EP0472639A1 - Accumulation de medicaments dans des liposomes au moyen d'un gradient protonique - Google Patents

Accumulation de medicaments dans des liposomes au moyen d'un gradient protonique

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Publication number
EP0472639A1
EP0472639A1 EP19900908748 EP90908748A EP0472639A1 EP 0472639 A1 EP0472639 A1 EP 0472639A1 EP 19900908748 EP19900908748 EP 19900908748 EP 90908748 A EP90908748 A EP 90908748A EP 0472639 A1 EP0472639 A1 EP 0472639A1
Authority
EP
European Patent Office
Prior art keywords
composition according
liposome
buffer solution
lipid
pharmaceutical agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19900908748
Other languages
German (de)
English (en)
Other versions
EP0472639A4 (en
Inventor
Thomas D. Madden
Michael J. Hope
Colin P. S. Tilcock
Pieter R. Cullis
P. Richard Harrigan
Barbara S. Mui
Marcel B. Bally
Linda Tai
Lawrence D. Mayer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Elan Pharmaceuticals LLC
Original Assignee
Liposome Co Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Liposome Co Inc filed Critical Liposome Co Inc
Publication of EP0472639A1 publication Critical patent/EP0472639A1/fr
Publication of EP0472639A4 publication Critical patent/EP0472639A4/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A61K9/1278—Post-loading, e.g. by ion or pH gradient

Definitions

  • the present invention relates to pharmaceutical composi ⁇ tions and methods of making liposome containing compositions which exhibit characteristics of uptake which may be greater than expected by the relationship defined by the Henderson-Hasselbach equation.
  • the present invention also relates to liposomal composi ⁇ tions wherein the liposome comprises in part a membrane- stabilizing component, for example, cholesterol, which exhibits favorable characteristics in preventing rapid release of a pharmaceutical agent after it has been formulated in liposomes.
  • a membrane- stabilizing component for example, cholesterol
  • the present invention also relates to novel sustained release liposomal compositions comprising the bronchodilators metaproterenol, isoproterenol and terbutaline.
  • the present invention also relates to minimum buffering capacity required to achieve liposomal encapsulation of phrmaceutical agents with maintenance of a major portion of the initial pH gradient.
  • the therapeutic properties of many drugs may be dramati ⁇ cally improved by the administration in a liposomally encapsu ⁇ lated form [See, for example P.N. Shek and R.F. Barber, Mod. M ⁇ d. Canada. 41, 314-382, (1986)].
  • a liposomally encapsu ⁇ lated form See, for example P.N. Shek and R.F. Barber, Mod. M ⁇ d. Canada. 41, 314-382, (1986)].
  • amphotericin B and doxorubicin [Lopez- Bernstein, et al., J. Infect. Pis. P 151, 704-710, (1985) and Rah ⁇ man, et al.. Cancer Res.. 40, 1532 (1980)] toxicity is reduced while efficacy is maintained or even increased.
  • liposo ally encapsulated agents may be fortuitous and likely results from the altered pharmacokinetics and biodistribution of the entrapped drug [Ostro, et al., Amer. J. H ⁇ sp. Phar .. in press.
  • the transmembrane distribution of the drug is generally determined by the proton gradient which modu ⁇ lates drug leakage by changes in the buffering capacity of the intravesicular medium.
  • the use of proton and other ion gradients to trap drugs which are non-zwitterionic weak bases has been shown to be practical for adriamycin, the local anaesthetics dibucaine and dopa ine and other drugs.
  • Advantages of this system include efficient drug trapping, slower rates of drug release than passively trapped drug, and higher drug to lipid ratios than can otherwise be achieved.
  • the liposomes can be prepared in the absence of the drug, problems with drug release during storage, or drug degradation during liposomal preparation can be avoided.
  • Intraliposomal drug accumulation in response to pH gradi ⁇ ents is believed to occur in a manner similar to that of other weak bases, for example, the pH gradient probe methylamine. Methylamine equilibrates across liposomal membranes in the uncharged form, and re-ionizes according to the Henderson- Hasselbach relationship of the pH of its environment. The equi ⁇ librium distribution reflects the transmembrane pH gradient, and its redistribution can be used to measure these gradients.
  • Liposomes are completely closed lipid bilayer membranes which contain entrapped aqueous volume. Liposomes are vesicles which may be unilamellar (single membrane) or multilammelar (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer) .
  • the bilayer is composed of two lipid monolayers having a hydrophobic "tail” region and a hydrophilic "head” region. In the membrane bilayer, the hydrophobic (nonpolar) "tails” of the lipid monolayers orient toward the center of the bilayer, whereas the hydrophilic (polar) "heads” orient toward the aqueous phase.
  • the basic structure of liposomes may be made by a variety of techni ⁇ ques known in the art.
  • Liposomal encapsulation could potentially provide numerous beneficial effects for a wide variety of pharmaceutical agents and the remote loading technique should prove instrumental in realizing the potential of liposo ally encapsulated agents.
  • a high trapping efficiency for loading liposomes results in very little drug being lost during the encapsulation process, an advantage that proves to be important when dealing with expensive drugs.
  • the use of liposomes to administer drugs has raised problems with regard both to drug encapsulation and drug release during therapy. For example, even with the present use of "remote loading" systems, there is a con ⁇ tinuing need to increase trapping efficiencies so as to minimize the lipid load presented to the patient.
  • Figure 1 shows the accumulation of itoxantrone by EPC vesicles exhibiting a proton gradient with an internal aqueous buffer system comprising 300 mM citrate, pH 4.0 and an external buffer system comprising 300 mM NaCl, 20 mM HEPES, pH 7.5. Accumulation was rapid and complete and evidenced no release over several hours.
  • Figure 2A shows the response of timolol uptake in EPC vesicles (partial accumulation, uptake stable) .
  • the level of uptake is about 100 nmoles/umole (about 50% of available drug) .
  • Figure 2B shows the response of quinacrine, which is similar to timolol in EPC vesicles.
  • the level of uptake is about 80 nmoles/umole lipid after 30 minutes.
  • Figure 3A shows the response of quinidine uptake in EPC vesicles (complete accumulation, rapid release) . ithin 30 minutes about 50% of the drug leaks back out of the vesicles.
