U.S. patent application number 17/348985 was filed with the patent office on 2021-10-14 for compositions and methods for glucose transport inhibition.
The applicant listed for this patent is OHIO UNIVERSITY. Invention is credited to STEPHEN BERGMEIER, XIAOZHUO CHEN.
Application Number | 20210317068 17/348985 |
Document ID | / |
Family ID | 1000005654953 |
Filed Date | 2021-10-14 |
United States Patent
Application |
20210317068 |
Kind Code |
A1 |
CHEN; XIAOZHUO ; et
al. |
October 14, 2021 |
COMPOSITIONS AND METHODS FOR GLUCOSE TRANSPORT INHIBITION
Abstract
Glucose deprivation is an attractive strategy in cancer research
and treatment. Cancer cells upregulate glucose uptake and
metabolism for maintaining accelerated growth and proliferation
rates. Specifically blocking these processes is likely to provide
new insights to the role of glucose transport and metabolism in
tumorigenesis, as well as in apoptosis. As solid tumors outgrow the
surrounding vasculature, they encounter microenvironments with a
limited supply of nutrients leading to a glucose deprived
environment in some regions of the tumor. Cancer cells living in
the glucose deprived environment undergo changes to prevent glucose
deprivation-induced apoptosis. Knowing how cancer cells evade
apoptosis induction is also likely to yield valuable information
and knowledge of how to overcome the resistance to apoptosis
induction in cancer cells. Disclosed herein are novel anticancer
compounds that inhibit basal glucose transport, resulting in tumor
suppression and new methods for the study of glucose deprivation in
animal cancer research.
Inventors: |
CHEN; XIAOZHUO; (ATHENS,
OH) ; BERGMEIER; STEPHEN; (ATHENS, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OHIO UNIVERSITY |
ATHENS |
OH |
US |
|
|
Family ID: |
1000005654953 |
Appl. No.: |
17/348985 |
Filed: |
June 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16522314 |
Jul 25, 2019 |
11072576 |
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17348985 |
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15995645 |
Jun 1, 2018 |
10385005 |
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16522314 |
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14935902 |
Nov 9, 2015 |
10000443 |
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15995645 |
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13071386 |
Mar 24, 2011 |
9181162 |
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14935902 |
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61317062 |
Mar 24, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07C 235/56 20130101;
C07C 205/32 20130101; C07D 231/12 20130101; C07D 235/18 20130101;
A61K 31/09 20130101; C07D 233/64 20130101; C07C 43/23 20130101;
C07D 311/82 20130101; C07D 311/16 20130101; C07C 39/367 20130101;
C07D 249/08 20130101; C07D 209/08 20130101; C07C 211/48 20130101;
C07D 319/20 20130101; C07C 43/253 20130101; A61K 45/06
20130101 |
International
Class: |
C07C 205/32 20060101
C07C205/32; C07C 211/48 20060101 C07C211/48; C07C 39/367 20060101
C07C039/367; C07C 43/23 20060101 C07C043/23; C07C 235/56 20060101
C07C235/56; C07D 209/08 20060101 C07D209/08; C07D 231/12 20060101
C07D231/12; C07D 233/64 20060101 C07D233/64; C07D 235/18 20060101
C07D235/18; C07D 249/08 20060101 C07D249/08; C07D 319/20 20060101
C07D319/20; A61K 31/09 20060101 A61K031/09; A61K 45/06 20060101
A61K045/06; C07C 43/253 20060101 C07C043/253; C07D 311/16 20060101
C07D311/16; C07D 311/82 20060101 C07D311/82 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This work was sponsored in part by the National Science
Foundation through a Partnership for Innovation Grant
(HER-0227907). The United States government may have certain rights
in the invention.
Claims
1. A compound of formula (I): ##STR00029## wherein R.sub.1 is
selected from the group consisting of hydrogen, alkyl, benzyl,
aryl, and heteroaryl; wherein R.sub.2 is selected from the group
consisting of hydrogen, alkyl, benzyl, aryl, heteroaryl, and
fluorescent tags; and wherein R.sub.3 is selected from the group
consisting of hydrogen, halo, alkyl, benzyl, aryl, heteroaryl,
amino, cyano, and alkoxy; or a salt thereof.
2. The compound of claim 1, wherein R.sub.1 and R.sub.2 are
aryl.
3. The compound of claim 2, wherein R.sub.1 and R.sub.2 are
independently selected from the group consisting of 2-, 3-, and
4-hydroxyphenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and
3,5-dihydroxyphenyl, 2,3,4-, 2,3,5-, 2,3,6-, and
3,4,5-trihydroxyphenyl, 2,3,4,5- and 2,3,4,6-tetrahydroxyphenyl,
and perhydroxyphenyl.
4. The compound of claim 2, wherein R.sub.1 and R.sub.2 are
independently selected from the group consisting of methoxyphenyl,
dimethoxyphenyl, and trimethoxyphenyl.
5. The compound of claim 1, wherein R.sub.1 is aryl and R.sub.2 is
a fluorescent tag.
6. The compound of claim 5, wherein R.sub.1 is selected from the
group consisting of 2-, 3-, and 4-hydroxyphenyl, 2,3-, 2,4-, 2,5-,
2,6-, 3,4-, and 3,5-dihydroxyphenyl, 2,3,4-, 2,3,5-, 2,3,6-, and
3,4,5-trihydroxyphenyl, 2,3,4,5- and 2,3,4,6-tetrahydroxyphenyl,
and perhydroxyphenyl; and wherein R.sub.2 is selected from the
group consisting of coumarins, dansyl, rhodamine, fluorescein,
carboxynaphthofluorescein, and fluorescent proteins.
7. The compound of claim 1, wherein R.sub.1 and R.sub.2 are
3-hydroxyphenyl and R.sub.3 is hydrogen.
8. The compound of claim 1, wherein R.sub.1 and R.sub.2 are
3,4,5-trihydroxyphenyl and R.sub.3 is hydrogen.
9. The compound of claim 1, wherein R.sub.1 and R.sub.2 are
3,4,5-trimethoxyphenyl and R.sub.3 is hydrogen.
10. The compound of claim 1, wherein R.sub.1 and R.sub.2 are
2,6-dimethoxyphenyl and R.sub.3 is hydrogen.
11. The compound of claim 1, wherein R.sub.1 and R.sub.2 are
3,4-dimethoxyphenyl and R.sub.3 is hydrogen.
12. The compound of claim 1, wherein R.sub.1 and R.sub.2 are
3-methoxyphenyl and R.sub.3 is hydrogen.
13. A method of treating cancer, the method comprising:
administering to a subject in need of such treatment a
therapeutically effective amount of a basal glucose transport
inhibitor compound of formula (I) or a pharmaceutically acceptable
salt thereof: ##STR00030## wherein R.sub.1 is selected from the
group consisting of hydrogen, alkyl, benzyl, aryl, and heteroaryl;
wherein R.sub.2 is selected from the group consisting of hydrogen,
alkyl, benzyl, aryl, heteroaryl, and fluorescent tags; and wherein
R.sub.3 is selected from the group consisting of hydrogen, halo,
alkyl, benzyl, aryl, heteroaryl, amino, cyano, and alkoxy; and
whereby administration of said basal glucose transport inhibitor
compound of formula (I) or a pharmaceutically acceptable salt
thereof to the subject treats said cancer by inhibiting basal
glucose transport in said subject, and wherein the cancer is
selected from lung cancer, colon cancer, breast cancer, and
cervical cancer.
14. The method of claim 13, wherein the cancer upregulates basal
glucose transport.
15. The method of claim 13, wherein R.sub.1 and R.sub.2 are
independently selected from the group consisting of 2-, 3-, and
4-hydroxyphenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and
3,5-dihydroxyphenyl, 2,3,4-, 2,3,5-, 2,3,6-, and
3,4,5-trihydroxyphenyl, 2,3,4,5- and 2,3,4,6-tetrahydroxyphenyl,
and perhydroxyphenyl, and R.sub.3 is hydrogen.
16. The method of claim 13, wherein R.sub.1 and R.sub.2 are
3-hydroxyphenyl and R.sub.3 is hydrogen.
17. The method of claim 13, wherein the basal glucose transport
inhibitor compound of formula (I) or pharmaceutically acceptable
salt thereof is administered by at least one of the following
methods: oral, topical, intra-arterial, intrapleural, intrathecal,
intraventricular, subcutaneous, intraperitoneal, intraveneous,
intravesicular, and gliadel wafers.
18. The method of claim 13, wherein the subject is a human.
19. The method of claim 13, further comprising administering to the
subject in need of such treatment a second cancer drug.
20. The method of claim 19, wherein the second cancer drug is
selected from the group consisting of methotrexate, doxorubicin
hydrochloride, fluorouracil, everolimus, imiquimod, aldesleukin,
alemtuzumab, pemetrexed disodium, palonosetron hydrochloride,
chlorambucil, aminolevulinic acid, anastrozole, aprepitant,
exemestane, nelarabine, arsenic trioxide, ofatumumab, bevacizumab,
azacitidine, bendamustine hydrochloride, bexarotene, bleomycin,
bortezomib, cabazitaxel, irinotecan hydrochloride, capecitabine,
carboplatin, daunorubicin hydrochloride, cetuximab, cisplatin,
cyclophosphamide, clofarabine, ifosfamide, cytarabine, dacarbazine,
decitabine, dasatinib, degarelix, denileukin difitox, denosumab,
dexrazoxane hydrochloride, docetaxel, rasburicase, epirubicin
hydrochloride, oxaliplatin, eltrombopag olamine, eribulin mesylate,
erlotinib hydrochloride, etoposide phosphate, raloxifene
hydrochloride, toremifane, fulvestrant, letrozole, filgrastim,
fludarabim phosphate, pralatrexate, gefitinib, gemcitabine
hydrochloride, gemcitibine-cisplatin, gemtuzumab ozogamicin,
imatinib mesylate, trastuzamab, topotecan hydrochloride,
ibritumomab tiuxetan, romadepsin, ixabepilone, palifermin,
lapatinib ditosylate, lenalidomide, leucovorin calcium, leuprolide
acetate, liposomal procarbazine hydrochloride, temozolomide,
plerixafor, acetidine, sorafenib tosylate, nilotinib, tamoxifen
citrate, romiplostim, paclitaxel, pazopanib hydrochloride,
pegaspargase, prednisone, procarbazine hydrochloride, proleukin,
rituximab, romidepsin, sunitinib malate, thalidomide, temsirolimus,
toremifene, pantiumumab, vinblastine sulfate, vincristine,
vorinostat, zoledronic acid, and any combination thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. Utility
patent application Ser. No. 16/522,314, filed Jul. 25, 2019, which
is a continuation of U.S. Utility patent application Ser. No.
15/995,645, filed Jun. 1, 2018, now U.S. Pat. No. 10,385,005, which
is a continuation of U.S. Utility patent application Ser. No.
14/935,902, filed Nov. 9, 2015, now U.S. Pat. No. 10,000,443, which
is a a continuation of U.S. Utility patent application Ser. No.
13/071,386, filed Mar. 24, 2011, now U.S. Pat. No. 9,181,162, which
claims priority to and any other benefit of U.S. Provisional
Application No. 61/317,062, filed Mar. 24, 2010, the entire
disclosures of which are incorporated herein by reference.
BACKGROUND
[0003] Cancer has overtaken cardiovascular diseases as the number
one killer in America since 2008 and it was estimated that 565,650
Americans died of cancer in 2008 alone. Different theories have
been proposed for the cause of cancer and numerous strategies have
been formulated and explored for combating the disease. The death
rates for some cancers such as breast cancer have significantly
reduced in the past three decades primarily due to earlier
detection rather than treatments, while those of other cancers,
such as lung and pancreatic cancer, actually increased. Novel
approaches are absolutely and urgently required for further
improvement in existing cancer therapies and for treating those
cancers for which there are no effective therapies yet. Glucose
deprivation may have the potential to become one such novel and
effective anticancer strategy due to recent progress made in
understanding of the Warburg Effect, the increased and "addicted"
reliance of cancer cells on increased glucose transport and glucose
metabolism, primarily glycolysis.
[0004] One of the common features of almost all cancers and also
potentially one of their common weaknesses is the increased glucose
uptake and increased dependence on glucose as either a source of
building blocks for cell growth and proliferation, a source for
energy, or both. Although cancer is not a single disease, different
cancers, particularly solid malignant tumors, do share some common
characteristics. One such common characteristic is that they all
grow faster than normal cells and hence require more synthetic
precursors and more energy to maintain their accelerated growth and
proliferation rates. Normal cells can utilize different chemicals,
such as amino acids, lipids and glucose as their energy
sources.
[0005] In contrast to typical cells, the preferred sources for
biosynthesis materials and energy for cancer cells is glucose. For
example, healthy colonocytes derive 60-70% of their energy supply
from short-chain fatty acids, particularly butyrate. Butyrate is
transported across the luminal membrane of the colonic epithelium
via a monocarboxylate transporter, MCT1. Carcinoma samples
displaying reduced levels of MCT1 were found to express the high
affinity glucose transporter, GLUT1, indicating that there is a
switch from butyrate to glucose as an energy/biosynthesis source in
colonic epithelia during transition to malignancy.
[0006] The strongest piece of evidence that almost all cancer cells
in vivo have increased glucose supply and metabolism as compared
with normal cells in the body has been provided by positron
emission tomography (PET) scans (FIG. 14). In the PET scan of
cancer, .sup.18F-labeled 2-deoxyglucose (2-DG or FDG) as a
non-metabolizable glucose analog was used as a tracer. The regions
that light up in the scan are organs, tissues, cells, and cancers
that trap more FDG. Brighter spots indicate a higher FDG
concentration. This specific PET scan, like many others, reveals
that both primary and metastatic cancers (near the lung and armpit)
contain higher FDG concentrations than surrounding normal cells,
providing strong evidence that cancer cells have increased glucose
uptake relative to normal cells. The PET scans on various cancer
types, including both primary and secondary metastatic cancers,
have shown that almost all of the studied tumors "trapped"
significantly more FDG as compared to the normal cells and tissues
surrounding the tumors. Furthermore, PET scan studies have
consistently correlated poor prognosis and increased tumor
aggressiveness with increased glucose uptake and upregulated
glucose transporters. Although various theories have been proposed
to explain the mechanisms by which glucose is used inside cancer
cells, there is a near-unanimous consensus in the field that
glucose uptake in almost all malignant tumors is increased
regardless of how glucose is used by cancer cells after it is taken
up. The increased glucose uptake and its accompanied increased
glucose metabolism by cancer cells can be, should be, and has been
becoming a general target for intensive basic and clinical research
and for developing novel anti-cancer therapies.
[0007] In the 1920s, Warburg discovered that, even in the presence
of abundant oxygen, cancer cells prefer to metabolize glucose by
glycolysis in cytosol than the oxidative phosphorylation in
mitochondria as in normal cells. This is seemingly paradoxical as
glycolysis is less efficient in generating ATP. It has been
suggested that such a switch to glycolysis confers cancer cells
some selective advantages for survival and proliferation in the
unique tumor microenvironment. Because of accelerated growth rates
and insufficient oxygen supply, a significant portion of cancer
cells in a nodule are in a hypoxic condition, forcing cancer cells
to make a shift toward glycolysis by increasing expression of
glucose transporters, glycolytic enzymes, and inhibitors of
mitochondrial metabolism. However, the Warburg Effect cannot be
explained solely by adaptation to hypoxia, since glycolysis is
preferred by cancer cells even when ample oxygen is present. Other
molecular mechanisms are likely to be involved.
[0008] Recent studies have shown that the phenomena observed in
Warburg effect, increase glucose consumption and decreased
oxidative phosphorylation, and accompanying drastically increased
lactate production can also be found in oncogene activation. Ras,
when mutated, was found to promote glycolysis. The activation of
Akt was found to increase the rate of glycolysis, partially due to
its ability to promote the expression of glycolytic enzymes through
HIF.alpha.. This was speculated as a major factor contributing to
the highly glycolytic nature of cancer cells. Myc, the
proto-oncogene and a transcription factor, has also been found to
upregulate the expression of various metabolic genes. Tumor
suppressors, such as p53, have also been found to be involved in
regulation of metabolism. All of these recent findings suggest that
the Warburg effect in cancer cells is not simply a result of
isolated changes in glycolysis alone, but is a biological
consequence of extensive communications made through known and
unknown cross-talk network among multiple signaling pathways. These
pathways are involved in cell growth, proliferation, and both
mitochondrial and glucose metabolism that respond to changes in
oxygen and nutrient supply. Understanding such extensive signaling
networks in the Warburg effect is essential for both understanding
and combating cancer.
[0009] Some of the most recent studies have focused on glycolytic
enzymes, particular on pyruvate kinase (PK). These studies have
shown that increased glucose transport and glycolysis in cancer
cells appear to be directed toward the generation of building
blocks (biosynthesis of macromolecules) in cancer cells, and making
preparations for cell division and proliferation rather than as a
means to provide bioenergy (ATP). Although aerobic glycolysis is
generally accepted as a metabolic hallmark of cancer, its causal
relationship with tumorigenesis is still unclear. Glycolysis genes
comprise one of the most upregulated gene sets in cancer. Among
genes significantly upregulated in tumors is PK, which regulates
the rate-limiting final step of glycolysis. Four isoforms of PK
exist in mammals: the L and R isoforms are expressed in liver and
red blood cells; the M1 isoform is expressed in most adult tissues;
and the M2 isoform is a splice variant of M1 expressed during
embryonic development. Notably, it has been reported that tumor
tissues exclusively express the embryonic M2 isoform of pyruvate
kinase. Because of its almost ubiquitous presence in cancer cells,
PKM2 has been designated as tumor specific, and its presence in
human plasma is currently being used as a molecular marker for the
diagnosis of various cancers. Both normal proliferating cells and
tumor cells express PKM2. PKM2 regulates the proportions of glucose
carbons that are channeled to synthetic processes (inactive dimeric
form) or used for glycolytic energy production (highly active
tetrameric form, a component of the glycolytic enzyme complex). In
cancer cells, the dimeric form of PKM2 is always predominant. The
switch between the tetrameric and dimeric form of PKM2 allows tumor
cells to survive in environments with varying oxygen and nutrient
supplies. The transition between the two forms regulates the
glycolytic flux in tumor cells. These findings suggest that PKM2 is
a metabolic sensor which regulates cell proliferation, cell growth
and apoptotic cell death in a glucose supply-dependent manner.
Nuclear translocation of PKM2 was found to be sufficient to induce
cell death that is caspase-independent and isoform-specific. These
results show that the tumor marker PKM2 plays a general role in
caspase-independent cell death of tumor cells and thereby defines
this glycolytic enzyme as a novel target for cancer therapy
development.
[0010] Two recent studies demonstrate that PKM2 is regulated by
binding to phospho-tyrosine motifs, leading to promotion of
increased cell growth and tumor development. PKM2 enhances the use
of glycolytic intermediates for macromolecular biosynthesis and
tumor growth. These findings illustrate the distinct advantages of
this metabolic phenotype in cancer cell growth. It appears that the
expression of PKM2 and switch from oxidative phosphorylation to
aerobic glycolysis is absolutely required for maintaining cancer
growth and proliferation. Thus, inhibiting glycolysis as well as
PKM2 may constitute a new and effective anticancer strategy. These
new findings are significant in that they have almost completely
changed our conventional understanding of the biological functions
of the Warburg effect in cancer, which was believed to be for
biosynthesis of ATP under hypoxic conditions.
[0011] Glucose is an essential substrate for metabolism in most
cells. Because glucose is a polar molecule, transport through
biological membranes requires specific transport proteins.
Transport of glucose through the apical membrane of intestinal and
kidney epithelial cells depends on the presence of secondary active
Na.sup.+/glucose symporters, SGLT-1 and SGLT-2, which concentrate
glucose inside the cells, using the energy provided by co-transport
of Na.sup.+ ions down their electrochemical gradient. Facilitated
diffusion of glucose through the cellular membrane is otherwise
catalyzed by glucose carriers (protein symbol GLUT, gene symbol
SLC2 for Solute Carrier Family 2) that belong to a superfamily of
transport facilitators (major facilitator superfamily) including
organic anion and cation transporters, yeast hexose transporter,
plant hexose/proton symporters, and bacterial sugar/proton
symporters. Molecule movement by such transport proteins occurs by
facilitated diffusion. This characteristic makes these transport
proteins energy independent, unlike active transporters which often
require the presence of ATP to drive their translocation mechanism,
and stall if the ATP/ADP ratio drops too low.
[0012] Basal glucose transporters (GLUTs) function as glucose
channels and are required for maintaining the basic glucose needs
of cells. These GLUTs are constitutively expressed and functional
in cells and are not regulated by (or sensitive to) insulin. All
cells use both glycolysis and oxidative phosphorylation in
mitochondria but rely overwhelmingly on oxidative phosphorylation
when oxygen is abundant, switching to glycolysis at times of oxygen
deprivation (hypoxia), as it occurs in cancer. In glycolysis,
glucose is converted to pyruvate and 2 ATP molecules are generated
in the process (FIG. 15). Cancer cells, because of their faster
proliferation rates, are predominantly in a hypoxic (low oxygen)
state. Therefore, cancer cells use glycolysis (lactate formation)
as their predominant glucose metabolism pathway. Such a glycolytic
switch not only gives cancer higher potentials for metastasis and
invasiveness, but also increases cancer's vulnerability to external
interference in glycolysis since cancer cells are "addicted" to
glucose and glycolysis. The reduction of basal glucose transport is
likely to restrict glucose supply to cancer cells, leading to
glucose deprivation that forces cancer cells to slow down growth or
to starve. Thompson's group found that activated Akt led to
stimulated aerobic glucose metabolism in glioblastoma cell lines
and that the cells then died when glucose was withdrawn. This
provides direct evidence that cancer cells are very sensitive to
glucose concentration change and glucose deprivation could induce
death in cancer cells.
[0013] In normal cells, as shown in FIG. 15, extracellular glucose
is taken up by target cells through one or more basal glucose
transporters (GLUTs). GLUTs used by cells depend on cell types and
physiological needs. For example, GLUT1 is responsible for low
level of basal glucose transport in all cell types. All GLUT
proteins contain 12 transmembrane domains and transport glucose by
facilitating diffusion, an energy-independent process. GLUT1
transports glucose into cells probably by alternating its
conformation. According to this model, GLUT1 exposes a single
substrate-binding site toward either the outside or the inside of
the cell. Binding of glucose to one site triggers a conformational
change, releasing glucose to the other side of the membrane.
