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1. FIELD OF THE INVENTION
The present invention relates to methods and compositions for the
entrapment of compounds in liposomes composed of salt forms of organic
acid derivatives of sterols that are capable of forming bilayers.
Sterols such as cholesterol or other lipids, to which a hydrophilic moiety
such as a salt form of an organic acid is attached, can be used to prepare
suspensions of multilamellar or small unilamellar vesicles. The sterol
liposomes of the present invention may be prepared with or without the use
of organic solvents. These vesicles may entrap water-soluble compounds,
partially water-soluble compounds, and water-insoluble compounds.
The sterol vesicles described herein are particularly useful for the
entrapment of biologically active compounds or pharmaceutical compounds
which can be administered in vivo. Alternatively, the sterol liposomes of
the present invention may be used in vitro. For instance, the cholesterol
hemisuccinate liposomes described herein may be used in vitro in divalent
cation-dependent assay systems.
2. BACKGROUND OF THE INVENTION
2.1. LIPOSOMES
Liposomes are completely closed bilayer membranes containing an
encapsulated aqueous phase. Liposomes may be any variety of multilamellar
vesicles (onion-like structures characterized by concentric membrane
bilayers each separated by an aqueous layer) or unilamellar vesicles
(possessing a single membrane bilayer).
Two parameters of liposome preparations are functions of vesicle size and
lipid concentration: (1) Captured volume, defined as the volume enclosed
by a given amount of lipid, is expressed as units of liters entrapped per
mole of total lipid (1 mol.sup.-1). The captured volume depends upon the
radius of the liposomes which in turn is affected by the lipid composition
of the vesicles and the ionic composition of the medium. (2) Encapsulation
efficiency, defined as the fraction of the aqueous compartment sequestered
by the bilayers, is expressed as a percentage. The encapsulation
efficiency is directly proportional to the lipid concentration; when more
lipid is present, more solute can be sequestered within liposomes. (See
Deamer and Uster, 1983, Liposome Preparation: Methods and Mechanisms, in
Liposomes, ed. M. Ostro, Marcel Dekker, Inc., N.Y., pp. 27-51.)
The original method for liposome preparation (Bangham et al., 1965, J. Mol.
Biol. 13: 238-252) involved suspending phospholipids in an organic solvent
which was then evaporated to dryness leaving a waxy deposit of
phospholipid on the reaction vessel. Then an appropriate amount of aqueous
phase was added, the mixture was allowed to "swell," and the resulting
liposomes which consisted of multilamellar vesicles (hereinafter referred
to as MLVs) were dispersed by mechanical means. The structure of the
resulting membrane bilayer is such that the hydrophobic (non-polar)
"tails" of the lipid orient toward the center of the bilayer while the
hydrophilic (polar) "heads" orient towards the aqueous phase. This
technique provided the basis for the development of the small sonicated
unilamellar vesicles (hereinafter referred to as SUVs) described by
Papahadjopoulos and Miller (1967, Biochim. Biophys. Acta. 135: 624-638).
Both MLVs and SUVs, however, have limitations as model systems.
In attempts to increase captured volume or encapsulation efficiency a
number of methods for the preparation of liposomes comprising physpholipid
bilayers have been developed; however, all methods require the use of
organic solvents. Some of these methods are briefly described below.
An effort to increase the encapsulation efficiency involved first forming
liposome precursors or micelles, i.e., vesicles containing an aqueous
phase surrounded by a monolayer of lipid molecules oriented so that the
polar head groups are directed towards the aqueous phase. Liposome
precursors are formed by adding the aqueous solution to be encapsulated to
a solution of polar lipid in an organic solvent and sonicating. The
liposome precursors are then emulsified in a second aqueous phase in the
presence of excess lipid and evaporated. The resultant liposomes,
consisting of an aqueous phase encapsulated by a lipid bilayer are
dispersed in aqueous phase (see U.S. Pat. No. 4,224,179 issued Sep. 23,
1980 to M. Schneider).
