U.S. patent application number 12/778054 was filed with the patent office on 2011-01-27 for methods for treatment of metabolic disorders using epimetabolic shifters, multidimensional intracellular molecules, or environmental influencers.
Invention is credited to John Patrick McCook, Niven Rajin Narain, Rangaprasad Sarangarajan.
Application Number | 20110020312 12/778054 |
Document ID | / |
Family ID | 43085533 |
Filed Date | 2011-01-27 |
United States Patent
Application |
20110020312 |
Kind Code |
A1 |
Narain; Niven Rajin ; et
al. |
January 27, 2011 |
METHODS FOR TREATMENT OF METABOLIC DISORDERS USING EPIMETABOLIC
SHIFTERS, MULTIDIMENSIONAL INTRACELLULAR MOLECULES, OR
ENVIRONMENTAL INFLUENCERS
Abstract
Methods and formulations for treating metabolic disorders in
humans using epimetabolic shifters, multidimensional intracellular
molecules or environmental influencers are described.
Inventors: |
Narain; Niven Rajin;
(Cambridge, MA) ; McCook; John Patrick; (Frisco,
TX) ; Sarangarajan; Rangaprasad; (Rutland,
MA) |
Correspondence
Address: |
MCCARTER & ENGLISH, LLP BOSTON
265 Franklin Street
Boston
MA
02110
US
|
Family ID: |
43085533 |
Appl. No.: |
12/778054 |
Filed: |
May 11, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61177241 |
May 11, 2009 |
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61177243 |
May 11, 2009 |
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61177244 |
May 11, 2009 |
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61177245 |
May 11, 2009 |
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61177246 |
May 11, 2009 |
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Current U.S.
Class: |
424/94.5 ;
435/7.21; 514/251; 514/276; 514/345; 514/356; 514/460; 514/47;
514/557; 514/561; 514/567; 514/568; 514/570; 514/574; 514/62;
514/625; 514/678 |
Current CPC
Class: |
C12Q 1/6883 20130101;
G01N 2800/52 20130101; A61K 31/122 20130101; G01N 2570/00 20130101;
A61P 3/10 20180101; C12Q 2600/158 20130101; A61P 3/08 20180101;
G01N 33/5735 20130101; G01N 33/57484 20130101; G01N 2800/042
20130101; A61P 13/12 20180101; C12Q 2600/136 20130101; C12Q 2600/16
20130101; A61P 3/04 20180101; A61P 9/12 20180101; A61P 7/02
20180101; C12Q 1/6886 20130101; A61P 3/00 20180101; A61P 3/06
20180101; A61P 9/10 20180101; A61P 1/16 20180101; A61P 35/02
20180101; A61P 35/04 20180101; A61P 43/00 20180101; A61K 31/00
20130101; A61P 9/00 20180101; C12Q 2600/112 20130101; A61P 9/04
20180101; A61P 35/00 20180101; C12Q 2600/106 20130101 |
Class at
Publication: |
424/94.5 ;
514/678; 514/567; 514/561; 514/570; 514/345; 514/625; 514/568;
514/557; 514/47; 514/276; 514/356; 514/62; 514/574; 514/460;
514/251; 435/7.21 |
International
Class: |
A61K 38/45 20060101
A61K038/45; A61K 31/122 20060101 A61K031/122; A61K 31/198 20060101
A61K031/198; A61K 31/192 20060101 A61K031/192; A61K 31/44 20060101
A61K031/44; A61K 31/16 20060101 A61K031/16; A61K 31/194 20060101
A61K031/194; A61K 31/191 20060101 A61K031/191; A61K 31/7076
20060101 A61K031/7076; A61K 31/51 20060101 A61K031/51; A61K 31/455
20060101 A61K031/455; A61K 31/7008 20060101 A61K031/7008; A61K
31/19 20060101 A61K031/19; A61K 31/351 20060101 A61K031/351; A61K
31/525 20060101 A61K031/525; G01N 33/53 20060101 G01N033/53; A61K
31/7084 20060101 A61K031/7084; A61P 3/10 20060101 A61P003/10; A61P
3/04 20060101 A61P003/04 |
Claims
1. A method for treating, alleviating symptoms of, inhibiting
progression of, or preventing a metabolic disorder in a mammal, the
method comprising: administering to the mammal in need thereof a
therapeutically effective amount of a pharmaceutical composition
comprising at least one environmental influencer (env-influencer),
wherein the environmental influencer selectively elicits, in a
disease cell of the mammal, a cellular metabolic energy shift
towards normalized mitochondrial oxidative phosphorylation.
2. The method of claim 1, wherein the environmental influencer does
not substantially elicit, in normal cells of the mammal, the
cellular metabolic energy shift towards mitochondrial oxidative
phosphorylation.
3. The method of claim 1, wherein the mammal is human (or a
non-human mammal).
4. The method of claim 1, wherein the metabolic disorder is
responsive or sensitive to treatment by Coenzyme Q10 or its
metabolites or analogs thereof.
5. The method of claim 1, wherein the metabolic disorder is
characterized by a dysregulated mitochondrial oxidative
phosphorylation function that leads to altered gene regulation
and/or protein-protein interactions which contribute to or causally
lead to the metabolic disease.
6. The method of claim 1, wherein the environmental influencer
comprises: (a) benzoquinone or at least one molecule that
facilitates the biosynthesis of the benzoquinone ring, and (b) at
least one molecule that facilitates the synthesis of and/or
attachment of isoprenoid units to the benzoquinone ring.
7. The method of claim 6, wherein said at least one molecule that
facilitates the biosynthesis of the benzoquinone ring comprises:
L-Phenylalanine, DL-Phenylalanine, D-Phenylalanine, L-Tyrosine,
DL-Tyrosine, D-Tyrosine, 4-hydroxy-phenylpyruvate,
3-methoxy-4-hydroxymandelate (vanillylmandelate or VMA), vanillic
acid, pyridoxine, or panthenol.
8. The method of claim 6, wherein said at least one molecule that
facilitates the synthesis of and/or attachment of isoprenoid units
to the benzoquinone ring comprises: phenylacetate,
4-hydroxy-benzoate, mevalonic acid, acetylglycine, acetyl-CoA, or
farnesyl.
9. The method of claim 1, wherein the environmental influencer
comprises: (a) one or more of L-Phenylalanine, L-Tyrosine, and
4-hydroxyphenylpyruvate; and, (b) one or more of 4-hydroxy
benzoate, phenylacetate, and benzoquinone.
10. The method of claim 1, wherein the environmental influencer:
(a) inhibits Bcl-2 expression and/or promotes Caspase-3 expression;
and/or, (b) inhibits cell proliferation.
11. The method of claim 1, wherein the environmental influencer is
a multidimensional intracellular molecule (MIM).
12. The method of claim 11, wherein the MIM is selected from: alpha
ketoglutarate/alpha ketoglutaric acid, Malate/Malic acid,
Succinate/Succinic acid, Glucosamine, Adenosine, Adenosine
Diphosphate, Glucuronide/Glucuronic acid, Nicotinic Acid, Nicotinic
Acid Dinucleotide, Alanine/Phenylalanine, Pyridoxine, Thiamine, or
Flavin Adenine Dinucleotide.
13. The method of claim 1, wherein the environmental influencer is
an epimetabolic shifter (epi-shifter).
14. The method of claim 13, wherein the epimetabolic shifter is
selected from: Transaldolase, Transketolase, Succinyl CoA synthase,
Pyruvate Carboxylase, or Riboflavin.
15. The method of claim 13, wherein the epimetabolic shifter is
coenzyme Q10.
16. The method of claim 1, wherein the concentration of the
environmental influencer in the tissues of the human being treated
is different than that of a control standard of human tissue
representative of a healthy or normal state.
17. The method of claim 1, wherein the form of the environmental
influencer administered to the human is different than the
predominant form found in systemic circulation in the human.
18. The method of claim 1, wherein the amount sufficient to treat
the metabolic disorder in the human up-regulates or down-regulates
mitochondrial oxidative phosphorylation.
19. The method of claim 18, wherein the amount sufficient to treat
the metabolic disorder in the human modulates anaerobic use of
glucose and/or lactate biosynthesis.
20. The method of claim 1, wherein the treatment occurs via an
interaction of the env-influencer with HNF4alpha.
21. The method of claim 1, wherein the treatment occurs via an
interaction of the env-influencer with transaldolase.
22. The method of claim 1, wherein the metabolic disorder is
selected from the group consisting of diabetes, obesity,
pre-diabetes, Metabolic Syndrome and any key elements of a
metabolic disorder.
23. The method of claim 22, wherein the metabolic disorder is
diabetes, and the env-influencer affects beta cell function,
insulin metabolism, and/or glucagon deposition.
24. The method of claim 22, wherein the metabolic disorder is
obesity, and the env-influencer affects beta cell oxidation in the
mitochondria, decrease in adipocyte size, and/or control of
cortisol levels.
25. The method of claim 22, wherein the metabolic disorder is a
cardiovascular disease, and the env-influencer affects decrease in
smooth muscle cell proliferation in the tunica media, lipid
peroxidation, thromboxane-ax2 synthesis, TNF.alpha., IL-1B,
platelet aggregation, decrease in nitric oxide (NO) production,
plaque deposition and/or normalized glycemic control.
26. The method of claim 22, wherein said key elements of a
metabolic disorder is selected from the group consisting of
impaired fasting glucose, impaired glucose tolerance, increased
waist circumference, increased visceral fat content, increased
fasting plasma glucose, increased fasting plasma triglycerides,
decreased fasting high density lipoprotein level, increased blood
pressure, insulin resistance, hyperinsulinemia, cardiovascular
disease, arteriosclerosis, coronary artery disease, peripheral
vascular disease, cerebrovascular disease, congestive heart
failure, elevated plasma norepinephrine, elevated
cardiovascular-related inflammatory factors, elevated plasma
factors potentiating vascular endothelial dysfunction,
hyperlipoproteinemia, arteriosclerosis or atherosclerosis,
hyperphagia, hyperglycemia, hyperlipidemia, and hypertension or
high blood pressure, increased plasma postprandial triglyceride or
free fatty acid levels, increased cellular oxidative stress or
plasma indicators thereof, increased circulating hypercoagulative
state, hepatic steatosis, hetaptic steatosis, renal disease
including renal failure and renal insufficiency.
27. The method of claim 1, further comprising administering an
additional therapeutic agent.
28. The method of claim 27, wherein the additional therapeutic
agent is selected from the group consisting of diabetes
mellitus-treating agents, diabetic complication treating agents,
anti-hyperlipemic agents, hypotensive or antihypertensive agents,
anti-obesity agents, diuretics, chemotherapeutic agents,
immunotherapeutic agents and immunosuppressive agents.
29. A method for selectively augmenting mitochondrial oxidative
phosphorylation, in a disease cell of a mammal in need of treatment
for a metabolic disorder, the method comprising: administering to
said mammal a therapeutically effective amount of a pharmaceutical
composition comprising at least one env-influencer, thereby
selectively augmenting mitochondrial oxidative phosphorylation in
said disease cell of the mammal.
30. The method of claim 29, further comprising up-regulating the
expression of one or more genes selected from the group consisting
of the molecules listed in Tables 2-4 & 6-28 & 63-68 having
a positive fold change; and/or down-regulating the expression of
one or more genes selected from the group consisting of the
molecules listed in Tables 2-4 and 6-28 & 63-68 having a
negative fold change.
31. The method of claim 29, further comprising modulating the
expression of one or more genes selected from the group consisting
of HNF4-alpha, Bcl-xl, Bcl-xS, BNIP-2, Bcl-2, Birc6, Bcl-2-L11,
XIAP, 20 BRAF, Bax, c-Jun, Bmf, PUMA, cMyc, transaldolase 1, COQ1,
COQ3, COQ6, prenyltransferase, 4-hydrobenzoate, neutrophil
cytosolic factor 2, nitric oxide synthase 2A, superoxide dismutase
2, VDAC, Bax channel, ANT, Cytochrome c, complex 1, complex II,
complex III, complex IV, Foxo 3a, DJ-1, IDH-1, Cpt1C and Cam Kinase
II.
32. The method of anyone of claims 29-31, wherein the metabolic
disorder is selected from the group consisting of diabetes,
obesity, pre-diabetes, Metabolic Syndrome and any key elements of a
metabolic disorder.
33. The method of anyone of claims 29-31, wherein said key elements
of a metabolic disorder is selected from the group consisting of
impaired fasting glucose, impaired glucose tolerance, increased
waist circumference, increased visceral fat content, increased
fasting plasma glucose, increased fasting plasma triglycerides,
decreased fasting high density lipoprotein level, increased blood
pressure, insulin resistance, hyperinsulinemia, cardiovascular
disease, arteriosclerosis, coronary artery disease, peripheral
vascular disease, cerebrovascular disease, congestive heart
failure, elevated plasma norepinephrine, elevated
cardiovascular-related inflammatory factors, elevated plasma
factors potentiating vascular endothelial dysfunction,
hyperlipoproteinemia, arteriosclerosis or atherosclerosis,
hyperphagia, hyperglycemia, hyperlipidemia, and hypertension or
high blood pressure, increased plasma postprandial triglyceride or
free fatty acid levels, increased cellular oxidative stress or
plasma indicators thereof, increased circulating hypercoagulative
state, hepatic steatosis, hetaptic steatosis, renal disease
including renal failure and renal insufficiency.
34. The method of claim 29, further comprising administering an
additional therapeutic agent.
35. The method of claim 34, wherein the additional therapeutic
agent is selected from the group consisting of diabetes
mellitus-treating agents, diabetic complication treating agents,
antihyperlipemic agents, hypotensive or antihypertensive agents,
antiobesity agents, diuretics, chemotherapeutic agents,
immunotherapeutic agents and immunosuppressive agents.
36. A method of identifying an agent that is effective in treating
a metabolic disorder, the method comprising: (1) selecting an
environmental influencer; (2) identifying an environmental
influencer capable of shifting the metabolic state of a cell; and
(3) determining whether the environmental influencer is effective
in treating the metabolic disorder; thereby identifying an agent
that is effective in treating a metabolic disorder.
37. The method of claim 36, wherein an environmental influencer is
identified as capable of shifting the metabolic state of a cell by
measuring changes in any one or more of mRNA expression, protein
expression, lipid or metabolite concentration, levels of
bioenergetic molecules, cellular energetics, mitochondrial function
and mitochondrial number.
38. The method of claim 36, wherein an environmental influencer
effective in treating a metabolic disorder is capable of reducing
glucose levels or lipid levels in a patient.
39. A composition comprising an agent identified according to the
method of any one of claims 36-38.
40. A kit comprising the composition of claim 39.
41. A method of reducing glucose levels in a patient comprising
administering to the patient an effective amount of the composition
of claim 39.
42. A method of reducing lipid levels in a patient comprising
administering to the patient an effective amount of the composition
of claim 39.
43. A method for treating, alleviating symptoms of, inhibiting
progression of, or preventing a Coenzyme Q10 responsive disorder in
a mammal, the method comprising: administering to the mammal in
need thereof a therapeutically effective amount of pharmaceutical
composition comprising at least one environmental influencer
(env-influencer), wherein the environmental influencer selectively
elicits, in a disease cell of the mammal, a cellular metabolic
energy shift towards levels of glycolysis and mitochondrial
oxidative phosphorylation observed in a normal cell of the mammal
under normal physiological conditions.
44. The method of claim 43, wherein the Coenzyme Q10 responsive
disorder is a metabolic disorder.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 61/177,241, filed May 11, 2009, entitled "Methods
for Treatment of Oncological Disorders Using an Epimetabolic
Shifter (Coenzyme Q10)" (Attorney Docket No.: 117732-00601), U.S.
Provisional Application No. 61/177,243, filed May 11, 2009,
entitled "Methods for Treatment of Oncological Disorders Using
Epimetabolic Shifters, Multidimensional Intracellular Molecules or
Environmental Influencers" (Attorney Docket No.: 117732-00701),
U.S. Provisional Application No. 61/177,244, filed May 11, 2009,
entitled "Methods for the Diagnosis of Oncological Disorders Using
Epimetabolic Shifters, Multidimensional Intracellular Molecules or
Environmental Influencers" (Attorney Docket No.: 117732-00801),
U.S. Provisional Application No. 61/177,245, filed May 11, 2009,
entitled "Methods for Treatment of Metabolic Disorders Using
Epimetabolic Shifters, Multidimensional Intracellular Molecules or
Environmental Influencers" (Attorney Docket No.: 117732-00901), and
U.S. Provisional Application No. 61/177,246, filed May 11, 2009,
entitled "Methods for the Diagnosis of Metabolic Disorders Using
Epimetabolic Shifters, Multidimensional Intracellular Molecules or
Environmental Influencers" (Attorney Docket No.: 117732-01001), the
entire contents of each of the aforementioned applications are
hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The invention relates to the treatment, prevention, and
reduction of metabolic disorders, such as diabetes and obesity.
[0003] As the levels of blood glucose rise postprandially, insulin
is secreted and stimulates cells of the peripheral tissues
(skeletal muscles and fat) to actively take up glucose from the
blood as a source of energy. Loss of glucose homeostasis as a
result of dysregulated insulin secretion or action typically
results in metabolic disorders such as diabetes, which may be
co-triggered or further exacerbated by obesity. Because these
conditions are often fatal, strategies to restore adequate glucose
clearance from the bloodstream are required.
[0004] Although diabetes may arise secondary to any condition that
causes extensive damage to the pancreas (e.g., pancreatitis,
tumors, administration of certain drugs such as corticosteroids or
pentamidine, iron overload (e.g., hemochromatosis), acquired or
genetic endocrinopathies, and surgical excision), the most common
forms of diabetes typically arise from primary disorders of the
insulin signaling system. There are two major types of diabetes,
namely type 1 diabetes (also known as insulin dependent diabetes
(IDDM)) and type 2 diabetes (also known as insulin independent or
non-insulin dependent diabetes (NIDDM)), which share common
long-term complications in spite of their different pathogenic
mechanisms.
[0005] Type 1 diabetes, which accounts for approximately 10% of all
cases of primary diabetes, is an organ-specific autoimmune disease
characterized by the extensive destruction of the insulin-producing
beta cells of the pancreas. The consequent reduction in insulin
production inevitably leads to the deregulation of glucose
metabolism. While the administration of insulin provides
significant benefits to patients suffering from this condition, the
short serum half-life of insulin is a major impediment to the
maintenance of normoglycemia. An alternative treatment is islet
transplantation, but this strategy has been associated with limited
success.
[0006] Type 2 diabetes, which affects a larger proportion of the
population, is characterized by a deregulation in the secretion of
insulin and/or a decreased response of peripheral tissues to
insulin, i.e., insulin resistance. While the pathogenesis of type 2
diabetes remains unclear, epidemiologic studies suggest that this
form of diabetes results from a collection of multiple genetic
defects or polymorphisms, each contributing its own predisposing
risks and modified by environmental factors, including excess
weight, diet, inactivity, drugs, and excess alcohol consumption.
Although various therapeutic treatments are available for the
management of type 2 diabetes, they are associated with various
debilitating side effects. Accordingly, patients diagnosed with or
at risk of having type 2 diabetes are often advised to adopt a
healthier lifestyle, including loss of weight, change in diet,
exercise, and moderate alcohol intake. Such lifestyle changes,
however, are not sufficient to reverse the vascular and organ
damages caused by diabetes.
[0007] Coenzyme Q10, also referred to herein as CoQ10, Q10,
ubiquinone, or ubidecarenone, is a popular nutritional supplement
and can be found in capsule form in nutritional stores, health food
stores, pharmacies, and the like, as a vitamin-like supplement to
help protect the immune system through the antioxidant properties
of ubiquinol, the reduced form of CoQ10. CoQ10 is art-recognized
and further described in International Publication No. WO
2005/069916, the entire disclosure of which is incorporated by
reference herein.
[0008] CoQ10 is found throughout most tissues of the human body and
the tissues of other mammals. The tissue distribution and redox
state of CoQ10 in humans has been reviewed in a review article by
Bhagavan and Chopra (2006 Free Radical Research 40(5):445-453). The
authors report that "as a general rule, tissues with high-energy
requirements or metabolic activity such as the heart, kidney, liver
and muscle contain relatively high concentrations of CoQ10." The
authors further report that "[a] major portion of CoQ10 in tissues
is in the reduced form as the hydroquinone or uniquinol, with the
exception of brain and lungs," which "appears to be a reflection of
increased oxidative stress in these two tissues." In particular,
Bhagavan report that in heart, kidney, liver, muscle, intenstine
and blood (plasma), about 61%, 75%, 95%, 65%, 95% and 96%,
respectively, of CoQ10 is in the reduced form. Similarly,
Ruiz-Jiminez et al. (2007 J. Chroma A, 1175, 242-248) report that
when human plasma was evaluated for Q10 and the reduced form of Q10
(Q10H2), the majority (90%) of the molecule was found in the
reduced form.
[0009] CoQ10 is very lipophilic and, for the most part, insoluble
in water. CoQ10 is very lipophilic and, for the most part,
insoluble in water. Due to its insolubility in water, limited
solubility in lipids, and relatively large molecular weight, the
efficiency of absorption of orally administered CoQ10 is poor.
Bhagavan and Chopra report that "in one study with rats it was
reported that only about 2-3% of orally-administered CoQ10 was
absorbed." Bhagavan and Chopra further report that "[d]ata from rat
studies indicate that CoQ10 is reduced to ubiquinol either during
or following absorption in the intestine."
[0010] Given that the strategies currently available for the
management of diabetes are suboptimal, there is a compelling need
for treatments that are more effective and are not associated with
such debilitating side-effects.
SUMMARY OF THE INVENTION
[0011] The present invention is partly based on the finding that
mitochondrial dysfunction is associated with a wide range of
diseases, including metabolic diseases (such as diabetes and
obesity), and that certain endogenous molecules, such as CoQ10,
hold the key to the successful diagnosis, treatment, and prevention
of such metabolic diseases. The invention is also partly based on
the finding that these key endogenous molecules play important
roles in maintaining normal mitochondrial function by directly
influencing oxidative phosphorylation, and that restoring or
promoting more normalized mitochondrial osidative phosphorylation
can effectively treat or prevent the progression of metabolic
diseases. The invention is further based on the discovery that a
class of environmental enfluencers (e.g., CoQ10) can selectively
elicit, in disease cells of the metabolic diseases, a cellular
metabolic energy shift towards more normalized mitochondrial
oxidative phosphorylation. These environmental influencers are
capable of modulating intracellular targets that serve as key
indices of metabolic disorders (such as diabetes), in a manner
representative of therapeutic endpoints.
[0012] The present invention is further based, at least in part, on
the discovery that application of endogenous Coenzyme Q10 (also
referred to as CoQ10 or Q10 herein) to cells results in an
apoptotic response. The apoptotic response is preferentially
induced in cancer cells. A time and dose response of mitochondrial
Q10 levels was observed, wherein after 48 hours, the level of Q10
in cell mitochondria was increased by six fold. The invention is
further based on the surprising and unexpected discovery that the
Q10 is maintained in the supplied oxidized form (pro-oxidant) and
not converted to the reduced (anti-oxidant) form of Q10H2 in any
significant amounts. The invention is based on the further
discovery that a significant number of proteins and mRNA levels are
modulated in cells treated with Q10. These modulated proteins were
found to be clustered into several cellular pathways, including
apoptosis, cancer biology and cell growth, glycolysis and
metabolism, molecular transport, and cellular signaling.
[0013] Applicants' data described herein has provided insight into
the mechanism of action of Q10. In particular, while not wishing to
be bound by theory, Applicants' discoveries indicate that Q10
induces a metabolic shift to the cell microenvironment. Many
diseases are known to be associeated with an altered metabolic
state. For example, differential metabolism is known to occur in
cancer cells (the Warurg effect), whereby most cancer cells
predominantly produce energy by glycolysis followed by lactic acid
fermentation in the cytosol, rathe than by oxidative
phosphorylation (oxidation of pyruvate) in the mitochondria. In
another example, metabolic disorders, such as diabetes and obesity,
are associated with an altered glucose metabolism.
[0014] Accordingly, the invention provides, in a first aspect, a
method for treating, alleviating symptoms of, inhibiting
progression of, or preventing a CoQ10 responsive disorder in a
mammal, the method comprising: administering to the mammal in need
thereof a therapeutically effective amount of pharmaceutical
composition comprising at least one environmental influencer
(env-influencer), wherein the environmental influencer selectively
elicits, in a disease cell of the mammal, a cellular metabolic
energy shift towards levels of glycolysis and mitochondrial
oxidative phosphorylation observed in a normal cell of the mammal
under normal physiological conditions.
[0015] In one embodiment, the CoQ10 responsive disorder is a
metabolic disorder.
[0016] The invention provides, in another aspect, a method for
treating, alleviating symptoms of, inhibiting progression of, or
preventing a metabolic disorder in a mammal, the method comprising
administering to the mammal in need thereof a therapeutically
effective amount of a pharmaceutical composition comprising at
least one environmental influencer (env-influencer), wherein the
environmental influencer selectively elicits, in a disease cell of
the mammal, a cellular metabolic energy shift towards normalized
mitochondrial oxidative phosphorylation.
[0017] In one embodiment, the environmental influencer does not
substantially elicit, in normal cells of the mammal, the cellular
metabolic energy shift towards mitochondrial oxidative
phosphorylation.
[0018] In one embodiment, the mammal is human (or a non-human
mammal).
[0019] In one embodiment, the metabolic disorder is responsive or
sensitive to treatment by Coenzyme Q10 or its metabolites or
analogs thereof.
[0020] In one embodiment, the metabolic disorder is characterized
by a dysregulated mitochondrial oxidative phosphorylation function
that leads to altered gene regulation and/or protein-protein
interactions which contribute to or causally lead to the metabolic
disease.
[0021] In one embodiment, the environmental influencer comprises
(a) benzoquinone or at least one molecule that facilitates the
biosynthesis of the benzoquinone ring, and (b) at least one
molecule that facilitates the synthesis of and/or attachment of
isoprenoid units to the benzoquinone ring.
[0022] In one embodiment, said at least one molecule that
facilitates the biosynthesis of the benzoquinone ring comprises:
L-Phenylalanine, DL-Phenylalanine, D-Phenylalanine, L-Tyrosine,
DL-Tyrosine, D-Tyrosine, 4-hydroxy-phenylpyruvate,
3-methoxy-4-hydroxymandelate (vanillylmandelate or VMA), vanillic
acid, pyridoxine, or panthenol.
[0023] In one embodiment, said at least one molecule that
facilitates the synthesis of and/or attachment of isoprenoid units
to the benzoquinone ring comprises: phenylacetate,
4-hydroxy-benzoate, mevalonic acid, acetylglycine, acetyl-CoA, or
farnesyl.
[0024] In one embodiment, the environmental influencer comprises
(a) one or more of L-Phenylalanine, L-Tyrosine, and
4-hydroxyphenylpyruvate; and (b) one or more of 4-hydroxy benzoate,
phenylacetate, and benzoquinone.
[0025] In one embodiment, the environmental influencer: (a)
inhibits Bcl-2 expression and/or promotes Caspase-3 expression;
and/or (b) inhibits cell proliferation.
[0026] In one embodiment, the environmental influencer is a
multidimensional intracellular molecule (MIM). In one embodiment,
the MIM is selected from: alpha ketoglutarate/alpha ketoglutaric
acid, Malate/Malic acid, Succinate/Succinic acid, Glucosamine,
Adenosine, Adenosine Diphosphate, Glucuronide/Glucuronic acid,
Nicotinic Acid, Nicotinic Acid Dinucleotide, Alanine/Phenylalanine,
Pyridoxine, Thiamine, or Flavin Adenine Dinucleotide. In one
embodiment, the multidimensional intracellular molecule is selected
from the group consisting of acetyl Co-A, palmityl Co-A,
L-carnitine, and amino acids, e.g., tyrosine, phenylalanine, and
cysteine.
[0027] In one embodiment, the environmental influencer is an
epimetabolic shifter (epi-shifter). In one embodiment, the
epimetabolic shifter is selected from Transaldolase, Transketolase,
Succinyl CoA synthase, Pyruvate Carboxylase, or Riboflavin. In one
embodiment, the epimetabolic shifter is selected from the group
consisting of coenzyme Q10, vitamin D3 and extracellular matrix
components. In one embodiment, the epimetabolic shifter is coenzyme
Q10. In one embodiment, the extracellular matrix components are
selected from the group consisting of fibronectin, immunomodulators
(e.g., TNF.alpha. or an interleukin), angiogenic factors, and
apoptotic factors.
[0028] In one embodiment, a population of humans are treated and at
least 25% of the population had a systemic environmental influencer
level that was therapeutic for the disorder being treated. In other
embodiments, a population of humans are treated and at least 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95% or more of the population had a systemic
Coenzyme Q10 level that was therapeutic for the disorder being
treated. It should be understood that ranges having any one of
these values as the upper or lower limits are also intended to be
part of this invention, e.g., 10% to 25%, 15% to 35%, 25% to 50%,
35% to 60%, 40% to 70%, 50% to 75%, 60% to 85% or 70% to 90%.
[0029] In one embodiment, the metabolic disorder being treated is
not a disorder typically treated via topical administration with
the expectation of systemic delivery of an active agent at
therapeutically effective levels.
[0030] In one embodiment, the concentration of the environmental
influencer in the tissues of the human being treated is different
than that of a control standard of human tissue representative of a
healthy or normal state.
[0031] In one embodiment, the form of the environmental influencer
administered to the human is different than the predominant form
found in systemic circulation in the human. In one embodiment, the
environmental influencer is administered to the human in oxidized
form.
[0032] In one embodiment, the amount sufficient to treat the
metabolic disorder in the human up-regulates or down-regulates
mitochondrial oxidative phosphorylation.
[0033] In one embodiment, the amount sufficient to treat the
metabolic disorder in the human modulates anaerobic use of glucose
and/or lactate biosynthesis.
[0034] The invention provides, in another aspect, a method for
treating or preventing a metabolic disorder in a human, comprising
administering an environmental influencer to the human in an amount
sufficient to treat or prevent the metabolic disorder, wherein the
environmental influencer is administered such that it is maintained
in its oxidized form during treatment, thereby treating or
preventing the metabolic disorder.
[0035] In one embodiment, the form of the environmental influencer
administered to the human is different than the predominant form
found in systemic circulation in the human.
[0036] The invention provides, in still another aspect, a method
for treating or preventing a metabolic disorder in a human
comprising selecting a human subject suffering from a metabolic
disorder; and administering to said human a therapeutically
effective amount of an env-influencer capabable of augmenting
mitochondrial oxidative phosphorylation and, optionally, blocking
anaerobic use of glucose, thereby treating or preventing the
metabolic disorder.
[0037] The invention provides, in another aspect, a method for
selectively augmenting mitochondrial oxidative phosphorylation, in
a disease cell of a mammal in need of treatment for a metabolic
disorder, the method comprising: administering to said mammal a
therapeutically effective amount of a pharmaceutical composition
comprising at least one env-influencer, thereby selectively
augmenting mitochondrial oxidative phosphorylation in said disease
cell of the mammal.
[0038] In one embodiment of the methods of the invention, the
method further comprises upregulating the expression of one or more
genes selected from the group consisting of the molecules listed in
Tables 2-4 & Tables 6-28 & Tables 63-68 with a positive
fold change; and/or downregulating the expression of one or more
genes selected from the group consisting of the molecules listed in
Tables 2-4 & Tables 6-28 & Tables 63-68 with a negative
fold change, thereby treating or preventing the metabolic disorder.
In one embodiment, the method further comprises modulating the
expression of one or more genes selected from the group consisting
of HNF4-alpha, Bcl-x1, Bcl-xS, BNIP-2, Bcl-2, Birc6, Bcl-2-L11,
XIAP, BRAF, Bax, c-Jun, Bmf, PUMA, cMyc, transaldolase 1, COQ1,
COQ3, COQ6, prenyltransferase, 4-hydrobenzoate, neutrophil
cytosolic factor 2, nitric oxide synthase 2A, superoxide dismutase
2, VDAC, Bax channel, ANT, Cytochrome c, complex 1, complex II,
complex III, complex IV, Foxo 3a, DJ-1, IDH-1, Cpt1C and Cam Kinase
II.
[0039] In one embodiment of the methods of the invention, the
treatment occurs via an interaction of the environmental influencer
with a molecule selected from the group consisting of the molecules
listed in Tables 2-4 & 6-28 & 63-68. In one embodiment, the
treatment occurs via an interaction of the environmental influencer
with a protein selected from the group consisting of HNF4-alpha,
Bcl-xl, Bcl-xS, BNIP-2, Bcl-2, Birc6, Bcl-2-L11 (Bim), XIAP, BRAF,
Bax, c-Jun, Bmf, PUMA, cMyc, transaldolase 1, COQ1, COQ3, COQ6,
prenyltransferase, 4-hydrobenzoate, neutrophil cytosolic factor 2,
nitric oxide synthase 2A, superoxide dismutase 2, VDAC, Bax
channel, ANT, Cytochrome c, complex 1, complex II, complex III,
complex IV, Foxo 3a, DJ-1, IDH-1, Cpt1C and Cam Kinase II. In one
embodiment, the treatment occurs via an interaction of the
env-influencer with HNF4alpha. In one embodiment, the treatment
occurs via an interaction of the env-influencer with
transaldolase.
[0040] In one embodiment of the methods of the invention, the
metabolic disorder is selected from the group consisting of
diabetes, obesity, pre-diabetes, Metabolic Syndrome and any key
elements of a metabolic disorder.
[0041] In one embodiment, the metabolic disorder is diabetes, and
the env-influencer affects beta cell function, insulin metabolism,
and/or glucagon deposition.
[0042] In one embodiment, the metabolic disorder is obesity, and
the env-influencer affects beta cell oxidation in the mitochondria,
decrease in adipocyte size, and/or control of cortisol levels.
[0043] In one embodiment, the metabolic disorder is a
cardiovascular disease, and the env-influencer affects decrease in
smooth muscle cell proliferation in the tunica media, lipid
peroxidation, thromboxane-ax2 synthesis, TNF.alpha., IL-1B,
platelet aggregation, decrease in nitric oxide (NO) production,
plaque deposition and/or normalized glycemic control.
[0044] In one embodiment, key elements of a metabolic disorder
include impaired fasting glucose, impaired glucose tolerance,
increased waist circumference, increased visceral fat content,
increased fasting plasma glucose, increased fasting plasma
triglycerides, decreased fasting high density lipoprotein level,
increased blood pressure, insulin resistance, hyperinsulinemia,
cardiovascular disease, arteriosclerosis, coronary artery disease,
peripheral vascular disease, cerebrovascular disease, congestive
heart failure, elevated plasma norepinephrine, elevated
cardiovascular-related inflammatory factors, elevated plasma
factors potentiating vascular endothelial dysfunction,
hyperlipoproteinemia, arteriosclerosis or atherosclerosis,
hyperphagia, hyperglycemia, hyperlipidemia, and hypertension or
high blood pressure, increased plasma postprandial triglyceride or
free fatty acid levels, increased cellular oxidative stress or
plasma indicators thereof, increased circulating hypercoagulative
state, hepatic steatosis, hetaptic steatosis, renal disease
including renal failure and renal insufficiency.
[0045] In one embodiment of the methods of the invention, the
method further comprises administering an additional therapeutic
agent, e.g., diabetes mellitus-treating agents, diabetic
complication-treating agents, antihyperlipemic agents, hypotensive
or antihypertensive agents, anti-obesity agents, diuretics,
chemotherapeutic agents, immunotherapeutic agents and
immunosuppressive agents. In one embodiment, the metabolic disorder
is selected from the group consisting of diabetes, obesity,
pre-diabetes, Metabolic Syndrome and any key elements of a
metabolic disorder. In one embodiment, a key element of a metabolic
disorder is selected from the group consisting of impaired fasting
glucose, impaired glucose tolerance, increased waist circumference,
increased visceral fat content, increased fasting plasma glucose,
increased fasting plasma triglycerides, decreased fasting high
density lipoprotein level, increased blood pressure, insulin
resistance, hyperinsulinemia, cardiovascular disease,
arteriosclerosis, coronary artery disease, peripheral vascular
disease, cerebrovascular disease, congestive heart failure,
elevated plasma norepinephrine, elevated cardiovascular-related
inflammatory factors, elevated plasma factors potentiating vascular
endothelial dysfunction, hyperlipoproteinemia, arteriosclerosis or
atherosclerosis, hyperphagia, hyperglycemia, hyperlipidemia, and
hypertension or high blood pressure, increased plasma postprandial
triglyceride or free fatty acid levels, increased cellular
oxidative stress or plasma indicators thereof, increased
circulating hypercoagulative state, hepatic steatosis, hetaptic
steatosis, renal disease including renal failure and renal
insufficiency.
[0046] In one embodiment of the methods of the invention, the
method further comprises administering an additional therapeutic
agent, e.g., diabetes mellitus-treating agents, diabetic
complication-treating agents, antihyperlipemic agents, hypotensive
or antihypertensive agents, anti-obesity agents, diuretics,
chemotherapeutic agents, immunotherapeutic agents and
immunosuppressive agents.
[0047] The invention provides, in another aspect, a method of
identifying an agent that is effective in treating a metabolic
disorder, the method comprising selecting an environmental
influencer; identifying an environmental influencer capable of
shifting the metabolic state of a cell; and determining whether the
environmental influencer is effective in treating the metabolic
disorder; thereby identifying an agent that is effective in
treating a metabolic disorder.
[0048] In one embodiment, an environmental influencer is identified
as capable of shifting the metabolic state of a cell by measuring
changes in any one or more of mRNA expression, protein expression,
lipid or metabolite concentration, levels of bioenergetic
molecules, cellular energetics, mitochondrial function and
mitochondrial number.
[0049] In one embodiment, the environmental influencer effective in
treating a metabolic disorder is capable of reducing glucose levels
or lipid levels in a patient.
[0050] The invention provides, in still another aspect, a method of
identifying a Multidimensional Intracellular Molecule, comprising
contacting a cell with an endogenous molecule; monitoring the
effect of the endogenous molecule on a cellular microenvironment
profile; and identifying an endogenous molecule that induces a
change to the cellular microenvironment profile; thereby
identifying a Multidimensional Intracellular Molecule.
[0051] In one embodiment, the method further comprises comparing
the effects of the endogenous molecule on the cellular
microenvironment profile of a diseased cell and a normal control
cell; and identifying an endogenous molecule that differentially
induces a change to the cellular microenvironment profile of the
diseased cell as compared to the normal control cell; thereby
identifying a MIM.
[0052] In one embodiment, the effect on the cellular
microenvironment profile is monitored by measuring a change in the
level or activity of a cellular molecule selected from the group
consisting of mRNA, protein, lipid and metabolite.
[0053] The invention provides, in still another aspect, a method of
identifying an Epimetabolic shifter, comprising comparing molecular
profiles for two or more cells or tissues, wherein the two or more
cells or tissues display differential disease states; identifying a
molecule from the moleculer profiles for which a change in level
correlates to the disease state; introducing the molecule to a
cell; and evaluating the ability of the molecule to shift the
metabolic state of a cell, wherein a molecule capable of shifting
the metabolic state of a cell is identified as an Epimetabolic
shifter.
[0054] In one embodiment, the molecular profile is selected from
the group consisting of a metabolite profile, lipid profile,
protein profile or RNA profile.
[0055] In one embodiment, the molecule does not negatively effect
the health or growth of a normal cell.
[0056] The invention provides, in another aspect, a composition
comprising an agent identified according to any of the methods of
the invention. The invention further provides, in a related aspect,
a kit comprising a composition of the invention.
[0057] The invention provides, in another aspect, a method of
reducing glucose levels in a patient comprising administering to
the patient an effective amount of a composition of the invention.
The invention provides, in a related aspect, a method of reducing
lipid levels in a patient comprising administering to the patient
an effective amount of a composition of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] FIG. 1: Sensitivity of SK-MEL-28 to 24 hours of Q10
treatment measured by the amount of early and late apoptotic
cells.
[0059] FIG. 2: Sensitivity of SKBR3 to 24 hours of Q10 treatment
measured by the amount of early and late apoptotic cells.
[0060] FIG. 3: Sensitivity of PaCa2 to 24 hours of Q10 treatment
measured by the amount of early and late apoptotic cells.
[0061] FIG. 4: Sensitivity of PC-3 to 24 hours of Q10 treatment
measured by the amount of early and late apoptotic cells.
[0062] FIG. 5: Sensitivity of HepG2 to 24 hours of Q10 treatment
measured by the amount of early and late apoptotic cells.
[0063] FIG. 6: Sensitivity of MCF-7 to 24 hours of Q10 treatment
measured by the amount of early and late apoptotic cells.
[0064] FIG. 7: Measurement of apoptotic cells upon 24 hour
treatment with Q10, as measured by Apostrand ELISA method.
[0065] FIG. 8: Example gel analysis of 2-D gel electrophoresis.
Spots excised for identification are marked.
[0066] FIG. 9: Network of interaction between proteins identified
by 2-D gel electrophoresis as being modulated by Q10 in SK-MEL-28
cells.
[0067] FIG. 10: The pentose phosphate pathway adapted from
Verhoeven et al. (Am. J. Hum. Genet. 2001 68(5):1086-1092).
[0068] FIG. 11: 2-D gel of the mitochondrial enriched material of
SK-MEL-28 cells. Spots excised and identified by mass spectrometry
characterization are marked.
[0069] FIG. 12: Comparative plot of the relative amounts of Q10
present in SK-MEL-28 mitochondria following the exogenous addition
of 100 .mu.M Q10 into the culture medium.
[0070] FIG. 13: Apoptosis pathway mapping known processes.
[0071] FIG. 14: Western blot analysis of Bcl-x1.
[0072] FIG. 15: Western blot analysis of SK-MEL-28 sample set
proved with a Vimentin antibody.
[0073] FIG. 16: Western blot analysis of cell lysis from a number
of cell lines, evaluated with five antibodies targeting oxidative
phosphorylation complexes (MitoSciences #MS601).
[0074] FIG. 17: Western blot comparison of F1-alpha levels.
[0075] FIG. 18: Western blot comparison of Q10 response with
C-III-Core 2.
[0076] FIG. 19: Western blot comparison of Q10 response with
C-II-30.
[0077] FIG. 20: Western blot comparison of Q10 response with
C-IV-COX II.
[0078] FIG. 21: Western blot comparison of Q10 response with C-1-20
(ND6).
[0079] FIG. 22: Western blot analysis of a variety of cell types
against five mitochondrial protein.
[0080] FIG. 23: Western blot comparison of Q10 response with
Complex V protein C-V-.alpha..
[0081] FIG. 24: Western blot comparison of Q10 response with
C-III-Core 1.
[0082] FIG. 25: Western blot comparison of Q10 response with Porin
(VDAC1).
[0083] FIG. 26: Western blot comparison of Q10 response with
Cyclophilin D
[0084] FIG. 27: Western blot comparison of Q10 response with
Cytochrome C.
[0085] FIG. 28: Theoretical model of Q10 (spheres) inserted into
the lipid binding channel of HNF4alpha (1M7W.pdb) in the Helix 10
open conformation.
[0086] FIG. 29: OCR in HDFa cells in various glucose conditions in
normoxic and hypoxic conditions.
[0087] FIG. 30: OCR in HASMC cells in various glucose conditions in
normoxic and hypoxic conditions.
[0088] FIG. 31: OCR values in MCF-7 breast cancer cells in the
absence and presence of 31510 and stressors.
[0089] FIG. 32: OCR values in PaCa-2 pancreatic cancer cells in the
absence and presence of 31510 and stressors.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0090] As used herein, each of the following terms has the meaning
associated with it in this section.
[0091] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0092] The term "including" is used herein to mean, and is used
interchangeably with, the phrase "including but not limited
to".
[0093] The term "or" is used herein to mean, and is used
interchangeably with, the term "and/or," unless context clearly
indicates otherwise.
[0094] The term "such as" is used herein to mean, and is used
interchangeably, with the phrase "such as but not limited to".
[0095] A "patient" or "subject" to be treated by the method of the
invention can mean either a human or non-human animal, preferably a
mammal.
[0096] "Therapeutically effective amount" means the amount of a
compound that, when administered to a patient for treating a
disease, is sufficient to effect such treatment for the disease.
When administered for preventing a disease, the amount is
sufficient to avoid or delay onset of the disease. The
"therapeutically effective amount" will vary depending on the
compound, the disease and its severity and the age, weight, etc.,
of the patient to be treated.
[0097] "Preventing" or "prevention" refers to a reduction in risk
of acquiring a disease or disorder (i.e., causing at least one of
the clinical symptoms of the disease not to develop in a patient
that may be exposed to or predisposed to the disease but does not
yet experience or display symptoms of the disease).
[0098] The term "prophylactic" or "therapeutic" treatment refers to
administration to the subject of one or more of the subject
compositions. If it is administered prior to clinical manifestation
of the unwanted condition (e.g., disease or other unwanted state of
the host animal) then the treatment is prophylactic, i.e., it
protects the host against developing the unwanted condition,
whereas if administered after manifestation of the unwanted
condition, the treatment is therapeutic (i.e., it is intended to
diminish, ameliorate or maintain the existing unwanted condition or
side effects therefrom).
[0099] The term "therapeutic effect" refers to a local or systemic
effect in animals, particularly mammals, and more particularly
humans caused by a pharmacologically active substance. The term
thus means any substance intended for use in the diagnosis, cure,
mitigation, treatment or prevention of disease or in the
enhancement of desirable physical or mental development and
conditions in an animal or human. The phrase
"therapeutically-effective amount" means that amount of such a
substance that produces some desired local or systemic effect at a
reasonable benefit/risk ratio applicable to any treatment. In
certain embodiments, a therapeutically-effective amount of a
compound will depend on its therapeutic index, solubility, and the
like. For example, certain compounds discovered by the methods of
the present invention may be administered in a sufficient amount to
produce a reasonable benefit/risk ratio applicable to such
treatment.
[0100] By "patient" is meant any animal (e.g., a human), including
horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea
pigs, rats, mice, lizards, snakes, sheep, cattle, fish, and
birds.
[0101] "Metabolic pathway" refers to a sequence of enzyme-mediated
reactions that transform one compound to another and provide
intermediates and energy for cellular functions. The metabolic
pathway can be linear or cyclic.
[0102] "Metabolic state" refers to the molecular content of a
particular cellular, multicellular or tissue environment at a given
point in time as measured by various chemical and biological
indicators as they relate to a state of health or disease.
[0103] The term "microarray" refers to an array of distinct
polynucleotides, oligonucleotides, polypeptides (e.g., antibodies)
or peptides synthesized on a substrate, such as paper, nylon or
other type of membrane, filter, chip, glass slide, or any other
suitable solid support.
[0104] The terms "disorders" and "diseases" are used inclusively
and refer to any deviation from the normal structure or function of
any part, organ or system of the body (or any combination thereof).
A specific disease is manifested by characteristic symptoms and
signs, including biological, chemical and physical changes, and is
often associated with a variety of other factors including, but not
limited to, demographic, environmental, employment, genetic and
medically historical factors. Certain characteristic signs,
symptoms, and related factors can be quantitated through a variety
of methods to yield important diagnostic information.
[0105] The term "expression" is used herein to mean the process by
which a polypeptide is produced from DNA. The process involves the
transcription of the gene into mRNA and the translation of this
mRNA into a polypeptide. Depending on the context in which used,
"expression" may refer to the production of RNA, protein or
both.
[0106] The terms "level of expression of a gene" or "gene
expression level" refer to the level of mRNA, as well as pre-mRNA
nascent transcript(s), transcript processing intermediates, mature
mRNA(s) and degradation products, or the level of protein, encoded
by the gene in the cell.
[0107] The term "modulation" refers to upregulation (i.e.,
activation or stimulation), downregulation (i.e., inhibition or
suppression) of a response, or the two in combination or apart. A
"modulator" is a compound or molecule that modulates, and may be,
e.g., an agonist, antagonist, activator, stimulator, suppressor, or
inhibitor.
[0108] The term "intermediate of the coenzyme biosynthesis pathway"
as used herein, characterizes those compounds that are formed
between the chemical/biological conversion of tyrosine and
Acetyl-CoA to uqiquinone. Intermediates of the coenzyme
biosynthesis pathway include 3-hexaprenyl-4-hydroxybenzoate,
3-hexaprenyl-4,5-dihydroxybenzoate,
3-hexaprenyl-4-hydroxy-5-methoxybenzoate,
2-hexaprenyl-6-methoxy-1,4-benzoquinone,
2-hexaprenyl-3-methyl-6-methoxy-1,4-benzoquinone,
2-hexaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone,
3-Octaprenyl-4-hydroxybenzoate, 2-octaprenylphenol,
2-octaprenyl-6-metholxyphenol,
2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinone,
2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone,
2-decaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone,
2-decaprenyl-3-methyl-6-methoxy-1,4-benzoquinone,
2-decaprenyl-6-methoxy-1,4-benzoquinone,
2-decaprenyl-6-methoxyphenol,
3-decaprenyl-4-hydroxy-5-methoxybenzoate,
3-decaprenyl-4,5-dihydroxybenzoate, 3-decaprenyl-4-hydroxybenzoate,
4-hydroxy phenylpyruvate, 4-hydroxyphenyllactate,
4-hydroxy-benzoate, 4-hydroxycinnamate and
hexaprenydiphosphate.
[0109] As used herein, the phrase "anaerobic use of glucose" or
"anaerobic glycolysis" refers to cellular production of energy by
glycolysis followed by lactic acid fermentation in the cytosol. For
example, many cancer cells produce energy by anaerobic
glycolysis.
[0110] As used herein, the phrase "aerobic glycolysis" or
"mitochondrial oxidative phosphorylation" refers to cellular
production of energy by glycolysis followed by oxidation of
pyruvate in mitochondria.
[0111] As used herein, the phrase "capable of blocking anaerobic
use of glucose and augmenting mitochondrial oxidative
phosphorylation" refers to the ability of an environmental
influencer (e.g., an epitmetabolic shifter) to induce a shift or
change in the metabolic state of a cell from anaerobic glycolysis
to aerobic glycolysis or mitochondrial oxidative
phosphorylation.
[0112] Reference will now be made in detail to preferred
embodiments of the invention. While the invention will be described
in conjunction with the preferred embodiments, it will be
understood that it is not intended to limit the invention to those
preferred embodiments. To the contrary, it is intended to cover
alternatives, modifications, and equivalents as may be included
within the spirit and scope of the invention as defined by the
appended claims.
II. Environmental Influencers
[0113] The present invention provides methods of treating metabolic
disorders by administration of an Environmental influencer.
"Environmental influencers" (Env-influencers) are molecules that
influence or modulate the disease environment of a human in a
beneficial manner allowing the human's disease environment to
shift, reestablish back to or maintain a normal or healthy
environment leading to a normal state. Env-influencers include both
Multidimensional Intracellular Molecules (MIMs) and Epimetabolic
shifters (Epi-shifters) as defined below.
