WO2013072332A1 - Procédés d'utilisation d'antagonistes de récepteurs du goût amer - Google Patents

Procédés d'utilisation d'antagonistes de récepteurs du goût amer Download PDF

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Publication number
WO2013072332A1
WO2013072332A1 PCT/EP2012/072553 EP2012072553W WO2013072332A1 WO 2013072332 A1 WO2013072332 A1 WO 2013072332A1 EP 2012072553 W EP2012072553 W EP 2012072553W WO 2013072332 A1 WO2013072332 A1 WO 2013072332A1
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WIPO (PCT)
Prior art keywords
compound
formula
seq
bitter
acid
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PCT/EP2012/072553
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English (en)
Inventor
Jay Patrick Slack
Thomas Scott Mccluskey
Christopher Allen HARTZEL
Amy Marie ODLEY
Zhonghua Jia
Ioana Maria Ungureanu
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Givaudan Sa
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Publication of WO2013072332A1 publication Critical patent/WO2013072332A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
    • A23F3/00—Tea; Tea substitutes; Preparations thereof
    • A23F3/06—Treating tea before extraction; Preparations produced thereby
    • A23F3/14—Tea preparations, e.g. using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • A23L27/31—Artificial sweetening agents containing amino acids, nucleotides, peptides or derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • A23L27/33—Artificial sweetening agents containing sugars or derivatives
    • A23L27/36—Terpene glycosides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • A23L27/33—Artificial sweetening agents containing sugars or derivatives
    • A23L27/37—Halogenated sugars
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/86—Addition of bitterness inhibitors
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins

Definitions

  • the field relates to compounds that act as antagonists to the T2R family of bitter taste receptors, in particular to compositions comprising and methods of using [2-(fitran-3- yl)ethyl]-trimethyI-hexahydronaphthalene-carboxylic acid and related compounds in free or orally acceptable base addition salt form to mask or decrease a subject's perception of bitter taste.
  • T2R also known as the TAS2R
  • TAS2R T2R
  • T2R family belongs to the family A of GPCRs including VI r pheremone receptors and opsin, receptors.
  • T2R41 , T2R4.2, T2R45, T2R48, T2R60 receptor ligands for four of the five of the .25 T2R members (T2R41 , T2R4.2, T2R45, T2R48, T2R60) are at present unknown
  • T2R41 has been, recently shown, to bind to cyclamate (see for example, WO 201 1/138455). The fact that T2Rs are coexpressed in a.
  • T2Rs form heterooligomers like Tl Rs for sweet and. umami taste.
  • the bitter receptor gene family in vertebrates, retains a relatively conserved family size of about 15-33 functional genes; among mammalian species there are multiple orthologues of those genes. Possibly a contributing factor as to why small molecule development in this area has been slow to develop, is that the region where the most T2R family sequence divergence is observed happens to occur in the extracellular domain where ligand binding may be likely to take place.
  • T2R antagonists have been difficult to develop as T2R receptors have shown preference for activation by single specific ligands.
  • mT2R5, hT2R43, and hT2R47 show specificity for single ligands (cyclohexamide, aristolochic acid, and denatoniuni. respectively) (Sainz E.. et al Biochem. J. Immediate Publications, doi, 10.1042/BJ20061744 (2007)).
  • Orthologs hT2R4 and mT2R8 responded to high concentrations of the structurally disparate compounds PROP and denatoniuni, but those two compounds are the only active agonists out of a battery of fifty-five compounds tested (Chaiidreshakar, J. et al. Cell 100, 703-71 1 (2000)).
  • two possible exceptions to the high selectivity exhibited by T2Rs appear to be hT2R7 (Sainz E., et al Biochem. J. Immediate Publications, doi, 10.1042/BJ20061744 (2007)) and hT2R14 (Behrens. M., Biochem, Biophys. Res. Comm. 319, 479-484 (2004)).
  • bitter taste perception Being able to manipulate bitter taste perception would be extremely useful.
  • the lingering aftertaste that accompanies some commercially desirable artificial sweeteners which show greatly enhanced sweetness compared with sucrose and have very low caloric value, could be due to the sweeteners activation or binding to certain T2R bitter taste receptors.
  • such sulfonyl amide sweeteners as saccharin and acesulfame K. both of which possess lingering bitter aftertaste (see Kuhn et al (2004); Journal of Neuroscience 24 (45); 10260-10265).
  • the ability to modulate bitter taste receptors in the context of artificial sweeteners. could markedly improve the quality of the taste of the food product wherein the artificial sweetener has been incorporated.
  • bitterness is a major technical hurdle in formulating pharmaceuticals, which can be essential to ensure patient compliance.
  • Certain foods or beverages that are inherently bitter could be improved by the addition of a compound that could modulate bitter taste receptor signal transduction.
  • utraceutical compositions or nutritional supplements may have a bitter taste for example due to the presence of amino acids or minerals. Alleviating bitterness would be useful also for inducing animals to eat food that is healthy, cheap and nourishing, but has a bitter taste.
  • a compound would need to decrease the perception of bitterness not only at one T2R receptor but via a number of those same T2R family of receptors.
  • Compounds of the Invention e.g. of Formula 1 below, or [2-(furan-3-yl)ethyl]-trimethyl-hexahydronaphthalene-carboxylic acid compounds, e.g.. hardwickiic acid and isomers thereof, in free or orally acceptable base addition salt form, are useful to modulate multiple bitter taste receptors (T2R), and the invention thus provides methods and products utilizing Compounds of the Invention to reduce the perception of bitter flavors and/or to enhance the perception of sweet flavors, ; hereinafter described,
  • Ri is H, -CH 3 or (wherein the dotted line is a double bond) is not present;
  • R 2 is -CH 3 , -(CO)OR 9 , -CHO, or -C3 ⁇ 4OH;
  • R 3 is -CH 3 or H
  • RA is -CH 3 or H
  • R 5 is H or -CH3
  • R 7 is -CH 3 ;
  • R 7 and Rg together are furanyl
  • R « is H or Ci.4 alkyl, e.g., methyl
  • the Compound of the Invention may be selected from, e.g.. Compounds 1 , 2, 3, 4, 5, 6, and 7:
  • the Compound of the Invention is a [2-(furan-3-yl)ethyl]- trimethyl-hexahydronaphthalene-carboxylic acid, e.g., selected from hardwickiic acid and isomers thereof, in free or orally acceptable base addition salt form, e.g. selected from (-)- hardwickiic acid (Compound 1), (-)-epi-hardwickiic acid (Compound 2), and the hardwickiic acid structural isomer Compound 7;
  • the compounds of Formula 1 are compounds of Formula l :
  • R 2 , R 3 . R$, R?. Rg and Rg are as hereinbefore set forth for Formula 1 , and R ⁇ Is methyl.
  • the Compound of the Invention may be selected from
  • each in free or orally acceptable base addition salt form is orally acceptable base addition, salt
  • a base addition, salt is meant a salt formed by reacting a carboxylic acid bitter taste antagonist with a base to form a salt, wherein the cation, provided by the base is safe and palatable for oral ingestion, at concentrations to be administered, e.g., a sodium or potassium salt.
  • the Compound of the Invention is (-)-hardwickiic acid in. free or orally acceptable base addition salt form.
  • isolated as used herein in relation to Compounds of the Invention is meant isolated from or synthesized independently from .an environment where the Compounds of the Invention could naturally occur, e.g., being substantially free of plant alkaloids and/or plant terpenoids which are not Compounds of the Invention but which would normally be present in a plant or a crude plant extract containing a Compound of the Invention. This is important not only because it is desirable where possible to use well characterized materials that can be precisely controlled, but also because these plant alkaloids and terpenoids frequently have a strong bitter taste, which would impair the effectiveness of the Compounds of the Invention.
  • the Compound of the Invention is a receptor-binding antagonist (ligand) to one or more T2R bitter receptors.
  • ligand receptor-binding antagonist
  • T2R bitter taste receptors
  • a Compound of the Invention e.g., a Compound of the Invention, e.g.
  • (-)- hardwickiic acid in free or orally acceptable base addition salt form, in combination with, a comestible product, e.g., a food, drink, confectionary, oral care, or oral pharmaceutical product, to enhance the taste of the product.
  • a comestible product e.g., a food, drink, confectionary, oral care, or oral pharmaceutical product
  • the invention contemplates a method of using a Compound, of the Invention in combination with, a comestible product.
  • a Compound of the Invention can. be used as a pretreatment before the ingestion of a bitter substance, or administered contemporaneously with a bitter substance.
  • the invention provides for Method I, wherein Method I is a method of modulating bitter taste perception in a subject, comprising orally administering an.
  • hT2R50 Any of the preceding methods, wherein the Compound of the Invention modulates bitter taste perception in a subject via binding to one or more bitter taste receptors selected from the group consisting of: hT2RJ 0, hT2R14, hT2R16, hT2R38. h.T2R.44, hT2R46, hT2R50; hT2R3, hT2R4. hT2R7, hT2R8. hT2R13, hT2R31 , hT2R39 and h.T2R43; 15.
  • the Compound of the Invention is an antagonist to at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen of the bitter taste receptors in. the group consisting of hT2R10, liT2R14, hT2R16. hT2R38. hT2R44, hT2R46, hT2R50; h!2R3, hT2R4, liI2R7, h.T2R8, hT2Rl 3, hT2R31. hT2R39 and h.T2R43;
  • the Compound of the Invention is an antagonist to at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen of the bitter taste receptors in the group consisting of hT2R10 (SEQ ID No. 15 or SEQ ID No 62), hT2R14 (SEQ ID No 19 or SEQ ID No 64), hT2R16 (SEQ ID No 21 or SEQ ID No 65), hT2R38 (SEQ ID No 29 or SEQ ID No 69), hT2R44 (SEQ ID No 39), hT2R46 (SEQ ID No 43 or SEQ ID No 73).
