DESC:TECHNICAL FIELD:
The present invention relates to a novel synergistic bioactive composition for modulating choline dehydrogenase activity, useful in managing the treatment of neural tube defects (NTDs), attention and cognitive dysfunction, infant processing speed, visuospatial memory, congenital disabilities, and attention deficit hyperactivity disorder. Particularly, the bioactivet composition comprises exogenous blend of 2-Hydroxy-N,N,N-trimethylethanaminium or precursors thereof and Methylene tetrahydrofolate reductase (MTHFR) regulators and salts thereof in specific ratio along with pharmaceutically acceptable excipients.

BACKGROUND:
According to the American Congress of Obstetricians and Gynaecologists (ACOG), a woman’s intake of nutrients and vitamins during pregnancy should come from a variety of foods, including Proteins, Carbohydrates, Vitamins, Minerals, Fats. Prenatal vitamins are essential during pregnancy to support both women health and the health of growing baby. Some of the nutrients include folic acid, iron, calcium, vitamin D, choline, omega-3 fatty acids, B vitamins, and vitamin C. vitamin B9, is necessary for DNA synthesis and cell division. Maternal nutrition during pregnancy and infant nutrition in the early postnatal period (lactation) are critically involved in the development and health of the newborn infant.
Nutritional requirements increase tremendously during pregnancy & lactation. Nutrients containing methyl-donors influence epigenetic programs and has importance in understanding of not only developmental abnormalities but also for understanding the origin of chronic diseases. Various animal studies have examined the effect of nutrient containing methyl-group-supplemented diets of mother on offspring epigenome, health, and longevity.
Choline is important for regulation of gene expression, the biosynthesis of lipoproteins and membrane phospholipids and for the biosynthesis of the neurotransmitter acetylcholine; glycine betaine plays important roles as a primary intracellular osmo-protectant and as methyl donor for the biosynthesis of methionine from homocysteine, a required step for the synthesis of the ubiquitous methyl donor S-adenosyl methionine. It is observed that choline can modulate methylation because, via betaine homocysteine methyltransferase (BHMT). Choline is an essential dietary nutrient with functions like as a source of labile one carbon units (CH3, methyl); as a component of lipids including phosphatidylcholine, sphingomyelin, and lipid mediators such as platelet activating factor; and as a component of the neurotransmitter acetylcholine.
It is reported that plasma free choline assessed early in the second trimester of gestation was positively associated with betaine, dimethylglycine, and methionine, indicating that choline is further metabolized and contributes methyl groups for regeneration of methionine. [PLoS ONE 7(8): e43448, 2012]. Recently, choline dehydrogenase CHD has generated considerable medical attention due to its association with various human pathologies, including male infertility, homocysteinuria, breast cancer and metabolic syndrome. [Archives of Biochemistry and Biophysics 537 (2013) 243–252].
Human choline dehydrogenase is a nuclear encoded, mitochondrial enzyme involved in choline metabolism. From a medical point of view, human CHD is of great interest due to its association with various pathologies, including male infertility, homocystinuria (HCU) and cancer. It is observed that maternal methyl-group donor intake before and during pregnancy could possibly induce epigenetic alterations in offspring genes related to metabolism and genes important to maintain DNA methylation patterns. [Clin Epigenetics. 2017; 9: 16].
In humans and mammals, glycine betaine also plays an important role as a methyl group donor involved, for example, in the methylation of homocysteine to methionine.
Human CHD catalyses the oxidation of choline to betaine aldehyde, which is further oxidized to glycine betaine. Human CHD is important for the catabolic utilization of choline because choline is involved in the stimulation of cholinergic neuronal activity and in restoring phosphatidylcholine levels in the neuronal membrane, thus displaying a neuroprotective action relevant for diseases such as memory and cognitive deficits. One of the studies suggests that maternal dietary and supplemental intake of methyl-group donors, especially in early gestation, can influence infant buccal DNA methylation in genes related to metabolism, growth, appetite regulation, and DNA methylation reactions. Nutrition in the early postnatal period, lactation, can influence infant DNA methylation levels.
Methionine supplementation of the maternal diet improves fetal growth through enhanced protein synthesis. Therefore, need to be approached with caution because any imbalance may worsen rather than improve the supply of amino acids. During pregnancy, methionine continues to be metabolized by a series of metabolic pathways that have the nonprotein amino acid homocysteine as a central component.
By acquiring a methyl group from either methylated bioactive compound, homocysteine is converted back to methionine, completing the cycle. Methionine is dependent on the availability of choline, betaine, and folic acid in the diet. Hyperhomocysteinemia during pregnancy, which is a consequence of perturbations in methionine and/or folate metabolism, has been implicated in adverse outcomes such as neural tube defects, preeclampsia, spontaneous abortion, and premature delivery.
Children with homocystinuria, present with a clinical syndrome of dislocated optic lens, premature osteoporosis, mental retardation, in addition to atherothrombosis of medium and small arterial vessels. Further elevated levels of homocysteine are associated with premature diabetic retinopathy, osteoporosis-induced bone fractures, and dementia-type disorders.
Randomized clinical trials have shown that oral supplementation with the combination of folic acid, B6-, and B12-vitmains substantially lowers circulating homocysteine levels, but does not appear to improve outcome cognition defects. Limited choline intake during pregnancy and lactation increases the susceptibility of the mother to develop choline deficiency [Adv Nutr 2022;13:2445–2457]. Higher gestational choline levels protect fetal brain development in maternal infection [J Pediatr. 2019 May; 208: 198–206.e2.].
Emerging evidence suggests that choline plays a vital role in epigenetic regulation, cellular signaling, and lipid metabolism. Further research is needed to fully understand choline's role in fetal development and to establish evidence-based recommendations for optimal choline intake during pregnancy.
It is also known that hyperhomocysteinemia in women is associated with pregnancy complications such as recurrent pregnancy loss and pregnancy hypertension disorders, and is negatively associated with birth outcomes such as low birth weight and preterm birth, although there are some inconsistencies in the literature in this regard [Hum. Reprod. Update. 2013;19:640–655].
Further maternal homocysteine is associated with neural tube defects and continued this line of research in humans by revealing associations between maternal one-carbon metabolism and embryonic size and growth disturbed homocysteine and folate metabolism is implicated in many different diseases, including congenital birth defects like congenital heart disease, cleft lip and palate, stillbirths, late pregnancy complications, different kinds of neurodegenerative and psychiatric diseases, osteoporosis and cancer.
Methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme in folate/homocysteine pathway. It catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5 methylene tetrahydrofolates, which donates methyl group for the conversion of homocysteine to methionine. Normal development of fetal brain and spinal cord depends on nutrients derived from the maternal diet. For example, maternal diets deficient in folate or choline result in decreased neurogenesis and increased apoptosis in fetal brain [J Nutr. 2010 Jun; 140(6): 1162–1166].
Therefore, the need arises to develop a stable bioactive composition that has impact on one-carbon metabolism status during conception and gestation on embryonic development and offspring health.

