US20110020312A1 - Methods for treatment of metabolic disorders using epimetabolic shifters, multidimensional intracellular molecules, or environmental influencers - Google Patents

Methods for treatment of metabolic disorders using epimetabolic shifters, multidimensional intracellular molecules, or environmental influencers Download PDF

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US20110020312A1
US20110020312A1 US12/778,054 US77805410A US2011020312A1 US 20110020312 A1 US20110020312 A1 US 20110020312A1 US 77805410 A US77805410 A US 77805410A US 2011020312 A1 US2011020312 A1 US 2011020312A1
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metabolic
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Niven Rajin Narain
John Patrick McCook
Rangaprasad Sarangarajan
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Definitions

  • 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.
  • the invention relates to the treatment, prevention, and reduction of metabolic disorders, such as diabetes and obesity.
  • 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.
  • type 1 diabetes also known as insulin dependent diabetes (IDDM)
  • type 2 diabetes also known as insulin independent or non-insulin dependent diabetes (NIDDM)
  • 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.
  • 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.
  • Coenzyme Q10 also referred to herein as CoQ10, Q10, ubiquinone, or ubidecarenone
  • CoQ10 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.
  • 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.”
  • 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.
  • 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.”
  • 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.
  • 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.
  • Applicants' data described herein has provided insight into the mechanism of action of Q10.
  • Q10 induces a metabolic shift to the cell microenvironment.
  • Many diseases are known to be correlated with an altered metabolic state.
  • 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.
  • metabolic disorders such as diabetes and obesity, are associated with an altered glucose metabolism.
  • 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.
  • env-influencer environmental influencer
  • the CoQ10 responsive disorder is a metabolic disorder.
  • 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.
  • env-influencer environmental influencer
  • the environmental influencer does not substantially elicit, in normal cells of the mammal, the cellular metabolic energy shift towards mitochondrial oxidative phosphorylation.
  • the mammal is human (or a non-human mammal).
  • the metabolic disorder is responsive or sensitive to treatment by Coenzyme Q10 or its metabolites or analogs thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the environmental influencer : (a) inhibits Bcl-2 expression and/or promotes Caspase-3 expression; and/or (b) inhibits cell proliferation.
  • the environmental influencer is a multidimensional intracellular molecule (MIM).
  • 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.
  • 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.
  • the environmental influencer is an epimetabolic shifter (epi-shifter).
  • the epimetabolic shifter is selected from Transaldolase, Transketolase, Succinyl CoA synthase, Pyruvate Carboxylase, or Riboflavin.
  • the epimetabolic shifter is selected from the group consisting of coenzyme Q10, vitamin D3 and extracellular matrix components.
  • the epimetabolic shifter is coenzyme Q10.
  • the extracellular matrix components are selected from the group consisting of fibronectin, immunomodulators (e.g., TNF ⁇ or an interleukin), angiogenic factors, and apoptotic factors.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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.
  • the amount sufficient to treat the metabolic disorder in the human up-regulates or down-regulates mitochondrial oxidative phosphorylation.
  • the amount sufficient to treat the metabolic disorder in the human modulates anaerobic use of glucose and/or lactate biosynthesis.
  • 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.
  • the form of the environmental influencer administered to the human is different than the predominant form found in systemic circulation in the human.
  • 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.
  • 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.
  • 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.
  • 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.
  • genes selected from the group consisting of HNF4-alpha, Bcl-x1, Bcl-xS, BNIP-2, Bcl-2, Birc6, Bcl-2-L
  • 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.
  • 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, C
  • the metabolic disorder is selected from the group consisting of diabetes, obesity, pre-diabetes, Metabolic Syndrome and any key elements of a metabolic disorder.
  • the metabolic disorder is diabetes
  • the env-influencer affects beta cell function, insulin metabolism, and/or glucagon deposition.
  • the metabolic disorder is obesity
  • the env-influencer affects beta cell oxidation in the mitochondria, decrease in adipocyte size, and/or control of cortisol levels.
  • the metabolic disorder is a cardiovascular disease
  • the env-influencer affects decrease in smooth muscle cell proliferation in the tunica media, lipid peroxidation, thromboxane-ax2 synthesis, TNF ⁇ , IL-1B, platelet aggregation, decrease in nitric oxide (NO) production, plaque deposition and/or normalized glycemic control.
  • 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.
  • 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.
  • 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.
  • the metabolic disorder is selected from the group consisting of diabetes, obesity, pre-diabetes, Metabolic Syndrome and any key elements of a metabolic disorder.
  • 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 insuff
  • 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.
  • 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.
  • 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.
  • 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.
  • the environmental influencer effective in treating a metabolic disorder is capable of reducing glucose levels or lipid levels in a patient.
  • 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.
  • 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.
  • 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.
  • 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.
  • the molecular profile is selected from the group consisting of a metabolite profile, lipid profile, protein profile or RNA profile.
  • the molecule does not negatively effect the health or growth of a normal cell.
  • 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.
  • 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.
  • FIG. 1 Sensitivity of SK-MEL-28 to 24 hours of Q10 treatment measured by the amount of early and late apoptotic cells.
  • FIG. 2 Sensitivity of SKBR3 to 24 hours of Q10 treatment measured by the amount of early and late apoptotic cells.
  • FIG. 3 Sensitivity of PaCa2 to 24 hours of Q10 treatment measured by the amount of early and late apoptotic cells.
  • FIG. 4 Sensitivity of PC-3 to 24 hours of Q10 treatment measured by the amount of early and late apoptotic cells.
  • FIG. 5 Sensitivity of HepG2 to 24 hours of Q10 treatment measured by the amount of early and late apoptotic cells.
  • FIG. 6 Sensitivity of MCF-7 to 24 hours of Q10 treatment measured by the amount of early and late apoptotic cells.
  • FIG. 7 Measurement of apoptotic cells upon 24 hour treatment with Q10, as measured by Apostrand ELISA method.
  • FIG. 8 Example gel analysis of 2-D gel electrophoresis. Spots excised for identification are marked.
  • FIG. 9 Network of interaction between proteins identified by 2-D gel electrophoresis as being modulated by Q10 in SK-MEL-28 cells.
  • FIG. 10 The pentose phosphate pathway adapted from Verhoeven et al. (Am. J. Hum. Genet. 2001 68(5):1086-1092).
  • 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.
  • FIG. 12 Comparative plot of the relative amounts of Q10 present in SK-MEL-28 mitochondria following the exogenous addition of 100 ⁇ M Q10 into the culture medium.
  • FIG. 13 Apoptosis pathway mapping known processes.
  • FIG. 14 Western blot analysis of Bcl-x1.
  • FIG. 15 Western blot analysis of SK-MEL-28 sample set proved with a Vimentin antibody.
  • FIG. 16 Western blot analysis of cell lysis from a number of cell lines, evaluated with five antibodies targeting oxidative phosphorylation complexes (MitoSciences #MS601).
  • FIG. 17 Western blot comparison of F1-alpha levels.
  • FIG. 18 Western blot comparison of Q10 response with C-III-Core 2.
  • FIG. 19 Western blot comparison of Q10 response with C-II-30.
  • FIG. 20 Western blot comparison of Q10 response with C-IV-COX II.
