Solid formulations of tetrahydrofolates

Abstract

The present invention relates to tertrahydrofolate formulations in the form of a granulated mixture consisting of a waxy agent and a desiccant.

Description

[0001] SOLID FORMULATIONS OF TETRAHYDROFOLATES

[0002] The present invention relates to tetrahydrofolate formulations in the form of a granulated mixture comprising a waxy agent and a desiccant.

[0003] Technical field of invention

[0004] Folic acid, namely N-[4-[[(2 -amino- 1,4-dihy dro-4-oxo-6- pteridinyl)methyl]amino]benzoyl]-L-glutamic acid, and folate, the anion thereof, and its precursors dihydrofolic and tetrahydrofolic acid and the respective anions thereof, are water-soluble forms of vitamin B9. They are found naturally in foodstuffs, mainly as conjugates, in particular in liver, kidneys, yeast, fruit and green leafy vegetables, and can also be taken as supplements.

[0005] B-complex vitamins help the body to convert carbohydrates to glucose, which is metabolised to produce energy. Said vitamins are essential in the breakdown of fats and proteins, and play an important part in maintaining muscle tone in the digestive tract and promoting the health of the nervous system, skin, hair, eyes, mouth and liver.

[0006] Folates are known to be necessary for DNA replication, and consequently for the production and maintenance of new cells. This is particularly important during periods of rapid cell division and growth, such as childhood and pregnancy. A folate deficiency therefore hinders DNA synthesis and cell division, clinically affecting bone marrow in particular, a site of rapid cell turnover. As the synthesis of RNA and proteins is not hindered, large red blood cells, namely megaloblasts, are produced, causing macrocytic anaemia such as megaloblastic anaemia (as in the case of coeliac disease) and anaemias of nutritional origin or arising during pregnancy or childhood. Both adults and children therefore need folate to produce normal red blood cells and prevent anaemia. Folate also helps to prevent DNA modifications that can lead to cancer.

[0007] Folic acid, like the above-mentioned derivatives thereof, are available on the market and are prepared synthetically. Folic acid presents as a yellow or yellowish-orange crystalline powder, and is slightly water-soluble and insoluble in alcohol; it is readily soluble in dilute solutions of alkali hydroxides and carbonates.

[0008] Tetrahydrofolic acid and the derivatives thereof are highly unstable, especially due to their liability to oxidise. The 5 -methyltetrahydrofolic acid present in nature is solely in the S form; the R form is biochemically inactive, and is excreted through the kidneys.

[0009] In particular, 5 -methyltetrahydrofolic acid is important as a pharmaceutical ingredient, mainly in oncology, as a concomitant treatment with methotrexate and 5 -fluorouracil, and in the treatment of anaemia caused by folic acid deficiency associated with pregnancy, with antibiotic treatment, etc. FR 2137186 describes water-soluble ammonium salts of folic acids.

[0010] Calcium salts can be described as the most relatively stable derivatives among the folates and reduced folates; US 5817659 and US 6441168 describe crystalline salts, preferably calcium salts, of 5-methyl-(6R,S)-, -(6S)- or - (6R)-tetrahydrofolic acid having water of crystallisation of at least one equivalent per equivalent of said acid.

[0011] EP 2 245 032 describes the crystalline or amorphous water-soluble salts of D- glucosamine and D-galactosamine folates, in particular 5-methyl-(6S)-tetrahydrofolate D- glucosamine salt.

[0012] Description of the invention

[0013] It has now been found that the stability of tetrahydrofolates is advantageously improved when they are formulated in granules comprising a waxy excipient as binder, and a desiccant.

[0014] According to a first aspect, the object of the present invention is therefore a granulate comprising a tetrahydrofolate, a waxy excipient and a desiccant.

[0015] The term “tetrahydrofolate” refers to tetrahydrofolic acid, unsubstituted or substituted with a 5-methyl-, 5-formyl-, 10-formyl-, 5,10-methylene- or 5,10-methenyl moiety, in a (6R,S), (6S) or (6R) configuration, and the salts or derivatives thereof.

[0016] The sodium, calcium or glucosamine salts in amorphous or crystalline form are preferred, in particular the D-glucosamine salts disclosed in EP 2 245 032.