In another attempt to maximize the encapsulation efficiency,
Papahadjopoulos (U.S. Pat. No. 4,235,871 issued Nov. 25, 1980) describes a
"reverse-phase evaporation process" for making oligolamellar lipid
vesicles also known as reverse-phase evaporation vesicles (hereinafter
referred to as REVs). According to this procedure, the aqueous material to
be encapsulated is added to a mixture of polar lipid in an organic
solvent. Then a homogeneous water-in-oil type of emulsion is formed and
the organic solvent is evaporated until a gel is formed. The gel is then
converted to a suspension by dispersing the gel-like mixture in an aqueous
media. The REVs produced consist mostly of unilamellar vesicles (large
unilamellar vesicles, or LUVs) and some oligolamellar vesicles which are
characterized by only a few concentric bilayers with a large internal
aqueous space.
Much has been written regarding the possibilities of using liposomes for
drug delivery systems. See, for example, the disclosures in U.S. Pat. No.
3,993,754 issued on Nov. 23, 1976, to Yeuh-Erh Rahman and Elizabeth A.
Cerny, and U.S. Pat. No. 4,145,410 issued on Mar. 20, 1979, to Barry D.
Sears. In a liposome drug delivery system the medicament is entrapped
during liposome formation and then administered to the patient to be
treated. The medicament may be soluble in water or in a non-polar solvent.
Typical of such disclosures are U.S. Pat. No. 4,235,871 issued Nov. 25,
1980, to Papahadjopoulos and Szoka and U.S. Pat. No. 4,224,179, issued
Sep. 23, 1980 to M. Schneider. When preparing liposomes for use in vivo it
would be advantageous (1) to eliminate the necessity of using organic
solvents during the preparation of liposomes; and (2) to maximize the
encapsulation efficiency and captured volume so that a greater volume and
concentration of the entrapped material can be delivered per dose.
2.2. WATER-SOLUBLE STEROLS
A variety of sterols and their water soluble derivatives have been used for
cosmetic, pharmaceutical and diagnostic purposes. Of the water soluble
sterols, for example, branched fatty acid cholesterol esters, steroid
esters and PEG-phytosterols have been used in cosmetic preparations
(European Patent Application No. 28,456; U.S. Pat. No. 4,393,044; and
Schrader, Drug and Cosmetic Industry, September 1983, p.33 and October
1983, p.46). Thakkar and Kuehn (1969, J. Pharm. Sci. 58(7): 850-852)
disclose the solubilization of steroid hormones using aqueous solutions of
steroidal non-ionic surfactants, specifically ethoxylated cholesterol
(i.e., PEG-cholesterol) at a concentration of 1-5%. However, the
effectiveness or utility of the solubilized steroid hormones in -vivo was
not demonstrated. A number of water soluble cholesterols have been
prepared and used as water-soluble standards for the determination of
cholesterol levels in biological fluids (U.S. Pat. Nos. 3,859,047;
4,040,784; 4,042,330; 4,183,847; 4,189,400; and 4,224,229). Shinitzky et
al. (1979, Proc. Natl. Acad. Sci. USA 76:5313-5316) incubated tumor cells
in tissue culture medium containing a low concentration of cholesterol and
cholesteryl hemisuccinate. Incorporation of cholesterol or cholesteryl
hemisuccinate into the cell membrane decreased membrane fluidity and
increased membrane-lipid microviscosity.
Cholesterol and other sterols, have also been incorporated into
phospholipid liposome membranes in order to alter the physical properties
of the lipid bilayers. For example, in a recent abstract, Ellens et al.
(1984, Biophys. J. 45: 70a) discuss the effect of H.sup.+ on the stability
of lipid vesicles composed of phosphatidylethanolamine and cholesteryl
hemisuccinate. In fact, Brockerhoff and Ramsammy (1982, biochim. Biophys.
