Polyethylene glycol modified ceramide lipids and liposome uses thereof

The present invention provides a novel class of polyethylene glycol modified ceramide lipids. The lipids can be used to form liposomes optionally containing various biological agents or drugs, such as anti-cancer agents. In addition, methods of use for the liposomes are provided.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to novel polyethylene glycol (PEG) 
derivatized lipids, their method of preparation and their use in liposomes 
or other lipid-based carriers. More specifically, the present invention 
includes PEG-Ceramide lipids and their inclusion in liposomes for use in 
drug delivery. 
2. The Relevant Art 
Liposomes are vesicles comprised of concentrically ordered lipid bilayers 
which encapsulate an aqueous phase. Liposomes form when lipids, molecules 
which typically comprise a polar head group attached to one or more long 
chain aliphatic tails, such as phospholipids, are exposed to water. Upon 
encountering such media the lipids aggregate to form a structure in which 
only the polar head groups are exposed to the external media to form an 
external shell inside which the aliphatic tails are sequestered. See, 
e.g., Lehninger, PRINCIPLES OF BIOCHEMISTRY (Worth, 1982). Liposomes can 
entrap a variety of bioactive or pharmaceutical agents for delivery of 
these agents to cells and tissues in vivo. See, e.g., U.S. Pat. No. 
5,185,154 to Lasic, et al.; European Patent Application No. 526,700 to 
Tagawa, et al.; and U.S. Pat. No. 5,013,556 to Woodle, et al. 
Liposomes can alter the biodistribution and rate of delivery of an 
encapsulated bioactive agent in a number of ways. For example, drugs 
encapsulated in liposomes are protected from interactions with serum 
factors which may chemically degrade the drug. The size of the liposome 
compared to the free drug also affects its access to certain sites in the 
body; this property can be advantageous in limiting drug delivery to 
certain sites. Uptake by the reticuloendothelial system (RES) can be 
inhibited by including factors on the liposome surface that inhibit 
protein association with the liposome or liposome interactions with RES 
cells, for example, by using PEG-lipids with other lipids such as 
ganglioside GM.sub.1. See, Woodle, supra. 
A variety of liposome structures can be formed using one or more lipids. 
Typical classes of liposome structures include small unilamellar vesicles 
(SUVs), large unilamellar vesicles (LUVs), or multilamellar vesicles 
(MLVs). The construction of liposomes and their application as delivery 
systems is described in the art. See, e.g., LIPOSOMES, Marc J. Ostro, ed. 
(Marcel Dekker 1983). 
Liposomes have been prepared by derivatizing existing lipid systems to form 
new liposome structures. For example, polyethyleneglycol (PEG) derivatized 
lipids have been developed. See Woodle, supra. 
Typically, PEG-lipids are prepared by derivatization of the polar head 
group of a diacylglycerophospholipid, such as 
distearoylphosphatidylethanolamine (DSPE), with PEG. These phospholipids 
usually contain two fatty acyl chains bonded to the 1- and 2- position of 
glycerol by ester linkages. Unfortunately, these acyl groups are 
susceptible to cleavage under acidic or basic conditions. The resulting 
hydrolytic products, such as analogs of lysophospholipid and 
glycerophosphate, do not remain associated with the bilayer structure of 
the liposome. Such dissociation may weaken the integrity of the liposome 
structure, leading to significant leakage of the bioactive agent or drug 
from the liposome and contributing to instability during storage, and thus 
shortened shelf-life of the liposome product. In addition, the loss of 
these hydrolysis products, such as PEG-lysophospholipid, from the liposome 
would negate the benefits otherwise resulting from the presence of the 
PEG-phospholipid. 
Lipid stability is important in the development of liposomal drug delivery 
systems. This is especially relevant when a transmembrane pH gradient is 
used to entrap or encapsulate the bioactive agent in the liposome, as very 
acidic (pH 2-4) or basic (pH 10-12) conditions may be used to achieve 
efficient drug uptake and retention. Therefore, it is desirable to develop 
PEG-lipids that are less susceptible to hydrolysis, thereby, increasing 
the liposome circulation longevity. 
SUMMARY OF THE INVENTION 
In one aspect, the present invention includes novel PEG-lipids such as the 
PEG-modified ceramide lipids of Formula I: 
##STR1## 
wherein: 
R.sup.1, R.sup.2, and R.sup.3 are independently hydrogen, C.sub.1 -C.sub.6 
alkyl, acyl, or aryl; 
R.sup.4 is hydrogen, C.sub.1 -C.sub.30 alkyl, C.sub.2 -C.sub.30 alkenyl, 
C.sub.2 -C.sub.30 alkynyl, or aryl; 
R.sup.5 is hydrogen, alkyl, acyl, aryl, or PEG; 
X.sup.1 is --O--, --S--, or --NR.sup.6 --, where R.sup.6 is hydrogen, 
C.sub.1 -C.sub.6 alkyl, acyl or aryl; or when R.sup.5 is PEG and b is 1, 
X.sup.1 is also --Y.sup.1 -alk-Y.sup.2 --; 
Y is --NR.sup.7 --, where R.sup.7 is hydrogen, C.sub.1 -C.sub.6 alkyl, acyl 
or aryl, or Y is --O--, --S-- or --Y.sup.1 -alk-Y.sup.2 --, wherein 
Y.sup.1 and Y.sup.2 are independently amino, amido, carboxyl, carbamate, 
carbonyl, carbonate, urea, or phosphoro; and alk is C.sub.1 -C.sub.6 
alkylene; 
PEG is a polyethylene glycol with an average molecular weight from about 
550 to about 8,500 daltons optionally substituted by C.sub.1 -C.sub.3 
alkyl, alkoxy, acyl or aryl; wherein a is 0 or 1; and b is 1 unless 
R.sup.5 is PEG wherein b is 0 or 1. 
More preferred are those compounds wherein R.sup.1, R.sup.2, R.sup.3, and 
R.sup.5 are hydrogen; R.sup.4 is alkyl; X.sup.1 is O, Y is succinate; and 
PEG has an average molecular weight of about 2,000 or about 5,000 daltons 
and is substituted with methyl at the terminal hydroxyl position. 
Also preferred are those compounds wherein R.sup.1, R.sup.2, R.sup.3, and 
R.sup.5 are hydrogen, R.sup.4 is alkyl; X.sup.1 is O; Y is --NH--; and PEG 
has an average molecular weight of about 2,000 or about 5,000 daltons and 
is substituted with methyl at the terminal position. 
Other preferred lipid compounds are those wherein R.sup.1, R.sup.2, 
R.sup.3, and R.sup.5 are hydrogen; R.sup.4 is C.sub.7 -C.sub.23 alkyl, 
X.sup.1 is O; Y is succinate; and PEG has an average molecular weight of 
about 2,000 daltons and is substituted with methoxy at the terminal 
hydroxyl position; more preferred are those lipid compounds wherein 
R.sup.4 is C.sub.13 -C.sub.19 alkyl. 
In another aspect, the present invention includes liposomes or other 
lipid-based carriers including the above-described PEG-Ceramide lipids. 
Preferred liposome compositions include the preferred lipids described 
above. In construction of the liposomes, various mixtures of the described 
PEG-Ceramide lipids can be used in combination and in conjunction with 
other lipid types, such as DOPE and DODAC, as well as DSPC, SM, Chol and 
the like, with DOPE and DODAC preferred. Typically, the PEG-Ceramide will 
comprise about 5 to about 30 mol % of the final liposome construction, but 
can comprise about 0.0 to about 60 mol % or about 0.5 to about 5 mol %. 
More preferred lipid compositions are those wherein a drug or a biological 
agent is encapsulated within the liposome. The invention also includes 
lipid complexes whereby the PEG-Ceramide lipid comprises about 0.01 to 
about 90 mol % of the complex. 
In still another aspect, the present invention includes methods for 
delivering therapeutic agents such as drugs and vaccines to a patient in 
need thereof comprising administering to the patient a therapeutically 
effective amount of such therapeutic agent in a liposome or a lipid-based 
carrier of the invention. Also provided are kits for preparing labeled 
liposomes, containing the PEG-Ceramide lipids, and pharmaceutical 
formulations containing liposomes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
The PEG-modified ceramide lipids of Formula I enhance the properties of 
liposomes by increasing the circulation longevity or lifetime of the 
liposome; preventing aggregation of the liposomes during covalent protein 
coupling, such as for targeting; preventing aggregation of liposomes 
incorporating targeting moieties or drugs, such as antibodies, and DNA; 
promoting drug retention within the liposome; and/or increasing bilayer or 
other stability of the liposome when low pH is required for encapsulation 
of the bioactive agents. These PEG-Ceramide lipids also reduce leakage due 
to hydrolysis of the fatty acyl chains of the liposome bilayer and are 
more stable than other lipid forms. 
DEFINITIONS 
As used herein, the term "alkyl" denotes branched or unbranched hydrocarbon 
chains, such as, e.g., methyl, ethyl, n-propyl, iso-propyl, n-butyl, 
sec-butyl, iso-butyl, tert-butyl, and 2-methylpentyl. These groups may be 
optionally substituted with one or more functional groups which are 
attached commonly to such chains, such as, e.g., hydroxyl, bromo, fluoro, 
chloro, iodo, mercapto or thio, cyano, alkylthio, heterocycle, aryl, 
heteroaryl, carboxyl, carbalkoyl, alkyl, alkenyl, nitro, amino, alkoxyl, 
amido, and the like to form alkyl groups such as trifluoromethyl, 
3-hydroxyhexyl, 2-carboxypropyl, 2-fluoroethyl, carboxymethyl and 
cyanobutyl and the like. 
