U.S. patent application number 13/260553 was filed with the patent office on 2012-02-09 for glycolipids as treatment for disease.
This patent application is currently assigned to SENEB BIOSCIENCES, INC.. Invention is credited to Shawn DeFrees.
Application Number | 20120035120 13/260553 |
Document ID | / |
Family ID | 42781521 |
Filed Date | 2012-02-09 |
United States Patent
Application |
20120035120 |
Kind Code |
A1 |
DeFrees; Shawn |
February 9, 2012 |
GLYCOLIPIDS AS TREATMENT FOR DISEASE
Abstract
This invention provides compounds, compositions, and methods for
treating a disorder selected from cancer, hyperinsulinemia,
hypoglycemia, hyperinsulinemia with hypoglycemia, atypical
Parkinson's disease, Huntington's disease, multiple systems
atrophy, GM3 synthase deficiency, GM2 synthase deficiency or
tauopathy.
Inventors: |
DeFrees; Shawn; (North
Wales, PA) |
Assignee: |
SENEB BIOSCIENCES, INC.
North Wales
PA
|
Family ID: |
42781521 |
Appl. No.: |
13/260553 |
Filed: |
March 25, 2010 |
PCT Filed: |
March 25, 2010 |
PCT NO: |
PCT/US10/28725 |
371 Date: |
September 26, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61163371 |
Mar 25, 2009 |
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61180098 |
May 20, 2009 |
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61180346 |
May 21, 2009 |
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61220151 |
Jun 24, 2009 |
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61244735 |
Sep 22, 2009 |
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61293200 |
Jan 7, 2010 |
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Current U.S.
Class: |
514/25 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/70 20130101; A61P 25/28 20180101; A61K 31/715 20130101;
A61K 31/7028 20130101; A61K 31/7032 20130101; A61P 25/14 20180101;
A61P 3/10 20180101 |
Class at
Publication: |
514/25 |
International
Class: |
A61K 31/7028 20060101
A61K031/7028; A61P 25/14 20060101 A61P025/14; A61P 35/00 20060101
A61P035/00; A61P 3/10 20060101 A61P003/10; A61K 31/7032 20060101
A61K031/7032; A61K 31/715 20060101 A61K031/715 |
Claims
1. A method of treating a subject having a disorder associated with
a ganglioside deficiency, said method comprising administering to
the subject a therapeutic amount of a compound of the formula:
##STR00008## wherein Z is O, S, C(R.sup.10).sub.2 and NR.sup.2, X
is H, D, --OR.sup.11, --NR.sup.11R.sup.12, --SR.sup.11, and
--CHR.sup.11R.sup.12, and R.sup.10, R.sup.11 and R.sup.12
independently selected from H, D, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted heterocycloalkyl, --C(.dbd.B)R.sup.13,
--C(.dbd.B)-Z-R.sup.13, --SO.sub.2R.sup.13, and --SO.sub.3
functional moieties; B, B', and Z independently selected from O,
NR.sup.14 or S; Y is selected from H, D, --OR.sup.15, --SR.sup.15,
--NR.sup.15R.sup.16, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl, and
substituted or unsubstituted heterocycloalkyl moieties; R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.13, R.sup.14 independently selected from H, D,
--OR.sup.17, --NR.sup.17R.sup.18, --SR.sup.19, and
--CHR.sup.19R.sup.20, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted heterocycloalkyl moieties; R.sup.15,
R.sup.16, R.sup.17, R.sup.18, and R.sup.19 independently selected
from substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heterocycloalkyl moieties, --C(.dbd.B')R.sup.20,
--C(.dbd.B')--Z--R.sup.20, --SO.sub.2R.sup.20, and --SO.sub.3
functional moieties; R.sup.20 is selected from H, D, --OR.sup.21,
--SR.sup.21, --NR.sup.21R.sup.22, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted heterocycloalkyl moieties; R.sup.21
and R.sup.22 are independently selected from H, D, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heterocycloalkyl moieties;
n is an integer from 1 to 30.
2. The method of claim 1, wherein the disorder is selected from
cancer, hyperinsulinemia, hypoglycemia, hyperinsulinemia with
hypoglycemia, Huntington's disease, multiple systems atrophy, GM3
synthase deficiency, GM2 synthase deficiency or tauopathy.
3. The method of claim 1, wherein the glycolipid is selected from
the group consisting of LacC er, GA2, GA1, GM1b, GD1c, GD1.alpha.,
GM3, GM2, GM1a, GD1a, GT1a, GT1a, GD3, GD2, GD1b, GT1b, GQ1b,
GQ1b.alpha., GT3, GT2, GT1c, GQ1c, GP1c, and GP1c.alpha..
4. The method of claim 2, wherein the affected disorder has a
deficiency in tissue level of any one or more of the gangliosides
selected from GA2, GA1, GM1b, GD1c, GD1.alpha., GM3, GM2, GM1a,
GD1a, GT1a, GT1.alpha., GD3, GD2, GD1b, GT1b, GQ1b, GQ1b.alpha.,
GT3, GT2, GT1c, GQ1c, GP1c, and GP1c.alpha..
5. The method of claim 1, wherein the saccharide moiety of a
ganglioside selected from GM3, GM2, GM1a, GD1a, GT1a, or
GT1.alpha..
6. The method of claim 1, wherein the saccharide is a mono-, di-,
tri-, tetra-, penta-, hexa-, or hepta-saccharide.
9. The method of claim 1, wherein the fatty acid has at least one
cis double bond.
10. The method of any one of claims 1, wherein the saccharide is
selected from the group consisting of Neu5Ac.alpha.3Gal.beta.4Glc-;
GalNAc.beta.4(Neu5Ac.alpha.3)Gal.beta.4Glc-;
Gal.beta.3GalNAc.beta.4(Neu5Ac.alpha.3)Gal.beta.4Glc-;
Neu5Ac.alpha.3Gal.beta.3GalNAc.beta.4(Neu5Ac.alpha.3)Gal.beta.4Glc-;
and
Neu5Ac.alpha.8Neu5Ac.alpha.3Gal.beta.3GalNAc.beta.4(Neu5Ac.alpha.3)Gal.be-
ta.4Glc-.
11. The method of claim 1, wherein the fatty acid is
.alpha.-hydroxylated.
12. The method of claim 1, wherein the subject is human.
13. The method of claim 1, wherein the cancer is a hematologic
cancer or a solid tumor.
14. The method of any of claim 1 wherein the disorder is associated
with a deficiency in a ganglioside level and the compound of
formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) has the
saccharide moiety of the deficient ganglioside.
16. The method of claim 14, wherein the medicament is formulated
for parenteral administration.
17. The method of claim 14, wherein the fatty acid moiety is
.alpha.-hydroxylated.
18. The method of claim 14 wherein the glycolipid comprises an
.alpha.-hydroxylated fatty acid moiety.
19. A pharmaceutical composition comprising a compound of Formula
(1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) and a
pharmaceutically acceptable excipient for use in treating a
disorder selected from cancer, hyperinsulinemia, hypoglycemia,
hyperinsulinemia with hypoglycemia, Huntington's disease, multiple
systems atrophy, GM3 synthase deficiency, GM2 synthase deficiency
or tauopathy.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Patent Application No. 61/163,371
filed on Mar. 25, 2009, U.S. Provisional Patent Application No.
61/293,200, filed Jan. 7, 2010, U.S. Provisional Patent Application
No. 61/244,735, filed Sep. 22, 2009, U.S. Provisional Patent
Application No. 61/220,151, filed Jun. 24, 2009, U.S. Provisional
Patent Application No. 61/180,346, filed May 21, 2009 and U.S.
Provisional Patent Application No. 61/180,098, filed May 20, 2009,
which are incorporated herein by reference in their entirety for
all purposes.
FIELD OF THE INVENTION
[0002] This invention relates to methods of treating and
amliorating diseases of cancer, hyperinsulinemia, hypoglycemia and
atypical parkinson's disease, tauopathies, and other neurological
diseases, and/or a ganglioside deficiency state or disorder by
administering a suitable glycolipid, ganglioside or ganglioside
analog.
BACKGROUND OF THE INVENTION
[0003] Besides the administration of antibodies or tyrosine kinase
inhibitors for malignancies, the typical treatment for many cancers
is the administration of radiation therapy and chemotherapeutic
agents. However, side effects are a limiting factor in radiation
treatments. Combination chemotherapy has some success in reaching
partial or complete responses. Unfortunately, the remissions
obtained through chemotherapy are often not durable.
[0004] Congenital hyperinsulinism (CHI, OMIM 256450) is a genetic
disorder of pancreatic .beta.-cell function characterized by
failure to suppress insulin secretion in the presence of
hypoglycemia, resulting in brain damage or death if inadequately
treated.
[0005] Neuroendocrine tumors including such cancers as insulinoma,
hepatomas, mesotheliaoma and fibrosarcoma may also cause
hyperinsulinemia accompanied by hypoglycemia.
[0006] Gangliosides are sialic acid containing glycosphingolipids.
Gangliosides are normal components of plasma membranes which are
particularly abundant in the nervous system. In humans,
gangliosides are most abundant in the gray matter of the brain,
particularly in nerve endings. As a result, the addition of one or
more glycolipids for cancer (e.g., during immunotherapy, such as
antibodies or tyrosine kinase inhibitors), patients suffering from
hyperinsulinemia, hypoglycemia or hypersinsulinemia with
hypoglycemia or neurological disorders (e.g., Huntington's Disease,
multiple systems atrophy, atypical Parkinson's disease,
tauopathies) may reduce the mortality and/or morbidities associated
with each disease. Attempts have been made to use gangliosides in
the treatment of disorders of the nervous system. This has led to
the development of synthetic gangliosides as well as natural
ganglioside containing compositions for use in the treatment of
disorders of the nervous system (see, U.S. Pat. Nos. 4,476,119;
4,593,091; 4,639,437; 4,707,469; 4,713,374; 4,716,223; 4,849,413;
4,940,694; 5,045,532; 5,135,921; 5,183,807; 5,190,925; 5,210,185;
5,218,094; 5,229,373; 5,260,464; 5,264,424; 5,350,841; 5,424,294;
5,484,775; 5,519,007; 5,521,164; 5,523,294; 5,677,285; 5,792,858;
5,795,869; and 5,849,717).
[0007] There exists a need in the art for ganglioside compounds and
methods of treatment which use the compounds to treat diseases such
as cancer, hyperinsulinemia, atypical
[0008] Parkinson's disease, multiple systems atrophy, Huntington's
disease, tauopathies or ganglioside deficient states or disorders,
including neurological conditions. Ideally, such compounds act in a
manner similar to, or better than, the natural gangliosides for the
prophylaxis, treatment and cure of such states or disorders.
BRIEF SUMMARY OF THE INVENTION
[0009] In various aspects, this invention provides compounds,
compositions, and methods for treating congenital and
non-congenital diseases. Exemplary diseases treatable according to
the invention include those characterized by ganglioside
deficiency. Exemplary diseases treatable according to the invention
include cancer (e.g., in combination with one or more of
antibodies, antibody fragments and antibody conjugates, tyrosine
kinase inhibitors), atypical Parkinson's disease, Parkinson's
Disease, other neurological diseases and conditions (e.g., multiple
systems atrophy, Huntington's disease, GM3 synthase deficiency, GM2
synthase deficiency); a tauopathy (e.g., a neurological disorder or
condition associated with an increased aggregation of tau protein),
hyperinsulinemia, hypoglycemia or hyperinsulinemia with
hypoglycermia.
[0010] In a first aspect, the invention provides novel synthetic
compositions of formula (1)-(5):
##STR00001##
including pharmaceutically acceptable salts, isomers, hydrates,
solvates, and prodrugs thereof in which Z can be O, S,
C(R.sup.10).sub.2 and NR.sup.2, X can be H, D, --OR.sup.11,
--NR.sup.11R.sup.12, --S.sup.11, and --CHR.sup.11R.sup.12, and
R.sup.10, R.sup.11 and R.sup.12 can be independently selected from
H, D, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heterocycloalkyl, --C(.dbd.B)R.sup.13,
--C(.dbd.B)--Z--R.sup.13, --SO.sub.2R.sup.13, and --SO.sub.3
functional moieties. Further, a novel ganglioside of the present
invention can have B, B', and Z independently selected from O,
NR.sup.14 or S, and Y can be selected from H, D, --OR.sup.15,
--SR.sup.15, --NR.sup.15R.sup.16, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl, and
substituted or unsubstituted heterocycloalkyl moieties. Further
still, a novel ganglioside of the invention can have R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.13, R.sup.14, independently selected from H, D,
--OR.sup.17, --NR17R.sup.18, --SR.sup.19, and --CHR.sup.19R.sup.20,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heterocycloalkyl moieties. Further R.sup.15, R.sup.16, R.sup.17,
R.sup.18, and R.sup.19 are independently selected from substituted
or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heterocycloalkyl moieties,
--C(.dbd.B,)R.sup.20, --C(.dbd.B')R.sup.20, --SO.sub.2R.sup.20, and
--SO.sub.3 functional moieties. Further, R.sup.20 is selected from
H, D, --OR.sup.21, --SR.sup.21, --NR.sup.21R.sup.22, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heterocycloalkyl moieties.
Further, R.sup.21 and R.sup.22 are independently selected from H,
D, substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heterocycloalkyl moieties. Further, double bonds can be E, Z or a
mixture of E and Z.
[0011] In some embodiments in which X is NHR that contains an amide
(i.e., R is hydrogen), a substituted or unsubstituted fatty acid
amide of from 1 to 40 carbon atoms in length or from 2 to 40 carbon
atoms in length or C(O)CHCl.sub.2; saccharide is a moiety of a
naturally occurring mammalian ganglioside or glycosphingolipid or
sulfatide; Y is substituted or unsubstituted alkyl, alkenyl, or
alkynyl from 12 to 40 carbon atoms in length; and R''' is hydrogen
or hydroxy. In some embodiments, the Y member is unsubstituted. In
some embodiments, the Y member consists of an unsubstituted linear
or branched alkyl chain or an unsubstituted linear or branched
alkenyl chain. In some embodiments, the Y member is alkenyl having
from 2 to 4 double bonds in a branched or linear carbon chain. In
some embodiments, the R fatty acid is unsubstituted. In other
embodiments, the R fatty acid member is from 14 to 24 carbon atoms
in length. In some exemplary embodiments, the R fatty acid is
unsubstituted and the Y member consists of an unsubstituted linear
or branched alkyl chain or an unsubstituted linear or branched
alkenyl chain. In a further set of such embodiments, R is a long
chain saturated or polyunsaturated fatty acid; in some further
embodiments the long chain polyunsaturated fatty acid is DHA, EPA,
or GLA. In some embodiments of the above, R is an .alpha.-hydroxy
fatty acid (e.g., .alpha.-hydroxy palmitic acid) joined at the
amide bond to the sphingoid base. In some embodiments, R is a
.alpha.-hydroxy fatty acid (e.g., .alpha.-hydroxy palmitic acid)
joined at the amide bond to a 4,8-sphingadiene base of from 16, 18
or 20 carbons in length.
