COMPOSITION FOR THE PREVENTION AND TREATMENT OF FOLATE AND/OR VITAMIN B12 DEFICIENCY CONDITIONS, PARTICULARLY HYPERHOMOCYSTEINEMIA

Abstract

A composition for oral use comprising folic acid or derivatives and precursors thereof, an alkalizing agent selected from the group consisting of Magnesium Oxide, Calcium Oxide, Zinc Oxide and mixtures thereof, and optionally Vitamin B12 or derivatives and precursors thereof. The composition is intended for use in the treatment and/or prevention of the disease states caused by deficiency of folate and/or vitamin B12, particularly hyperhomocysteinemia.

Claims

1. An oral composition comprising folic acid or derivatives and precursors thereof, and an alkalizing agent selected from the group consisting of Magnesium Oxide, Calcium Oxide, Zinc Oxide, and mixtures thereof.

2. A method of treatment of conditions caused by folate deficiency, said method comprising orally administering a composition comprising folic acid or derivatives and precursors thereof; and an alkalizing agent selected from the group consisting of Magnesium Oxide, Calcium Oxide, Zinc Oxide and mixture thereof.

3. The method according to claim 2, wherein the condition caused by folate deficiency is hyperhomocysteinemia.

4. The method according to claim 2, wherein the composition comprising folic acid or derivatives and precursors thereof; and an alkalizing agent selected from the group consisting of Magnesium Oxide, Calcium Oxide, Zinc Oxide and mixture thereof is administered in combination with antiblastic drug therapies or antimetabolites.

5. A method of prevention of foetal neural tube defects comprising the oral administration, during gestation, of a composition comprising folic acid or derivatives and precursors thereof; and an alkalizing agent selected from the group consisting of Magnesium Oxide, Calcium Oxide, Zinc Oxide, and mixtures thereof.

6. The oral composition according to claim 1, wherein the alkalizing agent and folic acid or derivatives and precursors thereof are in a weight ratio between 20:1 and 200:1.

7. The oral composition according to claim 1, wherein the folic acid derivatives and precursors are selected from the group consisting of: folic acid salts, folinic acid or salts thereof, 5-methyltetrahydrofolate (MTHF) or salts thereof, and mixtures thereof.

8. A method of treating diseases caused by deficiency of folate or Vitamin B12, said method comprising: orally administering a composition comprising folic acid or derivatives and precursors thereof; an alkalizing agent selected from the group consisting of Magnesium Oxide, Calcium Oxide, Zinc Oxide and mixtures thereof; and Vitamin B12 or derivatives/precursors thereof.

9. The method of treatment according to claim 8, wherein the disease states caused by deficiency of folate or Vitamin B12 are selected from the group consisting of: anemias, and polyneurites.

10. The oral composition according to claim 1, further comprising Vitamin B12 or derivatives/precursors thereof, wherein said derivatives/precursors are selected from the group consisting of: Cyanocobalamin, Methylcobalamin, 5′-deoxyadenosylcobalamin, hydroxycobalamin and mixtures thereof.

11. The oral composition according to claim 1, in the form of solid formulation selected from the group consisting of: tablets and granulates; or hydro-polyol-based liquid formulation.

12. The oral composition according to claim 1, further comprising other active ingredients selected from the group consisting of: other B vitamins, L-Methionine, S-Adenosylmethionine, Betaine, Choline, Iron at oxidation state 2.sup.+ or 3.sup.+ in the form of organic or inorganic salts, and mixtures thereof.

13-14. (canceled)

15. The oral composition according to claim 1, wherein said alkalizing agent is Zinc Oxide.

16. The method according to claim 2, wherein said alkalizing agent is Zinc Oxide.

17. The method according to claim 5, wherein said alkalizing agent is Zinc Oxide.

18. The method according to claim 8, wherein said alkalizing agent is Zinc Oxide.

Description

DESCRIPTION OF THE FIGURES

[0050] FIG. 1: Methionine cycle.

[0051] FIG. 2: Structural Cobalamin core. With R=—CH3 Methylcobalamin, with R=—CN, Cyanocobalamin, with R=-deoxyadenosyl, 5-deoxyadenosylcobalamin.

[0052] FIG. 3: Diagram of Cobalamin absorption in the intestine.

DETAILED DESCRIPTION OF THE INVENTION

[0053] As mentioned above, the object of the invention is a composition for oral use comprising, as an active ingredient, folic acid or the derivatives and precursors thereof, and as an alkalizing agent, a metal oxide selected from the group consisting of Magnesium Oxide, Calcium Oxide, Zinc Oxide and mixtures thereof.

