This invention provides a compound having the formula R.sup.1 --Y.sup.1 --CHZ.sup.1 --CH(NY.sup.2 Y.sup.3)--CH.sub.2 --Z.sup.2, wherein: R.sup.1 is a straight-chained alkyl, alkenyl or alkynyl group having from 8 to 19 carbon atoms in the aliphatic chain; Y.sup.1 is --CH.dbd.CH--, --C.tbd.C-- or --CH(OH)CH(OH)--; Z.sup.1 is OH or a conversion-inhibiting group; Z.sup.2 is a conversion-inhibiting group; Y.sup.2 is H, a phenyl group, an alkyl-substituted phenyl group having from 1 to about 6 carbons in the alkyl chain, or an alkyl chain having from 1 to 6 carbons; Y.sup.3 is H or a group having the formula --C(O)R.sup.2 or --S(O).sub.2 R.sup.2 ; R.sup.2 is a straight-chained alkyl, alkenyl or alkynyl group having from 1 to 23 carbon atoms in the chain; and when Z.sup.2 is an amino, R.sup.2 is an aliphatic chain having from 1 to 9 or from 19 to 23 carbon atoms in the aliphatic chain.

FIELD OF THE INVENTION 
This invention is directed to pharmaceutically active sphingolipid 
compounds, to liposomes containing pharmaceutically active sphingolipid 
compounds, and to methods of using such compounds and liposomes, 
particularly for the treatment of animals afflicted with cancers. 
Cell death in multicellular organisms can be an accidental response to 
external trauma, or it can be a programmed response to internal or 
external stimuli. Necrosis, or accidental cell death, is most often seen 
when cells die uncontrollably as a result of sudden and severe injury to 
an organism, e.g., by physical or chemical trauma, sustained hyperthermia 
or ischemia (see, e.g., J. Cohen, Immunol. Today. 14(3):126 (1993)); J. 
Marx, Science 259:750 (1993)). Plasma membrane damage can cause cells to 
lose their ability to regulate their osmotic pressure, and cell rupture 
can thereby result. The consequent leakage of cell contents can cause 
further injury to surrounding cells and can invoke an inflammatory 
response to clear away the cellular debris. 
Apoptosis, by contrast, describes a programmed series of events resulting 
in cell death by fragmentation into membrane-bound particles; these 
particles are then phagocytosed by other cells (see, e.g., Stedman's 
Medical Dictionary (Illustrated), supra). Cells typically undergo 
apoptosis in physiologically determined circumstances such as the 
elimination of self-reactive T cells, the death of cells (e.g., 
neutrophils) with short half-lives, involution of growth factor-deprived 
cells, morphogenetic cell death during embryonic development and the 
deaths of cellular targets of cell-mediated cytotoxicity (see, e.g., J. 
Cohen, supra). 
Cells undergoing apoptosis can break up into apoptotic bodies, which are 
cellular fragments that retain their membranes and are able to regulate 
their osmotic pressures. Unlike necrotic cells, there is usually no 
leakage of cellular contents and hence, no invocation of an inflammatory 
response. Apoptotic cells typically have disrupted plasma membranes and 
condensed, disrupted nuclei. Nuclear chromatin in these cells is 
fragmented randomly between nucleosomes, as the result of endonuclease 
activation during apoptosis. 
Although transcription in apoptotic cells ceases, cell death occurs more 
rapidly than would be expected from the cessation of transcription alone. 
This indicates that cellular processes in addition to transcription 
termination are likely to be involved in apoptosis. Gene expression itself 
may actually be required for the occurrence of the morphological changes 
associated with apoptosis (see, e.g., J. Cohen, supra). Alternatively, 
inhibition of transcription termination may itself induce apoptosis. 
Furthermore, apoptosis of some cells does not appear to be affected one 
way or the other by the inhibition of protein synthesis. Expression of the 
bcl-2 oncogene, for example, can inhibit the apoptosis otherwise induced 
by different stimuli, and may thereby contribute to cancer development. 
Accordingly, inhibition of bcl-2 expression may be required to induce 
apoptosis (see, e.g., J. Marx, supra; J. Cohen, supra; G. Williams and C. 
Smith, Cell 74:777 (1993); M. Barinaga, Science 259:762 (Feb. 5, 1993)). 
C-myc protein is known to stimulate cell proliferation; however, it may 
also stimulate apoptosis in the absence of additional proliferative 
stimuli. p53, which is thought to suppress tumor growth, may also 
stimulate apoptosis. C-fas, a transmembrane protein homologous to Tumor 
Necrosis Factor (TNF), can also induce apoptosis, as can TNF itself. 
TNF is a monokine protein produced by monocytes and macrophages. There are 
two known structurally and functionally related TNF proteins, TNF-.alpha. 
and TNF-.beta., both of which bind to the same cell surface receptors. 
Binding to these receptors by TNF leads to the activation of multiple 
signal transduction pathways, including the activation of sphingomyelinase 
(see, e.g., M. Raines et al., J. Biol. Chem. 268(20):14572 (1993); L. 
Obeid et al., Science 259:1769 (Mar. 12, 1993); H. Morishige et al., 
Biochim. Biophys. Acta. 1151:59 (1993); J. Vilcek and T. Lee, J. Biol. 
Chem. 266(12):7313 (1991); Dbaibo et al., J. Biol. Chem. 268(24):17762 
(1993); R. Kolesnik, Trends Cell Biol. 2:232 (1992); J. Fishbein et al., 
J. Biol. Chem. 268(13):9255 (1993)). 
Applicants have found that increases in ceramide concentrations can 
stimulate apoptosis. Ceramides are a class of sphingolipids comprising 
fatty acid derivatives of a sphingoid, e.g., sphingosine, base (see, e.g., 
Stedman's Medical Dictionary (Illustrated), 24th edition (J. V. Basmajian 
et al., eds.), Williams and Wilkins, Baltimore (1982), p. 99)). Different 
ceramides are characterized by different fatty acids linked to the 
sphingoid base. For example, stearic acid can be attached to the amide 
group of sphingosine to give rise to the ceramide CH.sub.3 
(CH.sub.2).sub.12 CH.dbd.CH--CHOH--CH(CH.sub.2 OH)--NH--CO--(CH.sub.2) 
.sub.16 CH.sub.3. Shorter- or longer-chain fatty acids can also be linked 
to the sphingoid base. Applicants have also found that attachment of 
certain chemical groups to sphongolipids and ceramides so as to form 
analogs of such compounds can inhibit bioconversion of ceramides to 
sphingomyelins, and can thereby lead to an apoptosis stimulating increase 
in ceramide concentrations. 
Ceramides are found in all eukaryotic cell membranes, and are known to 
participate in a variety of critical cellular processes. Furthermore, 
certain sphingolipid compounds have been found to play a role in 
prevention of cellular proliferation (). However, none of these references 
teach applicants' chemical compounds and liposomes, or their use in 
stimulating cell death. 
SUMMARY OF THE INVENTION 
Provided herein is a compound having the formula R.sup.1 --Y.sup.1 
--CHZ.sup.1 -CH(NY.sup.2 Y.sup.3)--CH.sub.2 --Z.sup.2, wherein: R.sup.1 is 
a straight-chained alkyl, alkenyl or alkynyl group having from 8 to 19 
carbon atoms in the aliphatic chain; Y.sup.1 is --CH.dbd.CH--, --C.tbd.C-- 
or --CH(OH)CH(OH)--; Z.sup.1 is OH or a conversion-inhibiting group; 
Z.sup.2 is a conversion-inhibiting group; Y.sup.2 is H, a phenyl group, an 
alkyl-substituted phenyl group having from 1 to about 6 carbons in the 
alkyl chain, or an alkyl chain having from 1 to 6 carbons; Y.sup.3 is H or 
a group having the formula --C(O)R.sup.2 or --S(O).sub.2 R.sup.2 ; R.sup.2 
is a straight-chained alkyl, alkenyl or alkynyl group having from 1 to 23 
carbon atoms in the chain; and wherein when Z.sup.2 is an amino, R.sup.2 
is an aliphatic chain having from 1 to 9 or from 19 to 23 carbon atoms in 
the aliphatic chain. Preferably, R.sup.1 is an alkyl group, more 
preferably, CH.sub.3 (CH.sub.2).sub.12 --, Y.sup.1 is --CH.dbd.CH--, 
Y.sup.2 is H, Y.sup.3 is --C(O)R.sup.2 and R.sup.2 is an alkyl chain. 
