Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR UTILIZING NEUTRAL LIPIDS TO MODIFY
IN VIVO R~''~.F~~E FROM MQhTIVESICUhAR LIPOSOMES
Background of the Invention
This invention relates to liposomal formulations
of compounds such as drugs. More particularly this
invention relates. to methods of modifying the .in vivo
rate of felease of encapsulated compounds from
multivesicular liposomes by the choice of the neutral
lipid in the. liposomal formulation.
When phospholipids and many other amphipathic
lipids are dispersed gently in an aqueous medium they
swell, hydrate, and spontaneously form multilamellar
concentric bilayer vesicles with layers of aqueous media
separating the lipid bilayers. These systems are
commonly referred to as multilamellar liposomes or
multilameilar vesicles (MLV), and usually have diameters
of from 0.2 to 5 um. Sonication of MLV results in the
formation of small unilamellar vesicles (SUV) bounded by
a single lipid bilayer with diameters usually in the
range of from 20 to 100 nm, containing an aqueous
solution. Multivesicular liposomes (MVL) differ from MLV
and SUV in the way they are manufactured, in the random,
non-concentric arrangement of aqueous-containing chambers
within the liposo~e, and in the inclusion of neutral
lipids necessary to form the MVL.
Various types of lipids differing in chain length,
saturation, and head group have been used in liposomal
drug formulations for years, including the unilamellar,
multilamellar, and multivesicular liposomes mentioned
CA 02564120 2006-11-07
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above. The neutral lipids used in the manufacture of
multivesicular liposomes to date have been primarily
limited to triolein and tricaprylin.
One of the major goals of the field is to develop
liposomal formulations for controlled in vivo release of
drugs and. other active agents of interest. Certain drugs
need to be released fairly rapidly upon emplacement of
the liposomal depot, and others require a relatively slow
rate of release over a sustained period of time.
Heretofore, the rate of release of a biologically active
compound from a liposomal formulation has been modified
by selection of the amphipathic lipid, the accepted
membrane forming lipid, or by manipulation of the
phospholipid/cholesterol molar ratio. Alternatively,
such compounds as an acid or an osmolality spacer have
been included in the aqueous solution for encapsulation
to aid in modifying the rate of release of the
encapsulated biologically active compound.
The control of release rates from liposomal
formulations is complicated by the fact that many
biologically active agents, such as proteins, need to be
stored at reduced temperatures, i.e., about 4°C, to
retain full activity. Unfortunately, some liposomal
formulations that display excellent release rates at in
vivo temperatures disintegrate rather rapidly at such
storage temperatures.
Thus, the need exists for more and better methods
for selecting liposomal formulations that maximize
control over the rate of release of the encapsulated
active compound while simultaneously affording shelf life
stability for long periods of time at storage
temperatures of about 9°C, for example 2 to 10°C.
CA 02564120 2006-11-07
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Summary of the Invention
In general, the invention features a method for
modifying the rate of release of a biologically active
compound, such as a drug, that is encapsulated in a
multivesicular liposomal formulation by utilizing in the
formulation a neutral lipid component having a selected
molar ratio of a slow release neutral lipid to a fast
release neutral lipid, wherein the proportion of the fast
release neutral lipid in the molar ratio is increased to
increase the rate of release of the biologically active
compound. Alternatively, the proportion of the slow
release neutral lipid in the molar ratio can be increased
to decrease the rate of release of the biologically
active compound. Generally, the slow: fast neutral lipid
molar ratio is in the range from about 1:1 to 0:1, for
example 1:4 to 1:100, or 1:4 to 1:27, and the molar ratio
of the neutral lipid component to the total lipid
component (all the lipids in the liposome) is in the
range from about 0.01 to about 0.21.
For modifying the in vivo release rate, the
melting point of the neutral lipid component preferably
is at or below the in vivo temperature at which the
formulation is to be used, as well as at or below the
temperature at which the formulation is to be stored.
Slow release neutral lipids useful in the new
method of this invention are, for example, triolein,
tripalmitolein, trimyristolein, trilaurin, and tricaprin
with triolein being most preferred. Useful fast release
neutral lipids include, e.g., tricaprylin and tricaproin,
and mixtures thereof. However, tricaproin and other
similar lipids are usually not used as the sole neutral
lipid in a formulation of multivesicular liposomes
intended for use in vivo.
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Tn another embodiment, the present invention
provides a liposomal composition comprising a
therapeutically effective amount of a biologically active
compound encapsulated in a multivesicular liposome
formulation wherein the formulation comprises a neutral
lipid component comprising a molar ratio of slow release
neutral lipid to fast release neutral lipid from about
1:1 to 1:100, for example from about 1:3 to 1:54, 1:4 to
1:27, or from about 1:4 to 1:18.
In yet another embodiment, the invention features
a method for selecting a multivesicular liposomal
formulation for encapsulating a selected biologically
active compound so as to obtain a desired release rate
profile of the active compound in vitro and/or a desired
therapeutic release rate in- vivo. In this embodiment of
the invention, a family of MVL formulations that
encapsulate the selected biologically active compound is
prepared wherein each member of the family utilizes a
neutral lipid component having a different slow: fast
neutral lipid molar ratio, generally in the range from
1:0 to 0:1. For example, a family of formul-ations
utilizing slow: fast neutral lipid molar ratios of 1:0,
1:1, 1:4, 1:18, 1:27, 1:100, 0:1 can be prepared. Each
member of the family of formulations is incubated in the
medium in which the desired rate of release is to be.
obtained, i.e., either ,in a storage medium at storage
temperature or in human plasma, a plasma-like medium, or
in a physiological medium into which the physiologically
active substance is, to be released,. such as cerebrospinal
fluid (CSF) at body temperature. By this means a release
rate profile is obtained for each formulation. Then the
formulation having the slow: fast neutral lipid ratio that
yields the desired release rate profile under the .desired
CA 02564120 2006-11-07
conditions for the selected biologically active substance
is selected.
Unless otherwise defined, all technical and
scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art
to which this invention belongs: Although methods and
materials similar or equivalent to those described herein
can be used in the practice or testing of the present
invention, the preferred methods and materials are
described below.
In case of
conflict, the present specification, including
definitions, will control. In addition, the materials,
methods, and examples are illustrative only and not
intended to be limiting.
The practice of this invention provides the
advantage that a formulation of multivesicular liposomes
can be selected to have relatively rapid release in vivo
that .does not compromise the desire of slow release at
storage conditions. ~~e method of the invention,
therefore, provides a rationale for obtaining a desired
release rate through selection of the neutral Lipid
component used in manufacture of the MVL without
compromising the desire for the formulation to have a
slow release at storage conditions. Further, the control
over the rate of release under in vivo conditions
operates more or less independently of the composition of
the aqueous phase encapsulated or the combination of the
other lipids in the formulations.
This finding is particular to multivesicular
liposomes since other types of liposomes do not contain
neutral lipids in the lipid component and/or do not
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incorporate the neutral lipids into closely packed, non-
concentric vesicles.
Other features and advantages of the invention
will be apparent from the following detailed description,
and from the claims.
Brief Description of the Drawincss
Figure 1 is a graph comparing the release rates of
recombinant, human granulocyte colony stimulating factor
(rhu G-CSF) from multivesicular liposomes (MVL)
~0 formulated with different molar ratios of triolein to
tricaprylin (O, 100:0;n,50:50~ O, 25:75 0, 10:90:' 0,
0:100)' as the neutral lipid when incubated at 37°C in 60$
human plasma in saline solution.
Figure 2 is a graph showing the results of a
pharmacodynamic study comparing the duration of the rhu
G-CSF dependent elevation in peripheral blood leucocyte
number of golden Syrian hamsters following subcutaneous
injection of 100 ug/kg of rhuG-CSF in saline solution
(D), in a triolein-only, 100:0, MVL formulation (O), in a
triolein-only MVL formulation solubilized pre-injection
with Tween'~' 20 (n), and in a tricaprylin-only, 0:100, MVL
formulation (D).
Figure 3 is a graph comparing the release rates of
rhu GM-CSF from MVL formulated with different molar
ratios of triolein to tricaprylin (n, 100:0: O, 25:75; D,
10:90) as the neutral lipid when incubated at 37°C in 60$
human plasma in saline solution.
Figure 4 is a graph showing a pharmacokinetic
study of levels of rhu GM-CSF in venous blood, plasma
samples of mice following a subcutaneous injection of 1
mg/kg rhu GM-CSF in the triolein-only, 100:0, MVL
formulation (O) and in the 25:75, triolein:tricaprylin
CA 02564120 2006-11-07
MVL formulation (D). The rhu GM-CSF level in EDTA-plasma
was determined by ELISA. LOQ is the limit of
quantitation.
Figure 5 is a graph showing the release rates of
rhu IGF-1 from multivesicular liposomes formulated with
various triglycerides as the neutral lipid component when
incubated at 37°C in 60~ human plasma in saline solution.
The neutral lipids used in MVL manufacture were: (D)
triolein, (O) tripalmitolein, (n) tricaprylin, (D)
trilaurin, and (0).tricaprin.
Figure 6 is a graph showing the results of a
pharmacokinetic study of levels of rhu IGF-1 in serum of
rats following subcutaneous injection of 6.25 mg/kg rhu
IGF-1 in the tripalmitolein MVL formulation (O).and in
the tricaprylin MVL formulation (n). The rhu IGF-1 level
in serum was determined by ELISA.
Figure 7 is a graph showing the release of rhu
insulin and coencapsulant 1'C-sucrose from MVL
manufactured with the neutral lipid, triolein or
tricaprylin when incubated at 37°C in saline solutions
containing 60~ human plasma. (D) sucrose retained by
triolein MVL formulation; (O) insulin retained by
triolein MVL.formulation; (o) sucrose retained by
tricaprylin MVL formulation: and (O) insulin retained by
tricaprylin MVL formulation.
Figure 8 is a graph comparing the release rates of
morphine from MVL formulated with different molar ratios
of triolein to tricaprylin as the neutral lipid when
incubated at 37°C in 60g human plasma in saline solution.
(D) 10:0, triolein:tricaprylin, l0 batches; (0,0) 1:4,
triolein:tricaprylin, 2 batches; (e) 1:9,
triolein:tricaprylin: (D) 0:10, triolein:tricaprylin.
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_ g
Figure 9 shows results of a pharmacokinetic study
of morphine MVL formulations manufactured with different
molar ratios of triolein to tricaprylin and injected into
the epidural space of Beagle dogs. Morphine release by
the MVL was determined by measuring the level of morphine
in the adjacent, dura membrane-separated, cerebral spinal
fluid. (~) 5 mg morphine (sulfate) in saline solution;
(O) 40 mg of morphine in the 10:0, triolein:tricaprylin
MVL formulation; (O) 20. mg of morphine in the 1:4,
triolein:tricaprylin MVL formulations (~) 20 mg of
morphine in the 1:9, triolein:tricaprylin MVL
formulation; and (e) 20 mg of morphine in the 0:10,
triolein:tricaprylin MVL formulation.
figure 10 is a graph summarizing in an Arrhenius
-~--~~~P~fl fI-ERA--tea-r~s-fo-rmati-on--tfr~-resuits of a -study
determining the effect of storage temperature on release
rate of morphine from 2 batches of the 1:9,
triolein:tricaprylin MVL formulation. The MVL were
stored in normal saline solution at 4°C, 26°C, 37°C, and
41°C. The ordinate is the rate. of morphine release from
MVL expressed in terms of amount released per day as a
percentage of total amount of morphine in the MVL
suspension; the abscissa is 1/storage temperature x 10'
( °K) _i .
Figure 11 is a graph showing cytosine arabinoside
(AraC) retained in MVL incubated in human plasma at 37°C.
The MVL formulations used as the neutral lipid component
(~) triolein only; (O) 1:4, triolein:tricaprylin; (O)
1:9, triolein:tricapry~in; (x) 1:18 triolein:tricaprylin;
(e) 1:27, triolein:tricaprylin: and (0) tricaprylin only.
Figure 12 is a graph showing the effect the
neutral lipid ratio on release of cytosine arabinoside
from MVh incubated in human plasma at 37°C when the MVL
CA 02564120 2006-11-07
_ g _
were formulated with the major phospholipid component,
DOPC, replaced with DSPC. The formulations were (0) DOPC
and triolein only (e) DOPC 1:9, triolein:tricaprylin;
(0) DOPC and tricaprylin only (O) DSPC and triolein
only; (O) DSPC 1:9, triolein:tricaprylin; and (+) DSPC
and tricaprylin only.
Figure 13 is a graph showing the effect of the
neutral lipid ratio on release of 14C-sucrose from MVL
incubated in human plasma at 37°C when the MVL were
formulated with the major phopholipid component, DOPC,
replaced with DEPC. The DOPC formulations were (~) .
triolein only: (e) 1:9, triolein:tricaprylin: and (O)
tricaprylin only. The DSPC formulations were (~)
triolein only; (+) 1:9, triolein:tricaprylin; and (0).
tricaprylin only.
