Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/4741—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having oxygen as a ring hetero atom, e.g. tubocuraran derivatives, noscapine, bicuculline
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives

Definitions

• the present invention relates to methods and compositions for treating metabolic and cardiovascular disorders such as hyperlipidemia, obesity, diabetes, insulin resistance, glucose intolerance, hyperglycemia, metabolic syndrome and hypertension, as well as conditions or complications associated with these metabolic and cardiovascular disorders in mammals.
• metabolic and cardiovascular disorders such as hyperlipidemia, obesity, diabetes, insulin resistance, glucose intolerance, hyperglycemia, metabolic syndrome and hypertension, as well as conditions or complications associated with these metabolic and cardiovascular disorders in mammals.
• Metabolic disorders particularly glucose and lipid regulatory disorders
• diabetes is caused by a combination of insulin resistance and defective secretion of insulin by pancreatic- ⁇ cells.
• Individuals with insulin resistance often have abdominal obesity, dyslipidemia, hypertension, glucose intolerance and a prothrombitic state. This group of symptoms is commonly termed "Metabolic syndrome.”
• obese individuals as a whole are at higher risk for acquiring insulin resistance.
• the breakdown of a metabolic pathway thus can trigger myriad disorders such as hyperlipidemia, obesity, diabetes, insulin resistance, glucose intolerance, hyperglycemia, metabolic syndrome and hypertension which may in turn trigger further metabolic dysfunction putting individuals at risk for additional complications and premature morbidity.
• Metabolic disorders that effect glucose and lipid metabolism such as hyperlipidemia, obesity, diabetes, insulin resistance, hyperglycemia, glucose intolerance, metabolic syndrome and hypertension have long term health consequences leading to chronic conditions including cardiovascular disease and premature morbidity.
• Such metabolic and cardiovascular disorders may be interrelated, aggravating or triggering each other and generating feedback mechanisms that are difficult to interrupt.
• the invention achieves these objects and satisfies additional objects and advantages by providing novel and surprisingly effective methods and compositions for treating and/or preventing metabolic and cardiovascular disorders including, but not limited to, metabolic syndrome, hyperlipidemia, hypercholesterolemia, obesity, diabetes, insulin resistance, hyperglycemia, glucose intolerance and hypertension in mammalian subjects employing berberine and related compounds, derivatives, metabolites, prodrugs, and proto-berberine compounds and derivatives according to formula I, below.
• each of Rl, R2, R3, R4, R8, R9, RlO, Rl 1, R12 and/or R13 may independently, collectively, or in any combination that yields an active (e.g., anti-dyslipidemic, anti- hyperlipidemic, anti-hyperglycemic, anti-hypertensive, LDL-modulatory, LDLR stability increasing, LDLR-modulatory, AMP-activated protein kinase modulatory or insulin receptor (InsR) modulatory) compound according to this disclosure, be a hydrogen, halogen, hydroxy, alkyl, alkoxy for example methoxy, nitro, amino, trifiuoromethyl, cycloalkyl, (cycloalkyl)alkyl, alkanoyl, alkanoyloxy, aryl, aroyl, aralkyl, nitrile, dialkylamino, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl
• Ri, R 4 , R 8 , Ri i, Ri 2 and Rj 3 are hydrogen and R 2 , R 3j R 9 and R] 0 are independently selected from hydrogen, hydroxy, methyl or methoxy.
• R 2 and R 3 may together form a methylenedioxy group.
• the R group may be selected from any of the stated groups so as to be the same or different.
• two or more R groups may be linked, for example to form methylenedioxy.
• Exemplary candidate compounds in this context include compounds of Formula I, wherein: R 1 is selected from methyl, ethyl, hydroxyl, or methoxy; R 2 is selected from H, methyl, ethyl, methene; R 3 is selected from H, methyl, ethyl, methene; R 4 is selected from methyl, ethyl, hydroxyl, or methoxy; R 8 is selected from straight or branched (Cl -C6) alkyl (e.g., substitution selected from methyl, ethyl, n-propyl, 1 -methylethyl, n-butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, n-pentyl, 2-methylbutyl, 1,1- dimethylpropyl, 2,2 dimethylpropyl, 3-methylbutyl, n-hexyl, 1 -methylpentyl, 1,1-dimethylbutyl, 2,2-di
• each of Rj, R 4 R 8 R 9 R 10 , Rn, Ra and/or R !3 may independently, collectively, or in any combination that yields an active (e.g., anti-dyslipidemic, anti-hyperlipidemic, anti- hyperglycemic, anti-cholesterolemic, anti-hypertensive, LDL-modulatory, LDLR-modulatory, LDLR stabilizing, AMP-activated protein kinase modulatory or insulin receptor (InsR) modulatory) compound according to this disclosure, be a hydrogen, halogen, hydroxy, alkyl, alkoxy, nitro, amino, acetyl, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, formyl, alkanoyl, alkanoyloxy, aryl, aroyl, aralkyl, nitrile, dialkylamino, alkenyl, alkynyl,
• R group When more than one R group is present, the R group may be selected from any of the stated groups so as to be the same or different.
• Ri, R 4, R 8> R 9, R] 0 , Ri i, R) 2 and R 13 are each hydrogen.
• Useful berberine compounds, berberine-related compounds, berberine salts, berberine derivatives, berberine prodrugs, berberine metabolites, and proto-berberine compounds, salts, prodrugs, metabolites and derivatives, of any of the exemplary Formulae herein, within the formulations and methods of the invention include, but are not limited to, salts of berberine and related or derivative compounds, for example, berberine sulfate, berberine hydrochloride, berberine chloride, palmatine chloride, palmatine, oxyberberine, dihydroberberine, 8- cyanodihydroberberine, (-)-canadine, tetrahydroberberine N-oxide, tetrahydroberberine, N- methyltetrahydroberberinium iodide, 6-protoberberine, 9-ethoxycarbonyl berberine, 9-N,N
• berberine may be demethylated.
• demethylated beberine may be further reacted to form acetyl, terbutyl formyl, tertiarybutylformylacetyl, 3- phenylacryloyl, chloracetyl or chloroacetylpropanyl forms of berberine.
• Other useful forms of derivatives for use within the invention include other pharmaceutically acceptable active salts of said compounds, as well as active isomers, enantiomers, polymorphs, glycosylated derivatives, solvates, hydrates, and/or prodrugs of said compounds.
• compositions for use within the invention use metabolites of berberine of the formulas VI to IX, below, as well as pharmaceutically acceptable active salts of said compounds, active isomers, enantiomers, polymorphs, glycosylated derivatives, solvates, hydrates, and/or prodrugs of said compounds.
• Useful berberine and related compounds, metabolites, prodrugs, and derivatives within the formulations and methods of the invention include, but are not limited to, berberrubine, thalifendine, demethyleneberberine, jatorrhizine, as well as salts of berberine, other berberine metabolites, related or derivative compounds, for example, berberine sulfate, berberine hydrochloride, berberine chloride, palmatine chloride, palmatine, oxyberberine, dihydroberberine, 8-cyanodihydroberberine, (-)-canadine, tetrahydroberberine N-oxide, tetrahydroberberine, N-methyltetrahydroberberinium iodide, 6-protoberberine, 9-ethoxycarbonyl berberine, 9-N,N-dimethylcarbamoyl berberine
• compositions and methods of the invention employ a berberine compound or a berberine related or derivative compound of Formula I or Formula XXVII to treat and/or prevent symptoms of metabolic and cardiovascular disorders or another disease or condition associated with metabolic disorders, such as a cardiovascular disease.
• Mammalian subjects amenable for treatment with berberine compounds, berberine- related compounds, berberine salts, berberine derivatives, berberine prodrugs, berberine metabolites, and proto-berberine compounds, salts, prodrugs, metabolites and derivatives, of any of the exemplary Formulae herein (collectively referred to herein as the "active compound"), according to the methods of the invention include, but are not limited to, subjects with hyperlipidemia and subjects with elevated cholesterol and/or elevated triglycerides, including subjects presenting with, or at elevated risk for developing, elevated LDL, elevated cholesterol, and/or elevated triglyceride levels; subjects with hyperglycemia; subjects with elevated blood glucose levels; subjects with diabetes; subjects with insulin resistance; subjects with elevated blood pressure; subjects with obesity; subjects with decreased insulin sensitivity; subjects in a prothrombotic state; subjects in a proinflammatory state.
• subjects with hyperlipidemia and subjects with elevated cholesterol and/or elevated triglycerides including subjects
• a metabolic correcting effective amount or, alternatively, an anti- dyslipidemic, anti-hyperlipidemic, anti-cholesterolemic, anti-hyperglycemic, anti-hypertensive, LDL-modulatory, LDLR-modulatory, LDLR stabilizing, insulin receptor (InsR) modulatory, AMP-activated protein kinase modulatory effective amount
• insulin receptor (InsR) modulatory AMP-activated protein kinase modulatory effective amount
• active compounds including any of the herein described berberine compounds, berberine-related compounds, berberine salts, berberine derivatives, berberine prodrugs, berberine metabolites, and proto-berberine compounds, salts, prodrugs, metabolites and derivatives, including but not limited to active comounds of any of the exemplary Formulae herein, sufficient to prevent or reduce metabolic and cardiovascular disorders including metabolic syndrome, hyperlipidemia, obesity, diabetes,
• the therapeutically useful methods and formulations of the invention will effectively use a subject compound as noted above, including any active, pharmaceutically acceptable salt of said compounds, as well as active isomers, metabolites, enantiomers, polymorphs, solvates, hydrates, prodrugs, and/or combinations thereof.
• Berberine is employed as an illustrative embodiment of the invention within the examples herein below.
• mammalian subjects are effectively treated, prophylactically and/or therapeutically, by administering to the subject a cholesterol-controlling effective amount of any of the berberine compounds, berberine-related compounds, berberine salts, berberine derivatives, berberine prodrugs, berberine metabolites, and proto-berberine compounds, salts, prodrugs, metabolites and derivatives, including a compound of any of the exemplary Formulae herein, sufficient to prevent or reduce elevated cholesterol, or one or more associated symptoms or condition(s), in the subject.
• therapeutically useful methods and formulations of the invention may likewise employ berberine, a berberine related compound, metabolite, prodrug, or derivative compound, i.e., any of the herein described "active compounds” (including any of these compunds' pharmaceutically acceptable salts, isomers, enantiomers, polymorphs, solvates, hydrates, prodrugs, and/or combinations thereof).
• active compounds including any of these compunds' pharmaceutically acceptable salts, isomers, enantiomers, polymorphs, solvates, hydrates, prodrugs, and/or combinations thereof.
• combinatorial formulations and methods employ an effective amount of any of the active berberine compounds (i.e., any of the subject berberine compounds, berberine-related compounds, berberine salts, berberine derivatives, berberine prodrugs, berberine metabolites, and proto-berberine compounds, salts, prodrugs, metabolites and derivatives, including a compound of any active compound of any of the exemplary Formulae described herein) in combination with one or more secondary or adjunctive active agent(s) that is/are combinatorially formulated or coordinately administered with the subject compound to yield lipid lowering and/or glucose lowering effective response (or, alternatively, an anti-dyslipidemic, anti-hyperlipidemic, anti-hypercholesterolemic, anti- hyperglycemic, anti-metabolic syndrome, insulin sensitivity increasing, insulin resistance decreasing, anti-diabetic, anti-obesity, anti-hypertens
• active berberine compounds i.e.
• Exemplary combinatorial formulations and coordinate treatment methods in this context employ the subject compound in combination with one or more additional, lipid and/or glucose lowering agent(s) or other indicated, secondary or adjunctive therapeutic agents.
• the secondary or adjunctive therapeutic agents used in combination with, e.g., berberine in these embodiments may possess direct or indirect lipid and/or glucose lowering activity and/or hypertension decreasing activity, including cholesterol lowering activity, insulin resistance decreasing activity, insulin sensitivity increasing activity or glucose regulating activity, alone or in combination with, e.g., berberine, or may exhibit other useful adjunctive therapeutic activity in combination with, e.g., berberine.
• Useful adjunctive therapeutic agents in these combinatorial formulations and coordinate treatment methods include, for example, anti-hyperlipidemic agents; anti-dyslipidemic agents; plasma HDL-raising agents; anti-hypercholesterolemic agents, including, but not limited to, cholesterol-uptake inhibitors; cholesterol biosynthesis inhibitors, e.g., HMG-CoA reductase inhibitors (also referred to as statins, such as lovastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, pitavastatin, and atorvastatin); HMG-CoA synthase inhibitors; squalene epoxidase inhibitors or squalene synthetase inhibitors (also known as squalene synthase inhibitors); acyl- coenzyme A cholesterol acyltransferase (ACAT) inhibitors, including, but not limited to, melinamide; probucol; nico
• Such agents may be referred to in whole or in part as metabolic disorder therapeutics, metabolic syndrome therapeutics, anti-obesity therapeutics, anti- hypercholesterolemia therapeutics, anti-diabetic therapeutics, insulin resistance therapeutic agents, anti-hyperglycemia agents, anti-hyperlipidemia agents, insulin sensitivity increasing agents, anti-hypertensive agents, and/or blood glucose lowering therapeutic agents.
• Adjunctive therapies may also be used including, but not limited, physical treatments such as changes in diet, psychological counseling, behavior modification, exercise and surgery including, but not limited to, gastric partitioning procedures, jejunoileal bypass, stomach stapling, gastric bands, vertical banded gastroplasty, laparoscopic gastric banding, roux-en-Y gastric bypass, biliopancreatic bypass procedures and vagotomy.
• Some herbal remedies may also be employed effectively in combinatorial formulations and coordinate therapies for treating metabolic disorders, for example curcumin, gugulipid, garlic, vitamin E, soy, soluble fiber, fish oil, green tea, carnitine, chromium, coenzyme QlO, anti-oxidant vitamins, grape seed extract, pantothine, red yeast rice, and royal jelly.
• metabolic disorders for example curcumin, gugulipid, garlic, vitamin E, soy, soluble fiber, fish oil, green tea, carnitine, chromium, coenzyme QlO, anti-oxidant vitamins, grape seed extract, pantothine, red yeast rice, and royal jelly.
• Figure 1 is a drawing of the promoter region of the LDL receptor gene. Three direct repeats and two TATA-like sequences are identified with the promoter region. The cis-acting element of sterols is located on repeat 2, whereas the regulatory element for cytokine OM (SIRE) overlaps the TATA-like sequences.
• SIRE cytokine OM
• Figure 2 is a schematic representation of intracellular regulation of LDL receptor gene expression, including regulation by berberine.
• Figures 3 A and B are quantitative RT-PCR of LDLR mRNA levels in human liver BEL- 7402 cells twenty-four hours after being treated with berberine (A) or berberine sulfate (B).
• Figure 4 is a measurement using flow cytometry of the concentration of the protein level of LDLR expressed on the cell surface of BEL-7402 cells twenty-four hours after treatment with
• BBR berberine
• Figures 5 A-C are charts of the decrease in serum cholesterol (A) and LDL (B) in hamsters after treatment with varying concentrations of berberine (BBR) and the decrease of
• Figure 6 is a depiction of the concentration of total LDLR mRNA and protein extracts as measured by quantitative real time RT-PCR (A) and Western blot (B) in hamsters sacrificed four hours after the last treatment with berberine.
• Figure 7 is a Western Blot showing the concentration of the precursor (P) and mature (M) forms of SREBP2 using a monoclonal antibody to SREBP2 in HepG2 cells.
• Figure 8 is (A) a northern blot showing LDLR expression in HepG2 cells treated with either lovastatin (Lov) alone or in combination with berberine (BBR) for 24 hours and (B) a chart of real-time RT-PCR of the same cells.
• Figure 9 is a chart showing the increase in LDLR promoter activity in the presence of Berberine (BBR), GW707 (GW), and oncostatin M (OM).
• BBR Berberine
• GW707 GW
• OM oncostatin M
• Figure 10 is (A) a northern blot showing concentrations of LDLR mRNA in HepG2 cells treated with berberine (BBR) in the presence of different lengths of time of actinomycin D treatment and (B) a plot of normalized LDLR mRNA signals as a percentage of LDLR mRNA remaining.
• Figure 11 is a schematic representation of the LDLR mRNA 3' UTR and the chimeric Luc-LDLR 3' UTR constructs.
• Figure 12 is a northern blot of analysis of Luc-LDLR fusion mRNA in (A) control cells and cells treated with (B) berberine or dimethylsulfoxide (C) as a control.
