U.S. patent number RE39,042 [Application Number 09/976,936] was granted by the patent office on 2006-03-28 for etherlipid-containing multiple lipid liposomes.
This patent grant is currently assigned to The Liposome Company, Inc.. Invention is credited to Imran Ahmad, Suresh K. Bhatia, Andrew S. Janoff, Eric Mayhew.
United States Patent |
RE39,042 |
Mayhew , et al. |
March 28, 2006 |
Etherlipid-containing multiple lipid liposomes
Abstract
Described herein are liposomes containing etherlipids of the
formula: ##STR00001## as well as a phosphatidylcholine, a sterol,
and a headgroup-derivatized lipid. These liposomes are useful in a
variety of therapeutic regimens, including the treatment of cancers
and inflammatory disorders.
Inventors: |
Mayhew; Eric (Seattle, WA),
Janoff; Andrew S. (Yardley, PA), Ahmad; Imran
(Wadsworth, IL), Bhatia; Suresh K. (New Delhi,
IN) |
Assignee: |
The Liposome Company, Inc.
(Princeton, NJ)
|
Family
ID: |
26689872 |
Appl.
No.: |
09/976,936 |
Filed: |
October 11, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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08602669 |
Feb 16, 1996 |
5762958 |
|
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Reissue of: |
09017440 |
Feb 2, 1998 |
05965159 |
Oct 12, 1999 |
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Current U.S.
Class: |
424/450;
428/402.2 |
Current CPC
Class: |
A61K
9/1272 (20130101); Y10T 428/2984 (20150115) |
Current International
Class: |
A61K
9/127 (20060101) |
Field of
Search: |
;424/450,1.21,9.321,9.51
;428/402.2 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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4132345 |
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Apr 1993 |
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DE |
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4408011 |
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Nov 1995 |
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DE |
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1583661 |
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Jan 1981 |
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GB |
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072294 |
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Jul 1984 |
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JP |
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93/04673 |
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Mar 1993 |
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WO |
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93/08202 |
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Apr 1993 |
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WO |
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94/27580 |
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Dec 1994 |
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WO |
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|
Primary Examiner: Kishore; Gollamudi S.
Attorney, Agent or Firm: Burns, Doane, Swecker & Mathis,
L.L.P.
Parent Case Text
This application is a CIP of Ser. No. 08/602,659 filed Feb. 16,
1996 now U.S. Pat. No. 5,762,958.
Claims
What is claimed is:
1. A liposome having a lipid bilayer which comprises: (a) a
phosphatidylcholine; (b) a sterol; (c) a headgroup-derivatized
lipid comprising a phosphatidylethanolamine linked at the
ethanolamine group to a dicarboxylic acid; and, (d) an etherlipid
having the formula: ##STR00005## wherein R.sub.1 is Y.sub.1
Y.sub.2, Y.sub.2 is CH.sub.3 or CO.sub.2H, Y.sub.1 is
(CH.sub.2).sub.n1
(CH.dbd.CH).sub.n2(CH.sub.2).sub.n3(CH.dbd.CH).sub.n4
(CH.sub.2).sub.n5(CH.dbd.CH).sub.n6
(CH.sub.2).sub.n7(CH.dbd.CH).sub.n8(CH.sub.2).sub.n9, the sum of
n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer of from 3 to 23, n1 is
zero or an integer of from 1 to 23, n3 is zero or an integer of
from 1 to 20, n5 is zero or an integer of from 1 to 17, n7 is zero
or an integer of from 1 to 14, n9 is zero or an integer of from 1
to 11, and each of n2, n4, n6 and .[.8.]. .Iadd.n8 .Iaddend.is
independently zero or 1; wherein Z is oxygen or sulfur and R.sub.2
is CH.sub.3; wherein R.sub.3 is
--O--P(O).sub.2--O--CH.sub.2CH.sub.2N(CH.sub.3).sub.3; and wherein
the phosphatidylethanolamine-dicarboxylic acid comprises from about
5 mole percent to about 20 mole percent of the lipid bilayer and
the etherlipid comprises from greater than about 10 mole percent to
less than about 30 mole percent of the lipid bilayer.
2. The liposome of claim 1 which is a unilamellar liposome having a
diameter of from greater than about 50 nm to less than about 200
nm.
3. The liposome of claim 1, wherein the phosphatidylcholine is an
unsaturated or partially unsaturated phosphatidylcholine.
4. The liposome of claim 3, wherein the phosphatidylcholine is
dioleoyl phosphatidylcholine.
5. The liposome of claim 1, wherein the sterol is cholesterol.
6. The liposome of claim 1, wherein the headgroup derivatized lipid
comprises a phosphatidylethanolamine selected from the group
consisting of dipalmitoyl phosphatidylethanolamine, palmitoyloleoyl
phosphatidylethanolamine and dioleoyl phosphatidylethanolamine.
7. The liposome of claim 6, wherein the headgroup derivatized lipid
comprises dioleoyl phosphatidylethanolamine.
8. The liposome of claim 1, wherein the headgroup-derivatized lipid
comprises a dicarboxylic acid selected from the group consisting of
glutaric acid, sebacic acid, succinic acid and tartaric acid.
9. The liposome of claim 8, wherein the dicarboxylic acid is
glutaric acid.
10. The liposome of claim 1, wherein the headgroup-derivatized
lipid comprises dioleoyl phosphatidylethanolamine and glutaric
acid.