The term "alkylene" refers to divalent alkyl as defined above, e.g., 
methylene (--CH.sub.2 --), propylene (--CH.sub.2 CH.sub.2 CH.sub.2 --), 
chloroethylene (--CHClCH.sub.2 --), 2-thiobutene (--CH.sub.2 
CH(SH)CH.sub.2 CH.sub.2 --), 1-bromo-3-hydroxyl-4-methylpentene 
(--CHBrCH.sub.2 CH(OH)CH(CH.sub.3)CH.sub.2 --) and the like. 
The term "alkenyl" denotes branched or unbranched hydrocarbon chains 
containing one or more carbon-carbon double bonds. 
The term "alkynyl" refers to branched or unbranched hydrocarbon chains 
containing one or more carbon-carbon triple bonds. 
The term "aryl" denotes a chain of carbon atoms which form at least one 
aromatic ring having preferably between about 6-14 carbon atoms, such as, 
e.g., phenyl, naphthyl, indenyl, and the like, and which may be 
substituted with one or more functional groups which are attached commonly 
to such chains, such as, e.g., hydroxyl, bromo, fluoro, chloro, iodo, 
mercapto or thio, cyano, cyanoamido, alkylthio, heterocycle, aryl, 
heteroaryl, carboxyl, carbalkoyl, alkyl, alkenyl, nitro, amino, alkoxyl, 
amido, and the like to form aryl groups such as biphenyl, iodobiphenyl, 
methoxybiphenyl, anthryl, bromophenyl, iodophenyl, chlorophenyl, 
hydroxyphenyl, methoxyphenyl, formylphenyl, acetylphenyl, 
trifluoromethylthiophenyl, trifluoromethoxyphenyl, alkylthiophenyl, 
trialkylammoniumphenyl, amidophenyl, thiazolylphenyl, oxazolylphenyl, 
imidazolylphenyl, imidazolylmethylphenyl and the like. 
The term "acyl" denotes groups --C(O)R, where R is alkyl or aryl as defined 
above, such as formyl, acetyl, propionyl, or butyryl. 
The term "alkoxy" denotes --OR--, wherein R is alkyl. 
The term "amido" denotes an amide linkage: --C(O)NH--. 
The term "amino" denotes an amine linkage: --NR-- wherein R is hydrogen or 
alkyl. 
The term "carboxyl" denotes --C(O)O--, and the term "carbonyl" denotes 
--C(O)--. 
The term "carbonate" indicates --OC(O)O--. 
The term "carbamate" denotes --NHC(O)O--, and the term "urea" denotes 
--NHC(O)NH--. 
The term "phosphoro" denotes --OP(O)(OH)O--. 
Structure and Preparation of Lipid Compounds 
The compounds of the invention are synthesized using standard techniques 
and reagents. It will be recognized that the compounds of the invention 
will contain various amide, amine, ether, thio, ester, carbonate, 
carbamate, urea and phosphoro linkages. Those of skill in the art will 
recognize that methods and reagents for forming these bonds are well known 
and readily available. See, e.g., March, ADVANCED ORGANIC CHEMISTRY (Wiley 
1992), Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); and 
Furniss, VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY 5th ed. (Longman 
1989). It will also be appreciated that any functional groups present may 
require protection and deprotection at different points in the synthesis 
of the compounds of the invention. Those of skill in the art will 
recognize that such techniques are well known. See, e.g., Green and Wuts, 
PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley 1991). 
A general sequence of reactions for forming the compound of the invention 
is illustrated below in Reaction Scheme I. As shown therein, ceramide 
derivative 1 is reacted with the PEG derivative PEG-Y.sup.1 -alk-RG. 
R.sup.1 -R.sup.4 and Y.sup.1 have their meanings as defined above. RG is a 
group which reacts with X.sup.2 to form the desired linkage Y.sup.2 
between PEG and the ceramide derivative (i.e., --Y.sup.1 -alk-Y.sup.2 --). 
Thus, it will be appreciated that the identities of RG and X.sup.2 will be 
complementary to each other and defined in such a way as to provide the 
desired linkage. For example, where RG is a nucleophilic center, such as 
--SH, --OH, or --NH.sub.2, X.sup.2 may be oxygen derivatized to form a 
good leaving group, such as --OTs where Ts represents the tosyl group, or 
halogen. Conversely, X.sup.2 may be a nucleophilic center, e.g., --SH, 
--OH, or --NH.sub.2, and RG a group which is reactive toward nucleophilic 
attack, e.g., carboxyl activated with dicyclohexylcarbodiimide (DCC) or 
acyl chloride (--COCl). By suitable choice of RG and X.sup.2, the desired 
amido, amine, ether, ester, thioether, carboxyl, carbamate, carbonyl, 
carbonate, urea or phosphoro coupling between the linker and the ceramide 
may be obtained. Finally any protecting groups, e.g., R.sup.8, remaining 
on the intermediate 2 are converted to form the desired PEG-Ceramide 
derivative 3. 
##STR2## 
An exemplary synthesis of the PEG-Ceramide lipids of the invention wherein 
Y.sup.1 and Y.sup.2 are carboxyl is illustrated below in Reaction Scheme 
II. To eliminate the potential problem of crosslinkage formation, PEG is 
capped at one end by an unreactive group such as methoxy or ethoxy. The 
second hydroxy group at the other terminal of the PEG molecule is either 
activated with a suitable reagent such as cyanuric acid, 
1,1'-carbonyldiimidazole (CDI) or tresyl halide. Alternatively the 
terminal hydroxyl group may first be converted to a derivative that can be 
readily reacted with ceramide in the presence of appropriate condensation 
reagents, such as the succinate or amine. In other alternative methods, 
the hydroxy groups on ceramide can be selectively activated for 
conjugation with PEG, or the two compounds can be linked in a concerted 
coupling reaction by established coupling procedures. 
In the example shown, the primary hydroxyl group of ceramide available 
commercially from Sigma Chemical Company (St. Louis, Mo.) and Avanti Polar 
Lipids Inc. (Alabaster, Ala.)! is reacted with a hydroxyl protecting group 
of the type which favors reaction at primary alcohols over secondary and 
tertiary alcohols. Preferred protecting groups are those which are 
considered sterically hindered, such trityl chloride (TrCl) which 
comprises three phenyl rings attached to a central carbon atom. However, 
other protecting groups are known in the art (see, Green and Wuts supra). 
This reaction is performed using standard techniques and conditions. 
Following the protection of the C.sub.1 hydroxyl group, the secondary 
alcohol at C.sub.3 is protected with a second protecting group. The second 
protecting group should be one which is reactive towards more hindered 
secondary alcohols, but which is not removed under conditions effective to 
remove the protecting group blocking the C.sub.1 alcohol. A preferred 
protecting group is benzyl (Bn). Again, other suitable protecting group 
combinations will be apparent to those of skill in the art. 
Once both of the hydroxyl groups are protected, the C.sub.1 --OH protecting 
group is removed under conditions which do not affect the protecting group 
at the C.sub.3 alcohol. The free hydroxyl function is then reacted with 
the PEG derivative Me(PEG)OC(O)CH.sub.2 CH.sub.2 CO.sub.2 H with 
dicyclohexylcarbodiimide (DCC) and 4-N,N'-dimethylaminopyridine (DMAP) to 
form the desired PEG-Ceramide derivative. 
The protecting group at C.sub.3 can be removed, if desired, to permit other 
reactions at this site to obtain other substituent groups. 
##STR3## 
In another approach, shown in Reaction Scheme III below, Y.sup.1 is a 
carboxyl ester group --OC(O)-- and Y.sup.2 is an amido --C(O)NH--. As 
shown in the scheme, the 1-amino analog of ceramide can be prepared by 
derivitization of the C.sub.1 hydroxyl group first to the corresponding 
C.sub.1 alkyl sulfonate (e.g., methyl sulfonate or 
2,2,2-trifluoroethanesulfonate). The latter is converted to the amino 
analog directly with ammonia or through an azide intermediate as shown. 
The 1-amino-ceramide is then coupled to the N-hydroxysuccinamide (NHS) 
ester of MePEG-S to form a MePEG-S-ceramide with an amide linkage. 
##STR4## 
Alternatively, the group Y may be --NR.sup.7 --, where R.sup.7 is hydrogen, 
alkyl, acyl, or aryl; or Y may be --O-- or --S--. Such embodiments may be 
formed using well known methods and reagents. For example, the embodiment 
wherein Y is --NH-- can be made by the synthesis pathway shown in Reaction 
Scheme IV below. There, the 1-mesyl-ceramide described above is reacted 
with the amino analog of (MePEG-NH.sub.2) to form the desired 
MePEG-Ceramide conjugate having an amino linkage. 
##STR5## 
Both the C.sub.1 and C.sub.3 hydroxy functions in ceramide can be activated 
with a reagent such as CDI to form the corresponding bis-imidazolyl 
formate. The latter is then reacted with the amino group of MePEG-NH.sub.2 
to form a conjugate with two MePEG molecules bonded to one ceramide. 
Either one or two PEG molecules can be selected to attach to each 
ceramide, allowing a more flexible arrangement for introducing specific 
properties to a liposomal system. 
The group Y=Y.sup.1 -alk-Y.sup.2 can be formed from readily available 
starting materials using known techniques. Preferred embodiments include 
those wherein Y.sup.1 and Y.sup.2 are both carbonyl (--C(O)--) or where 
one of Y.sup.1 or Y.sup.2 is carbonyl and the other is amido (--C(O)NH--). 
