UA66354C2 - Methods and compositions for reducing incidence of necrotizing enterocolitis - Google Patents

Abstract

Enteral formulas that contain long-chain polyunsaturated fatty acids (PUFAs), e.g. arachidonic acid (AA) and docosahexaenoic acid (DHA) essentially free of cholesterol, and a process for making such enteral compositions are described. More particularly, the invention relates to methods for reducing the incidence of necrotizing enterocolitis by administering compositions which provide n-6 and n-3 long chain PUFAs; phospholipids and/or choline. Compositions from egg yolk lipids are presently preferred as they contain n-6 and n-3 long chain PUFAs and are predominantly in a phosphatidylchoine form. This is believed to provide a synergistic effect. Also disclosed is a process of making such a composition that provides improved organoleptic and stability properties.

Description

Description of the invention

This invention relates mainly to baby food mixtures containing polyunsaturated fatty acids with a long chain (POFA), the process of obtaining such enteral compositions and methods of reducing the incidence of necrotizing enterocolitis. More specifically, this invention relates to enteral compositions consisting of long-chain FISH arachidonic acid (AA) and docosahexaenoic acid (DHA), which are substantially free of cholesterol and can be derived from egg yolk lipids. Long-chain ROS derived from egg yolk exist mainly in the phospholipid form. The process of obtaining such compositions provides 70 improved organoleptic and stable properties. Enteral administration of such compositions can reduce the incidence of necrotizing enterocolitis.

Technical level

Long-chain RPOs in infant formulas or formulations have been the subject of various sources

For example, US Patent No. 4,670,285 ("Clandinin") describes a special blend of fats suitable for use in 12 infant formulas. More specifically, the Clandinin fat mixture contains at least one Co or Co» n-6 fatty acid and Co or Co» n-3 fatty acid. These fatty acids are described as such that, when present in a certain, defined amount, they prevent the occurrence of harmful effects on the infant who is fed a fat mixture. Sor or Co» p-b6 fatty acids are present in a total amount of about 0.13 to 5.695 by weight of all fatty acids in the product. Co0 or Co3 n-3 fatty acids, if present, are included in a total amount of about 0.013 to 3.3395 by weight of all fatty acids in the product. Clandinin contains egg lipids with p-6 and p-3 fatty acids; however, the egg lipid used in Clandinin also contains large amounts of cholesterol. In addition, this invention uses from 75 to 95 parts by weight of egg yolk lipid with coconut oil or soybean oil. The terminology used in the Clandinin invention for fatty acids will be used here. p 29 Patent UUO 93/20717 describes a formula for babies that contains no more than a non-irritating amount of free He) long-chain (C48-Co») fatty acids and triglycerides. This application also describes that the use of lower alkyl esters, such as ethyl esters of such fatty acids in infant formula, significantly prevents free fatty acid damage to the intestinal epithelium of the infant, but does not interfere with the absorption and conversion of a portion of the fatty acid. M 30 US patent No. 4,918,063, issued to I ispiepregdeg, describes compositions containing unique mixtures of phospholipids and neutral lipids for the prevention and/or treatment of ulcers and inflammation of the intestines. This patent describes mixtures of saturated or unsaturated phospholipids together with saturated or unsaturated triglycerides and/or sterols that provide effective protection against ulcers in experimental animal models. This patent Ge) also describes the inclusion of polyvalent cations or antioxidants in the lipid mixture to increase activity. International Publication MoUuO 96/10922, owned by Kopp et al., describes a fat mixture for feeding babies, which is characterized by the fact that arachidonic acid and docosahexaenoic acid are present in the fat mixture in the form of phospholipids.

European patent application Mo0 376 628 B1, issued to Totageii, describes a composition consisting entirely of "vegetable fats" that uses randomized palm oil or randomized palm C 50 oleic oil as the sole source of palmitic acid. It is also described that all compositions with fats of vegetable origin are particularly suitable for use in formula for preterm infants (or with

With" low birth weight). Fat compositions for premature babies, according to the application

Totageii, include medium chain triglycerides (MCT) containing random palmitic acid oil, lauric acid oil, oleic acid oil and linoleic acid oil. 45 Although the references discussed above have made important contributions, there remains a need for an infant formula containing egg phospholipids as a source of long-chain PPAs in nutritional concentrations.

Ge") There is also a need for methods of obtaining mixtures for baby food containing egg phospholipids, so that the mixtures have acceptable organoleptic properties. Especially such compositions are used in infant formulas for term and/or premature babies, whose needs for long-chain SL 20 FISH have been determined for the necessary development of the nervous system and for the development of visual acuity. In addition, there may be a protective effect on the intestines.

T» Necrotizing enterocolitis (NEC) is a serious problem in infants weighing less than approximately 1500 grams at birth. Despite three decades of research, the exact etiology and pathophysiology of MES remains unclear. MES is a fatal disease characterized by ischemic necrosis of digestive tract structures and intestinal pneumatosis, which often leads to intestinal perforation. Prematurely

HF) an infant born with MES has clinical signs of thermal instability, lethargy, gastric retention, vomiting, gastric distension, overt or occult stool hemorrhage, and radiographic evidence of intestinal pneumatosis, portal air, or pneumoperitonitis. An attack of apnea begins, shock and sclera quickly follow, and, as a rule, death. 60 Various observations were made by many authors and factors affecting these diseases were identified. (Mie, Reaiig. SGt. Moi. At., Argii, 1996, 43(2): 409-32). The following observations and factors are given as examples:

Niadeoie ei ai., Mesgoiigipd Epiegosoiiiz oi (She Mem/rogp, Kemiem/u yTog (Pe Siipisiap. Opiop-Med-Sap. 1991

Zer-Osi 120(5)3: 334-8, suggests that the pathogenesis of MES includes ischemia of the mesentery, underdevelopment of the gastrointestinal tract, enteral (artificial) nutrition and even, possibly, infection;

Sariap ei aiYi., Koie oi Riaieiei Asiimaipu Rasiog apa Titog Mesgoviv Rasiog-AIrna in Meopaiia! Mesgoiiipd

Epiegosoiyiz, doigpa! oi Reiaigisv, dipe, 1990, 960-964, found that the amount of platelet activation factor and o-factor of tumor necrosis is increased in patients with MES;