Acta. 691:227-232) reported that bilayers can be constructed which consist
entirely of cholesterol, provided a stabilizing hydrophilic anchor is
supplied. Multilamellar and unilamellar cholesterol liposomes were
prepared in a conventional manner described above evaporating to dryness
the cholesterol derivatives (i.e., cholesterol-phosphocholine,
cholesterol-polyethylene glycol, or cholesterol-SO.sub.4) dispersed in an
organic solvent leaving a lipid film deposited in the reaction vessel. The
lipid films were sonicated under 2 ml water using an ultrasonic
homogenizer with a microtip. Formation of multilamellar vesicles required
10 minutes sonication, whereas formation of small unilamellar vesicles
required 4 hours of sonication. The resulting suspensions of multilamellar
liposomes were milky whereas the suspensions of unilamellar liposomes were
transparent.
However, the ability to efficiently entrap bioactive agents in sterol
vesicles which are suitable for administration in vivo to provide for the
administration of higher doses of water-soluble agents and to facilitate
the administration of water-insoluble agents has not heretofore been
explored.
3. SUMMARY OF THE INVENTION
The present invention involves methods and compositions for the entrapment
of various compounds in liposomes, the bilayers of which comprise salt
forms of organic acid derivatives of sterols. Entrapment of a compound is
defined herein as the encapsulation of a water-soluble compound in the
aqueous compartment of the liposome or the entrapment of a water-insoluble
compound within the sterol bilayer. The tris(hydroxymethyl)aminomethane
salt (tris-salt) form of organic acid derivatives of sterols are
particularly useful as the vesicle bilayer ingredient.
The method for preparing the sterol vesicles involves adding to an aqueous
buffer a salt form of an organic acid derivative of a sterol capable of
forming closed bilayers in an amount sufficient to form completely closed
bilayers which entrap an aqueous compartment. A suspension of
multilamellar vesicles is formed by shaking the mixture. The formation of
vesicles is facilitated if the aqueous buffer also contains the counterion
of the salt in solution. Furthermore, if the dissociated salt form of the
organic acid derivative of a sterol is negatively charged at neutral pH,
the aqueous buffer should be essentially free of divalent or multivalent
cations. Similarly, when the dissociated salt form of the organic acid
derivative of a sterol is positively charged at neutral pH, the aqueous
buffer should be essentially free of multivalent anions. The application
of energy to the suspension, e.g., sonication, or extrusion of the
vesicles through a French pressure cell (French Press) or through a porous
filter of the appropriate pore size, will convert the multilamellar sterol
vesicles to unilamellar vesicles.
In order to entrap a water-soluble compound, a partially water-soluble
compound or a water-insoluble compound in the sterol vesicles of the
present invention, a number of approaches are possible. Compounds which
either partition into the sterol bilayers (e.g., water-insoluble
compounds) or water-soluble compounds may be added to the aqueous phase
before formation of the vesicles in order to entrap the agent within the
vesicles during formation. Alternatively, compounds which are
water-insoluble or lipid soluble may be added to the suspension of sterol
vesicles after the vesicles are formed, in which case the compound
partitions into the sterol bilayers. In another embodiment, a
water-soluble compound and the salt-form of an organic acid derivative of
a sterol may be added to an organic solvent so that both are solubilized
(co-solubilized). The organic solvent may then be evaporated leaving a
film containing a homogeneous distribution of the water-insoluble compound
and the sterol derivative. Sterol liposomes entrapping the water-insoluble
compounds are formed when an aqueous buffer is added to the film with
shaking.
The sterol liposomes of the present invention are particularly advantageous
when used to entrap water-insoluble bioactive agents or those that are
sparingly soluble in water. This enables the administration in vivo of
water-insoluble drugs; and furthermore, it allows for the administration
in vivo of high concentrations of the water insoluble compounds, because
it allows alteration of the dose:volume ratio. The sterol vesicles of the
present invention offer similar advantages when used to entrap water
soluble bioactive agents. In addition, the sterol vesicles of the present
invention may be used in diagnostic assays in vitro.
