U.S. patent application number 13/993946 was filed with the patent office on 2014-02-20 for liposomal formulation of nonglycosidic ceramides and uses thereof.
This patent application is currently assigned to LUDWIG INSTITUTE FOR CANCER RESEARCH LTD.. The applicant listed for this patent is Vincenzo Cerundolo, Simon Eastman. Invention is credited to Vincenzo Cerundolo, Simon Eastman.
Application Number | 20140050780 13/993946 |
Document ID | / |
Family ID | 45524965 |
Filed Date | 2014-02-20 |
United States Patent
Application |
20140050780 |
Kind Code |
A1 |
Cerundolo; Vincenzo ; et
al. |
February 20, 2014 |
LIPOSOMAL FORMULATION OF NONGLYCOSIDIC CERAMIDES AND USES
THEREOF
Abstract
The invention provides liposomes containing nonglycosidic
ceramides within their bilayers, and compositions thereof. These
liposomes activate murine iNKT cells and induce dendritic cell (DC)
maturation, both in vitro and in vivo at an efficacy that is
comparable to their corresponding soluble nonglycosidic ceramides.
Also provided are methods for treating diseases using the liposomes
and compositions of the invention.
Inventors: |
Cerundolo; Vincenzo;
(Oxford, GB) ; Eastman; Simon; (Delta,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cerundolo; Vincenzo
Eastman; Simon |
Oxford
Delta |
|
GB
CA |
|
|
Assignee: |
LUDWIG INSTITUTE FOR CANCER
RESEARCH LTD.
New York
NY
|
Family ID: |
45524965 |
Appl. No.: |
13/993946 |
Filed: |
December 22, 2011 |
PCT Filed: |
December 22, 2011 |
PCT NO: |
PCT/US11/66840 |
371 Date: |
November 6, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61426725 |
Dec 23, 2010 |
|
|
|
61442755 |
Feb 14, 2011 |
|
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Current U.S.
Class: |
424/450 ;
424/184.1; 424/277.1 |
Current CPC
Class: |
A61K 39/001156 20180801;
A61K 9/1272 20130101; A61K 9/1277 20130101; A61K 45/06 20130101;
A61K 39/001195 20180801; A61K 39/001191 20180801; A61K 39/00117
20180801; A61K 39/001184 20180801; A61K 39/001188 20180801; A61K
39/001189 20180801; A61K 39/39 20130101; A61K 39/0011 20130101;
A61K 39/001186 20180801; A61K 2039/55555 20130101 |
Class at
Publication: |
424/450 ;
424/184.1; 424/277.1 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 45/06 20060101 A61K045/06; A61K 39/00 20060101
A61K039/00 |
Claims
1.-114. (canceled)
115. A liposome comprising: (a) threitolceramide present in an
amount of about 3 wt. % to about 12 wt. %, based upon the total
weight of the liposome; (b) a first lipid present in an amount of
about 20 wt. % to about 30 wt. %, based upon the total weight of
the liposome; and (c) a second lipid present in an amount of about
65 wt. % to about 75 wt. %, based upon the total weight of the
liposome.
116. The liposome of claim 115, wherein the first lipid comprises a
phosphotidylcholine.
117. The liposome of claim 116, wherein the phosphatidylcholine is
selected from the group consisting of
1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC),
1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC),
1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC),
1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1-myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine (MPPC),
1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC),
1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC),
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC),
1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC),
1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC),
1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC), egg
phosphatidylcholine (EPC), and mixtures thereof.
118. The liposome of claim 117, wherein the phosphtidylcholine
comprises egg phosphatidylcholine (EPC).
119. The liposome of claim 115, wherein the second lipid comprises
phosphatidylglycerol.
120. The liposome of claim 119, wherein the phosphatidylglycerol is
selected from the group consisting of 1,2-dierucoyl
phosphatidylglycerol (DEPG), 1,2-dilauroyl phosphatidylglycerol
(DLPG), 1,2-dimyristoyl phosphatidylglycerol (DMPG), 1,2-dioleoyl
phosphatidylglycerol (DOPG), 1,2-dipalmitoyl phosphatidylglycerol
(DPPS), 1,2-distearoyl phosphatidylglycerol (DSPG),
1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG), egg
phosphatidylglycerol (EPG), salts of any of the foregoing, and
mixtures thereof.
121. The liposome of claim 120, wherein the phosphatidylglycerol
comprises egg phosphatidylglycerol (EPG).
122. The liposome of claim 115, further comprising at least one
antigen.
123. The liposome of claim 122, wherein the at least one antigen
comprises at least one tumor antigen.
124. The liposome of claim 123, wherein the tumor antigen comprises
a member selected from the group consisting of P1A, MUC1, MAGE-A1,
MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8,
MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1, GAGE-2, GAGE-3,
GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE,
LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4
(MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7, MAGE-C2,
LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i,
NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, XAGE, antigenic
fragments thereof, and mixtures thereof.
125. The liposome of claim 115, further comprising at least one
adjuvant.
126. The liposome of claim 115, further comprising at least one
therapeutic agent or antigen.
127. The liposome of claim 126, wherein the at least one
therapeutic agent or antigen is selected from the group consisting
of an immune modulator, a Toll-like receptor agonist, a Nod ligand,
an anti-viral agent, an antifungal agent, an antibiotic, an
antiviral antibody, a cancer immune therapeutic, a chemotherapy
agent, a kinase inhibitor, a cytotoxic agent, an anti-asthmatic
agent, an antihistamine agent, an anti-inflammatory agent, a
vaccine adjuvant, a second liposome, an artificial antigen
presenting cell, a cytokine or chemokine blocking antibody, P1A,
MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6,
MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1,
GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1,
RAGE-1, CAGE, LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3
(MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase,
MAGE-C1/CT7, MAGE-C2, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3,
SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A,
XAGE, antigenic fragments thereof, and combinations thereof.
128. The liposome of claim 115, having a diameter of less than
about 100 nm.
129. A composition comprising the liposome of claim 115 and a
pharmaceutically acceptable diluent, excipient or carrier.
130. The composition of claim 129, wherein the liposome is present
in the composition in an amount of about 1 mg/mL to about 20
mg/mL.
131. The composition of claim 129, wherein the composition is
formulated for parenteral, intrathecal, transdermal, rectal, oral,
or nasal administration.
132. The composition of claim 129, further comprising at least one
therapeutic agent.
133. The composition of claim 132, wherein the therapeutic agent is
within the liposome.
134. A method of stimulating an immune response in a mammalian
subject comprising administering to the subject the liposome of
claim 115.
135. The method of claim 134, further comprising administering a
therapeutic agent to the mammalian subject.
136. A method of treating a mammalian subject having cancer
comprising administering to said subject a therapeutically
effective amount of the liposome of claim 115.
137. The method of claim 136, wherein the cancer is selected from
the group consisting of basal cell carcinoma, breast cancer
leukemia, Burkitt's lymphoma, colon cancer, esophageal cancer,
bladder cancer, gastric cancer, head and neck cancer,
hepatocellular cancer, Hodgkin's lymphoma, hairy cell leukemia,
Wilms' tumor, thyroid cancer, thymoma and thymic carcinoma,
testicular cancer, T-cell lymphoma, prostate cancer, non-small cell
lung cancer, liver cancer, renal cell cancer, melanoma, and
combinations thereof.
138. The method of claim 136, further comprising administering to
the subject at least one further therapeutic agent, therapy, or
antigen selected from the group consisting of a chemotherapeutic
agent, a radiotherapeutic agent, radiation therapy, an immune
modulator, a Toll-like receptor agonist, a Nod ligand, an
anti-viral agent, an antifungal agent, an antibiotic, an antiviral
antibody, a cancer immune therapeutic, a chemotherapy agent, a
kinase inhibitor, a cytotoxic agent, an anti-asthmatic agent, an
antihistamine agent, an anti-inflammatory agent, a vaccine
adjuvant, a second liposome, an artificial antigen presenting cell,
a cytokine or chemokine blocking antibody, P1A, MUC1, MAGE-A1,
MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8,
MAGE-A9, MAGE-A10, MAGE-All, MAGE-A12, GAGE-1, GAGE-2, GAGE-3,
GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE,
LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4
(MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7, MAGE-C2,
LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i,
NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, XAGE, antigenic
fragments thereof, and a combination thereof.
139. A method of reducing the growth or metastatic spread of a
tumor in a mammalian subject comprising administering to said
subject a therapeutically effective amount of a combination therapy
comprising the liposome of claim 115 or a composition comprising
the liposome and at least one further therapeutic agent, therapy,
or antigen selected from the group consisting of a chemotherapeutic
agent, a radiotherapeutic agent, radiation therapy, an immune
modulator, a Toll-like receptor agonist, a Nod ligand, an
anti-viral agent, an antifungal agent, an antibiotic, an antiviral
antibody, a cancer immune therapeutic, a chemotherapy agent, a
kinase inhibitor, a cytotoxic agent, an anti-asthmatic agent, an
antihistamine agent, an anti-inflammatory agent, a vaccine
adjuvant, a second liposome, an artificial antigen presenting cell,
a cytokine or chemokine blocking antibody, P1A, MUC1, MAGE-A1,
MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8,
MAGE-A9, MAGE-A10, MAGE-All, MAGE-A12, GAGE-1, GAGE-2, GAGE-3,
GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE,
LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4
(MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7, MAGE-C2,
LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i,
NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, XAGE, antigenic
fragments thereof, and a combination thereof.
140. A method of treating an infection in a subject, said infection
caused by an infectious agent selected from the group consisting of
a virus, a microbe or bacteria, or a parasite, comprising
administering to said subject a therapeutically effective amount of
the liposome of claim 115.
141. A method of making the liposome of claim 115 comprising: (a)
preparing a stock solution of threitolceramide in an organic
solvent; (b) combining an aliquot of the stock solution with a
mixture containing a first lipid and a second lipid to form a lipid
solution; (c) diluting the lipid solution with an aqueous solution;
(d) forming multi-lamellar vesicles (MLVs) from the diluted lipid
solution; and (e) downsizing the MLVs to about 50 nm to about 150
nm at a temperature above the Tc of the lipids, wherein the
threitolceramide is present in an amount of about 3 wt. % to about
12 wt. %, based upon the total weight of the liposome; the first
lipid is present in an amount of about 20 wt. % to about 30 wt. %,
based upon the total weight of the liposome; and the second lipid
present in an amount of about 65 wt. % to about 75 wt. %, based
upon the total weight of the liposome.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of priority of
U.S. Provisional Application No. 61/426,725, filed Dec. 23, 2010
and U.S. Provisional Application No. 61/442,755, filed Feb. 14,
2011. The disclosure of each priority application is incorporated
herein by reference in its entirety.
TECHNICAL FIELD OF THE INVENTION
[0002] The invention generally relates to liposomes that contain
nonglycosidic ceramides, compositions that comprise the liposomes,
methods of making the liposomes, and methods of using the liposomes
to treat diseases.
BACKGROUND OF THE INVENTION
[0003] Invariant natural killer T (iNKT) cells have important tumor
immunosurveillance properties, and their activation with
pharmacological agents is associated with tumour clearance in
animal models. The archetypal iNKT cell agonist,
.alpha.-galactoseceramide (.alpha.-GalCer, shown below) is a
glycolipid that is currently being used in a limited number of
clinical trials for the treatment of lung, myeloma, and head and
neck tumors. Treatment with .alpha.-GalCer often results in
side-effects, such as activation induced anergy of iNKT cells (i.e.
long-term unresponsiveness to repeated .alpha.-GalCer treatment)
and dendritic cell (DC) lysis by iNKT cells following presentation
of .alpha.-GalCer. Loss of circulating levels of iNKT cells could
represent a therapeutically significant limitation with
iNKT-cell-based therapies if multi-dosing regimens are
required.
.alpha.-Galactosylceramide
##STR00001##
[0005] A group of nonglycosidic ceramides that substantially mimic
the binding properties of .alpha.-GalCer with the human CD1d
molecule, but differs significantly in the interaction with T-cell
receptors (TCR) compared to .alpha.-GalCer is described in U.S.
Patent Application Publication No. 2009/0239813 and Silk et al., J.
of Immunol. 180:6452-6456 (2008), each incorporated herein by
reference. These compounds, such as threitolceramide (TC) and
glycerolceramide, are able to sensitize murine and human iNKT cells
and, like .alpha.-GalCer, maintain potent anti-tumor responses in
animal models. Some of these nonglycosidic ceramides display poor
water solubility, however, which can limit their use clinically. A
need exists for the effective delivery of nonglycosidic ceramides
for clinical applications.
SUMMARY OF THE INVENTION
[0006] The present invention has many facets, including new
liposomes; compositions (including pharmaceutical compositions)
containing the liposomes, and optionally containing additional
agents; methods of making the liposomes and compositions; and
methods of using the liposomes and compositions, including
therapeutic and prophylactic methods of preventing, ameliorating,
treating, and curing diseases.
[0007] In one aspect, described herein are liposomes that contain a
lipid bilayer membrane surrounding an aqueous core, wherein the
lipid bilayer comprises:
[0008] (a) a nonglycosidic ceramide present in an amount of about 1
wt. % to about 50 wt. %, based on the total weight of the liposome
or the lipid bilayer; and
[0009] (b) one to five lipids present in an amount of about 50 wt.
% to about 99 wt. %, based on the total weight of the liposome or
the lipid bilayer.
[0010] In some embodiments, the liposomes described herein have a
diameter of less than about 100 nm. In some embodiments, the
liposomes are about 50 nm to about 150 nm in diameter, or about 75
nm to 125 nm in diameter, or about 75 nm to about 100 nm in
diameter. In some embodiments, the liposomes described herein are
produced by a method that includes extrusion through an 80 nm
filter. In some embodiments, the nonglycosidic ceramide is present
in an amount of about 2 wt. % to about 20 wt. %, or about 2 wt. %
to about 10 wt. %, based on the total weight of the liposome or the
lipid bilayer. In some embodiments, the one to five lipids is
present in an amount of about 80 wt. % to about 98 wt. %, based on
the total weight of the liposome or the lipid bilayer.
[0011] In some embodiments, the nonglycosidic ceramide is a
compound of Formula I, or a pharmaceutically acceptable salt
thereof:
##STR00002##
[0012] wherein R.sup.1 is a hydrophobic moiety than can occupy the
C' channel of human CD1d, and fills at least about 30%, or at least
about 35%, of the volume of the C' channel that is occupied by the
(CH.sub.2).sub.13CH.sub.3 group of .alpha.-galactosylceramide
(.alpha.-GalCer) when the .alpha.-GalCer is bound to human CD1d; or
R.sup.1 is a C.sub.1-C.sub.25 hydrocarbon chain;
[0013] R.sup.2 is a hydrophobic moiety that can occupy the A'
channel of human CD1d, and fills at least about 30%, or at least
about 40%, of the volume of the A' channel that is occupied by the
(CH.sub.2).sub.24CH.sub.3 group of .alpha.-GalCer when the
.alpha.-GalCer is bound to human CD1d; or R.sup.2 is a
C.sub.1-C.sub.30 hydrocarbon chain;
[0014] R.sup.3 is H or OH (e.g., H);
[0015] R.sup.4 and R.sup.5 either are both H or together form a
single bond, with the proviso that when R.sup.4 and R.sup.5
together form a single bond, R.sup.3 is H;
[0016] X is
##STR00003##
[0017] Y is CH.sub.2 or
##STR00004##
[0018] R.sup.6 and R.sup.7 each independently are H, OH, or phenyl,
with the proviso that either one of R.sup.6 or R.sup.7 is H and the
other is H, OH, or phenyl, or one of R.sup.6 or R.sup.7 is OH and
the other is phenyl;
[0019] R.sup.8 is H, OH, or OSO.sub.3H; and
[0020] n is 1, 2, 3, or 4 (e.g., 1, 2, or 3).
[0021] In some variations, X is
##STR00005##
and Y is CH.sub.2. In some variations, one of R.sup.6 and R.sup.7
is H, and the other is either H or OH.
[0022] In some variations, R.sup.1 is a linear C.sub.5-C.sub.14
hydrocarbon chain, for example, a linear C.sub.11-C.sub.14
hydrocarbon chain. In some variations, R.sup.2 is a linear
C.sub.8-C.sub.25 hydrocarbon chain. In additional or alternative
variations, R.sup.1, R.sup.2, or a combination thereof comprise at
least 1 double bond (e.g., 1, 2, or 3 double bonds). Optionally, at
least one of the double bonds comprises Z stereochemistry.
[0023] In some variations, the nonglycosidic ceramide is selected
from the group consisting of arabinitolceramide, glycerolceramide,
threitolceramide, threitolceramide C.sub.14 acyl,
threitol-22-(Z)-ceramide, 4-deoxy-4-phenyl-threitolceramide,
4-deoxy-4-phenyl-threitol-22-(Z)-ceramide, glycerol-phosphate
ceramide, inositolceramide, inositolceramide C15 acyl,
myoinositolceramide salt, 4-phenyl threitolceramide, 4-phenyl
threitol-22-(Z)-ceramide, threitol-(19Z,22Z)-ceramide, and mixtures
thereof. In some exemplary embodiments, the nonglycosidic ceramide
is arabinitolceramide, glycerolceramide, threitolceramide, e.g.,
threitolceramide.
[0024] In some embodiments, at least one of the one to five lipids
is a phospholipid, such as, for example, a phosphatidylcholine, a
phosphatidic acid, a phosphatidylethanolamine, a
phosphatidylglycerol, a phosphatidylserine, a phosphatidylinositol,
an inositol phosphate, and mixtures thereof.
[0025] In some embodiments, the liposomes described herein further
comprise at least one antigen (e.g., a viral antigen, a bacterial
antigen, a fungal antigen, a tumor antigen, and mixtures thereof).
In some embodiments, the at least one antigen comprises a tumor
antigen. In some exemplary embodiments, the at least one antigen
comprises a full length protein antigen, a long peptide antigen,
and a short peptide antigen. In some embodiments, the antigen
comprises a hydrophilic antigen.
[0026] In some embodiments, the liposome includes at least one
antigen in the aqueous core of the liposome. Examples of such
antigens include hydrophilic tumor antigens, which include, but are
not limited to, NY-ESO-1, tyrosinase, MAGE-3 and Melan-A.
[0027] In some embodiments, the liposome includes at least one
antigen within the lipid bilayer. Examples of such antigens include
hydrophobic tumor antigens.
[0028] In some embodiments, the liposome includes at least one
antigen that is noncovalently associated with the lipid bilayer. In
some embodiments, the liposome includes at least one antigen that
is covalently linked to the lipid bilayer, such as, for example,
covalently attached to a phospholipid in the lipid bilayer.
[0029] In some embodiments, the liposome comprises an antigen with
a net negative charge. In some of these embodiments when the
liposome comprises an antigen with a net negative charge, the
liposome comprises at least two lipids wherein at least one of the
lipids is cationic. Nonlimiting examples of the cationic lipid
include a trimethyl sphingosine, a trimethyl phytosphingosine, a
pyridinium ceramide, a
3.beta.-[N--(N',N'-dimethylaminoethane)-carbamoyl]cholesterol
hydrochloride, a 1,2-dioleoyl-3-trimethylammonium-propane, a
1,2-dioleoyl-3-trimethylammonium-propane, a
1,2-dimyristoyl-3-trimethylammonium-propane, a
1,2-dipalmitoyl-3-trimethylammonium-propane, a
1,2-stearoyl-3-trimethylammonium-propane, a
1,2-dioleoyl-3-dimethylammonium-propane, a
1,2-dimyristoyl-3-dimethylammonium-propane, a
1,2-dipalmitoyl-3-dimethylammonium-propane, a
1,2-distearoyl-3-dimethylammonium-propane, a
dimethyldioctadecylammonium (Bromide Salt), a
2-dilauroyl-sn-glycero-3-ethylphosphocholine, a
1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, a
1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine, a
1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, a
1,2-distearoyl-sn-glycero-3-ethylphosphocholine, a
1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, a
1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, a lysyl
phosphatidylglycerol, and mixtures thereof.
[0030] In some embodiments, the liposome comprises an antigen with
a net positive charge, such as, for example, NY-ESO-1. In some of
these embodiments when the liposome comprises an antigen with a net
positive charge, the liposome comprises at least two lipids wherein
at least one of the lipids is anionic. In some embodiments, the
anionic lipid is selected from the group consisting of anionic
sphingosine, an anionic phospholipid, a phosphatidylinositol, an
inositol phosphate, a cardiolipin, a bis(monoacylglycero)phosphate,
an anionic detergent that is not a sphingolipid or a phospholipid,
a liponucleotide, a TLR-4 agonist, a diacylglycerol pyrophosphate,
and mixtures thereof.
[0031] In some embodiments, the tumor antigen comprises a member
selected from the group consisting of MUC1, MAGE, BAGE, RAGE, CAGE,
SSX-2, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, antigenic
fragments thereof, and mixtures thereof. In some embodiments, the
tumor antigen comprises a member selected from the group consisting
of P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MACE-A5, MAGE-A6,
MAGE-A7, MAGE-A8, MAGE-A9, MACE-A10, MAGE-A11, MACE-A12, GAGE-1,
GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1,
RAGE-1, CAGE, LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3
(MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase,
MAGE-C1/CT7, MAGE-C2, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3,
SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A,
XAGE, antigenic fragments thereof, and mixtures thereof.
[0032] In some embodiments, the liposomes includes at least one
zwitterionic lipid. In some embodiments, the liposome comprises at
least three lipids.
[0033] In some exemplary embodiments, the liposome described herein
comprises:
[0034] (a) a nonglycosidic ceramide present in an amount of about 1
wt. % to about 20 wt. %, based on the total weight of the liposome
or the lipid bilayer;
[0035] (b) a first lipid present in an amount of about 15 wt. % to
about 55 wt. %, or about 20 wt. % to about 30 wt. %, based on the
total weight of the liposome or the lipid bilayer; and,
[0036] (c) a second lipid present in an amount of about 35 wt. % to
about 75 wt. %, or about 65 wt. % to about 75 wt. %, based on the
total weight of the liposome or the lipid bilayer, wherein the
second lipid is anionic.
[0037] In some of these exemplary embodiments, the first lipid and
the second lipid have a weight ratio of about 1 to about 3. In some
of these embodiments, the nonglycosidic ceramide is present in an
amount of about 2 wt. % to about 15 wt. %, or about 3 wt. % to
about 12 wt. %, for example, about 5 wt. % or about 10 wt. %, based
on the total weight of the liposome or the lipid bilayer.
[0038] In some embodiments, the first lipid is a zwitterionic
lipid. In some embodiments, the zwitterionic lipid is a
phospholipid, for example phosphatidylcholine. In some exemplary
embodiments, the phosphatidylcholine is selected from the group
consisting of 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC),
1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC),
1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC),
1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1-myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine (MPPC),
1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC),
1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC),
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC),
1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC),
1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC),
1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC), egg
phosphatidylcholine (EPC), and mixtures thereof. In some
embodiments, the zwitterionic lipid is selected from the group
consisting of EPC, soy phosphatidylcholine,
dioleoylphosphatidylcholine (DOPC) and
palmitoyloleoylphosphatidylcholone (POPC).
[0039] In some embodiments, the second lipid is an anionic lipid.
In some embodiments, the anionic lipid is a phospholipid, for
example, phosphatidylglycerol. In some exemplary embodiments, the
phosphatidylglycerol is selected from the group consisting of
1,2-dierucoyl phosphatidylglycerol (DEPG), 1,2-dilauroyl
phosphatidylglycerol (DLPG), 1,2-dimyristoyl phosphatidylglycerol
(DMPG), 1,2-dioleoyl phosphatidylglycerol (DOPG), 1,2-dipalmitoyl
phosphatidylglycerol (DPPS), 1,2-distearoyl phosphatidylglycerol
(DSPG), 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG), egg
phosphatidylglycerol (EPG), salts of any of the foregoing (e.g.,
sodium, ammonium, or sodium/ammonium), and mixtures thereof, (e.g.,
egg phosphatidylglycerol). In some embodiments, the liposome
comprises a third lipid. In some embodiments, the third lipid is
selected from the group consisting of a fatty acid, a glycerolipid,
a phospholipid, a sphingolipid, a sterol lipid, a prenol lipid, a
saccharolipid, and mixtures thereof. In some exemplary embodiments
the third lipid is cholesterol, dimethyldioctadecyl ammonium
bromide, and a mixture thereof.
[0040] In some of these exemplary embodiments, the liposome further
comprises a tumor antigen comprising a net positive charge. In some
embodiments, the tumor antigen is NY-ESO-1 and is present in an
amount of about 1 .mu.g to about 1 mg per 2 mg of the liposome
(e.g., about 400 .mu.g per 2 mg of the liposome).
[0041] In some of these exemplary embodiments, the liposome further
comprises at least one adjuvant, wherein the at least one adjuvant
is in the core of the liposome, in the lipid bilayer, covalently
attached to the lipid bilayer, non-covalently associated with the
lipid bilayer, or combinations thereof.
[0042] In some of these exemplary embodiments, the liposome further
comprises at least one therapeutic agent in the core of the
liposome. Examples of the therapeutic agent include an immune
modulator, a Toll-like receptor agonist, a Nod ligand, an
anti-viral agent, an antifungal agent, an antibiotic, an antiviral
antibody, a cancer immune therapeutic, a chemotherapy agent, a
kinase inhibitor, a cytotoxic agent, an anti-asthmatic agent, an
antihistamine agent, an anti-inflammatory agent, a vaccine
adjuvant, a second liposome, an artificial antigen presenting cell,
a cytokine or chemokine blocking antibody, and combinations
thereof.
[0043] In some embodiments, the liposome described herein
comprises:
[0044] (a) a nonglycosidic ceramide present in an amount of about 5
wt. % or about 10 wt. %, based on the total weight of the liposome
or the lipid bilayer;
[0045] (b) egg phosphatidylcholine in an amount of about 20 wt. %
to about 30 wt. %, based on the total weight of the liposome or the
lipid bilayer; and,
[0046] (c) egg phosphatidylglycerol present in an amount of about
65 wt. % to about 75 wt. %, based on the total weight of the
liposome or the lipid bilayer.
[0047] In another aspect, described herein is a composition
comprising the liposome described herein and a pharmaceutically
acceptable excipient, carrier, or adjuvant. In some embodiments,
the liposome is present in the composition in an amount of about 1
mg/mL to about 20 mg/mL, (e.g., about 13 mg/mL). In some
embodiments, the composition further comprises an antigen (e.g., a
viral antigen, a bacterial antigen, a tumor antigen, and mixtures
thereof) present in an admixture with the liposome. In some
embodiments, the antigen comprises a tumor antigen, for example,
MUC1, MAGE, BAGE, RAGE, CAGE, SSX-2, NY-ESO-1, PRAME, PSMA,
tyrosinase, melan-A, antigenic fragments thereof, and mixtures
thereof. In some embodiments, the antigen comprises a tumor antigen
selected from the group consisting of P1A, MUC1, MAGE-A1, MAGE-A2,
MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,
MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4,
GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-4, RAGE-1, CAGE, LB33/MUM-1,
NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4),
brain glycogen phosphorylase, MAGE-C1/CT7, MAGE-C2, LAGE-1, SSX-1,
SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME,
PSMA, tyrosinase, melan-A, XAGE, antigenic fragments thereof, and
mixtures thereof. In some exemplary embodiments, the tumor antigen
is NY-ESO-1. The NY-ESO-1 can be present in the composition in a
concentration of about 0.01 mg/mL to about 5 mg/mL.
[0048] In some embodiments, the composition further includes a
therapeutic agent present in an admixture with the liposome. In
some variations, the therapeutic agent is selected from the group
consisting of an immune modulator, a Toll-like receptor agonist, a
Nod ligand, an anti-viral agent, an antifungal agent, an
antibiotic, an antiviral antibody, a cancer immune therapeutic, a
chemotherapy agent, a kinase inhibitor, a cytotoxic agent, an
anti-asthmatic agent, an antihistamine agent, an anti-inflammatory
agent, a vaccine adjuvant, a second liposome, an artificial antigen
presenting cell, a cytokine or chemokine blocking antibody, and
mixtures thereof.
[0049] The composition described herein can be formulated for
parenteral, intrathecal, transdermal, rectal, oral, or nasal
administration. Further, the composition described herein can be
formulated for administration in a form selected from the group
consisting of a tablet, a capsule, a powder, a suppository, a
lozenge, a soft gelatin capsule, a transdermal patch, an aerosol, a
dragee, a cream, a drop, a liquid suspension, an emulsion, or an
ointment.
[0050] In another aspect, described herein is a method for
stimulating an immune response in a mammalian subject by
administering to the subject a liposome or a composition described
herein.
[0051] In yet another aspect, described herein is a method of
treating a viral infection, a microbial infection, a parasitic
infection, an autoimmune disease, an allergy, or asthma in a
mammalian subject in need thereof comprising administering to the
subject the liposome or composition described herein in an amount
effective to treat said viral infection, microbial infection,
parasitic infection, autoimmune disease, allergy, or asthma.
[0052] In some embodiments, the method comprises treating a viral
infection caused by a virus is selected from the group consisting
of a hepatitis virus, a liver tropic virus, a skin tropic virus, a
lung tropic virus, an immune tropic virus, and combinations
thereof. In some variations, the virus is hepatitis B virus (HBV),
hepatitis C virus (HBC), human papilloma virus (HPV), herpes
simplex virus (HSV), influenza virus, respiratory syncytial virus
(RSV), human immunodeficiency virus (HIV), Epstein-Barr virus
(EBV), cytomegalovirus (CMV), and combinations thereof.
[0053] In some embodiments, the method comprises treating a
microbial infection selected from the group consisting of a
bacterial infection of the lung, a bacterial infection of the gut,
a bacterial infection of the skin, or combinations thereof.
[0054] In some embodiments where the method is a method of treating
an infection caused by an infectious agent selected from a virus, a
microbe or bacteria, or a parasite, the method further comprises
administering an antigen to the mammalian subject prior to,
concurrently with, or after administration of the liposome, wherein
the antigen is an antigen that elicits an immune response to the
infectious agent.
[0055] In some embodiments, the method comprises treating an
autoimmune disease selected from the group consisting of psoriasis,
Crohn's disease, connective tissue disease, multiple sclerosis,
systemic lupus erythematosus, rheumatoid arthritis, autoimmune
pulmonary inflammation, Guillain Barre syndrome, autoimmune
thyroiditis, insulin dependent diabetes mellitis, myasthenia
gravis, graft versus host disease, and autoimmune inflammatory eye
disease.
[0056] In yet another aspect described herein is a method of
treating cancer in a mammalian subject. In this method, the subject
is administered a therapeutically effective amount of a liposome or
composition described herein. In some variations, at least one
further therapeutic or therapy selected from the group consisting
of a chemotherapeutic agent, a radiotherapeutic agent, and
radiation therapy is administered to the subject. The further
therapeutic can be administered concurrently with the liposome or
composition described herein or separately.
[0057] In some variations of this aspect, the cancer is selected
from the group consisting of basal cell carcinoma, breast cancer
leukemia, Burkitt's lymphoma, colon cancer, esophageal cancer,
bladder cancer, gastric cancer, head and neck cancer,
hepatocellular cancer, Hodgkin's lymphoma, hairy cell leukemia,
Wilms' tumor, thyroid cancer, thymoma and thymic carcinoma,
testicular cancer, T-cell lymphoma, prostate cancer, non-small cell
lung cancer, liver cancer, renal cell cancer, melanoma, and
combinations thereof.
[0058] In some embodiments, described herein is a method of
reducing the growth or metastatic spread of a tumor in a mammalian
subject. In this method, the subject is administered a
therapeutically effective amount of a liposome or composition
described herein. In some variations, at least one further
therapeutic agent or therapy selected from the group consisting of
a chemotherapeutic agent, a radiotherapeutic agent, and radiation
therapy is administered to the subject.
[0059] In yet another aspect, described herein is a method of
making a liposome comprising:
[0060] (a) preparing a stock solution of a nonglycosidic ceramide
in an organic solvent;
[0061] (b) combining an aliquot of the stock solution with a
mixture of one to five lipids to form a lipid solution;
[0062] (c) diluting the lipid solution with an aqueous
solution;
[0063] (d) forming multi-lamellar vesicles (MLVs); and,
[0064] (e) downsizing the MLVs to about 50 nm to about 150 nm at a
temperature above the Tc of the lipids by, for example,
extrusion.
[0065] The following numbered paragraphs each succinctly define one
or more exemplary variations of the invention.
[0066] 1. A liposome comprising:
[0067] a lipid bilayer membrane surrounding an aqueous core,
wherein the lipid bilayer comprises:
[0068] (a) a nonglycosidic ceramide present in an amount of about 1
wt. % to about 50 wt. %, based on the total weight of the liposome
or the lipid bilayer; and
[0069] (b) one to five lipids present in an amount of about 50 wt.
% to about 99 wt. %, based on the total weight of the liposome or
the lipid bilayer.
[0070] 2. The liposome of paragraph 1, wherein the diameter of the
liposome is less than about 100 nm.
[0071] 3. The liposome of paragraph 1, wherein the diameter of the
liposome is about 50 nm to about 150 nm.
[0072] 4. The liposome of paragraph 1, wherein the diameter of the
liposome is about 75 nm to about 125 nm.
[0073] 5. The liposome of paragraph 1, wherein the diameter of the
liposome is about 75 nm to about 100 nm.
[0074] 6. The liposome of any one of paragraphs 1 to 5, wherein the
liposome is produced by a method that includes extrusion through an
80 nm filter.
[0075] 7. The liposome of any one of paragraphs 1 to 6, wherein the
nonglycosidic ceramide is present in an amount of about 2 wt. % to
about 20 wt. %, based on the total weight of the liposome or the
lipid bilayer.
