Stable liquid formulations containing (6S)-5-methyltetrahydrofolic acid or salts thereof

Abstract

The present invention relates to a stable liquid formulation comprising (6S)-5-methyltetrahydrofolic acid or a salt thereof, a method for obtaining the stable liquid formulation, and the use of the stable liquid formulation as a liquid vitamin formulation or a raw material for a liquid vitamin formulation.

Description

[Technical Field]

[0001] The present invention relates to a stable liquid formulation comprising (6S)-5-methyltetrahydrofolic acid or a salt thereof, a method for preparing the liquid formulation, and the use of the liquid formulation as a liquid vitamin formulation or as a raw material for a liquid vitamin formulation.

[0002] As used herein, (6S)-5-methyltetrahydrofolic acid, also known as L-5-methyltetrahydrofolic acid, L-5-MTHF, (6S)-5-methyltetrahydrofolate, or (6S)-5-MTHF, refers to levomefolic acid, which naturally occurs as the isomer N-[4-({[(6S)-2-amino-5,6,7,8-tetrahydro-4-hydroxy-5-methylpteridin-6-yl]methyl}amino)benzoyl]-L-glutamic acid. It is the major bioactive form of folate utilized at the cellular level for DNA replication, the cysteine ​​cycle, and homocysteine ​​regulation. It is also the major form of tetrahydrofolic acid present in the circulation and transported across tissue membranes and the blood-brain barrier. Within cells, (6S)-5-methyltetrahydrofolate is utilized in the methylation of homocysteine ​​to produce methionine and tetrahydrofolate (THF). THF is the direct acceptor of one-carbon units in the synthesis of thymidine-DNA, purines (RNA and DNA), and methionine. The oxidized, unmethylated form of folic acid is a synthetic form of folate that requires enzymatic reduction by dihydrofolate reductase (DHFR) to become biologically active. [Background technology]

[0003] Folate plays an essential role in regulating amino acid and nucleic acid metabolism and one-carbon metabolism. Thus, folate plays an important role in the body, supporting normal cell division, normal DNA, and gene expression. Folate cannot be produced by the human body and must be obtained through the diet. Adequate folate intake is essential for cell division and normal development, and plays an important role in promoting the nervous system and preventing birth defects, vascular and cardiovascular disease, and cancer. Folate deficiency is recognized as underlying many dysfunctions and abnormalities in some populations and at specific times in life, such as during reproductive years and pregnancy.

[0004] Therefore, folic acid supplementation is highly recommended for populations at risk for deficiency due to certain conditions. Folic acid is a synthetic form of vitamin B9 that is typically found in foods and supplements (see Heffernan et al., Nutrition & Metabolism, 2021, 18, 104, 1-11). However, in the body, synthetic folic acid must be reduced by cellular metabolism to the bioactive (6S)-5-methyltetrahydrofolate, which is present in the blood and is the bioavailable form of folate required to convert the amino acid homocysteine ​​to the essential methionine. The human body utilizes methionine to synthesize proteins and other important compounds. However, common polymorphisms in the gene encoding methylenetetrahydrofolate reductase (MTHFR) are associated with reduced activity of this enzyme. Individuals with this genetic defect are unable to convert folic acid taken as a dietary supplement to the bioactive (6S)-5-methyltetrahydrofolate. MTHFR polymorphisms have also been associated with an increased risk of several pathologies, including cancer. For those who unknowingly carry these MTHFR polymorphisms, dietary supplementation with folic acid will be ineffective and ultimately result in critical unmetabolized folic acid circulating in the body.

[0005] In recent years, extensive research has been conducted into the physiological activity of L-5-MTHF and its salts in humans, particularly in preventing MTHFR polymorphisms. L-5-MTHF and its salts are promising candidates for use as a supplement to prevent folate deficiency in humans, especially during pregnancy (see N. Miraglia et al., Alternative Therapies, May / June 2022, 28, 4, 12-17).

[0006] Synthetic folic acid is stable under most conditions of food processing and storage. In contrast, reduced tetrahydrofolates, such as 5-formyltetrahydrofolate, 5-methyltetrahydrofolate (L-5-MTHF), or tetrahydrofolic acid, are susceptible to degradation. Various oxidative and nonoxidative degradation mechanisms of folic acid have been extensively studied and documented (see Gazzali et al., Eur. J. Pharm. Sci., 2016, 93, 419-430; Fitzhugh, Pteridines, 1993, 4, 187-191). It should be noted that not only do different folate species differ in their sensitivity to oxygen, but their oxidative degradation pathways are also fundamentally different. For example, in the case of folic acid, exposure to UV light is typically required to initiate the oxidative degradation process. Furthermore, the pH value plays a crucial role for various folates, which is particularly important when preparing samples for analysis (see DeBrouwer et al., Phytochem. Anal., 2007, 18, 496-508; Jastrebova et al., Eur. Food Res. Technol., 2013, 237, 747-754). For example, the use of two antioxidants, ascorbic acid in combination with 2-mercaptoethanol or 2,3-dimercapto-1-propanol, and a near-neutral pH have been shown to be advantageous in reducing the loss of 5-formyl-tetrahydrofolate during sample preparation. Additionally, various antioxidants, such as cinnamic acid, flavonoids, catechol, stilbene, p-benzoquinone, isoprenoids, curcumin, oleic acid, and linoleic acid, have been investigated for their potential to prevent the photodegradation of follic acid (see Wusigale et al., J. Agric. Food Chem. 2020, 68, 340-350).

[0007] While L-5-MTHF is reasonably stable in solid crystalline form, it decomposes relatively rapidly in the amorphous state, especially in solution, reducing the available effective amount. The initial step in oxidative degradation produces L-5-methyl-5,6-dihydrofolate, L-5-methyl-5,8-dihydrofolate, and 4α-hydroxy-5-methyl-5,6,7,8-tetrahydrofolate (see Ratanathien et al., J. Clin. Path., 1977, 30, 438-448; Blair et al., J. C. S. Perkin II, 1975, 18-21). Further reactions result in irreversible chemical rearrangements of the pteroic acid heterocycle (see Whiteley et al., Biochem. Biophys. Res. Comm., 1981, 101, 4, 1259-1265). The resulting pyrazino-s-triazine derivative (L-Mefox) has no vitamin activity. Considering the susceptibility of naturally occurring folic acid to degradation, it is not surprising that the folic acid content of foods can be significantly reduced during cooking or even during storage at ambient temperature (see Indrawati et al., J. Agric. Food Chem., 2004, 52, 486-492).