These groups can be formed from commercially available diacids, such as 
malonic acid (CH.sub.2 (CO.sub.2 H).sub.2), succinic acid (HO.sub.2 
CCH.sub.2 CH.sub.2 CO.sub.2 H), glutaric acid (HO.sub.2 CCH.sub.2 CH.sub.2 
CH.sub.2 CO.sub.2 H) and adipic acid (HO.sub.2 CCH.sub.2 CH.sub.2 CH.sub.2 
CH.sub.2 CO.sub.2 H) and the like; as well as substituted diacids, such as 
tartaric acid (HO.sub.2 CCH(OH)CH(OH)CO.sub.2 H), 3-methylglutaric acid 
(HO.sub.2 CCH.sub.2 CH(CH.sub.3)CH.sub.2 CO.sub.2 H) and the like, using 
methods well known in the chemical arts. Acyl derivatives, such as acyl 
chlorides, e.g., 3-carbomethoxypropiponyl chloride (ClC(O)C.sub.2 H.sub.4 
CO.sub.2 CH.sub.3), and amides corresponding to these compounds are 
available commercially or can be formed using known procedures. 
PEG is a linear, water-soluble polymer of ethylene oxide repeating units 
with two terminal hydroxyl groups. PEGs are classified by their molecular 
weights; for example, PEG 2000 has an average molecular weight of about 
2,000 daltons, and PEG 5000 has an average molecular weight of about 5,000 
daltons. PEGs are commercially available from Sigma Chemical Co. and other 
companies and include: 
monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene 
glycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidyl 
succinate (MePEG-S--NHS), monomethoxypolyethylene glycol-amine 
(MePEG-NH.sub.2), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), 
and monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). 
The attachment of PEG to the linker Y may be performed using methods and 
materials known in the art. Generally, a hydroxyl or amino moiety of the 
PEG group, is reacted with suitable derivative of Y so as to form the 
desired coupling. For example, reaction of a free hydroxyl functionality 
of MePEG-OH with an acyl chloride derivative, such as 
3-carbomethoxypropiponyl chloride (ClC(O)C.sub.2 H.sub.4 CO.sub.2 
CH.sub.3), available commercially from Aldrich Chemical Co., Milwaukee, 
Wis., provides Me(PEG)-OC(O)C.sub.2 H.sub.4 CO.sub.2 CH.sub.3. The methyl 
ester can be further derivatized, e.g., to the acyl chloride or amide, 
using standard procedures. Alternatively, Me(PEG)OC(O)CH.sub.2 CH.sub.2 
CO.sub.2 H may be formed from Me(PEG)-OH and succinic anhydride as shown 
below: 
##STR6## 
Still other methods will be apparent to those of skill in the art. 
To couple PEG directly to the ceramide, the hydroxy function in MePEG can 
be directly activated with reagent such as CDI to form the corresponding 
imidazolyl formate. The latter is then reacted with a nucleophile, such as 
one or both alcohol functions of ceramide, to form a conjugate with a 
carbonate linkage. Alternatively, coupling of the MePEG imidazolyl formate 
with 1-aminoceramide will result in the formation of a MePEG-Ceramide 
adduct with a carbamate linkage. 
Commercial ceramides, which are N-acyl fatty acids of sphingosines, may be 
obtained by phospholipase C cleavage of the phosphocholine in the 
respective sphingomyelin precursors, which are extracted from egg yolks 
and brain tissue. The sphingomyelin lipids differ in the composition of 
the fatty amide chains, such as in the carbon chain length and the number 
of double bonds. The following ceramides are commercially available from 
Sigma Chemical Co. and Avanti Polar Lipids Inc.: (1) Type III: from bovine 
brain (approximately 99%); (2) Type IV: bovin brain (approximately 99%); 
(3) from brain (approximately 99%); and (4) from egg (approximately 99%). 
The fatty amide chains differ in composition based upon the source of the 
sphingomyelin, as shown in Table I: 
TABLE I 
______________________________________ 
FATTY ACID CONTENT OF TISSUE DERIVED SPHINGOMYELIN 
Fatty Acid 
Egg Sphingomyelin 
Brain Sphingomyelin 
______________________________________ 
16:0 77.70 2.38 
18:0 7.44 57.99 
20:0 1.83 6.08 
22:0 3.98 9.16 
24:0 1.86 7.04 
24:1 2.80 14.71 
______________________________________ 
A wide variety of ceramide derivatives may be synthesized from common 
starting materials using known techniques. For example, starting from 
commercially available erythritol (Aldrich, Milwaukee, Wis.), any ceramide 
derivative may be synthesized as illustrated in Reaction Scheme V below. 
Selective protection of erythritol using known methods provides the 
starting material shown in Reaction Scheme V, wherein the C.sub.1 and 
C.sub.3 carbons are protected as the benzyl (Bn) derivatives, the C.sub.2 
carbon is protected as the methylmethoxy (MOM) ether and the C.sub.4 
carbon is protected as the 3,4-dimethoxybenzyl (DMPM) ether derivative. 
Selective removal of the DMPM group using dichlorodicyanoquinone (DDQ) 
provides the corresponding alcohol which can be oxidized using standard 
methods to form the aldehyde as shown (see, e.g., Larock, supra). 
Reaction of the aldehyde with the Wittig reagent Et.sub.3 P.sup.+ Cl.sub.4 
H.sub.29 Br.sup.- provides the trans olefin preferentially. 
Alternatively, reaction with the triphenylphosphine derivative Ph.sub.3 
P.sup.+ C.sub.14 H.sub.29 Br.sup.- provides the cis olefin predominantly. 
Again, other methods of olefin formation will be apparent to those of 
skill in the art. Removal of the MOM protecting group, followed by 
conversion of the alcohol using sodium azide (NaN.sub.3) and lithium 
aluminum hydride (LiAlH.sub.4) provides the desired amine which is reacted 
with an acyl chloride to produce the amide shown. Reaction of the amide 
with boron trichloride (BCl.sub.3) in methylene chloride (CH.sub.2 
Cl.sub.2) using a temperature gradient from -78.degree. C. to 0.degree. 
C., followed by reaction with methanol (MeOH) at -78.degree. C., provides 
the desired diol. Other equivalent methods of synthesis will be apparent 
to those of skill in the art. Additional information regarding the 
synthesis of sphingolipids and optically active ceramides can be found in 
Schmidt, et al. ANGEW. CHEM. INT. Ed. Engl (1987) 26:793; Kiso, et al. J. 
CARBOHYDR. CHEM. (1986) 5:335; and Nicolaou, et al. J. AMER. CHEM. SOC. 
(1988) 110:7910. 
Ceramides of varying fatty amide chain lengths also can be prepared by 
reacting the amine of Scheme V with various acyl chlorides R.sup.4 
C(O)Cl! or other acyl or acid derivatives, whereby the carbon chain length 
is based upon the particular acyl group used. Typically and preferably, 
the carbon chain length is from about 8 to about 24, without any double 
bonds present, e.g., an alkyl chain. Most preferred are those ceramides 
designated as 20:0, which designates a 20 carbon length chain with no 
double bonds, i.e., a completely saturated C.sub.20 alkyl as the fatty 
amide chain. Alternatively, ceramides with specific acyl chains of 
homogenous composition can be prepared by conjugation of a suitably 
activated carboxylic compounds, such as N-hydroxysuccinimide (NHS) ester 
of fatty acid, with the amino function of D-sphingosine. For example, an 
acyl chloride of eicosanoic acid (also known as arachidic acid) will 
provide a chain 20 carbons (C20) in length for the resulting amide side 
chain. Other acids preferably include: octanoic acid (also known as 
caprylic acid) for C8; myristic acid for C14; palmitic acid (also known as 
hexadecanoic acid) for C16; and tetracosanoic acid (also known as 
lignoceric acid) for C24. Ceramides with fatty amide chain lengths of 14 
to 20 carbons are especially preferred. Most preferred are those 14 or 20 
carbons in length. (It is understood that R4 is one carbon shorter in 
length than the starting acyl chloride or acid.) 
##STR7## 
Liposome Preparation 
After the lipids of Formula I are prepared, they can be utilized in 
liposome structures incorporating or entrapping one or more bioactive 
agents, wherein one or more of the lipid compounds comprise the liposome. 
For example, the fatty amide chain can have various lengths on the 
ceramide portion of the lipid, and a mixture of the various resulting 
lipid compounds forms the desired liposome. In addition, non-PEG ceramide 
lipids can be used to construct the liposome in conjunction with the 
lipids of Formula I. 
A variety of methods are available for preparing liposomes as described in, 
e.g., Szoka et al., 9 ANN. REV. BIOPHYS. BIOENG. 467 (1980); U.S. Pat. 
Nos. 4,235,871, 4,501,728, 4,837,028; the text LIPOSOMES Ch. 1 (supra) and 
Hope et al., 40 CHEM. PHYS. LIP. 89 (1986). One method produces 
multilamellar vesicles of heterogeneous sizes. In this method, the 
vesicle-forming lipids are dissolved in a suitable organic solvent or 
solvent system and dried under vacuum or an inert gas to form a thin lipid 
film. Alternatively, the lipids may be dissolved in an organic solvent 
such as tert-butyl alcohol or benzene:methanol (95:5 v/v) and lyophilized 
to form a homogeneous lipid mixture, which is in a more easily hydrated 
powder-like form. The dry lipid mixture is covered with an aqueous 
buffered solution and allowed to hydrate, typically over a 15-60 minute 
period with agitation. The size distribution of the resulting 
multilamellar vesicles can be shifted toward smaller sizes by hydrating 
the lipids under more vigorous agitation conditions. Full hydration of the 
lipids may be enhanced by freezing in liquid nitrogen and thawing to about 
50.degree. C. This cycle is usually repeated about five times. 