OBJECT OF THE INVENTION;
The primary object of the invention is to provide novel therapeutical approach for improving fetal development.
Another object of the invention is to provide effective combination of bioactive components to balance one carbon metabolism by regulating homocysteine and methionine cycle.
Yet another object of the invention is to provide bioavailable oral compositions for treating congenital birth defects.
Yet another object of the invention is to provide cost-effective, side-effect-free composition for modulating CHD activity.

SUMMARY OF THE INVENTION:
To meet the above objects, the inventors of the instant invention carried out thorough experiments to establish significant effects of the active ingredients or vitamin or quaternary ammonium compounds or nutrients or methyl donors that ameliorate therapeutic efficacy in the treatment of congenital birth defects/ fetal development.
The present invention provides a bioactive composition for modulating Choline Dehydrogenase [CHD] activity comprising therapeutically active exogenous blend of 2-Hydroxy-N,N,N-trimethylethanaminium or its precursors and salts thereof and Methylenetetrahydrofolate reductase (MTHFR) regulators and salts thereof, along with pharmaceutically acceptable excipients,

ABBREVIATIONS:
CHD: Choline dehydrogenase
NTD: Neural tube defects
MTHFR: Methylenetetrahydrofolate reductase

BRIEF DESCRIPTION OF THE FIGURES:
Figure 1 illustrates CHD protein activity in cmp (counts per minutes) for G1 to G6 test groups.
Figure 2 illustrates CHD protein detection by Western blot analysis for G1 to G6 test groups.
Figure 3 illustrates Choline plasma serum level in pg/ml for G1 to G6 test groups.

