CROSSLINKED STARCH DERIVATIVE-BASED MATRIX

Abstract

The invention relates to a water insoluble solid crosslinked dextrin-based matrix, wherein the crosslinking agent is sodium trimetaphosphate (STMP), its use and method of preparation.

Claims

1. A method of preparing a water insoluble crosslinked dextrin-based matrix, comprising the following steps: a) providing at least one dextrin or at least one dextrin and at least one cyclodextrin, b) forming the water insoluble crosslinked dextrin-based matrix by crosslinking said dextrin or dextrin and cyclodextrin with sodium trimetaphosphate (ST MP) in an aqueous medium containing an alkaline agent, and c) recovering a mixture of the water insoluble crosslinked dextrin-based matrix and the aqueous medium.

2. The method according to claim 1, wherein the at least one dextrin is a maltodextrin.

3. The method according to claim 1, wherein the at least one dextrin is a pyrodextrin.

4. The method according to claim 1, wherein the crosslinking is carried out in the absence of any organic solvent.

5. A water insoluble crosslinked dextrin-based matrix, wherein at least one dextrin is or at least one dextrin and at least one cyclodextrin are is/are crosslinked with sodium trimetaphosphate.

6. The cross-linked dextrin-based matrix according to claim 5, wherein the at least one dextrin is a maltodextrin.

7. The cross-linked dextrin-based matrix according to claim 5, wherein the at least one dextrin is a pyrodextrin.

8. Use of the cross-linked dextrin-based matrix according to claim 1 as carrier for organic compounds.

9. Use according to claim 8, wherein the organic compounds are selected from active ingredients, in particular chosen from pharmaceutically active ingredients, bioactive ingredients, and food active ingredients.

10. Use according to claim 9, wherein the active ingredient is insulin.

11. An oral delivery system comprising a water insoluble cross-linked dextrinbased matrix according to claim 1 and an active ingredient, wherein the matrix is loaded with the active ingredient.

12. The oral delivery system according to claim 11, wherein the active ingredient is an active ingredient, in particular chosen from pharmaceutically active ingredient, a bioactive ingredient or a food active ingredient active ingredient.

13. The oral delivery system according to claim 12, wherein the active ingredient is insulin.

14. Use of the water insoluble cross-linked dextrin-based matrix according to claim 1 for capturing pollutants in water or air.

Description

FIGURES

[0060] FIG. 1 shows the insulin release over time at pH = 1.2 from an insulin loaded matrix according to the invention.

[0061] FIG. 2 shows the insulin release over time at pH = 6.8 from an insulin loaded matrix according to the invention.

EXAMPLES

[0062] The following starting materials were used for the synthesis of the cross-linked matrices: [0063] KLEPTOSE Linecaps®17 (Roquette Freres): pea maltodextrin. [0064] Stabilys® A025 (Roquette Freres): maize pyrodextrin. [0065] Stabilys® A053 (Roquette Freres): maize pyrodextrin. [0066] Sodium trimetaphosphate (STMP, Na.sub.3P.sub.3O.sub.9, CAS No. 7785-84-4): Sigma Aldrich, 95% purity.

Example 1: Synthesis of a Maltodextrin-Based Matrix According to the Invention Crosslinked with 60% STMP

[0067] In a glass reactor equipped with a mechanical stirrer, was introduced 105.2 g of Linecaps 17 (residual moisture 4.9 weight%, 100 g dry substance).

[0068] 20 weight % of NaOH based on dry weight of starch (20 g, 0.5moles) were added under stirring using 10% NaOH solution (200 g).

[0069] Reaction was left under stirring at room temperature (~20-25° C.) for 3.5 hours.

[0070] 60 weight % sodium trimetaphosphate based on dry weight of starch (60 g, 0.196 mole) were added under stirring. The reaction mixture was left for 1.5 h.

[0071] After several minutes, jellification of the mixture was observed and stirring was stopped.

[0072] After that time, recovery of the crude material was performed.

[0073] The solid was crushed and dispersed in a sufficient amount of water to obtain a stirred suspension. Neutralization of the crude was made by addition of HCI until residual pH reaches 6.5.

[0074] The mixture was centrifuged for 15 minutes at 4700 rpm using a VWR Mega Star 1.6 centrifuge. The supernatant was removed and the obtained matrix was washed with demineralized water. After 15 minutes of stirring, the mixture was centrifuged for 15 minutes at 4700 rpm using the same centrifuge as before. The supernatant was removed and the obtained gel was washed with demineralized water 2 more times.