Following liposome preparation, the liposomes may be sized to achieve a 
desired size range and relatively narrow distribution of liposome sizes. A 
size range of about 0.05-0.20 microns allows the liposome suspension to be 
sterilized by filtration through a conventional filter, using typically a 
0.22 micron filter. The filter sterilization method can be carried out on 
a high through-put basis if the liposomes have been sized down to about 
0.05-0.20 microns. 
Several techniques are available for sizing liposomes, such as the sizing 
method described in U.S. Pat. No. 4,737,323. For example, sonicating a 
liposome suspension either by bath or probe sonication produces a 
progressive size reduction down to small unilamellar vesicles less than 
about 0.05 microns in size. Homogenization is another method which relies 
on shearing energy to fragment large liposomes into smaller ones. In a 
typical homogenization procedure, multilamellar vesicles are recirculated 
through a standard emulsion homogenizer until selected liposome sizes, 
typically between about 0.01 and 0.5 microns, are observed. In both 
methods, the particle size distribution can be monitored by conventional 
laser-beam particle size discrimination. 
Extrusion of liposome through a small-pore polycarbonate membrane or an 
asymmetric ceramic membrane is also an effective method for reducing 
liposomes to a relatively well-defined size distribution. Typically, the 
suspension is cycled through the membrane one or more times until the 
desired liposome size distribution is achieved. The liposomes may be 
extruded through successively smaller-pore membranes, to achieve a gradual 
reduction in liposome size. For use in the present invention, liposomes 
having a size of from about 0.05 microns to about 0.20 microns are 
preferred. Liposome preparations are also described by Deamer et al., in 
Liposomes (supra) LIPOSOME PREATIONS: METHODS AND MECHANISMS. 
Liposome size distributions also may be determined by quasi-elastic light 
scattering techniques. See Bloomfield, 10 Ann. Rev. Biophys. Bioeng. 421 
(1981). 
Use of Liposomes as Delivery Vehicles 
The liposomes prepared by using the lipid compounds of this invention can 
be labeled with markers that will facilitate diagnostic imaging of various 
disease states including tumors, inflamed joints or lesions. Typically, 
these labels will be radioactive markers, although fluorescent labels can 
also be used. The use of gamma-emitting radioisotopes is particularly 
advantageous as they easily can be counted in a scintillation well 
counter, do not require tissue homogenization prior to counting, and can 
be imaged with gamma cameras. 
Gamma- or positron- emitting radioisotopes are typically used, such as 
.sup.99 Tc, .sup.24 Na, .sup.51 Cr, .sup.59 Fe, .sup.67 Ga, .sup.86 Rb, 
.sup.111 In, .sup.125 I, and .sup.195 Pt as gamma-emitting; and such as 
.sup.68 Ga, .sup.82 Rb, .sup.22 Na, .sup.75 Br, .sup.122 I and .sup.18 F 
as positron-emitting. 
The liposomes also can be labelled with a paramagnetic isotope for purposes 
of in vivo diagnosis, as through the use of magnetic resonance imaging 
(MRI) or electron spin resonance (ESR). See, for example, U.S. Pat. No. 
4,728,575. 
Liposomes are a valuable system for the controlled delivery of drugs. As 
discussed earlier, liposomes formulated from PEG-lipids are especially 
advantageous, since they are more stable and have an increased half-life 
in circulation over conventional liposomes. Using liposomes as drug 
carriers allows more control of the site or rate of release of the drug, 
enabling more precision to be obtained in regulating the blood and organ 
levels of drug and/or its metabolites. Thus, drug dosages needed to 
produce clinical effects can be reduced which in turn reduces toxicity. 
Toxicity concerns are particularly valid in cancer chemotherapy where the 
dose levels required for beneficial effects and the doses that result in 
significant toxicity are very close. Thus, for cancer chemotherapy the use 
of liposome carriers for antitumor drugs can provide significant 
therapeutic advantages. 
Depending on the capture volume within the liposome and the chemical and 
physical properties of the bioactive agents, compatible bioactive agents 
can be simultaneously encapsulated in a single liposome. Simultaneous 
delivery of two or more synergistic drugs in this manner will ensure the 
delivery of these drugs to the same location in the body and maintain the 
drugs in close proximity to act together, thus greatly facilitating 
therapy. 
A wide variety of bioactive agents, pharmaceutical substances, or drugs can 
be encapsulated within the interior of the relatively impermeable bilayer 
membranes of the liposomes where these substances can be protected from 
the environment during transit to their target areas. These substances 
include antitumor agents, antibiotics, immunomodulators, anti-inflammatory 
drugs and drugs acting on the central nervous system (CNS). Especially 
preferred antitumor agents include actinomycin D, vincristine, 
vinblastine, cystine arabinoside, anthracyclines, alkylative agents, 
platinum compounds, antimetabolites, and nucleoside analogs, such as 
methotrexate and purine and pyrimidine analogs. Considering the preferred 
uptake of intravenously injected liposomes by the bone marrow, lymphoid 
organs, liver, spleen and lungs, and the macrophage cell, neoplasms and 
other diseases involving these organs can be effectively treated by 
PEG-derived liposome-entrapped drug. (See Daoud et al., "Liposomes In 
Cancer Therapy", 3 ADV DRUG DELIVERY REVIEWS 405-418, 1989.) 
Another clinical application of liposomes is as an adjuvant for 
immunization of both animals and humans. Protein antigens such as 
diphtheria toxoid, cholera toxin, parasitic antigens, viral antigens, 
immunoglobulins, enzymes, histocompatibility antigens can be incorporated 
into or attached onto the liposomes for immunization purposes. 
Liposomes are also particularly useful as carriers for vaccines that will 
targeted to the appropriate lymphoid organs to stimulate an immune 
response. 
Liposomes have been used as a vector to deliver immunosuppressive or 
immunostimulatory agents selectively to macrophages. In particular, 
glucocorticoids useful to suppress macrophage activity and lymphokines 
that activate macrophages have been delivered in liposomes. 
Liposomes with targeting molecules can be used to stimulate or suppress a 
cell. For example, liposomes incorporating a particular antigen can be 
employed to stimulate the B cell population displaying surface antibody 
that specifically binds that antigen. Similarly, PEG-stabilized liposomes 
incorporating growth factors or lymphokines on the liposome surface can be 
directed to stimulate cells expressing the appropriate receptors for these 
factors. Such an approach can be used for example, in stimulating bone 
marrow cells to proliferate as part of the treatment of cancer patients 
following radiation or chemotherapy which destroys stem cells and actively 
dividing cells. 
Liposome-encapsulated antibodies can be used to treat drug overdoses. The 
tendency of liposomes having encapsulated antibodies to be delivered to 
the liver has a therapeutic advantage in clearing substances such as toxic 
agents from the blood circulation. It has been demonstrated that whereas 
unencapsulated antibodies to digoxin caused intravascular retention of the 
drug, encapsulated antibodies caused increased splenic and hepatic uptake 
and an increased excretion rate of digoxin. 
Liposomes comprising PEG-lipids also find utility as carriers in 
introducing lipid or protein antigens into the plasma membrane of cells 
that lack the antigens. For example, histocompatibility antigens or viral 
antigens can be introduced into the surface of viral infected or tumor 
cells to promote recognition and killing of these cells by the immune 
system. 
In certain embodiments, it is desirable to target the liposomes of the 
invention using targeting moieties that are specific to a cell type or 
tissue. Targeting of liposomes using a variety of targeting moieties, such 
as ligands, cell-surface receptors, glycoproteins, and monoclonal 
antibodies, has been previously described. See U.S. Pat. Nos. 4,957,773 
and 4,603,044. The targeting moieties can comprise the entire protein or 
fragments thereof. 
Targeting mechanisms generally require that the targeting agents be 
positioned on the surface of the liposome in such a manner that the target 
moiety is available for interaction with the target; for example, a cell 
surface receptor. The liposome is designed to incorporate a connector 
portion into the membrane at the time of liposome formation. The connector 
portion must have a lipophilic portion that is firmly embedded and 
anchored into the membrane. It must also have a hydrophilic portion that 
is chemically available on the aqueous surface of the liposome. The 
hydrophilic portion is selected so as to be chemically suitable with the 
targeting agent, such that the portion and agent form a stable chemical 
bond. Therefore, the connector portion usually extends out from the 
liposome's surface and is configured to correctly position the targeting 
agent. In some cases it is possible to attach the target agent directly to 
the connector portion, but in many instances, it is more suitable to use a 
third molecule to act as a "molecular bridge." The bridge links the 
connector portion and the target agent off of the surface of the liposome, 
making the target agent freely available for interaction with the cellular 
target. 
Standard methods for coupling the target agents can be used. For example, 
phosphatidylethanolamine, which can be activated for attachment of target 
agents, or of derivatized lipophilic compounds, such as lipid-derivatized 
bleomycin, can be used. Antibody-targeted liposomes can be constructed 
using, for instance, liposomes that incorporate protein A. See Renneisen 
et al., 265 J. Biol. Chem. 16337-16342 (1990) and Leonetti et al., 87 
Proc. Natl. Acad. Sci. (USA) 2448-2451 (1990). Other examples of antibody 
conjugation are disclosed in U.S. patent application Ser. No. 08/316,394, 
filed Sep. 30, 1994, now abandoned, the teachings of which are 
incorporated herein by reference. Examples of targeting moieties also can 
include other proteins, specific to cellular components, including 
antigens associated with neoplasms or tumors. Proteins used as targeting 
moieties can be attached to the liposomes via covalent bonds. See Heath, 
Covalent Attachment of Proteins to Liposomes, 149 Methods in Enzymology 
111-119 (Academic Press, Inc. 1987). Other targeting methods include the 
biotin-avidin system. 
