Pharmaceutical compositions for the controlled release of weakly acidic drugs and uses thereof

Abstract

To reduce potential side effects and extend the therapeutic effect of weakly acidic drugs, a liposome composition without an initial burst release is provided. [Solution] Provided is a pharmaceutical composition comprising at least one liposome, the liposome comprising an outer lipid bilayer containing at least one vesicle-forming phospholipid and less than 15 mol% of a sterol; and an inner aqueous medium containing a weakly acidic drug and a weakly acidic salt. This pharmaceutical composition reduces the burst release of the weakly acidic drug. Also provided is use of the pharmaceutical composition for treating respiratory diseases and reducing the side effects of weakly acidic drugs.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Application No. 62 / 731,101, filed September 14, 2018, the entire disclosure of which is incorporated herein by reference.

[0002] Field The present invention relates to a method for treating a weakly acidic drug comprising administering to a subject a weakly acidic drug comprising administering to a subject a weakly acidic drug, the method comprising administering to a subject a weakly acidic drug comprising administering to a subject a weakly acidic drug; Disclosed herein are pharmaceutical compositions for maintaining blood flow. [Background technology]

[0003] background Liposomes are microstructures composed of natural or synthetic lipid bilayers that form internal compartments that act as reservoirs for therapeutic agents. Various liposome compositions have been designed as drug delivery vehicles with different sizes, permeabilities, and stabilities, all of which are designed to provide sustained drug release. However, these sustained-release liposome compositions generally exhibit a high initial burst of drug release, resulting in increased side effects and / or subtherapeutic plasma drug levels during the burst release.

[0004] The release profile of liposome compositions depends on the structure of the liposome membrane, which affects liposome performance. Therefore, controlling the release profile is an important prerequisite for effectively using liposomes as drug delivery vehicles. For example, adding cholesterol to the outer lipid bilayer increases membrane rigidity and stability and decreases the permeability of the lipid bilayer (S. Kaddah et al., Food Chem Toxicol. 2018 Mar;113:40-48). S. Kaddah et al. The release of encapsulated drugs decreases with increasing cholesterol content (up to 30%) in the liposome bilayer. E. Corvera et al. (Biochim Biophys Acta. 1992 Jun 30;1107(2):261-70) suggested that the addition of low concentrations of cholesterol (5-8%) to DMPC and DPPC liposomes decreases liposome stability and increases membrane permeability.

[0005] There remains a need for liposomal compositions that do not have an initial burst release to reduce potential side effects and extend the therapeutic effect of weakly acidic drugs. The present invention addresses these and other needs. Summary of the Invention

[0006] Brief Summary of the Invention The present invention relates to a pharmaceutical composition comprising one or more liposomes suspended in an external medium. The liposome comprises (a) an external lipid bilayer comprising at least one vesicle-forming phospholipid and less than 15 mole % of a sterol, and (b) an internal aqueous medium comprising a weakly acidic drug and a weakly acidic salt, wherein less than 65% by weight of the weakly acidic drug is administered during administration of the pharmaceutical composition. The pharmaceutical composition is provided such that the composition is released into the external medium within one hour of administration.

[0007] The present invention also discloses a method for treating respiratory disorders, comprising administering the pharmaceutical compositions described herein.

[0008] Also provided is a method for reducing the side effects of a weakly acidic drug, comprising administering an effective amount of the pharmaceutical composition described herein to a subject in need of taking the weakly acidic drug.

[0009] As used in this patent, the terms "invention," "the invention," "this invention," and "the present invention" shall mean the invention of this patent and The term "overview" is intended to broadly refer to all subject matter of the following claims. It should be understood that descriptions containing these terms do not limit the subject matter described herein or the meaning or scope of the following claims. The embodiments of the invention encompassed by this patent are defined by the following claims, not this Summary. This Summary is a high-level overview of various aspects of the invention and introduces some concepts that are further described in the Detailed Description section below. This 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 reference to the entire specification, some or all of the drawings, and appropriate portions of each claim.

[0010] The invention will become more apparent from the following drawings and detailed description. [Brief explanation of the drawings]

[0011] Specific embodiments of the present invention are described in detail below with reference to the following drawings: [Figure 1] FIG. 1 is a line graph showing the logarithm of the mean plasma iloprost concentration in rats administered a liposomal composition comprising iloprost, bicarbonate, and HP-β-CD (LL021b3A2), a liposomal composition comprising iloprost, bicarbonate, and RM-β-CD (LL021m3A2), or an iloprost solution. [Figure 2]FIG. 2 is a line graph showing the ratio of the area under the plasma concentration-time curve from time 0 to a specific time (AUCt) to the area under the plasma concentration-time curve from time 0 to infinity (AUCinf) for a liposomal composition comprising iloprost, bicarbonate, and HP-β-CD (LL021b3A2), a liposomal composition comprising iloprost, bicarbonate, and RM-β-CD (LL021m3A2), or an iloprost solution. DETAILED DESCRIPTION OF THE INVENTION

[0012] Detailed Description As used herein, the articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0013] All numbers are modified by the term "about." As used herein, the term "about" refers to a range of plus or minus 10% of the particular value.

[0014] The term "comprise" or "comprising" generally means that one or more features, ingredients, or components may be present. Used in the sense of include / including.