• Figure 13 is a schematic representation of the constructs containing the deletions of ARE and UCAU motifs (B) and a chart illustrating the responses of the wt pLuc/UTR-2 and deletion constructs to berberine treatment as determined by real-time RT-PCR analysis.
• Figure 14 is a western blot of cellular proteins harvested from (A) Bel-7402 cells or (B) HepG2 cells that were untreated or treated with berberine (BBR) at a dose of 5 ⁇ g/ml for different lengths of time as indicated and (C) a western blot of HepG2 cells treated for 1 hour at the indicated concentrations.
• BBR berberine
• Figure 15 (A) is a chart depicting a dose dependent increase in the expression of InsR mRNA in human hepatoma cells treated with berberine as measured using real time PCR and (B) confirmed by slot blot.
• Figure 16 (A) is a chart depicting the time-dependent effect of berberine on InsR mRNA expression in human hepatoma cells over 24 hours as confirmed by (B) slot blot.
• Figure 17 (A-F) are graphs depicting increased cell surface InsR expression in Caucasian liver cell line HepG2 when treated with (A) IgG, (B) O ⁇ g/ml of berberine, (C) 2.5 ⁇ g/ml of berberine, (D) 5 ⁇ g/ml of berberine, (E) 10 ⁇ g/ml of berberine, and (F) 15 ⁇ g/ml of berberine.
• Figure 18 (A-F) are graphs depicting increased cell surface InsR expression in Asian liver cell line Bel-7402 when treated with (A) IgG, (B) O ⁇ g/ml of berberine, (C) 2.5 ⁇ g/ml of berberine, (D) 5 ⁇ g/ml of berberine, (E) 10 ⁇ g/ml of berberine, and (F) 15 ⁇ g/ml of berberine.
• Figure 19 (A-B) are charts showing that (A) berberine increases glucose consumption in the presence of InsR expression and insulin and that (B) silencing InsR expression abolishes the glucose consumption effect.
• Figure 20 (A-D) are charts showing that treatment of human liver cells with 7.5 ⁇ g/ml of berberine increases the surface expression of both InsR and LDLR over time.
• Figure 21 are (A) a slot blot of the amount of InsR mRNA in HepG2 cells untreated (column C) or treated with berberine (column BBR) and then treated with actinomycin and normalized with ACTB and (B) a chart of the data plotted as a the percentage of the InsR mRNA remaining.
• Figure 22 is a chart showing the dose dependent increase of Luc mRNA in pG13-1.5kIRP transfected cells incubated with varying concentrations of berberine (BBR) for eight hours.
• Figure 23 is a chart showing RT-PCR measurements of the amount of InsR and LDLR mRNA in HepG2 cells treated with calphostin (CaI), berberine (BBR) or a combination of calphostin and berberine.
• Figure 24 is a chart showing the relative amounts of InsR and LDLR mRNA as measured by RT-PCR in HepG2 cells treated with UO 126 (U), berberine (BBR) or a combination of UO 126 and berberine.
• Figure 25 is a picture of a gel of phosphorylated and nonphosphorylated substrates in cell lysates of HepG2 cells treated with berberine (BBR) for 0, 0.25, 1, 2, and 4 hours and (B) a chart of the quantification of protein kinase C (PKC) activity using densitometry and expressed as the number of picomoles of phosphate transferred to the substrate per minute per milligram of sample protein.
• BBR berberine
• Figure 26 is a chart showing luciferase activity representing normalized InsR promoter activity in pGL3-1.5kIRP transfected HepG2 cells treated with calphostin (CaI), berberine (Ber), phorbol 12-myristate 13 -acetate (PMA) or combinations as shown.
• CaI calphostin
• Ber berberine
• PMA phorbol 12-myristate 13 -acetate
• Figure 27 is a graph of the decline in the fasting blood glucose of hyperglycemic rats treated with berberine (BBR).
• Figure 28 is a chart of liver InsR and LDLR mRNA of rats treated with berberine (BBR) as calculated by RT-PCR.
• Figure 29 is a chart of dose-dependent induction of InsR mRNA expression in HepG2 cells incubated with berberine for eight hours as measured by RT-PCR with the amount of InsR mRNA in untreated cells defined as "1" and the amounts of InsR mRNA from berberine (BBR) treated cells plotted relative to that value.
• Figure 30 (A) is a picture of a gel showing the phosphorylated and nonphosphorylated substrates in cell lysates of liver samples of rats treated with berberine (BBR) and (B) a chart of the quantification of PKC activity using densitometry and expressed as the number of picomoles of phosphate transferred to the substrate per minute per milligram of sample protein.
• Figure 31 is a chart showing the decrease in the level of fasting serum insulin in hyperglycemic rats on a high fat and high cholesterol (HFHC) diet when they were treated with berberine (BBR).
• Figure 32 is a chart of the increase in insulin sensitivity index (ISI) in hyperglycemic rats on a four week HFHC diet when treated with berberine (BBR).
• ISI insulin sensitivity index
• Figure 33 is a chart of the decrease in serum lipid levels in hyperlipidemic rats treated with varying concentrations of berberine (BBR).
• Figure 34 is a schematic of InsR and LDLR expression and their upregulation by berberine (BBR).
• Figure 35 is a chart of serum insulin levels in hyperglycemic patients as measured before and after two months of therapy with berberine.
• Figure 36 is a chart of InsR expression on the surface of peripheral blood lymphocytes
• PBL hyperglycemic patients as measured before and after two months of therapy with berberine.
• Figure 37 (A-H) are charts showing the negative correlation between InsR expression on the surface of peripheral blood lymphocytes and fasting blood glucose levels in eight patients 0,
• Figure 38 is a diagram of cholesterol synthesis by the body.
• Figure 39 is a chart depicting the effect of various amounts of berberine (BBR) and demethylberberine (LlOl) on LDLR mRNA levels in cells from the human hepatoma cell line
• Figure 40 is a chart depicting LDL changes in LDL-R mRNA levels in human hep G2 cells treated with berberine and berberine metabolites.
• Figure 41 is a chart depicting an increase in insulin receptor activity in human hepG2 cells treated with berberine and berberine metabolites.
• Figure 42 is a chart depicting the increase in the production of LDL receptors and INS receptors in cells treated with berberine and berberine metabolites.
• the instant invention provides novel methods and compositions for preventing and/or treating metabolic and cardiovascular disorders including but not limited to metabolic syndrome, hyperlipidemia, hypercholesterolemia, obesity, diabetes, insulin resistance, hyperglycemia, hypertension and elevated cholesterol in mammalian subjects, including individuals and in vitro, ex vivo, and in vivo mammalian cells, tissues, and organs.
• the methods and compositions are effective to prevent or treat diseases caused by metabolic and cardiovascular disorders including cardiovascular disease.
• cardiovascular disease is intended to include a range of symptoms, conditions, and/or diseases including atherosclerosis, coronary artery disease, pulmonary embolism, diabetic cardiomyopathy, angina pectoris, carotid artery disease, strokes, peripheral vascular disease, cerebral arteriosclerosis, myocardial infarction, high blood pressure, cerebral infarction, restenosis following balloon angioplasty, intermittent claudication, dyslipidemia post-prandial lipidemia and xanthoma, and all conventionally targeted symptoms arising from or associated with the foregoing diseases and conditions.
• diseases including atherosclerosis, coronary artery disease, pulmonary embolism, diabetic cardiomyopathy, angina pectoris, carotid artery disease, strokes, peripheral vascular disease, cerebral arteriosclerosis, myocardial infarction, high blood pressure, cerebral infarction, restenosis following balloon angioplasty, intermittent claudication, dyslipidemia post-prandial lipidemia and xanthoma, and all conventionally targeted symptoms
• Active compounds and formulations of the invention employ any of the "active compounds” described herein, selected from berberine compounds, berberine-related compounds, berberine salts, berberine derivatives, berberine prodrugs, berberine metabolites, and proto-berberine compounds, salts, prodrugs, metabolites and derivatives, of any of the exemplary Formulae herein, including glycosylated derivatives, demethylated derivatives, and all active pharmaceutically acceptable salts, solvates, isomers, enantiomers, polymorphs, metabolites and prodrugs of these compounds and combinations thereof.
• active compounds for use within any of the herein- described aspects, examples or embodiments of the invention may be selected from any of the active compounds represented within any of the distinct exemplary classes of compounds descrbed by Formulae I-XIII, XXVI-XXVIII, or XXXII-XXXVI, including any of the species within these distinct classes of active compounds.
• Active compounds of the invention can be employed as novel glucose or lipid lowering agents.
• Glucose lowering formulations and methods provided herein employ berberine, a berberine related compound, berberine metabolite, berberine prodrug, or berberine derivative compound of any of the Formulae herein, such as Formula I or Formula II, any active compound of formulae VI-IX , any active compound of Formulae I- V, XXVI-XXVIII, or XXXII-XXXVI, including any of the species within these classes of active compounds (encompassing all complexes, salts, solvates, isomers, enantiomers, polymorphs, metabolites and prodrugs of these compounds and combinations thereof) as novel glucose lowering agents.
• Insulin sensitivity increasing formulations and methods provided herein employ berberine, a berberine related compound, berberine metabolite, berberine prodrug, or berberine derivative compound of any of the Formulae herein, such as Formula I or Formula II, any active compound of formulae VI-IX , any active compound of Formulae I-V, XXVI-XXVIII, or XXXII-XXXVI, including any of the species within these classes of active compounds, and active combinations thereof, as novel insulin sensitivity increasing agents.
• Insulin resistance decreasing formulations and methods provided herein employ berberine, a berberine related compound, berberine metabolite, berberine prodrug, or berberine derivative compound of any of the Formulae herein, such as Formula I or Formula II, any active compound of formulae VI-FX , any active compound of Formulae I-V, XXVI-XXVIII, or XXXII-XXXVI, including any of the species within these classes of active compounds, and combinations thereof, as novel insulin resistance decreasing agents.
• Anti-obesity formulations and methods provided herein employ berberine, a berberine related compound, berberine metabolite, berberine prodrug, or berberine derivative compound of any of the Formulae herein, such as Formula I or Formula II, any active compound of formulae VI-IX , any active compound of Formulae I- V, XXVI-XXVIII, or XXXII-XXXVI, including any of the species within these classes of active compounds, and active combinations thereof, as novel anti-obesity agents.
• Anti-hypertensive formulations and methods provided herein employ berberine, a berberine related compound, berberine metabolite, berberine prodrug, or berberine derivative compound of any of the Formulae herein, such as Formula I or Formula II, any active compound of formulae VI-IX , any active compound of Formulae I-V, XXVI-XXVIII, or XXXII-XXXVI, including any of the species within these classes of active compounds, and active combinations thereof, as novel hypertension lowering agents.
• Metabolic syndrome treating formulations and methods provided herein employ berberine, a berberine related compound, berberine metabolite, berberine prodrug, or berberine derivative compound of any of the Formulae herein, such as Formula I or Formula II, any active compound of formulae VI-IX , any active compound of Formulae I-V, XXVI-XXVIII, or XXXII-XXXVI, including any of the species within these classes of active compounds, and active combinations thereof, as novel metabolic syndrome treating agents.
• berberine within the formulations and methods, berberine, a berberine related compound, berberine metabolite, berberine prodrug, or berberine derivative compound of any of the Formulae herein, such as Formula I or Formula II, any active compound of formulae VI-IX , any active compound of Formulae I-V, XXVI-XXVIII, or XXXII-XXXVI, including any of the species within these classes of active compounds, and active combinations thereof, as disclosed herein is effectively used to treat metabolic and cardiovascular disorders in mammalian subjects suffering metabolic and cardiovascular disorders and conditions associated with metabolic and cardiovascular disorders including but not limited to, fatty liver, reproductive abnormalities, growth abnormalities, arterial plaque accumulation, osteoarthritis, gout, joint pain, respiratory problems, skin conditions, sleep apnea, idiopathic intracranial hypertension, lower extremity venous stasis disease, gastro-esophageal reflux, urinary stress incontinence, kidney damage, cardiovascular diseases such as
• a broad range of mammalian subjects are amenable to treatment using the formulations and methods of the invention. These subjects include, but are not limited to, human and other mammalian subjects presenting with metabolic and cardiovascular disorders or diseases aggravated or triggered by metabolic and cardiovascular disorders such as fatty liver, reproductive abnormalities, growth abnormalities, arterial plaque accumulation, osteoarthritis, gout, joint pain, respiratory problems, liver dysfunction, skin conditions, sleep apnea, idiopathic intracranial hypertension, lower extremity venous stasis disease, gastro-esophageal reflux, urinary stress incontinence, kidney damage, cardiovascular diseases such as atherosclerosis, coronary artery disease, enlarged heart, peripheral vascular disease, diabetic cardiomyopathy, pulmonary embolism, angina pectoris, carotid artery disease, stroke, cerebral arteriosclerosis, myocardial infarction, cerebral infarction, restenosis following balloon angioplasty, intermittent claudication, dyslipidemia post-pran
• Human and mammalian subjects amenable to treatment using the formulations and methods of the invention further include those displaying symptoms of metabolic and cardiovascular diseases including, but not limited to, weight gain, elevated glucose levels, elevated lipid levels, frequent urination, increased thirst, dehydration, weight loss, blurred vision, fatigue, coma, fatty liver, reproductive abnormalities, growth abnormalities, arterial plaque accumulation, osteoarthritis, gout, joint pain, respiratory problems, skin conditions, sleep apnea, idiopathic intracranial hypertension, gastro-esophageal reflux, urinary stress incontinence, kidney damage, shortness of breath, chest pain, leg pain, tiredness, vision changes, blood in urine, nosebleeds, irregular heartbeat, loss of balance or coordination, weakness, or vertigo.
• metabolic and cardiovascular diseases including, but not limited to, weight gain, elevated glucose levels, elevated lipid levels, frequent urination, increased thirst, dehydration, weight loss, blurred vision, fatigue, coma, fatty liver, reproductive abnormalities, growth abnormalities
• berberine within the methods and compositions of the invention, berberine, a berberine related compound, berberine metabolite, berberine prodrug, or berberine derivative compound of any of the Formulae herein, such as Formula I or Formula II, any active compound of formulae VI-IX , any active compound of Formulae I- V, XXVI-XXVIII, or XXXII-XXXVI, including any of the species within these classes of active compounds, and active combinations thereof, as disclosed herein is/are effectively formulated or administered as an anti-hyperlipidemia or cholesterol lowering agent effective for treating hyperlipidemia and/or related disorders.
• berberine chloride is demonstrated for illustrative purposes to be an anti- hyperlipidemia effective agent in pharmaceutical formulations and therapeutic methods, alone or in combination with one or more adjunctive therapeutic agent(s).
• the present disclosure further provides additional, pharmaceutically acceptable berberine related and derivative compounds in the form of a native or synthetic compound, including complexes, derivatives, including glycosylated derivatives, salts, solvates, isomers, enantiomers, polymorphs, metabolites and prodrugs of the compounds disclosed herein, and combinations thereof, which are effective as lipid lowering therapeutic agents within the methods and compositions of the invention.
• a relatively constant level of cholesterol in the body is maintained primarily by controlling the level of de novo synthesis which is regulated in part by the dietary intake of cholesterol. Slightly less than half of the cholesterol in the body is synthesized de novo with about 20-25% of total daily production occurring in the liver. Other sites of synthesis include the intestines, adrenal glands and reproductive organs. Cholesterol synthesis occurs in the cytoplasm and microsomes through the conversion of acetyl CoA.
• acetyl-CoA utilized for cholesterol biosynthesis is derived from either an oxidation reaction (e.g., fatty acids or pyruvate) in the mitochondria and is transported to the cytoplasm, or derived from cytoplasmic oxidation of ethanol by acetyl-Co A synthetase.
• an oxidation reaction e.g., fatty acids or pyruvate
• cytoplasmic oxidation of ethanol acetyl-Co A synthetase.
• Hyperlipidemia is an abnormal increase in serum lipids in the bloodstream. It is generally classified as primary hyperlipidemia, which is caused by genetic defects; or secondary hyperlipidemia, which is caused by various disease states, drugs and/or dietary factors. Hyperlipidemia may also result from a combination of primary and secondary causes of hyperlipidemia. Deficiencies or failures of LDL regulatory mechanisms can result in hyperlipidemia.
• Hyperlipidemia is a known causal factor for development of atherosclerosis and other cardiovascular and peripheral vascular diseases.
• Primary hyperlipidemia is generally caused by genetic defects, and secondary hyperlipidemia generally caused by secondary factors such as disease, drugs and/or dietary factors. Hyperlipidemia can also result from a combination of primary and secondary causes.