[0076] 8. The liposome of any one of paragraphs 1 to 6, wherein the
nonglycosidic ceramide is present in an amount of about 2 wt. % to
about 10 wt. %, based on the total weight of the liposome or the
lipid bilayer.
[0077] 9. The liposome of any one of paragraphs 1 to 8, wherein the
one to five lipids are present in an amount of about 80 wt. % to
about 98 wt. %, based on the total weight of the liposome or the
lipid bilayer.
[0078] 10. The liposome of any one of paragraphs 1-9, wherein the
nonglycosidic ceramide is a compound of Formula I, or a
pharmaceutically acceptable salt thereof:
##STR00006##
[0079] wherein R.sup.1 is a hydrophobic moiety than can occupy the
C' channel of human CD1d, and fills at least about 30%, of the
volume of the C' channel that is occupied by the
(CH.sub.2).sub.13CH.sub.3 group of .alpha.-galactosylceramide
(.alpha.-GalCer) when the .alpha.-GalCer is bound to human
CD1d;
[0080] R.sup.2 is a hydrophobic moiety that can occupy the A'
channel of human CD1d, and fills at least about 30%, of the volume
of the A' channel that is occupied by the
(CH.sub.2).sub.24--CH.sub.3 group of .alpha.-GalCer when the
.alpha.-GalCer is bound to human CD1d;
[0081] R.sup.3 is H or OH;
[0082] R.sup.4 and R.sup.5 either are both H or together form a
single bond, with the proviso that when
[0083] R.sup.4 and R.sup.5 together form a single bond, R.sup.3 is
H;
[0084] X is
##STR00007##
[0085] Y is CH.sub.2 or
##STR00008##
[0086] R.sup.6 and R.sup.7 each independently are H, OH, or phenyl,
with the proviso that either one of R.sup.6 or R.sup.7 is H and the
other is H, OH, or phenyl, or one of R.sup.6 or R.sup.7 is OH and
the other is phenyl;
[0087] R.sup.8 is H, OH, or OSO.sub.3H; and
[0088] n is 1, 2, 3, or 4.
[0089] 11. The liposome of paragraph 10, wherein R.sup.1 fills at
least about 35% of the occupied volume of the C' channel, R.sup.2
fills at least about 40% of the occupied volume of the A' channel,
or a combination thereof.
[0090] 12. The liposome of paragraph 1, wherein the nonglycosidic
ceramide is a compound of Formula I, or a pharmaceutically
acceptable salt thereof:
##STR00009##
[0091] wherein R.sup.1 is a C.sub.1-C.sub.25 hydrocarbon chain;
[0092] R.sup.2 is a C.sub.1-C.sub.30 hydrocarbon chain;
[0093] R.sup.3 is H or OH;
[0094] R.sup.4 and R.sup.5 either are both H or together form a
single bond, with the proviso that when R.sup.4 and R.sup.5
together form a single bond, R.sup.3 is H;
[0095] X is
##STR00010##
[0096] Y is CH.sub.2 or
##STR00011##
[0097] R.sup.6 and R.sup.7 each independently are H, OH, or phenyl,
with the proviso that either one of R.sup.6 or R.sup.7 is H and the
other is H, OH, or phenyl, or one of R.sup.6 or R.sup.7 is OH and
the other is phenyl;
[0098] R.sup.8 is H, OH, or OSO.sub.3H; and
[0099] n is 1, 2, 3, or 4.
[0100] 13. The liposome of any one of paragraphs 10 to 12, wherein
R.sup.1 is a linear C.sub.5-C.sub.14 hydrocarbon chain.
[0101] 14. The liposome of paragraph 13, wherein R.sup.1 is a
linear C.sub.11-C.sub.14 hydrocarbon chain.
[0102] 15. The liposome of any one of paragraphs 10 to 14, wherein
R.sup.2 is a linear C.sub.8-C.sub.15 hydrocarbon chain.
[0103] 16. The liposome of any one of paragraphs 10 to 15, wherein
at least one of R.sup.1 and R.sup.2 comprises at least 1 double
bond.
[0104] 17. The liposome of paragraph 16, wherein at least one of
R.sup.1 and R.sup.2 comprises 1, 2, or 3 double bonds.
[0105] 18. The liposome of any one of paragraphs 16-17, wherein the
double bond comprises Z stereochemistry.
[0106] 19. The liposome of any one of paragraphs 10 to 18, wherein
X is
##STR00012##
and Y is CH.sub.2.
[0107] 20. The liposome of paragraph 19, wherein X is
##STR00013##
[0108] 21. The liposome of any one of paragraphs 10 to 20, wherein
n is 1, 2, or 3.
[0109] 22. The liposome of any one of paragraphs 10 to 21, wherein
R.sup.3 is H.
[0110] 23. The liposome of any one of paragraphs 10 to 22, wherein
R.sup.6 and R.sup.7 are each H.
[0111] 25. The liposome of any one of paragraphs 10 to 22, wherein
one of R.sup.6 or R.sup.7 is H and the other is OH.
[0112] 25. The liposome of any one of paragraphs 1 to 9, wherein
the nonglycosidic ceramide is selected from the group consisting of
arabinitolceramide, glycerolceramide, threitolceramide,
threitolceramide C14 acyl, threitol-22-(Z)-ceramide,
4-deoxy-4-phenyl-threitolceramide,
4-deoxy-4-phenyl-threitol-22-(Z)-ceramide, glycerol-phosphate
ceramide, inositolceramide, inositolceramide C15 acyl,
myoinositolceramide salt, 4-phenyl threitolceramide, 4-phenyl
threitol-22-(Z)-ceramide, threitol-(19Z,22Z)-ceramide, and mixtures
thereof.
[0113] 26. The liposome of paragraph 25, wherein the nonglycosidic
ceramide is selected from the group consisting of
arabinitolceramide, glycerolceramide, threitolceramide, and
mixtures thereof.
[0114] 27. The liposome of paragraph 25, wherein the nonglycosidic
ceramide is threitolceramide.
[0115] 28. The liposome of any one of paragraphs 1-27, wherein at
least one of the one to five lipids is a phospholipid.
[0116] 29. The liposome of paragraph 28, wherein the phospholipid
is selected from the group consisting a phosphatidylcholine, a
phosphatidic acid, a phosphatidylethanolamine, a
phosphatidylglycerol, a phosphatidylserine, a phosphatidylinositol,
an inositol phosphate, and mixtures thereof.
[0117] 30. The liposome of any one of paragraphs 1-29 further
comprising at least one antigen.
[0118] 31. The liposome according to paragraph 30, wherein the at
least one antigen comprises a member selected from the group
consisting of a viral antigen, a bacterial antigen, a fungal
antigen, a tumor antigen, and mixtures thereof.
[0119] 32. The liposome of paragraph 30, wherein the at least one
antigen comprises at least one tumor antigen.
[0120] 33. The liposome of according to any one of paragraphs 30 to
32, wherein the at least one antigen comprises a full length
protein antigen, a long peptide antigen, and a short peptide
antigen.
[0121] 34. The liposome of any one of paragraphs 30 to 33, wherein
the at least one antigen includes a hydrophilic antigen.
[0122] 35. The liposome according to any one of paragraphs 30 to
34, wherein the liposome includes at least one antigen in the
aqueous core of the liposome.
[0123] 36. The liposome of any one of paragraphs 30 to 35, wherein
the liposome includes at least one antigen within the lipid
bilayer.
[0124] 37. The liposome of any one of paragraphs 30 to 36, wherein
the liposome includes at least one antigen attached to the outside
of the lipid bilayer.
[0125] 38. The liposome of any one of paragraphs 30 to 37, wherein
the liposome includes at least one antigen that is noncovalently
associated with the lipid bilayer.
[0126] 39. The liposome of any one of paragraphs 30 to 38, wherein
the liposome includes at least one antigen that is covalently
linked to the lipid bilayer.
[0127] 40. The liposome of paragraph 39, wherein the liposome
includes at least one antigen that is covalently attached to a
phospholipid in the lipid bilayer.
[0128] 41. The liposome of any one of paragraphs 30 to 40, wherein
the liposome comprises at least one antigen with a net negative
charge.
[0129] 42. The liposome of paragraph 41, wherein the liposome
comprises at least two lipids, wherein at least one of the lipids
is cationic.
[0130] 43. The liposome of paragraph 42, wherein the cationic lipid
selected from the group consisting of a trimethyl sphingosine, a
trimethyl phytosphingosine, a pyridinium ceramide, a
3.beta.-[N--(N',N'-dimethylaminoethane)-carbamoyl]cholesterol
hydrochloride, a 1,2-dioleoyl-3-trimethylammonium-propane, a
1,2-dioleoyl-3-trimethylammonium-propane, a
1,2-dimyristoyl-3-trimethylammonium-propane, a
1,2-dipalmitoyl-3-trimethylammonium-propane, a
1,2-stearoyl-3-trimethylammonium-propane, a
1,2-dioleoyl-3-dimethylammonium-propane, a
1,2-dimyristoyl-3-dimethylammonium-propane, a
1,2-dipalmitoyl-3-dimethylammonium-propane, a
1,2-distearoyl-3-dimethylammonium-propane, a
dimethyldioctadecylammonium (Bromide Salt), a
2-dilauroyl-sn-glycero-3-ethylphosphocholine, a
1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, a
1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine, a
1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, a
1,2-distearoyl-sn-glycero-3-ethylphosphocholine, a
1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, a
1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, a lysyl
phosphatidylglycerol, and mixtures thereof.
[0131] 44. The liposome of any one of paragraphs 30 to 43, wherein
the liposome comprises at least one antigen with a net positive
charge.
[0132] 45. The liposome of paragraph 44, wherein the tumor antigen
comprises a member selected from the group consisting of MUC1,
MAGE, BAGE, RAGE, CAGE, SSX-2, NY-ESO-1, PRAME, PSMA, tyrosinase,
melan-A, antigenic fragments thereof, and mixtures thereof.
[0133] 45.1 The liposome of paragraph 44, wherein the tumor antigen
comprises a member selected from the group consisting of P1A, MUC1,
MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7,
MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1, GAGE-2,
GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1,
CAGE, LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3),
MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7,
MAGE-C2, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5,
SCP-i, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, XAGE, antigenic
fragments thereof, and mixtures thereof.
[0134] 46. The liposome of paragraph 44 or 45, wherein the liposome
comprises at least two lipids, wherein at least one of the lipids
is anionic.
[0135] 47. The liposome of paragraph 46, wherein the anionic lipid
is selected from the group consisting of anionic sphingosine, an
anionic phospholipid, a phosphatidylinositol, an inositol
phosphate, a cardiolipin, a bis(monoacylglycero)phosphate, an
anionic detergent that is not a sphingolipid or a phospholipid, a
liponucleotide, a TLR-4 agonist, a diacylglycerol pyrophosphate,
and mixtures thereof.
[0136] 48. The liposome of any one of paragraphs 1 to 47, wherein
the liposome includes at least one zwitterionic lipid.
[0137] 49. The liposome of any one of paragraphs 1 to 48 comprising
at least three lipids.
[0138] 50. The liposome of any one of the paragraphs 1-40
comprising: [0139] (a) a nonglycosidic ceramide present in an
amount of about 1 wt. % to about 20 wt. %, based on the total
weight of the liposome or the lipid bilayer; [0140] (b) a first
lipid present in an amount of about 15 wt. % to about 55 wt. %,
based on the total weight of the liposome or the lipid bilayer;
and, [0141] (c) a second lipid present in an amount of about 35 wt.
% to about 75 wt. %, based on the total weight of the liposome or
the lipid bilayer, wherein the second lipid is anionic.
[0142] 51. The liposome of paragraph 50 further comprising a tumor
antigen comprising a net positive charge.
[0143] 52. The liposome of paragraph 50, wherein the nonglycosidic
ceramide is present in an amount of about 2 wt. % to about 15 wt.
%, based on the total weight of the liposome or the lipid
bilayer.
[0144] 53. The liposome of paragraph 52, wherein the nonglycosidic
ceramide is present in an amount of about 3 wt. % to about 12 wt.
%, based on the total weight of the liposome or the lipid
bilayer.
[0145] 54. The liposome of paragraph 53, wherein the nonglycosidic
ceramide is present in an amount of about 5 wt. %, based on the
total weight of the liposome or the lipid bilayer.
[0146] 55. The liposome of paragraph 53, wherein the nonglycosidic
ceramide is present in an amount of about 10 wt. %, based on the
total weight of the liposome or the lipid bilayer.
[0147] 56. The liposome of any one of paragraphs 50-55, wherein the
first lipid is a zwitterionic lipid.
[0148] 57. The liposome of paragraph 56, wherein the zwitterionic
lipid is a phosphosolipid.
[0149] 58. The liposome of paragraph 57, wherein the phosphoslipid
is phosphatidylcholine.
[0150] 59. The liposome of paragraph 58, wherein the
phosphatidylcholine is selected from the group consisting of
1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC),
1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC),
1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC),
1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1-myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine (MPPC),
1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC),
1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC),
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC),
1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC),
1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC),
1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC), egg
phosphatidylcholine (EPC), and mixtures thereof.
[0151] 60. The liposome of any one of paragraphs 50-59, wherein the
first lipid comprises egg phosphatidylcholine.
[0152] 61. The liposome of any one of paragraphs 50-60, wherein the
second lipid comprises a phospholipid.
[0153] 62. The liposome of paragraph 61, wherein the second lipid
comprises phosphatidylglycerol.
[0154] 63. The liposome of paragraph 62, wherein the
phosphatidylglycerol is selected from the group consisting of
1,2-dierucoyl phosphatidylglycerol (DEPG), 1,2-dilauroyl
phosphatidylglycerol (DLPG), 1,2-dimyristoyl phosphatidylglycerol
(DMPG), 1,2-dioleoyl phosphatidylglycerol (DOPG), 1,2-dipalmitoyl
phosphatidylglycerol (DPPS), 1,2-distearoyl phosphatidylglycerol
(DSPG), 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG), egg
phosphatidylglycerol (EPG), salts of any of the foregoing, and
mixtures thereof.
[0155] 64. The liposome of any one of paragraphs 50-63, wherein the
second lipid comprises egg phosphatidylglycerol.
[0156] 65. The liposome of any one of paragraphs 50-64, wherein the
first lipid is present in an amount of about 20 wt. % to about 30
wt. % and the second lipid is present in an amount of about 65 wt.
% to about 75 wt. %, based on the total weight of the liposome or
the lipid bilayer.
[0157] 66. The liposome of any one of paragraphs 50-65, wherein the
weight ratio of the first lipid to the second lipid is about 1 to
about 3.
[0158] 67. The liposome of any one of paragraphs 50-66, wherein the
tumor antigen comprises a member selected from the group consisting
of MUC1, MAGE, BAGE, RAGE, CAGE, SSX-2, NY-ESO-1, PRAME, PSMA,
tyrosinase, melan-A, antigenic fragments thereof, and mixtures
thereof.
[0159] 67.1. The liposome of any one of paragraphs 50-66, wherein
the tumor antigen comprises a member selected from the group
consisting of P1A, MUC1, MAGE-A2, MAGE-A3, MAGE-M, MAGE-A5,
MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12,
GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8,
BAGE-1, RAGE-1, CAGE, LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3
(MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase,
MAGE-C1/CT7, MAGE-C2, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3,
SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A,
XAGE, antigenic fragments thereof, and mixtures thereof.
[0160] 68. The liposome of paragraph 67, wherein the tumor antigen
is NY-ESO-1 and is present in an amount of about 1 .mu.g to about 1
mg per 2 mg of the liposome.
[0161] 69. The liposome of paragraph 68, wherein the NY-ESO-1 is
present in an amount of about 400 .mu.g per 2 mg of the
liposome.
[0162] 70. The liposome of any one of paragraphs 1-69, further
comprising at least one adjuvant, wherein the at least one adjuvant
is in the core of the liposome, in the lipid bilayer, covalently
attached to the lipid bilayer, non-covalently associated with the
lipid bilayer, or combinations thereof.
[0163] 71. The liposome of any one of paragraphs 1-70 further
comprising at least one therapeutic agent in the core of the
liposome.
[0164] 72. The liposome of paragraph 71, wherein the at least one
therapeutic agent is selected from the group consisting of an
immune modulator, a Toll-like receptor agonist, a Nod ligand, an
anti-viral agent, an antifungal agent, an antibiotic, an antiviral
antibody, a cancer immune therapeutic, a chemotherapy agent, a
kinase inhibitor, a cytotoxic agent, an anti-asthmatic agent, an
antihistamine agent, an anti-inflammatory agent, a vaccine
adjuvant, a second liposome, an artificial antigen presenting cell,
a cytokine or chemokine blocking antibody, and combinations
thereof.
[0165] 73. The liposome of any one of the paragraphs 1-72
comprising:
[0166] (a) a nonglycosidic ceramide present in an amount of about 5
wt. % or about 10 wt. %, based on the total weight of the liposome
or the lipid bilayer;
[0167] (b) egg phosphatidylcholine in an amount of about 20 wt. %
to about 30 wt. %, based on the total weight of the liposome or the
lipid bilayer; and,
[0168] (c) egg phosphatidylglycerol present in an amount of about
65 wt. % to about 75 wt. %, based on the total weight of the
liposome or the lipid bilayer;
[0169] wherein the diameter of the liposome is less than about 100
nm.
[0170] 74. A composition comprising the liposome of any one of
paragraphs 1-72 and a pharmaceutically acceptable diluent,
excipient, carrier, or adjuvant.
[0171] 75. The composition of paragraph 74, wherein the liposome is
present in the composition in an amount of about 1 mg/mL to about
20 mg/mL.
[0172] 76. The composition of paragraph 75, wherein the liposome is
present in an amount of about 13 mg/mL.
[0173] 77. The composition of any one of paragraphs 74-76 further
comprising at least one antigen admixed with the liposome.
[0174] 78. The composition according to paragraph 77, wherein the
at least one antigen is selected from the group consisting of a
viral antigen, a bacterial antigen, a tumor antigen, and mixtures
thereof.
[0175] 79. The composition of paragraph 77, wherein the at least
one antigen comprises a tumor antigen.
[0176] 80. The composition of paragraph 79, wherein the tumor
antigen comprises a member selected from the group consisting of
MUC1, MAGE, BAGE, RAGE, CAGE, SSX-2, NY-ESO-1, PRAME, PSMA,
tyrosinase, melan-A, antigenic fragments thereof, and mixtures
thereof.
[0177] 80.1. The composition of paragraph 79, wherein the tumor
antigen comprises a member selected from the group consisting of
P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6,
MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1,
GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1,
RAGE-1, CAGE, LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3
(MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase,
MAGE-C1/CT7, MAGE-C2, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3,
SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A,
XAGE, antigenic fragments thereof, and mixtures thereof.
[0178] 81. The composition of paragraph 80, wherein the tumor
antigen is NY-ESO-1 and is present in the composition in a
concentration of about 0.1 mg/mL to about 5 mg/mL.
[0179] 82. The composition of any one of paragraphs 74-81, further
comprising at least one adjuvant in an admixture with the
liposome.
[0180] 83. The composition of any one of paragraphs 74-82 further
comprising at least one therapeutic agent present in an admixture
with the liposome.
[0181] 84. The composition of paragraph 83, wherein the at least
one therapeutic agent is selected from the group consisting of an
immune modulator, a Toll-like receptor agonist, a Nod ligand, an
anti-viral agent, an antifungal agent, an antibiotic, an antiviral
antibody, a cancer immune therapeutic, a chemotherapy agent, a
kinase inhibitor, a cytotoxic agent, an anti-asthmatic agent, an
antihistamine agent, an anti-inflammatory agent, a vaccine
adjuvant, a second liposome, an artificial antigen presenting cell,
a cytokine or chemokine blocking antibody, and combinations
thereof.
[0182] 85. The composition of any one of paragraphs 74-84, wherein
the composition is formulated for parenteral, intrathecal,
transdermal, rectal, oral, or nasal administration.
[0183] 86. The composition of paragraph 85, wherein the composition
is formulated for intravenous, intraperitoneal, subcutaneous, or
intramuscular administration.
[0184] 87. A method of stimulating an immune response in a
mammalian subject comprising administering to the subject a
liposome according to any one of paragraphs 1-73 or a composition
according to any one of paragraphs 74-86.
[0185] 88. The method of paragraph 87, further comprising
administering an antigen to the mammalian subject prior to,
concurrently with, or after administration of the liposome, wherein
the immune response is an immune response to the antigen.
[0186] 89. A method of treating a viral infection, a microbial
infection, a parasitic infection, an autoimmune disease, an
allergy, or asthma in a mammalian subject in need thereof
comprising administering to the subject the liposome of any one of
paragraphs 1-73 or a composition according to any one of paragraphs
74-86 in an amount effective to treat said viral infection,
microbial infection, parasitic infection, autoimmune disease,
allergy, or asthma.
[0187] 90. The method of paragraph 89, comprising treating a viral
infection caused by a virus selected from the group consisting of a
hepatitis virus, a liver tropic virus, a skin tropic virus, a lung
tropic virus, an immune tropic virus, and combinations thereof.
[0188] 91. The method of paragraph 90, wherein the virus is
hepatitis B virus (HBV), hepatitis C virus (HBC), human papilloma
virus (HPV), herpes simplex virus (HSV), influenza virus,
respiratory syncytial virus (RSV), human immunodeficiency virus
(HIV), Epstein-Ban virus (EBV), cytomegalovirus (CMV), and
combinations thereof.
[0189] 92. The method of paragraph 89, comprising treating a
microbial infection selected from the group consisting of a
bacterial infection of the lung, a bacterial infection of the gut,
a bacterial infection of the skin, and combinations thereof.
[0190] 93. The method of any one of paragraphs 89 to 92 that is a
method of treating an infection caused by an infectious agent
selected from a virus, a microbe or bacteria, or a parasite,
wherein the method further comprises administering an antigen to
the mammalian subject prior to, concurrently with, or after
administration of the liposome, wherein the antigen is an antigen
that elicits an immune response to the infectious agent.
[0191] 94. The method of paragraph 89, comprising treating an
autoimmune disease selected from the group consisting of psoriasis,
Crohn's disease, connective tissue disease, multiple sclerosis,
systemic lupus erythematosus, rheumatoid arthritis, autoimmune
pulmonary inflammation, Guillain Bane syndrome, autoimmune
thyroiditis, insulin dependent diabetes mellitis, myasthenia
gravis, graft versus host disease, and autoimmune inflammatory eye
disease.
[0192] 95. A method of treating a cancer in a mammalian subject
comprising administering to said subject a therapeutically
effective amount of a liposome according to any one of paragraphs
1-73 or a composition according to any one of paragraphs 74-86.
[0193] 96. The method of paragraph 95, wherein the cancer is
selected from the group consisting of basal cell carcinoma, breast
cancer leukemia, Burkitt's lymphoma, colon cancer, esophageal
cancer, bladder cancer, gastric cancer, head and neck cancer,
hepatocellular cancer, Hodgkin's lymphoma, hairy cell leukemia,
Wilms' tumor, thyroid cancer, thymoma and thymic carcinoma,
testicular cancer, T-cell lymphoma, prostate cancer, non-small cell
lung cancer, liver cancer, renal cell cancer, melanoma, and
combinations thereof.
[0194] 97. The method of paragraph 95 or 96, further comprising the
step of administering to the subject at least one further
therapeutic or therapy selected from the group consisting of a
chemotherapeutic agent, a radiotherapeutic agent, and radiation
therapy.
[0195] 98. The method of paragraph 97, wherein the liposome and the
further therapeutic or therapy are administered concurrently.
[0196] 99. The method of paragraph 97, wherein the liposome and the
further therapeutic or therapy are administered separately.
[0197] 100. The method according to any one of paragraphs 95 to 99,
further comprising administering a tumor antigen to the mammalian
subject prior to, concurrently with, or after administration of the
liposome.
[0198] 101. A method of reducing the growth or metastatic spread of
a tumor in a mammalian subject comprising administering to said
subject a therapeutically effective amount of a combination therapy
comprising a liposome according to any one of paragraphs 1-73 or a
composition according to any one of paragraphs 74-86 and at least
one further therapeutic or therapy selected from the group
consisting of a chemotherapeutic agent, a radiotherapeutic agent,
and radiation therapy.
[0199] 102. Use of a liposome according to any one of paragraphs
1-73 or a composition according to any one of paragraphs 74-86 for
treating cancer in a mammalian subject, or in the manufacture of a
medicament for treating cancer in a mammalian subject.
[0200] 103. Use of a liposome according to any one of paragraphs
1-73 or a composition according to any one of paragraphs 74-86 for
treating an autoimmune disease in a mammalian subject, or in the
manufacture of a medicament for treating an autoimmune disease in a
mammalian subject.
[0201] 104. Use of a liposome according to any one of paragraphs
1-73 or a composition according to any one of paragraphs 74-86 for
treating a disorder selected from the group consisting of a viral
infection, a bacterial infection, an allergy, a parasitic infection
and asthma in a mammalian subject, or in the manufacture of a
medicament for treating a disorder selected from the group
consisting of a viral infection, a bacterial infection, an allergy,
a parasitic infection and asthma in a mammalian subject.
[0202] 105. Use of a liposome according to any one of paragraphs
1-73 or a composition according to any one of paragraphs 74-86 for
stimulating an immune response in a mammalian subject, or in the
manufacture of a medicament for stimulating an immune response in a
mammalian subject.
[0203] 106. The method or use of any one of paragraphs 87-105,
wherein the mammalian subject is human.
[0204] 107. A method of making a liposome of any one of paragraphs
1-73, comprising:
[0205] (a) preparing a stock solution of a nonglycosidic ceramide
in an organic solvent;
[0206] (b) combining an aliquot of the stock solution with a
mixture of one to five lipids to form a lipid solution;
[0207] (c) diluting the lipid solution with an aqueous
solution;
[0208] (d) forming multi-lamellar vesicles (MLVs); and,
[0209] (e) downsizing the MLVs to about 50 nm to about 150 nm at a
temperature above the Tc of the lipids.
[0210] 108. The method of paragraph 107, wherein the aqueous
solution further comprises an antigen.
[0211] 109. The method of any one of paragraphs 107-108, wherein
the MLVs are downsized using extrusion.
[0212] 110. The method of paragraph 109, wherein the extrusion is
through an 80 nm filter.
[0213] 111. The method of any one of paragraphs 107-110, wherein
the MLV's are downsized to liposomes less than 100 nm in size.
[0214] 112. The method of any one of paragraphs 107-111 further
comprising mixing the liposomes with a pharmaceutically acceptable
carrier.
[0215] The foregoing summary is not intended to define every aspect
of the invention, and additional aspects are described in other
sections, such as the Detailed Description. The entire document is
intended to be related as a unified disclosure, and it should be
understood that all combinations of features described herein are
contemplated, even if the combination of features are not found
together in the same sentence, or paragraph, or section of this
document.
[0216] In addition to the foregoing, the invention includes, as an
additional aspect, all embodiments of the invention narrower in
scope in any way than the variations specifically mentioned above.
With respect to aspects of the invention described or claimed with
"a" or "an," it should be understood that these terms mean "one or
more" unless context unambiguously requires a more restricted
meaning. With respect to elements described as one or more within a
set, it should be understood that all combinations within the set
are contemplated. If aspects of the invention are described as
"comprising" a feature, embodiments also are contemplated
"consisting of" or "consisting essentially of" the feature.
[0217] Aspects of the invention described as methods of treatment
should also be understood to include first or subsequent "medical
use" aspects of the invention or "Swiss use" of compositions for
the manufacture of a medicament for treatment of the same disease
or condition.
[0218] Although the applicant(s) invented the full scope of the
claims appended hereto, the claims appended hereto are not intended
to encompass within their scope the prior art work of others.
Therefore, in the event that statutory prior art within the scope
of a claim is brought to the attention of the applicants by a
Patent Office or other entity or individual, the applicant(s)
reserve the right to exercise amendment rights under applicable
patent laws to redefine the subject matter of such a claim to
specifically exclude such statutory prior art or obvious variations
of statutory prior art from the scope of such a claim. Variations
of the invention defined by such amended claims also are intended
as aspects of the invention. Additional features and variations of
the invention will be apparent to those skilled in the art from the
entirety of this application, and all such features are intended as
aspects of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0219] FIG. 1A shows that nonglycosidic ceramide containing
liposomes activate iNKT cells in vitro in splenocytes prepared from
wild-type mice. FIG. 1B shows that the nonglycosidic ceramide
containing liposomes caused minimal activation in vitro using
splenocyteS prepared from CD.sup.-/- (NKT-deficient (mice (negative
control).
[0220] FIG. 2 shows a graph indicating that the non-glycosidic
ceramide-containing liposomes induced dendritic cell maturation in
vivo, as measured by CD86 expression, with much greater CD86
expression observed in wild-type mice than CD1d.sup.-/-
(NKT-deficient) mice.
[0221] FIGS. 3A and 3B shows serum IL-4 and IFN-.gamma.,
respectively, levels 2 hours after injection of either wild-type
mice or CD1d.sup.-/- (NKT-deficient) mice with liposomes.
[0222] FIG. 4 shows the level of IFN-.gamma. released by invariant
natural killer (iNTK) cells that were assayed with dendritic cells
pulsed for 40 hours with different concentrations of liposomes.
[0223] FIG. 5 is a graph showing the viability of lipid pulsed
dendritic cells after iNKT interaction.
[0224] FIG. 6 shows the level of IFN-.gamma. released by invariant
natural killer (iNTK) cells that were assayed with dendritic cells
pulsed 24 hours with different concentrations of liposomes.
[0225] FIG. 7 is a graph showing the viability of liposome-pulsed
dendritic cells with and without maturation cocktails for 24 or 48
hours.
[0226] FIG. 8 is a graph showing that upregulation of CD80 on
lipid-pulsed dendritic cells is increased in the presence of the
maturation cocktail.
[0227] FIG. 9 is a graph showing the functionality of
liposome-pulsed dendritic cells in the presence of maturation
cocktail (TNF.alpha., IL-1.beta., IL-6 and PGE2).
[0228] FIG. 10 is a graph showing the superior binding of
EPC:EPG:TC formulation to iNKT TCR tetramer as determined by a
human C.sup.1R.CD1d binding assay.
[0229] FIGS. 11 and 12 are graphs showing the adjuvant properties
of EPC:EPG:TC formulation by showing the levels of
CD8+Kb-tetramer+lymphocytes in the blood and spleen of tested
mice.
DETAILED DESCRIPTION OF THE INVENTION
[0230] The present application is based on the discovery that
nonglycosidic ceramides can be incorporated into liposomes, and
that these liposomes perform comparably to their soluble (i.e.,
free, non-liposomal) nonglycosidic ceramide counterparts. The
liposomes described herein were found to activate murine iNKT cells
and induce dendritic cell (DC) maturation, both in vitro and in
vivo at an efficacy that is at least comparable to their
corresponding soluble nonglycosidic ceramides. The liposomes
described herein were also found to expand an andogenous T cell
repertoire, which recognizes the ovalbulmin (OVA) pepetide SIINFEKL
(SEQ ID NO: 1), in an in vivo mouse model.
[0231] Liposomes are lipid-containing vesicles that include one or
several concentric lipid bilayers enclosing an aqueous core, and
can be used to deliver both hydrophilic and hydrophobic bioactive
agents to the body. Small, hydrophilic bioactive agents can be
encapsulated within the aqueous core (cavity) of a liposome. Both
charged, hydrophilic bioactive agents and uncharged, hydrophobic
bioactive agents can be associated with the membrane of a liposome
through electrostatic or hydrophobic interactions, respectively, or
through a covalent bond. Liposomes can deliver bioactive agents to
the body by, for example, fusion of the liposome with other
bilayers, such as the cell membrane, diffusion of the compound out
of the liposome, or digestion of the liposome within
macrophages.
[0232] As described in U.S. Pat. No. 7,060,291, incorporated herein
by reference, liposomes can have a variety of sizes, e.g., an
average diameter as low as 25 nm or as high as 10,000 nm or more.
Size is affected by a number of factors, such as, for example,
lipid composition and method of preparation, and is determined by a
number of techniques, such as quasi-elastic light scattering.
Various methodologies can be used to prepare liposomes of a smaller
size from larger liposomes, such as sonication, homogenization,
French Press application, and milling. Extrusion (see, e.g., U.S.
Pat. No. 5,008,050, incorporated herein by reference) can be used
to produce liposomes having a predetermined mean size by forcing
the liposomes, under pressure, through filter pores of a defined,
selected size. Tangential flow filtration, as described in PCT
Application No. WO 1989/008846, incorporated herein by reference,
also can be used to regularize the size of liposomes (i.e., produce
a population of liposomes having less size heterogeneity, and a
more homogeneous, defined size distribution).
[0233] Using liposomes to deliver of bioactive agents is highly
advantageous. Unlike micellar delivery vehicles, liposomes are
physically stable. The liposomal lipid composition can be tailored
to deliver specific bioactive agents to targeted locations, which
limits the potential toxicity of the bioactive agents. Further,
liposomes allow the delivery of known amounts of bioactive agents
because they minimize loss of the bioactive agent from, for
example, degradation, removal by non-target organs, and
precipitation, as seen with detergents. Liposomes also provide a
method for co-delivering adjuvants and antigens in particular
ratios that otherwise cannot occur by, for example, coinjection, as
described in U.S. Pat. Nos. 7,850,990 and 7,842,676, each
incorporated herein by reference.
[0234] Schwendener et al., Methods Mol. Biol. 605:163-175 (2010),
incorporated herein by reference, discloses liposomal vaccine
formulations for the entrapment of antigenic peptides and antigen
encoding plasmid DNAs. Inoue et al., Biochemistry 19(13):2574-2581
(1971), incorporated herein by reference, discloses liposomes
(e.g., 1:1 equimolar mixture of lecithin and sphingomyelin)
comprising galactrocerbroside or cytolipin K. U.S. Pat. No.
