阅读说明：本技术 用于控制释放弱酸药物的医药组合物及其用途 (Pharmaceutical composition for controlled release of weak acid drugs and use thereof ) 是由 甘霈 林一峰 陈可洁 于 2019-09-12 设计创作，主要内容包括：本发明提供一种医药组合物,包含至少一种脂质体,该脂质体包括外部脂质双层,所述外部脂质双层包括至少一囊泡形成磷脂质及小于15摩尔％的固醇；以及包括弱酸药物及弱酸盐的内部水性介质。该医药组合物减少弱酸药物的爆发释放。更提出一种本发明所揭露的医药组合物用于治疗呼吸系统疾病,并减少弱酸药物的副作用的用途。(The present invention provides a pharmaceutical composition comprising at least one liposome comprising an outer lipid bilayer comprising at least one vesicle-forming phospholipid and less than 15 mole% sterol; and an internal aqueous medium comprising a weak acid drug and a weak acid salt. The pharmaceutical composition reduces burst release of the weak acid drug. Further, the present invention provides a use of the pharmaceutical composition for treating respiratory diseases and reducing the side effects of weak acid drugs.)

1. A pharmaceutical composition, comprising:

one or more liposomes suspended in an external medium, the liposomes comprising:

(a) an outer lipid bilayer comprising at least one vesicle-forming phospholipid and less than 15 mole% sterol (sterol); and

(b) an internal aqueous medium comprising a weak acid drug and a weak acid salt;

wherein less than 65% of the weak acid drug is released to the external medium within 1 hour after administration of the pharmaceutical composition.

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

3. The pharmaceutical composition of claim 1, wherein the outer lipid bilayer is substantially free of sterols.

4. The pharmaceutical composition of claim 1, wherein the sterol is selected from the group consisting of cholesterol, cholesterol hexasuccinate (cholesteryl), ergosterol (ergosterol), lanosterol (lanosterol), and combinations thereof.

5. 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.

6. The pharmaceutical composition of claim 1, wherein the salt of a weak acid is a carboxylate (carboxylic acid salt) or bicarbonate salt (bicarbonate salt).

7. The pharmaceutical composition of claim 6, wherein the carboxylate salt is selected from the group consisting of formate (formate), acetate (acetate), propionate (propionate), butyrate (butyrate), isobutyrate (isobutrate), valerate (valerate), isovalerate (isovalate), benzoate (benzoate), and combinations thereof.

8. The pharmaceutical composition of claim 6, wherein the bicarbonate is selected from the group consisting of potassium bicarbonate (potassium bicarbonate), sodium bicarbonate (sodium bicarbonate), calcium bicarbonate (calcium bicarbonate), magnesium bicarbonate (magnesium bicarbonate), cesium bicarbonate (magnesium bicarbonate), lithium bicarbonate (lithium bicarbonate), nickel bicarbonate (nickel bicarbonate), iron bicarbonate (ferrous bicarbonate), or a combination thereof.

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

10. The pharmaceutical composition of claim 9, wherein the molar ratio of the weak acid drug to the cyclodextrin (drug/CD ratio) is less than or equal to 0.06.

11. The pharmaceutical composition of claim 9, wherein the molar ratio of the weak acid drug to the cyclodextrin (drug/CD ratio) is less than or equal to 0.03.

12. The pharmaceutical composition of claim 1, wherein the weak acid drug is a prostaglandin (prostaglandin), prostacyclin receptor agonist (prostacyclin receptor agonist), steroid, non-steroidal anti-inflammatory drug (NSAID), anticoagulant (anticoagulant), Endothelin (ET) receptor agonist, or a combination thereof.

13. The pharmaceutical composition of claim 12, wherein the prostaglandin is iloprost (iloprost).

14. The pharmaceutical composition of claim 12, wherein the Endothelin (ET) receptor agonist is ambrisentan (ambrisentan).

15. A method of treating a respiratory disease comprising the step of administering the pharmaceutical composition of claim 1.

16. A method of reducing the side effects of a weak acid drug comprising the step of administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 1.

17. The method of claim 16, wherein the weak acid is inhaled to reduce side effects of the weak acid drug in the upper respiratory tract.

Technical Field

Cross reference to related applications

This application claims the benefit of U.S. application No.62/731,101 filed on 9, 14, 2018, the entire disclosure of which is incorporated herein by reference.

Disclosed herein is a pharmaceutical composition comprising at least one liposome, wherein the liposome encapsulates a weak acid drug; the small amount of sterol in the liposome outer lipid bilayer can reduce or avoid burst release of the weak acid drug and/or maintain release of the weak acid drug.

Background

Liposomes are composed of natural or synthetic lipid bilayers forming microstructures with an internal barrier (reservoir) as a reservoir for therapeutic agents. A wide variety of liposomal compositions have been designed with drug delivery vehicles of varying size, permeability, and stability, all of which are designed to provide sustained drug release. However, these sustained release liposome compositions often initially exhibit burst drug release, resulting in higher side effects during the burst release and/or plasma drug concentrations outside the therapeutic range (therapeutic window).

