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1. FIELD OF THE INVENTION
The present invention relates to methods for preparing liposome suspensions
characterized by high encapsulation efficiencies and high lipid
concentrations.
2. REFERENCES
Cafiso, D. S., Biochim Biophys Acta 649:129 (1981).
Deamer, D., et al, Biochim Biophys Acta 443:629 (1976).
Gabizon, A., et al, Cancer Research 42:4734 (1982).
Poznansky, M. L., et al, Pharm Revs 36(4):277 (1984).
Schieren, H., et al, Biochim Biophys Acta 542:137 (1978).
Szoka, F. Jr., et al, Proc Nat Acad Sci (USA) 75:4194 (1978).
Szoka, F. Jr., et al, Ann Rev Biophys Bioeng 9:467 (1980).
3. BACKGROUND OF THE INVENTION
Liposomes provide several advantages in drug delivery. When administered
parenterally, either by the intravenous or intramuscular route, liposomes
can 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 allowing less
frequent drug administration. Liposome drug delivery systems are reviewed
in Poznansky.
The use of liposomes for drug delivery by inhalation has also been studied,
as reported in co-owned U.S. patent application for "Liposome Inhalation
and Method", Ser. No. 737,221, filed May 22, 1985, and now abandoned. 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.
The compatibility of 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",
Ser. No. 890,815, filed July 28, 1986.
Several methods for preparing liposomes with entrapped drug are known. In
one method, 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 multilamellar
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 intravenously (Gabizon).
One of the drawbacks of the MLV method is relatively poor encapsulation
efficiency of water-soluble drugs. Typically when the vesicles are
prepared by addition of an aqueous drug solution, only about 5-15% of the
total drug added to the lipid film is encapsulated in the vesicles,
Liposome sizing, if needed, reduces the percentage of free drug still
more, since liposome sizing methods generally result in some loss of
encapsulated material.
Alternative methods for preparing liposomes with higher encapsulation
efficiencies have been reported. One of these is a solvent injection, in
which a lipid-in-solvent solution is injected into an aqueous medium
(Deamer, Schieren, Cafiso). The method produces relatively uniform
unilamellar vesicles with encapsulation efficiencies (trapping volumes) of
between about 20-45 percent. The higher trapping volumes are presumably
related to formation of relatively large unilamellar structures.
Increased encapsulation efficiencies can also be achieved in a reverse
evaporation phase method of liposome preparation (Szoka, 1978, 1980). Here
a 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. Encapsulation efficiencies for water-soluble compounds are
typically between about 30-50 percent of the compound present in the
original aqueous medium.
In both the solvent-injection and REV procedures, it may be necessary to
reduce liposome sizes, to permit liposome sterilization by filtration
and/or to improve the targeting properties of the liposomes. As with MLVs,
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 undesired side effects when administered in
free form. The benefit of administering a water-soluble drug in
predominantly liposomal form is illustrated in co-owned patent application
for "Liposome Inhalation Method and System", Ser. No. 737,221, filed May
22, 1985, and now abandoned. Here it was shown that the systemic side
effects of metaproteranol sulfate (MPS) were substantially reduced when
the drug was delivered by inhalation in predominantly
liposome-encapsulated form.
In the case of water-soluble drugs, where known liposome preparation
methods yield at best 30-50% encapsulation, higher encapsulation levels
(above 50% encapsulated drug) can be achieved by treating the liposomes to
remove free drug. This can be done, conventionally, by molecular sieve
chromatography, centrifugation, or diafiltration. In all of these methods,
the bulk phase suspension medium containing the free drug is replaced by
drug-free bulk medium.
One drawback of this approach is the additional processing required to
remove free drug and, if desired, reclaim the removed drug. A second
limitation, in the case of a water-soluble, liposome-permeable drug, is
that the liposome composition must be administered before the drug can
re-equilibrate between encapsulated and bulk-phase compartments in the
suspension. The second problem has been addressed in co-owned patent
application for "Liposome Concentrate and Method", Ser. No. 860,528, filed
May 5, 1986, and now abandoned. According to this invention, 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 suspension 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. Despite its advantages, the
liposome paste approach involves loss of free drug material, and
additional processing of the liposome suspension.