• Primary hyperlipidemias include familial hyperchylomicronemia, familial hypercholesterolemia, familial combined hyperlipidemia, familial dysbetaliproteinemia, familial hypertriglyceridemia, and familial defective apolipoprotein B- 100.
• Familial hyperchylomicronemia is a genetic disorder which results in a deficiency in an enzyme, LP lipase, that breaks down fat molecules. The LP lipase deficiency can cause the accumulation of large quantities of fat or lipoproteins in the blood.
• Familial hypercholesterolemia is caused by one or more mutations in the LDL receptor gene that result(s) in a malfunctioning LDL receptor or even complete absence of the LDL receptor.
• Familial combined hyperlipidemia also known as multiple lipoprotein-type hyperlipidemia, can result in periodic elevation of cholesterol and triglyceride levels and a decrease in HDL levels.
• Familial defective apolipoprotein B-100 is an autosomal dominant genetic abnormality caused by a single nucleotide mutation that substitutes glutamine for arginine. This mutation leads to a reduced affinity of LDL particles for the LDL receptor, increasing plasma levels of LDL and total cholesterol.
• Familial dysbetalipoproteinemia also referred to as Type III hyperlipoproteinemia, results in moderate to severe elevations of serum triglyceride and cholesterol levels with abnormal apolipoprotein E function.
• familial hypertriglyceridemia the concentration of plasma VLDL is elevated. This can cause mild to moderately elevated triglyceride levels (and usually not elevated cholesterol levels) and can often be associated with low plasma HDL levels.
• Secondary hyperlipidemia can be triggered by diseases such as uncontrolled diabetes mellitus (insulin-dependent diabetes mellitus and non-insulin-dependent diabetes mellitus) (Bianchi, R., et al, Diab. Nutr. Metabl.
• Elevated lipoprotein levels can additionally contribute to the development of secondary hyperlipidemia. Elevated lipoprotein levels, regardless of cause, are associated with a number of disease states, including atherosclerosis, coronary artery disease, angina pectoris, carotid artery disease, stroke, cerebral arteriosclerosis, myocardial infarction, cerebral infarction, restenosis following balloon angioplasty, high blood pressure, intermittent claudication, dyslipidemia, post-prandial lipidemia and xanthoma.
• Obesity is defined as having a body weight that is 20 to 25 percent over the recommended body weight, taking into account a person's particular age, height, and sex.
• Obesity is a well- established risk factor for a number of potentially life-threatening diseases such as coronary heart disease, osteoarthritis, gout, atherosclerosis, joint pain, sexual and fertility problems, respiratory problems, skin conditions, hypertension, diabetes, stroke, pulmonary embolism, sleep apnea, idiopathic intracranial hypertension, lower extremity venous stasis disease, gastro-esophageal reflux, urinary stress incontinence, and cancer. It also complicates numerous chronic conditions such as respiratory disease, osteoarthritis, osteoporosis, gall bladder disease, and dyslipidemias.
• the compositions and methods of the present invention are effective in the treatment of all types of hyperlipidemia, regardless of cause.
• liver dysfunction One cause of hyperlipidemia is liver dysfunction. In normal humans, when dietary cholesterol is increased, de novo synthesis of cholesterol decreases. However in cases of liver dysfunction, this mechanism fails and cholesterol synthesis continues, increasing cholesterol levels in the body and leading to hyperlipidemia. Liver dysfunction can result from genetic conditions, inflammatory disorders, toxins such as drugs and alcohol, diseases such as hepatitis, immunological disorders, vascular disorders or metabolic conditions. Regardless of the cause, liver damage can have a systemic effect on the function of metabolic processes and the regulation of blood glucose and serum lipid levels, exacerbating chronic disease states and leading to increased risks for further disease and morbidity. [0084] Certain types of adverse diets also interfere with hepatic control of cholesterol synthesis.
• LDL concentrations in plasma are regulated in part by the LDL receptor which captures LDL particles from the bloodstream and draws them inside the cell, clearing them from the bloodstream when there is too much and releasing them when more LDL is needed.
• Transcriptional regulation of the LDL receptor gene is controlled through the sterol regulatory element-binding protein pathway (SREBP).
• Bile acid sequestrants, cholesterol biosynthesis inhibitors, and cholesterol absorption inhibitors all influence the SREBP pathway and subsequently upregulate LDL receptor expression.
• the statins competitively inhibit 3-hydroxy- 3-methyl-glutaryl-CoA reductase (HMG-CoA reductase) and block cholesterol biosynthesis in the liver.
• Hormones, cytokines, growth factors and second messengers also regulate transcription of the LDL receptor gene as outlined in Table 1, below. Post-transcriptional control of the LDL receptor gene is also a target for pharmaceutical intervention. It has been determined in the present invention that berberine is capable of upregulating LDL receptor expression through a post-transcriptional and sterol independent mechanism in hepatocytes ( Figure 2).
• EBP CCAAT/enhancer binding protein
• Egrl early growth response gene 1
• ERK extracellular signal-regulated kinase
• IL interleukin
• LDL low density lipoprotein
• OM oncostatin M
• PKC protein kinase C
• PMA phorbol-12-myristate- 13 -acetate
• SIRE sterol- independent regulatory element
• SRE sterol regulatory element
• SREBP sterol regulatory element-binding protein
• TNF tumor necrosis factor
• UTR untranslated region.
• each of the forgoing agents identified in Table 1 that possess activity for regulating LDL receptor expression are useful in combination with the berberine, berberine related compound, berberine metabolite, berberine prodrug, or berberine derivative compound of any of the Formulae herein, such as Formula I or Formula II, any active compound of formulae VI-IX , any active compound of Formulae I- V, XXVI-XXVIII, or XXXII-XXXVI, including any of the species within these classes of active compounds, and active combinations thereof, in various combinatorial formulations and coordinate administration methods as described in detail below.
• active compounds of the invention exercise post- transcriptional control of the LDL receptor as illustrated in Figure 2.
• the active compound simultaneously elevates InsR expression through the PKC system as illustrated in Figure 34.
• one or more of the "active compound(s) i.e., berberine, a berberine related compound, berberine metabolite, berberine prodrug, or berberine derivative compound of any of the Formulae herein, such as Formula I or Formula II, any active compound of formulae VI-IX , any active compound of Formulae I-V, XXVI-XXVIII, or XXXII-XXXVI, including any of the species within these classes of active compounds, and active combinations thereof) is/are additionally effectively formulated or administered as a glucose lowering, insulin resistance decreasing and/or insulin sensitivity increasing compound effective for treating hyperglycemia and/or related disorders.
• berberine chloride is demonstrated for illustrative purposes to be a glucose lowering effective agent in pharmaceutical formulations and therapeutic methods, alone or in combination with one or more adjunctive therapeutic agent(s).
• the present disclosure further provides additional, berberine related and derivative compounds, in the form of a native or synthetic compound, including complexes, derivatives, including glycosylated derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of the compounds disclosed herein, and combinations thereof, which are effective as glucose lowering therapeutic agents within the methods and compositions of the invention.
• PKC inhibitor calphostin C eliminated the stimulating effect of berberine on the promoter of the InsR gene and InsR mRNA transcription, indicating that PKC is required for the effect of berberine on InsR gene transcription.
• the examples further demonstrate that PKC is a part of the activation mechanism for the InsR gene promoter.
• the 18% reduction of serum triglyceride in these patients reflects at least partially an improved glycogen synthesis from the glucose pool. Since berberine also lowers serum lipids that influence sugar metabolism, the reduction of glucose and triglycerides in the circulation represents a synergistic effect of berberine on the activation of both InsR and LDLR expression. As illustrated in figure 34, berberine increases the LDLR expression through activation of ERK pathway in lever cells, and also elevates InsR expression through the PKC system. The two signal pathways closely collaborate in producing a full cellular response against lipid/glucose related metabolic disorders. In the study described in the examples below, 50% of the type II diabetes patients in the berberine treatment group also had hyperlipidemia.
• Essential hypertension is the clinical expression of a disordered interaction between the genetic, physiological, and biochemical systems that usually maintain cardiovascular homeostasis. The multifactorial nature of essential hypertension has made it difficult to completely isolate the action of any one of these systems from the actions of the others. The relation between insulin metabolism/resistance and essential hypertension has the potential to provide insight into the mechanisms that operate this complex interaction. (DeFronzo, Diabetes Care 14: 173-194 (1991). Insulin increases renal sodium retention while increasing free water clearance.
• Insulin resistance is associated with increased sympathetic nervous system activity and stimulation of vascular smooth muscle growth. Additionally, insulin levels have been found to be significantly higher in adult patients with essential hypertension and borderline hypertension than in normotensive control patients. This is true regardless of the technique used to measure insulin and glucose level and independent of age, sex, and ethnic group. Numerous studies have confirmed the association between weight gain, percent body fat, and insulin resistance. However, there has also been found to be an interaction between insulin and hypertension that is independent of obesity. (Steinberger, Circulation. 107:1448 (2003). Treatment with berberine and berberine related and derivative compounds of Formula XXVII of the present invention is effective in reducing hypertension, regardless of the cause.
• Metabolic syndrome also known as syndrome X, dysmetabolic syndrome, obesity syndrome, insulin resistance syndrome and Reaven's syndrome, is a collection of risk factors estimated to effect over 50 million Americans. While there are no well-accepted criteria for diagnosing metabolic syndrome, it is generally characterized by abdominal obesity, atherogenic dislipidemia, elevated blood pressure, insulin resistance or glucose intolerance, prothrombotic state and a proinflammatory state. People with metabolic syndrome are at increased risk of coronary heart disease, stroke, peripheral vascular diseases, fatty liver, skin lesions, reproductive abnormalities, growth abnormalities, type II diabetes, and accelerated atherosclerosis as well as other diseases related to the buildup of arterial plaques formed by lipoproteins.
• Berberine is a quaternary alkaloid widely distributed in nine plant families of the structure of the compound of the following Formula II.
• Berberine can be found in Hydrastis canadensis (goldenseal), Coptis chinensis (Coptis, goldenthread, also known as the Chinese herb Huanglian), Berberis aquifolium (Oregon grape), Berberis vulgaris (barberry), Berberis aristata (tree turmeric), Chinese Isatis, Mahonia swaseyi, Yerba mansa (Anemopsis californica), and Phellodendron amurense.
• compositions comprising berberine (or berberine analogs, related compounds, proto-berberine compounds and/or derivatives) for use within the invention.
• useful berberine compounds, berberine related, proto-berberine and derivative compounds for use within the invention will typically have a structure as illustrated in Formula I or Formula XXVII, although functionally equivalent analogs, complexes, conjugates, and derivatives of such compounds will also be appreciated by those skilled in the art as within the scope of at least certain aspects of this invention.
• Useful berberine compounds, berberine related compounds, berberine metabolites, berberine prodrugs, proto-berberine and derivative compounds for use within the invention according to Formula I will also typically have a structure wherein Ri, R 2, R 3 , R 4 , R 8 , R9, Rio, Ri 1, R12 and/or R 13 is selected (each independently, and in any combination yielding an active compound as described) from a hydrogen, halogen, hydroxy, alkyl, alkoxy, nitro, amino, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, alkanoyl, alkanoyloxy, aryl, aroyl, aralkyl, nitrile, dialkylamino, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, carboxyalkyl, alkoxyalkyl,
• Rj is selected from methyl, ethyl, hydroxyl, or methoxy
• R 2 is selected from H, methyl, ethyl, methene
• R 3 is selected from H, methyl, ethyl, methene
• R 4 is selected from a hydrogen atom, methyl, ethyl, hydroxyl, or methoxy, an alkyl group having 1 to 8 carbons , or an alkenyl group having 3 to 8 carbons
• R 8 is selected from straight or branched (C1-C6) alkyl (e.g., substitution selected from methyl, ethyl, n-propyl, 1-methylethyl, n-butyl, 1-methylpropyl, 2- methylpropyl, 1,1-dimethyl
• R 2 and R 3 may together form a methylenedioxy group.
• Additional candidate compounds for use within the compositions and methods will be readily produced and selected according to the further disclosure provided herein below. Further description is provided in U.S. Patent Application No. 11/784,294 filed April 4, 2006.
• each of the R 1 , R 2 , R 3 , R 4 , Rg, R 9 , Ri 0 , Rn, R 12 , and/or Rj 3 groups indicated in Formula I or the R 1 , R 4, R 8, R 9, R 10 , Rn, R12 and/or Rj 3 in Formula XXVII can be optionally (independently, collectively, or in any combination yielding an active compound as described) substituted as described and defined in the following passages.
• R x is selected from methyl, ethyl, hydroxyl, or methoxy
• R 2 is selected from H, methyl, ethyl, methene
• R 3 is selected from H, methyl, ethyl, methene
• R 4 is selected from methyl, ethyl, hydroxyl, or methoxy
• R 8 is selected from straight or branched (C1-C6) alkyl (e.g., substitution selected from methyl, ethyl, n-propyl, 1 -methylethyl, n-butyl, 1 -methylpropyl, 2-methylpropyl, 1,1- dimethylethyl, n-pentyl, 2-methyl
• Ri, R 4 , R 8 , Rn, Ri 2 and Rj 3 are hydrogen and R 2, R 3, R 9 and Ri 0 are independently selected from hydrogen, hydroxy, methyl or methoxy. In some embodiments, R 2 and R 3 may together form a methylenedioxy group. Additional candidate compounds for use within the compositions and methods will be readily produced and according to the further disclosure provided herein below and in U.S. Patent Application No. 11/784,294 filed April 4, 2006 and U.S. Patent Application No. 11/229,339, each of which is incorporated by reference herein in their entirety.
• each of the Ri, R 2 , R 3 , R 4 , R 8 , R9, Rio, Ri 1, and/or Ri 2 groups indicated in the formulas herein can be optionally (independently, collectively, or in any combination yielding an active compound as described) substituted as described and defined in the following passages.
• halogen refers to bromine, chlorine, fluorine or iodine. In one embodiment, the halogen is fluorine. In another embodiment, R 9 , Ri 0 , Ri 1 , Ri 2 and/or Rn may independently be chlorine or bromine.
• the term "hydroxy" as used herein refers to -OH or -O " .
• acetyl refers to -CH 3 CO.
• alkene refers to unsaturated hydrocarbons that contain carbon-carbon double bonds. Examples of such alkene groups include ethylene, propene, and the like. In one embodiment, R 2 and/or R 3 may independently be methene.
• alkyl refers to straight- or branched-chain aliphatic groups containing 1-20 carbon atoms, preferably 1-7 carbon atoms and most preferably 1-6 carbon atoms.
• R 1 , R 2 , R 3 , R 4 , R 8 and/or R 13 may independently be methyl or ethyl groups.
• R 8 and/or Rj 3 may independently be n-propyl, 1 -methylethyl, n-butyl, 1 -methylpropyl, 2-methylpropyl, n-pentyl, 2-methylbutyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3- methylbutyl, m-hexyl, 1-methylpentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3-methylpentyl, 1- 2-dimethylbutyl, 1,3 -dimethyl or 1 -methyl -2ethylpropyl.
• alkoxy includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom, hi one embodiment, the alkoxy group contains 1 to 6 carbon atoms.
• Embodiments of alkoxy groups include, but are not limited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups.
• Rg 1 Ri 0 , Rji, and/or Ri 2 may independently be methoxy or ethoxy groups.
• Ri is a methoxy group.
• substituted alkoxy groups include halogenated alkoxy groups.
• the alkoxy groups can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,
• halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, and trichloromethoxy.
• Ri, R 4 , R % Rio, Rn and/or Ri 2 may independently be an hydroxyl group.
• nitro as used herein alone or in combination refers to a -NO 2 group.
• amino refers to the group -NRR', where R and R' may independently be hydrogen, alkyl, aryl, alkoxy, or heteroaryl.
• aminoalkyl as used herein represents a more detailed selection as compared to “amino” and refers to the group — NRR', where R and R' may independently be hydrogen or (C 1 -C 4 )alkyl.
• trimeromethyl as used herein refers to -CF 3 .
• trifluoromethoxy refers to -OCF 3.
• cycloalkyl refers to a saturated cyclic hydrocarbon ring system containing from 3 to 7 carbon atoms that may be optionally substituted. Exemplary embodiments include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. In certain embodiments, the cycloalkyl group is cyclopropyl. In another embodiment, the (cycloalkyl)alkyl groups contain from 3 to 7 carbon atoms in the cyclic portion and 1 to 4 carbon atoms in the alkyl portion, hi certain embodiments, the (cycloalkyl)alkyl group is cyclopropylmethyl. The alkyl groups are optionally substituted with from one to three substituents selected from the group consisting of halogen, hydroxy and amino.