[0008] The stabilization of liquid formulations containing L-5-MTHF and its salts, particularly over the medium and long term, remains highly desirable, and such liquid formulations may be used in the following areas: (1) liquid vitamin formulations in the field of dietary supplements, such as dietary supplements for populations with high or suboptimal folate requirements, e.g., athletes (sports nutrition), women who are pregnant or lactating, or who are seeking pregnancy (pre- and lactation nutrition), children, the elderly, and others who have difficulty swallowing solid dosage forms, or for those wishing to improve their health (vitamin shots and vitamin drinks); (2) liquid vitamin formulations for the fortification of foods such as cereals and dairy products (e.g., flour and yogurt); (3) liquid vitamin formulations for life science applications, such as liquid vitamin formulations for stabilizing cell culture media; (4) pharmaceutical and / or cosmetic formulations, such as eye drops; and (5) non-pharmaceutical formulations for over-the-counter (OTC) products, such as bandages and wound dressings (wound healing). Of particular interest herein are industrially produced liquid vitamin formulations, which may or may not be based on natural food sources, and which may be available in the form of sprays, drops, or drink bottles.

[0009] However, in connection with the above, it must be emphasized that the ability to stabilize folic acid depends on many different factors, such as the chemical nature of the folic acid (reduced or non-reduced folic acid, substituted or non-substituted folic acid, type of substitution, etc.), the chemical nature of the stabilizer, the nature of the formulation (solid, liquid, dispersion, emulsion, etc.), the presence of additives and / or cosolvents, etc. Moreover, further physical parameters also play an important role, such as temperature, exposure to radiation (e.g., light), pressure, pH, etc. As mentioned above, various different degradation pathways of folic acid are known in the prior art, which are significantly different from each other both chemically and mechanistically. In light of this, those skilled in the art are well aware that in the field of folic acid stabilization, the results of a particular application cannot be simply transferred to another application. Each case must be considered individually.

[0010] For example, a comparative study of the pressure and temperature stability of 5-methyltetrahydrofolic acid in model systems and foods showed that ascorbic acid enhances the stability of 5-MTHF. This study showed that (i) the decomposition of 5-MTHF in model systems follows first-order kinetics, (ii) the heat resistance of 5-MTHF increases with increasing pH up to pH 7, (iii) 5-MTHF is relatively stable to pressure at temperatures below 40°C, and (iv) ascorbic acid enhances both the heat resistance and pressure resistance of 5-MTHF (see Indrawati et al., J. Agric. Food Chem., 2004, 52, 486-492). However, this study did not address the industrial production of medium- and long-term stabilized liquid formulations containing L-5-MTHF and its salts, as described in the present invention.

[0011] A 2020 review discussed the stability of folic acid and its interactions with biomolecules, including antioxidants, carbohydrates, proteins, and nucleic acids (see Wusigale et al., J. Agric. Food Res., 2020, 2, 100039, 1-7). It found that folic acid decomposes under light, heat, oxygen, and acidic pH, and that antioxidants can further preserve folic acid. This is particularly true for fruits and vegetables rich in antioxidants, such as ascorbic acid and polyphenols, which can further preserve folic acid. This effect has been confirmed in food systems with ascorbic acid and 5-MTHF. In this case, 0.1–0.5 mg / g of ascorbic acid was found to reduce the degradation of 0.1–0.6 μg / g of folic acid in foods (i.e., orange juice, kiwi puree, carrot juice, and asparagus). However, this review does not address the industrial production of medium- and long-term stabilized liquid formulations containing L-5-MTHF and its salts as described in the present invention.

[0012] Furthermore, numerous publications have described the stabilization of liquid or solid formulations containing folic acid together with natural antioxidants. However, these publications relate either to solid formulations containing folic acid (see U.S. Patent No. 8,747,915 and U.S. Patent Application Publication No. 2004 / 0001817) or to liquid formulations containing folic acid (see U.S. Patent Application Publication No. 2019 / 0060336; U.S. Patent Application Publication No. 2007 / 0190209; U.S. Patent Application Publication No. 2009 / 0061023; WO 2015 / 099842; and EP 0954986). None of these publications specifically address L-5-MTHF, which is fundamentally different from folic acid in terms of stability and degradation mechanism. Furthermore, none of these publications address the medium- to long-term stability of liquid formulations containing L-5-MTHF. However, liquid L-5-MTHF formulations are useful in the applications (1), (2), (3), (4), and (5) listed above, especially for populations who have difficulty swallowing solid dosage forms, such as children and the elderly.

[0013] Stability is a crucial property and factor for formulations containing compounds that can easily decompose under the influence of certain parameters. Chemical stability studies have been conducted in both solution and solid states. It is well established that stability in solution and solid states can differ qualitatively and quantitatively. Furthermore, the stability of amorphous and crystalline substances, such as lyophilized products, can also differ in the solid state. The chemical stability of active ingredients and their formulations has been extensively studied by exposure to various stressors, such as high temperature and / or humidity. These studies provide useful information on degradation products and also help develop the intrinsic stability and critical design specifications of formulations containing active ingredients. The most common degradation pathways of active ingredients include hydrolysis, oxidation, and photochemical degradation, among others. [Prior art documents] [Patent documents]

[0014] [License 1] U.S. Patent No. 8747915 [License 2] U.S. Patent and Trademark Publication No. 2004 / 0001817 [License 3] U.S. Patent and Trademark Publication No. 2019 / 0060336 [License 4] U.S. Patent and Trademark Publication No. 2007 / 0190209 [Patent Document 5] U.S. Patent and Trademark Publication No. 2009 / 0061023 [License 6] International Publication No. 2015 / 099842 [License 7] European Patent Publication No. 0954986 [Non-licensed literature]