Conversion-inhibiting groups can have the formula --X.sup.2 X.sup.3 or 
--O--X.sup.2 X.sup.3, wherein X.sup.2 is selected from the group 
consisting of CH.sub.2 --, C(CH.sub.3).sub.2 --, Si(PO.sub.4).sub.2 --, 
Si(CH.sub.3).sub.2 --, SiCH.sub.3 PO.sub.4 --, C(O)-- and S(O).sub.2 -- 
and wherein X.sup.3 is selected from the group consisting of --C(O)H, 
--CO.sub.2 H, --CH.sub.3, --C(CH.sub.3).sub.3, --Si(CH.sub.3).sub.3, 
--SiCH.sub.3 (C(CH.sub.3).sub.3).sub.2, --Si(C(CH.sub.3).sub.3).sub.3, 
--Si(PO.sub.4).sub.2 C(CH.sub.3).sub.3, a phenyl group, an 
alkyl-substituted phenyl group having from 1 to 6 carbons in the alkyl 
chain, an alkyl chain having from 1 to 6 carbons, an amino moiety, a 
chlorine, a flourine, or a group having the formula C(R.sup.3 R.sup.4)OH; 
each of R.sup.3 and R.sup.4 is independently an alkyl chain having from 1 
to 6 carbons, a phenyl group or an alkyl-substituted phenyl group having 
from 1 to 6 carbons in the alkyl chain. Preferably, the 
conversion-inhibiting group is --OC(O)CH.sub.3, --OC(O)CH.sub.2 CH.sub.2 
CH.sub.3, --OC(O)CH(CH.sub.3)CH.sub.3, or --OSi(CH.sub.3).sub.2 
C(CH.sub.3).sub.3, more preferably, --OSi(CH.sub.3).sub.2 
C(CH.sub.3).sub.3. Conversion-inhibiting groups can also have the formula 
--X.sup.1 or --OX.sup.1, wherein X.sup.1 is C(O)H, CO.sub.2 H, CH.sub.3, 
C(CH.sub.3).sub.3, Si(CH.sub.3).sub.3, SiCH.sub.3 
(C(CH.sub.3).sub.3).sub.2, Si(C(CH.sub.3).sub.3).sub.3, Si(PO.sub.4).sub.2 
C(CH.sub.3).sub.3, a phenyl group, an alkyl-substituted phenyl group 
having from 1 to 6 carbons in the alkyl chain, an alkyl chain having from 
1 to 6 carbons, an amino moiety, a flourine, a chlorine, or a group having 
the formula C(R.sup.3 R.sup.4)OH, and each of R.sup.3 and R.sup.4 is 
independently an alkyl chain having from 1 to 6 carbons. 
Preferably, the compound of this invention has the formula CH.sub.3 
(CH.sub.2).sub.12 --CH.dbd.CH--CH.sub.2 Z.sup.1 --CH (NHY.sup.3)--CH.sub.2 
--Z.sup.2. Y.sup.3 is then a group having the formula --C(O)R.sup.2, more 
preferably, --C(O)(CH.sub.2).sub.4 CH.sub.3. Z.sup.2 is preferably 
--OSi(CH.sub.3).sub.2 C(CH.sub.3).sub.3, --OSi (PO.sub.4).sub.2 
C(CH.sub.3).sub.3, --C(O)CH.sub.3 or --OC(O)CH.sub.2 CH.sub.2 CH.sub.3. 
Also provided herein is a pharmaceutical composition comprising the 
compound of this invention and a pharmaceutically acceptable carrier; the 
composition can also comprise an additional bioactive agent. Further 
provided is a method of administering a bioactive compound to an animal, 
preferably a human, which comprises administering to the animal this 
composition; the method can comprise administering an additional bioactive 
agent to the animal. 
The animal can be afflicted with a cancer, wherein the method comprises 
administering an amount of the composition which comprises an anticancer 
effective amount of the compound. Typically, the anticancer effective 
amount of the compound is at least about 0.1 mg of the compound per kg of 
body weight of the animal. Gemerally, the anticancer effective amount is 
from about 1 mg per kg to about 50 mg per kg. Treatable cancers include, 
without limitation, brain, breast, lung, ovarian, colon, stomach or 
prostate cancers, and can be sarcomas, carcinomas, neuroblastomas, or 
gliomas. Drug resistant cancers can also be treated. 
Provided herein is a liposome having a bilayer which comprises a lipid and 
a compound having the formula R.sup.1 --Y.sup.1 --CHZ.sup.1 --CH(NY.sup.2 
Y.sup.3)--CH.sub.2 --Z.sup.2, wherein R.sup.1 is a straight-chained alkyl, 
alkenyl or alkynyl group having from 5 to 19 carbon atoms in the chain; 
Y.sup.1 is --CH.dbd.CH--, --C.tbd.C-- or --CH(OH)CH(OH)--; each of Z.sup.1 
and Z.sup.2 is independently OH or a conversion-inhibiting group; Y.sup.2 
is H, a phenyl group, an alkyl-substituted phenyl group having from 1 to 6 
carbons in the alkyl chain, or an alkyl chain having from 1 to 6 carbons; 
Y.sup.3 is H or a group having the formula --R.sup.2, --C(O)R.sup.2 or 
--S(O).sub.2 R.sup.2 ; R.sup.2 is a straight-chained alkyl, alkenyl or 
alkynyl group having from 1 to 23 carbon atoms; and wherein the bilayer 
comprises at least about five mole percent of the compound. Y.sup.3 is 
preferably R.sup.2, which is preferably, --(CH.sub.2).sub.3 CH.sub.3, 
--(CH.sub.2).sub.5 CH.sub.3, --(CH.sub.2).sub.7 CH.sub.3, or 
--(CH.sub.2).sub.9 CH.sub.3, and more preferably, R.sup.2 is 
--(CH.sub.2).sub.5 CH.sub.3, or --C(O)R.sup.2, which is preferably 
--C(O)(CH.sub.2).sub.4 CH.sub.3. Preferably in the liposome of this 
invention, at least one of Z.sup.1 and Z.sup.2 is a conversion-inhibiting 
group, such as --OC(O)CH.sub.3, --OC(O)CH.sub.2 CH.sub.2 CH.sub.3, 
--OC(O)CH(CH.sub.3)CH.sub.3, or --OSi(CH.sub.3).sub.2 C(CH.sub.3).sub.3. 
More preferably, the conversion-inhibitng group is --OSi(CH.sub.3).sub.2 
C(CH.sub.3).sub.3. Most preferably, the liposome comprises a compound 
having the formula CH.sub.3 --(CH.sub.2).sub.12 --CH.dbd.CH--CH.sub.2 
Z.sup.1 --CH(NHY.sup.3)--CH.sub.2 Z.sup.2. 
Preferably, the liposomal bilayer comprises at least about 10 mole percent 
of the compound. The bilayer can comprise vitamin D.sub.3 ; such bilayers 
preferably comprise about 1 mole percent of vitamin D.sub.3. The bilayer 
can also comprise a headgroup-modified lipid. The liposome can comprise an 
additional bioactive agent, and can be dehydrated. 
Also provided herein is a pharmaceutical composition comprising the 
liposome of this invention and a pharmaceutically acceptable carrier. 
Further provided is a method of administering a compound to an animal 
which comprises administering to the animal the composition. The method 
can be used to treat an animal afflicted with a cancer, wherein a dose of 
the composition is administered and wherein the dose comprises an 
anticancer effective amount of the liposome. Typically, the dose comprises 
at least about 1 mg of the liposome per kg of body weight of the animal. 
Generally, the dose comprises from about 1 mg per kg to about 1000 mg per 
kg. 