4. SUMMARY OF THE INVENTION
It is a general object of the invention to provide a liposome processing
method which largely overcomes above-discussed problems and limitations of
prior art liposome-preparation methods.
More specifically, it is an object of the invention to provide a method for
producing liposomes in which the efficiency of encapsulation of a
water-soluble compound is at least about 50% and up to 70% or higher.
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 additional 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 predominantly--that is,
more than 50%--in liposome-encapsulated form. In practicing the method, a
solution of vesicle-forming lipids in a chlorofluorocarbon solvent are
infused in liquid form into an aqueous medium, under pressure,
temperature, and agitation conditions at which lipid frothing is largely
prevented, and at an infusion rate that produces predominantly
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 .mu.m/ml. The infused solvent is removed at
substantially the same rate that it is introduced, and is removed
completely when the selected lipid concentration is reached.
With continued solvent infusion up to a final liposome concentration of at
least about 250 .mu.m/ml, trapping efficiencies of between about 60-70
percent can be achieved. The high-concentration suspension is suitable for
use as a liposome paste, e.g., as a storage form for drug-containing
liposomes, may be readily converted to a paste by additional water
removal, or diluted with drug-free buffer to a desired liposome
concentration.
In another aspect, the invention includes a method of preparing a
concentrated liposome suspension having a lipid concentration of greater
than about 200 .mu.m/ml and liposome sizes no greater than about 0.6
microns. In this method, 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
predominantly oligolamellar vesicles. The compound to be encapsulated is
dissolved either in the aqueous medium or in the chlorofluorocarbon
solvent. During the infusing step, 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 .mu.m/ml--and liposome size range is reached.
The concentrated material may be sterilized by filtration through a 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 predomnantly encapsulated form.
In yet another aspect, 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. By continual filtration, recirculation, and
infusion mixing 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.
These and other objects and features of the invention will become more
fully apparent when the following detailed description of the invention is
read in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates in diagrammatic form, a lipid processing system used in
practicing the invention;
FIG. 2 illustrates additional components in the FIG. 1 system for use in
sizing liposomes by extrusion during liposome preparation;
FIG. 3 illustrates portions of an alternate embodiment of a liposome
processing system, for use in preparation of liposomes having defined size
ranges;
FIG. 4 shows plots of encapsulation efficiency as a function of lipid
concentration in two different processing methods carried out according to
the invention; and
FIGS. 5A-5D are representations of photomicrographs of liposomes taken
during a liposome preparation method at lipid concentrations of 50
.mu.m/ml (4A), 100 .mu.m/ml (4B), 150 .mu.m/ml (4C), and 200 .mu.m/ml
(4D).
DETAILED DESCRIPTION OF THE INVENTION
I. High-Encapsulation Processing
A. Processing System
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.degree. C. and 45.degree. 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. Thus if
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 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.
B. Processing Components
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.degree.-10.degree. C. As
defined herein 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" (CCl.sub.3 F), "Freon 12" (CCl.sub.2 F.sub.2), "Freon 21"
(CHFCl.sub.2), "Freon 22" (CHClF.sub.2), "Freon 113" (CCl.sub.2
FCClF.sub.2), "Freon 114" (CClF.sub.2 CClF.sub.2), and "Freon 115"
(CClF.sub.2 CF.sub.3). A preferred solvent is trichlorofluoromethane
("Freon 11"), whose boiling point is 23.8.degree. C. at 1 atm, or a
mixture of trichlorofluoromethane and dichlorofluoromethane ("Freon 21"),
whose boiling point is 8.9.degree. C. at 1 atm. Where the compound to be
encapsulated cannot be included in the aqueous medium used in forming the
suspension, and is not readily soluble in a pure chlorofluorocarbon
solvent, 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 amount 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 may be suitable. However, experiments conducted in
support of the present invention indicate that the presence of between
about 5-10% charged lipid, such as phosphatidylglycerol (PG), leads to
smaller, more uniformly sized liposomes during the coarse of liposome
formation. 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.
In addition, 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. 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. As indicated above, 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. As an example, studies conducted in support of the present
invention, and discussed below, show that the water-soluble compound
propranolol causes liposome disruption when originally dissolved in the
aqueous medium used in the solvent injection method. However, when
dissolved in the lipid solvent (Freon 11:ethanol, 10:1), liposomes with
very high encapsulated propranolol are formed.
