GREEN TEA CATECHINS EUTECTIC SYSTEM

Abstract

The present invention concerns a stable liquid eutectic system of an active ingredient chosen from catechins, anthocyanins, and procyanidins, and pharmaceutical, dermo-cosmetic or nutraceutical compositions comprising said eutectic system.

Claims

1. A stable liquid eutectic system comprising: an active ingredient chosen from catechins; a choline salt; a polar protic solvent or aprotic polar solvent which is pharmaceutically, dermo-cosmetically or nutraceutically acceptable; wherein the molar ratio between the active ingredient, the choline salt, and the solvent is 0.25-3.5:1:5-75; and wherein the concentration of the active ingredient is at least 100 mg/mL.

2. The stable liquid eutectic system according to claim 1, said system consisting of: an active ingredient chosen from catechins; a choline salt; a polar protic solvent or aprotic polar solvent which is pharmaceutically, dermo-cosmetically or nutraceutically acceptable; wherein the molar ratio between the active ingredient, the choline salt, and the solvent is 0.25-1.5:1:5-50; wherein the concentration of the active ingredient is at least 100 mg/mL.

3. The stable liquid eutectic system according to claim 1, wherein the active ingredient is green tea catechins, chosen from epicatechins, epigallocatechin, epicatechin-3-gallate, epigallocatechin gallate, or a mixture thereof.

4. The stable liquid eutectic system according to claim 1, wherein the choline salt is chosen from choline chloride, choline hydroxide, choline tartrate, choline dihydrogen citrate, choline acetate, and choline sulphate.

5. The stable liquid eutectic system according claim 1, wherein the solvent is an aprotic solvent chosen from the classes of solvents of sulfoxides, ketones, esters, amides, dimethyl sulfoxide, dimethylacetamide, or a polar protic solvent chosen from water, ethanol, propylene glycol, glycerol.

6. The stable liquid eutectic system according to claim 1, wherein said system is epigallocatechin gallate—choline chloride or choline tartrate—an aprotic polar solvent in a molar ratio of 1.0-1.5:1:6-10, preferably in a molar ratio of 1.0-1.3:1:6-10.

7. The stable liquid eutectic system according to claim 1, wherein said system is epigallocatechin gallate—choline chloride or choline tartrate—a polar protic solvent in a molar ratio of 0.25-1:1:15-50.

8. A pharmaceutical, dermo-cosmetic or nutraceutical composition, comprising a stable liquid eutectic system as defined according to claim 1, and a pharmaceutically, dermo-cosmetically or nutraceutically acceptable carrier.

9. The pharmaceutical, dermo-cosmetic or nutraceutical composition according to claim 8, comprising: 0.1% -15.0% wt, compared to total weight of composition, of the eutectic system, and; a pharmaceutically, dermo-cosmetically or nutraceutically acceptable polar aprotic co-solvent or polar protic co-solvent.

10. The pharmaceutical, dermo-cosmetic or nutraceutical composition according to claim 9, comprising: 0.1% -15.0% wt, compared to total weight of composition, of the eutectic system; 0.1-1.0 mmol/l of at least one chelating agent; water or water saline buffer; optionally at least one another antioxidant; and wherein said solution is at a pH in a range of 2.0-6.0, preferably 3.0-5.0.

11. The pharmaceutical, dermo-cosmetic or nutraceutical composition according to claim 8, said composition further comprises vitamin C.

12. The pharmaceutical composition according to claim 8, said composition is formulated in a self-emulsifying drug-delivery system, a self-micro emulsifying drug-delivery system or an oil-free system.

13. The pharmaceutical composition according to claim 8, for use as a medicament in the treatment of cancers, cardiovascular disorder, diabetes, neurodegenerative diseases, or dermatological diseases.

14. The composition according claim 8, said composition is formulated to be a semi-solid or pasty topical composition.

15. An oral care product's composition according to claim 8, said composition is formulated to be a liquid, semi-solid or pasty topical composition.

16. A unit dose formulated with a stable liquid eutectic system comprising: an active ingredient chosen from catechins; a choline salt; a polar protic solvent or aprotic polar solvent which is parmaceutically, dermo-cosmetically or nutraceutically acceptable; wherein the molar ratio between the active ingredient, the choline salt, and the solvent is 0.25-3.5:1.5-75; and wherein the concentration of the active ingredient is at least 100 mg/mL, or of the pharmaceutical, or dermo-cosmetic composition or a nutraceutical composition according to claim 8.