  • hT2R10 SEQ ID No. 15 or SEQ ID No 62
  • hT2R14 SEQ ID No 19 or SEQ ID No 64
  • hT2R16 SEQ ID No 21 or SEQ ID No 65
  • hT2R38 SEQ ID No 29 or SEQ ID No 69
  • hT2R50 (SEQ ID No 51 or SEQ ID No 74); hT2R3 (SEQ ID No 56), hT2R4 (SEQ ID No 57), hT2R7 (SEQ ID No 59), hT2R8 (SEQ ID No 60), h.T2R13 (SEQ ID No 63), hT2R31 (SEQ ID No 68), hT2R39 (SEQ ID No 70) and hT2R43 (SEQ ID No 72);
  • the Compound of the Invention is an antagonist to at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen of the bitter taste receptors in the group consisting of hT2R10 (SEQ ID No.
  • hT2R 14 (SEQ ID No 19 or SEQ ID No 64), hT2R16 (SEQ ID No 21 or SEQ ID No 65), hT2R38 (SEQ ID No 29 or SEQ ID No 69), hT2R44 (SEQ ID No 39), liT2R46 (SEQ ID No 43 or SEQ ID No 73), hT2R50 (SEQ ID No 51 or SEQ ID No 74); hT2R3 (SEQ ID No 56), hT2R4 (SEQ ID No 57), hT2R7 (SEQ ID No 59), hT2R8 (SEQ ID No 60), hT2R13 (SEQ ID No 63), hT2R31 (SEQ ID No 68), hT2R39 (SEQ ID No 70) and hT2R43 (SEQ ID No 72); or variants thereof with at least 80%, 81%, 82%, 82%.
  • hT2R10 SEQ ID No. 15 or SEQ ID No 62
  • hT2R14 SEQ ID No 19 or SEQ ID No 64
  • hT2R1 SEQ ID No 21 or SEQ ID No 65
  • hT2R38 SEQ ID No 29 or SEQ ID No 69
  • hT2R44 SEQ ID No 39
  • hI2R46 SEQ ID No 43 or SEQ ID No 73
  • hT2R50 SEQ ID No 51 or SEQ ID No 74
  • hT2R3 SEQ ID No 56
  • ttT2R4 SEQ ID No 57
  • hT2R7 SEQ ID No 59
  • hT2R8 SEQ ID No 60
  • hT2R13 SEQ ID No 63
  • hT2R31 SEQ ID No 56
  • ttT2R4 SEQ ID No 57
  • hT2R7 SEQ ID No 59
  • hT2R8 SEQ ID No 60
  • the Compound of the Invention is an antagonist to at least three, e.g., at least six, of the bitter taste receptors in the group consisting of hT2Rl, hT2R!0, taT2R14, hT2R16. hT2R38, hT2R44, hT2R46, and hT2R50; 1 .
  • the Compound of the Invention is an antagonist to at least three, e.g., at least six. of the bitter taste receptors in the group consisting of hT2R10. hT2R14. hT2R16, h.T2R38, hT2R44, hT2R46. and hT2R50;
  • the Compound of the Invention is an antagonist to at least three, e.g., at least six, of the bitter taste receptors in the group consisting of hT2R10. hT2R14, hT2R16, hT2R38. hT2R44, hT2R46. hT2R50; hT2R3, hT2R4, hT2R7, hT2R8, hT2R13. hT2R31 , hT2R39 and hT2R43;
  • the Compound of the Invention is an antagonist to at least four, e.g., at least eight, of the bitter taste receptors in the group consisting of hT2R10, h.T2R14, hT2R16. HT2R38, h.T2R44, hT2R46, hT2R50; iiT2R3, hT2R4. hT2R7, h ' I " 2R8, hT2R13, hT2R31 , hT2R39 and hT2R43;
  • any of the preceding methods wherein the Compound of the Invention is administered with a pharmaceutical or medicinal product e.g., a liquid for oral administration, or a chewable or quick-dissolve tablet.
  • a pharmaceutical or medicinal product e.g., a liquid for oral administration, or a chewable or quick-dissolve tablet.
  • the Compound of the Invention is administered with a nonsteroidal anti-inflammatory agent, a decongestant, an antihistamine, and/or an antibiotic: 28.
  • any of the preceding methods wherein the Compound of the Invention is administered with a sweetener e.g., an artificial sweetener, e.g., a sulfonyl amide sweetener, e.g.. saccharin, sodium cyclamate or acesulfame potassium;
  • a sweetener e.g., an artificial sweetener, e.g., a sulfonyl amide sweetener, e.g.. saccharin, sodium cyclamate or acesulfame potassium;
  • a second bitter masking agent e.g., an agent selected from the group consisting of: sucralose, zinc gluconate, ethyl maltol. glycine, acesulfame k, aspartame, saccharin, f uctose, xylitol. malitol. isomalt; salt, spray dried licorice root, glycyrrhizin.
  • the invention provides the use of a Compound of the Invention as a screening assay to identify compounds which are bitter receptor antagonists, for example:
  • a competitive assay to identify novel bitter receptor antagonists comprising providing a Compound of the Invention, optionally in labeled form, and measuring binding of the Compound of the Invention to a. bitter receptor in the presence and absence of a test compound;
  • bitter receptor is one or more of h.T2Rl, hT2R10, hT2R14, hT2R16, hT2R38 s hT2R44. hI2R46 ; and/or hT2R50; or
  • bitter receptor is one or more of hT2Rl 0, hT2R14, hT2R16, hT2R38, hT2R44, hT2R46. and/or hT2R50; or
  • bitter receptor is one or more of hT2R10, hT2R14, hT2R16, hT2R38, h!2R44, hT2R46. h.T2R50, hT2R3, hT2R4, hT2R7. hT2R8, hT2Rl 3, hT2R31 , hT2R39 and/or hT2R43.
  • the invention also provides a Compound of the Invention in labeled form, e.g., radiolabeled form.
  • the invention provides Compound of the Invention, optionally in radiolabeled form, in combination or association with suitable diluent or carrier for an assay, e.g., selected from dimethylsulfoxide, ethanol, dichloromethane. methanol, saline buffer solution (optionally comprising sugar and additional ions), and serum free media.
  • suitable diluent or carrier for an assay e.g., selected from dimethylsulfoxide, ethanol, dichloromethane. methanol, saline buffer solution (optionally comprising sugar and additional ions), and serum free media.
  • the invention provides a comestible product, e.g., a food, drink, confectionary, oral care, or oral pharmaceutical product, comprising a taste-enhancing amount of a Compound of the Invention, e.g. (-)-hardwickiic acid, in free or orally acceptable base addition salt form, e.g. providing a combination as set forth in Method I and the following Methods.
  • a comestible product e.g., a food, drink, confectionary, oral care, or oral pharmaceutical product
  • a taste-enhancing amount of a Compound of the Invention e.g. (-)-hardwickiic acid
  • free or orally acceptable base addition salt form e.g. providing a combination as set forth in Method I and the following Methods.
  • the invention provides a product for addition to a food or drink, comprising a Compound of the Invention, e.g. (-)-hardwickiic acid, in free or orally acceptable base addition salt form, and optionally further comprising flavorings, spices and or sweeteners.
  • a Compound of the Invention e.g. (-)-hardwickiic acid
  • free or orally acceptable base addition salt form e.g., a Compound of the Invention, e.g. (-)-hardwickiic acid, in free or orally acceptable base addition salt form, and optionally further comprising flavorings, spices and or sweeteners.
  • the invention provides a Compound of the Invention, e.g, (-)-hardwickiic acid, in free or orally acceptable base addition salt form, e.g., in combination with a sweetener, for example a sweetener selected from sugars, e.g., sucrose, lactose and/or fructose; artificial sweetening agents, e.g., selected from steviol glycosides, aspartame, sucralose, neotame, acesulfame potassium, sodium cyclamate, and saccharin; sugar alcohols, e.g. sorbitol and/or xylitol; and combinations of any of the foregoing.
  • a sweetener selected from sugars, e.g., sucrose, lactose and/or fructose
  • artificial sweetening agents e.g., selected from steviol glycosides, aspartame, sucralose, neotame, acesulf
  • the invention provides a Compound of the Invention, e.g. (-)- hardwickiic acid, in free or orally acceptable base addition salt form, in combination with an artificial sweetener having a bitter aftertaste, e.g., a sulfonyl amide sweetener, e.g., selected from saccharin, sodium cyclamate and acesulfame potassium.
  • an artificial sweetener having a bitter aftertaste e.g., a sulfonyl amide sweetener, e.g., selected from saccharin, sodium cyclamate and acesulfame potassium.
  • artificial sweeteners include saccharin, sucralose, Ace rM and NeotameTM.
  • natural sweeteners include tevioside, Luo Han Guo extract, mogrosides.
  • glycyrrhizin perillartine, naringin dihydrochalcone, neohesperidine dihydrochalcone. mogroside V, rubusoside, rubus extract, and rebaudioside A.
  • the proportion of compound used will depend on the sweetener, the compound, the use to which the sweetener will be put. and the effect desired. This means that the proportion may vary between very wide limits. The skilled person can easily determine an appropriate proportion in every case, using onl routine experimentation and the ordinary skill of the art. However, as a general, non-limiting guideline, the proportion of compound ma vary between 0.5 ppm and 40 ppm, more particularly in certain embodiments, between about 1 ppm and about 10 ppm of the comestible product.
  • Yet another aspect of the invention provides for methods of using compounds that modulate multiple bitter taste receptors (T2R or TAS2R) in conjunction with other compounds that also function to mask bitter taste. It is contemplated by the present invention that a Compound of the invention could be used in conjunction with, at least, one other compound that functions to mask bitter taste perception in a free or fixed combination, e.g. in combination with one or more of the bitter-masking agents listed in Method 1.1.33.
  • T2R or TAS2R Compounds that modulate one or more bitter taste receptors
  • modulator include any substance or compound that alters an activity of a protein of interest, for example, a bitter taste receptor.
  • a substance or compound is a modulator even if its modulating activity changes under different conditions or concentrations or with respect to different forms of a protein of interest, for example, a bitter taste receptor.
  • a modulator can alter the structure, conformation, biochemical or biophysical properties or functionality of a bitter taste receptor.
  • a modulator may for example, alter directly or indirectly the activity of a receptor or the interaction of a receptor with its ligands.
  • modulator includes genetically modified versions of bitter taste receptors with altered activity as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like.
  • modulator can be an agonist (potentiator or activator) or antagonist (inhibitor or blocker), including partial agonists or antagonists, selective agonists or antagonists, competitive (or unsurmoun table/insurmountable) antagonists and inverse agonists, and can also be an allosteric modulator.