• alkanoyl and alkanoyloxy refer, respectively, to --C(O)- alkyl groups and -O-C(O)-alkyl groups, each optionally containing 2-5 carbon atoms. Specific embodiments of alkanoyl and alkanoyloxy groups are acetyl and acetoxy, respectively.
• aryl refers to monocyclic or bicyclic aromatic hydrocarbon groups having from 6 to 12 carbon atoms in the ring portion, for example, phenyl, naphthyl, biphenyl and diphenyl groups, each of which may be substituted with, for example, one to four substituents such as alkyl; substituted alkyl as defined above, halogen, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, cycloalkyloxy, alkanoyl, alkanoyloxy, amino, alkylamino, dialkylamino, nitro, cyano, carboxy, carboxyalkyl, carbamyl, carbamoyl and aryloxy.
• Specific embodiments of aryl groups in accordance with the present invention include phenyl, substituted phenyl, naphthyl, biphenyl, and diphenyl.
• aroyl refers to an aryl radical derived from an aromatic carboxylic acid, such as optionally substituted benzoic or naphthoic acids.
• nitrile or "cyano” as used herein refers to the group -CN.
• dialkylamino refers to an amino group having two attached alkyl groups that can be the same or different.
• alkenyl refers to a straight or branched alkenyl group of 2 to 10 carbon atoms having 1 to 3 double bonds.
• Preferred embodiments include ethenyl, 1-propenyl, 2- propenyl, 1-methylethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-2-propenyl, 1-pentenyl, 2- pentenyl, 4-pentenyl, 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 1-heptenyl, 2-heptenyl, 1- octenyl, 2-octenyl, 1,3-octadienyl, 2-nonenyl, 1,3-nonadienyl, 2-decenyl, etc.
• alkynyl refers to a straight or branched alkynyl group of 2 to 10 carbon atoms having 1 to 3 triple bonds.
• exemplary alkynyls include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2- pentynyl, 4-pentynyl, 1-octynyl, 6-methyl-l-heptynyl, and 2-decynyl.
• hydroxyalkyl alone or in combination, refers to an alkyl group as previously defined, wherein one or several hydrogen atoms, preferably one hydrogen atom has been replaced by a hydroxyl group. Examples include hydroxymethyl, hydroxyethyl and 2- hydroxyethyl.
• aminoalkyl refers to the group -NRR', where R and R' may independently be hydrogen or (d-C 6 )alkyl.
• alkylaminoalkyl refers to an alkylamino group linked via an alkyl group (i.e., a group having the general structure — alkyl-NH-alkyl or — alkyl-N(alkyl)(alkyl)).
• alkyl group i.e., a group having the general structure — alkyl-NH-alkyl or — alkyl-N(alkyl)(alkyl)
• alkyl group i.e., a group having the general structure — alkyl-NH-alkyl or — alkyl-N(alkyl)(alkyl)
• alkyl group i.e., a group having the general structure — alkyl-NH-alkyl or — alkyl-N(alkyl)(alkyl)
• Such groups include, but are not limited to, mono- and di-(Ci-C 8 alkyl)aminoC]-C 8 alkyl, in which each
• dialkylaminoalkyl refers to alkylamino groups attached to an alkyl group. Examples include, but are not limited to, N,N-dimethylaminomethyl, N,N- dimethylaminoethyl N,N-dimethylaminopropyl, and the like.
• dialkylaminoalkyl also includes groups where the bridging alkyl moiety is optionally substituted.
• haloalkyl refers to an alkyl group substituted with one or more halo groups, for example chloromethyl, 2-bromoethyl, 3-iodopropyl, trifluoromethyl, perfluoropropyl, 8-chlorononyl and the like.
• carboxyalkyl refers to the substituent -R'-COOH wherein R' is alkylene; and carbalkoxyalkyl refers to ⁇ R' ⁇ COOR wherein R' and R are alkylene and alkyl respectively.
• alkyl refers to a saturated straight- or branched- chain hydrocarbyl radical of 1 -6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n- butyl, t-butyl, n-pentyl, 2-methylpentyl, n-hexyl, and so forth.
• Alkylene is the same as alkyl except that the group is divalent.
• alkoxyalkyl refers to an alkylene group substituted with an alkoxy group.
• methoxyethyl [CH 3 OCH 2 CH 2 --] and ethoxymethyl (CH 3 CH 2 OCH 2 -] are both C 3 alkoxyalkyl groups.
• alkanoylamino refers to alkyl, alkenyl or alkynyl groups containing the group -C(O)- followed by --N(H)--, for example acetylamino, propanoylamino and butanoylamino and the like.
• carbonylamino refers to the group -NR-CO-CH 2 -R', where R and
• R' may be independently selected from hydrogen or (Ci-C 4 )alkyl.
• carbamoyl refers to -0--C(O)NH 2 .
• R and R' can be hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, cycloalkyl, aryl, heterocyclo, or heteroaryl.
• alkylsulfonylamino refers to refers to the group -NHS(O) 2 R 3 wherein
• furyl refers to a heterocyclic group, having the formula C 4 H 3 O, which may be either the alpha or beta isomer
• benzotriazolyl refers to a monovalent group having a benzene group fused to a triazolyl group.
• the formula for a benzotriazolyl group is C 6 H 4 N 3 -.
• benzyloxy refers to an 0--CH 2 Ph substituent, wherein Ph is phenyl or a substituted phenyl.
• methylenedioxy refers to a -0-CH 2 -O- group.
• polystyl refers to the group CH 2 CH.
• glycosylation means the attachment of an oligosaccharide group, preferably, though not limited to, attachment to an nitrogen or oxygen.
• oligosaccharide as used herein is defined as encompassing 1 to 20 saccharides. Mono-, di-, and trisaccharides are specifically included in the definition of oligosaccharides. [00141] All value ranges expressed herein, are inclusive over the indicated range. Thus, a range of R between 0 to 4 will be understood to include the values of 1, 2, 3, and 4.
• derivative forms of the berberine compound of formula II may be formed through demethylation
• derivatives of berberine may be demethylated as shown in formula XXXII, below:
• R 10 may be a hydrogen, halogen, hydroxy, alkyl, alkoxy, nitro, amino, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, alkanoyl, alkanoyloxy, aryl, aroyl, aralkyl, nitrile, dialkylamino, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, carboxyalkyl, alkoxyalkyl, carboxy, alkanoylamino, carbamoyl, carbamyl, carbonylamino, alkylsulfonylamino, heterocyclo group, methylenedioxy, acetyl, formyl, phenylacryloyl, haloacetyl, haloacetylpropanyl, alkylacetyl
• the berberine compound of formula XXXII may be hydrogenated, in further embodiments, the berberine compound of formula XXXII may be demethylated and hydrogenated such that it forms compounds such as or derived from canadine of the structure shown below.
• berberine compounds, berberine related, proto-berberine and derivative compounds for use within the invention will in certain embodiments have structure as illustrated below in Formula XXXIII, although functionally equivalent analogs, complexes, conjugates, and derivatives of such compounds will also be appreciated by those skilled in the art as within the scope of at least certain aspects of this invention.
• Additional berberine compounds, berberine related, proto-berberine and derivative compounds for use within the invention according to Formula XXXIII or derivatives of Formula XXXIII as shown in Formulas XXXIV, XXXV and XXXVI below will typically have a structure wherein Ri, R 2i R 3, R 4, R 8, R 9, Rio, Ri 1 , R 12 and/or R13 is selected (each independently, and in any combination yielding an active compound as described) from a hydrogen, halogen, hydroxy, hydroxyalkyl, alkyl, alkoxy, nitro, amino, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, alkanoyl, alkanoyloxy, aryl, aroyl, aralkyl, nitrile, dialkylamino, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,
• X represents an inorganic acid ion, organic acid ion, or halide, more particularly, nitrate, sulfate, acetate, tartrate, maleate, succinate, citrate, fumarate, aspartate, salicylate, glycerate, ascorbate, fluoride, chloride, iodide or bromide.
• Z represents an alkyl group having 5 to 12 carbons, or an alkenyl group having 4 to 6 carbons, a N-benzotriazolyl group, a quinolinyl group, a furyl group, a substituted furyl group, or a radical represented by the formula:
• Z 1 , Z 2 , Z 3 , Z 4 and Z 5 which may be the same or different from each other, represent a hydrogen atom, halogen, an alkyl group having 1 to 5 carbons, a trifluoromethyl group, a phenyl group, a substituted phenyl group, a nitro group, an alkoxy group having 1 to 4 carbons, a methylenedioxy group, a trifluoro-methoxy group, a hydroxy group, a benzyloxy group, a phenoxy group, a vinyl group, a benzenesulfonylmethyl group or a methoxycarbonyl group; and A and B which may also be the same or different from each other, represent carbon or nitrogen.
• Berberine compounds and derivatives of Formula IV are exemplified by the compounds in the table 3 below. Table 3: Exemplary compositions of berberine compounds and derivatives of formula IV.
• berberine chloride exemplifies this type of compound having the structure of formula XXV below.
• Table 4 Exemplary forms of protoberberine compounds and derivatives of formula XXVI.
• illustrative structural modifications according to Formula XXVII above will be selected to provide useful candidate compounds for treating and/or preventing hyperglycemia, hyperlipidemia, hypercholesterolemia, insulin resistance, obesity, diabetes, metabolic syndrome and hypertension in mammalian subjects
• Ri is selected from methyl, ethyl, hydroxyl, or methoxy
• R 4 is selected from methyl, ethyl, hydroxyl, or methoxy
• Rg is selected from straight or branched (C1-C6) alkyl (e.g., substitution selected from methyl, ethyl, n-propyl, 1-methylethyl, n-butyl, 1-methylpropyl, 2-methylpropyl, 1,1- dimethylethyl, n-pentyl, 2-methylbutyl, 1,1-dimethylpropyl, 2,2 dimethylpropyl, 3-methylbutyl, n-hexyl, 1-methylpentyl
• each of the Ri, R 4, R 8; R 9 R 10 , Rn, Ri 2 , and/or R] 3 groups indicated in the formulas herein can be optionally (independently, collectively, or in any combination yielding an active compound as described) substituted as described above.
• Rj, R 4, R 8, R 9> Ri 0 , Ri i , Ri 2 and Ri 3 are hydrogen.
• berberine may be demethylated to form the structure of formula XXVIII.
• derivatives of berberine may be demethylated as shown in formula XXXII, below:
• Rio may be a hydrogen, halogen, hydroxy, alkyl, alkoxy, nitro, amino, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, alkanoyl, alkanoyloxy, aryl, aroyl, aralkyl, nitrile, dialkylamino, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, carboxyalkyl, alkoxyalkyl, carboxy, alkanoylamino, carbamoyl, carbamyl, carbonylamino, alkylsulfonylamino, heterocyclo group, methylenedioxy, acetyl, formyl, phenylacryloyl, haloacetyl, haloacetylpropanyl, alkylacetyl,
• each of Ri, R 2 , R 3 , R 41 R 81 R 9, R 10 , Rn, Ri 2 and/or Ri 3 may independently, collectively, or in any combination that yields an active (e.g., anti-dyslipidemic, anti-hyperlipidemic, anti- hyperglycemic, anti-hypertensive, LDL-modulatory, LDLR stability increasing, LDLR- modulatory, AMP-activated protein kinase modulatory or insulin receptor (InsR) modulatory) compound according to this disclosure, be a hydrogen, halogen, hydroxy, hydroxyalkyl, alkyl, alkoxy, nitro, amino, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, alkanoyl, alkanoyloxy, aryl, aroyl, aralkyl, nitrile, dialkylamino, alkenyl, alkynyl, hydroxyalkyl,
• the R group may be selected from any of the stated groups so as to be the same or different.
• two or more R groups may be joined together, for example, R 2 and R 3 may be combined to form a methylenedioxy group.
• Ri is selected from hydrogen, methyl, ethyl, hydroxyl, or methoxy
• R 4 is selected from hydrogen, methyl, ethyl, hydroxyl, or methoxy
• R 8 is selected from hydrogen, straight or branched (C1-C6) alkyl (e.g., substitution selected from methyl, ethyl, n-propyl, 1 -methylethyl, n-butyl, 1- methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, n-pentyl, 2-methylbutyl, 1,1-dimethylpropyl, 2,2 dimethylpropyl, 3-methylbutyl, n-hexyl, 1 -methylpentyl, 1,1-di
• R 2 and R 3 may together form a methylenedioxy.
• Additional candidate compounds for use within the compositions and methods of the invention are provided wherein each of the R 1 , R 4 , R ⁇ R 9> Ri 0 , Rj i , Ri 2 , and/or Ri 3 groups indicated in the formulas herein can be optionally (independently, collectively, or in any combination yielding an active compound as described) substituted as described above.
• Ri, R 4, R 8> R 9, Rio, Ri l, Ri 2 and Rj 3 are hydrogen.
• Additional candidate compounds for use within the compositions and methods will be readily produced and selected according to the further disclosure provided herein below.
• An exemplary embodiment of Formula XXXIII is canadine shown below as Formula XXXXIV or derivatives thereof.
• compounds of Formula XXXXIII may have the structure illustrated by Formulas XXXIV, XXXV, or XXXVI, below.
• each of R 1 , R 4, R 8, Rg, Ri 0 , Ri i, Ru and/or R 13 may independently, collectively, or in any combination that yields an active (e.g., anti-dyslipidemic, anti-hyperlipidemic, anti- hyperglycemic, anti-cholesterolemic, anti-hypertensive, LDL-modulatory, LDLR-modulatory, LDLR stabilizing, AMP-activated protein kinase modulatory or insulin receptor (InsR) modulatory) compound according to this disclosure, be hydrogen, halogen, hydroxy, hydroxyalkyl, alkyl, alkoxy, nitro, amino, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, alkanoyl, alkanoyloxy, aryl, aroyl, aralkyl, nitrile, dialkylamino, alkenyl
• an active e.g., anti-dyslipidemic,
• R group When more than one R group is present, the R group may be selected from any of the stated groups so as to be the same or different. In other embodiments, the two or more R groups may be linked, for example, R 2 and R 3 may be combined to form a methylenedioxy group. In certain exemplary embodiments, Rj, R 4 , R 8, R 9, Rj 0 , R 11 ⁇ R] 2 and Ri 3 are each hydrogen.
• Useful berberine and related compounds and derivatives and proto-berberine compounds and derivatives within the formulations and methods of the invention include those forms delineated above, including, but not not limited to, salts of berberine and related or derivative compounds, for example, berberine sulfate, berberine hydrochloride, berberine chloride, palmatine chloride, palmatine, oxyberberine, dihydroberberine, 8- cyanodihydroberberine, (-)-canadine, tetrahydroberberine N-oxide, tetrahydroberberine, N- methyltetrahydroberberinium iodide, 6-protoberberine, 9-ethoxycarbonyl berberine, 9-N,N- dimethylcarbamoyl berberine and 12-bromo berberine, berberine azide, and berberine betaine.
• salts of berberine and related or derivative compounds
• berberine may be demethylated.
• demethylated beberine may be further reacted for form acetyl, terbutyl formyl, tertiarybutylformylacetyl, 3- phenylacryloyl, chloracetyl or chloroacetylpropanyl forms of berberine.
• Other useful forms of derivatives for use within the invention include other pharmaceutically acceptable active salts of said compounds, as well as active isomers, enantiomers, polymorphs, glycosylated derivatives, solvates, hydrates, and/or prodrugs of said compounds.
• berberine, related berberine and proto-berberine compounds and derivative compounds may be generated by any methods known to those skilled in the art, exemplary compounds for use within the invention may also be generated, for example, according to Routes 1 , 2, 3, and 4 described herein, below.
• Routes 1 , 2, 3, and 4 described herein, below.
• Lipid lowering compositions comprise an active compound as described herein
• berberine including berberine, a berberine related compound, berberine metabolite, berberine prodrug, or berberine derivative compound of any of the Formulae herein, such as Formula I or Formula II, any active compound of formulae VI-IX , any active compound of Formulae I-V, XXVI- XXVIII, or XXXII-XXXVI, including any of the species within these classes of active compounds, and active combinations thereof) in a lipid lowering effective amount for prophylaxis and/or treatment of hyperlipidemia, hypercholesterolemia and elevated triglycerides in a mammalian subject.