1. Multidimensional Intracellular Molecule (MIM)
[0114] The term "Multidimensional Intracellular Molecule (MIM)", is
an isolated version or synthetically produced version of an
endogenous molecule that is naturally produced by the body and/or
is present in at least one cell of a human. A MIM is characterized
by one or more, two or more, three or more, or all of the following
functions. MIMs are capable of entering a cell, and the entry into
the cell includes complete or partial entry into the cell, as long
as the biologically active portion of the molecule wholly enters
the cell. MIMs are capable of inducing a signal transduction and/or
gene expression mechanism within a cell. MIMs are multidimensional
in that the molecules have both a therapeutic and a carrier, e.g.,
drug delivery, effect. MIMs also are multidimensional in that the
molecules act one way in a disease state and a different way in a
normal state. For example, in the case of CoQ-10, administration of
CoQ-10 to a melanoma cell in the presence of VEGF leads to a
decreased level of Bc12 which, in turn, leads to a decreased
oncogenic potential for the melanoma cell. In contrast, in a normal
fibroblast, co-administration of CoQ-10 and VEFG has no effect on
the levels of Bc12. Preferably, MIMs selectively act in cells of a
disease state, and have substantially no effect in (matching) cells
of a normal state. Preferably, MIMs selectively renders cells of a
disease state closer in phenotype, metabolic state, genotype,
mRNA/protein expression level, etc. to (matching) cells of a normal
state.
[0115] In one embodiment, a MIM is also an epi-shifter. In another
embodiment, a MIM is not an epi-shifter. The skilled artisan will
appreciate that a MIM of the invention is also intended to
encompass a mixture of two or more endogenous molecules, wherein
the mixture is characterized by one or more of the foregoing
functions. The endogenous molecules in the mixture are present at a
ratio such that the mixture functions as a MIM.
[0116] MIMs can be lipid based or non-lipid based molecules.
Examples of MIMs include, but are not limited to, CoQ10, acetyl
Co-A, palmityl Co-A, L-carnitine, amino acids such as, for example,
tyrosine, phenylalanine, and cysteine. In one embodiment, the MIM
is a small molecule. In one embodiment of the invention, the MIM is
not CoQ10. MIMs can be routinely identified by one of skill in the
art using any of the assays described in detail herein.
[0117] In some embodiments, MIMs include compounds in the Vitamin B
family, or nucleosides, mononucleotides or dinucleotides that
comprise a compound in the Vitamin B family. Compounds in the
vitamin B family include, for example, thiamine (vitamin B1),
niacin (also known as nicotinic acid or Vitamin B3), or pyridoxine
(vitamin B6) as well as provitamins such as panthenol (provitamin
B5). In some embodiments, the MIM is selected from thiamine, niacin
and pyridoxine. Nucleosides, mononucleotides or dinucleotides that
comprise a compound in the vitamin B family include, for example,
nucleosides, mononucleotides or dinucleotides which include an
adenine or a niacin (nicotinic acid) molecule. In some embodiments,
the MIM is selected from adenosine, adenosine diphosphate (ADP),
flavin adenosine dinucleotide (FAD, which comprises parts of
vitamin B2 and ADP) and nicotinic acid dinucleotide.
[0118] In other embodiments, the MIMs include amino acids. Examples
of amino acids include, for example, tyrosine (e.g., L-tyrosine),
cysteine, phenylalanine (e.g., L-phenylalanine) and alanine. In
some embodiments, the amino acid is phenylalanine or alanine. In
some embodiments, the MIMs include amino acid derivatives such as
4-hydroxyphenylpyruvate or acetylglycine.
[0119] In some embodiment, the MIM is a glucose analog, e.g., a
glucose molecule wherein one --OH or --CH.sub.2OH substituent has
been replaced with a --COOH, a --COO.sup.- or an --NH.sub.2
substituent. Examples of glucose analogs include glucosamine,
glucuronic acid, glucuronide and glucuronate.
[0120] In some embodiments, the MIM is selected from compounds of
formula (I):
##STR00001##
[0121] wherein
[0122] n is an integer of 0 or 1;
[0123] R.sup.1, R.sup.2, R.sup.3 and R.sup.4, when present, are
each independently selected from hydrogen and hydroxyl or R.sup.1
and R.sup.2 are taken together with the carbon on which they are
attached to form a carbonyl (C.dbd.O) group;
[0124] W is --COOH or --N(CH.sub.3).sub.3.sup.+; and
[0125] X is hydrogen, a negative charge or a alkali metal cation,
such as Na.sup.+ or.
[0126] It is to be understood that when n is 0, the CHR.sup.3 group
is bonded to the W substituent.
[0127] In some embodiments, W is --N(CH.sub.3).sub.3.sup.+. In some
embodiments, the MIM is a carnitine, such as L-carnitine.
[0128] In some embodiments, the MIM is a dicarboxylic acid. In some
embodiments, W is --COOH. In some embodiments, R.sup.3 is hydrogen.
In some embodiments, n is 0. In some embodiments, R.sup.1 and
R.sup.2 are each independently hydrogen. In some embodiments, W is
--COOH, R.sup.3 is hydrogen, n is 0 and R.sup.1 and R.sup.2 are
each independently hydrogen. In some embodiments, n is 1. In some
embodiments R.sup.1 and R.sup.2 are taken together with the carbon
on which they are attached to form a carbonyl (C.dbd.O) group. In
some embodiments, R.sup.4 is hydrogen. In some embodiments, R.sup.4
is hydroxyl. In some embodiments, W is --COOH, R.sup.3 is hydrogen,
n is 1 and R.sup.1 and R.sup.2 are taken together with the carbon
on which they are attached to form a carbonyl (C.dbd.O) group.
[0129] In some embodiments, the MIM is an intermediate of the Krebs
Cycle, the excess of which drives the Krebs Cycle towards
productive oxidative phosphorylation. Exemplary Krebs Cycle
intermediates that are MIMs include succinic acid or succinate,
malic acid or malate, and .alpha.-ketoglutaric acid or
.alpha.-ketoglutarate.
[0130] In some embodiments, the MIM is a building block of CoQ10,
which has the following structure:
##STR00002##
[0131] Thus, building blocks of CoQ10 include, but are not limited
to, phenylalanine, tyrosine, 4-hydroxyphenylpyruvate,
phenylacetate, 3-methoxy-4-hydroxymandelate, vanillic acid,
4-hydroxybenzoate, mevalonic acid, farnesyl,
2,3-dimethoxy-5-methyl-p-benzoquinone, as well as the corresponding
acids or ions thereof. In some embodiments, the MIM is selected
from phenylalanine, tyrosine, 4-hydroxyphenylpyruvate,
phenylacetate and 4-hydroxybenzoate.
(i) Methods of Identifying MIMS
[0132] The present invention provides methods for identifying a
MIM. Methods for identifying a MIM involve, generally, the
exogenous addition to a cell of an endogenous molecule and
evaluating the effect on the cell, e.g., the cellular
microenvironment profile, that the endogenous molecule provides.
Effects on the cell are evaluated at one or more of the cellular,
mRNA, protein, lipid, and/or metabolite level to identify
alterations in the cellular microenvironment profile. In one
embodiment, the cells are cultured cells, e.g., in vitro. In one
embodiment, the cells are present in an organism. The endogenous
molecule may be added to the cell at a single concentration or may
be added to the cell over a range of concentrations. In one
embodiment, the endogenous molecule is added to the cells such that
the level of the endogenous molecule in the cells is elevated
(e.g., is elevated by 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5
fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2.0 fold, 3.0 fold,
4.0 fold, 5.0 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35
fold, 40 fold, 45 fold, 50 fold or greater) as compared to the
level of the endogenous molecule in a control, untreated cell.
[0133] Molecules that induce a change in the cell as detected by
alterations in, for example, any one or more of morphology,
physiology, and/or composition (e.g., mRNA, protein, lipid,
metabolite) may be evaluated further to determine if the induced
changes to the cellular microenvironment profile are different
between a disease cellular state and a normal cellular state. Cells
(e.g., cell culture lines) of diverse tissue origin, cell type, or
disease state may be evaluated for comparative evaluation. For
example, changes induced in the cellular microenvironment profile
of a cancer cell may be compared to changes induced to a
non-cancerous or normal cell. An endogenous molecule that is
observed to induce a change in the microenvironment profile of a
cell (e.g., induces a change in the morphology, physiology and/or
composition, e.g., mRNA, protein, lipid or metabolite, of the cell)
and/or to differentially (e.g., preferentially) induce a change in
the microenvironment profile of a diseased cell as compared to a
normal cell, is identified as a MIM.
[0134] MIMs of the invention may be lipid based MIMs or non-lipid
based MIMs. Methods for identifying lipid based MIMs involve the
above-described cell based methods in which a lipid based
endogenous molecule is exogenously added to the cell. In a
preferred embodiment, the lipid based endogenous molecule is added
to the cell such that the level of the lipid based endogenous
molecule in the cell is elevated. In one embodiment, the level of
the lipid based endogenous molecule is elevated by 1.1 fold, 1.2
fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold,
1.9 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 10 fold, 15 fold,
20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold or
greater as compared to the level in an untreated control cell.
Formulation and delivery of the lipid based molecule to the cell is
dependent upon the properties of each molecule tested, but many
methods are known in the art. Examples of formulation and delivery
of lipid based molecules include, but are not limited to,
solubilization by co-solvents, carrier molecules, liposomes,
dispersions, suspensions, nanoparticle dispersions, emulsions,
e.g., oil-in-water or water-in-oil emulsions, multiphase emulsions,
e.g., oil-in-water-in-oil emulsions, polymer entrapment and
encapsulation. The delivery of the lipid based MIM to the cell can
be confirmed by extraction of the cellular lipids and
quantification of the MIM by routine methods known in the art, such
as mass spectrometry.
[0135] Methods for identifying non-lipid based MIMs involve the
above-described cell based methods in which a non-lipid based
endogenous molecule is exogenously added to the cell. In a
preferred embodiment, the non-lipid based endogenous molecule is
added to the cell such that the level of the non-lipid based
endogenous molecule in the cell is elevated. In one embodiment, the
level of the non-lipid based endogenous molecule is elevated by 1.1
fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold,
1.8 fold, 1.9 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 10
fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45
fold, 50 fold or greater as compared to the level in an untreated
control cell. Formulation and delivery of the non-lipid based
molecule to the cell is dependent upon the properties of each
molecule tested, but many methods are known in the art.
[0136] Examples of formulations and modes of delivery of non-lipid
based molecules include, but are not limited to, solubilization by
co-solvents, carrier molecules, active transport, polymer
entrapment or adsorption, polymer grafting, liposomal
encapsulation, and formulation with targeted delivery systems. The
delivery of the non-lipid based MIM to the cell may be confirmed by
extraction of the cellular content and quantification of the MIM by
routine methods known in the art, such as mass spectrometry.
2. Epimetabolic Shifters (Epi-Shifters)
[0137] As used herein, an "epimetabolic shifter" (epi-shifter) is a
molecule (endogenous or exogenous) that modulates the metabolic
shift from a healthy (or normal) state to a disease state and vice
versa, thereby maintaining or reestablishing cellular, tissue,
organ, system and/or host health in a human. Epi-shifters are
capable of effectuating normalization in a tissue microenvironment.
For example, an epi-shifter includes any molecule which is capable,
when added to or depleted from a cell, of affecting the
microenvironment (e.g., the metabolic state) of a cell. The skilled
artisan will appreciate that an epi-shifter of the invention is
also intended to encompass a mixture of two or more molecules,
wherein the mixture is characterized by one or more of the
foregoing functions. The molecules in the mixture are present at a
ratio such that the mixture functions as an epi-shifter. Examples
of epi-shifters include, but are not limited to, coQ-10; vitamin
D3; ECM components such as fibronectin; immunomodulators, such as
TNFa or any of the interleukins, e.g., IL-5, IL-12, IL-23;
angiogenic factors; and apoptotic factors.
[0138] In some embodiments, the epi-shifter is an enzyme, such as
an enzyme that either directly participates in catalyzing one or
more reactions in the Krebs Cycle, or produces a Krebs Cycle
intermediate, the excess of which drive the Krebs Cycle. In some
embodiments, the enzyme is an enzyme of the non-oxidative phase of
the pentose phosphate pathway, such as transaldolase, or
transketolase. In other embodiments, the enzyme is a component
enzyme or enzyme complex that facilitates the Krebs Cycle, such as
a synthase or a ligase. Exemplary enzymes include succinyl CoA
synthase (Krebs Cycle enzyme) or pyruvate carboxylase (a ligase
that catalyzes the reversible carboxylation of pyruvate to form
oxaloacetate (OAA), a Krebs Cycle intermediate).
[0139] In some embodiments, the epi-shifter is a building block of
CoQ10. Building blocks of CoQ10 include, but are not limited to,
phenylalanine, tyrosine, 4-hydroxyphenylpyruvate, phenylacetate,
3-methoxy-4-hydroxymandelate, vanillic acid, 4-hydroxybenzoate,
mevalonic acid, farnesyl, 2,3-dimethoxy-5-methyl-p-benzoquinone, as
well as the corresponding acids or ions thereof. In some
embodiments, the epi-shifter is selected from phenylalanine,
tyrosine, 4-hydroxyphenylpyruvate, phenylacetate and
4-hydroxybenzoate.
[0140] In some embodiments, the epi-shifter is a compound in the
Vitamin B family. Compounds in the vitamin B family include, for
example, riboflavin (vitamin B2), or analogs thereof. Epi-shifters
also include any analogs or pro-drugs that may be metabolized in
vivo to any of the endogenous MIMs, such as those described
herein.
[0141] In one embodiment, the epi-shifter also is a MIM. In one
embodiment, the epi-shifter is not CoQ10. Epi-shifters can be
routinely identified by one of skill in the art using any of the
assays described in detail herein.
(i) Methods of Identifying Epi-Shifters
[0142] Epimetabolic shifters (epi-shifter) are molecules capable of
modulating the metabolic state of a cell, e.g., inducing a
metabolic shift from a healthy (or normal) state to a disease state
and vice versa, and are thereby capable of maintaining or
reestablishing cellular, tissue, organ, system and/or host health
in a human. Epi-shifters of the invention thus have utility in the
diagnostic evaluation of a diseased state. Epi-shifters of the
invention have further utility in therapeutic applications, wherein
the application or administration of the epi-shifter (or modulation
of the epi-shifter by other therapeutic molecules) effects a
normalization in a tissue microenvironment and the disease
state.
[0143] The identification of an epimetabolic shifter involves,
generally, establishing a molecular profile, e.g., of metabolites,
lipids, proteins or RNAs (as individual profiles or in
combination), for a panel of cells or tissues that display
differential disease states, progression, or aggressiveness A
molecule from the profile(s) for which a change in level (e.g., an
increased or decreased level) correlates to the disease state,
progression or aggressiveness is identified as a potential
epi-shifter.
[0144] In one embodiment, an epi-shifter is also a MIM. Potential
epi-shifters may be evaluated for their ability to enter cells upon
exogenous addition to a cell by using any number of routine
techniques known in the art, and by using any of the methods
described herein. For example, entry of the potential epi-shifter
into a cell may be confirmed by extraction of the cellular content
and quantification of the potential epi-shifter by routine methods
known in the art, such as mass spectrometry. A potential
epi-shifter that is able to enter a cell is thereby identified as a
MIM.
[0145] To identify an epi-shifter, a potential epi-shifter is next
evaluated for the ability to shift the metabolic state of a cell.
The ability of a potential epi-shifters to shift the metabolic
state of the cell microenvironment is evaluated by introducing
(e.g., exogenously adding) to a cell a potential epi-shifter and
monitoring in the cell one or more of: changes in gene expression
(e.g., changes in mRNA or protein expression), concentration
changes in lipid or metabolite levels, changes in bioenergetic
molecule levels, changes in cellular energetics, and/or changes in
mitochondrial function or number. Potential epi-shifters capable of
shifting the metabolic state of the cell microenvironment can be
routinely identified by one of skill in the art using any of the
assays described in detail herein. Potential epi-shifters are
further evaluated for the ability to shift the metabolic state of a
diseased cell towards a normal healthy state (or conversely, for
the ability to shift the metabolic state of a normal cell towards a
diseased state). A potential epi-shifter capable of shifting the
metabolic state of a diseased cell towards a normal healthy state
(or of shifting the metabolic state of healthy normal cell towards
a diseased state) is thus identified as an Epi-shifter. In a
preferred embodiment, the epi-shifter does not negatively impact
the health and/or growth of normal cells.
[0146] Epimetabolic shifters of the invention include, but are not
limited to, small molecule metabolites, lipid-based molecules, and
proteins and RNAs. To identify an epimetabolic shifter in the class
of small molecule endogenous metabolites, metabolite profiles for a
panel of cells or tissues that display differential disease states,
progression, or aggressiveness are established. The metabolite
profile for each cell or tissue is determined by extracting
metabolites from the cell or tissue and then identifying and
quantifying the metabolites using routine methods known to the
skilled artisan, including, for example, liquid-chromatography
coupled mass spectrometry or gas-chromatography couple mass
spectrometry methods. Metabolites for which a change in level
(e.g., an increased or decreased level) correlates to the disease
state, progression or aggressiveness, are identified as potential
epi-shifters.
[0147] To identify epimetabolic shifters in the class of endogenous
lipid-based molecules, lipid profiles for a panel of cells or
tissues that display differential disease states, progression, or
aggressiveness are established. The lipid profile for each cell or
tissue is determined by using lipid extraction methods, followed by
the identification and quantitation of the lipids using routine
methods known to the skilled artisan, including, for example,
liquid-chromatography coupled mass spectrometry or
gas-chromatography couple mass spectrometry methods. Lipids for
which a change in level (e.g., an increase or decrease in bulk or
trace level) correlates to the disease state, progression or
aggressiveness, are identified as potential epi-shifters.
[0148] To identify epimetabolic shifters in the class of proteins
and RNAs, gene expression profiles for a panel of cells or tissues
that display differential disease states, progression, or
aggressiveness are established. The expression profile for each
cell or tissue is determined at the mRNA and/or protein level(s)
using standard proteomic, mRNA array, or genomic array methods,
e.g., as described in detail herein. Genes for which a change in
expression (e.g., an increase or decrease in expression at the mRNA
or protein level) correlates to the disease state, progression or
aggressiveness, are identified as potential epi-shifters.
[0149] Once the molecular profiles described above are established
(e.g., for soluble metabolites, lipid-based molecules, proteins,
RNAs, or other biological classes of composition), cellular and
biochemical pathway analysis is carried out to elucidate known
linkages between the identified potential epi-shifters in the
cellular environment.
[0150] This information obtained by such cellular and/or
biochemical pathway analysis may be utilized to categorize the
pathways and potential epi-shifters.
[0151] The utility of an Epi-shifter to modulate a disease state
can be further evaluated and confirmed by one of skill in the art
using any number of assays known in the art or described in detail
herein. The utility of an Epi-shifter to modulate a disease state
can be evaluated by direct exogenous delivery of the Epi-shifter to
a cell or to an organism. The utility of an Epi-shifter to modulate
a disease state can alternatively be evaluated by the development
of molecules that directly modulate the Epi-shifter (e.g., the
level or activity of the Epi-shifter). The utility of an
Epi-shifter to modulate a disease state can also be evaluated by
the development of molecules that indirectly modulate the
Epi-shifter (e.g., the level or activity of the Epi-shifter) by
regulating other molecules, such as genes (e.g., regulated at the
RNA or protein level), placed in the same pathway as the
Epi-shifter.
[0152] The Epimetabolomic approach described herein facilitates the
identification of endogenous molecules that exist in a cellular
microenvironment and the levels of which are sensed and controlled
through genetic, mRNA, or protein-based mechanisms. The regulation
response pathways found in normal cells that are triggered by an
Epi-shifter of the invention may provide a therapeutic value in a
misregulated or diseased cellular environment. In addition, the
epimetabolic approach described herein identifies epi-shifters that
may provide a diagnostic indication for use in clinical patient
selection, a disease diagnostic kit, or as a prognostic
indicator.
[0153] In certain embodiments, the MIMS and Epi-shifters disclosed
herein exclude those that are conventionally used as a dietary
supplement. In certain embodiments, these MIMS and/or Epi-shifter
that are disclosed herein are of pharmaceutical grade. In certain
embodiments, the MIMS and/or Epi-shifter of pharmaceutical grade
has a purity between about 95% and about 100% and include all
values between 95% and 100%. In certain embodiments, the purity of
the MIMS and/or Epi-shifter is 95%, 96%, 97%, 98%, 99%, 99.1%,
99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.9 or
100%. In certain embodiments, the MIMS and/or Epi-shifter is free
of endotoxins. In other embodiments, the MIMS and/or Epi-shifter is
free of foreign protein materials. In certain embodiments, the MIMS
and/or Epi-shifter is CoQ10.
III. Assays Useful for identifying MIMs/Epi-Shifters
[0154] Techniques and methods of the present invention employed to
separate and identify molecules and compounds of interest include
but are not limited to: liquid chromatography (LC), high-pressure
liquid chromatography (HPLC), mass spectroscopy (MS), gas
chromatography (GC), liquid chromatography/mass spectroscopy
(LC-MS), gas chromatography/mass spectroscopy (GC-MS), nuclear
magnetic resonance (NMR), magnetic resonance imaging (MRI), Fourier
Transform InfraRed (FT-IR), and inductively coupled plasma mass
spectrometry (ICP-MS). It is further understood that mass
spectrometry techniques include, but are not limited to, the use of
magnetic-sector and double focusing instruments, transmission
quadrapole instruments, quadrupole ion-trap instruments,
time-of-flight instruments (TOF), Fourier transform ion cyclotron
resonance instruments (FT-MS) and matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF
MS).
Quantification of Bioenergetic Molecule Levels:
[0155] Environmental influencers (e.g., MIMs or Epi-shifters) may
be identified by changes in cellular bioenergetic molecule levels
(e.g., ATP, pyruvate, ADP, NADH, NAD, NADPH, NADP, acetylCoA,
FADH2) of cells to which a candidate epi-shifter has been applied.
Exemplary assays of bioenergetic molecule levels use colorometric,
fluorescence, and/or bioluminescent-based methods. Examples of such
assays are provided below.
[0156] Levels of ATP within cells can be measured with a number of
assays and systems known in the art. For example, in one system,
cytoplasmic ATP released from lysed cells reacts with luciferin and
the enzyme luciferase to produce light. This bioluminescence is
measured by a bioluminometer and the intracellular ATP
concentration of the lysed cells can be calculated (EnzyLight.TM.
ATP Assay Kit (EATP-100), BioAssay Systems, Hayward, Calif.). In
another system, for example, both ATP and its dephosphorylated
form, ADP, are calculated via bioluminescence; after ATP levels are
calculated, ADP is transformed into ATP and then detected and
calculated using the same luciferase system (ApoSENSOR.TM. ADP/ATP
Ratio Assay Kit, BioVision Inc., Mountain View, Calif.).
[0157] Pyruvate is an important intermediate in cellular metabolic
pathways. Pyruvate may be converted into carbohydrate via
gluconeogenesis, converted into fatty acid or metabolized via
acetyl CoA, or converted into alanine or ethanol, depending upon
the metabolic state of a cell. Thus detection of pyruvate levels
provides a measure of the metabolic activity and state of a cell
sample. One assay to detect pyruvate, for example, uses both a
colorimetric and fluorimetric to detect pyruvate concentrations
within different ranges (EnzyChrom.TM. Pyruvate Assay Kit (Cat#
EPYR-100), BioAssay Systems, Hayward, Calif.).
[0158] Environmental influencers (e.g., MIMs or Epi-shifters) may
influence the process of oxidative phosphorylation carried out by
mitochondria in cells, which are involved in the generation and
maintenance of bioenergetic molecules in cells. In addition to
assays that detect changes in cellular energetics in cell cultures
and samples directly (described below), assays exist that detect
and quantify the effects of compounds on discrete enzymes and
complexes of mitochondria in cells. For example, the MT-OXC
MitoTox.TM. Complete OXPHOS Activity Assay (MitoSciences Inc.,
Eugene, Oreg.) can detect and quantify the effects of compounds
applied directly to complexes Ito V extracted from mitochondria.
Assays for the detection and quantification of effects on
individual mitochondrial complexes such as NADH dehydrogenase
(Complex I), cytochrome c oxidase (Complex IV) and ATP synthase
(Complex V) are also available (MitoSciences Inc., Eugene,
Oreg.).
Measurement of Cellular Energetics:
[0159] Environmental influencers (e.g., MIMs or Epi-shifters) may
also be identified by changes in cellular energetics. One example
of the measurement of cellular energetics are the real-time
measures of the consumption of molecular oxygen and/or the change
in pH of the media of a cell culture. For example, the ability of a
potential epi-shifter to modulate the metabolic state of a cell may
be analyzed using, for example, the XF24 Analyzer (Seahorse, Inc.).
This technology allows for real time detection of oxygen and pH
changes in a monolayer of cells in order to evaluate the
bioenergetics of a cell microenvironment. The XF24 Analyzer
measures and compares the rates of oxygen consumption (OCR), which
is a measure of aerobic metabolism, and extracellular acidification
(ECAR), which is a measure of glycolysis, both key indicators of
cellular energetics.
Measurement of Oxidative Phosphorylation and Mitochondrial
Function
[0160] Oxidative Phosphorylation is a process by which ATP is
generated via the oxidation of nutrient compounds, carried out in
eukaryotes via protein complexes embedded in the membranes of
mitochondria. As the primary source of ATP in the cells of most
organisms, changes in oxidative phosphorylation activity can
strongly alter metabolism and energy balance within a cell. In some
embodiments of the invention, environmental influencers (e.g., MIMs
or Epi-shifters) may be detected and/or identified by their effects
on oxidative phosphorylation. In some embodiments, environmental
influencers (e.g., MIMs or Epi-shifters) may be detected and/or
identified by their effects on specific aspects of oxidative
phosphorylation, including, but not limited to, the electron
transport chain and ATP synthesis.
[0161] The membrane-embedded protein complexes of the mitochrondria
that carry out processes involved in oxidative phosphorylation
perform specific tasks and are numbered I, II, III and IV. These
complexes, along with the trans-inner membrane ATP synthase (also
known as Complex V), are the key entities involved in the oxidative
phosphorylation process. In addition to assays that can examine the
effects of environmental influencers (e.g., MIMs or Epi-shifters)
on mitochondrial function in general and the oxidative
phosphorylation process in particular, assays are available that
can be used to examine the effects of an epi-shifter on an
individual complex separately from other complexes.
[0162] Complex I, also known as NADH-coenzyme Q oxidoreductase or
NADH dehydrogenase, is the first protein in the electron transport
chain. In some embodiments, the detection and quantification of the
effect of an epi-shifter on the production of NAD by Complex I may
be performed. For example, the complex can be immunocaptured from a
sample in a 96-well plate; the oxidation of NADH to NAD takes place
concurrently with the reduction of a dye molecule which has an
increased absorbance at 450 nM (Complex I Enzyme Activity
Microplate Assay Kit, MitoSciences Inc., Eugene, Oreg.).
[0163] Complex IV, also known as cytochrome c oxidase (COX), is the
last protein in the electron transport chain. In some embodiments,
the detection and quantification of the effect of an epi-shifter on
the oxidation of cytochrome c and the reduction of oxygen to water
by Complex IV may be performed. For example, COX can be
immunocaptured in a microwell plate and the oxidation of COX
measured with a colorimetric assay (Complex IV Enzyme Activity
Microplate Assay Kit, MitoSciences Inc., Eugene, Oreg.).
[0164] The final enzyme in the oxidative phosphorylation process is
ATP synthase (Complex V), which uses the proton gradient created by
the other complexes to power the synthesis of ATP from ADP. In some
embodiments, the detection and quantification of the effect of an
epi-shifter on the activity of ATP synthase may be performed. For
example, both the activity of ATP synthase and the amount of ATP
synthase in a sample may be measured for ATP synthase that has been
immunocaptured in a microwell plate well. The enzyme can also
function as an ATPase under certain conditions, thus in this assay
for ATP synthase activity, the rate at which ATP is reduced to ADP
is measured by detecting the simultaneous oxidation of NADH to
NAD.sup.+. The amount of ATP is calculated using a labeled antibody
to ATPase (ATP synthase Duplexing (Activity+Quantity) Microplate
Assay Kit, MitoSciences Inc., Eugene, Oreg.). Additional assays for
oxidative phosphorylation include assays that test for effects on
the activity of Complexes II and III. For example, the MT-OXC
MitoTox.TM. Complete OXPHOS System (MitoSciences Inc., Eugene,
Oreg.) can be used to evaluate effects of a compound on Complex II
and III as well as Complex I, IV and V, to provide data on the
effects of a compound on the entire oxidative phosphorylation
system.
[0165] As noted above, real-time observation of intact cell samples
can be made using probes for changes in oxygen consumption and pH
in cell culture media. These assays of cell energetics provide a
broad overview of mitochondrial function and the effects of
potential environmental influencers (e.g., MIMs or Epi-shifters) on
the activity of mitochondria within the cells of the sample.
[0166] Environmental influencers (e.g., MIMs or Epi-shifters) may
also affect mitochondrial permeability transition (MPT), a
phenomena in which the mitochondrial membranes experience an
increase in permeability due to the formation of mitochondrial
permeability transition pores (MPTP). An increase in mitochondrial
permeability can lead to mitochondrial swelling, an inability to
conduct oxidative phosphorylation and ATP generation and cell
death. MPT may be involved with induction of apoptosis. (See, for
example, Halestrap, A. P., Biochem. Soc. Trans. 34:232-237 (2006)
and Lena, A. et al. Journal of Translational Med. 7:13-26 (2009),
hereby incorporated by reference in their entirety.)
[0167] In some embodiments, the detection and quantification of the
effect of an environmental influencer (e.g., MIM or epi-shifter) on
the formation, discontinuation and/or effects of MPT and MPTPs are
measured. For example, assays can detect MPT through the use of
specialized dye molecules (calcein) that are localized within the
inner membranes of mitochondria and other cytosolic compartments.
The application of another molecule, CoCl.sub.2, serves to squelch
the fluorescence of the calcein dye in the cytosol. CoCl.sub.2
cannot access, however, the interior of the mitochondria, thus the
calcein fluorescence in the mitochondria is not squelched unless
MPT has occurred and CoCl.sub.2 can access the interior of the
mitochondra via MPTPs. Loss of mitochondrial-specific fluorescence
signals that MPT has occurred. Flow cytometry can be used to
evaluate cellular and organelle fluorescence (MitoProbe.TM.
Transition Pore Assay Kit, Molecular Probes, Eugene, Oreg.).
Additional assays utilize a fluorescence microscope for evaluating
experimental results (Image-iT LIVE Mitochondrial Transition Pore
Assay Kit, Molecular Probes, Eugene, Oreg.).
[0168] Measurement of Cellular Proliferation and Inflammation
[0169] In some embodiments of the invention, environmental
influencers (e.g., MIMs or Epi-shifters) may be identified and
evaluated by their effects on the production or activity of
molecules associated with cellular proliferation and/or
inflammation. These molecules include, but are not limited to,
cytokines, growth factors, hormones, components of the
extra-cellular matrix, chemokines, neuropeptides,
neurotransmitters, neurotrophins and other molecules involved in
cellular signaling, as well as intracellular molecules, such as
those involved in signal transduction.
[0170] Vascular endothelial growth factor (VEGF) is a growth factor
with potent angiogenic, vasculogenic and mitogenic properties. VEGF
stimulates endothelial permeability and swelling and VEGF activity
is implicated in numerous diseases and disorders, including
rheumatoid arthritis, metastatic cancer, age-related macular
degeneration and diabetic retinopathy.
[0171] In some embodiments of the invention, an environmental
influencer (e.g., MIM or Epi-shifter) may be identified and
characterized by its effects on the production of VEGF. For
example, cells maintained in hypoxic conditions or in conditions
mimicking acidosis will exhibit increased VEGF production. VEGF
secreted into media can be assayed using an ELISA or other
antibody-based assays, using available anti-VEGF antibodies
(R&D Systems, Minneapolis, Minn.). In some embodiments of the
invention, an Epi-shifter may be identified and/or characterized
based on its effect(s) on the responsiveness of cells to VEGF
and/or based on its effect(s) on the expression or activity of the
VEGF receptor.
[0172] Implicated in both healthy immune system function as well as
in autoimmune diseases, tumor necrosis factor (TNF) is a key
mediator of inflammation and immune system activation. In some
embodiments of the invention, an Epi-shifter may be identified and
characterized by its effects on the production or the activity of
TNF. For example, TNF produced by cultured cells and secreted into
media can be quantified via ELISA and other antibody-based assays
known in the art. Furthermore, in some embodiments an environmental
influencer may be identified and characterized by its effect(s) on
the expression of receptors for TNF (Human TNF RI Duoset, R&D
Systems, Minneapolis, Minn.).
[0173] The components of the extracellular matrix (ECM) play roles
in both the structure of cells and tissues and in signaling
processes. For example, latent transforming growth factor beta
binding proteins are ECM components that create a reservoir of
transforming growth factor beta (TGF.beta.) within the ECM.
Matrix-bound TGF.beta. can be released later during the process of
matrix remodeling and can exert growth factor effects on nearby
cells (Dallas, S. Methods in Mol. Biol. 139:231-243 (2000)).
[0174] In some embodiments, an environmental influencer (e.g., MIM
or Epi-shifter) may be identified or characterized by its effect(s)
on the creation of ECM by cultured cells. Researchers have
developed techniques with which the creation of ECM by cells, as
well as the composition of the ECM, can be studied and quantified.
For example, the synthesis of ECM by cells can be evaluated by
embedding the cells in a hydrogel before incubation. Biochemical
and other analyses are performed on the ECM generated by the cells
after cell harvest and digestion of the hydrogel (Strehin, I. and
Elisseeff, J. Methods in Mol. Bio. 522:349-362 (2009)).
[0175] In some embodiments, the effect of environmental influencer
(e.g., MIM or epi-shifter) on the production, status of or lack of
ECM or one of its components in an organism may be identified or
characterized. Techniques for creating conditional knock-out (KO)
mice have been developed that allow for the knockout of particular
ECM genes only in discrete types of cells or at certain stages of
development (Brancaccio, M. et al. Methods in Mol. Bio. 522:15-50
(2009)). The effect of the application or administration of an
epi-shifter or potential epi-shifter on the activity or absence of
a particular ECM component in a particular tissue or at a
particular stage of development may thus be evaluated.
Measurement of Plasma Membrane Integrity and Cell Death
[0176] Environmental influencers (e.g., MIMs or Epi-shifters) may
be identified by changes in the plasma membrane integrity of a cell
sample and/or by changes in the number or percentage of cells that
undergo apoptosis, necrosis or cellular changes that demonstrate an
increased or reduced likelihood of cell death.
[0177] An assay for lactate dehydrogenase (LDH) can provide a
measurement of cellular status and damage levels. LDH is a stable
and relatively abundant cytoplasmic enzyme. When plasma membranes
lose physical integrity, LDH escapes to the extracellular
compartment. Higher concentrations of LDH correlate with higher
levels of plasma membrane damage and cell death. Examples of LDH
assays include assays that use a colorimetric system to detect and
quantify levels of LDH in a sample, wherein the reduced form of a
tetrazolium salt is produced via the activity of the LDH enzyme
(QuantiChrom.TM. Lactate Dehydrogenase Kit (DLDH-100), BioAssay
Systems, Hayward, Calif.; LDH Cytotoxicity Detection Kit, Clontech,
Mountain View, Calif.).
[0178] Apoptosis is a process of programmed cell death that may
have a variety of different initiating events. A number of assays
can detect changes in the rate and/or number of cells that undergo
apoptosis. One type of assay that is used to detect and quantify
apoptosis is a capase assay. Capases are aspartic acid-specific
cysteine proteases that are activated via proteolytic cleavage
during apoptosis. Examples of assays that detect activated capases
include PhiPhiLux.RTM. (OncoImmunin, Inc., Gaithersburg, Md.) and
Caspase-Glo.RTM. 3/7 Assay Systems (Promega Corp., Madison, Wis.).
Additional assays that can detect apoptosis and changes in the
percentage or number of cells undergoing apoptosis in comparative
samples include TUNEL/DNA fragmentation assays. These assays detect
the 180 to 200 base pair DNA fragments generated by nucleases
during the execution phase of apoptosis. Exemplary TUNEL/DNA
fragmentation assays include the In Situ Cell Death Detection Kit
(Roche Applied Science, Indianapolis, Ind.) and the DeadEnd.TM.
Colorimetric and Fluorometric TUNEL Systems (Promega Corp.,
Madison, Wis.).
[0179] Some apoptosis assays detect and quantify proteins
associated with an apoptotic and/or a non-apoptotic state. For
example, the MultiTox-Fluor Multiplex Cytotoxicity Assay (Promega
Corp., Madison, Wis.) uses a single substrate, fluorimetric system
to detect and quantify proteases specific to live and dead cells,
thus providing a ratio of living cells to cells that have undergone
apoptosis in a cell or tissue sample.
[0180] Additional assays available for detecting and quantifying
apoptosis include assays that detect cell permeability (e.g.,
APOPercentage.TM. APOPTOSIS Assay, Biocolor, UK) and assays for
Annexin V (e.g., Annexin V-Biotin Apoptosis Detection Kit,
BioVision Inc., Mountain View, Calif.).
IV. Treatment of Metabolic Disorders
[0181] In some embodiments, the compounds of the present invention,
e.g., the environmental influencers, e.g., MIMs or epi-shifters,
described herein, may be used to treat a Coenzyme Q10 responsive
state in a subject in need thereof. The language "Coenzyme Q10
responsive state," or "CoQ10 responsive state," includes diseases,
disorders, states and/or conditions which can be treated,
prevented, or otherwise ameliorated by the administration of
Coenzyme Q10. Without wishing to be bound by any particular theory,
and as described further herein, it is believed that CoQ10
functions, at least partially, by inducing a metabolic shift to the
cell microenvironment, such as a shift towards the type and/or
level of oxidative phosphorylation in normal state cells.
Accordingly, in some embodiments, CoQ10 responsive states are
states that arise from an altered metabolism of cell
microenvironment. In one embodiment, the CoQ10 responsive disorder
is a metabolic disorder. Coenzyme Q10 responsive states include,
for example, metabolic disorders such as obesity, diabetes,
pre-diabetes, Metabolic Syndrome, satiety, and endocrine
abnormalities. Coenzyme Q10 responsive states further include other
metabolic disorders as described herein.
[0182] In some embodiments, the compounds of the present invention,
e.g., the MIMs or epi-shifters described herein, share a common
activity with Coenzyme Q10. As used herein, the phrase "share a
common activity with Coenzyme Q10" refers to the ability of a
compound to exhibit at least a portion of the same or similar
activity as Coenzyme Q10. In some embodiments, the compounds of the
present invention exhibit 25% or more of the activity of Coenzyme
Q10. In some embodiments, the compounds of the present invention
exhibit up to and including about 130% of the activity of Coenzyme
Q10. In some embodiments, the compounds of the present invention
exhibit about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,
40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,
53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,
66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%,
104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%,
115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%,
126%, 127%, 128%, 129%, or 130% of the activity of Coenzyme Q10. It
is to be understood that each of the values listed in this
paragraph may be modified by the term "about." Additionally, it is
to be understood that any range which is defined by any two values
listed in this paragraph is meant to be encompassed by the present
invention. For example, in some embodiments, the compounds of the
present invention exhibit between about 50% and about 100% of the
activity of Coenzyme Q10. In some embodiments, the activity shared
by Coenzyme Q10 and the compounds of the present invention is the
ability to induce a shift in cellular metabolism. In certain
embodiments, the activity shared by of CoQ10 and the compounds of
the present invention is measured by OCR (Oxygen Consumption Rate)
and/or ECAR (ExtraCellular Acidification Rate).
[0183] The present invention provides methods for treating,
alleviating symptoms of, inhibiting progression of, or preventing a
CoQ10 responsive disorder in a mammal, the method comprising
administering to the mammal in need thereof a therapeutically
effective amount of pharmaceutical composition comprising at least
one environmental influencer (env-influencer), wherein the
environmental influencer selectively elicits, in a disease cell of
the mammal, a cellular metabolic energy shift towards levels of
glycolysis and mitochondrial oxidative phosphorylation observed in
a normal cell of the mammal under normal physiological
conditions.
[0184] The present invention further provides methods for treating,
alleviating symptoms of, inhibiting progression of, or preventing a
metabolic disorder in a mammal, the method comprising administering
to the mammal in need thereof a therapeutically effective amount of
a pharmaceutical composition comprising at least one environmental
influencer (env-influencer), wherein the environmental influencer
selectively elicits, in a disease cell of the mammal, a cellular
metabolic energy shift towards normalized mitochondrial oxidative
phosphorylation.
[0185] The present invention further provides methods for
selectively augmenting mitochondrial oxidative phosphorylation, in
a disease cell of a mammal in need of treatment for a metabolic
disorder, the method comprising administering to said mammal a
therapeutically effective amount of a pharmaceutical composition
comprising at least one env-influencer, thereby selectively
augmenting mitochondrial oxidative phosphorylation in said disease
cell of the mammal.
[0186] The present invention further provides methods of treating
or preventing a metabolic disorder in a human, comprising
administering an environmental influencer to the human in an amount
sufficient to treat or prevent the metabolic disorder, thereby
treating or preventing the metabolic disorder.
[0187] The present invention further provides methods of treating
or preventing an metabolic disorder in a human, comprising
selecting a human subject suffering from an metabolic disorder, and
administering to said human a therapeutically effective amount of
an env-influencer capable of blocking anaerobic use of glucose and
augmenting mitochondrial oxidative phosphorylation, thereby
treating or preventing the metabolic disorder.
[0188] The present invention still further provides a method for
treating or preventing a metabolic disorder in a human, comprising
administering an environmental influencer (env-influencer) to the
human in an amount sufficient to treat or prevent the metabolic
disorder, wherein the environmental influencer (env-influencer) is
administered such that it is maintained in its oxidized form during
treatment, thereby treating or preventing the metabolic
disorder.
[0189] By "a metabolic disorder" is meant any pathological
condition resulting from an alteration in a patient's metabolism.
Such disorders include those associated with aberrant whole-body
glucose, lipid and/or protein metabolism and pathological
consequences arising therefrom. Metabolic disorders include those
resulting from an alteration in glucose homeostasis resulting, for
example, in hyperglycemia. According to this invention, an
alteration in glucose levels is typically an increase in glucose
levels by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
or even 100% relative to such levels in a healthy individual.
Metabolic disorders can detrimentally affect cellular functions
such as cellular proliferation, growth, differentiation, or
migration, cellular regulation of homeostasis, inter- or
intra-cellular communication; tissue function, such as liver
function, muscle function, or adipocyte function; systemic
responses in an organism, such as hormonal responses (e.g., insulin
response). Metabolic disorders include, but are not limited to,
obesity, diabetes (also referred to herein as diabetes mellitus)
(e.g., diabetes type I, diabetes type II, MODY, and gestational
diabetes), pre-diabetes, Metabolic Syndrome, satiety, and endocrine
abnormalities, e.g., of aging. Further examples of metabolic
disorders include, but are not limited to, hyperphagia, hypophagia,
triglyceride storage disease, Bardet-Biedl syndrome, Lawrence-Moon
syndrome, Prader-Labhart-Willi syndrome, Kearns-Sayre syndrome,
anorexia, medium chain acyl-CoA dehydrogenase deficiency, and
cachexia. In some embodiments the metabolic disorder is a Coenzyme
Q10 responsive state.
[0190] By "treating, reducing, or preventing a metabolic disorder"
is meant ameliorating such a condition before or after it has
occurred. As compared with an equivalent untreated control, such
reduction or degree of prevention is at least 5%, 10%, 20%, 40%,
50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard
technique. Diabetes mellitus is a heterogeneous group of metabolic
diseases which lead to chronic elevation of glucose in the blood
(hyperglycemia). Diabetes is characterized by pancreatic islet
destruction or dysfunction leading to loss of glucose regulation.
The two major types of diabetes mellitus are Type I, also known as
"insulin-dependent diabetes" ("IDDM") or "juvenile-onset diabetes",
and Type II, also known as "non-insulin dependent" ("NIDDM") or
"maturity-onset diabetes".
[0191] "Type I diabetes" refers to a condition that results from an
autoimmune-mediated destruction of pancreatic .beta. cells with
consequent loss of insulin production, which results in
hyperglycemia. Type I diabetics require insulin replacement therapy
to ensure survival. While medications such as injectable insulin
and oral hypoglycemics allow diabetics to live longer, diabetes
remains the third major killer, after heart disease and cancer.
However, these medications do not control blood sugar levels well
enough to prevent swinging between high and low blood sugar levels,
with resulting damage to the kidneys, eyes, and blood vessels. Data
from the Diabetes Control and Complications Trial (DCCT) show that
intensive control of blood glucose significantly delays
complications of diabetes, such as retinopathy, nephropathy, and
neuropathy, compared with conventional therapy consisting of one or
two insulin injections per day. Intensive therapy in the DCCT
included multiple injection of insulin three or more times per day
or continuous subcutaneous insulin infusion (CSII) by external
pump. Insulin pumps are one of a variety of alternative approaches
to subcutaneous multiple daily injections (MDI) for approximating
physiological replacement of insulin.
[0192] "Type 2 diabetes" refers to the condition in which a patient
has a fasting blood glucose or serum glucose concentration greater
than 125 mg/dl (6.94 mmol/L). Type II diabetes is characterized by
hyperglycemia in the presence of higher-than-normal levels of
plasma insulin (hyperinsulinemia) and represents over 90% of all
cases and occurs most often in overweight adults over 40 years of
age. Progression of Type II diabetes is associated with increasing
concentrations of blood glucose, coupled with a relative decrease
in the rate of glucose-induced insulin secretion. In Type II
diabetes, tissue processes which control carbohydrate metabolism
are believed to have decreased sensitivity to insulin and therefore
occur not from a lack of insulin production, but a decreased
sensitivity to increased glucose levels in the blood and an
inability to respond by producing insulin. Alternatively, diabetes
may result from various defects in the molecular machinery that
mediate the action of insulin on its target cells, such as a lack
of insulin receptors on their cell surfaces. Treatment of Type II
diabetes therefore frequently does not require administration of
insulin but may be based on diet and lifestyle changes, augmented
by therapy with oral hypoglycemic agents such as, for example,
sulfonylurea.
[0193] "Pre-diabetes" refers to a condition where a patient is
pre-disposed to the development of type 2 diabetes. Pre-diabetes
extends the definition of impaired glucose tolerance to include
individuals with a fasting blood glucose within the high normal
range.gtoreq.100 mg/dL (Meigs et al., Diabetes 2003 52:1475-1484)
and fasting hyperinsulinemia (elevated plasma insulin
concentration).
[0194] "Obesity" refers to the condition where a patient has a BMI
equal to or greater than 30 kg/m.sup.2. "Visceral obesity" refers
to a waist to hip ration of 1.0 in male patients and 0.8 in female
patients. In another aspect, visceral obesity defines the risk for
insulin resistance and the development of pre-diabetes.
[0195] "Overweight" refers to a patient with a BMI greater than or
25 kg/m.sup.2 and less than 30 kg/m.sup.2. "Weight gain" refers to
the increase in body weight in relationship to behavioral habits or
addictions, e.g., overeating or gluttony, smoking cessation, or in
relationship to biological (life) changes, e.g., weight gain
associated with aging in men and menopause in women or weight gain
after pregnancy.
[0196] "Metabolic Syndrome" (MS), also referred to as Syndrome X,
refers to a metabolic disorder that affects other pathways and
systems in the body. Originally, Metabolic Syndrome was defined as
a cluster of metabolic disorders (including obesity, insulin
resistance, hypertension, and dyslipidemia primarily
hypertriglyceridemia), that synergize to potentiate cardiovascular
disease. More recently (2001), the U.S. National Cholesterol
Education Program (NCEP) has classified "Metabolic Syndrome" as
meeting any three out of the following five criteria: fasting
glucose level of at least 110 mg/dl, plasma triglyceride level of
at least 150 mg/dl (hypertriglycerdemia), HDL cholesterol below 40
mg/dl in men or below 50 mg/dl in women, blood pressure at least
130/85 mm Hg (hypertension), and central obesity, with central
obesity being defined as abdominal waist circumference greater than
40 inches for men and greater than 35 inches for women. Presently,
there are three other internationally recognized definitions for
Metabolic Syndrome as follows: 1) World Health Organization 2)
American Heart Association/National Heart, Lung and blood Institute
(AHA/NHLBI) and 3) International Diabetes Federation (IDF). The
definitions of Metabolic Syndrome by the WHO, AHA/NHLBI and IDF are
very similar to the definition of the NECP and all use the same
metabolic parameters to define the syndrome, but the WHO also
includes assessment of insulin fasting insulin levels (Moebus S et
al, Cardiovascular Diabetology, 6: 1-10, 2007; Athyros V G et al,
Int. J. Cardiology, 117: 204-210, 2007). Yet subtle differences in
the thresholds for these metabolic parameters required to be
classified as having the syndrome among these different definitions
can result in different classification of a particular subject as
having or not having the syndrome according to these different
definitions. Also, the prevalence of cardiovascular disease (CVD)
with MS varies by the definition used. (Moebus S et al,
Cardiovascular Diabetology, 6: 1-10, 2007; Athyros V G et al, Int.
J. Cardiology, 117: 204-210, 2007). The American Diabetes
Association estimates that 1 in every 5 overweight people suffer
from Metabolic Syndrome.
[0197] In other aspects, the metabolic syndrome is described by
accepted synonyms, which includes, but is not limited to, syndrome
X, insulin resistance syndrome, insulin-resistant hypertension, the
metabolic hypertensive syndrome, dysmetabolic syndrome. Components
of the metabolic syndrome include, but are not limited to, glucose
intolerance, impaired glucose tolerance, impaired fasting serum
glucose, impaired fasting blood glucose, hyperinsulinemia,
pre-diabetes, obesity, visceral obesity, hypertriglyceridemia,
elevated serum concentrations of free fatty acids, elevated serum
concentrations of C-reactive protein, elevated serum concentrations
of lipoprotein(a), elevated serum concentrations of homocysteine,
elevated serum concentrations of small, dense low-density
lipoprotein (LDL)-cholesterol, elevated serum concentrations of
lipoprotein-associated phospholipase (A2), reduced serum
concentrations of high density lipoprotein (HDL)-cholesterol,
reduced serum concentrations of HDL(2b)-cholesterol, reduced serum
concentrations of adiponectin, and albuminuria (see: Pershadsingh
HA. Peroxisome proliferator-activated receptor-gamma: therapeutic
target for diseases beyond diabetes: quo vadis? Expert Opin
Investig Drugs. (2004) 13:215-28, and references cited
therein).
[0198] The "key elements" of the foregoing metabolic disorders
include but are not limited to, impaired fasting glucose or
impaired glucose tolerance, increased waist circumference,
increased visceral fat content, increased fasting plasma glucose,
increased fasting plasma triglycerides, decreased fasting high
density lipoprotein level, increased blood pressure, insulin
resistance, hyperinsulinemia, cardiovascular disease (or components
thereof such as arteriosclerosis, coronary artery disease,
peripheral vascular disease, or cerebrovascular disease),
congestive heart failure, elevated plasma norepinephrine, elevated
cardiovascular-related inflammatory factors, elevated plasma
factors potentiating vascular endothelial dysfunction,
hyperlipoproteinemia, arteriosclerosis or atherosclerosis,
hyperphagia, hyperglycemia, hyperlipidemia, and hypertension or
high blood pressure, increased plasma postprandial triglyceride or
free fatty acid levels, increased cellular oxidative stress or
plasma indicators thereof, increased circulating hypercoagulative
state, hepatic steatosis, hetaptic steatosis, renal disease
including renal failure and renal insufficiency.