5,543,152, incorporated herein by reference, discloses liposomes
composed of sphingomyelin and cholesterol having an acidic
intraliposomal pH that can deliver lipophilic drugs (e.g., alkaloid
compounds) to mammalian hosts.
[0235] U.S. Pat. No. 5,705,385, incorporated herein by reference,
discloses charge-neutralized lipid-nucleic acid particles useful
for the delivery of nucleic acids. The particles comprise lipids
that are both non-cationic and cationic, and can optionally include
polyethylene glycol(PEG)-modified ceramide (e.g., 1-15 wt. %, based
on the total weight of the particle). The PEG-ceramide prevents
particle aggregation, provides a means for increasing circulation
lifetime, and provides a means for increasing the delivery of the
lipid-nucelic acid particles to the target tissues. Holland et al.,
Biochemistry 35:2618-2624 (1996), incorporated herein by reference,
discloses large, unilamellar vesicles (LUVs) comprising equimolar
amounts of phosphatidylethanolamine and phosphatidylserine, along
with egg ceramide-PEG(2000 Da) conjugates. The ceramide-PEG was
found to inhibit LUV fusion. Benoit et al., Vaccine 25:7754-7762
(2007), incorporated herein by reference, discloses a
liposome-based vaccine comprising dioleoylphosphatidylcholine
(DOPC), a recombinant protein consisting of a fragment of
thioredoxin fused in frame to a portion of the RSV G protein
(Trx-G), and .alpha.-galactosylceramide in amounts of 1 mg/mouse,
7.5 .mu.g/mouse, and 4 .mu.g/mouse, respectively. Mui et al.,
Journal of Pharmacology and Experimental Therapeutics
298(3):1185-1192 (2001), incorporated herein by reference,
discloses liposome formulations (e.g., 100 nm in diameter)
comprising plasmids and CpG that contain pegylated ceramides.
Shabbits and Mayer, Biochem. Biophys. Acta 1612(1):98-106 (2003)
and Shabbits and Mayer, Anticancer Res. 23(5A):3663-3669 (2003),
each incorporated herein by reference, disclose ceramide-containing
liposomes. Tamura et al., Biochem. Biophys. Res. Commun.
369(2):485-492 (2008), incorporated herein by reference, disclose
.alpha.-GalCer-liposomes. Ishii et al., Front Biosci. 13:6214-6228
(2008), incorporated herein by reference, discloses
.alpha.-GalCer-OVA-liposomes. Stover et al., Clin. Cancer Res.
11(9):3465-3474 (2005); Stover and Kester, Journal of Pharmacology
and Experimental Therapeutics 307(2):468-475; Zolnik et al., Drug
Metabolism and Disposition 36:1709-1715 (2008); Tran et al., Clin.
Cancer Res. 14(11):3571-3581 (2008); Khazanov et al., Langmuir
24(13):6965-6980 (2008); and Tran et al., Pigment Cell Melanoma
Res. 22(4):388-399 (2009), each incorporated herein by reference,
disclose neutral liposomal formulations, either pegylated or
non-pegylated, comprising C.sub.6-ceramides.
[0236] As used herein, the term "nonglycosidic ceramide" refers to
ceramide that does not comprise a sugar moiety.
[0237] As used herein, the term "soluble," when modifying a
nonglycosidic ceramide, refers to a nonglycosidic ceramide that is
dissolved or suspended in a composition in a form that is not
incorporated into the bilayer of a liposome. In some embodiments,
the soluble nonglycosidic ceramide is solubilized or suspended in a
surfactant. In some exemplary embodiments, the nonglycosidic
ceramide can be dissolved in a chloroform/methanol/water solution
(e.g., 10:10:3) at, for example, about 10 mg/mL and then diluted to
a final volume of about 200 .mu.g/mL using vehicle solution
comprised of, for example, NaCl (about 150 mM) and polyoxyethylene
(20) sorbitan monolaurate (i.e., Tween 20, 0.5%).
[0238] As used herein the term "pharmaceutically acceptable salt"
refers to salts of compounds that retain biological activity of
interest of the parent compound, such as therapeutic activity, and
which are not biologically or otherwise undesirable.
Pharmaceutically acceptable base addition salts can be prepared
from inorganic and organic bases. Salts derived from inorganic
bases, include by way of example only, sodium, potassium, lithium,
ammonium, calcium and magnesium salts. Salts derived from organic
bases include, but are not limited to, salts of primary, secondary
and tertiary amines. Pharmaceutically acceptable acid addition
salts may be prepared from inorganic and organic acids. Salts
derived from inorganic acids include hydrochloric acid, hydrobromic
acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
Salts derived from organic acids include acetic acid, propionic
acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic
acid, succinic acid, maleic acid, fumaric acid, tartaric acid,
citric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,
salicylic acid, and the like.
[0239] As used herein an "effective" amount or a "therapeutically
effective amount" refers to a nontoxic amount of an agent that is
effective to provide one or more desired effects in vivo, when
administered to a subject in need of prophylaxis or therapy. The
amount that is "effective" will vary from subject to subject,
depending on the age and general condition of the individual, mode
of administration, and the like. An "effective" amount in any
individual case may be determined using routine experimentation,
such as dose-response studies.
[0240] As used herein, the term "treating" includes prophylaxis of
the specific disorder or condition, or alleviation of the symptoms
associated with a specific disorder or condition and/or preventing
or eliminating said symptoms, and/or eliminating the condition
(cure). "Prophylaxis" refers to a preventative therapy and its
effectiveness can be shown by comparison between a population that
receives the prophylaxis and a population that only receives a
negative control. Effective prophylaxis results in reduced
incidence and/or reduced severity compared to the control
population. In the context of an infection, treating refers to
slowing the spread of the infection and/or stopping the spread of
the infection and/or reducing or eliminating the amount of the
infective agent in a host. In the context of a cancer, an effective
treatment slows the growth of the cancer and/or reduces its harmful
effect on a patient and/or increases the life span of a cancer
patient and/or increases the span of quality life of the patient;
or causes shrinkage of a tumor or reduction in the number of cancer
cells, or results in elimination of the cancer. In the context of
vaccination, treatment causes the body to produce an immune
response that inhibits or prevents future infection by an infective
agent, or reduces the infection's severity or duration or negative
effects if infection occurs.
Liposomes
[0241] In one aspect, described herein is a liposome comprising a
lipid bilayer membrane surrounding an aqueous core, wherein the
lipid bilayer comprises (a) a nonglycosidic ceramide present in an
amount of about 1 wt. % to about 50 wt. %, and (b) one to five
lipids (e.g., 1, 2, 3, 4, or 5 lipids) present in an amount of
about 50 wt. % to about 99 wt. %, based on the total weight of the
liposome or the lipid bilayer.
[0242] In some embodiments, the liposome has a diameter of less
than about 100 nm. In some embodiments, the liposome has a diameter
of about 50 nm to about 150 nm, or about 60 nm to about 140 nm, or
about 70 nm to about 130 nm, or about 75 to nm to about 125 nm, or
about 75 nm to about 100 nm, for example, about 100 nm. In some
embodiments, the liposome has a diameter of 50 nm, about 55 nm,
about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm,
about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm,
about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130
nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm.
[0243] Stated another way, in some embodiments the liposome has a
diameter defined by a size range, with the lower end of the size
range being any size selected from about 25 nm, 30 nm, 35 nm, 40
nm, 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52 nm, 53 nm,
54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63
nm, 64 nm, 65 nm, 66 nm, 67 nm, 68 nm, 69 nm, 70 nm, 71 nm, 72 nm,
73 nm, 74 nm, 75 nm, 76 nm, 77 nm, 78 nm, 79 nm, 80 nm; and with
the upper end of the size range being any size selected from about
175 nm, 170 nm, 165 nm, 160 nm, 155 nm, 150 nm, 145 nm, 144 nm, 143
nm, 142 nm, 141 nm, 140 nm, 139 nm, 138 nm, 137 nm, 136 nm, 135 nm,
134 nm, 133 nm, 132 nm, 131 nm, 130 nm, 129 nm, 128 nm, 127 nm, 126
nm, 125 nm, 124 nm, 123 nm, 122 nm, 121 nm, 120 nm, 119 nm, 118 nm,
117 nm, 116 nm, 115 nm, 114 nm, 113 nm, 112 nm, 111 nm, 110 nm, 109
nm, 108 nm, 107 nm, 106 nm, 105 nm, 104 nm, 103 nm, 102 nm, 101 nm,
100 nm, 99 nm, 98 nm, 97 nm, 96 nm, 95 nm, 94 nm, 93 nm, 92 nm, 91
nm, or 90 nm.
[0244] The nonglycosidic ceramide can be any nonglycosidic ceramide
known to one skilled in the art. Nonlimiting examples of
nonglycosidic ceramides are described in U.S. Patent Application
Publication No. 2009/0239813 and Silk et al., J. of Immunol.
180:6452-6456 (2008), the disclosure of which are incorporated
herein by reference in their entireties. Liposomes that include two
or more nonglycosidic ceramides are specifically contemplated.
[0245] In some embodiments, the nonglycosidic ceramide is a
compound of Formula I or a pharmaceutically acceptable salt
thereof:
##STR00014##
[0246] wherein R.sup.1 is a hydrophobic moiety than can occupy the
C' channel of human CD1d, and fills at least about 30% of the
volume of the C' channel that is occupied by the
(CH.sub.2).sub.13CH.sub.3 group of .alpha.-galactosylceramide
(.alpha.-GalCer) when the .alpha.-GalCer is bound to human CD1d; or
R.sup.1 is a C.sub.1-C.sub.25 hydrocarbon chain;
[0247] R.sup.2 is a hydrophobic moiety that can occupy the A'
channel of human CD1d, and fills at least about 30% of the volume
of the A' channel that is occupied by the
(CH.sub.2).sub.24--CH.sub.3 group of .alpha.-GalCer when the
.alpha.-GalCer is bound to human CD1d; or R.sup.2 is a
C.sub.1-C.sub.30 hydrocarbon chain;
[0248] R.sup.3 is H or OH, for example, H;
[0249] R.sup.4 and R.sup.5 either are both H or together form a
single bond, with the proviso that when R.sup.4 and R.sup.5
together form a single bond, R.sup.3 is H;
[0250] X is
##STR00015##
[0251] Y is CH.sub.2 or
##STR00016##
[0252] R.sup.6 and R.sup.7 each independently are H, OH, or phenyl,
with the proviso that either one of R.sup.6 or R.sup.7 is H and the
other is H, OH, or phenyl, or one of R.sup.6 or R.sup.7 is OH and
the other is phenyl;
[0253] R.sup.8 is H, OH, or OSO.sub.3H; and
[0254] n is 1, 2, 3, or 4, for example, 1, 2, or 3.
[0255] In some embodiments, R.sup.1 fills at least about 35%, at
least about 60%, at least about 80%, or at least about 90% of the
occupied volume of the C' channel. In some embodiments, R.sup.2
fills at least about 40%, at least about 50%, at least about 60%,
at least about 70%, or at least about 80% of the occupied volume of
the A' channel.
[0256] The conditions for a substantially full occupation of both
the A' and C' channels of human CD1d, as exhibited by
.alpha.-GalCer, are described in detail in Koch et al, Nature
Immunology, 6(8) 819-826 (2005), incorporated herein by reference.
In Koch et al., cavities were identified as surfaces that are
accessible to water molecules (radius, 1.4 .ANG.), but not to large
probes (radius, 6 .ANG.) with the program VOLUMES (R. Esnouf,
University of Oxford, Oxford, UK). The open nature of the pockets
at the TCR recognition surface required imposition of a
self-consistent definition for the outer limit of the pocket, and
on this basis the authors calculated the pocket volumes for mouse
CD1d, CD1a and CD1b, as well as human CD1d. Although these
calculations resulted in some differences in absolute values from
those reported before, the same relative trends were noted.
[0257] In some embodiments, R.sup.1 (i.e., the sphingosine chain)
does not exceed 13 carbon-carbon single bonds in length. For
example, R.sup.1 can be a linear C.sub.5-C.sub.13 hydrocarbon
chain, a linear C.sub.11-C.sub.13 hydrocarbon chain, or a linear
C.sub.12-C.sub.13 hydrocarbon chain. In some embodiments, R.sup.2
(i.e., the acyl chain) does not exceed 25 carbon-carbon single
bonds in length. In some embodiments, R.sup.2 comprises at least 1
carbon atom, at least 5 carbon atoms, at least 8 carbon atoms, at
least 9 carbon atoms, at least 10 carbon atoms, at least 11 carbon
atoms, at least 12 carbon atoms, at least 13 carbon atoms, at least
14 carbon atoms, at least 15 carbon atoms, at least 16 carbon
atoms, at least 17 carbon atoms, at least 18 carbon atoms, at least
19 carbon atoms, at least 20 carbon atoms, at least 21 carbon
atoms, at least 22 carbon atoms, at least 23 carbon atoms, at least
24 carbon atoms, or at least 25 carbon atoms. For example, R.sup.2
can be a linear C.sub.1-C.sub.25 hydrocarbon chain, a linear
C.sub.5-C.sub.25 hydrocarbon chain, a linear C.sub.1-C.sub.16
hydrocarbon chain, C.sub.1-C.sub.18 hydrocarbon chain or a linear
C.sub.8-C.sub.25 hydrocarbon chain.
[0258] In some embodiments, the compounds of Formula I bind to
human CD1d with good shape complementarity, (i.e., with a S.sub.c
greater than 0.50, greater than 0.55, or greater than 0.60). Shape
complementarity analysis was made using the program SC
(http://www.ccp4.ac.uk/ccp4i_main.html). Using this analysis, the
26 carbon acyl chain and the 18 carbon sphingosine chain of
.alpha.-GalCer were found to fit into the A' and C' channels of
human CD1d, respectively, with a shape complementarity (S.sub.c) of
0.61. The total volume of these cavities (1,400 A.sup.3) in the
human CD1d binding groove is essentially filled by the hydrocarbon
chains. The 26 carbon acyl chain fits into the A' pocket by
adopting a counterclockwise circular curve, as viewed from above
the binding groove, filling the pocket. The 18 carbon sphingosine
chain adopts a straighter conformation to fit into the C' pocket,
and terminates at the end of the binding groove. It is likely that
.alpha.-GalCer has the maximum lipid chain lengths that are able to
fit into the antigen-binding groove of human CD1d.
[0259] From x-ray diffraction studies and modeling experiments, it
appears that when the R.sup.2 group is shorter than the maximum
length, the remaining space can be occupied by spacer molecules,
which occur naturally in the body and are sufficiently available to
occupy vacant spaces in the CD1d molecule. Such spacer molecules
include, e.g., lipids. Therefore, compounds of Formula I that
include an R.sup.2 group that is significantly smaller than what is
needed for maximum occupation of the A' channel will still bind
well to the CD1d molecule.
[0260] Binding studies reported in the literature teach that
carbon-carbon double bonds may be substituted for several of the
carbon-carbon single bonds on the 26 carbon acyl chain and on the
18 carbon sphingosine chain of .alpha.-GalCer, provided that the
hydrophobic moieties are still able to occupy the conformations
necessary for binding with their respective channels. Some studies
have shown, for example, that the channels are able to accept
relatively bulky hydrophobic residues, such as phenyl.
[0261] In some embodiments, at least one of R.sup.1 or R.sup.2
comprises at least 1 double bond, or 1, 2 or 3 double bonds. In
some embodiments, R.sup.1 and/or R.sup.2 comprises at least one
double bond with Z stereochemistry. For example, R.sup.2 can
comprise at least 1 double bond that has Z stereochemistry.
[0262] In some embodiments, X is
##STR00017##
and Y is CH.sub.2. In some exemplary embodiments, X is
##STR00018##
[0263] In some embodiments, R.sup.6 and R.sup.7 are each H. In
alternative embodiments, one of R.sup.6 or R.sup.7 is H and the
other is OH.
[0264] The compounds of Formula I can exhibit tautomerism. Further,
the compounds of Formula I can also contain one or more asymmetric
carbon atoms, and can exist as pure stereoisomers or as a mixture
of stereoisomers. In some embodiments, the compounds of Formula I
exhibit the same stereochemistry as .alpha.-GalCer.
[0265] Pharmaceutically acceptable salts of the compound of Formula
I may be prepared by reacting the compound of Formula I with an
appropriate acid or base in the presence of a suitable solvent, as
known to one of ordinary skill in the art. Suitable
pharmaceutically acceptable salts include salts with suitable
bases, such as, alkali metals (e.g., sodium and potassium), and
alkaline earth metals, (e.g., calcium and magnesium) salts. The
salts of the compound of Formula I can be converted to the free
base form using any method known to one skilled in the art.
[0266] In some embodiments, the nonglycosidic ceramide is selected
from the group consisting of arabinitolceramide, glycerolceramide,
threitolceramide, threitolceramide C.sub.14 acyl,
threitol-22-(Z)-ceramide, 4-deoxy-4-phenyl-threitolceramide,
4-deoxy-4-phenyl-threitol-22-(Z)-ceramide,
glycerol-phosphateceramide, inositolceramide, inositolceramide
C.sub.15 acyl, myoinositolceramide salt, 4-phenyl threitolceramide,
4-phenyl threitol-22-(Z)-ceramide, threitol-(19Z,22Z)-ceramide, and
mixtures thereof. For example, the nonglycosidic ceramide can
include arabinitolceramide, glycerolceramide, threitolceramide, and
mixtures thereof (e.g., threitolceramide).
[0267] The nonglycosidic ceramide (or combination of ceramides) can
be present in the bilayer of the liposome in any amount that
provides a liposome having a similar efficacy as a corresponding
soluble, nonglycosidic ceramide that is not incorporated within a
liposome, or provides a superior efficacy; or provides a
therapeutic window (between minimum effective concentration and
toxic concentration). In some embodiments, the nonglycosidic
ceramide is present in the bilyaer of the liposome in an amount of
about 1 wt. % to about 50 wt. %, or about 2 wt. % to about 20 wt.
%, or about 2 wt. % to about 10 wt. %, or about 3 wt. % to about 12
wt. %, based on the total weight of the liposome or the lipid
bilayer. In some embodiments, the nonglycosidic ceramide is present
in an amount of about 1 wt. % to about 30 wt. %, or about 20 wt. %
to about 50 wt. %, or about 3 wt. % to about 8 wt. %, or about 4
wt. % to about 13 wt. %, based on the total weight of the liposome
or lipid bilayer. For example, the nonglycosidic ceramide can be
present in an amount of about 1 wt. %, about 2 wt. %, about 3 wt.
%, about 4 wt. %, about 5 wt. %, about 6 wt. %, about 7 wt. %,
about 8 wt. %, about 9 wt. %, about 10 wt. %, about 11 wt. %, about
12 wt. %, about 13 wt. %, about 14 wt. %, about 15 wt. %, about 16
wt. %, about 17 wt. %, about 18 wt. %, about 19 wt. %, about 20 wt.
%, about 21 wt. %, about 22 wt. %, about 23 wt. %, about 24 wt. %,
about 25 wt. %, about 26 wt. %, about 27 wt. %, about 28 wt. %,
about 29 wt. %, about 30 wt. %, about 31 wt. %, about 32 wt. %,
about 33 wt. %, about 34 wt. %, about 35 wt. %, about 36 wt. %,
about 37 wt. %, about 38 wt. %, about 39 wt. %, about 40 wt. %,
about 41 wt. %, about 42 wt. %, about 43 wt. %, about 44 wt. %,
about 45 wt. %, about 46 wt. %, about 47 wt. %, about 48 wt. %,
about 49 wt. %, or about 50 wt. %, based on the total weight of the
liposome or the lipid bilayer. In some exemplary embodiments, the
nonglycosidic ceramide is present is an amount of about 5 wt. % or
about 10 wt. %, based on the total weight of the liposome or the
lipid bilayer. A weight percentage of nonglycosidic ceramide is
chosen to produce an effective immunomodulatory response with
minimal toxic side effects. If too little nonglycosidic ceramide is
present relative to total lipid, the liposome will not stimulate a
desired response. If too much nonglycosidic ceramide is present
relative to total lipid, the excess nonglycosidic ceramides will
compete for binding sites and may cause the liposomes to block each
other from binding. Further, too much nonglycosidic ceramide may
result in toxicity issues.
[0268] The one to five lipids each can be any lipid that is capable
of forming a liposome with the nonglycosidic ceramide described
herein, as long as the resulting liposome has at least a similar
efficacy as a corresponding soluble nonglycosidic ceramide that is
not incorporated within a liposome. The one to five lipids each can
independently have a net positive charge (i.e., a cationic lipid),
a net negative charge (i.e., an anionic lipid), no charges (i.e, a
nonionic lipid), or an equal number of positive and negative
charges (i.e., a zwitterionic lipid). The lipids can include those
lipids that form a bilayer themselves or with the nonglycosidic
ceramide, and those lipids that do not form a bilayer alone, but
they can be included as part of a stable bilayer made from one or
more other lipids.
[0269] A cationic lipid can be included in the liposome if, e.g.,
the resulting liposome will function as a delivery agent for a
negatively-charged bioactive agent, such as for nucleic acids.
Other advantages for including a cationic lipid include an
increased association with cells due to ionic attraction to the
negatively-charged surface of the cell, increased
incorporation/association of acidic proteins, and potential for
activation of immune cells, thereby enhancing the effect of the
nonglycosidic ceramide on the cell.
[0270] An anionic lipid can be included in the liposome if, e.g.,
the resulting liposome will function as a delivery agent for a
positively-charged bioactive agent. Other advantages for including
an anionic lipid include increased association with the target,
increased incorporation/association of basic proteins, and
potential for activation of immune cells, thereby enhancing the
effect of the nonglycosidic ceramide on the cell.
[0271] A nonionic lipid also can be included in the liposome if,
e.g., the resulting liposome.
[0272] A zwitterionic lipid can be included in the liposome if,
e.g., the resulting liposome will function as a delivery agent for
a neutral compound. Other advantages for including a zwitterionic
lipid include stabilization of the lipid bilayer formation and
formation of a permeability barrier allowing for encapsulation of
small molecules (e.g., small molecule immunomodulators).
[0273] In some embodiments, at least one of the one to five lipids
is selected from the group consisting of a sphingolipid; a
phospholipid; a sterol; a cationic lipid that is not a
sphingolipid, a phospholipid, or a sterol; a neutral lipid that is
not a sphingolipid, a phospholipid, or a sterol; a detergent that
is not a sphingolipid, a phospholipid, or a sterol; a bioactive
lipid that is not a sphingolipid, a phospholipid, or a sterol;
coenzyme A, or a derivative of any of the foregoing.
[0274] In some embodiments, the sphingolipid is selected from the
group consisting of a sphingosine, a ceramide, a sphingomyelin, a
ganglioside, a glycosphingolipid, a phosphosphingolipid, a
phytosphingosine, a sphingolipid receptor agonist, a sphingolipid
receptor antagonist, a sphingolipid metabolism inhibitor, a
bioactive ceramide, and derivatives thereof.
[0275] In some embodiments, the sphingosine is selected from the
group consisting of natural sphingosine, synthetic sphingosine,
phosphorylated sphingosine (S1P), and methylated sphingosine.
[0276] In some embodiments, the natural sphingosine is
D-erythro-sphingosine (e.g., from porcine brain or chicken
egg).
[0277] In some embodiments, the synthetic sphingosine is selected
from the group consisting of sphingosine (d18:1), sphingosine
(d17:1), sphingosine (d20:1), L-threo-sphingosine (d18:1),
1-deoxysphingosine, and 1-desoxymethylsphingosine. In some
embodiments, the sphinganine is selected from the group consisting
of sphinganine (d18:0), sphinganine (d17:0), sphinganine (d20:0),
1-deoxysphinganine, 1-desoxymethylsphinganine, and
L-threo-dihydrosphingosine (d18:0) (Safingol). In some embodiments,
the phosphorylated sphingosine is selected from the group
consisting of sphingosine-1-phosphate (d18:1),
sphingosine-1-phosphate (DMA Adduct), sphingosine-1-phosphate
(d17:1), sphingosine-1-phosphate (d20:1), sphinganine-1-phosphate
(d18:0), sphinganine-1-phosphate (d17:0), and
sphinganine-1-phosphate (d20:0). In some embodiments, the
methylated sphingosine is selected from the group consisting of
monomethyl sphingosine (d18:1), dimethyl sphingosine (d18:1),
dimethyl sphingosine (d17:1), trimethyl sphingosine (d18:1),
trimethyl sphingosine (d17:1), dimethyl sphinganine (d18:0),
trimethyl sphinganine (d18:0), dimethyl sphingosine-1-phosphate
(d18:1), and dimethyl sphinganine-1-phosphate (d18:0).
[0278] In some embodiments, the ceramide is selected from the group
consisting of natural ceramide, synthetic ceramide, a ceramide
phosphate, a 1-O-acyl-ceramide, a dihydroceramide, a
dihydroceramide phosphate, and a 2-hydroxy ceramide.
[0279] In some embodiments, the natural ceramide is ceramide from,
for example, porcine brain or egg.
[0280] In some embodiments the synthetic ceramide is selected from
the group consisting of N-octadecanoyl-D-erythro-sphingosine (C18),
N-hexadecanoyl-D-erythro-sphingosine (C16)
N-acetoyl-D-erythro-sphingosine (C2 Ceramide, d18:1/2:0),
N-butyroyl-D-erythro-sphingosine (C4 Ceramide, d18:1/4:0),
N-hexanoyl-D-erythro-sphingosine (C6 Ceramide, d18:1/6:0),
N-octanoyl-D-erythro-sphingosine (C8 Ceramide, d18:1/8:0),
N-decanoyl-D-erythro-sphingosine (C10 Ceramide, d18:1/10:0),
N-lauroyl-D-erythro-sphingosine (C12 Ceramide, d18:1/12:0),
N-myristoyl-D-erythro-sphingosine (C14 Ceramide, d18:1/14:0),
N-palmitoyl-D-erythro-sphingosine (C16 Ceramide, d18:1/16:0),
N-heptadecanoyl-D-erythro-sphingosine (C17 Ceramide, d18:1/17:0),
N-stearoyl-D-erythro-sphingosine (C18 Ceramide, d18:1/18:0),
N-oleoyl-D-erythro-sphingosine (C18:1 Ceramide, d18:1/18:1(9Z)),
N-arachidoyl-D-erythro-sphingosine (C20 Ceramide, d18:1/20:0),
N-behenoyl-D-erythro-sphingosine (C22 Ceramide, d18:1/22:0),
N-lignoceroyl-D-erythro-sphingosine (C24 Ceramide, d18:1/24:0),
N-nervonoyl-D-erythro-sphingosine (C24:1 Ceramide,
d18:1/24:1(15Z)), N-acetoyl-D-erythro-sphingosine (C17 base) (C2
Ceramide, d17:1/2:0), N-octanoyl-D-erythro-sphingosine (C17 base)
(C8 Ceramide, d17:1/8:0), N-stearoyl-D-erythro-sphingosine (C17
base) (C18 Ceramide, d17:1/18:0), N-oleoyl-D-erythro-sphingosine
(C17 base) (C18:1 Ceramide, d17:1/18:1(9Z)),
N-arachidoyl-D-erythro-sphingosine (C17 base) (C20 Ceramide,
d17:1/20:0), N-lignoceroyl-D-erythro-sphingosine (C17 base) (C24
Ceramide, d17:1/24:0), and N-nervonoyl-D-erythro-sphingosine (C17
base) (C24:1 Ceramide, d17:1/24:1(15Z)).
[0281] In some embodiments, the ceramide phosphate is selected from
the group consisting of N-acetoyl-ceramide-1-phosphate (ammonium
salt) (C2 Ceramide-1-Phosphate, d18:1/2:0),
N-octanoyl-ceramide-1-phosphate (ammonium salt) (C8
Ceramide-1-Phosphate, d18:1/8:0), N-lauroyl-ceramide-1-phosphate
(ammonium salt) (C12 Ceramide-1-Phosphate, d18:1/12:0),
N-palmitoyl-ceramide-1-phosphate (ammonium salt) (C16
Ceramide-1-Phosphate, d18:1/16:0), N-oleoyl-ceramide-1-phosphate
(ammonium salt) (C18:1 Ceramide-1-Phosphate, d18:1/18:1(9Z)),
N-lignoceroyl-ceramide-1-phosphate (ammonium salt) (C24
Ceramide-1-Phosphate, 18:1/24:0), N-acetoyl-ceramide-1-phosphate
(C17 base) (ammonium salt) (C2 Ceramide-1-Phosphate, d17:1/2:0),
and N-octanoyl-ceramide-1-phosphate (C17 base) (ammonium salt) (C8
Ceramide-1-Phosphate, d17:1/8:0).
[0282] In some embodiments, the 1-O-acyl-ceramide is
1-oleoyl-N-heptadecanoyl-D-erythro-sphingosine
N-acetoyl-D-erythro-sphinganine (C2 Dihydroceramide,
d18:0/2:0).
[0283] In some embodiments, the dihydroceramide is selected from
the group consisting of N-hexanoyl-D-erythro-sphinganine (C6
Dihydroceramide, d18:0/6:0), N-octanoyl-D-erythro-sphinganine (C8
Dihydroceramide, d18:0/8:0), N-palmitoyl-D-erythro-sphinganine (C16
Dihydroceramide, d18:0/16:0), N-stearoyl-D-erythro-sphinganine (C18
Dihydroceramide, d18:0/18:0), N-oleoyl-D-erythro-sphinganine (C18:1
Dihydroceramide, d18:0/18:1(9Z)),
N-lignoceroyl-D-erythro-sphinganine (C24 Dihydroceramide,
d18:0/24:0), and
N-nervonoyl-D-erythro-sphinganine-D-erythro-sphinganine (C24:1
Dihydroceramide, d18:0/24:1(15Z)).
[0284] In some embodiments, the dihydroceramide phosphate is
N-palmitoyl-D-erythro-dihydroceramide-1-phosphate (ammonium salt)
(C16 Dihydroceramide-1-Phosphate, d18:0/16:0) or
N-lignoceroyl-D-erythro-dihydroceramide-1-phosphate (ammonium salt)
(C24 Dihydroceramide-1-Phosphate, d18:0/24:0).
[0285] In some embodiments, the 2-hydroxy ceramide is selected from
the group consisting of
N-(2'-(R)-hydroxylauroyl)-D-erythro-sphingosine (12:0(2R--OH)
Ceramide), N-(2'-(S)-hydroxylauroyl)-D-erythro-sphingosine
(12:0(2S--OH) Ceramide),
N-(2'-(R)-hydroxypalmitoyl)-D-erythro-sphingosine (16:0(2R--OH)
Ceramide), N-(2'-(S)-hydroxypalmitoyl)-D-erythro-sphingosine
(16:0(2S--OH) Ceramide),
N-(2'-(R)-hydroxyheptadecanoyl)-D-erythro-sphingosine (17:0(2R--OH)
Ceramide), N-(2'-(S)-hydroxyheptadecanoyl)-D-erythro-sphingosine
(17:0(2S--OH) Ceramide),
N-(2'-(R)-hydroxystearoyl)-D-erythro-sphingosine (18:0(2R--OH)
Ceramide), N-(2'-(S)-hydroxystearoyl)-D-erythro-sphingosine
(18:0(2S--OH) Ceramide),
N-(2'-(R)-hydroxyoleoyl)-D-erythro-sphingosine (18:1(2R--OH)
Ceramide), N-(2'-(S)-hydroxyoleoyl)-D-erythro-sphingosine
(18:1(2S--OH) Ceramide),
N-(2'-(R)-hydroxyarachidoyl)-D-erythro-sphingosine (20:0(2R--OH)
Ceramide), N-(2'-(S)-hydroxylarachidoyl)-D-erythro-sphingosine
(20:0(2S--OH) Ceramide),
N-(2'-(R)-hydroxybehenoyl)-D-erythro-sphingosine (22:0(2R--OH)
Ceramide), N-(2'-(S)-hydroxylbehenoyl)-D-erythro-sphingosine
(22:0(2S--OH) Ceramide),
N-(2'-(R)-hydroxylignoceroyl)-D-erythro-sphingosine (24:0(2R--OH)
Ceramide), N-(2'-(S)-hydroxyllignoceroyl)-D-erythro-sphingosine
(24:0(2S--OH) Ceramide),
N-(2'-(R)-hydroxynervonoyl)-D-erythro-sphingosine (24:1(2R--OH)
Ceramide), and N-(2'-(S)-hydroxylnervonoyl)-D-erythro-sphingosine
(24:1(2S--OH) Ceramide).
[0286] In some embodiments, the sphingomyelin is selected from the
group consisting of a natural sphingomyelin (e.g., from porcine
brain, chicken egg, or bovine milk),
N-acetyl-D-erythro-sphingosylphosphorylcholine (02:0 SM,
d18:1/2:0), N-hexanoyl-D-erythro-sphingosylphosphorylcholine (06:0
SM, d18:1/6:0), N-lauroyl-D-erythro-sphingosylphosphorylcholine
(12:0 SM, d18:1/12:0),
N-lauroyl-D-erythro-sphinganylphosphorylcholine (12:0 Dihydro SM,
d18:0/12:0), N-palmitoyl-D-erythro-sphingosylphosphorylcholine
(16:0 SM, d18:1/16:0),
N-heptadecanoyl-D-erythro-sphingosylphosphorylcholine (17:0 SM, d
18:1/17:0), N-stearoyl-D-erythro-sphingosylphosphorylcholine (18:0
SM, d18:1/18:0), N-oleoyl-D-erythro-sphingosylphosphorylcholine
(18:1 SM, d18:1/18:1(9Z)),
N-lignoceroyl-D-erythro-sphingosylphosphorylcholine (24:0 SM),
N-nervonoyl-D-erythro-sphingosylphosphorylcholine (24:1 SM),
sphingosylphosphorylcholine (Lyso SM, d18:1),
sphingosylphosphorylcholine (C17 base) (Lyso SM, d17:1), and
sphinganine phosphorylcholine (Lyso SM (dihydro), d18:0).
