HK1066489A - Methods, compounds, and compositions for reducing body fat and modulating fatty acid metabolism - Google Patents

Description

Methods, compounds and compositions for reducing body fat and modulating fatty acid metabolism

Cross reference to related applications

This application claims priority from U.S. patent application No. 60/336,289 filed on day 10/31 in 2001 and U.S. patent application No. 60/279,542 filed on day 3/27 in 2001. The contents of which are each incorporated herein by reference.

Statement regarding rights to inventions sponsored by federal government for research and development

The invention was carried out with government support under grant No. DA12653 granted by the national institutes of health. The government has certain rights in this invention.

Field of the invention

The present invention relates to fatty acid ethanolamines, their homologs and analogs, and their use as pharmacologically active agents for reducing body fat, reducing food consumption, and modulating lipid metabolism.

Background

Obesity is a worldwide health challenge and has reached a level of vigilance in the united states and other developed countries. Adults of about 97,000,000 in the united states are overweight. Of which 40,000,000 are obese. Obesity and overweight increase the risk of many diseases. Hypertension, type 2 diabetes, dyslipidemia, coronary heart disease, stroke, gallbladder disease, osteoarthritis, sleep apnea, and other respiratory problems as well as endometrial, breast, prostate, and colon cancers are all associated with higher body weight. Higher weight people are also afflicted with higher mortality rates for all causes. According to the national institutes of health, approximately 280,000 adult deaths per year in the united states are attributed in part to obesity.

Weight loss is desirable for obese and overweight people. Weight loss can help prevent many of these consequences, particularly diabetes and cardiovascular disease (CVD). Weight loss can also reduce blood pressure in very hypertensive and non-hypertensive patients, reduce serum triglyceride levels, and increase cholesterol in the beneficial High Density Lipoprotein (HDL) form. Weight loss also typically slightly reduces the levels of total serum cholesterol and Low Density Lipoprotein (LDL) cholesterol. Weight loss can also reduce blood glucose levels in overweight and obese people.

Although weight loss is desirable, it is difficult to achieve. There are many methods of treating overweight and obesity and maintaining weight loss. However, the bounce is severe. Approximately 40% of women and 24% of men are trying to best aggressively lose weight at any given time. These therapeutic approaches include low calorie and low fat diets; increase physical exercise; behavioral therapy aimed at reducing food intake; drug treatment; surgery and combinations of the above.

The relative absence of medication for weight loss. Drugs such as sibutramine, dexfenfluramine, orlistat, norephedrine, phentolamine or fenfluramine promote weight loss in obese adults over an extended period of time. Generally, however, the safety of agents for treating weight loss by long-term administration of drugs is not clear. For example, fenfluramine and dexfenfluramine have recently been withdrawn from the market due to concerns about valvular heart disease found in patients. In the face of invaluable drugs and the high prevalence of obesity and overweight, there is a need for new pharmacological methods and compositions to improve and maintain weight loss.

Fatty Acid Ethanolamines (FAEs) are unusual constituents of animal and vegetable lipids, and their concentration in unactivated cells is generally low. (Bachur et al, J.biol.chem., 240: 1019-1024 (1965); Schmid et al, chem.Phys.lipids, 80: 133-142 (1996); Chapman, K.D., chem.Phys.lipids, 108: 221-229 (2000)). However, FAE biosynthesis can be rapidly increased by a variety of physiological and pathological stimuli, including exposure to fungal pathogens in tobacco cells (Chapman et al, Plant Physiol., 116: 1163-. The mechanism of stimulus-dependent FAE production in mammalian tissues is believed to involve two synergistic responses: membrane phospholipid cleavage, N-acylphosphatidylethanolamine (NAPE), catalyzed by unknown phospholipase D; and NAPE synthesis, catalyzed by N-acyltransferase (NAT) activity controlled by calcium ions and cyclic AMP (Di Marzo et al, Nature, 372: 686-.

The fact that both plant and animal cells release FAEs in a stimulus-dependent manner suggests that these compounds may play a significant role in cellular communication. Further support for this view comes from the discovery that polyunsaturated FAE, anandamide (anandamide) is an endogenous ligand for the cannabinoid (cannabinoid) receptor (Deven et al, Science, 258: 1946-⁹-tetrahydrocannabinol (Δ)⁹-THC) (see, for review, reference (Pertwee, R.G., Exp. Opin. invest. drugs, 9: 1553-.

Two observations made it impossible for other FAEs to also participate in cannabinoid neurotransmission. The FAE family contains a large proportion of saturated and monounsaturated species, such as palmitoylethanolamide and oleoylethanolamide, which have insignificant and cannabinoid receptor effects (Deven et al, Science, 258: 1946-1949 (1992)); griffin et al, j. pharmacol. exp. ther., 292: 886-894.(2000)). Second, when the pharmacological properties of FAEs, such as palmitoylethanolamide, are studied in detail, these properties and Δ are found⁹Those of-THC are different and independent of the activation of the known atypical cannabinol receptor (Calignano et al, Nature, 394: 277-281 (1998)). Thus, the biological significance of FAEs remains elusive.

Oleoylethanolamide (OEA) is a natural analogue of endogenous cannabinoids anandamide. Like anandamide, OEA is produced in hormone-dependent cells and is rapidly eliminated by enzymatic digestion, suggesting a role in cell signaling. However, unlike anandamide, OEA does not activate the cannabinoid receptor, and its biological function is essentially unknown so far.

As well as maintaining weight loss, there is a need for additional methods and agents for treating obesity and overweight. The present invention addresses this need by providing novel methods and pharmaceutical compositions that are related to our present discovery that Oleoylethanolamide (OEA) and other fatty acid ethanolamine compounds (e.g., palmitoylethanolamide, elaidoylethanolamide) are capable of reducing appetite, food intake, body weight and body fat and altering fat metabolism.

Summary of The Invention

The present invention provides compounds, compositions and methods for reducing body fat and treating or preventing obesity and overweight, as well as diseases associated with these health problems in mammals. In one aspect, the invention provides methods for reducing body fat or weight, treating or preventing obesity or overweight and reducing food intake by administering a pharmaceutical composition comprising a fatty acid alkanolamide compound, a homolog or analog thereof, administered in a sufficient dose to reduce body fat, weight or prevent body fat or weight gain. In another aspect, the present invention is directed to the production of fatty acid amine compounds, homologs and analogs thereof, pharmaceutical compositions thereof, and uses of these methods.

In other embodiments, the fatty acid moiety of the fatty acid alkanolamide or ethanolamine compound, homologs, and analogs thereof may be saturated or unsaturated, and if unsaturated, may be mono-or polyunsaturated.

In some embodiments, the fatty acid moiety of the fatty acid alkanolamide or ethanolamine compound, homologs, and analogs thereof is selected from the group consisting of oleic acid, palmitic acid, elaidic acid, unsaturated hexadecene, linoleic acid, alpha-linolenic acid, and gamma-linolenic acid. In particular embodiments, the fatty acid moiety has from 12 to 20 carbon atoms.

Other embodiments are provided by varying the fatty acid moiety of the fatty acid amide compound, homolog or analog thereof. These embodiments include the introduction of saturated or unsaturated lower (C) on the hydroxyl group of an alkanolamide or ethanolamine moiety₁-C₃) Alkyl to form the corresponding lower alkyl ether. In another embodiment, the hydroxyl group and C of the alkanolamide or ethanolamine moiety₂To C₆The carboxyl groups of the substituted or unsubstituted carboxylic acids combine to form the corresponding fatty acid cycloaliphatic groups. Such embodiments include fatty acid alkanolamides and fatty acid ethanolamines and organic carboxylic acid lipidation, such as acetic acid, propionic acid, and butyric acid. In one embodiment, the fatty acid alkanolamide is an oleic acid alkanolamide. In a further embodiment, the fatty acid alkanolamide is oleoylethanolamide.

In yet another embodiment, the fatty acid ethanolamine compound, homologue, or analogue thereof further comprises a substituted or unsubstituted lower (C) covalently bonded to the fatty acid ethanolamine nitrogen atom₁-C₃) An alkyl group.

In another aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound, or a pharmaceutically acceptable salt thereof, having the formula:

in the formula, n is 0 to 5, and the sum of a and b is 0 to 4. Z is selected from the group consisting of-C (O) N (R)⁰)-；-(R⁰)NC(O)-；-OC(O)-；-(O)CO-；O；NR⁰And S, wherein R⁰And R²Independently selected from substituted or unsubstituted alkyl, hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted lower (C)₁-C₆) Acyl, homo-alkyl (homoalkyl) and aryl. Up to four hydrogen atoms of either or both the fatty acid moiety and the ethanolamine moiety of the compound may be substituted with methyl groups or double bonds. In addition, the molecular bond between carbons c and d may be unsaturated or saturated. In some embodiments, fatty acid ethanolamines such as those of the above formula are naturally occurring compounds.

In other aspects of the invention, the methods and compositions employ fatty acid ethanolamine and fatty acid alkanolamide compounds, homologs and analogs thereof to reduce body weight when administered to a test animal (e.g., rat, mouse, rabbit, hamster, guinea pig).

In still other aspects, the present invention contemplates methods of using aryl thiazolidinedione compounds, heteroaryl and arylhydroxyacetic acid type compounds to reduce body fat, weight and appetite.

Other aspects of the invention provide methods for applying and administering compounds and compositions to a subject to reduce body weight or reduce appetite or reduce food intake or cause hypophagia in a mammal (e.g., a human, cat, or dog). The subject compositions may be administered by a variety of routes, including orally.

Brief Description of Drawings

Figure 1 starvation increased circulating levels of oleoylethanolamide in rats: (a) time course of the effect of fasting on plasma oleoylethanolamide (oleoylethanolamide OEA) levels; (b) effect of water deprivation (18 hours) on plasma oleoylethanolamide levels; (c) effect of fasting (18 hours) on oleoylethanolamide levels in cerebrospinal fluid (CSF); (d) time course of the effect of fasting on plasma anandamide (anandamide, AEA) levels; (e) effect of water deprivation (18 hours) on plasma anandamide levels; (f) effect of fasting (18 hours) on anandamide levels in cerebrospinal fluid (CSF). Results are expressed as mean s.e.m.; (. about.), P < 0.05; p < 0.01, n of each group is 10.

FIG. 2 adipose tissue is the main source of circulating oleoylethanolamide: starvation caused changes in acyltransferase (NAT) and Fatty Acid Amide Hydrolase (FAAH) activity in various tissues of rats. (a) Fat (b) brain (c) liver (d) stomach (e) small intestine. Empty fences, freely feeding animals; the pens were filled and the animals fasted for 18 hours. The activity was pmol/mg protein/min. (x), P < 0.05, n ═ 3.

FIG. 3 adipose tissue is the main source of circulating oleoylethanolamide: changes in NAPE and oleoylethanolamide (oleoylethanolamide OEA) caused by starvation were achieved in fat and liver tissues. (a) Oleoylethanolamide precursors have the structure of the base-1-palmitoyl-2-arachidonoyl-sn-glycero-phosphoethanolamine-N-oleyl acid (left panel, NAPE1) and the base-1-palmitoyl-2-arachidonoyl-sn-glycero-phosphoethanolamine-N-oleyl acid (right panel, NAPE 2); (b) NAPE1 (left panel, M/z-987, deprotonated molecule, [ M-H ] in free-fed (top) and 18-hour fasted rats (bottom)]^-) NAPE2 (right panel, M/z 1003, [ M-H ═ M-H)]^-) Representative HPLC/MS traces of characteristic selective ions (c) fasting (18 hours) increased the volume of NAPE material in fat but decreased its volume in the liver. Quantification of all identifiable NAPE substances, including oleoylethanolamide precursors NAPE1 and NAPE2 and PEA precursor NAPE 3; (d) fasting (18 hours) increases the volume of oleoylethanolamide in fat and liver. Empty fences, free-feeding movementsAn agent; the pens were filled and the animals fasted for 18 hours. (. about.), P < 0.05, student's t test; n is 3.

FIG. 4 inhibition of food intake by oleoylethanolamide/pranamide selectivity: (a) dose-dependent effects of oleoylethanolamide (oleoylethanolamide/OEA/pranamide) (e.g. open plot), elaidoylethanolamide (open circle plot), PEA (triangle plot), oleic acid (solid plot) and anandamide (solid circle plot) on food intake of rats fasted for 24-hours. Vehicle alone (70% DMSO in salt, 1ml/kg) had no significant effect on short-term intake of things; (b) time course of the effect of oleoylethanolamide (20mg/kg) (plot) or vehicle (diamond plot) on food intake hypophagic; (c) effect of excipient (V), lithium chloride (LiCl, 0.4M, 7.5ml/kg) or oleoylethanolamide (20mg/kg) in a conditional sample rejection assay. Empty fences, water intake; filling the fence and taking saccharin. The effect of excipient (V) or oleoylethanolamide (5 or 20mg/kg) on: (d) water intake (expressed as ml/4 h); (e) body temperature; (f) ability to jump in a hot plate painless experiment; (g) the percentage time it takes to open the arm in the elevation plus puzzlement anxiety experiment; (h) the number of crossover points in open field activity experiments; (i) the amount of reaction to food motion. P < 0.05, each group n-8-12.

FIG. 5 Effect of subchronic (Subchronic) oleoylethanolamide administration on food intake and body weight: (a) effect of oleoylethanolamide (oleoylethanolamide OEA) (5mg/kg once daily) (empty hurdle) or excipients (5% Tween 80/5% propylene glycol in sterile salt; filled hurdles) on cumulative food intake; (b) time course of the effect of oleoylethanolamide (triangle) or vehicle (square) on body weight change; (c) the time course of the effect of oleoylethanolamide or excipients on net body weight change; (d) effect of Oleoylethanolamine (5mg/kg) or vehicle on cumulative Water intake. (. about.), P < 0.05; p < 0.01, n of each group is 10.

FIG. 6 the effect of peripheral sensory fibers in anorexia induced by oleoylethanolamide. Excipient (V), oleoylethanolamide (oleoylethanolamide/OEA/pranamide) (5mg/kg), CCK-8 (10. mu.g/kg) and CP-93129(1mg/kg), a centrally activated 5-HT_1BStimulation of receptorsAnimal, effect on food intake, a is control rats, c is rats given capsaicin. (b) Control rats and (d) rats given capsaicin ingested water. (. about.), P < 0.05; each group has n-8-12.