  • Figure 3B shows the effect of added cholesterol to the uptake of quinidine and the stability of the pH gradient.
  • Figure 4 shows the effect of physostigmine to transmem ⁇ brane pH gradient. Under the conditions used to assess drug uptake (200 uM physostigmine) only a small decrease in the measured pH is observed.
  • Figure 5 shows the entrapment of metaproterenol, ter- butaline and isoproterenol in response to pH gradients using egg phosphatidylcholine 200 nm extruded liposomes.
  • Figure 6 demonstrates the effect of drug uptake on the residual pH gradient as measured by methylamine redistribution in the presence and absence of isoproterenol. As shown, when the internal and external pH is 7.4 or 4.0 (no gradient), the methylamine does not detect any pH gradient.
  • Figure 7 shows the effect of temperature on the Entrap ⁇ ment of Metaproterenol in response to pH gradients at 21°C, 37°C and 60 * C.
  • Figure 8 shows the influence of cholesterol on the accumulation of metaproterenol in response to a pH gradient.
  • Figure 9 shows the influence of varying the external drug concentration on the level of metaproterenol uptake.
  • Figure 10 shows the effect of internal buffering capacity on drug uptake.
  • the present invention relates to liposomal compositions having a pH gradient which exhibit markedly increased accumula- tion of pharmaceutical agents above that expected from the Henderson-Hasselbach relationship by formulating the liposomes utilizing a first internal aqueous buffer and a second external aqueous buffer wherein the concentration of the pharmaceutical agent exceeds its solubility product in the internal buffer fol ⁇ lowing uptake. Therefore, preferably, the pharmaceutical agent exhibits a solubility within the liposome which is less than the final concentration of agent within the liposome.
  • the solubility of the pharmaceutical agent is less than about 20 mM and most preferably less than about 10 mM.
  • the internal buffer solution has a buffer strength of at least about 50 mMol, preferably about 100 to about 300 mMol and most preferably about 300.
  • the present invention also relates to liposomal composi ⁇ tions comprising in part, membrane-stabilizing components, for example, cholesterol, among other lipids, to prevent the rapid release of certain pharmaceutical agents from liposomes which do not contain the membrane-stabilizing components.
  • liposomes preferably comprise a mixture of phosphatidylcholine and cholesterol in a molar weight ratio of about 55:45.
  • the present invention also relates to liposomal composi ⁇ tions having a pH gradient containing bronchodilators selected from the group consisting of metaproterenol, terbutaline and isoproterenol. It has been shown that the above agents, which heretofore have not been formulated in such liposomes, will accumulate into liposomes to an appreciable extent to produce effective, stable liposomal compositions useful for treating con ⁇ ditions requiring sustained release of bronchodilators. Such liposomal compositions comprising bronchodilator formulations may be useful for treating a number of conditions, including asthma.
  • compositions are expected to have a longer residence time in the lung than the same free drug, thus obtaining concentrations of bronchodilator at the site of activity within the lung for a period longer than for compositions presently available.
  • Such compositions may be formulated as aerosols within a pharmaceuti ⁇ cally acceptable solution for administration of bronchodilators directly into the lungs for treatment of acute asthma attacks.
  • the brochodilator compositions of the present invention have been shown to effectively accumulate in liposomes comprising eggphosphatidylcholine (EPC) as well as a mixture of phosphatidylcholine and cholesterol (55:45, w:w) .
  • EPC eggphosphatidylcholine
  • the liposomes comprising the mixture of phosphatidylcholine and cholesterol accumulate the bronchodilator to about the same relative extent as the EPC liposomes, although the time need for accumulation is longer for the cholesterol containing liposomes.
  • the liposome compositions of the present invention have a drug to lipid molar ratio ranging from about 0.5% up to about 50%.
  • the liposomes of the present invention may comprise phospholipids such as egg phosphatidylcholine (EPC) , hydrogenated soy phosphatidylcholine, distearoylphosphatidyl- choline, dimyristoylphosphatidylcholine, or diarachidonoyl- phosphatidylcholine, among others, and may additionally comprise a number of steroidal compositions, as well as other composi ⁇ tions.
  • EPC egg phosphatidylcholine
  • hydrogenated soy phosphatidylcholine distearoylphosphatidyl- choline
  • dimyristoylphosphatidylcholine dimyristoylphosphatidylcholine
  • diarachidonoyl- phosphatidylcholine among others, and may additionally comprise a number of steroidal compositions
  • the liposomes range in size from about 0.05 to greater than 2 microns, with a preferred range being about 0.05 to about 0.3 microns. Most preferably, the liposomes are unilamellar and range in size from about 0.1 to about 0.3 microns. These unilamellar liposomes may be homogeneous or uni odal with regard to size distribution.
  • the liposomes of the present invention may be administered via oral, parenteral, buccal, topical, and trans- dermal routes of administration, among other routes of adminis ⁇ tration.
  • the present invention utilizes efficient trapping of pharmaceutical agents in liposomes exhibiting a transmembrane ionic gradient, preferably a transmembrane pH gradient, which can result in an accumulation of the agent in an amount significantly higher than otherwise expected from the Henderson-Hasselbach relationship.
  • Liposome compositions of the present invention comprise at least one lipid, a phrmaceutical agent accumulated therein, an internal buffer solution wherein the solubility of the pharmaceutical agent within the buffer solution is less than the concentration of the agent within the liposome and an external buffer solution wherein the solubility of the pharmaceutical agent is preferably at least about 0.2 mM.
  • the terms pharmaceutical agent and drug are synonymous.
  • the liposomes of the present invention may be formed by any of the methods known in the art, but preferably they are formed according to the procedures disclosed in Bailey, et al., PCT Application No. US86/01102, published February 27, 1986 and Mayer, et al. PCT Application No. US88/00646, published Sep- tember 7, 1988. These techniques allow the loading of liposomes with ionizable pharmaceutical agents to achieve interior con ⁇ centrations considerably greater than otherwise expected from the drugs' solubility in aqueous solution at neutral pH and/or con ⁇ centrations greater than can be obtained by passive entrapment techniques.
  • transmembrane ion (pH) gradient is created between the internal and external membranes of the liposomes and the pharmaceutical agent is loaded into the liposomes by means of the ion (pH) gradient, which drives the uptake.