Figure 14 is a graph showing the effect of using
tricaproi~ as the neutral .lipid component alone or in
combination with triolein on the release of 14C-sucrose
from MVL incubated in human plasma at 37°C. The
formulations were (O) triolein only; (o) 1:4
triolein:tricaproin; (O) 1:9 triolein:tricaproin; (0)
1:18 triolein:tricaproin; and (+) tricaproin only.
Figure 15 is a graph showing the in vitro release
of a 15-mer oligonucleotide from MVL prepared with
triolein (D) or tricaprylin (O) as the neutral lipid.
The MVL were incubated in rat cerebral spinal fluid (CSF)
at 37°C.
Figure 16 is a graph showing the release during
incubation in human plasma of 14C-sucrose from MVL also
containing E. coli plasmid PBR 322 and lysine-
hydrochloride. Sucrose release was used as a surrogate
CA 02564120 2006-11-07
- ~~ -
indicator of plasmid release. The MVL were manufactured
with either (O) triolein or (o) tricaprylin as the
neutral lipid.
Figure 17 is a graph showing the release of 14C-
sucrose during incubation in human plasma of MVL
manufactured with tripalmitolein and tricaprylin as the
neutral lipid component. The neutral lipid ratio used in
the MVL formulations were (O) tripalmitolein only; (+)
1:1 tripalmitolein:tricaprylin; (0) 1:2
tripalmitolein:tri-caprylin; (Q) 1:9
tripalmitolein:tricaprylin; (o) 1:9
tripalrnitolein:tricaprylin; and (~) tricaprylin only.
Figure 18 is a graph showing the amount of
tetracaine remaining at the subcutaneous injection site
of mice injected with tetracaine containing MVL
manufactured with a neutral lipid component of either (~)
triolein; (X) 10:90, triolein:tricaprylin; (+) 5:95,
triolein:tricaprylin; (O) 2:98, triolein:tricaprylin;
(D) 1:99, triolein:tricaprylin; or (O) tricaprylin.
Figures 19A-D are graphs showing the release of
MVL-encapsulated, biologically active compounds during
storage in normal saline at temperatures of 2 - 8
(nominal 4), 25, 32, and 37°C. The MVL were manufactured
with either tricaprylin (Figures 19A and 19C); or
tricaprin (Figures 19B and 19D); as the neutral lipid
component and with either cytosine arabinoside (Figures
19A and 19B); or morphine, Figures 19C and 19D) as the
biologically active compound.
Figure 20 is an Arrhenius plot of the graphs in
Figures 19A and 19B showing the dependence of cytosine
arabinoside release rate on the storage temperature of
MVL manufactured with (D) tricaprylin, melting point (mp)
8°C; or (O) tricaprin, mp 31.5°C, as the neutral lipid
CA 02564120 2006-11-07
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component. The abscissa is the inverse of storage
temperature,°K.
Detailed Description
A method is provided for modifying the rate of
release of a biologically active compound, such as a
drug, encapsulated in a inultivesicular liposomal
formulation by selection of the neutral lipid, or
combination of neutral lipids, used to manufacture the
multivesicular liposomes (MVL).
There are at least three types of liposomes. The
term "multivesicular liposomes (MVL)" as used throughout
the specification and claims means man-made; 1-200 um
particles partially comprised of lipid membranes
enclosing multiple non-concentric aqueous chambers. In
contrast, "multilamellar liposomes or vesicles" (MLV)
have multiple "onion-skin" concentric membranes, in
between which are shell-like concentric aqueous
compartments. Multilamellar liposomes characteristically
have mean diameters in the micrometer range, usually from
0.5 to 25 pm. The term "unilamellar liposomes or
vesicles (ULV)" as used herein refers to liposomal
structures having a single aqueous chamber, usually with
a mean diameter range from about 20 to 500 nm.
Multilamellar and unilamellar liposomes can be
made by several relatively simple methods. The prior art
describes a number of techniques for producing ULV and
MLV (for example U.S. Patent No. 4,522,803 to Lenk;
4,310,506 to Baldeschweiler; 4,235,871 to
Papahadjopoulos; 4,224,179 to Schneider, 4,078,052 to
Papahadjopo~los: 4,394,372 to Taylor 4,308,166 to
Marchetti; 4,485,054 to Mezei; and 4,508,703 to
Redziniak).
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By contrast, production of multivesicular
liposomes requires several process steps. .Briefly, the
preferred method for making MVh is as follows: The first
step is making a "water-in-oiI" emulsion by capturing in
a lipid component composed of at least one amphipathic
lipid and at least one neutral lipid in one or more
volatile organic, solvents for the lipid component, an
immi.scible first aqueous component and a biologically
active substance to be encapsulated,'and optionally
adding, to either or both the lipid component and the
first aqueous component, an acid or other excipient for
modulating the release rate of the encapsulated
biologically active substances from the MVZ. The mixture
is emulsified, and then mixed with a second immiscible
aqueous component to form a second emulsion. The
turbulence required for formation of the second emulsion
'is provided either mechanically, by ultrasonic energy,
nozzle atomization; and the like, or by combinations
thereof, to form solvent spherules suspended in the
second aqueous component. The solvent spherules contain
multiple aqueous droplets with the substance to be
encapsulated dissolved in them (see Kim et al., Biochem.
Biophys. Acta, 728:339-348, 1983). For a comprehensive
review of various methods of ULV and MLV preparation,
refer to Szoka, et a1. Ann. Rev. Biophys. Bioeng. 9:465=
508, 1980.
The term "solvent spherule" as used throughout the
specification and claims means a microscopic spheroid
droplet of organic solvent, within which are multiple
smaller droplets of aqueous solution. The solvent
spherules are suspended and totally immersed in a second
aqueous solution.
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The term "neutral lipid" means an oil or fat that
has no membrane-forming capability by itself and lacks a
hydrophilic "head" group.
The term "amphipathic lipid" means a molecule that
has a hydrophilic "head" group and hydrophobic "tail"
group and has membrane-forming capability.
The term "zwitterionic lipid" means an amphipathic
lipid with a net charge of zero at pH 7.4.
The term "anionic lipid" means an amphipathic
lipid with a net negative charge at pH 7.4..
The term "cationic lipid" means an amphipathi.c
lipid with a net positive charge at pH 7.4.
As used herein, the "shelf life" of a liposomal
formulation is related to the rate of release of the
encapsulated substance from a liposomal formulation in a
storage solution, for instance normal saline~(0.9~ sodium
chloride), at a storage temperature, for instance at 4°G.
In general, for making multivesicular liposomes,
it i~s required that at least one amphipathic lipid and
one neutral lipid be included in the lipid component.
The amphipathic lipids can be zwitterionic, anionic, or
cationic lipids..Examples of zwitterionic amphipathic
lipids are phosphatidylcholines,
phosphatidylethanolamines, sphingomyelins etc. Examples
of anionic amphipathic lipids are phosphatidylglycerols,
phosphatidylserines, phosphatidylinositols, phosphatidic
acids, etc. Examples of cationic amphipathic lipids are
diacyl trimethylammoniumpropane and ethyl .
phosphatidylcholine. Examples of neutral lipids are
triolein and tripalmitoiein, trimy~istolein and
tricaprylin. In the new method, the release rate of the
biologically active compound is modified by utilizing in
manufacture of the multivesicular liposomes a neutral
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lipid component that provides the desired rato of release
in the type of fluid in which the MVI. are to be used.
For in vivo use, therefore, the release rate of the MVZ
must be determined in plasma or a plasma-like medium
because the release rate of the biologically active
compound from the MVZ for certain neutral lipids can
differ greatly depending on whether the release is into
saline or plasma:
As used herein, the term "neutral lipid component"
means the neutral lipid, or mixture of neutral lipids,
used in manufacture of the multivesicular liposomes.
As used herein, the term "plasma-like medium,"
means a synthetic solution that includes in addition to
normal saline, at least some of the protein~or lipid.
constituents of blood plasma or components of other
biological fluids, such as cerebro-spinal fluid (CSF), or
interstitial fluids. For instance, normal saline
containing citrated human plasma or bovine serum albumin
(BSA) is an example of a "plasma-like medium".as the term
is used herein.
The term "in vivo conditions" means actual
injection or emplacement of MVL into a living body, and
includes so-called "ex vivo" incubation of MVL in plasma
or a plasma-like medium at body temperature (i.e., 37°C
for humans).
Although the neutral lipid component can comprise
a single neutral lipid, generally the neutral lipid
component comprises a mixture of a slow release neutral
lipid and a fast release neutral lipid in a molar ratio
range from about 1:1 to 1:100, e.g., from about 1:4 to
1:18, wherein the rate of release of the biologically
active compound decreases in proportion with the increase
in the ratio of the slow release neutral lipid.to the
CA 02564120 2006-11-07
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fast release neutral lipid. For convenience, the molar
ratio of the slow release neutral lipid to the fast
release neutral lipid is referred to herein as "the
slow: fast neutral lipid molar ratio."
The "slow release neutral lipid" used in the
practice of this invention can be selected from
triglycerides having monounsaturated fatty acid ester
moieties containing from about 14 to 18 carbons in the
acyl chain and generally having a molecular weight from
about 725 to 885, and those with saturated fatty acid
ester-moieties containing --from -about--10----to--12--ca~rboris--in
the acyl chain and generally having a molecular weight
from about 725 to 885; and mixtures thereof. Cholesterol
esters such as cholesterol oleate and esters of propylene
glycol. The preferred slow release neutral lipids for
use in the method of this invention are triolein,
tripalmitolein, trimyristolein, trilaurin, and tricaprin,
with triolein or trip.almitolein being most preferred.
When in vivo use is contemplated, trilaurin (mp 46.5°C)
and other neutral lipids with a melting point above 37°C
are generally used in the practice of this invention only
in mixture with one or more other neutral lipids wherein
the mixture has a melting point temperature at or below,
and preferably below 37°C. One skilled in the art will
know how to determine the melting point of a mixture of
lipids, such as a mixture of triglycerides.
The "fast release neutral lipid" used in the
practice of this invention can be selected from
triglycerides having monounsaturated fatty acid ester
moieties containing from about 6 to 8 carbons in the acyl
chain and having a molecular weight from about 387 to
471, and mixtures thereof. However, it has surprisingly
been discovered that the use of a neutral lipid component
CA 02564120 2006-11-07
- 16 -
in MVZ containing one or more neutral lipids. with an acyl
chain of six or less carbons (especially use of
tricaproin as the sole neutral lipid) results in rapid.
release of the encapsulated compounds upon contact with
the in vivo environment. Therefore neutral lipids with
an acyl chain of six or less carbons should be used only
in combination with one or more neutral lipids having a
longer chain aryl moiety. The preferred fast release
neutral lipids are tricaprylin, and mixtures~of
l0 tricaprylin and tricaproin, or mixed chain Cs to C8
triglycerides. Propylene glycol diesters witf~ eight or
ten carbon acyl moieties, cholesterol oleate, and
cholesterol octanoate can also be used as neutral lipids.
A factor of equal importance to the molar ratio of
neutral lipids is selection of a neutral lipid component
having a melting point below the temperature~at which the
MVZ is to be stored and/or used. As many biologically
active compounds_,highl~ desirable_in_therapeutic
applications require low storage temperature to prevent
rapid deterioration, the neutral lipid component should
be selected to have a melting point below the desired
storage temperature as well as below the temperature at
which the formulation will be used in vivo.
The results of storage temperature tests were
determined by inspecting the particles microscopically
and measuring by~chemical assay the amounts of
encapsulated material released. As is seen in these
studies (Figure 20), MVhs stored at a temperature below
the melting point of the neutral lipid component undergo
a structural reorganization of the'membranes that is, in
some cases, accompanied by rapid release ("dumping") of
the encapsulated contents. This phenomenon is referred
to herein as "the melting point effect." The time to
CA 02564120 2006-11-07
_ 17 _
onset of the melting point effect depends upon the
neutral lipid composition and the ingredients of the
encapsulated aqueous phase. Certain encapsulated
compounds, such as IZ-2, appear to interact with MVLs in
such a way as to delay by hours or even days, the onset
of "freezing" at temperatures below the melting point of
the neutral lipid, perhaps by influencing formation in
the MVL of an intermediate, "meta-stable" state.
However, even formulations with such delayed onset
eventually undergo the morphological transition
characteristic of the melting point effect. Examples 15
and 16 below illustrate the melting point effect on the
release rates of encapsulated biologically active
compounds from MVL stored at a temperature below the
melting point (Figures 19A-D and 20). To distinguish
between the two states of morphological transition, the
delayed onset effect is referred to herein as "the
temperature effect."
In selecting the neutral lipid component in
accordance with the practice of this invention, it is
generally preferred that the neutral lipid component have
a melting point at or above the temperature at which the
MVh are to be stored and/or used (to prevent rapid loss
of the encapsulated active compound either during storage
or during in vivo use) due to the melting point effect.