[0075] The final matrix was recovered and precipitated in Ethanol under stirring. The obtained white powder was filtered and dried under vacuum. Product was recovered with 59% yield (based on the dry weight of product recovered / the initial amount of dry Linecaps + STMP introduced in the reaction mixture).

Example 2: Synthesis of a Maltodextrin-Based Matrix According to the Invention Crosslinked with 50% STMP

[0076] In a glass reactor equipped with a mechanical stirrer, was introduced 105.2 g of Linecaps 17 (residual moisture 4.9%, 100 g dry substance).

[0077] 20 weight % of NaOH based on dry weight of starch (20 g, 0.5 moles) were added under stirring using 10% NaOH solution (200 g).

[0078] Reaction was left under stirring at room temperature (~20-25° C.) for 3.5 hours.

[0079] 50 weight % sodium trimetaphosphate based on dry weight of starch (50 g, 0.163 mole) were added under stirring. The reaction mixture was left for 1.5h.

[0080] After several minutes, jellification of the mixture was observed and stirring was stopped.

[0081] After that time, recovery of the crude material was performed.

[0082] The solid was crushed and dispersed in a sufficient amount of water to obtain a stirred suspension. Neutralization of the crude was made by addition of HCl until residual pH reaches 6.5.

[0083] The reaction mixture was centrifuged for 15 minutes at 4700 rpm using a VWR Mega Star 1.6 centrifuge. The supernatant was removed and the obtained gel was washed with demineralized water. After 15 minutes of stirring, the mixture was centrifuged for 15 minutes at 4700 rpm using the same centrifuge as before. The supernatant was removed and the obtained matrix was washed with demineralized water 2 more times.

[0084] The final matrix was recovered and precipitated in Ethanol under stirring. The obtained white powder was filtered and dried under vacuum. Product was recovered with 54% yield (based on the dry weight of product recovered / the initial amount of dry Linecaps + STMP introduced in the reaction mixture).

Example 3: Synthesis of a Maltodextrin-Based Matrix According to the Invention Crosslinked with 40% STMP

[0085] In a glass reactor equipped with a mechanical stirrer, was introduced 525,8 g of Linecaps 17 (residual moisture 4.9%, 500 g dry substance).

[0086] 16 weight % of NaOH based on dry weight of starch (80 g, 2 moles) were added under stirring using 10% NaOH solution (800 g).

[0087] Reaction was left under stirring at room temperature (~20-25° C.) for 3.5 hours.

[0088] 40 weight % sodium trimetaphosphate based on dry weight of starch (200 g, 0.653 mole) were added under stirring. The reaction mixture was left for 1.5h.

[0089] After several minutes, jellification of the mixture was observed and stirring was stopped.

[0090] After that time, recovery of the crude material was performed.

[0091] The solid was crushed and dispersed in a sufficient amount of water to obtain a stirred suspension. Neutralization of the crude was made by addition of HCl until residual pH reaches 6.5.

[0092] The reaction mixture was centrifuged for 15 minutes at 4700 rpm using a VWR Mega Star 1.6 centrifuge. The supernatant was removed and the obtained gel was washed with demineralized water. After 15 minutes of stirring, the mixture was centrifuged for 15 minutes at 4700 rpm using the same centrifuge as before. The supernatant was removed and the obtained matrix was washed with demineralized water 2 more times.

[0093] The final matrix was recovered and precipitated in Ethanol under stirring. The obtained white powder was filtered and dried under vacuum. Product was recovered with 59% yield (based on the dry weight of product recovered / the initial amount of dry Linecaps + STMP introduced in the reaction mixture).

Example 4: Synthesis of a Maltodextrin-Based Matrix According to the Invention Crosslinked with 25% STMP

[0094] In a glass reactor equipped with a mechanical stirrer, was introduced 525,8 g of Linecaps 17 (residual moisture 4.9%, 500 g dry substance).

[0095] 10 weight % of NaOH based on dry weight of starch (50 g, 1,25 moles) were added under stirring using 10% NaOH solution (500 g).

[0096] 50 g of demineralized water were added to the reaction mixture to allow good stirring conditions.

[0097] Reaction was left under stirring at room temperature (~20-25° C.) for 3.5 hours.