Results of transgenic and knockout animal studies support an
important role for these transporters in the control of glucose
utilization, glucose storage and glucose sensing. The GLUT proteins
differ in their kinetics and are tailored to the needs of the cell
types they serve. Although more than one GLUT protein may be
expressed by a particular cell type, cancers frequently over
express GLUT1, which is a high affinity glucose transporter, and
its expression level is correlated with invasiveness and metastasis
potentials of cancers, indicating the importance of upregulation of
glucose transport in cancer cell growth and in the severity of
cancer malignancy. GLUT1 expression was also found to be
significantly higher than that of any other glucose transporters.
In one study, all 23 tumors tested were GLUT1-positive and GLUT1
was the major glucose transporter expressed. In addition, both FDG
uptake and GLUT1 expression appear to be associated with increased
tumor size. In several tumors including NSCLC, colon cancer,
bladder cancer, breast cancer, and thyroid cancers, increased GLUT1
expression not only confers a malignant phenotype but also predicts
for inferior overall survival. Based on all these observations, it
is conceivable that inhibiting cancer growth through basal glucose
transport inhibition may be an effective way to block cancer growth
and improve on prognosis and survival time.
[0014] Evidence indicates that cancer cells are more sensitive to
glucose deprivation than normal cells. Numerous studies strongly
suggest that basal glucose transport inhibition induces apoptosis
and blocks cancer cell growth. First, anti-angiogenesis has been
shown to be a very effective way to restrict cancer growth and
cause cancer ablation. In essence, the anti-angiogenesis approach
is to reduce new blood vessel formation and achieve blood vessel
normalization inside and surrounding the tumor nodules. This
severely limits the nutrients necessary for tumor growth from
reaching the cancer cells. One of the key nutrients deprived by
anti-angiogenesis is glucose. In this sense, inhibition of basal
glucose transport can be viewed as an alternative approach to
anti-angiogenesis therapy in restricting nutrient supply to cancer
cells. Thus, the success of the anti-angiogenesis strategy
indirectly supports the potential efficacy of limiting glucose
supply to cancer cells as a related but novel strategy. Second,
inhibitors of various enzymes involved in glycolysis, have been
used to inhibit different steps in the glycolysis process, and
shown to have significant anti-cancer efficacies. The glycolytic
enzymes that have been targeted include: hexokinase, an enzyme that
catalyzes the first step of glycolysis; ATP citrate lyase; and more
recently pyruvate dehydrogenase kinase (PDK). Among glycolysis
inhibitors tested, 3-bromopyruvate and a hexokinase inhibitor were
found to completely eradicate advanced glycolytic tumors in treated
mice. Compounds targeting mitochondrial glycolytic enzyme lactate
dehydrogenase A (LDH-A) have shown significant anti-cancer activity
both in vitro and in vivo. This result suggests a strong connection
between mitochondrial function and cytosolic glycolysis. 2-DG, the
tracer used in PET scans for locating metastasis, has been used as
a glucose competitor and a glycolytic inhibitor in anti-cancer
clinical trials. These and other related studies have also shown
that these inhibitors induced apoptosis in cancer cells as a cancer
cell killing mechanism. Two important conclusions can be drawn from
all these published studies. (1) The compounds that inhibit various
steps of glycolysis reduce cancer cell growth both in vitro and in
vivo, and (2) inhibiting one of the various steps of glycolysis
induces apoptosis in cancer cells and is an effective anti-cancer
strategy. They also strongly suggest that inhibiting glucose
transport, the step immediately before glycolysis and the first
rate-limiting step for glycolysis and all glucose metabolism inside
cells, should produce biological consequences to cancer cells
similar to or potentially more severe as glycolysis inhibition. In
addition, glucose transport may potentially be a better target than
downstream glycolysis targets because 1) glucose transporters are
known to be highly upregulated in cancer cells, 2) by restricting
the glucose supply at the first step and thus, creating an absolute
intracellular glucose shortage, it will prevent any potential
intracellular glucose-related compensatory/salvage pathways that
cancer cells may use for self-rescue and avoidance of cell
death.
[0015] For inhibiting basal glucose transport to become a
successful anti-cancer strategy, it must kill cancer cells without
significantly harming the normal cells. Some experimental
observations indicate that this is indeed the case. Because cancer
cells favor the use of glucose as the energy source and glycolysis
is upregulated in cancer cells, compounds that inhibit glycolysis
may kill cancer cells while sparing normal cells, which can use
fatty acids and amino acids as alternative energy sources.
[0016] It has recently been reported that the addition of
anti-GLUT1 antibodies to various lung and breast cancer cell lines
significantly reduced the glucose uptake rate and proliferation of
cancer cells, leading to induction of apoptosis. Furthermore, the
antibodies potentiated the anti-cancer effects of cancer drugs such
as cisplatin, paclitaxel and gefitinib. These results clearly
indicate that agents that inhibit GLUT1-mediated glucose transport
are effective either when working alone or when used in combination
with other anti-cancer therapeutics to inhibit cancer cell growth
and induce apoptosis in cancer cells. These findings are further
supported by two recent publications in which glucose transport
inhibitor fasentin was found to sensitize cancer cells to undergo
apoptosis induced by anticancer drugs cisplatin or paclitaxel and
anticancer compound apigenin was found to down-regulate GLUT1 at
mRNA and protein levels. Down-regulation of GLUT1 was proposed as
the potential anticancer mechanism for apigenin. All these new
findings point to the direction that glucose transport inhibitors
are likely to sensitize and synergize with other anticancer drugs
to further enhance anticancer efficacy of the drugs. Disclosed
herein are compounds and methods that are 2-5 times more potent
than either fasentin or apigenin in inhibiting basal glucose
transport and induction of apoptosis.
[0017] In one recent study using glucose deprivation, cells growing
in high concentrations of growth factors were found to show an
increased susceptibility to cell death upon growth factor
withdrawal. This susceptibility correlated with the magnitude of
the change in the glycolytic rate following growth factor
withdrawal. To investigate whether changes in the availability of
glycolytic products influence mitochondrion-initiated apoptosis,
glycolysis was artificially limited by manipulating the glucose
levels in cell culture media. Like growth factor withdrawal,
glucose limitation resulted in Bax translocation, a decrease in
mitochondrial membrane potential, and cytochrome c release to the
cytosol. In contrast, increasing cell autonomous glucose uptake by
over-expression of GLUT1 significantly delayed apoptosis following
growth factor withdrawal. These results suggest that a primary
function of growth factors is to regulate glucose uptake and
metabolism and thus maintain mitochondrial homeostasis and enable
anabolic pathways required for cell growth. It was also found that
expression of the three genes involved in glucose uptake and
glycolytic commitment, GLUT1, hexokinase 2, and phosphofructokinase
1, was rapidly declined to nearly undetectable levels following
growth factor withdrawal. All of these studies suggest that glucose
deprivation has been a very valuable and frequently used method for
studying cancer. Intracellular glucose deprivation can also be
created by inhibition of basal glucose transport. The difference
between glucose deprivation resulted from glucose removal from cell
culture media and from inhibition of glucose transport/glucose
metabolism is that glucose removal generates initially a glucose
deprived extracellular environment while inhibition of glucose
transport/glucose metabolism generates a glucose deprived
intracellular environment without changing or even increasing
extracellular glucose concentration. The use of glucose transport
inhibitors should be able to supplement and substitute traditional
glucose deprivation. Furthermore, traditional glucose deprivation
by decreasing extracellular glucose concentration cannot be used in
animals while inhibitors of glucose transport can, creating a new
approach in studying cancer in vivo and in treating cancer.
SUMMARY
[0018] Disclosed herein are compounds of formula (I), in which
R.sub.1 is selected from a group consisting of hydrogen, alkyl,
benzyl, aryl, and heteroaryl moieties; R.sub.2 is selected from the
group consisting of hydrogen, alkyl, benzyl, aryl, heteroaryl, and
fluorescent tags; R.sub.3 is selected from the group consisting of
hydrogen, halo, alkyl, benzyl, aryl, heteroaryl, amino, cyano, and
alkoxy; or a salt thereof. In some embodiments, the two R.sub.1
groups may be independently selected, and hence different as
recognized by one of skill in the art. In other embodiments, when
the R.sub.1 groups are different, R.sub.1 may be represents as
R.sub.1' and R.sub.1'' to indicate a difference between R.sub.1
moieties.
##STR00001##
[0019] In some embodiments, the compound of formula (I) may be
further defined to include species where R.sub.1 is an aryl
functionality selected from the group consisting of 2-, 3-, and
4-hydroxyphenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and
3,5-dihydroxyphenyl, 2,3,4-, 2,3,5-, 2,3,6-, and
3,4,5-trihydroxyphenyl, 2,3,4,5- and 2,3,4,6-tetrahydroxyphenyl,
and perhydroxyphenyl. In other embodiments, the compound of formula
(I) may be further defined where R.sub.2 is a fluorescent tag
selected from the group consisting of coumarins, dansyl, rhodamine,
fluorescein, and carboxynaphthofluorescein. In some embodiments,
the compound of formula (I) is consisting of a molecule, in which
R.sub.1 and R.sub.2 are equal to 3-hydroxyphenyl and R.sub.3 is a
hydrogen atom.
[0020] Disclosed herein are compounds of formula (II), in which
R.sub.1 is selected from the group consisting of hydrogen, alkyl,
benzyl, aryl, and heteroaryl; R.sub.2 is selected from the group
consisting of hydrogen, alkyl, benzyl, aryl, and heteroaryl; X is
selected from the group consisting of hydrogen, halo, alkyl,
benzyl, aryl, heteroaryl, amino, cyano, and alkoxy; Y is selected
from the group consisting of hydrogen, halo, alkyl, benzyl, aryl,
heteroaryl, amino, cyano, and alkoxy; or a salt thereof.
##STR00002##
[0021] In some embodiments, the compound of formula (II) may be
further defined to include species where R.sub.1 is an aryl
functionality selected from the group consisting of 2-, 3-, and
4-hydroxyphenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and
3,5-dihydroxyphenyl, 2,3,4-, 2,3,5-, 2,3,6-, and
3,4,5-trihydroxyphenyl, 2,3,4,5- and 2,3,4,6-tetrahydroxyphenyl,
and perhydroxyphenyl. In other embodiments, the compound of formula
(II) is consisting of a molecule in which R.sub.1 and R.sub.2 are
equal to 3-hydroxyphenyl, X is equal to fluorine, and Y is equal to
hydrogen.
[0022] Disclosed herein is a series of compounds of formula (III),
in which R.sub.1 is selected from the group consisting of hydrogen,
halo, alkyl, benzyl, amino, nitro, cyano, and alkoxy; R.sub.2 is
selected from the group consisting of hydrogen, halo, alkyl,
benzyl, amino, nitro, cyano, and alkoxy; R.sub.3 is selected from
the group consisting of hydrogen, halo, alkyl, benzyl, amino,
nitro, cyano, and alkoxy; X is selected from the group consisting
of carbon, oxygen, nitrogen and sulfur; and Y is selected from the
group consisting of carbon, oxygen, nitrogen and sulfur; or, a salt
thereof.
##STR00003##
[0023] In some embodiments, the compound of formula (III) may be
selected from the group consisting of the following compounds;
##STR00004## ##STR00005##
In some embodiments, the compound of formula (III) may be selected
from the group consisting of the following compounds;
##STR00006## ##STR00007##
and where X is selected from the group consisting of H, 3-Cl, 3-F,
3-CN, 4-F, 4-CN, 4-NO.sub.2, 4-SO.sub.2Me, and 4,5-Cl.sub.2. In
other embodiments, the compound of formula (III) is consisting of a
molecule in which R.sub.1, R.sub.2, and R.sub.3 are hydrogen, and X
and Y are oxygen.
[0024] Disclosed herein are compounds of formula (IV), in which
R.sub.1 is selected from the group consisting of hydrogen, halo,
alkyl, benzyl, amino, nitro, cyano, and alkoxy; R.sub.2 is selected
from the group consisting of hydrogen, alkyl, benzyl, aryl, and
heteroaryl; R.sub.3 is selected from the group consisting of
hydrogen, alkyl, benzyl, aryl, and heteroaryl; or a salt thereof.
In some embodiments, the compound of formula (IV) is a molecule, in
which R.sub.1 is chlorine, and R.sub.2 and R.sub.3 are
2-nitro-5-hydroxyphenyl groups.
##STR00008##
[0025] Disclosed herein are methods for the treating cancer
involving the administration of a therapeutically effective amount
of a compound selected from the group consisting of formula (I),
formula (II), formula (III), and formula (IV) to a subject in need
of such treatment.
[0026] In some embodiments, the cancer is a solid malignant tumor
that upregulates basal glucose transport via a biological shift
from oxidative phosphorylation to glycolysis in a process known as
the Warburg effect. In some embodiments, administration of the
compound to a human subject may be by any method selected from the
group consisting of oral, topical, intra-arterial, intrapleural,
intrathecal, intraventricular, subcutaneous, intraperitoneal,
intraveneous, intravesicular, and gliadel wafers.
[0027] In some embodiments, the compound of formula (I), formula
(II), formula (III), and formula (IV) may be administered to a
human subject or patient in combination with one or multiple
chemotherapeutic agents as a means to enhance the efficacy of one
or more of the therapeutically useful compounds. In other
embodiments, the chemotherapeutic agent that the compound of
formula (I), formula (II), formula (III), and formula (IV) may be
administered in combination with is selected from the group
consisting of methotrexate, doxorubicin hydrochloride,
fluorouracil, everolimus, imiquimod, aldesleukin, alemtuzumab,
pemetrexed disodium, palonosetron hydrochloride, chlorambucil,
aminolevulinic acid, anastrozole, aprepitant, exemestane,
nelarabine, arsenic trioxide, ofatumumab, bevacizumab, azacitidine,
bendamustine hydrochloride, bexarotene, bleomycin, bortezomib,
cabazitaxel, irinotecan hydrochloride, capecitabine, carboplatin,
daunorubicin hydrochloride, cetuximab, cisplatin, cyclophosphamide,
clofarabine, ifosfamide, cytarabine, dacarbazine, decitabine,
dasatinib, degarelix, denileukin difitox, denosumab, dexrazoxane
hydrochloride, docetaxel, rasburicase, epirubicin hydrochloride,
oxaliplatin, eltrombopag olamine, eribulin mesylate, erlotinib
hydrochloride, etoposide phosphate, raloxifene hydrochloride,
toremifane, fulvestrant, letrozole, filgrastim, fludarabim
phosphate, pralatrexate, gefitinib, gemcitabine hydrochloride,
gemcitibine-cisplatin, gemtuzumab ozogamicin, imatinib mesylate,
trastuzamab, topotecan hydrochloride, ibritumomab tiuxetan,
romadepsin, ixabepilone, palifermin, lapatinib ditosylate,
lenalidomide, leucovorin calcium, leuprolide acetate, liposomal
procarbazine hydrochloride, temozolomide, plerixafor, acetidine,
sorafenib tosylate, nilotinib, tamoxifen citrate, romiplostim,
paclitaxel, pazopanib hydrochloride, pegaspargase, prednisone,
procarbazine hydrochloride, proleukin, rituximab, romidepsin, Talc,
sorafenic tosylate, sunitinib malate, thalidomide, temsirolimus,
toremifene, pantiumumab, vinblastine sulfate, vincristine,
vorinostat, and zoledronic acid.
[0028] Additional features and advantages will be set forth in part
in the description that follows, and in part will be obvious from
the description, or may be learned by practice of the inventions.
The objects and advantages of the inventions will be realized and
attained by means of the elements and combinations particularly
pointed out in the appended claims.
[0029] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and not restrictive of the invention, as
claimed.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0030] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate some
embodiments of the inventions, and together with the description,
serve to explain principles of the inventions.
[0031] FIG. 1 shows the initial glucose transport inhibitors 1a and
2a.
[0032] FIG. 2 shows glucose uptake results for the possible
hydrolyzed products from 1p, 9a, and 2a. These possible hydrolyzed
products do not show significant inhibition of basal glucose
uptake, suggesting that the inhibition was due to the original
compounds, not the hydrolyzed products.
[0033] FIG. 3 shows the molecular structures of .alpha.-PGG and
.beta.-PGG. PGG has a glucose core that is linked to five galloyl
groups through ester bonds that are formed between the hydroxyl
groups of glucose and gallic acids. .alpha.-PGG and .beta.-PGG are
structural isomers. .alpha.-PGG and its derivatives are hydrophilic
and are likely to work extracellularly on cell membrane
proteins.
[0034] FIG. 4 shows the structure of novel inhibitors of basal
glucose transport WZB-25, WZB-26, and WZB-27.
[0035] FIG. 5 shows .alpha.-PGG treatment induces cell death in
cancer cells and the cell death was primarily mediated by
apoptosis. A. .alpha.-PGG treatment led to 70-80% reduction of cell
viability as measured by a cell viability (MTT) assay. B.
.alpha.-PGG treatment resulted in more than 2-fold increase in
apoptosis in HeLa cells as measured by an apoptosis (ELISA) assay.
C. .alpha.-PGG treatment of HeLa cells led to a decrease in G1
phase cells but a significant increase (.about.3-fold) in apoptotic
cells as determined by an antibody-coupled flow cytometry
study.
[0036] FIG. 6 shows p53 activation and inactivation as determined
by Western blot analyses. When HeLa, RKO, or MCF-7 cells were
treated with 25 .mu.M .alpha.-PGG, p53 protein was found not to be
activated in HeLa cells but was activated in RKO cells.
[0037] FIG. 7 shows .alpha.-PGG and its derivatives inhibit glucose
uptake in HeLa, RKO, and MCF-7 cancer cells. Cells were treated
with .alpha.-PGG for 20 minutes before .sup.3H-labeled 2-DG. Thirty
minutes after the addition of 2-DG, cells were harvested, lysed,
and counted for their respective glucose uptake. Samples: 1. Mock;
2. Insulin (100 nM); 3. .alpha.-PGG (30 .mu.M); 4. WZB-25 (30
.mu.M); 5. WZB-26 (30 .mu.M); and 6. WZB-27 (30 .mu.M).
[0038] FIG. 8 shows that .alpha.-PGG and its derivatives inhibit
basal glucose transport in HeLa cells in dose-dependent manners. A.
.alpha.-PGG inhibits basal glucose transport in HeLa cells in a
dose-dependent manner. B. WZB-25 inhibits basal glucose transport
in HeLa cells in a dose-dependent manner. C. WZB-27 inhibits basal
glucose transport in HeLa cells in a dose-dependent manner.
[0039] FIG. 9 shows a time course of glucose transport inhibition
induced by .alpha.-PGG or .alpha.-PGG derivatives. The glucose
uptake assay was conducted the same way as previously described
except that the glucose uptake was terminated at different times
(from 1, 5, 10, and up to 30 minutes).
[0040] FIG. 10 shows .alpha.-PGG induces Akt while .alpha.-PGG
derivatives do not induce Akt. CHO cells overexpressing the insulin
receptor were treated with .alpha.-PGG or its derivatives. After
treatment, cells were lysed and proteins were analyzed by the
antibody specific for phosphorylated Akt.
[0041] FIG. 11 shows PGG-derived compounds induce more cell death
in cancer cells than in their normal cell counterparts. The left
panel shows compounds were used to treat human lung cancer cells
(H1299) or normal lung cells (NL20) at 25 mM. Forty-eight hours
after the treatment, cell viability assay was performed to
determine percentage of cell killing. Cells without compound
treatment were used as controls (100% baseline). The right panel
shows compounds used to treat human breast cancer cells (MCF-7) and
normal breast cells (MCF-12A). Cell viability assay was performed
at the same conditions as for the lung cancer cells in the left
panel.
[0042] FIG. 12 shows cell viability and apoptotic assays. A. Cell
viability assay in RKO colon cancer cells with .alpha.-PGG, W25
(WZB-25), W27 (WZB-27), and C7 treatments. RKO cells contain high
levels of p53 while RKO E6 cells contain much lower levels of p53.
B. Apoptosis assay in H1299 lung cancer cells using cleaved PARP
protein (89 kDa) as an indicator for Caspase 3 since PARP is a
substrate of activated Caspase 3.
[0043] FIG. 13 shows a schematic presentation of potential
mechanisms for cancer cell killing.
[0044] FIG. 14 shows a PET scan of primary and metastatic human
cancer.
[0045] FIG. 15 shows a schematic presentation of glucose transport
in normal cells.
[0046] FIG. 16 shows new PGG derivatives.
[0047] FIG. 17 shows the structure of two tested compound
inhibitors. Compounds WZB-27 and WZB-115 are polyphenolic compounds
derived from PGG. Unlike PGG, they do not have insulin-like glucose
uptake stimulatory activity. On the contrary, they only possess
potent glucose transport inhibitory activity and anticancer
activity as demonstrated in glucose uptake and MTT cell viability
assays.
[0048] FIG. 18 shows cell viability and apoptosis assays
(W25=WZB-25, W27=WZB-27). A. Cell viability assay in RKO colon
cancer cells. RKO cells contain high levels of p53 while RKO E6
cells contain much lower levels of p53. B. .alpha.-PGG induces
stronger viability-lowering effect in RKO than in RKO-E6 cells. RKO
cells have higher level of p53 while RKO-E6 is p53 deficient.
***p<0.001, **p<0.01. C. Apoptosis assay in A549 lung cancer
cells using cleaved PARP protein (89 kDa) as an indicator for
Caspase 3 since PARP is a substrate of activated Caspase 3. Low
glucose (5% of normal) treated samples served as positive
controls.
[0049] FIG. 19 shows cancer cells express more GLUT1 protein and
are inhibited more by compounds in glucose uptake more their
non-cancerous counterparts. Cancer cells and their non-cancerous
counterparts were treated with or without compounds and then
measured for their respective glucose uptakes. A. Glucose uptake
assay of H1299 lung cancer cells and their non-cancerous NL20 cells
treated with or without WZB-27. B. Glucose uptake assay of MCF7
cancer cells and their non-cancerous MCF12A cells. C. Western blot
analysis of GLUT1 protein expression in cancer and non-cancerous
cells using antibody specific against GLUT1 (H43 fragment).
.beta.-actin served as protein loading control.
[0050] FIG. 20 shows blood glucose levels after compound injection.
Compound W27 (=WZB-27) or W115 (=WZB-115) was injected IP into
fasting Balb/c healthy mice and blood glucose levels were measured
multiple times post injection. N=5 per group. PBS+DMSO group was
the vehicle control.