The melting point temperature of a neutral lipid
component comprising a mixture of neutral lipids, and
hence the temperature of the melting point effect on MVL
containing the neutral lipid component, can readily be
determined by preparing the mixture of interest and
subjecting it to progressively lower temperatures until
the mixture is observed t-o "freeze." One method of
performing this procedure is disclosed in J.B. Rossell,
CA 02564120 2006-11-07
Advances in Lipid Research 5: 353-408, 1967. One skilled
in the art will be aware of other approaches that can be
used to obtain this information. However, it should be
remembered that the melting point effect upon the
liposomal formulation as a whole can be influenced by the
other lipids in the MVh as well as by the ingredients in
the first aqueous solution.
Table 1 below lists the number of carbons in the
acyl chains, the molecular weight, and melting point
temperatures of representative neutral lipids that can be
used in the practice of this invention.
Table 1
The Physical Properties of hipids
Neutral
1 5 Mol. Viscosity
M.P.
Neutra-1 lipid Fatty Acid Ester. Wt. cP C
TriGlycerides
Triolein C18:1 9C 885 74 5
Tripalmitolefn C16:1 9C 801' s5
2 0 TrimyristoleinC19:1 9C 725 s5
Trilawcin C12 639 95.5
Trfcaprin C10 555 31.5
Trfcaprylin C8 471 20-28 8.3
Tricaproin C6 387 s0
2 5 Captex 355 C8,C10 mixed Avg. 496 55
Propylene Glycol
_ Diester
Captex'~M200 C8,C10 mixed Avg. 345 9-13 s5
3 0 Cholesterol Ester
Cholesterol Oleate C18:1 9C 651 99-47
Cholesterol Octanoate C8 512 110
In one method of the invention, the release rate
35 of the biologically active compound is modified by
utilizing in manufacture of the multivesicular liposomes
a neutral lipid component comprising triolein or
tripalmitolein, or a mixture thereof, as the slow release
neutral lipid, and selecting a molar ratio of the slow
40 release neutral lipid to a fast release neutral lipid in
the range from about 1:0 to 0.1. The rate of release of
CA 02564120 2006-11-07
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the active compound increases with the increase in the
proportion of the fast release neutral lipid in the molar
ratio. Generally the molar ratio of the neutral lipid.
component to the sum of all the lipids in the MVh
formulation is in the range from about 0.01 to about
0.21. The preferred fast release neutral lipid for use in
such formulations with triolein and/or tripalmitolein is
tricaprylin.
In addition to the melting point effect, which is
contributed in part by the neutral lipid component, the
characteristics of the aqueous phase encapsulated in the
MVL, particularly the chemical interaction of the active
compound with the lipids in the MVL, can also influence
the rate of release of the biologically active compound.
To take this additional factor into account during
formulation, in one embodiment this invention provides a
method of tailoring the neutral lipid component to the
aqueous phase of interest.
In the first step of the method, to determine how
the neutral lipid component functions with any specific
aqueous phase (i.e., one containing a biologically active
compound of interest), a family of MVZ formulations is
made containing the aqueous phase of interest, wherei-n
each member of the family of formulations contains a
different slow:fast release neutral lipid molar ratio of
the selected slow and fast release neutral lipids such
that the family as a whole represents a graded
progression of such ratios, for example 1:1, 1:2, 1:4,
1:9, 1:18 1:27, 1:100, etc.
The in vivo release rates corresponding to the
various slow:fast neutral lipid molar ratios embodied in
the individual members of the family of MVZ formulations
is determined by separately incubating each member of the
CA 02564120 2006-11-07
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family in vitro in plasma or a plasma-like medium at in
vivo temperature, i:e. 37°C for humans, for a period of
hours, or even days.
Any of the methods illustrated in the Examples, or
others known to one of skill in the arts, can be used to
determine at progressive time points the cumulative
amount of one or more substances) encapsulated with the
aqueous phase that has been released during incubation.
For ease in making this determination, a radioactive
substance, such as 1°C sucrose, can be included in the
aqueous.phase at the time of encapsulation. However, it
is preferred to select the biologically active compound
of interest as the substance whose release rate is
monitored and recorded. It is recommended that the
release rate information be obtained in this manner for
each member of the family of formulations being tested.
From the release rate information determined by
this procedure, a graph showing a release curve, or
"release rate profile" can be plotted for each member of
the family of formulations to show its individual in vivo
release characteristics, with the ordinate of the graph
indicating the cumulative amount of the substance of
interest that has either been released or retained; and
the abscissa indicating progressive time points at which
the amount released or retained is measured. A
corresponding family of rate release curves is thus
generated, with each curve of the family illustrating the
release characteristics of its corresponding slow: fast
neutral lipid molar ratio when used with the aqueous
phase being tested.
The skilled practitioner can then select the
formulation having the most desirable release
characteristics for the particular therapeutic
CA 02564120 2006-11-07
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application of interest to obtain the desired control
over release of the substance of interest (i.e., a
biologically active compound) so as to deliver a
therapeutically effective amount of the active compound
to the individual to be administered the MVL formulation.
A skilled practitioner can thus select a MVL formulation,
in particular one having the most advantageous slow: fast
neutral lipid molar ratio; for delivering a
therapeutically effective dose over the optimum period of
time so as to maximize the therapeutic effect of the drug
or other biologically active compound administered during
therapy.
For instance, if it is desired to produce a MVL
formulation that releases a particular active compound in
vivo in a relatively short period of time, i.e., over
several hours after administration, the neutral lipid
component. that yields a release curve indicating such
delivery characteristics when stored in vitro in plasma,
or a plasma-like composition, at about 37°C will be
selected. The proportion of the fast release neutral
lipid in the ratio will be comparatively large in this
circumstance. On the other hand, when it is desired to
produce a MVL formulation that releases its active
compound in vivo over a relatively long period of time,
i.e., over tens of hours after administration, even up to
200 hours post administration, the neutral lipid
component that yields a release curve indicating such
delivery characteristics when incubated under in vivo
conditions will be selected. In this case the proportion
of the slow release neutral lipid in the molar ratio will
be comparatively large, and in some instances the neutral
lipid component will contain no fast release neutral
lipid at all.
CA 02564120 2006-11-07
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The shelf-life stability of the formulations
should also be determined by incubation of the
formulations in the contemplated storage medium at
whatever storage temperature is required to assure
integrity of the biologically active compound for a
suitable period of time. For convenience, the self-life
stability tests can also be conducted in plasma or a
plasma-like medium, but one skilled in the art will be
able to substitute a different suitable storage medium,
such as normal saline, for use with the biologically
active ,compound of interest, if desired. Since many
biologically active compounds require storage at
temperatures in the range from about 2 to 8°C, it is
recommended that the shelf-life stability tests be
conducted at a temperature in this range.
These procedures are illustrated in the Examples
of this application. For instance, in formulations
containing mixtures of triolein and tricaprylin, when the
neutral lipid component was held constant and incremental
increases in the ratio of trioiein to tricaprylin were
made, MVL formulations characterized by increasingly
slower release were obtained, as is illustrated in
Example 14 (Figure' 18). In Example 13, a graded family
of formulations encapsulating sucrose and lysine-HC1 and
containing mixtures of tripalmitolein and tricaprylin
were prepared. A graded family of formulations was
created with tripalmitolein:tricaprylin molar ratios of
0:1, 1:0, 1:9, 1:4, 1:2, and 1:l by holding constant the
amount of tripalmitolein and making incremental increases
in the amount of tricaprylin. In this Example,
increasingly more rapid release was obtained for each
incremental increase in the proportion of tricaprylin in
the neutral lipid component.
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It should particularly be noted that,. unlike most
other types of liposomes, accurate in vivo release
characteristics regarding MVL formulations cannot be
predicted from in vitro release studies conducted in
saline. For instance, MVL formulated with tricaproin, a
triglyceride having only 6 carbons in its acyl moieties,
as the only neutral lipid were not stable under in vivo
conditions (in solutions containing plasma or serum
albumin at 37°C, but were stable under storage conditions
(in saline at 2-8°C for up to a week).' However,
formulations containing tricaproin:tr'iolein molar ratios
of 4:1, 9:1, and 18:1 were stable under in vivo
conditions for at least 4 days, and yielded a graded set
of release rate curves (Figure 14).
In another embodiment, the present invention
provides liposomal compositions comprising a
therapeutically effective amount of a biologically active
compound encapsulated in a multivesicular liposome
formulation wherein the formulation comprises a neutral
20'lipid component with a molar ratio of slow release
neutral lipid to fast release neutral lipid in the molar
ratio range from about 0:1 to 1:0, for example l:1 to
1:100 and generally from about 4:1 to 27:1. The molar
ratio of the neutral lipid component to the sum of the
lipids in the MVL formulation is generally in the range
from about 0.01 to about 0.21.
The preferred amphipathic lipids for use in making
the multivesicular liposomes are phospholipids with even
numbers of carbons in the carbon chain because such
phospholipids are natural lipids found in the body and do
not produce toxic metabolites. A representative list of
amphipathic lipids preferred for use in the practice of
this invention follows. Also included are abbreviations
CA 02564120 2006-11-07
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that may be used to refer to particular phospholipids in
this application.
DOPC or DC18:1PC = i,2.-dioleoyl-sn-glycero-3-phosphocholine
DhPC or DC12:OPC = 1,2-dilauroyl-sn-glycero-3-phosphocholirle
DMPC or DC14:OPC = 1,2-dimyristoyl-sn-glycero-3-phosphocholine
DPPC or DC16:OPC= 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
DSPC or DC18:OPC = 1,2-distearoyl-sn-glycero-3-phosphocholine
DAPC or DC20:OPC =
1,2-diarachidoyl-sn-glycero-3-phosphocholine
DBPC or DC22:OPC = 1,2-dibehenoyl-sn-glycero-3-phosphochoiine
DC16:1PC = 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine
DC20:1PC = 1,2-dieicosenoyl-sn-glycero-3-phosphocholine
DEPC or DC22:lPC = 1,2-dierucayl-sn-glycero-3-phosphocholine
DPPG = 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol
DOPG = 1,2-dioleoyl-sn-g.lycero-3-phosphoglycerol
The term "biologically active compound" as used herein
means a chemical compound that is known in the art as
having utility for modulating biological processes so as
to achieve a desired effect in modulation or treatment of
an undesired existing condition in a living being, such
as a medical, agricultural or cosmetic effect.. Thus,
biologically active compounds are generally selected from
the broad categories of medicaments, pharmaceuticals,
radioisotopes, agricultural products, and cosmetics.
Therapeutic biologically active compounds, or
drugs for encapsulation in the methods and compositions
of this invention include anti-neoplastic agents, anti-
infective agents, hormones, anti-depressives, antiviral
agents, anti-nociceptive agents, anxiolytics and
biologics.
Representative examples of anti-neoplastic agents
usefuh in the compositions and methods of the present
CA 02564120 2006-11-07
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invention include methotrexate, taxol, tumor necrosis
factor, chlorambucil, interleukins, etoposide,
cytarabine, fluorouracil and vinblastine.
Representative examples of anti-infective agents
useful in the compositions and methods of the present
invention include pentamidine, metronidazole, penicillin,
cephalexin, tetracyclin and.chloramphenicol.
Representative examples of anti-viral agents
useful in the composition and methods of the present
invention include dideoxycytidine, zidovudine, acyclovir,
interferons, dideoxyinosine and ganciclovir. '
Representative examples of anxiolytics and
sedatives useful in the compositions and methods of the
invention include benzodiazepines such as diazepam,
barbiturates such as phenobarbital and other compounds
.such as buspirone and haloperidol. '
Representative examples of hormones useful in the
compositions and methods of the present invention include
estradiol, prednisone, insulin, growth hormone,
erythropoietin, and prostaglandins.
Representative examples of anti-depressives useful
in the compositions and methods of the present invention
include fluoxetine, trazodone, imipramine, and doxepin.
Representative examples of anti-nociceptives
useful in the compositions and methods of the present
invention include hydromorphine, oxycodone, fentanyl,
morphine and meperidine.
The term "biologics" encompasses nucleic acids
(DNA and RNA), proteins and peptides, and includes
compounds such as cytokines, hormones (pituitary and
hypophyseal hormones), growth factors, vaccines etc. Of
particular interest are interleukin-2, insulin-like
growth factor-1, interferons, insulin, heparin,
CA 02564120 2006-11-07
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leuprolide, granulocyte colony stimulating factor (GCSF),
granulocyte-macrophage colony stimulating factor (GM-
CSF), tumor necrosis factor, inhibin, tumor growth factor
alpha and beta, Mullerian inhibitory substance,
calcitonin, and hepatitis B vaccine.
The biologically active compound can be employed
in the present-invention in various forms, such as
molecular complexes or biologically acceptable salts.
Representative examples of such salts are succinate,
hydrochloride, hydrobromide, sulfate, phosphate, nitrate,
borate, acetate, maleate, tartrate, salicylate, metal
salts (e. g., alkali or alkaline earth), ammonium or amine
salts (e. g., quaternary ammonium), and the like.
Furthermore, derivatives of the active substances-such as
esters, aides, and ethers which have desirable retention
and release characteristics, but which are readily
hydrolyzed in vivo by physiological pH or enzymes, can
also be employed.