In some cases, the diagnostic targeting of the liposome can subsequently be 
used to treat the targeted cell or tissue. For example, when a toxin is 
coupled to a targeted liposome, the toxin can then be effective in 
destroying the targeted cell, such as a neoplasmic cell. 
Once the encapsulated bioactive agents or the liposomes themselves are 
taken up by the cell, the bioactive agents also can be targeted to a 
specific intracellular site of action if target recognizing moieties are 
incorporated into the agent. For example, protein agents to be delivered 
to the nucleus may comprise a nuclear localization signal sequence 
recombinantly engineered into the protein or the signal sequence may be on 
a separate protein or peptide covalently attached to the primary protein. 
Likewise, non-protein drugs destined for the nucleus may have such a 
signal moiety covalently attached. Other target recognizing moieties that 
can be recombinantly engineered into or covalently attached to protein 
components to be delivered by liposomes include ligands, receptors and 
antibodies or fragments thereof. 
The present invention also provides a kit for preparing labeled liposomes. 
The kit will typically be comprised of a container that is 
compartmentalized for holding the various elements of the kit. One 
compartment can contain the materials for preparing the label just prior 
to use. A second compartment can contain the liposomes with or without a 
pH buffer to adjust the composition pH to physiological range of about 7 
to about 8. The liposomes also can be provided in freeze-dried form for 
reconstitution at the time of use. Also included within the kit will be 
other reagents and instructions for use. 
Liposomes comprising the lipid compounds of this invention can be 
formulated as pharmaceutical compositions or formulations according to 
standard techniques using acceptable adjuvants or carriers. Preferably, 
the physiologically pharmaceutical compositions are administered 
parenterally, i.e., intravenously, subcutaneously, or intramuscularly. 
Suitable formulations for use in the present invention are found in 
REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., 18th ed. 1990). 
Preferably, the compositions are administered intravenously. Therefore, 
this invention provides for compositions for intravenous administration 
which comprise a solution of liposomes suspended in a 
physiologically-acceptable adjuvant or carrier, preferably an aqueous 
carrier, such as water, buffered water, isotonic saline, and the like. The 
compositions may be sterilized by conventional, well-known sterilization 
techniques, or may be sterile filtered. The resulting aqueous solutions 
may be packaged for use as is, or lyophilized; the lyophilized preparation 
being combined with a sterile aqueous solution prior to administration. 
The compositions can contain pharmaceutically-acceptable auxiliary 
substances as required to approximate the appropriate physiological 
conditions, such as pH adjusting and buffering agents, tonicity adjusting 
agents, wetting agents, and the like. Such agents include sodium acetate, 
sodium lactate, sodium chloride, potassium chloride, calcium chloride, 
sorbitan monolaurate, triethanolamine oleate, and the like. 
The concentration of liposomes useful in pharmaceutical compositions can 
range from about 0.05%, usually about 2-5%, or as much as about 10-30% by 
weight of the composition and the range of concentration is selected in 
accordance with the mode of administration and bioactive agent contained 
within the liposomes. 
Since the present liposomes made from PEG-Ceramide lipids are less 
susceptible to hydrolysis, they have a prolonged half-life resulting in 
prolonged circulation. Additionally, the liposome pharmaceutical 
composition can include lipid-protective agents that protect the liposomes 
against free-radical and lipid-peroxidative damage upon storage. Such 
protective agents include alpha-tocopherol and water-soluble, 
iron-specific chelators, such as ferrioxamine. 
Use of Lipids or Lipid-Based Carriers as Delivery Vehicles 
Cationic lipids may be used in the delivery of therapeutic genes or 
oligonucleotides intended to induce or to block production of some protein 
within the cell. Nucleic acid is negatively charged and must be combined 
with a positively charged entity to form a lipid complex suitable for 
formulation and cellular delivery. 
Cationic lipids have been used in the transfection of cells in vitro and in 
vivo (Wang C-Y, Huang L. pH-sensitive immunoliposomes mediate target 
cell-specific delivery and controlled expression of a foreign gene in 
mouse. PROC. NATL. ACAD. SCI USA, 1987; 84:7851-7855 and Hyde S. C., Gill 
D. R. Higgins C. F., et al. Correction of the ion transport defect in 
cystic fibrosis transgenic mice by gene therapy. NATURE. 
1993;362:250-255.) The efficiency of this transfection has often been less 
than desired, for various reasons. One is the tendency for cationic lipids 
complexed to nucleic acid to form unsatisfactory carriers. These carriers 
are improved by the inclusion of PEG lipids. 
Cationic lipids useful in producing lipid-based carriers for gene and 
oligonucleotide delivery are LIPOFECTIN (U.S. Pat. Nos. 4,897,355; 
4,946,787; and 5,208,036 by Eppstein et al.) and LIPOFECTACE (U.S. Pat. 
No. 5,279,883 by Rose). Both agents, as well as other transfecting 
cationic lipids, are available from Life Technologies, Inc. in 
Gaithersburg, Md. 
The invention will be better understood by reference to the following 
examples, which are intended to illustrate aspects of the invention, but 
the invention is not to be considered as limited thereto. 
EXAMPLE 1 
N-Eicosanoyl-D-Sphingosine C20:0-Ceramide! 
N-Hydroxysuccinimide (NHS) ester of eicosanoic acid was synthesized using 
the procedure of Lapidot et al. (J. Lipid Res., 1967, 8:142). 428 mg of 
the ester was added to a solution of D-sphingosine (Avanti Polar Lipids, 
Inc.) (300 mg) in anhydrous methylene chloride (CH.sub.2 Cl.sub.2, 16 ml) 
and triethylamine (118 mg) with stirring under nitrogen (N.sub.2) at 
20.degree. C. for 4 hours. Analysis by thin layer chromatography (t.l.c.) 
(silica gel, CHCl.sub.3 :CH.sub.3 OH:H.sub.2 O--65:25:4 v/v or CHCl.sub.3 
:CH.sub.3 OH--90:10 v/v) indicated most of the D-sphingosine had reacted. 
If necessary, another small portion of NHS-ester of eicosanoic acid 
(20-30 mg) may be added to complete the acylation of D-sphingosine!. The 
reaction mixture was cooled in ice and diluted with CH.sub.2 Cl.sub.2 (60 
ml), H.sub.2 O (30 ml) and neutralized with 1N HCl. The CH.sub.2 Cl.sub.2 
layer was washed with H.sub.2 O (2.times.30 ml) and dried (MgSO.sub.4) 
before evaporation to dryness in vacuo. The residue was recrystallized 
twice from acetone to give the pure product, N-eicosanoyl-D-sphingosine 
(428 mg), as a white solid. T.l.c. showed a single spot and .sup.1 H-NMR 
spectrum was consistent with the expected structure. 
EXAMPLE 2 
Monomethoxypolyethylene Glycol.sub.2000 -Succinate (MePEG.sub.2000 -S) 
Monomethoxypolyethylene glycol with an average molecular weight of 2000 
daltons (MePEG.sub.2000) (Sigma Chemical Co.), (4 g) dissolved in CH.sub.2 
Cl.sub.2 (30 ml) was treated with succinic anhydride (600 mg), 
triethylamine (400 mg) and 4-dimethylaminopyridine (DMAP) (250 mg), and 
stirred under N.sub.2 at 20.degree. C. for 16 hours. The reaction solution 
was diluted with CH.sub.2 Cl.sub.2 (60 ml), cooled in ice and H.sub.2 O 
(50 ml) added. The mixture was acidified with 1N HCl and the organic layer 
separated. The aqueous layer was further extracted with CH.sub.2 Cl.sub.2 
(2.times.30 ml). The combined organic extracts were dried (MgSO.sub.4) and 
then evaporated to dryness. The crude product was purified on a silica gel 
(G60) column eluted with a solvent system of CH.sub.2 Cl.sub.2 containing 
2 to 8% methanol. Fractions collected were analyzed by t.l.c. (silica gel, 
CHCl.sub.3 :CH.sub.3 OH-88:12 v/v) and those containing the pure product 
(MePEG.sub.2000 -S) with R.sub.f value of 0.4 were pooled and 
concentrated. Trituration of the product with diethyl ether gave 
MePEG.sub.2000 -S as a white solid (3.2 g). 
EXAMPLE 3 
Monomethoxypolyethylene Glycol.sub.5000 -Succinate (MePEG.sub.5000 -S) 
The titled compound was prepared from monomethoxypolyethylene glycol with 
an average molecular weight of 5000 daltons (MePEG.sub.5000) (Sigma 
Chemical Co.) in a similar procedure as described above for MePEG.sub.2000 
-S. 
EXAMPLE 4 
1-O-(MePEG.sub.2000 -S)-(C20: 0-Ceramide) 
C20:0-Ceramide (60 mg), dicyclohexylcarbodiimide (DCC) (28 mg) and DMAP (13 
mg) were dissolved in warm anhydrous CH.sub.2 Cl.sub.2 (6 ml). 
MePEG.sub.2000 -S (230 mg) in anhydrous CH.sub.2 Cl.sub.2 (1 ml) was added 
dropwise to the above solution with stirring under N.sub.2 at 25.degree. 
C. for 6 hours. The precipitated dicyclohexylurea (DCU) was filtered off 
and the filtrate concentrated in vacuo. Trituration of the solid residue 
with diethyl ether removed most of the DCC, DMAP and unreacted 
C20:0-ceramide. The resulting crude product was chromatographed on a short 
silica gel column (G60) eluted with CH.sub.2 Cl.sub.2 :CH.sub.3 OH-98:2 
(v/v). Fractions containing the product were combined and evaporated to 
dryness in vacuo. The resulting solid was dissolved in distilled H.sub.2 O 
(2 ml) and dialysed at 4.degree. C. against distilled water overnight. The 
pure product was obtained as a white powder (160 mg) by lyophilization. 