[0017] Examples of preferred tetrahydrofolates include D-glucosamine (6R,S)- tetrahydrofolate, D-glucosamine (6S)-tetrahydrofolate, D-glucosamine (6R)-tetrahydrofolate; D-galactosamine (6R,S)-tetrahydrofolate, D-galactosamine (6S)-tetrahydrofolate, D- galactosamine (6R)-tetrahydrofolate; D-glucosamine 5-methyl-(6R,S)-tetrahydrofolate, D- glucosamine 5-methyl-(6S)-tetrahydrofolate, D-glucosamine 5-methyl-(6R)-tetrahydrofolate; D-galactosamine 5-methyl-(6R,S)-tetrahydrofolate, D-galactosamine 5-methyl-(6S)- tetrahydrofolate, and D-galactosamine 5-methyl-(6R)-tetrahydrofolate. Particularly preferred is D-glucosamine 5-methyl-(6S)-tetrahydrofolate.

[0018] The term “waxy excipient” refers to fatty compounds with low melting points, in particular mono- and diglycerides of fatty acids, stearic acid, stearin or mixtures thereof.

[0019] Stearic acid preferably meets the specifications reported in the USP monograph, identified as additive E-570 by the European Pharmacopoeia.

[0020] The mono- and diglycerides of fatty acids consist of mixtures of mono-, di- and triesters of glycerol with fatty acids present in oils and edible fats. They may contain small amounts of free fatty acids and glycerol.

[0021] Stearic acid and mono- and diglycerides of fatty acids, alone or mixed together, are preferred.

[0022] The desiccant is selected from rice starch, com starch, sodium carboxymethylcellulose and microcrystalline cellulose, preferably sodium carboxymethylcellulose and microcrystalline cellulose.

[0023] The granules according to the invention are useful to prepare pharmaceutical, nutraceutical and food formulations.

[0024] The concentration by weight of tetrahydrofolate in the formulations according to the invention ranges from 10% to 75%, preferably from 35% to 55%.

[0025] The concentration by weight of waxy excipient in the granules according to the invention ranges from 2% to 50%, preferably from 5% to 25%.

[0026] The concentration by weight of desiccant in the granules according to the invention ranges from 5% to 70%, preferably from 25% to 60%.

[0027] In a second aspect thereof, the invention provides a process for the preparation of granules which comprises: heating the mixture of tetrahydrofolates, waxy excipients and desiccants under stirring in a granulator until the melting point of the waxy excipient is reached; eliminating the residual moisture at low pressure; cooling the mixture under stirring to a temperature below the melting point of the waxy excipient; optionally sieving the granulate obtained in the preceding step.

[0028] In more detail, the granulation process involves the following steps: loading the active ingredient and the other excipients (desiccant and waxy binder) into the process chamber of a thermostated granulator such as the High Shear Mixer; activating the spindle movement and increasing the temperature of the process chamber until the melting point of the waxy excipient is reached; when the melting point of the waxy excipient has almost been reached (e.g. at 50°C), producing a vacuum in the process chamber to eliminate the residual moisture retained from the mixture of ingredients; when the mixture has melted, reducing the thermostat temperature to below the melting point of the mixture (e.g. to 25°C); reducing the spindle speed during the product cooling step; optionally, using a granulating mill fitted with a specific grid to sieve the granulate at the desired size.

[0029] The granules according to the invention are characterised by the stability of the active ingredients at temperatures of up to 40°C for 90 days or more, with relative humidity of 75% (RH), without any noteworthy variations in colour.

[0030] The invention is described in greater detail in the examples below.

[0031] Example 1 Evaluation of compatibility between desiccant excipients and 5-methyl- tetrahydrofolate (MTHF) glucosamine salt (Quatrefolic®).

[0032] The experiment is designed to establish the compatibility of the active ingredient (5MTHF glucosamine salt) with excipients commonly used as diluents, desiccants and binders for compression.

[0033] A sample of Quatrefolic® powder was mixed in equal parts (weight / weight) with each of the excipients listed in the table below, and the homogeneous mixture was then sampled to determine the moisture content (LOD). Each mixture was then distributed evenly in Petri dishes (3.5 g per dish) and photographed. All the dishes were incubated in a controlled-humidity climate chamber at 30°C, 65% RH. A dish containing Quatrefolic® alone was used as comparator.