[0015] The term "subject" refers to a vertebrate animal having a respiratory disease or a vertebrate animal for the treatment of a respiratory disease. The term "subject" can refer to a vertebrate that is thought to require treatment. Subjects include warm-blooded animals such as mammals, primates, and more preferably humans. Non-human primates are also subjects. The term subject includes domestic animals such as cats and dogs, livestock (e.g., cows, horses, pigs, sheep, goats, and the like), and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, and the like). Thus, the present specification encompasses veterinary applications and medical preparations.

[0016] The term "treating" refers to both therapeutic treatment and prophylactic or preventative measures. Subjects in need of treatment include those already suffering from a respiratory disease or related disorder. The subject may be a subject susceptible to or in need of prevention of a respiratory disease or related disorder.

[0017] As used herein, the term "weak acid drug" encompasses its pharmaceutically acceptable salts and protonated forms, unless otherwise specified or apparent from the context. In one embodiment, the weak acid drug contains at least one functional group selected from the group consisting of a carboxyl group (-COOH), a hydroxyl group (-OH), a phosphate group (-PO), and any combination thereof. In other embodiments, the weak acid drug has a pKa of 1 or greater but less than about 7, 2 or greater but less than about 6, 2 to 6.9, or 2.5 to 6. The weak acid drug may also contain one or more functional groups in addition to the carboxyl group (-COOH), hydroxyl group (-OH), and phosphate group (-PO); such additional functional groups should not significantly alter the acidity of the drug from that of its non-functionalized counterpart. In one embodiment, the weak acid drug is used to treat pulmonary hypertension. In other embodiments, the weakly acidic drug is a prostaglandin, a prostacyclin receptor agonist, a glucocorticoid, or a nonsteroidal anti-inflammatory drug. Table 1 provides non-limiting examples of weakly acidic drugs of the present invention.

[0018] [Table 1-1]

[0019] [Table 1-2]

[0020] As used herein, "encapsulation" and "loaded" The terms "encapsulated" and "entrapped" can be used interchangeably and are not intended to be limiting unless expressly stated. It refers to the incorporation or association of a biologically active agent (eg, iloprost) in the internal aqueous medium of a some.

[0021] The present disclosure provides a pharmaceutical composition comprising one or more liposomes suspended in an external medium, the liposomes comprising (a) an external lipid bilayer comprising at least one vesicle-forming phospholipid and less than 15 mole % of a sterol, and (b) an internal aqueous medium comprising a weakly acidic drug and a weakly acidic salt, wherein less than 65 weight % of the weakly acidic drug is released into the external medium within one hour of administering the pharmaceutical composition.

[0022] In a specific embodiment, the sterols in the outer lipid bilayer are less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 mol %. In an exemplary embodiment, the outer lipid bilayer is substantially free of sterols.

[0023] The encapsulation rate of the weakly acidic drug in the pharmaceutical composition is greater than about 70%, 75%, or 80%.

[0024] The pharmaceutical composition provides a low burst release of the encapsulated weakly acidic drug. In one embodiment, less than about 70%, 69%, 68%, 67%, 66%, or 65% of the weakly acidic drug is released within one hour of administration of the pharmaceutical composition. As a result, the side effects of the weakly acidic drug at the target site (e.g., cough, throat irritation, sore throat, nosebleed, hemoptysis, and upper respiratory tract wheezing) are reduced compared to a pharmaceutical composition having 15 mol% or more of sterol in the outer lipid bilayer. Furthermore, the pharmaceutical composition extends the release of the weakly acidic drug and reduces the frequency of administration.

[0025] In one embodiment, the burst release of a weakly acidic drug from the disclosed pharmaceutical composition is further reduced by the addition or encapsulation of a cyclodextrin in the internal aqueous medium. Non-limiting examples of cyclodextrins include α-CD, β-CD, γ-CD, 2-hydroxypropyl β-CD (HP-β-CD), sulfobutyl ether β-CD (SBE-β-CD), randomly methylated β-CD (RM-β-CD), or a combination thereof. Preferably, the cyclodextrin is HP-β-CD, RM-β-CD, or a combination thereof. In one specific embodiment, the molar ratio of the weakly acidic drug to the cyclodextrin (drug / CD ratio) is about 0.06, 0.055, 0.05, 0.045, 0.04, 0.035, or 0.03 or less.

[0026] Also disclosed is a method for treating respiratory diseases, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition disclosed herein, wherein the amount of sterol in the outer lipid bilayer is less than 15 mol%.The burst release of the weakly acidic drug in the pharmaceutical composition disclosed herein is reduced compared to a pharmaceutical composition having 15 mol% or more of sterol in the outer lipid bilayer.Non-limiting examples of respiratory diseases include pulmonary hypertension and interstitial lung disease.

[0027] Further disclosed is the use of a pharmaceutical composition disclosed herein for treating a respiratory disorder, or the use of a pharmaceutical composition disclosed herein for the manufacture of a medicament for treating a respiratory disorder.

[0028] The present invention also relates to a method for reducing the side effects of a weakly acidic drug, comprising administering to a subject in need of taking the weakly acidic drug an effective amount of a pharmaceutical composition disclosed herein, wherein the sterol in the outer lipid bilayer is less than 15 mol %.

