COMPOSITIONS COMPRISING CHOLINE AND DERIVATIVES THEREOF, USES THEREOF AND PROCESSES FOR THEIR PREPARATION

Abstract

The invention provides compositions comprising choline and water soluble derivatives thereof, processes for their preparation and uses thereof.

Claims

1-28. (canceled)

29. A method for one or more of: (i) optimizing, improving, promoting, increasing or maintaining plasma levels of one or more of ferritin, iron, growth hormone, insulin-like growth factor, insulin-like growth factor binding protein or ketone bodies in a subject; (ii) optimizing, improving, promoting, increasing or maintaining one or more of plasma lipid profile, plasma triglyceride levels and plasma cholesterol levels in a subject; (iii) promoting optimal growth of a subject, improving statural growth of a subject or a combination of the same; and (iv) preventing, delaying or reducing subject's obesity, reducing subject's body mass index or a combination of the same, said method comprising administering to said subject a composition comprising at least one water soluble choline compound selected from a group consisting of GPC, PCh and choline; wherein said at least one water soluble choline compound is derived from mammalian milk, and wherein the concentration of said at least one water soluble choline compound is at least 0.5% w/w of the composition.

30. A method for one or more of: (i) optimizing, improving, promoting, increasing or maintaining plasma levels of one or more of ferritin, iron, growth hormone, insulin-like growth factor, insulin-like growth factor binding protein or ketone bodies in a subject; (ii) optimizing, improving, promoting, increasing or maintaining one or more of plasma lipid profile, plasma triglyceride levels and plasma cholesterol levels in a subject; (iii) promoting optimal growth of a subject, improving statural growth of a subject or a combination of the same; and (iv) preventing, delaying or reducing subject's obesity, reducing subject's body mass index or a combination of the same, said method comprising administering to said subject a composition comprising at least one water soluble choline compound selected from a group consisting of GPC, PCh and choline; wherein said at least one water soluble choline compound is derived from at least one natural source, and wherein the concentration of said at least one water soluble choline compound is at least 0.5% w/w of the composition.

31. The method according to claim 29 wherein PCh comprises at least 0.02% w/w of the composition, and wherein GPC comprises at least 0.5% w/w of the composition.

32. The method according to claim 29, wherein GPC comprises between about 20% w/w to about 100% w/w of the at least one water soluble choline compound, and PCh and choline comprise between about 1% w/w to about 60% w/w of at least one water soluble choline compound.

33. The method according to claim 29 wherein the composition comprises at most 50 ppm of at least one of TMA, TMAO, ethylene oxide, glycidol or any combination of the same.

34. A method for one or more of: (i) optimizing, improving, promoting, increasing or maintaining plasma levels of one or more of ferritin, iron, growth hormone, insulin-like growth factor, insulin-like growth factor binding protein or ketone bodies in a subject; (ii) optimizing, improving, promoting, increasing or maintaining one or more of plasma lipid profile, plasma triglyceride levels and plasma cholesterol levels in a subject; (iii) promoting optimal growth of a subject, improving statural growth of a subject or a combination of the same; and (iv) preventing, delaying or reducing subject's obesity, reducing subject's body mass index or a combination of the same, said method comprising administering to said subject a composition comprising at least two water soluble choline compounds selected from a group consisting of GPC, PCh and choline; wherein said at least two water soluble choline compounds comprise at least 0.5% w/w of the composition; and wherein PCh and choline together comprise at least 1% w/w of said water soluble choline compounds.

35. The method according to claim 34, wherein PCh comprises between about 1% w/w to 70% w/w of said water soluble choline compounds.

36. The method according to claim 34, wherein said at least two water soluble choline compounds comprise at least 1% w/w of the composition.

37. The method according to claim 34, comprising GPC, PCh and choline.

38. The method according to claim 34, wherein said GPC comprises between 20% w/w to about 100% w/w of said water soluble choline compounds.

39. The method according to claim 34, wherein said PCh comprises between 1% w/w to about 70% w/w of said water soluble choline compounds.

