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Cancer Patent Abstract
A treatment for cancer, and in particular, of therapeutic compounds
which block the ability of cytokines and chemokines to promote metastasis
of malignant cells. The therapeutic compound comprises a carboxylated
and/or sulfated oligosaccharide, preferably in a substantially purified
form, which is a heparin or heparan-sulfate derived saccharide compound.
In one embodiment of the present invention, the carbohydrate or
oligosaccharide has a molecular weight of no more than about 3000
daltons, preferably lying in the range of about 400 to about 2000
daltons, most preferably between about 400 and about 1100 daltons.
Generally, substances of the present invention inhibit tumor cell
migration, as determined by biological assays, and comprise molecules
of various sugar units of which the basic unit of activity is associated
with a disaccharide. However, larger oligosaccharide chains of up
to about 10 sugar units, containing the basic disaccharide unit
of activity can also function to inhibit such activity.
Cancer Patent Claims
What is claimed is:
1. A method for treating a malignancy selected from the group consisting
of breast cancer, lung cancer, bone cancer, bladder cancer, rhabdomyosarcoma,
angiosarcoma, adenocarcinoma, prostate cancer, colon cancer, squamous
cell carcinoma of the cervix, ovarian cancer, malignant fibrous
histiocytoma, skin cancer, leiomyosarcoma, astrocytoma, glioma and
heptocellular carcinoma in a subject; wherein the method comprises
administering a pharmaceutically effective amount of a therapeutic
agent to the subject, said therapeutic agent comprising an oligosaccharide,
wherein said oligosaccharide has a molecular weight of less than
about 3000 daltons and comprises a disaccharide of formula (I) or
its pharmaceutically acceptable salt: ##STR00012## in which X.sub.1
is hydrogen or sulfate; X.sub.2 is hydrogen or sulfate; and X.sub.3
is sulfate or acetyl, provided that if X.sub.3 is sulfate, then
at least one of X.sub.1 or X.sub.2 is sulfate and if X.sub.3 is
acetyl, then both X.sub.1 and X.sub.2 are sulfates.
2. The method of claim 1, wherein said oligosaccharide is an N-sulfated-4-deoxy-4-en-iduronoglucosamine
having at least one other sulfate group and pharmaceutically acceptable
salts thereof.
3. The method of claim 1, wherein said oligosaccharide is an N-acetylated-4-deoxy-4-en-iduronoglucosamine
having at least two sulfate groups and pharmaceutically acceptable
salts thereof.
4. The method of claim 1, wherein said oligosaccharide is a disaccharide
of formula (I) or its pharmaceutically acceptable salt: ##STR00013##
in which X.sub.1 is hydrogen or sulfate; X.sub.2 is hydrogen or
sulfate; and X.sub.3 is sulfate or acetyl, provided that if X.sub.3
is sulfate, then at least one of X.sub.1 or X.sub.2 is sulfate and
if X.sub.3 is acetyl, then both X.sub.1 and X.sub.2 are sulfates.
5. The method of claim 1, wherein said oligosaccharide is an N-sulfated-4-deoxy-4-en-glucuronoglucosamine
having at least one other sulfate group or a pharmaceutically acceptable
salt thereof.
6. The method of claim 1, wherein said oligosaccharide is a sulfated
disaccharide.
7. The method of claim 1, wherein said oligosaccharide is a sulfated
disaccharide.
8. The method of claim 1, wherein said oligosaccharide comprises
at least one of Po912, DS 1145, DS 1020, DS 8767, Po821, DS 9267,
DS 9517 and DS 0895.
9. The method of claim 8, wherein said oligosaccharide comprises
Po912.
10. The method of claim 1, wherein the malignancy is a metastatic
tumor.
11. The method of claim 1, wherein the malignancy is lung cancer.
12. The method of claim 1, wherein said oligosaccharide is administered
in an amount in a range of from about 1 to about 1000 micrograms
of oligosaccharide per Kg of subject, weight per weight.
13. The method of claim 1, wherein said cancer is metastatic.
14. The method of claim 13, wherein said oligosaccharide is a sulfated
glucosamine derivative and pharmaceutically acceptable salts thereof.
15. The method of claim 14, wherein said oligosaccharide is a sulfated
disaccharide.
16. The method of claim 13, wherein said oligosaccharide is an
N-acetylated-4-deoxy-4-en-iduronoglucosamine having at least two
sulfate groups and pharmaceutically acceptable salts thereof.
17. The method of claim 15, wherein said oligosaccharide is a disaccharide
of formula (I) or its pharmaceutically acceptable salt: ##STR00014##
in which X.sub.1 is hydrogen or sulfate; X.sub.2 is hydrogen or
sulfate; and X.sub.3 is sulfate or acetyl, provided that if X.sub.3
is sulfate, then at least one of X.sub.1 or X.sub.2 is sulfate and
if X.sub.3 is acetyl, then both X.sub.1 and X.sub.2 are sulfates.
18. The method of claim 15, wherein said oligosaccharide is an
N-sulfated-4-deoxy-4-en-glucuronoglucosamine having at least one
other sulfate group or a pharmaceutically acceptable salt thereof.
19. The method of claim 12, wherein said oligosaccharide comprises
at least one of Po912, DS 1145, DS 1020, DS 8767, DS Po821, DS 9267,
DS 9517 and DS 0895.
20. The method of claim 13, wherein said oligosaccharide comprises
at least one of Po912, DS 1145, DS 1020, DS 8767, Po821, DS 9267,
DS 9517 and DS 0895.
21. The method of claim 20, wherein said oligosaccharide comprises
Po912.
22. The method of claim 19, wherein said oligosaccharide is DS
1145.
23. The method of claim 13, wherein said oligosaccharide alters
localization of tumor cells to treat the metastatic cancer.
24. The method of claim 13, wherein said oligosaccharide alters
homing activity of tumor cells to treat the metastatic cancer.
25. The method of claim 13, wherein said oligosaccharide interferes
with the CXCR4 7TM-GPCR signaling pathway.
26. The method of claim 1, wherein said oligosaccharide has a molecular
weight lying in the range of from about 400 daltons to about 2000
daltons.
27. The method of claim 26, wherein said oligosaccharide has a
molecular weight lying in the range of from about 400 to about 1100
daltons.
28. The method of claim 1, wherein said malignancy is selected
from the group consisting of breast cancer, bone cancer, bladder
cancer, rhabdomyosarcoma, angiosarcoma, adenocarcinoma, prostate
cancer, colon cancer, squamous cell carcinoma of the cervix, ovarian
cancer, malignant fibrous histiocytoma, skin cancer, leiomyosarcoma,
astrocytoma, glioma and heptocellular carcinoma.
