WO2004012727A1 - Compositions comprenant des acides gras polyinsatures (pufas) utiles pour reguler l'appetit et gerer la masse corporelle - Google Patents

Compositions comprenant des acides gras polyinsatures (pufas) utiles pour reguler l'appetit et gerer la masse corporelle Download PDF

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WO2004012727A1
WO2004012727A1 PCT/US2003/023708 US0323708W WO2004012727A1 WO 2004012727 A1 WO2004012727 A1 WO 2004012727A1 US 0323708 W US0323708 W US 0323708W WO 2004012727 A1 WO2004012727 A1 WO 2004012727A1
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chain
pufa
long
dha
brain
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PCT/US2003/023708
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Nancy A. Auestad
Tina D. Wolf
Yung-Sheng Huang
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Abbott Laboratories
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
    • A61K31/232—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms having three or more double bonds, e.g. etretinate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents

Definitions

  • This invention relates to products, including nutritional supplements and formulas, that contain long chain polyunsaturated fatty acids (LCPs or LC-PUFAs), specifically n-3 LCPs; and to methods of using such products to control appetite and help treat and or prevent obesity and conditions of overweight, especially in a pediatric population.
  • LCPs or LC-PUFAs long chain polyunsaturated fatty acids
  • n-3 LCPs specifically n-3 LCPs
  • Treatment strategies include increasing physical activity and voluntary restriction of calories, in order to affect a negative energy balance.
  • Pharmaceutical interventions have also been attempted.
  • Prevention strategies emphasize balanced nutrition with a regimen of physical activity.
  • the present invention tests whether the quality of ingested lipids may play a role in regulation of appetite through n-6 and n-3 fatty acyl compounds formed in brain.
  • Endocannabinoids are a class of naturally occurring compounds that exhibit cannabimimetic properties such as: analgesia, hyperphasia, alteration of cognition and motor control, among other physiological effects including appetite.
  • analgesia a fatty acyl derivative that to bind to the cannabinoid receptors, better known as CBi and CB 2 .
  • CBi and CB 2 fatty acyl derivatives that to bind to the cannabinoid receptors.
  • These fatty acyl derivatives are families of compounds, N- acylethanolamines ( ⁇ AEs) and monoacylglycerols (MAGs; Mechoulam et al, 1998).
  • Arachidonyl ethanolamine 20:4n-6 ⁇ AE
  • Arachidonyl ethanolamine 20:4n-6 ⁇ AE
  • 20:4n-6 MAG is made up of arachidonic acid and glycerol and has recently been demonstrated to increase food intake when injected into rat brain (Kirkham et al, 2002).
  • Other fatty acyl compounds in the n-6 and n-3 families also bind to the CB receptor, namely those with >20 carbons and at least 3 double bonds (Mechoulam et al, 1998).
  • Arachidonic acid (AA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3) can be made in vivo through the process of desaturation and elongation of the essential fatty acids, linoleic acid and linolenic acid, or obtained from the diet.
  • Studies with animal models during the 'brain growth spurt' have shown that varying the levels of dietary essential fatty acids and long-chain n-6 and n-3 polyunsaturated fatty acids results in corresponding changes in the long-chain n-6 and n-3 fatty acids in brain, particularly AA and DHA (Ward et al, 1998 and 1999; de la Presa Owens and Innis, 1999 and 2000).
  • One recent study has demonstrated in formula fed piglets that dietary AA and DHA result in increases in corresponding n-6 and n-3 NAEs and some monoacylglycerols in brain (MAGs; Berger et al, 2001).
  • CB ⁇ carmabinoid receptor
  • Overweight is defined by the Centers for Disease Control as an increased body weight in relation to height compared to accepted standards for desirable weight; obesity is defined as an excessive amount of body fat in relation to lean muscle mass (CDC, 2002). Overweight and obesity are more commonly defined as having a body mass index (weight/height ) of between 25 and 29.9 or >30, respectively (CDC, 2002).
  • the regulation of food intake is a highly complex process controlled to a large extent by the hypothalamus in the brain.
  • Neural control of energy intake for maintenance of body weight involves a complex integration of neuronal, hormonal, sensory, and thermoregulatory signals from the periphery and within various regions in brain (Williams et al, 2000; Hovel, 2001; van Dijk et al, 2000; Berthoud, 2000).
  • leptin and insulin are hormones known to provide the brain with information about the amount of fat stored in the body (van Dijk et al, 2000). Thus leptin and insulin help to regulate food intake.
  • Leptin is a peptide hormone secreted from adipose cells. The amount of leptin secreted has been shown to be directly proportional to the amount of fat in storage.
  • Insulin is also a peptide hormone that is secreted from pancreatic B cells and plays a central role in controlling glucose homeostasis and lipid utilization and storage. The amount of insulin secreted at any given time is also directly proportional to the size of body fat stores. Both leptin and insulin act through receptors in the hypothalamus of the brain.
  • ⁇ 9 -tetrahydrocarmabinol The active ingredient in cannabis, ⁇ 9 -tetrahydrocarmabinol (THC), has appetite stimulating effects, and is prescribed by some doctors to help patients retain weight (Mechoulam and Fride, 2001). Due to the biological effects of ⁇ 9 -THC, which are mediated by a specific cannabinoid receptor, referred to as the CB ⁇ receptor, researchers began to look for endogenous compounds, endocannabinoids. In the early 1990s, a family of bioactive fatty-acyl compounds that exhibited neuromodulator activity at the cannabinoid receptor was identified (Devane et al, 1992; Hanus et al, 1993). Later another family was identified, monoacylglycerols or MAGs, that exhibited neuromodulatory activity at cannabinoid receptors (Sugiura et al, 1995; Mechoulam et al, 1995).
  • CBi and CB 2 receptors cannabinoid receptors
  • CBi receptors are found primarily in the brain, with some mRNA expressed also in the peripheral organs (adrenal gland, heart, lung, prostate, uterus, ovary, testis, bone marrow, thymus, tonsils, and testis).
  • CB receptors have been found in immune system cells (Buckley et al, 1998).
  • this compound anandamide more commonly referred to now as N-arachidonyl ethanolamine (20:4n-6 ⁇ AE). They purified 20:4n-6 ⁇ AE and tested the cannabimimetic pharmacological activity by measuring the ability to inhibit the twitch response of isolated murine vas deferentia, a standard model to investigate the mode of action of psychotropic agents.
  • the structure of anandamide was determined by mass spectrometry and nuclear magnetic resonance.
  • the chemical name for 20:4n-6 ⁇ AE is [5,8,11,14-eicosatetraenamide, (N-2-hydroxyethyl)-(all-Z)].
  • Ananadamide and its effects are also described in WO 2001/24645 Al (Nestle, 2001).
  • 20:4n-6 MAG has also been shown to bind to the CBi and CB 2 receptors and exhibit cannabimimetic activities both in vitro and in vivo. While most of the research on the specific roles of the endocannabinoids that bind to the CB ⁇ -receptor in brain has been associated with 20:4n-6 NAE, other fatty acyl NAEs and MAGs also bind to the CBi- receptor (Mechoulam et al, 1998) and may play a role in central nervous system regulation of food intake (Di Marzo et al, 2001 ; Berger et al, 2001 ; Kirkham et al, 2002).
  • NAE has been found in many species including rat, pig, cow, and human, and in many tissues (Schmid et all995; Felder et al, 1996; Kondo et al, 1998; Bisogno et al, 1999;
  • NAEs have been found in tissues where C i receptors are found, including brain, kidney, spleen, testis, skin, blood plasma, and uterus. They are present in concentrations ranging from none detected to 29 pmol/g in rat brain (Mechoulam et al,
  • NAE biosynthesis involves the Ca 2+ -dependent transfer of a fatty acyl chain from the sn- 1 position of a phosphatidylcholine to the primary amine of phosphatidylethanolamine, forming N-acylphosphatidylethanolamine (NAPE) and lyso-phosphatidylcholine (Patricelli and Cravatt 2001). NAPE is subsequently hydrolyzed by a phospholipase D-like enzyme to yield the corresponding NAE and phosphatidic acid. These two reactions are thought to be tightly coordinated.
  • the proposed mechanism for MAG biosynthesis is similar to that for NAE as has been shown to be Ca + -dependent (Mechoulam et al, 1998).