As used herein the term "therapeutically effective
amount" means the amount of a biologically active
compound necessary to induce a desired pharmacological
effect. The amount can vary greatly according to the
effectiveness of a particular active substance, the age,
weight, and response of the individual host as well as
the nature and severity of the host's symptoms.
Accordingly, there is no upper or lower critical
limitation upon the amount of the active substance: The
therapeutically effective amount to be employed in the
present invention can readily be determined by those
skilled in the art.
It is believed that the neutral lipid component in
MVL, which is unique to MVL among liposoma~l formulations,
interacts with the in vivo environment in such a way as
CA 02564120 2006-11-07
_ 2~ _
to affect the rate at which compounds encapsulated within
the MVL are released. In particular, MVL having a
neutral lipid component comprised of triglycerides with
less than 6 carbons in the acyl moiety interact with the
in vivo environment so as to become completely
destabilized virtually upon contact with blood plasma.
For this reason, saline solutions do not accurately mimic
the effect of the .in vivo environment on the drug release
characteristics of MVL, but it. has been discovered that
in vitro release studies conducted using blood plasma or
a plasma-like medium can be used to accurately determine
the in vivo release characteristics of an MVL
formulation. .
The following examples illustrate the manner~in
which the invention can be practiced. It is understood,
however, that the examples are for the purpose of
illustration, and the invention is not to be regarded as
li~i~W'~.. to c'.~ ny v~f t~'re ~peWifiv '.v'S'iat2ria-l~u' .~v~'
v'~'~?d3:t3oii
therein:
Example 1
G-CSF-containing MVL
1. Manufacture
For manufacture of multivesicular liposomes (MVL)
containing granulocyte colony stimulating factor (G-CSF),
the lipid combination solution contained (per ml
chloroform): 11 mg DOPC, 2.3 mg DPPG, 8.7 mg cholesterol
and either 2.4 mg (2.7 umol) triolein or 1.3 mg (2.7
umol) tricaprylin. Lipid solutions which contained four
different molar ratios of the neutral lipids triolein and
tricaprylin were prepared by mixing appropriate volumes
of the triolein and tricaprylin containing lipid
CA 02564120 2006-11-07
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solutions. The molar ratios of triolein to tricaprylin
were 100:0, 50:50, 25:75, 10:90., and 0:100:
The first aqueous phase solution for MVL
formulations contained 174 mM glycine, 100 mM HC1, O.OOlo
Tween80T"', and 100 ug/ml G-CSF, 1 ml was added to a vial
containing 1 ml of the lipid combination and emulsified
by fixing the capped vial in a horizontal configuration
to the head of a vortex mixer (Scientific Products) and
shaking at maximum speed (2400 oscillations/min.) for 6
minutes.
The final emulsion (2 ml) was divided~and
transferred to two vials containing 2.5 ml 3.2$ glucose
and 40 mM lysine. The emulsion was dispersed into .
microscopic droplets by fixing the capped vial in a
horizontal configuration to the head of a vortex mixer
and shaking for 3 seconds at 2400 oscillatioris/min. The
contents of the vial were transferred to a flask
containing 5 ml of 3.2$~glucose, 40 mM lysine. The
chloroform was removed from the microscopic droplets or.
spherules by transferring the flask to a 37°C gyrorotary
water bath and flushing the surface of the suspension
with nitrogen gas at a flow rate of 10 - 15 cfh for 10
minutes. Multivesicular liposomes in suspension
containing encapsulated G-CSF were obtained.
The particle suspensions were diluted 1:4 with
normal saline, and the particles were harvested by
centrifugation at 800 X g for 10 minutes. The
supernatant solution was removed by aspiration, and the
particles were washed twice by resuspension in fresh,
normal saline solution and centrifugation. The final
washed product was resuspended at 25~ packed-particle
volume per total volume and stored at 2-8°C for
subsequent studies.
CA 02564120 2006-11-07
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2. In vitro release rate
The in vitro release rate of the MVL prepared as
described above was determined by incubation in saline
solutions containing 70$ citrated-human plasma and 0.10
sodium azide at 37°C. G-CSF was determined in the
centrifuge-collected particle fraction by solubilization
of samples in 50~ IPA and quantitation by high pressure
liquid chromatography and UV detection using known
methods.
The dependence of the in vitro release rate of G-
CSF on the composition of the neutral lipid component
used in manufacture of the multivesicular liposomes is
shown in Figure 1. As the triolein to tricaprylin molar
ratio was increased, the rate of release of G-CSF from
the multivesicular liposomes decreased.
3. In vivo release rate
The triolein and tricaprylin MVL formulations of
G-CSF were evaluated in a pharmacodynamic model in Syrian
Golden Hamsters. Exogenous G-CSF stimulates neutrophil
(granulocyte) production which can be evaluated by
assaying blood samples for an increase in peripheral
blood neutrophil number and correspondingly an increase
in peripheral blood leukocyte number.
Therefore, the formulations containing tricaprylin
or triolein were evaluated for release in a
pharmacodynamic hamster model that measured peak and
duration of excess leukocyte (granulocyte) production
caused by rhu G-CSF (Cohen et a1. 1987, Proc. Natl. Acad.
Sci. USA, 89:2484-2488). In these studies the
subcutaneous injection of a solution of G-CSF was used as
a control for bioequivalence of the encapsulated protein.
The rapid-release formulation containing tricaprylin was
CA 02564120 2006-11-07
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also used as a control for bioequivalence of the
encapsulated protein. First, the peak and duration of
granulocyte increase upon subcutaneous injection of
hamsters with formulations containing all tricaprylin
(slow: fast molar ratio of 0:.100) was compared against
that caused by a solution of unencapsulated G-CSF and the
longer-release duration triolein formulation (slow: fast
mole ratio of 100:0). For each formulation; 3 to S
hamsters received 75-100 ug G-CSF per kg. The results
l0 (Figure 2); are as predicted by the in vitro release
studies. The tricapryiin formulation provided a rapid
release of G-CSF and stimulated granulocyte production
similar to that of the unencapsulated G-CSF solution.
The triolein formulation had a lower (and later) peak and
longer duration.
As a further control, a group of hamsters were
injected with the triolein formulation solubilized with
detergent (Tween 20~') immediately prior to injection.
The granulocyte number found at.24 hours for the
solubilized formulation was. similar to that observed
resulting from injections of the G-CSF-solutions or the
formulation containing only tricaprylin as the neutral
lipid (Figure 2).
Example 2
1. Manufacture
Mul.tivesicular liposomes containing granulocyte/
macrophage-colony stimulating factor (GM-CSF) were
manufactured as described in Example 1, but using neutral
lipid molar ratios of triolein to tricaprylin of 100:0,
25:75 and 10:90.
CA 02564120 2006-11-07
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2. In vitro release in human plasma at 37°C
The suspensions were prepared and incubated in
human plasma at 37°C as described in Example 1. GM-CSF
remaining encapsulated was determined by solubilization
of the particle fraction.in 50o IPA and quantitation by
high pressure liquid chromatography and UV detection
using known methods.
The results of the in vitro release assay (Figure
3) demonstrate that the graded replacement of triolein
with tricaprylin results in graded increases in release
rate.
3. In vitro pharmacokinetics
BAZBc mice (aged 7 to 8 weeks and weighing
approximately 20 grams) were administered subcutaneous
injections of liposomes containing GM-CSF manufactured
with either 100 triolein or a 25:75 ratio of triolein to
tricaprylin. Blood samples were collected before the
injection and at 1, 2, 4, and 7 days post injection, and
the plasma was assayed for GM-CSF concentration using an
ELISA kit. The formulation manufactured with 25:75
triolein to tricaprylin provided a higher peak level and
a shorter duration of detectable rhu GM-CSF compared to
the formulation manufactured with lOD~ triolein as the
neutral lipid (Figure 4). These results are as predicted
by the in vitro assay in human plasma at. 37°C, and show
that the rate of release of encapsulated GM-CSF is
increased by addition of tricaprylin to the neutral lipid
component.
CA 02564120 2006-11-07
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Example 3
Depo/IGF-1
1. Manufacture
Multivesicular liposomes were manufactured with
rhu IGF (recombinant human insulin-like growth factor),
with '"C-sucrose as an osmotic spacer, and ammonium
citrate as a buffer. The lipid combination solution
contained (per liter) 10.4 g DOPC, 2.1 g DPPG, 7.7 g
cholesterol, and the triglyceride component was either
2.1 g triolein, 1.9 g tripalmitolein, 1.5 g trilaurin,
1.3 g tricaprin, or 1.1 g tricaprylin (molar ratio,
0.34:0.07:0.52:0.06).
The first aqueous phase solution contained (per
ml) 16 mg rhu IGF-1 (Chiron), 7g sucrose, and 20 mM
ammonium citrate, pH 5. A first emulsion was made by
high-speed mixing of 5 ml of the lipid combination
solution with 5 ml of the aqueous phase solution at 9000
rpm for 9 minutes at 25-27°C. The emulsion was sheared
into microdroplets (spherules) by addition of 30 ml 40 mM
lysine, 4~ glucose solution to the mixing vessel and
mixing at 6000 rpm for 1 min. The chloroform was removed
from the microscopic droplets or spherules by
transferring the suspension to a flask containing 70 ml
of 40 mM lysine, 3.2~ glucose solution, placing the flask
in 37°C gyrorotary water bath, and flushing the surface
of the suspension with nitrogen gas at a flow rate of 70
cfh for 20 minutes to obtain the MVh particles in
suspension.
The suspensions were diluted 1:4 with normal
saline, and the particles were harvested by
centrifugation at 800 X g fo= 10 minutes at room
temperature. The supernatant solution was removed by
CA 02564120 2006-11-07
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aspiration, and the particles were washed twice by
resuspension in fresh, normal saline solution and
centrifugation. -
2. In vitro release rates
The final washed product was resuspended at 25%
packed-particle volume per total volume and stored at a
temperature of 2-8, 25, 32, or 37°C for either 24 or 48
hours. The pellet fraction from the 25,000 X G X 2
minute centrifugation was solubilized and assayed by HPLC
1O for IGF-1 retained. Also, each of the IGF-1-containing
formulations stored at 2-8°C was evaluated within 24
hours in~the in vitro release assay described in Example
1 and the results are summarized in Figure 5. The
formulations manufactured with triolein (C18:1)or
tripalmitolein (C16:1) showed a similar release rate
profile.. Greater than 70% of the encapsulated IGF-1
released in 7 days. The formulation manufactured with
tricaprylin (C8) demonstrated a rapid release rate
profile with nearly complete release of IGF-1 in 2 days.
The formulations manufactured with tricaprin (C10) or
trilaurin (C12) released IGF-1 markedly more slowly than
the standard triolein formulation, less than 50% released
in 7 days. These results suggest that the acyl chain
length of the triglyceride is not directly correlated
with. the rate of release of encapsulated IGF-1 in vitro,
because the release of the tricaprylin (C8) formulations
was more rapid than that of the tricaprin (C10) or
trilaurin (C12) formulations.
It should be noted that storage of the trilaurin
formulations in saline for only a few days at 2-8°C
resulted in morphological reorganization .of the particles
and an accompanying complete release of encapsulated
CA 02564120 2006-11-07
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materials. This effect would not be expected from the
stability exhibited in the in vitro release assay at
higher temperatures. This catastrophic effect was
related to storage of the formulation at temperatures
significantly lower than the freezing point of the
neutral lipid. This effect is demonstrated in Examples
16 and l7 below.
3. In vivo pharmacokinetics
Male,~Sprague-Dawley rats (weighing.250 to 30.0g)
were given subcutaneous injections of the multivesicular
liposome formulation containing IGF-1 that were
manufactured with either tri.caprylin or tripalmitolein as
the neutral lipid. The tricaprylin formulation
demonstrated a higher peak level of plasma rhu IGF-1 and
a short release duration compared to the formulation
manufactured with tripalmitolein (Figure 6). This result
confirms the prediction of rapid release of
multivesicular liposomes manufactured with tricaprylin
based upon results of the in vitro release assay.
Example 4
1. Manufacture
For manufacture of multivesicular liposomes
containing rhu-insulin, the lipid combination solution
contained (per ml chloroform): 11 mg DOPC, 2.3 mg DPPG,
8.7 mg cholesterol and either 2.4 mg (2.7 umol) triolein
or 1.3 mg (2.7 umol) tricaprylin as indicated.
The first aqueous phase solution formulations
contained 7.5~ ~4C-sucrose, 20 mM citric acid, 50 mM HC1
and 5 mg/ml rhu-Insulin (E. Coli, Sigma Chemical Co., St.