[0098] 25 weight % sodium trimetaphosphate based on dry weight of starch (125 g, 0.408 mole) were added under stirring. The reaction mixture was left for 1.5 h.

[0099] After several minutes, jellification of the mixture was observed and stirring was stopped.

[0100] After that time, recovery of the crude material was performed.

[0101] The solid was crushed and dispersed in a sufficient amount of water to obtain a stirred suspension. Neutralization of the crude was made by addition of HCl until residual pH reaches 6.5.

[0102] The reaction mixture was centrifuged for 15 minutes at 4700 rpm using a VWR Mega Star 1.6 centrifuge. The supernatant was removed and the obtained gel was washed with demineralized water. After 15 minutes of stirring, the mixture was centrifuged for 15 minutes at 4700 rpm using the same centrifuge as before. The supernatant was removed and the obtained matrix was washed with demineralized water 2 more times.

[0103] The final matrix was recovered and precipitated in Ethanol under stirring. The obtained white powder was filtered and dried under vacuum. Product was recovered with 58% yield (based on the dry weight of product recovered / the initial amount of dry Linecaps + STMP introduced in the reaction mixture).

Example 5: Synthesis of a Maltodextrin-Based Matrix According to the Invention Crosslinked with 20% STMP

[0104] In a glass reactor equipped with a mechanical stirrer, was introduced 525,8 g of Linecaps 17 (residual moisture 4.9%, 500 g dry substance).

[0105] 8 weight % of NaOH based on dry weight of starch (40 g, 1 mole) were added under stirring using 10% NaOH solution (400 g).

[0106] 140 g of demineralized water were added to the reaction mixture to allow good stirring conditions.

[0107] Reaction was left under stirring at room temperature (~20-25° C.) for 3.5 hours.

[0108] 20 weight % sodium trimetaphosphate based on dry weight of starch (100 g, 0.327 mole) were added under stirring. The reaction mixture was left for 1.5 h.

[0109] After several minutes, jellification of the mixture was observed and stirring was stopped.

[0110] After that time, recovery of the crude material was performed.

[0111] The solid was crushed and dispersed in a sufficient amount of water to obtain a stirred suspension. Neutralization of the crude was made by addition of HCl until residual pH reaches 6.5.

[0112] The reaction mixture was centrifuged for 15 minutes at 4700 rpm using a VWR Mega Star 1.6 centrifuge. The supernatant was removed and the obtained gel was washed with demineralized water. After 15 minutes of stirring, the mixture was centrifuged for 15 minutes at 4700 rpm using the same centrifuge as before. The supernatant was removed and the obtained matrix was washed with demineralized water 2 more times.

[0113] The final matrix was recovered and precipitated in Ethanol under stirring. The obtained white powder was filtered and dried under vacuum. Product was recovered with 57% yield (based on the dry weight of product recovered / the initial amount of dry Linecaps + STMP introduced in the reaction mixture).

Example 6: Synthesis of Pyrodextrin-Based Matrices According to the Invention Crosslinked with 60% STMP

[0114] Example 6a: In a glass reactor equipped with a mechanical stirrer, was introduced a 20 weight % dry matter slurry composed of 100 g of dry Stabilys® A053 (amount calculated after determination of the residual moisture, 100 g dry substance) and 400 g of demineralized water. This preparation was cooked at 95° C. the slurry became a paste (yellow color). After 30 minutes of cooking at 95° C., the reaction mixture was cooled down to 25° C. prior to addition of sodium hydroxide solution

[0115] 20 weight % of NaOH based on dry weight of starch (20 g, 0.5 moles) were added under stirring using 10% NaOH solution (200 g).

[0116] Reaction was left under stirring at room temperature (~20-25° C.) for 3.5 hours.

[0117] 60 weight % sodium trimetaphosphate based on dry weight of starch (60 g, 0.196 mole) were added under stirring. The reaction mixture was left for 1.5 h.

[0118] After several minutes, jellification of the mixture was observed and stirring was stopped.

[0119] After that time, recovery of the crude material was performed.

[0120] The solid was crushed and dispersed in a sufficient amount of water to obtain a stirred suspension. Neutralization of the crude was made by addition of HCl until residual pH reaches 6.5.

[0121] The reaction mixture was centrifuged for 15 minutes at 4700 rpm using a VWR Mega Star 1.6 centrifuge. The supernatant was removed and the obtained matrix was washed with demineralized water. After 15 minutes of stirring, the mixture was centrifuged for 15 minutes at 4700 rpm using the same centrifuge as before. The supernatant was removed and the obtained matrix was washed with demineralized water 2 more times.