Kiedtap ei ai.,, Siovigidia az RafSodepz in MeopaiaI Mesgoiigiipd Epiegosoiiiv, dotsgpai oi Rediaigisv,

Atsdyzi, 1979, 287-289, reported on the isolation of Siovigidia rephipdepz in infants with MES;

Ovieiad ey a, Eapu Epiegai Reedipd YuOoez My AiYesi Ipsidepse Mesgoiiigipd Epiegosoiiv, Reaiagisv,

MoI.77, Mo.3. Magsp 1986, 275-280, reports that dilute, early enteral calories do not promote MES;

Ve ei aiY.,, MeopaiaiI Mesgoiiigipd EpiegosoiYiiz, Appaiv oi Zygdegu, MoiI.187, daptsagu 1978, Mo.1, 1-7. suggests using a combination of antimicrobial therapy to treat infants with MES;

Wow! ei ai., Mesgoiiigipd Epiegosoiiiv ip (She Megu Gom Vii UUeidni Ipitapi: Ehrgezzei Vgeavzi MIiiK Reeadipd

Sotraged m/y Raghepieega! Reedipud, Agspimez oi Oivease ip Spyapooa, 1982. 57, 274-276 reported that the incidence of MES in low birth weight infants was reduced by delaying the initiation of 75 enteral nutrition.

Ripeg et al., Miyatip E apa Mesgoiiigiipd Eepiegosoiiv, Reaiagisv, MoiI.73, Mo.3, Magsp 1984, suggests that the administration of vitamin E to reduce the severe effects of enterolental fibroplasia may be associated with the disease of MES.

Vgom/up eii ai.,, Rhemepiipud Mesgoiiigiipd EpiegosoYiiv ip Meopafez, ZAMA, Mo.24, 1978, MoiI.240, Mo.22, 2o 2452-2454 reports that MES can be virtually eliminated by using a slowly progressive diet.

KoziozKe, Raiiodepevziv apa Rhemepiipud Mesgoiigipud Epiegosoiiiv: A Nuroiyeziz Vazei op Reggopai

Orzeguayop apa Kemiemzh/oi (She and Megaishge, Reaiaigisv, Moi.74, Mo.b6, JuOes.1984, 1086-1092, the hypothesis that MES occurs when two of three pathological events coincide: (1) intestinal ischemia; (2) colonization of Ha pathological bacteria, and (3) an excess of protein substrate in the intestinal cavity.

KoziozKe, mentioned above, also reports that MES rarely occurs in infants who were exclusively breastfed. In humans, milk from the mammary glands plays a significant role in the passive immunization of the neonatal intestine and contains factors that contribute to the development of Vigiydobrasiegicht in the intestinal flora. It is also reported that the beneficial composition of human milk can be adversely altered by freezing, pasteurization, or storage.

Thus, debate continues regarding the etiology and treatment of MEC, and there remains a need for compositions and methods that are better for treating and/or reducing the morbidity of this devastating and often fatal disease. co

Brief description of the invention

This invention has many aspects. First, the invention contemplates a method for reducing the disease of ee, c5 necrotizing enterocolitis in an infant who is susceptible to necrotizing enterocolitis, the aforementioned method, which (Te) consists of administering an effective amount of at least one long-chain PORA selected from the group C 20 p-6 fatty acids, Co» n-b6 fatty acids, Sod n-3 fatty acids and Co» 5-3 fatty acids. For example, the introduction of arachidonic acid, n-6 fatty acids, proved to be effective. Administration can be enteral or parenteral. It is even better when a combination of n-6 and n-3 fatty acids, such as arachidonic acid, is used

Acids and docosahexaenoic acid. Enteral administration is at least 1.0 mg of p-b6 fatty acids per kilogram of the infant's weight per day. An even better embodiment uses a combination of n-6 and n-3 fatty acids in a weight ratio of about 2:1 to about 4:1 and administration of at least 5.0mg of long-chain p-b6 fatty acids per day.

The method may be embodied by feeding a sufficient amount of an enteral composition containing arachidonic acid and docosahexaenoic acid to administer to the above-mentioned infant from about 1.0 to

Ge) about bOmg per kg per day of arachidonic acid and about 0.25 to about 35 mg per kg per day of b» docosahexaenoic acid. More typically, the amount is from about 5.0 to about 4mg of arachidonic acid and from about 1.5 to about 20mg of docosahexaenoic acid per kg per day. It is better when it is massive

Ge) the ratio of arachidonic acid to docosahexaenoic acid is from about 2 to about 4. sl 50 Preferably, long-chain polyunsaturated fatty acids are in the form of phospholipids, in particular, phosphatidylcholine. Such phospholipids are present in high concentrations in egg lecithins and egg phosphatides. c" Thus, the invention further provides a method for reducing the incidence of necrotizing enterocolitis in infants, said method comprising feeding said infant a sufficient amount of an enteral nutritional composition that contains protein, carbohydrate, and phospholipids to supply at least 1.0mg of p-6 polyunsaturated fatty acids with a long chain per day. It is better if the food contains at least

0.5 Bmg per day of n-3 polyunsaturated fatty acids with a long chain in the form of arachidonic acid and i) docosahexaenoic acid, respectively. In another aspect, the invention provides a method for reducing the incidence of necrotizing enterocolitis in human infants, said method comprising administering to the infant phospholipids in an amount effective to reduce the incidence of necrotizing enterocolitis.

Typically, the aforementioned phospholipids are administered to create a concentration of between about 60 and about 2400 μmol, preferably between about 200 and about 1500 μmol, most preferably between about 400 and about 10000 μmol of phospholipids per kg per day. The source of phospholipids is not critical; phospholipids obtained from egg lecithin are suitable for the present invention. Phospholipids can also be readily obtained from 65 animal membranes, including the globular membranes of milk fat. Other sources rich in phospholipids include soybean and other seed oils. When using egg lecithin phospholipids, the best amount for enteral administration is sufficient to produce at least 1.0 mg of long-chain p-6 fatty acids per day. Mainly, egg phospholipid supplies arachidonic acid as a significant part of p-b fatty acids, as well as docosahexaenoic acid and/or other long-chain p-3 fatty acids in the ratios mentioned above. It

May have a synergistic effect. However, it should be noted that the length and saturation of the fatty acids attached to the glycerol as a base in this "phospholipid aspect of the invention are not critical, and fatty acids other than 1 SRBAv may be used in this invention.