  • a phosphoinositide-specific phospholipase C causes the release of diacylglycerol and a inositol-triphosphate, which is subsequently hydrolyzed to yield MAG by sn-1 -diacylglycerol lipase (Ameri, 1999).
  • NAEs and MAGs After release from the phospholipid membrane, NAEs and MAGs are available to bind to the CBI receptor. They are also hydrolyzed rapidly by a membrane bound enzyme called fatty acyl amide hydrolase (FAAH) or sometimes referred to as 'anandamide [20:4n-6 NAE] hydrolase' (Patricelli and Cravatt, 2001; Goparaju et al, 1998; Giang and Cravatt, 1997). Giuffrida et al (2001) have proposed that 20:4n-6 NAE and 20:4n-6 MAG are hydrolyzed by a two-step process involving enzymatic hydrolysis after transport by a specific carrier into the site of degradation.
  • FAAH fatty acyl amide hydrolase
  • a carrier-mediated transport of NAEs and MAGs into cells has been proposed based on a fast rate of action, temperature dependence, saturability, and substrate selectivity.
  • FAAH is the key enzyme involved in hydrolysis of these endocannabinoids.
  • FAAH appears to be a general hydrolytic enzyme, acting on many biologically active lipids and esters (Giuffrida et al, 2001).
  • 20:4n-6 NAE is hydrolyzed to free arachidonic acid and ethanolamine by FAAH.
  • 20:4n-6 MAG is broken down into free arachidonic acid and glycerol through FAAH enzymatic action.
  • Another mechanism of degradation for MAGs has been suggested, possibly a monoacylglycerol lipase, although this has not been firmly established.
  • Ward et al demonstrated 'dose' related effects of feeding varying amounts of 20:4n-6 and 22:6n-3 in a rat milk formula.
  • Rat pups were fed one of three levels of 20:4n-6 and 22:6n-3 (0%, 0.4%, or 2.4% total fatty acids) using a 3 x 3 design from postnatal day 5 through 18 by gastrostomy tube.
  • the formulas contained what were considered adequate amounts of the essential fatty acids, 10% of total fatty acids as 18:2n-6 and 1% as 18:3n-3.
  • the red blood cell and brain phospholipid membrane fatty acids generally reflected the fatty acid composition of the supplemented formula fed.
  • NAEs and MAGs in brain 20:4n-6 NAE increased 4-fold, 20:5n-3 NAE increased 5-fold, 22:5n-3 and 22:6n-3 NAE increased 9-10-fold, 22:4n-6 MAG and 22:6n-3 MAG increased nearly 2-fold; whereas 20:4n-6 MAG did not increase.
  • dietary fatty acids modulate NAE levels by changing levels of NAE precursors or by providing substrate for biosynthesis.
  • mice were randomly assigned to vehicle or 20:4n-6 ⁇ AE treatment. The mice were given food cakes weighed before and after feeding, including spillage. The mice were fed for 7 days for 2.5 hours per day (between 9am and 12pm). Ten minutes before feeding, 0.001, 0.7, or 4 mg/kg of 20:4n-6 ⁇ AE in vehicle or vehicle alone was injected intraperitoneally in a volume of O.lmL/lOg of body weight.
  • the 0.001 mg/kg 20:4n-6 ⁇ AE treated group also showed improved cognitive function and reversal of most effects of severe food restriction.
  • the other two 20:4n-6 ⁇ AE treated groups did not show any significant change.
  • Fride et al studied the effects of blocking CBi receptor activity in suckling mouse pups.
  • mice On postnatal day 1 or 2, mice were injected intraperitoneally with 20 mg/kg of a CB ! receptor antagonist (SR141716A). The researchers observed overwhelming effects on mortality. Injecting the antagonist on postnatal day 1 resulted in death in all rat pups by day 4 and injecting it on postnatal day 2 resulted in death in 50% of the rat pups.
  • mouse pups were injected with 20 mg/kg of the antagonist daily from postnatal day 2 through day 8. All of the pups immediately stopped gaining weight and died by day 8.
  • CBi receptor knockout mice and wild-type controls were given an injection intraperitoneally of vehicle or the CBi receptor antagonist after fasting for 18 hours.
  • CBi receptor knockout mice given vehicle ate significantly less than wild-type controls.
  • the CB preceptor antagonist decreased food intake in wild-type controls to the level of food intake of the CBi receptor knockout mice given vehicle; administration of the antagonist to the CBi receptor knockout mice resulted in no changes in food intake.
  • Arbuckle LD and Innis SM Docosahexaenoic acid is transferred through maternal diet to milk and to tissues of natural milk-fed piglets. J Nutr, 123(10):1668-1675, (1993). Auestad N, Korsak RA, Bergstrom JD, and Edmond J. Milk-substitutes comparable to rat's milk; their preparation, composition, and impact on development and metabolism in the artificially reared rat. British Journal of Nutrition, 61: 495-518, (1989).
  • Di Marzo V De Petrocellis L, Bisogno T, and Melck D. Metabolism of anandamide and 2- arachidonoylglycerol: an historical overview and some recent developments. Lipids, 34:S319-S325, (1999). Di Marzo V, Goparaju SK, Wang L, Liu J, Batkal S, Jaral Z, Fezza F, Miura GI, Pahniter RD, Sugiura T, and Kunos G. Leptin-regulated endocannabinoids are involved in maintaining food intake. Nature, 410:822-825, (2001).
  • Fride E Ginzburg Y, Breuer A, Bisogno T, Di Marzo N, Mechoulam R. Critical role of the endogenous cannabinoid system in mouse pup suckling and growth. European Journal of Pharmacology, 419:207-214, (2001).
  • Giang DK and Cravatt BF Molecular characterization of human and mouse fatty acid amide hydrolases. Proc. ⁇ atl. Acad. Sci., 94:2238-2242, (1997). Giuffrida A, Beltramo M, and Piomelli D. Mechanisms of endocannabinoid inactivation: biochemistry and pharmacology. Journal of Pharmacology and Experimental Therapeutics, 298(1):7-14, (2001).
  • Peripheral signals conveying metabolic information to the brain: short-term and long-term regulation of food intake and energy homeostasis. Exp Bio Med, 226(11):963-977, (2001).
  • Heird WC Parental feeding behavior and children's fat mass. Am J Clin ⁇ utr, 75:451 -452, (2002).
  • Arachidonoylglycerol an endogenous cannabinoid receptor agonist: identification as one of the major species on monoacylglycerols in various rat tissues, and evidence for its generation through Ca 2+ -dependent and -independent mechanisms. FEBS Letters, 429:152-156, (1998).
  • Makrides M Neumann MA, Byrad RW, Simmer K, and Gibson RA. Fatty acid composition of brain, retina, and erythrocytes in breast- and formula-fed infants. Am J Clin Nutr, 60(2): 189-194, (1994). McLaughlin CR, Martin BR, Compton DR, and Abood ME. Cannabinoid receptors in developing rats: detection of mRNA and receptor binding. Drug and Alcohol Dependence, 36:27-31, (1994).
  • Arachidonylglycerol a possible endogenous cannabinoid ligand in brain. Biochem. Biophys. Res. Commun, 215:89-97, (1995).
  • Wainwright PE Effects of ⁇ -linolenic acid and docosahexaenoic acid in formulae on brain fatty acid composition in artificially reared rats. Lipids, 34: 1057-1063,
  • the invention comprises a method for decreasing the appetite of a mammal comprising enterally administering to said mammal an amount of long-chain n-3 PUFA effective to decrease the appetite of said mammal.
  • the invention comprises a method for antagonizing the CBi receptor in the brain of a mammal comprising enterally administering to said mammal an amount of long-chain n-3 PUFA effective to antagonize the CBi receptor activity in the brain of said mammal.
  • the invention comprises a method for decreasing the incidence of obesity or overweight status in a population of mammals comprising enterally administering to at least some members of said population an amount of long-chain n-3 PUFA effective to modulate negatively the appetite of said mammal.
  • the invention comprises a method for increasing serum leptin levels in humans or other mammals, preferably to reduce appetite as a result of, in whole or in part, the serum leptin level increase, more preferably to also reduce the incidence or extent of obesity in such humans or other mammals.
  • the leptin level increase is preferably determined by postprandial or fasting serum measurement following administration of a DHA-containing nutritional composition (or other long chain n-3 PUFA-containing composition) relative to a similar measurement following administration of a similar nutritional composition but without DHA (or other similar long chain n-3 PUFA).
