Liposomal compositions comprising a weak acid drug and uses thereof

Abstract

The present invention relates to pharmaceutical compositions comprising a weak acid drug, wherein bicarbonate is used to achieve high incorporation of the drug into liposomes and better therapeutic efficacy. Also disclosed are methods of using the pharmaceutical compositions disclosed herein to treat respiratory diseases.

Description

[0001] This invention is a divisional application of the patent application filed on July 23, 2018, with application number "201880049079.3" and invention title "Liposome Composition Containing a Weak Acid Drug and Its Use Thereof".

[0002] Cross-referencing of related applications

[0003] This application claims the benefits of U.S. Application No. 62 / 536,034, filed July 24, 2017, and U.S. Application No. 62 / 595,207, filed December 6, 2017, the entire disclosure of which is incorporated herein by reference. Technical Field

[0004] This invention relates to pharmaceutical compositions having high drug encapsulation or loading, methods for manufacturing the same, and uses of the pharmaceutical compositions disclosed herein.

[0005] Prior Technology

[0006] Liposomes are vesicles formed by a lipid bilayer containing an internal aqueous medium. Liposomes have been used as carriers for various therapeutic agents to provide improved delivery properties, such as enhanced blood circulation time, reduced cytotoxicity, sustained drug release, and delivery of specific drugs to selected tissues. When using liposomes for therapeutic drug delivery, a high drug encapsulation rate is required.

[0007] Currently, many drug loading methods are available for incorporating drugs into liposomes. For weakly acidic drugs, liposomes with a higher inside / lower outside pH gradient have been disclosed for drug loading, for example, in US Patent No. 5,939,096. The pH gradient is established by weak acid salts, including carboxylic acids such as formic acid, acetic acid, propionic acid, butyric acid, valeric acid, and their substituted derivatives. WO 96 / 25147 discloses the use of bicarbonate for drug loading. However, later studies have suggested that the use of bicarbonate in liposomal formulations can lead to gas buildup, which can damage and destabilize liposomes and cause premature drug release (Nahire). and a l. pH-triggered echogenicityand contents release from liposomes, Mol Pharm. 2014 Nov 3;11(11):4059-68; and Chen et alA thermoresponsive bubble-generating liposomal system for triggering Localized Extracellular Drug Delivery, ACS Nano. 2013 Jan 22;7(1):438-46), due to its destructive and unstable effects on liposomes, it is recommended that bicarbonate be avoided as a loading agent.

[0008] Many problems remain unresolved regarding the use of conventional liposomes for weakly acidic drugs, including poor drug loading and inadequate control of release rates. The need for liposome suspensions with high drug loading efficiency to increase therapeutic efficacy remains unmet. This invention addresses this and other needs. Summary of the Invention

[0009] In one embodiment, the present invention discloses a pharmaceutical composition comprising one or more liposomes, the liposomes comprising (a) a lipid bilayer comprising at least one vesicle-forming lipid; and (b) an internal aqueous medium within the lipid bilayer comprising a bicarbonate and a weakly acidic drug, wherein the liposomes are suspended in an external medium, and the molar ratio of the drug to the bicarbonate is about 0.1:1 to 1:1.

[0010] Another embodiment of the invention provides a pharmaceutical composition comprising one or more liposomes, the liposomes comprising (a) a lipid bilayer comprising at least one vesicle-forming lipid; and (b) an internal aqueous medium within the lipid bilayer comprising a bicarbonate and a weakly acidic drug, wherein the liposomes are suspended in an external medium, and the concentration of the bicarbonate is from about 50 mM to less than about 1000 mM.

[0011] A third embodiment of the present invention provides a pharmaceutical composition comprising one or more liposomes, the liposomes comprising (a) a lipid bilayer comprising at least one vesicle-forming lipid; and (b) an internal aqueous medium within the lipid bilayer comprising a bicarbonate and a weakly acidic drug, wherein the liposomes are suspended in an external medium, and the pH of the external medium is higher than the pKa of the weakly acidic drug.

[0012] A fourth embodiment of the present invention provides a pharmaceutical composition comprising one or more liposomes, the liposomes comprising (a) a lipid bilayer comprising at least one vesicle-forming lipid; and (b) an internal aqueous medium within the lipid bilayer comprising bicarbonate and prostacyclin, wherein the liposomes are suspended in an external medium, and the molar ratio of prostacyclin to bicarbonate is from about 0.1:1 to about 1:1.

[0013] A fifth embodiment of the present invention provides a pharmaceutical composition comprising one or more liposomes, the liposomes comprising (a) a lipid bilayer comprising at least one vesicle-forming lipid; and (b) an internal aqueous medium within the lipid bilayer comprising bicarbonate and prostaglandin, wherein the liposomes are suspended in an external medium, and the concentration of bicarbonate is from about 50 mM to less than about 1000 mM.

[0014] In yet another embodiment, the pharmaceutical composition comprises one or more liposomes, each liposome comprising (a) a lipid bilayer containing at least one vesicle-forming lipid; and (b) an internal aqueous medium within the lipid bilayer containing bicarbonate and prostaglandin, wherein the pH of the external medium is higher than the pKa of the provided prostaglandin.

[0015] The present invention further provides a method for preparing the pharmaceutical composition disclosed herein, comprising the following steps: (i) Prepare a lipid solution by forming a lipid using at least one vesicle; (ii) The lipid solution from step (i) is mixed with bicarbonate to form at least one liposome comprising a lipid bilayer and an inner aqueous medium, wherein the liposome is suspended in an outer medium. (iii) Adjusting the bicarbonate concentration in the external medium to produce a lower pH in the external medium of the liposomes and a higher pH in the internal aqueous medium of the liposomes, wherein the pH of the external medium is higher than the pKa of the weak acid drug; and (iv) Add a weak acid drug to the external medium.