Cancer Patent Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This Application claims priority from U.S. patent application Ser.
No. 09/495,723, filed on Feb. 1, 2000, which is a divisional of
U.S. patent application Ser. No. 08/486,127, filed on Jun. 7, 1995
and now issued as U.S. Pat. No. 6,020,323, on Feb. 1, 2000, which
is a continuation of U.S. patent application Ser. No. 08/436,330,
filed on May 10, 1995, now U.S. Pat. No. 5,861,382, issued on Jan.
19, 1999, which claims priority from PCT Application No. PCT/US93/10868,
filed on Nov. 9, 1993, and which is a continuation in part of U.S.
patent application Ser. No. 08/096,739, filed on Jul. 23, 1993,
abandoned, which is a continuation in part of U.S. patent application
Ser. No. 07/974,750, filed on Nov. 10, 1992, abandoned, which is
a continuation in part of U.S. patent application Ser. No. 07/878,188,
filed on May 1, 1992, abandoned; all of which are owned in common
with the present application, and all of which are hereby incorporated
by reference as if fully set forth herein.
FIELD OF THE INVENTION
The present invention is of therapeutic compounds for cancer, and
in particular, of therapeutic compounds which block the ability
of cytokines and chemokines to promote metastasis of malignant cells,
and which are heparin and heparan-sulfate derived saccharide compounds.
BACKGROUND OF THE INVENTION
Cancer and Metastasis
Cancer is a growing problem in the world, particularly in the western
countries. The increase in cancer cases and in cancer-related mortality
may be attributed, at least in part, to an overall decrease in the
rate of deaths from other causes, such as infectious disease. Therefore,
new treatments for cancer are becoming increasingly important, both
in order to extend the lifespan and also to increase quality of
life.
The mechanism basis of the ability of metastatic cells to home
and proliferate in the parenchyma of certain organs, such as the
liver, and to develop organ-specific metastases remain largely unknown.
For metastasis to occur, the malignant cells must escape from the
primary tumor, circulate through the blood stream and subsequently
arrest and develop in the target tissues. Recently, it was shown
that metastatic breast carcinomas utilize the SDF-1/CXCR4 chemokine/chemokine
receptor pathway for metastasis (1-3).
In recent years, chemokines, molecules that actively modulate the
onset and progression of the immune response, and their cellular
receptors have received increasing attention due to their critical
role in the progression of immune disease states such as Asthma,
Atherosclerosis, Graft Rejection, AIDS, and Multiple Sclerosis (MS).
Chemokines are a family of structurally related proteins that have
an essential role in the recruitment and activation of cells from
the immune system. Thus, chemokines can be considered as master
regulators of the body's immune response repertoire. Because of
their varied activities, chemokines are potentially valuable targets
for therapeutic intervention in a wide range of diseases (4).
Several research groups have shown anti-tumor activity with a variety
of chemokines overexpressed in tumor cells. More specifically, anti
tumor activity was shown for MCP-3, MIP-1alpha, Rantes, lymphotactin,
TCA-3, and MIP-3alpha (5). The chemokine receptor CXCR-4 has been
shown to function as the major co-receptor for HIV-1/2 on T cells,
as well as the CD4-independent receptor for HIV-2 (6). The murine
CXCR-4-predicted amino acid sequence is 91% identical to human CXCR-4.
CXCR-4 is expressed on human CD34+ stem cells, PBLs, monocytes,
and neutrophils (7). Stromal cell-derived factor 1 alpha/beta (SDF-1),
the ligand for CXCR-4, is a powerful chemoattractant for T cells
and CD34+ cells and can inhibit HIV infection of these cells (8).
Human and murine SDF-1 differ by one amino acid and are cross-reactive.
SDF-1 is produced in high levels in the bone marrow, lymph node
(LN) and spleen (9-11). In contrast to pro-inflammatory chemokines,
SDF-1 expression is not regulated by stimuli generated by viral
or bacterial infections, suggesting a major role for SDF-1 in steady-state
homeostatic processes, such as leukocyte trafficking (12).
SDF-1 can induce the arrest of rolling CD34.sup.+ on human endothelium
under shear flow in vitro, and that in vivo, human bone marrow endothelial
cells express SDF-1 (13). Furthermore, by increasing the expression
level of CXCR4 on CD34+ progenitors, their ability to migrate to
and engraft in the bone marrow is improved (14). Overexpression
of human CXCR4 on murine T cells led to enhanced numbers of these
cells in the murine BM and to a dramatic decrease in their numbers
in the circulation (15). In addition, injection of SDF-1 into the
murine spleen and bone marrow was shown to increase the homing of
FDCP-mix cells to the spleen and the homing of human CD34+ cells
to the bone marrow (16). These results suggest that an increase
in the concentration of SDF-1 within the bone marrow microenvironment
or enhanced expression of CXCR4 on effector T cells may stimulate
the homing and retention of these cells to the bone marrow.
The process of metastasis requires at least three consecutive steps
in which chemokines may be involved. First, chemokines may facilitate
the interaction of tumor cells with endothelial cells. Second, following
the transendothelial migration of tumor cells chemokines can direct
the intra-tissue localization of tumors. Thereafter chemokines may
stimulate the growth of tumor cells after metastasis.
Saccharide-based Compounds
Heparin is a glycosaminoglycan, a polyanionic sulfated polysaccharide,
which is used clinically to prevent blood clotting as an antithrombotic
agent. In animal models, heparin has been shown to reduce the ability
of autoimmune T lymphocytes to reach their target organ (Lider,
O. et al., Eur. J. Immunol. (1990) 20:493-499). Heparin was also
shown to suppress experimental autoimmune diseases in rats and to
prolong the allograft survival in a model of skin transplantation
in mice, when used in low doses (5.mu.g for mice and 20.mu.g for
rats) injected once a day (Lider, O. et al., J. Clin. Invest. (1989)
83:752-756).
The mechanisms behind the observed effects are thought to involve
inhibition-of release by T lymphocytes of enzyme(s) necessary for
penetration of the vessel wall, primarily the enzyme heparanase
that specifically attacks the glycosaminoglycan moiety of the sub-endothelial
extracellular matrix (ECM) that lines blood vessels (Naparstek,
Y. et al., Nature (1984) 310:241-243). Expression of the heparanase
enzyme is associated with the ability of autoimmune T lymphocytes
to penetrate blood vessel walls and to attack the brain in the model
disease experimental autoimmune encephalomyelitis (EAE).