17. A food or a drink comprising a stable liquid eutectic system according to claim 1.

18. The food or the drink according to claim 17, which is a product of a coffee capsule, or a tea capsule, or any type of ready-to-use powder concentrates to be dissolved in water or other beverages, said product comprising at least 10 mg of epigallocatechin gallate per gram of product, from the eutectic system according to claim 1.

19. A food or a drink comprising a nutraceutical composition according to claim 8, said food or drink comprises at least 10 mg of epigallocatechin gallate, from a eutectic system comprising: an active ingredient chosen from catechins; a choline salt; a polar protic solvent or aprotic polar solvent which is pharmaceutically, dermo-cosmetically or nutraceutically acceptable; wherein the molar ratio between the active ingredient, the choline salt, and the solvent is 0.25-3.5:1:5-75; and wherein the concentration of the active ingredient is at least 100 mg/mL.

Description

FIGURES

[0193] FIG. 1 compares HPLC profiles of an aqueous EGCG composition INV1 (formulation showed in table 2 below) exposed to alkali conditions at initial time (1A) and after a 6-day exposure (1B) and that of reference EGCG water suspension also after a 6-day exposure in the same stress conditions (1C).

[0194] FIG. 2 compares HPLC profiles of the EGCG composition INV1 exposed to 70° C. at initial time (2A) and after a 6-day exposure (2B) and that of reference EGCG water suspension also after a 6-day exposure in the same stress conditions (2C).

[0195] FIG. 3 compares HPLC profiles of the EGCG composition INV1 exposed to photolysis conditions at initial time (3A) and after a 6-day exposure (3B) and that of reference EGCG water suspension also after a 6-day exposure in the same stress conditions (3C).

[0196] FIG. 4 shows ATR-IR spectra of eutectic system formed by EGCG, choline and DMSO (4A) and that of EGCG powder (4B).

[0197] FIG. 5 compares in vitro cumulative percentage of EGCG permeation over time of a EGCG cream formulation INV2 (represented in the figure by diamond) and that of control formulation (represented in the figure by square) through Franz diffusion cells membrane.

[0198] FIG. 6 shows the evolution over time of plasma EGCG concentrations in primates after oral administration of a control formula (capsules dosed to administer 27 mg EGCG per kg) and the aqueous EGCG composition INV1 (27 mg EGCG per mL).

EXAMPLES

Example 1: EGCG Solubility and Stability

1. Materials and Methods

1.1 Tested EGCG Composition

[0199] An EGCG aqueous composition according to the present invention comprising 26.7 mg/mL of EGCG is prepared by using an EGCG-choline chloride-DMSO eutectic system of the present invention. This composition is named composition INV1 in the description.

[0200] The reference standard suspension of EGCG used for the stress stability testing, or long-term and accelerated stability studies is a water suspension containing uniquely EGCG at a concentration of 26.6 mg/mL. The standard suspension is extemporaneously prepared by dispersing an appropriate amount of an isolated EGCG extract (>97% w/w of the extract dry) in distilled water to afford the said concentration.

1.1.1 EGCG-Choline Chloride-DMSO Liquid Eutectic System Formula

Formulation

[0201] A EGCG-choline chloride-DMSO liquid eutectic system according to the invention is prepared according to the formulation given in Table 1 below.

TABLE-US-00001 TABLE 1 example of the formula of a liquid eutectic system EGCG-choline chloride-DMSO Ingredients For 1 mL volume For 1 l volume EGCG 595 mg 0.595 kg DMSO 507 mg 0.507 kg Choline chloride 140 mg 0.140 kg

[0202] EGCG used for preparing said system is an isolated EGCG extract (>97% w/w of the extract dry). Prior to its use, the active substance was thoroughly controlled to ensure its quality comply with Monograph 2668 (Pharmeuropa June 2017).

Protocol of Preparation

[0203] An example of manufacturing process of said liquid eutectic system is given below.

Stage 1: Preparation of the EGCG-Choline Chloride-DMSO Liquid Eutectic System

[0204] Step1: a suitable quantity of DMSO according to Table 1 is poured into a 1-liter Pyrex glass vessel.

[0205] Step2: a suitable quantity of choline chloride as mentioned in table 1 is added to the solvent and the mixture is stirring until choline chloride solubilizes.