[0016] A method for treating respiratory diseases is also provided, comprising the step of administering the pharmaceutical composition disclosed herein to an individual in need.

[0017] The present invention also provides the pharmaceutical compositions disclosed herein for the treatment and / or preventive treatment of respiratory diseases.

[0018] The present invention also includes the use of a pharmaceutical composition disclosed herein for the manufacture of a medicament for the treatment and / or preventive treatment of respiratory diseases.

[0019] The following description will set forth details of one or more embodiments of the invention. Other features or advantages of the invention will become apparent from the detailed description of the following embodiments and from the appended claims. The inclusion of these terms in the description should not be construed as limiting the subject matter of the application described herein or limiting the meaning or scope of the following claims. The summary is a high-level overview of various aspects of the invention and leads to some concepts further described in the detailed description section below. The summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used alone to determine the scope of the claimed subject matter. The subject matter should be understood by referring to the appropriate portions of the specification and the claims throughout.

[0020] 1. A pharmaceutical composition comprising: One or more liposomes, the liposomes comprising: (a) A lipid bilayer comprising at least one vesicle-forming lipid; and (b) The internal aqueous medium within the lipid bilayer, which contains bicarbonate and a weak acid drug, The lipid system is suspended in an external medium, and the concentration of the bicarbonate is from about 50 mM to less than about 1000 mM.

[0021] 2. The pharmaceutical composition of claim 1, wherein the molar ratio of the weak acid drug to the bicarbonate is about 0.1:1 to about 1:1.

[0022] 3. The pharmaceutical composition of claim 1, wherein the pH of the external medium is higher than the pKa of the weak acid drug.

[0023] 4. The pharmaceutical composition of claim 1, wherein the concentration of the bicarbonate is from about 250 mM to about 800 mM.

[0024] 5. The pharmaceutical composition of claim 1, wherein the bicarbonate provides a pH gradient between the internal aqueous medium and the external medium, and the pH of the internal aqueous medium is at least one unit higher than the pH of the external medium.

[0025] 6. The pharmaceutical composition of claim 1, wherein the coating of the weak acid drug is at least 80%.

[0026] 7. The pharmaceutical composition of claim 1, wherein the vesicle-forming lipid is a mixture of a first phospholipid and a second phospholipid, or a mixture of the first phospholipid and a charged lipid.

[0027] 8. The pharmaceutical composition as described in item 7, wherein the first phospholipid is selected from phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylserine (PS), and any combination thereof, and the second phospholipid is a polyethylene glycol (PEG) modified phospholipid, a positively charged phospholipid, or a negatively charged phospholipid, and the charged lipid is a positively charged lipid or a negatively charged lipid.

[0028] 9. The pharmaceutical composition of claim 8, wherein the first phospholipid is selected from hydrogenated soybean phosphatidylcholine (HSPC), distearyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), or combinations thereof, and the second phospholipid is selected from distearyl phosphatidylglycerol (DSPG), dipalmitoyl phosphatidylglycerol (DPPG), dimyristoyl phosphatidylglycerol (DMPG), polyethylene glycol distearyl phosphatidylethanolamine (PEG-DSPE), or combinations thereof.

[0029] 10. The pharmaceutical composition of claim 8, wherein the first phospholipid is selected from hydrogenated soybean phosphatidylcholine (HSPC), distearylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), or a combination thereof, and the charged lipid is stearylamine, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (3 β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol), N4-Cholesteryl-Spermine (GL67), dimethyldioctadecylammonium (DDAB), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), ethylphosphocholine (ethyl PC), or combinations thereof.

[0030] 11. The pharmaceutical composition described in paragraph 7, further comprising a sterol selected from cholesterol, cholesterol hemisuccinate, ergosterol, lanosterol, and combinations thereof.

[0031] 12. The pharmaceutical composition of claim 11, wherein the molar percentage of the first phospholipid:cholesterol:the second phospholipid or the charged lipid is 50-70:20-45:0.1-10.

[0032] 13. The pharmaceutical composition of claim 1, wherein the bicarbonate is selected from potassium bicarbonate, sodium bicarbonate, calcium bicarbonate, magnesium bicarbonate, cesium bicarbonate, lithium bicarbonate, nickel bicarbonate, ferrous bicarbonate, or combinations thereof.

[0033] 14. The pharmaceutical composition of claim 1, wherein the weak acid drug is a prostaglandin, a steroid, a nonsteroidal anti-inflammatory drug (NSAID), or an anticoagulant.

[0034] 15. A method for treating a respiratory disease, comprising the step of administering a pharmaceutical composition comprising one or more liposomes, wherein the liposomes comprise: (a) a lipid bilayer comprising at least one vesicle-forming lipid; and (b) The internal aqueous medium within the lipid bilayer, which contains bicarbonate and a weak acid drug, The liposome is suspended in an external aqueous medium, and the concentration of the bicarbonate is from about 50 mM to less than about 1000 mM.

[0035] 16. A pharmaceutical composition comprising one or more liposomes, the liposomes comprising: (a) a lipid bilayer comprising at least one vesicle-forming lipid; and (b) The internal aqueous medium within the lipid bilayer, which contains bicarbonate and prostaglandin, The liposome is suspended in an external aqueous medium, and the concentration of the bicarbonate is from about 50 mM to less than about 1000 mM. Implementation

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] As used in this article, the articles “a” and “an” refer to one or more (i.e., at least one) grammatical objects. For example, “an element” means one or more elements.

[0038] The terms "comprise" or "comprising" are generally used to indicate the inclusion or inclusion of one or more features, components, or elements. The terms "comprise" or "comprising" encompass the terms "consists" or "consisting of."

[0039] All figures are modified by the term "about". As used in this article, "about" refers to a range of ±10% of a specific value.

