TAXIFOLIN FORMULATION COMPRISING THIAMINE

Abstract

The present invention relates to formulations of taxifolin with thiamine as a dosage form for oral administration, in particular as dietary supplements or foods for special medical purposes/FSMPs.

Claims

1. A formulation for oral administration comprising. (i) taxifolin or a pharmaceutically acceptable salt, derivative or prodrug thereof, (ii) thiamine or a pharmaceutically acceptable salt, derivative or prodrug thereof, and (iii) at least one excipient selected from a) β-cyclodextrin and derivatives thereof, and b) a basic (co)polymer of methacrylic acid and/or methacrylate, wherein taxifolin is present (a) as a complex with the β-cyclodextrin or derivative thereof, or (b) as a solid dispersion with the basic (co)polymer of methacrylic acid and/or methacrylate.

2. The formulation according to claim 1, wherein taxifolin is present as a complex with β-cyclodextrin or a derivative thereof, preferably in a molar ratio of about 1:1, and wherein derivatives are selected from substituted ß-cyclodextrins that are substituted on one or more hydroxyl groups, in particular on the C6 carbon atom of one or more glucose units, preferably with —O—C.sub.1-18 alkyl or —O—C.sub.1-18 hydroxyalkyl groups.

3. The formulation according to claim 1, wherein taxifolin is present as a solid dispersion with the basic (co)polymer of methacrylic acid and/or methacrylate, preferably in a weight ratio of taxifolin:basic (co)polymer of methacrylic acid and/or methacrylate in the range of 1:1 to 1:3, wherein the basic (co)polymer of methacrylic acid and/or methacrylate is preferably selected from Eudragit® E and Eudraguard Protect®.

4. The formulation according to claim 1, wherein thiamine is present as mononitrate or hydrochloride, preferably in microencapsulated form.

5. The formulation according to claim 1, wherein taxifolin is present in the form of an extract of larch wood, preferably an extract of Dahurian larch (Larix gmelinii), wherein the extract may optionally comprise one or more additional flavonoids, preferably aromadendrin and/or eriodictyol.

6. The formulation according to claim 5, wherein the taxifolin content in the extract is at least 88%, preferably 90-97%, more preferably 90-93%.

7. The formulation according to claim 1, wherein taxifolin is present in an amount of 50-500 mg, more preferably 50-150 mg, and/or thiamine is present in an amount of 0.1-250 mg, preferably 1-100 mg, particularly preferably 5-50 mg.

8. The formulation according to claim 1, wherein the taxifolin:thiamine ratio is in the range of 700:1 to 1:1, preferably in the range of 100:1 to 3:1, more preferably in the range of 20:1 to 5:1, and most preferably in the range of 10:1.

9. The formulation according to claim 1, further comprising a water soluble polymer, preferably selected from polyethylene glycol, polyvinyl alcohol, poloxamer and mixtures thereof.

10. The formulation according to claim 1, further comprising one or more pharmacologically acceptable excipients and/or carriers, and/or one or more further ingredients, preferably selected from choline, vitamins, in particular B vitamins, vitaminoids, minerals, trace elements, amino acids and pharmaceutically acceptable salts, derivatives and prodrugs thereof.

11. The formulation according to claim 1, wherein the formulation is present in the form of powder, granules, capsule, tablet, chewable tablet, effervescent tablet, coated tablet, sachet or solution/suspension, wherein the formulation may consist of one or more dosage units, wherein preferably at least one dosage unit is in the form of compressed material.

12. The formulation according to claim 1, for the use as a medicament.

13. The formulation according to claim 1, for the use in the prevention or treatment of alcohol intoxication, consequential conditions and secondary diseases associated with alcohol consumption, or alcoholism.

14. The formulation for use according to claim 13, wherein consequential conditions associated with alcohol consumption comprise hangovers.

15. The formulation for use according to claim 1, wherein consequential conditions and diseases associated with alcohol consumption comprise damage due to alcohol intoxication, in particular neurological damage as well as liver damage.

16. The formulation for use according to claim 13, wherein the treatment of alcoholism comprises alcohol dishabituation and/or alcohol withdrawal.

Description

FIGURES

[0060] FIG. 1a .sup.1H-NMR-Spectres of taxifolin complexes with various cyclodextrins

[0061] FIG. 1b relevant overlays

[0062] FIG. 1c Assigning the peaks of the spectres to the various taxifolin protons

[0063] FIG. 2: Labeled dissolution graph showing the dissolution behavior of cyclodextrin complexes [0064] 3: Taxifolin/β-CD complex [0065] 2: Eudragit® E solid dispersion [0066] 1: Taxifolin (reference)

[0067] FIG. 3: Thin layer chromatographic separation of various compositions with taxifolin and thiamine

EXAMPLES

1. H.SUP.1.-NMR Spectrometric Study of Various Cyclodextrin Complexes

[0068] In order to qualitatively detect the complex formation in aqueous solution, .sup.1H NMR spectroscopy was used. This allows the characteristic spectra of taxifolin and the cyclodextrin to be determined. When a complex is formed, a shift of certain signals occurs. In addition, the exact three-dimensional structure of the complex and the conformation of the flavonoid in the cyclodextrin cavity can be determined.