[0206] Step3: an appropriate amount of EGCG according to table 1 is added to the mixture from step 2 while maintaining stirring until any particles or lumps are completely dissolved.

[0207] Stage 2: Filling Into Multiple-Dose Bottle

[0208] The solution is filled into 15 mL single-dose amber glass bottles. Filled bottles are closed with caps with a polyethylene seal.

1.1.2 Aqueous EGCG Composition Formula at 26.7 mg/mL

Formulation

[0209] The liquid eutectic system obtained before is used for preparing the aqueous EGCG composition named INV1 comprising 26.7 mg/mL of EGCG. The formulation of this composition is given in Table 2 below.

TABLE-US-00002 TABLE 2 example EGCG 26.7 mg/mL solution formula Quantities 26.67 mg/mL EGCG aqueous solution Unit formula Formula for 20 L (15 mL) of solution EGCG 400 mg 0.533 kg DMSO 340 mg 0.453 kg Choline chloride  95 mg 0.127 kg Citric acid  4.5 mg 0.006 kg Purified water Qs 15 mL Qs 20 L

[0210] EGCG used for preparing said solution is an isolated EGCG extract (>97% w/w of the extract dry). Prior to its use, the active substance was thoroughly controlled to ensure its quality comply with Monograph 2668 (Pharmeuropa June 2017).

Protocol of Preparation

[0211] An example of manufacturing process of said aqueous solution is given below.

Stage 1: Preparation of the EGCG-Choline Chloride-DMSO Liquid Eutectic System

[0212] Step1: a suitable quantity of DMSO according to Table 2 is poured into a 20-liter Pyrex glass vessel.

[0213] Step2: a suitable quantity of choline chloride as mentioned in table 2 is added to the solvent and the mixture is stirring until choline chloride solubilizes.

[0214] Step3: an appropriate amount of EGCG according to Table 2 is added to the mixture from step 2 while maintaining stirring until any particles or lumps are completely dissolved.

Stage 2: Aqueous Solution Preparation

[0215] Step1: purified water is added to reach ⅔ of the final volume while maintaining agitation until a clear and homogeneous aqueous solution is obtained.

[0216] Step2: an appropriate amount of citric acid according to Table 2 is added to the mixture from step 1. The mixture is stirred until the citric acid is completely dissolved.

Stage 3: Filtration

[0217] Filtration is carried out by a 1pm Millipore® filtration cartridge. The filtered solution is stocked in a 20 liter-stainless steel tank with nitrogen inlet for inerting the solution and the headspace of the container.

Stage 4: Filling Into Multiple-Dose Bottle

[0218] The solution is filled into 15 mL single-dose amber glass bottles. Filled bottles are closed with caps with a polyethylene seal.

1.2 Stress Stability Testing

[0219] 1 mL of the reference standard suspension in water and 1 mL of the aqueous composition INV1 were exposed to alkali, thermal and photolysis stress conditions up to 6 days. These conditions, described in Table 3, were chosen to assess the stability of EGCG because they are known to cause its degradation. At the end of exposure, the solutions were diluted at 1/100 in distilled water and analyzed by high performance liquid chromatography (HPLC). The results are displayed in FIGS. 1-3.

TABLE-US-00003 TABLE 3 Stress conditions to assess EGCG stability Stress Exposure conditions Descriptions duration Basic Dilution of samples to be tested at Up to condition ½ in a 0.1 mol/L NaOH solution. 6 days Photolysis The source used produces an output Up to conditions similar to the D65/ID65 emission 6 days standard. These irradiation conditions provide an overall illumination of not less than 1.2 million lux hours. Thermolysis 70° C. Up to conditions 6 days

1.3 Long-Term and Accelerated Stability Testing

Storage Conditions and Sampling Protocols

[0220] The tested batch is the aqueous composition INV1 prepared according to the formulation aforementioned in Table 2.

[0221] Reference standard solution of EGCG is a water solution containing uniquely EGCG at a concentration of 0.26 mg/mL.

[0222] The tested batch has been stored in accordance with ICH (The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) guideline Q1A(R2) (https://www.ich.org/products/guidelines/quality/quality-single/article/stability-testing-of-new-drug-substances-and-products.html). Details of the sampling time points for each condition are presented in Table 4. It is diluted 1/100 in distilled water prior to HPLC analysis.