  • a modulator may change the ability of another modulator to affect the function of a protein of interest, for example, a bitter taste receptor.
  • the Compounds of the Invention are antagonists of one or more of the bitter taste receptors selected from the group of human bitter taste receptors consisting of hT2 10, hT2R14, hT2R16, hT2R38, hT2R44, hT2R46, hT2R50; hT2R3. hT2R4. hT2R7, hT2R8, hT2R13, hT2R31, hT2R39 and hT2R43.
  • inhibitor refers to a compound or substance that decreases or blocks one or more activities of a protein of interest, for example, a bitter taste receptor by, for example binding to, partially or totally block stimulation, preventing, delaying activation, inactivating, desensitizing or down-regulating bitter taste receptor and/or taste transduction.
  • An. "inhibitor”, “antagonist” or “blocker” attenuates the effect of an. agonist. It may be competitive (or surmountable), i.e. it binds reversibly to a region of the receptor in common with an agonist, but occupies the site without activating the effector mechanism.
  • a competitive antagonist may be overcome by increasing the concentration of agonist, thereby shifting the equilibrium and increasing the proportion of receptors which the agonist occupies.
  • antagonists may be unsurmountable or insurmountable where no amount of agonist can completely overcome the inhibition once it has been established.
  • Unsurmountable or insurmountable antagonists may bind covalently to the agonist binding site (competitive irreversible antagonists), in which case there is a period before the covalent bond forms during which competing ligands can prevent the inhibition.
  • Other types of unsurmountable antagonists act allosterically at a different site on the receptor or an associated ion channel.
  • bitter taste receptor refers to a compound or substance that decreases the downstream, signaling response associated with the bitter taste receptor.
  • decreasing taste receptor activity can result in a change in. the amount or distribution of an. intracellular molecule or the activity of an enzyme which is part of the intracellular signaling pathway for the bitter receptor.
  • intracellular molecule include, but are not. limited to, free calcium, cyclic adenosine monophosphate (cAMP), inositol mono-, di- or tri-phosphate.
  • the 5 enzyme include, but are not limited to. adenylate cyclase,. phospholipase-C and G-protein coupled receptor kinase.
  • the Compounds of the Invention are isolated antagonists of one or more of the bitter taste receptors selected from the group o human bitter taste receptors consisting of 10 h.T2R10, hT2R14, hT2R16, hT2R38, hT2R44, h.T2R46, hT2R50; hT2R3. hT2R4, hT2R7, hT2R8. hT2R13, hT2R31. hT2R39 and hT2R43.
  • an "isolated antagonist" of hTAS2R bitter taste receptor activity is a substance which is isolated from or synthesized independently from an environment where 15 the antagonist (eg a Compounds of the Invention.) could naturally occur, e.g., being substantially free of plant alkyloids and/or plant terpenoids which, for example, are not Compounds of the Invention but which would normally be present in a plant or a crude plant extract containing a Compound of the Invention.
  • the isolated antagonist reduces the activity of hTAS2R stimulated by the hTAS2R bitter taste receptor agonist, preferably selected from the group consisting of the agonists (for example, those used in Examples 1 and 2 and as defined in Tables 1, 3, 7 and 7) and structurally related agonists thereof. Preferably this reduction is by at least 10%
  • the extent of the lowering of the hTAS2R bitter taste receptor activity, caused by the antagonist is determined in the presence of said agonist, e.g. one of the compounds indicated above in any one or more of Tables 1, 3, 6 and 7 or a structurally related 30 compound, which may be added prior, concomitantly or after addition of the antagonist.
  • said agonist e.g. one of the compounds indicated above in any one or more of Tables 1, 3, 6 and 7 or a structurally related 30 compound, which may be added prior, concomitantly or after addition of the antagonist.
  • the identified antagonist exerts this inhibitory activity, if present in the same molar. 2-fold, 5-fold, 10-fold. 50-fold or 100-fold molar concentration as the agonist.
  • the antagonist exerts its antagonizing action when it is contacted prior, concomitantly or after, preferably concomitantly, to contacting the hTAS2R polypeptide, the host ceil genetically engineered with a polynucleotide encoding an hTAS2R polypeptide as defined above in. any one or more of Tables 1-3 and 6-7 or a vector containing a polynucleotide as defined above to express hTAS2R polypeptide with said hTAS2R agonist.
  • the antagonist is contacted at the same molar concentration as said hTAS2R agonist.
  • potentiator refers to a compound or substance that increases one or more activities of a protein of interest, for example, a bitter taste receptor, by for example, binding to, stimulating, increasing, opening, activating, facilitating, enhancing activating, sensitising, or up regulating bitter taste receptor signal transduction.
  • the terms “potentiator”, “agonist” or “activator” in the context of taste receptors refer to a compound or substance that increases the downstream signaling response associated with the bitter taste receptor.
  • partial agonist means an agonist which, no matter how high a concentration is applied, is unable to produce maximal activation of the receptors.
  • the receptor reserve may vary between tissues, depending on the number of receptors in the particular tissue and the efficiency of coupling between them and their effector mechanism, Consequently, a partial agonist in one tissue may appear to act as a full agonist in a tissue with a higher receptor reserve.
  • the molar concentration of an agonist which produces 50% of the maximum possible response for that agonist is termed its EC50 value.
  • Other percentage values eg EC25. EC 0 and EC90
  • the IC50 is the molar concentration which produces 50% of the maximum possible inhibition.
  • increasing the bitter taste receptor activity can result in change in the amount or distribution of an intracellular molecule or the activity of an enzyme which is part of the intracellular signaling pathway for the bitter receptor.
  • the intracellular molecule include, but are not limited to, free calcium, cyclic adenosine monophosphate (cAMP), inositol mono-, di- or tri-phosphate.
  • cAMP cyclic adenosine monophosphate
  • the enzyme include, but are not limited to, adenylate cyclase, phospholipase-C, G-protein coupled receptor kinase.
  • a method of identifying a modulator of a bitter receptor function comprises; a) exposing a cell or cell line that stably expresses a bitter taste receptor to a test compound; and b) detecting a change in a function of the bitter taste receptor.
  • the detecting utilizes an assay that measures intracellular free calcium.
  • the intracellular free calcium is measured using one or more calcium-sensitive fluorescent dyes, a fluorescence microscope, and optionally a fluorescent plate reader, wherein at least one fluorescent dye binds free calcium.
  • the intracellular free calcium is monitored by realtime imaging using one or more calcium-sensitive fluorescent dyes, wherein at least one fluorescent dye binds free calcium.
  • the screening method as defined above is useful in identifying potential antagonists of any one or more of the known human bitter taste receptors.
  • the term “potential antagonist”, comprises any perceivable chemical substance or combination thereof having a structure according to the compound formula (I) in a non- purified, partially purified or purified state.
  • the potential antagonist is selected on the basis of its antagonizing behaviour.
  • the "potential antagonist” is a compound structurally related to the Compound of Formula 1 of the present invention.
  • the potential bitter taste receptor hTAS2R antagonist is used in the method of the invention has a different structure than any of the preferred bitter taste receptor hTAS2R agonists (bitter substances).
  • a modulator identified using a screening method as defined above is a structurally related antagonist to any one of the Compounds of the Invention,
  • the term "structurally related antagonist” is a substance, which is derived from the compound of Formula ⁇ of the present invention, respectively, by 1. 2, 3, 4, 5 or 6 steps of chemical modification and which lowers the specific hT.AS2R bitter taste receptor activity compared to the activity determined in the presence of an agonist specific for the specific TAS2R receptor, respectively, by at least 10% (e.g. at least 10%, 15%. 20%, 30%, 40%, 50%, 60%. 70%, 80%, 90%, 95%, 98%, 99%, 99,5% or 100%).
  • the antagonistic derivative exerts this action, when it is contacted prior, concomitantly or after. preferably concomitantly, to the contacting of the hTAS2Rpolypeptide.
  • the host cell expressing the hIAS2R polypeptide, or the vector comprising the hTAS2R polypeptide, with a hTAS2R agonist.
  • the Compound of the Invention is (-)-hardwickiic acid which is used in a method of modulating bitter taste perception in a subject so that bitter taste perception is modulated in the subject.
  • (-)-Hardwickiic acid is a known diterpenoid compound, which has surprisingly been found to be a natural hTAS2R antagonist.
  • Hardwickiic acid has been found in many plants, e.g. Hardwickia pinnata. Soli da o jimcea, Solidago arguta.
  • (-)-hardwickiic acid exists, for example, as a 20% w/w of black currant buds absolute extract. It exhibits insecticidal activity against Aphis compora, and also antimicrobial activity. While plant alkaloids and terpenoids typically present in extracts comprising hardwickiic acid tend to be very bitter tasting, it is surprisingly found that hardwickiic acid itself, and structurally related compounds, e.g., of Formula 1 supra, are antagonists rather than agonists of the bitter receptors, and moreover exhibit inhibition of a variety of human T2R receptors. This is in contrast to other T2R receptor antagonists that have merely shown affinity to only one or a few receptors.
  • the Compounds of the Invention are exceptional in that it is not common for a compound to be a promiscuous or broad spectrum inhibitor of bitter receptors.
  • a blackcurrant bud absolute extract as a bitter taste masking agent.
  • the w/w content of (-)-hardwickiic acid in the blackcurrant bud absolute extract is about 20% (w/w).
  • the blackcurrant bud absolute extract is a fractionated extract.
  • the blackcurrant bud absolute extract is a Rib s nigrum extract.
  • the blackcurrant bud absolute extract has a mixture of (- )-hardwickii acid and (-)-epi-hardwickiic acid in the ratio of about 4: 1 (eg from about 120mg to about 29mg respectively). This is the first time that (-)-epi-hardwickiic acid has been isolated from a Blackcurrant source.
  • a Compound of the Invention e.g., (-)-hardwickiic acid
  • the efficacy of a Compound of the Invention can be increased when the compound is applied prior to the agonist.
  • the present invention also contemplates a use of a Compound of the Invention, e.g., (-)-hardwickiic acid, wherein the inhibition of T2R receptors is reversible.
  • the sample isolated from blackcurrant absolute extract is a mixture of two enantiomers.
  • the minor epi-isomer of (-)-hardwtckiic acid ((-)-epi-hardwickiic acid (Compound 2 of Formula 1)) is also an inhibitor of T2R44, T2R46, T2R50.