[0069] In order to achieve complex formation in solution, taxifolin and the respective cyclodextrin (β/CAVAMAX W7, HP-β or γ) were weighed at a molar ratio of 1:1, dissolved in D.sub.2O/DMSO (80/20 v/v) and stirred for 3 h at room temperature and 600 rpm. In the following, the sample was measured. The reference solutions (taxifolin, β-CD, HP-β-CD and γ-CD) were dissolved in D.sub.2O/DMSO (80/20 v/v) only and then measured. The results are shown in FIG. 1.

[0070] Discussion: Due to the signal shifts, the results clearly indicate complex formation in solution. However, the results can also be used to accurately predict the location of the flavonoid in the CD cavity. This is because the protons, which exhibit a signal shift due to complex formation are embedded in the CD cavity. Here, there are clear differences between β-CD/HP-β-CD and γ-CD.

##STR00009##

[0071] In β-CD and HP-β-CD, the signals of the protons H2′, H5′ and H6′ are shifted, which indicates that ring B is embedded in the CD cavity. This is also consistent with the prevailing view that β-CDs mainly include monocyclic aromatics because of their ring size. Based on .sup.1H-NMR spectroscopy, the following conformation of the flavonoid in the β-CD/HP-β-CD cavity can be predicted:

##STR00010##

[0072] However, it is interesting to note that in the HP-β-CD complex, the signals of the protons H6 and H8 combine to form a common peak. This is probably due to hydrogen bonding between the hydroxypropyl residue of cyclodextrin and various residues on ring A.

[0073] In γ-CD, in particular the signals of protons H6 and H8 are shifted, however, although less pronounced, also those of protons H2 and H3 are shifted. This indicates that rings A and C in part are embedded in the CD cavity. This is also consistent with the prevailing view that γ-CD mainly includes polycyclic aromatics due to the ring size. Based on .sup.1H-NMR spectroscopy, the following conformation of flavonoid in the γ-CD cavity can be predicted:

##STR00011##

[0074] The different position of the flavonoid in the CD cavity naturally influences the interactions of the flavonoid with thiamine. Only a complex with β-cyclodextrin can prevent undesired redox reactions during storage time, whereas γ-cyclodextrin has no influence on this.

2. Preparation of Cyclodextrin/Taxifolin Complexes

[0075] Different methods to prepare the complexes were examined and compared:

Spray Drying β-CD (SD β).

[0076] 10000 mg of taxifolin and 37300 mg of β-cyclodextrin were each weighed out in a molar ratio of 1:1 and placed in a shared beaker. Correspondingly 940 ml of distilled water (25° C., 5% w/v) was added to the β-CD-taxifolin mixture now, followed by stirring at 25° C. for 30 min with a high-shear mixer (3000 min-1) until a concentrated suspension was formed. This suspension was stirred for 24 h at 600 rpm and 25° C. in the absence of oxygen to complete the complex formation. The solution was vacuum filtered (0.45 μm membrane filter) to remove undissolved flavonoid and cyclodextrin residues, and the filtrate was then spray dried.

[0077] Parameters: V=900 ml, T(in)=125° C.; pump rate: 20%; aspirator: 100%, spray gas: 55 mm; T(out)=71° C.

Freeze Drying β-CD (FD β).

[0078] 1000 mg of taxifolin and 3730 mg of β-cyclodextrin were each weighed at a molar ration of 1:1 and placed in a shared beaker. Accordingly, to the β-CD/taxifolin mixture (5% w/v) 94 ml distilled water was then added and stirred for 30 min at 30° C. with a homogenizer (3000 min-1) until a suspension was formed. This suspension was stirred for 24 h at 600 rpm and 25° C. in the absence of oxygen to complete the complex formation. The solution was vacuum filtered (0.45 μm membrane filter) to remove undissolved flavonoid and cyclodextrin residues, and the filtrate was then cooled to −80° C. for 24 h in centrifuge tubes to freeze it. In the following, the tubes were placed in the freeze dryer and the pressure was adjusted to 0.05 mbar and the temperature was adjusted to −30° C. Under these conditions, the solution was freeze dried for 96 h.

Freeze Drying γ-CD (FD-γ).

