Note: Descriptions are shown in the official language in which they were submitted.
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A METHOD FOR PREPARING LIPOSOMES AND USES THEREOF
FIELD OF THE INVENTION
This invention relates to liposome technology and in particular to methods of
preparing liposomes.
LIST OF PRIOR ART
The following is a list of prior art, which is considered to be pertinent for
describing the state of the art in the field of the invention.
1. W000/09089 - Liposomal bupivacaine compositions prepared using an
ammonium sulfate gradient (Bolotin EM et al.);
2. US 4,532,089 - Method of preparing giant size liposomes
(MACDONALD ROBERT C);
3. US 5,192,549 - Method of amphiphatic drug loading in liposomes by pH
gradient (Barenholz et al.)
4. US 5,807,572 - Multivesicular liposomes having a biologically active
substance encapsulated therein in the presence of a hydrochloride (Kim S. et
al.);
5. Kim S. and Martin, G.M. Biochim. Biophys. Acta 646:1-9 (1981)i
6. US 5,723,147 - Multivesicular liposomes having a biologically active
substance encapsulated therein in the presence of a hydrochloride (Kim S. et
al.):
BACKGROUND OF THE INVENTION
Liposomes are one of the most potential drug carriers available currently.
Liposomes contain both a hydrophobic bilayer, which may encapsulate
hydrophobic
substances, and an aqueous core, which may encapsulate other substances (e.g.
hydrophilic or amphiphatic compounds).
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Liposomal encapsulation of therapeutic compounds has shown significant
promise in controlled drug delivery. For example, some lipid-based
formulations
provide a longer half-life in vivo, superior tissue targeting, or decreased
toxicity. In
efforts to develop more effective therapeutic treatments, attempts have been
made to
encapsulate a variety of therapeutic compounds in liposomes. For example, many
anticancer or antineoplastic drugs have been encapsulated in liposomes. These
include
alkylating agents, nitrosoureas, cisplatin, antimetabolites, vinca alkaloids,
camptothecins, taxanes and anthracyclines. Studies with liposomes containing
a.nthracycline antibiotics have clearly shown reduction of cardiotoxicity.
Liposomal fonnulations of drugs modify the drug pharmacokinetics of the free
drug counterpart, which is not liposome-encapsulated. For a liposomal drug
formulation, drug pharmacokinetics is largely determined by the rate at which
the
carrier is cleared from the blood and the rate at which the drug is released
from the
carrier. Considerable efforts have been made to identify liposomal carrier
compositions
that show slow clearance from the blood, and long-circulation carriers have
been
described in numerous scientific publications and patents. Efforts have also
been made
to control drug leakage or release rates from liposomal carriers, using for
example,
various lipid components or a transmembrane potential to control release.
Liposomes are prepared by many methods and the obtained vesicles may vary
significantly in terms of diameter and number of bilayers. Liposomes may be
classified
as small or large unilamellar vesicles (SUV, LUV), multilamellar vesicles
(MLV) and
multivesicular vesicles (MVV) or large multivesicular vesicles (LMVV), which
contain
several vesicles and, consequently, several separate aqueous phases [Kulkarni,
S.B., et
al. J. Microencapsu112:229-246 (1995)]. The vesicles-in-vesicles are formed
during the
preparation of multivesicular vesicles (MVV) [Szoka, F. and Papahadjopoulos,
D. Proc.
Natl. Acad. Sci. USA 75:4194-4198(1978)] and the conversion of MLVs into
freeze-
thawed vesicles (FT MLV) [Kim S. and Martin, G.M. Biochim. Biophys. Acta 646:1-
9
(1981)], which are structurally similar to MVVs [Kramer, J.M.H., et al.
Biochemistry
17:3932-3935 (1997)]. MVVs and FT MLVs encapsulate far more aqueous phase
volume than SUVs and MLVs, but the structures of MVVs and FT MLVs (LMVV) are
large, i.e. 0.5-15 m in diameter.
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The majority of the liposome preparation methods are based on either dry lipid
hydration or the evaporation of an organic solvent in which the thus dried
lipids are
added into an aqueous solution. The methods based on dry lipid hydration are
typically
multi-step processes (organic solvent evaporation from lipid solution, lipid
drying,
hydration, calibration, and possibly other steps). The methods based on the
injection of
ethanol or ether lipid solution into the buffer results in small vesicles,
useful primarily
only as model membranes [Batzri, S. and Korn, E., Biochim. Biophys. Acta
298:1015-
1019 (1973); Kramer, J.M.H., et al. Biochemistry 17:3932-3935 (1997)]. There
are
several multi-step methods based on the aqueous phase dispergation in organic,
lipid
solubilizing solvents, and on the organic solvent evaporation directly during
vesicle
formation. The reverse-phase evaporation method is the oldest of these [Szoka,
F. and
Papahadjopoulos, D. Proc. Natl. Acad. Sci. USA 75:4194-4198 (1978)]. In
accordance
with the original procedure, an aqueous phase is dispergated in an organic
solvent (ethyl
ether, halothane, chloroform, methylene chloride or other) to form a water-in-
oil
einulsion by sonicating the mixture of both of these phases. Next, the
emulsion is
transferred to a rotary evaporator, and the solvent is reinoved under reduced
pressure.
At this stage, some of the aqueous phase droplets combine and form the
environment
where buffer droplets, enveloped in lipid membrane, are suspended. Then, the
aggregates are centrifuged and the supernatant is filtered to obtain LUVs,
smaller than
the defined sizes of a filter's pores. Another method using double emulsion
characteristics is based on aqueous emulsion formation in chloroform and ethyl
ether
solutions of liposomal lipids by mechanical agitation with a shaker. Constant
bubbling
of nitrogen agitates the combined emulsions, and consequently unilamellar or
multivesicullar vesicles are formed [Kim S. and Martin, G.M. Biochim. Biophys.
Acta
646:1-9 (1981), and US Patent No. 5,723,147].
Disclosed in US Patent No. 5,723,147 are multivesicular liposomes containing
biologically active substances, the multivesicular liposomes having a defined
size
distribution, adjustable average size, adjustable internal chamber size and
number, and a
modulated rate of the biologically active substance. The process used to form
such
multivesicular liposomes comprises dissolving a lipid component in volatile
organic
solvents, adding an immiscible aqueous component containing at least one
biologically
active substance to be encapsulated, and adding a hydrochloride effective to
control the
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release rate of the biologically active substance from the multivesicular
liposome to
either or both of the organic solvents and the lipid component, making a water-
in-oil
emulsion from the two components, immersing the emulsion into a second aqueous
component, dividing the emulsion into small solvent spherules which contain
even
smaller aqueous chambers, and then removing the solvents to give an aqueous
suspension of multivesicular liposomes encapsulating biologically active
substances.
A further method of preparing LMVV is also described in W000/09089.
According to this publication liposomal bupivacaine compositions are prepared
using an
ammonium salt (e.g. sulfate) gradient loading procedure, at a pH which
prevents
precipitation of the drug from the loading solution. The liposomes may be
large
multivesicular liposomes (referred to as GMV) which are prepared by vortexing
a dry
lipid film with an aqueous solution of ammonium salt, homogenizing the
resulting
suspension to form a suspension of SW and repeatedly (at least 5 times) freeze-
thawing the suspension of SUV in liquid nitrogen followed by water.
SUMMARY OF THE INVENTION
The present invention is aimed at providing a simplified and more cost-
effective
method for preparing liposomes.