Provided herein is a liposome having a bilayer which comprises a lipid and 
a compound having the formula R.sup.1 --Y.sup.1 --CHZ.sup.1 --CH(NY.sup.2 
Y.sup.3)--CH.sub.2 --Z.sup.2, wherein: R.sup.1 is a straight-chained 
alkyl, alkenyl or alkynyl group having from 5 to 19 carbon atoms in the 
chain; Y.sup.1 is --CH.dbd.CH--, --C.tbd.C-- or --CH(OH)CH(OH)--; each of 
Z.sup.1 and Z.sup.2 is independently OH or a conversion-inhibiting group. 
Y.sup.2 is H, a phenyl group, an alkyl-substituted phenyl group having 
from 1 to 6 carboins or an alkyl chain having from 1 to 6 carbons; Y.sup.3 
is H or a group having the formula --R.sup.2, --C(O)R.sup.2 or 
--S(O).sub.2 R.sup.2 ; R.sup.2 is a straight-chained alkyl, alkenyl or 
alkynyl group having from 1 to 23 carbon atoms; and wherein the bilayer 
comprises an anticancer-effective amount of the compound. Also provided is 
a pharmaceutical composition comprising this liposome and a 
pharmaceutically acceptable carrier. Further provided is a method of 
treating an animal afflicted with a cancer which comprises administering 
to the animal this composition.

DETAILED DESCRIPTION OF THE INVENTION 
Provided herein is a compound having the formula R.sup.1 --Y.sup.1 
--CHZ.sup.1 --CH(NY.sup.2 Y.sup.3)--CH.sub.2 --Z.sup.2, wherein: R.sup.1 
is a straight-chained alkyl, alkenyl or alkynyl group having from 8 to 19 
carbon atoms in the aliphatic chain; Y.sup.1 is --CH.dbd.CH--, --C.tbd.C-- 
or --CH(OH)CH(OH)--; Z.sup.1 is OH or a conversion-inhibiting group; 
Z.sup.2 is a conversion-inhibiting group; Y.sup.2 is H, a phenyl group, an 
alkyl-substituted phenyl group having from 1 to about 6 carbons in the 
alkyl chain, or an alkyl chain having from 1 to 6 carbons; Y.sup.3 is H or 
a group having the formula --C(O)R.sup.2 or --S(O).sub.2 R.sup.2 ; R.sup.2 
is a straight-chained alkyl, alkenyl or alkynyl group having from 1 to 23 
carbon atoms in the chain; and wherein when Z.sup.2 is an amino, R.sup.2 
is an aliphatic chain having from 1 to 9 or from 19 to 23 carbon atoms in 
the aliphatic chain. Preferably, R.sup.1 is an alkyl group, more 
preferably, CH.sub.3 (CH.sub.2).sub.12 --, Y.sup.1 is --CH.dbd.CH--, 
Y.sup.2 is H, Y.sup.3 is --C(O)R.sup.2 and R.sup.2 is an alkyl chain, more 
preferably an alkyl chain having from 6 to 8 carbons. Most preferably, 
R.sup.1 and R.sup.2 together comprise from about 15 to about 25 carbons, 
wherein R.sup.1 preferably comprises 13 carbons and R.sup.2 preferably 
comprises 6 to 8 carbons. Without intending to be limited by theory, it is 
believed that total carbon chain length of a lipid is an important factor 
in determing the ability of the lipid to insert itself into biological 
membranes. 
Without intending for this invention in any way to be limited by theory, it 
is believed that sphingosines and ceramides can act as signal transducers 
or secondary messengers in cells, i.e., that intracellular levels are 
increased in response to external stimuli, and that this increase results 
in enhanced protein kinase and phosphatase activities (see, e.g., M. 
Raines et al., supra; R. Kolesnik et al., supra; G. Dbaibo et al., supra; 
and J. Fishbein et al., supra). Activated protein kinases and phosphatases 
can activate cellular processes which lead to cell death. Accordingly, it 
can be therapeutically desirable to increase intracellular concentrations 
of sphingosines and ceramides in cancer cells. 
Sphingosines and ceramides are formed in animal cells by the combination of 
palmitoyl CoA (CH.sub.3 (CH.sub.2).sub.14 --CO--S--CoA) and serine to give 
dehydrosphinganine (CH.sub.3 (CH.sub.2).sub.14 Co--CH(NH.sub.3)--CH.sub.2 
OH and CO.sub.2 (see, e.g., L. Stryer, Biochemistry (2nd edition), W. H. 
Freeman and Co., New York, pp. 461-462)). Dehydrosphinganine is converted 
to dihydrosphingosine (CH.sub.3 (CH.sub.2).sub.14 
--CH(OH)--CH(NH.sub.3)--CH.sub.2 OH) which is then converted to 
sphingosine (CH.sub.3 (CH.sub.2).sub.12 
CH.dbd.CH--CH(OH)--CH(NH.sub.3)--CH.sub.2 OH). A fatty acid is then linked 
to the amide group of sphingosine to give rise to a ceramide (CH.sub.3 
(CH.sub.2).sub.12 CH.dbd.CH--CHOH--CH(CH.sub.2 OH)--NH--CO--R, where R is 
a fatty acid chain). A phosphorylcholine group (PO.sub.4 CH.sub.2 CH.sub.2 
--N(CH.sub.3).sub.3) can be attached to the ceramide at its hydroxyl group 
to produce a sphingomyelin (CH.sub.3 (CH.sub.2).sub.12 CH.dbd.CH--CHOH--CH 
(CH.sub.2 PO.sub.4 CH.sub.2 CH.sub.2 --N(CH.sub.3).sub.3)--NH--CO--R). 
Sphingomyelinase can catalyze the hydrolytic removal of the 
phosphorylcholine from the sphingomyelin to give rise to a ceramide (see, 
e.g., FIG. 1). Reverse hydrolysis of the ceramide can give rise to a 
sphingomyelin. 
Blockage or inhibition of this "reverse hydrolysis" step, that is, 
conversion of a ceramide to the corresponding sphingomylein, can lead to 
increased intracellular ceramide levels. "Conversion-inhibiting groups" 
are attached to sphingosines and ceramides to inhibit sphingomylein 
formation therefrom. Such groups are generally not found atached to 
sphingosines and ceramides, or their biosynthetic precursors, in animal 
cells 
The compounds of this invention are synthesized by a number of routes well 
known to and readily practiced by ordinarily skilled artisans, given the 
teachings of this invention (see, for example, below, wherein "rf" refers 
to one of the following references: 1: J. Am. Chem. Soc., 94:6190 (1972); 
2: J. Org. Chem. 59:668 (1994); 3: Angew. Chem., Intl. Ed. (English), 
17:569 (1978); 4: Vogel's Textbook of Practical Organic Chemistry (5th 
ed.), pp. 769-780); 5: J. Org. Chem. 40:574 (2975); .6:.J. Org. Chem. 
59:182 (1994); 7: J. Org. Chem. 25:2098 (1960); 8: Synthesis (1985): pp. 
253-268; 9: J. Chem. Soc. (1953): p. 2548; 10: J. Am. Chem. Soc. 90: 4462, 
4464 (1968); 11: Oxidations in Organic Chemistry (Am. Chem. Soc, 
Washington, D.C. (1990), pp. 60-64; 12: J. Med. Chem. 30 1326 (1987); 13: 
Synth. Commun. 9:757 (1979); 14: The Chemistry of Amides (J. Wiley & Sons, 
New York (2970)), pp. 795-801; 15: J. Med. Chem. 37:2896 (1994);4: J. Med. 
Chem, 30:1326 (1987); 16: Rec. Chem. Prog. 29:85 (1968); and 17: 
Phospholipids Handbook (Marcell Dekker, Inc., New York (1993), p. 97); the 
contents of these are incorporated herein by reference). 
For example, such artisans would use a sphingosine or a ceramide as their 
starting material. Alkyl, alkenyl or alkynyl chains of varying length can 
be attached thereto, or removed therefrom, by known means. 
