Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CERAMIDE DERIVATIVES AND METHOD OF USE
Field of the Invention
The invention relates to ceramide derivatives, pharmaceutical compositions
comprising the
ceramide derivatives, and therapeutic uses of the ceramide derivatives as
agents against cancer,
metabolic diseases such as diabetes, inflammatory conditions and viral
infections.
Background of the Invention
Ceramides are a class of naturally occurring splungolipids normally formed by
intracellular
hydrolysis of sphingomyelin, and are found in all animal cells. Sphingolipid
metabolites
participate in signal transduction and cell regulation possibly as first or
second messengers.
Although neither the direct targets nor the mechanisms of action of ceramides
are fully understood,
there are several pieces of evidence which suggest that ceramides play an
important role as a
regulatory component of apoptosis induced by tumor necrosis factor-alpha (TNF-
a), Fas ligand,
ionizing radiation, and chemotherapeutic agents. Intracellular ceramide is
elevated during the
induction of apoptosis in a wide variety of cellular systems and addition of
cell-permeable
ceramide analogs induces apoptosis in many cell lines, providing evidence that
ceramide
generation plays a direct role in the apoptotic response.
Apoptosis 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 Dictionar~(Illustrated)). 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,
Immuhol. Today, 14(3):126-130 (1993)).
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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, Science 259: 760-761 (1993); J. Cohen,
supra; G.
Williams and C. Smith, Cel174:777 (1993); M. Barinaga, Science 259: 762
(February 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-a and TNF-(3,
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.
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Raines et al., J. Biol. Chern. 268 (20):14572 (1993); L. Obeid et al., Science
259:1769 (March 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., .l: 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)).
In general, apoptosis occurs in two phases, an initial eommitment phase
followed
by an execution phase resulting in the condensation of nuclear chromatin,
fragmentation
of DNA, and alterations to the cell membrane. Caspases, a family of cysteine
proteases,
play a critical role in the execution phase of apoptosis; they participate in
a cascade that is
triggered in response to proapoptotic signals and in cleavage of intracellular
substrates,
resulting in disassembling cells. Based on their function in the cascade,
caspases can be
grouped into two categories, initiators and effectors. Different initiator
caspases such as
caspase 8 and 9 mediate distinct sets of signals. Caspase 8 binds with the
death effector
domain DED of the Fas receptor and a cofactor FADD (Fas-associated protein
with death
domain). The activation of caspase 9 requires several cofactors such as
cytochrome c,
dATP, and APAF-1 and through the caspase recruitment domain (CARD). Several
pieces of evidence on the role of cerarnides during caspase cascade using
caspase
inhibitors to prevent sphingolipid-induced apoptosis suggest ceramide
formation is
associated with the execution phase of apoptosis. Modulation of cellular
ceramide levels
could be a useful approach to modulate cell regulation and apoptosis.
It was previously reported that increases in cerarnide 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.), WiIIiams and Wilkins, Baltimore
(1982), p.
99). Different ceramides are eharaeterized 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 CH3(CH2) 12CH=CH-CHOH-CH(CH20H)-NH-CO-(CH2) 16CH3.
Shorter- or longer-chain fatty acids can also be linked to the sphingoid base.
Applicants
have previously reported that attachment of certain chemical groups to
sphingolipids and
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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, there has been no recognition in the prior art of the specific
halogentated
ceramides of the invention and their use in treating neoplastic diseases,
metabolic
conditions such as diabetes, inflammations and viral infections.
Short-chain (C2-C6), cell-permeable ceramides have been shown to directly
affect
endogenous ceramide levels, these short-chain ceramides mainly have been used
for studying
ceramide-mediated cellular functions. Further, though some reports have
suggested that
halogenated ceramides may be potentially useful as therapeutics for various
pathologies of the
nervous system, there has been no indication that halogenated ceramide
derivatives would exhibit
enhanced anti-neoplastic activity as compared to non-halogenated analogs, such
as C2 and C6
ceramides.
Summary of the Invention
Briefly, the invention involves a compound of the formula I:
HO OH
R
X
QTY
M~ ~Z
wherein:
R is CnH2n+i, where n is an integer of from 1-18;
A is CH2-CH2, CH2-CH(OH), or cis, trans or cis+trans CH=CH;
M is C(O) or CH2;
Q is C, OC or S(02)N;
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Y is H, OH, Cl-C6 alkyl, C(O)OH, aryl, phenyl, NH2, NO2, C6H5, a halogen, NH-
PI, (O)
or (S);
Z is H, C6H5, alkyl, NH2, NH-Pl or C(O)OH, wherein when Y is (O) or (S), Z is
not
present, and wherein when X, Y and Z are present as different moieties, the
compound
has an R, an S, or any combination of the R and S configurations about the a-
carbon;
X is F, Cl, Br, I, C6H5, O-Si(CH3)3, O-Si(C4H9)3, O-Si(C6H5)3, Cl-C17 alkyl, -
CH2-O-Xl, or
Rz N_Pi Xi Xi I t
R1~R3 Rl )k Xi ~~ N
~~ ~,~'
Pi
i X1 ~ X1 XI
X1 w I w I '~.~ N Xi N~Pi
_ 1 ~Xz ~Xi
~N ~ Xl ~'-O \ / Xl / \ / X ~x. \ X3 O~ \ /
,~"" O o
~Xi ~Xz X X O Xi ~ NiXi
O~ \ / ~ / \ X3 ~4 \ / 1 \ /
Xi NiXi Xi
w I NI ~ ~ i_
k is integer from 0 to 6;
Rl, RZ, and R3 are each independently H, Cl-C6 alkyl, Cl-C6 alkenyl, C2-C6
cycloalkyl, or aryl;
Xl is H, OH, Cl-C6 alkyl, Cl-C6 O-alkyl, Cl-C6 S-alkyl, CZ-C6 cycloalkyl,
aryl,
phenyl, a halogen, NO2, CN, C(O)H, C(O)OH, C(O)OCH3, COCH3, CH20H, NH2, N3,
NHCH3, CONH2, N(CH2CHa)2C12, B(OH)2, furyl or aryl sulfonate;
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X2 is -O-CH2-, and X3 is -O-CH2- or -O-, such that X2 and X3 taken together
form a heterocyclic ring; and
Pl is H or an amino protecting group. The ceramide derivative can be one or
any
combination of the D-erythro, D-threo, L-erythro and L-threo configurations,
as well as
one or any combination of (+) and (-) optical isomers.
The following combinations of substituents are preferred for formula I:
Each of Y and Z is independently H or C6H5.
M is C(O), X is Br or a Cl-C17 alkyl group, Q is C, Z is H, C6H5, an alkyl
group or
C(O)OH.
M is C(O), and X is
R2
R1 R
3
M is C(O), X is
Xl
'~ ~ \ /
Xl is H, Cl-C6 alkyl, F, Cl, Br, I, OH, OCH3, N02, CN, C(O)OH, C(O)OCH3, C1-C6
O-
alkyl, C1-C6 S-alkyl, phenyl or aryl;
Y is H, CH3, NH2, or N02; and
Z is H.
M is C(O), X is
~X~
"'"/-~\~ Xs
6
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Y is H, CH3, NH2, or N02; and
Z is H.
M is C(O), X is
X25
\ , X3
Y is H, CH3, NHa, or N02; and
Z is H.
M is C(O), X is
X4 X
1
\ /
X1 is H, C1-C6 alkyl, F, C1, Br, I, OH, OCH3, NOZ, CN, C(O)OH, C(O)OCH3, C1-C6
O
alkyl, Cl-C6 S-alkyl, phenyl or a~.yl;
X4 is H, a halogen, OH, OCH3, N02, CN, Cl-C6 alkyl or Ca-C6 cycloalkyl;
Y is H, CH3, NH2, or N02; and
Z is H.
M is C(O), X is
~ I X1
w
Xl is H, Cl-C6 alkyl, F, Cl, Br, I, OH, OCH3, N02, CN, C(O)OH, C(O)OCH3, C1-C6
O-
alkyl, Cl-C6 S-alkyl, phenyl or aryl;
Y is H; and
Z is H, CI-C6 alkyl or Ca-C6 cycloalkyl.
M is C(O), X is
7
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X1
\ /
0
i
X1 is H, C1-C6 alkyl, F, C1, Br, I, OH, OCH3, N02, CN, C(O)OH, C(O)OCH3, C1-C6
O-
alkyl, Cl-C6 S-alkyl, phenyl or aryl;
Y is H; and
Z is H, Cl-C6 alkyl or C2-C6 cycloalkyl.