40. The method according to claim 34, wherein choline comprises between about 1% w/w to 25% w/w of said water soluble choline compounds.

41. The method according to claim 34, wherein at least one of said GPC, PCh or choline is derived from a natural source.

42. The method according to claim 34, wherein at least one of said GPC, PCh or choline is derived from mammalian milk

43. The method according to claim 34, wherein the molar concentration of PCh is greater than the molar concentration of GPC.

44. The method according to claim 29, wherein said composition is a pharmaceutical, a dietary supplement, a medical food or a nutraceutical composition.

45. The method according to claim 29, wherein the subject is an infant.

Description

DETAILED DESCRIPTION OF EMBODIMENTS

EXAMPLE 1

Production of Compositions According To The Invention

A. Extraction and Concentration of Water Soluble Choline Compounds Using Ethanol as a Solvent to Obtain a Powder Product.

[0215] Whey stream from dairy production was concentrated by evaporation of water and then crystalized to yield lactose crystals that were separated by filtration from their mother liquor. Mother liquor was partially demineralized by nanofiltration membrane and dried by means of spray drying to obtain dry powder.

[0216] Ten grams of the dried powder were mixed with 200 ml of ethanol 90% (with 10% water) for 2 hours at 40 C. to obtain slurry. The slurry was filtered through Buchner filter in order to separate between the filtrate and the solids. The filtrate was evaporated, under reduced pressure, using rotary evaporator to obtain dry powder. The dry powder obtained contained 5.5% GPC and 0.52% PCh (as determined by .sup.31P-NMR).

B. GPC Enrichment Using Ion Exchange Columns.

[0217] Ten grams of the final product from Example 1A (containing 5.5% GPC and 0.52% PCh) were dissolved in 40 ml of water and then were passed through a glass column containing 100 ml of strong anionic exchange resin (Doc2001). The solution coming out of the first column was then transferred through a strong cationic resin (0017) column and was neutralized by a weak anionic resin (D301) column, in order to remove minerals and to obtain a neutralized filtrate. All resins were obtained from JIANGSU SUQING WATER TREATMENT ENGINEERING GROUP CO. Finally, the neutralized filtrate was evaporated using a rotary evaporator, under reduced pressure, to receive a product containing 20.5% GPC and 0.03% of PCh (as determined by .sup.31P-NMR).

C. GPC Enrichment Using a Silica Chromatography Procedure

[0218] Ten grams of the final product of Example 1A (containing 5.5% GPC) were dissolved in 20 ml of ethanol:water (80%:20% v/v) solution and were loaded on a silica gel chromatography column filled with 70 grams of Davisil LC60A 20-45 m silica from the Grace Company. 500 ml ethanol:water (80%:20% v/v) was then transferred through the column in order to separate a lactose containing fraction from a GPC containing fraction. After the first 200 ml of the ethanol containing solvent was applied to the column, the collection of GPC containing fractions of the filtrate commenced. The GPC containing fractions were then evaporated using rotary evaporator, under reduced pressure, to obtain a dry powder, The dried powder obtained was injected to the HPLC with ELS detector against a sample of the product of Example 1A, in the same concentration. The relative peak area of the GPC was about 5 fold higher in the purified product compared to the product of example 1A.

D. Purification of Choline Compounds From Whey Stream With Methanol as a Solvent to Obtain a Powder Product.

[0219] Whey stream from dairy production was used to produce whey protein concentrate by diafiltration using Ultra filtration membranes. The permeate from the membranes was demineralized by means of electrodialysis. The mineral free stream was dried by spray dryer to produce a powder. Five grams of this dried powder were mixed with 40 ml of methanol for 2 hours at 25 C. The whole sample was then centrifuged for 5 minutes at 6,000 RPM in order to separate between the solution and the solids. The solution was evaporated using rotary evaporator, under reduced pressure, to receive dry powder. The dry powder obtained contained 7.6% GPC and 0.4% PCh (By .sup.31P-NMR).