In accordance with a first of its aspects, there is provided by the present
invention a method for preparing liposomes comprising:
(a) providing a dry liposome-forming lipid or a dry mixture comprising a
liposome-forming lipid;
(b) dissolving the liposome-forming lipid or said mixture comprising a
liposome-forming lipid of step (a) with a protic organic solvent to form a
solution or dispersion of the lipid or mixture of lipids;
(c) adding the solution or dispersion to an ion-containing aqueous solution
to form a liposome suspension;
wherein the liposomes thus formed are capable of encapsulating an agent
at a mole/mole ratio between the agent and the liposome-forming lipid of
greater
than 1Ø
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The invention also provides a method for preparing liposomes consisting
of:
(a) providing a dry liposome-forming lipid or a dry mixture comprising a
liposome-forming lipid;
(b) dissolving the liposome-forming lipid or said mixture comprising a
liposome-forming lipid with a protic organic solvent to form a solution or
dispersion of the lipid or mixture of lipids;
(c) adding the solution or dispersion to an ion-containing aqueous solution
to form a liposome suspension;
wherein the liposomes thus formed are capable of encapsulating an agent
at a mole/mole ratio between the agent and the liposome-forming lipid of
greater
than 1Ø
Further, the invention provides a method for preparing liposomes
comprising:
(a) providing a dry liposome-forming lipid or a dry mixture comprising a
liposome-forming lipid;
(b) dissolving the liposome-forming lipid or said mixture comprising a
liposome-forming lipid with a protic organic solvent to form a solution or
dispersion of the lipid or mixture of lipids;
(c) adding the solution or dispersion to an ion-containing aqueous solution
to form a liposome suspension;
wherein the liposomes thus formed are capable of encapsulating an agent
at a ratio between the agent and the liposome-forming lipid of greater than
1.0;
with the proviso that said method does not comprise one or more of the
following steps:
- drying said solution or dispersion to form a dry lipid film; and/or
- down-sizing liposomes in said liposome suspension to form small
unilamellar vesicles (SUV).
The liposomes may be used to carry active agents such as drugs. Thus, in
accordance with a further aspect, the invention provides a method for
preparing agent-
carrying liposoines coinprising or consisting of: .
(i) providing a liposome suspension by the method of the invention, and
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(ii) incubating said liposome suspension with a solution comprising said
agent to form agent-carrying liposomes, wherein said agent-carrying liposomes
comprise a mole/mole ratio between said agent and said liposome-formiuig lipid
of greater than 1Ø
In a preferred embodiment the method of the invention for preparing agent
carrying liposomes excludes drying said solution or dispersion to form a dry
lipid film;
and/or down-sizing liposomes in said liposome suspension to form small
unilamellar
vesicles (SUV).
The invention also provides pharmaceutical compositions comprising a
physiologically acceptable carrier and agent-carrying liposomes whenever
prepared by
any of the methods of the invention.
Further, the invention provides a method for treating a subject in need
comprising administering to said subject an amount of agent-carrying liposomes
whenever prepared by the method of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention concerns a method for preparing liposomes with a high
agent-to-lipid ratio (by amount in moles of each), which, as appreciated by
those versed
in the art, may have benefits in terms of prolonged delivery, safety and other
pharmacological and pharmacokinetic parameters. The simplicity of the
proposed'
approach resides, inter alia, in the elimination of several steps which
hitherto have been
considered essential components in the preparation of liposomes of similar
characteristics and loading capabilities.
The terms "liposome" and "vesicle" are well known in the art and are used
interchangeably herein, except where otherwise specifically stated or required
by
context.
The method of the present invention comprises: providing a dry liposome-
forming lipid or a dry mixture comprising one or more liposome-forming lipids;
dissolving the lipid or the dry mixture comprising the same with a protic
organic solvent
to form a solution or dispersion of the lipid(s); and adding the solution or
dispersion
thus formed to an ion-containing aqueous solution, resulting in the formation
of a
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liposomal suspension. It has been found that such liposomes are capable of
loading an
agent at a higli agent-to-lipid mole/mole ratio, a high ratio being greater
than 1Ø As
fi.uther discussed below, the active agent may be loaded into the liposome by
different
methods, such as incubation of the liposome suspension with an aqueous
solution of the
agent.
In accordance with the invention, a"lzigh agent-to-lipid ratio" is determined
by
the mole/mole or weight/weight ratio of the agent to the liposome-fonning
lipid(s). In
the following description, unless otherwise stated, the term "ratio" denotes a
mole/mole
ratio. A high agent-to-lipid (agent/lipid) ratio in accordance with the
invention is to be
understood as any ratio being at least greater than 1.0, preferably greater
than 1.5, more
preferably greater than 1.8. It has been found that under suitable conditions
which are
within the scope of the present invention the ratio may even be greater than
2Ø
It is noted that conventional methods for preparing liposomes loaded with an
agent (e.g., a drug) with relatively high agent/lipid ratio (i.e., greater
than 1.0) comprise
the following features, which had previously been considered to be essential
components in the preparation of liposomes of similar characteristics and
loading
capabilities:
- the liposome-forming lipid needs to be dried and then rehydrated prior to
the formation of liposomes therefrom; and/or
- the liposomes need to be down-sized, e.g. by vortexing, by
ultrasonication, by extrusion and/or by like processes, to form small
unilamellar
vesicles (SUV), before obtaining (following a suitable manipulation of the
SUV)
the final liposomes into which the agent is to be loaded; and/or
- the SUVs require at least five freezing and thawing (F&T) cycles in
order to obtain liposomes capable of exhibiting a desirably high agent-to-
lipid
ratio;
- the selection of specific (and thus limiting) organic solvents which do not
freeze under lyophilization conditions (temperatures of 0 C or higher).
As shown herein, the method of the present invention provides agent-carrying
liposomes with a high agent/lipid ratio without the need to perform any of the
above
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steps in order to achieve the desired high loading. In other words, by
eliminating the
need to dry the lipids prior to liposome formation, and/or the need to down-
size the
liposomes, and/or the need to perform multiple (more than 1) F&T cycles, a
much
simpler, cost-effective method is provided. Such a simplified method may have
many
advantages, in particular with respect to large-scale production of agent-
carrying
liposomes.
The liposomes of the present invention are formed from liposome-forming
lipids. "Liposonze forming lipids" (or "vesicle fornzing lipids ") are
amphiphilic
molecules essentially characterized by a packing parameter of 0.74 - 1.0,
inclusive, or
by a lipid mixture having an additive packing parameter (the sum of the
packing
parameters of each component of the liposome multiplied by the mole fraction
of each
component) in the range between 0.74 and 1, inclusive.
Further, "liposome forming lipids" in-accordance with the invention are lipids
having a glycerol backbone wherein at least one, preferably two, of the
hydroxyl groups
at the head group is substituted by one or more of an acyl, an alkyl or
alkenyl group, a
phosphate group, preferably an acyl chain (to form an acyl or diacyl
derivative), a
combination of any of the above, and/or derivatives of the above, and may
contain a
chemically reactive group (such as an amine, acid, ester, aldehyde or alcohol)
at the
headgroup, thereby providing a polar head group. Sphingolipids, and especially
sphingomyeliuis, are good alteniatives to glycerophospholipids.
Typically, a substituting chain, e.g. an acyl, alkyl and/or alkenyl chain, is
between about 14 to about 24 carbon atoms in length, and has varying degrees
of
saturation, thus resulting in fully, partially or non-hydrogenated liposome-
forming
lipids. Further, the liposome-forming lipid may be of a natural source, semi-
synthetic or
a fully synthetic lipid, and may be neutral, negatively or positively charged.
