Note: Descriptions are shown in the official language in which they were submitted.
CA 02533010 2005-09-06
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ENHANCED DELIVERY OF SPHINGOLIPIDS
Cross-Reference to Related Applications
[0001] This application claims benefit of U.S. Serial No. 60/453,002 filed
7 March 2003. The contents of this application are incorporated herein by
reference.
Technical Field
[0002] The invention relates to compositions and methods for improved delivery
of
sphingolipids, such as ceramide. More particularly, the invention concerns
delivery
systems that ensure the intracellular uptake of effective amounts of
sphingolipids when
the sphingolipids are delivered to an intended target by providing a
formulation
comprising delivery vehicles.
Background Art
[0f03] The progression and treatment of many life-threatening diseases such as
cancer, immune disorders and cardiovascular disorders is influenced by
multiple
molecular mechanisms, including those that regulate the balance between cell
proliferation and cell death. In particular, hyperproliferative disorders such
as cancer
result from an increase in cell proliferation that is often coupled with a
decrease in
programmed cell death (apoptosis). Key molecules being examined for their role
in
regulating intracellular processes that affect this balance are ceramides and
related
sphingolipids such as sphingosine. These molecules are known to be involved in
regulating a number of cellular responses to both environmental and
pharmacological
extracellular stimuli. Such responses include, differentiation, inhibition of
growth, cell
senescence and apoptosis (Kolesnick, R.N., and Kronke, M., Afznu. Rev.
Physiol. (1998)
60:643-665; Hannun, Y.A., Sciefzce (1996) 274:1855-1859; and Ariga, T., et
al., J. Lipid
Res. (1998) 39:1-16).
[0004] The role of ceramide in apoptosis and reduced growth may be
particularly
important for the treatment of cancer. One dramatic demonstration of the
relationship
between ceramide and cancer was reported by Selzner and colleagues who showed
that
the cellular content of ceramide in human colon cancer is reduced by more than
50%
relative to that of healthy colon mucosa (Selzner, et. al., Cancer Res. (2001
) 61 (3):1233-
1240). Similarly, alterations in metabolism of ceramide to the noncytotoxic
metabolite
CA 02533010 2005-09-06
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glucosylceramide have been implicated in the multidrug resistance (MDR)
phenomenon
associated with numerous cancers (Shabbits and Mayer, Mol. Cancer Then. (2002)
1 (3):205-213).
[0005] Ceramides are a class of lipid second messengers comprising a
sphingosine
backbone and are found in all eukaryotic membranes. They are generated within
a cell
from the hydrolysis of sphingomyelin or de novo biosynthesis. Different
ceramides are
characterized by different fatty acids linked to the sphingoid base. Pei, et
al.,
WO 95/21175, have shown that analogs of sphingolipids and ceramides that
inhibit
conversion of ceramides to sphingomyelins lead to enhanced apoptosis as a
result of the
increased intracellular ceramide content. In fact, much of the research
focusing on
ceramides has been directed at altering their intracellular levels. Of
particular interest is
the observation in several systems that transformed cell types are
hypersensitive to the
effects of ceramide perturbation, suggesting that strategies to kill tumor
cells by
increasing their ceramide content should have a favorable therapeutic index.
[0006] Increasing intracellular ceramide levels has been achieved with
multiple
endogenous methods that inhibit ceramide catabolism, such as those performed
by Pei, et
al., as well as exogenous administration of cell-permeable (short-chain)
ceramides.
Traditionally, these short-chain ceramides (C2 or C6) are efficiently
incorporated into
liposomes comprised of phosphatidylcholine or phosphatidylethanolamine and are
effectively delivered to the cytosol of a target cell. Pei, et al., showed
that C2- and
C6-ceramide or related analogs, incorporated into various liposomal
formulations
comprising egg phosphatidylcholine or sphingomyelin and cholesterol, exhibited
significant cytotoxic effects both in vitro and in vivo.
[0007] More recently, however, it has been demonstrated in vitro that the
effects of
short-chain ceramides on the activity of particular intracellular molecules
are different
from those of natural ceramide, indicating that the cell-permeable short-chain
ceramide
compounds many not completely mimic the natural product (Huang, et al.,
Biophys. J.
(1999) 77:1489-1497). Thomas, et al., have also indicated that the ceramide
species
involved in apoptosis of Jurkat cells is the long-chain, C~6-ceramide (Thomas,
et al., J.
Biol. Cheni. (1999) 274:30580-30588).
[0008] Research relating to the naturally occurnng ceramides and other long-
chain
sphingolipids has been limited because of their hydrophobic nature making them
cell-
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WO 2004/078677 PCT/CA2004/000344
impermeable and also difficult to formulate at effective amounts into delivery
vehicles,
such as liposomes. Physicochemical characterization of long-chain ceramides in
lipid
bilayers have shown that greater than about 20 mol % of these ceramides leads
to
extensive liposomal aggregation. Holopainen, et al., Chem. Phys. ofLipids
(1997)
88:1-13) prepared dimyristoylphosphatidylcholine (DMPC) or 1-palmitoyl-
2[(pyren-1-
yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) liposomes incorporating
ceramides
ranging from C16-C2~ at up to 20 mol % in order to study thermal phase
behaviors with
Differential Scanning Calorimetry and Fluorescence Spectroscopy. Liposomes
incorporating more than 20 mol % could not be studied due to extensive
aggregation.
The researchers did not investigate alternate liposome formulations that could
relieve this
aggregation nor did they determine if the ceramide-containing liposomes
exhibited a
cytotoxic effect. Similarly, using Nuclear Magnetic Resonance imaging, Hsueh
and
colleagues examined the effect of increasing ceramide concentrations on 1-
palmitoyl-2-
oleoyl-sn-glycero-3-phosphocholine (POPC) membranes (Hsueh, et al.~ Biophys.
J.
(2002) 82:3089-3095). They found that increasing the level of C16-ceramide
above 20
mol % resulted in the formation of unstable (or metastable) liposomes which
exhibited
significant hydration complexities.
[0009] Despite the many liposomal formulations designed for delivery of
sphingolipids, a composition suitable for long-chain sphingolipids has not
been achieved.
It is especially important to accomplish this since it has been suggested that
manipulation
of ceramide levels may also enhance the effectiveness of some cancer
therapies.. VJanebo
and coworkers showed that the addition of ceramide enhances taxol-mediated
apoptotic
death of Tu138 head and neck tumor cells (Mehta, et al., Cancer
Chemother°. Pharnaacol.
(2000) 46(2):85-92). Because a variety of anti-cancer drugs are known to
elevate
endogenous ceramide levels, combinations of sphingolipids with anti-cancer
drugs may
result in significantly improved chemotherapy regimes.
Disclosure of the Invention
[0010] It has been found that formulations of the present invention can
incorporate as
much as 50 mole % C16-ceramide without aggregation and that these formulations
result
in a significant Increase in Life Span (ILS) of a host when administered in
vivo. The
delivery vehicles described herein provide effective delivery of
sphingolipids, even those
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that are substantially insoluble in water. The delivery vehicles comprise at
least one acid
derivative of a sterol.