European Patent Application EP 0114589 (Folkman et al.) describes
a composition for inhibition of angiogenesis in mammals in which
the active agents consist essentially of (1) heparin or a heparin
fragment which is a hexasaccharide or larger and (2) cortisone or
hydrocortisone or the 11-.alpha. isomer of hydrocortisone. According
to the disclosure, heparin by itself or cortisone by itself are
ineffective; only the combination of both gives the desired effects.
Although there is no proof in the literature that there is a connection
between angiogenesis and autoimmune diseases, the description on
page 5 of the patent application connects angiogenesis with psoriasis
and with arthritis, indicating the use of high doses of 25,000 units
to 47,000 units of heparin per day (i.e., about 160 to about 310
mg per day).
Horvath, J. E. et al., in Aust. N.Z.J. Med. (1975) 5(6):537-539,
describe the effect of subanticoagulant doses of subcutaneous heparin
on early renal allograft function. The daily dosage is high (5000
U or about 33 mg) and the conclusion of the study is that heparin
in subanticoagulant doses has no effect on early graft function
or graft survival and that it may be associated with increased hemorrhagic
complications.
Toivanen, M. L. et al., Meth. and Find. Exp. Clint. Pharmacol.
(1982) 4(6):359-363, examined the effect of heparin in high dosage
(1000 U/rat or about 7 mg/rat) in the inhibition of adjuvant arthritis
in rats and found that heparin enhanced the severity of the rat
adjuvant arthritis.
PCT Patent: Application PCT/AU88/00017 published under No. WO88/05301
(Parish et al.) describes sulphated polysaccharides that block or
inhibit endoglycosylase activity, such as heparanase activity, for
use as antimetastatic and anti-inflammatory agents. Heparin and
heparin derivatives, such as periodate oxidized, reduced heparins,
that had negligible anticoagulant activity, were shown to have antimetastatic
and anti-inflammatory activity when used in dosages within, the
range of 1.6-6.6 mg per rat daily, administered by constant infusion
(corresponding to 75-308 mg daily for an adult human patient).
Heparin and heparan sulfate are closely related glycosaminoglycan
macromolecules. The degradation products of these polymeric macromolecules,
which are termed low molecular weight heparins (LMWH), may have
the same or greater pharmacological effects on the blood clotting
system as the parent macromolecules. Furthermore, because there
is extensive but incomplete post-synthetic processing of the polymer's
basic disaccharide subunit, glucuronic acid and N-acetyl glucosamine,
the LMWH will be a heterogeneous mixture not only of sizes but also
of chemical compositions (See Goodman and Gilman's The Pharmacological
Basis of Therapeutics, 8th Ed., (Pergamon Press, New York, 1990)
pp. 1313-1315. Methods to obtain low molecular weight products from
heparin, which are useful as anticoagulants, are described in the
art. These methods seek to optimize the persistence in vivo or the
extent of hemorrhagic side effects of their products (See, for example,
Alpinro R. R., et al., U.S. Pat. No. 5,010,063; Choay, J., et al.,
U.S. Pat. No. 4,990,502; Lopez, L. L., et al., U.S. Pat. No. 4,981,955).
Others teach the use of affinity chromatographic methods to obtain
low molecular weight products (See, for example, Rosenberg, R. D.,
et al., U.S. Pat. No. 4,539,398 and Jordan, R. E., et al., U.S.
Pat. No. 4,446,314).
Psuja, P., as reported in Folio Haematol. (Leipz), (1987) 114:429-436,
studied the effect of the heterogeneity of heparins on their interactions
with cell surfaces. Psuja reported that there are moderate affinity
receptors for LMWH found on cultured endothelial cells, but he-determined
that the upper limit of the fraction of LMWH bound to these receptors
was less than 1% of total LMWH.
Other workers have demonstrated effects of LMWH on the metabolism
of a variety of cultured cell types. Asselot-Chapel, C., et al.,
in Biochem. Pharmacol. (1989) 38:895-899 and Biochem. Biophys. Acta,
(1989) 993:240-244, report that LMWH cause cultured smooth muscle
cells to decrease the ratio of type III to type I collagen and fibronectin
synthesis. Rappaport, R. in U.S. Pat. No. 4,889,808, teaches that
LMWH can cause human diploid pulmonary fibroblasts, cultured in
the absence of serum, to respond to LMWH by increased secretion
of tissue plasminogen activator and related proteins.
Effects of LMWH on complex multicellular systems have been reported,
for example in Folkman et al. and Lider et al., in EPO Application
0114589 and J. Clin. Invest. (1989) 83:752:756. In addition, Diferrante,
N., in published International Application WO 90/03791, teaches
the use of LMWH to inhibit the reproduction of HIV in cultures of
C8166 transformed human lymphocytes (ALL). However, none of the
prior art experiments that have studied the effects of LMWH on cellular
metabolism has considered that the heterogeneity of LMWH may produce
antagonistic effects. Furthermore, none has shown or suggested a
regulatory effect on cytokine activity based on the use of substantially
pure oligosaccharide substances.
Cahalon et al. (International Immunology, vol. 9, p, 1517-1522,
1997; see also Lider et al., Proc. Natl. Acad. Sci. USA, vol 92,
p. 5037-5041, 1995) describe the ability of heparin disaccharides
to inhibit tumor necrosis factor alpha production by macrophages.
These disaccharides are also able to stop the immunologically based
inflammation process in rodents. Also, disaccharides derived from
heparin or heparan sulfate were shown to block IL-8 and IL-1.beta.
secretion by intestinal epithelial cells (Chowers et al., Gastroenterology,
vol 120, p. 449-459, 2001) and to modulate chemokine-induced T-cell
adhesion to the extracellular matrix (Hershkoviz et al., Immunology,
vol 99, p. 87-93, 2000).
SUMMARY OF THE INVENTION
The background art does not teach or suggest the use of LMWH, or
substances derived from such compounds, for the treatment of cancer.
In particular, the background art does not teach or suggest the
use of such compounds for prevention of metastasis and/or for the
prevention of induction of cell migration. Furthermore, the background
art does not teach or suggest the use of glucosamine derivatives
for treatment of cancer, and/or prevention of metastasis and/or
for the prevention of induction of cell migration. In particular,
the teachings of the background art with regard to the inhibition
of T-cell adhesion do not teach or suggest any efficacy against
malignancies, or against the migration of malignant cells.
