsulated compounds have a drug-to-drug molar ratio that exhibits a non-antagonistic therapeutic effect on the relevant cells or tumor homogenates.

[0106] The lipid-based delivery carriers of the present disclosure can be used not only for parenteral administration but also for local, nasal, subcutaneous, intraperitoneal, intramuscular, aerosol, or oral delivery by applying the delivery carrier onto or within a natural or synthetic implantable device located at or near the target site for therapeutic purposes or medical imaging, etc. The lipid-based delivery carriers of the present disclosure are preferably used for parenteral administration and most preferably for intravenous administration.

[0107] The preferred embodiments described herein are not intended to be exhaustive or to limit the scope of the invention to the precise forms disclosed. The selection and description of these embodiments are intended to best explain the principles, applications, and practical uses of the invention so that those skilled in the art can understand its teachings.

[0108] I. Poorly Soluble Compounds

[0109] As described above, the present invention provides liposomes encapsulating one, two, or more therapeutic agents, wherein at least one therapeutic agent has poor water solubility. In the context of the present invention, the term "poor water solubility" means insoluble in water or having very limited solubility in water, more specifically, a water solubility of less than or equal to 1 mg / mL, sometimes preferably 0.5 mg / mL, and sometimes more preferably 0.2 mg / mL. The water solubility used herein refers to the solubility of the compound measured at ambient temperature (usually about 20 - 25°C and pH neutral). If the solubility varies with temperature within the range of 20 - 25°C, it should be considered "poor water solubility" when the solubility is not higher than 1 mg / mL, sometimes preferably 0.5 mg / mL, and sometimes more preferably 0.2 mg / mL at any point within this temperature range. In an exemplary embodiment, the water solubility of the compound is measured under neutral conditions, with a pH of about 7.0, sometimes preferably between pH 6.9 and pH 7.1, sometimes more preferably between pH 6.8 and pH 7.2, and sometimes more preferably between pH 6.5 and pH 7.5.

[0110] "Active pharmaceutical ingredient" refers to the active ingredient in a pharmaceutical product that produces the desired therapeutic effect on the body. Exemplary active pharmaceutical ingredients include the following: afatinib, abemaciclib, abiraterone, acalabrutinib, alectinib, amitinib, alpelisib, anlotinib, apatinib, avapritinib, axitinib, baricitinib, belinostat, binimetinib, bortezomib, bosutinib, brigatinib, bupivacaine, cabozantinib, capecitabine, carfilzomib, crizotinib, dabrafenib, dacomitinib, dasatinib, delanzomib, docetaxel, doxorubicin, duvelisib, enasidenib, encorafenib, entrectinib, erdafitinib, erlotinib, everolimus, ficlatuzumab, fotanib, fruquintinib, gefitinib, gemcitabine, gilteritinib, glasdegib, icotinib, ibrutinib, idarubicin, idelalisib, imatinib, ivosidenib, ixazomib, ixabepilone, lapatinib, larotrectinib, lenalidomide, lenvatinib, lorlatinib, marizomib, mistolactone, mitoxantrone, neratinib, nidasulodil, nilotinib, nintedanib, niraparib, olaparib, oportuzomib, osimertinib, paclitaxel, palbociclib, panobinostat, pazopanib, pemetrexed, pemigatinib, pexidartinib, ponatinib, pralsetinib, quizartinib, radotinib, regorafenib, ribociclib, ripretinib, rivastigmine, romidepsin, rucaparib, ruxolitinib, selpercatinib, selumetinib, sirolimus, sonidegib, sorafenib, sunitinib, talazoparib, tazemetostat, temsirolimus, tepotinib, tivozanib, tofacitinib, topotecan, trametinib, tucatinib, tucidinostat, upadacitinib, vandetanib, vemurafenib, venetoclax, vinorelbine, vismodegib, vorinostat, zanubrutinib and its free base, medicinal salts and derivatives of these pharmaceutical compounds.

[0111] However, the above list of compounds is not intended to limit the scope of the present disclosure. In fact, the compound encapsulated in the liposome can be any amphiphilic weak base or amphiphilic weak acid with poor water solubility. In addition, compounds with poor water solubility other than drugs or medicaments are also covered in the present disclosure.

[0112] Generally, the "weak base" and "weak acid" used above refer to compounds that are only partially protonated or deprotonated in water, respectively. Examples of protonating agents include compounds having an amino group, which can be protonated in an acidic medium; and compounds that are zwitterionic in a neutral medium and can also be protonated in an acidic environment. Examples of deprotonating agents include compounds having a carboxyl group, which can be deprotonated in a basic medium; and compounds that are zwitterionic in a neutral medium and can also be deprotonated in a basic environment.

[0113] The above content implies that an aqueous solution of a weak amphiphilic acid or base compound contains both charged and uncharged compound forms. Only the uncharged form is likely to cross the liposome membrane.

[0114] When the compounds used in the present invention (i.e., the active pharmaceutical ingredients) contain relatively basic or acidic functional groups, salts of such compounds are also included within the scope of the present invention. The salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid or base (pure or in a suitable inert solvent). Examples of salts of the relatively acidic compounds of the present invention include sodium salts, potassium salts, calcium salts, ammonium salts, organic amino salts or magnesium salts, or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid (pure or in a suitable inert solvent). Examples of acid addition salts include salts derived from inorganic acids, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, hydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid or phosphorous acid, etc.; and salts derived from organic acids, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid or methanesulfonic acid, etc. Also included are salts of amino acids, such as arginine salts, etc.; and salts of organic acids, such as glucuronic acid or galacturonic acid, etc. Certain specific compounds of the present invention contain both basic and acidic functional groups, such that the compounds can be converted into base addition salts or acid addition salts.

[0115] An exemplary compound is an organic molecule having a molecular weight between about 50 daltons and 5000 daltons and specifically between 100 daltons and 1000 daltons.

[0116] II. Determining the in vitro non-antagonistic drug ratio

[0117] In another embodiment of the present invention, two or more drug compounds can be encapsulated into liposomes in a synergistic or additive (i.e., non-antagonistic) drug ratio. At least one of the compounds has poor water solubility. The therapeutically effective and non-antagonistic ratio of the compounds is determined by evaluating the biological activity or effect of the drugs on relevant cell cultures and / or tumor homogenates from biopsies of individual patients within a certain concentration range. Any method capable of determining the therapeutic drug ratio that maintains the desired therapeutic effect can be used. For example, unless otherwise specified, the Chou-Talalay median effect method (Chou, TC, J. Theor. Biol., 1976, 39:253-276) is employed in the examples of the present invention.

[0118] Basic experimental data are usually determined using in vitro cultured cells. Sometimes, it is preferred to use the combination index (CI) as an alternative parameter for the concentration range, which is plotted as a function of the fraction affected (Fa). The term "fraction affected" refers to the proportion of cells whose growth is affected by a specific drug dose in an in vitro assay. The fraction affected is used to calculate the combination index as described by the Chou and Talalay procedure. Preferred drug combinations are those that exhibit synergistic or additive effects over a substantial range of Fa values. A combination is selected if it does not exhibit antagonistic effects over at least about 5% of the concentration range (where more than 1% of the cells are affected, i.e., the Fa range is greater than 0.01). Sometimes, it is preferred that a larger proportion of the total concentration exhibits a favorable CI; for example, 5% over the Fa range of 0.2 - 1.0. Sometimes it is more preferred that about 10% of this range exhibits a favorable CI. Sometimes even more preferably, about 20%, more than about 50%, or at least about 70% of the Fa value range from 0.2 to 1.0 is used in the composition. Combinations that show synergistic effects over a large range of Fa values can be re-evaluated at different drug ratios to determine the optimal ratio to enhance the strength of the non-antagonistic interaction and to widen the Fa value range over which synergism is observed.

[0119] Although it is desirable to have synergistic effects over the entire concentration range in which the cells are affected, it has been observed that in many cases, when using spectrophotometry (e.g., MTT assay), the results are more reliable when the Fa value is in the range of 0.2 - 0.8. Thus, although the synergistic effects exhibited by the combinations of the present invention are set to exist over a wide range of 0.01 or higher, sometimes it is preferred to establish synergistic effects when the Fa value is in the range of 0.2 - 0.8. However, other more sensitive detection methods can also be used to evaluate synergistic effects when the Fa value is greater than 0.8, such as bioluminescence or colony-forming ability assays.

[0120] The optimal combination ratio can further be used as a single drug unit to determine synergistic or additive interactions with a third drug. In addition, a three-drug combination can be used as a unit to determine non-antagonistic interactions with a fourth drug, and so on.

[0121] As described above, in vitro studies of cell cultures will be performed using "relevant" cells. The choice of cells depends on the intended therapeutic use of the drug. The inclusion of a composition within the scope of the present invention can be based on the demonstration of the required non-antagonistic effect by just one relevant cell line or cell culture type.

[0122] For example, in a preferred embodiment of the present disclosure, the drug combination is intended for anti-cancer treatment. In a common embodiment, the drug combination is intended for multiple cancers, such as multiple myeloma, lung cancer, non-small cell lung cancer, leukemia or lymphoma treatment, breast cancer, triple-negative breast cancer, gastrointestinal cancer, colorectal cancer, and renal cell carcinoma. Then, appropriate cells to be tested and test properties will be selected. Specifically, tumor cell lines are suitable subjects for study, and measuring cell death or cell arrest is a suitable endpoint. As will be further discussed below, other target cells and criteria other than cytotoxicity or cell arrest can be employed when attempting to find suitable non-antagonistic combinations for other indications.

[0123] For assays involving anti-tumor drugs, cell lines can be obtained from standard cell line repositories (such as NCI or ATCC), academic institutions, or other organizations (including commercial sources). Some preferred cell lines include one or more cell lines identified by the NCI / NIH Developmental Therapeutics Program. The tumor cell line screening used in this program has currently identified approximately 60 different tumor cell lines, including leukemia, melanoma, and lung, colon, brain, ovarian, breast, prostate, gastric, and renal cell carcinomas. Within the desired concentration range, the required non-antagonistic effect only needs to be demonstrated on a single cell type; however, sometimes at least two cell lines are preferred, and sometimes three cell lines, five cell lines, or even ten cell lines are more preferred to show this effect. The cell lines can be established tumor cell lines or primary cultures obtained from patient samples. The cell lines can be from any species, but the preferred source is mammalian, especially human. The cell lines can be genetically modified by selection under various laboratory conditions.

[0124] In a preferred embodiment, a given effect (Fa) refers to cell death or cell arrest resulting from the application of a cytotoxic drug to a cell culture. In the present disclosure, cell death or viability can be measured by the MTT assay. The non-antagonistic ratio of two or more drugs for disease indications other than cancer can be determined, and this information can be used to prepare therapeutic formulations of two or more drugs for treating these diseases. Regarding in vitro assays, many measurable endpoints can be selected to define drug synergy, provided that these endpoints are therapeutically relevant to a specific disease. As described above, in vitro studies of cell cultures will be performed using "relevant" cells. The selection of cells will depend on the intended therapeutic use of the drug. "Tumor homogenates" generated by homogenizing tumor samples into single cells can be used for in vitro studies of individual patient biopsy samples or whole tumors. In a preferred embodiment, a given effect (Fa) refers to cell death or cell arrest resulting from the application of a cytotoxic agent to a "relevant" cell culture. Cell death or viability can be measured using a variety of methods known in the art.

[0125] In one embodiment, an anthracycline drug (i.e., doxorubicin) is used in combination with a proteasome inhibitor (i.e., carfilzomib) to produce a synergistic effect, and the synergistic drug molar ratio is determined by the above-mentioned combination index (CI)-based method. The combination of an anthracycline drug and a proteasome inhibitor is known to have a synergistic effect. Specifically, doxorubicin and bortezomib have been shown to have a synergistic effect and have been approved by the FDA for combined use in the treatment of multiple myeloma (Mitsiades, N., Blood, 2003, 101:2377-80). Carfilzomib is a second-generation proteasome inhibitor that has reduced off-target effects compared to bortezomib and can eliminate the dose-limiting side effects of bortezomib, such as peripheral neuropathy (Demo, SD, Cancer Res., 2007, 67:6383-91). In a preferred embodiment, the molar ratio of carfilzomib to doxorubicin showing a synergistic therapeutic effect is between 1:50 and 1:1000.

[0126] In a specific embodiment, two protein kinase inhibitors, i.e., afatinib and dasatinib, are used in combination to produce a synergistic effect, and the synergistic drug molar ratio is determined by the CI-based method. The combination of afatinib and dasatinib may affect the SFK / FAK, PI3K / PTEN / Akt, Ras / Raf / MEK / ERK, and JAK / Stat signaling pathways and ultimately reverse the drug resistance generated by single-agent treatment of cancer cells (Wang, M., Oncotarget, 2018, 9:16533-46). In a preferred embodiment, the molar ratio of afatinib to dasatinib showing a synergistic therapeutic effect is between 1:30 and 30:1.