[0015] [Non-licensed Document 1] Heffernan et al.,Nutrition&Metabolism,2021,18,104,1-11 [Non-licensed Document 2] N.Miraglia et al.,Alternative Therapies,May / June 2022,28,4,12-17 [Non-licensed Document 3] Gazzali et al.,Eur.J.Pharm.Sci.,2016,93,419-430 [Non-licensed Document 4] Fitzhugh,Pteridines,1993,4,187-191 [Non-licensed Document 5] DeBrouwer et al.,Phytochem.Anal.,2007,18,496-508 [Non-licensed Document 6] Jastrebova et al.,Eur.Food Res.Technol.,2013,237,747-754 [Non-licensed Document 7] Wusigale et al., J.Agric.Food Chem. 2020, 68, 340 - 350 [[Non - Patent Document 8]] Ratanathien et al., J.Clin.Path., 1977, 30, 438 - 448 [[Non - Patent Document 9]] Blair et al., J.C.S.Perkin II, 1975, 18 - 21 [[Non - Patent Document 10]] Whiteley et al., Biochem.Biophys.Res.Comm., 1981, 101, 4, 1259 - 1265 [[Non - Patent Document 11]] Indrawati et al., J.Agric.Food Chem., 2004, 52, 486 - 492 [[Non - Patent Document 12]] Wusigale et al., J.Agric.Food Res., 2020, 2, 100039, 1 - 7 [[Summary of the Invention]] [[Problems to be Solved by the Invention]]

[0016] The object of the present invention is to overcome the drawbacks and shortcomings of the prior art and to provide a liquid formulation comprising (6S)-5-methyltetrahydrofolic acid or a salt thereof, which (a) exhibits medium-term and / or long-term stability at ambient and elevated temperatures, (b) comprises a substance that is generally recognized as safe (GRAS), and (c) is particularly suitable for use in the following (1) to (5): (1) liquid vitamin preparations in the field of dietary supplements, for example, for populations with high or below-met folic acid requirements, for example, for athletes (sports nutrition), pregnant or lactating women, or women who are breastfeeding or breastfeeding. (1) Liquid vitamin formulations for the fortification of foods such as cereals and dairy products (e.g., flour and yogurt); (2) Liquid vitamin formulations for life science applications, such as liquid vitamin formulations for stabilizing cell culture media; (3) Pharmaceutical and / or cosmetic formulations, such as eye drops; and (4) Non-pharmaceutical formulations for over-the-counter (OTC) products, such as bandages and wound dressings (wound healing). Liquid formulations containing (6S)-5-methyltetrahydrofolic acid or its salts can be used in the form of sprays, drops, or drink bottles.

[0017] Therefore, an object of the present invention is to provide a liquid formulation containing (6S)-5-methyltetrahydrofolic acid or a salt thereof, which exhibits the above-mentioned beneficial effects (a) to (c).A further object of the present invention is to provide a liquid formulation containing (6S)-5-methyltetrahydrofolic acid or a salt thereof, which exhibits the above-mentioned beneficial effects (a) to (c), and which can be used as a liquid vitamin preparation or an ingredient for a liquid vitamin preparation.

[0018] Finally, it is an object of the present invention to provide a method for preparing such a liquid formulation.

[0019] The aim of stability testing is to provide evidence of how product quality changes over time under the influence of various environmental factors such as temperature, humidity and light, and to establish appropriate shelf life and recommended storage conditions for products containing active ingredients. [Means for solving the problem]

[0020] It has surprisingly been found that a liquid formulation comprising (6S)-5-methyltetrahydrofolic acid or a salt thereof; one or more antioxidants selected from grape seed extract, rosemary extract, or components thereof; and a liquid formulation vehicle overcomes the known drawbacks described above and enables the preparation of a stable liquid formulation comprising (6S)-5-methyltetrahydrofolic acid or a salt thereof. Such liquid formulations are particularly suitable for use in: (1) liquid vitamin formulations in the field of dietary supplements, e.g., dietary supplements for populations with high or unmet folic acid requirements, such as athletes (sports nutrition), women who are pregnant or lactating or who are trying to become pregnant (pre- and lactation nutrition), children, the elderly, and others who have difficulty swallowing solid dosage forms, or for those wishing to improve their health (vitamin shots and vitamin drinks); (2) liquid vitamin formulations for the fortification of foods such as cereals and dairy products (e.g., flour and yogurt); (3) liquid vitamin formulations for life science applications, such as liquid vitamin formulations for stabilizing cell culture media; (4) pharmaceutical and / or cosmetic formulations, such as eye drops; and (5) non-pharmaceutical formulations for over-the-counter (OTC) products, such as bandages and wound dressings (wound healing).

[0021] The liquid formulations of the present invention exhibit medium- and / or long-term stability at ambient and elevated temperatures and contain ingredients that are generally recognized as safe (i.e., GRAS) substances. The liquid formulations of the present invention can be used in the form of sprays, drops, or drink bottles.

[0022] It has surprisingly been found that the addition of grape seed extract, rosemary extract, or one or more antioxidants selected from these components provides medium- to even long-term stabilization to liquid formulations containing (6S)-5-methyltetrahydrofolic acid or its salts. In particular, the decrease in the reduced folic acid content in the formulation due to conversion or decomposition is significantly slowed down or even stopped. As a result, the content of available reduced folic acid remains constant for a longer period, making it possible to produce vitamin-containing formulations with a stable L-5-MTHF content over a certain period.

[0023] The resulting liquid formulations are stable for several months, preferably up to 3 months, more preferably up to 6 months, even more preferably up to 12 months, even more preferably up to 18 months, and most preferably for more than 18 months, without significant loss of active ingredient, i.e., the amount of L-5-MTHF is maintained at 40% or more, preferably 45% or more, more preferably 50% or more, even more preferably 55% or more, even more preferably 60% or more, even more preferably 65% ​​or more, even more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, and most preferably 95% or more after a specified period of time. This allows the liquid formulations of the present invention to be stored and used to prepare formulations or preparations containing L-5-MTHF.