[0051] FIG. 21 shows a combination of glucose inhibitors and
anticancer drugs further reduces cancer cell viability. Anticancer
drugs cisplatin (2.5 .mu.M for W27 (=WZB-27) study or 5 .mu.M for
W115 (=WZB-115) study) or taxol (2.5 .mu.M) was used to treat
either H1299, A549 lung cancer cells or MCF7 breast cancer cells in
the absence or presence of 10 .mu.M of WZB-115 or 30 .mu.M of
WZB-27. Cell viability was measured by the MTT assay. The presence
of the compounds significantly increased cancer cell death induced
by either cisplatin or taxol. This experiment was repeated three
times and the results were presented as means.+-.standard
deviations.
[0052] FIG. 22 shows a comparison study between in house compound
inhibitors with fasentin in glucose uptake inhibition and cell
viability in cancer cells. Known basal glucose compound fasentin
and compound inhibitors generated in house were compared side by
side in both glucose uptake assay and cell viability (MTT) assay.
Glucose uptake or cell viability of mock treated samples was
arbitrarily assigned a value of 100%. A. Glucose uptake assay in
H1299 cancer cells. Concentration for all compounds was 30 .mu.M.
B. Cell viability assays in three different cancer cell lines.
Concentration for all compounds was 60 .mu.M.
[0053] FIG. 23 shows an NCI anticancer activity screening results
for compound WZB-115 in 59 cancer cell lines. WZB-115 was sent to
NCI for anticancer activity screening using 59 cancer cell lines,
including 9 cancer types. The test was done at a single
concentration: 10 .mu.M. Growth rates of mock treated cancer cells
were used as baseline 100%. Any growth rate that is smaller than
100% indicates an inhibition. Because of its promising anticancer
activity profile, NCI has recommended that the compound be tested
again using five different concentrations to determine its
IC.sub.50s in these cancer cell lines.
[0054] FIG. 24 shows a general scheme for identifying improved
basal glucose transport inhibitors.
[0055] FIG. 25 shows several lead compounds and areas for
structural modification.
[0056] FIG. 26 shows an initial set of proposed linkage
analogs.
[0057] FIG. 27 shows energy-minimized structures of proposed
linkage analogs.
[0058] FIG. 28 shows biosteric analogs of the phenol group.
[0059] FIG. 29 shows an initial set of core aromatic rings.
[0060] FIG. 30 shows a comparison of tumor sizes of human lung
cancer A549 grafted on nude mice. Photos were taken 8 weeks after
the treatment. Male NU/J nude mice (7-8 weeks old) were used and
purchased from The Jackson Laboratory (Bar Harbor, Me.) and
provided the Irradiated Teklad Global 19% protein rodent diet from
Harlan Laboratories (Indianapolis, Ind.). To determine the in vivo
efficacy of compound WZB-117 against human NSCLC tumor xenograft
growth, exponentially growing A549 cells were harvested and
re-suspended in PBS to achieve a final concentration of
5.times.10.sup.6 cells in 25 .mu.l suspension. Each mouse was
injected subcutaneously in the right flank with 25 .mu.l cell
suspension. At this time, mice were randomly divided into two
groups: control group (n=10) treated with PBS/DMSO (1:1, v/v), and
WZB-117 treatment group (n=10), treated with WZB-117 (15 mg/kg).
Compound WZB-117 was dissolved in PBS/DMSO (1:1, v/v). Mice were
given intraperitoneal injection with either PBS/DMSO mixture or
compound WZB-117 (15 mg/kg) daily since the day of tumor cell
inoculation. A. Tumor growth curve. Animal tumor study indicated
that, by daily injection of WZB-117 at the dose of 10 mg/kg body
weight, the tumor size of the compound treated tumors were on
average approximately 75% smaller than that of the mock treated
mice. B. Mouse tumor photos. Tumor-bearing mice on the left were
mock-treated while the mice on the right were treated with WZB-117.
C. Mouse body weight measurements. D. Body weight compositions of
the WZB-117 treated mice compared to those of mock-treated
mice.
[0061] FIG. 31 shows WZB-117 inhibition of glucose transport by
inhibiting Glut1. A. and B. WZB-117 inhibits glucose transport in
red blood cells (RBC) in a dose-dependent fashion. C. and D.
WZB-117 inhibits glucose transport in RBC derived "inside out"
vesicles (IOV) in a dose-dependent fashion. E. WZB-117 inhibits
glucose transport in RBC derived "right side out" (ROV)
vesicles.
[0062] FIG. 32 shows WZB-117 treatment of cancer cells induce ER
stress, apoptosis and change in glycolytic enzymes. A. WZB-117
treatment upregulates ER stress protein BiP in a similar way as
glucose deprivation. B. WZB-117 treatment induces cleavage of PARP,
suggesting the apoptosis induction mediated by p53. C. WZB-117
treatment upregulates the key glycolytic enzyme PKM2 in cancer
cells in a similar manner as the glucose deprivation.
[0063] FIG. 33 shows food intake of mice treated with or without
WZB-117. There was no change of food intake between the PBS/DMSO
and the WZB-117 treated group during the study. Food intake of each
group was measured every 7 days since tumor cell inoculation.
[0064] FIG. 34 shows blood glucose measurement of tumor-bearing
nude mice treated with or without WZB-117. Blood glucose level of
each mouse was measured by a blood glucose monitor, and it was
measured right before the IP injection of WZB-117 (15 mg/kg) and
every 30 minutes after injection. Food was available to mice during
the measurement. Data was expressed in average.+-.standard
deviation. No significant difference in blood glucose levels was
found between untreated and compound WZB-117 injection group
immediately after the compound injection or during or after the
animal study.
[0065] FIG. 35 shows that compound WZB-117 kills significantly more
cancer cells than non-cancerous cells. A. A549 lung cancer and B.
MCF7 breast cancer cells were treated with or without WZB-117 for
48 hr, and then measured for their respective viability rates with
the MTT assays. Mock-treated cells served as controls (100%
viability) for comparison. Noncancerous NL20 and MCF12A cells were
treated the same way for comparison.
[0066] FIG. 36 shows the anticancer activity of WZB-117 as
demonstrated by clonogenic assays. Three cancer cell lines A549,
H1299 (lung cancers) and MCF7 (breast cancer) grown in culture
dishes were treated with WZB-117 or a weaker inhibitor WZB-134 or
no compound (mock) for 48 hrs. Then the treated cells were allowed
to grow back in compound-free normal cell culture medium for 2
weeks and then stained with crustal violet and counted for number
of survived clones. The fewer and smaller the stained spots
(clones), the higher the inhibition.
[0067] FIG. 37 shows the structures of several novel glucose
transport inhibitors WZB-115, 117, and 173. Compound 117 is an
analog of 115 while 173 is an ether-bond analog. Compounds WZB-117
and WBZ-173 are derived from compound WZB-115, which is a
polyphenolic model compound used in our previous cancer studies.
WZB-115 was derived from a natural anticancer and antidiabetic
compound called penta-galloyl-glucose (PGG). WZB-117 and WZB-173
are very similar structurally to 115 but are both structurally
simplified and functionally optimized compared to WZB-115. As a
result, WZB-117 and WZB-173 are more potent in their anticancer
activities than WZB-115 and are also structurally more stable than
115 in solution and cell culture media.
DETAILED DESCRIPTION
[0068] The present inventions will now be described by reference to
some more detailed embodiments, with occasional reference to the
accompanying drawings. These inventions may, however, be embodied
in different forms and should not be construed as limited to the
embodiments set forth herein. Rather, these embodiments are
provided so that this disclosure will be thorough and complete, and
will fully convey the scope of the inventions to those skilled in
the art.
[0069] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which these inventions belong. The
terminology used in the description of the inventions herein is for
describing particular embodiments only and is not intended to be
limiting of the inventions. As used in the description of the
inventions and the appended claims, the singular forms "a," "an,"
and "the" are intended to include the plural forms as well, unless
the context clearly indicates otherwise. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety.
[0070] Unless otherwise indicated, all numbers expressing
quantities of ingredients, reaction conditions, and so forth used
in the specification and claims are to be understood as being
modified in all instances by the term "about." Accordingly, unless
indicated to the contrary, the numerical parameters set forth in
the following specification and attached claims are approximations
that may vary depending upon the desired properties sought to be
obtained by the present inventions. At the very least, and not as
an attempt to limit the application of the doctrine of equivalents
to the scope of the claims, each numerical parameter should be
construed in light of the number of significant digits and ordinary
rounding approaches.
[0071] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the inventions are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in their respective testing measurements.
Every numerical range given throughout this specification will
include every narrower numerical range that falls within such
broader numerical range, as if such narrower numerical ranges were
all expressly written herein.
[0072] In addition, where features or aspects of the invention are
described in terms of Markush groups or other grouping of
alternatives, those skilled in the art will recognize that the
invention is also thereby described in terms of any individual
member or subgroup of members of the Markush group or other
group.
Definitions
[0073] "Alkyl" shall refer to any chemical compound that consists
only of the elements hydrogen and carbon, wherein the atoms are
linked together exclusively through single bonds. The term alkyl
may also be extended to mean any chemical compound that consists
only of the elements hydrogen, fluorine, and carbon, wherein the
atoms are linked together exclusively through single bonds. This
class of fluorinated compounds may also be referred to as
"fluoroalkanes," "fluoroalkyl," "fluoroalkyl groups," and
"fluorocarbons." Examples of hydrocarbons include, but are not
limited to methyl, ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl,
sec-butyl, i-butyl, t-butyl, cyclobutyl, n-pentyl, i-pentyl,
neo-pentyl, cyclopentyl, n-hexyl, cyclohexyl, thexyl, n-heptyl,
n-octyl, n-decyl, and adamantyl. Examples of fluorinated compounds
include, but are not limited to fluoromethyl, difluoromethyl,
trifluoromethyl, 1,1,1-trifluoroethyl, 2,2-difluoroethyl, and
perfluoroethyl.
[0074] "Benzyl" shall be used to describe the substituent or
molecular fragment possessing a structure related to
RC.sub.6H.sub.4CH.sub.2-- of an organic compound. A substituted
benzyl compound may also be described as any aryl or heteroaryl
ring system attached to a methylene (--CH.sub.2--) subunit.
Examples of benzyl groups include, but are not limited to benzyl;
2, 3, and 4-halobenzyl; 2, 3, and 4-alkylbenzyl; 2, 3, and
4-cyanobenzyl; 2, 3, and 4-ketobenzyl; 2, 3, and 4-carboxybenzyl;
2, 3, and 4-aminobenzyl; 2, 3, and 4-nitrobenzyl; 2, 3, and
4-hydroxybenzyl; 2, 3, and 4-alkoxybenzyl; disubstituted benzyl,
and trisubstituted benzyl derivatives.
[0075] "Aryl" shall mean any functional group on a compound that is
derived from a simple aromatic ring. Examples include, but are not
limited to phenyl; 2-, 3-, and 4-hydroxyphenyl; 2,3-, 2,4-, 2,5-,
2,6-, 3,4-, and 3,5-dihydroxyphenyl; 2,3,4-, 2,3,5-, 2,3,6-, and
3,4,5-trihydroxyphenyl; 2,3,4,5- and 2,3,4,6-tetrahydroxyphenyl;
perhydroxyphenyl; 2, 3, and 4-halophenyl; 2, 3, and 4-alkylphenyl;
2, 3, and 4-cyanophenyl; 2, 3, and 4-ketophenyl; 2, 3, and
4-carboxyphenyl; 2, 3, and 4-aminophenyl; 2, 3, and 4-nitrophenyl;
2, 3, and 4-hydroxyphenyl; 2, 3, and 4-alkoxyphenyl; disubstituted
phenyl, and trisubstituted phenyl derivatives.
[0076] "Heteroaryl" shall mean any functional group on a compound
that is derived from a heteroaromatic ring. Heteroaromatic species
contain a heteroatom, or an atom other than hydrogen or carbon,
including, oxygen, nitrogen, sulfur, phosphorous, silicon, and
boron. Examples include, but are not limited to furans,
benzofurans, thiophenes, benzothiophenes, pyrroles, indoles, and
borabenzenes.
[0077] "Halo" and "halogen" shall refer to any element of the
periodic table from Group 17, consisting of fluorine, chlorine,
bromine, iodine, and astatine.
[0078] "Amine" and "amino" shall refer to any organic compound or
functional group that contains a basic nitrogen with a lone pair of
electrons. Amines are derived from ammonia and may be primary,
secondary, and tertiary. Examples of amines include, but are not
limited to ammonia, methylamine, dimethlamine, trimethylamine,
ethylamine, diethlamine, triethylamine, ethyldimethylamine,
isopropylamine, diisopropylamine, diisopropylethylamine,
diphenylamine, dibenzylamine, t-butylamine, analine, and
pyridine.
[0079] "Cyano" shall be considered synonymous with the organic
functionality nitrile, which contains a carbon triple-bonded to a
nitrogen atom. Cyanides tend to be highly toxic in nature, and are
typically found as salts.
[0080] "Alkoxy" shall mean an alkyl group singly bonded to an
oxygen. The range of alkoxy groups is great, ranging from methoxy
to any number of arylalkoxy groups. Examples of alkoxy groups
include, but are not limited to methoxy, ethoxy, n-propoxy,
i-propoxy, n-butoxy, t-butoxy, and phenoxy.
[0081] "Fluorescent tags," "fluorescent molecule," "fluorophore,"
and "fluorescent labels" shall mean any portion of a molecule that
scientists or researchers have attached chemically to aid in the
detection of the molecule to which it has been attached. Examples
of fluorescent tags include, but are not limited to coumarins,
dansyl, rhodamine, fluorescein, carboxynaphthofluorescein, and
fluorescent proteins.
[0082] A "salt" shall refer to an ionic species resulting from the
pairing of an anionic derivative of one of the compounds from
formulas 1, 2, 3, and 4 with a cationic species. The cationic
species may include, but is not limited to lithium, sodium,
potassium, rubidium, cesium, beryllium, magnesium, calcium,
strontium, barium, aluminum, copper, zinc, iron, chromium,
manganese, nickel, palladium, platinum, indium, rhodium, and
arsenic.
[0083] "Therapeutically effective" when used to describe an amount
of a compound applied in a method, refers to the amount of a
compound that achieves the desired biological effect, for example,
an amount that leads to the inhibition of basal glucose
transport.
[0084] "Inhibit" or "stop" shall mean reduce, inhibit, damage,
eliminate, kill, or a combination thereof.
[0085] "Lowering" in the context of basal glucose transport shall
mean to reduce the efficiency of glucose transport within a cancer
cell.
[0086] Structure of .alpha.-PGG-Derived Generation 1 Compounds;
WZB-25, WZB-26, and WZB-27
[0087] The methods for the synthesis of compounds WZB-25, WZB-26,
and WZB-27 are as follows:
##STR00009##
[0088] Acid chloride 2 (863 mg, 1.88 mmol) was added to a solution
of 1,6-anhydro-.beta.-D-glucose (100 mg, 0.62 mmol) in anhydrous
acetonitrile (25 mL) at room temperature. DMAP (241 mg, 1.97 mmol)
was added to the reaction mixture after stirred for 30 min at room
temperature, the mixture was stirred for 24 h and the solvent was
removed, the crude was purified by chromatography on silica gel
giving 730 mg of 3 in 83% yield. .sup.1H NMR (CDCl3) .delta.
7.60-7.31 (m, 51H), 5.88 (s, 1H), 5.69 (t, 1H, J=3.0 Hz), 5.29 (d,
3H, J=5.3 Hz), 5.23 (s, 4H), 5.11-5.02 (m, 13H), 4.98 (d, 1H, J=6.2
Hz), 4.28 (d, 1H, J=7.4 Hz), 3.97 (q, 1H, J=6.2, 7.4 HZ).
[0089] 10% palladium on carbon (21 mg, 0.02 mmol) was added to a
solution of 3 (420 mg, 0.29 mmol) in anhydrous THF (20.0 mL), the
mixture was stirred under hydrogen gas atmosphere for overnight at
room temperature. The mixture was filtered through Celite, the
filtrate was diluted with methanol and dichloromethane and filtered
through Celite three times until the solution was clear. The
solvent was removed and gave crude 4 in 64% yield.
##STR00010##
[0090] Acid chloride 2 (941 mg, 2.05 mmol) was added to a solution
of 3-methoxycatechol (140 mg, 1.00 mmol) in anhydrous acetonitrile
(10 mL) at room temperature. DMAP (268 mg, 2.20 mmol) was added to
the reaction mixture after stirred for 30 min at room temperature,
the mixture was stirred for 2 days and the solvent was removed, the
crude was purified by chromatography on silica gel (25% EA in
hexane) giving 708 mg of 6 in 72% yield. .sup.1H NMR (CDCl3)
.delta. 7.64 (d, 4H, J=16.7 Hz), 7.51-7.33 (m, 31H), 7.16 (d, 1H,
J=8.2 Hz), 7.06 (d, 1H, J=8.4 Hz), 5.18 (d, 4H, J=7.0 Hz), 5.06 (d,
8H, J=11 Hz), 3.96 (s, 3H); .sup.13C NMR (CDCl3) .delta. 164.0,
163.6, 153.1, 152.8, 152.3, 144.1, 143.2, 137.6, 137.5, 136.5,
136.4, 132.3, 128.6, 128.4, 128.3, 128.2, 128.1, 128.0, 127.8,
127.7, 126.6, 123.9, 123.8, 115.4, 110.2, 109.7, 109.6, 75.3, 71.2,
56.4.
[0091] 10% palladium on carbon (43 mg, 0.04 mmol) was added to a
solution of 6 (500 mg, 0.51 mmol) in anhydrous THF (20.0 mL), the
mixture was stirred under hydrogen gas atmosphere for 12 h at room
temperature. Then the mixture was filtered through Celite, the
filtrate was concentrated and purified by chromatography on silica
gel giving 9.5 mg of 7. Most of compound 7 was decomposed on silica
gel. .sup.1H NMR (CDCl3) .delta. 8.30 (brs, 6H), 7.31-7.13 (m, 5H),
7.04 (d, 1H, J=8.1 Hz), 6.95 (d, 1H, J=8.3 Hz), 3.83 (s, 3H).
##STR00011##
[0092] Acid chloride 2 (1.40 g, 3.05 mmol) was added to a solution
of pyrogallol (126 mg, 1.00 mmol) in anhydrous acetonitrile (15 mL)
at room temperature. DMAP (391 mg, 3.20 mmol) was added to the
reaction mixture after stirred for 30 min at room temperature, the
mixture was stirred for 24 h and the solvent was removed, the crude
was purified by chromatography on silica gel (25% EA in hexane as
eluent) giving 570 mg of 9 in 41% yield. .sup.1H NMR (CDCl3)
.delta. 7.53 (s, 4H), 7.49-7.20 (m, 50H), 5.10 (s, 4H), 5.01 (s,
8H), 4.94 (s, 2H), 4.85 (s, 4H); .sup.13C NMR (CDCl3) .delta.
163.6, 163.0, 152.7, 144.2, 143.6, 143.3, 137.4, 137.3, 136.3,
136.1, 135.2, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9,
127.8, 126.3, 123.6, 123.1, 120.9, 109.6, 75.2, 75.1, 71.2.
[0093] 10% palladium on carbon (16 mg, 0.024 mmol) was added to a
solution of 6 (260 mg, 0.29 mmol) in anhydrous THF (15.0 mL), the
mixture was stirred under hydrogen gas atmosphere for overnight at
room temperature. Then the mixture was filtered through Celite, the
filtrate was concentrated and purified by chromatography on silica
gel (25% EA in hexane) giving 11.3 mg of 10. Compound 10 was
decomposed on the column. .sup.1H NMR (CDCl3) .delta. 8.24 (brs,
4H), 7.47-7.41 (m, 1H), 7.34-7.32 (m, 2H), 7.17 (s, 4H), 7.09 (s,
2H), 2.92 (brs, 5H).
[0094] Evaluation of Generation 1 Compounds Derived from
.alpha.-PPG
[0095] Compounds 1a and 2a were initially prepared as potential
anti-diabetic analogs of .alpha.-PGG (FIG. 1). Given the tight SAR
of this class of compounds, it was hypothesized that a more rigid
scaffold (i.e., a benzene ring) might enhance the activity. Rather
than possessing insulin-like activity, these two compounds were
surprisingly and serendipitously found to inhibit basal glucose
transport on cervical (HeLa), colon (RKO), and breast (MCF-7)
cancer cells at a concentration of 30 .mu.M (Table 1).
TABLE-US-00001 TABLE 1 Glucose transport inhibitory activity (%) of
1a and 2a in different cancer cell lines Compound Hela RKO MCF-7 1a
46.3 .+-. 3.3 57.2 .+-. 4.4 33.0 .+-. 0.5 2a 36.0 .+-. 4.9 58.1
.+-. 0.1 60.4 .+-. 2.4
[0096] Compounds 1a and 2a also inhibited basal glucose transport
in H1299 cells by 58.4.+-.6.3% and 86.1.+-.1.0%, respectively
(Table 2), as measured by a standard glucose uptake assay compared
to non-compound treated cells controls (considered as 0%
inhibition). Tested in an MTT cell proliferation assay in H1299
cells, their inhibitory activities on cancer cell growth were found
to be 36.0.+-.6.1% and 39.9.+-.5.0%, respectively (non-compound
treated cell controls were considered as 0% inhibition).
[0097] Given the potential utility of the inhibition of glucose
transport for development of novel anticancer agents,
structure-activity relationship of these compounds as both
inhibitors of glucose transport and cancer cell proliferation were
investigated. Based on these two compounds a number of derivatives
were prepared in order to understand the need for the
trihydroxyphenyl ester and the need for three of these esters on
the central aromatic ring.
[0098] The desired analogs were prepared by the acylation of a
series of di- and tri-hydroxy benzenes with a group of substituted
benzoyl halides. A group of mono-, di-, and trihydroxybenzoyl
halides as well as methoxybenzoyl halides were chosen as acylating
agents. The synthesis of the hydroxybenzoyl halides is outlined in
Scheme 1. Commercially available phenols 3a-f were perbenzylated
and the resulting esters hydrolyzed and the acid converted to the
acid chlorides 5a and benzyloxybenzoyl chlorides 5b, 5c, 5d, 5e,
and 5f. The requisite methoxy-substituted benzoyl halides were
prepared from the commercially available carboxylic acids.