  • the epi-isomer differs from the major form of (-)-hardwickiic acid (Compound 1 of Formula 1) in that the epi-isomer is itself a TAS2 14 agonist.
  • a blackcurrant bud absolute extract comprising a. mixture of (-)-hardwickiic acid and (-)-epi hardwickiic acid in the ratio of about 1 : 1, 2: 1, 3: 1, 4: 1. 5: 1 , 6: 1, 7: 1 , 8: 1, 9: I , 10: L or about I: 2, 1 : 3, 1 : 4, 1: 5, 1 : 6. 1 : 7, 1 : 8, 1 : 9, 1 : 10.
  • a blackcurrant bud absolute extract comprising about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, (w/w) of (-)- hardwickiic acid.
  • the content of hardwickiic acid in. a product can be enriched easily and the ratio can be different depending on the source of raw material and the way of processing. Additionally, the ratio of the isomers may be changed or even reversed if the products produced by biosynthetic methods, such as cell culture or synthetic biology.
  • a blackcurrant bud absolute extract as a bitter taste ligand which modulates bitter taste receptor activity by decreasing bitterness signal transduction activity across multiple bitter taste receptors selected from the group consisting of hT2R10, hT2R14, hT2R16, hT2R38, hT2R44, hT2R46, hT2R50; hI2R3, h.T2R4, hT2R7, hT2R8, h.T2R13, h.T2R31 , h.T2R39 and hT2R43.
  • the blackcurrant bud absolute extract contains about 20% (w/w) of (-)- hardwickiic acid.
  • the blackcurrant bud absolute extract comprises a mixture of (-)-hardwickiic acid and (-)-epi hardwickiic acid in the ratio of about 4:1.
  • T2R refers to the family of taste receptors responsible for the perception of bitter taste in mammals
  • T2R may refer to one or multiple bitter taste receptors.
  • T2R is used interchangeably with the term “TAS2R”
  • human bitter taste is mediated by about 25 members of the human.
  • bitter receptors are also important in a series of physiological contexts.
  • bitter receptor agonists elicit a secretory response in entero-endocrine cells in vitro and in animals in vivo, and induce neuronal activation. Therefore, all of the bitter receptor family members are important clinical targets for managing a variety of conditions associated with detection of bitter tastants.
  • Bitter receptors are G protein coupled receptors (GPCRs) expressed at the surface of taste receptor cells and. are coupled to secondary messenger pathways.
  • GPCRs G protein coupled receptors
  • TAS2R receptors can be coupled to transducing or gustducin for example, through which they can activate both phospodiesterases and a phospholipase C (PLC) p2-dependent pathway to increase intracellular Ca 2+ concentration.
  • PLC phospholipase C
  • bitter receptor refers to any one of the G protein coupled receptors that is expressed at the surface of a taste receptor cell and that mediates bitter taste perception via secondary messenger pathways.
  • functional bitter receptor refers to a bitter receptor that responds to a known activator or a known inhibitor in substantially the same way as the bitter receptor in a cell that normally expresses the bitter receptor without engineering.
  • Bitter receptor behavior can be determined, by, for example, physiological activities and pharmacological responses.
  • Physiological activities include, but are not limited to, the sense of bitter taste.
  • the perception of the bitter taste can be measured, by, for example, using a 2-alternative forced choice paradigm (2-AFC), coupled with anchored taste intensity ratings in order to quantify the reduction in bitterness (see, for example, the disclosure and teachings in Slack et al (2010) Curr Biol 20(1.2) 1 104-1109).
  • Pharmacological responses include, but are not limited to. a change in. the amount or distribution of an intracellular molecule or the activity of an enzyme which is part of the intracellular signaling pathway for the bitter receptor when a bitter receptor is contacted with a modulator.
  • a pharmacological response may include an increase in intracellular free calcium when the bitter receptor is activated, or a decrease in intracellular free calcium when the bitter receptor is blocked.
  • a representative list of human bitter taste receptors and their accession numbers is provided in Table 2 below.
  • hT2R39 and h ' I ' 2R43 can be found using the Genbank Accession numbers and Genbank Version numbers listed in Table 2 below (see also US7579453B2 and WO 2005/007891 and Table 1 on pages 17-1 of WO2004/029087).
  • the terms "polypeptide” and “protein” are used interchangeably herein and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification.
  • protein "variant" is to be understood as a polypeptide which differs in comparison to the polypeptide from which it is derived by one or more changes in the amino acid sequence.
  • the polypeptide from which a variant is derived is also known, as the parent polypeptide.
  • a variant is constructed artificially, preferably by gene- technological means.
  • the polypeptide from which the variant is derived is a wild-type protein or wild-type protein domain.
  • the variants usable in the present invention may also be derived from homologs, orthologs, or paralogs of the parent polypeptide or from artificially constructed variants, provided that the variant exhibits at least one biological activity of the parent polypeptide.
  • the changes in the amino acid sequence may be amino acid exchanges, insertions, deletions, N-termmal truncations, or C- terminal truncations, or any combination of these changes, which may occur at one or several sites.
  • a variant usable in the present invention exhibits a total number of up to 200 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 15. 20, 25, .30, 35, 40. 45, 50, 55, 60. 65. 70, 75. 80. 85, 90, 95, 100, 110, 120, 130. 140. 150, 160, 170, 1 80. 190. or 200) changes, in the amino acid sequence (i.e. exchanges, insertions, deletions, N-terminal truncations, and/or C -terminal truncations).
  • the amino acid exchanges may be conservative and/or non-conservative.
  • a variant usable in the present invention differs from the protein or domain from which it is derived by up to 1 , 2, 3, 4, 5, 6, 7, 8. 9, 10. 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid exchanges, preferably conservative amino acid changes.
  • Variants may additionally or alternatively comprise deletions of amino acids, which may be N-terminal truncations, C -terminal truncations or internal, deletions or any combination of these.
  • Such variants comprising N-terminal truncations, C-terminal truncations and/or internal deletions are referred to as "deletion variants" or "fragments" in the context of the present application.
  • deletion variant and “fragment” are used interchangeably herein.
  • a deletion variant may be naturally occurring (e.g. splice variants) or it may be constructed artificially, preferably by gene-technological means.
  • the protein or protein domain from, which the deletion variant is derived is a wild-type protein.
  • the deletion variants of the present invention may also be derived from homologs. orthologs, or paralogs of the parent polypeptide or from artificially constructed variants, provided that the deletion variants exhibit at least one biological activity of the parent polypeptide.
  • a deletion variant (or fragment) has a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25. 30. 35, 40. 45, 50, 55, 60, 65, 70. 75, 80, 85, 90, 95, or 100 .amino acids at its N- terminus and/or at its C-terminus and/or internally as compared to the parent polypeptide.
  • a “variant” as used herein, can alternatively or additionally be characterised by a certain degree of sequence identity to the parent polypeptide from which it is derived.
  • a variant of the present invention may have a sequence identity of at least 50%, 55%, 60%, 65%, 70%, 75% identity to the respective reference polypeptide or to the respective reference polynucleotide.
  • the expression "at least 50%, 55%. 60%, 65%, 70%, 75% sequence identity” is used throughout the specification with regard to polypeptide and polynucleotide sequence comparisons.
  • a variant in the context of the present invention exhibits "at least 80% sequence identity" to its parent polypeptide.
  • the sequence identity is over a continuous stretch, of 20, 30, 40, 45, 50. 60. 70. 80, 90, 100 or more amino acids.
  • the expression "at least 80% sequence identity” is used throughout the specification with regard to polypeptide and polynucleotide sequence comparisons. This expression preferably refers to a sequence identity of at least 80%, at least 81 %. at least 82%. at least 83%, at least 84%. at least 85%, at least 86%. at least 87%. at least 88%. at least 89%. at least 90%. at least 1%, at least 92%.
  • the polypeptide in question and the reference polypeptide exhibit the indicated sequence identity over a continuous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids.
  • the polynucleotide in question and the reference polynucleotide exhibit the indicated sequence identity over a continuous stretch of 60, 90, 120, 135, 150, 180, 210, 240, 270, 300 or more nucleotides.
  • sequence identity is to be calculated with reference to the longer of the two sequences to be compared, if not specifically indicated otherwise. If the reference sequence is indicated, the sequence identity is determined on the basis of the full length of the reference sequence indicated by SEQ ID. if not specifically indicated otherwise. For example, a peptide sequence consisting of 30 amino acids compared to the amino acids of full length hTAS2R with 299 amino acid residues may exhibit a maximum sequence identity percentage of 10.03% (30/299) while a sequence with a length of 150 amino acids may exhibit a maximum sequence identity percentage of 50.17% (150/299).
  • the similarity of nucleotide and .amino acid sequences can be determined via sequence alignments.
  • sequence alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci, USA 90: 5873-5877), with hmmalign (HMMER package, http://hmmer.wusti.edu ) or with the CLUSTAL algorithm (Thompson, J, D., Higgins. D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80) available e.g. on http://www.ebi.
  • ac.uk/Tools/clustalw/ or on http://www.ebi.ac.uk Tools/clustalw2/index.html or on http://npsa-pbil.ibcp.fr/cgi- Preferred parameters used are the default parameters as they are set on http://www.ebi.ac.uk/Tools/clustalw/ or http://www.ebi.ac.uk Tools/clustalw2/index.html.
  • the grade of sequence identity may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX).
  • BlastZ or BlastX
  • Gapped BLAST is utilized as described in Altschul el al (1997) Nucleic Acids Res. 25: 3389-3402.
  • Sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle- LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1 : 154- 162) or Markov random fields.
  • Shuffle- LAGAN Brudno M., Bioinformatics 2003b, 19 Suppl 1 : 154- 162
  • Markov random fields Markov random fields.
  • Hybridization can also be used as a measure of sequence identity or homology between two nucleic acid sequences.
  • hT2R bitter taste receptors HT2R1 , h.T2R10, hT2R!4, hT2R16, hT2R38, hT2R44. hT2R46, hT2R50; hT2R3, hT2R4. hT2R7, h ' I ' 2R8. hT2R13, hT2R31 , liT2R39 and hT2R43, to DNA or RNA from a test source (e.g.
