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

Definitions

• the present invention relates to methods for preparing liposome suspensions characterized by high en ⁇ capsulation efficiencies and high lipid concentrations.
• Liposomes provide several advantages in drug delivery. When administered parenterally, either by the intravenous or intramuscular route, liposomes ca -provide controlled "depot" release of encapsulated drug over an extended time period, and reduce the side effects of the drug, by limiting the concentration of free drug in the bloodstream. Liposomes can alter the tissue distribution of and uptake of drugs, in a therapeutically favorable way, and can increase the convenience of therapy, by al ⁇ lowing less frequent drug administration. Liposome drug delivery systems are reviewed in Poznansky.
• liposomes for drug delivery by inhalation has also been studied, as reported in co-owned US patent application for "Liposome Inhalation and Method", Serial No. 737,221, filed May 22, 1985.
• the inhalation, liposomes can be tailored, according to lipid composition, to release an entrapped drug at a selected release rate which, may vary, in half life, from a few hours to several days. Further, to the extent the drug is sequestered in the liposomes, side effects related to rapid uptake into the respiratory tract and bloodstream are reduced.
• liposomes with both lipophilic and hydrophilic drugs, and the ability to vary lipid composition to achieve a selected drug release rate are also advantageous in administering a drug topically or to mucosal tissue.
• An added advantage of liposome for drug delivery to mucosal tissue is that the liposome surfaces can be modified for increased tissue stickiness, to enhance the residence time of the liposomes at the target tissue site. This feature is described in co-owned patent application for "Liposomes with Enhanced Retention on Mucosal Tissues", Serial No. 890, 815, filed July 28, 1986.
• vesicle forming lipids are deposited as a thin film on the sides of a flask, and slowly rehydrated by addition of an aqueous buffer.
• the drug to be entrapped may be included either in the lipid film (in the case of a lipophilic drug), or in the aqueous hydration medium (in the case of a hydrophilic drug) .
• the liposomes that form are ultilamellar vesicles (MLVs) having heterogeneous sizes between about 0.05 and 10 microns.
• the MLVs may be subsequently processed, typically by homogenization, sonication, or membrane extrusion, to produce smaller, more uniformly sized suspension.
• Liposome sizing down to about 0.2-0.4 microns is generally preferred. Liposomes in this size range can be sterilized by passage through a 0.45 micron depth filter, have less tendency to aggregate, and also may show more favorable organ distribution when administered intra ⁇ venously (Gabizon) .
• One of the drawbacks of the MLV method is relatively poor encapsulation efficiency of water-soluble drugs.
• lipid-in-solvent solution is mixed with an aqueous medium, and emulsified to form a water-in-oil emulsion.
• Removal of the lipid solvent produces a reverse-phase lipid gel which is then agitated, preferably in the presence of added aqueous medium, to form reverse-phase evaporation vesicles (REVs) characterized by relatively large sizes and one to a few bilayer shells.
• REVs reverse-phase evaporation vesicles
• liposome sizing leads to a loss of encapsulated material. Since the advantages of liposome drug delivery depend on entrapment of the drug by liposomes, it is generally desirable to administer ' a drug in predominantly liposome entrapped form, i.e., at least about 50 percent of the drug is associated with the liposomes. This is particularly true where the drug is known to cause un- desired side effects when administered in free form.
• a dilute suspension of liposomes containing a water-soluble, liposome-permeable drug are concentrated to a lipid paste containing at least about 50% and preferably about 70% encapsulated aqueous volume, which also represents the percentage of drug which is encapsulated in the liposomes.
• the suspension is stored in concentrated form; and-diluted shortly before use, i.e., the drug in the diluted suspen ⁇ sion is administered in a non-equilibrated, predominantly encapsulated form.
• the removal of free drug and liposome concentration can be accomplished in a single step by ultrafiltration, centrifugation, or the like.
• the liposome paste approach involves loss of free drug material, and additional processing of the liposome suspension.
• Another object of the invention is to provide a method for producing a concentrated liposome suspension which exists in paste-or near-paste form without ad ⁇ ditional dehydration processing.
• a related object is to provide a method for producing such a paste which can be readily sterilized by filter sterilization.
• Still another object of the invention is to provide a liposome processing method which can be adapted to produce liposomes in a narrow size range, such as 0.1- 0.4 microns, while maintaining encapsulation above 50 percent.
• the invention includes, in one aspect, a method of preparing a suspension of liposomes containing a water- soluble compound predominantl — hat is, more than 50%- *- in liposome-encapsulated form.
• a solution of vesicle-forming lipids in a chlorofluorocarbon solvent are infused in liquid form into an aqueous medium, under pressure, temperature, and agita ⁇ tion conditions at which lipid frothing is largely prevented, and at an infusion rate that produces pre ⁇ dominantly oligolamellar vesicles.
• the compound to be encapsulated is dissolved either in the aqueous medium or in the lipid solvent. Solvent infusion is continued until the lipid concentration in the aqueous medium is between about about 150-500 um/ml.
• the infused solvent is removed at substantially the same rate that it is introduced, and is removed completely when the selected lipid concentra ⁇ tion is reached.
• trap ⁇ ping efficiencies of between about 60-70 percent can be achieved.
• the high-concentration suspension is suitable .
• the invention includes a method of preparing a concentrated liposome suspension having a lipid concentration of greater than about 200 um/ ml and liposome sizes no greater than about 0.6 microns.