E. Purification of GPC From Lactosalt Optitase

[0220] Dairy salts fraction called Lactosalt Optitase (Armor) containing about 85% salts, 5% moisture and 0.5% protein was purified by electrodialysis. Purification was performed using a PCCell ED 64-4 Electrodialysis cell unit. This unit has a 10 parallel cell pair stack structure. The active size of each membrane is 88 cm (active area of 0.0064 m2). Hence, total active area is 0.064 m.sup.2.

[0221] For the electrolyte circuit, a 0.25 M solution of sodium sulfate was used. The anolyte and the catholyte chambers were connected in series. A circulating NaCl solution served as the concentrate. Its initial concentration was around 1000 mg/lit. 1 liter of solution containing 10 gr of Lactosalt Optitase dissolved in demineralized water was fed to the circulating chamber. The voltage was pre-set at its highest value (36.5 Volts for the stack). Recirculation was stopped when further significant decreases in conductivity were no longer noted in the salts solution. Sample of the purified solution was dried by rotary evaporator, under reduced pressure, to dryness. The dried product obtained was injected to the HPLC with ELS detector against a sample of the same concentration of the original Lactosalt Optitase. The relative peak area of the GPC was about 10 fold higher in the purified product compared to the raw material.

F. Purifeation of GPC From Krill Meal

[0222] 200 gr. of Superba Krill meal were mixed together with 1 liter of methanol for one hour at 25 C. The whole solution was then filtered through Buchner filter in order to separate between the filtrate and the solids. The filtrate was evaporated using rotary evaporator to obtain 35 gr of oil. Thirty five ml of purified water and thirty five ml of butanol were added to the oil and the solution was mixed for few minutes. Phase separation was achieved in a separatory funnel. The bottom phase was evaporated to obtain 2.78 gr. which contained 7.1% GPC (by .sup.31P-NMR).

G. Preparation of Gummy Bears Containing GPC

[0223] 0.4 gr of Citric acid and 0.37 gr of Trisodium citrate were dissolved in 30 ml water using agitation. The solution was heated up to 75 C., followed by the addition of 5 gr of white sugar (Sucrose) and 1.5 gr of Citrus Pectin. The mixture was heated up to 100 C., and agitated at 100 C. for 2-3 minutes. 30 gr of glucose syrup 80% and 50 gr white sugar (Sucrose) were added and the mixture was heated up to 108 C. under continuous agitation until full dissolution and 78 Bx is achieved (about 40-50 minutes). The solution was cooled down to 1.00 C. and 1.06 gr product of example No. 1A were added. Agitation was continued at 100 C. for 2-3 minutes, and the following flavorings and colorings were added: 0.5 gr Adipic acid, 0.15 gr strawberry flavor essence, 0.5 ml Lemon juice, 0.15 gr shade ruby red essence, 0.77 gr Citric acid 50%. Agitation was continued at 100 C. for 2-3 more minutes, than heat source was stopped and product obtained was poured into molds at 90-100 C. Molds were placed in an air conditioned room for about 48 hours for drying.

EXAMPLE 2

GPC Stability Of Natural Vs. Synthetic Choline Composition in Stomach Model

[0224] More than 90% of orally administrated GPC is absorbed from the intestine. Once absorbed, GPC is rapidly circulated to all organs and taken up into the cells. It is thus desirable that GPC will be minimally affected by gastrointestinal conditions and remain intact without any modifications which might affect its activity and efficacy.

[0225] In order to test the gastrointestinal stability of natural (mammalian milk derived) water soluble choline compounds according to the invention, in comparison with synthetic, conventional choline compounds an in vitro gastric (stomach) model was used.

[0226] In vitro gastric model was conducted as previously described by Kanner and Lapidot 2001 using simulated gastric fluid (SGF) as an artificial dissolution medium intended to represent stomach conditions. SGF was prepared according to the U.S. Pharmacopoeia by dissolving 0.2% w/w sodium chloride and 0.32% w/w of purified pepsin (derived from porcine stomach mucosa) in acidic water, pH about 1.2.

[0227] Choline compounds were purified from mother liquor from lactose crystallization. The purification included two stages: first stage of membrane purification and a second stage of crystallization.