DETAILED DESCRIPTION OF THE INVENTION:
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully interpreted and comprehended. However, any skilled person or artisan will appreciate the extent to which such embodiments could be generalized in practice.
It is further to be understood that all terminology used herein is for the purpose of describing particular embodiment only and is not intended to be limiting in any manner or scope. Unless defined otherwise, all technical and scientific expressions used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertain.
In describing and claiming the embodiments of the present invention, the following terminology will be used in accordance with the definitions set out below which are known in the state of art.
The singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Also, the term ‘composition’ does not limit the scope of the invention for multiple compositions that can be illustrated for best mode of the invention.
The term “pharmaceutically/nutraceutically acceptable salt,” as used herein, represents those salts which are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio.
Particularly the term “pharmaceutically acceptable salts” refers to the relatively non-toxic, inorganic, and organic acid addition salts of compounds, amino acid salt, sugar-based salt, alkali or alkaline earth metal salts, as well as hydrates, solvates, co-crystals, polymorphs and the like of the salts.
The salts are preferably selected from chloride, bromide, calcium, sodium, hydroxide, phosphate, acetate, fumarate, lactate, maleate, sulphate, tartrate, citrate, hydrates carbonate, gluconate, mesylate, hydrochloride, hydrobromide and glucosamine.
The inventors of the present invention astonishingly found that the modulation of CHD activity improves the fetal development by potentiating choline requirement and balancing the one carbon metabolism during the pregnancy. To meet the requirement, the inventors developed novel composition that synergistically acts on CHD enzyme and improves the fetal development.
All modifications and substitutions that come within the meaning of the description and the range of their legal equivalents are to be embraced within their scope. A description using the transition “comprising” allows the inclusion of other elements to be within the scope of the invention.
As used herein, the term (MTHFR) pertains to methylenetetrahydrofolate reductase which is a key regulatory enzyme in methionine and homocysteine metabolism. It is rate-limiting folate cycle enzyme which regulates folate cycle.
The term ‘NTD’ relates to neural tube defects which are birth defects of the brain, spine or spinal cord that happen in fetuses within the first month of pregnancy. The two most common NTDs are spina bifida (a spinal cord defect) and anencephaly (a brain defect).
In a preferred embodiment, the invention provides synergistic bioactive composition for modulating choline dehydrogenase activity.
In another preferred embodiment, the present invention provides composition comprising synergistic combination of biologically active compounds wherein the combination comprises exogenous blend of 2-hydroxy-N,N,N-trimethylethanaminium and/or precursors thereof and methylenetetrahydrofolate reductase (MTHFR) regulators and salts thereof, present in suitable weight ratio along with pharmaceutically acceptable excipients.
In another embodiment, the present invention provides a synergistic bioactive composition comprising combination of 2-Hydroxy-N,N,N-trimethylethanaminium and/or precursors thereof which is present in the range of 0.1-2000 mg and Methylenetetrahydrofolate reductase (MTHFR) regulators which is present in the range of 0.1-1000 mg along with pharmaceutically acceptable excipients/carriers.
In another embodiment, the present invention provides composition comprising synergistic combination, wherein 2-Hydroxy-N,N,N-trimethylethanaminium or its precursors are present in the range of 1-2000 mg ; preferably 1-1000 mg; more preferably 1-500 mg by weight of total composition.
In yet another embodiment, the 2-Hydroxy-N,N,N-trimethylethanaminium precursors are selected from the group consisting of 2-Amino-3-hydroxypropanoic acid, ß-Aminoethyl alcohol, 2-Methylaminoethanol and N,N-Dimethylaminoethanol.
In yet another embodiment, 2-Hydroxy-N,N,N-trimethylethanaminium may include phospholipid salt. Phospholipid conjugated to a hydrophobic or hydrophilic molecule is safe and biocompatible. The phosphate salt include calcium;2-(trimethylazaniumyl)ethyl phosphate; chloride, 2-(trimethylazaniumyl)ethyl phosphate, 2-(trimethylazaniumyl)ethyl phosphate; chloride.
In one more embodiment, the present invention provides composition comprising synergistic combination, wherein the methylenetetrahydrofolate reductase (MTHFR) regulators and salts thereof are present in the range of 0.1-500 mg; preferably 0.1-200 mg; more preferably 0.1-100 mg by weight of total composition.
In further embodiment, the methylenetetrahydrofolate reductase (MTHFR) regulators are selected from group consisting of (2S)-2-[[4-[(2-amino-4-oxo-3H-pteridin-6-yl)methylamino] benzoyl]amino] pentanedioic acid; (2S)-2-[[4-[(2-Amino-4-oxo-1H-pteridin-6-yl)methylamino]benzoyl] amino]pentanedioic acid ; (2S)-2- [ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino] benzoyl] amino] pentanedioic acid ; (2S)-2-[[4-[(2-amino-4-oxo-5,6,7,8-tetrahydro-3H-pteridin-6-yl)methylamino]benzoyl]amino]pentanedioic acid; (2S)-2-({4-[(6aR)-3-amino-1-oxo-1H,2H,5H,6H,6aH,7H,8H,9H-imidazolidino[1,5-f]pteridin-8-yl]phenyl} formamido) pentanedioic acid; (2S)-2-[[4-[(2-amino-5-methyl-4-oxo-3,6,7,8-tetrahydropteridin-6-yl)methylamino]benzoyl]amino]pentanedioic acid; (2S)-2-(4-(((2-Amino-5-methyl-4-oxo-3,4,5,6,7,8-hexahydropteridin-6- yl)methyl)amino)benzamido)pentanedioic acid and salts thereof.
In another embodiment, the present invention provides composition comprising synergistic combination of 2-Hydroxy-N,N,N-trimethylethanaminium and/or precursors thereof and Methylenetetrahydrofolate reductase (MTHFR) regulators ; wherein (MTHFR) regulators or salts thereof are optionally complexing with trimethylglycine.
Particularly 2-Hydroxy-N,N,N-trimethylethanaminium and its precursors maintains the methyl group metabolism simultaneously, (MTHFR) regulators maintain methionine cycle and inhibits activity of CHD to meets the dietary requirements for choline and thus prevents common birth defects. Moreover, the composition reduces the elevated NTD risks associated with lower levels of total choline.
In a preferred embodiment, the present invention provides composition comprising synergistic combination of 2-hydroxy-N,N,N-trimethylethanaminium or precursors thereof and methylenetetrahydrofolate reductase (MTHFR) regulators; wherein 2-hydroxy-N,N,N-trimethylethanaminium or precursors thereof and (MTHFR) regulators or salts thereof are present in the weight ratio of 1: 0.001 to 1:1 along with pharmaceutically acceptable excipients.
In another embodiment, the present invention provides composition comprising synergistic combination of 2-hydroxy-N,N,N-trimethylethanaminium or precursors thereof and methylenetetrahydrofolate reductase (MTHFR) regulators ; wherein (MTHFR) regulators or salts thereof are optionally complexing with trimethylglycine.
In another embodiment, the present invention provides composition comprising synergistic combination of 2-Hydroxy-N,N,N-trimethylethanaminium and precursors thereof and Methylenetetrahydrofolate reductase (MTHFR) regulators which facilitates normal development (neurogenesis) of hippocampus of the fetus. Particularly it supports fetal brain development.
In another embodiment, the present bioactive composition modifies epigenome via methylation to support integrity and stability of fetal and placental genomes.
The CHD enzymatic reaction achieves the dual physiological role of regulating the concentration of free choline in cells and bodily fluids and synthesizing a metabolite that is relevant to both osmo protective and methylating processes. The elevated activity of choline preserves the membrane integrity through phosphatidylcholine; promotes myelin sheath formation through sphingomyelin and synaptogenesis through acetylcholine.
In another embodiment, the present invention provides stable composition that provides bio active nutrients useful for fetal brain and spinal cord development, moreover the composition is useful to improve neural development of fetus by reducing incidence of birth defects. The maternal intake of the present composition not only improves several pregnancy outcomes but also protects against certain neural and metabolic insults.
The composition is useful supplement for improving cell survival, synaptic plasticity, and normal brain development of fetus. It further reduces cortisol induced stress conditions.
In another embodiment, the present invention provides synergistic bioactive composition wherein two ingredients work intracellularly in synergistic way to improve one carbon metabolic cycle through modulating activity of CHD. Further the said composition is useful in the treatment of neural tube defects (NTDs), attention and cognitive dysfunction, infant processing speed, visuospatial memory, congenital disabilities, and autism. It is also involved in the production of red blood cells and supports optimal fetal growth and development.
In yet another embodiment, the present invention provides composition that is useful to improve cognitive function and memory in maternal and foetus.
The term "therapeutically effective amount " denotes an amount that reduces the risk, potential, possibility or occurrence of a disease or disorder, or provides advanced alleviation, mitigation, and/or reduction or restoration or modulation, regulation of at least one indicator/biomarker (e.g., blood or serum CRP level), and/or minimize at least one clinical symptom related to neural tube defects.
The term ‘subject in need thereof’ pertains to a human, preferably female; more preferably female with choline and folate deficiency. Particularly female including but not limited to adult, women planning for pregnancy, women before conception, pregnant women, conceive, lactating women, women of reproductive age.
In the context of the present invention, the term “treatment” refers to alleviate, mitigate, prophylaxis, attenuate, manage, regulate, modulate, control, minimize, lessen, decrease, downregulate, up regulate, moderate, inhibit, restore, suppress, limit, block, decrease, prevent, inhibit, stabilize, ameliorate, cure, heal birth defects, fetal growth.
Notably, the instant composition is non-hazardous, non-toxic, and safe for human consumption without any severe adverse effects, therefore the present composition can also be used as preventive therapy/ adjuvant therapy/ add-on therapy/ combination/ adjunctive therapy in a subject in need thereof.
Certain compounds of the present invention exist in unsolvated forms as well as solvated forms, including hydrated forms. Further, some compounds of the present invention exist in multiple crystalline or amorphous forms (“polymorphs”). Compounds of the present invention are formulated in isomeric, geometric, enantiomeric or stereoisomeric forms.
The active compounds may be in the hygroscopic or non-hygroscopic form based on water affinity of the compound salts.
In general, all physical forms are of use in the methods contemplated by the present invention and are intended to be within the scope of the present invention.
Compound or pharmaceutically acceptable salts includes, hydrates, halides like chloride, bromide, metal salts like calcium, sodium, potassium, hydroxide, phosphate; polymorphs, solvates, enantiomers or racemates. Some of the crystalline forms of the compound exist as polymorphs and as such are intended to be included in the present disclosure. In addition, some of the compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are intended to be encompassed by some embodiments.
In one of the embodiments, the present invention provides a synergistic bioactive composition that is present in effective amount along with pharmaceutically acceptable excipients.
As used herein, the term “pharmaceutically acceptable carriers, diluents or excipients” is purported to mean, without limitation, any adjuvant, carrier, excipient, sweetening agent, diluents, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, emulsifier, or encapsulating agent, encapsulating polymeric delivery systems or polyethylene glycol matrix which is acceptable for use in the subject, preferably humans. Excipients may also include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, fragrances, glidants (flow enhancers), lubricants, preservatives, sorbents, suspending or dispersing agents, sweeteners, surfactant, anticaking agent, food additives, or waters of hydration, salts.
In another embodiment, the present invention relates to synergistic bioactive composition, which can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. The preferable route of administration includes but not limited to sublingual, rectal, topical, parenteral, nasal, or oral.
In some embodiment, the present synergistic bioactive composition can be administered to the subject in need thereof, in the form which is suitable for oral use, such as a tablet, capsule (in the form of delayed release, extended release, sustained release, enteric coated release); hard gelatin capsules, hard gel, soft gelatin capsules in an oily vehicle, veg capsule, hard or soft cellulose capsule, granulate for sublingual use, effervescent or carbon tablets, aqueous or oily solution, suspension or emulsion, encapsulate, matrix, coat, beadlets, nanoparticles, caplet, granule, particulate, agglomerate, spansule, chewable tablet, lozenge, troche, solution, suspension, rapidly dissolving film, elixir, gel, tablets, pellets, granules, capsules, lozenges, aqueous or oily solutions, suspensions, emulsions, sprays or reconstituted dry powdered form with a liquid medium or syrup; for topical use including transmucosal and transdermal use, such as a cream, ointment, gel, aqueous or oil solution or suspension, salve, parch or plaster; for nasal use, such as a snuff nasal spray or nasal drops; for vaginal or rectal use, such as a suppository; for administration by inhalation, such as a finely divided powder or a liquid aerosol; for sub-lingual or buccal use, such as a tablet, capsule, film, spray. Further, the composition can be formulated for parenteral use including intravenous, subcutaneous, intramuscular, intravascular, infusion, intraperitoneal, intracerebral, intracerebroventricular, or intradermal.