[0011] Thus, in one aspect, the invention relates to methods for administering
sphingolipids, including hydrophobic sphingolipids, using delivery vehicle
compositions
comprising an acid derivative of a sterol. Incorporation of the acid-
derivatized sterol in
the delivery vehicle allows for increased encapsulation of sphingolipids
without causing
aggregation of the delivery vehicles, resulting in enhanced intracellular
delivery.
[0012] In another aspect, the invention provides a delivery vehicle-containing
composition for administration comprising an effective amount of a
sphingolipid and an
acid derivative of a sterol. Sphingolipids may include long-chain, hydrophobic
ceramides. Another aspect of the invention is directed to a method to deliver
an effective
amount of a sphingolipid to a desired target by administering the compositions
of the
invention.
[0013] The present invention provides liposomes incorporating at least one
acidic
lipid and a sphingolipid. The inclusion of an acidic lipid allows the liposome
to remain
stable at physiological pH and to destabilize upon delivery to a low pH target
site such as
endosomes and tumors. Destabilization of the liposome allows for increased
availability
of the sphingolipid at a target site. In preferred embodiments, delivery of
the sphingolipid
is enhanced by the incorporation of a lipid that has a net negative charge at
physiological
pH and neutral at reduced pH. Conversion of the acidic lipid to its neutral
form at low pH
triggers liposome destabilization thereby allowing for release of the
sphingolipid from the
bilayer along with encapsulated contents, if present. Preferably the acidic
lipid is a
derivatized sterol. More preferably, the acid lipid is cholesteryl
hemisuccinate.
[0014] The delivery vehicles may further comprise one or more encapsulated
active
agents.
Brief Describtion of the Drawings
[0015] FIGURE 1 is a graph showing the cytotoxicity of various acyl chain
length
free ceramide lipids on wild-type (A) and MDR-1 gene transfected (B)
MDA435/LCC6
human breast cancer cells. Cells were incubated with the indicated ceramide
concentrations for 72 hours and cell viability was measured using the MTT
assay. Data
are averaged means from three triplicate experiments. Each value represents
the mean
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WO 2004/078677 PCT/CA2004/000344
from at least three independent experiments; error bars indicate the Standard
Error of the
Mean (S.E.M).
[0016] FIGURE 2 is a graph showing the cytotoxicity of various acyl chain
length
free ceramide lipids on J774 rnurine macrophage cells. Cells were incubated
with the
indicated ceramide concentrations for 72 hours and cell viability was measured
using the
MTT assay. Data are averaged means from three triplicate experiments. Each
value
represents the mean from at least three independent experiments; error bars
indicate the
S.E.M.
[0017] FIGURE 3 is a graph showing the cellular uptake of free C6- and C16-
ceramide
by wild-type (A) and MDR-1 gene transfected (B) MDA435/LCC6 cells. Cells were
incubated with 10 ~,M C6-ceramide or 50 ~M C16-ceramide for the times
indicated.
[~4C]C6- or [14C]C16-ceramide was added at 0.1 ~Ci/nmole ceramide for
quantitation by
scintillation counting. Cellular protein content was measured
spectrophotometrically
(Abs 562 nm) using the micro BCA protein assay kit. Data are averaged means
from two
triplicate experiments; error bars indicate the S.E.M. .
[001] FIGURE 4 is a graph showing the cytotoxicity of control (DPPC/CHEMS,
50:50) and C16-ceramide (C16-ceramide/CHEMS, 50:50) liposomes on J774 murine
macrophage cells. Cells were incubated with the indicated concentrations of
liposomes
for 72 hours and cell viability was measured using the MTT assay. The
indicated
liposome concentration represents total lipid for control liposomes and was
corrected for
ceramide content for ceramide-containing liposomes. Data are averaged means
from
three triplicate experiments; error bars indicate the S.E.M.
[0019] FIGURE 5 is a graph showing the cellular uptake of C16-ceramide/CHEMS
(50:50) liposomes by J774 murine macrophage cells. Uptake of
[3H]cholesterylhexadecyl
ether ([3H]CHE) bulk liposomal lipid and [14C]C~6-ceramide are expressed as a
percent of
the total radioactivity added, normalized to 105 cells. Data are averaged
means from two
triplicate experiments; error bars indicate the S.E.M.
[0020] FIGURE 6 is a graph showing the antitumor activity of C~6-
cer/CHEMS/PEG2000-DSPE (47.5:47.5:5) versus DPPC/CHEMS/PEG2000-DSPE
(47.4:47.5:5) liposomes in the J774 ascites tumor model. On day zero, 1x106
cells were
inoculated intraperitoneally (i.p.) into female Balb/c mice (12 per group) and
saline,
control liposomes or ceramide liposomes were administered by intravenous
(i.v.) bolus
CA 02533010 2005-09-06
WO 2004/078677 PCT/CA2004/000344
injection on days 1, 5 and 9 at the lipid concentrations indicated. Arrows
indicate the
days of treatment administration. Animals were weighed and monitored daily for
survival.
[0021] FIGURE 7 is a graph showing the antitumor activity of C16-
cer/CHEMS/PEG2000-DSPE (47.5:47.5:5) versus DPPC/CHEMS/PEG2000-DSPE
(47.4:47.5:5) liposomes in the J774 ascites tumor model. On day zero, 1x106
cells were
inoculated intraperitoneally (i.p.) into female Balb/c mice (12 per group) and
saline,
control liposomes or ceramide liposomes were administered i.p. on days 1, 5
and 9 at the
lipid concentrations indicated. Arrows indicate the days of treatment
administration.
Animals were weighed and monitored daily for survival.
Modes of Carrying Out the Invention
[0022] The invention provides compositions comprising delivery vehicles that
include
at least one acid-derivative of a sterol that are useful in delivering
sphingolipids,
especially long-chain sphingolipids. The acid-derivative sterol aids in
incorporating high
levels of said sphingolipid. The acid-derivatized sterol may be pH sensitive
in that it is
negative at physiological pH and neutral at lower pH.
[0023] Preferably delivery vehicles included herein will incorporate high
levels of
sphingolipids. The delivery vehicles will contain greater than 20 mol % of
sphingolipids,
more preferably, greater than 30 mol % of said sphingolipids, more preferably
more than
40 mol % or 50 mol %, the base for these percentages is total lipid.
[0024] In further embodiments of the invention, the above described delivery
vehicles
incorporate one or more additional active agents. Any therapeutic, cosmetic or
diagnostic
agent may be included.
[0025] In another aspect of the invention, delivery vehicles which comprise a
pH
sensitive, "acidic lipid", and a sphingolipid are provided.
[0026] The delivery vehicles of the present invention may be used not only in
parenteral administration but also in topical, nasal, subcutaneous,
intraperitoneal,
intramuscular, or oral delivery or by the application of the delivery vehicle
onto or into a
natural or synthetic implantable device at or near the target site for
therapeutic purposes
or medical imaging and the like. Preferably, the delivery vehicles of the
present invention
are used in parenteral administration, most preferably, intravenous
administration.
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[0027] The preferred embodiments herein described are not intended to be
exhaustive
or to limit the scope of the invention to the precise forms disclosed. They
are chosen and
described to best explain the principles of the invention and its application
and practical
use to allow others skilled in the art to comprehend its teachings.