[0040] The term "subject" can refer to a vertebrate with a respiratory disease, or a vertebrate believed to require treatment for a respiratory disease. Subjects include warm-blooded mammals, such as primates, and preferably humans. Non-human primates are also considered subjects. The term "subject" includes domesticated animals such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, goats), and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs). Therefore, this article considers veterinary uses and pharmaceutical preparations.

[0041] As used herein, "substantially free" means that the pharmaceutical composition contains less than 5%, 4%, 3%, 2%, or 1% of the specific substance. In some embodiments, the pharmaceutical composition does not contain the specific substance.

[0042] As used herein, "liposome" refers to a microscopic vesicle or particle consisting of one or more lipid bilayers surrounding an internal aqueous medium. For liposome formation to occur, at least one "vesicle-forming lipid" is required, which is an amphipathic lipid capable of forming or incorporating into the lipid bilayer. Any suitable vesicle-forming lipid can be used to form the lipid bilayer that constitutes the liposome. Vesicle-forming lipids include, but are not limited to, phospholipids, such as phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylethanolamine (PE), or phosphatidylserine (PS), as well as charged lipids, such as positively or negatively charged lipids.

[0043] The lipid bilayer of the liposome comprises at least one vesicle-forming lipid and a sterol selected from, but not limited to, cholesterol, cholesterol hemisuccinate, ergosterol, lanosterol, and combinations thereof. In an exemplary embodiment, the sterol is cholesterol.

[0044] In some embodiments, the vesicle-forming lipid is a mixture of a first phospholipid and a second phospholipid. In some embodiments, the first phospholipid is phosphatidylcholine (PC), selected from the group comprising hydrogenated egg phosphatidylcholine (HEPC), hydrogenated soybean phosphatidylcholine (HSPC), dipalmitoylphosphatidylcholine (DPPC), distearyloylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine, dimyristoylphosphatidylcholine (DMPC), egg phosphatidylcholine (EPC), soybean phosphatidylcholine (SPC), and oleoylpalmitoylphosphatidylcholine. Palmitoylphosphatidylcholine, dioleoyl phosphatidylcholine (DOPC), dipetroselinoyl phosphatidylcholine, palmitoylelaidoyl phosphatidylcholine, palmoyloleoyl phosphatidylcholine, dilauroyl phosphatidylcholine (DLPC), diundecanoyl phosphatidylcholine, didecanoyl phosphatidylcholine, dinonanoyl phosphatidylcholine, and combinations thereof.In other embodiments, the second phospholipid is a polyethylene glycol modified phospholipid having a molecular weight of about 500 Daltons to about 10,000 Daltons, such as 1,2-disteaarolyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (1,2-distearoly-). sn -glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000], DSPE-PEG2000), and negatively charged phospholipids, such as distearyloyl phosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG), or dioleoylphosphatidylglycerol (DOPG). In exemplary embodiments, the molar percentage of the first phospholipid:cholesterol:second phospholipid is 50-70:20-45:0.1-10, 50-70:20-45:0.5-8, or 55-65:25-40:1-6.

[0045] In other embodiments, the vesicle-forming lipid is a mixture of a first phospholipid and a charged lipid. In an exemplary embodiment, the vesicle-forming lipid is a mixture of a first phospholipid, a second phospholipid, and a charged lipid. The charged lipid comprises stearamide, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol), N... 4 -cholesterol-spermine (N 4-Cholesteryl-Spermine (GL67), dimethyldioctadecylammonium (DDAB), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), ethylphosphocholine (ethyl PC), or combinations thereof. In another exemplary embodiment, the molar percentage of the first phospholipid:cholesterol:charged lipid is 50-70:20-45:0.1-10, 50-70:20-45:0.5-8, or 55-65:25-40:1-6.

[0046] In one embodiment, the molar percentage (%) of HSPC, cholesterol, and DSPG in the lipid bilayer is 50-70:20-45:0.1-10, 50-70:20-45:0.1-5, or 55-65:25-40:0.5-8. In another embodiment, the molar percentage (%) of HSPC, cholesterol, and DSPE-PEG2000 in the lipid bilayer is 50-70:20-45:0.1-10, 50-70:20-45:0.1-5, or 55-65:25-40:0.5-8. In yet another embodiment, the molar percentage (%) of DPPC:Chol:DSPE-PEG2000 in the lipid bilayer is 50-70:20-45:0.5-8, 50-70:20-45:0.1-5, or 55-65:25-40:1-6.

[0047] In one embodiment, the lipid bilayer of the liposome may further comprise at least one vesicle-forming lipid and a surfactant, which may be a nonionic surfactant, a cationic surfactant, or a zwitterionic surfactant. Nonionic surfactants have no charged groups in their head form. Cationic surfactants carry a net positive charge in their head, while zwitterionic surfactants are electrically neutral but carry both positive and negative charges on different atoms.

[0048] Non-limiting examples of nonionic surfactants include nonionic water-soluble monoglycerides, nonionic water-soluble diglycerides, and nonionic water-soluble triglycerides; nonionic water-soluble mono- and nonionic water-soluble di-fatty acid esters of polyethylene glycol; nonionic water-soluble dehydrated sorbitan fatty acid esters (e.g., sorbitan monooleate, such as TWEEN 20 (polyoxyethylene 20 sorbitan monooleate), SPAN 80); and nonionic water-soluble triblock copolymers (e.g., poly(ethylene oxide) / poly(propylene oxide) / poly(ethylene oxide) triblock copolymers, such as POLOXAMER 406 (PLURONIC F-127) or derivatives thereof).

[0049] Non-limiting examples of cationic surfactants include dimethyldialkylammonium bromide or dodecyltrimethylammonium bromide.

[0050] Non-limiting examples of zwitterionic surfactants include 3-(N,N-dimethylpalmitolammonio)-propanesulfonate.

[0051] In some embodiments, liposomes are substantially free of ionophores, which are substances that promote the transport of hydrogen ions (H+). + ) or hydroxide ions (OH-) - Compounds transported across liposome membranes.