The present invention affords a number of advantages in that the sterol
vesicles:
(1) are formed easily and rapidly;
(2) have high encapsulation efficiencies as compared with phospholipid
MLVs;
(3) do not require the use of organic solvents for their preparation
(although the sterol vesicles of the present invention can be prepared
using organic solvents); and
(4) can entrap a bioactive or pharmaceutical agent, which when administered
in vivo, is released and metabolized. The fate of the entrapped agent in
vivo depends upon the mode of administration.
The present invention is further directed to a composition comprising the
tris(hydroxymethyl)aminomethane salt of cholesteryl hemisuccinate and an
antifungal compound, particularly when the antifungal agent is miconazole,
terconazole or econazole, isoconazole, tioconazole, bifonazole,
clotrimazole, ketoconazole, butaconazole, itraconazole, oxiconazole,
fenticonazole, nystain, naftifine, amphotericin B, zinoconazole or
ciclopirox olamine. The composition can be used to treat a fungal
infection and can be administered topically including orally or
intravaginally.
The present invention includes a composition comprising the
tris(hydroxymethyl)aminomethane salt of cholesteryl hemisuccinate and a
peptide, particularly a hydrophobic peptide, human growth hormone, bovine
growth hormone, porcine growth hormone or insulin. The composition can be
administered to increase milk production or to increase or initiate growth
of a mammal.
4. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 graphically demonstrates the inverse relationship of the captured
solute (chromium) and the concentration of cholesterol hemisuccinate used
to prepare the multilamellar liposomes.
FIG. 2 represents the X-ray diffraction patterns obtained for four
different CHS-MLV preparations, those gently hydrated; (FIG. 2a) 68.9% CHS
by weight, (FIG. 2b) 59% CHS by weight and (FIG. 2c) 20.2% CHS by weight,
and (FIG. 2d) a 20.7% CHS preparation prepared by mixing the dry lipid
vortically with the buffer.
FIG. 3 represents the electron spin resonance data for CHS-multilamellar
vesicles and EPC-multilamellar vesicles.
FIG. 4 graphically demonstrates the swelling profiles of cholesterol
hemisuccinate liposomes and egg phosphatidylcholine liposomes in aqueous
buffers of various tonicity.
FIG. 5 graphically illustrates the effectiveness of indomethacin entrapped
in cholesterol hemisuccinate liposomes in reducing joint swelling when
administered intramuscularly.
FIG. 6 represents the organ distribution of .sup.14 C-diazepam administered
intravenously in mice either unencapsulated (free) or encapsulated in
CHS-SUVs.
FIG. 7 A, B and C, represent the organ distribution of .sup.51 Chromium
administered intravenously in mice either, encapsulated in CHS-MLVs (FIG.
7A) or encapsulated in EPC-SPLVs (FIG. 7B) or unencapsulated (FIG. 7C).
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention describes methods and compositions for the entrapment
of water-soluble, partially water-soluble, or water-insoluble compounds in
liposomes, the bilayers or which comprise salt forms of organic acid
derivatives of sterols that are capable of forming closed bilayers.
Accordingly, the sterol liposomes of the present invention can be prepared
to (1) entrap a water-soluble compound in the aqueous compartment; or (2)
entrap a water-insoluble compound which partitions into the sterol
bilayers; or (3) both entrap a water.TM.soluble compound and entrap a
water-insoluble compound in one liposome preparation.
Any salt form of an organic acid derivative of a sterol which is capable of
forming completely closed bilayers in aqueous solutions (i.e., liposomes)
may be used in the practice of the invention. The suitability of a
particular salt-form of an organic acid derivative of a sterol depends
upon its ability to sequester a water-soluble compound so that the
compound is not in contact with the outside environment.
