WO2018206719A1 - Compounds for upregulating ucp1 expression and promoting transdifferentiation into brown adipose tissue - Google Patents

Compounds for upregulating ucp1 expression and promoting transdifferentiation into brown adipose tissue Download PDF

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WO2018206719A1
WO2018206719A1 PCT/EP2018/062136 EP2018062136W WO2018206719A1 WO 2018206719 A1 WO2018206719 A1 WO 2018206719A1 EP 2018062136 W EP2018062136 W EP 2018062136W WO 2018206719 A1 WO2018206719 A1 WO 2018206719A1
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formula
compound
composition
compounds
use according
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PCT/EP2018/062136
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French (fr)
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Karin Brecht BRÜNGGER
Harald Heider
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Evolva Sa
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    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/02—Ethers
    • C07C43/20—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring
    • C07C43/23—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring containing hydroxy or O-metal groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C65/01—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups
    • C07C65/105—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups polycyclic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C65/01—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups
    • C07C65/19—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups having unsaturation outside the aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C65/21—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing ether groups, groups, groups, or groups
    • C07C65/28—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing ether groups, groups, groups, or groups having unsaturation outside the aromatic rings

Definitions

  • the invention disclosed herein relates to treatment of metabolic diseases such as obesity, diabetes, and other related diseases or conditions. This can be done by inducing browning of adipose tissue and/or up-regulation of uncoupling Protein 1 (UCPl) in brown adipose tissue deposits or by administration of specific compounds and compositions.
  • UCPl uncoupling Protein 1
  • the invention provides compounds and compositions that significantly upregulate and/or induce expression of UCPl and/or promote
  • Mammalian white adipose tissue is used as a store of energy.
  • Brown adipose tissue is a specialized form of adipose tissue found mainly in newborns and hibernating mammals. This specialized tissue can generate heat by uncoupling the respiratory chain of oxidative phosphorylation within mitochondria. The process of uncoupling means that when protons transit down the electrochemical gradient across the inner mitochondrial membrane, energy is released as heat rather than being used to generate ATP.
  • This thermogenic process may be vital in neonates exposed to cold, which require this thermogenesis to keep warm, as they are unable to shiver, or take other actions to keep themselves warm. Recent studies have identified the presence of brown adipocytes in adult humans (Virtanen et al., N.
  • Brown adipocytes appearing in white adipose tissue are often called inducible brown adipocytes (or beige, brown-in-white, or BRITE adipocytes). The appearance of these inducible brown adipocytes in white adipose tissue may also involve a
  • Uncoupling Proteins are mitochondrial transmembrane proteins that decrease the proton gradient generated in oxidative phosphorylation, by increasing the permeability of the inner mitochondrial membrane, thereby allowing protons that have been pumped into the intermembrane space to return to the mitochondrial matrix.
  • UCPl also known as thermogenin, is an exclusive hallmark of brown adipocytes in mammals.
  • UCP1 functions as a proton transporter, allowing the dissipation of the energy of the proton gradient generated by the respiratory chain, and thereby uncoupling oxidative phosphorylation. This results in heat generation, instead of ATP production (“non-shivering thermogenesis").
  • the ability of UCPl-expressing adipocytes to produce heat and thus increase energy expenditure may also alter energy expenditure and body mass in humans.
  • brown fat to burn energy instead of storing it, has important health implications, notably relating to weight loss, obesity, diabetes, and other pathologic conditions.
  • conversion or transdifferentiation of white adipose tissue into brown adipose tissue would be a useful tool in establishing and maintaining metabolic health and warding off or combating obesity and diabetes.
  • This process the acquisition by white adipose tissue of characteristics of brown adipose tissue— is also commonly referred to as "browning.”
  • SPPARMs Selective PPARy modulators
  • PPARy ligands devoid of the detrimental side effects of the classical glitazones.
  • Selective PPARy modulators (SPPARMs)have been shown to bind in distinct manners to the ligand-binding pocket of PPARy, leading to alternative receptor conformations and cofactor recruitment/displacement, as well as differential gene expression leading to differential biological responses.
  • SPPARMs have been shown to display favorable pharmacological features, with insulin-sensitizing and glucose-lowering properties without increasing body weight (DePaoli et al., Diabetes Care 37, 1918-1923 (2014; Gregoire et al., Mol. Endocrinol. 23 :975-988 (2009); Schupp et al., Diabetes 54: 3442-3452 (2005)).
  • WISSENSCHAFTEN E.V. ; BOYCE THOMPSON INSTITUTE FOR PLANT RESEARCH INC.) 6 November 2014 discloses amorfrutin analogues suitable to selectively modulate the PPAR3gamma.
  • WO 02/14252 A2 HOPPE, D.C. and INMAN, W.D. 21 February 2002 discloses several hypoglycemically active stilbenoids. These all have a fully aromatic backbone, and thus have a fixed three-dimensional structure. The double bond in the linker portion between the two aromatic moieties, does not allow rotation around, and thus lock the two aromatic moieties into a plane.
  • WEIDNER C. et al. : "Amorfrutins are potent antidiabetic dietary natural products", PNAS, 2012, Vol. 109, no. 19, pages 7257-7262 discloses an amorfrutin 1 tested in a mouse model for insulin resistance.
  • WO 02/13811 A2 INSMED CORPOATION 21 February 2002 hypoglycemically active stilbenoids. These all have a fully aromatic backbone, and thus have a fixed three- dimensional structure.
  • WO02/013809 A2 (INMAN, W. D. and HOPPE, D.C.) 21 February 2002 several hypoglycemically active stilbenoids. These all have a fully aromatic backbone, and thus have a fixed three-dimensional structure.
  • WO 2010/094760 Al (DSM IP ASSETS B.V.) 26 August 2010 discloses a compound A which has a fully aromatic backbone, and thus have a fixed three-dimensional structure.
  • EP 2796442 Al (INSTITUTE OF MEDICINAL BIOTECHNOLOGY; CHINESE ACADEMY OF MEDICAL SCIENCES) 29 October 2014 discloses among others an amorfrutin as an anti-viral compound lOv. TONTONOZ, P. and SPIEGELMAN, B.M. :"Fat and Beyond : The Diverse Biology of PPARy", ANNUAL REVIEWS OF BIOCHEMISTRY, 2008, Vol. 77, pages 289-312 discloses rosiglitazone. This molecule does not have a recognition site for
  • amorfrutin 1 The exact mechanism by which the amorfrutins exert their beneficial effects on conditions of metabolic disorders have not been elucidated yet. Partially those effects may be due to the fact that as dislosed herein for amorfrutin 1, it does not promote, in contrast to rosiglitazone, differentiation of pre-adipocytes into mature adipocytes (see figure 9B in this patent application).
  • compositions comprising one or more compounds, and methods for the treatment of obesity, diabetes, and/or other related diseases or conditions by converting white adipose cells or adipose precursor cells into BRITE (brown in white) cells, inducing UCP1 in such BRITE cells, and/or inducing UCP1 in brown adipose tissue deposits without concomitantly increasing overall differentiation of pre-adipocytes into adipocytes.
  • BRITE blue in white
  • An object of the present invention relates to a compound for the regulation of metabolism in a mammal.
  • Ri is -H, -CO2H, -OH, -CO2CH3, -CO2C2H5, -C0 2 CH(CH 3 )2, -CONH2, CONHCH3, or CON(CH 3 ) 2 ;
  • R 2 is -H, -OH, -OCH3, or -OC2H5;
  • R2 and Rs taken together with the atoms to which they are connected represent a group of the formula
  • R3 and Rs together with the atoms to which they are connected represent a group of the formula
  • Px6 is alkyl or a group of the formula
  • Re represents -H, -OH, -OCH3, -OC2H5, or ;
  • Rg represents -H, -OH, -OCH3, -OC2H5, or
  • Preferred compounds of Formula I include those where R6 represents propyl.
  • the compound an Amorfrutin is the compound an Amorfrutin.
  • the present invention is the compound Amorfrutin 1, having the chemical structure of Formula II: (Formula II).
  • Amorfrutin 1 has been tested in the examples of the present disclosure, and shown to upregulate Amorfrutin 1 and furthermore to have a positive effect on the WAT to BAT transdifferentiation.
  • the compound Amorfrutin 2 having the chemical structure of Formula III
  • Formula V and Formula VI are also known as "Radula compound 1" and “Radula compound 2", respectively.
  • Radula 1 is also known as stilbene carboxylate 1 (figure IE)
  • Radula 2 is also known as stilbene carboxylate 2 (figure IF). These are tested in Figure 6C of the present disclosure.
  • composition comprising the compound of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI and/or Formula VII, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
  • nucleic acid means one or more nucleic acids.
  • terms like "preferably”, “commonly”, and “typically” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.
  • the term “substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation.
  • the term “substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
  • the term “about” in association with a numerical value means that the numerical value can vary plus or minus by 5% or less of the numerical value.
  • alkyl means a straight or branched chain hydrocarbon containing from 1 to 14 carbons (unless otherwise noted).
  • alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2- dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, undecyl, dodecyl, tridecyl, and the like.
  • alkoxy refers to an alkyl group of the indicated number of carbon atoms attached to the parent molecular moiety through an oxygen bridge.
  • alkoxy groups include, for example, methoxy, ethoxy, propoxy and isopropoxy.
  • the oxygen atom in the 5-membered ring is contributed by the R3 group.
  • the oxygen atom in the 5- and 6-membered rings is contributed by the R5 group and the R3 group contributes the carbon atom directly attached to the parent ring.
  • the present invention provides compositions comprising one or more compounds, and methods for the treatment of obesity, diabetes, and/or other related diseases or conditions by converting white adipose cells or adipose precursor cells into BRITE (brown in white) cells, inducing UCPl in such BRITE cells, and/or inducing UCPl in brown adipose tissue deposits.
  • An object of the present invention relates to a compound for the regulation of metabolism in a mammal.
  • Ri is -H, -CO2H, -OH, -CO2CH3, -CO2C2H5, -C0 2 CH(CH 3 ) 2 , -CONH2, -CONHCH3, or CON(CH 3 ) 2 ;
  • R 2 is -H, -OH, -OCH3, or -OC2H5;
  • R 3 is -H
  • R3 and R 4 taken to ether with the atoms to which they are connected represent
  • R3 and Rs together with the atoms to which they are connected represent a group of the formula
  • R6 is alkyl or a group of the formula
  • R.7 represents -H, -OH, -OCH 3 ,
  • Rg represents -H, -OH, -OCH3,
  • Preferred compounds of Formula I include those where R6 represents propyl .
  • the compound an Amorfrutin is the compound an Amorfrutin.
  • Amorfrutin 1 has been tested in the examples of the present disclosure, and shown to upregulate Amorfrutin 1 and furthermore to have a positive effect on the WAT to BAT transdifferentiation.
  • Formula V and Formula VI is also known as "Radula compound I” and “Radula compound ⁇ ”, respectively. These are tested in Figure 6C of the present disclosure.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula VII, as disclosed herein, or a compound derived from this compound by medicinal chemistry and/or biochemistry, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
  • composition of the present invention comprising a compound of Formula (X) :
  • R 4 is -H
  • R7, Rs, Rg represents -H
  • composition of the present invention comprising a compound of Formula (X) :
  • R 2 is -OH;
  • R3 and R5 together with the atoms to which they are connected represent a group that has a ring structure;
  • R 4 is -H
  • R6 is alkyl or a group of the formula
  • R7, Rs, Rg represents -H
  • One embodiment of the present invention relates to a composition comprising a compound or more compounds of the present invention.
  • composition comprising a compound selected from one or more of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula I, as disclosed herein, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
  • the invention also provides a pharmaceutical composition comprising a compound of Formula II, as disclosed herein, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
  • the invention also provides a pharmaceutical composition comprising a compound of Formula III, as disclosed herein, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula IV, as disclosed herein, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
  • the invention also provides a pharmaceutical composition comprising a compound of Formula V, as disclosed herein, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
  • the invention also provides a pharmaceutical composition comprising a compound of Formula VI, as disclosed herein, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula VII, as disclosed herein, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
  • compositions particularly pharmaceutical compositions, comprising one or a plurality of the compounds or compositions set forth herein, useful in the practice of any of the disclosed methods or uses.
  • the amount of the compounds is preferably in a pharmaceutically effective amount. This document shows that such compounds could promote the conversion or transdifferentiation/conversion of white adipose tissue into brown adipose tissue. In addition, this document describes, that such compounds were able to dramatically upregulate expression of UCP1. It is believed that expression and activity of UCP1 is what drives the aforementioned conversion or transdifferentiation to brown
  • adipocytes One or more compounds of the invention can be used in the compositions or methods described herein.
  • one aspect of the present invention relates to the one or more compounds of the present invention for use as a medicament.
  • the medicament can be for delay in body mass increase.
  • the medicament can also be for delay in body fat increase.
  • the medicament may also be for improvement of glucose tolerance.
  • the medicament can be for improvement of insulin sensitivity, and can furthermore be for increase in interscapular brown adipose tissue.
  • medicament may also be for use in the treatment of diabetes and/or obesity.
  • the administration of one or more compounds of the invention to a subject can be used to increase levels of UCP-1 expression (or other BAT related genes) and to induce white adipocytes to convert or transdifferentiate to brown adipocytes or BRITE.
  • One or more compounds of the invention can induce conversion or transdifferentiation of white adipose cells to brown adipose cells. Transdifferentiation is where a differentiated (non-stem) cell is changed to another type of differentiated cell without being de-differentiated into a stem cell, for example, where a white adipose cell is changed to a brown adipose cell or BRITE.
  • the invention provides a composition for conversion or transdifferentiation of white adipose cells to thermogenic adipose cells, brown adipose cells or brown in white (BRITE) adipose cells, or for weight reduction, in a subject or an animal.
  • the composition is able to : (1) upregulate UCP1 expression and other BAT-related genes and/or (2) promote the conversion or transdifferentiation of white adipocyte tissue into brown adipose tissue.
  • Thermogenic adipocytes are called brown adipocytes if localized in brown adipose tissue or BRITE (brown-in-white adipocytes, also called beige adipocytes or inducible brown adipocytes) if localized in white adipose tissue.
  • BRITE brown-in-white adipocytes, also called beige adipocytes or inducible brown adipocytes
  • White adipocytes may be converted or transdifferentiated to brown adipocytes.
  • the present invention is white adipose tissue (WAT) converted to brown adipose tissue (BAT).
  • WAT white adipose tissue
  • BAT brown adipose tissue
  • the regulation of the metabolism can result in the treatment of diabetes and/or obesity.
  • the expression of UCPl can be upregulated in the mammal that has had the one or more of the compounds of the present invention administered.
  • the expression of UCPl can be upregulated in the target cell. This cell may be in vivo, in vitro or ex vivo.
  • One or more brown adipose tissue specific genes selected from cell death-inducing DFFA (DNA fragmentation factor-alpha)-like effector A (CIDEA), carnitine
  • CPTIB palmitoyltransferase IB
  • PPARGC1A peroxisome proliferator-activated receptor gamma
  • cytochrome C can be upregulated.
  • An embodiment of the invention provides methods for upregulating UCPl expression. These methods comprise contacting a white adipocyte with one or more compositions of the invention, wherein the white adipocyte upreguiates UCPl expression, for a time and at a concentration sufficient to upregulate expression of UCPl . Upregulation of UCPl expression can promote conversion of the white adipocyte into a brown adipocyte, such as a BRITE adipocyte.
