Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Pharmaceutical Composition
Field of the Invention
This invention relates to therapeutic and diagnostic compositions, and more
particularly to pharmaceutical compositions, and methods of use thereof, which
demonstrate
selective uptake into cells characterized by hyperproliferation, including
cancer cells, and
which modulate the regulation or perturbation of the structure, expression,
and/or activity of
enzymes, thereby facilitating the detection and treatment or destruction of
these cells. More
specifically, these agents target and perturb the activity or regulation
thereof of the altered
mitochondrial energy metabolism observed in such locales as the modified
pyruvate
dehydrogenase (PDH) complex associated with most cancers.
Background of the Invention
Mitochondria are the primary control centers for energy production and
cellular life-
and-death processes in eukaryotes. While various mechanisms of communication
with the
rest of the cell exist, reversible phosphorylation is an important means of
regulating
mitochondrial functions. (Pagliarini D.J. and Dixon J.E. (2006). Mitochondrial
modulation:
reversible phosphorylation takes center stage? TRENDS in Biochem. Sci. 31:26-
34, passim,
herein incorporated by reference) The steadily increasing number of reported
mitochondrial
kinases, phosphatases, and phosphoproteins suggests that phosphorylation is
likely to emerge
as a common theme in the regulation of mitochondrial processes. Pathological
or genetic
changes associated in mitochondrial enzyme structure, function and activity
regulation
contribute to, and may be important targets for, the treatment of disease.
In line with their role as both a point of convergence and regulator of
diverse cellular
functions, mitochondria have crucial roles in apoptosis, production of
reactive oxygen species
(ROS), and numerous metabolic processes, including the production of more than
90% of
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cellular ATP. Furthermore, as cells grow and divide, new mitochondria have to
be made, this
process itself requiring careful coordination of nuclear and mitochondrial DNA
transcription
and translation. Finally, as cellular energy needs change, mitochondria must
respond rapidly
by tuning their ATP output. Hence, mitochondria require a complex system of
communication with cellular functions. As evident from FIGURE 1, signal
molecules to and
from mitochondria include ions, gases, metabolites, hormones, transcription
factors and
proteins. Consequently, the recognition of mitochondria as centers for
receiving, integrating,
and transmitting cellular signals is an important advance in the design and
testing of
pharmaceuticals.
A cornerstone of signalling in mitochondrial regulation is reversible
phosphorylation.
However, while the first demonstration of a protein kinase event was reported
in 1954, and
the fact that a core mitochondrial function can be regulated by reversible
phosphorylation
was discovered nearly four decades ago, reports of mitochondrial
phosphorylation events are
scarce. This is so despite the recognition in the 1980s and 1990s of numerous
signal
transduction phosphorylation cascades that traverse the plasma membrane and
extend through
the cytosol to the nucleus. Such paucity of knowledge may be due partly to the
fact that the
bulk of the mitochondrial protein machinery lies behind two lipid bilayers,
which seemingly
places mitochondrial proteins out of the reach of cytosolic signalling
cascades. In any event,
it has been not widely accepted that the mitochondrion regulates signalling by
reversible
phosphorylation in a manner key to disease management. Nevertheless, as seen
in Table 1,
by 2006 more than 60 proteins in all mitochondrial compartments (i.e., the
matrix, inner
membrane, intermembrane space, and outer membrane, including the cytoplasmic-
facing
outer surface) had been identified as phosphoproteins implicated in a wide
spectrum of
mitochondrial functions. Mounting data further demonstrates the importance of
reversible
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phosphorylation of mitochondrial targets and the use of compositions targeting
the same for
the improved treatment of cancer.
Table 1. Mitochondrial phosphoproteinse
No. Protein Location P site Source Function Refs
1 PDC Eta M Ser Various Acetyl-CoA formation [11)
2 PDC E10 M Tyr Human sperm Acetyl-CoA formation (8]
3 PDC E3 M Tyr Hamster sperm Regulation of the PDC (8]
4 PDK isoform 2a M Ser/Thr Rat TCA. 1271
Aconitase M 7 Bovine/potato TCA [22)
6 NAD-isocitrate dehydrogenase M ? Bovine/potato TCA [221.
7 NAD-malete dehydrogenase M ? Potato TCA. (221
8 NAD-malic enzyme M ? Potato TCA [221
9 Succinyl-CoA-ligase x subunit M ? Rat/potato TCA [22,471
Succinyl-CoA-ligase (i subunit M 7 Rat/potato TCA [22,47)
11 Formate dehydrogenase M Ser/Thr Potato TCA [22,231
12 Aconitase M Tyr. Guinea pig synaptosomes TCA (81
13 BCKAD M Ser Various AA metabolism [311
14 BCKAD kinase M Ser Rat AA Metabolism 154]
HSP22 M Ser Corn Chaperone [391
16 HSP 90 M 7 Potato Chaperone. [22]
17 Chaperonin 60 M 7 Potato Chaperone (221
18 mthsp75 M Tyr Rat hepatome cells Chaperone [29]
19 TRAP-1` M Tyr Human sperm Chaperone [81
CYP2E1' M/IM? Ser COS Detoxification [43]
21 CYP2B10 M/IM? Ser COS Detoxification 1181
22 GSTA 4-4 M . Ser/Thr COS Detoxification (44]
23 DBP M 7 Yeast mRNAturnover [381
24 MnSOD M 7 Potato Oxidative stress defense [22)
EF-Tu M Thr? Rabbit heart Protein synthesis (32]
26 Creatine kinase M 7 Bovine Synthesis of phosphocreatine [451
27 MTERFh M ? Rat Transcription termination [42]
28 Abf2p M Ser/Thr7 Yeast mtDNA maintenance [26j
29 MtTBP5 M ? Yeast mtDNA maintenance (501
NOK M/IMS Ser/His Pisumsativum Nucleoside triphosphate balance [49]
31 StAR IMS Ser COS-1 Steroid hormone synthesis [19]
32 Axmito` M? Tyr Rat Regulation of PLAs? (531
33 SSATe M? Ser? Rat Acetylation of spermidine [21]
34 Sab OM Ser Rat cardiac myocytes SH3 domain-binding protein [28]
CPT-I OM Ser Rat (t-OX [351
36 MtGAT` OM Ser/Thr? Rat Glycerolipid biosynthesis [411
37 BAD` OM Ser FLS.12 cells Apoptosis [30)
38 BCL-2 OM Ser Jurkat Apoptosis [17,521
39 BCI-XL OM Thr U-937 cells Apoptosis 136)
CREB M/IM Ser? Rat mtDNA transcription? (24]
41 VDAC OM Tyr Guinea pig synaptosomes transport 18,451
42 Cl: ESSS iM Ser . Bovine OXPHOS 1251
43 Cl: 10 kDa IM Ser Bovine OXPHOS [25]
44 CI 42 kOa (2 sites) IM Ser & Thr Bovine OXPHOS [45,55)
CIII core I . IM Tyr Human sperm OXPHOS (81
46 CIII core II IM ? Bovine OXPHOS- [451
47 CIV I IM. Tyr Bovine OXPHOS [371
48 CIV Il IM Tyr Osteoclasts OXPHOS [40)
49' CIV 111 IM Ser/Thr? Bovine OXPHOS [34]
CIV IV IM ? Rat OXPHOS 1461
51 CIV Vba . IM Ser/Thr? Bovine OXPHOS 134)
52 CV z IM ? Bovine/potato OXPHOS 1221
53 CV p IM Thr Human sk mus/bovine OXPHOS [331
54 CV d IM ? Potato OXPHOS [481
CV b IM 7 Potato OXPHOS 1481
56 ScIRP IM ? Rat OXPHOS 1201
57 SDH-Fp IM 7 Bovine/potato. OXPHOS 1221
58 bcl complex, [i-MPP subunit IM 7 Potato OXPHOS [22)
59 CIII core I IM Tyr Human sperm OXPHOS [8]
NAD(P) transhydrogenaso IM 7 Bovine H* pump 1451
61 ANT IM 7 Bovine Transport (451
62 Phosphate carrier protein IM 7 Bovine Transport [451
63 Aldose reductase' 7 SOFT? Various cell lines Osmoregulation [511
'Abbreviations: AA, amino acid IS, serine;T, threonine; Y, tyrosine); ANT,
adenine nucleotide transporter; Axmito, mitochondria) annexin; fl-Ox, p-
oxidation; BCKAD,
branched chain ketoacid dehydrogenase; CI-CV. respiratory chain complexes 1-5:
CPT, carnitine palmitoyltransferese; CREB, cAMP-responsive element (Cre)-
binding
protein: CYP, cytochrome P450; DBP, dodecamer-binding protein; EF. elongation
factor; GST. glutathione S-transterase; HSP, heat shock protein; IM. Inner
membrane: IMS.
intermembrane space; M. matrix: MnSOD. manganese super oxide dismutase; mTERF,
mitochondria) transcription termination factor; m1GAT. mitochondrial glycerol-
3-
phosphatase aceryltransferase; mthsp, mitochondrialHSP; mtTBP; mitochondrial
telomere-binding protein; NDK, nucleoside diphosphate kinase: OM. outer'
membrane:
OXPHOS; oxidative phosphorylation; P site; site of phosphoryiation: POC E1/3,
pyruvate dehydrogenase complex,E1/3 subunit: PDK, pyruvate dehydrogenase
kinase: PLA,
phospholipaseA; ScIRP, subunit c-immunoreactive peptide; SSAT,
spermidine/spermina scetyltransfarase: StAR, steroidogenic acute regulatory
protein: TCA. tricarboxylie
acid cycle: TRAP-1, tumor-necrosis factor type 1 receptor-associated protein:
VOAC. voltage-dependent anion channel.
Phosphorylation is observed only in.vitru on recombinant protein.
`Protein translocetes to mitochondria (i.e. protein is not a resident
mitochondrial protein).
5
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The largest kinase and phosphatase families in the human genome, the protein
kinases
(PKs) and protein tyrosine phosphatases (PTPs), possess more than 500 and more
than 100
members, respectively. Together with smaller families of kinases and
phosphatases, these
signalling molecules comprise nearly three percent of all proteins encoded in
the human
genome. Similar to the aforementioned phosphoproteins, kinases and
phosphatases have
been implicated in mitochondrial functions in a surprising number of studies,
and so far at
least 25 kinases and eight phosphatases have been reported to localize to
mitochondria, as
seen in FIGURE 2. These kinases and phosphatases are clearly not restricted to
one group or
family; rather, they represent nearly every known mammalian kinase and
phosphatase
subgroup, reflecting the range of signalling pathways that are likely to
influence the
mitochondrion. These signalling molecules include kinases and phosphatases
varying in
substrate specificity, (e.g., tyrosine kinases, classic PTP subgroups,
serine/threonine kinases,
and dual-specific PTPs); in catalytic mechanisms (e.g., cysteine-based PTPs,
aspartic acid-
based PTPs, and metal-dependent phosphatases); and in evolutionary
conservation (e.g.,
bacterially-related pyruvate dehydrogenase kinases (PDKs) and phosphatases
(PDPs),
branched chain ketoacid dehydrogenase kinase (BCKDK) and phosphatase (BCKDP),
and
many mammalian-specific enzymes).