• a lipid lowering effective amount will comprise an amount of the active compound which is therapeutically effective, in a single or multiple unit dosage form, over a specified period of therapeutic intervention, to measurably alleviate one or more symptoms of hyperlipidemia or elevated cholesterol in the subject, and/or to alleviate one or more symptom(s) of a cardiovascular disease or condition in the subject.
• these compositions are effective within in vivo treatment methods to alleviate hyperlipidemia.
• Lipid lowering compositions of the invention typically comprise a lipid lowering effective amount or unit dosage of the active compound, which may be formulated with one or more pharmaceutically acceptable carriers, excipients, vehicles, emulsifiers, stabilizers, preservatives, buffers, and/or other additives that may enhance stability, delivery, absorption, half-life, efficacy, pharmacokinetics, and/or pharmacodynamics, reduce adverse side effects, or provide other advantages for pharmaceutical use.
• Lipid lowering effective amounts including cholesterol lowering effective amounts and/or triglyceride lowering effective amounts of the active compound will be readily determined by those of ordinary skill in the art, depending on clinical and patient-specific factors.
• Suitable effective unit dosage amounts of the active compounds for administration to mammalian subjects, including humans, may range from 10 to 1500 mg, 20 to 1000 mg, 25 to 750 mg, 50 to 500 mg, 150 to 500 mg, 100 to 200mg, 200 to 400mg, or 400 to 600 mg.
• the anti-hyperlipidemia or hypolipidemia effective dosage of a berberine derivative compound of Formula XXVII may be selected within narrower ranges of, for example, 10 to 25 mg, 30-50 mg, 75 to 100 mg, 100 to 250 mg, or 250 to 500 mg.
• dosages of 10 to 25 mg, 30-50 mg, 75 to 100 mg, 100 to 250 mg, or 250 to 500 mg, are administered one, two, three, four, or five times per day.
• dosages of 50-75 mg, 100-200 mg, 250-400 mg, or 400-600 mg are administered once or twice daily.
• dosages are calculated based on body weight, and may be administered, for example, in amounts from about 0.5mg/kg to about 100mg/kg per day, lmg/kg to about 75mg/kg per day, lmg/kg to about 50mg/kg per day, 2mg/kg to about 50mg/kg per day, 2mg/kg to about 30mg/kg per day or 3mg/kg to about 30mg/kg per day.
• Glucose lowering compositions comprise an active compound as described above, or pharmaceutical formulations comprising the active compound in a glucose lowering effective amount effective for prophylaxis and/or treatment of hyperglycemia in a mammalian subject.
• a glucose lowering effective amount will comprise an amount of the active compound which is therapeutically effective, in a single or multiple unit dosage form, over a specified period of therapeutic intervention, to measurably alleviate one or more symptoms of hyperglycemia in the subject, and/or to alleviate one or more symptom(s) of a cardiovascular disease or condition in the subject.
• these compositions are effective within in vivo treatment methods to alleviate hyperglycemia.
• Insulin sensitivity increasing and insulin resistance decreasing compositions comprising an active compound or pharmaceutical formulation thereof in an insulin sensitivity increasing and/or insulin resistance decreasing effective amount, which is effective for prophylaxis and/or treatment of insulin resistance in a mammalian subject.
• a insulin sensitivity increasing and/or insulin resistance decreasing effective amount will comprise an amount of the active compound which is therapeutically effective, in a single or multiple unit dosage form, over a specified period of therapeutic intervention, to measurably alleviate one or more symptoms of insulin resistance in the subject, and/or to alleviate one or more symptom(s) of a cardiovascular disease or condition in the subject.
• these compositions are effective within in vivo treatment methods to alleviate insulin resistance.
• Glucose lowering or insulin sensitivity increasing/insulin resistance decreasing compositions of the invention typically comprise a glucose lowering effective amount or unit dosage of the active compound, which may be formulated with one or more pharmaceutically acceptable carriers, excipients, vehicles, emulsifiers, stabilizers, preservatives, buffers, and/or other additives that may enhance stability, delivery, absorption, half-life, efficacy, pharmacokinetics, and/or pharmacodynamics, reduce adverse side effects, or provide other advantages for pharmaceutical use.
• Glucose lowering effective amounts of the active compound will be readily determined by those of ordinary skill in the art, depending on clinical and patient- specific factors.
• Suitable effective unit dosage amounts of the active compounds for administration to mammalian subjects, including humans, may range from 10 to 1500 mg, 20 to 1000 mg, 25 to 750 mg, 50 to 500 mg, or 150 to 500 mg.
• the anti- hyperglycemic effective dosage of the active compound may be selected within narrower ranges of, for example, 10 to 25 mg, 30-50 mg, 75 to 100 mg, 100 to 250 mg, or 250 to 500 mg.
• These and other effective unit dosage amounts may be administered in a single dose, or in the form of multiple daily, weekly or monthly doses, for example in a dosing regimen comprising from 1 to 5, or 2-3, doses administered per day, per week, or per month.
• dosages of 10 to 25 mg, 30-50 mg, 75 to 100 mg, 100 to 250 mg, or 250 to 500 mg are administered one, two, three, four, or five times per day.
• dosages of 50-75 mg, 100-200 mg, 250-400 mg, or 400-600 mg are administered once or twice daily.
• dosages are calculated based on body weight, and may be administered, for example, in amounts from about 0.5mg/kg to about 100mg/kg per day, lmg/kg to about 75mg/kg per day, lmg/kg to about 50mg/kg per day, 2mg/kg to about 50mg/kg per day, 2mg/kg to about 30mg/kg per day or 3mg/kg to about 30mg/kg per day.
• compositions of the invention comprising an anti-hyperlipidemia and/or anti-hyperglycemic effective amount of an active compound as described will be routinely adjusted on an individual basis, depending on such factors as weight, age, gender, and condition of the individual, the acuteness of the hyperlipidemia and/or related symptoms, whether the administration is prophylactic or therapeutic, and on the basis of other factors known to effect drug delivery, absorption, pharmacokinetics, including half-life, and efficacy.
• An effective dose or multi-dose treatment regimen for the instant lipid lowering formulations will ordinarily be selected to approximate a minimal dosing regimen that is necessary and sufficient to substantially prevent or alleviate hyperlipidemia and cardiovascular diseases in the subject, and/or to substantially prevent or alleviate one or more symptoms associated with hyperlipidemia in the subject.
• a dosage and administration protocol will often include repeated dosing therapy over a course of several days or even one or more weeks or years.
• An effective treatment regime may also involve prophylactic dosage administered on a day or multi-dose per day basis lasting over the course of days, weeks, months or even years.
• An effective dose or multi-dose treatment regimen for the instant glucose lowering formulations will ordinarily be selected to approximate a minimal dosing regimen that is necessary and sufficient to substantially prevent or alleviate hyperglycemia in the subject, and/or to substantially prevent or alleviate one or more symptoms associated with hyperglycemia in the subject.
• a dosage and administration protocol will often include repeated dosing therapy over a course of several days or even one or more weeks or years.
• An effective treatment regime may also involve prophylactic dosage administered on a day or multi-dose per day basis lasting over the course of days, weeks, months or even years.
• Various assays and model systems can be readily employed to determine the therapeutic effectiveness of anti-hyperlipidemia treatment according to the invention. For example, blood tests to measure total cholesterol as well as triglycerides, LDL and HDL levels are routinely given. Individuals with a total cholesterol level of greater than 200 mg/dL are considered borderline high risk for cardiovascular disease. Those with a total cholesterol level greater than 239 mg/dL are considered to be at high risk. An LDL level of less than 100 mg/dL is considered optimal. LDL levels between 130 to 159mg/dL are borderline high risk.
• LDL levels between 160 to 189 mg/dL are at high risk for cardiovascular disease and those individuals with an LDL greater than 190 mg/dL are considered to be at very high risk for cardiovascular disease.
• Triglyceride levels of less than 150 mg/dL are considered normal. Levels between 150-199 mg/dL are borderline high and levels above 200 are considered to put the individual at high risk for cardiovascular disease.
• Lipid levels can be determined by standard blood lipid profile tests. Effective amounts of the compositions of the invention will lower elevated lipid levels by at least 10%, 20%, 30%, 50% or greater reduction, up to a 75-90%, or 95% or greater.
• Effective amounts will also move the lipid profile of an individual towards the optimal category for each lipid, i.e., decrease LDL levels from 190mg/dl to within 130 to 159mg/dL or even further to below 100 mg/dL. Effective amounts may further decrease LDL or triglyceride levels by about 10 to about 70 mg/dL, by about 20 to about 50 mg/dL, by about 20 to about 30 mg/dL, or by about 10 to about 20 mg/dL. [00166] Individuals may also be evaluated using a hs-CRP (high-sensitivity C-reactive protein) blood test. Those with a hs-CRP result of less than 1.0 mg/L are at low risk for cardiovascular disease.
• hs-CRP high-sensitivity C-reactive protein
• compositions of the present invention will lower hs-CRP results below 3.0mg/L. Effective amounts of the compositions of the present invention can lower hs-CRP results by about 0.5 to about 3.0mg/L, and further by about 0.5 to about 2.0mg/L.
• An effective amount of a berberine related or derivative compound of Formula XXVII of the present invention will lower the hs-CRP level from over 3.0 mg/L to between 1.0 and 3.0 mg/1, more preferably to about 1.0 mg/L to about 0.6 mg/L.
• Therapeutic effectiveness may be determined, for example, through a change in body fat as determined by body fat measurements.
• Body fat measurements may be determined by a variety of means including, but not limited to, determinations of skinfold thickness, bioelectrical impedance, air displacement plethysmography, underwater weighing, DEXA scans, measurement on a scale or calculation of body mass index (BMI).
• BMI body mass index
• Percentages of weight due to body fat for normal men are between 10-20%. In athletes, the normal range is between 6-10%. In women, the normal range is between 15-25% and in athletic women it is between 10-15%. Effective amounts of the compounds of the present invention will decrease body fat percentages from above 20-25%. Effective amounts may also decrease body fat percentages to within the normal ranges for that individual. Effectiveness may also be demonstrated by a 2-50%, 10-40%, 15-30%, 20-25% decrease in body fat. [00169] Skinfold measurements measure subcutaneous fat located directly beneath the skin by grasping a fold of skin and subcutaneous fat between the thumb and forefinger and pulling it away from the underlying muscle tissue.
• the thickness of the double layer of skin and subcutaneous tissue is then read with a caliper.
• the five most frequently measured sites are the upper arm, below the scapula, above the hip bone, the abdomen, and the thigh.
• Skinfold measurements are used to determine relative fatness, changes in physical conditioning programs, and the percentage of body fat in desirable body weight. Effective amounts of berberine related or derivative compound of Formula XXVII will decrease body fat percentages by 2-50%, 10- 40%, 15-30%, 20-25%, 30-40% or more. [00170] Body fat percentages can also be determined by body impedance measurements.
• Body impedance is measured when a small electrical signal is passed through the body carried by water and fluids. Impedance is greatest in fat tissue, which contains only 10-20% water, while fat-free mass, which contains 70-75% water, allows the signal to pass much more easily.
• the impedance measurements along with a person's height, weight, and body type (gender, age, fitness level), it is possible to calculate the percentage of body fat, fat- free mass, hydration level, and other body composition values. Effective amounts of the active compound will decrease body fat percentages by 2-50%, 10-40%, 15-30%, 20-25%, 30-40% or more.
• Hydrostatic or underwater weighing is another method for determining lean muscle mass and body fat percentages.
• DEXA or dual energy x-ray absorptiometry scans determine whole body as well as regional measurements of bone mass, lean mass, and fat mass. Total fat mass is expressed in kg and as a percentage of body mass. These are calculated by integrating the measurements for the whole body and different automatic default regions such as arms, trunk, and legs.
• Body fat percentages may further be determined by air displacement plethysmography.
• Air displacement plethysmography determines the volume of a subject to be measured by measuring the volume of air displaced by the subject in an enclosed chamber. The volume of air in the chamber is calculated through application of Boyle's Law and/or Poisson's Law to conditions within the chamber. More particularly, in the most prevalent method of air displacement plethysmography used for measuring human body composition (such as disclosed in U.S. Pat. No. 4,369,652, issued to Gundlach, and U.S. Pat. No. 5,105,825, issued to Dempster), volume perturbations of a fixed frequency of oscillation are induced within a measurement chamber, which perturbations lead to pressure fluctuations within the chamber.
• the amplitude of the pressure fluctuations is determined and used to calculate the volume of air within the chamber using Boyle's Law (defining the relationship of pressure and volume under isothermal conditions) or Poisson's law (defining the relationship of pressure and volume under adiabatic conditions).
• Body volume is then calculated indirectly by subtracting the volume of air remaining inside the chamber when the subject is inside from the volume of air in the chamber when it is empty.
• body composition can be calculated based on the measured subject volume, weight of the subject, and subject surface area (which, for human subjects, is a function of subject weight and subject height), using known formulas defining the relationship between density and human fat mass.
• Body Mass Index has been recognized by the U.S. Department of Health as a reference relationship between a person's height and weight and can be used to determine when extra weight above an average or normal weight range for a person of a given height can translate into and signal increased probability for additional health risks for that person. While BMI does not directly measure percent of body fat, higher BMIs are usually associated with an increase in body fat, and thus excess weight.
• a desired BMI range is from about 18 kg/m 2 to about 24 kg/m 2 , wherein a person is considered to have a healthful weight for the person's height and is neither overweight nor underweight.
• a person with a BMI above 24 kg/m 2 such as from about 25 kg/m 2 to about 30 kg/m 2 , is considered to be overweight, and a person with a BMI above about 30 kg/ m 2 is considered to be obese.
• a person with a BMI above about 40 kg/m is considered to be morbidly obese.
• an individual who has a BMI in the range of about 25 kg/m 2 to about 35 kg/m 2 , and has a waist size of over 40 inches for a man and over 35 inches for a woman is considered to be at especially high risk for health problems.
• Effectiveness of berberine related or derivative compound of Formula XXVII may be demonstrated by a reduction in the body mass index from a range between 40kg/m 2 to about 30 kg/m 2 to 25 kg/m 2 to about 24 kg/m 2 .
• a compound of the present invention may also reduce BMI from a range above 30 kg/m 2 to a range between 30 kg/m 2 to 25 kg/m 2 and more preferably to about 24 kg/m 2 .
• Effectiveness may further be demonstrated by a decrease in body weight from 1-25%, 3 -15%, 2-50%, 10-40%, 15-30%, 20-25%.
• Effectiveness may additionally be demonstrated by a decrease in BMI by 2-50%, 10- 40%, 15-30%, 20-25%, 30-40% or more. Effective amounts of the active compound will lower an individual's BMI to within about 18 kg/m 2 to about 24 kg/m 2 .
• Therapeutic effectiveness of active compounds of the invention may also be determined by changes in the waist/hip ratio.
• the waist/hip ratio is determined by dividing the circumference of the waist by the circumference of the hip. Women should have a waist/hip ratio of 0.8 or less and men should have a waist/hip ratio of 0.95 or less. Effective amounts of the active compound will lower the waist/hip ratio by about 2-50%, 10-40%, 15-30%, 20-25% or more.
• the waist/hip ratio of a female subject may be lowered to 0.8 or less and the ratio of a male subject to a ratio of 0.95 or less.
• effective amounts of active compounds will decrease weight by about 2-50%, 10-40%, 15-30%, 20-25% or more, or will yield a waist circumference decrease of a similar percentage, e.g., to decrease waist circumference by about 2-50%, 10-40%, 15-30%, 20-25% or more.
• Therapeutic effectiveness may also be demonstrated with a decrease in fasting glucose.
• a fasting glucose test measures blood glucose after an overnight fast. Fasting glucose levels of 100 to 125 mg/dL are above normal. Effective amounts of active compounds of the invention will decrease fasting glucose levels by about 2-50%, 10-40%, 15-30%, 20-25% or more, or will lower the fasting glucose level from above 125 mg/dL to a range between 70 to 99 mg/dL.
• An effective amount of a composition of the present invention may further lower fasting glucose levels by about 1 to about 5 mg/dL, by about 1 to about 10 mg/dL, by about 5 to about 20 mg/dL, by about 5 to about 30 mg/dL, by about 20 to about 60 mg/dL or more.
• Therapeutic effectiveness may further be demonstrated by a glucose tolerance test.
• a glucose tolerance test is taken after an overnight fast and 2 hours after consumption of a glucose solution.
• An effective amount of an active compound of the invention will lower glucose levels by about 2-50%, 10-40%, 15-30%, 20-25% or more, or from above 200 mg/dL to a range between 140 to 200 mg/dL, and more preferably to below 140 mg/dL.