  • brown adipose tissue related genes can also be upregulated or downregulated, including but not limited to cell death-inducing DFFA (DNA fragmentation factor-alpha)-like effector A (CIDEA)), carnitine palmitoyltransferase IB (CPTIB), peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PPARGC1A), cytochrome C, creatine kinase,
  • DFFA DNA fragmentation factor-alpha
  • CPTIB carnitine palmitoyltransferase IB
  • PPARGC1A peroxisome proliferator-activated receptor gamma
  • cytochrome C creatine kinase
  • CKMT1A/B Potassium channel subfamily K member 3
  • KCNK3 Potassium channel subfamily K member 3
  • IGHM immunoglobulin heavy constant mu
  • COBL cordon-bleu WH2 repeat protein
  • CYFIP2 Cytochrome P450 1A2
  • myosin myosin
  • heavy chain 11 smooth muscle
  • MYH 11 smooth muscle
  • angiotensinogen serpin peptidase inhibitor, clade A, member 8
  • AGT Creatine kinase S-type
  • TGM2 transglutaminase 2
  • ARG2 arginase 2
  • CA12 carbonic anhydrase XII
  • HMGCS2 3- hydroxy-3-methylglutaryl-CoA synthase 2
  • C4A/B Complement C4-A
  • IGA8 immunoglobulin lambda variable 2-14
  • IGLV2-14 immunoglobulin lambda variable 2-14
  • the compounds of the invention upregulate expression of one or more other brown adipose tissue specific genes CIDEA, CPTIB, PPARGC1A, and cytochrome C. In some embodiments, the compounds of the invention upregulate expression of CIDEA. In some embodiments, the compounds of the invention upregulate expression of CPTIB. In some embodiments, the compounds of the invention upregulate expression of PPARGC1A. In some embodiments, the compounds of the invention upregulate expression of cytochrome C.
  • the compounds of the invention upregulate expression of one or more other brown adipose tissue specific genes CIDEA, CPT1B, PPARGC1A, and cytochrome C.
  • Upregulation is an increase in the amount of protein, levels of mRNA expression, or both from a target gene by about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100% or more (or any range between about 10% and 100%) as compared to a control not contacted with a compound.
  • Downregulation is a decrease in the amount of protein, levels of mRNA expression, or both from a target gene by about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or more including 100% (or any range between about 10% and 100%) as compared to a control not contacted with a compound.
  • Administration of one or more compounds of the invention can induce or upregulate expression or biological function of brown adipose tissue-specific genes, such as UCP- 1, by at least about 1.5, by at least about 1.6, or about 1.75, or about 2.0, or about 2.5, or at least about 3.0 fold higher than the expression level or biological function of a control sample that is not contacted with the one or more of said compounds.
  • brown adipose tissue-specific genes such as UCP- 1
  • the compound of the invention is administered to a mammal so that the upregulation of expression of UCPl in a mammal cell is more than 5-fold.
  • the upregulation can be more than 10-fold; or more than 20-fold.
  • the upregulation may also be more than 50-fold or more than 100-fold.
  • the upregulation may even be more than 200-fold.
  • the compound of the invention is administered to a mammal so as to provide a plasma concentration of more than 0.05 ⁇ ; more specifically more than 1.0 ⁇ ; more than 3.0 ⁇ ; more than 5.0 ⁇ ; more than 10 ⁇ ; more than 20 ⁇ , or even more than 25 ⁇ .
  • the amount of compound administered may be 0.05 ⁇ - 30 ⁇ .
  • the amount of compound administered may also be 0.05 ⁇ - 20 ⁇ .
  • the amount of compound administered can be 1 ⁇ - 10 ⁇ .
  • the amount of compound administered may be 5 ⁇ - 200 ⁇ .
  • the amount of compound administered may also be 3 ⁇ - 25 ⁇ .
  • the amount of compound administered would usually be less than 30 ⁇ .
  • the amount may also be less than 20 ⁇ or less than 10 ⁇ .
  • the compound of the invention is administered to a human patient in need of treatment, in a daily dose of more than 0.1 mg/kg body mass ⁇ ; more specifically more than 0.5 mg/kg; more than 1.0 mg/kg; more than 5.0 mg/kg; more than 10 mg/kg; more than 50 mg/kg, or even more than 100 mg/kg.
  • the daily dose may be 0.1 mg/kg to 200 mg/kg body mass ⁇ .
  • the daily dose may be less than 5.0 mg/kg, or less than 10 mg/kg;
  • the daily dose may also be less than 50 mg/kg, or less than 100 mg/kg, or even less
  • brown adipose tissue-specific genes can be tested by, for example, western blot or quantitative RT-PCR (qPCR). Additionally, administration of one or more compounds of the invention can suppress or downregulate expression or biological function of brown adipose tissue-specific genes, by at least about 1.5, or about 1.75, or about 2.0, or about 2.5, or about 3.0 fold lower than the expression level or biological function of a control sample that is not contacted with the one or more of said compounds.
  • the brown adipocyte can be a BRITE adipocyte.
  • the brown adipocytes can comprise thermogenic adipocytes.
  • the mammal can be an overweight or obese mammal.
  • the mammal can be a human subject.
  • An aspect of the present invention relates to a method for regulation of the
  • metabolism in a mammal comprising a step of administering to the mammal a pharmaceutically effective amount of one or more compositions comprising one or more compounds of the present invention.
  • An aspect of the present invention relates to a method of treating diabetes in a mammal, comprising a step of administering to the mammal, comprising a step of administering to the mammal a pharmaceutically effective amount of one or more compositions comprising one or more compound of the present invention.
  • An aspect of the present invention relates to a method of upregulating UCP1 expression, comprising contacting a white adipocyte with a pharmaceutical composition comprising a compound of Formula II and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the white adipocyte upregulates UCP1 expression.
  • An aspect of the present invention relates to a method of upregulating UCP1 expression, comprising contacting a white adipocyte with a pharmaceutical composition comprising a compound of Formula III and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the white adipocyte upregulates UCP1 expression.
  • An aspect of the present invention relates to a method of upregulating UCP1 expression, comprising contacting a white adipocyte with a pharmaceutical composition comprising a compound of Formula IV and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the white adipocyte upregulates UCP1 expression.
  • An aspect of the present invention relates to a method of upregulating UCP1 expression, comprising contacting a white adipocyte with a pharmaceutical composition comprising a compound of Formula V and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the white adipocyte upregulates UCP1 expression.
  • An aspect of the present invention relates to a method of upregulating UCP1 expression, comprising contacting a white adipocyte with a pharmaceutical composition comprising a compound of Formula VI and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the white adipocyte upregulates UCP1 expression.
  • An aspect of the present invention relates to a method of upregulating UCP1 expression, comprising contacting a white adipocyte with a pharmaceutical composition comprising a compound of Formula VII and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the white adipocyte upregulates UCP1 expression.
  • An aspect of the present invention relates to a method of upregulating UCP1 expression, comprising contacting a white adipocyte with a pharmaceutical composition comprising a compound having a chemical formula of any one of the chemical formulae set forth in Figure 1A, Figurel B, Figure 1C, Figure ID, Figure IE, and Figure IF, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the white adipocyte upregulates UCP1 expression.
  • An aspect of the present invention relates to a method of inducing conversion or transdifferentiation of white adipocytes into brown adipocytes, comprising contacting the white adipocytes with one or more compositions comprising a compound of the present invention, wherein the white adipocytes are converted or transdifferentiated into brown adipocytes.
  • An aspect of the present invention relates to a method of depleting existing fat stores in a mammal, comprising administering to the mammal one or more compositions comprising a compound of the present invention.
  • the compounds of the present invention may be effective in treating diseases such as obesity, as well as diabetes, by promoting burning of excessive body energy stores. Further, these data indicate that treatment with such compounds may represent a dramatic improvement over existing treatments.
  • Body energy stores include, for example ATP and creatine phosphate, carbohydrates (available, inter alia, as serum glucose, liver glycogen, and muscle glycogen), fat (available, inter alia, as serum fatty acids, serum triglycerides, muscle triglycerides, and adipose triglycerides) and protein (available, inter alia, as muscle protein).
  • carbohydrates available, inter alia, as serum glucose, liver glycogen, and muscle glycogen
  • fat available, inter alia, as serum fatty acids, serum triglycerides, muscle triglycerides, and adipose triglycerides
  • protein available, inter alia, as muscle protein.
  • the compounds of the invention burn excessive body energy fat stores and/or excessive body energy adipose triglyceride stores.
  • excessive body energy stores of the subject are burned is the uses or methods of the present invention.
  • An aspect of the present invention relates to a method for inducing thermogenesis in an adipocyte in a mammal, comprising a step of administering to the animal a thermogenesis-inducing amount of one or more compositions comprising a compound of the present invention, wherein thermogenesis in an adipocyte in the mammal is induced.
  • An aspect of the present invention relates to a method for treating obesity in a mammal, comprising a step of administering to the animal a pharmaceutically effective amount of one or more compositions comprising a compound of the present invention, and wherein the administration of the one or more compositions
  • An aspect of the present invention relates to a method of treating diabetes in mammal, comprising a step of administering to the animal a pharmaceutically effective amount of one or more compositions comprising a compound of the present invention, and wherein the administration of the one or more compositions
  • the invention provides a diet composition, containing compounds or compositions, including pharmaceutical compositions set forth herein, wherein in particular embodiments said diet comprises one or more of those compounds or compositions of the invention.
  • two or more compounds in combination can be administered at a lower percentage than one type of compound alone to achieve a similar effect as when one compound is used alone.
  • One, two or more said compounds e.g., 1, 2, 3, 4, 5, 10 or more
  • the diet can comprise, by weight, about 0.1% to about 1%, or about 0.1 to about 5%, or about 5% to about 40% or more, or about 10% to about 40%; or about 10% to about 30%; or about 15% to about 25%; or about 20% to about 25% of said compounds or compositions set forth herein.
  • Dietary supplements can be tablets, capsules, softgels, gelcaps, liquids, or powder that supplement the diet, but are not considered a substitute for food.
  • a dietary supplement can comprise about 0.001, or about 0.001, or about 0.01, or about 0.1, or about 1, or about 5, or about 10, or about 20 or more grams of one or more said compounds.
  • a dietary supplement can be administered to a subject 1, 2, 3, 4, 5 or more times a day.
  • the invention also provides a diet composition for converting or transdifferentiating white adipose cells to thermogenic adipose cells, brown adipose cells or brown in white (“BRITE") adipose cells, or de novo generation of brown adipose cells from precursor cells, or for weight reduction, in an animal comprising about 0.01% to about 40%, or about 3.5% to about 40%; or about 10% to about 30%; or about 15% to about 25%; or about 20% to about 25% compounds or compositions set forth herein (i.e., about 0.01, or about 0.1, or about 1, or about 3.5, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20, or about 25, or about 30, or about 35, or about 40%, or more by weight of compound) and about 60% to about 99.99% (i.e., about 99.99, 99.9, 99, 96.5, 95, 90,
  • a low-calorie diet is a diet that comprises about 3%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80% less calories than the normal recommended amount of calories for a certain species of a given age, weight, gender, and general health condition.
  • the invention also provides a diet composition for converting or transdifferentiating white adipose cells to thermogenic adipose cells, brown adipose cells or brown in white (BRITE) adipose cells, or de novo generation of brown adipose cells from precursor cells, or for weight reduction, in an animal comprising about 0.01% to about 40%, or about 3.5% to about 40%; or about 10% to about 40%; or about 10% to about 30%; or about 15% to about 25%; or about 20% to about 25% of the compounds or compositions set forth herein (i.e., about 0.01, 0.1, 1, 3.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40%, or more by weight of compound) and about 60% to about 99.99% (i.e., about 99.99, 99.9, 99, 96.5, 95, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 75, 70, 65, or 60%
  • the low-fat diet can be a low saturated fat diet.
  • a low-fat diet is a diet that comprises about 3%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about80% less fat than the normal recommended amount of fat in a diet for a given species of a given age, weight, gender, and general health condition.
  • a low-fat diet in humans can comprise a diet consisting of about 0%, or about 3%, or about 5%, or about 7%, or about 10%, or about 13%, or about 15%, or about 20%, or about 25% fat.
  • the invention also provides a diet composition for converting or transdifferentiating white adipose cells to thermogenic adipose cells, brown adipose cells or brown in white (BRITE) adipose cells, or de novo generation of brown adipose cells from precursor cells, or for weight reduction, in an animal comprising about 0.01% to about 40%, or about 3.5% to about 40%; or 10% to about 40%; or about 10% to about 30%; or about 15% to about 25%; or about 20% to about 25% of compounds or compositions set forth herein (i.e., about 0.01, 0.1, 1, 3.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40%, or more by weight of compound) and about 60% to about 99.99% (i.e., about 99.99, 99.9, 99, 96.5, 95, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 75, 70, 65, or 60% ) of
  • a low carbohydrate diet is a diet that comprises about 3%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80% less carbohydrates than the normal recommended amount of carbohydrates for a certain species of a given age, weight, gender, and general health condition.
  • One or more compounds as set forth herein can be administered with an adjunctive weight loss therapy such as an exercise regimen, a low-fat diet, a low-calorie diet, a low-carbohydrate diet, surgical intervention such as gastroplasty, gastric partitioning, gastric bypass, behavioral therapy, pharmacotherapy (e.g., use of sibutramine, MERIDIA® (sibutramine HCI monohydrate, reductil), XENICAL® (orlistat), metformin (GLUCOPHAGE®), exenatide (BYETTA®), pramlintide (SYMLIN®), phentermine combined with tupiramate (QSYMIA®) rimonabant (acomplia), catecholamines, bupropion, topirmate, phentamine, amphetamines, locaserin (BELVIQ®) and combinations thereof), natural dietary aids and/or over-the-counter (OTC) weight-loss products, and combinations thereof.
  • Behavior therapy includes strategies that help in overcoming barriers to compliance with dietary therapy and/or exercise therapy.
  • Such strategies include, for example, self-monitoring of eating habits and exercise, stress management, stimulus control, problem-solving (e.g., self-corrections of problem areas related to eating and exercise), contingency management (e.g., use of rewards for specific desirable actions, cognitive restructuring (e.g., modification of unrealistic goals and inaccurate beliefs), and social support.
  • compositions can be formulated as pharmaceutical compositions.
  • Pharmaceutical compositions comprise a pharmaceutically acceptable carrier or diluent and a pharmaceutically effective amount of compound.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990). Additionally, components such as preservatives, stabilizers, dyes, antioxidants, suspending agents, wetting agents, pH buffering agents, preservatives, and flavoring agents can be present in a pharmaceutical composition.
  • a pharmaceutical composition can also comprise an excipient, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride and the like.
  • compositions can be formulated as tablets, capsules, lozenges, elixirs, suppositories, sterile solutions, inhalants or nasal sprays, liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid, injectables, topical sprays, powers, gels, or emulsions.
  • the pharmaceutical compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents.
  • compositions can be utilized in vivo, in a mammalian patient, such as a human, or in vitro or ex vivo.
  • the pharmaceutical compositions can be administered to a subject in a variety of ways, including topically, parenterally, intravenously, percutaneous, subcutaneously, intramuscularly, intravenous, intrathecal, colonically, rectally, nasally or intraperitoneally. Such methods can also be used in testing the activity of compounds in vivo or in vitro or ex vivo.