  • the long-chain n-3 PUFA preferably comprises DHA, and more preferably is administered to a child or adult in a daily amount of from about 84 to about 15,832 mg.
  • a preferred long-chain n-3 PUFA is DHA; and this may be administered independent of AA.
  • the long-chain n-3 PUFA is administered during a growth phase.
  • the long-chain n-3 PUFA is administered prior to or in conjunction with an appetite-impacting stimulus.
  • the preferred effective dosing levels are about 8 to about 396 mg/kg/day for an infant, (preferably about 127 to 165 mg/kg/day); about 84 to about 11610 mg/day for a child up to age 15 and about 84 to about 15,832 mg/day for an adult. More preferred levels are included herein.
  • the invention comprises a method for modulating the appetite of a mammal comprising enterally administering to said mammal an amount of long-chain n-3 PUFA and an amount of long-chain n-6 PUFA in relative amounts effective to modulate the appetite of said mammal.
  • the long-chain n-3 PUFA preferably comprises DHA and the long-chain n-6 PUFA preferably comprises AA.
  • the long-chain n-3 PUFA is administered during a growth phase.
  • the long-chain n-3 PUFA is administered prior to or in conjunction with an appetite-impacting stimulus.
  • the preferred effective dosing levels are about 8 to about 396 mg/kg/day for an infant, (preferably about 127 to 165 mg/kg/day); about 84 to about 11610 mg/day for a child up to age 15 and about 84 to about 15,832 mg/day for an adult. More preferred levels are included herein.
  • Fatty acids are an important component of nutrition. Fatty acids are carboxylic acids and are classified based on the length and saturation characteristics of the carbon chain. Long chain fatty acids have from 16 to 24 or more carbons and may also be saturated or unsaturated. In longer fatty acids there may be one or more points of unsaturation, giving rise to the terms "monounsaturated” and "polyunsaturated", respectively. Long chain polyunsaturated fatty acids, (LCP's or LC-PUFAs) having 20 or more carbons are of particular interest in the present invention.
  • LCP's or LC-PUFAs Long chain polyunsaturated fatty acids having 20 or more carbons are of particular interest in the present invention.
  • LC-PUFAs are categorized according to the number and position of double bonds in the fatty acids according to a nomenclature well understood by the biochemist. There are two main series or families of LC-PUFAs, depending on the position of the double bond closest to the methyl end of the fatty acid: the n-3 series contains a double bond at the third carbon, while the n-6 series has no double bond until the sixth carbon. Thus, arachidonic acid (“AA” or "ARA”) has a chain length of 20 carbons and 4 double bonds beginning at the sixth carbon. As a result, it is referred to as "20:4 n-6".
  • DHA docosahexaenoic acid
  • AA and DHA are of particular importance in the present invention.
  • LCPs are the Cl 8 fatty acids that are precursors in these biosynthetic pathways, as is described in US Patent 5,223,285.
  • LA linoleic
  • GLA intermediates ⁇ -linolenic
  • DHGLA dihomo- ⁇ - linolenic
  • ⁇ - linolenic (18:3n-3, "ALA” intermediates stearodonic (18:4n-3) and EPA (20:5n-3) are important precursors to DHA (22:6n-3).
  • a glyceride is such an ester of one or more fatty acids with glycerol (1,2,3-propanetriol). If only one position of the glycerol backbone molecule is esterified with a fatty acid, a "monoglyceride” is produced; if two positions are esterified, a “diglyceride” is produced; and if all three positions of the glycerol are esterified with a "triglyceride” or "triacylglycerol" is produced.
  • a phospholipid is a special type of diglyceride, wherein the third position on the glycerol backbone is bonded to a nitrogen containing compound such as choline, serine, ethanolamine, inositol, etc., via a phosphate ester.
  • Triglycerides and phospholipids are often classified as long chain or medium chain, according to the fatty acids attached thereto.
  • a "source" of fatty acids may include any of these forms of glycerides from natural or other origins.
  • Lipid is a general term describing fatty or oily components. In nutrition, lipids provide energy and essential fatty acids and enhance absorption of fat soluble vitamins. The type of lipid consumed affects many physiological parameters such as plasma lipid profile, cell membrane and organ lipid composition and synthesis of mediators of the immune response such as prostaglandins and thromboxanes. Other physiological effects of lipids are described in the background.
  • Sources of longer LCPs include dairy products like eggs and butterfat; marine oils, such as cod, menhaden, sardine, tuna and many other fish; certain animal fats, lard, tallow and microbial oils such as fungal and algal oils as described in detail in US Patents 5,374,657, 5,550,156, and 5,658,767.
  • fish oils are a good source of DHA and they are commercially available in "high EPA” and "low EPA” varieties, the latter having a high DHA:EPA ratio, preferably at least 3:1.
  • Algal oils such as those from dinoflagellates of the class Dinophyceae, notably Crypthecodinium cohnii are also sources of DHA (including DHASCO TM), as taught in US Patents 5,397,591, 5,407,957, 5,492,938, and 5,711,983.
  • DHA including DHASCO TM
  • the genus Mortierella, especially M. alpina , and Pythium insidiosum are good sources of AA, including ARASCOTM as taught by US Patent 5,658,767 and as taught by Yamada, et al. J. Dispersion Science and Technology, 10(4&5), pp561-579 (1989), and Shinmen, et al. Appl. Microbiol. Biotechnol. 31 :11-16 (1989).
  • LCPs LCP-containing oils
  • Desaturase and elongase genes have been identified from many organisms and these might be engineered into plant or other host cells to cause them to produce large quantities of LCP-containing oils at low cost.
  • the use of such synthetic or recombinant oils are also contemplated in the present invention.
  • the present invention is utilized in combination with an environmental stress or stimulus.
  • an environmental stress or stimulus Studies in rodents have shown that mild to moderate stressors result in increased food intake, while a more severe stress does not (Harris et al 2000).
  • the effect of stress on food intake depends on the duration of the stressor and includes both physical and psychological stressors. Mild stressors known to elicit increased food intake in rats include tail pinch, a brief period of restraint or handling, food restriction, and sleep deprivation.
  • the food restriction periods in the present study represent such mild stressors that elicited an appetitive response. This was most apparent following the overnight 40% food restriction period on day 19, and less so following the overnight fast on day 20.
  • the differences in appetitive response following the different food restriction paradigms may be explained by limited sample size, an adaptive response to the fasting/feeding paradigm, or the latter (overnight fast) exceeded a mild/moderate stress threshold.
  • the dietary fatty acids of the present invention may be given in many forms, including but not limited to, nutritional products, dietary supplements, pharmaceuticals or other products. They may be used at any age, for example by infants, children or adults. There may be particular value in using them during periods of rapid growth, such as infancy, childhood and adolescence.
  • the dietary fatty acids of the invention may be incorporated into a nutritious "vehicle or carrier" which includes but is not limited to the FDA statutory food categories: conventional foods, foods for special dietary uses, dietary supplements and medical foods.
  • Nutritional products contain macronutrients, ie. fats, proteins and carbohydrates, in varying relative amounts depending on the age and condition of the intended user, and often contain micronutrients such as vitamins, minerals and trace minerals.
  • the term "nutritional product” includes but is not limited to these FDA statutory food categories: conventional foods, foods for special dietary uses, medical foods and infant formulas.
  • Foods for special dietary uses are intended to supply a special dietary need that exists by reason of a physical, physiological, pathological condition by supplying nutrients to supplement the diet or as the sole item of the diet.
  • a “medical food” is a food which is formulated to be consumed or administered enterally under the supervision of a physician and which is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation.
  • a "dietary supplement” is a product intended to supplement the diet by ingestion in tablet, capsule or liquid form and is not represented for use as a conventional food or as a sole item of a meal or the diet.
  • Infant formula refers to nutritional formulations that meet the standards and criteria of the Infant Formula Act, (21 USC ⁇ 350(a) et. seq.) and are intended to replace or supplement human breast milk. Although such formulas are available in at least three distinct forms (powder, liquid concentrate and liquid ready-to-feed (“RTF”), it is conventional to speak of the nutrient concentrations on an "as fed” basis and therefore the RTF is often described, it being understood that the other forms reconstitute or dilute according to manufacturer's directions to essentially the same composition and that one skilled in the art can calculate the relevant composition for concentrated or powder forms.