[0127] In another embodiment, afatinib is used in combination with another protein kinase inhibitor, ceritinib, to produce a synergistic effect, and the synergistic drug molar ratio is determined by the CI-based method. The combined inhibition of Abl / Src family kinases (dasatinib) and anaplastic lymphoma kinase (ceritinib) can improve the therapeutic effect (van Erp, A., Target Oncol., 2017, 12:815-826). In a preferred embodiment, the molar ratio of afatinib to ceritinib showing a synergistic therapeutic effect is between 1:30 and 30:1.

[0128] III. Solubilizer

[0129] As described above, in the exemplary embodiments of the present invention, a complex between a drug compound and a solubilizer (or sometimes referred to as a solubility enhancer, solubility improver, solubilizing agent, etc.) is added to the external aqueous medium of the liposomal formulation to increase the absorption rate and efficiency of a poorly water-soluble drug from the external medium into the aqueous core of the liposome.

[0130] According to one embodiment of the present invention, there is provided a method as described above, which uses solubilizers selected from the following: complexing agents, cosolvents, surfactants, and emulsifiers. The solubilizers generally increase the solubility of poorly soluble drugs in an external aqueous medium by at least two-fold, preferably five-fold, and more preferably ten-fold.

[0131] Exemplary solubilizers include the following: α-, β-, and γ-cyclodextrins, and the cyclodextrins can be modified with alkyl-, hydroxyalkyl-, dialkyl-, and preferably sulfoalkyl ether-modified cyclodextrins; polyvinylpyrrolidone (povidone) of different molecular weights; polyethylene glycol (PEG) of different molecular weights; hydroxypropylmethylcellulose (HPMC); polyvinyl alcohol-polyethylene glycol graft copolymer ( IR); chitosan; hydroxypropylcellulose; polyvinyl alcohol (PVA); poly(2-hydroxyethyl methacrylate); methacrylic acid copolymer ( S100 sodium salt and L100 sodium salt); poloxamer, a nonionic triblock copolymer composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide) and two hydrophilic chains of polyoxyethylene (polyethylene oxide); polyethylene glycolated glyceride (Labrasol); polyoxyethylene sorbitan monoesters (Tween); sorbitan esters (Span); polyoxyethylene stearate; poly(β-benzyl-L-aspartic acid)-β-poly(ethylene oxide); poly(caprolactone)-b-poly(ethylene oxide); ethanol; dimethyl sulfoxide; oleic acid, and combinations thereof.

[0132] However, the above list of solubilizers is not intended to limit the scope of the present invention. In fact, any molecule capable of increasing the water solubility of poorly soluble compounds is encompassed by the present invention.

[0133] In one exemplary embodiment, the solubilizer is selected from the sodium salt of sulfobutyl ether-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and polyvinylpyrrolidone.

[0134] IV. Liposomes

[0135] As used herein, the term "liposome" is consistent with its ordinary meaning and refers to a nanoscale lipid vesicle in which an internal aqueous medium is enclosed by a bilayer membrane mainly composed of phospholipids or any similar amphiphilic lipid. The liposomes in the present invention can be unilamellar vesicles, such as small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs), as well as multilamellar vesicles (MLVs). Generally, the particle size of these liposomes is in the range of 20 nm to 300 nm, specifically in the range of 50 nm to 200 nm. The present invention places no particular limitation on the liposome membrane structure. The term "liposome membrane" refers to the phospholipid bilayer membrane that separates the internal aqueous medium from the external aqueous medium.

[0136] Exemplary liposomal membranes useful in the present disclosure may be formed from a variety of vesicle-forming lipids, typically including diacyl chain lipids such as phospholipids, diglycerides, diacylglycolipids, monoacyl lipids (such as sphingomyelin and glycosphingolipids), cholesterol and its derivatives, and combinations thereof. As defined herein, a phospholipid is an amphiphilic substance having a hydrophobic group formed from a long-chain alkyl chain and a hydrophilic group containing a phosphate ester moiety. Phospholipids include phosphatidic acid, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and mixtures thereof. Preferably, the phospholipid is selected from hydrogenated egg phosphatidylcholine (HEPC), soy phosphatidylcholine (SPC), egg yolk phosphatidylcholine (EYPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DUPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphate-rac-(1-glycerol) sodium salt (DPPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate-rac-(1-glycerol) sodium salt (POPG), N-(3-maleimid-1-oxopropyl)-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE-Mal), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and the like, and mixtures thereof. Other diacyl phosphatidylcholines, diacyl phosphatidylethanolamines, and diacyl phosphatidylserine phospholipids, sterol-modified lipids, cationic lipids, and zwitterionic lipids may also be used.In some embodiments, the acyl groups in these lipids are acyl groups derived from fatty acids having a carbon chain length of 10-24 carbon atoms (e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl).

[0137] The liposomal membrane according to the present disclosure may also comprise ionophores such as nigericin and A23187.

[0138] In the compositions of the present invention, the exemplary liposome phase transition temperature ranges from -20 °C to 100 °C, specifically from 20 °C to 80 °C. The phase transition temperature is the temperature required to induce a change in the physical state of the lipids constituting the liposome from an ordered gel phase (where the hydrocarbon chains are fully extended and closely packed) to a disordered liquid crystal phase (where the hydrocarbon chains are randomly oriented and in a fluid state). Above the phase transition temperature of the liposome, the permeability of the liposomal membrane increases. Selecting phospholipids with a phase transition temperature higher than the exposure environment or conditions of use can provide a leak-free liposomal composition, i.e., the concentration of the poorly soluble drug in the internal aqueous medium can remain stable during exposure to the environment. Alternatively, when high membrane permeability is required, such as during remote drug loading, the ambient temperature is set to be higher than the lipid phase transition temperature, so that the drug can diffuse through the lipid membrane and be encapsulated within the core of the liposome.

[0139] It is well known in the art that the phase transition temperature of liposomes, as well as other parameters, is affected by the choice of phospholipids and the addition of steroids. Sterols can be selected from the non-limiting list including lanosterol, stigmasterol, cholesterol, cholesterol derivatives, ergosterol, and ergosterol derivatives, etc. Non-limiting examples of cholesterol derivatives include 5α-cholestanol, 5α-coprostanol, cholesterol-(2'-hydroxy)ethyl ether, cholesterol-(4'-hydroxy)butyl ether, 6-ketocholestanol, thiocholesterol, cholesterol acetate, cholesterol sulfate, cholesterol-3,5-diene, 5α-coprostan, cholestenone, 5α-cholestenone, cholesterol laurate, etc., and mixtures thereof. Thus, in one embodiment of the present invention, there is provided a method according to any one of the foregoing, wherein the liposome comprises one or more components selected from different phospholipids and cholesterol in different molar ratios to alter the overall transition temperature of the liposome as well as the stability of the liposome during storage and in plasma. It is well known in the art that a lower cholesterol content in the lipid composition results in lower stability of the liposome in plasma.

[0140] In the exemplary lipid compositions used in the present disclosure, the phospholipid or phospholipid combination accounts for at least 10 mol%, specifically at least 30 mol% of the total lipids present in the liposome; cholesterol or its derivatives account for 5 mol% to 50 mol% of the total lipids present in the liposome.

[0141] Polyethylene glycol (PEG)-lipid conjugates have been widely used to improve the circulation time of liposome-encapsulated therapeutic compounds, thereby enhancing the accumulation of liposomes at the target disease site and avoiding detection by the human immune system. One or more PEG-lipid conjugates can be incorporated into liposomes to perform the above functions. PEG-conjugated lipids can be selected from the following non-limiting list: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] sodium salt (mPEG2000-DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] sodium salt (PEG2000-DPPE), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG), distearoyl-rac-glycero-PEG2000 (PEG2000-DSG), methoxypolyethylene glycol oxy(2000)-N,N-ditetradecylethylacetamide (ALC-0159), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] sodium salt (DOPE-PEG1000-amine), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] sodium salt (DOPE-PEG2000-amine), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000] sodium salt (DOPE-PEG1000-COOH), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] sodium salt (DOPE-PEG2000-COOH), cholesterol-(polyethylene glycol-600) (PEG600-Chol), polyethylene glycolated ceramide, polyethylene glycolated phosphatidic acid, polyethylene glycolated phosphatidylethanolamine, polyethylene glycolated dialkylamine, polyethylene glycolated diacylglycerol, polyethylene glycolated dialkylglycerol, polyethylene glycolated glycerol ester, polyethylene glycolated sterol, etc. and mixtures thereof. In some embodiments, the average molecular weight of the PEG chain is 2000 atomic mass units. Other polymer-conjugated liposomes can also be used to extend the blood circulation time of liposomes. For example, polyglycerol modification, polyacrylamide modification, polydimethylacrylamide modification, polyvinylpyrrolidone modification, hyaluronic acid modification, heparin modification, polysialic acid modification, etc.

[0142] In one embodiment, the polymer-conjugated lipid (e.g., PEG-lipid) that inhibits liposome aggregation accounts for 0 mol% to 10 mol% of the total lipids present in the liposome.

[0143] In one embodiment, the preferred PEG-lipids are mPEG2000-DSPE and PEG2000-DMG.

[0144] For the preparation of liposomes, typically, multilamellar vesicles with relatively large particle sizes and a broad particle size distribution are first prepared through a lipid hydration step, in which the selected lipids are hydrated and dispersed in an aqueous solution. After the hydration step, a particle size reduction treatment is usually carried out to reduce the particle size and narrow the particle size distribution to the desired range. In addition, after the particle size reduction step, the lamellar structure of the liposomes can be reduced; in other words, unilamellar vesicles can be generated through this process. A liposome suspension with a particle size range of approximately 20 - 200 nm can be sterilized by filtration through a conventional filter (usually a 0.22 or 0.45 micron filter). There are various techniques known in the art for reducing the size of liposomes. For example, by bath or probe sonication of the liposome suspension, its size can be gradually reduced until small unilamellar vesicles (SUVs) with a size less than approximately 50 nanometers are formed. Homogenization is another method that relies on shear energy to break large liposomes into smaller liposomes. In a typical homogenization process, multilamellar vesicles are processed through a homogenizer for multiple cycles at a defined pressure until the desired liposome size is reached, usually between approximately 50 nm and 500 nm. Extrusion of liposomes through a small pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing the liposome size to a relatively well-defined size distribution. Typically, the liposome suspension is extruded through a membrane with a defined pore size one or more times until the desired liposome size and size distribution are achieved. Liposomes can be extruded through membranes with gradually decreasing pore sizes, thereby gradually reducing the liposome size. Alternatively, microfluidic techniques can be used to prepare liposomes of a limited size, in which lipids are rapidly mixed with an aqueous medium in an organic solvent (such as ethanol or an ethanol - aprotic solvent mixture) such that the organic solvent / water ratio is less than 30%, the microchannel size is less than 300 microns, preferably the width is less than 150 microns, and the height is less than 50 microns. Then the organic solvent is removed from the liposomes by dialysis. Other available size determination methods, such as reverse evaporation and freeze - thaw methods, are known to those skilled in the art.

[0145] The size of the exemplary liposomes for each embodiment of the present invention is from about 20 nanometers to about 50 microns. In one exemplary embodiment, the diameter of the liposomes is from about 30 nanometers to about 150 nanometers.

[0146] In certain processes, temperature control may be crucial for achieving the desired results. For example, in the liposome hydration and particle size reduction steps, the temperature should be higher than the phase transition temperature of all lipids used in the liposome formulation. In some embodiments, the temperature during liposome hydration and particle size reduction is at least 50 °C or higher.

[0147] V. Remote Loading of Poorly Soluble Compounds

[0148] The internal aqueous medium is typically the original medium in which liposomes are prepared during the hydration step and is initially encapsulated after liposome formation. This original medium contains one or more trappers for remote loading of drug compounds. The original medium may also contain one or more buffers for maintaining the internal pH of the liposomes. After the liposome size reduction step, the external liquid medium is replaced with a medium of different composition using a medium exchange process. There are various techniques known in the art for this purpose, such as dialysis, ultracentrifugation, size exclusion chromatography, etc. After the medium exchange step, the original medium remains encapsulated within the internal aqueous core, but the external liquid medium becomes of other composition, such as other charged substances. Thus, an ion gradient can be generated across the liposome bilayer membrane. Liposomes having a transmembrane ion gradient (i.e., a trapper gradient) after such treatment can be used for remote loading of therapeutic compounds. "Active loading" and "remote loading" are synonyms and can be used interchangeably.