  • FIG. 21 Western blot comparison of Q10 response with C-1-20 (ND6).
  • FIG. 22 Western blot analysis of a variety of cell types against five mitochondrial protein.
  • FIG. 23 Western blot comparison of Q10 response with Complex V protein C-V- ⁇ .
  • FIG. 24 Western blot comparison of Q10 response with C-III-Core 1.
  • FIG. 25 Western blot comparison of Q10 response with Porin (VDAC1).
  • FIG. 26 Western blot comparison of Q10 response with Cyclophilin D
  • FIG. 27 Western blot comparison of Q10 response with Cytochrome C.
  • FIG. 28 Theoretical model of Q10 (spheres) inserted into the lipid binding channel of HNF4alpha (1M7W.pdb) in the Helix 10 open conformation.
  • FIG. 29 OCR in HDFa cells in various glucose conditions in normoxic and hypoxic conditions.
  • FIG. 30 OCR in HASMC cells in various glucose conditions in normoxic and hypoxic conditions.
  • FIG. 31 OCR values in MCF-7 breast cancer cells in the absence and presence of 31510 and stressors.
  • FIG. 32 OCR values in PaCa-2 pancreatic cancer cells in the absence and presence of 31510 and stressors.
  • an element means one element or more than one element.
  • 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.
  • “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.
  • Preventing 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).
  • 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).
  • the unwanted condition e.g., disease or other unwanted state of the host animal
  • 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.
  • 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.
  • a therapeutically-effective amount of a compound will depend on its therapeutic index, solubility, and the like.
  • 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.
  • patient 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.
  • animal e.g., a human
  • horses dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats, mice, lizards, snakes, sheep, cattle, fish, and birds.
  • Metal 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.
  • Metal 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • intermediate of the coenzyme biosynthesis pathway 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
  • 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.
  • the phrase “aerobic glycolysis” or “mitochondrial oxidative phosphorylation” refers to cellular production of energy by glycolysis followed by oxidation of pyruvate in mitochondria.
  • 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.
  • an environmental influencer e.g., an epitmetabolic shifter
  • 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.
  • Multidimensional Intracellular Molecule (MIM)
  • MIM Multidimensional Intracellular Molecule
  • 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.
  • 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.
  • co-administration of CoQ-10 and VEFG has no effect on the levels of Bc12.
  • MIMs selectively act in cells of a disease state, and have substantially no effect in (matching) cells of a normal state.
  • 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.
  • a MIM is also an epi-shifter. In another embodiment, a MIM is not an epi-shifter.
  • 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.
  • MIMs can be lipid based or non-lipid based molecules.
  • 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.
  • the MIM is a small molecule.
  • 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.
  • 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).
  • 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.
  • 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.
  • the MIMs include amino acids.
  • amino acids include, for example, tyrosine (e.g., L-tyrosine), cysteine, phenylalanine (e.g., L-phenylalanine) and alanine.
  • the amino acid is phenylalanine or alanine.
  • the MIMs include amino acid derivatives such as 4-hydroxyphenylpyruvate or acetylglycine.
  • the MIM is a glucose analog, e.g., a glucose molecule wherein one —OH or —CH 2 OH substituent has been replaced with a —COOH, a —COO ⁇ or an —NH 2 substituent.
  • glucose analogs include glucosamine, glucuronic acid, glucuronide and glucuronate.
  • the MIM is selected from compounds of formula (I):
  • n is an integer of 0 or 1;
  • R 1 , R 2 , R 3 and R 4 when present, are each independently selected from hydrogen and hydroxyl or R 1 and R 2 are taken together with the carbon on which they are attached to form a carbonyl (C ⁇ O) group;
  • W is —COOH or —N(CH 3 ) 3 + ;
  • X is hydrogen, a negative charge or a alkali metal cation, such as Na + or.
  • W is —N(CH 3 ) 3 + .
  • the MIM is a carnitine, such as L-carnitine.
  • the MIM is a dicarboxylic acid.
  • W is —COOH.
  • R 3 is hydrogen.
  • n is 0.
  • R 1 and R 2 are each independently hydrogen.
  • W is —COOH, R 3 is hydrogen, n is 0 and R 1 and R 2 are each independently hydrogen.
  • n is 1.
  • R 1 and R 2 are taken together with the carbon on which they are attached to form a carbonyl (C ⁇ O) group.
  • R 4 is hydrogen.
  • R 4 is hydroxyl.
  • W is —COOH, R 3 is hydrogen, n is 1 and R 1 and R 2 are taken together with the carbon on which they are attached to form a carbonyl (C ⁇ O) group.
  • 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 ⁇ -ketoglutaric acid or ⁇ -ketoglutarate.
  • the MIM is a building block of CoQ10, which has the following structure:
  • 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.
  • the MIM is selected from phenylalanine, tyrosine, 4-hydroxyphenylpyruvate, phenylacetate and 4-hydroxybenzoate.
  • 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.
  • the cells are cultured cells, e.g., in vitro.
  • 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.
  • 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.
  • 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
  • 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 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
  • 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
  • to differentially e.g., preferentially induce a change in the microenvironment profile of a diseased cell as compared to a normal cell
  • MIM an endogenous molecule that is observed to induce a change in the microenvironment profile of a cell
  • 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.
  • 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.
  • 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.
  • lipid based molecules examples 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.
  • 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.
  • 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.
  • 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.
  • non-lipid based molecules examples 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.
  • an “epimetabolic 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the enzyme is an enzyme of the non-oxidative phase of the pentose phosphate pathway, such as transaldolase, or transketolase.
  • 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).
  • Krebs Cycle enzyme succinyl CoA synthase
  • pyruvate carboxylase a ligase that catalyzes the reversible carboxylation of pyruvate to form oxaloacetate (OAA), a Krebs Cycle intermediate.
  • 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.
  • the epi-shifter is selected from phenylalanine, tyrosine, 4-hydroxyphenylpyruvate, phenylacetate and 4-hydroxybenzoate.
  • 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.
  • 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.
  • Epimetabolic shifters 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.
  • 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 molecular profile e.g., of metabolites, lipids, proteins or RNAs (as individual profiles or in combination)
  • 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.
  • 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.
  • 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.
  • the epi-shifter does not negatively impact the health and/or growth of normal cells.
  • Epimetabolic shifters of the invention include, but are not limited to, small molecule metabolites, lipid-based molecules, and proteins and RNAs.
  • 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
  • a change in level correlates to the disease state, progression or aggressiveness
  • 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.
  • 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
  • a change in expression e.g., an increase or decrease in expression at the mRNA or protein level
  • This information obtained by such cellular and/or biochemical pathway analysis may be utilized to categorize the pathways and potential epi-shifters.
  • 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.
  • molecules that indirectly modulate the Epi-shifter e.g., the level or activity of the Epi-shifter
  • genes e.g., regulated at the RNA or protein level
  • 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.
  • 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.
  • 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.
  • 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).