[0079] 1000 mg of taxifolin and 4266 mg of γ-cyclodextrin were each weighed out at a molar ratio of 1:1 and placed in a shared beaker. Accordingly, 265 ml distilled water (2.5% w/v, 25° C.) was then added to the γ-CD-taxifolin mixture and stirred for 30 min with a homogenizer (3000 min-1) until a clear solution was formed. The solution was vacuum filtered (0.45 μm membrane filter) to remove undissolved flavonoid and cyclodextrin residues, and the filtrate was then cooled to −80° C. for 24 h in centrifuge tubes to freeze it. In the following, the tubes were placed in the freeze dryer and the pressure was adjusted to 0.05 mbar and the temperature was adjusted to −30° C. Under these conditions, the solution was freeze-dried for 96 h.

Phys. Mix 1:1 β-CD and γ-CD, Respectively.

[0080] Taxifolin and β-cyclodextrin or γ-cyclodextrin were weighed out at a molar ratio of 1:1 and mixed together in a mortar.

3. DSC Analyses of the Cyclodextrin Complexes

[0081] In order to be able to quantitatively determine the efficiency of the encapsulation method, various measurement methods are available. One very popular method is Differential Scanning calorimetry (DSC), which can be used to determine the residual content of the free active substance on the basis of characteristic endothermic peaks (approx. 240° C. for taxifolin). Since the active substance/cyclodextrin complex has a different decomposition or melting point, the absence of the “active substance peak” can thus be used to indirectly infer high encapsulation efficiency.

[0082] Therefore, the comparison of the sample peaks with the peaks of the pure active substance, the pure cyclodextrin and an equimolar physical mixture (Phys. Mix 1:1) is of particular importance. The latter serves as a reference for the samples, since in a physical mixture the drug is present in its free, uncomplexed form (encapsulation efficiency=0%). A complete absence of the drug peak at 240° C. corresponds to an encapsulation efficiency of 100%. Based on the area of the characteristic drug peak of the individual samples, they can be compared with each other and with the physical mixture. The main advantage of this measurement method is, on the one hand, the quite high precision and, above all, the possibility of measuring the samples in solid state. This prevents the complex equilibrium from being influenced or readjusted by water or other solvents.

[0083] In the case of the β-cyclodextrin samples SD β and FD β, characteristic drug peaks cannot be detected any more. Moreover, the intensity of the broad endothermic peak decreases significantly between 70° C. and 100° C. compared to the reference samples (Phys. Mix 1:1). This indicates that less water escapes from the β-cyclodextrin cavity during heating as it is occupied by the flavonoid. Therefore, from the DSC thermograms, it appears that in these samples the flavonoid is completely present as a β-CD complex and the encapsulation efficiency is close to 100%.

[0084] The thermogram of the γ-CD complex is fundamentally different from the thermograms of the β-CD complexes. Although the γ-CD complex sample does also not have a characteristic drug peak that coincides with the physical mixture (Phys. Mix. 1:1). This indicates complete encapsulation, as no free flavonoid can be detected anymore. But instead, this sample shows a broad peak in the range of 245° C.-250° C., whose area clearly exceeds that of the physical mixture. This peak indicates the decomposition of the supramolecular complex agglomerate. These agglomerates lead to poor dissolution behavior in that a “spring parachute effect” occurs due to the formation of supramolecular agglomerates, whereby the complex precipitates out of the solution after dissolution.

4. Saturation Solubility of Cyclodextrin Complexes in Distilled Water (HPLC)

[0085] The final most important point to compare the manufacturing methods is the solubility in distilled water. This is because the solubility of the complex has a direct influence on the bioavailability, since only dissolved complexes/active substances can pass through the epithelial cells of the GI tract. In addition, the samples were analyzed for related substances to detect possible breakdown of the active substance during the preparation process.

Method:

Reference Measurement (Taxifolin)

[0086] 10 mg of taxifolin (Lavitol® 98.9% purity) were added to a vial containing 5 ml distilled water to produce a saturated solution and was shaken for 60 min. The solution was then transferred to a vial by syringe with HPLC filter (0.22 μm) and then measured in undiluted state (HPLC DAD-254 nm).

Sample Measurement

[0087] 500 mg of the sample were added to a vial containing 6 ml distilled water to produce a saturated solution and was shaken for 60 min. In the following, the solution was transferred to a vial using a syringe with HPLC filter (0.22 μm), diluted 10:1 with distilled water to avoid supersaturation and then measured (HPLC DAD-254 nm). Based on the peak area taking into account the dilution, the taxifolin concentration was calculated in mg/ml.

