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WO2005002524A2 - Composes, compositions et utilisations therapeutiques de composes de type oleoylethanolamide et de modulateurs des ppar$g(a) - Google Patents

Composes, compositions et utilisations therapeutiques de composes de type oleoylethanolamide et de modulateurs des ppar$g(a) Download PDF

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Publication number
WO2005002524A2
WO2005002524A2 PCT/US2004/021394 US2004021394W WO2005002524A2 WO 2005002524 A2 WO2005002524 A2 WO 2005002524A2 US 2004021394 W US2004021394 W US 2004021394W WO 2005002524 A2 WO2005002524 A2 WO 2005002524A2
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oea
compounds
pparα
compound
fatty acid
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WO2005002524A3 (fr
Inventor
Jin Fu
Silvana Gaetani
Daniele Piomelli
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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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
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids

Definitions

  • FAE biosynthesis can be rapidly enhanced, however, in response to a wide variety of physiological and pathological stimuli, including exposure to fungal pathogens in tobacco cells(Chapman et al., Plant Physiol., 116:1163-1168 (1998)), activation of neurotransmitter receptors in rat brain neurons (Di Marzo et al., Nature, 372:686-691 (1994); Giuffrida et al., Nat. Neuroscl, 2:358-363 (1999)) and exposure to metabolic stressors in mouse epidermal cells (Berdyshev et al., Biochem. J., 346:369-374 (2000)).
  • the specificity for PPAR ⁇ 15 compound in activation assays for each of PPAR ⁇ , PPAR ⁇ and PPAR/3 and selecting the compound which has at least a 5 fold specificity for PPAR ⁇ over either or both of PPAR ⁇ and PPAR/3 under comparable or physiological assay conditions.
  • the identified PPAR ⁇ selective compound can then be tested in an animal model by administering the compound to a subject and determining body fat reduction in the subject.
  • the fatty acid moiety of the fatty acid alkanolamide or ethanolamide compound, homologue, or analog may be saturated or unsaturated, and if unsaturated may be monounsaturated or polyunsaturated.
  • Vehicle alone (70% DMSO in saline, 1 ml per kg, i.p.) had no significant effect on acute food intake;
  • time course of the hypophagic effects of oleoylethanolamide (20 mg per kg, i.p.) (squares) or vehicle (lozenges) on food intake
  • Empty bars water intake; filled bars, saccharin intake.
  • Fig. 9 Activation of human PPAR ⁇ -GAL4 chimeric receptors by OEA.
  • a Concentration-dependent effects of OEA on PPAR ⁇ (closed circles), PPAR ⁇ (open triangles), PPAR ⁇ (closed squares) and RXR (open lozenges)
  • OEA reduces feeding in wild-type mice, but not in mice deficient for PPAR- ⁇ .
  • Asterisk, P ⁇ 0.05; n 8-12 per group.
  • FIG. 12 Synthetic PPAR- ⁇ agonists mimic the satiety-inducing actions of OEA.
  • Fig. 17 Effect of subhronic OEA administration (5 mg/kg, once daily for 2 weeks, i.p.) on food intake and body weight gain over the two week period. Black circles, OEA. Open squares, vehicle.
  • OEA selectively engages with high affinity the peroxisome proliferator-activating receptor alpha (PPAR ⁇ ), a ligand-operated transcription factor that regulates multiple aspects of lipid metabolism.
  • PPAR ⁇ peroxisome proliferator-activating receptor alpha
  • substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents which would result from writing the structure from right to left, e.g., -CH 2 O- is intended to also recite -OCH 2 -.
  • Compounds of the invention may contain one or more asymmetric centers and can thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers.
  • the present invention is meant to comprehend all such isomeric forms of the inventive compounds.
  • Some of the compounds described herein contain olefmic double bonds, and unless specified otherwise, are meant to include both E and Z geometric isomers.
  • tautomers Some of the compounds described herein may exist with different points of attachment of hydrogen, referred to as tautomers. Such an example may be a ketone and its enol form known as keto-enol tautomers. The individual tautomers as well as mixture thereof are encompassed by the inventive formulas.
  • Fatty acid refers to a saturated or unsaturated substituted or unsubstituted, branched or unbranched alkyl group having a carboxyl substituent. Preferred fatty acids are C 4 -C 22 acids. Fatty acid also encompasses species in which the carboxyl substituent is replaced with a -CH 2 - moiety.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (be.
  • alkyl groups examples include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4- pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
  • alkyl unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as “heteroalkyl.”
  • Alkyl groups which are limited to hydrocarbon groups are termed "homoalkyl".
  • heteroalkylene by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH 2 - CH 2 -S-CH 2 -CH 2 - and -CH 2 -S-CH 2 -CH 2 -NH-CH 2 -.
  • heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O) 2 R'- represents both -C(O) 2 R'- and -R'C(O) 2 -.
  • heterocycloalkyl examples include, but are not limited to, 1 -(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4- morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1 -piperazinyl, 2-piperazinyl, and the like.
  • -NR'R is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF 3 and -CH 2 CF 3 ) and acyl (e.g., -C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 OCH 3 , and the like).
  • BMI Body Mass Index
  • fatty acid oxidation relates to the conversion of fatty acids (e.g., oleate) into ketone bodies.
  • Fatty acid amide hydrolase is the enzyme primarily responsible for the hydrolysis of anandamide in vivo. It also is responsible for the hydrolysis of OEA in vivo. Inhibitors of the enzyme are well known to one of ordinary skill in the art.
  • Oleoylethanolamide refers to a natural lipid of the following structure:
  • Such compounds include those compounds whose affinity for the PPAR ⁇ receptor is at least 5-fold, 10-fold, or 50-fold greater than that for a cannabinoid receptor (e.g., CBi or CB 2 receptor).
  • OEA is an example of a preferred OEA-like compound.
  • An OEA-like modulator or OEA like agonist is a PPAR ⁇ agonist having a selective affinity for the PPAR ⁇ receptor at least 5-fold greater (e.g., having a concentration which produces a half-maximal effect which is at least 5-fold lower) than for either or both PPAR/3 or PPAR ⁇ as measured under comparable bioassay conditions in vivo or in vitro or in any bioassay as described herein.
  • Particularly preferred OEA-like modulators have a selective affinity of at least 5-fold, 10-fold, 50-fold or 100-fold greater for PPAR ⁇ than for PPAR/3 or PPAR ⁇ .
  • Such preferred OEA-like compounds are particularly preferred if they produce a half-maximal effect on the PPAR ⁇ receptor under physiological conditions at a concentration of 1 micromolar or less, 100 nanomolar or less, 10 nanomolar or less, or 1 nanomolar or less, or from 1 micromolar to 1.0 nanomolar, or less.
  • Such OEA-like compounds can include, but are not limited to, fatty acid alkanolamides, their homologues and their analogues. Also particularlyt preferred are OEA and compounds of Formula I or Formula VI.
  • weight loss refers to loss of a portion of total body weight.
  • the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, buffers and excipients, including phosphate-buffered saline solution, ' water, and emulsions (such as an oil water or water/oil emulsion), and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers and their formulations are described in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., Easton, 19th ed. 1995). Preferred pharmaceutical carriers depend upon the intended mode of administration of the active agent. Typical modes of administration are described below. [0097] The term "effective amount" means a dosage sufficient to produce a desired result on health.