There are a variety of synthetic liposome-forming lipids and naturally
occurring
liposome-forming lipids, including the phospholipids (which are preferred
lipids in
accordance with the invention), such as phosphatidylcholine (PC),
phosphatidylinositol
(PI), phosphatidyl glycerol (PG), dimyristoyl phosphatidyl glycerol (DMPG),
egg yolk
phosphatidylcholine (EPC), 1-palmitoyl-2-oleoylphosphatidyl choline (POPC),
distearoylphosphatidylclioline (DSPC), dimyristoyl phosphatidylcholine (DMPC),
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phosphatidic acid (PA), pliosphatidylserine (PS), 1-palmitoyl-2-
oleoylphosphatidyl
choline (POPC), and sphingophospholipids such as sphingomyelins (SM) having 12-
to
24-carbon-atom acyl or alkyl chains. The above-described lipids and
phospholipids,
whose hydrocarbon chain (e.g., acyl/alkyUalkenyl chains) have varying degrees
of
saturation, can be obtained commercially or prepared according to published
methods.
Other suitable lipids which may be included in the liposomes are
glyceroglycolipids,.
sphingoglycolipids and sterols (such as cliolesterol or plant sterol). The
liposomes
formed in accordance with the invention may comprise a mixture of lipids. The
above-
described list of lipids for use in accordance with the invention is not
exhaustive and
non-limiting, and thus, other lipids not disclosed herein may be used in
accordance with
the invention.
In accordance with one embodiment, the liposome-forming lipids are selected
from those having a T,,, (gel to liquid crystalline phase transition
temperatures). above
45 C, such as, without being limited thereto, phosphatidylcholine (PC) and
derivatives
thereof having an acyl chain with 16 or more carbon atoms. One preferred
example of a
PC derivative is hydrogenated soy PC (HSPC) having a Tm of 52 C. The liposome-
forming lipids may additionally or alternatively comprise sphingomyelins of
various N-
acyl chains, such as N-stearoyl sphingomyelin.
Cationic lipids (mono- and polycationic) are also suitable for use in the
liposomes of the invention, where the catioiiic lipid can be included as a
minor
component of the lipid composition or as a major or sole component. Such
cationic
lipids typically have a lipophilic moiety, such as a sterol, an acyl or diacyl
chain, and
the lipid typically has an overall net positive charge. Preferably, the head
group of the
lipid carries the positive charge. Monocationic lipids may include, for
example: 1,2-
dimyristoyl-3-trimethylammonium propane (DMTAP); 1,2-dioleyloxy-3-
(trimethylamino) propane (DOTAP); N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-
dimethyl-
N-hydroxyethylanunonium bromide (DMRIE); N-[l-(2,3,-dioleyloxy)propyl]-N,N-
dimethyl-N-hydroxy ethyl ammonium bromide (DORIE); N-[1-(2,3-dioleyloxy)
propyl]-N,N,N-trimethylammoniuin chloride (DOTMA); 3 (3 [N-(N',N'-
dimethylaminoethane) carbamoyl] cholesterol (DC-Chol); and
dimethyl-dioctadecylammonium (DDAB).
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Examples of polycationic lipids may include a lipophilic moiety similar to
those
described for monocationic lipids, to which the polycationic moiety is
attached.
Exemplary polycationic moieties include spermine or spennidine (as exemplified
by
DOSPA and DOSPER), or a peptide, such as polylysine or other polyamine lipids.
For
example, the neutral lipid (DOPE) can be deriva.tized witli polylysine to form
a cationic
lipid. Polycationic lipids include, without being limited thereto, N-[2-[[2,5-
bis[3-
aminopropyl)amino] - 1 -oxopentyl] amino]ethyl]-N,N-dimethyl-2,3-bis[(1-oxo-9-
octadecenyl)oxy]-1-propanatninium (DOSPA), and ceramide carbamoyl spermine
(CCS).
Further, the liposomes nlay also include a lipid derivatized with a
hydrophilic
polymer to form new entities known by the term lipopolymers. Lipopolymers
preferably
comprise lipids modified at their head group with a polymer having a molecular
weight
equal to or above 750 Da. The head group may be polar or apolar; however it is
preferably a polar head group to which a large (>750 Da), highly hydrated (at
least 60
molecules of water per head group), flexible polymer is attached. The
attachment of the
hydrophilic polymer head group to the lipid region may be a covalent or non-
covalent
attachment; however it is preferably via the formation of a covalent bond
(optionally via
a linker). The outermost surface coating of hydrophilic polymer chains is
effective to
provide a liposome with a long blood circulation lifetime in vivo. The
lipopolymer may
be introduced into the liposome in two different ways either by: (a) adding
the
lipopolymer to a lipid inixture, thereby forming the liposome, where the
lipopolymer
will be incorporated and exposed at the inner and outer leaflets of the
liposome bilayer
[Uster P.S. et al. FEBBS Letters 386:243 (1996)]; or (b) first preparing the
liposome,
and then incorporating the lipopolymers into the external leaflet of the pre-
formed
liposome either by incubation at a temperature above the Tm of the lipopolymer
and
liposome-forming lipids, or by short-term exposure to microwave irradiation.
Preparation of vesicles composed of liposome-forming lipids and lipids such as
phosphatidylethanolamines (which are not liposome-forming lipids) and
derivatization
of such lipids with hydrophilic polymers (thereby forming lipopolymers) which
in most
cases are not liposome-forming lipids. Examples have been described, for
example, by
Tirosh et al. [Tirosh et al., Biopys. J., 74(3):1371-1379 (1998)] and in U.S.
Patent Nos.
5,013,556; 5,395,619; 5,817,856; 6,043,094; and 6,165,501; incorporated herein
by
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reference; and in WO 98/07409. The lipopolymers may be non-ionic lipopolymers
(also
referred to at times as neutral lipopolymers or uncharged lipopolymers) or
lipopolymers
having a net negative or a net positive charge.
There are numerous polymers which may be attached to lipids. Polymers
typically used as lipid modifiers include, without being limited thereto,
polyethylene
glycol (PEG), polysialic acid, polylactic acid (also termed polylactide),
polyglycolic
acid (also termed polyglycolide), apolylactic-polyglycolic acid, polyvinyl
alcohol,
polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline,
polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polyaspartamide,
polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide,
polyvinylmethylether, polyhydroxyethyl acrylate, and derivatized celluloses
such as
hydroxymethylcellulose or hydroxyethylcellulose. The polymers may be employed
as
homopolymers or as block or random copolymers.
While the lipids derivatized into lipopolymers may be neutral, negatively
charged, or positively charged, i.e. there is no restriction regarding a
specific (or no)
charge, the most commonly used and commercially available lipids derivatized
into
lipopolyiners are those based on phosphatidyl ethanolamine (PE), usually
distearylphosphatidylethanolamine (DSPE).
A specific family of lipopolymers which may be employed by the invention
includes monomethylated PEG attached to DSPE (with different lengths of PEG
chains,
the metllylated PEG referred to herein by the abbreviation PEG), in which the
PEG
polymer is linked to the lipid via a carbamate linkage resulting in a
negatively charged
lipopolymer. Other lipopolymers are the neutral methyl polyethyleneglycol
distearoylglycerol (mPEG-DSG) and the neutral methyl polyethyleneglycol
oxycarbonyl-3-amino-1,2-propanediol distearoylester (mPEG-DS) [Garbuzenko O.
et
al., Langmuir. 21:2560-2568 (2005)]. The PEG moiety preferably has a molecular
weight of the PEG head group from about 750 Da to about 20,000 Da. More
preferably,
the molecular weight of the headgroup is from about 750 Da to about 12,000 Da,
and it
is most preferably between about 1,000 Da to about 5,000 Da. One specific PEG-
DSPE
employed herein is a PEG moiety with a molecular weight of 2,000 Da,
designated
herein 20ooPEG-DSPE or akPEG-DSPE.