[0054] According to a preferred embodiment, the alkalizing agent is Zinc Oxide.

[0055] It should be noted that, preferably, the folic acid derivatives and precursors are selected from the group consisting of: salts of folic acid, folinic acid or its salts, 5-methyltetrahydrofolate (MTHF) or its salts, and mixtures thereof.

[0056] According to a preferred embodiment, the folic acid salts are selected from sodium salt and calcium salt.

[0057] According to a preferred embodiment, the folinic acid salts are selected from sodium salt and calcium salt.

[0058] According to a preferred embodiment, the salts of 5-methyltetrahydrofolate (MTHF) are selected from sodium salt, calcium salt and glucosamine salt.

[0059] An important aspect of the present invention is the use of oxides and not of the hydrated forms thereof as alkalizing agents; in fact, hydroxides are known alkalizing agents, characterized by a more vigorous neutralizing activity, immediate but not modulable over time.

[0060] The unhydrated form of the oxides has the advantage of allowing a controlled formation of the corresponding hydrates when the composition comes into contact with gastric fluids; this “controlled release” of the hydrates allows to raise the pH at the gastric level (pH between 4.5 and 5.5 for at least 60 minutes) to values compatible with the solubilization of folic acid or the derivatives and precursors thereof; it should be noted that such an alkalinization effect would not be possible, for such a prolonged period, using the corresponding hydroxides of the alkalizing agents indicated above.

[0061] It should be noted that preferably the composition is suitable for use as a medicament (medicinal specialty), nutraceutical, food supplement or food for special medical purposes.

[0062] According to a preferred embodiment, the oral composition is intended for use in the prevention and/or treatment of disease states caused by folate deficiency; it should be noted that, for the purposes of the present invention, hyperhomocysteinemia falls within the definition of folate deficiency disease state.

[0063] As already mentioned above, the use of antiblastic drugs or antimetabolites is sometimes responsible for a depletion of folic acid; in this sense, antiblastic or antimetabolic drug therapies induce a folate deficiency disease state.

[0064] The composition is therefore preferably suitable for use in association with antiblastic drug therapies or antimetabolites, preferably in association with therapeutic agents inhibiting Dihydrofolate Reductase, preferably in association with Methotrexate.

[0065] It should be noted that folate deficiency disease states can also occur in the event of inadequate diets or intestinal malabsorption; folic acid deficiency in the early stages of pregnancy significantly increases the risk of foetal malformations, in particular neural tube defects (NTDs). Furthermore, folate deficiency can be associated with other adverse pregnancy outcomes (intrauterine growth retardation, premature delivery).

[0066] In this sense, the composition of the invention is suitable for use in the prevention of neural tube defects during pregnancy.

[0067] The neural tube is an embryonic structure from which the central nervous system develops (brain, braincase, spine, etc.). When the neural tube does not close properly and completely during the first weeks of pregnancy, the newborn develops severe congenital malformations known as neural tube defects (NTDs).

[0068] It should be noted that NTDs are preferably selected from the group consisting of: spina bifida, anencephaly and encephalocele.

[0069] Spina bifida is due to an incomplete closure of the underside of the neural tube. Spina bifida also has very different consequences, ranging from problems which can be corrected with surgery to serious physical and mental disabilities. In the latter case, paralysis of the lower limbs, difficulty controlling the internal organs (intestine and bladder), developmental and learning difficulties and mental retardation, sometimes hydrocephalus, can occur. Children with spina bifida most often survive to adulthood.

[0070] Anencephaly is a condition in which the brain develops incompletely or does not develop at all as a result of incomplete closure of the upper part of the neural tube. Children with anencephaly die before or immediately after birth.

[0071] Encephalocele is a condition in which a part of the brain, more or less severely malformed, forms a hernia from a skull closure defect. Encephalocele can have an unfortunate outcome and only in a limited percentage of cases does normal psychomotor development occur.sup.6.

[0072] According to a preferred embodiment, the alkalizing agent and the folic acid or derivatives/precursors thereof are in a weight ratio between 20:1 and 200:1, preferably between 50:1 and 100:1, preferably between 65:1 and 90:1, preferably between 75:1 and 90:1, preferably between 78:1 and 88:1

[0073] It should be noted that, preferably, the amount of folic acid or the derivatives and precursors thereof per dosage unit is between 100 and 5000 μg, preferably between 200 and 1000 μg, preferably between 200 and 800 μg, preferably between 200 and 400 μg, preferably between 300 and 400 μg.