[0012] In other embodiments of any of the above, the saccharide
moiety is selected from galactosyl glycosphingolipids, digalactosyl
glycosphingolipids, sulfated glycosphingolipids, glucosyl
glycosphingolipids, lactosyl glycosphingolipids, the lacto series
glycosphingolipids, the neo-lacto series of glycosphingolipids,
globoside glycosphingolipids, or the ganglioside glycosphingolipids
(Chen, "Handbook of Carbohydrate Engineering", Chapter 1, pages
1-48, CRC Press, Yarema, ed., (2005)). In preferred embodiments,
the saccharide moiety is a glycosphingolipid selected from GA2,
GA1, GM1b, GD1c, GD1.alpha., GM3, GM2, GM4, GM1a, GD1a, GT1a,
GT1.alpha., GD3, GD2, GD1b, GT1b, GQ1b, GQ1b.alpha., GT3, GT2,
GT1c, GQ1c, GP1c, sulfatide, globoside, sialyl paraglobosides and
GPlc.alpha.. In a particularly preferred embodiment, the compound
is the ganglioside GM1 or another compound of the above formula
having the saccharide moeiety of GM1. In other embodiments, the
compound is the ganglioside GM3 or GD3 or another compound of the
above formula having the saccharide moeiety of GM3 or GD3. In still
other embodiments, the compound or its saccharide moiety
corresponds to that of GT1b, sulfatide, or, more preferably,
Gb3.
[0013] In some exemplary embodiments, the present invention also
provides a novel ganglioside compound in which the saccharide
component can be
##STR00002##
and such saccharide moieties may or may not be deacetylated. These
structures are optionally incorporated into the compounds of
formulae 1-5, above.
[0014] The invention further provides pharmaceutical compositions
including at least one compound of the invention and a
pharmaceutically acceptable carrier.
[0015] In some exemplary embodiments of any of the above, the
compound is of formula (6)-(10):
##STR00003##
wherein n is an integer from 1 to 40, NHR is NH.sub.2 or an amide
of a saturated, unsaturated, or polyunsaturated (i.e., having at
least two double bonds in the fatty acid chain) fatty acid having
from 0 to 40 carbons in the chain. In some embodiments, n is an
integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16 or 17; More particularly, in some
embodiments, n can be 7, 10, 11, 13 or 14. In some embodiments, the
fatty acid is unsubstituted or alpha-hydroxylated. In other
embodiments the long chain fatty acid is substituted. In other
embodiments still, the R fatty acid member has from 14 to 28 carbon
atoms in the fatty acid chain. In some embodiments, the long chain
fatty acid is an unsubstituted or an alpha-hydroxylated C16, C18,
C20, C22, or C24 fatty acid. In some embodiments, the fatty acid is
an unsubstituted omega-3 or omega-6 or omega 9 fatty acid which may
be optionally substituted with an alpha-hydroxyl group. In some
further embodiments of such, the fatty acid is a C16, C18, or C18
fatty acid. In some embodiments, the compound comprises a
4,8-sphingadiene backbone. In some further embodiments of any of
the above, the long chain fatty acid is a polyunsaturated fatty
acid. In some embodiments of the above, the long chain fatty acid
is an .alpha.-hydroxy fatty acid (e.g., a substituted or
unsubstituted .alpha.-hydroxy palmitic acid or stearic acid) joined
at the amide bond to a sphingoid base. In other embodiments of any
of the above, R' is the saccharide moiety of a glycosphingolipid
selected from GA2, GA1, GM1b, GD1c, GD1.alpha., GM3, GM2, GM4,
GM1a, GD1a, GT1a, GT1.alpha., GD3, GD2, GD1b, GT1b, GQ1b,
GQ1b.alpha., GT3, GT2, GT1c, GQ1c, GP1c, Gb3, Gb4,
sailyl-paragloboside, globoside, sulfatide and GP1c.alpha..
Accordingly, the saccharide moiety can be a mono-, di-, tri-,
tetra-, penta-, hexa-, hepta-, octa-, nano-, or deca-saccharide. In
some embodiments, the saccharide moiety can be the moiety of GM3,
GM2, GM1a, GD1a, GT1a, or GT1.alpha.. In exemplary embodiments, the
compound of formula (1)-(10) has the stereoisomerism of a
corresponding naturally occurring glycolipids. In some embodiments,
wherein the glycolipid has one or more double bond, the double-bond
may be independently cis or trans, or a mixture thereof. In some
embodiments, the double bonds are in the trans configuration.
[0016] In yet other embodiments, the compounds according to the
invention, include the compounds of formulae (1), (2), (3), (4),
(5), (6), (7), (8), (9) or (10) comprise an omega-3 fatty acid
(e.g., DHA (docahexaenoic acid), EPA (eiocosapentaenoic acid)) or
an omega-6 fatty acid (e.g., GLA (gamma linolenic acid)) as the
polyunsaturated fatty acid. In a further embodiment of such
compounds, n is 7, 10, 11, 12, 13 or 14. In a still further
embodiment, the saccharide moiety can be the oligosaccharide moiety
of GM3, GM2, GMla, GD1a, GT1a, sialyl-paragloboside or
GT1.alpha..
[0017] In another embodiment of the compounds of formulae (1), (2),
(3), (4), (5), (6), (7), (8), (9) or (10), the polyunsaturated
fatty acid has non-conjugated double bonds. In a further embodiment
of such, the polyunsaturated fatty acid is arachidonic acid. In yet
other embodiments, the polyunsaturated fatty acid has 2, 3, 4, 5,
or 6 non-conjugated double bonds;
[0018] or at least two, three, four, or five non-conjugated double
bonds. In still further embodiments, n is 7, 10, 11, 12, 13 or
14.
[0019] In another embodiment of compounds of formulae (1)-(10), the
saccharide moiety, is selected from the glycans of the glycolipid
families of Gala (Ga) (e.g., galactosyl-ceramides, sulfatides,
sulfated digalactosides), Gluco, Globo (Gb), Isoglobo (iGb), Lacto
(Lc), Neolacto (nLc), Ganglio (Gg), Isoganglio (iGg), Lactoganglio
(LcGg) and Gangliosides (for example the a-, b- and c-series). In a
further set of such embodiments, the fatty acid is palmitic,
stearic or oleic acids.
[0020] In another embodiment of compounds of formula (1)-(10), the
saccharide moiety, is selected from the group consisting of [0021]
Neu5Ac.alpha.3Gal.beta.4Glc-; [0022]
GalNAc.beta.4(Neu5Ac.alpha.3)Gal.beta.4Glc-; [0023]
Gal.beta.3GalNAc.beta.4(Neu5Ac.alpha.3)Gal.beta.4Glc-; [0024]
Neu5Ac.alpha.3Gal.beta.3GalNAc.beta.4(Neu5Ac.alpha.3)Gal.beta.4Glc-;
and [0025]
Neu5Ac.alpha.8Neu5Ac.alpha.3Gal.beta.3GalNAc.beta.4(Neu5Ac.alpha.3-
)Gal.beta.4Glc-. In a further set of such embodiments, the fatty
acid is DHA, EPA, or GLA.
[0026] In another aspect, the invention provides pharmaceutical
compositions for treating a disorder selected from cancer,
malignancy, hyperinsulinemia, hypoglycemia and hyperinsulinemia
with hypoglycemia, a neurological disorder (e.g., Huntington's
Disease, GM3 synthase deficiency, GM2 synthase deficiency,
Parkinson's Disease, multiple systems atrophy, atypical Parkinson's
Disease, a tauopathy) comprising a compound of the invention and a
pharmaceutically acceptable excipient. In another embodiment, the
pharmaceutical compositions are in unit dose format and each unit
dose provides a therapeutically effective amount of one or more
compounds for use according to the invention. The compositions, are
formulated for oral, intraperitoneal, intranasal, topical,
transcutaneous, subcutaneous, or intravenous administration.
[0027] In some embodiments, the above compositions can comprise a
plurality of glycolipid analogs wherein each has a saccharide
moiety of a different glycolipid. In some such embodiments, the
administered glycosphingolipid consist of at least two or three
such glycosphingolipids. In still other embodiments, the
administered glycosphingolipid consist of at least 80%, 90%, 95%,
or 98% of a single glycosphingolipid having a saccharide moiety of
a naturally occurring mammalian glycosphingolipid.
[0028] In yet another aspect, the invention provides methods for
treating a disease by administering a therapeutically effective
amount of a glycolipid. This method optionally includes the
administration of a therapeutically effective amound of at least a
second therapeutic agent to a subject in need thereof. Exemplary
glycolipids include, for example, those of the present invention
and a those disclosed and/or claimed in any of U.S. patent
applications Ser. Nos. 10/485,195; 10/485,892; 10/487,841;
60/452,796; 11/666,577; 60/626,678; and 10/547,566 which are each
incorporated herein by reference particularly with respect to such
compound subject matter
[0029] Exemplary indications for this treatment include
malignancies that are resistant or unresponsive to one or more
antibodies or tyrosine kinase inhibitors, hematologic and solid
tumors, congenital and non-congenital hyperinsulinemia,
hypoglycemia, hyperinsulinemia with hypoglycemia, Parkinson's
disease, Huntington's disease, multiple systems atrophy, GM3
synthase deficiency, GM2 synthase deficiency, Parkinson's Disease,
multiple systems atrophy, atypical Parkinson's Disease, and a
tauopathy.
[0030] In another embodiment, the present invention provides a
method of treating or inhibiting development of a post-prandial
hypoglycemia, fasting hypoglycemia, neonatal hypoglycemia,
hypoglycemia secondary to dialysis. The method includes the step of
administering to the subject a therapeutically effective amount of
a glycolipid. The post-prandial hypoglycemia treated or inhibited
by methods and compositions of the present invention is, in another
embodiment, associated with a Nissen fundoplication or
gastric-bypass surgery
[0031] In another embodiment, administration of a therapeutically
effective amount of a glycolipid to a subject suppresses insulin
secretion by the subject.
[0032] Methods of the invention can be used for treating different
cancers, both solid tumors and soft-tissue tumors alike.
Non-limiting examples of cancers amendable to the treatment of the
invention include breast cancer, colorectal cancer, rectal cancer,
non-small cell lung cancer (NSCLC), non-Hodgkins lymphoma (NHL),
renal cell cancer, prostate cancer, liver cancer, pancreatic
cancer, soft-tissue sarcoma, Kaposi's sarcoma, sarcoma, renal cell
carcinoma, carcinoid carcinoma, head and neck cancer, glioblastoma,
melanoma, ovarian cancer, gastric cancer, mesothelioma, and
multiple myeloma. In certain aspects, the cancers are metastatic.
In other aspects, the cancers are non-metastatic.
[0033] In another embodiment, a compound of the invention utilized
in methods and compositions of the present invention exhibits an
improvement in a desirable biological property relative to the
natural glycolipid. In another embodiment, the biological property
is improved biological half-life. In another embodiment, the
biological property is improved affinity for the insulin receptor.
In another embodiment, the biological property is improved potency
for antagonism of the insulin receptor. In another embodiment, the
biological property is any other desirable biological property
known in the art. Each possibility represents a separate embodiment
of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 is a schematic diagram of two methods for synthesis
of the ganglioside GM2 by enzymatic synthesis using as the starting
material lactosylceramide obtained from bovine buttermilk.
[0035] FIG. 2 is a schematic diagram of two methods for
synthesizing the ganglioside GD2 from lactosylceramide obtained
from bovine buttermilk.
[0036] FIG. 3 is a collection of three routes for synthesizing a
GM2 ganglioside using a plant glucosylceramide as the starting
material.
[0037] FIG. 4 is a collection of three routes for synthesizing GM2
and other gangliosides starting from a glucosylceramide.
[0038] FIG. 5 is a scheme used for synthesis of the ganglioside GM2
from lactosylceramide via deacylation, two consecutive enzymatic
glycosylations, and final chemical acylation.
[0039] FIG. 6. HPLC monitoring of an endoglycanase reaction(s).
Transfer of the fluorinated GM1 sugar donor was monitored using an
HPLC reverse phase method on a Chromolith RP-8e column with eluants
of 0.1% trifluoroacetic acid (TFA) in acetonitrile (ACN) to 0.1%
TFA in H.sub.2O. Exemplified results of HPLC monitoring of a
glycosynthase reaction for a Rhodococcus E351S mutant is depicted.
FIG. 6A--Lyso-GM3 and Sphingosine; FIG. 6B--Triton X-100 and
Sphingosine; FIG. 6C--Reaction-low enzyme concentration; and FIG.
6D--Reaction-high enzyme concentration.
[0040] FIG. 7. Effect of glycolipids, GM3 and
.alpha.-2,3-sialylparagloboside (SPG), on glucose uptake upon
insulin stimulation of primary human adipocytes.
DETAILED DESCRIPTION
[0041] The present invention provides novel glycolipid compositions
and methods of using these compositions to treat a congenital or
non-congenital disorder, such as a disease characterized by a
ganglioside deficiency. Exemplary disorders include cancer,
atypical Parkinson's disease; Huntington's disease, multiple
systems atrophy, GM3 synthase deficiency, GM2 synthase deficiency,
hyperinsulinemia, hypoglycemia, hyperinsulinemia with hypoglycemis,
a tauopathy, or another neurological disorder or condition
associated with an increased aggregation of tau protein. The method
includes administering to a subject in need thereof of a
composition of the invention in a therapeutically effective
amount.
[0042] Exemplary compounds of the invention have improved
structures over lyso-GM1, GM1(stearate) and GM3 (stearate) when
injected or given orally. These structures exhibit improved
biodistribution, (e.g., brain delivery), improved pharmacokinetics,
and improved oral bioavailability.
[0043] In some embodiments, the compound of Formula (1) to (10) has
a saccharide moiety selected from those set forth in the following
table of gangliosides:
TABLE-US-00001 Neu5Ac3Gal4GlcCer GM3 GalNAc4(Neu5Ac3)Gal4GlcCer GM2
Gal3GalNAc4(Neu5Ac3)Gal4GlcCer GM1a Neu5Ac3Gal3GalNAc4Gal4GlcCer
GM1b Neu5Ac8Neu5Ac3Gal4GlcCer GD3 GalNAc4(Neu5Ac8Neu5Ac3)Gal4GlcCer
GD2 Neu5Ac3Gal3GalNAc4(Neu5Ac3)Gal4GlcCer GD1a
Neu5Ac3Gal3(Neu5Ac6)GalNAc4Gal4GlcCer GD1.alpha.