In yet another aspect, the invention provides a method for reducing the incidence of necrotizing enterocolitis in human infants, the aforementioned method comprising administering to the infant choline in an amount effective to 7/0 reduce the incidence of necrotizing enterocolitis. Typically, the aforementioned choline is administered to create a concentration between about 60 and about 1800 μmol; more preferably between about 150 and about 1200 µmol choline per kg per day. The source of choline is not critical; phosphatidylcholine may be preferable, and phosphatidylcholine derived from egg lecithin is suitable for the present invention. Other sources enriched with choline or phosphatidylcholine include soybean and other seed oils. When using egg lecithin choline, it is better to /5 INTRODUCE a combination of p-6 and/or p-3 fatty acids to obtain at least 1.0 mg of long-chain p-b6 fatty acids per day. Egg lecithin contains mainly arachidonic acid as a significant part of p-6 fatty acids and also docosahexaenoic acid and/or other long-chain p-3 fatty acids in the ratios mentioned above. This can lead to a synergistic effect. However, as with the phospholipid aspect, the length and saturation of the fatty acids attached to the glycerol as the base of any phosphatidylcholine used in the invention is not critical, and fatty acids other than | SRIORA, can be used in this invention.

Alternatively, choline from non-phospholipid sources can be used.

In addition, due to the beneficial effect observed in enterocolitis-susceptible infants, it can be assumed that the beneficial effect is also observed in adults. Thus, a further aspect of the invention is the use of any of the above-mentioned compositions for the treatment or prevention of ulcerative colitis and related intestinal diseases in adults. Dosage should take into account the increased weight of the adult patient and other factors known in the industry. and)

Other aspects of the invention, including mixtures for feeding an infant and processes for their preparation, are described in the application. For example, in yet another aspect of the invention, the process of preparing an infant formula consisting of egg yolk phospholipids consists of: (a) obtaining a dried egg phosphatide powder substantially free of cholesterol; (b) dispersing the aforementioned phospholipid fraction in the aqueous phase to form a phospholipid dispersion; (c) mixing the aforementioned phospholipid dispersion with suspensions of other components of the aforementioned infant formula. ise)

Preferably, according to the process, the dispersion in the aqueous phase contains from about 2 to about 1595% by weight of egg phosphatide and more preferably, if the egg phosphatide powder is added to the water at a temperature of from about 20 to about 507°C. This aspect can be used to obtain a formula for feeding infants containing arachidonic acid and docosahexaenoic acid in the form of phospholipids, the above formula for feeding infants is obtained according to the process.

In yet another aspect, the invention provides an infant formula consisting of protein, carbohydrates and lipids, which is improved and characterized by the content of lipids consisting of medium-chain triglycerides and egg phospholipid, wherein the aforementioned egg phospholipid is present in the amount from ;" about 195 by weight to about 4095 by weight of lipid content, wherein the aforementioned egg phospholipid is substantially free of cholesterol. Typically, egg phospholipid is present in an amount from 5 to 3095 by weight of the lipid content of infant formula, and arachidonic acid in a concentration from about 10 to about 31 mg per 100 kcal. Even better, the infant formula also includes docosahexaenoic acid at a concentration of about 3 to about 1 mg per 100 kcal, and arachidonic acid and docosahexaenoic acid

Me, present in a ratio of about 4:1 to about 2:1. 2) Detailed description of the invention General terminology

Fatty acids are hydrocarbon chains of various lengths that have a carboxylic acid at one end, thus making them somewhat polar and hydrophilic at that point, and at the same time hydrophobic to a degree that depends on the length of the hydrocarbon chain. Fatty acids are divided into categories depending on the length of the hydrocarbon chain. For example, chains of less than approximately carbon atoms are considered "short"; chains of about 6-18 carbon atoms are "medium" and chains of 20 or more carbon atoms are considered "long". Fatty acids may also have one or more double bonds, which are sites of "unsaturation" in the hydrocarbon chain. As used herein, the term "long-chain fatty acids" refers to fatty acids (F, acids with twenty or more carbon atoms that have at least two carbon-carbon double bonds (polyunsaturated). The number of double bond positions in fatty acids is generally for example, arachidonic acid ("AA" or "AKA") has a chain length of 20 carbon atoms and 4 boron double bonds starting at the sixth carbon atom from the end where the methyl radical is located. As a result, it has the name "C20:4 p-6". Similarly, docosahexaenoic acid ("DOH") has a chain length of 22 carbon atoms with b double bonds starting from the third carbon atom from the end where the methyl radical is located, and has the name "C22:6b p-3". Less common long-chain RORAs are also known, and some of them are listed in Tables I and IM (below the solid line). 65 "Glycerides" are complexes of lipids having a glycerol skeleton, esterified fatty acids. "Triglice ryd" (ie, "triglyceride") has esterified fatty acids, one for each hydroxyl group of glycerol. Di- and monoglycerides have, respectively, two and one esterified fatty acids. A phosphoglyceride (ie "phospholipid" or "phosphatide" - all terms are used interchangeably) differs from a triglyceride in that it has a maximum of two esterified fatty acids, while the third position of the glycerol base is esterified with phosphoric acid to form "phosphatidic acid". In nature, phosphatidic acid is usually combined with alcohol, which gives the molecule a very polar head. Two such alcohols that commonly occur in nature are choline and ethanolamine. "Lecithin" is a phosphatidic acid conjugated with an amino alcohol, "choline", also known as "phosphatidylcholine". Lecithins differ in fatty acid content and can be obtained from, for example, eggs and soy. Cephalin (phosphatidylethanolamine), phosphatidylserine, and phosphatidylinositol are other phosphoglycerides. 70 Phospholipids are commonly found in the membranes of all living systems. The traditional source of phospholipids is egg yolk and soybean oil. Phospholipids can also be obtained from the brain, kidney, heart and lungs of mammals; or from the membranes of milk fat globules. In addition, sources of microbial origin (single-cell oils) can be used, such as oils from algae and fungi, which can be used, in particular, for the AA and ONA fatty acid components of phospholipids.