TABLE-US-00002 TABLE 2 Compounds prepared, their induced inhibitory
activities in basal glucose transport and cell growth in H1299 lung
cancer cells Glucose Cell growth Compound Yield.sup.a transport
inhibition.sup.b # Ar/Ar' X Y (%) inhibition.sup.b (%) (%) 1a
3,4,5-(OH).sub.3--C.sub.6H.sub.2 OMe H 69 58.4 .+-. 1.0.sup.c 36.0
.+-. 6.1 2a 3,4,5-(OH).sub.3--C.sub.6H.sub.2 -- -- 78 86.1 .+-. 1.0
39.9 .+-. 5.0 1b 3,4,5-(OH).sub.3--C.sub.6H.sub.2 H Cl 69 84.4 .+-.
0.1 -- 1c 3,4,5-(OH).sub.3--C.sub.6H.sub.2 F H 78 81.1 .+-. 1.1 --
1d 3,4,5-(OH).sub.3--C.sub.6H.sub.2 H H 65 32.1 .+-. 6.2 -- 9a
3,4,5-(OMe).sub.3--C.sub.6H.sub.2 -- -- 86 41.1 .+-. 1.5 -- 7a
3,4,5-(OMe).sub.3--C.sub.6H.sub.2 OMe H 90 33.3 .+-. 3.6 22.2 .+-.
3.4 7b 3,4,5-(OMe).sub.3--C.sub.6H.sub.2 H Cl 89 1.7 .+-. 1.1 20.6
.+-. 2.1 7c 3,4,5-(OMe).sub.3--C.sub.6H.sub.2 F H 87 7.9 .+-. 2.6
19.6 .+-. 3.6 9b 2,6-(OMe).sub.2--C.sub.6H.sub.3 -- -- 69 75.8 .+-.
4.2 -- 9c 3,4-(OMe).sub.2--C.sub.6H.sub.3 -- -- 78 0 .+-. 3.9 10.0
.+-. 1.1 9d 3-(OMe)--C.sub.6H.sub.4 -- -- 92 66.2 .+-. 1.8 -- 7d
3-(OMe)--C.sub.6H.sub.4 OMe H 96 32.1 .+-. 0.2 18.3 .+-. 4.3 2b
3,5-(OH).sub.2--C.sub.6H.sub.3 -- -- 81 98.7 .+-. 0.8 41.0 .+-. 5.5
1e 3,5-(OH).sub.2--C.sub.6H.sub.3 OMe H 78 69.4 .+-. 3.0 -- 1f
3,5-(OH).sub.2--C.sub.6H.sub.3 H Cl 87 95.2 .+-. 0.2 38.8 .+-. 6.9
1g 3,5-(OH).sub.2--C.sub.6H.sub.3 F H 81 94.0 .+-. 0.8 35.0 .+-.
7.6 2c 3,4-(OH).sub.2--C.sub.6H.sub.3 -- -- 81 94.7 .+-. 0.4 42.2
.+-. 6.1 1h 3,4-(OH).sub.2--C.sub.6H.sub.3 OMe H 78 0 32.4 .+-. 2.7
1i 3,4-(OH).sub.2--C.sub.6H.sub.3 H Cl 87 94.4 .+-. 0.4 45.2 .+-.
7.6 1j 3,4-(OH).sub.2--C.sub.6H.sub.3 F H 81 88.7 .+-. 1.8 41.5
.+-. 5.5 2d 2-(OH)--C.sub.6H.sub.4 -- -- 88 74.7 .+-. 2.0 -- 1k
2-(OH)--C.sub.6H.sub.4 OMe H 91 50.5 .+-. 7.6 18.0 .+-. 4.4 1l
2-(OH)--C.sub.6H.sub.4 H Cl 92 51.6 .+-. 5.9 26.6 .+-. 2.3 1m
2-(OH)--C.sub.6H.sub.4 F H 89 51.0 .+-. 6.6 -- 2e
4-(OH)--C.sub.6H.sub.4 -- -- 81 88.7 .+-. 2.5 34.8 .+-. 7.7 1n
4-(OH)--C.sub.6H.sub.4 OMe H 78 86.5 .+-. 2.8 36.6 .+-. 6.7 1o
4-(OH)--C.sub.6H.sub.4 H Cl 87 79.0 .+-. 8.7 -- 1p
4-(OH)--C.sub.6H.sub.4 F H 84 92.6 .+-. 0.7 35.9 .+-. 4.7 1q
4-(OH)--C.sub.6H.sub.4 H H 82 57.5 .+-. 2.9 -- 2f
3-(OH)--C.sub.6H.sub.4 -- -- 81 99.7 .+-. 0.1 59.3 .+-. 4.9 1r
3-(OH)--C.sub.6H.sub.4 OMe H 78 79.0 .+-. 8.7 -- 1s
3-(OH)--C.sub.6H.sub.4 H Cl 87 93.1 .+-. 1.7 44.5 .+-. 5.2 1t
3-(OH)--C.sub.6H.sub.4 F H 81 92.8 .+-. 0.1 40.8 .+-. 5.6
.sup.aOverall yield from 6 to 8. .sup.bUntreated cells served as
negative controls (0% inhibition). .sup.cData were presented as
mean .+-. standard deviation.
##STR00012##
[0099] In terms of the core aromatic ring, pyrogallol 8 (present in
2a) and 3-methoxycatechol 6a (X=OMe, Y=H, present in 1a) were
chosen. Two halogen substituted phenols, 6b (X=H, Y=Cl) and 6c
(X=F, Y=H) were chosen to provide a p-donor (similar to the methoxy
group) but electron withdrawing group. An unsubstituted catechol 6d
(X, Y=H) was included. As shown in Scheme 2, each of these phenols
was then coupled to each of the acid chlorides 5a-f and several
methoxy-substituted benzoyl chlorides. After coupling, the
benzyloxy esters were deprotected via catalytic hydrogenation. The
overall yields are shown in Table 2.
##STR00013##
[0100] As shown in Table 2, compounds were tested in a standard
glucose uptake assay. Briefly, H1299 cancer cells were treated with
or without the compounds (30 .mu.M) in triplicates for 10 min
before the glucose uptake assay. Cellular glucose uptake was
measured by incubating cells in the glucose-free KRP buffer with
0.2 Ci/mL [.sup.3H]2-deoxyglucose (specific activity, 40 Ci/mmol)
for 30 min in the absence or presence of compounds. After the cells
were washed with ice-cold PBS and lysed by 0.2 N NaOH, the cell
lysates were transferred to scintillation counting vials and the
radioactivity in the cell lysates was quantified by liquid
scintillation counting. Cell growth measurements were performed
using an MTT assay in hexads in a 96-well tissue culture plate with
5,000 cells plated in each well. The cells were incubated in
presence or absence of the compounds (30 .mu.M) for 48 h. After
incubation, cell viabilities were assayed using a 96-well
SPECTRAMAX.TM. absorbance/fluorescence plate reader (Molecular
Devices).
[0101] In some embodiments, comparison of compounds 1a and 2a to
derivatives in which the core aromatic ring was substituted with a
fluorine or chlorine (1b and 1c), revealed that both of these
compounds have similar activity in the glucose transport inhibition
assay as compound 2a and were significantly better than 1a. Clearly
the halogen substitution on the core aromatic ring is important. An
unsubstituted core aromatic ring 1d showed lower levels of
inhibition relative to 1a and 2a. In order to determine the
necessity of the phenolic hydroxyl groups we prepared several
derivatives of 1a and 2a in which the OH group was replaced with a
methoxyl group (9a-d, 7a-d). These compounds showed uniformly lower
levels of inhibition of glucose transport. Only compounds 9b and 9d
(2,6-dimethoxybenzoyl and 3-methoxybenzoyl) showed moderate levels
of inhibition.
[0102] In some embodiments, the phenolic hydroxyl groups were
systematically removed preparing a series of di-hydroxyl and
mono-hydroxyl derivatives. The 3,5-dihydroxy and 3,4-dihydroxyl
derivatives overall showed good levels of glucose transport
inhibition. In both series the tribenzoyl derivatives (2b and 2c)
as well as the fluoro- and chloro-substituted derivatives showed
>90% inhibition while the methoxy-substituted derivatives (1e
and 1h) showed little to no inhibition. All of the compounds
showing >90% glucose transport inhibition also show .about.40%
decrease in cancer cell growth rate.
[0103] In some embodiments, removing an additional hydroxyl group
provided a set of monohydroxyl compounds at the 2-, 3-, and
4-positions. The 2-hydroxyl series showed uniformly poorer
inhibition of glucose transport. The 4-hydroxyl series while
showing poorer inhibition of glucose transport did provide
compounds with >85% inhibition of glucose transport. These
compounds showed a >35% decrease in cancer cell growth rate. The
3-hydroxyl series showed excellent inhibition of glucose transport
with the tribenzoyl derivative 2f showing >99% inhibition of
glucose transport. This compound also showed the highest level of
cell growth inhibition at .about.60%. By analyzing all data in
Table 2, it was found that the linear correlation coefficient
R=0.817 (R2=0.667), indicating that approximately 2/3 (66.7%) of
the inhibitory activity of cancer cell growth came from the
inhibitory activity of basal glucose transport.
[0104] In general, the presence of a hydroxy group at the
3-position of the pendant benzoyl group is important for both
inhibitions of glucose transport and cancer cell growth. This
3-hydroxy group can be attached to a chloro- or fluoro-substituted
core benzene ring or be part of a tribenzoyl system. Unsubstituted
or electron-donating substituents lead to significant decreases in
the inhibition of glucose transport activity.
[0105] In other embodiments, the hydrolysis products (8, 6c, 10,
11, and 12) of the phenolic esters may exhibit glucose uptake
inhibition activity. As shown in FIG. 2, none of these compounds
had any glucose uptake inhibition activity. Similarly, none of
these compounds showed any activity in anticancer screens. In
summary, a library of multiphenolic ester compounds as novel
inhibitors of basal glucose transport and anticancer agents with a
potential new target, basal glucose transport, were
synthesized.
[0106] Potent and Selective Inhibitors of Basal Glucose Transport
have been Identified Through an SAR Study
[0107] During a structure activity relationship (SAR) study in
which more than 80 PGG analogs were synthesized and analyzed by
glucose uptake assay and functional assays, a group of inhibitors
for glucose uptake, which had an opposite activity as PGG, were
also identified. It was serendipitously discovered that these
inhibitors caused inhibition of cancer cell growth and cancer cell
death. With these new findings, it was decided to chemically
synthesize more potent and selective inhibitors and use them for
cancer study.
[0108] Given the tight SAR of the initial set of PGG analogs, it
was hypothesized that a more rigid scaffold upon which the galloyl
group was appended might provide enhanced potency and selectivity.
Several new analogs of the PGG class of compounds were prepared.
Two compounds were based upon an aromatic nucleus (as opposed to
the glucose nucleus). The hypothesis for all compounds was that the
rigid central core would prevent any conformational mobility
associated with the glucose nucleus. When these compounds (WZB-26
and WZB-27) were assayed, it was found that they possessed basal
glucose transport inhibitory activity without any insulin-like
activity. Thus, these compounds are selective inhibitors of basal
glucose transport of animal cells. When these compounds were used
to treat different cancer cell lines, they were found to also
inhibit basal glucose transport in cervical (HeLa), colon (RKO),
and breast (MCF7) cancer cells. This result suggests that these
compounds are general basal glucose transport inhibitors;
inhibiting all three cancer cell lines tested. Based on these two
compounds a number of derivatives were prepared in order to
understand the need for the trihydroxyphenyl ester and the need for
three of these esters on the central aromatic ring (FIG. 16). In
brief, a series of di- or trihydroxybenzenes were acylated with a
protected hydroxybenzoic acid. After coupling the protecting group
(benzyl) was removed to provide the target compounds, either a
tribenzoyl derivative (3) or a phenyl substituted dibenzoyl
derivative (5).
[0109] Compounds WZB-26 and WZB-27 were the first compounds
prepared. Both compounds showed similar inhibition of basal glucose
transport (.about.85%). Initially, the substitution pattern on the
central aromatic ring was of interest. As WZB-27 has three galloyl
group while WZB-26 has only 2 but an additional methoxy group.
Preparation of multiple compounds was completed to explore the
impact other substituents on the central aromatic ring would have
upon the levels of inhibition. In some embodiments, the compounds
prepared may be designated WZB-89 (F substitution), WZB-90 (Cl
substitution), and WZB-110 (H substitution). Both WZB-89 and WZB-90
showed similar levels of activity to WZB-26 and -27 while the
unsubstituted WZB-101 showed dramatically decreased levels of
activity. Clearly some type of it-donor group in the 3- or
4-position as seen in WZB-26, -27, -89, and -90 is beneficial to
activity. Both WZB-26 and WZB-27 have three phenolic hydroxyl
groups. In order to determine if these hydroxyl groups were acting
as H-bond donors or H-bond acceptors, 4 analogs in which the OH
group was replaced with an OMe group (WZB-76, -81, -101, -102) were
prepared. The activity of these compounds was dramatically
decreased, clearly indicating the need for an H-bond donor (i.e. a
phenolic OH). Next, the need for the galloyl group (i.e.
3,4,5-trihydroxybenzoyl) was examined by a systematic removal of
the hydroxy group. A series of dihydroxy (3,5-dihydroxy and
3,4-dihydroxy) and monohydroxy (2-OH, 3-OH, and 4-OH) derivatives
were prepared. In all cases analogs with a methoxy group or no
substitution on the central aromatic ring provided significantly
lower levels of inhibition regardless of the number or position of
hydroxyl groups on the pendant benzoyl ester. Of the analogs
prepared the 3,5-dihydroxy (WZB-111, WZB-113, WZB-114), the
3,4-dihydroxy (WZB-119, WZB-121, WZB-112) and the 3-monohydroxy
(WZB-115, WZB-117, WZB-118) derivatives showed inhibition levels of
95-99%. As there is an interest in the pharmaceutical industry to
develop simpler, lower molecular weight inhibitors, compounds
WZB-27 and WZB-115 were used for biological studies first and
better compounds would be employed in later synthesis and assays.
Table 3 indicates that we have systematically synthesized more than
100 different compounds to study SAR of the compounds with an
objective of making more potent and selective inhibitors to basal
glucose transport. As shown in Table 3, some of these inhibitors
are the best basal glucose transport inhibitors reported to date
and may be useful for future clinical studies.
TABLE-US-00003 TABLE 3 Analogs prepared and basal glucose transport
inhibition Compound # X Y (OR).sub.n % inhibition.sup.a WZB-26 OMe
H 3,4,5-(OH).sub.3 84.0 .+-. 1.9 WZB-27 NA, tribenzoyl analog 3
3,4,5-(OH).sub.3 86.1 .+-. 1.0 WZB-89 F H 3,4,5-(OH).sub.3 81.0
.+-. 1.1 WZB-90 H Cl 3,4,5-(OH).sub.3 84.4 .+-. 0.1 WZB-110 H H
3,4,5-(OH).sub.3 32.1 .+-. 6.2 WZB-76 NA, tribenzoyl analog 3
3,4,5-(OMe).sub.3 41.1 .+-. 1.5 WZB-81 OMe H 3,4,5-(OMe).sub.3 33.3
.+-. 3.6 WZB-101 F H 3,4,5-(OMe).sub.3 7.9 .+-. 2.6 WZB-102 H Cl
3,4,5-(OMe).sub.3 1.7 .+-. 1.1 WZB-111 NA, tribenzoyl analog 3
3,5-(OH).sub.2 99.5 .+-. 0.3 WZB-112 OMe H 3,5-(OH).sub.2 69.4 .+-.
3.0 WZB-112 OMe H 3,5-(OH).sub.2 69.4 .+-. 3.0 WZB-113 F H
3,5-(OH).sub.2 96.7 .+-. 1.6 WZB-114 H Cl 3,5-(OH).sub.2 96.1 .+-.
0.1 WZB-119 NA, tribenzoyl analog 3 3,4-(OH).sub.2 94.7 .+-. 0.4
WZB-120 OMe H 3,4-(OH).sub.2 0 WZB-121 F H 3,4-(OH).sub.2 88.7 .+-.
1.8 WZB-122 H Cl 3,4-(OH).sub.2 94.4 .+-. 0.4 WZB-91 NA, tribenzoyl
analog 3 4-OH 88.7 .+-. 2.5 WZB-92 F H 4-OH 92.6 .+-. 0.7 WZB-93
OMe H 4-OH 86.5 .+-. 2.8 WZB-94 H Cl 4-OH 79.0 .+-. 8.7 WZB-103 H H
4-OH 57.5 .+-. 2.9 WZB-115 NA, tribenzoyl analog 3 3-OH 99.7 .+-.
0.1 WZB-116 OMe H 3-OH 79.0 .+-. 8.0 WZB-117 F H 3-OH 98.3 .+-. 8.0
WZB-118 H Cl 3-OH 96.5 .+-. 0.4 WZB-127 NA, tribenzoyl analog 3
2-OH 74.7 .+-. 2.0 WZB-128 OMe H 2-OH 50.5 .+-. 7.6 WZB-129 F H
2-OH 51.0 .+-. 6.6 WZB-130 Cl H 2-OH 51.6 .+-. 5.9 .sup.aCompounds
were tested at 30 .mu.M in H1299 cells by the glucose uptake assay.
Cells without being treated by any compound served as negative
controls (0% inhibition).
[0110] .alpha.-PGG Induces Apoptosis in Human Colon, Cervical, and
Breast Cancer Cells
[0111] When .alpha.-PGG was used to treat RKO (colon), HeLa
(cervical), and MCF-7 (breast) human cancer cells, it was found
that the treatment resulted in pronounced cell death (FIG. 5A), and
the cell death was caused primarily, if not exclusively, by
apoptosis (FIGS. 5B & 5C). .alpha.-PGG does not cause much
apoptosis in normal (non-cancer) cells (data not shown), indicating
the compound shows increased cytotoxicity more towards cancer
cells.
[0112] PGG-Derived Compounds Induce Cell Death Preferentially in
Cancer Cells than their Normal Counterparts
[0113] Cancer cells heavily depend on glucose as their preferred
energy source and glucose deprivation has been proposed as an
anti-cancer strategy. In order for these compounds to be effective
anti-cancer agents, they must be able to kill more cancer cells
than normal cells. Cell killing (or cell viability) assays revealed
that some of these compounds, particularly WZB-27 (=W27),
preferentially kill cancer cells (NSCLC H1299 and breast carcinoma
MCF7) than their non-cancerous cell counterparts (NL20 or MCF12A
cells, Table 3). These results suggest that these compounds have
excellent potential to be anti-cancer agents. Based on this
observation, it was speculated that optimal compounds and drug
concentration can be determined that will minimally impact normal
cells while causing maximal damage to cancer cells.
[0114] Comparative assays using WZB-27, WZB-115, as well as two
known anticancer drugs, cisplatin and taxol, were completed. In
Table 4, the percent cell death in cancer cell lines as well as
normal cell lines is shown. Compounds WZB-27 and WZB-115 kill
approximately the same percent of lung cancer cell line H1299 as
taxol while killing significantly less of the normal lung cell
line, NL20. Comparing the breast cancer cell line MCF7, WZB-27 and
WZB-115 kill somewhat fewer cells relative to taxol but more than
cisplatin. Both WZB-27 and WZB-115 kill fewer of the normal breast
cell line MCF12A than taxol or cisplatin. WZB-27 kills fewer of the
normal breast cell line MCF12A than taxol or cisplatin while
WZB-115 kills no more normal cells than either cisplatin or taxol.
The results shown in table 4 suggest that compounds have
cytotoxicities in cancer cells comparable or better than cisplatin
and/or taxol while they exhibit less cytotoxicities in
non-cancerous ("normal") cells than the anticancer drugs.
TABLE-US-00004 TABLE 4 Comparison of % cell death in cancer vs.
normal cells induced by compounds.sup.a, b, c Compound H1299 NL20
MCF7 MCF12A Cisplatin 26.4 .+-. 3.8 58.4 .+-. 7.0 27.0 .+-. 1.9
69.6 .+-. 2.9 Taxol 45.6 .+-. 4.9 53.8 .+-. 4.2 61.4 .+-. 7.1 73.7
.+-. 5.6 WZB-27 52.3 .+-. 9.4 0 48.9 .+-. 5.7 21.1 .+-. 8.9 WZB-115
61.3 .+-. 3.6 34.1 .+-. 8.7 51.6 .+-. 8.3 66.0 .+-. 8.6 .sup.aThis
test is done using a standard MTT assay to measure viable cells
after compound treatment. .sup.bConcentration used in the test was
the IC.sub.50 for each compound. .sup.cNon-compound treated cells
were used as controls (0% death)
[0115] Cancer Cell Lines Overexpress GLUT1 and Compounds Inhibit
More Glucose Uptake in Cancer Cells than their Non-Cancerous Cell
Counterparts
[0116] To determine the possible causes for increased killing in
cancer cells than in their non-cancerous cell counterparts,
comparison studies were conducted to determine the effect of
compound treatment on glucose uptake. It was found that compound
WZB-27 produced larger reductions in glucose uptake in cancer cell
line H1299 and MCF7 as compared to the reduction in non-cancerous
cell lines NL20 or MCF12A (FIGS. 19A and 19B). Western blot
analysis revealed that these same cancer cell lines express
significantly higher levels of GLUT1 protein than their
non-cancerous counterparts (FIG. 19C). The larger reduction in
glucose uptake observed in cancer cell lines was correlated with
higher GLUT1 levels in these cells.
[0117] .alpha.-PGG Activates p53 in RKO (Colon) Cells but not in
HeLa (Cervical) or MCF-7 (Breast) Cancer Cells
[0118] After it was found that .alpha.-PGG induced apoptosis in the
three cancer cell lines, knowledge of the mechanism of apoptosis
was sought (i.e., is apoptosis related to p53 status or not).
Western blot analysis using anti-p53 antibody revealed that
.alpha.-PGG led to activation of p53 in RKO cells but not in HeLa
or MCF-7 cells (FIG. 6). This result suggests that the apoptosis
induced in HeLa and MCF-7 cells was p53-independent. This result is
both interesting and important in that it shows that .alpha.-PGG
can induce apoptosis in certain cancer cells using a
p53-independent mechanism, which should be effective in inducing
apoptosis in more than 50% of all human cancers in which p53 is
mutated and non-functional.
