U.S. patent application number 13/139170 was filed with the patent office on 2011-10-06 for glucose-peg conjugates for reducing glucose transport into a cell.
This patent application is currently assigned to Agency for Science, Technology and Research. Invention is credited to Nandanan Erathodiyil, Karthikeyan Narayanan, Andrew Chwee Aun Wan, Jackie Y. Ying.
Application Number | 20110243851 13/139170 |
Document ID | / |
Family ID | 42242962 |
Filed Date | 2011-10-06 |
United States Patent
Application |
20110243851 |
Kind Code |
A1 |
Narayanan; Karthikeyan ; et
al. |
October 6, 2011 |
GLUCOSE-PEG CONJUGATES FOR REDUCING GLUCOSE TRANSPORT INTO A
CELL
Abstract
There is provided a glucose-PEG conjugate comprising a PEG
moiety conjugated to a linear glucose moiety at the C1 position of
the glucose moiety. The glucose-PEG conjugate may be used to reduce
glucose transport into a cell and may be used to treat a
proliferative disorder.
Inventors: |
Narayanan; Karthikeyan;
(Singapore, SG) ; Wan; Andrew Chwee Aun;
(Singapore, SG) ; Ying; Jackie Y.; (Singapore,
SG) ; Erathodiyil; Nandanan; (Singapore, SG) |
Assignee: |
Agency for Science, Technology and
Research
Connexis
SG
|
Family ID: |
42242962 |
Appl. No.: |
13/139170 |
Filed: |
December 11, 2009 |
PCT Filed: |
December 11, 2009 |
PCT NO: |
PCT/SG2009/000477 |
371 Date: |
June 10, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61193640 |
Dec 11, 2008 |
|
|
|
Current U.S.
Class: |
424/9.1 ; 435/29;
435/375; 514/23; 536/53 |
Current CPC
Class: |
C07C 235/08 20130101;
A61K 31/16 20130101; A61K 31/69 20130101; A61K 31/08 20130101; A61P
35/00 20180101; G01N 33/5091 20130101; C08G 65/00 20130101; C07H
15/08 20130101 |
Class at
Publication: |
424/9.1 ; 536/53;
435/375; 435/29; 514/23 |
International
Class: |
A61K 49/00 20060101
A61K049/00; C07H 15/12 20060101 C07H015/12; C07H 15/26 20060101
C07H015/26; C12N 5/071 20100101 C12N005/071; C12N 5/09 20100101
C12N005/09; C12Q 1/02 20060101 C12Q001/02; A61K 31/7008 20060101
A61K031/7008; A61P 35/00 20060101 A61P035/00 |
Claims
1. A glucose-PEG conjugate comprising ##STR00001## wherein each n
is independently from 2 to 500.
2. The glucose-PEG conjugate of claim 1 further comprising a
detectable label.
3. The glucose-PEG conjugate of claim 2 wherein the detectable
label is a PET label, an SPECT label, an MRI label, a quantum dot
label, a coloured label, a fluorescent label, a radiolabel or a
label that may be detected by an antibody or antibody fragment.
4. The glucose-PEG conjugate of claim 2 comprising ##STR00002##
5. A method of reducing glucose transport into a cell comprising
contacting the cell with a glucose-PEG conjugate, the glucose-PEG
conjugate comprising a PEG moiety conjugated to a linear glucose
moiety at the C1 position of the glucose moiety.
6. The method of claim 5 wherein the PEG moiety comprises linear,
branched, dendritic, hyperbranched, star or comb PEG.
7. The method of claim 5 wherein the PEG moiety is conjugated to
the C1 position of the glucose moiety via an amine linkage.
8. The method of claim 5 wherein the PEG moiety is terminated at
one or both ends with an end group.
9. The method of claim 5 wherein the glucose-PEG conjugate further
comprises a linker moiety connecting the PEG moiety to the glucose
moiety.
10. The method of claim 5 wherein the glucose-PEG conjugate is the
glucose-PEG conjugate of claim 1.
11. The method of claim 5 wherein the glucose-PEG conjugate further
comprises a detectable label.
12. The method of claim 11 wherein the detectable label is a PET
label, an SPECT label, an MRI label, a quantum dot label, a
coloured label, a fluorescent label, a radiolabel or a label that
may be detected by an antibody or antibody fragment.
13. The method of claim 10 wherein the glucose-PEG conjugate
comprises ##STR00003##
14. The method of claim 5 wherein the cell is a hyper-proliferative
cell.
15. A method of imaging a hyper-proliferative cell comprising
contacting a hyper-proliferative cell with a glucose-PEG conjugate
of claim 2; and detecting the detectable label.
16. The method of claim 15 wherein detecting involves fluorescence
microscopy, positron emission tomography imaging, single photon
emission computed tomography imaging or magnetic resonance
imaging.
17-18. (canceled)
19. The method of claim 15 wherein the cell is associated with a
proliferative disorder.
20. The method of claim 19 wherein contacting comprises
administering an effective amount of the glucose-PEG conjugate at
the site of a hyper-proliferating cell in a subject.
21. The method of any one of claims 14 to 20 further comprising
contacting the cell with a chemotherapeutic agent.
22. A pharmaceutical composition comprising a glucose-PEG conjugate
of claim 1.
23-36. (canceled)
37. The method of claim 5 wherein the cell is associated with a
proliferative disorder.
38. The method of claim 37 wherein contacting comprises
administering an effective amount of the glucose-PEG conjugate at
the site of a hyper-proliferating cell in a subject.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims benefit of, and priority from, U.S.
provisional patent application No. 61/193,640, filed on Dec. 11,
2008, the entire contents of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to glucose-polyethylene glycol
conjugates and the use of such conjugates to reduce glucose
transport into a cell.
BACKGROUND OF THE INVENTION
[0003] One of the key characteristics of cancer cells is the
increased rate of proliferation. Often the rate of proliferation
exceeds de novo vascular formation. As a result, tumor cells may
out-grow the available blood supply, and shortage of blood leads to
hypoxia (low oxygen level). To overcome hypoxia, tumor cells tend
to rely on glycolysis for ATP production (17). High glycolysis rate
is characteristic of solid tumors, and is associated with an
over-expression of glucose transporters (GLUTs) and glycolytic
enzymes.
[0004] Thus, a high rate of glucose uptake and increased glucose
metabolism are involved in maintaining proliferation of tumor cells
(18). This phenomenon is commonly known as Warburg effect (19). It
was observed that the initial high level of anaerobic glycolysis
resulted in the accumulation of lactate (20). The enzyme (lactate
dehydrogenase) responsible for the conversion of pyruvate to
lactate was also observed to be elevated in the cancer cells (20).