  • "Standard" or “Term” infant formula refers to infant formula intended for infants that are born full term as a first feeding.
  • the protein, fat and carbohydrate components provide, respectively, from about 8 to 10, 46 to 50 and 41 to 44% of the calories; and the caloric density ranges narrowly from about 660 to about 700 kcal/L (or 19-21 Cal/fl.oz.), usually about 675 to 680 (20 Cal/fl.oz.).
  • the distribution of calories among the fat, protein and carbohydrate components may vary somewhat among different manufacturers of term infant formula.
  • SIMILACTM Ross Products Division, Abbott Laboratories
  • ENFAMILTM Mead Johnson Nutritionals
  • GOOD STARTTM(Carnation) are examples of term infant formula.
  • Nutrient-enriched formula refers to infant formula that is fortified relative to "standard” or "term” formula.
  • the primary defining characteristic that differentiates nutrient-enriched formulas is the caloric density; a secondary factor is the concentration of protein.
  • a formula with a caloric density above about 700 Kcal/L or a protein concentration above about 18 g/L would be considered “nutrient-enriched”.
  • Nutrient- enriched formulas typically also contain higher levels of calcium (e.g. above about 650 mg/L) and/or phosphorus (e.g. above about 450 mg/L). Examples include Similac NEOSURETM and Similac Special CareTM formulas.
  • Dietary supplements are soft gels, capsules, powders, tablets, liquids and other dosage forms with specific nutrients that are generally intended to support the normal structure and function of the body. Dietary supplements may be formulated with suitable excipients and carriers, much like standard pharmaceutical products.
  • Soft gels are widely used in the pharmaceutical industry as an oral dosage form containing many different types of pharmaceutical and vitamin products. Soft gels are available in a great variety of sizes and shapes, including round shapes, oval shapes, oblong shapes, tube shapes and other special types of shapes such as stars. The finished capsules or soft gels can be made in a variety of colors, with or without opacifiers. Soft gels are predominantly employed for enclosing liquids, more particularly oily solutions, suspensions or emulsions. Filling materials normally used are vegetable, animal or mineral oils, liquid hydrocarbons, volatile oils and polyethylene glycols.
  • the soft gelatin capsules can be manufactured using techniques well known to those skilled in the art.
  • U.S. Patents 4,935,243, 4,817,367 and 4,744,988 are directed to the manufacturing of soft gelatin capsules. Manufacturing variations are certainly well known to those skilled in the pharmaceutical sciences.
  • these comprise an outer shell primarily made of gelatin, a plasticizer, and water, and a fill contained within the shell.
  • the fill may be selected from any of a wide variety of substances that are compatible with the gelatin shell.
  • a gelatin capsule manufacturing system is comprised of three main systems: a sheet forming unit, a capsule forming unit, and a capsule recovery unit.
  • Melted gelatin is formed into sheets of desired thickness which is inserted between a pair of die rolls fitted with the desired die heads in the capsule-forming unit.
  • a fill nozzle is positioned so as to discharge the desired amount of fill liquid between two gelatin sheets.
  • the discharging timing is adjusted so that the recess formed by the die heads are filed with fill liquid as the gelatin sheets are brought into contact with each other, which allows filled capsules to be formed.
  • Die roll scraping brushes remove the formed gelatin capsules from the die heads.
  • the gelatin capsules are subsequently collected into a bulk container for storage prior to filing into the desired container.
  • Fro dry-filled capsules the two halves of the shell may be formed separately and sealed after filling. Tablets are generally formed by compression of the active ingredient, often as a
  • pharmaceutically acceptable salt along with binders, lubricants and other excipients in a die and mold. Additional details of capsule and tablet formation can be obtained in any of several texts on this topic, including Remington's Pharmaceutical Sciences, XV edition (1975). Pharmaceutically acceptable salts are well-known in the art. For example, S. M.
  • Representative acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsufonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate.
  • the basic nitrogen-containing groups can be quarternized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.
  • lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
  • dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates
  • long chain halides such as decyl,
  • Basic addition salts can be prepared in situ during the final isolation and purification of the compounds by reacting a carboxylic acid-containing moiety with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal action or with ammonia or an organic primary, secondary or tertiary amine.
  • a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal action or with ammonia or an organic primary, secondary or tertiary amine.
  • Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like and nontoxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine and the like.
  • Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like.
  • the level of a particular fatty acid in a formula is typically expressed as percent of the total fatty acids. This percentage multiplied by the absolute concentration of total fatty acids in the formula (either as g/L or g/100 kcal) gives the absolute concentration of the fatty acid of interest (in g/L or g/100 kcal, respectively). Total fatty acids may be estimated as about 95%) of total fat to account for the weight of the glycerol backbone. Conversion from mg/100 kcal to mg/L is a simple calculation dependant on the caloric density as is known to those skilled in the art.
  • Nutritional compositions enriched in DHA according to the invention may provide from 100%), in the case of a sole source feeding such as infant formula, to less than about 5% of daily caloric intake, in the case of a conventional snack food. If formula is fed to newborns it may be complemented with some human milk. And as the infant gets to about 2-4 months, solid foods often begin to supply some of the calories and the amount of formula may decrease as a percent of total caloric intake. Any nutritive or caloric component of supplements or pharmaceuticals is usually de minimis and disregarded.
  • DHA dosing with a mild stressor as noted above.
  • the preferred time to feed the DHA enriched diets is when accretion of the long- chain n-6 and n-3 fatty acids is the fastest — i.e. during infancy, childhood and adolescence. The most rapid rate of brain growth occurs in infancy. However, brain growth and neuronal maturation continues until about 12-20 years of age The fatty acid content in adult brains also can be affected by diet in adults but over a longer time frame. Table B, below gives ranges and preferred ranges for effective dosing of DHA in accordance with the invention.
  • the effective dose of the ingredient, DHA does not differ whether given as part of a nutritional product, as a supplement or as a pharmaceutical.
  • a range of effective dosing for a child age 1-5 years is 84 to 9499 mg per day, preferably 84 to 3800 mg per day and most preferably 253 to 2322 mg per day.
  • Comparable values for an adult are 84-15832, preferably 84 - 6333, most preferably 253 - 3166. Note that values are given in mg /day for children and adults, and in mg/kg/day for infants.
  • comparable values for an infant up to about 6 months of age are: 8 - 380 mg/kg/day, preferably 8 -165 mg/kg/day, and most preferably 13 - 89 mg/kg/day.
  • liquid and powder nutritional products of the present invention can be manufactured by generally conventional techniques known to those skilled in the art. Briefly, three slurries are prepared, blended together, heat treated, standardized, spray dried (if applicable), packaged and sterilized (if applicable).
  • a carbohydrate/mineral slurry is prepared by first heating water to an elevated temperature with agitation. Minerals are then added. Minerals may include, but are not limited to, sodium citrate, sodium chloride, potassium citrate, potassium chloride, magnesium chloride, tricalcium phosphate, calcium carbonate, potassium iodide and trace mineral premix.
  • a carbohydrate source such as one or more of lactose, corn syrup solids, sucrose and/or maltodextrin is dissolved in the water, thereby forming a carbohydrate solution.
  • a source of dietary fiber, such as soy polysaccharide, may also be added. The completed carbohydrate/mineral slurry is held under agitation at elevated temperature until it is blended with the other slurries, preferably for no longer than about twelve hours.
  • An oil slurry is prepared by combining and heating the basic oil blend.
  • the basic oil blend typically contains some combination of soy, coconut, palm olein, high oleic safflower or sunflower oil and medium chain triglycerides.
  • Emulsifiers such as diacetyl tartaric acid esters of mono, diglycerides, soy mono, diglycerides, and soy lecithin may be used.
  • Any or all of the oil-soluble vitamins A, D, E ( natural R,R,R form or synthetic) and K may be added individually or as part of a premix.
  • Beta carotene which can function as an in vivo antioxidant, may also be added, as may a stabilizer such as carrageenan.
  • Oils containing specific LCPs important to this invention can be added to the oil slurry. Care must be used with these LCPs since they easily degrade and become rancid.
  • the completed oil slurry is held under agitation until it is blended with the other slurries, preferably for a period of no longer than about twelve hours.