[0024] Thus, the present invention relates to the following embodiments: (a) (6S)-5-methyltetrahydrofolic acid or a salt thereof; (b) one or more antioxidants selected from grape seed extract, rosemary extract, or components thereof; (c) a liquid formulation medium; and A liquid formulation comprising:

[0025] A method for preparing a liquid formulation according to the present invention, comprising the steps of: (i) and (ii); or (i') and (ii') of: (i) dissolving (6S)-5-methyltetrahydrofolic acid or a salt thereof in a liquid formulation medium; and (ii) adding one or more antioxidants selected from grape seed extract, rosemary extract, or components thereof; or (i') dissolving one or more antioxidants selected from grape seed extract, rosemary extract, or components thereof in a liquid formulation medium; and (ii') adding (6S)-5-methyltetrahydrofolic acid or a salt thereof;

[0026] Use of the liquid formulation according to the invention as a liquid vitamin preparation or as a raw material for a liquid vitamin preparation.

[0027] Preferred embodiments of the invention are set out in the dependent claims as follows. [Brief explanation of the drawings]

[0028] [Figure 1] The long-term stability of L-5-MTHF in Example 1-2 (■) and Reference Example 1-1 (●) at 25°C and 60% relative humidity (RH) is shown. The graph shows the content (%) of L-5-MTHF monosodium salt over time, as measured by LC-MS / MS according to the AOAC 2013.13 method. [Figure 2] The long-term stability of L-5-MTHF in Example 1-1 (▲), Example 1-2 (■), and Reference Example 1-1 (●) is shown at 40°C and 75% relative humidity (RH). The graph shows the content (%) of L-5-MTHF monosodium salt over time, as measured by LC-MS / MS according to the AOAC 2013.13 method. DETAILED DESCRIPTION OF THE INVENTION

[0029] definition In the present invention, the term "formulation vehicle" as used herein refers to one or more compounds that function as a solvent, emulsifier, suspending agent, carrier, and / or matrix for (6S)-5-methyltetrahydrofolic acid or its salt and other components of the liquid formulation. The formulation vehicle typically consists of a compound or a mixture of several compounds that are inert to (6S)-5-methyltetrahydrofolic acid or its salt and other components contained in the liquid formulation. In the present invention, the formulation vehicle is liquid and is the main component of the liquid formulation. Exemplary formulation vehicles include water or a mixture of water and ethanol.

[0030] For purposes of the present invention, the term "antioxidant," as used herein, refers to a substance that inhibits oxidation, a chemical reaction that can generate free radicals. Oxidation can result in the breakdown of oxidation-sensitive compounds. Antioxidants are often added to industrial products, such as fuels and lubricants, to prevent oxidation, and to foods, particularly to prevent spoilage, such as the rancidity of fats and oils. Dietary antioxidants include vitamins A, C, and E.

[0031] In the present invention, the term "medium-term stabilization" as used herein means stabilization over a period of 3 to 6 months.

[0032] In this application, the term "long-term stabilization," as used herein, refers to stabilization for a period of more than 6 months, preferably 9 months or more, and more preferably 12 months or more. Preferred periods of long-term stabilization are greater than 6 months to 24 months, greater than 6 months to 18 months, 9 to 18 months, and 12 to 18 months.

[0033] In the present invention, the term "ambient temperature" as used herein refers to a temperature in the range of about 20°C to about 25°C, preferably a temperature of about 20°C.

[0034] Throughout this specification and the claims that follow, unless otherwise indicated, the word "comprise" and variations such as "comprises" or "comprising" are understood to mean the inclusion of a stated compound, or class or group of compounds, or group of steps, but not the exclusion of other elements, steps, or groups of elements or steps.

[0035] Liquid formulations The present invention relates to a liquid formulation comprising: (a) (6S)-5-methyltetrahydrofolic acid or a salt thereof; (b) one or more antioxidants selected from grape seed extract, rosemary extract, or components thereof; (c) Liquid formulation medium.

[0036] Preferably, the liquid formulations of the present invention comprise one, two, three, four, five, six, seven, eight, nine, or ten antioxidants selected from grape seed extract, rosemary extract, or components thereof. More preferably, the liquid formulations of the present invention comprise one, two, three, four, or five antioxidants selected from grape seed extract, rosemary extract, or components thereof. Even more preferably, the liquid formulations of the present invention comprise one, two, or three antioxidants selected from grape seed extract, rosemary extract, or components thereof. Most preferably, the liquid formulations of the present invention comprise one antioxidant selected from grape seed extract, rosemary extract, or components thereof.

[0037] In a preferred embodiment of the present invention, the antioxidant in the liquid formulation is selected from grape seed extract or its components. Grape seed extract is an industrial derivative of whole grape seeds and contains proanthocyanidins. The properties and components of grape seed extract are described in Chen et al., Journal of Functional Foods, 73, 2020, 104113, 1-18. The antioxidants present in grape seed extract include (poly)phenolic compounds such as catechin, epicatechin, proanthocyanidins, kaempferol, quercetin, and gallic acid.

[0038] Preferably, the antioxidant selected from grape seed extract or its components is selected from the group consisting of catechin, epicatechin, epicatechin gallate, epigallocatechin gallate, gentisic acid, syringic acid, protocatechuic acid, gallic acid, caffeic acid, procyanidin B1, procyanidin B2, procyanidin B3, procyanidin B4, procyanidin B1-3-O-gallate (B1-G), procyanidin B2-3-O-gallate (B2-G), procyanidin B2-3'-O-gallate (B2-G'), procyanidin C1, kaempferol, and quercetin. More preferably, the antioxidant selected from grape seed extract or its components is selected from the group consisting of catechin and caffeic acid.

[0039] In another preferred embodiment of the present invention, the antioxidant in the liquid formulation is selected from rosemary extract or its components. Rosemary extract is a plant extract used as a food additive. The properties and components of rosemary extract are described in Xie et al., Eur. J. Lipid Sci. Technol., 2017, 119, 1600439, 1-10. The antioxidants present in rosemary extract include (poly)phenolic compounds such as carnosic acid, carnosol, and rosmarinic acid.

[0040] Preferably, the antioxidant selected from rosemary extract or its components is selected from the group consisting of carnosic acid, carnosol, and rosmarinic acid, more preferably, the antioxidant selected from rosemary extract or its components is selected from the group consisting of carnosic acid and rosmarinic acid.