M is C(O), X is
O _ Xi
\ /
X1 is H, C1-C6 alkyl, F, C1, Br, I, OH, OCH3, N02, CN; C(O)OH, C(O)OCH3, C1-C6
O-
alkyl, C1-C6 S-alkylr phenyl or aryl;
Y is H; and
Z is H, C1-C6 alkyl or C2-C6 cycloalkyl.
M is C(O), X is
X1
\ /
Xl is H, C1-C6 alkyl, F, C1, Br, I, OH, OCH3, NOZ, CN, C(O)OH, C(O)OCH3, C1-C6
O-
alkyl, Cl-C6 S-alkyl, phenyl or aryl;
Y is H, Ct-C~ alkyl, aryl, phenyl, OH, C(O)OH, NH2, N02, (O) or (S);
Z is H or C(O)OH, wherein when Y is (O) or (S), Z is not present.
M is C(O), X is
Xi
-O \ /
8
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Xl is H, Cl-C6 alkyl, F, Cl, Br, I, OH, OCH3, N02, CN, C(O)OH, C(O)OCH3, Cl-C6
O-
alkyl, Cl-C6 S-alkyl, phenyl or aryl;
Y is H, Cl-C6 alkyl, aryl, phenyl, OH, C(O)OH, NH2, N02, (O) or (S);
Z is H or C(O)OH, wherein when Y is (O) or (S), Z is not present.
M is C(O); X is -CH2-O-XI;
Xl is H, Cl-C6 alkyl, F, Cl, Br, I, OH, OCH3, N02, CN, C(O)OH, C(O)OCH3, Ci-C6
O-
alkyl, Cl-C6 S-alkyl, phenyl or aryl;
Y is H, Cl-C6 alkyl, aryl, phenyl, OH, C(O)OH, NH2, N02, (O) or (S);
Z is H or C(O)OH, wherein when Y is (O) or (S), Z is not present.
M is C(O), X is
NiX~
Xl is H, Cl-C6 alkyl, Cl-C6 O-alkyl, Cl-C6 S-alkyl, COCH3, CH20H, phenyl or
aryl;
Y is H, Cl-C6 alkyl, aryl, phenyl, OH, C(O)OH, NHZ, N02, (O) or (S);
Z is H or C(O)OH, wherein when Y is (O) or (S), Z is not present.
M is C(O), X is
X1
X1 is H, C1-C6 alkyl, C1-C6 O-alkyl, C1-C6 S-alkyl, COCH3, CH20H, phenyl or
aryl; and
Z is NH2 or NH-Pl.
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M is C(O), X is
X1
N
X1 is H, C~-C6 alkyl, C1-C6 O-alkyl, CI-C6 S-alkyl, COCH3, CHzOH, phenyl or
aryl;
Y is H, OH, a halogen, NH2, NH-Pl, (O) or (S).
M is C(O), X is
X~
O ~ \~/
to
and X1 is H, Cz-C6 alkyl, C1-C6 O-alkyl, C1-C6 S-alkyl, COCH3, CH20H, phenyl
or aryl.
P1 is Boc, Fmoc, Troc, silyl, sulfonyl, acetyl or benzyl.
The invention also encompasses a pharmaceutical composition comprising the
ceramide
derivative described above, and a liposome having a lipid bilayer that
comprises a lipid and the
ceramide derivative described above. The invention encompasses methods of
treating an animal
afflicted with cancer, an inflammatory disease or a viral infection by
administering an anti-cancer,
anti-inflammatory or anti-viral effective amount of the ceramide derivative to
the animal.
Brief Description of the Drawings
Figs. lA-1H are a series of fluorescence micrographs showing 2-Br C2 ceramide
induced
apoptosis in human tumor cell lines. Control (untreated) U937 (Fig. 1A), MCF7
(Fig. 1E),
MCF7/ADR (Fig 1G) or 2-Br C2 ceramide treated cells: U937, with 2 ~M fox 2 hr
(Fig 1B), 5 ~M
for 2 hr (Fig. 1C), 5 ~M for 3 hr (Fig. 1D), MCF7, with 25 ~M for 20 hr (Fig.
1F), MCF7/ADR,
with 1 ~M for 20 hr (Fig. 1H), were labeled irz situ with biotin-dUTP and
counterstained with
to
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fluoresceinated avidin. Only a few cells were labeled in untreated samples and
the unlabeled cells
are barely visible. The photographs were taken using a fluorescence microscope
with 40X
objective lens.
Figs. 2A-2D are a series of graphical depictions of 2-Br C2 ceramide induced
apoptosis in
U937 cells. For Fig. 2A, cells were treated with 0, 2, or 5 p M of 2-Br C2
ceramide for the
indicated hours and then processed using TUNEL assay. The dUTP-biotin labeled
(apoptotic) cells
were distinguished from unlabeled ones and quantified using flow cytometry.
Mean values +_ SE
from at least three independent experiments are shown in 5 pM treated samples.
Histograms shown
are from a representative experiment, in which cells were either untreated
(Fig. 2,B), treated with
100 ~M C2 ceramide (Fig. 2C), or 5 ~M 2-Br C2 ceramide (Fig. 2D) for 5 hr.
Cells were double
stained with Propidium Iodide for DNA content (X-axis) and Biotin-dUTP for
apoptotic cells (Y-
axis). R2 and R3 regions represent the non-apoptotic and apoptotic
populations, respectively.
Total event contains 15,000 cells and doublets were gated out of the statistic
regions.
Figs. 3A and 3B are plots showing caspase inhibitors preventing U937 cells
from
undergoing 2-Br C2 ceramide induced apoptosis. For Fig. 3A, various peptide
inhibitors were
incubated with cells an hour prior to drug treatment. Cells were further
treated with 2-Br C2
ceramide for additional 3 hrs and processed using TUNEL assay as described
previously. Fig. 3B
indicates ZIETD-FMK inhibition of 2-Br C2 ceraxnide induced apoptosis in a
dose-dependent
manner. Increasing doses of ZIETD-FMK were added to cells prior to 2-Br C2
ceramide
treatment. Data shown are representative of two independent experiments.
Figs. 4A and 4B are graphs showing caspase activation by 2-Br C2 ceramide
treatment.
U937 cells were treated with 5 ~M 2-Br C2 ceranude for indicated times were
processed for
cytosolic extract as described in materials and methods. 50 p g of total
protein from the extracts
were used in all samples and the volume was normalized with lysis buffer.
lETDase (Fig 4A) and
DEVDase (Fig. 4B) activity in extracts was measured with the fluorogenic
substrate, Ac-IETD-
AFC and Ac-DEVD-AFC, respectively.
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Detailed Description of the Invention
The present invention comprises novel ceramide derivatives that show
significant
anti-neoplastic activity. The ceramide derivatives are of the formula I:
HO OH
R
X
~y
~ M~Q~Z
I
wherein:
R is C"HZn+1, where n is an integer of from 1-1~;
A is CHZ-CH2, CH2-CH(OH), or cis, traps or cis+trans CH=CH;
M is C(O) or CHa;
Q is C, OC or S(02)N;
Y is H, OH, Cl-C6 alkyl, C(O)OH, aryl, phenyl, NH2, N02, C6H5, a halogen, NH-
Pl, (O)
or (S);
Z is H, C6H5, alkyl, NH2, NH-Pl or C(O)OH, wherein when Y is (O) or (S), Z is
not
present, and wherein when X, Y and Z are present as different moieties, the
compound
has an R, an S, or any combination of the R and S configurations about the oc-
carbon;
X is F, Cl, Br, I, C6H5, O-Sl(CH3)3, O-Si(C4H9)3, O-Si(C6H5)3~ Cl-C17 ~kYh -
CH2'O-X1~
Or
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Rz N_PI Xl Xl y
RyR Ri ~ ~X1 ~~ N
3
Pi
X1 ~ X1 X1
X1 ~ I w I ~~ N Xi N~PI
~ I XI 1 _ XI ~X2 ~Xl
O \ / X / \ / .w, \ X3 O o \ /
""" O 1
/~/ O Xi iXi
HN \-/ Xl ' XZ X Xl \ /
O~ ~J/~Y3 ~4 \ /
/X1 Xl
Xl ~ ~ N ~ i
I
x1
k is an integer from 0 to 6;
Rl, R2, and R3 are each independently H, Cl-C6 alkyl, Cl-C6 alkenyl, CZ-C6
cycloalkyl, or aryl;
Xl is H, OH, Cl-C6 alkyl, Cl-C6 O-alkyl, Cl-C6 S-alkyl, C2-C6 cycloalkyl,
aryl,
phenyl, a halogen, NO2, CN, C(O)H, C(O)OH, C(O)OCH3, COCH3, CH20H, NHZ, N3,
NHCH3, CONHZ, N(CH2CH2)ZC12, B(OH)2, furyl or aryl sulfonate;
X2 is -O-CH2-, and X3 is -O-CHa- or -O-, such that X2 and X3 taken together
form a heterocyclic ring; and
P1 is H or an amino protecting group. The ceramide derivative can be one or
any
combination of the D-erythro, D-threo, L-erythro or L-threo confirmations, but
is
preferably D-erythro. Additionally, the ceramide derivatives can consist of a
(+) optical
isomer exclusively, a (-) optical isomer exclusively, or any combination of
(+) and (-)
optical isomers.