  • the transmembrane gradient is generated by creating a concentration gradient for one or more charged species, for exam ⁇ ple Na+, Cl ⁇ , K+, Li+, OH ⁇ and preferably H+, across the liposome membranes, such that the ion gradient drives the uptake of ionizable pharmaceutical agents across the membranes.
  • transmembrane ion (H+) gradients are preferably employed to produce the ion gradient and load the pharmaceutical agents, which tend to have weakly basic nitrogen groups, into the liposomes.
  • liposomes are preferably first formed in an aqueous buffer solution.
  • the first solution is either acidic or basic, depending upon whether the pharmaceutical agent to be loaded produces a charged species at basic or acidic pH.
  • a charged species is produced at low pH, i.e., a pH of about 2.0 to 5.0, preferably a pH of about 4.0.
  • the buffer solution external to the liposomes is then modified to a pH significantly above the pH of the internal buffer solution, preferably at least about 3.0 to 4.0 pH units above the internal buffer solution.
  • the modification of the external buffer results in a pH gradient which drives the accumulation of pharmaceutical agent within the liposome.
  • the internal buffer solution may differ from the external buffer solution only in the difference in pH.
  • uncharged pharmaceutical agent will pass through the lipid layer(s) of the liposome much more readily than will charged (protonated, in the case of weakly basic pharmaceutical agents) agent.
  • uncharged pharmaceutical agent in the external buffer will readily pass through the liposome into the internal buffer, become protonated, and remain within the liposome as a "trapped" protonated molecule which does not readily pass through the liposome layer(s) .
  • Pharmaceutical agent will thus concentrate in the liposome as a function of the pH gradient between the inter ⁇ nal and external buffer solutions.
  • liposome compositions are preferred which are formed utilizing a first internal buffer solution of either basic (pH about 8 to 10) or acidic (pH about 3.0 to 5.0) character and a second external buffer solution, the pH of which is preferably between about 6.5 and 8.0, preferably 7.4.
  • the high or low pH of the internal buffer relative to a neutral pH of the external buffer produces a transmembrane gradi ⁇ ent which acts to drive the accumulation of the agent in the liposome.
  • internal buffer solutions useful in embodi ⁇ ments of the present invention are chosen so that the pharmaceutical agent to be accumulated has a solubility within the internal buffer solution which is less than the total agent to be accumulated in the liposome.
  • the solubility of the pharmaceutical agent in the internal buffer solution is no greater than about 65 mM, preferably no greater than about 20 mM and most preferably no greater than about 10 mM.
  • the internal buffer solution is also chosen to maximize the buffer strength of the internal solution. It is believed that the buffer strength of the internal buffer solution is also important to the total accumulation of agent within the liposome and internal buffer solutions are chosen to maximize this strength. Of course, the solubility of the agent within the internal buffer solution is also a most important factor in determining accumulation. Therefore, where a buffer solution is to be chosen, it is both the solubility factor and the buffer strength factor which should be maximized in choosing useful buffer solutions. In the present invention, it has been determined that the buffer strength of the internal buffer solu ⁇ tion should be at least about 50 mM, preferably about 100 mM to about 300 mM and most preferably about 300 mM.
  • Lipids which can be used in the liposome formulations of the present invention include synthetic or natural phospholipids and may include phosphatidylcholine (PC) , phosphatidylethanolamine (PE) , phosphatidylserine (PS) , phosphatidylglycerol (PG) , phosphatidic acid (PA) , phosphatidylinositol(PI) , sphingomyelin (SPM) and cardiolipin, among others, either alone or in combination.
  • the phospholipids useful in the present invention may also include dimyristoyl- phosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) .
  • distearylphosphatidylcholine DSPC
  • dipal itoylphosphatidylcholine DPPC
  • soy phosphatidylcholine HSPC
  • Dimyristoyl- phosphatidylcholine DMPC
  • diarachidonoylphosphatidylcholine DAPC
  • T c transition temperaturres
  • lipids such as DSPC (T c of about 65°C) , DPPC (T c of about 45'C) and DAPC (T c of about 85'C)
  • T c transition temperaturres
  • lipids are preferably heated to about their T c or temperatures slightly higher, e.g., up to about 5°C higher than the T c , in order to make these liposomes.
  • egg phosphatidylcholine is used.
  • a steroidal component may be added to the liposome.
  • Any of the above-mentioned phospholipids may be used in combination with at least one additional component selected from the group consisting of cholesterol, cholestanol, coprostanol or cholestane.
  • PEG- cholesterols polyethylene glycol derivatives of cholesterol
  • CHS cholesterol hemisuccinate
  • organic acid derivatives of alpha-tocopherol hemisuccinate, (THS) may also be used.
  • CHS- and THS-containing liposomes and their tris salt forms may generally be prepared by any method known in the art for preparing lipsomes containing sterols Any of the above-mentioned sterols may be used in liposomes, so long as the resultant phospholipid-sterol mixture yields stable liposomes.
  • sterols Any of the above-mentioned sterols may be used in liposomes, so long as the resultant phospholipid-sterol mixture yields stable liposomes.
  • PCT Publication No. 87/02219, published April 23, 1987, entitled “Alpha Tocopherol-Based Vehicles” relevant portions of which are incoporated by reference herein.
  • cholesterol in an amount equal to about 30 mole% to about 45 mole% by weight of the lipid comprising the liposome is preferably used in combination with any of the above-named phospholipids or phospholipid/steroid combinations.
  • Such com ⁇ positions should, in general, prevent the undesired rapid release of accumulated pharmaceutical agent from the liposome.
  • Any com ⁇ bination of membrane-stabilizing component and lipid may be used which prevents rapid release of pharmaceutical agents from the liposome, and one of ordinary skill in the art will be able to modify the membrane-stabilizing component and the phospholipid to formulate liposomes which prevent rapid release of the pharmaceutical agent.
  • liposomes comprising a mixture of about 45 mole % by weight cholesterol and about 55 mole % by weight phosphatidylcholine are used in this aspect of the present invention.
  • any number of pharmaceutical agents which show a proclivity to release rapidly from liposomes may be used in this aspect of the present invention, it has been determined that the agents quinine, diphenhydramine and quinidine are especially prone to rapidly release from liposomes and thus liposomal formulations comprising these agents preferably com ⁇ prise cholesterol in an amount equal to about 30 to 45 mole % and preferably about 45 mole % of the lipid plus membrane-stabilizing component.