Synergistic bioactive composition of the present invention is suitable for oral administration and can be presented as discrete units such as capsules (e.g., soft-gel capsules), cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid, syrup; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredients can also be presented in the form of a bolus, electuary or paste, bioactive bar, energy bars (candy bars), powder, energy drink, ready to drink, granule sachet.
Further, the present composition can be formulated in the form of age-appropriate pediatric oral dosage forms such as syrup, minitablets, chewable formulations, orodispersible films, orodispersible tablets and bioadhesive buccal tablets. It can also be prepared in the form of snack, confectionery food products, sachet, gummies.
In another embodiment, the synergistic bioactive composition of the present invention is non-toxic, cost effective, enriched with bioactive ingredients, and provides safeguard against problems associated with choline deficiency without any adverse effect.
In another embodiment of the present invention, the diluents are selected from starches, hydrolyzed starches, partially pregelatinized starches, anhydrous lactose, cellulose powder, lactose monohydrate, sugar alcohols such as sorbitol, xylitol and mannitol, silicified microcrystalline cellulose, ammonium alginate, calcium carbonate, calcium lactate, dibasic calcium phosphate (anhydrous/ dibasic dehydrate/ tribasic), calcium silicate, calcium sulphate, cellulose acetate, corn starch, pregelatinized starch, dextrin, ß-cyclodextrin, methylated-ß-cyclodextrin, dextrates, dextrose, erythritol, ethyl cellulose, fructose, fumaric acid, glyceryl palmitostearate, magnesium carbonate, magnesium oxide, maltodextrin, maltose, medium-chain triglycerides, polydextrose, polymethacrylates, sodium alginate, sodium chloride, sterilizable maize, sucrose, sugar spheres, talc, trehalose, xylitol, vehicles like petrolatum, dimethyl sulfoxide and mineral oil or the like.
In some embodiment of the present invention, the diluent in the composition/formulation is present in a range of 1% to 30% by weight of the total composition/formulation.
In yet another embodiment of the present invention, the binder is selected from disaccharides such as sucrose, lactose, polysaccharides and their derivatives like starches, cellulose, or modified cellulose such as microcrystalline cellulose and cellulose ethers such as hydroxypropyl cellulose (HPC); hydroxypropyl methyl cellulose (HPMC); sugar alcohols such as xylitol, sorbitol, or mannitol; protein like gelatin; synthetic polymers such as polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), starch, acacia, agar, alginic acid, calcium carbonate, calcium lactate, carbomers, carboxymethylcellulose sodium, carrageenan, cellulose acetate phthalate, chitosan, copovidone, corn starch, pregelatinized starch, cottonseed oil, dextrates, dextrin, dextrose, ethyl cellulose, guar gum, hydrogenated vegetable oil, mineral oil, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxyl ethyl methyl cellulose, hydroxypropyl cellulose, inulin, cellulose, methyl cellulose, polyvinylpyrrolidone and polyethylene glycol, lactose, liquid glucose, hypromellose, magnesium aluminium silicate, maltodextrin, maltose, methyl-cellulose, microcrystalline cellulose, pectin, poloxamer, polydextrose, polymethacrylates, povidone, sodium alginate, stearic acid, sucrose, sunflower oil, various animal vegetable oils, and white soft paraffin, paraffin, flavorants, colorants and wax.
In another embodiment, the present invention provides synergistic bioactive composition which is useful in the treatment of neural tube defects (NTDs), attention and cognitive dysfunction, infant processing speed, visuospatial memory, congenital disabilities, and autism.
In a further embodiment of the present invention, the binder in the composition/formulation is present in a range of 0.1 to 30% by weight of the composition/formulation.
In some embodiment, the antioxidant is selected from tocopherol (vitamin E), sesamol, guaiac resin, methionine, beta-carotene, lycopene, lutein, zeaxanthin, butylated hydroxy anisole (BHA), butylated hydroxytoluene (BHT), sodium ascorbate, sodium metabisulfite (SMB), l-carnosine, propyl gallate (PG), tertiary butyl hydroquinone, cysteine (CYS), citric acid, tartaric acid, phosphoric acid and ascorbic acid.
In another embodiment of the present invention, the amount of antioxidant in the composition/formulation is present in the range of 0.1 to 10% by wt. of the composition/ formulation.
In another embodiment of the present invention, the lubricant is selected from magnesium stearate, zinc stearate, calcium stearate, glycerin monostearate, glyceryl behenate, glyceryl palmitostearate, hydrogenated castor oil, hydrogenated vegetable oil, light mineral oil, magnesium lauryl sulphate, medium-chain triglycerides, mineral oil, myristic acid, palmitic acid, poloxamer, polyethylene glycol, sodium benzoate, sodium chloride, sodium lauryl sulphate, sodium stearyl fumarate, stearic acid, talc, potassium, or sodium benzoate or the like.
In another embodiment of the present invention, the lubricant in the composition/formulation is present in a range of 0.1% to 10.0% by weight of the total composition/formulation.
In another embodiment of the present invention, the solubilizing agent is selected from polysorbate 80, sodium lauryl sulphate, anionic emulsifying wax, nonionic emulsifying wax, glyceryl monooleate, phospholipids, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polyoxylglycerides, sorbitan esters, triethyl citrate, vitamin E, polyethylene glycol succinate, microcrystalline cellulose, carboxymethylcellulose sodium, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, hypromellose, hypromellose, acetate succinate, lecithin, polyethylene alkyl ethers, aluminum oxide, poly(methylvinyl ether/maleic anhydride), calcium carbonate, crospovidone, cyclodextrins, fructose, hydroxpropyl betadex, oleyl alcohol, povidone, benzalkonium chloride, benzethonium chloride, benzyl alcohol, benzyl benzoate, cetylpyridinium chloride, inulin, meglumine, poloxamer, pyrrolidone, sodium bicarbonate, starch, stearic acid, sulfobutylether beta cyclodextrin, tricaprylin, triolein, docusate sodium, glycine, alcohol, self-emulsifying glyceryl monooleate, cationic benzethonium chloride, cetrimide, xanthan gum, lauric acid, myristyl alcohol, butylparaben, ethylparaben, methylparaben, propylparaben, sorbic acid or the like.
In another embodiment of the present invention, the amount of solubilizing agent or surfactant in thecomposition/formulation ranges from 0.1% to 10% by weight of the composition/formulation.
In a preferred embodiment of the present invention, the solubilizing agent or surfactant is present in a range of 0.1% to 5.0% by weight of the composition/formulation.
In one of the embodiments of the present invention, the glidant is selected from colloidal silicon dioxide, magnesium stearate, fumed silica (colloidal silicon dioxide), starch, talc, calcium phosphate tribasic, cellulose powdered, hydrophobic colloidal silica, magnesium oxide, zinc stearate, magnesium silicate, magnesium trisilicate, silicon dioxide or the like.
In another embodiment of the present invention, the glidant in the composition/formulation is present in a range of 0.1% to 5.0% by weight of the total composition/formulation.
In some embodiment of the present invention, the stabilizers are selected from the group consisting of alginate, agar, carrageen, gelatin, guar gum, gum arabic, locust bean gum, pectin, starch, xanthan gum, trehalose and likewise.
In some embodiment of the present invention, the stabilizer in the composition/formulation is present in a range of 0.1% to 10.0% by weight of the total composition/ formulation.
In some embodiment of the present invention, the plasticizers added to coating of the formulation are selected from the group consisting of propylene glycol, glycerol, glyceryl triacetate (triacetin), triethyl citrate, acetyl triethyl citrate, diethyl phthalate, acetylated monoglycerides, castor oil, mineral oil and like thereof.
In some embodiment of the present invention, the plasticizer in the composition/formulation is present in a range of 0.1% to 5.0% by weight of the total composition/ formulation.
In some embodiment of the present invention, the solvent is selected from water, alcohol, isopropyl alcohol, propylene glycol, mineral oil, benzyl alcohol, benzyl benzoate, flavored glycol, carbon dioxide, castor oil, corn oil (maize), cottonseed oil, dimethyl ether, albumin, dimethylacetamide, ethyl acetate, ethyl lactate, medium-chain triglycerides, methyl lactate, olive oil, peanut oil, polyethylene glycol, polyoxyl, castor oil, propylene carbonate, pyrrolidone, safflower oil, sesame oil, soybean oil, sunflower oil, water-miscible solvents, organic polar or non-polar solvents or mixtures thereof.
In a preferred embodiment of the present invention, the solvent in the composition/formulation is used in a quantity sufficient to make the weight of the composition/formulation 100% by weight.
The additional additives include a polymer, a plasticizer, a sweetener, and a powdered flavor, a preservative, a colorant, a surfactant, and other excipients. The powdered flavor composition includes a flavourant associated with a solid carrier. Coating materials such as synthetic polymers, shellac, corn protein (zein) or other polysaccharides, gelatin, fatty acids, waxes, shellac, plastics, and plant fibers and like thereof are used.
In a preferred embodiment of the present invention, the additives are used in a range of 0.1 to 10% w/w of unit dose.
In yet another embodiment, the present invention provides the composition/formulation comprising a therapeutic blend of 2-Hydroxy-N,N,N-trimethylethanaminium or precursors thereof and Methylene tetrahydrofolate reductase (MTHFR) regulators and salts thereof along with pharmaceutical excipients, wherein the pharmaceutical excipients are selected from a diluent, a binder, a lubricant, a glidant, an additive, a surfactant, a stabilizer or mixtures thereof.
In a preferred embodiment, the present invention provides the composition/formulation wherein the pharmaceutically acceptable excipients are selected from a group consisting of the diluent is present in a range of 1 to 30%; the binder present is present in a range of 0.1 to 25%; the lubricant is present in a range of 0.1 to 10.0 %; the glidant is present in a range of 0.1 to 5.0%; the additive is present in a range of 0.1 to 10%; the surfactant is present in a range of 0.1 to 5.0%; the stabilizer is present in a range of 0.1 to 5.0%; %; the antioxidant is present in a range of 0.1 to 5.0%; and the plasticizer is present in a range of 0.1 to 5.0%; by weight of total composition.
In further embodiment, compositions containing compounds of the present invention, can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. Preferred unit dosage formulations are those containing an effective dose, or an appropriate fraction thereof, of the active ingredient, or a pharmaceutically acceptable salt thereof.
The magnitude of a prophylactic or therapeutic dose typically varies with the nature and severity of the condition to be treated and the route of administration. The dose, and perhaps the dose frequency, will also vary according to the age, body weight and response of the individual patient.
In general, the total daily dose (in single or divided doses) ranges from about 1 mg per day to about 2500 mg per day, preferably about 10 mg per day to about 1500 mg per day.
In certain embodiments, the present invention provides the potent composition wherein the effective unit dose for an oral administration is formulated in a range of 10 to 1500 mg.
It is further recommended that women planning for pregnancy, initially receive low doses and that the dosage be titrated based on individual physiological responses and/or pharmacokinetics. It can be necessary to use dosages outside these ranges in some cases, as will be apparent to those in the art.
The present composition can be used as infant formula as well as adult formula by varying the concentration of active ingredients. Further, it is noted that the dietician or nutritionist or certified physician, medical practitioner knows how and when to interrupt, adjust or terminate therapy in conjunction with an individual patient's response.
The use of any and all examples, or exemplary language (e.g., such as) provided herein, is intended merely to better illuminate the invention, and does not pose a limitation on the scope of the invention unless otherwise claimed.
Various other examples of compositions and modifications or adaptations thereof can be devised by a person skilled in the art after reading the foregoing preferred embodiments without departing from the spirit and scope of the invention. All such further examples, modifications and adaptations are included within the scope of the invention.
It will be appreciated by those versed in the art that the present invention makes available novel and useful bioactive compositions, which have effects in several administration forms. Also, it will be understood by those with knowledge in the dietary supplement and nutraceutical art, that many embodiments of this invention may be made without departing from the spirit and scope of the invention, and the invention is not to be construed as limited, as it embraces all equivalents therein.
The invention may be further illustrated by the following examples, which are for illustrative purposes only and should not be construed as limiting the scope of the invention in anyway.
The present disclosure is therefore to be considered as in all respects illustrative and not restrictive, the scope of the invention being indicated by the appended claims and examples, and all changes or alterations which come within the ambit of equivalency are intended to be encompassed therein.