[0041] In yet another embodiment of the present invention, the antioxidant selected from grape seed extract, rosemary extract, or components thereof is selected from the group consisting of catechin, epicatechin, epicatechin gallate, epigallocatechin gallate, gentisic acid, syringic acid, protocatechuic acid, gallic acid, caffeic acid, procyanidin B1, procyanidin B2, procyanidin B3, procyanidin B4, procyanidin B1-3-O-gallate (B1-G), procyanidin B2-3-O-gallate (B2-G), procyanidin B2-3'-O-gallate (B2-G'), procyanidin C1, kaempferol, quercetin, carnosic acid, carnosol, and rosmarinic acid. Preferably, the antioxidant selected from grape seed extract, rosemary extract, or components thereof is selected from the group consisting of catechin, caffeic acid, carnosic acid, and rosmarinic acid.

[0042] Preferably, in an embodiment of the liquid formulation according to the present invention, the (6S)-5-methyltetrahydrofolic acid or a salt thereof is selected from the group consisting of (6S)-5-methyltetrahydrofolic acid, (6S)-5-methyltetrahydrofolic acid calcium salt (Metafolin®), (6S)-5-methyltetrahydrofolic acid monosodium salt (Arcofolin®), (6S)-5-methyltetrahydrofolic acid disodium salt, (6S)-5-methyltetrahydrofolic acid L-isoleucine ethyl ester salt, (6S)-5-methyltetrahydrofolic acid L-leucine ethyl ester salt, and (6S)-5-methyltetrahydrofolic acid L-valine ethyl ester salt.

[0043] More preferably, in an embodiment of the liquid formulation according to the present invention, the (6S)-5-methyltetrahydrofolic acid or a salt thereof is (6S)-5-methyltetrahydrofolic acid monosodium salt (Arcoforin®).

[0044] In liquid formulation embodiments according to the present invention, the liquid formulation medium is preferably water.

[0045] Alternatively, in a preferred embodiment of the liquid formulation according to the present invention, the liquid formulation medium is a mixture of water and ethanol, and the content of ethanol in the mixture is preferably in the range of 0.01 to 10.0% by volume, more preferably 0.01 to 5.0% by volume, even more preferably 0.01 to 1.0% by volume, and most preferably 0.01 to 0.5% by volume, based on the total volume of the formulation medium.

[0046] In an embodiment of the liquid formulation according to the present invention, the concentration of (6S)-5-methyltetrahydrofolic acid or a salt thereof, calculated as the free acid, is preferably in the range of 0.0001 to 10.0 mg / mL, preferably 0.001 to 6.00 mg / mL, more preferably 0.001 to 4.00 mg / mL of the liquid formulation.

[0047] Typical concentrations of (6S)-5-methyltetrahydrofolic acid or its salts, calculated as the free acid, in sprays or drops range from 1.00 to 10.0 mg / mL, preferably 2.00 to 7.00 mg / mL, more preferably 3.00 to 5.00 mg / mL, and most preferably about 4.00 mg / mL. Typical concentrations of (6S)-5-methyltetrahydrofolic acid or its salts, calculated as the free acid, in vitamin shots range from 0.001 to 1.00 mg / mL, preferably 0.01 to 0.50 mg / mL, more preferably 0.05 to 0.10 mg / mL, and most preferably about 0.08 mg / mL. Typical concentrations of (6S)-5-methyltetrahydrofolic acid or its salts, calculated as the free acid, in vitamin drinks range from 0.0001 to 0.100 mg / mL, preferably 0.0005 to 0.010 mg / mL, more preferably 0.001 to 0.005 mg / mL, and most preferably about 0.0016 mg / mL.

[0048] In an embodiment of the liquid formulation according to the present invention, it is preferred that the concentration of the antioxidant is in the range of 0.0001 to 300 mg / mL, preferably 0.001 to 150 mg / mL, more preferably 0.004 to 34.0 mg / mL of the liquid formulation.

[0049] In an embodiment of the liquid formulation according to the present invention, the molar equivalent of the antioxidant relative to (6S)-5-methyltetrahydrofolic acid or a salt thereof is preferably in the range of 0.01 to 4500 equivalents, preferably 0.01 to 2250 equivalents, more preferably 0.01 to 450 equivalents.

[0050] In a preferred embodiment of the liquid formulation according to the present invention, the liquid formulation further comprises L-ascorbic acid or a salt thereof, preferably sodium L-ascorbate, at a concentration of 0.001 to 200 mg / mL, preferably 0.01 to 100 mg / mL, more preferably 0.1 to 10 mg / mL, calculated as the free acid, relative to the liquid formulation.

[0051] Preferably, the molar equivalent of L-ascorbic acid or a salt thereof to the antioxidant is in the range of 0.0001 to 2000 equivalents, preferably 0.001 to 1000 equivalents, more preferably 0.01 to 100 equivalents.

[0052] In a preferred embodiment of the liquid formulation according to the present invention, the liquid formulation further comprises tocopherol or a derivative thereof, preferably tocopherol or tocopherol polyethylene glycol succinate (tocophersolan), more preferably tocopherol polyethylene glycol succinate (tocophersolan). Preferably, the concentration of tocopherol or a derivative thereof is in the range of 0 to 200 mg / mL, preferably 0.001 to 50.0 mg / mL, more preferably 0.01 to 10.0 mg / mL, based on the liquid formulation.

[0053] In an embodiment of the liquid formulation according to the present invention, it is preferred that the liquid formulation further comprises one or more compounds selected from the group consisting of L-isoleucine ethyl ester, L-leucine ethyl ester, and L-valine ethyl ester. These compounds can improve the solubility properties of the individual components contained in the liquid formulation. Furthermore, these compounds are particularly suitable as additives for dietary supplements, preferably dietary supplements for athletes.

[0054] In an embodiment of the liquid formulation according to the present invention, the liquid formulation has a pH in the range of 5.0 to 10.0, preferably 5.5 to 9.0, more preferably 6.0 to 8.0, and most preferably 6.4 to 8.0. The liquid formulation according to the present invention may contain a buffer to stabilize a specific pH. Preferred buffers include citrate buffer, aspartate buffer, and potassium dihydrogen phosphate (KH2PO4) buffer.