[0228] Synthetic water soluble choline compound was produced from soy lecithin by a reaction using Sodium methoxide as a catalyst to obtain GPC and methyl esters. Several purification steps were performed in order to separate between the methyl esters and GPC. In addition, a mineral removal step was performed using ion exchangers.

[0229] Both natural and synthetic water soluble choline compounds (compositions are detailed in Table 2) were incubated with SGF at 37 C. in a shaking bath for 180 minutes and then the samples were analyzed for GPC levels by HPLC.

[0230] The results, presented in Table 2, show that a composition containing about 5% w/w of natural, water soluble, choline compound from dairy source did not demonstrate any GPC degradation (GPC level remained constant) following 180 minutes incubation in gastric model conditions. On the other hand, the composition which contained synthetic, water soluble, choline compound, demonstrated degradation of about 5.4% of the GPC (from 30.50 mg GPC to 28.84 mg GPC).

[0231] These results demonstrate that compositions containing water soluble choline compounds according to the invention are more stable in gastric conditions than other compositions which contain conventional choline compounds.

TABLE-US-00002 TABLE 2 Composition A B Source of water Natural (mammalian Synthetic soluble choline source) compound % PCh and Choline 12.5% <1% from total choline compounds (w/w) at baseline after after at time 0 180 min at time 0 180 min GPC (mg GPC/ml 35.35 35.48 30.50 28.84 SGF) % GPC Degradation 0 5.4

EXAMPLE 3

The Effect of Different Water Soluble Choline Compounds on Factors Related to Infant Growth

[0232] Study Design

[0233] The ability of different water soluble choline compounds to affect blood levels of factors related to infant growth was investigated in an animal model of neonatal Sprague Dawley rats aged 3-5 days.

[0234] Animals were randomly assigned to one of three diets, twelve rats per group.

[0235] Animals within a litter were randomly assigned across treatments.

[0236] The study groups were:

[0237] Group A: Rats fed with formula containing choline

[0238] Choline chloride was purchased from Sigma Chemical Company.

[0239] Group B. Rats fed with formula containing GPC and phosphocholine (wherein Phosphocholine>GPC). Synthetic water soluble choline compound was produced from soy lecithin by a reaction using sodium methoxide as a catalyst to obtain GPC and methyl esters. Several purification steps were performed in order to separate between the methyl esters and GPC. In addition, a mineral removal step was performed using ion exchangers. Phosphocholine chloride calcium salt tetrahydrate was purchased from Sigma Chemical Company.

[0240] Group C: Rats fed with formula containing GPC (wherein GPC>Phosphocholine) from bovine milk natural source. Choline compounds were purified from mother liquor from lactose crystallization. The purification included two stages: Ion exchange purification using a strong cationic resin (0017) column and weak anionic resin (D301) column (all resins were obtained from JIANGSU SUQING WATER TREATMENT ENGINEERING GROUP CO.) and chromatographic purification based on UBK535K resin from Diaion Company.

[0241] All formula-based diets contained the same added choline equivalent levels that originated from different water soluble choline compounds. The water soluble choline compound composition of each diet is detailed in Table 3.

TABLE-US-00003 TABLE 3 Group A Group B Group C Source of the choline compound synthetic synthetic natural Total choline equivalent (mg/liter 679 680 679 formula) Choline (mg/liter formula) 678.4 NA NA Choline (M) 6525 NA NA GPC (mg/liter formula) NA 826.65 1676 GPC (M) NA 3217 6525 Phosphocholine (mg/liter NA 610.4 1.35 formula) Phosphocholine (M) NA 3317 2 GPC out of composition (% w/w) NA 39.7 24.8 GPC out of choline compound NA 57.5 99 (% w/w) Choline compound out of 100 69 24.8 composition (% w/w) Choline + phosphocholine 100 41.9 <1 compound out of choline compound composition (% w/w) NA = Not Applicable

[0242] Gastrostomy tube fed infant rats: The gastrostomy tube fed rat pup is model to mimic infants fed formula, using tube feeding to overcome the difficulties in bottle feeding of neonatal rats. The formulas were prepared to resemble the composition of rat milk with ingredients modified to meet the study objectives, The model enables complete control of the volume and thus nutrient intake. This avoids any difficulties due to variable intake across treatment groups. The animals were reared by milk feeding from 3-5 to 18-20 days of age. Formula volume which was administered to the rats was calculated daily based on the animal weight.