Gal3GalNAc4(Neu5Ac8Neu5Ac3)Gal4GlcCer GD1b
Neu5Ac8Neu5Ac3Gal3GalNAc4(Neu5Ac3)Gal4GlcCer GT1a
Neu5Ac3Gal3GalNAc4(Neu5Ac8Neu5Ac3)Gal4GlcCer GT1b
Gal3GalNAc4(Neu5Ac8Neu5Ac8Neu5Ac3)Gal4GlcCer GT1c
Neu5Ac8Neu5Ac3Gal3GalNAc4(Neu5Ac8Neu5c3)Gal4GlcCer GQ1b
[0044] Additionally the invention provides for the use of other
glycolipids in treating a disorder selected from cancer, atypical
Parkinson's disease; Huntington's disease, multiple systems
atrophy, GM3 synthase deficiency, GM2 synthase deficiency, a
tauopathy, or another neurological disorder or condition associated
with an increased aggregation of tau protein including, for
instance, the glycolipids of disclosed herein. These compounds to
be administered to treat a disorder (e.g., cancer, atypical
Parkinson's disease; Huntington's disease, multiple systems
atrophy, GM3 synthase deficiency, GM2 synthase deficiency, a
tauopathy, or another neurological disorder or condition associated
with an increased aggregation of tau protein) include compounds
according to the formula:
##STR00004##
wherein Z, X, Y and R.sup.1 have their identities disclosed herein
including in those references incorporated herein by reference
(e.g., U.S. Patent Application Publication No. 2007-0275908; U.S.
Patent Application Publication No. US 2005/0239741; U.S. Patent
Application Publication No. US 2005/0245735; and U.S. Patent
Application Publication No. US 2005/0032742).
[0045] These compounds also include the neutral glycosphingolipids
and neutral glycosyl sphingosines disclosed U.S. Patent Application
Publication No. US 2005/0245735 which is incorporated by reference
in its entirety.
[0046] These compounds also include sialylated oligosaccharide
glycolipids disclosed in U.S. Patent Application Publication No. US
2005/0032742 which is incorporated herein by reference. In some
embodiments, the oligosaccharide is a glycosylated ganglioside,
ceramide, or sphingosine or an analogue of same.
[0047] Other compounds of use according to the invention include
those disclosed in commonly owned U.S. Patent Application
Publication Nos. US 2005/0245735; US 2005-0032742; US 2005-0239741;
US 2007-0275908; and US 2008-0125392, each of which is incorporated
by reference therein in its entirety for all purposes.
[0048] In exemplary embodiments, a compound of Formula (1), (2),
(3), (4), (5), (6), (7), (8), (9) or (10) associates with the
insulin receptor inhibiting cellular signalling. In a more
preferred embodiment, the glycolipid inhibits the upregulation and
transport of Glut-4 or Glut-2 to the cell surface. In another
embodiment, the glycolipid reduces glucose uptake by the cell. In
another exemplary embodiment, the saccharide of Formula (1), (2),
(3), (4), (5), (6), (7), (8), (9) or (10) is a sugar of GM3,
sialylparagloboside. Further, administration of compounds from
Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) based
on the glycan structures of sialylparagloboside, GD1a or GM3 to a
subject with hypoinsulinemia, hypoglycemia or hypoinsulinemia with
hypoglycemia will reduce the hypoglycemic state, decrease insulin
secretion, or reduce the morbidities and mortalities associated
with the disease state of the subject.
[0049] In some embodiments, the compound selected from Formula (1),
(2), (3), (4), (5), (6), (7), (8), (9) or (10) is administered to a
subject or patient to replace a missing or low level glycolipid in
the subject. For example, type II diabetes is associated with
reduced levels of sulfatide (Buschard, Diabetes, 55: 2826-2834
(2006)). Supplementation of sulfatide (e.g., ceramide fatty acid of
C16:0) inhibited glucose stimulation of insulin secretion by
activation of the K.sub.ATP channels, in vitro. In addition, the
exogenous sulfatide increased the expression of adiponectin while
reducing inflammatory responses by decreasing the levels of
TNF-alpha, IL-6 and IL-8 in human adippose tissue, in vitro (Bruun,
Mol Cell Endocrinol, 263:142-148 (2007)). In a preferred
embodiment, a compound selected from Formula (1), (2), (3), (4),
(5), (6), (7), (8), (9) or (10) will reduce insulin secretion from
congenital or noncongenital diseases. In a more preferred
embodiment, the congenital disease is Congenital Hyperinsulinemia
(e.g., Familial Hyperinsulinemia). In an even more preferred
embodiment, a compound from Formula (1), (2), (3), (4), (5), (6),
(7), (8), (9) or (10) contains the saccharide moiety of sulfatide
or GM4.
[0050] In another aspect, the invention provides methods of
treating cancer, atypical Parkinson's disease; Huntington's
disease, multiple systems atrophy, GM3 synthase deficiency, GM2
synthase deficiency, a tauopathy, or another neurological disorder
or condition associated with an increased aggregation of tau
protein a subject by administering to the subject one or more of
the compounds selected from formula (1) to (10) in which the
compound has a saccharide moiety of a deficient ganglioside or of a
ganglioside which is downstream of a deficient ganglioside in the
subject. In some embodiments, the ganglioside deficiency state may
be mediated, exacerbated or caused by the condition or disease to
be treated or by a reduced level of activity or absence of any of
the enzymes involved in mammalian ganglioside synthesis or
anabolism (e.g., ceramide glycosyl transferases,
galactosyltransferases, sialyltransferases, and GalNAc
transferases).
[0051] In certain aspects, the present invention provides a method
for treating hyperinsulinemia, by administering effective amounts
of compounds from Formula (1), (2), (3), (4), (5), (6), (7), (8),
(9) or (10) and combining this treatment with the administration of
drugs that reduce insulin or insulin like growth factor (e.g.,
IGF-1 and IGF-2) secretion, drugs and methods for increasing
glucose levels, diet, diuretic, chemotherapy, radiation and
surgery.
[0052] In an exemplary embodiment, the treatment uses a compound of
Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) in
combination with one or more of additional treatment methods
including sulfonylureas (e.g., diazoxide), somatostatin analogs
(e.g., octreotide.RTM.), calcium channel blockers (e.g.,
nifedipine), glucagon, IGF-1, insulin, glucocorticoids, growth
hormone, glucagon like peptides (e.g., GLP-1, exendin-4, exendin
peptide derivatives, exendin peptide antagonists and extended
formulations thereof), glucose (e.g., IV, oral or gastrostomy),
pancreatectomies, and tumor resection.
[0053] In some embodiments the glycolipid is capable of inhibiting
cell proliferation, intiating apoptosis or is capable of killing
the malignant cells independent of any additional antineoplastic
therapies including antibodies. For example, GM3 can be used to
treat certain CNS malignancies including glioma and bladder cancer
(PCT Patent Application Serial No. WO9852577; Fujimoto, J
Neuro-Oncology, 71: 99-106 (2005); Watanabe, Cancer Res, 62:
3850-3854 (2002), 9-O-acetyl-GD1b can treat CNS malignancies and
breast cancer (Abad-Rodriguez, J Neurochemistry, 74: 2547-2556
(2006), sialyl-paragloboside can inhibit leukemias and Burkitt
lymphoma (Schaade, Med Microbiol Immunol, 188: 23-29 (1999) and
GD1b, GT1b and GQ1b suppress the growth of melanoma (Kanda, J
Invest Dermatol, 117: 284-293 (2001)).
[0054] In certain aspects, the present invention provides a method
for treating cancer, by administering effective amounts of
compounds from Formula (1) to (10) combined with one or more
antibodies directed against the tumor or tumor enviroment and
combining this treatment with the administration of radiation
treatment or chemotherapeutic agents to a patient susceptible to,
or diagnosed with cancer. A variety of chemotherapeutic agents may
be used in the combined treatment methods of the invention.
[0055] In one embodiment, the present invention can be used for
increasing the duration of survival of a human patient susceptible
to or diagnosed with a cancer, increasing progression free survival
of a human patient susceptible to or diagnosed with a cancer,
increasing response rate in a group of human patients susceptible
to or diagnosed with a cancer, increases the duration of response
in a subject with cancer, or to treat the signs and symptoms,
ameliorate, or slow the progression of the malignancy when
administered with an antibody directed towards an antigen or
tyrosine kinase inhibitor. In an even more preferred embodiment,
the response of the malignancy to the combination of agents is
synergistic.
[0056] As will be understood by those of ordinary skill in the art,
the appropriate doses of therapeutic and chemotherapeutic agents
will be generally around those already employed in clinical
therapies wherein the therapeutics are administered alone or in
combination with other therapeutics. Variation in dosage will
likely occur depending on the condition being treated. The
physician administering treatment will be able to determine the
appropriate dose for the individual subject.
Definitions
[0057] In accordance with the invention and as used herein, the
following terms are defined with the following meanings, unless
explicitly stated otherwise.
[0058] The terms "cancer", "cancerous", "malignancy" and
"malignant: refer to or describe the physiological condition in
mammals that is typically characterized by unregulated cell growth.
Examples of cancer include, but are not limited to, carcinoma,
lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
More particular examples of such cancers include squamous cell
cancer (e.g., epithelial squamous cell cancer), lung cancer
including small-cell lung cancer, non-small cell lung cancer,
adenocarcinoma of the lung and squamous carcinoma of the lung,
cancer of the peritoneum, hepatocellular cancer, gastric or stomach
cancer including gastrointestinal cancer, pancreatic cancer,
glioblastoma, cervical cancer, ovarian cancer, liver cancer,
bladder cancer, cancer of the urinary tract, hepatoma, breast
cancer, colon cancer, rectal cancer, colorectal cancer, endometrial
or uterine carcinoma, salivary gland carcinoma, kidney or renal
cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic
carcinoma, anal carcinoma, penile carcinoma, melanoma, multiple
myeloma and B-cell lymphoma, Hodgkin's disease, non-Hodgkins
lymphoma, anaplastic large T-cell lymphoma, acute myelogenous
leukemia (AML), chronic lympocytic leukemia (CLL), cutaneous T-cell
lymphoma, follicular lymphoma, Burkitt's lymphoma, peripheral
T-cell lympoma, brain, as well as head and neck cancer, and
associated metastases. Examples of head and neck cancers include,
but are not limited to, adenoid cystic carcinomas localized, for
example to the tongue, parotid gland, nose, palate, skin, neck,
submandibular gland, glottis, sinus, epiglottis, buccal space,
nerves, larynx, mouth, pharynx, or cheek.
[0059] "Treatment" is an intervention performed with the intention
of preventing the development or altering the pathology of a
disorder. Accordingly, "treatment" refers to both therapeutic
treatment and prophylactic or preventative measures. Those in need
of treatment include those already with the disorder as well as
those in which the disorder is to be prevented.
[0060] Atypical Parkisonian diseases or syndromes refers to a group
of disorders whose clinical features overlap those of idiopathic
Parkinson's disease. The four major Parkinsonian syndromes embrace
three important neurodegenerations, multiple system atrophy,
progressive supranuclear palsy, and corticobasal degeneration, and
a lacunar cerebrovascular disorder, vascular parkinsonism (see,
Gilman S., Clin. Geriatr. Med 22:827-42 (2006). In embodiments, the
conditions to be treated according to the invention can be any one
of these four major syndromes.
[0061] The term "congenital disease" as used herein refers to a
disease or disorder that is inherited genetically. The term is
intended to include familial hyperinsulinemia (OMIM no. 256450,
601820, 606762, 602485, 609968, 609975, 610021) (e.g., persistent
hyperinsulinemic hypoglycemia of infancy, ABCC8-related
hyperinsulinism, GCK-related hyperinsulinism, HADHSC-related
hyperinsulinism, GLUD1-related hyperinsulinism, KCNJ11-related
hyperinsulinism, insulin receptor-related hyperinsulinism,
UCP2-related hyperinsulinism, exercise induced hyperinsulinism,
diffuse hyperinsulinism), Beckwith-Wiedemann Syndrome, Congenital
Disorders of Glycosylation (CDG)(e.g., CDG-Ia-n, CDG-IIa-h), type
II diabetes, polycystic kidney disease, hypopituitarism,
hyperinsulinism, glycogen storage disease (Type I and III), organic
acidurias (e.g., maple syrup urine disease,
3-hydroxymethylglutaryl-CoA lysase deficiency, glutaric acidemia
type I and II, 3-methylglutaconic aciduria type 1, mevalonic
aciduria, alpha-methylacetoacetic aciduria, short-chain acyl-CoA
dehydrogenase deficiency, 3-methylcrotonyl-CoA carboxylase I
deficiency, long-chain acyl-CoA dehydrogenase deficiency,
carnitine-acylcarnitine translocase deficiency, carnitine
palmitoyltransferase II deficiency, medium chain acyl-CoA
dehydrogenase deficiency), phosphoenolpyruvate carboxykinase
deficiency, disorders of fatty acid oxidation (e.g.,
3-hydroxyacyl-CoA dehydrogenase deficiency, very long chain
acyl-CoA dehydrogenase deficiency, trifunctional protein
deficiency), familial leucine sensitive hypoglycemia, insulin
secreting tumors, insulin like growth factor (e.g., IGF-1, IGF-2)
secreting tumors, Addison's disease, reactive hypoglycemia and
idiopathic postprandial syndrome, Doege-Potter syndrome,
hyperinsulin hyperammonia syndrome, multiple endocrine neoplasia,
neurofibromatosis type 1, adrenal insufficiency (e.g., adrenal
hyperplasias including ACTH-independent macronodular adrenal
hyperplasia), Simpson-Golabi-Behmel syndrome type 1, idiopathic
ketotic hypoglycemia, and reactive hypoglycemia and idiopathic
postprandial syndrome. The term is also intended to include GM3
synthase deficiency (OMIM no. 609056; Amish Infantile Epilepsy
Syndrome) in which the sialyltransferase (ST3Gal5; SIAT9) that
prepares GM3 in the ganglioside biosynthetic pathway has decreased
or absent biosynthetic activity. The term also is intended to
include GM2 synthase deficiency [GM3 (Hematoside)
Sphingolipodystrophy] in which the GalNAc-transferase (B4Galnt1)
that prepares GA2, GM2 and GD2 in the glycolipid biosynthetic
pathway has decreased or absent biosynthetic activity. The term is
also intended to include Huntington's Disease (OMIM no. 143100)
which has a glycolipid deficient phenotype that results in a
reduction in the amount of higher glycolipids (e.g., GM2, GM1, GD2,
GD1a, GT1a, GT1b, GQ1b)(Neurobiol Dis, 27:265-277 (2007)).