  • a protein in water slurry is prepared by first heating water to an appropriate elevated temperature with agitation.
  • the protein source is then added to the water with agitation.
  • this protein source is intact or hydrolyzed milk proteins (e.g. whey, casein), intact or hydrolyzed vegetable proteins (e.g. soy), free amino acids and mixtures thereof.
  • any known source of amino nitrogen can be used in this invention.
  • the completed protein slurry is held under agitation at elevated temperature until it is blended with the other slurries, preferably for a period no longer than about two hours.
  • some protein may be mixed in a protein-in-fat emulsion rather than protein-in- water.
  • the protein in water and carbohydrate/mineral slurries are blended together with agitation and the resultant blended slurry is maintained at an elevated temperature.
  • the oil slurry is added to the blended slurry from the preceding step with agitation.
  • the LCP oils can be added directly to the blend resulting from combining the protein, carbohydrate/mineral and oil slurries.
  • the pH of the completed blend is adjusted to the desired range.
  • the blended slurry is then subjected to deaeration, ultra-high temperature heat treatment, emulsification and homogenization, then is cooled to refrigerated temperature.
  • appropriate analytical testing for quality control is conducted. Based on the analytical results of the quality control tests, and appropriate amount of water is added to the batch with agitation for dilution.
  • a vitamin solution containing water soluble vitamins and trace minerals (including sodium selenate), is prepared and added to the processed slurry blend with agitation.
  • a separate solution containing nucleotides is prepared and also added to the processed blended slurry with agitation.
  • the pH of the final product may be adjusted again to achieve optimal product stability.
  • the completed product is then filled into the appropriate metal, glass or plastic containers and subjected to terminal sterilization using conventional technology.
  • the liquid product can be sterilized aseptically and filled into plastic containers.
  • a carbohydrate/mineral slurry is prepared as was described above for liquid product manufacture.
  • a protein in water slurry is prepared as was described above for liquid product manufacture.
  • the carbohydrate/mineral slurry, protein in water slurry and oil slurry are blended together in a similar manner as described for liquid product manufacture.
  • LCPs are then added to the blended slurry with agitation.
  • the LCPs are slowly metered into the product as the blend passes through a conduit at a constant rate just prior to homogenization (in-line blending).
  • the processed blend may be evaporated to increase the solids level of the blend to facilitate more efficient spray drying.
  • the blend then passes through a preheater and a high pressure pump and is spray dryed using conventional spray drying technology.
  • the spray dryed powder may be agglomerated, and then is packaged into metal or plastic cans or foil/laminate pouches under vacuum, nitrogen, or other inert environment.
  • Pharmaceutical dosage forms may be useful for both drug and dietary supplement forms. They are well known to those skilled in the art, and include tablets, capsules, pills, powders, and other forms. Methodologies for making each of these dosage forms is well known and, except as noted in an earlier section, will not be repeated here.
  • the plan was to artificially rear rat pups on different n-6 and n-3 formulas and then assess food intake after weaning them onto a semi-solid food.
  • One group of rats was reared and tested in February and the other in April 2002.
  • the intent was to combine the February and April results, however methodological problems (described below) limited the reliability of some of the results from February.
  • the April dataset is reliable and complete.
  • the artificial rearings and food intake studies took place at the University of California, Los Angeles, in Dr. John Edmond' s laboratory, kindly under the care of Rose Korsak, who were both blind to the composition of the different rat milk formulas and feeding groups.
  • the dietary levels of AA and DHA in the present study were chosen based on published data from Ward et al (1998) and Wainwright et al (1999) who studied similar levels of AA and DHA using the same gastronomy reared rat model.
  • the levels of AA and DHA studied were 0%> and 2.5% total fatty acids alone or in combination; a fourth group was fed no AA or DHA (Table 3.1 and Table 3.2). This phase of the study is the AA and DHA (feeding) rearing phase.
  • the base formula was designed to contain marginally adequate levels of linoleic acid and undetectable levels of ⁇ -linolenic acid as has been used in studies of rats (Wainwright et al, 1999) and piglets (de la Presa Owens and Innis, 1999 and 2000) to maximize differences in brain levels of AA and DHA among the four experimental groups of rats. 3.1.2 Reference Groups
  • rat milk formula groups were also studied.
  • One reference group was a normal suckling group in which the rat pups remained with the dam until day 20 when brain tissue was obtained.
  • the normal suckling rats were not included in the food intake phase of the experiment.
  • a second reference group of suckling rats served as an experimental design reference group.
  • rat pups were removed from the dam and included in the feeding measurements on days 19 and 20. These rats were not weaned or introduced to the mash diet before the initiation of the food intake phase. 3.1.3 Statistical Analyses
  • Pregnant Sprague-Dawley rats were obtained from Charles River Laboratories (Wilmington, MA) on day 14 of gestation. They were housed under a controlled temperature environment with a 12-hour light/dark cycle. Rat pups were born on day 21 of gestation within a 24 hour time period. The day of birth was designated day 0. Male rat pups were removed from dams on postnatal day 6 and artificially reared on rat milk formulas to day 18. This procedure has been described in detail in the literature by Sonnenberg et al, 1982; Smart et al, 1983 and 1984; and Auestad et al, 1989. Similar procedures have been also been described by Ward et al, 1998, and Wainwright et al, 1999.
  • rat pups were randomly assigned to one of the four experimental rat milk formula groups (Table 3.2).
  • the rat pups were lightly anesthetized, fitted with an intragastric cannula, and placed individually in pint-sized plastic containers free floating in a waterbath maintained at 36 ⁇ 2°C.
  • the cannulae for individual rat pups were each connected to syringes filled with one of the four experimental rat milk formulas using polyethylene tubing.
  • the rat pups were fed by intermittent, intragastric infusion, from day 6 to day 18.
  • the formula was delivered to the rat pups for 20 or 30 min each hour, depending on the age of the rat, using a programmable pump housed in a bench-top refrigerator.
  • the pump settings were modified daily to deliver specific quantities of rat milk formula to the rat pups to support normal growth.
  • the study protocol is shown in Table 3.3. 3.2.2 Rat Milk Formula Composition
  • Rat milk formulas were prepared as described in the literature (Auestad et al, 1989; Ward et al, 1998) except that the protein source was whey and casein powders (kindly provided by Ross Products Division of Abbott Laboratories). Briefly, a premilk base consisting of casein, whey, and water was prepared first. Then, a fat blend (see Table 3.4), lactose, minerals, vitamins, and additional nutrients as found in rat milk were added to the premilk base and mixed using a Polytron homogenizer (see Table 3.5). The fat blends used in preparation of the rat milk were formulated to provide marginal amounts of linoleic acid, linolenic acid, and different amounts of AA and DHA.
  • Rat Milk Formula (AA and DHA Rearing Phase) 100 80 80 20 0 0
  • Table 3.3 Calorie sources during AA and DHA rearing phase and food intake phase of experiments. Feeding protocol for the four rat milk formula groups. Experimental rat milk formula was randomly assigned on postnatal day 6 and fed through day 18. The rat milk formula contained no AA or DHA; no AA, 2.5% DHA; 2.5% AA, no DHA; or 2.5% AA, 2.5% DHA. Mash was a semi-solid food and was introduced to all four groups on day 16 and fed exclusively during the food intake phase. The mash met the AIN-93 recommendations for nutrients and contained no AA or DHA.
  • Vitamin Mix (Supplementary) 1.375
  • Table 3.5 Ingredients in the rat milk formula. Ingredients are listed in gram per 2.5 liters. 'Copper sulfate solution was 30.9g CuSCy5H 2 0/L H 2 0; 2 Zinc sulfate solution was 379.3g ZnS0 4 -7H 2 0/L H 2 0 (as described in Auestad et al, 1989).
  • the fatty acid composition of the rat milk formulas was determined by gas chromatographic analysis and results are shown in Tables 3.6.
  • the target percentages of the fatty acids linoleic acid, linolenic acid, AA and DHA, were achieved with concentrations at or near expected targets.
  • the other prepared formulas with added AA ech contained approximately 2.5% AA as expected.
  • the artificially reared rat pups were weighed daily. Weights at the beginning and end of the AA and DHA rearing phase as well as weights during the food intake phase will be reported.
  • the food mash used in the February experiment was prepared by mixing a fat-free powder meal (Bioserv Inc., Frenchtown, NJ), a fat blend (coconut oil:MCT oil, 70:30, w/w), and water until the consistency was crumbly.