T.l.c. (silica gel, CHCl.sub.3 :CH.sub.3 OH-90:10 v/v showed a single spot 
(R.sub.f 0.5). .sup.1 H-NMR spectrum of the product was consistent with 
the structure of 1-O-(MePEG.sub.2000 -S)-(C20:0-ceramide). PEG.sub.2000 
Ceramide! 
EXAMPLE 5 
1-O-(MePEG.sub.2000 -S) -(Egg Ceramide) PEG.sub.2000 Ceramide! 
Egg ceramide (Avanti Polar Lipids, Inc.) (108 mg), DCC (48 mg), and DMAP 
(25 mg) were dissolved in warm anhydrous CH.sub.2 Cl.sub.2 (8 ml). 
MePEG.sub.2000 -S (460 mg) in anhydrous CH.sub.2 Cl.sub.2 (2 ml) was added 
dropwise to the above solution with stirring under N.sub.2 at 25.degree. 
C. for 6 hours. The precipitated DCU was filtered off and the filtrate 
concentrated in vacuo. Trituration of the solid residue with diethyl ether 
removed most of the residual reagents and small amount of unreacted egg 
ceramide. The crude product was chromatographed on a short silica gel 
column (G60) eluted with CH.sub.2 Cl.sub.2 :CH.sub.3 OH-98:2 (v/v). 
Fractions containing the product were combined and evaporated to dryness 
in vacuo. The resulting solid was dissolved in distilled water (2 ml) and 
dialysed at 4.degree. C. against distilled water overnight. Lyophilization 
of the solution gave the pure product as a white powder (338 mg). T.l.c. 
(silica gel, CHCl.sub.3 OH-90:10 v/v) showed a single spot (R.sub.f 0.5) 
.sup.1 H-NMR spectrum of the product was in agreement with the structure 
of 1-O-(MePEG.sub.2000 -S)-(egg ceramide). 
EXAMPLE 6 
1-O-(MePEG.sub.5000 -S)-(egg Ceramide) PEG.sub.5000 Ceramide! 
The titled compound was prepared from MePEG.sub.5000 -S (550 mg) and egg 
ceramide (54 mg) in a procedure similar to that described above for 
1-O-(MePEG.sub.2000 -S)-(egg ceramide) using DCC (28 mg) and DMAP (13 mg) 
as the condensation reagents in anhydrous CH.sub.2 Cl.sub.2 (6 ml). 
Similar purification by column chromatography (silica gel) and dialysis 
gave the pure product 1-O-(MePEG.sub.5000 -S)-(egg ceramide) as a white 
powder (310 mg). 
EXAMPLE 7 
Plasma Clearance of Specific Liposomes 
In this example, the plasma clearance for 100 nm liposomes prepared of 
Distearylphosphatidylcholine (DSPC)/Cholesterol (Chol) (55:45 mol %; open 
circles); DSPC/Chol/PEG.sub.2000 Ceramide (50:45:5 mol %; filled circles), 
and DSPC/Chol/PEG.sub.5000 Ceramide (50:45:5 mol %; filled squares) was 
determined. The results are shown in FIG. 1A. Lipid mixtures were prepared 
in chloroform (CHCl.sub.3) and subsequently dried under a stream of 
nitrogen gas. The resulting lipid film was placed under high vacuum for at 
least 2 hours prior to hydration with 150 mM sodium chloride and 20 mM 
Hepes (pH 7.4) (Hepes buffered saline solution). Liposomes were then 
prepared by extrusion through 100 nm pore size filters using an Extruder 
pre-heated to 65.degree. C. prior to extrusion. The resulting liposomes 
exhibited a mean diameter of approximately 120 nm. These liposomes were 
diluted such that mice (female CD1) could be given an i.v. dose of lipid 
equivalent to 50 .mu.moles/kg in an injection volume of 200 .mu.l. At 
various time points indicated in FIG. 1A, blood samples were taken by 
nicking the tail vein and collecting 25 .mu.l of blood into a EDTA 
(ethylenediaminetetraacetic acid) coated capillary tube. The amount of 
lipid in the resulting sample was determined by measuring the amount of 
3H!-cholesteryl hexadecyl ether present. This non-exchangeable, 
non-metabolizable lipid marker was incorporated into the liposomes prior 
to formation of the lipid film. 
EXAMPLE 8 
Plasma Clearance of Specific Liposomes 
This example illustrates the plasma clearance for 100 nm liposomes prepared 
of DSPC/Chol (55:45 mol %; open circles), Sphingomyelin/Chol (55:45 mol %; 
filled circles), and Sphingomyelin/Chol/PEG.sub.2000 Ceramide (50:45:5 mol 
%; open squares). The results are presented in FIG. 1B. Lipid mixtures 
were prepared in chloroform (CHCl.sub.3) and subsequently dried under a 
stream of nitrogen gas. The resulting lipid film was placed under high 
vacuum for at least 2 hours prior to hydration with a 300 mM citrate 
buffer (pH 4.0). Liposomes were then prepared by extrusion through 100 nm 
pore size filters using an Extruder pre-heated to 65.degree. C. prior to 
extrusion. The resulting vesicles were diluted with 150 mM NaCl, 20 mM 
Hepes, pH 7.4 and the pH adjusted to 7.4 by titration with 500 mM sodium 
phosphate. The sample was then heated at 60.degree. C. for 10 minutes. The 
resulting liposomes exhibited a mean diameter of approximately 120 nm. 
These liposomes were diluted such that mice (female BDF1) could be given 
an i.v. dose of lipid equivalent to 20 mg/kg in an injection volume of 200 
.mu.l. At the time points indicated in FIG. 1B, blood samples were taken 
by cardiac puncture. The amount of lipid in the resulting sample was 
determined by measuring the amount of 3H!-cholesteryl hexadecyl ether 
present. This non-exchangeable, non-metabolizable lipid marker was 
incorporated into the liposomes prior to formation of the lipid film. 
EXAMPLE 9 
Vincristine Retention 
In this example, vincristine retention by Sphingomyelin/Chol (55:45 mol %) 
liposomes within the circulation was shown not to be affected by 
incorporation of PEG.sub.2000 Ceramide. In contrast, similar formulations 
prepared with PEG.sub.2000 -Phosphatidylethanolamine (PEG-PE) exhibit 
significantly reduced drug retention. The results were obtained by 
measuring both liposomal lipid (14C!-cholesterylhexadecyl ether) and drug 
(3H!-Vincristine) in plasma collected form BDF1 mice given an i.v. 
injection of liposomal vincristine (2 mg drug/kg). Samples were injected 
in a volume of 200 .mu.l. The liposomes were prepared as described below. 
The dry lipid was hydrated with 300 mM citrate -buffer, pH 4.0. Following 
extrusion, the vesicles (100 mg/ml) were added to a solution of 
vincristine (Oncovin; 1 mg/ml) to achieve a drug:lipid weight ratio of 
0.1:1. The exterior pH of the liposome/vincristine mixture was raised to 
pH 7.0-7.2 by titration with 500 mM sodium phosphate and immediately the 
sample was heated at 60.degree. C. for 10 minutes to achieve encapsulation 
of the vincristine. At the time points following i.v. administration in 
mice, shown in FIG. 2, blood samples were taken by cardiac puncture. The 
amount of vincristine and the amount of lipid were measured by use of 
appropriately labeled markers. The ratio of drug to lipid was then 
determined and plotted as a percentage of the original drug to lipid 
ratio. The DSPC/Chol liposome is represented by open circles, the SM/Chol 
liposome by filled circles; the SM/Chol/PEG.sub.2000 -ceramide by open 
squares; and the SM/CHOL/PEG-PE by filled squares. 
EXAMPLE 10 
Various PEG-Ceramide Acyl Chain Lengths and Effects on Retention Time 
Methods 
One hundred (100) mg of total lipid was dissolved in CHCl.sub.3 with 5 
.mu.Ci of .sup.14 H-cholesterylhexadecyl ether (Amersham custom 
synthesis). Lipid preparations consisted of egg sphingomyelin 
(SM)/cholesterol/PEG.sub.2000 -ceramide (SM/chol/PEG-Ceramide; 55/40/5, 
mol/mol/mol) or of egg sphingomyelin/cholesterol/PEG.sub.2000 
-distearolyphosphatidylethanolamine (SM/chol/PEG-DSPE; 55/40/5, 
mol/mol/mol). The PEG.sub.2000 -ceramides used in this study had fatty 
amide chain lengths of C8, C14, C20 or C24 or were synthesized from egg 
ceramide (egg-CER). Bulk CHCl.sub.3 was removed under a stream of nitrogen 
gas, then residual solvent was removed by placing the lipid film under 
high vacuum overnight. 
Liposomes were prepared by hydration of the lipid film with 1.0 mL of 0.3M 
citrate (pH 4.0) using extensive vortexing and brief heating to 65.degree. 
C. (Aliquots of this suspension were removed for determination of the 
specific activity.) The resulting lipid suspension was freeze/thaw cycled 
5 times between -196.degree. C. and 65.degree. C. Large unilamellar 
liposomes were produced by extrusion technology; the lipid suspension was 
passed through two stacked 0.1 .mu.m filters at 65.degree. C. using The 
Extruder (Lipex Biomembranes, Vancouver, B.C.). 
As a separate operation, 2.0 mg of vincristine (as 2.0 mL of vincristine 
sulfate at 1.0 mg/mL; David Bull Laboratories, Mulgrave, Australia) was 
labelled by the addition of 5 .mu.Ci of .sup.3 H-vincristine (Amersham) 
and aliquots removed for the determination of vincristine specific 
activity. 