[0052] Solvents used to dissolve vesicles to form lipids for the preparation of liposomes can be, for example, methanol, ethanol, diethyl ether, and combinations thereof. Alternatively, the solvent can be subsequently removed by supercritical fluid, and preferably used in minimal amounts to reduce the time required for the organic solvent removal step.

[0053] According to the present invention, liposomes are prepared in a medium containing bicarbonate. Specifically, when vesicle-forming lipids are contacted with a medium containing bicarbonate, a liposome suspension is formed.

[0054] As used herein, "bicarbonate salt" is a pharmaceutically acceptable salt compound that preferably comprises both anionic and cationic components of bicarbonate. In one embodiment, the cationic component of the salt compound is a metal. Non-limiting examples of metals include metals of Group IA or IIA, such as potassium (K), sodium (Na), calcium (Ca), magnesium (Mg), cesium (Cs), and lithium (Li), or metals other than Group IA or IIA metals, such as ferrous iron (Fe) and nickel (Ni). Examples of bicarbonates include, but are not limited to, potassium bicarbonate, sodium bicarbonate, calcium bicarbonate, magnesium bicarbonate, cesium bicarbonate, lithium bicarbonate, nickel bicarbonate, ferrous iron bicarbonate, or any combination thereof.

[0055] Liposomes in suspension are reduced in size. The size of a liposome typically refers to its diameter. Liposome size reduction can be achieved by a variety of methods, such as extrusion, sonication, homogenization, or milling, which are well-known and can be performed by someone with ordinary knowledge in the art. Extrusion involves passing liposomes under pressure through a filter with defined pore sizes one or more times. The filter is typically made of polycarbonate, but can also be made of any durable material that does not interact with the liposomes and is strong enough to allow extrusion under sufficient pressure. The size of liposomes can be reduced by sonication, which uses acoustic energy to break down or break down the liposomes, spontaneously transforming them into smaller liposomes. For example, sonication can be performed by immersing a glass tube containing a liposome suspension in the acoustic center of a bath sonicator, or by using a probe sonicator in which acoustic energy generated by the vibration of a titanium probe is brought into direct contact with the liposome suspension. In this invention, liposomes typically have a diameter of about 50 nm to 500 nm, for example, about 500 nm or less, about 400 nm or less, about 300 nm or less, about 200 nm or less, or about 100 nm or less.

[0056] After scaling, the bicarbonate concentration in the external medium is adjusted to provide a pH gradient between the internal aqueous medium and the external medium. This can be done in various ways, such as by exchanging the external medium with a suitable buffer that lacks bicarbonate, such as citrate buffer (H3C6H5O) and phosphate buffer (H3PO4), or by methods such as diafiltration, dialysis, ultrafiltration, or tangential flow filtration.

[0057] In one embodiment, bicarbonate provides a lower pH gradient externally and a higher pH gradient internally between the external medium and the internal aqueous medium of the liposome. In another embodiment, the pH of the internal aqueous medium is at least one unit higher than the pH of the external medium. In yet another embodiment, the pH of the internal aqueous medium is about 7, 8, 9, or 10, and the pH of the external medium is less than 7, less than 6, less than 5, less than 4, less than 3, about 3 to 7, about 3.5 to 6.5, or about 4 to 6. In yet another exemplary embodiment, the pH of the external medium is higher than the pKa of the weak acid drug.

[0058] The prepared liposomes can be stored for extended periods prior to drug loading and administration to an individual. For example, liposomes can be stored under refrigeration for an extended period prior to drug loading. Alternatively, liposomes can be dehydrated, stored, and then rehydrated and loaded with one or more active ingredients (drugs) when needed. Liposomes can also be dehydrated after loading with one or more weakly acidic drugs. Dehydration can be performed by a number of methods available and known in the art. In some embodiments, liposomes are dehydrated using standard freeze-drying equipment, i.e., under low-pressure conditions. Additionally, liposomes can be frozen, for example, using liquid nitrogen. Prior to dehydration, sugars can be added to the liposome environment, for example, to the buffer containing the liposomes, to ensure the stability and integrity of the liposomes during dehydration. Examples of sugars include, but are not limited to, maltose, lactose, sucrose, trehalose, dextrose, sorbitol, mannitol, xylitol, or combinations thereof.

[0059] Liposome suspensions with a lower external / higher internal pH gradient as described above are prepared for drug loading. Typically, a weakly acidic drug to be loaded is added to the external medium of the liposomes and reacted under certain conditions to load the drug into the internal aqueous medium of the liposomes, which allows the weakly acidic drug to diffuse into the internal aqueous medium of the liposomes, until the desired loading concentration and encapsulation ratio (the percentage of internal drug / encapsulated drug relative to the total amount of drug in the composition) are reached.

[0060] As used herein, weak acid drugs, unless otherwise stated or apparent from the context, also comprise pharmaceutically acceptable salts and their protonated forms. In one embodiment, the weak acid drug comprises at least one functional group selected from carboxyl (-COOH), hydroxyl (-OH), phosphate (-PO4), and any combination thereof. In another embodiment, the weak acid drug has a pKa of less than about 7, less than about 6, between 1 and less than about 7, between 2 and less than about 6, between 2 and 6.9, or between 2.5 and 6. In addition to the carboxyl (-COOH), hydroxyl (-OH), and phosphate (-PO4) groups described above, the weak acid drug may also comprise one or more functional groups; these additional functional groups should not significantly alter the drug acidity from the unfunctionalized counterpart. Table 1 shows non-limiting examples of the weak acid drugs of the present invention.