Results:

[0088]

TABLE-US-00001 Solubility Rel. taxifolin Substances Name in mg/ml Peak Taxifolin reference 0.7496 3430343 Phys. Mix 1:1 β-CD 22.3834 — Phys. Mix 1:1 γ-CD 5.1944 SD β 24.8682 — FD β 23.7192 — FD γ 5.0152

[0089] The saturation solubility of the flavonoid was increased by inclusion complexes with β-CD and also, to a lesser extent, with γ-CD. This effect is particularly pronounced in the spray-dried SD β formulation. However, the saturation solubilities of the γ-CD complexes are significantly lower than those of the β-CD complexes.

[0090] The physical 1:1 mixtures also provided very good results, which can be attributed to the complex formation in solution. The physical mixture actually represents the maximum possible upper limit for solubility enhancement, since here the complex can form under maximum saturation, i.e., optimal conditions.

[0091] Nevertheless, the taxifolin concentration of the SD β formulation exceeds this value. This is probably due to supersaturation of the solution due to the small particle size and thus large surface area of the material.

5. Agglomeration in Complexes with γ-CD

[0092] An important point to consider, especially for γ-cyclodextrins, is possible agglomeration of the complexes. This problem has an enormous influence on the solubility and dissolution behavior of the product. In this case, the complexes arrange themselves into supramolecular complexes in a solid crystal structure. This massively reduces the surface area and also the hydration of the individual complexes. Consequently, even if there is high solubility of the complexes in theory, a turbid, characteristically opalescent suspension is formed.

[0093] In order to be able to demonstrate the solubility restriction by agglomeration properly, experiments were carried out with chaotropic substances. These substances prevent the formation of hydrogen bonds, which stabilize the complexes in the highly ordered structure. At the same time, the highly ordered structure of the solvent water is broken, and thereby hydrophobic effects are reduced.

[0094] Specifically, an opalescent suspension of a γ-CD complex was prepared (250 mg of γ-CD complex powder in 20 ml distilled water) again, and then 10 g urea was added. The suspension completely cleared after only 10 min of stirring at 600 rpm without increasing the temperature. By breaking up the aggregates, the solubility could be considerably increased.

[0095] These agglomerates do not occur in β-CD, so only β-CD is suitable to ensure optimal resorption of the flavonoid and thiamine. This is due to the instant-release behavior of this formulation, whereby negative interactions of the taxifolin with intestinal thiamine transporters can be reduced.

6. Ternary Complexes β-Cyclodextrin

[0096] In order to examine which water-soluble polymers are particularly suitable for improving the stability and dissolution capacity of flavonoid-cyclodextrin complexes, a screening was carried out. For this purpose, first, a supersaturated taxifolin/β-CD complex solution was prepared by adding an excess of equimolar taxifolin/β-CD complex and subsequent heating to 35° C. and filtering off. Subsequently, various water-soluble polymers were added (0.25% w/v) and choline bitartrate as well as L-carnitine tartrate were added (taxifolin:choline/carnitine cations ratio 1:0.85) to examine the influence of polymers/alkylammonium cations on complex formation and solubility, respectively. The solution was allowed to stand for 96 h and then recrystallization was compared with the reference solution.

TABLE-US-00002 Polymer used Recrystallization after (0.25% w/v) 96 h Remark No polymer (reference) Distinct Reference PVP K30 Very strongly distinct Deterioration PVP/VA Very strongly distinct Deterioration HPMC Very distinct Deterioration MC Distinct No change Carbomer Distinct No change Poloxamer 188 Not present Improvement PEG 6000 Not present Improvement PVA Merely present Improvement PEG/PVA (Kollicoat ® IR) Not present Improvement Xanthan Distinct No change Gellan Distinct No change Eudragit E100 Very distinct Solution in 0.1N HCl, Deterioration Chitosan Distinct Solution in 0.1N HCl, no change Pectin Distinct No change Na-CMC Very distinct Deterioration Alginic acid Distinct No change Collagen Hydrolysat Distinct No change Maltodextrin Distinct No change Choline bitartrate Not present Improvement L-Carnitine tartrate Distinct No change

[0097] The results clearly show that polymers with prominent H-bridge acceptors (PVP, PVP/VA, Eudragit E100 and cellulose derivatives) lead to breakdown due to a too strong interaction with the drug. The polymer-drug complex precipitates and Ks decreases. In addition, no interaction could be detected for typical biopolymers, either with regard to the active substance or with the cyclodextrin, and, therefore, the dissolution behavior of the active substance is not changed.

[0098] In contrast, PEG 6000, Kollicoat IR and Poloxamer 188 are of particular interest. These polymers are composed of ethylene oxide blocks and show very promising properties. The interaction with the hydroxy groups of the flavonoid are not so strong that precipitation occurs. At the same time, the polymers also interact with the hydroxy groups of the cyclodextrin. This increases the complex stability. The same can be seen with polyvinyl alcohol (PVA). However, the interaction of the hydroxyl groups of the polymer with the flavonoid and the cyclodextrin is less pronounced than with the ethylene oxide polymers.