EXAMPLES:
Having described the basic aspects of the present invention, the following non-limiting examples illustrate specific embodiments thereof. Those skilled in the art will appreciate that many modifications may be made in the invention without changing the essence of invention.
Example 1: Various compositions/formulations.
i. Composition 1: Tablet / Capsule.
Ingredient mg per unit dose
2-Hydroxy-N,N,N-trimethylethanaminium 450
(2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino] benzoyl] amino] pentanedioic acid 1
Magnesium Stearate 0.1-10
Hydroxypropyl Methylcellulose 0.5-10
Microcrystalline Cellulose 0.1-10
Polyvinylpyrrolidone 0.1-10
Starch 0.1-5
Talc 0.1-5
Mannitol 0.1-2
Propylene Glycol QS
Water QS
Average weight 460-520 mg

ii. Composition 2: Tablet / Capsule.
Ingredient mg per unit dose
2-Hydroxy-N,N,N-trimethylethanaminium 450
calcium;(2S)-2-[[4-[[(6S)-2-amino-5-methyl-4-oxo-3,6,7,8-tetrahydropteridin-6-yl]methylamino]benzoyl]amino]pentanedioate 1
Magnesium Stearate 0.1-5
Ascorbic acid 0.1-5
Microcrystalline Cellulose 0.1-10
Colloidal Silicon dioxide 0.1-5
Hydroxypropyl Methylcellulose 0.1-5
PVPP 0.1-5
Talc 0.1-5
Tween 80 0.1-5
Mannitol 0.1-1
Alcohol QS
Water QS
Average weight 460-520 mg