FIG. 7 oleoylethanolamide/pranamide increased the expression of c-fos mRNA in discrete brain sites associated with energy homeostasis and feeding behavior: (a) pseudo-color images of autoradiographs show that oleoylethanolamide (right panel) causes a significant and selective increase in c-fos mRNA labeling in the Paraventricular (PVN) and Supraoptic (SO) hypothalamic nuclei, as assessed by in situ hybridization. A representative portion from vehicle-treated rats is shown on the left. The density of the mark is shown by color: blue < green < yellow < red. (b) Quantification of c-fos cRNA markers in the forebrain regions [ PVN, SO, Arch (Arc), layer II piriform cortex (pir), ventral lateral thalamus (VI) and S1 anterior branch layer (S1FL) ] of rats treated with vehicle, oleoylethanolamide, oleic acid (c) autoradiographs showing increased expression of 35Sc-fos mRNA in the solitary nucleus pulposus (NST) of oleoylethanolamide-treated rats; the insertion, the c-fos cRNA marker in NST (shown as red) was confirmed by its localization and the relative proximity of the efferent nuclei (hypoglossal and dorsal nuclei of the vagus nerve), which express the enzyme acetylcholine transferase mRNA (chat) (shown as purple) (d) oleoylethanolamide increased the expression of c-fos mRNA in NST but not in hypoglossal nuclei (HgN). P < 0.0001, n of each group is 5.

FIG. 8 Effect of OEA, Oleic Acid (OA), AEA, PEA and methyl-OEA on fatty acid oxidation in soleus muscle.

Detailed Description

The present invention relates to the surprising discovery that OEA and other fatty acid alkanolamide compounds reduce food intake, body weight and body fat and modulate fatty acid oxidation. It has been surprisingly found that Oleoylethanolamide (OEA), a natural lipid for which the biological function is not known so far in mammals, is an effective body fat reducing and weight controlling compound when administered to experimental animals. U.S. patent application 60/279,542, filed 3/27/2001, assigned to the same assignee and incorporated herein by reference in its entirety, discloses that OEA and OEA-like compound agents reduce body fat and appetite in mammals.

In addition to the OEA found as the prototype, other fatty acid alkanolamide compounds and homologs have also been found to be active.

OEA can be used as a model for the development of other fatty acid alkanolamide-like compounds for use in reducing fat in the treatment of obesity, including weight loss, reducing appetite or food intake. The present invention provides such other compounds as described below.

OEA administration was found to reduce appetite, food intake and body weight, and this finding could be used to identify other fatty acid ethanolamine compounds, homologs or analogs thereof as agents for weight and appetite control. The present invention provides such a medicament.

Definition of

Abbreviations used herein have their ordinary meaning in chemical and biological articles.

Substituents are distinguished by their general chemical formula, written left to right, and they likewise contain chemically identical substituents, written right to left, of the structure, e.g. -CH₂O-can also be expressed as-OCH₂-。

The term "composition", such as a pharmaceutical composition, is intended to encompass a product comprising the active ingredient and the inert ingredient as a carrier, as in any product, formed by the combination, complexation or polymerization of any two or more of the ingredients, directly or indirectly, or formed by the decomposition of one or more of the ingredients, or by other reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention include any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier. The term "pharmaceutical composition" means a compound, including an animal or human, suitable for use as a medicament by a subject. A pharmaceutical composition generally comprises an effective amount of an active agent and a pharmaceutically acceptable carrier.

The compounds of the present invention contain one or more asymmetric centers and thus give rise to racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. The present invention is meant to encompass all such isomeric forms of the compounds of the invention.

The compounds described herein contain olefinic double bonds and are meant to include both E and Z geometric isomers unless otherwise indicated.

Some of the compounds described herein may bind hydrogen atoms at different sites, due to tautomers. Such an example may be a ketone and its enol form, known as keto-enol tautomers. The formula of the present invention also includes individual tautomers and mixtures thereof.

The compounds of the present invention include diastereomers of the enantiomers. Diastereomers, for example, methanol or ethyl acetate or mixtures thereof. The pair of enantiomers thus obtained may be resolved into the individual stereoisomers by conventional methods, for example using optically active acids as solvents.

Alternatively, enantiomers of any of the compounds of the invention may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known structure.

As used herein, the term "heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).

As used herein, "alkanol" refers to a saturated or unsaturated, substituted or unsubstituted, branched or unbranched alkyl group having a hydroxyl substituent or substituents derived from hydroxyl moieties, e.g., ethers, esters. The alkanol is also more suitably substituted with a nitrogen, sulphur or oxygen-position substituent, which is contained in the Z bond (formula I), between the "fatty acid" and the alkanol.

As used herein, "fatty acid" refers to a saturated or unsaturated, substituted or unsubstituted, branched or unbranched alkyl group, which may have a carboxyl substituent. The desired fatty acid is C₄-C₂₂And (4) acid. Fatty acids also include the class of carboxyl substituents substituted by-CH₂-partial substitution.

The term "alkyl" by itself or as part of another substituent, unless otherwise specified, is a straight or branched chain, or cyclic alkyl or compound thereof, which may be fully saturated, mono-or polyunsaturated, and may contain 2-and polyvalent groups, having the indicated number of carbon atoms. (e.g. C)₁-C₁₀Meaning 1 to 10 carbon atoms). Examples of saturated alkyl groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, homologs and isomers thereof, e.g., n-pentyl, n-hexyl, n-heptyl, n-octyl and the like. Unsaturated alkyl groups contain one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, ethenyl, 2-propenyl, butenyl, 2-isopentenyl, 2- (butadienyl), 2, 4-pentadienyl, 3- (1, 4-pentadienyl), ethynyl, 1-and 3-propynyl, 3-butynyl, and higher homologs and isomers. The term "alkyl" unless otherwise indicated, also includes those derivatives of alkyl as specifically defined below, such as "heteroalkyl". Alkyl groups are limited to hydrocarbon groups and are called "homoalkyl".

The term "alkylene" by itself or as part of another substituent refers to a divalent radical derived from an alkane, such as, but not limited to, -CH₂CH₂CH₂CH₂Further included are those groups described below as "heteroalkylene". Typically, an alkyl (or alkylene) group will contain 1 to 24 carbon atoms, and it is desirable in the present invention for those groups to contain 10 or fewer carbon atoms. ' Low priceAlkyl "or" lower alkylene "is a shorter chain alkyl or alkylene group, typically containing 8 or fewer carbon atoms.

The terms "alkoxy", "alkylamino" and "alkylthio" (thioalkoxy) are used in their customary sense and refer to those alkyl groups which are bonded to the molecular residue via an oxygen atom, an amino group or a sulfur atom, respectively.

The term "heteroalkyl," by itself or with other terms, unless otherwise stated, refers to a stable straight or branched chain, or cycloalkyl group, or compound thereof, containing the stated number of carbon atoms and at least one heteroatom selected from O, N, Si and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. The heteroatoms O, N, Si and S may be present anywhere within the heteroalkyl group or at the site of attachment of the alkyl group to the molecular residue. Examples include, but are not limited to, -CH₂-CH₂-O-CH₃，-CH₂-CH₂-NH-CH₃，-CH₂-CH₂-N(CH₃)-CH₃，-CH₂-S-CH₂-CH₃，-CH₂-CH₂，-S(O)-CH₃，-CH₂-CH₂-S(O)₂-CH₃，-CH＝CH-O-CH₃，-Si(CH₃)₃，-CH₂-CH＝N-OCH₃and-CH ═ CH-N (CH)₃)-CH₃. The two hetero atoms may be consecutive, e.g. -CH₂-NH-OCH₃and-CH₂-O-Si(CH₃)₃. Similarly, the term "heteroalkylene" by itself or as part of another substituent refers to a divalent radical derived from a heteroalkyl group, such as, but not limited to, -CH₂-CH₂-S-CH₂-CH₂-and-CH₂-S-CH₂-CH₂-NH-CH₂-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Further, for alkylene and heteroalkylene linked groups, the orientation of the linked group does not include the formula in which it is writtenAnd (3) direction. For example, of the formula-C (O)₂R' -simultaneously represents-C (O)₂R '-and-R' C (O)₂-。

The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent "alkyl" and "heteroalkyl" cyclic forms, respectively, unless otherwise indicated. In addition, for heterocycloalkyl, a heteroatom may occupy the site of attachment of the heterocycle to the molecular residue. Examples of cycloalkyl groups include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1- (1, 2, 5, 6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-alkyl, tetrahydrofuran-3-alkyl, tetrahydrothiophen-2-alkyl, tetrahydrothiophen-3-alkyl, 1-piperazinyl, 2-piperazinyl, and the like.

The term "halo" or "halogen", by itself or as part of another substituent, unless otherwise specified, refers to a fluorine, chlorine, bromine, iodine atom. In addition, terms such as "halogenated hydrocarbon" include monohalogenated hydrocarbons and polyhalogenated hydrocarbons. For example, the term "halogenated hydrocarbon (C)₁-C₄) "includes, but is not limited to, trifluoromethane, 2, 2, 2-trifluoroethane, 4-chlorobutane, 3-bromopropane, and the like.

Unless otherwise indicated, the term "aryl" refers to polyunsaturated, aromatic hydrocarbon substituents, which can be monocyclic or polycyclic (desirably 1 to 3 rings), fused together or covalently bonded. The term "heteroaryl" means that the aryl group contains 1 to 4 heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. Heteroaryl groups may be bonded to the molecular residue through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalyl, 5-quinoxalyl, 3-quinolyl and 6-quinolyl. The substituents for the aryl and heteroaryl ring systems noted above are selected from the group of acceptable substituents described below.

For simplicity, the term "aryl" encompasses aryl and heteroaryl rings as defined above. Thus, the term "aralkyl" is meant to include those groups in which an aryl group is combined with an alkyl group (e.g., benzyl, phenethyl, picolyl, and the like) including those groups in which a carbon atom (e.g., methylene) in the alkyl group is replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3- (1-naphthyloxy) propyl, and the like).

Each of the above terms (e.g., "alkyl," "heteroalkyl," "aryl," "heteroaryl") is meant to include both substituted and unsubstituted forms of the recited group. The ideal substituents for each group are provided below.

Alkyl, heteroalkyl substituents (including those often mentioned such as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl) may be one or more groups selected from, but not limited to: -OR ', -O, ═ NR ', -N-OR ', -NR ' R ", -SR ', -halogen, -SiR ' R '" -oc (O) R ', -c (O) R ', -CO₂R’、-CONR’R”、-OC(O)NR’R”、-NR”C(O)R’、-NR’-C(O)NR”R*、-NRC(O)₂R’、-NR-C(NR’R”R*)＝NR””、-NR-C(NR’R”)＝NR*、-S(O)R’、-S(O)₂R’、-S(O)₂NR’R”、-NRSO₂R’、-CN、-NO₂The number may be from O- (2m '+ 1) where m' is the total number of carbon atoms in such a group. R ', R ", R'", R "" each preferably denote hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroaryl, such as aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy or aralkyl. If the compounds of the present invention contain more than one R group, each R group is chosen independently, e.g., more than one of these groups is selected to provide the R ', R ", R'", R "" alternative. When R' and R "are bonded to the same nitrogen atom, they may be bonded to the nitrogen atom to form a 5-, 6-or 7-atom ring. For example, -NR' R "refers to, but is not limited to, 1-pyrrolyl and 4-morpholinyl. From the above discussion of substituents, one of skill in this article would mean that the term "alkyl" refers to those groups having carbon atoms bonded thereto rather than hydrogen atoms such as halogenated hydrocarbons (e.g., -CF)₃and-CH₂CF₃) And acyl groups (e.g., -C (O) CH)₃，-C(O)CF₃，-C(O)CH₂OCH₃Etc.).

Similar to the alkyl substituents described, the aryl and heteroaryl substituents are also varied and are selected from, for example: -halogen, -OR ', - (O), - (NR ') R ", -SR ', halogen, -SiR ' R '", -oc (O) R ', -c (O) R ', -CO₂R’、-CONR’R”、-OC(O)NR’R”、-NR”C(O)R’、-NR’-C(O)NR”R*、-NRC(O)₂R’、-NR-C(NR’R”R*)＝NR””、-NR-C(NR’R”)＝NR*、-S(O)R’、-S(O)₂R’、-S(O)₂NR’R”、-NRSO₂R’、-CN、-NO₂、-R’、-N₃、-CH(Ph)₂Fluorine (C)₁-C₄) Alkoxy and fluorine (C)₁-C₄) Alkyl, a number varying from 0 to all open valences on the aromatic ring system; wherein R ', R ", R'" and R "" are preferably independently selected from hydrogen, (C)₁-C₈) Alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl) - (C)₁-C₄) Alkyl and (unsubstituted aryl) oxy- (C)₁-C₄) An alkyl group. When the compounds of the invention contain multiple R groups, each R group is selected individually as more than one of these groups present R ', R ", R'", R "".

The term "body fat reduction" refers to subtracting a portion of body fat.

The formula (BMI) for calculating the body mass index is [ weight (pounds)/height (inches) ] × 703. The BMI is a fixed number for adult discrimination points, regardless of age and gender, using the following rules: the BMI of an overweight adult individual is between 25.0 and 29.9. The BMI of obese adults is 30.0 or higher. The BMI of a lighter adult is less than 18.5. The BMI for adults in the normal weight range is between 18.5 and 25. The BMI differentiation points for children under 16 years old are defined as a percentage: overweight is when the BMI is greater than 85% of the same age, and obesity is when the BMI is greater than 95% of the same age. A lighter body weight is a BMI of less than 5% at the same age. BMI in children in the normal weight range is greater than 5% and less than 85%.

The term "fatty acid oxidation" relates to the conversion of fatty acids (e.g., oleates) to ketone bodies.

The term "hepatocyte" refers to the original cells produced from liver tissue. Hepatocytes may be freshly isolated from liver tissue or cell cords established.

The term "modulate" refers to the induction of any change, including an increase or decrease (e.g., a modulator of fatty acid oxidation increases or decreases the rate of fatty acid oxidation).

The term "muscle cell" refers to a cell derived from a muscle tissue host cell. Muscle cells can be freshly isolated from muscle tissue or cell cords established.