To determine definitively that entrapment within the aqueous compartment of
any liposome has occurred, the following criteria have been established
(See Sessa and Weissmann, 1970, J. Biol. Chem. 245: 3295) (a) there must
be a clear separation of free from sequestered compound as assayed by gel
filtration; (b) there must be no hydrophobic or charge-charge interaction
between the outermost vesicle bilayer and the entrapped compound since
this may result in a failure to achieve separation of the free compound
from the liposomes by molecular sieving, thereby artificially increasing
the apparent sequestration or encapsulation efficiency. To eliminate this
possibility it must be shown that the water-soluble compound added to a
suspension of previously formed liposomes does not coelute with preformed
liposomes; (c) disruption of gel-filtered liposomes by use of detergents
or other membrane perturbants must induce a shift in the gel filtration
pattern of the sequestered molecule from a position coincident with the
liposome peak to one that coelutes with the free molecule.
Generally any sterol which can be modified by the attachment of an organic
acid may be used in the practice of the present invention. For example,
such sterols include but are not limited to cholesterol, vitamin D,
phytosterols (including but not limited to sitosterol, campesterol,
stigmasterol, and the like), steroid hormones, and the like.
Organic acids which can be used to derivatize the sterols include but are
not limited to the carboxylic acids, dicarboxylic acids, polycarboxylic
acids, hydroxy acids, amino acids and polyamino acids. Because the salt
forms increase the water solubility of organic acids, any organic acid may
be used to derivatize the sterols; however an advantage may be obtained if
the organic acid moiety itself is water soluble. Such water-soluble
organic acid moieties include but are not limited to water-soluble
aliphatic carboxylic acids such as acetic, propionic, butyric, valeric
acids and the like (N.B., up to four-carbon acids are miscible with water;
the five-carbon free acid is partly soluble and the longer chain free
acids are virtually insoluble); water-soluble aliphatic dicarboxylic acids
such as malonic, succinic, glutaric, adipic, pimelic, maleic and the like
(N.B., the shorter chains are appreciably more soluble in water;
borderline solubility in water occurs at C.sub.6 to C.sub.7); and
water-insoluble aromatic dicarboxylic acids such as hemimellitic,
trimesic, succinic, and the like; polycarboxylic acids; water-so hydroxy
acids such as glycolic, lactic, mandelic, glyceric, malic, tartaric,
citric, and the like (N.B., .alpha.-hydroxy acids containing a branched
chain attached to the .alpha.-carbon of the carbonyl group would be less
susceptible to hydrolysis and, therefore, advantageous in the practice of
the present invention); and any of the amino acids and polyamino acids.
The organic acid can be linked to an hydroxyl group of the sterol via an
ester or an ether bond using conventional methods (see, for example, U.S.
Pat. Nos. 3,859,047; 4,040,784; 4,042,330; 4,183,847; and 4,189,400). The
salt forms of the derivatized sterols can be prepared by dissolving both
the organic acid derivative of the sterol and the counterion of the salt
(e.g., the free base of the salt) in an appropriate volatile solvent, and
removing the solvent by evaporation or a similar technique leaving a
residue which consists of the salt form of the organic acid derivative of
the sterol. Counterions that may be used include, but are not limited to,
tris, 2-amino-2-methyl-1,3-propanediol, 2-aminoethanol, bis-tris propane,
triethanolamine, and the like to form the corresponding salt. In fact, the
free base of an ionizable bioactive agent such as miconazole free base and
the like may be used as the counterion. Thus, the bioactive agent can be
used as a counterion.
The sterol liposomes of the present invention may be prepared by adding to
an aqueous phase a salt form of an organic acid derivative of a sterol
capable of forming bilayers so that the derivatized sterol is present in
an amount sufficient to form vesicles (i.e., completely closed bilayers
containing an entrapped aqueous compartment). The preparation is then
shaken until a milky suspension of multilamellar sterol vesicles is
formed. In the preferred embodiment, the aqueous phase should contain the
salt in solution to facilitate vesicle formation. Furthermore, if the
dissociated salt form of the organic acid derivative of the sterol is
negatively charged at neutral pH, the aqueous buffer should be essentially
free of multivalent cations. Similarly, when the dissociated salt form of
the organic acid derivative is positively charged at neutral pH, the
aqueous buffer should be essentially free of multivalent anions.