  • the desired result may comprise a subjective or objective improvement in the recipient of the dosage.
  • a subjective improvement may be, for instance, decreased appetite or craving for food.
  • An objective improvement may be, for instance, decreased body weight, body fat, or food, decreased food consumption, decreased food seeking behavior, or improved serum lipid profile, or a decreased likelihood of developing a disease or harmful health condition.
  • a "prophylactic treatment” is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs of a disease, wherein treatment is administered for the purpose of decreasing the risk of developing a pathology associated with the disease.
  • the compounds of the invention may be given, for instance, as a prophylactic treatment to prevent undesirable or unwanted weight gain.
  • a “therapeutic treatment” is a treatment administered to a subject who exhibits signs or symptoms of pathology, wherein treatment is administered for the purpose of diminishing or eliminating those pathological signs.
  • Diseases or conditions mediated by PPAR ⁇ or responsive to administration of a PPAR ⁇ modulator include, but are not limited to, each of obesity, an appetite disorder, overweight, a metabolic disorder, cellulite, Type I and Type II diabetes, hyperglycemia, dyslipidemia, Syndrome X, insulin resistance, diabetic dyslipidemia, anorexia, bulimia, anorexia nervosa, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, artherogenesis, artherosclerosis, an inflammatory disorder or condition, Alzheimers disease, Crohn's disease, vascular inflammation, an inflammatory bowel disorder, rheumatoid arthritis, asthma, thrombosis or cachexia.
  • Appetency disorders or “appetite disorders” are understood as meaning disorders associated with a substance and especially abuse of a substance and/or dependency on a substance, disorders of food behaviors, especially those liable to cause excess weight, irrespective of its origin, for example: bulimia, appetency for sugars, non-insulin-dependent diabetes.
  • Appetizing substances are therefore understood as meaning substances to be taken into the body and for which an appetite or craving for such consumption by any route of entry.
  • Appetizing substances includes, foods, and their appetizing ingredients such as sugars, carbohydrates, or fats, as well as drinking alcohol or drugs of abuse or excess consumption.
  • An "appetite' may be directed toward such substances as foods, sugars, carbohydrates, fats, as well as ethanol or drugs of abuse or addiction or excess consumption (e.g., tobacco, CNS depressants, CNS stimulants).
  • An agonist is a ligand of a receptor which activates the receptor or causes signal transduction upon binding to the receptor.
  • OEA is an example of a PPAR ⁇ receptor agonist.
  • An antagonist is a ligand of a receptor which binds to the receptor but does not appreciably activate the receptor or appreciably cause signal transduction.
  • An antagonist may block the ability of an agonist to bind and activate a receptor or otherwise reduce the activity of the receptor under physiological conditions.
  • a peroxisome proliferator activated receptor is a member of a family of nuclear receptors, distinguished in ⁇ , ⁇ , and ⁇ subtypes as described herein.
  • a specific or selective PPAR activator is a compound that preferentially binds and activates one PPAR subtype over another.
  • a specific activator of PPAR ⁇ is OEA.
  • a specific or selective binder is a compound that preferentially binds one PPAR subtype over another.
  • a specific binder of PPAR ⁇ is OEA.
  • OEA-like compounds may possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all intended to be encompassed within the scope of the present invention.
  • any enantiomer of such a compound of the invention may be obtained by stereospecific synthesis using optically pure starting materials of known configuration.
  • OEA-like compounds and OEA-like modulators of the invention include, but are not limited to fatty acid ethanolamide compounds, and their homologues.
  • OEA- like compounds ane OEA-like modulators are contemplated. These compounds include compounds having the following general formula:
  • the compound may also be substituted by methyl group or a double bond.
  • the molecular bond between carbons c and d may be unsaturated or saturated.
  • the fatty acid ethanolamide of the above formula is a naturally occurring compound.
  • the alkyl subsitutents are each homoalkyl.
  • the compounds of Formula la have n from 0 to 5; and a sum of a and b that is from 0 to 4; and members R 1 and R 2 independently selected from the group consisting of hydrogen, substituted or unsubstituted Ci -C 6 alkyl, lower substituted or unsubstituted (C ⁇ -C 6 ) acyl, homoalkyl, and substituted or unsubstituted aryl.
  • R 1 and R 2 independently selected from the group consisting of hydrogen, substituted or unsubstituted Ci -C 6 alkyl, lower substituted or unsubstituted (C ⁇ -C 6 ) acyl, homoalkyl, and substituted or unsubstituted aryl.
  • up to eight hydrogen atoms of the fatty acid portion and alkanolamine (e.g., ethanolamine) portion of compounds of the above formula may also be substituted by methyl or a double bond.
  • the above compounds particularly include those in which the fatty acid moiety comprises oleic acid, elaidic acid, or palmitic acid.
  • Such compounds include oleoylethanolamide, elaidylethanolamide and palmitoylethanolamide.
  • the molecular bond between carbons c and d is unsaturated and no other hydrogen atoms are substituted.
  • the members R 1 and R 2 are independently selected from the group consisting of hydrogen, substituted or unsubstituted Ci -C 3 alkyl, and substituted or unsubstituted lower (CrC 3 ) acyl.
  • the methyl substituted compounds of the above formula include particularly those compounds where R 1 and R 2 are both H: (R)l '-methyloleoylethanolamide, S(l ')- methyloleoylethanolamide, (R)2'-methyloleoylethanolamide, (S)2'- methyloleoylethanolamide, (R)l -methyloleoylethanolamide, and (S)l- methyloleoylethanolamide.
  • Exemplary compounds of formula II include those compounds where the alkanolamine portion is ethanolamine, compounds where R 2 is H, and compounds where a and b are each 1 , and compounds where n is 1.
  • the compounds of Formula II have n from 1 to 5 and a sum of a and b from 1 to 3.
  • the member R 2 is selected from the group consisting of hydrogen, substituted or unsubstituted d -C 6 alkyl, and substituted or unsubstituted lower (C ⁇ -C 6 ) acyl.
  • up to four hydrogen atoms of either or both the fatty acid portion and alkanolamine (e.g., ethanolamine) portion of compounds of the above formula may also be substituted by methyl or a double bond.
  • the compounds of Formula III have n from 1 to 5; and the sum of a and b from 0 to 4.
  • the member R 2 is selected from the group consisting of hydrogen, substituted or unsubstituted C ⁇ -C 6 alkyl, lower (C C ⁇ ) acyl, homoalkyl, and aryl. Up to four hydrogen atoms of either or both the fatty acid portion and alkanol (e.g., ethanol) portion of compounds of the above formula may also be substituted by methyl or a double bond.
  • the compounds of Formula IV have an n from 1 to 5 and a sum of a and b that can be from 0 to 4.
  • the member R is selected from the group consisting of hydrogen, substituted or unsubstituted Ci -C 6 alkyl, substituted or unsubstituted lower (Ci- C 6 ) acyl, alkyl, and substituted and unsubstituted aryl. Up to four hydrogen atoms of either or both the fatty acid portion and alkanol (e.g., ethanol) portion of compounds of the above formula may also be substituted by methyl or a double bond.
  • the compounds of Formula IV have n from 1 to 3; and the sum of a and b can be from 1 to 3.