[0099] This showed that the use of water-soluble polymers can increase the complex stability and improve the dissolution behavior.

[0100] Furthermore, a significant improvement was observed when choline bitartrate was added, however, this was not the case for the structurally related L-carnitine.

[0101] Thereby, it could be demonstrated that not every alkylammonium cation, but only choline cations are suitable for this purpose. This can be explained by the structure-breaking influence of the alkylammonium group on the hydrogen bonds of the solvent and the associated single-salt effect. As regards carnitine, on the other hand, the hydroxyl group as well as the carboxyl group act as structure-forming elements which can form H-bridges and counteract the effect of the alkylammonium group. It was found that in choline compounds, on the other hand, the structure-breaking component predominates and leads to an improvement in solubility and physicochemical properties, respectively, especially in taxifolin/β-CD formulations as well as in solid dispersions of taxifolin/basic polymethacrylate.

[0102] In order to achieve this positive effect, it is already sufficient to physically mix or combine the water-soluble polymer/choline compound and the final flavonoid/CD complex in an oral dosage form, since a ternary complex is formed after dissolution in solution and the positive effect of the choline cation unfolds, respectively. However, integration of the polymer can also occur before or during complex formation. For example, small amounts of the polymer can be added to the complex solution before spray or freeze drying.

7. Preparation of Solid Dispersions with Eudragit® E

Common Solvent Evaporation 2:1 (CSE 2:1)

[0103] 2000 mg of Eudragit® E100 was weighed out and dissolved in 30 ml ethanol. Subsequently, 1000 mg of taxifolin were weighed out and dissolved in 15 ml ethanol. Hereafter, both solutions were mixed and stirred at 600 rpm and at room temperature for 30 min. At last, the clear, light amber solution was dried in a dry place protected from light. After powdering, the solid dispersion was stored airtight and protected from light.

XRD Analysis

[0104] The XRD method is considered the method of choice for detecting the complete, amorphous embedding of an active substance in the polymer matrix. For this purpose, the crystallinity of the sample is determined, which provides conclusions about the arrangement of the molecules of the active substance. Since contrary to the active substance, the polymer matrix is amorphous, crystalline peaks indicate incomplete embedding. If, however, the sample is amorphous, a solid solution is present.

[0105] In addition, amorphous samples usually show significantly better dissolution behavior than crystalline samples, which is why an increase in bioavailability is possible with an amorphous sample.

[0106] Result: It can be taken from the diffraction diagrams that both taxifolin and the physical mixture of taxifolin/Eudragit® E100 are crystalline. As expected, the polymer is amorphous. The physical mixture also shows superimposed X-ray diffraction patterns of taxifolin and Eudragit® E100. Furthermore, all three formulations are amorphous and do not differ from the reference polymer.

[0107] Discussion: The results of the XRD analyses indicate that a solid dispersion is present at CSE 2:1, with the flavonoid taxifolin being fully embedded in the polymer matrix.

FITR Analyses

[0108] FT-IR spectroscopy is applied in order to analyze the molecular interactions between the functional groups of the flavonoid and the basic polymethacrylate.

[0109] Initially, the peak is broadened at 3435 cm.sup.−1, which is due to the presence of a protonated ammonium group as the R—N+-H stretching vibration absorbs in exactly this region, thus broadening the band. This indicates that the tertiary amino group of the polymer is present in protonated form. Also, as regards the peaks at 2770 cm.sup.−1 and 2820 cm.sup.−1 a significant loss of intensity or even complete disappearance occurs, which implies that the tertiary amino group of the polymer is involved in ionic interactions with the flavonoid.

[0110] There exist strong ionic interactions between the tertiary amino groups of the polymer and the phenolic hydroxyl groups of the flavonoid, whereby the tertiary amino groups are protonated to cationic ammonium groups and the hydroxyl groups of the flavonoid are deprotonated to resonance-stabilized phenolate ions.

8. Solubility of the Solid Dispersion with Eudragit E

[0111] The last most important point in order to compare the preparation methods is the solubility in simulated gastric juice. The solubility of the complex has a direct influence on the bioavailability, because only dissolved active substances can pass through the epithelial cells of the GI tract.

Reference Measurement (Taxifolin)

[0112] 10 mg of taxifolin (Lavitol® 98.9% purity) was added to a vial containing 5 ml 0.1N HCl to produce a saturated solution and was shaken for 60 min. Subsequently, the solution was transferred to a vial by means of a syringe with HPLC filter (0.22 μm) and then measured.