TABLE-US-00004 TABLE 4 Sampling and storage protocol for primary batch studies Storage conditions/container Time 2-8° C. 25° C./60% RH (months) Pack Pack 1 + + 2 + + 3 + + 4 + + 5 + + 6 + + 9 + + 12 + + 18 + + Pack: 15 mL-single-dose amber glass bottle (Type III) with a polyethylene cap with polyethylene seal.

Stability Tests and Acceptance Limits

[0223] Monitoring appropriate chemical, physical and microbial characteristics during the stability studies and at each time point has allowed assessing the stability of EGCG aqueous solution.

[0224] The chemical stability of EGCG was evaluated by HPLC, validated assay method as a stability indicating method. Said procedure is suitable for the EGCG investigational product assay using an external standard. The assay of EGCG is achieved by comparing the response of a sample solution with the response of EGCG reference standard solution prepared at a similar nominal concentration and analysed in the same way. The sample and reference standard solutions are analysed by gradient reversed phase HPLC and UV detection using a suitable column and chromatography conditions.

[0225] HPLC assay uses a liquid chromatograph fitted with a UV detector (variable wavelength or diode array detector) and equipped with column of Interchim® VKR5 C18 (250×4.6.0 mm, 5 μm particle size), or equivalent.

[0226] Acetonitrile, acetic acid and pure water are used for prepare mobile phase A (5/95/0.07 v/v/v Acetonitrile/Water/Acetic acid) and mobile phase B (50/50/0.05 v/v/v Acetonitrile/Water/Acetic acid).

Chromatography Procedure

[0227] Analyse using suitable chromatographic conditions, for example, those given in Table 5.

TABLE-US-00005 TABLE 5 Chromatographic conditions for the assay of EGCG Parameter Setting Mobile phase A Prepared as described before Mobile phase B Prepared as described before Flow rate 1 mL/min (or as appropriate) Oven temperature 32° C. Detection wavelength 275 nm Injection volume 20 μL Gradient programme The gradient program was set as follows: Temps (min) A (%) B (%) Approximate 0 90 10 equilibration time 10 80 20 Approximate 16 60 40 retention time 20 50 50 of EGCG 25 50 50 27 60 40 30 90 10 33 90 10 45 min ≈ 15 min

System Suitability Criteria

[0228] All system suitability criteria are calculated according to the current European Pharmacopoeia “Chapter 2.2.46”. Specific parameters of the procedure may be modified within pharmacopoeial and validated limits, if necessary, to achieve system suitability.

[0229] System suitability criteria should be met are displayed in Table 6.

TABLE-US-00006 TABLE 6 System suitability criteria System suitability test Acceptance criteria Tailing factor Confirm that the tailing factor of the EGCG peak from a reference standard solution injection is not greater than 1.5. System repeatability Confirm that the relative standard deviation for 3 injections from a reference standard solution is not greater than 2.0%

Calculation

[0230] The percentage of quantity of remained EGCG in diluted sample solution compared to original quantity in that sample is calculated according to following formula

[00001]$\mathrm{EGCG}\left(\%\mathrm{of}\mathrm{original}\mathrm{quantity}\right)=\frac{\mathrm{Wstd}\times \mathrm{Pstd}\times \mathrm{Asam}\times D}{\mathrm{Astd}\times \mathrm{Vstd}\times 26.27}\times 100$

Wherein:

[0231] A.sub.std=Peak area due to EGCG in reference standard solution
A.sub.sam=Peak area due to EGCG in diluted aqueous composition INV1
D=Dilution factor
W.sub.std=Weight of EGCG taken for the reference standard solution (mg)
P.sub.std=Purity of reference standard solution (% w/w)
V.sub.std=Volume of reference standard solution (mL)

1.5 ATR-IR Spectres Image

[0232] ATR-IR spectra image of eutectic system EGCG/choline chloride/DMSO is obtained by FTIR-spectroscopy. The experiments were performed on a Perkin-Elmer Spectrum BX FT-IR system based on diffuse reflectance sampling accessories with FT-IR Spectrum v2.00 software. The spectra of the stressed samples were recorded at room temperature in the wavenumber range of 400-4000 cm.sup.−1 using ATR cell.