In complete contrast to reported methods for multilamellar vesicle
formation (e.g., phospholipid vesicles or the cholesterol liposomes of
Brockerhoff and Ramsammy, 1982, Biochim. Biophys. Acta. 691: 227-232), the
method for the formation of the sterol multilamellar vesicles of the
solvents. Furthermore, unlike the method of Brockerhoff and Ramsammy
(supra) sonication is not necessary to form the sterol multilamellar
vesicles. In fact, sonication of the milky suspension of sterol
multilamellar vesicles of the present invention, or the use of a French
press (SLM-Aminco, Urbana, Ill.) followed by sonication may be used to
convert the milky suspension of multilamellar sterol vesicles to a clear
suspension of unilamellar sterol vesicles. Similarly, multiple extrusions
of the multilamellar sterol vesicles at moderate pressures through a
filter having a pore size of equal to or less than 100 nm in diameter can
be employed to obtain unilamellar sterol vesicles. This extrusion
technique is described in detail in co-pending application Ser. No.
622,690 filed Jun. 20, 1984 abandoned in favor of U.S. Pat. application
Ser. No. 788,017, filed Oct. 16, 1985, now abandoned in favor of U.S.
patent application Ser. No. 310,495 filed Feb. 13, 1989, now U.S. Pat. No.
5,008,050 by Cullis et al. for "Extrusion Technique for Producing
Unilamellar Vesicles" which is incorporated by reference herein.
As previously explained, the tris-salt form of any organic acid derivative
of a sterol may be advantageously used in the practice of the present
invention. For example, the tris-salt form of a sterol hemi-dicarboxylic
acid such as a sterol hemisuccinate or a mixture of sterol hemisuccinates
are particularly useful for forming the vesicle bilayers of the steroidal
liposomes to be administered in vivo. For instance, when using cholesterol
hemisuccinate, 2.5 to 700 .mu.moles of the tris-salt form may be added to
2.0 ml aqueous buffer containing Tris-HCl (tris(hydroxymethyl)aminomethane
hydrochloride) in order to form vesicles; in this case the aqueous buffer
should be essentially free of divalent or multivalent cations.
According to the present invention, the entrapment of water-soluble
compounds, water-insoluble compounds, or sparingly soluble compounds in
liposomes composed of the salt form of organic acid derivatives of sterols
may be accomplished in a number of ways:
(1) A water-insoluble compound can be added to a suspension of sterol
liposomes (either multilamellar sterol vesicles or unilamellar sterol
vesicles), which were prepared as described above using an appropriate
salt form of an organic acid derivative of a sterol. The compound is
entrapped in the liposomes because it partitions into the sterol bilayers.
This embodiment may be conveniently carried out as follows: the
water-insoluble compound may be dissolved in an appropriate organic
solvent which is then evaporated leaving a film or residue of the
compound. When an aqueous suspension of previously formed sterol liposomes
is added to the residue, the residue will be entrapped in the bilayers of
the sterol liposomes.
(2) A water-insoluble compound and the salt form of an organic acid
derivative of a sterol can both be co-solubilized in an organic solvent
which is then evaporated off leaving a film comprising a homogeneous
distribution of the water-insoluble compound and the sterol derivative. A
suspension of multilamellar sterol vesicles containing the entrapped
compound is formed with an aqueous phase is added to the film with
shaking. The multilamellar vesicles may be converted to unilamellar
vesicles as previously described.