[0149] During the active loading process, the complex formed by the solubilizer and the water-insoluble drug can facilitate the transfer of the compound from the external aqueous medium across the liposome membrane into the internal aqueous medium. Once the compound enters the internal aqueous core, it can form a complex with the trapper through various interactions (such as ionic interactions, hydrophobic interactions, hydrogen bonding), and this newly formed complex can serve as a driving force to further recruit more drugs to be loaded into the liposomes through the active loading process.

[0150] Surprisingly, during the active drug loading process, the pH value of the external liquid medium of the liposomes affects the drug encapsulation efficiency. To obtain a high drug encapsulation efficiency, an optimal external pH range is required. Without being bound by any particular theory, it is believed that drug loading at an external pH below the optimal range results in a high content of ionized compounds in the external medium, which may hinder the transmembrane diffusion of the drug because only the neutral form of the compound can freely cross the lipid membrane. On the other hand, drug loading at an external pH above the optimal range results in drug solubility problems even in the presence of a solubilizer. In this case, the poorly water-soluble drug cannot be sufficiently dissolved in the external medium, leading to a low drug encapsulation efficiency.

[0151] The optimal drug loading pH value of each compound in the external medium depends on the physicochemical properties (such as pKa) of the target drug compound and the solubilizer selected. In an exemplary embodiment, after the active drug loading step, about 90% or more of the drug is encapsulated in the aqueous compartment of the liposomes, and about 10% or less of the drug forms a complex with the solubilizer located outside the liposomes.

[0152] For the remote loading of poorly water-soluble weak amphiphilic bases, a pH gradient needs to be established across the lipid membrane. Surprisingly, we observed that the pH of the external medium of the liposome needs to be lower than that of the internal medium. This is completely opposite to the conventional pH gradient method reported previously for the remote loading of weak bases (Madden, D., Lipid Chemistry and Physics, 1990, 53:37 - 46), where the pH of the external medium is higher than that of the internal medium. Without being bound by any particular theory, the pH gradient requirement proposed herein is determined by considering the degree of ionization of the compound in the external medium, the solubility of the compound, and the drug and lipid degradation caused by acidic or basic conditions.

[0153] In an exemplary embodiment for the active loading of poorly soluble weak amphiphilic bases, the pH of the external medium is 8.0, 7.0, 6.0, 5.0, 4.0, 3.0, 2.0, 1.0, 0.5, 0.25, or 0.1 units lower than the pH of the internal medium. In one embodiment, the pH of the internal medium is between 5.0 and 10.0, and the pH of the external medium is between 2.0 and 5.0.

[0154] In one embodiment, during the lipid hydration step of preparing liposomes, a mixture of one or more buffers is added to the aqueous solution so that during the drug loading step, these buffers can remain in the aqueous core inside the liposomes, thereby maintaining the pH inside the liposomes and avoiding significant internal pH fluctuations during drug encapsulation. Exemplary buffers include: acetic acid, citric acid, histidine, HEPES, lactic acid, succinic acid, phosphates, tromethamine (Tris), etc.

[0155] In one embodiment, no buffer is added to the aqueous solution during the lipid hydration step of preparing liposomes. In this case, there is no buffer in the aqueous core inside the liposomes.

[0156] In one embodiment, the preferred pH of the external medium for the active loading of carfilzomib is in the range of 3.0 to 5.0, and the preferred pH of the internal medium is in the range of 5.0 to 10.0.

[0157] After drug loading, the distribution of solubilizers in liposomes (external, internal, or both) depends to a large extent on the physicochemical properties of the molecules used, such as molecular weight, lipid membrane permeability, pKa value, and charge. The external medium pH value during the drug loading process affects the above properties of the solubilizer. In one embodiment using sodium sulfobutyl ether-β-cyclodextrin (SBE-β-CD) as a solubilizer, without being bound by any particular theory, it is believed that when the external pH is lower than its pKa value, most of the sulfonic groups on SBE-β-CD are neutral, so this molecule can carry poorly soluble compounds and diffuse into the lipid inner core together in the form of a drug / cyclodextrin complex. In this case, both the interior and exterior of the liposome should contain SBE-β-CD.

[0158] In another embodiment, sodium salt of SBE-β-cyclodextrin is used as a solubilizer, but the external medium pH value during the drug loading process is higher than its pKa value. At this time, most of the sulfonic groups on SBE-β-cyclodextrin carry negative charges, which will hinder its penetration through the lipid membrane. In this case, SBE-β-cyclodextrin should only stay outside the liposome.

[0159] Trapping agents can establish transmembrane pH and ion gradients and may form precipitates, aggregates, or gels with therapeutic agents, thereby retaining the compounds in liposomes. Suitable trapping agents can be anionic, cationic, amphoteric, or nonionic surfactants, including but not limited to substances containing carboxylates, polyphosphates, sulfonates (including long-chain alkyl sulfonates and alkyl aryl sulfonates), and sulfates. Cationic trapping agents include quaternary ammonium compounds, such as benzalkonium chloride, benzethonium chloride, cetrimide, stearyldimethylbenzylammonium chloride, polyoxyethylene, and coconut amine, etc.

[0160] More specific examples of trapping agents include ammonium salts, metal salts, and ammonium salts or substituted ammonium salts of the following substances: polyanionic sulfated cyclodextrins, sulfobutyl ether cyclodextrins, polyanionic sulfated sugars, polyphosphates, etc.

[0161] Specifically, trapping agents include ammonium salts or substituted ammonium salts of the following polyanionic sulfated sugars: sucrose octasulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin sulfate, and sulfated hyaluronic acid, fucoidan, galactan, carrageenan, rhamnan sulfate, galactan, mannuronic acid fucan, arabinogalactan sulfate, mannan sulfate, sulfated isorhamnan, and xylomannan sulfate, etc.

[0162] Specifically, trapping agents include ammonium salts or substituted ammonium salts of sulfobutyl ether cyclodextrins in the following forms: sulfobutyl ether-α-cyclodextrin, sulfobutyl ether-β-cyclodextrin, sulfobutyl ether-γ-cyclodextrin, etc.

[0163] Specifically, the scavenger includes the ammonium salts or substituted ammonium salts of the following polyphosphates: phytic acid, triphosphoric acid, polyphosphoric acid, and cyclic metaphosphates.

[0164] Specifically, the counterions of the above polyanions include ammonium and substituted ammonium, and further include the protonated forms of the following substances: triethylamine, triethanolamine, tris(hydroxymethyl)aminomethane or tromethamine, diethanolamine, ethylenediamine, tributylamine, 1,4-diazabicyclo[2.2.2]octane, diethylethanolamine, diethylethanolamine, ethanolamine, morpholine, etc.

[0165] The metal scavenger includes the salt forms of the following ions: calcium, copper, zinc, magnesium, manganese, nickel, and cobalt. The counterions of the metal include acetate, carbonate, citrate, sulfate, chloride, halide, gluconate, bromide, and hydroxide.

[0166] More specifically, the scavengers for liposome-loading of weak amphiphilic bases include the following: ammonium sulfate, triethylammonium sucrose octasulfate (TEA-SOS), triethylammonium sulfobutylether-β-cyclodextrin (TEA-SBE-β-CD); tris(hydroxymethyl)aminomethane salt of sulfobutylether-β-cyclodextrin (Tris-SBE-β-CD), triethylammonium salt of phytic acid or inositol hexaphosphate (TEA-IP6), copper gluconate, copper sulfate, copper chloride, and zinc sulfate.

[0167] Specifically, the scavengers for liposome-loading of weak amphiphilic acids include: calcium acetate, magnesium acetate, and zinc acetate.

[0168] One or more therapeutic drug compounds can be remotely loaded into liposomes. At least one of the drugs has poor water solubility. The encapsulation of the drug combination can be completed in the same liposome carrier or in different liposomes. In the former case, multiple combined compounds can be added to the liposomes simultaneously for drug loading. For example, drugs A and B can be dissolved together in the presence of a cosolvent to form an aqueous solution, and then the drug solution can be mixed with the liposomes for loading. Alternatively, the drug compounds can also be loaded into the same liposome sequentially to avoid possible interference with the loading efficiency and / or problems related to drug degradation. For example, drug A can be dissolved in the presence of a cosolvent to form an aqueous solution, and then it can be mixed with the liposomes for loading. After a specified time, drug B is added to the same liposome pre-loaded with drug A for combined drug loading.

[0169] When it is necessary to encapsulate drugs into separate liposomes, the lipid composition of each liposome can vary greatly to achieve coordinated pharmacokinetics. By changing the composition of the liposome carrier, the release rate of the encapsulated drug can be matched, thereby delivering the desired proportion of the drug to the tumor site. Methods for changing the release rate include increasing the acyl chain length of the vesicle-forming lipids to enhance drug retention, controlling the exchange of surface-grafted hydrophilic polymers (such as the polyethylene glycol group on mPEG-DSPE) from the liposome membrane, and incorporating membrane hardeners such as sterols or sphingomyelin into the membrane. Those skilled in the art should understand that if it is desired to administer a specific drug ratio and the second drug has poor retention in the liposome composition of the first drug (e.g., DMPC / cholesterol), the pharmacokinetics can be improved by encapsulating the second drug in a liposome composition with a longer acyl chain (e.g., DSPC / cholesterol). When encapsulated in separate liposomes, it should be readily understood that the ratio of the two drugs can be determined for an individual patient by appropriately combining the amounts of the drugs encapsulated in each liposome before administration, thereby providing optimal therapeutic activity for the individual patient.

[0170] Temperature control during the drug loading step is crucial to ensure that the compound can effectively penetrate the lipid membrane of the liposome, and this temperature should be higher than the phase transition temperature of all lipids in the liposome formulation. In a preferred embodiment, the drug loading temperature is higher than 50 °C.

[0171] After the drug loading step, a free drug removal process can be optionally employed to remove unencapsulated drug compounds from the external medium of the liposome suspension. Free drug removal techniques are similar to the aforementioned medium exchange processes, such as dialysis, ultracentrifugation, size exclusion chromatography, etc. The final external medium of the liposome suspension usually contains a buffer and an osmotic pressure regulator. Examples of buffers include acetic acid, citric acid, histidine, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), lactic acid, succinic acid, phosphates, tris(hydroxymethyl)aminomethane (Tris), etc. The pH value of the final external medium of the liposome suspension is between 5.0 and 10.0, preferably between 6.0 and 8.0. When the pH value of the final external medium of the liposome suspension is below 5.0 or above 10.0, the product may undergo severe lipid degradation in a short time when stored in liquid form. Therefore, the pH value of the final external medium is preferably close to neutral. Examples of osmotic pressure regulators include sucrose, glucose, mannitol, trehalose, sodium chloride, etc.

[0172] In an exemplary embodiment, after the free drug removal step, about 98% or more of the agent is encapsulated in the aqueous compartment of the liposome, and less than 2% of the agent is located outside the liposome.

[0173] To ensure product stability, in the final production step, the liposomes can be dehydrated under reduced pressure using standard freeze-drying (lyophilization) equipment or equivalent devices. In various embodiments, the liposomes and their surrounding medium are frozen at a low temperature (e.g., -20 to -80 °C), then dehydrated and placed under reduced pressure conditions. To ensure that the liposomes do not lose most of their contents during dehydration, one or more protective sugars are typically used to interact with the lipid vesicle membrane and maintain its integrity when the water in the system is removed. A variety of sugars can be used, including trehalose, maltose, sucrose, glucose, lactose, and dextran. Generally, disaccharides have been found to be more effective than monosaccharides, with disaccharides such as trehalose and sucrose being the most effective. Other more complex sugars can also be used. For example, aminoglycoside drugs (including streptomycin and dihydrostreptomycin) have been found to protect liposomes during dehydration. Typically, one or more sugars are included in the internal or external medium of the lipid vesicles. Most preferably, the sugars are included in both the internal and external media so that they can interact with the inner and outer surfaces of the liposome membrane. Encapsulating the sugar in the internal medium is achieved by adding one or more sugars to a buffer solution that is encapsulated within the lipid vesicles during the liposome formation process (i.e., the lipid hydration step). In these embodiments, the external medium used during the active loading process should also preferably contain one or more protective sugars.