The present invention is of therapeutic compounds for treatment
of cancer, and in particular, of therapeutic compounds which block
the ability of cytokines and chemokines to promote metastasis of
malignant cells. The therapeutic compound comprises a carboxylated
and/or sulfated oligosaccharide, preferably in a substantially purified
form, which is a heparin or heparan-sulfate derived saccharide compound.
In one embodiment of the present invention, the carbohydrate or
oligosaccharide has a molecular weight of no more than about 3000
daltons, preferably lying in the range of about 400 to about 2000
daltons, most preferably between about 400 and about 1100 daltons.
Generally, substances of the present invention which inhibit tumor
cell migration, as determined by biological assays (described more
fully, below), comprise molecules of various sugar units of which
the basic unit of activity is associated with a disaccharide. However,
larger oligosaccharide chains of up to about 10 sugar units, containing
the basic disaccharide unit of activity can also function to inhibit
such activity.
The substances of the present invention may be obtained from natural
sources, including living organisms. For example, active substances
have been isolated and purified from low molecular weight heparin
(LMWH) fractions, as well as extracellular matrices that have been
degraded by the action of an enzyme, e.g., heparanase derived from
animals (mammals) or microorganisms (bacteria). Yet another source
of active substances is enzyme-treated heparin (e.g., endoglycosylase-degraded
heparin).
A preferred class of oligosaccharides is the glucosamine derivatives,
particularly those derivatives which are sulfated. More preferably,
the oligosaccharides are N-sulfated 4-deoxy-4-en-iduronoglucosamine
having at least one other sulfate group, or an N-acetylated 4-deoxy-4-en-iduronoglucosamine
having at least two sulfate groups, as well as pharmaceutically
acceptable salts thereof. Most preferably, the oligosaccharides
are disaccharides of formula (I) or its pharmaceutically acceptable
salt:
##STR00001## in which X.sub.1 is hydrogen or sulfate; X.sub.2 is
hydrogen or sulfate; and X.sub.3 is sulfate or acetyl, provided
that if X.sub.3 is sulfate, then at least one of X.sub.1 or X.sub.2
is sulfate and if X.sub.3 is acetyl, then both X.sub.1 and X.sub.2
are sulfates.
Non-limiting examples of preferred saccharide compounds for use
in the present invention are given in the table below. The reference
"Sigma (number)" refers to the product number for this
compound, which may be ordered from Sigma Chemicals (USA). The reference
"CAS number (number)" refers to the CAS index number for
these compounds, according to which the compound may be obtained
from a chemical supplier. Please note that the structures of these
compounds are given below, in an Appendix.
TABLE-US-00001 List of Heparin and Heparan sulfate Disaccharides
Po912 Sigma H 9392, (.alpha.-.DELTA.UA-2S-[1.fwdarw.4]-GlcNS), produced
by the action of heparinase I and II on heparin, CAS number 136098-03-8.
Po821 A novel DS, Synthetic, (GlcNS,6S-[1.fwdarw.4]-GlcA-2S), produced
by the action of heparanase on heparan sulfate. DS 1020 Sigma H
1020, (.alpha.-.DELTA.UA-[1.fwdarw.4]-GlcNS-6S), produced by the
action of heparinase II on heparin. DS 9267 Sigma H 9267, (.alpha.-.DELTA.UA-2S-[1.fwdarw.4]-GlcNS-6S),
produced by the action of heparinase I and II on heparin, CAS number
136098- 10-7. DS 9517 Sigma H 9517, (.alpha.-.DELTA.UA-2S-[1.fwdarw.4]-GlcNAc-6S),
produced by the action of heparinase II on heparin. DS 8892, Sigma
H 8892, (.alpha.-.DELTA.UA-2S-[1.fwdarw.4]-GlcN-6S), produced by
the action of heparinase on heparin. DS 8767 Sigma H 8767, (.alpha.-.DELTA.UA-2S-[1.fwdarw.4]-GlcNAc),
produced by the action of heparinase II on heparin. DS 0895 Sigma
H 0895, (.alpha.-.DELTA.UA-[1.fwdarw.4]-GlcNAc), produced by the
action of heparinase II and III on heparin. DS 1145 Sigma H 1145,
(.alpha.-.DELTA.UA-[1.fwdarw.4]-GlcNS), produced by the action of
heparinase II and III on heparin. Abbreviation: .DELTA.UA = 4-deoxy-.sub.L-threohex-4-enopyranosyluronic
acid. GlcA = .beta.-D-glucopyranoside uronic acid. GlcN = D-glucosamine.
Ac = Acetyl. NS, 2S, 6S, = N-sulfo, 2-sulfate and 6-sulfate respectively.
Hereinafter, the term "substantially purified form" means
that specific steps have been taken to remove non-active components,
or components that have an opposing effect, from the oligosaccharide
substances and to isolate the active moiety or moieties from mixtures
or supernatants, such as those obtained from enzymatic degradation.
Specifically, the substances claimed in the present invention are
obtained from a rigorous chromatographic process, in which low-pressure
size-exclusion gel chromatography (i.e., chromatography on Sephadex
columns) is but an initial step in the purification scheme. Subsequent
to the low-pressure separation, high-pressure liquid chromatographic
(HPLC) techniques are used to isolate individual component oligosaccharides.
Preferably, these steps have resulted in the purification of the
individual active substances to substantial homogeneity.
Such a preferred purification step may include, for example, passing
mixtures containing the active substance (e.g., fractions obtained
from low pressure gel chromatography) through gel permeation HPLC
or strong anion exchange (SAX) HPLC columns. Thus, substances comprising
oligosaccharides selected from the group consisting of di-, tri-,
tetra-, penta-, or hexasaccharides, preferably disaccharides, have
been observed and isolated. The oligosaccharides of the present
invention are carboxylated and/or sulfated and are, therefore, negatively
charged. Particular embodiments of the invention preferentially
include disaccharides having three negatively charged groups. Those
that exhibit a specific inhibitory activity possess a molecular
weight ranging from about 400 to about 2000, preferably, about 400
to about 1100.
When purified these substances or the compositions that contain
them are substantially free of other substances that exert the opposite
or antagonistic effect. Thus, a substance exhibiting inhibitory
activity ("down" regulation) in a substantially purified
form would be substantially free not only of other substances, in
general, but of other substances that exhibit augmentation or retard
the inhibitory activity of the "down" regulator. The situation
would, of course, be reversed in the case of an augmentative substance
(i.e., "up" regulators), in which the substance would
be substantially free of other substances, particularly those that
"down" regulate or antagonize augmentation.