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. As will be seen below,
the minimum concentration of lipids in the final liposome suspension is
about 150 .mu.m/ml, and 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. Preferably, the lipid
solution is made up to between about 200-700 .mu.m/ml. Here it is noted
that 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 aqueous 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
inhalation, 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,
cemetidine, naphazoline, lodoxamide, and phenylepinephrine, exemplary of
relatively small compounds that may may be diffusable through liposome
bilayer membranes. 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 preferably that which is
desired in the encapsulated volume in the liposomes.
In addition, 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.
C. Processing Operation
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. Initially, the lipid solution is added to feed tank 18, and the
aqueous medium, to chamber 12, and the two solutions are equilibrated, by
temperature baths 26, 14, respectively, to chamber and tank temperatures,
above and below the boiling point of the lipid solvent, at the selected
pressure. Preferably, where the lipid solvent in "Freon 11", the lipid
solvent and aqueous medium are equilibrated to and maintained during
operation at about 4.degree. C. and 20.degree. C., respectively.
With the mixer operating a preferred speed of between about 850 revolutions
per minute, and the vacuum in the mixing chamber set at between about 200
and 400 mbar, pump 22 is activated to supply the cooled lipid solvent into
the aqueous medium contained in the mixing chamber. As noted above, 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 encapsulation
volume per unit lipid. If the infusion rate is too rapid, the lipid
material tends to froth, with loss of lipid material and poor
encapsulation. Frothing can also be caused by over-rapid removal of
solvent vapor from the chamber. Therefore if frothing is observed, and the
solvent infusion rate is no greater than that noted above, the vacuum in
the system should be reduced until frothing is largely eliminated.
Under the processing conditions described above, 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. FIG. 5A shows a typical field of liposomes formed when the total
lipid concentration in the mixing chamber has reached 50 .mu.m/ml. The
larger liposomes seen in the figure are between about 10-15 microns, and
the smaller ones, about 1.5 microns or less. Determination of the percent
of encapsulated water-soluble material, according to methods described in
Example I, show that the total entrapped volume in the 50 .mu.m/ml
preparation is between about 30-35% (Example I).
According to an important aspect of the invention, continued addition of
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 .mu.m/ml or greater. The general increase in
encapsulation efficiency, as a function of lipid concentration is seen in
FIG. 4. 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 encapsulated 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.
Where 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 FIGS. 5B-5D, which show the general appearance of the
liposome suspension at 100, 150, and 200 .mu.m/ml, respectively. At 100
.mu.m/ml lipid, the general size reduction with respect to the suspension
at 50 .mu.m/ml is easily seen, and at 150 .mu.m/ml lipid concentration,
almost all of the liposomes are about 1.5 microns or smaller. Further
lipid increase to 200 .mu.m/ml did not significantly change the liposome
size distribution. Thus, in addition to a high encapsulation efficiency,
the method of the invention produces a relatively homogeneous size
distribution of liposomes with maximum liposomes sizes less than about 1.5
microns.
It is noted that the gradual decrease in liposome size seen in the method
is related to the presence of charged lipid components, and in the case of
Example I, to the presence of 5 mole percent phosphatidylglycerol (PG).
Examples III and IV below describe solvent unfusion methods involving
uncharged lipids, either PC alone or PC and cholesterol. In both examples,
final liposome sizes were heterogeneous, and between about 0.1 and 10
microns.
The lipsosome suspension becomes quite viscous at a lipid concentration
greater than about 300-400 .mu.m/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 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. Alternatively, the liposomes may be filter sterilized before
in vivo administration. Here the liposomes must be further sized down to
maximum sizes of about 0.4 microns. In a preferred sizing method, 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 U.S. patent
application for "Liposome Extrusion Method", Ser. No. 829,710, filed Feb.
28, 1986 and now U.S. Pat. No. 4,737,323, 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 .mu.m/ml. Table 2 in the example shows the
encapsulation 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 .mu.m/ml, is about 45%. Thus the combined
liposome preparation method and subsequent extrusion is limited in lipid
concentration to about 350 .mu.m/ml, and encapsulation efficiency to less
than 50%. These limitations are overcome in the high-concentration method
described below.