11. The liposome of claim 1, wherein R.sub.1 is
(CH.sub.2).sub.n1CH.sub.3 and Z is O.
12. The liposome of claim 11, wherein the etherlipid is:
##STR00006##
13. The liposome of claim 12, wherein the phosphatidylcholine is
dioleoyl phosphatidylethanolamine, the sterol is cholesterol and
the headgroup derivatized lipid comprises dioleoyl
phosphatidylethanolamine and glutaric acid.
14. The liposome of claim 13, wherein the bilayer comprises about
20 mole percent of the etherlipid, about 10 mole percent of the
headgroup-derivatized lipid, about 30 mole percent cholesterol and
about 40 mole percent dioleoyl phosphatidylcholine.
15. The liposome of claim 1, comprising an additional bioactive
agent.
16. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and the liposome of claim 1.
.Iadd.17. A liposome having a lipid bilayer which comprises: (a) a
phosphatidylcholine; (b) a sterol; (c) a headgroup-derivatized
lipid comprising a phosphatidylethanolamine linked at the
ethanolamine group to a dicarboxylic acid; and, (d) an etherlipid
having the formula: ##STR00007## wherein R.sub.1 is Y.sub.1
Y.sub.2, Y.sub.2 is CH.sub.3 or CO.sub.2H, Y.sub.1 is
(CH.sub.2).sub.n1(CH.dbd.CH).sub.n2(CH.sub.2).sub.n3(CH.dbd.CH).sub.n4(CH-
.sub.2).sub.n5(CH.dbd.CH).sub.n6(CH.sub.2).sub.n7(CH.dbd.CH).sub.n8(CH.sub-
.2).sub.n9, the sum of n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer
of from 3 to 23, n1 is zero or an integer of from 1 to 23, n3 is
zero or an integer of from 1 to 20, n5 is zero or an integer of
from 1 to 17, n7 is zero or an integer of from 1 to 14, n9 is zero
or an integer of from 1 to 11, and each of n2, n4, n6 and n8 is
independently zero or 1; wherein Z is NH.sub.2,C(O)O.sub.2 or
HNC(O); R.sub.2 is CH.sub.3; R.sub.3 is
--O--P(O).sub.2--O--CH.sub.2CH.sub.2N(CH.sub.3).sub.3; and wherein
the phosphatidylethanolamine-dicarboxylic acid comprises from about
5 mole percent to about 20 mole percent of the lipid bilayer and
the etherlipid comprises from greater than about 10 mole percent to
less than about 30 mole percent of the lipid bilayer..Iaddend.
.Iadd.18. The liposomes of claim 17 which is a unilamellar liposome
having a diameter of from greater than about 50 nm to less than
about 200 nm..Iaddend.
.Iadd.19. The liposome of claim 17, wherein the phosphatidylcholine
is an unsaturated or partially unsaturated
phosphatidylcholine..Iaddend.
.Iadd.20. The liposome of claim 19, wherein the phosphatidylcholine
is dioleoyl phosphatidylcholine..Iaddend.
.Iadd.21. The liposome of claim 17, wherein the sterol is
cholesterol..Iaddend.
.Iadd.22. The liposome of claim 17, wherein the headgroup
derivatized lipid comprises a phosphatidylethanolamine selected
from the group consisting of dipalmitoyl phosphatidylethanolamine,
palmitoyloleoyl phosphatidylethanolamine and dioleoyl
phosphatidylethanolamine..Iaddend.
.Iadd.23. The liposome of claim 22, wherein the headgroup
derivatized lipid comprises dioleoyl
phosphatidylethanolamine..Iaddend.
.Iadd.24. The liposome of claim 17, wherein the
headgroup-derivatized lipid comprises a dicarboxylic acid selected
from the group consisting of glutaric acid, sebacic acid, succinic
acid and tartaric acid..Iaddend.
.Iadd.25. The liposome of claim 24, wherein the dicarboxylic acid
is glutaric acid..Iaddend.
.Iadd.26. The liposome of claim 17, wherein the
headgroup-derivatized lipid comprises dioleoyl
phosphatidylethanolamine and glutaric acid..Iaddend.
.Iadd.27. The liposome of claim 17, wherein R.sub.1 is
(CH.sub.2).sub.n1CH.sub.3 and Z is C(O)O..Iaddend.
.Iadd.28. The liposome of claim 17, wherein R.sub.1 is
(CH.sub.2).sub.n1CH.sub.3 and Z is NH..Iaddend.
.Iadd.29. The liposome of claim 17, wherein R.sub.1 is
(CH.sub.2).sub.n1CH.sub.3 and Z is HNC(O)..Iaddend.
.Iadd.30. The liposome of claim 17, comprising an additional
bioactive agent..Iaddend.
.Iadd.31. A pharmaceutical composition comprising a
pharmaceutically acceptable carrier and the liposome of claim
17..Iaddend.
Description
Etherlipids are synthetic analogues of platelet activating factor
(PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), an effector
generally believed to be involved in a variety of physiological
processes, such as inflammation, the immune response, allergic
reactions and reproduction. Etherlipids have been shown to be
effective antitumor agents in animals, and are believed to be
selectively cytotoxic to a broad variety of cancer cells (see, for
example, Dietzfelbinger et al. (1993); Zeisig et al. (1993); Powis
et al. (1990); Berdel (1991); Bhatia and Hadju (1991); Reed et al.