Most of these signalling molecules possess other non-mitochondrial roles in
the cell
and are primarily found to exist outside mitochondria. The impetus for, or
mechanism of,
their translocation to mitochondria is poorly understood for most proteins.
What is clear,
however, is that kinases and phosphatases, like the phosphoproteins listed
earlier, are present
in all compartments of the mitochondrion, as evident in FIGURE 3, and that
their activities
impinge on diverse mitochondrial functions.
A few signalling molecules, however, seem to localize primarily to
mitochondria. In
addition to PDKs and PDPs, this group includes the PTEN-induced kinase PINK 1,
the dual-
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specific PTP targeted to the mitochondrion PTPMT 1, and the aspartic-acid-
based
phosphatase/ATPase Tim50. Although the substrates for many of these proteins
are currently
unknown, it is clear from biological and genetic data that they possess
crucial functions in
mitochondria. For example, although its submitochondrial localization remains
to be
5 determined, P1NK1 is targeted to mitochondria by an N-terminal signal
sequence. This
kinase, which shares high sequence homology with the Ca+2/calmodulin-regulated
kinase
family, seems to be involved in pro-survival activities. Similarly, PTPMTI was
recently
identified as the first PTP that is localized primarily inside the
mitochondrion and, like
PINK1, is targeted to the mitochondrion by an N-terminal signal peptide and is
found tightly
associated with the matrix face of the inner mitochondrial membrane. PTPMT 1
is highly
expressed in pancreatic B cells, whose mitochondria have the important
function of coupling
glucose metabolism to the secretion of insulin. Finally, Tim50, a key
component of the TIM
(translocase of the inner membrane) complex, has sequence homology to the CTD
family of
aspartic-acid-based phosphatases/ATPases. Tim50, like other members of this
family, might
function as an ATPase, but it has been also shown to possess phosphatase
activity against the
phosphotyrosine analog para-nitrophenyl phosphate in vitro. Given the above,
it can be seen
that not only are kinases and phosphatases recruited to mitochondria from
elsewhere in the
cell, but the mitochondrion itself seems to possess a contingent of resident
signalling
molecules.
Although the effect of phosphorylation on most known mitochondrial
phosphoproteins is unclear, with the kinases and phosphatases responsible
sometimes
remaining unidentified, a few phosphorylation events have been partially
characterized.
These examples, like the phosphoproteins and signalling molecules discussed
earlier, are not
restricted to one area of the mitochondrion.
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Phosphorylation on the mitochondrial outer membrane has a crucial role in
regulating
apoptosis. A particularly well-defined event is the phosphorylation of BAD, a
proapoptotic
member of the BCL-2 family. It has been shown that PKA, after treatment with
the pro-
survival cytokine interleukin-3, translocates to the outer membrane. Once
anchored to an A-
kinase anchoring protein (AKAP) on the outer membrane, PKA phosphorylates BAD
on Ser
112, contributing to the inactivation and disassociation of BAD from
mitochondria, a process
depicted in FIGURE 3A. Phosphorylation of BAD on Ser 136 by p70S6 kinase and
on Ser
155 by in unidentified kinase has also been implicated in the inactivation of
BAD.
The most well-established example of reversible phosphorylation acting as a
regulatory mechanism in healthy cell mitochondria is that of the PDH complex
in the matrix,
a simplified cartoon of which is illustrated in FIGURE 3B. This complex
catalyzes the
conversion of glycolysis-derived pyruvate to acetyl-coenzyme A (CoA), the main
precursor
to the tricarboxylic acid (TCA) cycle. As the link between these two major
energy-producing
pathways, the PDH complex must be properly regulated for the maintenance of
cellular
glucose homeostasis.
Since its identification as the first mitochondrial phosphoprotein, the PDH
complex
and its regulation by reversible phosphorylation have been studied
exhaustively.
Phosphorylation and dephosphorylation of the PDH complex are carried out by
PDKs and
PDPs, respectively. At least four PDK isoforms and two PDP isoforms are known,
all of
which are associated with the E2 subunit of the PDH complex. The
phosphorylation events
occur on three separate serine residues of the El subunit, each leading to
significant
inactivation of this complex. Notably, there is now at least one report of a
PDK itself being
phosphorylated. This phosphorylation, carried out by PKC, has been shown to
inactivate
PDK, potentially demonstrating an additional level of PDH complex regulation
by reversible
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phosphorylation. Thus, the PDH complex is a prime example of mitochondria
using
phosphorylation to add a level of regulation to an otherwise conserved
process.
As indicated by the mitochondrial tyrosine kinases and phosphatases listed
earlier,
phosphorylation within this organelle is not limited to serine and threonine
residues. An
example of tyrosine phosphorylation that affects mitochondrial energetics is
seen in the
regulation of cytochrome c oxidase (COX) in the inner membrane, depicted in
FIGURE 3C.
COX, as the terminal enzyme in the respiratory chain, coordinately reduces
oxygen to water
while pumping protons across the inner membrane. Similar to the PDH complex,
COX is
allosterically regulated by ATP and ADP, as well as the thyroid hormone T2 and
possibly
Ca+2 ions. In addition to these forms of regulation, it has been shown that
COX becomes
phosphorylated in a cAMP-dependent manner both in vitro and in HepG2 cells in
vivo. COX
comprises thirteen subunits and has been crystallized as a dimer. The
phosphorylation site
has been identified as Tyr 304 of subunit 1, which is located at the dimer
interface on the
intermembrane. The phosphorylation event markedly inhibits COX activity,
perhaps by
disrupting dimer formation.
In a second example of tyrosine phosphorylation of COX, a portion of the non-
receptor tyrosine kinase c-Src, similar to the Lyn tyrosine kinase localizes
inside
mitochondria and leads to tyrosine phosphorylation of COX on an unidentified
site of subunit
II in osteoclasts. The result of the phosphorylation event is opposite to that
seen for subunit I,
leading to enhanced COX activity.
An important aspect of mitochondrial signalling is how kinases and
phosphatases are
themselves regulated. Numerous kinases that primarily reside elsewhere in the
cell but
become targeted to the mitochondrion, such as Abl, Akt, GSK3I3 and PKC6, seem
to do so
only in their active state. Thus, the extent of some kinase activities within
mitochondria
might simply be dictated by the number of enzymes that are imported into the
organelle.
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For resident signalling molecules, however, different regulatory means must be
in
place. Although these processes remain to be determined, it is likely that
second messengers
will have a key role. The activities of the PDK and PDP isoforms are known to
be controlled
by ions and small molecules such as Mgt+, Cat+, K+ and ADP. The
characterization of
mitochondrial nitric oxide synthases and the recent discovery of a soluble
adenylate cyclase
in mitochondria provide further opportunities for second messengers to
contribute to
regulating mitochondrial signalling molecules. Finally, ROS, which have been
established as
a means of regulating signalling molecules elsewhere in the cell, will almost
certainly be
involved in regulating kinases and phosphatases in mitochondria, where the
bulk of reactive
oxygen species is produced. Relative expression levels of isoforms of kinase
and
phosphatase may play an important role in pathology and linked to other signal
transduction
events associated with disease. Changes at the gene and expression level may
also correlate
with such changes.
Even after several thorough proteomic surveys, it is estimated that only two-
thirds of
the mammalian mitochondrial proteome is known. Much of the remaining third is
likely to
be comprised of low-abundance proteins, such as signalling proteins, which
were below the
detection level of these mass spectrometric analyses. What is also clear from
these studies is
the high variability in protein content among mitochondria from different
tissues. For
example, it has been found that only -50% of the proteins in their proteomics
effort were
conserved across the four tissues examined (i.e., brain, heart, liver and
skeletal muscle). It is
likely that different mitochondrial signalling pathways not only will vary
from tissue to tissue
in the same way but might very well contribute to this observed mitochondrial
diversity.
Nonetheless, there is more than sufficient evidence to conclude that
reversible
phosphorylation is involved in the regulation of mitochondrial processes. With
over 60
reported phosphoproteins, 30 kinases and phosphatases, and various auxiliary
signalling
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proteins, the mitochondrion is certainly an underappreciated site for
signalling by reversible
phosphorylation, and in fact such regulation may be useful for the treatment
of
hyperproliferative diseases such as cancer.
The vast majority of fast-growth tumor cells exhibits profound genetic,
biochemical,
and histological differences with respect to nontransformed cells. Many of
these are
associated with altered energy metabolism in comparison to the tissue of
origin. The most
notorious and well-known energy metabolism alteration in tumor cells is an
increased
glycolytic capacity even in the presence of a high 02 concentration, a
phenomenon known as
the Warburg effect.
Warburg originally proposed that the driving force of the enhanced glycolysis
in
tumor cells was the energy deficiency caused by an irreversible damage of the
mitochondrial
function in which, similarly to anaerobic muscle, glucose is converted through
glycolysis to
lactate, which is later secreted. It has been proposed that this increase in
the glycolytic flux
in tumor cells is a metabolic strategy to ensure survival and growth in
environments with low
02 concentrations, such as the partial hypoxia observed in poorly-oxygenated
solid tumors.
In particular, since the concentration of 02 is lower than 20 M in many human
hypoxic
tumors, oxidative phosphorylation is limited therein. Consequently, glycolysis
seems to be
the main energy pathway in solid tumors (e.g., slow-growing melanomas and
mammary
adenocarcinoma).
A proportional relationship between the rate of cellular proliferation and the
rate of
ATP supply has been established for fast-growth tumor cells. Some authors have
proposed
that the glycolytic activity correlates with the degree of tumor malignancy,
so that the
glycolytic rate is greater in highly de-differentiated and fast-growing tumors
than in slower-
growing tumors or normal cells. In fact, a high level of lactate has been
proposed as a
predictor of malignancy. That these events are linked to additional signal
transduction events
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and genetic changes is likely and examples include hypoxic inducing factor and
the
production and release of angiogenic factors.