The term "obesity" means that body weight exceeds 20% of theoretical body weight as determined by body mass index.

Oleoylethanolamide (OEA) refers to a natural lipid of the following structure:

in the formula, "Me" represents a methyl group.

The term "weight loss" refers to the subtraction of a portion of body weight.

The term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, buffer, excipient, including phosphate buffered saline, water and emulsions (e.g., oil/water or water/oil emulsions), 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). The ideal pharmaceutical carrier depends on the mode of administration of the active agent. Characteristic modes of administration are described below.

The term "effective amount" means a dosage sufficient to produce the desired result. The desired result includes an objective or subjective improvement by the subject. The objective improvement may be a decrease in appetite or the desire to crave food. The subjective improvement may be a reduction in body weight, body fat or food or a reduction in food consumption or a reduction in foraging behavior.

"prophylactic treatment" is a treatment administered to a subject who exhibits no signs of disease or only early signs of disease, in order to reduce the risk of developing disease associated with increased body weight or body fat. The compounds of the invention may be used as prophylactic treatments to prevent undesirable or unwanted weight gain.

"therapeutic treatment" refers to a treatment administered to a subject exhibiting pathological signs, wherein the purpose of the treatment is to reduce or eliminate those pathological signs.

The term "controlling body weight" encompasses a reduction in body weight or a reduction in body weight gain over a period of time.

The methods, compounds and compositions of the present invention are generally effective for reducing or controlling body fat and body weight in a mammal. For example, the methods, compounds and compositions of the invention are useful for reducing appetite or hypophagia in a mammal. The methods, compounds and compositions of the present invention also help prevent or reduce diseases associated with overweight or obesity by increasing weight and body fat reduction.

The methods, compounds and compositions of the present invention include modulators of lipid metabolism, particularly fat and fatty acid metabolism.

Compounds of the invention

Certain compounds of the invention will have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers and individual isomers are all included within the scope of the present invention.

Such compounds of the invention will be separated into diastereomeric pair enantiomers by partial recrystallization from a suitable solvent which may be methanol or ethyl acetate or mixtures thereof. The pair of enantiomers thus obtained may be resolved into stereoisomers by conventional methods, for example using an optional active acid as solvent.

Alternatively, enantiomers of the compounds of the invention may be obtained by stereospecific synthesis using pure starting materials, optionally of unknown structure.

The compounds of the present invention have an abnormal proportion of atomic isotopes at one or more of their atoms. For example, the compound may be labelled with an isotopically radioactive label, such as tritium or C-14. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

The instant compounds may be isolated in the form of their pharmaceutically acceptable acid additive salts, such as salts derived from the use of inorganic or organic acids. Such acids include hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, formic acid, acetic acid, trifluoroacetic acid, propionic acid, maleic acid, succinic acid, malonic acid, and the like. In addition, the specific compounds containing an acid function may be present in the form of their inorganic salts, wherein the counterions may be selected from sodium, potassium, lithium, calcium, magnesium and others, and may also be derived from organic bases. The term "pharmaceutically acceptable salt" refers to salts formulated from pharmaceutically acceptable non-toxic bases or acids, including inorganic bases or acids and organic bases or acids.

Prodrugs of the compounds, which if administered, undergo chemical transformation by metabolic processes prior to becoming the active drug, are also encompassed by the present invention. In general, such prodrugs are derivatives of the compounds which are stably converted in vivo into the effective compounds of the present invention. The conventional procedure for selecting and configuring suitable prodrug derivatives is described, for example, in "Design of produgs", h. The invention also encompasses active metabolites of this compound.

A fatty acid alkanolamide compounds, homologs, and analogs

The compounds of the present invention include body fat reducing fatty acid alkanolamide compounds, including fatty acid ethanolamine compounds, as well as homologs thereof and specific analogs of fatty acid alkanolamides. Such compounds are identified and defined by exhibiting the ability to reduce appetite, food intake, and or reduce body weight or body fat by administration to a living experimental animal.

Such diversity of fatty acid alkanolamide compounds, homologs, and analogs is contemplated. The compounds of the present invention have the following general formula:

in this formula, n is 0 to 5 and the sum of a and b is 0 to 4. Z is selected from the group consisting of-C (O) N (R)⁰)-；-(R⁰)NC(O)-；-OC(O)-；-(O)CO-；O；NR⁰And S, wherein R⁰And R²Independently selected from substituted or unsubstituted alkyl, hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted lower (C)₁-C₆) Acyl, homo-alkyl and aryl. Up to four hydrogen atoms of either or both the fatty acid moiety and the alkanolamide (e.g., ethanolamine) moiety of the compound may also be substituted with methyl groups or double bonds. In addition, the molecular bond between carbons c and d may be unsaturated or saturated. In some embodiments, fatty acid ethanolamines such as those of the above formula are naturally occurring compounds.

The invention also includes compounds of the formula:

in one embodiment, n of the compound of formula Ia is 0 to 5, and the sum of a and b is 0 to 4; wherein R is¹And R²Independently selected from hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted lower (C)₁-C₆) Acyl, homo-alkyl and substituted or unsubstituted aryl. In this embodiment, up to four hydrogen atoms of the fatty acid moiety and alkanolamide (e.g., ethanolamine) moiety of the compound may be substituted with methyl or double bonds. In addition, the molecular bond between carbons c and d may be unsaturated or saturated. In some embodiments where there is an acyl group, the acyl group may be propionic acid, acetic acid or butyric acid and is attached by esterification as R²Or acylated with a linkage such as R¹。

In another embodiment, the above compounds contain characteristic fatty acid moieties such as oleic acid, elaidic acid, palmitic acid. Such compounds include oleoylethanolamide, elaidoylethanolamide, palmitoylethanolamide.

Oleoylethanolamide

In another embodiment, the compound of formula Ia is n is 1 to 3, the sum of a and b is 1 to 3; wherein R is¹And R²Independently selected from hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted lower (C)₁-C₆) An acyl group. In this embodiment, up to four hydrogen atoms of the fatty acid moiety and alkanolamide (e.g., ethanolamine) moiety of the compounds of the above formula may also be substituted with methyl or double bonds. In addition, the molecular bond between carbon c and d may beUnsaturated or saturated. In a further embodiment, the molecular bond between carbons c and d may be unsaturated and no other hydrogen atoms are substituted. In still further embodiments thereof, R¹And R²Independently selected from hydrogen, substituted or unsubstituted C₁-C₃Alkyl, substituted or unsubstituted lower (C)₁-C₃) An acyl group.

Typical compounds provide monomethyl substituted compounds, including ethanolamines of formula Ia. Such compounds include:

(R) 1' -methyl group

(S) 1' -methyl group

(R) 2' -methyl group

(S) 2' -methyl group

(R) 1-methyl group

(S) 1-methyl group

Methyl-substituted compounds of the above formula characteristically include those compounds R¹And R²Are all H: (R)1 '-methylfatty acid ethanolamine, (S) 1' -methylfatty acid ethanolamine, (R)2 '-methylfatty acid ethanolamine, (S) 2' -methylfatty acid ethanolamine, (R) 1-methylfatty acid ethanolamine, (S) 1-methylfatty acid ethanolamine.

Trans OEA-type compound

The compounds of the present invention also include various analogs of OEA. These compounds include trans-OEA compounds of the general formula:

in some embodiments, the present invention provides compounds of formula II. Typical compounds of formula II have n from 1 to 5 and the sum of a and b is from 0 to 4. In such embodiments, R²Selected from hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted lower (C)₁-C₆) Acyl, homo-alkyl and aryl. In addition, up to four hydrogen atoms of the fatty acid moiety and alkanolamide (e.g., ethanolamine) moiety of the compounds of the above formula may be substituted with methyl or double bonds.

Typical compounds of formula II include those compounds whose alkanolamide position is ethanolamine, R²Is H, a and b are both 1, and n is 1.

One embodiment of the compounds of formula II is

Trans OEA

In another embodiment, the compound of formula II has n from 1 to 5 and the sum of a and b is from 1 to 3. In this embodiment, R²Selected from hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted lower (C)₁-C₆) An acyl group. In addition, up to four hydrogen atoms of either or both the fatty acid moiety and the alkanolamide (e.g., ethanolamine) moiety of the compounds of the above formula may be substituted with methyl groups or double bonds.

Oleic acid alkanol ester compound

The compounds of the present invention also include alkanol oleates having the general formula:

in some embodiments, n of the compound of formula III is 1 to 5 and the sum of a and b is 0 to 4. R²Selected from hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted lower (C)₁-C₆) Acyl, homo-alkyl and aryl. Up to four hydrogen atoms of either or both of the fatty acid moiety and the alkanol (e.g., ethanol) moiety of the compounds of the above formula may be substituted with methyl or double bonds.

In some embodiments, n of the compound of formula III is 1 to 3, and the sum of a and b is 1 to 3. R²Selected from hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted lower (C)₁-C₆) An acyl group. The fatty acid moiety and alkanol (e.g., ethanol) moiety of the compounds of the above formula have up to four hydrogen atoms that may be substituted with methyl or double bonds.

The compounds of formula III include those compounds, their R²Is hydrogen, a and b are both 1, and n is 1. Examples of compounds of formula III include ethyl oleate:

the compounds of formula III also comprise monomethyl-substituted ethyl oleate, such as (R or S) -2' -methyl ethyl oleate; (R or S) -1' -methyl oleic acid ethyl ester; (R or S)) -1' -methyl oleic acid ethyl ester; the method comprises the following steps:

oleic acid alkyl alcohol ether

The compounds of the present invention also include oleic acid alkyl alcohol ethers, of the general formula:

in some embodiments, n of the compound of formula IV is 1 to 5 and the sum of a and b is 0 to 4. R²Selected from hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted lower (C)₁-C₆) Acyl, alkyl and substituted or unsubstituted aryl. Up to four hydrogen atoms of either or both of the fatty acid moiety and the alkanol (e.g., ethanol) moiety of the compounds of the above formula may be substituted with methyl or double bonds.

In a further embodiment, n of the compound of formula IV is 1 to 3 and the sum of a and b is 1 to 3. R²Selected from hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted lower (C)₁-C₆) An acyl group. Up to four hydrogen atoms of either or both of the fatty acid moiety and the alkanol (e.g., ethanol) moiety of the compounds of the above formula may be substituted with methyl or double bonds.

The compounds of formula IV include those compounds, their R²Is hydrogen, a and b are both 1, and n is 1. Examples of compounds of formula IV include the following (R or S) -1 '-oleic acid ethanol esters and (R or S) -2' -oleic acid ethanol esters:

fatty acid alkanolamide analogs with polar head modifications

The compounds of the present invention also include various OEA polar head analogs. These compounds include compounds containing a fatty acid moiety, having the general formula:

in some embodiments, the sum of compounds a and b of formula IV is 0 to 4. In a further embodiment, the sum of a and b is 1 to 3. In these embodiments, up to four hydrogen atoms of the compounds of the above formula may be substituted with methyl or double bonds. In addition, the molecular bond between carbons c and d may be unsaturated or saturated. A particularly preferred embodiment is an oleic acid fatty acid moiety:

r of the above structure³The group may be selected from any one of the following:

HO-(CH₂)_z-NH-wherein z is from 1 to 5, the alkyl moiety of which is an unbranched methylene chain. For example:

H₂N-(CH₂)_z-NH-wherein z is from 1 to 5, the alkyl moiety of which is an unbranched methylene chain. For example:

HO-(CH₂)_x-NH-wherein x is from 1 to 8, the alkyl portion of which is branched or cyclic. For example:

other R³Polar head groups include, for example, compounds having furan, dihydrofuran, tetrahydrofuran functional groups:

in the above structure, z may be 1 to 5.

Compounds of the present invention include, for example, those having R³Polar head group, based on pyrrole (pyrole), pyrrolidine and pyrrole rings:

in the compounds of the above structure, z may be 1-5.

Other typical polar head groups include various imidazoles and oxazoles, such as:

in the compounds of the above structure, z may be 1-5.

Azole-pyrimidine polar head groups are also typical:

fatty acid alkanolamides with no polar tail changes

The compounds of the present invention include a variety of alkanolamide and ethanolamine compounds, all of which have a variety of flexible, non-polar tails. These compounds include those of the formula wherein R represents an ethanolamine moiety, an alkanolamide moiety or a stable analog thereof. In the case of ethanolamine, the ethanolamine moiety is preferably bonded via its amino group rather than oxygen.

In the above structures, m is 1 to 9 and p alone is 1 to 5.