(1991); Workman (1991); Workman et al. (1991); Bazill and Dexter
(1990); Berdel (1990); Counsell et al. (1990); Tritton and Hickman
(1990); Muschiol et al. (1990); Layton et al. (1980); Runge et al.
(1980); Great Britain Patent No. 1,583,661; U.S. Pat. No.
3,752,886). Etherlipids have also been shown to be antimetastatic
and anti-invasive, and to be capable of cell differentiation
induction.
Mechanisms of etherlipid cytotoxicity, while not definitively
established, appear to involve action at, and possible disruption
of, the cell membrane. The selective cytotoxicity of etherlipids
may involve intracellular accumulation and differential activity of
alkyl cleavage enzymes. Etherlipids may also be selective
inhibitors of phosphatidylinositol phospholipase C and protein
kinase C activities, as well as of phosphatidylcholine
biosynthesis. Hence, etherlipids are potentially quite useful as
therapeutic agents. However, their administration can also lead to
hemolysis, hepatic dysfunction and gastrointestinal disorders.
Applicants have found that certain liposomal formulations of
etherlipids can buffer these toxicities without inhibiting
anticancer efficacy, and thereby can provide a more therapeutically
useful basis for etherlipid administration.
SUMMARY OF THE INVENTION
This invention provides a liposome comprising a bilayer having a
lipid component which comprises: (a) a phosphatidylcholine; (b) a
sterol; (c) a headgroup derivatized lipid and, (d) an etherlipid.
The headgroup-derivatized lipid, comprising a
phosphatidylethanolamine linked to a moiety selected from the group
consisting of dicarboxylic acids, polyethylene glycols,
gangliosides and polyalkylethers, comprises from about 5 mole
percent to about 20 mole percent of the bilayer's lipid component;
the etherlipid comprises from about 10 mole percent to about 30
mole percent of the lipid component.
Preferably, the phosphatidylcholine is dioleoyl phosphatidylcholine
("DOPC"), the sterol is cholesterol ("chol"), the
headgroup-derivatized lipid is dioleoyl
phosphatidylethanolamine-glutaric acid ("DOPE-GA") and the
etherlipid is ##STR00002## also known as "EL-18,""ET-18-OCH.sub.3,"
or "edelfosine"). Most preferably, the liposome is a unilamellar
liposome having a diameter of from greater than about 50 nm to less
than about 200 nm, and the liposome's bilayer has a lipid component
comprising about 20 mole percent of the etherlipid, about 10 mole
percent of the headgroup-derivatized lipid, about 30 mole percent
cholesterol and about 40 mole percent dioleoyl
phosphatidylcholine.
Also provided herein is a pharmaceutical composition comprising a
pharmaceutically acceptable carrier and such liposomes. Further
provided is a method of treating a mammal afflicted with a cancer,
including, but not limited to: a lung, brain, colon, ovarian or
breast cancers, the method comprising administering the
pharmaceutical compositions of this invention to the mammal.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Time Course of Carboxyfluorescein Leakage from Liposomal
Edelfosine Formulations Incubated at 48 deg. Celsius in PBS. ELL 28
(uppermost curve, "ELL" indicating "etherlipid liposome"):
Distearoyl phosphatidylcholine ("DSPC"); cholesterol ("CHOL");
dioleoyl phosphatidylethanolamine-glutaric acid ("DOPE-GA");
edelfosine "EL," standing for "etherlipid" (the respective molar
ratio of these lipid components being 4:3:1:2); ELL 30 (second from
top curve): EPC:CHOL:DOPE-GA:EL (4:3:1:2); ELL 25 (middle curve):
DOPE:CHOL:DOPE-GA:EL (3:3:1:3); ELL 12 (second from bottom curve):
DOPC:CHOL:DOPE-GA:EL (4:3:1:2); and, ELL 20 (bottom curve):
DOPE:CHOL:DOPE-GA:EL (4:3:1:2). Y-axis: % CF Leakage; x-axis: time
(seconds).
FIG. 2. Comparison of Hemolytic Activity and CF Leakage in
Etherlipid Liposomes. From top-to-bottom: ELL 20--ELL 12--ELL
25--ELL 30--ELL 28 (y=34231.times..sup.-2.1614; R.sup.2=0.96).
Y-axis: HI.sub.10; x-axis: % CF leakage upon incubation in PBS.
FIG. 3. Stability of Etherlipid Liposomal Formulations on
Incubation in 0.5% Serum at 37 Degrees Celsius. Y-axis: time
(minutes); x-axis (from left-to-right): ELL 28, ELL 40, ELL 30; ELL
25; ELL 12; ELL 20. Inset: Y-axis: time (minutes); x-axis: ELL 28,
ELL 40, ELL 30.
DETAILED DESCRIPTION OF THE INVENTION
This invention provides a liposome comprising a bilayer having a
lipid component which comprises: (a) a phosphatidylcholine; (b) a
sterol; (c) a headgroup derivatized lipid containing a
phosphatidylethanolamine and a moiety selected from the group
consisting of dicarboxylic acids, gangliosides, polyethylene
glycols and polyalkylethers, which headgroup-derivatized lipid
comprises from about 5 mole percent to about 20 mole percent of the
bilayer's lipid component; and, (d) an etherlipid having the
following formula: ##STR00003## the etherlipid comprising from
greater than about 10 mole percent, to less than about 30 mole
percent, of the bilayer's lipid component.