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Preparation of liposomes including such derivatized lipids has also been
described where typically between 1-20 mole percent of such a derivatized
lipid is
included in the liposome formulation.
In addition to liposome-forming lipids (such as PCs and sphingomyelins),
cholesterol and phopshatidylethanolamines can be included in the liposomal
fonnulation (e.g. to decrease a membrane's free volume and thereby
permeability and
leakage of an agent encapsulated therein). In accordance with one embodiment,
the
liposomes comprise cholesterol. In accordance with a further embodiment, the
lipid/cholesterol mole/mole ratio is within the range of between about 80:20
to about
50:50. A more specific mole/mole ratio is about 60:40.
The liposome may include other constituents. For example, charge-inducing
lipids such as phosphatidyl glycerol may also be incorporated into the
liposome bilayer
to decrease vesicle-vesicle fusion, and to increase interaction of the
liposomewith cells.
Buffers at a pH suitable to make the pH of the surface of the liposomes close
to neutral
can decrease hydrolysis. Addition of an antioxidant, such as vitamin E, or
chelating
agents, such as Desferal or DTPA, may be used.
The liposome-forming lipid and other lipid and non-lipid components (if used)
are dissolved in a protic organic solvent. In the context of the present
invention, a protic
organic solvent is typically an alcohol, preferably C2 to C4 alcohols. The
solvent is
preferably miscible in water. Non-limiting examples of protic organic solvents
include
methanol, ethanol, and tertiary butanol (tert-butanol). Ethanol is a preferred
solvent.
The solvent dissolves the lipid or mixture of lipids to form a solution or
dispersion. By the terms "dissolves" or "solution" it is to be understood that
the lipid is
preferably homogeneously mixed within the solvent; nonetheless, non-homogenous
mixtures may be formed and used in the context of the present invention (in a
dispersion).
The formed solution or dispersion is then added to an aqueous solution
comprising ions. The term "ions-conzprisitzg aqueous solution" is used herein
to denote
an aqueous solution which comprises a salt, such as ammonium sulfate, calcium
acetate,
etc., dissolved therein. The different types of ions and salts are discussed
in detail below
with respect to pH or ion gradient formation.
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The method of the invention is also characterized in that a high lipid
concentration may be employed. It has been found that a high agent/lipid ratio
(above
1.0) may be achieved even with lipid concentration in the formed liposomes
being
above 20 inM, above 30 mM, above 50 mM, above 90 mM and even up to 97-100 mM,
inclusive. In accordance with one embodiment, the lipid concentration is
between 30
mM and 100 mM, inclusive.
Variations in ratios between these liposoine constituents dictate the
pharmacological properties of the liposome. For example, stability of the
liposomes,
which is a major concern for various types of vesicular applications, may be
dictated by
selecting specific liposome constituents. Evidently, the stability of
liposomes should
meet the same standards as conventional pharxnaceuticals. Chemical stability
involves
prevention of both the hydrolysis of ester bonds in the phospholipid bilayer
and the
oxidation of unsaturated sites in the lipid chain. Chemical instability can
lead to
physical instability or leakage of encapsulated drug from the bilayer and
fusion and
aggregation of vesicles. Chemical instability also results in short blood
circulation time
of the liposome, which affects the effective access to and interaction with
the target.
Different types of liposomes may be prepared by the method of the present
invention, including, without being limited thereto, multilamellar vesicles
(MLV), small
unilamellar vesicles (SUV), large unilamellar vesicles (LUV), sterically
stabilized
liposomes (SSL), multivesicular vesicles (MVV), and large multivesicular
vesicles
(LMVV). The different types of liposomes may be obtained by applying one or
more
additional treatment steps to liposomes formed by the above-described steps.
For
example, LMVV may be obtained by performing one, and optionally more, F&T
cycles
on the liposomes suspension. SUV may be obtained by any known technique to
down-
size liposomes (such as vortexing, ultrasonication, extrusion, etc.). Those
versed in the
art will know how to select the appropriate additional treatment step(s) in
order to
convert the liposomes formed by the method of the present invention to other
liposomal
forms and structures.
In accordance with one embodiment, the method of the invention preferably
provides MLVs. Following one F&T cycle, the MLVs are converted to LMVVs.
Additional F&T cycles may be performed.
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In accordance with an embodiment, at least one F&T cycle is performed. In
accordance with another embodiment, between 5 to 9 F&T steps are performed.
The
F&T cycle(s) convert(s) the MLV to LMVV which are then incubated with the
agent to
be loaded therein.
The liposome suspension thus formed comprises an amount of the protic organic
solvent. The amount may vary, depending on the solvent and the type of the
liposoxnes
thus formed. The amount of the organic solvent will be such that the liposomes
are not
converted to micelles (or micellae). For example, when using ethanol as the
organic
solvent, transformation from liposomes to inicelles will typically occur at
30% solvent
(by volume). Thus, ethanol levels in the liposomal suspension may be as high
as about
25% by volume. In accordance with one embodiment, ethanol level is about 10%
by
volume.
An active agent to be loaded into the liposomes may be any substance, e.g., a
low or high molecular weight compound, having a utility in therapy or
diagnostics. In
accordance with one embodiment, the active substance is an amphiphatic weak
acid or
an amphiphatic weak base. In accordance with a preferred embodiment, the agent
is an
amphiphatic weak acid drug or amphipathic weak base drug.
Amphiphatic weak base drugs include, among others, the following non-limiting
list: tempamine (TMN) doxorubicin, epirubicin, daunorubicin, carcinomycin, N-
acetyladriamycin, rubidazone, 5-imidodaunomycin, N-acetyldaunomycine, all
anthracyline drugs, daunoryline, topotecan, irinotecan propranolol,
pentamidine,
dibucaine, bupivacaine, tetracaine, procaine, chlorpromazine, vinblastine,
vincristine,
mitoniycin C, pilocarpine, physostigmine, neostigmine, chloroquine,
amodiaquine,
chloroguanide, primaquine, mefloquine, quinine, pridinol, prodipine,
benztropinemesylate, trihexyphenidyl llydrochloride, propra.nolol, timolol,
pindolol,
quinacrine, benadryl, promethazine, dopamine, L-DOPA serotonin, epinephrine,
codeine, meperidine, methadone, morphine, atropine, decyclomine, methixene,
propantheline, imipramine, amitriptyline, doxepin, desipraniine, quinidine,
propranolol,
lidocaine, bupivacaine, chlorpromazine, promethazine, perphenazine, acridine
orange,
opiates such as morphine, and others.
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In accordance with one embodiment, the amphiphatic weak base is an analgesic
drug. Some analgesic drugs are listed above and include lidocaine and
bupivacaine.
These drugs are also specifically exemplified herein below.
Amphiphatic weak acid drugs include, without being limited thereto, ibuprofen,
toluetin, indomethacin, phenylbutazone, mecloferamic acid, piroxicam,
citrofloxacin,
prostaglandins, fluoresgein, carboxyfluorescein, methyl perdnisolone
hemisuccinate
(MPS), paracetamol (acetaminophen), aspirin (acetyl salicylic acid) and other
NSAIDs,
and nalidixic acid.
Further, there may be interest in having glucocorticosteroids as an agent
loaded
in liposomes and treating these liposomes prior to administration with empty
liposomes.
A non-limiting list of glucocorticoids may be found at the internet site
http://www.steraloids.com/, incorporated herein in its entirety by reference.