  • LC liquid chromatography
  • HPLC high-pressure liquid chromatography
  • MS mass spectroscopy
  • GC gas chromatography
  • LC-MS liquid chromatography/mass spectroscopy
  • GC-MS gas chromatography/mass spectroscopy
  • NMR nuclear magnetic resonance
  • MRI magnetic resonance imaging
  • FT-IR Fourier Transform InfraRed
  • ICP-MS inductively coupled plasma mass spectrometry
  • 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).
  • TOF time-of-flight instruments
  • FT-MS Fourier transform ion cyclotron resonance instruments
  • MALDI-TOF MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
  • Environmental influencers 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.
  • bioenergetic molecule levels e.g., ATP, pyruvate, ADP, NADH, NAD, NADPH, NADP, acetylCoA, FADH2
  • Exemplary assays of bioenergetic molecule levels use colorometric, fluorescence, and/or bioluminescent-based methods. Examples of such assays are provided below.
  • 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 (EnzyLightTM ATP Assay Kit (EATP-100), BioAssay Systems, Hayward, Calif.).
  • both ATP and its dephosphorylated form are calculated via bioluminescence; after ATP levels are calculated, ADP is transformed into ATP and then detected and calculated using the same luciferase system (ApoSENSORTM ADP/ATP Ratio Assay Kit, BioVision Inc., Mountain View, Calif.).
  • 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 (EnzyChromTM Pyruvate Assay Kit (Cat# EPYR-100), BioAssay Systems, Hayward, Calif.).
  • Environmental influencers 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.
  • 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.
  • the MT-OXC MitoToxTM 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.
  • Environmental influencers 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.
  • 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.
  • OCR oxygen consumption
  • ECAR extracellular acidification
  • 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.
  • ATP oxidative phosphorylation
  • changes in oxidative phosphorylation activity can strongly alter metabolism and energy balance within a cell.
  • environmental influencers e.g., MIMs or Epi-shifters
  • environmental influencers e.g., MIMs or Epi-shifters
  • environmental influencers e.g., MIMs or Epi-shifters
  • 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.
  • 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.
  • Complex I also known as NADH-coenzyme Q oxidoreductase or NADH dehydrogenase, is the first protein in the electron transport chain.
  • the detection and quantification of the effect of an epi-shifter on the production of NAD by Complex I may be performed.
  • 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.).
  • Complex IV also known as cytochrome c oxidase (COX)
  • COX cytochrome c oxidase
  • 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.
  • 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.).
  • 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.
  • 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 + .
  • 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.
  • the MT-OXC MitoToxTM 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.
  • MIMs mitochondrial permeability transition
  • MPTP mitochondrial permeability transition pores
  • 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.
  • 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.
  • 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 2 serves to squelch the fluorescence of the calcein dye in the cytosol.
  • CoCl 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 2 can access the interior of the mitochondra via MPTPs.
  • Flow cytometry can be used to evaluate cellular and organelle fluorescence (MitoProbeTM 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.).
  • environmental influencers e.g., MIMs or Epi-shifters
  • MIMs may be identified and evaluated by their effects on the production or activity of molecules associated with cellular proliferation and/or inflammation.
  • 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.
  • VEGF Vascular endothelial growth factor
  • an environmental influencer e.g., MIM or Epi-shifter
  • MIM vascular endothelial growth factor
  • 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.).
  • 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.
  • tumor necrosis factor is a key mediator of inflammation and immune system activation.
  • an Epi-shifter may be identified and characterized by its effects on the production or the activity of TNF.
  • TNF produced by cultured cells and secreted into media can be quantified via ELISA and other antibody-based assays known in the art.
  • 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.).
  • ECM extracellular matrix
  • latent transforming growth factor beta binding proteins are ECM components that create a reservoir of transforming growth factor beta (TGF ⁇ ) within the ECM.
  • TGF ⁇ transforming growth factor beta
  • Matrix-bound TGF ⁇ 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)).
  • an environmental influencer e.g., MIM or Epi-shifter
  • MIM MIM or Epi-shifter
  • 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)).
  • the effect of environmental influencer e.g., MIM or epi-shifter
  • the effect of environmental influencer e.g., MIM or epi-shifter
  • 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.
  • Environmental influencers 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.
  • LDH lactate dehydrogenase
  • 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 (QuantiChromTM Lactate Dehydrogenase Kit (DLDH-100), BioAssay Systems, Hayward, Calif.; LDH Cytotoxicity Detection Kit, Clontech, Mountain View, Calif.).
  • 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® (OncoImmunin, Inc., Gaithersburg, Md.) and Caspase-Glo® 3/7 Assay Systems (Promega Corp., Madison, Wis.).
  • TUNEL/DNA fragmentation 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 DeadEndTM Colorimetric and Fluorometric TUNEL Systems (Promega Corp., Madison, Wis.).
  • Some apoptosis assays detect and quantify proteins associated with an apoptotic and/or a non-apoptotic state.
  • the MultiTox-Fluor Multiplex Cytotoxicity Assay 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.
  • Additional assays available for detecting and quantifying apoptosis include assays that detect cell permeability (e.g., APOPercentageTM APOPTOSIS Assay, Biocolor, UK) and assays for Annexin V (e.g., Annexin V-Biotin Apoptosis Detection Kit, BioVision Inc., Mountain View, Calif.).
  • assays that detect cell permeability e.g., APOPercentageTM APOPTOSIS Assay, Biocolor, UK
  • assays for Annexin V e.g., Annexin V-Biotin Apoptosis Detection Kit, BioVision Inc., Mountain View, Calif.
  • the compounds of the present invention 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.
  • 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.
  • CoQ10 responsive states are states that arise from an altered metabolism of cell microenvironment.
  • 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.
  • the compounds of the present invention e.g., the MIMs or epi-shifters described herein, share a common activity with Coenzyme Q10.
  • 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.
  • the compounds of the present invention exhibit 25% or more of the activity of Coenzyme Q10.
  • the compounds of the present invention exhibit up to and including about 130% of the activity of Coenzyme Q10.
  • 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%,
  • the compounds of the present invention exhibit between about 50% and about 100% of the activity of Coenzyme Q10.
  • the activity shared by Coenzyme Q10 and the compounds of the present invention is the ability to induce a shift in cellular metabolism.
  • 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).
  • 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.
  • env-influencer environmental influencer
  • 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.
  • env-influencer environmental influencer
  • 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.
  • 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.
  • 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.
  • 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.
  • an environmental influencer env-influencer
  • 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.
  • 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.
  • the metabolic disorder is a Coenzyme Q10 responsive state.
  • treating, reducing, or preventing a metabolic disorder is meant ameliorating such a condition before or after it has occurred.
  • 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.
  • Type I also known as “insulin-dependent diabetes” (“IDDM”) or “juvenile-onset diabetes”
  • IDDM insulin-dependent diabetes
  • NIDDM non-insulin dependent diabetes
  • 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.
  • DCCT Diabetes Control and Complications Trial
  • 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.
  • 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.
  • 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.
  • 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).
  • “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.
  • Weight 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.
  • Metabolic Syndrome 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.
  • NASH National Cholesterol Education Program
  • NEP National Cholesterol Education Program
  • 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)
  • central obesity with central obesity being defined as abdominal waist circumference greater than 40 inches for men and greater than 35 inches for women.
  • Metabolic Syndrome 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).
  • 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.