Abbreviations
[0028] DMPC: dimyristoylphosphatidylcholine;
PPDPC: 1-palmitoyl-2 [(pyren-1-yl)] decanoyl-sn-glycero-3-phosphocholine;
POPC: l -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine;
DSPC: distearoylphosphatidylcholine; DSPE: distearoylphosphatidylethanolamine;
DOPE: dioleoylphosphatidylethanolamine; DPPC: dipalmitoylphosphatidylcholine;
DPPG: 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; DSPE-PEG350 or
PEG350-DSPE: distearoylphosphatidylethanolamine-N-[polyethylene glycol 350].
[0029] Chol or CH: cholesterol; CHEMS or CHS: cholesteryl hemisuccinate;
cer: ceramide.
Acid-Derivatized Sterols
[0030] "Acid-Derivatized Sterol" refers to a steroid that is coupled to an
acidic group
- i. e., a group that is negatively charged at physiological pH. Preferably
the steroid
contains a hydroxyl group and the acid to be coupled is sufficiently
bifunctional to form
an ester with the alcohol and retain its acidic characteristics. However,
other coupling
means may be used.
[0031] Cholesterol and other sterols are routinely used in delivery vehicle
preparations, such as liposomes, in order to broaden the range of temperatures
at which
phase transition occurs, with phase transition disappearing at high
cholesterol levels.
Many acid-derivatized sterols have also been incorporated into a number of
cosmetic,
diagnostic and pharmaceutical preparations. Particular acid-derivatized
cholesterols, such
as cholesteryl phosphate and cholesteryl hemisuccinate; are known to retain
many of the
properties that cholesterol exhibits in model membrane systems (Lai, et al.,
Biochemistry
(1995) 24(7):1646-1653). However, these cholesterol esters also display unique
properties in the extent to which they interact or bind with phospholipids
(Lai, et al.,
Biochen~isty (1995) 24(7):1654-1661). The attachment of the charged ester is
known to
greatly enhance the partitioning of cholesterol and other sterols into
phospholipid
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WO 2004/078677 PCT/CA2004/000344
bilayers. This has been beneficial for many cosmetic formulations that have
incorporated
hydrophilic sterols (see European Patent No. 28,456 and U.S. Patent No.
4,393,044).
[0032] It is well known that many acid-derivatized sterols can readily self
assemble
into mufti- or single lamellar vesicles without the use of organic solvents.
These vesicles
display high trapping efficiencies and captured volumes. The present invention
describes
delivery vehicle compositions comprised of at least one acid-derivatized
sterol, preferably
an organic acid derivative of cholesterol, which allows for enhanced
incorporation of
sphingolipids into said delivery vehicles. Preferably, the derivatized sterol
is capable of
self-assembling into a closed bilayer; however, any acid-derivatized sterol
may be used in
the practice of the invention. The suitability of a particular sterol
derivative depends
upon its ability to allow for stable association of high levels of
sphingolipids, preferably
hydrophobic sphingolipids, with delivery vehicles of the invention. Generally
any sterol
which can be modified by the attachment of an organic acid may be used in the
practice
of the invention. Non-limiting examples of such sterols include cholesterol,
Vitamin D,
phytosterols, steroid hormones and the like.
[0033] ~rganic acids which can be used to derivatize the sterols include, but
are not
limited to, di- and polycarboxylic acids, hydroxy acids, amino acids and
polyamino acids.
Particular organic acid moieties that are water-soluble themselves may be more
advantageous in increasing the hydrophilicity of the acid-derivatized sterol.
Non-limiting
examples of such moieties include dicarboxylic acids such as malonic,
succinic, glutaric,
adipic, pimelic, malefic and the like; and aromatic dicarboxylic acids such as
hemimellitic,
trimesic, and the like; hydroxy acids such as glycolic, lactic, mandelic,
glyceric, malic,
tartaric, citric and the like; and any amino or polyamino acid. The
derivatized acid can be
linked to the hydroxyl group of the sterol preferably via an ester bond using
conventional
methods (see for example, U.S. patent Nos. 3,859,047; 4,040,784 or 4,189,400).
If the
carboxylic acid contains only a single carboxyl group, other reactive groups
present in the
molecule can be used to couple the acid moiety to any reactive functional
group on the
sterol. In some cases, such as formation as phosphate esters, the multivalent
nature of the
acid itself is sufficient.
[0034] A further advantage of acid-derivatized sterols is their sensitivity to
pH.
Generally, such sterol derivatives have a net negative charge at physiological
pH which
enhances the stability of delivery vehicles incorporating them. Therefore,
delivery
CA 02533010 2005-09-06
WO 2004/078677 PCT/CA2004/000344
vehicles of the invention remain stable in the blood compartment
(physiological pH) and
destabilize upon delivery to a low pH target site, such as endosomes or
tumors, where
they become protonated. This destabilization allows for release of the
contents, which
may allow more rapid uptake. It should be apparent to those knowledgeable in
the.art
that other acidic lipids, with similar pH sensitivities, could be included in
the vehicles of
this invention. Thus another aspect of the invention provides delivery
vehicles
comprising an acidic lipid and a sphingolipid. Preferably the acidic lipid is
a sterol
derivative. More preferably, the acidic lipid is a derivative of cholesterol.
Even more
preferably, the acidic lipid is cholesteryl hemisuccinate.
Sphyngolipids
[0035] Sphingolipids, as described herein, are compounds that comprise long-
chain
bases containing a secondary amine and one or more hydroxyl groups. The most
commonly found long-chain bases are sphingosine (4-sphingenine), sphinganine
and
4-hydroxy sphinganine. Sphingosine is most commonly found in mammalian cells
and
has the formula CH3(CH2)izCH=CHCHOHCHNH+3CH2~H. The other two long-chain
bases are those wherein the CH=CH is reduced to -CHZCH2- or hydrated to -
CH2CHOH.
The latter two are commonly called dihydrosphingosine and phytosphingosine; as
implied
by the name, the last named long-chain base is most commonly found in plants.
These
long-chain bases can, themselves, be considered sphingolipids, but most
commonly,
sphingolipids include forms of these bases wherein the amino and/or hydroxyl
groups are
derivatized. The ceramides are members of the sphingolipid class which are
acylated at
the amino group. In addition, one or more hydroxyl groups, typically the
primary
hydroxyl group, can further be derivatized, for example with phosphocholine to
yield
sphingomyelin.
[0036] As used herein, "sphingolipid" there refers to the derivatized and
underivatized forms of sphingosine and its related compounds which have the
essential
features of containing a secondary amine and at least one hydroxyl group.
Included in the
sphingolipids, therefore, are derivatives of sphingosine, derivatives of
phytosphingosine,
derivatives of dihydrosphingosine, and related long-chain bases and
specifically includes
the ceramides. The ceramides are defined as sphingolipid derivatives which
comprise
acyl groups coupled to the amino group to form an amide. "Derivatives of
ceramide"
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WO 2004/078677 PCT/CA2004/000344
refer to further substitutions on the hydroxyl groups of ceramides. They are
also
sphingolipids.