[0034] At the end of the experiment, all the dishes were photographed again and, where possible, sampled to determine the degree of moisture.

[0035] Table 1 : excipients mixed with Quatrefolic®.

[0036] X: colour darker at end of experiment

[0037] = : colour substantially stable at end of experiment.

[0038] As the active ingredient absorbs moisture under the test conditions, becoming deliquescent, it was impossible to determine the water content at the end of the experiment; the same applies to some of the mixtures with excipients. Moreover, some compositions were discarded because they had acquired a much darker colour at the end of the experiment.

[0039] Surprisingly, some excipients normally used for other functions (e.g. as diluents, gelling agents etc.) exhibited the ability to reduce the natural hygroscopicity of the active ingredient. Among said excipients, microcrystalline cellulose, the two samples of sodium carboxymethylcellulose, corn starch and rice starch are preferable, because they maintain a stable colour.

[0040] Example 2 Evaluation of compatibility between excipients and 5 -methyl tetrahydrofolate calcium salt (Extrafolate-S®).

[0041] The procedure described in Example 1 was repeated with the same excipients, but using 5-methyl tetrahydrofolate calcium salt, Extrafolate-S®, as active ingredient.

[0042] The results are reported in the Table below.

[0043] Table 2: excipients mixed with Extrafolate-S®

[0044] X: colour darker at end of experiment than at the start

[0045] = : colour substantially stable at end of experiment.

[0046] In the case of Extrafolate-S, the mixtures with excipient maintained an acceptable colour; moreover, the increase in moisture of the active ingredient is minor. Once again, microcrystalline cellulose exhibits unexpected desiccant properties.

[0047] Example 3 Preparation of granulates of Quatrefolic® and excipients

[0048] Mixtures of Quatrefolic® with low-melting-point excipients are prepared, and granulated by a process that comprises: a. loading the excipients and Quatrefolic® into a mixer fitted with a thermostatic j acket; b. activating the stirring blade for the time required to distribute the various ingredients evenly in the mixture c. activating heating, maintaining the mixture under constant stirring, until the granulating excipient melts (60-70°C) d. applying a vacuum, while continuing to mix the mixture e. when the melting point of the granulating excipient (65-70°C) has been exceeded, deactivating the heating and maintaining the granulate under slight movement f. cooling until a product temperature lower than 30°C is reached, and optionally relieving the vacuum g. optionally, sieving the granulate to obtain the preferred size.

[0049] The granulates containing the compositions reported below were obtained in this way; the operating temperature did not exceed 70°C in any case. The residual moisture data at the start and end of processing are reported in Table 3.

[0050] Table 3: granulates prepared with Quatrefolic®.

[0051] To identify the best waxy granulating agent, the three excipients described above were examined. Before and after the granulation process, LOD% (Loss on Drying) analysis was conducted to establish whether the waxy granulating agent used, as well as binding the desiccant to the folate, protects the latter against environmental moisture.

[0052] The Quatrefolic® granulates use the excipients sodium carboxymethylcellulose / mono- and di glycerides of fatty acids in the proportion of about 4: 1

[0053] Example 4 Stability of the Quatrefolic® granulates

[0054] At the end of the preparation, all samples of the preceding example were photographed and placed in a climate chamber for an accelerated stability test; in this case they were stored at 40°C, 75% RH and at 53°C, 75% RH for 7 days in plastic envelopes, packaged in aluminium bags. At the end of the test they were photographed again to observe any differences in colour.

[0055] Table 4: residual moisture of granulates prepared with Quatrefolic®.

[0056] X: colour darker at end of experiment than at the start = : colour substantially stable at end of experiment. As demonstrated by the data obtained, Quatrefolic stored at 53°C, despite the protection provided by the aluminium bag, absorbs a considerable amount of moisture.

[0057] However, the same behaviour was not observed for the granulates according to the invention, which absorbed a minimal amount of moisture (granulate with stearic acid) or even a smaller amount of moisture than expected (granulate with mono / diglycerides of fatty acids).

[0058] The granulates prepared as described in Example 3 were analysed by HPLC to determine the active ingredient (5 -methyl tetrahydrofolate) content in an accelerated stability study at 53°C for 17 days. The results are shown in the Table below.