Non-
limiting examples include: prednisolone hemisuccinate, methylprednisolone
hemisuccinate, dexamethasone hemisuccinate, allopregnanolone hemisuccinate;
beclomethasone 21-hemisuccinate; betamethasone 21-hemisuccinate; boldenone
hemisuccinate; prednisolone hemisuccinate; sodium salt; prednisolone 21-
hemisuccinate; nandrolone hemisuccinate; 19-nortestosterone hemisuccinate;
deoxycorticosterone 21-hemisuccinate; dexametliasone hemisuccinate;
dexamethasone
hemisuccinate: spermine; corticosterone hemisuccinate; cortexolone
hemisuccinate.
In general, there is a variety of loading methods available for preparing
liposomes with entrapped active agents, including passive entrapment and
active
(reniote) loading. The term "entrapment" as used herein denotes any form of
loading of
the agent onto the liposomes, such that at least a substantial part of the
agent is
encapsulated within the interior aqueous core of the liposomes. Within the
interior core
the agent may be free or associated to the inner surface of the lipid bilayer.
Thus, in the
context of the present invention the term "entrapment" may at times be used
interchangeably with the terms "encapsulation" or "Carfying" or "loadinb".
The passive entrapment method is most suited for entrapment of lipophilic
drugs
in the liposome membrane and for entrapment of agents having high water
solubility.
In the case of ionizable hydrophilic or amphiphatic agents, even greater agent-
loading efficiency can be achieved by loading the agent into liposomes against
a
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transmembrane pH or ion gradient [Nichols, J.W., et al., Biochim. Biophys.
Acta
455:269-271 (1976); Cramer, J., et al., Biochemical and Biophysical Research
Communications 75(2):295-301 (1977)]. This loading method, generally referred
to as
remote loading, typically involves an agent that is amphiphatic in nature and
has an
ionizable group which is loaded by adding it to a suspension of liposomes
having a
higher inside/lower outside H+ and/or ion gradient.
The liposomes employed in the context of the present invention are preferably
loaded by the remote loading principle. Remote loading occurs due to pH or ion
gradient, such as ammonium or ammonium-like (with non-organic, organic or
polymeric anions, e.g. alkylamine) gradient aggregation due to a high intra-
liposome
concentration of the agent and the formation of an agent-counter-ion salt
within the
liposome. Excess of the counter-ion occurs when the NH3 is released from the
liposomes. Remote loading via an ammonium salt is based on the large
difference in
permeability of the neutral ammonia gas molecule (1.3ac10'1 cm/s) and the
charged
anion (<10-10cm/s). Typically, the pH of the intra-liposome aqueous phase
composed of
an ammonium salt solution may be decreased by lowering the external
concentration of
ammonium and ainmonia [Haran G., et al., Biochim Biophys. Acta 1151:201-215,
(1993)]. The decrease of intra-liposomal pH results from the release from the
liposome
of the unprotonated ammonia compound (NH3) leaving within the liposome protons
(H) and the counter-ion (e.g. HS04 ; S04 2); thereby an excess of the counter-
anions
over NH4+ is created within the liposome.
Reduction of the pH inhibits ammonia formation and thereby iuzhibits its
release
from the liposome. When adding to the external medium of the liposome an
agent, e.g., an
amphiphatic wealc base, it freely crosses the lipid bilayer in its uncharged
form and
accumulates in its charged (having low permeability) form in the internal
aqueous
compartment (after being protonated by the free H+) [Schuldiner, et al., Eur.
J. Bichem
25:64-70 (1972); Nicolas and Deamer, Biochem. Biophys Acta 455:269-271
(1976)].
Evidently, this accumulation raises the internal pH and thus ammonia is again
formed and
released from the liposome, resulting in the reduction of internal pH and so
forth, until an
effective loading of the agent is accomplished. It is thus noted that for
loading of
amph.iphatic weak bases, it is preferable that the liposome have a low inter-
liposomaUhigh
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intra-liposomal trans-membrane gradient, such as anunonium salt gradient (e.g.
ammonium sulfate).
When the agent is a weak amphiphatic acid, it is preferable that the liposomes
have
a high inter-liposome/low intra-liposome transmembrane gradient. Such a
gradient may be
achieved using an aqueous solution of acetate salt such as calcium acetate. In
this case the
acetate ion gradient is the driving force while the Ca+2 ions, which have very
low
permeability through the liposome membrane, act as counter-ions of the weak
amphiphatic
acid within the aqueous phase, thereby stabilizing the loading and enabling
better control
over the release rate of the loaded weak amphiphatic acid. [Clerc, S. and
Barenholz, Y.,
Loading of atnpliiphatic weak acids into liposomes in response to
transmembrane calcium
acetate gradients. Biochim. Biophys. Acta 1240:257-265 (1995)].
The equilibrium between charged (protonated) and uncharged agents enables the
slow leakage of the uncharged weak base from the liposomes at a rate which is
dependent on the permeability coefficient. Shifting the equilibrium via
formation of
aggregates (formed between the loaded charged agent and the counter-ion within
the
liposome) further improves the retention of the agent inside the liposome, and
as now
being disclosed, may function as a tool for controlling the release of the
agent from the
liposome.
In accordance with the present invention, the H+ and/or ion gradient is formed
by dissolving the liposome-forming lipid or the mixture coinprising a liposome-
forming
lipid and other lipids (not necessarily liposome-forming lipids, e.g.,
cholesterol) with a
protic organic solvent to form a solution or suspension of said lipid and then
adding the
lipid solution to an ion-containing aqueous solution to form a liposome
suspension. The
liposome suspension is then incubated with a solution comprising the agent to
be loaded
and an H+ or ion concentration suitable for achieving a respective W or ion
gradient
between the inter-liposomal compartment and the intraliposomal surrounding. In
the case
of amphiphatic weak acids or amphiphatic weak bases (as the active agent/drug)
there is an
ionizable group, and thus the agent is loaded by adding it to a suspension of
liposomes
prepared so as to have an inside/outside pH gradient. As a result, the
uncharged species of
3o amphiphatic weak acid or amphiphatic weak base diffuses trough the liposome
membrane.
However, due to the different pH inside the interliposomal aqueous phase,
being more
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acidic (for weak bases) or more alkaline (for weak amphiphatic acids) with
respect to the
intraliposomal surrounding, the agent is protonated or deprotonated,
respectively, to
become a charged species.
In accordance with one embodiment, the liposomes formed by the method of the
invention may be prepared by using an aqueous buffer containing an ammonium
salt,
such as ammonium sulfate, ammonium phosphate, anunonium citrate, etc.,
typically
about 0.1 M to 0.3 M ammonium salt, at a suitable pH, e.g., about 5.5 to 7.5.
The
gradient can also be produced by including sulfated polymers in the aqueous
solution
added to the lipid solution. For example, such sulfated polymers may include
dextran
sulfate ammonium salt, heparin sulfate aiumonium salt or sucralfate. After
liposome
formation, the external medium may be exchanged for one lacking ammonium ions.
In
this approach, during loading, the amphiphatic weak base is exchanged with
ammonium
ion.
An H+/ion gradient may also be achieved by including in the liposomes with a
selected ionophore. To illustrate, liposomes prepared to contain valinomycin
in the
liposome bilayer are prepared in a potassium buffer, after which the external
medium is
then exchanged with a sodium buffer, creating a potassium inside/sodium
outside
gradient. Movement of potassium ions in an inside-to-outside direction in turn
generates
a lower inside/higher outside H-' or ion gradient, presumably due to movement
of
protons into the liposomes in response to the net electronegative charge
across the
liposome membranes [Deamer, D. W., et al., Biochim. et Biophys. Acta 274:323
(1972)].
A similar approach is to add the lipid to an aqueous solution having a higlz
concentration of magnesium sulfate. The magnesium sulfate gradient is created
by
dialysis against 20 mM HEPES buffer, pH 7.4, in sucrose. Then, an A23187
ionophore
is added, resulting in outwards transport of the magnesium ion in exchange for
two
protons for each magnesium ion, plus establishing a inner liposome high/outer
liposome
low proton gradient [Senske DB et al. Biochim. Biophys. Acta 1414: 188-204
(1998)].