[0061] Table 1. Characteristics of the weak acid drug applicable to the present invention

[0062]

[0063] The present invention further provides a method for manufacturing the pharmaceutical composition disclosed herein, comprising the following steps: (i) Use at least one vesicle to form a lipid to prepare a lipid solution; (ii) The lipid solution from step (i) is mixed with bicarbonate to form at least one liposome comprising a lipid bilayer and an inner aqueous medium, wherein the liposome is suspended in an outer medium; (iii) Adjusting the bicarbonate concentration in the external medium to produce a lower pH in the external medium of the liposomes and a higher pH in the internal aqueous medium of the liposomes, wherein the pH of the external medium is higher than the pKa of the weak acid drug; and (iv) Add a weak acid drug to the external medium.

[0064] In some embodiments, step (i) of the method comprises dissolving at least one vesicle-forming lipid in an organic solvent to form a lipid solution. The vesicle-forming lipid may be a mixture of a first phospholipid and a second phospholipid, or a mixture of a first phospholipid and a charged lipid. In step (ii), the lipid solution is then mixed with an aqueous buffer solution of a metal group bicarbonate to form a suspension comprising an external medium and at least one liposome, wherein the liposome is suspended in the external medium. In step (iii), the concentration of bicarbonate in the external medium is adjusted by replacing it with another buffer solution lacking bicarbonate to create a lower external / higher internal pH gradient between the external medium and the internal medium of the liposome. In step (iv), a weakly acidic drug is added to the external medium of the suspension, and the resulting suspension is reacted under predetermined conditions to load the drug into the internal medium of the liposome.

[0065] In some embodiments, in step (iii) of the method, the concentration of bicarbonate in the internal aqueous medium is from about 50 mM to less than about 1000 mM.

[0066] In some embodiments, in step (iii) of the method, the pH of the external medium is higher than the pKa of prostaglandin. In other embodiments, the pH of the external medium is at least one unit lower than the pH of the internal aqueous medium.

[0067] In some embodiments, in step (iv) of the method, the molar ratio of the drug to bicarbonate in the internal aqueous medium is from about 0.1:1 to 1:1.

[0068] In addition, the present invention provides a method for loading a weak acid drug into the internal medium of a liposome using the freeze-drying or freezing of the liposomes disclosed herein.

[0069] In some embodiments, the concentration of bicarbonate in the internal aqueous medium of the liposome is 50 mM or more, 100 mM or more, 150 mM or more, 200 mM or more, 250 mM or more, 300 mM or more, 350 mM or more, 400 mM or more, 450 mM or more, 500 mM or more, 600 mM or more, 700 mM or more, 800 mM or more, and at most less than 1000 mM. In some embodiments, the concentration of bicarbonate in the aqueous buffer solution is from 50 mM to less than 1000 mM, from 50 mM to 800 mM, from 200 mM to less than 1000 mM, from 200 mM to 800 mM, or from 200 mM to 600 mM, from 250 mM to less than 1000 mM, from 250 mM to 800 mM, or from 250 mM to 600 mM, or from 300 mM to 600 mM. In exemplary embodiments, the concentration of bicarbonate in the internal aqueous medium of the liposomes can be measured by any method known or to be known in the art, or by the following steps: (a) separating the bicarbonate in the external medium from the internal aqueous medium by dialysis or size exclusion chromatography; (b) dissolving the liposome suspension in the external medium, which does not contain bicarbonate, with methanol; and defining the concentration of bicarbonate in the internal aqueous medium by ion chromatography. In some embodiments, the external medium is substantially free of bicarbonate.

[0070] In the embodiments, the pharmaceutical composition of the present invention has a drug coating rate of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.

[0071] In another embodiment, the molar ratio of the drug to the bicarbonate (hereinafter, drug / salt molar ratio) in the internal aqueous medium of the liposome is from about 0.1:1 to about 1:1. In exemplary embodiments, the drug / salt ratio is from about 0.1:1 to 0.9:1, from about 0.1:1 to 0.8:1, from about 0.1:1 to 0.7:1, from about 0.1:1 to 0.6:1, or from about 0.1:1 to 0.5:1.

[0072] In yet another embodiment, the concentration of the weak acid drug encapsulated in the internal medium of the liposome is from 1 mM to 800 mM or more. In some embodiments, the concentration of the drug encapsulated in the internal medium of the liposome is 5 mM or more, 10 mM or more, 20 mM or more, 40 mM or more, 50 mM or more, 80 mM or more, or 100 mM or more, and up to 800 mM.

[0073] According to the present invention, the pharmaceutical compositions described herein have a high drug coating rate, an increased molar ratio of the weak acid drug to bicarbonate of about 0.1 to about 1, and a high concentration of coated drug. Without being bound by any particular theory, it is believed that the pharmaceutical compositions described herein with a high drug coating or drug loading can deliver more drug to the site of action, prolong the effect of the coated drug, and more effectively alleviate the signs or symptoms of disease.

[0074] The pharmaceutical compositions of the present invention, having a high drug encapsulation or high drug loading, as described herein, can be administered in therapeutically effective amounts to individuals in need of treatment for respiratory diseases. Examples of respiratory diseases include, but are not limited to, pulmonary hypertension, chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, lower respiratory tract infections, bronchiectasis, bronchitis, bronchiolitis, or croup. In an exemplary embodiment, the respiratory disease is pulmonary hypertension and the weak acid drug is prostaglandin or a prostaglandin. In another exemplary embodiment, the respiratory disease is COPD and the weak acid drug is a steroid or a prostaglandin. In yet another exemplary embodiment, the respiratory disease is asthma and the weak acid drug is a steroid. In yet another exemplary embodiment, the respiratory disease is a lower respiratory tract infection and the weak acid drug is an antibiotic.