[0287] In some embodiments, the ganglioside is selected from the
group consisting of GM1 ganglioside ammonium salt (e.g., from ovine
brain), GM3 ganglioside ammonium salt (e.g., from bovine milk), GD3
ganglioside ammonium salt (e.g., from bovine milk), and total
ganglioside extract ammonium salt (e.g., from porcine brain).
[0288] In some embodiments, the glycosphingolipid is selected from
the group consisting of a natural glycosphingolipid, a glycosyl
sphingolipid, a galactosyl sphingolipid, a lactosyl sphingolipid, a
sulfatide, and .alpha.-galactosyl ceramide (.alpha.GalCer).
[0289] In some embodiments, the natural glycosphingolipid is
selected from the group consisting of a a cerebroside (e.g., from
porcine brain), a glucocerebroside (e.g., from soy), a sulfatide
(ammonium salt) (e.g., from porcine brain), a GM1 ganglioside
(ammonium salt) (e.g., from ovine brain), a ganglioside GM1 (e.g.,
from ovine brain), and a total ganglioside extract (ammonium salt)
(e.g., from porcine brain).
[0290] In some embodiments, the glycosyl sphingolipid is selected
from the group consisting of
D-glucosyl-.beta.1-1'-D-erythro-sphingosine (Glucosyl(.beta.)
Sphingosine, d18:1),
D-glucosyl-.beta.-1,1'N-octanoyl-D-erythro-sphingosine (C8
Glucosyl(.beta.) Ceramide, d18:1/8:0),
D-glucosyl-.beta.-1,1'N-lauroyl-D-erythro-sphingosine (C12
Glucosyl(.beta.) Ceramide, d18:1/12:0),
D-glucosyl-.beta.-1,1'N-palmitoyl-D-erythro-sphingosine (C16
Glucosyl(.beta.) Ceramide, d18:1/16:0),
D-glucosyl-.beta.-1,1'N-stearoyl-D-erythro-sphingosine (C18
Glucosyl(.beta.) Ceramide, d18:1/18:0),
D-glucosyl-.beta.-1,1'N-oleoyl-D-erythro-sphingosine (C18:1
Glucosyl(.beta.) Ceramide, d18:1/18:1(9Z)), and
D-glucosyl-.beta.1-1'-N-nervonoyl-D-erythro-sphingosine (C24:1
Glucosyl(.beta.) Ceramide, d18:1/24:1(15Z)).
[0291] In some embodiments, the galactosyl sphingolipid is selected
from the group consisting of
D-galactosyl-.beta.1-1'-D-erythro-sphingosine (Galactosyl(.beta.)
Sphingosine, d18:1),
N,N-dimethyl-D-galactosyl-.beta.1-1'-D-erythro-sphingosine
(Galactosyl(.beta.)Dimethyl Sphingosine, d18:1),
D-galactosyl-.beta.-1,1'N-octanoyl-D-erythro-sphingosine (C8
Galactosyl(.beta.) Ceramide, d18:1/8:0),
D-galactosyl-.beta.-1,1'N-lauroyl-D-erythro-sphingosine (C12
Galactosyl(.beta.) Ceramide, d18:1/12:0),
D-galactosyl-.beta.-1,1'N-palmitoyl-D-erythro-sphingosine (C16
Galactosyl(.beta.) Ceramide, d18:1/16:0), and
D-galactosyl-.beta.-1,1'N-nervonoyl-D-erythro-sphingosine (C24:1
Galactosyl(.beta.) Ceramide, d18:1/24:1(15Z)).
[0292] In some embodiments, the lactosyl sphingolipid is selected
from the group consisting of
D-lactosyl-.beta.1-1'-D-erythro-sphingosine (Lactosyl(.beta.)
Sphingosine, d18:1),
D-lactosyl-.beta.-1,1'N-octanoyl-D-erythro-sphingosine (C8
Lactosyl(.beta.) Ceramide, d18:1/8:0),
D-lactosyl-.beta.1-1'-N-octanoyl-L-threo-sphingosine (C8
L-threo-Lactosyl(.beta.) Ceramide, d18:1/8:0),
D-lactosyl-.beta.-1,1'N-lauroyl-D-erythro-sphingosine (C12
Lactosyl(.beta.) Ceramide, d18:1/12:0),
D-lactosyl-.beta.-1,1'N-palmitoyl-D-erythro-sphingosine (C16
Lactosyl(.beta.) Ceramide, d18:1/16:0),
D-lactosyl-.beta.-1,1'N-lignoceroyl-D-erythro-sphingosine (C24
Lactosyl(.beta.) Ceramide, d18:1/24:0), and
D-lactosyl-.beta.1-1'-N-nervonoyl-D-erythro-sphingosine (C24:1
Lactosyl(.beta.) Ceramide, d18:1/24:1).
[0293] In some embodiments, the sulfatide is selected from the
group consisting of
3-O-sulfo-D-galactosyl-.beta.1-1'-N-lignoceroyl-D-erythro-sphingosine
(ammonium salt) (e.g., from porcine brain), 3-O--
sulfo-D-galactosyl-.beta.1-1'-N-lauroyl-D-erythro-sphingosine
(ammonium salt) (C12 Mono-Sulfo Galactosyl(.beta.) Ceramide,
d18:1/12:0),
3-O-sulfo-D-galactosyl-.beta.1-F--N-heptadecanoyl-D-erythro-sphingosine
(ammonium salt) (C17 Mono-Sulfo Galactosyl(.beta.) Ceramide,
d18:1/17:0),
3-O-sulfo-D-galactosyl-.beta.1-1'-N-lignoceroyl-D-erythro-sphingosine
(ammonium salt) (C24 Mono-Sulfo Galactosyl(.beta.) Ceramide
(d18:1/24:0),
3-O-sulfo-D-galactosyl-.beta.1-1'-N-nervonoyl-D-erythro-sphingosine
(ammonium salt) (C24:1 Mono-Sulfo Galactosyl(.beta.) Ceramide,
d18:1/24:1), and
3,6-di-O-sulfo-D-galactosyl-.beta.1-1'-N-lauroyl-D-erythro-sphingosine
(ammonium salt) (C12 Di-Sulfo Galactosyl(.beta.) Ceramide,
d18:1/12:0).
[0294] In some embodiments, the phosphospingolipid is selected from
the group consisting of D-erythro-sphingosyl phosphoethanolamine
(Sphingosyl PE, d18:1), N-lauroyl-D-erythro-sphingosyl
phosphoethanolamine (C17 base) (C12 Sphingosyl PE, d17:1/12:0), and
D-erythro-sphingosyl phosphoinositol (Sphingosyl PI).
[0295] In some embodiments, the phytosphingosine is selected from
the group consisting of 4-hydroxysphinganine (Saccharomyces
Cerevisiae) (D-ribo-Phytosphingosine), 4-hydroxysphinganine (C17
base) (D-ribo-phytosphingosine, C17 base),
4-hydroxysphinganine-N,N-dimethyl (Saccharomyces Cerevisiae)
(Phytosphingosine-N,N-Dimethyl),
4-hydroxysphinganine-N,N,N-trimethyl(methyl sulfate salt)
(Saccharomyces cerevisiae) (Phytosphingosine-N,N,N-Trimethyl),
4-hydroxysphinganine-1-phosphate (Saccharomyces Cerevisiae)
(D-ribo-Phytosphingosine-1-Phosphate),
4-hydroxysphinganine-N,N-dimethyl-1-phosphate (ammonium salt)
(Saccharomyces Cerevisiae)
(Phytosphingosine-N,N-Dimethyl-1-Phosphate), N-acetoyl
4-hydroxysphinganine (Saccharomyces Cerevisiae) (N-02:0
Phytosphingosine), N-octanoyl 4-hydroxysphinganine (Saccharomyces
Cerevisiae) (N-08:0 Phytosphingosine), N-stearoyl
4-hydroxysphinganine (Saccharomyces Cerevisiae) (N-18:0
Phytosphingosine), and 4-hydroxysphinganine-1-phosphocholine
(Saccharomyces Cerevisiae) (Phytosphingosine Phosphocholine).
[0296] In some embodiments, the sphingolipid receptor agonist or
antagonist is selected from the group consisting of (S)-phosphoric
acid mono-[2-amino-3-(4-octyl-phenylamino)-propyl]ester (VPC
24191), (R)-phosphoric acid
mono-[2-amino-2-(6-octyl-1H-benzoimiazol-2-yl)-ethyl]ester (VPC
23153), (R)-phosphoric acid
mono-[2-amino-2-(3-octyl-phenylcarbamoyl)-ethyl]ester (VPC 23019),
R)-3-Amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (TFA
salt) (W146), and
(S)-3-amino-4-(3-hexylphenylamino)-4-oxobutylphosphonic acid (TFA
salt) (W140).
[0297] In some embodiments, the sphingolipid metabolism inhibitor
is selected from the group consisting of 1-deoxysphingosine
(m18:1), 1-desoxymethylsphingosine (m17:1), 1-deoxysphinganine
(m18:0), 1-desoxymethylsphinganine (m17:0),
N-[(1R,2S)-2-hydroxy-1-hydroxymethyl-2-(2-tridecyl-1-cyclopropenyl)ethyl]-
octanamide (GT-11),
1R,2R-(+)-1-phenyl-2-palmitoylamino-3-N-morpholine-1-propanol
(D-threo-PPMP), L-threo-dihydrosphingosine (d18:0) (Safingol),
N-lauroyl-1-deoxysphingosine (m18:1/12:0)
(N--C12-deoxysphingosine), N-palmitoyl-1-deoxysphingosine
(m18:1/16:0) (N--C16-deoxysphingosine),
N-nervonoyl-1-deoxysphingosine (m18:1/24:1)
(N--C24:1-deoxysphingosine), N-lauroyl-1-deoxysphinganine
(m18:0/12:0) (N--C12-deoxysphinganine),
N-palmitoyl-1-deoxysphinganine (m18:0/16:0)
(N--C16-deoxysphinganine), N-nervonoyl-1-deoxysphinganine
(m18:0/24:1) (N--C24:1-deoxysphinganine),
N-lauroyl-1-desoxymethylsphingosine (m17:1/12:0)
(N--C12-desoxymethylsphingosine),
N-palmitoyl-1-desoxymethylsphingosine (m17:1/16:0)
(N--C16-desoxymethylsphingosine),
N-nervonoyl-1-desoxymethylsphingosine (m17:1/24:1)
(N--C24:1-desoxymethylsphingosine),
N-lauroyl-1-desoxymethylsphinganine (m17:0/12:0)
(N--C12-desoxymethylsphinganine),
N-palmitoyl-1-desoxymethylsphinganine (m17:0/16:0)
(N--C16-desoxymethylsphinganine), and
N-nervonoyl-1-desoxymethylsphinganine (m17:0/24:1)
(N--C24:1-desoxymethylsphinganine).
[0298] In some embodiments, the bioactive ceramide is selected from
the group consisting of
D-erythro-2-N-[6'-(1''-pyridinium)-hexanoyl]-sphingosine bromide
(C6-Pyridinium-Ceramide),
D-erythro-N-[2-(1,3-dihydroxy-4E-octadecene)]-N'-hexadecane-urea
(C16-Urea-Ceramide), and
D-erythro-N-[2-(1,3-dihydroxy-4E-octadecene)]-N'-hexane-urea-sphingosine
(C6-Urea-Ceramide).
[0299] In some embodiments, the phospholipid is selected from the
group consisting of a phosphatidylcholine, a phosphatidic acid, a
phosphatidylethanolamine, a phosphatidylglycerol, a
phosphatidylserine, a phosphatidylinositol, an inositol phosphate,
a platelet activing factor (PAF), a cardiolipin, an ether
phospholipid, a plasmalogen, an oxidized phospholipid, a
bis(monoacylglycero)phosphate (BMP), a phospholipid for supported
monolayers, and a sterol modified phospholipid.
[0300] In some embodiments, the phosphatidylcholine is selected
from the group consisting of a natural phosphatidylcholine, a
saturated synthetic phosphatidylcholine, an unsaturated synthetic
phosphatidylcholine, a mixed acyl phosphatidylcholine, a lyso
phosphatidylcholine, and an alkylphosphocholine.
[0301] In some embodiments, the natural phosphatidylcholine is
L-.alpha.-phosphatidylcholine
[0302] (e.g., from chicken egg, soy, hydrogenated soy, bovine
heart, porcine brain, or bovine liver).
[0303] In some embodiments, the saturated, synthetic
phosphatidylcholine is selected from the group consisting of
1,2-dipropionyl-sn-glycero-3-phosphocholine (03:0 PC),
1,2-dibutyryl-sn-glycero-3-phosphocholine (04:0 PC),
1,2-dipentanoyl-sn-glycero-3-phosphocholine (05:0 PC),
1,2-dihexanoyl-sn-glycero-3-phosphocholine (06:0 PC, DHPC),
1,2-diheptanoyl-sn-glycero-3-phosphocholine (07:0 PC, DHPC),
1,2-dioctanoyl-sn-glycero-3-phosphocholine (08:0 PC),
1,2-dinonanoyl-sn-glycero-3-phosphocholine (09:0 PC),
1,2-didecanoyl-sn-glycero-3-phosphocholine (10:0 PC),
1,2-diundecanoyl-sn-glycero-3-phosphocholine (11:0 PC),
1,2-dilauroyl-sn-glycero-3-phosphocholine (12:0 PC, DLPC),
1,2-ditridecanoyl-sn-glycero-3-phosphocholine (13:0 PC),
1,2-dimyristoyl-sn-glycero-3-phosphocholine (14:0 PC, DMPC),
1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (15:0 PC),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (16:0 PC, DPPC),
1,2-diphytanoyl-sn-glycero-3-phosphocholine (4ME 16:0 PC),
1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (17:0 PC),
1,2-distearoyl-sn-glycero-3-phosphocholine (18:0 PC, DSPC),
1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (19:0 PC),
1,2-diarachidoyl-sn-glycero-3-phosphocholine (20:0 PC),
1,2-dihenarachidoyl-sn-glycero-3-phosphocholine (21:0 PC),
1,2-dibehenoyl-sn-glycero-3-phosphocholine (22:0 PC),
1,2-ditricosanoyl-sn-glycero-3-phosphocholine (23:0 PC), and
1,2-dilignoceroyl-sn-glycero-3-phosphocholine (24:0 PC).
[0304] In some embodiments, the unsaturated, synthetic
phosphatidylcholine is selected from the group consisting of
1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (14:1 (.DELTA.9-Cis)
PC), 1,2-dimyristelaidoyl-sn-glycero-3-phosphocholine (14:1
(.DELTA.9-Trans) PC),
1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (16:1 (.DELTA.9-Cis)
PC), 1,2-dipalmitelaidoyl-sn-glycero-3-phosphocholine (16:1
(.DELTA.9-Trans) PC),
1,2-dipetroselenoyl-sn-glycero-3-phosphocholine (18:1
(.DELTA.6-Cis) PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1
(.DELTA.9-Cis) PC, DOPC),
1,2-dielaidoyl-sn-glycero-3-phosphocholine (18:1 (.DELTA.9-Trans)
PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (18:2 (Cis) PC,
DLPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3 (Cis)
PC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (20:1 (Cis) PC),
1,2-diarachidonoyl-sn-glycero-3-phosphocholine (20:4 (Cis) PC),
1,2-dierucoyl-sn-glycero-3-phosphocholine (22:1 (Cis) PC),
1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (22:6 (Cis) PC),
and 1,2-dinervonoyl-sn-glycero-3-phosphocholine (24:1 (Cis)
PC).
[0305] In some embodiments, the mixed acyl phosphatidylcholine is
selected from the group consisting of
1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (14:0-16:0 PC),
1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (14:0-18:0 PC),
1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine (16:0-02:0 PC),
1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (16:0-14:0 PC),
1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (16:0-18:0 PC),
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0-18:1 PC),
1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2 PC),
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (16:0-20:4
PC), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine
(16:0-22:6 PC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine
(18:0-14:0 PC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine
(18:0-16:0 PC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine
(18:0-18:1 PC), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine
(18:0-18:2 PC),
1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (18:0-20:4
PC), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine
(18:0-22:6 PC), 1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine
(18:1-14:0 PC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine
(18:1-16:0 PC), and 1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine
(18:1-18:0 PC).
[0306] In some embodiments, the lyso phosphatidylcholine is
selected from the group consisting of
L-.alpha.-lysophosphatidylcholine from chicken egg (Egg Lyso PC),
L-.alpha.-lysophosphatidylcholine from soy (Soy Lyso PC),
1-alkyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso PAF, from Heart
PC), 1-hexanoyl-2-hydroxy-sn-glycero-3-phosphocholine (06:0 Lyso
PC), 1-heptanoyl-2-hydroxy-sn-glycero-3-phosphocholine (07:0 Lyso
PC), 1-octanoyl-2-hydroxy-sn-glycero-3-phosphocholine (08:0 Lyso
PC), 1-nonanoyl-2-hydroxy-sn-glycero-3-phosphocholine (09:0 Lyso
PC), 1-decanoyl-2-hydroxy-sn-glycero-3-phosphocholine (10:0 Lyso
PC), 1-undecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (11:0 Lyso
PC), 1-lauryl-2-hydroxy-sn-glycero-3-phosphocholine (12:0 Lyso PC),
1-tridecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (13:0 Lyso PC),
1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (14:0 Lyso PC),
1-petadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (15:0 Lyso
PC), palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (16:0 Lyso
PC), 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (17:0
Lyso PC), 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (18:0
Lyso PC), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (18:1 Lyso
PC), 1-nonadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (19:0
Lyso PC), 1-arachidoyl-2-hydroxy-sn-glycero-3-phosphocholine (20:0
Lyso PC), 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine (22:0
Lyso PC), 1-lignoceroyl-2-hydroxy-sn-glycero-3-phosphocholine (24:0
Lyso PC), and 1-hexacosanoyl-2-hydroxy-sn-glycero-3-phosphocholine
(26:0 Lyso PC).
[0307] In some embodiments, the alkylphosphatidylcholine is
selected from the group consisting of dodecylphosphocholine (DPC),
tetradecylphosphocholine, and hexadecylphosphocholine.
[0308] In some embodiments, the phosphatidic acid is selected from
the group consisting of a natural phosphatidic acid, a saturated
synthetic phosphatic acid, an unsaturated synthetic phosphatidic
acid, a lyso phosphatidic acid, a cyclic phosphatidic acid, a
lysophosphatidic acid receptor agonist, and a lysophosphatidic acid
antagonist.
[0309] In some embodiments, the natural phosphatidic acid is
L-.alpha.-phosphatidic acid (sodium salt) (e.g., from chicken egg
or soy).
[0310] In some embodiments, the saturated, synthetic phosphatidic
acid is selected from the group consisting of
1,2-dihexanoyl-sn-glycero-3-phosphate (sodium salt) (06:0 PA),
1,2-dioctanoyl-sn-glycero-3-phosphate (sodium salt) (08:0 PA),
1,2-didecanoyl-sn-glycero-3-phosphate (sodium salt) (10:0 PA),
1,2-dilauroyl-sn-glycero-3-phosphate (sodium salt) (12:0 PA),
1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (14:0 PA),
1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (16:0 PA),
1,2-diphytanoyl-sn-glycero-3-phosphate (sodium salt) (4ME 16:0 PA),
1,2-diheptadecanoyl-sn-glycero-3-phosphate (sodium salt) (17:0 PA),
and 1,2-distearoyl-sn-glycero-3-phosphate (sodium salt) (18:0
PA).
[0311] In some embodiments, the unsaturated, synthetic phosphatidic
acid is selected from the group consisting of
1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt (18:1 PA),
1,2-dilinoleoyl-sn-glycero-3-phosphate (sodium salt) (18:2 PA),
1,2-diarachidonoyl-sn-glycero-3-phosphate (sodium salt) (20:4 PA),
and 1,2-didocosahexaenoyl-sn-glycero-3-phosphate (sodium salt)
(22:6 PA).
[0312] In some embodiments, the lyso phosphatidic acid is selected
from the group consisting of
1-hexanoyl-2-hydroxy-sn-glycero-3-phosphate (ammonium salt) (06:0
Lyso PA), 1-myristoyl-2-hydroxy-sn-glycero-3-phosphate (ammonium
salt) (14:0 Lyso), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate
(ammonium salt) (16:0 Lyso PA),
1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphate (ammonium salt)
(17:0 Lyso PA), 1-stearoyl-2-hydroxy-sn-glycero-3-phosphate
(ammonium salt) (18:0 Lyso PA),
1-oleoyl-2-hydroxy-sn-glycero-3-phosphate (ammonium salt) (18:1
Lyso PA), 1-arachidonoyl-2-hydroxy-sn-glycero-3-phosphate (ammonium
salt) (20:4 Lyso PA),
1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphate (ammonium salt) (C16
LPA), 1-O-octadecyl-2-hydroxy-sn-glycero-3-phosphate (ammonium
salt) (C18 LPA), and
1-(9Z-octadecenyl)-2-hydroxy-sn-glycero-3-phosphate (ammonium salt)
(C18:1 LPA).
[0313] In some embodiments, the cyclic phosphatidic acid is
selected from the group consisting of
1-palmitoyl-sn-glycero-2,3-cyclic-phosphate (ammonium salt) (16:0
Cyclic LPA), 1-heptadecanoyl-glycero-2,3-cyclic-phosphate (ammonium
salt) (17:0 Cyclic LPA), 1-oleoyl-sn-glycero-2,3-cyclic-phosphate
(ammonium salt) (18:1 Cyclic LPA),
1-O-hexadecyl-sn-glycero-2,3-cyclic-phosphate (ammonium salt) (C16
Cyclic LPA), and
1-O-(9Z-octadecenyl)-sn-glycero-2,3-cyclic-phosphate (ammonium
salt) (C18:1 Cyclic LPA).
[0314] In some embodiments, the lysophosphatidic acid agonist or
antagonist is selected from the group consisting of
1-oleoyl-2-methyl-sn-glycero-3-phosphothionate (ammonium salt)
(S-OMPT),
N-{(1R)-2-hydroxy-1-[(phosphonooxy)methyl]ethyl}(9Z)octadec-9-enamide
(ammonium salt) (VPC 31143 (R)),
N-{(1S)-2-hydroxy-1-[(phosphonooxy)methyl]ethyl}(9Z)octadec-9-enamide
(ammonium salt) (VPC 31144 (S)), (S)-phosphoric acid
mono-{2-octadec-9-enoylamino-3-[4-(pyridin-2-ylmethoxy)-phenyl]-propyl}es-
ter (ammonium salt) (VPC 32183 (S)), (S)-phosphoric acid
mono-[3-(4-benzyloxy-phenyl)-2-octadec-9-enoylamino-propyl]ester
(ammonium salt) (VPC 12249 (S)), (R)-phosphoric acid
mono-{2-octadec-9-enoylamino-3-[4-(pyridin-2-ylmethoxy)-phenyl]-propyl}es-
ter (ammonium salt) (VPC 32179 (R)), N-palmitoyl-serine phosphoric
acid (ammonium salt) (N--P Serine PA), and N-palmitoyl-tyrosine
phosphoric acid (ammonium salt) (N--P Tyrosine PA).
[0315] In some embodiments, the phosphatidylethanolamie is selected
from the group consisting of a natural phosphatidylethanolamine, a
saturated synthetic phosphatidylethanolamine, an unsaturated
synthetic phosphatidylethanolamine, a mixed acyl
phosphatidylethanolamine, and a lyso phosphatidylethanolamine.
[0316] In some embodiments, the natural phosphatidylethanolamine is
L-.alpha.-phosphatidylethanolamine (e.g., from chicken egg, bovine
heart, porcine brain, bovine liver, soy, or E. coli) or
transphosphatidylated L-.alpha.-phosphatidylethanolamine (e.g.,
from chicken egg) (Egg Trans PE).
[0317] In some embodiments, the saturated, synthetic
phosphatidylethanolamine is selected from the group consisting of
1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (06:0 PE),
1,2-dioctanoyl-sn-glycero-3-phosphoethanolamine (08:0 PE),
1,2-didecanoyl-sn-glycero-3-phosphoethanolamine (10:0 PE),
1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (12:0 PE),
1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (14:0 PE),
1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine (15:0 PE),
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (16:0 PE),
1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (4ME 16:0 PE),
1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine (17:0 PE), and
1,2-distearoyl-sn-glycero-3-phosphoethanolamine (18:0 PE).
[0318] In some embodiments, the unsaturated, synthetic
phosphatidylethanolamine is selected from the group consisting of
1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (16:1 PE),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (18:1 (.DELTA.9-Cis)
PE, DOPE), 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (18:1
(.DELTA.9-Trans) PE),
1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (18:2 PE),
1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine (18:3 PE),
1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine (20:4 PE), and
1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine (22:6
PE).
[0319] In some embodiments, the mixed acyl phosphatidylethanolamine
is selected from the group consisting of
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1
PE), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine
(16:0-18:2 PE),
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine
(16:0-20:4 PE),
1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine
(16:0-22:6 PE),
1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:1
PE), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine
(18:0-18:2 PE),
1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine
(18:0-20:4 PE), and
1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine
(18:0-22:6 PE).
[0320] In some embodiments, the lyso phosphatidylethanolamine is
selected from the group consisting of
L-.alpha.-lysophosphatidylethanolamine (e.g., from chicken egg or
porcine brain),
1-myristoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (14:0 Lyso
PE), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (16:0
Lyso PE), 1-stearoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine
(18:0 Lyso PE), and 1-oleo
yl-2-hydroxy-sn-glycero-3-phosphoethanolamine (18:1 Lyso PE).
[0321] In some embodiments, the phosphatidylglycerol is selected
from the group consisting of a natural phosphatidylglycerol, a
saturated synthetic phosphatidylglycerol, an unsaturated synthetic
phosphatidylglycerol, a mixed acyl phosphatidylglycerol, a lyso
phosphatidylglycerol, and a phosphatidylglycerol platelet activing
factor.
[0322] In some embodiments, the natural phosphatidylglycerol is
L-.alpha.-phosphatidylglycerol (sodium salt) (e.g., from E. coli,
chicken egg, or soy).
[0323] In some embodiments, the saturated, synthetic
phosphatidylglycerol is selected from the group consisting of
1,2-dihexanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)
(06:0 PG), 1,2-dioctanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)
(sodium salt) (08:0 PG),
1,2-didecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (10:0 PG),
1,2-dilauroyl-sn-glycero-3-phospho-(1'-rac-glycerol) (12:0 PG),
1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (14:0 PG),
1,2-dipentadecanoyl-sn-3-phospho-(1'-rac-glycerol) (15:0 PG),
1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (16:0 PG),
1,2-diphytanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (4ME 16:0
PG), 1,2-diheptadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)
(17:0 PG), and
1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (18:0
PG).
[0324] In some embodiments, the unsaturated, synthetic
phosphatidylglycerol is selected from the group consisting of
1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (18:1
(.DELTA.9-Cis) PG),
1,2-dielaidoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (18:1
(.DELTA.9-Trans) PG),
1,2-dilinoleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (18:2 PG),
1,2-dilinolenoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (18:3 PG),
1,2-diarachidonoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (20:4
PG), and
1,2-didocosahexaenoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (22:6
PG).
[0325] In some embodiments, the mixed acyl phosphatidylglycerol is
selected from the group consisting of,
1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)
(16:0-18:1 PG),
1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)
(16:0-18:2 PG),
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-(1'-rac-glycerol)
(16:0-20:4 PG),
1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phospho-(1'-rac-glycerol)
(16:0-22:6 PG),
1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)
(18:0-18:1 PG),
1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)
(18:0-18:2 PG),
1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-(1'-rac-glycerol)
(18:0-20:4 PG), and
1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospho-(1'-rac-glycerol)
(18:0-22:6 PG).
[0326] In some embodiments, the lyso phosphatidylglycerol is
selected from the group consisting of
1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol)
(sodium salt) (14:0 Lyso PG),
1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol)
(sodium salt) (16:0 Lyso PG),
1-stearoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium
salt) (18:0 Lyso PG), and
1-oleoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium
salt) (18:1 Lyso PG).
[0327] In some embodiments, the phosphatidylglycerol platelet
activing factor is 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG)
(C16-02:0 DG).
[0328] In some embodiments, the phosphatidylserine is selected from
the group consisting of a natural phosphatidylserine, a saturated
synthethic phosphatidylserine, an unsaturated synthetic
phosphatidylserine, a mixed acyl phosphatidylserine, and a lyso
phosphatidylserine.
[0329] In some embodiments, the natural phosphatidylserine is
L-.alpha.-phosphatidylserine (sodium salt) (e.g., from porcine
brain or soy).
[0330] In some embodiments, the saturated, synthetic
phosphatidylserine is selected from the group consisting of
1,2-dihexanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (06:0
PS), 1,2-dioctanoyl-sn-glycero-3-phospho-L-serine (sodium salt)
(08:0 PS), 1,2-didecanoyl-sn-glycero-3-phospho-L-serine (sodium
salt) (10:0 PS), 1,2-dilauroyl-sn-glycero-3-phospho-L-serine
(sodium salt) (12:0 PS),
1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt) (14:0
PS), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt)
(16:0 PS), 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium
salt) (4ME 16:0 PS),
1,2-diheptadecanoyl-sn-glycero-3-phospho-L-serine (sodium salt)
(17:0 PS), and 1,2-distearoyl-sn-glycero-3-phospho-L-serine (sodium
salt) (18:0 PS).
[0331] In some embodiments, the unsaturated, synthetic
phosphatidylserine is selected from the group consisting of,
1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (18:1 PS,
DOPS), 1,2-dilinoleoyl-sn-glycero-3-phospho-L-serine (sodium salt)
(18:2 PS), 1,2-diarachidonoyl-sn-glycero-3-phospho-L-serine (sodium
salt) (20:4 PS), and
1,2-didocosahexaenoyl-sn-glycero-3-phospho-L-serine (sodium salt)
(22:6 PS).
[0332] In some embodiments, the mixed acyl phosphatidylserine is
selected from the group consisting of
1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt)
(16:0-18:1 PS, POPS),
1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (sodium salt)
(16:0-18:2 PS),
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine (sodium
salt) (16:0-20:4 PS),
1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phospho-L-serine (sodium
salt) (16:0-22:6 PS),
1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt)
(18:0-18:1 PS),
1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (sodium salt)
(18:0-18:2 PS),
1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine (sodium
salt) (18:0-20:4 PS), and
1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospho-L-serine (sodium
salt) (18:0-22:6 PS).
[0333] In some embodiments, the lyso phosphatidylserine is selected
from the group consisting of L-.alpha.-lysophosphatidylserine
(sodium salt) (e.g., from porcine
brain),.sub.--1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-L-serine
(sodium salt) (16:0 Lyso PS),
1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (sodium salt)
(18:0 Lyso PS), and
1-oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (sodium salt)
(18:1 Lyso PS).
[0334] In some embodiments, the phosphatidylinositol is selected
from the group consisting of natural phosphatidylinositol, a
saturated synthethic phosphatidylinositol, an unsaturated synthetic
phosphatidylinositol, a mixed acyl phosphatidylinositol, and a lyso
phosphatidylserine.
[0335] In some embodiments, the natural phosphatidylinositol is
L-.alpha.-phosphatidylinositol (sodium salt) (e.g., from bovine
liver or soy), L-.alpha.-phosphatidylinositol-4-phosphate (ammonium
salt) (e.g., from porcine brain) (Brain PI(4)P), or
L-.alpha.-phosphatidylinositol-4,5-bisphosphate (ammonium salt)
(e.g., from porcine brain) (Brain PI(4,5)P2).
[0336] In some embodiments, the saturated, synthetic
phosphatidylinositol is selected from the group consisting of
1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-myo-inositol) (ammonium
salt) (16:0 PI),
1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol) (ammonium salt)
(18:1 PI),
1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3'-phosphate)
(ammonium salt) (18:1 PI(3)P,
1,2-dioctanoyl-sn-glycero-3-phospho-(1'-myo-inositol-4'-phosphate)
(ammonium salt) (8:0 PI(4)P),
1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-5'-phosphate)
(ammonium salt) (18:1 PI(5)P),
1,2-dihexanoyl-sn-glycero-3-phospho-(1'-myo-inositol-3',5'-bisphosphate)
(ammonium salt) (06:0 PI(3,5)P2),
1,2-dioctanoyl-sn-glycero-3-phospho-(1'-myo-inositol-3',4'-bisphosphate)
(ammonium salt) (08:0 PI(3,4)P2),
1,2-dioctanoyl-sn-glycero-3-phospho-(1'-myo-inositol-3',5'-bisphosphate)
(ammonium salt) (08:0 PI(3,5)P2), and
1,2-dioctanoyl-sn-glycero-3-phospho-(1'-myo-inositol-4',5'-bisphosphate)
(ammonium salt) (08:0 PI(4,5)P2).
[0337] In some embodiments, the unsaturated, synthetic
phosphatidylinositol
1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3',4'-bisphosphate)
(ammonium salt) (18:1 PI(3,4)P2),
1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3',5'-bisphosphate)
(ammonium salt) (18:1 PI(3,5)P2),
1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-4',5'-bisphosphate)
(ammonium salt) (18:1 PI(4,5)P2),
1,2-dihexanoyl-sn-glycero-3-phospho-(1'-myo-inositol-3',4',5'-trisphospha-
te) (ammonium salt) (06:0 PI(3,4,5)P3),
1,2-dioctanoyl-sn-glycero-3-phospho-(1'-myo-inositol-3',4',5'-trisphospha-
te) (ammonium salt) (08:0 PI(3,4,5)P3),
1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3',4',5'-trisphosphate-
) (ammonium salt) (18:1 PI(3,4,5)P3),
1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-(1'-myo-inositol-3',5'-bis-
phosphate) (ammonium salt) (18:0-20:4 PI(3,5)P2),
1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-(1'-myo-inositol-4',5'-bis-
phosphate) (ammonium salt) (18:0-20:4 PI(4,5)P2), and
1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-(1'-myo-inositol-3',4',5'--
trisphosphate) (ammonium salt) (18:0-20:4 PI(3,4,5)P3).