Sample Measurement

[0113] A saturated solution of the sample was prepared in 0.1 molar HCL solution at room temperature. In the following, the solution was transferred to a vial by means of a syringe with an HPLC filter (0.22 μm), diluted accordingly, and the taxifolin concentration of the solution was determined by HPLC.

TABLE-US-00003 Initial Solubility weight Dilution of taxifolin Name mg factor in mg/ml Taxifolin reference 10.34 — 0.6927 CSE 2:1 1331.63 66.6 15.00

[0114] Discussion: By formulating a solid dispersion with basic polymethacrylates the saturation solubility of taxifolin can be increased. This is particularly due to the fact that the flavonoid is embedded in amorphous form in the polymer in all three formulations, which is confirmed by both FT-IR and XRD analyses.

9. Dissolution Behavior of Formulations with Cyclodextrin or Basic Polymethacrylate

[0115] In order to examine the dissolution behavior of the final formulations, dissolution studies of the cyclodextrin and eudragitol formulations against pure taxifolin were carried out. Here, the instant-release formulations are expected to significantly improve the dissolution behavior of the flavonoid, as the pure taxifolin dissolves quite slowly due to its stable crystalline structure and low water solubility.

[0116] Due to the solid dispersion with Eudragit® E the crystalline structure is dissolved (see XRPD analyses) and thus water solubility is increased. In the case of the CD complexes, the crystalline structure is also dissolved by encapsulating each individual taxifolin molecule, and at the same time the water solubility and wettability are increased by the CD acting as a “Trojan horse”. Both should lead to an improvement in dissolution behavior.

[0117] The instant-release formulation is considered optimal when 85% of the drug has dissolved within the first 15 min. Since gastric emptying when fasting is a first order reaction (50% emptying in 10-20 min), 85% dissolution within the first 15 min, it can be assumed that the formulation behaves like a solution and, thus, behaves optimally. Thereby, optimal absorption behavior of thiamine and taxifolin can be ensured when administered at the same time.

[0118] Method: In order to determine the dissolution behavior, the usual pharmacopoeial procedure was chosen.

[0119] USP Apparatus II (paddle); 100 rpm; medium: 500 ml 0.1N HCl; 2 vessels per sample (N=2); 7 sampling points: 0 min, 5 min, 10 min, 15 min, 20 min, 30 min, 60 min; weight: formulation as powder corresponding to 100 mg of taxifolin; detection by HPLC.

[0120] The following formulations were tested: [0121] taxifolin (Ametis Lavitol®, 98.8% purity) [0122] Eudragit® E solid dispersion formulation [0123] β-cyclodextrin formulation

[0124] Here, the pure taxifolin represents the reference value.

Results:

[0125]

TABLE-US-00004 Taxifolin release (reference) Initial Sampling weight Mean Time Vessel mg Release Release 5 min 1 106.52 28.2% 29% 2 108.20 29.2% 10 min 1 106.52 46.4% 46% 2 108.20 44.8% 15 min 1 106.52 61.1% 60% 2 108.20 58.7% 20 min 1 106.52 70.4% 69% 2 108.20 67.5% 30 min 1 106.52 80.8% 79% 2 108.20 77.4% 60 min 1 106.52 90.8% 92% 2 108.20 93.1% Initial Sampling weight Mean Time Vial mg Release Release Release taxifolin/β-CD complex 5 min 1 586.93 100.1% 100% 2 587.81 100.5% 10 min 1 586.93 100.6% 100% 2 587.81 100.2% 15 min 1 586.93 103.2% 103% 2 587.81 103.1% 20 min 1 586.93 100.1% 100% 2 587.81 100.5% 30 min 1 586.93 100.5% 101% 2 587.81 101.1% 60 min 1 586.93 100.3% 101% 2 587.81 101.3% Release Eudragit ® E solid dispersion 5 min 1 301.21 82.2% 82.2%  2 303.27 (150.0%) 10 min 1 301.21 84.8%  85% 2 303.27 84.5% 15 min 1 301.21 86.6%  86% 2 303.27 86.2% 20 min 1 301.21 85.5%  85% 2 303.27 84.4% 30 min 1 301.21 85.3%  85% 2 303.27 84.9% 60 min 1 301.21 85.0%  85% 2 303.27 84.8% Note: At time of sampling 5 min, vial 2, a particle was drawn through the filter, which dissolved before the measurement. Therefore, this measurement point was therefore not taken into account.

[0126] Discussion: Taxifolin in its free form shows a typical dissolution behavior with continuous release. The results are shown in FIG. 2. However, the release after 15 min is only 60% and thus does not meet the requirement of an instant-release formulation (min. 85% after 15 min). This means that reduced thiamine resorption is to be expected. Both the solid dispersion in Eudragit® E and the β-cyclodextrin formulation meet the requirements and are, therefore, considered optimal instant-release formulations.