Typical compounds are:

another typical compound is an ethanolamine analog with a nonpolar tail of the formula:

typical compounds include analogs of fatty acid alkanolamides. Such analogs include those compounds taught in U.S. patent No. 6,200,998 (incorporated herein by reference). This reference teaches the general formula of the compounds:

in the above formula, Ar is as set forth in U.S. Pat. No. 6,200,998¹Is (1) arylene or (2) heteroaryl, wherein phenylene and heteroarylene are substituted from R^aOptionally substituted with 1-4 groups selected from (a); ar (Ar)²Is (1) n-substituted aryl or (2) n-substituted heteroaryl, wherein the n-substituent is selected from R; both aryl and heteroaryl are further optionally independently selected from R^a1-4 groups of (A). X and Y are each independently O, S, N-R^bOr CH₂(ii) a Z is O or S; n is 0 to 3; r is (1) C optionally substituted by 1-4 groups selected from halogen_3-10Alkyl and C_3-6Cycloalkyl, (2) C_3-10Alkenyl or (3) C_3-8A cycloalkenyl group; r^aIs (1) C_1-15Alkanol group (2) C_1-15Alkyl (3) C_2-15Alkenyl (4) C_2-15Alkynyl (5) halo (6) OR^b(7) Aryl or (8) heteroaryl, wherein said alkyl, alkenyl, alkynyl, alkanol group is selected from R^cOptionally substituted by 1 to 5 groups selected from R^dOptionally substituted with 1 to 5 groups of (a); r^bIs (1) hydrogen (2) C_1-10Alkyl (3) C_2-10Alkenyl (4) C_2-10Alkynyl (5) aryl (6) heteroaryl (7) alkyl C_1-15Heteroalkyl (8) heteroaryl C_1-15Alkyl (9) C_1-15Alkanol group (10) C_3-8Cycloalkyl wherein alkyl, alkenyl, alkynyl are all independently selected from R^cOptionally substituted with 1-4 groups of (A), cycloalkyl, aryl, heteroaryl are all independently selected from R^dOptionally substituted with 1 to 4 groups of (a); or R^cIs (1) halogen, (2) aryl, (3) heteroaryl, (4) CN, (5) NO₂，(6)OR^f，(7)S(O)_mR^fM is 0, 1 or 2 if R^fM is 1 or 2 when not H, (8) NR^fR^f，(9)NR^fCOR^f，(10)NR^fCO₂R^f，(11)NR^fCON(R^f)₂，(12)NR^fSO₂R^fIf R is^fNot H, (13) COR^f，(14)CO₂R^f，(15)CON(R^f)₂，(16)SO₂N(R^f)₂，(17)OCON(R^f)₂Or (18) C_3-8Cycloalkyl, wherein said cycloalkyl, aryl, heteroaryl is substituted by 1-3 halogen radicals or C_1-6Alkyl is optionally substituted; r^dIs (1) from R^cA group selected from (2) C_1-10Alkyl group, (3) C_2-10Alkenyl, (4) C_2-10Alkynyl, (5) aryl C_1-10Alkyl or (6) heteroaryl C_1-10Alkyl, wherein alkyl, alkenyl, alkynyl, aryl, heteroaryl are all independently selected from R^eOptionally substituted with; r^eIs (1) halogen, (2) amino, (3) carboxyl, (4) C_1-4Alkyl, (5) C_1-4Alkoxy, (6) hydroxy, (7) aryl, (8) aryl C_1-4Alkyl or (9) aryloxy;R^fis (1) hydrogen, (2) C_1-10Alkyl group, (3) C_2-10Alkenyl, (4) C_2-10Alkynyl, (5) aryl, (6) heteroaryl, (7) alkyl C_1-15Heteroalkyl group, (8) heteroaryl C_1-15Alkyl, (9) C_1-15Alkanol group, (10) C_3-8Cycloalkyl wherein alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkanol, cycloalkyl are all selected from R^eOptionally substituted with 1-4 groups.

The analogs taught in U.S. patent No. 5,859,051 are also preferred. These analogs have the following general formula:

in the embodiment of formula VII, R is as set forth in U.S. Pat. No. 5,859,051¹Is selected from H, C_1-6Alkyl radical, C_5-10Aryl and C_5-10Heteroaryl, said alkyl, aryl or heteroaryl being optionally substituted with 1-3R^aGroup substitution; r¹Selected from H, C_1-15Alkyl radical, C_2-15Alkenyl radical, C_2-15Alkynyl, C_3-10Cycloalkyl, said alkyl, alkenyl, alkynyl, cycloalkyl optionally substituted with 1-3R^aGroup substitution; r³Selected from H, NHR¹NH aryl, C_1-15Alkyl radical, C_3-10Cycloalkyl radical, C_2-15Alkenyl radical, C_1-15Alkoxy, CO₂Alkyl, OH, C_2-15Alkynyl, C_5-10Aryl radical, C_5-10Heteroaryl, said alkyl, cycloalkyl, alkenyl, alkynyl, aryl and heteroaryl optionally substituted with 1-3R^aGroup substitution; (Z-W-) is Z-CR⁶R⁷-, Z-CH ═ CH-, or:

R⁸selected from the group consisting of CR⁶R⁷，O，NR⁶And S (O)_P；R⁶And R⁷Independently selected from H, C_1-6An alkyl group; b is selected from: 1) the 5 and 6 membered heterocyclic ring contains 0-2 double bonds, 1 heteroatom selected from O, S, N, the heteroatom being substituted at any position of the 5 or 6 membered heterocyclic ring, the heterocyclic ring being optionally substituted with 1-3R^aSubstituted or unsubstituted; 2) the 5 and 6 membered carbocycles contain 0-2 double bonds, the carbocycles optionally being substituted with 1-3R at any position of the 5 and 6 membered carbocycles^aSubstituted or unsubstituted; 3) the 5 and 6 membered heterocyclic ring contains 0-2 double bonds, 3 heteroatoms selected from O, S, N, substituted at any position of the 5 or 6 membered heterocyclic ring, the heterocyclic ring optionally substituted with 1-3R^aSubstituted or unsubstituted; x¹And X²Independently selected from: H. OH, C_1-15Alkyl radical, C_2-15Alkenyl radical, C_2-15Alkynyl, halogen, OR³、ORCF₃、C_5-10Aryl radical, C_5-10Aralkyl radical, C_5-10Heteroaryl and C_1-10Aryl, said alkyl, alkenyl, alkynyl, aryl and heteroaryl being optionally substituted with 1-3R^aGroup substitution; r^aRepresents a constituent element selected from the group consisting of halogen, acyl, aryl, heteroaryl, CF₃、OCF₃、-O-、CN、CO₂、R³、OR³；SR³、＝N(OR)、S(O)R³、SO₂R³、COR³、CO₂R³、CON(R³)₂、SO₂N(R³)₂、OCON(R³)₂Said aryl and heteroaryl being optionally substituted by 1-3 halogens or C_1-6Alkyl substitution; y is selected from S (O)_P、-CH₂-、-C(O)-、-C(O)NH-、-NR-、-O-、-SO₂NH-、-NHSO₂；Y¹Selected from O and C; z is selected from CO₂R³、R³CO₂R³、CONHSO₂Me、CONHSO₂、CONH₂And 5- (1H-tetrazole); t and v are each 0 or 1, such that t + v ═ 1Q is a linear saturated or unsaturated hydrocarbon containing 2 to 4 carbon atoms, p is 0 to 2, with the proviso that Z is CO₂R³And B is a 5-membered heterocycle containing O, R³And does not represent a methyl group.

Additional analogs suitable for practicing the methods and compositions of the invention include the compounds taught in U.S. patent nos. 5,847,008, 6,090,836, and 6,090,839. All incorporated herein by reference in their entirety to the extent they do not conflict with the present invention.

A number of other suitable analogues are taught in us patent No. 6,274,608. Aryl and heteroaryl acetic acids and glycolic acid analogs are taught, for example, in U.S. patent No. 6,160,000; substituted 5-aryl-2, 4-thiazolidinedione analogues are taught in U.S. patent No. 6,200,998; other possible analogs such as polyunsaturated fatty acids and eicosanoic acids are known (see, e.g., formin, BM, Chen, J, and EvansRM, PNAS) 94: 4312-4317. These publications are incorporated by reference in their entirety to the extent that they do not conflict with the present invention, and their compounds can be screened by the following methods to provide, for example, compounds useful for reducing body fat and weight, regulating fat metabolism, and reducing appetite in accordance with the present invention.

Synthesis of fatty acid alkanolamides

Compounds useful in the practice of the present invention are stably synthesized and purified using the methods recognized herein. In a typical synthetic scheme (scheme 1), a carboxylic acid and an ethanolamine (or an O-protected derivative thereof) are reacted in the presence of a dehydrating agent, such as dicyclohexylcarbodiimide in a suitable solvent. The fatty acid alkanolamides are isolated by, for example, extraction, recrystallization, precipitation, chromatography, and the like. If the final product is an O-protected adduct, it is typically deprotected by methods known in the art to provide a fatty acid adduct having free hydroxyl groups.

Those skilled in the art will recognize that many variations of the above illustrations are useful. For example, an activated derivative, such as halogenated aryl and activated ester of an acid may be used. Similarly, ethylene glycol (preferably mono-O-protected) may be substituted for aminoethanol, resulting in an ester linkage between the two molecular components.

Trans esters and trans amides are also stably synthesized by known methods. For example, a hydroxycarboxylic acid is reacted with a long chain alkyl group (e.g., C) in the presence of a dehydrating agent₄-C₂₂) Amine or hydroxyl derivatives. In certain reaction pathways, it is desirable to protect the hydroxyl moiety of the hydroxycarboxylic acid.

The ethers and thiols are formulated by methods well known to those skilled in the art, for example, Williamson synthesis. For example, a long chain alkyl alcohol or thiol is deprotonated by a base, such as NaH, reactive alcohol derivatives, such as halogens, tosyl, mesylethanols or their protected derivatives, and the resulting anion reacts to form an ether or thiol.

The method described above and variations thereof can be found IN the following text as RECENT DEVELOPMENT IN THESYYNESIS OF FATTY ACID DERIVATIVES, Knothe G eds, Amer. oil Chemists Society 1999; complex machinery summary CHEMISTRY AND OTHER SECONDARYMETABOLITES INCLUDING FATTY ACIDS AND THEIR DERIVATIVES, Nakanishi K, Pergamon Press, 1999; ORGANIC SYNTHSIS COLLECTED VOLUMES I-V, John Wiley and Sons; (ii) COMPENDIUM OF ORGANIC SYNTHETIC METHODS, Vol.1-6, Wiley, Interscience, 1984; ORGANIC FUNCTIONAL GROUP PREPARATION, volumes I-III, Academic Press, 1983; greene T, PROTECTING GROUPSIN ORGANIC SYNTHSIS, second edition, Wiley, Interscience, 1991.

Application method, pharmaceutical composition and medicine thereof

Application method

The compounds, compositions and methods of the invention (e.g., fatty acid alkanolamides, fatty acid ethanolamine compounds, analogs and homologs) are useful for preventing weight or body fat gain in mammals, including dogs, cats and particularly humans. Weight loss is for cosmetic and therapeutic purposes. These compounds are also used to reduce appetite or elicit hypophagia.

The compounds, compositions and methods of the invention are also useful for preventing weight gain and body fat gain in individuals having a weight within the normal range. The compounds are also useful in other healthy individuals who do not require any pharmaceutical intervention to treat diseases associated with diabetes or hypertension or cancer. In some embodiments, the individual being treated is protected from a disease associated with abnormal levels of sugar or lipids or metabolic abnormalities, or from a threat of cardiovascular and cerebrovascular disease. Such individuals may be non-diabetic and have normal levels of blood glucose, and may also have normal levels of blood lipids (e.g., cholesterol) or triglycerides. Such individuals are protected from atherosclerosis and possibly other diseases such as cancer or other tumors, disorders including insulin resistance, syndrome X and pancreatitis.

In other embodiments, the subject is an overweight or obese human in need of weight or body fat reduction. In these embodiments, the methods, compounds and compositions of the invention can be administered to increase weight loss and also to prevent weight gain, so long as a normal weight range for a person's sex, age and height is achieved. The compounds are also useful in other healthy individuals who do not require any medication to treat conditions associated with diabetes or hyperlipidemia or cancer. Such individuals may also be protected from risk factors for cardiovascular and cerebrovascular disease. In some embodiments, the individual receiving treatment is free of a disease associated with carbohydrate (e.g., glucose) or lipid metabolism. Such individuals do not have diabetes and have blood glucose levels in the normal range, and their blood lipid (e.g., cholesterol, HDL, LDL, total cholesterol) or triglyceride levels in the normal range. Such individuals do not require treatment for atherosclerosis.

The compounds, methods and compositions of the present invention can be administered to inhibit the stomachs of mammals including cats, dogs and humans. In some embodiments, such compounds are applied to other healthy individuals who do not require any pharmaceutical intervention to treat any disease. In some embodiments, the individual does not need prophylactic or palliative treatment of any disease, including cancer, diabetes, or hyperlipidemia. In some embodiments, the individual being treated is free of a disease associated with abnormal blood glucose or lipid levels. In additional embodiments, the individual being treated is protected from cardiovascular and cerebrovascular disease. Such individuals do not have diabetes and have blood glucose levels in the normal range, and blood lipid (e.g., cholesterol) or triglyceride levels in the normal range. Such individuals are protected from atherosclerosis.

The compounds, methods and compositions of the present invention can be administered to modulate lipid metabolism (e.g., increase fat catabolism) in mammals, including cats, dogs and humans. In some embodiments, the use of such compounds reduces appetite in otherwise healthy individuals. In some embodiments, the individual receiving treatment is free of diseases associated with blood glucose or lipid metabolism (e.g., diabetes, hypercholesterolemia, low HDL levels, or high LDL levels). Such individuals do not have diabetes and blood glucose ranges at normal levels. Their blood lipid or triglyceride levels were within the normal range. Such individuals are protected from atherosclerosis.

Treatment with the compounds and compositions of the present invention may be prescribed by the degree or amount of weight loss achieved over a period of time, or by the individual achieving a BMI in the normal range. Treatment with the compounds and compositions of the present invention is reduced when a prescribed amount and degree of weight loss is achieved or when the individual achieves a BMI within normal ranges.

The compounds and compositions of the present invention may be administered alone for the purpose of reducing body weight or appetite.

Pharmaceutical composition

In another aspect, the invention provides a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the invention comprise a compound of the invention as an active ingredient or a pharmaceutically acceptable salt thereof, and also a pharmaceutically acceptable carrier and optionally other therapeutic ingredients.

The compositions include those 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 mode of administration for a particular situation will depend in part on the nature and severity of the condition being treated, as well as on the nature of the active ingredient. A typical route of administration is oral. The compositions may conveniently be presented in unit dosage form and formulated by any of the methods well known in the pharmaceutical arts.

In practical applications, the compounds of the present invention may be synthesized according to conventional pharmaceutical compound techniques as the active ingredient in intimate admixture with a pharmaceutical carrier. The carrier may take a wide variety of forms depending on the form of formulation desired for administration, e.g., oral or parenteral (including intravenous). In formulating the compositions for oral dosage form, any of the usual media may be employed, such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like, as well as in oral liquid formulations such as suspensions, elixirs, and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like, and in the form of oral solids such as powders, hard and soft capsules and tablets, solid formulations being preferred over liquid formulations.

Because of their ease and ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case carriers are obviously employed. If desired, the tablets may be coated by standard aqueous or non-aqueous techniques. Such compositions can contain at least 0.1% active compound. The percentage of active compound in these compositions will, of course, vary and will be approximately between 2% and 60% of the weight of the unit. The amount of active compound in the composition for such therapeutic use is such that a therapeutically effective dose is obtained. The active compounds may also be administered intranasally, e.g., in the form of droplets or a spray.

Tablets, pills, capsules and the like also contain a binder such as gum, tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrators such as corn starch, potato starch, alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose or saccharin. When a dosage unit form is a capsule, it contains, in addition to materials of the above type, a liquid carrier such as a fatty oil.