[0055] Additionally, the liquid formulations of the present invention may contain minerals of the types and amounts found in mineral water, including, but not limited to, magnesium, calcium, sodium, potassium, bicarbonate, sulfate, silica, chloride, and nitrate.

[0056] The liquid formulation of the present invention is preferably provided in a dosage form for use as a spray, drops, or a drinking bottle. Such a drinking bottle may be either a simple drinking bottle containing a liquid vitamin formulation as a vitamin shot or vitamin drink, or a drinking bottle containing a liquid vitamin formulation as a vitamin shot or vitamin drink, and further containing one or more solid formulations (such as tablets) of additional vitamins and / or minerals (collectively also referred to as micronutrients), which are separated from the liquid vitamin formulation. For example, such solid formulations may be placed in a separate portion of the drinking bottle, such as a cap.

[0057] method Further provided is a method for preparing a liquid formulation according to the present invention, the method comprising the following steps (i) and (ii); or (i') and (ii'): (i) dissolving (6S)-5-methyltetrahydrofolic acid or a salt thereof in a liquid formulation medium; and (ii) adding one or more antioxidants selected from grape seed extract, rosemary extract, or components thereof; or (i') dissolving one or more antioxidants selected from grape seed extract, rosemary extract, or components thereof in a liquid formulation medium; and (ii') adding (6S)-5-methyltetrahydrofolic acid or a salt thereof;

[0058] In a preferred embodiment of the method for preparing a liquid formulation according to the invention, the formulation medium is water.

[0059] In another preferred embodiment of the method for preparing a liquid formulation according to the present invention, the formulation medium is a mixture of water and ethanol, preferably with an ethanol content in the range of 0.01 to 10.0% by volume, preferably 0.01 to 5.0% by volume, more preferably 0.01 to 1.0% by volume, and most preferably 0.01 to 0.5% by volume, based on the total volume of the formulation medium.

[0060] In a preferred embodiment of the method for preparing a liquid formulation according to the present invention, the one or more antioxidants are added in step (ii) as a solution, preferably comprising the same liquid formulation medium as in step (i).

[0061] In a preferred embodiment of the method for preparing a liquid formulation according to the present invention, (6S)-5-methyltetrahydrofolic acid or a salt thereof is added in step (ii') as a solution, preferably comprising the same liquid formulation medium as in step (i').

[0062] The preferred, more preferred, even more preferred and most preferred features disclosed for the formulation above can be applied to further preferred, more preferred, even more preferred and most preferred features of the method for preparing the liquid formulation according to the invention.

[0063] use The present invention relates to the use of a liquid formulation according to the invention as a liquid vitamin preparation or as a raw material for a liquid vitamin preparation.

[0064] Preferably, the liquid vitamin formulation is a dietary supplement, preferably a dietary supplement for populations with high or unmet folate requirements, more preferably a dietary supplement for athletes, women who are pregnant or lactating or seeking to become pregnant, people who have difficulty swallowing solid dosage forms, and / or people wishing to improve their health.

[0065] Preferably, dietary supplement is in the dosage form for use as spray, drops or drink bottle.This drink bottle can be either a simple drink bottle that contains liquid vitamin preparation as vitamin shot or vitamin drink, or a drink bottle that contains liquid vitamin preparation as vitamin shot or vitamin drink, and also contains one or more solid preparations (such as tablets) of additional vitamins and / or minerals (also referred to as micronutrients), and this solid preparation is separated from the liquid vitamin preparation.For example, this solid preparation can be placed in a separate part of drink bottle, such as cap.

[0066] Preferably, the liquid vitamin formulation is a liquid vitamin formulation for the fortification of foods, preferably cereals and dairy products.

[0067] Preferably, the liquid vitamin formulation is a liquid vitamin formulation for life science applications, preferably for stabilizing cell culture media.

[0068] Preferably, the liquid vitamin formulation is a liquid pharmaceutical and / or liquid cosmetic formulation, preferably an eye drop.

[0069] Preferably, the liquid vitamin formulation is a liquid non-medicinal formulation for over-the-counter (OTC) products, preferably bandages and wound dressings.

[0070] The present invention is further illustrated by the following examples, which should not be construed as limiting. Those skilled in the art will recognize that various modifications, additions, and variations can be made to the present invention without departing from the spirit and scope of the invention, as defined in the appended claims. [Example]

[0071] Analysis method The L-5-MTHF content in samples prepared from the liquid formulations was determined by LC-MS / MS according to the method of AOAC 2013.13.

[0072] Preparation of liquid formulations [Example 1-1] Preparation of a liquid formulation containing L-5-MTHF monosodium salt (Arcoforin®), sodium L-ascorbate, and grape seed extract (GSE) in mineral water (Evian®)

[0073] Reagents and equipment used L-5-MTHF monosodium salt, bulk (Arcoforin®) (batch: ESY0008-XX); mineral water (Evian®); 20 mL GC-HS vials; sodium L-ascorbate; grape seed extract (GSE) solution (2%, 46.2% ethanol, 100% in deionized water) (Mibelle AG Biochemistry, CH-5033 Buchs); analytical balance with printer; pH meter; 500 mL volumetric flask; 100 mL volumetric flask; 50 mL volumetric flask; 500 mL graduated cylinder

[0074] Testing Procedure: 1) Add 0.5 mL of grape seed extract (GSE) solution (2%) to 50 mL of water to prepare a diluted solution of grape seed extract (GSE) (1:100, 0.02%). The concentration of grape seed extract (GSE) is 0.2 mg / mL. This solution can be stored in a refrigerator (cool, dark place) and reused as needed. 2) Prepare Solution 1 containing 35 mg of L-5-MTHF monosodium salt (Arcoforin®) in 100 mL of water. The concentration of L-5-MTHF monosodium salt is 0.3500 mg / mL. 3) Add 3 mL of Solution 1, 500 mg of sodium L-ascorbate, and 10 mL of a diluted solution of grape seed extract (GSE) (1:100, 0.02%) to a 500 mL volumetric flask and fill to 500 mL with mineral water (Evian®). The concentration of L-5-MTHF monosodium salt is 0.0021 mg / mL, the concentration of sodium L-ascorbate is 1.0000 mg / mL, and the concentration of grape seed extract (GSE) is 0.004 mg / mL. Step 3) should be performed immediately after step 2). 4) Measure the pH of the prepared solution. 5) Divide this solution into 17 x 20 mL GC-HS vials (20 mL each). 6) Seal the GC-HS vial with crimping pliers. 7) Label two vials as "Example 1-1, Initial," nine vials as "Example 1-1, 25°C / 60%RH," and six vials as "Example 1-1, 40°C / 75%RH."