The phrase "regulatory effect" includes both the up regulation
or down regulation of any process affecting the availability or
resulting activity in vivo or in vitro of cytokines which are generally
functional to promote or otherwise support migration of malignant
cells, including but not limited to, the cytokines IL-1, IL-6, and
TNF-alpha and the chemokines IL-8, SDF-1, IP.sub.--10, MIG, I-TAC
etc. Thus, compositions of the present invention may exert a regulatory
effect on the host production of such a cytokine, on the host secretion
of such a cytokine, on the extracellular availability of such a
cytokine, or on the active forms of such a cytokine in a host. For
instance, but not wishing to be limited by theory, the instant invention
may act to elicit the secretion of a substance, such as a protein,
which may bind to such a cytokine, change its conformation, and,
consequently, affect its biological activity. It is also possible
that the compositions of the present invention may, in penetrating
a malignant cell, bind to particular oligonucleotide sequences and,
thus, affect transcriptional or translational processes that ultimately
alter protein synthesis. The compositions may also work through
binding to cell surface receptors.
To simplify the following discussion, reference will be made, among
others, to the "secretion of active cytokine" or the regulation
of the "activity of a cytokine" with the understanding
that a much broader meaning is to be attached to these phrases which
encompasses the actual mechanism that is responsible for or the
actual manner by which the observed augmentation or inhibition of
the cytokine activity is effected by the substances and compositions
of the present invention.
Hereinafter, the term "biologically active" refers to
molecules, or complexes thereof, which are capable of exerting an
effect in a biological system.
BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES
The invention is herein described, by way of example only, with
reference to the accompanying drawings and tables, wherein:
FIG. 1 shows the inhibition of migration of human T cells by DS
II (9267);
FIG. 2 demonstrates that immobilized SDF-1 stimulates T cell adhesion
to VCAM-1, which in turn is inhibited by DS 9267;
FIG. 3 shows that DS-II (9267) inhibits SDF-1alpha induced Ca.sup.++
mobilization in RBL-2H3 cells;
FIG. 4 shows that co-stimulation of PBLs with SDF-1alpha and DS-9267
results in down-regulation of Pyk-2 and ERK1/2 phosphorylation;
FIG. 5 is a graph showing the effect of treatment of mice with
DS Po912;
FIG. 6 is a graph showing the effect of treatment of mice with
DS Po821;
FIG. 7 is a graph showing the effect of treatment of mice with
DS 1020;
FIG. 8 is a graph showing the effect of treatment of mice with
DS 9267;
FIG. 9 is a graph showing the effect of treatment of mice with
DS 9517;
FIG. 10 is a graph showing the effect of treatment of mice with
DS 8892;
FIG. 11 is a graph showing the effect of treatment of mice with
DS 8767;
FIG. 12 is a graph showing the effect of treatment of mice with
DS 0895;
FIG. 13 is a graph showing the effect of treatment of mice with
DS 1145;
FIG. 14 is a graph showing comparative effects of the treatment
of mice with different compounds at day 49 after injection with
tumor cells; and
FIG. 15 is a graph showing comparative effects of the treatment
of mice with different compounds on the weight of lungs.
Table 1 shows survival of mice at day 49 after injection with the
tumor cells; and
Table 2 shows weights of lungs of the mice at day 50 after injection
with the tumor cells.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is of therapeutic compounds for the treatment
of cancer, and in particular, of therapeutic compounds which block
the ability of cytokines and chemokines to promote metastasis of
malignant cells. The therapeutic compound comprises a carboxylated
and/or sulfated oligosaccharide, preferably in a substantially purified
form, which is a heparin or heparan-sulfate derived saccharide compound.
Hereinafter, the term "heparin or heparan-sulfate derived"
refers to any oligosaccharide obtained from, or otherwise structurally
homologous to, any portion of heparin or heparan-sulfate.
In one embodiment of the present invention, the carbohydrate or
oligosaccharide has a molecular weight of no more than about 3000
daltons, preferably lying in the range of about 400 to about 2000
daltons, most preferably between about 400 and about 1100 daltons.
Generally, substances of the present invention which inhibit tumor
cell migration, as determined by biological assays (described more
fully, below), comprise molecules of various sugar units of which
the basic unit of activity is associated with a disaccharide. However,
larger oligosaccharide chains of up to about 10 sugar units, containing
the basic disaccharide unit of activity can also function to inhibit
such activity.
A preferred class of oligosaccharides is the glucosamine derivatives,
particularly those derivatives which are sulfated. More preferably,
the oligosaccharides are N-sulfated 4-deoxy-4-en-iduronoglucosamine
having at least one other sulfate group, or an N-acetylated 4-deoxy-4-en-iduronoglucosamine
having at least two sulfate groups, as well as pharmaceutically
acceptable salts thereof. Alternatively and more preferably, the
oligosaccharides are N-sulfated or N-acetylated 4-deoxy-4-en-glucuronoglucosamine
or a pharmaceutically acceptable salt thereof. Such compounds, if
N-sulfated, have at least one other sulfate group and, if N-acetylated,
have at least two sulfate groups.
Most preferably, the oligosaccharides are disaccharides of formula
(I) or its pharmaceutically acceptable salt:
##STR00002## in which X.sub.1 is hydrogen or sulfate; X.sub.2 is
hydrogen or sulfate; and X.sub.3 is sulfate or acetyl, provided
that if X.sub.3 is sulfate, then at least one of X.sub.1 or X.sub.2
is sulfate and if X.sub.3 is acetyl, then both X.sub.1 and X.sub.2
are sulfates.
Illustrative but non-limiting examples of such compounds are disclosed
in the previously incorporated patents/applications, which were
incorporated by reference above.
The present invention also discloses methods for treating malignancies.
Hereinafter, the term "treatment" includes both the prevention
of the genesis of the malignancy, as well as the substantial reduction
or elimination of malignant cells and/or symptoms associated with
the development and metastasis of malignancies. Malignancies for
which the therapeutic agents of the present invention are useful
include all metastatic tumors. Examples of tumors for which such
a treatment would be effective include, but are not limited to,
breast cancers such as infiltrating duct carcinoma of the breast
or other metastatic breast cancers, lung cancers such as small cell
lung carcinoma, bone cancers, bladder cancers such as bladder carcinoma,
rhabdomyosarcoma, angiosarcoma, adenocarcinoma of the colon, prostate
or pancreas, or other metastatic prostate or colon cancers, squamous
cell carcinoma of the cervix, ovarian cancer, malignant fibrous
histiocytoma, skin cancers such as malignant melanoma, lymphomas
and leukemia, leiomyosarcoma, astrocytoma, glioma and heptocellular
carcinoma.