  • the member R 2 is selected from the group consisting of hydrogen, substituted or unsubstituted Ci -C 6 alkyl, and substituted or unsubstituted lower (C ⁇ -C 6 ) acyl. Up to four hydrogen atoms of either or both the fatty acid portion and alkanol (e.g., ethanol) portion of compounds of the above formula may also be substituted by methyl or a double bond.
  • Compounds of Formula IV include those compounds where R is H, compounds where a and b are each 1, and compounds where n is 1.
  • Examples of compounds according to Formula IV include the following (R or S) l'-oleoylethanol ethers and (R or S)-2'- oleoylethanol ethers:
  • OEA-like compounds and OEA-like modulators of the invention include compounds having a variety of polar head analogs of OEA. These compounds include compounds having a fatty acid moiety of the general formula:
  • the compounds of Formula V have a sum of a and b that can be from 0 te 4. In other embodiments, the sum of a and b is from 1 to 3. In these embodiments, up to four hydrogen atoms of the compounds of the above formula may also be substituted by methyl or a double bond. In addition, the molecular bond between carbons c and d may be unsaturated or saturated. A particularly preferred embodiment is that of the oleic acid fatty acid moiety:
  • R 3 group of the above structures may be selected from any of the following:
  • x is from 1 to 8, and the alkyl portion thereof may be branched or cyclic.
  • Additional polar head groups for R 3 include, for instance, compounds having furan, dihydrofuran and tetrahydro furan functional groups:
  • z can be from 1 to 5.
  • Other exemplary polar head groups include a variety of imidazole and oxazoles, for example:
  • m is from 1 to 9 and p is independently from 1 to 5.
  • An exemplary compound is:
  • Another exemplary compound is an ethanolamine analog with an apolar tail of the following structural formula:
  • OEA-like compound and OEA-like modulators of the invention of the invention include those disclosed in U.S. Patent Application No. 10/112509 filed March, 27, 2002, assigned to the same assignee as the present application, which is incorporated herein by reference.
  • the fatty acid moiety of the fatty acid alkanolamide or ethanolamide compound, homologue, or analog may be saturated or unsaturated, and if unsaturated may be monounsaturated or polyunsaturated.
  • hydroxyalkylamide moiety of the fatty acid amide compound, homologue or analog include the introduction of a substituted or unsubstituted lower (d-C 3 ) alkyl group on the hydroxyl group of an alkanolamide or ethanolamide moiety so as to form the corresponding lower alkyl ether.
  • the hydroxy group of the alkanolamide or ethanolamide moiety is bound to a carboxylate group of a C 2 to C 6 substituted or unsubstituted alkyl carboxylic acid to form the corresponding ester of the fatty acid ethanolamide.
  • Such embodiments include fatty acid alkanolamide and fatty acid ethanolamides in ester linkage to organic carboxylic acids such as acetic acid, propionic acid, and butanoic acid.
  • the fatty acid alkanolamide is oleoylalkanolamide.
  • the fatty acid alkanolamide is oleoylethanolamide.
  • the fatty acid ethanolamide compound, homologue, or analog further comprises a substituted or unsubstituted lower alkyl (d-C 3 ) group covalently bound to the nitrogen atom of the fatty acid ethanolamide.
  • the compound of the invention is fatty acid alkanolamide compound or homologue satisfying the following formula VI:
  • n is any number from 0 to 5 and the sum of a and b can be any number from 0 to 4.
  • Z is a member selected from -C(O)N(R 0 )-; -(R°)NC(O)-; -OC(O)-; -(O)CO-; O; NR°; and S, in which R° and R 2 are independently selected from the group consisting of unsubstituted or unsubstituted alkyl, hydrogen, substituted or unsubstituted d - C 6 alkyl, substituted or unsubstituted lower (C ⁇ -C 6 ) acyl, homoalkyl, and aryl.
  • the compound may also be substituted by methyl group or a double bond.
  • the molecular bond between carbons c and d may be unsaturated or saturated.
  • the fatty acid ethanolamide of the above formula is a naturally occurring compound.
  • the alkyl subsitutents are each homoalkyl, or its pharmaceutically acceptable salt.
  • Further embodiments of the compounds of Formula VI have substituents as set forth for compounds of Formula I above.
  • a H atom attached to a carbon atom of a compound of the above formula is replaced with a halogen atom, preferably a CI atom or a F atom.
  • Ethers and mercaptans can be prepared by methods well-known to those of skill in the art, e.g., Williamson synthesis.
  • a long chain alkyl alcohol or thiol is deprotonated by a base, e.g, NaH, and a reactive alcohol derivative, e.g., a halo, tosyl, mesyl alcohol, or a protected derivative thereof is reacted with the resulting anion to form the ester or mercaptan.
  • a base e.g, NaH
  • a reactive alcohol derivative e.g., a halo, tosyl, mesyl alcohol, or a protected derivative thereof is reacted with the resulting anion to form the ester or mercaptan.
  • OEA-like modulators need not be an OEA-like compound (e.g., OEA, fatty acid amide or homolog thereof).
  • the OEA-like modulator is a compound such as taught in U.S. Patent No. 6,200,998 (hereby incorporated by reference) that are PPAR ⁇ activators. This reference teaches PPAR agonist compounds of the general formula:
  • R a is (1) d- 15 alkanoyl, (2) d- 15 alkyl, (3) C 2 - 15 alkenyl, (4) C 2 -i 5 alkynyl, (5) halo, (6) OR b , (7) aryl, or (8) heteroaryl, wherein said alkyl, alkenyl, alkynyl, and alkanoyl are optionally substituted with from 1-5 groups selected from R e (defined below), and said aryl and heteroaryl optionally substituted with 1 to 5 groups selected from R d (defined below) ;
  • R b is (1) hydrogen, (2) d-io alkyl, (3) C 2 - 10 alkenyl, (4) C 2 - ⁇ O alkynyl, (5) aryl, (6) heteroaryl, (7) aryl C 1 - 15 alkyl, (8) heteroaryl
  • R 1 is selected from a group consisting of: H, C 1 -1 5 alkyl, d-1 5 alkenyl, - 15 alkynyl and - t o cycloalkyl, said alkyl, alkenyl, alkynyl, and cycloalkyl optionally substituted with 1 to 3 groups of R a (defined below);
  • R 3 is selected from a group consisting of: H, NHR 1 , NHacyl, C MS alkyl, C 3 - ⁇ o cycloalkyl, d- 15 alkenyl, C 1 - 15 alkoxy, CO 2 alkyl, OH, d-15 alkynyl, d-.o aryl, C 5 -10 heteroaryl said alkyl, cycloalkyl, alkenyl, alkynyl, aryl and heteroaryl optionally substituted with 1 to 3 groups of R a ; (Z-W-) is Z
  • R 8 is selected from the group consisting of CR 6 R 7 , O, NR 6 , and S(O ;
  • R 6 and R 7 are independently selected from the group consisting of H, C ⁇ - 6 alkyl;
  • B is selected from the group consisting of: 1) a 5 or 6 membered heterocycle containing 0 to 2 double bonds, and 1 heteroatom selected from the group consisting of O, S and N, the heteroatom being substituted at any position on the five or six membered heterocycle, the heterocycle being optionally unsubstituted or substituted with 1 to 3 groups of R a ; 2) a 5 or 6 membered carbocycle containing 0 to 2 double bonds, the carbocycle optionally unsubstituted or substituted with 1 to 3 groups of R a at any position on the five or six membered carbocycle; and 3) a 5 or 6 membered heterocycle containing 0 to 2 double bonds, and 3 heteroatoms selected from the group consisting of O, N, and S, which are
  • Additional compounds suitable for practicing the inventive methods include compounds taught in U.S. Patent No. 5,847,008, U.S. Patent No 6,090,836 and U.S. Patent No. 6,090,839, U.S. Patent No. 6,160,000 each of which is herein incorporated by reference in its entirety to the extent not inconsistent with the present disclosure.