Many other materials may be present as coatings or to modify the physical form of the dosage unit. For example, 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, xylem and p-phenylene as preservatives, a dye and a flavoring agent such as cherry red or orange. To prevent degradation during transport through the upper GI route, the composition may be formulated as an enteric coating.

Administration of drugs

The compounds of the invention may also be administered parenterally. Solutions or suspensions of these active compounds may be formulated in a suitable mixture of water and a surfactant such as hydroxypropyl cellulose. Colloidal solutions can also be formulated in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary storage and use environments, these formulations contain preservatives to prevent microbial growth.

Pharmaceutical forms suitable for injectable use include sterile injectable or colloidal solutions. In all cases the form must be sterile and must be fluid to the extent that convenient injection is achieved. It must be stable in the environment of manufacture and storage and to prevent the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or colloidal solution medium comprising, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof, and vegetable oils.

The compounds of the present invention are effective over a wide dosage range. For example, in treating adults, the dose is approximately 10-1000mg, requiring approximately 100-500mg or 1-100 mg. About 0.05-100mg, more preferably 0.1-100mg, is used daily. The most preferred dosage is 0.1-70mg per day. In selecting the regimen for a patient, it is generally initially necessary to administer a dose of 2-70mg per day, with the lowest possible dose being reduced, perhaps starting at 0.1-10mg per day, when the condition is being managed. For example, in treating adults, about 0.05-100mg per day may be used, with about 0.1-100mg per day being preferred. The determined dosage will depend on the mode of administration, the mode of administration desired for the treatment, the subject being treated and his weight, and the preferences and experience of the physician or veterinarian in charge.

In general, the compounds of the invention may be formulated in unit dosage form, preferably containing from 0.1 to 100mg of the active ingredient per unit dose of a pharmaceutically acceptable carrier. In general, dosage forms suitable for oral, nasal, pulmonary, or transdermal administration include approximately 0.001-100mg, more preferably 0.01-50mg of the compound in admixture with pharmaceutically acceptable carriers and diluents. For storage and use, these formulations preferably contain a preservative to prevent the growth of microorganisms.

Administration of a suitable dose of a candidate compound may be by any method known herein, e.g., oral or rectal, parenteral, intraperitoneal, intravenous, subcutaneous, subdermal, intranasal, or intramuscular administration. In some embodiments, the method of administration is transdermal. Suitable dosages for candidate compounds may be determined empirically as known herein. An appropriate or therapeutic dose is a dose that is sufficient to cause a loss of body fat and weight in the animal over a period of time. Candidate compounds may be administered as needed to reduce body fat and body weight, for example hourly, every 6, 8, 12 or 18 hours, daily or weekly.

Formulations suitable for oral administration include (a) solutions, e.g., small packets of nucleic acid, such as water, saline, or PEG400, in an effective amount suspended in a diluent; (b) capsules, sachets or tablets, each containing a defined amount of active ingredient, such as a liquid, solid, granules or gelatin; (c) suspensions in suitable liquids; and (d) a suitable emulsion. Tablet forms include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, buffering agents, wetting agents, preservatives, flavoring agents, dyes, disintegrating agents, and a pharmaceutically compatible carrier. The diamond form contains the active ingredient in a flavoring agent, such as sucrose, as a lozenge contains the active ingredient in an inert base, such as gelatin and glycerin or sucrose and gum arabic emulsions, gels, and the like, except for the active ingredients and carriers known herein.

Injectable solutions and suspensions may be formulated from sterile powders, granules, and tablets of the kind previously described. Formulations suitable for parenteral administration, for example, intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal and subcutaneous routes, including aqueous and nonaqueous, isotonic sterile injections. It contains antioxidants, buffers, bacteriostats and solutes which render the formulation and the subject's blood isotonic, as well as aqueous and non-aqueous sterile suspensions which include suspending agents, solubilizers, thickening agents, stabilizers and preservatives.

For the route of intradermal administration, methods of intradermal administration are disclosed in Remington's Pharmaceutical Sciences, 17 th edition (Gennaro et al eds., Mack Publishing Co., 1985). Dermal or dermal plaques are the preferred route of intradermal administration of the compounds of the present invention. The plaque preferably provides an absorption enhancer, such as DMSO, to increase absorption of the compound. Other routes of intradermal drug delivery are disclosed in U.S. patent nos. 5,962,012 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 into the skin. Plaques will provide multiple dosing regimes, each involving an accumulation regime or a single regime. The accumulation system design may have, for example, 4 layers: an adhesive layer in direct contact with the skin, a control membrane, which controls the dispersion of drug molecules, the accumulation of drug molecules and a water resistant base. Such a design delivers a uniform dose of drug over a specific period of time, the delivery rate having to be below the saturation limit for different types of skin.

Single design, characteristically only three layers: an adhesive layer, a matrix polymer containing the compound, and a waterproof base.

Such a design results in a skin-saturating amount of drug. Thus, the skin controls the transmission. When the drug amount decreases below the saturation level in the plaque, the delivery rate decreases.

The compounds of the invention may be used in combination with other compounds of the invention or other medicaments which are useful in dieting or in the treatment, prevention, inhibition or reduction of body fat. Such compounds may therefore be administered, by commonly used routes and dosages, either simultaneously or sequentially with a compound of the present invention. When the compounds of the present invention are applied simultaneously with one or more other drugs, pharmaceutical compositions comprising such other drugs and unit dosage forms of the compounds are preferred. When used in combination with one or more other active ingredients, the compounds of the present invention and other active ingredients will have a lower ridge than when used alone. Accordingly, the pharmaceutical compositions of the present invention include compositions comprising one or more other active ingredients in addition to those disclosed above.

Identification of Compounds of the invention

Candidate compounds, as described above, can be screened by methods known herein. For example, the body fat reducing compounds may be identified in vivo using animal bioassay techniques well known to those of ordinary skill in the art. The experimental compound, and the appropriate excipients or controls of energy, can be administered to the experimental subject by a number of routes (e.g., oral route, parenteral route) and the subject's weight monitored during the course of treatment. The subject of the experiment is a human or an experimental animal (e.g., rat, mouse).

The effect of the compounds on appetite or eliciting hypophagia or reducing food intake can be assessed, for example, by monitoring food consumption in the experimental subject. (e.g., measuring the amount eaten or uneaten as a weight of food or energy content). The effect of the compounds on appetite was also assessed by subjective methods including using questionnaires of the subject's level of appetite or food craving. The effect of the test compound on lipid metabolism can be assessed by monitoring blood lipids and fatty acid oxidation. The techniques for such evaluations are well known to those of ordinary skill in the art. This consideration may be acute, subacute, chronic or sub-chronic depending on the duration of administration and or the effect of the administration.

The reduction in body weight can be measured, for example, by directly measuring changes in body fat or body weight of an animal. The animal may be selected from mouse, rat, guinea pig or rabbit. The animal can be an ob/ob mouse, db/db mouse or Zucker mouse or other animal models of body weight related diseases. Clinical studies have also been conducted in humans.

Combinatorial chemical libraries

Recently, attention has focused on the use of combinatorial chemistry libraries to aid in the production of new chemical compound examples. Combinatorial chemistry libraries are collections of various compounds produced by chemical synthesis or by the biosynthesis of a combination of numerous building blocks such as reagents. For example, linear combinatorial chemical libraries, such as polypeptide libraries, are formed by combining a series of chemical building blocks called amino acids, by each possible method, at a particular compound length (e.g., the number of amino acids in a polypeptide compound). Hundreds of thousands of chemical compounds can be synthesized by combinatorial mixing of such chemical building blocks. For example, a critic has found that systematic, combinatorial mixing of 100 switchable chemical building blocks leads to the theoretical synthesis of 1 million tetrameric or 100 million pentamer compounds (Gallop et al J. Med. chem.37 (9): 1233 (1994)).

Formulating and screening combinatorial chemistry libraries is well known to those skilled in the art. Such combinatorial chemistry libraries include, but are not limited to, benzodiazepines (U.S. Pat. No. 5,288,514), diversomers? Such as hydantoins, benzodiazepines and dipeptides (Hobbs et al PNAS USA 90: 6909(1993)), organic synthesis of similar small compound libraries (Chen eds.) J.Amer.chem.Soc.116: 2661(1994), oligocarbamates (Cho et al, Science 261: 1303(1993)), and/or peptidylphosphonates (Campbell et al J. org. chem.59: 658(1994)) and libraries of small organic molecules (see, e.g., benzodiazepines (Baum C & EN, Jan 18, page 33(1993)), thiazolidinediones and metazanones (U.S. Pat. No. 5,549,974), tetrahydropyrrole (U.S. Pat. Nos. 5,525,735 and 5,519,134), benzodiazepines (U.S. Pat. No. 5,288,514), and the like.

The equipment used to formulate combinatorial libraries is commercially available (see, e.g., 357MPS, 390MPS, Advanced Chemtech, Louisville KY, Symphony, Rainin, Wobum, MA, 433A Applied Bio-systems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA).

Many well-known automated systems have also been used to develop liquid phase chemistry. These systems include automated workstations, like automated synthesis equipment manufactured by Takeda chemical company, Inc. (Osaka, Japan) and many automated systems using robotic arms (Zymate II, Zymate, Hopkinton, Mass; Orca, Hewlett packard, Palo Alto, Calif.), which mimic the manual synthesis operations performed by chemists. Any of the above devices is suitable for use with the present invention. The nature and implementation of modifications to these devices will be apparent to those skilled in the relevant art so that they may operate as discussed herein. In addition, many combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, n.j., Asinex, moshow, Ru, Tripos, inc.st.louis, MO, ChemStar, ltd., moshow, Ru, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

High throughput assay for chemical libraries

The compound assays described herein must be amenable to high throughput screening. Preferred assays therefore find activation of transcription by the test compound (e.g., activation of an mRNA product), activation of protein expression by the test compound, or by binding of the test compound to a gene product; or the effects on fatty acid regulation as described below.

High throughput assays or binding assays for the presence, absence or quantification of a particular protein product are well known to those skilled in the art. Thus, for example, U.S. Pat. No. 5,559,410 discloses a high-yield screening method for proteins, and U.S. Pat. Nos. 5,576,220 and 5,541,061 disclose high-yield screening methods for ligand/antibody binding.

In addition, high throughput screening systems are commercially available (see, e.g., Zymark corp., hopkinton MA; Air technologies, Mentor, OH; Beckman Instruments, inc. fullerton, CA; Pre-precision systems, inc., nature, MA, etc.). These systems typically perform the entire steps automatically including all sample and reagent pipetting, liquid dispensing, incubation for a period of time and reading on a calibrator microplate appropriate for the assay. These structural systems provide high throughput and fast start-up, yet a high degree of flexibility and customization. Manufacturers of such systems provide detailed specifications for a variety of high throughput. Thus, for example, Zymark corporation provides technical bulletins describing the discovery of screening systems for gene transcription regulation, ligand binding, and the like.

Determining whether a compound affects food intake, body weight, body fat, appetite, foraging behavior or modulates fatty acid oxidation

The compounds of the invention may be administered to animals to determine whether they affect food intake and body weight, body fat, appetite, foraging behavior or modulate fatty acid oxidation.

The animal may be, for example, an obese or normal guinea pig, rat, mouse or rabbit. Suitable rats include, for example, Zucker rats. Suitable rats include, for example, plain rats, ALS/LtJ, C3.SW-H-2b/SnJ, (NON/LtJxNZO/HlJ) Fl, NZO/HlJ, ALR/LtJ, NON/LtJ, KK. Cg-AALR/LtJ, NON/LtJ, KK. Cg-Ay/J, B6.HRS (BKS) -Cpefat/+, B6.12P2-Gcktm/Efr, B6.V-Lepob, BKS. Cg-m +/+ Leprdb and C57BL/6J diet-induced obesity.

Administration of a suitable amount of a candidate compound may be by any method known herein, e.g., oral or rectal, parenteral, e.g., intraperitoneal, intravenous, subcutaneous, subdermal, intranasal, or intramuscular. Preferred methods of administration may be intraperitoneal or oral. As known herein, a suitable effective amount of a candidate compound can be determined empirically. A suitable effective amount is an amount sufficient to result in a reduction in body fat or weight or a reduction in food consumption of the animal over a period of time. Candidate compounds may be administered as needed to reduce body fat and body weight, for example hourly, every 6, 8, 12 or 18 hours, daily or weekly.

Formulations suitable for oral administration include (a) solutions, e.g., an effective amount of the candidate compound suspended in a diluent, such as water, saline, or PEG 400; (b) capsules, sachets or tablets, each containing a defined amount of active ingredient, such as a liquid, solid, granules or gelatin; (c) suspensions in suitable liquids; and (d) a suitable emulsion. Tablet forms include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphate, cereal starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, mica, magnesium stearate, stearic acid and other excipients, pigments, fillers, binders, diluents, buffering agents, wetting agents, preservatives, flavoring agents, dyes, disintegrating agents and pharmaceutically compatible carriers. The diamond form contains the active ingredient in a flavoring agent, such as sucrose, as a lozenge contains the active ingredient in an inert base, such as gelatin and glycerin or sucrose and gum arabic emulsions, gels, and the like, except for the active ingredients and carriers known herein.

Injectable solutions and suspensions may be formulated from sterile powders, granules, and tablets of the kind previously described. Formulations suitable for parenteral administration include, for example, aqueous or non-aqueous, isotonic sterile injection solutions containing antioxidants, buffers, bacteriostats and solutes which render the formulation and the subject's blood isotonic, and aqueous and non-aqueous sterile suspensions including suspending agents, solubilizers, thickeners, stabilizers and preservatives.

The animal is administered in a dose sufficient to cause a change in body weight, body fat and/or fatty acid oxidation over a period of time. Such dosages can be determined based upon the efficacy of the particular candidate compound employed and the condition of the animal,animal body weight or surface parts are also contemplated. The size of the dose is also determined by the presence, nature and extent of side effects associated with administration of the candidate compound; and also LD dependent on candidate compounds₅₀(ii) a And side effects of candidate compounds at various concentrations. In general, the dosage is in the range of 0.1 to 50mg/kg, preferably 1 to 25mg/kg, most preferably 1 to 20mg/kg body weight. Determination of dose response relationships is well known to one of ordinary skill in the art.