[0037] In some instances, the derivatives of sphingosine and ceramide or of
other
long-chain bases are designed to block the metabolism of ceramides. This
function is
useful in enhancing the levels of ceramides in cells, and thus enhancing the
apoptotic
potential of these cells. Accordingly, these derivatives, that block ceramide
metabolism,
are useful antitumor agents.
[0038] Sphingolipids may be hydrophilic or hydrophobic. Hydrophobic
sphingolipids generally contain greater than 6 carbon atoms in at least one
acyl chain.
Sphingolipids that induce apoptosis or sphingolipids that mediate opposing
pathways may
be used. Preferably sphingolipids of the invention are hydrophobic. More
preferably
they are long-chain ceramides or ceramide derivatives. Even more preferably,
they are
therapeutically active and physiologically relevant ceramides or derivatives
thereof.
[0039] Many sphingolipids that are sphingosine and ceramide derivatives can be
generated with hydroxyl-replacement groups that block the bioconversion of
ceramide to
sphingolipids such as sphingomyelin, ceramide-1-phosphate, sphingosine,
sphingosine-1-
phosphate and glucosylceramide and thus result in enhanced intracellular
ceramide
content. Many such derivatives are detailed in Pei, et al., WO 95/21175 and
U.S.
5,681,589 are incorporated herein by reference. Studies have indicated that
this is
possible without inhibiting the signaling properties of the ceramide molecule.
By
inhibiting the metabolism of ceramide, the metabolic stability of the lipid
can be
increased thereby stimulating apoptosis. Either hydroxyl group may be modified
with a
hydroxyl-replacement group. Any hydroxyl-replacement group that can
effectively
inhibit conversion of ceramide to one or more metabolites can be employed.
Replacement of the amide moiety by a sulfonamide-group is one possibility to
enhance
the metabolic stability of the lipid. Enhanced catabolic stability may likely
be achieved
by incorporation of this modification into the ceramide structure. This should
also
prevent formation of sphingosine-1-phosphate which is an additional signaling
substance
metabolically derived from ceramide.
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Types of Delivery Vehicles
[0040] Delivery vehicles for use in this invention include lipid carriers,
liposomes,
lipid micelles, lipoprotein micelles, lipid-stabilized emulsions,
cyclodextrins, polymer
nanoparticles, polymer microparticles, block copolymer micelles, polymer-lipid
hybrid
systems, derivatized single chain polymers, and the like, all containing a
sphingolipid and
an acid-derivatized sterol. The carriers can be prepared with additional lipid
or polymer
components conventionally employed in the art. For example, the lipid carriers
may
comprise surface stabilizing hydrophilic polymer-lipid conjugates such as
polyethylene
glycol-DSPE, to enhance circulation longevity (see Example 3). Optionally,
negatively
charged lipids such as phosphatidylglycerol (PG) and phosphatidylinositol (PI)
can be
included in lipid carrier formulations to increase the circulation longevity
of the delivery
vehicle. These lipids may be employed to replace hydrophilic polymer-lipid
conjugates
as surface stabilizing agents. Lipid carriers of the invention may also
contain therapeutic
lipids in addition to bioactive sphingolipids. Examples include ether lipids,
phosphatidic
acid, phosphonates and phosphatidylserine.
[0041] In addition to the sphingolipid, additional therapeutic agents may be
included.
Particularly preferred are antitumor agents such as DNA damaging agents, DNA
repair
inhibitors, topoisomerase I inhibitors, S/G2 and G2/M cell cycle checkpoint
inhibitors,
G1/early-S checkpoint inhibitors and CDI~ inhibitors, G2M checkpoint
inhibitors,
receptor tyrosine kinase inhibitors, apoptosis-inducing agents, cell cycle
control
inhibitors, hormones and anti-angiogenic agents.
[0042] DNA damaging agents include, for example, chlorambucil, carboplatinum
and
doxorubicin; DNA repair inhibitors include aminopterin derivatives, 5-
fluorouracil, and
methotrexate; topoisomerase I inhibitors include irinotecan and camptothecin;
topoisomerase II inhibitors include deoxydoxorubicin and etoposide; S/G2 and
G2/M
checkpoint inhibitors include bleomycin and dolastatin; G~/early-S checkpoint
and cyclin
dependent kinase inhibitors include flavopiridol and hydroxyurea; G2/M
checkpoint
inhibitors include bleomycin and vincristine; receptor tyrosine kinase
inhibitors include
AG-1478 and lavendustin A. Various other therapeutic compounds could also be
included; however, the combined effect of a sphingolipid and an antitumor
agent on
tumor cells is particularly advantageous.
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[0043] Tumors that can be treated include lymphomas, carcinomas, and solid
tumors
of various organs.
[0044] Delivery vehicles of the invention may further comprise targeting
ligands.
Such ligands may be individually incorporated into the delivery vehicle or
conjugated to
components comprising the delivery vehicle, such as lipids or polymers.
"Targeting
ligands" are molecules, typically naturally-occurring, that bind to cell-
surface molecules
in both specific and non-specific interactions. Non-specific binding is
generally through
charge-charge interactions, whereas specific binding occurs through cell-
surface
receptors. This type of receptor-ligand binding can initiate internalization
processes.
Internalization can occur through phagocytosis, endocytosis or receptor-
mediated
endocytosis. Preferably, ligands are chosen such that they bind to specific
cell-surface
receptors known as "internalizing receptors." Binding to these receptors leads
to
receptor-mediated endocytosis which results in the receptor, ligand and any
ligand-
associated material being internalized within an endosome or lysosome of the
cell. In this
way, targeting ligands attached to delivery vehicles of the invention will
thus result in
enhanced delivery of the sphingosine-containing delivery vehicles to the low
pH
environment of the endosome or lysosome. This will lead to destabilization of
the
delivery vehicle due to protonation of the associated acid-derivatized sterol
which will
allow for increased intracellular delivery of the sphingosine. The use of
targeting ligands
aimed at internalizing receptors is particularly useful for cancer treatment
as many of
these receptors are overexpressed on cancer cells. Several studies of
different cancers,
including breast cancer, have correlated expression of the transferrin
receptor (TfR) to
tumor grade and metastatic potential. Other non-limiting examples of
internalizing
receptors are low-density lipoprotein receptor (LDL-R), epidermal growth
factor receptor -
(EGF-R), folate receptor (FR), and cluster designation (CD) molecules, such as
CD3.
[0045] The targeting agents may be ligands specific for cell surface
receptors,
immunoglobulins or fragments thereof, and the like. These targeting agents can
be
coupled to the delivery vehicles using methods generally known in the art.
[0046] Preferred lipid earners for use in this invention are liposomes.
Liposomes can
be prepared as described in Liposomes: Rational Design (A.S. Janoff, ed.,
Marcel Dekker,
Inc., New York, NY), or by additional techniques known to those knowledgeable
in the
art. Suitable liposomes for use in this invention include large unilamellar
vesicles
12
CA 02533010 2005-09-06
WO 2004/078677 PCT/CA2004/000344
(LUVs), multilariiellar vesicles (MLVs), small unilamellar vesicles (SUVs) and
interdigitating fusion liposomes.