However, lactate dehydrogenase also helps in the survival of the
cells under hypoxic conditions by anaerobic glycolysis (20).
Increased glycolytic enzyme activities have been reported in
cancerous cells (21, 22), and have been observed with the
progression of cancer from primary breast tumor to the metastatic
stage (23, 24).
[0005] Transport of glucose across the membrane of cells is
facilitated by proteins called glucose transporters (GLUTs). 13
GLUTs have been identified to date, and have been categorized into
three classes. Class I includes GLUT1 to GLUT4, class II includes
GLUT5, GLUT7, GLUT9 and GLUT11, and class III includes GLUT6,
GLUT8, GLUT10, GLUT12 and GLUT13. The different transporters have
different kinetics and affinities towards glucose and other
hexoses. The expression level of the different GLUTS in various
tissues varies depending on the metabolic consumption of glucose by
the particular tissue type. GLUT1 is a ubiquitously expressed GLUT
and GLUT1 and GLUT3 expression levels have been found to be much
higher in cancerous cells than in normal cells (1). This
overexpression has been observed in a wide variety of different
cancer cell types (2-14). Extensive studies with breast cancer
patients indicated increased GLUT 1 activity among the patients
(15, 16).
[0006] Metabolic targeted cancer therapy is a relatively new field
in cancer therapeutic research and is designed to take advantage of
the inherent hyper-metabolic characteristics of cancer cells.
[0007] Certain previous studies focused on developing therapeutic
drugs based on the metabolism of cancer cells. Glufosfamide is a
small molecule generated by the conjugation of ifosfamide and
glucose. This compound enters the cancer cells through the GLUT
proteins. It breaks down in the cell, leading to the release of the
toxin (ifosfamide) inside the cell (25-28).
[0008] Other groups have used different glucose analogs in an
attempt to reduce the glucose metabolism in cancer cells (29, 30).
Of the various compounds, 2-deoxy-glucose (2-DG) has been shown to
be a promising analog. 2-DG is an orally administered glucose
analog that inhibits the glycolysis pathway of ATP production in
cancer cells. 2-DG accumulates in the cancer cells because the
phosphorylated 2-DG cannot be processed by glycolytic enzymes (31,
32). 2-DG can elicit 50% apoptotic cells at a concentration of 4 mM
in the SkBr3 (human breast cancer) cell line (33). In vivo animal
studies with 2-DG have shown that the growth of the tumor was
inhibited with supplemental 2-DG (31, 34).
[0009] Noguchi et al. have used anti-sense against GLUT-1 to
suppress tumor growth in MKN45 (gastric cancer) cell line.
Comparison of tumor development in nude mice demonstrated that the
cells expressing anti-sense GLUT-1 develop tumor much more slowly
than the wild-type cells (35).
[0010] In a more recent study, monoclonal antibody against GLUT-1
was shown to induce growth arrest and apoptosis in breast cancer
and lung cancer cell lines. 50% and 75% reductions in cell growth
were found in lung cancer and breast cancer cell lines,
respectively (36).
SUMMARY OF THE INVENTION
[0011] The present invention relates to novel glucose-polyethylene
glycol (glucose-PEG) conjugates and their use to reduce glucose
transport into a cell. Thus, the glucose-PEG conjugates may be
useful to reduce cellular proliferation, particularly in respective
of proliferative disorders, such as cancer. The glucose-PEG
conjugates of the present invention may also be labelled with a
detectable label, and thus may be useful for imaging of
hyper-proliferative cells such as cancer cells.
[0012] The present invention takes advantage of the overexpression
of GLUTs in hyper-proliferative cells in order to control cellular
proliferation, potentially leading to the death of the
hyper-proliferative cells. The glucose-PEG conjugate may be used to
target and induce apoptosis in hyper-proliferating cells, such as
cancer cells, as a result of reduced glucose transport into the
cells. Cancer cells often thrive on glycolytic enzymes that break
down glucose into ATP in an anaerobic process. Glucose uptake by
the GLUTs has been shown to be high in many cancerous cells and
tissues. The glucose-PEG conjugates of the present invention are
able to bind to the GLUTs, thus taking advantage of the GLUT
overexpression in hyper-proliferative cells such as cancer cells.
The glucose-PEG conjugates are not transported into the cells and
thus binding of the glucose-PEG conjugates to a GLUT reduces the
availability of GLUTs for transporting glucose into the cell,
potentially triggering apoptosis.
[0013] Thus, the glucose-PEG conjugates of the present invention
may be useful to control proliferation of hyper-proliferative cells
such as cancer cells, including tumor cells within a tumor core
where blood vascularization may be limited. Direct injection into
the tumor core may reduce proliferation of the tumor while having
minimal effect on surrounding healthy tissue.
[0014] In one aspect, the invention provides a glucose-PEG
conjugate comprising a PEG moiety conjugated to a linear glucose
moiety at the C1 position of the glucose moiety.
[0015] Conjugation of the PEG moiety to the C1 position of the
glucose moiety may occur via an amine linkage.
[0016] The PEG moiety may comprise a linear, branched, dendritic,
hyperbranched, star or comb PEG, and may be terminated at one or
both ends with an end group.
[0017] The glucose-PEG conjugate may further comprise a linker
moiety connecting the PEG moiety to the glucose moiety.
[0018] The glucose-PEG conjugate may also further comprise a
detectable label. The detectable label may be a PET label, an SPECT
label, an MRI label, a quantum dot label, a coloured label, a
fluorescent label, a radiolabel or a label that may be detected by
an antibody or antibody fragment.
[0019] In another aspect, the invention provides a method of
reducing glucose transport into a cell comprising contacting the
cell with a glucose-PEG conjugate as described herein. The cell may
be a hyper-proliferative cell.
[0020] In another aspect, the invention provides a method of
imaging a hyper-proliferative cell comprising contacting a
hyper-proliferative cell with a glucose-PEG conjugate as described
herein comprising a detectable label; and detecting the detectable
label.
[0021] The detecting may involve fluorescence microscopy, positron
emission tomography imaging, single photon emission computed
tomography imaging or magnetic resonance imaging.
[0022] In the above described methods, the cell may be an in vitro
cell. Alternatively, the cell may be an in vivo cell, including a
cell that is associated with a proliferative disorder. Thus,
contacting may include administering an effective amount of the
glucose-PEG conjugate at the site of a hyper-proliferating cell in
a subject.