Louis, MO). A first emulsion was made by high-speed
CA 02564120 2006-11-07
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mixing of 4 ml of the lipid combination solution with 4
ml of the aqueous phase solution at 9000 rpm for 8
minutes at 25-27°C. The emulsion was sheared into
microdroplets (spherules) by addition of a solution to
the mixing vessel of 20 ml 20 mM lysine, 4~ glucose, and
mixing at 3000 rpm for 1 min. The chloroform was removed
from the microscopic droplets or spherules by
transferring the suspension to a flask containing 30 ml
of a solution of 20 mM lysine, 4~ glucose at 37°C in a
gyrorotary water bath and flushing the~surface of the
suspension with nitrogen gas at a flow rate of 70 cubic
feet per hour for 20 minutes to obtain the MVL particles
in suspension.
The suspensions were diluted 1:4 with normal
saline, and the particles were harvested by
centrifugation at 800 X g for 10 minutes. The
supernatant solution was removed by aspiration, and the
particles were washed twice by resuspension in fresh,
normal saline solution and centrifugation. The final
washed product was resuspended at 250 ~,acked-particle
volume per total volume and stored at 2-8°C for
subsequent studies.
2. In vitro release rates
The "in vitro" release assay was performed by
diluting 1 volume of stored suspension with 3.2 volumes
of citrated, human plasma, and placing 0.6 ml of this
suspension into microfuge tubes which were stoppered and
incubated under dynamic conditions at 37°C. After 1, 3,
6, and 7 days, the tubes were centrifuged at 14,000 X g X
2 minutes, and the supernatant solution was transferred
to another tube for bioassay. The pellet fractions of
centrifuged samples were incubated in 1~ Nonidet~ NP-40
CA 02564120 2006-11-07
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(CalBiochem, San Diego, CA), 50 mM trifluoroacetic acid
for 10 minutes at 37°C. The 1°C sucrose and insulin
retained by the pellet fraction was determined,
respectively, by scintillation counting and reverse phase
HPZC using known methods. The results of these studies
showed that the multivesicular liposomes manufactured
with triolein as the neutral lipid released both sucrose
and insulin in an equivalent and linear fashion, with
nearly complete release in 7 days. ~By contrast, the
formulation manufactured with tricaprylin 'retained only
to 25~ of the encapsulated sucrose and insulin after
only 1 day of incubation in plasma (Figure 7).
Example 5
I: Manufacture
15 For manufacture of multivesicular liposomes
containing morphine, a GMP-validatable, scalable process
was used. The neutral lipid component molar ratios of
triolein to tricaprylin were. 1:4 or 1:9. Control
formulations contained triolein or tricaprylin as the
20 sole neutral lipid. The lipid combination solution
contained (per liter) 10.2 g~DOPC, 2.0 g DPPG, 7.6 g
cholesterol, 2.1 g to 1.1 g triglyceride depending on
the molar ratio of triolein:tricaprylin, (molar ratio,
0.34:Ø07Ø52:0.06) .
The first' aqueous phase solution contained (per
liter) 21 g morphine sulfate pentahydrate, 0.01 N
hydrochloric acid. A first~emulsion was made by high-
speed mixing of 0.62 1 of lipid combination solution with
0.9 1 of aqueous phase solution at 8000 rpm for 30
minutes at 25-27°C. The emulsion was sheared into
microdroplets (spherules) by transferring to a second
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mixing vessel containing 15 liter 4 rnM Lysine, 5.9%
glucose solution; and mixing at 1250-1300 rpm for 1.5 min
at 45°C. Chloroform was removed from spherules by
sparging the suspension for 50 min at 45°C in stepped
intervals: 17 min at 15 1/min, 5 min at 40 1/min, 28 min
at 10 1/min. The MVL particles were thus obtained in
suspension. The particles in the final suspension were
concentrated and washed free of unencapsulated morphine
by either cross-flow- or dia-filtration with 25 1 of
normal saline solution. The final washed product was
stored at 2-8°C for subsequent studies.
2. In vitro release profiles
The in vitro release assays were performed by a
1:9 dilution of suspensions which contained 8 to 15 mg of
encapsulated morphine sulfate per ml into human plasma.
The suspensions were incubated at 37°C under dynamic
conditions. After 1, 2, 4, and 7 days, samples were
diluted 1:4 with normal saline, the particles were
sedimented by centrifugation~at 800 X g X 10 min and the
particle fraction was assayed to determine the amount of
morphine retained by solubilization of the pellet
fraction with 50% IPA and assay by UV spectrophotometry
using known methods.
Figure 8 shows the release rate profile of
morphine release for these formulations into human
plasma. As shown in earlier examples, as the ratio of
the triolein to tricaprylin increases, the~rate and
extent of morphine release decreases.
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'3. In vivo pharmacokinetics
The in vivo release of the multivesicular
liposomes containing morphine manufactured with triolein
and tricaprylin alone or in combination was evaluated in
the Beagle dog using an epidural injection,
pharmacokinetic model. In these studies the level of
morphine released from the liposomal formulation injected
at a epidural site was determined in the plasma and at an
adjacent intrathecal site (CSF, cerebral spinal fluid)
separated from the epidural site by the spinal meningeal
membrane. Only the CSF results are shown (Figure 9).
A correlation is found between the-in vitro
release results summarized in Figure 8 and the release
rate profiles observed in the dog model (Figure 9): The
in vivo tricaprylin-containing formulation releases very
rapidly, with nearly complete release in 24 hours. The
Mean Residence Time (MRT) for the 100% tricaprylin
formulation was similar to that for injection of
unencapsulated morphine, i.e., 2.3 h vs. 1.6 h. The
inclusion of a small amount of triolein during
manufacturing, i.e., 1:9 or 1:4 triolein:tricaprylin
molar ratio slows the release rate, extending the release
duration over 4 to 5 days, with MRTs of 13.2 h and 15.3
h, respectively. The tricaprylin containing formulations
all release more rapidly than the formulation using
triolein as the only neutral lipid, which had a MRT of 69
h in this model.
4. Storaae Stability
The step-wise alteration in the neutral lipid
composition of the MVL to provide a family of
formulations with step-wise increase in the release rate
profiles of bioactive compound does not seem to
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compromise the storage stability of the formulations
provided that the formulations are stored near or above
the melting point of the neutral lipid combination.
Batches of the 10:90 triolein:tricaprylin morphine MVh
formulation, described above and which release rapidly
both in vitro and in vivo, were evaluated for dependence
of the release rate on temperature of storage. The MVL
suspensions in saline storage solution were incubated at
the normal storage temperature, 2-8°C (4°C.nominal) and at
elevated temperatures, 26, 37, and 41°C. The results of
these studies are shown in Figure 1D, an Arrhenius.plot.,
which plots the logarithm of the-release rate against
1/Temperature (°K). The slope of the plot appears to be
continuous. The release rate profile at high temperature
supports the observation that the MVL stored in saline
solution at 2-8°C have a very slow release (about 1~ of
the encapsulated morphine per 100 days) and therefore can
be expected to have a shelf-life in excess of a year if a
criteria of S or 10~ release of encapsulated material is
the limit for shelf-life. Further, it is also evident.
that a physiological matrix such as plasma (Figure 9) or
the in vivo environment of the epidural space (Figure 9)
greatly accelerates the release when compared to the
release in saline solution at 37°C, i.e., 50~ of the
encapsulated morphine released per day, versus less than
1°s of encapsulated morphine per day in the latter.
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Examt~le 6
Cytosine Arabinoside
1. Manufacture
Multivesicular liposomes containing cytosine
arabinoside (AraC) were manufactured with various
substitutions for the phosphatidylcholine component of
the lipid organic phase solution. The substitutions were
performed to determine the effect on the release rate
profile of changing the acyl component in the major
phospholipid in a.MVL formulation containing either.
triolein or tricaprylin as the neutral lipid.
In this first example, DOPC is used to demonstrate
that the release rate in a physiological medium from MVL
formulations containing AraC/HC1 in the aqueous phase can
be modified by adjusting the triolein to tricaprylin
ratio. The lipid combinations contained (per 1200 ml)
122.4 ml DOPC at 100 mg/ml, 2:4 g DPPG; 9:12°g_
cholesterol, 2.5 g triolein or 1.3 g tricaprylin, or a
mixture thereof yielding a molar ratio of triolein to
tricaprylin of 1:4, 1:9, 1:18, or 1:27.
The first aqueous phase solution contained 20
mg/ml cytosine arabinoside, 0.1 N Hcl. A first emulsion
was made by high speed mixing of 10 ml lipid combination
and 10 ml of first.aqueous phase solution at 9000 rpm for
9 min. The emulsion was sheared into microdroplets by
transfer of first emulsion to second mixing vessel
containing 200 ml of 3.2~ glucose, 40 mM lysine and
mixing at 2100 rpm for 2.5 minutes. The chloroform was
removed by transferring suspension~to two, 1-liter flasks
and flushing at 70 cfh. The final suspension was diluted
1:2 with saline and particles collected by centrifugation
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at 800 X g X 10 min washed 3 times, resuspended to 33~
lipocrit, and then adjusted to 10 mg/ml.
2. In vitro release into human plasma
The in vitro release rate profiles of the cytosine
arabinoside (AraC) MVL formulations were obtained by
dilution of the suspension 1:9 into human plasma and
incubation of samples at 37°C under dynamic conditions.
At time points of 1, 2, 4, and 7 days, 1.2 ml of saline
was added to triplicate 0.3 ml samples, and the particle
fraction was collected by centrifugation at 16,000 X g X
2 min. The supernatant fraction was removed by
aspiration, and the particle fraction was resuspended in
1 ml of 50°s isopropyl alcohol, vortexed, incubated at
37°C for 10 min, centrifuged and then 0.06 or 0.2 ml of
the supernatant was added to 1.0 ml of 0.1 N HC1.
Cytosine arabinoside was determined by U.V. samples at
280 nm.
The in vitro release profiles measuring AraC
retained by the formulations of multivesicular liposomes
are shown in Figure 11. Release from the formulation
having only tricaprylin as the neutral lipid was rapid.
As the ratio of tricaprylin to triolein used in the
manufacture of the MVL formulation increases, the rate of
release of cytosine arabinoside by the MVL incubated in
plasma decreases. Thus, a family of release rate curves
with predictably increasing release rates is created by
incremental increases in the amount of triolein in the
triolein to tricaprylin ratio.
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Example 7
Effect of Substitution of Higher Transition
Temperature PhosphatidylCholines or Longer Chain Length
for DOPC in Formulations Containing Tricaprylin
The ranking in a family of in vitro release rate
profiles (as obtained in Example 6) with the greater
molar ratio of tricaprylin to triolein resulting in v
faster release both in vivo and in in vitro models
(plasma at 37°C), remains consistent when a
phospha.tidylcholine of higher phase transition
temperature or of increased chain length is substituted
for DOPC in the formulations of Example 6. In this
example distearoylphosphatidylcholine (DSPC, mp 55°C) was
substituted for DOPC (mp 0°C).
1. Manufacture
The manufacturing parameters were adjusted to
accommodate the higher mp of DSPC by performing
emulsification at 50°C. The manufacture of emulsions
used for the DOPC control formulations was performed at
ambient temperature. In addition, the HC1 concentration
of the first aqueous phase solution was increased 36%
over that in Example 6.
The lipid combination solution contained (per
liter) 10.2 g DOPC or 10.3 g DSPC, 2.1 g DPPG, 7.7 g
cholesterol, and the triglyceride component was either
1.1 g tricaprylin or 2.1 g triolein (molar ratio of
0.34:0.07:0.52:0.06). Mixtures of triolein and
tricaprylin were blended to provide the ratios of 1:4,
1:9, 1:18, 1:2 triolein to tricaprylin for use in the
formulations.
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The first aqueous phase contained (per ml) 20 mg
cytosine arabinoside and 136 mM Hcl. An emulsion was
made by high-speed mixing of 10 ml of the lipid
combination solution with 10 ml of the aqueous phase
solution using a 2 cm diameter blade in a stainless steel
vessel at 9000 rpm for 8 minutes at room temperature (for
formulations with DOPC) or 55°C (for formulations with
DSPC). The emulsion was sheared into microdroplets by
transfer into a 400 ml glass Mason jar containing 200 ml
of 5$ glucose and 40 mM lysine'and, using a 4 cm diameter
blade, mixing at 4000 rpm for 1.5 minutes at the same
temperature as used for the first emulsification.
Chloroform was removed by placing the container in a 37°C
gyrorotary water bath.and flushing the'surface of the
suspension with nitrogen gas at a flow rate of 70 cfh for
minutes.
The suspensions were diluted 1:4 with normal
saline and the particles were collected by centrifugation
at 800 x g for 10 minutes at room temperature. The
20 supernatant was removed by aspiration, and then the
particles were washed twice by resuspension in normal
saline solution and centrifugation. The final washed.
pellet was resuspended in normal saline solution and
adjusted to 10 mg/ml of cytosine arabinoside.
2. In vitro release profiles
The in vitro release assay of AraC from the MVLs
was performed in human plasma as described in Example 6
above. As shown in Figure 12, in MVL in which the higher
melting point DSPC was substituted for DOPC, a family of
graded release rate profiles was obtained-by varying the
molar ratio of tricaprylin to triolein, with the faster
release formulations containing a higher molar ratio of
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tricaprylin to triolein. As has been shown previously
for other formulations of MVZ, an increase in
hydrochloric acid concentration in the aqueous phase
solution (as compared with that contained in the MVZ of
Example 6) can slow the release of the active substance
in vitro.