[0174] In one embodiment, drug-loaded liposomes can be prepared according to the following steps:

[0175] h) Form a lipid dispersion in a solution containing the trapping agent (e.g., TEA-SOS) and optionally a buffer (e.g., HEPES);

[0176] i) Reduce the liposome particle size at an elevated temperature (e.g., 50 °C or above) by extruding the liposomes through a polycarbonate membrane with a defined pore size (e.g., 100 nm);

[0177] j) Substantially remove the trapping agent outside the liposomes to obtain drug-free liposomes, e.g., by dialysis;

[0178] k) Dissolve the active pharmaceutical ingredient in an aqueous solution in the presence of a solubilizing agent. When encapsulating two or more compounds, at least one of the drugs has poor water solubility;

[0179] l) Incubate the unloaded liposomes with the drug or combination drug solution containing the solubilizing agent at an elevated temperature (e.g., 50 °C or above) to form drug-loaded liposomes by active loading;

[0180] m) Optionally, remove the unloaded drug and solubilizing agent outside the liposomes by dialysis or ultracentrifugation or size exclusion chromatography; and

[0181] n) Optionally, a dry form of the liposomal product is formed by lyophilization.

[0182] VI. Methods of treatment

[0183] In one aspect, the present disclosure provides a method of treating a proliferative disease (e.g., cancer) in a subject (e.g., a human), the method comprising administering to the subject a composition comprising a pharmaceutical formulation of the present disclosure in an amount effective to treat the disease, thereby treating the proliferative disease.

[0184] In one embodiment, the pharmaceutical formulation is administered in combination with one or more additional anti-cancer agents (e.g., chemotherapeutic agents described herein or combinations of chemotherapeutic agents) and radiation therapy.

[0185] In one embodiment, the cancer is a cancer described herein. For example, the cancer can be bladder cancer (including accelerated bladder cancer and metastatic bladder cancer), breast cancer (e.g., estrogen receptor positive breast cancer, estrogen receptor negative breast cancer; HER-2 positive breast cancer; HER-2 negative breast cancer, progesterone receptor positive breast cancer, progesterone receptor negative breast cancer; estrogen receptor negative, HER-2 negative and progesterone receptor negative breast cancer (i.e., triple negative breast cancer); inflammatory breast cancer), colon cancer (including colorectal cancer), kidney cancer (e.g., transitional cell carcinoma), liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer, lung adenocarcinoma and squamous cell carcinoma). Genitourinary tract, such as ovarian cancer (including fallopian tube cancer and peritoneal cancer), cervical cancer, prostate cancer, testicular cancer, kidney cancer and ureteral cancer, lymphatic system cancer, rectal cancer, laryngeal cancer, pancreatic cancer (including pancreatic exocrine cancer), esophageal cancer, gastric cancer, gallbladder cancer, thyroid cancer, skin cancer (including squamous cell carcinoma), brain cancer (including glioblastoma multiforme), head and neck cancer (e.g., occult primary cancer) and soft tissue cancer (e.g., Kaposi's sarcoma (e.g., AIDS-related Kaposi's sarcoma), leiomyosarcoma, angiosarcoma and histiocytoma).

[0186] In one exemplary embodiment, the cancer is multiple myeloma or a solid tumor. In one embodiment, the pharmaceutical formulation of the present invention comprises carfilzomib as a poorly soluble therapeutic agent.

[0187] In one aspect, the present invention describes a method of treating a disease or disorder associated with inflammation (e.g., an allergic reaction or an autoimmune disease) in a subject (e.g., a human), the method comprising: administering to the subject an amount of a composition comprising a pharmaceutical formulation of the present disclosure effective to treat the disorder, thereby treating the disease or disorder associated with inflammation.

[0188] In one embodiment, the disease or disorder associated with inflammation is the disease or disorder described herein. For example, the disease or disorder associated with inflammation can be, for example, multiple sclerosis, rheumatoid arthritis, psoriatic arthritis, degenerative joint disease, spondyloarthropathy, gouty arthritis, systemic lupus erythematosus, juvenile arthritis, rheumatoid arthritis, osteoarthritis, osteoporosis, diabetes (e.g., insulin-dependent diabetes or juvenile diabetes), dysmenorrhea, cystic fibrosis, inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, mucous colitis, ulcerative colitis, gastritis, esophagitis, pancreatitis, peritonitis, Alzheimer's disease, shock, ankylosing spondylitis, gastritis, conjunctivitis, pancreatitis (acute or chronic), multiple organ injury syndrome (e.g., secondary to sepsis or trauma), myocardial infarction, atherosclerosis, stroke, reperfusion injury (e.g., due to cardiopulmonary bypass or renal dialysis), acute glomerulonephritis, vasculitis, thermal injury (i.e., sunburn), necrotizing enterocolitis, granulocyte transfusion-related syndrome, and / or Sjogren's syndrome. Skin inflammatory diseases include, for example, eczema, atopic dermatitis, contact dermatitis, urticaria, scleroderma, psoriasis, and skin diseases with an acute inflammatory component. In some embodiments, the autoimmune disease is an organ-tissue autoimmune disease (e.g., Raynaud's syndrome), scleroderma, myasthenia gravis, transplant rejection, endotoxin shock, sepsis, psoriasis, eczema, dermatitis, multiple sclerosis, autoimmune thyroiditis, uveitis, systemic lupus erythematosus, Addison's disease, autoimmune polyglandular disease (also known as autoimmune polyglandular syndrome), or Graves' disease.

[0189] In another embodiment, the pharmaceutical formulations or the methods described herein of the present invention can be used to treat or prevent allergic and respiratory diseases, including asthma, bronchitis, pulmonary fibrosis, allergic rhinitis, oxygen toxicity, emphysema, chronic bronchitis, acute respiratory distress syndrome, and any chronic obstructive pulmonary disease (COPD). The pharmaceutical formulations, granules, or compositions of the present invention can be used to treat chronic hepatitis infections, including hepatitis B and hepatitis C.

[0190] In one aspect, the present disclosure describes a method of treating a cardiovascular disease (e.g., heart disease) in a subject (e.g., a human), the method comprising administering to the subject a pharmaceutical formulation of the present disclosure in an amount effective to treat the condition, thereby treating the cardiovascular disease.

[0191] In one embodiment, the cardiovascular disease is a disease or disorder as described herein. For example, the cardiovascular disease can be cardiomyopathy or myocarditis; such as idiopathic cardiomyopathy, metabolic cardiomyopathy, alcoholic cardiomyopathy, drug-induced cardiomyopathy, ischemic cardiomyopathy, and hypertensive cardiomyopathy. In addition, the pharmaceutical preparations, particles, compositions, and methods described herein can be used to treat or prevent atherosclerotic diseases (macrovascular diseases) of the major blood vessels, such as the aorta, coronary arteries, carotid arteries, cerebrovascular arteries, renal arteries, iliac arteries, femoral arteries, and popliteal arteries. Other vascular diseases that can be treated or prevented include diseases associated with platelet aggregation, retinal arterioles, glomerular arterioles, neurovascular, cardiac arterioles, and capillary beds associated with the eye, kidney, heart, central, and peripheral nervous systems. In addition, other diseases that can be treated with the pharmaceutical preparations of the present disclosure include vascular restenosis (e.g., after coronary intervention) and diseases associated with abnormal high- and low-density cholesterol levels.

[0192] In one embodiment, the pharmaceutical preparation of the present invention can be administered to a subject who is undergoing or has undergone angioplasty. In one embodiment, the pharmaceutical preparation, particle, or composition of the present invention is administered to a subject who is undergoing or has undergone angioplasty and has had a stent implanted. In some embodiments, the pharmaceutical preparation, particle, or composition of the present invention can be used as a support for a stent or a coating for a stent.

[0193] In one aspect, the present disclosure provides a method of treating a subject (e.g., a human) having a kidney-related disease or disorder (e.g., a kidney disease), the method comprising: administering to the subject an amount of the pharmaceutical preparation of the present disclosure effective to treat the disorder, thereby treating the disease or disorder associated with the kidney disease.

[0194] In one embodiment, the kidney-related disease or disorder is a disease or disorder as described herein. For example, the kidney-related disease or disorder can be, for example, acute renal failure, acute nephritic syndrome, painless nephropathy, atherosclerotic embolic nephropathy, chronic renal failure, chronic nephritis, congenital nephrotic syndrome, end-stage renal disease, Goodpasture's syndrome, interstitial nephritis, kidney injury, kidney infection, renal injury, kidney stones, lupus nephritis, membranoproliferative glomerulonephritis (GN1), membranoproliferative glomerulonephritis (GN2), membranous nephropathy, minimal change nephropathy, necrotizing glomerulonephritis, nephroblastoma, nephrocalcinosis, nephrogenic diabetes insipidus, nephrosis (nephrotic syndrome), polycystic kidney disease, post-streptococcal glomerulonephritis. Reflux nephropathy, renal artery embolism, renal artery stenosis, renal papillary necrosis, type I renal tubular acidosis, type II renal tubular acidosis, renal hypoperfusion, renal vein thrombosis.

[0195] In one exemplary embodiment, the present invention provides a method for treating metal poisoning or metal overload. Examples of metal-related diseases or disorders include iron overload disorders (e.g., thalassemia or sickle cell anemia), copper overload disorders (e.g., Wilson's disease), and radioactive isotope contamination (e.g., occurring after contamination with plutonium, uranium, and other radioactive isotopes).

[0196] Definitions

[0197] Unless otherwise defined, all professional terms, symbols, and other scientific terms or terminology used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In certain instances, terms with commonly understood meanings are defined herein for clarity and / or convenience of reference, and the inclusion of such definitions herein is not necessarily to be construed as materially different from the commonly understood meaning in the art. Many of the techniques and procedures described or cited herein are well understood by those skilled in the art and are commonly carried out using conventional methods. Unless otherwise indicated, procedures involving the use of commercially available kits and reagents are generally carried out according to the protocols and / or parameters defined by the manufacturer. All patents, applications, published applications, and other publications cited herein are hereby incorporated by reference in their entirety. If the definitions set forth in this section are contrary to or inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated by reference herein, the definitions set forth in this section shall control.

[0198] As used herein, the singular forms "a" and "the" include plural referents and vice versa, and any plural form also includes the singular referent, unless the context clearly dictates otherwise.

[0199] The term "about" or "approximately" generally encompasses values within a range of plus or minus 10% of the indicated value. For example, "about 10%" may represent a range from 9% to 11%, and "about 20%" may represent a range from 18% to 22%. Sometimes, "about" preferably encompasses values within a range of plus or minus 5% of the indicated value. Alternatively, "about" also encompasses values within a range of plus or minus 5% of the indicated value. When "about" is used before a range, it applies to both the lower and upper limits of the range.

[0200] The term "substantially" means "mostly" or "essentially" as understood by one of ordinary skill in the art, and if measurable quantitatively, it refers to at least 90%, preferably at least 95%, more preferably at least 98%.

[0201] Unless otherwise specified, terms such as "comprising," "having," "containing," and "including" shall be construed as open-ended terms (i.e., meaning "including but not limited to").

[0202] As used herein, the term "synergism" refers to the interaction between two or more drugs such that the total effect of the drugs is greater than the sum of the effects of each drug alone.

[0203] The synergy ratio refers to the molar ratio at which two or more drugs, when used in combination, can produce a synergistic effect.

[0204] As used herein, the term "synergistic cytotoxicity" refers to the interaction between two or more drugs, the total effect of which is greater than the sum of the effects of each drug alone. This total effect results in the killing of cells and ultimately the shrinkage of tumors.

[0205] As used herein, "synergistic cytostatic effect" refers to the interaction between two or more drugs such that the overall effect of the drugs is greater than the sum of the effects of each drug alone. This overall effect results in the inhibition of tumor growth but does not directly kill cells.

[0206] The term "additive effect" refers to the combined effect produced by two or more drugs, which is equal to the sum of the effects of each drug alone.

[0207] The additive ratio refers to the molar ratio at which two or more drugs, when used in combination, achieve an additive effect.

[0208] The term "non-antagonistic ratio" refers to the synergy ratio and the additive ratio.

[0209] As used herein, the term "antagonism" refers to a therapeutic response to two or more drugs that is lower than the expected therapeutic response when the effects of the individual known drugs are added together.

[0210] As used herein, the term "antagonistic ratio" refers to the molar ratio at which two or more drugs, when used in combination, can produce an antagonistic effect.

[0211] The "combination index" is a parameter used to determine the degree of drug interaction. The combination index (CI) can be calculated based on the median effect analysis algorithm described by Chou and Talalay (TC Chou and P. Talalay, Adv. Enzyme Reg., 1984, 22: 27-55). A CI value < 0.9 indicates synergism between the drugs; 0.9 ≤ CI ≤ 1.1 indicates an additive effect; CI > 1.1 indicates antagonism.

[0212] The "affected fraction" refers to the proportion of cells affected by a specific drug dose in an in vitro assay. This proportion is used to calculate the combination index, and the specific method can be found in the Chou and Talalay method.

