CA1291425C - Method of producing liposome - Google Patents
Method of producing liposomeInfo
- Publication number
- CA1291425C CA1291425C CA000533545A CA533545A CA1291425C CA 1291425 C CA1291425 C CA 1291425C CA 000533545 A CA000533545 A CA 000533545A CA 533545 A CA533545 A CA 533545A CA 1291425 C CA1291425 C CA 1291425C
- Authority
- CA
- Canada
- Prior art keywords
- liposomes
- drug
- organic solvent
- gel
- parts
- 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.)
- Expired - Lifetime
Links
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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/829—Liposomes, e.g. encapsulation
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T428/00—Stock material or miscellaneous articles
- Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
- Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]
- Y10T428/2984—Microcapsule with fluid core [includes liposome]
Abstract
Abstracts of The Disclosure Liposomes with an increased drug trap can be prepared by adding a readily volatile organic solvent to a drug-containing liquid with liposomes dispersed therein to cause gel formation and then removing said organic solvent by evaporation.
Description
Method of Producinq Liposome This invention relates to a method of producing liposomes. More particularly, the invention is to provide liposomes with an increased drug trap ratio in a practically advantageous manner.
The so-far known liposome species include M~V (multi-lamellar vesicle), SUV (small unilamellar vesicle) and LUV
(large unilamellar vesic].e; also called REV: reverse-phase evaporation vesicle) and a presentation of their characteristics and the methods of their production can be found in "Cell Engineering, Vol. 2, No. 9, 1136 (1983)", for instance. A further liposome species differing from the above three species as called SPLV (stable plurilamellar vesicle) has recently become known (Japanese Patent Application under PCT laid open under Kohyo Mo. 59-500952).
Liposomes of these kinds as obtained by the so-far known methods have drawbacks with resspects to their manufacturing and their application as drug dosage forms.
The MLV, when applied for drug encapsulation, gives a low drug trap ratio and, moreover, has poor stability. The ~UV
gives a low drug trap ratio, too. Some kinds of drugs can hardly be incorporated into the SUV. The REV is known to be a liposome species giving relatively high drug trap ratios. Its production is performed by dissolving a phospholipid in an organic solvent, adding an aqueous layer to give a w/o emulsion and further evaporating the organic solvent from said emulsion (Japanese Patent ~pplication laid open under Kokai No. 55-118415). This process involves the step of preparing a fine w/o emulsion using an organic solvent. Therefore, the following problems, among others, may be pointed out in the REV preparation: 1) it needs a ultrasonic emulsification step, 2) it needs a large quantity of an organic solvent and 3) it needs fine adjustment of the vacuum during the solvent evaporation process. Any o-f the points makes lt difflcult to produce the REV
in a large scale at a time. The SPLV process encounters a similar problem that it needs a larger quantity of organic solvent as compared with lipids and also a problem of necessity of using reetricted kinds of lipids which have low phase transition temper-atures, such as egg yolk leci-thin.
Thus, -the liposomes, when produced by the sofar known methods, still have problems as mentioned above, i.e. the problems of their manufacturing and of their application as microcapsules for drugs. Therefore, -the present inventors conducted intensive investigations and, as a result, they found out a method of pro-ducing stable liposomes with an increased drug trap ratio and good reproducibility, irrespective of smallness or largeness o~ the production scale, which comprises adding a small amount of a readily volatile organic solvent to an MLV produced by a known method or to SUV obtained by ultrasonic treatment and then removing the organic solvent from the resulting gel by evaporation under ordinary reduced pressure or in a ni-trogen gas stream.
Further studies have now resulted in completion of the present invention.
The present invention thus consists of a method of producing liposomes with increased drug trap, which comprise~:
(1) preparing liposomes with or without a drug incorporated therein from a bilayer-Eorming lipid, said liposomes being dis-persed in a drug-containing liquid, 4;~
- 2a - 24205-733 (2) adding a readily volatile organic solvent to the disper-sion to cause gel formation, and (3) then removing said organic solvent by evaporation to reconstitute liposomes.
The liposomes may be those produced by any of the known methods and, in particular, MLV or SUV liposomes are used advan-tageously. For instance, the MLV to be used in practicing the invention can be obtained by a known methocl, for example, 5-100 parts by volume of an aqueous drug solution is added to 1 part by weight of a lipid in the thin film form which is previously obtained by the film method, and then MLV is obtained by stirring the mixture of the lipid and the drug solution with a vortex mixer. In this instance, the drug concentration can suitably be selected depending on the kind thereof. The SVV can be obtained by comminuting the above MLV, for example by treatment on a probe-type ultrasonic generator (20 KHz). The thus obtained M~V or SUV can be used as a starting material for the production of the liposomes in the present invention. Any other vesicles capable of forming lipid bilayers can also be used as starting materials for the production of the liposomes in the present invention in the same manner as the above~
mentioned M~V and SUV, even when said vesicles are low in a ; drug trap ratio or unstable. As such starting materials, thPre may be mentioned, for example, liposomes obtained by the ether addition method-~Biochimica et Biophysica Acta, Vol. 443, page 629 (1976)], liposomes obtained by dispersing a lyophilizate in water (Japanese Patent Application laid open under Kokai No. 53-14251~) and liposomes obtained by the freezing-thawing method (Japanese Patent Application laid open under Kokai No. 51-86117).
In addition, drug-free liposomes, which are prepared from the lipid and the drug-free solution, can also be used as a starting material. Such drug-free liposomes may be dispersed in a drug-containing liquid.
Regarding the drug to be used in the practice o~ the invention, there is no particular limitationi it may be a hydrophilic drug or a lipophilic one. However, the method of the invention is most preferably applicable to hydrophilic drugs having an "octyl alcohol/water-distribution ratio" lower than 10 as the logarithmic value.
Examples of such hydrophilic drug include various antiinflammatory analgesicsr lymphokines, anticancer agents, immunopotentiators, physiologically active ~. 2~3~4~5 _ 4 _ 24205-733 peptides, antibiotics, antiprotozoa agents, enzymes, antiallergic drugs and so on. More detailed examples of the hydrophilic drug are lymphokines such as interferons and interleukin 2, antiinflammatory analgesic peptides such as manganese-containing superoxide dismutase (SOD), superoxide dismutase-PEG (SOD-PEG) which is a derivative of SOD (Japanese Patent Application laid-open No. 58-16~85 and European Patent Publication No. 0070656), lipomodulins;
anticancer agents such as cisplatin, mitomycin C, 5-FU, adriamycin, actinomycin and bleomycin; immunopotentiators such as muramyldipeptides and muramyltripeptides;
physiologically active peptides such as thyroid hormone-releasing hormone (TRH), leuprolide, insulin and DN--1417 (Japanese Patent Application laid-open No. 57-132658 and European Patent Publication No. 0094157); ~-lactam antibiotics such as sulbenicillin, cefotiam, cefmenoxime and sulfazecin; aminoglycoside antibiotics such as gentamicinr streptomycin and kanamycin; vitamins (e.~.
cyanocobalamin), antiprotozoa~ agents (e.g. meglumine antimonate); enzymes (e.g. alkaline phosphatase);
anticoagulants (e.g. heparin); antiallergics such as amoxanox; and other general-use drugs (e.g. glutathione).
The lipid material to be used in the practice of the invention may be any one that can form lipid bilayers. For example, there may be mentioned phospholipids derived ~rom egg yolk, soybean or other animal or vegetable tissues, such as phosphatidylcholines, phosphatidyle~hanolamines, phosphatidylinositols, phosphatidylserines and sphingomyelins; mixtures o these, such as egg yolk lecithin; hydro~ennated lecithins;
dipalmitoylphosphatidylcholines, semisynthetic distearoylphosphatidylcholines; and other lipids than the abo~e-mentioned phospholipids, such as cholesterol, monoglycerides, diglycerides and triglycerides. These may be used either singly or in admixture.
The so-far known liposome species include M~V (multi-lamellar vesicle), SUV (small unilamellar vesicle) and LUV
(large unilamellar vesic].e; also called REV: reverse-phase evaporation vesicle) and a presentation of their characteristics and the methods of their production can be found in "Cell Engineering, Vol. 2, No. 9, 1136 (1983)", for instance. A further liposome species differing from the above three species as called SPLV (stable plurilamellar vesicle) has recently become known (Japanese Patent Application under PCT laid open under Kohyo Mo. 59-500952).
Liposomes of these kinds as obtained by the so-far known methods have drawbacks with resspects to their manufacturing and their application as drug dosage forms.
The MLV, when applied for drug encapsulation, gives a low drug trap ratio and, moreover, has poor stability. The ~UV
gives a low drug trap ratio, too. Some kinds of drugs can hardly be incorporated into the SUV. The REV is known to be a liposome species giving relatively high drug trap ratios. Its production is performed by dissolving a phospholipid in an organic solvent, adding an aqueous layer to give a w/o emulsion and further evaporating the organic solvent from said emulsion (Japanese Patent ~pplication laid open under Kokai No. 55-118415). This process involves the step of preparing a fine w/o emulsion using an organic solvent. Therefore, the following problems, among others, may be pointed out in the REV preparation: 1) it needs a ultrasonic emulsification step, 2) it needs a large quantity of an organic solvent and 3) it needs fine adjustment of the vacuum during the solvent evaporation process. Any o-f the points makes lt difflcult to produce the REV
in a large scale at a time. The SPLV process encounters a similar problem that it needs a larger quantity of organic solvent as compared with lipids and also a problem of necessity of using reetricted kinds of lipids which have low phase transition temper-atures, such as egg yolk leci-thin.
Thus, -the liposomes, when produced by the sofar known methods, still have problems as mentioned above, i.e. the problems of their manufacturing and of their application as microcapsules for drugs. Therefore, -the present inventors conducted intensive investigations and, as a result, they found out a method of pro-ducing stable liposomes with an increased drug trap ratio and good reproducibility, irrespective of smallness or largeness o~ the production scale, which comprises adding a small amount of a readily volatile organic solvent to an MLV produced by a known method or to SUV obtained by ultrasonic treatment and then removing the organic solvent from the resulting gel by evaporation under ordinary reduced pressure or in a ni-trogen gas stream.
Further studies have now resulted in completion of the present invention.