[0023] The above described methods may further comprise contacting
the cell with a chemotherapeutic agent.
[0024] In another aspect, the invention provides a pharmaceutical
composition comprising a glucose-PEG conjugate as described
herein.
[0025] In another aspect, the invention provides use of a
glucose-PEG conjugate as described herein, including use in the
preparation of a medicament, for reducing glucose transport into a
cell.
[0026] In another aspect, the invention provides use of a
glucose-PEG conjugate as described herein, including use in the
preparation of a medicament, for treating a proliferative disorder
in a subject.
[0027] In another aspect, the invention provides use of a
glucose-PEG conjugate as described herein comprising a detectable
label, including use in the preparation of a composition, for
imaging a hyper-proliferative cell.
[0028] Other aspects and features of the present invention will
become apparent to those of ordinary skill in the art upon review
of the following description of specific embodiments of the
invention in conjunction with the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The figures illustrate, by way of example only, embodiments
of the present invention, are as described below.
[0030] FIG. 1: Structure of an exemplary glucose-PEG-BODIPY. The
PEG moiety is depicted as only two repeating ethyloxo units.
However, the PEG moiety may be larger.
[0031] FIG. 2: Structure of an exemplary glucose-PEG-OH,
synthesized by conjugating glucose with an amino-terminated PEG and
then reaction with caprolactone. As with FIG. 1, the PEG moiety may
be larger than depicted.
[0032] FIG. 3: Structure of an exemplary glucose-branched PEG
synthesized by conjugating glucose with an amino-terminated
branched PEG and then reaction with caprolactone. Each n is
independently greater than or equal to 1.
[0033] FIG. 4: Synthetic scheme for synthesis of
glucose-PEG-BODIPY.
[0034] FIG. 5: Fluorescence image of MCF-7 cells labeled with GPB.
MCF-7 cells grown overnight were incubated with 500 .mu.M of GPB in
the growth medium for 30 min. They were washed with PBS before
fluorescence imaging. Scale bar=500 .mu.m.
[0035] FIG. 6: Competition assay. Competition binding assay was
performed in the presence of different concentrations of glucose
added to the medium containing GPB (200 .mu.M). The cells were
incubated for 30 min at 37.degree. C. The cells were washed with
PBS, and the total intensity was measured using a plate reader. The
total intensity for the control cells with GPB and without glucose
was taken as 100%.
[0036] FIG. 7: Dose-dependent cell death in MCF-7 cells. MCF-7
cells were seeded onto 96-well plates at least 24 h prior to the
experiment. Different concentrations of GPB were added and further
cultured for 7 days. The medium was changed everyday along with the
specified GPB concentration. MTT assay was performed to assess the
cell viability. Control cells received no GPB treatment. Cell
viability was normalized with that of the control cells.
[0037] FIG. 8: Effect of PEG on MCF-7 cells. The MCF-7 cells were
treated with 200 .mu.M and 400 .mu.M of PEG, and 200 .mu.M of GPB.
The cells were treated as described in FIG. 3, followed by the MTT
assay.
[0038] FIG. 9: Effect of GPP on MCF-7 cells. The MCF-7 cells were
cultured on a 96-well plate. 200 .mu.M and 400 .mu.M of GPP were
added to the cells, and the cell viability was analyzed after 7
days of culture. The medium was changed everyday with the specified
GPP concentration. Value for the control cells without GPP was
taken as 100% viable.
[0039] FIG. 10: Binding of GBP to GLUT1. Human breast cancer cells
(MCF-7) were incubated with 200 .mu.M of GPB in the presence of the
increasing concentrations of un-modified glucose. After 30 minutes
the fluorescence intensity was measured using a plate reader.
[0040] FIG. 11: Gene expression in lung cancer cells treated with
GBrP. Human lung cancer cells (H1299) were treated for 3 days with
200 .mu.M of modified glucose (GBrP). Control cells were prepared
without treatment. Total RNA was extracted from the cells and
reverse transcribed. A PCR array containing primers to identify the
apoptotic pathway was used to identify the apoptosis pathway
between control and treated cells in lung cancer cells.
[0041] FIG. 12: Gene expression in prostate cancer cells treated
with GBrP. Human prostrate cancer cells (DU145) were treated for 3
days with 200 .mu.M of modified glucose (GBrP). Control cells were
prepared without treatment. Total RNA was extracted from the cells
and reverse transcribed. A PCR array containing primers to identify
the apoptotic pathway was used to identify the apoptosis pathway
between control and treated cells in prostate cancer cells.
[0042] Table 1: Effect of GBrP and branched PEG (BrP) was tested on
different cancer cell lines along with normal breast epithelial
cells (MCF-10A). Cells were treated as described in FIG. 3 with 200
.mu.M of either BrP or GBrP. MTT assay was carried out to monitor
the viability of the cells. Values obtained for the cells without
any treatments were kept as 100%.
DETAILED DESCRIPTION
[0043] Thus, there is provided a glucose-PEG conjugate, having the
PEG moiety conjugated to a linear glucose moiety at the C1 position
of the glucose.
[0044] The glucose moiety of the glucose-PEG conjugate is a linear
glucose, meaning that the glucose is in an open form and has not
cyclised. Cyclic glucose is the form of glucose typically found in
biological systems, in which the C1 to C5 carbons, together with an
oxygen atom, form a six-membered ring. In contrast, in the
glucose-PEG conjugate, the glucose is linear, leaving the C1
position available for conjugation to the PEG moiety. The linear
glucose is still able to bind to the GLUTs, but the linear form of
glucose is not typically found in biological systems, and does not
tend to enter glycolytic metabolic pathways.
[0045] The PEG moiety may be any PEG moiety. As will be
appreciated, polyethylene glycol or poly(ethylene glycol) refers to
a polymer made up of monomers of ethylene glycol condensed to form
the polymer.
[0046] The PEG used may be monodispersed, meaning the PEG
preparation or solution used to form the conjugate has chains of
uniform length, or may be polydispersed, meaning the PEG
preparation or solution used has chains of varying length. The PEG
moiety of the glucose-PEG conjugate may be of any length, for
example from 2 to 500 repeating units, or having an average
molecular weight of from about 300 g/mol to about 10,000,000 g/mol.
In various embodiments, the PEG may be (all average molecular
weight) PEG 200, PEG 300, PEG 400, PEG 600, PEG 800, PEG 1000, PEG
1500, PEG 2000, or PEG 3350.