[0075] As used herein, the term "treating" generally refers to the administration or administration of the pharmaceutical composition described herein, containing at least one weak acid drug, to an individual suffering from a respiratory disease, its symptoms or condition, or the course of the disease, for the purpose of curing, healing, alleviating, resolving, altering, remedying, ameliorate, improving, or influencing the disease, its symptoms or condition, its induced disabilities, or its course. The term "therapeutically effective amount," as used herein, refers to the amount of weak acid drug that imparts a therapeutic effect in an individual. Therapeuticly effective amount can vary depending on various factors, such as route and frequency of administration, body weight, and age. Those skilled in the art can determine the dosage for each case based on the disclosure herein, established methods, and their own experience.

[0076] Specifically, the liposome compositions containing weak acid drugs described herein are administered by inhalation, parenteral injection (i.e., intra-arterial, intravenous, intraperitoneal, subcutaneous, intravitreal, intrathecal, intra-articular, intramuscular, intra-articular, intramuscular, intra-articular, or via aerosol dispersal. Aerosol administration methods include intranasal and pulmonary administration. In some embodiments, the liposome compositions of the present invention are administered by a single rapid intravenous injection or infusion into a vein or peritoneum.

[0077] The present invention is further illustrated by the following embodiments, which are provided for illustrative purposes and not for limitation. In view of this disclosure, those skilled in the art will understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention and still obtaining the same or similar results.

[0078] Example 1: Effects of different salts on drug loading

[0079] 1. Materials and Methods

[0080] 1.1 PGE1 liposomes prepared using sodium acetate (NaC2H3O2)

[0081] Liposome colloidal suspensions were prepared using ethanol injection technology. Simply put, 496.8 mg of hydrogenated soybean phosphatidylcholine (HSPC), 154.65 mg of cholesterol, and 42.15 mg of 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) were dissolved in 2.86 ml of ethanol. At 60°C... o Under appropriate stirring, the ethanol lipid solution was injected into 17.4 ml of sodium acetate (200 mM or 400 mM). Liposomes formed upon contact with the sodium acetate. The liposome suspension was then extruded through 200 nm and 100 nm polycarbonate membranes (6 times each). Liposomes with a diameter of approximately 100 nm were obtained and dialyzed against citrate buffer (pH 5.5) to establish a higher internal / lower external pH gradient between the external medium and the internal aqueous medium of the liposomes. The empty liposome suspension was then stored at 4°C prior to drug loading. o C.

[0082] PGE1 (alprostadil) at room temperature (25°C) o C) Add the drug to the liposome suspension described in the preceding paragraph at various drug / lipid molar ratios and hold for 20 minutes. Store the PGE1-loaded liposome suspension at 4°C. o C.

[0083] 1.2 PGE1 liposomes prepared using sodium bicarbonate (NaHCO3)

[0084] Except for using sodium bicarbonate (e.g., 200 mM, 400 mM or other concentrations) instead of sodium acetate (200 mM or 400 mM), prepare liposome suspensions according to the method described in section 1.1.

[0085] 1.3 Quantitative determination of drugs

[0086] The amount of drug encapsulated in liposomes was quantified using a WatersAlliance® HPLC system equipped with a photodiode array (PDA) detector. Simply put, 100 μL aliquots of the sample solution were injected directly into the HPLC system at a flow rate of 1.0 mL / min into a mobile phase of a mixture of methanol and phosphate buffer (6.7 mM, pH 3.0) at a volume ratio of 80:20. Separation was performed in a C18 column (Phenomenex Prodigy 5U ODS-3150 x 4.6 mm), followed by chromatography at 65 °C. oThe reaction proceeds after mixing with 1N KOH at C. For example, the peak value of PGE1 was detected at 280 nm. The residence time was approximately 4.6 minutes.

[0087] 1.4 Coverage rate

[0088] Encapsulation efficiency (EE), which is the percentage of drug encapsulated (or loaded) in the internal aqueous medium of liposomes relative to the total amount of drug present in the liposome suspension (i.e., drug in the external medium and drug loaded into the internal aqueous medium of liposomes), is quantified as described below. Simply put, the liposome suspension is subjected to column chromatography. The liposome suspension is eluted through a G-25 column to separate free or unrelated drug (i.e., drug in the external medium) from the liposomes containing encapsulated drug in the internal aqueous medium. The amount of free drug is determined using a Waters HPLC system with PDA detection, and the amount of drug encapsulated in the internal aqueous medium of the liposomes is determined using the same method after the liposomes are disrupted with methanol (90% methanol and 10% liposome suspension). Encapsulation efficiency is calculated using the following formula. , Where L is the amount of drug encapsulated in the internal aqueous medium of the liposome, and F is the amount of free drug in the external medium.

[0089] In addition, the internal drug concentration (mM) is calculated as follows.

[0090] Internal drug concentration (mM) = [drug[ [g / mL] x EE% / molecular weight] / [trapped volume] ]

[0091] ( Under specific conditions, when the liposome particle size is approximately 110 nm and the phospholipid concentration is 10 mM, the internal volume of each milliliter of liposome suspension is 0.0314 mL. The internal volume can be calculated using methods known in the art (Xue et al., International Journal of Pharmaceutics (2012) 423(2), pp. 410-418.).

[0092] 1.5 Average particle size and polydispersity index (PdI)

[0093] The average particle size of liposomes was assessed using dynamic light scattering. The polydispersity index (PdI), representing the size distribution of liposomes, was determined using the same assessment technique as the average particle size on a particle size analyzer (Beckman Coulter Delsa™ Nano C particle size analyzer).

[0094] 2. Results

[0095] 2.1 Sodium bicarbonate provides a high drug encapsulation rate.

[0096] Table 2 shows the drug encapsulation efficiency (EE) using sodium bicarbonate and sodium acetate as loading salts, as well as the molar ratio of drug to salt in liposome suspensions.

[0097] Table 2. Effects of bicarbonate and acetate on drug loading.

[0098] a. The liposome composition contains 10 mM of phospholipids (HSPC:Chol:mPEG-DSPE = 3:1.91:0.072).