[0075] [Example 1-2] Preparation of a liquid formulation containing L-5-MTHF monosodium salt (Arcoforin®), sodium L-ascorbate, and rosemary extract (RME) in mineral water (Evian®)

[0076] Reagents and equipment used L-5-MTHF monosodium salt, bulk (Arcoforin®) (Batch: ESY0008-XX); mineral water (Evian®); 20 mL GC-HS vials; sodium L-ascorbate; rosemary extract (RME) (30% rosmarinic acid) (Gustav Parmentier GmbH, D-60320 Frankfurt am Main); analytical balance with printer; pH meter; 500 mL volumetric flask; 100 mL volumetric flask; 500 mL graduated cylinder

[0077] Testing Procedure: 1) Prepare Solution 1 containing 35 mg of L-5-MTHF monosodium salt (Arcoforin®) in 100 mL of water. The concentration of L-5-MTHF monosodium salt is 0.3500 mg / mL. 2) Add 3 mL of Solution 1, 500 mg of sodium L-ascorbate, and 1750 mg of rosemary extract (RME) (30% rosmarinic acid) to a 500 mL volumetric flask and fill to 500 mL with mineral water (Evian®). The concentrations of L-5-MTHF monosodium salt are 0.0021 mg / mL, the concentration of sodium L-ascorbate is 1.0000 mg / mL, and the concentration of rosemary extract (RME) (30% rosmarinic acid) is 3.5000 mg / mL. Step 2) should be performed immediately after step 1). 3) Measure the pH of the prepared solution. 4) Divide this solution into 17 x 20 mL GC-HS vials (20 mL each). 5) Seal the GC-HS vial with crimping pliers. 6) Label two vials as "Example 1-2, Initial," nine vials as "Example 1-2, 25°C / 60%RH," and six vials as "Example 1-2, 40°C / 75%RH."

[0078] [Reference Example 1-1] Preparation of a liquid formulation containing L-5-MTHF monosodium salt (Arcoforin®) and sodium L-ascorbate in mineral water (Evian®)

[0079] Reagents and equipment used L-5-MTHF monosodium salt, bulk (Arcoforin®) (Batch: ESY0008-XX); mineral water (Evian®); 20 mL GC-HS vial; sodium L-ascorbate; analytical balance with printer; pH meter; 500 mL volumetric flask; 100 mL volumetric flask; 500 mL graduated cylinder

[0080] Testing Procedure: 1) Prepare Solution 1 containing 35 mg of L-5-MTHF monosodium salt (Arcoforin®) in 100 mL of water. The concentration of L-5-MTHF monosodium salt is 0.3500 mg / mL. 2) Add 3 mL of solution 1 and 500 mg of sodium L-ascorbate to a 500 mL volumetric flask and fill to 500 mL with mineral water (Evian®). The concentration of L-5-MTHF monosodium salt is 0.0021 mg / mL, and the concentration of sodium L-ascorbate is 1.0000 mg / mL. Step 2) should be performed immediately after step 1). 3) Measure the pH of the prepared solution. 4) Divide this solution into 17 x 20 mL GC-HS vials (20 mL each). 5) Seal the GC-HS vial with crimping pliers. 6) Label two vials as "Reference Example 1-1, Initial", nine vials as "Reference Example 1-1, 25°C / 60%RH", and six vials as "Reference Example 1-1, 40°C / 75%RH".

[0081] [Table 1]

[0082] For long-term stability testing, nine vials were stored in a climate chamber at 25°C and 60% relative humidity (RH), and six vials were stored in a climate chamber at 40°C and 75% relative humidity (RH). Periodically, samples were taken, and the content of L-5-MTHF monosodium salt was measured by HPLC according to the general procedure described above. The results are shown in Figures 1 and 2.

[0083] As can be seen from Figure 1, Example 1-2 demonstrates unexpectedly improved long term stability at 25°C and 60% RH compared to Reference Example 1-1.

[0084] As can be seen from Figure 2, Examples 1-1 and 1-2 demonstrate unexpectedly improved long-term stability at 40°C and 75% RH compared to Reference Example 1-1.

Claims

1. (a) (6S)-5-methyltetrahydrofolic acid or a salt thereof; (b) one or more antioxidants selected from grape seed extract, rosemary extract, or components thereof; (c) a liquid formulation medium; and A liquid formulation comprising:

2. 2. The liquid formulation according to claim 1, characterized in that the antioxidant is selected from grape seed extracts or components thereof.

3. The antioxidants include catechin, epicatechin, epicatechin gallate, epigallocatechin gallate, gentisic acid, syringic acid, protocatechuic acid, gallic acid, caffeic acid, and procyanidin B １ , procyanidin B ２ , procyanidin B ３ , procyanidin B ４ , procyanidin B １ -3-O-gallate (B １ -G), procyanidin B ２ -3-O-gallate (B ２ -G), procyanidin B ２ -3'-O-gallate (B ２ -G'), procyanidin C １ 3. A liquid formulation according to claim 1 or 2, characterized in that the hydroxybenzoates are selected from the group consisting of kaempferol and quercetin.

4. 2. A liquid formulation according to claim 1, characterized in that the antioxidant is selected from rosemary extract or its components.

5. 5. A liquid formulation according to claim 1 or 4, characterized in that the antioxidant is selected from the group consisting of carnosic acid, carnosol and rosmarinic acid.