"Liposomes" are self-assembling structures comprising one or more
lipid bilayers, each of which surrounds an aqueous compartment and
comprises two opposing monolayers of amphipathic lipid molecules.
Amphipathic lipids comprise a polar (hydrophilic) headgroup region
covalently linked to one or two non-polar (hydrophobic) acyl
chains. Energetically unfavorable contacts between the hydrophobic
acyl chains and the aqueous medium are generally believed to induce
lipid molecules to rearrange such that the polar headgroups are
oriented towards the aqueous medium while the acyl chains reorient
towards the interior of the bilayer. An energetically stable
structure is formed in which the acyl chains are effectively
shielded from coming into contact with the aqueous medium.
Liposomes can have a single lipid bilayer (unilamellar liposomes,
"ULVs"), or multiple lipid bilayers (multilamellar liposomes,
"MLVs"), and can be made by a variety of methods (for a review,
see, for example, Deamer and Uster (1983)). These methods include
without limitation: Bangham's methods for making multilamellar
liposomes (MLVS); Lenk's, Fountain's and Cullis' methods for making
MLVs with substantially equal interlamellar solute distribution
(see, for example, U.S. Pat. Nos. 4,522,803, 4,588,578, 5,030,453,
5,169,637 and 4,975,282); and Papahadjopoulos et al.'s
reverse-phase evaporation method (U.S. Pat. No. 4,235,871) for
preparing oligolamellar liposomes. ULVs can be produced from MLVs
by such methods as sonication (see Papahadjopoulos et al. (1968))
or extrusion (U.S. Pat. No. 5,008,050 and U.S. Pat. No. 5,059,421).
The etherlipid liposome of this invention can be produced by the
methods of any of these disclosures, the contents of which are
incorporated herein by reference.
Various methodologies, such as sonication, homogenization, French
Press application and milling can be used to prepare liposomes of a
smaller size from larger liposomes. Extrusion (see U.S. Pat. No.
5,008,050) can be used to size reduce liposomes, that is 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 (see WO89/008846), can
also be used to regularize the size of liposomes, that is, to
produce liposomes having a population of liposomes having less size
heterogeneity, and a more homogeneous, defined size distribution.
The contents of these documents are incorporated herein by
reference. Liposome sizes can also be determined by a number of
techniques, such as quasi-electric light scattering, and with
equipment, e.g., Nicomp.RTM. particle sizers, well within the
possession of ordinarily skilled artisans.
The liposomes of this invention can be unilamellar or
multilamellar. Preferably the liposomes are unilamellar and have
diameters of less than about 200 nm, more preferably, from greater
than about 50 nm to less than about 200 nm; such liposomes are
preferably produced by a method comprising the steps of: dissolving
lipids in a suitable organic solvent so as to establish a lipidic
solution; removing the organic solvent from the resulting lipidic
solution; adding an aqueous solution so as to form liposomes; and,
then extruding the resulting liposomes through a suitable
filter.
Liposomes of the 50-200 nm size are preferred because they
generally believed to circulate longer in mammals than do larger
liposomes, which are more quickly recognized by the mammals'
reticuloendothelial systems ("RES"), and hence, more quickly
cleared from the circulation. Longer circulation can enhance
therapeutic efficacy by allowing more liposomes to reach their
intended site of actions, e.g., tumors or inflammations. Small
unilamellar liposomes, i.e., those generally less than 50 nm in
diameter, carry amounts of bioactive agents which may be, in some
cases, too low to be of sufficient therapeutic benefit.
R.sub.1 of the etherlipid, the chain attached at the carbon #1
position of its glycerol backbone by way of an oxygen, has the
formula Y.sub.1.[.y.sub.2.]. .Iadd.Y.sub.2.Iaddend.. Y.sub.2 is
CH.sub.3 or CO.sub.2H, but preferably is CH.sub.3. Y.sub.1 is
--(CH.sub.2).sub.n1(CH.dbd.CH).sub.n2(CH.sub.2).sub.n3(CH.dbd.CH).sub.n4(-
CH.sub.2).sub.n5 (CH.dbd.CH).sub.n6
(CH.sub.2).sub.n7(CH.dbd.CH).sub.n8(CH.sub.2).sub.n9; the sum of
n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer of from 3 to 23; that
is, the acyl chain is from 4-24 carbon atoms in length, n1 is equal
to zero or is an integer of from 1 to 23; n3 is equal to zero or is
an integer of from 1 to 20; n5 is equal to zero or is an integer of
from 1 to 17; n7 is equal to zero or is an integer of from 1 to 14;
n9 is equal to zero or is an integer of from 1 to 11; and each of
n2, n4, n6 and .[.8.]. .Iadd.n8 .Iaddend.is independently equal to
zero or 1.
The hydrocarbon chain is preferably saturated, that is, it
preferably has no double bonds between adjacent carbon atoms, each
of n2, n4, n6 and n8 then being equal to zero. Accordingly, Y.sub.1
is preferably (CH.sub.2).sub.n1. More preferably, R.sub.1 is
(CH.sub.2).sub.n1CH.sub.3, and most preferably, is
(CH.sub.2).sub.17CH.sub.3. Alternatively, the chain can have one or
more double bonds, that is, it can be unsaturated, and one or more
of n2, n4, n6 and n8 can be equal to 1. For example, when the
unsaturated hydrocarbon has one double bond, n2 is equal to 1, n4,
n6 and n8 are each equal to zero and Y.sub.1 is
(CH.sub.2).sub.n1CH.dbd.CH (CH.sub.2).sub.n3. n1 is then equal to
zero or is an integer of from 1 to 21, and n3 is also zero or is an
integer of from 1 to 20, at least one of n1 or n3 not being equal
to zero.