[0062] The term "noncongenital disease" as used herein refers to a
disease or disorder that is not inherited genetically. The term is
intended to include type II diabetes, acquired adrenal
insufficiency, premature birth, sepsis, acquired hypopituitarism,
gastric dumping syndrome, insulin secreting tumors, insulin like
growth factor (e.g., IGF-1, IGF-2) secreting tumors,
immunopathologic hypoglycemia, bowel bypass surgery or resection,
reactive hypoglycemia and idiopathic postprandial syndrome,
acquired hypoglycemia resulting from dialysis, hypoglycemia
resulting from organ (e.g., kidney, liver, heart, lung)
transplantation, medication or toxin induced hypoglycemia,
asphyxia, hypocalcemia, opoid withdrawal and Nissen
fundoplication.
[0063] The compounds for use according to the present invention may
also contain unnatural proportions of atomic isotopes at one or
more of the atoms that constitute such compounds. For example, the
compounds may be radiolabeled with radioactive isotopes, such as
for example tritium (.sup.3H), iodine-125 (.sup.125I) or carbon-14
(.sup.14C). All isotopic variations of the compounds (e.g., D,
.sup.13C, .sup.18O) for use according to the present invention,
whether radioactive or not, are intended to be encompassed within
the scope of the present invention.
[0064] The term "substituted" as used herein means that a hydrogen
atom has been replaced with another monovalent group (e.g., halo,
haloalkyl, hydroxy, thiol, alkoxy, thiohaloalkyl, amino, and the
like), or that two hydrogen atoms of the same atom have been
replaced by a divalent group.
[0065] The terms "halo" or "halogen" as used herein refer to Cl,
Br, F or I. The term "haloalkyl" and the like, refer to an alkyl
group, as defined herein, wherein at least one hydrogen atom of the
alkyl group is replaced by a Cl, Br, F or I. A mixture of different
halo atoms may be used if more than one hydrogen atom is replaced.
For example, a haloalkyl includes chloromethyl (--CH.sub.2Cl) and
trifluoromethyl (--CF.sub.3) and the like.
[0066] The term "alkyl," by itself or as part of another
substituent, means, unless otherwise stated, a straight or branched
chain, or cyclic hydrocarbon radical, or combination thereof, which
is saturated, and can include di- and multivalent radicals, having
the number of carbon atoms designated (i.e., C.sub.1-C.sub.10 means
one to ten carbons). Examples of saturated hydrocarbon radicals
include, but are not limited to, groups such as methyl, ethyl,
n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl,
cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and
isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and
the like.
[0067] The term "alkylene" by itself or as part of another
substituent means a divalent radical derived from an alkane, as
exemplified, but not limited, by
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. Typically, an alkyl (or
alkylene) group will have from 1 to 24 carbon atoms, with those
groups having 10 or fewer carbon atoms being preferred in the
present invention. A "lower alkyl" or "lower alkylene" is a shorter
chain alkyl or alkylene group, generally having six or fewer carbon
atoms.
[0068] The terms "alkyl," "alkenyl" and "alkynyl" are meant to
include both substituted and unsubstituted forms of the indicated
radical. The terms also include, but are not limited to, forms of
the radicals having 3 or fewer or 6 or fewer carbon atoms.
Preferred substituents for each type of radical are provided
below.
[0069] Substituents for the alkyl, alkenyl, alkynyl radicals can be
one or more of a variety of groups selected from, but not limited
to: --OR.sup.a, .dbd.O, .dbd.NR.sup.a, .dbd.N--OR.sup.a,
--NR.sup.aR.sup.b, --SR.sup.a, --halogen, --OC(O)R.sup.a,
--C(O)R.sup.a, --CO.sub.2R.sup.a, --CONR.sup.aR.sup.b,
--OC(O)NR.sup.aR.sup.b, --NR.sup.bC(O)R.sup.a,
--NR.sup.c--C(O)NR.sup.bR.sup.a, --NR.sup.bC(O).sub.2R.sup.a,
--NR.sup.e--C(NR.sup.aR.sup.bR.sup.c).dbd.NR.sup.d,
--NR.sup.d--C(NR.sup.aR.sup.b).dbd.NR.sup.c, --S(O)R.sup.a,
S(O).sub.2R.sup.a, --S(O).sub.2NR.sup.aR.sup.b,
--NR.sup.bSO.sub.2R.sup.a, --CN and --NO.sub.2 in a number ranging
from zero to (2m'+1), wherein m' is the total number of carbon
atoms in such radical. R.sup.a, R.sup.b, R.sup.c, R.sup.d and
R.sup.e each preferably independently refer to hydrogen or
unsubstituted alkyl. When a compound of the invention includes more
than one of any R.sup.a, R.sup.b, R.sup.c, R.sup.d or R.sup.e
group, each of those groups are independently selected as well. For
example, if there are two or more R.sup.a groups in a formula, each
of those are independently selected. When R.sup.a and R.sup.b are
attached to the same nitrogen atom, they can optionally be combined
with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For
example, --NR.sup.aR.sup.b is meant to include, but not be limited
to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of
substituents, one of skill in the art will understand that the term
"alkyl" is meant to include, but not be limited to, groups
including carbon atoms bound to groups other than hydrogen groups,
such as haloalkyl (e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and
acyl (e.g., --C(O)CH.sub.3, --C(O)CF.sub.3,
--C(O)CH.sub.2OCH.sub.3, and the like). Preferred substituents are
lower alkyl, lower alkoxy, hydroxy, and halo. The term "lower"
indicates C.sub.1 to C.sub.6 carbons in the chain. In some
embodiments, the total number of substituents for the alkyl,
alkenyl, or alkynyl radical are independently in a number which is
0, 1, 2, 3 or 4. In a further embodiment, these substituents are
independently selected from the group consisting of lower alkyl,
lower alkoxy, hydroxy, and halo.
[0070] In some embodiments, according to the invention, the
compound of the invention has a saccharide moiety with the
structure: [0071] Neu5Ac3Gal4Glc- (as in GM3); [0072]
GalNAc4(Neu5Ac3)Gal4Glc- (as in GM2) [0073]
Gal3GalNAc4(Neu5Ac3)Gal4Glc- (as in GM1a) [0074]
Neu5Ac3Gal3GalNAc4Gal4Glc- (as in GM1b) [0075]
Neu5Ac8Neu5Ac3Gal4Glc- (as in GD3) [0076]
GalNAc4(Neu5Ac8Neu5Ac3)Gal4Glc- (as in GD2) [0077]
Neu5Ac3Gal3GalNAc4(Neu5Ac3)Gal4Glc- (as in GD1a) [0078]
Neu5Ac3Gal3(Neu5Ac6)GalNAc4Gal4Glc- (as in GD1a. [0079]
Gal3GalNAc4(Neu5Ac8Neu5Ac3)Gal4Glc- (as in GD lb) [0080]
Neu5Ac8Neu5Ac3Gal3GalNAc4(Neu5Ac3)Gal4Glc- (as in GT1a) [0081]
Neu5Ac3Gal3GalNAc4(Neu5Ac8Neu5Ac3)Gal4Glc- (as in GT1b) [0082]
Gal3GalNAc4(Neu5Ac8Neu5Ac8Neu5Ac3)Gal4Glc- (as in GT1c) [0083]
Neu5Ac8Neu5Ac3Gal3GalNAc4(Neu5Ac8Neu5c3)Gal4Glc- (as in GQ1b) See,
Nomenclature of Glycolipids, IUPAC-IUB Joint Commission on
Biochemical Nomenclature (Recommendations 1997); Pure Appl. Chem.
69: 2475-2487 (1997); Eur. J. Biochem 257: 293-298 (1998))
(www.chem.qmw.ac.uk/iupac/misc/glylp.html).
[0084] The term "sphingoid," as used herein, includes sphingosines,
phytosphingosines, sphinganines, ceramides, and the like. Both
naturally occurring and synthetically produced compounds are
included. The naturally occurring sphingoid base has an alkyl chain
length of from 14 to 22 carbons long, but is preferably 18 carbons
long. Synthetic sphingoid bases can be longer or shorter (e.g.,
having from 2 to 40 carbons in the alkyl chain). A ceramide is an
N-acetylated sphingoid base. The fatty acids of ceramides vary in
chain lengths (14 to 40 carbons in the chain) and the presence or
absence of a hydroxyl group at the .alpha.- or .omega.-carbon
atom.
[0085] The term "glycosphingolipid" is a carbohydrate-containing
derivative of a sphingoid or ceramide. The carbohydrate residue is
attached by a glycosidic linkage to O-1 of the sphingoid or
ceramide.
[0086] As used herein, the term "heteroatom" is meant to include
oxygen (O), nitrogen (N), and sulfur (S).
[0087] Ganglioside analog refers to gangliosides in which the
saccharyl moiety, the base (e.g., sphingoid-like backbone), or the
fatty acid-derived hydrocarbon is of a structure other than that
found in naturally occurring ganglioside. Unless indicated
otherwise, the term ganglioside is meant to include ganglioside
analogs as well as naturally occurring gangliosides.
[0088] The term "sialic acid" (abbreviated "Sia") refers to any
member of a family of nine-carbon carboxylated sugars. The most
common member of the sialic acid family is N-acetyl-neuraminic acid
(2-keto-5-acetamindo-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic
acid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member
of the family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in
which the N-acetyl group of NeuAc is hydroxylated. Another sialic
acid family member is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano
et al., J. Biol. Chem. 261: 11550-11557 (1986); Kanamori et al., J.
Biol. Chem. 265: 21811-21819 (1990). Also included are
9-substituted sialic acids such as a 9-O--C.sub.1-C.sub.6
acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetylNeu5Ac,
9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of
the sialic acid family, see, e.g., Varki, Glycobiology 2: 25-40
(1992); Sialic Acids: Chemistry, Metabolism and Function, R.
Schauer, Ed. (Springer-Verlag, New York (1992). The synthesis and
use of sialic acid compounds in a sialylation procedure is
described in, for example, International Patent Application
Publication No. WO 92/16640, published Oct. 1, 1992.
[0089] In some embodiments, the compound of formula (1)-(10) is a
glycolipid compound of a formula:
##STR00005## ##STR00006## ##STR00007##
in which each n is independentlyas set forth above. In some
embodiments of the above, each n is 1.
[0090] The terms "deficiency" and "deficiency state" refer to a
large relative reduction in the levels of the referenced
ganglioside or enzyme or enzyme activity in tissues or samples from
a subject. Such deficiency states may generally exist, for
instance, when the levels of a subject glycolipid are zero or
undetectable or less than two-thirds, one-half, one-fourth,
one-tenth, one-twentieth, or one-one hundredth of the levels for
the comparable well-matched control group with a similar
malignancy.
[0091] The term "fatty acid" refers to an aliphatic monocarboxylic
acid which may be substituted or unsubstituted and saturated or
unsaturated and have from 1 to 40 carbon atoms.
[0092] In some embodiments, the fatty acid is from 2 to 40 carbon
atoms in length, including the carboxyl moiety carbon. In further
embodiments, the fatty acid is 14 to 24 carbon atoms in length and
is saturated or unsaturated. In other embodiments, the fatty acid
is unsubstituted and from 14 to 24 carbons in length and is
saturated or unsaturated. If unsaturated, the double bond may be in
the cis or trans conformation, or be a mixture thereof. In some
embodiments, the fatty acid is unsaturated with at least one of the
double bonds is in the trans configuration. Unsaturated fatty acids
may be polyunsaturated, for instance, having from 1, 2, 3, or 4
double bonds. Exemplary fatty acids are stearic acid and oleic acid
as well as the omega-3, omega-6, and omega 9 fatty acids of from 14
to 24 carbons in length. In some embodiments, the fatty acid is
optionally substituted with an .alpha.-hydroxy or an .alpha.-alkoxy
group or an acylated .alpha.-hydroxy group (acetyl .alpha.-hydroxy)
which acylated compound may function as a prodrug of a compound of
formulae (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10). The
aliphatic moiety may be linear or branched. In preferred
embodiments of any of the above, it is linear.
[0093] "Commercial scale" refers to gram scale production of a
compound of formulae (1), (2), (3), (4), (5), (6), (7), (8), (9) or
(10) in a single reaction. In preferred embodiments, commercial
scale refers to production of greater than about 50, 75, 80, 90,
100, 125, 150, 175, or 200 grams of a compound of formula (1), (2),
(3), (4), (5), (6), (7), (8), (9) or 10). In some embodiments, the
methods of use and manufacture of the pharmaceutical compositions
involve the use of such compounds produced on a commercial
scale.
Method of Preparing the Compounds
[0094] The compounds of formula (1), (2), (3), (4), (5), (6), (7),
(8), (9) or (10) can be made by any method available to one of
ordinary skill in the art. The saccharide moiety of the compounds
for use according to the invention can be prepared by any means
known in the art including those methods described in U S. Pat.
Nos. 5,922,577; 6,284,493; and 6,331,418, each of which is
incorporated by reference herein in its entirety. Additional
particularly suitable methods for making compounds for use
according to the invention are described in U.S. Patent Application
Publication No. US 2009-0170155; U.S. Patent Application
Publication No. US 2009-0170155; and U.S. Patent Application
Publication No. US 2009-0170155; which are assigned to the same
assignee as the instant application; and in U.S. Pat. Nos.
6,440,703 and 6,030,815; and PCT International Patent Application
Publications Nos. WO2004/080960, WO2003/016469, WO2005/118798 and
WO2004/080960. Each of these references is incorporated herein by
reference particularly with respect to their teachings as to how to
synthesize various glycolipids.
[0095] The compounds of the invention and of use in the methods of
the invention can be prepared using, unless otherwise indicated,
conventional methods and protocols in chemistry and enzymology
known in the art. For example, compounds for use according to the
invention may be prepared by chemical and enzymatic processes as
outlined further below.
[0096] Additional suitable synthetic approaches are disclosed in WO
2004/080960 A2 which is incorporated by reference herein in its
entirety.