  • compound compositions When administered by a drinking solution, compound compositions, including pharmaceutical compositions comprise one or more compounds dissolved in water, with appropriate pH adjustment.
  • the compounds can be dissolved in distilled water, tap water, spring water, juice, milk, or any other beverage.
  • the pH can be adjusted to between about 3.5 and about 8.5.
  • Sweeteners may be added, e.g., about 0.1, or about 1, or about 5, or about 10% (w/v) sucrose or other natural or artificial sweetener.
  • compositions of this invention can also include: acids and bases to adjust the pH ; tonicity imparting agents such as sorbitol, glycerin and dextrose;
  • antimicrobial preservatives such as other parahydroxy benzoic acid esters, sorbate, benzoate, propionate, chlorbutanol, phenylethyl alcohol, benzalkonium chloride, and mercurials; viscosity imparting agents such as sodium carboxymethylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, polyvinyl alcohol and other gums; suitable absorption enhancers; stabilizing agents such as antioxidants, like bisulfate and ascorbate, metal chelating agents such as sodium edetate and drug solubility enhancers such as polyethylene glycols.
  • the dosage of compounds or compositions set forth herein can range broadly depending upon the desired affects, the therapeutic indication, route of administration and purity and activity of the compound or composition. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the test compound.
  • the pharmaceutically effective amount of a compound or composition set forth herein required as a dose will depend on the route of administration, the type of animal being treated, and the physical characteristics of the specific animal under consideration.
  • the dose can be tailored to achieve a desired effect, but will depend on such factors as weight, gender, diet, metabolic rate, age, side effects, concurrent medication, health status, a property or nature of the compound itself, such as the administration vehicle or method, and other factors which those skilled in the medical arts will recognize.
  • a dosage for administration of a compound to a subject can be from about 1 mg/kg of body weight/day to about 1,000 mg/kg of body weight/day.
  • a dosage for administration of one or more compounds or compositions set forth herein to a subject can be from about 1, or about 5, or about 10, or about 20, or about 25, or about 50, or about 75, or about 100, or about 200, or about 300, or about 400, or about 500, or about 600, or about 700, or about 800, or about 900, or about 1,000 or more mg/kg of body weight/day, or any range between about 1 to about 1,000 mg/kg/day such as, for example about 1 mg/kg/day to about 1,000 mg/kg/day, or about 10 mg/kg/day to about 500 mg/kg/day, or about 50 mg/kg/day to about 200 mg/kg/day.
  • a dosage for administration of one or more compounds or compositions set forth herein to a subject can also be from about 1,000, or about 900, or about 800, or about 700, or about 600, or about 500, or about 400, or about 300, or about 200, or about 100, or about 75, or about 50, or about 25, or about 20, or about 10, or about 1 or less mg/kg of body weight/day.
  • the dosage of amorfrutin 1, amorfrutin 2, DHPC, Rohla compound 1, Rohla compound 2, Formula (I), Formula (x), Formula (Y), or amorfrutin 3 is about 1 g/kg/day.
  • the dosage of amorfrutin 1, DHPC, amorfrutin 2, Rohla compound 1, Rohla compound 2, Formula (I), Formula (x), Formula (Y), or amorfrutin 3 is about 100 to about 1000 mg/kg/day. In some other embodiments, the dosage of amorfrutin 1, amorfrutin 2, DHPC, Rohla compound 1, Rohla compound 2, Formula (I), Formula (x), Formula (Y), or amorfrutin 3 is about 10 to about 100 mg/kg/day. In some other embodiments, the dosage of amorfrutin 1, amorfrutin 2, DHPC, Rohla compound 1, Rohla compound 2, Formula (I), Formula (x), Formula (Y), or amorfrutin 3 is about 500 to about 1000 mg/kg/day.
  • the administration of one or more of the compounds or compositions set forth herein can be used to increase levels of UCP1 expression and to induce white adipose tissue to transdifferentiate or convert to brown adipose tissue.
  • Administration of such compounds or compositions set forth herein to a subject can be useful to treat, prevent, or ameliorate several diseases and conditions, and to treat, prevent or ameliorate symptoms of several diseases and conditions as described herein.
  • compositions of the invention can also be used to convert or
  • thermogenic adipose cells brown adipose cells or brown in white (“BRITE") adipose cells or to de novo generate brown adipose cells from precursor cells, or to reduce weighting an animal such as an overweight, obese, immature, or mature animal.
  • Weight loss of about 3%, or about 5%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 75% can be achieved.
  • the invention provides methods for converting or transdifferentiating white adipose cells to thermogenic adipose cells, brown adipose cells or brown in white (“BRITE") adipose cells, or de novo generation of brown adipose cells from precursor cells, or for weight reduction, in an animal comprising administering to the animal one or more compounds as described above in an amount and for a time sufficient to have the desired effect in the animal.
  • BRITE brown in white
  • Methods and compositions of the invention can be used to deplete existing fat stores (i.e. white adipose tissue or white adipocytes) in a subject.
  • the subject is overweight or obese.
  • One or more compounds or compositions set forth herein can be administered to the subject, for example an overweight or obese subject.
  • Fat is depleted or reduced when the fat is reduced by about 1, or about 5, or about 10, or about 15, or about 20, or about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90 % or more as compared to a control subject that is not administered one or more compounds or compositions set forth herein.
  • the subject, animal or patient can be, for example, a human, a non-human primate, a rat, a mouse, a rabbit, a guinea pig, a bovine, a pig, a sheep, a goat, a dog, a cat, a horse, a bird, a fish or an invertebrate.
  • the subject, animal, or patient is healthy and has no underlying health problems or issues.
  • the subject, animal, or patient has one or more overweight or obesity health problems and no other health problems or issues.
  • Figure 1 shows a generic structure encompassing the compounds of the invention (Formula I; Figure 1A), Amorfrutin 1 (Formula II; Figure IB), Amorfrutin 2 (Formula III; Figure 1C) and Amorfrutin 3 (Formula IV; Figure ID), Stilbene carboxylate 1 (Formula V, Figure IE), and stilbene carboxylate 2 (Formula VI, Figure IF).
  • Figure 2 shows the scheme of the 3T3-L1 differentiation protocol used in Example 1.
  • Figure 3 shows the scheme of the 3T3-L1 differentiation protocol used in Example 1.
  • Figure 3 shows expression levels of UCPl in 3T3-L1 cells upon adipocyte
  • Figure 4 shows the morphology of 3T3-L1 cells upon adipocyte differentiation from Example 1.
  • Figure 5 shows the scheme of the human mesenchyme adipose-derived stem cells (hMADS) differentiation protocol used in Example 2.
  • Figure 6A shows expression levels of UCPl and FABP4 in hMADS upon adipocyte differentiation from Example 2.
  • Figure 6B shows expression level of UCPl in hMADS treated with the stilbene carboxylates.
  • Figure 6C shows expression level of UCPl in hMADS treated with Radula compounds 1 and 2 (a mixture of both compounds), and 2,4-dihydroxy-6-(2-phenylethyl)-benzoic acid (DHPC).
  • Figure 7 shows expression levels of UCPl by western blot in hMADS upon adipocyte differentiation from Example 2.
  • Figure 8 shows expression levels of UCPl by western blot in hMADS upon adipocyte differentiation from Example 2.
  • Figure 8 shows high magnification images of the morphology of hMADS upon adipocyte differentiation from Example 2.
  • Figure 9A shows the scheme of human mesenchyme adipose-derived stem cells
  • hMADS hMADS differentiation protocol using, apart from Rosiglitazone, either Amorfrutin l, 2 or 3 for white adipose cell induction.
  • Figure 9B shows that Amorfrutins are only weak inducers of white adipogenesis, in contrast to rosiglitazone which has a strong induction effect on white adipogenesis.
  • Rosiglitazone treatment increases body mass in knockout mice more rapidly than high fat diet only in example 4.
  • Amorfrutin 1 ("edelweiss") in the HFD slightly decreases body mass increase in WT mice.
  • rosiglitazone significantly accelerates fat mass increase in both WT and knockout mice.
  • resveratrol slows down fat mass increase in knockout mice, but not in WT mice.
  • the oral glucose tolerance test strated with an oral glucose (1,4 mg/kg lean weight) administration (by oral gavage).
  • Amorfrutin 1 turned out to have a general beneficial effect on insulin sensitivity.
  • protein concentration in the lysate was determined using the Lowry methods. We could see that interscapular brown adipose tissue increases in WT mice upon rosiglitazone and upon amorfrutin 1 treatment.
  • Amorfrutin compounds used in these Examples were obtained commercially from AnalytiCon Discovery (Potsdam, Germany). Additionally, these compounds may also be obtained by routine methods known by one of ordinary skill in the art. For example, Amorfrutin may be isolated by extraction from natural sources, such as a plant (Amorpha fruticosa), as described by Dat, N. T. et al. (2008) "Phenolic
  • Amorfrutins may also be synthesized by methods known by one of ordinary skill in the art (see, e.g., Weidner et al. (2012) "Amorfrutins are potent antidiabetic dietary natural products” Proc Natl Acad Sci USA 109 : 7257-7262, SI Appendix, SI Methods).
  • Example 1 Amorfrutin 1 induces differentiation and expression of BAT-specific genes in 3T3-L1 cell line
  • Mouse embryonic fibroblast cells (3T3-L1 cells; ATCC) were cultured in DMEM (high glucose), containing 10% fetal calf serum ("growth medium”) at 37°C in a humidified C02 atmosphere. Cells were seeded at high density and were grown until they reached complete confluency. Then, at day 0, adipogenic differentiation was stimulated by adding 10 ⁇ g/ml insulin, 0.5 mM isobutylmethylxanthine (IBMX), and 1.0 ⁇ Dexamethasone.
  • IBMX isobutylmethylxanthine
  • Quantitative PCR (qPCR) analyses were performed on RotorGene Real-Time PCR System (Qiagen), using a FastStart Universal SYBR Green Master Mix (Roche) and the following mouse-specific primers: Mm_Ucpl_l_SG QuantiTect primer assay (Cat. Number QT00097300), Mm_Actb_2_SG QuantiTect primer assay (Cat. Number QT01136772).
  • Example 2 Up-regulation of UCP1 expression upon candidate compound treatment - Amorfrutin 1 and similar compounds induces differentiation and expression of BAT- specific genes in hMADS cell line hMADS, human multipotent adipose-derived stem cells, were cultured in growth medium composed of DMEM containing low glucose (Sigma) supplemented with 10% FCS (Sigma, Lot No.
  • hMADS of passage 13- 15 were seeded at the density of approximately 10,000 per cm2 in growth medium containing 2.5 ng/ml FGF2 (Invitrogen). After three days growth medium was replaced by fresh growth medium lacking FGF2.
  • hMADS Two days later (Day 0) hMADS were induced to differentiate in the presence of differentiation medium composed of DMEM/Ham's F12 (1 : 1) (Sigma) supplemented with 10 transferrin, 0.2 nM triiodothyronine (T3), 10 nM insulin, and 1 ⁇ dexamethasone, 0.5 mM isobutylmethylxanthine (IBMX).
  • differentiation medium composed of DMEM/Ham's F12 (1 : 1) (Sigma) supplemented with 10 transferrin, 0.2 nM triiodothyronine (T3), 10 nM insulin, and 1 ⁇ dexamethasone, 0.5 mM isobutylmethylxanthine (IBMX).
  • RNA and protein were incubated for another four days in presence of differentiation medium containing either 0.3 ⁇ Amorfrutin 1, 0.1 ⁇ Rosiglitazone, or DMSO.
  • RNA and protein was isolated following the TriReagent protocol (Sigma).
  • cDNA was synthesized using iScript cDNA synthesis kit (BioRad) and thereafter quantitative PCR was performed on a CFX connect (BioRad) with the SsoAdvanced Universal SYBR Green Supermix 1000 (BioRad) using 10 ng of cDNA per reaction.
  • the following primers were used : For UCPl : QuantiTec primer assay (Qiagen), for 36B4: EVPR5825 and EVPR5826, for FABP4: EVPR4690 and EVPR4691.
  • Proteins (20 ⁇ g) were separated on 4-20% Mini-PROTEAN® TGXTM gels (BioRad) and transferred onto PVDF membranes using Trans-Blot Transfer Pack Mini (BioRad). Immunoblotting was performed using goat-anti-human UCPl antibody (Santa Cruz) or rabbit-anti-human Tom20 (Santa Cruz) as loading control, followed by HRP-conjugated secondary antibodies (Sigma). All antibodies were diluted in Tris-buffered saline containing 0.1% Tween and 5% milk powder (RapiLait, Migros). Protein bands were visualized by incubating membranes with Amersham ECL Prime Western Blotting Detection Reagent and subsequent visualization with the Image Quant LAS4000 (GE Healthcare).
  • Non-differentiated hMADS do not significantly differentiate into white adipocytes upon Amorfrutin treatment ( Figure 9A).
  • the lipid content also does not massively increase in hMADS upon Amorfrutin treatment ( Figure 9B), in contrast to the situation in Rosiglitazone-treated hMADS.
  • amorfrutins including Amorfrutin 1, the Radula compounds and DHPC all have the potential to promote conversion or transdifferentiation of human adipocyte precursors into brown adipocytes, as they all inducethe BAT-specific gene UCPl .
  • Amorfrutin 1 delays body mass increase in WT mice but not in UCP1 Kos.
  • Amorfrutin 1 delays fat mass increase in WT mice but not in UCP1 Kos.
  • Amorfrutin 1 improves glucose tolerance in UCP1 Kos.
  • Amorfrutin 1 has a beneficial effect on insulin sensitivity of WT as well as UCP1 KOs. Amorfrutin 1 increases interscapular brown adipose tissue in WT mice.
  • mice used in this study WTs and UCPlKOs— are on C57BI/6 background.
  • UCP1 KO mice progeny of those described in Enerback et al., 1997) have been backcrossed to C57BI/6 for 10 generations, and after intercrossing, they were maintained as a strain, in parallel with the wild-type C57BI/6 mice. The mice were kept at
  • thermoneutrality had access to food and water ad libitum. Mice were sacrificed 4 weeks after start of high fat diet feeding. Animal studies were approved by the Ethical Committee of the University of Sweden.
  • Amorfrutin 1 is a carboxylated, O-methylated, and prenylated dihydropinosylvin found in the roots of Glycyrrhiza foetida, in the fruits of Amorpha fruticosa, and in the moss Rohla complanata.
  • Resveratrol is a stilbenoid found in the skin of grapes, blueberries, raspberries, mulberries, lingonberry, and senna.
  • Rosiglitazone is a purely chemical compound that is used here as a control compound. It has recently been shown to upregulate UCP1 in vivo in white adipocytes of mice (Pardo et al., 2011).
  • the high fat diet (HFD) was formulated by Research Diets Inc, located in New
  • mice were kept at room temperature until they reached an age of 9 weeks. Then they were transferred to 30°C in order to keep them at thermoneutrality (figure 12).
  • Rosiglitazone significantly accelerates fat mass increase in both WT and knockout mice (figure 18).
  • AF1 slightly slows down fat mass increase in both WT and knockout mice (figure 19).
  • Resveratrol slows down fat mass increase in knockout mice, but not in WT mice (figure 20).