Z is oxygen, sulfur, NH, or --NHC(O)--, Z then being connected to
the methyl group by way of either the nitrogen or carbonyl carbon.
Z can also be --OC(O)--, it then being connected to the methyl
group by way of either the oxygen or carbonyl carbon atom.
Preferably, Z is O; accordingly, this invention's glycerol-based
etherlipids preferably have a methoxy group at the sn-2 position of
their glycerol backbone.
R.sub.2 is an alkyl group, or a halogen-substituted alkyl group,
having the formula
(C(X.sub.1).sub.n10(X.sub.2).sub.n11).sub.n12CX.sub.3X.sub.4X.sub.5,
wherein each of X.sub.1, X.sub.2, X.sub.3, X.sub.4, and X.sub.5 is
independently hydrogen or a halogen, but is preferably hydrogen.
n10 is equal to zero, 1 or 2; n11 is equal to zero, 1, or 2; and
n12 is equal to zero or an integer of from 1 to 23, but is most
preferably, zero, R.sub.2 then being CX.sub.3X.sub.4X.sub.5.
X.sub.3, X.sub.4, and X.sub.5 are most preferably H, R.sub.2 then
being CH.sub.3. Accordingly, the etherlipid preferably has a methyl
group attached to its carbon #2. However, R.sub.2 can then also be
CH.sub.2F, CHF.sub.2 or CF.sub.3. When n12 is not zero, the sum of
n10+n11 is equal to 2, n12 is preferably equal to 1, and R.sub.2 is
preferably CH.sub.2CH.sub.3, CH.sub.2CF.sub.3 or
CF.sub.2CF.sub.3.
Most preferably, the etherlipid is one in which Y.sub.2 is
CH.sub.3, R.sub.1 is (CH.sub.2).sub.n1CH.sub.3, R.sub.2 is CH.sub.3
and Z is O. The preferred etherlipid is therefore: ##STR00004##
that is, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine
("ET-18-OCH.sub.3" or "edelfosine").
Preferably, the phosphatidylcholine ("PC") is partially or wholly
unsaturated, that is, it has two acyl chains, at least one of which
has at least one double bond between adjacent carbon atoms. More
preferably, presently, the PC is dioleoyl phosphatidylcholine
("DOPC"). The liposome's lipid bilayer also contains a sterol,
which generally affects the fluidity of lipid bilayers (see, for
example, Lewis and McElhaney (1992) and Darnell et al. (1986)).
Accordingly, sterol interactions with surrounding hydrocarbon
chains generally inhibit emigration of these chains from the
bilayer. The sterol of the liposomes of this invention is
preferably, but not necessarily, cholesterol, and can also be a
variety of other sterolic compounds.
A "headgroup-derivatized" lipid is a lipid which, when present in a
liposomal lipid bilayer with an etherlipid, can buffer the toxicity
of the etherlipid. That is, the derivatized lipid can decrease the
etherlipid's toxicity, such that it is generally less toxic than
the free form of the etherlipid. Headgroup-derivatized lipids
generally are amphipathic lipids comprising hydrophobic acyl
chains, and a phosphorylethanolamine group to which a suitable
chemical moiety has been attached. Acyl chains are those which can
adopt compatible packing configurations with the hydrophobic
portions of other lipids present in the bilayer, and which can
interact with an etherlipid such that release of the etherlipid
from the bilayer is inhibited and etherlipid toxicity is buffered;
these are saturated or unsaturated, straight-chained or branched,
and typically contain from 4 to 24 carbon atoms in a straight
chain. Preferred acyl chains are palmitate or oleate chains; hence
preferred headgroup-modified lipids are dipalmitoyl
phosphatidylethanolamine ("DPPE"), palmitoyloleoyl
phosphatidylethanolamine ("POPE") or dioleoyl
phosphatidylethanolamine ("DOPE"); most preferably, presently, the
lipid is DOPE.
Chemical moieties suitable for attachment to such lipids are those,
such as dicarboxylic acids, gangliosides, polyethylene glycols,
polyalkyl ethers and the like, which can be attached to the amino
group of a phosphorylethanolamine, and which give rise to lipids
having toxicity buffering, circulation-enhancing properties. Means
of identifying suitable chemical moieties, for example by
subjecting derivatized lipids to in vitro and in vivo toxicity
testing, are well known to, and readily practiced by, ordinarily
skilled artisans given the teachings of this invention. Means of
attaching chemical moieties to phosphorylethanolamine groups are
also well known to, and readily practiced by, ordinarily skilled
artisans.
Toxicity buffering capacities of headgroup-derivatized lipids can
be determined by a number of in vitro and in vivo testing methods
well known to, and readily practiced by, ordinarily skilled
artisans, given the teachings of this invention. For example,
etherlipid-induced red blood cell (RBC) hemolysis can be examined
in vitro by combining an etherlipid with an RBC suspension,
incubating the combination, and then quantitating the percentage of
RBC lysis.