  • a pelleted food mash with the same nutrient composition was prepared (Research Diets Inc., Princeton, NJ) for the April experiment.
  • the pellets were extremely dense and hard and there were concerns that the weanling rats may not readily eat the solid pellets. Therefore, the pellets were crushed into powder, mixed with water, and formed into %" to 54" semi-solid balls which were used in the food intake phase.
  • Results are expressed in % total fatty acids.
  • the fat blend was coconut oil:MCT oil (70:30, w/w). 'February food mash was made with fat-free powder to which the fat blend was added along with water to form a crumbly consistency. 2 April food mash was made from pellets and contained the same fat blend; the pellets were crushed and formed into V" to V" mash balls by adding a small amount of water. 3 Fatty acids present in 0.5% or less are not reported except where indicated by '*' for clarity.
  • the rat pups were calorie restricted with 20% of caloric requirements from the rat milk formula and 20% from the wet mash. The formulas were diluted with water to 20% the initial calorie content. Water intake thus was not restricted to keep the animals properly hydrated.
  • the rat pups were stimulated to urinate and then weighed. The intragastric cannulae were removed, and then rat pups were placed in individual cages containing water bottles and approximately 15g of 'crumbly' mash in ceramic dishes (February experiment) or 'mash balls' added directly to the bottom of the cages (April experiment).
  • the cages had clear plastic bottoms and sides, were approximately 8 inches wide x 12 inches long, and were enclosed with a wired, slanted top that held a water bottle. Every two hours for eight hours all the remaining mash was weighed to determine the amount of food eaten. Three mash 'controls' were included to measure weight loss due to evaporation during the food intake phase. The rats were weighed again at the end of the food intake phase. The mash was then removed from the cages and the rats fasted for the next 18 hours with free access to water. At 9:00 am on day 20, the rats were again stimulated to urinate, weighed, and placed in their cages with access to water and approximately 15g of mash. The amount of food eaten and final weights of the rats were determined after 2 hours. 3.4 TISSUE COLLECTION
  • the rat pups were sacrificed by decapitation on day 20 after the food intake phase and final body weights were taken.
  • the brain was removed, weighed, and quickly frozen (within 5 minutes) in liquid nitrogen. Brain tissue was stored in a -70°C freezer. Blood was collected from the neck stump, mixed with heparin, placed on ice, and centrifuged to ensure adequate phase separation to prepare plasma. Plasma was stored at -70°C.
  • Brain and plasma samples were shipped overnight on dry ice from UCLA to Ross Products Division of Abbott Labs, Columbus, Ohio, and arrived completely frozen. The shipped samples were inspected for damage and signs of thawing and immediately transferred to a - 70°C freezer for storage until analysis. Plasma samples were obtained but not analyzed as a component of this thesis.
  • the fatty acid composition of three lipid fractions in brain was determined.
  • Phospholipid fatty acid methyl esters were determined similar to the methods described by Ward et al (1999).
  • Gas chromatography-mass spectrometry (GC/MS), liquid chromatography-mass spectrometry (LC/MS/MS), as well as HPLC methods for measuring MAG and NAE fatty acids have been described (Berger et al, 2001; Kempe et al, 1996; Fontana et al, 1995; Felder et al, 1996; Wang et al, 2001).
  • a less costly and simpler method for measuring these fatty acids in brain tissue was developed.
  • Total lipid was extracted from rat brains using the Folch extraction method, typical for lipid extraction (Folch et al, 1957).
  • the total lipid extract from each rat brain was separated into neutral lipid and phospholipid fractions using a silica cartridge.
  • the neutral lipid fraction was further separated into MAG and NAE fractions using High Performance Liquid Chromatography (HPLC).
  • Fatty acid composition of the MAG, NAE, and phospholipid fractions was determined using Gas-Liquid Chromatography (GLC) after derivatizing to the corresponding fatty acid methyl esters.
  • the fatty acid composition results correspond to total fatty acids in the membrane phospholipids in brain, and the MAG and NAE fatty acid results represent the concentration of these fatty acyl derivatives in rat brains.
  • Arachidonyl ethanolamide and docosatetraeonyl ethanolamide were from Cayman Chemical Co. (Ann Arbor, MI).
  • Docosatrieonyl chloride and fatty acid standards were from Nu-Chek Prep, Inc. (Elysan, MN).
  • Ethanolamine and boron trifluoride-methanol complex (BF 3 ) were from Sigma-Aldrich (Milwaukee, WI).
  • Dichloromethane, methanol, chloroform, hexane, ethyl acetate, and isopropyl alcohol were from Burdick & Jackson (Muskegon, MI), petroleum ether was from Mallinckrodt (Paris, KY), and formic acid was from J.T.
  • the GLC fatty acid standard was prepared. Briefly, a representative mixture of fatty acid methyl esters (>98% purity) was accurately weighed into a tared 100-mL pear-shaped flask in a specific order to ensure proper blending. After all of the fatty acid methyl esters were added and mixed, the flask was weighed for a final weight of the standard. One hundred milligrams of standard were added to ampules, flushed with nitrogen, sealed with a propane flame, and stored in the -20°C freezer until use. The GLC stock standard was prepared by quantitatively transferring the contents of one ampule to a 25 -mL volumetric flask and diluting to volume with hexane. The GLC working standard was prepared by diluting the GLC stock standard 1 :3 (v/v) with hexane and injecting between 1 and 5 ⁇ onto the GLC.
  • Rat brain samples stored frozen at -70°C, were separated into the two hemispheres; one half was used for determination of fatty acids in phospholipid, MAG, and NAE fractions and the other half was refrozen at -70°C.
  • the half brain for analysis was transferred to a 50-mL glass centrifuge tube. Eight mL of methanol was added and the sample homogenized using a Polytron Dispersing and Mixing System (Kinematica, Switzerland) until well blended. The homogenizer probe was rinsed with 2 mL methanol added directly into the centrifuge tube. Twenty mL of chloroform was then added to the sample and mixed vigorously by shaking. The sample was left undisturbed at room temperature for at least 1 hour.
  • Each sample of the reconstituted brain extract was loaded onto a Silica Plus SEP-PAK cartridge (Waters/Millipore, Milford, MA) using a disposable glass pipette.
  • the test tube containing the brain extract was rinsed twice with 500 ⁇ L of chloroform, which was then loaded onto the cartridge to ensure that all of the extract was transferred to the cartridge.
  • Neutral lipids were eluted with 15 mL chloroforrmmethanol (99:1, v/v) and phospholipids were eluted with 15 mL of methanol.
  • the neutral lipid eluant was filtered through a syringe filter (German Acrodisc® CR PTFE, 0.45 ⁇ or 0.2 ⁇ , 25mm; Ann Arbor, MI) attached to the bottom of the SEP-PAK cartridge. Each eluant was collected into test tubes and evaporated to dryness under N 2 .
  • a syringe filter German Acrodisc® CR PTFE, 0.45 ⁇ or 0.2 ⁇ , 25mm; Ann Arbor, MI
  • the resuspended neutral lipid fraction extract i.e. chloroforrmmethanol SEP- PAK elution
  • 250 ⁇ L of the resuspended neutral lipid fraction extract i.e. chloroforrmmethanol SEP- PAK elution
  • the MAG and NAE fractions collected from the HPLC for each rat brain sample were then evaporated to dryness under N 2 . 3.5.8 Methylation Procedure
  • the MAG and NAE fractions were resuspended in hexane :isopropyl alcohol: ethyl acetate (80:10:10, v/v/v) and transferred to 2-mL amber screw cap vials. The fractions were again evaporated to dryness under a stream of N 2 at room temperature.
  • the samples containing MAGs, NAEs, and phospholipids were then methylated by addition of excess boron trifluoride-methanol complex, BF 3 , under N 2 . After capping tightly with teflon-lined caps, the samples were placed on a heating block at 95°C for 20 minutes. The samples were cooled to room temperature and opened very carefully. The MAG and NAE r4samples were transferred in methanol to 15mL test tubes. Then, 2 mL of 0.9% saline and 4mL hexane were added to the samples and they were mixed vigorously by shaking.
  • the hexane layer was removed using disposable glass pipettes, transferred to clean 15 mL test tubes, and evaporated to dryness under N 2 .