[0213] "Relevant" cells refer to at least one cell culture or cell line suitable for testing the expected biological effect. Since these drugs are used as anti-tumor drugs, "relevant" cells refer to cell lines identified by the Developmental Therapeutics Program (DTP) of the National Cancer Institute (NCI) / National Institutes of Health (NIH) of the United States for use in their anti-cancer drug development projects. Currently, DTP screening uses 60 different human tumor cell lines. It is necessary to demonstrate that the drug has the expected activity against at least one such cell line.

[0214] "Tumor homogenate" refers to cells generated by homogenizing patient biopsy tissue or tumors. Extraction of intact tumors or tumor biopsy tissue can be done by a qualified doctor using standard medical techniques, and the tissue can be homogenized into individual cells in the laboratory using a variety of known methods in the art.

[0215] As used herein, the term "trapping agent" refers to a compound that is present in the aqueous compartment of a liposome and is used to trap and retain one or more drugs within the same location within the liposome.

[0216] The term "poor water solubility" means insoluble in water or having extremely low solubility, more specifically, having a solubility in water of less than 1 mg / mL. As used herein, "water solubility" refers to the solubility of a compound measured at ambient temperature (usually about 20 - 25 °C). In an exemplary embodiment, the water solubility of a compound is measured at pH = 7.0.

[0217] As used above, the terms "weak base" and "weak acid" refer to compounds that are only partially protonated or deprotonated in water, respectively. Examples of protonating agents include compounds having an amino group that can be protonated in an acidic medium; and compounds that are zwitterionic in a neutral medium and can also be protonated in an acidic environment. Examples of deprotonating agents include compounds having a carboxyl group that can be deprotonated in a basic medium; and compounds that are zwitterionic in a neutral medium and can also be deprotonated in a basic environment.

[0218] The term "zwitterion" refers to a compound capable of carrying a positive charge and a negative charge simultaneously on different atoms.

[0219] As used in the present disclosure, the term "amphiphilic" generally refers to a compound having both a lipophilic moiety and a hydrophilic moiety.

[0220] As used herein, the term "complexing agent" is a solubilizing agent, a water-soluble compound that forms a water-soluble inclusion complex with a poorly water-soluble reagent, thereby increasing the water solubility of the poorly water-soluble compound.

[0221] The term "encapsulation efficiency" is defined by the following formula:

[0222]

[0223] "Effective amount" means the amount of the pharmaceutical preparation in the present invention that, upon administration to a subject in a single dose or multiple doses, can effectively treat cells, or cure, relieve, mitigate, or improve the symptoms of a disease. The effective amount of the composition can vary depending on factors such as the individual's disease state, age, sex, and body weight, as well as the ability of the compound to elicit the desired response in the individual. The effective amount also refers to the amount at which any toxic or harmful effects of the composition are offset by the therapeutic beneficial effects.

[0224] As used herein, the term "prevent" when used in the context of administering an agent to a subject means causing the subject to receive a certain therapy, such as administering the pharmaceutical preparation in the present invention, such that the onset of at least one symptom of the disease is delayed compared to the situation where the therapy is not received.

[0225] The term "subject" as used herein is intended to include human and non-human animals. Exemplary human subjects include human patients or normal subjects suffering from a disease (such as the diseases described herein). The term "non-human animal" includes all vertebrates, such as non-mammals (such as chickens, amphibians, reptiles) and mammals. For example, non-human primates, domestic animals, and / or agricultural animals, such as sheep, dogs, cats, cows, pigs, etc.

[0226] The term "treat" or "treating a subject suffering from a disease" as used herein means causing the subject to receive a certain therapy, such as administering the pharmaceutical preparation of the present disclosure, so that at least one symptom of the disease is cured, healed, relieved, mitigated, altered, remedied, improved, or ameliorated. Treatment includes administering an effective dose to relieve, mitigate, alter, remedy, improve, ameliorate, or affect the disease or its symptoms. The treatment can inhibit the worsening or exacerbation of the disease symptoms.

[0227] The term "pharmaceutically acceptable" describes a substance that has no biological or other adverse effects, i.e., does not cause unacceptable levels of adverse biological effects or interact in a harmful manner.

[0228] The term "liposome lamellarity" refers to the number of lipid bilayers in a liposome, which affects the encapsulation efficiency and drug release kinetics.

[0229] The term "liposome" as used herein has the same meaning as its ordinary meaning, referring to nanoscale lipid vesicles that are enclosed by a bilayer membrane mainly composed of phospholipids or any similar amphiphilic lipid and enclose an internal aqueous medium. The liquid medium outside the bilayer membrane in which the liposome is suspended is called the external liquid medium.

[0230] As used herein, the term "unilamellar vesicle" refers to a spherical vesicle composed of a single lipid bilayer membrane that defines a closed aqueous compartment. The bilayer membrane consists of two layers of lipids: an inner layer and an outer layer. The hydrophilic heads of the outer layer lipid molecules face the external aqueous environment, and the hydrophobic tails point downward towards the interior of the liposome. The inner layer lipids are located directly beneath the outer layer lipids, with the heads of the lipid molecules facing the aqueous interior of the liposome and the tails facing the tails of the outer layer lipid molecules.

[0231] As used herein, the term "multilamellar vesicle" refers to a liposome composed of multiple lipid bilayer membranes that define multiple closed aqueous compartments. These membranes are arranged concentrically, with the different membranes separated by aqueous compartments, like an onion.

[0232] The term "total lipids" refers to all lipids and lipid derivatives used in a formulation, including phospholipids (e.g., HSPC, DSPC, DPPC, DMPC, and DSPG), sterols (e.g., cholesterol), and phospholipids conjugated with polyethylene glycol (e.g., mPEG-DSPE).

[0233] Release refers to the drug encapsulated in the liposome passing through the lipid membrane that constitutes the liposome and then being released outside the liposome.

[0234] As used herein, the term "encapsulation" refers to enclosing the inner phase, usually forming a lumen isolated from the external medium. Thus, the components of the inner phase / lumen are "encapsulated" as described herein. The inner phase described herein that is encapsulated or enclosed refers to the lipid bilayer and the aqueous phase. The amount of therapeutic drug loaded into the liposome lumen and thus not absorbable by the external medium until the liposome is triggered to release can be considered "encapsulated" within the liposome.

[0235] The phrases "co-encapsulation" and "co-entrapment" used herein refer to the situation where two or more therapeutic agents are encapsulated within a liposome.

[0236] As used herein, "active drug loading" or "remote drug loading" refers to a drug loading technique used in the preparation of liposomal drug formulations. Commonly used active drug loading methods in the art include transmembrane pH gradient drug loading techniques and transition metal drug loading techniques. The former uses ammonium salts or substituted ammonium salts of monoanions or polyanions as drug trappers, which are pre-loaded into liposomes before encapsulating therapeutic drugs. Based on the equilibrium determined by the pH gradient, the therapeutic drug can "actively" diffuse into the aqueous compartment of the liposome and interact with the pre-loaded drug trapper to form precipitation, aggregation, or gelation, which serves as another driving force to encapsulate the therapeutic drug within the liposome. The transition metal drug loading technique uses transition metals to drive the absorption of drugs by liposomes through complexation or coordination. Generally speaking, compared with passive loading techniques, the use of active loading techniques can achieve a higher encapsulation efficiency of therapeutic agents (e.g., >90%).

[0237] The average particle size refers to the average diameter of liposomes and can be measured by an instrument based on dynamic light scattering.

[0238] The term "substituted ammonium" refers to the substitution of one or more hydrogen atoms in the ammonium ion by alkyl or other organic groups, forming a substituted ammonium ion.

[0239] The term "triple-negative breast cancer" refers to breast cancer in which cancer cells lack estrogen or progesterone receptors and are unable to produce sufficient human epidermal growth factor receptor 2 (HER2) protein. That is, the cancer cells are "negative" in all three of the above receptor tests.

[0240] The term "non-small cell lung cancer" (NSCLC) refers to any type of epithelial lung cancer other than small cell lung cancer (SCLC). The most common types of NSCLC are squamous cell carcinoma, large cell carcinoma, and adenocarcinoma, but there are also several other less common types, and all types may have uncommon histological variations.

[0241] The term "renal cell carcinoma" (RCC) refers to a type of kidney cancer that originates from the inner wall of the proximal convoluted tubule. The proximal convoluted tubule is part of the tiny tubes in the kidney responsible for transporting the primary urine. RCC is the most common type of kidney cancer in adults, accounting for about 90%-95% of cases.

[0242] The term "drug-resistant cancer" refers to cancer types that are resistant to specific therapeutic drugs. Drug resistance means that cancer cells do not respond to drugs that normally can kill or weaken cancer cells. Drug resistance may exist before treatment (intrinsic resistance) or may occur during or after treatment (acquired resistance). In cancer treatment, many factors can lead to drug resistance to anticancer drugs. For example, DNA mutations or other gene mutations may change the way drugs enter cancer cells or the way they are broken down within cancer cells. Drug resistance may result in ineffective cancer treatment or cancer recurrence.

[0243] The following abbreviations are used in this application:

[0244] AE: Adverse event;

[0245] AFA: Afatinib;

[0246] API(s): Active pharmaceutical ingredient(s);

[0247] CAR: Carfilzomib;

[0248] CD: Cyclodextrin;

[0249] CER: Ceritinib;

[0250] Chol: Cholesterol;

[0251] CI: Combination index;

[0252] DAS: Dasatinib;

[0253] DDPC: 1,2-Didecanoyl-sn-glycero-3-phosphocholine;

[0254] DEPC: 1,2-Dierucoyl-sn-glycero-3-phosphocholine;

[0255] DLPC: 1,2-Dilauroyl-sn-glycero-3-phosphocholine;

[0256] DMPC: 1,2-Dimyristoyl-sn-glycero-3-phosphocholine;

[0257] DPPC: 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine;

[0258] DMPG: 1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol;

[0259] DPPG: 1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol;

[0260] DSPC: 1,2-Distearoyl-sn-glycero-3-phosphocholine;

[0261] DSPG: 1,2-Distearoyl-sn-glycero-3-phosphoglycerol;

[0262] DOX: Doxorubicin;

[0263] EDTA: Ethylenediaminetetraacetic acid;

[0264] ED 75 and ED 90 : The effective doses required to affect 75% and 90% of the cells in cell culture;

[0265] Fa: The affected part;

[0266] GIST: Gastrointestinal stromal tumor;

[0267] HBS: HEPES Buffered Saline (20 mM HEPES, 150 mM NaCl, pH 7.4);

[0268] HEPES: N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid;

[0269] HSPC: Hydrogenated L-α-phosphatidylcholine;

[0270] HP-β-CD: Hydroxypropyl-β-cyclodextrin;

[0271] LUV: Large unilamellar vesicles;

[0272] MLV: Multilamellar Vesicle;

[0273] mPEG-2000-DSPE, sodium salt: N-(carbomethoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, with polyethylene glycol having an average molecular weight of 2000;

[0274] MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2-H tetrazolium bromide;

[0275] NSCLC: Non-Small Cell Lung Cancer;

[0276] PEG-2000-DMG: 1-Monomethoxypolyethylene glycol-2,3-dimyristoyl glycerol, where the average molecular weight of polyethylene glycol is 2000;

[0277] PG: Phosphatidylglycerol;

[0278] PSPC: 1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine;

[0279] RCC: Renal Cell Carcinoma;

[0280] SBE-α-CD: Sulfobutylether-α-cyclodextrin;

[0281] SBE-β-CD: Sulfobutylether-β-cyclodextrin;

[0282] SBE-γ-CD: Sulfobutylether-γ-cyclodextrin;

[0283] SMPC: 1-Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine;

[0284] SOPC: 1-Stearoyl-2-oleoyl-sn-glycero-3-phosphocholine;

[0285] SOS: Sucrose octasulfate;

[0286] SPPC: 1-Stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine;

[0287] SUV: Small Unilamellar Vesicle;

[0288] TEA: Triethylamine;

[0289] TEA-phytate: Triethylammonium phytate;

[0290] TEA-SOS: Triethylammonium sucrose octasulfate;

[0291] TEA-SBE-β-CD: Triethylammonium sulfobutylether-β-cyclodextrin;

[0292] Tris: Tris(hydroxymethyl)aminomethane;

[0293] Tris-SBE-β-CD: Tris(hydroxymethyl)aminomethane salt of sulfobutylether-β-cyclodextrin;

[0294] Tris-SOS: Tris(hydroxymethyl)aminomethane salt of sucrose octasulfate.

[0295] Examples

[0296] The following examples are provided to illustrate, but not limit, the disclosed invention.