  • a mammalian cell is an indication of the presence of the DNA or RNA for any one of the following hT2R bitter taste receptors: hT2Rl . hT2R10, hT2R14, hT2R16, hT2R38, hT2R44, hT2R46, hT2R50; hT2R3, h.T2R4, hT2R7, hT2R8. h.T2R13. hT2R31 , hT2R39 and hT2R43 in the test source.
  • Hybridization conditions are known to those skilled in the art and can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y., 6.3.1 - 6.3.6, 1991.
  • “Moderate hybridization conditions” are defined as equivalent to hybridization in 2X sodium chloride/sodium citrate (SSC) at 30°C, followed by a wash in IX SSC, 0.1 % SDS at 50°C.
  • Highly stringent conditions are defined as equivalent to hybridization in 6X sodium chloride/sodium citrate (SSC) at 45°C followed by a wash in 0.2 X SSC, 0.1 % SDS at 65°C.
  • Nucleotide sequences encoding any one or more of the following human bitter taste receptors can be found in Table 2.
  • Constant substitutions may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved.
  • the 20 naturally occurring amino acids can. be grouped into the following six standard amino acid groups:
  • substitutions are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide.
  • glycine and proline may be substituted for one another based on their ability to disrupt a-helices.
  • Some preferred conservative substitutions within the above six groups are exchanges within the following sub-groups: (i) Ala. Val. Leu and He: (ii) Ser and Thr; (ii) Asn and Gin; (iv) Lys and Arg; and (v) Tyr and Phe.
  • non-conservative substitutions or “non-conservative amino acid exchanges” are defined as exchanges of an amino acid b another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.
  • the term "derivative" of a polypeptide refers to a polypeptide that has been chemically modified so that it comprises other chemical groups than the 20 naturally occurring amino acids. Examples of such other chemical groups include without limitation glycosylated amino acids and phosphoryiated amino acids.
  • the polypeptide from which the derivative derives is also known as the parent polypeptide. This parent polypeptide can be a naturally occurring protein but can also be a protein variant as defined above. Chemical modifications of a polypeptide may provide advantageous properties as compared to the parent polypeptide, e.g. one or more of enhanced stability, increased biological half- life, or increased water solubility. Chemical modifications applicable to the derivatives 5 usable in the present invention include without limitation: PEGylation. glycosylation of non-glycosylated parent polypeptides, or the modification of the glycosylation pattern present in the parent polypeptide.
  • a variant is regarded as a 15 variant within the context of the present application, if it exhibits the relevant activity to a degree of at least 10% of the activity of the parent polypeptide.
  • a derivative is regarded as a derivative within the context of the present application, if it exhibits the relevant biological activity to a degree of at least 10% o the activity of the parent polypeptide.
  • bitter taste receptor activity i.e. the ability of the receptors described herein (hT2Rl , hT2R10, hT2R14. hT2R16, hT2R38. hT2R44, hT2R46, hT2R50; hT2R3, hT2R4. hT2R7, hT2R8, h.T2R13, hT2R31. HT2R39 and hT2R43); to be stimulated by bitter substances, such as the 25 bitter receptor agonists as recited herein in Tables 1. 3, 6 and 7 of Examples 1 and 2).
  • hT2R46, hT2R50; hT2R3, hT2R4, hT2R7, hT2R8 5 hT2R13, hT2R31 , hT2R39 and hT2R43 are described immediately below and in several other passages of this specification and in the specific Examples,
  • One way of detecting the bitter taste receptor activity of any one or more of the following bitter taste receptors, hT2Rl , h.T2R10, hT2R14. hT2R16, hT2R38, h.T2R44, hT2R46, hT2R50; hT2R3, h.T2R4, h.T2R7, hT2R8, hT2R13, hT2R31, hT2R39 and hT2R43 is by measuring a change in concentration of an intracellular messenger, e.g. Ca 2 ⁇ IP 3 , or cAMP.
  • an intracellular messenger e.g. Ca 2 ⁇ IP 3 , or cAMP.
  • All functional assays may be performed by samples containing cells expressing the receptor on their surfaces or on isolated cell membrane fractions. Useful cells are described herein. Instead of samples with separate cells or cell membranes, tissues from transgenic animals may be used. Isolated cells or isolated tissues naturally expressing any one or more of the following bitter taste receptors: hT2Rl , hT2R10. hT2R14, hT2R16, hT2R38, hT2R44, hT2R46. hT2R50; hT2R3. hT2R4. hT2R7, hT2R8, hT2R13, hT2R31 , hT2R39 and hT2R43 can be used in the methods described herein.
  • any one or more of the following bitter taste receptors can be expressed using stable or transient expression systems. The generation of a stable cell line is well known.
  • cells transiently expressing any one or more of the following bitter taste receptors hT2Rl, hT2R10, hT2R14, hT2R16. hT2R38, hT2R44, hT2R46.
  • hT2R50 hT2R3, hT2R4, hT2R7, hT2R8, hT2R13, hT2R31 , hT2R39 and h.T2R43, can be used, or for example, HEK293T/Gal6-gustducin 44 cells transiently expressing any one or more of the following bitter taste receptors: hT2Rl, hT2R10, hT2R14, hT2R16, h.T2R38, M2R44, hT2R46, hT2R50; h.T2R3, hT2R4.
  • Suitable eucaryotic cells include, for example, without limitation, mammalian cells, yeast cells, or insect cells (including Sf9), amphibian cells (including melanophore cells), or worm cells including cells of Caenorhabditis (including Caenorhabditis elegans).
  • Suitable mammalian cells include, for example, without limitation, COS cells (including Cos-1 and Cos-7), CHO cells, HEK293 celts, HEK293T ceils, HEK293 T-RexTM cells, or other transfectable eucaryotic cell lines.
  • Suitable bacterial cells include without limitation E. coli.
  • Cells may be transfected with any one or more of the following bitter taste receptors: h.T2R.l, hT2R10 5 hT2R14, hT2R16, h.T2R38, hT2R44, hT2R46, hT2R50; hT2R3.
  • G-protein G alpha 16-gustducin 44 also known as G.sub.alpha.16 gust(ducin)44, G.sub.alpha.l6gust(ducin)44, Gctl6gust(ducin)44, GaI6gust(d cin)44, Gal6-gustducin 44, or as used herein-below.
  • G16gust44 which provides for enhanced coupling to taste GPCRs, is described in detail in WO2004/055048 and in US Patent No, 7919236.
  • G 16 or G 15 may also be used. Any one or more of the following bitter taste receptors: hT2Rl , hT2Rl 0, hT2R14, h ' T2Rl 6, hT2R38, hT2R44, hT2R46, hT2R50; h.T2R3, hT2R4, h.T2R7, hT2R8.
  • hT2R13, h ' T2R3 l, hT2R39 and hT2R43 can be expressed in a cell with a G-protein that links the receptor to a signal transduction pathway, for example, the phospholipase C signal transduction pathway, or signal transduction pathways including, for example, the following: adenylate cyclase, guanylate cyclase, phospholipase C. IP3, GTPase/GTP binding, arachinoid acid, cAMP/cGMP, DAG, protein kinase c (PKC), MAP kinase tyrosine kinase, or ERK kinase.
  • any suitable reporter gene may be linked to a bitter taste receptor-activation responsive promoter and used to determine the specific bitter taste receptor activity, as described in more detail hereinunder.
  • One preferred way of measuring the "bitter taste receptor activity" of any of these receptors is the ability to release intracellular calcium in a heterologous cell expression system like, for example, (HEK293T/G16gust44) cells that stably expresses a chimeric G-protein consisting of Gal6 and 44 carboxy-terminal amino acids of a-gustducin, in response to bitter tastants, which is dependent on the expression of polypeptides encoded by the polynucleotides of the present invention.
  • the amount of intracellular calcium, released can be monitored by, for example, the in vitro FLIPR assay described herein but also by the measurement of one of a variety of other parameters including, for example, IP 3 or cAMP level.
  • G-protein coupled receptor activity comprises without limitation electrophysiological methods, transcription assays, which measure, e.g. activation or repression of reporter genes which are coupled to regulatory sequences regulated via the respective G-protein coupled signalling pathway, such reporter proteins comprise, e.g., CAT or LUC; assays measuring internalization of the receptor; or assays in frog melanophore systems, in which pigment movement in. melanophores is used as a readout for the activity of adenylate cyclase or phospholipase C (PLC), which in turn, are coupled via G-proteins to exogenously expressed, receptors (see, for example, McClintock T.S.
  • the Compounds of Formula I of the present invention are useful in offsetting the bitter taste o ⁇ bitter tastant compounds which include but are not limited to: Acteoside, Adhumulon.e,Adlupu.lone, Aesculetin, Aesculin, L- Alanine, L-aJanyl-L-aianyl-L- Alanine, L-alanyl-L-isoleucyl-Alanine L-, L-valyl-L-val l-Amarogentin, Amaropanin Amaroswerin.
  • EpicatecbJn gallate Chaconine, alpha-Chaconine, beta2-ChIoramphemcol s Cholic Acid, Ciehoriin, Cohumulone, Colupuione, Cryptochlorogenic Acid, gamma-lactone, Cucurbitacin B, Cucurbitacin D, Cyclo Alanine-glycine, Cyclo Alanine-phenylanaline, Cyclo Alanine- valine, Cyclo(L-arginylglycyl-L-protyl-L-prolyl-L-phenylalanyl-L-isoIeucyl-L-vaIyl) ⁇ Cyclo Asparagine-phenylalanine, Cyclo Glycine-phenylalanine.
  • Cycloheximide Cyclo Lucine-Tryptophan, Cyclopent(b)azepin-8(l H)-one, 7-Methyl-2,3,6,7-Tetrahydro- Cyclopent(b)azepin-8( 1 H)-one, 2 ,3 ,6,7-tetrahydro-7-hyd.rox.y-7-meth.yl.-C clopent-2-en- 1 - one. 2,5-dih.ydroxy-5-methy!-3-(l -piperidinyl)-
  • Cyclopent-2-en-l -one 2,5-dihydroxy-5-methyl-3-(l -pyrrolidinyl.) Cyclopent-2-en-l-one, 2,3-di-l -pyrrolidinyl-Cyclopent-2-en-l -one, 5-hydrox y-5-methyl-2.3-di- 1 -piperidinyl- Cyclopent-2-en-l -one, 5-hydroxy-5-methyl-2 J 3-di-l -pyrrolidinyl-Cyclopent-2-en-l-one, 5- methyl-2,3-di- 1 -pyrroIidinyl-Cyclopent-2-en- 1 -one.