Conversion-inhibiting groups can also be attached to the sphingosines and 
ceramides by known means. These include, without limitation, 
oxidation/reduction, substitution, condensation and alkylation reactions, 
as well as other generally accepted means for attaching and removing 
chemical groups from a compounds and for converting compounds from one 
form to another. Such reactions are generally formed using generally 
accepted solvents and are performed under readily determinable conditions. 
##STR1## 
Specific compounds can be synthesized as follows. Synthesis of sily ether 
of ceramide: a mixture of ceramide and t-butyldimethylsilyl chloride (1 
equivalent) and imidazole (2 equivalent) in DMF is stirred under N.sub.2 
at room temperature overnight. The solvent is then removed under a stream 
of N.sub.2 and residue is dissolved in CH.sub.2 Cl.sub.2, washed (H.sub.2 
O), dried (MgSO.sub.4) and concentrated to dryness. The residue is 
purified over silica gel (AcOEt: Hexan=:1:3). Synthesis of 1-ester 
ceramide: The mixture of ceramide and Ac2O (1 equivalent) and catalytic 
amount of dimethyl amino pyridine in dry CH2Cl2 is stirred at room 
temperature for 1 hour and the reaction is checked by TLC (AcOEt). The 
mixture is then concentrated. The crude product is purified over silica 
gel (AcOEt: Hexane=2:3.5) Oxidation of C3-OH of ceramide to ketone: 1-OAc 
ceramide is dissolved in acetone and cooled in an ice-bath. Jone's reagent 
is dropwised slowly till the orange color persists. The reaction is 
quenched by isopropanol, and NaHCO.sub.3 is added and stirred for 5 
minutes. The solution is filtered and concentrated to dryness. The crude 
product was purified by preparative TLC (ACOET: Hexane=1:2.5). Reduction 
of ceramide to sphingosine analogs: To an ice-cold stirred solution of 
ceramide in anhydrous THF is added LiAlH.sub.4 and the mixture is stirred 
at room temperature under N.sub.2 for 24 hours. Under ice cooling, the 
reaction mixture is quenched by addition of saturated aqueous NaHCO.sub.3. 
The resulting slurry is filtered and washed with THF. The solution is 
concentrated and the residue is brought into CH.sub.2 Cl.sub.2, washed 
with H.sub.2 O, dried (MgSO.sub.4) and concentrated to dryness. The 
residue is then purified over preparative TLC (silica gel) CH.sub.2 
Cl.sub.2 : MeOH: TEA=8:1:0.08. Synthesis of 4,5-diol ceramide: To a 
solution of ceramide in a mixture of Me.sub.2 CO distilled H.sub.2 O and 
t-BuOH, N-Methyl morpholine N-oxide (NMO, 1.2 equivalent) and OsO.sub.4 
(catalytic amount) in THF are added. The reaction mixture is stirred at 
45.degree. C. for 6 hours, quenched by solid NaHCO.sub.3, and the mixture 
is then stirred for 15 minutes. The suspension is filtered, and the 
filtrate dissolved in THF; the solution is then washed with brine. The 
organic solution is separated, dried and concentrated to dryness. The 
residue is purified over preparative TLC (THF). 
Suitable conversion-inhibiting groups can be identified by a number of 
meanns readily practiced by ordinarily skilled artisans, given the 
motivation by this invention to identify such groups. For example, and 
without limitation, such artisans can select a chemical moiety, and attach 
it to a sphingosine or ceramide as described above. The artisans can then 
readily determine the relative rate at which such a compound undergoes 
hydrolysis, and the rate at which a sphingmyelin is formed from the 
compound. Rates of hydrolysis are themselves readily determinable by 
ordinarily skilled artisans, for example and without limitation, by 
attaching a radioactive moiety to a sphingosine or ceramide and then 
following the rate of hydrolytic cleavage of the moiety by chromatographic 
means. Rates of sphingomyelin formation are also readily determinable, for 
example and without limitation, by combining radioactive phosphorylcholine 
with a conversion-inhibiting group-containing compound in an enzyme system 
capable of attaching the phosphorylcholine to the compound, and then using 
chromatographic means to assess the rate at which the phosphorylcholine is 
added. Preferred conversion-inhibiting groups are those which most inhibit 
hydrolysis and phosphorylcholine attachment. 
Conversion-inhibiting groups can have the formula --X.sup.2 X.sup.3 or 
--O--X.sup.2 X.sup.3, wherein X.sup.2 is selected from the group 
consisting of CH.sub.2 --, C(CH.sub.3).sub.2 --, Si(PO.sub.4).sub.2 --, 
Si(CH.sub.3).sub.2 --, SiCH.sub.3 PO.sub.4 --, C(O)-- and S(O).sub.2 -- 
and wherein X.sup.3 is selected from the group consisting of --C(O)H, 
--CO.sub.2 H, --CH.sub.3, --C(CH.sub.3).sub.3, --Si(CH.sub.3).sub.3, 
--SiCH.sub.3 (C(CH.sub.3).sub.3).sub.2, --Si(C(CH.sub.3).sub.3).sub.3, 
--Si(PO.sub.4).sub.2 C(CH.sub.3).sub.3, a phenyl group, an 
alkyl-substituted phenyl group having from 1 to 6 carbons in the alkyl 
chain, an alkyl chain having from 1 to 6 carbons, an amino moiety, a 
chlorine, a flourine, or a group having the formula C(R.sup.3 R.sup.4)OH; 
each of R.sup.3 and R.sup.4 is independently an alkyl chain having fron 1 
to 6 carbons, a phenyl group or an alkyl-substituted phenyl group having 
from 1 to 6 carbons in the alkyl chain. Preferably, the 
conversion-inhibiting group is --OC(O)CH.sub.3, --OC(O)CH.sub.2 CH.sub.2 
CH.sub.3, --OC(O)CH(CH.sub.3)CH.sub.3, or --OSi(CH.sub.3).sub.2 
C(CH.sub.3).sub.3 (TBDMS) more preferably, --OSi(CH.sub.3).sub.2 
C(CH.sub.3).sub.3. 
Conversion-inhibiting groups can also have the formula --X.sup.1 or 
--OX.sup.1, wherein X.sup.1 is C(O)H, CO.sub.2 H, CH.sub.3, 
C(CH.sub.3).sub.3, Si(CH.sub.3).sub.3, SiCH.sub.3 
(C(CH.sub.3).sub.3).sub.2, Si(C(CH.sub.3).sub.3).sub.3, Si(PO.sub.4).sub.2 
C(CH.sub.3).sub.3, a phenyl group, an alkyl-substituted phenyl group 
having from 1 to 6 carbons in the alkyl chain, an alkyl chain having from 
1 to 6 carbons, an amino moiety, a flourine, a chlorine, or a group having 
the formula C(R.sup.3 R.sup.4)OH; each of R.sup.3 and R.sup.4 is 
independently an alkyl chain having from 1 to 6 carbons. 
Conversion-inhibiting groups include attachment of chemical moities to 
sphingosines and ceramides by ether, silyl ether, ester, acetal and 
sulfonate linkages. 
Preferably, the compound of this invention has the formula CH.sub.3 
(CH.sub.2).sub.12 --CH.dbd.CH--CH.sub.2 Z.sup.1 --CH (NHY.sup.3)--CH.sub.2 
--Z.sup.2. Y.sup.3 is then prefeably a group having the formula 
--C(O)R.sup.2, more preferably, --C(O)(CH.sub.2).sub.4 CH.sub.3. Z.sup.2 
is preferably --OSi(CH.sub.3).sub.2 C(CH.sub.3) .sub.3, 
--OSi(PO.sub.4).sub.2 C(CH.sub.3).sub.3, --C(O)CH.sub.3 or --OC(O)CH.sub.2 
CH.sub.2 CH.sub.3. 
Also provided herein is a pharmaceutical composition comprising the 
compound of this invention and a pharmaceutically acceptable carrier; the 
composition can also comprise an additional bioactive agent. 