  • compositions of these publications which are each herein incorporated by reference in their entirety to the extent not inconsistent with the present disclosure can be screened by the methods provide below to provide the PPAR ⁇ specific agonists of the invention which are useful, for instance, in reducing body fat. and body weight, modulating fat catabolism, and reducing appetite according to the present disclosure.
  • PPAR ⁇ specific agonists of the invention which are useful, for instance, in reducing body fat. and body weight, modulating fat catabolism, and reducing appetite according to the present disclosure.
  • compositions include compositions suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic), pulmonary (nasal or buccal inhalation), or nasal administration, although the most suitable route in any given case will depend in part on the nature and severity of the conditions being treated and on the nature of the active ingredient.
  • An exemplary route of administration is the oral route.
  • the compositions may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy.
  • the FAAH inhibitors , OEA-like compounds, and OEA-like modulators of the invention can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous).
  • any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, hard and soft capsules and tablets, with the solid oral preparations being preferred over the liquid preparations.
  • oral liquid preparations such as, for example, suspensions, elixirs and solutions
  • carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, hard and soft capsules and tablets, with the solid oral preparations being preferred over the liquid preparation
  • tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers can be employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques. Such compositions and preparations can contain at least 0.1 percent of active compound. The percentage of active compound in these compositions may, of course, be varied and may conveniently be between about 2 percent to about 60 percent of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a therapeutically effective dosage will be obtained.
  • the active compounds can also be administered intranasally as, for example, liquid drops or spray.
  • compositions may be present as coatings or to modify the physical form of the dosage unit.
  • tablets may be coated with shellac, sugar or both.
  • a syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry or orange flavor.
  • the composition may be an enteric coated formulation.
  • compositions of the invention may also be administered parenterally.
  • Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
  • dosages from about 0.05 to about 100 mg, preferably from about 0.1 to about 100 mg, per day may be used.
  • the exact dosage will depend upon the mode of administration, on the therapy desired, form in which administered, the subject to be treated and the body weight of the subject to be treated, and the preference and experience of the physician or veterinarian in charge.
  • the FAAH inhibitors, OEA-like compounds, and OEA-like modulators can be dispensed in unit dosage form comprising preferably from about 0.1 to about 100 mg of active ingredient together with a pharmaceutically acceptable carrier per unit dosage.
  • dosage forms suitable for oral, nasal, pulmonary or transdermal administration comprise from about 0.001 mg to about 1000 mg, preferably from about 0.1 mg to about 100 mg of the compounds admixed with a pharmaceutically acceptable carrier or diluent.
  • these preparations preferably contain a preservative to prevent the growth of microorganisms.
  • Administration of an appropriate amount of the compounds may be by any means known in the art such as, for example, oral or rectal, parenteral, intraperitoneal, intravenous, subcutaneous, subdermal, intranasal, or intramuscular. In some embodiments, administration is transdermal.
  • An appropriate amount or dose of the candidate compound may be determined empirically as is known in the art. For example, with respect to body fat or loss of body weight, an appropriate or therapeutic amount is an amount sufficient to effect a loss of body fat or a loss in body weight in the animal over time.
  • the candidate compound can be administered as often as required to effect a loss of body fat or loss in body weight, for example, hourly, every six, eight, twelve, or eighteen hours, daily, or weekly
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
  • liquid solutions such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or PEG 400
  • capsules, sachets or tablets each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin
  • suspensions in an appropriate liquid such as water, saline or PEG 400
  • Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers.
  • Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • a flavor e.g., sucrose
  • an inert base such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Formulations suitable for parenteral admimstration such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • transdermal routes of administration methods for transdermal administration of drugs are disclosed in Remington's Pharmaceutical Sciences, 17th Edition, (Gennaro et al. Eds., Mack Publishing Co., 1985).
  • Dermal or skin patches are a preferred means for transdermal delivery of the compounds of the invention. Patches preferably provide an absorption enhancer such as DMSO to increase the absorption of the compounds.
  • Other methods for transdermal drug delivery are disclosed in U.S. Patents No. 5,962,012, 6,261,595, and 6,261,595. Each of which is incorporated by reference in its entirety.
  • Preferred patches include those that control the rate of drug delivery to the skin.
  • Patches ⁇ ray provide a variety of dosing systems including a reservoir system or a monolithic system, respectively.
  • the reservoir design jnay, for example, have four layers: the adhesive layer that directly contacts the skin, the control membrane, which controls the diffusion of drug molecules, the reservoir of drug molecules, and a water-resistant backing.
  • Such a design delivers uniform amounts of the drug over a specified time period, the rate of delivery has to be less than the saturation limit of different types of skin.
  • the monolithic design typically has only three layers: the adhesive layer, a polymer matrix containing the compound, and a water-proof backing.
  • This design brings a saturating amount of drug to the skin. Thereby, delivery is controlled by the skin. As the drug amount decreases in the patch to below the saturating level, the delivery rate falls.
  • the FAAH inhibitors, OEA-like compounds and OEA-like modulators of the invention may be used in combination with other compounds of the invention or with other drugs that may also be useful in dieting or the treatment, prevention, suppression or amelioration of body fat. Such other drugs may be administered, by a route and in an amount commonly used therefore, contemporaneously or sequentially with a compound of the invention.
  • a FAAH inhibitor or OEA-like compound or OEA-like modulator of the invention is used contemporaneously with one or more other drugs, a pharmaceutical composition in unit dosage form containing such other drugs and the compound is preferred.
  • the compound of the present invention and the other active ingredients may be used in lower doses than when each is used singly. Accordingly, the pharmaceutical compositions of the present invention include those that contain one or more other active ingredients, in addition to the compounds disclosed above.
  • the pharmaceutically or physiologically acceptable salts include, but not limited to, a metal salts such as sodium salt, potassium salt, lithium salt and the like; alkaline earth metals such as calcium salt, magnesium salt and the like; organic amine salts such as triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N.N'-dibenzylethylenediamine salt and the like; inorganic acid salts such as hydrochloride, hydrobromide, sulfate, phosphate and the like; organic acid salts such as formate, acetate, trifluoroacetate, maleate, tartrate and the like; sulfonates such as methanesulfonate, benzenesulfonate, p-toluenesulfonate, and the like; amino acid salts such as arginate, asparginate, glutamate and the like.
  • a metal salts such as sodium salt
  • the invention provides a method of treating, controlling or preventing one or more diseases, disorders, or conditions in a subject mediated by the PPAR ⁇ receptor or responsive to administration of a PPAR ⁇ modulator by administering one or more of an agent selected from the group consisting of a FAAH inhibitor, an OEA-like compound and an OEA-like modulator.