As depicted in FIGURE 4, for years, the tricarboxylic acid (TCA) cycle was
regarded
as only being biologically significant only for its role in the production of
ATP as an energy
5 source for the organism. However, recent studies have shown that TCA cycle
activity also
affects signal transduction pathway functions, including cell growth and
apoptosis decisions,
and that the pertinent glycolytic and TCA cycle enzymes are able to be
upregulated or down-
regulated. There is also a direct correlation between tumor progression and
the activities of
the glycolytic enzymes hexokinase and phosphofructokinase (PFK) 1, which are
greatly
10 increased in fast-growth tumor cells. Accordingly, it has been postulated
that tumor cells
which exhibit deficiencies in their oxidative capacity are more malignant than
those that have
an active oxidative phosphorylation. No matter whether under hypoxic or
aerobic conditions,
then, cancer tissue's reliance on glycolysis is associated with increased
malignancy.
The role of lipoic acid in the PDH complex of healthy cells has been well
studied.
The PDH complex has three central subunits, El, E2, and E3 (pyruvate
dehydrogenase,
dihydrolipoyl transacetylase, and dihydrolipoamide dehydrogenase,
respectively). These
complexes have a central E2 core, with the other subunits surrounding this
core to form the
complex. In the gap between these two subunits, the lipoyl domain ferries
intermediates
between the active sites. The lipoyl domain itself is attached by a flexible
linker to the E2
core. Upon formation of a hemithioacetal by the reaction of pyruvate and
thiamine
pyrophosphate, this anion attacks the S I of an oxidized lipoate species that
is attached to a
lysine residue. Consequently, the lipoate S2 is displaced as a sulfide or
sulfhydryl moiety,
and subsequent collapse of the tetrahedral hemithioacetal ejects thiazole,
releasing the TPP
cofactor and generating a thioacetate on the S I of the lipoate. At this
point, the lipoate-
thioester functionality is translocated into the E2 active site, where a
transacylation reaction
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transfers the acetyl from the "swinging arm" of lipoate to the thiol of
coenzyme A. This
produces acetyl-CoA, which is released from the enzyme complex and
subsequently enters
the TCA cycle. The dihydrolipoate, still bound to a lysine residue of the
complex, then
migrates to the E3 active site, where it undergoes a flavin-mediated oxidation
back to its
lipoate resting state, producing FADH2 (and ultimately NADH) and regenerating
the lipoate
back into a competent acyl acceptor. Should this lipoate species be
interrupted, then, there
would be no flow of electrons to FADH2 or generation of acetyl-CoA, and, as a
consequence,
a toxic buildup of pyruvate within the cell. In cancer cells the production of
acetoin has been
suggested to be a requirement for cellular detoxification and survival.
As stated previously, the activity of the PDH complex in mitochondria is
highly
regulated by a variety of allosteric effectors and by covalent modification.
PDH activity is
regulated by its state of phosphorylation, being most active in the
dephosphorylated state.
PDH phosphorylation is catalyzed by PDK. PDK activity is enhanced by an
increase in the
level of ATP, NADH, and acetyl-CoA. Negative effectors of PDK are ADP, NAD+,
CoA-
SH, and pyruvate, the levels of which increase when ATP levels fall. While the
regulation of
PDP, the enzyme which activates PDH through dephosphorylation, is not
completely
understood, it is known that Mg+2 and Ca+2 activate PDP.
Two products of the complex, NADH and acetyl-CoA, are negative allosteric
effectors of PDH-a, the dephosphorylated active form of PDH. These effectors
reduce the
affinity of the enzyme for pyruvate, thus limiting the flow of carbon through
the PDH
complex. In addition, NADH and acetyl-CoA are powerful positive effectors of
PDK, the
enzyme that inactivates PDH by converting it to the phosphorylated PDH-b form.
Since
NADH and acetyl-CoA accumulate when the cell energy charge is high, it is not
surprising
that high ATP levels also up-regulate PDK activity, reinforcing down-
regulation of PDH
activity in energy-rich cells. However, since pyruvate is a potent negative
effector of PDK,
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when pyruvate levels rise, PDH-a will be favored even with high levels of NADH
and acetyl-
CoA.
Concentrations of pyruvate which maintain PDH-a are sufficiently high so that,
in
ATP-rich cells, the allosterically down-regulated, high Km form of PDH is
nonetheless
capable of converting pyruvate to acetyl-CoA. With large amounts of pyruvate
in cells
having high ATP and NADH levels, pyruvate carbon will be directed to the two
main storage
forms of carbon (glycogen via gluconeogenesis and fat production via fatty
acid synthesis)
where acetyl-CoA is the principal carbon donor. Although the regulation of PDP-
b is not
well understood, it is quite likely regulated to maximize pyruvate oxidation
under diminished
ATP concentrations and to minimize PDH activity under high ATP concentrations.
Tumor cells cannot indefinitely build up pyruvate and associated aldehydes and
radicals, such as acetaldehyde, superoxide, hydrogen peroxide, and hydroxyl
radicals, as
these molecules are cytotoxic at high levels through such mechanisms as
drastically lowering
cellular pH. It has thus been described, for AS-30D and Ehrlich hepatomas,
that a significant
fraction of mitochondrial pyruvate is decarboxylated to an active acetaldehyde
by the El
component of the PDH complex via bound (3-hydroxyethylthiamine pyrophosphate.
This
active acetaldehyde is in turn condensed with a second acetaldehyde,
ultimately deacidifying
or reducing the original pyruvate, by using either the amino acid glutamine or
lipoic acid, to
generate acetoin (3-hydroxybutanone), a compound which both competitively
inhibits PDH
and which is less toxic to the cell than its pyruvate precursor (e.g., by
maintaining pH
homeostasis within the cell). Despite the importance of acetoin in the pathway
of tumor cell
detoxification as a result of pyruvate buildup due to the tumor cell's
reliance of glycolysis as
a source of ATP production, however, there is little reference in the prior
art to the effects of
blocking the production of acetoin on tumor cell viability.
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Recent studies suggest that forcing cancer cells into more aerobic metabolism
suppresses tumor growth. The transition to Warburg metabolism therefore
requires shutting
down the PDH complex. In this transition, there is enhanced signalling by
hypoxia-inducing
factor (HIF) in cancer cells, not surprising given HIF's significant role in
the metabolism of
glucose, as shown in FIGURE 5. Mutations that directly or indirectly instigate
HIF signalling
in fact appear to be a common mechanism in the development of cancer. HIF
induces the
overexpression of PDK1, which then acts to lower PDH complex activity.
Phosphorylation
by PDK1 can be particularly effective for maintaining an inactive PDH complex
since this
isoform uniquely phosphorylates three serine residues in the alpha subunit of
El, the first
subunit of the PDH complex. Reactivation of El requires the removal of all
three phosphate
groups. Furthermore, PDH complex activation may lead to the enhanced ROS
production,
which may in turn lead to apoptosis. However, alterations in PDK1 observed in
cancer may
not only be due to changes in its concentration but also to changes in its
activity and possibly
in its amino acid sequence, even between one tumor type or one patient to
another.
Additionally, PDK1 may form different complexes with various molecules
associated with
tumors depending upon tumor type. Thus, inhibition of PDK may be a potential
target in
generating apoptosis in tumors. However, to date, known PDKI inhibitors have
been
demonstrated to cause maximally only 60% inhibition of this isozyme.
While traditional chemotherapy targets dividing, proliferating cells, all
clinically-
accepted chemotherapeutic treatments use large drug doses that also induce
profound damage
to normal, proliferative host cells. Therefore, more selective targeting is
required for the
treatment of cancer. Another problem associated with chemotherapy is that, in
many tumor
types, there is either inherent or acquired resistance to antineoplastic
drugs. Overall,
traditional chemotherapy currently offers little long-term benefit for most
malignant tumors
and is often associated with adverse side-effects that diminish the length or
quality of life.
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14
Hence, radical new approaches are required that can provide long-term
management of
tumors while permitting a decent quality of life.
Certainly, drug efficacy, delivery, and side-effects are problems that need to
be solved
in developing new chemotherapies. In solid tumors, delivery to a hypoxic
region may be
difficult when the drug does not permeate through the different cellular
layers easily. To
eliminate these uncertainties, it seems relevant to design anticancer agents
having metabolic
inhibition constants in, at least, the submicromolar range. It may be argued
that cancer cells
are genetic and phenotypically heterogeneous from line to line. However, all
tumor cell lines
depend on glycolysis and oxidative phosphorylation for ATP supply.
Concentrating on the
Warburg effect allows for designing drugs based on the physico- and
biochemical energetic
differences between tumor and normal cells to facilitate the design of
delivery and
therapeutic strategies that selectively affect solely tumor metabolism and
growth, without
affecting healthy host tissue and organ functionality.
US Patents 6,331,559 and 6,951,887 to Bingham et al., as well as US
Provisional
Application No. 60/912,598 by Bingham et al., all herein incorporated by
reference, disclose
a novel class of lipoic acid derivative therapeutic agents which selectively
target and kill both
tumor cells and certain other types of diseased cells. These patents further
disclose
pharmaceutical compositions, and methods of use thereof, comprising an
effective amount of
such lipoic acid derivatives along with a pharmaceutically acceptable carrier.
However,
while these patents describe the structures of and general use for these
lipoic acid derivatives,
there is no indication in either patent that these derivatives are useful in
modulating the
structure and/or expression level, and/or regulating the activity, of the PDH
complex.
As it has been demonstrated that the structure and/or activity of the PDH
complex is a
critical determinant of tumor activity, then, it would be beneficial to
provide for a
PCT/US08/04410 09-09-2008 PFf lU .jZsRM8ORM 93AO10
pharmaceutically-acceptable modulator of the phosphorylation state of the PDH
complex,
and methods of use thereof.
Obiects of the Invention and Industrial Applicability
5 Consequently, it is an object of the present invention to provide a
pharmaceutical
composition to be used in the treatment or diagnosis of a disease, condition,
or syndrome
characterized by cellular hyperproliferation, such as cancer, which exhibits
selective activity
in tumor cells.
It is a further object of the present invention to provide a pharmaceutical
composition
10 to be used in the treatment or diagnosis of such an aforementioned disease,
condition, or
syndrome which causes minimal side effects upon administration.
It is a still further object of the present invention to provide a
pharmaceutical
composition to be used in the treatment or diagnosis of such an aforementioned
disease,
condition, or syndrome which is easily manufactured at the least possible cost
and is capable
15 of being stored for the longest possible period.
It is a still further object of the present invention to provide a
pharmaceutical
composition to be used in the treatment or diagnosis of such an aforementioned
disease,
condition, or syndrome which modulates mitochondrial energy metabolism,
especially via the
phosphorylation state of the PDH complex in tumor cell mitochondria.