[0338] In some embodiments, the lyso phosphatidylinositol is
selected from the group consisting of
L-.alpha.-lysophosphatidylinositol (sodium salt) (e.g., from bovine
liver or soy), and
1-oleoyl-2-hydroxy-sn-glycero-3-phospho-(1'-myo-inositol) (ammonium
salt) (18:1 Lyso PI).
[0339] In some embodiments, the inositol phosphate is selected from
the group consisting of D-myo-inositol-1,3,4-trisphosphate
(ammonium salt) (IP3(1,3,4)), D-myo-inositol-1,3,5-triphosphate
(ammonium salt) (IP3(1,3,5)), and D-myo-inositol-1,4,5-triphosphate
(ammonium salt) (IP3(1,4,5)), and
D-myo-inositol-1,3,4,5-tetraphosphate (ammonium salt)
(IP4(1,3,4,5)).
[0340] In some embodiments, the platelet activating factor or
analog thereof is selected from the group consisting of
1-alkyl-2-acetoyl-sn-glycero-3-phosphocholine (PAF) (e.g., from
heart PC), 1-alkyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso
PAF), (e.g., from heart PC),
1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16-02:0 PC),
1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine (C18-02:0 PC),
1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (C16 Lyso PAF),
1-O-heptadecyl-2-hydroxy-sn-glycero-3-phosphocholine (C17 Lyso
PAF), 1-O-octadecyl-2-hydroxy-sn-glycero-3-phosphocholine (C18 Lyso
PAF), 1-O-(9Z)octadecenyl-2-hydroxy-sn-glycero-3-phosphocholine
(C18:1 Lyso PAF),
1-O-hexadecyl-2-butyryl-sn-glycero-3-phosphocholine (C16-04:0 PC),
1-O-octadecyl-2-butyryl-sn-glycero-3-phosphocholine (C18-04:0 PC),
1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphocholine (C16-18:1 PC),
1-O-hexadecyl-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycero-3-phosphocholine
(C16-20:3 PC),
1-O-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine (C16-20:4
PC), 1-O-hexadecyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine
(C16-20:5 PC), 3-O-hexadecyl-2-acetyl-sn-glycero-1-phosphocholine
(C16-22:6 PC), 3-O-hexadecyl-2-acetyl-sn-glycero-1-phosphocholine
(C16-02:0 PC), 1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine
(16:0-02:0 PC), and
1-myristoyl-2-(4-nitrophenylsuccinyl)-sn-glycero-3-phosphocholine
(14:0 NPS PC).
[0341] In some embodiments, the cariolipin is selected from the
group consisting of natural cardiolipin, a saturated synthethic
cardiolipin, an unsaturated synthetic cardiolipin, and a lyso
cardiolipin. In some embodiments, the natural cardiolipin is
cardiolipin (sodium salt) (e.g., from bovine heart or E. coli). In
some embodiments, the saturated, synthetic cardiolipin is
1',3'-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol
(ammonium salt) (14:0 CA, (ammonium salt) or
1',3'-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol (sodium
salt) (14:0 CA, sodium salt). In some embodiments, the unsaturated,
synthetic cardiolipin is
1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol (sodium
salt) (18:1 CA). In some embodiments, the lyso cardiolipin is
monolysocardiolipin (sodium salt) (e.g., from bovine heart)
(Monolyso Heart Calif.) or dilysocardiolipin (sodium salt) (e.g.,
from bovine heart) (Dilyso Heart Calif.).
[0342] In some embodiments, the ether phospholipid is selected from
the group consisting of 1,2-di-O-hexyl-sn-glycero-3-phosphocholine
(06:0 Diether PC), 1,2-di-O-dodecyl-sn-glycero-3-phosphocholine
(12:0 Diether PC), 1,2-di-O-tridecyl-sn-glycero-3-phosphocholine
(13:0 Diether PC), 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine
(14:0 Diether PC), 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine
(16:0 Diether PC), 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine
(18:0 Diether PC),
1,2-di-O-(9Z-octadecenyl)-sn-glycero-3-phosphocholine (18:1 Diether
PC), 1-O-octadecyl-2-methyl-sn-glycero-3-phosphocholine (18:0-1:0
Diether PC), 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine (4ME
16:0 Diether PC),
1,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine (4ME 16:0
Diether PE), 1,2-di-O-phytanyl-sn-glycerol (4ME 16:0 Diether DG),
1,2-di-O-tetradecyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium
salt) (14:0 Diether PG), 1-O-hexadecyl-sn-glycerol (HG) (1-C16
Ether MG), and 1-O-hexadecyl-2-O-methyl-sn-glycerol (PMG)
(1-C16-2-C1 DG).
[0343] In some embodiments, the plasmalogen is selected from the
group consisting of
1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphocholine
(C18(Plasm)-18:1 PC),
1-(1Z-octadecenyl)-2-arachidonoyl-sn-glycero-3-phosphocholine
(C18(Plasm)-20:4 PC),
1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphocholine
(C18(Plasm)-22:6 PC),
1-O-1'-(Z)-octadecenyl-2-hydroxy-sn-glycero-3-phosphocholine
(C18(Plasm) LPC),
1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphoethanolamine
(C18(Plasm)-18:1 PE),
1-(1Z-octadecenyl)-2-arachidonoyl-sn-glycero-3-phosphoethanolamine
(C18(Plasm)-20:4 PE),
1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine
(C18(Plasm)-22:6 PE),
1-O-1'-(Z)-octadecenyl-2-hydroxy-sn-glycero-3-phosphoethanolamine
(C18(Plasm) LPE),
[0344] In some embodiments, the oxidized phospholipid is selected
from the group consisting of
1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine
(POVPC),
1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine
(16:0-09:0 (ALDO) PC),
1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC),
1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PAzPC),
1-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (C16-09:0
(COOH)PC), and (E)-4R-hydroxynonenal-dimethylacetal
(4-HNE-dimethylacetal).
[0345] In some embodiments, the bis(monoacylglycero)phosphate is
selected from the group consisting of
bis(monomyristoylglycero)phosphate (S,R Isomer) (ammonium salt)
(14:0 BMP (S,R)), bis(monooleoylglycero)phosphate (S,R Isomer)
(ammonium salt) (18:1 BMP (S,R)),
sn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho-sn-1'-(3'-oleoyl-2'-hydroxy)-g-
lycerol (ammonium salt) (18:1 BMP(S,S)),
sn-(1-oleoyl-2-hydroxy)-glycerol-3-phospho-sn-3'-(1'-oleoyl-2'-hydroxy)-g-
lycerol (ammonium salt) (18:1 BMP(R,R)),
sn-[2,3-dioleoyl]-glycerol-1-phospho-sn-1'-[2',3'-dioleoyl]-glycerol
(ammonium salt) (18:1 BDP(S,S)),
sn-(3-myristoyl-2-hydroxy)-glycerol-1-phospho-sn-3'-(1',2'-dimyristoyl)-g-
lycerol (ammonium salt) (14:0 Hemi BMP (S,R)), and
sn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho-sn-3'-(1',2'-dioleoyl)-glycero-
l (ammonium salt) (18:1 Hemi BMP (S,R)).
[0346] In some embodiments, the phospholipid for supported
monolayers is
1-palmitoyl-2-[16-(acryloyloxy)palmitoyl]-sn-glycero-3-phosphorylcholine
(16:0-16:0 (acrylate) PC) or
1-myristoyl-2-(14-carboxymyristoyl)-sn-glycero-3-phosphocholine
(14:0-14:0(COOH)PC).
[0347] In some embodiments, the sterol modified phospholipid is
selected from the group consisting of
1-palmitoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine
(PChemsPC),
1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine
(OChemsPC), and
1-palmitoyl-2-cholesterylcarbonoyl-sn-glycero-3-phosphocholine
(PChcPC),
1,2-dicholesterylhemisuccinoyl-sn-glycero-3-phosphocholine
(DChemsPC).
[0348] In some embodiments, the sterol is selected from the group
consisting of a natural sterol, a substituted oxysterol, a
derivative of a substituted oxysterol, and a vitamin (e.g., 25-OH
Vitamin D2 or 25-OH Vitamin D3). In some embodiments, the natural
sterol is selected from the group consisting of cholesterol,
desmosterol, zymosterol, stigmasterol, lathosterol, lanosterol,
lanostenol, 14-demethyl-lanosterol, and cholesterol sulfate.
[0349] In some embodiments, the oxysterol or derivative of an
oxysterol is A-ring substituted, B-ring substituted, D-ring
substituted, side chain substituted, double substituted,
cholestanoic acid, cholestenoic acid, deuterated, fluorinated,
sulfonated, or fluorescent. In some embodiments, the A-ring
substituted oxysterol is selected from the group consisting of
4.beta.-hydroxycholesterol, a cholestanol (e.g.,
trihydroxycholestanoic acid), and cholestenone. In some
embodiments, the B-ring substituted oxysterol is selected from the
group consisting of 7-ketocholesterol,
5.alpha.,6.alpha.-epoxycholestanol,
5.beta.,6.beta.-epoxycholestanol, 7.alpha.-hydroxycholesterol,
7.beta.-hydroxycholesterol, 6.alpha.-hydroxy-5.alpha.-cholestanol,
and 7-dehydrocholesterol. In some embodiments, the D-ring
substituted oxysterol is selected from the group consisting of
15-ketocholestene, 15-ketocholestane, 15.alpha.-hydroxycholestene,
15.beta.-hydroxycholestene, 15.alpha.-hydroxycholestane, and
15.beta.-hydroxycholestane. In some embodiments, the side chain
substituted sterol is selected from the group consisting of
22(R)-hydroxycholesterol, 22(S)-hydroxycholesterol,
24(R)-hydroxycholesterol, 24(S)-hydroxycholesterol,
24(S),25-epoxycholesterol, 24(R/S),25-epoxycholesterol,
25-hydroxycholesterol, and 27-hydroxycholesterol. In some
embodiments, the double substituted oxysterol is selected from the
group consisting of 3.beta.,27-dihydroxy-5-cholesten-7-one,
7.alpha.,27-dihydroxy-4-cholesten-3-one,
7.alpha.,27-dihydroxycholesterol, and
7.beta.,27-dihydroxycholesterol. In some embodiments, the
cholestenone is selected from the group consisting of
3.beta.-hydroxy-7-oxo-5-cholestenoic acid,
7.alpha.-hydroxy-3-oxo-4-cholestenoic acid,
3.alpha.,7.alpha.-dihydroxy-5-cholestenoic acid, and
3.beta.,7.beta.-dihydroxy-5-cholestenoic acid.
[0350] In some embodiments, at least one lipid of the one to five
lipids is a cationic lipid that is not a sphingolipid, a
phospholipid, or a sterol. In some embodiments, the cationic lipid
is selected from the group consisting of
3.beta.-[N--(N',N'-dimethylaminoethane)-carbamoyl]cholesterol
hydrochloride (DC-Cholesterol.HCl),
1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (18:1 TAP,
DOTAP), 1,2-dioleoyl-3-trimethylammonium-propane(methyl sulfate
salt) (18:1 TAP, DOTAP, MS Salt),
1,2-dimyristoyl-3-trimethylammonium-propane (chloride salt) (14:0
TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (chloride salt)
(16:0 TAP), 1,2-stearoyl-3-trimethylammonium-propane (chloride
salt) (18:0 TAP), Transfection Reagent I (i.e., contains DOTAP:DOPE
in 1:1 w/w ratio), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP)
(18:1 DAP), 1,2-dimyristoyl-3-dimethylammonium-propane (14:0 DAP),
1,2-dipalmitoyl-3-dimethylammonium-propane (16:0 DAP),
1,2-distearoyl-3-dimethylammonium-propane (18:0 DAP),
dimethyldioctadecylammonium (Bromide Salt) (18:0 DDAB),
1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (chloride salt)
(12:0 EPC, Cl Salt),
1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (chloride salt)
(14:0 EPC, Cl Salt),
1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (Tf salt) (14:1
EPC, Tf Salt), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine
(chloride salt) (16:0 EPC, Cl Salt),
1,2-distearoyl-sn-glycero-3-ethylphosphocholine (chloride salt)
(18:0 EPC, Cl Salt), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine
(chloride salt) (18:1 EPC, Cl Salt), and
1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (chloride
salt) (16:0-18:1 EPC, Cl Salt).
[0351] In some embodiments, at least one lipid of the one to five
lipids is a neutral lipid that is not a sphingolipid, a
phospholipid, or a sterol. In some embodiments, the neutral lipid
is a diacyl glycerol or an analog of a diacyl glycerol, a lysosomal
PLA2 substrate, a glycosylated diacyl glycerol, a prostaglandin, a
prenol, or a N-acylglycine (NAGly).
[0352] In some embodiments, the diacyl glycerol or an analog of a
diacyl glycerol is selected from the group consisting of
1,2-dioctanoyl-sn-glycerol (08:0 DG), 1,2-didecanoyl-sn-glycerol
(10:0 DG), 1,2-dilauroyl-sn-glycerol (12:0 DG),
1,2-dimyristoyl-sn-glycerol (14:0 DG), 1,2-dipalmitoyl-sn-glycerol
(16:0 DG), 1-2-dioleoyl-sn-glycerol (18:1 DG),
1-palmitoyl-2-oleoyl-sn-glycerol (16:0-18:1 DG),
1-stearoyl-2-linoleoyl-sn-glycerol (18:0-18:2 DG),
1-stearoyl-2-arachidonoyl-sn-glycerol (18:0-20:4 DG),
1-stearoyl-2-docosahexaenoyl-sn-glycerol (18:0-22:6 DG),
1-oleoyl-2-acetyl-sn-glycerol (18:1-2:0 DG),
1,2-di-O-phytanyl-sn-glycerol (4ME 16:0 Diether DG),
1,2-dipalmitoyl ethylene glycol (16:0 Ethylene Glycol), and
1-2-dioleoyl ethylene glycol (18:1 Ethylene Glycol).
[0353] In some embodiments, the lysosomal PLA2 substrate is
selected from the group consisting of 1-O-hexadecyl-sn-glycerol
(HG) (1-C16 Ether MG), 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG)
(C16-02:0 DG), and 1-O-hexadecyl-2-O-methyl-sn-glycerol (PMG)
(1-C16-2-C1 DG).
[0354] In some embodiments, the glycosylated diacyl glycerol is
1,2-diacyl-3-O-(.alpha.-D-glucopyranosyl)-sn-glycerol (E. coli)
(MGlc-DAG).
[0355] In some embodiments, the prostaglandin is selected from the
group consisting of prostaglandin E.sub.1 (PGE1), prostaglandin
F.sub.1.alpha. (PGF1.alpha.), prostaglandin F.sub.1.beta.
(PGF1.beta.), prostaglandin A.sub.1 (PGA1), and prostaglandin
B.sub.1 (PGB1).
[0356] In some embodiments, the prenol is selected from the group
consisting of Coenzyme Q6 (S. cerevisiae) (CoQ6), a dolichol
mixture (13.about.21), and a polyprenol mixture (13.about.21).
[0357] In some embodiments, the N-acylglycine is selected from the
group consisting of lauroyl L-carnitine (C12 Carnitine), palmitoyl
L-carnitine (C16 Carnitine), oleoyl L-carnitine
(C18:1(.DELTA.9-cis)), N-palmitoylglycine, N-oleoylglycine, and
N-arachidonoylglycine.
[0358] In some embodiments, at least one lipid of the one to five
lipids is a detergent that is not a sphingolipid, a phospholipid,
or a sterol. In some embodiments, the detergents is a nonionic
detergent, a zwitterionic detergent, or an anionic detergent. In
some embodiments, the nonionic detergent is selected from the group
consisting of
3.alpha.-hydroxy-7.alpha.,12.alpha.-di-((O-.beta.-D-maltosyl)-2-hydroxyet-
hoxy)-cholane, n-dodecyl-.beta.-D-maltoside,
n-octyl-.beta.-D-glucoside, and n-nonyl-.beta.-D-glucoside. In some
embodiments, the zwitterionic detergent is
3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)
or
3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate
(CHAPSO). In some embodiments, the anionic detergent is cholesteryl
hemisuccinate (CHEMS).
[0359] In some embodiments, at least one lipid of the one to five
lipids is a bioactive lipid that is not a sphingolipid, a
phospholipid, or a sterol. In some embodiments, the bioactive lipid
is an adjuvant, a liponucleotide, a TLR-4 agonist (e.g.,
di[3-deoxy-D-manno-octulosonyl]-lipid A (ammonium salt) (Kdo2-Lipid
A)), an endocannabinoid/anadamide, a lysyl phosphatidylglycerol, a
diacylglycerol pyrophosphate, or an inhibitor of phosphipase D.
[0360] In some embodiments, the adjuvant is selected from the group
consisting of Lipid A Detoxified (Salmonella Minnesota R595),
phosphorylated hexaacyl disaccharide (MPLA, PHAD.TM.),
D-(+)-trehalose 6,6'-dibehenate (22:0 Trehalose), and
dimethyldioctadecylammonium (Bromide Salt) (18:0 DDAB).
[0361] In some embodiments, the liponucleotide is
1,2-dipalmitoyl-sn-glycero-3-(cytidine diphosphate) (ammonium salt)
(16:0 CDP DG) or 1,2-dioleoyl-sn-glycero-3-(cytidine diphosphate)
(ammonium salt) (18:1 CDP DG).
[0362] In some embodiments, the endocannabinoid/anadamide is
selected from the group consisting of 2-arachidonoyl glycerol
(2-AG), 2-oleoyl glycerol (2-OG), 2-O-arachidonyl glyceryl ether
(2-AG Ether), 2-O-oleyl glyceryl ether (2-OG Ether), N-palmitoyl
L-serine methyl ester (N-16:0 L-Serine), N-palmitoyl L-serine
(N-16:0 L-Serine), N-oleoyl L-serine methyl ester (N-18:1 L-Serine
MeEster), N-oleoyl L-serine (N-18:1 L-Serine), N-arachidonoyl
L-serine methyl ester (N-20:4 L-Serine MeEster), N-arachidonoyl
L-serine (N-20:4 L-Serine), 10Z-heptadecenoylethanolamide (C17:1
anandamide), 9Z-octadecenoylethanolamide (C18:1 anandamide),
5Z,8Z,11Z,14Z-eicosatetraenoylethanolamide (C20:4 anandamide), and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-arachidonoyl(ammonium
salt) (18:1 PE-N-20:4).
[0363] In some embodiments, the lysyl phosphatidylglycerol is
1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))]
(chloride salt) (16:0 Lysyl PG) or
1,2-dioleoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))]
(chloride salt) (18:1 Lysyl PG).
[0364] In some embodiments, the diacylglycerol pyrophosphate is
dioctanoylglycerol pyrophosphate (ammonium salt) (08:0 DGPP) or
dioleoylglycerol pyrophosphate (ammonium salt) (18:1 DGPP).
[0365] In some embodiments, the inhibitor of phospholipase D is
selected from the group consisting of
N-(2-{4-[2-oxo-2,3-dihydro-1H-benzo(d)imidazol-1-yl]piperidin-1-yl}ethyl)-
-2-naphthamide (VU0155056),
N-{2-[4-oxo-1-phenyl-1,3,8-triazaspiro(4.5)decan-8-yl]ethyl}quinoline-3-c-
arboxamide (VU0285655-1), and
(1R,2R)--N-([S]-1-{4-[5-bromo-2-oxo-2,3-dihydro-1H-benzo(d)imidazol-1-yl]-
piperidin-1-yl}propan-2-yl)-2-phenylcyclopropanecarboxamide
(VU0359595).
[0366] In some embodiments at least one lipid of the one to five
lipids is a coenzyme A, a saturated acyl coenzyme A, an unsaturated
acyl coenzyme A, or a derivative of a coenzyme A. In some
embodiments, the saturated acyl coenzyme A is selected from the
group consisting of 03:0 Coenzyme A, 04:0 Coenzyme A, 06:0 Coenzyme
A, 08:0 Coenzyme A, 10:0 Coenzyme A, 12:0 Coenzyme A, 13:0 Coenzyme
A, 14:0 Coenzyme A, 14:0 Ether Coenzyme A, 15:0 Coenzyme A, 16:0
Coenzyme A, 16:0 Ether Coenzyme A, 4ME 16:0 Coenzyme A, 17:0
Coenzyme A, 18:0 Coenzyme A, 18:0(.alpha.-OH) Coenzyme A, 19:0
Coenzyme A, 20:0 Coenzyme A, 21:0 Coenzyme A, 22:0 Coenzyme A, 23:0
Coenzyme A, 24:0 Coenzyme A, 25:0 Coenzyme A, and 26:0 Coenzyme
A.
[0367] In some the unsaturated acyl coenzyme A is selected from the
group consisting of 16:1 (n7) Coenzyme A, 17:1 (n7) Coenzyme A,
18:1 ether Coenzyme A, 18:1(n7) Coenzyme A, 18:1 (n9) Coenzyme A,
18:1 (n12) Coenzyme A, 18:2 (n6) Coenzyme A, 18:3 (n3) Coenzyme A,
18:3 (n6) Coenzyme A, 20:4 Coenzyme A, 20:5 Coenzyme A, 22:6
Coenzyme A, and 24:1 Coenzyme A.
[0368] In some embodiments, the derivative of coenzyme A is
selected from the group consisting of 12:0 Biotinyl Coenzyme A,
16-NBD-16:0 Coenzyme A, 12:0 Biotinyl Coenzyme A, 16-NBD-16:0
Coenzyme A, 04:0 Pyrene Coenzyme A, and 04:0 Pyrene Coenzyme A.
[0369] In some embodiments, the fatty acid portion of any lipid
described herein is modified by, for example, halogenation (e.g.,
bromination or fluorination), biotinylation, oxidation,
acetylation, methylation, or a mixture thereof.
[0370] In some embodiments, the headgroup of any lipid described
herein is modified with, for example, a succinyl group, a glutaryl
group, a dodecanoyl group, an amino group (e.g., caproylamine,
dodecanoylamine), a thiol (e.g., thioethyl), a maleimido group, a
pyridyldithio group, a biotinyl group, a N-cyanide group, a
polyalkylene glycol moiety (e.g., methoxypolyethylene glycol,
300-5000 MW), a galloyl group, a dinitrophenyl group, a
homocysteine group, a glycosyl group (e.g., lactosyl), a chelator
(e.g., N-diethylenetriaminepentaacetic acid (DTPA) or
N-(5-amino-1-carboxylpentyl)miniodiacectic acid)succinic acid), or
an alkyl phosphate (e.g., phosphatidylmethanol,
phosphatidylethanol, phosphatidylpropanol, phosphatidylbutanol,
phosphoethanolamine-N-methyl, and
phosphoethanolamine-N,N-dimethyl).
[0371] In some embodiments, at least one lipid of the one to five
lipids is a zwitterionic lipid. A zwitterionic lipid can be
included in a liposome if, e.g., the resulting liposome will
function as a delivery agent for a neutral compound. A zwitterionic
lipid also provides stabilization to the lipid bilayer and forms a
permeability barrier that allows for encapsulation of small
molecules (e.g., small molecule immunomodulators). The zwitterionic
lipid can be any zwitterionic lipid that is capable of forming a
liposome with the nonglycosidic ceramide described herein, as long
as the resulting liposome has at least a similar efficacy as a
corresponding soluble nonglycosidic ceramide that is not
incorporated within a liposome. In some embodiments, the
zwitterionic lipid is saturated. In some embodiments, the
zwitterionic lipid is unsaturated.
[0372] In some embodiments, the zwitterionic lipid is selected from
the group consisting of a zwitterionic sphingolipid (e.g., a
phosphorylated sphingosine, a sphingomyelin, a phosphosphingolipid,
a sphingolipid receptor agonist, and a sphingolipid receptor
antagonist), a zwitterionic phospholipid (e.g., a
phosphatidylcholine, a phosphatidylethanolamine, a
phosphatidylserin, a platelet activing factor phospholipid, an
ether phospholipid, a plasmalogen, an oxidized phospholipid, a
phospholipid for supported monolayers, and a sterol modified
phospholipid), and a zwitterionic detergent that is not a
sphingolipid or a phospholipid.
[0373] In some embodiments, the zwitterionic lipid is a
phospholipid. Examples of zwitterionic phospholipids include any of
the herein described zwitterionic phosphatidylcholines (e.g.,
natural; saturated, synthetic; unsaturated, synthetic; mixed acyl;
lyso; alkylphosphocholine), zwitterionic phosphatidylethanolamines
(e.g., natural; saturated, synthetic; unsaturated, synthetic; mixed
acyl; lyso), zwitterionic phosphatidylserines (e.g., natural;
saturated, synthetic; unsaturated, synthetic; mixed acyl; lyso),
zwitterionic platelet activing factor phospholipids, zwitterionic
ether phospholipids, zwitterionic plasmalogens, zwitterionic
oxidized phospholipids, zwitterionic phospholipids for supported
monolayers, and zwitterionic aterol modified phospholipids. In some
embodiments, the zwitterionic phospholipid is a zwitterionic
phosphatidylcholine (e.g., natural; saturated, synthetic;
unsaturated, synthetic; mixed acyl; lyso; alkylphosphocholine), a
zwitterionic phosphatidylethanolamine (e.g., natural; saturated,
synthetic; unsaturated, synthetic; mixed acyl; lyso), or a
zwitterionic phosphatidylserine (e.g., natural; saturated,
synthetic; unsaturated, synthetic; mixed acyl; lyso).
[0374] In some embodiments, at least one lipid of the one to five
lipids is a zwitterionic phosphatidylcholine, such as, for example,
any of the phosphatidylcholine lipids previously described herein.
In some exemplary embodiments, the zwitterionic phosphatidylcholine
is selected from the group consisting of
1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC),
1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC),
1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC),
1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1-myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine (MPPC),
1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC),
1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC),
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC),
1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC),
1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC),
1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC), egg
phosphatidylcholine (EPC), and mixtures thereof, for example, egg
phosphatidylcholine (EPC).
[0375] In some embodiments, at least one lipid of the one to five
lipids is anionic. An anionic lipid can be included in a liposome
if, e.g., the resulting liposome will function as a delivery agent
for a positively-charged compound. An anionic lipid also provides
increased association with the target, increased
incorporation/association of basic proteins, and potential for
activation of immune cells, thereby enhancing the effect of the
nonglycosidic ceramide on the cell. The anionic lipid can be any
anionic lipid that is capable of forming a liposome with the
nonglycosidic ceramide described herein, as long as the resulting
liposome has a similar efficacy as a corresponding soluble
nonglycosidic ceramide that is not incorporated within a liposome.
In some embodiments, the anionic lipid is saturated. In some
embodiments, the anionic lipid is unsaturated.
[0376] In some embodiments, the anionic lipid is selected from the
group consisting of an anionic sphingosine (e.g., a dimethyl
sphingosine-1-phosphate, a ceramide phosphate, a dihydroceramide
phosphate, a ganglioside, and a sulfatide), an anionic phospholipid
(e.g., a phosphatidic acid, a phosphatidylglycerol, a
phosphatidylinositol, an inositol phosphate, a cardiolipin, a
bis(monoacylglycero)phosphate, an anionic detergent that is not a
sphingolipid or a phospholipid, and an anionic bioactive lipid,
such as, for example, an adjuvant, a liponucleotide, a TLR-4
agonist, and a diacylglycerol pyrophosphate.
[0377] In some embodiments, the anionic lipid is a phospholipid.
Examples of anionic phospholipids include any of the herein
described anionic phosphatidic acids (e.g., natural; saturated,
synthetic; unsaturated, synthetic; lyso; cyclic, lysophosphatidic
acid receptor agonist; lysophosphatidic acid receptor antagonist),
anionic phosphatidylglycercols (e.g, natural; saturated, synthetic;
unsaturated, synthetic; mixed acyl; lyso; platelet activing factor
phospholipids), and anionic phosphatidylinositols (e.g., natural;
saturated, synthetic; unsaturated, synthetic; lyso; inositol
phosphates). In some embodiments, the anionic lipid is
phosphatidylglycerol. In some exemplary embodiments, the anionic
phosphatidylglycerol is selected from the group consisting of
1,2-dierucoyl phosphatidylglycerol (DEPG), 1,2-dilauroyl
phosphatidylglycerol (DLPG), 1,2-dimyristoyl phosphatidylglycerol
(DMPG), 1,2-dioleoyl phosphatidylglycerol (DOPG), 1,2-dipalmitoyl
phosphatidylglycerol (DPPS), 1,2-distearoyl phosphatidylglycerol
(DSPG), 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG), egg
phosphatidylglycerol (EPG), salts of any of the foregoing (e.g.,
sodium, ammonium, or sodium/ammonium), and mixtures thereof (e.g.,
egg phosphatidylglycerol).
[0378] The amount of lipid that can be present in the liposome,
excluding any nonglycosidic ceramide, is about 50 wt. % to about 99
wt. %, or about 80 wt. % to about 98 wt. %, based on the total
weight of the liposome or the lipid bilayer. In some embodiments,
the lipid is present in an amount of about 50 wt. % to about 75 wt.
%, or about 75 wt. % to about 99 wt. %, or about 60 wt. % to about
80 wt. %, or about 90 wt. % to about 98 wt. %, based on the total
weight of the liposome or the lipid bilayer. For example, the lipid
can be present in an amount of about 50 wt. %, about 51 wt. %,
about 52 wt. %, about 53 wt. %, about 54 wt. %, about 55 wt. %,
about 56 wt. %, about 57 wt. %, about 58 wt. %, about 59 wt. %,
about 60 wt. %, about 61 wt. %, about 62 wt. %, about 63 wt. %,
about 64 wt. %, about 65 wt. %, about 66 wt. %, about 67 wt. %,
about 68 wt. %, about 69 wt. %, about 70 wt. %, about 71 wt. %,
about 72 wt. %, about 73 wt. %, about 74 wt. %, about 75 wt. %,
about 76 wt. %, about 77 wt. %, about 78 wt. %, about 79 wt. %,
about 80 wt. %, about 81 wt. %, about 82 wt. %, about 83 wt. %,
about 84 wt. %, about 85 wt. %, about 86 wt. %, about 87 wt. %,
about 88 wt. %, about 89 wt. %, about 90 wt. %, about 91 wt. %,
about 92 wt. %, about 93 wt. %, about 94 wt. %, about 95 wt. %,
about 96 wt. %, about 97 wt. %, about 98 wt. %, or about 99 wt. %,
based on the total weight of the liposome or the lipid bilayer.
Antigen-Carrying Liposomes
[0379] In some embodiments, the liposomes described herein comprise
at least one antigen, such as, for example, a viral antigen, a
bacterial antigen, a fungal antigen, a tumor antigen, or mixtures
thereof. The exact amount of the antigen with respect to the
liposome depends on the composition and purpose of the antigen, and
can be determined by one skilled in the art. In these embodiments,
the liposomes described herein can be used as immunostimulants or
adjuvants to produce a protective immune response, such as a B-cell
response, an IgG antibody response, a T-cell response, or a CTL
response to the administered antigen.
[0380] The antigen can be any antigenic material that is suitable
for treatment of a particular disease. In exemplary embodiments,
the liposome described herein comprises an antigen and is used to
treat cancer. The antigen can be a full length protein antigen, a
long peptide antigen (i.e, a peptide that comprises at least 25
amino acids, such as 27-75, 25-50, 25-40, or 25-30 amino acids), or
a short peptide antigen (i.e., a peptide that comprises 6-25 amino
acids, such as 6-25, 8-25, 10-25, or 15-20 amino acids). In these
embodiments, the antigen can be a tumor associated peptide or
protein that induces or enhances immune response and is derived
from tumor associated genes and encoded proteins including, for
example, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6,
MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-All, MAGE-A12, MAGE-A13,
GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8,
BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2),
MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (AGE-B4), tyrosinase, brain glycogen
phosphorylase, Melan-A, MAGE-C1, MAGE-C2, NY-ESO-1, LAGE-1, SSX-1,
SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7,
alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin,
cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1
fusion protein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2,
HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-2, and 3, neo-PAP, myosin
class I, OS-9, pml-RAR.alpha. fusion protein, PTPRK, K-ras, N-ras,
Triosephosphate isomeras, GnTV, Herv-K-mel, Lage-1, Mage-C2, NA-88,
/Lage-2, SP17, and TRP2-Int2, (MART-I), gp100 (Pmel 17), TRP-1,
TRP-2, MAGE-1, MAGE-3, p15(58), CEA, NY-ESO (LAGE), SCP-1,
Hom/Mel-40, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET,
IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human
papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5,
MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA
19-9, CA 72-4, CAM 17.1, NuMa, K-ras, .beta.-Catenin, CDK4, Mum-1,
p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F,5T4, 791Tgp72,
.alpha.-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA
27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5,
G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\170K,
NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin
C-associated protein), TAAL6, TAG72, TLP, and TPS. For example,
antigenic peptides characteristic of tumors include those listed in
International Patent Application Publication No. WO 20000/020581
and U.S. Patent Application Publication No. 2010/0284965, which are
each incorporated herein by reference. In some exemplary
embodiments, the antigen is a tumor antigen selected from the group
consisting of MUC1, MAGE, BAGE, RAGE, CAGE, SSX-2, NY-ESO-1, PRAME,
PSMA, tyrosinase, melan-A, and mixtures thereof. In some
embodiments, the tumor antigen is selected from the group
consisting of P1A, MUC1, MAGE-A1, MAGE-A2. MAGE-A3, MAGE-A4,
MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11,
MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5. GAGE-6, GAGE-7,
GAGE-8, BAGE-1, RAGE-1, CAGE, LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2),
MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen
phosphorylase, MAGE-C1/CT7, MAGE-C2, LAGE-1, SSX-1, SSX-2
(HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA,
tyrosinase, melan-A, XAGE and mixtures thereof. In some variations,
the tumor antigen is a mammalian protein. In some variations, the
tumor antigen is a human protein. In some variations, the full
length protein is employed as the antigen. In some variations,
peptides comprising an antigenic fragment of these proteins is used
as the tumor antigen.