[0047] The PEG moiety may be a linear PEG. Alternatively, the PEG
may be a branched PEG. Branched PEG includes any PEG having one or
more branches of PEG groups extending from a PEG backbone, and
includes specific arrangements or degrees of branching, such as a
hyperbranched PEG, a dendritic PEG, a star PEG, or a comb PEG. In a
particular embodiment, the PEG is a branched PEG.
[0048] The PEG used to form the conjugate may be terminated at one
or both ends with an end group, for example an amino group, a
hydroxyl group, a methyl ether group, a caprolactone group, a
carbohydrate moiety or one or more amino acids.
[0049] The PEG moiety is conjugated to the linear glucose moiety at
the C1 position of the glucose molecule. The C1 position in free
linear glucose is part of an aldehyde functional group, that is, in
free linear glucose the C1 carbon atom is bonded to an oxy
functional group and a hydrogen atom, as well as to the C2 carbon
atom. In the conjugate, the oxy portion of the aldehyde group may
be removed by the conjugation of the PEG moiety, or may be involved
in the conjugation reaction and thus may be converted to another
functional group, for example the oxy group may become an oxo
group, forming an ether linkage to the PEG moiety. Thus, the PEG
moiety (or an end group on the PEG moiety) may be bonded directly
to the C1 carbon or may be bonded to the oxygen of the aldehyde
group. In particular embodiments, the PEG used to form the
conjugate has a free primary amino group at least at the terminus
that is to be conjugated to the C1 position of the linear glucose,
and following conjugation, the oxy group is replaced by a secondary
amino group connecting the linear glucose moiety to the PEG moiety
via an amine linkage.
[0050] The PEG moiety, including an end group, may be directly
attached to the linear glucose moiety at the C1 position, or may be
attached via a linker moiety. For example, a linker that is
connected to the C1 position of glucose (or via reaction with the
aldehyde functionality at the C1 position), may also be attached to
the PEG moiety, including an end group on the PEG moiety, thus
linking together the glucose moiety and the PEG moiety via an
attachment at the C1 carbon, as described above.
[0051] Optionally, the glucose-PEG conjugate may include a
detectable label. The label may be attached anywhere on the
glucose-PEG conjugate, including attached to the PEG moiety, and
may be attached for example at the free end of the PEG moiety or
(end group) not conjugated to the glucose moiety.
[0052] The detectable label may be any label that is detectable
using known detection methods, including imaging methods. For
example, the detectable label may be a PET label, an SPECT label,
an MRI label, a quantum dot label, a coloured label, a fluorescent
label, a radiolabel or a label that may be detected by an antibody
or antibody fragment. Such labels are known and are readily
available. For example, fluorescent labels include BODIPY, FITC,
Rhodamine, TRITC, Texas Red, cyanine dyes (e.g. Cy3 or Cy5) or
Alexa fluors. Radiolabels include moieties or groups having at
least one radioactive isotope, and include moieties or groups
having a positron emitting radioactive isotope or a gamma emitting
radioactive isotope.
[0053] In particular, radiolabels such as PET labels useful for PET
scanning may include an unstable positron-emitting isotope. Such
isotopes may be synthesized in a cyclotron by bombarding nitrogen,
carbon, oxygen, or fluorine with protons. Examples of the isotopes
used for PET labels include .sup.15O (half-life: 2 min), .sup.18F
(half-life: 110 min), and .sup.11C (half-life: 20 min). Positron or
photon emitting atoms such as .sup.18F, .sup.11C, .sup.125I,
.sup.123I, .sup.16N, .sup.15O, .sup.3H, .sup.133Xe, .sup.111In,
.sup.68Ga and other isotopes of metals such as technetium, or
copper may be used in PET labels or SPECT labels. MRI labels
include T1 (Gd) and T2 (Fe.sub.3O.sub.4) contrast agents.
[0054] In particular embodiments, the glucose-PEG conjugate
comprises the conjugate depicted in FIG. 1, FIG. 2 or FIG. 3. In
particular embodiments, the glucose-PEG conjugate is the conjugate
depicted in FIG. 1, FIG. 2 or FIG. 3. For FIG. 3, each n is
independently 1 or greater, or from 1 to 500. In other embodiments,
the glucose-PEG conjugate has the arrangement as depicted in FIG. 1
or FIG. 2, but having the PEG moiety longer than depicted in the
relevant Figure, up to 500 repeating ethylene glycol units.
[0055] The glucose-PEG conjugates may be synthesized using standard
known organic synthesis methods. Linear glucose and various PEGs
are readily commercially available. Conjugation may be readily
performed using known reactions to reaction an appropriate
functional group on the terminus of the PEG molecule or on a linker
molecule with the free aldehyde located at the C1 position of the
linear glucose molecule. Similarly, standard chemical coupling
reactions may, be used to attach any detectable label, including
attachment to the PEG moiety, either before or after conjugation to
the glucose moiety.
[0056] Where the PEG used is terminated with an end group having a
free amino group or where a linker molecule with a free amino group
is used, conjugation of the amino group with the C1 carbon atom may
be performed in accordance with Example 1 set out below, and the
synthetic scheme described in FIG. 4.
[0057] As stated above, it appears that the glucose-PEG conjugate
binds to the GLUTs but is not transported into the cell. Without
being limited to any particular theory, the glucose-PEG conjugate
appears to bind to the GLUTs, blocking binding and transport of
glucose, thus reducing the cell's internal glucose supply available
for glycolysis, and thus reducing the proliferation rate of the
cell. The cell surface of the normal cells contains glucose
transporters along with transporters for other saccharides such as
fructose and galactose. However, in hyper-proliferating cells such
as cancer cells, glucose transporters are the primary transporters
and the cancer cells depend on the glucose intake for their ATP
generation and oxygen production under anaerobic conditions.
Blocking the glucose receptors in hyper-proliferating cancer cells
may lead to cell death, as was found in studies involving siRNA and
antibody based blockage of the glucose receptors leading to cell
death in proliferating cancer cells.
[0058] Since the linear form of glucose used in the glucose-PEG
conjugate typically does not participate in glycolytic metabolic
pathways, even if the conjugate were to degrade in vivo to release
the linear glucose moiety from the conjugate, no cyclic glucose (or
cyclizable glucose) would be made available and thus would not
result in increased glycolysis by cells. This is in contrast to
other glucose analogues and conjugates, which typically use cyclic
glucose or glucose which is able to cyclise due to availability of
the C1 aldehyde functionality to form the cyclic form of glucose.