Summary of the Invention
To achieve the aforementioned aims, the present invention broadly provides a
pharmaceutical composition useful for treating, diagnosing, or preventing a
disease,
condition, or syndrome characterized by an alteration of the phosphorylation
state of at least
one enzyme and/or enzyme complex, or subunit thereof, such as the PDH complex,
including
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16
those characterized by cellular hyperproliferation, such as cancer, or
symptoms thereof, in
warm-blooded animals, including humans, wherein the pharmaceutical composition
comprises an effective amount of at least one lipoic acid derivative,
including those as
described in US Patents 6,331,559 and 6,951,887 and US Provisional Application
No.
60/912,598, all herein incorporated by reference, and at least one
pharmaceutically-
acceptable carrier thereof.
By inhibiting mitochondrial energy metabolism, the lipoic acid derivatives of
the
present invention cause both the loss of mitochondrial membrane potential and
other
mitochondrial consequences in the diseased cell, resulting in the irreversible
initiation of cell
death. The lipoic acid derivatives of the present invention may also inhibit
mitochondrial
energy metabolism by the activation of PDKs and/or inhibition of PDPs or by
inhibiting the
conversion of pyruvate to the less-toxic molecule acetoin through inhibition
of the activity of
the El subunit of the PDH complex. The inhibition of acetoin synthesis will
distort other
processes, including redox balance and may also cause the production of toxic
by-products,
including acetaldehyde, superoxide, hydrogen peroxide, and hydroxyl radical,
these by-
products themselves consequently causing irreversible damage to the
mitochondrion of the
diseased cell.
The pharmaceutical composition of the present invention may modulate the
effects of
PDKI, PDK2, PDK3, PDK4, and the mutants or isoforms of each thereof. The
pharmaceutical compound may also modulate the effects of PDP1, PDP2 and the
isoforms of
each thereof.
The pharmaceutical composition of the present invention may also modulate the
expression level of the phosphorylase, kinase, and dehydrogenase enzyme
constituents found
in the PDH complex. This modulation may occur at the transcriptional,
translational, or post-
translational stage, including epigenetic silencing of the appropriate genes.
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O
17
As a compound derived from a molecule fundamentally associated with the TCA
cycle, and by extension glycolysis, the pharmaceutical composition of the
present invention
demonstrates selective uptake into tumor cells. Furthermore, such selective
tumor cell uptake
minimizes the side effects the administration of this pharmaceutical
composition would have
on healthy non-transformed cells and tissue.
In one embodiment of the present invention, the lipoic acid derivatives have
the
general formula (I):
R1 R2
S
(CH2)x R3 (1)
wherein R, and R2 are independently selected from the group consisting of
hydrogen,
alkyl CnH2õ+i, alkene Cõ H2,,, alkenyl Cõ H2i_,, alkyine CõH2õ-2, alkynyl
G,H2r_3, alkyl sulfide
CH3(CH2)n-S-, disulfide alkyl CH3CHr-S-S-, thiocarbamic ester (CH2)õ C=NH-,
and
semithioacetal CH3CH(OH)--S-, wherein n is 1-10 and t is 0-9, aromatic, acyl
defined as
R4C(O)-, heteroaryl, imidoyl defined as R5C(=NH)-, organometallic aryl, alkyl-
organometallic aryl, semiacetal R6CH(OH)-S-, amino acids, carbohydrates,
nucleic acids,
lipids, and multimers and combinations thereof;
wherein R, and R2 as defined above can be unsubstituted or substituted;
wherein R3 is selected from a group consisting of amino acids, carbohydrates,
nucleic
acids, lipids, and multimers thereof;
wherein R4 is selected from the group consisting of hydrogen, alkenyl,
alkynyl,
alkylaryl, heteroaryl, alkylheteroaryl and organometallic aryl, any of which
can be substituted
or unsubstituted;
wherein R5 is selected from the group consisting of hydrogen, alkenyl,
alkynyl, aryl,
alkylaryl, heteroaryl, and alkylheteroaryl, any of which can be substituted or
unsubstituted;
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18
wherein R6 is CC13, CF3, or COON;
and wherein x is 0-16;
or salts thereof.
In a second embodiment of the present invention, the lipoic acid derivatives
are
defined by a second general formula (II):
CI(CH2)x R3 (II)
wherein M is a covalent bond, -[C(R,)(R2)]Z , or a metal chelate or other
metal
complex where the metal is not palladium;
wherein R, and R2 are independently selected from the group consisting of
hydrogen,
acyl R4C(O)-, alkyl CõH2õ+,, alkenyl defined as C,,,H2m4, alkynyl defined as
CmH2m-3, aryl,
heteroaryl, alkyl sulfide CH3(CH2)õ--S-, imidoyl defined as R4C(=NH)-,
hemiacetal defined
as R6CH(OH)-S-, amino acids, carbohydrates, nucleic acids, lipids, and
multimers and
combinations thereof,
wherein R, and R2 as defined above can be unsubstituted or substituted;
wherein R3 is selected from a group consisting of amino acids, carbohydrates,
nucleic
acids, lipids, and multimers thereof;
wherein R4 and R5 are independently selected from the group consisting of
hydrogen,
alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl, and
heterocyclyl, any of which
can be substituted or unsubstituted;
wherein R5 is selected from the group consisting of CCl3, CF3 or COOH;
and wherein x is 0-16, z is 0-5, n is 0-10 and m is 2-10;
or salts thereof.
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MUO
Ainjdj9.D9:Zd8OjO
19
Furthermore, as any or all of these general structures may be metabolized
within the
cell or mitochondrion, it is expressly intended that metabolites of the above-
referenced
structures are within the scope of the present invention.
In a further aspect of the present invention, there is provided a method of
diagnosing,
treating, or preventing a disease, condition, syndrome, or symptoms thereof,
which includes
an alteration of the phosphorylation state of at least one enzyme and/or
enzyme complex, or
subunit thereof, such as the PDH complex, including those characterized by
cellular
hyperproliferation, such as cancer, in warm-blooded animals, including humans,
wherein the
method comprises administering to such an animal an effective amount of the
pharmaceutical
composition disclosed herein.
In a still further aspect of the present invention, there is provided a method
of
diagnosing and predicting benefit in a patient presenting symptoms of a
disease, condition, or
syndrome, or symptoms thereof, which includes an alteration of the
phosphorylation state of
at least one enzyme and/or enzyme complex, or subunit thereof, such as the PDH
complex,
including those characterized by cellular hyperproliferation, such as cancer,
comprising
obtaining a sample of cells from the patient, administering an effective
amount of the
pharmaceutical composition of the present invention to the cells in vitro, and
obtaining the
results therefrom.
Brief Description of the Figures
The following drawings are illustrative of embodiments of the invention and
are not
intended to limit the scope of the application as encompassed by the entire
specification and
claims.
FIGURE 1 depicts general signal transduction molecules targeted both into and
out
from mitochondria and the effects thereof.
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VUtS/MW 09.K&P10
FIGURE 2 shows a list of mitochondrial kinases and phosphatases and the
locations
within the mitochondrion thereof.
FIGURE 3A illustrates the phosphorylation of BAD by PKA in the outer
mitochondrial membrane, and the effects of reversible phosphorylation
thereupon.
5 FIGURE 3B presents the conversion of pyruvate into acetyl-CoA in the
mitochondrial
matrix by the action of the PDH complex, and the effects of reversible
phosphorylation
thereupon.
FIGURE 3C shows the action of COX in reducing oxygen to water and pumping
protons across the inner mitochondrial membrane, and the effects of reversible
10 phosphorylation thereupon.
FIGURE 4 illustrates the structures of substrates and products in the
glycolytic
production of pyruvate, also showing ATP and NADH generation and associated
enzymes.
FIGURE 5 shows the regulation of glucose metabolism by HIF-1.
FIGURE 6A illustrates the difference in energy metabolism between normal
tissue
15 and cancer tissue in vivo.
FIGURE 6B depicts the differences between the biogenic forms of lipoic acid in
the
PDH complex and the lipoic acid derivatives forming part of the pharmaceutical
composition
of the present invention.
FIGURE 6C presents the regulation of the PDH complex by lipoyl residue effects
on
20 PDK.
FIGURE 7 shows the effects of the pharmaceutical composition of the present
invention on xenograft tumor growth.
FIGURE 8 shows the effect of treatment with the pharmaceutical composition of
the
present invention on three tumor cell types and a non-transformed cell.
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21
FIGURE 9A shows ATP levels in lung cancer cells after treatment with the
pharmaceutical composition of the present invention at or above the lethal
threshold.
FIGURE 9B compares the pharmaceutical composition of the present invention's
inhibition of ATP synthesis in pyruvate-containing media versus glucose-
containing media.
FIGURE 9C compares the pharmaceutical composition of the present invention's
inhibition of ATP synthesis in breast cancer cells to that in normal breast
cells.
FIGURE 9D compares the pharmaceutical composition of the present invention's
inhibition of ATP synthesis with that of lipoic acid and an inactive form of
the present
invention in lung cancer cells.
FIGURE 10 illustrates the pharmaceutical composition of the present
invention's
effects on tumor cell mitochondrial levels of the PDH complex and alpha-
ketoglutarate
(aKDH) dehydrogenase enzymatic activities.
FIGURE I 1A shows Western analyses of two-dimensional gels of extracts from
lung
cancer cells treated or mock-treated with the pharmaceutical composition of
the present
invention.
FIGURE 11 B shows enlargements of paired two-dimensional gel samples treated
and
mock-treated with the pharmaceutical composition of the present invention.
FIGURE 12A depicts the regulatory role of PDKs as modulated by endogenous
lipoate covalently bound to the PDH complex E2 subunit.
FIGURE 12B depicts a possible mechanism for differential inactivation of tumor
cell
PDH complex by the pharmaceutical composition of the present invention.
FIGURE 13 presents the effects of the pharmaceutical composition of the
present
invention on mitochondrial membrane potential in H460 lung cancer cells.
FIGURE 14 shows Western blot analysis results wherein cell death pathways in
diverse tumor cell types by the pharmaceutical composition of the present
invention.