The release profile of a liposome composition (release profile) depends on the structure of the liposome membrane and affects the performance of the liposomes. Therefore, controlled release profiles are an important prerequisite for the effective use of liposomes as drug delivery vehicles. For example, cholesterol is added to the outer lipid bilayer to increase membrane rigidity, stability and reduce permeability of the lipid bilayer (S.Kaddah et al, Food Chem toxicol.2018 Mar; 113: 40-48). Kaddah et al indicate that release of the coated drug decreases with increasing (up to 30%) cholesterol in the lipid bilayer. Corvera et al (Biochim Biophys acta.1992 Jun 30; 1107(2):261-70) teach that the addition of low concentrations of cholesterol (5-8%) to DMPC and DPPC liposomes reduces liposome stability and increases membrane permeability.

There remains a need for a liposome composition that does not exhibit an initial burst release to reduce potential side effects and prolong the therapeutic effect of weak acid drugs. The present invention addresses this need and other needs.

Disclosure of Invention

The present invention provides a pharmaceutical composition comprising one or more liposomes suspended in an external medium, the liposomes comprising: (a) an outer lipid bilayer comprising at least one vesicle-forming phospholipid and less than 15 mole% sterol (sterol); and (b) an internal aqueous medium comprising a weak acid drug and a weak acid salt, wherein less than 65% of the weak acid drug is released to the external medium within 1 hour after administration of the pharmaceutical composition.

Also disclosed are methods of treating respiratory diseases comprising the step of administering a pharmaceutical composition as described herein.

Also provided are methods for reducing the side effects of a weak acid drug, comprising the step of administering to an individual in need of ingestion of a weak acid drug an effective dose of a pharmaceutical composition as disclosed herein.

The terms "invention", "this invention" and "present invention" as used in this patent application are intended to refer broadly to all the subject matter of the present patent application and the claims that follow. Statements containing these terms should not be understood to limit the subject matter of the applications described herein or to limit the meaning or scope of the claims that follow. Embodiments of the invention covered by this patent application are defined by the following claims, not this summary. This summary is a high-level overview of various aspects of the invention, and is intended to introduce a selection of concepts that are further described below in the detailed description. 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 of the application should be understood by reference to appropriate portions of the entire specification, any or all of the drawings, and claims.

The invention will become more apparent upon reading the following figures and embodiments.

Brief Description of Drawings

Exemplary embodiments of the present invention will be described in detail with reference to the following drawings.

FIG. 1 shows a liposome composition comprising iloprost (iloprost), bicarbonate and HP- β -CD (LL021b3A 2); a liposome composition comprising iloprost, bicarbonate, and RM- β -CD (LL021m3a 2); or iloprost solution, log plot of mean plasma iloprost concentration in rats.

FIG. 2 shows a liposome composition comprising iloprost, bicarbonate and HP- β -CD (LL021b3A 2); a liposome composition comprising iloprost, bicarbonate, and RM- β -CD (LL021m3a 2); or iloprost solution from time zero to specific time point (AUC)_t) The area under the plasma concentration-time curve of (a) versus time from the zero time point to infinity (AUC)_inf) A plot of the area ratio under the plasma concentration-time curve of (a).

Detailed description of the preferred embodiments

The articles "a" and "an" as used herein refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

All numbers are modified by the term "about". The term "about" as used herein refers to a range of ± 10% of a particular value.

The terms "comprises" and "comprising" are used in a generic sense to refer to the inclusion/inclusion of one or more features, elements or components in a layer.

The term "subject" may refer to a vertebrate having a respiratory disease, or a vertebrate believed to be in need of treatment for a respiratory disease. The subject comprises a warm-blooded animal such as a mammal, such as a primate, and more preferably a human. Non-human primates are also subjects. The term subject includes domesticated animals such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, etc.). Accordingly, veterinary uses and pharmaceutical formulations are contemplated herein.

The term "treatment" refers to both therapeutic treatment (therapeutic treatment) and prophylactic (therapeutic) or preventative measures (prophylactic measures). Those in need of treatment include those already having a respiratory disease or related condition and those prone to have a respiratory disease or related condition or those in whom a respiratory disease is to be prevented.

The weak acid drugs used herein also include pharmaceutically acceptable salts thereof and charged forms thereof (programmed forms) unless the context clearly indicates or is otherwise indicated to the contrary. In one embodiment, the weak acid drug comprises a compound selected from the group consisting of carboxyl (-COOH), hydroxyl (-OH), phosphate (-PO)₄) And any combination thereof. In another embodiment, the weak acid drug has a pKa 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 above carboxyl (-COOH), hydroxyl (-OH), phosphate (-PO)₄) In addition, the weak acid drug may also contain one or more functional groups; these additional functional groups should not significantly alter the acidity of the drug from the corresponding portion of the drug that is not functionalized. In one embodiment, the weak acid drug is used to treat pulmonary hypertension (pulmony hypertension). In another embodiment, the weak acid drug is a prostaglandin (prostagladin), prostacyclin receptor agonist (prostacyclin receptor agonist), glucocorticosteroid (glucocorticosteroid), or a non-steroidal anti-inflammatory drug (non-steroidal anti-inflammatory drug). Table 1 shows non-limiting examples of weak acid drugs of the present invention.