  • Cyclo Proline-leucine Cyclo Proline-methionine, Cyclo Proline-phenylalanine, Cyclo Proline-proline, Cyclo Proline-valine, Cyclo Valine-phenylalanine, Cynaratriol, Cynaropicrin, Cynaropicrin, Daidzein, Daidzin Denatonium benzoate, Denatonium saccharide, Dhurrin, Dihydroxybenzoic Acid, 2,3-Dihydroxybenzoic Acid, 2,4-Ethyl b-L- arabinoside, Ethyl alpha-D-Glucoside.
  • Ethyl beta-D-Glucoside Eustomoroside, Eustomoside, Gallic Acid, Epigallocatechin, Epigallocatechin gallate.
  • Gaudichaudioside F Gelidoside, Genistein, Genistin, Gentiopicroside, Gentistic Acid, Gentomoside.
  • Hymenoside C Hymenoside D, Hymenoside E, Hymenoside F, Isohumulone, cis-Isohumulone, trans-Isoleucine, L-Isolupanine, Isosparteine, beta-Isosparteine, 10,17-Dioxo-beta-Isosparteine, 10-oxo-beta-Lactucin, L- Leucine, L-alanyl-L-alanyI-L-Leucine,N-[(2R)-6-amino-2-[(4S)-2,5-dioxo-4- (phenylmethyl)- 1 -imidazolidinylj-1 -oxohexyl]-L-leucyl-L-methionyl-N-methyl-L- phenylalanyl-, (4-l)-lactam, L-Leucine, glycyl-L-alanyl-Leu
  • Lupoxes C Lupulone, Luputrione, Mellein, 6-Methoxy-Methionine, L- Methyl alpha-L-arabinoside, Methyl beta-L-arabinoside, Methyl beta-D-Glucoside, Methyl alpha- D-Glucoside 2,3-Di-isoleucine, Methyl alpha-D-Glucoside 2,3-Di-leucine.
  • Olivieroside C Perrottetin H, Phenylalanine, L-Phenyl alpha-D-galactoside, Phenyl alpha-D-glucosidc, Phenyl beta-D-glucoside.
  • Phenylthiourea Phlomisoside II, Piperidine-2-carboxylic acid, 4-[(2-carboxy-2-hydroxyethyl)thio]-Piperidinecarboxylic acid-2. 4-[(2-carboxy-2-hydroxyethyl)thio]-Prehumulone, Prelupulone.
  • Propyl beta-D- fructoside Propyl alpha-D-glucoside.
  • Theaflavin Gallate A Theaflavin Gal late B.
  • Toraatidine Tomatine, alpha-Tricyclodehydroisohumulone, Trifloroside, Trihydroxybenzoic Acid, 2,4,6-Tryptophan.
  • the Compounds of Formula 1 of the present invention are useful, e.g., in methods as hereinbefore described, to offset the bitter taste of common food ingredients such as potassium, chloride, ammonium chloride, sodium chloride, magnesium chloride, halide salts, naringin, caffeine, urea, magnesium sulfate, saccharin, acetosulfames, aspirin, potassium benzoate, potassium bicarbonate, potassium carbonate, potassium nitrate, potassium nitrite, potassium sulfate, potassium sulfite, potassium glutamate, food preservatives in their physiologically acceptable salts, antibiotics, unsweetened chocolate, cocoa beans, yogurt, preservatives, flavor enhancers, dietary supplements, supplemental amino acids, gelling agents, pH control agents, nutrients, processing aids, bodying agents, dispersing agents, stabilizers, colorings, coloring diluents, anticaking agents, antimicrobial agents, formulation aids, leavening agents, surface active agents,
  • denuding agents general purpose buffers, thickeners, cooked out juice retention agents, color fixatives in meat and meat products, colorfixatives in poultry and poultry products, dough conditioners, maturing agents, yeast foods, mold retardants, emuisifiers, texturizers, binders, water correctives, miscellaneous and general purpose food additives, tableting aids, lye peeling agents, washing water agents, oxidizers, antioxidants, enzymes, extenders, fungicides, cake mixes, coffee, tea, dry mixes, non-dairy creamers, salts, animal glue adjuvant, cheese, nuts, meat and meat products, poultry and poultry product, pork and pork products, fish and fish products, vegetable and vegetable products, fruit and fruit products, smoked products such as meat, cheese fish, poultry, and vegetables, whipping agents, masticatory substances in chewing gums, dough strengthened, animal feed, poultry feed, fish feed, pork feed, defoaming agents, juices, liquors, substances or drinks containing alcohol, beverages including but not limited to
  • the invention contemplates one or more comestibles having a bitter ingredient together with a Compound of the Invention in an amount sufficient to reduce or eliminate the bitter taste of the bitter ingredient.
  • the comestibles are breads, biscuits, pancakes, cakes, pretzels, snack foods, baked goods etc, prepared using, for example, potassium bicarbonate or potassium carbonate in place of the sodium salts as leavening agents, comprising a bitter-reducing amount of the Compound of the Invention, e.g. any of the specific compounds disclosed above.
  • the Compound of the Invention can be typically present in an amount ranging from about 0.001% to about 1% by weight of the food product, typically l %-50% by weight of a supplement to be added to a food product.
  • a method for the production of a food, a food precursor material or additive employed in the production of a foodstuff comprising the step of admixing an antagonist selected from the group consisting of: (i) a compound of Formula 1 which is an isolated compound of Formula 1 in free or orally acceptable base addition salt form; (ii) an antagonist identified in a screening assay as described herein; or (iii) an antagonist structurally related to the antagonists of (i) or (ii); with the comestible product, the food, the food precursor material or the additive employed in the production of the foodstuff.
  • a method for the production of a nutraceutical or a pharmaceutical composition comprising the step of admixing an antagonist selected from the group consisting of: (i) a Compound of Formula 1 which is an isolated Compound of Formula 1 in free or orally acceptable base addition salt form; (ii) an antagonist identified in a screening assay as described herein; or (Hi) an antagonist structurally related to the antagonists of (i) or (ii); with an active agent and optionally with a pharmaceutically acceptable carrier and/or adjuvant.
  • T2R bitter receptor agonists can include but is not limited to any one of the 58 natural bitter compounds disclosed in Meyerhof et at (2010) Chem Senses 35: 157-170. These compounds include limonin, Naringm, Neofaesperidine, Coumarin, Caffeine, Brucine, Thiamine, Riboflavin, Quinine. Simgrin, Campher, Arbutin, Absinthin and others from Table 1 of that publication all of which are incorporated herein by reference.
  • Other natural compounds of interest include but are not limited to sucralose, aceK, saccharin, stevioside, Reb A, Sesquiterpene lactones from lettuce such as laetucopicrin, Amygdalin D found in almonds. Menthol and Cucurbitacins from cucumber and others disclosed in paragraph 1.1.33 above.
  • a a food, a food precursor material or additive employed in the production of a foodstuff producible according to any of the methods for the production of a food, a food precursor material or additive employed in the production of a foodstuff.
  • nutraceutical or a pharmaceutical composition producible by any of the methods for the production of a nutraceutical or a pharmaceutical composition and wherein the nutraceutical or a pharmaceutical composition comprises at least one nutraceutically or pharmaceutically active agent and optionally one or more pharmaceutically acceptable carrier and/or adjuvant.
  • an "ingestible composition” includes any substance intended for oral consumption either alone or together with another substance.
  • the ingestible (or comestible) composition includes both “food or beverage products” and “non-edible products”.
  • food or beverage products it is meant any edible product intended for consumption by humans or animals, including solids, semi-solids, or liquids (e.g.. beverages).
  • non-edible products includes supplements, nutraceuticals, functional food products (e.g.. any fresh or processed food claimed to have a health-promoting and/or disease-preventing properties beyond the basic nutritional function of supplying nutrients), pharmaceutical and over the counter medications, oral care products such as dentifrices and mouthwashes, cosmetic products such as lip balms and other personal care products.
  • the ingestible (or comestible) composition also includes a pharmaceutical, medicinal or alternatively in a formulation, e.g., a pharmaceutical or medicinal formulation or a food or beverage product or formulation.
  • the Compounds of the present invention can also be provided, individually or in combination, with any ingestible composition known or later discovered.
  • ingestible or comestible products include, but not limited to, all food products, food additives, nutraceuticals. pharmaceuticals and any product placed in the mouth including chewing gum, oral, care products, and oral hygiene products including but not limited to. cereal products, rice products, tapioca products, sago products, baker ' s products, biscuit products, pastry products, bread products, confectionery products, dessert products, gums, chewing, gums, flavored or flavor-coated straws, flavor or flavor-coated food/beverage containers, chocolates, ices, honey products, treacle products, yeast products, baking-powder, salt and spice products, savoury products, mustard products, vinegar products, sauces (condiments), tobacco products, cigars, cigarettes, processed foods, cooked fruits and vegetable products, meat and meat products, jellies, jams, fruit sauces, egg products, milk and dairy products, yoghurts, cheese products, butter and butter substitute products, milk substitute products, soy products, edible oils and fat products,
  • Oral care products by which is meant any product applied to or taken into the oral cavity for the purposes of cleaning, freshening, healing, deodorising the cavity or any part thereof, may include, but are not limited to. toothpastes, tooth gels, tooth powders, tooth whitening products, mouthwashes, lozenges, dental floss, toothpicks, anti-plaque and anti-gingivitis compositions, throat lozenges, throat drops, inflammatory compositions, compositions for treatment of nasal symptoms, cold symptoms and upper gastrointestinal tract distress. compositions for cold relief, for alleviating discomfort of hot flash, and gargle compositions.
  • the invention provides a product and a method substantially as described herein and with reference to the accompanying Tables and Figures.
  • the examples provided in the detailed description are merely examples, which should not be used to limit the scope of the claims in any claim construction or interpretation.