Exaiaple 8
Sucrose
1. Manufacture
In this example, the effect of tr'icaprylin on the
release rate was tested for formulations containing 4~
sucrose as the active agent in formulations having
dieucrylphosphatidylcholine (DEPC, mp (0°C), a 22 carbon
chain length phosphatidylcholine, substituted for
dioleolyphosphatidylcholine (DOPC, mp (0°C), a T8 carbon
chain length phosphatidylcholine.
The lipid solution contained (per liter) either
10.2 g DOPC or 11.0 g DEPC and 2.1 g DPPG, 7.7 g
cholesterol, and the triglyceride component was either
1.1 g tricaprylin or 2.1 g triolein (molar ratio of
0.34:0.07:0.52:0.06). For mixtures of the neutral lipid,
triolein- and tricaprylin-containing lipid combinations
were blended to provide these triolein to tricaprylin
molar ratios.
The first aqueous phase contained 4% Sucrose
(Spectrum USP/NF, Los Angeles, CA) and was spiked with 40
uL of 1°C sucrose. A first emulsion was made by high-
speed mixing of 5 ml of the lipid combination solution
with 5 ml of the aqueous phase solution using a 2 cm
diameter blade in a stainless steel vessel at 9000 rpm
for l0 minutes at room.temperature. The emulsion was
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sheared into microdroplets by transfer to a 400 ml glass
Mason jar containing 200 ml of 4% glucose and 4 mM lysine
for 2 minutes, using a 4 cm diameter blade and mixing at
3500 rpm. Chloroform was removed from the droplets by
transferring the suspension to a 1 liter culture flask
placed in 37°C gyrorotary water bath and flushing the
surface of the suspension with nitrogen gas at a flow
rate of 70 cfh for 20 minutes.
The particle suspensions were diluted 1:2 with
normal saline; and the particles were collected by
centrifugation at 800 x g for 10 minutes at room
temperature. The supernatant was removed by aspiration,
and the particles were washed twice by resuspension in
normal saline solution and centrifugation. The final,
washed particle pellet was resuspended in normal saline
solution and adjusted to approximately 33% lipocrit.
2. In vitro plasma release studies
For in vitro, plasma release studies, the
suspensions were diluted 1:10 in human plasma with 0.1%
sodium azide..Triplicate aliquots of 300~uL were
incubated in 1.5 ml screw-top Eppendorf tubes and
harvested on days 0, 1, 2, 3, and 4. Samples were
harvested by pulling tubes from the incubator at random,
labeling the tube with day of pull, diluting the contents
with 1.2 ml of normal saline solution, and centrifuging
at 27,000 x g for 5 minutes in a microfuge. The
supernatant was carefully aspirated away from the
particle pellet. The pellet fraction was resuspended in
1 ml of 50% IPA by vortexing, incubating at 37°C for 10
minutes, and then vortexing. A 50 u1 sample was then
diluted with 3 ml of scintillation fluid in a
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scintillation vial, the vial was shaken vigorously and
sucrose was determined by scintillation counting.
As can be seen from the data contained in Figure
13, the formulation containing only tricaprylin released
all encapsulated sucrose within one day when incubated in
plasma. The ratio of triolein: tricaprylin of 1:18 was
required to achieve an intermediate rate of release for
an MVL formulation with this aqueous phase when DEPC
replaced DOPC.
Example 9
In this example, tricaproin (C6) was substituted
for tricaprylin (C8) as a release rate modifying neutral
lipid.
- 1: Manufacture
The lipid combination solution contained (per
liter) a combination of 10.2 g, 2.1 g DPPG, 7.7 g
cholesterol, and the triglyceride component was either
0.9 g tricaproin or 2.1 g triolein (molar ratio of
0.34:0.07:0.52:0.06). Mixtures of the lipid solutions
containing triolein'and tricaproin, were blended to
provide lipid solutions containing molar ratios of
triolein to caproin of 1:4, 1:9 and 1:18. These
formulations were manufactured and tested as described in
Example 8.
The first aqueous phase contained 4~ Sucrose
(Spectrum USP/NF) and was spiked with 40 uL of 1'C Sucrose
(ICN lot#54661027). A first emulsion was made by high-
speed mixing of 5 ml of the lipid combination solution
with 5 ml of the aqueous phase solution using a 2 cm
diameter blade in a stainless steel vessel at 9000 rpm
for 10 minutes at room temperature. The second
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emulsification was performed in a 400 ml glass Mason jar
containing 200 ml of 4% glucose and 4 mM lysine, using a
4 cm diameter blade and mixing at 3500 rpm for 2 minutes.
Chloroform was removed by transferring the contents of
the jar to a 1 liter culture flask placed in 37°C
gyrorotary water bath and flushing the surface of the
suspension with nitrogen gas at a flow rate of 70 cfh for
20 minutes.
The suspensions were diluted 1:2 with normal
saline, and the particles were harvested by
centrifugation at 800 x g for 10 minutes at room
temperature. The supernatant was removed by aspiration,
and the particles were washed twice by resuspension in
normal saline solution and centrifugation. The final
washed pellet was resuspended in normal saline solution
and adjusted to approximately 33% lipocrit wherein
lipocrit.in percent is the volume occupied by the
liposomes divided by the total volume of the liposome
suspension multiplied by one hundred. The yield for each
variation in the neutral lipid was greater than 50%.
Following manufacture the free sucrose concentration at
33% lipocrit was approximately 3% of the total sucrose
concentration.
2. In vitro release profiles
. For in vitro plasma release studies, the
suspensions were diluted 1:10 in human plasma.
Triplicate aliquots of 300 uI. were incubated dynamically
in 1.5 ml screw-top Eppendorf tubes and harvested on days
0,1,2,3, and 4. Samples were harvested by pulling tubes
from the incubator at random, labeling the tube with the
day of the pull, diluting the contents with 1.2 ml of
normal saline solution, and centrifuging at 27,000 x g
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for 5 minutes in microfuge. The supernatant was
carefully aspirated away from the pellet. The pellet
fraction was resuspended in 1 ml of 50$ IPA by vortexing,
incubating at 37°C for 10 minutes, and then vortexing
again. A 50 u1 sample was then diluted with 3 ml of
scintillation fluid in a scintillation vial, the vial was
shakew vigorously, and 14C-sucrose radioactivity was
determined by scintillation counting.
As shown in Figure 14, the substitution of
tricaproin for tricaprylin in mixtures containing
triolein as a release rate modifying neutral. lipid
combination results in formulations having a graded
family of release rates for various triolein:tricaproin
molar ratios. The greater the molar ratio~of tricaproin
to triolein, the more rapid the release of sucrose.
There is a distinguishing difference between tricaproin
and tricaprylin containing formations. With this aqueous
phase, i.e., containing sucrose as the encapsulated
active agent, the multivesicular particles manufactured
using only tricaproin underwent a physical transformation
and released their contents within 5 minutes of dilution
into human plasma at room temperature. Dilution into
saline solution containing 0.5~ bovine serum albumin had
the same effect. By contrast, the formulations
containing only tricaprylin as the neutral lipid
generally required 12 or more hours of incubation in
plasma at 37°C for complete release of the active agent.
Despite the instability of the tricaproin only -
formulations in human plasma or saline at room
temperature. The formulations were stable during storage
at 2-8°C in saline for at least a week.
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Example 10
Antisense Oligonucleotides
This example illustrates use of the method of the
invention to encapsulate antisense oligonucleotides.
1. Manufacture
The first aqueous phase solution contained (per
ml) 5, 10 or 20 mg of an IL-6 antisense oligonucleotide
(donated by Leu Neckers, NIH, Bethesda, MD), which in
some studies was biotinylated, and 5~ mannitol.
Hydrochloric acid (0.1 ml of 1 N) was added to a
vial containing 1 ml of the lipid combination of Example
1 and the combination was emulsified by fixing the capped
vial in a horizontal configuration to the head of a
.vortex mixer (Scientific Products) and shaking at 2400
oscillations/min for 1 minute. The remainder of the
first aqueous phase solution (0.9 ml 5~ mannitol
containing 10 mg antisense oligonucleotide) was added to
the vial, and emulsification was continued for 5 minutes:
The final emulsion (2 ml) was divided and
transferred to two vials containing 2.5 ml 3.2~ glucose,
and 40 mM lysine. The emulsion was dispersed into
microscopic droplets by fixing the capped vial in a
horizontal configuration to the head of a vortex mixer
and shaking for 3 seconds at approximately 1200 rpm. The
contents of the vial were transferred to a flask
containing 5 ml of 3.2~ glucose, 40 mM lysine, and the
chloroform was removed from the microscopic droplets or
spherules by transferring the flask to a 37°C gyrorotary
water bath, and flushing the surface of the suspension
with nitrogen gas at a flow rate of 15 cubic feet per
hour for 10~ minutes.
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The suspensions were diluted 1:4 with normal
saline and the particles were harvested by centrifugation
at 800 X g for 10 minutes. The supernatant solution was
removed by aspiration, and the particles were washed.
twice by resuspension in fresh, normal saline solution
and centrifugation. The final washed product was
resuspended at 25~ packed-particle volume per total
volume and stored at 2-8°C for subsequent studies.
The recovery of encapsulated oligonucleotide was
determined by diluting a sample of the~suspension 1:1
with 0.1% SDS, incubating,the sample in a boiling water
bath for 2 minutes, and then diluting the sample
(typically 1/20) into 0.1 N NaOH for determination of UV
absorption using a wavelength scan from 320 to 212
nmeters. The concentration of oligonucleotide in the
sample was calculated by subtracting the measured A320
absorbance value from~the A257 absorbance value. A
sample of the first aqueous phase~solution served as the
standard, 17.6 (A257-1 cm) units per mg/ml.
2. In vitro release profiles
The in vitro release characteristics of
multivesicular particles containing oligonucleotides were
determined by measuring the amount of o7:igonucleotide
remaining with particle fraction when incubated at 37°C
in rat cerebral spinal fluid (CSF). Samples stored in
normal saline were resuspended in the storage solution,
and centrifuged at 750 X g for 10 minutes. The saline
supernatant solution was removed by aspiration, and the
particles were resuspended in rat CSF to concentrations
of 0.25 to 0.5 mg of the oiigonucleotide per ml of CSF.
The samples were incubated at 37 °C under static
conditions. After 0, 1, 2, 3, and 7 days, samples of the
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particle/CSF suspensions were removed, diluted 10-fold in
saline, centrifuged, and the supernatant was aspirated
away from the particle fraction. The particle fraction
was incubated in 0.1% SDS and diluted with 0.1 N NaOFi,
and a UV absorbance spectrum of the oligonucleotide was
obtained over 340 to 210 nmeter range. The .amount of
oligonucleotide retained by the particle fraction was
determined as above.
As shown in Figure 15, the tricaprylin formulation
did not release in vitro more rapidly, but rather had a
greater overall release; the triolein-containing .-
formulation stopped releasing the drug in rut CSF after
about 2.5 days as compared with continued release by the
tricaprylin-containing formulation.
3. In vivo pharmacokinetics
When the antisense MVL formulations manufactured
with tricaprylin as the neutral lipid were tested in vivo
by intrathecal injection in rats and samples of the CSF
subsequently taken were examined microscopically, no
particles were evident in CSF after two days. The MVL
particles manufactured with triolein were evident at two
days but had a "shrunken" appearance. There was no
evidence of free, native oligonucleotide by a 3'-end
labeling assay.
To improve the sensitivity of the in vivo assay
for oligonucleotide concentration in CSF, the
oligonucleotide was biotinylated and the biotinylated
oligonucleotide was formulated as described above in MVL
using either triolein or tricaprylin as the neutral
lipid. The recoveries of encapsulated biotinylated
oligonucleotide were 78% for triolein and 82% for
tricaprylin-containing MVL. When studied in vivo,
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biotinylated oligonucleotide, found free in the CSF
released from the tricaprylin-containing liposomes was
below the limit of detection after 2 days. Free,
biotinylated oligonucleotide was present in the rat
sample CSF 4 days after administration of the triolein-
containing formulations at a concentration of
approximately 0.S uM (results not shown). The in vivo
data are consistent with the tricaprylin MVL rapidly
releasing antisense oligonucleotide in vivo, and the
triolein formulation providing a more sustained release
of the oligonucleotides.
Example 1l
Plasmid-containing MVL
1. Manufacture
For manufacture of multivesicular liposomes
encapsulating the E. coli PBR322 plasmid, the lipid
combination solution contained (per liter) 10.4 g DOPC,
2.1 g DPPG, ?.7 g cholesterol, 2.16 g triolein (mw
885.40) or 1.15 g tricaprylin (mw 470.7) (DOPC . DPPG .
Cholesterol . triglyceride molar ratio,
0.34:0.07:0.52:0.06).