6. The antioxidants include catechin, epicatechin, epicatechin gallate, epigallocatechin gallate, gentisic acid, syringic acid, protocatechuic acid, gallic acid, caffeic acid, and procyanidin B １ , procyanidin B ２ , procyanidin B ３ , procyanidin B ４ , procyanidin B １ -3-O-gallate (B １ -G), procyanidin B ２ -3-O-gallate (B ２ -G), procyanidin B ２ -3'-O-gallate (B ２ -G'), procyanidin C １ 2. The liquid formulation according to claim 1, characterized in that the hydroxybenzoates are selected from the group consisting of kaempferol, quercetin, carnosic acid, carnosol, and rosmarinic acid.

7. 7. The liquid formulation of any one of claims 1 to 6, wherein the (6S)-5-methyltetrahydrofolic acid or a salt thereof is selected from the group consisting of (6S)-5-methyltetrahydrofolic acid, (6S)-5-methyltetrahydrofolic acid calcium salt, (6S)-5-methyltetrahydrofolic acid monosodium salt, (6S)-5-methyltetrahydrofolic acid L-isoleucine ethyl ester salt, (6S)-5-methyltetrahydrofolic acid L-leucine ethyl ester salt, and (6S)-5-methyltetrahydrofolic acid L-valine ethyl ester salt.

8. The liquid formulation according to any one of claims 1 to 7, wherein the (6S)-5-methyltetrahydrofolic acid or a salt thereof is (6S)-5-methyltetrahydrofolic acid monosodium salt.

9. 9. The liquid formulation according to any one of claims 1 to 8, wherein the liquid formulation medium is water or a mixture of water and ethanol, preferably having an ethanol content in the range of 0.01 to 10.0% by volume relative to the total amount of the formulation medium.

10. 10. The liquid formulation according to any one of claims 1 to 9, wherein the concentration of (6S)-5-methyltetrahydrofolic acid or a salt thereof, calculated as the free acid, ranges from 0.0001 to 10.0 mg / mL, preferably from 0.001 to 6.00 mg / mL, more preferably from 0.001 to 4.00 mg / mL of the liquid formulation.

11. 11. The liquid formulation according to any one of claims 1 to 10, wherein the concentration of the antioxidant ranges from 0.0001 to 300 mg / mL, preferably from 0.001 to 150 mg / mL, more preferably from 0.004 to 34.0 mg / mL of the liquid formulation.

12. 12. The liquid formulation according to any one of claims 1 to 11, wherein the molar equivalent of the antioxidant to (6S)-5-methyltetrahydrofolic acid or a salt thereof is in the range of 0.01 to 4500 equivalents, preferably 0.01 to 2250 equivalents, more preferably 0.01 to 450 equivalents.

13. The liquid formulation according to any one of claims 1 to 12, wherein the liquid formulation further comprises L-ascorbic acid or a salt thereof.

14. 14. The liquid formulation according to any one of claims 1 to 13, further comprising L-ascorbic acid or a salt thereof, wherein the concentration of L-ascorbic acid or a salt thereof, calculated as the free acid, ranges from 0.001 to 200 mg / mL, preferably from 0.01 to 100 mg / mL, more preferably from 0.1 to 10 mg / mL of the liquid formulation.

15. 15. The liquid formulation according to any one of claims 1 to 14, further comprising L-ascorbic acid or a salt thereof, wherein the molar equivalents of L-ascorbic acid or its salt to the antioxidant range from 0.0001 to 2000 equivalents, preferably from 0.001 to 1000 equivalents, more preferably from 0.01 to 100 equivalents.

16. The liquid formulation according to any one of claims 1 to 15, wherein the liquid formulation further comprises tocopherol or a derivative thereof.

17. 17. The liquid formulation of any one of claims 1 to 16, wherein the liquid formulation further comprises one or more compounds selected from the group consisting of L-isoleucine ethyl ester, L-leucine ethyl ester, and L-valine ethyl ester.

18. 18. The liquid formulation according to any one of the preceding claims, wherein the pH of the liquid formulation is in the range of 5.0 to 10.0, preferably 5.5 to 9.0, more preferably 6.0 to 8.0, and most preferably 6.4 to 8.

0.

19. A liquid formulation according to any one of claims 1 to 18, wherein the liquid formulation is in a dosage form for use as a spray, drops or a drinking bottle.

20. A method for preparing a liquid formulation according to any one of claims 1 to 19, comprising the following steps (i) and (ii); or (i') and (ii'): (i) dissolving (6S)-5-methyltetrahydrofolic acid or a salt thereof in a liquid formulation medium; and (ii) adding one or more antioxidants selected from grape seed extract, rosemary extract, or components thereof; or (i') dissolving one or more antioxidants selected from grape seed extract, rosemary extract, or components thereof in a liquid formulation medium; and (ii') adding (6S)-5-methyltetrahydrofolic acid or a salt thereof;

21. 21. A method for preparing a liquid formulation according to claim 20, wherein the formulation medium is water or a mixture of water and ethanol, preferably with an ethanol content ranging from 0.01 to 10.0% by volume relative to the total amount of the formulation medium.

22. Use of a liquid formulation according to any one of claims 1 to 19 as a liquid vitamin preparation or as a raw material for a liquid vitamin preparation.

23. 23. The use of claim 22, wherein the liquid vitamin formulation is a dietary supplement, preferably a dietary supplement for populations with high or unmet folate requirements, more preferably a dietary supplement for athletes, women who are pregnant or lactating or who wish to become pregnant, people who have difficulty swallowing solid dosage forms, and / or people wishing to improve their health.

24. 24. The use according to claim 23, wherein the liquid vitamin formulation is in a dosage form for use as a spray, drops, or drink bottle.

25. 23. The use according to claim 22, wherein the liquid vitamin formulation is a liquid vitamin formulation for food fortification.

26. 23. The use according to claim 22, wherein the liquid vitamin formulation is a liquid vitamin formulation for life science applications.

27. 23. The use according to claim 22, wherein the liquid vitamin formulation is a liquid pharmaceutical and / or liquid cosmetic formulation.

28. 23. The use of claim 22, wherein the liquid vitamin formulation is a liquid non-pharmaceutical formulation for over-the-counter (OTC) products.

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