[0297] Example 1

[0298] Preparation of polyanion-based trappers

[0299] The encapsulating agent for loading active drugs in liposomes plays a crucial role in the encapsulation, retention, and dissolution characteristics of the active drug. The preparation method of such polyanion encapsulating agents will be introduced in detail below.

[0300] Preparation of ammonium salts or substituted ammonium salts of SOS and SBE-β-CD: First, load sulfonated polystyrene-divinylbenzene copolymer cation exchange resin beads into an ion exchange column. Then, equilibrate the resin with about 1N HCl and rinse with deionized water until the pH of the eluate is close to neutral. After that, add a solution of sucrose octasulfate (SOS) or SBE-β-CD to the column and elute with deionized water. Then, titrate the eluate containing the hydrogen form of SOS or SBE-β-CD with triethylamine to a pH of 4.0 - 7.0. The resulting polyanion salts are named triethylammonium SOS (TEA-SOS) or triethylammonium SBE-β-CD (TEA-SBE-β-CD), respectively. In other cases, ammonium hydroxide or tris(hydroxymethyl)aminomethane (Tris) or triethanolamine (TEOA) is used as the base to titrate the hydrogen form of SOS or SBE-β-CD to form the corresponding polyanion salts.

[0301] Example 2

[0302] General protocol for preparing drug-loaded liposomes by active loading for compounds with poor water solubility

[0303] The general steps for preparing drug-loaded liposomes by active drug loading are as follows: (1) Lipid hydration and particle size reduction (2) Dialysis (3) Active drug loading, and finally (4) Removal of free drug.

[0304] The specific steps are as follows: Dissolve DSPC, cholesterol, PEGylated lipid (such as mPEG-DSPE) and optionally DSPG in ethanol, and add it to an aqueous solution containing any one of the following trappers: ammonium sulfate, TEA-SOS, TEA-SBE-β-CD or TEA-phytate, at a temperature of 50 - 70 °C. Optionally, a buffer (such as HEPES) can be added to the above aqueous solution. Subsequently, mix the organic phase and the aqueous phase under vigorous stirring for about 30 minutes to form multilamellar vesicles (MLV). Then, reduce the size of the liposome suspension (such as by extrusion) through a 100 nm and / or 50 nm polycarbonate membrane at a high temperature (50 - 70 °C) to obtain the desired liposome particle size (70 - 120 nm) and particle size distribution (PDI < 0.2), and then quickly cool the liposomes to room temperature. After that, remove the trapper outside the liposomes by dialysis with deionized water. Based on the above method, unloaded liposomes containing trappers and optionally buffers are prepared.

[0305] To load drugs into liposomes, first, a drug solution needs to be prepared and then mixed with liposomes. For this purpose, add and dissolve a poorly soluble compound (such as carfilzomib) in an aqueous solution containing a buffer (such as citric acid) with a pH of 2.0 - 6.0 and a solubilizer (such as sodium salt of SBE-β-cyclodextrin). In some cases, at this step, two or more drug compounds are dissolved in the buffer solution to achieve combined drug loading. Subsequently, mix the drug solution with the above-prepared drug-free liposomes and carry out drug loading at 50 - 70 °C for 1 hour. This active drug loading method usually can make the drug encapsulation efficiency higher than 90%. The unloaded drugs and external solubilizers can be removed from the liposomes by one of the following methods: dialysis, size exclusion chromatography, or ultracentrifugation, or other suitable methods. After the free drug removal step, the encapsulation efficiency is generally higher than 99%. Finally, the liposome drug product is aseptically filtered and filled into type I borosilicate glass bottles and stored at 2 - 8 °C.

[0306] Example 3

[0307] General protocol for physicochemical characterization of liposomes

[0308] Particle size and Zeta potential. Use Nano-S90 to measure the hydrodynamic particle size, polydispersity index (PDI), and Zeta potential (i.e., the surface charge of particles) of the liposome drug product. ZetaSizer (Malvern Instruments, UK). Before measurement, each sample is fully diluted with distilled water.

[0309] Morphological characterization.The size and morphology of liposomes were examined using a cryogenic transmission electron microscope (Cryo-TEM), specifically a cryo-TEM-Titan Krios 80 / 300 Kev transmission electron microscope (ThermoFisher Scientific).

[0310] Drug loading and encapsulation. The drug content (determination) of the liposome product was determined by dissolving a known amount of the loaded liposomes in an aqueous solution of Triton-X100, and then the drug content was quantified by HPLC-UV analysis. The method for determining the free drug content was to first separate the free drug from the liposomes by size exclusion chromatography (SEC), and then quantify the unloaded drug content in the corresponding fraction by HPLC-UV analysis. The drug encapsulation efficiency (EE%) was calculated by subtracting the free drug content from the total drug content and then dividing by the total drug content.

[0311] In vitro drug release study. The in vitro release of the drug-loaded liposomes was evaluated by a dialysis-based method. For example, first, a certain volume of the liposome product (about 1 - 2 mL) was added to a dialysis bag (molecular weight cut-off 10 kDa) and pre-hydrated overnight in phosphate buffer saline (PBS) at pH 7.4. Then the dialysis bag was placed in a glass container containing 150 mL of PBS (pH 7.4). Dissolution studies were carried out with gentle stirring at 37°C. Aliquots (about 1 mL) of the release medium were sampled at predetermined time intervals, and the container was replenished with an equal volume of fresh medium. The drug content of the specific compound was determined by HPLC-UV method. Then a cumulative drug release curve was generated based on the drug content released at each time point.

[0312] Example 4

[0313] Preparation of carfilzomib liposomes by active drug loading method

[0314] Example 2 describes the detailed steps for preparing drug-loaded liposomes. Among them, four different types of trappers were used (Table 1), namely (1) TEA-SOS, (2) TEA-SBE-β-CD, (3) TEA-phytate, and (4) ammonium sulfate. By changing the concentration of the trapper, different molar ratios of sulfate or sulfo or phosphate to API were applied for each trapper studied (Table 1). All liposomal drug products in Table 1 contained the following composition: (1) lipid composition: DSPC (59 mol%), cholesterol (40 mol%), and mPEG-DSPE (1 mol%); (2) the sodium salt of 100 mg / ml SBE-β-CD was used as a solubilizer to dissolve carfilzomib in all drug loads, and (3) 10 mM citric acid with a pH of 6.50. During drug loading, the external medium pH was 3.50. Generally, the higher the molar ratio of sulfate, sulfo-salt, or phosphate to API, the higher the drug encapsulation efficiency. Another conclusion of Table 1 is that the encapsulation efficiency using polyanion as a trapper (>60%) is significantly higher than that using ammonium sulfate (<30%).

[0315] Table 1. Physicochemical properties of carfilzomib-loaded liposomes prepared using sodium salt of SBE-β-cyclodextrin as a solubilizer and different types of encapsulating agents. All liposomes contained the following lipid components: DSPC, cholesterol, and mPEG-DSPE.

[0316]

[0317] In some cases, DSPG was also included in the lipid composition with the aim of improving the physical stability of the liposomes. In the example shown in Table 2, the lipid composition was as follows: DSPC (58.5 mol%), cholesterol (39.5 mol%), mPEG-DSPE (1 mol%), and DSPG (1 mol%). The final formulation contained the following: (1) 100 mg / ml sodium salt of SBE-β-CD was used as a solubility enhancer to dissolve carfilzomib during drug loading; (2) 10 mM citric acid with a pH of 6.50. During drug loading, the external medium pH was 4.0. Here, the method of dialysis to remove free drug was adopted to remove unloaded carfilzomib, so that the encapsulation efficiency of the final drug product reached more than 99%.

[0318] Table 2. Physicochemical properties of carfilzomib liposomes. Free drug removal process was adopted. All liposomes contained the following lipid components: DSPC, cholesterol, mPEG-DSPE, and DSPG.

[0319]

[0320] Example 5

[0321] Effect of Drug-Loading pH on the Encapsulation Efficiency of Carfilzomib

[0322] The specific preparation method of the drug-loaded liposomes is shown in Example 2. Using sodium SBE-β-cyclodextrin as a solubilizer, six drug-loading pH values (Table 3) were studied, namely pH 5.0, pH 4.75, pH 4.50, pH 4.0, pH 3.50, and pH 3.0. The finally obtained liposomal drug products all contained the following components: (1) lipid composition: DSPC (59 mol%), cholesterol (40 mol%), and mPEG-DSPE (1 mol%); (2) 100 mg / ml sodium SBE-β-cyclodextrin; (3) buffer with a pH of 6.50. As can be seen from Table 3, when the drug-loading pH value was 4.0, the encapsulation efficiency was the highest. Deviating from the above value for the drug-loading pH led to a decrease in the drug encapsulation efficiency.

[0323] Table 3. Effect of drug-loading pH on EE%. All liposomes contained the following lipid composition: DSPC, cholesterol, and mPEG-DSPE. Using TEA-SOS as an encapsulating agent.

[0324]

[0325] Example 6

[0326] Study on the Drug Loading of Carfilzomib Liposomes

[0327] The detailed preparation steps of the drug-loaded liposomes are shown in Example 2. Using sodium SBE-β-cyclodextrin as a solubilizer. The effect of different drug-to-lipid weight ratios in the feed on the drug loading of liposomes was investigated. The finally prepared liposomal drug products contained the following components: (1) lipid component: DSPC (59 mol%), cholesterol (40 mol%), and mPEG-DSPE (1 mol%); (2) 100 mg / ml sodium SBE-β-cyclodextrin; (3) buffer with a pH of 6.50. When loading the drug, the external medium pH was 4.0. As shown in Table 4 below, the highest drug loading of the liposomes could reach nearly 30 wt%.

[0328] Table 4. Study on drug loading. High drug loading of carfilzomib can be achieved in liposomes. Using TEA-SOS as an encapsulating agent.

[0329]

[0330]

[0331] Example 7

[0332] Preparation of Dasatinib Liposomes by Active Drug Loading Method

[0333] The specific preparation steps of the drug-loaded liposomes are shown in Example 2. SBE-β-cyclodextrin sodium salt was used as a solubilizer. Different encapsulants were used to encapsulate dasatinib, and the product characterization results are shown in Table 5. The final liposomal drug products all contained the following components: (1) lipid components: DSPC (58.5 mol%), cholesterol (39.5 mol%), mPEG-DSPE (1 mol%), and DSPG (1 mol%); (2) 100 mg / ml SBE-β-CD sodium salt; and (3) 10 mM citric acid (pH 6.50). The external medium pH was 3.50 during drug loading. The results showed that the encapsulation efficiency of the TEA-SOS encapsulant for dasatinib was the highest.

[0334] Table 5. Physicochemical properties of dasatinib liposomes. All liposomes contained the following lipid components: DSPC, cholesterol, mPEG-DSPE, and DSPG.

[0335]

[0336] Example 8

[0337] Preparation of ceritinib liposomes by active drug loading method

[0338] The specific preparation steps of the drug-loaded liposomes are shown in Example 2. SBE-β-cyclodextrin sodium salt was used as a solubilizing agent. Different encapsulants were used to encapsulate ceritinib, and the product characterization results are shown in Table 6. The final liposomal drug products all contained the following components: (1) lipid composition: DSPC (58.5 mol%), cholesterol (39.5 mol%), mPEG-DSPE (1 mol%), and DSPG (1 mol%); (2) 100 mg / ml SBE-β-cyclodextrin sodium salt; (3) buffer pH = 6.50. During drug loading, the external medium pH was 3.50. The results showed that the encapsulation efficiency of the three encapsulants for ceritinib was close to 100%.

[0339] Table 6. Physicochemical properties of ceritinib-loaded liposomes. All liposomes contained the following lipid components: DSPC, cholesterol, mPEG-DSPE, and DSPG.

[0340]

[0341] Example 9

[0342] In vitro cytotoxicity studies to determine drug synergy

[0343] For combination drug regimens, two or more drugs in combination may exhibit synergistic, additive, or antagonistic effects, depending on the molar ratio of the drugs. To quantitatively study the interactions between these drugs, this study employed a method based on the combination index (CI), which follows the method reported previously (Chou, TC, J. Theor. Biol. (1976) 39:253-276).

[0344] A general protocol for cell culture and liposome pharmacodynamic evaluation is described below. Using standard cell culture techniques, adherent cancer cell lines in the logarithmic growth phase were collected. The cell concentration was determined using a hemocytometer and then diluted to the target cell concentration with the appropriate medium. The cells were then seeded into 96-well plates. The well plate map design included treatment groups, cell-only control groups (no drug treatment), and medium-only control groups (no cells and no drug treatment). The cell seeding concentration was optimized so that after 48 hours of cell seeding, MTT analysis of untreated control cells produced an absorbance value of approximately 1.0 at 590 nm.