Reduction of body fat

The reduction in body weight can be characteristically determined by directly measuring the change in body fat or weight loss. Body fat and body weight of the animals are determined before, during, and after administration of the candidate compound. The change in body fat is measured by any method herein, for example, measuring fat folds with calipers, bioelectrical impedance, hydrostatic weighing, or dual x-ray absorption spectroscopy. Preferred animals exhibit at least a 2%, 5%, 10% or 15% reduction in body weight. The reduction in body weight is determined prior to candidate compound administration and measured at intervals during and after treatment. Preferably, body weight is measured once every 5 days, more preferably every 4 days, more preferably every 3 days, more preferably every 2 days, more preferably every 1 day.

Alterations in fatty acid metabolism

Changes in fatty acid metabolism can be determined, for example, by observing fatty acid oxidation in cells of major adipose burning tissues such as liver (Beynen et al Diabetes 28: 828(1979)), muscle (Chiasson Lab. Ant of Rat (1980)), heart (Flink et al J.biol.chem.267: 9917(1992)), and adipocytes (Rodbell J.biol.chem.239: 375(1964)), which can be from primary cultures or cell cords. The cells may be formulated into a primary culture by methods known herein, including, for example, enzymatic digestion and dissection. Suitable cell lines are known to those of skill in the art. Suitable hepatocyte cords include, for example, Fao, MH1C1, H-4-II-E, H4TG, H-4-II-E-C3, McA-RH7777, McA-RH8994, N1-S1 Fudr, N1-S1, ARL-6, Hepa1-6, Hepa-1C1C7, Bcll, tao BpRcl, NCTC clone 1469, PLC/PRF/5, Hep 3B2.1-7[ Hep 3B ], Hep G2[ HepG2], SK-HEP-1, WCH-17. Suitable osteocyte cords are, for example, L6, L8, C8, NOR-10, BLO-11, BC3H1, G-7, G-8, C2C12, P19, Sol8, SJRH30[ RMS 13], QM 7. Suitable cardiac cell lines are H9c2(2-1), P19, CCD-32Lu, CCD-32Sk, Girardi, FBHE. Suitable adipocyte cords are NCTC clone 929[ derivatives of Strain L; l-929; l cells ], NCTC2071, L-M, L-M (TK-) [ LMTK-; LM (tk-) ], A9 (negative derivatives of APRT and HPRT StrainL), NCTC clone 2472, NCTC clone 2555, 3T3-L1, J26, J27-Neo, J27-B7, MTKP97-12 pMp97B [ TKMp97-12], L-NGC-5HT2, Ltk-11, L-alpha-1B, L-alpha-2A, L-alpha-2C, B82.

The fatty acid oxidation rate can be controlled by¹⁴C oleic acid is oxidized to ketone bodies (Guzman and Geelen biochem. J.287: 487(1982)) and/or¹⁴Oxidation of C oleic acid to CO₂(Fruebis PNAS 98: 2005 (2001)); blazquez et al j. neurochem 71: 1597 (1998)). Lypolysis can be determined by obtaining the release of fatty acids or glycerol using a suitable labeling precursor or spectrophotometric assay (Serradeil-Le Gal FEBS Lett 475: 150 (2000)). To analyze¹⁴C oleic acid is oxidized to ketone bodies and fresh, isolated cells or cultured cell cords can be incubated with oleic acid for a suitable period of time, such as 30, 60, 90, 120 or 180 minutes. Can measure¹⁴C in the incubation medium to determine their oleic acid oxidation rate. Oleic acid oxidation can be expressed as nanomolar numbers of oleic acid produced in x minutes/g cell. To analyze the release of lyssisol/glycerol, fresh isolated cells or cords of cultured cells may be washed and then incubated for a suitable period of time. The amount of glycerol released into the incubation medium may provide a factor for the lyopis.

Examples

The following examples are intended to be illustrative and not limiting. Those skilled in the art will appreciate that many of the unimportant parameters may be transformed or modified to produce similar results.

Example 1: synthesis of fatty acid ethanolamine compounds, homologs, and analogs.

The process of formulating fatty acid ethanolamines from ethanolamines and the corresponding fatty acyl groups is relatively simple and known to one of ordinary skill in the art. For example, fatty acid ethanolamine may be synthesized by reacting a fatty acid or a chlorinated fatty acid with ethanolamine, as described by Abadjj et al (Abadjj, v., Lin, s.y.taha, g., Griffin, g., Stevenson, l.a.pertwee, r.g.).&Makriyannis, A.J.Med.chem.37, 1889-. Fatty acids can be formulated using procedures similar to sergarevich and Carroll (sergarevich, B.&Carroll, K.K.J.lipid Res.7, 277-284 (1966)). The radiolabeled fatty acid ethanolamine may be prepared by the reaction of an acid chloride (Nu-Check Prep, Elysian, MN)³H]Ethanolamine (10-30 Ci/mmol; American Radiolabed Chemicals.St.Louis) as Desarnaud, F., Cadas, H.&Piomelli, D. (1995) J.biol.chem.270, 6030-. The compound can be purified by flash column chromatography or HPLC. The identity of the compounds can be established by using NMR and/or gas chromatography-mass spectrometry and thin layer chromatography.

Primary reagents and materials can be purchased from Avanti Polar Lipids, Cayman Chemicals (Ann Arbor, MI), Nu-Check Prep, Research Biochemicals, or Sigma. Briefly, unlabeled or labeled fatty acyl ethanolamines can be synthesized by reacting the corresponding fatty acid chlorides with unlabeled or labeled ethanolamine according to Giuffrida, A. et al (see Giuffrida, S.G. and Rubin, R.P.eds., 113-133, CRC Press LLC, Boca Raton, Florida) and Deven et al (Deven W., Hanus, L. et al Science 258, 1946-1949 (1992)). The fatty acid chloride can be dissolved in methylene chloride (10mg/ml) and reacted with ethanolamine at 0.4 ℃ for 15 minutes. The reaction was stopped after adding purified water. Only after active stirring can the phases be separated. The upper aqueous phase was discarded. The organic phase material was washed twice with water. These washes remove unreacted ethanolamine. This method provides quantitative formulation of fatty acylethanolamines. The ethanolamine is concentrated to dryness under a stream of nitrogen and reconstituted in an organic solvent such as dichloromethane at a concentration of 20 mM. The resulting fatty acylethanolamine solution can be stored at-20 ℃ until needed for use.

Fatty acid carboxylic acid groups, primary and secondary amino groups, and primary ethanolamine are well known to one of ordinary skill in the art. Fatty acid ethanolamines have many substituents at the ethanolamine position and therefore can be formulated in a number of ways, but most preferably the formulation is started with the corresponding substituted ethanolamine and fatty acid moiety. Such substituted ethanolamines would include alkylaminoethanol esters and acylaminoethanol esters, as well as secondary alkylethanolamines. Alternatively, a specific fatty acid ethanolamine may be synthesized by adding an appropriate substituent to the corresponding fatty acid ethanolamine.

Example 2: method for screening fatty acid ethanolamine and other compounds of the present invention in living body

Male Wistar rats (200-350g) were used. The procedure must comply with NIH regulations, which are detailed in the management and use guidelines for laboratory animals and in the European Community guidelines 86/609/EEC study of managed animals.

Chemical substances, FAEs and²H₄]FAEs are synthesized in the laboratory (Giuffrida et al, "Lipid SeconddMessengers" (Laychock, S.G).&Rubin, r.p. eds.) 113-133(CRC Press LLC, Boca Raton, FL, 1998)); 1, 2-dioleyl-sn-glycero-phosphoethanolamine-N-oleyl is purchased from Avanti Polarlipids (Alabaster, AL); SR141716A is supplied by RBI (Natick, MA), which is part of the NIMH (N01MH30003) chemical synthesis project; SR144528 is a generous gift of Sanofi Recherche; all other drugs were from Tocris (Ballwin, MO) or Sigma (Saint Louis, MO). FAE was dissolved in dimethyl sulfoxide (DMSO) and administered as a 70% DMSO sterile saline solution (acute treatment) or 5% Tween 80/5% propylene glycol sterile saline solution (chronic treatment) (1 mg/kg). Capsaicin was administered as a 10% Tween 80/10% ethanol/80% saline solution; SR141716A, SR144528, CCK-8 and CP-93129 were in a 5% Tween 80/5% ethylene glycol acrylate solution (1 mg/kg).

In all biochemical experiments, rats were killed after various prolonged fasts and tissues were collected between 1400 and 1600 h. The microsomal fraction was formulated as described above (Desarnaud et al, J.biol.chem., 270: 6030-. Using 1, 2-¹⁴C]NAT measurements were performed using palmitic acid-sn-glycerophosphorylcholine as a substrate (108mCi/mmol, Amersham, Piscataway, NJ) (Cadas et al H., J. Neurosci., 17: 1226-1242 (1997)). FAAH was determined according to (Desarnaud et al, J.biol.chem., 270: 6030-³H]Anandamide (arachidonic acid- [1-³H]Ethanolamine); 60 Ci/mmol; ARC, st.louis, MO) was included in the substrate and radioactivity was measured in the aqueous phase after chloroform extraction.

Plasma was prepared from blood obtained by cardiac puncture (Giuffrida et al, anal. biochem., 280: 87-93(2000)) CSF using a 27G1/2 needle (precision, USA) from a large pool. FAEs and NAPE were extracted from the tissue with methanol/chloroform and separated using a column chromatography (Giuffrida et al, "Lipid Second memories" (Laychock, S.G. & Rubin, R.P. eds.) 113-133(CRC Press LLC, Boca Raton, FL, 1998)). FAEs were quantified by HPLC/MS using an isotope dilution method (Giuffrida et al, anal. biochem., 280: 87-93 (2000)). Individual NAPE species were identified and quantified by HPLC/MS using an external standard method (Calignano et al, Nature, 408: 96-101 (2000)).

Blood chemistry plasma beta-hydroxybutyrate and glycerol were determined using commercial tools (Sigma, St, Louis, MO). Plasma prolactin, adrenalone and progesterone were quantified by radioimmunoassay (Navarro et al, J.Neuroreport, 8: 491-.

Feeding experiment acute experiment food intake was determined in 24-h fasted rats (Navarro et al, J.neurohem., 67: 1982-. And (5) subacute experiments. Rats fed Ad libitum received vehicle injections for 3 days. On day 4, animals were divided into the same two groups, and they were injected with vehicle or OEA (5mg/kg at 1900 h) daily for 7 consecutive days while measuring body weight, food intake and water intake.

Rats were deprived of water for 24-h and then habituated to drink from graded bottles during the 30 minute experiment, which lasted 4 days. On day 5, the water was replaced with a 0.1% saccharin solution and the animals were injected 30 minutes later with vehicle, OEA (20mg/kg) or lithium chloride (0.4M, 7.5 ml/mg). During the next two days, water consumption was recorded over a 30 minute experimental time. Then, animal water, saccharin and the drinking amount were measured again.

Rats were trained to eat on a lever according to a set supplementation schedule of comparative example 1(FR1), however each rat was restricted to 20g of food per day (Rodriguez de Fonseca et al, acta pharmacol. Once a stable response was obtained, the animals were trained to obtain FR5, and rested for 2 minutes for food supplementation and to maintain their limited access to food. When a stable baseline was obtained, these animals were used to test the effect of vehicle or OEA (1, 5 or 20mg/kg) administered 15 minutes before the lever feeding. The test time was 60 minutes.

And (4) measuring other behaviors. After administration of the excipient or OEA (20mg/kg), the weightlifting and maze experiment (aged plus maze test) was performed as described (Navarro et al, J.Neuroreport, 8: 491-496 (1997)). The level of activity in the open field (Beltramo et al, neurosci, 20: 3401-. A digital thermometer was used to determine rectal temperature (Martin-Calderon et al, Eur. J. Pharmacol., 344: 77-86 (1998)).

Rats are typically treated and injected for 5 days. On day 6, vehicle or drug OEA (10mg/kg) or oleic acid (10mg/kg) was administered. After 60 minutes the rats were killed by decapitation under anesthesia.Using c-fos^{3 5}The S-labeled cRNA probe of (Guthrie et al, Proc. Natl. Acad. Sci. U.S.A., 90: 3329. sup. 3333(1993)) and acetylcholine transferase (ChAT) (Lauterborn et al, Brain Res. mol. BrainRes., 17: 59-69(1993)) were subjected to in situ hybridization analysis. The average hybridization density was determined by at least 3 tissue fractions per rat. Statistical significance was assessed using an analysis of variation (ANOVA) followed by assay paired controls after Tukey-Kramer.

Data analysis results are expressed as mean ± s.e.m of n different experiments. Significance of differences between groups was assessed using (ANOVA), followed by Student-Newman-Keuls later experiments, unless indicated otherwise.

Example 3: effect of starvation on rat OEA and other FAE levels

In a certain embodiment, the present invention provides a method of treatment wherein an individual is in need of weight and/or body fat reduction and is tested for OEA levels before and/or during fasting. Individuals with low OEA levels before fasting or after fasting response are particularly targeted for this treatment.

Rats are fasted when FAE levels in heart blood are determined periodically by High Performance Liquid Chromatography (HPLC) and electrospray Mass Spectrometry (MS). Plasma OEA remained at baseline levels during the first 12 hours of fasting, increased significantly between 18 and 24 hours, and returned to normal at 30 hours (fig. 1 a). After water deprivation (fig. 1b) or administration of inhibitors such as brake and Lipopolysaccharide (LPS) [ pmol/ml; 10.3 plus or minus 0.8; 8.4 +/-1.6 minutes after 15 minutes of braking; 60 minutes (1mg/kg), 7.0. + -. 0.7 after LPS injection; no such effect was found when n-6-9. Plasma PEA concentrations were not significantly affected by any of these treatments (data not shown), however anandamide rapidly declined when food was removed, remaining at baseline levels below the duration of the entire experiment (fig. 1 d). Following braking (in pmol/ml; control, 3.6 ± 0.4; braking, 1.1 ± 0.5; n ═ 7-8; P < 0.01), LPS treatment (control, 2.0 ± 0.5; LPS, 0.2 ± 0.2; n ═ 6; P < 0.01) and insignificant water deprivation (fig. 1e), anandamide levels also decreased. These results indicate that circulating OEA levels are increased transiently during fasting. This reaction is selective to OEA, anandamide and other FAEs do not occur, and an increase in blood glycerol and β -hydroxybutyrate (table 1) occurs simultaneously, indicating that the energy metabolism is shifted from carbohydrate-based fuels to fatty acid-based fuels (Cahill, g.f., clin. endocrinol. metab., 5: 397-.