Chicken eggs are a relatively rich source of lipids. Approximately 3395 of the yolk of a chicken egg consists of lipid, of which approximately 6790 is triglyceride, 2895 is phospholipid, and the rest is mainly cholesterol (percentage by weight). These figures are approximate and vary depending on the diet, breed and condition of the chicken. Of the phospholipid fraction, approximately 7595 is phosphatidylcholine and the remaining approximately 2095 is phosphatidylethanolamine. The choline component is approximately 15 to 3095 of each phospholipid molecule, depending on the attached fatty acid.

Thus, the choline content of egg phospholipid can vary from about 1095 to about 25905 by weight; or from about 3 to about 795% total egg lipid by weight.

Compositions

The compositions used in the invention contain p-b and/or p-3 long-chain FISH. The source of long-chain ROBs is not decisive. Known sources of long-chain ROS include fish or marine oil, egg yolk lipids and phospholipids, single-cell oils (eg, algae and mushroom oils), and it is also clear to those skilled in the art that some sources are better. , than others, to i) achieve the greatest number of special long-chain FISH. Other edible, semi-refined, or purified sources of long-chain RIORAs are obvious. New sources of long-chain ROS can be developed by genetic engineering of vegetable and oil plants, and the use of such recombinant products is also contemplated by the present invention.

Long-chain PORA can be found in the composition in the form of esters of free fatty acids; mono-, di- and o-triglycerides; phosphoglycerides, including lecithins; and/or their mixtures. It may be better if the long-chain PORA are in the form of phospholipids, in particular, phosphatidylcholine. Nowadays, the preferred source, at least when the processing route is such that organoleptic properties and cholesterol levels are acceptable, are egg yolk phospholipids, possibly due to the high phospholipid and/or phosphatidylcholine content of the egg yolk. n-β6 and/or n-3 fatty long-chain ROS can be administered in the form of an intravenous (ie, parenteral) solution, such as choline and phosphatidylcholine. It is better if the intravenous solution contains an effective amount of PORA, phospholipid and/or choline in the appropriate daily dose of the parenteral solution. "

The exact concentration therefore varies widely depending on the intended infusion dose and is much more concentrated in a bolus or low-volume parenteral product than in a hydrated or parenteral nutritional product. Parenteral compositions generally include pharmaceutical;" acceptable carriers and excipients such as buffers, preservatives, etc. n-6 and/or n-3 fatty long-chain RORBA, choline and phospholipid can, as an alternative, be introduced in the form of an enteral composition. Enteral compositions containing long-chain PORA, choline or phospholipid,

Ge" can be in the form of a solution or emulsion of the active ingredient; or in the form of a food medium consisting of protein, carbohydrates, other fats, minerals and vitamins. Enteral compositions containing

Me. active components, can provide either additional or complete nutrition. The concentration of 2) long-chain FISH in an enteral composition can vary from almost 10095 by weight (as in the case of a bolus emulsion) to 0.595 by weight (as in the case of formulas for feeding infants with full nutrition) o composition depending on the route of administration and purpose. In complete infant formula, the concentration can be even lower if enough is added to the formula to deliver an effective amount of long-chain RORBA.

The best embodiment of the present invention relates to an improved formula for feeding infants for complete nutrition, suitable for feeding even premature infants. Such a best egg composition consists of protein, carbohydrates, and lipids, wherein about b to about 4095% by weight of the total lipid is egg phospholipid, which is substantially free of cholesterol. The term "substantially free" means

F) that the cholesterol content of egg phospholipid is less than 0.195 by weight, preferably less than 0.0595 by weight, of total lipid.

Those skilled in the art will readily understand what is meant by infant formula. When diluted or reconstituted, if the original form is a concentrate or powder, in the ready-to-use state, a typical infant formula contains approximately 10-35g of protein per liter of formula; 20-50g of lipid per liter of formula; 60-110g of carbohydrates per liter of formula and other various ingredients such as vitamins, minerals, fibers, emulsifiers, etc. For the purpose of understanding the ingredients of infant formula and its preparation, the following US patents are incorporated herein by reference: 1) US Patent No. 5492899, issued by Mazvgog ei a).; 2) 65 US patent No. 5021245, issued to Wojciech A.; 3) US patent No. 5234702, issued to Kai?

Mo5602109, issued by the Mazvog of Ukraine; and 5) US patent No. 4,670,268 issued to Maptots. More particularly, this embodiment of the invention includes an infant formula containing approximately 40-50g of lipid per liter of formula, wherein the lipid is a mixture of medium chain triglycerides and egg phospholipid that is substantially free of cholesterol. Typically, the lipid mixture contains from about 1-40% by weight, more preferably from about 5% to about 30% by weight, of egg phospholipid. This embodiment is particularly designed to provide long-chain FISH selected from n-3 fatty acids and n-β6 fatty acids, phospholipids and/or choline in amounts beneficial to infants.

Method of obtaining

Since chicken egg yolks contain triglycerides and phosphatides, it may be preferable to treat 7/0 egg yolks with organic solvents in such a way as to separate the phosphatides from the triglycerides, sterols (eg, cholesterol), and other components. A variety of methods described in the literature are suitable for this separation, at least on a laboratory scale. Alternatively, such egg phosphatides are substantially free of cholesterol and are commercially available in dry powder form from RITapzienyi, Ips. (U/a0 Kedap, I) under MoR-123 in the catalog.

Next, egg phosphatide is included in the enteral composition of the present invention. Given the lipid content, it was expected that the introduction of egg phosphatides into infant formula would be easy to do at the oil stage. However, it was discovered that these lipid-lipid dispersions were unacceptable, and that obtaining an aqueous dispersion of egg phosphatide led to an improved product. Aqueous dispersions of about 2-1590 wt.%, preferably from about 3 to about 895 wt.%, were prepared in water at a temperature of between cool to 2°C ambient temperature (about 20-25") for best results. The warmer the water formed, the less acceptable organoleptic properties.

Separately, carbohydrate, protein, and lipid suspensions containing the nutrient macronutrient are prepared as is known in the art, and these suspensions are mixed at a temperature of approximately 130 to 140°C. Immediately prior to homogenization, the phosphatide dispersion is mixed with the remainder of the infant formula.