TABLE 1 Weak acid drugs suitable for use in the present invention

As used herein, the terms "encapsulation", "loaded" and "encapsulation" are used interchangeably and refer to the incorporation (incorporation) or association (association) of a bioactive agent (e.g., iloprost) in the internal aqueous medium of a liposome.

The present invention provides a pharmaceutical composition comprising one or more liposomes suspended in an external medium, the 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 comprising a weak acid drug and a weak acid salt, wherein less than 65% by weight of the weak acid drug is released into the outer medium within 1 hour after administration of the pharmaceutical composition.

In an exemplary embodiment, the sterol in the outer lipid bilayer is less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 mole%. In another exemplary embodiment, the outer lipid bilayer is substantially free of sterols.

In the pharmaceutical composition, the weak acid drug has a coating efficiency greater than about 70%, 75%, or 80%.

The pharmaceutical composition reduces the burst release of the coated weak acid drug. In one embodiment, less than about 70%, 69%, 68%, 67%, 66%, or 65% of the weak acid drug is released within 1 hour after drug administration. As a result, side effects of the weak acid drug at the target site (e.g., cough (cough), throat irritation (throat irritation), sore throat (pharyngel pain), epistaxis (epistaxis), hemoptysis (hemoptysis), and wheezing (wheezing)) are reduced compared to pharmaceutical compositions having 15 mole% or more sterols in the outer lipid bilayer. In addition, the pharmaceutical composition prolongs the release of the weak acid drug and reduces the frequency of administration.

In one embodiment, burst release of the weak acid drug from the disclosed pharmaceutical composition is further reduced by the addition or coating of cyclodextrin (cyclodextrin) in the internal aqueous medium. Non-limiting examples of cyclodextrins include alpha-CD, beta-CD, gamma-CD, 2-hydroxypropyl beta-CD (HP-beta-CD), sulfobutyl ether (sulfobutyl) beta-CD (SBE-beta-CD), randomly methylated beta-CD (RM-beta-CD), or combinations thereof. Preferably, the cyclodextrin is HP- β -CD, RM- β -CD, or a combination thereof. In an exemplary embodiment, the molar ratio of weak acid salt to the cyclodextrin (drug/CD ratio) is less than or equal to about 0.06, 0.055, 0.05, 0.045, 0.04, 0.035, or 0.03.

Also disclosed are methods for treating a respiratory disease comprising the step of administering to a subject in need thereof an effective amount of the pharmaceutical composition disclosed herein, wherein the sterol content in the outer lipid bilayer is less than 15 mole%. The pharmaceutical compositions disclosed herein have a reduced burst release of weak acid drug compared to pharmaceutical compositions having equal to or greater than 15 mole% sterol in the outer lipid bilayer. Non-limiting examples of respiratory diseases include pulmonary hypertension and interstitial lung disease (interstitial lung disease).

Further disclosed is the use of a pharmaceutical composition disclosed herein for the treatment of a respiratory disease or the use of a pharmaceutical composition disclosed herein for the preparation of a medicament for the treatment of a respiratory disease.

The present invention is also directed to a method of reducing side effects of a weak acid drug comprising administering to a subject in need of ingestion of a weak acid drug an effective amount of a pharmaceutical composition described herein, wherein the content of sterols in the outer lipid bilayer is less than 15 mole%.

In some embodiments, the pharmaceutical compositions disclosed herein are administered by inhalation (inhalation) to reduce the side effects of weak acid drugs in the upper respiratory tract.

A. Liposome component

The term "liposome" as used herein refers to a microscopic vesicle or particle composed of one or more lipid bilayers surrounding an internal aqueous medium. To form liposomes, it is desirable that there be at least one "vesicle-forming lipid," which is an amphiphilic lipid capable of forming or being incorporated into a lipid bilayer, and any suitable vesicle-forming lipid can be used to form the lipid bilayer constituting 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.

The lipid bilayer of the liposome comprises at least one vesicle-forming lipid and 0 (zero) to less than 15 mole% of a sterol (e.g., 0-14.99 mole%) selected from the group consisting of cholesterol, cholesterol hexasuccinate (cholesterol hexasuccinate), ergosterol (ergosterol), lanosterol (lanosterol), and any combination thereof. In an exemplary embodiment, the sterol is cholesterol.