[0381] Small, hydrophilic antigens can be encapsulated within the
core of the liposome. Small or large hydrophobic antigens can be
noncovalently associated (e.g., through hydrophobic interactions)
with the nonpolar portion of the lipid bilayer, small or large
charged antigens can be attached (e.g., through electrostatic
interactions) to a charged portion on the outside of the lipid
bilayer, and small or large hydrophobic or hydrophilic antigens
also can be covalently linked to any portion of the liposomal
membrane.
[0382] In embodiments when the liposome described herein is
carrying an antigen, the lipid used to form the liposome is
dictated by the size and charge of the antigen (e.g., small,
hydrophilic, hydrophobic, positively-charged, or negatively-charged
antigens).
[0383] In embodiments when a positively-charged antigen is
noncovalently associated with the liposomal membrane, at least one
lipid of the one to five lipids preferably has a net negative
charge and is selected from the group consisting of an anionic
sphingosine (e.g., a dimethyl sphingosine-1-phosphate, a ceramide
phosphate, a dihydroceramide phosphate, a ganglioside, and a
sulfatide), an anionic phospholipid (e.g., a phosphatidic acid, a
phosphatidylglycerol, a phosphatidylinositol, an inositol
phosphate, a cardiolipin, a bis(monoacylglycero)phosphate, an
anionic detergent that is not a sphingolipid or a phospholipid, and
an anionic bioactive lipid, such as, for example, an adjuvant, a
liponucleotide, a TLR-4 agonist, and a diacylglycerol
pyrophosphate.
[0384] In embodiments when a negatively-charged antigen is
noncovalently associated with the liposomal membrane, at least one
lipid of the one to five lipids preferably has a net positive
charge. In these embodiments, the cation lipid can be cationic
sphingosine (e.g., a trimethyl sphingosine, a trimethyl
phytosphingosine, and a pyridinium ceramide), a cationic lipid that
is not a sphingosine (e.g.,
3.beta.-[N--(N',N'-dimethylaminoethane)-carbamoyl]cholesterol
hydrochloride (DC-Cholesterol.HCl),
1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (18:1 TAP,
DOTAP), 1,2-dioleoyl-3-trimethylammonium-propane (methyl sulfate
salt) (18:1 TAP, DOTAP, MS Salt),
1,2-dimyristoyl-3-trimethylammonium-propane (chloride salt) (14:0
TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (chloride salt)
(16:0 TAP), 1,2-stearoyl-3-trimethylammonium-propane (chloride
salt) (18:0 TAP), Transfection Reagent I (i.e., contains DOTAP:DOPE
in 1:1 w/w ratio), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP)
(18:1 DAP), 1,2-dimyristoyl-3-dimethylammonium-propane (14:0 DAP),
1,2-dipalmitoyl-3-dimethylammonium-propane (16:0 DAP),
1,2-distearoyl-3-dimethylammonium-propane (18:0 DAP),
dimethyldioctadecylammonium (Bromide Salt) (18:0 DDAB),
1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (chloride salt)
(12:0 EPC, Cl Salt),
1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (chloride salt)
(14:0 EPC, Cl Salt),
1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (Tf salt) (14:1
EPC, Tf Salt), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine
(chloride salt) (16:0 EPC, Cl Salt),
1,2-distearoyl-sn-glycero-3-ethylphosphocholine (chloride salt)
(18:0 EPC, Cl Salt), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine
(chloride salt) (18:1 EPC, Cl Salt), and
1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (chloride
salt) (16:0-18:1 EPC, Cl Salt), and a cationic bioactive lipid
(e.g., dimethyldioctadecylammonium (Bromide Salt) (18:0 DDAB), a
lysyl phosphatidylglycerol).
[0385] Exemplary lipids that can be used to produce a liposome
having a bilayer that can non-covalently associate with a
hydrophobic antigen include, but are not limited to,
phosphatodylglycerol (PG), phosphatidylinositol (PI),
phosphatidylserine (PS), cardiolipin and phosphatidic acid
(PA).
Adjuvants
[0386] In some embodiments, the liposomes described herein further
comprise at least one adjuvant, wherein the at least one adjuvant
is in the core of the liposome, in the lipid bilayer, covalently
attached to the lipid bilayer, non-covalently associated with the
lipid bilayer, or combinations thereof. The term "adjuvant" as used
herein refers to a substance that enhances the pharmacological
effect of a drug or increases the immune response to an antigen.
Adjuvants may enhance the immunological response by providing a
reservoir of antigen (extracellularly or within macrophages),
activating macrophages and stimulating specific sets of
lymphocytes. Adjuvants of many kinds are well known in the art.
Exemplary adjuvants known in the art include, but are not limited
to, mineral salts, e.g., aluminium hydroxide and aluminum or
calcium phosphate gels; oil emulsions and surfactant based
formulations, e.g., MF59 (microfluidised detergent stabilized
oil-in-water emulsion), saponins, including but not limited to,
QS21 (purified saponin), QA-21 (a pure saponin purified from
Quillja saponaria extract), DQS21, described in PCT Publication No.
WO 96/33739, QS-7, QS-17, QS-18 and QS-LI (So et al., MO1. Cells,
7:178-186, 1997); AS02 [SBAS2] (oil-in-water emulsion+MPL+QS-21),
Montanide ISA-51 and ISA-720 (stabilised water-in-oil emulsion);
particulate adjuvants, e.g., virosomes (unilamellar liposomal
vehicles incorporating influenza haemagglutinin), ASO.sub.4
([SBAS4] Al salt with MPL), ISCOMS (structured complex of saponins
and lipids), polylactide co-glycolide (PLG); microbial derivatives
(natural and synthetic), e.g., monophosphoryl lipid A (MPL), Detox
(MPL+M. Phlei cell wall skeleton), AGP [RC-529] (synthetic acylated
monosaccharide), DC_Chol (lipoidal immunostimulators able to self
organize into liposomes), OM-174 (lipid A derivative), CpG motifs
(synthetic oligonucleotides containing immunostimulatory CpG
motifs), modified LT and CT (genetically modified bacterial toxins
to provide non-toxic adjuvant effects and endogenous human
immunomodulators, e.g., hGM-CSF or hIL-12 (cytokines that can be
administered either as protein or plasmid encoded), Immudaptin (C3d
tandem array); inert vehicles, such as gold particles; a congener
obtained after purification and acid hydrolysis of Salmonella
ninnesota Re 595 lipopolysaccharide; ISCOMATRIX adjuvant (a
cage-like structure composed of saponin, phosphilipid, and
cholesterol, see e.g., Marakovsky et al., Clin. Cancer Res.,
10:2879-2890, 2004); incomplete Freund's adjuvant; complete
Freund's adjuvant; CpG oligonucleotides (see e.g., Kreig et al.,
Nature 374:546-549, 1995) and other immunostimulatory
oligonucleotides including poly-IC and poly-ICLC (Hiltonol.RTM.);
and various water-in-oil emulsions prepared from biodegradeable
oils such as squalene and/or tocopherol
[0387] In some embodiments, the liposomes described herein further
comprise a TLR agonist, such as, for example, poly I:C (TLR3), MPL
(TLR4), imiquimod (TLR7), R848 (TLR8) or CpG (TLR9) to produce an
enhanced immune stimulation and resulting protection from
conditions in which it is desirable for the immune system to
respond effectively such as infectious disease or cancer.
Lipid Bilayer-Coated Particles
[0388] In another aspect, the invention relates to lipid
bilayer-coated particles comprising a nonglycosidic ceramide.
Poly(D,L-lactide-co-glycolide) (PLGA) encapsulated perfluorocarbon
particles coated with DEAE-dextran, poly-L-lysine, and mouse
anti-human antibodies against DC-SIGN are described in Srinivas et
al., Biomaterials 31:7070-7077 (2010), incorporated herein by
reference. Lipid bilayer-coated particles that comprise a
glycosidic ceramide in the lipid bilayer (e.g., .alpha.-GalCer) are
described in Banal et al., Nature Immunology 11(4):303-314 (2010)
and Barral et al., PNAS 105(24):8345-8350 (2008), each incorporated
herein by reference.
[0389] The lipid bilayer coating is comprised of the at least one
to five lipids previously described herein and a nonglycosidic
ceramide, as previously described herein. The weight percentages
and ratios of the one to five lipids and the nonglycosidic ceramide
are as previously described herein. The particle can be any
particle that can be coated with a lipid bilayer. Nonlimiting
examples of the particle include inorganic particles (e.g., silica
particles) and organic particles (e.g., acrylic polymer bead,
poly-glutamic acid particles, PLGA particles).
[0390] In some embodiments, the particle has a diameter of about 20
nm to about 500 nm, 25 nm to about 300 nm, about 50 nm to about 250
nm, about 60 nm to about 200 nm, about 70 nm to about 150 nm, about
75 to nm to about 125 nm, about 75 nm to about 100 nm, for example,
about 100 nm. In some embodiments, the liposome has a diameter of
about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm,
about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm,
about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm,
about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120
nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about
145 nm, about 150 nm, about 155 nm, about 160 nm, about 165 nm,
about 170 nm, about 175 nm, about 180 nm, about 185 nm, about 190
nm, about 195 nm, about 200 nm, about 205 nm, about 210 nm, about
215 nm, about 220 nm, about 225 nm, about 230 nm, about 235 nm,
about 240 nm, about 245 nm, about 250 nm, about 260 nm, about 265
nm, about 270 nm, about 275 nm, about 280 nm, about 285 nm, about
290 nm, about 295 nm, or about 300 nm.
[0391] Stated another way, in some embodiments the particle has a
diameter defined by a size range, with the lower end of the size
range being any size selected from about 25 nm, 30 nm, 35 nm, 40
nm, 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52 nm, 53 nm,
54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63
nm, 64 nm, 65 nm, 66 nm, 67 nm, 68 nm, 69 nm, 70 nm, 71 nm, 72 nm,
73 nm, 74 nm, 75 nm, 76 nm, 77 nm, 78 nm, 79 nm, 80 nm; and with
the upper end of the size range being any size selected from about
300 nm, 295 nm, 290 nm, 285 nm, 280 nm, 275 nm, 270 nm, 265 nm, 260
nm, 255 nm 250 nm, 245 nm, 240 nm, 235 nm, 230 nm, 225 nm, 220 nm,
215 nm, 210 nm, 205 nm, 200 nm, 195 nm, 190 nm, 185 nm, 180 nm, 175
nm, 170 nm, 165 nm, 160 nm, 155 nm, 150 nm, 145 nm, 144 nm, 143 nm,
142 nm, 141 nm, 140 nm, 139 nm, 138 nm, 137 nm, 136 nm, 135 nm, 134
nm, 133 nm, 132 nm, 131 nm, 130 nm, 129 nm, 128 nm, 127 nm, 126 nm,
125 nm, 124 nm, 123 nm, 122 nm, 121 nm, 120 nm, 119 nm, 118 nm, 117
nm, 116 nm, 115 nm, 114 nm, 113 nm, 112 nm, 111 nm, 110 nm, 109 nm,
108 nm, 107 nm, 106 nm, 105 nm, 104 nm, 103 nm, 102 nm, 101 nm, 100
nm, 99 nm, 98 nm, 97 nm, 96 nm, 95 nm, 94 nm, 93 nm, 92 nm, 91 nm,
or 90 nm.
[0392] The lipid bilayer of the lipid bilayer-coated particles can
further comprise an antigen, adjuvant, or a mixture thereof, as
previously described herein.
Methods of Preparing Liposomes
[0393] The liposomes described herein can be produced using any
method known to one skilled in the art (e.g., solvent dilution
method), as long as the method results in the production of
liposomes having a diameter and having the other desired properties
described herein.
[0394] As described by AVANTI.RTM. Polar Lipids, Inc, (worldwide
web at avantilipids.com), liposomes are formed when thin lipid
films or lipid cakes are hydrated and stacks of liquid crystalline
bilayers become fluid and swell. The hydrated lipid sheets detach
during agitation and self-close to form large, multilamellar
vesicles (LMV) which prevents interaction of water with the
hydrocarbon core of the bilayer at the edges. After particle
formation occurs, their size is reduced through the introduction of
energy, such sonic energy (e.g., through sonication) or mechanical
energy (e.g., through extrusion). Although the properties of lipid
formulations can vary depending on the composition (cationic,
anionic, neutral lipid species), the same preparation method can be
used for all lipid vesicles regardless of composition. The general
elements of the procedure involve preparation of the lipid for
hydration, hydration with agitation, and sizing to a homogeneous
distribution of vesicles.
[0395] Lipids are added to a stock solution of the nonglycosidic
ceramide and they are mixed in an organic solvent to assure a
homogeneous mixture of lipids and to obtain a clear lipid solution.
This process is typically accomplished using chloroform or
chloroform:methanol mixtures. Alternatively, the lipid(s) can be
dissolved in tert-butanol or cyclohexane. The lipid solutions are
typically prepared using a concentration of about 10 mg to about 20
mg of lipid per mL of organic solvent, although higher
concentrations may be used if the lipid solubility and mixing are
acceptable. After the lipids are thoroughly mixed in the organic
solvent, the solvent is removed to yield a lipid film. If the
volume of the organic solvent in the lipid solution is small (<1
mL), the solvent may be evaporated using a dry nitrogen or argon
stream in a fume hood. If the volume of the organic solvent in the
lipid solution is large, the organic solvent can be removed by
rotary evaporation, which yields a thin lipid film on the sides of
a round bottom flask. The lipid film is thoroughly dried to remove
residual organic solvent by placing the vial or flask on a vacuum
pump overnight. The lipid solution is transferred to containers
that can withstand sudden temperature changes without cracking, and
then frozen either by placing the containers on a block of dry ice
or by swirling the container in a dry ice-acetone or alcohol (e.g.,
ethanol or methanol) bath. After freezing completely, the frozen
lipid cake is placed on a vacuum pump and lyophilized until dry
(e.g., about 1 to about 3 days, depending on volume). The thickness
of the lipid cake should be no more than the diameter of the
container being used for lyophilization. Dry lipid films or cakes
can be removed from the vacuum pump, the container tightly closed
and taped, and then stored frozen until ready to hydrate.
[0396] Hydration of the dry lipid film/cake is accomplished by
adding an aqueous medium to the container of dry lipid and then
agitating the container. The temperature of the hydrating medium
should be above the gel-liquid crystal transition temperature (Tc
or Tm) of the lipid with the highest Tc before adding it to the dry
lipid. After addition of the hydrating medium, the lipid suspension
should be maintained above the Tc for the duration of the hydration
period. For high transition lipids, agitation of the suspension,
while maintaining the suspension above the Tc, can occur by
transferring the lipid suspension to a round bottom flask, placing
the flask on a rotary evaporation system without a vacuum, and
spinning the round bottom flask in a warm water bath that is at a
temperature above the Tc of the lipid suspension. Use of a rotary
evaporation apparatus allows the lipid to hydrate in its fluid
phase with adequate agitation. Although, hydration time may
slightly differ among lipid species and structure, a hydration time
of about 1 hour with vigorous shaking, mixing, or stirring is
typically recommended. Optionally, the vesicle suspension is
allowed to stand overnight (i.e., aging) prior to downsizing the
vesicles, which makes the sizing process easier and improves the
homogeneity of the size distribution. Aging is not recommended for
high transition lipids because lipid hydrolysis increases with
elevated temperatures. The hydration medium is generally determined
by the application of the lipid vesicles. Suitable hydration media
include distilled water, buffer solutions, saline, and
nonelectrolytes such as sugar solutions. In embodiments where the
resulting liposomes comprise antigens encapsulated within their
aqueous cores, the hydration media includes one or more antigens.
The concentration of the one or more antigens in the hydration
media depends on the antigens used and can be determined by one
skilled in the art. Methods of encapsulating antigens in liposomes
are known to one skilled in the art and described in, for example,
Gregoriadis, "Liposome Technology: Interactions of Liposomes with
the Biological Mili," 3.sup.rd ed., Informa Healthcare USA, Inc.,
New York, N.Y. (2007), incorporated herein by reference.
[0397] Physiological osmolality (290 mOsm/kg) can be used for in
vivo applications (e.g., 0.9% saline, 5% dextrose, and 10%
sucrose). During hydration some lipids form complexes unique to
their structure. For example, highly charged lipids can form a
viscous gel when hydrated with low ionic strength solutions. This
problem can be alleviated by addition of salt or by downsizing the
lipid suspension. Poorly hydrating lipids, such as
phosphatidylethanolamine, have a tendency to self aggregate upon
hydration. Lipid vesicles containing more than about 60 mol %
phosphatidylethanolamine form particles having a small hydration
layer surrounding the vesicle. The small hydration layer is
insufficient to allow the particles to repel each other when they
are in close proximity, and the two membranes fall into an energy
well where they adhere and form aggregates. The aggregates settle
out of solution as large flocculates. These flocculates will
disperse on agitation but reform upon sitting.
[0398] The product of hydration is a large, multilamellar vesicle
(LMV) analogous in structure to an onion, with each lipid bilayer
separated by a water layer. The spacing between lipid layers is
dictated by composition with poly-hydrating layers being closer
together than highly charged layers, which separate based on
electrostatic repulsion. Once a stable, hydrated LMV suspension has
been produced, the particles can be downsized by a variety of
techniques,
[0399] One method of downsizing LMVs is by using sonic energy, for
example, by sonication. Disruption of LMV suspensions by sonication
typically produces small, unilamellar vesicles (SUV) with diameters
in the range of about 15 to about 50 nm. The most common
instrumentation for the preparation of sonicated particles are bath
and probe tip sonicators, although cup-horn sonicators can be used
as well. Probe tip sonicators can have the disadvantages of causing
overheating of the lipid suspension, which results in degradation.
Sonication tips also can release titanium particles into the lipid
suspension that must be removed by centrifugation prior to use. Use
of bath sonicators avoids these problems. Bath sonication of an LMV
dispersion is accomplished by placing a test tube containing the
suspension in a bath sonicator (or placing the tip of the sonicator
in the test tube) and sonicating for 5-10 minutes above the Tc of
the lipid. The lipid suspension should begin to clarify to yield a
slightly hazy transparent solution. The haze is due to light
scattering induced by residual large particles remaining in the
suspension. These particles can be removed by centrifugation to
yield a clear suspension of SUV. Mean size and distribution of the
SUV is influenced by composition and concentration, temperature,
sonication time and power, volume, and sonicator tuning. Due to the
high degree of curvature of these membranes, SUV are inherently
unstable and will spontaneously fuse to form larger vesicles when
stored below their phase transition temperature.
[0400] Another method of downsizing LMVs is through extrusion.
Lipid extrusion is a technique in which a lipid suspension is
forced through a polycarbonate filter with a defined pore size to
yield particles having a diameter near the pore size of the filter
used. Prior to extrusion through the final pore size, LMV
suspensions are disrupted either by several freeze-thaw cycles or
by prefiltering the suspension through a larger pore size
(typically about 0.2 .mu.m to about 1.0 .mu.m). Disruption of the
LMV suspensions before extruding them through a final pore size
helps prevent the membranes from fouling and improves the
homogeneity of the size distribution of the final suspension. As
with all procedures for downsizing LMV dispersions, the extrusion
should be done at a temperature above the Tc of the lipid. Attempts
to extrude below the Tc are typically unsuccessful because the
membrane has a tendency to foul with rigid membranes, which cannot
pass through the pores. Extrusion through filters with 100 nm pores
typically yields large, unilamellar vesicles (LUV) with a mean
diameter of 120-140 nm. Mean particle size also depends on lipid
composition and is reproducible from batch to batch.
[0401] Section 5.3 of Gad, Pharmaceutical Manufacturing Handbook:
Production and Processes, John Wiley & Sons, Inc., Hoboken,
N.J. (2008), incorporated herein by reference, also provides
examples of liposome preparation methods (e.g., sonication,
homogenization, dehydrated-rehydrated vesicle (DRV), reverse-phase
evaporation (REV), and extrusion). Colas et al., Micron 38:841-847
(2007), incorporated herein by reference, describes liposome
preparation using the Mozafari method. Other methods for producing
liposomes are described in Riaz, Pakisant Journal of Pharmaceutical
Sciences 19(1):65-77 (1996), incorporated herein by reference.
[0402] In some exemplary embodiments, an extruder (e.g., LIPEX.TM.
by Northern Lipids) is used to form homogeneous populations of the
liposomes described herein. In this process, an aqueous suspension
of lipids is forced at about 100 to about 700 psi through a
polycarbonate filter with a defined pore size at a controlled
temperature.
[0403] The size of the liposomes described herein can be determined
using, for example, dynamic light scattering.
Exemplary Liposome Embodiments
[0404] In some embodiments, provided herein is a liposome
comprising (a) a nonglycosidic ceramide present in an amount of
about 1 wt. % to about 20 wt. %, (b) a first lipid present in an
amount of about 15 wt. % to about 55 wt. %, and (c) a second lipid
present in an amount of about 35 wt. % to about 75 wt. %, based on
the total weight of the liposome or the lipid bilayer. In some
embodiments, the liposome has a diameter less than about 100 nm, as
previously described herein. In some embodiments, the liposome has
a diameter of about 50 nm to about 150 nm, as previously described
herein.
[0405] The nonglycosidic ceramide can be any nonglycosidic ceramide
known to one skilled in the art, as previously described herein. In
some embodiments, the nonglycosidic ceramide is a compound of
Formula I or a pharmaceutically acceptable salt thereof, as
previously described herein. In some embodiments, the nonglycosidic
ceramide is selected from the group consisting of
arabinitolceramide, glycerolceramide, threitolceramide,
threitolceramide C.sub.14 acyl, threitol-22-(Z)-ceramide,
4-deoxy-4-phenyl-threitolceramide,
4-deoxy-4-phenyl-threitol-22-(Z)-ceramide,
glycerol-phosphateceramide, inositolceramide, inositolceramide
C.sub.15 acyl, myoinositolceramide salt, 4-phenyl threitolceramide,
4-phenyl threitol-22-(Z)-ceramide, threitol-(19Z,22Z)-ceramide, and
mixtures thereof. For example, the nonglycosidic ceramide can
include arabinitolceramide, glycerolceramide, threitolceramide, and
mixtures thereof (e.g., threitolceramide).
[0406] In some embodiments, the nonglycosidic ceramide is present
in the bilayer of the liposome in an amount of about 1 wt. % to
about 50 wt. %, based on the total weight of the liposome or the
lipid bilayer, as previously described herein. In some embodiments,
the nonglycosidic ceramide is present in an amount of about 2 wt. %
to about 20 wt. %, or about 3 wt. % to about 12 wt. %, based on the
total weight of the liposome or the lipid bilayer. In some
embodiments, the nonglycosidic ceramide is present in an amount of
about 1 wt. % to about 30 wt. %, or about 20 wt. % to about 50 wt.
%, or about 3 wt. % to about 8 wt. %, or about 4 wt. % to about 13
wt. %, based on the total weight of the liposome or the lipid
bilayer. For example, the nonglycosidic ceramide can be present in
an amount of about 1 wt. %, about 2 wt. %, about 3 wt. %, about 4
wt. %, about 5 wt. %, about 6 wt. %, about 7 wt. %, about 8 wt. %,
about 9 wt. %, about 10 wt. %, about 11 wt. %, about 12 wt. %,
about 13 wt. %, about 14 wt. %, about 15 wt. %, about 16 wt. %,
about 17 wt. %, about 18 wt. %, about 19 wt. %, about 20 wt. %,
about 21 wt. %, about 22 wt. %, about 23 wt. %, about 24 wt. %,
about 25 wt. %, about 26 wt. %, about 27 wt. %, about 28 wt. %,
about 29 wt. %, about 30 wt. %, about 31 wt. %, about 32 wt. %,
about 33 wt. %, about 34 wt. %, about 35 wt. %, about 36 wt. %,
about 37 wt. %, about 38 wt. %, about 39 wt. %, about 40 wt. %,
about 41 wt. %, about 42 wt. %, about 43 wt. %, about 44 wt. %,
about 45 wt. %, about 46 wt. %, about 47 wt. %, about 48 wt. %,
about 49 wt. %, or about 50 wt. %, based on the total weight of the
liposome or the lipid bilayer. In some exemplary embodiments, the
nonglycosidic ceramide is present is an amount of about 5 wt. % or
about 10 wt. %, based on the total weight of the liposome or the
lipid bilayer.
[0407] The first lipid and the second lipid each can independently
be a cationic lipid, an anionic lipid, a nonionic lipid, or a
zwitterionic lipid, as previously described herein. In some
embodiments, the first lipid is a zwitterionic lipid. The first
lipid can be any zwitterionic lipid described herein, such as, for
example a zwitterionic phospholipid. In some embodiments, the first
lipid is a zwitterionic phosphatidylcholine. In some exemplary
embodiments, the zwitterionic phosphatidylcholine is selected from
the group consisting of 1,2-didecanoyl-sn-glycero-3-phosphocholine
(DDPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC),
1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC),
1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1-myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine (MPPC),
1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC),
1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC),
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC),
1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC),
1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC),
1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC), egg
phosphatidylcholine (EPC), and mixtures thereof, for example, egg
phosphatidylcholine (EPC).
[0408] The first lipid can be present in an amount of about 5 wt. %
to about 75 wt. %, or about 15 wt. % to about 55 wt. %, for
example, about 20 wt. % to about 30 wt. %, based on the total
weight of the liposome or the lipid bilayer. In some embodiments,
the first lipid is present in an amount of about 5 wt. % to about
25 wt. %, or about 10 wt. % to about 35 wt. %, or about 25 wt. % to
about 45 wt. %, or about 50 wt. % to about 65 wt. %, or about 55
wt. % to about 75 wt. %, based on the total weight of the liposome
or the lipid bilayer. For example, the first lipid can be present
in an amount of about 5 wt. %, about 6 wt. %, about 7 wt. %, about
8 wt. %, about 9 wt. %, about 10 wt. %, about 11 wt. %, about 12
wt. %, about 13 wt. %, about 14 wt. %, about 15 wt. %, about 16 wt.
%, about 17 wt. %, about 18 wt. %, about 19 wt. %, about 20 wt. %,
about 21 wt. %, about 22 wt. %, about 23 wt. %, about 24 wt. %,
about 25 wt. %, about 26 wt. %, about 27 wt. %, about 28 wt. %,
about 29 wt. %, about 30 wt. %, about 31 wt. %, about 32 wt. %,
about 33 wt. %, about 34 wt. %, about 35 wt. %, about 36 wt. %,
about 37 wt. %, about 38 wt. %, about 39 wt. %, about 40 wt. %,
about 41 wt. %, about 42 wt. %, about 43 wt. %, about 44 wt. %,
about 45 wt. %, about 46 wt. %, about 47 wt. %, about 48 wt. %,
about 49 wt. %, about 50 wt. %, about 51 wt. %, about 52 wt. %,
about 53 wt. %, about 54 wt. %, about 55 wt. %, about 56 wt. %,
about 57 wt. %, about 58 wt. %, about 59 wt. %, about 60 wt. %,
about 61 wt. %, about 62 wt. %, about 63 wt. %, about 64 wt. %,
about 65 wt. %, about 66 wt. %, about 67 wt. %, about 68 wt. %,
about 69 wt. %, about 70 wt. %, about 71 wt. %, about 72 wt. %,
about 73 wt. %, about 74 wt. %, or about 75 wt. %, based on the
total weight of the liposome or the lipid bilayer. In some
exemplary embodiments, the first lipid is present in an amount of
about 20 wt. %, about 21 wt. %, about 22 wt. %, about 23 wt. %,
about 24 wt. %, about 25 wt. %, about 26 wt. %, about 27 wt. %,
about 28 wt. %, about 29 wt. %, or about 30 wt. %, based on the
total weight of the liposome or the lipid bilayer.
[0409] In some embodiments, the second lipid is an anionic lipid.
The second lipid can be any anionic lipid described herein, such
as, for example, an anionic phospholipid. In some embodiments, the
second lipid is an anionic phosphatidylglycerol. In some exemplary
embodiments, the second lipid is selected from the group consisting
of 1,2-dierucoyl phosphatidylglycerol (DEPG),
1,2-dilauroylphosphatidylglycerol (DLPG), 1,2-dimyristoyl
phosphatidylglycerol (DMPG), 1,2-dioleoyl phosphatidylglycerol
(DOPG), 1,2-dipalmitoyl phosphatidylglycerol (DPPS), 1,2-distearoyl
phosphatidylglycerol (DSPG), 1-palmitoyl-2-oleoyl
phosphatidylglycerol (POPG), egg phosphatidylglycerol (EPG), salts
of any of the foregoing (e.g., sodium, ammonium, or
sodium/ammonium), and mixtures thereof (e.g., egg
phosphatidylglycerol).
[0410] The second lipid can be present in an amount of about 20 wt.
% to about 90 wt. %, or about 35 wt. % to about 75 wt. %, such as,
for example, about 65 wt. % to about 75 wt. %, based on the total
weight of the liposome or the lipid bilayer. In some embodiments,
the second lipid is present in an amount of about 25 wt. % to about
45 wt. %, or about 30 wt. % to about 65 wt. %, or about 45 wt. % to
about 75 wt. %, or about 50 wt. % to about 85 wt. %, based on the
total weight of the lipsome or the lipid bilayer. For example, the
second lipid can be present in an amount of about 20 wt. %, about
21 wt. %, about 22 wt. %, about 23 wt. %, about 24 wt. %, about 25
wt. %, about 26 wt. %, about 27 wt. %, about 28 wt. %, about 29 wt.
%, about 30 wt. %, about 31 wt. %, about 32 wt. %, about 33 wt. %,
about 34 wt. %, about 35 wt. %, about 36 wt. %, about 37 wt. %,
about 38 wt. %, about 39 wt. %, about 40 wt. %, about 41 wt. %,
about 42 wt. %, about 43 wt. %, about 44 wt. %, about 45 wt. %,
about 46 wt. %, about 47 wt. %, about 48 wt. %, about 49 wt. %,
about 50 wt. %, about 51 wt. %, about 52 wt. %, about 53 wt. %,
about 54 wt. %, about 55 wt. %, about 56 wt. %, about 57 wt. %,
about 58 wt. %, about 59 wt. %, about 60 wt. %, about 61 wt. %,
about 62 wt. %, about 63 wt. %, about 64 wt. %, about 65 wt. %,
about 66 wt. %, about 67 wt. %, about 68 wt. %, about 69 wt. %,
about 70 wt. %, about 71 wt. %, about 72 wt. %, about 73 wt. %,
about 74 wt. %, about 75 wt. %, about 76 wt. %, about 77 wt. %,
about 78 wt. %, about 79 wt. %, about 80 wt. %, about 81 wt. %,
about 82 wt. %, about 83 wt. %, about 84 wt. %, about 85 wt. %,
about 86 wt. %, about 87 wt. %, about 88 wt. %, about 89 wt. %, or
about 90 wt. %, based on the total weight of the liposome or the
lipid bilayer.
[0411] In some embodiments, the liposome comprises at least three,
at least four, or five lipids, as previously described herein.
[0412] It has surprisingly been found that liposomes containing egg
phosphatidylcholine (EPC) and egg phosphatidylglycerol (EPG) in a
weight ratio of about 0.4 to about 3.5, for example, about 0.5:2.0,
or about 1:2.5, or about 0.5:4, or about 1:3, and a nonglycosidic
ceramide, e.g., threitolceramide, in a weight ratio to total lipid
of about 1:99 to about 30:70, for example about 2:98, 3:97, 4:96,
5:95, 6:94, 7:93, 8:92, 9:91, 10:90, 11:89, 12:88, 13:87, 14:86.
15:85, 16:84, 17:83, 18:82, 19:81, 20:80, 21:79, 22:78, 23:77,
24:76, 25:75, 26:74, 27:73, 28:72, 29:71, or 30:70 provide superior
results with respect to sensitization of murine and human iNKT
cells and potent anti-tumor responses in animal models. In some
exemplary embodiments, the liposomes described herein comprise an
EPC:EPG weight ratio of about 1:3 and a weight ratio of
nonglycosidic ceramide to total lipid of about 5:95 or about
10:90.
[0413] The EPC:EPG:threitolceramide (TC) liposomes described herein
provide superior ability to activate murine invariant natural
killer cells (iNKT) and human cells, as measured by IFN.gamma.
release, when compared to liposomes comprising either
dimethyldioctadecyl ammonium bromide (DDAP) or cholesterol (CHOL)
in place of EPG. For example, EPC:EPG:TC and EPC:DDAB:TC liposomal
formulations were both able to activate iNKT cells to the same
extent as soluble threitolceramide, while a EPC:CHOL:TC liposomal
formulation was not (FIG. 1A). The EPC:DDAB:TC formulations had the
disadvantage of appearing to aggregate on cultured cells.
[0414] The liposomes described herein also provide superior
dendritic cell (DC) maturation, as measured by CD86 and CD40
release, and iNKT cell activation, as measured by IL-4 and
IFN.gamma. release when administered in vivo. Although all of the
tested threitolceramide-containing liposomal formulations were able
to induce DC maturation to the same extent as soluble
threitolceramide, the EPC:DDAB:TC liposomal formulation also
induced DC maturation in CD1d-/- mice (FIG. 2).
[0415] The liposomes described herein were also able to provide
superior dendritic cell maturation when administered in vitro (see
FIGS. 5-9). Results provided in FIG. 4 indicate that the EPC:EPG:TC
formulation, particularly at 10% threitolceramide, was superior to
all tested formulations (and easier to handle than EPC:DDAB:TC,
data not shown).