1.6 Solubility

[0233] The Crystal16 equipment of Technobis BV has been used to determine the solubility of said EGCG eutectic system in ultrapure water. In the Crystal16, cloud points and clear points of sixteen 1 mL solution aliquots can be measured in parallel and automatically, based on turbidity. The temperature at the point the suspension becomes a clear solution upon heating (at 0.5° C. per minute) is generally taken as the saturation temperature of the measured sample, of which the composition is established beforehand. The maximum error (overshoot) in the saturation temperature measured in this way, because the conditions are not strictly at equilibrium, is in the order of <0.5° C.

2. Results

2.1 Solubility of EGCG According to the Invention

[0234] The EGCG liquid eutectic system were extremely soluble and no temperature ramp has been used. Samples were kept at 25° C. with 600 rpm stirring. Water solutions were prepared with said liquid eutectic system of the following concentrations: 60, 75, 100.8, 120, 150, 199.9, 310.7 and 407.3 mg/mL. According to Table 1, the effective concentrations of EGCG in these water solutions are respectively the follows: 28.8, 36, 48.4, 57.6, 72, 95.9, 149.1, 195.5 mg/mL.

[0235] All solutions became completely clear after several minutes of stirring, indicating that all concentrations were basically soluble. Compared to the intrinsic solubility of EGCG in water at room temperature, i.e. 4.6 mg/mL, the

[0236] EGCG eutectic system of the present invention allows EGCG to gain solubility by a factor of at least 32.

2.2 Stability of EGCG Solution According to the Invention

[0237] Stability tests as described in section 1.2 are carried out for the EGCG aqueous composition INV1 and an EGCG-based suspension alone as reference. After six days of exposure to the various stress conditions mentioned in Table 3, the samples were subjected to HPLC analysis. The reference EGCG-based suspension alone contains practically no more EGCG in solution when exposed to alkali conditions (FIG. 1C), losses 49.5% EGCG when subjected to high temperature (FIG. 2C) and losses 22.2% under photolysis conditions (FIG. 3C), whereas the chromatographic profiles of the product resulting from the heat treatment, alkali treatment or photolysis treatment of the EGCG aqueous composition INV1 (FIGS. 1B, 2B, 3B) is identical to that of the starting solution (FIGS. 1A, 2A, 3A), namely that it represents the same as the peak due to EGCG and that of another polyphenol of the green tea initially present.

[0238] The results of long-term stability performed on the EGCG aqueous composition INV1 are given in Table 7 below.

TABLE-US-00007 TABLE 7 long-term stability test Relative content of EGCG with respect to the initial time (%) for each storage conditions Time (months) 2-8° C. 25° C./60% RH 1 >99% >99% 3 >99% >99% 6 >99% >99% 9 Analysis planned Analysis planned 12 Analysis planned Analysis planned 18 Analysis planned Analysis planned

[0239] Long-term stability studies, up to now, show that the EGCG levels present in EGCG aqueous composition INV1 (26.7 mg/mL) remained unchanged at both storage conditions, i.e. 2-8° C. and 25° C./60% HR.

[0240] These results suggest that the formulations developed were able to protect EGCG from auto-oxidation, hydrolysis and photolysis.

2.3 Structure of Eutectic System

[0241] ATR-IR spectres image shows the presence of broad absorption in the O—H stretch IR regions and the absence of O—H free stretch peaks (FIG. 4A), that suggests that with OH functions DMSO and choline chloride as acceptors, EGCG would form eutectic system with DMSO and choline chloride by strong hydrogen bonds OH—O.

[0242] H-bonding may have caused vibrational modes to vibrate at lower frequencies and/or relative intensities than before, as long as the atoms involved in the vibrational modes are actively participating in H-bonding.

Example 2: In Vitro Permeability Study

[0243] In this study, Franz diffusion cells were used to compare the membrane passage of EGCG from a cream control formulation and a cream formulation of the present invention.

1. Materials and Methods

[0244] The tested EGCG topical formulation named INV2 is obtained by mixing the eutectic liquid EGCG eutectic system presented in Table 1 with a commercially available moisturizing serum chosen at random on the market. The control EGCG-containing cream was made from EGCG powder directly mixed with the same serum. Both formulations contain an equivalent amount of 1% w/w EGCG.

Permeability Assessment Using Franz Diffusion Cells

[0245] Drug release and skin permeation were determined using Franz diffusion cells.