[0047] Sphingolipids and acid-derivatized sterols may be prepared as liposomes
of
the invention using standard methods described in the art. Said liposomes may
further
comprise one or more lipids commonly used in the preparation of liposomes as
well as
other non-lipid components. It should be readily apparent to those
knowledgeable in the
art that a number of lipid combinations could be employed to generate delivery
vehicles
of the present invention.
[0048] The internal compartment of the liposomes can optionally encapsulate
one or
more therapeutic agents. This provides for the preparation of a delivery
vehicle that
contains encapsulated therapeutic agents in addition to a bioactive
sphingolipid, thereby
allowing for the treatment of diseases that result from multiple molecular
mechanisms.
This is of particular significance as synergistic effects between exogenous
ceramide and
anticancer treatments have been reported (Mehta, et al., Ca~acer Chemother.
Phaf°macol.
(2000) 46:85-92).
[0049] Various methods may also be utilized to encapsulate active agents in
liposomes. Examples of suitable loading techniques include conventional
passive and
active entrapment methods. Passive methods of encapsulating active agents in
liposomes
involve encapsulating the agent during the preparation of the liposomes. This
includes a
passive entrapment method described by Bangham, et al. (J. Mol. Biol. (1965)
12:238).
This technique results in the f~rmation of multilamellar vesicles (MLVs) that
can be
converted to large unilamellar vesicles (LUVs) or small unilamellar vesicles
(SUVs) upon
extrusion. Additional suitable methods of passive encapsulation include an
ether
injection technique described by Deamer and Bangham (Biochina. Biophys. Acta
(1976)
443:629) and the Reverse Phase Evaporation technique as described by Szoka and
Paphadjopoulos (P.N.A.S. (1978) 75:4194).
[0050] Active methods of encapsulation include the pH gradient loading
technique
described in U.S. patent Nos. 5,616,341, 5,736,155 and 5,785,987. A preferred
method of
pH gradient loading is the citrate-base loading method utilizing citrate as
the internal
buffer at a pH of 4.0 and a neutral exterior buffer. Other methods employed to
establish
and maintain a pH gradient across a liposome involve the use of an ionophore
that can
insert into the liposome membrane and transport ions across membranes in
exchange for
13
CA 02533010 2005-09-06
WO 2004/078677 PCT/CA2004/000344
protons (see U.S. patent No. 5,837,282). A recent technique utilizing
transition metals to
drive the uptake of drugs into liposomes via complexation in the absence of an
ionophore
may also be used. This technique relies on the formation of a drug-metal
complex rather
than the establishment of a pH gradient to drive uptake of drug.
[0051] Passive and active methods of entrapment may also be coupled in order
to
prepare a liposome formulation containing more than one encapsulated agent. pH
sensitive liposomes are dissolved in Drurnnaond, D et al. P~og Lipid Res.
(2000).39: 409-
460.
Therapeutic Uses of Sphin~olipid-Containing Delivery Vehicle Compositions
[0052] These delivery vehicle compositions may be used to treat a variety of
diseases
or conditions in warm-blooded animals and in avian species. Examples of
medical uses
of the compositions of the present invention include treating cancer, treating
cardiovascular diseases such as hypertension, cardiac arrhythmia and
restenosis, treating
bacterial, viral, fungal or parasitic infections, treating and/or preventing
diseases through
the use of the compositions of the present inventions as vaccines, treating
inflammation or
treating autoimmune diseases.
[0053] In one embodiment, delivery vehicle compositions in accordance with
this
invention are preferably used to treat neoplasms. Delivery of formulated
sphingolipids to
a tumor site is achieved by administration of liposomes or other particulate
delivery
systems. Preferably liposomes have a diameter of less than 200 nm. Tumor
vasculature
is generally leakier than normal vasculature due to fenestrations or gaps in
the endothelia.
This allows the delivery vehicles of 200 nm or less in diameter to penetrate
the
discontinuous endothelial cell layer and underlying basement membrane
surrounding the
vessels supplying blood to a tumor. Selective accumulation of the delivery
vehicles into
tumor sites following extravasation leads to enhanced sphingolipid delivery
and
therapeutic effectiveness.
[0054] Other cosmetic or diagnostic uses, depending upon the particular
properties of
a preparation, may be envisioned by those skilled in the art. For example,
because of
their sensitivity to divalent cations, cholesteryl hemisuccinate liposomes of
the present
invention may be made to entrap indicator dyes which are sensitive to divalent
cations for
use in colorimetric diagnostic assays.
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CA 02533010 2005-09-06
WO 2004/078677 PCT/CA2004/000344
Administering Delivery Vehicle Compositions
[0055] As mentioned above, the delivery vehicle compositions of the present
invention inay be administered to warm-blooded animals, including humans as
well as to
domestic avian species. For treatment of human ailments, a qualified physician
will
determine how the compositions of the present invention should be utilized
with respect
to dose, schedule and route of administration using established protocols.
Such
applications may also utilize dose escalation should agents encapsulated in
delivery
vehicle compositions of the present invention exhibit reduced toxicity to
healthy tissues
of the subject.
[0056] Preferably, the pharmaceutical compositions of the present invention
are
administered parenterally, i.e., intraarterially, intravenously,
intraperitoneally,
subcutaneously, or intramuscularly. More preferably, the pharmaceutical
compositions
are administered intravenously or intraperitoneally by a bolus injection. For
example, see
Rahman, et al., U.S. patent No. 3,993,754; Sears, U.S. patent No. 4,145,410;
Papahadjopoulos, et al., U.S. patent No. 4,235,871; Schneider, U.S. patent No.
4,224,179;
Lenk, et al., U.S. patent No. 4,522,803; and Fountain, et al., U.S. patent No.
4,588,578,
incorporated by reference.
[0057] In other methods, the pharmaceutical or cosmetic preparations of the
present
invention can be contacted with the target tissue by direct application of the
preparation to
the tissue. The application may be made by topical, "open" or "closed"
procedures. By
"topical", it is meant the direct application of the sphingolipid preparation
to a tissue
exposed to the environment, such as the skin, oropharynx, external auditory
canal, and the
like. "Open" procedures are those procedures that include incising the skin of
a patient
and directly visualizing the underlying tissue to which the pharmaceutical
preparations
are applied. This is generally accomplished by a surgical procedure, such as a
thoracotomy to access the lungs, abdominal laparotomy to access abdominal
viscera, or
other direct surgical approach to the target tissue. "Closed" procedures are
invasive
procedures in which the internal target tissues are not directly visualized,
but accessed via
inserting instruments through small wounds in the skin. For example, the
preparations
may be administered to the peritoneum by needle lavage. Alternatively, the
preparations
may be administered through endoscopic devices.