(3) A water-soluble compound or a water-insoluble compound can be entrapped
in the sterol liposomes by adding the compound to the aqueous phase which
is used in the preparation of the sterol vesicles; i.e., the compound can
be added to the aqueous phase before or simultaneously with the addition
of the salt form of an organic acid derivative of a sterol. In this case,
a water-insoluble compound becomes entrapped when it partitions into the
bilayers during vesicle formation; whereas a water-soluble compound
becomes entrapped in the aqueous compartment of the sterol vesicles during
vesicle formation. In either case, the multilayer vesicles can be
converted to unilamellar vesicles as previously described.
(4) If the bioactive agent is ionizable, the free base of the bioactive
agent may be used as the counterion to prepared the salt form of the
organic acid derivative of a sterol. The sterol liposomes may be prepared
by any of the methods previously described herein using the bioactive
agent-salt form of the organic acid derivative of the sterol. For example,
the free base of miconazole, an anti-fungal compound, may be used to make
the salt derivatives in this embodiment of the present invention.
Using any of the four method described above, both a water-soluble compound
and a water-insoluble compound may be entrapped in one sterol liposome
preparation.
According to the methods described above for the entrapment of
water-insoluble compounds using the sterol vesicles of the present
invention, it is not required that the vesicles remain intact once a
water-insoluble compound partitions into the bilayers. In fact, it is
conceivable that once the compound partitions into the bilayers the
vesicles will be disturbed or disrupted leading to the leakage or release
of aqueous entrapped compounds.
According to one embodiment of the present invention, sterol liposomes are
prepared using the tri-salt form of cholesterol hemisuccinate as follows:
4.5 to 200 mg of the tris-salt form of cholesterol hemisuccinate is added
per ml or aqueous buffer containing 0.01M Tris-HCl, 0.14M NaCl. The
mixture is shaken and a milky suspension of cholesterol hemisuccinate
multilamellar vesicles forms. The vesicles may be pelleted by
centrifugation and washed repeatedly with the aqueous buffer. The
suspension of cholesterol hemisuccinate multilamellar vesicles (CHS-MLVs)
may be sonicated (e.g. in a bath-type sonicator) in order to form
cholesterol hemisuccinate small unimellar vesicles (CHS-SUVs).
Alternatively, the CHS-MLVs may be passed through a French pressure cell (
a French Press) at 40,000 psi or the CHs-MLVs may be passed through two
100 nm Nucleopore (TM) filters at 300-400 pa in order to form CHS-SUVs.
The cholesterol hemisuccinate vesicles (whether MLVs or SUVs) are unstable
in the presence of divalent cations; i.e. upon exposure to divalent
cations the entrapped aqueous compartment and water-soluble compounds are
released. Thus, the aqueous medium used in the preparation or during
storage of the vesicles should be essentially free of divalent cations.
The compounds which are entrapped according to the method of the present
invention may be used in various ways. For example, if the compound is a
bioactive agent, the sterol liposome entrapped compound may be
administered in vivo. This facilitates the in vivo delivery of bioactive
agents which are normally insoluble or sparingly soluble in aqueous
solutions. Entrapment in liposomes composed of the salt form of organic
acid derivatives of sterols enables ease in the administration of such
insoluble compounds at a higher dose:volume ratio. In fact, the sterol
vesicles of the present invention are particularly advantageously used in
vivo because the vesicles may be used to entrap one or more bioactive
agents for delivery in vivo. Furthermore, the vesicles of the present
invention offer an advantage over conventional lipid vesicles or liposomes
when used in vivo because they can be prepared without using organic
solvents. The fate of the entrapped agent in vivo depends upon the route
or mode of administration. For instance, when the sterol liposome
entrapped agent is administered intravenously the clearance of the agent
in vivo follows a pathway different from that of non-entrapped agent or
that of an agent entrapped in conventional liposomes composed of
phospholipids (i.e., MLVs, SUVs, REVs, LUVs). On the other hand,
intramuscular administration of the sterol liposome entrapped agent
results in a sustained release of the agent in vivo.
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