Such cyclic glucose analogues and conjugates may contribute to the
glucose supply within a cell should the analogue or conjugate be
degraded to release free glucose.
[0059] Furthermore, the PEG portion of the conjugate is highly
soluble, and is generally biologically inert, biodegradable,
non-toxic and non-immunogenic. Thus, the glucose-PEG conjugated
described herein is useful for reducing glucose uptake by a cell,
including in in vivo contexts.
[0060] Thus, in another aspect, there is provided a method of
reducing glucose transport into a cell comprising contacting the
cell with a glucose-PEG conjugate as described herein. Contacting
the cell with the conjugate thus allows the conjugate to bind to
the GLUTs on the surface of the hyper-proliferative cell.
[0061] Glucose transport into a cell refers to the process of
moving glucose from the exterior of a cell into the interior of the
cell, including that mediated by GLUTs. Reducing glucose transport
in a cell refers to lessening the amount of glucose that is taken
up into the cell, including via transport by GLUTs. Reducing
includes lessening as well as completely blocking glucose
transport, including glucose transport by GLUTs. Reducing may lead
to slowing of proliferation by the cell so that the cell still
proliferates but not as quickly as in the absence of the
glucose-PEG conjugate, or may lead to cessation of proliferation,
or even cell death, including apoptotic cell death.
[0062] The term cell as used herein refers to and includes a single
cell, a plurality of cells or a population of cells where context
permits, unless otherwise specified. Similarly, reference to cells
also includes reference to a single cell where context permits,
unless otherwise specified.
[0063] The cell may be any cell, including an in vitro cell, a cell
in culture, an in vivo cell, or an ex vivo cell explanted from a
subject.
[0064] The cell may be derived from any organism that expresses
GLUTs and that undergoes anaerobic glycolysis, for example an
animal, including a mammal, including a human. The cell may be a
primary cell or it may be a cell from an established cell line,
including a cancer cell line.
[0065] The cell may be a hyper-proliferative cell. As used herein,
a hyper-proliferative cell or hyper-proliferating cell is a cell in
which proliferation is uncontrolled or is increased relative to a
healthy cell. A healthy cell is a cell of the same cell type but
that is not hyper-proliferating or in which proliferation is under
normal cellular controls. The hyper-proliferative cell may be a
cell associated with a proliferative disorder, including a cell
within a solid tumor, and may be a cell that is being treating with
a further cancer therapy.
[0066] A cell is associated with a proliferative disorder if that
cell is a cell that is abnormally proliferating so as to result in
the disorder in a subject in which the cell is located, or if the
disorder is characterized by the proliferation of such a cell.
[0067] A proliferative disorder is a disease or disorder in which a
cell of a subject is abnormally proliferating, resulting in
uncontrolled growth and division of the cell, which in a healthy
individual would not be proliferating or would be proliferating in
a controlled manner. The proliferative disorder may be
characterized by the proliferation of malignant or non-malignant
cell populations, including in a solid tumor. Such disorders
include cancer including breast cancer, liver cancer, gastric
cancer, bladder cancer, colon cancer, prostate cancer, lung cancer,
nasopharyngeal carcinoma, cervical carcinoma, skin cancer.
[0068] Where the cell is an in vitro cell, including a cell in
culture and/or an explanted cell, contacting the cell with the
glucose-PEG conjugate may comprise adding the conjugate to the
buffer solution or growth medium in which the cell is
contained.
[0069] The glucose-PEG conjugate may be added to the buffer
solution or growth medium at a concentration of about 0.01 mM or
greater, about 0.02 mM or greater, about 0.05 mM or greater or
about 1.0 mM or greater. Alternatively, the glucose-PEG conjugate
may be added to the buffer solution or growth medium at a
concentration of from about 0.01 mM to about 20 mM, about 0.02 to
about 10 mM, or about 0.02 to about 0.5 mM.
[0070] If desired, the glucose-PEG conjugate may comprise a
detectable label as described above, in order to allow for
confirmation that the conjugate is binding to the GLUTs on the
exterior surface of the cell by detecting the location of the
conjugate after contacting with a cell. Thus, the method in certain
embodiments may comprise detecting the glucose-PEG conjugate after
contacting with the cell. The detecting may include imaging of the
cell, including using known fluorescent imaging techniques in
vitro.
[0071] Where the cell is an in vivo cell, contacting the cell with
the glucose-PEG conjugate may comprise administering an effective
amount of the conjugate to a subject.
[0072] The term "effective amount" as used herein means an amount
effective, at dosages and for periods of time necessary to achieve
the desired result, for example, to reduce glucose transport in the
cell or to treat the specific proliferative disorder.
[0073] Since reducing glucose transport into a hyper-proliferative
cell may result in starvation of the cell, resulting in slowing of
proliferation, cessation of proliferation, or even cell death, the
method may include treatment of a proliferative disorder in a
subject.
[0074] The term "treating" a proliferative disorder refers to an
approach for obtaining beneficial or desired results, including
clinical results. Beneficial or desired clinical results can
include, but are not limited to, alleviation or amelioration of one
or more symptoms or conditions, diminishment of extent of disease,
stabilization of the state of disease, prevention of development of
disease, prevention of spread of disease, delay or slowing of
disease progression, delay or slowing of disease onset,
amelioration or palliation of the disease state, and remission
(whether partial or total). "Treating" can also mean prolonging
survival of a patient beyond that expected in the absence of
treatment. "Treating" can also mean inhibiting the progression of
disease, slowing the progression of disease temporarily, although
more preferably, it involves halting the progression of disease
permanently.
[0075] The subject is any animal in need of treatment of a
proliferative disorder, including a mammal, including a human.
[0076] The conjugate may be administered to the subject using
standard techniques known in the art. The conjugate may be
administered systemically, or may be administered directly at the
site at which the proliferating cell that is associated with the
proliferative disorder is located. Delivery to the site includes
topical administration, injection or surgical implantation,
including at a site of a tumor. Delivery may be performed using a
drug delivery device, to allow sustained delivery of the conjugate
according to a desired release profile. Drug delivery devices
including transdermal systems as well as devices for
implantation.
[0077] The concentration and amount of the conjugate to be
administered will vary, depending on the proliferative disorder to
be treated, the type of cell associated with the proliferative
disorder, the type of conjugate that is administered, the mode of
administration, and the age and health of the subject.