[0119] This result suggests that, unlike in RKO cells, the
apoptosis induced by .alpha.-PGG in HeLa cells and MCF-7 was not
mediated by p53 or p53 signaling pathway. Thus, apoptosis induced
by .alpha.-PGG in HeLa cells is p53-independent.
[0120] PGG and its Derivatives Inhibit Basal Glucose Transport in
Human Cancer Cell Lines
[0121] Inhibition of basal glucose transport was speculated as a
cause for cancer cell death induced by .alpha.-PGG. PGG derivatives
were synthesized and tested along with .alpha.-PGG in different
human cancer cell lines. These derivatives were found to inhibit
basal glucose transport in cervical, colon, and breast cancer cell
lines (FIG. 7).
[0122] Insulin had no effect on the glucose uptake, suggesting that
the glucose measured was basal glucose transport, not
insulin-mediated glucose transport. Based on this observation, we
further hypothesized that the pronounced glucose transport
inhibition might be the cause of apoptosis, particularly in HeLa
cells.
[0123] .alpha.-PGG and its Derived Compounds Inhibit Basal Glucose
Transport in HeLa Cells in a Dose-Dependent Manner
[0124] Different concentrations of .alpha.-PGG or its derived
compounds, WZB-25 and WZB-27, were used to determine if the
inhibition of the basal glucose transport was dose-dependent. The
experimental results indicated that .alpha.-PGG and its derivatives
inhibited the basal glucose transport in a dose-dependent manner
and .alpha.-PGG appears to be slightly more potent than its
derivatives in inhibiting the transport (FIG. 8). It was also found
that 30 .mu.M of any compound led to approximately 50% inhibition
of basal glucose transport in HeLa cells (FIG. 8), suggesting that
all 4 compounds were about equally effective in inhibiting basal
glucose transport in HeLa cells. This result led us to conclude
that we could either use .alpha.-PGG or substitute .alpha.-PGG with
its derivatives to do the glucose transport inhibition study. We
have also found that reducing glucose concentration in cell culture
media also reduces cell growth rates and induces apoptosis in HeLa
and MCF-7 cells in a glucose concentration-dependent manner (data
not shown). These results clearly show that .alpha.-PGG and its
derivatives WZB-25 and WZB-27 inhibit basal glucose transport and
the inhibition is dose-independent.
[0125] HeLa cells were incubated with .alpha.-PGG or its
derivatives (WZB-25 or WZB-27) at various concentrations for 20 min
before .sup.3H-labeled 2-DG was added to the cells for 30 min.
After 30 min of 2-DG incubation, cells were harvested, lysed, and
counted for their respective glucose uptake (intracellular 2-DG
counts) with a scintillation counter. The results show that
.alpha.-PGG starts to inhibit glucose transport at 5 while other
derivatives start to inhibit the transport at about 10 .mu.M and
all three compounds show dose-responsive inhibition profiles. Error
bars in FIG. 8 represent standard deviations of the measurements.
Samples were done in triplicates and the experiment was repeated
three times.
[0126] Unlike .alpha.-PGG (an Insulin Mimetic), .alpha.-PGG
Derivatives do not Induce Akt Phosphorylation
[0127] So far, it has been shown that .alpha.-PGG and its
derivatives inhibited basal glucose (FIG. 7) and exhibited very
similar dose-response (FIG. 8). However, these derivatives are much
smaller than .alpha.-PGG in molecular weight and it is expected
that these derivatives will be cleaner than .alpha.-PGG in that
they are more selective and do not have as many activities
unrelated to the basal glucose transport inhibition. Previous data
showed that .alpha.-PGG binds and activates the insulin receptor
(IR), and induces the phosphorylation of Akt, a protein factor
involved in IR signaling. Since the derivatives are simpler in
structure and smaller in size than .alpha.-PGG, it was speculated
that these derivatives would not induce Akt phosphorylation, which
was confirmed by Western blot analyses on compounds-treated CHO
cells that overexpress IR (FIG. 10).
[0128] Akt is a key factor in the insulin receptor signaling
pathway. Therefore, .alpha.-PGG derivatives WZB-25 through WZB-27
appear to be more selective than .alpha.-PGG in that they do not
induce activities unrelated to basal glucose transport. They should
generate glucose transport inhibition and apoptosis data that are
even easier to analyze and interpret than those of .alpha.-PGG.
[0129] Basal Glucose Transport Inhibitors Induce Apoptosis and Cell
Death Using a p53-Independent Signaling Pathway
[0130] To determine if anti-cancer compounds W25 and W27 kill
cancer cells using a p53 dependent or a p53-independent pathway, a
cell killing assay was performed in RKO and RKO E6 cell lines. The
difference between the two cell lines is that RKO cells contain
much higher levels of p53 than RKO E6 cells. The fact that W25 and
W27 killed about the same amounts of RKO cells and RKO E6 cells in
the assay (FIG. 12A) suggests that p53 in the cancer cell lines did
not play any important roles in the cell killing. Furthermore, the
PARP assay revealed that, in the W25 and W27 treated H1299 cells,
the intact 116 kDa PARP protein was cleaved into a 89 kDa protein
(FIG. 12B), indicating that the caspase 3 was activated and caspase
3 apoptosis pathway is active in the compound treated lung cancer
cells. These results suggest that the compounds kill cancer cells
using a p53-independent and caspase 3-dependent apoptosis
pathway.
[0131] Although it was found that there was no significant
difference between the viability of RKO and RKO-E6 cells after they
were treated with compound WZB-25 or WZB-27 (FIG. 18A), a
significant difference in viability between the two cell lines was
observed when they were treated by .alpha.-PGG (FIGS. 18A and 18B),
suggesting that the apoptosis induced by WZB-25 or WZB-27 is
p53-independent, since p53 levels in the two cell lines did not
affect cell viability. In contrast, the apoptosis induced by
.alpha.-PGG is p53-dependent since its treatment led to very
different cell viability in the same two cell lines (FIG. 18B).
This finding is important because it is known that more 50% of all
human cancers harbor p53 mutations. These inhibitors should be able
to exert their anticancer effects on all human cancers regardless
their p53 status.
[0132] Furthermore, a Western blot analysis revealed that, in the
WZB-27 and WZB-115 treated A549 cells, the intact 116 kDa PARP
protein was cleaved into a 89 kDa protein (FIG. 18C), indicating
that the caspase 3 was activated and caspase 3 apoptosis pathway is
active in the compound treated lung cancer cells. More
interestingly, cells growing in cell culture media containing 5% of
normal glucose concentration (1.25 mM vs. 25 mM normal) also
demonstrated the cleaved 89 kDa band (FIG. 18C), indicating the
glucose withdrawal resulted in the same PARP cleavage. These
results suggest that the compounds WZB-27 and WZB-115 induce
apoptosis in these cancer cell lines using a p53-independent and
caspase 3-dependent apoptosis pathway while .alpha.-PGG does this
in a different p53-dependent mechanism. It also reveals that cell
treatments by the compound inhibitors produced the same PARP
cleavage as glucose withdrawal, providing initial experimental
evidence that compound treatment mimics the effects produced by
glucose withdrawal. This is important since it suggests that the
methods of inhibition of basal glucose transport by inhibitors and
glucose withdrawal by reducing glucose concentration in cell growth
media may be interchangeable when in producing certain biological
effects in cells.
[0133] Mechanism of Compounds in Cancer Cell Killing
[0134] Potential mechanisms for cancer cell killing are graphically
presented in FIG. 13. According to the hypothesis, extracellular
glucose is taken up by cancer cells through glucose transporter 1
(GLUT1). Compound .alpha.-PGG and its derivatives inhibit basal
glucose transport by inhibiting GLUT (most likely to be GLUT1). The
inhibition of basal glucose transport results in reduction of
intracellular glucose concentration and an increase of
intracellular free Zn.sup.2+; these changes, through an unknown
mechanism(s), induces ER stress and eventually apoptosis (FIG.
11).
[0135] Glucose Transport Inhibitors Induced Mild and Temporary
Hyperglycemia in Fasting Mice
[0136] Inhibitors to basal glucose transport are likely to increase
blood glucose level when used in animals because the movement of
blood glucose into target cells, primarily muscle and fat cells, is
partially blocked by the inhibitors. To find out the intensity and
duration of the induced hyperglycemia and other potential side
effects in animals, we performed animal studies by injecting two
lead compounds WZB-27 and WZB-115 separately and intraperitoneally
(IP) into fasting mice and observing blood glucose changes,
movement, and behaviors of the compound injected animals. As
expected, the compound-injected mice showed mild and temporary
hyperglycemia compared to the vehicle-injected group (PBS+DMSO) and
the hyperglycemia went away approximately 3 hrs after the compound
injection (FIG. 20). Both compounds WZB-27 and WZB-115 showed very
similar blood glucose profiles (FIG. 20). At IC.sub.50 (10 mg/kg
for WZB-27 and 1.5 mg/kg for WZB-115), both compounds induced mild
and temporary hyperglycemia. Interestingly, at a concentration of
3.times.IC.sub.50, neither compound induced higher hyperglycemia.
Instead, injection of 3.times.IC.sub.50 (30 mg/kg for WZB-27 and 5
mg/kg for WZB-115) resulted in blood glucose levels very similar to
that of the vehicle injected group (FIG. 20). This experiment was
repeated once and similar results were obtained. Although the
mechanism for normoglycemia at higher compound concentrations was
currently unclear, it was concluded that these compounds do not
produce severe hyperglycemia in mice. No other noticeable changes
in animal movement, activity, and behavior were observed in this
experiment.
[0137] To further address the concerns of animal side effects, a
longer-term multiple compound injections at higher concentration
were performed. Animals were injected once a day at a concentration
of 6.times.IC.sub.50 or 10.times.IC.sub.50 for one week. Similar to
the single day experiment, no noticeable side effects were observed
except mild and temporary hyperglycemia in both groups and some
weight loss for the 6.times.IC.sub.50 group (.ltoreq.10% of total
body weight). There was no significant difference between blood
glucose of the compound treated group at the end of the one week
study and that of the vehicle treated group. These results strongly
suggest that inhibition of basal glucose transport may not be very
toxic and are relatively safe to mice. The anticancer animal
studies disclosed herein can be carried out to determine the in
vivo anticancer efficacy of the compounds.
[0138] Comparison of Intracellular Glucose Levels in Glucose
Deprivation Induced by Either Glucose Removal or by Inhibition of
Basal Glucose Transport
[0139] Glucose deprivation experiments, in which glucose in the
cell culture media is partially removed, have been frequently done.
However, the intracellular glucose level changes have not been
measured often during or after the deprivation. In order to compare
the effects of glucose withdrawal and basal glucose transport
inhibition on intracellular glucose levels, a comparison study will
be conducted.
[0140] H1299 and MCF7 will be incubated in 24-well plates in their
regular media with a glucose concentration of 25 mM overnight and
intracellular glucose concentrations of the cells should be in
equilibrium with the extracellular glucose concentration. The
glucose concentration in cell growth media of some wells is reduced
by mixing regular glucose-containing medium with glucose-free
medium at different ratios to achieve the following final glucose
concentrations 25 mM, 10 mM, 2.5 mM, 1 mM, 0.25 mM (1% of the
original concentration). The media will be supplemented with of
.sup.3H-deoxyglucose at 1/50 of the cold glucose concentration and
then added to cells. The cells will be incubated for 30, 60, and
120 min, and the intracellular .sup.3H-DG will be measured by
scintillation counter after media removal and cell lysis as in a
standard glucose uptake assay. These samples treated by glucose
removal can be considered as positive controls of the experiment.
For comparison, cancer cells in wells with regular growth medium
supplemented with .sup.3H-DG will be treated with different
concentrations of known glucose transport inhibitors fasentin,
apigenin, or anti-GLUT1 antibody and then have their intracellular
.sup.3H-DG levels measured at the same times post treatment as the
glucose removal samples. Dose response and time response curves can
be generated from these data and then compared. These curves will
reveal if the glucose removal and inhibition of basal glucose
transport lead to the same or different intracellular glucose
concentrations. Our in house glucose transport inhibitor WZB-27 and
WZB-115 will also be used in the experiment and will be compared to
those samples treated by fasentin, apigenin, or anti-GLUT1
antibody. Cells not treated by either glucose withdrawal or
compound (but with same amount of radioactive .sup.3H-DG) will
serve as untreated baseline (negative) controls.
[0141] Measurement of GLUT1 Protein and mRNA Levels During Glucose
Removal or Inhibition of Basal Glucose Transport
[0142] It is known that GLUT1 protein and mRNA are down-regulated
when glucose is withdrawn. However, it is not known if the
inhibition of basal glucose transport induced by fasentin or our
inhibitor compounds also results in GLUT1 down-regulation. To
answer this question, cancer cells H1299 and MCF7 cells are treated
with (1) glucose withdrawal (removal from cell culture media) at
multiple glucose concentrations, and (2) inhibition of basal
glucose transport by the compounds (fasentin, apigenin, anti-GLUT1
antibody, and compounds WZB-27 and WZB-115) at multiple compound
concentrations. After treatments for 1, 2, 4, 8, 12, 24 or 48 hrs,
the cells are harvested and total cellular proteins are isolated. A
western blot analysis is carried out using anti-GLUT1 antibody
(from Santa Cruz) to compare GLUT1 protein levels in the samples of
two different treatments. Each GLUT1 protein band will be
quantified using densitometry and then normalized with its own
.beta.-actin protein loading control, and compared to GLUT1 level
in the untreated control samples. The comparison between treated
samples and untreated samples will tell us whether the treatments
lead to down-regulation of GLUT1 protein. The comparison between
samples of glucose removal and samples treated by compounds will
show whether these two treatments result in differences in GLUT1,
while the comparison between fasentin/apigenin/anti-GLUT1 antibody
treated samples and those treated by WZB-27/WZB-115 will reveal the
similarity and difference among these compounds.
[0143] In the same experiment, total RNA will also be isolated.
Commercially available primers unique to GLUT1 will be purchased
and used in real-time PCR to quantify GLUT1 mRNA levels in each
treatment conditions. GAPDH and/or .beta.-actin mRNA will be used
as internal RNA control. Comparisons will be made among these
treatments with untreated samples (control) and between glucose
removal and inhibition of basal glucose transport by compounds.
These comparisons will show whether GLUT1 is also down-regulated at
the mRNA level and whether these two treatments lead to different
GLUT1 mRNA expression results.
[0144] Measurement of Glycolysis Rates Under Different Experimental
Conditions
[0145] H1299 lung cancer and MCF7 breast cancer cells will be
treated with or without glucose removal or inhibitor of basal
glucose transport. Glycolysis rates of each treatment condition
will be measured by monitoring the conversion of 5-.sup.3H-glucose
to .sup.3H.sub.2O, as described previously. Briefly, 10.sup.6 of
H1299 and MCF7 cells are washed once in PBS prior to re-suspension
in 1 ml of Krebs buffer and incubation for 30 min at 37.degree. C.
Cells are then pelleted, re-suspended in 0.5 ml of Krebs buffer
containing glucose (10 mM, if not specified), and spiked with 10
.mu.Ci of 5-.sup.3H-glucose. Following incubation for 1 h at
37.degree. C., triplicate 50-.mu.l aliquots are transferred to
uncapped PCR tubes containing 50 .mu.l of 0.2 N HCl (for stopping
the reaction), and a tube is transferred to a scintillation vial
containing 0.5 ml of H.sub.2O such that the water in the vial and
the contents of the PCR tube are not allowed to mix. The vials will
be sealed, and diffusion is allowed to occur for a minimum of 24 h
(to reach equilibrium). The amounts of diffused and undiffused
.sup.3H are determined by scintillation counting. Appropriate
.sup.3H-glucose-only (no cell) and .sup.3H.sub.2O-only controls
will be included in the assay, enabling the calculation of
.sup.3H.sub.2O in each sample and thus the rate of glycolysis.
Glucose utilization rate will be calculated as .sup.3H H.sub.2O
formed from .sup.3H-glucose, expressed in the term of pmol of
glucose utilized/10.sup.6 cancer cells from the formula:
Glucose .times. .times. utilized .times. .times. ( pmol ) = [ 3
.times. H ] .times. water .times. .times. formed .times. .times. (
d.p.m. ) sp . .times. radioactivity .times. .times. of .times. [ 5
- 3 .times. H ] .times. glucose .times. .times. (d.p.m./pmol )
##EQU00001##
[0146] These measurements will enable us to determine how glucose
withdrawal and compound inhibition affect glycolysis rates in
cancer cells. Untreated cancer cells will be used as baseline
controls. Non-cancerous cell counterparts NL-20 (normal lung) cells
and MCF12A (normal breast) cells will also be used as controls for
comparison.
[0147] Glycolytic Enzymes Alteration During Glucose Withdrawal or
Basal Glucose Transport Inhibition
[0148] It has been shown that glucose withdrawal resulted in
down-regulation of glycolytic enzymes such as hexokinase and
pyruvate kinase (PK). PKM2 has been found to be very important for
tumorigenesis and exclusively expressed in cancer or proliferating
cells. It is still unclear if inhibition of basal glucose transport
by fasentin or our compound inhibitors also leads to similar
results. To answer this question, H1299 and MCF7 cancer cells
growing in 24-well cell culture plates will be treated with or
without anti-GLUT1 antibody, fasentin and our inhibitor compounds
WZB-27 and WZB-115 at their respective IC.sub.50 and IC.sub.70.
After 24 and 48 hr treatment, cells will be harvested and total
protein is isolated. Western blot analyses will be performed to
compare the levels of PKM2 in the treated and untreated samples
using PK antibodies (Cell Signaling). This study will enable us to
know exactly what happens to PKM2 when cancer cells are treated
with compound inhibitors. Same cancer cells treated with or without
glucose withdrawal will be included in the study for
comparison.
[0149] The activity of hexokinase, the first enzyme in glycolysis,
will also be studied by a similar method described for PK.
Antibodies against hexokinase are commercially available. In
addition, the enzymatic activity of hexokinase will also be
measured and used as an indication of changes in glycolysis as
hexokinase is the enzyme catalyzing the first rate-limiting step of
glycolysis. Hexokinase activity will be measured using a published
protocol. Briefly, the activity will be determined
spectrophotometrically at 30.degree. C. by coupling the formation
of glucose 6-phosphate with its removal via glucose-6-phosphate
dehydrogenase, during which the absorbance of NADPH at 340 nm
changes. Activity is expressed in mUs, 1 mU defined as the
formation of 1 nmol NADPH/min. Enzyme was dissolved in TrisMgCl2
buffer, pH 8.0 to obtain a rate of 0.02-0.04 AA/min. The assay
medium contained 0.05M TrisMgCl.sub.2 buffer, pH 8.0, 15 mM
MgCl.sub.2, 16.5 mM ATP, 6.8 mM NAD, 0.67 mM glucose, and 1.2
units/ml glucose-6-phosphate dehydrogenase. Incubate in the
spectrophotometer at 30.degree. C. for 6-8 minutes to achieve
temperature equilibration and establish blank rate, if any. At zero
time, add 0.1 ml of diluted hexokinase solution and mix thoroughly.
Record increase in absorbance at 340 nm for 3-4 minutes. Determine
AA/min from initial linear portion of curve.
[0150] Calculation will be performed using the formula shown
below:
Units/mg .times. .times. protein = .DELTA. .times. .times. A 340
.times. / .times. min 6.22 .times. mg .times. .times. enzyme
.times. / .times. ml .times. .times. reaction .times. .times.
mixture ##EQU00002##
[0151] Studies Related to Protein Factors Signal Transduction
During Glucose Deprivation Induced by Inhibition of Basal Glucose
Transport
[0152] The activation of Akt was found to increase the rate of
glycolysis partially due to its ability to promote the expression
of glycolytic enzymes through HIF.alpha.. This was speculated as a
major factor contributing to the highly glycolytic nature of cancer
cells. It would be also interesting to find out how changes in
glucose transport and glycolysis affect expression of Akt. In this
experiment, cancer cell H1299 and MCF7 will be treated with or
without anti-GLUT1 antibody, fasentin, WZB-27, or WZB-115 at their
respective IC.sub.50. Differentially treated cells will be
harvested 1, 2, 4, 8, 24 hrs after the treatment. Total proteins
from each sample will be isolated and analyzed by western blots
using antibodies specifically against Akt. Akt has multiple
phosphorylation sites and different antibodies will be used to
distinguish Akt phosphorylated at different sites. Treated samples
will be compared to untreated samples and samples with different
treatments will also be compared. Changes in intensity of total Akt
protein as well as changes in different phosphorylated forms of Akt
will specify how inhibition of basal glucose transport affects
expression and phosphorylation of Akt. Another protein factor
involved cell growth signaling pathway, AMPK, which has been found
to affect glycolysis, will also be studied the same way as Akt.
[0153] Inhibitors to Glucose Transport Sensitize and Synergize with
Anticancer Drugs in Cancer Cell Killing
[0154] The inhibitors disclosed herein target basal glucose
transport while other anticancer drugs target pathways or processes
not directly related to glucose transport. As a result, it was
speculated that glucose transport inhibitors could potentiate or
synergize with other anticancer drugs in their cancer killing
activity when used together. This would be consistent with the
recent finding that anti-GLUT1 antibody sensitizes and enhances the
anticancer activity of anticancer drugs and our inhibitors should
do the same. This has been shown to be the case in a compound study
in H1299 or A549 cells lung cancer cells, which were treated with
WZB-115 or (WZB-115+cisplatin) or (WZB-115+taxol) (FIG. 21).
Addition of WZB-115 to either cisplatin or taxol led to
significantly more cancer cell killing than that induced by drugs
alone. Similar results have been obtained for WZB-27 as well (FIG.
21).
[0155] This result suggests that glucose inhibitors such as WZB-115
or WZB-27 could significantly enhance the cytotoxic activity of
anticancer drugs. This is accomplished by the compound's
independent anticancer activity or the sensitizing activity of the
compound to anticancer drugs or both. Further mechanistic study
should be able to determine the real mechanism(s). This result,
similar to what others found in their glucose transport inhibitor
studies, strongly suggests that these compounds can be used alone
to inhibit cancer growth or used together with other anticancer
drugs to further increase the anticancer efficacy of the drugs.
This also suggests that we may not have to use these inhibitors at
very high concentrations as long as they are co-administered along
with other anticancer drugs.