[0416] When the liposomes described herein were tested for their
effect on iNKT T-Cell Receptor (TCR) priming in vitro, the
EPC:EPG:TC liposomal formulation was found to bind with the
greatest efficiency to the iTCR, and better than soluble
threitolceramide. In contrast, the EPC:EPG:CHOL and EPC:EPG:DDAB
liposomal formulations bound weakly to the iTCR in comparison to
the EPC:EP:TC formulation and soluble threitolceramide (FIG.
10).
[0417] In some embodiments, the liposome in this embodiment further
comprises a positively-charged antigen, as previously described
herein. The antigen can be a full length protein antigen, a long
peptide antigen, or a short peptide antigen, as previously
described herein. In exemplary embodiments, the liposome comprises
a positively-charged antigen and is used to treat cancer. In these
embodiments, the positively-charged antigen can be a
positively-charged tumor associated peptide or protein that induces
or enhances an immune response and is derived from tumor associated
genes and encoded proteins. In some embodiments, the tumor antigen
is selected from the group consisting of MUC1, MAGE, BAGE, RAGE,
CAGE, SSX-2, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, and
mixtures thereof. In some embodiments, the tumor antigen is
selected from the group consisting of P1A, MUC1, MAGE-A1, MAGE-A2,
MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,
MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4,
GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE, LB33/MUM-1,
NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4),
brain glycogen phosphorylase, MAGE-C1/CT7, MAGE-C2, LAGE-1, SSX-1,
SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME,
PSMA, tyrosinase, melan-A, XAGE, antigenic fragments thereof, and
mixtures thereof.
[0418] In some exemplary embodiments, the positively-charged
antigen is NY-ESO-1. The NY-ESO-1 can be the full length protein, a
long peptide, or a short peptide. In some embodiments, NY-ESO-1 is
SLLMWITQC (SEQ ID NO: 2), or a variant thereof, as disclosed in
U.S. Patent Application Publication No. 2006/0094661, incorporated
herein by reference. A variant of NY-ESO-1 can include, for
example, a peptide comprising 1 or more amino acid substitutions of
SEQ ID NO: 2. One, two, or three substitutions are specific
embodiments contemplated. The substituted NY-ESO-1 preferably still
generates an immune response to tumors that express the relevant
naturally occurring tumor antigen. Amino acid substitutions in
variant NY-ESO-1 may be "conservative" or "non-conservative". As
used herein, "conservative" amino acid substitutions are
substitutions wherein the substituted amino acid has similar
structural or chemical properties, and "non-conservative" amino
acid substitutions are those in which the charge, hydrophobicity,
or bulk of the substituted amino acid is significantly altered.
[0419] Examples of conservative amino acid substitutions include
those in which the substitution is within one of the five following
groups: 1) small aliphatic, nonpolar or slightly polar residues
(Ala, Ser, Thr, Pro, Gly); 2) polar, negatively charged residues
and their amides (Asp, Asn, Glu, Gln); polar, positively charged
residues (H is, Arg, Lys); large aliphatic, nonpolar residues (Met,
Leu, Be, Val, Cys); and large aromatic resides (Phe, Tyr, Trp).
Examples of non-conservative amino acid substitutions are those
where 1) a hydrophilic residue, e.g., seryl or threonyl, is
substituted for (or by) a hydrophobic residue, e.g., leucyl,
isoleucyl, phenylalanyl, valyl, or alanyl; 2) a cysteine or proline
is substituted for (or by) any other residue; 3) a residue having
an electropositive side chain, e.g., lysyl, arginyl, or histidyl,
is substituted for (or by) an electronegative residue, e.g.,
glutamyl or aspartyl; or 4) a residue having a bulky side chain,
e.g., phenylalanine, is substituted for (or by) a residue that does
not have a side chain, e.g., glycine.
[0420] The substitution can include a standard amino acid
substitution, a non-standard amino acid substitution, or both. A
non-standard amino acid substitution can include, for example, Tyr,
Val, Leu, Ala, Be, Met, Nle, Nva, Trp, Phe, Asp, Asn, Ser, Abu, and
D-stereoisomer of a standard amino acid. In some embodiments, the
substitution can include a modified terminal amino acid. For
example, the terminal amino acid can be an amidated C-terminal
amino acid or the addition of an amino acid to the C-terminus of
the peptide. Examples of variants of NY-ESO-1 can be found in U.S.
Patent Application Publication No. 2006/0094661, the disclosure
which is incorporated by reference.
[0421] In these embodiments, the NY-ESO-1 can be present in an
amount of about 1 .mu.g to about 1 mg, e.g., about 1 .mu.g, about
10 .mu.g, about 20 .mu.g, about 30 .mu.g, about 40 .mu.g, about 50
.mu.g, about 60 .mu.g, about 70 .mu.g, about 80 .mu.g, about 90
.mu.g, about 100 .mu.g, about 110 .mu.g, about 120 .mu.g, about 130
.mu.g, about 140 .mu.g, about 150 .mu.g, about 160 .mu.g, about 170
.mu.g, about 180 .mu.g, about 190 .mu.g, about 200 .mu.g, about 210
.mu.g, about 220 .mu.g, about 230 .mu.g, about 240 .mu.g, about 250
.mu.g, about 260 .mu.g, about 270 .mu.g, about 280 .mu.g, about 290
.mu.g, about 300 .mu.g, about 310 .mu.g, about 320 .mu.g, about 330
.mu.g, about 340 .mu.g, about 350 .mu.g, about 360 .mu.g, about 370
.mu.g, about 380 .mu.g, about 390 .mu.g, about 400 .mu.g, about 410
.mu.g, about 420 .mu.g, about 430 .mu.g, about 440 .mu.g, about 450
.mu.g, about 460 .mu.g, about 470 .mu.g, about 480 .mu.g, about 490
.mu.g, about 500 .mu.g, about 510 .mu.g, about 520 .mu.g, about 530
.mu.g, about 540 .mu.g, about 550 .mu.g, about 560 .mu.g, about 570
.mu.g, about 580 .mu.g, about 590 .mu.g, about 600 .mu.g, about 610
.mu.g, about 620 .mu.g, about 630 .mu.g, about 640 .mu.g, about 650
.mu.g, about 660 .mu.g, about 670 .mu.g, about 680 .mu.g, about 690
.mu.g, about 700 .mu.g, about 710 .mu.g, about 720 .mu.g, about 730
.mu.g, about 740 .mu.g, about 750 .mu.g, about 760 .mu.g, about 770
.mu.g, about 780 .mu.g, about 790 .mu.g, about 800 .mu.g, about 810
.mu.g, about 480 .mu.g, about 830 .mu.g, about 840 .mu.g, about 850
.mu.g, about 860 .mu.g, about 870 .mu.g, about 880 .mu.g, about 890
.mu.g, about 900 .mu.g, about 910 .mu.g, about 920 .mu.g, about 930
.mu.g, about 940 .mu.g, about 590 .mu.g, about 960 .mu.g, about 970
.mu.g, about 980 .mu.g, about 990 .mu.g, or about 1000 .mu.g per 2
mg of the liposome. In some embodiments, the NY-ESO-1 is present in
an amount of about 1 .mu.g to about 200 .mu.g, or about 300 .mu.g
to about 600 .mu.g, or about 700 .mu.g to about 800 .mu.g, or about
100 .mu.g to about 500 .mu.g, or about 500 .mu.g to about 800
.mu.g, about 10 .mu.g to about 100 .mu.g per 2 mg of liposome. In
some exemplary embodiments, the NY-ESO-1 is present in an amount of
about 400 .mu.g per 2 mg of liposome.
Pharmaceutical Compositions and Routes of Administration
[0422] In some embodiments, described herein is a composition
comprising a liposome described herein and pharmaceutically
acceptable diluent, carrier, excipient, or mixtures thereof.
Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W.
Martin (Mack Publishing Co., Easton, Pa., 1980), which is
incorporated by reference in its entirety, discloses various
components used in formulating pharmaceutically acceptable
compositions and known techniques for the preparation thereof.
Except insofar as any conventional agent is incompatible with the
pharmaceutical compositions, its use in pharmaceutical compositions
is contemplated. Supplementary active ingredients also can be
incorporated into the compositions.
[0423] In some embodiments, the composition is sterile and has a
purity level of, for example, at least about 90%, at least about
91%, at least about 92%, at least about 93%, at least about 94%, at
least about 95%, at least about 96%, at least about 97%, at least
about 98% or at least about 99%.
[0424] In some embodiments, the composition comprises a liposome
described herein present in the composition in a concentration of
about 0.5 mg/mL to about 30 mg/mL (e.g., about 1 mg/mL to about 20
mg/mL (e.g., about 1 mg/mL, about 2 mg/mL, about 3 mg/mL, about 4
mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL,
about 8 mg/mL, about 10 mg/mL, about 11 mg/mL, about 12 mg/mL,
about 13 mg/mL, about 14 mg/mL about 15 mg/mL, about 16 mg/mL,
about 17 mg/mL, about 18 mg/mL, about 19 mg/mL, about 20 mg/mL,
about 21 mg/mL, about 22 mg/mL, about 23 mg/mL, about 24 mg/mL
about 25 mg/mL, about 26 mg/mL, about 27 mg/mL, about 28 mg/mL,
about 29 mg/mL, or about 30 mg/mL) In some exemplary embodiments,
the concentration of the liposome is about 13 mg/mL. In some
embodiments, the liposome is present in the composition in an
amount of about 1 mg/mL to about 10 mg/mL, or about 5 mg/mL to
about 15 mg/mL, or about 10 mg/mL to about 20 mg/mL, or about 15
mg/mL to about 30 mg/mL.
[0425] In some embodiments, the composition comprises a liposome
described herein wherein the concentration of the nonglycosidic
ceramide in the liposome is about 0.05 mg/mL to about 1 mg/mL
(e.g., about 0.1 mg/mL to about 0.9 mg/mL, or about 0.2 mg/mL to
about 0.8 mg/mL). In some embodiments, the concentration of the
nonglycosidic ceramide in the liposome is about 0.05 mg/mL, about
0.10 mg/mL, about 0.15 mg/mL, about 0.20 mg/mL, about 0.25 mg/mL,
about 0.30 mg/mL, about 0.35 mg/mL, about 0.40 mg/mL, about 0.45
mg/mL, about 0.50 mg/mL, about 0.55 mg/mL, about 0.60 mg/mL, about
0.65 mg/mL, about 0.70 mg/mL, about 0.75 mg/mL, about 0.80 mg/mL
about 0.85 mg/mL, about 0.90 mg/mL, about 0.95 mg/mL, or about 1
mg/mL
[0426] In some embodiments, the composition comprising a liposome
described herein further comprises an antigen (e.g., a viral
antigen, a bacterial antigen, a tumor antigen, and mixtures
thereof) present in an admixture with the liposome. The antigen can
be a tumor antigen, as previously described herein. In some
embodiments, the antigen is selected from the group consisting of
P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAAGE-A6.
MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1,
GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1,
RAGE-1, CAGE, LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3
(MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase,
MAGE-C1/CT7, MAGE-C2, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3,
SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A,
XAGE, antigenic fragments thereof, and mixtures thereof., as
previously described herein. In some exemplary embodiments, the
antigen is NY-ESO-1, which can be present in a concentration of
about 0.01 mg/mL to about 5 mg/mL, or about 0.1 to about 1 mg/mL,
for example about 0.3 mg/mL to about 0.6 mg/mL (e.g., about 0.01
mg/mL to about 1 mg/mL, or about 1 mg/mL to about 3 mg/mL, or about
0.1 mg/mL to about 2 mg/mL, or about 2 mg/mL to about 5 mg/mL. For
example, NY-ESO-1 can be present in the composition in an admixture
with the liposome in an amount of about 0.01 mg/mL, about 0.05
mg/mL, about 0.1 mg/mL, about 0.15 mg/mL, about 0.2 mg/mL, about
0.25 mg/mL, about 0.3 mg/mL, about 0.35 mg/mL, about 0.4 mg/mL
about 0.45 mg/mL, about 0.5 mg/mL, about 0.55 mg/mL, about 0.6
mg/mL, about 0.65 mg/mL, about 0.7 mg/mL, about 0.75 mg/mL, about
0.8 mg/mL, about 0.85 mg/mL, about 0.9 mg/mL about 0.95 mg/mL,
about 1 mg/mL, about 1.5 mg/mL about 2 mg/mL, about 2.5 mg/mL about
3 mg/mL, about 3.5 mg/mL, about 4 mg/mL, about 4.5 mg/mL, or about
5 mg/mL.
[0427] In some embodiments, the composition comprising the liposome
described herein further comprises a therapeutic agent present in
an admixture with the liposome or within the aqueous core of the
liposome. In some embodiments, the therapeutic agent is selected
from the group consisting of an immune modulator (e.g., an
anti-CD40 antibody, an anti-CD40L antibody, an anti-CTLA4 blocking
antibody, or soluble LAG-3 based immune modulators), a Toll-like
receptor agonist (e.g., MPL, CpG, single-stranded RNA, nucleotides,
nucleotide analogs like CL087, or loxoribine,
polyinosine-polycytidylic acid (poly I:C), flagellin, resiquimod,
imiquimod, and gardiquimod), a Nod ligand (e.g., muramyl dipeptide,
murabutide, peptidoglycan), an anti-viral agent (e.g., oseltamivir
phosphate), an antifungal agent (e.g., amphotericin B), an
antibiotic, an antiviral antibody (e.g., palivizumab), a cancer
immune therapeutic (e.g., herceptin, alemtuzumab, gemtuzumab,
rituximab, ibritumomab tiuxetan, and other monoclonal antibody
based cancer treatments), a chemotherapy agent, a kinase inhibitor
(e.g., imatinib and erlotinib), a cytotoxic agent (e.g.,
cyclophosphamide), an anti-asthmatic agent, an antihistamine agent,
an anti-inflammatory agent, a vaccine adjuvant (e.g., virus-like
particles), a second liposome, an artificial antigen presenting
cell, a cytokine or chemokine blocking antibody (e.g., infliximab,
adalimumab, basiliximab), and mixtures thereof.
[0428] In some embodiments, the composition comprising the liposome
further comprises at least one adjuvant present in an admixture
with the liposome.
[0429] The composition comprising the liposomes described herein
can be formulated for a standard route of administration, including
parenteral, such as intravenous, intraperitoneal, subcutaneous or
intramuscular, intrathecal, transdermal, rectal, oral, nasal or by
inhalation. Parenteral injection or extended infusion, e.g. over a
period of 1, 2, 3, 4, 5, 6, 7, 8, 12 or 24 hours is possible.
[0430] Compositions described herein can be formulated for
administration in a form selected from the group consisting of a
tablet, a capsule, a powder, a suppository, a lozenge, a soft
gelatin capsule, a transdermal patch, an aerosol (e.g., pressurized
or non-pressurized powder), a dragee, a cream (e.g., an
oil-in-water emulsion or a water-in-oil emulsion), a drop, a liquid
suspension, an emulsion, and an ointment.
[0431] The dose of the liposomes that is administered will vary
with the exact composition of the liposome. In general the
liposomes are administered to result in a daily dosage of the
nonglycosidic ceramide of about 0.5 .mu.g to about 30 mg, or about
1 .mu.g to about 20 mg per kg of animal body weight, e.g., about
0.5 .mu.g, about 1 .mu.g, about 10 .mu.g, about 20 .mu.g, about 30
.mu.g, about 40 .mu.g, about 50 .mu.g, about 60 .mu.g, about 70
.mu.g, about 80 .mu.g, about 90 .mu.g, about 100 .mu.g, about 110
.mu.g, about 120 .mu.g, about 130 .mu.g, about 140 .mu.g, about 150
.mu.g, about 160 .mu.g, about 170 .mu.g, about 180 .mu.g, about 190
.mu.g, about 200 .mu.g, about 210 .mu.g, about 220 .mu.g, about 230
.mu.g, about 240 .mu.g, about 250 .mu.g, about 260 .mu.g, about 270
.mu.g, about 280 .mu.g, about 290 .mu.g, about 300 .mu.g, about 310
.mu.g, about 320 .mu.g, about 330 .mu.g, about 340 .mu.g, about 350
.mu.g, about 360 .mu.g, about 370 .mu.g, about 380 .mu.g, about 390
.mu.g, about 400 .mu.g, about 410 .mu.g, about 420 .mu.g, about 430
.mu.g, about 440 .mu.g, about 450 .mu.g, about 460 .mu.g, about 470
.mu.g, about 480 .mu.g, about 490 .mu.g, about 500 .mu.g, about 510
.mu.g, about 520 .mu.g, about 530 .mu.g, about 540 .mu.g, about 550
.mu.g, about 560 .mu.g, about 570 .mu.g, about 580 .mu.g, about 590
.mu.g, about 600 .mu.g, about 610 .mu.g, about 620 .mu.g, about 630
.mu.g, about 640 .mu.g, about 650 .mu.g, about 660 .mu.g, about 670
.mu.g, about 680 .mu.g, about 690 .mu.g, about 700 .mu.g, about 710
.mu.g, about 720 .mu.g, about 730 .mu.g, about 740 .mu.g, about 750
.mu.g, about 760 .mu.g, about 770 .mu.g, about 780 .mu.g, about 790
.mu.g, about 800 .mu.g, about 810 .mu.g, about 480 .mu.g, about 830
.mu.g, about 840 .mu.g, about 850 .mu.g, about 860 .mu.g, about 870
.mu.g, about 880 .mu.g, about 890 .mu.g, about 900 .mu.g, about 910
.mu.g, about 920 .mu.g, about 930 .mu.g, about 940 .mu.g, about 590
.mu.g, about 960 .mu.g, about 970 .mu.g, about 980 .mu.g, about 990
.mu.g, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg,
about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about
11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16
mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21
mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26
mg, about 27 mg, about 28 mg, about 29 mg, or about 30 mg. In some
embodiments, the liposomes are administered to result in a daily
dosage of the nonglycosidic ceramide in an amount of about 1 .mu.g
to about 200 .mu.g, or about 1 mg to about 10 mg, or about 700
.mu.g to about 2 mg, or about 800 .mu.g to about 28 mg, or about 10
.mu.g to about 30 mg, about 10 .mu.g to about 5 mg. In some
embodiments, the liposomes are administered in divided doses 1 to 4
times daily or in a sustained release form.
[0432] In some embodiments, the composition described herein
comprises a lipid-bilayer coated particle, as previously described
herein, in place of, or in addition to, a liposome.
Therapeutic and Prophylactic Uses
[0433] In yet another aspect, described herein is a method of
treating a viral or microbial infection, a parasitic infection, an
autoimmune disease, an allergy, or asthma in a mammalian subject in
need thereof comprising administering to the subject the liposome
described herein or the composition described herein in an amount
effective to treat said infection autoimmune disease, allergy, or
asthma. In a preferred embodiment, the mammalian subject is a human
subject. Practice of methods described herein in other mammalian
subjects, especially mammals that are conventionally used as models
for demonstrating therapeutic efficacy in humans (e.g., primate,
porcine, canine, or rabbit animals), is also contemplated. Routes
of administration are described in the preceding section. Repeated
administration is contemplated to maintain a sustained response for
long term management of the disease or condition, or until a cure
is achieved. Standard dose-response studies are used to optimize
dose and dosing schedule.
[0434] In some embodiments, the liposomes described herein or
compositions described herein can be used to treat a disorder
caused by a virus. Exemplary viruses include, but are not limited
to, a hepatitis virus, a liver tropic virus, a skin tropic virus, a
lung tropic virus, an immune tropic virus, and combinations
thereof. For example, the liposomes described herein or composition
described herein can be used to treat disorders associated with a
virus selected from the group consisting of hepatitis B virus
(HBV), hepatitis C virus (HBC), human papilloma virus (HPV), herpes
simplex virus (HSV), influenza virus, respiratory syncytial virus
(RSV), human immunodeficiency virus (HIV), Epstein-Barr virus
(EBV), cytomegalovirus (CMV), and combinations thereof.
[0435] In some embodiments, the liposomes described herein or
compositions described herein are used to treat a microbial
infection, such as a bacterial infection of the lung with e.g.,
Haeemophilius influenza or mycobacteria, e.g., Mycobacterium
tuberculosis, a bacterial infection of the gut with e.g.,
Helicobacter pylori, a bacterial infection of the skin with e.g.,
Staphylococcus aureus, and combinations thereof.
[0436] In some embodiments, the liposomes described herein or
compositions described herein can be used to treat an autoimmune
disease. Autoimmune disorders which may be treated using a liposome
disclosed herein include, but are not limited to, psoriasis,
Crohn's disease, connective tissue disease, multiple sclerosis,
systemic lupus erythematosus, rheumatoid arthritis, autoimmune
pulmonary inflammation, Guillain Barre syndrome, autoimmune
thyroiditis, insulin dependent diabetes mellitis, myasthenia
gravis, graft versus host disease and autoimmune inflammatory eye
disease.
[0437] In one aspect, the liposomes described herein or
compositions described herein are used to treat cancer. In such
embodiments, the liposome described herein or composition described
herein is administered in a mammalian subject in need thereof, in
amount effective to treat cancer. The term "cancer" generally
refers to tumors, including both primary and metastasized tumors.
In some embodiments, the tumor is a solid tumor.
[0438] The disclosed methods are useful for, for example,
inhibiting cancer growth, including complete cancer remission, for
inhibiting cancer metastasis, and for promoting cancer resistance.
The term "cancer growth" generally refers to any one of a number of
indices that suggest change within the cancer to a more developed
form. Thus, indices for measuring an inhibition of cancer growth
include but are not limited to a decrease in cancer cell survival,
a decrease in tumor volume or morphology (for example, as
determined using computed tomographic (CT), sonography, or other
imaging method), a delayed tumor growth, a destruction of tumor
vasculature, improved performance in delayed hypersensitivity skin
test, an increase in the activity of cytolytic T-lymphocytes, and a
decrease in levels of tumor-specific antigens.
[0439] The term "cancer resistance" refers to an improved capacity
of a subject to resist cancer growth, in particular growth of a
cancer already had. In other words, the term "cancer resistance"
refers to a decreased propensity for cancer growth in a
subject.
[0440] In one aspect, the cancer comprises a solid tumor, for
example, a carcinoma and a sarcoma. Carcinomas include malignant
neoplasms derived from epithelial cells which infiltrate, for
example, invade, surrounding tissues and give rise to metastases.
Adenocarcinomas are carcinomas derived from glandular tissue, or
from tissues that form recognizable glandular structures. Another
broad category of cancers includes sarcomas and fibrosarcomas,
which are tumors whose cells are embedded in a fibrillar or
homogeneous substance, such as embryonic connective tissue. The
invention also provides methods of treatment of cancers of myeloid
or lymphoid systems, including leukemias, lymphomas, and other
cancers that typically are not present as a tumor mass, but are
distributed in the vascular or lymphoreticular systems. Further
contemplated are methods for treatment of adult and pediatric
oncology, growth of solid tumors/malignancies, myxoid and round
cell carcinoma, locally advanced tumors, cancer metastases,
including lymphatic metastases. The cancers listed herein are not
intended to be limiting. Age (child and adult), sex (male and
female), primary and secondary, pre- and post-metastatic, acute and
chronic, benign and malignant, anatomical location cancer
embodiments and variations are contemplated targets. Cancers are
grouped by embryonic origin (e.g., carcinoma, lymphomas, and
sarcomas), by organ or physiological system, and by miscellaneous
grouping. Particular cancers may overlap in their classification,
and their listing in one group does not exclude them from
another.
[0441] Carcinomas that may targeted include adrenocortical, acinar,
acinic cell, acinous, adenocystic, adenoid cystic, adenoid squamous
cell, cancer adenomatosum, adenosquamous, adnexel, cancer of
adrenal cortex, adrenocortical, aldosterone-producing,
aldosterone-secreting, alveolar, alveolar cell, ameloblastic,
ampullary, anaplastic cancer of thyroid gland, apocrine, basal
cell, basal cell, alveolar, comedo basal cell, cystic basal cell,
morphea-like basal cell, multicentric basal cell, nodulo-ulcerative
basal cell, pigmented basal cell, sclerosing basal cell,
superficial basal cell, basaloid, basosquamous cell, bile duct,
extrahepatic bile duct, intrahepatic bile duct, bronchioalveolar,
bronchiolar, bronchioloalveolar, bronchoalveolar, bronchoalveolar
cell, bronchogenic, cerebriform, cholangiocelluarl, chorionic,
choroids plexus, clear cell, cloacogenic anal, colloid, comedo,
corpus, cancer of corpus uteri, cortisol-producing, cribriform,
cylindrical, cylindrical cell, duct, ductal, ductal cancer of the
prostate, ductal cancer in situ (DCIS), eccrine, embryonal, cancer
en cuirasse, endometrial, cancer of endometrium, endometroid,
epidermoid, cancer ex mixed tumor, cancer ex pleomorphic adenoma,
exophytic, fibrolamellar, cancer fibro'sum, follicular cancer of
thyroid gland, gastric, gelatinform, gelatinous, giant cell, giant
cell cancer of thyroid gland, cancer gigantocellulare, glandular,
granulose cell, hepatocellular, Hurthle cell, hypernephroid,
infantile embryonal, islet cell carcinoma, inflammatory cancer of
the breast, cancer in situ, intraductal, intraepidermal,
intraepithelial, juvenile embryonal, Kulchitsky-cell, large cell,
leptomeningeal, lobular, infiltrating lobular, invasive lobular,
lobular cancer in situ (LCIS), lymphoepithelial, cancer medullare,
medullary, medullary cancer of thyroid gland, medullary thyroid,
melanotic, meningeal, Merkel cell, metatypical cell,
micropapillary, mucinous, cancer muciparum, cancer mucocellulare,
mucoepidermoid, cancer mucosum, mucous, nasopharyngeal,
neuroendocrine cancer of the skin, noninfiltrating, non-small cell,
non-small cell lung cancer (NSCLC), oat cell, cancer ossificans,
osteoid, Paget's, papillary, papillary cancer of thyroid gland,
periampullary, preinvasive, prickle cell, primary intrasseous,
renal cell, scar, schistosomal bladder, Schneiderian, scirrhous,
sebaceous, signet-ring cell, cancer simplex, small cell, small cell
lung cancer (SCLC), spindle cell, cancer spongiosum, squamous,
squamous cell, terminal duct, anaplastic thyroid, follicular
thyroid, medullary thyroid, papillary thyroid, trabecular cancer of
the skin, transitional cell, tubular, undifferentiated cancer of
thyroid gland, uterine corpus, verrucous, villous, cancer villosum,
yolk sac, squamous cell particularly of the head and neck,
esophageal squamous cell, and oral cancers and carcinomas.
[0442] Sarcomas that may be targeted include adipose, alveolar soft
part, ameloblastic, avian, botryoid, sarcoma botryoides, chicken,
chloromatous, chondroblastic, clear cell sarcoma of kidney,
embryonal, endometrial stromal, epithelioid, Ewing's, fascial,
fibroblastic, fowl, giant cell, granulocytic, hemangioendothelial,
Hodgkin's, idiopathic multiple pigmented hemorrhagic, immunoblastic
sarcoma of B cells, immunoblastic sarcoma of T cells, Jensen's,
Kaposi's, kupffer cell, leukocytic, lymphatic, melanotic, mixed
cell, multiple, lymphangio, idiopathic hemorrhagic, multipotential
primary sarcoma of bone, osteoblastic, osteogenic, parosteal,
polymorphous, pseudo-kaposi, reticulum cell, reticulum cell sarcoma
of the brain, rhabdomyosarcoma, rous, soft tissue, spindle cell,
synovial, telangiectatic, sarcoma (osteosarcoma)/malignant fibrous
histiocytoma of bone, and soft tissue sarcomas.
[0443] Lymphomas that may targeted include AIDS-related,
non-Hodgkin's, Hodgkin's, T-cell, T-cell leukemia/lymphoma,
African, B-cell, B-cell monocytoid, bovine malignant, Burkitt's,
centrocytic, lymphoma cutis, diffuse, diffuse, large cell, diffuse,
mixed small and large cell, diffuse, small cleaved cell,
follicular, follicular center cell, follicular, mixed small cleaved
and large cell, follicular, predominantly large cell, follicular,
predominantly small cleaved cell, giant follicle, giant follicular,
granulomatous, histiocytic, large cell, immunoblastic, large
cleaved cell, large nocleaved cell, Lennert's, lymphoblastic,
lymphocytic, intermediate; lymphocytic, intermediately
differentiated, plasmacytoid; poorly differentiated lymphocytic,
small lymphocytic, well differentiated lymphocytic, lymphoma of
cattle; MALT, mantle cell, mantle zone, marginal zone,
Mediterranean lymphoma mixed lymphocytic-histiocytic, nodular,
plasmacytoid, pleomorphic, primary central nervous system, primary
effusion, small b-cell, small cleaved cell, small concleaved cell,
T-cell lymphomas; convoluted T-cell, cutaneous t-cell, small
lymphocytic T-cell, undefined lymphoma, u-cell, undifferentiated,
aids-related, central nervous system, cutaneous T-cell, effusion
(body cavity based), thymic lymphoma, and cutaneous T cell
lymphomas.
[0444] Leukemias and other blood cell malignancies that may be
targeted include acute lymphoblastic, acute myeloid, acute
lymphocytic, acute myelogenous leukemia, chronic myelogenous, hairy
cell, erythroleukemia, lymphoblastic, myeloid, lymphocytic,
myelogenous, leukemia, hairy cell, T-cell, monocytic, myeloblastic,
granulocytic, gross, hand minor-cell, basophilic, hemoblastic,
histiocytic, leukopenic, lymphatic, Schilling's, stem cell,
myelomonocytic, monocytic, prolymphocytic, promyelocytic,
micromyeloblastic, megakaryoblastic, megakaryoctyic, rieder cell,
bovine, aleukemic, mast cell, myelocytic, plamsa cell, subleukemic,
multiple myeloma, nonlymphocytic, chronic myelogenous leukemia,
chronic lymphocytic leukemia, polycythemia vera, lymphoma,
Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade
forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy
chain disease, myelodysplastic syndrome, and myelodysplasia and
chronic myelocytic leukemias.
[0445] Brain and central nervous system (CNS) cancers and tumors
that may be targeted include astrocytomas (including cerebellar and
cerebral), brain stem glioma, brain tumors, malignant gliomas,
ependymoma, glioblastoma, medulloblastoma, supratentorial primitive
neuroectodermal tumors, visual pathway and hypothalamic gliomas,
primary central nervous system lymphoma, ependymoma, brain stem
glioma, visual pathway and hypothalamic glioma, extracranial germ
cell tumor, medulloblastoma, myelodysplastic syndromes,
oligodendroglioma, myelodysplastic/myeloproliferative diseases,
myelogenous leukemia, myeloid leukemia, multiple myeloma,
myeloproliferative disorders, neuroblastoma, plasma cell
neoplasm/multiple myeloma, central nervous system lymphoma,
intrinsic brain tumors, astrocytic brain tumors, gliomas, and
metastatic tumor cell invasion in the central nervous system.
[0446] Gastrointestimal cancers that may be targeted include
extrahepatic bile duct cancer, colon cancer, colon and rectum
cancer, colorectal cancer, gallbladder cancer, gastric (stomach)
cancer, gastrointestinal carcinoid tumor, gastronintestinal
carcinoid tumors, gastrointestinal stromal tumors, bladder cancers,
islet cell carcinoma (endocrine pancreas), pancreatic cancer, islet
cell pancreatic cancer, prostate cancer rectal cancer, salivary
gland cancer, small intestine cancer, colon cancer, and polyps
associated with colorectal neoplasia.
[0447] Lung and respiratory cancers that may be targeted include
bronchial adenomas/carcinoids, esophagus cancer esophageal cancer,
esophageal cancer, hypopharyngeal cancer, laryngeal cancer,
hypopharyngeal cancer, lung carcinoid tumor, non-small cell lung
cancer, small cell lung cancer, small cell carcinoma of the lungs,
mesothelioma, nasal cavity and paranasal sinus cancer,
nasopharyngeal cancer, nasopharyngeal cancer, oral cancer, oral
cavity and lip cancer, oropharyngeal cancer; paranasal sinus and
nasal cavity cancer, and pleuropulmonary blastoma.
[0448] Urinary tract and reproductive cancers that may be targeted
include cervical cancer, endometrial cancer, ovarian epithelial
cancer, extragonadal germ cell tumor, extracranial germ cell tumor,
extragonadal germ cell tumor, ovarian germ cell tumor, gestational
trophoblastic tumor, spleen, kidney cancer, ovarian cancer, ovarian
epithelial cancer, ovarian germ cell tumor, ovarian low malignant
potential tumor, penile cancer, renal cell cancer (including
carcinomas), renal cell cancer, renal pelvis and ureter
(transitional cell cancer), transitional cell cancer of the renal
pelvis and ureter, gestational trophoblastic tumor, testicular
cancer, ureter and renal pelvis, transitional cell cancer, urethral
cancer, endometrial uterine cancer, uterine sarcoma, vaginal
cancer, vulvar cancer, ovarian carcinoma, primary peritoneal
epithelial neoplasms, cervical carcinoma, uterine cancer and solid
tumors in the ovarian follicle), superficial bladder tumors,
invasive transitional cell carcinoma of the bladder, and
muscle-invasive bladder cancer.
[0449] Skin cancers and melanomas (as well as non-melanomas) that
may be targeted include cutaneous t-cell lymphoma, intraocular
melanoma, tumor progression of human skin keratinocytes, basal cell
carcinoma, and squamous cell cancer. Liver cancers that may be
targeted include extrahepatic bile duct cancer, and hepatocellular
cancers. Eye cancers that may be targeted include intraocular
melanoma, retinoblastoma, and intraocular melanoma Hormonal cancers
that may be targeted include: parathyroid cancer, pineal and
supratentorial primitive neuroectodermal tumors, pituitary tumor,
thymoma and thymic carcinoma, thymoma, thymus cancer, thyroid
cancer, cancer of the adrenal cortex, and ACTH-producing
tumors.