CA 02533010 2005-09-06
WO 2004/078677 PCT/CA2004/000344
[0058] Pharmaceutical compositions comprising delivery vehicles of the
invention are
prepared according to standard techniques and may comprise water, buffered
water, 0.9%
saline, 0.3% glycine, 5% dextrose and the like, including glycoproteins for
enhanced
stability, such as albumin, lipoprotein, globulin, and the like. These
compositions may be
sterilized by conventional, well-known sterilization techniques. The resulting
aqueous
solutions may be packaged for use or filtered under aseptic conditions and
lyophilized,
the lyophilized preparation being combined with a sterile aqueous solution
prior to
administration. The compositions may contain pharmaceutically acceptable
auxiliary
substances as required to approximate physiological conditions, such as pH
adjusting and
buffering agents, tonicity adjusting agents and the like, for example, sodium
acetate,
sodium lactate, sodium chloride, potassium chloride, calcium chloride, and the
like.
Additionally, the delivery vehicle suspension may include lipid-protective
agents which
protect lipids against free-radical and lipid-peroxidative damages on storage.
Lipophilic
free-radical quenchers, such as alpha-tocopherol and water-soluble iron-
specific
chelators, such as ferrioxamine, are suitable.
[0059] The concentration of delivery vehicles in the pharmaceutical
formulations can
vary widely, such as from less than about 0.05%, usually at or at least about
2-5% to as
much as 10 to 30% by weight and will be selected primarily by fluid volumes,
viscosities,
and the like, in accordance with the particular mode of administration
selected. For
example, the concentration may be increased to lower the fluid load associated
with
treatment. Alternatively, delivery vehicles composed of irritating lipids may
be diluted to
low concentrations to lessen inflammation at the site of administration. For
diagnosis, the
amount of delivery vehicles administered will depend upon the particular label
used, the
disease state being diagnosed and the judgment of the clinician.
[0060] Preferably, the pharmaceutical compositions of the present invention
are
administered intravenously. Dosage for the delivery vehicle formulations will
depend on
the ratio of drug to lipid and the administrating physician's opinion based on
age, weight,
and condition of the patient.
[0061] In addition to pharmaceutical compositions, suitable formulations for
veterinary use may be prepared and administered in a manner suitable to the
subject.
Preferred veterinary subjects include mammalian species, for example, non-
human
16
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WO 2004/078677 PCT/CA2004/000344
primates, dogs, cats, cattle, horses, sheep, and domesticated fowl. Subjects
may also
include laboratory animals, for example, in particular, rats, rabbits, mice,
and guinea pigs.
[0062] The following examples are offered to illustrate but not to limit the
invention.
EXAMPLES
Materials
[0063] All phospholipids and ceramides were obtained from Avanti Polar Lipids
(Alabaster, AL). Cholesterol, cholesteryl hemisuccinate and MTT reagent were
obtained
from Sigma-Aldrich Canada (Oakville, ON, Canada). [3H]cholesterylhexadecyl
ether
([3H]CHE) was purchased from Perkin Elmer (Boston, MA). [14C]C6- and [14C]C16-
ceramide were purchased from American Radiolabeled Chemicals (St. Louis, MO).
Dulbecco's Modified Eagle's Medium (DMEM) and Hank's Balanced Salt Solution
(without pH indicator; Hank's) were obtained from Stem Cell Technologies
(Vancouver,
BC, Canada). Fetal bovine serum was purchased from Hyclone (Logan, UT). L-
glutamine and trypsin-EDTA were obtained from Gibco BRL (Burlington, ON,
Canada).
The Micro BCA Protein Assay kit was purchased from Pierce (Rockford, IL).
Tissue
culture flasks, incubation plates and cell scrapers were obtained from Falcon
(Becton
Dickinson, Franklin Lakes, NJ).
Cell Lines and Culture
[0064] Human estrogen receptor negative MDA435/LCC6 wild-type and MDR-1
gene transfected MDA435/LCC6MDRi multidrug resistant breast cancer cell lines
were
obtained from Dr. Robert Clark, Georgetown University; Washington, D.C. J774
murine
macrophage cells were obtained from ATCC (Rockville, MD). All cells were grown
as
adherent monolayer cultures in 25-cm2 Falcon flasks in DMEM supplemented with
10%
fetal bovine serum and 1 % L-glutamine. Cells were maintained at 37°C
in humidified air
with 5% C02. Cells were sub-cultured weekly using 0.25% trypsin with 1 mM EDTA
(MDA435/LCC6) or gentle cell scraping (J774).
17
CA 02533010 2005-09-06
WO 2004/078677 PCT/CA2004/000344
Preparation of Liposomes
[0065] Lipids were weighed into individual test tubes and dissolved in 1 mL of
chloroform (DPPC, DSPC, CHEMS, Chol), ethanol (C2-, C6-, C8-, Coo-, C~4-
ceramide) or
chloroform:methanol (2:1, volume/volume; C16-ceramide). C~6-ceramide required
brief
heating at 65°C to achieve complete dissolution. Appropriate volumes of
each lipid were
transferred to a single tube in order to achieve the desired ratio of each
lipid component.
All ratios indicated are on a mole:mole basis. [3H]CHE was incorporated at 1
p,Ci/mg
lipid as a non-exchangeable, non-metabolizeable lipid marker to facilitate
liposome
quantitation. For the preparation of ceramide-containing liposomes, [14C]C6-
ceramide or
[i4C]C16-ceramide was incorporated into the formulation at 0.5 ~,Ci/mg
ceramide. The
mixtures were evaporated with vortexing and heating under a stream of nitrogen
gas and
subjected to vacuum drying for a minimum of 4 hours to produce a homogenous
lipid
film. The lipid film was hydrated in 1 ml of warm Hepes buffered saline (HBS;
20 mM
Hepes/1 SO mM NaCl; pH 7.4) with vortexing. Homogenously sized liposomes were
then
produced following a 10 cycle extrusion through three stacked 100 nm
polycarbonate
filters (IVucleopore, Canada) at 65°C for non-ceramide formulations and
95°C for
ceramide formulations, using an extrusion apparatus (Lipex Biomembranes,
Vancouver,
BC, Canada). The resulting mean liposome diameter obtained following extrusion
was
within a range of 91-132 nm, depending on lipid composition, as determined by
quazi-
elastic light scattering using the Nicomp 270 submicron particle sizer model
370/270.
Liposome and ceramide concentrations were determined by liquid scintillation
counting.
Cytotoxicity Assay
[0066] Cell suspensions were diluted 1:1 with trypan blue, counted with a
hemocytometer and seeded into 96-well microtitre plates at 1.5x106 cells/well
in 0.2 ml
complete medium. The perimeter wells were not used and contained 0.2 ml
sterile water.
The cells were allowed to adhere for 24 hours at 37°C, after which the
medium was
aspirated and replaced with 0.1 ml fresh medium. Free ceramide, control
liposome or
ceramide-liposome stocks were diluted into complete medium and added to cells
in 0.1
ml to achieve the desired final concentration. The C~6-ceramide stock was kept
warm and
diluted into warm medium prior to addition to the cells, and remained in
solution at all
times. After 72 hours the cell viability was assessed using a conventional 3-
(4,5-
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CA 02533010 2005-09-06
WO 2004/078677 PCT/CA2004/000344
dimethylthiazol-2-yl)-2,5-diphenyl tehazolium bromide (MTT) dye reduction
assay.