"Pharmaceutically acceptable carriers" as used herein are generally 
intended 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, Naim (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 administering a bioactive compound to an 
animal, preferably a human, which comprises administering to the animal 
this composition; the method can comprise administering an additional 
bioactive agent to the animal. The administration, which can be by any 
means generally accepted for administering pharmaceutical products to 
animals, is generally intravenous administration. 
The animal can be afflicted with a cancer, wherein the method comprises 
administering an amount of the composition which comprises an anticancer 
effective amount of the compound. Treatable cancers include, without 
limitation, brain, breast, lung, ovarian, colon, stomach or prostate 
cancers, and can be sarcomas, carcinomas, neuroblastomas, or gliomas, 
amongst others. Drug resistant cancers can also be treated. 
"Anticancer effective amounts" of the compound of this invention are 
generally amounts effective to inhibit, ameliorate, lessen or prevent 
establishment, growth, metastasis or invasion of one or more cancers in 
animals to which the compound has 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. Typically, the anticancer effective amount of the compound 
is at least about 0.1 mg of the compound per kg of body weight of the 
animal. Gemerally, the anticancer effective amount is from about 1 mg per 
kg to about 50 mg per kg. 
Provided herein is a liposome having a bilayer which comprises a lipid and 
a compound having the formula R.sup.1 --Y.sup.1 --CHZ.sup.1 --CH(NY.sup.2 
Y.sup.3)--CH.sub.2 --Z.sup.2, wherein: R.sup.1 is a straight-chained 
alkyl, alkenyl or alkynyl group having from 5 to 19 carbon atoms in the 
chain; Y.sup.1 is --CH.dbd.CH--, --C.tbd.C-- or --CH(OH)CH(OH)--; each of 
Z.sup.1 and Z.sup.2 is independently OH or a conversion-inhibiting group; 
Y.sup.2 is H, a phenyl group, an alkyl-substituted phenyl group having 
from 1 to 6 carbons in the alkyl chain, or an alkyl chain having from 1 to 
6 carbons; Y.sup.3 is H or a group having the formula --R.sup.2, 
--C(O)R.sup.2 or --S(O).sub.2 R.sup.2 ; R.sup.2 is a straight-chained 
alkyl, alkenyl or alkynyl group having from 1 to 23 carbon atoms; and 
wherein the bilayer comprises at least about five mole percent of the 
compound. 
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. These 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 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 muiltilamellar 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 
Paphadjopoulos et al.'s reverse-phase evaporation method (U.S. Pat. No. 
4,235,871) for preparing oligolamellar liposomes. Unilamellar vesicles can 
be produced from MLVs by such methods as sonication (see Paphadjopoulos et 
al. (1968)) or extrusion (U.S. Pat. No. 5,008,050 and U.S. Pat. No. 
5,059,421). The ether lipid lipasome 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, milling and extrusion can be used to size reduce liposomes, 
that is to prepare liposomes of a smaller size from larger liposomes. 
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 liposome of this invention can 
be unilamellar or multilamellar. 
Liposomal bilayers can comprise a variety of ampipathic lipids, including 
those which are saturated or unsaturated, and which typically have acyl 
chains of from 10 to 24 carbons. Suitable polar groups include, without 
limitation, phosphorylcholine, phosphorylethanolamine, phosphorylserine, 
phosphorylglycerol and phosphorylinositiol. Suitable acyl chains include, 
without limitation, laurate, myristate, palmitate, stearate and oleate 
chains. Liposomal bilayers can further comprise sterols, such as 
cholesterol. Sterols generally affect the fluidity of lipid bilayers, 
typically increasing the fluidity of bilayer hydrocarbon chains below the 
gel-to-liquid transition temperature (Tm), and decreasing fluidity above 
the Tm (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. 
Preferably, the liposomal bilayer comprises at least about 10 mole percent 
of the compound. When Y.sup.3 is R.sup.2, it is then preferably, 
--(CH.sub.2).sub.3 CH.sub.3, --(CH.sub.2).sub.5 CH.sub.3, 
--(CH.sub.2).sub.7 CH.sub.3, or --(CH.sub.2).sub.9 CH.sub.3, and more 
preferably, (CH.sub.2).sub.5 CH.sub.3. When Y.sup.3 is --C(O)R.sup.2, it 
is then preferably --C(O)(CH.sub.2).sub.4 CH.sub.3. Preferably, the 
liposome comprises a compound having the formula CH.sub.3 
--(CH.sub.2).sub.12 --CH.dbd.CH--CH.sub.2 Z.sup.1 
--CH(NHY.sup.3)--CH.sub.2 Z.sup.2 ; more preferably, the compound 
comprises at least one conversion-inhibiting group, such as 
--OC(O)CH.sub.3. --OC(O)CH.sub.2 CH.sub.2 CH.sub.3, 
--OC(O)CH(CH.sub.3)CH.sub.3, or --OSi(CH.sub.3).sub.2 C (CH.sub.3).sub.3. 
More preferably, the conversion-inhibitng group is --OSi(CH.sub.3).sub.2 
C(CH.sub.3).sub.3 (TBDMS). 
Intracellular ceramide levels can also be increased by administration of 
vitamin D3, either separately from administration of the compounds and 
liposomes of this invention, or more preferably, in connection with the 
administration of liposomes. Without intending in any way to be limited by 
theory, it is believed that vitamin D3 can stimulate sphingomyelinase to 
convert sphingomyelins to ceramides. Preferably, bilayers containing 
vitamin D3 contain about 1 mole percent of vitamin D.sub.3. 
The liposome can comprise an additional bioactive agent. 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 include therapeutic and imaging agents. 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-labelled 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 comprise a headgroup-modified lipid. 
"Headgroup-modified lipids" are lipids which, when incorporated into the 
lipid bilayers of liposomes can inhibit clearance of the liposomes from 
the circulatory systems of animals to which they have been administered. 
Liposomes are cleared from an animal's body by way of its 
reticuloendothelial system (RES) which consists of fixed and circulating 
macrophages. Avoiding RES clearance can allow liposomes to remain in the 
circulation longer, meaning that less of the drug need be administered to 
achieve desired serum levels. Enhanced circulation times can also allow 
targeting of liposomes to non-RES containing tissues. Liposomal surfaces 
can become coated with serum proteins when administered to animals, i.e., 
liposomes can be opsonized. Rates of clearance by the RES can be related 
to the rate and level of opsonization; accordingly, clearance can be 
inhibited by modifying the outer surface of liposomes such that binding of 
serum proteins is generally inhibited. This can be accomplished by 
minimizing or shielding negative surface charges, which can promote 
protein binding, or by otherwise presenting a steric hindrance to the 
binding of serum proteins. 
Effective surface modification, that is, alterations to the outer surfaces 
of liposomes which result in inhibition of opsonization and RES uptake, 
can be accomplished by incorporating headgroup-modified lipids into 
liposomal bilayers. "Headgroup-modified lipids" as used herein are 
amphipathic lipids whose polar headgroups have been derivatized by 
attachment thereto of a chemical moiety, e.g., polyethylene glycol, a 
polyalkyl ether, a ganglioside, an organic dicarboxylic acid or the like, 
which can inhibit the binding of serum proteins to liposomes such that the 
pharmacokinetic behavior of the vesicles in the circulatory systems of 
animals is altered (see, e.g., Blume et al., Biochim. Biophys. Acta. 
1149:180 (1993); Gabizon et al., Pharm. Res. 10(5):703 (1993); Park et al. 
Biochim. Biophys Acta. 1108:257 (1992); Woodle et al., U.S. Pat. No. 
5,013,556; Allen et al., U.S. Pat. Nos. 4,837,028 and 4,920,016; U.S. Ser. 
No. 142,691, filed Oct. 25, 1993 now abandoned; the contents of these 
disclosures are incorporated herein by reference). 
The amount of a headgroup-modified lipid incorporated into the liposome 
depends upon a number of factors well known to the ordinarily skilled 
artisan, or within his purview to determine without undue experimentation. 
These include, but are not limited to: the type of lipid and the type of 
headgroup modification; the type and size of the liposome; and the 
intended therapeutic use of the liposomal formulation. Typically, the 
concentration of the headgroup-modified lipid in the lipid bilayer of the 
liposome is at least about five mole percent, desirably, about ten mole 
percent. 