Toxicity-buffering can also be assessed by determining the
etherlipid's therapeutic window "TW," which is a numerical value
derived from the relationship between the compound's induction of
hemolysis and its ability to inhibit the growth of tumor cells. TW
values are determined in accordance with the formula
HI.sub.5/GI.sub.50 (wherein "HI.sub.5" equals the concentration of
compound inducing the hemolysis of 5% of the red blood cells in a
culture, and wherein "GI.sub.50" equals the dose of compound
inducing fifty percent growth inhibition in a population of cells
exposed to the agent). The higher an agent's HI.sub.5 value, the
less hemolytic is the agent--higher HI.sub.5's mean that greater
concentrations of compound are required to be present in order for
the compound to induce 5% hemolysis. Hence, the higher its
HI.sub.5, the more therapeutically beneficial is a compound,
because more of it can be given before inducing the same amount of
hemolysis as an agent with a lower HI.sub.5. By contrast, lower
GI.sub.5's indicate better therapeutic agents--a lower GI.sub.50
value indicates that a lesser concentration of an agent is required
for 50% growth inhibition. Accordingly, the higher is its HI.sub.5
value and the lower is its GI.sub.50 value, the better are a
compound's agent's therapeutic properties.
Generally, when a bioactive agent's TW is less than 1, it cannot be
used effectively as a therapeutic agent. That is, the agent's
HI.sub.5 value is sufficiently low, and its GI.sub.50 value
sufficiently high, that it is generally not possible to administer
enough of the agent to achieve a sufficient level of tumor growth
inhibition without also attaining an unacceptable level of
hemolysis. Etherlipid liposomes having bilayers that also comprise
headgroup-derivatized lipids have TS's of greater than 1.
Preferably, the TW of an etherlipid in a liposomal bilayer also
comprising a headgroup-derivatized lipid is greater than about 1.5,
more preferably, greater than about 2, and still more preferably,
greater than about 3.
Headgroup-derivatized lipids can also be circulation-enhancing
lipids, that is, the modifications directed to lipid toxicity
buffering can also afford circulation enhancement. Accordingly,
headgroup-derivatized lipids can inhibit clearance of liposomes
from the circulatory systems of animals to which they have been
administered. Liposomes are generally believed to be cleared from
an animal's body by way of its reticuloendothelial system (RES).
Avoiding RES clearance means that the frequency of liposome
administration can be reduced, and that less of a
liposome-associated bioactive agent need be administered to achieve
desired serum levels of the agent. Enhanced circulation times can
also allow targeting of liposomes to non-RES containing
tissues.
Liposome outer surfaces are believed to become coated with serum
proteins, such as opsonins, in animals' circulatory systems.
Without intending in any way to be limited by theory, it is
believed that liposome clearance can be inhibited by modifying the
outer surface of liposomes such that binding of serum proteins
thereto is generally inhibited. Effective surface modification,
that is, alterations to the outer surfaces of liposomes which
result in inhibition of opsonization and RES uptake, is believed to
be accomplished by incorporating into liposomal bilayers lipids
whose polar headgroups have been derivatized by attachment thereto
of a chemical moiety which can inhibit the binding of serum
proteins to liposomes such that the pharmacokinetic behavior of the
liposomes in the circulatory systems of animals is altered (see,
e.g., Blume et al. (1993); Gabizon et al. (1993); Park et al.
(1992); Woodle et al. U.S. Pat. No. 5,013,556; and, U.S. Pat. No.
4,837,028).
Presently, dicarboxylic acids, such as glutaric, sebacic, succinic
and tartaric acids, are preferred components of
headgroup-derivatized lipids. Most preferably, the dicarboxylic
acid is glutaric acid ("GA"). Accordingly, preferred
headgroup-derivatized lipids include
phosphatidylethanolamine-dicarboxylic acids such as dipalmitoyl
phosphatidylethanolamine-glutaric acid ("DPPE-GA"), palmitoyloleoyl
phosphatidylethanolamine-glutaric acid ("POPE-GA") and dioleoyl
phosphatidylethanolamine-glutaric acid ("DOPE-GA"). Most
preferably, presently, the derivatized lipid is DOPE-GA.
The liposomes of this invention can comprise one or more additional
lipids, that is, lipids in addition to the phosphatidylcholine,
sterol, headgroup-derivatized lipid and etherlipid already present
in the liposomes' bilayers. Additional lipids are selected for
their ability to adapt compatible packing conformations with the
other lipid components of the bilayer such that the lipid
constituents are tightly packed, and release of the lipids from the
bilayer is inhibited. Lipid-based factors contributing to
compatible packing conformations are well known to ordinarily
skilled artisans and include, without limitation, acyl chain length
and degree of unsaturation, as well as the headgroup size and
charge. Accordingly, suitable additional lipids, including various
phosphatidylethanolamines ("PE's") such as egg
phosphatidylethanolamine ("EPE") or dioleoyl
phosphatidylethanolamine ("DOPE") can be selected by ordinarily
skilled artisans without undue experimentation.
Preferred embodiments of this invention have the
phosphatidylcholine being DOPC, the sterol being cholesterol
("chol"), the headgroup-derivatized lipid being DOPE-GA and the
etherlipid being ET-18-OCH.sub.3. Most preferably, presently, the
liposome comprises DOPC, chol, DOPE-GA and ET-18-O-CH.sub.3 in a
respective molar ratio of 4:3:1:2, wherein DOPC comprises 40 mole %
of the bilayer lipid component, chol 30% mole, DOPE-GA 10 mole %
and the etherlipid 20 mole %. Preferably, the liposomes are
unilamellar and have an average diameter of from about 50 nm to
about 200 nm, "average" meaning that the median diameter of a
population of this invention's liposomes is between about 50 and
200 nm.