• An effective amount of an active compound may alternatively lower glucose levels by about 1 to about 5 mg/dL, by about 1 to about 10 mg/dL, by about 5 to about 20 mg/dL, by about 5 to about 30 mg/dL, by about 20 to about 60 mg/dL or more.
• Therapeutic effectiveness may additionally be demonstrated by a hyperinsulinemic euglycemic clamp study.
• insulin and glucose are infused intravenously at several different doses to determine what levels of insulin control different levels of glucose.
• insulin is infused at 0.06 units per kg body weight per minute.
• glucose 20% is infused to maintain blood sugar levels between 5 and 5.5 mmol/1.
• the rate of glucose infusion is determined by checking the blood sugar levels every 5 minutes. The rate of glucose infusion during the last 30 minutes of the test determines insulin sensitivity. If high levels (7.5 mg/min or higher) are required, the patient is insulin-sensitive.
• a glycohemoglobin test is a blood test that measures the amount of glucose bound to hemoglobin. The results reflect the amount of glycohemoglobin divided by the total amount of hemoglobin multiplied by 100 (to produce a percentage).
• An effective amount of an active compound of the invention will decrease the hemoglobin AIc % to less than 14%, preferably to between 8 and 10%, more preferably to between 5 and 8%, more preferably to between 6 and 8% and most preferably to between 4 to 6%, or may decrease the hemoglobin Alc% by about 2- 50%, 10-40%, 15-30%, 20-25% or more. [00180] Therapeutic effectiveness may also be calculated through an insulin suppression test.
• an effective amount of active compounds of the present invention will increase insulin sensitivity by about 2-50%, 10-40%, 15-30%, 20-25% or more.
• Therapeutic effectiveness may additionally be determined by the C13 glucose breath test in which glucose labeled with non-radioactive C 13, is ingested and the byproduct of its metabolism 13 CO 2 is detected in expired air. In insulin resistant states glucose uptake would be impaired and the production of l CO 2 would therefore also be impaired.
• An effective amount of an active compound of the present invention will increase 13 CO 2 by about 2-50%, 10-40%, 15- 30%, 20-25% or more.
• Therapeutic effectiveness may further be determined by a random plasma glucose test.
• An effective amount of active compound will decrease blood glucose from above 200mg/dL to a range between 140 to 200 mg/dL, and more preferably to below 140 mg/dL.
• therapeutic effectiveness may be determined by a CIGMA test in which 180 mg/mhv'/m "2 of glucose is infused for 120 min at a rate of 5 mg/kg with blood samples are taken at 110, 115 and 120 minutes. An effective amount of an active compound will increase glucose clearance by 2-50%, 10-40%, 15-30%, 20-25% or more.
• therapeutic effectiveness may be determined by a FSIVGTT test in which an intravenous glucose bolus (0.3g/kg) is administered followed by a 5 minute insulin infusion 20 minutes later.
• Blood samples are tested for glucose every two minutes for the first 20 minutes and samples are tested for glucose and insulin levels at 22, 24, 26, 28, 30, 33, 36, 40, 50, 60 , 70, 80, 100, 120, 140, 160 and 180 minutes.
• An effective amount of active compound will increase glucose clearance by 2-50%, 10-40%, 15-30%, 20-25% or more.
• Effectiveness may further be determined by blood pressure testing. Effective amounts of active compounds of the present invention will lower blood pressure from above 150/100 mm Hg to less than 120/80 mm Hg, preferably to between about 139/89 mm to about 120/80mm Hg, most preferably to less than 120/80mm Hg. Preferably the methods and compositions of the present invention will lower blood pressure to between 110/60 mm Hg to about 120/70 mmHg.
• Therapeutic effectiveness may also be determined by a D-dimer test.
• An effective amount of an active compound will decrease the amount of d-dimer in a sample to about 0-300 ng/ml, or may decrease the d-dimer level in a sample by about 2-50%, 10-40%, 15-30%, 20-25% or more.
• compositions and methods of the invention may also be demonstrated by a significant decrease or improvement in the complications or symptoms of metabolic and cardiovascular disorders including fatty liver, reproductive abnormalities, growth abnormalities, arterial plaque accumulation, osteoarthritis, gout, joint pain, respiratory problems, skin conditions, sleep apnea, idiopathic intracranial hypertension, lower extremity venous stasis disease, gastroesophageal reflux, urinary stress incontinence, kidney damage, cardiovascular diseases such as atherosclerosis, coronary artery disease, enlarged heart, diabetic cardiomyopathy, angina pectoris, carotid artery disease, peripheral vascular disease, stroke, cerebral arteriosclerosis, myocardial infarction, cerebral infarction, restenosis following balloon angioplasty, intermittent claudication, dyslipidemia postprandial lipidemia, high blood pressure and xanthoma.
• metabolic and cardiovascular disorders including fatty liver, reproductive abnormalities, growth abnormalities, arterial plaque accumulation, osteoarthritis, gout, joint pain, respiratory problems, skin conditions
• compositions and methods of the invention may also be demonstrated by a decrease in the symptoms of cardiovascular disease including shortness of breath, chest pain, leg pain, tiredness, confusion vision changes, blood in urine, nosebleeds, irregular heartbeat, loss of balance or coordination, weakness, or vertigo.
• test subjects administered the active compound will exhibit a 5%, 10%, 20%, 30%, 50% or greater reduction, up to a 75- 90%, or 95% or greater, reduction, in one or more symptom(s) caused by, or associated with, hyperlipidemia, hyperglycemia, elevated cholesterol, hypertension, metabolic syndrome, obesity, diabetes, elevated glucose and/or a targeted cardiovascular disease or condition in the subject, compared to placebo-treated or other suitable control subjects.
• combinatorial lipid lowering formulations and coordinate administration methods employ an effective amount of an active compound as described herein in combination with one or more secondary or adjunctive agent(s) that is/are combinatorially formulated or coordinately administered with the active compound to yield a combined, multi-active agent anti-hyperlipidemia composition or coordinate treatment method.
• exemplary combinatorial formulations and coordinate treatment methods employ the berberine related or derivative compound in combination with the one or more secondary anti-hyperlipidemia agent(s), or with one or more adjunctive therapeutic agent(s) that is/are useful for treatment or prophylaxis of the targeted (or associated) disease, condition and/or symptom(s) in the selected combinatorial formulation or coordinate treatment regimen.
• combinatorial glucose lowering formulations and coordinate administration methods employ an effective amount of the active compound and one or more secondary or adjunctive agent(s) that is/are combinatorially formulated or coordinately administered with the active compound to yield a combined, multi-active agent anti-hyperglycemic composition or coordinate treatment method.
• exemplary combinatorial formulations and coordinate treatment methods in this context employ the active compound in combination with the one or more secondary anti-hyperglycemic agent(s), or with one or more adjunctive therapeutic agent(s) that is/are useful for treatment or prophylaxis of the targeted (or associated) disease, condition and/or symptom(s) in the selected combinatorial formulation or coordinate treatment regimen.
• combinatorial hypertension lowering formulations and coordinate administration methods employ an effective amount of active compound and one or more secondary or adjunctive agent(s) that is/are combinatorially formulated or coordinately administered with the active compound to yield a combined, multi-active agent anti-hypertensive composition or coordinate treatment method.
• exemplary combinatorial formulations and coordinate treatment methods employ active compound in combination with the one or more secondary anti-hypertensive agent(s), or with one or more adjunctive therapeutic agent(s) that is/are useful for treatment or prophylaxis of the targeted (or associated) disease, condition and/or symptom(s) in the selected combinatorial formulation or coordinate treatment regimen
• active compound is formulated, or coordinately administered, in combination with one or more secondary or adjunctive therapeutic agent(s), to yield a combined formulation or coordinate treatment method that is combinatorially effective or coordinately useful to treat hyperlipidemia, hyperglycemia, hypertension, metabolic syndrome, diabetes, obesity, insulin resistance and/or one or more symptom(s) of a metabolic disorder or condition in the subject.
• Exemplary combinatorial formulations and coordinate treatment methods in this context employ active compound in combination with one or more secondary or adjunctive therapeutic agents selected from, e.g.,
• the secondary or adjunctive therapeutic agents used in combination with, e.g., berberine in these embodiments may possess direct or indirect lipid and/or glucose lowering activity and/or hypertension decreasing activity, including cholesterol lowering activity, insulin resistance decreasing activity, insulin sensitivity increasing activity or glucose regulating activity, alone or in combination with, e.g., berberine, or may exhibit other useful adjunctive therapeutic activity in combination with, e.g., berberine.
• Useful adjunctive therapeutic agents in these combinatorial formulations and coordinate treatment methods include, for example, anti- hyperlipidemic agents; anti-dyslipidemic agents; plasma HDL-raising agents; anti- hypercholesterolemic agents, including, but not limited to, cholesterol-uptake inhibitors; cholesterol biosynthesis inhibitors, e.g., HMG-CoA reductase inhibitors (also referred to as statins, such as lovastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, pitavastatin, and atorvastatin); HMG-CoA synthase inhibitors; squalene epoxidase inhibitors or squalene synthetase inhibitors (also known as squalene synthase inhibitors); acyl-coenzyme A cholesterol acyltransferase (ACAT) inhibitors, including, but not limited to, melinamide; probucol; nicotinic acid and
• Such agents may be referred to in whole or in part as metabolic disorder therapeutics, metabolic syndrome therapeutics, anti-obesity therapeutics, anti-hypercholesterolemia therapeutics, anti-diabetic therapeutics, insulin resistance therapeutic agents, anti-hyperglycemia agents, insulin sensitivity increasing agents, anti-hypertensive agents, and/or blood glucose lowering therapeutic agents.
• Adjunctive therapies may also be used including, but not limited, physical treatments such as changes in diet, psychological counseling, behavior modification, exercise and surgery including, but not limited to, gastric partitioning procedures, jejunoileal bypass, stomach stapling, gastric bands, vertical banded gastroplasty, laparoscopic gastric banding, roux-en-Y gastric bypass, biliopancreatic bypass procedures and vagotomy.
• Some herbal remedies may also be employed effectively in combinatorial formulations and coordinate therapies for treating metabolic disorders, for example curcumin, gugulipid, garlic, vitamin E, soy, soluble fiber, fish oil, green tea, carnitine, chromium, coenzyme QlO, anti-oxidant vitamins, grape seed extract, pantothine, red yeast rice, and royal jelly.
• metabolic disorders for example curcumin, gugulipid, garlic, vitamin E, soy, soluble fiber, fish oil, green tea, carnitine, chromium, coenzyme QlO, anti-oxidant vitamins, grape seed extract, pantothine, red yeast rice, and royal jelly.
• the active compound may be administered, simultaneously or sequentially, in a coordinate treatment protocol with one or more of the secondary or adjunctive therapeutic agents contemplated herein.
• active compound is administered coordinately with a non- berberine lipid lowering agent; a non-berberine glucose lowering agent; a non-berberine insulin sensitivity increasing agent; a non-berberine anti-diabetic agent; a non-berberine cholesterol lowering agent; a non-berberine triglyceride lowering agent, a non-berberine insulin resistance lowering agent; a non-berberine anti-hypertensive agent; or a non-berberine anti-obesity agent, or any other secondary or adjunctive therapeutic agent contemplated herein, using separate formulations or a combinatorial formulation as described above (i.e., comprising both active compound and a non-berberine therapeutic agent).
• This coordinate administration may be done simultaneously or sequentially in either order, and there may be a time period while only one or both (or all) active therapeutic agents individually and/or collectively exert their biological activities.
• a distinguishing aspect of all such coordinate treatment methods is that the active compound exerts at least some lipid lowering activity, some glucose lowering activity, and/or some hypertension lowering activity which yields a favorable clinical response in conjunction with a complementary agent, or distinct, clinical response provided by the secondary or adjunctive therapeutic agent.
• the coordinate administration of the active compound with the secondary or adjunctive therapeutic agent will yield improved therapeutic or prophylactic results in the subject beyond a therapeutic effect elicited by the active compound or the secondary or adjunctive therapeutic agent administered alone. This qualification contemplates both direct effects, as well as indirect effects.
• the active compound will be coordinately administered (simultaneously or sequentially, in combined or separate formulation(s)), with one or more secondary therapeutic agents as described above.
• the pharmaceutical compositions of the present invention may be administered by any means that achieve their intended therapeutic or prophylactic purpose. Suitable routes of administration for the compositions of the invention include, but are not limited to, oral, buccal, nasal, aerosol, topical, transdermal, mucosal, injectable, slow release, controlled release, iontophoresis, sonophoresis, and including all other conventional delivery routes, devices and methods. Injectable methods include, but are not limited to, intravenous, intramuscular, intraperitoneal, intraspinal, intrathecal, intracerebroventricular, intraarterial, subcutaneous and intranasal routes.
• compositions of the present invention may further include a pharmaceutically acceptable carrier appropriate for the particular mode of administration being employed.
• Dosage forms of the compositions of the present invention include excipients recognized in the art of pharmaceutical compounding as being suitable for the preparation of dosage units as discussed above. Such excipients include, without intended limitation, binders, fillers, lubricants, emulsifiers, suspending agents, sweeteners, flavorings, preservatives, buffers, wetting agents, disintegrants, effervescent agents and other conventional excipients and additives.
• the compositions of the invention can be administered in a controlled release form by use of a slow release carrier, such as a hydrophilic, slow release polymer.
• Controlled release agents in this context include, but are not limited to, hydroxypropyl methyl cellulose, having a viscosity in the range of about 100 cps to about 100,000 cps or other biocompatible matrices such as cholesterol.
• Compositions of the invention will often be formulated and administered in an oral dosage form, optionally in combination with a carrier or other additive(s).
• Suitable carriers common to pharmaceutical formulation technology include, but are not limited to, microcrystalline cellulose, lactose, sucrose, fructose, glucose, dextrose, or other sugars, di-basic calcium phosphate, calcium sulfate, cellulose, methylcellulose, cellulose derivatives, kaolin, mannitol, lactitol, maltitol, xylitol, sorbitol, or other sugar alcohols, dry starch, dextrin, maltodextrin or other polysaccharides, inositol, or mixtures thereof.
• Exemplary unit oral dosage forms for use in this invention include tablets, which may be prepared by any conventional method of preparing pharmaceutical oral unit dosage forms can be utilized in preparing oral unit dosage forms.
• Oral unit dosage forms such as tablets, may contain one or more conventional additional formulation ingredients, including, but not limited to, release modifying agents, glidants, compression aides, disintegrants, lubricants, binders, flavors, flavor enhancers, sweeteners and/or preservatives.
• Suitable lubricants include stearic acid, magnesium stearate, talc, calcium stearate, hydrogenated vegetable oils, sodium benzoate, leucine carbowax, magnesium lauryl sulfate, colloidal silicon dioxide and glyceryl monostearate.
• Suitable glidants include colloidal silica, fumed silicon dioxide, silica, talc, fumed silica, gypsum, and glyceryl monostearate.
• Substances which may be used for coating include hydroxypropyl cellulose, titanium oxide, talc, sweeteners and colorants.
• compositions of the invention can be prepared and administered in any of a variety of inhalation or nasal delivery forms known in the art.
• Devices capable of depositing aerosolized purified berberine related or derivative compounds of Formula XXVII formulations in the sinus cavity or pulmonary alveoli of a patient include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Methods and compositions suitable for pulmonary delivery of drugs for systemic effect are well known in the art. Additional possible methods of delivery include deep lung delivery by inhalation.
• Suitable formulations wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, may include aqueous or oily solutions of berberine related or derivative compounds of Formula XXVII and any additional active or inactive ingredient(s).
• Topical compositions may comprise the active compound along with one or more additional active or inactive component(s) incorporated in a dermatological or mucosal acceptable carrier, including in the form of aerosol sprays, powders, dermal patches, sticks, granules, creams, pastes, gels, lotions, syrups, ointments, impregnated sponges, cotton applicators, or as a solution or suspension in an aqueous liquid, non-aqueous liquid, oil-in-water emulsion, or water-in-oil liquid emulsion.
• a dermatological or mucosal acceptable carrier including in the form of aerosol sprays, powders, dermal patches, sticks, granules, creams, pastes, gels, lotions, syrups, ointments, impregnated sponges, cotton applicators, or as a solution or suspension in an aqueous liquid, non-aqueous liquid, oil-in-water emulsion, or water-in-oil liquid e
• compositions may comprise the active compound dissolved or dispersed in a portion of water or other solvent or liquid to be incorporated in the topical composition or delivery device.