  • Body fat mass was measured with in vivo magnetic resonance imaging using an EchoMRI-lOOTM instrument (Houston, USA). Glucose tolerance
  • the oral glucose tolerance test strated with an oral glucose (1,4 mg/kg lean weight) administration (by oral gavage). Blood glucose levels were measured with a blood glucometer (Accu-Check Avia, Roche) before glucose challenge, and 15, 30, 60, and 90 minutes afterwards. Mice were fasted for 8h prior to glucose administration. In UCPl knockouts all three compounds improve glucose tolerance, whereas in WT mice only rosiglitazone could improve glucose tolerance (figures 21 and 22).
  • mice were killed by C02. Interscapular depots of brown adipose tissue and inguinal depots of white adipose tissue (one lobe each) were dissected and weighted. Tissues were homogenized in modified RIPA buffer (50 mM Tris-Hcl, pH 7.4, 1% Triton X-100, 150 mM NaCI, 1 mM EDTA) with freshly added phosphatase inhibitors (ImM Na3V04 and 10 mM NaF) and protease inhibitor cocktail (Complete Mini, Roche). Homogenates were centrifuged at 14000 g for 15 min to discard top fat layer. Protein concentration in the lysate was determined using the Lowry methods. We could see that
  • interscapular brown adipose tissue increases in WT mice upon rosiglitazone and upon amorfrutin 1 treatment (figure 24).
  • the increase in IBAT total protein was not accompanied by elevated amounts of total UCPl (not shown).
  • the increase in IBAT may still be beneficial as BAT secretes Neuregulin 4 (Nrg4). This growth factor is enriched in brown adipose tissue. Therefore, more BAT may lead to higher secretion of Nrg4. It has been shown that transgenic mice overexpressing Nrg4 were protected against diet-induced insulin resistance, independent of UCPl expression (Wang et al. 2014). Items
  • a composition comprising a compound of Formula I:
  • Ri is -H, -CO2H, -OH, -CO2CH3, -CO2C2H5, -C0 2 CH(CH 3 )2, -CONH2, -CONHCH3, or CON(CH 3 ) 2 ;
  • R 2 is -H, -OH, -OCH3, or -OC2H5;
  • R2 and Rs taken together with the atoms to which they are connected represent a group of the formula
  • R.3 and R 4 taken to ether with the atoms to which they are connected represent
  • R3 and Rs together with the atoms to which they are connected represent a group of the formula
  • R6 is alkyl or a group of the formula
  • R? represents -H, -OH, -OCH3, -OC2H5 , or
  • Rg represents -H, -OH, -OCH3, -OC2H5, or
  • composition for use in regulation of the metabolism in a mammal for use in regulation of the metabolism in a mammal.
  • the compound is an
  • R 2 is -OH ;
  • R3 and Rs together with the atoms to which they are connected represent a group that has a ring structure
  • R 4 is -H ;
  • R6 is alkyl or a group of the formula
  • R7, Rs, Rg represents -H, 7.
  • R6 is alkyl or a group of the formula
  • R7, Re, R9 represents -H
  • composition for use according to any one of items 1-5 wherein the compound is in form of a pharmaceutical composition comprising the compound of Formula I and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
  • the regulation of the metabolism results in depletion of existing fat stores of the mammal.
  • WAT white adipose tissue
  • BAT brown adipose tissue
  • composition for use according to any one of items 1-15 wherein one or more brown adipose tissue specific genes selected from cell death-inducing DFFA (DNA fragmentation factor-alpha)-like effector A (CIDEA), carnitine palmitoyltransferase IB (CPT1B), peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PPARGC1A), and cytochrome C are upregulated.
  • DFFA DNA fragmentation factor-alpha
  • CPT1B carnitine palmitoyltransferase IB
  • PPARGC1A peroxisome proliferator-activated receptor gamma
  • cytochrome C coactivator 1 alpha
  • a method for regulation of the metabolism in a mammal comprising a step of administering to the mammal a pharmaceutically effective amount of one or more compositions comprising a compound of Formula I :
  • a method of treating diabetes in a mammal comprising a step of administering to the animal mammal, comprising a step of administering to the mammal a
  • a method of upregulating UCP1 expression comprising contacting an adipocyte precursor cell or white adipocyte with a pharmaceutical composition comprising a compound of Formula II and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the adipocyte precursor cell or white adipocyte upregulates UCP1 expression.
  • a method of upregulating UCP1 expression comprising contacting an adipocyte precursor cell or white adipocyte with a pharmaceutical composition comprising a compound of Formula III and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the adipocyte precursor cell or white adipocyte upregulates UCP1 expression.
  • a method of upregulating UCP1 expression comprising contacting an adipocyte precursor cell or white adipocyte with a pharmaceutical composition comprising a compound of Formula IV and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the adipocyte precursor cell or white adipocyte upregulates UCP1 expression.
  • a method of upregulating UCP1 expression comprising contacting an adipocyte precursor cell or white adipocyte with a pharmaceutical composition comprising a compound having a chemical formula of any one of the chemical formulae set forth in Figure 1A through IF and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the adipocyte precursor cell or white adipocyte
  • a method of inducing conversion or transdifferentiation of adipocyte precuri cells into brown adipocytes comprising contacting the adipocyte precursor cells one or more compositions comprising a compound of Formula I :
  • a method of inducing conversion or transdifferentiation of white adipocytes into brown adipocytes comprising contacting the white adipocytes with one or more compositions comprising a compound of Formula I :
  • a method of depleting existing fat stores in a mammal comprising administering to the mammal one or more compositions comprising a compound of Formula I : Formula I wherein Ri-R.6 are as recited in item 1.
  • thermogenesis-inducing amount of one or more compositions comprising a compound of Formula I :
  • a method for treating obesity in a mammal comprising a step of administering to the animal a pharmaceutically effective amount of one or more compositions comprising a compound of Formula I :
  • a method of treating diabetes in mammal comprising a step of administering to the animal a pharmaceutically effective amount of one or more compositions comprising a compound of Formula I :
  • Formula I wherein R1-R6 are as recited in item 1, and wherein the administration of the one or more compositions upregulates UCP1 expression in the mammal.

Abstract

The invention disclosed herein relates generally to the field of treating obesity, diabetes, and other related diseases or conditions. This can be done by inducing Uncoupling Protein 1 (UCP1) in brown adipose tissue deposits or by administration of specific compounds and compositions. The invention provides compounds and compositions that upregulate and/or induce expression of UCP1 and promote transdifferentiation or conversion of white adipose tissue into brown adipose tissue without concomitantly increasing overall differentiation of pre-adipocytes into adipocytes.

Description

Compounds for upregulating UCPl expression and promoting transdifferentiation into brown adipose tissue
FIELD OF THE INVENTION
The invention disclosed herein relates to treatment of metabolic diseases such as obesity, diabetes, and other related diseases or conditions. This can be done by inducing browning of adipose tissue and/or up-regulation of uncoupling Protein 1 (UCPl) in brown adipose tissue deposits or by administration of specific compounds and compositions. The invention provides compounds and compositions that significantly upregulate and/or induce expression of UCPl and/or promote
transdifferentiation or conversion of white adipose tissue into brown adipose tissue, without inducing differentiation of adipose stem cells into white adipocytes.
BACKGROUND OF THE INVENTION
Mammalian white adipose tissue is used as a store of energy. Brown adipose tissue is a specialized form of adipose tissue found mainly in newborns and hibernating mammals. This specialized tissue can generate heat by uncoupling the respiratory chain of oxidative phosphorylation within mitochondria. The process of uncoupling means that when protons transit down the electrochemical gradient across the inner mitochondrial membrane, energy is released as heat rather than being used to generate ATP. This thermogenic process may be vital in neonates exposed to cold, which require this thermogenesis to keep warm, as they are unable to shiver, or take other actions to keep themselves warm. Recent studies have identified the presence of brown adipocytes in adult humans (Virtanen et al., N. Engl. J. Med. 360 : 1518-1525 (2009); Nedergaard et al., Am. J. Physiol. Endocrinol. Metab. 293(2) : E444-452 (2007); van Marken Lichtenbelt et al., N. Engl. J. Med. 360(15) : 1500-1508 (2009)). Brown adipocytes appearing in white adipose tissue are often called inducible brown adipocytes (or beige, brown-in-white, or BRITE adipocytes). The appearance of these inducible brown adipocytes in white adipose tissue may also involve a
transdifferentiation/conversion processes of white-to-brown adipocytes (Rosenwald et al., Nature Cell Biology 15 : 659 (2013)).
Uncoupling Proteins (UCPs), including Uncoupling Protein 1 (UCPl), are mitochondrial transmembrane proteins that decrease the proton gradient generated in oxidative phosphorylation, by increasing the permeability of the inner mitochondrial membrane, thereby allowing protons that have been pumped into the intermembrane space to return to the mitochondrial matrix. UCPl, also known as thermogenin, is an exclusive hallmark of brown adipocytes in mammals. Here, UCP1 functions as a proton transporter, allowing the dissipation of the energy of the proton gradient generated by the respiratory chain, and thereby uncoupling oxidative phosphorylation. This results in heat generation, instead of ATP production ("non-shivering thermogenesis"). The ability of UCPl-expressing adipocytes to produce heat and thus increase energy expenditure may also alter energy expenditure and body mass in humans.
Accordingly, the ability of brown fat to burn energy instead of storing it, has important health implications, notably relating to weight loss, obesity, diabetes, and other pathologic conditions. As such, the conversion or transdifferentiation of white adipose tissue into brown adipose tissue would be a useful tool in establishing and maintaining metabolic health and warding off or combating obesity and diabetes. This process— the acquisition by white adipose tissue of characteristics of brown adipose tissue— is also commonly referred to as "browning."
Studies have shown that a variety of stimuli can induce the conversion of white adipose tissue into brown adipose tissue (reviewed e.g. by Song et al., Exp Mol Med 49(7) : e353 (2017)), amongst them PPARy agonists. PPARy is expressed in several tissues but most abundantly in white adipocytes where it acts as a key messengers responsible for the translation of nutritional and metabolic stimuli into expression changes of genes involved in lipid and glucose metabolism (Ahmadian et al., Nat. Med. 19 : 557-566 (2013)). PPARy has also been shown to be highly expressed in brown adipocytes and to induce the expression of UCP1 (Digby et al., Diabetes 47(1) : 138-141 (1998)).
Despite the proven efficacy of PPARy agonists to normalize glycaemia and decrease insulin as well as free fatty acid serum levels in conditions of type 2 diabetes, these drugs possess a number of deleterious class-related side effects, including peripheral edema, increased risks of congestive heart failure, and increased rate of bone fracture. One side effect associated with all TZDs is an increase in peripheral fat mass associated with a significant gain weight in both rodents and humans (Gitlin et al., Ann Intern Med 129(l) :36-38 (1998); Singh and Loke, Expert Opin Drug Saf
7(5) : 579-85 (2008)). For this reason, there is a considerable need for identifying antidiabetic PPARy ligands devoid of the detrimental side effects of the classical glitazones.. Selective PPARy modulators (SPPARMs)have been shown to bind in distinct manners to the ligand-binding pocket of PPARy, leading to alternative receptor conformations and cofactor recruitment/displacement, as well as differential gene expression leading to differential biological responses. In animal and human trials SPPARMs have been shown to display favorable pharmacological features, with insulin-sensitizing and glucose-lowering properties without increasing body weight (DePaoli et al., Diabetes Care 37, 1918-1923 (2014; Gregoire et al., Mol. Endocrinol. 23 :975-988 (2009); Schupp et al., Diabetes 54: 3442-3452 (2005)).
A family of isoprenoid-substituted dihydropinosylvin derivatives have been identified as PPARy agonists, showing effects in diabetic and obese mouse models (Weidner et al., Proc Natl Acad Sci U S A. 109(19) : 7257-62 (2012)). WO 2017/177593 Al (MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER
WISSENSCHAFTEN E.V. ; BOYCE THOMPSON INSTITUTE FOR PLANT RESEARCH INC.) 6 November 2014 discloses amorfrutin analogues suitable to selectively modulate the PPAR3gamma. WO 02/14252 A2 (HOPPE, D.C. and INMAN, W.D.) 21 February 2002 discloses several hypoglycemically active stilbenoids. These all have a fully aromatic backbone, and thus have a fixed three-dimensional structure. The double bond in the linker portion between the two aromatic moieties, does not allow rotation around, and thus lock the two aromatic moieties into a plane.
WEIDNER, C. et al. : "Amorfrutins are potent antidiabetic dietary natural products", PNAS, 2012, Vol. 109, no. 19, pages 7257-7262 discloses an amorfrutin 1 tested in a mouse model for insulin resistance. WO 02/13811 A2 (INSMED CORPOATION) 21 February 2002 hypoglycemically active stilbenoids. These all have a fully aromatic backbone, and thus have a fixed three- dimensional structure.
WO02/013809 A2 (INMAN, W. D. and HOPPE, D.C.) 21 February 2002 several hypoglycemically active stilbenoids. These all have a fully aromatic backbone, and thus have a fixed three-dimensional structure.
WO 2010/094760 Al (DSM IP ASSETS B.V.) 26 August 2010 discloses a compound A which has a fully aromatic backbone, and thus have a fixed three-dimensional structure. EP 2796442 Al (INSTITUTE OF MEDICINAL BIOTECHNOLOGY; CHINESE ACADEMY OF MEDICAL SCIENCES) 29 October 2014 discloses among others an amorfrutin as an anti-viral compound lOv. TONTONOZ, P. and SPIEGELMAN, B.M. :"Fat and Beyond : The Diverse Biology of PPARy", ANNUAL REVIEWS OF BIOCHEMISTRY, 2008, Vol. 77, pages 289-312 discloses rosiglitazone. This molecule does not have a recognition site for
PPARgamma. The exact mechanism by which the amorfrutins exert their beneficial effects on conditions of metabolic disorders have not been elucidated yet. Partially those effects may be due to the fact that as dislosed herein for amorfrutin 1, it does not promote, in contrast to rosiglitazone, differentiation of pre-adipocytes into mature adipocytes (see figure 9B in this patent application). SUMMARY OF THE INVENTION
The present invention provides compositions comprising one or more compounds, and methods for the treatment of obesity, diabetes, and/or other related diseases or conditions by converting white adipose cells or adipose precursor cells into BRITE (brown in white) cells, inducing UCP1 in such BRITE cells, and/or inducing UCP1 in brown adipose tissue deposits without concomitantly increasing overall differentiation of pre-adipocytes into adipocytes.
An object of the present invention relates to a compound for the regulation of metabolism in a mammal.
In one embodiment of the present invention is the compound of Formula I :
Figure imgf000005_0001
Formula I Wherein Ri is -H, -CO2H, -OH, -CO2CH3, -CO2C2H5, -C02CH(CH3)2, -CONH2, CONHCH3, or CON(CH3)2;
R2 is -H, -OH, -OCH3, or -OC2H5;
Figure imgf000006_0001
R2 and Rs taken together with the atoms to which they are connected represent a group of the formula
Figure imgf000006_0002
R3 and R4 taken together with the atoms to which they are connected represent
Figure imgf000006_0003
R3 and Rs together with the atoms to which they are connected represent a group of the formula
Figure imgf000007_0001
Px6 is alkyl or a group of the formula
Figure imgf000007_0002
represents -H, -OH, -OCH3, -OC2H5,
Re represents -H, -OH, -OCH3, -OC2H5, or ; and
Rg represents -H, -OH, -OCH3, -OC2H5, or
Figure imgf000007_0003
Preferred compounds of Formula I include those where R6 represents propyl.
Other preferred compounds of Formula I include those where R6 represents a grou of the formula
Figure imgf000007_0004
In one embodiment of the present invention is the compound an Amorfrutin.