• a solution of vesicle-forming lipids in a chlorofluorocarbon solvent is infused, as above, into an aqueous medium, under pressure, temperature, and agitation conditions conditions at which lipid frothing is largely prevented, and at an infusion rate that produces pre ⁇ dominantly oligolamellar vesicles.
• the compound to be encapsulated is dissolved either in the aqueous medium or in the chlorofluorocarbon solvent.
• the aqueous suspension is circulated through an extrusion device effective to size the liposomes to between 0.1-0.6 microns.
• Solvent infusion with continued extrusion and solvent removal, is continued until a final desired liposome concentration- *-which may be as high as 300-500 um/ml- *-and liposome size range is reached.
• the concentrated material may be sterilized by filtration through a 0.45 or 0.22 micron depth filter. The two methods, when combined, are useful in producing concentrated liposome suspensions (a) having liposome sizes less than about 0.4 microns and (b) water- soluble drug in predominantly encapsulated " form.
• the liposome suspension is filtered during the solvent infusion and sizing steps, to remove liposomes below a selected size range, and these liposomes are recirculated and mixed with newly infused lipid-in-solvent, during which a portion of the small liposomes are converted to larger ones.
• the final liposome suspension can be made substantially free of the smaller liposomes, without sacrificing other advantages of the invention, such as high encapsulation efficiency and high, lipid concentration.
• FIG 1 illustrates in diagrammatic form, a lipid processing system used in practicing the invention?
• Figure 2 illustrates additional components in the Figure 1 system for use in sizing liposomes by extru- sion during liposome preparation
• Figure 3 illustrates portions of an alternate embodiment of a liposome processing system, for use in preparation of liposomes having defined size ranges?
• Figure 4 shows plots of encapsulation efficiency as a function of lipid concentration in two different processing methods carried out according to the invention.
• Figures 5A-5D are representations of photomicrographs of liposomes taken during a liposome preparation method at lipid concentrations of 50 um/ml
• FIG. 1 shows a processing system, indicated generally at 10- used in preparing liposomes with high encapsulation efficiencies, according to the method of the invention.
• the system includes a sealed, solvent-infusion chamber 12 which, during operation, contains a given volume of aqueous medium in which the liposomes are formed.
• the working volume of the chamber may range from 100 ml or less, for small-volume processing, up to 100 liters or more, for scale-up liposome production.
• the particular system which will be described herein is designed for preparation of up to about 4 liters of liposome suspension in a single batch, and the solvent- infusion chamber has a total volume of about 5 liters. It will be understood that the entire system can be scaled up or down, to accomodate larger chamber volumes.
• Chamber 12 is maintained at a constant temperature during operation by a temperature bath 14 which circulates water or other suitable coolant at a desired temperature through a jacket 16 surrounding the chamber.
• the bath is operable to maintain the temperature of the liquid contents of the chamber above the boiling point of the lipid solvent, and typically at a selected temperature between about 5°C and 45°C.
• the lipid-in-solvent solution is infused into the chamber from a sealed solvent feed tank 18 connected to the chamber through a feeder line 20 and an in-line feed pump 22.
• the solvent material is introduced into the chamber through a nozzle 24 which is positioned preferably just below the surface of the aqueous medium.
• Pump 22 is operable to infuse the solvent solution at a rate which is between about 0.5-2 ml, and preferably about 1 ml, per minute per 100 ml aqueous medium in the mixing chamber.
• the mixing chambers contains 270 ml of aqueous buffer
• the pump is operable to infuse between about 1.4- 5.4, and preferably about 2.7 ml solvent per minute into the chamber.
• the solvent in the tank and feeder line are maintained at a selected " temperature below the solvent boiling point during operation by a temperature bath 26 which circulates a cooled liquid, such as refrigerated water through a jacket 28 surrounding the feed tank and a jacket-like sleeve (not shown) surrounding the feeder line.
• a temperature bath 26 which circulates a cooled liquid, such as refrigerated water through a jacket 28 surrounding the feed tank and a jacket-like sleeve (not shown) surrounding the feeder line.
• a mixer 30 which includes a blade 32 extending into the chamber is used in.mixing the liquid contents of the chamber during operation.
• the blade speed is controlled by a rheostat 34, and is preferably operable produce blade rotation of between about 400 and 1,500 revolutions per minute.
• the pressure in the chamber is maintained during operation to a vacuum of about 200 mbar, by a vacuum pump 36.
• the pump is connected to the chamber, as shown, through a condenser 38 where solvent drawn off by the pump is condensed.
• the condensed solvent is collected in a solvent-recovery tank 40.
• a temperature bath 42 supplies cooled liquid, such as refrigerated water, through condensing coils 44 in the condenser, and through a water jacket 46 surrounding tank 40.
• the lipid-in-solvent solution contains vesicle- forming lipids dissolved in a chlorofluorocarbon solvent whose boiling point is preferably below room temperature, and more preferably, between about 2-10 C.
• a chlorofluorocarbon is a chlorinated, fluorinated carbon or hydrocarbon which has the above boiling point characteristics and which can serve as a lipid solvent.
• Typical chlorofluorocarbons include "Freon 11" (CC1 3 F), "Freon 12" (CC1 2 F 2 ), "Freon 21" (CHFC1 2 ),
• Freon 22 CHC1F 2
• Freon 113 CC1 2 FCC1F 2
• Freon 114 CC1F 2 CC1F 2
• Freon 115 CC1F 2 CF 3
• a preferred solvent is trichlorofluoromethane ("Freon 11"), whose boiling point is 23.8° C at 1 atm, or a mixture of trichlorofluoromethane and dichlorofluoromethane (“Freon 21”), whose boiling point is 8.9° C at 1 atm.