[0097] If the acceptor is a ceramide, the enzymatic step is
optionally preceded by hydrolysis of the fatty acid moiety from the
ceramide. Methods of removing a fatty acid moiety from a
glycosphingolipid are known to those of skill in the art. Standard
carbohydrate and glycosphingolipid chemistry methodology can be
employed, such as that described in, for example, Paulson et al.,
Carbohydrate Res. 137: 39-62 (1985); Beith-Halahmi et al.,
Carbohydrate Res. 5: 25-30 (1967); Alais and Veyrieries,
Carbohydrate Res. 207: 11-31; (1990); Grudler and Schmidt,
Carbohydrate Res. 135: 203-218 (1985); Ponpipom et al.; Tetrahedron
Lett. 1717-1720 (1978); Murase et al., Carbohydrate Res. 188: 71-80
(1989); Kameyama et al. Carbohydrate Res. 193: cl-c5(1989);
Hasegawa et al. J. Carbohydrate Chem. 10: 439-459(1991);
Schwarzmann and Sandhoff, Meth. Enzymol. 138: 319-341 (1987);
Guadino and Paulson, J. Am. Chem. Soc. 116: 1149-1150 (1994)
(including supplemental material, which is also incorporated herein
by reference). For example, the fatty acid moiety can be removed by
base hydrolysis. Once the glycosylation reactions are completed,
the same or a different fatty acid can be attached to the product
of the glycosylation reactions.
[0098] Methods for coupling a fatty acid are generally known in the
art and examples are discussed herein. The N-acyl group (NHR) of a
compound selected from formulae (1) to (10) can be derived from a
wide variety of polyunsaturated fatty acids (or corresponding
activated derivative, e.g., active ester, acid halide, etc.).
Acylation can be carried out in the conventional way, for example,
by reacting the starting products with an acylating agent,
particularly with a reactive functional derivative of the acid,
whose residue is to be introduced.
[0099] The invention also provides metal or organic base salts of
the glycosphingolipid compounds for use according to the present
invention having free carboxy functions, and these also form part
of the invention. Also forming part of the invention are acid
addition salts of glycosphingolipid derivatives, which contain a
basic function, such as a free amino function, for example, esters
with aminoalcohols.
[0100] Another suitable method of synthesizing a glycolipid of
formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) can use
wild type or mutant endoglycoceramidase as taught in WO/2005/118798
which is incorporated herein by reference in its entirety with
respect to the methods of synthesis and enzymes used therein.
[0101] Further modifications can be made to the glycolipids
synthesized using the endoglycoceramide synthase of the present
invention. Exemplary methods of further elaborating glycolipids
produced using the present invention are set forth in WO 03/017949;
PCT/US02/24574; US2004063911 (although each is broadly directed to
modification of peptides with glycosyl moieties, the methods
disclosed therein are equally applicable to the glycolipids and
method of producing them set forth herein). Moreover, the
glycolipid compositions of the invention can be subjected to
glycoconjugation as disclosed in WO 03/031464 and its progeny
(although each is broadly directed to modification of peptides with
glycosyl moieties, the methods disclosed therein are equally
applicable to the glycolipids and method of producing them set
forth herein).
[0102] The products produced by the above processes can be used
without purification. However, for some applications it is
desirable to purify the compounds. Standard, well-known techniques
for purification of substrates are generally suitable. For example,
thin or thick layer chromatography, column chromatography, ion
exchange chromatography, or membrane filtration can be used.
Moreover, membrane filtration, preferably utilizing a reverse
osmotic membrane, or one or more column chromatographic techniques,
can be utilized. For instance, membrane filtration wherein the
membranes have molecular weight cutoff of about 3,000 to about
10,000 can be used to remove proteins such as glycosyl
transferases. Nanofiltration or reverse osmosis can then be used to
remove salts and/or purify the product saccharides (see, e.g., WO
98/15581).
[0103] Another exemplary purification strategy makes use of a
membrane in conjunction with an organic solvent. Both glycolipids
and glycosphingolipids can be purified by this method. Moreover,
any of the intermediate enzyme reaction products described herein
can be purified according to this method. The method includes
concentrating a reaction product in a membrane purification system
with the addition of an organic solvent.
[0104] Suitable solvents include, but are not limited to alcohols
(e.g., methanol), halocarbons (e.g., chloroform), and mixtures of
hydrocarbons and alcohols (e.g., xylenes/methanol). In a preferred
embodiment, the solvent is methanol. The concentration step can
concentrate the reaction product to any selected degree.
[0105] In yet another embodiment, the invention provides a
pharmaceutical formulation comprising a glycolipid as set forth or
incorporated by reference herein, e.g., of Formula (1), (2), (3),
(4), (5), (6), (7), (8), (9) or (10), in admixture with a
pharmaceutically accecptable carrier. The pharmaceutical
compositions of the invention are suitable for use in a variety of
drug delivery systems. Suitable formulations for use in the present
invention are found in Remington's Pharmaceutical Sciences, Mace
Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief
review of methods for drug delivery, see, Langer, Science 249:
1527-1533 (1990).
[0106] The pharmaceutical compositions can be administered by a
number of routes, for instance, the parenteral, subcutaneous,
intravenous, intranasal, topical, oral or local routes of
administration, such as by aerosol or transdermally, for
prophylactic and/or therapeutic treatment. Commonly, the
pharmaceutical compositions may be administered parenterally, e.g.,
intravenously. Preparations for parenteral administration include
sterile aqueous or non-aqueous solutions, suspensions, and
emulsions. Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers
include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media.
[0107] The compositions containing the glycolipid compounds can be
administered for prophylactic and/or therapeutic treatments. In
therapeutic applications, compositions are administered to a
subject already suffering from a disease, as described above, in an
amount sufficient to cure or at least partially arrest the symptoms
of the disease and its complications. Amounts effective for this
use will depend, as discussed further below, on the particular
compound, the severity of the disease and the weight and general
state of the subject, as well as the route of administration, but
generally range from about 0.5 mg to about 2,000 mg of substrate
per day for a 70 kg subject, with dosages of from about 5 mg to
about 200 mg of the compounds per day being more commonly used.
[0108] In prophylactic applications, compositions containing the
compound for use according to the invention are administered to a
subject susceptible to or otherwise at risk of a particular
disease. Such an amount is defined to be a "prophylactically
effective dose." In this use, the precise amounts again depend on
the subject's state of health and weight, and the route of
administration but generally range from about 0.5 mg to about 2,000
mg per 70 kilogram subject, more commonly from about 5 mg to about
200 mg per 70 kg of body weight.
[0109] Single or multiple administrations of the compositions can
be carried out with dose levels and pattern being selected by the
treating physician. In any event, the pharmaceutical formulations
should provide a quantity of the substrates of this invention
sufficient to effectively treat the subject.
[0110] Labeled substrates can be used to determine the locations at
which the substrate becomes concentrated in the body due to
interactions between the desired oligosaccharide determinant and
the corresponding ligand. For this use, the compounds can be
labeled with appropriate radioisotopes, for example, .sup.125I,
.sup.14C, or tritium, or with other labels known to those of skill
in the art.
[0111] The dosage ranges for the administration of the compounds
for use according to the invention are those large enough to
produce the desired effect. Generally, the dosage will vary with
the age, condition, sex and extent of the disease in the subject
and can be determined by one of skill in the art. The dosage can be
adjusted by the individual physician monitoring the therapy.
[0112] Additional pharmaceutical methods may be employed to control
the duration of action. Controlled release preparations may be
achieved by the use of polymers to conjugate, complex or adsorb the
glycosphingolipid.
[0113] In an exemplary embodiment, the invention provides a method
of treating Parkinson's disease by providing neuroprotection from
damage caused by toxic metabolites of dopamine. The method
comprises administering to a subject in need thereof an amount of a
glycolipid as set forth herein, which is sufficient to provide the
neuroprotection. In an exemplary embodiment, the administration
results in an amelioration of Parkinson's symptoms or a cure of the
disease.
[0114] In another exemplary embodiment, the invention provides a
method of binding a protein implicated in a disease state.
Exemplary proteins include tau protein, beta-amyloid proteins,
.alpha.-synuclein and huntingtin. The method comprises
administering to a subject in need thereof an amount of a
glycolipid as set forth herein, which is sufficient to protein
implicated in the disease state. In an exemplary embodiment, the
binding yields an amelioration or cure of the disease state by
reducing or eliminating protein in the form in which it is
implicated in the disease state.
Methods of Analyzing Glycosphingolipids and Identifying a
Ganglioside Deficiency State in a Subject
[0115] A ganglioside deficiency state can be identified by assaying
blood, plasma, CSF or other tissue samples from the subject for the
pertinent ganglioside. A deficiency state exists when a particular
ganglioside is absent or less than two-thirds that of an otherwise
comparable control population. In some embodiments, the deficiency
state exists when tissue levels of the pertinent ganglioside are
one-half, one-quarter, one-tenth or one-twentieth of a normal,
control, or reference level.
[0116] Many methods of analyzing biological samples for ganglioside
and glycosphingolipid content are known to one of ordinary skill in
the art. The use of such methods has been exemplified by Simpson,
et al., Nature Genetics 36(11):1225 (2004) using HPLC-based methods
developed by Neville, et al., Anal Biochem. 331(2):275-82 (2004)
which are each incorporated herein by reference in their entirety,
and particularly, with respect to such methods. These methods
provide a rapid and sensitive method to both analyze and
characterize the full complement of glycosphingolipid structures
present in various cells and tissues. The method characterizes the
oligosaccharides released from glycosphingolipids following
ceramide glycanase digestion.
[0117] Additional methods for analyzing gangliosides are taught by
Anumula and Dhume, Glycobiology 8:685 (1998), Svennerholm L, et
al., Biochim Biophys Acta 617:97-109 (1980) and Wang B. et al.,
Comp Biochem Physiol A Mol Integr Physiol 119:435-9 (1998); and
Wang B., e al., Am J Clin Nutr. 78(5):1024-9 (2003). Such methods
can also be used to identify and quantify compounds for use
according to the invention in tissue samples.
[0118] Glycolipid deficiency states suitable for treatment can also
be identified by measuring the levels of the pertinent glycolipid
in a sample from the subject and comparing the measured level with
levels of the pertinent ganglioside measured or already established
for suitable control populations as would be known to one of
ordinary skill in the art. Referent levels of glycolipid in plasma
for a control population have been reported previously (Simpson,
Nature Genetics, 36:1225-1229 (2004)).
[0119] It is contemplated that when used to treat various diseases
such as cancer, atypical Parkinson's disease; Huntington's disease,
multiple systems atrophy, GM3 synthase deficiency, GM2 synthase
deficiency, hyperinsulinemia, hypoglycemia, hyperinsulinemia with
hypoglycemis, a tauopathy, or another neurological disorder or
condition associated with an increased aggregation of tau protein,
the compounds of the invention can be combined with other
therapeutic agents suitable for the same or similar diseases.
[0120] The compounds, antibodies and chemotherapeutic agents of the
invention are administered to a human patient, in accord with known
methods, such as intravenous administration as a bolus or by
continuous infusion over a period of time, by intramuscular,
intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,
intrasynovial, intrathecal, oral, topical, or inhalation routes.
Intravenous or subcutaneous administration of the antibody is
preferred.
[0121] In one embodiment, the treatment of the present invention
involves the combined administration of a compound from Formula (1)
to (10), an antibody and one or more chemotherapeutic agents.
Preparation and dosing schedules for chemotherapy are also
described in Chemotherapy Service Ed., M. C. Perry, Williams &
Wilkins, Baltimore, Md. (1992). The chemotherapeutic agent may
precede, or follow administration of the compound, the antibody or
may be given simultaneously therewith.
[0122] A typical therapeutically effective dosage of a compound of
the invention ranges from about 0.1 mg/kg to about 1000 mg/kg,
preferably from 0.1 mg/kg to about 100 mg/kg, more preferably from
about 0.1 mg/kg to about 30 mg/kg, more preferably from about 0.1
mg/kg to about 10 mg/kg, and more preferably 0.1 mg/kg to about 3
mg/kg. Advantageously, the compounds for use according to the
invention , alone or as part of a pharmaceutical composition, maybe
administered several times daily, and other dosage regimens may
also be useful. A compound of the invention may be administered on
a regimen in a single or multidose (e.g., 2 to 4 divided daily
doses or applications). The regimen may be tailored over time
according to the response of the subject. The dosage regimen may be
hourly, daily, weekly, monthly, acute, subacute, subchronic or
chronic. Up to 30 g (e.g., 1 to 2, 2 to 5, 5 to 10, 10 to 20, or 20
to 30 g), for instance, may be administered in one dose.
[0123] The effectiveness of the compounds for use according to the
invention may be determined using screening protocols known in the
art. The biological properties of the compounds for use according
to the invention can be readily characterized by methods that are
well known in the art including, for example, in vitro screening
protocols (e.g., cell cultures and in vivo studies for
effectiveness). Exemplary methods for identifying or screening
suitable compounds for use according to the invention are set forth
below.
[0124] The invention is further described with reference to the
following Examples. The Examples are provided for the purpose of
illustration only and the invention not be construed as being
limited to these Examples, but rather should be construed to
encompass any and all variations which become evident as a result
of the teaching provided herein.
EXAMPLE 1
Synthesis of
[5-N-acetyl-.alpha.-neuraminyl)-(2.fwdarw.3)]-[.beta.-D-galactopyranosyl--
(1.fwdarw.4)-O-D-glucopyranose] by Fermentation
[0125] The synthesis of this compound uses the process described by
Samain (Samain and Priem, Method for Producing
Oligopolysaccharides. WO 01/04341 (2001); Priem, Glycobiology,
12:235 (2002); Cottaz, EP Application No. 06291569.9 (2006)) with
E. coli JM107-nanA-(Nst-01, pBBnsy) strain using lactose and sialic
acid (Kok, J Chem Soc Perkin Trans, 1:2811-2815 (1996)) acid as
exogeneous acceptors.
[0126] E. coli JM107-nanA-(Nst-01, pBBnsy) strain was grown at low
cell density culture in shake flask fermentation. When the OD540 is
about 1, isopropyl 1-thio-.beta.-galactopyranoside (IPTG) and the
substrates Neu5Ac and lactose (2 equivalents) were added via a
sterile filter and the culture is shaken at 28.degree. C. for 16
hr. After 16 hr, the cells were recovered by centrifugation at 8000
g and 4.degree. C. for 10 min. The supernatant was separated for
further TLC analysis while the pellet is resuspended in H.sub.2O
and boiled for 30 min. The resulting suspension was again
centrifuged (8000 g, 4.degree. C. for 10 min). The supernatant was
mixed with activated charcoal, filtered through Celite and is
washed with distilled water. The adsorbed oligosaccharides were
elutetd with 50% aq. EtOH. The oligosaccharide was further purified
using a DOWEX exchange ions resin (DOWEX 1.times.4 50) and silica
gel column chromatography. The solution of product eluted from the
silica gel chromatography was evaporated to dryness to yield a
solid that is examined by NMR and MS to confirm the structure.