[0099] b. Internal drug concentration (mM) = [drug concentration] [g / mL] x EE% / Molecular weight] / [Capture volume (mL)] ] In the examples, when the liposome particle size was about 110 nm and the phospholipid concentration was 10 mM, the capture volume in each milliliter of liposome suspension was 0.0314 mL.

[0100] The results in Table 2 show that, compared with sodium acetate, sodium bicarbonate increases the drug encapsulation rate of weak acid drugs and significantly increases the drug concentration in the aqueous medium inside liposomes. Specifically, when using sodium acetate as a carrier, the molar ratio of drug to sodium acetate is less than 0.1:1, and the molar ratio cannot be further increased by doubling the concentration of sodium acetate. When using sodium bicarbonate as a carrier, the molar ratio of drug to salt is greater than 0.1:1.

[0101] Example 2: Effects of pharmaceutical compositions using bicarbonate for drug loading on human physiological conditions

[0102] According to section 1.1 of Example 1, liposome suspensions loaded with PGE1 were prepared using sodium bicarbonate of various concentrations. The liposome suspensions were cultured in an external buffer having an osmotic pressure between 280 mOsm / kg and 300 mOsm / kg at 25°C. o C and 37 o Cultured at C for 20 minutes to simulate human physiological conditions.

[0103] Table 3. Effect of temperature on pharmaceutical compositions containing sodium bicarbonate of various concentrations.

[0104] In human physiological conditions (37) o Culturing liposomes in a formulation with a bicarbonate concentration higher than approximately 1000 mM (C and 280 mOsm / Kg to 300 mOsm / Kg) produces more carbon dioxide (CO2) and leads to liposome leakage. Leaking liposomes release bicarbonate and drug from the internal aqueous medium into the external medium, increasing the pH of the external medium to above 6.5.

[0105] Example 3: Effect of different bicarbonate concentrations on the amount of drug loaded

[0106] Liposome suspensions were prepared according to the method in section 1.1 of Example 1, and PGE1 was loaded with different concentrations of sodium bicarbonate (50 mM, 150 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM, 600 mM and 800 mM).

[0107] Table 4 shows the effect of bicarbonate concentration on drug loading.

[0108] Table 4

[0109] Phospholipids 5mM phospholipids The results suggest that for a fixed amount of phospholipid, bicarbonate at concentrations of 50 mM to 800 mM can achieve drug encapsulation rates exceeding 90% and drug-to-salt molar ratios exceeding 0.1:1. A concentration of 500 mM bicarbonate provides the highest drug encapsulation rate and drug-to-salt molar ratio.

[0110] Example 4: Using bicarbonate to load different weak acid drugs

[0111] A first liposome suspension (4-1) containing HSPC, cholesterol and DSPE-mPEG2000 in a molar ratio of 3:2:0.075 was prepared according to the method in Example 1.

[0112] A second liposome suspension (4-2) comprising HSPC, cholesterol and DSPG in a molar ratio of 3:2:0.075 was prepared according to the method in Example 1.

[0113] A third liposome suspension (4-3) comprising DPPC, cholesterol and DSPE-mPEG2000 in a molar ratio of 3:2:0.075 was prepared according to the method in Example 1.

[0114] A fourth liposome suspension (4-4) comprising DSPC, DPPC, cholesterol and mPEG2000-DSPE in a molar ratio of 2.7:0.3:2:0.075 was prepared according to the method in Example 1.

[0115] A fifth liposome suspension (4-5) comprising HSPC, cholesterol, mPEG2000-DSPE and stearamide in a molar ratio of 3:2:0.075:0.025 was prepared according to the method in Example 1.

[0116] A sixth liposome suspension (4-6) comprising HSPC, DMPC, cholesterol, and mPEG2000-DSPE in a molar ratio of 2.7:0.3:2:0.075 was prepared according to the method in Example 1.

[0117] Various weak acid drugs are treated with 200mM to 400mM bicarbonate at 25°C. o C or 40 o The reaction was carried out at C for more than 20 minutes to load the drug into the first, second, and third liposome suspensions. The drug-loaded liposome suspensions were stored at 4°C. o C.

[0118] Table 5 shows that bicarbonate systems are effective carriers for various weak acid drugs encapsulated in various liposome formulations, exhibiting high drug encapsulation rates (above 80%) and drug-to-salt molar ratios greater than 0.1:1.

[0119] Table 5. Drug loading data for various weak acid drugs using bicarbonate as a carrier.

[0120] a. At 25 o C. Drug loading at 40°C; b. At 40°C o Drug loading under C

[0121] Example 5: The effect of the pH of the external medium of liposome suspension on drug loading

[0122] A first liposome suspension (5-1) containing HSPC, cholesterol, and mPEG2000-DSPE in a molar ratio of 3:2:0.075, and a second liposome suspension (5-2) and a third liposome suspension (5-3) containing HSPC, cholesterol, and mPEG2000-DSPE in a molar ratio of 3:2:0.28 were prepared. Treprostacyclin was loaded into the liposome suspension containing 200 mM bicarbonate.

[0123] The pH of the external medium for the first liposome suspension (5-1) is higher than the pKa of the drug (the pKa of treprostol is 4.5), while the pH of the external medium for the second liposome suspension (5-2) and the third liposome suspension (5-3) is lower than the pKa of the drug.

[0124] Table 6 illustrates the crucial role of the pH of the external medium in drug loading for liposome suspensions. Specifically, compared to liposome suspensions with an external medium pH below the drug's pKa, liposome suspensions with an external medium pH above the drug's pKa exhibited significantly higher drug encapsulation (90.6% vs. 27.3% and 33.5%). Similarly, compared to liposome suspensions with an external medium pH below the drug's pKa (where the molar ratio of drug to salts was less than 0.1:1), liposome suspensions with an external medium pH above the drug's pKa had a molar ratio of drug to salts greater than 0.1:1.