[0122] The final matrix was recovered and precipitated in ethanol under stirring. The obtained white powder was filtered and dried under vacuum. Product was recovered with 58% yield (based on the dry weight of product recovered / the initial amount of dry Stabilys® A053 + STMP introduced in the reaction mixture).

[0123] Example 6b: Example 3a was repeated by replacing the 20 weight % dry matter slurry of Stabilys® A053 with a 15 weight % dry matter slurry of Stabilys® A025 (75 g of Stabilys® A025 in 425 g of demineralized water). The product was recovered with 68% yield (based on the dry weight of product recovered / the initial amount of dry Stabilys® A025 + STMP introduced in the reaction mixture).

Example 7: Solubility of Matrices According to the Invention

[0124] Solubility of the matrices of Examples 1 to 3 in water was determined according to the following protocol:

[0125] 250 mg of each matrix was taken in a vial. To each vial, 5 mL of deionized water were added. All samples were stirred periodically and kept under constant observation.

[0126] The swelling and dissolution of each sample was observed for up to 72 hours. For checking solubility, the viscosity and transparency of the supernatant liquid (water) was carefully observed through a magnifying glass. The swelling of samples and dissolution can be clearly differentiated by this visual evaluation.

[0127] For each matrix, the test was carried out at pH 7, pH 5 (addition of HCl) and pH 9 (addition of NaOH).

[0128] All of the samples were insoluble under the test conditions. However, they showed important swelling.

Example 8: Swelling Capacity of Matrices According to the Invention

[0129] The Swelling Index (SI) of the matrices of Examples 2 to 4 was determined according to the following protocol:

[0130] 1 g (dry weight) of product was dispersed in 100 ml of demineralized water in a graduated cylinder, and left 24 h for swelling. After 24 h of contact the mixture of gel dispersed water was centrifuged to separate the supernatant and the bottom layer (swollen gel). The swollen gel was weighed to determine the amount of water absorbed.

[0131] The SI was calculated as described above.

[0132] The results are presented in table 1 below.

TABLE-US-00001 Matrix SI % Ex. 2 1640 Ex. 3 1530 Ex. 4 1080

Example 9: pH Value, Average Diameter and Polydispersity of Matrices According to the Invention

[0133] The pH value of the matrices of Examples 2, 3, 4, and 5 was determined using a pH meter (Orion model 420A).

[0134] The average diameter and polydispersity index of the matrices of Examples 2, 3, 4, and 5 were determined by Laser Light Scattering using a 90plus Instrument (Brookhaven, NY, USA) and zeta potential was determined by electrophoretic mobility using the same instrument.

[0135] The analyses were carried out on matrix suspensions that were prepared as follows:

[0136] 1. Preparation of a suspension starting from coarse powder in distilled water at the concentration of 10 mg/ml under stirring at room temperature.

[0137] 2. Dispersion of the suspension using a high shear homogenizer (Ultraturrax®, IKA, Konigswinter, Germany) for 10 minutes at 24000 rpm.

[0138] 3. Use of high pressure homogenization for 90 minutes at a back-pressure of 500 bar, using an EmulsiFlex C5 instrument (Avastin, USA) for further size reduction.

[0139] 4. Purification of homogenized nanosuspension by dialysis (Spectrapore, cellulose membrane, cutoff 12000 Da) to remove synthesis residues potentially present.

[0140] 5. Storage of the nanosuspensions at 4° C.

[0141] The results are presented in table 2 below.

TABLE-US-00002 Matrix pH Average diameter (nm) Polydispersity Index (PI) Zeta potential (mV) Example 2 8.24 246.7 0.171 38.34±2.93 Example 3 6.58 259.2 0.224 21.39±1.25 Example 4 6.44 301.7 0.224 -30.27±1.29 Example 5 6.50 224.2 0.377 -18.25±1.5

Example 10: Methylene Blue Loading Capacity of Matrices According to the Invention

[0142] Methylene blue was used as a model for an organic cationic compound in order to demonstrate the ability of the matrixes of the invention to retain organic cationic compounds.

[0143] 2 g (dry weight) of crosslinked matrix was dispersed in 100 ml of 10.sup.-5 M methylene blue solution in water and left 24 h for swelling. After 24 h of contact the mixture of gel dispersed in aqueous methylene blue solution was centrifuged to separate the supernatant and the bottom layer (blue colored swollen gel).