In one embodiment of the present invention is the compound Amorfrutin 1, having the chemical structure of Formula II:
Figure imgf000008_0001
(Formula II).
Amorfrutin 1 has been tested in the examples of the present disclosure, and shown to upregulate Amorfrutin 1 and furthermore to have a positive effect on the WAT to BAT transdifferentiation. In one embodiment of the present invention is the compound Amorfrutin 2, having the chemical structure of Formula III
Figure imgf000008_0002
(Formula III).
In one embodiment of the present invention is the compound Amorfrutin 3, having the chemical structure of Formula IV:
Figure imgf000008_0003
In one embodiment of the present invention is the compound stilbene carboxylate 1, having the chemical structure of Formula V:
Figure imgf000009_0001
(Formula V).
In one embodiment of the present invention is the compound stilbene carboxylate 2, havin the chemical structure of Formula VI :
Figure imgf000009_0002
(Formula VI).
Formula V and Formula VI are also known as "Radula compound 1" and "Radula compound 2", respectively. Radula 1 is also known as stilbene carboxylate 1 (figure IE) and Radula 2 is also known as stilbene carboxylate 2 (figure IF). These are tested in Figure 6C of the present disclosure.
Figure imgf000009_0003
(Formula VII)
Formula VII is also known as 2,4-dihydroxy-6-(2-phenylethyl)-benzoic acid (= DHPC).
Chemical synthesis of 2,4-dihydroxy-6-(2-phenylethyl)-benzoic acid (=DHPC, CAS registry number 16929-93-4) is feasible, but comprises 11 steps, complex chemistry, harsh conditions and precious catalysts. In Akagawa and Kugo, 2017 (Chem Commun (Camb). l ;53(62) : 8645-8648) a potential route for chemical synthesis of this compound is described. Harris and Carney; Journal of the American Chemical
Society (1967), 89(25), 6734-41, and Douglas and
Money; Tetrahedron (1967), 23(9), 3545-55 also relate to DHPC. In one embodiment of the present invention is the compound in form of a
pharmaceutical composition comprising the compound of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI and/or Formula VII, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
DETAILED DESCRIPTION OF THE INVENTION
Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to a "nucleic acid" means one or more nucleic acids. It is noted that terms like "preferably", "commonly", and "typically" are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.
For the purposes of describing and defining the present invention it is noted that the term "substantially" is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue. The term "about" in association with a numerical value means that the numerical value can vary plus or minus by 5% or less of the numerical value.
As used herein, the term "alkyl" means a straight or branched chain hydrocarbon containing from 1 to 14 carbons (unless otherwise noted). Examples of "alkyl" include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2- dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, undecyl, dodecyl, tridecyl, and the like.
The term "alkoxy" refers to an alkyl group of the indicated number of carbon atoms attached to the parent molecular moiety through an oxygen bridge. Examples of alkoxy groups include, for example, methoxy, ethoxy, propoxy and isopropoxy.
The term "carboxy" as used herein means a -CO2H group. In each of the following groups which are represented by R2 and R5 taken together,
Figure imgf000011_0001
the oxygen atom in the 5- and 6-membered rings is contributed by the R2 group and the R5 group contributes the carbon atom directly attached to the parent ring. roup which is represented by R3 and R4 taken together,
Figure imgf000011_0002
the oxygen atom in the 5-membered ring is contributed by the R3 group.
In each of the following groups which are represented by R3 and R5 taken togethe
Figure imgf000011_0003
the oxygen atom in the 5- and 6-membered rings is contributed by the R5 group and the R3 group contributes the carbon atom directly attached to the parent ring.
The present invention provides compositions comprising one or more compounds, and methods for the treatment of obesity, diabetes, and/or other related diseases or conditions by converting white adipose cells or adipose precursor cells into BRITE (brown in white) cells, inducing UCPl in such BRITE cells, and/or inducing UCPl in brown adipose tissue deposits. An object of the present invention relates to a compound for the regulation of metabolism in a mammal.
In one embodiment of the present invention is the compound of Formula I :
Figure imgf000012_0001
Formula I
Wherein Ri is -H, -CO2H, -OH, -CO2CH3, -CO2C2H5, -C02CH(CH3)2, -CONH2, -CONHCH3, or CON(CH3)2;
R2 is -H, -OH, -OCH3, or -OC2H5;
R3 is -H,
Figure imgf000012_0002
Figure imgf000013_0001
R.2 and Rs taken together with the atoms to which they are connected represent a roup of the formula
Figure imgf000013_0002
R3 and R4 taken to ether with the atoms to which they are connected represent
Figure imgf000013_0003
R3 and Rs together with the atoms to which they are connected represent a group of the formula
Figure imgf000013_0004
R6 is alkyl or a group of the formula
Figure imgf000013_0005
wherein R.7 represents -H, -OH, -OCH3,
Rs represents -H, -OH, -OCH3, an
Rg represents -H, -OH, -OCH3,
Figure imgf000014_0001
Preferred compounds of Formula I include those where R6 represents propyl .
Other preferred compounds of Formula I include those where R6 represents a group of the formula
Figure imgf000014_0002
In one embodiment of the present invention is the compound an Amorfrutin.
In one embodiment of the present invention is the compound Amorfrutin 1, having the chemical structure of Formula II :
Figure imgf000014_0003
(Formula II).
Amorfrutin 1 has been tested in the examples of the present disclosure, and shown to upregulate Amorfrutin 1 and furthermore to have a positive effect on the WAT to BAT transdifferentiation.
In one embodiment of the present invention is the compound Amorfrutin 2, having the chemical structure of Formula III
Figure imgf000015_0001
In one embodiment of the present invention is the compound Amorfrutin 3, having the chemical structure of Formula IV:
Figure imgf000015_0002
(Formula IV).
In one embodiment of the present invention is the compound stilbene carboxylate 1, having the chemical structure of Formula V:
Figure imgf000015_0003
(Formula V).
In one embodiment of the present invention is the compound stilbene carboxylate 2, having the chemical structure of Formula VI :
Figure imgf000016_0001
(Formula VI).
Formula V and Formula VI is also known as "Radula compound I" and "Radula compound Π", respectively. These are tested in Figure 6C of the present disclosure.
Figure imgf000016_0002
(Formula VII)
Formula VII is also known as 2,4-dihydroxy-6-(2-phenylethyl)-benzoic acid (= DHPC).
Thus, the invention also provides a pharmaceutical composition comprising a compound of Formula VII, as disclosed herein, or a compound derived from this compound by medicinal chemistry and/or biochemistry, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
In one embodiment of the present invention is the composition of the present invention comprising a compound of Formula (X) :
Figure imgf000017_0001
Formula (X) wherein
Figure imgf000017_0002
R4 is -H
Figure imgf000017_0003
wherein
R7, Rs, Rg represents -H,
In one embodiment of the present invention is the composition of the present invention comprising a compound of Formula (X) :
Figure imgf000017_0004
Formula (X) wherein
R2 is -OH; R3 and R5 together with the atoms to which they are connected represent a group that has a ring structure;
R4 is -H; and
R6 is alkyl or a group of the formula
Figure imgf000018_0001
wherein
R7, Rs, Rg represents -H,
One embodiment of the present invention relates to a composition comprising a compound or more compounds of the present invention.
In one embodiment of the present invention is the compound in form of a
pharmaceutical composition comprising a compound selected from one or more of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
The invention also provides a pharmaceutical composition comprising a compound of Formula I, as disclosed herein, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
The invention also provides a pharmaceutical composition comprising a compound of Formula II, as disclosed herein, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents. The invention also provides a pharmaceutical composition comprising a compound of Formula III, as disclosed herein, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
The invention also provides a pharmaceutical composition comprising a compound of Formula IV, as disclosed herein, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
The invention also provides a pharmaceutical composition comprising a compound of Formula V, as disclosed herein, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents. The invention also provides a pharmaceutical composition comprising a compound of Formula VI, as disclosed herein, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
The invention also provides a pharmaceutical composition comprising a compound of Formula VII, as disclosed herein, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents.
The invention further provides compositions, particularly pharmaceutical compositions, comprising one or a plurality of the compounds or compositions set forth herein, useful in the practice of any of the disclosed methods or uses.
The amount of the compounds is preferably in a pharmaceutically effective amount. This document shows that such compounds could promote the conversion or transdifferentiation/conversion of white adipose tissue into brown adipose tissue. In addition, this document describes, that such compounds were able to dramatically upregulate expression of UCP1. It is believed that expression and activity of UCP1 is what drives the aforementioned conversion or transdifferentiation to brown
adipocytes. One or more compounds of the invention can be used in the compositions or methods described herein.
Furthermore, this document shows that such compounds are capable of delay body mass increase, delay body fat increase, improve glucose tolerance, improve insulin sensitivity and increase interscapular brown adipose tissue. This can be seen in example 4. Thus, one aspect of the present invention relates to the one or more compounds of the present invention for use as a medicament.
The medicament can be for delay in body mass increase. The medicament can also be for delay in body fat increase. The medicament may also be for improvement of glucose tolerance. The medicament can be for improvement of insulin sensitivity, and can furthermore be for increase in interscapular brown adipose tissue. The
medicament may also be for use in the treatment of diabetes and/or obesity.
The administration of one or more compounds of the invention to a subject can be used to increase levels of UCP-1 expression (or other BAT related genes) and to induce white adipocytes to convert or transdifferentiate to brown adipocytes or BRITE. One or more compounds of the invention can induce conversion or transdifferentiation of white adipose cells to brown adipose cells. Transdifferentiation is where a differentiated (non-stem) cell is changed to another type of differentiated cell without being de-differentiated into a stem cell, for example, where a white adipose cell is changed to a brown adipose cell or BRITE.
The invention provides a composition for conversion or transdifferentiation of white adipose cells to thermogenic adipose cells, brown adipose cells or brown in white (BRITE) adipose cells, or for weight reduction, in a subject or an animal. The composition is able to : (1) upregulate UCP1 expression and other BAT-related genes and/or (2) promote the conversion or transdifferentiation of white adipocyte tissue into brown adipose tissue.
Thermogenic adipocytes are called brown adipocytes if localized in brown adipose tissue or BRITE (brown-in-white adipocytes, also called beige adipocytes or inducible brown adipocytes) if localized in white adipose tissue. The regulation of the metabolism can result in depletion of existing fat stores of the mammal.
White adipocytes may be converted or transdifferentiated to brown adipocytes.
In one embodiment of the present invention is white adipose tissue (WAT) converted to brown adipose tissue (BAT). The regulation of the metabolism can result in the treatment of diabetes and/or obesity. The expression of UCPl can be upregulated in the mammal that has had the one or more of the compounds of the present invention administered. The expression of UCPl can be upregulated in the target cell. This cell may be in vivo, in vitro or ex vivo. One or more brown adipose tissue specific genes selected from cell death-inducing DFFA (DNA fragmentation factor-alpha)-like effector A (CIDEA), carnitine
palmitoyltransferase IB (CPTIB), peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PPARGC1A), and cytochrome C can be upregulated.
An embodiment of the invention provides methods for upregulating UCPl expression. These methods comprise contacting a white adipocyte with one or more compositions of the invention, wherein the white adipocyte upreguiates UCPl expression, for a time and at a concentration sufficient to upregulate expression of UCPl . Upregulation of UCPl expression can promote conversion of the white adipocyte into a brown adipocyte, such as a BRITE adipocyte. One or more additional brown adipose tissue related genes can also be upregulated or downregulated, including but not limited to cell death-inducing DFFA (DNA fragmentation factor-alpha)-like effector A (CIDEA)), carnitine palmitoyltransferase IB (CPTIB), peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PPARGC1A), cytochrome C, creatine kinase,
mitochondrial 1A (CKMT1A/B), Potassium channel subfamily K member 3 (KCNK3), immunoglobulin heavy constant mu (IGHM), cordon-bleu WH2 repeat protein (COBL), cytoplasmic FMR1 interacting protein 2 (CYFIP2), Cytochrome P450 1A2 (CYP1A2), myosin, heavy chain 11, smooth muscle (MYH 11), angiotensinogen (serpin peptidase inhibitor, clade A, member 8) (AGT), Creatine kinase S-type (CKMT2),
transglutaminase 2 (TGM2), arginase 2 (ARG2), carbonic anhydrase XII (CA12), 3- hydroxy-3-methylglutaryl-CoA synthase 2 (mitochondrial) (HMGCS2), Complement C4-A (C4A/B), integrin, alpha 8 (ITGA8), and immunoglobulin lambda variable 2-14 (IGLV2-14).
In some embodiments, the compounds of the invention upregulate expression of one or more other brown adipose tissue specific genes CIDEA, CPTIB, PPARGC1A, and cytochrome C. In some embodiments, the compounds of the invention upregulate expression of CIDEA. In some embodiments, the compounds of the invention upregulate expression of CPTIB. In some embodiments, the compounds of the invention upregulate expression of PPARGC1A. In some embodiments, the compounds of the invention upregulate expression of cytochrome C.
In some embodiments, the compounds of the invention upregulate expression of one or more other brown adipose tissue specific genes CIDEA, CPT1B, PPARGC1A, and cytochrome C.
Upregulation is an increase in the amount of protein, levels of mRNA expression, or both from a target gene by about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100% or more (or any range between about 10% and 100%) as compared to a control not contacted with a compound.
Downregulation is a decrease in the amount of protein, levels of mRNA expression, or both from a target gene by about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or more including 100% (or any range between about 10% and 100%) as compared to a control not contacted with a compound.
Administration of one or more compounds of the invention can induce or upregulate expression or biological function of brown adipose tissue-specific genes, such as UCP- 1, by at least about 1.5, by at least about 1.6, or about 1.75, or about 2.0, or about 2.5, or at least about 3.0 fold higher than the expression level or biological function of a control sample that is not contacted with the one or more of said compounds.
In one embodiment the compound of the invention is administered to a mammal so that the upregulation of expression of UCPl in a mammal cell is more than 5-fold. The upregulation can be more than 10-fold; or more than 20-fold. The upregulation may also be more than 50-fold or more than 100-fold. The upregulation may even be more than 200-fold.
In a further embodiment the compound of the invention is administered to a mammal so as to provide a plasma concentration of more than 0.05 μΜ; more specifically more than 1.0 μΜ; more than 3.0 μΜ; more than 5.0 μΜ; more than 10 μΜ; more than 20 μΜ, or even more than 25 μΜ. The amount of compound administered may be 0.05 μΜ - 30 μΜ. The amount of compound administered may also be 0.05 μΜ - 20 μΜ. The amount of compound administered can be 1 μΜ - 10 μΜ. The amount of compound administered may be 5 μΜ - 200 μΜ. The amount of compound administered may also be 3 μΜ - 25 μΜ.
The amount of compound administered would usually be less than 30 μΜ. The amount may also be less than 20 μΜ or less than 10 μΜ. In a further embodiment the compound of the invention is administered to a human patient in need of treatment, in a daily dose of more than 0.1 mg/kg body mass μΜ; more specifically more than 0.5 mg/kg; more than 1.0 mg/kg; more than 5.0 mg/kg; more than 10 mg/kg; more than 50 mg/kg, or even more than 100 mg/kg. Thus, the daily dose may be 0.1 mg/kg to 200 mg/kg body mass μΜ. The daily dose may be less than 5.0 mg/kg, or less than 10 mg/kg; The daily dose may also be less than 50 mg/kg, or less than 100 mg/kg, or even less
than 200 mg/kg.