• transdermal route of administration may be enhanced by the use of a dermal penetration enhancer known to those skilled in the art.
• Formulations suitable for such dosage forms incorporate excipients commonly utilized therein, particularly means, e.g. structure or matrix, for sustaining the absorption of the drug over an extended period of time, for example, 24 hours.
• Transdermal delivery may also be enhanced through techniques such as sonophoresis.
• Yet additional compositions of are designed for parenteral administration of the active compound(s), e.g.
• compositions of the invention may optionally contain anti -oxidants, buffers, bacteriostats and/or solutes which render the formulation isotonic with the blood of the mammalian subject; and aqueous and non-aqueous sterile suspensions which may include suspending agents and/or thickening agents.
• the formulations may be presented in unit-dose or multi-dose containers. Additional compositions and formulations of the invention may include polymers for extended release following parenteral administration.
• the parenteral preparations v may be solutions, dispersions or emulsions suitable for such administration.
• the subject agents may also be formulated into polymers for extended release following parenteral administration.
• Pharmaceutically acceptable formulations and ingredients will typically be sterile or readily sterilizable, biologically inert, and easily administered. Such polymeric materials are well known to those of ordinary skill in the pharmaceutical compounding arts.
• Parenteral preparations typically contain buffering agents and preservatives, and injectable fluids that are pharmaceutically and physiologically acceptable such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like. Extemporaneous injection solutions, emulsions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
• Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as described herein above, or an appropriate fraction thereof, of the active ingredient(s).
• compositions of the invention may comprise an active compound encapsulated for delivery in microcapsules, microparticles, or microspheres, prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacylate) microcapsules, respectively; in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules); or within macroemulsions.
• the methods and compositions of the invention may employ pharmaceutically acceptable salts, e.g., acid addition or base salts of the above-described active compounds.
• Suitable acid addition salts are formed from acids which form non-toxic salts, for example, hydrochloride, hydrobromide, hydroiodide, sulphate, hydrogen sulphate, nitrate, phosphate, and hydrogen phosphate salts.
• Additional pharmaceutically acceptable salts include, but are not limited to, metal salts such as sodium salts, potassium salts, cesium salts and the like; alkaline earth metals such as calcium salts, magnesium salts and the like; organic amine salts such as triethylamine salts, pyridine salts, picoline salts, ethanolamine salts, triethanolamine salts, dicyclohexylamine salts, N 5 N'- dibenzylethylenediamine salts and the like; organic acid salts such as acetate, citrate, lactate, succinate, tartrate, maleate, fumarate, mandelate, acetate, dichloroacetate, trifluoroacetate, oxalate, and formate salts; sulfonates such as methanesulfonate, benzenesulfonate, and p- toluenesulfonate salts; and amino acid salts such as arginate, asparginate, glutamate, tartrate, and
• Suitable base salts are formed from bases that form non-toxic salts, for example aluminum, calcium, lithium, magnesium, potassium, sodium, zinc and diethanolamine salts.
• the methods and compositions of the invention employ prodrugs of berberine related or derivative compounds of any of the Formulae described herein.
• Prodrugs are considered to be any covalently bonded carriers which release the active parent drug in vivo.
• Examples of prodrugs useful within the invention include esters or amides with hydroxyalkyl or aminoalkyl as a substituent, and these may be prepared by reacting such compounds as described above with anhydrides such as succinic anhydride.
• the invention disclosed herein will also be understood to encompass methods and compositions comprising related or derivative compounds of of any of the Formulae described herein using in vivo metabolic products of the said compounds (either generated in vivo after administration of the subject precursor compound, or directly administered in the form of the metabolic product itself). Such products may result for example from the oxidation, reduction, hydrolysis, amidation, esterif ⁇ cation, glycosylation and the like of the administered compound, primarily due to enzymatic processes. Accordingly, the invention includes methods and compositions of the invention employing compounds produced by a process comprising contacting any active compound as described herein with a mammalian subject for a period of time sufficient to yield a metabolic product thereof.
• Such products typically are identified by preparing a radiolabeled compound of the invention, administering it parenterally in a detectable dose to an animal such as rat, mouse, guinea pig, monkey, or to man, allowing sufficient time for metabolism to occur and isolating its conversion products from the urine, blood or other biological samples.
• the invention disclosed herein will also be understood to encompass diagnostic compositions for diagnosing the risk level, presence, severity, or treatment indicia of, or otherwise managing a hyperlipidemia and/or cardiovascular disease or condition in a mammalian subject, comprising contacting a labeled (e.g., isotopically labeled, fluorescent labeled or otherwise labeled to permit detection of the labeled compound using conventional methods) active compound as described herein to a mammalian subject (e.g., to a cell, tissue, organ, or individual) at risk or presenting with one or more symptom(s) of hyperlipidemia and/or cardiovascular disease, and thereafter detecting the presence, location, metabolism, and/or binding state (e.g., detecting binding to an unlabeled binding partner involved in LDL receptor physiology/metabolism) of the labeled active compound using any of a broad array of known assays and labeling/detection methods.
• a labeled e.g., isotopically labeled,
• the invention disclosed herein will also be understood to encompass diagnostic compositions for diagnosing the risk level, presence, severity, or treatment indicia of, or otherwise managing a metabolic disorder disease or condition in a mammalian subject, comprising contacting a labeled (e.g., isotopically labeled, fluorescent labeled or otherwise labeled to permit detection of the labeled compound using conventional methods) active compound to a mammalian subject (e.g., to a cell, tissue, organ, or individual) at risk or presenting with one or more symptom(s) of metabolic disorders, and thereafter detecting the presence, location, metabolism, and/or binding state (e.g., detecting binding to an unlabeled binding partner involved in InsR receptor physiology/metabolism) of the labeled compound using any of a broad array of known assays and labeling/detection methods.
• a labeled e.g., isotopically labeled, fluorescent labeled or otherwise labeled to permit detection of the label
• the invention disclosed herein will further be understood to encompass diagnostic compositions for diagnosing the risk level, presence, severity, or treatment indicia of, or otherwise managing a hyperglycemic disease or condition in a mammalian subject, comprising contacting a labeled (e.g., isotopically labeled, fluorescent labeled or otherwise labeled to permit detection of the labeled compound using conventional methods) active compound to a mammalian subject (e.g., to a cell, tissue, organ, or individual) at risk or presenting with one or more symptom(s) of hyperglycemia, and thereafter detecting the presence, location, metabolism, and/or binding state (e.g., detecting binding to an unlabeled binding partner involved in InsR receptor physiology/metabolism) of the labeled compound using any of a broad array of known assays and labeling/detection methods.
• a labeled e.g., isotopically labeled, fluorescent labeled or otherwise labeled
• the invention disclosed herein will additionally be understood to encompass diagnostic compositions for diagnosing the risk level, presence, severity, or treatment indicia of, or otherwise managing insulin resistance in a mammalian subject, comprising contacting a labeled (e.g., isotopically labeled, fluorescent labeled or otherwise labeled to permit detection of the labeled compound using conventional methods) active compound to a mammalian subject (e.g., to a cell, tissue, organ, or individual) at risk or presenting with one or more symptom(s) of insulin resistance, and thereafter detecting the presence, location, metabolism, and/or binding state (e.g., detecting binding to an unlabeled binding partner involved in InsR receptor physiology/metabolism) of the labeled compound using any of a broad array of known assays and labeling/detection methods.
• a labeled e.g., isotopically labeled, fluorescent labeled or otherwise labeled to permit detection of the labeled compound using conventional
• the invention disclosed herein will also be understood to encompass diagnostic compositions for diagnosing the risk level, presence, severity, or treatment indicia of, or otherwise managing a hypertensive disease or condition in a mammalian subject, comprising contacting a labeled (e.g., isotopically labeled, fluorescent labeled or otherwise labeled to permit detection of the labeled compound using conventional methods) active compound to a mammalian subject (e.g., to a cell, tissue, organ, or individual) at risk or presenting with one or more symptom(s) of hypertension, and thereafter detecting the presence, location, metabolism, and/or binding state (e.g., detecting binding to an unlabeled binding partner involved in InsR receptor physiology/metabolism) of the labeled compound using any of a broad array of known assays and labeling/detection methods.
• a labeled e.g., isotopically labeled, fluorescent labeled or otherwise labeled to permit detection of
• the invention disclosed herein will further be understood to encompass diagnostic compositions for diagnosing the risk level, presence, severity, or treatment indicia of, or otherwise managing diabetes in a mammalian subject, comprising contacting a labeled (e.g., isotopically labeled, fluorescent labeled or otherwise labeled to permit detection of the labeled compound using conventional methods) active compound to a mammalian subject (e.g., to a cell, tissue, organ, or individual) at risk or presenting with one or more symptom(s) of diabetes, and thereafter detecting the presence, location, metabolism, and/or binding state (e.g., detecting binding to an unlabeled binding partner involved in InsR receptor physiology/metabolism) of the labeled compound using any of a broad array of known assays and labeling/detection methods.
• a labeled e.g., isotopically labeled, fluorescent labeled or otherwise labeled to permit detection of the labeled compound using conventional methods
• the invention disclosed herein will also be understood to encompass diagnostic compositions for diagnosing the risk level, presence, severity, or treatment indicia of, or otherwise managing a metabolic syndrome disease or condition in a mammalian subject, comprising contacting a labeled (e.g., isotopically labeled, fluorescent labeled or otherwise labeled to permit detection of the labeled compound using conventional methods) active compound to a mammalian subject (e.g., to a cell, tissue, organ, or individual) at risk or presenting with one or more symptom(s) of metabolic syndrome, and thereafter detecting the presence, location, metabolism, and/or binding state (e.g., detecting binding to an unlabeled binding partner involved in InsR receptor physiology/metabolism) of the labeled compound using any of a broad array of known assays and labeling/detection methods.
• a labeled e.g., isotopically labeled, fluorescent labeled or otherwise labeled to permit detection of the label
• the experiments further demonstrate the novel and powerful uses for berberine compounds and berberine related and derivative compounds in decreasing insulin resistance, increasing glucose consumption, and decreasing serum insulin.
• berberine acts on the insulin receptor (InsR) through a second pathway that differs from the pathway that leads to an increase in LDLR expression
• InsR insulin receptor
• HepG2 cells treated with berberine had an increased expression of InsR.
• both hyperglycemic rats and humans treated with berberine had decreased levels of blood glucose and increased levels of InsR.
• berberine decreased the levels of cholesterol, triglycerides and LDL protein in all of the treated animals.
• a dose of 50/mg/kg/day of berberine reduced LDL by 26% and a dose of 100 mg/kg/day reduced LDL by 42%.
• Reductions in serum LDL were observed by day 5 and became significant by day 7 at both doses (Figure 5).
• LDLR mRNA and protein expressions were examined by quantitative real-time RT-PCR and western blot analysis.
• reverse transcription with random primers using Superscript II at 42°C for 30 minutes with 1 ⁇ g of total RNA was performed using the ABI Prism 7900-HT Sequence Detection System and Universal MasterMix (Applied Biosystems, Foster City, CA).
• LDLR and GAPD mRNA expression levels were determined using the human LDLR and GAPD Pre-developed TaqMan Assay Reagents (Applied Biosystems).
• LDLR mRNA and protein levels were elevated in all berberine treated hamsters in a dose dependent manner. There was a 3.5 fold increase in mRNA and a 2.6 fold increase in protein in hamster livers treated with 100 mg/kg/day of berberine.
• BBR did not change kidney functions (as determined by measurements of creatine, blood urea nitrogen, and total bilirubin in treated and placebo subjects), but substantially improved liver function — reducing levels of alanine aminotransaminase, aspartate aminotransaminase, and gama glutamyl transpeptidase, by approximately 48%, 36%, and 41%, respectively.
• the placebo group showed no significant changes in these parameters.
• Bel-7402 cells were treated with 0, 0.5, 1, 2.5, 5, ⁇ g/ml of berberine or 2.5, 7.5 and 15 ⁇ g/ml of berberine sulfate. The cells were then centrifuged and washed and LDLR mRNA was extracted. LDLR mRNA levels were then measured using scan quantitative RT- PCR, ( Figure 3 A and B). As can be seen in Figure 3 A and B, treatment with berberine and berberine sulfate increased LDLR mRNA expression in a dose dependent fashion with 5 ⁇ g/ml berberine increasing LDLR mRNA expression 2.3 fold. Berberine also increased LDLR protein expression on the surface of BEL-7402 cells.
• Bel-7402 cells treated with 5 ⁇ g/ml of berberine were detached with cell removal buffer containing EDTA, washed and resuspended in FACS solution (PBS with 0.5% BSA and 0.02% sodium azide) at a density of 1 x 10 6 cells/ml. Cells were then incubated with monoclonal antibody to LDLR (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a final dilution of 1 :50 and left at room temperature for 1 hour. The cells were then reacted with isotope matched, nonspecific mouse IgG as a control for nonspecific staining.
• FACS solution PBS with 0.5% BSA and 0.02% sodium azide
• the cells were then washed and stained with FITC conjugated goat antibody to mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, 1 :100 dilution) and the fluorescence intensity was analyzed by FACS (FACSort, Becton Dickinson, Franklin Lakes, NJ). As can be seen in Figure 4, berberine increased cell surface LDLR protein expression 4 times.
• Rats were fed a high fat high cholesterol (HFHC) diet for 10 days, and then divided into groups of seven. The rats were then administered berberine or simvastatin, or a combination of berberine and simvastatin orally for 25 days. After 25 days, serum cholesterol, triglyceride and LDL-c levels were measured. As can be seen in Table 8, treatment with berberine significantly decreased the cholesterol, triglycerides and LDL-c levels in the rats and was more effective than simvastatin in lowering triglyceride and LDL-c levels. The combination of simvastatin, and berberine lowered the cholesterol, triglyceride and LDL-c levels further than either alone.
• HFHC high fat high cholesterol
• HepG2 cells were cultured with either berberine hydrochloride or GW707 as a positive control for 8 hours. Total cell lysates from untreated cells or cells treated with either berberine or GW707 were then harvested and analyzed by Western blot. As can be seen in Figure 7, GW70 substantially increased the amount of the mature form of SREB-2, whereas berberine had no effect. These data indicate that berberine effectively increases LDLR expression by a mechanism distinct from that used by statins, thereby further evincing that this novel drug and its related and derivative compounds will provide useful anti-hyperlipidemic formulations and methods with minimal side effects attributed to other known anti- hyperlipidemic drugs.
• HepG2 cells were cultured in LPDS medium and were then untreated, treated with lovastatin at 0.5 and l ⁇ M concentrations with or without berberine for 24 hours, or were treated with berberine alone.
• berberine and lovastatin had additive stimulation effects on LDLR mRNA expression, which data evince general utility of the novel, combinatorial formulations and coordinate treatment methods describe herein above.
• HepG2 cells were transfected with the reporter construct pLDLR234Luc, which contains the SRE-I motif and the sterol-independent regulatory element that mediates the cytokine oncostatin M-induced transcription of the LDLR gene. After transfection, cells were cultured in 0.5% lipoprotein depleted fetal bovine serum (LPDS) or LPDS and cholesterol medium followed by an 8 hour treatment with berberine (lO ⁇ g), GW707 (2 ⁇ g) or oncostatin M (50 ng/ml). As can be seen in Figure 9, LDLR promoter activity was strongly elevated by GW707 and oncostatin M under both culturing conditions. Berberine had no effect, further evincing that this compound operates via a different mechanism of LDLR regulation compared to other known drugs possessing anti-hyperlipidemic activity.
• LPDS lipoprotein depleted fetal bovine serum
• GW707 2 ⁇ g
• oncostatin M 50 ng/ml
• HepG2 cells were cultured and then left alone or treated with berberine for 15 hours. After 15 hours, actinomycin D (5 ⁇ g/ml) was added to cells at 0, 20, 40, 60, 90, 120, or 150 minutes. Total mRNA was isolated and analyzed by Northern blot for the amount of LDLR mRNA. As can be seen in Figure 10, berberine prolonged the turnover rate of LDLR transcript by approximately threefold. In contrast, the mRNA stability of HMG-CoA reductase was not altered by berberine.
• LDLR 3'UTR Three consecutive fragments of LDLR 3'UTR were inserted into a cytomegalovirus promoter driven Luc plasmid (pLuc) at the 3' end of the Luc coding sequence before the SV40 polyadenylation signal.