[0450] Miscellaneous other cancers that may be targeted include
advanced cancers, AIDS-related, anal cancer adrenal cortical,
aplastic anemia, aniline, betel, buyo cheek, cerebriform,
chimney-sweeps, clay pipe, colloid, contact, cystic, dendritic,
cancer a deux, duct, dye workers, encephaloid, cancer en cuirasse,
endometrial, endothelial, epithelial, glandular, cancer in situ,
kang, kangri, latent, medullary, melanotic, mule-spinners',
non-small cell lung, occult cancer, paraffin, pitch workers', scar,
schistosomal bladder, scirrhous, lymph node, small cell lung, soft,
soot, spindle cell, swamp, tar, and tubular cancers.
[0451] Miscellaneous other cancers that may be targeted also
include carcinoid (gastrointestinal and bronchal) Castleman's
disease chronic myeloproliferative disorders, clear cell sarcoma of
tendon sheaths, Ewing's family of tumors, head and neck cancer, lip
and oral cavity cancer, Waldenstrom's macroglobulinemia, metastatic
squamous neck cancer with occult primary, multiple endocrine
neoplasia syndrome, multiple myeloma/plasma cell neoplasm, Wilms'
tumor, mycosis fungoides, pheochromocytoma, sezary syndrome,
supratentorial primitive neuroectodermal tumors, unknown primary
site, peritoneal effusion, malignant pleural effusion,
trophoblastic neo-plasms, and hemangiopericytoma.
[0452] In some embodiments, the liposomes described herein can be
used to treat an allergic or asthmatic condition. Exemplary
allergic or asthmatic conditions include, but are not limited to,
anaphylaxis, serum sickness, drug reactions, food allergies, insect
venom allergies, mastocytosis, allergic rhinitis, hypersensitivity
pneumonitis, urticaria, angioedema, eczema, atopic dermatitis,
allergic contact dermatitis, erythema multiforme, Stevens Johnson
syndrome, allergic conjunctivitis, atopic keratoconjunctivitis,
venereal keratoconjunctivitis, giant papillary conjunctivitis and
contact allergies), such as asthma (particularly allergic asthma)
or other respiratory problems.
[0453] In another aspect, described herein is a method of
stimulating an immune response in a mammalian subject comprising
administering to the subject a liposome or composition described
herein. In some embodiments, the liposome (or composition
comprising the liposome) is administered directly to the subject in
the same manner as a vaccine. In some embodiments, the liposomes
described herein are useful for the induction of an immune response
to a tumor antigen, one or more pathogenic organisms, or other
antigen as described herein. The liposomes can be administered
alone or in combination with therapeutic agent or prophylactic
treatment. It is contemplated that administration of the liposome
in combination with a therapeutic agent or prophylactic treatment
will enhance the therapeutic or protective effect. In certain
embodiments the vaccine will be a booster or at least the second
exposure of a subject to such a vaccine and can comprise the same
or different antigenic determinant relative to an initial
administration.
[0454] In another variation of the inventon, any of the methods
described herein with respect to a liposome can be modified such
that a lipid bilayer-coated particle is used in place of, or in
addition to, a liposome.
Combination Therapy
[0455] In some embodiments, a standard of care treatment is
administered with a liposome or lipid bilayer-coated particle
described herein. In the context of methods of described herein,
"standard of care treatment" refers to a treatment that is
generally accepted by clinicians for a certain type of patient
diagnosed with a type of illness. For all varieties of cancers and
neoplastic disorders described herein, for example, an aspect of
the disclosure is to improve standard of care therapy with
co-therapy with the liposomes or lipid bilayer-coated particles
described herein.
[0456] In some embodiments, the standard of care treatment is
selected from the group consisting of a cytokine, a
chemotherapeutic agent, a radiotherapeutic agent, and radiation
therapy.
[0457] Cytokines that are effective in inhibiting tumor
growth/metastasis are contemplated for use in the combination
therapy. Such cytokines, lymphokines, or other hematopoietic
factors include, but are not limited to, M-CSF, GM-CSF, TNF, IL-1,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNF.alpha.,
TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell
factor, and erythropoietin.
[0458] Any chemotherapeutic or radiotherapeutic agent may be
suitable for use in combination with a liposome or lipid
bilayer-coated particle described herein. Examples of suitable
chemotherapeutic and radiotherapeutic agents include, but are not
limited to: an anti-metabolite; a DNA-damaging agent; a cytokine
useful as a chemotherapeutic agent; a covalent DNA-binding drug; a
topoisomerase inhibitor; an anti-mitotic agent; an anti-tumor
antibiotic; a differentiation agent; an alkylating agent; a
methylating agent; a hormone or hormone antagonist; a nitrogen
mustard; a radiosensitizer; a photosensitizer; a radiation source,
optionally together with a radiosensitizer or photosensitizer; or
other commonly used therapeutic agents.
[0459] In some embodiments, the therapeutic agent is a
chemotherapeutic agent. Exemplary chemotherapeutic agents useful in
methods described herein are listed in Table 1 below.
TABLE-US-00001 TABLE 1 Alkylating agents Epipodophylotoxins
Hormones and antagonists Nitrogen mustards etoposide
Adrenocorticosteroids/antagonists mechlorethamine teniposide
prednisone and equivalents cyclophosphamide Antibiotics
dexamethasone ifosfamide actimomycin D ainoglutethimide melphalan
daunomycin (rubido-mycin) Progestins chlorambucil doxorubicin
(adria-mycin) hydroxyprogesterone caproate Nitrosoureas
mitoxantroneidarubicin medroxyprogesterone acetate carmustine
(BCNU) bleomycinsplicamycin megestrol acetate lomustine (CCNU)
(mithramycin) Estrogens semustine (methyl-CCNU) mitomycinC
dietbylstilbestrol Ethylenimine/Methyl-melamine dactinomycin
ethynyl estradiol/equivalents thriethylenemelamine (TEM) Enzymes
Antiestrogen triethylene thiophosphoramide L-asparaginase tamoxifen
(thiotepa) Biological response modifiers Androgens
hexamethylmelamine interferon-alpha testosterone propionate (HMM,
altretamine) IL-2 fluoxymesterone/equivalents Alkyl sulfonates
G-CSF Antiandrogens busulfan GM-CSF flutamide Triazines
Differentiation Agents gonadotropin-releasing dacarbazine (DTIC)
retinoic acid derivatives hormone analogs Antimetabolites
Radiosensitizers leuprolide Folic Acid analogs metronidazole
Nonsteroidal antiandrogens methotrexate misonidazole flutamide
Trimetrexate desmethylmisonidazole Photosensitizers Pemetrexed
pimonidazole hematoporphyrin derivatives Multi-targeted antifolate
etanidazole Photofrin .RTM. Pyrimidine analogs nimorazole
benzoporphyrin derivatives 5-fluorouracil RSU 1069 Npe6
fluorodeoxyuridine EO9 tin etioporphyrin (SnET2) gemcitabine RB
6145 pheoboride-a cytosine arabinoside SR4233 bacteriochlorophyll-a
(AraC, cytarabine) nicotinamide naphthalocyanines 5-azacytidine
5-bromodeozyuridine phthalocyanines 2,2'-difluorodeoxy-cytidine
5-iododeoxyuridine zinc phthalocyanines Purine analogs
bromodeoxycytidine 6-mercaptopurine Miscellaneous agents
6-thioguanine Platinium coordination complexes azathioprine
cisplatin 2'-deoxycoformycin Carboplatin (pentostatin) oxaliplatin
erythrohydroxynonyl-adenine Anthracenedione (EHNA) mitoxantrone
fludarabine phosphate Substituted urea 2-chlorodeoxyadenosine
hydroxyurea (cladribine, 2-CdA) Methylhydrazine derivatives Type I
Topoisomerase Inhibitors N-methylhydrazine (MIH) camptothecin
procarbazine topotecan Adrenocortical suppressant irinotecan
mitotane(o,p'- DDD) Natural products ainoglutethimide Antimitotic
drugs Cytokines paclitaxel interferon (*, *, *) Vinca alkaloids
interleukin-2 vinblastine (VLB) vincristine vinorelbine Taxotere
.RTM. (docetaxel) estramustine estramustine phosphate
[0460] In some embodiments, the combination therapy comprising
administration of (a) a liposome or lipid bilayer-coated particle
described herein and a therapeutic agent selected from the group
consisting of an immune modulator (e.g., an anti-CD40 antibody, an
anti-CD40L antibody, an anti-CTLA4 blocking antibody, or soluble
LAG-3 based immune modulators), a Toll-like receptor agonist (e.g.,
MPL, CpG, single-stranded RNA, nucleotides, nucleotide analogs like
CL087, or loxoribine, polyinosine-polycytidylic acid (poly I:C),
flagellin, resiquimod, imiquimod, and gardiquimod), a Nod ligand
(e.g., muramyl dipeptide, murabutide, peptidoglycan), an anti-viral
agent (e.g., oseltamivir phosphate), an antifungal agent (e.g.,
amphotericin B), an antibiotic, an antiviral antibody (e.g.,
palivizumab), a cancer immune therapeutic (e.g., herceptin,
alemtuzumab, gemtuzumab, rituximab, ibritumomab tiuxetan, and other
monoclonal antibody based cancer treatments), a chemotherapy agent,
a kinase inhibitor (e.g., imatinib and erlotinib), a cytotoxic
agent (e.g., cyclophosphamide), an anti-asthmatic agent, an
antihistamine agent, an anti-inflammatory agent, a vaccine adjuvant
(e.g., virus-like particles), a second liposome or lipid
bilayer-coated particle, an artificial antigen presenting cell, a
cytokine or chemokine blocking antibody (e.g., infliximab,
adalimumab, basiliximab), and mixtures thereof.
[0461] Treatment with the liposomes or lipid bilayer-coated
particles described herein may precede or follow the administration
of the standard of care treatment or therapeutic agent by intervals
ranging from minutes to weeks. In embodiments where the standard of
care treatment (or therapeutic agent) and a liposome or lipid
bilayer-coated particle described herein are administered
separately, one would generally ensure that a significant period of
time did not expire between the times of each delivery, such that
the standard of care treatment (or therapeutic agent) and the
liposome or lipid bilayer-coated particle would still be able to
exert an advantageously combined effect. In such instances, it is
contemplated that one would administer both modalities within about
12-24 hours of each other and, more preferably, within about 6-12
hours of each other, with a delay time of only about 12 hours being
most preferred. In some situations, it may be desirable to extend
the time period for treatment significantly, however, where several
days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or
8) lapse between the respective administrations. Repeated
treatments with one or both agents is specifically
contemplated.
[0462] The invention is further described in the following
Examples. The Examples serve only to illustrate the invention and
are not intended to limit the scope of the invention in any
way.
EXAMPLES
Example 1
Manufacture of Liposomes
[0463] This example describes a general procedure for the
manufacture of liposomes containing two lipids and a nonglycosidic
ceramide.
[0464] The liposomes described herein were manufactured using the
Solvent Dilution method. In this method, the lipids are dissolved
in an appropriate solvent, which is then slowly diluted in an
aqueous buffer to form multilamellar vesicles (MLV's). In some
embodiments, an antigen is added to the aqueous buffer when
antigen-encapuslated lipsomes are desired. The resulting material
is passed through an extruder with a specific pore size to generate
large unilamellar vesicles (LUV's). The LUV suspension is
diafiltered against buffer to remove solvent, and if desired, to
concentrate the liposomes to the final concentration. Finally, the
material is filtered through one or more sterilizing grade filters
before being filled into vials.
[0465] Specifically, a stock solution (5 mg/mL) of the
nonglycosidic ceramide was prepared by dissolving 25 mg of the
nonglycosidic ceramide in 5.0 mL t-butanol/water (95/5, v/v), with
heating at 60.degree. C. Appropriate lipids were weighed and an
aliquot of the nonglycosidic ceramide stock solution was added to
the lipids to obtain the desired nonglycosidic ceramide to lipid
ratio. The volume of mixture was then adjusted with t-butanol:water
(95:5, v:v) to a total volume of 1.0 mL, and the mixture heated at
60.degree. C. to dissolve the lipids. An aliquot of Rh-PE (5 mg/mL
in ethanol) was added (at 0.25 mol %) to the dissolved
lipid/nonglycosidic ceramide solution. The resulting solution was
diluted into 9 mL of 145 mM NaCl-10 mM phosphate buffer pH 6.5
(PBS), pre-heated to 60.degree. C. to produce multi-lamellar
vesicles (MLVs) at 5.0 mg/mL total lipid, 10% solvent. The MLVs
were then extruded at 60.degree. C. through two stacked 80 nm
polycarbonate filters, using a 10 mL LIPEX.TM. extruder (Northern
Lipids), to produce large unilamellar vesicles (LUVs), with a
target vesicle size of 100 nm or less. Using a SPECTRUM.TM.
cartridge (55 cm.sup.2, 500,000 molecular weight cut-off (MWCO)),
the sample was concentrated to approximately 5 mL, then diafiltered
against 10 volumes phosphate buffered saline (PBS) to remove the
solvent. The sample was then sterile filtered by passing it through
a 0.2 .mu.m SARTORIUS.TM. MINISART.RTM. syringe filter and aliquots
transferred to clean depyrogenated vials, which were then stoppered
and capped. Because the lipids were sterile filtered, they are
acceptable for clinical use. Samples were stored at 2-8.degree. C.
This process can also be practiced under industrial conditions
using the GMP process.
Example 2
Method of Manufacturing Liposomes with Different Components
[0466] This example describes a procedure for the manufacture of
liposomes containing egg phosphatidylcholine, egg
phosphatidylglycerol, and theritol ceramide (EPC:EPG:TC).
[0467] A stock solution (5 mg/mL) of threitolceramide was prepared
by dissolving 25 mg in 5.0 mL t-butanol/water (95/5, v/v), with
heating at 60.degree. C. Appropriate lipids (see Table 2) were
weighed and an aliquot of the threitolceramide stock solution was
added to the lipids to obtain the desired threitolceramide to lipid
ratio. The volume of the mixture was then adjusted with
t-butanol:water (95:5, v:v) to a total volume of 1.0 mL, and the
mixture heated at 60.degree. C. to dissolve the lipids. An aliquot
of Rh-PE (5 mg/mL in ethanol) was added (at 0.25 mol %) to the
dissolved lipid/threitolceramide solution. The resulting solution
was diluted into 9 mL of 145 mM NaCl-10 mM phosphate buffer pH 6.5
(PBS), pre-heated to 60.degree. C. to produce multi-lamellar
vesicles (MLVs) at 5.0 mg/mL total lipid, 10% solvent. The MLVs
were then extruded at 60.degree. C. through two stacked 80 nm
polycarbonate filters, using a 10 mL LIPEX.TM. extruder (Northern
Lipids), to produce large unilamellar vesicles (LUVs), with a
target vesicle size of 100 nm or less. Using a SPECTRUM.TM.
cartridge (55 cm.sup.2, 500,000 molecular weight cut-off (MWCO)),
the sample was concentrated to approximately 5 mL, then diafiltered
against 10 volumes phosphate buffered saline (PBS) to remove the
solvent. The sample was then sterile filtered by passing it through
a 0.2 .mu.m SARTORIUS.TM. MINISART.RTM. syringe filter and aliquots
transferred to clean depyrogenated vials, which were then stoppered
and capped. Samples were stored at 2-8.degree. C. The lipid
compositions described herein in Table 2 were supplied by Northern
Lipid Inc.
TABLE-US-00002 TABLE 2 Liposome Compositions (total volume of 5 mL)
Name Vesicle Size wt/wt/wt TC Content (mg/mL) EPC/EPG 41.8 .+-.
13.3 25/75 0 EPC/EPG/TC (2) 44.8 .+-. 14.2 24.5/73.5/2 0.134
EPC/EPG/TC (5) 47.7 .+-. 16.2 23.8/71.3/5 0.385 EPC/EPG/TC (10)
69.0 .+-. 24.5 22.5/67.5/10 0.794 EPC/CHOL/TC (2) 93.1 .+-. 43.7
54/36/10 0.663 EPC/DDAB/TC (2) 69.7 .+-. 23.4 54/36/10 0.683 EPC =
egg phosphatidylcholine; EPG = egg phosphatidylglycerol; CHOL =
cholesterol; DDAB = dimethyldioctadecyl ammonium bromide; TC =
threitolceramide
Example 3
Non-Glycosidic Ceramide-Containing Liposomes Activate Natural
Killer T Cells in Vitro
[0468] This Example illustrates ability of the
threitolceramide-containing liposomes to activate murine invariant
natural killer cells (iNKT) and human cells, as measured by
IFN.gamma. release.
[0469] Splenocytes were prepared from either wild type (WT) or CD
1d.sup.-/- (NKT cell deficient) mice and cultured in the presence
of titrating amounts (10 .mu.g/ml, 1 .mu.g/ml, 100 ng/ml) of
non-glycosidic ceramide containing liposomes (EPC:DDAB:TC(10);
EPC:EPG:TC(2); EPC:EPG:TC(5); EPC:EPG:TC(10) and EPC:CHOL:TC(10)),
control liposome (EPC:EPG), soluble threitolceramide or soluble
.alpha.-GalCer for 72 hours. Soluble threitolceramide and soluble
.alpha.-GalCer were prepared by dissolution in a
chloroform/methanol/water solution (10:10:3) at 10 mg/mL. The
resulting solution was diluted to a final volume of about 200
.mu.g/mL using vehicle solution comprised of NaCl (about 150 mM)
and polyoxyethylene (20) sorbitan monolaurate (i.e., Tween 20,
0.5%). Supernatants were removed from cultures and the amount of
IFN.gamma. produced by the cells was quantified by ELISA. Briefly,
ELISA plates were coated with 1D1K antibody (Mabtech) and left
overnight at 4.degree. C. The plates were washed with 0.05% Tween
20 in PBS (v/v). To eliminate any non-specific antigen binding, the
plates were coated (i.e. blocked) with 200 .mu.L of 10% FCS in PBS
(v/v) and incubated for 2 h at 37.degree. C. 100 .mu.L of reaction
supernatant was transferred to the ELISA plate and the standard was
added starting at 50 ng ml.sup.-1. The plate was then incubated at
4.degree. C. overnight. The following day, the plates were washed
with 0.05% Tween 20 in PBS and the biotinylated anti-cytokine
detecting mAb was added at 50 .mu.L/well and incubated at room
temperature for 2 h. After incubation, the plates were washed eight
times with 0.05% Tween 20 in PBS. 100 .mu.L of working dilution of
avidin-peroxidase was added per well after which the plates were
incubated at room temperature for 2 h. The plates were once again
washed eight times with 0.05% Tween 20 in PBS. 100 .mu.l of
tetramethylbenzidine (TMB) agent was added per well. Reaction was
stopped with 50 .mu.l solution of 0.5 M H.sub.2SO.sub.4 and the
optical density of each well was measured immediately using a
microplate reader (Bio-Rad, model 680) set to 450 nm.
[0470] The results are provided below in Table 3.
TABLE-US-00003 Assayed material IFN.gamma. (ng/ml) EPC:EPG
(Control) 0.31 EPC:EPG:TC (2) 3.89 EPC:EPG:TC (5) 5.61 EPC:EPGTC
(10) 7.35 EPC:Chol:TC (10) 0.45 EPC:DDAB:TC (10) 3.56 DOPC:Chol:TC
0.37 Soluble TC 3.77 Soluble .alpha.-GalCer 9.21
[0471] Results indicated that the EPC:EPG and EPC:DDAB:TC liposomes
were both able to activate iNKT cells to the same extent as soluble
threitolceramide (FIG. 1A). iNKT cell activation was not observed
with the EPC:EPG control liposome (FIG. 1B). Likewise, IFN.gamma.
in the CD.sup.-/- splenocyte culture was also not observed with the
EPC:EPG control liposome. The EPC:DDAB:TC formulations appeared to
aggregate on the cultured cells.
Example 4
Non-Glycosidic Ceramide Containing Liposomes Induced Natural Killer
T Cell Activation In Vivo
[0472] This example illustrates the in vivo effect of
threitolceramide-containing liposomes on dendritic cell (DC)
maturation, as measured by CD86 and CD40 expression, and iNKT cell
activation, as measured by IL-4 and IFN.gamma. release.
[0473] WT (n=3) and CD1d.sup.-/- (n=1) mice were administered 1
.mu.g of liposomes (EPC:DDAB:TC(10); EPC:EPG:TC(2); EPC:EPG:TC(5);
EPC:EPG:TC(10) and EPC:CHOL:TC(10)) or control liposome (EPC:EPG),
soluble threitolceramide or soluble .alpha.-GalCer by intravenous
injection.
[0474] DC Maturation:
[0475] 18 hours post-injection, the spleens of the mice were
harvested and the phenotype of DC maturation was examined by flow
cytometry as determined by the median fluorescent intensity (MFI)
of CD86 and CD40 expression.
[0476] Activation of NKT Cells:
[0477] Serums were obtained by tail venipuncture 2 and 18 hours
post-injection to detect IL-4 and IFN-.gamma. levels by ELISA.
[0478] The results are provided below in Table 4.
TABLE-US-00004 CD86 (DC CD40 (DC INF.gamma. IL-4 maturation)
maturation) (serum) (serum) Assayed Material Median Median ng/ml
ng/ml EPC:EPG (Control) 7.9 16.8 0 0 EPC:EPG:TC (2) 46.8 26.9 0.27
0.63 EPC:EPG:TC (5) 61.2 32.8 1.93 0.81 EPC:EPGTC (10) 58.7 31.3
1.09 0.71 EPC:Chol:TC (10) 68.4 39.1 3.32 0.53 EPC:DDAB:TC (10)
36.6 34.6 13.1 0.90 DOPC:Chol:TC 62.6 32.9 2.07 0.62 Soluble TC
60.1 28.9 0.99 0.12 Soluble .alpha.-GalCer 91.6 39.1 8.47 0.83
[0479] Results indicated that all threitolceramide-containing
liposomes were able to induce DC maturation to the same extent as
soluble threitolceramide but the EPC:DDAB:TC liposome also induced
DC maturation in CD1d-/- mice (FIG. 2).
[0480] The production of iNKT-cell associated cytokines following
activation was either comparable to or greater than soluble
threitolceramide at 2 hours (IL-4; FIG. 3A) and 18 hours
(IFN.gamma.; FIG. 3B) using the liposomes.
Example 5
Non-Glycosidic Ceramide-Containing Liposomes Induce Dendritic Cell
Maturation In Vitro
[0481] Peripheral blood mononuclear cells (PBMCs) were isolated
from healthy mice buffy coats by density gradient centrifugation
over Lymphoprep (Nycomed). Monocytes were positively selected using
anti-CD14 mAb-coated magnetic beads (MACS; Miltenyi Biotec) and
were then cultured in 6-well plates in either X-Vivo 15+2% Human AB
serum with 800 U ml.sup.-1 granulocyte monocyte colony stimulating
factor (GM-CSF) and 500 U ml.sup.-1 IL-4 for 4 days to produce
immature DCs. On day 4, liposomes identified in Table 2 (at
concentration of 1 .mu.g ml.sup.-1 or 100 ng ml.sup.-1), soluble
threitolceramide ml.sup.-1) or soluble .alpha.-GalCer (100 ng
ml.sup.-1) were added to the immature DCs, followed by the addition
of maturation cocktail (IL-1.beta., IL-6, TNF-.alpha., and PGE2)
after 3 hours. DCs were harvested and assayed with iNKT cells
overnight, the supernatants were taken and an IFN.gamma. ELISA, the
results of which are set forth in FIG. 6. Briefly, ELISA plates
were coated with 1D1K antibody (Mabtech) and left overnight at
4.degree. C. The plates were washed with 0.05% Tween 20 in PBS
(v/v). To eliminate any non-specific antigen binding, the plates
were coated (i.e. blocked) with 200 .mu.L of 10% FCS in PBS (v/v)
and incubated for 2 h at 37.degree. C. 100 .mu.L of reaction
supernatant was transferred to the ELISA plate and the standard was
added starting at 50 ng ml.sup.-1. The plate was then incubated at
4.degree. C. overnight. The following day, the plates were washed
with 0.05% Tween 20 in PBS and the biotinylated anti-cytokine
detecting mAb was added at 50 .mu.L/well and incubated at room
temperature for 2 h. After incubation, the plates were washed eight
times with 0.05% Tween 20 in PBS. 100 .mu.L of working dilution of
avidin-peroxidase was added per well after which the plates were
incubated at room temperature for 2 h. The plates were once again
washed eight times with 0.05% Tween 20 in PBS. 100 .mu.l of
tetramethylbenzidine (TMB) agent was added per well. Reaction was
stopped with 50 .mu.l solution of 0.5 M H.sub.2SO.sub.4 and the
optical density of each well was measured immediately using a
microplate reader (Bio-Rad, model 680) set to 450 nm.
[0482] The presence of IL12-p40 was also detected by ELISA, the
results of which are set forth in FIG. 9.
[0483] DCs were also analyzed by FACS after addition of liposomes
for expression of the co-stimulatory molecule CD80 (FIG. 8) and for
cell viability (FIGS. 5 and 7).
[0484] Results provided in FIG. 4 indicate that the EPC:EPG:TC
formulation particularly at 10% threitolceramide was superior to
all tested formulations (and easier to handle than EPC:DDAB:TC,
data not shown).
Example 6
Human iNKT Cell TCR Binding Assay
[0485] This example illustrates the effect of the
threitolceramide-containing liposomes on iNKT T-Cell Receptor (TCR)
priming in vitro.
[0486] The efficiency of binding of liposomes to human CD1d
molecule and recognition by the iNKT T-cell-receptor (TCR) was
investigated in vitro. C1R human-CD 1d expressing cells were
cultured overnight with various dilutions of the liposomes
identified above in Table 2, soluble .alpha.-GalCer or soluble
threitolceramide (5 .mu.g/mL to 1 ng/mL). The cells were washed and
incubated with a fluorescently labelled iTCR tetramer. Binding of
iTCR to lipid-CD1d complex was determined by flow cytometry.
[0487] The results are provided in FIG. 10. Results indicated that
the EPC:EPG:TC formulation bound with the greatest efficiency,
better than soluble threitolceramide. In contrast, the EPC:EPG:CHOL
and EPC:EPG:DDAB liposomes bound weakly to the iTCR in comparison
to the EPC:EP:TC formulation and soluble threitolceramide (FIG.
10).
Example 7
Adjuvant Properties of EPC:EPG Liposome Formulations
[0488] This Example illustrates the expansion of an endogenous T
cell repertoire that recognizes the ovalbulmin (OVA) peptide,
SIINFEKL (SEQ ID NO: 1), in an in vivo mouse using a
threitolceramide-containing liposome.
[0489] Having established in Example 6 that the EPC:EPG:TC
formulation was the most comparable to soluble TC, the ability of
the EPC:EPG:TC (10%) liposome to expand an endogenous T cell
repertoire that recognizes the ovalbulmin (OVA) peptide, SIINFEKL
(SEQ ID NO:1), in an in vivo B6 mouse model was determined. A
protocol similar to that which would be used clinically was
developed. In this protocol, bone-marrow-derived-DC (BMDC) cultures
containing GM-CSF and IL-4 were generated at day -6. At day -1,
BMDC were left untreated or pulsed with either EPC:EPG (control
liposome), EPC:EPG:TC (10%) or soluble threitolceramide, before
being matured with LPS overnight. The following day, SIINFEKL
peptide (SEQ ID NO: 1) was added to cultures for 3 hours, cells
were then washed, and 1.times.10.sup.6 BMDC injected intravenously.
At day 7 post injection, mice were harvested and the percentage of
CD8+Kb tetramer positive cells were determined by flow cytometry in
the blood and spleen. Results indicated that the degree of CD8+Kb
tetramer+cells expansion using the liposome was directly comparable
to that expanded by the soluble threitolceramide (FIG. 11).
[0490] In another protocol, 800 .mu.g of the OVA peptide and either
soluble aGalCer, soluble TC, control liposome (EPC:EPG) or a
EPC:EPG:TC liposome were injected into B6 mice intravenously. 7
days post injection, OVA-specific T cells were analyzed in the
blood serum and the spleen. Results are provided in FIG. 12.
Example 8
Preparation of Antigen-Associated Liposomes
[0491] The following Example evaluates the effects of the
association of NY-ESO-1 with the EPC/EPG/TC liposome as described
in Example 1.
[0492] Briefly, .about.1 mg/ml of NY-ESO-1 (in 4M urea, 50 mM
glycine, 145 mM NaCl, 10 mM phosphate, pH 6.5) was diluted to 1
mg/ml by combining 3 mL of NY-ESO-1 solution with 0.18 mL 4M urea,
50 mM glycine, 145 mM NaCl, 10 mM phosphate, pH 6.5. The diluted
NY-ESO1 solution was combined with the EPC/EPG/TC liposomes and the
samples were diluted as needed (see Table 5 below) for
determination of vesicle size. Vesicle size (dual angle) was
determined using a Malvern Nanosizer. Upon dilution of the NY-ESO-1
into the liposome suspension, the NY-ESO-1 binds ionically to the
negatively charged surface of the liposomes. This is evidenced by
the very low level of association of NY-ESO-with neutral (EPC)
liposomes (<6%) and the increased association with liposomes in
direct correlation to the molar content of anionic lipid (EPG) in
the liposomes. Hydrophobic interactions may also contribute to the
interaction of NY-ESO-1 with the liposomes but ionic interactions
are clearly dominant.
TABLE-US-00005 TABLE 5 NY- Required TC:NY- ESO-1 PBS dilution for
Lipid:protein ESO-1 Sample X (mL) (mL) Liposomes (mL) size Mass
Ratio Mass Ratio 1 0.5 0.5 0 None N/A N/A 2 0.5 0.384 0.116 None
1:1 0.1:1 3 0.5 0.355 0.145 None 2:5:1 0.25:1 4 0.5 0.209 0.291
2.5X 5:1 0.5:1 5 0.5 0.064 0.436 3.75X 7.5:1 0.75:1 6 0.43 0.07 0.5
4X 10:1 1:1 (urea, gly, PBS) 7 0 0.25 0.25 4X N/A N/A (urea, gly,
PBS)
[0493] Table 6 below provides the vesicle size of the liposomes
within each sample when mixed with NY-ESO-1. An increase in vesicle
size indicates interaction of the NY-ESO-1 antigen with the
liposomes. Without wishing to be bound by theory, it is
contemplated that this interaction involves frosslinking of the
anionic liposomes by the multivalent (positive charges) of
NY-ESO-1.
TABLE-US-00006 TABLE 6 Peak 1 Peak 2 Peak 3 Sample Z-Avg. PDI
Diameter Width Diameter Width Diameter Width 1 72.86 0.402 67.19
(88.2%) 30.05 315.3 (5.5%) 82.85 5298 (4.5%) 403.4 2 3217 0.339
3145 (100%) 468.3 3 3867 0.427 1837 (100%) 255.3 4 1593 0.073 1673
(100%) 292.2 5 1038 0.218 1283 100%) 349.9 6 755.9 0.591 1291
(89.9%) 468.1 104.2 (10.1%) 15.26 7 62.29 0.102 69.77 (100%)
24.3
[0494] Results indicated that there were no obvious physical
changes (i.e., no precipitate, no increase in turbidity) when
NY-ESO-1 or the liposomes were diluted with PBS. In contrast, when
NY-ESO-1 was combined with the liposomes, there was a significant
increase in turbidity in all samples, with no apparent difference
between samples with varying amounts of lipid. However, after
standing at room temperature for a period of time, sample 2 was
distinctly flocculant, sample 3 showed some degree of flocculance,
and samples 4-6 appeared turbid but homogenous. Large flocculates
are difficult to inject and would be expected to have variable
efficacy.
[0495] The samples were then centrifuged for 5 minutes at
1000.times.g. After centrifugation, no pellet was observed in
samples 1 or 7 (NY-ESO-1 only, liposomes only, respectively).
Samples 2 and 3 both had complete pelleting with clear supernatant.
Sample 4 had a more loosely packed pellet with clear supernatant.
Samples 5 and 6 had no distinct pellets, but appeared to be less
homogenous.
Example 9
Activity of Antigen-Associated Liposomes
[0496] The liposomes described herein comprising full length
NY-ESO-1 protein (prepared according to a method provided in
Example 8) are intravenously injected into HHD mice crossed with
C57BL6 mice (F1 mice). HLA A2 restricted responses specific for the
NY-ESO-1 157-165 peptide are assessed by either HLA A2 tetramer
staining or by an ELISPOT assay. Because NY-ESO-1 encodes a H-2
I-Ab epitope (Lopes L. J. Virol 82:86-95, 2007, incorporated herein
by reference), the experiment is repeated using C57BL/6 mice and
I-Ab restricted responses are assessed by ELISPOT assays. Control
mice receive recombinant full length NY-ESO-1 protein alone, or
with soluble threitolceramide. In another variation the
experimental and control mice receive their respective regimen in
combination with TLR ligands (e.g., CpG). Anti NY-ESO-1 specific
antibody responses are measured by ELISA. The expansion of
NY-ESO-1-specific immune responses is demonstrated.
[0497] Mice are challenged with H-2b tumor cells or transplanted
tumors transduced with lentiviral vectors encoding the full length
NY-ESO-1 protein, with or without transfected HLA-A2 cDNAs. Tumor
growth or shrinkage and survival times are assessed.
Sequence CWU 1
1
218PRTArtificial SequenceSynthetic peptide 1Ser Ile Ile Asn Phe Glu
Lys Leu 1 5 29PRTArtificial SequenceSynthetic peptide 2Ser Leu Leu
Met Trp Ile Thr Gln Cys 1 5
* * * * *
References