In some embodiments, the vesicle-forming lipid is a mixture of a first phospholipid and a second phospholipid. In particular embodiments, the first phospholipid is a Phosphatidylcholine (PC) selected from the group consisting of Hydrogenated Egg Phosphatidylcholine (HEPC), hydrogenated soybean phosphatidylcholine (hydrogenated soyay phosphatidylcholine, HSPC), Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), diarachioyl phosphatidylcholine (dipachyoylphosphatidylcholine), Dimyristoylphosphatidylcholine (DMPC), egg phosphatidylcholine (egg phosphatidylcholine, dyEPC), soybean phosphatidylcholine (soyaphosphatidylcholine, SPC), oleoylphosphatidylcholine (palmitoylphosphatidylcholine), palmitoylphosphatidylcholine (palmitoylphosphatidylcholine, phosphatidylcholine), palmitoylphosphatidylcholine (phosphatidylcholine), palmitoylphosphatidylcholine (phosphatidylcholic), and phosphatidylcholic), palmitoylphosphatidylcholine (phosphatidylcholic), and phosphatidylinositol (palmitoylphosphatidylcholine (phosphatidylinositol), phosphatidylinositol, HPC), and phosphatidylinositol (palmitoylphosphatidylcholine, HPC), and phosphatidylinositol (palmitoyl) and phosphatidylinositol, Dilauroyl phosphatidylcholine (DLPC), diundecenoyl phosphatidylcholine (didecanoyl phosphatidylcholine), didecanoyl phosphatidylcholine (dinonylphosphatidylcholine), and any combination thereof. In other embodiments, the second phospholipid is a polyethylene glycol modified phospholipid comprising polyethylene glycol having a molecular weight of about 500 daltons (dalton) to about 10,000 daltons, such as 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (1, 2-distearoyl-sn-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000], DSPE-PEG2000), a negatively charged phospholipid, such as Distearoylphosphatidylglycerol (DSPG), Dipalmitoylphosphatidylglycerol (Dipalmitoylphosphatidylglycerol, DPPG), or dimyristoylphosphatidylglycerol (dimyristoylphosphatidylglycerol, DMPG). In one exemplary embodiment, the first phospholipid: cholesterol: the molar percentage of the second phospholipid is 75-99: 0 to 14.9: 0.1 to 25.

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 (stearylamine), 1,2-dioleoyl-3-trimethylammonium-propane (1, 2-dioleoyl-3-trimethyllammonium-propane, DOTAP), 3 beta- [ N- (N ', N' -dimethylaminoethane) -carbamoyl]Cholesterol (3 beta- [ N- (N ', N' -dimethylionothane) -carbamoyl]cholesterol，DC-Cholesterol)、N⁴-cholesteryl-spermine (N)⁴-cholestyryl-hormone, GL67), dioctadecyldimethylammonium (DDAB), 1,2-di-O-octadecenyl-3-trimethylammonium propane (1, 2-di-O-octadecenyl-3-trimethylammoniumpropane, DOTMA), ethyphosphatidylcholine (ethyl PC), or combinations thereof. In another exemplary embodiment, the first phospholipid: cholesterol: the molar percentage of the charged lipid is 75-99: 0 to 14.9: 0.1 to 25.

In one embodiment, the mole% of HSPC, cholesterol and DSPG in the lipid bilayer is 75-99: 0 to 14.9: 0.1 to 25. In another embodiment, the mole% of HSPC, cholesterol and DSPE-PEG2000 in the lipid bilayer is 75-99: 0 to 14.9: 0.1 to 25.

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

Non-limiting examples of nonionic surfactants include nonionic water-soluble monoglycerides, nonionic water-soluble diglycerides, and nonionic water-soluble triglycerides; nonionic water-soluble monoglyceride and nonionic water-soluble diglyceride of polyethylene glycol (polyethylene glycol); nonionic water-soluble sorbitan fatty acid esters (e.g., sorbitan monooleate such as TWEEN 20 (polyoxyethylene 20sorbitan 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).

Non-limiting examples of cationic surfactants include dimethyldialkylammonium bromide (dimethyldodecylammonium bromide) or dodecyltrimethylammonium bromide (dodecamethylenelammonium bromide).

A non-limiting example of a zwitterionic surfactant includes 3- (N, N-dimethylpalmitylammonium) -propanesulfonate (3- (N, N-dimethyl palmitylammonio) -propanesulfonate).

According to the invention, liposomes are prepared in a medium comprising a weak acid salt to provide a pH gradient between the inner aqueous medium and the outer medium of the liposome. When vesicle-forming phospholipids and less than 15 mole% of sterols are contacted with a medium containing a weak acid salt, a liposome suspension is formed.

The liposomes in suspension are subjected to size reduction. The size of a liposome is generally referred to as its diameter. Size reduction of liposomes can be accomplished by a number of methods, such as extrusion, sonication, homogenization techniques, or milling techniques, which are well known and can be performed by those of ordinary skill in the art. Extrusion involves passing the liposomes under pressure through a filter having a defined pore size one or more times. The filter is typically made of polycarbonate (polycarbonate), but may 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 the liposomes can be reduced by sonic treatment, which applies sonic energy to break or cut the liposomes, which spontaneously reforms smaller liposomes. For example, the sound wave treatment may be performed by immersing a glass tube containing the liposome suspension in the center of the sound wave generated in a bath type sound wave device, or may be performed by using a probe type sound wave device that generates sound wave energy by vibration of a titanium probe in direct contact with the liposome suspension. In the present invention, the liposomes typically have a diameter of about 50nm to 500nm, for example about 500nm or less, about 400nm or less, about 300nm or less, about 200nm or less or about 100nm or less.

After sizing, the concentration of the weak acid salt in the external medium is adjusted to provide a pH gradient between the internal aqueous medium and the external medium, which can be performed in a variety of ways, for example by mixing the external medium with, for example, a citric acid buffer (H)₃C₆H₅O) and phosphate buffer (H)₃PO₄) The suitable buffer lacking the weak acid salt of (a) is exchanged by methods such as diafiltration, dialysis, ultrafiltration or tangential flow filtration.