Such treatment may optionally and preferably be performed by systemic
administration of the therapeutic compound according to the present
invention. A preferred route of administration is oral, but alternative
routes of administration include, but are not limited to, intranasal,
intraocular, sub-cutaneous and parenteral administration. Such treatment
may be performed topically, for example for skin malignancies, including
but not limited to, metastatic melanoma. Other routes of administration,
and suitable pharmaceutical formulations thereof, are described
in greater detail below.
The following description is divided into sections for ease of
discussion only and without any intention of being limiting. Section
1 describes illustrative and preferred compounds according to the
present invention. Section 2 describes tests performed to demonstrate
the efficacy of these compounds. Section 3 describes exemplary formulations
and methods of use of these compounds for treatment of malignancies.
Section 1: Illustrative Saccharide Compounds
The present invention encompasses a number of preferred saccharide
compounds. The therapeutic compound comprises a carboxylated and/or
sulfated oligosaccharide, preferably in a substantially purified
form, which is a heparin or heparan-sulfate derived saccharide compound.
In one embodiment of the present invention, the carbohydrate or
oligosaccharide has a molecular weight of no more than about 3000
daltons, preferably lying in the range of about 400 to about 2000
daltons, most preferably between about 400 and about 1100 daltons.
Generally, substances of the present invention which inhibit tumor
cell migration, as determined by biological assays (described more
fully, below), comprise molecules of various sugar units of which
the basic unit of activity is associated with a disaccharide. However,
larger oligosaccharide chains of up to about 10 sugar units, containing
the basic disaccharide unit of activity can also function to inhibit
such activity.
The substances of the present invention may be obtained from natural
sources, including living organisms. For example, active substances
have been isolated and purified from low molecular weight heparin
(LMWH) fractions, as well as extracellular matrices that have been
degraded by the action of an enzyme, e.g., heparanase derived from
animals (mammals) or microorganisms (bacteria). Yet another source
of active substances is enzyme-treated heparin (e.g., endoglycosylase-degraded
heparin).
Non-limiting examples of tested saccharide compounds are given
in the table below. The reference "Sigma (number)" refers
to the product number for this compound, which may be ordered from
Sigma Chemicals (USA). The reference "CAS number (number)"
refers to the CAS index number for these compounds, according to
which the compound may be obtained from a chemical supplier. Please
note that the structures of these compounds are given at the end,
in an Appendix.
TABLE-US-00002 List of Heparin and Heparan sulfate Disaccharides
Po912 Sigma H 9392, (.alpha.-.DELTA.UA-2S-[1.fwdarw.4]-GlcNS), produced
by the action of heparinase I and II on heparin, CAS number 136098-03-8.
Po821 A novel DS, Synthetic, (GlcNS,6S-[1.fwdarw.4]-GlcA-2S), produced
by the action of heparanase on heparan sulfate. DS 1020 Sigma H
1020, (.alpha.-.DELTA.UA-[1.fwdarw.4]-GlcNS-6S), produced by the
action of heparinase II on heparin. DS 9267 Sigma H 9267, (.alpha.-.DELTA.UA-2S-[1.fwdarw.4]-GlcNS-6S),
produced by the action of heparinase I and II on heparin, CAS number
136098- 10-7. DS 9517 Sigma H 9517, (.alpha.-.DELTA.UA-2S-[1.fwdarw.4]-GlcNAc-6S),
produced by the action of heparinase II on heparin. DS 8892, Sigma
H 8892, (.alpha.-.DELTA.UA-2S-[1.fwdarw.4]-GlcN-6S), produced by
the action of heparinase on heparin. DS 8767 Sigma H 8767, (.alpha.-.DELTA.UA-2S-[1.fwdarw.4]-GlcNAc),
produced by the action of heparinase II on heparin. DS 0895 Sigma
H 0895, (.alpha.-.DELTA.UA-[1.fwdarw.4]-GlcNAc), produced by the
action of heparinase II and III on heparin. DS 1145 Sigma H 1145,
(.alpha.-.DELTA.UA-[1.fwdarw.4]-GlcNS), produced by the action of
heparinase II and III on heparin. Abbreviation: .DELTA.UA = 4-deoxy-.sub.L-threohex-4-enopyranosyluronic
acid. GlcA = .beta.-D-glucopyranoside uronic acid. GlcN = D-glucosamine.
Ac = Acetyl. NS, 2S, 6S, N-sulfo, 2-sulfate and 6-sulfate respectively.
EXAMPLE 1
Testing of Compounds In vitro
Compounds according to the present invention, as described in the
table given above, were tested in vitro for their ability to block
the migration of cells in response to the chemokine SDF-1. The experiments
are described in greater detail below.
Materials and Methods
Human T cells were pretreated with DS II (9267) or DS-B (control
DS 8767) (100 ng/ml, 30 min), both obtained from Sigma-Aldrich Chemicals
(USA). The cells were then placed on FN-coated membranes in the
upper wells of a chemotaxis chamber that contained SDF-1alpha (250
ng/ml) in the lower compartment. Migrating cells were collected
from the lower wells after 1.5 hr (see FIG. 1 for results).
The effect of immobilized SDF-1 or inactive SDF-1 (co-coated at
2.mu.g/ml with sVCAM-1 as explained in Materials and Methods) on
the accumulation of T cells before and after treatment with DS 9267
was examined (see FIG. 2 for results).
The effect of disaccharide compounds according to the present invention
on SDF-1alpha induced Ca.sup.++ mobilization in RBL-2H3 cells was
examined. Flue-3 labeledRBL-2H3 cells were treated with SDF-1.alpha.
in the presence or absence of DS-II pretreatment, and Ca.sup.2+
influx was measured (see FIG. 3).
Serum-starved PBLs were stimulated with either SDF-1.alpha. alone
(250 ng/ml) or together with DS-9267 (100 ng/ml). Cell lysates were
then resolved on SDS-PAGE gels, transferred to a nitrocellulose
membrane and blotted with antibodies raised aginst ERK1/2, phospho-ERK1/2,
Pyk-2 and phospho Pyk-2. Densitometric analysis is presented (see
FIG. 4).
Chemokine and chemotaxis assays were performed as follows. Chemotaxis
experiments were assayed by using Costar (Cambridge, Mass., 6.5
mm/diameter, 5 .mu.m/pore) transwell plates. 100 .mu.l chemotaxis
buffer (RPMI 1640, 1% FCS) containing 2.times.10.sup.5 T cells.sup.+
were added to the upper chamber, and 0.6 ml of chemotaxis buffer
with or without SDF-1alpha was added to the bottom chamber. After
4 hours, migrating (bottom chamber) and non migrating (upper chamber)
cells were counted for 30 seconds using a FACSort (B.D).