Preferably, before adding the phosphatide dispersion to the final mixture (immediately before homogenization), air is removed from the phosphatide dispersion under moderate vacuum. The removal of air may be accomplished by any mechanism, but an atomizing deaerator at about 15 inches (38.1 cm) of mercury has given satisfactory results. This additional step improves the organoleptic and olfactory properties of the final product even more than filtration with activated carbon atoms or a combination of both (see Example 1PI).

To obtain parenteral compositions used in this invention, generally accepted technologies for obtaining sterile parenteral mixtures for baby food can be used. It may be found that in this case it is better not to use egg phosphatides, but instead to use triglycerides or fatty acid esters, which can be found in recombinant or cell-derived sources and oils. Gee)

Industrial use

The compositions of the present invention are useful for nutritional support of infants and/or adults. Supplementation of long-chain RIGAs, particularly n-6 and n-3 fatty acids and best of all AA and ONA, has been generally accepted as beneficial “for infant neuron development and visual acuity, although negative results have also been found in the literature.

Compositions useful in this invention include those containing any or all of the following: - c (a) long-chain RIOBA selected from p-b and p-C fatty acids, typically in the form of phospholipid or c phosphatidylcholine; "» (b) polar phospholipids, regardless of the nature or length of the attached fatty acids; (c) choline, preferably phosphatidylcholine.

For example, the improved egg phospholipid mixture described in detail in the Examples provides high levels of each of these components and has surprisingly been found to significantly reduce disease in

MES in MES-sensitive infant populations. In a special embodiment, the method of reducing the disease on MES b is carried out through the introduction of arachidonic acid (AA, 20:4 p-b6) or, even better, AA in combination with docosahexaenoic acid (ONA, 22:6 p-3).

In more detail, this aspect of the invention provides a method for reducing necrotizing enterocolitis in an infant susceptible to necrotizing enterocolitis, the aforementioned method comprising

Chl" introduction of an effective amount of at least one long-chain PORA selected from the group of Cr n-6 fatty acids,

Co» n-6 fatty acids, Co n-3 fatty acids and Co» n-3 fatty acids. Administration is carried out at a level of at least 1.Omg p-3 fatty acids per kilogram of the baby's weight per day. Even better, an embodiment uses a combination of n-6 and n-C fatty acids at a weight ratio of about 2:1 to about 4:1.

A method for reducing the incidence of necrotizing enterocolitis in human infants, mentioned above, is further described

F, the method consists of giving the baby egg phospholipids in the amount of at least 1.0 mg of long-chain p-6 fatty acids per day. Preferably, the egg phospholipids contain AA as a significant portion of p-b6 fatty acids and preferably also contain ONA and/or other long-chain p-3 fatty acids in the ratios mentioned above. An additional aspect of the present invention relates to the enteral administration of phospholipids to humans, in particular phospholipids containing AA and/or ONA, which easily increase the level of AA and ONA fatty acids in blood serum in humans relative to compositions containing AA and ONA triglycerides.

A more convenient criterion for the administration of the compositions according to the invention is the daily administration in mg per kg of the infant's body weight. The following Table presents the basic norms for minimum, best and ideal target 65 levels that differ for each composition useful in the present invention.

The wide range exists due to the fact that not all infants who will benefit most from the present invention will consume equal volumes of formula. Those who consume less get less of each ingredient. The ideal range of doses is approximately 100 kcal. There are also different points of view on how to determine the choline content.

Table A shows that the best dose is about 2-4 times more AA than ONA. It is also observed that the minimum amount of phospholipid and choline is the same. This is achieved when the entire phospholipid is phosphatidylcholine. If other nitroalcohols replace choline (eg, ethanolamine, serine, or inositol), the ratio of choline to total phospholipid is reduced.

AA and/or ONA can be administered as separate components or together or in combination with other ingredients such as protein, lipid, carbohydrate, vitamins and minerals. Nutritional support for low birth weight infants is parenteral (intravenous feeding) or enteral. Thus, an appropriate amount of long-chain RIOBA can be incorporated into a parenteral nutrition solution or added to a conventional enteral formula for feeding low birth weight infants. It is best if the method of the present invention is carried out through enteral administration of infant feeding mixtures containing AA with and ONA to infants with low birth weight. Such a formula for feeding babies, in addition, consists of the right amount of carbohydrate and protein and the right combination of minerals and vitamins. As an example, an infant formula for use in the methods of the present invention is a modified Zityas Zresia formula! Sage U (Kovv

Rhodysiv Oimiviop oi ArBroi I arogayugies, Soishtryv, Opio), which is described in detail in Example II.

An additional aspect of the present invention is a method of increasing the amount of arachidonic acid and docosahexaenoic acid in human blood serum, the above-mentioned method consists of the step of administering to a person the above-mentioned mixture for feeding babies, containing AA and ONA in the form of phospholipids. at

Recent studies by applicants of the present invention have shown that administration of long-chain FISH to infants susceptible to MES reduces the incidence of MES and may also reduce the level or severity of MES. Applicants have also found that administration of phospholipids from animal or plant sources is also effective for ee,

Reduction of MES disease in MES-susceptible infant populations. Gee)

Example 1

Egg yolk phosphatide was obtained from RIapziyei, Ips. (U/aykedap, II - Sayaiyud Mo. P-123) and used in the following Examples. The content of fatty acids and cholesterol of this egg phosphatide is given in Table I. Also given is the sum of all p-3 and all p-6b "long-chain" ROS. "- with: with" cholesterol in egg yolk lecithin Sat 00011111 To

Fo svo 01111111 vv sila 11111111111111ova

F VVA 00111111111111111111100 C SIT 00000088 ha of Sutte 00000000 0000 0000 Oz BUZIA 30000008.

Vetstuvdtya 00000007"

VeisvogAa tu 831

Those skilled in the art will appreciate that the specific amount of different fatty acids contained in egg yolk lipid 65 will vary depending on the breed, diet and age of the hen. In addition, the extraction procedure used in

RIapz (enpi for obtaining phosphatide) was used to obtain the results in the Examples in the material,

containing ultra-low cholesterol when treated with fatty acids, which are very useful in the field of nutrition.