The weak acid salt provides an external lower and internal higher pH gradient between the external medium and the internal aqueous medium of the liposome. In one embodiment, the pH of the inner aqueous medium is at least 0.1 units higher than the pH of the outer medium. In another embodiment, the pH of the inner aqueous medium is at least 1 unit higher than the pH of the outer medium. In another embodiment, the pH of the inner aqueous medium is about 7, 8, 9, or 10 and the pH of the outer 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 another exemplary embodiment, the pH of the external medium is above the pKa of the weak acid salt drug.

Non-limiting examples of salts of weak acids include carboxylates (carboxylic acid salt) or bicarbonates (bicarbonate salt).

As used herein, "bicarbonate" refers to a pharmaceutically acceptable salt-like compound comprising an anionic and a cationic component of bicarbonate. In one embodiment, the cationic component of the salt-like 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 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 (potassium bicarbonate), sodium bicarbonate (sodium bicarbonate), calcium bicarbonate (calcium bicarbonate), magnesium bicarbonate (magnesium bicarbonate), cesium bicarbonate (magnesium bicarbonate), lithium bicarbonate (lithium bicarbonate), nickel bicarbonate (nickel bicarbonate), iron bicarbonate (ferrous bicarbonate), or any combination thereof.

As used herein, "carboxylic acid salt" includes, but is not limited to, formate (formate), acetate (acetate), propionate (propionate), butyrate (butyrate), isobutyrate (isobutryate), valerate (valerate), isovalerate (isovalerate), or combinations thereof. In an exemplary embodiment, the acetate salt is sodium acetate (sodium acetate), calcium acetate (calcium acetate), or a combination thereof.

The concentration of bicarbonate or carboxylate is 50mM or more, 100mM or more, 150mM or more, 200mM or more, 250mM or more, 300mM or more, 350mM or more, 400mM or more, 450mM or more, 500mM or more, 600mM or more, 700M or more, 800mM or more than 900mM, less than 1000mM, from 50mM to 800mM, from 200mM to less than 1000mM, from 200mM to 800mM, or from 200mM to 600mM, from 250mM to less than 1000mM, from 250mM to 800mM, or from 250mM to 600mM, from 300mM to 600mM, from 200mM to 600mM

The liposomes prepared can be stored for a long period of time prior to loading with the weak acid drug and administration to a subject. For example, liposomes can be stored under refrigerated conditions for extended periods of time prior to loading with the weak acid drug. Alternatively, liposomes can be dehydrated, stored, and then rehydrated and loaded with a weak acid drug prior to administration. Liposomes can also be dehydrated after loading with a weak acid drug. Dehydration can be performed by a number of methods available and known in the art. In some embodiments, the liposomes are dehydrated using standard freeze drying equipment, i.e., dehydrated under low pressure conditions. In addition, liposomes can be frozen, for example, using liquid nitrogen. Before dehydration, sugars can be added to the liposome environment, for example to the buffer containing the liposomes, to ensure 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.

The liposomal suspension with less than 15 mole% sterol or substantially no sterol as described above is ready for loading with the weak acid drug. Typically, the weak acid drug is added to the external medium of the liposomes and the resulting suspension is reacted to diffuse the weak acid drug into the internal aqueous medium of the liposomes until the desired loading concentration and coating efficiency (percentage of the amount of internal/coated weak acid drug relative to the total amount of weak acid drug in the pharmaceutical composition) is achieved.

B. Relationship between sterol content in outer lipid bilayer and controlled release profile

The pharmaceutical composition of the present invention having less than 15 mole percent (e.g., 0-14.99 mole%) of sterols in the liposome outer lipid bilayer reduces burst release of the coated weak acid drug and thus reduces the side effects of the weak acid drug. In addition, a sufficient amount of the weak acid drug can be released from the pharmaceutical composition to achieve a desired therapeutic effect, and the release profile is unexpectedly extended compared to pharmaceutical compositions having greater than 15 mole percent sterol in the liposome outer lipid bilayer.

As used herein, the term "burst release" refers to a rapid and/or slightly uncontrolled release of greater than 70%, 69%, 68%, 67%, 66%, or 65% of the coated weak acid drug from the pharmaceutical composition within 1 hour (60 minutes) of administration of the pharmaceutical composition.

The term "extended release" as used herein may be used interchangeably with "controlled release", "delayed release", "modified release", "prolonged release", "programmed release", "preset release", "timed release", "rate controlled", or "sustained release", and refers to release of less than 50%, 45%, or 40% of a weak acid drug within 1 hour after administration of a pharmaceutical composition.

In one embodiment, the sustained release profile of the pharmaceutical composition is based on In Vitro Release (IVR) assays and/or in vivo pharmacokinetic studies of the encapsulated weak acid drug.

In certain embodiments, the pharmaceutical composition has a release profile in which less than about 70%, 69%, 68%, 67%, 66% or 65% by weight of the coated weak acid drug is released within 1 hour from the time point of administration of the pharmaceutical composition based on an In Vitro Release (IVR) assay and/or in vivo pharmacokinetic studies.