Laminar flow adhesion assays were performed as follows. Soluble
VCAM-1 adhesion molecule was diluted at indicated concentrations
in coating medium (PBS buffered with 20 mM bicarbonate pH 8.5) and
adsorbed as 20 microliter spots on polystyrene plates (a polystyrene
60.times.15 mm petri dish, Becton Dickinson, Lincoln Park, N.J.)
either for 2 hr at 37.degree. C. sVCAM-1 was coated at 1-10 microgram/ml
in the presence of 2 microgram/ml HSA carrier. The plates were then
washed three times with PBS and blocked with HSA (20 mg/ml in PBS)
for 2 hrs at room temperature. To co-coat the adhesive spots with
SDF-1, washed plates were coated with 10 microgram/ml SDF-1 in PBS
for 30 min at room temperature, before being blocked with HSA. The
accumulation of T cells before and after treatment with DS 9267
was examined (see FIG. 2 for results).
Results
Disaccharide (DS) molecules were found to inhibit T cell migration
in response to the chemokine SDF-1.alpha. (FIG. 1). Furthermore,
these molecules were able to block the chemokine-mediated interaction
of cells with endothelial ligands such as VCAM-1 (FIG. 2). The same
molecules inhibit SDF-1alpha induced Ca.sup.++ mobilization in the
mast cell leukemia RBL-2H3 cells (FIG. 3).
Without wishing to be limited to a single hypothesis, these results
support the possibility that disaccharides, such as those which
may optionally be produced by the enzymatic degradation of HS or
heparin, exert their activity by interfering with the CXCR4 7TM-GPCR
signaling pathway. This hypothesis was therefore examined by studying
the phosphorylation and activation of Pyk-2 and ERK1/2. Co-stimulation
of PBLs with SDF-1alpha and DS results in down-regulation of Pyk-2
and ERK1/2 phosphorylation (FIG. 4). Ca.sup.++ mobilization and
phosphorylation of Pyk-2 and ERK1/2 are key regulatory events that
regulate cell migration and proliferation of tumor cells. Thus,
these additional results further support the possibility that disaccharides
according to the present invention exert their anti-migratory effect
at least partially by interfering with the CXCR4 7TM-GPCR signaling
pathway.
Regardless of the exact pathway or pathways through which the effect
is exerted, the above results demonstrate that the saccharide compounds
of the present invention are able to block migration of cells.
EXAMPLE 2
Testing of Compounds In vivo
Compounds according to the present invention, as described in the
table given above, were tested in vivo. Briefly, mice were inoculated
with tumor cells obtained from lung carcinoma. The mice were then
treated with the compounds according to the present invention. Treatment
with the different disaccharides, except for DS8892, resulted in
a significant increase in the survival rate of the mice. Treatment
with DS Po912 and DS 1145 appeared to provide the best treatment,
at least in terms of inhibition of mortality.
Materials and Methods
Disaccharide Compounds
The disaccharide compounds were obtained from Sigma-Aldrich Chemicals
(USA), with the exception of Po821, which was prepared by the inventors
as previously described, in the patents/applications which were
incorporated by reference (see for example U.S. Pat. Nos. 5,861,382
and 6,020,323; all of which were previously incorporated by reference).
Chemokine and chemotaxis assays were performed as follows. Chemotaxis
experiments were assayed by using Costar (Cambridge, Mass., 6.5
mm/diameter, 5 .mu.m/pore) transwell plates. 100 .mu.l chemotaxis
buffer (RPMI 1640, 1% FCS) containing 2.times.10.sup.5 T cells.sup.+
were added to the upper chamber, and 0.6 ml of chemotaxis buffer
with or without SDF-1 was added to the bottom chamber. After 4 hours,
migrating (bottom chamber) and non migrating (upper chamber) cells
were counted for 30 seconds using a FACSort (B.D).
Experimental Animals
C57BL/6, 12 months old male mice were obtained from the Jackson
Laboratory.
Cells
Syngeneic, 3LL lewis lung carcinoma, variant D122, was obtained
from the Weizmann Institute of Science. This tumor line develops
metastases in the lungs after intravenous (iv) inoculation.
Cells were cultured in DMEM medium containing high glucose and
supplemented with 10% FCS, glutamine and penicillin.
Tumor Cell Inoculation
Cells were incubated 24 hr before inoculation into the mice with
the different disaccharides (300 ng/ml) in culture medium. Then,
the cells were washed and injected i. v., (500,000 cells per mouse)
in 100 .mu.l PBS containing 30 ng of the corresponding disaccharide.
The number of dead mice was counted each day after the inoculation
was performed. The mice were sacrificed at day 50 and the weights
of their lung were determined.
Disaccharide Treatment
Each group, injected by one of the disaccharides, contained 16
mice. Control mice received PBS alone. The different disaccharides,
were injected to the mice subcutaneously (30 ng per mouse in 0.1
ml of PBS), one day before and one and two days after cell inoculation.
Results
Treatment with the different disaccharides, except for DS8892 (FIG.
10), resulted in a significant increase in the survival of the mice.
Treatment with DS Po912 (FIG. 5) and DS 1145 (FIG. 13) appears to
be the best treatment in terms of inhibition of mortality. DS Po912
increased significantly the mice survival to 68.75% from 6.2% (Table
1 and FIG. 14) and DS 1145 increased it to 62.6% (Table 1). DS 1020
(FIG. 7) and DS 8767 (FIG. 11) increased significantly the mice
survival to 50% from 6.2% (Table 1 and FIG. 14). DS Po821 (FIG.
6), DS 9267 (FIG. 48), DS 9517 (FIG. 9) and DS 0895 (FIG. 12) significantly
increased survival of the mice to 43.75% (Table 1 and FIG. 14).
In addition to measuring survival, some mice were sacrificed on
day 50 and their lungs were examined and weighted. Increase in lung
weight is a sign for tumor development; the heavier the lungs, the
greater the amount of metastasis. The results, shown in Table 2,
shows a substantially decrease in lung weight in mice receiving
the treatment of DS Po821, DS 1020 and DS Po912.
Discussion
The best anti-tumor treatment was seen with DS Po912 as well as
all the other tested DS, although to a lesser degree. In contrast,
DS 8892 did not inhibit the tumor development at all. Without wishing
to be limited to a single hypothesis, since the DS treatment was
at the very early stage of development of the experimental metastasis,
the DS may modulate the homing and tissue localization of tumor
cells.