Yet another approach is described in US 5,939,096, incorporated herein by
reference. In brief, that method employs a proton shuttle mechanism involving
the salt
of a weak acid, such as acetic acid, of which the protonated form translocates
across the
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liposome membrane to generate a higher inside/lower outside H+ or ion
gradient. An
amphiphatic weak acid compound is then added to the medium to the pre-formed
liposomes. This amphiphatic weak acid accumulates in liposomes in response to
this
gradient, and may be retained in the liposomes by cation (i.e. calcium ions)-
promoted
precipitation or low permeability across the liposome membrane; namely, the
amphiphatic weak acid is exchanged with the acetic acid.
In the case of a weak base, the H+/ ion gradient may be formed by using salts
having a counter-ion selected from, without being limited thereto: hydroxide;
sulfate;
phosphate; glucuronate; citrate; carbonate; bicarbonate; nitrate; cyanate;
acetate;
benzoate; bromide; chloride; other inorganic or organic anions; an anionic
polymer such
as dextran sulfate, dextran phosphate, dextran borate, carboxyniethyl dextran
and the
like; as well as polyphosphates. In the case of a weak acid the counter-ion
may be
calcium, magnesium, sodium, a.mmonium and other inorganic and organic cations,
or a
cationic polymer such as dextran spermine, dextran spermidine, aminoethyl
dextran,
trimethyl ammonium dextran, diethylaminoethyl dextran, polyethyleneimine
dextran and
the like. The counter-ion may be present in the form of a free small ion or
attached to a
polymer, or in both forms simultaneously. A specific embodiment for liposomes
carrying weak amphiphatic acids is those in which the high inter-liposomal/low
intra-
liposomal trans-membrane gradient is formed by using an acetate salt, such as
calcium
acetate, sodium acetate or potassium acetate. Ca2+ acetate is a preferred
acetate salt.
The release rate of the loaded agent from liposomes was shown to be dependent
on
a variety of factors, including, without being limited thereto, the counter
ion which forms a
salt with the active agent (see in this connection W003/032947, "A method for
preparing
liposome formulations with a predefined release profile", incorporated herein
its entirety
by reference), temperature, medium-related properties (medium composition,
ionic
strengtli, pH), liposome-related properties (membrane lipid composition,
liposome type,
number of lamellae, liposome size, physical state of phospholipid membrane
i.e., liquid-
disordered (LD), liquid-ordered (LO), solid-ordered (SO)), and loaded-molecule-
related
properties (lipophilicity, hydrophilicity, size) [Haran G., et al., Biochim
Biophys. Acta
1151:201-215, (1993)].
The invention also provides pharmaceutical compositions comprising a
physiologically acceptable carrier and an amount of the agent-carrying
liposomes
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prepared in accordance with the invention, the amount being effective to treat
or prevent
a disease or disorder.
The pharmaceutical composition may be provided as a single dose, however it
may be preferably administered to a subject in need of treatment over an
extended
period or tiine (e.g. to produce a cumulative effective amount), in a single
daily dose for
several days, in several doses a day, etc. The treatment regimen and the
specific
formulation to be administered will depend on the type of disease to be
treated and may
be determined by various considerations, known to those skilled in the art of
medicine,
e. g. physicians.
The term "effective amount" or "amount effective" is used herein to denote the
amount of the agent which, when loaded in the liposome, is sufficient in a
given
therapeutic regimen to achieve a desired therapeutic effect with respect to
the treated
disease or disorder. The amount is determined by such considerations as may be
known
in the art and depends on the type and severity of the condition to be treated
and the
treatment regime. The effective amount is typically determined in
appropriately
designed clinical trials (dose range studies) and the person versed in the art
will know
how to properly conduct such trials in order to determine the effective
amount. As
generally known, an effective amount depends on a variety of factors,
including the
mode of administration, type of vehicle carrying the amphipathic weak
acid/base, the
reactivity of the active agent (the weak amphiphatic acid or base), the
liposome's
distribution profile within the body, a variety of pharmacological parameters
such as
lzalf-life in the body after being released from the liposome, undesired side
effects, if
any, factors such as age and gender of the treated subject, etc.
The term "administering" (or "adnzinistration") is used to denote the
contacting
or dispensing, delivering or applying of the liposomal formulation to a
subject by any
suitable route of delivery thereof to the desired location in the subject,
including oral,
parenteral (including subcutaneous, intramuscular and intravenous, intra-
arterial,
intraperitoneal, etc.) and intranasal administration, as well as intrathecal
and infusion
techniques.
According to one embodiment, the composition used in accordance with the
invention is in a form suitable for injection. The requirements for effective
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pharmaceutical vehicles for injectable formulations are well known to those of
ordinary
skill in the art [See Pharnzaceutics and Phai rnacy Practice, J.B. Lippincott
Co.,
Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP
Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)].
Further, the invention concerns a method of treating a subject for a disease
or
disorder, the method comprising administering to said subject an amount of
agent-cartying liposomes prepared by the method of the invention.
As used herein the term "treatment" (or "treating") denotes curing of an
undesired pathological condition or prevention of a condition from developing.
For the
purpose of curing, the term "treatment" includes ameliorating undesired
symptoms
associated with the condition, slowing down progression of the condition,
delaying the
onset of a progressive stage of the condition, slowing down deterioration of
such
symptoms, enhancing onset of a remission period of the condition, if existing,
delayi.ng
onset of a progressive stage, improving survival rate or more rapid recovery
from the
condition, lessening the severity of or curing the condition, etc. Treatment
also includes
prevention of a disease or disorder. The term "prevention" includes, without
being
limited thereto, administering an amount of the composition to prevent the
condition
from developing or to prevent irreversible damage caused by the condition, to
prevent
the manifestation of symptoms associated with the condition before they occur,
to
inhibit the progression of the condition etc.
It is noted that the forms "a", "an" and "the" as used in the specification
include
singular as well as plural references unless the context clearly dictates
otherwise. For
example, the term "a lipid" includes one or more, of the same or different
lipids.
Similarly, reference to the plural includes the singular, unless the context
clearly
dictates otherwise.
Further, as used herein, the term "comprising" is intended to mean that the
liposome includes the recited constituents, but does not exclude others which
may be
optional in the formation or composition of the liposome, such as
antioxidants,
cryoprotectants, etc. The term "consisting essentially of' is used to define a
substance,
e.g. liposome, that includes the recited constituents but excludes other
constituents that
may have an essential significant effect on a parameter of the substance
(e.g., in the case
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of liposomes, the stability, release or lack of release of the agent from the
liposome as
well as on other parameters characterizing the liposomes). "Consistittg of'
shall thus
mean excluding more than trace amounts of such other constituents. Embodiments
defined by each of these transition terms are within the scope of this
invention.
Further, all numerical values, e.g. when referring the amounts or ranges of
the
elements constituting the composition or liposome components, are
approximations
which are varied (+) or (-) by up to 20%, at times by up to 10% from the
stated values.
It is to be understood, even if not always explicitly stated, that all
numerical
designations are preceded by the term "about".
It is appreciated that certain features of the invention, which are, for
clarity,
described in the context of separate embodiments, may also be provided in
combination
in a single embodiment. Conversely, various features of the invention, which
are, for
brevity, described in the context of a single embodiment, may also be provided
separately or in any suitable sub-combination.
Although the invention has been described in conjunction with specific
einbodiments thereof, it is evident that many alternatives, modifications and
variations
will be apparent to those skilled in the art, and it is explicitly intended
that the invention
include such alternatives, modifications and variations.