The present invention thus consists of a method of producing liposomes with increased drug trap, which comprise~:
(1) preparing liposomes with or without a drug incorporated therein from a bilayer-Eorming lipid, said liposomes being dis-persed in a drug-containing liquid, 4;~
- 2a - 24205-733 (2) adding a readily volatile organic solvent to the disper-sion to cause gel formation, and (3) then removing said organic solvent by evaporation to reconstitute liposomes.
The liposomes may be those produced by any of the known methods and, in particular, MLV or SUV liposomes are used advan-tageously. For instance, the MLV to be used in practicing the invention can be obtained by a known methocl, for example, 5-100 parts by volume of an aqueous drug solution is added to 1 part by weight of a lipid in the thin film form which is previously obtained by the film method, and then MLV is obtained by stirring the mixture of the lipid and the drug solution with a vortex mixer. In this instance, the drug concentration can suitably be selected depending on the kind thereof. The SVV can be obtained by comminuting the above MLV, for example by treatment on a probe-type ultrasonic generator (20 KHz). The thus obtained M~V or SUV can be used as a starting material for the production of the liposomes in the present invention. Any other vesicles capable of forming lipid bilayers can also be used as starting materials for the production of the liposomes in the present invention in the same manner as the above~
mentioned M~V and SUV, even when said vesicles are low in a ; drug trap ratio or unstable. As such starting materials, thPre may be mentioned, for example, liposomes obtained by the ether addition method-~Biochimica et Biophysica Acta, Vol. 443, page 629 (1976)], liposomes obtained by dispersing a lyophilizate in water (Japanese Patent Application laid open under Kokai No. 53-14251~) and liposomes obtained by the freezing-thawing method (Japanese Patent Application laid open under Kokai No. 51-86117).
In addition, drug-free liposomes, which are prepared from the lipid and the drug-free solution, can also be used as a starting material. Such drug-free liposomes may be dispersed in a drug-containing liquid.
Regarding the drug to be used in the practice o~ the invention, there is no particular limitationi it may be a hydrophilic drug or a lipophilic one. However, the method of the invention is most preferably applicable to hydrophilic drugs having an "octyl alcohol/water-distribution ratio" lower than 10 as the logarithmic value.
Examples of such hydrophilic drug include various antiinflammatory analgesicsr lymphokines, anticancer agents, immunopotentiators, physiologically active ~. 2~3~4~5 _ 4 _ 24205-733 peptides, antibiotics, antiprotozoa agents, enzymes, antiallergic drugs and so on. More detailed examples of the hydrophilic drug are lymphokines such as interferons and interleukin 2, antiinflammatory analgesic peptides such as manganese-containing superoxide dismutase (SOD), superoxide dismutase-PEG (SOD-PEG) which is a derivative of SOD (Japanese Patent Application laid-open No. 58-16~85 and European Patent Publication No. 0070656), lipomodulins;
anticancer agents such as cisplatin, mitomycin C, 5-FU, adriamycin, actinomycin and bleomycin; immunopotentiators such as muramyldipeptides and muramyltripeptides;
physiologically active peptides such as thyroid hormone-releasing hormone (TRH), leuprolide, insulin and DN--1417 (Japanese Patent Application laid-open No. 57-132658 and European Patent Publication No. 0094157); ~-lactam antibiotics such as sulbenicillin, cefotiam, cefmenoxime and sulfazecin; aminoglycoside antibiotics such as gentamicinr streptomycin and kanamycin; vitamins (e.~.
cyanocobalamin), antiprotozoa~ agents (e.g. meglumine antimonate); enzymes (e.g. alkaline phosphatase);
anticoagulants (e.g. heparin); antiallergics such as amoxanox; and other general-use drugs (e.g. glutathione).
The lipid material to be used in the practice of the invention may be any one that can form lipid bilayers. For example, there may be mentioned phospholipids derived ~rom egg yolk, soybean or other animal or vegetable tissues, such as phosphatidylcholines, phosphatidyle~hanolamines, phosphatidylinositols, phosphatidylserines and sphingomyelins; mixtures o these, such as egg yolk lecithin; hydro~ennated lecithins;
dipalmitoylphosphatidylcholines, semisynthetic distearoylphosphatidylcholines; and other lipids than the abo~e-mentioned phospholipids, such as cholesterol, monoglycerides, diglycerides and triglycerides. These may be used either singly or in admixture.
4~
_ 5 _ 24205-733 In accordance with the invention, a gel is then formed by adding a readily volatile organic solvent to a "drug~
containing liquid phase with liposomes dispersed therein"
such as mentioned above.
The readily volatile organic solvent to be used here may be any one that has high affinity for lipids and can evaporate more readily than water. The miscibility with water does not matter. Generally, organic solvents having a boiling point not higher than 100C are used advantageously. For example, there may be mentioned ethers (e.g. diethyl ether, isopropyl ether), chloroform, acetone and alcohols (e.g. methyl alcohol, ethyl alcohol). These solvents may be used alone or as a mixture. Among them, diethyl ether and acetone are particularly preferred.
The readily volatile organic solvent is added generally in amount of about 1-30 parts by volume per 10 parts by volume of the drug-containing liquid with liposomes dispersed therein, preferably in an amount of 2-10 parts by volume on the same basis. The mixture is then stirred and/or shaken with a vortex mixer or some other ;~ appropriate apparatus generally for tens of ~econds to severaI minutes to give a homogeneous gel. As a result of this treatment, the vesicle structure of the liposomes used as a raw material is relaxed or disintegrated by the organic solvent, whereby a uniform gel state is produced.
The readily volatile organic solvent is then removed by evaporation. As the method of evaporation, there may be mentioned the vacuum evaporation method using a rotary evaporator, which is in common use, or a vibrating vacuum evaporator and the evaporation method comprising blowing a ~9~
nitrogen gas stream. In particular, in blowing a nitrogen gas stream, application of vibrations from the outside usin~ a vortex mixer or a bath-type sonicator can result in more efficient removal of the organic solvent The gel formation and organic solvent removal by evaporation are preferably carried out at a temperature not lower than the phase transition point o~ the lipid used. Thus, for instance, temperatures not lower than room temperature are preferable for egg yolk lecithin and soybean lecithin, temperatures not lower than 50C for dipalmitoylphosphatidylcholines, and temperatures not lower than 60C for distearoylphosphatidyl-cholines.
The removal of the organic solvent leads to reconstitution of liposomes and, on that occasion, increased drug trap ratios are achieved as compared with the case of MLV or S W used as the starting material Thus, the liposomes with increased drug trap ratios are obtained according to the invention.
The thus~obtained liposomes with a drug included therein may, if necessary, be comminuted by using an ultrasonic homogenizer or some other homogenizer or be adjusted to a favorable grain size by filtration through a filter. Although the liposomes can be used as they are, they may be made up into such dosage forms as injections, oral preparations and suppositories after removing that portion of the drug which remains unincluded in the liposome~, by centrifugation, gel filtration or dialysis, for instance.
The liposomes obtained by the production method according to the invention show higher drug trap ratios as compared with those obtained by the conventional MLV, SUV
or REV method and are advantageous form the practical viewpoint in utilizing them as microcapsules. Furthermore, the method according to the invention has the foilowing features as compared with the conventional production methods:
1~9~L42S
a) The preparation of a w/o emulsion as required in the REV process is not always necessary.
b) The fine adjustment of the degree of vacuum in distilling off the organic solvent as required in the REV process is unnecessary.
c) While the REV process is limited in liposome production size per batch, the method according to the invention can be easily conducted on an increased scale. 0 d) Unlike the case of REV or SPLV, the use of an organic solvent in large amounts is not necessary.
e) Many kinds of organic solvents can be used. Even when the drug is one that can be readily inactivated by an organic solvent, the use of a hydrophilic organic 15- solvent such as acetone or ethyl alcohol can prevent such inactivation. For these and other reasons, it is possible to select an appropriate organic solvent while taking the characteristics of the drug to be included into consideration. 0 f) Generally, when a lipid relatively low in solubility in an organic solvent, for example a lipid having a high phase transition point (in particular, a lipid of the saturated type, such as DPPC or DSPC) r is used, the organic solvent is needed in large amounts for the dissolution of the lipid as in the REV process. In accordance with the present method, however, any lipid can be used without presenting any particular problems.
~lX~25 Examples The following working examples and test examples will illustrate the invention in further detail.
Example 1 ~ thin~lipid~film was formed on the glass wall of a 50-ml eggplant-shaped flask using 10 ml of a chloroform solution containing 1% of a 9:1 (w/w) mixture of dipalmitoyl-phosphatidylcholine and distearoylphosphatidylcholine. To the flask was added 3 ml of a 50 mM solution of 6-carboxy-fluorescein (6-CF) as adjusted to pH 7. The flask contents were stirred and shaken well in a ~ath-type sonicator main-tained at 55C to cause inclusion of 6-CF. Thus were pro-duced MLV liposomes dispersed in the 6-CF solution. To the thus obtained M~V dispersion was added 3 ml of ethyl ether at the same temperature as above. Stirring and shaking of the mixture with a vortex mixer for about 1 minute gave a ~lX~4~
homogeneous gel. The ethyl ether in the gel was removed by evaporation caused by blowing N2 gas against the gel with vibration by means of the sonicator. About 3 ml of a dis-persion of liposomes with 6-CF included therein was obtain-ed. This was diluted with 2 ml of physiological saline andthe dilution was filtered through a 1.2-micron filter (~crodisc ~ ; Gelman) and then dialyzed against 1,000 ml of physiological saline using a dialyzing membrane (Spectrapor ~ ; Spectrum Medical) for 24 hours to give liposomes with a 6-CF trap ratio of 28.9%.
Example 2 The procedure of Example 1 was followed using a chloro-form solution containing 1~ of a 7:3 (w/w) mix~ure of di-palmi~oylphosphatidylcholine and distearoylphosphatidyl-choline and a processing temperature of 60C to giveliposomes with a 6-CF trap ratio of 32.2~.
Example 3 The procedure of Example 1 was followed using a chloro-form solution containing 1% of distearoylphosphatidylcholine and a processing temperature of 70C to give liposomes with a 6-CF trap ratio of 37.7%.