The liposome can comprise an additional bioactive agent, that is, a
bioactive agent in addition to the etherlipid. A "bioactive agent"
is any compound or composition of matter that can be administered
to animals, preferably humans. Such agents can have biological
activity in animals; the agents can also be used diagnostically in
the animals. Bioactive agents which may be associated with
liposomes include, but are not limited to: antiviral agents such as
acyclovir, zidovudine and the interferons; antibacterial agents
such as aminoglycosides, cephalosporins and tetracyclines;
antifungal agents such as polyene antibiotics, imidazoles and
triazoles; antimetabolic agents such as folic acid, and purine and
pyrimidine analogs; antineoplastic agents such as the anthracycline
antibiotics and plant alkaloids; sterols such as cholesterol;
carbohydrates, e.g., sugars and starches; amino acids, peptides,
proteins such as cell receptor proteins, immunoglobulins, enzymes,
hormones, neurotransmitters and glycoproteins; dyes; radiolabels
such as radioisotopes and radioisotope-labeled compounds;
radiopaque compounds; fluorescent compounds; mydriatic compounds;
bronchodilators; local anesthetics; and the like.
Liposomal bioactive agent formulations can enhance the therapeutic
index of the bioactive agent, for example by buffering the agent's
toxicity. Liposomes can also reduce the rate at which a bioactive
agent is cleared from the circulation of animals. Accordingly,
liposomal formulation of bioactive agents can mean that less of the
agent need be administered to achieve the desired effect.
Additional bioactive agents preferred for the liposome of this
invention include antimicrobial, anti-inflammatory and
antineoplastic agents, or therapeutic lipids, for example,
ceramides. Most preferably, the additional bioactive agent is an
antineoplastic agent.
Liposomes can be loaded with one or more biologically active agents
by solubilizing the agent in the lipid or aqueous phase used to
prepare the liposomes. Alternatively, ionizable bioactive agents
can be loaded into liposomes by first forming the liposomes,
establishing an electrochemical potential, e.g., by way of a pH
gradient, across the outermost liposomal bilayer, and then adding
the ionizable agent to the aqueous medium external to the liposome
(see Bally et al. U.S. Pat. No. 5,077,056 and WO86/01102).
The liposome of this invention can be dehydrated, stored and then
reconstituted such that a substantial portion of its internal
contents are retained. Liposomal dehydration generally requires use
of a hydrophilic drying protectant such as a disaccharide sugar at
both the inside and outside surfaces of the liposome bilayers (see
U.S. Pat. No. 4,880,635). This hydrophilic compound is generally
believed to prevent the rearrangement of the lipids in the
liposome, so that the size and contents are maintained during the
drying procedure and through subsequent rehydration. Appropriate
qualities for such drying protectants are that they be strong
hydrogen bond acceptors, and possess stereochemical features that
preserve the intramolecular spacing of the liposome bilayer
components. Alternatively, the drying protectant can be omitted if
the liposome preparation is not frozen prior to dehydration, and
sufficient water remains in the preparation subsequent to
dehydration.
Also provided herein is a pharmaceutical composition comprising a
pharmaceutically acceptable carrier and the liposome of this
invention. "Pharmaceutically acceptable carriers" as used herein
are those media generally acceptable for use in connection with the
administration of lipids and liposomes, including liposomal
bioactive agent formulations, to animals, including humans.
Pharmaceutically acceptable carriers are generally formulated
according to a number of factors well within the purview of the
ordinarily skilled artisan to determine and account for, including
without limitation: the particular liposomal bioactive agent used,
its concentration, stability and intended bioavailability; the
disease, disorder or condition being treated with the liposomal
composition; the subject, its age, size and general condition; and
the composition's intended route of administration, e.g., nasal,
oral, ophthalmic, topical, transdermal, vaginal, subcutaneous,
intramammary, intraperitoneal, intravenous, or intramuscular (see,
for example, Nairn (1985)). Typical pharmaceutically acceptable
carriers used in parenteral bioactive agent administration include,
for example, D5W, an aqueous solution containing 5% weight by
volume of dextrose, and physiological saline. Pharmaceutically
acceptable carriers can contain additional ingredients, for example
those which enhance the stability of the active ingredients
included, such as preservatives and anti-oxidants.
Further provided is a method of treating a mammal afflicted with a
cancer, e.g., a brain, breast, lung, colon or ovarian cancer, or a
leukemia, lymphoma, sarcoma, carcinoma, which comprises
administering the pharmaceutical composition of this invention to
the mammal, etherlipids being believed to be selectively cytotoxic
to tumor cells. Generally, liposomal etherlipids can be used to
treat cancers treated with free, that is, nonliposomal,
etherlipids. However, encapsulation of an etherlipid in a liposome
can enhance its therapeutic index, and therefore make the liposomal
etherlipid a more effective treatment.