  • CVD cardiovascular disease
  • 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 (
  • 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
  • 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).
  • HOMA-IR homeostasis model assessment to insulin resistance
  • “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).
  • 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).
  • hyperglycemia high blood sugar
  • 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.
  • hypoglycemia low blood sugar
  • 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.
  • 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 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.
  • a metabolic disorder e.g., diabetes or obesity
  • 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.
  • 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).
  • a biological sample e.g., blood, serum, or urine
  • HbAlc Hemoglobin Alc
  • 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.
  • 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.
  • 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.
  • 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.
  • HbAlc hemoglobin Alc
  • a person without diabetes typically has an HbAlc value that ranges between 4% and 6%.
  • 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%.
  • 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.
  • 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.
  • 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.
  • pre-diabetes 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.
  • 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).
  • 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.
  • Metabolic Syndrome also known as Syndrome X
  • 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)
  • central obesity is defined as abdominal waist circumference greater than 40 inches for men and greater than 35 inches for women.
  • HbAlc hemoglobin Alc
  • the therapeutic methods of the invention are effective in reducing glucose levels or lipid levels in a patient.
  • 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.
  • 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.
  • 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).
  • the agent of the invention increases more than one of the activities associated with the clearance of glucose.
  • 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.
  • 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.
  • 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.
  • an environmental influencer e.g., epi-shifter
  • an environmental influencer e.g., epi-shifter
  • cell-based screening assays that identify agents that increase glucose uptake may be employed.
  • 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.
  • 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.
  • cells 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a drug target may include HNF4A, Transaldolase, NM23 and BSCv.
  • the drug target is TNF4A.
  • the drug target is transaldolase.
  • the drug target is NM23.
  • the drug target is BSCv. Screening assays useful for identifying modulators of identified drug targets are described below.
  • 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.
  • 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.
  • 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
  • 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.
  • a compound is selected based on its ability to increase expression and/or activity of a therapeutic target of the invention.
  • 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).
  • a compound is selected based on its ability to decrese expression and/or activity of a therapeutic target of the invention.
  • 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).
  • 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.
  • compounds e.g., marker substrates
  • compounds can be labeled with 131 I, 125 I, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
  • 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.
  • 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.
  • 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.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatment as described above.
  • the environmental influencers of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject.
  • the pharmaceutical composition comprises an environmental influencer of the invention and a pharmaceutically acceptable carrier.
  • 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.
  • pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • 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.
  • 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.
  • 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 e.g., ointments or pasts.
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • the environmental influencers of the present invention can be administered by a variety of methods known in the art.
  • the preferred route/mode of administration is subcutaneous injection, intravenous injection or infusion.
  • the route and/or mode of administration will vary depending upon the desired results.
  • 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.
  • 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.
  • the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • the environmental influencer is administered by intravenous infusion or injection.
  • the environmental influencer is administered by intramuscular or subcutaneous injection.
  • the environmental influencer is administered topically.
  • 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.
  • 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.
  • 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.
  • 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.
  • the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • 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).
  • 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
  • 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.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • 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.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • 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
  • 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.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • 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.
  • 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.
  • 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.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • 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.
  • 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.
  • 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.
  • 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.
  • injection is preferred, including intramuscular, intravenous, intraperitoneal, intranodal, and subcutaneous.
  • 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.
  • the compound(s) may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • 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.
  • 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.
  • 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 50 (the dose lethal to 50% of the population) and the ED 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 50 /ED 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 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.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 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.
  • 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.
  • Such agents may be formulated and administered systemically or locally.
  • Techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, 18 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.
  • compositions described above may be administered to a subject in any suitable formulation.
  • an environmental influencer e.g., CoQ10
  • the environmental influencer e.g., CoQ10
  • the environmental influencer, e.g., CoQ10 might be delivered by other methods.
  • 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.
  • compositions may be administered in a single bolus, multiple injections, or by continuous infusion (for example, intravenously or by peritoneal dialysis).
  • 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.
  • Such agents may be formulated and administered systemically or locally.
  • Techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, 18. 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.
  • 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.
  • physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions 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.
  • compositions 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).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coating.
  • suitable coating 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.
  • compositions 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.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • 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.
  • 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.
  • the remaining component of the composition is water, which is necessarily purified, e.g., deionized water.
  • 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.
  • 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 (AzoneTM, 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-tallow
  • the present invention provides CoQ10 compositions and methods of preparing the same.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the CoQ10 cream comprises about 3% of Coenzyme Q10.
  • the CoQ10 cream comprises about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% of Coenzyme Q10.
  • 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.
  • 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.
  • the CoQ10 cream comprises about 3% of Coenzyme Q10.
  • the CoQ10 cream comprises about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% of Coenzyme Q10.
  • 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.
  • 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.
  • 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.
  • the Phase A ingredients include Alkyl C 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 A ingredients include Al
  • Trolamine refers to Trolamine NF, Triethanolamine, TEAlan®, TEAlan 99%, Triethanolamine, 99%, Triethanolamine, NF or Triethanolamine, 99%, NF. These terms may be used interchangeably herein.
  • 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 lactic acid US
  • methods 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.
  • a pharmaceutical composition comprising CoQ10 cream 3%.
  • the cream includes a phase A having C 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
  • a pharmaceutical composition comprising CoQ10 cream 3%.
  • 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
  • a pharmaceutical composition comprising CoQ10 cream 1.5%.
  • the cream includes a phase A having C 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%
  • a pharmaceutical composition comprising CoQ10 cream 1.5%.
  • 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 5.000% w/w, cet
  • 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.
  • an environmental influencer and/or a pharmaceutical composition thereof is administered concurrently with the administration of another therapeutic agent.
  • a compound and/or pharmaceutical composition thereof is administered prior or subsequent to administration of another therapeutic agent.
  • 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.
  • 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
  • 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, lycopen
  • 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).
  • 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,
  • 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).
  • angiotensin II antagonists e.g., losartan, candesartan cilexetil,
  • 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.
  • central antiobesity agents e.g., dexfenflu
  • 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
  • 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.
  • 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
  • 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.
  • IL-1 interferon, interleukin (IL) and the like
  • IL-12 interleukin
  • 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.
  • 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.
  • glucosteroid e.g., dexamethasone and the like
  • metoclopramide-based agents e.g., metoclopramide-based agents
  • tetrahydrocannabinol-based agents e.g., lipid metabolism improving agents
  • 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.
  • 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.
  • results described by Applicants herein identified the endogenous molecule CoQ10 and, in particular, the oxidized form of CoQ10, as a MIM.
  • 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.
  • 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.
  • a 500 ⁇ M Coenzyme Q10 (5% isopropanol in cell growth media) was prepared as follows. A 10 mL 500 ⁇ M Coenzyme Q10 stock was made fresh every time.
  • 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.
  • 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 ⁇ M Q10, and 100 ⁇ 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.
  • 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 metalastatic skin melanoma
  • HEKa kerantinocytes, skin control
  • HEMa melanocyte, skin control
  • nFIB neonatal fibroblasts
  • HEP-G2 liver cancer
  • SkBr-3 breast cancer, Her2 overexpressed
  • MCF-7 breast cancer, p53 mutation
  • PC-3 prostate cancer
  • SBH cell line SkBr-3 (human breast adenocarcinoma)
  • 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.