  • any buffer solution may be used for the internal and external buffer solutions in this aspect of the present invention regardless of the solubility of the pharmaceutical agent therein.
  • the preferred buffer solutions are chosen so that the solubility of the pharmaceutical agent is less than the concentration of the agent within the . liposome, preferably is less than 20mM and most preferably is less than 10 mM as is the case with other embodiments of the present invention.
  • MLVs multilamellar vesicles
  • SPLVs stable plurila ellar vesicles
  • REVs reverse phase evapora ⁇ tion vesicles
  • MLVs are extruded through filters forming large unilamellar vesicles (LUVs) of sizes dependent upon the filter size utilized.
  • LUVs large unilamellar vesicles
  • polycarbonate filters of 30, 50, 60, 100, 200 or 800 nm pores may be used. In this method, disclosed in Cullis, et al., PCT Publication Ho.
  • the liposome suspension may be repeatedly passed through the extrusion device resulting in a population of liposomes of homogeneous size dis ⁇ tribution.
  • the filtering may be performed through a straight-through membrane filter (a Nucleopore polycarbonate fil ⁇ ter) or a tortuous path filter (e.g. a Nucleopore filter mem- brafil filter (mixed cellulose esters) of 0.1 u size), or by alternative size reduction techniques such as homogenization.
  • the size of the liposomes may vary from about 0.03 to above about 2 microns in diameter; preferably about 0.05 to 0.3 microns and most preferably about 0.1 to about 0.2 microns.
  • the size range includes liposomes that are MLVs, SPLVs, or LUVs.
  • the preferred liposomes are those which are unilam ⁇ ellar liposomes of about 0.1 to about 0.2 microns.
  • lipids may be used to form liposomes having a gel to liquid crystalline T c above ambient temperature.
  • an extruder having a heating barrel or thermojacket may be employed.
  • Such a device serves to increase the liposome suspension temperature allowing extrusion of the LUVs.
  • the lipids which are used with the ther oj cketed extruder are, for example, DSPC, DPPC, DMPC and DAPC or mixtures thereof, which may include cholesterol in certain embodiments for preventing the rapid release of pharmaceutical agents from the liposome.
  • Liposomes containing DSPC are generally extruded at about 65*C, DPPC at about 45'C and DAPC at about 85*C (about 5°C above the lipid T c ) .
  • the preferred liposome for use in the pres ⁇ ent invention are LUVs of about 0.06 to about 0.3 microns in size.
  • a homogeneous popu ⁇ lation of vesicles is one comprising substantially the same size liposomes, and may have a Gaussian distribution of particle sizes.
  • Such a population is said to be of uniform size distribu ⁇ tion, and may be unimodal with respect to size.
  • the term "unimodal" refers to a population having a narrow polydispersity of particle sizes, and the particles are of a single "mode".
  • a liposomal population is unimodal if, when measured by quasi elastic light scattering methods, the population approxi ⁇ mates to a Gaussian distribution, and if a second order poly ⁇ nomial will fit the natural logarithm of the autocorrelation function of a sample (Koppel, 1972, J. Chem. Phvs.. 57:4814). The closer this fit, the better the measure of unimodality. The closeness of this fit may be determined by how close the chi square (chi 2 ) value of the sample is to unity. A chi 2 value of 2.0 or less is indicative of a unimodal population.
  • size reduction techniques may be employed in prac- tiing the present invention.
  • homgenization or mill- ing techniques may successfully be employed. Such techniques may yield liposomes that are homogeneous or unimodal with regard to size distribution.
  • Liposomes may be prepared which encapsulate the first aqueous buffer solution having the characteristics described hereinabove.
  • this first aqueous buffer solution will surround the liposomes as they are formed, resulting in the buffer solution being internal and external to the liposomes.
  • the original external buffer solution may be acidified or basified sp that the concentration of charged species differs from the internal buffer, or alterna ⁇ tively, the external buffer may be replaced by a new external medium having different charge species.
  • the replacement of the external medium can be accomplished by various techniques, such as, by passing the liposome preparation through a gel filtration column, e.g., a Sephadex column, which has been equilibrated with the new medium, or by dialysis or related techniques.
  • a gel filtration column e.g., a Sephadex column
  • organic solvents may also be used to suspend the lipids.
  • Suitable organic solvents for use in the present invention include those with a variety of polarities and dielectric properties, which solubilize the lipids, for example, chloroform, methanol, ethanol, dimethylsul- foxide (DMSO) , methylene choloride, and solvent mixtures such as benzene: ethanol (70:30), among others.
  • DMSO dimethylsul- foxide
  • solvent mixtures such as benzene: ethanol (70:30)
  • Sol ⁇ vents are generally chosen on the basis of their biocompatability, low toxicity, and solubilization abilities.
  • One preferred embodiment of the present invention is a 3 component liposomal-pharmaceutical agent treatment system which allows for highly efficient entrapment of the agent at the clini ⁇ cal site.
  • the pharmaceutical agent is one that loads in response to a transmembrane pH gradient where the interior of the liposome is acid
  • the first component of the system (Vial 1) com ⁇ prises liposomes in an acidic buffer solution, in which for exam ⁇ ple, citric acid buffer (300 mmol., pH about 3.8 to 4.2, preferably 4.0) or another buffer in which the ionized form of the pharmaceutical agent to be trapped is only marginally soluble (solubility less than the final concentration of agent within the liposome, preferably no greater than about 20 mM and most preferably no greater than about 10 mM) .
  • the second component of the system comprises a basic buffer solution, for exam ⁇ ple, a sodium carbonate or sodium bisphosphate solution at about 0.5 M, pH about 10 to 12, preferably about pH 11.5, which serves to become part of the external buffer solution of the liposome formulation.
  • a solubility within the external buffer solution of at least about 0.2 mMol.
  • the third component (Vial 3) is the pharmaceutical agent.
  • the above-described treat ⁇ ment system may be provided as a 3-vial system, the first vial containing the liposomes in acidic medium, the second vial con ⁇ taining the base, and the third vial containing the pharmaceuti ⁇ cal agent as described hereinabove.