TABLE 1 plasma beta-hydroxybutyrate (beta-HBA) levels in fasted rats

beta-HBA glycerol

Free feeding 1.2 + -0.44.6 + -0.9

Fasting for 2 hours 1.2 + -0.25.3 + -0.6

Fasting for 4 hours 0.8 + -0.19.1 + -1.8

Fasting for 8 hours 1.3 + -0.26.3 + -0.4

Fasting for 12 hours 4.6 ± 0.8 × 7.6 ± 1.0

Fasting for 18 hours 6.8 ± 0.4 × 8.4 ± 0.4

Fasting for 24 hours 9.1 ± 1.2 × 8.4 ± 0.3

The concentration is expressed in mg/dl units per group by P < 0.05, n is 3

The levels of OEA in cerebrospinal fluid were not significantly affected by fasting (fig. 1c), implying that plasma OEA is produced outside the CNS in a surge. To test this hypothesis, the effect of fasting on OEA metabolism in various tissues of rats was investigated. The biochemical pathways by which animal cells produce and degrade OEA and other FAEs are thought to involve three major enzymatic reaction steps. Calcium-activated NAT activity transfers fatty acid groups from the sn-1 site of a donor phospholipid to the main amino site of phosphatidylethanolamine, thus generating NAPE2(Schmid et al, chem. Phys. lipids, 80: 133-142 (1996); Piomelli et al, neurobiol. Dis., 5: 462-473(1998)), which is ultimately decomposed into fatty acids and ethanolamine by intracellular Fatty Acid Amide Hydrolase (FAAH) (Schmid et al, J. biol. chem., 260: 14145-14149 (1985); Cravatt et al, Nature, 384: 83-87 (1996)). Fasting (18h) was accompanied by a significant increase in NAT activity in white adipose tissue (fig. 2a), but not in brain, stomach and kidney (fig. 2b, d, data not shown). In liver, intestine and skeletal muscle, NAT activity was decreased due to fasting (fig. 2c, d, data not shown). These enzymatic changes are accompanied by changes in the corresponding NAPE tissue contents. NAPE of many molecular species appears in rat tissues, including OEA precursor base-1-palmitoenyl-2-arachidonoyl-sn-glycero-phosphoethanolamine-N-oleate (NAPE 1; FIG. 3a) and base-1-palmitoyl-2-arachidonoyl-sn-glycero-phosphoethanolamine-N-oleate (NAPE 2; FIG. 3 a); and PEA precursor base 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphoethanolamine-N-palmitoyl (not shown). Consistent with NAT activity assays, fasting increased NAPE levels in fat and decreased liver levels (fig. 3b, c).

Since NAPE biosynthesis and FAE formation are tightly coupled processes (Cadas et al, H., J. Neurosci., 17: 1226-1242(1997)), fasting is expected to increase OEA and other FAEs levels in fat, but not in other tissues. Accordingly, starved rats contained more OEA and PEA in the fat than free-fed rats (fig. 3d, data not shown), however, this difference was not seen in brain, stomach and intestine (data not shown). Contrary to what we expect, however, fasting rats also contained higher amounts of OEA and PEA in the liver than the freely fed rats (data not shown in fig. 3 d). This discrepancy may be due to accumulation of FAE in the liver, which is consistent with the presumed role of this organ in FAE reuptake and metabolism (Bauchur et al, J.biol.chem., 240: 1019-1411024 (1965); Schmid et al, J.biol.chem., 260: 14145-14149 (1985)).

The catalytic hydrolysis of FAAH to fatty acids and ethanolamine by the enzyme is a key step in the FAE degradation process (Bauchur et al, J.biol.chem., 240: 1019-1024 (1965); Schmid et al, J.biol.chem., 260: 14145-14149 (1985); Cravatt et al, Nature, 384: 83-87 (1996); Desarnaud et al, J.biol.chem., 270: 6030-6035 (1995)). Fasting greatly reduced FAAH activity in lipid cell membranes, but had no effect on FAAH activity in brain, liver, stomach, intestine, kidney and skeletal muscle (fig. 2a-e do not show data). Fasting therefore increased the levels of OEA and other FAEs in white fat in two mutually promoting ways, which differ in the other reactions occurring on the mechanisms and during lipolysis: activation of NAT activity leads to increased biosynthesis of NAPEs and FAEs, whereas inhibition of FAAH activity prolongs the survival of newly synthesized FAEs. Although many tissues have an effect on normal levels of OEA in the bloodstream, the dynamic biochemical changes observed in fat underscore the critical role of this tissue in producing OEA during starvation.

Example 4 inhibition of food intake by OEA and other FAEs

A 24 hour fast may be used to evaluate the effect of systemic administration of OEA on food intake in rats. In this system, OEA resulted in a dose-and time-dependent inhibition of food intake (fig. 4a, b). To illustrate the selectivity of this reaction, a variety of OEA analogs were tested for their ability to produce hypophagia.

Anandamide and oleic acid had no effect.

Palmitoylethanolamide is active but significantly weaker than OEA.

Elaeoylethanolamide, an unusual OEA analogue, had similar potency to OEA (figure 4 a).

These results indicate that OEA reduces diet in a structurally selective manner, and that other fatty acid ethanolamine-like compounds can also be identified for use in accordance with the present invention.

Example 5 specificity of cannabinoid receptor agonists

The essential molecules of OEA hypophagia are different from those involved in Anandamide and its known target reaction for cannabinoids (Khanolkar et al, Life Sci., 65: 607-616 (1999)). The cannabinoid receptor agonist did not affect OEA hyphagia in vivo, and OEA did not bind to the cell membrane of rats instead of cannabinoid in vivo. Thus, although it has a structural and biogenic relationship with Anandamide, OEA does not rely on the endogenous cannabinoid system to produce anorexia.

Example 6 sustained weight loss

In some embodiments, the compounds of the present invention provide sustained body fat or weight loss upon prolonged administration to a mammal. This effect is advantageous when many drugs inhibit diet after acute administration, but treatment is prolonged and ineffective (Blundell, J., Trends Pharmacol. Sci., 12: 147-.

OEA was subacute administered to rats. Daily injection of OEA (5mg/kg) resulted in a gradual small but significant decrease in food intake for 7 days, accompanied by significant inhibition of body weight gain (FIG. 5b, c). OEA did not affect water intake (fig. 5 d). The effect of OEA on body weight was only partly explained by its modest reduction in food consumption, suggesting that other factors, such as stimulation of energy expenditure or inhibition of energy accumulation, also contribute to this effect.

Example 7 peripheral sites of action of FAEs

In one aspect of the invention, it provides compounds having a peripheral site of action. Such as sites that are beneficial in reducing central nervous system side effects.

While peripheral administration is useful, OEA has no effect following direct injection into the ventricles (table 2), suggesting that the primary site of action of this compound is outside the CNS. Further elaborated, adult rats, after administration of the nerve agent, capsaicin, have chemical destruction of sensory fibers and other peripheral nerves in their vagus nerve (Kaneko et al, am. J. physiol., 275: G1056-G1062 (1998)). Capsaicin-treated rats did not respond to peripherally applied cholecystokinin-8 (CCK-8) (FIGS. 6a, c) in the controlled groupMore water was taken (FIG. 6b, d) and the chemosensory reflex of the cornea was lost (data not shown), indicating that the neurotoxin destroyed the afferent sensory nerves (MacLean, D.B., Regul. Pept., 11: 321-. The treated animals also did not respond to OEA (10mg/kg), but generally responded to the compound CP-93129, which is targeted at 5-HT in the CNS_1BReceptors (FIGS. 6a, c) (Lee et al, Psychopharmacology, 136: 304-307 (1998)). These findings support the hypothesis that OEA produces hyphagia by acting at peripheral sites and that this action is a desirable sensory fiber.

TABLE 2 Effect of pranamide in the ventricles of brain on food intake

60 minutes 120 minutes 240 minutes

Excipient 5.8 + -0.68.0 + -0.59.5 + -0.5

prana 0.4μg 4.8±0.4 6.6±0.4 8.4±0.4

prana 2μg 4.9±0.4 6.6±0.6 8.7±0.5

prana10μg 5.9±0.2 8.1±0.4 9.6±0.7

pranamide/OEA (prana, μ g/animal) or vehicle (DMSO, 5 μ l) was administered to 24-hour fasted rats 15 minutes prior to food administration with n ═ 12 per group

The compounds of the present invention use peripheral sensory input to suppress appetite. Peripheral sensory input associated with appetite suppression is linked to a number of CNS structures, including the solitary bundle Nucleus (NST) in the brainstem and the segmental paraventricular nucleus (PVN) in the hypothalamus (Schwarts et al, Nature, 404: 661-. To clarify the brain pathways involved in OEA-induced hypophagia, mRNA levels of the activity control gene c-fos (Curran et al, Oncogene, 2: 79-84(1987)) were mapped by in situ hybridization after systemic administration of OEA, oleic acid or vehicle. OEA (10mg/kg) caused a highly localized increase in c-fosmRNA levels in PVN, supraoptic nuclei (FIG. 7a) and NST (FIG. 7c) compared to the control group. This increase was specific for these regions, in which case the expression of c-fos in other regions of the brain was not significantly affected by OEA treatment (FIG. 7b, d). OEA was found to promote the expression of c-fosmRNA in NST (which transmits vagal sensory CNS) and PVN (the major site of central energy signal synthesis) (Schwartz et al, Nature, 404: 661-671(2000)), consistent with the physiological role of this lipid as a peripheral transmitter of anorexia.

OEA is likely to reduce diet by causing a non-specific behavioral inhibition state. As is true, that OEA must cause a specific taste shift, which can be gradually caused in rats by a number of toxic substances (Green et al, Science 173: 749-751(1971)), including lithium chloride (FIG. 4 c). However, the maximum dose of OEA (20mg/kg) had little effect in this assay (fig. 4c), indicating that the compound was not transformed. Many additional observations support the behavioral characteristics of OEA. OEA did not alter water intake, body temperature, pain threshold (fig. 4d-f) or activity of the hypothalamus-pituitary-adrenal (HPA) axis (table 3). Furthermore, OEA does not produce anxiety (fig. 4g) and although it inhibits exercise and motor responses to food, it does so at much higher doses than those producing hypophagia (fig. 4 h-i). This pharmacological profile distinguishes OEA from other appetite suppressants such as amphetamine and glucagon-like peptide 1, all of which often include anorexia, hyperactivity, anxiety and activation of the HPA axis, as well as endogenous cannabinoide, which stimulates food intake in semi-satiated animals, increases pain threshold, decreases body temperature and activity of the HPA axis (Pertwee, r.g., exp.opin.invest.drugs, 9: 1553-flaker 1571 (2000)).

TABLE 3 Effect of OEA on plasma hormone levels

B PRL LH

Excipient 212 + -2410.8 + -2.75.3 + -0.9

prana20 280±61 8.2±3.2 6.2±1.5

In table 3, plasma levels of adrenalone (B), Prolactin (PRL) and Luteinizing Hormone (LH) were determined by radioimmunoassay of plasma samples collected 60 minutes after injection of vehicle or pranamide (prana mg/kg) and reported in ng/ml, n-6-9/group.

OEA causes hypophagia at physiologically relevant doses. After administration of half the maximum effective dose (5mg/kg) for 1 hour, circulating levels of OEA (16.1 + -2.6 pmol/ml) were significantly higher than baseline levels (10.1 + -1.1; P < 0.05, student's t-test; n-5), but lower than those in 18-hour fasted animals (FIG. 1 a). Thus, the levels of OEA achieved in the blood during fasting are sufficient to produce a significant behavioral response.

Example 8 identification of body fat reducing Compounds of the invention

The following example illustrates how appetite suppressants can be identified using OEA as a positive control. The synthesis of OEA, the determination of body fat reduction and fatty acid oxidation are specifically discussed.

Synthesis of OEA

Oleoyl chloride was purchased from Nu-Check Prep (Elysian, MN) or formulated according to standard procedures. Oleoyl chloride was dissolved in methylene chloride (10mg/ml) and allowed to react with the equivalent of five ethanolamines at 4 ℃ for 15 minutes. The reaction was terminated by adding pure water. After vigorous stirring and separation of the phases, the upper aqueous phase was discarded and the organic phase was washed twice with water to remove unreacted ethanolamine. The resulting OEA is in N₂Concentrated to dryness in gas, reconstituted to 20mM chlorinated form and stored at-20 ℃ until use.

Determination of body fat loss by candidate Compounds

The ability of a compound to reduce body fat can be assessed in a number of ways. For example, rats are administered appropriate doses of OEA and/or candidate compounds by intraperitoneal injection. OEA and candidate compounds can be in 70% DMSO sterile saline solution, 5% Tween 80/5% propylene glycol sterile saline solution, or 10% Tween 80/10% ethanol/80% saline solution. OEA at 5mg/kg was used as a positive control. The amount of candidate compound administered may vary from 1-25 mg/kg. Typical doses of 1, 2, 5, 10, 15 and 20mg/kg of each candidate compound were administered to different groups of rats to determine which dose was optimal. Injections were administered 30 minutes prior to the animal's staple food for 7-14 days.

Determination of the effect of candidate compounds on total body weight body fat can be measured directly by using a skin caliper. The skin of the rat dorsal upper, abdominal, thoracic, front and rear legs can be clamped with calipers and measured before and during and every 48 hours after administration of OEA and/or candidate compounds. The difference in measured values at least two clamp sites reflects changes in the overall lipid of the rat.