  • Such conditions include, but are not limited to, diabetes mellitus, hyperglycemia, obesity, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, vascular restenosis, irritable bowel syndrome, pancreatitis, abdominal obesity, adipose cell tumors, adipose cell carcinomas, Syndrome X, polycystic ovarian syndrome, and other disorders where insulin resistance is a component; metabolic disorders, excess body fat, cellulite, Type II diabetes, insulin resistance, artherogenesis, an inflammatory disorder or condition, Alzheimers disease, Crohn's disease, a vascular inflammation, an inflammatory bowel disorder, rheumatoid arthritis, asthma, thrombosis and cachexia.
  • the subject is human.
  • the modulator is a fatty acid alkanolamide.
  • the compound is a modulator of Formula I or Formula VI.
  • the FAAH inhibitors, OEA-like compounds e.g., fatty acid alkanolamides, fatty acid ethanolamide compounds, analogs, and homologues with PPAR ⁇ modulatory activity
  • OEA-like modulators their compositions and methods of administration can be used to reduce body fat and or body weight in mammals, including dogs, cats, and especially humans.
  • the weight loss may be for aesthetic or therapeutic purposes.
  • the compounds may also be used to reduce appetite or induce hypophagia.
  • the FAAH inhibitors, OEA-like compounds and/or OEA-like modulators, and their compositions can be administered to subjects (e.g., humans) to prevent weight gain or body fat increases in individuals within a normal weight range.
  • the compounds may be used in otherwise healthy individuals who are not otherwise in need of any pharmaceutical intervention for diseases related to diabetes or hyperlipidemia or cancer.
  • the individuals to be treated are free of diseases related to disturbances in sugar or lipid levels or metabolism or free of risk factors for cardiovascular and cerebrovascular disease.
  • the individuals may be non-diabetic and have blood sugar levels in the normal range.
  • the individuals may also have blood lipids (e.g., cholesterol) or triglyceride levels in the normal jange.
  • the individuals may be free of atherosclerosis.
  • the individuals may be free of other conditions such as cancer or other tumors, disorders involving insulin resistance, Syndrome X, and pancreatitis.
  • the subjects are overweight or obese persons in need of body fat and/or body weight reduction.
  • the methods, compounds, and compositions of the invention can be administered to promote weight loss and also to prevent weight gain once a body weight within the normal range for a person of that sex and age and height has been achieved.
  • the FAAH inhibitors, OEA-like compounds and/or OEA-like modulators may be used in otherwise healthy individuals who are not in need of any pharmaceutical treatment of a disorder related to diabetes, hyperlipidemia, or cancer.
  • the individuals may also otherwise free of risk factors for cardiovascular and cerebrovascular diseases.
  • the individuals to be treated are free of diseases related to sugar (e.g., glucose) or lipid metabolism.
  • the individuals may be non-diabetic and have blood sugar levels in the normal range.
  • the individuals may also have blood lipids (e.g., cholesterol, HDL, LDL, total cholesterol) or triglyceride levels in the normal range.
  • the individuals may not need to be in treatment for atherosclerosis.
  • the FAAH inhibitors, OEA-like compounds and/or OEA-like modulators compositions of the invention may also be administered to suppress appetite in mammals, including cats, dogs, and humans.
  • the compounds may be used in otherwise healthy individuals who are not in need of pharmaceutical interventions for any disease.
  • the individuals do not need preventive or ameliorative therapy for diseases, including cancer, diabetes, or hyperlipidemia.
  • the individuals to be treated are free of diseases related to abnormal sugar or lipid levels.
  • the individuals may be free of risk factors for cardiovascular or cerebrovascular disease.
  • the individuals may be non-diabetic and have blood sugar levels in the normal range.
  • the individuals may also have blood lipids (e.g., cholesterol) or triglyceride levels in the normal range.
  • the individuals may be free of atherosclerosis.
  • the individuals to be treated are free of diseases related to sugar or lipid metabolism (e.g., diabetes, hypercholesterolemia, low HDL levels or high LDL levels).
  • the individuals may be non-diabetic and have blood sugar levels in the normal range.
  • the individuals may also have blood lipids (e.g., cholesterol) or triglyceride levels in the normal range.
  • the individuals may be free of atherosclerosis. [0195] ;
  • Treatment with the FAAH inhibitors, OEA-like compounds and/or OEA-like modulators of the invention may be prophylactic or to prevent progression of harm preventable by activation of PPAR ⁇ receptors.
  • the duration and frequency of treatment can be according to the severity of the disease or condition, its chronicity, and responsiveness to treatment.
  • a treatment may be short-term over days or weeks or chronic for months to years.
  • One of ordinary skill in the art will be able to determine when a subject is responding favorably to an administered agent (e.g., by measuring blood lipids, weight, blood sugar or insulin levels, inflammatory cytokines, or other objective and subjective signs or symptoms of the subject diseases and conditions).
  • treatment with the compounds and compositions of the invention may be reduced or terminated once a predetermined parameter as been reached has been accomplished. For instance, with respect to weight loss as an objective, the administration can be terminated when the desired amount amount of weight loss has been accomplished or when the individual achieves a BMI within the normal range.
  • the FAAH inhibitor, OEA-like compound and/or OEA-like modulator is administered with a second agent, including but not limited to, an agent selected from the group consisting of insulin sensitizers, PPAR ⁇ .
  • glitazones glitazones, troglitazone, pioglitazone, englitazone, MCC-555, BRL49653, biguanides, metformin, phenformin, insulin, insulin mimetics, sulfonylureas, tolbutamide, glipizide, ⁇ .-glucosidase inhibitors, acarbose, cholesterol lowering agents, HMG-CoA reductase inhibitors, lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rivastatin, other statins, sequestrants, cholestyramine, colestipol, dialkylaminoalkyl derivatives of a cross-linked dextran, nicotinyl alcohol, nicotinic acid, a nicotinic acid salt, PPAR/3 agonists, fenof ⁇ bric acid derivatives, gemfibrozil, clo
  • an appropriate amount of the FAAH inhibitor, OEA-like compound or OEA-like modulator or pharmaceutical composition(s) thereof may be by any means known in the art such as, for example, oral or rectal, intraparenteral such as, for example, intraperitoneal, intravenous, subcutaneous, subdermal, intranasal, or intramuscular. Preferably administration is intraperitoneal.
  • An appropriate amount of the candidate compound may be determined empirically as is known in the art. For example, with respect to weight loss, as the objective, an appropriate amount is an amount sufficient to effect a loss of body fat or a loss in body weight in the animal over time.
  • the candidate compound can be administered as often as required to effect a loss of body fat or loss in body weight, for example, hourly, every six, eight, twelve, or eighteen hours, daily, or weekly.
  • PPAR ⁇ specific binding compounds have at least 5- 10 fold, preferably 10-100 fold, more preferably 100-500 fold, most preferably greater than 1000 fold specificity for PPAR ⁇ compared to other PPAR subtypes.
  • Mammalian PPAR subtypes e.g., rat, mouse, hamster, rabbit, primate, guinea pig
  • human PPAR subtypes are preferably used.