• the solvent may include .up to about 10-20 percent (v/v) of a solvent such as ethanol which is miscible with both the chlorofluorocarbon and water. Minor amounts of other organic solvents which are either volatilized under the selected conditions of solvent infusion, or which are tolerated in low concentrations in the aqueous suspension of liposomes may also be included.
• the vesicle-forming lipids are selected from known vesicles forming lipids which generally include phospholipids and sterols.
• a list of phospholipids used commonly in liposome preparation is given on page 471 of Szoka, 1980.
• Neutral lipid components such as egg phosphotidylcholine (egg PC), egg PC/cholesterol mixtures m y be suitable.
• egg PC egg phosphotidylcholine
• PG phosphatidylglycerol
• One preferred lipid composition described in Examples I and II, includes 55 mole percent egg PC, 5 mole percent PG, and 40 mole percent cholesterol.
• the lipid solution may contain lipophilic protective agents, such as alpha-tocopherol, and/or lipophilic drug compounds which are to be entrapped in the lipid bilayer phase of the liposomes.
• lipophilic protective agents such as alpha-tocopherol
• lipophilic drug compounds which are to be entrapped in the lipid bilayer phase of the liposomes.
• Representative lipophilic compounds which can be administered in liposome-entrapped form include protaglandins, amphotericin B, progesterone, isosorbide dinatrate, testosterone, nitroglycerin, estradiol, cortisone, dexamethasome and related esters, and betamethasone valerate.
• the lipid solvent may also contain the water-soluble compound to be encapsulated, where such cannot be included in the aqueous medium used in forming the liposomes.
• the concentration of lipids in the lipid-in- solvent solution is adjusted to achieve a desired concentration of lipids in the aqueous medium after introduction of a selected volume of the solution.
• the minimum concentration of lipids in the final liposome suspension is about 150 um/ml
• the total volume of lipid solution added to the aqueous medium is between about one-half and twice that of the total volume of aqueous medium in the mixing chamber.
• the lipid solution is made up to between about 200- 700 um/ml.
• a mixed chlorofluorocarbon solvent such as an equal-volume mixture of "Freon 11" and "Freon 21" may be preferred for high-concentration lipid solutions.
• the concentration of lipids in the solution is adjusted accordingly, so that a desired amount of lipid is added to the aqueous medium, within this volume mixing range.
• the aqueous medium is typically a buffered aque ⁇ ous solution having a pH between about 6.0 and 7.5, and usually containing the water-soluble pharmaceutical agent or compound which is to encapsulated in the liposomes.
• the pharmaceutical agent may be any drug, hormone, peptide, vitamin, or other pharmaceutical agent which is relatively soluble in the aqueous medium and which can be released from liposomes at a controlled rate, when the liposomes administered parenterally, topically, by inhala ⁇ tion, or other route.
• the controlled release may be by passage of the agent through the liposomal membrane, in the case of a liposome-permeable agent, or by liposome breakdown, in the case of a liposome-impermeable drug.
• Representative water-soluble drugs include terbutaline, albuterol, atropine methyl, cromylyn sodium, propranolol, flunoisolide, ibuprofin, gentamycin, tobermycin, pentamidine, penicillin, theophylline, bleomycin, etoposide, captoprel, n-acetyl cycteine, verampimil, fluorouracil, iodouridine, trifluorouridine, vidarabine, azidothymidine, ribavirin, phosphonoformate,- phosphonoacetate, acyclovir, ce etidine, naphazoline, lodoxamide, and phenylepinephrin , exemplary
• Suitable water-soluble, liposome-impermeable compounds include peptide hormones, enzymes, enzyme inhibitors, apolipoproteins, and higher molecular weight carbohydrates.
• Representative compounds in this class include calcitonin, atriopeptin, alpha-1 antitrypsin, interferon, oxytocin, vasopressin, insulin, interleukin-2, superoxide dismutase, tissue plasminogen activator, plasma factor 8, epidermal growth factor, tumor necrosis factor, lung surfactant protein, and lipocortin.
• the concentration of drug in the aqueous medium is prefer ⁇ ably that which is desired in the encapsulated volume in the liposomes.
• the aqueous medium may contain soluble protective agents, such as chelating agents, which reduce oxidative, lipid hydrolysis, or drug degradative effects which may occur on storage.
• This section describes the method used in producing liposomes in which the encapsulation efficiency of a water-soluble compound is greater than about 50%, and as high as 65% or more.
• the operation is described with respect to the processing system and components detailed in Sections IA and IB above.
• the lipid solu ⁇ tion is added to feed tank 18, and the aqueous medium, to chamber 12, and the two solutions are equilibrated, by temperature baths 14, 24, respectively, to chamber and tank temperatures, above and below the boiling point of the lipid solvent, at the selected pressure.
• the lipid solvent in "Freon 11” the lipid solvent and aqueous medium are equilibrated to and maintained dur- ing operation at about 4°C and 20°C, respectively.
• pump 22 is activated to supply the cooled lipid solvent into the aqueous medium contained, in the mixing chamber.
• the solvent is infused just below, and preferably between about 1 and 3 cm below the lipid surface in the chamber, and is supplied to the chamber at a preferred rate of about 1 ml per minute per 100 ml aqueous medium. If the infusion rate is too slow, the lipid vesicles which form tend to be more multilamellar in structure, which tends to reduce en ⁇ capsulation volume per unit lipid.