[0127] β-CD releases the flavonoid very quickly and already achieves 100% release at the first measuring point. Furthermore, there is no recrystallization in the sense of a “spring parachute effect” as occurs in γ-CD complexes, but the release is constantly 100%.

[0128] The Eudragit® E formulation also achieves a very rapid release of the flavonoid, with 82.2% of the flavonoid already in solution at the first measuring point. Here, too, there is no recrystallization and no precipitation of the taxifolin from the solution, but the release of the taxifolin is limited to a maximum of 85%.

[0129] Both formulations thus fulfil the requirements for optimal instant-release formulations, which allow the formulation of a taxifolin/thiamine combination.

[0130] In addition, both formulations allow good storage stability by unwanted redox reactions between the taxifolin and the thiamine during the storage period being able to be avoided. This is due to the inclusion of the catechol group in the β-CD formulation, while ionic interactions between the hydroxyl groups of the catechol group and the aminoalkyl moiety of the polymer are decisive in the solid dispersion in basic polymethacrylate.

10. Stability Experiments Thiamine

[0131] Stability experiments were carried out in order to investigate the interactions between thiamine and taxifolin and the influence of galenic formulations in more detail. Contrary to the breakdown of taxifolin, the breakdown of thiamine is not accompanied by a color change and is therefore more difficult to detect. However, the possible breakdown products, in particular thiamine disulfide as well as under certain circumstance also thiochrome, have completely different physicochemical properties, which can be exploited by thin-layer chromatography.

[0132] Method: First, four mixtures were prepared in a mortar consisting of I) 1000 mg taxifolin and 127 mg thiamine HCl II) 5266 mg taxifolin/γ-CD complex (FD-γ) and 127 mg thiamine HCl III) 4730 mg taxifolin/β-CD complex (FD β) and 127 mg thiamine HCl and IV) 3030 mg taxifolin/Eudragit® E CSE 2: 1 and 127 mg thiamine HCl, wherein each formulation contained 1000 mg taxifolin and the thiamine HCl corresponded to 100 mg thiamine (taxifolin:thiamine ratio 10:1).

[0133] The mixtures were placed in glass petri dishes and stored open in a climate-controlled cabinet at 40° C. and 75% humidity for 3 months (Accelerated Stability Test).

[0134] In the following, the samples were divided in half and the amount corresponding to 50 mg of thiamine was weighed out in each case (564 mg of taxifolin/thiamine, 2697 mg of FD-γ/thiamine, 2429 mg FD β/Thiamine and 1579 mg of Eudragit® E CSE 2:1/thiamine). Subsequently, each sample was extracted with 50 ml of solvent having a temperature of 45° C. (ethanol for the taxifolin-pur, FD-γ and FD β mixture and petroleum ether for the Eudragit® E CSE 2:1 mixture) in order to dissolve the thiamine breakdown products, and then filtered. The final solutions contained the equivalent amount of breakdown product of 50 mg of thiamine/50 ml solvent.

[0135] Besides, a reference solution was prepared containing the equivalent concentration of thiamine disulfide (53 mg of thiamine disulfide hydrate in 50 ml ethanol and petroleum ether, respectively).

[0136] In the following, silica gel DC plates were loaded with 5 μl per sample each and placed in a DC chamber along with a running medium consisting of ethanol:acetone:acetonitrile 4:2:1. The plates were dried thereafter and sprayed with Dragendorff reagent. The Dragendorff reagent was chosen because it specifically stains basic tertiary amines due to the potassium tetraiodobismuthate complex it contains. This allows selective staining of thiamine as well as its breakdown products thiamine disulfide and thiochrome.

[0137] Results: Thin layer chromatography resulted in a clean separation of the substances (FIG. 3), in particular a clear, semi-quantitative detection of thiamine disulfide. Taxifolin was entrained with the running medium and is visible near the running centerline due to oxidation in air, but thiamine HCl remained at the starting line due to its hydrophilicity instead. Thiamine disulfide was cleanly separated and had an Rf value in the optimum range of 0.22 to 0.27.

[0138] Thiamine disulfide could be detected in the taxifolin/thiamine and FD-γ/thiamine mixtures, whereby less breakdown was visible in the FD-γ than in the pure taxifolin/thiamine sample. In contrast, no thiamine disulfide or other breakdown product was present in the FD β sample or in the Eudragit® E CSE 2:1 sample. Thiochrome could not be detected in any sample under UV light.