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22
Detailed Description of the Invention
The present invention is generally directed to pharmaceutical compositions for
treating, diagnosing, or preventing a disease, condition, or syndrome, or
symptoms thereof,
which includes an alteration of the phosphorylation state of at least one
enzyme and/or
enzyme complex, or subunit thereof, such as the PDH complex, including those
characterized
by cellular hyperproliferation, such as cancer, or symptoms thereof, in warm-
blooded
animals. Such animals include those of the mammalian class, such as humans,
horses, cattle,
domestic animals including dogs and cats, and the like, subject to disease and
other
pathological conditions and syndromes characterized by cellular
hyperproliferation, including
cancer. The pharmaceutical composition of the present invention comprises an
effective
amount of at least one lipoic acid derivative, including those described in US
Patents
6,331,559 and 6,951,887 and US Provisional. Application No. 60/912,598, also
known as a
thioctan, and a pharmaceutically-acceptable carrier or excipient therefor. As
a molecule
which is not only a derivative of one which is found normally within
mitochondria but also
one which is instrumental to the increased glycolytic activity of tumor cells
as seen' in the
Warburg effect, the lipoic acid derivatives of the present invention are
particularly well-
suited for the selective delivery into and effective concentration within the
mitochondria of
cells and tissues characterized by hyperproliferation, such as tumorous ones,
thereby sparing
normal cells and tissue from the effects of the composition.
The pharmaceutical composition of the present invention may modulate the
effects of
PDKI, PDK2, PDK3, PDK4, and the isoforms of each thereof via reversible
phosphorylation.
The pharmaceutical composition may also modulate the effects of PDP1, PDP2,
and the
isoforms and/or mutants of each thereof also by reversible phosphorylation.
Such modulation
may occur through either promotion or inhibition of kinase or phosphatase
activity.
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23
By inhibiting mitochondrial energy metabolism, the lipoic acid derivatives of
the
present invention cause both the loss of mitochondrial membrane potential and
other
mitochondrial consequences in the diseased cell, resulting in the irreversible
initiation of cell
death. The lipoic acid derivatives of the present invention may also inhibit
mitochondrial
energy metabolism by the activation of PDKs and/or inhibition of PDPs or by
inhibiting the
conversion of pyruvate to the less-toxic molecule acetoin through inhibition
of the activity of
the El subunit of the. PDH complex. The inhibition of acetoin synthesis will
distort other
processes, including redox balance and may also cause the production of toxic
by-products,
including acetaldehyde, superoxide, hydrogen peroxide, and hydroxyl radical,
these by-
products themselves consequently causing irreversible damage to the
mitochondrion of the
diseased cell.
In a first embodiment of the present invention, the lipoic acid derivatives
are defined
by a first general formula (1):
R1 R2
I I
S
(CH2)x R3 (I)
wherein R, and R2 are independently selected from the group consisting of
hydrogen,
alkyl CõH2,,+i, alkene Cõ H2,,, alkenyl CõHZõ_i, alkyne CõH2r-2, alkynyl Cõ
H2,,.3, alkyl sulfide
CH3(CH2)õ-S-, disulfide alkyl CH3CH1---S--S-, thiocarbamic ester (CH2)õC=NH-,
and
semithioacetal CH3CH(OH}-S-, wherein n is 1-10 and t is 0-9, aromatic, acyl
defined as
R4C(O)-, heteroaryl, imidoyl defined as R5C(=NH)-, organometallic aryl, alkyl-
organometallic aryl, semiacetal R6CH(OH)-S-, amino acids, carbohydrates,
nucleic acids,
lipids, and multimers and combinations thereof;
wherein R, and R2 as defined above can be unsubstituted or substituted;
AMENDED SHEET - IPEA/US
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24
wherein R3 is selected from a group consisting of amino acids, carbohydrates,
nucleic
acids, lipids, and multimers thereof;
wherein R4 is selected from the group consisting of hydrogen, alkenyl,
alkynyl,
alkylaryl, heteroaryl, alkylheteroaryl and organometallic aryl, any of which
can be substituted
or unsubstituted;
wherein R5 is selected from the group consisting of hydrogen, alkenyl,
alkynyl, aryl,
alkylaryl, heteroaryl, and alkylheteroaryl, any of which can be substituted or
unsubstituted;
wherein R6 is CC13, CF3, or COOH;
and wherein x is 0-16;
or salts thereof.
In a second embodiment of the present invention, the lipoic acid derivatives
are
defined by a second general formula (1I):
SIM,S
(CH )x R
2 s (II)
wherein M is a covalent bond, -[C(R,)(R2)]Z-, or a metal chelate or other
metal
complex where the metal is not palladium;
wherein R, and R2 are independently selected from the group consisting of
hydrogen,
acyl R4C(O)-, alkyl CõH2õ+1, alkenyl defined as CmH2m.,, alkynyl defined as
CmH2m_3i aryl,
heteroaryl, alkyl sulfide CH3(CH2)õ-S-, imidoyl defined as R4C(=NH)-,
hemiacetal defined
as R6CH(OH)-S-, amino acids, carbohydrates, nucleic acids, lipids, and
multimers and
combinations thereof;
wherein R, and R2 as defined above can be unsubstituted or substituted;
wherein R3 is selected from a group consisting of amino acids, carbohydrates,
nucleic
acids, lipids, and multimers thereof;
AMENDED SHEET - IPEA/US
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wherein R4 and R5 are independently selected from the group consisting of
hydrogen,
alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl, and
heterocyclyl, any of which
can be substituted or unsubstituted;
wherein R5 is selected from the group consisting of CC13r CF3 or COOH;
5 and wherein x is 0-16, z is 0-5, n is 0-10 and m is 2-10;
or salts thereof.
Furthermore, as any or all of these general structures may be metabolized
within the
cell or mitochondrion, it is expressly intended that metabolites of the above-
referenced
structures are within the scope of the present invention.
10 The pharmaceutical composition of the present invention may also modulate
the
expression levels of the phosphatase, kinase, and dehydrogenase enzyme
constituents found
in the PDH complex. This modulation may occur at the transcriptional,
translational, or post-
translational stage, including epigenetic silencing of the appropriate genes.
The compositions of the present invention may further include a
pharmaceutically-
15 acceptable carrier or excipients. Examples of pharmaceutically-acceptable
carriers are well
known in the art and include those conventionally used in pharmaceutical
compositions, such
as, but not limited to, antioxidants, buffers, chelating agents, flavorants,
colorants,
preservatives, absorption promoters to enhance bioavailability, antimicrobial
agents, and
combinations thereof. The amount of such additives depends on the properties
desired,
20 which can readily be determined by one skilled in the art.
The pharmaceutical compositions of the present invention may routinely contain
salts,
buffering agents, preservatives, and compatible carriers, optionally in
combination with other
therapeutic ingredients. When used in medicine, the salts should be
pharmaceutically
acceptable, but non-pharmaceutically-acceptable salts may conveniently be used
to prepare
25 pharmaceutically-acceptable salts thereof and are not excluded from the
scope of the
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26
invention. Such pharmacologically- and pharmaceutically-acceptable salts
include, but are
not limited to, those prepared from the following acids: hydrochloric,
hydrobromic, sulfuric,
nitric, phosphoric, maleic, acetic, palicylic, p-toluene sulfonic, tartaric,
citric, methane
sulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzene
sulfonic. Also,
pharmaceutically-acceptable salts can be prepared as alkaline metal or
alkaline earth salts,
such as sodium, potassium or calcium salts of the carboxylic acid group.
The present invention additionally provides methods for treating or diagnosing
a
patient with therapeutic or diagnostic agents by delivering an effective
amount of at least one
therapeutic or diagnostic agent to cells for implementing the prevention,
diagnosis, or
treatment of a disease, condition, or syndrome, or symptoms thereof, which
includes an
alteration of the phosphorylation state of at least one enzyme and/or enzyme
complex, or
subunit thereof, including those characterized by cellular hyperproliferation.
Modulating the
PDH complex as an improved treatment of cancer is especially contemplated,
including
treatment of primary tumors by the control of tumor cell proliferation,
angiogenesis,
metastatic growth, apoptosis, and treatment of the development of
micrometastasis after or
concurrent with surgical removal; and radiological or other chemotherapeutic
treatment of a
primary tumor. The pharmaceutical composition of the present invention is
useful in such
cancer types as primary or metastatic melanoma, lymphoma, sarcoma, lung
cancer, liver
cancer, Hodgkin's and non-Hodgkin's lymphoma, leukemias, uterine cancer,
cervical cancer,
bladder cancer, kidney cancer, colon cancer, and adenocarcinomas such as
breast cancer,
prostate cancer, ovarian cancer, and pancreatic cancer.
For therapeutic and diagnostic applications, the pharmaceutical composition
can be
administered directly to a patient when combined with a pharmaceutically-
acceptable carrier.
This method may be practiced by administering the therapeutic or diagnostic
agent alone or
in combination with an effective amount of another therapeutic or diagnostic
agent, which
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may be, but is not limited to, a glycolytic inhibitor, a microtubule-
interacting agent, a
cytostatic agent, a folic acid inhibitor, an alkylating agent, a topoisomerase
inhibitor, a
tyrosine kinase inhibitor, podophyllotoxin or derivatives thereof, an
antitumor antibiotic, a
chemotherapeutic agent, an apoptosis-inducing agent, an anti-angiogenic agent,
nitrogen
mustards, nucleic acid intercalating agents, and combinations thereof. Such
therapeutic
agents may further include other metabolic inhibition reagents. Many such
therapeutic agents
are known in the art. The combination treatment method provides for
simultaneous,
sequential, or separate use in treating such conditions as needed to amplify
or ensure patient
response to the treatment method.
The methods of the present invention may be practiced using any mode of
administration that is medically acceptable, and produces effective levels of
the active
compounds without causing clinically unacceptable adverse effects. Although
formulations
specifically suited for parenteral administration are preferred, the
compositions of the present
invention can also be formulated for inhalational, oral, topical, transdermal,
nasal, ocular,
pulmonary, rectal, transmucosal, intravenous, intramuscular, subcutaneous,
intraperitoneal,
intrathoracic, intrapleural, intrauterine, intratumoral, or infusion
methodologies or
administration, in the form of aerosols, sprays, powders, gels, lotions,
creams, suppositories,
ointments, and the like. If such a formulation is desired, other additives
well-known in the art
may be included to impart the desired consistency and other properties to the
formulation.
Those skilled in the art will recognize that the particular mode of
administering the
therapeutic or diagnostic agent depends on the particular agent selected;
whether the
administration is for treatment, diagnosis, or prevention of a disease,
condition, syndrome, or
symptoms thereof, the severity of the medical disorder being treated or
diagnosed; and the
dosage required for therapeutic efficacy. For example, a preferred mode of
administering an
anticancer agent for treatment of leukemia would involve intravenous
administration,
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whereas preferred methods for treating skin cancer could involve topical or
intradermal
administration.