• the liposomes formed are largely oligolamellar, i.e., contain predominantly one or only a few bilayers. In the initial phases of the method, the liposomes are heterodisperse in size, ranging from submicron sizes to 10 microns or greater.
• Figure 5A shows a typical field of liposomes formed when the total lipid concentration in the mixing chamber has reached 50 um/ml.
• the larger liposomes seen in the figure are between about 10-15 microns, and the smaller ones, about 1.5 microns or less.
• Determina ⁇ tion of the percent of encapsulated water-soluble material, according to methods described in Example I, show that the total entrapped volume in the 50 um/ml preparation is between about 30-35% (Example I).
• lipids into the aqueous suspension results in a continued increase in the percent of encapsulated water-soluble marker (entrapped volume) up to a maximum of between about 60-65 percent encapsulation, at a lipid concentration of about- 300 um/ml or greater.
• the general increase in encapsulation efficiency, as a function of lipid concentration is seen in Figure 3.
• the upper curve (solid circles) in the figure is a plot from one of the processing runs described in Example I.
• the dotted line in the graph shows the lipid concentration at which 50% encapsulation efficiency is reached.
• the en ⁇ capsulated compound is fluorescein, representative of a relatively small, water-soluble compound which is originally contained in the aqueous buffer used in forming the liposome suspension.
• the vesicle-forming lipids include charged lipid components, such as PG
• continued addition of lipid- in-solvent to the mixing chamber produces a gradual size reduction of the larger liposomes in the suspension.
• This effect is seen in Figures 5B-5D, which show the general. appearance of the liposome suspension at 100, 150, and 200 um/ml, respectively.
• the general size reduction with respect to the suspension at 50 um/ml is easily seen, and at 150 um/ml lipid concentration, almost all of the liposomes are about 1.5 microns or smaller.
• Further lipid increase to 200 um/ml did not significantly change the liposome size distribution.
• the method of the invention produces a relatively homogeneous size distribu- tion of liposomes, with maximum liposomes sizes less than about 1.5 microns.
• the lipsosome suspension becomes quite viscous at a lipid concentration greater than about 300-400 um/ml, and further introduction of lipids becomes difficult or impossible.
• the concentrated suspension has a paste-like consistency which is suitable for several applications which utilize liposome paste material, as will be considered in Section IV below.
• the paste ⁇ like material can be further dehydrated, to increase en ⁇ capsulation efficiencies up to about 70% or higher, ac- cording to methods described in co-owned US patent ap ⁇ plication for "Liposome Concentrate and Method", Serial No. 860,528, filed May 7, 1986.
• the process may be carried out under sterile conditions, using sterile lipid and aqueous components, and by presterilizing the vessels and connective tubing . in the system which are in contact with the liquid components.
• the liposomes may be filter sterilized before in vivo administration.
• the liposomes must be further sized down to maximum sizes of about 0.4 microns.
• the liposomes are extruded through a defined pore size membrane, such as a polycarbonate membrane with a 0.4 micron pore size (Szoka, 1982), or an asymmetric ceramic membrane, as described in co-owned US patent application for "Liposome Extrusion Method", Serial No. 829,710, filed February 28, 1986, and also discussed below.
• Example II describes the polycarbonate membrane extrusion method as it is applied to liposome suspensions having one of a number of lipid concentrations between 100-350 um/ml.
• Table 2 in the example shows the en ⁇ capsulation efficiencies measured for each of the several preparations following extrusion.
• a comparison of this data with the encapsulation data in Table 1 (Example I) indicates that the extrusion process results in a significant loss of encapsulated material, which is presumably due to larger vesicles breaking and reforming smaller ones during extrusion.
• the highest encapsulation which can be achieved by the method, at a maximum lipid concentration of about 350 um/ml, is about 45%.
• Example III describes the use of the present method for producing liposomes with high encapsulation of calcitonin, representative of a water-soluble liposome- impermeable compound which is originally contained in the aqueous medium.
• the calcitonin liposomes were prepared by infusing a solution of uncharged lipids in "Freon 11" into an aqueous solution of calcitonin, to a final lipid concentration of about 300 umole/ml.
• the encapsulation efficiency of the procedure was greater than 60%. Because uncharged lipid components were used, the final liposome sizes ranged up to about 10 microns, as discussed above.
• Example IV describes the use of the present method for producing liposomes encapsulated propranolol, representative of a water-soluble which is originally included in the lipid solvent.
• the propranolol liposomes were prepared by infusing a solution PC and propranolol in "Freon ll”:ethanol, 10:1 (v/v) into an aqueous buffer. The presence of ethanol in the lipid solvent was necessary for solubilizing the drug in a chlorofluorocarbon solvent (both “Freon 11" and “Freon 21" were tested).
• the infusion process was continued to a final lipid concentration of about 300 umole/ml, at which about 75% of the drug was encapsulated in the liposomes formed.
• the liposome sizes were heterodisperse, having sizes up to about 10 microns.
• the ethanol remaining in the liposome suspension after removal of the "Freon" solvent can be removed, if desired, by diafiltration, molecular sieve chromatography, or the like. -However, the presence of the ethanol in the suspen ⁇ sion does not appear to effect liposome stability or reduce encapsulation efficiency. _
• the high-concentration method is designed for producing a liposome suspension having (a) a lipid concentration preferably above about 200 um/ml, and up to about 500 um/ml and (b) liposome sizes less than about 0.4 microns.