Example 4 The procedure of Example 1 was followed using 1 ml of a chloroform solution contalning 10% of egg yolk lecithin in lieu of the semisynthetic lecithin used in Example 1 with further modifications such that ethyl ether ~asl~used in an amount of 1 ml and that room temperature was used as the 4~5 processing temperature. Thus were obtained liposomes with a 6-CF trap ratio of 30.1~.
Example 5 Several batches of MLV were produced by the method of Example 4. To a 30-ml portion of MLV as obtained by com-bining such batches was added 10 ml of ethyl ether and the mixture was processed in the same manner as in Example 4 to give liposomes with an improved 6-CF trap ratio.
Example 6 Liposomes were obtained with an improved 6-CF trap ratio in the same manner as in Example 4 except that 3 ml of acetone was used in lieu of the ethyl ether used in Example 4.
Example 7 An SUV obtained by comminuting the MLV produced in Example 4 by means of a probe-type ultrasonic shaker was used and processed in the same manner as in Example 4 to give liposomes with an improved 6-CF trap ratio.
Example 8 A thin lipid film was formed on the glass wall of a 50-ml eggplant-shaped flask using 1.5 ml of a chloroform solution containing 1~ of a 9:1 (w/w) mixture of di-palmitoylphosphatidylcholine and distearoylphosphatidyl-choline. To the flask was added 0.5 ml of an aqueous solution of interleukin 2 (content: 308 ~g protein/ml;
kind of solution: 5 mM ammonium acetate solution contain-ing human serum albumin, pH 5.0). The flask contents were stirred and shaken well in a bath-type sonicator maintained ~9~
at 55C to thereby cause inclusion of interleukin 2. Thus was obtained an MLV dispersion in the above drug solution.
Then, 0.5 ml of ethyl ether was added to this MLV disper-sion at the same temperature and the resultant mixture was stirred and shaken with a vortex mixer for about l minute to give a homogeneous gel. The ethyl ether in the gel was removed by evaporation caused by blowing N2 gas against the gel with further vibration by means of the sonicator.
There was obtained about 3 ml of a dispersion o~ liposomes with interleukin 2 included therein. After further addi-tion to this lO ml of the interleukin-free solution used for preparing the above aqueous interleuXin 2 solution (dispersant for interleukin 2 liposomes) for dilution, the whole mixture was centrifuged on an ultracentrifuge (Sorvall(R); Sorvall) at 30,000 r.p.m. for 30 minutes to give liposomes with an improved interleukin 2 trap ratio.
Example 9 A thin lipid film was formed on the glass wall of a 50-ml eggplant-shaped flask using 10 ml of a chloroform solution containing 1~ of a 9:1 (w/w) mixture of dipalmitoyl-phosphatidylcholine and distearoylphosphatidylcholine. To the flask was added 3 ml of an a~ueous solution of manganese-SOD (content: 6 mg protein/ml; kind of solution: 67 mM
phosphate buffer, pH 7.0), and the flask contents were stirred and shaken well in a bath-type sonicator maintained at 55C to give a dispersion of MLV liposomes in said drug solution with manganese-SOD included therein. To the -!L42~ii thus obtained MLV dispersion, there was added 3 ml of ethyl ether, and the resultant mixture was stirred with shaking by a vortex mixer for about 1 minute ~o give a homogeneous gel. The ethyle ether in the gel was removed by evaporation caused by blowing N2 gas against the gel with further vib-ration by means of the sonicator to give about 3 ml of a dispersion of liposomes with manganese-SOD included therein.
To this was further added 10 ml of physiological saline, followed by centrifugation on an ultracentrifuge (Sorvall ~ ;
Sorvall) at 30,000 r.p.m. for 30 minutes. There were ob-tained liposomes with a manganese-SOD trap ratio of 25.3~.
Example 10 A thin lipid film was formed on the glass wall of a 50-ml eggplant-shaped ~lask using 10 ml of a chloroform solution containing 1% of a 9:1 (w/w) mixture of dipalmitoyl-phsophatidylcholine and distearoylphosphatidylcholine. To the flask was then added 3 ml of an aqueous solution of manganese-SOD-PEG(5000) (content: 0.5 mg protein/ml; kind of so].ution: 67 mM phosphate buffer, pH 7.2). The flask contents were stirred and shaken well in a bakh-type sonicator maintained at 55C to gi~e a dispersion of MLV
liposomes with manganese-SOD-P~G(5~00) included therein.
To the thus-obtained MLV dispersion, there was added 3 ml of ethyl ether at the same temeprature and the mixture was stirred with shaking b~ a vortex mixer for about 1 minute to give a homogeneous gel. The ethyl ether in the gel was L4~;
removed by evaporation caused by blowing N2 gas against the gel with vibration by means of the sonicator, to give about 3 ml of a dispersion of liposomes with manganese~SOD-PEG~5000) included therein. To this was further added 10 ml of physiological saline and the resultant mixture was sub]ected to centrifugation in the same manner as in Example 8 to give liposomes with a manganese-SOD-PEG(5000) trap ratio of 27.0%.
Example 11 A thin lipid film was formed on the glass wall of a 50-ml eggplant-shaped flask using 10 ml of a chloroform solution containing 1% of a 9:1 Iw/w) mixture of dipalmitoyl-phosphatidylcholine and distearoylphsophatidylcholineO To the flask was added 3 ml of an aqueous solution of cisplatin (content: 0.5 mg/ml; kind of solution: physiological saline).
The flask contents were stirred and shaken well in a bath-type sonicator maintained at 55C to give a dispersion of MLV liposomes in said drug solution with the aqueous cisplatin solution included therein. Ethyl ether (3 ml) was added to the thus-obtained MLV dispersion at the same temperature and the mixture was stirred with shaking by a vortex mixer for about 1 minutes to give a homogeneous gel.
The ethvl ether in the gel was then removed by evaporation caused by blowing N2 gas against the gel with vibration by means of the sonicator to give about 3 ml of a dispersion of liposomes with cisplatin included therein. To this was added 2 ml of physiological saline and the mixture was 42~;
filtered through a 1.2-micron filter (Acrodisc ~ ; Gelman) and further dialyzed against 1~000 ml of physiological saline for 24 hours using a dialyzing membrane (Spectrapor ~ ;
Spectrum Medical).: Thus were obtained liposomes with a S cisplatin trap ratio of 29.8%.
Example 12 The procedure of Example 11 was followed using a chloro-form solution containing 1% of a 7:3 (w/w) mixture of di-palmitoylphosphatidylcholine and distearoylphosphatidyl-choline and a processing temperature of 60C to give lipo-somes with a cisplatin trap ratio of 32.4%.
Example 13 The procedure o~ Example 11 was followed using a chloro-form solution containing 1% of distearoylphosphatidylcholine and a processing temperature of 70C to give liposomes with a cisplatin trap ratio of 35.2~.
Example 14 MLV-type liposomes with no drug included therein were produced by adding 2 ml of physiological saline to the thin lipid film prepared in the same manner as in Example 1, fol-lowed by processing in the same manner as in Example 1.
Thereto was added 1 ml of 50 mM 6-CF solution (pH 7), and an MLV dispersion in the 6-CF solution was prepared without inclusion of 6-CF. Using the thus-obtained MLV dispersion and following the procedure of Example 1, there was obtained liposomes with a 6-CF trap ratio of 21.3%.
Example 15 A thin lipid film was formed on the glass wall of a 50-ml eggplant-shaped flask using 10 ml oE a choloroform solution containing 1% of a 9:1 (w/w) mixture of dipalmitoyl-phosphatidylcholine and distearoylphosphatidylcholine. Tothe flask was added 3 ml of a 50 mM soltution of 6-carboxy-fluorescein (6-CF) as adjusted to pH 7. The flask contents were stirred and shaken well in a bath-type sonicator main-tained at 55C to cause inclusion of 6-CF. Thus were pro-duced MLV liposomes dispersed in the 6-CF solution~ To the thus obtained MLV dispersion was added 1 ml of ethyl alcohol at the same temperature as above. Stirring and shaking of the mixture with a vortex mixer for about 1 minute gave a homogeneous gel. The ethyl alcohol in the gel was removed by evaporation caused by blowing N2 gas against the gel with vibration by means of the sonicator, ten 0.2 ml portion of water being added to the gel. About 3 ml of a dispersion of liposomes with 6-CF included therein was obtained. This was diluted with 2 ml of physiological saline and the dilution was filtered through a 1.2-micron filter (Acrodisc ~ ; Gelman) and the dialyzed against 1,000 ml of physiological saline using a dialyzing membrane (Spectrapor ~ ; Spectrum Medical) Eor 24 hours to give liposomes with a 6-CF trap ratio oE 13.5%.
Test Example 1 To 3 ml of the M~V dispersion prepared by the method ~.X9~L4~5 described in Example 4, there was added ethyl ether in an amount within the range of 0.1 ml to 1.0 ml to thereby investigate the dependency of the ability thereof to cause gel formation and the ability to form liposomes upon removal of the ethyl ether on the amount of the ethyl ether used. As a result, it was found that the minimum amount of ethyl ether as required for gel formation and production of liposomes in accordance with the invention is 0.3 ml, as shown in Table 1.
Table 1 Amount of ethyl ether used and ability to cause gel formation and liposome formation Amount of ethyl ether (ml~ 0.10.2 0.3 0.4 0.51.0 Gel formation - - + + + +
Formation of lipo-somes according - - + ~ + +
to the~inuention .
- ...... No gel formation ~ ...... Partial gel formation + ...... Good gel formation Te~t Example_ The liposomes according to the invention with 6-CF in-cluded therein as produced in Examples 1, 2, 3 and 4 were assayed for 6-CF content by measuring the intensity of the fluorescence of 6-CF (1) and the 6-CF trap ratios cal-culated on the basis of the fl~uorescence measurement results t2) were compared with the trap ratios for the MLVs 2~
produced in the respective examples and for the liposomes with 6~CF included'therein as prepared separately by'the REV
method (3) so as to have the same lipid structure. As a result, the liposomes according to the invention all showed higher trap ratios, as shown in Table 2.
tl) Determination of 6-CF content in liposomes: Lipo-somes (0.1 ml) were diluted lO0-fold with phosphate-buffered physiological saline (PBS), a 0.1-ml portion of the dilution was further diluted 100-fold with PBS containing 0.02% of Triton X-lO0, and the dilution was heated at 60C for 30 minutes to thereby cause disruption of liposomes. The resul-tant solution was measured for fluorescence intensity (Hitachi model F3000 fluorescence spectrometer; excitation wavelength 494 nm; measurement wavelength 515 nm) and the total 6-CF content in ~he liposome dispersion ~inclusive of free 6-CF occurring unincorporated into liposomes) was determined. Separately, 0.1 ml of the liposome dispersion was diluted 10,000-fold with PBS, a 2.5-ml portion of the dilution was filtered'through an ultracentrifugal filter tCentrisart ~ SM13249E; Sartorius) and the filtrate was measured for fluorescence intensity; the content of free 6-CF occurring in the liposome dispersion in the unincluded state was thus determined.