An amount of the composition comprising an anticancer effective
amount of the etherlipid, typically from about 0.1 to about 1000 mg
of the lipid per kg of the mammal's body, is administered,
preferably intravenously. For the purposes of this invention,
"anticancer effective amounts" of liposomal etherlipids are amounts
effective to inhibit, ameliorate, lessen or prevent establishment,
growth, metastasis or invasion of one or more cancers in animals to
which the etherlipids have been administered. Anticancer effective
amounts are generally chosen in accordance with a number of
factors, e.g., the age, size and general condition of the subject,
the cancer being treated and the intended route of administration,
and determined by a variety of means, for example, dose ranging
trials, well known to, and readily practiced by, ordinarily skilled
artisans given the teachings of this invention. Antineoplastic
effective amounts of the liposomal etherlipid of this invention are
about the same as such amounts of free, nonliposomal, etherlipids,
e.g., from about 0.1 mg of the etherlipid per kg of body weight of
the mammal being treated to about 1000 mg per kg.
Preferably, the liposome administered is a unilamellar liposome
having an average diameter of from about 50 nm to about 200 nm. The
anti-cancer treatment method can include administration of one or
more bioactive agents in addition to the liposomal etherlipid,
these additional agents preferably, but not necessarily, being
included in the same liposome as the etherlipid. The additional
bioactive agents, which can be entrapped in liposomes' internal
compartments or sequestered in their lipid bilayers, are
preferably, but not necessarily, anticancer agents or cellular
growth promoting factors.
The liposomes are also effective as anti-inflammatory agents.
This invention will be better understood from the following
examples. However, those of ordinary skill in the art will readily
understand that these examples are merely illustrative of the
invention as defined in the claims which follow thereafter.
EXAMPLES
Example 1
Preparation
Liposomes were prepared with edelfosine (ET-18-O-CH.sub.3, 5
mg/ml), various other lipids obtained from Avanti Polar Lipids,
Birmingham, Ala., and cholesterol (Sigma Chemical Co.). Briefly,
the lipids were dissolved in an organic solvent, such as
chloroform, at various mole ratios. The organic solvent was then
removed, and the dried lipids were rehydrated, e.g., with
Dulbecco's phosphate-buffered saline (D-PBS) (Gibco BRL Life
Technologies, Grand Island, N.Y.). The resulting liposomes were
extruded through 0.1 micron Nuclepore.RTM. filters (see, for
example, Mayer et al., 1985). Liposome sizes were then determined
by light scattering, using a Nicomp.RTM. Model 370 Submicron
Particle Sizer.
Example 2
Red Blood Cell ("RBC") Hemolysis Assay
A 4% suspension of red blood cells (RBCs), 0.5 ml, was washed three
times in PBS and then incubated with free (non-liposomal)
etherlipid or liposomal etherlipid, prepared as described above.
These samples were vortexed on a 37 deg. C. agitator for 20 hours,
and were then centrifuged for 10 minutes at 3000 rpm. 0.2 ml of the
resulting supernatant was diluted to 1 ml with water, and the
percentage hemolysis in the sample was quantitated by
spectrophotometric examination at 550 nm.
Results from these studies are presented in Table 1 (see below),
wherein the concentration (.mu.M) of edelfosine required to cause
10% RBC hemolysis ("HI.sub.10") in each formulation is set forth.
The table's first column is a shorthand designation of the
particular formulation, "ELL" standing for "etherlipid liposome."
The second column indicates the components of the formulation
tested, including dioleoyl phosphatidylethanolamine "(DOPE"),
cholesterol ("CHOL"), dioleoyl-phosphatidylethanolamine-glutaric
acid ("DOPE-GA"), dioeloyl phosphatidylcholine ("DOPC"),
palmitoyloleoyl phosphatidylcholine ("POPC"), distearoyl
phosphatidylcholine ("DSPC"), egg phosphatidylcholine ("EPC") and
edelfosine ("EL," for etherlipid). The respective molar ratios of
the various lipid components are also set forth. The last row of
the table gives the HI.sub.10 value for edelfosine alone, i.e., not
incorporated in a liposome.
TABLE-US-00001 TABLE 1 Formulation Composition HI.sub.10 ELL 20
DOPE:CHOL:DOPE-GA:EL 1726 .+-. 160 4 3 1 2 ELL 12
DOPC:CHOL:DOPE-GA:EL 670 .+-. 60 4 3 1 2 ELL 40
POPC:CHOL:DOPE-GA:EL 65 .+-. 6 4 3 1 2 ELL 28 DSPC:CHOL:DOPE-GA:EL
32 .+-. 3 4 3 1 2 ELL 25 DOPE:CHOL:DOPE-GA:EL 537 .+-. 50 3 3 1 3
ELL 30 EPC:CHOL:DOPE-GA:EL 314 .+-. 30 4 3 1 2 Edelfosine --- 5
.+-. 1
Example 3
Fluorescence Spectroscopy
Liposomes were prepared as described above, and in the presence of
an aqueous solution of 0.1 M 6-carboxyfluorescein ("CF"); free CF
was then removed by gel filtration. CF efflux from liposomes over
time was monitored by measuring, at 520 nm (excitation at 490 nm),
increases in CF fluorescence in the aqueous phase external to the
liposomes, upon their incubation in PBS at 48 deg. C. Fluorescence
values, presented in FIG. 1 herein, are expressed as a percentage
increase in CF fluorescence relative to the total CF fluorescence
found after disrupting liposomes with Triton X-100.
FIG. 2 herein compares hemolytic activity and CF leakage in various
liposomal formulations described in Table 1, upon incubation of the
liposomes in PBS at 48 deg. C. for 25 minutes. FIG. 3 compares the
time required for 50% CF leakage in various liposomal formulations,
upon their incubation in 0.5% serum at 37 deg. C.
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