[0078] Optionally, the method also involves imaging of
hyper-proliferative cells in vivo. Thus, as described above, the
conjugate may comprise a detectable label, including a fluorescent
marker, an MRI label, a PET label, a SPECT label, a radiolabel or a
quantum dot, suitable for detection using in vivo imaging
techniques.
[0079] Since the conjugate can influence the growth of
hyper-proliferative cells, the conjugate may be used in combination
with other cancer treatments, including to target drug-resistant
cancer cells to make such cells more susceptible to treatment by
cancer treatments such as with chemotherapeutic agents.
[0080] Thus, the reducing and/or treating may be further
accomplished in combination with a chemotherapeutic agent. In
combination with a chemotherapeutic agent means that the reducing
and/or treating occurs in a time period during which a
chemotherapeutic agent is contacted with or administered to the
cell. The reducing of glucose transport and the contacting with or
administration of the chemotherapeutic agent may occur
simultaneously or sequentially, and the respective time period for
each may be conterminous or may be overlapping provided that the
benefit or effect of the chemotherapy treatment is ongoing in the
cell concomitantly with the reducing. The reducing and the
administration each may be achieved in one or more discrete
treatments or may be performed continuously for a given time period
required in order to achieve the desired result.
[0081] The cell may be further contacted with the chemotherapeutic
agent in a manner similar to that described above for contacting
with the conjugate, depending on the nature of the chemotherapeutic
agent. The cell may be contacted with the chemotherapeutic agent
prior to, following, or simultaneously with the conjugate.
[0082] The chemotherapeutic agent may be a compound that is
typically administered to a cell and which has a cytotoxic or
cytostatic effect. The chemotherapeutic agent may be an agent that
induces apoptosis, such as p53-dependent apoptosis, or that induces
cell cycle arrest, including p53-dependent cell cycle arrest, in a
cell that is abnormally proliferating, even in the absence of the
conjugate. The chemotherapeutic agent may also be an agent that
activates p53 or p21 in an abnormally proliferating cell but that
does not induce apoptosis in the cell, due to a property of the
abnormally proliferating cell, for example an alteration or
mutation in p53 or in the p53 pathways. Treatment of the cell with
the chemotherapeutic agent in combination with the conjugate
induces cell death, or increases sensitivity to cell death, at a
level greater than that which is observed in the absence of the
conjugate.
[0083] The chemotherapeutic agent may be a DNA damaging agent or a
genotoxic agent that can activate p53-dependent apoptosis or
p53-dependent cell cycle arrest in a proliferating cell. The
chemotherapeutic agent may be, without limitation, a small
molecule, a peptide or a protein, an anthracycline, an alkylating
agent, an alkyl sulfonate, an aziridine, an ethylenimine, a
methylmelamine, a nitrogen mustard, a nitrosourea, an antibiotic,
an antimetabolite, a folic acid analogue, a purine analogue, a
pyrimidine analogue, an enzyme, a podophyllotoxin, a
platinum-containing agent or a cytokine. The chemotherapeutic agent
may be chosen as a chemotherapeutic agent that is known to be
effective against the particular cellular proliferative disorder
and cell type. In certain embodiments the chemotherapeutic agent is
cisplatin, paclitaxel, Adriamycin (ADR), 5-fluorouracil (5-FU),
etoposide, or camptothecin or a derivative or analog thereof.
[0084] Also contemplated are various uses of the glucose-PEG
conjugate, including use of the conjugate for reducing glucose
transport into a cell, or for treating a proliferative disorder in
a subject. The use may include use in the manufacture of a
medicament or pharmaceutical composition.
[0085] Thus, to aid in administration to a subject, the glucose-PEG
conjugate may be formulated as an ingredient in a pharmaceutical
composition. Therefore, in a further embodiment, there is provided
a pharmaceutical composition comprising a glucose-PEG conjugate,
and may further include a pharmaceutically acceptable diluent. The
invention in one aspect therefore also includes such pharmaceutical
compositions for use in reducing glucose transport in a cell and/or
for use in treating a proliferative disorder.
[0086] The pharmaceutical compositions may routinely contain
pharmaceutically acceptable concentrations of salt, buffering
agents, preservatives and various compatible carriers. For all
forms of delivery, the glucose-PEG conjugate may be formulated in a
physiological salt solution.
[0087] The pharmaceutical composition can be prepared by known
methods for the preparation of pharmaceutically acceptable
compositions suitable for administration to patients, such that an
appropriate quantity of the glucose-PEG conjugate, and any
additional active substance or substances, is combined in a mixture
with a pharmaceutically acceptable vehicle. Suitable vehicles are
described, for example, in Remington's Pharmaceutical Sciences
(Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., USA 1985). On this basis, the pharmaceutical
compositions include, albeit not exclusively, solutions of the
glucose-PEG conjugate, in association with one or more
pharmaceutically acceptable vehicles or diluents, and contained in
buffer solutions with a suitable pH and iso-osmotic with
physiological fluids. A person skilled in the art would know how to
prepare suitable formulations.
[0088] The proportion and identity of the pharmaceutically
acceptable diluent is determined by chosen route of administration,
compatibility with live cells, and standard pharmaceutical
practice. Generally, the pharmaceutical composition will be
formulated with components that will not significantly impair the
properties of the glucose-PEG conjugate to reduce glucose transport
into a cell.
[0089] The pharmaceutical compositions may additionally contain
other therapeutic agents useful for treating the particular
proliferative disorder, for example a cytotoxic agent, for example
a chemotherapeutic agent.
[0090] The pharmaceutical composition may be administered to a
subject in a variety of forms depending on the selected route of
administration, as will be understood by those skilled in the
art.
[0091] In different embodiments, the composition is administered
topically, surgically or by injection (subcutaneously,
intravenously, intramuscularly, etc.) directly at the desired site
where the cells that are proliferating in an uncontrolled manner
are located in the patient, including at or within a tumor.
[0092] The dose of the pharmaceutical composition that is to be
used depends on the particular condition being treated, the
severity of the condition, the individual patient parameters
including age, physical condition, size and weight, the duration of
the treatment, the nature of concurrent therapy (if any), the
specific route of administration and other similar factors that are
within the knowledge and expertise of the health practitioner.
These factors are known to those of skill in the art and can be
addressed with minimal routine experimentation.
[0093] In another aspect, there is provided a method of imaging a
hyper-proliferative cell, including both in vitro and in vivo.
[0094] Terms used in describing this method are those as defined
above, unless otherwise indicated.