[0156] Inhibitors are More Potent than Known Inhibitor Fasentin in
Both Glucose Uptake Inhibition and in Reducing Cancer Viability
[0157] Fasentin is a published inhibitor of basal glucose transport
and known GLUT1 inhibitor. A comparison of reported inhibitors with
fasentin was completed to determine if the compounds of interest
exhibited similar biological activities to fasentin. It has been
found that, similar to fasentin, our inhibitors induce reduction of
glucose uptake. Some compounds such as WZB-115, 131, and 133
demonstrated stronger inhibition than fasentin in cancer cells
(FIG. 22A). In addition, addition of these compounds (WZB-131 and
133) to different cancer cell lines resulted in more cancer cell
death than fasentin at the same concentration (FIG. 22B).
[0158] These results demonstrated that (a) these compounds exhibit
biological activities similar to fasentin, they are true inhibitors
of basal glucose transport like fasentin, (b) they are more potent
than fasentin in both activities tested. All these results indicate
that these compounds are fasentin-like and are inhibitors of
glucose transport but they possess more potent anticancer
activities. As a result, they can be used in studies of glucose
transport, glycolysis, and apoptosis of cancer cells as potentially
superior inhibitors than fasentin.
[0159] Anticancer Activity Screening of Compound WZB-115 in 59
Cancer Cell Lines Done by NCI
[0160] In order to further evaluate the lead compound WZB-115, the
compound was sent to the National Cancer Institute (NCI) for
screening its anticancer activities in a total of 59 cancer cell
lines (FIG. 23). The screening results indicate that, (a) among 59
cancer cell lines and at 10 .mu.M, the compound reduced the growth
rates of 51 cell lines by more than 10% (<90% of the growth
rates of the controls). (b) The compound reduced the growth rates
of 21 cancer cell lines by more than 50% (or 35.6% of the 59 lines
tested, FIG. 23). This result suggests that, in these 21 lines, the
IC.sub.50 of WZB-115 is lower than 10 .mu.M. (c) The compound shows
anticancer activities in all cancer types although it may be more
effective in certain cancer types than others. (d) Large variations
in activities are observed among cancer cell lines both within a
single cancer type or among different cancer types. As a result,
this compound is less likely to be very cytotoxic to normal cells
than those compounds that are equally cytotoxic to all cancer cell
lines. This is also consistent to our observation in animal
injection tests (FIG. 20). Because of these promising results, the
compound is recommended by NCI for a second round of screening
using 5 different concentrations to determine its IC.sub.50s in all
these different cancer cell lines.
[0161] In summary, the preliminary results indicate that these
compounds are inhibitors of basal glucose transport in all cancer
cell lines tested (Table 3 and FIG. 19). The compound treatment led
to apoptosis that is p53-independent and caspase 3-dependent (FIG.
18). The compound treatment caused significantly more cell death in
cancer cells than in their normal cell counterparts (Table 4).
Their preferential cancer cell killing also indicates that these
cancer cell lines are more sensitive to the compound treatment than
their normal cell counterparts, strongly suggesting these compounds
are significantly more toxic to cancer cells to normal cells. They
only cause mild and temporary hyperglycemia in animals (FIG. 20)
without other noticeable side effects. Furthermore, they potentiate
and synergize with existing anticancer drugs (FIG. 21). The
addition of the compounds led to phenotypic changes in cancer cells
as induced by glucose deprivation (FIG. 18C). These compounds form
a novel group of molecular tool and anticancer agents since they
inhibit a new target, basal glucose transport. Fasentin and these
compounds can be used for studying how glucose deprivation affects
glycolysis and how changes in glycolysis affect other changes such
as apoptosis.
[0162] Initial Animal Efficacy and Safety Study
[0163] The ability of compounds WZB-27 and WZB-115 to
inhibit/reverse tumor growth in nude mice, as well as the clinical
safety of the compounds will be determined. Compounds will be use
to treat nude mice (Jackson Labs) with cancer grown from H1299
(lung cancer) and MCF-7 (breast cancer) cells. Five millions of
cancer cells will be injected subcutaneously into the flank of each
of 30 nude mice. The tumor cell-injected mice will be randomly
split into three groups: ten for compound treatment, ten for drug
(e.g. WZB-117) treatment (positive controls) and another ten
receive vehicle (solvent) treatment. After tumors become palpable
and visible (.about.5-7 days), the compound treatment will begin. A
molar concentration of IC.sub.50 will be chosen for each compound
for the treatment. Compounds will be dissolved in DMSO or other
compatible solvent right before injection. The solvent that
dissolve the compounds will be used in the solvent treatment group.
The animal study design is shown in Table 5.
TABLE-US-00005 TABLE 5 Design of initial animal study (same study
in both H1299 and MCF-7 tumor mice) Group/ group Treatment
Parameters function N = Treatment duration measured 1. Negative 10
Solvent, once a day 4-5 wks Tumor size, control blood glucose, body
2. Fasentin, 10 Fasentin at IC.sub.50*, 4-5 wks Same as above
positive as above control 3. WZB-27 10 WZB-27 at IC.sub.50, 4-5 wks
Same as above as above 4. WZB-115 10 WZB-115 at IC.sub.50, 4-5 wks
Same as above as above *IC.sub.50 is determined from cancer cell
viability study. IC.sub.50 is different for each cancer cell
line
[0164] The IP injection of compounds will be performed once every
day for 4-5 weeks depending upon tumor growth rates. Tumor sizes
will be measured with calipers twice a week and recorded as
LW.sup.2/2=volume in mm.sup.3 (L=length, W=width) and compared to
those of tumors on solvent injected control mice. Body weight of
the mice is measured once a week. Body weight is an indication of
the health status of the treated mice. In order to determine how
compound treatment affects blood glucose levels, blood glucose will
also be measured immediately prior to the compound injection and 1,
2, and 4 hr after the injection and the blood glucose levels will
be compared to those of the solvent injected mice. Once the glucose
levels are measured, a decision will be made on if the blood
glucose monitoring should be continued or can be terminated. The
compound treatment lasts 3-5 weeks until the tumors grown in the
solvent treated mice become large (>5% but <10% of the body
weight, or .gtoreq.20 mm in the largest length measurement). The
animal study will be carried out and terminated in accordance to
the rules and regulations of NIH and of our university IACUC.
Tumor-bearing mice will be euthanized at the end of the study,
according to the related rules by NIH and DOA. The average size of
the tumors in the treated groups will be compared to the untreated
control group to show treatment efficacy and statistical
differences. The better of the two compounds, based on combined
consideration of anticancer efficacy and toxicity to mice
(primarily its effect on blood glucose levels and body weight
changes and/or other unexpected side effects), will be chosen for
the dose response animal study described below.
[0165] Determination of Dose Response of Compound Treatment in
Animals
[0166] Although anticancer efficacy may be shown in the initial
animal study, the dose used in the study is definitely not optimal
for each compound. In order to further determine the better dose,
at which anticancer efficacy is maximized but the side effects are
still tolerable, a dose response animal study will be conducted
(Table 6).
TABLE-US-00006 TABLE 6 Determination of compound dose response
Group/group Treatment Parameter function N Treatment duration
measured 1. Negative control 10 Solvent 4-5 wks Tumor size, blood
glucose levels, body weight 2. Fasentin low dose 10 Fasentin at
IC.sub.50 4-5 wks Same as above 3. Fasentin high dose 10 Fasentin
at 3x IC.sub.50 4-5 wks Same as above 4. WZB-27 low dose 10 WZB-27
at IC.sub.50 4-5 wks Same as above 5. WZB-27 high dose 10 WZB-27 at
3x IC.sub.50 4-5 wks Same as above 6. WZB-115 low dose 10 WZB-115
at IC.sub.50 4-5 wks Same as above 7. WZB-115 high dose 10 WZB-115
at 3x IC.sub.50 4-5 wks Same as above
[0167] For compounds Fasentin, WZB-27 and WZB-115, two doses will
be tried: IC.sub.50 and 3.times.IC.sub.50. After this experiment,
The dose response for these compounds will be known. We will also
know which compound performs the best in terms of reduction of
tumor size and side effects, and if the 3.times.IC.sub.50 dose can
be well tolerated by nude mice or not. This study will be done
using both H1299 and MCF-7 cancer cell models. From the results of
this experiment, one of the two compounds, WZB-27 or WZB-115
designated as compound to be determined (Compound.sub.tbd), and its
better dose, will be selected for the next round of animal
study.
[0168] Does Inhibitor of Basal Glucose Transport Potentiate and
Synergize with Cancer Drugs in the Anticancer Activity
[0169] Synergistic and potentiating effects were found between
fasentin and anticancer drugs (Ref). In our preliminary studies,
the similar effects were also observed (FIG. 21). However, it is
unclear if such synergistic effects can also be found in animals.
To that end, a large animal study will be conducted (Table 7).
TABLE-US-00007 TABLE 7 Design of animal study for determination of
synergy between inhibitors and cancer drugs Group/group Treatment
Parameter function N Treatment duration measured 1. Negative
Control 10 Solvent 4-5 wks Tumor size, blood glucose levels, body
weight 2. Fasentin 10 Fasentin at C.sub.tbd* 4-5 wks Same as above
3. Compound 10 Compound.sub.tbd** at C.sub.tbd 4-5 wks Same as
above 4. Cisplatin 10 Cisplatin at IC.sub.70 4-5 wks Same as above
5. Taxol 10 Taxol at IC.sub.70 4-5 wks Same as above 6. Fasentin +
cisplatin 10 Fasentin at C.sub.tbd + 4-5 wks Same as above
Cisplatin at IC.sub.70 7. Compound + cisplatin 10 Compound at
C.sub.tbd + 4-5 wks Same as above Cisplatin at IC.sub.70 8.
Fasentin + taxol 10 Fasentin at 4-5 wks Same as above C.sub.tbd +
taxol at IC.sub.70 9. Compound + taxol 10 Compound at 4-5 wks Same
as above C.sub.tbd + taxol at IC.sub.70 *C.sub.tbd = concentration
to be determined as described herein; **Compound.sub.tbd = compound
to be determined as described herein.
[0170] How Compound Inhibitor Treatment Affects Levels of
Proteins/Enzymes Involved in Glycolysis, Cell Growth Signal
Transduction, and Apoptosis
[0171] To better understand how inhibitors of basal glucose
transport inhibit tumor growth in vivo, tumors treated with or
without compound inhibitors will be removed from tumor mice
euthanized at the end of animal studies described above and are
immediately frozen by liquid nitrogen for late analysis. For tumor
analysis, proteins will be extracted from removed tumors and
quantified. Protein samples will then be subjected to PAGE followed
by western blotting analysis using antibodies specifically against
p53, Akt, PKM2, hexokinase, and caspases. The intensities of these
proteins from compound treated samples will be compared to those of
tumor samples that are treated by solvent (vehicle). Protein
.beta.-actin and/or GAPDH will be used as protein loading controls
for normalizing protein bands. These western blots will enable us
to gain the protein expression changes in the compound treated
tumors, which can potentially facilitate the final elucidation of
anticancer mechanism(s) of these compound inhibitors in vivo.
[0172] Synthesis of Generation 2 Compounds
[0173] Disclosed herein is the design and synthesis of a second
generation of basal glucose transporters based upon alteration of
the linkage between the parent aromatic ring and the phenolic
aromatic substituents. Previously, a small library of polyphenolic
esters were synthesized and evaluated. The generation 1 compounds
were shown to inhibit basal glucose transport in H1299 lung cancer
cells, and also inhibited cancer cell growth in H1299 cells.
WZB-115 was selected as the lead compound from this library.
Unfortunately, WZB-115 failed long-term stability assays in animal
models. The degradation rate of WZB-115 and WZB-117 in human serum
was established, both compounds degraded completely after 48 hours.
Thus, more stable analogs need to be designed and synthesized.
[0174] Disclosed herein are the structures of novel glucose
transport inhibitors WZB-115, WZB-117, and WZB-173 (FIG. 37).
Compound WZB-117 is an analog of WZB-115 while WZB-173 is an
ether-bond analog. Compounds WZB-117 and WBZ-173 (FIG. 37) are
derived from compounds WZB-115, which is a polyphenolic model
compound used in our generation 1 cancer studies. WZB-115 was
derived from a natural anticancer and antidiabetic compound called
penta-galloyl-glucose (PGG). WZB-117 and WZB-173 are very similar
structurally to WZB-115 but are both structurally simplified and
functionally optimized compared to WZB-115. As a result, WZB-117
and WZB-173 are more potent in their anticancer activities than
WZB-115 and are also structurally more stable than WZB-115 in
solution and cell culture media.
[0175] Synthesis of Multi Phenolic Ether Derivatives
[0176] 3-(Methoxymethoxy)benzyl chloride, the precursor for
predesigned polyphenolic derivatives, was synthesized in high yield
over four steps. Comercially available 3-hydroxybenzoic acid was
treated with sulfuric acid in methanol to elicit a Fischer
esterification. Protection of the phenol was accomplished by
treatment of the phenolate with MOMCl. Reduction of the ester
followed by a modified Appel reaction afforded the desired compound
in greater than 65% over four steps (Scheme 2).
##STR00014##
[0177] The synthesis of the polyphenolic ethers, amines, and amides
were accomplished via S.sub.N2-type reactions or nucleophilic acyl
substitution reactions (Scheme 3 and 4). Synthesis of the second
generation basal glucose transport inhibitors could be accomplished
by one who is skilled in the art without further description or
disclosure of further experimental detail.
##STR00015##
TABLE-US-00008 Compd # X.sup.1 (OH)n X.sup.2 (OR)n Yield.sup.a
WZB-131 OH 1,2-(OH).sub.2 3-OH--C.sub.6H.sub.4CH.sub.2O
1,2-(3-OH--C.sub.6H.sub.4CH.sub.2O).sub.2 54 WZB-132 OMe
1,2-(OH).sub.2 OMe 1,2-(3-OH--C.sub.6H.sub.4CH.sub.2O).sub.2 70
WZB-133 Cl 3,4-(OH).sub.2 Cl
3,4-(3-OH--C.sub.6H.sub.4CH.sub.2O).sub.2 65 WZB-134 F
1,2-(OH).sub.2 F 1,2-(3-OH--C.sub.6H.sub.4CH.sub.2O).sub.2 46
WZB-137 Cl 1,3-(OH).sub.2 Cl
1,3-(3-OH--C.sub.6H.sub.4CH.sub.2O).sub.2 57 WZB-141 Cl
2,4-(OH).sub.2 Cl 2,4-(3-OH--C.sub.6H.sub.4CH.sub.2O).sub.2 72
.sup.ayield is based on two steps
##STR00016##
TABLE-US-00009 Compd # X R.sup.2 a Y Yield WZB-125 Cl H H.sub.2,
Pd/C CO 83 WZB-138 Cl H 1) H.sub.2, Pd/C; 2) LAH CH.sub.2 75
WZB-142 Cl Me 1) Mel, NaH; 2) H.sub.2, Pd/C CO 71 WZB-145 Cl Me 1)
Mel, NaH; 2) H.sub.2, Pd/C; 3) LAH CH.sub.2 66 WZB-124 F H H.sub.2,
Pd/C CO 86 WZB-139 F H 1) H.sub.2, Pd/C; 2) LAH CH.sub.2 78 WZB-143
F Me 1) Mel, NaH; 2) H.sub.2, Pd/C CO 70 WZB-144 F Me 1) Mel, NaH;
2) H.sub.2, Pd/C; 3) LAH CH.sub.2 65 The separate yield is based on
the step(s) shown in reaction conditions above
[0178] Evaluation of Generation 2 Compounds
[0179] Compounds WZB-134, WZB-141 and WZB-144 inhibited basal
glucose transport in H1299 cells by 92.5.+-.2.2%, 96.5.+-.0.5%, and
78.2.+-.1.4%, respectively (Table 12), as measured by a standard
glucose uptake assay compared to non-compound treated cells
controls (considered as 0% inhibition). Tested in an MTT cell
proliferation assay in H1299 cells, their inhibitory activities on
cancer cell growth were found to be 10.6.+-.2.0%, 39.5.+-.1.8%, and
14.5.+-.7.6%, respectively (non-compound treated cell controls were
considered as 0% inhibition).
TABLE-US-00010 TABLE 12 Polyphenolic ethers, amine, and amide
induced inhibitory activities in basal glucose transport and cell
growth in H1299 lung cancer cells Glucose transport Cell growth
Compound # inhibition.sup.a (%) inhibition.sup.b (%) WZB-124 .sup.
89.7 .+-. 2.5.sup.b 5.7 .+-. 2.1 WZB-125 83.7 .+-. 0.7 5.9 .+-. 1.4
WZB-131 95.0 .+-. 1.6 12.9 .+-. 2.6 WZB-132 60.0 .+-. 7.7 6.0 .+-.
2.2 WZB-133 91.2 .+-. 0.8 14.3 .+-. 2.7 WZB-134 92.6 .+-. 2.2 10.6
.+-. 2.0 WZB-137 86.2 .+-. 1.6 -- WZB-138 52.2 .+-. 9.7 -- WZB-139
39.2 .+-. 0.8 -- WZB-141 96.5 .+-. 0.5 39.5 .+-. 1.8 WZB-142 82.7
.+-. 6.8 14.2 .+-. 9.0 WZB-143 85.7 .+-. 3.6 31.1 .+-. 7.9 WZB-144
78.2 .+-. 1.4 14.5 .+-. 7.6 WZB-145 73.2 .+-. 3.8 38.2 .+-. 6.6
.sup.aUntreated cells served as negative controls (0% inhibition).
.sup.bData were presented as a mean .+-. standard deviation.
[0180] Antitumor Study. Anticancer Activity of WZB-117 Against
Human Lung Cancer A549 Grrafted on Nude Mice
[0181] This animal tumor study indicated that, by daily injection
of WZB-117, the tumor size of the compound treated tumors were on
average approximately 75% smaller than that of the mock treated
group although the variation of the tumor sizes were quite large
(FIG. 30A). This result was qualitatively similar to that of a
tumor study using antiglycolytics. Noteworthy, two of the ten
compound treated tumors disappeared during the treatment and they
never grew back even at the end of the study (FIG. 30B). Body
weight measurement and analysis revealed that the mice treated with
WZB-117 lost 2-3 grams of weight compared to the mock-treated mice
with most of the weight loss in the fat tissue (Table 8). Blood
counts and analysis showed that some key blood cells such as
lymphocytes and platelets were significantly changed in the
compound treated mice, but they were still in the normal ranges
(Table 9). All these results indicate that the treatment of WZB-117
was effective in reducing tumor sizes and the treatment was
relatively well tolerated by the mice. One of the concerns for
using basal glucose transport inhibitors is that the inhibitor may
cause hyperglycemia in the injected patient. It has been found that
the injection of WZB-117 produced mild and temporary hyperglycemia
that disappeared within a period of 2 hours after the compound
administration, and it did not result in permanent
hyperglycemia.
[0182] Anticancer Mechanism Study. Anticancer Compound WZB-117
Inhibition of Glut1
[0183] To demonstrate that inhibitors of glucose transport induce
ER stress, 30 .mu.M of inhibitor WZB-117 was used to treat A549
cells (with glucose deprivation being the control). The Western
blot of the proteins isolated from the treated cells shows that
glucose regulated protein-78 (GRP78, also called BiP), one of the
key ER stress markers, was significantly upregulated at 48 and 72
hrs after the inhibitor treatment (FIG. 32), indicating that the
inhibitor indeed induces ER stress in cancer cells. Glucose
concentration in regular cell culture medium was 25 mM, and 2.5 mM
(10% of the regular concentration) was used as the glucose
deprivation control condition. The inhibitor also led to
qualitatively similar results in BiP upregulation as glucose
deprivation (FIG. 32).
[0184] Glucose deprivation induces upregulation of ER stress
protein BiP. Lung cancer A459 cells were treated by either glucose
deprivation or by inhibitor 117 for various times and then proteins
of the cells were analyzed by using anti-BiP antibody. .beta.-actin
serves as a protein control (FIG. 32A).
[0185] Previously, it was found that our anti-glucose transport
compounds inhibited glucose transport in all the cancer cell lines
tested. It was speculated that the target of these inhibitors is
Glut1 since Glut1 is responsible for basal glucose transport in
almost all cell types. In order to test this hypothesis, RBC was
chosen as a cell model to study because RBC has been known to
express only Glut1, not any other glucose transporters. The glucose
uptake assays revealed that WZB-117 indeed inhibited the glucose
transport in RBC (FIG. 31A), supporting the notion that WZB-117
inhibits glucose transport by inhibiting Glut1. To further
eliminate other possibilities, the glucose uptake assays were
repeated in RBC-derived vesicles, in which all the intracellular
proteins and enzymes were removed and only membrane-bound and
tightly associated proteins left. The assay result showed that
WZB-117 continued to inhibit glucose transport in these vesicles,
indicating that intracellular proteins are not needed for the
inhibition and providing strong evidence that Glut1 is the target
of the inhibition (FIG. 31C-E).
[0186] In order to determine the protein target of the inhibition
of basal glucose transport by our compound, human red blood cells
(RBC) were used. The selection of RBC was based on (1) Glut1 was
hypothesized as the most probable target and RBC express Glut1 as
their only glucose transporter, (2) RBC are an established model
for and have been frequently used in studying glucose
transport.
[0187] It was found that, in addition its inhibition of basal
glucose transport in all the cancer cell lines, our compound
inhibits glucose transport in RBC (FIG. 31), indicative that the
compound acts on Glut1 for the inhibition and supportive that Glut1
is the target of the inhibition.
[0188] To further the target identification, vesicles were prepared
from the ruptured RBC called ghosts. These small sealed vesicles
were formed from plasma membrane of RBC under different salt
conditions and they demonstrated two distinct orientations: inside
out or right side out. The right side out vesicles (ROV) exhibit
the same membrane orientation as RBC while inside out vesicles
(IOV) show opposite membrane orientation as the membrane of RBC.
However, since Glut1 is a glucose uniporter and can transport
glucose in both directions, Glut1 located on either IOV or ROV
should be able to transport glucose down the glucose gradient. As
expected, the glucose uptake assays showed that our compound could
inhibit Glut1-mediated glucose transport in both IOV and ROV (FIG.
31C-E), providing strong supporting evidence for our hypothesis
that Glut1 is the target of the action of the inhibition by our
compound. The reason that the compound showed more inhibition in
IOV than in ROV was due to the presence of Mg.sup.++ in the ROV
preparation. Mg.sup.++ is not previously known for its interference
with Glut1 function.
[0189] The observation that the compound worked on both ROV and IOV
also suggests that the compound may interact with the excellular
portion of Glut1. The extracellular portion of Glut1 is located
intracelluarly (intravesicularlly) in IOV. The compound is likely
to cross the vesicle membrane and interact with the intravesicular
part of Glut1, inhibiting the glucose transport down the gradient.