• the method allows for direct preparation of high concentration liposomes suspension which are readily sterilized by filtration through a 0.45 micron depth filter.
• the liposomes When produced in the presence of a water-soluble compound, the liposomes have an encapsulation efficiency of up to 50-60%.
• Figure 2 illustrates a modification of system 10 for use in practicing the high-concentration method.
• the modified system which is indicated generally at 48 in Figure 2, contains all of the components of system 10 which are shown in Figure 1, including mixing chamber 12, surrounding water-jacket 16, and mixing blade 32 seen in Figure 2.
• the system further includes a liposome- extrusion shunt containing a valve 50, a pump 52, and an in-line extrusion device 54.
• the shunt is preferably designed to circulate suspension in the mixing chamber at a rate which is at least about 5-10% of the total volume in the mixing chamber per minute. That is, the shunt is designed to process the entire suspension volume in the mixing chamber at least every ten-to-twenty minutes.
• the pump is preferably designed to develop up to several hundred psi pressure, at the volume level just mentioned.
• the extrusion device is a filter device equipped with a 0.2-0.6 micron pore size polycarbonate filter, of the type noted in Sec ⁇ tion IC This method is effective to size liposomes ap ⁇ proximately to the largest filter pore size, which may be selected from a pore size of 0.1 micron up to 2 micron or larger.
• the extrusion device is an asymmetric ceramic filter of the type constructed of a series of concentric ceramic layers which progress from finer to coarser ceramic mesh on proceeding from an inner to an outer annular space.
• Filters of this type may be obtained commercially in cartridge from the Norton Co (San Diego, CA) , and are available in inner pore (mesh) sizes which are effective in trapping particle size of 0.2, 0.4, or 1.0 microns.
• the use of this type of filter for ef ⁇ ficient liposome sizing has been described in the above- cited US patent application for "Liposome Extrusion Method".
• the high-concentration processing method uses lipid-in-solvent and aqueous medium components like those used in in the high-encapsulation method described in Sec ⁇ tion I.
• the aqueous medium may, but does not necessarily contain a water-soluble pharmaceutical compound for en ⁇ capsulation in the liposomes. That is, the liposomes may be formulated to contain either a lipophilic compound (which is preferably included in the lipid-in-solvent _ _
• the system is operated substantially as described in Section IC. Additionally, when the lipid concentration in the mixing chamber reaches a given concentration, the extrusion shunt is opened, and material in the mixing chamber is circulated through the extrusion device, at a suitable flow rate. Although the shunt may be placed in operation from the time of initial solvent infusion into the chamber, it is generally not necessary to begin extrusion until the lipid concentration in the chamber reaches 50-100 um/ml. Below this concentration, vesicles being formed with charged lipid components are becoming progressively smaller as infusion proceeds, as noted above. Above this concentration, liposome extrusion becomes more difficult, requiring higher pressure and producing less efficient sizing due to slower extrusion rates.
• the shunt is preferably oper ⁇ ated at a flow rate which processes the entire volume of the suspension in about 20 minutes or less. More gener ⁇ ally, the shunt is operated at a flow rate which allows about 5-10 passes of aqueous volume during the infusion process.
• the integrated liposome sizing step in the method significantly reduces the viscosity of the suspension, at higher lipid concentrations, and this feature allows infu- sion of additional lipid into the suspension to levels which are substantially higher than those achievable without integrated sizing.
• lipid infusion can be continued up to a final lipid concentra ⁇ tion of about 500 um/ml, at which the encapsulation ef- ficiency for water-soluble compounds is greater than 50%.
• the vesicle suspension would be too viscous to extrude above a lipid concentration of about 300 um/ml, even at high extrusion pressure.
• Example V illustrates the use of the method for producing a liposome suspension having (a) a final lipid concentration of 500 um/ml, (b) liposome sizes less than about 0.2 microns, and (c) an encapsulation efficiency of trapped water-soluble material of about 55%.
• the suspension was monitored at increasing lipid concentrations to determine encapsulation of a water-soluble marker.
• the encapsulation data are given in the table in Example V, and plotted (open circles) in Figure 4.
• the extrusion process produces lower encapsulation efficiencies, at comparable lipid concentrations than the system described in Section I.
• encapsulation efficiencies above 50% can be achieved.
• greater encapsulation efficiency for liposome- permeable compounds
• This section describes a system and method for producing a suspension of liposomes which are' pre ⁇ dominantly in a size range greater than 0.08 microns, and preferably between 0.1 and a selected size less than 1.0 microns, e.g., 0.4 microns.
• the suspension may also be characterized by high encapsulation efficiency and/or high lipid concentration, when the method is combined with one or both of the methods described in Sections I and II above.
• Figure 3 shows a system 60 designed for produc ⁇ ing uniform-size liposomes according to this aspect of the invention.
• the system will be described with respect to four functional subsystems I-IV which are shown in dotted lines in the figure. These sub ⁇ systems are designed for (I) mixing buffer and lipid-in- solvent solutions; (II) solvent injection and removal; (III) down-sizing filtration; and (IV) diafiltration and concentration.
• the solvent mixing subsystem (I) includes a pair of feed tanks 62, 64 used in supplying a lipid-in-solvent solution and aqueous buffer, respectively. Both tanks are pressurized with filtered nitrogen gas to prevent back flow into the tanks and to provide an inert atmosphere within the system.
• the lipid-in-solvent solution is pumped from tank 62 to a mixing chamber 66 by metering pump 68. Buffer is fed to the mixing chamber under pres ⁇ sure and its flow is regulated by valve 70.