For the liposomal encapsulation of vincristine, 5-6 mg of each lipid was 
removed to a glass test tube and labelled vincristine added to achieve a 
vincristine/lipid ratio of 0.1/1.0 (wt/wt). This mixture was equilibrated 
for 5-10 minutes at 65.degree. C., then vincristine encapsulation was 
initiated by the addition of sufficient 0.5M Na.sub.2 HPO.sub.4 to bring 
the solution pH to 7.0-7.5. Vincristine uptake was allowed to proceed for 
10 minutes at 65.degree. C., and the sample then cooled to room 
temperature and diluted with 150 mM NaCl, 20 mM Hepes (pH 7.5) (HBS) to 
the final concentration required for in vivo testing. The uptake of 
vincristine into the liposomes was determined by the centrifugation of 100 
.mu.L of liposomes on a 1.0 mL mini-column of Sephadex G-50 
pre-equilibrated in HBS. The eluate was assayed for vincristine/lipid 
ratio by liquid scintillation counting (LSC). 
Female BDF1 mice were administered i.v. by tail vein injection with 
liposomal vincristine at a dose of 20 mg lipid/kg, or 2 mg vincristine/kg. 
At 1, 4 and 24 hours after administration, blood was recovered by cardiac 
puncture into EDTA-containing Micro-Tainer tubes. Plasma was obtained by 
centrifugation at 2000 g for 15 minutes, and aliquots were assayed for 
lipid and vincristine content by LSC. 
All data represent the means (.+-.standard error) from 3 mice per time 
point, i.e., 9 animals/group. The half-lives of lipid, vincristine and the 
vincristine/lipid ratio were obtained from the slope of the semi-log plot 
of concentration vs. time. All r.sup.2 values for the linear regression of 
these slopes were&gt;0.98. Experiments with the C20 chain length of 
PEG.sub.2000 -ceramide were performed twice, and are presented separately. 
Results 
The results presented in FIG. 3 (Lipid T.sub.1/2) show that the half-life 
of SM/cholesterol liposomes containing 5 mol % PEG.sub.2000 -DSPE is 
approximately two-fold greater than SM/cholesterol liposomes containing 
PEG.sub.2000 -ceramides (PEG-Ceramide), regardless of the chain length. 
For the PEG-Ceramides, there was no significant influence of fatty acyl 
chain length on circulation longevity in these vincristine-loaded 
liposomes. 
The results presented in FIG. 4 (vincristine/lipid T.sub.1/2) indicate that 
there is a significant influence of acyl chain length on vincristine 
retention in the liposomes during circulation in the plasma. Specifically, 
C20 PEG-Ceramide was retained significantly better than both shorter (C8, 
C14, egg-CER and C24) chain lengths of PEG-Ceramide and also better than 
PEG-DSPE. The C20 chain lengths of PEG-Ceramide had half-life values for 
the vincristine/lipid ratio of 28-30 hours; about twice as long as those 
observed for the poorest vincristine retaining formulations at 15 hours 
(C8 PEG-Ceramide and PEG-DSPE). 
The combined result of lipid circulation longevity and drug retention 
within these liposomes is the circulation half-lives of vincristine (see 
FIG. 5). Amongst the PEG-Ceramides, the C20 chain length resulted in the 
greatest circulation lifetime for the vincristine (9.5-10.5 hours T1/2 vs. 
7-9 hours for the C8, C14, C24 and egg-CER chain lengths). In the samples 
containing PEG-DSPE, the combined influence of longer liposome circulation 
lifetime (FIG. 3) contrasted with poor vincristine retention (FIG. 4), 
resulted in overall drug half-life very similar to the C20 PEG-Ceramide. 
EXAMPLE 11 
Fusogenic Liposomes 
The ability of amphipathic polyethyleneglycol (PEG) derivatives to 
stabilize fusogenic liposomes containing a cationic lipid in vivo were 
examined in this study. A freeze-fracture electron microscope analysis of 
liposomes composed of dioleoylphosphatidylethanolamine (DOPE) and 
N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) showed that inclusion 
of amphipatic PEG derivatives, PEG-DSPE and PEG-Ceramide (PEG-Ceramide) 
effectively prevented liposome aggregation in the presence of mouse serum. 
Biodistribution of fusogenic liposomes composed of DOPE and DODAC, 
additionally containing an amphipathic polyethyleneglycol (PEG) 
derivative, were then examined in mice using .sup.3 H-labelled 
cholesterylhexadecylether as a lipid marker. Amphipathic PEG derivatives 
included PEG-DSPE and various PEG-Ceramide (PEG-Cer) with different acyl 
chain length ranging from C8 to C24. DOPE/DODAC liposomes (85:15, mol/mol) 
were shown to be cleared rapidly from the blood and accumulate exclusively 
in the liver. Inclusion of amphipathic PEG derivatives at 5.0 mol % of the 
lipid mixture resulted in increased liposome levels remaining in the blood 
and concomitantly decreased accumulation in the liver. Among various 
amphipathic PEG derivatives, PEG-DSPE shows the highest activity in 
prolonging the circulation time of DOPE/DODAC liposomes. The activity of 
PEG-Ceramide is directly proportional to the acyl chain length: the longer 
the acyl chain, the higher the activity. The activity of PEG-Ceramide 
(C20) exhibiting the optimal acyl chain length depends on its 
concentration of the lipid mixture with the maximal circulation time 
obtained at 30 mol % of the lipid mixture. While inclusion of amphipathic 
PEG derivatives in the lipid composition generally results in increased 
circulation time of DOPE/DODAC liposomes, the presence of a cationic 
lipid, DODAC, appeared to promote their rapid clearance from the blood. 
The preparations and uses of DODAC liposomes are disclosed in U.S. patent 
application Ser. No. 08/316,399, filed Sep. 30, 1994, now abandoned, the 
teachings of which are incorporated herein by reference. 
Fusogenic liposomes incorporating bilayer stabilizing components are 
disclosed in U.S. patent application Ser. No. 08/316,407, filed Sep. 30, 
1994, now abandoned, and U.S. patent application Ser. No. 08/485,608, 
filed Jun. 7, 1995 (Attorney Docket Number 016303-001310), the teachings 
of which are incorporated herein by reference. 
MATERIALS AND METHODS 
Liposome Preparation 
Small unilamellar liposomes composed of DOPE and DODAC additionally 
containing amphipathic PEG derivatives at various ratios were prepared by 
the extrusion method. Briefly, the solvent-free lipid mixture containing 
.sup.3 H-labelled CHE, as a nonexchangeable and nonmetabolizable lipid 
marker, was hydrated with distilled water overnight. Normally, the 
liposome suspension (5 mg lipid per ml) was extruded, at room temperature, 
10 times through stacked Nucleopore membranes (0.1 .mu.m pore size) using 
an extrusion device obtained from Lipex Biomembranes, Inc. to generate 
liposomes with homogeneous size distributions. Liposome size was 
determined by quasi-elastic light scattering using a particle sizer and 
expressed as average diameter with standard deviation (SD). 
Liposome Biodistribution Study 
.sup.3 H-labelled liposomes with various lipid compositions were injected 
i.v. into female CD-1 mice (8-10 weeks old) at a dose of 1.0 mg lipid per 
mouse in 0.2 ml of distilled water. At specified time intervals, mice were 
killed by overexposure to carbon dioxide, and blood was collected via 
cardiac puncture in 1.5-ml microcentrifuge tubes and centrifuged (12000 
rpm, 2 min, 4.degree. C.) to pellet blood cells. Major organs, including 
the spleen, liver, lung, heart, and kidney, were collected, weighed, and 
homogenized in distilled water. Fractions of the plasma and tissue 
homogenates were transferred to glass scintillation vials, solubilized 
with Solvable (NEN) at 50.degree. C. according to the manufacturer's 
instructions, decolored with hydrogen peroxide, and analyzed for .sup.3 H 
radioactivity in scintillation fluid in a Beckman counter. Data were 
expressed as percentages of the total injected dose of .sup.3 H-labelled 
liposomes in each organ. Levels of liposomes in the plasma were determined 
by assuming that the plasma volume of a mouse is 5.0% of the total body 
weight. 
RESULTS AND DISCUSSION 
Freeze-Fracture Electron Microscopic Studies 
Liposomes composed of DOPE/DODAC (85:15, mol/mol), DOPE/DODAC/PEG-Ceramide 
(C20) (80:15:5, mol/mol), and DOPE/DODAC/PEG-DSPE (80:15:5, mol/mol) were 
prepared by the extrusion method and had similar average diameters (100 
nm). Freeze-fracture electron micrographs of the three liposomal 
formulations showed unilamellar liposomes with relatively narrow size 
ranges. However, preincubation of DOPE/DODAC liposomes in 50% mouse serum 
at 37.degree. C. for 30 minutes resulted in their massive aggregations. On 
the other hand, both DOPE/DODAC/PEG-Ceramide (C20) and DOPE/DODAC/PEG-DSPE 
liposomes did not show any aggregation when these liposomes were 
pretreated with mouse serum. Thus, these results show the effectiveness of 
amphipathic PEG derivatives in preventing serum-induced rapid aggregations 
of DOPE/DODAC liposomes. 
Biodistribution of DOPE/DODAC Liposomes Containing Amphipathic PEG 
Derivatives 
DOPE/DODAC liposomes with or without amphipathic PEG derivatives were 
prepared to include .sup.3 H-labelled cholesterol hexadecylether as a 
lipid marker, and their biodistribution was examined in mice at 1 hour 
after injection. Liposomes tested in this study were composed of 
DOPE/DODAC (85:15, mol/mol), DOPE/DODAC/PEG-Ceramide (80:15:5, mol/mol), 
and DOPE/DODAC/PEG-DSPE (80:15:5, mol/mol). To also examine the effect of 
the hydrophobic anchor on biodistribution of liposomes, various 
PEG-Ceramide derivatives with different acyl chain lengths were used. 