[0029] In some embodiments, the pharmaceutical compositions disclosed herein are administered by inhalation to reduce the side effects of weakly acidic drugs in the upper respiratory tract.

[0030] A. Liposome Components As used herein, the term "liposome" refers to microscopic vesicles or particles composed of one or more lipid bilayers that enclose an internal aqueous medium. The formation of a liposome requires the presence of at least one "vesicle-forming lipid," which forms or is incorporated into the lipid bilayer. Suitable vesicle-forming lipids can be used to form the lipid bilayer that constitutes the liposome. Vesicle-forming lipids include, but are not limited to, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylethanolamine (PEA), and the like. Lipids include phospholipids such as phospholipids (PE) or phosphatidylserine (PS), and charged lipids such as positively charged lipids or negatively charged lipids.

[0031] The lipid bilayer of the liposome comprises at least one vesicle-forming lipid and 0 (zero) to 15 mol % of a sterol (e.g., 0 to 14.99 mol %), where the sterol is selected from the group consisting of, but not limited to, cholesterol, cholesterol hexasuccinate, ergosterol, lanosterol, and any combination thereof. In a specific embodiment, the sterol is cholesterol.

[0032] In some embodiments, the vesicle-forming lipids are a mixture of a first phospholipid and a second phospholipid. In certain embodiments, the first phospholipid is a phosphatidylcholine (PC) selected from the group consisting of hydrogenated egg phosphatidylcholine (HEPC), hydrogenated soy phosphatidylcholine (HSPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine, dimyristoylphosphatidylcholine (DMPC), egg phosphatidylcholine (EPC), soy phosphatidylcholine (SPC), oleoylpalmitoylphosphatidylcholine, dioleoylphosphatidylcholine (DOPC), dipetroselinoylphosphatidylcholine, palmitoyleleidoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine, dilauroylphosphatidylcholine (DLPC), diundecanoylphosphatidylcholine, didecanoylphosphatidylcholine, dinonanoylphosphatidylcholine, and any combination thereof. In other embodiments, the second phospholipid is a polyethylene glycol-modified phospholipid containing polyethylene glycol having a molecular weight of about 500 to about 10,000 daltons, such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), or a negatively charged phospholipid such as distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG), or dioleoylphosphatidylglycerol (DOPG). In a specific embodiment, the molar ratio of first phospholipid:cholesterol:second phospholipid is 75-99:0-14.9:0.1-25.

[0033] In other embodiments, the vesicle-forming lipid is a mixture of a first phospholipid and a charged lipid. In a specific embodiment, the vesicle-forming lipid is a mixture of a first phospholipid, a second phospholipid, and a charged lipid. The charged lipid includes stearylamine, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[N-(N,N-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), N 4 -cholesteryl-spermine (GL67), dimethyldioctadecylammonium (DDAB), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), ethylphosphocholine (ethylPC), or a combination thereof. In another specific embodiment, the molar ratio of first phospholipid:cholesterol:charged lipid is 75-99:0-14.9:0.1-25.

[0034] In one embodiment, the molar percentages of HSPC, cholesterol, and DSPG in the lipid bilayer are 75-99:0-14.9:0.1-25. In another embodiment, the molar percentages of HSPC, cholesterol, and DSPE-PEG2000 in the lipid bilayer are 75-99:0-14.9:0.1-25.

[0035] In one embodiment, the outer lipid bilayer of the liposome may further comprise a surfactant, which may be a nonionic surfactant, a cationic surfactant, or a zwitterionic surfactant. Nonionic surfactants have no formally charged groups in their heads. Cationic surfactants have a net positive charge in their heads. Zwitterionic surfactants are electrically neutral but have formal positive and negative charges on different atoms.

[0036] Non-limiting examples of nonionic surfactants include nonionic water-soluble mono-, di-, and triglycerides; nonionic water-soluble mono- and di-fatty acid esters of polyethylene glycol; nonionic water-soluble sorbitan fatty acid esters (e.g., sorbitan monooleates such as TWEEN 20 (polyoxyethylene 20 sorbitan monooleate), SPAN 80); 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.

[0037] Non-limiting examples of cationic surfactants include dimethyldialkylammonium bromide or dodecyltrimethylammonium bromide.

[0038] A non-limiting example of a zwitterionic surfactant is 3-(N,N-dimethylpalmitylammonio)-propanesulfonate.

[0039] According to the present invention, liposomes are prepared in a medium containing a weak acid salt to create a pH gradient between the internal aqueous medium of the liposome and the external medium. When vesicle-forming phospholipids and less than 15% of the sterol are contacted with a medium containing a weak acid salt, a liposome suspension is formed.

[0040] The liposomes in suspension are subjected to size reduction. Liposome size usually refers to their diameter. Liposome size reduction can be achieved by many methods, such as extrusion, sonication, homogenization, or milling techniques, which are well known and can be performed by those skilled in the art. Extrusion involves passing the liposomes through a filter with a defined pore size under pressure one or more times. The filter is usually made of polycarbonate, but may be made of a durable material that does not interact with the liposomes and is strong enough to allow extrusion under sufficient pressure. Liposome size can be reduced by sonication. Sonication uses sound energy to disrupt or shear the liposomes, which will spontaneously reform into smaller liposomes. For example, sonication can be performed by immersing a glass tube containing a liposome suspension in a sonic epicenter generated by a bath-type sonicator, or by using a probe-type sonicator. In this case, the sonic energy is generated by the vibration of a titanium probe that is in direct contact with the liposome suspension. In the present invention, the liposomes typically have a diameter of about 50 nm to 500 nm, such as 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.