Example II

In this example, "experimental" and "control" infant formulas were obtained, respectively, with the egg phosphatide of Example I! and without him. Control composition courtesy of Zitias Zresias! Sage? (Bovv Rhodysiv

Oimiziop oi ABroiii Iarogaigiez, Soishtrives, OPpio) and was obtained using the following list of ingredients contained in the formula for feeding infants, which has the composition shown in Tables 1I-IM below:

Water (Kozpeg), nonfat milk, hydrolyzed corn starch, lactose, fractionated coconut oil 70 (medium-chain triglycerides), whey protein concentrate, soybean oil, coconut oil, tribasic calcium phosphate, potassium citrate, sodium citrate, magnesium chloride, ascorbic acid acid, mono- and diglycerides, soy lecithin, calcium carbonate, Irish moss, choline chloride, iron sulfate, m-inositol, taurine, niacinamide, I-carnitine, os-tocopherol acetate, zinc sulfate, calcium pantothenate, potassium chloride, copper sulfate , riboflavin and vitamin A palmitate, thiamine chloride hydrochloride, pyridoxine hydrochloride, 75 biotin, folic acid, manganese sulfate, phylloquinone, vitamin C, sodium selenite and cyanocobalamin.

Typically, the protein, carbohydrate, lipid, vitamin, and mineral suspensions are prepared separately and then mixed prior to homogenization, as generally described in previously filed US patents relating to the preparation of infant formulas.

In the experimental infant formula, the egg phosphatide of Example I was incorporated into the formula during preparation. First, egg phosphatides were dispersed in water at 257C to obtain 895 dispersion. Immediately prior to homogenization, the phosphatide dispersion was mixed with protein, carbohydrate, vitamin, mineral, and other lipid suspensions to produce an "experimental" infant formula containing the composition shown in Tables II-IM below. The amount of each ingredient is given "per liter" and "per kcal" because it is well known in the industry that the resulting infant formula has a higher or lower caloric density than the standard 20kcal per fluid ounce (28.3g). about experimental mixtures for feeding babies

Preferred range Units per liter" | Units per 10Ocal Z with M vii ini o

Fo

F zv o 154.76-209.36 dam 0111 zt 000 «

Phosphorus, mg 720-970 85.71-119.46 is Pon pei z s mak 1 yuyu 1 » y 119.05-174.88 dai 0111111 yuyu 100 1

F

Fo sp t" zv about her in vv

Choline, mg 81-243 9.64-29.93

Energy (kcal) viv 01 "allowed 24kcal per ounce (28.3g) of liquid.

The following Table III describes the lipid content (variable only) used in the control and experimental products. It can be seen that the two infant formulas have the same total amount of lipid, but differ fundamentally in the replacement of egg phospholipid with a portion of medium-chain triglycerides.

This substitution yields significantly higher amounts of phospholipids and choline, as well as additional long-chain ROS.

Table of PI

Lipids for control and experimental formulas for feeding infants phospholipid egg 9 9(4 ug/l) 2 o "MST-triglycerides with an average chain length I" fractionated "I same N/V-not detected

IF)

Table IM shows the composition of fatty acids for control and experimental infant feeding formulas. This is the sum of the fatty acid components of egg lecithin and Zytias Zresia infant formula!

Saget (Se)

From Table IM ish

The average content of fatty acids by weight is 95%, 01%, 01%, 02%, 0%, 0%, 0%, 0000231, 0000231, 0000231, 0000231, 0000231, 0000231, 00969002

Zomrystinovya 0000385000000004080 iv your no s ts HY NO

F votelmtyuolenia 551 00007650

Zvptalmtoolenova | 003 | 042

F 20000011 111300 o Tomararinova | 004. | 1009 i. yaz 171111 iy 164 pl i PO PI PO i» TOstearynova 0000268 1389 18:1 p-9-oleic acid 11.25 SHHI 182 p-in-linoleic acid. 010001000016361010000 1887

TZpblinolenova 1001230 at 1V3 pz EE U NI NI tva Ag, at 20yu-arachidonova 102! 10 20:11 to nt shi HU SHI bo 202 po lova | ro02 203 p ni i NO A 20:4 p-vaAA AS om go pz A 20:53 nya p p PO POI bo 22:0-behenova 0.07 012 gavpo | 1 1-1 177 tot gavpz 1-11 zavpz-onA 11101 morovi. | Ltd

WeisROgatZ 1011021

Vyssseogape | 1710 1 homv

Greetings 1

All 70171177 vvi | 777 fl

The inclusion of egg phosphatide resulted in 0.2195 by weight of the total lipid mixture of long-chain p-3 fatty acids and 0.4895 by weight of the total lipid mixture of long-chain p-b fatty acids. More specifically, 0.1495 by mass of the total fat mixture was ONA and 0.4195 by mass of the total lipid mixture was AA. At 1000 kcal/kg/day per 1 kg infant, this infant formula contained approximately 22 mg AA and approximately 7 mg ONA per day. Of course, this ratio is only one possible ratio in the invention.

Example II

In this experiment, changes in the process of reducing organoleptic deficiencies associated with the use of egg phospholipids were evaluated. Isolation of egg phospholipids useful in the present invention often results in egg phosphatides that have somewhat undesirable organoleptic properties for use in infant formula. The situation can be improved by creating a product that is not undesirable for the infant or the caregiver. This process of improving the final product is described below.

Several food mixtures were prepared as in Example II, except that 6% by weight of the fat mixture was egg phospholipid, which was preheated using various procedures. Phospholipid from eggs was dispersed in a portion of the mixture of oils described in Example II, or in a portion of water. The oil dispersions were unacceptable and could not be used even after heating to about 957C. Dispersing the phospholipid in ambient to warm water was easy, and this dispersion is the best way to create an aqueous dispersion.

The main batch of 390 by weight dispersion of egg phospholipid was obtained by mixing phospholipid in 907 water "Ezo for about 1 hour. Part of this dispersion was passed: (1) only through the deaerator; (2) through a carbon filter only; (3) deaerator and carbon filter; or (4) no processing. what)

The activated carbon filter contained 80 g of activated carbon, and the deaerator was operated at medium vacuum (15 inches (38.1 cm Hg)). Portions of batches were passed through a filter 3 times and once through a deaerator. After both procedures, the batch was first passed through a filter, then through a deaerator. The treated batches were then added to the appropriate infant formula immediately prior to sample homogenization and packaging.