This is consistent with the chemical properties of the compound,
which indicates the hydrophobicity of the compound is likely to
allow the compound to cross the vesicle membrane and function
intravesicularlly in artificial vesicles or intracellularly in
intact cells.
[0190] Generation 2 Compounds Induce Cell Death Preferentially in
Cancer Cells Opposed to their Normal Counterparts
[0191] Cancer cells depend on glucose as their energy source and
basal glucose transport inhibition has been proposed as an
anti-cancer strategy. In order for these compounds to be effective
anti-cancer agents, they must be able to kill more cancer cells
than normal cells. Compound WZB117 kills significantly more cancer
cells than non-cancerous cells. A549 lung cancer and MCF7 breast
cancer cells (FIG. 35) were treated with or without WZB117 for 48
hr, and then measured for their respective viability rates with the
MTT assays. Mock-treated cells served as controls (100% viability)
for comparison. Noncancerous NL20 and MCF12A cells were treated the
same way for comparison. These results suggest that compound
WZB-117 has potential to serve as an anti-cancer agent. FIG. 36
suggests that compound WZB-117 exhibits significantly more
cytotoxicities towards cancer cells than towards non-cancerous
(i.e., "normal") cells.
Examples
General Experimental Protocols
Experimental Protocols. Chemical and Synthetic
[0192] General Scheme for Identifying Improved Basal Glucose
Transport Inhibitors
[0193] Identification of improved small molecule anticancer agents
that act as inhibitors of basal glucose transport is outlined in
FIG. 24. Upon design and synthesis of potential basal glucose
uptake inhibitors, the compounds will be examined in both a basal
glucose inhibition assay and an apoptosis assay at a single
concentration. Compounds that exceed a baseline value will be
further examined and IC.sub.50 values determined. The results of
these assays will be used to guide the design of additional
agents.
[0194] Pharmacological Evaluation of Inhibitors of Basal Glucose
Transport
[0195] All compounds will be evaluated in a series of
pharmacological assays designed to ascertain the ability of the
compounds to both inhibit basal glucose uptake and kill cancer
cells. Initial evaluation of compounds will focus upon elimination
of compounds that are unstable in serum or lack appropriate
activity.
[0196] Compounds will be assayed for their ability to inhibit basal
glucose uptake. All compounds will be tested at a concentration of
10 .mu.M first and their inhibitory activity on basal glucose
transport will be measured and compared to that of WZB-27. Cell
lines H1299 (lung cancer) and MCF-7 (breast cancer) grown in
24-well cell culture plates will be treated with or without the
compounds for 10 min before the glucose uptake assay. Cellular
glucose uptake will be measured by incubating cells in glucose-free
RPMI 1640 buffer with 0.2 Ci/mL [.sup.3H]2-deoxyglucose (specific
activity, 40 Ci/mmol) for 30 min in the absence and presence of
compounds. After removal of the buffer, the cells are washed with
ice-cold PBS, cells will be lysed and transferred to scintillation
vials and the radioactivity in the cell lysates will be quantified
by liquid scintillation counting. Only those compounds that show
comparable or stronger inhibitory activity than that of WZB-27 will
be selected for the next assay. Known glucose transport inhibitors
Fasentin (IC.sub.50.gtoreq.50 .mu.M) and apigenin
(IC.sub.50.gtoreq.60 .mu.M) will be used as positive controls for
comparison.
[0197] Initial assays will also determine the ability of these
compounds to kill cancer cells and to leave normal cells untouched.
Those compounds that pass the criteria of the glucose uptake assay
for inhibitory activity of basal glucose transport described above
will be tested in a subsequent cell killing (viability) assay.
Compounds will be individually added to H1299 (and its normal cell
counterpart NL20) and MCF-7 (and its normal cell counterpart
MCF-12A) cells grown in 24-well plates at a concentration of 30
.mu.M and incubated at 37.degree. C. in a cell culture incubator
for 48 hrs. After incubation, the cells in each well will be
measured for its viability by an MTT assay.
[0198] Further investigation of compounds will be conducted pending
that the compounds of interest inhibit glucose uptake greater than
WZB-27, cause a decrease in cancer cell viability of at least 50%,
and cause no more than a 20% decrease in normal cell viability.
Further assays for compounds meeting the minimal initial screening
requirements will include determination of an IC.sub.50 for glucose
uptake inhibition and an EC.sub.50 for cell killing by including
multiple concentration assay points such as 0.1, 0.3, 1, 3, 10, 30,
and 50 .mu.M.
[0199] Synthesis of More Potent and Selective Inhibitors of Basal
Glucose Transport
[0200] The general goal is to generate more potent and selective
inhibitors of glucose uptake. These compounds should have a lower
molecular weight, improved water solubility, and good stability.
All compounds will be prepared on a 15-20 mg scale and will be
purified to >90% purity as analyzed by HPLC, LCMS and
.sup.1H/.sup.13C NMR. Each compound will be stored in a bar-coded
vial as a 50 mM solution in DMSO. This scale will provide ample
material for initial screens, as well as follow-up screening if
necessary.
[0201] As shown in FIG. 25, lead compounds (WZB-115, WZB-117, and
WZB-118) have four distinct molecular areas of interest, the
central aromatic core, the pendant aryl groups, the linker, and the
substitution on the central aromatic core. Each of these four areas
will be examined in order to determine its significance and to
develop more potent analogs. Initial focus will be placed upon the
development of non-hydrolyzable linkers that retain the activity of
the parent ester. This will be key for carrying out in vivo
efficacy studies. Concurrently, the replacement of the pendant
phenol with bioisosteric replacements will be examined in an effort
to improve bioavailability. The substitution and identity of the
central aromatic core will also be examined in an effort to improve
potency.
[0202] Linker Modifications
[0203] Contemplated herein are a series of analogs (FIG. 26) in
which the linker between the central aromatic core and the pendant
aromatic rings has been modified. These investigations into
modifications of the linker are focused on the replacement of the
potentially labile ester linkage. These studies are significant in
that the linker may have a profound influence on the conformational
relationship between the central aromatic core and the pendant
aromatic ring. In addition more hydrolytically stable analogs will
be of great utility in in vivo studies. Three analogs with all
carbon linkages between the core aromatic ring and the pendant
aromatic ring (6, 7, 8) will be prepared. Sulfur-linked analogs
will also be prepared, which will include a sulfide, sulfoxide, and
sulfone linker (9, 10, 11). An ether analog with an oxygen linkage
between the core aromatic ring and the pendant aromatic rings (12)
will be prepared. Amide-linked analog 13 and two bicycle-linked
derivatives (14, 15) will be prepared. All of these analogs are
based upon the WZB-117 structure. This core has been chosen based
upon the activity of the parent compound and the ease of synthesis
based on the WZB-117 core relative to the WZB-27 core.
[0204] Contemplated herein are the energy-minimized structures of
the proposed analogs are shown in FIG. 14. Several analogs stand
out in their similarity to the parent WZB-117. These include alkene
7, sulfoxide 10, sulfone 11, and amide 13. All of these analogs
have the pendant aromatic rings on opposite sides of the core
aromatic ring. As might be expected alkane 8, sulfide 9, and ether
12 share strong similarities. The highly flexible nature of these
analogs suggest that they can adopt a wide variety of conformers
including that of the active WZB-117. Alkyne 6 and benzimidazole 14
are similar as they share a relatively planar overall structure.
Dioxin 15 is unique, having a single well-defined conformation with
both pendant aromatic rings on the same side of the core aromatic
ring. Obviously, the alkyne and alkene derivatives with a very
rigid linker provide unique structures unlike any of the others.
The synthesis and assay of this set of analogs will provide
information on optimal conformations and determine appropriate
hydrolytically stable linkers.
[0205] The first set of analogs contemplated herein will contain a
carbon linkage between the pendant hydroxyphenyl ring and the core
fluoro phenyl ring and will be prepared using a single synthetic
sequence. Starting from the known dibromo fluorobenzene 16, two
Sonagashira couplings with terminal alkyne 17 will be achieved to
provide an alkyne linked intermediate. Alkyne 17 can be prepared
from the requisite aldehyde or via a Sonagashira coupling of the
bromide. Deprotection of the hydroxyl group with acid should
provide target analog 6. The alkyne will then be partially reduced
to provide cis olefin analog 7. Finally, a complete hydrogenation
of the olefin will be effected with H.sub.2 and Pd to provide
carbon-linked analog 8.
##STR00017##
[0206] Also contemplated herein are a series of sulfur-linked
analogs, including compounds 10 and 11, which will provide
bioisosteric and hydrolytically stable analogs of the parent
esters. Additionally, the three analogs (9-11) will alter the
acidity of the phenol groups. The synthesis of this series starts
with the dibromination of commercially available fluoride 18. The
dibromide will then be used to alkylate thiophenol 19 to provide
the first analog. Two sequential oxidations will then provide the
sulfoxide (10) and the sulfone (11).
##STR00018##
[0207] The synthesis of the ether-linked derivative starts with the
dialkylation of diol 20 with benzyl bromide (21) to provide a
protected bis ether. Bromide 21 can be readily prepared via the
reduction/bromination of the corresponding acid. Deprotection of
the phenolic hydroxyl groups with acid will then provide the target
ether-linked analog 12.
##STR00019##
[0208] The amide derivative provides a conformationally restricted
analog relative to the parent ester as well as providing a more
hydrolytically stable derivative. The synthesis is quite
straightforward and simply requires the acylation of commercially
available diamine 23 with the requisite acid. Removal of the MOM
group will provide analog 13.
##STR00020##
[0209] Dioxin derivative 14 will provide a highly conformationally
restricted analog. As shown below, this compound will be prepared
via a condensation of .alpha.-bromoketone 27 and diol 20. Bromide
27 can be prepared via the coupling of Weinreb amide 24 with
Grignard reagent 25. This will provide ketone 26, which can be
readily brominated to provide key .alpha.-bromoketone 27.
Condensation of 27 with diol 20 followed by stereoselective
reduction of the intermediate oxonium ion provides the
cis-disubstituted dioxin 14.
##STR00021##
[0210] Benzimidazole analog 15 provides an alternate conformation
relative to dioxin 14. In addition the benzimidazole ring should
provide improved water solubility relative to the parent WZB-117.
Compound WZB-117 has a C log P of 2.96 (lower numbers indicating
greater water solubility) while analog 15 has a C log P of 2.72.
Again, this compound should be hydrolytically stable. Diamine 22
will be condensed with aldehyde 28 to provide substituted
benzimidazole 29. Acylation followed by removal of the benzyl
protecting groups should provide the target compound 15.
##STR00022##
[0211] This set of compounds will provide an initial structure
activity relationship for glucose uptake inhibition relative to the
linker group. Several analogs have been proposed that address the
stability of the ester group as well as the conformation of the
pendant aromatic rings. These analogs represent only the initial
targets for modification of the ester linkage and further
modifications will be made to extend these studies as warranted by
the pharmacological activity of this set.
[0212] Also contemplated herein are the following compounds,
wherein R is selected from the group consisting of H, Me, Et, and
iPr:
##STR00023## ##STR00024##
[0213] Pendant Bioisosteric Replacements
[0214] The 3-hydroxyphenyl group has been identified as the optimal
pendant aromatic group. Phenols are known to often have poor
bioavailability and short duration of activity due the facile
metabolism, conjugation and excretion of this group. Given the need
to develop more metabolically stable analogs, a series of phenol
bioisosteres will be examined. Dozens of such bioisosteres have
been reported in the literature.
[0215] Contemplated herein are two general sets of analogs, the
acetamido group and a series of heterocyclic bioisosteres.
3-Aminobenzoic acid will be converted to an acetamide,
methansulfonamide, and a urea and then couple to diol 20 to provide
analogs 31, 32, and 33, respectively. An additional analog (34),
based on the replacement of a phenol with a hydroxymethyl group as
seen in the 0 adrenergic blocker albuterol, will also be prepared.
Four different heterocyclic derivatives (35-38) will be prepared by
coupling the commercially available heterocyclic carboxylic acid
with diol 20. This set of compounds will provide information on the
ability to replace the phenolic group with potentially more
metabolically stable moieties.
[0216] Also contemplated herein are the following compounds,
wherein X is selected from the group consisting of H, 3-Cl, 3-F,
3-CN, 4-F, 4-CN, 4-NO.sub.2, 4-SO.sub.2Me, and 4,5-Cl.sub.2:
##STR00025## ##STR00026##
[0217] Optimization of the Central Aromatic Core
[0218] Also contemplated herein is a third group of analogs, in
which the central aromatic ring from WZB-117 and WZB-118 will be
modified. This study will allow for the optimization of the
activity of WZB-117 and WZB-118 analogs through changes in the
substitution (i.e. the F or Cl group) on the aromatic ring. The
introduction of a chloro- or fluoro-group to the aromatic core and
the removal of one of the benzoyl groups has resulted in improved
potency and a lower molecular weight. It has been demonstrated that
the 3-fluoro and 4-chloro derivatives (when counting starting with
the furthest O-benzoyl group) have the best activity. In terms of
generating additional analogs, this means that the diols shown in
FIG. 29 will be critical to further scaffold elaboration. A Topliss
tree type approach has been employed to generate many of these
derivatives. These derivatives will be acylated with acid chloride
23 and then deprotected as shown previously. These compounds are
commercially available or may be easily prepared from a
commercially available precursor by one who is skilled in the art.
Compounds 39, 42, 46, and 43 are commercially available. Nitriles
40 and 44 can be prepared via an oxidative conversion of the
corresponding aldehyde to the nitrile. Nitro derivatives 41 and 45
may be prepared by nitration with a zeolite supported copper
nitrate reagent.
[0219] This set of compounds will provide valuable insight into
optimal substitution on the central aromatic ring and provide
directions for the synthesis of generation 4 and 5 analogs. This
set of derivatives is a small sample of potential analogs that will
be synthesized as a result of positive biological outcomes from
generation 2 analogs, and further generations of analogs may be
synthesized based upon the combination of results from several
specific modifications.
Experimental Protocols. Biological
[0220] Compounds. Powders of compounds were stored at -20.degree.
C. and solutions were freshly prepared before each experiment.
Compounds were dissolved in DMSO to make 10 mM stock solution. In
most studies, 30 .mu.M WZB-27 and 10 .mu.M WZB-115 were used for
cell treatment.
[0221] Cell lines and cell culture. Human non-small cell lung
cancer (NSCLC) cell lines H1299 and A549, human duct epithelial
breast cancer MCF7, and human non-tumorigenic NL20 lung and MCF12A
breast cells were purchased from ATCC. H1299, A549 and MCF7 cells
were maintained in Dulbecco's Modified Eagle Medium (DMEM) with 10%
fetal bovine serum. NSCLC cell lines A549, H358, H226, and H1650
are grown in Ham's F12K containing 10% FBS. NL20 cells were
maintained in a Ham's F12 medium, supplemented with 0.1 mM
nonessential amino acids, 0.005 mg/ml insulin, 10 ng/ml epidermal
growth factor, 0.001 mg/ml transferring, 500 ng/ml hydrocortisone,
and 4% fatal bovine serum. MCF12A cells were cultured in a 1:1
mixture of DMEM and Ham's F12 medium, with 20 ng/ml human epidermal
growth factor, 100 ng/ml cholera toxin, 0.01 mg/ml bovine insulin,
500 ng/ml hydrocortisone and 5% horse serum. All cells were grown
at 37.degree. C. in a humid atmosphere with 5% CO2. Cells were
treated with compound WZB-27 or WZB-115 at concentration of 30
.mu.M or 10 respectively, for 24 or 48 hours. Untreated cells were
used as control.
[0222] Cell lysate preparation and Western blot analyses. Protocol
1. Cells were harvested from plates, re-suspended with 3.times.
sample buffer, and boiled for 5 min. Approximately 50 .mu.g of
protein extract was loaded after protein concentration measurement
by Pierce bicinchoninic acid (BCA) protein assay (Pierce
Biotechnology, Inc. Rockford, Ill.). Samples were run on a 10%
Bio-Tris NuPAGE gel (Invitrogen) and transferred to a
polyvinylidene difluoride membrane (PVDF, Biorad). The membrane was
incubated with antibodies specific to p53, or PARP, or
.beta.-actin. Specific protein bands were visualized after the
development of film. Antibodies for p53 were purchased from Santa
Cruz Biotechnology, Inc. (Santa Cruz, Calif., USA). The PARP
antibody and .beta.-actin antibody were purchased from Cell
Signaling Technology, Inc. (Danvers, Mass., USA).
[0223] Cell lysate preparation and Western blot analyses. Protocol
2. Lysates from cells are prepared by NP40 lyses. Samples are
boiled in equal volume of 2.times.SDS sample buffer, and separated
on 8% polyacrylamide gels. After semi-dry transfer to supported
nitrocellulose membranes, the blots are probed with monoclonal
antibody to GLUT1 from R&D systems. The proteins are detected
by using an enhanced chemiluminescence assay system from Amersham
Biosciences.
[0224] Real time RT-PCR protocol. Total RNA was isolated using
Trizol (Invitrogen, Carlsbad, Calif.). In order to eliminate any
carryover of genomic DNA, total RNA was treated with DNAse using
the DNA-free kit (Ambion, Austin, Tex.). cDNA was synthesized from
total RNA using the Advantage RT for PCR (BD Biosciences, Palo
Alto, Calif.). One mg of the total RNA was used in a 50 .mu.l
reaction mixture with the random hexamer primer. Real time primers
and TaqMan.RTM. probes for GAPDH were purchased from Biosource
(Camarillo, Calif.), and were used according to the manufacturer's
instructions. Three ml of cDNA template were used in 25 .mu.l of
real time PCR reaction with ABI TaqMan.RTM. Universal Master Mix
(Applied Biosystems, Branchburg, N.J.). The GLUT1 detection is done
with Sybr.RTM. green dye and the Quntitect Sybr.RTM. Green kit
according to the manufacturer's instructions, using 1 .mu.l of cDNA
template in a 25 .mu.l reaction volume.
[0225] Glucose uptake assay. Cancer cells are treated with or
without the compounds for 10 min before the glucose uptake assay.
Cellular glucose uptake will be measured by incubating cells in
glucose-free RPMI 1640 with 0.2 Ci/mL [.sup.3H]2-deoxyglucose
(specific activity, 40 Ci/mmol) for 30 min in the absence and
presence of compounds. After the cells are washed with ice-cold PBS
and lysed, the cell lysates will be transferred to scintillation
counting vials and the radioactivity in the cell lysates is
quantified by liquid scintillation counting.
[0226] Cell cycle analysis. After being treated by compounds or
medium with low concentration of glucose, cells were harvested,
washed with cold PBS, and re-suspended in 70% cold ethanol. After
an overnight fixation, ethanol was removed and cells were treated
with propidium iodide, DNase-free RNase A, and PBS mix (4:1:95) for
30 min at 37.degree. C. The DNA content was analyzed by flow
cytometry (FACS, BD). Modfit software (Verity Software House) was
used to calculate the percentage of cells in each phase of the cell
cycle. Each sample was repeated three times.
[0227] Cell viability assay. MTT assays are performed by the
following well-established method. In a 96 well tissue culture
plate 10,000 cells are plated in each well. The cells are incubated
in presence or absence of the compounds for 18 h.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
is dissolved in PBS (10 mg/ml) and filter sterilized. Three hours
before the end of the incubation 20 .mu.L of MTT solution is added
to each well containing cells in a 96-well plate. The plate is
incubated in an incubator at 37.degree. C. for 3 h. Media is
aspirated gently and 200 .mu.L of DMSO is added to each well to
dissolve formazan crystals. The absorbance is measured at 550 nm
for cell viability.
[0228] Proliferation assay. MCF-7, H1299 and H1650 cells are plated
onto poly-d-lysine (Sigma) coated 8-well glass chamber slides
(10,000 cells per well). The cells are incubated with the compounds
for 24 h or 48 hr. The cells are fixed and stained using
5-Bromo-2'-deoxyuridine labeling and Detection kit from Roche
according to manufacturer's protocol.
[0229] Study for anticancer synergistic effect. H1299, and MCF7
cells were grown in 96-well plates. Cells were treated with either
cisplatin or taxol at their IC.sub.50s in presence or absence of
compound WZB-27 (30 .mu.M) or WZB-115 (10 .mu.M) at 37.degree. C.
for 48 hours. Viability of the treated cells was measured by the
MTT cell proliferation assay.
[0230] Apoptosis assay. MCF-7, H1299 cells are plated onto
poly-d-lysine (Sigma) coated 8-well glass chamber slides (10,000
cells per well). The cells incubated with the compounds of various
concentrations for 24 h. Cells without compound treatment serve as
controls. After 24 h of incubation cells are fixed and stained
according to manufacturer's instructions using Promega's DeadEnd
Colorimetric TUNEL system. The cooperative effect of drugs will
also be evaluated by adding 5 .mu.M of cisplatin or paclitaxel or
10 .mu.M gefitinib by the same procedure.
[0231] Immunofluorescence. GLUT1 monoclonal antibody will be
purchased from R&D Systems Inc (Minneapolis, Minn.). Cells are
plated onto poly-d-lysine (Sigma) coated 8-well glass chamber
slides (10,000 cells per well) for immunostaining. Cells are fixed
in 3.5% paraformaldehyde for 25 min, permeabilized in 0.2% Triton
X-100/PBS for 5 min, and blocked in 5% normal goat serum in PBS at
room temperature for 1 h. Primary antibody incubation is performed
overnight at 4.degree. C. After washing, secondary antibody
incubation is performed with goat anti-mouse IgG Alexa Fluor-488
for 30 min at room temperature. DAPI is detected using Vectashield
Mounting Medium with DAPI (Vector Laboratories, Inc.). Staining by
secondary antibodies only will be used as negative controls. Slides
are observed by fluorescence microscopy using fluorescent
microscope (40.times./0.75 numerical aperture) with a camera.
[0232] Statistical analyses. Samples of same experimental
conditions are in triplicate or more. Each experiment is repeated
at least once (animal studies are exceptions). Data will be
reported in mean.+-.standard deviation or standard error of means.
Data will be analyzed using unpaired Student t-test and with
one-way or two-way ANOVA with Turkey's post-hoc test depending on
the nature of the assays. Significance level was set at
p.ltoreq.0.05.