The following combinations of substituents are preferred for formula I:
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Each of Y and Z is independently H or C6H5.
M is C(O), X is Br or a C1-C17 alkyl group, Q is C, Z is H, C6H5, an alkyl
group or
C(O)OH.
M is C(O), and X is
R2
R1 R
3
MisC O ,Xis
Xi
Xl is H, Cl-C6 alkyl, F, Cl, Br, I, OH, OCH3, N02, CN, C(O)OH, C(O)OCH3, C1-C6
O-
alkyl, Cl-C6 S-alkyl, phenyl or aryl;
Y is H, CH3, NH2, or N02; and
Z is H.
M is C(O), X is
~X2
'v" \ X3
Y is H, CH3, NHa, or N02; and
Z is H.
M is C(O), X is
X2
\ X3
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Y is H, CH3, NH2, or N02; and
Z is H.
M is C(O), X is
X4 X
1
X1 is H, C1-C6 alkyl, F, C1, Br, I, OH, OCH3, NOZ, CN, C(O)OH, C(O)OCH3, C1-C6
O
alkyl, Cl-C6 S-alkyl, phenyl or aryl;
X4 is H, a halogen, OH, OCH3, N02, CN, Ci-C6 alkyl or C2-C6 cycloalkyl;
Y is H, CH3, NH2, or NO2; and
Z is H.
M is C(O), X is
Xl
w
X1 is H, C1-C6 alkyl, F, C1, Br, I, OH, OCH3, NO2, CN, C(O)OH, C(O)OCH3, C~-C6
O-
alkyl, Cl-C6 S-alkyl, phenyl or aryl;
Y is H; and
Z is H, Cl-C6 alkyl or CZ-C6 cycloalkyl.
M is C(O), X is
Xi
O~
O
I
X1 is H, C1-C6 alkyl, F, C1, Br, I, OH, OCH3, N02, CN, C(O)OH, C(O)OCH3, C1-C6
O-
alkyl, Cl-C6 S-alkyl, phenyl or aryl;
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Y is H; and
Z is H, Cl-C6 alkyl or C2-C6 cycloalkyl.
M is C(O), X is
O Xi
U
X1 is H, C1-C6 alkyl, F, C1, Br, I, OH, OCH3, NO2, CN, C(O)OH, C(O)OCH3, C1-C6
O-
alkyl, Cl-C6 S-alkyl, phenyl or aryl;
Y is H; and
Z is H, Cl-C6 alkyl or C2-C6 cycloalkyl.
M is C(O), X is
\ /
X1 is H, C1-C6 alkyl, F, C1, Br, I, OH, OCH3, N02, CN, C(O)OH, C(O)OCH3, C1-C6
O-
alkyl, C1-C6 S-alkyl, phenyl or aryl;
Y is H, Cl-C6 alkyl, aryl, phenyl, OH, C(O)OH, NH2, N02, (O) or (S);
Z is H or C(O)OH, wherein when Y is (O) or (S), Z is not present.
M is C(O), X is
X1
-O \ / 25
Xl is H, Cl-C6 alkyl, F, Cl, Br, I, OH, OCH3, N02, CN, C(O)OH, C(O)OCH3, Cl-C6
O-
alkyl, C~-C6 S-alkyl, phenyl or aryl;
Y is H, Cl-C6 alkyl, aryl, phenyl, OH, C(O)OH, NH2, NO~, (O) or (S);
Z is H or C(O)OH, wherein when Y is (O) or (S), Z is not present.
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M is C(O); X is -CH2-O-Xl;
Xl is H, Cl-C6 alkyl, F, Cl, Br, I, OH, OCH3, N02, CN, C(O)OH, C(O)OCH3, Cl-C6
O-
alkyl, Cl-C6 S-alkyl, phenyl or aryl;
Y is H, Cl-C6 alkyl, aryl, phenyl, OH, C(O)OH, NH2, N02, (O) or (S);
Z is H or C(O)OH, wherein when Y is (O) or (S), Z is not present.
M is C(O), X is
N~XQ
Xl is H, Cl-C6 alkyl, Cl-C6 O-alkyl, Cl-C6 S-alkyl, COCH3, CHZOH, phenyl or
aryl;
Y is H, CI-C6 alkyl, aryl, phenyl, OH, C(O)OH, NHZ, NOZ, (O) or (S);
Z is H or C(O)OH, wherein when Y is (O) or (S), Z~is not present.
M is C(O), X is
X1
,0
X1 is H, C1-C6 alkyl, C1-C6 O-alkyl, C1-C6 S-alkyl, COCH3, CHZOH, phenyl or
aryl; and
Z is NH2 or NH-Pl.
M is C(O), X is
x~
N
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WO 01/72701 PCT/USO1/09894
Xl is H, C1-C6 alkyl, C1-C6 O-alkyl, C1-C6 S-alkyl, COCH3, CH20H, phenyl or
aryl;
Y is H, OH, a halogen, NH2, NH-Pl, (O) or (S).
M is C(O), X is
Xi
O~~
and Xl is H, CI-C6 alkyl, C1-C6 O-alkyl, C1-C6 S alkyl, COCH3, CH20H, phenyl
or aryl.
P1 is Boc, Fmoc, Troc, silyl, sulfonyl, acetyl or benzyl.
The invention also encompasses a pharmaceutical composition comprising the
novel ceramide derivatives. The pharmaceutical composition may include a
pharmaceutically acceptable carrier. The invention also comprises a method of
treating
an animal afflicted with cancer, an inflammatory disease or a viral infection
by
administering an anti-cancer, anti-inflammatory or anti-viral effective amount
of the
ceramide derivative to the animal. An "anti-cancer effective amount" is an
amount that
slows or stops the proliferation of cancer cells or other abnormally-
proliferating cells, or
causes cancer cell death. An "anti-inflammatory effective amount" is an amount
that
slows or stops an inflammatory response in a patient. An "anti-viral effective
amount" is
an amount that slows or stops the proliferation of a virus, or causes the
death of a virus.
The methods used to synthesize the ceramide derivative are not particularly
limited, and various techniques well known to those of ordinary skill in the
art, may be
used, such as those described in R. Selinger and Y. Lapidot, J. Lipid Res.,
7:174-175
(I966), incorporated in its entirety herein by reference. Illustrative
examples of solution-
phase syntheses follow, though solid-phase and combinatorial techniques may
also be
used. The examples are not intended to limit the scope of the invention
defined in the
appended claims.
1s
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Sphingosines and ceramides are formed in animal cells by the combination of
palmitoyl CoA (CH3(CH2)14-CO-S-CoA) and serine to give dehydrosphinganine
(CH3(CH2)l4Co-CH(NH3)- CH20H and C02 (see, e.g., L. Stryer, Biochemistry (2nd
edition), W. H. Freeman and Co., New York, pp. 461-462). Dehydrosphinganine is
converted to dihydrosphingosine (CH3(CH2)14-CH(OH)-CH(NH3)-CH20H) which is
then converted to sphingosine (CH3(CH2)12CH=CH-CH(OH)-CH(NH2)-CH20H). A
fatty acid is then linked to the amide group of sphingosine to give rise to a
ceramide
(CH3(CH2)12CH=CH-CHOH-CH(CH20H)-NH-CO-R, where R is a fatty acid chain).