[0144] Determination of the concentration of residual methylene blue in the supernatant was determined using UV-visible spectrometry.

[0145] Methylene blue absorption capacity (in %) was calculated as the ratio of the quantity of methylene blue retained by the matrix / quantity of methylene blue initially introduced * 100. The quantity of methylene blue retained by the matrix corresponds to the difference between the quantity of methylene blue initially introduced and the quantity of methylene blue present in the supernatant.

[0146] The results are presented in table 2 below.

TABLE-US-00003 Matrix Methylene blue loading capacity % Ex. 1 93 Ex. 2 88 Ex. 6a (Stabilys® A053) 84 Ex. 6b (Stabilys® A023) 78

Example 11: Insulin Loading of Matrices According to the Invention

[0147] Insulin from bovine pancreas powder was used to prepare a 2 mg/mL solution in distilled water pH 2.3 adjusted using phosphoric acid. Insulin solution was added to pre-formed aqueous nanosuspensions of the crosslinked matrix (according to the protocol described in example 9) in a weight ratio insulin solution: nanosuspension of 1:5. The mixture was stirred at room temperature for 30 minutes and then centrifuged. The supernatant was separated from the sediment which was collected and freeze-dried.

[0148] According to this procedure, freeze-dried insulin-loaded matrices were prepared from the matrices of Examples 1 and 2.

Insulin Loading Capacity

[0149] The loading capacity was determined from the freeze-dried insulin loaded samples according to the following protocol.

[0150] 2-3 mg of freeze-dried insulin loaded crosslinked matrix was dispersed in 5 mL of distilled water. Sonication (15 minutes, 100 W) and centrifugation treatments were performed so as to allow the release of insulin from the system delivery. Then the supernatant was analyzed for the quantitative determination of insulin.

[0151] The quantitative determination of insulin was carried out by High Performance Liquid Chromatography (HPLC) (Perkin Elmer 250B, Waltham, MA) equipped with a spectrophotometer detector (Flexar UV/Vis LC, Perkin Elmer, Waltham, MA). An analytical column C18 (250 mm × 4.6 mm, ODS ultrasphere 5 .Math.m; Beckman Instruments, USA) was used. The mobile phase consisted of a mixture of 0.1 M sodium sulfate in distilled water and acetonitrile (72:28 v/v) filtered through a 0.45 .Math.m nylon membrane and ultrasonically degassed prior to use. Ultraviolet detection was fixed at 214 nm and the flow rate was set to 1 mL/min. The insulin concentration was calculated using external standard method from standard calibration curves. For this purpose, 1 mg of Insulin was weighted, placed in a 10 mL flask, and dissolved distilled water at pH 2.3 adjusted by phosphoric acid to obtain a mother solution. This solution was then diluted using the mobile phase and a series of standard solutions were prepared, consequently injected into the HPLC system. Linear calibration curves were obtained over the concentration range of 0.5-25 .Math.g/mL, linear plotted with regression coefficient of 0.999.

[0152] The insulin loading capacity (%) of the delivery systems was calculated as follows:

[00002]$\left[\text{weight of insulin}/\text{weight of freeze dried crosslinked matrix}\right]\times \text{100}\text{.}$

[0153] The results are shown in table 3 below.

TABLE-US-00004 Matrix Insulin loading capacity % Ex. 1 13.53 ± 0.55 Ex. 2 18.10 ± 0.68

Example 12: Insulin Release

In Vitro Drug Release Kinetics

[0154] The in vitro drug release experiment was conducted in a multi-compartment rotating disc (a diffusion cell system comprising a donor chamber separated by a membrane from the donor compartment), constituted from several donor cells on one side separated by a cellulose membrane (Spectrapore, cut-off 50 kDa) from the receiving cells on the other side. The freeze-dried insulin-loaded cross-linked matrix prepared in Example 11 from the matrix of Example 2 was placed in the donor cell (1 mL). The receiving cells were filled by phosphate buffered saline (PBS) solutions at pH 1.2 and pH 6.8 separately. The in vitro release studies were carried out during 24 hours, whereby the receiving phase was withdrawn at regular intervals and replaced with the same amount of new PBS solution. The sampling times investigated were 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 22 and 24 hours. The concentration of insulin in the withdrawn samples was later detected by HPLC.