[0243] Plasma and Tissue collection: Blood samples were centrifuged at 2000 g10 minutes, and plasma was recovered. In order to standardize tissue sample location from every animal, tissue samples harvest was done following the same protocol.

[0244] List of Analyses:

[0245] In plasma: cholesterol (total, VLDL+LDL, HDL), triglycerides, phosphatidycholine, free choline, glycerophosphocholine, phosphocholine, betaine, dimethylglycine (DMG), homocysteine, methionine, cysteine, Trimethylamine (TMA) and Trimethylamine N-oxide (TMAO), Ketone bodies, Folate, S-adenosyl methionine (SAM), S-adenosyl homocysteine (SAH), Growth hormone, Insulin-like growth factor 1 (IGF-1), IGF 3 binding protein (IGF3BP) and ferritin.

[0246] In urine: TMA and TMAO.

[0247] In liver: SAM, SAH and triglycerides.

[0248] Results

[0249] Plasma and urine samples were analyzed for levels of various lipids and metabolites and tissue samples were weighted. The results are presented in Table 4.

[0250] Plasma parameters: plasma levels of choline in groups B and C were higher in comparison with the choline levels in group A. Choline levels in group C were higher also in comparison with group B. Choline/betaine ratio and IGF-I levels increased in groups B and C in comparison with group A. Triglyceride and cholesterol levels were higher in group C in comparison with both group A and group B and better resembled the high triglyceride and cholesterol levels of suckling rats (128.41111.3 mg/dl and 158.536.0 mg/dl, respectively). TMAO levels in group B and C were lower in comparison with those of group A. Plasma SAM and SAH levels were higher in group C in comparison with both group A and B. Folate levels were lower in group C in comparison with groups A and B.

[0251] Urine parameters: TMA and TMAO levels in the urine of group B and C animals reduced in comparison with those of group A.

[0252] Tissue parameters: body and liver weights were lower in group C in comparison with those of group A and B.

TABLE-US-00004 TABLE 4 Group A Group B Group C Plasma parameters Choline (M) 11.5 3.5 12 3.4 13.7 4.0 Betaine (M) 151.3 43.2 122.4 31.9 148.5 30.9 DMG (M) 18.0 3.8 17.6 5.1 15.6 1.9 Ketones (mM) 0.5 0.2 0.4 0.2 0.4 0.2 Triglycerides (mg/dl) 99.5 55.7 92.1 45.5 127.9 86.6 Cholesterol (total) 77.1 20.9 88.5 10.2 89.0 17.7 (mg/dl) TMA (M) 0.2 0.1 0.1 0.1 0.2 0.1 TMAO (M) 1.2 0.4 0.9 0.4 1.0 0.3 IGF-I (ng/ml) 184.9 51.1 235.7 56.2 203.4 44.2 Plasma SAM (nM) 448.6 24.6 404.5 37.2 486.3 15.9 Plasma SAH (nM) 123.5 31.9 127.3 70.8 150.4 24.5 Folate (nM) 128.5 18.3 118.7 26.6 115.6 9.6 Homocysteine (M) ND ND ND Methionine (M) ND ND ND Cysteine (M) ND ND ND HDL (mg/dL) ND ND ND GPC (M) ND ND ND Phosphatidylcholine ND ND ND and lyso phosphatidylcholine (M) IGF3BP (mg/l) ND ND ND Ferritin (ng/ml) ND ND ND Urine parameters TMA (M) 8.0 9.6 2.4 1.4 4.6 3.1 TMAO (M) 158.3 79.0 73.3 54.0 139.1 89.0 Tissue parameters Body weight (gr) 41.3 5.9 48.2 5.8 39.7 5.1 Liver weight (gr) 1.7 0.4 2.1 0.3 1.6 0.3 Liver weight/100 gr 4.1 0.5 4.3 0.3 3.9 0.3 body weight Liver SAM (M) 120.3 6.1 114.1 5.3 115.4 7.6 Liver SAH (M) 18.9 0.9 17.5 1.0 17.4 1.1 *results are given as mean SD, ND = Not Determined