[0041] After sizing, the concentration of weak acid salts in the external medium is adjusted to provide a pH gradient between the internal aqueous medium and the external medium. This can be accomplished in a variety of ways, for example, by exchanging the external medium for a suitable buffer that does not contain weak acid salts, such as citrate buffer (H3C6H5O) or phosphate buffer (H3PO4), by methods such as diafiltration, dialysis, ultrafiltration, or tangential flow filtration.

[0042] The weak acid salt provides a lower external to higher internal pH gradient between the external medium and the internal aqueous medium of the liposome. In one embodiment, the pH of the internal aqueous medium is at least 0.1 units higher than the pH of the external medium. In another embodiment, the pH of the internal aqueous medium is at least 1 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 above the pKa of the weakly acidic drug.

[0043] Non-limiting examples of weak acid salts include carboxylates and bicarbonates.

[0044] As used herein, "bicarbonate salt" refers to a pharmaceutically acceptable salt compound comprising a bicarbonate anion and a cationic component. In one embodiment, the cationic component of the salt compound is a metal. Non-limiting examples of metals include Group IA or IIA metals, such as potassium (K), sodium (Na), calcium (Ca), magnesium (Mg), cesium (Cs), and lithium (Li), or metals outside of Group IA or IIA, such as iron (Fe) and nickel (Ni). Examples of bicarbonate salts 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.

[0045] As used herein, "carboxylate salt" includes, but is not limited to, formate, acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, or a combination thereof. In an exemplary embodiment, the acetate salt is sodium acetate, calcium acetate, or a combination thereof.

[0046] Bicarbonate or carboxylate concentrations are ≥ 50mM, ≥ 100mM, ≥ 150mM, ≥ 200mM, ≥ 250mM, ≥ 300mM, ≥ 350mM, ≥ 400mM, ≥ 450mM, ≥ 500mM, ≥ 600mM, ≥ 700mM, and ≥ 800mM. 900 mM, less than 1000 mM, 50 mM or more but less than 1000 mM, 50 mM to 800 mM, 200 mM or more but less than 1000 mM, 200 mM to 800 mM, or 200 mM to 600 mM, 250 mM or more but less than 1000 mM, 250 mM to 800 mM, or 250 mM to 600 mM, or 300 mM to 600 mM.

[0047] The prepared liposomes are suitable for loading weakly acidic drugs and administering them to subjects. For example, the liposomes may be stored under refrigeration for a significant period of time before loading with the weakly acidic drug. Liposomes may be dehydrated, stored, and subsequently rehydrated and loaded with a weakly acidic drug. Liposomes may also be dehydrated after loading with a weakly acidic drug. Dehydration can be performed by many methods available and known in the art. In some embodiments, liposomes are dehydrated using a standard freeze-drying apparatus, i.e., dehydration under low pressure conditions. Liposomes may also be frozen, for example, using liquid nitrogen. Prior to dehydration, saccharides may be added to the liposome environment, for example, a buffer containing the liposomes, to ensure the stability and integrity of the liposomes during dehydration. Examples of saccharides include, but are not limited to: However, maltose, lactose, sucrose, trehalose, dextrose, sorbitol, mannitol, xylitol, or a combination thereof may be used.

[0048] As described above, a liposome suspension containing less than 15 mol % of sterol or substantially no sterol is prepared for loading a weakly acidic drug. Specifically, the weakly acidic drug is added to the external medium of the liposome, and the resulting suspension is incubated to allow the weakly acidic drug to diffuse into the internal aqueous medium of the liposome until the desired loading concentration and encapsulation efficiency (the percentage of the internal / encapsulated amount of the weakly acidic drug relative to the total amount of the weakly acidic drug in the pharmaceutical composition) are achieved.

[0049] B. Relationship between sterol content of the outer lipid bilayer and controlled release profile Pharmaceutical compositions of the present invention having less than 15 mol% (e.g., 0-14.99 mol%) of sterol in the outer lipid bilayer of liposomes reduce the burst release of the encapsulated weakly acidic drug, thereby reducing the side effects of the weakly acidic drug. Furthermore, sufficient amounts of the weakly acidic drug are released from the pharmaceutical composition for the desired therapeutic effect, and the release profile is unexpectedly extended compared to the release profile of pharmaceutical compositions having more than 15 mol% of sterol in the outer lipid bilayer of liposomes.

[0050] As used herein, the term "burst release" refers to a rapid release of a pharmaceutical composition. refers to the rapid and / or somewhat uncontrolled release of greater than 70, 69, 68, 67, 66, or 65% of an encapsulated weakly acidic drug from a pharmaceutical composition within 1 hour (60 minutes) after administration of the composition.

[0051] As used herein, the term "extended release" can be used interchangeably with "controlled release," "delayed release," "modified release," "prolonged release," "programmed release," "time release," "rate controlled," or "sustained release," and refers to the release of less than 50, 45, or 40% of a weakly acidic drug within 1 hour of administration of the pharmaceutical composition.