• the wild-type Luc reporter plasmid pLuc was constructed by insertion of the Luc cDNA into the Hindlll and Xba sites of pcDNA3.1/Zeo(+). Addition of the LDLR 3/UTR was accomplished by PCR amplifying different regions of the
• a PCR reaction was performed to amplify a 550 base pair fragment of Luc coding region with 5' primer Luc-2up (5'- GCTGGAGAGCAACTGCARAAGGC-3') (SEQ ID NO.l) and the 3' primer Zwc-21o (5'- GCAGACCAGTAGATCCAGAGG-S') (SEQ ID NO:2) using pGL3-basic as the template.
• the PCR fragment was labeled with P and used in the northern blot analysis to measure expression of Luc mRNA and Zwc-LDLR 3'UTR chimeric fusion.
• ARE deletion constructs an Apa site at nt 3,384 was generated for deleting ARE3, and an Apal site at nt 3,334 for deleting ARE2 by site-directed mutagenesis using pLuc/UTR-2 as the template. Mutated plasmids were cut with Apal to remove the ARE- containing region and then the remaining vector was religated with the 5' proximal region of UTR-2. To create the UCAU motif deletion, two SacII sites for internal deletion of nt 3.062- 3,324 were generated using UTR-2 as the template. All constructs were sequenced and the correct clones were further propagated to isolate plasmid DNA.
• HepG2 or Bel-7402 cells were treated with berberine for 0.25, 0.5, 0.75, 1, 2, 8, and 24 hours respectively and tested for levels of activated ERK by western blotting using antibodies that only recognize the activated (phosphorylated) ERK.
• berberine rapidly activated ERK and the kinetics of ERK activation preceded the upregulation of LDLR expression by berberine ( Figure 14A and B).
• the activation of berberine is also dose dependent ( Figure 14C).
• the rabbit blood protein binding rate was measured by in vitro dialysis at a rate of
• mice were injected in the tail vein with
• rats were orally administered 3H- bereberine. Forty-eight hours after administration, excretions were tested for the presence of berberine. 2.7% of the oral dose was measured in the urine and 86% of the oral dose was measured in the fecal matter.
• Rats and mice were administered berberine through a variety of techniques, including orally, through subcutaneous injection, intraperitoneal injection and intravenous injection.
• Toxicity through subcutaneous injection was LD 50 7970-10690mg/kg.
• mice In mice, toxicity was achieved with an oral dose of LD 50 > 29586-4500 mg/kg.
• Toxicity through subcutaneous injection was LD 50 13.9-20 mg/kg.
• Toxicity through intraperitoneal injection was LD 50 30-32.2 mg/kg and LD 50 7.6-10.2 mg/kg with intravenous injection.
• rats were administered 300mg/kg of berberine orally for 182 days. No abnormalities were found in blood tests, blood biochemistry, urine analysis or histopathology
• Day 15 rats treated for 15 days
• the livers were dissected and stored in liquid nitrogen for RNA extraction, realtime RT-PCR and PKC activity assay. Total blood samples were also collected to assay fasting blood glucose and serum insulin levels. The insulin levels were analyzed using radioimmunoassay (Linco Research, St Charles, MO). The insulin sensitivity indexes (ISI) were calculated according to the formula: 10 4 / (fasting serum insulin X fasting blood glucose) (Hanson, Am. J. Epidemiol. 15 1(2), 190-198 (2000)). The insulin level and ISI of normal rats were also determined for comparison. As can be seen in Figure 32, the HFHC diet significantly reduced insulin sensitivity (p ⁇ 0.001).
• sample protein was mixed with 5 ⁇ l of PepTag® Cl peptide (specific substrate of PKC), 5 ⁇ l of reaction buffer and 5 ⁇ l of PKC activator solution in a 25 ⁇ l reaction system.
• the reactions were performed at 30°C for 30 minutes.
• the samples were loaded onto a 0.8% agarose gel.
• electrophoresis the phosphorylated and nonphosphorylated PepTag® Cl peptide were separated, with the phosphorylated ones negatively charged.
• the gels were photographed under an UV light. The bands containing the phosphorylated substrate were then excised and melted.
• the lipid profile of the rats was also measured after the 14 day treatment with berberine. As can be seen in figure 33, 150 mg/kg/day of berberine reduced cholesterol by 25%, LDL-c by 33% and triglyceride by 24% (p ⁇ 0.01, **0.01 and *0.05 respectively), as compared to the control animals administered the same HFHC diet.
• the therapeutic efficacy observed in this animal model reflects a synergistic effect of berberine on InsR and LDLR, which antagonizes insulin resistance and significantly improves sugar- and lipid- metabolism in vivo.
• Blood samples were taken both before and after two months of berberine treatment. Fasting blood levels of glucose, LDL-c, cholesterol, HDL-c, triglyceride and blood insulin were measured using standard methods routinely applied in hospitals. Liver and kidney functions were also monitored in the patients.
• FBG Fasting blood glucose
• BBR treatment lg/day, Bid, 2 months; Values are means ⁇ SEs.
• BMI Body-Mass-Index
• RNA samples 10 or 15 ⁇ g/ml of berberine respectively for eight hours.
• Total cellular RNA was isolated using the Ultraspec RNA lysis solution (Biotecxs Laboratory, Houston, TX) following the vender's protocols. lOpg of the RNA sample was transferred to nitrocellulose membrane via a slot-blot apparatus (Schleicher & Schuell, Keene, NH). The blots were fixed by baking at 80°C for 2h, followed by hybridization to a 0.89-kb long, 32P-labelled human InsR cDNA probe. The same membranes were then stripped and re-hybridized to a human ACTB probe as internal control. Quantitative real time RT-PCR assays were also done.
• RNA was reverse-transcribed into cDNA using the Reverse Transcription System (Promega, Madison, WI). Quantitative real-time PCR were performed with these cDNA using the Applied Biosystems 7500 Real-Time PCR System and TaqMan® Universal PCR Master Mix (Applied Biosystems, Foster City, CA). All of the 20 X TaqMan® Gene Expression Assay reagents containing gene-specific primers and TaqMan® probes for human or rat InsR, ACTB and LDLR were purchased from Applied Biosystems.
• RNA was isolated as described above for slot blot and RT-PCR assays of InsR mRNA and ACTB mRNA expression.
• the level of InsR mRNA increased 4 h after exposure of cells to berberine and reached the peak level of 2.5-fold of the control at 8 h; the expression of InsR mRNA remained high for at least 24 h ( Figure 16).
• siRNA transfection medium containing 6 ⁇ l of human InsR siRNA duplex (or control siRNA) and 6 ⁇ l of siRNA transfection reagent were well mixed at room temperature for 30 min, followed by loading onto washed HepG2 cells. After an 8 hr incubation, the transfection mixture was removed and fresh RPMI- 1640 medium supplied with 10% FBS was added. The cells were then incubated for 24 hr. Then, the culture medium was replaced with fresh medium and incubated for an additional 24 hr. At the end of incubation, FBS containing medium was removed and replaced with serum-free fresh RPMI- 1640 medium.
• InsR siRNA transfected or untransfected cells 7.5 ⁇ g/ml of berberine and/or 0.5 nM of insulin were added to the InsR siRNA transfected or untransfected cells. After 12 hr incubation, the amount of glucose in the sample medium was determined. The glucose consumption was calculated according to the following formula: glucose level of the fresh RPMI- 1640 minus glucose level of the used RPMIN- 1640. Inhibition of InsR mRNA and protein expression by InsR siRNA was confirmed by either real-time RT-PCR or Western-Blot analysis.
• HepG2 cells were treated or untreated with 7.5 ⁇ g/ml of berberine for 8h. Then, 5 ⁇ g/ml of actinomycin D was added to block the transcription. Total cellular RNA was harvested at 0, 2, 4, 6, or 8 hours after actinomycin D treatment, and slot-blotted to nitrocellulose membranes as described above. The membranes were respectively hybridized with InsR and ACTB specific probes as described above, and bands were quantitated through densitometry. The InsR mRNA levels were normalized to ACTB, and their remaining percentages are plotted against time and the decaying rate or half-life of InsR mRNA was calculated (Figure 21B).
• the InsR gene promoter contains a 1.8 kb long segment (Mitchell, Science 245, 371-378 (1989); Araki J. Biol. Chem. 262, 16186-16191 (1987)).
• the InsR promoter luciferase gene fusion plasmid (pGL3-1.5kIRP) was kindly provided by Dr. Araki E of the graduate School of Medical Sciences, Kumamoto University, Honjo, Kumamoto, Japan.
• 1.5 kb fragment of the human insulin receptor gene promoter was inserted into the Hind III site ofpGL3 -basic vector forming pGL3-l. SkIRP fusion plasmid (Nakamaru, Biochem Biophys Res Commun. 328 (2) 449-454 (2005)).
• HepG2 cells (2 X 105) were transfected with 1 ⁇ g of the pGL3-I.SkIRP using the
• HepG2 cells were pretreated with each of the inhibitors 1 hour prior to treatment with 7.5 ⁇ g of berberine for 8 hours. Total RNA was then isolated and the relative amount of InsR and LDLR mRNA was measured by quantitative RT-PCR as described in Example XVII. It was determined that the activity of berberine on InsR gene transcription was most sensitive to the PKC inhibitor calphostin C.
• pGL3- 1.5kIRP transfected HepG2 cells were either not treated, treated with 0.2 ⁇ M of calphostin C, 5 ⁇ g/ml of berberine or 0.5 ⁇ M of PKC activator phorbol-12-myristate- 13 -acetate (PMA) (Gandino, Oncogene, 5 (%), 721-725 (1990) or combinations thereof for eight hours.
• PMA PKC activator phorbol-12-myristate- 13 -acetate
• the bands containing the phosphorylated substrate were then excised and melted. They were transferred to a 96-well plate and quantified using densitometry according to the supplier's protocol.
• the catalytic activity of total PKC of a specific sample was expressed as pmol/min/mg, representing the number of picomoles of phosphate transferred to the substrate per minute per milligram of proteins of the sample.
• PKC activity was increased in liver cells treated with berberine in a time-dependent fashion; the elevation of PKC activity was first observed at 0.5 hr (after berberine treatment) and went up with time.
• the kinetics of PKC activation preceded the upregulation of InsR expression by berberine.
• Example XXII Effect of berberine on blood glucose in hyperglycemic patients with type II diabetes
• rosiglitazone (Glaxowelcome, UK), orally for two months. Of these 21 patients, ten had hyperlipidemia and four had hypertension. Metformin and rosiglitazone served as reference controls as they are standard treatments for type II diabetes. Statistical analysis of the baselines of fasting blood glucose, HbAIc and triglycerides showed no significant differences among the groups prior to treatment (p>0.05).
• Type 2 diabetes (normal range) (2 months) Type 2 diabetes Type 2 diabetes Type 2 diabetes
• FBG Fasting blood glucose
• Example XXIII Determination of an increase in InsR expression in patients treated with berberine
• PBL Peripheral blood lymphocytes
• InsR protein expressed on the surface of peripheral blood lymphocytes were stained with monoclonal antibody against human insulin receptor (Pharmagen, San Diego, CA) and analyzed in a flow cytometer (BD and Company, San Jose, CA)
• RNA samples from the human hepatoma cell line HepG2 were incubated with 0, 2.5, 5, and 10 ⁇ g/ml of berberine or demethylberberine (LlOl) respectively for eight hours.
• Total cellular RNA was isolated using the Ultraspec RNA lysis solution (Biotecxs Laboratory, Houston, TX) following the vender's protocols.
• lOpg of the RNA sample was transferred to nitrocellulose membrane via a slot-blot apparatus (Schleicher & Schuell, Keene, NH). The blots were fixed by baking at 80 0 C for 2h, followed by hybridization to a 0.89-kb long, 32P- labelled human InsR cDNA probe.
• RNA samples from the human hepatoma cell line HepG2 were incubated with 1 O ⁇ g of berberine, berberrubine, thalifendine, demthyleneberberine, or jatorrhizine for 8 hours at 37 0 C.
• Total cellular RNA was isolated using the Ultraspec RNA lysis solution (Biotecxs Laboratory, Houston, TX) following the vender's protocols. lOpg of the RNA sample was transferred to nitrocellulose membrane via a slot-blot apparatus (Schleicher & Schuell, Keene, NH).
• blots were fixed by baking at 80°C for 2h, followed by hybridization to a 0.89-kb long, 32P- labelled human InsR cDNA probe. The same membranes were then stripped and re-hybridized to a human ACTB probe as internal control. Quantitative real time RT-PCR assays were also done. For the RT-PCR assay, total cellular RNA was reverse-transcribed into cDNA using the Reverse Transcription System (Promega, Madison, WI). Quantitative real-time PCR were performed with these cDNA using the Applied Biosystems 7500 Real-Time PCR System and TaqMan® Universal PCR Master Mix (Applied Biosystems, Foster City, CA).
• InsR expression was also increased by treatment with the berberine metabolites though they were less effective than berberine itself. As shown in Figure 41 and Table 13, each of the berberine metabolites increased InsR expression at least 50% more than the control.
• Example XXIX Measurement of plasma concentration of Berberine and Berberrubine following administration of prodrugs of Berberrubine.
• 18 Wistar rats (180-200 gm each) were divided into six groups. Each group received a single oral dose (300mg/kg) of either berberine, berruberine, or a berruberine prodrug. Blood samples were taken 1 hour and 3 hours after administration of the dose.
• ester prodrugs were absorbed from the gut. As can be seen in Table 14, all four ester prodrugs were cleaved by blood esterases, resulting in serum levels of berberrubine. Additionally, administraton of berberrubine ester prodrugs resulted in higher serum levels of berberrubine than administration of berberine.
• Dresner, A Laurent, D., Marcucci, M., Griffin, MB., Dufour, S., Cline, G. W., Slezak, L.A,
• Eckel, RH Lipoprotein lipase. A multifunctional enzyme relevant to common metabolic diseases. N Engl J Med 320, 1060-1068 (1989).
• LDL LDL receptor up-regulator
• Estrogen receptor- ⁇ and SpI interact in the induction of the low density lipoprotein-receptor. J
• Haffner SM D'Agostino R Jr, Mykkanen L, Tracy R, Howard B, Rewers M, Selby J, Savage PJ,
• Hsu HY Nicholson AC, Hajjar DP. Basic fibroblast growth factor-induced low density lipoprotein receptor transcription and surface expression. Signal transduction pathways mediated by the bFGF receptor tyrosine kinase. J Biol Chem 269:9213-9220 (1994). Hua X, Nohturfft A, Goldstein JL, Brown MS. Sterol resistance in CHO cells traced to point mutations in SREBP cleavage activating protein (SCAP). Cell 87:415-426 (1996).
• SCAP SREBP cleavage activating protein
• LDLR low density lipoprotein receptor
• Mehta KD Role of mitogen-activated protein kinases and protein kinase C in regulating Io w- density lipoprotein receptor expression. Gene Expr 10:153-164 (2002).
• Bile acids enhance low density lipoprotein receptor gene expression via a MAPK cascade-mediated stabilization of mRNA. J Biol Chem 277:37229-37234 (2002).
• Ness GC Thyroid hormone. Basis for its hypocholesterolemic effect. J FIa Med Assoc 78:383- 385 (1991).
• G.M A new rat model of type II diabetes: the fat-fed, streptozotocin-treated rat. Metabolism
• Saltiel AR Kahn CR. Insulin signalling and the regulation of glucose and lipid metabolism.
• Seino S Seino M
• Nishi S Bell GI. Structure of the human insulin receptor gene and characterization of its promoter. Proc Natl Acad Sci U S A. 86(1):114-8 (1989).
• Singh RP Dhawan P, Golden C, Kapoor GS, Mehta KD.
• One-way cross-talk between p38 MAPK and p42/44 MAPK Inhibition of p38 MAPK induces low density lipoprotein receptor expression through activation of p42/44 MAPK cascade.
• Vasudevan AR Balasubramanyam A.
• Thiazolidinediones a review of their mechanisms of insulin sensitization, therapeutic potential, clinical efficacy, and tolerability. Diabetes Technol
• Wilson GM Roberts EA, Deeley RG. Modulation of LDL receptor mRNA stability by phorbol esters in human liver cell culture models. J Lipid Res 38:437-446 (1997).
• the human LDL receptor a cysteine-rich protein with multiple AIu sequences in its mRNA. Cell 39:27-38 (1984).
• SREBP-I a basic-helix-loop-helix-leucine zipper protein that controls transcription of the low density lipoprotein receptor gene. Cell 75:187-197 (1993).
• Zeng X Zeng X. Relationship between the clinical effects of berberine on severe congestive heart failure and its concentration in plasma studied by HPLC. Biomed Chromatogr. 13(7):442-

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