[0155] The results are shown in FIGS. 1 and 2. FIG. 1 shows the results up to 3h only as there was no evolution after 3 h.

[0156] The cross-linked matrix according to the invention prevents the release of insulin at gastric pH (FIG. 1, pH 1.2), whereas it allows insulin release at intestinal pH (FIG. 2, pH 6.8). In other terms the matrix according to the invention prevents the release of insulin at a pH at which it would be hydrolyzed due to the high acidity of the stomach, and allows the release of insulin in the intestine, where insulin absorption is desired. FIG. 2 also shows that the matrix according to the invention continues to release insulin over a period of several hours.

[0157] In addition, the cross-linked matrices according to the invention advantageously allow slow release of insulin. This means that insulin is potentially bioavailable during a longer period of time and that the cross-linked matrices according to the invention are less likely to provoke an abrupt increase of blood insulin after ingestion. The cross-linked matrices according to the invention thus reduce the risk of harmful hypoglycemia caused by abrupt increase of blood insulin.

In Vivo Experiments

[0158] The Freeze-dried insulin-loaded cross-linked matrix prepared in Example 11 from the matrix of Example 2 was administrated to a rat by oral gavage in the stomach. The insulin dose administered was 2.10 mg/kg. Blood samples were collected at different points in time.

[0159] The insulin was extracted from plasma samples obtained from the collected blood samples according to the following protocol. To each 100 .Math.l of plasma 100 .Math.l of PBS (pH 7.4), 50 .Math.l of acetonitrile, 20 .Math.l of ethyl paraben, and 3 ml of dichloromethane/n-hexane (1:1 v/v) were added. The mixture was vortexed for 2 min and then centrifuged at 5000 rpm for 10 min. The supernatant was transferred to a test tube. Then 300 .Math.l of 0.05 N HCl were added and the mixture was vortexed for 2 minutes under nitrogen flux. After complete evaporation of the organic phase under nitrogen flux, the remaining supernatant was centrifuged at 15000 rpm for 10 min. A clear supernatant was obtained. The supernatant samples were stored in a freezer at -18° C. and analyzed by HPLC and ELISA.

HPLC Analysis

[0160] HPLC analysis was carried out on a PerkinElmer 250B HPLC system and peaks were integrated using the Chromera software. The experimental HPLC conditions were as follows: [0161] Loop: 20.Math.l [0162] Flow: 1 ml/min [0163] Pressure: 180 bar* [0164] Column: Agilent TC-C18 (2) 5 .Math.m (4.6 mm×150 mm, USA) [0165] λ: 214 nm [0166] Instrument: PerkinElmer 250B, Waltham, MA [0167] Eluent: Mixture of 42 volumes of mobile phase A (solution of 28.4 g of anhydrous sodium sulphate dissolved in 1000 ml of water, pH= 2.3 using phosphoric acid) and 58 volumes of mobile phase B (mixture of 550 ml of mobile phase A and 450 ml of acetonitrile).

[0168] The results are presented in table 4.

TABLE-US-00005 Time (min) t.sub.R (min) Area Concentration (.Math.g/ml) Bovine Insulin Internal standard Bovine Insulin Internal standard Bovine Insulin t = 60 min 11.9 6.3 796731.4 66339.9 282.84 t = 180 min 12.3 6.2 77600.8 59487.6 32.13

ELISA Assay

[0169] The Elisa assay (Sigma Aldrich ELISA kit) was performed on the samples collected at 15, 60 and 360 min. They are listed in the following table together with the mean absorbance read at 450 nm measured with a Perkin Elmer instrument and the corresponding concentration (.Math.lU/ml). the results are presented in table 5.

TABLE-US-00006 Sample Mean absorbance Concentration* (.Math.lU/ml) Effective concentration (.Math.lU/ml) t=360 min 0.131 8.10 8.10 t=60 min 0.127 7.358 29.42** t=15 min 0.127 7.38 7.38 *calculated using the insulin calibration curve constructed following the protocol present in the “Certificate of Analysis” of the ELISA kit provided by SIGMA-ALDRICH. The linear regression equation was: y = 0.0061x + 0.0819 with the correlation coefficient of 0.954. **The sample was diluted 1:4.

[0170] The results of the HPLC analysis and the ELISA assay show that the administered insulin is found in the blood and thus confirm that the matrices according to the invention are useful for oral administration of insulin.