  • a similar treatment system may be provided for a pharmaceutical agent that loads in response to a transmembrane gradient wherein the internal buffer of the liposomes is relatively basic i.e., has a pH about 8.5-11.5.
  • the first component comprises liposomes in a relatively basic buffer, for example, sodium carbonate or sodium bisphosphate, at a pH of about 8.0-11.0, preferably about 10.
  • the second component com ⁇ prises a relatively acidic or neutral solution as the external buffer for the liposomes, for example, 150 mM NaCl buffer/150 mM HEPES buffer at a pH of about 7.4.
  • the third component comprises the pharmaceutical agent which is less ionized at the pH of the external buffer and is ionized at the pH of the internal buffer.
  • the liposomes may be heated prior to admixing with the drug.
  • the pharmaceutical agent is to be loaded into liposomes comprising at least about 30 mole % cholesterol to minimize the rapid release of the agent, it may be advantageous to heat the liposomes up to about 60 # C to facilitate loading.
  • the methods described in Mayer, et al. PCT Publication No. WO 88/06442, Sep ⁇ tember 7, 1988, relvant portions of which are incorporated by reference, herein may be modified for use with the agents of the present invention.
  • the pharmaceutical agent is entrapped in or associated with the liposome and then administered to the patient to be treated.
  • pharmaceutical agent, drug and agent are used interchangably.
  • U.S. Patent No. 3,993,754 Sears, U.S. Patent No. 4,145,410; Papahadjopoulos et al., U.S. Patent No. 4,235,871; Schneider, U.S. Patent No. 4,114,179; Lenk et al., U.S. Patent No. 4,522,803; and Fountain et al., U.S. Patent No. 4,588,578.
  • any number of different pharmaceutical agents and different pharmaceutical types may be entrapped in or associated with liposomes.
  • pharmaceutical agents useful in the present invention may include any agent which readily passes through a liposomal layer(s) and exhibits limited solubility in a buffer solution internal to.the liposome at an ion concentration or pH at which the pharmaceutical agent is in an ionized form.
  • Such agents may include antineoplastics; for example mitoxantrone, local anaesthetics; for example, lidocaine, dibucaine and chlorpromazine, bronchodilators; for example, metaproterenol, terbutaline and isoproterenol, beta-adrenergic blockers, for example propanolol, timolol and labetolol; antihypertensive agents, for example clonidine and hydralazine; anti-depressants, for example, imipramine, amitryptyline and doxepim, anti-convulsants, for example, phenytoin, anti-emetics, for example, procainamide and prochlorperazine; antihistamines, for example, diphenhydramine, chlorpheniramine and promethazine; anti-arrhythmic agents, for example, quinidine and disopyramide, anti-malarial agents, for example, chloroquine
  • internal buffers to be used in the liposomal compositions of the present invention are chosen using several criteria, the most important of which, after buffer strength, is the solubility characteristics of the pharmaceutical agent to be loaded in the buffer solution, as described hereinabove. It is preferred that the buffer system used as the internal buffer has a buffer strength of at least about 50 mM, preferably within the range of about 100 mM to about 300 mM, and most preferably about 300 mM.
  • the most preferred buffer solutions for use as the internal buffer system of the present invention are therefore characterized by their inability to solubilize the ionized, preferably protonated pharmaceutical agent, i.e., the ionized pharmaceutical agent is generally soluble in the buffer solution to an extent no greater.than about 65 mMol, preferably no greater than about 20mMol and most preferably no greater than about 10 mMol and which also have buffer strengths of at least about 50 mM, preferably about 100 to about 300 mM, most preferably about 300 mM.
  • the internal buffer solution precipitates the ionized species of the pharmaceutical agent out of solution.
  • the choice of buffer to use as the internal buffer solu ⁇ tion will vary depending upon the pharmaceutical agent chosen for loading.
  • One of ordinary skill in the art will be able to assess the relative solubilities of ionized species of a pharmaceutical agent and the buffer strength to determine the buffer solution to be used as the internal buffer solution.
  • any buffer solution having the characteristics generally described hereinabove may be used in the present invention, pro ⁇ vided that the solution is pharmaceutically compatible, i.e., the solution may be administered to the patient without deleterious affects.
  • Typical internal buffer solutions include citric acid, oxalic acid, succinic acid and other organic acid salts being preferred, among others.
  • Citric acid in a concentration ranging from about 100 mM to about 300 mM is preferred. Most preferably, the citric acid buffer solution has a concentration ranging from about 100 mM to about 300 mM.
  • Typical external buffer solutions may include NaCl, KC1, potassium phosphate, sodium bicarbonate, sodium carbonate, sodium bisphosphate, potassium sulfate and HEPES, and mixtures thereof, among others.
  • Loading efficiencies of pharmaceutical agents utilizing the present invention generally range from about 20% up to about 100%, preferably at least about 50%. In general, the loading efficiencies for pharmaceutical agents according to the present invention are greater than expected from the Henderson-Hasselbach relationship. Of course, not all agents readily accumulate in liposomes according to the Henderson-Hasselbach relationship, and certain agents (see Table 1, Example 1) appear, in certain cases, not to accumulate at all. This phenomenon may be the result of the pharmaceutical agent being too polar for penetration of the liposomes, or other factors.
  • the liposomes formed by the procedures of the present invention may be lyophilized or dehydrated at various stages of formation.
  • the lipid film may be lyophilized after removing the solvent and prior to adding the drug.
  • the lipid-drug film may be lyophilized prior to hydrating the liposomes.
  • Such dehydration may be carried out by exposure of the lipid or liposome to reduced pressure thereby removing all suspending solvent.
  • the liposomes may be dehydrated in the presence of a hydrophilic agent according to the procedures of Bally et al, PCT Publication No.
  • hydrated lipsome preparation may also be dehydrated by placing it in surrounding medium in liquid nitrogen and freezing it prior to the dehydration step.
  • Dehydration with prior freezing may be performed in the presence of one or more protective agents, such as sugars in the preparation according to the techniques of Bally, et al., PCT Application No. 86/01103 published February 27, 1986, relevant portions of which are also incorporated by reference herein.
  • the pharmaceuti ⁇ cal agent may be mixed with a sugar solution in a sugar: lipid weight/weight ratio ranging from about 0.5:1 to about 100:1, preferably about 20:1, without affecting the ability of the liposome to retain loaded agent during rehydration.