EXAMPLE 3
Methods and Compositions for Administration
The compounds according to the present invention, and their pharmaceutically
acceptable salts, hereinafter referred to as the "therapeutic
agents of the present invention", can be administered to a
subject by various ways, which are well known in the art.
Hereinafter, the term "subject" refers to the human or
lower animal to whom the therapeutic agent is administered. For
example, administration may be done topically (including opthalmically,
vaginally, rectally, intranasally and by inhalation), orally, or
parenterally, for example by intravenous drip or intraperitoneal,
subcutaneous, or intramuscular injection.
Formulations for topical administration may include but are not
limited to lotions, ointments, gels, creams, suppositories, drops,
liquids, sprays and powders. Conventional pharmaceutical carriers,
aqueous, powder or oily bases, thickeners and the like may be necessary
or desirable.
Compositions for oral administration include powders or granules,
suspensions or solutions in water or non-aqueous media, sachets,
capsules or tablets. Thickeners, diluents, flavorings, dispersing
aids, emulsifiers or binders may be desirable.
Formulations for parenteral administration may include but are
not limited to sterile aqueous solutions which may also contain
buffers, diluents and other suitable additives.
Dosing is dependent on the severity of the symptoms and on the
responsiveness of the subject to the therapeutic agent. Persons
of ordinary skill in the art can easily determine optimum dosages,
dosing methodologies and repetition rates.
Optionally, the therapeutic agent of the present invention is administered
in an amount in a range of from about 1 to about 1000 .mu.g of the
agent per Kg of subject, weight per weight.
The following example is an illustration only of a method of treating
a malignancy with the therapeutic agent of the present invention,
and is not intended to be limiting.
The method includes the step of administering a therapeutic agent,
in a pharmaceutically acceptable carrier, to a subject to be treated.
The therapeutic agent is administered according to an effective
dosing methodology, preferably until a predefined endpoint is reached,
such as the absence of a symptom of the malignancy in the subject,
and/or the prevention of the appearance of such a symptom in the
subject, and/or the reduction of the number of metastatic malignant
cells in the subject, and/or the prevention of the genesis of metastatic
tumors.
Examples of tumors for which such a treatment would be effective
include, but are not limited to, breast cancers such as infiltrating
duct carcinoma of the breast or other metastatic breast cancers,
lung cancers such as small cell lung carcinoma, bone cancers, bladder
cancers such as bladder carcinoma, rhabdomyosarcoma, angiosarcoma,
adenocarcinoma of the colon, prostate or pancreas, or other metastatic
prostate or colon cancers, squamous cell carcinoma of the cervix,
ovarian cancer, malignant fibrous histiocytoma, lymphoma and leukemia,
skin cancers such as malignant melanoma, leiomyosarcoma, astrocytoma,
glioma and heptocellular carcinoma.
It will be appreciated that the above descriptions are intended
only to serve as examples, and that many other embodiments are possible
within the spirit and the scope of the present invention.
Tables
TABLE-US-00003 TABLE 1 Survival at day 49 after 3LL injection DS
live mice dead mice survival (%) * p-value DS Po912 11 5 68.75 0.0003
DS Po821 7 9 43.75 0.014 DS 1020 8 8 50 0.005 DS 9267 7 9 43.75
0.014 DS 9517 7 9 43.75 0.014 DS 8892 4 12 25 0.14 DS 8767 8 8 50
0.005 DS 0895 7 9 43.75 0.014 DS 1145 10 6 62.5 0.0008 PBS (control)
1 15 6.2 * survival (%) = [live mice/total mice] .times. 100
TABLE-US-00004 TABLE 2 Weights of lungs at day 50 after 3LL injection
Treatment with DS Weights .+-. SD (gr.) Decrease (%) * DS Po912
0.37 .+-. 0.17 60.1 DS Po821 0.28 .+-. 0.07 69.3 DS 1020 0.28 .+-.
0.13 69.8 DS 9267 0.51 .+-. 0.07 44.8 DS 9517 0.43 .+-. 0.13 53.0
DS 8892 0.66 .+-. 0.18 28.8 DS 8767 0.44 .+-. 0.08 52.2 DS 0895
0.49 .+-. 0.14 46.7 DS 1145 0.59 .+-. 0.18 36.4 PBS 0.92 * Decrease
(%) = [1-(weights of treated group/weights of control group)] .times.
100
Appendix of Structures
TABLE-US-00005 List of Heparin and Heparan sulfate Disaccharides
Po912Sigma H 9392,(.alpha.-.DELTA.UA-2S-[1.fwdarw.4]-GlcNS), produced
by theaction of heparinase Iand II on heparin. ##STR00003## Po821Our
novel DS, Synthetic,(GlcNS,6S-[1.fwdarw.4]-GlcA-2S), produced bythe
action of heparanaseon heparan sulfate. ##STR00004## DS 1020Sigma
H 1020,(.alpha.-.DELTA.UA-[1.fwdarw.4]-GlcNS-6S), produced bythe
action of heparinaseII on heparin. ##STR00005## DS 9267Sigma H 9267,(.alpha.-.DELTA.UA-2S-[1.fwdarw.4]-GlcNS-6S),
produced bythe action of heparinase Iand II on heparin. ##STR00006##
DS 9517Sigma H 9517,(.alpha.-.DELTA.UA-2S-[1.fwdarw.4]-GlcNAc-6S),
producedby the action ofheparinase II on heparin. ##STR00007## DS
8892,Sigma H 8892,(.alpha.-.DELTA.UA-2S-[1.fwdarw.4]-GlcN-6S), produced
bythe action of heparinaseon heparin. ##STR00008## DS 8767Sigma
H 8767,(.alpha.-.DELTA.UA-2S-[1.fwdarw.4]-GlcNAc), produced bythe
action of heparinaseII on heparin. ##STR00009## DS 0895Sigma H 0895,(.alpha.-.DELTA.UA-[1.fwdarw.4]-GlcNAc),
produced bythe action of heparinaseII and III on heparin. ##STR00010##
DS 1145Sigma H 1145,(.alpha.-.DELTA.UA-[1.fwdarw.4]-GlcNS), produced
by theaction of heparinase IIand III on heparin. ##STR00011## Abbreviation:
.DELTA.UA = 4-deoxy-.sub.L-threohex-4-enopyranosyluronic acid. GlcA
= .beta.-D-glucopyranoside uronic acid. GlcN = D-glucosamine. Ac
= Acetyl. NS, 2S, 6S, = N-sulfo, 2-sulfate and 6-sulfate respectively.
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