Fifty microliters of 5 mg/ml MTT reagent in phosphate buffered saline (PBS)
was added
to each well. Viable cells with active mitochondria reduce the MTT to an
insoluble
purple formazan precipitate that is solubilized by the subsequent addition of
150 ~,1
dimethyl sulfoxide. The formazan dye was measured spectrophotometrically using
a
Dynex plate reader (570 nm). All assays were performed in triplicate. The
cytotoxic
effect of each treatment was expressed as percent cell viability relative to
untreated
control cells (% control) and is defined as: [(Abss~o treated cells)/( AbsS~o
control
cells)] x 100.
Lipid Uptake Studies
[0067] Cell suspensions were diluted 1:1 with trypan blue, counted with a
hemocytometer and seeded into 6-well Falcon plates at 2.5x105 cells/well in 2
ml
complete medium. The cells were allowed to adhere for 24 hours at 37°C,
after which the
medium was aspirated and replaced with 1 ml complete medium. Free ceramide,
control
liposome or ceramide-liposome stocks were diluted in l ml complete medium and
added
to each well to give the desired final concentration. Cells were incubated
with the
treatments for 1, 4 and 24 hours at 37°C. The incubation medium was
then aspirated and
cells were washed twice with 2 ml Hank's. Cells were gently scraped into 0.5
ml Hank's
and collected into glass scintillation vials using glass pipettes. Each well
was rinsed with
an additional 0.5 ml Hank's to remove residual cells. An aliquot of cells was
removed for
protein quantification and the remainder was counted for radioactivity by
scintillation
counting.
Spectrobhotometric Protein Quantification
[0068] The protein content of each cell aliquot was determined using the
Pierce micro
BCA protein assay according to the method included with the assay kit.
Briefly, a
standard curve was prepared using the supplied purified bovine serum albumin
(BSA)
diluted in distilled water to a final volume of 0.5 ml. Samples were prepared
using 5 p,1
cell suspension + 495 ~1 dH20. Micro BCA reagents A, B and C were added in the
specified ratios. All samples and standards were prepared in glass test tubes,
which were
heated in a 65°C waterbath for 1 hour and cooled to room temperature.
The absorbance
19
CA 02533010 2005-09-06
WO 2004/078677 PCT/CA2004/000344
at 562 nm of each sample was read against a dHaO reference. The protein
concentration
for each cell sample was determined using a standard curve prepared from the
known
BSA samples.
hz Vivo Evaluation of Antitumor Activity
[0069] Evaluation of the antitumor activity of C16-ceramide-containing
liposomes was
carried out in the J774 ascites tumor model. On day zero, 1x106 cells were
inoculated
intraperitoneally (i..p.) into female Balb/c mice (12 mice/group). Mice were
administered
saline control and liposomes either by intravenous bolus or intraperitoneally
on the days
indicated in the Examples below. Animals were weighed and monitored daily for
survival.
Example 1
The Inactivity of Free Lon:~-Chain Ceramides is Due to Lack of Intracellular
DeliverX
[0070] In order evaluate the relationship between the acyl chain length of
exogenous
ceramides and cytotoxicity, the MTT assay (see Methods) was carried out by the
addition
of free C2-, C6-, C8-, Coo- and C16-ceramide to human estrogen-negative
MDA435/LCC6
wild-type and MDR-1 gene transfected MDA435/LCC6MDRi breast cancer cells, over
a
range of 0-100 ~M final ceramide concentration. The 72-hour MTT cytotoxicity
results
shown in Figure 1 demonstrate that cytotoxic activity is dependent on ceramide
acyl
chain length. With the exception of C~-ceramide, as acyl chain length
increased the
cytotoxic activity decreased. This trend may be explained by the
hydrophobicity, and
thus cell-permeability characteristics, of the various ceramides. The C2-
ceramide, being
very hydrophilic, likely remains dispersed in the tissue culture media. The C6-
and C~-
ceramides were the most cytotoxic, with ICSO values in the 3-14 ~,M range. As
the chain
length increases to Clo-, C14- and C16- ceramide the hydrophobic nature also
increases,
and ICSO values of approximately 45 ~M are observed for Coo-ceramide and in
excess of
100 ~M are observed for C14- and C~6- ceramides. From these results, it was
concluded
that the C6-ceramide is the most potent exogenous ceramide form. Free
ceramides of
varying acyl chain length were also tested for cytotoxicity in the J774
macrophage cell
line. Similar results were obtained in the J774 cell line as with the wild-
type and resistant
CA 02533010 2005-09-06
WO 2004/078677 PCT/CA2004/000344
MDA435/LCC6 cell line, with increases in acyl chain length correlating with
decreases in
cytotoxicity (see Figure 2).
[0071] Cellular uptake studies were performed using radioactive C6- and C16-
ceramides to demonstrate that acyl chain length determines ceramide
cytotoxicity due to
differences in cell-permeability. Uptake studies were performed as set forth
in the
Methods employing the MDA435/LCC6 and MDA435/LCC6MDRi cell lines. Figure 3
shows that for both the wild-type and resistant cell lines [14C]C6-ceramides
levels steadily
increased to 7 pmole ceramide/~g protein over the 24 hour incubation period
while
[14C]C16-ceramide levels did not significantly increase and remained under 2
pmole
ceramide/~g protein over the 24 hour period. The lack of cell-associated
[14C]C16-
ceramide suggests that the long chain ceramide does not partition into the
cell membrane.
It is possible that these lipids form aggregate structures or bind to serum
proteins in he
culture medium, although no turbidity was observed. Nevertheless, the
Cl6~ceramide was
not found to be cell-associated, which correlates with its lack of cytotoxic
effect.
Example 2
The Intracellular Delivery of Long-Chain Ceramides is Enhanced Ifs hitro by
Formulation
in Cholesteryl Hemisuccinate (CHEMS) Liposomes
[0072] Although C6-ceramide is efficacious in vitro, C~6-ceramide is the more
physiologically relevant ceramide. This is based on the observation that both
short- and
long-term increases in C16-ceramide accumulation have been observed during
apoptosis
(Thomas, et al. (supra)). As the results in Example 1 suggest that the lack of
cytotoxic
effect of C16-ceramide may be due to poor cellular uptake, C16-ceramide was
formulated
into liposomes with the goal of increasing the intracellular delivery of this
lipid.
[0073] Liposomes containing various lipid components were made-as described in
the
Methods section and the formulation characteristics were monitored during
preparation.
The results are presented in Table 1 below:
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CA 02533010 2005-09-06
WO 2004/078677 PCT/CA2004/000344
Table 1.