  • the liposomes preferably range in size from about 0.1 to about 0.2 microns.
  • the sugar is annitol, or mannitol:glucose:lactose in a 2:1:1 w/w/w ratio.
  • the preparation is preferably heated for ten minutes at an elevated temperature, for example 60*C.
  • Other suitable methods may be used in the dehydration of the above- disclosed liposome preparations.
  • the liposomes may also be dehydrated without prior freezing.
  • the liposomes Once the liposomes have been dehydrated, they can be stored for extended periods of time until they are to be used.
  • the appropriate temperature for storage will depend on the lipid formulation of the liposomes and the temperature sensitivity of encapsulated materials.
  • various antineoplastic agents are heat labile, and thus dehydrated liposomes containing such agents should be stored under refrigerated conditions e.g. at about 4'C, so that the potency of the agent is not lost.
  • the dehydration process is preferably carried out at reduced temperatures, rather than at room temperature.
  • rehydration is accomplished by simply adding an aqueous solution, e.g. , dis ⁇ tilled water or an appropriate buffer, to the liposomes and allowing them to rehydrate.
  • the liposomes can be resuspended into the aqueous solution by gentle swirling of the solution.
  • the rehydration can be performed at room temperature or at other temperatures appropriate to the composition of the liposomes * and their internal contents.
  • the rehydrated liposomes can be used directly in the cancer therapy following known procedures for administering liposome encapsulated drugs.
  • ionizable antineoplastic agents can be incorporated into the rehydrated liposomes just prior to administration.
  • the concentration gradient used to generate the transmembrane pH gradient can be created either before dehydration or after rehydration using the external medium exchange techniques described above.
  • the high drug to lipid ratio liposomes may be dehydrated prior to establishing the transmembrane pH gradient, for example, dehydrated from their first external medium.
  • the pH gradient can be established by admixing the liposomes with the second external medium of relatively acidic or basic pH.
  • the antineoplastic agent can be admixed with the liposomes simultaneously with or following the establishment of the pH gradient.
  • the liposomes may be rehydrated by admixing them with an aqueous solution of neutral pH.
  • the rehydration step would proceed by adding sodium carbonate and the pharmaceutical agent, for example, propanolol.
  • the liposomes already contain the base (e.g. sodium carbonate) , and therefore already have the transmembrane pH gradient are rehydrated, water or another neutral aqueous solution, and doxorubicin are added.
  • rehydration proceeds using water or another aqueous solution.
  • a second pharmaceutical agent may be added, if desired.
  • Liposomes containing the pharmaceutical formulations of the present invention may be used therapeutically in mammals, especially humans, in the treatment of a number of disease states or pharmacological conditions which require sustained release formulations as well as repeated administration.
  • the mode of administration of the liposomes containing the pharmaceutical agents of the present invention may determine the sites and cells in the organism to which the compound may be delivered.
  • the liposomes of the present invention may be administered alone but will generally be administered in admix ⁇ ture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the preparations may be injected parenterally, for example, intravenously.
  • parenteral administration they can be used, for example, in the form of a sterile aqueous solution which may contain other solutes, for example, enough salts or glucose to make the solution isotonic.
  • the liposomes of the present invention may also be employed subcutaneously or intramuscularly. Other uses, depending upon the particular properties of the preparation, may be envisioned by those skilled in the art.
  • the liposomal for- mulations of the present invention can be used in the form of tablets, capsules, losenges, troches, powders, syrups, elixirs, aqueous solutions and suspensions, and the like.
  • carriers which can be used include lactose, sodium citrate and salts of phosphoric acid.
  • Various disintegrants such as starch, lubricating agents, and talc are commonly used in tablets.
  • useful diluents are lactose and high molecular weight polyethylene glycols.
  • aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.
  • the liposomal formulations of the present invention may be incorporated into dosage forms such as gels, oils, emulsions, and the like. These formulations may be administered by direct application as a cream, paste, ointment, gel, lotion or the like.
  • the prescribing physician will ultimately determine the appropriate dosage of the neoplastic drug for a given human subject, and this can be expected to vary according to the age, weight and response of the individual as well as the pharmacokinetics of the agent used. Also the nature and severity of the patient's disease state or pharmacological condition will influence the dosage regimen. While it is expected that, in general, the dosage of the drug in liposomal form will be about that employed for the free drug, in some cases, it may be necessary to administer dosages outside these limits.
  • EPC egg phosphatidylcholine
  • cholesterol 55:45 molar ratio
  • propanolol timolol
  • dibucaine chlorpromazine
  • lidocaine quinidine
  • pilocarpine physostigmine
  • dopamine imipramine
  • diphen- hydramine quinine
  • chloroquine quinacrine
  • dauorubicin vin- cristine and vinblastine
  • the radiolabels 3 H- dipal itoylphosphatidylcholine, 14 C- dipalmitoylphosphatidylcholine, 14 C-dopamine and 14 C-Imipramine were obtained from Amersham while 3 H-chlorpromazine, 3 H- propranolol, 14 C-pilocarpine, 1 C-chlorpromazine, 1 C-methylamine and 14 C-lidocaine were obtained from New England Nuclear.
  • the Liposome Company, Inc. (Princeton, N.J.) kindly provided l c- timolol. Salts and reagents used were of analytical grade.
  • EPC vesicles containing 3 H-Dipalmitoylphosphatidylcholine
  • EPC:cholesterol mixtures 55:45 molar ratio
  • EPC:cholesterol mixtures were prepared by colyophilization from benzene:methanol (95:5 v/v) .
  • the dry lipid was hydrated with 300 mM citrate pH 4.0 as internal buffer solution and the resultant MLVs were sub- jected to five freeze-thaw cycles employing liquid nitrogen to enhance solute distribution according to Mayer, et al. Biochim. Biophvs. Acta. 817, 193 (1986) .
  • Lipid concentrations were determined by liquid scintilla ⁇ tion counting of 3 H-DPPC or 14 C-DPPC using a Packard 2000 CA instrument. Similarly, pilocarpine, chlorpromazine, timolol, propranolol, imipramine, lidocaine and dopamine were quantified using tracer quantities of 3 H- or ⁇ - 4 C-radiolabel. Physostigmine was assayed by fluorescence spectroscopy following solubilization of vesicles in 60% ethanol (v/v) . The excitation and emission of wavelengths used were 305 and 350 nm, respectively.