The liposome of this invention can be dehydrated, stored and then 
reconstituted such that a substantial portion of their internal contents 
are retained in the liposomes. Liposomal dehydration generally requires 
use of a hydrophilic drying protectant (see U.S. Pat. Nos. 4,229,360 and 
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 rehydration, such 
that the liposomes can be reconstituted. 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. Saccharide sugars, preferentially 
mono- and disaccharides, are suitable drying protectants for liposomes. 
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 the 
liposome of this invention and a pharmaceutically acceptable carrier. 
Further provided is a method of administering a compound to an animal 
which comprises administering to the animal thiscomposition. The method 
can be used to treat an animal afflicted with a cancer, wherein a dose of 
the composition is administered and wherein the dose comprises an 
anticancer effective amount of the liposome. Typically, the dose comprises 
at least about 1 mg of the liposome per kg of body weight of the animal. 
Generally, the dose comprises from about 1 mg per kg to about 1000 mg per 
kg. 
Provided herein is a liposome having a bilayer which comprises a lipid and 
a compound having the formula R.sup.1 --Y.sup.1 --CHZ.sup.1 --CH(NY.sup.2 
Y.sup.3)--CH.sup.2 --Z.sup.2, wherein: R.sup.1 is a straight-chained 
alkyl, alkenyl or alkynyl group having from 5 to 19 carbon atoms in the 
chain; Y.sup.1 is --CH.dbd.CH--, --C.tbd.C-- or --CH(OH)CH(OH)--; each of 
Z.sup.1 and Z.sup.2 is independently OH or a conversion-inhibiting group; 
Y.sup.2 is H, a phenyl group, an alkyl-substituted phenyl group having 
from 1 to 6 carboins or an alkyl chain having from 1 to 6 carbons; Y.sup.3 
is H or a group having the formula --R.sup.2, --C(O)R.sup.2 or 
--S(O).sub.2 R.sup.2 ; R.sup.2 is a straight-chained alkyl, alkenyl or 
alkynyl group having from 1 to 23 carbon atoms; and wherein the bilayer 
comprises an anticancer-effective amount of the compound. Also provided is 
a pharmaceutical composition comprising this liposome and a 
pharmaceutically acceptable carrier. Further provided is a method of 
treating an animal afflicted with a cancer which comprises administering 
to the animal this composition. 
EXAMPLES 
Example 1 
Liposome Preparation 
Liposomes were prepared with the components, and at the mole ratios of 
components, indicated in Table 1 (see below) by the solvent evaporation 
method. For example, PC/Chol/C2-ceramide liposomes were prepared by 
dissolving 1.8242 mg bovine phosphatidylcholine (BPC), 0.4639 mg 
cholesterol (Chol) and 0.1366 mg C2-ceramide (C2) in a chloroform/methanol 
solvent mixture (2:1, volume/volume). The solvent was then evaporated to 
produce dried lipid, and the dried lipid was rehydrated with HEPES 
buffered saline (10 mM HEPES, 150 mM NaCl, pH 7.4). For vitamin 
D3-containing preparations, 0.0154 mg vitamin D3 was added to the lipid 
mixture. For C6-ceramide-containing preparations, 0.1590 mg C6 ceramide 
(C6) was substituted for the C2 ceramide. For sphingomyelin 
(SM)-containing preparations, 2.0470 mg SM, 0.4639 mg cholesterol and 
0.0154 mg vitamin D3 were used. Furthermore, the PC/Chol and PC/Chol/D3 
preparations were prepared with 2.1280 mg BPC, 0.4639 mg cholesterol and 
0.0154 mg vitamin D3. 
TABLE 1 
______________________________________ 
LIPOSOME PREATION 
COMPONENTS MOLAR RATIO 
______________________________________ 
PC:Chol:C2 6:3:1 
PC:Chol:C2:D3 6:3:1:0.1 
PC:Chol:C6 6:3:1 
PC:Chol:C6:D3 6:3:1:0.1 
SM:Chol 7:3 
SM:Chol:D3 7:3:0.1 
PC:Chol 7:3 
PC:Chol:D3 7:3:1:0.1 
______________________________________ 
PC: phosphatidylcholine; Chol: cholesterol; C2: C2 ceramide; D3: vitamin 
D3; C6: C6 ceramide; SM: sphingomyelin. 
Example 2 
Effect of Various Liposomal Ceramide/Sphingomyelin Formulations on the 
Growth of HL-60 Cells 
2.times.10.sup.5 HL-60 cells were incubated with egg 
phosphatidylcholine/cholesterol (EPC/Chol), EPC/Chol/C2-ceramide (C2), 
EPC/Chol/Vitamin D3 (D3), EPC/Chol/D3/C2, EPC/Chol/C6-ceramide (C6), 
EPC/Chol/D3/C6, sphingomyelin (SM)/Chol and SM/Chol/D3 liposomes, as well 
as with buffer (no liposomes; "control") and with egg 
phosphatidylcholine/cholesterol (EPC/Chol) liposomes. Incubation was at 37 
degrees C. in serum-free medium, supplemented with 5 mg/L insulin and 5 
mg/L transferrin, for 24 hours. Fetal calf serum was then added to the 
culture medium to a final concentration of 10%; the cells were then 
incubated for another 24 hours, after which the number of viable cells in 
each culture were counted using trypan blue staining and a hemocytometer. 
The number of viable cells was determined for lipid doses of 100 .mu.M and 
200 .mu.M, and is given in the figures (below) as the number of viable 
cells per ml of culture medium, times 10,000. Results are reported in FIG. 
3 and Table 2 (see below). 
Example 3 
Effect of Various Liposomal Ceramide/Sphingomyelin Formulations on the 
Growth of P388 Cells 
2.times.10.sup.5 P388 cells were incubated with various ceramide or 
sphingomyelin liposomal formulations (see Example 2, above), as well as 
with buffer alone and with egg phosphatidylcholine/cholesterol (EPC/Chol) 
liposomes, under the conditions given above. The number of viable cells in 
the cultures was determined for lipid doses of 50 .mu.M, 100 .mu.M and 200 
.mu.M. Results are reported in FIG. 4 and Tables 2 and 3. 
Example 4 
Effect of Various Liposomal Ceramide/Sphingomyelin Formulations on the 
Growth of U937 Cells 
2.times.10.sup.5 U937 cells were incubated with the various ceramide or 
sphingomyelin liposomal formulations indicated above (see Example 2), as 
well as with buffer alone and with egg phosphatidylcholine/cholesterol 
(EPC/Chol) liposomes, under the conditions given above. The number of 
viable cells in the cultures was determined for lipid doses of 50 .mu.M, 
100 .mu.M and 200 .mu.M. Results are reported in FIGS. 5 and 7, and Tables 
2 and 3. 
Example 5 
Effect of Various Liposomal Ceramide/Sphingomyelin Formulations on the 
Growth of RPMI-7666 Cells 
2.times.10.sup.5 RPMI-7666 cells were incubated with the various 
ceramide/sphingomyelin liposomal formulations indicated above (see example 
2), as well as with no liposomes (control) and with egg 
phosphatidylcholine/cholesterol (EPC/Chol) liposomes, under the conditions 
indicated above. The number of viable cells in the cultures was 
determined. Results are reported in FIGS. 6 and 7, and Tables 2 and 3. 
Example 6 
Effect of Various Liposomal Ceramide/Sphingomyelin Formulations on the 
Growth of CHO/K1 Cells 
2.times.10.sup.5 CHO/k1 cells were incubated with the various 
ceramide/sphingomyelin liposomal formulations indicated above (see example 
2), as well as with no liposomes (control) and with egg 
phosphatidylcholine/cholesterol (EPC/Chol) liposomes, under the conditions 
indicated above. The number of viable cells in the cultures was 
determined. Results are reported in Table 2. 