As used herein, "effective amount" refers to the dosage or multiple dosages of
the
therapeutic or diagnostic agent at which the desired therapeutic or diagnostic
effect is
achieved. Generally, an effective amount of the therapeutic or diagnostic
agent may vary
with the activity of the specific agent employed; the metabolic stability and
length of action
of that agent; the species, age, body weight, general health, dietary status,
sex and diet of the
subject; the mode and time of administration; rate of excretion; drug
combination, if any; and
extent of presentation and/or severity of the particular condition being
treated. The precise
dosage can be determined by an artisan of ordinary skill in the art without
undue
experimentation, in one or several administrations per day, to yield the
desired results, and
the dosage may be adjusted by the individual practitioner to achieve a desired
therapeutic
effect or in the event of any complication. Importantly, when used to treat
cancer, the dosage
amount of the therapeutic agent used should be sufficient to inhibit or kill
tumor cells while
leaving normal cells substantially unharmed.
The therapeutic or diagnostic agent included in the pharmaceutical
compositions of
the present invention can be prepared in any amount desired up to the maximum
amount that
can be administered safely to a patient. The amount of the diagnostic agent or
therapeutic
agent may range from less than 0.01 mg/mL to greater than 1000 mg/mL,
preferably about 50
mg/mL.
Generally, the pharmaceutical composition of the present invention will be
delivered
in a manner sufficient to administer to the patient an amount effective to
modulate the
structure and/or activity of the PDH complex. The dosage amount may thus range
from
about 0.3 mg/rn 2 to 2000 mg/m2, preferably about 60 mg/m2. The dosage amount
may be
administered in a single dose or in the form of individual divided doses, such
as from one to
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four or more times per day. In the event that the response in a subject is
insufficient at a
certain dose, even higher doses (or effective higher doses by a different,
more localized
delivery route) may be employed to the extent of patient tolerance. Multiple
doses per day
are contemplated to achieve appropriate systemic or targeted levels of the
therapeutic or
diagnostic agent.
In yet another embodiment of the present invention, the lipoic acid
derivatives of the
present invention may be used as diagnostic and predictive agents in vitro. As
stated earlier,
depending on the specific tumor cell or cell type in question, different
lipoic acid derivatives
may be more or less effective at inhibiting distinct tumor classes through the
modulation of
the PDH complex. Thus, for example, in cases where diagnosis or selection of
an appropriate
chemotherapeutic strategy may be difficult, testing of a culture of tumor
cells in vitro with
lipoic acid derivatives known to target specific tumor cell types provides an
alternative
approach for identifying tumor types and effective treatments.
Turning to the figures, FIGURE 6A illustrates one of the many likely
differences in
energy metabolism in normal tissues and tumor cells in vivo. Tumor cells often
rely more
heavily on cytoplasmic glycolysis than mitochondrial energy metabolism for ATP
generation
than do normal cells under corresponding conditions.. Changes in expression
and regulation
of the PDH complex are apparently part of this tumor-specific adaptation. The
decreases in
the levels of PDH catalytic components and/or increases in the levels of
inhibitory PDKs
producing these effects may render tumor cells much more vulnerable to agents
attacking the
PDH complex than are normal cells.
FIGURE 6B depicts the structures of lipoic acid as it catalyzes the normal
reactions
involved in synthesizing acetyl-CoA from pyruvate in the PDH complex. In vivo
lipoic acid
is joined through its carboxyl terminus in a non-peptide amide linkage to an
epsilon amino
group of a lysine in the E2 lipoyl domain active sites. Notice also that the
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oxidation/reduction/acetylation state of PDH E2-bound lipoate is monitored by
the kinases
and phosphatases that control PDH activity by controlling the phosphorylation
inactivation of
the Ela PDH subunit. This figure also depicts the structure of the three
representative lipoic
acid derivatives which may be used in the present invention. While CPI-613 and
CPI-045
5 have high anticancer potency, CPI-157 has little or no activity in cell
culture and is useful as
a control in several experiments.
FIGURE 6C presents the relationship between PDH complex components, including
E2 with its bound lipoates, El, and the regulatory PDK. High levels of acetyl-
lipoate or
dihydrolipoate (not diagrammed) activate PDKs which, in turn, suppress further
flux through
10 the PDH complex by inactivating Ela, the subunit catalyzing the first step
in PDH complex
catalysis. This process acts as a governor for carbon/energy flow through the
PDH complex,
and this regulatory process is apparently substantially altered to support the
variant energy
metabolism of tumor cells, as seen in FIGURE 3A.
FIGURE 7 shows the effects of the pharmaceutical composition of the present
15 invention on xenograft tumor growth. Cells were implanted subcutaneously on
the dorsal
flank as described in EXAMPLE 2. Mice were then injected with the drug (or
vehicle alone;
"mock") intraperitoneally starting on days as indicated in the figure. The
left panel shows a
pancreatic tumor model injected three times weekly with the present invention
at 1 mg/kg or
the vehicle control. This experiment is representative of two done with BxPC-3
cells and of
20 two done with AsPC-1 cells. The right three panels show an H460 lung tumor
model injected
with the concentrations indicated either once weekly (circles), three times
weekly (inverted
triangles), or five times weekly (triangles for vehicle treatment and squares
for drug
treatment). This experiment is representative of four done with H460 cells.
FIGURE 8 shows the effect of treatment with the pharmaceutical composition of
the
25 present invention on three tumor cell types and a non-transformed cell type
(MDCK) either at
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200-300 pM ("Treated") or mock treatment ("Mock treated"). Cells were treated
in
appropriate tissue culture media containing 10% serum or 48 hours. Extensive
cell death by
apoptosis or apoptosis-like pathways (see also FIGURE 11) in the three cancer
cell lines is
observed through the methodology described in EXAMPLE 2. In contrast, the non-
transformed MDCK cells are apparently unaffected by drug treatment at this
dose.
FIGURE 9A shows ATP levels in H460 lung cancer cells after treatment with the
pharmaceutical composition of the present invention at or above the lethal
threshold (200 M
in 10% serum). Dashed lines represent treatment at the concentrations
indicated. Solid lines
of corresponding texture represent treatment for the time indicated, followed
by removal of
the drug and 60 minutes of recovery in the drug-free medium. Block arrows
indicated
intervals of ATP recovery.
FIGURE 9B compares inhibition of ATP synthesis in media in which pyruvate (in
the
form of methyl-pyruvate) is the primary carbon source (dashed lines) and in
which glucose is
the primary carbon source (solid lines). Notice that the pharmaceutical
composition of the
present invention ultimately produces cell death at the same threshold
concentrations in both
media; however, early depletion of total cellular ATP levels is high in
pyruvate-containing
media and absent in glucose-containing media. Also, the onset of cell death is
more rapid in
the 300 M than the 200 M drug concentration.
FIGURE 9C compares the pharmaceutical composition of the present invention's
inhibition of ATP synthesis in SK-Br-3 breast cancer cells and HMEC normal
breast cells. In
contrast to the experiments whose results are depicted in FIGURES 6A and 6B,
these
experiments were done in serum-free medium (MEBM). As a result, the drug's
lethal
threshold is lower, approximately 50 M. Note that the small depression in ATP
levels in the
22-hour normal cell samples is not related to drug dose and reflects normal
experimental
variation.
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FIGURE 9D compares inhibition of ATP synthesis in H460 lung cancer cells by
the
pharmaceutical composition of the present invention (left graph), lipoic acid
(center graph),
and an inactive form of the present invention (right graph). As in FIGURE 9C,
these
experiments are done in serum-free medium so that the drug's lethal threshold
is
approximately 50pM.
FIGURE 10 illustrates the pharmaceutical composition of the present
invention's
effects (at 400 M in DMEM with 10% serum) on tumor cell mitochondria] levels
of PDH
(PDC) and (aKDH) enzymatic activities. Notice that PDH is strongly inhibited
whereas
aKDH is not. Enzyme activity levels are measured in extracts of purified
mitochondria using
resazurin reduction in response to added carbon source, as described in
EXAMPLE 2. The
background line corresponds to resazurin reduction in the absence of added
carbon source.
Next, in FIGURE 11 A, Western analyses of two-dimensional gels of extracts
from
H460 lung cancer cells treated (+) or mock treated (-) with the pharmaceutical
composition of
the present invention (at 400 M for 120 minutes in RPMI medium with 10% serum)
were
performed. The Western transfers were probed with a cocktail of monoclonal
antibodies
against Eta and E2 subunits of the PDH complex. The Western transfers are
aligned at E2.
Notice the substantially higher levels of hyper-phosphorylated and the reduced
levels of
hypo-phosphorylated forms of El in the drug-treated sample. The left vertical
white line
illustrates one of the criteria for aligning the gels, the mobility of the E2
subunit. The right
vertical white line passes through the less phosphorylated Eta form, the
presumptively-
enzymatically-active component.
FIGURE 11 B shows enlargements of paired two-dimensional gel samples treated
and
mock-treated with the pharmaceutical composition of the present invention.
Element A is an
enlargement of a portion of FIGURE 8A. Element B is SK-Br-3 breast cancer
cells mock-
treated (-) and treated (+) for 180 minutes with 80 M of the composition in
MEBM serum-
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free breast epithelial cell medium. Element C is SK-Br-3 breast cancer cells
mock-treated (-)
and treated (+) for 240 minutes with 80 M of the composition in MEBM serum-
free breast
epithelial cell medium. Element D is HMEC normal breast epithelial cells mock-
treated (-)
and treated (+) for 240 minutes with 80 M of the composition in MEBM serum-
free breast
epithelial cell medium. The vertical white line passes through the less
phosphorylated Ela
form, the presumptively-enzymatically-active component.
FIGURES 12A and 12B depict a working hypothesis for the strong, selective
anticancer effects of the pharmaceutical composition of the present invention
in vivo.
FIGURE 12A, for example, shows the regulatory role of PDKs as modulated by
endogenous
lipoate covalently bound to the PDC E2 subunit. PDKs normally inactivate the
PDC in
response to high levels of reduced and/or acetylated lipoate, a process that
is apparently
substantially modified in tumor cells.
Concomitantly, FIGURE 12B shows the large quantitative difference in the ratio
of
PDK to its substrate PDC-EI in the PDC, believed to distinguish normal and
tumor cells in
vivo. In normal cells the low level of PDK is thought to "walk" hand-over-hand
(through its
two dimeric subunits) around the PDH complex, gradually phosphorylating El.
This
phosphorylation is in steady state equilibrium with PDP dephosphorylation (not
diagrammed). On the working hypothesis diagrammed here, thioctans stimulate
PDKs
through the same sites that normally bind acetyl-lipoate and/or
dihydrolipoate, thereby
artificially stimulating one or more PDK isoform to inacative Ela. In cancer
cells, the much
higher levels of PDK might make this stimulation by thioctans much more
effective in
shutting down PDC enzymatic activity and mitochondrial energy metabolism.
The following non-limiting examples are provided to facilitate understanding
of the
pharmaceutical compositions of the present invention.