(2) Method of producing REV:
A 100-mg portion of the lipid used in each of Examples 1, 2, 3 and 4 was placed in a 100-ml eggplant-shaped flask and dissolved by adding lO ml of chloroform and 10 ml of isopropyl ether, followed by further addition of 3 ml of 4~
the 6-CF solution used in each example. The resultant mix-ture was emulsified in a probe-type ultrasonic shaker (20 KHz). Then, the organic solvents were removed by evaporation using a rotary evaporator (temperature condition: same as used in each example1 and the residual dispersion was dia-lyzed in the same manner as in each example to give an REV
with 6-CF included therein.
(3) Calculation of trap ratios:
.
Trap ratio (~) =
(Total amount of 6-CF (Amount of free 6 CF
in liposome dispersion) in liposome dispersion) - x 100 (Amount of 6-CF used i.n liposome production) Table 2 Comparison of trap ratios of 6-CF in liposomes (in ~) Liposomes according to Lipid construction the invention MLV REV
Dppce)/Dspcf) = 9/1 28.9 ) 12.5 ) 17.6 DPPC / DSPC = 7/3 32.2b) - 24.2 DSPC 37.7C) _ 27.2 Egg yolk lecithin 30.1d) - 23.0 _ a) Product of Example 1 b) Product of Example 2 c) Product of Example 3 d) Product of Example 4 e) Dipalmitoylphosphatidylcholine f) Distearoylphosphatidylcholine Test_Example_3 In this test example, the liposomes according to the invention with cisplatin included therein as produced in Examples ll, 12 and 13 were used. Each liposome dispersion was assayed for cisplatin by HPLC (column: Zorbax CN ;
eluent: n-hexane/isopropyl alcohol = 8/2; UV = 254 nm) for the adduct formed by reaction of cisplatin with diethyl-dithiocarbamate (DDTC) (l). The cisplatin trap ratios calculated on the basis of the results of such assay were compared with those for the MLVs produced in the respective examples and for the liposomes with cisplatin included therein as prepared separately by the REV method so as to at-tain the same lipid structure. Thus, the data shown in Table 3 were obtained and the liposomes according to the invention always showed higher trap ratios.
(l) Determination of cisplatin content in liposomes:
Liposomes (0.1 ml) were dispersed in 5 ml of physiolo-logical saline. A 2.5-ml portion of the dispersion was subjected to freezing-thawing treatment and the liposome disruption product was filtered through a Centrizart~ filter.
To 0.1 ml of the filtrate, there was added 2 ml o~ 0.1 N
NaOH solution. After allowing the mixture to stand at room temperature for 30 minutes, the adduct formed was extracted with 5 ml of n-hexane. The extract was assayed for cisplatin by HPLC under the conditions mentioned above. Then, the total cisplatin content in the liposome dispersion was cal-culated. Separately, about 2.5 ml of the remaining liposome dispersion in physiological saline was subjected to freezing-thawing treatment, followed by filtration through a Centrisart , filter and adduct formation under the same conditions as mentioned above. Thus was determined the content of free cisplatin remaining uninclud~d in liposomes in said liposome dispersion.
Table 3 Comparison of trap ratios of cisplatin in liposomes (in ~) Liposomes ac-Lipid construction cording to the MLV REV
DDPC/DSPC = 9:1 29.8b) 8.2a) 17.9 DPPC/DSPC = 7:3 32.4 ) - 24.6 DSPC 35.2C) - 26.6 ..... . _ .. . _ _ . _ _ . .. _ _ . . _ . ...
a) Product of Example 11 b) Product of Example 12 c) Product of Example 13 -
_ 5 _ 24205-733 In accordance with the invention, a gel is then formed by adding a readily volatile organic solvent to a "drug~
containing liquid phase with liposomes dispersed therein"
such as mentioned above.
The readily volatile organic solvent to be used here may be any one that has high affinity for lipids and can evaporate more readily than water. The miscibility with water does not matter. Generally, organic solvents having a boiling point not higher than 100C are used advantageously. For example, there may be mentioned ethers (e.g. diethyl ether, isopropyl ether), chloroform, acetone and alcohols (e.g. methyl alcohol, ethyl alcohol). These solvents may be used alone or as a mixture. Among them, diethyl ether and acetone are particularly preferred.
The readily volatile organic solvent is added generally in amount of about 1-30 parts by volume per 10 parts by volume of the drug-containing liquid with liposomes dispersed therein, preferably in an amount of 2-10 parts by volume on the same basis. The mixture is then stirred and/or shaken with a vortex mixer or some other ;~ appropriate apparatus generally for tens of ~econds to severaI minutes to give a homogeneous gel. As a result of this treatment, the vesicle structure of the liposomes used as a raw material is relaxed or disintegrated by the organic solvent, whereby a uniform gel state is produced.
The readily volatile organic solvent is then removed by evaporation. As the method of evaporation, there may be mentioned the vacuum evaporation method using a rotary evaporator, which is in common use, or a vibrating vacuum evaporator and the evaporation method comprising blowing a ~9~
nitrogen gas stream. In particular, in blowing a nitrogen gas stream, application of vibrations from the outside usin~ a vortex mixer or a bath-type sonicator can result in more efficient removal of the organic solvent The gel formation and organic solvent removal by evaporation are preferably carried out at a temperature not lower than the phase transition point o~ the lipid used. Thus, for instance, temperatures not lower than room temperature are preferable for egg yolk lecithin and soybean lecithin, temperatures not lower than 50C for dipalmitoylphosphatidylcholines, and temperatures not lower than 60C for distearoylphosphatidyl-cholines.
The removal of the organic solvent leads to reconstitution of liposomes and, on that occasion, increased drug trap ratios are achieved as compared with the case of MLV or S W used as the starting material Thus, the liposomes with increased drug trap ratios are obtained according to the invention.
The thus~obtained liposomes with a drug included therein may, if necessary, be comminuted by using an ultrasonic homogenizer or some other homogenizer or be adjusted to a favorable grain size by filtration through a filter. Although the liposomes can be used as they are, they may be made up into such dosage forms as injections, oral preparations and suppositories after removing that portion of the drug which remains unincluded in the liposome~, by centrifugation, gel filtration or dialysis, for instance.
The liposomes obtained by the production method according to the invention show higher drug trap ratios as compared with those obtained by the conventional MLV, SUV
or REV method and are advantageous form the practical viewpoint in utilizing them as microcapsules. Furthermore, the method according to the invention has the foilowing features as compared with the conventional production methods:
1~9~L42S
a) The preparation of a w/o emulsion as required in the REV process is not always necessary.
b) The fine adjustment of the degree of vacuum in distilling off the organic solvent as required in the REV process is unnecessary.
c) While the REV process is limited in liposome production size per batch, the method according to the invention can be easily conducted on an increased scale. 0 d) Unlike the case of REV or SPLV, the use of an organic solvent in large amounts is not necessary.
e) Many kinds of organic solvents can be used. Even when the drug is one that can be readily inactivated by an organic solvent, the use of a hydrophilic organic 15- solvent such as acetone or ethyl alcohol can prevent such inactivation. For these and other reasons, it is possible to select an appropriate organic solvent while taking the characteristics of the drug to be included into consideration. 0 f) Generally, when a lipid relatively low in solubility in an organic solvent, for example a lipid having a high phase transition point (in particular, a lipid of the saturated type, such as DPPC or DSPC) r is used, the organic solvent is needed in large amounts for the dissolution of the lipid as in the REV process. In accordance with the present method, however, any lipid can be used without presenting any particular problems.
~lX~25 Examples The following working examples and test examples will illustrate the invention in further detail.
Example 1 ~ thin~lipid~film was formed on the glass wall of a 50-ml eggplant-shaped flask using 10 ml of a chloroform solution containing 1% of a 9:1 (w/w) mixture of dipalmitoyl-phosphatidylcholine and distearoylphosphatidylcholine. To the flask was added 3 ml of a 50 mM solution of 6-carboxy-fluorescein (6-CF) as adjusted to pH 7. The flask contents were stirred and shaken well in a ~ath-type sonicator main-tained at 55C to cause inclusion of 6-CF. Thus were pro-duced MLV liposomes dispersed in the 6-CF solution. To the thus obtained M~V dispersion was added 3 ml of ethyl ether at the same temperature as above. Stirring and shaking of the mixture with a vortex mixer for about 1 minute gave a ~lX~4~
homogeneous gel. The ethyl ether in the gel was removed by evaporation caused by blowing N2 gas against the gel with vibration by means of the sonicator. About 3 ml of a dis-persion of liposomes with 6-CF included therein was obtain-ed. This was diluted with 2 ml of physiological saline andthe dilution was filtered through a 1.2-micron filter (~crodisc ~ ; Gelman) and then dialyzed against 1,000 ml of physiological saline using a dialyzing membrane (Spectrapor ~ ; Spectrum Medical) for 24 hours to give liposomes with a 6-CF trap ratio of 28.9%.
Example 2 The procedure of Example 1 was followed using a chloro-form solution containing 1~ of a 7:3 (w/w) mix~ure of di-palmi~oylphosphatidylcholine and distearoylphosphatidyl-choline and a processing temperature of 60C to giveliposomes with a 6-CF trap ratio of 32.2~.
Example 3 The procedure of Example 1 was followed using a chloro-form solution containing 1% of distearoylphosphatidylcholine and a processing temperature of 70C to give liposomes with a 6-CF trap ratio of 37.7%.
Example 4 The procedure of Example 1 was followed using 1 ml of a chloroform solution contalning 10% of egg yolk lecithin in lieu of the semisynthetic lecithin used in Example 1 with further modifications such that ethyl ether ~asl~used in an amount of 1 ml and that room temperature was used as the 4~5 processing temperature. Thus were obtained liposomes with a 6-CF trap ratio of 30.1~.