iii. Composition 3: Tablet / Capsule.
Ingredient mg per unit dose
2-Hydroxy-N,N,N-trimethylethanaminium 450
(2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino] benzoyl] amino] pentanedioic acid 0.5
Microcrystalline Cellulose 0.1-10
Silicon dioxide 0.1-5
Hydroxypropyl Methylcellulose 0.1-5
Stearic acid 0.1-5
Pregelatinized starch 0.1-10
Talc 0.1-5
Tween 80 0.1-5
Polydextrose 0.1-5
PEG QS
Water QS
Average weight 460-520 mg

iv. Composition 4: Tablet / Capsule.
Ingredient mg per unit dose
2-Hydroxy-N,N,N-trimethylethanaminium chloride 500
(2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino] benzoyl] amino] pentanedioic acid calcium 1
Microcrystalline Cellulose 0.1-5
Silicon dioxide 0.1-5
Glycerin 0.1-5
Ethyl Cellulose 0.1-2
Hydroxypropyl Methylcellulose 0.1-10
Magnesium Stearate 0.1-5
Polyvinylpolypyrrolidone 0.1-10
Talc 0.1-5
Polysorbate 20 0.1-2
Mannitol 0.1-2
IPA QS
Water QS
Average weight 510-600 mg

v. Composition 5: Tablet / Capsule.
Ingredient mg per unit dose
2-Hydroxy-N,N,N-trimethylethanaminium 220
(2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methyl
amino] benzoyl]amino] pentanedioic acid 0.5
Silicon Dioxide 0.1-2
Medium-chain triglycerides 0.1-5
Microcrystalline Cellulose 0.1-10
Magnesium Stearate 0.1-5
Polyvinylpyrrolidone 0.1-5
Talc 0.1-5
Corn Starch 0.1-5
Sodium ascorbate 0.1-2
Propylene glycol 0.1-1
Water QS
Average weight 230-300 mg

vi. Composition 6: Tablet / Capsule.
Ingredient mg per unit dose
Phospho 2-Hydroxy-N,N,N-trimethyl ethanaminium chloride 400
(2S)-2-({4-[(6aR)-3-amino-1-oxo-1H,2H,5H,6H,6aH,7H,8H,9H-imidazolidino[1,5-f] pteridin-8-yl]phenyl}formamido)pentanedioic acid 1
Microcrystalline Cellulose 0.1-10
Colloidal silicon dioxide 0.1-5
Hydroxypropyl Methylcellulose 0.1-5
Magnesium Stearate 0.1-5
Polyvinylpyrrolidone 0.1-5
Calcium Phosphate 0.1-5
Ascorbic Acid 0.1-1
Polysorbate 20 0.1-2
Talc 0.1-5
Sucrose 0.1-1
Mannitol 0.1-1
Glycerol 0.1-2
Average weight 410-470mg

vii. Composition 7: Tablet / Capsule.
Ingredient mg per unit dose
ß-Aminoethyl alcohol 300
((2S)-2-[[4-[(2-amino-5-methyl-4-oxo-3,6,7,8-tetrahydropteridin-6-yl)methylamino]benzoyl]amino]pentanedioic acid 1
Microcrystalline Cellulose 0.1-5
Silicon dioxide 0.1-5
Hydroxypropyl Methylcellulose 0.1-10
Magnesium Stearate 0.1-2
Zinc Stearate 0.1-5
Polyvinylpyrrolidone 0.1-3
Mineral Oil 0.1-2.5
Sodium benzoate 0.1-1
Ascorbic Acid 0.1-2
Polysorbate 20 0.1-1
Talc 0.1-1
Dextrose 0.1-1
Mannitol 0.1-1
Water QS
Average weight 310-360 mg

viii. Composition 8: Tablet / Capsule.
Ingredient mg per unit dose
2-Hydroxy-N,N,N-trimethylethanaminium 15
(2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino] benzoyl]amino] pentanedioic acid 15
Microcrystalline Cellulose 0.1-5
Silicon dioxide 0.1-5
Hydroxypropyl Methylcellulose 0.1-5
Magnesium Stearate 0.1-5
Zinc Stearate 0.1-5
Polyvinylpyrrolidone 0.1-5
Mineral Oil 0.1-2
Sodium benzoate 0.1-1
Ascorbic Acid 0.1-2
Polysorbate 20 0.1-1
Talc 0.1-5
Mannitol 0.1-1
Water QS
Average weight 35-50 mg

ix. Composition 9: Tablet / Capsule.
Ingredient mg per unit dose
2-Hydroxy-N,N,N-trimethylethanaminium chloride dihydrate 450
(2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino] benzoyl] amino] pentanedioic acid 1
Microcrystalline Cellulose 0.1-5
Silicon dioxide 0.1-5
Hydroxypropyl Methylcellulose 0.1-5
Magnesium Stearate 0.1-5
Zinc Stearate 0.1-5
Polyvinylpyrrolidone 0.1-5
Mineral Oil 0.1-2
Sodium Ascorbate 0.1-2
Polysorbate 20 0.1-1
Talc 0.1-5
Mannitol 0.1-1
Water QS
Average weight 460-530 mg