The first aqueous phase solution contained (per
liter) 42 g 14C-sucrose (0.6 uCi per ml) 100 mmol lysine,
84 mmol hydrochloric acid, pH 7.4 and 20 ug/ml PBR322
plasmid (Promega, Madison WI). A first emulsion was made
by high-speed mixing of 3 ml of lipid combination
solution with 3 ml of aqueous phase solution at 9000 rpm
for 9 minutes at 25-27°C. The emulsion was sheared into
microdroplets (spherules) by addition of 20 ml of a 20 mM
lysine, 4% glucose solution to the mixing vessel and
further mixing at 4000 rpm for 2 min. The chloroform was
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removed from the microscopic droplets or spherules by
transferring the suspension to a flask containing 30 ml
of 20 mM lysine, 4~ glucose solution at 37°C in a
gyrorotary water bath, and flushing the surface of the
suspension with nitrogen gas at a flow rate of 70 liter
per hr. for 20 minutes to form the MVL.
The MVL suspensions were diluted 1:4 with normal
saline, and the particles were harvested by
centrifugation at 800 X g for 10 minutes. The
supernatant solution was removed by aspiration and the
particles were washed twice by resuspension in fresh,
normal saline solution and centrifugation. The final
washed product was resuspended to 25~ packed-particle
volume per total volume, and stored at 2-8°C for
subsequent studies.
2. In vitro release urofile
The in vitro release assays were performed by a
1:2.5 dilution of suspensions which contained the
multivesicular liposomes encapsulating PBR322 plasmid and
1°C-sucrose into human plasma. Previous studies had
established that 1"C-sucrose release was an adequate
surrogate for estimating the release of the PBR322
plasmi.d from the MVL. The suspensions were incubated at
37°C under static conditions. At times of 0, 1,_2, 3; 6,
10, and 17 days, samples were diluted 1:4 with normal
saline, particles were sedimented by centrifugation at
800 X g X 10 min, and the particle fraction was assayed
by dissolving samples in a scintillation counting
solution and performing scintillation counting of the
amount of '9C-sucrose retained by the particle fraction.
The results of this study shown in Figure 16
indicate the PBR322 plasmid formulation using triolein as
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the neutral lipid was stable under simulated in vivo
conditions and released about 30~ of the encapsulated
1°C-sucrose over a span of 17 days; whereas, the
tricaprylin-formulated particles rapidly released their
contents upon contact with human plasma at 37°C.
Example 12
In the following example sucrose and lysine-HC1
used as excipients in the PBR322 plasmid formulation were
encapsulated without additional active agent to yield an
"excipient only" family of graded, sucrose release
formulations using tripalmitolein:tricaprylin molar
ratios as the neutral lipid.
1. Manufacture
For manufacture of multivesicular liposomes
encapsulating only sucrose-lysine HC1, the lipid
combination solution contained (per liter) 10.4 g DOPC,
2.1 g DPPG, 7.7 g cholesterol, 1.9 g to 1.0 g
triglyceride depending on the molar ratio of
tripalmitolein (mw 801, C16:1 9C):tricaprylin to yield a
DOPC:DPPG:cholesterol:triglyceride molar ratio of
0.34:0.07:0.52:0.06. The molar ratios of tripalmitolein
to tricaprylin prepared in the formulations of this
example were 0:1, 1:0 , 1:9, 1:4, 1:2, and 1.1.
The first aqueous phase solution contained (per
liter) 42 g 1°C-sucrose (1. 0 ~zCi per ml) , 100 mmol lysine,
90 mmol hydrochloric acid, pH 5.7. A first emulsion was
made by high-speed mixing of 5 ml of lipid combination
solution with 5 ml of aqueous phase solution at 9000 rpm
for 9 minutes at 25-27°C. The emulsion was sheared into
microdroplets (spherules) by addition of 20 ml of a 20 mM
CA 02564120 2006-11-07
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lysine, 4$ glucose solution to the mixing vessel, and
mixing at 5000 rpm for 1.5 min. The chloroform was
removed from the microscopic droplets or spherules by
transferring the suspension to a flask containing 30 ml
of the 20 mM lysine, 4~ glucose solution in a 37°C
gyrorotary water bath, and flushing the surface of the
suspension with nitrogen gas at a flow rate of 70 cubic
feet per hr. for 20 minutes to obtain the multivesicular
particles in suspension.
The suspensions were diluted 1:4 with normal
saline, and the particles were harvested by
centrifugation at 800 X g for 10 minutes. The
supernatant solution was removed by aspiration and the
particles were washed twice by resuspension in fresh,
normal saline solution and centrifugation. The final
washed product was resuspended at 25~ packed-particle
volume per total volume and stored at 2-8°C for
subsequent studies.
2. In vitro release profile
The "in vitro" release assays were performed by a
1:9 dilution into human plasma of suspensions which
contained multivesicular liposome encapsulating "C-
sucrose. The suspensions were incubated at 37°C under
dynamic gentle~mixing. At time points of 0, 1, 3, 5, 8,
and 12 days, samples were diluted 1:4 with normal saline,
the particles were sedimented by centrifugation at 800 X
g for 10 min, and the particle fraction was assayed by
dissolving in scintillation counting solution and
scintillation counting of the amount of 14C-sucrose
retained in the particles. The results of~these studies
(Figure 17) show that a 1:1 molar ratio of tripalmitolein
to tricaprylin provided a somewhat slower release rate
CA 02564120 2006-11-07
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than tripalmitolein alone. A graded family of release
rates was obtained having faster release by increasing
the proportion of tricaprylin in the neutral lipid
component.
Example 13
Anesthetics
1. Tetracaine MVL formulations
For manufacture of MVh encapsulating tetracaine,
the lipid combination solution contained (per ml
chloroform) 15.6 mg DOPC, 3.1 mg DPPG, 11.5 mg
cholesterol, and 12.6 mg to 6.6 mg triglyceride
depending on the molar ratio of triolein:tricaprylin.
The lipophilicity of tetracaine was found to require that
the concentration of lipid .(68 umol/ml) in the lipid
combination be increased 2 to 1.5 times higher to obtain
satisfactory MVL formulations. The-triglyceride was
enriched as well; the molar ratio of
DOPC:DPPG:cholesterol:triglyceride was 0.29 . 0.06 . 0.44
. 0.21. The molar ratios of triolein to tricaprylin
prepared in the formulations of this example were 1:0;
1:10; 0.5:10; 0.2:10; 0.1:10: 0.05:10; and 0:1. The first
aqueous phase solution for encapsulation of tetracaine
contained (per ml) 15 mg of tetracaine phosphate and 200
mg of alpha-cyclodextrin polymer.
An aliquot of first aqueous phase solution (1 ml)
was added to a vial containing 1 ml of the lipid
combination and emulsified by fixing the capped vial in a
horizontal configuration to the head of a vortex mixer
(Scientific Products), and shaking at 2400
oscillativns/min for 12 minutes.
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_ 5.~ _
The final emulsion (1 ml) was divided and
transferred to two vials containing 2.5 ml of a solution
of 3.2% glucose, 5 mM lysine. The emulsion was dispersed
into microscopic droplets by fixing the capped vial in a
horizontal configuration to the head of a vortex mixer
and shaking for 3 seconds at a setting of 600-800
oscillations/min. The contents of the vial were
transferred to a flask containing 50 ml of a solution of
3.2% glucose, 5 mM lysine, and the chloroform was removed
from the microscopic droplets or spherules by
transferring the flask to a 37°C gyrorotary water bath,
and flushing the surface of the suspension with nitrogen
gas at a flow rate of one liter per min. for 20 minutes.
The suspensions were diluted 1:4 by volume with
normal saline, and the particles were harvested by
centrifugation at 800 X g for 10 minutes. The
supernatant solution was removed by aspiration, and the
particles were washed twice by resuspension in fresh,
normal saline solution and centrifugation.
2. In vivo pharmacokinetics
The in vivo release characteristics of the
multivesicular particles were determined in BalbC mice
(aged 7 to 8 weeks: weighing approximately 20 grams) by
subcutaneous injection in the abdomen region (100 u1).
At time points of 0, 5, and 24 hours after injection,
mice were sacrificed, the subcutaneous tissue was
harvested, homogenized, and extracted, and extracts were
assayed by HPLC using UV detection for the amount of
tetracaine retained.
The desired in vivo release duration for the
tetracaine MVL was 24 hours. Formulations which
contained triolein only as the neutral lipid were found
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to be stable in vivo and released over too long a
duration..
By including tricaprylin in the neutral lipid
component, formulations with shorter duration and more
desirable pharmacokinetic profiles were obtained (Figure
18). The desired 24 hour release duration for the
anesthetic was provided by a formulation with a 1
triolein: 100 tricaprylin ratio.
The Relationship Betoveen.Neutral Lipid
Selection and Intended Stowage Temperature.
As shown in the following examples, the melting
point of the neutral lipid is an important consideration
in selection of the rate-modifying neutral lipid.
However, other factors must also be taken into account,
for example, the composition of the first aqueous phase
solution. The conclusion to these examples is that the
freezing (melting-or cloud) point of the neutral lipid or
neutral lipid mixture should be above or near the storage
temperature in order to assure storage stability. If
formulations are stored at temperatures significantly
lower than the freezing point of the neutral lipid or
mixture thereof, the MVL particles undergo a physical,
morphological transition, which results in loss of
internal structure and release of encapsulated materials.
This transition may occur within a few hours or over
several days or weeks, depending on the composition of
the first aqueous phase composition.
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Example 14
In the following example, MVZs were manufactured
with either tricaprylin or tricaprin as the neutral lipid
component. The freezing point of tricaprylin is 8°C and
that of tricaprin is 31 °C. The first aqueous phase
contained either cytosine arabinoside or morphine sulfate
in 0.1 N HCl. Aliquots of the final product were stored
in saline at 2- 8, 25, 32, and 37 °C. The release of
encapsulated materials was determined over the time of
storage.
1. Manufacture
For manufacture of MVZs, the lipid combination
solution contained (per liter) 10.4 g DOPC, 2.1 g DPPG,
7.7 g cholesterol, and the triglyceride component was
7.5 either 0.93 g tricaprylin (C8) or 1.1 g tricaprin (C10)
(molar ratio, 0.34:0.07:0.52:0.06). The first aqueous
phase solution contained either (per ml) 20 mg cytosine
arabinoside in 0.1 N HC1 or 20 mg morphine sulfate
pentahydrate in 0.1 N HC1.
For formulations encapsulating cytosine
arabinoside, the first emulsion was made by high-speed
mixing of 10 ml of lipid combination solution with 20 ml
of aqueous phase solution.at 9000 rpm for l4 minutes at
25-27°C with a high shear blade. For formulations
encapsulating morphine, the first emulsion was made by
high-speed mixing of 12.5 ml of the lipid combination
solution with 7.5 ml of the aqueous phase solution at
9000 rpm for 14 minutes at 25-27°C. The first emulsions
were sheared into microdroplets (spherules) by transfer
to a mixing chamber containing 200 ml of 40 mM lysine,
3.2o glucose solution, and mixing at 2100 rpm for 2.5
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min. The chloroform was removed from the microscopic
droplets or spherules by transferring the suspension to a
flask, placing the flask in 37°C gyrorotary water bath,
and flushing the surface of the suspension with nitrogen
S gas at a flow rate of 70 cfh for 20 minutes.
The suspensions were diluted 1:4 by volume with
normal saline, and the particles were harvested by
centrifugation at 800 X g for 10 minutes at room
temperature. The supernatant solution was removed by
aspiration, and the particles were washed twice by
resuspension in fresh, normal saline solution and
centrifugation. The final washed product was resuspended
at 33~ packed-particle volume and stored.
The characterization of yields of encapsulated and
free (supernatant) cytosine arabinoside and morphine from
the tricaprylin and tricaprin NIVZ formulations was
performed a few hours post-manufacture (Table 2). The
yield of encapsulated cytosine arabinoside and morphine
was acceptable, and the free (supernatant) concentrations
of cytosine arabinoside and morphine were low for both
the tricaprylin and tricaprin formulations.
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TABZE 2
First Aqueous Neutral Yield, % Storage Supernatant
Phase Active hipid of Initial Concen- Concen-
Agent Active tration, tration,
mg/ml mg/ml
Cytosine Tricaprylin 42 7.0 0.3
Arabinoside
.. Tricaprin 37 6.2 . 0.2
Morphine Sulfate Tricaprylin 46 11.2 0.3
Tricaprin 36 8.9 ~ 0.2
2. Storacre release profiles
The suspensions were stored at 2-8, 25, 32 or
37°C. At time points of 24, 48 and 200 hours the
suspensions were centrifuged at 25,000 X G for 2 minutes,
and supernatant solutions were analyzed for content of
_ cytosine arabinoside or morphine by 1:1 volume dilution
with 50% isopropyl alcohol, vortexing, incubation at 37°C
for 10 min, and centrifugation. The supernatant samples
were analyzed by. dilution into 0.1 N HC1 and the
concentration of cytosine arabinoside was measured by
absorbance determined at 280 nm, or dilution into 0.1 N
NaOH, and the morphine concentration was measured by
absorbance determined at 298 nm.