[0052] In one embodiment, the burst or sustained release profile of the pharmaceutical composition is based on in vitro release (IVR) assays and / or in vivo pharmacokinetic studies of the entrapped weakly acidic drug.

[0053] In certain embodiments, based on in vitro release (IVR) assays and / or in vivo pharmacokinetic studies, the pharmaceutical composition has a release profile in which less than about 70, 69, 68, 67, 66, or 65% by weight of the entrapped weakly acidic drug is released within 1 hour from the time the pharmaceutical composition is administered.

[0054] C. Administration The pharmaceutical compositions of the present invention may be administered to a cavity of a subject that does not come into direct contact with blood. Examples of routes of administration include, but are not limited to, inhalation, intratracheal injection, subcutaneous injection, intraarticular injection, intramuscular injection, intravitreal injection, and intrathecal injection.

[0055] The pharmaceutical compositions of the present invention may also be administered directly into the blood of a subject.

[0056] According to the present disclosure, the pharmaceutical composition may be administered one to three times daily, once every two days, or once every three days.

[0057] The present disclosure is further described in the following examples, however, it should be understood that the following examples are for illustrative purposes only and should not be construed as limiting the present disclosure in any way.

[0058] Example General experimental procedure: 1. Preparation of Iloprost Liposomal Composition Liposome colloidal suspensions were prepared using the ethanol injection technique. All lipid components, including the first phospholipid (HSPC) and the second phospholipid (DSPE-PEG2000 or DSPG) in a molar ratio of 98:2 or 98.5:1.5, were dissolved in 2.86 mL of ethanol solution at approximately 60 °C. The resulting lipid solution was added to 17.4 mL of ethanol containing (2-hydroxypropyl)-β-cyclodextrin (i.e., 45–120 mM) as needed. The liposomes were injected into a sodium bicarbonate solution (100–400 mM; pH 8.5) and mixed under vigorous stirring at 60°C for liposome hydration. The mixture was extruded 6–10 times through polycarbonate membranes with pore sizes of 0.2 or 0.1 μm to obtain a suspension of liposomes with an average particle size ranging from approximately 100 nm to 200 nm and a polydispersity index (PdI) of <0.2. The liposome suspension was dialyzed in a tangential flow filtration system against 10 mM sodium citrate buffer (pH 5.5) to obtain the liposomes. A transmembrane pH gradient exists between the internal aqueous medium of the some and the external medium. A pH gradient was formed (i.e., a higher pH gradient inside and a lower pH gradient outside). The suspension of liposomes with such a pH gradient was then stored at 4°C until the drug loading process.

[0059] Iloprost (purchased from Cayman Chemical, USA) was dissolved in 50 mM sodium citrate solution and added to the liposome suspension to a drug concentration of 1000-250 μg / mL, followed by incubation at 37°C for 30 minutes. The resulting product was adjusted with sodium citrate buffer (pH 5.5) to obtain an iloprost-loaded liposomal composition with an external pH of 5.5 and a phospholipid concentration of 10 mM in the liposome suspension.

[0060] 2. Preparation of Ambrisentan Liposomal Composition A liposome suspension was prepared according to Step 1 above, with or without (2-hydroxypropyl)-β-cyclodextrin. Ambrisentan (purchased from Cayman Chemical, USA) was dissolved in dimethyl sulfoxide (DMSO) and added to the liposome suspension to achieve a predetermined drug concentration of approximately 500 μg / mL, followed by incubation at 37°C for 30 minutes. The resulting product was adjusted with sodium citrate buffer (pH 5.5) to obtain an ambrisentan-loaded liposome composition with an external medium pH of 5.5 and a phospholipid concentration of 10 mM in the liposome suspension.

[0061] 3. Quantitative Characterization of Liposome Composition a. Encapsulated free iloprost / ambrisentan concentration The liposomal composition of iloprost or ambrisentan was TM The encapsulated drug was separated from the free drug by injection into a G-25 column (GE Healthcare). The iloprost or ambrisentan liposome composition was mixed with methanol (90% by volume methanol and 10% by volume liposome suspension) to form a liposome-methanol mixture.

[0062] The concentrations of encapsulated and free iloprost were determined by measuring 30 μL of liposome- The analysis was performed by injecting a mixture of methanol and acetonitrile. The mobile phase was a mixture of acetonitrile, methanol, and phosphate buffer (pH 2.5) in a volume ratio of 36:17:47, with a flow rate of 1.0 mL / min. Separation was performed at 25 °C using a C8 column with dimensions of 3.9 mm x 15.0 cm and 5.0 μm, and the absorbance peak was detected at 205 nm.

[0063] The concentrations of encapsulated and free ambrisentan were determined by measuring 1 μL of liposome-methanolide on a Waters Acquity UPLC system equipped with a mass detector (QDa). The analysis was performed by injecting a mixture of mobile phases. Mobile phase A contained 0.1% formic acid in acetonitrile, and mobile phase B contained 0.1% formic acid in ddH2O. The gradient conditions were as follows: 50% mobile phase A for 0.2 min, 10% mobile phase A by 2 min, and 50% mobile phase A by 5.5 min. Separation was performed using a C18 column with dimensions of 4.6 mm x 10.0 cm, 3.0 μm, at 35 °C and a flow rate of 1.0 mL / min. MS acquisition was performed using a 4.6 mm x 10.0 cm, 3.0 μm C18 column with dimensions of 4.6 mm x 10.0 cm, 3.0 μm, at 35 °C and a flow rate of 1.0 mL / min. For ambrisentan, [M+H] + The analysis was performed in SIR mode using the ion m / z 347.2.