Then a committee of expert scientists checked the original samples for "taste characteristics" (organoleptic properties). The results of the inspection are given in Table M. "and Z

Mixture for feeding: n » Results of organoleptic inspection.

Dispersion processing 1

F o : PE " Taste characteristics: o AA - Arachidonic acid

AAI - Arachidonic acid and long-acting acid Ratio: 0.5 - very weak; 1 - weak; 1.5 - weak-moderate; 2-dimensional; 2.5 - moderate-strong; and Z are strong.

Interestingly, the least aromatic sample was the one containing the dispersion that was only passed through the deaerator. The dispersion that was passed through an aerator and an activated carbon filter had the lowest score, (F) except for the control (no treatment of the phospholipid dispersion).

Formulas for feeding infants obtained according to Examples ІІ and ІІ were given to infants in a study conducted in Meopaia! Migvzegu oi Opimegeiu oi Teppezzee Mem/rogp Sepieg under the leadership of Og. Zyvap E.

Sapvop with financial support from Kovzv Rgodisiv Oimiviop oi ArbBoi I aroghaigiez (Research AE78), MISNYUO grant KO1-YANO31329, and Mayopa grant! Eue Ipziyshe KO1-EMO8770. The parameters of the study included: growth, development of neurons and visual acuity. Long-chain ROS are considered physiologically important for brain and eye development and rapidly accumulate in fetal tissues in the last trimester of pregnancy. Thus, preterm 65 infants do not exhibit normal amounts of long-chain RORA relative to full-term infants.

Enrollment criteria: Low birth weight, less than 1500g (range 750-1375g) without evidence of cardiac, respiratory, gastrointestinal or other systemic diseases is the basis for enrollment in this clinical trial. The infant also had no birth asphyxia or clinical complications related to blood group incompatibility. Mothers of enrolled infants had no prenatal infections with proven adverse effects on the fetus. The fact that the mother abuses medical drugs was considered an exclusion criterion. All babies started oral feeding on the 7th day of life.

During the clinical study, all 120 infants were registered on the 7th day of life. With the exception of one infant who was transferred to another hospital immediately after enrollment (control), all other infants (n-119) 7/0 were cared for in the same hospital. The infants were assigned (randomly) to 1 of 3 groups, two of which received the control infant formula during their hospitalization, the other received the experimental infant formula (see Example II). Infants who were lost during hospitalization were replaced by other infants from this group for treatment. Due to the study design, most infants were fed the control formula. The total number of infants fed the control formula was more than double the number of infants fed the experimental formula.

Results: An unexpected result was that more cases of necrotizing enterocolitis (NEC) were observed in the control groups than in the experimental groups. Table MI shows the total number of births according to treatment (control/experimental ratio) and the number of births in each group that had MES. MES was considered present or suspected when clinical signs and symptoms 2o were consistent with this disease, such as abdominal distension, gastric residues, vomiting, heme-positive stool, the presence of mucus in the stool, and the presence of C-reactive protein 20.5 mg/dL ( Rotsygsugoiv ei ai., "Zidpiysapse oi

Zegia! S-geasiime Rgoiveip Kezropsez in MeopaiaI Ipiesiop apa O(Sheg Oiveazev", Reaiaig., 1993, 92:431-435).

MES was proven in 15 of the control infants and only in 1 of the experimental group. and cm o 770 control Ecoleriment ch y sosopdeyujuvvnniachesi y " or suspect on MES so

Statistical analysis of these data using Fisher's exact test (two-tailed test) showed that the number of infants with confirmed MES in the control treatment group(s) was significantly higher (p-0.039) than the number of infants in the experimental treatment group with MES. eh

Example M

In this experiment, the inclusion of AA and ONA in parenteral (intravenous) feeding was determined. The parenteral solution may contain various components known in the field, with AA and ONA in the form of phospholipid, triglycerides or methyl esters. AA and ONA may be the sole active ingredients, C 740 mixed with conventional parenteral carriers and excipients, or, better still, AA and ONA are included in a parenteral formula for infant feeding to supplement or supply the total nutritional

From" supporting the baby. Typical parenteral nutritional solutions contain an amount of lipid equal to approximately 2g/kg/day. The amount of AA and ONA in the lipid mixture is better to create by introducing 10 to ZOmg/kg/day of AA and 3-15mg/kg/day for ONA.

Example MI b In this experiment, egg lecithin in the experimental infant formula of Example II

Ge") was substituted for soy lecithin in an amount approximately 10 times greater than in control infant formulas. Soy lecithin, like other phospholipids obtained from plant sources, does not contain polyunsaturated acids with a long chain; however, the polar nature of phospholipids and their ability to readily enter the gut mucosa may provide protection to the gut lining, thus producing results comparable to those of experimental infant formulas

T" of Example II. In addition, soy lecithin contains linoleic acid (18:2 p-6 - a dietary irreplaceable precursor

AA) and linolenic acid (18:3 p-3 - a dietary irreplaceable precursor of ONA).

Example MY 29 In this experiment, the use of phospholipids containing AA and ONA in infant formula is compared with

GF) with triglycerides containing AA and ONA. The infant formula of Example II is compared with the same infant formula where the egg phospholipid is replaced by a mixture of microbial triglycerides containing an amount of AA and ONA comparable to the egg phospholipid formula.

Healthy, full-term infants were enrolled in a clinical study to determine the amount of AA and 60 ONA in blood serum after enteral administration. Infants fed phospholipid infant formula were expected to have serum AA and ONA levels more similar to those of breast-fed infants than those fed a control infant formula containing AA and ONA in the form of triglycerides. This experiment should demonstrate that phospholipids containing AA and ONA are a better form of administration than triglycerides containing AA and ONA. Thus, improved enteral mixtures and methods of increasing the amount of AA and ONA in blood serum are proposed in this invention.

Modifications and alternative embodiments of the compositions and methods of the present invention will be apparent to those skilled in the art in light of the foregoing description. Accordingly, this description is compiled only as an illustration, with the aim of teaching specialists how to implement it. The full scope of the invention, for which protection is created, is described in the attached Formula.