  • 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° C. with 5% CO 2 . Details of additional cell line and growth conditions are outlined in the table below.
  • SK-MEL28 cells were treated with 100 ⁇ 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 ⁇ L of isopropanol. The resulting solution was warmed in a 65° 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 ⁇ M Q10.
  • control vehicle 9.5 mL of media was added to 500 ⁇ 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.
  • SCC cells were treated with 100 ⁇ 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° C. until the RNA was isolated at XTAL as described below.
  • First strand cDNA was synthesized from 1 ⁇ g of total RNA using the RT2 First Strand Synthesis kit (SABiosciences, Frederick Md.) as per manufacturer's recommendations.
  • 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 ⁇ M Q10 or 100 ⁇ M Coenzyme Q10. After 24 hours, the presence of apoptotic cells was estimated on a PCA96 flow cytometer (Guava Technologies, Hayward, Calif.).
  • 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.).
  • 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.
  • 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.
  • 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.
  • the proteins 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.
  • 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.
  • 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° 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.
  • SKMEL-28 cells were plated at a density of 1 ⁇ 10 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.
  • 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° C.-15 second denaturation step and 60° 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.
  • 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.
  • PaCa2 cells showed an increase in both early and late apoptotic cells (between 5-10% of gated cells) with 50 ⁇ M Q10 and 100 ⁇ M Q10 after 24 hours of Q10 application.
  • PC-3 cells also showed an increase in both early and late apoptotic population with 50 ⁇ M and 100 ⁇ 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 ⁇ M and 100 ⁇ M Q10.
  • HepG2 cells were also sensitive to 50 ⁇ 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 ⁇ M or 100 ⁇ M Q10 treatment. The results are depicted in FIGS. 1-6 .
  • a second apoptosis assay was evaluated using the ApoStrandTM ELISA based method that measures single-stranded DNA.
  • the ApoStrandTM 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).
  • ssDNA single-stranded DNA
  • 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.
  • 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 ⁇ M Q10.
  • 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.
  • 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.
  • SCC Squamous Cell Carcinoma
  • SCC cells were treated with 100 ⁇ 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.
  • 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).
  • 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.
  • 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).
  • BSCv may a have a potential role in cancer and diabetes.
  • 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.
  • NDK nucleoside diphosphate
  • 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.
  • the skin cancer SKMEL-28 cells were treated with 100 ⁇ 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° 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 ⁇ L aliquots (4-5 aliquots per sample) and stored at ⁇ 80° C.
  • 2-D gel electrophoresis was performed on proteins solubilized from two aliquots of the SK-MEL-28 mitochondria enriched samples treated with 100 ⁇ 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 ).
  • Acyl-CoA thioesterase 7 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.
  • 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.
  • PEP phosphoenolpyruvate
  • 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.
  • 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.
  • the levels of cholesterol was also measured to confirm that the differences were not due to sample size errors.
  • 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.
  • a significant increase in Q10 levels was obtained at 19 hours ( ⁇ 3-fold) with an even larger increase by the 48 hour time point ( ⁇ 6-fold) ( FIG. 12 ).
  • RT-PCR real-time polymerase chain reaction
  • 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.
  • 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.
  • each mRNA may have different rates at which it is degraded or its translation inefficiently, thus resulting in differing amounts of protein.
  • 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.
  • 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 ⁇ M Q10 treatment.
  • the genes in the Apoptosis array that appear to be regulated in SCC cells are described in Table 7.
  • Genes that appear to be regulated in both SK-MEL-28 cells and in SCC cells are described in Table 8.
  • 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
  • protein 3 CARD6 Caspase recruitment domain family Down regulated at 6 hours.
  • 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.
  • 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.
  • Bcl-xL 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 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).
  • BCL2L11 BNIP2, BAG1, HRK, BAK1, BCL2, BCL2L1
  • 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.
  • E2F-1 transcription factors within the body involved in cell cycle regulation and synthesis of DNA in mammalian cells
  • 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 .
  • BCL2L11 BNIP2, BAG1, HRK, BAK1, BCL2, BCL2L1
  • p73 may be an oncoprotein, but may involve different mechanism that the related p53 protein
  • RT-PCR real-time polymerase chain reaction
  • Table 10 lists the genes that are regulated in SK-MEL28 cells with 100 ⁇ 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.
  • 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.
  • 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.
  • NME5 Non-metastatic cells 5
  • 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.
  • SIRT2 Sirtuin sient mating type Up regulation at 16 Nucleus Transcription factor. information regulation 2 hours homolog
  • SOD1 Superoxide dismutase 1
  • soluble (amyotrophic hours lateral sclerosis 1 (adult))
  • SOD2 Superoxide dismutase 2
  • SOD3 Superoxide dismutase 3
  • Down Regulation at Extracellular Pro-apoptotic extracellular 48 hours space SRXN1 Sulfiredoxin 1 homolog S.
  • 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.
  • NCF1 neutral cytosolic factor 1
  • 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: a Rho guanosine triphosphatase (GTPase), usually Rac1 or Rac2 (Rac stands for Rho-related C3 botulinum toxin substrate)
  • GTPase Rho guanosine triphosphatase
  • Rac1 or Rac2 Rho-related C3 botulinum toxin substrate
  • 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
  • Phosphatidylinositol 3,4,5-trisphosphate-dependent RAC exchanger 1 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.
  • Nitric oxide synthase 2A inducible, hepatocytes
  • 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.
  • SOD2 Superoxide dismutase 2
  • mitochondrial 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.
  • IDC idiopathic cardiomyopathy
  • FOXM1 Forkhead box M1
  • Plk1, cyclin B2, Nek2 and CENPF G2/M-specific genes
  • CENPF CENPF
  • the level of mRNA present in SKMEL-28 cells treated with 100 ⁇ M Q10 were compared to the levels in untreated cells at various time points using real-time PCR methods (RT-PCR).
  • RT-PCR real-time PCR methods
  • 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.
  • 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.
  • NCF1 neutral cytosolic factor 1
  • 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:
  • Rho guanosine triphosphatase usually Rac1 or Rac2 (Rac stands for Rho-related C3 botulinum toxin substrate)
  • 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
  • Phosphatidylinositol 3,4,5-trisphosphate-dependent RAC exchanger 1 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.
  • Nitric oxide synthase 2A inducible, hepatocytes
  • 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.
  • FOXM1 Down regulated protein
  • Plk1, cyclin B2, Nek2 and CENPF G2/M-specific genes
  • CENPF 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).
  • BCC basal cell carcinoma
  • 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.
  • 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 Nucle
  • 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
  • PYGL Phosphorylase glycogen
  • liver Down regulated (Hers disease, glycogen storage at 6 hours. disease type VI)
  • STXBP2 Syntaxin binding protein 2 Down regulated at 6 hours.
  • TNF Tumor necrosis factor (TNF Down regulated superfamily, member 2) at 6 hours.
  • 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.
  • 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.
  • 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.)
  • 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)).
  • 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).
  • Hepatocyte nuclear factor 4 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.
  • RCC renal cell carcinoma
  • 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.