• the two tanks, the mixing chamber, and the lines connecting the tanks and the chamber are maintained at a temperature below the boiling point of the lipid solvent by suitable tank jackets and tubing heat exchangers, as indicated.
• the solvent injection subsystem (II) includes a heat jacketed vacuum chamber 72 which is connected to the mixing chamber through a heat-jacketed static mixer 74.
• the mixer functions to mix the lipid/and buffer solutions as they pass from the mixing chamber into the vacuum chamber, and delivers the mixed material into the chamber as a fine spray through a spray nozzle 75.
• a heated fluid circulating through the mixer jacket serves to warm the solvent/buf er mixture in the mixer above the boiling point of the solvent before injection into the vacuum chamber.
• the vacuum chamber is evacuated to a selected pressure by a vacuum source 77.
• Chamber 72 communicates through a drain 76 with a high-pressure pump 78 which functions to continuously recirculate fluid contained within the chamber through a closed loop containing a three-way flow regulating valve 80, a conduit 82 and mixer 74.
• conduit 82 sup ⁇ plies material into the mixer downstream of the mixing chamber.
• the downstream end region of the conduit is heat jacketed, to cool the material being supplied to the mixer below the boiling point of the lipid solvent.
• the down-sizing subsystem (III) includes a filter 84, such as a polycarbonate or ceramic filter of the type described above, which is effective to reduce liposome sizes to a selected size range, preferably below about 1-2 microns.
• the fourth subsystem is composed of a holding tank 88 equipped with a bottom drain 90 which is connected to the intake of a pump 92 which serves to circulate the fluid contained in the holding tank through diafiltration filter 94.
• the diafiltration filter pore size is prefer- ably selected to retain liposomes larger than about 0.2 microns.
• the retentate from the diafiltration filter (larger liposomes) is returned to the holding tank and the filtrate passes through a three-way valve 96 which either diverts the filtrate (smaller liposomes) to drain in the case of concentration operations or to the three way flow regulating valve 70 for diafiltration and up-sizing operations. During these latter operations, the filtrate is fed to mixing chamber 66 by adjusting valve 70.
• buffer may be supplied directly from tank 64 to tank 88 through a valve 98.
• Operation of the System can be divided into five phases: (1) start-up, (2) injection/solvent removal, (3) down-sizing, (4) diafiltration/up-sizing, and (5) diafiltration and concentration.
• the lipid-in- solvent solution is held in its feed tank while aqueous buffer is allowed to pass from tank 64 into vacuum chamber 72, until the chamber is about half full.
• the chamber is heated by its external heat jacket until the buffer in the chamber is above the boiling temperature of the lipid solvent.
• the pressure in the chamber is lowered by vacuum source 77.
• the heated buffer contained in chamber 66 is then circulated by pump 78 through conduit 82 and mixer 74 and back into chamber 72.
• valve 80 is set to direct the entire flow from pump 78 through conduit 82, but as described below, during the down-sizing and diafiltration/up-sizing phases of operation the valve is set to meter a portion of the loop flow through a sizing filter and into the diafiltration loop circulation.
• the heated buffer circulates through conduit 82, it is cooled by the heat exchanger in the conduit below the boiling point of the lipid solvent.
• the recirculated buffer passes through the static mixer which here serves two purposes: it provides thorough mixing of the lipid-in- solvent/buffer mixture with the fluid recirculating through the loop (during the injection/solvent removal phase) and it heats the mixture to a temperature above the boiling point of the lipid solvent (which facilitates solvent removal in the vacuum chamber) .
• the start up phase ends as temperatures of the fluids and pressures within the vacuum chamber and process lines equilibrate at the appropriate values.
• liposomes are produced as the lipid solvent is removed from the lipid-in-solvent/buffer mixture.
• the lipid-in-solvent solution in tank 62 is introduced into chamber 62 by the metering pump 68, and aqueous buffer held in tank 64 is fed into the mixing chamber under pressure.
• the two solution mix in mixing chamber 66 and the lipid-in-solvent/buffer mixture is subsequently mixed with the aqueous buffer being recirculated through the solvent removal subsystem immediately upstream of static mixer.
• all solutions are at a temperature below the boiling point of the lipid solvent.
• the temperature is raised to a point above the boiling point of the lipid solvent (at the pressure set in the vacuum chamber) .
• the mixture is sprayed into the vacuum chamber as a fine mist by nozzle 80. Since the lipid solvent will vaporize at the temperature and pressure present in the chamber, the bulk of the solvent is stripped from the mixture as the droplets fall through the chamber.
• the vaporized solvent is condensed by a condenser (not shown) connected to the vacuum system.
• the lipid which is left behind as the solvent is removed, forming liposomes ranging in size between about 0.1 to 20 microns in diameter.
• lipid-in-solvent/buffer mixture injection coupled with solvent removal continues until the lipid concentration in the aqueous phase reaches the desired level, whereupon valve 80 is adjusted to divert a fraction of the fluid supplied from pump 78 to the down ⁇ sizing filter subsystem. A balance is struck so that the same volume of buffer entering through the injection system is diverted to the down-sizing filter subsystem. _ ? _
• the liposomes formed during the injection phase in the vacuum chamber are sized by passage (extrusion) through filter 84.
• the suspension of reduced-size liposomes is combined with the fluid circulating through the diafiltration system by adjusting valve 86.