All publications, patents and patent applications mentioned in this
specification
are herein incorporated in their entirety by reference into the specification.
DETAILED DESCRIPTION OF SOME NON-LIMITING EXEMPLARY
EMBODIMENTS
Materials
Hydrogenated soy phosphatidylcholine (hereinafter referred to by the
abbreviation HSPC) was obtained from Lipoid, Ludwigsahfen, Germany.
Cholesterol was obtained from Sigma.
Bupivacaine hydrochloride (hereinafter referred to by the abbreviation BUP)
was obtained from Orgamol, Evionnar, Switzerland.
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Lidocaine hydrochloride (hereinafter referred to by the abbreviation LID) was
obtained from Sigma. -
Dimyristoylphosphatidylcholine (hereinafter referred to by the abbreviation
DMPC) was obtained from Lipoid, Ludwigsahfen, Germany.
Dipalrnitoylphosphatidylcholine (hereinafter referred to by the abbreviation
DPPC) was obtained from Lipoid, Ludwigsahfen, Germany.
Liposome preparation and characterization
Preparation ofmultilamellar vesicles (MLV)
MLV liposomes were prepared by weighing 450 mg of dry HSPC and 154 mg of
dry cholesterol (a 60:40 mole ratio). The dry phospholipid/cholesterol mixture
was then
dissolved in 1 ml ethanol at 80 C and the dissolved mixture was added to an
aqueous
solution of (NH4)2SO4 (250 mM, prepared by adding 297 mg of ammonium sulfate
to 9
ml of water), to obtain a preparation having a final phospholipid
concentration of
60 mM. Ethanol volume was 10% of final volume. The thus obtained MLVs were
heated at 65 C for 45 min.
Preparation of large multi-vesicular vesicles (LMM
MLV prepared as above were freeze-thawed either once or more (up to a total of
10 freeze-thawing cycles). Freezing was performed using liquid nitrogen (-196
C) and
thawing was performed using a water bath (37 C). Freezing time was
proportional to
the volume of liposome preparation such that for each milliliter of
preparation, one
minute freezing was executed (i.e. for 10 ml, 10 minute freezing took place).
To create a transmembrane ammonium sulfate gradient, the liposome
preparation was centrifuged 4 times sequentially in normal saline (4 C, 1000
g, 5 min).
This is effective to create an inside-to-outside ammonium ion gradient across
the
liposomal membrane. The ammonium ion concentration gradient provides the
driving
force for loading of amphiphilic weak bases such as Bupivacaine (BUP). The
presence
of a transmembrane pH gradient was verified by determining the distribution of
amphiphatic weak base acridine orange (AO), as described in Haran, G. et al.
Biochim.
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Biophys. Acta 1151:201-215 (1993) and Clerc S., Barenholz Y. Anal Biochem.
259(1):104-11 (1998).
Drug loading to LAIVT Liposome
The drug, BUP or LID was remote-loaded into the liposomes by incubating the
liposome preparation with 4.5% of appropriate drug solution (50 mg/mi solution
of
drug) at 60 C for 45 min.
Non-entrapped drug was removed from LMVV suspension by centrifugation in
normal saline (4 C, 1000 g, 5 min). The pH of the fmal medium was about 5.5.
This pH
was retained to ensure the drug's solubility and prevent precipitation.
The amount of trapped and free drug after one day of storage and for at least
one
month after storage was measured using high performance liquid chromatography
(HPLC) (Grant G. et al. Pharm. Res., 18 N3336-343 (2001) aild Grant G. et al.
Anasthesiology, 101:133-137 (2004)); the amount of phospholipid in the
liposomal
formulation was determined using the Bartlett method (Shmeeda, H. et al.
Methods
Enzymol. 367:272-292 (2003)). The drug-to-lipid ratio was calculated from the
parameters obtained.
Characterization of druk-loaded liposomes
Drug-to-Lipid Ratio
The drug-to-lipid ratio obtained in the liposomal formulation prepared as
described above was greater than 2 (mole drug/mole lipid > 2).
Liposome Size
The size of the liposomes was determined using laser Fraunhofer diffraction
(LS
13320 Laser Diffraction Particle Sizer Analyzer, Beckman Coulter UK). The
instrument's Software expresses particle size as the volume median diameter.
The mean
size of LMVV was - 8.5 6.5 micron. The level of transmembrane pH gradient
according to AO distribution was - 89% verifying a low inter-liposome /high
intra-
liposome transmembrane pH gradient, being larger than 3.
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Kinetics of Drug Loaded Liposome
Kinetics of drug leakage from liposomes was measured at 4 C during storage for
one month (for BUP) or three months (for LID) after preparation. Tables 1 and
2
provide parameters indicating leakage at the different time points.
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Table 1: Kinetics of BUP leakage from liposomes.
Days after Peak area g/m1 Mg/mi mM BUP % free %
mixing BUP BUP BUP liposomal
BUP
1 20.63 54.56 5.46 18.94 - 100*
2.5 6.97 0.70 2.42 12.8 87.2
7 3.76 10.28 1.03 3.57 18.8 81.2
9 4.8 13.01 1.30 4.52 23.8 76.2
11 4.48 12.2 1.22 4.23 22.3 77.7
13 4.07 11.10 1.11 3.85 20.3 79.7
4.49 12.20 1.22 4.24 22.4 77.6
27 2.59 7.21 0.72 2.50 13.2 86.8
29 4.87 13.20 1.32 4.58 24.2 75.8
31 4.18 11.38 1.14 3.95 20.9 79.1
33 4.89 13.25 1.32 4.60 24.3 75.7
34 5.28 14.27 1.43 4.96 26.2 73.8
36 4.59 12.46 1.25 4.33 22.8 77.2
38 3.14 8.65 0.87 3.00 15.9 84.1
46 5.51 14.87 1.49 5.16 27.3 72.7
53 4.76 12.91 1.29 4.48 23.7 76.3
57 5.51 14.87 1.48 5.13 27.1 72.9
60 6.33 17.03 1.70 5.90 31.1 68.9
65 6.57 17.66 1.76 6.11 32.2 67.8
69 6.74 18.10 1.81 6.29 33.2 66.8
74 7.17 19.23 1.92 6.68 35.2 64.8
78 7.71 20.65 2.06 7.17 37.8 62.2
61 7.82 20.94 2.09 7.27 38.4 61.6
63 7.77 20.81 2.08 7.22 38.1 61.9
68 8.83 23.59 2.36 8.19 43.2 56.8
* liposomal + residual free BUP (after wash)
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Table 2: Kinetics of LID leakage from liposomes
Day after Peak area gg/ml LID mg/ml mM LID % free /a
mixing LID LID liposomal
LID
1 24.54 53.0 5.3 19.6 - 100*
3 1.15 1.8 0.2 0.7 3.5 97
7 1.73 3.1 0.3 1.2 5.9 94
14 1.51 2.6 0.3 1.0 5.0 95
24 1.59 2.8 0.3 1.0 5.3 95
36 1.97 3.6 0.4 1.3 6.9 93
* liposomal + residual free LID (after wash)
The effect of number offi'eeze-thaw cycles on df ug/lipid ratio and drug
release level
The effect of number of freezing-thawing cycles on LMVV properties was
studied. Two independent experiments were performed. In the second experiment
a
batch of liposomes that were not freeze-thawed (MLV) were examined.
Transmembrane
pH gradient and BUP loading were prepared as described above. According to AO
distribution measurements the transmembrane pH gradient was the same for all
1o preparations, i.e., - 85% - 90%.
The results presented in Table 3 show the percentage of free, non-encapsulated
BUP in the composition relative to the total BUP is provided after preparation
(overnight) and following one week of storage.