  • the hydrophobic tail would extend out of the internal cavity and would then interact with the elongated helix 10.
  • 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.
  • 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.
  • 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 ⁇ M Q10.
  • 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 ⁇ M Q10.
  • 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.
  • 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 inhibits the ability of Bcl-2 to inhibit cell death and renders cells more susceptible to apoptotic cell death.
  • 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 ⁇ M Q10.
  • FIGS. 16-21 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.
  • Angiogenesis Related Genes Identified as being Modulated at the mRNA Level by Treatment of Pancreatic Cancer Cells (PaCa2) with 100 ⁇ M Q10
  • 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.
  • 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.
  • HepG2 (liver cancer) cells were treated with either the vehicle for 24 hours or 100 ⁇ M Q10 for different times. The treatment was initiated on 1 ⁇ 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 ⁇ g of total RNA (determined by measurement at 260 nm). The maximum volume that can be used per reverse transcription is 8 ⁇ 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 ⁇ g of RNA.
  • the arrays provided an initial analysis of trends and patterns in HepG2 gene regulation with 100 ⁇ 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).
  • PPARGC1A and PRKAA1 had normalized or were slightly upregulated, while SNAP25 was downregulated by approximately 2 fold.
  • HepG2 (liver cancer) cells were treated with either the vehicle for 24 hours or 100 ⁇ M Q10 for different times. The treatment was initiated on 1 ⁇ 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 ⁇ g of total RNA (determined by measurement at 260 nm). The maximum volume that can be used per reverse transcription is 8 ⁇ 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 ⁇ g of RNA.
  • the arrays provided an initial analysis of trends and patterns in HepG2 gene regulation with 100 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ANGPTL4 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • CASP9, BAG1 and TNFRSF1A were upregulated by approximately 55, 1 and 1 fold, respectively, over control.
  • a selected Epi-shifter e.g., CoQ10
  • a metabolic disorder e.g., diabetes
  • cell based assays that monitor an increase in insulin-stimulated glucose uptake in vitro are employed.
  • 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).
  • Prees media 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).
  • DM1 differentiation medium-1
  • DM2 differentiation medium-2
  • Differentiation media-1 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 was prepared by supplementing DMEM with 10% FBS, L-glutamine, pen/strep, and insulin (120 nM).
  • 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.
  • 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.
  • D-(+)-glucose (“cold” glucose, not radiolabeled) was added to DPBS mix to a final concentration of 10 mM.
  • 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.
  • a base e.g., sodium hydroxide at a final concentration of 0.5N
  • a detergent e.g., sodium dodecyl sulphate (SDS) diluted to a final concentration of 0.1% w/v
  • 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.
  • DM1 differentiation medium-1
  • DM2 differentiation medium-2
  • DM2 is removed and replaced with fresh Prees media.
  • Candidate compounds are added at this time, allowing an incubation period of approximately 48 hours.
  • cells now at day 9 to 15 of differentiation
  • DPBS magnesium sulfate
  • Hepes 10 mM
  • fresh DPBS containing insulin 10 nM
  • 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.
  • 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.
  • 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.
  • 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.
  • a candidate molecule e.g., environmental influencer
  • 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 ApostrandTM 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.
  • 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.
  • 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.
  • potential changes to levels of specific proteins induced by the candidate MIM may be evaluated by Western blot analysis.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Candidate MIMs observed to induce changes in cell composition 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
  • a disease state e.g., diabetes or obestity
  • a normal state e.g., diabetes or obestity
  • 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.
  • 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.
  • TAL transaldolase 1
  • Coenzyme 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.
  • the results described herein identified the endogenous molecule CoQ10 as an epi-shifter.
  • the results identified CoQ10 as inducing a shift in the metabolic state, and partially restoration of mitochondrial function, in cells.
  • 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.
  • 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.
  • 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.
  • Q10 is acting as an Epi-shifter by shifting the metabolic state of a cell.
  • 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.
  • control cell lines e.g., primary culture of keratinocytes and melanocytes
  • cancer cell lines e.g., SK-MEL-28, a non-metastatic skin melanoma; SK-MEL-2, a meta
  • 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 ApostrandTM 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.
  • 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.
  • 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.
  • potential changes to levels of specific proteins induced by the candidate MIM may be evaluated by Western blot analysis.
  • proteins with increased or decreased levels in the various cell lines are identified and evaluated.
  • 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.
  • candidate Epi-shifters are evaluated based on their ability to modualate genes known to be associated with or involved in cellular metabolic processes.
  • candidate Epi-shifters are evaluated based on their ability to modulate genes known to be associated with, for example, diabetes or obesity.
  • 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.
  • 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.
  • 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.
  • a candidate Epi-shifter that enhances, increases or augments insulin-stimulated glucose uptake in cells is identified as an Epi-shifter.
  • Vitamin D3 or 1 ⁇ , 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).
  • VDR ubiquitous nuclear vitamin D receptor
  • vitamin D3 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).
  • 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).
  • pro-apoptotic proteins e.g., BAX, BAK and BAD
  • 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.
  • VEGF vascular endothelial growth factor
  • 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).
  • 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).
  • ROS reactive oxygen species
  • refs damage of mitochondria
  • 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.
  • any deviation in expression of proteins and mitochondrial respiration rates in cancer cell lines 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.
  • 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.
  • OXPHOS mitochondrial oxidative phosphorylation
  • TCA Tricarboxylic acid cycle
  • Citric Acid Cycle mitochondrial oxidative phosphorylation
  • 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 ⁇ M and 100 ⁇ 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.
  • the gels were run for 50 minutes using 1 ⁇ 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).
  • 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 ⁇ -15′; 2 ⁇ 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 ⁇ -15′; 2 ⁇ 5′ each) and probed with the secondary antibody (antirabbit; 1:10,000 dilution) for 1 h on the orbital tilting shaker at room temperature.
  • the blots were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • 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 ⁇ -15′; 2 ⁇ 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 ⁇ -15′; 2 ⁇ 5′ each) and probed with the secondary antibody (antimouse; 1:10,000 dilution) for 1 h on the orbital tilting shaker at room temperature.
  • the blots were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • 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 ⁇ M or 100 ⁇ 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.
  • 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 ⁇ -15′; 2 ⁇ 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.
  • the blot was washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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 ⁇ -15′; 2 ⁇ 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.
  • 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 ⁇ -15′; 2 ⁇ 5′ each).
  • the blot was washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • 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 ⁇ -15′; 2 ⁇ 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 ⁇ -15′; 2 ⁇ 5′ each) and probed with the secondary antibody (antimouse; 1:10,000 dilution) for 1 h on the orbital tilting shaker at room temperature.
  • the blots were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • 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 ⁇ M and 100 ⁇ M) and harvested 48 hours of treatment.
  • the cells were additionally treated with either 5 mM glucose (“5G”) or 22 mM glucose (“22G”).
  • 5G 5 mM glucose
  • 22G 22 mM glucose
  • 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.
  • 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.
  • the cells used in this experiment were HepG2 cells that were treated or not with Coenzyme Q10 at two different concentrations, 50 or 100 ⁇ 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 are Lactate Dehydrogenase Levels in HepG2 Cells.
  • 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 ⁇ -15′; 2 ⁇ 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.