• liposomes smaller than the pore size of the diafiltration filter enter the filtrate and are delivered to the mixing chamber following passage through valve 96 and valve 70.
• the filtrate is exposed to the lipid-in-solvent solution being introduced from tank 62.
• the solvent content in the mixing chamber is sufficient to extract the lipid from the small liposomes present in the filtrate.
• This lipid is combined with the lipid entering in the lipid-in-solvent solution and is injected into the lipid- in-solvent/buffer injection and solvent removal subsystem for formation into new liposomes.
• fresh buffer may be introduced from tank 64 into the filtrate material, to offset the loss of volume in the retentate from the diafiltration filter, or, alternatively, by not adding additional buffer to the filtrate, the system can be operated in a mode which concentrates the liposome suspension during operation.
• valve 96 is adjusted to divert the filtrate from the diafiltration filter to a drain and valve 98 is adjusted to deliver the same volume of fresh buffer to tank 84 as is being removed by diafiltration.
• the final concentra ⁇ tion phase of operation follows the procedure used during diafiltration, with the exception that valve 98 is closed so that no fresh buffer is added.
• the product in tank 88 becomes progressively more concentrated as filtrate is • — o —
• the process is terminated when the liposome suspension reaches the desired concentration.
• One liposome-based diagnostic test is based on complement-mediated liposome lysis, in response to the presence or absence of an analyte of interest.
• the liposomes used in the test contain a surface bound ligand which is designed to bind to or compete with the analyte of interest, and an encapsulated reporter, such as a fluorophore, chromophore, or enzyme, whose release from the lysed liposomes can be quantitated to determine the concentration of analyte in the reaction sample.
• the methods described herein provide two important advantages in the preparation of liposomes suit ⁇ able for this type of assay.
• the liposomes formed are predominantly uni *-and oligolamellar, and are therefore are more sensitive to lysis than multilamellar liposomes.
• the encapsulated reporter is an enzyme
• the liposomes can be formed with little loss of enzyme, under conditions which are gener- ally non-disruptive of enzyme activity.
• Liposomes having sizes less than about 0.4 microns and and predominance of liposome-entrapped drug are ideally suited for parenteral administration of the drug, either by intravenous or intramuscular route.
• the present invention provides a simple, efficient method for preparing liposome suspensions of this type.
• encapsulation rates of up to about 75%, in the case of unsized liposomes, and up to about 55%, in the - case of concurrently sized liposomes, for a water-soluble compound can be achieved without additional sizing and dehydration processing.
• the high encapsulation efficiency is especially advantageous in forming liposomes with en- capsulated peptides or hormones, since the total amount of non-encapsulated material is relatively small, thus avoid ⁇ ing or reducing compound-recovery problems.
• the ability to produce sized liposomes at high concentration may be an added advantage in some parenteral uses.
• An example is the use of liposomes for controlled drug releasd from an intramuscular site, as reported in co-owned U.S. patent application for "Controlled-Release Liposome Delivery System", Serial No. 828,153, filed 2/10/ 86.
• the system is based in part on the discovery that release of encapsulated drug from administered liposomes can be increased in a controlled manner by increasing the amount of liposome lipid injected into the site. It can be appreciated that the high concentration formulations produce by the present nvention have the potential for greater release times than prior art formulations.
• paste formulations provide a ideal storage form for liposomes where the entrapped drug is water-soluble and liposome-permeable, e.g., where drug equilibration of, the drug between encapsulated and bulk aqueous compartments occurs.
• the present invention provides a simple means for forming such a paste directly, or with little additional water removal.
• the paste is diluted, preferably to between about 10-30 volume percent, and the diluted suspension is atomized in a form suitable for inhalation, before significant drug equilibration can occur.
• the liposomes can be administered in dry, particulized form, also as detailed in the above- cited co-owned patent application.
• the liposomes are prepared in the presence of a bulking agent, such as mannitol, sorbitol, or the like r dried by dehydration, and particulized, for example, by grinding.
• the particles can be atomized in powdered form or suspended in a chlorofluorocarbon solvent for atomization.
• the liposome paste formulations which are producible by the invention are suited for use .as topical ointments and creams, without additional processing.
• the liposomes when applied topically, can provide controlled release of a variety of topical medications, such as anti ⁇ bacterial or anti-fungal agents, and steroids, and can also serve as a source of moisturizing lipids.
• the paste formula- tions would provide advantages of enhanced retention at the ocular surface, by virtue of the high viscosity of the material, and controlled drug release, by virtue of the high percentage of encapsulated drug.
• PC Phosphatidylcholine
• CH cholesterol
• PG phosphatidylglycerol
• PC 55 mole percent
• cholesterol 45 mole percent
• PG PG (5 mole percent) were dissolved in 270 ml of Freon, to a final lipid concentration of 500 um/ml.
• the processing system illustrated in Figure 1 and described above in Section IA was used for liposome preparation, except that in-line extrusion was not employed.
• the lipid-in-solvent solution (270 ml) was placed in the solvent feed tank and equilibrated to 4 C by means of the circulating temperature bath.
• the aqueous CF solution (270 ml) was placed in the hydration tank and equilibrated to 20°C.
• the lipid solution was infused into the hydra ⁇ tion tank at a flow rate of 2.7 ml/minute; i.e., at 1 ml/ min/100 ml of aqueous medium, with the speed control mixer in the hydration tank set at about 850 revolutions/ minute.
• the tank was maintained at negative pressure (about 200 mbar), and the solvent that boiled off was condensed and fed into a solvent recovery tank. The amount of solvent in the tank quickly reached equilibrium.