[0095] The method of imaging includes contacting a cell with a
glucose-PEG conjugate as described herein, the conjugate comprising
a detectable label; and detecting the detectable label in order to
image the hyper-proliferative cell.
[0096] As indicated above, the detectable label is any label that
is detectable using standard detection methods, including imaging
methods. In vitro imaging methods include fluorescence microscopy
techniques. Imaging methods for in vivo imaging include magnetic
resonance imaging (MRI) including functional magnetic resonance
imaging (fMRI) techniques, positron emission tomography (PET)
imaging techniques, and single photon emission computed tomography
(SPECT) imaging techniques.
[0097] The glucose-PEG conjugate is contacted with the
hyper-proliferative cell in a manner as described above to allow
the conjugate to bind to the GLUTs on the surface of the
hyper-proliferative cell. For example, for an in vitro cell, the
glucose-PEG conjugate may be added to a buffer or culture medium
containing the cell. For example, for an in vivo cell, the
glucose-PEG conjugate may be administered to a subject in which a
hyper-proliferative cell is desired to be imaged, as described
above, including by topical administration, injection or surgical
implantation, including at a site of a tumor. As described above,
inclusion of the conjugate in a composition may aid in
administration of the conjugate to a subject.
[0098] Once contacted with the cell, the hyper-proliferative cell
may be incubated with the glucose-PEG conjugate prior to detecting.
The incubation may be for any period of time so as to allow for the
conjugate to bind to the GLUTs on the hyper-proliferative cell, for
example for 5 minutes or longer, for 15 minutes or longer, for 30
minutes or longer or for 1 hour or longer.
[0099] Following contacting and optional incubation, the method
comprises detecting the detectable label. The method of detecting
will depend on the detectable label used. Standard detection
methods may be used to detect the detectable label, including the
above-mentioned fluorescence microscopy techniques, MRI techniques,
PET imaging techniques, and SPECT imaging techniques. Such methods
are known to a skilled person and may be performed in accordance
with standard, known methods.
[0100] The imaging method may be used in vitro to identify a
hyper-proliferative cell within a population of cells, or to
identify conditions that induce hyper-proliferation within a
population of cells. The imaging method is also useful for in vivo
imaging, and may be used to identify a hyper-proliferative cell
within a subject, including within a solid tumor, or may be used to
monitor treatment progression within a subject.
[0101] Also contemplated are uses based on the above methods, such
as use of a glucose-PEG conjugate as described herein for imaging a
hyper-proliferative cell, or use of a glucose-PEG conjugate as
described herein in the manufacture of a composition for imaging a
hyper-proliferative cell.
[0102] The present methods and uses are further exemplified by way
of the following non-limited examples.
EXAMPLES
Example 1
[0103] The following example demonstrates that glucose modified
with poly(ethylene glycol) (PEG) reduces cell proliferation and
induces apoptosis in human breast cancer cell line, MCF-7.
[0104] Methods
[0105] Synthesis of Glucose-PEG-BODIPY (GPB): Glucose was modified
with PEG-conjugated to BODIPY. BODIPY was used as a red fluorescent
indicator for cell imaging. A flame dried reaction vial (2 mL) was
charged with a solution of glucose-PEG-NH.sub.2 (32 mg, 0.1 mmol)
and BODIPY 650/665-X (64 mg, 0.1 mmol) under argon, and the mixture
was cooled in an ice bath at 0.degree. C. Dry dimethylformamide
(DMF; Aldrich, 1 mL) was added dropwise and was stirred at
0.degree. C. for 2 h under argon. The reaction mixture was then
brought to room temperature, and continuously stirred for 24 h
under argon in the dark. The reaction was monitored by
reverse-phase high-pressure liquid chromatography (HPLC; Waters
Corporation, USA). After completion of the reaction, DMF was
removed under reduced pressure and the blue residue was purified by
reverse-phase flash column chromatography using a Combiflash
separating system (ISCO Combiflash Companion, USA). The desired
fractions were collected and lyophilized to obtain the
FR-BODIPY-glucose as a bluish green solid (75 mg, 90%). The final
compound was soluble in deionized (DI) water and in a mixture of
ethanol and water (volume ratio=1:1). Mass spectral analysis showed
a molecular ion peak of 841 (M+1). The structure of
glucose-PEG-BODIPY is shown in FIG. 1 and the synthesis scheme,
including the preparation of glucose-PEG-NH.sub.2, is depicted in
FIG. 4.
[0106] Synthesis of Glucose-PEG-OH: A dry reaction vial (10 mL) was
charged with glucose-PEG-NH.sub.2 (31 mg, 0.1 mmol) and dry DMF (2
mL), and was stirred under argon until complete dissolution was
observed. The solution was then cooled to 0.degree. C. in an ice
bath. A solution of caprolactone (12 mg, 0.11 mmol) in DMF (1 mL)
was added dropwise, and the reaction mixture was stirred overnight
and brought to room temperature under argon. After completion of
the reaction, as monitored by HPLC and liquid chromatography-mass
spectrometry (LC-MS), the solvent was evaporated and purified by
precipitation from methanol-diethylether mixture to give the
glucose-PEG-OH as a light yellow solid (40 mg, 95%). Mass spectral
analysis showed a molecular ion peak of 427 (M+1). The structure of
glucose-PEG-OH is shown in FIG. 2.
[0107] Conjugation of Glucose to Branched PEG: A flame dried
reaction flask (25 mL) was charged with branched PEG-NH.sub.2
(PEG-tetrapropylamine, molecular weight=20 kDa, 500 mg, 0.025 mmol)
and glucose (5 mg, 0.0275 mmol) in dry methanol (10 mL), and the
mixture was heated at 60.degree. C. under argon for 24 h. The
reaction mixture was cooled to 0.degree. C., and reduced with
sodium borohydride. The conjugated glucose-branched PEG was
purified by precipitation method. The free amino groups in the
branched PEG were protected by reaction with caprolactone. The
final conjugated product was purified by exhaustive filtration
through an Amicon membrane filter (polyethersulfone (PES) membrane,
10 kDa molecular weight cutoff), and lyophilized to obtain a
product as a colorless sticky solid. A representative structure of
glucose-branched PEG is shown in FIG. 3.
[0108] Cell Culture: Different cancer cell lines, MCF-7 (breast
cancer), H1299 (lung cancer), HepG2 (liver cancer), DU145 (prostate
cancer), Caco2 (colon cancer) and AGS (gastric cancer) were
obtained from American Type Culture Collection (ATCC). Normal
breast epithelial cell line (MCF-10A) was used for comparison. The
cells were maintained in the growth medium as described by ATCC.