[0064] b. Encapsulation efficiency (EE) and drug-to-cyclodextrin ratio: The total concentration of drug (iloprost or ambrisentan) in the liposomal composition includes the encapsulated drug in the internal aqueous medium (L) and the free drug in the external medium (F).

[0065] The drug encapsulation efficiency (EE) was calculated as the percentage of encapsulated drug (L) in the internal aqueous medium of the liposome relative to the total amount of drug (L+F), see the following formula:

[0066]

number

[0067] The ILO / CD ratio of the iloprost liposomal composition and the AMB / CD ratio of the ambrisentan liposomal composition were calculated using the following formula:

[0068]

number

[0069] c. Average particle size and polydispersity index (PdI): The mean particle size of liposomes was evaluated by dynamic light scattering. The polydispersity index (PdI), which indicates the size distribution of liposomes, was measured using a Beckman Coulter Delsa™ Nano C particle analyzer. The average particle size was measured using a tickle analyzer using the same evaluation method as for the average particle size.

[0070] Example 1: In vitro release (IVR) profiles of iloprost liposomal compositions with different amounts of sterol A. In vitro release (IVR) assay Iloprost liposome compositions were formulated and the concentration of iloprost was analyzed according to the procedures in the General Experimental Procedures section above. The liposomes had a mean particle size of 100-200 nm and a PdI of less than 0.20.

[0071] Various IVR assays can be used to evaluate the IVR profile. The actual IVR assay will be known or apparent to those skilled in the art depending on the iloprost in the claimed liposome composition. The iloprost release profile from liposomes was obtained by 10-fold dilution of iloprost-loaded liposome solution with a starting phospholipid concentration of 10 mM into simulated lung fluid (SLF) [Dissolution Technologies 2011, 18, 15-28] at 37°C with a shaking speed of 100 rpm. The percentage of iloprost released at each time point (Release %) was calculated using the following formula: The encapsulation efficiency (EE) after encapsulation was calculated by comparing it with the initial (T0) encapsulation efficiency.

[0072]

number

[0073] result: The physicochemical properties and IVR profiles of iloprost liposome compositions with different amounts of sterol are shown in Table 1.

[0074] [Table 2]

[0075] Table 1 shows that an EE of >90% was achieved using sodium bicarbonate salt, and that iloprost liposome compositions containing less than 15 mol% cholesterol released less than 65% of the iloprost within 1 hour of SLF incubation, while iloprost liposome compositions containing 15 mol% or more cholesterol released more than 70% of the iloprost within 1 hour of SLF incubation at 37°C.

[0076] Example 2: In vitro release (IVR) profiles of ambrisentan liposomal compositions with different amounts of sterol Ambrisentan liposome compositions were formulated and the concentration of ambrisentan was analyzed according to the procedures in the General Experimental Procedures section above. The liposomes had an average particle size of 100-200 nm and a PdI of less than 0.20.

[0077] result: The physicochemical properties and IVR profiles of ambrisentan liposomal compositions with different amounts of sterol are shown in Table 2.

[0078] [Table 3]

[0079] Table 2 shows that >90% EE was achieved using sodium bicarbonate salt, and less than 50% of ambrisentan was released within 1 hour of SLF incubation at 37°C for ambrisentan liposomal compositions containing less than 15 mol% cholesterol.

[0080] Example 3: In vitro release (IVR) profiles of iloprost liposomal compositions with and without cyclodextrin (CD) An in vitro study was conducted to evaluate the effect of cyclodextrin ((2-hydroxypropyl)-β-cyclodextrin (HP-β-CD)) in the internal aqueous medium of the liposomes on the release profile of the iloprost liposomal composition of Example 1.

[0081] result: The physicochemical properties and IVR profiles of iloprost liposomal compositions with and without cyclodextrin (HP-β-CD) are shown in Table 3.

[0082] [Table 4]

[0083] Table 3 shows that the addition of cyclodextrin further reduced the burst release (less than 60% of the iloprost was released within 1 hour of SLF incubation at 37°C) while maintaining the release attributes of the iloprost liposomal composition (less than 40% of the iloprost was released within 1 hour of SLF incubation at 37°C).

[0084] Example 4: Encapsulation efficiency of iloprost liposome compositions using different weak acid salts Effect of different weak acid salts on the encapsulation efficiency of the iloprost liposome composition of Example 1 In vitro studies were conducted to evaluate the effectiveness of iloprost loading in this example using sodium bicarbonate solution (400 mM) and sodium acetate solution.

[0085] result: The encapsulation efficiency of iloprost liposome compositions using different weak acid salts is shown in Table 4.

[0086] [Table 5]

[0087] Table 4 shows that >80% EE was achieved using bicarbonate and acetate salts, and the presence of cyclodextrin in the internal aqueous medium further reduced the burst release and sustained the release of iloprost from the liposomal composition.