Experimental Protocols. Animal Studies
[0233] The ability of compounds to inhibit/reverse tumor growth in
nude mice, as well as the clinical safety of the compounds will be
determined. Only compounds that meet the following criteria will be
considered for animal studies; IC.sub.50 of <10 .mu.M in the
glucose uptake inhibition, and an EC.sub.50 of <10 .mu.M in cell
killing assays as well as significant less killing in normal
cells.
[0234] For the anticancer efficacy and safety animal study, each
selected compound will be use to treat nude mice with cancer grown
from H1299 (lung cancer) and MCF-7 (breast cancer) cells. Five
millions of cancer cells will be injected subcutaneously into the
flank of each of 15 nude mice. The tumor cell-injected mice will be
randomly split into three groups: five for compound treatment, five
for drug (e.g. WZB-117) treatment (positive controls) and another
five receive vehicle (solvent) treatment. After tumors become
palpable and visible (.about.2-3 weeks), the compound treatment
will begin. A molar concentration of EC.sub.50 will be chosen for
each compound for the treatment. Compounds will be dissolved in
DMSO or other compatible solvent.
[0235] The IP injection of compounds will be performed 3 times a
week for 3-5 weeks depending upon tumor growth rates. Tumor sizes
will be measured with calipers twice a week and recorded as
W.times.L.times.H=volume in mm.sup.3 and compared to those of
tumors on non-compound injected control mice. Body weight of the
mice is measured once a week. In order to determine how compound
treatment affects blood glucose levels, blood glucose will also be
measured immediately prior to the compound injection and 1 hr after
the injection and the blood glucose levels will be compared to
those of the non-compound injected mice. The compound treatment
lasts 3-5 weeks until the tumors grown in the untreated mice become
large (>5% but <10% of the body weight). The animal study
will be carried out and terminated in accordance to the rules and
regulations of NIH and of our university IACUC. Tumor-bearing mice
will be euthanized at the end of the study, according to the
related rules by NIH and DOA. The average size of the tumors in the
treated groups will be compared to the untreated control group to
show treatment efficacy and statistical differences.
[0236] The best compound(s), based on combined consideration of
anticancer efficacy and toxicity (primarily its effect on blood
glucose levels and body weight changes and/or other unexpected side
effects), will be chosen for a larger scale animal study described
below.
[0237] Investigation of the Molecular Mechanism of Action of Basal
Glucose Transport Inhibitors
[0238] Anticancer Activities in More Cancer Cell Lines
[0239] Contemplated herein is a cancer cell line screen including
multiple lung and breast cancer cell lines that will be tested with
compounds to determine their anticancer activities via the MTT
assay to demonstrate that the compounds' cancer cell killing
activity is an activity that is cell line-independent. H1299 cells
are derived from non-small cell lung cancers (NSCLC). Other NSCLC
cell lines such as A549, H358, H226, and H1650 will also be tested.
Similarly, the compounds will also be tested in breast carcinoma
cell line T47D along with MCF-7 to determine if the compounds
exhibit similar anti-cancer activity in the additional breast
cancer cell lines. Normal cells of same tissues (NL20 for normal
lung tissue and MCF-12A for normal breast tissue) will be included
in the study to establish compounds' increased cytotoxicity and
killing to cancer cells than to their normal counterparts. To
correlate inhibition of basal glucose transport with cytotoxicity
of the compounds, glucose uptake rates of cancer and normal cell
lines will be measured and compared using the glucose uptake assay.
The cancer cell lines are expected to exhibit higher glucose uptake
rates than their normal cell counterparts due to their higher
energy needs. Increased cell killing in cancer cells opposed to
normal cells may be due to the inhibition of basal glucose
transport that is crucial for cancer cell proliferation and
survival. This study will also further strengthen the notion that
the basal glucose transport is the target of the anticancer action
of the compounds disclosed in this patent and related publications.
The enzymatic activity of hexokinase, the first enzyme involved in
glycolysis, will also be measured with standard assays in the
compound treated cells using untreated cells as a control to
determine how glycolysis is affected by the compound treatment in
these cancer cells.
[0240] In addition to the cell lines described, select compounds
will be submitted to the Developmental Therapeutics Program's NCI
60-Cell Line Screen.
[0241] Also contemplated herein is the theory that the basal
glucose tansporters disclosed will potentiate the chemotherapeutic
effects of other anti-cancer agents. In order to determine if the
compounds can potentiate anti-cancer activity of other anticancer
drugs, 5 .mu.M of cisplatin or paclitaxel or 10 .mu.M gefitinib
will be added to H1299 and MCF-7 cells in the absence and presence
of the compound for 48 hrs. After incubation, cell viability will
be determined by both MTT and the cell proliferation assays (see
general procedures at the end of this section for details).
(Drug+compound) treated samples will be compared to those samples
treated by drugs alone. Increased cell death in the (drug+compound)
treated samples indicates that the compound could potentiate the
anticancer activity of the drugs.
[0242] Additional Receptor Binding Assays
[0243] Contemplated herein are a series of receptor binding assays
which will be used to determine the action target of the basal
glucose transport inhibitor disclosed in this application. In order
to investigate the action target of the compound, a binding
competition study will be carried out. Anti-GLUT1 antibodies will
be added to H1299 or MCF-7 cells in the absence and presence of
increasing amount of the compound at 37.degree. C. for 1 hr. After
co-incubation, unbound antibodies and compound will be removed by
washing. The treated cells will be incubated with a secondary
antibody (goat anti-mouse IgG Alexa Fluor-488) that interacts with
the bound anti-GLUT1 antibodies. A "chromogenic" reaction will be
performed after the secondary antibody binding. The intensity of
fluorescence generated by the bound secondary antibodies should be
proportional to the GLUT1-bound primary antibodies. The intensity
of the fluorescence of differently treated cells will be quantified
and compared. The decrease of the intensity in the
antibody/compound treated samples suggests that the presence of the
compound decreases the binding of GLUT1 antibodies to GLUT1 and
further strongly suggests that the compound bind to GLUT1 located
on the cell membrane. On the other hand, if no competition is
found, it does not necessarily mean that the compound does not bind
to GLUT1. It may also mean that the compound binds to a place on
GLUT1 that is different from the binding site of anti-GLUT1
antibody.
[0244] To further determine how compounds work, the anti-GLUT1
antibody of a fixed concentration will be added to cancer cells in
a glucose uptake assay in the absence and presence of the compounds
of various concentrations. In a similar study, the compound
concentration can be fixed and the antibody's concentration can be
varied. These glucose uptake assays are to determine whether the
basal glucose transport inhibitory activities of anti-GLUT1
antibody and the compounds are additive or synergistic to each
other. If the activities are additive to each other, it may suggest
that these two agents act, probably but not necessarily, on the
same target. If the activities are synergistic, it is more likely
that these two molecules act on different targets. It is also
possible that the effects may not change or even decrease when
compounds are added with GLUT1 Ab.
[0245] Contemplated herein is a method to directly show the binding
of the compound to GLUT1, which will be accomplished by the
inclusion of a fluorescent tag as a moiety on any lead compounds.
It is anticipated that more potent and selective analogs will be
identified prior to the preparation of fluorescent tracers. Two
approaches will be used to identify the necessary fluorescent
probes. As an illustrative example we will show a synthesis based
on the current lead compounds. In the first approach we will
replace a pendant aromatic ring with a fluorescent tag. Depending
upon the SAR for these compounds this could be the optimal approach
in that we can use fluorescent tags similar to the pendant aromatic
rings. Thus a significant change in affinity to the biological
target would be decreased. As shown below, we would monoprotect
triol 1, and then introduce two esters onto the free hydroxyl
groups. The protected hydroxyl would be deprotected to provide 48.
The fluorescent coumarin 49 would be coupled to provide the target
fluorescent probe 50.
##STR00027##
[0246] A second approach will be to simply label an active compound
with a fluorescent tag. For example we would couple WZB-113 with
any of a number of commercially available fluorescent carboxylic
acids (flRCOOH=49 or rhodamine, or carboxynaphthofluorescein).
Based on the activity of WZB-113 relative to WZB-117 only one of
the two hydroxyl groups is likely involved in significant
non-covalent interactions. Thus we can use one of these hydroxyl
groups to attach the fluorescent tag.
##STR00028##
[0247] All fluorescently tagged compounds will first be evaluated
for their ability to both inhibit basal glucose transport and
induce apoptosis. If the fluorescent analogs do not act in a
similar manner to the parent compound new derivatives will be
prepared.
[0248] Varying concentrations of the fluorescent compound, will be
used to incubate with GLUT1 pre-bound to the bottom of a 96-well
plate with a protocol similar to that previously used for insulin
receptor binding of the polyphenolic compound PGG. After an
overnight incubation at 4.degree. C. with shaking, the unbound
compound will be washed off. The fluorescence intensity of
different samples will be measured with a 96-well plate reader
(SPECTRA Max M2, software: SoftMax Pro). A GLUT1 binding saturation
curve can be generated from intensities corresponding to different
compound concentration. The GLUT1 binding affinity (K.sub.a) can
also be generated from this binding experiment. A binding
displacement curve can also be generated from an assay in which an
increasing amount of regular compound is added to GLUT1 pre-bound
to 96 well plates while fluorescent compound is kept at a fixed
concentration. At higher concentrations, the regular compound will
compete with fluorescent compound for the same binding site on
GLUT1, reducing the fluorescent intensity proportionally to the
increased concentrations of the regular compound. The binding
affinity of the compound (K.sub.d) can be derived from the
displacement curve using computer software.
[0249] Relationships and Signaling Pathways Linking Inhibition of
Basal Glucose Transport and Induction of Apoptosis in Cancer Cell
Lines
[0250] Although it has been shown that addition of compounds leads
to inhibition of basal glucose transport inhibition and apoptosis
(cancer cells), the cause-effect relationship between the
inhibition and apoptosis has not been established. To determine the
relationship, cell samples treated with anti-GLUT1 antibody will be
used as a positive control. Since the anti-GLUT1 antibody has only
one target, GLUT1, the apoptosis induced by the addition of the
antibody is the "direct effect" caused by the antibody. In this
study, the apoptosis induced by the compounds will be compared side
by side with the apoptosis induced by the antibody. The parameters
compared include: 1) onset time of the apoptosis induced; 2) dose
responses of apoptosis; 3) apoptosis inducing mechanism. It has
been disclosed that the apoptosis induced by compounds WZB-25 and
WZB-27 is p53-independent (FIG. 24A). If the apoptosis induced by
the antibody is also p53-independent (p53 is not significantly
changed by the antibody treatment), then this evidence would
support the fact that these compounds induce apoptosis in cancer
cells using a mechanism similar to that of the antibody. If the
apoptosis induced by the antibody is p53-dependent (p53 is
activated), it indicates that the antibody uses a mechanism in
apoptosis induction different than the one used by the compounds.
This will strongly suggest that compounds inhibit a different
target than GLUT1.
[0251] Also contemplated herein is a proteomic approach to study
the link between inhibition of basal glucose transport and
induction of apoptosis. The selected compound will be used to treat
H1299 and MCF-7 cells for 24 hrs with untreated cells as negative
controls. Total proteins will be isolated from the treated and the
untreated cells. Protein samples will be treated with protease
inhibitor, TBP and sample buffer which contains urea, thiourea, and
CPHAS for 2 hours. Then IAA will be added. Twenty min later, IAA
will be added to the samples again. After treatment, samples will
be loaded to strips and the strips with samples will be kept in
room temperature for 2 hours before they are placed in the first
dimension gel electrophoresis instrument for the first dimensional
protein separation. After finishing the first dimensional protein
separation, the strip will be loaded on SDS-PAGE gels for the
secondary protein separation. After the separation, gels will be
fixed overnight with fixing buffer containing ethanol, acetic acid,
and SDS. Then the gels will be washed with washing buffer
containing acetic acid and SDS before they are stained with sypro
orange for 2 hours for spot detection. The 2-D gel results will be
analyzed with software of PDQuest.
[0252] Protein spots will picked automatically under control of a
camera and transferred to a 96-well microtiter plate with holes in
the bottom. The microtiter plate with all gel spots will be
transferred to an automatic digester (Tecan GmbH) to wash the gel
pieces, digest the protein with trypsin at 50.degree. C. for 2 hrs.
The digest will be done with 2 mM ammoniumhydrogen carbonate buffer
(pH 7.8) to reduce the salt content and the and 0.5 .mu.L of the
extracted peptides are automatically spotted on a MALDI target,
whereas the remaining 20 .mu.L are stored in a microtiter plate.
Usually about 90% of all protein spots can be already identified
from the MALDI-TOF/TOF mass spectra by combining a peptide mass
fingerprint with the tandem mass spectra of the top five peptide
signals. Alternatively, the sample stored in the microtiter plate
can be analyzed my nanoRP-HPLC-nano-ESIQqTOF-MS/MS, which usually
gives a better sequence coverage and often a better confidence
level. Overall proteins can be identified at the 100 fmol
level.
[0253] Twenty up-regulated protein spots and twenty down-regulated
spots on a single gel, as compared to the untreated control gel,
will be selected for MS analysis and amino acid sequence
determination. Considering the intrinsic variations of the system,
only those spots that are either up-regulated by 2-fold or more or
down-regulated by 2-fold or more will be chosen. After the amino
acid sequence of the N-terminus of a protein is determined, the
identity of the protein will be uncovered by comparing the sequence
with the protein sequences in the data bank. By knowing which
proteins are up-regulated or down-regulated by the compound
treatment, these proteins will be categorized into different groups
and different metabolic and/or signaling pathways, which should
enable us to identify pathways that are activated or inactivated by
the compound, providing clues for how the inhibition of basal
glucose transport leads to eventual induction of cancer cell
apoptosis.
[0254] In Vivo Anticancer Studies
[0255] Although the compounds' anticancer activity in cancer cell
lines has been established in preliminary studies and in a recent
GLUT1 antibody study in multiple cancer cell lines, it has also
been tested in animal models, which is an intermediate step for
moving cancer research from laboratory to clinics. Secondly,
inhibiting basal glucose transport may induce hyperglycemia in the
treated animals. To address the question of in vivo efficacy and
safety, a compound selected based on its improved IC.sub.50 (basal
glucose transport), EC.sub.50 (cancer cell killing), and
maintained/improved target selectivity (improved killing in cancer
cells without increased killing in normal cells), as well as
anticancer efficacy and safety findings from animal study described
above will be used in this animal study.
[0256] The objectives of the proposed animal study are to determine
if the compound treatment reduces cancer growth and if the compound
treatment is safe to the tumor-bearing mice. The effective and safe
doses will be chosen based on cell killing assays on cancer cell
lines and tolerable cytotoxicity in the normal counterparts of the
cancer cells tested as well as in a short term pilot animal study
similar to the one described above, in which the cell
study-determined compound dose and 2.times. and 4.times. doses will
be tested in nude mice (3 per group). In the pilot study, the
compound will be administered to mice once a day for five days and
the compound-injected mice will be monitored for signs of side
effects (hyperglycemia immediately after compound treatment and
with time, reduction and difficulty in movement, loss of body
weight). A safe dose will be selected and an animal study will be
performed as follows:
[0257] Because human cancer cell line(s) will be used,
immune-deficient nude mice will be used. A total of 30 nude mice
will be used, 10 mice per group. These mice will be randomly
selected into each group. Group 1 is the negative control group,
which will be inoculated with cancer cells but without receiving
subsequent compound treatment; Group 2 will be the low dose
compound treatment group and group 3 will be high dose compound
treatment group. Five million cells of either H1299 or MCF-7 cell
lines will be injected subcutaneously (SubQ) into the flank of nude
mice. We decided to use H1299 NSCLC and MCF-7 cells as our cancer
models were based on these considerations: (a) although the
inhibitors showed anticancer activity in all the cancer cell lines
we tested, they showed either the higher anticancer activity or
higher cancer cell:normal cell killing ratios or both among all
cell lines tested, (b) NSCLC and breast cancers are two cancers
that have been considered major targets of current cancer research
and therapeutic treatment. In addition, GLUT1 has been found
over-expressed in these two cancer types.
[0258] The compound treatment will start when tumors become
palpable and visible. The compound at a concentration of 10 mg/kg
of body weight will be intraperitoneally (IP) injected, one
injection for every other day (except the weekends) for the entire
study. The negative control mice will be injected exactly the same
way but with vehicle (the solution in which the compound is
dissolved). Tumor size will be measured twice a week with calipers,
and dimensions of length, width and height of the tumors will be
measured and recorded (L.times.W.times.H) as tumor volumes. The
compound treatment lasts 3-5 weeks until the tumors grown in the
untreated mice become large (>5% but <10% of the body
weight). The animal study will be carried out and terminated in
accordance to the rules and regulation of NIH and our university
IACUC. Tumor-bearing mice will be euthanized at the end of the
study according to the related rules by NIH and DOA. The average
size of the tumors in the treated groups will be compared to the
untreated control group to show treatment efficacy and statistical
differences. The tumor size will also be compared between the high
dose group and the low dose group to show the dose response of the
treatment. The food intake, body weight, as well as blood glucose
levels will also be measured twice a week to monitor the animal
health and to compare these health parameters with the untreated
control group. At the end of the animal study, after animal
euthanasia, tumors will be removed from the compound treated mice
and from the untreated control mice. Total proteins will be
isolated from the tumors and their respective p53 and caspase 3
will be measured to determine if more apoptosis is induced in the
compound treated mice and if there is any change in the activated
p53. This is to determine if the in vivo anticancer mechanism is
the same as observed in cancer cell lines.
[0259] Animal Tumor Treatment Study. Protocol
[0260] 1. Study from November 2009 to January 2010, 10 weeks; 2.
Nude mice (immunodeficient), ten mice per group; 3. Tumor
model--Human lung cancer A549 (NIH recognized and recommended),
5.times.10.sup.6 cells injected into the flank of each mouse
subcutaneously; 4. Treatment with or without compound WZB-117, PI
injection daily for all 10 weeks, dose=15 mg/kg body weight; 5.
Weekly measurements: tumor size, body weight, food intake; and 6.
Other indicators measured: blood glucose, serum insulin, body
composition, and blood cell counts. FIGS. 30-34 and Tables 8-11
show the results of this study.
TABLE-US-00011 TABLE 8 Body mass composition Body weight Fat Fluid
Lean Treatment (g) g % g % g % Control 29.30 .+-. 1.55 2.84 .+-.
0.61 9.74 .+-. 2.29 1.78 .+-. 0.38 6.02 .+-. 1.07 23.61 .+-. 1.64
80.54 .+-. 2.22 WZB117 28.48 .+-. 0.17 1.05 .+-. 0.17* 3.69 .+-.
0.61* 1.80 .+-. 0.12 6.32 .+-. 0.40 24.40 .+-. 0.72 85.69 .+-.
2.40
[0261] Minispec data indicated that the difference in body weight
between the control and WZB-117 treated group was primarily due to
decrease in fat tissue in the WZB-117-treated mice. Body mass
composition of each mouse was measured by the Minispec NMR Analyzer
mq7.5 (Bruker, Billerica, Mass.) after 70 days of compound WZB-117
treatment. Results were analyzed by an OPUS program (Bruker).
TABLE-US-00012 TABLE 9 Blood cell count analysis of differently
treated mice No tumor no PBS + DMSO WZB 117 CBC treatment treatment
treatment Normal parameters group group group range WBC (K/.mu.l)
10.1 .+-. 3.3 9.1 .+-. 1.6 5.9 .+-. 0.3 1.8-10.7 LYMPH (K/.mu.l)
7.2 .+-. 2.2 49 .+-. 1.6 2.0 .+-. 0.7 0.9-9.3 RBC (M/.mu.l) 10.0
.+-. 0.3 8.7 .+-. 1.7 8.8 .+-. 2.0 6.36-9.42 HGB (g/dL) 15.9 .+-.
0.5 13.7 .+-. 2.0 13.8 .+-. 2.6 11.0-15.1 PLT (K/.mu.l) 1522.5 .+-.
159.7 1427.9 .+-. 327.0 2214.0 .+-. 192.3 592-2972
[0262] Blood was collected after 70 days treatment through mouse
tail vein using heparinized capillary tubes and then transferred to
EDTA containing microfuge tubes. Blood was analyzed using Hemavet
950 hematology system from Drew Scientific (Dallas, Tex.). CBC
analyses indicated that, compared to the PBS-DMSO-treated group,
WZB-117 treated group showed reduced counts in WBC, lymphocyte, as
well as increased platelet count. However, these changes for the
treated group were still in the normal ranges.
TABLE-US-00013 TABLE 10 Serum insulin levels in differently treated
mice. Treatment Serum insulin (mg/l) No tumor no treatment 0.971
.+-. 0.373 PBS+DMSO 0.743 .+-. 0.104 WZB117 0.572 + 0.319 Normal
range 0.5-10
[0263] Serum from each mouse was obtained by centrifuging the blood
from mouse tail vein at 10,000 rpm for 10 minutes and keeping the
supernatant. Serum insulin level of each mouse was measured using
Mercodia Ultrasensitive Mouse Insulin ELISA (Uppsala, Sweden).
Compared to the PBS-DMSO-treated group, WZB-117 treated group
showed reduced circulating insulin in serum. However, the change
was still in the normal range. Considering that there was no
significant difference in blood glucose levels between untreated
and compound WZB-117 injection group, the reduced but normal serum
insulin level indicated the normal function of pancreas after 70
days treatment of compound WZB-117.
TABLE-US-00014 TABLE 11 HPLC analysis of compound stability in
human serum Compound concentration (%) Time after incubation with
serum Compound 0 h 1 h 2 h 4 h 8 h 16 h 24 h 48 h WZB-115 100 41.6
18.9 18.4 0.6 -- 0 0 WZB-117 100 25.1 15.2 10.1 7.7 7.8 5.4 5.0
WZB-141 100 100.3 -- 103.5 -- -- 109.7 87.4 WZB-149 100 86.4 50.7
43.9 50.9 65.0 51.0 62.4
[0264] A total of 2.5 ml of compound solution was added to 22.5 ml
of human serum and the mixture was incubated at 37 C for various
times. The final concentration of the compound in serum was 6.8 mM.
After incubation, 200 ml of acetonitrile was added to the mixture,
centrifuged to remove proteins before HPLC analysis. The relative
concentration of samples at 0 h (in serum but without incubation)
was arbitrarily assigned a value of 100% and all other samples were
compared to the 0 h samples. Analysis indicated that ether
bond-containing compounds WZB-141 and WZB-149 were much more stable
in serum than ester bond-containing compounds WZB-115 and
WZB-117.
* * * * *