  • Figure 1 shows the chemical structure of Compounds of Formula 1 ; and the chemical structure of Compound 1 ((-)- hardwickiic acid) of Formula 1 and Compound 2 ((-)-epi- hardwickiic acid) of Formula 1 ;
  • Figure 2 shows the chemical structure of Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 of Formula 1 ; and the Compound of Formula l a; and
  • Figure 3 shows the inhibition by (-)-hardwickiic acid (15 ⁇ ) of the response of multiple hTAS2Rs (hTAS2Rl , hTAS2R10. hTAS2R14, hTAS2R16, hTAS2R38, hTAS2R44, hTAS2R46 and hTAS2R50) to the bitter agonists listed in Table 1.
  • the data in. Figure 3 is expressed as a percent of the receptor response to agonist alone.
  • the hTAS2R sequences and the h.TAS2R receptor/agonist combination used to generate the data in Figure 3 are shown in Table 1 below.
  • SEQ ID No, 1 is the polypeptide sequence and SEQ ID No. 2 is the nucleotide sequence for human TAS2R1 which is disclosed in Genbank Accesssion Number AF227129 and Genbank Version AF227129.1 , GI: 7262604.
  • SEQ ID No. 3 is the polypeptide sequence and SEQ ID No. 4 is the nucleotide sequence for human TAS2R3 which is disclosed in Genbank Accesssion Number AF227130 and Genbank Version AF227130.1, GI:7262606.
  • SEQ ID No. 5 is the polypeptide sequence and SEQ ID No. 6 is the nucleotide sequence for human.
  • TAS2R4 which is disclosed in Genbank Accesssion Number AF227131 and Genbank Version AF227131.1 , GE7262608.
  • SEQ ID No. 7 is the polypeptide sequence and SEQ ID No. 8 is the nucleotide sequence for human TAS2R5 which is disclosed in Genbank Accesssion Number AF22 132 and Genbank Version AF227132.1, GI;7262610.
  • SEQ ID No. 9 is the polypeptide sequence and SEQ ID No. 10 is the nucleotide sequence for human TAS2R7 which is disclosed in Genbank Accesssion Number AF227133 and Genbank Version AF227133.1. GI:7262612.
  • SEQ ID No. 1 1 is the polypeptide sequence and SEQ ID No. 12 is the nucleotide sequence for human.
  • TAS2R8 which is disclosed in Genbank Accesssion Number AF2271.34 and Genbank Version. AF227134.1, 01:7262614.
  • SEQ ID No. 13 is the polypeptide sequence and SEQ ID No. 14 is the nucleotide sequence for human TA.S2R9 which is disclosed in Genbank Accesssion Number AF.227135 and Genbank Version AF2271.35.1 , 01:7262616.
  • SEQ ID No. 1 5 is the polypeptide sequence and SEQ ID No. 16 is the nucleotide sequence for human TAS2R10 which is disclosed in Genbank Accesssion Number AF227136 and Genbank Version AF227136..L GI:7262618.
  • SEQ ID No, 17 is the polypeptide sequence and SEQ ID No, 18 is the nucleotide sequence for human TAS2R 13 which is disclosed in Genbank Accesssion Number AF227137 and
  • SEQ ID No. 19 is the polypeptide sequence and SEQ ID No. 20 is the nucleotide sequence for human TAS2R14 which is disclosed in Genbank Accesssion Number AF227138 and Genbank Version AF227138. 1. GI:7262622.
  • SEQ ID No. 21 is the polypeptide sequence and SEQ ID No. 22 is the nucleotide sequence for human TAS2R16 which is disclosed in Genbank Accesssion Number AF227139 and Genbank Version AF22713 .
  • L GI:7262624 is the nucleotide sequence for human TAS2R16 which is disclosed in Genbank Accesssion Number AF227139 and Genbank Version AF22713 .
  • SEQ ID No. 23 is the polypeptide sequence and SEQ ID No. 24 is the nucleotide sequence for human TAS2R20 which is disclosed in Genbank Accesssion Number AF494236 and Genbank Version AF494236.1, GF20336528.
  • SEQ ID No. 25 is the polypeptide sequence and SEQ ID No. 26 is the nucleotide sequence for human TAS2R30 which is disclosed in Genbank Accesssion Number AF494233 and AF494233.1. GI:20336522.
  • SEQ ID No. 27 is the polypeptide sequence and SEQ ID No. 28 is the nucleotide sequence for human TAS2R31 which is disclosed in Genbank Accesssion Number AF4 4228 and Genbank Version AF494228.L GE20336512.
  • SEQ ID No. 29 is the polypeptide sequence and SEQ ID No. 30 is the nucleotide sequence for human TAS2R38 which is disclosed in Genbank Accesssion Number AF494231 and Genbank Version. AF494231.1 , GF20336518.
  • SEQ ID No. 31 is the polypeptide sequence and SEQ ID No. 32 is the nucleotide sequence for human TAS2R39 which is disclosed in Genbank Accesssion Number AF494230 and Genbank Version AF494230.1 , GI:20336516.
  • SEQ ID No. 33 is the polypeptide sequence and SEQ ID No. 34 is the nucleotide sequence for human TAS2R40 which is disclosed in Genbank.
  • SEQ ID No. 35 is the polypeptide sequence and SEQ ID No. 36 is the nucleotide sequence for human TAS2R41 which is disclosed in Genbank Accesssion Number AF494232 and Genbank. Version AF494232.1 , GI:20336520.
  • SEQ ID No. 37 is the polypeptide sequence and SEQ ID No. 38 is the nucleotide sequence for human TAS2R43 which is disclosed in Genbank Accesssion Number AF494237 and Genbank Version AF494237. L GI:20336530.
  • SEQ ID No. 39 is the polypeptide sequence and SEQ ID No. 40 is the nucleotide sequence for human TAS2R.44 which is disclosed in Genbank Accesssion Number AF494228 and Genbank Version AF494228.1 , GI:20336512.
  • SEQ ID No. 41 is the polypeptide sequence and SEQ ID No. 42 is the nucleotide sequence for human TAS2R45 which is disclosed in Genbank Accesssion Number AF494266 and Genbank Version AF494266.1 , GE33321829.
  • SEQ ID No. 4 is the polypeptide sequence and SEQ ID No. 44 is the nucleotide sequence for human TAS2R46 which is disclosed in Genbank Accesssion Number AF494227 and Genbank Version AF494227. L GI:20336510.
  • SEQ ID No. 45 is the polypeptide sequence and SEQ ID No. 46 is the nucleotide sequence for human TAS2R47 which is disclosed in Genbank Accesssion Number AF494233 and. Genbank Version AF494233.1. GE20336522.
  • SEQ ID No. 47 is the polypeptide sequence and SEQ ID No. 48 is the nucleotide sequence for human TAS2R48 which is disclosed in Genbank. Accesssion Number AF4 4234 and Genbank Version AF494234.1. GI:20336524.
  • SEQ ID No. 49 is the polypeptide sequence and SEQ ID No. 50 is the nucleotide sequence for human TAS2R49 which is disclosed in Genbank. Accesssion Number AF494236 and Genbank Version. AF494236.1 , GI:20336528.
  • SEQ ID No. 51 is the polypeptide sequence and SEQ ID No. 52 is the nucleotide sequence for human TAS2R50 which is disclosed in Genbank Accesssion Number AP494235 and Genbank Version AF494235.L GI:20336526.
  • SEQ ID No. 53 is the polypeptide sequence and.
  • SEQ ID No. 54 is the nucleotide sequence for human TAS2R60 which, is disclosed in Genbank Accesssion Number AYl 14094 and Genbank Version AYl 14094.1, GI:28603637.
  • hTAS2Rs which can be employed in a process for isolating binding compounds and for identifying hTAS2R antagonists, with the exception of hTAS2R40, single nucleotide polymorphisms (SNPs) are known. Seventy nine of these are listed in Table 1 on pages 17-1.9 of WO 2004/029087, sixty one of which result in an. amino acid change.
  • SNPs single nucleotide polymorphisms
  • Polynucleotide or polypeptides that differ from those disclosed in SEQ ID Nos 1-54 above or SEQ ID Nos 55-74 (provided, below) by, for example, the nucleotide and amino acid changes as indicated in Table 1 on pages 17-19 of WO 2004/029087 can similarly be employed for the screening processes of the present invention.
  • high-throughput screening of antagonists is performed via calcium imaging using Fluo-4. Growth medium is discarded and the cells are incubated i the dark for 1 hour at 37° C in 50 ⁇ loading buffer consisting of 1.5 ⁇ Fluo-4 AM (Invitrogen. San. Diego CA) and 2.5 ⁇ probenicid (Sigma-Aldrich, St. Louis, MO, US) in DMEM (no FBS).
  • test compounds are prepared at a. final. concentration, of 15 ⁇ in the presence of the appropriate receptor agonist and assessed for their ability to decrease the hTAS2R receptor response to the agonist.
  • Candidate inhibitors that show significant inhibition of the agonist response during primary screening are selected and retested for their ability to inhibit hTAS2R receptor activation.
  • hTAS2R receptors and agonists are used: hTAS2Rl - 2.38 mM Chloramphenicol (Cas No 56-75-7)
  • hTAS2R50 - 10 ⁇ Andrographolide (Cas No 5508-58-7)
  • Table 3 hTAS2R sequence information and Agonist Cas no for Example 1
  • FIG. 3 shows the response of the hTAS2R receptors to agonist + 15 ⁇ (-)- hardwickiic acid (GIV237) and are expressed as a percent of the receptor response to agonist alone. It can be seen that (-)-hardwickiic acid effectively inhibits the response to bitter agonists at bitter receptors hTASRl O, hTASR14, hTASR16. hTASR38, hTASR44 5 hTASR46, and hTASRSO. This is an unusual finding,, as most bitter antagonists are specific for particular receptors and do not have activity across multiple receptors. Compounds 3, 4, 5 and. 6 are also tested and show inhibition of bitter receptors at concentrations of 20 micromolar or less.
  • Example 1 The data from Example 1 demonstrates that GIV237, which is derived from a botanical source, can inhibit several TAS2R bitter taste receptors (hTAS2 10. hTAS2R14. hTAS2R16. hTAS2R38. hTAS2R44. hTAS2R46. hTAS2R5G) in vitro at a concentration of 15 ⁇ .
  • TAS2R bitter taste receptors hTAS2 10.