[0088] Example 5: In vitro release (IVR) profiles and in vivo pharmacokinetic (PK) parameters of iloprost liposomal compositions with different iloprost to cyclodextrin (ILO / CD) ratios An in vitro study was conducted to evaluate the effect of different ILO / CD ratios on the IVR profile of iloprost liposomal compositions. The liposomal compositions of this study were prepared and their IVR profiles analyzed according to the procedures outlined in Example 1. An iloprost solution (20 μg / mL) was prepared by dissolving iloprost in a 2 mM solution of tromethamine, adjusted to a pH of approximately 8.4.

[0089] B. In Vivo Pharmacokinetic (PK) Studies of Iloprost Liposome Compositions In this in vivo PK study, three male Sprague-Dawley rats (purchased from BioLASCO Taiwan Co., Ltd.) in each group were anesthetized with isoflurane and placed on a hook around the upper incisors. Each rat was placed on an arched platform in a supine position at a 45-50° angle using a ribbon. A microspray aerosol tip (Microsprayer, PennCentury, Philadelphia, USA) was inserted into the tracheal carina, and the test sample (i.e., the composition listed in Table 5 or iloprost solution) was administered intratracheally at a predetermined dose of 60 μg / kg using a high-pressure syringe attached to the microspray aerosol device.

[0090] At predetermined time points (i.e., 5, 30 min, 1.5, 3, 6, 7, and 8 h after administration), blood samples were collected from each rat into heparin-coated tubes and placed on wet ice. The blood samples were then centrifuged at approximately 2500 × g for 15 min at 4 ± 2°C within 1 h of collection to separate the plasma from the blood cells. Approximately 0.1 mL of each plasma sample from each rat was added to a new storage tube and stored at -70 ± 2°C.

[0091] To measure plasma iloprost concentrations, 50 μL of plasma samples were transferred to wells of a 96-well plate, and then 150 μL of acetonitrile was added to each well. The mixture was vortexed for 1 min to disrupt the binding of plasma proteins to iloprost, followed by centrifugation at 3000 rpm for 5 min. The supernatant (150 μL) was mixed with an equal volume of HO and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) to determine the plasma iloprost concentration in rats.

[0092] result: IVR profile and PK parameters (C max ) are shown in Table 5, Figures 1 and 2.

[0093] [Table 6]

[0094] Table 5 shows that iloprost-liposome compositions with an ILO / CD ratio of less than 0.06 exhibit a reduced burst release profile (less than 68.7% of the iloprost is released within 1 hour of administration). More sustained release attributes (less than 45% of the iloprost is released within 1 hour of SLF incubation at 37°C) were observed in iloprost-liposome compositions with an ILO / CD ratio of less than 0.026. A similar trend was observed when cyclodextrin was added to the internal aqueous medium.

[0095] FIG. 1 shows the iloprost-liposome composition (LL021b3A2) of Table 6 at the indicated doses. Plasma mean values ​​in rats administered iloprost solution The logarithm of iloprost concentration versus administration time up to 24 hours is shown. There is no significant peak after administration of the iloprost-liposome composition, compared to the peak within 1 hour of administration of the iloprost solution. The reduced peak release may prevent side effects of the drug, e.g., the claimed Less local irritation in the upper respiratory tract upon direct contact with the liposome composition defined in the range of

[0096] FIG. 2 shows the area under the plasma concentration-time curve (AUC) from time zero to a specific time to determine the total exposure of iloprost over a period of time and to normalize the different doses of iloprost in each composition (iloprost-liposome composition or iloprost solution in Table 6). t ) and the area under the plasma concentration-time curve from time zero to infinity (AUC inf ) is shown. 100% of the iloprost was released within 1 hour of administration of the iloprost solution, while more than 80% of the iloprost was released within 24 hours of administration of the iloprost-liposome composition. These results indicate reduced drug accumulation at the target site and therefore fewer side effects.

[0097] Example 6: In vitro release (IVR) profiles and in vivo pharmacokinetic (PK) parameters of iloprost liposomal compositions containing different cyclodextrins (CD) Following the procedure outlined in Example 1, iloprost liposomal compositions containing (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD) or randomly methylated-β-cyclodextrin (RM-β-CD) were prepared and their IVR profiles evaluated.

[0098] result: Table 6 shows the physicochemical properties of iloprost liposomal compositions containing different CDs. Both HP-β-CD and RM-β-CD reduced the burst release of iloprost-liposomal compositions (less than 20% of iloprost was released within 1 hour of SLF incubation at 37°C).

[0099] [Table 7]