Levels of expression of brown adipose tissue-specific genes can be tested by, for example, western blot or quantitative RT-PCR (qPCR). Additionally, administration of one or more compounds of the invention can suppress or downregulate expression or biological function of brown adipose tissue-specific genes, by at least about 1.5, or about 1.75, or about 2.0, or about 2.5, or about 3.0 fold lower than the expression level or biological function of a control sample that is not contacted with the one or more of said compounds. The brown adipocyte can be a BRITE adipocyte. The brown adipocytes can comprise thermogenic adipocytes.
The mammal can be an overweight or obese mammal. The mammal can be a human subject.
An aspect of the present invention relates to a method for regulation of the
metabolism in a mammal, comprising a step of administering to the mammal a pharmaceutically effective amount of one or more compositions comprising one or more compounds of the present invention.
An aspect of the present invention relates to a method of treating diabetes in a mammal, comprising a step of administering to the mammal, comprising a step of administering to the mammal a pharmaceutically effective amount of one or more compositions comprising one or more compound of the present invention. An aspect of the present invention relates to a method of upregulating UCP1 expression, comprising contacting a white adipocyte with a pharmaceutical composition comprising a compound of Formula II and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the white adipocyte upregulates UCP1 expression.
An aspect of the present invention relates to a method of upregulating UCP1 expression, comprising contacting a white adipocyte with a pharmaceutical composition comprising a compound of Formula III and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the white adipocyte upregulates UCP1 expression.
An aspect of the present invention relates to a method of upregulating UCP1 expression, comprising contacting a white adipocyte with a pharmaceutical composition comprising a compound of Formula IV and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the white adipocyte upregulates UCP1 expression.
An aspect of the present invention relates to a method of upregulating UCP1 expression, comprising contacting a white adipocyte with a pharmaceutical composition comprising a compound of Formula V and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the white adipocyte upregulates UCP1 expression.
An aspect of the present invention relates to a method of upregulating UCP1 expression, comprising contacting a white adipocyte with a pharmaceutical composition comprising a compound of Formula VI and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the white adipocyte upregulates UCP1 expression.
An aspect of the present invention relates to a method of upregulating UCP1 expression, comprising contacting a white adipocyte with a pharmaceutical composition comprising a compound of Formula VII and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the white adipocyte upregulates UCP1 expression.
An aspect of the present invention relates to a method of upregulating UCP1 expression, comprising contacting a white adipocyte with a pharmaceutical composition comprising a compound having a chemical formula of any one of the chemical formulae set forth in Figure 1A, Figurel B, Figure 1C, Figure ID, Figure IE, and Figure IF, and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the white adipocyte upregulates UCP1 expression. An aspect of the present invention relates to a method of inducing conversion or transdifferentiation of white adipocytes into brown adipocytes, comprising contacting the white adipocytes with one or more compositions comprising a compound of the present invention, wherein the white adipocytes are converted or transdifferentiated into brown adipocytes. An aspect of the present invention relates to a method of depleting existing fat stores in a mammal, comprising administering to the mammal one or more compositions comprising a compound of the present invention.
Taken together, the data provided herein indicate that the compounds of the present invention may be effective in treating diseases such as obesity, as well as diabetes, by promoting burning of excessive body energy stores. Further, these data indicate that treatment with such compounds may represent a dramatic improvement over existing treatments.
Body energy stores include, for example ATP and creatine phosphate, carbohydrates (available, inter alia, as serum glucose, liver glycogen, and muscle glycogen), fat (available, inter alia, as serum fatty acids, serum triglycerides, muscle triglycerides, and adipose triglycerides) and protein (available, inter alia, as muscle protein).
In one embodiment of the invention, the compounds of the invention burn excessive body energy fat stores and/or excessive body energy adipose triglyceride stores. In an embodiment of the present invention is excessive body energy stores of the subject are burned is the uses or methods of the present invention.
An aspect of the present invention relates to a method for inducing thermogenesis in an adipocyte in a mammal, comprising a step of administering to the animal a thermogenesis-inducing amount of one or more compositions comprising a compound of the present invention, wherein thermogenesis in an adipocyte in the mammal is induced. An aspect of the present invention relates to a method for treating obesity in a mammal, comprising a step of administering to the animal a pharmaceutically effective amount of one or more compositions comprising a compound of the present invention, and wherein the administration of the one or more compositions
upregulates UCP1 expression in mammal animal.
An aspect of the present invention relates to a method of treating diabetes in mammal, comprising a step of administering to the animal a pharmaceutically effective amount of one or more compositions comprising a compound of the present invention, and wherein the administration of the one or more compositions
upregulates UCP1 expression in the mammal.
In an embodiment of the present invention is the methods of the present invention performed in vitro or ex vivo.
Diet Compositions and Supplements
In one embodiment, the invention provides a diet composition, containing compounds or compositions, including pharmaceutical compositions set forth herein, wherein in particular embodiments said diet comprises one or more of those compounds or compositions of the invention. In some instances, two or more compounds in combination can be administered at a lower percentage than one type of compound alone to achieve a similar effect as when one compound is used alone. One, two or more said compounds (e.g., 1, 2, 3, 4, 5, 10 or more) can be administered in a diet at about 0.01, or about 0.1, or about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 20, or about 30, or about 40% or more by weight of the total diet.
In one embodiment of the invention, the diet can comprise, by weight, about 0.1% to about 1%, or about 0.1 to about 5%, or about 5% to about 40% or more, or about 10% to about 40%; or about 10% to about 30%; or about 15% to about 25%; or about 20% to about 25% of said compounds or compositions set forth herein.
Compounds or compositions set forth herein can be mixed into the diet or
administered by other routes such as the use of dietary supplements. Dietary supplements can be tablets, capsules, softgels, gelcaps, liquids, or powder that supplement the diet, but are not considered a substitute for food. A dietary supplement can comprise about 0.001, or about 0.001, or about 0.01, or about 0.1, or about 1, or about 5, or about 10, or about 20 or more grams of one or more said compounds. A dietary supplement can be administered to a subject 1, 2, 3, 4, 5 or more times a day.
The invention also provides a diet composition for converting or transdifferentiating white adipose cells to thermogenic adipose cells, brown adipose cells or brown in white ("BRITE") adipose cells, or de novo generation of brown adipose cells from precursor cells, or for weight reduction, in an animal comprising about 0.01% to about 40%, or about 3.5% to about 40%; or about 10% to about 30%; or about 15% to about 25%; or about 20% to about 25% compounds or compositions set forth herein (i.e., about 0.01, or about 0.1, or about 1, or about 3.5, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20, or about 25, or about 30, or about 35, or about 40%, or more by weight of compound) and about 60% to about 99.99% (i.e., about 99.99, 99.9, 99, 96.5, 95, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 75, 70, 65, or 60%) of a diet that is low in calories. A low-calorie diet is a diet that comprises about 3%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80% less calories than the normal recommended amount of calories for a certain species of a given age, weight, gender, and general health condition.
The invention also provides a diet composition for converting or transdifferentiating white adipose cells to thermogenic adipose cells, brown adipose cells or brown in white (BRITE) adipose cells, or de novo generation of brown adipose cells from precursor cells, or for weight reduction, in an animal comprising about 0.01% to about 40%, or about 3.5% to about 40%; or about 10% to about 40%; or about 10% to about 30%; or about 15% to about 25%; or about 20% to about 25% of the compounds or compositions set forth herein (i.e., about 0.01, 0.1, 1, 3.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40%, or more by weight of compound) and about 60% to about 99.99% (i.e., about 99.99, 99.9, 99, 96.5, 95, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 75, 70, 65, or 60% ) of a diet that is low in fat. The low-fat diet can be a low saturated fat diet. A low-fat diet is a diet that comprises about 3%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about80% less fat than the normal recommended amount of fat in a diet for a given species of a given age, weight, gender, and general health condition. For example, a low-fat diet in humans can comprise a diet consisting of about 0%, or about 3%, or about 5%, or about 7%, or about 10%, or about 13%, or about 15%, or about 20%, or about 25% fat. The invention also provides a diet composition for converting or transdifferentiating white adipose cells to thermogenic adipose cells, brown adipose cells or brown in white (BRITE) adipose cells, or de novo generation of brown adipose cells from precursor cells, or for weight reduction, in an animal comprising about 0.01% to about 40%, or about 3.5% to about 40%; or 10% to about 40%; or about 10% to about 30%; or about 15% to about 25%; or about 20% to about 25% of compounds or compositions set forth herein (i.e., about 0.01, 0.1, 1, 3.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40%, or more by weight of compound) and about 60% to about 99.99% (i.e., about 99.99, 99.9, 99, 96.5, 95, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 75, 70, 65, or 60% ) of a diet that is low in carbohydrates. A low carbohydrate diet is a diet that comprises about 3%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80% less carbohydrates than the normal recommended amount of carbohydrates for a certain species of a given age, weight, gender, and general health condition. One or more compounds as set forth herein can be administered with an adjunctive weight loss therapy such as an exercise regimen, a low-fat diet, a low-calorie diet, a low-carbohydrate diet, surgical intervention such as gastroplasty, gastric partitioning, gastric bypass, behavioral therapy, pharmacotherapy (e.g., use of sibutramine, MERIDIA® (sibutramine HCI monohydrate, reductil), XENICAL® (orlistat), metformin (GLUCOPHAGE®), exenatide (BYETTA®), pramlintide (SYMLIN®), phentermine combined with tupiramate (QSYMIA®) rimonabant (acomplia), catecholamines, bupropion, topirmate, phentamine, amphetamines, locaserin (BELVIQ®) and combinations thereof), natural dietary aids and/or over-the-counter (OTC) weight-loss products, and combinations thereof. Behavior therapy includes strategies that help in overcoming barriers to compliance with dietary therapy and/or exercise therapy. Such strategies include, for example, self-monitoring of eating habits and exercise, stress management, stimulus control, problem-solving (e.g., self-corrections of problem areas related to eating and exercise), contingency management (e.g., use of rewards for specific desirable actions, cognitive restructuring (e.g., modification of unrealistic goals and inaccurate beliefs), and social support.
Administration Compounds or compositions as set forth herein can be formulated as pharmaceutical compositions. Pharmaceutical compositions comprise a pharmaceutically acceptable carrier or diluent and a pharmaceutically effective amount of compound. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990). Additionally, components such as preservatives, stabilizers, dyes, antioxidants, suspending agents, wetting agents, pH buffering agents, preservatives, and flavoring agents can be present in a pharmaceutical composition. A pharmaceutical composition can also comprise an excipient, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride and the like.
Pharmaceutical compositions can be formulated as tablets, capsules, lozenges, elixirs, suppositories, sterile solutions, inhalants or nasal sprays, liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid, injectables, topical sprays, powers, gels, or emulsions. The pharmaceutical compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents.
Pharmaceutical compositions can be utilized in vivo, in a mammalian patient, such as a human, or in vitro or ex vivo. The pharmaceutical compositions can be administered to a subject in a variety of ways, including topically, parenterally, intravenously, percutaneous, subcutaneously, intramuscularly, intravenous, intrathecal, colonically, rectally, nasally or intraperitoneally. Such methods can also be used in testing the activity of compounds in vivo or in vitro or ex vivo.
When administered by a drinking solution, compound compositions, including pharmaceutical compositions comprise one or more compounds dissolved in water, with appropriate pH adjustment. The compounds can be dissolved in distilled water, tap water, spring water, juice, milk, or any other beverage. The pH can be adjusted to between about 3.5 and about 8.5. Sweeteners may be added, e.g., about 0.1, or about 1, or about 5, or about 10% (w/v) sucrose or other natural or artificial sweetener.
The pharmaceutical compositions of this invention can also include: acids and bases to adjust the pH ; tonicity imparting agents such as sorbitol, glycerin and dextrose;
antimicrobial preservatives such as other parahydroxy benzoic acid esters, sorbate, benzoate, propionate, chlorbutanol, phenylethyl alcohol, benzalkonium chloride, and mercurials; viscosity imparting agents such as sodium carboxymethylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, polyvinyl alcohol and other gums; suitable absorption enhancers; stabilizing agents such as antioxidants, like bisulfate and ascorbate, metal chelating agents such as sodium edetate and drug solubility enhancers such as polyethylene glycols.
Dosage
The dosage of compounds or compositions set forth herein can range broadly depending upon the desired affects, the therapeutic indication, route of administration and purity and activity of the compound or composition. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the test compound. The pharmaceutically effective amount of a compound or composition set forth herein required as a dose will depend on the route of administration, the type of animal being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, gender, diet, metabolic rate, age, side effects, concurrent medication, health status, a property or nature of the compound itself, such as the administration vehicle or method, and other factors which those skilled in the medical arts will recognize.
A dosage for administration of a compound to a subject can be from about 1 mg/kg of body weight/day to about 1,000 mg/kg of body weight/day. For example, a dosage for administration of one or more compounds or compositions set forth herein to a subject can be from about 1, or about 5, or about 10, or about 20, or about 25, or about 50, or about 75, or about 100, or about 200, or about 300, or about 400, or about 500, or about 600, or about 700, or about 800, or about 900, or about 1,000 or more mg/kg of body weight/day, or any range between about 1 to about 1,000 mg/kg/day such as, for example about 1 mg/kg/day to about 1,000 mg/kg/day, or about 10 mg/kg/day to about 500 mg/kg/day, or about 50 mg/kg/day to about 200 mg/kg/day. A dosage for administration of one or more compounds or compositions set forth herein to a subject can also be from about 1,000, or about 900, or about 800, or about 700, or about 600, or about 500, or about 400, or about 300, or about 200, or about 100, or about 75, or about 50, or about 25, or about 20, or about 10, or about 1 or less mg/kg of body weight/day. In some embodiments, the dosage of amorfrutin 1 (Formula II), amorfrutin 2
(Formula III), Radula compound 1 (Formula V), Radula compound 2 (Formula VI) DHPC (Formula VII), Formula (I), Formula (x), Formula (Y), or amorfrutin 3 (Formula IV) is about 100 mg/kg/day. In some embodiments, the dosage of amorfrutin 1, amorfrutin 2, DHPC, Radula compound 1, Radula compound 2, Formula (I), Formula (x), Formula (Y), or amorfrutin 3 is about 1 g/kg/day. In some other embodiments, the dosage of amorfrutin 1, DHPC, amorfrutin 2, Radula compound 1, Radula compound 2, Formula (I), Formula (x), Formula (Y), or amorfrutin 3 is about 100 to about 1000 mg/kg/day. In some other embodiments, the dosage of amorfrutin 1, amorfrutin 2, DHPC, Radula compound 1, Radula compound 2, Formula (I), Formula (x), Formula (Y), or amorfrutin 3 is about 10 to about 100 mg/kg/day. In some other embodiments, the dosage of amorfrutin 1, amorfrutin 2, DHPC, Radula compound 1, Radula compound 2, Formula (I), Formula (x), Formula (Y), or amorfrutin 3 is about 500 to about 1000 mg/kg/day.
The administration of one or more of the compounds or compositions set forth herein can be used to increase levels of UCP1 expression and to induce white adipose tissue to transdifferentiate or convert to brown adipose tissue.
Administration of such compounds or compositions set forth herein to a subject can be useful to treat, prevent, or ameliorate several diseases and conditions, and to treat, prevent or ameliorate symptoms of several diseases and conditions as described herein.