A phosphorylcholine group (P04CH2CH2-N(CH3)3) can be attached to the ceramide
at
its hydroxyl group to produce a sphingomyelin (CH3 (CH2) 12CH=CH-CHOH-
CH(CH2P04CH2CH2-N(CH3)3)-NH-CO-R). Sphingomyelinase can catalyze the
hydrolytic removal of the phosphorylcholine from the sphingomyelin to give
rise to a
ceramide. Reverse hydrolysis of the ceramide can give rise to a sphingomyelin.
Without limiting ourselves by theory, the growth inhibitory data collected on
the
inventive ceramides and presented below suggest that there is a structure-
activity
relationship depending upon the nature of the X, Y, and Z groups attached.
Since
variations in potency are found with changes in short-chain residues (as is
the case with
2-Bromo Cx, 2 or 3-Methyl Cx, 2 or 3-Phenyl Cx ceramide series), it is likely
that
hydrophobicity plays a key role in distinguishing the cellular targets. Those
targets may
have active sites that could accommodate only smaller size hydrophobic
moieties.
Having a hydrophilic moiety tethered to the hydrophobic moiety (as is the case
with N-
Tyrosine (4-O-'Bu); N-Boc-Phenylalanine (4-N,N-dichloroethyl), and N-Boc-
Phenylalanine (4-CHZNH-iPropyl ceramide compounds) may assist in enhancing
cytotoxicity.
The following depiction suggests the theoretical concept by which target
binding is
increased in order to enhance biological activity.
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WO 01/72701 PCT/USO1/09894
HO OH
R
~ Hydrophilic
antenna
O
Hydrophobic
umbrella
The compounds of this invention, comprising alkyl chains of varying length,
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:
Vo~el'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 (I987); 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)). Fox example, such artisans would use a
sphingosine or a ceramide as their starting material. Alkyl chains of varying
length can
be attached thereto, or removed therefrom, by known means.
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
CA 02402769 2002-09-17
WO 01/72701 PCT/USO1/09894
and account for, including without limitation: the particular liposomal
bioactive agent
used, its concentration, stability and intended bioavailability; the disease,
disorder or
condition being treated with the liposomal composition; the subject, its age,
size and
general condition; and the composition's intended route of administration,
e.g., nasal,
oral, ophthalmic, topical, transdermal, vaginal, subcutaneous, intramammary,
intraperitoneal, intravenous, or intramuscular (see, for example, Nairn, J.G.,
Pharmaceutical Sciences, Mack Publishing Co. (1985)). Typical pharmaceutically
acceptable carriers used in parenteral bioactive agent administration include,
for example,
DSW, 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.
Example 1
General Procedure for the Synthesis of Ceramide Derivatives
In this and the following examples, sphingosine (synthetic and swine brain),
with
purity >98%, was obtained from Avanti Polar Lipids (Alabaster, AL) or Matreya,
Inc.
(Pleasant Gap, PA). All other reagents used in the synthesis were obtained
either from
Aldrich (Milwaukee, Wn or from Fluka (Ronkonkoma, NY). Solvents used for the
reactions and purification, unless stated otherwise, were obtained from
Aldrich.
As generally shown in Scheme 1 below, sphingosine was reacted with the
carboxylic anhydride or chloride and triethyl amine (Et3N) in methylene
chloride
(CH2C12) at room temperature. The time of completion of most of the reactions
was < 5
min. The reactions were monitored by thin layer chromatography (TLC) using
ceramide
(Cs, Sigma) as a standard. The Rfvalues for most of the derivatives were
within 0.4-0.6
in CHCl3/MeOH (90:10). After the reaction, if carboxylic acid was used, the
white
precipitate of dicyclohexylurea was filtered through a Celite pad. Solvent was
evaporated
under vacuo and the residue was purified by TLC in CHCI3:MeOH (93:7) as the
eluents.
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X
HO HO\ , Q iY HO OH
~Z
C13H27 \ ~H C13H27 ~ X
NH DCC, Et3N, CHZCI2
2 w M ~Z
Scheme 1 showing the general route to the synthesis of ceramide derivatives.
Example 2
Procedure for the Synthesis of N-2-Bromoacetyl Sphingosine (2-Br C2 Ceramide)
As generally shown in Scheme 2 below, sphingosine (200 mg, 0.67 mmol, Avanti
Polar Lipids) was added to 15 ml of anhydrous dichloromethane solution
containing 2-
bromoacetic acid (111 mg, 0.80 mmol; Aldrich Chemicals) and 1,3-
dicyclohexylcarbodiimide (165 mg, 0.8 mmol, Aldrich Chemicals). The reaction
mixture
was brought to 0° C with the aid of an ice-water bath and then
triethylamine (73 mg, 100
~,1, 1 mmol, Fluka) was added. After 30-min reaction at 0° C, the white
precipitate of
dicyclohexyl urea was filtered through a Celite bed, and the filtrate was
concentrated
under vacuo. The residue obtained was purified by preparative TLC (silica gel,
2000
micron plate, Analtech, Newark, DE) using chloroform:methanol (9:1) to yield
the
desired product with Rf= 0.5 (9:1, chloroform:methanol). Finally, the product
was
lyophilized from cyclohexane to yield 80 mg of white flaky powder, which was
characterized by 1H NMR and mass spectrometry (MS). The characteristic proton
signals, underlined, were at 8 (ppm in CDCl3): 7.2 (d, 1H, NH), 5.8 (m, 1H,
CH=CH), 5.5
(dd, 1H, CH=CH), 4.3 (s, 1H, CH-OH), 4.0 (dd, 2H, CH OH), 3.9 (s, 2H, CHZBr),
3.7
(m, 1H, CH-NH), 2.5 (bs, 1H, OH), 2.4 (s, 1H, OH), 2.1 (m, 2H, allylic CHZ),
1.6 (s, 2H,
homoallylic CH2), 1.3 (s, 20H, (CHZ)lo), 0.9 (app. t, 3H, ~-CH3). ESI-MS: m/z
420
(M.H+), parent m/z 402 (M-HZO).H+.
22
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WO 01/72701 PCT/USO1/09894
HO HO
C13H27 \ Bromoacetic acide
OH CisHz7 \ OH
NH2 DCC, Et3N, CHZC12
Br
O
Scheme 2
Other ceramides varying in acyl chain length from C4 to C2o were prepared by
the
same procedure as described above except the corresponding 2-bromo carboxylic
acids
were used. The resulting products were characterized by TLC and 1H NMR. 2-
chloro
and 2-iodo C2 ceramides were prepared by the same procedure as described above
except
2-chloro acetic acid and 2-iodo acetic acid were used, respectively, and
characterized by
TLC and 1H NMR.
Example 3
Anti-neopIastic activity evaluations
Various ceramides were prepared as described above and evaluated for anti-
neoplastic activity against several established tumor cell lines using
Sulforhodamine B
(SRB) assays (described below). The anti-neoplastic activity of the ceramides
against
particular leukemia lines was also evaluated as described below. HT29, human
colon
carcinoma, was obtained from the American type culture collection (Rockville,
MD) and
the following cell lines were obtained from DCT Tumor Repository (Frederick,
MD):
MCF7 human breast tumor, MCF7/ADR (MCF7 adriamycin resistant subline), A549
human non small cell lung cancer, P388 murine leukemia, P388/ADR (P388
adriamycin
resistant subline), and U937 human promyelocytic leukemia. All lines were
grown in
Complete Medium (RPMI 1640 medium containing 10% fetal bovine serum (GIBCO))
in
an atmosphere of 5% C02 at 37 °C. Depending on the cell type, 3,000 to
10,000 adherent
cells were plated onto 96-well plates in a volume of 100 ~.1 per well one day
prior to the
ceramide incubation. Ceramides were first dissolved in DMSO (dimethyl
sulfoxide) at a
stock concentration of 20 mM, which is 400 times the desired final maximum
test
23
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WO 01/72701 PCT/USO1/09894
concentration. The stock solutions were then diluted with complete medium to
twice the
desired final concentration. 100 ~,1 aliquots of each dilution were added to
the designated
wells. After 3 days incubation, cell growth was determined by SRB assay as
described in
Example 4. For some apoptosis studies, 20 ~M protease inhibitors (all
purchased from
Enzyme System Products, CA), Cbz-Val-Asp(O-methyl)-fluoromethyl ketone (ZVAD-
FMK), Cbz-Asp-Glu-Val-Asp(O-methyl)-fluorornethyl ketone (ZDEVD-FMK), Cbz-Ile-
Glu-Thr-Asp(O-methyl)-fluoromethyl ketone (ZIETD-FMK), Cbz-Leu-Glu-His-Asp(O-
methyl)-fluoromethyl ketone (ZLEHD-FMK), or Cbz-Phe-Ala(O-methyl)-fluoromethyl
ketone (ZFA-FMK), were incubated with cells one hour prior to ceramide
incubation
(Cbz = the protecting group benzylcarbomethoxy).