[0345] The culture plates seeded with cells were incubated in a standard cell culture incubator at 37 °C and 5% CO2 for 24 hours. The next day, single drugs or drug combinations diluted at specific molar drug ratios were prepared using the appropriate cell medium. Then, the cell medium in the 96-well plates was replaced with fresh medium containing the drug or drug combination. After incubation for another 24 hours, cell viability was evaluated by MTT assay according to the manufacturer's protocol. The relative survival rate was determined by subtracting the absorbance value of the medium-only wells from the drug-treated wells and then normalizing it to the drug-free control wells (cell-only control). Subsequently, the proportion of affected cells (fa) or the cell growth inhibition rate (%) in each well was calculated. Then, the effects of the drug combinations were calculated and processed using software called "CompuSyn" for drug synergy analysis. This program uses the median effect analysis algorithm to generate combination index values as a quantitative measure of the degree of synergy. According to this analysis method, CI < 0.9 indicates synergistic effect, 0.9 ≤ CI ≤ 1.1 indicates additive effect, and CI > 1.1 indicates antagonistic effect. The CI plot is usually drawn with CI on the y-axis and the proportion of affected cells (or affected fraction (Fa)) on the x-axis. The synergy rate of the drug combination was determined and used for subsequent studies.

[0346] Example 10

[0347] In vitro evaluation of the synergistic effect of afatinib and dasatinib in the treatment of cancer cells

[0348] To determine the synergistic molar ratio of afatinib and dasatinib (AFA / DAS), we tested the in vitro cytotoxic effects of different drug ratios on cancer cell lines. In the HCC827 non-small cell lung cancer cell line, the cytotoxicity was determined at AFA / DAS molar ratios of 5:1, 2.5:1, 1:1, 1:2.5, and 1:5, respectively. The cytotoxicity of AFA alone and DAS alone in the corresponding cell lines was used as a control.

[0349] The detailed procedures for cell culture, drug treatment, cytotoxicity determination by the MTT method, and calculation of the CI value have been described in Example 9. Figure 2A A representative graph showing the relationship between the CI value and cell growth inhibition (i.e., the fraction of affected cells, Fa) at different AFA / DAS ratios in the HCC827 cell line is presented. It was found that all the above-tested drug molar ratios had a synergistic effect on tumor cell growth inhibition.

[0350] Example 11

[0351] In vitro evaluation of the synergistic effect of dasatinib combined with ceritinib in the treatment of cancer cells

[0352] To determine the synergistic molar ratio of dasatinib / ceritinib (DAS / CER), we tested the in vitro cytotoxic effects of different drug ratios on cancer cell lines. In the HCC827 non-small cell lung cancer cell line, the cytotoxicity was determined at DAS / CER molar ratios of 5:1, 2.5:1, 1:1, 1:2.5, and 1:5, respectively. The cytotoxicity of DAS alone and CER alone in the corresponding cell lines was used as a control.

[0353] The detailed procedures for cell culture, drug treatment, cytotoxicity determination by the MTT method, and calculation of the CI value have been described in Example 9. Figure 2B A representative graph showing the relationship between the CI value and cell growth inhibition (i.e., the fraction of affected cells, Fa) at different DAS / CER ratios evaluated in the HCC827 cell line is presented. It was found that a synergistic effect on tumor cell growth inhibition was obtained at the following drug molar ratios: DAS:CER = 2.5:1, 1:1, and 1:2.5.

[0354] Example 12

[0355] In vitro evaluation of the synergistic effect of carfilzomib combined with doxorubicin in the treatment of cancer cells

[0356] To determine the molar ratio of carfilzomib and doxorubicin (CAR / DOX) with synergistic effects, we tested the cytotoxic effects of CAR / DOX at different drug ratios in vitro cancer cell lines. In the H929 myeloma cell line, the cytotoxic effects were determined at CAR / DOX molar ratios of 1:100, 1:250, and 1:500, respectively. The cytotoxic effects of CAR alone and DOX alone in the corresponding cell lines were used as controls.

[0357] For the specific procedures of cell culture, drug treatment, MTT assay for detecting cytotoxicity, and calculation of the CI value, see Example 9. Figure 2C A representative graph showing the relationship between the CI value and the cell growth inhibition rate (i.e., the fraction of affected cells, Fa) at different CAR / DOX ratios evaluated in the H929 cell line is presented. It was found that when the CAR / DOX molar ratio was 1:500, a synergistic tumor cell growth inhibitory effect was observed.

[0358] Example 13

[0359] Preparation of carfilzomib and doxorubicin co - loaded liposomes

[0360] For the specific preparation steps of the drug - loaded liposomes, see Example 2. Sodium SBE - β - cyclodextrin was used as a solubilizer. In the drug - loading step, carfilzomib and doxorubicin were dissolved in a citrate buffer containing 100 mg / ml sodium SBE - β - cyclodextrin according to the molar ratios shown in Table 7, and then the drug solution was mixed with the liposome suspension, and drug - loading was carried out according to the method of Example 2. In this example, doxorubicin is highly soluble in water, while carfilzomib is poorly soluble in water. During the drug - loading process, the external medium pH was 3.50. TEA - SOS was used as the encapsulating agent for the encapsulation of this combination drug, and the product characterization results are shown in Table 7. The final liposomal drug product contains the following composition: (1) Lipids: DSPC (58.5 mol%), cholesterol (39.5 mol%), mPEG - DSPE (1 mol%), and DSPG (1 mol%); (2) 100 mg / ml sodium SBE - β - CD; (3) 10 mM citric acid (pH 6.50). High drug encapsulation rates were obtained for both compounds.

[0361] Table 7. Physicochemical properties of carfilzomib (CAR) and doxorubicin (DOX) co - loaded liposomes, with the following lipid composition: DSPC, cholesterol, mPEG - DSPE, and DSPG.

[0362]

[0363] Example 14

[0364] Preparation of dasatinib and ceritinib co - loaded liposomes

[0365] For the specific preparation method of the drug-loaded liposomes, see Example 2. SBE-β-cyclodextrin sodium salt was used as a solubilizer. In the drug-loading step, dasatinib and ceritinib were dissolved in a citrate buffer containing 100 mg / ml SBE-β-cyclodextrin sodium salt according to the molar ratio shown in Table 8, and then the drug solution was mixed with the liposome suspension, and drug loading was carried out according to the method of Example 2. During the drug-loading process, the pH of the external medium was 3.50. In this example, both compounds were poorly soluble compounds. Different trappers (i.e., TEA-SOS, TEA-SBE-β-CD, and TEA-Phytate) were used for the encapsulation of the combined drug, and the product characterization results are shown in Table 8. The final liposomal drug product contained the following composition: (1) DSPC (58.5 mol%), cholesterol (39.5 mol%), mPEG-DSPE (1 mol%), and DSPG (1 mol%); (2) 100 mg / ml SBE-β-CD sodium salt; and (3) a buffer with a pH value of 6.50. The TEA-SOS trapper had a high drug encapsulation rate for both compounds.

[0366] Table 8. Physicochemical properties of dasatinib (DAS) and ceritinib (CER) co-loaded liposomes with the following lipid composition: DSPC, cholesterol, mPEG-DSPE, and DSPG.

[0367]

[0368]

[0369] Example 15

[0370] Preparation of afatinib and dasatinib co-loaded liposomes

[0371] The specific preparation steps of the drug-loaded liposomes are shown in Example 2. SBE-β-cyclodextrin sodium salt was used as a solubilizer. In the drug-loading step, afatinib and dasatinib were dissolved in a citrate buffer containing 100 mg / ml SBE-β-cyclodextrin sodium salt according to the molar ratio shown in Table 9, and then the drug solution was mixed with the liposome suspension, and drug loading was carried out according to the method of Example 2. In this example, afatinib is soluble in water and dasatinib is insoluble in water. During the drug-loading process, the external medium pH was 4.0. TEA-SOS was used as an encapsulating agent for the encapsulation of the combined drug, and the product characterization results are shown in Table 9. The final liposomal drug product composition was as follows: (1) DSPC (58.5 mol%), cholesterol (39.5 mol%), mPEG-DSPE (1 mol%) and DSPG (1 mol%); (2) 100 mg / ml SBE-β-CD sodium salt; (3) a buffer with a pH value of 6.50. Higher drug encapsulation rates were obtained for both compounds.

[0372] Table 9. Physicochemical properties of afatinib (AFA) and dasatinib (DAS) co-loaded liposomes with the following lipid composition: DSPC, cholesterol, mPEG-DSPE and DSPG.

[0373]

[0374] Example 16

[0375] Application of HP-β-CD as a solubilizer for the remote loading of carfilzomib

[0376] The detailed preparation method of the drug-loaded liposomes is shown in Example 2. In this example, hydroxypropyl-β-cyclodextrin (HP-β-CD) was used as a solubilizing agent, and the solubilization and drug-loading results were compared with those of SBE-β-CD (Table 9). The composition of all liposomes in Table 10 was as follows: (1) DSPC (58.5 mol%), cholesterol (39.5 mol%), mPEG-DSP (1 mol%) and DSPG (1 mol%); (2) 100 mg / ml HP-β-CD or 100 mg / mL SBE-β-CD sodium salt; (3) a buffer with a pH value of 6.50. During the drug-loading process, the external medium pH value was 4.0. The results (Table 10) showed that significantly higher EE% (i.e., 95%) could be obtained using SBE-β-CD, while the drug loading using HP-β-CD was only 57%. In addition, the preparation using SBE-β-CD showed a narrower liposome particle size distribution (i.e., 0.037). In contrast, the preparation using HP-β-CD showed a very wide particle size distribution (i.e., 0.341), indicating that HP-β-CD may cause liposome aggregation and have a negative impact on the stability and morphology of liposomes.

[0377] Table 10. Influence of solubilizer type on the encapsulation of carfilzomib (CAR).

[0378]

[0379] Example 17

[0380] Loading carfilzomib into liposomes under controlled pH

[0381] In this formulation, the inner compartment of the liposome contains a buffer agent, which aims to improve the product stability. Example 2 describes the detailed preparation steps of the drug-loaded liposomes. Among them, 65 mM TEA-SOS is used as a trap, and the inner compartment of the liposome also contains 25 mM HEPES buffer with a pH of 7.0. The lipid composition contains the following components: DSPC (59 mol%), cholesterol (40 mol%), and mPEG-DSPE (1 mol%). For drug loading, 100 mg / ml SBE-β-CD sodium salt and 10 mM citric acid are used to dissolve carfilzomib. After the drug loading step, the encapsulation efficiency reaches 93%. Then, the free drug removal process is applied by dialyzing the product in a HEPES buffer solution of pH 7.0 plus 0.9% NaCl. After the free drug removal step, the drug encapsulation efficiency of the final product is increased to 99.2%. The final drug product contains 1 mg / mL carfilzomib and 4 mg / mL total lipid. Analyzed by a Zetasizer particle size analyzer based on dynamic light scattering, its average particle size is 98.4 nm, and the polydispersity index is 0.044. The morphology of carfilzomib liposomes is characterized by cryo-TEM, and representative images are shown in Figure 3A and Figure 3B as shown. The dark area inside the liposome core reflects the encapsulation of the drug compound.

[0382] Example 18

[0383] Effect of the pH inside the liposome on the stability of carfilzomib

[0384] Prepare aqueous core carfilzomib liposomes containing 65 mM TEA-SOS and pH 5.20 according to the method described in Example 2. Prepare aqueous core carfilzomib liposomes containing 65 mM TEA-SOS and 25 mM HEPES and pH 7.0 based on the method described in Example 17. All other formulation components and process conditions of the above two formulations are the same. In both cases, the lipid composition contains the following components: DSPC (59 mol%), cholesterol (40 mol%), and mPEG-DSPE (1 mol%). In addition, the sodium salt of SBE-β-CD is used as a solubilizer for drug loading. As shown in Table 11, a total of 1.8% drug-related impurities were obtained in the final drug product at an internal pH of 5.20. In contrast, no drug-related impurities were found in the product when the pH value inside the liposome was 7.0.

[0385] Table 11. Effect of the pH value of the internal aqueous center of liposomes on the stability of carfilzomib

[0386]

[0387] Example 19

[0388] Pharmacokinetics (PK) study of carfilzomib liposomes in mice

[0389] Prepare carfilzomib-loaded liposomes according to the method of Example 17. Conduct in vivo pharmacokinetic studies in BALB / c nude mice. Inject the carfilzomib drug solution (free drug) and liposomal carfilzomib into the mice via the tail vein, and monitor the change in plasma concentration of carfilzomib in the two formulations over time by liquid chromatography-mass spectrometry (LC-MS). The injection dose of both drug formulations is 5.0 mg / kg. After intravenous administration, collect blood at predetermined time points (3 mice per time point) and place it in EDTA-coated microcontainers. Then centrifuge the samples to separate the plasma. Quantitatively determine the plasma concentration of carfilzomib by liquid chromatography-mass spectrometry (LC-MS). Then calculate the pharmacokinetic (PK) parameters based on the measured change in drug plasma concentration over time.