Example 5 Several batches of MLV were produced by the method of Example 4. To a 30-ml portion of MLV as obtained by com-bining such batches was added 10 ml of ethyl ether and the mixture was processed in the same manner as in Example 4 to give liposomes with an improved 6-CF trap ratio.
Example 6 Liposomes were obtained with an improved 6-CF trap ratio in the same manner as in Example 4 except that 3 ml of acetone was used in lieu of the ethyl ether used in Example 4.
Example 7 An SUV obtained by comminuting the MLV produced in Example 4 by means of a probe-type ultrasonic shaker was used and processed in the same manner as in Example 4 to give liposomes with an improved 6-CF trap ratio.
Example 8 A thin lipid film was formed on the glass wall of a 50-ml eggplant-shaped flask using 1.5 ml of a chloroform solution containing 1~ of a 9:1 (w/w) mixture of di-palmitoylphosphatidylcholine and distearoylphosphatidyl-choline. To the flask was added 0.5 ml of an aqueous solution of interleukin 2 (content: 308 ~g protein/ml;
kind of solution: 5 mM ammonium acetate solution contain-ing human serum albumin, pH 5.0). The flask contents were stirred and shaken well in a bath-type sonicator maintained ~9~
at 55C to thereby cause inclusion of interleukin 2. Thus was obtained an MLV dispersion in the above drug solution.
Then, 0.5 ml of ethyl ether was added to this MLV disper-sion at the same temperature and the resultant mixture was stirred and shaken with a vortex mixer for about l minute to give a homogeneous gel. The ethyl ether in the gel was removed by evaporation caused by blowing N2 gas against the gel with further vibration by means of the sonicator.
There was obtained about 3 ml of a dispersion o~ liposomes with interleukin 2 included therein. After further addi-tion to this lO ml of the interleukin-free solution used for preparing the above aqueous interleuXin 2 solution (dispersant for interleukin 2 liposomes) for dilution, the whole mixture was centrifuged on an ultracentrifuge (Sorvall(R); Sorvall) at 30,000 r.p.m. for 30 minutes to give liposomes with an improved interleukin 2 trap ratio.
Example 9 A thin lipid film was formed on the glass wall of a 50-ml eggplant-shaped flask using 10 ml of a chloroform solution containing 1~ of a 9:1 (w/w) mixture of dipalmitoyl-phosphatidylcholine and distearoylphosphatidylcholine. To the flask was added 3 ml of an a~ueous solution of manganese-SOD (content: 6 mg protein/ml; kind of solution: 67 mM
phosphate buffer, pH 7.0), and the flask contents were stirred and shaken well in a bath-type sonicator maintained at 55C to give a dispersion of MLV liposomes in said drug solution with manganese-SOD included therein. To the -!L42~ii thus obtained MLV dispersion, there was added 3 ml of ethyl ether, and the resultant mixture was stirred with shaking by a vortex mixer for about 1 minute ~o give a homogeneous gel. The ethyle ether in the gel was removed by evaporation caused by blowing N2 gas against the gel with further vib-ration by means of the sonicator to give about 3 ml of a dispersion of liposomes with manganese-SOD included therein.
To this was further added 10 ml of physiological saline, followed by centrifugation on an ultracentrifuge (Sorvall ~ ;
Sorvall) at 30,000 r.p.m. for 30 minutes. There were ob-tained liposomes with a manganese-SOD trap ratio of 25.3~.
Example 10 A thin lipid film was formed on the glass wall of a 50-ml eggplant-shaped ~lask using 10 ml of a chloroform solution containing 1% of a 9:1 (w/w) mixture of dipalmitoyl-phsophatidylcholine and distearoylphosphatidylcholine. To the flask was then added 3 ml of an aqueous solution of manganese-SOD-PEG(5000) (content: 0.5 mg protein/ml; kind of so].ution: 67 mM phosphate buffer, pH 7.2). The flask contents were stirred and shaken well in a bakh-type sonicator maintained at 55C to gi~e a dispersion of MLV
liposomes with manganese-SOD-P~G(5~00) included therein.
To the thus-obtained MLV dispersion, there was added 3 ml of ethyl ether at the same temeprature and the mixture was stirred with shaking b~ a vortex mixer for about 1 minute to give a homogeneous gel. The ethyl ether in the gel was L4~;
removed by evaporation caused by blowing N2 gas against the gel with vibration by means of the sonicator, to give about 3 ml of a dispersion of liposomes with manganese~SOD-PEG~5000) included therein. To this was further added 10 ml of physiological saline and the resultant mixture was sub]ected to centrifugation in the same manner as in Example 8 to give liposomes with a manganese-SOD-PEG(5000) trap ratio of 27.0%.
Example 11 A thin lipid film was formed on the glass wall of a 50-ml eggplant-shaped flask using 10 ml of a chloroform solution containing 1% of a 9:1 Iw/w) mixture of dipalmitoyl-phosphatidylcholine and distearoylphsophatidylcholineO To the flask was added 3 ml of an aqueous solution of cisplatin (content: 0.5 mg/ml; kind of solution: physiological saline).
The flask contents were stirred and shaken well in a bath-type sonicator maintained at 55C to give a dispersion of MLV liposomes in said drug solution with the aqueous cisplatin solution included therein. Ethyl ether (3 ml) was added to the thus-obtained MLV dispersion at the same temperature and the mixture was stirred with shaking by a vortex mixer for about 1 minutes to give a homogeneous gel.
The ethvl ether in the gel was then removed by evaporation caused by blowing N2 gas against the gel with vibration by means of the sonicator to give about 3 ml of a dispersion of liposomes with cisplatin included therein. To this was added 2 ml of physiological saline and the mixture was 42~;
filtered through a 1.2-micron filter (Acrodisc ~ ; Gelman) and further dialyzed against 1~000 ml of physiological saline for 24 hours using a dialyzing membrane (Spectrapor ~ ;
Spectrum Medical).: Thus were obtained liposomes with a S cisplatin trap ratio of 29.8%.
Example 12 The procedure of Example 11 was followed using a chloro-form solution containing 1% of a 7:3 (w/w) mixture of di-palmitoylphosphatidylcholine and distearoylphosphatidyl-choline and a processing temperature of 60C to give lipo-somes with a cisplatin trap ratio of 32.4%.
Example 13 The procedure o~ Example 11 was followed using a chloro-form solution containing 1% of distearoylphosphatidylcholine and a processing temperature of 70C to give liposomes with a cisplatin trap ratio of 35.2~.
Example 14 MLV-type liposomes with no drug included therein were produced by adding 2 ml of physiological saline to the thin lipid film prepared in the same manner as in Example 1, fol-lowed by processing in the same manner as in Example 1.
Thereto was added 1 ml of 50 mM 6-CF solution (pH 7), and an MLV dispersion in the 6-CF solution was prepared without inclusion of 6-CF. Using the thus-obtained MLV dispersion and following the procedure of Example 1, there was obtained liposomes with a 6-CF trap ratio of 21.3%.
Example 15 A thin lipid film was formed on the glass wall of a 50-ml eggplant-shaped flask using 10 ml oE a choloroform solution containing 1% of a 9:1 (w/w) mixture of dipalmitoyl-phosphatidylcholine and distearoylphosphatidylcholine. Tothe flask was added 3 ml of a 50 mM soltution of 6-carboxy-fluorescein (6-CF) as adjusted to pH 7. The flask contents were stirred and shaken well in a bath-type sonicator main-tained at 55C to cause inclusion of 6-CF. Thus were pro-duced MLV liposomes dispersed in the 6-CF solution~ To the thus obtained MLV dispersion was added 1 ml of ethyl alcohol at the same temperature as above. Stirring and shaking of the mixture with a vortex mixer for about 1 minute gave a homogeneous gel. The ethyl alcohol in the gel was removed by evaporation caused by blowing N2 gas against the gel with vibration by means of the sonicator, ten 0.2 ml portion of water being added to the gel. About 3 ml of a dispersion of liposomes with 6-CF included therein was obtained. This was diluted with 2 ml of physiological saline and the dilution was filtered through a 1.2-micron filter (Acrodisc ~ ; Gelman) and the dialyzed against 1,000 ml of physiological saline using a dialyzing membrane (Spectrapor ~ ; Spectrum Medical) Eor 24 hours to give liposomes with a 6-CF trap ratio oE 13.5%.
Test Example 1 To 3 ml of the M~V dispersion prepared by the method ~.X9~L4~5 described in Example 4, there was added ethyl ether in an amount within the range of 0.1 ml to 1.0 ml to thereby investigate the dependency of the ability thereof to cause gel formation and the ability to form liposomes upon removal of the ethyl ether on the amount of the ethyl ether used. As a result, it was found that the minimum amount of ethyl ether as required for gel formation and production of liposomes in accordance with the invention is 0.3 ml, as shown in Table 1.
Table 1 Amount of ethyl ether used and ability to cause gel formation and liposome formation Amount of ethyl ether (ml~ 0.10.2 0.3 0.4 0.51.0 Gel formation - - + + + +
Formation of lipo-somes according - - + ~ + +
to the~inuention .
- ...... No gel formation ~ ...... Partial gel formation + ...... Good gel formation Te~t Example_ The liposomes according to the invention with 6-CF in-cluded therein as produced in Examples 1, 2, 3 and 4 were assayed for 6-CF content by measuring the intensity of the fluorescence of 6-CF (1) and the 6-CF trap ratios cal-culated on the basis of the fl~uorescence measurement results t2) were compared with the trap ratios for the MLVs 2~
produced in the respective examples and for the liposomes with 6~CF included'therein as prepared separately by'the REV
method (3) so as to have the same lipid structure. As a result, the liposomes according to the invention all showed higher trap ratios, as shown in Table 2.
tl) Determination of 6-CF content in liposomes: Lipo-somes (0.1 ml) were diluted lO0-fold with phosphate-buffered physiological saline (PBS), a 0.1-ml portion of the dilution was further diluted 100-fold with PBS containing 0.02% of Triton X-lO0, and the dilution was heated at 60C for 30 minutes to thereby cause disruption of liposomes. The resul-tant solution was measured for fluorescence intensity (Hitachi model F3000 fluorescence spectrometer; excitation wavelength 494 nm; measurement wavelength 515 nm) and the total 6-CF content in ~he liposome dispersion ~inclusive of free 6-CF occurring unincorporated into liposomes) was determined. Separately, 0.1 ml of the liposome dispersion was diluted 10,000-fold with PBS, a 2.5-ml portion of the dilution was filtered'through an ultracentrifugal filter tCentrisart ~ SM13249E; Sartorius) and the filtrate was measured for fluorescence intensity; the content of free 6-CF occurring in the liposome dispersion in the unincluded state was thus determined.