Methods and compositions of the invention can also be used to convert or
transdifferentiate white adipose cells to thermogenic adipose cells, brown adipose cells or brown in white ("BRITE") adipose cells or to de novo generate brown adipose cells from precursor cells, or to reduce weighting an animal such as an overweight, obese, immature, or mature animal. Weight loss of about 3%, or about 5%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 75% can be achieved. In an embodiment, the invention provides methods for converting or transdifferentiating white adipose cells to thermogenic adipose cells, brown adipose cells or brown in white ("BRITE") adipose cells, or de novo generation of brown adipose cells from precursor cells, or for weight reduction, in an animal comprising administering to the animal one or more compounds as described above in an amount and for a time sufficient to have the desired effect in the animal.
Methods and compositions of the invention can be used to deplete existing fat stores (i.e. white adipose tissue or white adipocytes) in a subject. In one embodiment, the subject is overweight or obese. One or more compounds or compositions set forth herein can be administered to the subject, for example an overweight or obese subject. Fat is depleted or reduced when the fat is reduced by about 1, or about 5, or about 10, or about 15, or about 20, or about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90 % or more as compared to a control subject that is not administered one or more compounds or compositions set forth herein.
The subject, animal or patient can be, for example, a human, a non-human primate, a rat, a mouse, a rabbit, a guinea pig, a bovine, a pig, a sheep, a goat, a dog, a cat, a horse, a bird, a fish or an invertebrate. In one embodiment of the invention, the subject, animal, or patient is healthy and has no underlying health problems or issues. In another embodiment of the invention the subject, animal, or patient has one or more overweight or obesity health problems and no other health problems or issues. General
It should be understood that any feature and/or aspect discussed above in
connections with the compounds according to the invention apply by analogy to the methods described herein. The following figures and examples are provided below to illustrate the present invention. They are intended to be illustrative and are not to be construed as limiting in any way. BRIEF DESCRIPTION OF THE FIGURES
Figure 1
Figure 1 shows a generic structure encompassing the compounds of the invention (Formula I; Figure 1A), Amorfrutin 1 (Formula II; Figure IB), Amorfrutin 2 (Formula III; Figure 1C) and Amorfrutin 3 (Formula IV; Figure ID), Stilbene carboxylate 1 (Formula V, Figure IE), and stilbene carboxylate 2 (Formula VI, Figure IF).
Figure 2
Figure 2 shows the scheme of the 3T3-L1 differentiation protocol used in Example 1. Figure 3
Figure 3 shows expression levels of UCPl in 3T3-L1 cells upon adipocyte
differentiation from Example 1.
Figure 4
Figure 4 shows the morphology of 3T3-L1 cells upon adipocyte differentiation from Example 1.
Figure 5 shows the scheme of the human mesenchyme adipose-derived stem cells (hMADS) differentiation protocol used in Example 2.
Figure 6
Figure 6A shows expression levels of UCPl and FABP4 in hMADS upon adipocyte differentiation from Example 2.
Figure 6B shows expression level of UCPl in hMADS treated with the stilbene carboxylates.
Figure 6C shows expression level of UCPl in hMADS treated with Radula compounds 1 and 2 (a mixture of both compounds), and 2,4-dihydroxy-6-(2-phenylethyl)-benzoic acid (DHPC).
Figure 7
Figure 7 shows expression levels of UCPl by western blot in hMADS upon adipocyte differentiation from Example 2. Figure 8
Figure 8 shows high magnification images of the morphology of hMADS upon adipocyte differentiation from Example 2.
Figure 9
Figure 9A shows the scheme of human mesenchyme adipose-derived stem cells
(hMADS) differentiation protocol using, apart from Rosiglitazone, either Amorfrutin l, 2 or 3 for white adipose cell induction.
Figure 9B shows that Amorfrutins are only weak inducers of white adipogenesis, in contrast to rosiglitazone which has a strong induction effect on white adipogenesis. Skilled artisans will appreciate that elements in the Figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the Figures can be exaggerated relative to other elements to help improve understanding of the embodiment(s) of the present invention.
Figure 10
Rodent diet with 45 and 10 kcal % fat and same with rosiglitazone for example 4. Figure 11
The amount of compound mixed into the diet translated into the following uptake kinetics of compounds, based on reasonable food uptake assumptions in example 4. Figure 12
Timeline of the experiments for example 4.
Figure 13
UCPl knockout mice were eating slightly more than corresponding WT mice as shown in example 4.
Figure 14
In example 4 ingestion of compounds varied between 85% and 92% of the foreseen amount in WT mice. Figure 15
Rosiglitazone treatment increases body mass in knockout mice more rapidly than high fat diet only in example 4.
Figure 16
In example 4, Amorfrutin 1 ("edelweiss") in the HFD slightly decreases body mass increase in WT mice.
Figure 17
In example 4 resveratrol supplementation slightly slows down body mass increase in WT and knockout mice. Figure 18
In example 4, rosiglitazone significantly accelerates fat mass increase in both WT and knockout mice.
Figure 19
In example 4, AFl ("edelweiss") slightly slows down fat mass increase in both WT and knockout mice.
Figure 20
In example 4, resveratrol slows down fat mass increase in knockout mice, but not in WT mice.
Figure 21
In example 4, the oral glucose tolerance test (OGTT) strated with an oral glucose (1,4 mg/kg lean weight) administration (by oral gavage).
Figure 22
In example 4, In UCP1 knockouts all three compounds improve glucose tolerance, whereas in WT mice only rosiglitazone could improve glucose tolerance. Figure 23
In example 4, Amorfrutin 1 turned out to have a general beneficial effect on insulin sensitivity.
Figure 24
In example 4, protein concentration in the lysate was determined using the Lowry methods. We could see that interscapular brown adipose tissue increases in WT mice upon rosiglitazone and upon amorfrutin 1 treatment.
EXAMPLES
The Examples that follow are illustrative of specific embodiments of the invention and various uses thereof. They are set forth for explanatory purposes only and are not to be taken as limiting the invention.
The Amorfrutin compounds used in these Examples were obtained commercially from AnalytiCon Discovery (Potsdam, Germany). Additionally, these compounds may also be obtained by routine methods known by one of ordinary skill in the art. For example, Amorfrutin may be isolated by extraction from natural sources, such as a plant (Amorpha fruticosa), as described by Dat, N. T. et al. (2008) "Phenolic
Constituents of Amorpha fruticosa That Inhibit NF-KB Activation and Related Gene Expression" J. Nat. Prod. 71 : 1696-1700. Amorfrutins may also be synthesized by methods known by one of ordinary skill in the art (see, e.g., Weidner et al. (2012) "Amorfrutins are potent antidiabetic dietary natural products" Proc Natl Acad Sci USA 109 : 7257-7262, SI Appendix, SI Methods).
Example 1 : Amorfrutin 1 induces differentiation and expression of BAT-specific genes in 3T3-L1 cell line Mouse embryonic fibroblast cells (3T3-L1 cells; ATCC) were cultured in DMEM (high glucose), containing 10% fetal calf serum ("growth medium") at 37°C in a humidified C02 atmosphere. Cells were seeded at high density and were grown until they reached complete confluency. Then, at day 0, adipogenic differentiation was stimulated by adding 10 μg/ml insulin, 0.5 mM isobutylmethylxanthine (IBMX), and 1.0 μΜ Dexamethasone. At the same time 1.0 μΜ Rosiglitazone or 1.0 μΜ Amorfrutin, or corresponding amount of solvent (DMSO) was added. At day 3 the medium was changed to growth medium supplied with 0.2 μg/ml of insulin. This medium was changed every second day until day 10 of differentiation (Figure 2). On days 0, 3, and 10, total cellular RNA was isolated from a portion of the cells using an RNeasy kit (Qiagen). A high capacity cDNA reverse transcription kit (BioRad) was used for cDNA synthesis. Quantitative PCR (qPCR) analyses were performed on RotorGene Real-Time PCR System (Qiagen), using a FastStart Universal SYBR Green Master Mix (Roche) and the following mouse-specific primers: Mm_Ucpl_l_SG QuantiTect primer assay (Cat. Number QT00097300), Mm_Actb_2_SG QuantiTect primer assay (Cat. Number QT01136772).
Quantitative RT-PCR analysis revealed upregulation of the BAT-specific gene UCP1 upon treatment with Rosiglitazone and Amorfrutin 1 in comparison to the mock- treated control in the samples isolated on days 3 and 10 of 3T3-L1 differentiation . Moreover, Amorfrutin 1 was able to upregulate expression of UCP1 to comparable levels as seen with Rosiglitazone (Figure 3).
Microscopic analysis of the cell morphology revealed differentiated adipocytes, accumulating small lipid droplets on day 3 in the samples treated with Rosiglitazone and compound . On day 10, the majority of the cells treated with Rosiglitazone and compound showed morphology of differentiated adipocytes, while only single cells in the mock-treated control underwent differentiation (Figure 4). Example 2 : Up-regulation of UCP1 expression upon candidate compound treatment - Amorfrutin 1 and similar compounds induces differentiation and expression of BAT- specific genes in hMADS cell line hMADS, human multipotent adipose-derived stem cells, were cultured in growth medium composed of DMEM containing low glucose (Sigma) supplemented with 10% FCS (Sigma, Lot No. 078K3395), 2 mM L-glutamine (Sigma) and 10 mM HEPES (Lonza) at 37°C in a humidified 5% C02 atmosphere. For adipogenic differentiation, hMADS of passage 13- 15 were seeded at the density of approximately 10,000 per cm2 in growth medium containing 2.5 ng/ml FGF2 (Invitrogen). After three days growth medium was replaced by fresh growth medium lacking FGF2. Two days later (Day 0) hMADS were induced to differentiate in the presence of differentiation medium composed of DMEM/Ham's F12 (1 : 1) (Sigma) supplemented with 10
Figure imgf000038_0001
transferrin, 0.2 nM triiodothyronine (T3), 10 nM insulin, and 1 μΜ dexamethasone, 0.5 mM isobutylmethylxanthine (IBMX). To elucidate if candidate compounds were potent to promote adipocyte differentiation, differentiation medium lacking IBMX and dexamethasone was supplemented with either Amorfrutin 1, 2 or 3, stilbene carboxylate or Radula compound 1 and/or 2, or DH PC, or 0.1 μΜ Rosiglitazone, or DMSO as solvent control (Day 3). Differentiation was performed for 7 days (until Day 10) with medium changes on Day 5 and 7 (Figures 5 and 6). In this example (Figure 6b) "stilbene carboxylate" refers to the mixture of Radula compounds 1 and 2. Radula 1 is also known as stilbene carboxylate 1 (figure IE) and Radula 2 is also known as stilbene carboxylate 2 (figure IF).
From Day 11 to Day 15 cells were incubated with differentiation medium lacking IBMX, dexamethasone and candidate compounds (treatment pause).
To test for the potential of test compounds to induce white to brown adipocyte conversion cells were incubated for another four days in presence of differentiation medium containing either 0.3 μΜ Amorfrutin 1, 0.1 μΜ Rosiglitazone, or DMSO. Total RNA and protein was isolated following the TriReagent protocol (Sigma). cDNA was synthesized using iScript cDNA synthesis kit (BioRad) and thereafter quantitative PCR was performed on a CFX connect (BioRad) with the SsoAdvanced Universal SYBR Green Supermix 1000 (BioRad) using 10 ng of cDNA per reaction. The following primers were used : For UCPl : QuantiTec primer assay (Qiagen), for 36B4: EVPR5825 and EVPR5826, for FABP4: EVPR4690 and EVPR4691.
Quantitative RT-PCR analysis revealed up-regulation of BAT-specific genes UCPl and FABP4 upon treatment with Rosiglitazone and Amorfrutin 1 in comparison to the mock- treated control in the samples isolated on days 3 and 10 of hMADS differentiation. Moreover, Amorfrutin 1 was able to upregulate expression of BAT- specific genes to comparable levels with Rosiglitazone (Figure 6A).
Western blot was conducted in order to assess levels of UCPl protein upon treatment with Amorfrutin 1. Total protein from hMADS (treated as discussed above) was extracted using the TriReagent (Sigma). Proteins were precipitated with 3 volumes of ice-cold acetone. Protein pellets were washed 3 times with 0.3 M guanidine HCI in 95% EtOH containing 2.5% glycerol. Each wash was performed for 20 minutes. After another wash using 100% EtOH containing 2% glycerol the protein pellets were dried and then dissolved in 2% SDS. After cooking proteins at 95°C for 10 minutes insoluble material was removed by centrifugation at lO.OOOg for 10 minutes at 4°C.
Proteins (20 μg) were separated on 4-20% Mini-PROTEAN® TGX™ gels (BioRad) and transferred onto PVDF membranes using Trans-Blot Transfer Pack Mini (BioRad). Immunoblotting was performed using goat-anti-human UCPl antibody (Santa Cruz) or rabbit-anti-human Tom20 (Santa Cruz) as loading control, followed by HRP-conjugated secondary antibodies (Sigma). All antibodies were diluted in Tris-buffered saline containing 0.1% Tween and 5% milk powder (RapiLait, Migros). Protein bands were visualized by incubating membranes with Amersham ECL Prime Western Blotting Detection Reagent and subsequent visualization with the Image Quant LAS4000 (GE Healthcare).
Western blotting analysis performed on hMADS protein extracts detected a band of approximately 34 kDa, corresponding to the described molecular weight of UCPl in the samples treated with Rosiglitazone and Amorfrutins 1, 2 and 3 (Figure 7). No band was detected in the mock-treated control sample. These Western blot results corroborate to a certain extent the RT-PCR data results, in showing that Amorfrutins upregulate UCPl protein expression, however, to a significantly lower level as compared to Rosiglitazone. Microscopic analysis of the cell morphology revealed differentiated adipocytes, accumulating small lipid droplets, on day 10 in all samples treated with Rosiglitazone and Amorfrutin I. Within the mock-treated control, some isolated cells only underwent differentiation. Morphology of the differentiated adipocyte was even more pronounced on day 15 upon stimulation (Figure 8).
Non-differentiated hMADS do not significantly differentiate into white adipocytes upon Amorfrutin treatment (Figure 9A). The lipid content also does not massively increase in hMADS upon Amorfrutin treatment (Figure 9B), in contrast to the situation in Rosiglitazone-treated hMADS.
Taken together, the data from Example 2 demonstrate that amorfrutins, including Amorfrutin 1, the Radula compounds and DHPC all have the potential to promote conversion or transdifferentiation of human adipocyte precursors into brown adipocytes, as they all inducethe BAT-specific gene UCPl .
Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as particularly advantageous, it is contemplated that the present invention is not necessarily limited to these particular aspects of the invention.
Example 3 - In vivo study of Amorfrutin on body mass, glucose tolerance, insulin sensitivity and BAT. Executive summary
The major outcomes of the experiments were:
Amorfrutin 1 delays body mass increase in WT mice but not in UCP1 Kos.
Amorfrutin 1 delays fat mass increase in WT mice but not in UCP1 Kos.
Amorfrutin 1 improves glucose tolerance in UCP1 Kos.
Amorfrutin 1 has a beneficial effect on insulin sensitivity of WT as well as UCP1 KOs. Amorfrutin 1 increases interscapular brown adipose tissue in WT mice.
Mice
All mice used in this study— WTs and UCPlKOs— are on C57BI/6 background. UCP1 KO mice (progeny of those described in Enerback et al., 1997) have been backcrossed to C57BI/6 for 10 generations, and after intercrossing, they were maintained as a strain, in parallel with the wild-type C57BI/6 mice. The mice were kept at
thermoneutrality and had access to food and water ad libitum. Mice were sacrificed 4 weeks after start of high fat diet feeding. Animal studies were approved by the Ethical Committee of the University of Stockholm.