[0199] "Insulin resistance" refers to a condition in which
circulating insulin levels in excess of the normal response to a
glucose load are required to maintain the euglycemic state (Ford et
al., JAMA. 2002, 287:356-9). Insulin resistance and the response of
a patient with insulin resistance to therapy, may be quantified by
assessing the homeostasis model assessment to insulin resistance
(HOMA-IR) score, a reliable indicator of insulin resistance
(Katsuki et al., Diabetes Care 2001, 24:362-5). An estimate of
insulin resistance by the homeostasis assessment model (HOMA)-IR
score may be calculated by a formula disclosed in Galvin et al.,
Diabet Med 1992, 9:921-8 where HOMA-IR=[fasting serum insulin
(.mu.U/mL)].times.[fasting plasma glucose (mmol/L)/22.5].
[0200] "Hyperinsulinemia" is defined as the condition in which a
subject with insulin resistance, with or without euglycemia, in
which the fasting or postprandial serum or plasma insulin
concentration is elevated above that of normal, lean individuals
without insulin resistance, having a waist-to-hip ration<1.0
(for men) or <0.8 (for women).
[0201] The term "impaired glucose tolerance" (IGT) is used to
describe a person who, when given a glucose tolerance test, has a
blood glucose level that falls between normal and hyperglycemic.
Such a person is at a higher risk of developing diabetes although
they are not considered to have diabetes. For example, impaired
glucose tolerance refers to a condition in which a patient has a
fasting blood glucose concentration or fasting serum glucose
concentration greater than 110 mg/dl and less than 126 mg/dl (7.00
mmol/L), or a 2 hour postprandial blood glucose or serum glucose
concentration greater than 140 mg/dl (7.78 mmol/L) and less than
200 mg/dl (11.11 mmol/L).
[0202] The condition of "hyperglycemia" (high blood sugar) is a
condition in which the blood glucose level is too high. Typically,
hyperglycemia occurs when the blood glucose level rises above 180
mg/dl. Symptoms of hyperglycemia include frequent urination,
excessive thirst and, over a longer time span, weight loss.
[0203] The condition of "hypoglycemia" (low blood sugar) is a
condition in which the blood glucose level is too low. Typically,
hypoglycemia occurs when the blood glucose level falls below 70
mg/dl. Symptoms of hypoglycemia include moodiness, numbness of the
extremities (especially in the hands and arms), confusion,
shakiness or dizziness. Since this condition arises when there is
an excess of insulin over the amount of available glucose it is
sometimes referred to as an insulin reaction.
(i) Diagnosis of Metabolic Disorders
[0204] The methods and compositions of the present invention are
useful for treating any patient that has been diagnosed with or is
at risk of having a metabolic disorder, such as diabetes. A patient
in whom the development of a metabolic disorder (e.g., diabetes or
obesity) is being prevented may or may not have received such a
diagnosis. One in the art will understand that patients of the
invention may have been subjected to standard tests or may have
been identified, without examination, as one at high risk due to
the presence of one or more risk factors.
[0205] Diagnosis of metabolic disorders may be performed using any
standard method known in the art, such as those described herein.
Methods for diagnosing diabetes are described, for example, in U.S.
Pat. No. 6,537,806, hereby incorporated by reference. Diabetes may
be diagnosed and monitored using, for example, urine tests
(urinalysis) that measure glucose and ketone levels (products of
the breakdown of fat); tests that measure the levels of glucose in
blood; glucose tolerance tests; and assays that detect molecular
markers characteristic of a metabolic disorder in a biological
sample (e.g., blood, serum, or urine) collected from the mammal
(e.g., measurements of Hemoglobin Alc (HbAlc) levels in the case of
diabetes).
[0206] A patient who is being treated for a metabolic disorder is
one who a medical practitioner has diagnosed as having such a
condition. Diagnosis may be performed by any suitable means, such
as those described herein. A patient in whom the development of
diabetes or obesity is being prevented may or may not have received
such a diagnosis. One in the art will understand that patients of
the invention may have been subjected to standard tests or may have
been identified, without examination, as one at high risk due to
the presence of one or more risk factors, such as family history,
obesity, particular ethnicity (e.g., African Americans and Hispanic
Americans), gestational diabetes or delivering a baby that weighs
more than nine pounds, hypertension, having a pathological
condition predisposing to obesity or diabetes, high blood levels of
triglycerides, high blood levels of cholesterol, presence of
molecular markers (e.g., presence of autoantibodies), and age (over
45 years of age). An individual is considered obese when their
weight is 20% (25% in women) or more over the maximum weight
desirable for their height. An adult who is more than 100 pounds
overweight, is considered to be morbidly obese. Obesity is also
defined as a body mass index (BMI) over 30 kg/m.sup.2.
[0207] Patients may be diagnosed as being at risk or as having
diabetes if a random plasma glucose test (taken at any time of the
day) indicates a value of 200 mg/dL or more, if a fasting plasma
glucose test indicates a value of 126 mg/dL or more (after 8
hours), or if an oral glucose tolerance test (OGTT) indicates a
plasma glucose value of 200 mg/dL or more in a blood sample taken
two hours after a person has consumed a drink containing 75 grams
of glucose dissolved in water. The OGTT measures plasma glucose at
timed intervals over a 3-hour period. Desirably, the level of
plasma glucose in a diabetic patient that has been treated
according to the invention ranges between 160 to 60 mg/dL, between
150 to 70 mg/dL, between 140 to 70 mg/dL, between 135 to 80 mg/dL,
and preferably between 120 to 80 mg/dL.
[0208] Optionally, a hemoglobin Alc (HbAlc) test, which assesses
the average blood glucose levels during the previous two and three
months, may be employed. A person without diabetes typically has an
HbAlc value that ranges between 4% and 6%. For every 1% increase in
HbAlc, blood glucose levels increases by approximately 30 mg/dL and
the risk of complications increases. Preferably, the HbAlc value of
a patient being treated according to the present invention is
reduced to less than 9%, less than 7%, less than 6%, and most
preferably to around 5%. Thus, the HbAlc levels of the patient
being treated are preferably lowered by 10%, 20%, 30%, 40%, 50%, or
more relative to such levels prior to treatment.
[0209] Gestational diabetes is typically diagnosed based on plasma
glucose values measured during the OGTT. Since glucose levels are
normally lower during pregnancy, the threshold values for the
diagnosis of diabetes in pregnancy are lower than in the same
person prior to pregnancy. If a woman has two plasma glucose
readings that meet or exceed any of the following numbers, she has
gestational diabetes: a fasting plasma glucose level of 95 mg/dL, a
1-hour level of 180 mg/dL, a 2-hour level of 155 mg/dL, or a 3-hour
level of 140 mg/dL.
[0210] Ketone testing may also be employed to diagnose type I
diabetes. Because ketones build up in the blood when there is not
enough insulin, they eventually accumulate in the urine. High
levels of blood ketones may result in a serious condition called
ketoacidosis.
[0211] According to the guidelines of the American Diabetes
Association, to be diagnosed with Type 2 diabetes, an individual
must have a fasting plasma glucose level greater than or equal to
126 mg/dl or a 2-hour oral glucose tolerance test (OGTT) plasma
glucose value of greater than or equal to 200 mg/dl (Diabetes Care,
26:S5-S20, 2003).
[0212] A related condition called pre-diabetes is defined as having
a fasting glucose level of greater than 100 mg/dl but less than 126
mg/dl or a 2-hour OGTT plasma glucose level of greater than 140
mg/dl but less than 200 mg/dl. Mounting evidence suggests that the
pre-diabetes condition may be a risk factor for developing
cardiovascular disease (Diabetes Care 26:2910-2914, 2003).
Prediabetes, also referred to as impaired glucose tolerance or
impaired fasting glucose is a major risk factor for the development
of type 2 diabetes mellitus, cardiovascular disease and mortality.
Much focus has been given to developing therapeutic interventions
that prevent the development of type 2 diabetes by effectively
treating prediabetes (Pharmacotherapy, 24:362-71, 2004).
[0213] Obesity (commonly defined as a Body Mass Index of
approximately >30 kg/m.sup.2) is often associated with a variety
of pathologic conditions such as hyperinsulinemia, insulin
resistance, diabetes, hypertension, and dyslipidemia. Each of these
conditions contributes to the risk of cardiovascular disease.
[0214] Along with insulin resistance, hypertension, and
dyslipidemia, obesity is considered to be a component of the
Metabolic Syndrome (also known as Syndrome X) which together
synergize to potentiate cardiovascular disease. More recently, the
U.S. National Cholesterol Education Program has classified
Metabolic Syndrome as meeting three out of the following five
criteria: fasting glucose level of at least 110 mg/dl, plasma
triglyceride level of at least 150 mg/dl (hypertriglycerdemia), HDL
cholesterol below 40 mg/dl in men or below 50 mg/dl in women, blood
pressure at least 130/85 mm Hg (hypertension), and central obesity,
with central obesity being defined as abdominal waist circumference
greater than 40 inches for men and greater than 35 inches for
women.
(ii) Assessing Treatment Efficacy of a Metabolic Disorder
[0215] The skilled artisan will recognize that the use of any of
the above tests or any other tests known in the art may be used to
monitor the efficacy of the therapeutic treatments of the
invention. Since the measurements of hemoglobin Alc (HbAlc) levels
is an indication of average blood glucose during the previous two
to three months, this test may be used to monitor a patient's
response to diabetes treatment.
[0216] The therapeutic methods of the invention are effective in
reducing glucose levels or lipid levels in a patient. By "reducing
glucose levels" is meant reducing the level of glucose by at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative
to an untreated control. Desirably, glucose levels are reduced to
normoglycemic levels, i.e., between 150 to 60 mg/dL, between 140 to
70 mg/dL, between 130 to 70 mg/dL, between 125 to 80 mg/dL, and
preferably between 120 to 80 mg/dL. Such reduction in glucose
levels may be obtained by increasing any one of the biological
activities associated with the clearance of glucose from the blood.
Accordingly, an agent having the ability to reduce glucose levels
may increase insulin production, secretion, or action. Insulin
action may be increased, for example, by increasing glucose uptake
by peripheral tissues and/or by reducing hepatic glucose
production. Alternatively, the agent of the invention may reduce
the absorption of carbohydrates from the intestines, alter glucose
transporter activity (e.g., by increasing GLUT4 expression,
intrinsic activity, or translocation), increase the amount of
insulin-sensitive tissue (e.g., by increasing muscle cell or
adipocyte cell differentiation), or alter gene transcription in
adipocytes or muscle cells (e.g., altered secretion of factors from
adipocytes expression of metabolic pathway genes). Desirably, the
agent of the invention increases more than one of the activities
associated with the clearance of glucose. By "reducing lipid
levels" is meant reducing the level of lipids by at least 1%, 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative
to an untreated control.
[0217] By "alter insulin signaling pathway such that glucose levels
are reduced" is meant to alter (by increasing or reducing) any one
of the activities involved in insulin signaling such that the
overall result is an increase in the clearance of glucose from
plasma. For example, the env-influencer of the invention alters the
insulin signaling pathway causing an increase in insulin
production, secretion, or action, an increase in glucose uptake by
peripheral tissues, a reduction in hepatic glucose production, or a
reduction in the absorption of carbohydrates from the
intestines.
[0218] The ability of an environmental influencer, e.g.,
epi-shifter, to reduce glucose levels and thereby treat a metabolic
disorder may be assessed using standard assays known in the art.
For example, cell-based screening assays that identify agents that
increase glucose uptake may be employed. In particular,
differentiated adipocytes in cell culture can be employed to assess
the ability of the epi-shifter to increase glucose uptake upon
insulin stimulation, as detected by radiolabeled glucose. In
another exemplary assay, human myoblasts obtained by the
conditional immortalization of cells derived from a non-diabetic
subject can be used to screen the effect of agents on glycogen
synthesis, using insulin as a positive control. Prior to treatment,
cells are serum-starved, and are then incubated either with the
epi-shifter or control for a period of two hours in serum-free
media containing radiolabeled glucose, after which, glycogen
synthesis is measured. Exemplary assays are further described in
the Examples.
V. Therapeutic Targets for Metabolic Disorders
[0219] The present invention provides methods for identifying
therapeutic targets for metabolic disorders. The invention further
provides therapeutic targets identified by such methods. The
identification of a therapeutic target involves, generally, the
exogenous application of an Env-influencer or candidate
Env-influencer to a cell or panel of cell lines, and the subsequent
evaluation of changes induced to a treated cell as compared to a
control, untreated cell. Induced cellular changes which are
monitored include, but are not limited to, changes to the
morphology, physiology or composition, e.g., RNA, protein, lipid or
metabolite levels, of the cell. Induced cellular changes as a
result of treatment by a candidate Env-influencer can be monitored
by using any of the assays described herein. For example, changes
in gene expression at the mRNA level can be evaluated by real-time
PCR arrays, while changes in gene expression at the protein level
can be monitored by using antibody microarrays and 2-D gel
electrophoresis. Genes identified as being modulated by the
candidate Env-influencer (e.g., at the mRNA and/or protein level)
are then evaluated from a Systems Biology perspective using pathway
analysis (Ingenuity IPA software) and by a review of the known
literature. Genes identified as potential therapeutic targets are
next submitted to confirmatory assays such as Western blot
analysis, siRNA knock-down, or recombinant protein production and
characterization methods. Screening assays can then be used to
identify modulators of the targets. Modulators of the therapeutic
targets are useful as novel therapeutic agents for metabolic
disorders. Modulators of therapeutic targets can be routinely
identified using screening assays described in detail herein, or by
using routine methodologies known to the skilled artisan.
[0220] Genes identified herein as being modulated (e.g.,
upmodulated or downmodulated, at either the mRNA or protein level)
by the MIM/Epi-shifter, CoQ10, are drug targets of the invention.
Drug targets of the invention include, but are not limited to, the
genes subsequently listed in Tables 2-4 & 6-28 & 63-68
herein. Based on the results of experiments described by Applicants
herein, the key proteins modulated by Q10 are associated with or
can be classified into different pathways or groups of molecules,
including transcription factors, apoptotic response, pentose
phosphate pathway, biosynthetic pathway, oxidative stress
(pro-oxidant), membrane alterations, and oxidative phosphorylation
metabolism. The key proteins modulated by CoQ10, based on the
results provided herein, are summarized as follows. A key protein
modulated by CoQ10 and which is a transcription factor is
HNF4alpha. Key proteins that are modulated by CoQ10 and associated
with the apoptotic response include Bcl-xl, Bcl-xl, Bcl-xS, BNIP-2,
Bcl-2, Birc6, Bcl-2-L11 (Bim), XIAP, BRAF, Bax, c-Jun, Bmf, PUMA,
and cMyc. A key protein that is modulated by CoQ10 and associated
with the pentose phosphate pathway is transaldolase 1. Key proteins
that are modulated by CoQ10 and associated with a biosynthetic
pathway include COQ1, COQ3, COQ6, prenyltransferase and
4-hydroxybenzoate. Key proteins that are modulated by CoQ10 and
associated with oxidative stress (pro-oxidant) include Neutrophil
cytosolic factor 2, nitric oxide synthase 2A and superoxide
dismutase 2 (mitochondrial). Key proteins that are modulated by
CoQ10 and associated with oxidative phosphorylation metabolism
include Cytochrome c, complex I, complex II, complex III and
complex IV. Further key proteins that are directly or indirectly
modulated by CoQ10 include Foxo 3a, DJ-1, IDH-1, Cpt1C and Cam
Kinase II.
[0221] Accordingly, in one embodiment of the invention, a drug
target may include HNF4-alpha, Bcl-x1, Bcl-xS, BNIP-2, Bcl-2,
Birc6, Bcl-2-L11 (Bim), XIAP, BRAF, Bax, c-Jun, Bmf, PUMA, cMyc,
transaldolase 1, COQ1, COQ3, COQ6, prenyltransferase,
4-hydrobenzoate, neutrophil cytosolic factor 2, nitric oxide
synthase 2A, superoxide dismutase 2, VDAC, Bax channel, ANT,
Cytochrome c, complex 1, complex II, complex III, complex IV, Foxo
3a, DJ-1, IDH-1, Cpt1C and Cam Kinase II. In a preferred
embodiment, a drug target may include HNF4A, Transaldolase, NM23
and BSCv. In one embodiment, the drug target is TNF4A. In one
embodiment, the drug target is transaldolase. In one embodiment,
the drug target is NM23. In one embodiment, the drug target is
BSCv. Screening assays useful for identifying modulators of
identified drug targets are described below.
VI. Screening Assays
[0222] The invention also provides methods (also referred to herein
as "screening assays") for identifying modulators, i.e., candidate
or test compounds or agents (e.g., proteins, peptides,
peptidomimetics, peptoids, small molecules or other drugs), which
modulate the expression and/or activity of an identified
therapeutic target of the invention. Such assays typically comprise
a reaction between a therapeutic target of the invention and one or
more assay components. The other components may be either the test
compound itself, or a combination of test compounds and a natural
binding partner of a marker of the invention. Compounds identified
via assays such as those described herein may be useful, for
example, for treating or preventing a metabolic disorder.
[0223] The test compounds used in the screening assays of the
present invention may be obtained from any available source,
including systematic libraries of natural and/or synthetic
compounds. Test compounds may also be obtained by any of the
numerous approaches in combinatorial library methods known in the
art, including: biological libraries; peptoid libraries (libraries
of molecules having the functionalities of peptides, but with a
novel, non-peptide backbone which are resistant to enzymatic
degradation but which nevertheless remain bioactive; see, e.g.,
Zuckermann et al., 1994, J. Med. Chem. 37:2678-85); spatially
addressable parallel solid phase or solution phase libraries;
synthetic library methods requiring deconvolution; the `one-bead
one-compound` library method; and synthetic library methods using
affinity chromatography selection. The biological library and
peptoid library approaches are limited to peptide libraries, while
the other four approaches are applicable to peptide, non-peptide
oligomer or small molecule libraries of compounds (Lam, 1997,
Anticancer Drug Des. 12:145).
[0224] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem.
37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med.
Chem. 37:1233.
[0225] Libraries of compounds may be presented in solution (e.g.,
Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991,
Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556),
bacteria and/or spores, (Ladner, U.S. Pat. No. 5,223,409), plasmids
(Cull et al, 1992, Proc Natl Acad Sci USA 89:1865-1869) or on phage
(Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science
249:404-406; Cwirla et al, 1990, Proc. Natl. Acad. Sci.
87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; Ladner,
supra.).
[0226] The screening methods of the invention comprise contacting a
cell with a test compound and determining the ability of the test
compound to modulate the expression and/or activity of a
therapeutic target of the invention in the cell. The expression
and/or activity of a therapeutic target of the invention can be
determined as described herein. The expression and/or activity of a
therapeutic target of the invention can also be determined by using
routine methods known to the skilled artisan. In one embodiment, a
compound is selected based on its ability to increase expression
and/or activity of a therapeutic target of the invention. In one
embodiment, a compound is selected based on its ability increase
expression and/or activity of a therapeutic target selected from
the protein listed in Tables 2-4 & 6-28 & 63-68, wherein
the therapeutic target is upmodulated by CoQ10 (e.g., exhibits a
positive-fold change). In one embodiment, a compound is selected
based on its ability to decrese expression and/or activity of a
therapeutic target of the invention. In one embodiment, a compound
is selected based on its ability to decrease expression and/or
activity of a therapeutic target selected from the proteins listed
in Tables 2-4 & 6-28 & 63-68, wherein the therapeutic
target is downmodulated by CoQ10 (e.g., exhibits a negative-fold
change).
[0227] In another embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of a
therapeutic target of the invention or biologically active portions
thereof. In yet another embodiment, the invention provides assays
for screening candidate or test compounds which bind to a
therapeutic target of the invention or biologically active portions
thereof. Determining the ability of the test compound to directly
bind to a therapeutic target can be accomplished, for example, by
coupling the compound with a radioisotope or enzymatic label such
that binding of the compound to the drug target can be determined
by detecting the labeled marker compound in a complex. For example,
compounds (e.g., marker substrates) can be labeled with .sup.131I,
.sup.125I, .sup.35S, .sup.14C, or .sup.3H, either directly or
indirectly, and the radioisotope detected by direct counting of
radioemission or by scintillation counting. Alternatively, assay
components can be enzymatically labeled with, for example,
horseradish peroxidase, alkaline phosphatase, or luciferase, and
the enzymatic label detected by determination of conversion of an
appropriate substrate to product.
[0228] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein in an appropriate animal model. For example, an
agent capable of modulating the expression and/or activity of a
marker of the invention identified as described herein can be used
in an animal model to determine the efficacy, toxicity, or side
effects of treatment with such an agent. Alternatively, an agent
identified as described herein can be used in an animal model to
determine the mechanism of action of such an agent. Furthermore,
this invention pertains to uses of novel agents identified by the
above-described screening assays for treatment as described
above.
VII. Pharmaceutical Compositions and Pharmaceutical
Administration
[0229] The environmental influencers of the invention can be
incorporated into pharmaceutical compositions suitable for
administration to a subject. Typically, the pharmaceutical
composition comprises an environmental influencer of the invention
and a pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. Examples of pharmaceutically
acceptable carriers include one or more of water, saline, phosphate
buffered saline, dextrose, glycerol, ethanol and the like, as well
as combinations thereof. In many cases, it will be preferable to
include isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Pharmaceutically acceptable carriers may further include minor
amounts of auxiliary substances such as wetting or emulsifying
agents, preservatives or buffers, which enhance the shelf life or
effectiveness of the environmental influencer.
[0230] The compositions of this invention may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, creams, lotions, ointments or pasts, drops suitable for
administration to the eye, ear, or nose, liposomes and
suppositories. The preferred form depends on the intended mode of
administration and therapeutic application.
[0231] The environmental influencers of the present invention can
be administered by a variety of methods known in the art. For many
therapeutic applications, the preferred route/mode of
administration is subcutaneous injection, intravenous injection or
infusion. As will be appreciated by the skilled artisan, the route
and/or mode of administration will vary depending upon the desired
results. In certain embodiments, the active compound may be
prepared with a carrier that will protect the compound against
rapid release, such as a controlled release formulation, including
implants, transdermal patches, and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Many methods for
the preparation of such formulations are patented or generally
known to those skilled in the art. See, e.g., Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978. In one embodiment, the mode of
administration is parenteral (e.g., intravenous, subcutaneous,
intraperitoneal, intramuscular). In one embodiment, the
environmental influencer is administered by intravenous infusion or
injection. In another embodiment, the environmental influencer is
administered by intramuscular or subcutaneous injection. In a
preferred embodiment, the environmental influencer is administered
topically.
[0232] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the active compound (i.e., environmental influencer)
in the required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile,
lyophilized powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and spray-drying that yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by
including in the composition an agent that delays absorption, for
example, monostearate salts and gelatin.
[0233] Techniques and formulations generally may be found in
Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton,
Pa. For systemic administration, injection is preferred, including
intramuscular, intravenous, intraperitoneal, and subcutaneous. For
injection, the compounds of the invention can be formulated in
liquid solutions, preferably in physiologically compatible buffers
such as Hank's solution or Ringer's solution. In addition, the
compounds may be formulated in solid form and redissolved or
suspended immediately prior to use. Lyophilized forms are also
included.
[0234] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., ationd oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0235] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound. For
buccal administration the compositions may take the form of tablets
or lozenges formulated in conventional manner. For administration
by inhalation, the compounds for use according to the present
invention are conveniently delivered in the form of an aerosol
spray presentation from pressurized packs or a nebuliser, with the
use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the
dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of e.g., gelatin for use in
an inhaler or insufflator may be formulated containing a powder mix
of the compound and a suitable powder base such as lactose or
starch.
[0236] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0237] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0238] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0239] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration bile
salts and fusidic acid derivatives in addition, detergents may be
used to facilitate permeation. Transmucosal administration may be
through nasal sprays or using suppositories. For topical
administration, the compound(s) of the invention are formulated
into ointments, salves, gels, or creams as generally known in the
art. A wash solution can be used locally to treat an injury or
inflammation to accelerate healing.
[0240] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
[0241] For therapies involving the administration of nucleic acids,
the compound(s) of the invention can be formulated for a variety of
modes of administration, including systemic and topical or
localized administration. Techniques and formulations generally may
be found in Remmington's Pharmaceutical Sciences, Meade Publishing
Co., Easton, Pa. For systemic administration, injection is
preferred, including intramuscular, intravenous, intraperitoneal,
intranodal, and subcutaneous. For injection, the compound(s) of the
invention can be formulated in liquid solutions, preferably in
physiologically compatible buffers such as Hank's solution or
Ringer's solution. In addition, the compound(s) may be formulated
in solid form and redissolved or suspended immediately prior to
use. Lyophilized forms are also included.
[0242] In one embodiment, the compositions comprising an
Environmental influencer are administered topically. It is
preferable to present the active ingredient, i.e. Env-influencer,
as a pharmaceutical formulation. The active ingredient may
comprise, for topical administration, from about 0.001% to about
20% w/w, by weight of the formulation in the final product,
although it may comprise as much as 30% w/w, preferably from about
1% to about 20% w/w of the formulation. The topical formulations of
the present invention, comprise an active ingredient together with
one or more acceptable carrier(s) therefor and optionally any other
therapeutic ingredients(s). The carrier(s) should be "acceptable"
in the sense of being compatible with the other ingredients of the
formulation and not deleterious to the recipient thereof.
[0243] In treating a patient exhibiting a disorder of interest, a
therapeutically effective amount of an agent or agents such as
these is administered. A therapeutically effective dose refers to
that amount of the compound that results in amelioration of
symptoms or a prolongation of survival in a patient.
[0244] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
which exhibit large therapeutic indices are preferred. The data
obtained from these cell culture assays and animal studies can be
used in formulating a range of dosage for use in human. The dosage
of such compounds lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized.
[0245] For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. For example, a dose can be formulated in animal
models to achieve a circulating plasma concentration range that
includes the IC.sub.50 as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by
HPLC.
[0246] The exact formulation, route of administration and dosage
can be chosen by the individual physician in view of the patient's
condition. (See e.g. Fingl et al., in The Pharmacological Basis of
Therapeutics, 1975, Ch. 1 p. 1). It should be noted that the
attending physician would know how to and when to terminate,
interrupt, or adjust administration due to toxicity, or to organ
dysfunctions. Conversely, the attending physician would also know
to adjust treatment to higher levels if the clinical response were
not adequate (precluding toxicity). The magnitude of an
administrated dose in the management of the oneogenic disorder of
interest will vary with the severity of the condition to be treated
and to the route of administration. The severity of the condition
may, for example, be evaluated, in part, by standard prognostic
evaluation methods. Further, the dose and perhaps dose frequency,
will also vary according to the age, body weight, and response of
the individual patient. A program comparable to that discussed
above may be used in veterinary medicine.
[0247] Depending on the specific conditions being treated, such
agents may be formulated and administered systemically or locally.
Techniques for formulation and administration may be found in
Remington's Pharmaceutical Sciences, 18.sup.th ed., Mack Publishing
Co., Easton, Pa. (1990). Suitable routes may include oral, rectal,
transdermal, vaginal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections, just to name a few.
[0248] The compositions described above may be administered to a
subject in any suitable formulation. In addition to treatment of a
metabolic disorder with topical formulations of an environmental
influencer, e.g., CoQ10, in other aspects of the invention the
environmental influencer, e.g., CoQ10, might be delivered by other
methods. For example, the environmental influencer, e.g., CoQ10,
might be formulated for parenteral delivery, e.g., for
subcutaneous, intravenous, intramuscular, or intratumoral
injection. Other methods of delivery, for example, liposomal
delivery or diffusion from a device impregnated with the
composition might be used. The compositions may be administered in
a single bolus, multiple injections, or by continuous infusion (for
example, intravenously or by peritoneal dialysis). For parenteral
administration, the compositions are preferably formulated in a
sterilized pyrogen-free form. Compositions of the invention can
also be administered in vitro to a cell (for example, to induce
apoptosis in a cancer cell in an in vitro culture) by simply adding
the composition to the fluid in which the cell is contained.
[0249] Depending on the specific conditions being treated, such
agents may be formulated and administered systemically or locally.
Techniques for formulation and administration may be found in
Remington's Pharmaceutical Sciences, 18..sup.th ed., Mack
Publishing Co., Easton, Pa. (1990). Suitable routes may include
oral, rectal, transdermal, vaginal, transmucosal, or intestinal
administration; parenteral delivery, including intramuscular,
subcutaneous, intramedullary injections, as well as intrathecal,
direct intraventricular, intravenous, intraperitoneal, intranasal,
or intraocular injections, just to name a few.
[0250] For injection, the agents of the invention may be formulated
in aqueous solutions, preferably in physiologically compatible
buffers such as Hanks's solution, Ringer's solution, or
physiological saline buffer. For such transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0251] Use of pharmaceutically acceptable carriers to formulate the
compounds herein disclosed for the practice of the invention into
dosages suitable for systemic administration is within the scope of
the invention. With proper choice of carrier and suitable
manufacturing practice, the compositions of the present invention,
in particular, those formulated as solutions, may be administered
parenterally, such as by intravenous injection. The compounds can
be formulated readily using pharmaceutically acceptable carriers
well known in the art into dosages suitable for oral
administration. Such carriers enable the compounds of the invention
to be formulated as tablets, pills, capsules, liquids, gels,
syrups, slurries, suspensions. and the like, for oral ingestion by
a patient to be treated.
[0252] Agents intended to be administered intracellularly may be
administered using techniques well known to those of ordinary skill
in the art. For example, such agents may be encapsulated into
liposomes, then administered as described above. Liposomes are
spherical lipid bilayers with aqueous interiors. All molecules
present in an aqueous solution at the time of liposome formation
are incorporated into the aqueous interior. The liposomal contents
are both protected from the external microenvironment and, because
liposomes fuse with cell membranes, are efficiently delivered into
the cell cytoplasm. Additionally, due to their hydrophobicity,
small organic molecules may be directly administered
intracellularly.
[0253] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
Determination of the effective amounts is well within the
capability of those skilled in the art, especially in light of the
detailed disclosure provided herein. In addition to the active
ingredients, these pharmaceutical compositions may contain suitable
pharmaceutically acceptable carriers comprising excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically. The
preparations formulated for oral administration may be in the form
of tablets, dragees, capsules, or solutions. The pharmaceutical
compositions of the present invention may be manufactured in a
manner that is itself known, e.g., by means of conventional mixing,
dissolving, granulating, dragee-making, levitating, emulsifying,
encapsulating, entrapping or lyophilizing processes.
[0254] Formulations suitable for topical administration include
liquid or semi-liquid preparations suitable for penetration through
the skin to the site of where treatment is required, such as
liniments, lotions, creams, ointments or pastes, and drops suitable
for administration to the eye, ear, or nose. Drops according to the
present invention may comprise sterile aqueous or oily solutions or
suspensions and may be prepared by dissolving the active ingredient
in a suitable aqueous solution of a bactericidal and/or fungicidal
agent and/or any other suitable preservative, and preferably
including a surface active agent. The resulting solution may then
be clarified and sterilized by filtration and transferred to the
container by an aseptic technique. Examples of bactericidal and
fungicidal agents suitable for inclusion in the drops are
phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride
(0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for
the preparation of an oily solution include glycerol, diluted
alcohol and propylene glycol.
[0255] Lotions according to the present invention include those
suitable for application to the skin or eye. An eye lotion may
comprise a sterile aqueous solution optionally containing a
bactericide and may be prepared by methods similar to those for the
preparation of drops. Lotions or liniments for application to the
skin may also include an agent to hasten drying and to cool the
skin, such as an alcohol or acetone, and/or a moisturizer such as
glycerol or an oil such as castor oil or arachis oil.
[0256] Creams, ointments or pastes according to the present
invention are semi-solid formulations of the active ingredient for
external application. They may be made by mixing the active
ingredient in finely-divided or powdered form, alone or in solution
or suspension in an aqueous or non-aqueous fluid, with the aid of
suitable machinery, with a greasy or non-greasy basis. The basis
may comprise hydrocarbons such as hard, soft or liquid paraffin,
glycerol, beeswax, a metallic soap; a mucilage; an oil of natural
origin such as almond, corn, arachis, castor or olive oil; wool fat
or its derivatives, or a fatty acid such as stearic or oleic acid
together with an alcohol such as propylene glycol or macrogels. The
formulation may incorporate any suitable surface active agent such
as an anionic, cationic or non-ionic surface active such as
sorbitan esters or polyoxyethylene derivatives thereof. Suspending
agents such as natural gums, cellulose derivatives or inorganic
materials such as silicaceous silicas, and other ingredients such
as lanolin, may also be included.
[0257] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0258] Pharmaceutical preparations for oral use can be obtained by
combining the active compounds with solid excipient, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations such as, for example, maize
starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxy-methylcellulose, and/or polyvinyl pyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0259] Dragee cores are provided with suitable coating. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0260] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added.
[0261] The composition can include a buffer system, if desired.
Buffer systems are chosen to maintain or buffer the pH of
compositions within a desired range. The term "buffer system" or
"buffer" as used herein refers to a solute agent or agents which,
when in a water solution, stabilize such solution against a major
change in pH (or hydrogen ion concentration or activity) when acids
or bases are added thereto. Solute agent or agents which are thus
responsible for a resistance or change in pH from a starting
buffered pH value in the range indicated above are well known.
While there are countless suitable buffers, potassium phosphate
monohydrate is a preferred buffer.
[0262] The final pH value of the pharmaceutical composition may
vary within the physiological compatible range. Necessarily, the
final pH value is one not irritating to human skin and preferably
such that transdermal transport of the active compound, i.e. CoQ10
is facilitated. Without violating this constraint, the pH may be
selected to improve CoQ10 compound stability and to adjust
consistency when required. In one embodiment, the preferred pH
value is about 3.0 to about 7.4, more preferably about 3.0 to about
6.5, most preferably from about 3.5 to about 6.0.
[0263] For preferred topical delivery vehicles the remaining
component of the composition is water, which is necessarily
purified, e.g., deionized water. Such delivery vehicle compositions
contain water in the range of more than about 50 to about 95
percent, based on the total weight of the composition. The specific
amount of water present is not critical, however, being adjustable
to obtain the desired viscosity (usually about 50 cps to about
10,000 cps) and/or concentration of the other components. The
topical delivery vehicle preferably has a viscosity of at least
about 30 centipoises.
[0264] Other known transdermal skin penetration enhancers can also
be used to facilitate delivery of CoQ10. Illustrative are
sulfoxides such as dimethylsulfoxide (DMSO) and the like; cyclic
amides such as 1-dodecylazacycloheptane-2-one (Azone.TM., a
registered trademark of Nelson Research, Inc.) and the like; amides
such as N,N-dimethyl acetamide (DMA) N,N-diethyl toluamide,
N,N-dimethyl formamide, N,N-dimethyl octamide, N,N-dimethyl
decamide, and the like; pyrrolidone derivatives such as
N-methyl-2-pyrrolidone, 2-pyrrolidone, 2-pyrrolidone-5-carboxylic
acid, N-(2-hydroxyethyl)-2-pyrrolidone or fatty acid esters
thereof, 1-lauryl-4-methoxycarbonyl-2-pyrrolidone,
N-tallowalkylpyrrolidones, and the like; polyols such as propylene
glycol, ethylene glycol, polyethylene glycol, dipropylene glycol,
glycerol, hexanetriol, and the like; linear and branched fatty
acids such as oleic, linoleic, lauric, valeric, heptanoic, caproic,
myristic, isovaleric, neopentanoic, trimethyl hexanoic, isostearic,
and the like; alcohols such as ethanol, propanol, butanol, octanol,
oleyl, stearyl, linoleyl, and the like; anionic surfactants such as
sodium laurate, sodium lauryl sulfate, and the like; cationic
surfactants such as benzalkonium chloride, dodecyltrimethylammonium
chloride, cetyltrimethylammonium bromide, and the like; non-ionic
surfactants such as the propoxylated polyoxyethylene ethers, e.g.,
Poloxamer 231, Poloxamer 182, Poloxamer 184, and the like, the
ethoxylated fatty acids, e.g., Tween 20, Myjr 45, and the like, the
sorbitan derivatives, e.g., Tween 40, Tween 60, Tween 80, Span 60,
and the like, the ethoxylated alcohols, e.g., polyoxyethylene (4)
lauryl ether (Brij 30), polyoxyethylene (2) oleyl ether (Brij 93),
and the like, lecithin and lecithin derivatives, and the like; the
terpenes such as D-limonene, .alpha.-pinene, .beta.-carene,
.alpha.-terpineol, carvol, carvone, menthone, limonene oxide,
.alpha.-pinene oxide, eucalyptus oil, and the like. Also suitable
as skin penetration enhancers are organic acids and esters such as
salicyclic acid, methyl salicylate, citric acid, succinic acid, and
the like.
[0265] In one embodiment, the present invention provides CoQ10
compositions and methods of preparing the same. Preferably, the
compositions comprise at least about 1% to about 25% CoQ10 w/w.
CoQ10 can be obtained from Asahi Kasei N&P (Hokkaido, Japan) as
UBIDECARENONE (USP). CoQ10 can also be obtained from Kaneka Q10 as
Kaneka Q10 (USP UBIDECARENONE) in powdered form (Pasadena, Tex.,
USA). CoQ10 used in the methods exemplified herein have the
following characteristics: residual solvents meet USP 467
requirement; water content is less than 0.0%, less than 0.05% or
less than 0.2%; residue on ignition is 0.0%, less than 0.05%, or
less than 0.2% less than; heavy metal content is less than 0.002%,
or less than 0.001%; purity of between 98-100% or 99.9%, or 99.5%.
Methods of preparing the compositions are provided in the examples
section below.
[0266] In certain embodiments of the invention, methods are
provided for treating or preventing a metabolic disorder in a human
by topically administering Coenzyme Q10 to the human such that
treatment or prevention occurs, wherein the human is administered a
topical dose of Coenzyme Q10 in a topical vehicle where Coenzyme
Q10 is applied to the target tissue in the range of about 0.01 to
about 0.5 milligrams of coenzyme Q10 per square centimeter of skin.
In one embodiment, Coenzyme Q10 is applied to the target tissue in
the range of about 0.09 to about 0.15 mg CoQ10 per square
centimeter of skin. In various embodiments, Coenzyme Q10 is applied
to the target tissue in the range of about 0.001 to about 5.0,
about 0.005 to about 1.0, about 0.005 to about 0.5, about 0.01 to
about 0.5, about 0.025 to about 0.5, about 0.05 to about 0.4, about
0.05 to about 0.30, about 0.10 to about 0.25, or about 0.10 to 0.20
mg CoQ10 per square centimeter of skin. In other embodiments,
Coenzyme Q10 is applied to the target tissue at a dose of about
0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11,
0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22,
0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33,
0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44,
0.45, 0.46, 0.47, 0.48, 0.49 or 0.5 mg CoQ10 per square centimeter
of skin. In one embodiment, Coenzyme Q10 is applied to the target
tissue at a dose of about 0.12 mg CoQ10 per square centimeter of
skin It should be understood that ranges having any one of these
values as the upper or lower limits are also intended to be part of
this invention, e.g., about 0.03 to about 0.12, about 0.05 to about
0.15, about 0.1 to about 0.20, or about 0.32 to about 0.49 mg CoQ10
per square centimeter of skin.
[0267] In another embodiment of the invention, the Coenzyme Q10 is
administered in the form of a CoQ10 cream at a dosage of between
0.5 and 10 milligrams of the CoQ10 cream per square centimeter of
skin, wherein the CoQ10 cream comprises between 1 and 5% of
Coenzyme Q10. In one embodiment, the CoQ10 cream comprises about 3%
of Coenzyme Q10. In other embodiments, the CoQ10 cream comprises
about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% of Coenzyme Q10.
In various embodiments, the CoQ10 cream is administered at a dosage
of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,
6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10 milligrams of CoQ10
cream per square centimeter of skin. It should be understood that
ranges having any one of these values as the upper or lower limits
are also intended to be part of this invention, e.g., between about
0.5 and about 5.0, about 1.5 and 2.5, or about 2.5 and 5.5 mg CoQ10
cream per square centimeter of skin.
[0268] In another embodiment, the Coenzyme Q10 is administered in
the form of a CoQ10 cream at a dosage of between 3 and 5 milligrams
of the CoQ10 cream per square centimeter of skin, wherein the CoQ10
cream comprises between 1 and 5% of Coenzyme Q10. In one
embodiment, the CoQ10 cream comprises about 3% of Coenzyme Q10. In
other embodiments, the CoQ10 cream comprises about 1%, 1.5%, 2%,
2.5%, 3%, 3.5%, 4%, 4.5% or 5% of Coenzyme Q10. In various
embodiments, the CoQ10 cream is administered at a dosage of about
3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2,
4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0 milligrams of CoQ10 cream
per square centimeter of skin. It should be understood that ranges
having any one of these values as the upper or lower limits are
also intended to be part of this invention, e.g., between about 3.0
and about 4.0, about 3.3 and 5.3, or about 4.5 and 4.9 mg CoQ10
cream per square centimeter of skin.
[0269] Certain aspects of the invention provide methods for
treating or preventing a metabolic disorder in a human by topically
administering Coenzyme Q10 to the human such that treatment or
prevention occurs, wherein the Coenzyme Q10 is topically applied
one or more times per 24 hours for six weeks or more.
[0270] Certain aspects of the invention provide methods for the
preparation of a Coenzyme Q10 cream 3% which includes the steps of
preparing a Phase A, B, C, D and E and combining all the phases
such that an oil-in-water emulsion of 3% CoQ10 cream is formed.
[0271] In some embodiments, the Phase A ingredients include Alkyl
C.sub.12-15 benzoate NF at 4.00% w/w, cetyl alcohol NF at 2.00%
w/w, glyceryl stearate/PEG-100 at 4.5% w/w and stearyl alcohol NF
at 1.50% w/w while the Phase B ingredients include diethylene
glycol monoethyl ether NF at 5.00% w/w, glycerin USP at 2.00% w/w,
propylene glycol USP at 1.50% w/w, phenoxyethanol NF at 0.475% w/w,
purified water USP at 16.725% w/w and Carbomer Dispersion 2% at
40.00% w/w and the Phace C ingredients include lactic acid USP at
0.50% w/w, sodium lactate solution USP at 2.00% w/w, trolamine NF
at 1.30% w/w, and purified water USP at 2.50% w/w. Furthermore in
these embodiments the Phase D ingredients include titanium dioxide
USP at 1.00% w/w while the Phase E ingredients include CoQ10 21%
concentrate at 15% w/w.
[0272] The term "Trolamine," as used herein, refers to Trolamine
NF, Triethanolamine, TEAlan.RTM., TEAlan 99%, Triethanolamine, 99%,
Triethanolamine, NF or Triethanolamine, 99%, NF. These terms may be
used interchangeably herein.
[0273] In certain other embodiments, the Phase A ingredients
include capric/caprylic triglyceride at 4.00% w/w, cetyl alcohol NF
at 2.00% w/w, glyceril stearate/PEG-100 at 4.5% and stearyl alcohol
NF at 1.5% w/w while the Phase B ingredients include diethylene
glycol monoethyl ether NF at 5.00% w/w, glycerin USP at 2.00% w/w,
propylene glycol USP at 1.50% w/w, phenoxyethanol NF at 0.475% w/w,
purified water USP at 16.725% w/w and Carbomer Dispersion 2% at
40.00% w/w and the Phace C ingredients include lactic acid USP at
0.50% w/w, sodium lactate solution USP at 2.00% w/w, trolamine NF
at 1.30% w/w, and purified water USP at 2.50% w/w. Furthermore in
these embodiments the Phase D ingredients include titanium dioxide
USP at 1.00% w/w while the Phase E ingredients include CoQ10 21%
concentrate at 15% w/w.
[0274] In certain embodiments of the invention, methods are
provided for the preparation of a Coenzyme Q10 cream 3% which
include the steps of (1) adding the Phase A ingredients to a
suitable container and heating to 70-80 degrees C. in a water bath;
(2) adding the Phase B ingredients, excluding the Carbomer
Dispersion, to a suitable container and mixing to form a mixed
Phase B; (3) placing the Phase E ingredients into a suitable
container and melting them at 50-60 degrees C. using a water bath
to form a melted Phase E; (4) adding the Carbomer Dispersion to a
Mix Tank and heating to 70-80 degrees C. while mixing; (5) adding
the mixed Phase B to the Mix Tank while maintaining the temperature
at 70-80 degrees C.; (6) adding the Phase C ingredients to the Mix
Tank while maintaining the temperature at 70-80 degrees C.; (7)
adding the Phase D ingredients to the Mix Tank and then continue
mixing and homogenizing the contents of the Mix Tank; then (8)
stopping the homogenization and cooling the contents of the Mix
Tank to 50-60 degrees C.; then (9) discontinuing the mixing and
adding the melted Phase E to the Mix Tank to form a dispersion;
(10) mixing is then resumed until the dispersion is smooth and
uniform; then (11) cooling the contents of the Mix Tank to 45-50
degrees C.
[0275] In some other embodiments of the invention, a pharmaceutical
composition comprising CoQ10 cream 3% is provided. The cream
includes a phase A having C.sub.12-15 alkyl benzoate at 4.00% w/w
of the composition, cetyl alcohol at 2.00% w/w of the composition,
stearyl alcohol at 1.5% w/w, glyceryl stearate and PEG-100 at 4.5%
w/w; a phase B having glycerin at 2.00% w/w, propylene glycol at
1.5% w/w, ethoxydiglycol at 5.0% w/w, phenoxyethanol at 0.475% w/w,
a carbomer dispersion at 40.00% w/w, purified water at 16.725% w/w;
a phase C having triethanolamine at 1.300% w/w, lactic acid at
0.500% w/w, sodium lactate solution at 2.000% w/w, water at 2.5%
w/w; a phase D having titanium dioxide at 1.000% w/w; and a phase E
having CoQ10 21% concentrate at 15.000% w/w. In some embodiments
the Carbomer Dispersion includes water, phenoxyethanol, propylene
glycol and Carbomer 940.
[0276] In some other embodiments of the invention, a pharmaceutical
composition comprising CoQ10 cream 3% is provided. The cream
includes a phase A having Capric/Caprylic triglyceride at 4.00% w/w
of the composition, cetyl alcohol at 2.00% w/w of the composition,
stearyl alcohol at 1.5% w/w, glyceryl stearate and PEG-100 at 4.5%
w/w; a phase B having glycerin at 2.00% w/w, propylene glycol at
1.5% w/w, ethoxydiglycol at 5.0% w/w, phenoxyethanol at 0.475% w/w,
a carbomer dispersion at 40.00% w/w, purified water at 16.725% w/w;
a phase C having triethanolamine at 1.300% w/w, lactic acid at
0.500% w/w, sodium lactate solution at 2.000% w/w, water at 2.5%
w/w; a phase D having titanium dioxide at 1.000% w/w; and a phase E
having CoQ10 21% concentrate at 15.000% w/w. In some embodiments
the Carbomer Dispersion includes water, phenoxyethanol, propylene
glycol and Carbomer 940.
[0277] In some other embodiments of the invention, a pharmaceutical
composition comprising CoQ10 cream 1.5% is provided. The cream
includes a phase A having C.sub.12-15 alkyl benzoate at 5.000% w/w,
cetyl alcohol at 2.000% w/w, stearyl alcohol at 1.5% w/w, glyceryl
stearate and PEG-100 stearate at 4.500% w/w; a phase B having
glycerin at 2.000% w/w, propylene at 1.750% w/w, ethoxydiglycol at
5.000% w/w, phenoxyethanol at 0.463% w/w, a carbomer dispersion at
50% w/w, and purified water at 11.377% w/w; a phase C having
triethanolamine at 1.3% w/w, lactic acid at 0.400% w/w, sodium
lactate solution at 2.000% w/w, and water at 4.210% w/w; a phase D
having titanium dioxide at 1.000% w/w; and a phase E having CoQ10
21% concentrate at 1.500% w/w.
[0278] In some other embodiments of the invention, a pharmaceutical
composition comprising CoQ10 cream 1.5% is provided. The cream
includes a phase A having Capric/Caprylic triglyceride at 5.000%
w/w, cetyl alcohol at 2.000% w/w, stearyl alcohol at 1.5% w/w,
glyceryl stearate and PEG-100 stearate at 4.500% w/w; a phase B
having glycerin at 2.000% w/w, propylene at 1.750% w/w,
ethoxydiglycol at 5.000% w/w, phenoxyethanol at 0.463% w/w, a
carbomer dispersion at 50% w/w, and purified water at 11.377% w/w;
a phase C having triethanolamine at 1.3% w/w, lactic acid at 0.400%
w/w, sodium lactate solution at 2.000% w/w, and water at 4.210%
w/w; a phase D having titanium dioxide at 1.000% w/w; and a phase E
having CoQ10 21% concentrate at 1.500% w/w. In some embodiments the
Carbomer Dispersion includes water, phenoxyethanol and propylene
glycol.
1. Combination Therapies
[0279] In certain embodiments, an environmental influencer of the
invention and/or pharmaceutical compositions thereof can be used in
combination therapy with at least one other therapeutic agent,
which may be a different environmental influencer and/or
pharmaceutical compositions thereof. The environmental influencer
and/or pharmaceutical composition thereof and the other therapeutic
agent can act additively or, more preferably, synergistically. In
one embodiment, an environmental influencer and/or a pharmaceutical
composition thereof is administered concurrently with the
administration of another therapeutic agent. In another embodiment,
a compound and/or pharmaceutical composition thereof is
administered prior or subsequent to administration of another
therapeutic agent.
[0280] Examples of other therapeutic agents which can be used with
an environmental influencer of the invention include, but are not
limited to, diabetes mellitus-treating agents, diabetic
complication-treating agents, antihyperlipemic agents, hypotensive
or antihypertensive agents, anti-obesity agents, diuretics,
chemotherapeutic agents, immunotherapeutic agents immunosuppressive
agents, and the like.
[0281] Examples of agents for treating diabetes mellitus include
insulin formulations (e.g., animal insulin formulations extracted
from a pancreas of a cattle or a swine; a human insulin formulation
synthesized by a gene engineering technology using microorganisms
or methods), insulin sensitivity enhancing agents, pharmaceutically
acceptable salts, hydrates, or solvates thereof (e.g.,
pioglitazone, troglitazone, rosiglitazone, netoglitazone,
balaglitazone, rivoglitazone, tesaglitazar, farglitazar, CLX-0921,
R-483, NIP-221, NIP-223, DRF-2189, GW-7282TAK-559, T-131, RG-12525,
LY-510929, LY-519818, BMS-298585, DRF-2725, GW-1536, G1-262570,
KRP-297, TZD18 (Merck), DRF-2655, and the like), alpha-glycosidase
inhibitors (e.g., voglibose, acarbose, miglitol, emiglitate and the
like), biguanides (e.g., phenformin, metformin, buformin and the
like) or sulfonylureas (e.g., tolbutamide, glibenclamide,
gliclazide, chlorpropamide, tolazamide, acetohexamide,
glyclopyramide, glimepiride and the like) as well as other insulin
secretion-promoting agents (e.g., repaglinide, senaglinide,
nateglinide, mitiglinide, GLP-1 and the like), amyrin agonist
(e.g., pramlintide and the like), phosphotyrosinphosphatase
inhibitor (e.g., vanadic acid and the like) and the like.