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 .mu.mole/ml. As described in the
example, 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 11":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). It is also noted
that although propranolol is itself soluble in water, attempts to prepare
the liposomes by injecting a lipid solution into an aqueous solution of
the propranolol were unsuccessful, apparently because of the ability of
the propanolol to act as a detergent and disrupt the nascent liposomes
being formed during liposome formation.
The infusion process was continued to a final lipid concentration of about
300 .mu.mole/ml, at which about 75% of the drug was encapsulated in the
liposomes formed. As in the Example III preparation, which also involved
uncharged lipid components, 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 suspension does not appear to
effect liposome stability or reduce encapsulation efficiency.
II. High-Concentration Processing
A. Processing System and Components
The high-concentration method is designed for producing a liposome
suspension having (a) a lipid concentration preferably above about 200
.mu.m/ml, and up to about 500 .mu.m/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. When produced in the
presence of a water-soluble compound, the liposomes have an encapsulation
efficiency of up to 50-60%.
FIG. 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 FIG. 2, contains all of the components of system 10
which are shown in FIG. 1, including mixing chamber 12, surrounding
water-jacket 16, and mixing blade 32 seen in FIG. 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.
In one embodiment of the system, the extrusion device is a filter device
equipped with a 0.2-0.6 micron pore size polycarbonate filter, of the type
noted in Section IC. This method is effective to size liposomes
approximately to the largest filter pore size, which may be selected from
a pore size of 0.1 micron up to 2 micron or larger.
In another embodiment, 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 efficient
liposome sizing has been described in the above-cited U.S. patent
application for "Liposome Extrusion Method". According to an important
aspect of that invention, it was discovered that liposomes processed in an
inside-to-outside direction through a 1 micron ceramic filter were reduced
to the desired sizes below about 0.4 microns by only one or a few passes
through the membrane.
The ceramic filter device is advantageous in the present invention for the
reasons that (a) high extrusion pressures can be used, to achieve higher
processing rates, (b) a multiple filter cartridge can handle relatively
large volumes in a scale-up operation (c) membrane clogging can be avoided
by periodically operating the device in a reverse (outside-to-inside)
direction, and (d) the device can be sterilized in place by high
temperature or chemical treatment.
The high-concentration processing method uses lipid-in-solvent and aqueous
medium components like those used in in the high-encapsulation method
described in Section I. The aqueous medium may, but does not necessarily
contain a water-soluble pharmaceutical compound for encapsulation 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
solution), or an encapsulated water-soluble compound, or both.
B. Processing Operation
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 .mu.m/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. As indicated above, the
shunt is preferably operated at a flow rate which processes the entire
volume of the suspension in about 20 minutes or less. More generally, the
shunt is operated at a flow rate which allows about 5-10 passes of aqueous
volume during the infusion process.
Accordinq to an important feature of the method, the integrated liposome
sizing step in the method significantly reduces the viscosity of the
suspension, at higher lipid concentrations, and this feature allows
infusion of additional lipid into the suspension to levels which are
substantially higher than those achievable without integrated sizing. As
seen in Example V, lipid infusion can be continued up to a final lipid
concentration of about 500 .mu.m/ml, at which the encapsulation efficiency
for water-soluble compounds is greater than 50%. Without concurrent
sizing, the vesicle suspension would be too viscous to extrude above a
lipid concentration of about 300 .mu.m/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 .mu.m/ml, (b)
liposome sizes less than about 0.2 microns, and (c) an encapsulation
efficiency of trapped water-soluble material of about 55%. As in Examples
I and II, 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
FIG. 4. As seen, the extrusion process produces lower encapsulation
efficiencies, at comparable lipid concentrations than the system described
in Section I. However, because the method allows for higher lipid
concentrations, encapsulation efficiencies above 50% can be achieved. As
with the preparation described in Section I, greater encapsulation
efficiency (for liposome-permeable compounds) can be achieved by
additional dehydration, such as described in Section I.
III. Uniform Size Processing
This section describes a system and method for producing a suspension of
liposomes which are predominantly 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.
A. Processing System
FIG. 3 shows a system 60 desiqned for producing uniform-size liposomes
according to this aspect | | |