• an aliquot of the liposome suspension in the hydration tank was drawn off for assaying (a) lipid concentration, (b) liposome size distribution, and (c) encapsulation ef ⁇ ficiency.
• the suspension samples were withdrawn at times corresponding to calculated lipid concentrations, in the liposome suspension, of 50, 100, 150, 200, 250, 300, 350, and 400 um/ml, i.e., about every 12.5 minutes duting the solvent infusion process.
• the lipid concentration of the samples was determined by the method of Bartlett. Liposome sizes and size distributions were determined from electron photomicrographs. The suspensions were fixed and stained conventionally, and embedded in an epoxy block for sectioning. To determine encapsulation e ficiencies, the samples were pelleted by ultracentrifugation, and the liposome pellets resuspended to the pre-centrifugation volume in CF-free Tris buffer. The resuspended liposomes were solubilized by the addition of volumes of 10 % Triton-X 100, and the CF concentration was determined by fluorescence emission, at an excitation wavelength of 492 nm, and an emission wavelength at 520 nm.
• the morphologic appearance of the liposomes at sample concentrations of 50, 100, 150, and 200 um/ml are shown in Figures 4A-4B.
• the liposomes are quite heterodisperse, with sizes ranging from submicron sizes to 10 microns or greater.
• the reduc- tion of larger liposomes to more uniform sizes of about 1.5 microns or larger is seen.
• the suspension is substantially free of liposomes with sizes greater than about 1.5 microns.
• a second test examined encapsulation efficien- cies in the lipid concentration range 100-400 um/ml, i.e., the highest concentration being reached by infusing all of the lipid-in-solvent solution into the aqueous medium.
• the suspension had a paste ⁇ like consistency that would make solvent infusion of further lipid material difficult.
• increasing the lipid concentration further enhances encapsulation ef ⁇ ficiency, up to about 66% efficiency.
• the encapsulation efficiencies of the extruded liposomes were determined as above, by pelleting the liposomes, resuspending in CF-free buffer, and comparing total CF concentration in the liposomes with that in the original aqueous suspension.
• PHSPC Partially hydrogenated soy phosphatidylcholine
• PHSPC 67 mole percent
• cholesterol 33 mole percent
• alpha-T 0.1 mole percent
• the aqueous medium in the system was 50 ml of a solution of 125I-labelled calcitonin (200 ug/ ml) in acetate buffer (4g/l sodium acetate), pH 4.2.
• the solvent-infusion processing was carried out exactly as described in Example I, with the lipid solution being infused into the hydration tank at a flow rate of about 0.5 ml/minute, i.e., 1 ml/min/100 ml of aqueous medium.
• the final concentration of liposomes in the suspension, at the end of the processing, was 288 umole/ ml.
• Aliquots of the final suspension material were diluted 1:10, 1:20, or 1:40 with drug-free buffer and vortexed to obtain a uniform dispersion of the liposomes.
• the total recovery of radioactive material shown for two separate preparations in Table 3 below, indicates substantially 100% recovery of calcitonin material at each of the three dilutions.
• the diluted samples were centrifuged at high speed to pellet the liposomes, and the radioactivity in the pellet was measured and compared with the total radio ⁇ activity contained in the sample. The ratio of the two values was used to calculate percent encapsulation of calcitonin. The results are shown in Table 3 for each of the two preparations examined. As seen, the average percent encapsulation measured for each preparation was greater than about 60%. -
• the size distribution of the liposomes in the preparations was quite heterodisperse, showing a size range f om about 0.1 to 10.0 microns. This result indicates the degree of liposome size heterogeneity which can be expected in the method in the absence of charged lipid components. By contrast, where charged lipid components are used, as in Example I, maximum liposome sizes of about 1.5 microns can be expected.
• the solvent-infusion processing was carried out exactly as described in Example I, with the lipid solution being infused into the hydration tank at a flow rate of about 0.5 ml/minute, i.e., 1 ml/min/100 ml of aqueous medium.
• the final concentration of liposomes in the suspension, at the end of the processing, was 288 umole/ ml.
• An aliquot of the final suspension material was diluted 1:5 with buffer and vortexed to obtain a uniform dispersion of the liposomes.
• the diluted sample was centrifuged at high speed to pellet the liposomes, and the total radioactivity in the pellet was measured and compared with the total radioactivity contained in the sample.
• the ratio of the encapsulated to total material was about 75%.
• Suspensions of liposomes were prepared as in example I, with the following two modifications: (1) The lipid-in-solvent solution contained 500 um/ml, and (2) the aqueous liposome suspension in the hydration tank was circulated through the filter extrusion line in the system shown in Figure 1.
• This line includes a high-pressure pump and a sealed membrane chamber which here was equipped with a 0.4 micron polycarbonate filter.
• the pressure on the upstream side of the filter chamber was maintained at about psi, giving a flow rate, at the initial stages of operation, of about ml/minute.
• the liposome suspension was sufficiently fluid, even at the highest lipid concentra ⁇ tion (about 500 um/ml) to allow continued infusion.
• the method also allowed for extrusion of a more concentrated lipid suspension, i.e., 500 um/ml vs 350 um/ml, when the extrusion is carried out following liposome preparation by solvent infusion.
• the encapsulation efficiencies of the liposome material at various concentrations is shown in Table 4.
• the data show that concentrated formulations having (a) sizes less than about 0.4 microns, and (b) encapsulation efficiencies above 50% can be made directly by the method.

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