Claims (1)

The formula of the invention 10. . M. with. she, 1. A method of reducing the incidence of necrotizing enterocolitis in children, characterized in that the child prone to necrotizing enterocolitis is administered at least one n-6 polyunsaturated fatty acid in an amount of approximately 1.0 - 60 mg/kg per day together with at least one p -3 polyunsaturated fatty acid in the amount of approximately 0.25 - 35 mg/kg per day. 2. The method according to claim 1, which differs in that the aforementioned at least one p-6 polyunsaturated fatty acid is arachidonic acid. C. The method according to claim 2, which differs in that the above-mentioned administration is carried out enterally. 4. The method according to claim 2, which differs in that the above-mentioned administration is carried out parenterally. 5. The method according to claim 1, which is characterized by the fact that the above-mentioned n-3 polyunsaturated fatty acid is α-docosahexaenoic acid. 6. The method according to claim 1, which differs in that the above-mentioned n-b6 polyunsaturated fatty acid is arachidonic acid, and the above-mentioned n-3 polyunsaturated fatty acid is docosahexaenoic acid. 7. The method according to item b, which differs in that the above-mentioned administration is performed by feeding a sufficient amount of an enteral composition containing arachidonic acid and docosahexaenoic acid, and the above-mentioned child is administered from about 1.0 to about 60 mg/kg per day of arachidonic acid acid and about 0.25 to about 35 mg/kg per day of docosahexaenoic acid. (at) 8. The method according to claim 7, which is characterized by the fact that the mass ratio of arachidonic and docosahexaenoic acids is in the range from about 2: 1 to about 4: 1. 9. The method according to claim 1, which is characterized in that the above-mentioned p-b6 polyunsaturated fatty acid and the above-mentioned p-3 polyunsaturated fatty acid are long-chain fatty acids independently selected from at least one source from the group consisting of egg lecithin, oils from mushrooms, oils from algae and oils from marine organisms. co 10. The method according to claim 1, which differs in that the aforementioned n-6 polyunsaturated fatty acid and the aforementioned n-3 polyunsaturated fatty acid are used in the form of phospholipids. |se) 11. The method according to claim 5, which is characterized by the fact that the parenteral solution consists of at least 20 mg/l of arachidonic acid and at least 10 mg/l of docosahexaenoic acid. 12. The method according to claim 11, which is characterized in that the aforementioned solution consists of about 20-200 mg/l of arachidonic acid and about 10-50 mg/l of docosahexaenoic acid. 13. The method according to claim 1, which is characterized by the fact that the child is fed with a sufficient amount of the composition for "70 enteral nutrition, which contains protein, carbohydrate and phospholipids, while at the same time, at least 2.0 mg/kg of p-6 polyunsaturated fatty acids are administered and at least 0.5 mg/kg p-3 polyunsaturated fatty acids per day. 14. The method according to claim 13, which is characterized by the fact that at the above-mentioned feeding, at least 2.0 mg/kg of arachidonic acid and at least 0.5 mg/kg of docosahexaenoic acid per day are administered. 15. The method according to claim 14, which differs in that an enteral composition containing egg phospholipid is used for feeding. FO 16. A method of reducing the incidence of necrotizing enterocolitis in children, which is characterized by the fact that an effective amount of phospholipids is administered daily to a child prone to necrotizing enterocolitis, which is approximately 60 μmol/kg - 2400 μmol/kg of the child's body weight. c 17. The method according to claim 16, which is characterized by the fact that the specified introduction is performed using a sufficient amount of the food composition. o 18. The method according to claim 17, which is characterized by the fact that it includes the introduction of a sufficient amount of the above-mentioned "Their" food composition to provide an amount of phospholipids between about 200 μmol/kg and about 1500 μmol/kg per day. 19. The method according to claim 16, which differs in that the aforementioned phospholipids are obtained from egg lecithin. 20. The method according to claim 17, which differs in that the aforementioned phospholipids are administered in combination with at least one polyunsaturated fatty acid selected from the group consisting of arachidonic acid and (F; docosahexaenoic acid. GI 21. The method according to claim 20, which differs in that the aforementioned phospholipids are administered in combination with arachidonic acid and docosahexaenoic acid in an amount of from about 200 μmol/kg to about 1500 μmol/kg in phospholipids, from about 5.0 mg/kg to about 40 mg/kg of arachidonic acid and from about 1.5 mg/kg to about 20 mg/kg docosahexaenoic acid per day. 22. A method of reducing the incidence of necrotizing enterocolitis in children, which differs in that a child prone to necrotizing enterocolitis is administered an effective amount of choline daily, which is approximately 60 μmol/kg - 1800 μmol/kg of the child's body weight. 65 23. The method according to claim 22, which is characterized by the fact that the indicated introduction is carried out using a food composition that contains the above-mentioned choline in a sufficient amount. 24. The method according to claim 23, which is characterized in that it includes the introduction of a sufficient amount of the aforementioned nutritional composition to provide an amount of choline between about 150 μmol/kg and about 1200 μmol/kg per day. 25. The method according to claim 22, which differs in that the aforementioned choline is in the form of phosphatidylcholine. 26. The method according to claim 25, which differs in that the above-mentioned phosphatidylcholine is obtained from egg lecithin. 27. The method according to claim 23, characterized in that the aforementioned choline is administered in combination with at least one polyunsaturated fatty acid selected from the group consisting of arachidonic acid and docosahexaenoic acid. 70 28. The method of claim 27, wherein said choline is administered in combination with arachidonic acid and docosahexaenoic acid to provide from about 150 μmol/kg to about 1200 μmol/kg choline, from about 5.0 mg/kg to about 40 mg/kg of arachidonic acid and from about 1.5 mg/kg to about 20 mg/kg of docosahexaenoic acid per day. Official bulletin "Industrial Property". Book 1 "Inventions, useful models, topographies of integrated microcircuits", 2004, M 5, 15.05.2004. State Department of Intellectual Property of the Ministry of Education and Science of Ukraine. se what about "I I c) Ge) (Se) (Se) - a (e)) (e)) (95) 1 GT» ko bo b5