[0059] Table 5: HPLC analysis of granulates prepared with Quatrefolic®.

[0060] Assay value: 5-methyl tetrahydrofolic acid content compared with theoretical dose Purity: percentage area of 5MTHF in HPLC analysis.

[0061] The data reported above clearly demonstrate the importance of conducting the granulation process under high vacuum to stabilise the folate before binding the desiccant to it by melt granulation.

[0062] Example 5 Granulates prepared under vacuum, with desiccant.

[0063] Operating as described in Example 3, three mixtures were prepared having Quatrefolic® as active ingredient and two different desiccants and two low-melting-point waxy binders as excipients. The vacuum was applied at the mixing step, reaching a minimum of 25-35 mbars in each experiment.

[0064] The table below illustrates the residual moisture data (LOD %) at the start of the experiment (mixture of ingredients) and at the end of the process (homogeneous granulate).

[0065] Table 6: granulates prepared under vacuum with desiccant.

[0066] From the data shown in the table, it is clear that the process disclosed enables drying and granulation of the folate composition to be obtained simultaneously.

[0067] Example 6

[0068] The compositions described in Example 5 underwent stability tests in a climatic chamber at 53°C for rapid evaluation. The data for the non-granulated active ingredient were used as comparator. The results are shown in the tables below; the HPLC assay value is shown for the active ingredient, and the normalised assay value for a nominal amount of 400 micrograms of folate per dose is shown for the granulates.

[0069] Formula A: Quatrefolic® granulate with microcrystalline cellulose and stearic acid.

[0070] Formula B: Quatrefolic® granulate with CMC-Na and stearic acid.

[0071] Formula C: Quatrefolic granulate with microcrystalline cellulose and mono / diglycerides of fatty acids.

[0072] FORMULA D: Quatrefolic granulate with CMC-Na and mono / diglycerides of fatty acids.

[0073] Comparator: Quatrefolic® (non-granulated powder).

[0074] From the data reported in the Table, it is clear that the process disclosed produces a composition of folates which is more stable than that obtained by using the simple, nongranulated ingredient.

Claims

CLAIMS1. A granulate comprising a tetrahydrofolate, a waxy excipient and a desiccant.

2. A granulate according to claim 1 wherein the tetrahydrofolate is in acid, sodium, calcium or glucosamine salt form, amorphous or crystalline form.

3. A granulate according to claim 1 or 2 wherein the tetrahydrofolate is selected from D- glucosamine (6R,S)-tetrahydrofolate, D-glucosamine (6S)-tetrahydrofolate, D-glucosamine (6R)-tetrahydrofolate; D-galactosamine (6R,S)-tetrahydrofolate, D-galactosamine (6S)- tetrahydrofolate, D-galactosamine (6R)-tetrahydrofolate; D-glucosamine 5-methyl-(6R,S)- tetrahydrofolate, D-glucosamine 5-methyl-(6S)-tetrahydrofolate, D-glucosamine 5-methyl- (6R)-tetrahydrofolate; D-galactosamine 5-methyl-(6R,S)-tetrahydrofolate, D-galactosamine 5-methyl-(6S)-tetrahydrofolate, and D-galactosamine 5-methyl-(6R)-tetrahydrofolate.

4. A granulate according to any one of claims 1 to 3 wherein the waxy excipient is stearic acid or a mixture of mono / di glycerides of fatty acids.

5. A granulate according to any one of claims 1 to 4 wherein the desiccant is microcrystalline cellulose or sodium carboxymethylcellulose.

6. Pharmaceutical, nutraceutical or food formulations comprising a granulate according to claims 1 to 5 and optionally excipients.

7. A process for preparing granulates of claims 1-5 comprising: heating the mixture of folates, waxy excipients and desiccants under stirring in a granulator to the melting point of the waxy excipient; removing the residual moisture under low pressure; cooling the mixture under stirring to a temperature below the melting point of the waxy excipient; optionally sieving the granulate obtained from the previous step.

8. A process according to claim 7 wherein the tetrahydrofolate is a glucosamine salt.

9. A process according to claim 7 or 8 wherein the tetrahydrofolate is D-glucosamine 5- methyl-(6S)-tetrahydrofolate.