TABLE 2 
______________________________________ 
Survival of Various Cancer Lines (Without Serum) 
% Survival (By trypan blue exclusion assay) 
RPMI- 
Formulations 
7666 U-937 P-388 HL-60 CHO/k1 
______________________________________ 
BPC/CHOL/ 129.7, 113.4, 136, 87 138 
VD3 107.1 130 103 
Control 100 100 100 100 100 
BPC/CHOL/VD 
103, 90 63.5, 55 
49.6, 26 
37 73 
3/C-6 
FREE C-6 79 82.6 55.4 -- -- 
CERAMIDE 
FREE C6 m- 85.6 80.6 -- -- -- 
silyl-ester 
BPC/CHOL/VD 
93.7 49 46 47 90 
3/C-2 
______________________________________ 
Bioactive lipid dose = 20 uM 
TABLE 3 
______________________________________ 
Effect of Free and Liposomal ceramide on various cancer lines 
(With serum) 
% Survival (By trypan blue assay) 
Formulations RPMI-7666 U-937 P-388 
______________________________________ 
Control 100 100 100 
BPC/CHOL/VD3/C-6 
93.8 84.3 55.0 
82 (thy)* 75.1 (thy)* 
84.7 (thy)* 
FREE C-6 CERAMIDE 
89.2 97 84.5 
______________________________________ 
Bioactive lipid dose = 20 uM; *: growth inhibition measured by standard 
thymidine incorporation assay. 
Example 7 
Therapeutic Efficacy of Liposomal Ceramides in Mice 
CDF1 mice were each injected intraperitoneally with 2.5.times.10.sup.6 P388 
cells. Groups having ten mice each were intraperitoneally administered 
either a HEPES-buffered saline control (10 mM HEPES, 150 mM NaCl, pH 7.4), 
or liposomal vitamin D3, liposomes containing C2 ceramide or liposomes 
containing C6 ceramide, prepared in accordance with the procedures 
described in Example 1 (see above), at a lipid dose of 1.5 mg of lipid per 
kg of body weight of the mice, the administration being 24 hours 
administration of the p388 cells. Survival was assessed at various times 
post-liposome/control administration. Results are presented in FIG. 8. 
Example 8 
In Vitro Cytoxicity Studies 
These studies were performed using a sulforrhodamine B assay (see Monks et 
al., J. Natl. Cancer Inst. (U.S.) 83:757 (1987)). Compounds were dissolved 
in ethanol. Results from these studies, presented in the following tables, 
are expressed as Gl.sub.50 values, that is, concentration of a drug 
(micromolar) required to inhibit growth of fifty percent of the cells. 
TABLE 4 
__________________________________________________________________________ 
In Vitro Drug Sensitivity of Human and Mouse Cell Lines to 
Ceramide Derivatives 
72 Hour Drug Exposure [GI.sub.50 (uM) .+-. SD] 
Ceramide and 1-Acetate 
1,3-Diacetate 
1-Butyrate 
1-Isobutyrate 
derivatives 
C6-Ceramide 
C6-Ceramide 
C6-Ceramide 
C6-Ceramide 
C6-Ceramide 
__________________________________________________________________________ 
A549 12.0 .+-. 4.8 
12.7 .+-. 7.6 
6.1 .+-. 1.1 
6.2 .+-. 0.4 
7.9 .+-. 3.5 
MCF7 24.2 .+-. 8.5 
ND 15.5 .+-. 0.2 
17.9 .+-. 10.0 
ND 
MCF7/ADR 
36.025 .+-. 0.88 
ND 42.89 .+-. 3.6 
37.80 .+-. 8.06 
37.80 .+-. 4.67 
RPMI 7666 
8.5 .+-. 3.5 
9.0 .+-. 2.0 
ND ND ND 
U937 7.0 .+-. 1.7 
11.3 .+-. 1.0 
ND ND ND 
LEWIS 9.6 .+-. 4.4 
8.5 .+-. 0.4 
4.9 .+-. 0.5 
6.4 .+-. 2.4 
ND 
LUNG 
__________________________________________________________________________ 
ND Not Done; 
*One Experiment Only 
TABLE 5 
__________________________________________________________________________ 
In Vitro Drug Sensitivity of Human and Mouse Cell Lines to 
Ceramide Derivatives 
72 Hour Drug Exposure [GI.sub.50 (uM) .+-. SD] 
Ceramide and 1-TBDMS 
3-TBDMS 
1,3-DiTBDMS 
1-TBDMS-3- 
Derivatives 
C6-Ceramide 
C6-Ceramide 
C6-Ceramide 
C6-Ceramide 
Butyrate C6-Cer 
__________________________________________________________________________ 
A549 12.0 .+-. 4.8 
&gt;200 * &gt;200 * &gt;200 &gt;200 * 
RPMI 7666 
8.5 .+-. 3.5 
&gt;200 * &gt;200 * &gt;200 &gt;200 * 
U937 7.0 .+-. 1.7 
&gt;200 * &gt;200 * 134.5 .+-. 28.8 
&gt;200 * 
C3H10T1/2 
14.0 .+-. 2.5 
&gt;161.2 * 
&gt;200 * ND &gt;200 * 
LEWIS 9.6 .+-. 4.4 
&gt;200 * &gt;200 * &gt;200 &gt;200 * 
LUNG 
__________________________________________________________________________ 
ND Not Done; 
* One Experiment Only 
TABLE 6 
__________________________________________________________________________ 
In Vitro Drug Sensitivity of Human and Mouse Cell Lines to 
Ceramide Derivatives 
72 Hour Drug Exposure [GI.sub.50 (uM) .+-. SD] 
Ceramide and N-Hexyl 
1-Acetate-3-one 
4,5-Diol C6- 
Derivatives 
C6-Ceramide 
Sphingosine 
Sphingosine 
C6-Cer Ceramide 
__________________________________________________________________________ 
A549 12.0 .+-. 4.8 
20.2 .+-. 0.8* 
4.9 .+-. 0.1 
14.4 .+-. 0.1* 
25.1 .+-. 0.3* 
MCF7 24.2 .+-. 8.5 
20.4 .+-. 0.4* 
4.8 .+-. 0.1 
6.7 .+-. 0.1* 
20.7 .+-. 0.1* 
MCF7/ADR 
36.03 .+-. 0.88 
19.8 .+-. 0.0* 
5.57 .+-. 1.17 
14.7 .+-. 0.1* 
28.3 .+-. 1.1* 
CAKI 1 6.04 .+-. 0.23 
39.7 .+-. 0.8* 
6.84 .+-. 3.62 
ND ND 
OVCAR 3 15.15 .+-. 1.77 
44.6 .+-. 0.9* 
4.99 .+-. 0.05* 
15.3 .+-. 0.1* 
38.2 .+-. 2.3* 
HT 29 4.0 .+-. 0.2 
19.3 .+-. 0.1* 
5.2 .+-. 0.1* 
13.9 .+-. 0.2* 
14.6 .+-. 0.4* 
SKMEL 28 
13.3 .+-. 2.51 
15.6 .+-. 1.0* 
4.94 .+-. 0.66* 
ND ND 
P388 6.24 .+-. 0.3 
ND 2.59 .+-. 0.3* 
ND ND 
P388/ADR 
12.7 .+-. 1.7* 
ND 2.61 .+-. 1.7* 
ND ND 
LEWIS LUNG 
9.6 .+-. 4.4 
12.2 .+-. 0.1 
4.9 .+-. 0.1 
14.5 .+-. 0.1 
15.2 +/ 0.1 
__________________________________________________________________________ 
ND Not Done; 
* One Experiment Only 
TABLE 7 
__________________________________________________________________________ 
In Vitro Drug Sensitivity of Selected Sphingosine Derivatives on a 
Diverse 
Tumor Cell Line Panel 
72 Hour Drug Exposure 
N-C8 
N-C4 N-C6 N-C8 Sphingosine 
N-C10 
Cell Lines 
Sphingosine 
Sphingosine 
Sphingosine 
HCl Salt Sphingosine 
Cer-C6 
__________________________________________________________________________ 
A549 4.90 +/- 0.10 
4.90 +/- 0.06 
4.91 +/- 0.01 
4.97 +/- 0.19 
4.99 +/- 0.08 
8.08 +/- 0.06 
MCF 7 4.88 +/- 0.03 
4.74 +/- 0.09 
4.88 +/- 0.11 
4.90 +/- 0.02 
5.13 +/- 0.01 