Example 4
Cell Growth Assay I
Sulforhodamine B (SRB) assays were performed as described by Monk, A. et al.,
JNatl Cancer Ihst, 83:757-766 (1991), (incorporated herein by reference), with
minor
modifications. Following incubation with experimental ceramide derivatives or
control
compounds (i.e., without drug), cells were fixed with 50 p.1 of cold 50%
(wt/vol)
trichloroacetic acid (TCA) for 60 minutes at 4 °C. The supernatant was
discarded, and
the plates were washed six times with deionized water and air dried. The
precipitate was
stained with 100 p,1 SRB solution (0.4% wt/vol in 1 % acetic acid) for 10
minutes at room
temperature, and free SRB was removed by washing three times with 1 % acetic
acid, and
the plates were air dried. Bound SRB was solubilized with Tris buffer (10 mM),
and the
optical densities (ODs) were read using an automated plate reader (Bio-Rad,
Model 3550-
UV) at 490 nm. Background values were subtracted from the test data, and the
data were
calculated as % of control. The GISO represents the concentration of test
agent resulting in
50% of net growth compared to that of the control untreated samples. In this
assay, ODs
were also taken at time 0 (the time when drugs were added) from an extra
plate. If the
ODs of the tested sample were less than that the time 0 sample, cell death had
occurred.
Percentage growth was calculated as described by Peters, A. C. et al., Lipids,
32,:1045-
1054, 1997 (incorporated herein by reference). Briefly, percentage growth was
calculated
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WO 01/72701 PCT/USO1/09894
as follows: (T - To)/(C - To) x 100, where T = mean optical density of treated
wells at a
given drug concentration, To = mean optical density of time zero wells, and C
= mean
optical density of control wells. If T < To,which indicates that cell death
has occurred,
then percentage cell death was calculated as (T - To)/(To) x 100.
Example 5
Cell growth assay II
To determine the GIso values in the leukemia lines (suspension cells), cell
numbers were directly counted instead of using the SRB assay which determines
total cell
protein. One day prior to drug treatment, 40,000/ cell wells were seeded into
24-well
plates in a volume of 0.5 ml. Stock solutions were diluted with complete
medium to
twice the desired final concentrations, and then 0.5 ml aliquots of each
dilution were
added to the designated wells. After 3 days incubation, cell growth was
determined by
counting cell number using a Coulter counter (Z-M, Coulter). Cell counts were
also
taken at time 0 and subtracted from the test results to give net growth. The
GISo
represents the concentration of test agent resulting in 50% of net growth
compared to that
of the untreated control samples.
Various ceramides prepared as described above were evaluated using the
materials
and techniques described above. The evaluations are detailed in the examples
below.
Example 6
Ceramides of the following formula were prepared as described above:
HO OH
R \ X
Y
HN
O
with R=(CH2)laCH3, Z=H, and X and Y as defined in Tables 1 and 2 below. Thus,
when
both X and Y are H, the structure of the CZ ceramide is depicted. The GIso
values were
CA 02402769 2002-09-17
WO 01/72701 PCT/USO1/09894
determined by the SRB assay as described above. Cells were treated with the
ceramide
derivatives as indicated for 3 days and then fixed for the assay. The results
are reported
in Tables 1 and 2.
Table 1. The GIso of Various Ceramides in Human tumor lines
Ceramide Cen GHT(29 A-5490 GMCF-7I)MCF-7lADR
Death )
at 50
EtM
X,Y=H + 20.0* 31.7 36.6 29.0*
X=H; Y=(CHZ)3CH3+ 4.1 4.6 12.0 12.3
X=Br; Y=H + 3.2 5.2* 5.6* 1.2
X=Br; Y=CH3 + 3.6 5.4 8.1 6.9
X=Br; Y=CH2CH3+ 5.5 6.3 6.3 4.8
X=Br; Y=(CHZ)3CH3+ 3.3 4.2 4.7 5.3
X=Br; Y=(CHZ)SCH3+ nd 15.5 23.1 20.2
X=Br; Y=(CHZ)~CH3- nd < 50 < 50 nd
X=Br; Y=(CHZ)9CH3- nd > 50 > 50 nd
X=Br; Y=(CHz)isCHs- nd > 50 > 50 nd
X=Cl; Y=H + 14.8 16.8 16.8 11.9
X=I; Y=H + 7.1 7.8 8.3 6.0
Table 2. The GIso (~M) of 2-Br C2 ceramide and C2 ceramide in Leukemia cell
lines
Cell lines X=Br; Y=H X,Y=H
P388 0.2 0.01 30.6*
P388/ADR 0.6 0.06 24.3*
U937 1.0 0.2 22.0 0.9
unless otherwise marked, the results in'I'abtes 1 and 2 were the average of
three samples of duplicates (total of six data
points) from a single experiment. Those marked by * were the average of two
independent experiments (total of twelve
data points), while results presented as mean ~ S.E. were from three or more
independent experiments. Numbers
containing > or < were an estimated values based on % control in the
prescreening test rather than from a full scale GIS«
study. nd: not determined. (+): Cell death observed (when the OD of the test
samples is less than the time 0 sample)
at 50 ~M, the maximum test concentration. (-): Cell death not observed.
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WO 01/72701 PCT/USO1/09894
Although the only difference between 2-Br C2 ceramide and C2 ceramide (a cell-
permeable
synthetic ceramide) is the replacement of the hydrogen with bromine at the 2-
position of ceramide,
this modification greatly enhanced growth inhibition compared to the parent
compound. We
compared the GISO's of the two ceramides in several murine and human cancer
cell lines and
surprisingly found that 2-Br C2 ceramide is 5 to 50 times more potent compared
to C2 parent
ceramide. While the GISO's for C2 ceramide ranged from 20 to 30 ~M, the GI$o's
for 2-Br C2
ceramide were from 0.2-6 ~M. Interestingly, 2-Br-C2 ceramide was more potent
in leukemia (< 1
pM) than in solid tumor lines (1- 6 pM) and also active in drug resistant
lines (MCF7/ADR vs.
MCF7 and P388 vs. P388/ADR). While not limiting ourselves by theory, we
believe that the
enhanced potency of 2-Br-C2 ceramide compared to C2 cerami.de could result
from changes in cell
permeation, compartmentalization and cell metabolism, due to the altered
structure and
conformation of the ceramide. The 2-Cl- and 2-I- C2 ceramides showed less
activity than the 2,-Br
C2 ceramide against tumor cell growth, though the 2-I was surprisingly much
better than the 2-Cl.
As in the C2 ceramides, the 2-Br-C6 ceramide surprisingly showed more potency
than the
C6 ceramide, though the differences were not as dramatic as those between the
2.-Br-C2 and C2
ceramides. Potency decreased in the 2-Br-C8 ceramide, and the 2-Br-C10 to C16
ceramides were
relatively inactive in these ih vitYO tests.
Example 7
The effect of length of the hydrocarbon chain in the sphingoid base on 2-Br-C2
ceramides
anti-neoplastic activity was investigated by preparing, as described above,
ceramides of the
following formula:
HO OH
R ~ X
Y
HN
Z
O
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WO 01/72701 PCT/USO1/09894
with X=Br, Y=H, Z=H, and R as defined in Table 3 below. The GISO values were
determined by the
SRB assay as described above. Cells were treated with ceramide derivatives as
indicated for 3 days
and then fixed for the assay. The results are reported in Table 3.
Table 3. The GISO ~M) of 2-BrC2 ceraxnide-derivatives with various length
of h drocarbon chain in the s hin oid base
Cell Glso (wM)Glso Glso Glso (!~M)
Death HT-29 (wM) (!~M) MCF-7/ADR
at s0 A-549 MCF-7
wM
(CHz)4CH3 + 9.9 7.1 1.2 3.0
(~H2)6CH3 + 7.2 7.5 3.7 1.8
(CHz)sCH3 + 13.8 13.2 7.4 3.0
(CHz)toCHs + 17.4 24.4 12.3 6.3
(CHz)tzCH3 + 3.7 5.0 5.9 0.9
~CH2)I4CH3 ~ + 6.3 6.1 5.4 < 1
Numbers in Table 3 containing > or < are based on the highest or the lowest
test concentration in a full scale GIso study. (+): Cell
death observed (when the OD of the test samples is less than the time 0
sample) at 50 p.M, the maximum test concentration.