Compounds
Three different compounds were used throughout the study
Amorfrutin 1
Figure imgf000041_0001
Amorfrutin 1 is a carboxylated, O-methylated, and prenylated dihydropinosylvin found in the roots of Glycyrrhiza foetida, in the fruits of Amorpha fruticosa, and in the moss Radula complanata.
Resveratrol
Figure imgf000042_0001
Resveratrol is a stilbenoid found in the skin of grapes, blueberries, raspberries, mulberries, lingonberry, and senna.
Rosiglitazone
Figure imgf000042_0002
Rosiglitazone is a purely chemical compound that is used here as a control compound. It has recently been shown to upregulate UCP1 in vivo in white adipocytes of mice (Pardo et al., 2011).
Diet composition and predicted compound uptake
The high fat diet (HFD) was formulated by Research Diets Inc, located in New
Brunswick in USA. Diet "D12451" designates the background ("control") diet to which the different compounds were added (figure 10).
Compound uptake
The amount of compound mixed into the diet translated into the following uptake kinetics of compounds, based on reasonable food uptake assumptions can be seen in Figure 11.
Timeline of the experiments
Mice were kept at room temperature until they reached an age of 9 weeks. Then they were transferred to 30°C in order to keep them at thermoneutrality (figure 12).
Results Please note that in most graphs shown throughout this section amorfrutin 1 is designated with the code name "Edelweiss".
Compound uptake
Compound uptake was calculated indirectly by a) measuring uptake of food once a week throughout the entire feeding period (4 weeks) and b) multiplying this number by the amount of compound in the respective amount of food. Both mice cohorts, WT and UCPl KO, tended to reduce food uptake throughout the 4 weeks of HFD feeding. Therefore, the amount of compound ingested also slowly decreased during the 4 weeks period of feeding. We therefore calculated an average intake of compound based on the total food intake throughout the 4 weeks period.
Altogether, UCPl knockout mice were eating slightly more than corresponding WT mice (figure 13). Compound ingestion was almost exactly the amount foreseen for UCPl KO mice, whereas ingestion of compounds varied between 85% and 92% of the foreseen amount in WT mice, (figure 14).
Body mass and fat mass increase
Body mass. Rosiglitazone treatment increases body mass in knockout mice more rapidly than high fat diet only (figure 15). Amorfrutin 1 ("edelweiss") in the HFD slightly decreases body mass increase in WT mice (figure 16), whereas resveratrol supplementation slightly slows down body mass increase in WT and knockout mice (figure 17). As the number of analysed mice was rather low, results did not reach statistical significance. But there is a clear tendency towards decreased body mass gain upon amorfrutin 1 treatment.
Fat Mass increase
Rosiglitazone significantly accelerates fat mass increase in both WT and knockout mice (figure 18). AF1 ("edelweiss") slightly slows down fat mass increase in both WT and knockout mice (figure 19). Resveratrol slows down fat mass increase in knockout mice, but not in WT mice (figure 20). Body fat mass was measured with in vivo magnetic resonance imaging using an EchoMRI-lOOTM instrument (Houston, USA). Glucose tolerance
The oral glucose tolerance test (OGTT) strated with an oral glucose (1,4 mg/kg lean weight) administration (by oral gavage). Blood glucose levels were measured with a blood glucometer (Accu-Check Avia, Roche) before glucose challenge, and 15, 30, 60, and 90 minutes afterwards. Mice were fasted for 8h prior to glucose administration. In UCPl knockouts all three compounds improve glucose tolerance, whereas in WT mice only rosiglitazone could improve glucose tolerance (figures 21 and 22).
Insulin sensitivity
To analyse insulin sensitivity, blood samples were collected using capillary tubes
(Microvette CB300, Sarstedt) at times 0 ("fasted") and 15 minutes of OGTT ("glucose stimulated"). Plasma insulin levels were measured using an Ultra-sensitive mouse insulin ELISA kit (Chrystal Chem Inc.). Amorfrutin 1 turned out to have a general beneficial effect on insulin sensitivity (figure 23). Both WT and UCPl knockouts show an elevated insulin sensitivity upon AFl supplementation of the high fat diet. WT mice significantly profit from AFl supplementation, and UCPl knockouts probably also profit from UCPl supplementation, albeit this result did not reach statistical significance.
Brown adipose tissue
Mice were killed by C02. Interscapular depots of brown adipose tissue and inguinal depots of white adipose tissue (one lobe each) were dissected and weighted. Tissues were homogenized in modified RIPA buffer (50 mM Tris-Hcl, pH 7.4, 1% Triton X-100, 150 mM NaCI, 1 mM EDTA) with freshly added phosphatase inhibitors (ImM Na3V04 and 10 mM NaF) and protease inhibitor cocktail (Complete Mini, Roche). Homogenates were centrifuged at 14000 g for 15 min to discard top fat layer. Protein concentration in the lysate was determined using the Lowry methods. We could see that
interscapular brown adipose tissue increases in WT mice upon rosiglitazone and upon amorfrutin 1 treatment (figure 24). The increase in IBAT total protein was not accompanied by elevated amounts of total UCPl (not shown). The increase in IBAT may still be beneficial as BAT secretes Neuregulin 4 (Nrg4). This growth factor is enriched in brown adipose tissue. Therefore, more BAT may lead to higher secretion of Nrg4. It has been shown that transgenic mice overexpressing Nrg4 were protected against diet-induced insulin resistance, independent of UCPl expression (Wang et al. 2014). Items
1. A composition comprising a compound of Formula I:
Figure imgf000045_0001
Formula I Wherein
Ri is -H, -CO2H, -OH, -CO2CH3, -CO2C2H5, -C02CH(CH3)2, -CONH2, -CONHCH3, or CON(CH3)2; R2 is -H, -OH, -OCH3, or -OC2H5;
Figure imgf000045_0002
R2 and Rs taken together with the atoms to which they are connected represent a group of the formula
Figure imgf000046_0001
R.3 and R4 taken to ether with the atoms to which they are connected represent
Figure imgf000046_0002
R3 and Rs together with the atoms to which they are connected represent a group of the formula
Figure imgf000046_0003
R6 is alkyl or a group of the formula
Figure imgf000046_0004
wherein
R? represents -H, -OH, -OCH3, -OC2H5 , or
Rs represents -H, -OH, -OCH3, -OC2H5, or ancj
Rg represents -H, -OH, -OCH3, -OC2H5, or
Figure imgf000046_0005
for use in regulation of the metabolism in a mammal. 2. The composition for use according to item 1, wherein the compound is an
Amorfrutin.
3. The composition for use according to any one of items 1-2, wherein the Amorfrutin is Amorfrutin 1 having the chemical structure of Formula II :
Figure imgf000047_0001
(Formula II).
4. The composition for use according to any one of items 1-2, wherein the Amorfrutin is Amorfrutin 2 having the chemical structure of Formula III
Figure imgf000047_0002
(Formula III).
5. The composition for use according to any one of items 1-2, wherein the Amorfrutin is Amorfrutin 3, having the chemical structure of Formula IV:
Figure imgf000047_0003
(Formula IV). 6. The composition for use according to item 1, wherein the composition is comprising a compound of Formula (X) :
Figure imgf000048_0001
Formula (X)
wherein R2 is -OH ;
R3 and Rs together with the atoms to which they are connected represent a group that has a ring structure;
R4 is -H ;
and
R6 is alkyl or a group of the formula
Figure imgf000048_0002
wherein
R7, Rs, Rg represents -H, 7. The composition for use according to any one of items 1-2 and 6, wherein the compound has the chemical structure of Formula V:
Figure imgf000049_0001
(Formula V)
8. The composition for use according to any one of items 1-2 and 6, wherein the compound has the chemical structure of Formula VI :
Figure imgf000049_0002
(Formula VI)
9. The composition for use according to item 1, wherein the composition is comprising a compound of Formula (X) :
Formula (X)
Figure imgf000050_0001
wherein
Figure imgf000050_0002
R2 is -OH ;
Figure imgf000050_0003
R4 is -H ;
Figure imgf000050_0004
R6 is alkyl or a group of the formula
Figure imgf000050_0005
wherein
R7, Re, R9 represents -H,
10. The composition for use according to any one of items 1-5, wherein the compound is in form of a pharmaceutical composition comprising the compound of Formula I and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents. 11. The composition for use according to any one of items 1-10, wherein the regulation of the metabolism results in depletion of existing fat stores of the mammal.
12. The composition for use according any one of items 1-11, wherein white adipocytes or the adipocyte precursor cell are converted or transdifferentiated to brown adipocytes.
13. The composition for use according any one of items 1-2, wherein white adipose tissue (WAT) is converted to brown adipose tissue (BAT).
14. The composition for use according any one of items 1-13, wherein the regulation of the metabolism results in the treatment of diabetes and/or obesity.
15. The composition for use according to any one of items 1-14, wherein the expression of UCP1 is upregulated.
16. The composition for use according to any one of items 1-15, wherein one or more brown adipose tissue specific genes selected from cell death-inducing DFFA (DNA fragmentation factor-alpha)-like effector A (CIDEA), carnitine palmitoyltransferase IB (CPT1B), peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PPARGC1A), and cytochrome C are upregulated.
17. The composition for use according to any one of items 12-16, wherein the brown adipocyte is a BRITE adipocyte.
18. The composition for use according to any one of items 12-16, wherein the brown adipocytes comprise thermogenic adipocytes. 19. The composition for use according to any one of items 12-18, wherein the mammal is an overweight or obese mammal.
20. The composition for use according to any one of items 1-19, wherein the mammal is a human.
21. A method for regulation of the metabolism in a mammal, comprising a step of administering to the mammal a pharmaceutically effective amount of one or more compositions comprising a compound of Formula I :
Figure imgf000052_0001
Formula I wherein Ri-R.6 are as recited in item 1.
22. A method of treating diabetes in a mammal, comprising a step of administering to the animal mammal, comprising a step of administering to the mammal a
pharmaceutically effective amount of one or more compositions comprising a compound of Formula I :
Figure imgf000052_0002
Formula I wherein Ri-R.6 are as recited in item 1.
23. A method of upregulating UCP1 expression, comprising contacting an adipocyte precursor cell or white adipocyte with a pharmaceutical composition comprising a compound of Formula II and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the adipocyte precursor cell or white adipocyte upregulates UCP1 expression.
24. A method of upregulating UCP1 expression, comprising contacting an adipocyte precursor cell or white adipocyte with a pharmaceutical composition comprising a compound of Formula III and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the adipocyte precursor cell or white adipocyte upregulates UCP1 expression.
25. A method of upregulating UCP1 expression, comprising contacting an adipocyte precursor cell or white adipocyte with a pharmaceutical composition comprising a compound of Formula IV and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the adipocyte precursor cell or white adipocyte upregulates UCP1 expression.
26. A method of upregulating UCP1 expression, comprising contacting an adipocyte precursor cell or white adipocyte with a pharmaceutical composition comprising a compound having a chemical formula of any one of the chemical formulae set forth in Figure 1A through IF and one or a plurality of pharmaceutically acceptable excipients, carriers or diluents, wherein the adipocyte precursor cell or white adipocyte
upregulates UCP1 expression. 27. A method of inducing conversion or transdifferentiation of adipocyte precuri cells into brown adipocytes, comprising contacting the adipocyte precursor cells one or more compositions comprising a compound of Formula I :
Figure imgf000054_0001
Formula I wherein Ri-R.6 are as recited in item 1, wherein the adipocyte precursor cells are converted to brown adipocytes.
28. A method of inducing conversion or transdifferentiation of white adipocytes into brown adipocytes, comprising contacting the white adipocytes with one or more compositions comprising a compound of Formula I :
Figure imgf000054_0002
Formula I wherein R1-R6 are as recited in item 1, wherein the white adipocytes are converted or transdifferentiated into brown adipocytes.
29. A method of depleting existing fat stores in a mammal, comprising administering to the mammal one or more compositions comprising a compound of Formula I :
Figure imgf000055_0001
Formula I wherein Ri-R.6 are as recited in item 1.
30. The method of item 28 or 29, wherein excessive body energy stores of the subject are burned.
31. A method for inducing thermogenesis in an adipocyte in a mammal, comprising a step of administering to the animal a thermogenesis-inducing amount of one or more compositions comprising a compound of Formula I :
Figure imgf000055_0002
Formula I wherein Ri-R.6 are as recited in item 1, wherein thermogenesis in an adipocyte in the mammal is induced.
32. A method for treating obesity in a mammal, comprising a step of administering to the animal a pharmaceutically effective amount of one or more compositions comprising a compound of Formula I :
Figure imgf000056_0001
Formula I wherein Ri-R.6 are as recited in item 1, and wherein the administration of the one or more compositions upregulates UCP1 expression in mammal animal.
33. A method of treating diabetes in mammal, comprising a step of administering to the animal a pharmaceutically effective amount of one or more compositions comprising a compound of Formula I :
Figure imgf000056_0002
Formula I wherein R1-R6 are as recited in item 1, and wherein the administration of the one or more compositions upregulates UCP1 expression in the mammal.
34. The method according to items 21-33, wherein the method is performed in vitro or ex vivo.
35. The composition for use according to any one of items 1-2 or 9, wherein the compound has the chemical structure of Formula VII :
Figure imgf000057_0001
10

Claims

1. A composition comprising a compound of Formula (X) :
Figure imgf000058_0001
Formula (X) wherein
R2 is -OH ;
R3 and Rs together with the atoms to which they are connected represent a group that has a ring structure;
and
R6 is alkyl or a group of the formula
Figure imgf000058_0002
wherein
R7, Rs, Rg represents -H, for use in regulation of the metabolism in a mammal, wherein the administration of a composition comprising compound of Formula leads to at least 5-fold upregulation of the expression of UCPl in fat tissue as compared to untreated.
2. The composition for use according to claim 1, wherein the compound has the chemical structure of Formula V:
Figure imgf000059_0001
(Formula V)
3. The composition for use according to claim 1, wherein the compound has the chemical structure of Formula VI :
Figure imgf000059_0002
(Formula VI)
4. The composition for use according to any one of claims 1-3, wherein the fat cell are human multipotent adipose-derived stem cells (hMADS).
5. The composition for use according to any one of claims 1-4, wherein the regulation of the metabolism results in depletion of existing fat stores of the mammal.
6. The composition for use according any one of claims 1-5, wherein white adipocytes or the adipocyte precursor cell are converted or transdifferentiated to brown adipocytes.
7. The composition for use according any one of claims 1-6, wherein white adipose tissue (WAT) is converted to brown adipose tissue (BAT).
8. The composition for use according any one of claims 1-7, wherein the regulation of the metabolism results in the treatment of diabetes and/or obesity.
9. The composition for use according to any one of claims 1-9, wherein the mammal is an overweight or obese mammal.
10. The composition for use according to any one of claims 1-9, wherein the mammal is a human.
11. The composition for use according to any one of claims 1-10, wherein the administration of a composition comprising a compound of Formula (Y) leads to a plasma concentration of 0.05 μΜ to 30 μΜ.
12. The composition for use according to any one of claims 1-10, wherein the composition comprising a compound of Formula (Y) is administered to a human individual in need of treatment in a daily dose of more than 0.1 mg/kg body mass and less than 200 mg/kg body mass.
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