  • hTAS2R46 hTAS2R5G
  • the blackcurrant bud absolute extract was fractionated using column chromatography as follows: 12 grams of blackcurrant buds absolute were deposited on 5 g of silicagel. This sample was fractionated by column chromatography (330 g of silica gel) using a gradient of hexane/Methyl Tertiary-Butyl Ether (MTBE) (start 100% hexanes to 100% MTBE). The hardwickiic acid was eluted at 80% MTBE to provide 4.1 g of light yellowish semi -so lid. The major component was identified as harwickiic acid (CAS No 1782-65-6, MW 316, C20H28O3) by Gas Chromatography Mass Spectrometry (GCMS).
  • GCMS Gas Chromatography Mass Spectrometry
  • the NMR also displayed signals ascribable to a ⁇ -mono-substituted furan ring (CI 1 -C 16).
  • 1 was determined to be a furanoid clerodane-type diterpenoid, hardwickiic acid.
  • the stereochemistry was established by NOESY and the optical rotation.
  • the lack of nOe interaction between H-10 and the methyl group at C-5 indicated the trans junction between the two ring systems.
  • a strong nOe observed between the methyl groups at C-5 and C-9 indicated both methyl groups are on the same side of the molecule.
  • the structure of 2 was established by a combination of 2D-NMR including COSY, TOCSY, HSQC, and HMBC. Interpretation of COSY and. TOCSY revealed the proton coupling networks. Based on the proton assignment, an HSQC experiment identified the corresponding carbons.
  • a second NOESY was measured in pyridine- ⁇ in hoping that the overlapped methyl groups would be resolved and provide further information concerning the stereochemistry of the molecule. Fortunately, the two methyl groups were well, separated with 9-CH 3 being at 0.92 ppm and 8-CH3 at 0.76 ppm. Interpretation of the NOESY revealed that nOes were existed between the two methyl, groups at C-8 and C-9 indicating 8-CH3 was ⁇ -orientated. The nOes between 7 ⁇ and 8, 6a and 8 further supported the ⁇ orientation of the 8-CH3. The nOes between 8-Ci and 7a (strong), 8-CH3 and 7 ⁇ (weak) as well as 9-CH 3 and 7 ⁇ (strong) suggested the B-ring existed in.
  • Both (-)-hardwickiic acid (1) and (-)-t'/?/-hardwickiic acid (2) adopt a half-chair A-ring and chair B-ring with trans and cis fusions between the ring systems, respectively.
  • 2 has a folded structure. It is well-known that proton and carbon chemical shifts are very sensitive to their environment, and therefore, the change in the conformation has resulted in significant displacement of these.
  • the 20 individually expressed hTAS2Rs were challenged with varying concentrations (2 ⁇ . 6 ⁇ , 8.7 ⁇ , 13 ⁇ , 21.7 ⁇ and 26 ⁇ ) of (-)-hardwickitc acid (Compound 1 of Formula 1), in the presence of ' known agonists for the 21 hTAS2Rs as set out in Tables 6 and 7.
  • the results provided in Table 7 indicate which of the 21 hTAS2Rs are sensitive to (-)-hardwickiic acid and the percentage inhibition of signal activity at the various levels of (-)-hardwickiic acid which were used.
  • (-)-hardwickiic acid effectively inhibits the response to bitter agonists at bitter receptors hTAS2R3.
  • GIV237 Compound 1 of Formula 1 ((-)-hardwickiic acid, (hereinafter GIV237) on taste perception are administered in. test subjects with informed writte consent and after prior approval by the Givaudan Institutional Review Board, Human subjects are recruited and trained for the experiments. Initially, the Intrinsic sensory aspects of GIV237 are assessed followed by an examination of its potential to inhibit bitterness. Experiments are done with and without nose clips. For the study to examine the potential of GIV237 to inhibit bitterness, GIV237 is co-administered with bitter tastants to the subject in various concentrations based on the data obtained in Figure 3 and Table 7 and empirically determined in pilot experiments.
  • Acesulfame K and saccharin are commonly used as sugar substitutes for low calorie food products and their acceptance would be significantly increased by blocking the undesirable bitter tastes associated with them (Schiffman et al (1 95) Brain Res Bull 36; 505-513), Therefore it is useful to determine whether GIV237 can reduce the bitter taste of acesulfame K in vivo using structured human taste trials. Briefly, taste tests are performed with the GIV237 antagonist using a 2 -alternative forced choice method (2- AFC). An Acesulfame K samples with the antagonist was given, to the taste panelists together with the same sample without the antagonist, the panelists are asked to identify the bitterer sample within the pair.
  • 2-alternative forced choice method (2- AFC
  • panelists are asked to rate the perceived sweet taste intensity of the two solutions following selection of the sample that is most sweet.
  • a binomial analysis is used to determine whether perceived taste intensity ratings are statistically different between the samples.
  • a p ⁇ 0.05 is taken as significant in both statistical tests.
  • 15 panelists evaluate the effect of the G1V237 compound (25-30 ppm) on sodium saccharin solutions (3 mM), using the same 2-AFC procedure but without the taste intensity ratings. Each panelist performs two replicates of the test.
  • test Compound 1 (25 ppm) in sucralose (3 mM) solution is evaluated. Twenty ml of each solution (sucralose or sucralose with test Compound 1 are presented in random order to 15 bitter-sensitive panelists. In two replications, panelists are asked to select the solution they perceived as more bitter. A beta- binomial analysis is calculated from the forced-choice data to correct for overdispersion. A check is made to see if a significant (p ⁇ 0.Q5) majority of evaluations select test Compound I (GIV237) (25 ppm) in a 3 mM sucralose solution as being less bitter than a 3 mM sucralose solution.
  • bitter taste prevents mammals from ingesting food potentially contaminated with hitter-tasting toxins that are numerous and structurally diverse.
  • Bitter taste perception is mediated by a family of heptahelical G protein-coupled receptors, called Taste 2 Receptors (TAS2Rs or T2Rs).
  • TAS2Rs Taste 2 Receptors
  • TAS2Rs taste 2 Receptors
  • the present invention demonstrates that a natural compound from edible plants, can act as a broad spectrum (or promiscuous) bitter taste receptor ligand. that is able to modulate bitter taste receptor activity by decreasing or possibly completely abating bitterness signal transduction across multiple bitter taste receptors. This natural compound has utility in reducing the perception of bitter flavours and/or enhancing the perception of sweet flavours.

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Abstract

La présente invention concerne des compositions et des procédés d'utilisation d'un composé de Formule 1, par exemple, l'acide (-)-hardwickiique sous forme libre ou sous forme de sel d'addition de base oralement acceptable, pour masquer ou diminuer la perception d'un sujet du goût amer et/ou améliorer la perception du goût sucré.
PCT/EP2012/072553 2011-11-14 2012-11-14 Procédés d'utilisation d'antagonistes de récepteurs du goût amer WO2013072332A1 (fr)

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Cited By (9)

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EP3085248A1 (fr) 2015-04-22 2016-10-26 Analyticon Discovery GmbH Compositions contenant acide dehydro abietique
WO2017019890A1 (fr) * 2015-07-28 2017-02-02 Mars, Incorporated Procédés de criblage à l'aide de récepteurs t2r canins, et produits et compositions alimentaires pour animaux domestiques identifiés à l'aide de ces derniers
CN110049688A (zh) * 2016-12-15 2019-07-23 国际香料和香精公司 用于掩味的新型组合物
EP3643183A1 (fr) 2018-10-26 2020-04-29 Analyticon Discovery GmbH Préparations contenant des flavonoïdes c-méthylés
WO2021050394A1 (fr) * 2019-09-09 2021-03-18 Firmenich Incorporated Bloqueurs du récepteur de goût amer et procédés pour leur identification
WO2021219725A1 (fr) 2020-04-29 2021-11-04 Bunge Loders Croklaan B.V. Composition, produit de confiserie et procédé
US20220183313A1 (en) * 2019-03-29 2022-06-16 Suntory Holdings Limited Catechin-containing beverage, production method therefor, and method for reducing bitterness of catechin-containing beverage
WO2022236274A1 (fr) * 2021-05-07 2022-11-10 Firmenich Incorporated Cellules exprimant des récepteurs de l'amertume et leurs utilisations
EP4360470A1 (fr) 2022-10-27 2024-05-01 Dr Corbyn Ltd Composition et procédé pour réduire les sensations gustatives négatives des préparations à base de propolis

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EP3085248A1 (fr) 2015-04-22 2016-10-26 Analyticon Discovery GmbH Compositions contenant acide dehydro abietique
WO2016169869A1 (fr) 2015-04-22 2016-10-27 Analyticon Discovery Gmbh Préparations avec acide déhydroabiétique
WO2017019890A1 (fr) * 2015-07-28 2017-02-02 Mars, Incorporated Procédés de criblage à l'aide de récepteurs t2r canins, et produits et compositions alimentaires pour animaux domestiques identifiés à l'aide de ces derniers
CN110049688A (zh) * 2016-12-15 2019-07-23 国际香料和香精公司 用于掩味的新型组合物
CN110049688B (zh) * 2016-12-15 2023-01-03 国际香料和香精公司 用于掩味的新型组合物
EP3643183A1 (fr) 2018-10-26 2020-04-29 Analyticon Discovery GmbH Préparations contenant des flavonoïdes c-méthylés
WO2020084163A1 (fr) 2018-10-26 2020-04-30 Analyticon Discovery Gmbh Préparations comprenant des flavonoïdes c-méthylées
US20220183313A1 (en) * 2019-03-29 2022-06-16 Suntory Holdings Limited Catechin-containing beverage, production method therefor, and method for reducing bitterness of catechin-containing beverage
WO2021050394A1 (fr) * 2019-09-09 2021-03-18 Firmenich Incorporated Bloqueurs du récepteur de goût amer et procédés pour leur identification
WO2021219725A1 (fr) 2020-04-29 2021-11-04 Bunge Loders Croklaan B.V. Composition, produit de confiserie et procédé
WO2022236274A1 (fr) * 2021-05-07 2022-11-10 Firmenich Incorporated Cellules exprimant des récepteurs de l'amertume et leurs utilisations
EP4360470A1 (fr) 2022-10-27 2024-05-01 Dr Corbyn Ltd Composition et procédé pour réduire les sensations gustatives négatives des préparations à base de propolis

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