x. Composition 10: Tablet / Capsule.
Ingredient mg per unit dose
Trimethyl (2-phosphonooxyethyl) azanium ;chloride 450
calcium;(2S)-2-[[4-[[(6S)-2-amino-5-methyl-4-oxo-3,6,7,8-tetrahydropteridin-6-yl]methylamino]benzoyl]amino]pentanedioate 1
Microcrystalline Cellulose 0.1-5
Silicon dioxide 0.1-5
Hydroxypropyl Methylcellulose 0.1-5
Magnesium Stearate 0.1-5
Zinc Stearate 0.1-5
Polyvinylpyrrolidone 0.1-5
Mineral Oil 0.1-2
Sodium benzoate 0.1-1
Ascorbic Acid 0.1-2
Polysorbate 20 0.1-1
Talc 0.1-5
Mannitol 0.1-1
Water QS
Average weight 460-550 mg

xi. Composition 11: Tablet / Capsule.
Ingredient mg per unit dose
calcium;2-(trimethylazaniumyl)ethyl phosphate;chloride 450
(2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino] benzoyl] amino] pentanedioic acid 1
Microcrystalline Cellulose 0.1-5
Silicon dioxide 0.1-5
Hydroxypropyl Methylcellulose 0.1-5
Magnesium Stearate 0.1-5
Zinc Stearate 0.1-5
Polyvinylpyrrolidone 0.1-5
Mineral Oil 0.1-2
Sodium benzoate 0.1-1
Ascorbic Acid 0.1-2
Polysorbate 20 0.1-1
Talc 0.1-5
Mannitol 0.1-1
Water QS
Average weight 455-520 mg

xii. Composition 12: Tablet / Capsule.
Ingredient mg per unit dose
2-Hydroxy-N,N,N-trimethylethanaminium 250
calcium;(2S)-2-[[4-[[(6S)-2-amino-5-methyl-4-oxo-3,6,7,8-tetrahydropteridin-6-yl]methylamino]benzoyl]amino]pentanedioate 10
Magnesium Stearate 0.1-5
Ascorbic acid 0.1-5
Microcrystalline Cellulose 0.1-10
Colloidal Silicon dioxide 0.1-5
Hydroxypropyl Methylcellulose 0.1-5
Polyvinylpyrrolidone 0.1-5
Talc 0.1-5
Tween 80 0.1-5
Sucrose 0.1-2
Sorbitol 0.1-1
Alcohol QS
Water QS
Average weight 265-320 mg

Animal Study
Example :2 Measurement of Choline dehydrogenase (CHD) and Choline level.
a) CHD Assay
Table1- Group Design
Group No. Group description [ Human Dose] and Treatment Observations
G1 Control No treatment
G2 2-Hydroxy-N,N,N-trimethylethanaminium 2-Hydroxy-N,N,N-trimethylethanaminium –[450 mg] CHD and Choline Level – No substantial change
G3 (2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino]benzoyl]amino]pentanedioic acid (2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino]benzoyl]amino]pentanedioic acid –[1 mg] CHD and Choline Level – No substantial change
G4 2-Hydroxy-N,N,N-trimethylethanaminium + (2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino]benzoyl]amino]pentanedioic acid
[Lower Ratio] [ 1:0.001] [450 mg] 2-Hydroxy-N,N,N-trimethylethanaminium + [0.5 mg] (2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino]benzoyl]amino]pentanedioic acid CHD level decrease / Choline level increase
G5 2-Hydroxy-N,N,N-trimethylethanaminium + (2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino]benzoyl]amino]pentanedioic acid
[Higher Ratio] [ 1:1] [15 mg] 2-Hydroxy-N,N,N-trimethylethanaminium + [15 mg] (2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino]benzoyl]amino]pentanedioic acid CHD level decrease / Choline level increase
G6 2-Hydroxy-N,N,N-trimethylethanaminium + (2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino]benzoyl]amino]pentanedioic acid
[Optimum Ratio] [1:0.002]
[450 mg] 2-Hydroxy-N,N,N-trimethylethanaminium + [1 mg] (2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino]benzoyl]amino]pentanedioic acid CHD level decrease / Choline level increase

Method:
Female Mice Oocytes were obtained from females at 3–4 weeks of age.
A choline dehydrogenase activity was evaluated as a function of time following the removal of female mice oocytes from antral follicles. The effects of test groups and that of time are significant by two-way ANOVA (p = 0.0002), with G4, G5, G6 inhibiting the development of choline dehydrogenase activity.
To determine the CHD activity oocytes were cultured for 3 h in the presence of test groups at a level (50 µM) that inhibit CHD protein synthesis in oocytes. When CHD protein synthesis was inhibited, the choline dehydrogenase enzyme activity seems to be decreased.

Results:
i. CHD Activity:
The development of choline dehydrogenase activity in oocytes measured at 3 h after removal from the follicle. It was observed that test groups G4, G5, G6 significantly inhibits CHD activity (Fig. 1).
Table 2:
Groups CHD activity (CPM)
G1 594
G2 498
G3 578
G4 239
G5 233
G6 210
CPM: counts per millions
ii. Western Blotting Test:
CHD was assessed by Western blotting in female mice oocytes that had been cultured for 3 hr when choline dehydrogenase activity has developed utmost.
Mouse kidney lysate was run to indicate the position of the CHD band. GAPDH served as a loading control.
CHD protein was detected by Western blot analysis as a single band near the predicted size of 65-70 kDa, particularly 66 kDa.
Quantitating a western blot refers to the measurement of the signal emitted by CHD protein band(s). The signal intensity of the band is directly proportional to the concentration of CHD protein. By analyzing the intensity of the signal, we determined whether the expression of the CHD protein in test groups G2-G6 decreased relative to control (Fig. 2).
b) Biochemical Assay: Choline
Evaluation of test substance on pregnant female mice.
Test system details:
Test Species: Mice.
Strain: Pregnant Swiss Albino Mice.
Sex: Female.
Age:10-12 Weeks.
Body Weight: 20-30gms.
Total No. of Animals: 36 Animals (6 groups of 6 animals each).
Animal House conditions
Lighting: 12 / 12-hour light-dark cycle.
Temperature: 22 ± 3°C.
Relative Humidity: 30 to 70%.
Feed: Normal chow diet.
Drinking water: Mice were provided with ad libitum drinking water passed through water filter system.
Vehicle details : 0.5% of Carboxy Methyl Cellulose sodium was used as a vehicle for test formulation.
Group, Designation and Dose Levels:
Table 3: Animal grouping and treatment details
Groups Group Description Human Dose No. of animals
G1 Control No treatment 6
G2 2-Hydroxy-N,N,N-trimethylethanaminium 450 mg 6
G3 (2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino]benzoyl]amino]pentanedioic acid 1 mg 6
G4 2-Hydroxy-N,N,N-trimethylethanaminium + (2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino]benzoyl]amino]pentanedioic acid
[Lower Ratio] [ 1:0.001] 450 mg + 0.5 mg 6
G5 2-Hydroxy-N,N,N-trimethylethanaminium + (2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino]benzoyl]amino]pentanedioic acid
[Higher Ratio] [ 1:1] 15 mg + 15 mg 6
G6 2-Hydroxy-N,N,N-trimethylethanaminium + (2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino]benzoyl]amino]pentanedioic acid
[Optimum Ratio] [1:0.002]
450 mg + 1 mg 6