EXAMPLE 2
Synthesis of
1-Fluoro-[(5-N-acetyl-.alpha.-neuraminyl)-(2.fwdarw.3)]-[.beta.-D-galacto-
pyranosyl-(1.fwdarw.4)-.beta.-D-glucopyranoside]
[0127] The
[5-N-acetyl-.alpha.-neuraminyl)-(2.fwdarw.3)]-[.beta.-D-galacto-
pyranosyl-(1.fwdarw.4)-.beta.-D-glucopyranose], and DMAP are
dissolved in pyridine. Acetic anhydride was added dropwise and then
the reaction mixtureis stirred for 2 days. The reaction mixture was
concentrated and the residue treated with methanol. After 30 min,
the reaction mixture was concetrated again, the residue dissolved
in ethyl acetate and the organic solution washed with water, 5%
citric acid in water, water and brine. The organic solution was
dried with sodium sulfate, filtered and concetrated to dryness
yielding a solid used directly for the next step.
[0128] The solid is cooled to -30.degree. C. and then hydrogen
fluoride-pyridine was added. The reaction mixture was stirred for 1
hr allowing the reaction to slowly warm to room temperature and
then for 4 hrs at room temperature. The reaction mixture was cooled
to -10.degree. C. and is then slowly added to a cold solution of 6
N NaOH. Sodium carbonate is then carefully added until the pH of
the solution is about 8.0. The solution was then extracted with
CH.sub.2Cl.sub.2 (three times) and the combined organic layers
dried over sodium sulfate. Concentration provided a solid used
directly for the next step.
[0129] The solid was dissolved in anhydrous methanol and sodium
methoxide in methanol is added. The reaction mixture was stirred
overnight, treated with Dowex HCR-W2 resin (H.sup.+), the resin was
removed by filtration and the filtrate concentrated to dryness to
yield a solid. The product is charaterized by NMR and MS.
EXAMPLE 3
Synthesis of
[(5-N-acetyl-.alpha.-neuraminyl)-(2.fwdarw.3)]-[.beta.-D-galactopyranosyl-
-(1.fwdarw.4)-.beta.-D-glucopyranosyl-.beta.-(1.fwdarw.1')-D-erythro-sphin-
gosine]
[0130] The compound
1-fluoro-[(5-N-acetyl-.alpha.-neuraminyl)-(2.fwdarw.3)]-[.beta.-D-galacto-
pyranosyl-(1.fwdarw.4)-.beta.-D-glucopyranoside] and
D-erythro-sphingosine are coupled using the procedure as described
in Vaughan, J Am Chem Soc, 128:6300-6301 (2006). The reaction was
performed in 25 mM NaOAc (pH 5.0) containing 0.2% Triton X-100. A
typical reaction mixture contained approximately 10 mM
1-fluoro-[(5-N-acetyl-.alpha.-neuraminyl)-(2.fwdarw.3)]-[.beta.-D-galacto-
pyranosyl-(1.fwdarw.4)-.beta.-D-glucopyranoside], 20 mM of the
acceptor D-erythro-sphingosine, and 0.5 mg/mL of the appropriate
EGC mutant in a total reaction volume. When completed, the product
was purified using reversed phase (C-18) chromatography, the eluted
product was concentrated to dryness, dissolved in water and
freeze-dried to yield a white solid. The product is analyzed by NMR
and MS.
EXAMPLE 4
Synthesis of
[(5-N-acetyl-.alpha.-neuraminyl)-(2.fwdarw.3)]-[.beta.-D-galactopyranosyl-
-(1.fwdarw.4)-.beta.-D-glucopyranosyl]-.beta.-(1.fwdarw.1)-[2-N-stearoyl-D-
-erythro-sphingosine]
[0131] The
[(5-N-acetyl-.alpha.-neuraminyl)-(2.fwdarw.3)]-[.beta.-D-galact-
opyranosyl-(1.fwdarw.4)-.beta.-D-glucopyranosyl-.beta.-(1-1')-D-erythro-sp-
hingosine] was dissolved in anhydrous methanol that contains
triethylamine. Stearic anhydride was added as a solid to the
reaction mixture and the reaction stirred for 24 hrs. The product
was purified using silica gel chromatography eluting the product
with CHCl.sub.3/CH.sub.3OH. Concentration of the product fractions
provides a solid that is analyzed by NMR and MS.
EXAMPLE 5
Synthesis of
(.beta.-D-galactopyranosyl)-(1.fwdarw.3)-(.beta.-D-2-N-acetamido-galactop-
yranosyl)]-.beta.-(1.fwdarw.4)-[(5-N-acetyl-.alpha.-neuraminyl)]-(2.fwdarw-
.3)-(.beta.-D-galactopyranosyl)-(1.fwdarw.4)-.beta.-D-glucopyranose
by Fermentation
[0132] The synthesis of this compound used the process described by
Samain (Samain and Priem, Method for Producing
Oligopolysaccharides. WO 01/04341 (2001); Priem, Glycobiology,
12:235 (2002); Cottaz, EP Application No. 06291569.9 (2006)) with
E. coli JM107-nanA-(Nst-01, pBBnsy) strain using lactose.
[0133] E. coli JM107-nanA-(Nst-01, pBBnsy) strain is grown at low
cell density culture in shake flask fermentation. When the OD540
was about 1, isopropyl 1-thio-.beta.-galactopyranoside (IPTG) and
the substrates Neu5Ac and lactose (2 equivalents) were added via a
sterile filter and the culture was shaken at 28.degree. C. for 16
hr. After 16 hr, the cells were recovered by centrifugation at 8000
g and 4.degree. C. for 10 min. The supernatant was separated for
further TLC analysis while the pellet is resuspended in H.sub.2O
and boiled for 30 min. The resulting suspension is again
centrifuged (8000 g, 4.degree. C. for 10 min). The supernatant was
mixed with activated charcoal, filtered through Celite and was
washed with distilled water. The adsorbed oligosaccharides were
elutetd with 50% aq. EtOH. The oligosaccharide was further purified
using a DOWEX exchange ions resin (DOWEX 1.times.4 50) and silica
gel column chromatography. The solution of product eluted from the
silica gel chromatography was evaporated to dryness to yield a
solid that is examined by NMR and MS to confirm the structure.
EXAMPLE 6
Synthesis of
1-Fluoro-[(.beta.-D-galactopyranosyl)-(1.fwdarw.3)-(.beta.-D-2-N-acetamid-
o-galactopyranosyl)]-.beta.-(1.fwdarw.4)-[(5-N-acetyl-6-deoxy-.alpha.-neur-
aminyl)]-(2.fwdarw.3)-(.beta.-D-galactopyranosyl)-(1.fwdarw.4)-.beta.-D-gl-
ucopyranoside]
[0134] The
(.beta.-D-galactopyranosyl)-(1.fwdarw.3)-(.beta.-D-2-N-acetamid-
o-galactopyranosyl)]-.beta.-(1.fwdarw.4)-[(5-N-acetyl-.alpha.-neuraminyl)]-
-(2.fwdarw.3)-(.beta.-D-galactopyranosyl)-(1.fwdarw.4)-.beta.-D-glucopyran-
ose, and DMAP were dissolved in pyridine. Acetic anhydride was
added dropwise and then the reaction mixtureis stirred for 2 days.
The reaction mixture was concentrated and the residue treated with
methanol. After 30 min, the reaction mixture was concetrated again,
the residue dissolved in ethyl acetate and the organic solution
washed with water, 5% citric acid in water, water and brine. The
organic solution was dried with sodium sulfate, filtered and
concetrated to dryness yielding a solid used directly for the next
step.
[0135] The solid was cooled to -30.degree. C. and then hydrogen
fluoride-pyridine was added. The reaction mixture was stirred for 1
hr allowing the reaction to slowly warm to room temperature and
then for 4 hrs at room temperature. The reaction mixture was cooled
to -10.degree. C. and was then slowly added to a cold solution of 6
N NaOH. Sodium carbonate was then carefully added until the pH of
the solution was about 8.0. The solution was then extracted with
CH.sub.2Cl.sub.2 (three times) and the combined organic layers
dried over sodium sulfate. Concentration provides a solid used
directly for the next step.
EXAMPLE 7
Synthesis of
[(.beta.-D-galactopyranosyl)-(1.fwdarw.3)-(.beta.-D-2-N-acetamido-galacto-
pyranosyl)]-.beta.-(1.fwdarw.4)-[(5-N-acetyl-.alpha.-neuraminyl)]-(2.fwdar-
w.3)-(.beta.-D-galactopyranosyl)-(1.fwdarw.4)-(.beta.-D-glucopyranosyl)-.b-
eta.-(1.fwdarw.1)-O-[(1R,2R)-2-amino-1-(1,3-dioxolan-2
yl)-propane-1,3-diol)]
[0136] The compound
1-fluoro-[(.beta.-D-galactopyranosyl)-(1.fwdarw.3)-(.beta.-D-2-N-acetamid-
o-galactopyranosyl)]-.beta.-(1.fwdarw.4)-[(5-N-acetyl-.alpha.-neuraminyl)]-
-(2.fwdarw.3)-(.beta.-D-galactopyranosyl)-(1.fwdarw.4)-.beta.-D-glucopyran-
oside] and (1R,2R)-2-amino-1-(1,3-dioxolan-2 yl)-propane-1,3-diol)
is coupled using the procedure described in Example 3 using a
mutant EGC enzymes (Vaughan, J Am Chem Soc, 128:6300-6301 (2006)).
The reaction is performed in 25 mM NaOAc (pH 5.0) containing 0.2%
Triton X-100. A typical reaction mixture contained approximately 10
mM
1-fluoro-[(5-N-acetyl-.alpha.-neuraminyl)-(2.fwdarw.3)]-[.beta.-D-galacto-
pyranosyl-(1.fwdarw.4)-.beta.-D-glucopyranoside], 20 mM of the
acceptor D-erythro-sphingosine, and 0.5 mg/mL of the appropriate
EGC mutant in a total reaction volume. When completed, the product
is purified using reversed phase (C-18) chromatography, the eluted
product is concentrated to dryness, dissolved in water and
freeze-dried to yield a white solid. The product is analyzed by NMR
and MS.
EXAMPLE 8
Synthesis of
[(5-N-acetyl-.alpha.-neuraminyl)-(2.fwdarw.3)]-[.beta.-D-galactopyranosyl-
-(1.fwdarw.4)-.beta.-D-glucopyranosyl]-.beta.-(1.fwdarw.1)-O-[N-((2R,3S,E)-
-1,3-dihydroxy-5-phenylpent-4-ene-2-yl)-stearamide]
[0137] The
[(5-N-acetyl-.alpha.-neuraminyl)-(2.fwdarw.3)]-[.beta.-D-galact-
opyranosyl-(1.fwdarw.4)-.beta.-D-glucopyranosyl]-.beta.-(1.fwdarw.1')-O-[2-
-N-stearoyl-D-erythro-sphingosine], a water stable Grubb's catalyst
(Hong, J Am Chem Soc, 128:3508-3509 (2006) and styrene is stirred
in water/methanol. The reaction mixture is then concentrated to
dryness and purified by silica gel chromatography using
CHCl.sub.3/CH.sub.3OH. The eluted product fractions is concentrated
to afford the product as a solid that was characterized by NMR and
MS.
EXAMPLE 9
Synthesis of
[(.beta.-D-galactopyranosyl)-(1.fwdarw.3)-(.beta.-D-2-N-acetamido-galacto-
pyranosyl)]-.beta.-(1.fwdarw.4)-[(5-N-acetyl-.alpha.-neuraminyl)]-(2.fwdar-
w.3)-(.beta.-D-galactopyranosyl)-(1.fwdarw.4)-(.beta.-D-glucopyranosyl)-.b-
eta.-(1.fwdarw.1)-O-[(2R,3R,E)-2-N-stearamido-4-(dioctylamino)-6-phenylhex-
-5-ene-1,3-diol]
[0138] The
(5-N-acetyl-.alpha.-neuraminyl)-(2.fwdarw.3)-(.beta.-D-galactop-
yranosyl)-(1.fwdarw.4)-(.beta.-D-glucopyranosyl)-.beta.-(1.fwdarw.1')-O-[(-
1R,2R)-2-amino-1-(1,3-dioxolan-2
yl)-propane-1,3-diol)]-.beta.-(1.fwdarw.1')-O-[(2R,3R,E)-2-amino-4-(dioct-
ylamino)-6-phenylhex-5-ene-1,3-diol] is dissolved in anhydrous
methanol that contains triethylamine. Stearic anhydride is added as
a solid to the reaction mixture and the reaction stirred for 24
hrs. The product is purified using silica gel chromatography
eluting the product with CHCl.sub.3/CH.sub.3OH. Concentration of
the product fractions provides a solid that is analyzed by NMR and
MS.
[0139] The solid is dissolved in methanol, tartaric acid, and water
and stirred at room temperature for 24 hrs. The reaction mixture is
neutralized with sodium bicarbonate the product extracted with
CH.sub.2Cl.sub.2 (3 times). The organic layers are combined and
dried with NaSO.sub.4, filtered and concentrated to dryness. The
solid is used directly for the next step.
[0140] The product from the previous step (20 mg, 0.01 mmol) and
dioctylamine (6 mg, 0.024 mmol) are dissolved in dimethylformamide
(DMF) at room temperature. A solution of trans-2-phenylvinylboronic
acid (9 mg, 0.045 mmol) dissolved in methanol (5 mL) is then added.
The resulting solution is stirred at room temperature for three
days. The reaction mixture is then concentrated to dryness on a
rotovap and the residue purified by solid phase extraction using a
1 g HAX cartridge. The eluant is then purified using HPLC to afford
the product as a solid that is characterized by NMR and MS.
EXAMPLE 10
In Vitro Cell Proliferation
[0141] The efficacy of compounds of formula (1)-(10) compounds
alone and in combination with a kinase inhibitor is measured by a
cell proliferation assay employing the following protocol (Promega
Corp. Technical Bulletin TB288; Mendoza et al (2002) Cancer Res.
62:5485-5488). An aliquot of 100 .mu.L of cell culture containing
about 10.sup.4 cells (from the ATCC or DZM such as A549, NCI-H441,
NCI-1650, NCI-1975, PC-3, DiFi, HN5, SKBR3, MCF-7, HCT-116,
CCL-221, NCI-H125, SK-OV-3, MIA PaCa-2, T-47D) in medium are
deposited in each well of a 384-well, opaque-walled plate. Control
wells are prepared containing medium and without cells. The
compound are added to the experimental wells and incubated for 3-5
days. The plates are equilibrated to room temperature for
approximately 30 minutes and a volume of CellTiter-Glo Reagent
equal to the volume of cell culture medium present in each well is
added. The contents are mixed for 2 minutes on an orbital shaker to
induce cell lysis. The plate is incubated at room temperature for
10 minutes to stabilize the luminescence signal and the
luminescence recorded and reported in graphs as RLU=relative
luminescence units.