Though all of the 2-Br-C2 ceramide derivatives showed anti-neoplastic
activity, there was a
decrease in activity for the C14 and C16 (sphingoid base hydrocarbon chain)
ceramides. The C10,
C 12 and C20 (sphingoid base hydrocarbon chain) cerarnides surprisingly showed
greater potency
against the MCF-7 cell line than the "natural" (C18) ceramide.
Example 8
The activity of the L-threo and D-erythro conformational isomers of 2-Br-C2
ceramide (N-
2-Bromoacetyl sphingosine) against human tumor cells was investigated. GISO
values of the L-threo
and D-erythro isomers (prepared as described above) were determined by the SRB
assay as
described above, and the results are reported in Table 4.
Table 4. The GISn of ceramide conformational-isomers in human tumor cells
Compound Cell GIso (1tM)GIso Glso (~,M)Glso (p,M)
(pM)
(Ceramide) DeathHT-29 A-549 MCF-7 MCF-7/ADR
at
50
L-threo 2-BrC2- + 6.4 10.9 5.5 < 1
D-er thro 2-BrC2- + 3.7 5.0 5.9 0.9
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WO 01/72701 PCT/USO1/09894
The Table 4 results were the average of three samples of duplicates (total of
six data points) from a single experiment. Numbers
containing > or < were an estimated values based on % control in the
prescreening test rather than from a full scale GISO study. (+)
Cell death observed (when the OD of the test samples is less than the time 0
sample) at 50 ~M, the maximum test concentration.
As Table 4 indicates, the D-erythro isomer showed higher anti-tumor activity
against the
HT-29 and A-549 cell lines than the Lrthreo isomer. Both isomers were
similarly effective against
the MCF-7 and MCF-7/ADR lines.
Example 9
TUNEL Assay
To determine whether 2-Br C2 ceramide induced apoptosis, TUNEL (Terminal
transferase mediated dUTP nick end labeling) assays were performed as
described
Gorczyca et al., Cancer Res., 53: 1945-1951 (1993). Briefly, following drug
treatment, 2
x 106 cells were incubated with 1 % formaldehyde in PBS (pH 7.4) for 20 min at
room
temperature and then fixed with 70% ice- cold ethanol at -20 °C for 1
to 3 days. Samples
were rehydrated with PBS (pH 7.4) before suspension in 50 ~1 of a solution
containing lx
TdT buffer (25 mM Tris-HCI, 200 mM Potassium cacodylate), 2.5 mM cobalt
chloride,
0.5 n mole Biotin-16-dUTF, and 10 unit Terminal transferase (all above
chemicals
obtained from Roche Molecular Biochemicals) at 37 °C for 30 rains.
Samples were then
washed with cold PBS and resuspended in 100 ~l of a staining solution
(fluoresceinated
avidin (I:150), 4x saline sodium citrate buffer, 0.1% Triton X-100, and 5%
nonfat dry
milk) for 30 rains at room temperature in the dark. For flow cytometric
studies, cells
were double stained with propidium Iodide ( 10 ~ g/ml) in PBS with 0.1 % RNAse
overnight at 4 °C. To observe samples using fluorescence microscopy,
adherent cells
were plated onto chamber slides (Falcon, NJ) before drug treatment and
suspension cells
were spun down to slides using a Cytospin (800 rpm, 4 rains) after drug
treatment. The
reaction volume was adjusted to cover the slides, and mounting fluid (Vector,
CA) was
applied to the slides after assay. Photographs were taken using a fluorescence
microscope
with a 40X lens, and are shown in Fig. 1 ((A) control (untreated) U937; (B)
U937 treated
with 2 ~,M 2-Br C2 ceramide for 2 hr; (C) U937 treated with 5 ~,M 2-Br C2
ceramide for
2 hr; (D) U937 treated with 5 ~,M 2-Br C2 ceramide for 3 hr; (E) control
(untreated)
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WO 01/72701 PCT/USO1/09894
MCF7; (F) MCF7 treated with 25 ~.iM 2-Br C2 ceramide for 20 hr; (G) control
(untreated)
MCF7/ADR; (H) MCF7/ADR treated with 1 ~t,M 2-Br C2 ceramide for 20 hr).
The results shown in Fig. 1 reveal that 2- Br C2 ceraxnide induces apoptosis
in U937 cells
and in other solid tumor lines. The apoptotic (brighter) cells and condensed
chromatin were
observed in U937 cells as early as 2 hr following 2 ~M 2-Br C2 ceraznide
treatment (Fig. 1B), and
the number of apoptotic cells increased in a dose- and time- dependent manner
(Fig.lB-D). At an
early stage of apoptosis, chromatin granules were present within the nuclear
membrane, however,
by 3 hr of drug treatment, many cells seemed to have lost their integrity and
the nuclei had
disintegrated. Apoptosis was also observed in MCF7/ADR cells treated with the
GISO concentration
(1 ~M) of 2-Br CZ ceramide for 20 hr (Fig. 1H). In contrast to MCF7/ADR, the
parent MCF7 cell
line required a much higher drug concentration (4 X GISO) for apoptosis
induction (Fig. 1F) and we
only observed positive staining of whole nuclei rather than condensed
chromatin granules as
observed in the other lines. In addition, with longer treatment, these cells
became fragile and we
found increased cell loss during assays.
In order to quantify 2-Br C2 ceramide-induced apoptosis, the same type of
TUNEL assays was performed, and the results were quantified by flow cytometry.
U937
cells were treated with 2 or 5 pM of 2-Br C2 cerarnide for up to 24 hrs and %
of apoptotic
population was determined (Fig. 2A). Treatment with 2 ~M (2 times GI$o
concentration)
2-Br C2 ceramide induced a marginal increase of apoptotic cells (5- 8% above
control) in
24 hr. However, with 5 ~M treatment, this induction of apoptosis was detected
by 2 hr
after initiation of treatment where there were more than 10% cells undergoing
apoptosis
and this number increased to over 60% at 6 hr. Based on the results in Fig.
2A, cells were
treated with either 100 ~M of C2 ceramide (representative histogram shown in
Fig. 2C)
or 5 ~M of 2-Br C2 ceramide (representative histogram shown in Fig. 2D) for 5
hrs and
apoptosis studies were then performed and compared to the untreated control
(representative histogram shown in Fig. 2B). These cells were double stained
with
Propidium Iodide for DNA content (x-axis of Figs. 2B-C) and Biotin-dUTP for
apoptotic
cells (y-axis of Figs. 2B-C). Although both C2 and 2-Br C2 ceramides induced
apoptosis
CA 02402769 2002-09-17
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in all phases of cell cycle, 2-Br C2 ceramide was at least three times more
efficient in
inducing apoptosis (12% vs. 43%) and at a far lower concentration (Fig. 2C,
D).
Example IO
Signaling pathway of apoptosis activated by 2-Br C2 ceramide
To explore which signaling pathway of apoptosis was activated by 2-Br C2
ceramide,
several cell permeable caspase inhibitors were added to U937 cells 1 hr prior
to drug treatment at a
final concentration of 20 ~M. These inhibitors were previously shown to
inhibit programmed cell
death induced by various apoptogenic agents (Thornberry, Chem Biol, 5: R97-103
(1998)). We
used several inhibitors, ZVAD- FMK: a general inhibitor, ZDEVD- FMK: a
inhibitor for Group II
caspases and, to a lesser extent 8, and ZIETD- FMK: a specific inhibitor for
caspase 8, ZLEHD-
FMK: a specific inhibitor for caspase 9, and ZFA- FMK: a negative control, and
studied their
effects on 2-Br C2 induced apoptosis using the TUNEL assay. Because of the
possible degradation
of these peptide inhibitors, we only treated U937 cells with ceramides for
additional 3 hrs after
addition of the inhibitors before the harvest of cells for TUNEL assay. We
found while ZVAD-,
ZDEVD-, and ZIETD- FMK prevented 2-Br C2 ceramide-induced apoptosis, neither
ZLEHD-
FMK or the negative control (ZFA- FMK) affected (actually slightly enhanced)
this apoptosis (Fig.