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EXAMPLE 1
CHEMICAL SYNTHESIS OF THIOCTANS
Lipoic acid derivatives (i.e., thioctans) CPI-613 and CPI-157 were synthesized
by
using a modified procedure described in US 6,331,559 BI and US 6,951,887 B2
with 6, 8-
bismercaptooctanoic acid as the starting material. Thioctan CPI-045 was
synthesized as
described in US 6,331,559 B1.
Structure analyses for the three thioctans are below. Multiple independent
syntheses
of CPI-045 and CPI-613 were indistinguishable in their anti-cancer properties.
Purity of CPI-
613 used in the xenograft (FIGURE 7) and ATP measurements (FIGURE 9) was in
excess of
99%. All other preparations were greater than 98% pure.
CPI-613: 6,8-Bis-benzylsulfanyloctanoic acid: White crystalline solid, m.p. 65-
66 C
(lit.' 67.5-69 ); 'H-NMR (250 MHz, CDCI3): 6 7.15-7.4 (m, 10H), 3.66 (s, 2H),
3.64 (s, 2H),
2.52-2.62 (m, 1H), 2.50 (t, J = 7.6 Hz, 2H), 2.29 (t, J = 7.6 Hz, 2H), 1.2-1.8
(m, 8H); 13C-
NMR (62.9 MHz, CDC13): 8 179.6, 138.6, 138.5, 128.9, 128.8, 128.5, 128.4,
126.9, 44.1,
36.4, 35.1, 34.4, 33.8, 28.7, 26.0, 24.4.
CPI-157: 6,8-Bis-ethylsulfanyloctanoic acid: Colorless oil; TLC
(EtAc:Hexanes:HAc, 200:200:1 v/v): Rf = 0.60; 'H-NMR (300 MHz, CDC13): S 2.64-
2.76
(m, 1H), 2.65 (t, J = 7.5 Hz, 2H), 2.52 (q, J = 7.5 Hz, 2H), 2.49 (q, J = 7.2
Hz, 2H), 2.36 (t, J
= 7.4 Hz, 2H), 1.40-1.85 (m, 8H), 1.25 (t, J = 7.2 Hz, 3H), 1.22 (t, J = 7.5
Hz, 3H); 13C-NMR
(75 MHz, CDC13): S 180.0, 44.3, 34.6, 33.9, 28.9, 26.2, 25.9, 24.5, 24.2,
14.9, 14.7; IR
(film): 2963, 1708, 1449, 1423, 1283, 1263 cm''.
CPI-045: 6,8-Bis-benzoylsulfanyloctanoic acid: Colorless, viscous oil; TLC
(Hexanes:EtAc:HAc, 100:50:1 v/v): Rf = 0.30; 'H-NMR (250 MHz, CDC13): S 7.9-
8.1 (m,
4H), 7.38-7.60 (m, 6H), 3.8-4.0 (m, 1H), 3.0-3.3 (m, 2H), 2.34 (t, J = 7.1 Hz,
2H), 1.4-2.2 (m,
8H); 13C-NMR (62.9 MHz, CDC13): S 191.7, 191.5, 179.7, 137.0, 136.9, 133.3,
128.5,
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127.3, 127.1, 43.6, 35.0, 34.6, 33.8, 26.4, 26.2, 24.3; IR (film): 2973, 1710,
1704, 1667,
1665, 1662, 1448, 1207, 1175, 911, 773, 757, 733, 688, 648 cm
EXAMPLE 2
5 METHODS USED TO DETERMINE THIOCTAN ANTI-CANCER EFFECTS
Cells: Human tumor cell lines were obtained from ATCC and propagated according
to
ATCC recommendations. Human Mammary Epithelial Cells (HMEC), Small Airway
Epithelial Cells (SAEC), and Normal Human Epidermal Keratinocytes (NHEK)
primary cells
were obtained from LONZA Walkersville, Inc (Walkersville, MD). Each cell line
was
10 maintained and propagated in appropriate media developed by and bought from
the supplier
according to the supplier's instructions. Experiments reported here used
normal cells at
passage three to six.
Tumor growth inhibition studies: CD1-Nu/Nu female mice were implanted with
human BxPC-3 or AsPC-1 pancreatic tumor cells or H460 NSCLC by subcutaneous
(SC)
15 injection. Approximately 8-12 days later the mice were injected
intraperitoneally (IP) at
doses and schedule as indicated in the figure legend. Drug or vehicle was
injected at ca. 2ml
per 25gm of body weight. Drug concentration was 1.25mg/ml (ca. 3.1mM) or less.
The
vehicle/solvent consisted of triethanolamine in water at 25mM or less. The
vehicle injected
in mock treated animals was always identical to the solvent in which the
highest drug dose
20 for that experiment was injected. Mice were monitored daily for physical
condition and
mortality. Body weight and tumor volume were assessed daily before treatment,
and
approximately three times weekly during and after treatment. Mice were kept on
a 12 hour
light/dark cycle, were fed ad libitum and were housed at Stony Brook
University Animal
Facility in accordance with institutional guidelines.
25 Cell death assays: For most assessments of cell viability CellTiter-Glo
assay
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(Promega) was used at times sufficiently long not to be confounded by early
thioctan
inhibition of ATP synthesis. (FIGURE 9). In a typical experiment, cells were
plated in
black, clear bottom, 96-well plates at 5,000 cells per well. 18-25 hours
later, medium was
replaced with fresh medium containing drug solvent (triethanolamine in water
at 2.8mM in
serum-containing media and 0.7mM in serum-free media) or thioctan CPI-613 in
the same
solvent. The assay was performed at 24 or 48 hours after drug addition,
depending on drug
dose, according the manufacturer's directions.
In some cases, cells were plated in 48-well plates at 10,000 cells per well,
and
medium was replaced 18-25 hours later with fresh medium containing drug
solvent (-I%
EtOH final concentration) or different concentrations of thioctan CPI-045 in
the same
solvent. Cells remained in solvent or drug-containing medium for the remainder
of the
experiment. Plates were inspected at 24, 48 and 72 hours post drug addition,
and cell
numbers were estimated as a confluence percentage. Under these conditions,
thioctan-
induced cell death is highly apoptotic at near-threshold doses, and cell
number estimates are
very reliable indicators of death. (FIGURE 10) The integrity of cells
remaining at 72 hours,
if any, was tested by trypan blue exclusion.
Table 2 provides data regarding the action of thioctans against tumor cells in
vitro.
Listed are the human tumor and primary human cells we have investigated for
sensitivity to
CPI-613 and/or CPI-045 killing. "+" indicates that the cells underwent
apoptosis or necrosis-
like cell death at doses of approximately 200-300 M (in the presence of 10%
serum) and
approximately 50 pM in serum-free medium. (FIGURES 8 and 9) "--" indicates
that these
cells required approximately five-fold higher drug .doses to induce cell death
in the
corresponding medium. "nt" indicates non-tested combination. All tumor lines
were
analyzed in the appropriate media with 10% serum, as were the MDCK normal
cells in
FIGURE 8. In addition, HMEC, SAEC, NHKC primary human cells, and SK-BR-3, A549
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and H460 tumor lines were also analyzed in the appropriate serum-free media.
Primary cells
were contact inhibited and transformed cells were at comparable densities.
ATP assay: Cells were plated in black, clear bottom, 96-well plates at 5,000
cells per
well. 18-25 hours later, medium was replaced with fresh medium containing drug
solvent
(triethanolamine) or thioctan (CPI-613 or CPI-157) or lipoic acid for time
interval and at drug
concentration as indicated. Cell viability and integrity was assessed by
recovery after drug
withdrawal by trypan blue exclusion. ATP was measured using CellTiter-Glo
luminescence
assay (Promega) according to manufacturer's directions. All measurements were
performed
in duplicate and showed high consistency. The standard error of the mean
ranged from 0.1-
2% of the measured value. As a result, error bars were omitted from FIGURE 9.
Methyl
pyruvate medium in FIGURE 9 consisted of RPMI without glucose (Invitrogen),
supplemented with 10% dialyzed fetal bovine serum, 5mM HEPES (pH 7.4), and
10mM
methylpyruvate (Sigma-Aldrich), and the matched glucose medium was
conventional RPMI
(Invitrogen).
PDH and aKDH enzyme assays: Tumor cells were grown to 80% confluence in 15
cm plates and treated with CPI-613 as indicated. Mitochondria were isolated
according to the
method of Moreadith and Fiskum. 1 Mitochondria were lysed in 0.4% lauryl
maltoside. 50 l
of mitochondrial lysate was added to 96-well plates. 50 ul of reaction mix
(50mM Tris, pH
7.5, 2mM R-NAD+, 225uMv TPP, 2mM pyruvate or a-ketoglutarate, 150 M coenzyme
A,
2.6mM cysteine, 1mM MgCl2) was added to mitochondrial lysates, and the mixture
was
incubated for 45 minutes at 37 C. At this time, 15 M resazurin and 0.5U/ml
diaphorase were
added to the mixture and incubated for an additional five minutes. NADH
production was
monitored by measuring fluorescence using an excitation wavelength of 530nm
and an
emission wavelength of 590nm in a microplate reader (Fluorostar). All
measurements were
performed in duplicate and showed high consistency. The standard error of the
mean ranged
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from 0.3-4% of the measured value. As a result, error bars were omitted from
FIGURE 10.
E 1 a phosphorylation:
Cell lysates for 2-D gels: Cells were grown to 95% confluence in 60 mm dishes
and
treated with drug or solvent as indicated. Cells were lysed in situ with 450
l Lysis buffer A
[455 l Zoom 2D protein solubilizer I (Invitrogen), 2.5 l 1M Tris base, 5.t1
IOOX protease
inhibitor cocktail (Complete min, EDTA-free, Roche); 5 l 2M DTT]. Cell lysate
was
transferred to 1.5ml microfuge tubes and sonicated on ice for 15 passes at 50%
power. After
minute incubation at room temperature, 2.5 l of dimethylacrylamide (DMA, Sigma-
Aldrich) was added, and lysates were incubated for an additional 10 minutes. 5
t1 of 2M
10 DTT were added to neutralize excess DMA. Lysates were centrifuged at
16,000x g for 15
minutes.
2-D gels: We used Zoom Benchtop proteomics system (Invitrogen) according to
the
manufacturer's direction. Briefly, 30-50pl of lysate were mixed with 0.8111 pH
3-10
ampholytes, 0.75 1 2M DTT and brought up to 150 l with Zoom 2D protein
solubilizer 1.