[0282] Examples of agents for treating diabetic complications
include, but are not limited to, aldose reductase inhibitors (e.g.,
tolrestat, epalrestat, zenarestat, zopolrestat, minalrestat,
fidareatat, SK-860, CT-112 and the like), neurotrophic factors
(e.g., NGF, NT-3, BDNF and the like), PKC inhibitors (e.g.,
LY-333531 and the like), advanced glycation end-product (AGE)
inhibitors (e.g., ALT946, pimagedine, pyradoxamine,
phenacylthiazolium bromide (ALT766) and the like), active oxygen
quenching agents (e.g., thioctic acid or derivative thereof, a
bioflavonoid including flavones, isoflavones, flavonones,
procyanidins, anthocyanidins, pycnogenol, lutein, lycopene,
vitamins E, coenzymes Q, and the like), cerebrovascular dilating
agents (e.g., tiapride, mexiletene and the like).
[0283] Antihyperlipemic agents include, for example, statin-based
compounds which are cholesterol synthesis inhibitors (e.g.,
pravastatin, simvastatin, lovastatin, atorvastatin, fluvastatin,
rosuvastatin and the like), squalene synthetase inhibitors or
fibrate compounds having a triglyceride-lowering effect (e.g.,
fenofibrate, gemfibrozil, bezafibrate, clofibrate, sinfibrate,
clinofibrate and the like).
[0284] Hypotensive agents include, for example, angiotensin
converting enzyme inhibitors (e.g., captopril, enalapril, delapril,
benazepril, cilazapril, enalapril, enalaprilat, fosinopril,
lisinopril, moexipril, perindopril, quinapril, ramipril,
trandolapril and the like) or angiotensin II antagonists (e.g.,
losartan, candesartan cilexetil, olmesartan medoxomil, eprosartan,
valsartan, telmisartan, irbesartan, tasosartan, pomisartan,
ripisartan forasartan, and the like).
[0285] Antiobesity agents include, for example, central antiobesity
agents (e.g., dexfenfluramine, fenfluramine, phentermine,
sibutramine, amfepramone, dexamphetamine, mazindol,
phenylpropanolamine, clobenzorex and the like), gastrointestinal
lipase inhibitors (e.g., orlistat and the like), .beta.-3 agonists
(e.g., CL-316243, SR-58611-A, UL-TG-307, SB-226552, AJ-9677,
BMS-196085 and the like), peptide-based appetite-suppressing agents
(e.g., leptin, CNTF and the like), cholecystokinin agonists (e.g.,
lintitript, FPL-15849 and the like) and the like.
[0286] Diuretics include, for example, xanthine derivatives (e.g.,
theobromine sodium salicylate, theobromine calcium salicylate and
the like), thiazide formulations (e.g., ethiazide,
cyclopenthiazide, trichloromethiazide, hydrochlorothiazide,
hydroflumethiazide, bentylhydrochlorothiazide, penflutizide,
polythiazide, methyclothiazide and the like), anti-aldosterone
formulations (e.g., spironolactone, triamterene and the like),
decarboxylase inhibitors (e.g., acetazolamide and the like), a
chlorbenzenesulfonamide formulations (e.g., chlorthalidone,
mefruside, indapamide and the like), azosemide, isosorbide,
ethacrynic acid, piretanide, bumetanide, furosemide and the
like.
[0287] Chemotherapeutic agents include, for example, alkylating
agents (e.g., cyclophosphamide, iphosphamide and the like),
metabolism antagonists (e.g., methotrexate, 5-fluorouracil and the
like), anticancer antibiotics (e.g., mitomycin, adriamycin and the
like), vegetable-derived anticancer agents (e.g., vincristine,
vindesine, taxol and the like), cisplatin, carboplatin, etoposide
and the like. Among these substances, 5-fluorouracil derivatives
such as furtulon and neofurtulon are preferred.
[0288] Immunotherapeutic agents include, for example,
microorganisms or bacterial components (e.g., muramyl dipeptide
derivative, picibanil and the like), polysaccharides having immune
potentiating activity (e.g., lentinan, sizofilan, krestin and the
like), cytokines obtained by a gene engineering technology (e.g.,
interferon, interleukin (IL) and the like), colony stimulating
factors (e.g., granulocyte colony stimulating factor, erythropoetin
and the like) and the like, among these substances, those preferred
are IL-1, IL-2, IL-12 and the like.
[0289] Immunosuppressive agents include, for example, calcineurin
inhibitor/immunophilin modulators such as cyclosporine (Sandimmune,
Gengraf, Neoral), tacrolimus (Prograf, FK506), ASM 981, sirolimus
(RAPA, rapamycin, Rapamune), or its derivative SDZ-RAD,
glucocorticoids (prednisone, prednisolone, methylprednisolone,
dexamethasone and the like), purine synthesis inhibitors
(mycophenolate mofetil, MMF, CellCept(R), azathioprine,
cyclophosphamide), interleukin antagonists (basiliximab,
daclizumab, deoxyspergualin), lymphocyte-depleting agents such as
antithymocyte globulin (Thymoglobulin, Lymphoglobuline), anti-CD3
antibody (OKT3), and the like.
[0290] In addition, agents whose cachexia improving effect has been
established in an animal model or at a clinical stage, such as
cyclooxygenase inhibitors (e.g., indomethacin and the like) [Cancer
Research, Vol. 49, page 5935-5939, 1989], progesterone derivatives
(e.g., megestrol acetate) [Journal of Clinical Oncology, Vol. 12,
page 213-225, 1994], glucosteroid (e.g., dexamethasone and the
like), metoclopramide-based agents, tetrahydrocannabinol-based
agents, lipid metabolism improving agents (e.g., eicosapentanoic
acid and the like) [British Journal of Cancer, Vol. 68, page
314-318, 1993], growth hormones, IGF-1, antibodies against
TNF-.alpha., LIF, IL-6 and oncostatin M may also be employed
concomitantly with a compound according to the present
invention.
[0291] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references and published patents and patent applications cited
throughout the application are hereby incorporated by
reference.
EXEMPLIFICATION OF THE INVENTION
[0292] The invention now being generally described, it will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain aspects
and embodiments of the present invention, and are not intended to
limit the invention, as one skilled in the art would recognize from
the teachings hereinabove and the following examples, that other
assays, cell types, agents, constructs, or data analysis methods,
all without limitation, can be employed, without departing from the
scope of the invention as claimed.
[0293] The contents of any patents, patent applications, patent
publications, or scientific articles referenced anywhere in this
application are herein incorporated in their entirety.
[0294] The practice of the present invention will employ, where
appropriate and unless otherwise indicated, conventional techniques
of cell biology, cell culture, molecular biology, transgenic
biology, microbiology, virology, recombinant DNA, and immunology,
which are within the skill of the art. Such techniques are
described in the literature. See, for example, Molecular Cloning: A
Laboratory Manual, 3rd Ed., ed. by Sambrook and Russell (Cold
Spring Harbor Laboratory Press: 2001); the treatise, Methods In
Enzymology (Academic Press, Inc., N.Y.); Using Antibodies, Second
Edition by Harlow and Lane, Cold Spring Harbor Press, New York,
1999; Current Protocols in Cell Biology, ed. by Bonifacino, Dasso,
Lippincott-Schwartz, Harford, and Yamada, John Wiley and Sons,
Inc., New York, 1999; and PCR Protocols, ed. by Bartlett et al.,
Humana Press, 2003.
Example 1
Identification of CoQ10 as a MIM
[0295] In order to evaluate CoQ10 as a potential MIM, CoQ10 in
oxidized form was exogenously added to a panel of cell lines,
including both cancer cell lines and normal control cell lines, and
the changes induced to the cellular microenvironment profile for
each cell line in the panel were assessed. Changes to cell
morphology/physiology, and to cell composition, including both mRNA
and protein levels, were evaluated and compared for the diseased
cells as compared to normal cells. The results of these experiments
identified CoQ10 and, in particular, the oxidized form of CoQ10, as
a MIM.
[0296] In a first set of experiments, changes to cell
morphology/physiology were evaluated by examining the sensitivity
and apoptotic response of cells to CoQ10. A panel of skin cell
lines including a control cell lines (primary culture of
keratinocytes and melanocytes) and several skin cancers cell lines
(SK-MEL-28, a non-metastatic skin melanoma; SK-MEL-2, a metastatic
skin melanoma; or SCC, a squamous cell carcinoma; PaCa2, a
pancreatic cancer cell line; or HEP-G2, a liver cancer cell line)
were treated with various levels of Coenzyme Q10. The results of
these experiments demonstrated that the cancer cell lines exhibited
an altered dose dependent response as compared to the control cell
lines, with an induction of apoptosis and cell death in the cancer
cells only. Exemplary experiments are described in detail in
Example 3 below.
[0297] Assays were next employed to assess changes in the
composition of the cell following treatment with CoQ10. Changes in
gene expression at the mRNA level were analyzed using Real-Time PCR
array methodology. Exemplary experiments are described in detail in
Examples 6 and 9-13 below. In complementary experiments, changes in
gene expression at the protein level were analyzed by using
antibody microarray methodology, 2-dimensional gel electrophoresis
followed by protein identificuation using mass spectrometry
characterization, and by western blot analysis. Exemplary
experiments are described in detail below in Examples 4, 7 and 8,
respectively. The results from these assays demonstrated that
significant changes in gene expression, both at the mRNA and
protein levels, were induced in the cell lines examined due to the
addition of the oxidized form of CoQ10. Genes modulated by CoQ10
treatment were found to be clustered into several cellular
pathways, including apoptosis, cancer biology and cell growth,
glycolysis and metabolism, molecular transport, and cellular
signaling.
[0298] Experiments were carried out to confirm the entry of CoQ10
into cells and to determine the level and form of CoQ10 present in
the cells. In particular, the level of Coenzyme Q10, as well as the
form of CoQ10 (i.e., oxidized or reduced), present in the
mitochondria was determined by analyzing mitochondrial enriched
preparations from cells treated with CoQ10. The level of Coenzyme
Q10 present in the mitochondria was confirmed to increase in a time
and dose dependent manner with the addition of exogenous Q10. In a
surprising and unexpected result, CoQ10 was determined to be
present in the mitochondria primarily in oxidized form. In
addition, changes in levels of proteins from mitochondria enriched
samples were analyzed by using 2-D gel electrophoresis and protein
identification by mass spectrometry characterization. The results
from these experiments demonstrated that the levels of the oxidized
form of CoQ10 in the mitochondria over the time course examined
correlated with a wide variety of cellular changes, as evidenced by
the modulation of mRNA and protein levels for specific proteins
related to metabolic and apoptotic pathways. Exemplary experiments
are described in detail in Example 5 below.
[0299] The results described by Applicants herein identified the
endogenous molecule CoQ10 and, in particular, the oxidized form of
CoQ10, as a MIM. For example, the results identified CoQ10 as a
MIM, since CoQ10 was observed to induce changes in gene expression
at both the mRNA and protein level. The results identified CoQ10 as
having multidimentional character, since CoQ10 induced differential
changes in cell morphology/physiology and cell composition (e.g.,
differential changes in gene expression at both the mRNA and
protein level), in a disease state (e.g., cancer) as compared to a
normal (e.g., non-cancerous) state. Moreover, the results
identified CoQ10 as having multidimensional character in that CoQ10
was capable of entering a cell, and thus exhibited both therapeutic
and carrier effects.
Example 2
Methods for Identifying Disease Relevant Processes and Biomarkers
for Metabolic Disorders
[0300] From the cell based assays in which cell lines were treated
with a molecule of interest, the differences in treated vs
non-treated cells is evaluated by mRNA arrays, protein antibody
arrays, and 2D gel electrophoresis. The proteins identified from
comparative sample analysis to be modulated by the MIM or
Epi-shifter, are evaluated from a Systems Biology perspective with
pathway analysis (Ingenuity IPA software) and a review of the known
literature. Proteins identified as potential therapeutic or
biomarker targets are submitted to confirmatory assays such as
Western blot analysis, siRNA knock-down, or recombinant protein
production and characterization methods.
Materials and Methods for Examples 3-8
[0301] Coenzyme Q10 stock
[0302] A 500 .mu.M Coenzyme Q10 (5% isopropanol in cell growth
media) was prepared as follows. A 10 mL 500 .mu.M Coenzyme Q10
stock was made fresh every time.
Molecular Weight: 863.34
[0303] (0.0005 mol/L)(0.010 L)(863.34 g/mol)=0.004317 g
[0304] To make 10 mL of 500 .mu.M stock, 4.32 mg Coenzyme Q10 was
weighted out in a 15 mL falcon tube, and 500 .mu.L isopropanol was
added. The solution was warmed in a 50-60.degree. C. water bath
while swirling to dissolve completely. To this solution, 9.5 mL of
media (the same media in which the cells are grown) was added.
Cell Culture
[0305] Cells were obtained from the American Type Culture
Collection or Gibco. Cells were grown in DMEM/F-12 media
supplemented with 5% fetal bovine serum, 0.25 ug/mL Amphotericin,
100 ug/mL Streptomycin, and 100 U mL-1 penicillin. Cells were
maintained in an atmosphere of 95% air and 5% CO2 at 37 degrees
C.
Coenzyme Q10 Treatment and Total Protein Isolation
[0306] Cells were grown to 85% confluency prior to exposure with
Q10. Supplemented media was conditioned with Q10 to 50 and 100
micro molar concentrations. Flasks were treated with control, 50
.mu.M Q10, and 100 .mu.M Q10 in triplicate. Protein was isolated
from the treated and control flask after 4, 8, 12, and 24 hours.
For isolation of proteins, cells were washed three times with 5 mL
of ice cold PBS at a pH of 7.4. The cells were then scraped in 3 mL
of PBS, pelleted by centrifuge, and re-suspended in a lysis buffer
at pH 7.4 (80 mM TRIS-HCl, 1% SDS, with protease and phosphotase
inhibitors). Protein concentrations were quantified using the BCA
method.
Cell Lines
[0307] The cell lines listed below were propagated and a cell bank
established for each. Large scale production of cells for various
assays were performed and the material harvested for analysis. In
general, when a cell specific media was not required for
maintenance of cell lines, the media used for cell growth was
DMEMF-12 with 5% serum. Cells were typically grown to 75-80%
confluence (clear spacing) prior to splitting and use in cell
assays and standard practice methods followed. The following cell
lines were established for experiments:
SK-MEL-28 (non-metastatic skin melanoma) SK-MEL-2 (metastatic skin
melanoma) HEKa (kerantinocytes, skin control) HEMa (melanocyte,
skin control) nFIB (neonatal fibroblasts) HEP-G2 (liver cancer)
[SBH cell line] SkBr-3 (breast cancer, Her2 overexpressed) MCF-7
(breast cancer, p53 mutation) PC-3 (prostate cancer) [SBH cell
line] SkBr-3 (human breast adenocarcinoma)
NCI-ES-0808
[0308] SCC (squamous cell carcinoma)
PaCa-2
NIH-3T3
Cell Culture:
[0309] Cells were obtained for the American Type Culture Collection
or Gibco. Cells were grown in DMEM/F-12 media supplemented with 5%
fetal bovine serum, 0.25 ug/mL Amphotericin, 100 ug/mL
Streptomycin, and 100 U mL-1 penicillin. Cells were maintained in
an atmosphere of 95% air and 5% CO2 at 37 degrees C.
[0310] Skin malignant melanoma SK-MEL28 cells were grown and
maintained in DMEM/F12 with Glutamax (Invitrogen, Carlsbad Calif.)
supplemented with 5% FBS, amphotericin and penicillin/streptomycin.
Cells were grown at 37.degree. C. with 5% CO.sub.2. Details of
additional cell line and growth conditions are outlined in the
table below.
TABLE-US-00001 TABLE 1 Cell lines analyzed for sensitivity to Q10.
Cell Line Description Growth Conditions PaCa2 Pancreatic Carcinoma
DMEM/F12 with Glutamax + 10% FBS, 2.5% Horse Serum, amphotericin,
penicillin/ streptomycin. HepG2 Hepatocellular MEM with Earles
Salts supple- Carcinoma mented with 10% FBS, amphotericin,
penicillin/ streptomycin, sodium pyruvate and non-essential amino
acids. PC3 Prostate DMEM/F12 with Glutamax, Adenocarcinoma
supplemented with 5% FBS, amphotericin and penicillin/streptomycin.
SKBr3 Breast Cancer DMEM/F12 with Glutamax supplemented with 5% FBS
and amphotericin, penicillin/streptomycin. MCF-7 Breast Cancer
DMEM/F12 with Glutamax supplemented with 5% FBS and amphotericin,
penicillin/streptomycin.
Q10 Treatment of SKMEL28 Cells:
[0311] SK-MEL28 cells were treated with 100 .mu.M Q10 or the
control vehicle. The formulation of the Q10 was as follows. In a 15
mL capped tube, 4.32 mg of Q10 (supplied by Cytotech) was
transferred and then dissolved by the addition of 500 .mu.L of
isopropanol. The resulting solution was warmed in a 65.degree. C.
water bath and vortexed at high speed. The Q10/isopropanol solution
was made to a volume of 10 mL with the addition of equilibrated
cell culture media. The stock solution was then vortexed to ensure
maximum solubility of Q10. The stock solution was diluted (2 mL of
stock with 8 mL of media) to obtain a final concentration of 100
.mu.M Q10. For the control vehicle, 9.5 mL of media was added to
500 .mu.L of isopropanol. The control stock was further diluted (2
mL of stock) with 8 mL of media. Cells were harvested 6, 16, 24, 48
or 72 hours after the start of the treatment.
Q10 Treatment of SCC Cells:
[0312] SCC cells were treated with 100 .mu.M Q10 (prepared as
described above) either for 6 hours or 24 hours. The control cells
were untreated cells. Cells were harvested and pelleted at the
different times after treatment and the pellets were flash frozen
and stored at -80.degree. C. until the RNA was isolated at XTAL as
described below.
RNA Isolation:
[0313] Cells were lysed for RNA isolation at different treatment
times using the RNeasy Mini kit (Qiagen, Inc., Valencia Calif.) kit
following the manufacturer's instructions. RNA was quantified by
measuring Optical Density at 260 nm.
First Strand Synthesis:
[0314] First strand cDNA was synthesized from 1 .mu.g of total RNA
using the RT2 First Strand Synthesis kit (SABiosciences, Frederick
Md.) as per manufacturer's recommendations.
Real-Time PCR:
[0315] Products from the first strand synthesis were diluted with
water, mixed with the SYBR green master mix (SABiosciences,
Frederick Md.) and loaded onto PCR arrays. Real time PCR was run on
the PCR Arrays (Apoptosis Arrays, Diabetes Arrays, Oxidative stress
and Antioxidant defense Arrays and Heat Shock Protein Arrays.)
(SABiosciences, Frederick Md.) on a Biorad CFX96.
Determining Cell Line Sensitivity to Coenzyme Q10 by Nexin Assay
for Apoptosis:
[0316] The percentage of cells in early and late apoptosis was
quantified following 24 hours of Coenzyme Q10 treatment. Early and
late apoptosis was used as a marker to understand the differences
in sensitivity of various cancer cell lines to Coenzyme Q10. The
different cell lines tested were PaCa2, HepG2, PC-3, SKBr3, MCF-7
and SK- MEL28. Cells were allowed to adhere overnight in 96-well
plates. These cells were treated with either control vehicle, 50
.mu.M Q10 or 100 .mu.M Coenzyme Q10. After 24 hours, the presence
of apoptotic cells was estimated on a PCA96 flow cytometer (Guava
Technologies, Hayward, Calif.). In addition, some cells were
treated with 4 .mu.M Staurosporine for 2 hours as a positive
control for apoptosis. Cells were first washed with PBS and
detached with 50 .mu.L of Accumax (Innovative Cell Technologies,
San Diego, Calif.) at room temperature. The dissociation was
stopped by addition of culture medium containing 1% Pluronic F-68
(Sigma-Aldrich, St. Louis, Mo.). Then 100 .mu.L of Nexin reagent
(Guava Technologies, Hayward, Calif.) was added to each of the
wells. After 20 minutes of incubation in the dark, the assay was
performed in low binding plates to minimize reattachment of cells
to the substrate. The Nexin Reagent contains two dyes. Annexin-V-PE
which detects phosphotidyl serine on the outside of a cell; a
characteristic of early apoptotic cells. The second dye, 7-AAD
permeates only late apoptotic cells while being excluded from live
(healthy) and early apoptotic cells. The percentage of four
populations of cells; live, early apoptotic, late apoptotic and
debris was determined using the Cytosoft 2.5.7 software (Guava
Technologies, Hayward, Calif.).
Immunoblotting
[0317] Approximately 50 .mu.g of protein were assayed per sample by
immunoblotting. All treatments were run in triplicate with
controls. Proteins were separated on 12% TRIS-HCl gels, transferred
via electrophoresis to nitro-cellulose membranes and blocked using
a 5% milk and TBST solution prior to incubation with primary
antibodies. The primary antibodies were incubated overnight at 4
degrees C. in a 5% BSA and TBST solution. Secondary antibodies were
incubated for one hour at 4 degrees. All antibodies were purchased
from Cell Signaling Technology. Antibodies were used at a ratio of
1:1000, with the exception of PActin at a ratio of 1:5000. Blots
were developed and results were quantified using the NIH Java based
densitometer analysis software Image J. All blots were also probed
for and normalized to their respective PActin expression.
Two-Dimensional Electrophoresis
[0318] Before isoelectric focusing (IEF), samples were solubilized
in 40 mM Tris, 7 M urea, 2 M thiourea, and 1% C7 zwitterionic
detergent, reduced with tributylphosphine, and alkylated with 10 mM
acrylamide for 90 min at room temperature. After the sample was run
through a 10-kDa cutoff Amicon Ultra device with at least 3 volumes
of the resuspension buffer, consisting of 7 M urea, 2 M thiourea,
and 2% CHAPS to reduce the conductivity of the sample. One hundred
micrograms of protein were subjected to IEF on 11-cm pH 3 to 10, pH
4 to 7 or pH 6 to 11 immobilized pH gradient strips (GE, Amersham,
USA) to 100,000 volts hour. After IEF, immobilized pH gradient
strips were equilibrated in 6 M urea, 2% SDS, 50 mM Tris-acetate
buffer, pH 7.0, and 0.01% bromphenol blue and subjected to
SDS-polyacrylamide gel electrophoresis on 8 to 16% Tris-HCl Precast
Gel, 1 mm (Bio-Rad, USA). The gels were run in duplicate. They were
either fixed, stained in SYPRO Ruby, 80 mL/gel (Invitrogen, USA)
and imaged on Fuji FLA-5100 laser scanner or transferred onto PVDF
membrane.
[0319] Additional information was obtained for a control sample to
test the utility of protein identification through the use of
methods that utilize dPC (Protein Forest Inc.) selective pI
fractionation, followed by trypsin digestion of the dPC plug with
mass spec identification and semi-quantization (Nanomate or
LC/LTQ/MS). The dPC analysis performed with a control sample
demonstrated its utility in identifying a large subset of proteins.
The materials produced during the studies were archived so that
they may be utilized as a resource should the future need arise
2D Gel Image Analysis:
[0320] Analysis of all gel images was performed using Progenesis
Discovery and Pro (Nonlinear Dynamics Inc., Newcastle upon Tyne,
UK). After spot detection, matching, background subtraction,
normalization, and filtering, data for SYPRO Ruby gel images was
exported. Pairwise comparisons between groups were performed using
the Student's t test in Progenesis Discovery to identify spots
whose expression was significantly altered (p>0.05).
Antibody Array:
[0321] An antibody microarray (Panorama XP725 Antibody Array,
Sigma) was utilized to screen over 700 protein antibodies to assess
changes at the protein concentration level in Q10 treated cells
(SK-MEL-28, SCC). The expression of a protein in a cell extract is
detected when it is bound by a corresponding antibody spotted on
the slide. Prior to binding, the proteins are directly labeled with
a fluorescent dye which is used for fluorescent visualization and
quantitative analysis. The array is used for comparing protein
expression profiles of two samples (test versus reference samples),
each labeled with a different CyDye (Cy3 or Cy5) and the two
samples are applied simultaneously at equal protein concentrations
on the array. Fluorescent signal intensity for each sample is then
recorded individually at the wavelength corresponding to the dye
label of the sample and compared.
[0322] High doses of Coenzyme Q10 regulates expression of genes
involved in the apoptotic, diabetic and oxidative stress pathways
in cultured SKMEL-28 cells. Experimental details: SKMEL-28 cells
(ATCC Catalog #HTB-72) are non metastatic, skin melanoma cells that
were cultured in DMEM-F12 containing Glutamax (Invitrogen Cat#
10565-042) supplemented with 5% FBS, Penicillin, Streptomycin and
Amphotericin, were treated with the vehicle or 100 uM Coenzyme Q10
for varying amounts of time. Any changes in gene expression
consequent to Coenzyme Q10 treatment were quantified using Real
time PCR Arrays (Apoptosis Cat #PAHS-12, Diabetes Cat #PAHS-023 and
Oxidative Stress Cat #PAHS-065). (SABiosciences, Frederick,
Md.).
[0323] A stock concentration of 500 uM Coenzyme Q10 was prepared by
dissolving 4.32 mg in 500 ul of isopropanol which was further
diluted to 10 ml by addition of media. Alternate vortexing and
heating to 65.degree. C. dissolved the Coenzyme Q10. 2 ml of the
stock solution was diluted to 10 ml with media to get a 100 uM Q10
containing media that was used to treat cells. A vehicle was
prepared in parallel with a similar protocol except that the
Coenzyme Q10 was not added.
[0324] SKMEL-28 cells were plated at a density of 1.times.10.sup.5
cells/well in a 6-well plate. After 24 hours, when cells had
attached and were at 50% confluence, either the vehicle or 100 uM
Q10 was added. Cells were harvested by at 6, 16, 24, 48 or 72 hours
after Q10 treatment while the vehicle treated cells were harvested
after 24 hours. Cells were lysed for RNA isolation at different
treatment times using the RNeasy Mini kit (Qiagen, Inc., Valencia
Calif. Cat #74104) kit following the manufacturer's instructions
using a spin column and on-column DNase treatment. RNA was
quantified by measuring absorbance at 260 nm.
[0325] Real time PCR was preceded by first strand cDNA synthesis
using 0.4-lug of total RNA as the template using the RT2 First
Strand Synthesis kit (SABiosciences, Frederick Md. Cat# C-03) with
a genomic DNA elimination step as per manufacturer's
recommendations. Products from the first strand synthesis were
diluted with water, mixed with the SYBR green master mix
(SABiosciences, Frederick Md. Cat#PA-010-12) and loaded onto PCR
arrays that contain primer assays for 84 different genes linked
within a common pathway, 5 housekeeping genes used for
normalization, reverse transcription and PCR controls. Real time
PCR was run on a Biorad Cfx96. The amplification was initiated with
a hot start to activate the enzyme, followed by 40 cycles each of
(95.degree. C.-15 second denaturation step and 60.degree. C.-1
minute annealing and extension step) followed by a melting curve
program. Ct values, the output from the PCR thermocycler for all
treatment groups were organized on an excel spreadsheet and loaded
onto the comparative analysis software available at
http://www.sabiosciences.com/pcdarrayanalysis.php.
Purification of Mitochondria Enriched Samples:
[0326] Experimental details: SKMEL-28, NC1-ES0808 and NIH-3T3 cells
that were treated with 100 .mu.M Q10 for 24 or 48 hours along with
cells that were harvested at t=0 were harvested by washing and
scraping from T160 flasks. Cells were centrifuged, pelleted, flash
frozen and stored at -80.degree. C. until the mitochondria were
isolated. Cell pellets were thawed, resuspended and ruptured in
Dounce homogenizer. The homogenate was centrifuged and mitochondria
were isolated using reagents and the protocol recommended by the
Mitochondria Isolation kit for Cultured cells (MitoSciences, Eugene
Oreg., Cat # MS852). The mitochondrial fraction was aliquoted and
stored at -80.degree. C.
Coenzyme Q10 and Ubiquinol-10 Quantification Method:
[0327] A method for the simultaneous determination of Coenzyme Q10
(Q10) and the reduced form ubiquinol-10 (Q10H2) was implemented
based upon a recently published method (Ruiz-Jimenez, 2007, J.
Chromatogr. A, 1175, 242-248) through the use of LC-MS/MS with
electrospray ionization (ESI) in the positive ion mode. The highly
selective identification and sensitive quantitation of both Q10 and
Q10H2 is possible, along with the identification of other selected
lipids. An aliquot of the mitochondrial enriched samples from
SK-MEL-28 treated with 100 .mu.M Q10 was subjected to a
conventional pre-treatment based on protein precipitation (100
.mu.l of packed cells sonicated in 300 .mu.l of 1-propanol),
liquid-liquid extraction (add 100 .mu.l of water to supernatant and
extract X3 with 200 .mu.l of n-hexane), evaporation of combined
hexane extracts to dryness and reconstitution in 50 .mu.l of 95:5
methanol/hexane (v/v). Analysis was by LC-MS/MS on a Waters Quattro
II triple quadrupole mass spectrometer with a Prism RP 1.times.100
mm, 5 .mu.m particle size column (Keystone Scientific). Isocratic
elution with 4 mM ammonium formate in 20% isopropyl alcohol 80%
methanol at a flow rate of 50 .mu.l/min Ten .mu.l of each sample
was injected. MRM analysis was performed using m/z 882.7>197.00
(Q10H2) and m/z 880.80>197.00 (Q10) transitions with cone
voltage of 40 and collision energy of 30.
Example 3
Sensitivity of Cell Lines to CoQ10
[0328] A number of cell lines were tested for their sensitivity to
Q10 after 24 hours of application by using a reagent (Nexin
reagent) that contains a combination of two dyes, 7AAD and
Annexin-V-PE. The 7AAD dye will enter into cells with permeabilized
cell membranes; primarily those cells that are in late apoptosis.
Annexin-V-PE is a dye that binds to Phosphotidyl serine, which is
exposed on the outer surface of the plasma membrane in early
apoptotic cells. The Nexin reagent thus can be used to
differentiate between different populations of apoptotic cells in a
flow cytometer.
[0329] PaCa2 cells showed an increase in both early and late
apoptotic cells (between 5-10% of gated cells) with 50 .mu.M Q10
and 100 .mu.M Q10 after 24 hours of Q10 application. PC-3 cells
also showed an increase in both early and late apoptotic population
with 50 .mu.M and 100 .mu.M Q10, although the increase was less
when compared to PaCa2 cells. MCF-7 and SK-MEL28 cells showed an
increase only in early apoptotic population with 50 .mu.M and 100
.mu.M Q10. HepG2 cells were also sensitive to 50 .mu.M Q10
treatment, where there was an increase of about 20% of the gated
populated in the late apoptotic and early apoptotic stages. SKBr3
was the only cell line tested that did not show any significant
increases of early and late apoptosis with either 50 .mu.M or 100
.mu.M Q10 treatment. The results are depicted in FIGS. 1-6.
[0330] To provide additional confirmation that Q10 treatment causes
an apoptotic response in HepG2 liver cancer cells, a second
apoptosis assay was evaluated using the ApoStrand.TM. ELISA based
method that measures single-stranded DNA. The ApoStrand.TM. ELISA
is based on the sensitivity of DNA in apoptotic cells to formamide
denaturation and the detection of the denatured DNA with a
monoclonal antibody to single-stranded DNA (ssDNA). Treatment of
the liver cancer cell line HepG2 with 50 and 100 .mu.M Q10 resulted
in detectable apoptosis, with a dose-response of 17% and 32%,
respectively (FIG. 7). These results are consistent with the
observation of Q10 inducing apoptosis in other cancer cell lines
from other tissues (e.g., SCC, SKMEL-28, MCF-7, and PC-3).
Example 4
Proteomic Analysis of Cells Treated with Q10
[0331] Cell pellets of samples treated with Q10 were analyzed using
proteomic methods. The cell pellets were lysed and treated for use
in 2-D gel and Western blot analysis. Three cell types (SKMEL-28,
SCC, and nFib) were treated with Q10 and submitted to proteomic
characterization by 2-D gel electrophoresis.
Proteomic Analysis of SKMEL-28 Cells Treated with Q10
[0332] The first experimental set processed and evaluated by
Western blot and 2-D gel electrophoresis was the skin cancer cell
line SKMEL-28. This experimental set involved SK-MEL-28 cells
treated at 3, 6, 12, and 24 hours with 0, 50 or 100 .mu.M Q10.
[0333] The set of Q10 treated SK-MEL-28 samples were subjected to
2-D gel electrophoreses (FIG. 8) and were analyzed to identify
protein-level changes relative to the control samples. A
comparative analysis of 943 spots across all twenty-four gels was
performed, comparing the control sample against all of the treated
samples. The analysis included the identification of spot changes
over the time course due to increase, decrease, or
post-translational modification.
[0334] The analysis found thirty-two statistically significant
differential spot changes. From this, twenty non-redundant spots
were excised and submitted for protein identification by trypsin
digestion and mass spectrometry characterization. The characterized
peptides were searched against protein databases with Mascot and
MSRAT software analysis to identify the protein (Table 2).
TABLE-US-00002 TABLE 2 Proteins identified to have a differential
response to Q10 treatment in SKMEL-28 cell. Q10 Time Conc. 2D (hr)
(uM) Spot # Expression Difference Protein Name Type 3 50 528 down
1.234 cathepsin D CTSD peptidase 3 50 702 down 1.575 chaperonin
containing CCT3 other TCP1, subunit 3 3 50 74 down 1.383 eukaryotic
translation EIF3G translation initiation factor 3 regulator 3 50
829 down 1.074 Ribosomal protein P2 RPLP2 other 3 50 368 down 1.121
transaldolase 1 TALDO1 enzyme 6 50 452 up -1.464 eukaryotic
translation EIF6 translation initiation factor 6 regulator 6 50 175
up -1.32 Stomatin; HSPC322 STOM other 6 50 827 up -1.457 Tyrosine
3/Tryptophan YWHAZ enzyme 5-monooxygenase activation protein 6 50
139 up -1.628 Vimentin VIM other 6 50 218 up -1.416 Vimentin VIM
other 6 50 218 up -1.212 Vimentin VIM other 6 50 139 up -1.036
Vimentin VIM other 6 50 507 down 1.379 Lamin B1 LMNB1 other 6 50
571 down 1.832 mitochandrial import TOMM22 transporter receptor
Tom22 12 50 166 up -1.171 ALG-2 interacting PDCD6IP other protein 1
12 50 550 up -1.747 peptidylprolyl PPIA enzyme isomerase A 12 50
613 down 1.802 galectin-1 LGALS1 other 12 50 242 down 1.373
Phosphoglycerate mutase; PGAM2 phosphatase Posphomannomutase 2 24
50 326 down 1.385 glycyl-tRNA synthase GARS enzyme 24 50 419 down
1.451 Mago-nashi homolog MAGOH other 3 100 528 down -1.036
cathepsin D CTSD peptidase 3 100 702 down 1.151 chaperonin
containing CCT3 other TCP1, subunit 3 3 100 74 down 1.122
eukaryotic translation EIF3G translation initiation factor 3
regulator 3 100 829 down 1.145 Ribosomal protein P2 RPLP2 other 3
100 368 down 1.209 transaldolase 1 TALDO1 enzyme 6 100 139 up
-1.829 Vimentin VIM other 6 100 218 up -1.761 Vimentin VIM other 6
100 452 down 1.134 eukaryotic translation EIF6 translation
initiation factor 6 regulator 6 100 252 down 1.4 Sec 13 protein, ?
Keratin II 6 100 827 down 1.12 Tyrosine 3/Tryptophan YWHAZ enzyme
5-monooxygenase activation protein 12 100 76 up -1.679 galectin-1;
keratin II LGALS1 other
[0335] A key finding in this experiment was the decrease of
Transaldolase 1, which supports the premise that Q10 acts by
altering the metabolic state within the cancer cell. Transaldolase
1 is an enzyme in the pentose phosphate pathway (also known as the
hexose monophosphate shunt). Transaldolase (EC:2.2.1.2) catalyses
the reversible transfer of a three-carbon ketol unit from
sedoheptulose 7-phosphate to glyceraldehyde 3-phosphate to form
erythrose 4-phosphate and fructose 6-phosphate. This enzyme,
together with transketolase, provides a link between the glycolytic
and pentose-phosphate pathways. This is relevant to nucleotide and
NADPH synthesis, to facilitate production of reducing equivalents
for biosynthetic reactions and maintenance of a reducing
environment.
[0336] A recent publication (Basta, P., et. al. August 2008, Cancer
Detect Prevention, 32, 200-208) provided evidence of genetic
polymorphism in Transaldolase and was linked to squamous cell
carcinoma of the head and neck. Another recent publication (Qian,
Y., et. al. May 2008, Biochem J, 415, 123-134) identified
transaldolase deficiency as a modulator of mitochondrial
homoeostasis, Ca2+ fluxing and apoptosis.
[0337] From these initial results, the other proteins identified by
2-D gel electrophoresis as being modulated by Q10 in SK-MEL-28 were
analyzed for known relationships (FIG. 9). A functional evaluation
of these proteins revealed that there was a group involved in
14-3-3-mediated signaling (PDCP61P, YWHAZ, and VIM), along with
individual proteins linked to a variety of processes [cell cycle;
pentose phosphate pathway (TALDO1); ceramide signaling (CTSD);
aminoacyl-tRNA biosynthesis (GARS), and mitochondrial protein
import (TOM22)].
Proteomic Analysis of SCC Cells Treated with Q10
[0338] Another skin cancer cell line, Squamous Cell Carcinoma
(SCC), was also prepared and analyzed by 2-D gel electrophoreses as
a follow-up experiment the previous SK-MEL-28 analysis The SCC
cells were treated with 100 .mu.M Q10 for 6 hour or 24 hours before
harvesting. A control of untreated cells was also harvested. The
cell pellets were lysed and the samples were subjected to 2-D
electrophoresis (in duplicate). Analysis of over six hundred
protein spots in the comparative study was performed, comparing the
control sample against the six hour and twenty-four hour
treatments.
[0339] The top twenty-five statistically significant differential
spot changes were evaluated from the comparative analysis of the
2-D electrophoresis gels. From this, twelve spots were excised and
submitted for identification by trypsin digestion and mass
spectrometry characterization (results summarized in Table 3
below).
TABLE-US-00003 TABLE 3 Proteins identified to have a differential
response to 100 .mu.M Q10 treatment in SCC cells at 6 and 24 hours.
Spot Cellular Response # Protein Name localization Function (fold
change) 331 Transaldolase 1 TALDO1 Cytoplasm Enzyme Decrease (1.5)
at 6 and 14 hr 23 Human BSCv C20ORF3 Plasma strictosidine Decrease
(chromosome 20 membrane synthase (2.1) at 6 and reading frame 3) 24
hr 54 NM23 protein NME1 Nucleus, Kinase Increase (mitochondria?)
(-1.2) at 6 hr, decrease at 24 hr 116 two Human ESTs HSP70 Decrease
from MCF7 (2.6) at 6 hr, breast cancer cell further decrease line
(HSP 70) at 24 hr 176 Heat shock HSPB1 Cytoplasm Response to
Increase 27 kDa protein 1 environmental (-1.9) at 6 and stresses 24
hr 135 Keratin I KRT1 Cytoplasm intermediate Decrease filaments
(2.3) at 6 and 24 hr 50 Keratin 14 KRT14 Cytoplasm intermediate
Increase filaments (-1.6) at 6 and 24 hr 68 Keratin 13 KRT13
Cytoplasm intermediate Increase filaments (-1.5) at 6 and 24 hr 49
Proteasome PSMB7 Cytoplasm Proteasome Decrease Beta 7 subunit (1.6)
at 24 hr only 93 Proteasome PSME3 Cytoplasm peptidase Decrease
activator (1.3) at 24 hr subunit 3 only 66 Rho GDP ARHGDIA
Cytoplasm Inhibitor Decrease dissociation (1.5) at 6 hr inhibitor
(GDI) only alpha 1 Unknown? Decrease (9.5)
[0340] Transaldolase 1: As previously observed in the SKMEL-28
cells treated with Q10, the enzyme Transaldolase 1 was modulated
with a decrease in levels. This provides an independent
confirmation of the previously observation of a linkage between Q10
and alterations in transaldolase (and thus the metabolic state of
the cell).
[0341] Transaldolase is an enzyme in the non-oxidative phase of the
pentose phosphate pathway (FIG. 10). The pentose phosphate pathway
is critical in the metabolic state of cells for the generation of
nicotinamide adenine dinucleotide phosphate (reduced NADH), for
reductive biosynthesis, and in the formation of ribose which is an
essential component of ATP, DNA, and RNA. Transaldolase also links
the pentose phosphate pathway to glycolysis. Glycolysis is the
metabolic pathway by which cancer cells obtain the energy needed
for cell survival, as the mitochondrial process of oxidative
phosphorylation is not utilized. Q10 is an essential coenzyme
factor required for oxidatative phosphorylation and mitochondrial
ATP production.
[0342] BSCv: Spot 23 was a novel human protein from Chromosome 20
named BSCv. BSCv protein is also known as Adipocyte plasma
membrane-associated protein (Gene names: APMAP or C20orf3) and is
predicted to be a single-pass type II membrane protein with
sequence similarity to the strictosidine synthase family of
proteins. Q10 treatment caused a reduction in the levels of this
protein. This protein is not well characterized, nor has its
homology with strictosidine synthases been confirmed.
Interestingly, this protein has been associated with a role in
adipocyte differentiation (Albrektsen et al., 2001). Recent
proteomic studies of human omental adipose tissue identified BSCv
as one of nine proteins with differential expression for polcystic
ovary syndrome (PCOS) from morbidly obese women (Corton, 2008 Hum.
Reprod. 23: 651-661). As a cell surface protein that responds to
Q10, an antibody against BSCv would be useful as a biomarker. Based
on the current results and the literature available, BSCv may a
have a potential role in cancer and diabetes.
[0343] NM23A: Non-metastatic cells 1, protein (NM23A, also known as
NME1) is thought to be a metastasis suppressor. This gene (NME1)
was identified because of its reduced mRNA transcript levels in
highly metastatic cells. The protein has activity as a nucleoside
diphosphate kinase (NDK) and exists as a hexamer composed of `A`
(encoded by this gene) and `B` (encoded by NME2) isoforms.
Mutations in this gene have been identified in aggressive
neuroblastomas. NDK activities maintain an equilibrium between the
concentrations of different nucleoside triphosphates such as, for
example, when GTP produced in the citric acid (Krebs) cycle is
converted to ATP. The NDK complex is associated with p53 through
interaction with STRAP. It is noteworthy that STRAP is linked to
HNF4A. Thus, NM23A is a potential protein involved in pathways
important for cell control and disease treatment.
[0344] Rho GDP dissociation inhibitor (GDI) alpha: GDI Regulates
the GDP/GTP exchange reaction of the Rho proteins by inhibiting the
dissociation of GDP from them, and the subsequent binding of GTP to
them. The protein is upregulated in cancer cells.
Example 5
Mitochondrial Enrichment Analysis
[0345] Several lines of evidence suggested that a closer evaluation
of the role of mitochondrial proteins and cancer biology and Q10
response was warranted. First, there is the essential role of Q10
in the mitochondrial oxidative phosphorylation process for energy
production in normal cells. However, the metabolic shift that
occurs in cancer cells is to energy production through the
alternative pathway of glycolysis, which does not require Q10.
Second, the apoptotic response of cells requires mitochondrial
proteins to occur. Q10 has been established as stimulating
apoptosis in cancer cells (Bcl-2 family proteins, cytochrome c).
Finally, new mitochondrial proteins were identified as being
modulated by Q10 treatment, as exemplified by the modulation in
protein levels of the mitochondrial import receptor protein TOM22
(see experiments described herein).
Production of Mitochondrial Enriched Samples
[0346] The skin cancer SKMEL-28 cells were treated with 100 .mu.M
Q10 or a mock vehicle for 6, 19, or 48 hours. The cells were
harvested by washing and scraping the cells from T-160 flasks (4
for each time point). The cells were collected by centrifugation
and the pellets flash frozen and stored at -80.degree. C. The cell
pellets were resuspended and ruptured using a 2 mL Dounce
homogenizer. The reagents and method were obtained from a
Mitochondria Isolation Kit for Cultured Cells (MitoSciences, Cat#
MS852). The resultant mitochondria samples were divided into 75
.mu.L aliquots (4-5 aliquots per sample) and stored at -80.degree.
C.
Proteomic Analysis of Mitochondria Enriched Samples Isolated from
SK-MEL-28 Cells Treated with Q10
[0347] 2-D gel electrophoresis was performed on proteins
solubilized from two aliquots of the SK-MEL-28 mitochondria
enriched samples treated with 100 .mu.M Q10 for 6, 19, and 48 hours
(along with the corresponding mock vehicle controls). The samples
were subjected to 2-D electrophoresis (in duplicate). Analysis of
525 protein spots in the comparative study was performed, comparing
the control samples against the other time point samples (FIG.
11).
[0348] The nine statistically significant differential spot changes
were selected from the comparative analysis of the 2-D
electrophoresis gels. From these, 9 spots were excised and
submitted for identification by trypsin digestion and mass
spectrometry characterization
TABLE-US-00004 TABLE 4 Proteins identified to have a differential
response to Q10 treatment in SKMEL-28 mitochondria. Spot Response
(fold # Protein Name Function change) 11 Unknown protein ? ? Up
(1.3) at 6 hr, drop to low levels after this 131 Unknown, same as ?
? Down (1.3) at 6 hr, spot #11, modified drops more for 19 and 48
hr 279 acyl-CoA thioesterase ACOT7 Cleaves fatty acyl- Down (1.3)
at 6 hr, 7 isoform hBACHb CoA's into free fatty back to normal at
48 acids and CoA hr 372 Pyruvate kinase PKM2 catalyzes the Up (1.5)
at 6 hr, back production of to normal at 48 hr phosphoenolpyruvate
from pyruvate and ATP 110 ER60 protein PDIA3 Protein disulfide Up
at 19 and 48 hr isomerase 185 Keratin 10 KRT10 intermediate
filament Up only at 19 hr 202 Beta-Actin Structural protein Up only
at 19 hr 246 Malectin MLEC carbohydrate-binding Up only at 19 hr
protein of the endoplasmic reticulum and a candidate player in the
early steps of protein N-glycosylation 75 Coiled-coil domain CCDC58
Conserved hypothetical Up at 48 hr containing 58 protein- nuclear
pore forming
[0349] Acyl-CoA thioesterase 7: Acyl-CoA thioesterase 7 (ACOT7) is
a member of the enzyme family that catalyzes the hydrolysis of
fatty acyl-CoA to free fatty acid and CoA. This enzyme thus has a
role in the regulation of lipid metabolism and cellular signaling.
ACOT7 has a preference for long-chain acyl-CoA substrates with
fatty acid chains of 8-16 carbon atoms (C8-C16). The exact cellular
function is ACOT7 is not fully understood. The transcription of
this gene is activated by sterol regulatory element-binding protein
2, thus suggesting a function in cholesterol metabolism.
[0350] The results in this Example indicate that ACOT7 is
potentially involved in the metabolism of Q10, either directly or
indirectly. Thus, targeting ACOT7 could facilitate modulation of
intercellular levels of Q10 and thus impact cellular Q10
effects.
[0351] Pyruvate kinase: Pyruvate kinase is an enzyme involved in
the last step of glycolysis. It catalyzes the transfer of a
phosphate group from phosphoenolpyruvate (PEP) to ADP, yielding one
molecule of pyruvate and one molecule of ATP.
##STR00003##
The protein is presumably that of PKM2, the type 2 isoform, as this
was identified from the mitochondria enriched SK-MEL-28 sample.
This isoform is well known to be involved in tumor cell formation
and regulation.
Quantification of Q10 Levels in Mitochondria
[0352] A method for the simultaneous determination of Coenzyme Q10,
(Q10) and the reduced form ubiquinol-10 (Q10H2) was implemented
based upon a recently published method (Ruiz-Jimenez, 2007, J.
Chroma A, 1175, 242-248) through the use of LC-MS-MS with
electrospray ionization (ESI) in the positive mode. The highly
selective identification and sensitive quantitation of both Q10 and
Q10H2 is possible, along with the identification of other selected
lipids. An aliquot of the mitochondrial enriched samples from
SK-MEL-28 treated with 100 .mu.M Q10 were subject to a conventional
pre-treatment based on protein precipitation, liquid-liquid
extraction, evaporation to dryness and reconstitution with 95:5
methanol/hexane (v/v).
[0353] In this analysis, Q10, Q10H2, and Q9 were quantitated (Table
5). The levels of the related molecule Q9 were low, and near the
level of detection. The level of the untreated samples were
relatively consistent, with the 6 hour Q10 treated sample having
this same level. To control for sample variance in total material,
the levels of cholesterol was also measured to confirm that the
differences were not due to sample size errors. When the Q10 levels
were corrected against total protein values obtained by protein
extraction other aliquots of the same mitochondrial preps, the
relative ratios were comparative. Thus, a significant increase in
Q10 levels was obtained at 19 hours (.about.3-fold) with an even
larger increase by the 48 hour time point (.about.6-fold) (FIG.
12).
TABLE-US-00005 TABLE 5 HPLC-MS Quantification results for the
levels of Q10 present in mitochondrial enriched samples from
SK-MEL-28 cells treated with 100 .mu.M Q10 in the media. Peak Area
ng/Sample .mu.g/sample File Sample Injection Q9 Q10 Q9 Q10
Q10H.sub.2 Cholesterol 081204-05 100 ng Std 245,342 352792
081204-06 6 hr mock#1 10% 2560 32649 1.04 9.25 081204-07 Solvent
Blank#1 5 ul 3781 3174 1.54 0.9 081204-08 Solvent Blank#2 5 ul 2396
4399 0.98 1.25 081204-09 6 hr mock#2 20% 1572 36328 0.64 10.3
081204-10 Solvent Blank#3 10 ul 1722 2504 0.7 0.71 081204-11 48 hr
Q10 treated 20% 4879 164496 1.99 46.63 0.28 13.86 081204-12 48 hr
mock 20% 2412 25552 0.98 7.24 0.09 13.04 081204-13 6 hr Q10 treated
20% 692 25427 0.28 7.21 081204-14 19 hr Q10 treated 20% 1161 59164
0.47 16.77 081204-15 19 hr mock 20% 901 19999 0.37 5.67
[0354] A surprising result from this study was the finding that the
Q10 was supplied to the cells as the oxidized form. For the 48 hour
samples, the reduced form Q10H2 was also measured and found to be
present in significantly lower amounts (0.28 ng/sample of CoQ10H2
as compared to 46.63 ng/sample of CoQ10). There was a general
increase (3-fold) in the levels of Q10H2 in the Q10 treated 48 hour
sample, although the levels were near the presumed detection limit
of the assay. Interestingly, the oxidized form (Q10) can act as a
pro-oxidant in biological systems. According to the literature,
when human plasma was evaluated for Q10 and Q10H2, the majority
(90%) of the molecule was found in the reduced form of Q10H2
(Ruiz-Jimenez, 2007, J. Chroma A, 1175, 242-248) which can act as
an anti-oxidant.