  • Electrophoretic mobility shift assays can be used to determine whether test compounds bind to PPAR ⁇ and affect its electrophoretic mobility. (Forman, et al. (1997) PNAS 94:4312 and Kliewer, et al. (1994) PNAS 91:7355). Electrophoretic mobility shift assays involve incubating a PPAR-RXR with a test compound in the presence of a labeled nucleotide sequence. Labels are known to those of skill in the art and include, for example, isotopes such as, H, C, S, and P, and non-radioactive labels such as fluorescent labels or chemiluminescent labels.
  • Fluorescent molecules which can be used to label nucleic acid molecules include, for example, fluorescein isothiocyanate and pentafluorophenyl esters. Fluorescent labels and chemical methods of DNA and RNA fluorescent labeling have been reviewed recently (Proudnikov et al., 1996, Nucleic Acids Res. 24:4535-42).
  • the compound is a candidate PPAR ⁇ specific binding compound.
  • U.S. Patent 6,265,160 The incubation mixture is then electrophoretically separated and the resulting gel exposed to X-ray film.
  • the resulting autoradiograph may have one or more bands representing slowly migrating DNA-protein complexes. This control lane can indicate the mobility of the complex between the DNA probe and PPAR.
  • Monoclonal antibodies specific for PPAR subtypes can be used to identify PPAR ⁇ specific binding compounds in modified electrophoretic mobility shift assays.
  • Purified PPAR ⁇ , PPAR ⁇ or PPAR ⁇ can be incubated with an appropriate amount of a test compound in the presence of RXR.
  • the test compound need not be labeled.
  • PPAR subtype specific monoclonal antibodies can be incubated with the PPAR-RXR-test compound mixture.
  • test compounds that bind PPAR induce supershifting of the PPAR- RXR complex on a gel (Forman, et al. (1997), PNAS 94:4312) which can be detected by anti- PPAR monoclonal antibodies using a Western blot (immunoblot).
  • Western blots generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind PPAR subtypes.
  • a suitable solid support such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter
  • These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the anti-PPAR antibodies.
  • the particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the PPAR subtype specific ligand used in the assay.
  • the detectable group can be any material having a detectable physical or chemical property.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, electrical, optical or chemical means.
  • a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
  • Useful labels in the present invention include magnetic beads (e.g., DYNABEADSTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, or 32 P), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.).
  • magnetic beads e.g., DYNABEADSTM
  • fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like
  • radiolabels e.g., 3 H, 125 1, 35 S, 14 C, or 32 P
  • colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.).
  • the compounds induce reporter gene expression at levels at least 5-10 fold, more preferably 10-100 fold, more preferably 100-500 fold, more preferably 500-1000 fold, most preferably greater than 1000 fold greater than the negative control.
  • Example 2 Test Methods, Physiology and Pharmacological Activity of OEA-like Compounds and/or OEA-like modulators. [0235],. , >Animals. Male Wistar rats (200-350 g) were used. Procedures should meet NIH guidelines detailed in the Guide for the Care and Use of Laboratory Animals, and the European Communities directive 86/609/EEC regulating animal research.
  • FAE were dissolved in dimethylsulphoxide (DMSO) and administered in 70% DMSO in sterile saline (acute treatments) or 5% Tween 80/5% propylenglycol in sterile saline (subchronic treatments) (1 ml per kg, i.p.).
  • DMSO dimethylsulphoxide
  • Capsaicin was administered in 10% Tween 80/10% ethanol/80% saline; SR141716A, SRI 44528, CCK- 8 and CP-93129 in 5% Tween 80/5% propylenglycol/90% saline (1 ml per kg, i.p.).
  • NAT assays were performed using l,2-di[ 14 C]palmityl-s ⁇ z-glycerophosphocholine as a substrate (108 mCi/mmol, Amersham, Piscataway, NJ) (Cadas et al., H., J. Neuroscl, 17:1226-1242 (1997)).
  • FAAH assays were performed according to (Desarnaud et al., J. Biol. Chem., 270:6030-6035 (1995)), except that [ 3 H] anandamide (arachidonyl-[l- 3 H]ethanolamide; 60 Ci/mmol; ARC, St.
  • Plasma was prepared from blood obtained by cardiac puncture (Giuffrida et al., Anal. Biochem., 280:87-93 (2000)) and CSF was collected from the cisterna magna using a 27G 1/2 needle (Precisionglide, USA).
  • FAEs and NAPE were extracted from tissues with methanol/chloroform and fractionated by column chromatography (Giuffrida et al., "Lipid Second Messengers" (ed. Laychock, S.G. & Rubin, R.P.) 113-133 (CRC Press
  • Plasma -hydroxybutyrate and glycerol were measured using commercial kits (Sigma, St. Louis, MO). Plasma prolactin, corticosterone and luteinizing hormone were quantified by radioimmunoassay (Navarro et al., Neuroreport, 8:491-496 (1997)).
  • Results are expressed as mean ⁇ s.e.m of n separate experiments. The significance of differences among groups was evaluated using ANOVA followed by a Student-Newman-Keuls post hoc test, unless indicated otherwise.
  • Plasma PEA was not significantly affected by any of these treatments (data not shown), whereas anandamide decreased rapidly upon food removal, remaining lower than baseline for the entire duration of the experiment ( Figure 1 d).
  • Table 1 Plasma level of /3-hydroxybutyrate (/3-HBA) and glycerol in fasting rats.
  • ⁇ -HBA Glycerol Plasma level of /3-hydroxybutyrate (/3-HBA) and glycerol in fasting rats.
  • Food deprivation profoundly reduced FAAH activity in adipose membranes, but had no effect on FAAH activity in the brain, liver, stomach, intestines, ⁇ idney and skeletal muscle ( Figure 2 a-e and data not shown).
  • food deprivation may increase the levels of OEA and other FAEs in white fat in two synergistic ways, which are mechanistically distinct from other reactions occurring during lipolysis: stimulation of NAT activity may lead to increase the biosynthesis of NAPE and FAEs, while inhibition of FAAH activity may prolong the life span of newly synthesized FAEs.
  • stimulation of NAT activity may lead to increase the biosynthesis of NAPE and FAEs
  • inhibition of FAAH activity may prolong the life span of newly synthesized FAEs.
  • several tissues may contribute to the normal levels of OEA in the bloodstream, the dynamic biochemical changes observed in fat underscore the crucial role of this tissue in generating OEA during starvation.
  • OEA was subchronically administered to rats.
  • Daily injections of OEA (5 mg per kg, i.p.) for seven days resulted in a small, but significant decrease in cumulative food intake (Figure 5 a), which was accompanied by a profound inhibition of weight gain (Figure 5 b, c).
  • OEA did not affect water intake ( Figure 5 d).
  • the impact of OEA on body weight may only be partially explained by its moderate reduction of food consumption indicating that other factors, such as stimulation of energy expenditure or inhibition of energy accumulation, may contribute to this effect.
  • the invention provides OEA-like compounds and OEA-like modulators having a peripheral site of action. Such a site can be advantageous in reducing the likelihood of central nervous system side effects.
  • OEA may reduce eating by inducing a non-specific state of behavioral suppression.