  • the MAG and NAE methylation hexane extracts were reconstituted in 100 ⁇ L of hexane for analysis of the constituent fatty acids by GLC.
  • the dried phospholipid methylation hexane extracts were reconstituted in 10 mL of hexane and diluted 50 ⁇ L of reconstituted extract with 150 ⁇ L of hexane for analysis of fatty acid composition by GLC.
  • the fatty acid methyl esters were analyzed using a Hewlett Packard 6890 gas-liquid chromatograph (GLC) equipped with a flame ionization detector; and an Omegawax 320 fused silica column coated with polyethylene glycol, 0.32mm ID x 30m, 0.25mm film • thickness (Supelco, Inc.; Bellefonte, PA).
  • the gas chromatographic instrument settings were adjusted for optimum signal sensitivity similar to conditions described in Ward et al (1999). Five L of each sample was injected onto the gas chromatograph using an autosampler (Hewlett Packard 7673 A). Individual fatty acids were identified by co-elution with corresponding fatty acid methyl ester internal standards.
  • Fatty acid levels in the rat brain phospholipid fractions are reported as relative percent of total fatty acids, as is typically reported in the literature.
  • the specific amounts of individual NAE fatty acids in the brain lipid extract were quantified relative to the NAE internal standard, methyl docosatrienoate.
  • the amounts of individual MAG fatty acids in the brain lipid extract were quantified relative to the monoglyceride internal standard, methyl heptadecanoate.
  • MAG and NAE corresponding fatty acids are reported as ng/g and ⁇ g/g wet weight of rat brain, respectively.
  • Table 4.2 Average body and brain weight across experimental formula and suckling groups, ⁇ ats were fed rat milk formulas containing different amounts of AA and DHA (see Table 3.6) by gastrostomy tube at 100% of calories from postnatal days 6 to 16, 80% of calories on days 17 and 18, and 20% of calories for the last 18 hours before the food intake study on day 19. A semi-solid mash containing no AA or DHA was fed ad lib at 20%) of calories on days 17 and 18 and as the only dietary source for the food intake study. 2 Main effects of feeding AA or DHA were determined by ANOVA; (+) indicates main effect was increased weight and (-) indicates main effect was decreased weight.
  • Rats were fed rat milk formulas containing different amounts of AA and DHA (see Table 3.6) by gastrostomy tube at 100% of calories from postnatal days 6 to 16, 80%> of calories on days 17 and 18, and 20% of calories for the last 18 hours before the food intake study.
  • a mash containing no AA or DHA was fed ad lib at 20%> of calories on days 17 and 18 and as the only dietary source for the food intake study.
  • Food consumption was measured for 8 hours on da 19, then rats were fasted overnight after which 2 hr of food consumption was measured.
  • 2 Main effects of feeding AA or DHA were determined by ANOVA (+) indicates main effect was increased food consumption and (-) indicates main effect was reduced food consumption.
  • Table 4.4 shows results for fatty acid levels in brain phospholipid membranes expressed as % total fatty acids (i.e. g/lOOg total fatty acid).
  • % total fatty acids i.e. g/lOOg total fatty acid.
  • the effects of dietary n-6 and n-3 fatty acids on n-6 and n-3 fatty acid composition in brain in this study were similar to that shown previously in the literature (Ward et al, 1999; de la Presa Owens and Innis, 1999). There were no significant differences in saturated fatty acids among the groups. There was a significant main effect of AA on unsaturated fatty acids (C 18 : 1 and C20:l) in brain phospholipids.
  • Results for n-6 and n-3 NAEs are shown in Table 4.5 and expressed as ng/g brain. There was a significant main effect of AA increasing 20:4n-6 NAE in brain. There was also a significant main effect of AA increasing total n-6 NAE in brain. No other significant main effects were found. No significant main effects of dietary DHA on n- or n-3 fatty acids were found.
  • Results for n-6 and n-3 MAG are shown in Table 4.6 and expressed as ⁇ g/g brain. There were no significant main effects of dietary AA on n-6 or n-3 MAG. However, there was a significant main effect of DHA increasing 22:6n-3 MAG as well as increasing total n-3 MAG in brain.
  • ° food intake is summation of all food eaten on . days 19 and 20 of the April rearing. r, correlation. Spearman is a ranked data correlation.
  • an artificially reared rat model we modified the fatty acid composition of the brain phospholipid membrane through inclusion of different dietary n-6 and n-3 fatty acids as has been demonstrated in previous research (Ward et al, 1998 and 1999; Wainwright et al, 1999).
  • the artificially reared rat model is an excellent choice for modifying brain phospholipid composition as the feeding period occurs during a period of rapid brain growth.
  • Our fatty acid results for phospholipid membrane also were consistent with previous studies using similar diets marginally deficient in essential fatty acids (de la Presa Owens and Innis, 1999 and 2000).
  • Berger et al fed adequate levels of linoleic acid and linolenic acid and reported that dietary AA and DHA can only increase the levels of n-6 and n-3 NAEs when adequate essential fatty acids are present.
  • Kirkham et al (2002) recently reported differences in NAE and MAG levels in brain during fasting, feeding, and satiation. They found increased levels of 20:4n-6 NAE and MAG after fasting; decreases in 20:4n-6 MAG during eating, and no changes compared to controls during satiation.
  • n-6 and n-3 diets were fed by gastrostomy tube before the food intake studies and all rats were fed the same mash diet during the food intake study, the observed effects cannot be explained by olfactory or other characteristics of the food (mash). There may be other explanations for these observations such as effects of feeding the different diets on release or activity of hormones (e.g. insulin; leptin) and neurotransmitters (e.g. serotonin) known to be involved in the regulation of appetite. However, based on our data it is reasonable to conclude that these observed effects on food consumption may be mediated through changes in the n-6 and n-3 fatty acid composition of the brain phospholipid membrane and consequently in NAE- and MAG- fatty acid levels.
  • hormones e.g. insulin; leptin
  • neurotransmitters e.g. serotonin
  • the endogenously formed NAEs and MAGs act through the cannabinoid receptor (CBi). It is well established that increasing 20:4n-6 NAE leads to overeating. The effect of dietary DHA on food intake has not been previously been studied and the association with reduced food intake was unexpected. Other possibilities for dietary fatty acid induced effect on food intake will need to be evaluated, such as responses of leptin, insulin, and other hormones and neurotransmitters, to stimuli known to lead to food consumption (e.g. sleep deprivation) not studied here.
  • the levels of 20:4n-6 NAE and 20:4n-6 MAG levels reported here in satiated rats were very similar to those reported recently in satiated rats by Kirkham et al (2001). Given these similarities, it is reasonable to conclude that the newly developed methodology for quantifying MAGs and NAEs in brain is a viable alternative to the standard GC/MS method.

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Abstract

La présente invention concerne des produits, des produits à finalité nutritionnelle, des suppléments et des compositions diététiques qui contiennent des acides gras polyinsaturés à longues chaînes (LCP ou LC-PUFAs), notamment le DHA de type n-3 LCPs. Cette invention porte également sur l'utilisation de tels produits pour réguler l'appétit et faciliter le traitement et/ou prévenir l'obésité et les pathologies de surpoids, plus spécifiquement pour la population pédiatrique. Le DHA diététique peut agir au niveau central en tant qu'antagoniste du CB1 dans le cerveau pour s'opposer aux endocannabinoïdes qui augmentent la prise alimentaire. Ceci est particulièrement intéressant lorsque le DHA est administré pendant des périodes de croissance rapide du cerveau telles que la petite enfance, l'enfance et l'adolescence.