[0355] Thus, these results confirm and quantitate that the levels
of Q10 increase in the mitochondria upon the exogenous addition of
Q10 to the media. A surprising and unexpected discovery was that
Q10 was maintained in the supplied oxidized form (pro-oxidant) and
not converted to the reduced (anti-oxidant) form of Q10H2 in any
significant amounts.
Example 6
Real-Time PCR Arrays
Experiment 1
Apoptosis Array
[0356] As discussed above in Example 3, exposure of cancer cells to
Q10 induces a portion of these cells to die due to apoptotic
processes. To identify proteins that were involved in the Q10
response, real-time polymerase chain reaction (RT-PCR) methods were
employed to identify changes in the level of mRNA for
genes/proteins involved in targeted pathway arrays for
apoptosis.
[0357] Using PCR arrays as a screening tool, a spectrum of
molecular targets that would potentially offer an insight to the
mode of biological action of Q10 within the cells were thus
evaluated. Changes in mRNA levels were evaluated using real-time
PCR quantification to assess mRNA levels in pre-selected subsets
containing 80 pathway specific targets.
[0358] For the interpretation of mRNA results, the genes that were
altered in their mRNA transcription by a two-fold level were
identified and evaluated. The level of gene transcription to
produce mRNA only provides a rough estimate of potential changes in
the level of the expressed protein. The skilled artisan will
appreciate that each mRNA may have different rates at which it is
degraded or its translation inefficiently, thus resulting in
differing amounts of protein.
SkBr-3 Cells Treated with 50 um Q10 for 24 Hours
[0359] The assay method of RT-PCR was utilized to provide a measure
of mRNA level changes to a total of 84 apoptotic pathway related
proteins. The experiments with the real-time PCR apoptosis analysis
on SkBr3 with Q10 (24 hr) identified the following mRNA's being
affected: Bcl2, Bcl2L1, Bcl2L11, Birc6, Bax, Xiap, Hprt1, Apaf1,
Ab11, Braf. These results again provided supporting evidence for
the apoptotic response of cancer cells to Q10 treatment.
TABLE-US-00006 TABLE 6A Up-Down Symbol Regulation Unigene Refseq
Description Gname BCL2L1 13.1957 Hs.516966 NM_138578 BCL2-like 1
BCL-XL/S BNIP2 6.3291 Hs.646490 NM_004330 BCL2/adenovirus E1B 19
kDa BNIP-2/NIP2 interacting protein 2 BCL2 5.4717 Hs.150749
NM_000633 B-cell CLL/lymphoma 2 Bcl-2 BIRC6 4.7966 Hs.150107
NM_016252 Baculoviral IAP repeat- APOLLON/ containing 6 (apollon)
BRUCE BCL2L11 4.6012 Hs.469658 NM_006538 BCL2-like 11 (apoptosis
BAM/BIM facilitator) XIAP 4.3832 Hs.356076 NM_001167 X-linked
inhibitor of API3/BIRC4 apoptosis BRAF 4.3832 Hs.550061 NM_004333
V-raf murine sarcoma viral B-raf 1/ oncogene homolog B1 BRAF1 BAX
3.896 Hs.631546 NM_004324 BCL2-associated X protein Bax zeta APAF1
2.6244 Hs.708112 NM_001160 Apoptotic peptidase CED4/DKFZp
activating factor 1 781B1145 HPRT1 -160.6748 Hs.412707 NM_000194
Hypoxanthine HGPRT/HPRT phosphoribosyltransferase 1 (Lesch-Nyhan
syndrome)
[0360] Results that are consistent from three independent
experiments from SK-MEL-28 cells are summarized below in Table 6B.
Many genes are regulated in SCC cells as well with 100 .mu.M Q10
treatment. The genes in the Apoptosis array that appear to be
regulated in SCC cells are described in Table 7. We find that many
genes are regulated at 6 hours, both in SK-MEL-28 cells and in SCC
cells. By 24 hours, the regulation is decreased. Genes that appear
to be regulated in both SK-MEL-28 cells and in SCC cells are
described in Table 8.
TABLE-US-00007 TABLE 6B Genes in SK-MEL-28 cells regulated by 100
.mu.M Q10 treatment when analyzed by the Apoptosis Array. Possible
Symbol Description Regulation Location Functions ABL1 C-abl
oncogene 1, Down Regulated Nucleus Tyrosine Kinase receptor
tyrosine at 72 hours kinase BAG1 BCL2-associated Up Regulated at
Cytoplasm Anti-apoptotic, athanogene 48 hours glucocorticoid
receptor pathway BCL2 B-cell Down Regulated Cytoplasm Cell death
CLL/lymphoma 2 at 48 hours BCL2A1 BCL2-related Down Regulated
Cytoplasm Regulates protein A1 at 48 hours Caspases, phosphorylates
TP73 BCL2L1 BCL2-like 1 Down Regulated Cytoplasm Caspase Inhibitor
at 72 hours BCL2L10 BCL2-like 10 Down Regulated Cytoplasm Caspase
Activator (apoptosis at 48 hours facilitator) BCL2L11 BCL2-like 11
Down Regulated Cytoplasm Pro-Apoptotic, (apoptosis at 48 hours
Caspase3 facilitator) Activator BIRC3 Baculoviral IAP Down
Regulated Cytoplasm Anti-apoptotic repeat-containing 3 at 6 hours
BIRC8 Baculoviral IAP Down Regulated Cytoplasm Activates Caspase
repeat-containing 8 at 48 hours CARD8 Caspase recruitment Down
Regulated Nucleus Caspase Activator domain family, at 48 hours
member 8 CASP14 Caspase 14, Down Regulated Cytoplasm Apoptosis
related apoptosis-related at 48 hours cysteine peptidase cysteine
peptidase CASP5 Caspase 5, Down Regulated Cytoplasm Apoptosis
related apoptosis-related at 48 hours cysteine peptidase cysteine
peptidase CD40LG CD40 ligand (TNF Down Regulated Extracellular CD40
receptor superfamily, at 48 hours Space binding member 5, hyper-
IgM syndrome) CIDEA Cell death-inducing Up Regulated at Cytoplasm
Pro-Apoptotic DFFA-like effector 48 hours a FADD Fas (TNFRSF6)-
Down Regulated Cytoplasm Pro-Apoptotic associated via death at 6
hours domain FAS Fas (TNF receptor Up Regulated at Plasma
Pro-Apoptotic superfamily, 48 hours Membrane member 6) FASLG Fas
ligand (TNF Down Regulated Extracellular Pro-Apoptotic superfamily,
at 48 hours Space member 6) GADD45A Growth arrest and Up Regulated
at Nucleus Growth Arrest DNA-damage- 48 hours inducible, alpha HRK
Harakiri, BCL2 Down Regulated Cytoplasm Pro-Apoptotic interacting
protein at 48 hours (contains only BH3 domain) PYCARD PYD and CARD
Down Regulated Cytoplasm Apoptotic domain containing at 6 hours
Protease Activator TNF Tumor necrosis Up Regulated at Extracellular
TNF receptor factor (TNF 48 hours then Space binding superfamily,
down regulated member 2) TNFRSF10A Tumor necrosis Up Regulated at
Plasma Caspase Activator factor receptor 48 hours then Membrane
superfamily, down regulated member 10a TNFRSF10B Tumor necrosis
Down Regulated Plasma p53 signaling, factor receptor at 72 hours
Membrane caspase superfamily, activation. member 10b TNFRSF1A Tumor
necrosis Down Regulated Plasma Pro-apoptotic factor receptor at 72
hours Membrane superfamily, member 1A TNFRSF21 Tumor necrosis Down
Regulated Plasma Activates Caspase factor receptor at 48 hours
Membrane superfamily, member 21 CD27 CD27 molecule Down Regulated
Plasma Caspase Inhibitor at 48 hours Membrane TNFRSF9 Tumor
necrosis Down Regulated Plasma Pro-apoptotic factor receptor at 48
hours Membrane superfamily, member 9 TNFSF10 Tumor necrosis
Upregulated at 48 Extracellular Pro-apoptotic factor (ligand) hours
Space superfamily, member 10 TP73 Tumor protein p73 Down Regulated
Nucleus Transcription at 48 hours factor TRAF3 TNF receptor- Down
Regulated Cytoplasm Zinc-finger associated factor 3 at 48 hours
domain TRAF4 TNF receptor- Down Regulated Cytoplasm Zinc-finger
associated factor 4 at 48 hours domain
TABLE-US-00008 TABLE 7 Genes in SCC cells that are regulated by 100
.mu.M Q10 treatment when analyzed by the Apoptosis Array. Symbol
Description Regulation. AKT1 V-akt murine thymoma viral oncogene
Down regulated at 6 hours and homolog 1 then up regulated at 24
hours. BAG4 BCL2-associated athanogene 4 Up regulated at 24 hours.
BAX BCL2-associated X protein Up regulated at 24 hours. BCL2 B-cell
CLL/lymphoma 2 Up regulated at 24 hours. BCL2L1 BCL2-like 1 Down
regulated at 6 hours and then up regulated at 24 hours. BIRC3
Baculoviral IAP repeat-containing 3 Down regulated at 6 hours.
BNIP3 BCL2/adenovirus E1B 19kDa interacting Down regulated at 24
hours. protein 3 CARD6 Caspase recruitment domain family, Down
regulated at 6 hours. member 6 CASP6 Caspase 6, apoptosis-related
cysteine Up regulated at 24 hours. peptidase CASP7 Caspase 7,
apoptosis-related cysteine Up regulated at 24 hours. peptidase CD40
CD40 molecule, TNF receptor superfamily Down regulated at 6 hours.
member 5 FADD Fas (TNFRSF6)-associated via death Up regulated at 24
hours. domain GADD45A Growth arrest and DNA-damage-inducible, Up
regulated at 24 hours. alpha HRK Harakiri, BCL2 interacting protein
(contains Up regulated at 24 hours. only BH3 domain) TNFRSF21 Tumor
necrosis factor receptor Down regulated at 6 hours. superfamily,
member 21 TNFRSF25 Tumor necrosis factor receptor Down regulated at
6 hours and superfamily, member 25 then up regulated at 24 hours.
CD27 CD27 molecule Down regulated at 6 hours. TNFRSF9 Tumor
necrosis factor receptor Down regulated at 6 hours. superfamily,
member 9 TNFSF10 Tumor necrosis factor (ligand) superfamily, Up
regulated at 24 hours. member 10 CD70 CD70 molecule Down regulated
at 6 hours. TP53 Tumor protein p53 Up regulated at 24 hours. TP73
Tumor protein p73 Down regulated at 6 hours and then up regulated
at 24 hours. TRAF2 TNF receptor-associated factor 2 Up regulated at
24 hours.
TABLE-US-00009 TABLE 8 Genes from the apoptosis array regulated
with 100 .mu.M Q10 treatment in both SK-MEL-28 and SCC cells.
Symbol Description BCL2 B-cell CLL/lymphoma 2 BCL2L1 BCL2-like 1
(Bcl-xl) BIRC3 Baculoviral IAP repeat-containing 3 FADD Fas
(TNFRSF6)-associated via death domain GADD45A Growth arrest and
DNA-damage-inducible, alpha TNFRSF21 Tumor necrosis factor receptor
superfamily, member 21 CD27 CD27 molecule TNFRSF9 Tumor necrosis
factor receptor superfamily, member 9 TNFSF10 Tumor necrosis factor
(ligand) superfamily, member 10 TP73 Tumor protein p73 TRAF2 TNF
receptor-associated factor 2
[0361] Interestingly, the altered mRNA levels showed a significant
up-regulation in a series of apoptitic proteins, with Bcl-xl one of
the highest. This was also observed in the protein array
experiments on SK-MEL-28 cells.
[0362] Bcl-x1 is a transmembrane molecule in the mitochondria
(Bcl-x1 stands for "Basal cell lymphoma-extra large"). It is
involved in the signal transduction pathway of the FAS-L and is one
of several anti-apoptotic proteins which are members of the Bcl-2
family of proteins. It has been implicated in the survival of
cancer cells. However, it is known that alternative splicing of
human Bcl-x mRNA may result in at least two distinct Bcl-x mRNA
species, Bcl-xL and Bcl-xS. The predominant protein product (233
amino acids) is the larger Bcl-x mRNA, Bcl-xL, which inhibits cell
death upon growth factor withdrawal (Boise et al., 1993. Cell 74,
597-608). Bcl-xS, on the other hand, inhibits the ability of Bcl-2
to inhibit cell death and renders cells more susceptible to
apoptotic cell death. The employed assays utilized do not
distinguish which isoform of Bcl-x is being upregulated. The Bcl-x
isoform being upregulated by CoQ10 in these studies may be
determined by routine methods known in the art, e.g., by using
RT-PCR methods to evaluate the ratio of the two mRNA splicing
isoforms (Bcl-xL vs Bcl-sL).
[0363] From the survey of apoptotic related proteins it was
observed multiple pro- and anti-apoptotic factors were in the BCL-2
family or that interact with these factors have modulated
expression levels (BCL2L11, BNIP2, BAG1, HRK, BAK1, BCL2, BCL2L1).
These proteins govern mitochondrial outer membrane
permeabilization.
[0364] An early marker for apoptotic response is observed with the
upregulation of Caspase-9 (16 hour) which is consistent with
previous observations of apoptosis with caspase 3/7 proteins.
Induction of stress signaling pathways causes release of cytochrome
c from mitochondria and activation of apaf-1 (apoptosome), which in
turn cleaves the pro-enzyme of caspase-9 into the active form. Once
intiated caspase-9 goes on to cleave procaspase-3 &
procaspase-7 to trigger additional apoptotic pathways.
[0365] There is also a consistent linkage to the tumor necrosis
factor receptor family of proteins being modulated.
[0366] A strong down regulation of tumor protein p73 is also noted.
Analyses of many tumors typically found in humans including breast
and ovarian cancer show a high expression of p73 when compared to
normal tissues in corresponding areas. Recent finding are
suggesting that deregulated over expression of transcription
factors within the body involved in cell cycle regulation and
synthesis of DNA in mammalian cells (i.e.: E2F-1), induces the
expression of p73. The suggestion is that p73 may be an
oncoprotein, but may involve different mechanism that the related
p53 protein. A schematic showing mapping of the apoptosis pathway
is provided in FIG. 13.
SKMEL-28 Cells
[0367] From the survey of apoptotic related proteins it was
observed multiple pro- and anti-apoptotic factors were in the BCL-2
family or that interact with these factors have modulated
expression levels (BCL2L11, BNIP2, BAG1, HRK, BAK1, BCL2, BCL2L1).
These proteins govern mitochondrial outer membrane
permeabilization.
[0368] An early marker for apoptotic response is observed with the
upregulation of Caspase-9 (16 hour) which is consistent with
previous observations of apoptosis with caspase 3/7 proteins.
Induction of stress signaling pathways causes release of cytochrome
c from mitochondria and activation of apaf-1 (apoptosome), which in
turn cleaves the pro-enzyme of caspase-9 into the active form. Once
intiated caspase-9 goes on to cleave procaspase-3 &
procaspase-7 to trigger additional apoptotic pathways.
TABLE-US-00010 TABLE 9 Changes in mRNA levels for SKMEL-28 cells
treated with 100 .mu.M A10, evaluated by RT-PCR arrays focused
around apoptotic pathways. 6 hr 16 hr 24 hr 72 hr Refseq
Description Symbol Q10 Q10 Q10 Q10 NM_006538 BCL2-like 11 BCL2L11
2.13 2.41 1.92 2.51 (apoptosis facilitator) NM_000875 Insulin-like
growth IGF1R 1.77 1.09 1.33 1.25 factor 1 receptor NM_004048
Beta-2-microglobulin B2M 1.74 1.76 1.58 3.11 NM_003921 B-cell
CLL/lymphoma 10 BCL10 1.55 1.87 1.48 -3.11 NM_004330
BCL2/adenovirus E1B 19 kDa BNIP2 1.46 1.51 1.57 -1.61 interacting
protein 2 NM_005157 C-abl oncogene 1, receptor ABL1 1.42 2.77 -1.22
-2.03 tyrosine kinase NM_004323 BCL2-associated athanogene BAG1
1.41 1.44 -1.61 -2.45 NM_001229 Caspase 9, apoptosis-related CASP9
1.32 3.96 1.83 1.14 cysteine peptidase NM_003806 Harakiri, BCL2
interacting HRK 1.18 4.52 2.73 -1.14 protein (contains only BH3
domain) NM_001924 Growth arrest and DNA-damage- GADD45A 1.07 3.34
1.13 -2.36 inducible, alpha NM_001188 BCL2-antagonist/killer 1 BAK1
1.06 2.73 -1.00 -4.54 NM_004295 TNF receptor-associated TRAF4 -1.91
2.63 -1.58 -740.66 factor 4 NM_003842 Tumor necrosis factor
receptor TNFRSF10B -2.07 1.53 -1.81 -710.49 superfamily, member 10b
NM_000633 B-cell CLL/lymphoma 2 BCL2 -2.98 -1.63 -2.82 -11.36
NM_001242 CD27 molecule CD27 -3.40 -2.38 -1.35 -12.72 NM_014430
Cell death-inducing CIDEB -3.48 1.56 -3.69 -2.59 DFFA-like effector
b NM_001065 Tumor necrosis factor receptor TNFRSF1A -4.53 2.28
-3.30 1.22 superfamily, member 1A NM_005427 Tumor protein p73 TP73
-4.66 -9.80 -8.71 -26.96 NM_003844 Tumor necrosis factor receptor
TNFRSF10A -4.84 -5.26 -4.33 -11.84 superfamily, member 10a
NM_138578 BCL2-like 1 BCL2L1 -4.94 -1.80 -6.17 -7.04 NM_001165
Baculoviral IAP BIRC3 -13.68 -1.98 -2.42 -3.42 repeat-containing
3
[0369] There is a consistent linkage to the tumor necrosis factor
receptor family of proteins being modulated.
[0370] A strong down regulation of tumor protein p73 is also noted.
Analyses of many tumors typically found in humans including breast
and ovarian cancer show a high expression of p73 when compared to
normal tissues in corresponding areas. Recent finding are
suggesting that deregulated over expression of transcription
factors within the body involved in cell cycle regulation and
synthesis of DNA in mammalian cells (i.e.: E2F-1), induces the
expression of p73. The suggestion is that p73 may be an
oncoprotein, but may involve different mechanism that the related
p53 protein
Experiment 2
Real-time PCR Arrays using Oxidative Stress and Antioxidant Defense
Array
[0371] To identify proteins that were involved in the Q10 response,
real-time polymerase chain reaction (RT-PCR) methods were employed
to identify changes in the level of mRNA's for genes/proteins
involved in targeted pathway arrays for oxidative stress and
antioxidant defense.
[0372] Table 10 below lists the genes that are regulated in
SK-MEL28 cells with 100 .mu.M Q10 treatment. Results are given only
for those genes that are regulated in two independent experiments.
Although there is a significant amount of gene regulation seen at 6
hours, most significant changes in RNA levels are seen at 48
hours.
TABLE-US-00011 TABLE 10 Genes in SK-MEL-28 cells that are regulated
by 100 .mu.M Q10 treatement as seen in the Oxidative Stress and
Antioxidant Defense Arrays. Symbo1 Description Regulation Location
Possible Functions. ALB Albumin Down Regulation at Extracellular
Carrier protein, anti-apoptotic 48 hours space AOX1 Aldehyde
oxidase 1 Up regulation from Cytoplasm Produces free radicals, drug
16 hours metabolic process. APOE Apolipoprotein E Down Regulation
at Extracellular Lipid metabolism 48 hours space ATOX1 ATX1
antioxidant protein 1 Down Regulation at Cytoplasm Copper
metabolism homolog (yeast) 48 hours BNIP3 BCL2/adenovirus E1B Down
Regulation at Cytoplasm Anti-apoptotic 19 kDa interacting protein 3
48 hours CSDE1 Cold shock domain Down Regulation at Cytoplasm
Transcriptional regulation. containing E1, RNA- 48 hours binding
CYBA Cytochrome b-245, alpha Down Regulation at Cytoplasm
Apoptotic, polypeptide 48 hours CYGB Cytoglobin Down Regulation at
Cytoplasm Peroxidase, Transporter. 48 hours DHCR24
24-dehydrocholesterol Down Regulation at Cytoplasm Electron
carrier, binds to reductase 6 hours TP53, involved in apoptosis.
DUOX1 Dual oxidase 1 Up Regulation at 48 Plasma Calcium ion
binding, electron hours Membrane carrier. DUOX2 Dual oxidase 2 Down
Regulation at Unknown Calcium ion binding. 48 hours EPHX2 Epoxide
hydrolase 2, Down Regulation at Cytoplasm Arachidonic acide
cytoplasmic 48 hours metabolism. EPX Eosinophil peroxidase Down
Regulation at Cytoplasm Phenyl alanine metabolism, 48 hours
apoptosis. GPX2 Glutathione peroxidase 2 Down Regulation at
Cytoplasm Electron carrier, binds to (gastrointestinal) 48 hours
TP53, involved in apoptosis. GPX3 Glutathione peroxidase 3 Up
Regulation at 48 Extracellular Arachidonic acid metabolims,
(plasma) hours space up regulated in carcinomas. GPX5 Glutathione
peroxidase 5 Up Regulation at 48 Extracellular Arachidonic acid
metabolism. (epididymal androgen- hours space related protein) GPX6
Glutathione peroxidase 6 Down Regulation at Extracellular
Arachidonic acid metabolism. (olfactory) 48 hours space GSR
Glutathione reductase Down Regulation at Cytoplasm Glutamate and
glutathione 48 hours metabolism, apoptosis. GTF2I General
transcription factor Down Regulation at Nucleus Transcriptional
activator, II, i 6 hours transcription of fos. KRT1 Keratin 1
(epidermolytic Up Regulation at 48 Cytoplasm Sugar Binding.
hyperkeratosis) hours LPO Lactoperoxidase Down Regulation at
Extracellular Phenyl alanine metabolism. 48 hours space MBL2
Mannose-binding lectin Down Regulation at Extracellular Complement
signaling, (protein C) 2, soluble 48 hours space pattern
recognition in (opsonic defect) receptors. MGST3 Microsomal
glutathione S- Upregulation at 16 Cytoplasm Xenobiotic metabolism.
transferase 3 hours MPO Myeloperoxidase Down Regulation at
Cytoplasm Anti-apoptotic, phenyl 48 hours alanine metabolism. MPV17
MpV17 mitochondrial Down Regulation at Cytoplasm Maintenance of
inner membrane protein 6 hours mitochondrial DNA. MT3
Metallothionein 3 Down Regulation at Cytoplasm Copper ion binding.
48 hours NCF1 Neutrophil cytosolic factor Down Regulation Cyoplasm
Produces free radicals. 1, (chronic granulomatous from 6 hours
disease, autosomal 1) NCF2 Neutrophil cytosolic factor Up
Regulation at 48 Cytoplasm Electron carrier. 2 (65 kDa, chronic
hours granulomatous disease, autosomal 2) NME5 Non-metastatic cells
5, Down Regulation at Unknown Kinase, Purine and protein expressed
in 48 hours pyrimidine metabolism. (nucleoside-diphosphate kinase)
NOS2A Nitric oxide synthase 2A Down Regulation at Cytoplasm
Glucocorticoid receptor (inducible, hepatocytes) 48 hours
signaling, apoptosis. OXR1 Oxidation resistance 1 Down Regulation
at Cytoplasm Responds to oxidative stress. 48 hours PDLIM1 PDZ and
LIM domain 1 Up Regulation at 48 Cytoplasm Transcriptional
activator. (elfin) hours PIP3-E Phosphoinositide-binding Down
Regulation at Cytoplasm Peroxidase. protein PIP3-E 48 hours PRDX2
Peroxiredoxin 2 Down Regulation at Cytoplasm Role in phenyl alanine
6 hours metabolism. Role in cell death. PRDX4 Peroxiredoxin 4 Down
Regulation Cytoplasm Thioredoxin peroxidase. from 24 hours PREX1
Phosphatidylinositol 3,4,5- Down Regulation at Cytoplasm Forms
oxygen free radicals. trisphosphate-dependent 48 hours RAC
exchanger 1 PRG3 Proteoglycan 3 Down Regulation at Extracellular
Role in cell death. 48 hours space PTGS1 Prostaglandin- Down
Regulation at Cytoplasm arachidonic acid metabolism, endoperoxide
synthase 1 48 hours prostaglandin synthesis. (prostaglandin G/H
synthase and cyclooxygenase) PTGS2 Prostaglandin- Up Regulation at
48 Cytoplasm arachidonic acid metabolism, endoperoxide synthase 2
hours prostaglandin synthesis. (prostaglandin G/H synthase and
cyclooxygenase) PXDN Peroxidasin homolog Up Regulation at 48
Unknown binds to TRAF4, calcium ion (Drosophila) hours binding,
iron ion binding. PXDNL Peroxidasin homolog Down Regulation at
Unknown peroxidase, calcium ion (Drosophila)-like 48 hours binding,
iron ion binding. RNF7 Ring finger protein 7 Up Regulation at 16
Nucleus apoptotic, copper ion binding, hours ubiquitin pathway.
SGK2 Serum/glucocorticoid Down Regulation at Cytoplasm Kinase,
potasium channel regulated kinase 2 48 hours regulator. SIRT2
Sirtuin (silent mating type Up regulation at 16 Nucleus
Transcription factor. information regulation 2 hours homolog) 2 (S.
cerevisiae) SOD1 Superoxide dismutase 1, Up Regulation at 16
Cytoplasm Apoptotic, Caspase Activator. soluble (amyotrophic hours
lateral sclerosis 1 (adult)) SOD2 Superoxide dismutase 2, Up
regulation at 16 Cytoplasm Apoptotic, Regulated by mitochondrial
hours TNF. SOD3 Superoxide dismutase 3, Down Regulation at
Extracellular Pro-apoptotic extracellular 48 hours space SRXN1
Sulfiredoxin 1 homolog (S. Down Regulation at Cytoplasm DNA
binding, oxidoreductase cerevisiae) 48 hours TPO Thyroid peroxidase
Down Regulation at Plasma iodination of thyroglobulin, 48 hours
Membrane tyrosine metabolism, phenylalanine metabolism. TTN Titin
Down Regulation at Cytoplasm Actin cytoskeleton signaling, 48 hours
integrin signaling TXNDC2 Thioredoxin domain- Down Regulation at
Cytoplasm Pyrimidine metabolism containing 2 (spermatozoa) 48
hours
[0373] The Neutrophil cytosolic factor 2 (NCF2, 65 kDa, chronic
granulomatous disease, autosomal 2) was one of the initial top
induced mRNA's (observed at 6 hours). Subsequently at the 16 hour
time point and onward, Neutrophil cytosolic factor 1 (NCF1)
(chronic granulomatous disease, autosomal 1) was induced at very
high levels after an initial lag phase.
[0374] Neutrophil cytosolic factor 2 is the cytosolic subunit of
the multi-protein complex known as NADPH oxidase commonly found in
neutrophils. This oxidase produces a burst of superoxide which is
delivered to the lumen of the neutrophil phagosome.
[0375] The NADPH oxidase (nicotinamide adenine dinucleotide
phosphate-oxidase) is a membrane-bound enzyme complex. It can be
found in the plasma membrane as well as in the membrane of
phagosome. It is made up of six subunits. These subunits are: a Rho
guanosine triphosphatase (GTPase), usually Rac1 or Rac2 (Rac stands
for Rho-related C3 botulinum toxin substrate) [0376] Five "phox"
units. (Phox stands for phagocytic oxidase.) [0377] P91-PHOX
(contains heme) [0378] p22phox [0379] p40phox [0380] p47phox (NCF1)
[0381] p67phox (NCF2)
[0382] It is noted that another NADPH oxidase levels do not change.
The enzyme is NOX5, which is a novel NADPH oxidase that generates
superoxide and functions as a H+ channel in a Ca(2+)-dependent
manner
[0383] In addition Phosphatidylinositol
3,4,5-trisphosphate-dependent RAC exchanger 1(PREX1) was also
upregulated. This protein acts as a guanine nucleotide exchange
factor for the RHO family of small GTP-binding proteins (RACs). It
has been shown to bind to and activate RAC1 by exchanging bound GDP
for free GTP. The encoded protein, which is found mainly in the
cytoplasm, is activated by phosphatidylinositol-3,4,5-trisphosphate
and the beta-gamma subunits of heterotrimeric G proteins.
[0384] The second major early induced protein was Nitric oxide
synthase 2A (inducible, hepatocytes) (NOS2A). Nitric oxide is a
reactive free radical which acts as a biologic mediator in several
processes, including neurotransmission and antimicrobial and
antitumoral activities. This gene encodes a nitric oxide synthase
which is expressed in liver and is inducible by a combination of
lipopolysaccharide and certain cytokines.
[0385] Superoxide dismutase 2, mitochondrial (SOD2) is a member of
the iron/manganese superoxide dismutase family. It encodes a
mitochondrial protein that forms a homotetramer and binds one
manganese ion per subunit. This protein binds to the superoxide
byproducts of oxidative phosphorylation and converts them to
hydrogen peroxide and diatomic oxygen. Mutations in this gene have
been associated with idiopathic cardiomyopathy (IDC), premature
aging, sporadic motor neuron disease, and cancer.
[0386] An example of a down regulated protein is Forkhead box M1
(FOXM1), which is known to play a key role in cell cycle
progression where endogenous FOXM1 expression peaks at S and G2/M
phases. Recent studies have shown that FOXM1, regulates expression
of a large array of G2/M-specific genes, such as Plk1, cyclin B2,
Nek2 and CENPF, and plays an important role in maintenance of
chromosomal segregation and genomic stability. The FOXM1 gene is
now known as a human proto-oncogene. Abnormal upregulation of FOXM1
is involved in the oncogenesis of basal cell carcinoma (BCC). FOXM1
upregulation was subsequently found in the majority of solid human
cancers including liver, breast, lung, prostate, cervix of uterus,
colon, pancreas, and brain. Further studies with BCC and Q10 should
evaluate FOXM1 levels.
[0387] SKMEL-28 Cells
[0388] Further experiments were carried out using SKMEL-28 cells.
The level of mRNA present in SKMEL-28 cells treated with 100 .mu.M
Q10 were compared to the levels in untreated cells at various time
points using real-time PCR methods (RT-PCR). The PCR array
(SABiosciences) is a set of optimized real-time PCR primer assays
on 96-well plates for pathway or disease focused genes as well as
appropriate RNA quality controls. The PCR array performs gene
expression analysis with real-time PCR sensitivity and the
multi-gene profiling capability of a microarray.
TABLE-US-00012 TABLE 11 Listing and classification of mRNA levels
evaluated in the Oxidative Stress and Antioxidant Defense PCR
Array. After six hours of treatment with 100 .mu.M Q10 on SKMEL-28
cells, the largest changes to the mRNA levels are indicated by
highlighting the protein code (increased - bold; decreased -
underlined; or no change - grey). ##STR00004##
TABLE-US-00013 TABLE 12 Time course evaluation of 100 .mu.M
treatment of SKMEL-28. 6 hr 16 hr 24 hr 48 hr 72 hr Refseq Symbol
Description Q10 Q10 Q10 Q10 Q10 NM_000265 NCF1 Neutrophil cytosolic
factor 1, 0 high 3.3829 15.7838 31.5369 (chronic granulomatous
disease, autosomal 1) NM_012423 RPL13A Ribosomal protein L13a
-0.9025 3.1857 2.5492 4.9253 7.82 NM_020820 PREX1
Phosphatidylinositol -3.2971 2.867 0.3222 6.3719 7.476
3,4,5-trisphosphate-dependent RAC exchanger 1 NM_012237 SIRT2
Sirtuin (silent mating type -0.9025 4.0829 4.4766 5.7166 6.6257
information regulation 2 homolog) 2 (S. cerevisiae) NM_005125 CCS
Copper chaperone for -0.6206 3.0077 3.452 2.9801 6.1539 superoxide
dismutase NM_181652 PRDX5 Peroxiredoxin 5 -2.995 3.0454 3.5381
4.7955 6.0169 NM_016276 SGK2 Serum/glucocorticoid regulated 0 0 0
0.5995 5.937 kinase 2 NM_003551 NME5 Non-metastatic cells 5,
protein -0.6652 3.1138 3.3694 3.1549 5.782 expressed in
(nucleoside- diphosphate kinase) NM_004417 DUSP1 Dual specificity
phosphatase 1 -0.6998 0.5902 2.7713 3.321 5.5375 NM_001752 CAT
Catalase -0.8589 2.8424 0.1046 3.8557 5.3988 NM_000041 APOE
Apolipoprotein E -0.8212 3.2069 -0.9543 3.7694 5.3315 NM_000101
CYBA Cytochrome b-245, alpha polypeptide -0.3945 4.3475 3.9208
6.2452 5.0762 NM_000433 NCF2 Neutrophil cytosolic factor 2 1.2266
3.0077 0.0954 5.476 0 (65 kDa, chronic granulomatous disease,
autosomal 2) NM_000963 PTGS2 Prostaglandin-endoperoxide -0.6912
2.7046 2.6552 4.0553 -3.3022 synthase 2 (prostaglandin G/H synthase
and cyclooxygenase) NM_183079 PRNP Prion protein (p27-30) -0.2144
3.5236 2.9086 5.0837 -3.9396 (Creutzfeldt-Jakob disease,
Gerstmann-Strausler-Scheinker syndrome, fatal familial insomnia)
NM_004052 BNIP3 BCL2/adenovirus E1B 19 kDa -2.9376 3.3288 4.312
-18.2069 -4.8424 interacting protein 3 NM_000242 MBL2
Mannose-binding lectin (protein C) -0.3622 -1.9072 -3.0142 -1.1854
-6.4544 2, soluble (opsonic defect) NM_021953 FOXM1 Forkhead box M1
-0.8135 0.068 -0.9216 3.3655 -10.0953 The mRNA level changes were
monitored by RT-PCR methods and oxidative stress and antioxidant
defense proteins array was evaluated.
[0389] The Neutrophil cytosolic factor 2 (NCF2, 65 kDa, chronic
granulomatous disease, autosomal 2) was one of the initial top
induced mRNA's (observed at 6 hours). Subsequently at the 16 hour
time point and onward, Neutrophil cytosolic factor 1 (NCF1)
(chronic granulomatous disease, autosomal 1) was induced at very
high levels after an initial lag phase.
[0390] Neutrophil cytosolic factor 2 is the cytosolic subunit of
the multi-protein complex known as NADPH oxidase commonly found in
neutrophils. This oxidase produces a burst of superoxide which is
delivered to the lumen of the neutrophil phagosome. The NADPH
oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) is a
membrane-bound enzyme complex. It can be found in the plasma
membrane as well as in the membrane of phagosome. It is made up of
six subunits. These subunits are:
[0391] a Rho guanosine triphosphatase (GTPase), usually Rac1 or
Rac2 (Rac stands for Rho-related C3 botulinum toxin substrate)
[0392] Five "phox" (phagocytic oxidase) units. [0393] P91-PHOX
(contains heme) [0394] p22phox [0395] p40phox [0396] p47phox (NCF1)
[0397] p67phox (NCF2)
[0398] It is noted that another NADPH oxidase levels do not change.
The enzyme is NOX5, which is a novel NADPH oxidase that generates
superoxide and functions as a H+ channel in a Ca(2+)-dependent
manner
[0399] In addition Phosphatidylinositol
3,4,5-trisphosphate-dependent RAC exchanger 1(PREX1) was also
upregulated. This protein acts as a guanine nucleotide exchange
factor for the RHO family of small GTP-binding proteins (RACs). It
has been shown to bind to and activate RAC1 by exchanging bound GDP
for free GTP. The encoded protein, which is found mainly in the
cytoplasm, is activated by phosphatidylinositol-3,4,5-trisphosphate
and the beta-gamma subunits of heterotrimeric G proteins.
[0400] The second major early induced protein was Nitric oxide
synthase 2A (inducible, hepatocytes) (NOS2A). Nitric oxide is a
reactive free radical which acts as a biologic mediator in several
processes, including neurotransmission and antimicrobial and
antitumoral activities. This gene encodes a nitric oxide synthase
which is expressed in liver and is inducible by a combination of
lipopolysaccharide and certain cytokines.
[0401] An example of a down regulated protein is FOXM1, which is
known to play a key role in cell cycle progression where endogenous
FOXM1 expression peaks at S and G2/M phases. Recent studies have
shown that FOXM1, regulates expression of a large array of
G2/M-specific genes, such as Plk1, cyclin B2, Nek2 and CENPF, and
plays an important role in maintenance of chromosomal segregation
and genomic stability. The FOXM1 gene is now known as a human
proto-oncogene. Abnormal upregulation of FOXM1 is involved in the
oncogenesis of basal cell carcinoma (BCC). FOXM1 upregulation was
subsequently found in the majority of solid human cancers including
liver, breast, lung, prostate, cervix, uterus, colon, pancreas, and
brain.
Experiment 3
Real-Time PCR Arrays Using Heat Shock Array
[0402] Heat Shock Arrays were run for SCC cells and the data of
regulated genes is summarized below in Table 13.
TABLE-US-00014 TABLE 13 Genes from the Heat Shock Protein array
regulated with 100 .mu.M Q10 treatment in SCC cells. Symbol
Description Regulation. Location. Possible functions. CCT6B
Chaperonin Down regulated Cytoplasm Protein folding and containing
TCP1, at 24 hours protein complex subunit 6B (zeta 2) assembly.
DNAJA1 DnaJ (Hsp40) Up regulated at 6 Nucleus Responds to DNA
homolog, subfamily hours. damage and changes in A, member 1 protein
folding. DNAJB13 DnaJ (Hsp40) Down regulated Unknown Protein
folding and related, subfamily B at 6 hours. apoptosis. member 13
DNAJB5 DnaJ (Hsp40) Down regulated Unknown Binds to HSP, involved
in homolog, subfamily at 6 hours. protein folding and in B, member
5 protein complex assembly. DNAJC12 DnaJ (Hsp40) Down regulated
Unknown Binds to HSP, involved in homolog, subfamily at 6 hours.
protein folding and in C, member 12 protein complex assembly.
DNAJC4 DnaJ (Hsp40) Down regulated Cytoplasm Binds to HSP, involved
in homolog, subfamily at 6 hours. protein folding and in C, member
4 protein complex assembly. DNAJC5B DnaJ (Hsp40) Down regulated
Unknown Involved in protein homolog, subfamily at 6 hours. folding
responds to C, member 5 beta changes in protein folding. HSPA8 Heat
shock 70 kDa Up regulated at 6 Cytoplasm Regulates TNF, binds
protein 8 hours. BAG1, STUB1, TP53, involved in apoptosis. HSPH1
Heat shock Up regulated at 6 Cytoplasm Binds to HSPA8, 1051
kDa/1101 kDa hours. important for protein protein 1 folding,
responds to protein unfolding and stress.
Experiment 4
Real-Time PCR Arrays Using Diabetes Array
[0403] The experiments described in this example were performed to
test the overall hypothesis that Q10 would have an impact on
multiple genes and alter the metabolic state of a cell. The mRNA
from SKMEL-28 cells treated with 100 .mu.M Q10 was evaluated by
RT-PCR against a panel of target proteins involved in diabetes and
related pathways. Results from this experiment demonstrate that
several proteins involved in glycolyic pathways and insulin
processing are altered in their mRNA expression levels (summarized
in Table 14).
TABLE-US-00015 TABLE 14 Major mRNA level changes to SKMEL-28 cells
treated with 100 .mu.M Q10 for 16 hours. Fold Change after 16 hours
Refseq Description Symbol (100 .mu.M Q10) NM_000162 Glucokinase GCK
8.5386 (hexokinase 4) NM_178849 Hepatocyte nuclear HNF4A 8.421
factor 4, alpha NM_005249 Forkhead box G1 FOXG1 4.6396 NM_000599
Insulin-like growth factor IGFBP5 2.2721 binding protein 5
NM_001101 Actin, beta ACTB -2.0936 NM_002863 Phosphorylase,
glycogen; PYGL -2.65 liver (Hers disease, glycogen storage disease
type VI) NM_001065 Tumor necrosis factor TNFRSF1A -2.8011 receptor
super- family, member 1A NM_021158 Tribbles homolog 3 TRIB3 -2.8011
(Drosophila) NM_003749 Insulin receptor IRS2 -2.9404 substrate 2
NM_004578 RAB4A, member RAS RAB4A -3.1296 oncogene family NM_004176
Sterol regulatory element SREBF1 -3.5455 binding tran- scription
factor 1 NM_004969 Insulin-degrading IDE -4.4878 enzyme NM_005026
Phosphoinositide- PIK3CD -6.8971 3-kinase, catalytic, delta
polypeptide NM_000208 Insulin receptor INSR -8.6099 NM_003376
Vascular endothelial VEGFA -15.5194 growth factor A NM_001315
Mitogen-activated MAPK14 -74.3366 protein kinase 14
[0404] The results of this initial experiment show that the mRNA
levels for a variety of insulin related proteins were modulated in
both directions. The results indicate that Q10 would have an impact
on diabetic disease treatment and/or evaluation.
[0405] Further experiments were next conducted to confirm the
results above obtained from SK-MEL-28 cells treated with Q10. Many
of the genes in SK-MEL-28 cells are regulated as early as 6 hours
after Q10 treatment. However, the initial regulation becomes less
evident by 16 and 24 hours. Around 48 hours, we find that many of
the genes in the Diabetes array are again strongly regulated.
Results that are consistent from two or more or independent
experiments are summarized below in Table 15. SCC cells also
appeared to exhibit regulation in some genes, both at 6 and 24
hours after Q10 treatment. These results from SCC cells are
summarized in Table 16 while genes that are regulated both in
SK-MEL-28 cells and in SCC cells are summarized in Table 17.
TABLE-US-00016 TABLE 15 Genes in SK-MEL-28 cells regulated by 100
.mu.M Q10 treatment when analyzed by the Diabetes Array. Symbol
Description Regulation. Location Possible Function ADRB3
Adrenergic, beta-3-, Down Regulated Plasma cAMP signaling, G-
receptor at 48 hours membrane protein signaling CEACAM1
Carcinoembryonic Down Regulated Extracellular Anti-apoptotic,
antigen-related cell at 48 hours space positive regulation of
adhesion molecule 1 angiogenesis. (biliary glycoprotein) CEBPA
CCAAT/enhancer Up regulated at Nucleus Glucocorticoid binding
protein (C/EBP), 48 hours receptor signaling, alpha VDR/RXR
activation. CTLA4 Cytotoxic T-lymphocyte- Down Regulated Plasma T
cell receptor associated protein 4 at 48 hours Membrane signaling,
activates CASP8. DUSP4 Dual specificity Down Regulated Nucleus
Phosphatase phosphatase 4 at 48 hours ENPP1 Ectonucleotide Down
Regulated Plasma Negative regulator pyrophosphatase/ at 48 hours
membrane of the insulin phosphodiesterase 1 receptor pathway FOXC2
Forkhead box C2 (MFH- Down Regulated Nucleus Anti-apoptotic, 1,
mesenchyme at 48 hours transcription factor forkhead 1) G6PD
Glucose-6-phosphate Up regulated at Cytoplasm Pentose Phosphate
dehydrogenase 48 hours, then Pathway, down regulated Glutathione
metabolism. HMOX1 Heme oxygenase Down Regulated Cytoplasm Heme
oxygenase (decycling) 1 at 48 hours decycling ICAM1 Intercellular
adhesion Down Regulated Plasma Regulated by molecule 1 (CD54), at
48 hours membrane atorvastatin, human rhinovirus processes some
receptor caspases. IL4R Interleukin 4 receptor Down Regulated
Plasma Up regulation by at 48 hours membrane TP73, binds to IRS1
and IRS2 IRS1 Insulin receptor Up regulated at Plasma Binds Insulin
substrate 1 48 hours then membrane receptor down regulated IRS2
Insulin receptor Down Regulated Plasma IGF-1 signaling substrate 2
at 48 hours membrane NSF N-ethylmaleimide- Down Regulated Cytoplasm
GABA signaling sensitive factor at 48 hours PIK3CD
Phosphoinositide-3- Down Regulated Cytoplasm Kinase kinase,
catalytic, delta at 48 hours polypeptide PPARG Peroxisome
proliferator- Down Regulated Nucleus Transcriptional activated
receptor at 48 hours factor gamma PRKCB1 Protein kinase C, beta 1
Down Regulated Cytoplasm PKC family at 48 hours SELL Selectin L
(lymphocyte Down Regulated Plasma Activates RAS, adhesion molecule
1) at 48 hours membrane MAPK SREBF1 Sterol regulatory Up regulated
at Nucleus Transcriptional element binding 48 hours then factor
transcription factor 1 down regulated STXBP1 Syntaxin binding
protein Down Regulated Cytoplasm Present in myelin 1 at 48 hours
enriched fraction. TGFB1 Transforming growth Up regulated at
Extracellular Pro-apoptotic factor, beta 1 48 hours then space down
regulated NKX2-1 NK2 homeobox 1 Down Regulated Nucleus
Transcriptional at 48 hours activator TNF Tumor necrosis factor Up
regulated at Extracellular Pro-apoptotic (TNF superfamily, 48 hours
space member 2) TNFRSF1A Tumor necrosis factor Down Regulated
Plasma Pro-apoptotic receptor superfamily, at 72 hours membrane
member 1A VEGFA Vascular endothelial Up regulated at Cytoplasm
Kinase growth factor A 58 hours then down regulated
TABLE-US-00017 TABLE 16 Genes in SCC cells regulated by 100 .mu.M
Q10 treatment when analyzed by the Diabetes Array. Symbol
Description Regulation. G6PD Glucose-6-phosphate dehydrogenase Down
regulated at 6 hours. ICAM1 Intercellular adhesion molecule 1 Down
regulated (CD54), human rhinovirus receptor at 6 hours. INPPL1
Inositol polyphosphate Down regulated phosphatase-like 1 at 6
hours. NOS3 Nitric oxide synthase 3 Down regulated (endothelial
cell) at 6 hours. PIK3CD Phosphoinositide-3-kinase, Down regulated
catalytic, delta polypeptide at 6 hours. PPARA Peroxisome
proliferative activated Down regulated receptor, alpha at 6 hours.
PYGL Phosphorylase, glycogen; liver Down regulated (Hers disease,
glycogen storage at 6 hours. disease type VI) SREBF1 Sterol
regulatory element binding Down regulated transcription factor 1 at
6 hours. STXBP2 Syntaxin binding protein 2 Down regulated at 6
hours. TNF Tumor necrosis factor (TNF Down regulated superfamily,
member 2) at 6 hours. TNFRSF1A Tumor necrosis factor receptor Down
regulated superfamily, member 1A at 6 and 24 hours. VEGFA Vascular
endothelial Down regulated growth factor A at 6 hours.
TABLE-US-00018 TABLE 17 Genes from the diabetes array regulated
with 100 .mu.M Q10 treatment for both SK-MEL-28 and SCC cells.
Symbol Description. G6PD Glucose-6-phosphate dehydrogenase ICAM1
Intercellular adhesion molecule 1 (CD54), human rhinovirus receptor
PIK3CD Phosphoinositide-3-kinase, catalytic, delta polypeptide
SREBF1 Sterol regulatory element binding transcription factor 1 TNF
Tumor necrosis factor (TNF superfamily, member 2) TNFRSF1A Tumor
necrosis factor receptor superfamily, member 1A VEGFA Vascular
endothelial growth factor A
[0406] The mRNA levels for a variety of insulin related proteins
were modulated in both directions. Q10 has an impact on regulation
of cellular metabolism, and thus influences metabolic disregluation
diseases such as diabetes. Two proteins that were significantly
modulated are further discussed below.
[0407] Mitogen-activated protein kinase 14 (MAPK14):
Mitogen-activated protein kinase 14 (MAPK14) is a member of the MAP
kinase family. MAP kinases act as an integration point for multiple
biochemical signals, and are involved in a wide variety of cellular
processes such as proliferation, differentiation, transcription
regulation and development. Results from this experiment show that
the MAPK14 was significantly down-regulated.
[0408] Hepatocyte nuclear factor 4, alpha (HNF4A): HNF4 (Hepatocyte
Nuclear Factor 4) is a nuclear receptor protein mostly expressed in
the liver, gut, kidney, and pancreatic beta cells that is critical
for liver development. In humans, there are two isoforms of NHF4,
alpha and gamma encoded by two separate genes HNF4A and HNF4G
respectively. (See, e.g., Chartier F L, Bossu J P, Laudet V,
Fruchart J C, Laine B (1994). "Cloning and sequencing of cDNAs
encoding the human hepatocyte nuclear factor 4 indicate the
presence of two isoforms in human liver". Gene 147 (2):
269-72.)
[0409] HNF4 was originally classified as an orphan receptor.
However HNF4 was found later to be constitutively active by virtue
of being continuously bound to a variety of fatty acids. (See,
e.g., Sladek F (2002). "Desperately seeking . . . something". Mol
Cell 10 (2): 219-221 and Jump D B, Botolin D, Wang Y, Xu J,
Christian B, Demeure I (2005). "Fatty acid regulation of hepatic
gene transcription". J Nutr 135 (11)). The ligand binding domain of
HNF4, as with other nuclear receptors, adopts a canonical alpha
helical sandwich fold (see, e.g., Wisely G B, Miller A B, Davis R
G, Thornquest A D Jr, Johnson R, Spitzer T, Sefler A, Shearer B,
Moore J T, Miller A B, Willson T M, Williams S P (2002).
"Hepatocyte nuclear factor 4 is a transcription factor that
constitutively binds fatty acids". Structure 10 (9): 1225-34 and
Dhe-Paganon S, Duda K, Iwamoto M, Chi Y I, Shoelson S E (2002).
"Crystal structure of the HNF4 alpha ligand binding domain in
complex with endogenous fatty acid ligand". J Biol Chem 277 (41):
37973-6) and interacts with co-activator proteins. (See, e.g., Duda
K, Chi Y I, Shoelson S E (2004). "Structural basis for HNF-4-alpha
activation by ligand and coactivator binding". J Biol Chem 279
(22): 23311-6). Mutations in the HNF4-.alpha. gene have been linked
to maturity onset diabetes of the young (MODY). (See, e.g., Fajans
S S, Bell G I, Polonsky K S (2001). "Molecular mechanisms and
clinical pathophysiology of maturity-onset diabetes of the young".
N Engl J Med 345 (13): 971-80.)
[0410] Hepatocyte nuclear factor 4 (HNF4) is a tissue-specific
transcription factor known to regulate a large number of genes in
hepatocytes and pancreatic cells. Although HNF4 is highly expressed
in some sections of the kidney, little is known about its role in
this organ and about HNF4-regulated genes in the kidney cells. The
abundance and activity of HNF4 are frequently reduced in renal cell
carcinoma (RCC) indicating some tumor suppressing function of HNF4
in renal cells. Interestingly, many of the genes regulated by HNF4
have been shown to be deregulated in RCC microarray studies. These
genes (ACY1, WT1, SELENBP1, COBL, EFHD1, AGXT2L1, ALDH5A1, THEM2,
ABCB1, FLJ14146, CSPG2, TRIM9 and HEY1) are good candidates for
genes whose activity is changed upon the decrease of HNF4 in
RCC.