  • OEA should cause conditioned taste aversion, which can be readily provoked in rats by a number of noxious substances (Green et al., Science, 173:749-751
  • This pharmacological profile differentiates OEA from other appetite suppressants such as amphetamine and glucagon-like peptide 1 (whose effects often include aversion, hyperactivity, anxiety and activation of the HP A axis) and from the endogenous cannabinoid anandamide (which stimulates food intake in partially satiated animals, increases pain threshold, decreases body temperature and activates the HPA axis) (Pertwee, R. G., Exp. Opin. Invest. Drugs, 9:1553-1571 (2000)).
  • amphetamine and glucagon-like peptide 1 whose effects often include aversion, hyperactivity, anxiety and activation of the HP A axis
  • cannabinoid anandamide which stimulates food intake in partially satiated animals, increases pain threshold, decreases body temperature and activates the HPA axis
  • OEA-like compound or OEA-like modulator can be evaluated by a number of methods.
  • appropriate amounts OEA and/or candidate compounds are administered to rats via intraperitoneal injection.
  • the OEA and candidate compounds can be formulated in 70% DMSO in sterile saline, 5% Tween 80/5% polyethyleneglycol in sterile saline, or 10% Tween 80/10% ethanol 80% saline.
  • Five mg per kg of OEA can be used as the positive control.
  • Amounts of candidate compounds administered may range, for instance, from 1-25 mg per kg. Typically 1, 2, 5, 10, 15, and 20 mg per kg doses of each candidate compound can be administered to different sets of rats to determine which dose is optimal. Injections may be given 30 minutes before the animals' principal meal for 7- 14 days.
  • the effect of the candidate compound on total body fat can be determined by taking direct measurements of the rat's body fat using skin fold calipers. Skin on the rats' backs, abdomen, chest, front and rear legs can be pinched with calipers to obtain measurements before administration of OEA and/or candidate compounds and every 48 hours during and after administration of OEA and/or candidate compounds. Differences in measurements in at least two of the pinched sites reflect the change in the rat's total body fat.
  • the protein concentration of the cultured cells can be determined and cells seeded in 2 ml media so that 4-6 mg protein per ml is present in the reaction mixture.
  • Cells can be incubated for 10 minutes at 37°C with [ 14 C]- oleic acid (Amersham), in the presence or absence of 10 ⁇ M OEA, reactions may be stopped with 200 ⁇ l 2M perchloric acid and acid-soluble products extracted with chloro form/methanol/water (5:1:1, vol: vol: vol). The aqueous phase can be removed and washed twice more. Protein concentration can be determined using a Lowry assay.
  • OEA Effect of OEA on fatty acid metabolism.
  • This example illustrates the effect of OEA on fat metabolism and methods for studying the same.
  • Oleoylethanolamide (OEA) decreases body weight not only by suppressing appetite, but also by possibly enhancing body fat catabolism.
  • the effects of OEA on fatty acid oxidation in major body-fat burning tissues was examined.
  • OEA significantly stimulates fatty acid oxidation in primary cultures of liver, skeletal muscle (soleus) and heart cells, whereas it has no effect in brain-derived astroglial cell cultures.
  • OEA induces a significant mobilization of triacylglycerol stores from primary white adipose tissue cells.
  • Table 4 details the methods and effects of OEA on fatty acid oxidation in these cells. Structure-activity relationship experiments provide evidence that the effect of OEA on skeletal muscle fatty acid oxidation is specific ( Figure 8).
  • the effects of OEA are mimicked by the hydrolysis-resistant homologue methyl-OEA and -only partially by palmitoylethanolamide (PEA), but not by arachidonylethanolamide (AEA) or oleic acid (OA).
  • PDA palmitoylethanolamide
  • OA arachidonylethanolamide
  • these results show that lipid oxidation and mobilization are enhanced by OEA, and that the effects of OEA are restricted to peripheral sites.
  • OEA may activate local, sensory fibers, which may in turn inhibit feeding by engaging brain structures such as the NST and PVN.
  • the above results for Example 2 reveal an unexpected role for OEA in the peripheral regulation of feeding, and provide a framework to develop novel medicines for reducing body weight or body fat, for preventing body weight gain or body fat increase, for suppressing appetite or reducing food seeking behavior, or food intake, and for the treating eating disorders, overweight, or obesity.
  • These medicines would include not only OEA analogues and homologues but also agents which control OEA levels by acting upon the OEA formation and hydrolyzing systems and enzymes as disclosed above.
  • GW 7647 ⁇ 2-(4- ⁇ 2-[3-Cyclohexyl-l-(4-cyclohexyl-butyl)-ureido]-ethyl ⁇ - phenylsulfanyl)-2-methyl-propionic acid was synthesized as follows. Phenethylamine was reacted with 4-cyclohexyl-butyric acid in the presence of diisopropylcarbodiimide and hydroxybenzotriazole (HOBT) in CH 2 C1 2 .
  • HOBT hydroxybenzotriazole
  • GW501516 [2-Methyl-4-[4-methyl-2-(4-trifluoromethyl-phenyl)-thiazol-5- ylmethylsulfanyl]-phenoxy ⁇ -acetic acid was synthesized via basic hydrolysis of the corresponding ethyl ester, prepared by coupling 5-chloromethyl-4-methyl-2-(4- trifluoromethyl-phenyl)-thiazole with (4-mercapto-2-methyl-phenoxy)-acetic acid ethyl ester (Chao et al., 2001).
  • o-tolyloxy-acetic acid ethyl ester was treated with chlorosulfonic acid to give (4-chlorosulfonyl-2-methyl-phenoxy)-acetic acid ethyl ester (synthesized).
  • Reduction to (4-acetylsulfanyl-2-methyl-phenoxy)-acetic acid ethyl ester (zinc dust/NaOAc/Ac 2 O/glacial AcOH), followed by hydrolysis under mild basic conditions (py ⁇ olidine in ethanol) yielded the desired intermediate (4-mercapto-2-methyl-phenoxy)- acetic acid " ethyl ester.
  • OEA and other fatty acid ethanolamides can be prepared as described in Giuffrida et al., 2000). All other chemicals from Sigma (Saint Louis, Missouri) or Tocris (Ballwin, Missouri).
  • mice Male C57BL/6J mice, homozygous mice deficient for PPAR ⁇ (129S4/SvJae-RR-4R ⁇ a""' 00 " 2 ) and wild-type mice (129Sl/SvlmJ) were purchased from the Jackson Laboratory. Male Zucker rats (7 weeks of age) were obtained from Charles River. Male Wistar rats (325 ⁇ 30g) were from Charles River. Animals were maintained on a 12-h light/dark cycle (light off at 5:30 PM) with water and chow pellets (RMH 2500, Prolab) available ad libitum.
  • Transactivator plasmids pFA-PPAR ⁇ , pFA-PPAR ⁇ , pFA-PPAR ⁇ and pFA-RXR which encoded for the DNA-binding domain (DBD) of hPPAR ⁇ (499-1404), hPPAR ⁇ (412-1320), hPPAR ⁇ (610-1434) and hRXR (402-1389) fused to the DNA-binding domain (residues 1-147) of yeast GAL4 under control of the human cytomegalovirus (CMV) promoter were generated.
  • the plasmids contained a neomycin-resistance gene to provide stable selection with G418 (200 ⁇ g-ml "1 ; Calbiochem).