3A). A single agent, ZIETD-FMK which inhibits caspase 8 specifically, caused a
dose-dependent
inhibition of the apoptosis (Fig. 3B). More than 90% of 2-Br C2 ceramide-
induced apoptosis was
inhibited at as low as 5 ~ M ZIETD-FMK. These data suggest that the induction
of apoptosis by 2-
Br C2 ceramide is mediated through the caspase 8 rather than the caspase 9
cascade and it acts
earlier in the pathway. In contrast to a previous report (Sweeney et al., FEBS
Lett, 425:61-65
( 1998)) that ZIETD-FMK had almost no inhibitory effects on C2- or C6-
ceraxnide-induced
apoptosis in HL,60 cells, we found that 20 p M of ZIETD-FMK was sufficient to
inhibit 90% of the
apoptosis induced by C2 ceramide in U937 cells (data not shown). Our data
suggest that ceramide
analogs can act not only upstream of the effector caspases but also upstream
of the initiator
caspase.
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Example 11
Caspase activation in 2-Br C2 ceramide-treated cytosolic extract.
To investigate the specificity of the caspase activity induced by 2-Br C2
ceramide,
we used the caspase specific substrates, lETD- and DEVD-AFC. Cytosolic
extracts were
prepared cell cytosolic extracts were prepared according to Tamm et al.,
Cancer Res,
58:5315-5320 (1998), and reactions were carried out in 96 well plates as
described by
Cuvillier et al., JBiol Chen2, 273:2910-2916 (1998). 100 p1 of reaction buffer
(2X)
containing 100 pM of Z-DEVE-AFC or Z-IETD-AFC (Enzyme System Products, CA),
20 mM Hepes (pH 7.4), 200 mM NaCI, 1 mM EDTA, 0.2% CHAPS, 10 mM DTT, and
20% Sucrose was add to an equal volume of buffer A (20 mM Hepes, 10 mM KCI,
1.5
mM MgCl2, 1 mM EDTA, and 1 mM DTT) containing 50 ~g total protein. Enzymatic
hydrolysis of substrates was measured by release of AFC (amino-trifluoromethyl
coumarin) induring a 30-min period using a Cytofluor 4000 multi plate reader
(PerSeptive Biosystems, MA) (excitation at 395 nm and emission at 490 nm).
Caspase
activity was measure as arbitrary fluorescence units and converted to fold
increase over
basal level in untreated (control) cells. Background fluorescence of substrate
alone was
subtracted in each coordinated sample. The accumulation of IETD- and DEVD-
specific
protease activity from untreated and treated U937 cells was determined (Fig.
4A & B).
As can be seen, the activation of IETDase activity preceded that of DEVDase,
although
the latter had a greater and more sustained increase. This is in agreement
with the
concept that caspase 8 acts upstream in the signaling pathway and its
activation occurs
prior to the activation of effector caspases. This drug induced- activation of
caspase 8 is a
"transit" response; it peaked at 2 hr (2-fold), dropped below basal level
right after and
then gradually return to basal level (Fig. 4A). DEVDase activity reflects the
activity of
the down stream effectors, caspases 3, 6, and 7. The increase in DEVDase
activity slowly
followed that of lETDase, peaked at 3 hr, and remained elevated up to 6 hr
(Fig. 4B),
implying the down stream effectors remained active through the apoptosis
process. These
data further support our finding on the association of caspase 8 activation on
2-Br C2
ceramide-induced apoptosis. Scaffidi, et al., JBiol Chem, 274:22532-22538
(1999)
recent reported that only type 1I (eg. CEM and Jurkat), but not type I (eg.
SKW6.4 and
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H9) cells, are sensitive to C2 ceramide-induced apoptosis. Type I and type II
cells of
CD95 pathway differ in the kinetics and the levels of caspase 8 activation and
in whether
or not this activation bypasses mitochondria. Their data suggested that, in
type II cells,
C2 ceramide acts at the level of mitochondria, which is upstream of caspase 8
in the
apoptosis pathway. In agreement with their finding, we also found that
ceraxnide could
act upstream of caspase 8 in U937 cells.
Example 12
Anti-proliferative testing of Peptidomimetic and Non-peptidomimetic Ceramides
Peptidomimetic ceramides (peptidomimetac = containing one or more an amino
acid
groups) and non-peptidometic cerarnides were synthesized using the
combinatorial parallel
synthesis-technique described in Example 2. The ceramides obtained were then
tested for anti-
proliferative activity, and the results are reported in Tables 7 and 8 below.
The GISO values were
determined by the SRB assay as described above. A "+" indicates that cell
death was observed
(when the OD of the test samples is less than the time 0 sample) at 50 ~.tM,
the maximum test
concentration. "-" indicates that cell death was not observed at 50 ~.M.
Table 7: Ceramides analogs and their in vitro cytotoxicity properties
Non-Peptidomimetic HT29 A549 MCF-7 MCF-7/
Ceramide (~,M) (~,M) (~,M) ADR
(!~)
(R)-2-Phen 1 ro ano I 6.2 9.7 11.7 18.9
(S)-2-Phen 1 ro ano 1 6.6 6.1 6.5 8.9
(R)-3-Phen Ibutano 1 3.2 3.4 3.9 5.4
(S)-2-Phen lbutano I 7.9 11.5 12.2 17.3
3-Meth lbutano 1 (C4) 2.4 3.8 4.5 10.6
2-Meth 1c clo ro ano 2.8 5.7 6.3 10.5
1
()-2-Meth lbutano 1 3.5 3.9 6.2 3.8
2-Chloroacet 1 (C2) 14.8 16.8 16.8 11.9
2-Bromoacet 1 (C2) 5.2 3.3 6.8 0.8
2-Iodoacet 1 (C2) 7.1 7.8 8.3 6.0
L-threo-2-Bromoacet I 6.4 10.9 5.5 <1
(C2)
2-Bromoacet 1 (C2)-C10 10.9 7.6 3.6 3.2
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2-Bromoacet 1 (C2)-C12 16.8 11.9 7.7 2.7
2-Bromoacet 1 (C2)-C14 >20 >20 >20 3.1
2-Bromoacet 1 (C2)-C16 >20 >20 >20 7.6
2-Bromoacet 1 (C2)-C20 3.0 2.0 7.0 0.4
(S -2-Br-Pro ano 1 4.2 12.6 3.8 7.8
Pro ano 1 (C-3) 3.0 10.2 12.2 12.68
3,3-di-Phen 1 ro ano 2.9 10.2 10.2 13.6
1
Butano 1 (C4) 2.5 7.2 13.4 18.7
(S)-2 Meth lbutano I 1.5 5.2 4.7 5.2
(C4)