CONCLUSION

[0253] The above results demonstrate that animals in group C which were fed with formula containing choline compound derived from mammalian milk (bovine milk) demonstrated higher choline bioavailability, better lipid profile and a reduced body and liver weight in comparison with both groups A and B in which rats were fed with formula containing synthetic choline compounds. Furthermore, group C demonstrated plasma SAM and SAH increase and folate reduction in comparison with groups A and B. To addition, both groups B and C demonstrated an increase in plasma choline/betaine ratio and IGF-1 levels and a reduced TMA and TMAO levels in comparison with group A.

[0254] The improved choline bioavailability, improved plasma and urine profile and the reduced body and liver weight which were observed with group C (and to a lesser extent in group B) may be indicative of healthy growth of the animals and be associated with for example optimal growth and statural growth of infants. They may also be associated with lower risk to develop various diseases such as CVD, obesity and atherosclerosis and other beneficial functions.

Example 4

GPC Stability in Infant Formula Containing Water Soluble Choline Compounds According to the Invention in Comparison With Infant Formula Containing Conventional Water Soluble Choline Compounds.

[0255] Two types of infant formulas were prepared in pilot scale by the following method: Skimmed milk powder, lactose and concentrated whey protein (80%) were mixed into distilled water by a high speed agitator and warmed to 65-70 C. Following 5 minutes of mixing, different recipes of waters soluble choline compounds, minerals, nucleotides, amino acids and vitamins were added. Following an additional 15 minutes, an oil mixture containing vegetable oils including ARA (Arachidonic acid) oil and DHA (Docosahexaenoic acid) oil were added. Mixing continued for additional 15 minutes. Then, the mixture was homogenized by APV Rannie pressure homogenizer with two-stage assembly: 70 Bars at stage 1 and 240 Bars at stage 2. Next, the homogenized mixture was spray dried by typical Spray Dryer at a rate of 20 liter/hr with air inlet temp of about 180 C. and air outlet temp of about 80 C. Dried powder was collected and dry blended with a premix (about 0.37%) of minerals and elemental substances.

[0256] At the end of this stage the two formulas contained a different composition of choline compounds: while 348-80-1 contained a molar ratio of GPC>PCh, formula no. 348-80-6 contained a molar ratio of GPC<PCh.

[0257] 100 gr aliquots from each formula were packed under nitrogen environment in sealed airtight aluminum packages and stored at 25 C.2 (Humidity 60%5%) in temperature and moisture controlled storage chambers.

[0258] At baseline and following 12 months of storage, samples were analyzed for GPC and PCh content.

[0259] Table 5 shows stability results following 12 months at 25 C.2 (Humidity 60%5%). Sample 348-80-6, containing PCh>GPC ratio demonstrated minor GPC degradation level (about 4%) while formula 348-80-1, containing GPC>PCh ratio, resulted in about 38% GPC degradation. These results demonstrate that compositions containing water soluble choline compounds according to the invention are more stable than other compositions which contain conventional choline compounds.

TABLE-US-00005 TABLE 5 Formula 348-80-1 348-80-6 GPC-Phosphocholine Ratio GPC > GPC < Phosphocholine Phosphocholine % w/w PCh and choline from >1 >1 water soluble choline compounds % w/w Choline 0.09 0.013 % w/w PCh T0 NA 0.09 % w/w PCh Following 12 NA 0.09 month of storage at 25 C. 2 % w/w GPC T0 0.037 0.024 % w/w GPC Following 12 0.023 0.023 month of storage at 25 C. 2 % GPC degradation 38 4.2 Following 12 month of storage at 25 C. 2 % PCh degradation NA 0 Following 12 month of storage at 25 C. 2 NA = Not Applicable, T0 = baseline values