[0246] The experiments were conducted in three independent vertical Franz cells with a nominal volume of the acceptor compartment of 125 mL and a diffusion area of 1.767 cm.sup.2. For the donor compartment, an amount of 1 mL of each formulation (1% w/w EGCG) was initially set. The experiments were conducted in triplicate, carried out at 32° C. and 400 rpm for 24 h. Samples were evaluated at different time points, and data analysis was made by comparing the releasing efficiency (PE) values.

[0247] The assays were performed in an aqueous medium as acceptor phase mimicking physiological conditions corresponding to 0.15 mol/L PBS® buffer solution at pH 7.4. A regenerated cellulose membrane with molecular weight cut-off at 12 kDa was used.

[0248] Releasing efficiency was defined in terms of the mass flux (J), which describes the change of drug permeation with respect to time in aqueous systems. In this study, the mass flux (mol cm.sup.−2 h.sup.−1) was determined using the AUC of the permeation profile recorded at a specific time interval and is related to the rectangular area (R) described by 100% of the permeation process at the same time interval (24 h).

[0249] Mass flux can be calculated from:

[00002]$\mathrm{Flux}\left(J\right)=\frac{{\int }_0^t\mathrm{��}\mathrm{dt}}{{\mathrm{��}}_{100}t}\times 100\%$

where y.sub.100 is the AUC value assumed with a permeation of 100% in a time interval t, and y represents the AUC value of the permeated drug during the same time interval.

[0250] A 0.5 mL aliquot was withdrawn periodically at pre-set time from the abovementioned receiver cell, which was thereafter 10 times diluted with PBS buffer solution and filtered through 0.45 μm filter. EGCG content was determined by HPLC. The diffusion fluid of the same volume was pre-warmed at 32° C. The volume of withdrawn samples was replaced by pre-warmed diffusion fluid into the diffusion cell to keep the volume constant so that sink condition could be maintained.

[0251] The cumulative amounts of drug in the receptor during 24 h were plotted after administration of various tested and control formulations as shown in FIG. 5.

HPLC Analysis

[0252] The diluted samples were analysed by HPLC, validated assay method as a stability indicating method described above in Example 1. Said procedure is suitable for EGCG assay using an external standard. The assay of EGCG is achieved by comparing the response of a sample solution with the response of

[0253] EGCG reference standard solution prepared at a similar nominal concentration and analysed in the same way. The sample and reference standard solutions are analysed by gradient reversed phase HPLC and UV detection using a suitable column and chromatography conditions.

2. Results

[0254] Permeability study of the two formulations of EGCG (formulation INV2 versus control EGCG containing cream) was carried out using cellulosic membrane as diffusional membrane and simulated skin medium as diffusional fluid at different time points up to 24 h.

[0255] The cumulative amounts of drug in the receptor during 24 h were plotted after administration of various formulations as shown in FIG. 5. EGCG flux across the skin was significantly lower than that released from the formulation of the present invention (Table 8). EGCG of the present invention significantly increased in vitro drug permeation compared to the control.

[0256] These results clearly show that the formulation INV2 of present invention significantly improves the skin permeation of EGCG, paving the way for improved clinical efficacy of a EGCG containing dermo-cosmetic product or a pharmaceutical dermal product.

TABLE-US-00008 TABLE 8 AUC values for mass flux and lag time obtained from the EGCG permeation profiles of the tested and control formulations using cellulose membrane at 32° C. Type of 1% w/w EGCG in a marketed moisturizing serum Flux (μg cm.sup.−2 h.sup.−1) Lag-time (h) EGCG cream composition 1.29 1.72 INV2 EGCG powder in serum 0.81 1.65 (control)

Example 3: Plasma Concentrations of EGCG in Primates

[0257] This test was performed to compare plasma concentrations and total area under the curver (AUC.sub.total) obtained after oral administration in Cynomolgus monkeys of the EGCG aqueous composition INV1 of the present invention with that of EGCG containing capsules as control formulation.

1. In-Life Phase

[0258] The content of EGCG in tested capsules are adapted according to the weight of monkey to ensure an admiration of 26.67 mg EGCG/kg of monkey weight.