Lipid Composition Mole % Formulation Characteristics
C16-Cer
C16-cer/DSPC/Chol (15:40:45)15 hydrates well and extrudes with
>80%
efficiency
C~6-cer/DSPC/Chol (20:35:45)20 lipid film difficult to hydrate;
lipid aggregates
CI6-cer/DSPC/Chol (15:55:30)15 hydrates well and extrudes with
>80%
efficiency
C16-cer/DSPC/Chol (20:50:30)20 lipid film difficult to hydrate;
lipid aggregates
C~6-cer/Chol (50:50) 50 Lipid film difficult to hydrate;
lipid aggregates
C~6-cer/CHEMS (50:50) 50 Hydrates well and extrudes with
>80%
efficiency at concentration
< 10 mg/mL
C16-cer/DPPG/PEG350-DSPE30 Hydrates well and extrudes but
non-uniform,
(30:30:40) trimodal size distribution;
liposome aggregate
C~6-cer/DSPC/DOPE/PEG2000-20 Lipid film difficult to hydrate;
lipid aggregates
DSPE (20:35:35:10)
C~6-cer/DPPC/PEG2000-DSPE15 Hydrates well and extrudes with>80%
( 15 :75 :10) efficiency
[0074] As depicted in Table l, liposomes consisting of C16-cer/DSPC/Chol
(15:40:45
and 15:55:30 mole ratio) could be prepared with 15 mole % C16-cer. When the
mole % of
Cls-ceramide in these formulations was increased to 20 mole %, the lipid films
were
difficult to hydrate and lipid aggregates formed. Similarly, liposome
formulations
containing C16-cerlChol (50:50 mole ratio), C~6-cer/DPPG/PEG350-DSPE (30:30:40
mole ratio) and C16-cer/DSPC/DOPE/PEG2000-DSPE (20:35:35:10 mole ratio) could
not
be successfully, formulated. In contrast, C16-ceramide could be incorporated
into CHEMS
contaixiing liposomes up to 50 mole %, for a final liposome composition of C16-
ceramide/CHEMS of 50:50 (mole ratio). These liposomes were stable and
displayed a
mean diameter range of 97-132 nm.
[0075] The inventors next determined whether the stable formulation of C~6-
ceramide
into CHEMS liposomes translated into enhanced cytotoxicity in vitro. Liposomes
consisting of C~6-cer/CHEMS (50:50) and control liposomes consisting of
DPPC/CHEMS (50:50) were tested for cytotoxic effects in the J774 macrophage
cell line
by employing the MTT cytotoxicity assay as described in the Methods. As well
as
providing for the stable formulation of C~6-cer, the presence of the pH
sensitive CHEMS
lipid, imparts to the liposomes the ability to be destabilized in the acidic
environment of
endosomes and lysosomes. This is due to the protonation of the lipid when
exposed to
22
CA 02533010 2005-09-06
WO 2004/078677 PCT/CA2004/000344
low pH conditions. Thus, J774 macrophage cells were employed due to the
ability of this
cell line to endocytose liposomes thereby ensuring that the ceramide would be
specifically delivered to the endosome. Delivery of ceramide to the endosome
is
preferred as it is within this membrane compartment that natural ceramide is
produced
during the process of apoptosis.
[0076] The MTT cytotoxicity results shown in Figure 4 indicate that liposomes
composed of C16-cer/CHEMS (50:50) dramatically improve the cytotoxicity of C16-
ceramide in the J774 macrophage cell line. Specifically, while the ICSO value
of C16-
ceramide when exogenously applied to cells in its free form was well in excess
of 100 ~,M
in these cells, its formulation and delivery via CHEMS liposomes decreased its
ICso to
36.1 ~.M, bringing it into the range of cytotoxicity observed with free C6-
ceramide (14.4
p.M). This cytotoxic effect was ceramide-specific and was not attributed to
the CHEMS
lipid alone, as control DPPCICHEMS (50:50) liposomes were non-cytotoxic. The
non-
ceramide lipid of the control liposomes in this case was DPPC in order to
closely match
the 16 carbon acyl chain length of the ceramide
[0077) Cellular uptake studies were conducted as described in the Methods. The
results indicate that both the liposome and the ceramide components are
internalized, as
evidenced by uptake of the [3H]CHE and ['4C]C16-ceramide labels which both
approach
50% after 24 hours (Figure 5). This indicates that the C16-ceramide lipid is
being
delivered via liposomes (rather than by passive lipid exchange), and
demonstrates that
endosomal delivery of these liposomes can enhance ceramide-induced apoptosis.
Thus,
in addition to providing a formulation that can stably incorporate up to 50
mole % of the
long-chain C~6-ceramide, CHEMS also allows for increased cytotoxicity in J774
macrophage cell lines due to increased cellular uptake.
Example 3
The Ifa Vivo Anti-Tumor Activity of Long-Chain Ceramides is Enhanced
by Fornulation in CHEMS Liposomes
[0078] In order to determine whether the enhanced isa vitro cytotoxicity of
C~6-
ceramide in CHEMS-containing liposomes would also be observed in vivo, the
antitumor
activity of C~6-cer/CHEMS/PEG2000-DSPE (47.5:47.5:5) liposomes containing both
[3H]CHE and ['4C]C~6-ceramide radiolabels was evaluated in the J774 ascites
tumor
23
CA 02533010 2005-09-06
WO 2004/078677 PCT/CA2004/000344
model and compared to control CHEMS-containing liposomes prepared in the
absence of
C16-ceramide (DPPC/CHEMS/PEG2000-DSPE, 47.5:47.5:5) and containing the [3H]CHE
radiolabel. The lipid, DSPE-PEG2000 was incorporated into the liposomes in
order to
enhance the blood stability of the formulations.
[0079] Female Balb/c mice bearing the J774 ascites tumor were prepared as
described
in the Methods. Two studies were conducted where twelve mice were administered
saline, C16-cer/CHEMS/P.EG2000-DSPE liposomes and DPPC/CHEMS/PEG2000-DSPE
(control) liposomes as follows: a) intravenous bolus on days 1, 5 and 9 at
lipid
concentrations of 200 mg/kg; and b) intraperitoneally on days l, 5 and 9 at a
lipid
concentration of 200 mglkg for both ceramide-containing liposomes and control
liposomes. Results from these studies are presented in Figures 6 and 7,
respectively, and
the arrows in the figures indicate the days of treatment administration.
[0080] As demonstrated in Figure 6, the C16-ceramide-containing formulations
displayed increased antitumor effects in the J774 ascites tumor model in
relation to
control liposomes and saline when administered at a dose of 200 mg/kg by i.v.
bolus on
days 1, 5 and 9 (cell inoculation day 0). Under these treatment conditions,
the saline and
liposome control groups displayed median survival times of 21.7 and 22.5 days,
respectively, while the C~6-ceramide containing liposome treatment group had a
median
survival time of 28.3 days. This corresponded to a statistically significant
(p<0.001)
mean increase in lifespan (ILS) of 25.6% (Figure 6).
[0081] Direct intraperitoneal administration of the formulations to the site
of the
ascites tumor cells was carried out in order to investigate whether the
therapeutic
response might be improved beyond that observed following i.v. administration.
In this
study, saline, control liposomes and ceramide-containing liposomes were
administered
i.p. at a 200 mg/kg total lipid dose on days 1, 5, and 9. The survival curves
presented in
Figure 7 indicate that groups treated with saline or control liposomes had a
median
survival time of 21.6 and 22.1 days, respectively. The group treated i.p. with
ceramide-
liposomes at a dose of 200 mglkg total lipid showed a median survival time of
26.7 days,
which corresponded to a statistically significant (p<0.001) mean ILS of 26.7%.
[0082] Cumulatively, these results thus demonstrate that increases in the
survival rate
of mice bearing the J774 ascites tumor can be obtained by the administration
of liposomal
formulations comprising C16-ceramide and CHEMS.
24