[0411] In the structure of the ligand binding domain of HNF4alpha
(1M7W.pdb; Dhe-Paganon (2002) JBC, 277, 37973); a small lipid was
observed and which co-purified from E. coli production. The crystal
contains two conformations of the protein, where the elongated
helix 10 and short helix 12 have alternate conformations. Upon
examination of the lipid binding region, it was interesting to
observe that there are two exits regions. One exit region holds the
small lipids head group, and it is noted that several pocket
regions are co-localized with this exit port. A hypothesis would be
that Q10 binds specifically to this transcription factor. When Q10
in modeled into this lipid binding tunnel, the Q10 ring would fit
into the surface pocket (FIG. 28). A known loss-of-function
mutation (E276Q) would have the potential to order the residues
lining this surface pocket, and thus have a negative impact on the
putative Q10 binding.
[0412] In addition, with this Q10 binding model, the hydrophobic
tail would extend out of the internal cavity and would then
interact with the elongated helix 10. Thus, this interaction could
potential alter the conformation of the helix 10/12 group. This may
then alter the activation/inactivation equilibrium of the
transcription factor activity.
Example 7
Antibody MicroArray Analysis
[0413] The evaluation of protein concentration due to the presence
of Q10 was evaluated through the utilization of antibody microarray
methods. The microarray contained antibodies for over 700 proteins,
sampling a broad range of protein types and potential pathway
markers.
[0414] An initial experiment to assess changes at the protein
concentration level in cells treated with Q10 was conducted with an
antibody microarray (Panorama XP725 Antibody Array, Sigma) and
SK-MEL-28 cells treated for 6 or 24 hour. The cells were harvested
and extracted to obtain a soluble protein supernatant. Two portions
of protein (.about.1 mg total) from each sample (at 1 mg/mL) were
each label with fluorescent dye (Cy3 and Cy5, respectively). The
excess dye was removed from the protein and the material utilized
for the microarray incubations. To compare two time point samples,
equal amounts of protein were mixed, with each sample being of the
different label type (e.g., 3 hour extract labeled with Cy3 was
mixed with the 24 hour extract labeled with Cy5). After incubation
with the microarray chip (according to manufactures recommended
protocols), the chips were washed and dried. The microarrays were
scanned with a fluorescent laser scanner to measure the relative
fluorescence intensity of the Cy3 and Cy5 dyes.
TABLE-US-00019 TABLE 18 Proteins with increased levels in SK-MEL-28
cells after 24 hour treatment with 50 .mu.M Q10 Name Ratio Cdk1 0.1
DcR1 0.1 Protein Kinase Cb2 0.1 Tumor Necrosis Factor 0.1 Soluble
Receptor II BAD 0.1 Caspase13 0.2 FBI1 PAKEMON 0.2 Zyxin 0.2 Cdc25A
0.3 PIASx 0.3 Nerve Growth Factor b 0.3 Protein Tyrosine 0.3
Phosphatase PEST hBRM hSNF2a 0.4 GRP94 0.4 Calmodulin 0.4 Serine
Threonine Protein 0.4 Phosphatase 2C a b ARC 0.4 NeurabinII 0.4
Nitric Oxide 0.4 Synthase bNOS Serine Threonine Protein 0.4
Phosphatase 1b Heat Shock Protein 110 0.4 Serine Threonine Protein
0.4 Phosphatase 1g1 COX II 0.5 HSP70 0.5 BLK 0.5 Cytokeratin 8 12
0.5 BUBR1 0.5 FOXC2 0.5 Serine Threonine Protein 0.5 Phosphatase 2
A Bg MSH6 0.5 DR6 0.5 Rad17 0.5 BAF57 0.5 Transforming Growth 0.5
Factorb pan BTK 0.5 SerineThreonine Protein 0.5 Phosphatase 2 A/B
pan2 CNPase 0.5 SynCAM 0.5 Proliferating Cell Nuclear 0.5
Antigen
TABLE-US-00020 TABLE 19 Proteins with increased levels in SK-MEL-28
cells after 24 hour treatment with 50 .mu.M Q10 Name Ratio BclxL
4.2 BID 3.7 Bmf 3.7 PUMA bbc3 3.0 Zip Kinase 2.8 Bmf 2.8 DcR2 2.7
E2F1 2.7 FAK pTyr577 2.5 FKHRL1 FOXO3a 2.5 MTBP 2.5 Connexin 32 2.5
Annexin VII 2.4 p63 2.4 SUMO1 2.4 IAfadin 2.3 MDMX 2.3 Pyk2 2.3 RIP
Receptor 2.3 Interacting Protein RICK 2.3 IKKa 2.3 Bclx 2.3 Afadin
2.2 Proliferating Cell 2.2 Protein Ki67 Histone H3 pSer28 2.2 CASK
LIN2 2.2 Centrin 2.2 TOM22 2.1 Nitric Oxide Synthase 2.1
Endothelial eNOS Protein Kinase Ba 2.1 Laminin 2.1 Myosin Ib
Nuclear 2.1 Caspase 7 2.1 MAP Kinase 2 ERK2 2.1 KIF17 2.1 Claspin
2.1 GRP75 2.1 Caspase 6 2.1 ILP2 2.1 aActinin 2.1 Vitronectin 2.1
DRAK1 2.1 PTEN 2.1 Grb2 2.1 HDAC4 2.0 HDAC7 2.0 Nitric Oxide 2.0
Synthase bNOS HDAC2 2.0 p38 MAPK 2.0 Reelin 2.0 Protein Kinase Cd
2.0 cerbBS 2.0 hSNF5 INI1 2.0 Protein Kinase Ca 2.0 Glutamate
receptor 2.0 NMDAR 2a Leptin 2.0 Dimethyl Histone H3 2.0 diMeLys4
BID 2.0 MeCP2 2.0 Nerve growth factor 2.0 receptor p75 Myosin Light
2.0 Chain Kinase cRaf pSer621 2.0 GRP78 BiP 2.0 cMyc 2.0 Raf1 2.0
MTA2 MTA1L 2.0 Sir2 2.0 ATF2 pThr69 71 2.0 Protein Kinase C 2.0
Protein Kinase Cb2 2.0
[0415] In order to confirm the previously observed apoptosis
proteins, and to expand the evaluation into a larger number of
pro-apoptosis and anti-apoptosis proteins, two assay methods were
chosen which were capable of screening the broad family of proteins
potentially involved.
[0416] First, an antibody micro array (Panorama XP725 Antibody
Array, Sigma) was utilized to screen over 700 protein antibodies to
assess changes at the protein concentration level in SK-MEL-28
cells treated for 24 hours with 50 .mu.M Q10.
[0417] From the Antibody array experiments, on SKMEL-28 with Q10
(24 hr), the following are some of the identified proteins with
altered levels: Bcl-x1, Bmf, BTK, BLK, cJun (pSer63), Connexin 32,
PUMA bbc3, BID, Par4, cCb1. The key conclusion from this initial
study was that the expected pro-apoptosis proteins are altered.
Antibody Microarray for SK-MEL-28
[0418] An antibody micro array (Panorama XP725 Antibody Array,
Sigma) was utilized to screen over 700 protein antibodies to assess
changes at the protein concentration level in SK-MEL-28 cells
treated for 24 hours with 50 .mu.M Q10.
TABLE-US-00021 TABLE 20 Changes in protein levels in SKMEL-28
treated with 50 .mu.M Q10 Antibody SKMEL28 Q10/ SKMEL28/ HEKa Q10/
Number SKMEL28 HEKa HEKa Name (Sigma) control control control BclxL
B9429 2.46 1.04 1.83 PUMA bbc3 P4743 2.31 1.14 2.14 Bmf B1559 2.23
1.12 2.11 Bmf B1684 2.09 1.13 1.74 cJun pSer63 J2128 1.99 1.14 1.85
BLK B8928 1.94 1.05 1.51
[0419] From the Antibody array experiments, on SKMEL-28 with Q10
(24 hr), the following are some of the identified proteins with
altered levels: Bcl-x1, Bmf, BTK, BLK, cJun (pSer63), Connexin 32,
PUMA bbc3, BID, Par4, cCb1. These data confirm that the levels of
pro-apoptosis proteins are altered upon incubation with elevated
levels of exogenously added Q10.
[0420] Bcl-x1 ("Basal cell lymphoma-extra large") is a
transmembrane molecule in the mitochondria. It is involved in the
signal transduction pathway of the FAS-L and is one of several
anti-apoptotic proteins which are members of the Bcl-2 family of
proteins. It has been implicated in the survival of cancer cells.
However, it is known that alternative splicing of human Bcl-x mRNA
may result in at least two distinct Bcl-x mRNA species, Bcl-xL and
Bcl-xS. The predominant protein product (233 amino acids) is the
larger Bcl-x mRNA, Bcl-xL, which inhibits cell death upon growth
factor withdrawal (Boise et al., 1993. Cell 74, 597-608). Bcl-xS,
on the other hand, inhibits the ability of Bcl-2 to inhibit cell
death and renders cells more susceptible to apoptotic cell
death.
TABLE-US-00022 TABLE 21 Proteins with increased levels in SCC cells
after 24 hour treatment with 100 .mu.M Q10. Name Ratio PUMA bbc3
3.81 HDAC7 3.21 BID 3.12 MTBP 3.00 p38 MAP Kinase 2.93 NonActivated
PKR 2.87 TRAIL 2.86 DR5 2.86 Cdk3 2.82 NCadherin 2.71 Reelin 2.68
p35 Cdk5 Regulator 2.63 HDAC10 2.60 RAP1 2.59 PSF 2.56 cMyc 2.55
methyl Histone H3 2.54 MeLys9 HDAC1 2.51 F1A 2.48 ROCK1 2.45 Bim
2.45 FXR2 2.44 DEDAF 2.44 DcR1 2.40 APRIL 2.40 PRMT1 2.36 Pyk2
pTyr580 2.34 Vitronectin 2.33 Synaptopodin 2.32 Caspase13 2.30
Syntaxin 8 2.29 DR6 2.29 BLK 2.28 ROCK2 2.28 Sir2 2.25 DcR3 2.24
RbAp48 RbAp46 2.21 OGlcNAc Transferase 2.21 GRP78 BiP 2.20 Sin3A
2.20 p63 2.20 Presenilin1 2.19 PML 2.18 PAK1pThr212 2.17 HDAC8 2.16
HDAC6 2.15 Nitric Oxide Synthase 2.15 Inducible iNOS Neurofibromin
2.15 Syntaxin 6 2.13 Parkin 2.12 Rad17 2.11 Nitric Oxide 2.10
Synthase bNOS TIS7 2.09 OP18 Stathmin 2.08 (stathmin 1/ oncoprotein
18) phospho-b-Catenin 2.07 pSer45 NeurabinII 2.07 e Tubulin 2.07
PKB pThr308 2.07 Ornithine Decarboxylase 2.07 P53 BP1 2.06 Pyk2
2.05 HDAC5 2.05 Connexin 43 2.05 a1Syntrophin 2.04 MRP1 2.04 cerbB4
2.03 S Nitrosocysteine 2.03 SGK 2.02 Rab5 2.01 Ubiquitin Cterminal
2.01 Hydrolase L1 Myosin Ib Nuclear 2.00 Par4 Prostate 2.00
Apoptosis Response 4
TABLE-US-00023 TABLE 22 Proteins with reduced levels in SCC cells
after 24 hour treatment with 100 .mu.M Q10. Name Ratio AP1 0.68
Centrin 0.55 CUGBP1 0.67 Cystatin A 0.69 Cytokeratin CK5 0.60
Fibronectin 0.63 gParvin 0.70 Growth Factor 0.63 Independence1
Nerve Growth 0.60 Factor b ProCaspase 8 0.72 Rab7 0.62 Rab9 0.73
Serine Threonine Protein 0.71 Phosphatase 1g1 Serine Threonine
Protein 0.73 Phosphatase 2 A Bg SKM1 0.70 SLIPR MAGI3 0.67 Spectrin
a and b 0.70 Spred2 0.66 TRF1 0.74
Example 8
Western Blot Analysis
[0421] The first experiment processed and evaluated by Western blot
and 2-D gel electrophoresis was carried out on the skin cancer cell
line SKMEL-28. This experimental set involved SK-MEL-28 cells
treated at 3, 6, 12, and 24 hours with 50 or 100 .mu.M Q10.
[0422] A variety of cell types were evaluated by Western blot
analysis against an antibody for Bcl-xL (FIG. 14), an antibody for
Vimentin (FIG. 15), a series of antibodies for mitochondrial
oxidative phosphorylation function (FIGS. 16-21) and against a
series of antibodies related to mitochondrial membrane integrity
(FIGS. 22-27). The results from these experiments demonstrated that
several of the examined proteins were upregulated or downregulated
as a result of cell treatment with Q10.
Example 9
Diabetes Related Genes Identified as being Modulated at the mRNA
Level by Treatment of Pancreatic Cancer Cells (PaCa2) with 100 um
Q10
[0423] Diabetes arrays were run for samples treated with 100 uM Q10
at various times after treatment. Experiments were carried out
essentially as described above. The various genes found to be
modulated upon Q10 treatment are summarized in Table 23 below. The
results showed that the following genes are modulated by Q10
treatment: ABCC8, ACLY, ADRB3, CCL5, CEACAM1, CEBRA, FOXG1, FOXP3,
G6PD, GLP1R, GPD1, HNF4A, ICAM1, IGFBP5, INPPL1, IRS2, MAPK14, ME1,
NFKB1, PARP1, PIK3C2B, PIK3CD, PPARGC1B, PRKAG2, PTPN1, PYGL,
SLC2A4, SNAP25, HNF1B, TNRFSF1A, TRIB3, VAPA, VEGFA, IL4R and
IL6.
TABLE-US-00024 TABLE 23 ##STR00005## ##STR00006##
Example 10
Angiogenesis Related Genes Identified as being Modulated at the
mRNA Level by Treatment of Pancreatic Cancer Cells (PaCa2) with 100
.mu.M Q10
[0424] Angiogenesis arrays were run for samples treated with 100 uM
Q10 at various times after treatment. Experiments were carried out
essentially as described above. The various genes found to be
modulated upon Q10 treatment are summarized in Table 24 below. The
results showed that the following genes are modulated by Q10
treatment: AKT1, ANGPTL4, ANGPEP, CCL2, CDH4, CXCL1, EDG1, EFNA3,
EFNB2, EGF, FGF1, ID3, IL1B, 1L8, KDR, NRP1, PECAM1, PROK2,
SERPINF1, SPHK1, STAB1, TGFB1, VEGFA and VEGFB.
TABLE-US-00025 TABLE 24 ##STR00007##
Example 11
Apoptosis Related Genes Identified as being Modulated at the mRNA
Level by Treatment of Pancreatic Cancer Cells (PaCa2) with 100
.mu.M Q10
[0425] Apoptosis arrays were run for samples treated with 100 uM
Q10 at various times after treatment. Experiments were carried out
essentially as described above. The various genes found to be
modulated upon Q10 treatment are summarized in Table 25 below. The
results showed that the following genes are modulated by Q10
treatment: ABL1, AKT1, Bcl2L1, BclAF1, CASP1, CASP2, CASP6, CIDEA,
FADD, LTA, TNF, TNFSF10A and TNFSF10.
TABLE-US-00026 TABLE 25 ##STR00008##
Example 12
PCR Diabetes Arrays on Liver Cancer (HepG2) Cells
[0426] HepG2 (liver cancer) cells were treated with either the
vehicle for 24 hours or 100 .mu.M Q10 for different times. The
treatment was initiated on 1.times.105 cells per well, following
the procedure utilized in the PaCa2 cells (above, Examples 9-11).
However, the total amount of RNA that was extracted from these
samples was lower than expected. Reverse transcription is normally
done using 1 .mu.g of total RNA (determined by measurement at 260
nm). The maximum volume that can be used per reverse transcription
is 8 .mu.l. Since the RNA concentration was low, the RT-PCR array
analysis using the vehicle, and Q10 treated samples from 16 hours
and 48 hours was performed using 0.44 .mu.g of RNA. The arrays
provided an initial analysis of trends and patterns in HepG2 gene
regulation with 100 .mu.M Q10 treatment, as summarized in Table 26
below. The results showed that each of the genes PPARGC1A, PRKAA1
and SNAP25 were downregulated at 16 hours following treatment (by
approximately 20 fold, 6 fold and 5 fold, respectively). At 48
hours following treatment, PPARGC1A and PRKAA1 had normalized or
were slightly upregulated, while SNAP25 was downregulated by
approximately 2 fold.
TABLE-US-00027 TABLE 26 List of genes regulated in the Diabetes
Arrays when HepG2 cells were treated with 100 .mu.M Q10. Gene Gene
name Gene Function. PPARGC1A peroxisome proliferator- Involved in
cell death, activated receptor gamma, proliferation, cellular
coactivator 1 alpha respiration and transmembrane potential. PRKAA1
protein kinase, AMP- Regulates TP53 and is activated, alpha 1
involved in apoptosis, catalytic subunit regulates glycolysis,
regulates metabolic enzyme activities. SNAP25
synaptosomal-associated Plays in transport, protein, 25 kDa fusion,
exocytosis and release of molecules.
Example 13
PCR Angiogenesis Array on Liver Cancer (HEPG2) Cells
[0427] HepG2 (liver cancer) cells were treated with either the
vehicle for 24 hours or 100 .mu.M Q10 for different times. The
treatment was initiated on 1.times.105 cells per well, following
the procedure utilized in the PaCa2 cells (above Examples 9-11).
However, the total amount of RNA that was extracted from these
samples was lower than expected. Reverse transcription is normally
done using 1 .mu.g of total RNA (determined by measurement at 260
nm). The maximum volume that can be used per reverse transcription
is 8 .mu.l. Since the RNA concentration was low, the RT-PCR array
analysis using the vehicle, and Q10 treated samples from 16 hours
and 48 hours was performed using 0.44 .mu.g of RNA. The arrays
provided an initial analysis of trends and patterns in HepG2 gene
regulation with 100 .mu.M Q10 treatment, as summarized in Table 27
below. The various genes found to be modulated upon Q10 treatment
are summarized in Table 27 below. The results showed that each of
the genes ANGPTL3, ANGPTL4, CXCL1, CXCL3, CXCL5, ENG, MMP2 and
TIMP3 were upregulated at 16 hours following treatment (by
approximately 5.5, 3, 3, 3.2, 3, 3, 1 and 6.5 fold, 6 fold and 5
fold, respectively, over that of control). ID3 was downregulated at
16 hours following Q10 treatment, by approximately 5 fold over
control. At 48 hours following treatment, ANGPTL3, CXCL1, CXCL3,
ENG and TIMP3 were still upregulated (by approximately 3.5, 1.5,
3.175, 2 and 3 fold, respectively, over control), while ANGPTL4,
CXCL5, ID3 and MMP2 were downregulated by approximately 1, 1, 2 and
18 fold, respectively, over control.
TABLE-US-00028 TABLE 27 List of genes regulated in the Angiogenesis
Arrays when HepG2 cells were treated with 100 .mu.M Q10. Gene Gene
Name. Gene Function. ANGPTL3 angiopoietin-like 3 Predominantly
expressed in live, role in cell migration and adhesion, regulates
fatty acid and glycerol metabolism. ANGPTL4 angiopoietin-like 4
Regulated by PPARG, apoptosis inhibitor for vascular endothelial
cells, role lipid and glucose metabolism and insulin sensitivity.
CXCL1 chemokine (C-X-C motif) Role in cell proliferation and
migration ligand 1 (melanoma growth stimulating activity, alpha)
CXCL3 chemokine (C-X-C motif) Chemokine activation, hepatic stellar
cell ligand 3 activation, migration, proliferation. CXCL5 chemokine
(C-X-C motif) Produced along with IL8 when stimulated ligand 5 with
IL1 or TNFA. Role in chemotaxis, migration, proliferation. ENG
endoglin Binds to TGFBR and is involved in migration,
proliferation, attachment and invasion. ID3 inhibitor of DNA
binding 3, Regulates MMP2, Regulated by TGFB1, dominant negative
helix- Vitamin D3, Retinoic acid, VEGFA, involved loop-helix
protein in apoptosis, proliferation, differentiation, migration.
MMP2 matrix metallopeptidase 2 Hepatic stellate cell activation,
HIF (gelatinase A, 72 kDa signaling, binds to TIMP3, involved in
gelatinase, 72 kDa type IV tumorigenesis, apoptosis, proliferation,
collagenase) invasiveness, migration and chemotaxis. TIMP3 TIMP
metallopeptidase Regulates MMP2, ICAM1. Regulated by inhibitor 3
TGFB, EGF, TNF, FGF and TP53. Involved in apoptosis, cell-cell
adhesion and malignancy.
[0428] Proteins known to be involved in the process of angiogenesis
were components in the RT-PCR array. Angiogenesis is a critical
process by which cancer cells become malignant. Some of these
proteins are also implicated in diabetes.
[0429] ANGPTL3 and ANGPTL4: The literature related to ANGPTL3
connects this protein to the regulation of lipid metabolism. In
particular, the literature (Li, C. Curr Opin Lipidol. 2006 April;
17(2):152-6) teaches that both angiopoietins and angiopoietin-like
proteins share similar domain structures. ANGPTL3 and 4 are the
only two members of this superfamily that inhibit lipoprotein
lipase activity. However, ANGPTL3 and 4 are differentially
regulated at multiple levels, suggesting non-redundant functions in
vivo. ANGPTL3 and 4 are proteolytically processed into two halves
and are differentially regulated by nuclear receptors. Transgenic
overexpression of ANGPTL4 as well as knockout of ANGPTL3 or 4
demonstrate that these two proteins play essential roles in
lipoprotein metabolism: liver-derived ANGPTL3 inhibits lipoprotein
lipase activity primarily in the fed state, while ANGPTL4 plays
important roles in both fed and fasted states. In addition, ANGPTL4
regulates the tissue-specific delivery of lipoprotein-derived fatty
acids. ANGPTL4 is thus an endocrine or autocrine/paracarine
inhibitor of lipoprotein lipase depending on its sites of
expression.
[0430] Lipoprotein lipase is an enzyme that hydrolyzes lipids in
lipoproteins, such as those found in chylomicrons and very
low-density lipoproteins (VLDL), into three free fatty acids and
one glycerol molecule. Lipoprotein lipase activity in a given
tissue is the rate limiting step for the uptake of
triglyceride-derived fatty acids. Imbalances in the partitioning of
fatty acids have major metabolic consequences. High-fat diets have
been shown to cause tissue-specific overexpression of LPL, which
has been implicated in tissue-specific insulin resistance and
consequent development of type 2 diabetes mellitus.
[0431] The results in this Example indicate that Q10 is modulating
proteins involved in lipid metabolism and thus warrants further
investigation of ANGPTL3/ANGPTL4 and their related pathways. For
example, ANGPTL3/ANGPTL4 have been implicated to play a role in the
following pathways: Akt, cholesterol, fatty acid, HDL-cholesterol,
HNF1A, ITGA5, ITGA5, ITGAV, ITG83, L-trilodothynonine, LIPG, LPL,
Mapk, Nrth, NR1H3, PPARD, PTK2, RXRA, triacylglerol and
9-cis-retinoic acid.
Example 14
PCR Apoptosis Array on Liver Cancer (HEPG2) Cells
[0432] Apoptosis arrays were run for samples treated with 100 uM
Q10 for 16 and 48 hours as described above. However, the array for
48 hours was run choosing FAM as the fluorophore instead of SYBR.
Both FAM and SYBR fluoresce at the same wavelength.
[0433] The various genes found to be modulated upon Q10 treatment
are summarized in Table 28 below. The results showed that CASP9 was
upregulated at 16 hours following Q10 treatment, by approximately
61 fold over control, while BAG1 and TNFRSF1A were downregulated at
16 hours following treatment by approximately 6 and 4 fold,
respectively, over that of control. At 48 hours following
treatment, CASP9, BAG1 and TNFRSF1A were upregulated by
approximately 55, 1 and 1 fold, respectively, over control.
TABLE-US-00029 TABLE 28 List of genes regulated in the Apoptosis
Arrays when HepG2 cells were treated with 100 .mu.M Q10. Gene Gene
Name Gene Function. BAG1 BCL2-associated athanogene Involved with
Apoptosis CASP9 caspase 9, apoptosis- Apoptosis through release
related cysteine of cytochrome c. peptidase TNFRSF1A tumor necrosis
factor anti-apoptosis, binds receptor superfamily, many cell death
member 1A factors, regulates ICAM1
Example 15
Assessing Ability of Epi-Shifter to Treat Metabolic Disorder
[0434] In order to determine if a selected Epi-shifter, e.g.,
CoQ10, is capable of treating a metabolic disorder, e.g., diabetes,
cell based assays that monitor an increase in insulin-stimulated
glucose uptake in vitro are employed. In particular, differentiated
mouse adipocytes are used to identify agents that have the ability
to increase glucose uptake upon insulin stimulation, as detected by
scintillation counting of radiolabelled glucose (using, for
example, the Perkin Elmer 1450 Microbeta JET reader). These assays
are conducted as follows.
Materials and Methods:
Prees Media
[0435] Complete media, also referred to as "Prees" media, is
prepared as follows. Dulbecco's Modified Eagle's Medium (DMEM) is
supplemented with L-glutamine, penicillin-G and streptomycin
(pen/strep), and heat-inactivated fetal bovine serum (FBS) (heat
inactivated at 65.degree. C. for 30 minutes). Because serum can
affect the growth, adherence, and differentiation of cells, any new
lot of serum was first tested prior to use. Media was equilibrated
in the incubator (5% CO.sub.2) until the pH was within the proper
range (.about.7), as indicated by the red/orange color of the
indicator dye. If the media became pink (indicating a high pH), we
discarded the media as basic conditions can affect cells and
denature the insulin used in the differentiation medium-1 (DM1) and
the differentiation medium-2 (DM2).
Differentiation Medias
[0436] Differentiation media-1 (DM1) was prepared by supplementing
DMEM with 10% FBS, L-glutamine, pen/strep, IBMX (375.mu.M), insulin
(120 nM), and dexamethasone (188 nM). Differentiation media-2 (DM2)
was prepared by supplementing DMEM with 10% FBS, L-glutamine,
pen/strep, and insulin (120 nM).
Preparation of Gelatinized Plates
[0437] Cell culture plates are gelatinized as follows. Gelatin (1%
w/v in distilled water) was autoclaved and stored at room
temperature. The bottom of each cell culture well was covered
uniformly in the gelatin solution, ensuring that no bubbles are
formed. This solution was removed leaving behind a thin film of
gelatin. These plates are left to dry under the tissue culture
hood. Plates are next washed with PBS, after which a 0.5% glutaric
dialdehyde solution (glutaraldehyde in distilled water) was added
to the cell culture wells. After ten minutes, wells are washed
twice with DMEM containing pen-strep. Each washing step should last
for approximately five minutes.
Cell Culture
[0438] 3T3-L1 pre-adipocyte cells are split approximately every 2-3
days or upon reaching a confluence of approximately 60%.
Overconfluency may affect the ability of these cells to
differentiate into adipocytes.
Other Reagents
[0439] D-(+)-glucose ("cold" glucose, not radiolabeled) was added
to DPBS mix to a final concentration of 10 mM.
[0440] Lysis buffer, a mixture of a base (e.g., sodium hydroxide at
a final concentration of 0.5N) and a detergent (e.g., sodium
dodecyl sulphate (SDS) diluted to a final concentration of 0.1%
w/v) was freshly prepared each time (within one to two hours of
use). Prior to use, lysis buffer was warmed up to a temperature
exceeding that of room temperature for a period of approximately 30
minutes to avoid precipitation of the buffer.
Determination of Glucose Uptake
[0441] Pre-adipocyte 3T3-L1 cells are plated at a density of
approximately 5000 cells/well (in black NUNC 96 well plate). These
cells are differentiated into adipocytes in two separate steps.
Initially, cells are cultured in differentiation medium-1 (DM1)
(day 1 of adipocyte differentation) for a period of two to three
days. DM1 prevents proliferation and induces the expression of
adipocyte-specific genes. Cells are next cultured in
differentiation medium-2 (DM2) for 3 to 4 days, after which the
culture media is replaced by fresh DM2. The glucose uptake assay is
performed at day 9-15 of differentiation.
[0442] Two days prior to the experiment (at day 7-13 of
differentiation), DM2 is removed and replaced with fresh Prees
media. Candidate compounds are added at this time, allowing an
incubation period of approximately 48 hours. On the day of the
experiment, cells (now at day 9 to 15 of differentiation) are serum
starved for three hours in DPBS, magnesium sulfate (0.8 mM), and
Hepes (10 mM) at pH .about.7. After this incubation period, fresh
DPBS containing insulin (10 nM) is added to the adipocytes. Fresh
DPBS without any insulin are placed on cells that served as a
negative control. Following an incubation period of 25 minutes at
37.degree. C., radioactive glucose (labeled with .sup.14C, at a
final concentration of 0.04 mM, .about.0.26.mu.Ci.sup.14C-glucose
in each well) is added to the media for a period of 15 minutes at
room temperature. Media is next removed and cells are washed
thoroughly and lysed. Upon lysis, cells form a small, cloudy mass,
detached from the well bottom. 10% glacial acetic acid is added to
each well to neutralize the lysis reaction. Scintillation fluid is
next added to the wells and the incorporation of glucose is
determined by measuring the amount of radioactivity in each well
using the MicroBeta plate reader.
[0443] Using the foregoing experimental protocol, an Epi-shifter is
identified as capable of treating a metabolic disorder, e.g.,
diabetes, when the Epi-shifter enhances, increases or augments
insulin-stimulated glucose uptake in the cells in vitro.
Example 16
Identification of a MIM Associated with an Metabolic Disorder
[0444] In order to evaluate a candidate molecule (e.g.,
environmental influencer) as a potential MIM, the selected
candidate MIM is exogenously added to a panel of cell lines,
including both diseased (cancer) cell lines and normal control cell
lines, and the changes induced to the cellular microenvironment
profile for each cell line in the panel are assessed. Changes to
cell morphology, physiology, and/or to cell composition, including
for example, mRNA and protein levels, are evaluated and compared
for the diseased cells as compared to normal cells.
[0445] Changes to cell morphology/physiology are evaluated by
examining the sensitivity and apoptotic response of cells to the
candidate MIM. These experiments are carried out as described in
detail in Example 3. Briefly, a panel of cell lines consisting of
at least one control cell line and at least one cancer cell line
are treated with various concentrations of the candidate MIM. The
sensitivity of the cell lines to the potential MIM are evaluated by
monitoring cell survival at various times, and over the range of
applied concentrations. The apoptoic response of the cell lines to
the potential MIM are evaluated by using, for example, Nexin
reagent in combination with flow cytometry methodologies. Nexin
reagent contains a combination of two dyes, 7AAD and Annexin-V-PE,
and allows quantification of the population of cells in early and
late apoptosis. An additional apoptosis assay that measures
single-stranded DNA may be used, using for example Apostrand.TM.
ELISA methodologies. The sensitivity and apoptotic response of the
disease and control cell lines are evaluated and compared. A
molecule that displays differential cytotoxicity and/or that
differentially induces the apoptotic response in the diseased cells
as compared to the normal cells is identified as a MIM.
[0446] Changes in the composition of cells following treatment with
the candidate MIM are evaluated. Changes in gene expression at the
mRNA level are analyzed using Real-Time PCR array methodology.
These experiments are carried out as described in detail in
Examples 6 and 9-13. Briefly, the candidate MIM is exogenously
added to one or more cell lines including, for example a diseased
cell and a normal control cell line, and mRNA is extracted from the
cells at various times following treatment. The level of mRNAs for
genes involved in specific pathways are evaluated by using targeted
pathway arrays, including, for example, arrays specific for
apoptosis, oxidative stress and antioxidate defense, angiogenesis,
heat shock or diabetes. The genes that are altered in their mRNA
transcription by a two-fold level or greater are identified and
evaluated. A molecule that induces changes in mRNA levels in cells
and/or that induces differential changes in the level of one or
more mRNAs in the diseased cells as compared to the normal cells is
identified as a MIM.
[0447] In complementary experiments, changes in gene expression at
the protein level are analyzed by using antibody microarray
methodology, 2-dimensional gel electrophoresis followed by protein
identification using mass spectrometry characterization, and by
western blot analysis. These experiments are carried out as
described in detail in Examples 7, 4 and 8, respectively. Briefly,
the candidate MIM is exogenously added to one or more cell lines,
including, for example a diseased cell and a normal control cell
line, and soluble protein is extracted from the cells at various
times, e.g., 6 hours or 24 hours, following treatment. Changes
induced to protein levels by the candidate MIM are evaluated by
using an antibody microarray containing antibodies for over 700
proteins, sampling a broad range of protein types and potential
pathway markers. Further complementary proteomic analysis can be
carried by employing 2-dimensional (2-D) gel electrophoresis
coupled with mass spectrometry methodologies. The candidate MIM is
exogenously added to one or more cell lines, including, for example
a diseased cell and a normal control cell line, and cell pellets
are lysed and subjected to 2-D gel electrophoresis. The gels are
analyzed to identify changes in protein levels in treated samples
relative to control, untreated samples. The gels are analyzed for
the identification of spot changes over the time course of
treatment due to increased levels, decreased levels or
post-translational modification. Spots exhibiting statistically
significant changes are excised and submitted for protein
identification by trypsin digestiona do mass spectrometry
characterization. The characterized peptides are searched against
protein databases with, for example, Mascot and MSRAT software
analysis to identify the proteins. In addition to the foregoing 2-D
gel analysis and antibody microarray experiments, potential changes
to levels of specific proteins induced by the candidate MIM may be
evaluated by Western blot analysis. In all of the proteomic
experiments, proteins with increased or decreased levels in the
various cell lines are identified and evaluated. A molecule that
induces changes in protein levels in cells and/or that induces
differential changes in the level of one or more proteins in the
diseased cells as compared to the normal cells is identified as a
MIM.
[0448] Genes found to be modulated by treatment with a candidate
MIM from the foregoing experiments are subjected to cellular and
biochemical pathway analysis and can thereby be categorized into
various cellular pathways, including, for example apoptosis, cancer
biology and cell growth, glycolysis and metabolism, molecular
transport, and cellular signaling.
[0449] Experiments are carried out to confirm the entry of a
candidate MIM into cells, to determine if the candidate MIM becomes
localized within the cell, and to determine the level and form of
the candidate MIM present in the cells. These experiments are
carried out, for example, as described in detail in Example 5. For
example, to determine the level and the form of the candidate MIM
present in the mitochondria, mitochondrial enriched preparations
from cells treated with the candidate MIM are prepared and
analyzed. The level of the candidate MIM present in the
mitochondria can thereby be confirmed to increase in a time and
dose dependent manner with the addition of exogenous candidate MIM.
In addition, changes in levels of proteins from mitochondria
enriched samples are analyzed by using 2-D gel electrophoresis and
protein identification by mass spectrometry characterization, as
described above for total cell protein samples. Candidate MIMs that
are found to enter the cell and to be present at increased levels,
e.g., in the mitochondria, are identified as a MIM. The levels of
the candidate MIM in the cell, or, for example, specifically in the
mitochondria, over the time course examined can be correlated with
other observed cellular changes, as evidenced by, for example, the
modulation of mRNA and protein levels for specific proteins.
[0450] Candidate MIMs observed to induce changes in cell
composition, e.g., to induce changes in gene expression at the mRNA
or protein level, are identified as a MIM. Candidate MIMs observed
to induce differential changes in cell morphology, physiology or
cell composition (e.g., differential changes in gene expression at
the mRNA or protein level), in a disease state (e.g., diabetes or
obestity) as compared to a normal state are identified as a MIM
and, in particular, as having multidimensional character. Candidate
MIMs found to be capable of entering a cell are identified as a MIM
and, in particular, as having multidimensional character since the
candidate MIM thereby exhibits a carrier effect in addition to a
therapeutic effect.
Example 17
Identification of CoQ10 as an Epi-Shifter Associated with a
Metabolic Disorder
[0451] A panel of skin cell lines consisting of a control cell
lines (primary culture of keratinocytes and melanocytes) and
several skin cancers cell lines (SK-MEL-28, a non-metastatic skin
melanoma; SK-MEL-2, a metastatic skin melanoma; or SCC, a squamous
cell carcinoma; PaCa2, a pancreatic cancer cell line; or HEP-G2, a
liver cancer cell line) were treated with various levels of
Coenzyme Q10. The cancer cell lines exhibited an altered dose
dependent response when compared to the control cell lines, with an
induction of apoptosis and cell death in the cancer cells only.
Detailed exemplary experiments are presented in, e.g., Example 3
herein.
[0452] Assays were employed to assess changes in the mRNA and
protein levels composition of the above-identified cells following
treatment with CoQ10. Changes in mRNA expression were analyzed
using real-time PCR microarrays specific for each of apoptosis,
oxidative stress and antioxidants, angiogenesis and diabetes.
Changes in protein expression were analyzed using antibody
microarray analysis and western blot analysis. The results from
these assays demonstrated that significant changes in gene
expression, both at the mRNA and protein levels, were occurring in
the cell lines due to the addition of the Coenzyme Q10. Numerous
genes known to be associated with or involved in cellular metabolic
processes were observed to be modulated as a result of treatment
with CoQ10. For example, expression of the nuclear receptor protein
HNF4A was found to be upmodulated in cells following Q10 treatment.
Expression of transaldolase 1 (TAL) was also modulated in cells
treated with Q10. TAL balances the levels of NADPH and reactive
oxygen intermediate, thereby regulating the mitochondrial
trans-membrande potentional, which is a critical checkpoint of ATP
synthesis and cell survival. Of particular relevance to metabolic
disorders, numerous genes known to be associated with, e.g.,
diabetes, were identified as being regulated by Q10. Detailed
exemplary experiments are presented in, e.g., Examples 4, 6, 7, 8
and 9 herein.
[0453] Q10 is an essential cofactor for exidative phosphorylation
processes in the mitochondria for energy production. The level of
Coenzyme Q10, as well as the form of CoQ10, present in the
mitochondria was determined by analyzing mitochondrial enriched
preparations from cells treated with CoQ10. The level of Coenzyme
Q10 present in the mitochondria was confirmed to increase in a time
and dose dependent manner with the addition of exogenous Q10. The
time course correlated with a wide variety of cellular changes as
observed in modulation of mRNA and protein levels for specific
proteins related to metabolic and apoptotic pathways. Detailed
exemplary experiments are presented in, e.g., Example 5 herein.
[0454] The results described herein identified the endogenous
molecule CoQ10 as an epi-shifter. In particular, the results
identified CoQ10 as inducing a shift in the metabolic state, and
partially restoration of mitochondrial function, in cells. These
conclusions are based on the following interpretation of the data
described herein and the current knowledge in the relevant art.
[0455] Q10 is known to be synthesized, actively transported to,
enriched in, and utilized in the mitochondrial inner membrane. Q10
is also known to be an essential cofactor for oxidative
phosphorylation processes in the mitochondrial for energy
production. However, most cancer cells predominantly produce energy
by glycolysis followed by lactic acid fermentation in the cytosol,
rather than by oxidation of pyruvate in mitochondria like most
normal cells. The oxidative phosphorylation involves the electron
transport complexes and cytochrome c. Apoptosis involves the
disruption of the mitochondria, with permiabilization of the inter
mitochondrial membrane by pro-apoptitic factors. By utilizing a
different metabolic energy synthesis pathway, cancer cells are able
to mitigate the normal apoptosis response to abnormalities in the
cell. While not wishing to be bound by theory, Applicants propose
that Q10 is functioning by upregulating the oxidative
phosphorylation pathway proteins, thus switching the mitochondrial
function back to a state that would recognize the oncogenic defects
and trigger apoptosis. Thus, Q10 is acting as an Epi-shifter by
shifting the metabolic state of a cell.
Example 18
Identification of an Epi-Shifter Associated with a Metabolic
Disorder
[0456] A panel of skin cell lines consisting of control cell lines
(e.g., primary culture of keratinocytes and melanocytes) and cancer
cell lines (e.g., SK-MEL-28, a non-metastatic skin melanoma;
SK-MEL-2, a metastatic skin melanoma; or SCC, a squamous cell
carcinoma; PaCa2, a pancreatic cancer cell line; or HEP-G2, a liver
cancer cell line) are treated with various levels of a candidate
Epi-shifter. Changes to cell morphology/physiology are evaluated by
examining the sensitivy and apoptotic response of cells to the
candidate Epi-shifter. These experiments are carried out as
described in detail in Example 3. Briefly, the sensitivity of the
cell lines to the candidate Epi-shifter are evaluated by monitoring
cell survival at various times, and over a range of applied
concentrations. The apoptoic response of the cell lines to the
candidate Epi-shifter are evaluated by using, for example, Nexin
reagent in combination with flow cytometry methodologies. Nexin
reagent contains a combination of two dyes, 7AAD and Annexin-V-PE,
and allows quantification of the population of cells in early and
late apoptosis. An additional apoptosis assay that measures
single-stranded DNA may be used, using for example Apostrand.TM.
ELISA methodologies. The sensitivity and apoptotic response of the
disease and control cell lines are evaluated and compared.
Candidate Epi-shifters are evaluated based on their ability to
inhibit cell growth preferentially or selectively in cancer cells
as compared to normal or control cells. Candidate Epi-shifters are
further evaluated based on their ability to preferentially or
selectively induce apoptosis in cancer cells as compared to normal
or control cells.
[0457] Assays are employed to assess changes in the mRNA and
protein level composition of the above-identified cells following
treatment with the candidate Epi-shifter. Changes in mRNA levels
are analyzed using real-time PCR microarrays. These experiments are
carried out as described in detail in Examples 6 and 9-13. Briefly,
mRNA is extracted from the cells at various times following
treatment. The level of mRNAs for genes involved in specific
pathways are evaluated by using targeted pathway arrays, including,
arrays specific for apoptosis, oxidative stress and antioxidate
defense, angiogenesis, heat shock or diabetes. The genes that are
altered in their mRNA transcription by a two-fold level or greater
are identified and evaluated.
[0458] Changes in protein expression are analyzed using antibody
microarray analysis, 2-D gel electrophoresis analysis coupled with
mass spectrometry characterization, and western blot analysis.
These experiments are carried out as described in detail in
Examples 7, 4 and 8, respectively. Briefly, soluble protein is
extracted from the cells at various times, e.g., 6 hours or 24
hours, following treatment with the candidate Epi-shifter. Changes
induced to protein levels by the candidate Epi-shifter are
evaluated by using an antibody microarray containing antibodies for
over 700 proteins, sampling a broad range of protein types and
potential pathway markers. Further complementary proteomic analysis
can be carried out by employing 2-dimensional (2-D) gel
electrophoresis coupled with mass spectrometry methodologies. The
candidate Epi-shifter is exogenously added to the cell lines and
cell pellets are lysed and subjected to 2-D gel electrophoresis.
The gels are analyzed to identify changes in protein levels in
treated samples relative to control, untreated samples. The gels
are analyzed for the identification of spot changes over the time
course of treatment due to increased levels, decreased levels or
post-translational modification. Spots exhibiting statistically
significant changes are excised and submitted for protein
identification by trypsin digestion and mass spectrometry
characterization. The characterized peptides are searched against
protein databases with, for example, Mascot and MSRAT software
analysis to identify the proteins. In addition to the foregoing 2-D
gel analysis and antibody microarray experiments, potential changes
to levels of specific proteins induced by the candidate MIM may be
evaluated by Western blot analysis. In all of the proteomic
experiments, proteins with increased or decreased levels in the
various cell lines are identified and evaluated.
[0459] Candidate Epi-shifters are evaluated based on changes
induced to gene expression, at the mRNA and/or protein levels, in
the cell lines due to the addition of the candidate Epi-shifter. In
particular, candidate Epi-shifters are evaluated based on their
ability to modualate genes known to be associated with or involved
in cellular metabolic processes. Of particular relevance to
metabolic disorders, candidate Epi-shifters are evaluated based on
their ability to modulate genes known to be associated with, for
example, diabetes or obesity.
[0460] The level of the candidate Epi-shifter, as well as the form
of the candidate Epi-shifter, present in the cell or a particular
cell location is determined using routine methods known to the
skilled artisan. For example, the level of the candidate
Epi-shifter in mitochondria over time and over a range of doses is
determined by analyzing mitochondrial enriched preparations from
cells treated with the candidate Epi-shifter. The levels of the
candidate Epi-shifter in the mitochondria over the time course can
be compared and correlated with other cellular changes observed,
such as modulation of mRNA and protein levels for specific proteins
related to metabolic and apoptotic pathways.
[0461] Candidate Epi-shifters observed to induce a shift in the
metabolic state of a cell based on the results obtained from the
foregoing experiments are identified as Epi-shifters. For example,
a candidate Epi-shifter that enhances, increases or augments
insulin-stimulated glucose uptake in cells is identified as an
Epi-shifter.
Example 19
Identification of Vitamin D3 as an Epi-Shifter
[0462] Vitamin D3, or 1.alpha., 25-dihydroxyvitamin D3 (also known
as calcitriol), is a vitamin D metabolite that is synthesized from
vitamin D by a two-step enzymatic process. Vitamin D3 interacts
with its ubiquitous nuclear vitamin D receptor (VDR) to regulate
the transcription of a wide spectrum of genes involved in calcium
and phosphate homeostasis as well as in cell division and
differentiation. Vitamin D3 has been reported to have anticancer
effects in numerous model systems, including squamous cell
carcinoma, prostate adenocarcinoma, cancers of the ovary, breast
and lung (reviewed in Deeb et al. 2007 Nature Reviews Cancer
7:684-700).
[0463] The anticancer effects of vitamin D3 are reported to involve
multiple mechanisms, including growth arrest at the G1 phase of the
cell cycle, apoptosis, tumor cell differentiation, disruption of
growth factor-mediated cell survival signals, and inhibition of
angiogenesis and cell adhesion (reviewed in Deeb et al. 2007 Nature
Reviews Cancer 7:684-700). For example, with particular respect to
apoptosis, Vitamin D3 has been reported to induce apoptosis by
regulating key mediators of apoptosis, such as repressing the
expression of the anti-apoptotic, pro-survival proteins BCL2 and
BCL-XL, or inducing the expression of pro-apoptotic proteins (e.g.,
BAX, BAK and BAD) (Deeb et al. 2007). In a further example, with
particular respect to angiogenesis, Vitamin D3 has been reported to
inhibit the proliferation of some tumor-derived endothelial cells
and to inhibit the expression of vascular endothelial growth factor
(VEGF) that induces angiogenesis in tumors (reviewed in Masuda and
Jones, 2006 Mol. Cancer. Ther. 5(4): 797-8070). In another example,
with particular respect to cell cycle arrest, Vitamin D3 has been
reported to induce gene transcription of the cyclin-dependent
kinase inhibitor p21WAFI/CIPI and to induce the synthesis and/or
stabilization of the cyclin-dependent kinase inhibiotor p27KIPI
protein, both of which are critical for induction of G1 arrest.
(Deeb et al. 2007).
[0464] Based on the foregoing observations, Vitamin D3 is
identified as an Epi-shifter, i.e., owing to its ability to shift
the metabolic state of a cell. Vitamin D3 is an Epi-shifter owing
to its ability to induce apoptosis in a cell and, in particular,
based on its ability to differentially inhibit cell growth and
induce the apoptotic response in diseased (cancer) cells as
compared to normal cells (e.g., differentially modulate expression
of proteins, such as BCL-2, BCL-XL, and BAX, involved in apoptosis
in cancer cells as compared to normal cells).
Example 20
Western Analysis of Cells Treated with Coenzyme Q10
[0465] Over the past five decades enormous volume of information
has been generated implicating endogenous/exogenous factors
influencing specific processes as the underlying cause of malignant
transformations. Clinical and basic literature provides evidence
that changes in the DNA structure and function play a significant
role in the initiation and progression of cancer, defining cancer
as a genetic disease (Wooster, 2010; Haiman, 2010). In the early
1920s, Otto Warburg and other investigators involved in
characterizing fundamental changes in etiology of oncogenesis
described two major observations (a) the ability of cells to
transport and utilize glucose in the generation of ATP for energy
production in the presence of oxygen--also known as Warburg Effect
and (b) alterations in the mitochondrial structure and
function--including changes in the electron transport leading to a
decrease in the production of mitochondrial ATP. The past few years
has seen a resurgence in the investigating the central role of
cellular bioenergetics in the etiology of cancer i.e. viewing
cancer as a metabolic disease.
[0466] Historically, although mutations in genes has been thought
to be responsible for changes in gene expression, there is
accumulating literature in support of epigenetic processes playing
a critical role in influencing gene expression in supporting
carcinogenesis. This is evidenced by the observation that mutation
rate for most genes is low and cannot account for the numerous
(spectrum of) mutations found in the cancer cells. Epigenetic
alteration is regulated by methylation and modification of histone
tails, both changes inherently linked to the energy (nutrient)
status of the cells since they require the availability of
co-factors e.g. acetyl CoA requirement for histone acetylation
(ref). The biosynthesis of acetyl CoA depends on glycolysis and
Kreb's Cycle, directly linking the intracellular energy status to
regulation of gene expression and activity.
[0467] In normal cells, mitochondrial oxidative phosphorylation
generates sufficient
[0468] ATP to meet the energy demands for maintaining normal
physiological activities and cell survival. A consequence of
mitochondrial energy production is the generation of reactive
oxygen species (ROS), aberrant production of which leads to damage
of mitochondria (refs). It is well established that chronic ROS
generation by the mitochondria leads to cumulative accumulation of
genetic mutations, a phenomenon that has been implicated in the
etiology of carcinogenesis. It has been suggested that cancer cells
decrease mitochondrial respiration to minimize ROS generation, and
switch to glycolysis to sustain energy production. Thus, a
progressive shift of energy generation from oxidative
phosphorylation to glycolysis would be essential for a cell to
maintain energy production to maintain physiological functions and
could be associated with the progression of a normal cell phenotype
to that of a cancer cell. The progressive shift in cellular energy
(bioenergetic) profile in tandem with accumulated alteration
(mutations) in mitochondrial genetic make-up alters the cellular
metabolome. Changes in the whole cell metabolomic profile as a
consequence of mitochondrial phosphorylation to glycolysis
transition corresponds to an abnormal bioenergetic induced
metabolomic profile and is the underlying cause supporting
carcinogenesis. Targeted intervention using an endogenous molecule
to elicit a cellular metabolomic shift towards conditions of a
non-cancerous normal mitochondrial oxidative phosphorylation
associated cellular bioenergetic state represents a therapeutic
endpoint in the treatment of cancer.