Administration and Procedure:
All the animals were acclimatized for at least 5 days under controlled environmental condition. Total 36 animals were divided into six groups consisting of 6 animals per group.
Normal control (G1) group receiving vehicle, all treatment groups were administered with test substances; (G2)2-Hydroxy-N,N,N-trimethylethanaminium; ((G3)2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino]benzoyl]amino]pentanedioic acid; (G4) 2-Hydroxy-N,N,N-trimethylethanaminium + (2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino]benzoyl]amino]pentanedioic acid [Lower Ratio] [ 1:0.001]; (G5)2-Hydroxy-N,N,N-trimethylethanaminium + (2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino]benzoyl]amino]pentanedioic acid [Higher Ratio] [ 1:1]; (G6) 2-Hydroxy-N,N,N-trimethylethanaminium + (2S)-2-[ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino]benzoyl]amino] pentanedioic acid [1:0.002] respectively.
All the treatments were administered daily for 02 weeks. After the completion of two weeks animals were anesthetized and blood was collected for biochemical parameter analysis.
All data including body weight, feed consumption, were analyzed statistically using Graph-Pad Prism Software, version 5.01. All values were expressed as Average Mean. The significant difference between the treatment and control group were estimated using one-way analysis of variance (ANOVA) with Dunnett’s test. All results of the statistical analysis were summarized in below table.

Results:
Table 4: Choline (pg/ml)
Animals 1 2 3 4 5 6 Average
(Mean)

Groups
Group 1(G1) 47.47 48.18 44.06 41.27 45.67 43.26 44.99
Group 2(G2) 58.25 59.99 60.12 57.58 57.26 59.90 58.85
Group 3(G3) 53.15 52.60 54.02 53.87 54.55 52.28 53.41
Group 4(G4) 119.45 120.56 120.78 121.65 119.98 119.42 120.31
Group 5(G5) 129.91 130.12 129.56 130.34 129.39 130.06 129.90
Group 6(G6) 142.05 141.88 142.20 141.67 142.53 142.80 142.19

Discussion and Conclusion:
In the present study the compositions G4, G5 and G6 significantly decrease the CHD enzyme activity in vitro (Fig. 3) and simultaneously enhance the choline concentration which is useful for fetal development. Moreover, this study indicates that the combination in specific ratio inhibits the degradation of choline by lowering the activity of choline dehydrogenase an important enzyme in regulation of the concentration of free choline in tissues under physiological conditions.
It is concluded that the present composition in the range of the weight ratio of 1: 0.001 to 1:1 along with pharmaceutically acceptable excipients, show significant decrease in CHD activity and thus enhancing choline concentration in the subject.
,CLAIMS:1. A bioactive composition for modulating Choline Dehydrogenase [CHD] activity comprising:
therapeutically active exogenous blend of 2-Hydroxy-N,N,N-trimethylethanaminium or its precursors and salts thereof and Methylenetetrahydrofolate reductase (MTHFR) regulators and salts thereof along with pharmaceutically acceptable excipients.
2. The composition as claimed in claim 1, wherein the 2-Hydroxy-N,N,N-trimethylethanaminium precursors are selected from the group consisting of 2-Amino-3-hydroxypropanoic acid, ß-Aminoethyl alcohol, 2-Methylaminoethanol and N,N-Dimethylaminoethanol and salts thereof .
3. The composition as claimed in claim 1, wherein the Methylenetetrahydrofolate reductase (MTHFR) regulators are selected from the group consisting of (2S)-2-[[4-[(2-amino-4-oxo-3H-pteridin-6-yl)methylamino]benzoyl] amino] pentanedioic acid , (2S)-2-[[4-[(2-Amino-4-oxo-1H-pteridin-6-yl)methylamino]benzoyl]amino]pentanedioic acid ; (2S)-2- [ [4-[(2-Amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl) methylamino] benzoyl] amino] pentanedioic acid; (2S)-2-[[4-[(2-amino-4-oxo-5,6,7,8-tetrahydro-3H-pteridin-6-yl)methylamino]benzoyl]amino]pentanedioic acid ; (2S)-2-({4-[(6aR)-3-amino-1-oxo-1H,2H,5H,6H,6aH,7H,8H,9H-imidazolidino [1,5-f] pteridin-8-yl]phenyl}formamido)pentanedioic acid; (2S)-2-[[4-[(2-amino-5-methyl-4-oxo-3,6,7,8-tetrahydropteridin-6-yl)methylamino] benzoyl] amino] pentanedioic acid; (2S)-2-(4-(((2-Amino-5-methyl-4-oxo-3,4,5,6,7,8-hexahydropteridin-6- yl)methyl)amino)benzamido)pentanedioic acid and salts thereof.
4. The composition as claimed in claim 1, wherein 2-hydroxy-N,N,N-trimethylethanaminium or precursors and salts thereof are present in the range of 1-1000 mg by weight of the total composition.
5. The composition as claimed in claim 1, wherein the Methylenetetrahydrofolate reductase (MTHFR) regulators and salts thereof are present in the range of 0.1-100 mg by weight of the total composition.
6. The composition as claimed in claim 1, wherein the pharmaceutically acceptable excipients are selected from group consisting of the diluent is present in a range of 1 to 30%; the binder present is present in a range of 0.1 to 25%; the lubricant is present in a range of 0.1 to 10.0 %; the glidant is present in a range of 0.1 to 5.0%; the additive is present in a range of 1 to 10%; the surfactant is present in a range of 0.1 to 5.0%; the stabilizer is present in a range of 0.1 to 10.0%; %; the antioxidant is present in a range of 0.1 to 5.0%; and the plasticizer is present in a range of 0.1 to 5.0%; by weight of total composition.