Approximately 10,000 cells were seeded onto a 96-well plate.
Modified glucose compounds were added to the cells after 24 h of
seeding. The cells were further cultured in the presence of the
modified glucose compounds for up to 7 days. The medium was changed
everyday along with the specified concentration of modified
glucose. At the end of the treatment, the cells were assayed for
viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assay.
[0109] MTT Assay: MTT assay was performed using the TACS.TM. MTT
cell proliferation assay kit (Trevigen Inc, MD, USA). Briefly,
after treatment of cells, 10 .mu.l of MTT reagent was added and
incubated at 37.degree. C. for 4 h. The cells were lysed using
detergent for 4 h at room temperature. The absorbance was measured
in a standard plate reader at 570 nm.
[0110] Competition Assay: Competition assay was performed to
demonstrate the specificity of the GPB binding to the GLUTs. GPB
binding to the cell surface could be quantified by measuring the
fluorescence intensity. Addition of unlabeled glucose at different
concentrations (10 mM, 20 mM, 30 mM, 35 mM and 40 mM) to the media
competed with the GPB binding, leading to the differences in the
fluorescence intensity. Cells were incubated with GPB and various
concentrations of glucose for 30 min at 37.degree. C. The medium
was removed and the cells were washed briefly with PBS twice. The
fluorescence intensity was measured with an excitation of 488 nm
and an emission of 522 nm. The fluorescence intensity was
normalized by that of the control cells.
[0111] Imaging: The MCF-7 cells were seeded on a 6-well plate at
least 24 h prior to imaging. The cells were incubated with GPB (200
.mu.M) for 1 h. The cells were washed with PBS (without calcium and
magnesium) thrice. Live imaging was performed with fluorescence
microscopy. Metamorph (Molecular Devices, USA) and Image J
(freeware from NIH, USA) were used for image capturing and
processing.
[0112] Results
[0113] To test the hypothesis that modified glucose could be
utilized for cancer therapy, a fluorescent tag (BODIPY) was
conjugated onto glucose via PEG. This GPB was used in the initial
imaging studies. Fluorescence imaging of MCF-7 cells fed with GPB
clearly indicated that the GPB molecules were localized on the
plasma membrane of the cells (FIG. 5). The binding of the modified
GPB was challenged with different concentrations of glucose. The
competition binding assay suggested that glucose competed with the
GPB in binding to the GLUTs (FIG. 6).
[0114] Dose-dependent effect of GPB on the MCF-7 cells was
examined. The cell viability assay (MTT assay) was performed on
cells treated with different GPB concentrations. There was a
gradual dose-dependent decrease in the cell viability with
increasing GPB concentration (FIG. 7). .about.63% reduction in cell
viability was observed when the cells were treated with 200 .mu.M
of GPB (FIG. 8). PEG alone at concentrations of 200 .mu.M and 400
.mu.M did not have any effect on the MCF-7 cells (FIG. 8). However,
cells treated with 200 .mu.M of GPB showed a drastic reduction in
cell viability (.about.70%) (FIG. 8).
[0115] A new compound was also synthesized to replace the BODIPY in
GPB with another PEG molecule. This new compound has two PEGs
conjugated to the glucose (glucose-PEG-PEG or GPP). The cell
viability was reduced to 57% and 42% at 200 .mu.M and 400 .mu.M of
GPP, respectively (FIG. 9). In contrast, the cell viability was not
affected by 400 .mu.M of PEG-PEG.
[0116] A further strategy to increase the levels of cell death was
to further modify the glucose with a branched structure. Glucose
was coupled to a branched PEG (4-arm PEG or BrP) as described in
the methods. The resulting compound was designated as
glucose-conjugated branched PEG or GBrP. 200 .mu.M of GBrP could
reduce the viability of MCF-7 breast cancer cells by >90%, where
as 200 .mu.M of branched PEG only decreased the viability of the
same cell line by 7% (see Table 1). Thus, GBrP was even more
effective at inhibiting cell viability than GPB and GPP. In
contrast, Table 1 also shows that 200 .mu.M of GBrP reduced the
viability of normal breast cells by only 29%. This illustrates the
ability of GBrP to preferentially target cancer cells. GBrP was
also demonstrated to significantly inhibit the viability of various
different cancer cell lines (see Table 1).
[0117] A glucose competition assay was carried out to confirm that
the modified glucose-PEG is binding to the GLUT1 receptors.
Increasing concentrations of un-modified glucose were used. The
human breast cancer cells (MCF-7) were incubated with 200 .mu.M of
GPB in the presence of the increasing concentrations of un-modified
glucose. After 30 minutes the fluorescence intensity was measured
using a plate reader. As indicated in FIG. 10, the intensity of the
fluorescence decreases with the increase in un-modified glucose,
indicating that the un-modified glucose is competing for the GLUT1
receptor.
[0118] Gene expression analysis was carried out to understand the
apoptosis pathway during the modified glucose mediated cell death.
Human lung cancer cells (H1299) (FIG. 11) or human prostrate cancer
cells (DU145) (FIG. 12) were treated for 3 days with 200 .mu.M of
modified glucose (GBrP). Control cells were prepared without
treatment. Total RNA was extracted from the cells and reverse
transcribed. A PCR array containing primers to identify the
apoptotic pathway was used to identify the apoptosis pathway
between control and treated cells in lung or prostate cancer cells.
The amplification data is represented in a scatter plot with
allowable variations. As seen in FIGS. 11 and 12, AKT1 and ABL1
were down regulated while APAF1 and TP53BP were upregulated.
[0119] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. The
citation of any publication is for its disclosure prior to the
filing date and should not be construed as an admission that the
present invention is not entitled to antedate such publication by
virtue of prior invention.
[0120] As used in this specification and the appended claims, the
singular forms "a", "an" and "the" include plural reference unless
the context clearly dictates otherwise. As used in this
specification and the appended claims, the terms "comprise",
"comprising", "comprises" and other forms of these terms are
intended in the non-limiting inclusive sense, that is, to include
particular recited elements or components without excluding any
other element or component. Unless defined otherwise all technical
and scientific terms used herein have the same meaning as commonly
understood to one of ordinary skill in the art to which this
invention belongs.
[0121] All lists or ranges provided herein are intended to include
any sub-list or narrower range falling within the recited list or
range.
[0122] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
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