  • the blots were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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 ⁇ -15′; 2 ⁇ 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.
  • PLM2 Pyruvate Kinase Muscle form
  • 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 ⁇ -15′; 2 ⁇ 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.
  • the membranes were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 5′ each) and probed with the secondary antibody (antirabbit; 1:10,000 dilution) for 1 h on the orbital tilting shaker at room temperature.
  • the blots were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • 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 ⁇ -15′; 2 ⁇ 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 ⁇ -15′; 2 ⁇ 5′ each) and probed with the secondary antibody (antimouse; 1:10,000 dilution) for 1 h on the orbital tilting shaker at room temperature.
  • the blots were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • 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 ⁇ 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.
  • the levels of LDH and PDH were determined by probing the blots successively with primary antibodies for LDH and PDH, essentially as described above.
  • 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 ⁇ -15′; 2 ⁇ 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 ⁇ -15′; 2 ⁇ 5′ each) and probed with the secondary antibody (antimouse; 1:10,000 dilution) for 1 h on the orbital tilting shaker at room temperature.
  • the blots were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • 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.
  • 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.
  • 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 ⁇ M or 100 ⁇ M, and harvested after 24 or 48 hours of treatment.
  • HASMC samples were processed and the gels were run, transferred, stained and scanned essentially as described above.
  • the levels of actin were determined by probing the blots with a primary antibody for actin, essentially as described above.
  • 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 ⁇ -15′; 2 ⁇ 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 ⁇ -15′; 2 ⁇ 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.
  • PDHB antimouse Hif 1a and Caspase 3 antirabbit; 1:10,000 dilution
  • the blots were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • 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 ⁇ -15′; 2 ⁇ 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 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 ⁇ -15′; 2 ⁇ 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.
  • the blots were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • 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 ⁇ -15′; 2 ⁇ 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.
  • the membranes were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • the blots were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • the cells used were HepG2 cells that were treated or not with Coenzyme Q10 at two different concentrations, 50 ⁇ M or 100 ⁇ 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.
  • the levels of actin were determined by probing the blots with a primary antibody for actin, essentially as described above.
  • 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 ⁇ -15′; 2 ⁇ 5′ each) and probed with the primary antibody for Caspase 3 or MMP-6 in 5% BSA by incubation overnight at 4 deg C.
  • 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 ⁇ -15′; 2 ⁇ 5′ each) and probed with the secondary antibody (MMP-6 antimouse; Caspase 3 antirabbit; 1:10,000 dilution) for 1 h at room temperature.
  • the blots were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • 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 ⁇ -15′; 2 ⁇ 5′ each) and probed with the primary antibody for LDH in 5% BSA or 5% milk by incubation overnight at 4 deg C.
  • 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.
  • the membranes were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 5′ each) and probed with the secondary antibody (Jackson Immuno Research antigoat; 1:10,000 dilution; 305-055-045) for 1 h.
  • the blots were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • 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 ⁇ -15′; 2 ⁇ 5′ each) and probed with the primary antibody for Transaldolase or Hifla in 5% BSA by incubation overnight at 4 deg C.
  • 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.
  • the membranes were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • the blots were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • 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 ⁇ -15′; 2 ⁇ 5′ each) and probed with the primary antibody for IGFBP3 or TP53 in 5% BSA by incubation overnight at 4 deg C.
  • 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 ⁇ -15′; 2 ⁇ 5′ each) and probed with the secondary antibody (antirabbit; 1:10,000 dilution) for 1 h on the orbital tilting shaker at room temperature.
  • the blots were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • 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 ⁇ -15′; 2 ⁇ 5′ each) and probed with the primary antibody for Transaldolase or PDHB in 5% BSA by incubation overnight at 4 deg C.
  • 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.
  • the membranes were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • the blots were washed 3 times with TBS-T (1 ⁇ -15′; 2 ⁇ 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.
  • Isocitrate Dehydrogenase-1 Isocitrate Dehydrogenase-1 (IDH-1)
  • Isocitrate dehydrogenase is one of the enzymes that is part of the TCA cycle that usually occurs within the mitochondrial matrix.
  • IDH1 is the cytosolic form of the enzyme that catalyzes the oxidative decarboxylation of isocitrate to ⁇ -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).
  • ⁇ -ketoglutarate ( ⁇ -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.
  • ATP citrate Lyase is a homotetramer ( ⁇ 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).
  • Acetyl CoA generated from citrate by cytosolic ACL serves as a source for biosynthesis of new lipids and cholesterol during cell division.
  • 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.
  • PAM2 Pyruvate Kinase M2
  • 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.
  • 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.
  • LDH Lactate Dehydrogenase
  • LDH is an enzyme that catalyzes the interconversion of pyruvate and lactate with the simultaneous interconversion of NADH and NAD + . 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.
  • 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
  • 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
  • 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 beta (PDH-E1) is the first enzyme component that is part of the pyruvate dehydrogenase complex (PDC) that converts pyruvate to acetyl CoA.
  • PDC pyruvate dehydrogenase complex
  • 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.
  • 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.
  • 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).
  • the levels of effectors caspase-3 protein were measured in the cancer cell lines and in normal cell lines in response to Coenzyme Q10.
  • 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.
  • the expression of Caspase-3 in normal cells was moderately decreased in normal cells. The results are summarized in the tables below.
  • SDH Succinate Dehydrogenase
  • 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.
  • 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.
  • 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).
  • Hypoxia inducible factor 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.
  • OCR Oxygen Consumption Rates
  • ECAR Extracellular Acidification
  • 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.
  • stressors e.g., hyperglycemia, hypoxia, lactic acid
  • 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:
  • 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.
  • 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.
  • ECM extracellular matrix
  • stroma structural framework
  • fibroblasts are known to serve as tissue ambassadors of numerous functions such as wound healing and localized immunomodulation.
  • 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.
  • the HASMC human aortic smooth muscle cell
  • 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.
  • ATP adenosine triphosphate
  • 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.
  • normal smooth muscle cells can use a combination of mitochondrial OXPHOS and glycolysis to meet their energy requirements under normal physiological environment.
  • 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.
  • 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.
  • CoQ10 exposure is associated with changes in OCR rates in normal cells towards a physiological state that is native to a particular cell.
  • Table 51 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.
  • 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.
  • cancer cells e.g., MCF-7, PaCa-2
  • 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 ).
  • Table 53 describes the ECAR values in PaCa-2 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.
  • OCR and ECAR values 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.
  • HAEC normal human aortic endothelial cells
  • MCF-7 breast cancer
  • HepG2 liver cancer
  • highly metastatic PC-3 prostate cancer
  • Skmel-28 melanoma cells were cultured in DMEM/F12 supplemented with 5% Fetal Bovine Serum (FBS) and 1 ⁇ 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.
  • FBS Fetal Bovine Serum
  • 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.
  • 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.
  • 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 ⁇ M, 25 ⁇ M, and 100 ⁇ M. Initial studies added L-Phenylalanine to DMEM/F12 medium which contained a concentration of 0.4 M phenylalanine. For the 5 ⁇ M, 25 ⁇ M, and 100 ⁇ M the final concentration of the L-Phenylalanine in the medium was 0.405 M, 0.425 M, and 0.500 M, respectively.