[0125] Table 6. Effect of pH of the external medium on drug loading in liposome suspensions

[0126] Some drug sedimentation was observed during drug loading. Example 6: Therapeutic effects of pharmaceutical compositions with high drug loading The in vivo efficacy of the pharmaceutical composition of Example 4 (using a 4-2 liposome suspension of approximately 1.5 mg of treprostolin coated with 400 mM bicarbonate) against pulmonary hypertension was evaluated using 18 male Sprague Dawley rats, each weighing approximately 300-350 g.

[0127] To induce pulmonary hypertension, a pressure catheter was inserted into the pulmonary artery of each rat, with one end exposed at the nape of the neck and connected to a pressure sensor. Twenty-four hours after catheter insertion, the rats were transferred to a hypoxic chamber, where oxygen (O2) levels were reduced to 10% (FiO2 = 0.1) by increasing nitrogen (N2) levels. Once stable pulmonary hypertension was established, each rat was administered a single dose of one of three test substances: saline (N = 6), 6 μg / kg of free treprostacyclin (N = 6), or the pharmaceutical composition of Example 4 (liposomal treprostacyclin, N = 6) with 6 μg / kg of treprostacyclin. The test substances were administered at the trachea bifurcation of the rats using a microneedle. Mean pulmonary artery pressure (PAP) was measured at predetermined time points after administration of the test substances.

[0128] The results showed that liposomal troprostacyclin was more effective than free troprostacyclin in achieving the maximum reduction in mean PAP after injection (mean PAP reduction of 40% in the liposomal troprostacyclin group and 20% in the free troprostacyclin group).

Claims

1. A pharmaceutical composition comprising One or more liposomes, the liposomes containing (a) A lipid bilayer comprising at least one vesicle-forming lipid; and (b) An internal aqueous medium within the lipid bilayer, comprising bicarbonate at concentrations ranging from 50 mM ± 10% to 1000 mM ± 10% and pK a It is a weak acid drug with a concentration ranging from 1 ± 10% to 6 ± 10%. The liposome is suspended in an external medium, the pH of which is higher than the pK of the weakly acidic drug. a And less than 7, and wherein the bicarbonate provides a pH gradient between the internal aqueous medium and the external medium, and the pH of the internal aqueous medium is at least one unit higher than the pH of the external medium.

2. The pharmaceutical composition of claim 1, wherein the molar ratio of the weak acid drug to the bicarbonate is 0.1:1 to 1:

1.

3. The pharmaceutical composition of claim 1, wherein the concentration of the bicarbonate is from 250 mM ± 10% to 800 mM ± 10%.

4. The pharmaceutical composition of claim 1, wherein the coating of the weak acid drug is at least 80%.

5. The pharmaceutical composition of claim 1, wherein the vesicle-forming lipid is a mixture of a first phospholipid and a second phospholipid, or a mixture of a first phospholipid and a charged lipid.

6. The pharmaceutical composition of claim 5, wherein the first phospholipid is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylserine (PS), or any combination thereof, and the second phospholipid is a polyethylene glycol (PEG) modified phospholipid, a positively charged phospholipid, or a negatively charged phospholipid, and the charged lipid is a positively charged lipid or a negatively charged lipid.

7. The pharmaceutical composition of claim 6, wherein the first phospholipid is hydrogenated soybean phosphatidylcholine (HSPC), distearyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), or any combination thereof, and the second phospholipid is distearyl phosphatidylglycerol (DSPG), dipalmitoyl phosphatidylglycerol (DPPG), dimyristoyl phosphatidylglycerol (DMPG), polyethylene glycol distearyl phosphatidylethanolamine (PEG-DSPE), or any combination thereof.

8. The pharmaceutical composition of claim 6, wherein the first phospholipid is hydrogenated soybean phosphatidylcholine (HSPC), distearylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), or any combination thereof, and the charged lipid is stearylamine, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol), N... 4 -Cholesterol-spermine (N 4 -Cholesteryl-Spermine (GL67), dimethyldioctadecylammonium (DDAB), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), ethylphosphocholine (ethyl PC), or any combination thereof.

9. The pharmaceutical composition of claim 5, further comprising sterols.

10. The pharmaceutical composition of claim 9, wherein the sterol is selected from cholesterol, cholesterol hemisuccinate, ergosterol, lanosterol, and combinations thereof.

11. The pharmaceutical composition of claim 10, wherein the molar percentage of the first phospholipid:cholesterol:the second phospholipid or the charged lipid is 50-70:20-45:0.1-10.

12. The pharmaceutical composition of claim 1, wherein the bicarbonate is potassium bicarbonate, sodium bicarbonate, calcium bicarbonate, magnesium bicarbonate, cesium bicarbonate, lithium bicarbonate, nickel bicarbonate, ferrous bicarbonate, or any combination thereof.

13. The pharmaceutical composition of claim 1, wherein the weakly acidic drug is a prostaglandin, a steroid, a nonsteroidal anti-inflammatory drug (NSAID), or an anticoagulant.

14. The pharmaceutical composition of claim 13, wherein the prostaglandin is prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), epoprostenol, iloprost, beraprostenol, MRE-269, treprostinil, or Ralinepag.

15. The pharmaceutical composition of claim 1, comprising... One or more liposomes, the liposomes containing (a) A lipid bilayer comprising at least one vesicle-forming lipid; and (b) An internal aqueous medium within the lipid bilayer, comprising bicarbonate and prostaglandin at concentrations ranging from 50 mM ±10% to 1000 mM ±10%. The liposome is suspended in an external aqueous medium, the pH of which is between that of prostaglandin and its pK. a Between 7 ± 10%, the pH of the internal aqueous medium is at least one unit higher than the pH of the external aqueous medium.

16. The composition as defined in any one of claims 1 to 15, for treating respiratory diseases.

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