Coenzyme Q10 as a MIM Causing an Epi-Metabolomic Shift
[0469] The data presented herein demonstrates that treatment of
normal and cancer cells with Coenzyme Q10 is associated with
changes in the expression of proteins that regulate key biochemical
terminals within the glycolysis--mitochondrial oxidative stress
continuum. The combination of data describing assessment of protein
expression by western blotting and oxygen consumption rates
demonstrates that in normal cells, there is no significant
alteration in normal glycolytic and mitochondrial respiration rates
following exposure to Coenzyme Q10. Thus, the values for expression
of the proteins and mitochondrial respiration rates in normal cell
lines e.g. HDFa (normal human adult fibroblast), HASMC (normal
human aortic smooth muscle cell), nFib (normal fibroblast) and HeKa
(normal human keratinocytes) can be considered as representatives
of baseline physiological state. Any deviation in expression of
proteins and mitochondrial respiration rates in cancer cell lines,
e.g. HepG2 (liver cancer), PaCa-2 (pancreatic cancer), MCF7 (breast
cancer), SK-MEL (melanoma) and SCC-25 (squamous cell carcinoma), is
representative of alteration due to initiation/progression of the
disease, in this case cancer. The experimental evidence provides
support to the hypothesis that exposure of Coenzyme Q10 to cancer
cells is associated with cellular pathophysiological reorganization
that is reminiscent of normal cells. Specifically, the data
provided herein demonstrates that Coenzyme Q10 exposure in cancer
cells is associated with a shift in the glycolytic pathways and
mitochondrial oxidative phosphorylation responsible for induction
of global reorganization of cellular architecture to that observed
in normal cells.
[0470] In normal cells, the end-points of glycolytic output are
linked to mitochondrial oxidative phosphorylation (OXPHOS), i.e.
generation of pyruvate from glucose via the glycolytic pathway for
the entry into the Kreb's Cycle (also known as Tricarboxylic acid
cycle, TCA, or Citric Acid Cycle) to generate reducing equivalents
to support the mitochondrial OXPHOS for ATP production. Thus, in
normal cells the expression and functional orientation of gene
products involved in glycolysis is primed towards adequate
generation of pyruvate and its entry into the Kreb's Cycle.
Dysregulated expression and function of key proteins participating
in glycolysis and Kreb's Cycle pathways in cancer cells results in
enhanced glycolysis with a significant decrease in mitochondrial
function. Exposure of cancer cells to Coenzyme Q10, an endogenous
molecule that selectively influences the mitochondrial respiratory
chain, alters (normalizes) expression of proteins of the glycolyis
and Kreb's Cycle pathways to facilitate a bioenergetic shift such
that energy production (i.e. ATP generation) is restored to the
mitochondria.
EXPERIMENTAL PROCEDURE
Western Blot Experiment 1
[0471] The cells that were used for the experiment were HDFa, and
MCF-7 cells that were treated or not with Coenzyme Q10 at two
different concentrations, 50 .mu.M and 100 .mu.M, and harvested
after 24 hours of treatment. The whole cell pellets were
resuspended one at a time in 1 mL of C7 buffer and transferred to
labeled 15 mL tubes. The samples were then sonicated in the cold
room on ice using 6 sonic pulses with the setting at #14. The
samples were spun for a short time to 2500 g after sonication and
the samples transferred to 2 ml tubes. The pH was verified of each
sample (pH should be 9.0) using the foam remaining in the 50 mL
sample tubes.
[0472] Alkylation and reduction of samples was performed for each
sample by adding 10 ul of 1M acrylamide, 25 ul of tributylphoshene
and incubation for 90 mins with intermittent mixing. After
incubation, 10 ul of 1M DTT was added and the tubes were spun at
20,000 g at 20 deg C. for 10 minutes and transferred the
supernatant to labeled Amicon Ultra centrifugal filter units with a
10 k cut off (Millipore catalog #UFC 801024). The samples were spun
for 15 minutes at 2500 g in 2 intervals. The conductivity was
measured for Chaps alone as well as the samples using a
conductivity meter. If the conductivity of samples is high, then 1
ml of chaps was added for buffer exchange and spun again at 2500 g
until the volume was down to 250 ul. When the conductivity was 200
or less the samples were spun in 5 min intervals at 2500 g until
the volume of the supernatant was between 150-100 ul. The sample
supernatants were transferred to eppendorf tubes and Bradford assay
was performed using BSA as standard.
[0473] The samples were processed as per standard protocols as
described above and the amount of protein in each of the samples
was determined by Bradford assay. Sample volumes equivalent to 10
ug of protein were prepared as shown below with Lamelli Loading dye
(LDS) and MilliQ water were run on a 4-12% Bis-Tris Novex NuPAGE
gel (Invitrogen, cat #NP0323Box)
[0474] The gels were run for 50 minutes using 1.times. MOPS buffer
using a NOVEX Xcell Surelock system at 200 V. The gels were then
transferred for 1 hour using a NOVEX Xcell Surelock wet transfer
protocol at 30 V. The blots were stained with Simply Blue Safestain
from Invitrogen (LC6065).
IDH1 and ATP Citrate Lyase Levels in HDFa and MCF-7 Samples.
[0475] After transfer each of the blots was placed in between 2
Whatman Filter papers and dried for 15-20 minutes. After drying the
blots were labeled with the date, the type of samples and either
blot 1 or blot 2 using a HB pencil. The molecular weight markers
were outlined with the pencil and with single lines for the blue
and a doublet for the colored markers. The blots were activated
with methanol for 5 seconds, washed with water for 5 minutes, and
TBST for 15 minutes. The blots were blocked for 1 hour with 5%
blocking reagent in TBS-T at room temperature and then washed 3
times with TBS-T (1.times.-15'; 2.times.5' each). Blot 1 was probed
with the primary antibody for IDH1 (Cell Signaling #3997) in TBST
with 5% BSA (at 1:1000 dilutions) and blot 2 with the rabbit
polyclonal antibody for ATP Citrate Lyase in 5% BSA (Cell Signaling
#4332) at 1:1000 dilution by incubation overnight at 4 deg C. with
shaking. After the overnight incubation with primary antibodies,
the blots were washed 3 times with TBS-T (1.times.-15'; 2.times.5'
each) and probed with the secondary antibody (antirabbit; 1:10,000
dilution) for 1 h on the orbital tilting shaker at room
temperature. After 1 h of incubation with secondary antibodies, the
blots were washed 3 times with TBS-T (1.times.-15'; 2.times.5'
each) and then incubated with ECF reagent for 5 mins and then each
blot scanned with 5100 Fuji Laser scanner at 25 uM resolution, 16
bit, green laser, at 400V and at 500 V.
Actin Levels in HDFa and MCF-7 Samples.
[0476] The above blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The 2 blots were scanned in laser scanner to check for complete
stripping. The blots were then activated with methanol for 5
seconds, washed with water for 5 minutes, and TBST for 15 minutes.
The blots were blocked for 1 hour with 5% blocking reagent in TBS-T
at room temperature and then washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and probed with the antibody for
Actin in 5% BSA (Sigma catalog #A5316, clone AC-74) at 1:5000
dilutions for 1 hour at room temperature with shaking. After 1 hour
of incubation with primary antibody for Actin, the membranes were
washed 3 times with TBS-T (1.times.-15'; 2.times.5' each) and
probed with the secondary antibody (antimouse; 1:10,000 dilution)
for 1 h on the orbital tilting shaker at room temperature. After 1
h of incubation with secondary antibodies, the blots were washed 3
times with TBS-T (1.times.-15'; 2.times.5' each) and then incubated
with ECF reagent for 5 minutes and then each blot scanned with 5100
Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at
400V and at 500 V.
Western Blot Experiment 2
[0477] The cells used in this experiment were SKMEL28, SCC-25, nFib
and Heka that were treated or not with coenzyme Q10 at two
different concentrations, 50 .mu.M or 100 .mu.M, and harvested
after 3, 6 and/or 24 hours of treatment. The samples were processed
and run on a 4-12% Bis-Tris Novex NuPAGE gel as described above.
The gels were run, transferred and stained essentially as described
above.
Levels of IDH1 for the 4 Cell Lines
[0478] After transfer the blot was dried for 15-20 minutes,
activated with methanol for 5 seconds, washed with water for 5
minutes, and TBST for 15 minutes. The blot was blocked for 1 hour
with 5% blocking reagent in TBS-T at room temperature and then
washed 3 times with TBS-T (1.times.-15'; 2.times.5' each). This was
then probed with the primary antibody for IDH1 (Cell Signaling
#3997) in TBST with 5% BSA (at 1:1000 dilutions) by incubation
overnight at 4 deg C. with shaking. After the overnight incubation
with primary antibody for IDH1, the blot was washed 3 times with
TBS-T (1.times.-15'; 2.times.5' each) and probed with the secondary
antibody (antirabbit; 1:10,000 dilution) for 1 h at room
temperature. After 1 h of incubation with secondary antibodies, the
blot was washed 3 times with TBS-T (1.times.-15'; 2.times.5' each)
and then incubated with ECF reagent for 5 mins and then each blot
scanned with 5100 Fuji Laser scanner at 25 uM resolution, 16 bit,
green laser, at 400V and at 500 V.
ATP Citrate Lyase Levels in 4 Different Cell Lines.
[0479] The Isocitrate dehydrogenase blot was stripped by incubating
for 30 minutes with methanol, followed by two 10 minute washes with
TBS-T, then 30 minutes of incubation with stripping buffer at 50
deg C., and followed by two washes with 100 ml or more of TBS-T for
30' each. The blot was scanned in laser scanner to check for
complete stripping. The blot was activated with methanol for 5
seconds, washed with water for 5 minutes, and TBST for 15 minutes.
The blot was blocked for 1 hour with 5% blocking reagent in TBS-T
at room temperature and then washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each). This was then probed with the
rabbit polyclonal antibody for ATP Citrate Lyase in 5% BSA (Cell
Signaling #4332) at 1:1000 dilution overnight at 4 deg C. with
shaking. After the overnight incubation with primary antibody for
ATP Citrate Lyase, the membrane was washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and probed with the secondary
antibody (antirabbit; 1:10,000 dilution) for 1 h on the orbital
tilting shaker at room temperature. After 1 h of incubation with
secondary antibodies, the blot was washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and then incubated with ECF reagent
for 5 minutes and then each blot scanned with 5100 Fuji Laser
scanner at 25 uM resolution, 16 bit, green laser, at 400V and at
500 V.
Actin Levels in 4 Different Cell Lines.
[0480] The ATP Citrate Lyase blot was stripped by incubating for 30
minutes with methanol, followed by two 10 minute washes with TBS-T,
then 30 minutes of incubation with Stripping buffer at 50 deg C.,
and followed by two washes with 100 ml or more of TBS-T for 30'
each. The blot was scanned in laser scanner to check for complete
stripping. The blot was activated with methanol for 5 seconds,
washed with water for 5 minutes, and TBST for 15 minutes. The blot
was blocked for 1 hour with 5% blocking reagent in TBS-T at room
temperature and then washed 3 times with TBS-T (1.times.-15';
2.times.5' each) and probed with the antibody for Actin in 5% BSA
(Sigma catalog #A5316, clone AC-74) at 1:5000 dilutions for 1 hour
at room temperature with shaking. After 1 hour of incubation with
primary antibody for Actin, the membranes were washed 3 times with
TBS-T (1.times.-15'; 2.times.5' each) and probed with the secondary
antibody (antimouse; 1:10,000 dilution) for 1 h on the orbital
tilting shaker at room temperature. After 1 h of incubation with
secondary antibodies, the blots were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and then incubated with ECF reagent
for 5 minutes and then each blot scanned with 5100 Fuji Laser
scanner at 25 uM resolution, 16 bit, green laser, at 400V and at
500 V.
Western Blot Experiment 3
[0481] The cells used in this experiment were HepG2, HASMC, and
PACA2 cells that were treated or not with Coenzyme Q10 at two
different concentrations (50 .mu.M and 100 .mu.M) and harvested 48
hours of treatment. In this experiment (western blot experiment 3),
and in all of the experiments described below in this Example
(i.e., western blot experiments 4 through 9), the cells were
additionally treated with either 5 mM glucose ("5G") or 22 mM
glucose ("22G"). The samples derived from the cells were processed
and run on a 4-12% Bis-Tris Novex NuPAGE gel as described above.
The gels were run, transferred and stained essentially as described
above.
IDH1, ATP Citrate Lyase and Actin Levels in HASMC vs. PACA2 and
HepG2.
[0482] The levels of IDH1, ATP citrate lyase and actin levels were
determined by probing the blots with primary antibodies for IDH1,
ATP citrate lyase and actin, essentially as described above.
Western Blot Experiment 4
[0483] The cells used in this experiment were HepG2 cells that were
treated or not with Coenzyme Q10 at two different concentrations,
50 or 100 .mu.M, and harvested after 24 or 48 hours of treatment.
The samples were processed and run on a 4-12% Bis-Tris Novex NuPAGE
gel as described above. The gels were run, transferred and stained
essentially as described above.
Lactate Dehydrogenase Levels in HepG2 Cells.
[0484] After transfer each blot was dried for 15-20 minutes,
activated with methanol for 5 seconds, washed with water for 5
minutes, and TBST for 15 minutes. The blots were blocked for 1 hour
with 5% blocking reagent in TBS-T at room temperature and then
washed 3 times with TBS-T (1.times.-15'; 2.times.5' each) and
probed with the primary antibody for Lactate Dehydrogenase (abcam
ab2101; polyclonal) in 5% BSA (at 1:1000 dilutions) by incubation
overnight at 4 deg C. with shaking. After the overnight incubation
with primary antibody for Lactate Dehydrogenase, the blots were
washed 3 times with TBS-T (1.times.-15'; 2.times.5' each) and
probed with the secondary antibody (rabbit antigoat; 1:10,000
dilution) for 1 h at room temperature. After 1 h of incubation with
secondary antibodies, the blots were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and then incubated with ECF reagent
for 5 mins and then each blot scanned with 5100 Fuji Laser scanner
at 25 uM resolution, 16 bit, green laser, at 400V and at 500 V.
Pyruvate Kinase Muscle form (PKM2) Levels in HepG2 Cells.
[0485] The lactate dehydrogenase blots were stripped by incubating
for 30 minutes with methanol, followed by two 10 minute washes with
TBS-T, then 30 minutes of incubation with Stripping buffer at 50
deg C., and followed by two washes with 100 ml or more of TBS-T for
30' each. The 2 blots were scanned in laser scanner to check for
complete stripping. The blots were activated with methanol for 5
seconds, washed with water for 5 minutes, and TBST for 15 minutes.
The blots were blocked for 1 hour with 5% blocking reagent in TBS-T
at room temperature and then washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and probed with the rabbit
polyclonal antibody for Pyruvate Kinase M2 in 5% BSA (NOVUS
BIOLOGICALS catalog #H00005315-DO1P) at 1:500 dilution overnight at
4 deg C. with shaking. After the overnight incubation with primary
antibody for Pyruvate Kinase M2, the membranes were washed 3 times
with TBS-T (1.times.-15'; 2.times.5' each) and probed with the
secondary antibody (antirabbit; 1:10,000 dilution) for 1 h on the
orbital tilting shaker at room temperature. After 1 h of incubation
with secondary antibodies, the blots were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and then incubated with ECF reagent
for 5 minutes and then each blot scanned with 5100 Fuji Laser
scanner at 25 uM resolution, 16 bit, green laser, at 400V and at
500 V.
Pyruvate Dehydrogenase Beta Levels in HepG2 Cells.
[0486] The pyruvate kinase blots were stripped by incubating for 30
minutes with methanol, followed by two 10 minute washes with TBS-T,
then 30 minutes of incubation with Stripping buffer at 50 deg C.,
and followed by two washes with 100 ml or more of TBS-T for 30'
each. The 2 blots were scanned in laser scanner to check for
complete stripping. After making sure stripping of the antibody and
the ECF reagent has worked, the blots were activated with methanol
for 5 seconds, washed with water for 5 minutes, and TBST for 15
minutes. The blots are blocked for 1 hour with 5% blocking reagent
in TBS-T at room temperature and then washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and probed with the antibody for
Pyruvate Dehydrogenase in 5% BSA (ABNOVA catalog #H00005162-M03) at
1:500 dilutions) overnight at 4 deg C. with shaking. After the
overnight incubation with primary antibody for Pyruvate
Dehydrogenase, the membranes were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and probed with the secondary
antibody (antimouse; 1:10,000 dilution) for 1 h on the orbital
tilting shaker at room temperature. After 1 h of incubation with
secondary antibodies, the blots were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and then incubated with ECF reagent
for 5 minutes and then each blot scanned with 5100 Fuji Laser
scanner at 25 uM resolution, 16 bit, green laser, at 400V and at
500 V.
Actin Levels in HepG2 Cells.
[0487] The Pyruvate Dehydrogenase blots were stripped and then
reprobed for actin, essentially as described above.
Western Blot Experiment 5
[0488] The cells used in this experiment were MIAPACA2 (PACA2)
cells that were treated or not with Coenzyme Q10 at two different
concentrations, 50 or 100 .mu.M, and harvested after 24 or 48 hours
of treatment. The PACA2 samples were processed and the gels were
run, transferred, stained and scanned essentially as described
above.
Lactate Dehydrogenase (LDH) and Pyruvate Dehydrogenase (PDH) Levels
in PaCa2 Cells
[0489] The levels of LDH and PDH were determined by probing the
blots successively with primary antibodies for LDH and PDH,
essentially as described above.
Caspase 3 Levels in PaCa2 Cells.
[0490] The blots were stripped by incubating for 30 minutes with
methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The 2 blots were scanned in laser scanner to check for complete
stripping. The blots were activated with methanol for 5 seconds,
washed with water for 5 minutes, and TBST for 15 minutes. The blots
were blocked for 1 hour with 5% blocking reagent in TBS-T at room
temperature and then washed 3 times with TBS-T (1.times.-15';
2.times.5' each) and probed with the antibody for Caspase 3 in 5%
BSA (Santacruz Biotechnology # sc7272) at 1:200 dilutions)
overnight at 4 deg C. with shaking. After the overnight incubation
with primary antibody for Caspase 3, the membranes were washed 3
times with TBS-T (1.times.-15'; 2.times.5' each) and probed with
the secondary antibody (antimouse; 1:10,000 dilution) for 1 h on
the orbital tilting shaker at room temperature. After 1 h of
incubation with secondary antibodies, the blots were washed 3 times
with TBS-T (1.times.-15'; 2.times.5' each) and then incubated with
ECF reagent for 5 minutes and then each blot scanned with 5100 Fuji
Laser scanner at 25 uM resolution, 16 bit, green laser, at 400V and
at 500 V.
Western Blot Experiment 6
[0491] The cells that were used for this Western blot experiment
were PC-3, HepG2, MCF-7, HDFa and PACA2 that were treated or not
with a Coenzyme Q10 IV formulation and harvested after 24 hours of
treatment. The samples were processed and the gels were run,
transferred, stained and scanned essentially as described
above.
Capase 3 and Actin Levels in Different Cell Types.
[0492] The levels of Caspase 3 and actin were determined by probing
the blots successively with primary antibodies for Caspase 3 and
actin, essentially as described above.
Western Blot Experiment 7
[0493] The cells used in this experiment were Human Aortic Smooth
Muscle (HASMC) cells that were treated or not with Coenzyme Q10 at
two different concentrations, 50 .mu.M or 100 .mu.M, and harvested
after 24 or 48 hours of treatment. The HASMC samples were processed
and the gels were run, transferred, stained and scanned essentially
as described above.
Experimental Protocol for Actin:
[0494] The levels of actin were determined by probing the blots
with a primary antibody for actin, essentially as described
above.
Experimental Protocol for Hif 1Alpha, Caspase3 and PDHB:
[0495] The Actin blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The blots were scanned in laser scanner to check for complete
stripping. The blots were activated with methanol for 5 seconds,
washed with water for 5 minutes, and TBST for 15 minutes. The blots
were blocked for 1 hour with 5% blocking reagent in TBS-T at room
temperature and then washed 3 times with TBS-T (1.times.-15';
2.times.5' each) and probed with the primary antibody for Hif 1
alpha, Caspase 3 or PDHB in 5% BSA (at 1:200 by incubation
overnight at 4 deg C. with gentle shaking. The primary antibody for
Hif 1 alpha (Abcam ab2185; antirabbit) was at 1:500 dilution in 5%
BSA. The primary antibody for Caspase 3 (Santacruz sc7272;
antirabbit) was at 1:200 dilution in 5% BSA. The primary antibody
for Pyruvate Dehydrogenase beta (PDHB) (Novus Biologicals
H00005162-M03; antimouse) was at 1:500 dilution in 5% BSA. After
incubation with primary antibodies, the membranes were washed 3
times with TBS-T (1.times.-15'; 2.times.5' each) and probed with
the secondary antibody (PDHB antimouse; Hif 1a and Caspase 3
antirabbit; 1:10,000 dilution) for 1 h at room temperature. After 1
h of incubation with secondary antibodies, the blots were washed 3
times with TBS-T (1.times.-15'; 2.times.5' each) and then incubated
with ECF reagent for 5 minutes and then each blot scanned with 5100
Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at
400V and at 500 V.
Experimental Protocol for PKM2, SDHB and SDHC:
[0496] The above blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The blots were scanned in laser scanner to check for complete
stripping. The blots were activated with methanol for 5 seconds,
washed with water for 5 minutes, and TBST for 15 minutes. The blots
were blocked for 1 hour with 5% blocking reagent in TBS-T at room
temperature and then washed 3 times with TBS-T (1.times.-15';
2.times.5' each) and probed with the primary antibody for PKM2,
SDHB or SDHC in 5% BSA in TBS-T by incubation overnight at 4 deg C.
with gentle shaking. The primary antibody for SDHC (ABNOVA
H00006391-MO2; antimouse) was at 1:500 dilution. The primary
antibody for SDHB was from Abcam ab4714-200; antimouse; at 1:1000
dilution. The primary antibody for Pyruvate Kinase M2 (PKM2) was
from Novus Biologicals H00005315-D0IP; antirabbit; at 1:500
dilution. After incubation with primary antibodies, the membranes
were washed 3 times with TBS-T (1.times.-15'; 2.times.5' each) and
probed with the secondary antibody (SDHB & C antimouse; and
PKM2 antirabbit; 1:10,000 dilution) for 1 h on the orbital tilting
shaker at room temperature. After 1 h of incubation, the blots were
washed 3 times with TBS-T (1.times.-15'; 2.times.5' each) and
incubated with ECF reagent for 5 minutes and then each blot scanned
with 5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green
laser, at 400V and at 500 V.
Experimental Protocol for LDH and Bik:
[0497] The above blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The blots were scanned in laser scanner to check for complete
stripping. The blots were activated with methanol for 5 seconds,
washed with water for 5 minutes, and TBST for 15 minutes. The blots
were blocked for 1 hour with 5% blocking reagent in TBS-T at room
temperature and then washed 3 times with TBS-T (1.times.-15';
2.times.5' each) and probed with the primary antibody for LDH or
Bik in 5% BSA in TBS-T by incubation overnight at 4 deg C. with
gentle shaking. The primary antibody for LDH was from Abcam ab2101;
antigoat; at 1:1000 dilution. The primary antibody for Bik was from
Cell Signaling #9942; antirabbit; at 1:1000 dilution. After
incubation with primary antibodies, the membranes were washed 3
times with TBS-T (1.times.-15'; 2.times.5' each) and probed with
the secondary antibody (LDH antigoat; Jackson Laboratories) and Bik
antirabbit; 1:10,000 dilution) for 1 h on the orbital tilting
shaker at room temperature. After 1 h of incubation, the blots were
washed 3 times with TBS-T (1.times.-15'; 2.times.5' each) and
incubated with ECF reagent for 5 minutes and then each blot scanned
with 5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green
laser, at 400V and at 500 V.
Western Blot Experiment 9
[0498] The cells used were HepG2 cells that were treated or not
with Coenzyme Q10 at two different concentrations, 50 .mu.M or 100
.mu.M, and harvested after 24 or 48 hours of treatment. The HepG2
samples processed and the gels were run, transferred, stained and
scanned essentially as described above.
Experimental Protocol for Actin:
[0499] The levels of actin were determined by probing the blots
with a primary antibody for actin, essentially as described
above.
Experimental Protocol for Caspase3 and MMP-6:
[0500] The Actin blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The blots were activated with methanol for 5 seconds, washed with
water for 5 minutes, and TBST for 15 minutes. The blots were
blocked for 1 hour with 5% blocking reagent in TBS-T at room
temperature and then washed 3 times with TBS-T (1.times.-15';
2.times.5' each) and probed with the primary antibody for Caspase 3
or MMP-6 in 5% BSA by incubation overnight at 4 deg C. with gentle
shaking. The primary antibody for Caspase 3 (Abcam ab44976-100;
antirabbit) was at 1:500 dilution in 5% BSA. The primary antibody
for MMP-6 (Santacruz scMM0029-ZB5; antimouse) was at 1:100 dilution
in 5% BSA. After incubation with primary antibodies, the membranes
were washed 3 times with TBS-T (1.times.-15'; 2.times.5' each) and
probed with the secondary antibody (MMP-6 antimouse; Caspase 3
antirabbit; 1:10,000 dilution) for 1 h at room temperature. After 1
h of incubation with secondary antibodies, the blots were washed 3
times with TBS-T (1.times.-15'; 2.times.5' each) and then incubated
with ECF reagent for 5 minutes and then each blot scanned with 5100
Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at
400V and at 500 V.
Experimental Protocol for LDH:
[0501] The above blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The blots were activated with methanol for 5 seconds, washed with
water for 5 minutes, and TBST for 15 minutes. The blots ere blocked
for 1 hour with 5% blocking reagent in TBS-T at room temperature
and then washed 3 times with TBS-T (1.times.-15'; 2.times.5' each)
and probed with the primary antibody for LDH in 5% BSA or 5% milk
by incubation overnight at 4 deg C. with gentle shaking. The
primary antibody for LDH 080309b1 (Abcam ab2101; antigoat) was at
1:1000 dilution in 5% BSA. The primary antibody for LDH 080309b2
(Abcam ab2101; antigoat) was at 1:1000 dilution in 5% milk. After
incubation with primary antibodies, the membranes were washed 3
times with TBS-T (1.times.-15'; 2.times.5' each) and probed with
the secondary antibody (Jackson Immuno Research antigoat; 1:10,000
dilution; 305-055-045) for 1 h. After 1 h of incubation with
secondary antibodies, the blots were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and then incubated with ECF reagent
for 5 minutes and then each blot scanned with 5100 Fuji Laser
scanner at 25 uM resolution, 16 bit, green laser, at 400V and at
500 V.
Experimental Protocol for Transaldolase and Hif1a:
[0502] The above blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The blots were activated with methanol for 5 seconds, washed with
water for 5 minutes, and TBST for 15 minutes. The blots are blocked
for 1 hour with 5% blocking reagent in TBS-T at room temperature
and then washed 3 times with TBS-T (1.times.-15'; 2.times.5' each)
and probed with the primary antibody for Transaldolase or Hifla in
5% BSA by incubation overnight at 4 deg C. with gentle shaking. The
primary antibody for Transaldolase (Abcam ab67467; antimouse) was
at 1:500 dilution. The primary antibody for Hifla (Abcam ab2185;
antirabbit) was at 1:500 dilution. After incubation with primary
antibodies, the membranes were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and probed with the secondary
antibody (antimouse or antirabbit; 1:10,000 dilution) for 1 h on
the orbital tilting shaker at room temperature. After 1 h of
incubation with secondary antibodies, the blots were washed 3 times
with TBS-T (1.times.-15'; 2.times.5' each) and then incubated with
ECF reagent for 5 minutes and then each blot scanned with 5100 Fuji
Laser scanner at 25 uM resolution, 16 bit, green laser, at 400
& 500V.
Experimental Protocol for IGFBP3 and TP53:
[0503] The above blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The blots were activated with methanol for 5 seconds, washed with
water for 5 minutes, and TBST for 15 minutes. The blots are blocked
for 1 hour with 5% blocking reagent in TBS-T at room temperature
and then washed 3 times with TBS-T (1.times.-15'; 2.times.5' each)
and probed with the primary antibody for IGFBP3 or TP53 in 5% BSA
by incubation overnight at 4 deg C. with gentle shaking. The
primary antibody for IGFBP3 (Abcam ab76001; antirabbit) was at
1:100 dilution. The primary antibody for TP53 (Sigma Aldrich
AV02055; antirabbit) was at 1:100 dilution. After incubation with
primary antibodies, the membranes were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and probed with the secondary
antibody (antirabbit; 1:10,000 dilution) for 1 h on the orbital
tilting shaker at room temperature. After 1 h of incubation with
secondary antibodies, the blots were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and then incubated with ECF reagent
for 5 minutes and then each blot scanned with 5100 Fuji Laser
scanner at 25 uM resolution, 16 bit, green laser, at 400 &
500V.
Experimental Protocol for Transaldolase and PDHB:
[0504] The above blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The blots were activated with methanol for 5 seconds, washed with
water for 5 minutes, and TBST for 15 minutes. The blots were
blocked for 1 hour with 5% blocking reagent in TBS-T at room
temperature and then washed 3 times with TBS-T (1.times.-15';
2.times.5' each) and probed with the primary antibody for
Transaldolase or PDHB in 5% BSA by incubation overnight at 4 deg C.
with gentle shaking. The primary antibody for Transaldolase
(Santacruz sc51440; antigoat) was at 1:200 dilution. The primary
antibody for PDHB (Novus Biologicals H00005162-M03; antimouse) was
at 1:500 dilution. After incubation with primary antibodies, the
membranes were washed 3 times with TBS-T (1.times.-15'; 2.times.5'
each) and probed with the secondary antibody (antigoat or
antimouse; 1:10,000 dilution) for 1 h on the orbital tilting shaker
at room temperature. After 1 h of incubation with secondary
antibodies, the blots were washed 3 times with TBS-T (1.times.-15';
2.times.5' each) and then incubated with ECF reagent for 5 minutes
and then each blot scanned with 5100 Fuji Laser scanner at 25 uM
resolution, 16 bit, green laser, at 400 & 500V.
Results
Isocitrate Dehydrogenase-1 (IDH-1)
[0505] Isocitrate dehydrogenase is one of the enzymes that is part
of the TCA cycle that usually occurs within the mitochondrial
matrix. However, IDH1 is the cytosolic form of the enzyme that
catalyzes the oxidative decarboxylation of isocitrate to
.alpha.-ketoglutarate and generates carbon dioxide in a two step
process. IDH1 is the NADP dependent form that is present in the
cytosol and peroxisome. IDH1 is inactivated by Ser113
phosphorylation and is expressed in many species including those
without a citric acid cycle. IDH1 appears to function normally as a
tumor suppressor which upon inactivation contributes to
tumorigenesis partly through activation of the HIF-1 pathway
(Bayley 2010; Reitman, 2010). Recent studies have implicated an
inactivating mutation in IDH1 in the etiology of glioblasotoma
(Bleeker, 2009; Bleeker, 2010).
[0506] Treatment with Coenzyme Q10 increased expression of IDH1 in
cancer cell lines including MCF-7, SKMEL28, HepG2 and PaCa-2 cells.
There was a moderate increase in expression in the SCC25 cell
lines. In contrast cultures of primary human derived fibroblasts
HDFa, nFIB and the human aortic smooth muscle cells HASMC did not
demonstrate significant changes in the expression pattern of the
IDH1 in response to Coenzyme Q10. .alpha.-ketoglutarate
(.alpha.-KG) is a key intermediate in the TCA cycle, biochemically
synthesized from isocitrate and is eventually converted to succinyl
coA and is a druggable MIM and EpiShifter. The generation of
.alpha.-KG serves as a critical juncture in the TCA cycle as it can
be used by the cell to replenish intermediates of the cycle,
resulting in generation of reducing equivalents to increase
oxidative phosphorylation. Thus, Coenzyme Q10 mediated increase in
IDH1 expression would result in formation of intermediates that can
be used by the mitochondrial TCA cycle to augment oxidative
phosphorylation in cancer cells. The results are summarized in the
tables below.
TABLE-US-00030 TABLE 29 IDH1 in HDFa and MCF-7 Average Normalized
Composition Intensity HDF, Media 346 HDF24-50-Coenzyme Q10 519
HDF24-100-Coenzyme Q10 600 MCF, Media 221 MCF24-50-Coenzyme Q10 336
MCF24-100-Coenzyme Q10 649
TABLE-US-00031 TABLE 30 IDH1 in HASMC vs. HepG2 after Treatment
Normalized Amount - Composition Intensity HAS5g48-media 20
HAS5g48-50-Coenzyme Q10 948 HAS5g48-100-Coenzyme Q10 1864
HAS22G48-Media 1917 HAS22G48-50-Coenzyme Q10 1370
HAS22G48-100-Coenzyme Q10 1023 Hep5g48-Media 14892
Hep5g48-50-Coenzyme Q10 14106 Hep5g48-100-Coenzyme Q10 15774
Hep22G48-Media 16558 Hep22G48-50-Coenzyme Q10 15537
Hep22G48-100-Coenzyme Q10 27878
TABLE-US-00032 TABLE 31 IDH1 in HASMC vs. PACA2 after Treatment
Amount - Composition Normalized Intensity HAS5g48-media 562
HAS5g48-50-Coenzyme Q10 509 HAS5g48-100-Coenzyme Q10 627
HAS22G48-Media 822 HAS22G48-50-Coenzyme Q10 1028
HAS22G48-100-Coenzyme Q10 1015 PACA5g48-Media 1095
PACA5g48-50-Coenzyme Q10 1095 PACA5g48-100-Coenzyme Q10 860
PACA22G48-Media 1103 PACA22G48-50-Coenzyme Q10 1503
PACA22G48-100-Coenzyme Q10 1630
ATP Citrate Lyase (ACL)
[0507] ATP citrate Lyase (ACL) is a homotetramer (.about.126 kd)
enzyme that catalyzes the formation of acetyl-CoA and oxaloacetate
in the cytosol. This reaction is a very important first step for
the biosynthesis of fatty acids, cholesterol, and acetylcholine, as
well as for glucogenesis (Towle et al., 1997). Nutrients and
hormones regulate the expression level and phosphorylation status
of this key enzyme. Ser454 phosphorylation of ACL by Akt and PKA
has been reported (Berwick, DC M W et al., 2002; Pierce M W et al.,
1982).
[0508] The data describes the effect of Coenzyme Q10 on ATP citrate
Lyase is that in normal and cancer cells. It is consistently
observed that in cancer cells there is a dose-dependent decrease in
the expression of ACL enzymes. In contrast there appears to be a
trend towards increased expression of ACL in normal cells.
Cytosolic ACL has been demonstrated to be essential for histone
acetylation in cells during growth factor stimulation and during
differentiation. The fact that ACL utilizes cytosolic glucose
derived citrate to generate Acetyl CoA essential for histone
acetylation, a process important in the neoplastic process
demonstrates a role of Coenzyme Q10 induced ACL expression in
influencing cancer cell function. Acetyl CoA generated from citrate
by cytosolic ACL serves as a source for biosynthesis of new lipids
and cholesterol during cell division. Thus, Coenzyme Q10 induced
changes in ACL expression alters Acetyl CoA availability for
synthesis of lipids and cholesterol in normal versus cancer cells.
The results are summarized in the tables below.
TABLE-US-00033 TABLE 32 ATPCL in HDFa and MCF-7 Composition Average
Normalized Intensity HDF-Media ~15000 HDF-50-Coenzyme Q10 ~17500
HDF-100-Coenzyme Q10 ~25000 MCF-Media ~7500 MCF-50-Coenzyme Q10
~7500 MCF-100-Coenzyme Q10 ~12500
TABLE-US-00034 TABLE 33 ATP Citrate Lysase ~kd band in HASMC vs.
HepG2 Amount - Composition Normalized Intensity HAS5g48-media 24557
HAS5g48-50-Coenzyme Q10 23341 HAS5g48-100-Coenzyme Q10 25544
HAS22G48-Media 27014 HAS22G48-50-Coenzyme Q10 21439
HAS22G48-100-Coenzyme Q10 19491 Hep5g48-Media 28377
Hep5g48-50-Coenzyme Q10 24106 Hep5g48-100-Coenzyme Q10 22463
Hep22G48-Media 24262 Hep22G48-50-Coenzyme Q10 31235
Hep22G48-100-Coenzyme Q10 50588
TABLE-US-00035 TABLE 34 ATP Citrate Lysase ~kd band in HASMC vs.
PACA2 Amount - Composition Normalized Intensity HAS5g48-media 11036
HAS5g48-50-Coenzyme Q10 12056 HAS5g48-100-Coenzyme Q10 15265
HAS22G48-Media 18270 HAS22G48-50-Coenzyme Q10 15857
HAS22G48-100-Coenzyme Q10 13892 PACA5g48-Media 11727
PACA5g48-50-Coenzyme Q10 8027 PACA5g48-100-Coenzyme Q10 4942
PACA22G48-Media 8541 PACA22G48-50-Coenzyme Q10 9537
PACA22G48-100-Coenzyme Q10 14901
TABLE-US-00036 TABLE 35 ATP Citrate Lysase in HepG2 and PACA2 as %
of CTRL Amount - Composition Normalized Intensity PACA5g48-Media
1.00 PACA5g48-50-Coenzyme Q10 0.68 PACA5g48-100-Coenzyme Q10 0.42
PACA22G48-Media 1.00 PACA22G48-50-Coenzyme Q10 1.12
PACA22G48-100-Coenzyme Q10 1.74 Hep5g48-Media 1.00
Hep5g48-50-Coenzyme Q10 0.85 Hep5g48-100-Coenzyme Q10 0.79
Hep22G48-Media 1.00 Hep22G48-50-Coenzyme Q10 1.29
Hep22G48-100-Coenzyme Q10 2.09
Pyruvate Kinase M2 (PKM2)
[0509] Pyruvate Kinase is an enzyme involved in the glycolytic
pathway. It is responsible for the transfer of phosphate from
phosphoenolpyruvate (PEP) to adenosine diphosphophate (ADP) to
generate ATP and pyruvate. PKM2 is an isoenzyme of the glycolytic
pyruvate kinase, expression of which is characterized by the
metabolic function of the tissue i.e. M2 isoenzyme is expressed in
normal rapidly proliferating cells with high energy needs such as
embryonic cells and also expressed in few normal differentiated
tissues such as lung and pancreatic islet cells that require high
rate of nucleic acid synthesis. PKM2 is highly expressed in tumor
cells due to their dependence on glycolytic pathway for meeting
cellular energetic requirements. The PKM2 isoform normally thought
to be embryonically restricted is re-expressed in cancerous cells.
Cells expressing PKM2 favor a stronger aerobic glycolytic phenotype
(show a shift in metabolic phenotype) with increased lactate
production and decreased oxidative phosphorylation. Thus, decrease
in expression of PKM2 in cancer cells would shift or down-regulate
energy generation via the glycolytic pathway, a strategy that is
useful in the treatment of cancer. Data demonstrates variable
expression pattern of PKM2 in normal and cancer cells, with cancer
cells demonstrating higher levels of expression compared to normal.
Treatment of cells with Coenzyme Q10 altered expression pattern of
the PKM2 upper and lower band levels in normal and cancer cells. In
cancer cells tested, there was a dose-dependent decrease in the
PKM2 expression, and no major changes in normal cells were
observed. The results are summarized in the tables below.
TABLE-US-00037 TABLE 36 Pyruvate Kinase Muscle form 2 Upper Band in
HepG2 Normalized Volume Normalized Intensity Amount - Composition
(24 h) (48 h) 5g-Media 28386 413 5g-50-Coenzyme Q10 29269 303
5g-100-Coenzyme Q10 18307 354 22G-Media 25903 659 22G-50-Coenzyme
Q10 22294 562 22G-100-Coenzyme Q10 19560 601
TABLE-US-00038 TABLE 37 Pyruvate Kinase Muscle form 2 Lower Band
(58 KD) in HepG2 Normalized Volume Normalized Volume Amount -
Composition (24 h) (48 h) 5g-Media 10483 310 5g-50-Coenzyme Q10
11197 185 5g-100-Coenzyme Q10 7642 122 22G-Media 9150 306
22G-50-Coenzyme Q10 6302 344 22G-100-Coenzyme Q10 6904 465
TABLE-US-00039 TABLE 38 Pyruvate Kinase Muscle form 2 Upper Band in
HASMC Cells after Treatment Amount - Composition Normalized
Intensity 5g48-Media 608 5g48-50-Coenzyme Q10 811 5g48-100-Coenzyme
Q10 611 22G48-Media 516 22G48-50-Coenzyme Q10 595
22G48-100-Coenzyme Q10 496 22G24-Media 301 22G24-50-Coenzyme Q10
477 22G24-100-Coenzyme Q10 701
Lactate Dehydrogenase (LDH)
[0510] LDH is an enzyme that catalyzes the interconversion of
pyruvate and lactate with the simultaneous interconversion of NADH
and NAD.sup.+. It has the ability to convert pyruvate to lactate
(lactic acid) under low cell oxygen tension for generation of
reducing equivalents and ATP generation at the expense of
mitochondrial oxidative phosphorylation. Cancer cells typically
demonstrate increased expression of LDH to maintain the glycolytic
flux to generate ATP and reducing equivalents and reducing
mitochondrial OXPHOS. Thus, reducing the expression of the LDH in
cancer cells would shift metabolism from generation of lactate to
facilitate entry of pyruvate into the TCA cycle. Treatment with
Coenzyme Q10 reduced Lactate Dehydrogenase (LDH) expression in
cancer with minimal effect on normal cells, supporting a role for
Coenzyme Q10 in eliciting a shift in cancer cell bioenergetics for
the generation of ATP from glycolytic to mitochondrial OXPHOS
sources by minimizing the conversion of cytoplasmic pyruvate to
lactic acid. The results are summarized in the tables below.
TABLE-US-00040 TABLE 39 Lactate Dehydrogenase in HepG2 Normalized
Volume Normalized Volume Amount - Composition (24 h) (48 h)
5g-Media 7981 5997 5g-50-Coenzyme Q10 7900 5188 5g-100-Coenzyme Q10
6616 7319 22G-Media 9171 7527 22G-50-Coenzyme Q10 7550 6173
22G-100-Coenzyme Q10 7124 9141
TABLE-US-00041 TABLE 40 Lactate Dehydrogenase in HepG2 as % Control
from 2 Experiments Average Volume as a Amount - Composition % of
Control 5g24-Media 1.00 5g24-50-Coenzyme Q10 0.64 5g24-100-Coenzyme
Q10 1.06 5g48-Media 1.00 5g48-50-Coenzyme Q10 1.12
5g48-100-Coenzyme Q10 1.21 22G24-Media 1.00 22G24-50-Coenzyme Q10
1.21 22G24-100-Coenzyme Q10 1.44 22G48-Media 1.00 22G48-50-Coenzyme
Q10 0.95 22G48-100-Coenzyme Q10 0.67
TABLE-US-00042 TABLE 41 Lactate Dehydrogenase in PACA2 Normalized
Volume Normalized Volume Amount - Composition (24 h) (48 h)
5g-Media 2122 2360 5g-50-Coenzyme Q10 5068 2978 5g-100-Coenzyme Q10
3675 2396 22G-Media 4499 2332 22G-50-Coenzyme Q10 10218 2575
22G-100-Coenzyme Q10 7158 3557
Pyruvate Dehydrogenase--B (PDH-E1)
[0511] Pyruvate Dehydrogenase beta (PDH-E1) is the first enzyme
component that is part of the pyruvate dehydrogenase complex (PDC)
that converts pyruvate to acetyl CoA. PDH-E1 requires thiamine as
cofactor for its activity, performs the first two biochemical
reactions in the PDC complex essential for the conversion of
pyruvate to acetyl CoA to enter the TCA cycle in the mitochondria.
Thus, concomitant decreases in PKM2 and LDH expression along with
increase in expression of PDH-E1 in cancer cells would enhance the
rate of entry of pyruvate towards augmenting the mitochondrial
OXPHOS for generation of ATP. The data shows that for expression of
PDH-E1 in normal and cancer cell lines, the baseline expressions of
this enzyme is decreased in cancer compared to normal cells.
Treatment with Coenzyme Q10 is associated with progressive increase
in the expression of the PDH-E1 proteins in cancer cells with
minimal changes in the normal cells. The results are summarized in
the tables below.
TABLE-US-00043 TABLE 42 Pyruvate Dehydrogenase Beta in HepG2
Normalized Volume Normalized Volume Amount - Composition (24 h) (48
h) 5g-Media 517 100 5g-50-Coenzyme Q10 921 123 5g-100-Coenzyme Q10
433 205 22G-Media 484 181 22G-50-Coenzyme Q10 426 232
22G-100-Coenzyme Q10 340 456
TABLE-US-00044 TABLE 43 Pyruvate Dehydrogenase Beta in PACA2
Normalized Volume Normalized Volume Amount - Composition (24 h) (48
h) 5g-Media 323 375 5g-50-Coenzyme Q10 492 339 5g-100-Coenzyme Q10
467 252 22G-Media 572 276 22G-50-Coenzyme Q10 924 279
22G-100-Coenzyme Q10 1201 385
TABLE-US-00045 TABLE 44 Pyruvate Dehydrogenase Beta in HASMC after
Treatment Amount - Composition Normalized Volume 5g48-Media 140
5g48-50-Coenzyme Q10 147 5g48-100-Coenzyme Q10 147 22G48-Media 174
22G48-50-Coenzyme Q10 149 22G48-100-Coenzyme Q10 123 22G24-Media
140 22G24-50-Coenzyme Q10 145 22G24-100-Coenzyme Q10 150
Caspase 3
[0512] Control of the onset of apoptosis is often exerted at the
level of the initiator caspases, caspase-2, -9 and -8/10. In the
extrinsic pathway of apoptosis, caspase-8, once active, directly
cleaves and activates executioner caspases (such as caspase-3). The
active caspase-3 cleaves and activates other caspases (6, 7, and 9)
as well as relevant targets in the cells (e.g. PARP and DFF). In
these studies, the levels of effectors caspase-3 protein were
measured in the cancer cell lines and in normal cell lines in
response to Coenzyme Q10. It should be noted although control of
apoptosis is through initiator caspases, a number of signaling
pathways interrupt instead the transmission of the apoptotic signal
through direct inhibition of effectors caspases. For e.g. P38 MAPK
phosphorylates caspase-3 and suppresses its activity
(Alvarado-Kristensson et al., 2004). Interestingly, activation of
protein phosphates (PP2A) in the same study or protein kinase C
delta (PKC delta) (Voss et al., 2005) can counteract the effect of
p38 MAPK to amplify the caspase-3 activation and bolster the
transmission of the apoptotic signal. Therefore, events at the
level of caspase-3 activation or after Caspase 3 activation may
determine the ultimate fate of the cell in some cases.
[0513] Caspase-3 is a cysteine-aspartic acid protease that plays a
central role in the execution phase of cell apoptosis. The levels
of caspase 3 in the cancer cells were increased with Coenzyme Q10
treatment. In contrast the expression of Caspase-3 in normal cells
was moderately decreased in normal cells. The results are
summarized in the tables below.
TABLE-US-00046 TABLE 45 Caspase 3 in PACA2 Normalized Volume
Normalized Volume Amount-Composition (24 h) (48 h) 5g-Media 324 300
5g-50-Coenzyme Q10 325 701 5g-100-Coenzyme Q10 374 291 22G-Media
344 135 22G-50-Coenzyme Q10 675 497 22G-100-Coenzyme Q10 842
559
TABLE-US-00047 TABLE 46 Caspase 3 in HepG2 cells as % Control from
2 Experiments Normalized Volume as Amount-Composition a % of
Control 5g24-Media 1.00 5g24-50-Coenzyme Q10 1.08 5g24-100-Coenzyme
Q10 1.76 5g48-Media 1.00 5g48-50-Coenzyme Q10 1.44
5g48-100-Coenzyme Q10 0.95 22G24-Media 1.00 22G24-50-Coenzyme Q10
1.39 22G24-100-Coenzyme Q10 1.78 22G48-Media 1.00 22G48-50-Coenzyme
Q10 1.50 22G48-100-Coenzyme Q10 1.45
TABLE-US-00048 TABLE 47 Caspase 3 in HASMC after Treatment
Amount-Composition Normalized Volume 5g48-Media 658
5g48-50-Coenzyme Q10 766 5g48-100-Coenzyme Q10 669 22G48-Media 846
22G48-50-Coenzyme Q10 639 22G48-100-Coenzyme Q10 624 22G24-Media
982 22G24-50-Coenzyme Q10 835 22G24-100-Coenzyme Q10 865
Succinate Dehydrogenase (SDH)
[0514] Succinate dehydrogenase, also known as succinate-coenzyme Q
reductase is a complex of the inner mitochondrial membrane that is
involved in both TCA and electron transport chain. In the TCA, this
complex catalyzes the oxidation of succinate to fumarate with the
concomitant reduction of ubiquinone to ubiquinol. (Baysal et al.,
Science 2000; and Tomlinson et al., Nature Genetics 2002). Germline
mutations in SDH B, C and D subunits were found to be initiating
events of familial paraganglioma or leiomyoma (Baysal et al.,
Science 2000).
[0515] Western blotting analysis was used to characterize
expression of SDH Subunit B in mitochondrial preparations of cancer
cells treated with Coenzyme Q10. The results suggest that Coenzyme
Q10 treatment is associated with increase SDH protein levels in the
mitochondrion of the cells. These results suggest one of the
mechanisms of action of Coenzyme Q10 is to shift the metabolism of
the cell towards the TCA cycle and the mitochondrion by increasing
the levels of mitochondrial enzymes such as SDHB. The results are
summarized in the table below.
TABLE-US-00049 TABLE 48 Succinate Dehydrogenase B in NCIE0808
Mitopreps Average Normalized Composition-Time Volume Media 531 50
uM Coezyme Q10, 3 h 634 100 uM Coenzyme Q10, 3 h 964 50 uM Coenzyme
Q10, 6 h 1077 100 uM Coenzyme Q10, 6 h 934
Hypoxia Induced Factor-1
[0516] Hypoxia inducible factor (Hif) is a transcription factor
composed of alpha and beta subunits. Under normoxia, the protein
levels of Hif1 alpha are very low owing to its continuous
degradation via a sequence of post translational events. The shift
between glycolytic and oxidative phosphorylation is generally
considered to be controlled by the relative activities of two
enzymes PDH and LDH that determine the catabolic fate of pyruvate.
Hif controls this crucial bifurgation point by inducing LDH levels
and inhibiting PDH activity by stimulating PDK. Due to this ability
to divert pyruvate metabolism from mitochondrion to cytosol, Hif is
considered a crucial mediator of the bioenergetic switch in cancer
cells.
[0517] Treatment with Coenzyme Q10 decreased Hif1 alpha protein
levels after in mitochondrial preparations of cancer cells. In
whole cell lysates of normal cells, the lower band of Hif1a was
observed and showed a decrease as well. The results are summarized
in the tables below.