[0142] Alternatively, cells are seeded at optimal density in a 96
well plate and incubated for 4 days in the presence of test
compound. Alamar Blue.TM. is subsequently added to the assay
medium, and cells are incubated for 6 h before reading at 544 nm
excitation, 590 nm emission. EC.sub.50 values are calculated using
a sigmoidal dose response curve fit.
EXAMPLE 11
Identification of Glycolipids Capable of Cell Growth Inhibition
Against Lung Cancer Cells, Ovarian Cancer Cells and Colon Cancer
Cells
[0143] As cancer cell lines, human lung cancer A549 cells (ATCC No.
CCL-185), human ovarian cancer SK-OV-3 cells (ATCC No. HTB-77), and
human colon cancer HCT 116 cells (ATCC No. CCL-247) are used. For
the culture of A549 cells, Nutrient Mixture F-12K medium containing
10% fetal bovine serum, 100 units/mL penicillin, and 100 .mu.g/mL
streptomycin are used. For the culture of SK-OV-3 cells and HCT 116
cells, McCoy's 5A medium containing 10% fetal bovine serum, 100
units/mL penicillin, and 100 .mu.g/mL streptomycin are used. The
cells are cultured at 37.degree. C. in a 5% carbon dioxide
atmosphere.
[0144] A549 cells (1000 cells/well), SK-OV-3 cells (2000
cells/well), or HCT 116 cells (1000 cells/well) are seeded in each
well of 96-well plates, and cultured overnight. Test compounds of
formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) to be
tested are applied to the appropriate wells at a variety of
concentrations and at various time points beginning at 24, 18, 12,
6, and 0 hrs prior to kinase inhibitor addition at three different
concentrations. The cells are further cultured for 72 hours. Fifty
.sub.iut of labeling mixture of Cell Proliferation Kit II (XTT) are
added to each well, and the plates are incubated at 37.degree. C.
After 1 to 3 hours, the absorbance at 490 nm (reference wavelength:
655 nm) is measured with a plate reader. Growth ratios of the cells
in the wells treated with the test compounds are calculated based
on the growth ratio of the cells in the control well treated with
solvent (dimethyl sulfoxide (DMSO)) for 72 hours, which was defined
as 100%. From a plot of test compound concentrations and the cell
growth ratios at the concentrations, the concentration of 50%
growth inhibition, the GI.sub.50 value, are calculated.
EXAMPLE 12
Identification of Glycolipids Capable of Cell Growth Inhibition
Against Pancreatic Cancer Cells, Cervical Cancer Cells, Breast
Cancer Cells, Prostate Cancer Cells, Skin Cancer Cells, Head and
Neck Cancer Cells, Renal Cancer Cells and Liver Cancer Cells
[0145] As cancer cell lines, human pancreatic cancer MIA PaCa-2
cells (JCRB No. 0070), human cervical cancer HeLa cells (ATCC No.
CCL-2), human breast cancer MDA-MB-468 cells (ATCC No. HTB-132),
human prostate cancer DU 145 cells (ATCC No. HTB-81), human skin
cancer SK-MEL-28 cells (ATCC No. HTB-72), human head and neck
cancer KB cells (JCRB No. 9027), human renal cancer 786-0 cells
(ATCC No. CRL-1932), and human liver cancer Hep G2 cells (ATCC No.
HB-8065) are used. The cells are cultured at 37.degree. C. in a 5%
carbon dioxide atmosphere (except for the human breast cancer
MDA-MB-468 cells, which are cultured under the condition at
37.degree. C.) by using the mediums mentioned below,
respectively.
[0146] Cell Medium: Human pancreatic Minimum Essential Medium (MIA
PaCa-2) containing 10% fetal bovine serum, 0.1 mmol/L MEM
Non-Essential Amino Acids Solution, 100 units/mL penicillin and 100
.mu.g/mL streptomycin; Human cervical cancer Minimum
[0147] Essential Medium containing 10% fetal bovine serum, 0.1
mmol/L MEM Non-Essential Amino Acids Solution, 100 units/mL
penicillin and 100 .mu.g/mL streptomycin; Human breast cancer
Leibovitz's L-15 Medium (MDA-MB-468) containing 10% fetal bovine
serum, 100 units/mL penicillin, and 100 .mu.g/mL streptomycin;
Human prostate cancer Minimum Essential Medium containing 10% fetal
bovine serum, 0.1 mmol/L MEM Non-Essential Amino Acids Solution, 1
mmol/L Sodium Pyruvate Solution, 100 units/mL penicillin and 100
.mu.g/mL streptomycin; Human skin cancer Minimum Essential Medium
(SK-MEL-28) containing 10% fetal bovine serum, 0.1 mmol/L MEM
Non-Essential Amino Acids Solution, 100 units/mL penicillin and 100
.mu.g/mL streptomycin; Human head and neck Minimum Essential Medium
(KB cell 11095-080) containing 10% fetal bovine serum, 0.1 mmol/L
MEM Non-Essential Amino Acids Solution, 100 units/mL penicillin and
100 .mu.g/mL streptomycin; Human renal cancer RPMI 1640 Medium
(786-O cell) containing 10% fetal bovine serum, 10 mmol/L HEPES
Buffer Solution, 1 mmol/L Sodium Pyruvate Solution, 4.5 g/L
D-(+)-Glucose Solution, 100 units/mL penicillin and 100 .mu.g/mL
streptomycin; Human liver cancer Minimum Essential Medium (Hep G2
cell) containing 10% fetal bovine serum, 0.1 mmol/L MEM
Non-Essential Amino Acids Solution, 1 mmol/L Sodium Pyruvate
Solution, 100 units/mL penicillin and 100 .mu.g/mL
streptomycin.
[0148] In the same manner as that in Example 9, the cells are
seeded (500 to 4000 cells/well, respectively) in each well of
96-well plates, and the growth ratios of the cells treated with
test compounds of formula (1), (2), (3), (4), (5), (6), (7), (8),
(9) or (10) are applied to the appropriate wells at a variety of
concentrations and at various time points beginning at 24, 18, 12,
6, and 0 hrs prior to kinase inhibitor addition at three different
concentrations. The measurement of absorbance is performed at 1.5
to 3 hours after the addition of the XTT labeling mixture. From a
plot of test compound concentrations and the cell growth ratios at
the concentrations, the concentration of 50% growth inhibition, the
GI.sub.50 value, are calculated.
EXAMPLES 13
Identification of Glycolipids Capable of Preventing Tumor Growth in
a Breast Cancer Cell Xenograft Mouse Model
[0149] In an exemplary model system, nude (nu/nu) mice are
inoculated with MDA-MB-231 cells (human breast carcinoma) (10.sup.6
cells in 0.2 mL) s.c. in the right flank of the animals. The tumors
are staged to 200 mm.sup.3 and then treatment with the test
compound (1, 30 or 100 mg/Kg; IV or sc) and kinase inhibitor (IV or
sc)(e.g., lapatinib). Tumor volumes are obtained every other day
and the animals are sacrificed after 2 weeks of treatment. The
tumors are excised, weighed and paraffin embedded. Histological
sections of the tumors are analyzed, for example, by H and E,
anti-CD31, Ki-67, TUNEL, and CD68 staining
EXAMPLES 14
Identification of Glycolipids Capable of Treating Established
Burkitt's Lymphoma in SICD Mice
[0150] A xenotransplant model of the Raji Burkitt's lymphoma in
SCID mice is used. Raji cells after s.c. injection cause locally
growing tumors. Treatment is started when the tumors have reached a
size of 5 mm in diameter. At days 0, 7, and 15, cohorts of five
mice receive i.v. either PBS (control group) or in vitro
preactivated human PBLs. Four hrs after each PBL inoculation, the
mice are treated via tail vein injection either with no antibody,
or the following: compounds of the invention alone; compound (1, 50
or 100 mg/Kg) and kinase inhibitor; or compound (1, 50 or 100
mg/Kg) 3 hrs prior to administration of kinase inhibitor. Tumor
size is measured using a caliper every 2.sup.nd day. Animals are
followed until the s.c. tumors reached a maximal tolerated size of
15 mm in diameter and are then killed by cervical dislocation. The
days of sacrifice are recorded and are used for survival time
analysis.
EXAMPLES 15
Identification of Glycolipids Capable of Enhancing the Effects of
EGFR Kinase Inhibitors on the Survival of Mice after the Orthotopic
Transfer of Non-Small Lung Cancer Cells
[0151] The compounds of the invention were dissolved in PBS and
administered in Gefitinib (AstraZeneca Pharmaceuticals) was
dissolved in vehicle containing 1% Tween 80. AEE788 (Novartis
Pharmaceuticals) was dissolved in vehicle containing 90%
polyethylene glycol 300 and 10% 1-methyl-2-pyrrolidinone. Five days
after the implantation of the NCI-H441 or PC14-PE6 tumor cells into
their lungs, mice (8-10 per group) are randomized into treatment
groups and treated with: compounds of the invention (once daily,
s.c. or oral gavage; 1-100 mg/Kg); an EGFR1 inhibitor (e.g.,
gefitinib, po, once daily, 50 mg/Kg; AEE788 po, 3.times.weekly, 50
mg/Kg; lapatinib, p.o., once daily, 50 mg/Kg; erlotinib, p.o., once
daily, 50 mg/Kg); or compounds of the invention (once daily, s.c.
or oral gavage; 1-100 mg/Kg) in combination with an EGFR1 inhibitor
(selected from gefitinib, p.o., once daily, 50 mg/Kg; AEE788 po,
3.times.weekly, 50 mg/Kg; lapatinib, p.o., once daily, 50 mg/Kg or
erlotinib, p.o., once daily, 50 mg/Kg) or vehicle control. All of
the mice are killed and autopsied when control animals become
moribund and primary lung tumor weight (total tumor-bearing lung
weight minus the normal lung weight of 0.17 g), incidence and
volume of pleural effusion, and the presence of metastasis are
measured.
EXAMPLE 15
Ability of Glycolipids to Modify Glucose Transport
[0152] Glucose transport are determined in 3T3-L1 adipocytes or
primary human adipocytes as uptake of 2-deoxy-D[1-.sup.3H]glucose
by the method of Frost, J Biol Chem, 262:9872-9876 (1987). Cells
are grown on 13 mm plastic coverslips in 24-well culture dishes.
The cells are treated with the compounds of formula (1), (2), (3),
(4), (5), (6), (7), (8), (9) or (10) at concentrations of 1, 10,
50, 100 .mu.M) and incubated with the cells for at various time
points from 0, 30 min, 1 hr and 2 hrs prior to addition of
2-deoxy-D-[1-.sup.3H]glucose. The 2-deoxy-D-[1-.sup.3H]glucose is
then added to a final concentration of 50 mM (0.5 mCi/mL) and the
cells are incubated for 6 min. Glucose uptake is stopped by rinsing
the coverslips in three successive solutions of ice-cold buffer.
Nonspecific uptake is determined in the presence of 25 mM of
cytochalasin B. Coverslips are transferred to scintillation vials
and the cells are dissolved in 1% SDS. Radioactivity is measured
after adding 5 mL of scintillation fluid.
EXAMPLE 16
Ability of Glycolipids to Inhibit the Insulin Receptor
[0153] Human Lymphoid cells (IM9) are grown in culture and the
insulin receptor isolated as described (Nojiri, J Biol Chem,
266:4531-4537 (1991)). Compounds of formula (1), (2), (3), (4),
(5), (6), (7), (8), (9) or (10) at concentrations of 0, 0.1, 1, 10,
30, 100 and 200 .mu.M are added to a solution (50 mM HEPES, pH 7.4,
0.1% (v/v) Triton X-100; 30 .mu.L) containing the purified insulin
receptor. Insulin (26.3 IU/mg; 5 .mu.L) and MnCl.sub.2 (30 mM, 10
.mu.L) are then added to each tube and incubated at room
temperature for 1 hr. Phosphorylation is initiated by adding
ATP-[.gamma.-.sup.32P] (5 .mu.L; 25 .mu.Ci) and the reaction
mixture incubated at room temperature for 10 min. The reaction is
terminated by the addition of Laemmli's buffer (Laemmli, Nature,
227:680-685 (1970)) containing DTT (75 mg/mL) and heating in
boiling water for 3 min. The phosphoproteins are separted by
SDS-PAGE, the gel stained with Cibracron blue and an autoradiograph
taken. The .beta.-subunit of the insulin receptor is cut from the
gel and the amount of radioactivity quantitified using a liquid
scintillation counter. The amount of inhibition is then
plotted.
EXAMPLE 17
Ability of Glycolipids to Inhibit the Phosphorylation of the
Insulin Receptor in Mice
[0154] Experiments are performed in mice fasted for 14 h. The
compounds of Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or
(10) are administered IV, SC, oral gavage or IP to the mice at
doses of 0, 0.1, 1, 10, 30 or 100 mg/Kg. Insulin (5 units per
animal) is administered at 0 min, 30 min, 1 hr, 2 hr, 6 hr, 12 hr
or 24 hr after compound administration through the inferior vena
cava under anesthesia. Two minutes after injection, hind limb
muscle and adipose tissue are removed and incubated for 30 min on
ice with lysis buffer (PBS, pH 7.4/1% Nonidet P-40/0.5% sodium a
deoxycholate/0.1% SDS/1 mM PMS/10 mM sodium orthovanadate/3 g/ml
aprotinin) Lysates are immunoprecipitated with an anti-insulin
receptor subunit antibody. Immunoprecipitated samples are subjected
to SDS-PAGE and then transferred to nitrocellulose membranes. The
blots are probed sequentially, first with an antiphospho-tyrosine
antibody (PY99) and then with an anti-insulin receptor-subunit
antibody. Blots are developed by a chemiluminescence detection
system. The mice may be normal mice or mice that contain one or
more genes that have been deleted, mutated, knocked-in or a
combination thereof.