[0390] The pharmacokinetic results are as shown in the following table and Figure 4 indicated. Compared with the free drug solution, the in vivo half-life (T 1 / 2 ) of liposomal carfilzomib was extended by 10 times. Compared with the free drug solution, the volume of distribution (Vd ss ) of liposomal carfilzomib decreased by 152 times. In addition, the AUC of the former was also increased by 665 times compared with the latter. Overall, compared with the free drug solution, the blood circulation time of liposomal carfilzomib was significantly extended.

[0391] Table 12. Key PK parameters of free carfilzomib and liposomal carfilzomib in mice

[0392]

[0393]

[0394] The foregoing embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The above embodiments and examples can be variously modified. Since those skilled in the art will obviously make various modifications and variations to the above embodiments and examples based on the content of the present invention, such modifications and variations are within the spirit and scope of the present invention. All cited patent or non-patent documents are incorporated herein by reference in their entirety, but are not regarded as prior art.

Claims

1. A drug-loaded liposome particle comprising a core and an outer lipid bilayer membrane, wherein the lipid bilayer membrane comprises an inner layer having an inner surface surrounding the core and an outer layer forming the outer surface of the liposome particle; wherein the core comprises an aqueous liquid medium and one or more active pharmaceutical ingredients encapsulated by the bilayer membrane, wherein at least one of the active pharmaceutical ingredients has poor water solubility; wherein the aqueous liquid medium of the core comprises a trapping agent and optionally a buffer; and wherein the drug-loaded liposome particle has an average particle size between 10 nm and 450 nm, optionally between 25 nm and 300 nm or between 50 nm and 200 nm.

2. The drug-loaded liposome particle according to claim 1, wherein the lipid bilayer membrane comprises: a) phospholipids selected from phosphatidylcholine (e.g., HSPC, DSPC, DPPC, and DMPC), phosphatidylglycerol (e.g., DSPG, DPPG, and DMPG), phosphatidylinositol, glyceroglycolipids, glycosphingolipids (e.g., sphingomyelin), and combinations thereof, wherein the amount of the phospholipids is at least 10 mol% of the total lipids present in the liposome particle; b) cholesterol or a derivative thereof, in an amount of 5 mol% to 50 mol% of the total lipids present in the liposome particle; and c) a conjugated lipid that inhibits liposome aggregation, in an amount of 0 mol% to 10 mol% of the total lipids present in the liposome particle.

3. The drug-loaded liposome particle according to claim 2, wherein the conjugated lipid that inhibits liposome aggregation comprises a polyethylene glycol (PEG)-lipid conjugate.

4. The drug-loaded liposome particle according to claim 3, wherein the average molecular weight of the PEG is in the range of about 1,500 daltons to about 2,500 daltons; and wherein optionally, the PEG-lipid conjugate is mPEG2000- DSPE or PEG2000-DMG.

5. The drug-loaded liposome particle according to any one of claims 1 to 4, wherein the buffer is selected from acetic acid, citric acid, histidine, HEPES, lactic acid, succinic acid, phosphoric acid, tris(hydroxymethyl)aminomethane (Tris), salts thereof, and combinations thereof.

6. The drug-loaded liposome particle according to any one of claims 1 to 5, wherein the liquid medium in the core comprises a trapping agent, optionally selected from ammonium sulfate; ammonium salts or substituted ammonium salts of polyanionic sulfobutylether cyclodextrin; ammonium salts or substituted ammonium salts of polyanionic sulfated carbohydrates; ammonium salts or substituted ammonium salts of polyphosphoric acid; metal salts; and combinations thereof.

7. The drug-loaded liposome particles according to claim 6, wherein the ammonium salts of the polyanionized sulfobutylether cyclodextrin are selected from TEA-SBE-α-cyclodextrin, TEA-SBE-β-cyclodextrin, TEA-SBE-γ-cyclodextrin, Tris-SBE-α-cyclodextrin, Tris-SBE-β-cyclodextrin, Tris-SBE-γ-cyclodextrin; the ammonium salts of the polyanionized sulfated carbohydrate are selected from TEA-SOS, Tris-SOS; the ammonium salts of the polyphosphoric acid are selected from triethylammonium inositol hexaphosphate, tris(hydroxymethyl)aminomethane inositol hexaphosphate; and the metal salts are selected from acetates, carbonates, citrates, halides, sulfates, and gluconates of calcium, copper, zinc, magnesium, manganese, nickel, and cobalt.

8. The drug-loaded liposome particles according to any one of claims 1 to 7, wherein the active pharmaceutical ingredient is selected from afatinib, abemaciclib, abiraterone, acalabrutinib, alectinib, amitinib, alpelisib, anlotinib, apatinib, avapritinib, axitinib, baricitinib, belinostat, binimetinib, bortezomib, bosutinib, brigatinib, bupivacaine, cabozantinib, capecitabine, carfilzomib, crizotinib, dabrafenib, dacomitinib, dasatinib, delanzomib, docetaxel, doxorubicin, duvelisib, enasidenib, encorafenib, entrectinib, erdafitinib, erlotinib, everolimus, fedratinib, fostamatinib, fruquintinib, gefitinib, gemcitabine, gilteritinib, glasdegib, icotinib, ibrutinib, idarubicin, idelalisib, imatinib,ivosidenib, ixazomib, ixabepilone, lapatinib, larotrectinib, lenalidomide, lenvatinib, lorlatinib, marizomib, mitolactol, mitoxantrone, neratinib, nidrasodil, nilotinib, nintedanib, niraparib, olaparib, oprozomib, osimertinib, paclitaxel, palbociclib, panobinostat, pazopanib, pemetrexed, pemigatinib, pexidartinib, ponatinib, pralsetinib, quizartinib, radotinib, regorafenib, ribociclib, ripretinib, rivastigmine, romidepsin, rucaparib, ruxolitinib, selpercatinib, selumetinib, sirolimus, sonidegib, sorafenib, sunitinib, talazoparib, tazemetostat, temsirolimus, tepotinib, tivozanib, tofacitinib, topotecan, trametinib, tucatinib, tucidinostat, upadacitinib, vandetanib, vemurafenib, venetoclax, vinorelbine, vismodegib, vorinostat, zanubrutinib, and their free bases, pharmaceutical salts, derivatives, and mixtures.

9. The drug-loaded liposome particles according to any one of claims 1 to 7, wherein the active pharmaceutical ingredient is selected from: a) Carfilzomib encapsulated alone; b) Dasatinib encapsulated alone; c) Ceritinib encapsulated alone; d) Carfilzomib and doxorubicin encapsulated together; e) Dasatinib and ceritinib encapsulated together; f) Co-encapsulated afatinib and dasatinib; g) Carfilzomib and doxorubicin in a molar ratio of from about 1:50 to about 1:1000; h) Dasatinib and ceritinib in a molar ratio of from about 30:1 to about 1:30; and i) Afatinib and dasatinib in a molar ratio of from about 30:1 to about 1:

30.

10. A pharmaceutical composition comprising the drug-loaded liposome particles according to any one of claims 1 to 9 and a liposome dispersion liquid medium.

11. The pharmaceutical composition according to claim 10, wherein the liposome dispersion liquid medium comprises water, a buffer, and an osmotic pressure regulator, wherein optionally, the buffer is selected from acetic acid, citric acid, histidine, HEPES, lactic acid, succinic acid, phosphates, tromethamine (Tris), and their salts; and wherein optionally, the osmotic pressure regulator is selected from sucrose, glucose, mannitol, trehalose, and sodium chloride.

12. The pharmaceutical composition according to claim 10 or 11, wherein two or more active pharmaceutical ingredients are co-encapsulated in the core of the liposome particles and can be released to act in a synergistic manner to exert efficacy, wherein the synergistic manner includes that after the pharmaceutical composition is administered to a subject, the active pharmaceutical ingredients can maintain a synergistic molar ratio in the blood for at least one hour; and optionally wherein the synergistic molar ratio is such a molar ratio that when provided to cancer cells associated with cancer in an in vitro assay, within a drug concentration range of about 0.20 to about 0.80 for cell growth inhibition (i.e., the fraction of affected cells is in the range of about 20% to about 80%), it exhibits at least a 20% synergistic effect within the cell growth inhibition range.

13. A method of treating a disease or condition of a subject in need of treatment with a therapeutic agent, the method comprising administering to the subject a therapeutically effective amount of the drug-loaded liposome particles according to any one of claims 1 to 9 or the pharmaceutical composition according to any one of claims 10 to 12.

14. The method according to claim 13, wherein the subject is a cancer patient in need of treatment with two or more cancer drugs in a synergistic manner; wherein the cancer is bladder cancer (including accelerated and metastatic bladder cancer), breast cancer (e.g., estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer; HER-2 positive breast cancer; HER-2 negative breast cancer, progesterone receptor positive breast cancer, progesterone receptor negative breast cancer; estrogen receptor negative, HER-2 negative and progesterone receptor negative breast cancer (i.e., triple negative breast cancer); inflammatory breast cancer), colon cancer (including colorectal cancer), kidney cancer (e.g., transitional cell carcinoma), liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer, lung adenocarcinoma and squamous cell carcinoma). Genitourinary tract, such as ovary (including fallopian tube cancer and peritoneal cancer), cervical cancer, prostate cancer, testicular cancer, kidney cancer and ureteral cancer, lymphatic system cancer, rectal cancer, laryngeal cancer, pancreatic cancer (including pancreatic exocrine cancer), esophageal cancer, gastric cancer, gallbladder cancer, thyroid cancer, skin cancer (including squamous cell carcinoma), brain cancer (including glioblastoma multiforme), head and neck cancer (e.g., occult primary cancer) and soft tissue cancer (e.g., Kaposi's sarcoma (e.g., AIDS-related Kaposi's sarcoma), leiomyosarcoma, angiosarcoma and histiocytoma), multiple myeloma, and chronic myeloid leukemia.

15. A method for preparing liposomes loaded with one or more active pharmaceutical ingredients, comprising the following steps: a) preparing a lipid dispersion in a solution containing a trapping agent and, optionally, a buffer to form a suspension containing liposome particles; b) reducing the liposome particle size by heating the suspension to a high temperature (50 °C or above); c) substantially removing the trapping agent in the suspension outside the liposomes to obtain drug-free liposomes; d) dissolving one or more active pharmaceutical ingredients (APIs) in an aqueous solution in the presence of a solubilizing agent to obtain an API solution; e) incubating the drug-free liposomes in step c) with the API solution including the solubilizing agent in step d) at an elevated temperature (50 °C or above), thereby forming liposome particles, the liposome particles comprising an aqueous core loaded with one or more APIs, the API being encapsulated by a lipid bilayer membrane, wherein the drug-loaded liposome particles are suspended in an external liquid medium; f) optionally, further removing unloaded drug molecules and solubilizing agents outside the liposome particles obtained in step e) by dialysis, ultracentrifugation or / and size exclusion chromatography; and g) optionally, forming dry liposome particles loaded with one or more APIs by lyophilizing the liposome particles obtained in step e) or step f).

16. The method according to claim 15, wherein the solubilizing agent is selected from cyclodextrin and its derivatives (such as sulfobutyl ether-β-cyclodextrin or hydroxypropyl-β-cyclodextrin), polyvinylpyrrolidone, polyethylene glycol and its derivatives, sorbitol, nonionic surfactants, and their salts, or a combination thereof.

17. In the method according to claim 15, wherein at the start of the drug loading step e), the pH value of the liquid medium of the inner core of the liposome is in the range of about 5.0 to about 10.0, and the pH value of the external medium outside the liposome particles is in the range of about 2.0 to about 5.0; and the step e) of incubating the unloaded liposomes with the drug solution results in at least 50% of the total API being encapsulated within the aqueous inner core of the liposome particles and less than 50% of the total API molecules being present in the external liquid medium.

18. A therapeutic kit comprising a first container containing a plurality of drug-loaded liposome particles as described in any one of claims 1 to 9, and a second container containing a liposome dispersion liquid medium, wherein, The drug-loaded liposome particles and the liposome dispersion liquid medium can be mixed in the first container or the second container to form a dispersion for administration to a subject in need of treatment; or, alternatively, comprise a container containing the pharmaceutical composition according to any one of claims 10 to 12 for administration to a subject in need of treatment.

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