(2) Method of producing REV:
A 100-mg portion of the lipid used in each of Examples 1, 2, 3 and 4 was placed in a 100-ml eggplant-shaped flask and dissolved by adding lO ml of chloroform and 10 ml of isopropyl ether, followed by further addition of 3 ml of 4~
the 6-CF solution used in each example. The resultant mix-ture was emulsified in a probe-type ultrasonic shaker (20 KHz). Then, the organic solvents were removed by evaporation using a rotary evaporator (temperature condition: same as used in each example1 and the residual dispersion was dia-lyzed in the same manner as in each example to give an REV
with 6-CF included therein.
(3) Calculation of trap ratios:
.
Trap ratio (~) =
(Total amount of 6-CF (Amount of free 6 CF
in liposome dispersion) in liposome dispersion) - x 100 (Amount of 6-CF used i.n liposome production) Table 2 Comparison of trap ratios of 6-CF in liposomes (in ~) Liposomes according to Lipid construction the invention MLV REV
Dppce)/Dspcf) = 9/1 28.9 ) 12.5 ) 17.6 DPPC / DSPC = 7/3 32.2b) - 24.2 DSPC 37.7C) _ 27.2 Egg yolk lecithin 30.1d) - 23.0 _ a) Product of Example 1 b) Product of Example 2 c) Product of Example 3 d) Product of Example 4 e) Dipalmitoylphosphatidylcholine f) Distearoylphosphatidylcholine Test_Example_3 In this test example, the liposomes according to the invention with cisplatin included therein as produced in Examples ll, 12 and 13 were used. Each liposome dispersion was assayed for cisplatin by HPLC (column: Zorbax CN ;
eluent: n-hexane/isopropyl alcohol = 8/2; UV = 254 nm) for the adduct formed by reaction of cisplatin with diethyl-dithiocarbamate (DDTC) (l). The cisplatin trap ratios calculated on the basis of the results of such assay were compared with those for the MLVs produced in the respective examples and for the liposomes with cisplatin included therein as prepared separately by the REV method so as to at-tain the same lipid structure. Thus, the data shown in Table 3 were obtained and the liposomes according to the invention always showed higher trap ratios.
(l) Determination of cisplatin content in liposomes:
Liposomes (0.1 ml) were dispersed in 5 ml of physiolo-logical saline. A 2.5-ml portion of the dispersion was subjected to freezing-thawing treatment and the liposome disruption product was filtered through a Centrizart~ filter.
To 0.1 ml of the filtrate, there was added 2 ml o~ 0.1 N
NaOH solution. After allowing the mixture to stand at room temperature for 30 minutes, the adduct formed was extracted with 5 ml of n-hexane. The extract was assayed for cisplatin by HPLC under the conditions mentioned above. Then, the total cisplatin content in the liposome dispersion was cal-culated. Separately, about 2.5 ml of the remaining liposome dispersion in physiological saline was subjected to freezing-thawing treatment, followed by filtration through a Centrisart , filter and adduct formation under the same conditions as mentioned above. Thus was determined the content of free cisplatin remaining uninclud~d in liposomes in said liposome dispersion.
Table 3 Comparison of trap ratios of cisplatin in liposomes (in ~) Liposomes ac-Lipid construction cording to the MLV REV
DDPC/DSPC = 9:1 29.8b) 8.2a) 17.9 DPPC/DSPC = 7:3 32.4 ) - 24.6 DSPC 35.2C) - 26.6 ..... . _ .. . _ _ . _ _ . .. _ _ . . _ . ...
a) Product of Example 11 b) Product of Example 12 c) Product of Example 13 -
Claims (24)
1. A method of producing liposomes with increased drug trap, which comprises:
(1) preparing liposomes with or without a drug incorporated therein from a bilayer-forming lipid, said liposomes being dis-persed in a drug-containing liquid, (2) adding a readily volatile organic solvent to the disper-sion to cause gel formation, and (3) then removing said organic solvent by evaporation to reconstitute liposomes.
(1) preparing liposomes with or without a drug incorporated therein from a bilayer-forming lipid, said liposomes being dis-persed in a drug-containing liquid, (2) adding a readily volatile organic solvent to the disper-sion to cause gel formation, and (3) then removing said organic solvent by evaporation to reconstitute liposomes.
2. The method according to claim 1, wherein the liposomes are formed using phospholipid.
3. The method according to claim 2, wherein the liposomes are multilamellar vesicles (MLV) with or without drug trapped therein.
4. The method according to claim 2, wherein the liposomes are small unilamellar vesicles (SUV) with or without drug trapped therein.
5. The method according to claim 2, wherein the drug-con-taining liquid is prepared with a hydrophilic drug having an octyl alcohol/water-distribution ratio lower than 10 as the logalithmic value.
6. The method according to claim 5, wherein the drug is of antiinflammatory analgesics, lymphokines, anticancer agents, immunopotentiators, physiologically active peptides, antibiotics, antiprotozoa agents, enzymes or antiallergics.
7. The method according to claim 6, wherein the lymphokine is interleukin 2.
8. The method according to claim 6, wherein the anticancer agent is cisplatin.
9. The method according to claim 6, wherein the anti-infammatory analgesic is manganese superoxide dismutase or superoxide dismutase-PEG.
10. The method according to claim 1, 2 or 6, wherein the readily volatile organic solvent has a boiling point not higher than 100°C.
11. The method according to claim 1, 2 or 6, wherein the organic solvent is ethyl ether.
12. The method according to claim 1, 2 or 6, wherein the organic solvent is acetone.
13. The method according to claim 1, 2 or 6, wherein the organic solvent is ethyl alcohol.
14. The method according to claim 1, 2 or 6, wherein the organic solvent is added in amount of about 1 to 30 parts by volume per 10 parts by volume of the drug-containing liquid with liposomes dispersed.
15. The method according to claim 1, 2 or 6, wherein the organic solvent is added in amount of about 2 to 10 parts by volume per 10 parts by volume of the drug-containing liquid with liposomes dispersed.
16. A method of producing liposomes with increased drug trap, which comprises:
(1) thoroughly mixing and stirring 5 to 100 parts by weight of an aqueous solution containing a drug with 1 part by weight of a phospholipid in a thin film form, thereby preparing liposomes with the drug trapped therein, said liposomes being dispersed in the drug-containing aqueous solution, (2) adding a readily volatile organic solvent which has a high affinity to the phospholipid and can evaporate more readily than water to the aqueous dispersion in an amount sufficient to cause gel formation, thereby forming gel of the aqueous dispersion and the solvent, and (3) then removing the organic solvent from the gel by evaporation, thereby reconstituting the liposomes.
(1) thoroughly mixing and stirring 5 to 100 parts by weight of an aqueous solution containing a drug with 1 part by weight of a phospholipid in a thin film form, thereby preparing liposomes with the drug trapped therein, said liposomes being dispersed in the drug-containing aqueous solution, (2) adding a readily volatile organic solvent which has a high affinity to the phospholipid and can evaporate more readily than water to the aqueous dispersion in an amount sufficient to cause gel formation, thereby forming gel of the aqueous dispersion and the solvent, and (3) then removing the organic solvent from the gel by evaporation, thereby reconstituting the liposomes.
17. The method according to claim 16, wherein the organic solvent is ethyl ether, acetone or ethyl alcohol and is used in an amount of about 1 to 30 parts by volume per 10 parts by volume of the aqueous dispersion.
18. The method according to claim 17, wherein the phospholipid is phosphatidylcholine, phosphatidylsarine, phos-phatidylinositol, phosphatidylethanolamine, sphingomylin, or a mixture thereof.
19. The method according to claim 17, wherein the phos-pholipid is dipalmitoylphosphatidylcholine, distearoylphos-phatidylcholine or a mixture thereof.
20. The method according to claim 17, wherein the phos-pholipid is egg yolk lecithin.
21. The method according to claim 17, wherein the removal of the solvent from the gel is carried out by blowing nitrogen gas against the gel with vibration by means of a sonicator.
22. The method according to claim 18, 19 or 20, wherein the removal of the solvent from the gel is carried out by blowing nitrogen gas against the gel with vibration by means of a sonicator.
23. The method according to claim 17, 18 or 21, wherein the drug is a hydrophilic drug and of antiinflammatory analgesics, lymphokines, anticancer agents, immunopotentiators, physio logically active peptides, antibiotics antiprotozoa agents, enzymes or antiallergics.