(S)-2-Methylbutanoyl 12.7 21.8 36.8 50.0
(C4) C-
12
(S)-2-Methylbutanoyl 5.3 7.9 9.8 17.5
(C4) C-
14
(S)-2-Methylbutanoyl 2.2 3.7 4.4 4.9
(C4) C-
16
(S)-2-Methylbutanoyl 3.5 3.2 3.9 4.8
(C4) C-
20
R-3-Phen lbutano 1 (C4)<2 2.5 3.6 5.4
2-Meth 1 entano 1 (C5) 2.4 3.4 5.3 6.2
3-Meth I entano 1 (C5) 1.6 1.4 5.9 11.7
4-Meth 1 entano 1 (C5) 0.9 10.0 9.9 13.8
3-Methylbutanoyl (C4) 1.1 2 3.8 6.8
3-(Trimeth lsil 1) ro 2.6 3.3 4.6 <1
ionic
3,4-(Methylenedioxyphenyl)-6.2 10.8 12.6 >20/17
stet 1
oc-Phen lc clo entano 8.7 12.8 12.5 >20
1
2-Methox hen 1 ro ano 2.5 3.3 4.2 5.0
1
2-(4-Nito hen I)- ro 3.2 6.6 6.7 7.3
ano I
(Indole-3-stet 1) 5.6 7.4 7.2 10.9
(2-Norbornane carbox 3.6 3.9 14.7 20.2
1)
(Tos I-3- rrolcarbox 3.4 5.0 7.4 >10
I)
3 3,5-Trimeth lhexanoic2.9 2.4 4.2 8.3
3-Meth 1 ro ano 1 (C3) 1.5 2.9 4.0 5.3
2-Eth lhexanoic 3.3 4.2 7.6 7.7
traps-2 meth 1-2- entenoic3.1 6.1 11.2 12.6
Peptidomimetic CeramideHT29 A549 MCF-7 MCF-7/
(~.M) (~.M) (~.M) ADR
( M)
BOC- -fluoro hen lalanine3.3 3.6 4.9 6.9
BOC- -meth 1 hen lalanine3.1 3.6 5.6 7.2
BOC- -chloro hen lalanine3.9 5.1 6.2 7.1
BOC-p-cyanophenylalanine1.1 4.2 5.6 6.7
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NH2-D-Homo hen lalanine4.5 11.0 6.7 >20
Boc-Homo hen Ialanine 1.9 3.0 4.0 6.7
Boc-Phenylalanine (4-N,N-<2 4.5 4.0 5.6
dichloroeth I)
Fmoc-Phen Ialanine (4-I)7.1 8.7 15 18
Fmoc-Norar anine (BOC)23.1 7.7 10.3 10.3
NHZ-Phenylalanine (4-COO-6.7 6.1 4.2 9.3
taut I)
4-t-Butylcyclohexanecar-6.0 8.2 11.4 14.9
box lic (c/t)
Fmoc-Phenylalanine (4-CH2N-5.3 5.6 2.4 10.4
iso ro I)
NH2-Phenylalanine (4CH2N-2.8 3.0 2.0 >20
iso ro ano 1)
BOC-Phenylalanine [4-NH-(4-11.8 13.8 6.8 <2
Bromobenzenesulfon 1)]
N-Boc-Phenylalanine <2 8.1 8.1 7.3
(4-Nitro-
tetrah drofur o1)
Boc-c clohex lalanine 3.5 4.3 7.1 11.2
Boc-D-c clohex Ialanine3.3 3.6 8.1 16.5
NHa-L(c/t)-Cyclohexylalanine-3.4 4.2 2.5 10.3
(4NH-Boc)
NHa-o-T rosine (O-taut 1.8 1.9 1.1 11.2
1)
NHZ-m-T rosine (O-taut 1.9 3.1 2.5 5.4
1)
NH2- -T rosine (O-taut 0.8 2.4 3.3 4.2
I)
BOC-T rosine (3,5-Ia) 3.4 9.4 11 13.6
NH2-T rosine 4.6 5.6 5.4 >10
NHZ-T rosine (-O-Boc) 5.1 4.1 4.0 5.9
NH2-D-T rosine (O-taut 4.4 6.0 6.4 13.6
1)
NH2-Homot rosine 7.0 6.9 6.1 14.6
BOC-Norvalanine 2.5 4.2 6.1 6.5
NH2-Leucine 3.5 5.3 3.3 7.3
Boc-Leucine 1.0 2.9 4.8 6.8
Boc-Leucine-L sine 3.7 6.3 6.5 14.9
NHZ-Leucine L sine 46.2 >50 >50 >50
Boc-Norleucine 1.0 1.5 2.6 3.6
NHZ-a-Aib Cer 4.2 10.4 5.8 11,7
a-Aib-Boc-Phealanine 11.6 12.7 10.9 14.7
a-Aib-Boc-D-Homophenyala-6.1 11.6 9.8 13.6
nine
NH2-Dab(Boc) 3.9 7.3 9.2 6.8
NH2-Da (Boc) 3.0 3.8 5.0 8.4
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Table 8: Screening against MCF-7 and A549 tumor cell lines; the "+" sign
indicates the
activity at 50 EtM concentration, whereas the "-" sign indicates relatively
inactive.
Non-Peptidomimetic Ceramide conc.
MCF7/
A549
2-Chloro hen facet I +/+
1-Phen 1-1-c clo entano 1 +/+
1-Phen I 1-c clo ro ano I +/+
t-Bu 1 hen Ithio +/+
4-Meth 1 hen loxo +/+
2-Nitrophenylpyruvic +/+
2-Bromo hen 1 acet 1 +/+
S -2 Phen Icarbamo to ro ionic-/+
3-Eth 1-3-meth I lutaric -/-
Tol lsulfon 1 +/+
O-Meth I acet I -/-
O-Meth 1 Sulfon I -/+
DL -thioct 1 +/+
Tol lsulfon 1 +/+
R -3-Meth I-2-NitroMeth I-OPA +/+
2-Meth lsulfon I eth 1 +/+
12 3-Triazolecarbo 1 +/+
1-Meth lc clo ro 1 +/+
2-Bromo ro ano 1 C3 -/-
C anoacet I C2 +/+
S -2-Meth Ibut I C4 C-10 -/-
2 2-di-Phen 1 ro ano 1 C3 -/-
3-Nitro ro ano I C-3 +/+
-2-Phen 1 ro ano I C-3 +/+
t-Bu facet 1 C2 +/+
2-Bromoacet 1-13-di- 2-Bromohexadexano-/-
1
2-Pro I entano 1 +/+
4-Meth Ihi uric +/+
3 3 3-Tri hen 1 ro iono 1 +/+
2-Bromo-2-Meth I ro ano 1 +/+
3-Meth Ihi uric +/+
Peptidomimetic Ceramide conc.
MCF7/
A549
Boc-HistdineBom +/+
Boc-Phen 1 I tine +/+
NHZ-Phen 1 I tine +!+
Fmoc-Phen 1 1 tine 4-CHzNH-Boc+/+
NH2-Phen I 1 tine 4CH2 NH2 +/+
S -2-Meth Ibutano I C4 Alanine-/+
Boc-D-Homo hen lalanine -/+
S -2-Meth lbu 1 Phen 1 1 tine -/-
NHZ-D-Homophenylalarune -/+
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D-Homo hen lalanine-Boc-Phenylalanine-/-
NHZ-L-Phen lalanine (4-Phen +/+
1)
Boc-Phen lalanine-[4-Nitro] +l+
Boc- Phen lalanine 4-NH-Acet +/+
I]
Boc- Phen lalanine (4-NH-Fmoc)-/-
Boc-D-Phen lalanine-(4-Nitro) +/+
Phen lalanine (4-NH-Fmoc) -/-
Boc-Phen lalanine (4-NHZ) +/+
Boc-Phen laalanine (4-Nitro-tetrah+/+
drofur o1)
Fmoc-Phen lalanine (4-CHO) +/+
Fmoc-Phen lalanine (4-Gu(Boc)Z)-/+
Fmoc-Phenylalanine (4-CHZ-OTrityl)-/-
NHZ-Phen lalanine (4-CHZ-O-Trit-/-
1)
NHZ-Fhen lalanine-(4Gu(Boc)2) +/+
Boc-Phenylalanine [4-NH-(4-Chloro-3-Nitro)--/+
Benzenesulfon 1]
Boc-Phenylalanine [4-NH-(2-Bromobenz-+/+
enesulfon 1]
Boc-Phenylalanine (4-NH)-[(3-trifluoromethyl)--/-
Benzenesulfon 1]-Ceramide
Boc-Phen lalanine (4-NH2) +/+
Boc-Phenylalarune [4-NH-(4-(trifluoromethoxy)-/-
benzenesulfon I)
Boc-Phenylalanine [4-NH-(2,4,6-Triisopropyl)-/-
benzenesulfon 1]
Boc-Fhen lalanine 4-NH-(3-Furoic)]+/+
2-Bromoacet 1-Phen lalanine -/+
D-Homophenylalanine-Boc-D- -/-
Homo hen lalanine
NHZ-Homo hen lalarune +/+
Boc-T rosine (Bromobenz 1) -/-
NH2-Homot rosine (O-Trit I) -/-
Fmoc- -T rosine (O-'But 1) -/+
N-Fmoc-m-T rosine (O-'But 1) +/+
Boc-a-Abu +/+
Boc-a-Aib +/+
C clhex lalanine- uinaldic -/-
Fmoc-(c/t)C clohe lalanine-(4-NH-Boc)-/-
Boc-O-Benz 1-Serine-leucine +/+
NHZ-O-Benz 1-Serine-Leucine -/-
Boc-Leucine-L sine-OH -/-
NHZ-Dab -/-
.Although the invention has been described with reference to specific examples
and
embodiments, those of ordinary skill in the art will readily recognize that
various equivalent
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elements may be substituted without departing from the spirit and scope of the
invention defined in
the appended claims.
38