The results of these storage-temperature-effect
studies are shown in Figures 19A-D. Release rates from
the MVL containing tricaprylin (melting point 8°C) are
very slow at 2-8°C. Release rate increased with
increasing storage temperature as would be expected as
consistent with acceleration of release with elevation of
temperature (Figures 29A and 19C). The AraC-MVL
containing tricaprin (melting point 31°C), however,
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released the active agent at a very fast rate when stored
below the melting point of the triglyceride. With
storage of the tricaprin MVL at higher temperatures 25°
and 32°, the release rate decreased, but with storage at
37° again increased (Figure 19B). In fact, the
tricaprin-containing AraC-MVL particles stored below the
melting point temperature of the neutral lipid tricaprin
(31°C), were completely destabilized and released the
encapsulated morphine over a few hours (Figure 19B).
The results for the storage study of cytosine
arabinoside-containing MVL are shown in Figure 20 by an
Arrhenius plot. The tricaprylin MVL formulation showed
an expected continuous linear relationship when log of
the release rate is plotted versus temperature,
suggesting that a single process is responsible for
release. On the other hand, the plot of the data for the
tricaprylin formulation was discontinuous, indicating
that two processes are responsible for release in the
temperature range studied. At higher temperatures above
the freezing point of tricaprin, the slope was as
expected, i.e., rates increased with increased
temperature of storage. The second process; the melting
point effect, is observed below the freezing point of
tricaprin wherein, the apparent rate increased with
decreasing temperature.
Again, comparison of the release rate profiles
(19A-D) of particles having cytosine arabinoside
encapsulated as a first aqueous phase component with
particles having morphine sulfate encapsulated indicates
that onset of the destablilzing effect caused by storage
of formulations below the melting point of the neutral
lipid, called herein "the melting point effect," is
dependent on the composition of the first. aqueous phase
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solution. Onset of the melting point effect of tricaprin
MVL during storage was observed to be rapid with cytosine
arabinoside-containing formulations, (Figure 19B) but
required several days for the morphine sulfate-containing
formulations (Figure 19D). Further, the results of these
studies suggest that the further below the freezing point
the formulation is stored, the more rapid the onset of
the melting point effect.
Example 15
In this example, the aqueous phase contained
sucrose, cytosine arabinoside and 0.1 N HC1 as an osmotic
spacer. MVL were manufactured with triolein (mp 8°C),
tricaprin (mp 31°C) , or trilaurin (mp 46 °C) as the
neutral lipid. The final product was stored at a
temperature of 2-8°C, 22, or 37°C, and the release of
encapsulated.materials was recorded during the~time of
storage.
1. Manufacture
For manufacture of MVL encapsulating cytosine
arabinoside with sucrose as an osmotic spacer in the
presence of O.1 N HCl, the lipid combination solution
contained (per liter) 10:4 g DOPC, 2.1 g DPPG, 7.7 g
cholesterol, and the triglyceride component was either
1.1 g tricaprin (C10), 1.3 g trilaurin (C12), or 1.7 g
triolein (molar ratio of
DOPC:DPPG:cholesterol:triglyceride was
0.34:0.07:0.52:0.06).
The first aqueous phase solution contained (per
ml) 20 mg cytosine arabinoside and 51.3 mg sucrose in 0.1
N Hcl. A first emulsion was made by high-speed mixing of
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3 ml of lipid combination solution with 3 ml of aqueous
phase solution at 9000 rpm for 8 minutes at 25-27°C. The
emulsion was sheared into microdroplets (spherules) by
addition of 20 ml of a 20 mM lysine, 4~ glucose solution
to the mixing vessel and mixing at 5000 rpm for 1.5 min.
The chloroform was removed from the microscopic droplets.
or spherules by transferring the suspension to a flask
containing 70 ml of a 40 mM lysine, 3.2~ glucose
solution, placing the flask in 37 C gyrorotary water bath
and flushing the surface of the suspension with nitrogen
gas at a flow rate of 70 cfh for 20 minutes to form the
MVL particles in suspension.
The suspensions were diluted 1:4 by volume with
normal saline, and the particles were harvested by
centrifugation at 800 X g for 10 minutes at room
temperature. The supernatant solution was removed by
aspiration, and the particles were washed twice by
resuspension in fresh, normal saline solution and
centrifugation. The final washed product was resuspended
at 25~ packed-particle volume per total volume and stored
at 2-8, 22, and 37°C for 1 or 6 days (data not shown).
The suspension and the supernatant fraction obtained by
centrifugation at 25,000 X G 2 minutes were analyzed for
content of cytosine arabinoside by 1:1 volume dilution
with 50~ isopropyl alcohol, vortexing, incubation at 37°C
for 10 min, and centrifugation. Then 0.06 or 0.2 ml of
the sample was added to 1.0 ml of 0.1 N HC1, and cytosine
arabinoside concentration was measured by absorbance
determined at 280 nm.
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TAB?~E 3
Trigiyceride Storage Total Supernatant Ratio of
Component Temp AraC AraC @24h Supernatant
Encapsulated mg/ml to total
C mg/ml ~ (Sup/Total)
Triolein, 2-8 6.0 0_4 0.06
mp 5C
22 5.6 0.3 0.05
37 5.2 0.9 0.17
Trilaurin, 2-8 3.0 2.5 0.82
mp 46C
22 3.1 2Ø 0.65
37 3.0 2.3 0.78
Tricaprin, 2-8 5.6 5.0 0.90
mp 31C
--
22 5.9 4.8 0.90
37 6.7 0.6 0.09
As shown by the data in Table 3, the product yield
of the MVL formulated using each of the neutral lipids
was~at least 3.0 mg/ml, an acceptable yield. Inspection
of the particles with white light microscopy showed that
the particles were spherical and multivesiculated in
_ Z5 appearance immediately post-manufacture: However; within
24 hours of storage at temperatures below the melting
point of the triglyceride, the particles changed in
appearance. Associated with the change in appearance was
significant loss of the encapsulated AraC to the storage
solution (supernatant) as can be seen by the data in
column 3 of Table 3. .Noteworthy, the formulation
manufactured with tricaprin (mp. 31°C) did not change in
appearance, or lose significant AraC when stored at 37°C;
whereas the formulation with trilaurin (mp 46°C) did
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undergo a significant morphological change and loss of
encapsulated active agent. Thus in formulating MVL for
in vivo controlled and sustained delivery of the
encapsulated active agent, the neutral lipid selected
should usually have a melting point below or near the
intended storage temperature. A preferred formulation
would maintain a supernatant/total ratio of less than
0.10 during storage.
Examm~le 16
This example illustrates that certain aqueous
phase solutions are interactive with the lipid layer
membrane of MVhs and prevent freezing of the liposomes at
storage temperatures below the melting point of the
neutral lipid. In such cases; the morphological
transition of the particles and loss of encapsulated
materials associated with storage below the melting point
of the neutral lipid may occur very slowly, or disappear.
Instead the release of encapsulated materials is
associated with activation by storage temperature
increase.
1. Manufacture
For manufacture of MVh encapsulating rhu IGF with
"C_sucrose as the osmotic spacer and ammonium citrate as~
the buffer, the lipid combination solution contained (per
liter) 10.4 g DOPC, 2.1 g DPPG, 7.7 g cholesterol, and
the triglyceride component was 1.3 g tricaprin (mp 31°C,~
C10) (with the molar ratio of
DOPC:DPPG:cholesterol:triglyceride was
0.34:0.07:0.52:0.06).
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The first aqueous phase solution contained (per
ml) l6 mg rhu IGF-1 (Chiron, Foster City, CA), 7% 14C-
sucrose, and 20 mM ammonium citrate, pH 5. A first
emulsion was made by high-speed mixing of 5 ml of lipid
combination solution with 5 ml of aqueous phase solution
at 9000 rpm for 9 minutes at 25-27°C. The emulsion was
sheared into microdroplets (spherules) by addition of 30
ml of a 40 mM lysine, 4% glucose solution to~the mixing
vessel and mixing at 6000 rpm for 1 min. The chloroform
was removed from the microscopic droplets or spherules by
transferring the suspension to a flask containing 70 ml
of a 40 mM lysine, 3.2$ glucose solution, placing the
flask in a gyrorotary water bath at 37°C, and flushing
the surface of the suspension with nitrogen gas at a flow
rate of 70 cfh for 20 minutes to obtain the MVL
particles.
The suspensions were diluted 1:4 with normal
saline and the particles were harvested by centrifugation
at 800 X g for 10 minutes at room temperature. The
supernatant solution was removed by aspiration, and the
particles were washed twice by resuspension in fresh,
normal saline solution and centrifugation.
2. In vitro release rates
The final washed product was resuspended at 25%
packed-particle volume per total volume and stored at
temperatures of 2-8, 25, 32, and 37°C for 24 or 48 hours.
The pellet and supernatant fraction from the
centrifugation at 25,000 X G for 2 minutes was assayed
for content of IGF-1 (pellet fraction only) and 1'C-
sucrose.
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TnRS-F 4
Temperature, Retained @ Retained
24 hours 48 hours
C - i4C-Sucrose IGF-1 19C-Sucrose IGF-1
2-8 1.00 1.00 0.98 0.98
25 0.95 0.97 0.91 0.94
32 0.90 0.96 0.74 0.86
37 0.83 0.91 0.56 0.75
The results of these studies summarized in Table 4
show that the contents of the first aqueous phase
solution can prevent or slow the freezing effect which
was shown in Example 16 to be associated with storage of
MVZs containing trilaurin as the neutral lipid at a
temperature below its melting point of 46°C. The lipids
used and method of manufacture were identical in both
formulations; however, the encapsulated aqueous. phase
components were different. This suggests that the
composition of the first aqueous phase may modulate
either the freezing point of the triglyceride or the
melting point effect, for instance by preventing the
neutral lipid from transitioning to an unstable structure
or markedly slowing the freezing.
The release in plasma of the tricaprin- and:
trilaurin-containing MVL formulations (shown in Example
16) was markedly slower than from MVh manufactured with
tricaprylin, as shown in Table 4 and in later examples.
It should be noted that all the plasma release assays
conducted at 37°C were initiated within 24 hours of
manufacture. The storage at 2-8°C for.longer than a few
days of the trilaurin, but not the tricaprin-containing
formulations, resulted in morphological change in the
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appearance of the particles and release of encapsulated
materials consistent with the "melting point effect".
Example I7
Other neutral lipids
A study series was performed wherein the neutral
1-ipids were decane, dodecane; squalene, and alpha-
tocopherol to determine the scope of neutral lipids that
can be used to obtain multivesicular liposomal
formulations.
1. Manufacture
Multivesicular liposomes encapsulating a
combination of glycine, sucrose, and Tris-EDTA were made
wherein the lipid combination solution contained (per
liter) 10.4 g DOPC, 2.1 g DPPG, 7.7 g cholesterol, and
the triolein component (6 mol % of lipid) was replaced
with either decane, dodecane, squalene, or alpha-
tocopherol, (molar ratio of DOPC:DPPG:cholesterol:neutral
lipid was 0.34:0.07:0.52:0.06).
The first aqueous phase solution contained 200 mM
glycine, 50 mM Sucrose, 1.8 mM Tris base, and 0.5 mM
. EDTA, pH 7.44; with an osmolarity of 268 mOsmol. A first
emulsion was made by high-speed mixing of 3 ml of lipid
combination solution with 3 ml of aqueous phase solution
at 9000 rpm for 9 minutes at 25-27°C. The emulsion was
sheared into microdroplets (spherules) by the addition of
20 ml of a 20 mM lysine, 4$ glucose solution to the
mixing vessel and mixing at 4000 rpm for 1.0 min.
Examination of the spherule suspensions under the
microscope indicated that the spherules prepared with
each of these neutral lipids were normal in internal
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appearance as compared to controls prepared with
triolein, tripalmitolein, or trimyristolein as the
neutral lipid component.
The chloroform was removed from the microscopic
droplets or spherules by transferring the suspension to a
flask containing 30 ml of a 40 mM lysine, 3.2~ glucose.
solution, placing the flask in 37°C gyrorotary water
bath, and flushing the surface of the suspension with
nitrogen gas at a~flow rate of 70 cfh for 20 minutes to
obtain the MVh particles.
Only MVL prepared with squalene as the neutral
lipid survived the solvent removal step or subsequent
washing of the particles and gave rise to normal-looking
MVZ particles after the wasf~ step. During the solvent
removal step, decane, dodecane and alpha-tocopherol
spherules began to shrink, with lobes of light-refractile
material emanating from their surface. Abruptly, the
particles collapsed into a crenellated structure. In
some cases a pellet, albeit small as compared to
controls, was recovered from the wash step, and the
particles did not have the appearance of multivesicular
liposome compositions.
Other Embodiments
The foregoing description of the invention is
exemplary for purposes of illustration and explanation.
It should be understood that various modifications can be
made without departing from the spirit and scope of the
invention. Accordingly, the following claims are
intended to be interpreted to embrace all such
modifications.