C. Administration of drugs

The pharmaceutical composition of the present invention may be administered into a cavity (cavity) of an individual that is not in direct contact with the blood stream. Examples of routes of administration include, but are not limited to, inhalation, intratracheal injection, subcutaneous injection, intra-articular injection, intramuscular injection, intravitreal injection, and intrathecal injection.

The pharmaceutical compositions of the present invention may be administered directly into the bloodstream of a subject.

In accordance with the present disclosure, the pharmaceutical composition may be administered from once to three times a day, once every 2 days, or once every 3 days.

The present disclosure will be further described in the following examples. It should be understood, however, that the following examples are for illustrative purposes only and are not to be construed as limiting the practice of the present disclosure.

Examples of the invention

General experimental procedure:

1. preparation of iloprost liposome composition

The liposome colloidal suspension was prepared using ethanol injection technique. Will comprise a molar ratio of 98:2 or 98.5: 1.5 all lipid components of the first phospholipid (HSPC) and the second phospholipid (DSPE-PEG2000 or DSPG) were dissolved in 2.86mL of ethanol solution at about 60 ℃. The resulting lipid solution was injected into 17.4mL of sodium bicarbonate solution (100 to 400 mM; pH 8.5) and optionally containing (2-Hydroxypropyl) - β -cyclodextrin (2-hydroxypypropyl) - β -cyclodextrin) (e.g., 45 to 120mM), and mixed with vigorous stirring at 60 ℃ to effect liposome hydration. The mixture is extruded 6 to 10 times through a polycarbonate membrane having a pore size of 0.2 and/or 0.1 μm to obtain a liposome suspension having an average particle size in the range of about 100 to 200nm and a polydispersity index (PdI) of less than 0.2. The liposome suspension was dialyzed (diasized) against a tangential flow filtration system with 10mM sodium citrate buffer (pH 5.5) to form a transmembrane pH gradient between the inner and outer aqueous media of the liposomes (e.g., higher inner pH and lower outer media pH). Suspensions of liposomes with this pH gradient were stored at 4 ℃ prior to the drug loading process.

Iloprost (available from Cayman Chemical, USA) was dissolved in 50mM sodium citrate solution and added to the liposome suspension to reach a drug concentration of 1000 to 250 μ g/mL, and reacted at 37 ℃ for 30 minutes. The resulting product was adjusted with sodium citrate buffer (pH 5.5) to obtain an iloprost-loaded liposome composition having pH 5.5 in an external medium and a liposome suspension having a phospholipid concentration of 10 mM.

2. Preparation of ambrisentan liposome composition

Liposome suspensions were prepared according to step 1 above with or without (2-hydroxypropyl) - β -cyclodextrin. Ambrisentan (available from Cayman Chemical, USA) was dissolved in dimethyl sulfoxide (DMSO), then added to the liposome suspension to reach a drug concentration of about 500 μ g/mL, and reacted at 37 ℃ for 30 minutes. The resulting product was adjusted with sodium citrate buffer (pH 5.5) to obtain an ambrisentan-loaded liposome composition having pH 5.5 in an external medium and a liposome suspension having a phospholipid concentration of 10 mM.

3. Quantitative characterization of liposome compositions

a. Coated and free iloprost/ambrisentan concentrations:

liposome composition of iloprostOr the ambrisentan liposome composition is poured into the PD MiniTrap^TMG-25 column (GE Healthcare) to separate the coated drug from the free drug. Iloprost liposome composition or ambrisentan liposome composition is mixed with methanol (90 vol% methanol and 10 vol% liposome suspension) to form a liposome-methanol mixture.

The concentrations of coated and free iloprost were analyzed by injecting 30 μ L of the liposome-methanol mixture into a Waters Acquity HPLC system equipped with a photodiode array (PDA) detector. The mobile phase is 36: 17: 47 acetonitrile (acetonitrile), methanol and phosphate buffer (pH 2.5), and the flow rate of the mobile phase was 1.0 mL/min. A C8 column having a size of 3.9 mm. times.15.0 cm, 5.0 μm was used, separation was performed at 25 ℃ and an absorption peak at 205nm was detected.

The coated ambrisentan concentration as well as the free ambrisentan concentration were analyzed by injecting 1 μ L of the liposome-methanol mixture into a Waters Acquity UPLC system equipped with a mass detector (QDa). Mobile phase a contained 0.1% formic acid in acetonitrile and mobile phase B contained in ddH₂0.1% formic acid in O. The gradient conditions were as follows: 50% mobile phase a, 0.2 minutes, 10% mobile phase a to 2 minutes, and 50% mobile phase a to 5.5 minutes. Separation was carried out at 35 ℃ at a flow rate of 1.0mL/min using a C18 column having a size of 4.6mm X10.0 cm, 3.0 μm. Using [ M + H]⁺Ions were MS detected in SIR mode, m/z 347.2 for ambrisentan.

b. Coating rate (EE) and drug to cyclodextrin ratio:

the concentration of the total amount of drug (iloprost or ambrisentan) in the liposomal composition comprises drug coated in the inner aqueous medium (L) and free drug in the outer medium (F).