[0259] The treatment patterns of two tested formulations are listed below:

TABLE-US-00009 Name Form Content Other details EGCG Capsules Monkey 1: 145.8 mg Content pre-adjusted capsules of EGCG per capsule to the weight of the Monkey 2: 116.1 mg monkey. of EGCG per capsule 2 capsules for each Monkey 3: 108.0 mg monkey were of EGCG per capsule provided. However, only one capsule is to be administered to each monkey. Please rely on labeling. EGCG Solution 26.7 mg/mL The volume aqueous administered is to be composition adjusted according to INV1 the weight of the monkey. Shake well before sampling.

[0260] Formulations were stored at room temperature and well shaken before sampling.

2. Animals and Husbandry

[0261] The conditions of monkey breeding are summarized below.

TABLE-US-00010 Species: Monkey Strain/Breed: Cynomolgus Number: 3 Weight: About 3-6 kg Age: adult Status: Non naïve Supplier: Avogadro LS colony Acclimatization: At Housing: Per group of 3 and individual least 7 days condition during the blood sampling collection time until 3 h post dosing Daily light cycle: Temperature range: 20-24° C. 12 h day/12 h night Feeding conditions: Ration: Regular Overnight fasting: No Fate of animals: Return Necropsy: On unexpected dead animals to stock only

3. Study Design and Dosing Details

[0262] EGCG aqueous composition INV1 was orally administered by gavage using a gastro-oesophageal tube or a nasogastric tube and an appropriate syringe. After administration, the tube was rinsed with 5 mL of water and flushed with 5 mL of air to ensure that the entire dose will be delivered.

[0263] Capsule of EGCG was administered using a specific device to place the item on the back of the tongue.

[0264] The wash out period is at least 7 days.

TABLE-US-00011 Quantity to Formulation Formulation Target administer per Period administered concentration dose animal 1 EGCG 145.8 mg/capsule 27 mg/kg 1 capsule capsule 116.1 mg/capsule 108.0 mg/capsule 2 EGCG 26.7 mg/mL 27 mg/kg Monkey 1: 5.5 mL Eutectic Monkey 2: 4.3 mL solution Monkey 3: 4.1 mL

4. Blood Sampling and Processing

[0265]

TABLE-US-00012 Blood Volume 0.5 mL Tube: LH collection: Total number of samples: 108 Vein: Cephalic or femoral Sampling times: 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 5 h, 7 h, 12 h, 24 h Plasma Centrifugation: Number of aliquots: preparation: 2500 g/10 min/5° C. 2 (A & B) Distribution: Eppendorf tubes Aliquot volume: A: 100 μL B: remaining plasma (at least 100 μL) Storage until shipping: ca. −80° C.

5. Analytical Test

[0266] The analytical test was based on the method described by de Lourdes et al (J. Agric. Food Chem. 2007, 55, 8857-8863).

[0267] The molecular and daughter ions were selected for each molecule after direct infusion into the MS-MS system. The analytical method consisted of a precipitation of the proteins by addition of appropriate solvent followed by a LC-MS/MS analysis. According to the expected sensitivity, at least 8 calibration standards were used for the preparation of the calibration curve in plasma. The corresponding correlation coefficient were calculated and had to be higher than 0.75 to continue with the in vivo test.

[0268] The calibration range tested will be 4 to 5000 ng/mL of EGCG in plasma. The difference between the mean concentration observed and the nominal concentration was used to estimate the deviation of the method.

6. Results

[0269] EGCG plasma concentration and AUC.sub.total results are summarized in the table below (Table 9).

[0270] FIG. 6 illustrates the evolution of plasma EGCG concentrations over time.

[0271] As these results show, EGCG plasma concentration resulting from the administration of the composition INV1 is, at equal doses administered, much higher than that resulting from the administration of capsule containing 100% of

TABLE-US-00013 TABLE 9 Plasma concentration and PK parameters C.sub.max AUC0-24 AUCt AUCinf Treatment T.sub.max(h) (ng/mL) (ng/mL .Math. h) Kel(1/h) T½(h) (ng/mL .Math. h) (ng/mL .Math. h) % AUC.sub.extra r.sup.2 EGCG 2.33 510.56 1181 0.12 6.05 1155 1219 5.42 0.94 capsule (n = 3) EGCG 1 2996.1 6781 0.11 6.52 6781 6981 3.04 0.99 formulation (n = 3) ratio 0.4 5.9 5.7 0.9 1.1 5.9 5.7 0.6 1.1
the EGCG powder. Indeed, the total AUC ratio per dose of administration of the composition INV1 is 6 times higher than that of EGCG-containing administration.