EXAMPLE 18
Ability of Glycolipids to Modify Glucose Tolerance, Insulin
Tolerance and Hormone Levels in Mice with and without a High-Fat
Diet
[0155] Glucose and insulin tolerance tests are performed in fasted
mice (13-15 h) with i.p. injections of glucose (2 g of glucose per
kg of body weight) or insulin (0.75 units of insulin per kg of body
weight), respectively. For the glucose tolerance test, compounds of
Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) are
administered by IV, SC, oral gavage or IP at a dose of 0, 0.1, 1,
10 and 100 mg/Kg at timepoints of 0 min, 30 min, 1 hr, 2 hr, 6 hr,
12 hr or 24 hrs prior to glucose injection. Blood glucose values
are measured immediately before and 15, 30, 60, and 120 min after
glucose injection. For the insulin tolerance test, compounds of
Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) are
administered by IV, SC, oral gavage or IP at a dose of 0.1, 1, 10
and 100 mg/Kg at timepoints of 0 min, 30 min, 1 hr, 2 hr, 6 hr, 12
hr or 24 hrs prior to insulin injection. Blood glucose levels are
measured immediately before and 15, 30, and 60 min after insulin
injection. To induce glucose intolerance, 6- to 8-week-old mice are
placed on a 45% high-fat diet (commercial preparation) for 10 weeks
(Steppan, Nature, 409:307-312 (2001)). The mice may be normal mice
or mice that contain one or more genes that have been deleted,
mutated, knocked-in or a combination thereof.
EXAMPLE 19
Ability of Glycolipids to Improve Hypoglycemia by Initiating a
Hyperinsulinemic-Hypoglycemia State in Normal Mice
[0156] Animals are fasted overnight. The compounds of Formula (1),
(2), (3), (4), (5), (6), (7), (8), (9) or (10) are administered via
IV, SC, oral gavage or IP at a dose of 0, 0.1, 1, 10, 30 or 100
mg/Kg. A 120-minute hyperinsulinemic-hypoglycemic clamp is
conducted with a continuous infusion of human insulin at a rate of
15 pmol/Kg/min or higher to create a hyperinsulinemic hypoglycemic
state that is started at 0 min, 30 min, 1 hr, 2 hr, 6 hr, 12 hr and
24 hrs after compound administration. Blood samples (20 .mu.L) are
collected at 20- to 30-minute intervals for the immediate
measurement of plasma glucose concentration. Insulin-stimulated
whole-body glucose flux is estimated using a prime-continuous
infusion of HPLC-purified [3-.sup.3H]glucose (10 .mu.Ci bolus, 0.1
.mu.Ci/min) throughout the clamps. To estimate insulin-stimulated
glucose transport activity in individual tissues,
2-deoxy-D[1-.sup.14C]glucose (2-[14C]DG) is administered as a bolus
(10 .mu.Ci) at 45 minutes before the end of clamps. Blood samples
(20 .mu.L) are taken at 77, 80, 85, 90, 100, 110, and 120 minutes
after the start of clamps for the determination of plasma
[.sup.3H]glucose, .sup.3H.sub.2O, and 2-[.sup.14C]DG
concentrations. Additional blood samples (10 .mu.L) are collected
before the start and at the end of clamps for measurement of plasma
insulin concentrations. All infusions are done using microdialysis
pumps. At the end of clamps, animals are anesthetized with sodium
pentobarbital injection. Within 5 minutes, four muscles (soleus,
gastrocnemius, tibialis anterior, and quadriceps) from both
hindlimbs, epididymal adipose tissue, and liver are taken. Each
tissue, once exposed, is dissected out within 2 seconds, frozen
immediately using liquid N.sub.2-cooled aluminum blocks, and stored
at -70.degree. C. for later analysis.
[0157] Plasma glucose concentration during clamps are analyzed
using 10 .mu.L plasma by a glucose oxidase method on a Beckman
glucose analyzer II. Plasma insulin concentration are measured by
RIA using kits. For the determination of plasma [3-.sup.3H]glucose
and 2-[.sup.14C]DG concentrations, plasma is deproteinized with
ZnSO.sub.4 and Ba(OH).sub.2, dried to remove .sup.3H.sub.2O,
resuspended in water, and counted in scintillation fluid on dual
channels for separation of .sup.3H and .sup.14C. The plasma
concentration of .sup.3H.sub.2O is determined by the difference
between .sup.3H counts without and with drying. For the
determination of tissue 2-.sup.14C]DG-6-phosphate (2-DG-6-P)
content, tissue samples are homogenized, and the supernatants are
subjected to an ion-exchange column to separate 2-DG-6-P from 2-DG
(Ohshima, Am J Physiol, 246:E193-E197 (1984)). The radioactivity of
3H in muscle glycogen is determined by digesting muscle samples in
KOH and precipitating glycogen with ethanol (Kim, Diabetes,
45:446-453 (1996)). Skeletal muscle glycogen synthase activity is
measured using .sup.14C-UDPG.
EXAMPLE 20
Ability of Glycolipids to Improve Hypoglycemia in Mice Containing
Mutations or Deletions in Genes Related to Familial
Hyperinsulinemia
[0158] The compounds of Formula (1), (2), (3), (4), (5), (6), (7),
(8), (9) or (10) are administered to rats via i.v., s.c., p.o.
(oral gavage), or i.p. at doses of 0, 0.1, 1, 10, 30 and 100 mg/Kg.
Blood glucose levels and insulin levels are measured by drawing
blood from the animal at 0, 30 min, 1 hr, 1.5 hrs, 2 hrs, 4 hrs, 6
hrs, 12 hrs and 24 hrs after administration of the compound. Whole
blood is assayed for glucose content using the glucose
dehydrogenase based enzymatic assay and quantitated using the
Hemocue glucose meter. Insulin levels are assayed in 15 .mu.L of
serum using the Rat Insulin RIA kit according to manufacturer's
procedure. Intraperitoneal glucose tolerance tests are made on 12-
to 20-week-old mice, after 16 hr fast. Animals are injected i.p.
with glucose (1 g/Kg). Blood is isolated from the tail vein at
times indicated and assayed for glucose content as described above.
Insulin content is assayed in isolated islets of equal diameter
(.about.100 .mu.m). Islets are sonicated in distilled water, then
pelleted for 5 min at 1,000.times.g. Supernatant is removed and
diluted 1:6,000 for RIA as described above.
[0159] Exendin-(9-39) Significantly Raises Fasting Blood Glucose
Levels in SUR-1-/-Mice
EXAMPLE 21
Ability of Glycolipids to Raise Fasting Blood Glucose Levels in
SUR-1 -/-Mice
[0160] Twelve-18 week old male SUR-1-/- and wildtype littermates
undergo a baseline evaluation including fasting blood glucose
measurements and oral glucose tolerance testing, followed by
randomization to treatment with compounds of Formula (1), (2), (3),
(4), (5), (6), (7), (8), (9) or (10) (0, 0.1, 1, 10, 30 or 100
mg/Kg/day; i.v., s.c., p.o. (oral gavage) or i.p.) or vehicle (0.9%
NaCl, 1% BSA). Fasting blood glucose levels are determined after an
overnight fast on days 3 and 7 after initiation of the study. In
addition, oral glucose tolerance and insulin sensitivity are
evaluated during treatment.
EXAMPLE 22
Ability of Glycolipids to Modify the Insulin Secretion, In
Vitro
[0161] Pancreatic islets (10 per well), isolated as described in
Koster, Proc Natl Acad Sci USA, 99: 16992-16997 (2002), are
incubated in glucose-free DMEM supplemented with D-(+)-glucose (1,
7, or 16.7 mM) and either gliblenclamide (1 .mu.M), diazoxide (250
.mu.M) or one or more of the compounds of Formula (1), (2), (3),
(4), (5), (6), (7), (8), (9) or (10)(0.1, 1, 10, 30 and 100 .mu.M),
as indicated. Alteneratively, the islets are first incubated with
the compounds of Formula (1), (2), (3), (4), (5), (6), (7), (8),
(9) or (10) (0.1, 1, 10, 30 and 100 .mu.M) for 0, 30 min, 1 hr, 2
hrs, 4 hrs or 6 hrs prior to glucose, gliblenclamide or diazoxide
addition. Islets are incubated for 60 min at 37.degree. C., medium
removed and assayed for insulin content with Rat Insulin RIA kit.
Pancreatic Islets may be isolated from normal or genetically
modified animals or humans.
Example 23
Effect of Glycolipids in Regulating Insulin and Plasma Glucose
Levels in HI Patients
[0162] After an overnight fast, subject receives an intravenous
infusion or subcutaneous injection of a compound selected from
Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) (1, 30
or 100 mg/Kg/day). On the second day, the subject is fasted
overnight. Blood samples for glucose, insulin, C-peptide, and
glucagon are obtained at different intervals after compound
administration.
Example 24
Identification of Glycolipids Capable of Enhancing the
Anti-Proliferative Activity of Rituxin Using Non-Hodgkin's Lymphoma
Cells Cultured from Patients
[0163] Cancer cell samples are obtained from the affected
individual from peripheral blood or bone marrow aspirates. In this
examples, either B-cell CLL or MCL cells, depending on the
malignancy being studies, were enriched using immunomagnetic
sorting (Decker, Blood, 95:999-1006 (2000)). The cells are then
cultured in RPMI 1640, supplemented with 30% autologous serum, 50
IU/mL penicillin/streptomycin, glutamine, CpG oligonucleotide
DSP30, IL-2 and mercaptoethanol. Cells are plated in 96 well dishes
and growth for 48 hrs. Cells are then resuspended in human serum
and culture medium and the compound of formula (1), (2), (3), (4),
(5), (6), (7), (8), (9) or (10) to be tested is applied to the
culture at a variety of concentrations at various time points
beginning at 24, 18, 12, 6, and 0 hrs prior to antibody addition
(Rituxan). The Rituxan (anti-CD20) is then added and the cells
incubated for 24 hrs. Dead and viable cells are discriminated by
Annexin V/propidium iodide staining and assessed by flow
cytometry.
EXAMPLE 26
Identification of Glycolipids Capable of Cell Growth Inhibition
Against Lung Cancer Cells, Breast, Ovarian Cancer Cells and Colon
Cancer Cells
[0164] As cancer cell lines, MCF7, human lung cancer A549 cells
(ATCC No. CCL-185), human ovarian cancer SK-OV-3 cells (ATCC No.
HTB-77), and human colon cancer HCT 116 cells (ATCC No. CCL-247)
are used. For the culture of A549 cells, Nutrient Mixture F-12K
medium containing 10% fetal bovine serum, 100 units/mL penicillin,
and 100 .mu.g/mL streptomycin are used. For the culture of SK-OV-3
cells and HCT 116 cells, McCoy's 5A medium containing 10% fetal
bovine serum, 100 units/mL penicillin, and 100 .mu.g/mL
streptomycin are used. The cells are cultured at 37.degree. C. in a
5% carbon dioxide atmosphere.
[0165] A549 cells (1000 cells/well), SK-OV-3 cells (2000
cells/well), or HCT 116 cells (1000 cells/well) are seeded in each
well of 96-well plates, and cultured overnight. Test compounds of
formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) to be
tested are applied to the appropriate wells at a variety of
concentrations and at various time points beginning at 24, 18, 12,
6, and 0 hrs prior to antibody addition at three different
concentrations. The cells are further cultured for 72 hours. Fifty
.mu.L of labeling mixture of Cell Proliferation Kit II (XTT) are
added to each well, and the plates are incubated at 37.degree. C.
After 1 to 3 hours, the absorbance at 490 nm (reference wavelength:
655 nm) is measured with a plate reader. Growth ratios of the cells
in the wells treated with the test compounds are calculated based
on the growth ratio of the cells in the control well treated with
solvent (dimethyl sulfoxide (DMSO)) for 72 hours, which was defined
as 100%. From a plot of test compound concentrations and the cell
growth ratios at the concentrations, the concentration of 50%
growth inhibition, the GI.sub.50 value, are calculated.
EXAMPLES 27
Identification of Glycolipids Capable of Antiproliferative Activity
in Hematologic Cancers
[0166] WSU-NHL and Jurkat cells are seeded at 5,000 cells per well
in 96-well plates in quadruplicates. Proliferation assays are
performed for 96 hours. Tritiated thymidine (3H-TdR) incorporation
during the last 16 hours of incubation was used to assess DNA
synthesis.
[0167] The responses of the compound of formula (1), (2), (3), (4),
(5), (6), (7), (8), (9) or (10) to be tested is applied to the
culture at a variety of concentrations at various time points
beginning at 24, 18, 12, 6, and 0 hrs prior to antibody addition
(anti-CD70).
EXAMPLE 28
Protection of Cortical Cells from Apoptosis
[0168] To induce apoptosis, mouse cortical cells were cultured and
treated with 50 .mu.M hydrogen peroxide for three hours prior to
being treated with the ganglioside analogue.
[0169] The cells were also treated with the hydrogen peroxide
during treatment with the ganglioside analogue and post-treatment
for 48 h. Cell death was assayed using the MTT assay.
[0170] Approximately 30% of the cells treated with hydrogen
peroxide died as a result of the treatment. Treatment with GM1
(approx. 0.1 .mu.M) provided approximately 20% protection of the
cells from apoptosis.
EXAMPLE 29
Protection of Cortical Cells from Cell Death
[0171] To induce non-apoptotic cell death, mouse cortical cells can
be cultured and treated with 50 .mu.M hydrogen peroxide and
oligomycin (0.01 .mu.M) for three hours prior to being treated with
the ganglioside analogue. The cells are also treated with the
hydrogen peroxide and oligomycin during treatment with the
ganglioside analogue and post-treatment for 48 h. Cell death can be
assayed using the MTT assay.
[0172] Approximately 30% of the cells treated with hydrogen
peroxide can die as a result of the treatment. Treatment of the
cells with compounds for use according to the invention protects
approximately 20% protection of the cells from death.
EXAMPLE 30
Animal Model of Parkinson's and Atypical Parkinson's Disease
[0173] C57B1/6 mice 7-8 weeks of age can be treated with MPTP
(b.i.d., 20 mg/kg, s.c.). The mice also receive a daily
administration of saline, compound of formula (1), (2), (3), (4),
(5), (6), (7), (8), (9) or (10) (1, 10 and 30 mg/kg, o.s.) or (0.3.
to 3 mg/kg, i.p. or s.c.) for three weeks starting 24 h after the
last MPTP injection. The brains are removed and analyzed for
striatum and substantia nigra pars compacta dopamine levels. The
midbrain is fixed for TH immunohistochemistry and dopamine neuron
cell counts.
[0174] MPTP alone can cause approximately 76% loss of striatal
dopamine. GMla and compounds for use according to the invention
increase striatal dopamine levels to approximately the same
extent.
EXAMPLES 31
Animal Model of Huntington's Disease
[0175] Transgenic R6/2 mice, 40 day old, expressing human
Huntingtin protein are administered compounds selected from (1) to
(10) by intraperitoneal injection (3 or 30 mg/Kg) or oral gavage
(30 mg/Kg), daily, for six weeks. Twice weekly, both control mice,
receiving saline, and treated mice are assessed for disease
progression using the rotorod test by methods known in the art.
[0176] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all purposes
to the extent not inconsistent with the present disclosure.
* * * * *