The drug Encapsulation Efficiency (EE) is calculated as the percentage of drug encapsulated in the inner aqueous medium (L) of the liposomes relative to the total amount of drug (L + F), see the following equation:

EE(％)＝[L/(L+F)]×100

the ILO/CD ratio of the iloprost liposome composition and the AMB/CD ratio of the AMB liposome composition were calculated using the following formulas:

ILO/CD ratio { [ ILO ] × EE }/[ CD ]

AMB/CD ratio { [ AMB ] × EE }/[ CD ]

[ ILO ] (mM) concentration of total iloprost amount (L + F)

[ AMB ] (mM) ═ ambrisentan Total amount (L + F) concentration

EE (%) coating rate

[ CD ] (mM) ═ Cyclodextrin concentration

c. Average particle diameter and polydispersity index (PdI):

the mean particle size of the liposomes was assessed by dynamic light scattering. The polydispersity index (PdI), which represents the size distribution of the liposomes, was determined using a Beckman Coulter Delsa (TM) Nano C particle analyzer using the same evaluation technique as the mean particle size.

Example 1: in Vitro Release (IVR) curves of iloprost liposome compositions with different sterol contents

A. In Vitro Release (IVR) assay

Iloprost liposome compositions were formulated and analyzed for iloprost concentration according to the procedures of the general experimental procedures section supra. The average particle size of the liposome is 100-200nm, and PdI is less than 0.20.

Various IVR assays may be used to evaluate IVR curves. Depending on the iloprost in the subject liposome composition, the actual IVR assay is known or apparent to one of ordinary skill in the art. Iloprost-loaded liposome solution with initial phospholipid concentration of 10mM was diluted 10-fold in Simulated Lung Fluid (SLF) [ resolution Technologies 2011,18, 15-28%]Iloprost release profiles from liposomes were taken at 37 ℃ with a shaking speed of 100 rpm. The coating efficiency (EE) after reaction at a specific time point (T) is compared with the initial value (T) by using the following formula₀) Coating efficiency comparisons were used to calculate the percentage of iloprost released (% released) at each time point:

releasing_{at T}(％)＝(EE_{at T0}–EE_{at T})/EE_{at T0}

As a result:

physicochemical characterization and IVR curves of iloprost liposome compositions with different sterol contents are shown in table 1.

TABLE 1

Table 1 shows that > 90% EE was achieved with sodium bicarbonate, and that iloprost liposome compositions with less than 15 mole% cholesterol released less than 65% of iloprost within 1 hour of SLF reaction time, while iloprost liposome compositions with equal to or greater than 15 mole% cholesterol released greater than 70% of iloprost within 1 hour of SLF reaction time at 37 ℃.

Example 2: in Vitro Release (IVR) profiles of ambrisentan liposome compositions with different sterol contents

Ambrisentan liposome compositions were formulated and analyzed for ambrisentan concentration according to the procedure of the general experimental procedure section supra. The average particle size of the liposome is 100-200nm, and PdI is less than 0.20.

As a result:

the physicochemical characteristics and IVR curves of ambrisentan liposome compositions with different sterol contents are shown in table 2.

TABLE 2

Table 2 shows that > 95% EE was achieved with sodium bicarbonate salt and that the ambrisentan liposome composition with less than 15 mole% cholesterol released less than 50% iloprost within 1 hour of SLF reaction time at 37 ℃.

Example 3: in Vitro Release (IVR) curves of Liposome compositions of iloprost with or without Cyclodextrin (CD)

An in vitro study was conducted to evaluate the effect of (2-hydroxypropyl) - β -cyclodextrin (HP- β -CD) in the internal aqueous medium of the liposomes on the release profile of the iloprost liposome composition in example 1.

As a result:

the physicochemical characterization and IVR curves of iloprost liposome compositions with and without cyclodextrin (HP- β -CD) are shown in table 3.

TABLE 3

Table 3 shows that addition of cyclodextrin further reduced burst release (less than 60% iloprost was released within 1 hour of SLF reaction time at 37 ℃) and maintained the release profile of iloprost liposome compositions (less than 40% iloprost was released within 1 hour of SLF reaction time at 37 ℃).

Example 4: encapsulation efficiency of iloprost liposome compositions using different salts of weak acids

An in vitro study was conducted to evaluate the effect of different weak acid salts on the coating efficiency of the iloprost liposome composition of example 1. In this sample iloprost was loaded with sodium bicarbonate solution (400mM) and sodium acetate (sodium acetate).

As a result:

the coating efficiency of iloprost liposome compositions using different salts of weak acids is shown in table 4.

TABLE 4

Table 4 shows that bicarbonate and acetate achieved > 80% EE, and the presence of cyclodextrin in the internal aqueous medium further reduced burst release and maintained the release of iloprost from the liposome composition.

Example 5: in Vitro Release (IVR) profile and in vivo Pharmacokinetic (PK) parameters of iloprost liposome compositions with different iloprost to cyclodextrin (ILO/CD) ratios

In vitro studies were conducted to evaluate the effect of different ILO/CD ratios on the IVR curve of iloprost liposome compositions. The liposome compositions of this study were prepared according to the procedure outlined in example 1 and the IVR curves were analyzed. Iloprost solution (20 μ g/mL) was prepared by dissolving iloprost in 2mM trimethylamine (tromethamine) solution and adjusting the pH to approximately 8.4.