  • Quinacrine, chloroquine and quinine were also quantified from their fluorescence using excitation and emission wavelengths of 420 nm, 505 nm; 335 nm, 375 nm; and 335 n , 365 nm; respectively.
  • Vinblastine and vincristine were assayed by U.V. spec ⁇ troscopy from their absorbances at 262 nm and 297 nm, respec ⁇ tively, following solubilization of the vesicles in 80% ethanol.
  • Codeine was also measured by U.V. spectroscopy at 220 nm in this case after solubilization in 40 mM octyl-beta-D-glucopyranoside.
  • Mitoxantrone was quantified from its absorbance at 670 nm follow ⁇ ing solubilization of the vesicles in 2% Triton-XlOO.
  • Diphenhydramine was assayed by gas-liquid chromatography using a HP 9850 gas chromatograph fitted with a Chromatographic Specialties DB-225 (25% cyanopropyIphenyl) capillary column.
  • the helium carrier flow rate was 1 ml/min and detection was by flame ionization.
  • An internal standard, methylpentadecanoate, was used to quantify diphenhydramine following its extraction from the aqueous sample in diethylether and its separation from EPC by thin layer chro atography.
  • Transbilayer pH gradients were quantified employing the weak base methylamine ( 14 C-labelled) as previously described by Bally, et al., Chem. Phvs. Lipids. 47, 97, (1988).
  • the first category of pharmaceutical agents exhibited complete, stable uptake. Propanolol, dopamine, dauonorubicin, epirubicin, dibucaine, imipramine and doxorubicin exhibited the characteristics of this drug category. All of the drugs within this category exhibited uptake greater than predicted from the Henderson/Hasselbach equation. The accumulation of mitoxantrone by EPC liposomes exhibiting a proton gradient is shown in Figure 1.
  • the second category showed partial, but stable uptake.
  • Timolol, lidocaine, chlorpro azine, serotonin and chloroquine exhibited the characteristics of this drug category.
  • Timolol was loaded to the extent of about 100 nmoles/umole lipid (about 50% of available drug, see figure 2A) and quinacrine was loaded to a level of about 80 nmoles/umole lipid after 30 minutes (see figure 2B) . While accumulation is lower than in the first group of agents, nevertheless uptake is quite substantial. In the case of timolol, an internal concentration of about 65 mM is achieved against an external concentration of 100 uM.
  • the third category shows a partial uptake followed by a rapid release of agent from the liposome.
  • Figure 3 indicates a rapid virtually complete accumulation of quinidine into the vesicles and within 30 minutes about 50% of the agent has leaked back out of the vesicles ( Figure 3A) .
  • Other agents which leak back out of EPC vesicles include quinine, diphenhydramine, vin- blastine and vincristine.
  • the leakage rates vary considerably with vincristine and vinblastine loaded vesicles losing only 27% of initially sequestered ' drug over two-hours. This loss is asso ⁇ ciated with a corresponding reduction in residual change in pH as determined using methylamine.
  • a similar decrease in proton gra ⁇ der is observed as quinine and diphenhydramine are released from EPC vesicles.
  • the fourth category of pharmaceutical agents physostig ⁇ mine, codeine and pilocarpine exhibited no measurable response to the transmembrane pH gradient.
  • the suggestion that these agents cause a major increase in membrane permeability resulting in loss of ion gradient is not borne by the date from physostigmine (Fig ⁇ ure 4) .
  • Fig ⁇ ure 4 Under the conditions used to assess uptake of physostig ⁇ mine (200 uM) only a small decrease in measured change in pH was observed.
  • the factor that influences the level of drug uptake to the greatest extent is the solubility of the protonated species in the internal buffer.
  • the con ⁇ centration of protonated drug inside the vesicle exceeds its solubility product and precipitation occurs this will effectively reduce the transmembrane concentration gradient for the remaining solube fraction thus allowing further accumulation by the vesicles.
  • table 3 is shown the maximum apparent solubilities in 300mM citrate buffer, pH 5.0 for most of the drugs whose proton gradient dependent uptake was examined.
  • Drugs such as mitoxantrone, epirubicin, doxorubicin and daunorubicin which exhibit complete and stable uptake are relatively insoluble in the intravesicular medium.
  • dibucaine, propranolol and dopamine may also be loaded in liposomes in an amount significantly greater than predicted by the Henderson-Hasselbach equation. This is a sur ⁇ prising result considering that the three agents' apparent solubility is greater than the final internal concentration of the agent in the liposome.
  • EPC Egg phosphatidylcholine
  • Avanti Polar Lipids (Birmingham, Alabama)
  • 1 C-methylamine was purchased from New England Nuclear. All other chemicals and buffers were purchased from Sigma (St. Louis, MO.) and were used without purification.
  • LUVs Large Unilamellar Vesicles (LUVs) were produced by extru ⁇ sion according to the method of Hope, et al., Biochim. Biophvs. Acta. 817, 193 (1985) Briefly, LUVs were produced by extrusion of frozen and thawed lipid dispersions prepared in 300 mM citrate, pH 4.0, through 0.1 or 0.2 urn polycarbonate filters (Nucleopore) employing an extrusion device (Lipex Biomembranes, Vancouver, Canada) . Vesicles prepared by this technique employ ⁇ ing 0.1 um filters have trapped volumes of 1.5 uL/umole phospholipid as determined using 14 C or 22 Na and have an average diameter of 90 nm.
  • Phospholipid concentrations were determined by assay of lipid phosphorous as previously described by Fiske and Subbarrow, J. Biol. Chem.. 66, 375, (1925) .
  • Transmembrane pH gradients were established according to Example 1, and untrapped internal buffer removed by passing the LUVs down a Sephadex G-50 column equilibrated with the external buffer [150 mM NaCl, 20 mM HEPES, pH 7.4].
  • Induced pH gradients were determined by measur ⁇ ing the transmembrane distributions of 14 C-methylamine as described by Hope, et al. , supra. In short, methylamine was added to the vesicle system to a final concentration of 0.5 uCi/mL.