150 l of sample was loaded into 1PG runner, and pH 3-IONL IPG strips were
added. Strips
were soaked overnight at room temperature. A step protocol was used for
isolectric focusing
(250V, 20min.; 450V, 15min; 750V, 15 min 2000V, 30min). Strips were. treated
for 15
minutes in 1X loading buffer, followed by 15 minutes in lX loading buffer plus
160mM
iodoacetatic acid. Strips were electrophoresed on NuPAGE 4-12% Bis Tris ZOOM
gels
(Invitrogen).
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Table 2: Effect of thioctans against tumor and primary cells in vitro
brain, glioblastoma U-87 MG + +
brain, glioblastoma LN-229 + nt
breast, adenocarcinoma SK-Br-3 + +
breast, adenocarcinoma MCF7 nt +
bone, osteosarcoma Saos-2 nt +
cervical, adenocarcinoma HeLa + +
colorectal, adenocarcinoma SW480 nt +
hepatocellular, carcinoma Hep G2 + +
kidney, carcinoma A-498 + nt
E lung, carcinoma A459 + +
F-
lung, carcinoma H460 + +
muscle, rhabdomyosarcoma RD nt +
ovarian, carcinoma SKOV-3 + nt
pancreatic, adenocarcinoma AsPC-1 + nt
pancreatic, adenocarcinoma BxPC-3 + nt
prostate, carcinoma LnCaP nt +
uterine, sarcoma Mes SA + +
uterine, sarcoma, MDR Mes-SA/dx5 + +
mammary epithelial cells HMEC -- --
small airway epithelial cells SAEC -- nt
a keratinocytes NHKC nt --
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zUW
Westerns: Proteins were blotted onto PVDF 4.5 m membranes. PDH Ela and E2
were detected using mAbs (Invitrogen).
Caspase-3 and PARP cleavage: Cleaved caspase-3 was detected on Western blots
according to Roy and Nicholson.2 Briefly, after drug or solvent treatment
cells were scraped
5 and the medium/cell/apoptotic bodies mixture was centrifuged at 6000x g.
Pellet was lysed
with lysis buffer C (4M urea, 10% glycerol, 2% SDS, 0.003% BPB; 5% 2-
mercaptoethanol).
30 g of total cell lysate protein per well were loaded on 12% Bis-Tris gels.
Proteins blotted
onto PVDF 4.5 m membranes. Pro- and active-Caspase-3 were detected with anti-
caspase-3
mAb (mouse monocolonal [31A1067]; abcam). PARP cleavage was detected using
10 monoclonal anti-poly (ADP-ribose) polymerase antibody, clone C-2-10 (Sigma-
Aldrich).
Mitochondrial Ca+2 detection: Cells were seeded on 35 mm glass bottom plates
(BD
Biosciences) at 3x105, grown overnight and treated with drug or solvent as
indicated. Cells
were then loaded with calcium dye Fluo-4, X-Rhod-1 or Rhod-2 (4 M,
Invitrogen) in phenol
red-free media and incubated at 37 C for 10 minutes. Cells were washed once
with PBS, and
15 images were captured using an Axiovert 200M, (Zeiss) deconvolution
microscope at a fixed
exposure time, using FITC filter. Quantification of fluorescence was performed
using
software provided by the manufacturer. X-Rhod-1 and Rhod-2 gave similar
results (FIGURE
13), indicating that these dyes were measuring a mitochondrial Ca +2 signal.34
References:
20 1. Moreadith RW and Fiskum G. Isolation of Mitochondria from Ascites
Tumour-Cells Permeabilized with Digitonin. Analytical Biochemistry 137, 360-
367 (1984).
2. Roy S and Nicholson DW. Criteria for identifying authentic caspase
substrates during apoptosis. Apoptosis 322,110-125 (2000).
3. Gerencser AA and Adam-Vizi V. Selective, high-resolution fluorescence
25 imaging of mitochondria] Ca2+ concentration. Cell Calcium 30, 311-321
(2001).
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4. Gyorgy H, Gyorgy C, Das S, Garcia-Perez C, Saotome M, Roy SS, and Yi
MQ. Mitochondrial calcium signalling and cell death: Approaches for assessing
the role of
mitochondrial Ca2+ uptake in apoptosis. Cell Calcium 40, 553-560 (2006).
EXAMPLE 3
THIOCTANS PERTURB MITOCHONDRIAL MEMBRANE POTENTIAL AND Ca+2.
UPTAKE
The substrate effects on thioctan inhibition of ATP synthesis (FIGURE 9)
indicate
that the drug is interfering with the TCA cycle in the mitochondrial matrix.
If this is the case,
we anticipate that mitochondrial membrane potential' might be compromised at
lethal
threshold doses and above. Using the potential-sensitive dye TMRE we observe
the expected
effect. (FIGURE 13) Mitochondrial membrane potential declines rapidly with
initiation of
drug treatment. The kinetics of membrane potential decline is very similar to
the loss of ATP
synthesis in the presence of mitochondrial substrates. (FIGURE 9)
ATP depletion in mitochondria is known to provoke a homeostatic response that
includes the uptake of Ca+2 released from cytoplasmic stores, including the
endoplasmic
reticulum.2 Moreover, import of this Ca+2 into the mitochondrial matrix is
thought to require
the mitochondrial membrane potential. Thus, we anticipate that thioctan
treatment at or
above the lethal threshold may produce a sustained cytoplasmic release of Ca+2
with transient
mitochondrial uptake of the ion in view of progressively compromised membrane
potential.
Using X-Rhod-1 and Rhod-2 to measure mitochondrial Ca+2 and Fluo-4 to measure
cytoplasmic Ca+2, we observe these expected effects. (FIGURE 13)
By around two hours at CPI-doses at and slightly above the lethal threshold
(compare
FIGURES 9 and 13), as mitochondrial membrane potential declines, this initial
mitochondrial
Ca+2 transient decays. It is followed at 4-6 hours by a second large
mitochondrial Ca +2 peak
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presumably associated with initiation of the calcium-dependent cell death
pathways.3
References:
1. Garrett R and Grisham CM. Biochemistry. Thomson Brooks/Cole, Southbank,
Vic., Australia; Belmont, CA. (2007).
2. Graier WF, Frieden M, and Malli R. Mitochondria and Ca2+ signaling: old
guests, new functions. Pflugers Archiv-European Journal of Physiology 455, 375-
396
(2007).
3. Gyorgy H, Gyorgy C, Das S, Garcia-Perez C, Saotome M, Roy SS, and Yi
MQ. Mitochondrial calcium signalling and cell death: Approaches for assessing
the role of
mitochondrial Ca2+ uptake in apoptosis. Cell Calcium 40, 553-560 (2006).
EXAMPLE 4
THIOCTANS INDUCE DIVERSE CELL DEATH PROGRAMS
Reduction in mitochondrial energy metabolism is known to correlate with the
decision to enter a cell death pathway in some circumstances, though detailed
mechanisms
remain incompletely understood.' "3 At thioctan doses above threshold but
within -2-fold of
this minimal killing dose, all tested cancer cell types undergo cell death
that morphologically
resembles apoptosis predominantly. (FIGURE 8) Apoptotic death was confirmed
under
these conditions by conventional Annexin immunostaning and TUNEL DNA end-
labelling
assays (results not shown).
At higher drug doses (more than -2-fold over threshold), active thioctans
induce cell
death (as assessed by replating viability assays and trypan blue or propidium
exclusion)
without the morphological correlates of apoptosis, suggesting a necrosis-like
pathway (results
not shown).
These data confirm that the thioctan CPI-613 inhibition mitochondrial energy
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metabolism correlates precisely with induction of cell death.
The observation that diverse tumor cells, known or presumed to contain
inactivating
mutations for distinct and diverse cell death pathways4 are all killed at very
similar thioctan
doses (FIGURE 8 and Table 2) is striking. This observation suggests that these
drugs induce
a master signal that is capable of engaging multiple, potentially redundant
distal cell death
execution pathways.5
Consistent with this possibility, we find that the Z-VAD-FMK generic caspase
inhibitor subtly alters the morphology of cell death in thioctan treated
cells, but has no
discernible effect on the lethal threshold dose of the drug.
To further test the possibility that thioctan-induced cell death can proceed
through
multiple terminal execution mechanisms we examined caspase-3 and PARP-1
cleavage,
diagnostic of distinct cell death pathways.5 We find that both thioctans CPI-
613 and CPI-045
induce highly variable levels of these two cleavage events in different cells.
(FIGURE 14)
Collectively, these results indicate that the thioctans are able to induce a
strategic
commitment to death that, depending on drug dose and cell type, is agnostic
about the
terminal, tactical execution of that decision.
References:
1. Watabe M and Nakaki T. ATP depletion does not account for apoptosis
induced by inhibition of mitochondrial electron transport chain in human
dopaminergic cells.
Neuropharmacology 52, 536-541 (2007).
2. Yuneva M, Zamboni N, Oefner P, Sachidanandam R, and Lazebnik Y.
Deficiency in glutamine but not glucose induces MYC-dependent apoptosis in
human cells.
Journal of Cell Biology 178, 93-105 (2007).
3. Skulachev VP. Bioenergetic aspects of apoptosis, necrosis and mitoptosis.
Apoptosis 11, 473-485 (2006).
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UII87Q0 W :0 NAO' O
44
4. Johnstone RW, Ruefli AA, and Lowe SW. Apoptosis: A link between cancer
genetics and chemotherapy. Cell 108, 153-164 (2002).
5. Cregan SP, Dawson VL, and Slack RS. Role of AIF in caspase-dependent and
caspase-independent cell death. Oncogene 23, 2785-2796 (2004).
EXAMPLE 5
LIPOIC ACID DERIVATIVE ANALOG STRUCTURES
Various non-limiting examples of lipoic acid derivative analogs presented
below have
been manufactured and are herein disclosed.
OH
S_s s.
61-r ~ N
OH
S's S,
I \ I \
r__Y~ OH
.S S=S
6
OH
,S S
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n--~j OH
crs /S
If
ry--~j OH
S;piS
07/
The foregoing discussion discloses and describes merely exemplary embodiments
of
5 the present invention. One skilled in the art will readily recognize from
such discussion, and
from the accompanying claims, that various changes, modifications and
variations can be
made therein without departing from the spirit and scope of the invention as
defined in the
following claims. Furthermore, while exemplary embodiments have been expressed
herein,
others practiced in the art may be aware of other designs or uses of the
present invention.
10 Thus, while the present invention has been described in connection with
exemplary
embodiments thereof, it will be understood that many modifications in both
design and use
will be apparent to those of ordinary skill in the art, and this application
is intended to cover
any adaptations or variations thereof. It is therefore manifestly intended
that this invention be
limited only by the claims and the equivalents thereof.
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