B. In vivo Pharmacokinetic (PK) study of iloprost liposome compositions

In this in vivo PK study, 3 male Sprague Dawley rats per group (purchased from BioLASCO Taiwan co., Ltd.) were anesthetized with isoflurane (isoflurane) and their backs were firmly positioned on the arched platform in a dorsal position in a 45 ° to 50 ° plane using a tape tied to the upper incisors. A microspray nozzle (Microsprayer, penncuryry, philiadelphia, USA) was inserted into the tracheal bifurcation of each rat, and 60 μ g/kg of the test sample (i.e., the composition in table 5 or iloprost solution) was administered intratracheally in each rat using a high pressure syringe connected to a microspray device.

At predetermined time points (5, 30 minutes, 1.5, 3, 6, 7 and 8 hours post-administration), blood samples from each rat were collected into heparin coated tubes (heparin coated tubes) and stored on wet ice. The blood sample was then centrifuged at about 2500 Xg for 15 minutes at 4. + -. 2 ℃ within 1 hour after collection to separate the plasma from the corpuscular cells. Approximately 0.1mL of plasma samples from each rat were added to a new storage tube and stored at-70 ± 2 ℃.

To determine plasma iloprost concentrations, 50 μ L of plasma samples were transferred to wells of a 96-well plate, followed by addition of 150 μ L of acetonitrile per well. Mixing the obtained mixtureThe plasma protein was disrupted by shaking for 1 minute to disrupt the binding to iloprost, and then centrifuged at 3000rpm for 5 minutes. Supernatant (150. mu.L) was mixed with an equal volume of H₂O mixed and analyzed by liquid chromatography mass spectrometry (LC-MS/MS) to determine the iloprost concentration in plasma samples from rats.

As a result:

IVR curves and PK parameters (C) for iloprost liposome compositions with different ILO/CD ratios_max) Shown in table 5, fig. 1 and fig. 2.

TABLE 5

1: the outer lipid bilayer comprises 10mM lipid (HSPC/DSPE-mPEG 98:2)

2: BCN: bicarbonate salt

Table 5 shows that iloprost liposome compositions having ILO/CD ratios less than 0.06 exhibit reduced burst release profiles (less than 68.7% iloprost released within 1 hour from administration time). It is noted that the iloprost liposome composition having an ILO/CD ratio of less than 0.026 is more sustained release (less than 45% of iloprost is released within 1 hour from the time of SLF incubation at 37 ℃). Note that there is a similar tendency to add cyclodextrins in the internal aqueous medium.

Figure 1 shows the results of the log of the mean plasma iloprost concentration in rats administered a given dose of the iloprost liposome composition or iloprost solution of table 6(LL021b3a2/LL021m3a2) versus the administration time to 24 hours. There was no significant peak after administration of the iloprost liposome composition compared to the peak in 1 hour of administration of the iloprost solution. Reduced peak release avoids side effects of the drug, e.g., less local irritation in direct contact with the upper respiratory tract with the claimed liposome composition.

The liposome composition and iloprost solution depicted in figure 2 are from time zero to specific time points (AUC)_t) Area under plasma concentration-time curve ofFor time from zero hour to infinity (AUC)_inf) To determine the total exposure of iloprost over a period of time, and to normalize different doses of iloprost in each composition (iloprost liposome composition or iloprost solution of table 6). Greater than 80% of iloprost is released within 24 hours from the time of administration of the iloprost liposome composition, as compared to 100% of iloprost released within 1 hour of administration of the iloprost solution. The results show that drug accumulation at the target site is reduced and thus side effects are reduced.

Example 6: in Vitro Release (IVR) profile and in vivo Pharmacokinetic (PK) parameters of iloprost liposome compositions with different Cyclodextrins (CD)

Iloprost liposome compositions containing (2-hydroxypropyl) - β -cyclodextrin (HP- β -CD) or randomly methylated- β -cyclodextrin (RM- β -CD) were prepared and VR curves were evaluated according to the procedure outlined in example 1.

As a result:

table 6 shows the physicochemical characterization of iloprost liposome compositions with different CD. Both HP-beta-CD and RM-beta-CD reduced the burst release of the iloprost liposome composition (less than 20% of iloprost was released within 1 hour of SLF reaction time at 37 ℃).

TABLE 6

1: lipid composition (10mM lipid) HSPC/DSPE-mPEG 98: 2.

2: BCN: bicarbonate salt

In the description above, for the purposes of explanation, numerous specific details have been set forth in order to provide a thorough understanding of the embodiments. It will be apparent, however, to one having ordinary skill in the art that one or more other embodiments may be practiced without these specific details. It should also be understood that reference throughout this specification to "one embodiment", "an embodiment", and embodiments having an indication of ordinal numbers, etc., which represent particular features, structures or characteristics, may be included in the practice of the present disclosure. It should be further appreciated that various features are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of various inventive aspects, and that one or more features or specific details from one embodiment may be practiced with one or more features or specific details from another embodiment when the disclosure is carried out in an appropriate situation.