PCT/US2003/023708 2002-08-06 2003-07-30 Compositions comprenant des acides gras polyinsatures (pufas) utiles pour reguler l'appetit et gerer la masse corporelle WO2004012727A1 (fr)

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WO2005039566A1 (fr) * 2003-10-24 2005-05-06 Solvay Pharmaceuticals Gmbh Polytherapie de l'obesite impliquant des derives de 4,5-dihydro-1h-pyrazole presentant une activite antagoniste de cb1, et inhibiteurs de lipases
WO2005039550A2 (fr) * 2003-10-24 2005-05-06 Solvay Pharmaceuticals Gmbh Nouvelles utilisations medicales de composes a activite antagoniste de cb1 et traitement combine impliquant ces composes
WO2005039579A1 (fr) * 2003-10-24 2005-05-06 Solvay Pharmaceuticals Gmbh Traitement de l'obesite combinant des antagonistes de cb1 selectifs et des inhibiteurs de lipase
WO2005060954A1 (fr) * 2003-12-19 2005-07-07 Pronova Biocare As Utilisation d'une composition d'acides gras comprenant au moins un epa et un dha ou des combinaisons de ceux-ci
WO2007100561A2 (fr) * 2006-02-28 2007-09-07 Bristol-Myers Squibb Company Utilisation de dha et d'ara pour la preparation d'une composition destinee a la prevention ou au traitement de l'obesite
WO2007100562A2 (fr) * 2006-02-28 2007-09-07 Bristol-Myers Squibb Company Utilisation de dha et d'ara dans la préparation d'une composition permettant de réduire les niveaux de triglycérides
WO2007135141A1 (fr) * 2006-05-23 2007-11-29 Nestec S.A. Supplément maternel
US7550613B2 (en) 2005-05-04 2009-06-23 Pronova Biopharma Norge As Compounds
EP2076256A1 (fr) * 2006-10-03 2009-07-08 Michael D. Myers Compositions de substitution d'un repas et procédé de contrôle de poids
WO2010018856A1 (fr) * 2008-08-13 2010-02-18 持田製薬株式会社 Agent prophylactique/d’amélioration ou thérapeutique destiné aux maladies associées au récepteur des cannabinoïdes
WO2011090922A1 (fr) 2010-01-19 2011-07-28 Mead Johnson Nutrition Company Compensation nutritionnelle pour une alimentation de type occidental
US8058264B2 (en) 2004-10-25 2011-11-15 Abbott Products Gmbh Pharmaceutical compositions comprising CB1 cannabinoid receptor antagonists and potassium channel openers for the treatment of obesity and related conditions
US8399516B2 (en) 2006-11-01 2013-03-19 Pronova Biopharma Norge As Alpha-substituted omega-3 lipids that are activators or modulators of the peroxisome proliferators-activated receptor (PPAR)
US10251928B2 (en) 2014-11-06 2019-04-09 Mead Johnson Nutrition Company Nutritional supplements containing a peptide component and uses thereof
WO2021035179A1 (fr) 2019-08-21 2021-02-25 Coda Biotherapeutics, Inc. Compositions et méthodes de traitement de maladies neurologiques
US11109607B2 (en) 2013-11-18 2021-09-07 Gary Hall Oil-based compositions for enhancing oral health and general wellness in humans

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WO2005039550A2 (fr) * 2003-10-24 2005-05-06 Solvay Pharmaceuticals Gmbh Nouvelles utilisations medicales de composes a activite antagoniste de cb1 et traitement combine impliquant ces composes
WO2005039579A1 (fr) * 2003-10-24 2005-05-06 Solvay Pharmaceuticals Gmbh Traitement de l'obesite combinant des antagonistes de cb1 selectifs et des inhibiteurs de lipase
WO2005039550A3 (fr) * 2003-10-24 2007-03-22 Solvay Pharm Gmbh Nouvelles utilisations medicales de composes a activite antagoniste de cb1 et traitement combine impliquant ces composes
WO2005039566A1 (fr) * 2003-10-24 2005-05-06 Solvay Pharmaceuticals Gmbh Polytherapie de l'obesite impliquant des derives de 4,5-dihydro-1h-pyrazole presentant une activite antagoniste de cb1, et inhibiteurs de lipases
WO2005060954A1 (fr) * 2003-12-19 2005-07-07 Pronova Biocare As Utilisation d'une composition d'acides gras comprenant au moins un epa et un dha ou des combinaisons de ceux-ci
US9282760B2 (en) 2003-12-19 2016-03-15 Pronova Biopharma Norge As Use of a fatty acid composition comprising at least one of EPA and DHA or any combinations thereof
US8058264B2 (en) 2004-10-25 2011-11-15 Abbott Products Gmbh Pharmaceutical compositions comprising CB1 cannabinoid receptor antagonists and potassium channel openers for the treatment of obesity and related conditions
US7550613B2 (en) 2005-05-04 2009-06-23 Pronova Biopharma Norge As Compounds
US8034842B2 (en) 2005-05-04 2011-10-11 Pronova Biopharma Norge As Compounds
US8618165B2 (en) 2005-05-04 2013-12-31 Pronova Biopharma Norge As Compounds
WO2007100562A2 (fr) * 2006-02-28 2007-09-07 Bristol-Myers Squibb Company Utilisation de dha et d'ara dans la préparation d'une composition permettant de réduire les niveaux de triglycérides
CN101389322A (zh) * 2006-02-28 2009-03-18 布里斯托尔―迈尔斯斯奎布公司 二十二碳六烯酸和花生四烯酸在制备用于预防或治疗肥胖症的组合物中的用途
WO2007100562A3 (fr) * 2006-02-28 2008-04-03 Bristol Myers Squibb Co Utilisation de dha et d'ara dans la préparation d'une composition permettant de réduire les niveaux de triglycérides
RU2456985C2 (ru) * 2006-02-28 2012-07-27 Бристол-Маерс Сквибб Компани Способ увеличения сухой мышечной массы и уменьшения жировой ткани
WO2007100561A2 (fr) * 2006-02-28 2007-09-07 Bristol-Myers Squibb Company Utilisation de dha et d'ara pour la preparation d'une composition destinee a la prevention ou au traitement de l'obesite
WO2007100561A3 (fr) * 2006-02-28 2007-10-18 Bristol Myers Squibb Co Utilisation de dha et d'ara pour la preparation d'une composition destinee a la prevention ou au traitement de l'obesite
WO2007135141A1 (fr) * 2006-05-23 2007-11-29 Nestec S.A. Supplément maternel
AU2007253309B2 (en) * 2006-05-23 2011-09-29 Société des Produits Nestlé S.A. Maternal supplement
EP1886680A1 (fr) * 2006-05-23 2008-02-13 Nestec S.A. Supplément pour futures mamans
US8454950B2 (en) 2006-05-23 2013-06-04 Nestec S.A. Maternal supplement
RU2457836C2 (ru) * 2006-05-23 2012-08-10 Нестек С.А. Добавка к материнскому рациону
US9066915B2 (en) 2006-10-03 2015-06-30 Michael D. Myers Meal replacement compositions and weight control method
EP2076256A4 (fr) * 2006-10-03 2010-01-13 Michael Myers Compositions de substitution d'un repas et procédé de contrôle de poids
EP2076256A1 (fr) * 2006-10-03 2009-07-08 Michael D. Myers Compositions de substitution d'un repas et procédé de contrôle de poids
US8399516B2 (en) 2006-11-01 2013-03-19 Pronova Biopharma Norge As Alpha-substituted omega-3 lipids that are activators or modulators of the peroxisome proliferators-activated receptor (PPAR)
JPWO2010018856A1 (ja) * 2008-08-13 2012-01-26 持田製薬株式会社 カンナビノイド受容体関連疾患の予防／改善または治療剤
WO2010018856A1 (fr) * 2008-08-13 2010-02-18 持田製薬株式会社 Agent prophylactique/d’amélioration ou thérapeutique destiné aux maladies associées au récepteur des cannabinoïdes
EP2353595A1 (fr) 2010-01-19 2011-08-10 Mead Johnson Nutrition Company Compensation nutritionnelle pour régime de type occidental
WO2011090922A1 (fr) 2010-01-19 2011-07-28 Mead Johnson Nutrition Company Compensation nutritionnelle pour une alimentation de type occidental
US11077166B2 (en) 2013-03-15 2021-08-03 Mead Johnson Nutrition Company Nutritional supplements containing a peptide component and uses thereof
US11109607B2 (en) 2013-11-18 2021-09-07 Gary Hall Oil-based compositions for enhancing oral health and general wellness in humans
US10251928B2 (en) 2014-11-06 2019-04-09 Mead Johnson Nutrition Company Nutritional supplements containing a peptide component and uses thereof
US10933114B2 (en) 2014-11-06 2021-03-02 Mead Johnson Nutrition Company Nutritional supplements containing a peptide component and uses thereof
WO2021035179A1 (fr) 2019-08-21 2021-02-25 Coda Biotherapeutics, Inc. Compositions et méthodes de traitement de maladies neurologiques

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