These liposomal formulations had similar average diameters, ranging from 
89 to 103 nm. Table II below shows levels of liposomes in the blood, 
spleen, liver, lung, heart, and kidney, together with respective 
blood/liver ratios. DOPE/DODAC liposomes were shown to be cleared rapidly 
from the blood and accumulate exclusively in the liver with the 
blood/liver ratio of approximately 0.01. Inclusion of amphipathic PEG 
derivatives at 5.0 mol % in the lipid composition resulted in their 
increased blood levels and accordingly decreased liver accumulation to 
different degrees. DOPE/DODAC/PEG-DSPE liposomes showed the highest blood 
level (about 59%) and the lowest liver accumulation (about 35%) with the 
blood/liver ratio of approximately 1.7 at 1 hour after injection. Among 
various PEG-Ceramide derivatives with different acyl chain lengths, 
PEG-Ceramide (C20)-containing liposomes showed the highest blood level 
(about 30%) with the blood/liver ratio of approximately 0.58, while 
PEG-Ceramide (C8)-containing liposomes showed a lower blood level (about 
6%) with the blood/liver ratio of approximately 0.1. It appeared that, 
among different PEG-Ceramide derivatives, the activity in increasing the 
blood level of liposomes is directly proportional to the acyl chain length 
of ceramide; the longer the acyl chain length, the greater the activity. 
It also appeared that the optimal derivative for increasing the blood 
level of liposomes is PEG-Ceramide (C20). 
TABLE II 
__________________________________________________________________________ 
Effect of Amphipathic PEG Derivatives on Biodistribution of DOPE/DODAC 
Liposomes 
% injected dose 
PEG- Average 
Derivative 
Diameter (mm) 
Blood 
Liver 
Spleen 
Lung Heart 
Kidney 
Total Blood/Liver 
__________________________________________________________________________ 
None 103 (29) 
0.8 (0.4) 
64.4 (2.) 
3.1 (1.8) 
1.2 (0.2) 
0.2 (0.0) 
0.3 (0.0) 
70.0 (1.4) 
0.012 
PEG DSPE 
95 (26) 
59.1 (8.2) 
34.7 (2.1) 
2.9 (0.1) 
1.9 (0.8) 
1.7 (0.4) 
1.2 (0.5) 
101.4 (6.1) 
1.703 
PEG-Cer (C8) 
89 (24) 
6.5 (1.9) 
62.8 (3.4) 
4.2 (1.0) 
0.5 (0.3) 
0.3 (0.1) 
0.3 (0.1) 
74.6 (5.1) 
0.104 
PEG-Cer (C14) 
93 (25) 
5.9 (0.5) 
55.9 (1.0) 
3.3 (0.2) 
0.1 (0.0) 
0.1 (0.0) 
0.1 (0.0) 
65.4 (1.6) 
0.106 
PEG-Cer (C16) 
93 (24) 
13.9 (2.1) 
57.5 (2.0) 
2.6 (0.1) 
0.0 (0.0) 
0.2 (0.1) 
0.0 (0.0) 
74.3 (4.0) 
0.242 
PEG-Cer (C20) 
101 (24) 
29.8 (4.8) 
51.0 (2.2) 
1.9 (0.2) 
0.0 (0.0) 
0.3 (0.1) 
0.0 (0.0) 
82.8 (2.8) 
0.584 
PEG-Cer (C24) 
92 (28) 
26.7 (0.8) 
46.7 (7.6) 
5.7 (1.2) 
1.0 (0.2) 
0.9 (0.2) 
0.4 (0.1) 
81.5 (4.1) 
0.572 
__________________________________________________________________________ 
.sup.3 Hlabelled liposomes composed of DOPE/DODAC (75:15, mol/mol) 
additionally containing an indicated PEG derivative at 5.0 mol % of the 
lipid mixture were injected i.v. into mice. Biodistribution was examined 
at 1 h after injection and expressed as percentage of injected dose of 
liposomes with SD (n = 3). 
Optimizations of DOPE/DODAC Liposomes for Prolonged Circulation Times 
The effect of increasing concentrations of PEG-Ceramide (C20) in the lipid 
composition on biodistribution of DOPE/DODAC liposomes was examined. 
PEG-Ceramide (C20) was included in DOPE/DODAC liposomes at increasing 
concentrations (0-30 mol %) in the lipid composition, while the 
concentration of DODAC was kept at 15 mol % of the lipid mixture. 
Liposomes were prepared by the extrusion method and had similar average 
diameters ranging from 102 nm to 114 nm. Liposomes were injected i.v. into 
mice, and biodistribution was examined at 1 hour after injections. FIG. 6 
shows the liposome level in the blood and liver at 1 hour after injections 
as a function of the PEG-Ceramide (C20) concentration. Clearly, increasing 
the concentration of PEG-Ceramide in the lipid composition resulted in 
progressive increase in liposome levels in the blood, accompanied by 
decreased accumulation in the liver. The highest blood level (about 84% at 
1 hour after injection) was obtained for DOPE/DODAC/PEG-Ceramide (C20) 
(55:15:30, mol/mol) showing the blood/liver ratio of about 6.5. 
The effect of increasing concentrations of DODAC on the biodistribution of 
DOPE/DODAC liposomes also was examined. DOPE/DODAC liposomes containing 
either 10 mol % or 30 mol % PEG-Ceramide (C20) and various concentrations 
(15, 30, 50 mol %) were prepared by the extrusion method and had similar 
average diameters ranging from 103 to 114 nm. Biodistribution was examined 
at 1 hour after injections, and expressed as percentages of liposomes in 
the blood as a function of the DODAC concentration (FIG. 7). As shown in 
FIG. 7, increasing DODAC concentrations in the lipid composition resulted 
in decreased levels in the blood for both liposomal formulations. Thus, 
the presence of a cationic lipid, DODAC, in the lipid composition results 
in rapid clearance from the blood. Also, shown in FIG. 7 is that such a 
DODAC effect can be reversed by increasing the concentration of 
PEG-Ceramide (C20) in the lipid composition. 
FIG. 8 shows time-dependent clearances of DOPE/DODAC liposomes with or 
without PEG-Ceramide from the blood. Only a small fraction of injected 
DOPE/DODAC liposomes remained in the blood, while increasing the 
concentration of PEG-Ceramide (C20) in the lipid composition resulted in 
prolonged circulation times in the blood. Estimated half-lives in the 
.alpha.-phase for DOPE/DODAC/PEG-Ceramide (C20) (75:15:10, mol/mol) and 
DOPE/DODAC/PEG-Ceramide (C20) (55:15:30, mol/mol) were &lt;1 hour and 5 
hours, respectively. 
CONCLUSIONS 
The above studies indicate that there are several levels at which 
biodistribution of fusogenic liposomes containing a cationic lipid can be 
controlled by inclusion of amphipathic PEG derivatives. Data in Table II 
shows that the hydrophobic anchor of amphipathic PEG derivatives has an 
important role in determining biodistribution of DOPE/DODAC liposomes. 
Studies using various PEG-Ceramide derivatives with different acyl chain 
lengths showed that the longer the acyl chain length of PEG-Ceramide, the 
greater the activity in prolonging the circulation time of DOPE/DODAC 
liposomes. These results are consistent with the rate at which amphipathic 
PEG derivatives dissociate from the liposome membrane is directly 
proportional to the size of the hydrophobic anchor. Accordingly, 
PEG-Ceramide derivatives with a longer acyl chain can have stronger 
interactions with other acyl chains in the liposome membrane and exhibit a 
reduced rate of dissociation from the liposome membrane, resulting in 
stabilization of DOPE/DODAC liposomes for a prolonged period of time and 
thus their prolonged circulation time in the blood. 
In addition to the hydrophobic anchor of amphipathic PEG derivatives, the 
concentration of amphipathic PEG derivatives in the lipid membrane can 
also be used to control in vivo behavior of DOPE/DODAC liposomes. Data in 
FIG. 6 show that increasing the concentration of PEG-Ceramide (C20) in the 
lipid composition resulted in increased liposome levels in the blood. The 
optimal concentration of PEG-Ceramide (C20) in the lipid composition was 
found to be 30 mol % of the lipid mixture. It appeared that the 
circulation time of DOPE/DODAC/PEG-Ceramide (C20) liposomes is determined 
by the relative concentrations of two lipid compositions, DOPE and 
PEG-Ceramide, exhibiting opposite effects on liposome biodistribution. 
While amphipathic PEG derivatives show the activity in prolonging the 
circulation time of liposomes in -the blood, a cationic lipid, DODAC, 
shows the activity to facilitate liposome clearance from the blood. Thus, 
for the maximal circulation time in the blood, an appropriate 
concentration of amphipathic PEG derivatives and a minimal concentration 
of DODAC should be used. It should be noted, however, that an optimal 
liposome formulation for the prolonged circulation time in the blood is 
not necessarily the one suitable for an intended application in delivery 
of certain therapeutic agents. Both pharmacokinetic and pharmacodynamic 
aspects of fusogenic liposomes should be examined for different 
applications using different therapeutic agents. 
All publications and other references or patent documents herein are 
incorporated by reference. It is to be understood that the above 
description is intended to be illustrative and not restrictive. Many 
embodiments will be apparent to those of skill in the art upon reviewing 
the above description. The scope of the invention should, therefore, be 
determined not with reference to the above description, but should instead 
be determined with reference to the appended claims, along with the full 
scope of equivalents to which the claims are entitled.