  • bronchodilators, isoproterenol, metaproterenol and terbutaline were incubated with liposomes with a transmembranepH gradient prepared as above at the indicted temperatures in a 150 mM NaCl, 20 mM HEPES, pH 7.4 buffer containing 500 uM of the bronchodilator and 6 M of the phospholipid.
  • Control samples without a transmembrane pH gradient were incubated at pH 4.0 or 7.4 (both inside and outside the vesicles) to determine the degree of gradient-independent membrane binding.
  • the pH 4.0 con ⁇ trol consisted of the vesicles prepared as above, but the external buffer was 150 mM NaCl, 20 mM citrate, pH 4.0.
  • the vesicles were prepared with 150 mM NaCl, 20 mM HEPES pH 7.4, both internally and externally.
  • FIG. 5 shows the entrapment of the bronchodilators metaproterenol, terbutaline and isoproterenol in response to pH gradients using (EPC) 200 nm extruded liposomes.
  • EPC EPC 200 nm extruded liposomes.
  • Vesicles con ⁇ taining 300 mM citrate, pH 4.0 were incubated with a 500 uM drug solution at pH 7.4.
  • the liposomes accumulate the drug to levels of greater than 60 nmoles/umole lipid.
  • the logarithm of the ratios of the internal and external concentrations of the radioactive methylamine can be used to measure the transmembrane change in pH, because the methylamine probe does not dissipate the internal proton poool at these con ⁇ centrations.
  • the internal and external pH is 7.4 or 4.0 (no gradient) the methylamine does not detect any gradient ( Figure 6).
  • the methylamine distribution indicates a 3.0 unit pH gradient, in good agreement with the 3.4 pH unit gradient.
  • metaproterenol is added to the external buffer to a final con ⁇ centration of 500 uM, the gradient dissipates to about 2.3 pH units as the drug is accumulated. The date indicates that the 190 fold achieved by the drug approximates the residual proton gradient (pH of 2.3 units).
  • the rate of metaproterenol entrapment is increased by increased temperature (See Figure 7) , reaching steady-state levels after 2 hours at 21"C, but faster than 15 minutes when incubated at 60"C. The extent of drug uptake is not dramatically affected by the temperature of incubation.
  • the amount of entrapped drug also depends on the initial drug to lipid ratio (see Figure 9) .
  • the amount of drug entrapped by the vesicles reflects the imposed transmembrane pH gradient greater than 3 units.
  • the reionization of the drug in the vesicle interior causes a decrease in the internal pH ( Figure 6) .
  • Equilibrium levels of drug entrapment would therefore be expected to relect the final change in pH rather than the initial change in pH as the ionized drug overwhelms the internal buffering capa ⁇ city of the vesicles.

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Abstract

L'invention concerne des compositions pharmaceutiques et des procédés de production de compositions contenant des liposomes et présentant des caractéristiques de grande captation. Cette captation peut être supérieure à celle qu'on l'on suppose en appliquant la relation définie par l'équation de Henderson-Hasselbach. L'invention concerne également des compositions liposomiques dans lesquelles le liposome comprend en partie un composant de stabilisation de la membrane, par exemple du cholestérol, qui présente des caractéristiques favorables de prévention de libération rapide d'un agent pharmaceutique sélectionné dans le groupe comprenant la quinine, la quinidine, et la diphénhydramine après formulation dans les liposomes. L'invention concerne également de nouvelles compositions liposomiques comprenant les bronchodilatateurs, à savoir le métaprotérénol, l'isoprotérénol et la terbutaline. L'invention concerne également une capacité de tampon minimum requise pour obtenir une encapsulation liposomique des agents pharmaceutiques avec maintien d'une grande partie du gradient du pH initial.
EP19900908748 1989-05-15 1990-05-15 Accumulation of drugs into liposomes by a proton gradient Withdrawn EP0472639A4 (en)

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ATE210966T1 (de) * 1993-05-21 2002-01-15 Liposome Co Inc Reduzierung von durch liposome induzierte physiologischen gegenreaktionen
US5741516A (en) * 1994-06-20 1998-04-21 Inex Pharmaceuticals Corporation Sphingosomes for enhanced drug delivery
US5543152A (en) * 1994-06-20 1996-08-06 Inex Pharmaceuticals Corporation Sphingosomes for enhanced drug delivery
US7351427B2 (en) 1998-06-18 2008-04-01 Busulipo Ab Pharmaceutical composition, a method of preparing it and a method of treatment by use thereof
HUP0303719A2 (hu) * 2000-10-16 2004-03-01 Neopharm, Inc. Mitoxantron hatóanyag-tartalmú liposzómás gyógyszerkészítmények és eljárás az előállításukra
RU2369384C2 (ru) * 2003-11-20 2009-10-10 ЙЭм БАЙОСАЙЕНСИЗ ИНК. Стабильные липосомальные композиции
AT500143A1 (de) * 2004-04-22 2005-11-15 Sanochemia Pharmazeutika Ag Cholinesterase-inhibitoren in liposomen sowie deren herstellung und verwendung
US20050255154A1 (en) 2004-05-11 2005-11-17 Lena Pereswetoff-Morath Method and composition for treating rhinitis
US20090220583A1 (en) 2005-06-09 2009-09-03 Lena Pereswetoff-Morath Method and composition for treating inflammatory disorders
CN104837483B (zh) 2012-11-20 2017-09-01 光谱医药公司 制备治疗用途的脂质体封装式长春新碱的改进方法
MX2017014130A (es) 2015-05-04 2018-07-06 Versantis AG Metodo para preparar vesiculas con gradiente de ph de transmembrana.
TWI678213B (zh) 2015-07-22 2019-12-01 美商史倍壯製藥公司 用於長春新鹼硫酸鹽脂質體注射之即可使用的調配物
CN113041223B (zh) * 2019-12-26 2022-08-19 南京绿叶制药有限公司 一种局部麻醉剂脂质体制备方法
CN111729087A (zh) * 2020-07-24 2020-10-02 成都大学 一种选择性β2受体激动剂的脂质修饰物及其制备方法与用途
KR20240037280A (ko) * 2021-07-16 2024-03-21 셀라토 파마슈티칼즈, 인코포레이티드 리포솜 제형의 제조 방법

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