[0100] In the above description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the embodiments. However, it will be apparent to one skilled in the art that one or more other embodiments may be practiced without some of these specific details. Also, references such as "one embodiment," "an embodiment," ordinal numbers, etc. throughout this specification may be used to refer to the embodiments. It should be understood that reference to an embodiment means that a particular feature, structure, or characteristic can be included in the implementation of the disclosure. In the description, various features may be grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and facilitating understanding of various inventive aspects, and it should be further understood that one or more features or specific details of one embodiment can be implemented, if necessary, with one or more features or specific details of other embodiments in the implementation of the disclosure. The present invention encompasses the following aspects and configurations. (1) A pharmaceutical composition comprising one or more liposomes suspended in an external medium, Somerset (a) an outer lipid bilayer comprising at least one vesicle-forming phospholipid and less than 15 mole % sterol; and (b) an internal aqueous medium containing a weakly acidic drug and a weakly acidic salt; Including, A pharmaceutical composition, wherein less than 65% of the weakly acidic drug is released into the external medium within 1 hour of administering the pharmaceutical composition. (2) The pharmaceutical composition according to (1) above, wherein the outer lipid bilayer contains less than 10 mol% of a sterol. (3) The pharmaceutical composition according to (1) above, wherein the outer lipid bilayer is substantially free of sterol. (4) The pharmaceutical composition according to (1) above, wherein the sterol is selected from the group consisting of cholesterol, cholesterol hexasuccinate, ergosterol, lanosterol, and combinations thereof. (5) The pharmaceutical composition according to (1) above, wherein the vesicle-forming phospholipid 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 according to (1) above, wherein the weak acid salt is a carboxylate or bicarbonate. (7) The pharmaceutical composition according to (6) above, wherein the carboxylate is selected from the group consisting of formate, acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, benzoate, and combinations thereof. (8) The pharmaceutical composition according to (6), wherein the bicarbonate is selected from the group consisting of potassium bicarbonate, sodium bicarbonate, calcium bicarbonate, magnesium bicarbonate, cesium bicarbonate, lithium bicarbonate, nickel bicarbonate, ferrous bicarbonate, or a combination thereof. (9) The pharmaceutical composition according to (1) above, wherein the internal aqueous medium further comprises a cyclodextrin. (10) The pharmaceutical composition according to (9) above, wherein the molar ratio of the weakly acidic drug to cyclodextrin (drug / cyclodextrin ratio) is 0.06 or less. (11) The pharmaceutical composition according to (9) above, wherein the molar ratio of the weakly acidic drug to cyclodextrin (drug / cyclodextrin ratio) is 0.03 or less. (12) The pharmaceutical composition according to (1) above, wherein the weakly acidic drug is a prostaglandin, a prostacyclin receptor agonist, a steroid, a nonsteroidal anti-inflammatory drug (NSAID), an anticoagulant, an endothelin (ET) receptor antagonist, or a combination thereof. (13) The pharmaceutical composition according to (12) above, wherein the prostaglandin is iloprost. (14) The pharmaceutical composition according to (12) above, wherein the ET receptor antagonist is ambrisentan. (15) A method for treating a respiratory disease, comprising the step of administering the pharmaceutical composition described in (1) above. (16) A method for reducing the side effects of a weakly acidic drug, comprising the step of administering an effective amount of the pharmaceutical composition according to (1) above to a subject in need thereof. (17) The method according to (16) above, wherein the side effects of the weak acid drug in the upper respiratory tract are reduced by inhaling the weak acid.

Claims

1. 1. A pharmaceutical composition comprising one or more liposomes suspended in an external medium, said liposomes comprising: (a) an outer lipid bilayer comprising at least one vesicle-forming phospholipid and less than 15 mole % sterol; and (b) an inner aqueous medium containing a weakly acidic drug and a weakly acidic salt; Including, the pH of the internal aqueous medium is higher than the pH of the external medium; the weakly acidic drug is iloprost or ambrisentan; the external medium is a buffer solution; A pharmaceutical composition, wherein the pharmaceutical composition is incubated with simulated lung fluid (SLF) and less than 65% of the weakly acidic drug is released into the simulated lung fluid within 1 hour of said incubation.

2. The pharmaceutical composition of claim 1 , wherein the external medium is a citrate buffer or a phosphate buffer.

3. 2. The pharmaceutical composition of claim 1, wherein the outer lipid bilayer comprises less than 10 mol% sterol.

4. The pharmaceutical composition of claim 1 , wherein the outer lipid bilayer is sterol-free.

5. 2. The pharmaceutical composition of claim 1, wherein the sterol is cholesterol, cholesterol hexasuccinate, ergosterol, lanosterol, or any combination thereof.

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

7. The pharmaceutical composition of claim 1 , wherein the weak acid salt is a carboxylate or bicarbonate.

8. 8. The pharmaceutical composition of claim 7, wherein the carboxylate is formate, acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, benzoate, or any combination thereof.

9. 8. The pharmaceutical composition of claim 7, wherein the bicarbonate salt is potassium bicarbonate, sodium bicarbonate, calcium bicarbonate, magnesium bicarbonate, cesium bicarbonate, lithium bicarbonate, nickel bicarbonate, ferrous bicarbonate, or any combination thereof.

10. 10. The pharmaceutical composition of claim 1, wherein the internal aqueous medium further comprises a cyclodextrin.

11. 11. The pharmaceutical composition of claim 10, wherein the molar ratio of the weakly acidic drug to cyclodextrin (drug / cyclodextrin ratio) is 0.06 or less.

12. 11. The pharmaceutical composition of claim 10, wherein the molar ratio of the weakly acidic drug to cyclodextrin (drug / cyclodextrin ratio) is 0.03 or less.

13. 13. The pharmaceutical composition according to any one of claims 1 to 12, for the treatment of pulmonary hypertension or interstitial lung disease, wherein the weakly acidic drug is inhaled.

14. The pharmaceutical composition according to any one of claims 1 to 12 for reducing the side effects of a weakly acidic drug, wherein the weakly acidic drug is inhaled.

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