CA 02627657 2008-04-28
WO 2007/049278 PCT/IL2006/001229
_ 28 -
~ 0 00 v1 N N 00 N d l~ O -a O
00 N ~-- l~ 00 00
N (V ~ e~ CC
l0 tn "O O oo kn O 01 l~ O~ O
d d; v~ ~n IO O~ ll~ c~ r-: Oo
~Q ,;~ O O O O O O O O O O O
ett r
cd M ~ O 41 O Ln M M in
01 O~ N o0 00 ~O ~O N d oo d
y =~ - O~ cy ~p 00 O O d ~O M v1
o eH O
~ O~ O M o0 d l~ O l~ O O~ [~
M M d N d ~O oo ~O ~ ~
O y ~ O O O O O O O O O O O
Q 00 M tn 00 -- O O ~ ~I) O
C N -4 tn 00 O
~ -~ - .-- .~ (~j =- -- -- = - N
N M cM d~ N ~n
N ~ N M ~ cn tn M oo N l~
~ -~ O cV d t~ ~- M \O lp l~
p
bA N M a1 l~ 01 O M
M M M d d M M d d ~
P;~ o0 O c', 1~ N 00 N M a1
+~ OC o0 01 06 M -- N O O -- O
O
4D
E"4
~ ~ w w w w w~~~ C~3 w w w w w
M k/ ~ [~ ~ w -- M N l~ 01
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WO 2007/049278 PCT/IL2006/001229
-29-
Table 3 shows that the number of F&T had no significant effect on the drug-
lipid
ratio. Further, the size distribution of the liposomes was measured as a
function of the
number of F&T.
The number of freezing-thawing cycles had no significant effect on liposome
size distribution as determined based on the volume of vesicles using
Universal Liquid
Model of Beckman Coulter (using a combination of light diffraction and PIDS
which
enable size range measurements of 0.040 to 2000 m, optical model of
Fraunhofer.rf780d (for size distribution calculation) and with a precision
size standard
diameter of 1.27 m, Cat No. 64035 of Polyscience, Inc) (data not shown).
Specifically,
there was no significant difference between drug/lipid ratio in cases of 1-5
freeze-thaw
cycles in the first experiment and even 1-7 freeze-thaw cycles in the second
one. At the
same time the highest drug/lipid ratio was obtained after 9 freeze-thaw cycles
in both
experiments. The second preparation showed higher drug release levels.
The effect of the initial phospholipid concentration on drug/lipid ratio and
drug
release level
Stock solutions of different initial lipid concentrations were prepared and
the
effect of the number of freezing-thawing cycles (0, 1, and 9 F&T cycles) was
evaluated.
Two independent experiments were performed. In the first experiment BUP
leakage
from liposomes was measured two days after drug loading, and in the second
experiment, BUP leakage was determined during two weeks after drug loading.
Results
are presented in Table 4 (first experiment) and Table 5 (second experiment).
Table 4: The effect of the initial phospholipid (PL) concentration on
drug/lipid ratio and drug leakage
mM PL Mg/ml mM BUP Drug/lipid Free Free/total
BUP total total BUP(a)
MLV 26.2
initial
No. F&T
0 F&T 7.5 2.9 9.98 1.33 0.42 14.57
1 F&T 8.1 4.3 14.95 1.85 0.57 13.23
9 F&T 6.1 6.5 22.60 3.69 0.80 12.28
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WO 2007/049278 PCT/IL2006/001229
-30-
MLV 54.3
initial
0 F&T 13.1 5.4 18.61 1.42 0.51 9.48
1 F&T 12.9 6.7 23.40 1.82 0.74 11.03
9 F&T 11.7 7.6 26.37 2.26 1.15 15.16
MLV ~ 97.4
initial
0 F&T 18.9 7.8 27.10 1.43 0.83 10.67
1 F&T 14.9 12.8 44.45 2.99 1.34 10.49
9 F&T 15.4 12.2 42.20 2.75 1.86 15.31
MLV 118.4
initial
0 F&T 24.2 8.1 28.09 1.16 1.06 13.16
1 F&T 22.2 7.7 26.72 1.20 1.05 13.64
9F&T 34.6 10.9 37.84 1.09 3.32 30.45
~ PL denotes phospholipids
(2) As measured two days after preparation
(3) F&T denotes number of Freezing and Thawing cycles
CA 02627657 2008-04-28
WO 2007/049278 PCT/IL2006/001229
31
P-I y N ~ v~i o~0 ~ 0~0 N o~o oo
i~, e}, o ~n ~o cn o t~ d
N ~n o ~
y ~ rn 00
;-õ = I-o r-~ V~
W e~ ... o o~ o 0 0 ~ o 0
00 l~ N
G> r+ N 01 N~ ~n tn oo V~ N O
r~ Tr O C y l~ d' l(j s ~ tn d" M ~--~
00
ro
~.
c-n o ~ ~ o 0 0
_ .~ ... .i~ ~ 01 00 M 7
~
,z 00 M N N M 00 01 N 9
a V7 d N= O M = =
v~-~ ~ o o ~ ~ dM ~ o~o oMo O 1~-,
I o f~ o ~ o 0 0~ o 0 0 0=-~ o D'.
ci
00 o~ 00 00 I'D m
ri
cq 00 kn OM1, 0~1
a N t~ ~ N ~ ~ N N M d
N 0~ '~--~
o a
ry
N rn ct N ~f) 00 0 ~ ~--1 O M
y ~ C~j r-i ~ M M M O \O 00 \O 00
y
18 '~ '8 1~
~~ w w w~~ w w w~~ w w w
aN .~ o,-~ arn
CA 02627657 2008-04-28
WO 2007/049278 PCT/IL2006/001229
32
...
\ N
ia r"' ~ p~ M
cl~
Cet '--O -+
ir O
W-)
w 00 00 O
.
...,
~o 00
{ y y bA -- ~O cn
I O ~ ': ~ O O
~ N M M
N d' c
N 00
N
{
88 ol~ o~
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WO 2007/049278 PCT/IL2006/001229
- 33 -
According to the data presented in Tables 4 and 5 it is evident that
increa'sing the
initial phospholipid concentrations, as high as about 97 mM), affected the
drug/lipid
ratio in all preparations with MLV (no F&T cycles (0). In LMVV obtained after
9 F&T
cycles there was no significant effect of the different initial phospholipid
concentration
on the drug/lipid ratio. It is noted that the higher the initial lipid
concentration, the lower
the drug leakage (after two weeks). However, it is further noted that
additional increase
of the phospholipid initial concentration up to 118 mM resulted in a decreased
drug/lipid ratio.
Loading of BUP in liposomes has various advantages, amongst others, in
reducing toxicity of the drug in a free form. It is known that BUP, which is
an amide-
linked local anesthetic of high potency, results in cardiovascular and central
nervous
system toxicity when high concentrations of the drug gain access to the
circulation.
Thus, the systemic toxicity of standard BUP and LMVV BUP was evaluated by
determining the dose that was lethal in 50% of mice (LD50) following
intraperitoneal
injection (data not shown). To minimize the number of animals required, the up-
and-
down technique of Dixon and Massey was used. For all study solutions, 0.3
m1/10 g
animal weight was injected, and mice were observed for 6 hours after injection
for signs
of toxicity. The results showed that the LD50 for standard BUP was 71 mg/kg
and the
LD50 for LMVV BUP was 565 mg/kg. This eight-fold increase in LD50 in LMVV BUP
was consistent with a slow release of the drug from the liposomal depot. It
also shows
that the use of LMVV will enable the safer administration of a much greater
dose of
local anesthetic than currently permitted.