TABLE-US-00050 TABLE 49 Hif1 alpha Lower Band in HASMC Cells after
Treatment Amount-Composition Normalized Volume 5g48-Media 22244
5g48-50-Coenzyme Q10 21664 5g48-100-Coenzyme Q10 19540 22G48-Media
14752 22G48-50-Coenzyme Q10 17496 22G48-100-Coenzyme Q10 23111
22G24-Media 21073 22G24-50-Coenzyme Q10 18486 22G24-100-Coenzyme
Q10 17919
TABLE-US-00051 TABLE 50 Hif1 alpha Upper Band in HepG2 after
Treatment Amount-Composition Normalized Volume 5g24-Media 12186
5g24-50-Coenzyme Q10 8998 5g24-100-Coenzyme Q10 9315 5g48-Media
8868 5g48-50-Coenzyme Q10 8601 5g48-100-Coenzyme Q10 10192
22G24-Media 11748 22G24-50-Coenzyme Q10 14089 22G24-100-Coenzyme
Q10 8530 22G48-Media 8695 22G48-50-Coenzyme Q10 9416
22G48-100-Coenzyme Q10 5608
Example 21
Analysis of Oxygen Consumption Rates (OCR) and Extracellular
Acidification (ECAR) in Normal and Cancer Cells Treated with
CoQ10
[0518] This example demonstrates that exposure of cells to
treatment by a representative MIM/epi-shifter of the
invention--CoQ10--in the absence and/or presence of stressors
(e.g., hyperglycemia, hypoxia, lactic acid), is associated with a
shift towards glycolysis/lactate biosynthesis and mitochondrial
oxidative phosphorylation (as measured by ECAR and OCR values)
representative of values observed in a normal cells under normal
physiological conditions.
[0519] Applicants have demonstrated in the previous section that
treatment with CoQ10 in cancer cells is associated with changes in
expression of specific proteins that enhance mitochondrial
oxidative phosphorylation, with a concomitant decrease in
glycolysis and lactate biosynthesis. This example shows that a
direct measure of mitochondrial oxidative phosphorylation can be
obtained by measuring the oxygen consumption rates (OCR) in cell
lines using the SeaHorse XF analyzer, an instrument that measures
dissolved oxygen and extracellular pH levels in an in vitro
experimental model. (SeaHorse Biosciences Inc, North Billerica,
Mass.).
[0520] The pH of the extracellular microenvironment is relatively
acidic in tumors compared to the intracellular (cytoplasmic) pH and
surrounding normal tissues. This characteristic of tumors serves
multiple purposes, including the ability to invade the
extracellular matrix (ECM), a hallmark attribute of tumor
metastasis that subsequently initiates signaling cascades that
further modulate: [0521] tumor angiogenesis [0522] increased
activation of arrest mechanisms that control cell cycle turn-over
[0523] immuno-modulatory mechanisms that facilitate a cellular
evasion system against immunosurveillance [0524] metabolic control
elements that increase dependency on glycolytic flux and lactate
utilization [0525] dysregulation of key apopototic gene families
such as Bcl-2, IAP, EndoG, AIF that serve to increase
oncogenicity
[0526] While not wishing to be bound by any particular theory, the
acidic pH of the external microenvironment in the tumor is a
consequence of increase in hydrogen ion concentrations extruded
from the tumor cells due to the increased lactate production from
an altered glycolytic phenotype.
[0527] In this experiment, the OCR and extracellular acidification
rate (ECAR) in normal cells lines were obtained in the presence and
absence of CoQ10 to determine baseline values. It was observed that
in its native nutrient environment, the basal OCR rates in normal
cells lines are different, and are usually a function of the
physiological roles of the cells in the body.
[0528] For example, one set of experiments were conducted using the
non-cancerous cell line HDFa, which is a human adult dermal
fibroblast cell line. Fibroblasts are cells that primarily
synthesize and secrete extracellular matrix (ECM) components and
collagen that form the structural framework (stroma) for tissues.
In addition, fibroblasts are known to serve as tissue ambassadors
of numerous functions such as wound healing and localized
immunomodulation. Under normal physiological conditions, energy
requirements in normal fibroblasts are met using a combination of
glycolysis and oxidative phosphorylation--the glycolysis providing
the necessary nutrients for synthesis of ECM.
[0529] In contrast to HDFa, the HASMC (human aortic smooth muscle
cell) is found in arteries, veins, lymphatic vessels,
gastrointestinal tracts, respiratory tract, urinary bladder and
other tissues with the ability to undergo regulated
excitation-contraction coupling. The ability of smooth muscles such
as HASMC cells to undergo contraction requires energy provided by
ATP. These tissues transition from low energy modes wherein ATP may
be supplied from mitochondria to high energy modes (during
exercise/stress) where energy is provided by switching to
glycolysis for rapid generation of ATP. Thus, normal smooth muscle
cells can use a combination of mitochondrial OXPHOS and glycolysis
to meet their energy requirements under normal physiological
environment.
[0530] The differences in their respective physiological roles
(i.e., HDFa and HASMC) were observed in the resting OCR values
measured in these cells lines using the SeaHorse XF analyzer. FIGS.
29 and 30 describe the OCR in HDFa and HASMC cells grown in
physiologically normal glucose (about 4.6 mM) and high glucose
(hyperglycemic) conditions.
[0531] The baseline OCR values for HDFa in the absence of any
treatments under normal oxygen availability is approximately 40
pmoles/min (FIG. 29) in the presence of 5.5 mM glucose. This value
was slightly elevated when the cells were maintained at 22 mM
glucose. In contrast, in HASMC cells, the OCR values at 5.5 mM
glucose is approximately 90 pmoles/min, and the OCR value declined
to approximately 40 pmoles/min while at 22 mM glucose. Thus, under
hyperglycemic conditions, there is a differential response between
HDFa and HASMC, further demonstrating inherent differences in their
respective physiological make-up and function.
[0532] Treatment with CoQ10 in cells is associated with changes in
OCR that is representative of conditions observed at normal (5 mM)
glucose conditions. The complexity of physiological response is
compounded in the presence of low oxygen tension. Thus, CoQ10
exposure is associated with changes in OCR rates in normal cells
towards a physiological state that is native to a particular
cell.
[0533] Table 51 below describes the ECAR values (mpH/min) in HDFa
cells in the presence or absence of CoQ10 under normoxic and
hypoxic conditions at 5.5 mM and 22 mM glucose. It can be observed
that in normal cells, treatment with CoQ10 had minimal influence on
ECAR values, even though it influenced OCR in these cells. In high
glucose hypoxic conditions, treatment with CoQ10 was associated
with lowering of elevated ECAR to a value that was observed in
untreated normoxic conditions.
TABLE-US-00052 TABLE 51 ECAR values in HDFa cells in the absence
and presence of CoQ10 under normoxic and hypoxic conditions at 5.5
mM and 22 mM glucose Normoxia Hypoxia Normoxia Hypoxia (5.5 mM)
(5.5 mM) (22 mM) (22 mM) Treatment ECAR SEM ECAR SEM ECAR SEM ECAR
SEM Untreated 5 1.32 5 0.62 5 0.62 9 0.81 50 .mu.M 6 1.11 5 0.78 5
0.78 6 0.70 31510 100 .mu.M 6 0.76 5 1.19 5 1.19 8 1.07 31510
[0534] In Table 52 the measured baseline ECAR values (mpH/min) in
HASMC were higher compared to that of HDFa. Induction of hypoxic
conditions caused an increase in ECAR most likely associated with
intracellular hypoxia induced acidosis secondary to increased
glycolysis.
TABLE-US-00053 TABLE 52 ECAR values in HASMC cells in the absence
and presence of CoQ10 under normoxic and hypoxic conditions at 5.5
mM and 22 mM glucose Normoxic Hypoxic Normoxic Hypoxic (5.5 mM)
(5.5 mM) (22 mM) (22 mM) Treatment ECAR SEM ECAR SEM ECAR SEM ECAR
SEM Untreated 9 2.22 11 2.18 22 2.08 19 1.45 50 .mu.M 9 2.13 11
2.54 21 1.72 17 1.60 31510 100 .mu.M 9 1.72 13 2.30 22 1.64 17 1.47
31510
[0535] Treatment with CoQ10 was observed to be associated with a
downward trend of ECAR rates in hyperglycemic HASMC cells in
hypoxic conditions towards a value that would be observed in
normoxic normal glucose conditions. These data demonstrate the
presence of physiological variables that is inherent to the
physiological role of a specific type of cell, alterations observed
in abnormal conditions (e.g. hyperglycemia) is shifted towards
normal when treated with CoQ10.
[0536] In contrast, cancer cells (e.g., MCF-7, PaCa-2) are
inherently primed to culture at higher levels of glucose compared
to normal cells due to their glycolytic phenotype for maintenance
in culture. Treatment with CoQ10 caused a consistent reduction in
OCR values (FIG. 31 and FIG. 32).
[0537] The effects of CoQ10 on OCR values in MCF-7 and PaCa-2 cells
was similar to that of the normal HDFa and HASMC cells, wherein the
variable response was suggestive of a therapeutic response based on
individual metabolic profile of the cancer cell line.
TABLE-US-00054 TABLE 53 ECAR values in PaCa-2 cells in the absence
and presence of CoQ10 under normoxic and hypoxic conditions at 5.5
mM and 22 mM glucose Normoxia Hypoxia Normoxia Hypoxia (17 mM) (17
mM) (22 mM) (22 mM) Treatment ECAR SEM ECAR SEM ECAR SEM ECAR SEM
Untreated 21 5.97 16 3.41 24 4.35 36 5.65 50 .mu.M 13 3.08 12 1.66
20 5.15 25 4.58 31510 100 .mu.M 14 2.14 17 2.59 19 3.38 30 5.62
31510
[0538] Table 53 describes the ECAR values in PaCa-2 cells. In
contrast to normal cells, cancer cells are phenotypically primed to
use high glucose for ATP generation (enhanced glycolysis) resulting
in higher ECAR (Table 53, ECAR for untreated normoxia 17 mM) at 21
mpH/min. Treatment with CoQ10 produces a significant decrease in
ECAR rates under these conditions, most likely associated with a
decrease in the glycolysis generated lactic acid. The associated
decrease in OCR in these cells was likely associated with increased
efficiency of the mitochondrial OXPHOS.
[0539] A similar comparison of OCR and ECAR values (data not shown)
were determined in numerous other normal and cancer cells lines,
including: HAEC (normal human aortic endothelial cells), MCF-7
(breast cancer), HepG2 (liver cancer) and highly metastatic PC-3
(prostate cancer) cell lines. In all of the cell lines tested,
exposure to CoQ10 in the absence and/or presence of stressors
(e.g., hyperglycemia, hypoxia, lactic acid) was associated with a
shift in OCR and ECAR values representative of values observed in a
normal cells under normal physiological conditions. Thus, the
overall effect of CoQ10 in the treatment of cancer, including cell
death, is an downstream effect of its collective influence on
proteomic, genomic, metabolomic outcomes in concert with shifting
of the cellular bioenergetics from glycolysis to mitochondrial
OXPHOS.
Example 22
Building Block Molecules for the Biosynthesis of CoQ10
[0540] This example demonstrates that certain precursors of CoQ10
biosynthesis, such as those for the biosynthesis of the
benzoquinone ring, and those for the biosynthesis of the isoprenoid
repeats and their attachment to the benzoquinone ring ("building
block components"), can be individually administered or
administered in combination to target cells, and effect
down-regulation of the apoptosis inhibitor Bcl-2, and/or
up-regulation of the apoptosis promoter Caspase-3. Certain
precursors or combinations thereof may also inhibit cell
proliferation. The data suggests that such CoQ10 precursors may be
used in place of CoQ10 to achieve substantially the same results as
CoQ10 administration.
[0541] Certain exemplary experimental conditions used in the
experiments are listed below.
[0542] Skmel-28 melanoma cells were cultured in DMEM/F12
supplemented with 5% Fetal Bovine Serum (FBS) and 1.times. final
concentration of Antibiotics. The cells were grown to 85%
confluency and treated with building block components for 3, 6, 12
and 24 hours. The cells were then pelleted and a Western blot
analysis was performed.
[0543] The test building block components included
L-Phenylylalanine, DL-Phenylyalanine, D-Phenylylalanine,
L-Tyrosine, DL-Tyrosine, D-Tyrosine, 4-Hydroxy-phenylpyruvate,
phenylacetate, 3-methoxy-4-hydroxymandelate (vanillylmandelate or
VMA), vanillic acid, 4-hydroxy-benzoate, pyridoxine, panthenol,
mevalonic acid, Acetylglycine, Acetyl-CoA, Farnesyl, and
2,3-Dimethoxy-5-methyl-p-benzoquinone.
[0544] In the Western Blot Analysis, the cells were pelleted in
cold PBS, lysed, and the protein levels were quantified using a BCA
protein assay. The whole cell lysate was loaded in a 4% loading 12%
running Tris-HCl gel. The proteins were then transferred to a
nitrocellulose paper then blocked with a 5% milk Tris-buffered
solution for 1 hour. The proteins were then exposed to primary
antibodies (Bcl-2 and Caspase-3) overnight. The nitrocellulose
paper was then exposed to Pico Chemilluminescent for 5 min and the
protein expression was recorded. After exposure, actin was
quantified using the same method. Using ImageJ the levels of
protein expression were quantified. A t-Test was used to analyze
for statistical significance.
[0545] Illustrative results of the experiments are summarized
below.
[0546] Western Blot Analysis of Building Block component
L-Phenylalanine: Before proceeding to the synthesis pathway for the
quinone ring structure, L-Phenylalanine is converted to tyrosine. A
western blot analysis was performed to quantify any changes in the
expression of the apoptotic proteins in the melanoma cells. The
concentrations tested were 5 .mu.M, 25 .mu.M, and 100 .mu.M.
Initial studies added L-Phenylalanine to DMEM/F12 medium which
contained a concentration of 0.4 M phenylalanine. For the 5 .mu.M,
25 .mu.M, and 100 .mu.M the final concentration of the
L-Phenylalanine in the medium was 0.405 M, 0.425 M, and 0.500 M,
respectively. These final concentrations were tested on the
Skmel-28 cells for incubation periods of 3, 6, 12 and 24 hours. The
cells were grown to 80% confluency before adding the treatment
medium and harvested using the western blot analysis procedure as
described above. A statistically significant decrease in Bcl-2 was
observed for the 100 .mu.M L-Phenylalanine after 3 hours and 12
hours incubation. Fr the 5 .mu.M L-phenylalanine, a statistically
significant decrease in Bcl-2 was observed after 6 hours of
incubation. For the 25 .mu.M L-phenylalanine, a statistically
significant decrease in Bcl-2 and a statistically significant
increase in Caspase-3 were observed after 12 hours of incubation. A
statistically significant decrease in Bcl-2 indicates a change in
the apoptotic potential and a statistically significant increase in
Caspase-3 confirms the cells are undergoing apoptosis. There was a
constant trend for the decrease in Bcl-2 compared to the control
even though, due to sample size and standard deviation, these time
points were not statistically significant in this experiment.
[0547] Western Blot Analysis of Building Block component
D-Phenylalanine: D-Phenylalanine, a chemically synthetic form of
the bioactive L-Phenylalanine, was tested for comparison to
L-phenylalanine. For all three concentrations (5 .mu.M, 25 .mu.M,
and 100 .mu.M of D-Phenylalanine, there was a significant reduction
in Bcl-2 expression after 6 hours of incubation. In addition, for
the 5 .mu.M and 25 .mu.M, there was a significant reduction after 3
hours of incubation. For the 5 .mu.M and 100 .mu.M concentrations,
a significant increase in Caspase-3 expression was observed after 6
hours of incubation.
[0548] Western Blot Analysis of Building Block component
DL-Phenylalanine: DL-Phenylalanine was also tested for comparison
to L-Phenylalanine. Again, concentrations of 5 .mu.M, 25 .mu.M, and
100 .mu.M were tested on Skmel-28 cells. The incubation periods
were 3, 6, 12 and 24 hours. A statistically significant increase in
Caspase-3 was observed after 3 hours of incubation. A statistically
significant decrease in Bcl-2 was observed after 24 hours of
incubation. Although a decreasing Bcl-2 and increasing Caspase-3
trend at all other concentrations and incubation time points, they
were not statistically significant in this experiment.
[0549] Western Blot Analysis of Building Block component
L-Tyrosine: L-Tyrosine is a building block component for the
synthesis of quinone ring structure of CoQ10. Initial testing of
L-Tyrosine did not result in a high enough protein concentration
for western blot analysis. From this study concentrations under 25
.mu.M were tested for Western Blot Analysis. The DMEM/F12 medium
used contained L-Tyrosine disodium salt concentration of 0.398467
M. The initial concentration was increased by 500 nM, 5 .mu.M, and
15 .mu.M. A statistically significant increase in Caspase-3 was
observed for the 500 nM concentration after 12 hours of incubation.
A statistically significant increase in Caspase-3 was also observed
for the 5A statistically significant decrease in Bcl-2 was observed
for the 5 .mu.M concentration after 24 hours of incubation. A
statistically significant decrease in Bcl-2 was observed for the
500 .mu.M and 5 .mu.M concentrations after 24 hours of
incubation.
[0550] Western Blot Analysis of Building Block component
D-Tyrosine: D-Tyrosine, a synthetic form of L-Tyrosine, was tested
for comparison against the L-Tyrosine apoptotic effect on the
melanonal cells. Based on initial studies with L-Tyrosine,
concentrations below 25 .mu.M were chosen for the western blot
analysis. The concentrations tested were 1 .mu.m, 5 .mu.M, and 15
.mu.M. D-Tyrosine showed a reduction in Bcl-2 expression for the 5
.mu.M and 15 .mu.M concentrations for 12 and 24 hour time periods.
Caspase-3 was significantly increased for the concentration of 5
.mu.M for 3, 12 and 24 time periods. Also there was an increase in
Caspase-3 expression for the 1 .mu.M for 12 and 24 hour time
period. In addition there is an increase in Caspase-3 expression
for 5 .mu.M for the 12 hour time period.
[0551] Western Blot Analysis of Building Block component
DL-Tyrosine: DL-Tyrosine, a synthetic form of L-Tyrosine, was also
tested for comparison against L-Tyrosine's apoptotic effect on the
cells. There is a statistical decrease in Bcl-2 expression seen in
the 1 .mu.M and 15 .mu.M concentrations after 12 hours incubation
and for the 5 .mu.M after 24 hour of incubation. An increase in
Caspase-3 expression was also observed for the 5 .mu.M and 15 .mu.M
after 12 hours of incubation.
[0552] Western Blot Analysis of Building Block component
4-Hydroxy-phenylpyruvate: 4-Hydroxy-phenylpyruvate is derived from
Tyrosine and Phenylalanine amino acids and may play a role in the
synthesis of the ring structure. The concentration of 1 .mu.M, 5
.mu.M, and 15 .mu.M were tested for Bcl-2 and Caspase-3 expression.
For the 5 .mu.M and 15 .mu.M concentrations there is a significant
reduction in Bcl-2 expression after 24 hours of incubation and a
significant increase in Caspase-3 expression after 12 hours of
incubation.
[0553] Western Blot Analysis of Building Block component
Phenylacetate: Phenylacetate has the potential to be converted to
4-Hydroxy-benzoate, which plays a role in the attachment of the
side chain to the ring structure. The concentration tested were 1
.mu.M, 5 .mu.M, and 15 .mu.M. For phenylacetate there was a
decrease in Bcl-2 expression for the concentration of 5 .mu.M and
15 .mu.M after 12 hours and 24 hours of incubation. An increase in
Caspase-3 expression was observed for the concentration of 5 .mu.M
and 15 .mu.M after 12 hours and 24 hours of incubation.
[0554] Western Blot Analysis of Building Block component
3-methoxy-4-hydroxymandelate (vanillylmandelate or VMA): VMA is an
additional component for the synthesis of the CoQ10 quinone ring
structure. The concentrations tested were 100 nM, 250 nM, 500 nM, 1
.mu.M, 25 .mu.M, 50 .mu.M, and 100 .mu.M. Though no statistically
significant apoptotic effect was observed in this experiment, the
data indicated a downward trend of Bcl-2 expression.
[0555] Western Blot Analysis of Building Block component Vanillic
acid: Vanillic is a precursor for the synthesis of the quinone ring
and was tested at a concentration of 500 nm, 5 .mu.M, and 15 .mu.M.
A western blot analysis measured Bcl-2 and Caspase-3 expression.
Vanillic Acid was shown to significantly reduce Bcl-2 expression
for the concentrations of 500 nM and 5 .mu.M at the 24 hour
incubation time point. For the 15 .mu.M concentration there is a
reduction in Bcl-2 expression after 3 hours of incubation. For the
cells incubated with 15 .mu.M for 24 hours there was a significant
increase in Caspase-3 expression.
[0556] Western Blot Analysis of Building Block component
4-Hydroxybenzoate: 4-Hydroxybenzoate acid plays a role in the
attachment of the isoprenoid side chain to the ring structure. The
concentrations tested were 500 nM, 1 .mu.M, and 50 .mu.M. There was
a significant reduction in Bcl-2 expression for the 15 .mu.M
concentration after 24 hours of incubation.
[0557] Western Blot Analysis of Building Block component
4-Pyridoxine: Pyridoxine is another precursor building block for
the synthesis of the quinone ring structure of CoQ10. The
concentrations tested for this compound are 5 .mu.M, 25 .mu.M, and
100 .mu.M. The cells were assayed for their levels of Bcl-2 and
Caspase-3. Pyridoxine showed a significant reduction in Bcl-2 after
24 hours of incubation in melanoma cells.
[0558] Western Blot Analysis of Building Block component Panthenol:
Panthenol plays a role in the synthesis of the quinone ring
structure of CoQ10. The concentrations tested on melanoma cells
were 5 .mu.M, 25 .mu.M, and 100 .mu.M. This compound showed a
significant reduction in Bc1-2 expression for the 25 .mu.M
concentration.
[0559] Western Blot Analysis of Building Block component Mevalonic:
Mevalonic Acid is one of the main components for the synthesis of
CoQ10. This compound was tested at the concentrations of 500 nM, 1
.mu.M, 25 .mu.m, and 50 .mu.M. There was no significant reduction
in Bcl-2 expression or an increase in Caspase-3 expression in this
experiment.
[0560] Western Blot Analysis of Building Block component
Acetylglycine: Another route for the synthesis of CoQ10 is the
isoprenoid (side chain) synthesis. The addition of Acetylglycine
converts Coenzyme A to Acetyl-CoA which enters the mevalonic
pathway for the synthesis of the isoprenoid synthesis. The
concentrations tested were 5 .mu.M, 25 .mu.M, and 100 .mu.M. The
testing of Acetylglycine showed significant decrease in Bcl-2
expression after 12 hours of incubation for the concentration of 5
.mu.M and 25 .mu.M. A significant decrease in Bcl-2 was recorded
for the 100 .mu.M concentration at the 24 hour incubation time
point.
[0561] Western Blot Analysis of Building Block component
Acetyl-CoA: Acetyl-CoA is a precursor for the mevalonic pathway for
the synthesis of CoQ10. The concentrations tested were 500 nm, 1
.mu.M, 25 .mu.M, and 50 .mu.M. There was no significant observed
reduction in Bcl-2 or increase in Caspase-3 expression for the time
points and concentrations tested.
[0562] Western Blot Analysis of Building Block component L-Tyrosine
in combination with farnesyl: L-Tyrosine is one of the precursors
for the synthesis of the quinone ring structure for CoQ10. Previous
experiment tested the reaction of L-Tyrosine in medium with
L-Phenylalanine and L-Tyrosine. In this study L-Tyrosine was
examined in medium without the addition of L-Phenylalanine and
L-Tyrosine. In this study the final concentrations of L-Tyrosine
tested were 500 nM, 5 .mu.M, and 15 .mu.M. Farnesyl was tested at a
concentration of 50 .mu.M. There was no observed significant
response for the 3 and 6 hour time points.
[0563] Western Blot Analysis of Building Block component
L-Phenylalanine in combination with Farnesyl: L-Phenylalanine, a
precursor for the synthesis of the quinone ring structure, was
examine in combination with farnesyl in medium free of L-Tyrosine
and L-Phenylalanine. A western blot analysis was performed to assay
the expression of Bcl-2 and Caspase-3. The final concentrations of
L-Phenylalanine were: 5 .mu.M, 25 .mu.M, and 100 .mu.M. Farnesyl
was added at a concentration of 50 .mu.M. This study showed a
decrease in Bc1-2 expression for most of the concentrations and
combinations tested as depicted in the table below.
TABLE-US-00055 TABLE 54 L-Phenylalanine and/or Farnesyl L-Phenyl- 3
hr 6 hr 12 hr 24 hr alanine Bcl-2 Cas-3 Bcl-2 Cas-3 Bcl-2 Cas-3
Bcl-2 Cas-3 5 .mu.M X 5 .mu.M X X w/Farnesyl 25 .mu.M X X 25 .mu.M
X X w/Farnesyl 100 .mu.M X X X 100 .mu.M X w/Farnesyl
[0564] Cell Proliferation Assay of the Combination of
4-Hydroxy-Benzoate with Benzoquinone: This set of experiments used
a cell proliferation assay to assess the effect of combining
different building block molecules on cell proliferation.
[0565] The first study examined the effect of combining
4-Hydroxy-Benzoate with Benzoquinone. Cells were incubated for 48
hours, after which a cell count was performed for the live cells.
Each test group was compared to the control, and each combination
groups were compared to Benzoquinone control. The compounds were
statistically analyzed for the addition of Benzoquinone. The
following table summarizes the cell count results wherein the X
mark indicates a statistical decrease in cell number.
TABLE-US-00056 TABLE 55 4-Hydroxy-Benzoate and/or Benzoquinone
Compared to 4- Hydroxy to Compared to Compared compound w/o
Benzoquinone 4-Hydroxy to Ctrl Benzoquinone Control 500 nm X 500 nm
w/Benzo X X (35 .mu.M) 500 nm w/Benzo X X (70 .mu.M) 1 .mu.m X 1
.mu.m w/Benzo X X (35 .mu.M) 1 .mu.m w/Benzo X X (70 .mu.M) 50
.mu.m X 50 .mu.m w/Benzo X (35 .mu.M) 50 .mu.m w/Benzo X X X (70
.mu.M)
[0566] There is a significant decrease in cell number for the cells
incubated with 4-Hydroxybenzoic and benzoquinone and in
combination. For the combination of 50 .mu.M 4-Hydroxybenzoate in
combination with 70 .mu.M Benzoquinone there is significant
reduction in cell number compared to the Benzoquinone control. This
suggests a synergistic effect for this molar ratio.
[0567] Additional studies were performed testing additional molar
ratios. For the first test 4-Hydroxybenzoic were tested at
concentrations of 500 nM, 1 .mu.M, and 50 .mu.M. These
concentrations were tested in combination with
2,3-Dimethoxy-5-methyl-p-benzoquinone (Benzo). The concentration of
Benzo tested were 25 .mu.M, 50 .mu.M, and 100 .mu.M. Melanoma cells
were grown to 80% confluency and seeded in 6 well plates at a
concentration of 40K cells per well. The cells were treated with
CoQ10, 4-Hydroxybenzoate, Benzo, and a combination of
4-Hydroxybenzoate/Benzo.
[0568] A T-test was performed with p<0.05 as statistically
significant. An X signifies a statistical decrease in cell
number.
TABLE-US-00057 TABLE 56 4-Hydroxybenzoic and/or 2,3-Dimethoxy-5-
methyl-p-benzoquinone (Benzo) Ctrl vs Benzo 25 .mu.M X Ctrl vs
Benzo (B) 50 .mu.M Ctrl vs Benzo (B) 100 .mu.M X Ctrl vs
4-Hydroxybenzoate (HB) 500 nm X Ctrl vs HB 1 .mu.M X Ctrl vs HB 50
.mu.M X 500 nM HB vs 500 nM HB w/25 B X 500 nM HB vs 500 nM HB w/50
B X 500 nM HB vs 500 nM HB w/100 B X 1 uM HB vs 1 .mu.M HB w/25 B X
1 uM HB vs 1 .mu.M HB w/50 B X 1 uM HB vs 1 .mu.M HB w/100 B 50 uM
HB vs 50 .mu.M HB w/25 B X 50 uM HB vs 50 .mu.M HB w/50 B X 50 uM
HB vs 50 .mu.M HB w/100 B 500 nM HB w/25 B vs 25 B X 500 nM HB w/50
B vs 50 B X 500 nM HB w/100 B vs 100 B X 1 .mu.M HB w/25 B vs 25 B
X 1 .mu.M HB w/50 B vs 50 B X 1 .mu.M HB w/100 B vs 100 B 50 .mu.M
HB w/25 B vs 25 B X 50 .mu.M HB w/50 B vs 50 B X 50 .mu.M HB w/100
B vs 100 B
[0569] There is a significant decrease in cell proliferation for
the treatment medium containing HB. Moreover the combination of the
HB with benzoquinone showed a significant reduction in cell number
compare to the cells incubated with the corresponding benzoquinone
concentrations.
[0570] A cell proliferation assay was also performed on neonatal
fibroblast cells. The concentrations of HB tested were 500 nM, 5
.mu.M, and 25 .mu.M. HB was also tested in combination with
benzoquinone at a concentrations of 25 .mu.M, 50 .mu.M, and 100
.mu.M. Melanoma cells were seeded at 40 k cells per well and were
treated for 24 hours. The cells were trypsinized and quantified
using a coulter counter.
[0571] Statistical analysis did not show a significant reduction in
fibroblast cells. This indicates minimal to no toxicity in normal
cells.
[0572] Cell Proliferation Assay of the Combination of phenylacetate
and benzoquinone: Phenyl acetate is a precursor for the synthesis
of 4-Hydroxybenzoic acid (facilitates the attachment of the ring
structure. A cell proliferation assay was performed to assay the
effect of incubating phenylacetate in combination with CoQ10 and
Benzoquinone.
TABLE-US-00058 TABLE 57 Phenylacetate and/or Benzoquinone Ctrl and
25/25 .mu.M Ben X Ctrl and 25/50 .mu.M Ben X Ctrl and 25/100 .mu.M
Ben X Ctrl and 25/25 .mu.M Q-10 X Ctrl and 25/25 .mu.M Q-10 X Ctrl
and 25/50 .mu.M Q-10 X Ctrl and 25/100 .mu.M Q-10 X Ctrl and Ben 25
X Ctrl and Ben 50 X Ctrl and Ben 100 X Ctrl and Q-10 25 Ctrl and
Q-10 50 Ctrl and Q-10 100 X Ben 25 .mu.M and 500 nM/25 .mu.M Ben X
Ben 25 .mu.M and 5 nM/25 .mu.M Ben X Ben 25 .mu.M and 25 nM/25
.mu.M Ben X Ben 50 .mu.M and 500 nM/50 .mu.M Ben X Ben 50 .mu.M and
5 nM/50 .mu.M Ben X Ben 50 .mu.M and 25 nM/50 .mu.M Ben X Ben 100
.mu.M and 500 nM/100 .mu.M Ben Ben 100 .mu.M and 5 nM/100 .mu.M Ben
Ben 100 .mu.M and 25 nM/100 .mu.M Ben Q-10 25 .mu.M and 500 nM/25
.mu.M Q-10 X Q-10 25 .mu.M and 5 nM/25 .mu.M Q-10 X Q-10 25 .mu.M
and 25 nM/25 .mu.M Q-10 X Q-10 50 .mu.M and 500 nM/50 .mu.M Q-10 X
Q-10 50 .mu.M and 5 nM/50 .mu.M Q-10 X Q-10 50 .mu.M and 25 nM/50
.mu.M Q-10 X Q-10 100 .mu.M and 500 nM/100 .mu.M Q-10 X Q-10 100
.mu.M and 5 nM/100 .mu.M Q-10 X Q-10 100 .mu.M and 25 nM/100 .mu.M
Q-10 X
[0573] The data indicates the addition of phenylacetate in
combination with benzoquinone significantly decreases the cellular
proliferation. The combination with CoQ10 and phenylacetate
significantly decrease the cell number compared to incubation with
CoQ10 and benzoquinone alone.
[0574] Cell Proliferation Assay of the Combination of
4-Hydroxy-Benzoate with Farnesyl: 4-Hydroxy-Benzoate was incubated
in combination with Farnesyl. The summary of the results are listed
below. 4-Hydroxybenzoate groups were compared to the control and
Farnesyl control groups. The X signifies a statistical decrease in
cell number.
TABLE-US-00059 TABLE 58 4-Hydroxy-Benzoate and/or Farnesyl Compared
to 4- Hydroxy to Compared to 4-Hydroxy- Compared compound w/o
Farnesyl Benzoate to Ctrl Farnesyl Control 500 nm X 500 nm w/ X
Farnesyl (35 .mu.M) 500 nm w/ X Farnesyl (70 .mu.M) 1 .mu.m Error 1
.mu.m w/Farnesyl Error (35 .mu.M) 1 .mu.m w/Farnesyl Error (70
.mu.M) 50 .mu.m X 50 .mu.m w/ Farnesyl X (35 .mu.M) 50 .mu.m w/
Farnesyl X (70 .mu.M)
[0575] Cell Proliferation Assay of the Combination of
L-Phenylalanine with Benzoquinone: A cell proliferation assay was
performed to test the combination of L-Phenylalanine combined with
Benzoquinone. Below is a summary of the results of L-Phenylalanine
compared to the control and Benzoquinone control. The X signifies a
statistical decrease.
TABLE-US-00060 TABLE 59 L-Phenylalanine and/or Benzoquinone
Compared to L- Phenylalanine to Compared to Compared compound w/o
Benzoquinone L-Phenylalanine to Ctrl Benzoquinone Control 5 .mu.M 5
.mu.m w/Benzo X (50 .mu.M) 5 .mu.m w/Benzo X (100 .mu.M) 25 .mu.m
25 .mu.m w/Benzo X (50 .mu.M) 25 .mu.m w/Benzo X (100 .mu.M) 100
.mu.m 100 .mu.m w/Benzo X X X (50 .mu.M) 100 .mu.m w/Benzo X X X
(100 .mu.M)
[0576] A similar synergistic role is seen for the L-Phenylalanine
combined with Benzoquinone.
[0577] Cell Proliferation Assay of the Combination of
L-Phenylalanine with Farnesyl: Preliminary results for combination
cell proliferation study of L-Phenylalanine incubated in
combination with Farnesyl. The L-Phenylalanine were compared to the
control and Farnesyl control group. An X signifies a statistical
decrease in cell number.
TABLE-US-00061 TABLE 60 L-Phenylalanine and/or Farnesyl Compared to
L- Phenylalanine to Compared to Compared compound w/o Farnesyl
L-Phenylalanine to Ctrl Farnesyl Control 5 .mu.M 5 .mu.m w/Farnesyl
(50 .mu.M) 5 .mu.m w/Farnesyl (100 .mu.M) 25 .mu.m X 25 .mu.m
w/Farnesyl X X X (50 .mu.M) 25 .mu.m w/Farnesyl X X X (100 .mu.M)
100 .mu.m X 100 .mu.m w/ X X Farnesyl (50 .mu.M) 100 .mu.m w/ X
Farnesyl (100 .mu.M)
[0578] Cell Proliferation Assay of the Combination of L-Tyrosine
with Benzoquinone: L-Tyrosine was incubated in combination with
Benzoquinone after which a cell count was performed. The groups
were compared the control groups and Benzoquinone control
group.
TABLE-US-00062 TABLE 61 L-Tyrosine and/or Benzoquinone Compared to
L- Tyrosine to Compared to Compared compound w/o Benzoquinone
L-Tyrosine to Ctrl Benzoquinone Control 500 nm 500 nm w/Benzo (50
.mu.M) 500 nm w/Benzo (100 .mu.M) 5 .mu.m X 5 .mu.m w/Benzo X (50
.mu.M) 5 .mu.m w/Benzo X (100 .mu.M) 15 .mu.m X 15 .mu.m w/Benzo X
(50 .mu.M) 15 .mu.m w/Benzo x (100 .mu.M)
[0579] The addition of Benzoquinone did not amplify the effect of
L-Tyrosine on the cell number.
[0580] Cell Proliferation Assay of the Combination of L-Tyrosine
with Benzoquinone: This study examined the combination of
L-Tyrosine with Farnesyl. The groups were compared to control and
Farnesyl control groups.
TABLE-US-00063 TABLE 62 L-Tyrosine and/or Farnesyl Compared to L-
Tyrosine to Compared to Compared compound w/o Farnesyl L-Tyrosine
to Ctrl Farnesyl Control 500 nm 500 nm w/Farnesyl (50 .mu.M) 500 nm
w/Farnesyl (50 .mu.M) 5 .mu.m X 5 .mu.m w/Farnesyl X (50 .mu.M) 5
.mu.m w/Farnesyl X (100 .mu.M) 15 .mu.m X 15 .mu.m w/Farnesyl X (50
.mu.M) 15 .mu.m w/Farnesyl X (100 .mu.M)
[0581] Combining L-Tyrosine and Farnesyl does not appear to have a
synergistic effect on reducing the cell number in this
experiment.
[0582] The synthesis of the CoQ10 is divided into two main parts,
which consist of the synthesis of the ring structure and synthesis
of the side chain structure. Here, oncogenic cells were
supplemented with compounds which are precursors for the synthesis
of the side chain and the ring structure components. These results
have focused the study to 3 main components involved in the
synthesis of the ring structure and two compounds that play a role
in the attachment of the ring structure to the side chain
structure. The three compounds that have shown a significant
reduction in Bcl-2 and increase in Caspase-3 expression are: 1)
L-Phenylalanine, 2) L-Tyrosine and 3) 4-Hydroxyphenylpyruvate. The
two compounds involved with the attachment of the side chain to the
ring structure are: 1) 4-hydroxy benzoate and 2) Phenylacetate.
[0583] These results also showed that exogenous delivery of these
compounds in combination with 2,3 Dimethoxy-5-methyl-p-benzoquinone
(benzoquinone) significantly inhibits cell proliferation. This
indicates a supplementation of the ring structure with compounds
for the attachment of the side chain to the benzoquinone ring may
supplement an impaired CoQ10 synthesis mechanism. This may also
assist in the stabilization of the molecule to maintain the
functional properties required by cellular processes. Phenylacetate
is a precursor for the synthesis of 4-Hydroxybenzoate, which
exogenous delivery in combination with benzoquinone has a similar
effect in oncogenic cells.
Example 23
Modulation of Gene Expression by Coenzyme Q10 in Cell Model for
Diabetes
[0584] Coenzyme Q10 is an endogenous molecule with an established
role in the maintenance of normal mitochondrial function by
directly influencing oxidative phosphorylation. Experimental
evidence is presented that demonstrates the ability of Coenzyme Q10
in modulating intracellular targets that serve as key indices of
metabolic disorders, such as diabetes, in a manner representative
of therapeutic endpoints.
[0585] In order to understand how Coenzyme Q10 regulates expression
of genes associated with the cause or treatment of diabetes,
immortalized primary kidney proximal tubular cell line derived from
human kidney (HK-2) and primary cultures of the human aortic smooth
muscle cells (HASMC) were used as experimental models. The HK-2 and
HASMC cells are normally maintained in culture at 5.5 mM glucose,
which is a concentration that corresponds to a range considered
normal in human blood. However, in order to simulate a diabetic
environment, both cell lines were subsequently maintained at 22 mM
glucose, which corresponds to the range observed in human blood
associated with chronic hyperglycemia. The cells were subsequently
allowed to propagate over 3 passages so that the intracellular
regulation processes were functionally adapted to mimic a diabetic
state. The choice of cell line was based on the physiologic
influence of diabetes on renal dysfunction and progression to
end-stage renal disease (ESRD) in addition to the progressive
pathophysiology of a compromised cardiovascular function.
Effect of Coenzyme Q10 on Gene Expression in HK-2 Cells using the
Diabetes PCR Array
[0586] The Diabetes PCR array (SABiosciences) offers a screen for
84 genes simultaneously. The 4 treatments tested in this study
were: [0587] HK-2; [0588] HK-2H maintained 22 mM glucose; [0589]
HK2(H)+50 .mu.M Coenzyme Q10; and [0590] HK2(H)+100 .mu.M Coenzyme
Q10.
[0591] A stringent analysis of the Real time PCR data of the HK-2
samples on the Diabetes Arrays (Cat # PAHS-023E, SABiosciences
Frederick Md.) was made to exclude all results where gene
regulation was not at least a two-fold regulation over HK-2 normal
untreated cells with a p value of less than 0.05. Genes that were
observed to be regulated either by chronic hyperglycemia or by
Coenzyme Q10 are listed in Table 63 and their functions and
subcellular locations (derived from Ingenuity Pathway Analysis) are
listed in Table 64.
TABLE-US-00064 TABLE 63 HK-2(H) HK-2(H)-50 .mu.M HK-2(H)-100 .mu.M
Fold Coenzyme Q10 Coenzyme Q10 Genes regulation p value Fold
regulation p value Fold regulation p value CEACAM1 1.26 0.409 3.47
0.067 5.36 0.032 PIK3C2B 1.48 0.131 2.32 0.115 3.31 0.003 INSR
-1.09 0.568 2.51 0.103 2.88 0.024 TNF 2.00 0.005 2.57 0.042 2.81
0.020 ENPP1 -1.50 0.002 1.42 0.238 2.67 0.038 PRKCB -1.75 0.005
1.82 0.280 2.49 0.042 DUSP4 1.27 0.318 1.24 0.455 2.26 0.060 SELL
-1.58 0.219 1.77 0.042 2.06 0.021 SNAP25 -1.00 0.934 1.46 0.377
1.97 0.059
TABLE-US-00065 TABLE 64 Symbol Entrez Gene Name Location Type(s)
CEACAM1 carcinoembryonic antigen- Plasma trans- related cell
adhesion mole- Membrane membrane cule 1 (biliary glycoprotein)
receptor PIK3C2B phosphoinositide-3-kinase, Cytoplasm kinase class
2, beta polypeptide INSR insulin receptor Plasma kinase Membrane
TNF tumor necrosis factor (TNF Extracellular cytokine superfamily,
member 2) Space ENPP1 ectonucleotide pyrophos- Plasma enzyme
phatase/phosphodiesterase 1 Membrane PRKCB protein kinase C, beta
Cytoplasm kinase DUSP4 dual specificity phosphatase 4 Nucleus
phosphatase SELL selectin L Plasma other Membrane SNAP25
synaptosomal-associated Plasma transporter protein, 25kDa
Membrane
[0592] Among the detected RNA transcripts with modulated levels,
the Carcino Embryonic Antigen Cell Adhesion Molecule 1 (CEACAM1)
was identified as being highly upregulated in HK2(H) cells,
particularly with 100 .mu.M Coenzyme Q10 treatment. CEACAM-1, also
known as CD66a and BGP-I, is a 115-200 KD type I transmembrane
glycoprotein that belongs to the membrane-bound CEA subfamily of
the CEA superfamily. On the surface of cells, it forms noncovalent
homo- and heterodimers. The extracellular region contains three
C2-type Ig-like domains and one N-terminal V-type Ig-like domain.
Multiple splice variants involving regions C-terminal to the second
C2-type domain (aa 320 and beyond) exist. The lack of intact
CEACAM1 expression in mice has been proposed to promote the
metabolic syndrome associated with diabetes, while an increase in
expression of CEACAM1 is associated with increased insulin
internalization, which suggests an increase in insulin sensitivity
and glucose utilization (e.g., movement of glucose from blood into
the cells), thus mitigating insulin resistance, a hallmark
characteristic of type 2 diabetes mellitus.
[0593] As shown in Table 63, insulin receptor (INSR) expression was
also altered in diabetic HK-2 cells treated with Coenzyme Q10.
Without being bound by theory, the increase in expression of INSR
with Coenzyme Q10 treatment should enhance insulin sensitivity
(either alone or in addition to expression of CEACAM1) with the
potential to reverse a major physiologic/metabolic complication
associated with diabetes.
Effect of Coenzyme Q10 on Gene Expression in HK-2 Cells using
Mitochondrial Arrays
[0594] Differential expression of mitochondrial genes in diabetes
was assayed using the mitochondria arrays (Cat# PAHS 087E,
SABisociences Frederick Md.). Genes that were regulated by chronic
hyperglycemia and/or Coenzyme Q10 treatment are listed in Table 65
while their functions and location are included in Table 66.
TABLE-US-00066 TABLE 65 HK2 (H) HK-2(H) 50 .mu.M HK-2(H) 100 .mu.M
Genes untreated p value Coenzyme Q10 p value Coenzyme Q10 p value
GRPEL1 -1.5837 0.151255 -2.6512 0.04704 -1.933 0.139161 SLC25A3
-8.6338 0.071951 -8.2059 0.0425 -1.6984 0.995194 TOMM40 -2.3134
0.140033 -1.1567 0.115407 -1.9509 0.038762 TSPO -3.6385 0.111056
-6.7583 0.073769 -2.1104 0.167084
TABLE-US-00067 TABLE 66 Symbol Entrez Gene Name Location Type(s)
GRPEL1 GrpE-like 1, mito- Mitochondria other chondrial (E.coli)
SLC25A3 solute carrier family 25 Mitochondrial transporter
(mitochondrial carrier; membrane. phosphate carrier), member 3
TOMM40 translocase of outer Outer membrane ion channel
mitochondrial membrane of mitochondria. 40 homolog (yeast) TSPO
translocator protein Outer membrane transmembrane (18kDa) of
mitochondria. receptor
[0595] To date, the role of the four mitochondrial genes identified
(Table 65) in diabetic HK-2 cells treated with Coenzyme Q10 in
diabetes is uncharacterized.
Study 2: Effect of Coenzyme Q10 on Gene Expression in HASMC Cells
using the Diabetes PCR Array
[0596] The Diabetes PCR array (SABiosciences) offers a screen for
84 genes simultaneously. The 4 treatments tested in this study
were: [0597] HASMC; [0598] HASMC H maintained at 22 mM glucose;
[0599] HASMC (H)+50 .mu.M Coenzyme Q10; and [0600] HASMC (H)+100
.mu.M Coenzyme Q10.
[0601] A stringent analysis of the Real time PCR data of the HASMC
cell samples on the Diabetes Arrays (Cat #PAHS-023E, SABiosciences
Frederick Md.) was made to exclude all results where gene
regulation was not at least a two-fold regulation over HASMC normal
untreated cells with a p value of less than 0.05. Genes that were
observed to be regulated either by chronic hyperglycemia or by
Coenzyme Q10 are listed in Table 67.
TABLE-US-00068 TABLE 67 HASMC-(H)-50 .mu.M HASMC-(H)-100 .mu.M
Genes HASMC-(H) p value Coenzyme Q10 p value Coenzyme Q10 p value
AGT 1.3051 0.547507 -1.0169 0.781622 2.3027 0.030195 CCL5 -17.4179
0.013798 -5.3796 0.022489 -4.6913 0.022696 CEACAM1 -5.5629 0.012985
-5.3424 0.014436 -5.8025 0.012948 IL6 2.7085 0.049263 3.8172
0.012685 6.0349 0.000775 INSR 1.4649 0.207788 1.9622 0.081204
2.0801 0.016316 NFKB1 1.482 0.072924 1.3779 0.191191 2.0898
0.027694 PIK3C2B 2.0479 0.218276 1.4331 0.254894 2.6329 0.069422
SELL -1.9308 0.087513 1.2476 0.393904 4.0371 0.000177 TNF -1.814
0.108322 -3.2434 0.043526 -1.8489 0.133757
[0602] In HASMC cells, treatment of hyperglycemic cells with
Coenzyme Q10 resulted in the altered expression of genes involved
in regulating vascular function (AGT), insulin sensitivity
(CEACAM1, INSR, SELL) and inflammation/immune function (IL-6, TNF,
CCL5). Without being bound by theory, an increase in expression of
INSR may be associated with increased insulin sensitivity in HASMC
cells, which is a physiological property that would be beneficial
in the treatment of diabetes, while IL-6, in addition to its
immunoregulatory properties, has been proposed to affect glucose
homeostasis and metabolism, both directly and indirectly, by action
on skeletal muscle cells, adipocytes, hepatocytes, pancreatic
.beta.-cells and neuroendocrine cells. Upon activation, normal
T-cell express and secrete RANTES and chemokine(C-Cmotif) ligand
(CCL5). CCL5 is expressed by adipocytes, and serum levels of RANTES
are increased in obesity and type 2 diabetes. However, as shown in
Table 67, treatment of HASMC cells with Coenzyme Q10 causes a
significant decrease in the expression of CCL5. Based on the
foregoing data, it is expected that administration of Coenzyme Q10
will have a therapeutic benefit in the management of diabetes.
Effect of Coenzyme Q10 on Gene Expression in HASMC Cells Using
Mitochondrial Arrays
[0603] Differential expression of mitochondrial genes in diabetes
was assayed using the mitochondria arrays (Cat# PAHS 087E,
SABisociences Frederick Md.). Genes that were regulated by chronic
hyperglycemia and/or Coenzyme Q10 treatment are shown in Table
68.
TABLE-US-00069 TABLE 68 HASMC-(H) 50 .mu.M HASMC-(H)-100 .mu.M
Genes HASMC-(H) p value Coenzyme Q10 p value Coenzyme Q10 p value
BCL2L1 -1.6558 0.244494 -2.7863 0.008744 -2.3001 0.014537 MFN1
-1.4992 0.317009 -1.2585 0.021185 -2.2632 0.005961 PMAIP1 -4.7816
0.206848 -6.8132 0.000158 -4.352 0.000286 SLC25A1 -2.2051 0.020868
-1.834 0.00581 -3.0001 0.03285 SLC25A13 -2.0527 0.035987 -1.5
0.029019 -1.5245 0.043712 SLC25A19 -1.0699 0.417217 -1.4257
0.104814 -2.1214 0.007737 SLC25A22 -2.1747 0.007344 -1.9839 0.0013
-10.3747 0.003437 TIMM44 -1.3605 0.414909 -2.3214 0.004118 -1.9931
0.010206 TOMM40 -1.1982 0.428061 -2.0922 0.002195 -2.2684 0.003272
TSPO -1.402 0.304875 -2.0586 0.061365 -2.3647 0.044656
[0604] Treatment of hyperglycemic HASMC cells with Coenzyme Q10
resulted in altered expression of genes that regulate programmed
cell death or apoptosis (BCL2L1, PMIAP1 also known as NOXA),
transporter proteins (SLC25A1 [citrate transporter], SLC25A13
[aspartate-glutamate exchanger], SLC25A19 [thiamine pyrophosphate
transporter] and SLC25A22 [glutamate-hydrogen cotransporter]) and
mitochondrial matrix transport proteins (MFN1, TIMM44 and TOMM40).
The activities of these transporters play important role in the
regulation of precursors essential for the Kreb's cycle and
maintenance of mitochondrial oxidative phosphorylation. These
results indicate that exposure of diabetic HASMC cells to Coenzyme
Q10 is associated with changes in expression of cytoplasmic and
mitochondrial genes, which in turn is consistent with Coenzyme Q10
providing a therapeutic benefit in the treatment of diabetes.
[0605] A comparison of the data obtained by treating HASMC cells
and HK-2 cells with Coenzyme Q10 or in a hyperglycemic environment
reveals that 4 genes were commonly regulated by Coenzyme Q10 in
both cell lines (e.g., PIK3C2B and SELL in the gene expression
assay and TOMM40 and TSPO in the mitochondrial array assay). These
results demonstrate that treatment of cells with Coenzyme Q10 in a
diabetic environment is associated with altered expression of genes
that are known to be involved in the cause or treatment of
diabetes.
EQUIVALENTS
[0606] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments and methods described
herein. Such equivalents are intended to be encompassed by the
scope of the following claims.
* * * * *
References