[0074] It should be noted that, preferably, the amount of alkalizing agent per dosage unit is between 20 and 100 mg, preferably between 20 and 80 mg, preferably between 20 and 60 mg, preferably between 25 and 50 mg.

[0075] It should be noted that, with particular reference to Zinc Oxide as an alkalizing agent, the maximum daily amount administered is 25 mg (corresponding to about 15 mg Zinc).

[0076] According to a preferred embodiment, the composition for oral use further comprises Vitamin B12 or derivatives/precursors thereof; this embodiment is particularly suitable for use in the prevention and/or treatment of disease states caused by folate and/or Vitamin B12 deficiency.

[0077] Preferably, the disease states caused by folate deficiency and/or Vitamin B12 are selected from the group consisting of hyperhomocysteinemia, anaemias and polyneuritis; it should be noted that the composition for oral use is particularly suitable for the prevention and/or treatment of hyperhomocysteinemia.

[0078] Preferably, the oral composition is intended for the prevention and/or treatment of macrocytic megaloblastic anaemia (pernicious anaemia).

[0079] It should be noted that the preferred form of the oral composition of the invention comprises folic acid or derivatives and precursors thereof, Zinc Oxide as alkalizing agent and Vitamin B12 or derivatives and precursors thereof; the therapeutic uses for which such embodiment is intended are those described in the description of the present application, preferably hyperhomocysteinemia.

[0080] It should be noted that the derivatives and precursors of Vitamin B12 are preferably selected from the group consisting of: Cyanocobalamin, Methylcobalamin, 5′-deoxyadenosylcobalamin, hydroxocobalamin and mixtures thereof.

[0081] According to a preferred embodiment, the amount of Vitamin B12 or the derivatives and precursors thereof is between 1 and 2000 μs, preferably between 1 and 1000 μs, preferably between 5 and 1000 μs, preferably between 5 and 500 μs, preferably between 5 and 100 μs, preferably between 5 and 50 μg.

[0082] Preferably, the oral composition is made in solid formulation form selected from tablets and granules. Preferably, the solid formulations suitable for conveying the composition are selected from, for example, swallowable tablets, two-layer tablets, filmed tablets, mouth-dispersible tablets, coated granules and mouth-dispersible granules.

[0083] According to an alternative embodiment, the composition for oral use is prepared in the form of a liquid formulation based on hydro-polyol; preferably, the liquid formulations suitable for conveying the composition are for example drops or syrups.

[0084] According to a first particularly preferred embodiment, the oral composition is made in the form of a mouth-dispersible solid formulation, preferably a granulate or a tablet, comprising a core and a mouth-soluble coating, which envelops the core externally. In fact, such an embodiment, allows to convey the folic acid or the derivatives and precursors thereof, the alkalizing agent and Vitamin B12 or the derivatives and precursors thereof in the outer coating, so as to allow the first active ingredient a correct solubilization at the gastric level, by virtue of the presence of the alkalizing agent; the second active ingredient is allowed a correct conjugation with AC at the salivary level, with FIC at the gastric level, thus ensuring a correct absorption in the subsequent intestinal transit.

[0085] According to a second particularly preferred embodiment, the composition for oral use is prepared in the form of a swallowable tablet which rapidly disaggregates in gastric fluid; said swallowable tablet comprises a core which disaggregates into gastric fluid, and an outer coating which completely covers said core. It should be noted the disaggregation rate of the core can be modulated with the use of suitably selected excipients, such as Polyvinylpolypyrrolidone (PVPP), so as to allow the disaggregation thereof in the gastric fluid, on an empty stomach, preferably within 10 minutes; the coating can be selected from the traditional coating forms, known in the literature (Part 5, Chapter 46, “Coating of pharmaceutical dosage forms”, from “The Remington, The science and Practice of Pharmacy”, 21st edition—Lippincott Williams & Wilkins), such as coatings aimed at masking an unpleasant taste.

[0086] According to an alternative embodiment, the swallowable, rapid gastric disaggregation tablet is formulated so that the disaggregating core contains folic acid and alkalizing agent, preferably Zinc Oxide, while the outer coating contains Vitamin B12.

[0087] The rapid gastric disaggregating swallowable tablet allows to produce a partial neutralization of gastric pH resulting in ionization of folic acid or its derivatives and precursors, while ensuring a release of Vitamin B12 such that it can be conjugated with AC and FIC.

[0088] It should be noted that the core of such solid formulations described above can accommodate other active agents.