Determination of fatty acid Oxidation by candidate Compounds

The effect of the compounds on fatty acid oxidation can also be determined. The effect of a candidate compound on fatty acid metabolism can be determined by measuring fatty acid oxidation in the primary culture of hepatocytes. Hepatocytes may be used to determine the rate of oxidation of oleic acid to ketone bodies and carbon dioxide. Such cells can be isolated from adult rat liver by enzymatic digestion, as described by Beynen et al in Disbetes 28: 828 (1979). Cells are typically cultured in suspension and incubated in Krebs-Henseleit's bicarbonate medium consisting of bovine serum and glucose, consisting of Guzman&Gellen, biochem.j.287: 487 (1992). The protein concentration of the cultured cells can be determined and the cells inoculated in 2ml of medium so that 4-6mg protein/ml appears in the reaction mixture. A cell and [ alpha ], [ alpha¹⁴C]Oleic acid (Amershamy) was incubated for 10 min with or without 10. mu.M OEA and the reaction was stopped with 200. mu.l of 2M perchloric acid and acid-soluble product extracted from chloroform/methanol/water (5: 1, vol.: vol.). Water phase material quiltRemoving and washing twice or more. Protein concentration was determined using the Lowry assay. The rate of conversion of oleic acid to ketone bodies can be expressed as nmol of oleic acid per mg of protein oxidation per hour and can be determined using liquid scintillation counting. Accordingly, OEA increased the oxidation of 21+ -6% oleic acid (n ═ 4, p < 0.01 and student's t-test control incubations).

Example 9 Effect of OEA on fatty acid metabolism

Oleoylethanolamide (OEA) reduces body weight not only by suppressing appetite but also by possibly increasing body fat metabolism. OEA was investigated for fatty acid oxidation in major body fat burning tissues (soleus muscle, liver, cultured cardiomyocytes and astrocytes). OEA clearly stimulated fatty acid oxidation in primary hepatocyte cultures, skeletal muscle (soleus) and heart cells, however, it was not effective on brain-derived astrocyte cultures. In addition, OEA caused mobilization of stored triglycerides in the major white adipose tissue cells. Table 4 details the method and the effect of OEA on fatty acid oxidation in these cells. The structure-activity relationship experiment provided the fact that the effect of OEA on skeletal muscle fatty acid oxidation was specific (figure 8). Thus, the effect of OEA is mimicked by the hydrolysis-resistant homologue methyl-OEA, only partially by Palmitoylethanolamine (PEA), but not by arachidonic Acid Ethanolamine (AEA) or Oleic Acid (OA). Briefly, these results indicate that OEA increases lipid oxidation and mobilization, and that the effects of OEA are localized to peripheral sites.

TABLE 4

Cell/tissue	Liver cell	Soleus muscle	Cardiac muscle cell	Astrocytes	Adipocytes
						Original	Adult rat liver	Hind limb of adult rat	Newborn rat heart	Neogenesis rat cerebral cortex	Adult rat epididymis
Separation step	Enzymatic digestion (Beynen et al 1979)	Dissection (Chiasson1980)	Enzymatic digestion (Flink et al 1992)	Enzymatic digestion (McCarthy)&De Vellis，1980)	Enzymatic digestion (Rodbell, 1964)
						Type of culture	Cell suspension	Tissue suspension	Cell monolayer	Cell monolayer	Cell suspension
Thermal insulation medium	Krebs-Henseleit's bicarbonate plus BSA and glucose (Guzman)&Geelen，1992)	Krebs-Henseleit's hepes plus BSA and glucose (Fruebis et al, 2001)	High-glucose DMEM plus BSA (Wu et al 2000)	Hams F12/DMEM plus insulin, transferrin, progesterone, putrescine and selenite (Blazquez et al, 1998)	Krebs-Henseleit's hepes plus BSA and glucose (Rodbell, 1965)
						Metabolic parameters	[14C]Oxidation of oleic acid to ketone bodies (Guz-man)&Geelen1992)	[14C]Oxidation of oleic acid to carbon dioxide (Fruebis et al 2001)	[14C]Oxidation of oleic acid to carbon dioxide (Blazquez et al, 1998)	[14C]Oxidation of oleic acid to ketone bodies (Blazquez et al, 1998)	Lipidation (Glycerol Release) (Serradeil-LeGal et al 2000)
Incubation time (minutes)	10	30	30	30	30
						Activation Effect of 10 μ M OEA	21±6(＝4)	36±10(n＝4)	37±9(n＝3)	2±6(n＝3)	38±16(n＝3)
Statistical significance vs	P＜0.01	P＜0.01	P＜0.01	Meaningless	P＜0.01

Reference for application: beynen AC et al, Disbetes 28: 828-835 (1979); blazquez C et al, j. neurochem 71: 1597 1606 (1998); chiasson RB "laboratory dissection of white mice" WBC, Dubu-Que, Iowa (1980); funk IL et al, j.biol.chem 267: 9917-; FruebisJ et al, ProcNil Acad Sci USA 98: 2005-2010 (2001); guzman M et al, biochem J287: 487-492 (1992); McCarthy KD et al, J Cell Biol 85: 890-902 (1980); rodshelm j.biol.chem 239: 375-; rodbell M Ann NY Acad Sci 131: 302-314 (1965); Serradeil-Le Gal C et al, FEBS Left 475: 150-156 (2000); wu W et al j.biol.chem 275: 40133-40119(2000).

Example 10 Effect of endogenous OEA in the intestinal tract

The effect of feeding on intestinal OEA biosynthesis was examined. High quality liquid chromatography/mass spectrometry revealed that the small intestine tissue of free-fed rats contained significant amounts of OEA (354. + -. 86 pmmol)

And/g, n is 3). Intestinal OEA is a marked drop in levels after fasting, but returns to baseline levels after refeeding. In contrast, no such change was found in the stomach (pmol/g; control 210 ± 20, hunger, 238 ± 84 hunger/refeeding, 239 ± 60, n ═ 3). Changes in intestinal OEA levels are accompanied by parallel changes in NAT activity, which is involved in OEA formation, but no changes in the activity of the fatty acid amide hydrolase that catalyzes OEA hydrolysis. These findings indicate that starvation and feeding, in contrast, regulate OEA biosynthesis in the small intestine. As with the source of intra-abdominal OEA, plasma levels of OEA in starved rats were found to be higher in the portal vein than in the vena cava (pmol/ml, portal vein, 14.6 ± 1.8; vena cava, 10.3 ± 2.8; n ═ 5). Now, the effect of other abdominal tissues on the formation of OEA cannot be ruled out. These results suggest that many interventions are fed with the OEA system. According to this model, feeding activates NAT activity to increase OEA biosynthesis in the small intestine and possibly also in other abdominal tissues. The newly generated OEA activates local sensory fibers which, as feedback, inhibit feeding by intervening brain structures such as NST and PVN.

Our results reveal the unexpected role of OEA in the peripheral regulation of feeding and provide a framework for the development of new drugs for the treatment of weight and body fat loss, for the prevention of weight or body fat gain, for the suppression of appetite or for the reduction of foraging behaviour or food intake, for the treatment of eating disorders, overweight or obesity. These include not only OEA analogues and homologues, but also those which control OEA levels by acting on OEA forming and hydrolyzing systems and enzymes as described above.

All publications and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference and were not presently found to be inconsistent with this specification.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims (48)

1. A method of reducing food intake in a mammal, said method comprising administering fatty acid alkanolamides to said mammal, wherein said administering is in an amount effective to reduce food intake in said mammal.

2. The method of claim 1, wherein the fatty acid alkanolamide is oleoylethanolamide.

3. The method of claim 1, wherein the fatty acid alkanolamide comprises a fatty acid moiety covalently attached to an ethanolamine moiety through an amide linkage.

4. The method of claim 3, wherein the fatty acid moiety is mono-unsaturated or poly-unsaturated.

5. The method of claim 1, wherein said administering is via a transdermal patch.

6. The method of claim 4, wherein the fatty acid moiety is oleic acid.

7. The method of claim 3, wherein the fatty acid moiety contains 12-20 carbon atoms.

8. The method of claim 3, wherein the fatty acid is selected from the group consisting of elaidic acid, palmitoleic acid, palmitic acid, linoleic acid, alpha-linolenic acid, and gamma-linolenic acid.

9. The method of claim 3, wherein the hydroxyl group of the ethanolamine moiety is substituted with a lower (C)₁-C₃) Alkyl groups are substituted to form the corresponding esters.

10. The method of claim 3, wherein the hydroxyl group of the ethanolamine moiety and lower (C)₂-C₆) The carboxyl groups of the alkyl carboxylic acids combine to form the corresponding esters.

11. The method of claim 3, wherein the fatty acid ethanolamines further comprise a lower (C) covalently bonded to the nitrogen atom of the fatty acid ethanolamines₁-C₃) An alkyl group.

12. The method of claim 1, wherein the mammal is a human.

13. The method of claim 1, wherein the fatty acid alkanolamide is palmitoylethanolamide.

14. The method of claim 1, wherein the fatty acid alkanolamides do not activate the cannabinoid CB2 or the cannabinoid CB1 receptor.

15. The method of claim 1, wherein said fatty acid alkanolamides and a pharmaceutically acceptable carrier are administered by an oral, rectal, topical, or parenteral route.

16. A method of reducing or controlling body fat or body weight in a mammal, said method comprising administering to said mammal a body fat or body weight reducing effective amount of a compound of formula (la)

Or a pharmaceutically acceptable salt thereof, wherein n is 0-5, and the sum of a and b can be 0-4; z is selected from the group consisting of-C (O) N (R)⁰)-；-(R⁰)NC(O)-；-OC(O)-；-(O)CO-；O；NR⁰And S; wherein R is⁰And R²Independently selected from unsubstituted or unsubstituted alkyl, hydrogen, C₁-C₆Alkyl and lower (C)₁-C₆) Acyl, wherein up to four hydrogen atoms of the fatty acid moiety and alkanol moiety are substituted by methyl or a double bond, the bond between carbons c and d may be unsaturated or saturated.

17. The method of claim 16, wherein the compound is of formula (la):

or pharmaceutically thereofAcceptable salts, wherein n is 0 to 4 and the sum of a and b is 1 to 3; wherein R is¹And R²Independently selected from hydrogen and C₁-C₆Alkyl, lower (C)₁-C₆) Acyl, wherein up to four hydrogen atoms of the fatty acid moiety and alkanolamine moiety are substituted by methyl or double bonds, and the bond between carbons c and d may be unsaturated or saturated.

18. The method of claim 17, wherein R¹And R²Independently selected from hydrogen and C₁-C₃Alkyl and lower (C)₁-C₃) An acyl group.

19. The method of claim 17, wherein a-1 and b-1.

20. The method of claim 17, wherein n-1.

21. The method of claim 17, wherein R¹And R²Are all H.

22. The method of claim 17, wherein the bond between carbons c and d is a double bond.

23. The method of claim 17, wherein the compound is oleoylethanolamide.

24. The method of claim 17, wherein the compound is palmitoylethanolamide.

25. The method of claim 17, wherein the route of administration is parenteral, oral, transdermal, rectal, or intranasal.

26. The method of claim 17, wherein the mammal is a human.

27. The method of claim 16, wherein the compound is one of the following formulas:

wherein n is 1 to 5 and the sum of a and b is 0 to 4; r²Selected from hydrogen, C₁-C₆Alkyl and lower (C)₁-C₆) An acyl group; and up to four hydrogen atoms of the fatty acid moiety and the alkanol moiety may be substituted by methyl or double bonds.

28. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of formula (la):

wherein n is 0 to 5 and the sum of a and b is 0 to 4; z is selected from the group consisting of-C (O) N (R)⁰)-；-(R⁰)NC(O)-；-OC(O)-；-(O)CO-；O；NR⁰And S, wherein R⁰And R²Independently selected from unsubstituted or unsubstituted alkyl, hydrogen, C₁-C₆Alkyl and lower (C)₁-C₆) Acyl, wherein up to four hydrogen atoms of the fatty acid moiety and alkanol moiety are substituted by methyl or a double bond, the bond between carbons c and d may be unsaturated or saturated.

29. The composition of claim 28, wherein the compound is of formula (la):

or a pharmaceutically acceptable salt of the compound, wherein n is 1 to 3 and the sum of a and b is 1 to 3; wherein R is¹And R²Independently selected from hydrogen C₁-C₆Alkyl and lower (C)₁-C₆) Acyl, wherein up to four hydrogen atoms of the fatty acid moiety and alkanol moiety are substituted by methyl or a double bond, the bond between carbons c and d may be unsaturated or saturated.

30. The composition of claim 29, wherein the composition is in the form of a unit dose comprising an amount of the compound effective to reduce or control body weight.

31. The composition of claim 29, wherein the dose is about 10-1000 mg.

32. The composition of claim 29, wherein the dose is about 1-100 mg.

33. The composition of claim 29 wherein the dose is about 100 and 500 mg.

34. The composition of claim 29, wherein the compound is palmitoylethanolamide.

35. The composition of claim 29, wherein the composition is a topical, oral, parenteral composition.

36. The composition of claim 29, wherein R¹And R²Independently selected from hydrogen and C₁-C₃Alkyl and lower (C)₁-C₃) An acyl group.

37. A composition according to claim 29, wherein a-1, b-1 and n-1.

38. The composition of claim 29, wherein the compound is oleoylethanolamide.

39. The composition of claim 29, wherein R¹And R²Are all H.

40. The composition of claim 30, wherein the bond between carbons c and d is a double bond.

41. The composition of claim 30, wherein the compound is palmitoylethanolamide.

42. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of formula (la):

wherein the sum of a and b is 0 to 4, up to four hydrogen atoms of the fatty acid moiety of the above formula may also be substituted by methyl or a double bond, and the bond between carbons c and d may be unsaturated or saturated. R represents a group selected from linear or branched alkylamines, cycloalkylamines, furans, tetrahydrofurans, pyrroles, pyrrolidines and pyrimidines; wherein the compound is administered to the mammal in an amount effective to reduce food intake.

43. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount of fatty acid alkanolamides to reduce body weight after administration to a mammal

44. The composition of claim 46, wherein the fatty acid moiety is oleic acid.

45. The composition of claim 46, wherein the fatty acid moiety has 12-20 atoms.

46. The composition of claim 46, wherein the fatty acid moiety is selected from the group consisting of elaidic acid, palmitoleic acid, palmitic acid, linoleic acid, alpha-linolenic acid, and gamma-linolenic acid.

47. A composition according to claim 46, wherein the alkanolamine of the alkanolamide is ethanolamine.

48. The composition of claim 46, wherein the composition is an enteric coated oral formulation.