12.70 +/- 0.14 
MCF 7/ADR 
9.60 +/- 0.47 
4.69 +/- 0.10 
4.88 +/- 0.04 
4.82 +/- 0.18 
6.19 +/- 0.43 
26.1 +/- 0.85 
OVCAR 3 14.05 +/- 0.35 
4.99 +/- 0.05 
5.41 +/- 0.13 
5.23 +/- 0.01 
12.50 +/- 0.85 
15.15 +/- 1.77 
CAKI 1 6.88 +/- 0.52 
4.28 +/- 0.04 
4.64 +/- 0.07 
4.47 +/- 0.01 
6.18 +/- 0.01 
5.88 +/- 0.10 
SKMEL 28 
6.13 +/- 1.03 
4.94 +/- 0.06 
4.96 +/- 0.03 
4.95 +/- 0.02 
13.00 +/- 0.00 
11.55 +/- 0.07 
HT 29 6.49 +/- 0.11 
5.03 +/- 0.06 
5.72 +/- 0.24 
5.78 +/- 0.00 
14.50 +/- 0.14 
4.67 +/- 0.19 
LEWIS LUNG 
5.08 +/- 0.13 
4.61 +/- 0.04 
5.05 +/- 0.02 
4.82 +/- 0.18 
6.71 +/- 1.04 
5.81 
__________________________________________________________________________ 
+/- 0.31 
GI.sub.50 (uM) +/- SD 
TABLE 8 
__________________________________________________________________________ 
In Vitro Sensitivity of A549 and MCF 7/ADR to C6-Ceramide Derivatives at 
Different Drug Exposure Times 
Hour Post-Drug Addition Incubation 
__________________________________________________________________________ 
Drug A549 
Exposure 
1-Acetate 
1-Butyrate 
1-Isobutyrate 
N-C8 
Time C6-Ceramide 
C6-Ceramide 
C6-Ceramide 
Sphingosine 
__________________________________________________________________________ 
1 Hour Pulse 
92.9 +/- 1.3 
&gt;100 &gt;100 31.2 +/- 5.0 
4 Hour Pulse 
44.1 +/- 1.3 
52.9 +/- 2.5 
&gt;100 12.8 +/- 1.0 
8 Hour Pulse 
35.3 +/- 2.5 
36.5 +/- 0.8 
40.2 +/- 2.5 
6.7 +/- 0.2 
24 Hour 
7.2 +/- 0.2 
8.6 +/- 0.6 
8.4 +/- 0.7 
5.0 +/- 0.1 
Pulse 
48 Hour 
4.35 +/- 0.1 
4.7 +/- 0.1 
5.2 +/- 0.3 
4.8 +/- 0.1 
Pulse 
72 Hour 
5.1 +/- 0.1 
5.5 +/- 0.2 
5.6 +/- 0.2 
5:0 +/- 0.3 
Continuous 
Exposure 
__________________________________________________________________________ 
Drug MCF 7/ADR 
Exposure 
1-Acetate 
1-Butyrate 
1-Isobutyrate 
N-C8 
Time C6 Ceramide 
C6 Ceramide 
C6-Ceramide 
Sphingosine 
__________________________________________________________________________ 
1 Hour Pulse 
&gt;100 &gt;100 &gt;100 34.3 +/- 4.2 
4 Hour Pulse 
59.5 +/- 1.4 
79.1 +/- 5.3 
&gt;100 14.2 +/- 1.2 
8 Hour Pulse 
42.7 +/- 1.2 
44.0 +/- 1.1 
52.3 +/- 1.6 
8.9 +/- 0.2 
24 Hour 
19.5 +/- 0.8 
20.8 +/- 1.8 
22.7 +/- 1.1 
4.8 +/- 0.1 
Pulse 
48 Hour 
15.6 +/- 0.9 
17.2 +/- 0.1 
18.7 +/- 0.5 
4.62 +/- 0.1 
Pulse 
72 Hour 
16.7 +/- 1.7 
18.5 +/- 0.4 
19.6 +/- 1.0 
5.0 +/- 0.1 
Continuous 
Exposure 
__________________________________________________________________________ 
GI.sub.50 (uM) +/- SD 
Example 9 
In Vivo Toxicity Studies 
These studies were performed by intravenous injection of n-hexyl 
sphingosine, at the indicated dose (see below), into mice. Results are 
presented below. 
TABLE 9 
______________________________________ 
Toxicity of N-hexyl sphingosine after i.v injection in mice 
Dose Number of Animals 
(mg/kg) Dead 
______________________________________ 
Control (Tween-80) 
0/2 
100 2/2 
50 0/2* 
25 0/2** 
12.5 0/2 
______________________________________ 
*Immediate ataxia, decreased activity. Respiration: Gasping. 
. After 30 minute decreased activity was still observed. 
. After 24 hours no abnormality was observed. 
**Decrease activity immediately after injection. 
. After 30 minute no abnormality was observed. 
Example 10 
In Vivo Studies 
These studies were performed using P-388/adriamycin resistant leukemia 
cells. Mice were injected i.p. with 100,000 cells, and then treated on 
days 1, 3 and 5 post-injection with n-hexyl sphingosine. Results are 
presented in FIG. 14. 
Example 11 
Compound Synthesis 
Synthesis of sily ether of ceramide: The mixture of Ceramide and 
t-Butyldimethylsilyl chloride (1 equivalent) and imidazole (2 equivalent) 
in DMF was stirred under N2 at room temperature overnight. The solvent was 
removed under a stream of N2 and residue was dissolved in CH.sub.2 
Cl.sub.2, washed (H.sub.2 O), dried (MgSO.sub.4) and concentrated to 
dryness. The residue was purified over silica get (AcOEt: Hexan=1:3). 
Synthesis of 1-ester ceramide: The mixture of ceramide and Ac2O (1 
equivalent) and catalytic amount of dimethyl amino pyridine in dry CH2Cl2 
was stirred at room temperature for 1 hour and the reaction was checked by 
TLC (AcOEt). The mixture was concentrated. The crude product was purified 
over silica gel (AcOEt: Hexane=2:3.5). 
Oxidation of C3-OH of ceramide to ketone: 1-OAc ceramide was dissolved in 
acetone and cooled in ice-bath. Jone's reagent was dropwised slowly till 
the orange color persistent. The reaction was quenched by isopropanol, and 
NaHCO.sub.3 was added and stirred for 5 minutes. The solution was filtrate 
and concentrated to dryness. The crude product was purified by preparative 
TLC (ACOET: Hexane=1:2.5). 
Reduction of ceramide to sphingosine analogs: To an ice-cold stirred 
solution of ceramide in anhydrous THF was added LiAlH.sub.4 and the 
mixture was stirred at room temperature under N.sub.2 for 24 hours. Under 
ice cooling the reaction mixtures was quenched by addition of saturated 
aqueous NaHCO.sub.3. The resulting slurry was filtered and washed with 
THF. The solution was concentrated and the residue was brought into 
CH.sub.2 Cl.sub.2, washed with H.sub.2 O, dried (MgSO.sub.4) and 
concentrated to dryness. The residue was purified over preparative TLC 
(silica gel) CH2Cl2: MeOH: TEA=8:1:0.08. 
Synthesis of 4,5-diol ceramide: To a solution of ceramide in a mixture of 
Me.sub.2 CO distilled H.sub.2 O and t-BuOH, N-Methyl morpholine N-oxide 
(NMO, 1.2 equivalent) and OsO.sub.4 (catalytic amount) in THF were added. 
The reaction mixture was stirred at 45.degree. C. for 6 hours and it was 
quenched by solid NaHCO.sub.3 and the mixture was stirred for 15 minutes. 
The suspension was filtered and filtrate was dissolved in THF. The 
solution was washed with brine. The organic solution was separated, dried 
and concentrated to dryness. The residue was purified over preparative TLC 
(THF).