[0089] According to a preferred embodiment, the composition for oral use further comprises other active agents selected from the group consisting of: other B group vitamins, L-Methionine, S-Adenosylmethionine, Betaine, Choline, Iron at oxidation state 2+ and/or 3+ in the form of organic and inorganic salts, and mixtures thereof.

[0090] Such additional active agents can be conveyed into the core of the aforementioned oral solid dosage form of the mouth-dispersible or swallowable type.

[0091] It should be noted that such additional active agents can also be formulated, in the composition for oral use of the invention, in a slow release technological form.

[0092] According to a preferred embodiment, the composition for oral use also comprises excipients known to those skilled in the art for the manufacture of solid or liquid pharmaceutical forms for oral consumption (according to techniques known to those skilled in the art, as available in the literature (Part 5, chapters “Solutions Emulsions Suspensions and Extracts” and “Oral Solid Dosage Forms” of “Remington: The Science and Practice of Pharmacy”, David B. Troy, Paul Beringer, Lippincott Williams & Wilkins, 2006).

[0093] Merely by way of example, the excipients suitable for the manufacture of the composition object of the present invention are indicated below: [0094] filling agents such as corn starch, microcrystalline cellulose mannitol, sucrose, sorbitol, lactose, maltodextrins, [0095] gliding agents such as colloidal silica, precipitated amorphous silica, magnesium oxide and carbonate; [0096] lubricating agents such as magnesium stearate; [0097] disaggregating agents such as carboxymethylcellulose sodium salt (CMC Na), polyvinylpolypyrrolidone (PVPP); [0098] colouring and flavouring agents; [0099] hardness modulators such as sucrose, calcium phosphate; [0100] coating agents such as shellac; [0101] enteric coated modified release polymers such as methacrylic acid derivatives, hydroxypropylmethylcellulose, sodium alginate, crosslinked sodium carboxymethylcellulose; [0102] polyols such as sorbitol, mannitol, erythritol, xylitol, glycerol, [0103] pH correctors such as citric acid, ascorbic acid.

Experimental Part

[0104] Gastric Simulated Fluid (GSF) Test.

[0105] For all the experiments, physiological gastric conditions were reproduced taking into account the volume of gastric fluid on an empty stomach (35-40 mL) (Mudie D M et al. Quantification of gastrointestinal liquid volumes and distribution following a 240 mL dose of water in the fasted state. Mol Pharm. 2014 Sep. 2; 11(9):3039-47) and the fact that in the stomach, the salt concentration is different from that of blood plasma (0.25% vs 0.9%) (Sandra Klein. The Use of Biorelevant Dissolution Media to Forecast the In Vivo Performance of a Drug. AAPS J. 2010 September; 12(3): 397-406).

[0106] The effect of the alkalizing agent on gastric pH was simulated in vitro by preparing a 0.2% saline solution of NaCl and bringing the pH of the solution to around 1.2-2 with 37% hydrochloric acid diluted 1:5.

[0107] GSF was prepared using the components in table 1, all tests were performed simulating body temperature (37° C.) and performed in triplicate.

TABLE-US-00001 TABLE 1 Simulated gastric fluid (GSF) composition GSF composition pH 1.2-2 Quantity NaCl 0.2 37% HCl diluted 1:5 as needed up to pH 1.2-1.5 H.sub.2O as needed up to 100

[0108] 400 ml of GSF was stirred with magnetic stirrer and settled at 37° C. and the three-point digital pH meter was applied. 40 mg of Folic Acid was added to this solution.

[0109] The bright orange dispersion was stirred for 5 minutes at 200 rpm and then the stirring was stopped.

[0110] After 10 minutes, the complete precipitation of folic acid was observed at the bottom of the beaker, demonstrating the poor solubility thereof under the simulated body conditions.

[0111] The same experiment was repeated adding 30 mg of Zinc Oxide to the same amount of folic acid. The system was allowed to stir for 5 minutes at 200 rpm and then stirring was stopped, recording a pH of 5.2 and the formation of a translucent orange solution in the absence of precipitation on the bottom, a sign of the dissolution thereof. Such a pH was maintained in the range 5.0-5.5 also by simulating physiological acid secretion, combining 2 ml of 0.1 M HCl every 15 minutes for a total of 60 minutes.

[0112] In fact, Zinc Oxide prevents the lowering of the physiological pH thanks to a progressive formation mechanism of Zinc hydroxide which opposes acidification.

EXAMPLES

[0113] By way of non-limiting illustration, two examples of the inventive composition are given below.