  • the HeLa cells were cultured in Dulbecco's-modified Eagles's medium (DMEM) supplemented with fetal bovine serum (10%).
  • DMEM Dulbecco's-modified Eagles's medium
  • the cells were transfected with Fugene 6 (3 ⁇ l, Roche) containing the pFR-luc plasmid (l ⁇ g, Stratagene). Eighteen hours following transfection, the culture media was replaced with supplemented DMEM containing hygromycin (100 ⁇ g-mi "1 , Calbiochem).
  • the clonal cell line HLR was selected because it demonstrated the highest levels of luciferase activity and transfected it with transactivator plasmids to generate cell lines that also expressed the DNA-binding domain of PPAR ⁇ (HLR- ⁇ ), PPAR ⁇ (HLR- ⁇ ), PPAR ⁇ (HLR- ⁇ ), and RXR (HLR-rxr).
  • the cells were cultured in supplemented DMEM containing hygromycin and G418.
  • RNA isolation and cDNA synthesis [0280] Tissues were stored in RnaLaterTM (Ambion), extracted total RNA with TRIzolTM (Invitrogen) and quantified it with RibogreenTM (Molecular Probes). cDNA was synthesized by using Superscriptll RNase H-reverse transcriptase (Invitrogen).
  • PPAR ⁇ F: CTTCCCAAAGCTCCTTCAAAAA, R: CTGCGCATGCTCCGTG, P: TGGTGGACCTTCGGCAGCTGG;
  • PPAR ⁇ GATGACAGTGACCTGGCGCT
  • R AGGCCTGGCCGGTCTC
  • P TTCATCGCGGCCATCATTCTGTGT
  • PPAR ⁇ F: AGTGGAGACCGCCCAGG, R: GCAGCAGGTTGTCTTGGATGT, P: TTGCTGAACGTGAAGCCCATCGA;
  • FATP F: GCACAGCAGGTACTACCGCA
  • R GGCGGCACGCATGC
  • P TGCTGCCTTTGGCCACCATTCCTA
  • F TCACCATCACCTATGGACCCA
  • R TCCAGTTCGCACTCCTCCC
  • P AGTGGTCCGCAATGAGTTCACCCTG;
  • GAPDH F: TCACTGGCATGGCCTTCC, R: GGCGGCACGTCAGATCC, P: TTCCTACCCCCAATGTGTCCGTCG.
  • RNA levels were normalized by using glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) as an internal standard. mRNA levels were measured by generating six-point serial standard curves using mouse total RNA. Estimates of relative mRNA abundance (in arbitrary units) were made by using the value (Schmittgen et al., 2000). Relative quantifications of RNAs of interest were made by using the 2 ⁇ CT formula, in which ⁇ Cjwas calculated by subtracting the value for GAPDH from the C T value for the gene of interest. This formula was validated for each primer/probe set by using six-point serial standard curves. Feeding experiments [0283] Acute experiments.
  • Drugs or appropriate vehicles were administered at 5:00-5:30 PM to free-feeding mice, which were habituated to the experimental setting. Vehicles exerted no significant effect on feeding. Food intake and feeding microstructure was continuously monitored for 12 h using an automated system (ScriPro Inc, NY) (Gaetani et al., 2003).
  • vehicle saline/polyethylene glycol/Tween 80, 90/5/5; 1 ml-kg "1 ) or OEA (5 mg-kg "1 , once daily, i.p.
  • obese Zucker rats were treated for 2 weeks with vehicle or OEA (5 mg-kg "1 , once daily, i.p.), while maintaining them on a regular rodent chow (RMH 2500, Prolab). Food intake and body weight were measured daily.
  • the animals were fasted overnight, and tissues and blood samples collected for biochemical analyses.
  • modified HeLa cells which cannot metabolize OEA and other fatty acid ethanolamides (FAE) (Day et al., 2001), were genetically modified to stably express a luciferase reporter gene along with the ligand-binding domain of human PPAR ⁇ , PPAR ⁇ , PPAR ⁇ , or retinoid X receptor (RXR) fused to the yeast GAL4 DNA-binding domain (Lazennec et al., 2000). In standard transactivation assays, each of these cell lines responded to appropriate synthetic PPAR ⁇ agonists (data not shown).
  • concentration EC 50
  • n 16
  • EC 50 l.l ⁇ O.l ⁇ M

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Abstract

L'invention concerne des composés, des compositions et des méthodes de traitement des troubles et affections à médiation de PPARα. L'invention a également trait à la découverte qui a été faite concernant l'affinité endogène élevée de l'oléoyléthanolamide (OEA) et le fait qu'il est un ligand sélectif des PPARα. Les composés selon l'invention comprennent, entre autres, des agonistes spécifiques des PPARα partageant les propriétés de liaison aux récepteurs de l'OEA et des alkanolamides d'acides gras, ainsi que leurs homologues qui sont également des agonistes des PPARα. Ces composés de type OEA comprennent, entre autres, des composés de formule suivante : dans laquelle n est compris entre 0 et 5, la somme de a et de b pouvant être comprise entre 0 et 4 ; Z représente un élément choisi dans le groupe constitué par -C(O)N(R0); (R0)NC(O); OC(O); (O)CO; O; NR0; et S ; et dans laquelle R0 et R2 représentent des éléments choisis indépendamment dans le groupe constitué par alkyle substitué ou non substitué, hydrogène, alkyle C1-C6 et acyle (C1-C6) inférieur, jusqu'à huit atomes d'hydrogène étant facultativement substitués par méthyle ou par une double liaison, la liaison entre les carbones c et d pouvant être saturée ou insaturée. L'invention concerne également des sels pharmaceutiquement acceptables desdits composés.
PCT/US2004/021394 2003-07-02 2004-07-01 Composes, compositions et utilisations therapeutiques de composes de type oleoylethanolamide et de modulateurs des ppar$g(a) Ceased WO2005002524A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7083933B1 (en) 2003-05-09 2006-08-01 Prosidion Limited Methods for identification of modulators of OSGPR116 activity
WO2008042892A3 (fr) * 2006-10-02 2009-02-19 Organon Nv Procédé de traitement de troubles du métabolisme énergétique en empêchant l'activité d'un amide hydrolase d'acide gras
WO2023170552A1 (fr) * 2022-03-07 2023-09-14 Clearmind Medicine Inc. Compositions comprenant du meai et des n-acylethanolamines et leurs utilisations

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5679667A (en) * 1992-04-24 1997-10-21 Lifegroup S.P.A. Aminoalcohols-N-Acyl derivatives as therapeutical agents against the neurogenic endoneural edema of the peripheral nerve
CA2455555A1 (fr) * 2001-07-31 2003-02-13 The Scripps Research Institute Modele animal pour comportements neurologiques associes a l'amide d'acide gras

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7083933B1 (en) 2003-05-09 2006-08-01 Prosidion Limited Methods for identification of modulators of OSGPR116 activity
WO2008042892A3 (fr) * 2006-10-02 2009-02-19 Organon Nv Procédé de traitement de troubles du métabolisme énergétique en empêchant l'activité d'un amide hydrolase d'acide gras
WO2023170552A1 (fr) * 2022-03-07 2023-09-14 Clearmind Medicine Inc. Compositions comprenant du meai et des n-acylethanolamines et leurs utilisations

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