24. The method according to claim 17, 18 or 21, wherein the drug is interleukin 2, cisplatin, manganese superoxide dismutase or superoxide dismutase-PEG.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP76102/1986 | 1986-04-02 | ||
| JP7610286 | 1986-04-02 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1291425C true CA1291425C (en) | 1991-10-29 |
Family
ID=13595510
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000533545A Expired - Lifetime CA1291425C (en) | 1986-04-02 | 1987-04-01 | Method of producing liposome |
Country Status (8)
| Country | Link |
|---|---|
| US (1) | US4877561A (en) |
| EP (1) | EP0240346B1 (en) |
| JP (1) | JPH0751496B2 (en) |
| AT (1) | ATE55542T1 (en) |
| CA (1) | CA1291425C (en) |
| DE (1) | DE3764290D1 (en) |
| ES (1) | ES2016842B3 (en) |
| GR (1) | GR3001049T3 (en) |
Families Citing this family (87)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5916588A (en) * | 1984-04-12 | 1999-06-29 | The Liposome Company, Inc. | Peptide-containing liposomes, immunogenic liposomes and methods of preparation and use |
| US6090406A (en) * | 1984-04-12 | 2000-07-18 | The Liposome Company, Inc. | Potentiation of immune responses with liposomal adjuvants |
| US5059421A (en) * | 1985-07-26 | 1991-10-22 | The Liposome Company, Inc. | Preparation of targeted liposome systems of a defined size distribution |
| US6406713B1 (en) * | 1987-03-05 | 2002-06-18 | The Liposome Company, Inc. | Methods of preparing low-toxicity drug-lipid complexes |
| JPS63290824A (en) * | 1987-05-22 | 1988-11-28 | Sumitomo Chem Co Ltd | Therapeutic agent for viral infection |
| US5000886A (en) * | 1987-05-26 | 1991-03-19 | American Cyanamid Company | Silicone-hardened pharmaceutical microcapsules and process of making the same |
| WO1989005151A1 (en) * | 1987-12-04 | 1989-06-15 | The Liposome Company, Inc. | High integrity liposomes and method of preration and use |
| JPH01238537A (en) * | 1988-03-18 | 1989-09-22 | Toyo Jozo Co Ltd | Remedy for hepatic disorder |
| EP0440742B1 (en) * | 1988-10-27 | 1997-01-15 | The Regents Of The University Of Minnesota | Liposome immunoadjuvants containing il-2 |
| US5352435A (en) * | 1989-12-22 | 1994-10-04 | Unger Evan C | Ionophore containing liposomes for ultrasound imaging |
| US5733572A (en) * | 1989-12-22 | 1998-03-31 | Imarx Pharmaceutical Corp. | Gas and gaseous precursor filled microspheres as topical and subcutaneous delivery vehicles |
| US20020150539A1 (en) | 1989-12-22 | 2002-10-17 | Unger Evan C. | Ultrasound imaging and treatment |
| US5542935A (en) * | 1989-12-22 | 1996-08-06 | Imarx Pharmaceutical Corp. | Therapeutic delivery systems related applications |
| US5656211A (en) * | 1989-12-22 | 1997-08-12 | Imarx Pharmaceutical Corp. | Apparatus and method for making gas-filled vesicles of optimal size |
| US6001335A (en) * | 1989-12-22 | 1999-12-14 | Imarx Pharmaceutical Corp. | Contrasting agents for ultrasonic imaging and methods for preparing the same |
| US5773024A (en) * | 1989-12-22 | 1998-06-30 | Imarx Pharmaceutical Corp. | Container with multi-phase composition for use in diagnostic and therapeutic applications |
| US5776429A (en) * | 1989-12-22 | 1998-07-07 | Imarx Pharmaceutical Corp. | Method of preparing gas-filled microspheres using a lyophilized lipids |
| US6088613A (en) | 1989-12-22 | 2000-07-11 | Imarx Pharmaceutical Corp. | Method of magnetic resonance focused surgical and therapeutic ultrasound |
| US6551576B1 (en) | 1989-12-22 | 2003-04-22 | Bristol-Myers Squibb Medical Imaging, Inc. | Container with multi-phase composition for use in diagnostic and therapeutic applications |
| US5922304A (en) * | 1989-12-22 | 1999-07-13 | Imarx Pharmaceutical Corp. | Gaseous precursor filled microspheres as magnetic resonance imaging contrast agents |
| US5469854A (en) * | 1989-12-22 | 1995-11-28 | Imarx Pharmaceutical Corp. | Methods of preparing gas-filled liposomes |
| US6146657A (en) * | 1989-12-22 | 2000-11-14 | Imarx Pharmaceutical Corp. | Gas-filled lipid spheres for use in diagnostic and therapeutic applications |
| US5705187A (en) * | 1989-12-22 | 1998-01-06 | Imarx Pharmaceutical Corp. | Compositions of lipids and stabilizing materials |
| US5580575A (en) * | 1989-12-22 | 1996-12-03 | Imarx Pharmaceutical Corp. | Therapeutic drug delivery systems |
| US5305757A (en) * | 1989-12-22 | 1994-04-26 | Unger Evan C | Gas filled liposomes and their use as ultrasonic contrast agents |
| US5585112A (en) * | 1989-12-22 | 1996-12-17 | Imarx Pharmaceutical Corp. | Method of preparing gas and gaseous precursor-filled microspheres |
| US5820848A (en) * | 1990-01-12 | 1998-10-13 | The Liposome Company, Inc. | Methods of preparing interdigitation-fusion liposomes and gels which encapsulate a bioactive agent |
| JP3131923B2 (en) * | 1990-01-12 | 2001-02-05 | ザ リポソーム カンパニー，インコーポレイテッド | Finger processes-fused liposomes and gels |
| US5741513A (en) * | 1990-02-08 | 1998-04-21 | A. Natterman & Cie. Gmbh | Alcoholic aqueous gel-like phospholipid composition, its use and topical preparations containing it |
| US5665379A (en) * | 1990-09-28 | 1997-09-09 | Pharmacia & Upjohn Aktiebolag | Lipid particle forming matrix, preparation and use thereof |
| AR244546A1 (en) * | 1990-10-30 | 1993-11-30 | Gador Y Cia S A C I Lab Dr | Procedure for the preparation of liposomes insoluble in oil for in soft completely soluble gelatinous capsules. |
| ES2074879T3 (en) * | 1991-01-16 | 1995-09-16 | Schering Corp | USE OF INTERLEUKIN-10 IN ADOPTIVE CANCER IMMUNOTHERAPY. |
| US5205290A (en) | 1991-04-05 | 1993-04-27 | Unger Evan C | Low density microspheres and their use as contrast agents for computed tomography |
| US5874062A (en) * | 1991-04-05 | 1999-02-23 | Imarx Pharmaceutical Corp. | Methods of computed tomography using perfluorocarbon gaseous filled microspheres as contrast agents |
| EP0624095B2 (en) * | 1991-12-31 | 2005-07-20 | Zymogenetics, Inc. | Methods and compositions for reducing blood loss |
| SE9200951D0 (en) * | 1992-03-27 | 1992-03-27 | Kabi Pharmacia Ab | PHARMACEUTICAL COMPOSITION CONTAINING A DEFINED LIPID SYSTEM |
| ES2041597B1 (en) * | 1992-05-05 | 1994-06-01 | Bazaco Joan Freixas | NEW PROCEDURE FOR THE PREPARATION OF LIPOSOMES THAT CAPSULATE PHARMACEUTICAL AND COSMETIC PRODUCTS. |
| US7083572B2 (en) | 1993-11-30 | 2006-08-01 | Bristol-Myers Squibb Medical Imaging, Inc. | Therapeutic delivery systems |
| US5736121A (en) * | 1994-05-23 | 1998-04-07 | Imarx Pharmaceutical Corp. | Stabilized homogenous suspensions as computed tomography contrast agents |
| US6156337A (en) * | 1994-07-08 | 2000-12-05 | Opperbas Holding B.V. | Method for high loading of vesicles with biopolymeric substances |
| DE4430592A1 (en) * | 1994-08-20 | 1996-02-22 | Max Delbrueck Centrum | Liposomal preparation, its preparation and its use |
| US6743779B1 (en) | 1994-11-29 | 2004-06-01 | Imarx Pharmaceutical Corp. | Methods for delivering compounds into a cell |
| US5830430A (en) * | 1995-02-21 | 1998-11-03 | Imarx Pharmaceutical Corp. | Cationic lipids and the use thereof |
| US5997898A (en) * | 1995-06-06 | 1999-12-07 | Imarx Pharmaceutical Corp. | Stabilized compositions of fluorinated amphiphiles for methods of therapeutic delivery |
| US6033645A (en) * | 1996-06-19 | 2000-03-07 | Unger; Evan C. | Methods for diagnostic imaging by regulating the administration rate of a contrast agent |
| US6521211B1 (en) | 1995-06-07 | 2003-02-18 | Bristol-Myers Squibb Medical Imaging, Inc. | Methods of imaging and treatment with targeted compositions |
| US6139819A (en) * | 1995-06-07 | 2000-10-31 | Imarx Pharmaceutical Corp. | Targeted contrast agents for diagnostic and therapeutic use |
| US6231834B1 (en) | 1995-06-07 | 2001-05-15 | Imarx Pharmaceutical Corp. | Methods for ultrasound imaging involving the use of a contrast agent and multiple images and processing of same |
| US6447800B2 (en) | 1996-01-18 | 2002-09-10 | The University Of British Columbia | Method of loading preformed liposomes using ethanol |
| WO1997040679A1 (en) | 1996-05-01 | 1997-11-06 | Imarx Pharmaceutical Corp. | Methods for delivering compounds into a cell |
| US6414139B1 (en) | 1996-09-03 | 2002-07-02 | Imarx Therapeutics, Inc. | Silicon amphiphilic compounds and the use thereof |
| US5846517A (en) * | 1996-09-11 | 1998-12-08 | Imarx Pharmaceutical Corp. | Methods for diagnostic imaging using a renal contrast agent and a vasodilator |
| CA2263568C (en) * | 1996-09-11 | 2008-12-02 | Imarx Pharmaceutical Corp. | Methods for diagnostic imaging using a contrast agent and a renal vasodilator |
| US6120751A (en) | 1997-03-21 | 2000-09-19 | Imarx Pharmaceutical Corp. | Charged lipids and uses for the same |
| US6537246B1 (en) | 1997-06-18 | 2003-03-25 | Imarx Therapeutics, Inc. | Oxygen delivery agents and uses for the same |
| US6143276A (en) * | 1997-03-21 | 2000-11-07 | Imarx Pharmaceutical Corp. | Methods for delivering bioactive agents to regions of elevated temperatures |
| US6090800A (en) * | 1997-05-06 | 2000-07-18 | Imarx Pharmaceutical Corp. | Lipid soluble steroid prodrugs |
| US7452551B1 (en) | 2000-10-30 | 2008-11-18 | Imarx Therapeutics, Inc. | Targeted compositions for diagnostic and therapeutic use |