[0114] 1. Example of a Mouth-Dispersible Tablet Coated Externally with a Mouth-Soluble Film

TABLE-US-00002 Outer film Sodium Folate equal to 400 μg Folic Acid Cyanocobalamin  5.0 μg Zinc Oxide   35 mg Sucralose  1.0 mg Vanilla flavouring as needed iron oxide yellow (E172) as needed iron oxide red (E172) as needed Hypromellose as needed Glycerol as needed Demineralized water as needed Core Microcrystalline cellulose as needed Calcium phosphate as needed Com starch as needed Betaine  250 mg Vitamin B2 (phosphate salt) equal to 2.4 mg Riboflavin Vitamin B6   3 mg Precipitated amorphous silica 3% w/w Magnesium Stearate 2% w/w Carboxymethylcellulose 2% w/w

[0115] 1.1 Physical Chemical Parameters:

[0116] Weight (average 50 tablets): 1.050 g

[0117] Hardness (in Kg average 50 tablets): 12

[0118] 1.2 Preparation Method

[0119] Outer Film

[0120] Dissolve the Vitamin B12, sodium folate and sucralose in water and then disperse in the order Glycerin, hypromellose, vanilla flavouring, iron oxide red and yellow, zinc oxide under mechanical stirring 400 rpm until a homogeneous orange-red mixture is obtained.

[0121] Core

[0122] Weigh together Vitamins B2 and B6, half of the starch, cellulose and mix the powders together with a mechanical V-stirrer for at least 5 minutes. Separately combine the Betaine hydrochloride with the other half of the corn starch, silica and magnesium stearate and mix the powder mixture for at least 5 minutes. Combine the powder mixture with the vitamins with the one containing Betaine and stir the mixture in a V-stirrer for another 5 minutes. Sieve the powdered mixture with 80 mesh manual metal sieve. Proceed to compress the thus obtained powder in a semi-automatic tablet press. Proceed to film the cores thus obtained with the aqueous dispersion previously set up in the coating pan.

[0123] 2. Example of Oral Solution

TABLE-US-00003 Sodium Folate equal to 33.90 mg Folic acid Cyanocobalamin  42.4 mg Zinc Oxide  2.65 g Sucralose 120.0 mg Orange flavouring as needed Lemon flavouring as needed Glycerol   74 g Demineralized water as needed up to 100 g

[0124] 2.1 Chemical-Physical Parameters:

[0125] pH=8.5

[0126] density (measured with digital densimeter): 1.18

[0127] average weight 20 gtt=1.20 g

[0128] Folic acid for 20 gtt=400 μg

[0129] Cyanocobalamin for 20 gtt=500 μg

[0130] Zinc for 20 gtt=21 mg

[0131] 2.2. Preparation Method:

[0132] Weigh the Glycerin and disperse the Zinc Oxide, stirring with a mechanical stirrer at 300 rpm until a homogeneous and translucent dispersion is obtained. Dissolve in the order sodium folate, Vitamin B12 and Sucralose in the available water. Combine the aqueous solution with the glycerol dispersion, maintaining stirring until a fluid, translucent mixture is obtained. Finally, combine the flavourings and keep stirring for at least 5 minutes.

[0133] Divide the dispersion into 12 ml dropper bottles.

REFERENCES

[0134] 1. Bernhard Kräutler Biochemistry of B12-cofactors in Human Metabolism. Subcell Biochem, 56, 323-46 2012 [0135] 2. Renata Kozyraki, Olivier Cases. Vitamin B12 Absorption: Mammalian

[0136] Physiology and Acquired and Inherited Disorders. Biochimie, 95 (5), 1002-7. May 2013 [0137] 3. Younis I R, Stamatakis M K, Callery P S, Meyer-Stout P J. Influence of pH on the dissolution of folic acid supplements. Int J Pharm. 2009 Feb. 9; 367(1-2):97-102. [0138] 4. Esfandiar Heidarian, Massoud Amini, Mahmoud Parham, and Ashraf Aminorroaya. Effect of Zinc Supplementation on Serum Homocysteine in Type 2 Diabetic Patients with Microalbuminuria. Rev Diabet Stud. 2009 Spring; 6(1): 64-70. [0139] 5. Pakfetrat M, Shahroodi J R, Zolgadr A A, Larie H A, Nikoo M H, Malekmakan L. Effects of zinc supplement on plasma homocysteine level in end-stage renal disease patients: a double-blind randomized clinical trial. Biol Trace Elem Res. 2013 June; 153(1-3):11-5. [0140] 6. https://www.epicentro.iss.it/acido-folico/