[0139] Discussion: The taxifolin formulations with β-CD and basic polymethacrylate were the only formulations which were able to inhibit the breakdown of thiamine to thiamine disulfide. This is due to the encapsulation of the catechol group by β-CD or the ionic interactions between taxifolin and the basic polymethacrylate. The sample containing Eudragit® E had to be extracted with petroleum ether, as otherwise the polymer would also have dissolved and been stained by the Dragendorff reagent. By this extraction no polymer, thiamine HCl or taxifolin became visible in the Eudragit® E sample, as these are too polar for the extracting agent petroleum ether, in contrast to the lipophilic thiamine disulfide, which could be extracted in the reference solution. In addition, the thiamine HCl in the taxifolin/thiamine sample and in the FD-γ/thiamine sample runs slightly further than in the FD β sample. This is probably due to interactions between thiamine and the β-CD, which increase the hydrophilicity of the vitamin.

11. Stability Examination Taxifolin

[0140] In order to investigate the stability of the flavonoid taxifolin and the influence of thiamine and β-CD, experiments were also carried out in this regard. Since taxifolin forms red-brown oligomers upon breakdown, the detection is quite straightforward to carry out.

[0141] Method: Three aqueous solutions were prepared in beakers containing I) 100 mg taxifolin in 150 ml distilled water II) 100 mg of Taxifolin+13 mg of thiamine HCl in 150 ml distilled water and III) 100 mg taxifolin+13 mg of thiamine HCl+373 mg of β-CD in distilled water. Samples were stored open and protected from light at room temperature and the color of the solution was checked every 24 h.

[0142] Results: The results are summarized in the following table.

TABLE-US-00005 Time of Color of Sample color change solution Taxifolin (Ref.) 48 h Red-brown Taxifolin/thiamine 48 h Yellowish, brown staining after 72 h Taxifolin/thiamine/β-CD 96 h Yellow-brown

[0143] A delay in taxifolin oxidation by addition of thiamine or β-CD can be seen, with oxidation decreasing in the order taxifolin (ref.)>taxifolin/thiamine>taxifolin/thiamine/β-CD.

[0144] Discussion: Addition of thiamine can delay the breakdown of taxifolin, with thiamine disulfide and various breakdown products and/or adducts being formed in the process, causing the yellow coloration of the solution. This also confirms the beneficial combination in vivo, wherein thiamine can reduce oxidized taxifolin, and thus prolongs the effect. The addition of R-CD now delays taxifolin oxidation in the first step, which results in in delayed oxidation of thiamine.

12. Oral Dosage Form with β-Cyclodextrin, Thiamine and Choline

[0145] Dosage corresponds to 1 tablet, ingredients per tablet, oblong shape:

TABLE-US-00006 Ingredient Dosage/Tablet (mg) Taxifolin/β-CD complex spray-dried 500 (containing 20% taxifolin from larch extract) thiamine HCl 13 choline bitartrate 207 Microcrystalline cellulose 118 Polyethylene glycol 6000 25 Magnesium stearate 6

[0146] The parameters of the finished tablet are as listed below:

TABLE-US-00007 Parameter Result Height 6.05 mm Breadth 8.5 mm Depth 20 mm Mass 869 mg Pressure force for production 12 kN Tablet hardness (longitudinal) (N = 10) >280N Disintegration time (N = 6) 16.5 min. Abrasion/Friability (N = 10) 0.023%

[0147] The results illustrate that the taxifolin formulation with R-CD, choline and thiamine can also be easily produced on a large scale, whereby the parameters are in the optimal range. In addition, the thiamine can now be in microencapsulated form, for example.

13. Formulation with Basic Polymethacrylate and Thiamine

[0148] Dose corresponds to 1 hard capsule, ingredients per hard capsule size 0 (gelatin):

[0149] 200 mg of basic methacrylate copolymer (Eudraguard Protect®, Evonik Nutrition & Care GmbH), 100 mg of taxifolin-rich extract from Larix gmelinii (Lavitol® from Ametis JSC, taxifolin content 90.5%), 20 mg of silicon dioxide, 13 mg of thiamine hydrochloride (Food Grade, BASF).

[0150] The formulation with basic polymethacrylate is also easy to implement and can be produced on a large scale.

14. Formulation with β-Cyclodextrin+Thiamine Microencapsulated

[0151] Dose corresponds to 1 tablet, ingredients per tablet, oblong shape 21 mm×9 mm:

[0152] 740 mg of β-cyclodextrin (Food Grade, CycloLab R&D Ltd.), 200 mg of taxifolin-rich extract of Larix gmelinii (Lavitol®, Ametis JSC, taxifolin content 90.5%), 35 mg of silica, 30 mg of thiamine microencapsulated (33.3% thiamine HCl+66.6% carnauba wax white), 20 mg of polyethylene glycol 6000.