| US6416740B1 (en) | 1997-05-13 | 2002-07-09 | Bristol-Myers Squibb Medical Imaging, Inc. | Acoustically active drug delivery systems |
| US20020128248A1 (en) * | 1997-06-20 | 2002-09-12 | Metaphore Pharmaceuticals, Inc | SODm therapy for prevention and/or treatment of inflammatory disease |
| US6180620B1 (en) | 1997-06-20 | 2001-01-30 | G.D. Searle & Co. | Analgesic methods using synthetic catalysts for the dismutation of superoxide radicals |
| US20050171198A1 (en) * | 1997-06-20 | 2005-08-04 | Metaphore Pharmaceuticals, Inc. | SODm therapy for treatment, prevention, inhibition and reversal of inflammatory disease |
| US6548047B1 (en) | 1997-09-15 | 2003-04-15 | Bristol-Myers Squibb Medical Imaging, Inc. | Thermal preactivation of gaseous precursor filled compositions |
| NZ503513A (en) * | 1997-09-18 | 2004-12-24 | Skyepharma Inc | Sustained-release liposomal anesthetic compositions |
| US20020039596A1 (en) | 1997-11-14 | 2002-04-04 | Hartoun Hartounian | Production of multivesicular liposomes |
| US6123923A (en) * | 1997-12-18 | 2000-09-26 | Imarx Pharmaceutical Corp. | Optoacoustic contrast agents and methods for their use |
| US20010003580A1 (en) | 1998-01-14 | 2001-06-14 | Poh K. Hui | Preparation of a lipid blend and a phospholipid suspension containing the lipid blend |
| DE69940735D1 (en) | 1998-07-17 | 2009-05-28 | Pacira Pharmaceuticals Inc | BIODEGRADABLE ARRANGEMENTS FOR THE CONTROLLED RELEASE OF INCLUDED SUBSTANCES |
| AU2002307343A1 (en) * | 2001-04-17 | 2002-10-28 | Genencor International, Inc. | Method to bind enzyme to carrier using cationic copolymers and product produced thereby |
| IL158819A0 (en) * | 2001-05-31 | 2004-05-12 | Skyepharma Inc | Encapsulation of nanosuspensions in liposomes and microspheres |
| US7125568B2 (en) * | 2001-08-23 | 2006-10-24 | Sung Michael T | Lipophilic drug compositions |
| TWI235066B (en) * | 2001-10-03 | 2005-07-01 | Celator Technologies Inc | Liposome loading with metal ions |
| AU2002357396A1 (en) * | 2001-12-31 | 2003-07-24 | Elan Pharmaceuticals, Inc. | Efficient liposomal encapsulation under mild conditions |
| WO2003059280A2 (en) * | 2002-01-09 | 2003-07-24 | Elan Pharmaceuticals, Inc. | Efficient liposomal encapsulation |
| WO2003059279A2 (en) * | 2002-01-09 | 2003-07-24 | Elan Pharmaceuticals, Inc. | Efficient liposomal encapsulation under mild conditions |
| DE10203923B4 (en) * | 2002-01-31 | 2011-06-01 | Klinipharm Gmbh | A method for increasing the water solubility of lipophilic active ingredients, preparation of highly concentrated aqueous compositions of these active ingredients, such products and their use |
| WO2006068890A2 (en) * | 2004-12-14 | 2006-06-29 | Transave, Inc. | Lipid particles comprising bioactive agents, methods of preparing and uses thereof |
| WO2006071659A1 (en) * | 2004-12-29 | 2006-07-06 | Trustees Of Boston University | Delivery of h2 antagonists |
| KR100831627B1 (en) | 2006-05-11 | 2008-05-22 | (주)아모레퍼시픽 | Reverse-Phase Multi-Lamella Vesicle Capturing Aqueous Physiologically Active Ingredients |
| CA2807357A1 (en) | 2010-08-05 | 2012-02-09 | Geetanjali Chandrashekhar Chimote | Microparticle formulation for pulmonary drug delivery of anti-infective molecule for treatment of infectious diseases |
| CA2857604A1 (en) | 2011-12-22 | 2013-06-27 | Nuvo Research Gmbh | Liposomal chlorite or chlorate compositions |
| EP2891495B1 (en) * | 2012-08-28 | 2018-05-30 | National University Corporation Hokkaido University | Lipid membrane structure including bacterial cell component having dispersibility in non-polar solvent, and method for producing same |
| US9775789B2 (en) * | 2012-12-06 | 2017-10-03 | D & J Rosée Inc. | Topical liposome compositions containing phenolic anti-inflammatory agents and their methods of preparation |
| EP3506882B1 (en) * | 2016-08-30 | 2022-01-12 | Bracco Suisse SA | Preparation of size-controlled microparticles |
| US11033495B1 (en) | 2021-01-22 | 2021-06-15 | Pacira Pharmaceuticals, Inc. | Manufacturing of bupivacaine multivesicular liposomes |
| US11278494B1 (en) | 2021-01-22 | 2022-03-22 | Pacira Pharmaceuticals, Inc. | Manufacturing of bupivacaine multivesicular liposomes |
| US11357727B1 (en) | 2021-01-22 | 2022-06-14 | Pacira Pharmaceuticals, Inc. | Manufacturing of bupivacaine multivesicular liposomes |
Family Cites Families (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4217344A (en) * | 1976-06-23 | 1980-08-12 | L'oreal | Compositions containing aqueous dispersions of lipid spheres |
| CH624011A5 (en) * | 1977-08-05 | 1981-07-15 | Battelle Memorial Institute | |
| US4394448A (en) * | 1978-02-24 | 1983-07-19 | Szoka Jr Francis C | Method of inserting DNA into living cells |
| US4235871A (en) * | 1978-02-24 | 1980-11-25 | Papahadjopoulos Demetrios P | Method of encapsulating biologically active materials in lipid vesicles |
| US4241046A (en) * | 1978-11-30 | 1980-12-23 | Papahadjopoulos Demetrios P | Method of encapsulating biologically active materials in lipid vesicles |
| JPS55118415A (en) * | 1979-02-28 | 1980-09-11 | Pii Papahadojiyopo Dometoriosu | Method of encapsulating biologically active substance in lipoid cell |
| JPS5782310A (en) * | 1980-11-11 | 1982-05-22 | Tanabe Seiyaku Co Ltd | Production of liposome preparation |
| JPS5782311A (en) * | 1980-11-11 | 1982-05-22 | Tanabe Seiyaku Co Ltd | Production of liposome preparation |
| DE3269611D1 (en) * | 1981-07-02 | 1986-04-10 | Hoffmann La Roche | Process for preparing liposome solutions |
| WO1983003383A1 (en) * | 1982-03-29 | 1983-10-13 | Liposome Co Inc | Stable plurilamellar vesicles |
| US4515736A (en) * | 1983-05-12 | 1985-05-07 | The Regents Of The University Of California | Method for encapsulating materials into liposomes |
| US4565696A (en) * | 1983-08-03 | 1986-01-21 | The Regents Of The University Of California | Production of immunogens by antigen conjugation to liposomes |
| FR2561101B1 (en) * | 1984-03-13 | 1986-10-31 | Paris Sud Universite | PROCESS FOR THE PREPARATION OF LIPOSOMES AND LIPOSOMES THUS OBTAINED |
| JPS6150912A (en) * | 1984-08-16 | 1986-03-13 | Shionogi & Co Ltd | Production of liposome preparation |
-
1987
- 1987-03-30 JP JP62079512A patent/JPH0751496B2/en not_active Expired - Fee Related
- 1987-04-01 CA CA000533545A patent/CA1291425C/en not_active Expired - Lifetime
- 1987-04-02 US US07/033,498 patent/US4877561A/en not_active Expired - Lifetime
- 1987-04-02 EP EP87302866A patent/EP0240346B1/en not_active Expired - Lifetime
- 1987-04-02 DE DE8787302866T patent/DE3764290D1/en not_active Expired - Fee Related
- 1987-04-02 AT AT87302866T patent/ATE55542T1/en not_active IP Right Cessation
- 1987-04-02 ES ES87302866T patent/ES2016842B3/en not_active Expired - Lifetime
-
1990
- 1990-11-06 GR GR90400868T patent/GR3001049T3/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| EP0240346A3 (en) | 1987-11-19 |
| JPS6339810A (en) | 1988-02-20 |
| EP0240346B1 (en) | 1990-08-16 |
| ATE55542T1 (en) | 1990-09-15 |
| DE3764290D1 (en) | 1990-09-20 |
| US4877561A (en) | 1989-10-31 |
| JPH0751496B2 (en) | 1995-06-05 |
| EP0240346A2 (en) | 1987-10-07 |
| GR3001049T3 (en) | 1992-01-20 |
| ES2016842B3 (en) | 1990-12-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1291425C (en) | Method of producing liposome | |
| US4515736A (en) | Method for encapsulating materials into liposomes | |
| CA1205383A (en) | Method of encapsulating biologically active materials in multilamellar lipid vesicles (mlv) | |
| EP0243446B1 (en) | Alpha tocopherol-based vesicles | |
| US5234634A (en) | Method for preparing alpha-tocopherol vesicles | |
| CA1287581C (en) | Liposome composition | |
| US4855090A (en) | Method of producing high aqueous volume multilamellar vesicles | |
| EP1005326B1 (en) | Method for producing liposomes with increased percent of compound encapsulated | |
| EP0219922B1 (en) | Anthracycline antineoplastic agents encapsulated in phospholipid micellular particles | |
| US5049392A (en) | Osmotically dependent vesicles | |
| US5077057A (en) | Preparation of liposome and lipid complex compositions | |
| JP2579625B2 (en) | Multilamellar liposomes with improved uptake efficiency | |
| JPH0753661B2 (en) | Pro-liposome composition and method of making an aqueous dispersion of liposomes | |
| HU203278B (en) | Process for producing compositions containing liposomes | |
| EP0467954B1 (en) | Preparation of liposome and lipid complex compositions | |
| JPH0714865B2 (en) | Liposome preparation and method for producing the same | |
| US5252336A (en) | Liposome composition whose liposome membrane contains a polyoxyethylene derivative | |
| US4963363A (en) | Phospholipid delivery vehicle for aqueous-insoluble active ingredients | |
| US5173219A (en) | Uniform spherical multilamellar liposomes of defined and adjustable size distribution | |
| HUT73531A (en) | Ketoprofene liposomes | |
| EP0488142B1 (en) | Process for encapsulating solid or liquid lipophilic agents in phospholipid-liposomes and medicaments containing those liposomes | |
| JP3831958B2 (en) | Lipid mixed lipid and liposome dispersion | |
| KR950002880B1 (en) | Process for preparing liposomes | |
| JPH0114A (en) | Liposome formulation and its manufacturing method | |
| JPH0446129A (en) | New adjuvant for forming liposome |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKLA | Lapsed |