  • 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.
  • D-Phenylalanine a chemically synthetic form of the bioactive L-Phenylalanine, was tested for comparison to L-phenylalanine.
  • concentrations 5 ⁇ M, 25 ⁇ M, and 100 ⁇ M of D-Phenylalanine
  • Bcl-2 expression after 6 hours of incubation.
  • 5 ⁇ M and 25 ⁇ M there was a significant reduction after 3 hours of incubation.
  • 5 ⁇ M and 100 ⁇ M concentrations a significant increase in Caspase-3 expression was observed after 6 hours of incubation.
  • DL-Phenylalanine was also tested for comparison to L-Phenylalanine. Again, concentrations of 5 ⁇ M, 25 ⁇ M, and 100 ⁇ 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.
  • 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 ⁇ 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 ⁇ M, and 15 ⁇ M. A statistically significant increase in Caspase-3 was observed for the 500 nM concentration after 12 hours of incubation.
  • 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 ⁇ M were chosen for the western blot analysis. The concentrations tested were 1 ⁇ m, 5 ⁇ M, and 15 ⁇ M. D-Tyrosine showed a reduction in Bcl-2 expression for the 5 ⁇ M and 15 ⁇ M concentrations for 12 and 24 hour time periods. Caspase-3 was significantly increased for the concentration of 5 ⁇ M for 3, 12 and 24 time periods. Also there was an increase in Caspase-3 expression for the 1 ⁇ M for 12 and 24 hour time period. In addition there is an increase in Caspase-3 expression for 5 ⁇ M for the 12 hour time period.
  • DL-Tyrosine a synthetic form of L-Tyrosine
  • Bcl-2 expression There is a statistical decrease in Bcl-2 expression seen in the 1 ⁇ M and 15 ⁇ M concentrations after 12 hours incubation and for the 5 ⁇ M after 24 hour of incubation.
  • An increase in Caspase-3 expression was also observed for the 5 ⁇ M and 15 ⁇ M after 12 hours of incubation.
  • 4-Hydroxy-phenylpyruvate is derived from Tyrosine and Phenylalanine amino acids and may play a role in the synthesis of the ring structure.
  • concentration of 1 ⁇ M, 5 ⁇ M, and 15 ⁇ M were tested for Bcl-2 and Caspase-3 expression.
  • 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.
  • 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 ⁇ M, 5 ⁇ M, and 15 ⁇ M.
  • An increase in Caspase-3 expression was observed for the concentration of 5 ⁇ M and 15 ⁇ M after 12 hours and 24 hours of incubation.
  • VMA is an additional component for the synthesis of the CoQ10 quinone ring structure.
  • concentrations tested were 100 nM, 250 nM, 500 nM, 1 ⁇ M, 25 ⁇ M, 50 ⁇ M, and 100 ⁇ M. Though no statistically significant apoptotic effect was observed in this experiment, the data indicated a downward trend of Bcl-2 expression.
  • Vanillic is a precursor for the synthesis of the quinone ring and was tested at a concentration of 500 nm, 5 ⁇ M, and 15 ⁇ 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 ⁇ M at the 24 hour incubation time point. For the 15 ⁇ M concentration there is a reduction in Bcl-2 expression after 3 hours of incubation. For the cells incubated with 15 ⁇ M for 24 hours there was a significant increase in Caspase-3 expression.
  • 4-Hydroxybenzoate acid plays a role in the attachment of the isoprenoid side chain to the ring structure.
  • concentrations tested were 500 nM, 1 ⁇ M, and 50 ⁇ M. There was a significant reduction in Bcl-2 expression for the 15 ⁇ M concentration after 24 hours of incubation.
  • Pyridoxine is another precursor building block for the synthesis of the quinone ring structure of CoQ10. The concentrations tested for this compound are 5 ⁇ M, 25 ⁇ M, and 100 ⁇ 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.
  • Panthenol plays a role in the synthesis of the quinone ring structure of CoQ10.
  • concentrations tested on melanoma cells were 5 ⁇ M, 25 ⁇ M, and 100 ⁇ M. This compound showed a significant reduction in Bc1-2 expression for the 25 ⁇ M concentration.
  • Mevalonic Acid is one of the main components for the synthesis of CoQ10. This compound was tested at the concentrations of 500 nM, 1 ⁇ M, 25 ⁇ m, and 50 ⁇ M. There was no significant reduction in Bcl-2 expression or an increase in Caspase-3 expression in this experiment.
  • 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 ⁇ M, 25 ⁇ M, and 100 ⁇ M.
  • the testing of Acetylglycine showed significant decrease in Bcl-2 expression after 12 hours of incubation for the concentration of 5 ⁇ M and 25 ⁇ M. A significant decrease in Bcl-2 was recorded for the 100 ⁇ M concentration at the 24 hour incubation time point.
  • Acetyl-CoA is a precursor for the mevalonic pathway for the synthesis of CoQ10.
  • concentrations tested were 500 nm, 1 ⁇ M, 25 ⁇ M, and 50 ⁇ M. There was no significant observed reduction in Bcl-2 or increase in Caspase-3 expression for the time points and concentrations tested.
  • 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.
  • L-Tyrosine was examined in medium without the addition of L-Phenylalanine and L-Tyrosine.
  • the final concentrations of L-Tyrosine tested were 500 nM, 5 ⁇ M, and 15 ⁇ M.
  • Farnesyl was tested at a concentration of 50 ⁇ M. There was no observed significant response for the 3 and 6 hour time points.
  • 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 ⁇ M, 25 ⁇ M, and 100 ⁇ M.
  • Farnesyl was added at a concentration of 50 ⁇ M. This study showed a decrease in Bc1-2 expression for most of the concentrations and combinations tested as depicted in the table below.
  • 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.
  • a T-test was performed with p ⁇ 0.05 as statistically significant.
  • An X signifies a statistical decrease in cell number.
  • a cell proliferation assay was also performed on neonatal fibroblast cells.
  • the concentrations of HB tested were 500 nM, 5 ⁇ M, and 25 ⁇ M.
  • HB was also tested in combination with benzoquinone at a concentrations of 25 ⁇ M, 50 ⁇ M, and 100 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HK-2 and HASMC 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.
  • 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.
  • ESRD end-stage renal disease
  • the Diabetes PCR array (SABiosciences) offers a screen for 84 genes simultaneously.
  • the 4 treatments tested in this study were:
  • CEACAM1 Carcino Embryonic Antigen Cell Adhesion Molecule 1
  • HK2(H) cells particularly with 100 ⁇ 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.
  • insulin receptor (INSR) expression was also altered in diabetic HK-2 cells treated with Coenzyme Q10.
  • INSR insulin receptor
  • 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.
  • the Diabetes PCR array (SABiosciences) offers a screen for 84 genes simultaneously.
  • the 4 treatments tested in this study were:
  • 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).
  • AGT vascular function
  • CEACAM1, INSR, SELL insulin sensitivity
  • IL-6 inflammation/immune function
  • 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
  • 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 ⁇ -cells and neuroendocrine cells.
  • CCL5 chemokine(C-Cmotif) ligand
  • 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.

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