Matrices based on cross-linked starch derivatives

Abstract

The present invention relates to a water-insoluble solid cross-linked dextrin-based matrix in which the cross-linking agent is sodium trimetaphosphate (STMP), its use and method of preparation.

Description

[Background technology]

[0001] The present invention relates to a water-insoluble solid cross-linked dextrin-based matrix, in which the cross-linking agent is sodium trimetaphosphate (STMP), and to its use, for example, for the sustained release of active ingredients. The present invention also relates to a method for preparing said cross-linked dextrin-based matrix.

[0002] Description of the Prior Art Hydrogels are well known in industry, especially in the pharmaceutical and medical industries. Hydrogels are three-dimensional networks of chemically or physically crosslinked hydrophilic polymers. Hydrogels can be used in a variety of applications, including, but not limited to, tissue engineering, drug or siRNA loading and delivery, food thickeners, and water treatment.

[0003] Wintgens et al. (Carbohydrate Polymers 98 (2013) 896-904; Carbohydrate Polymers 132 (2015) 80-88) reported cyclodextrin and cyclodextrin / dextran-based hydrogels prepared by cross-linking with sodium trimetaphosphate. According to these papers, cyclodextrin-based hydrogels are promising materials for carriers of bioactive molecules, and cyclodextrin / dextran-based hydrogels are promising carriers of bioactive molecules and bone regeneration.

[0004] International Patent Application No. 2016 / 100861(A1) describes cross-linked polysaccharide polymers and their use as flowable hemostatic compositions. The exemplified compositions are based on maltodextrin cross-linked with epichlorohydrin. However, this application does not provide any operating procedures that would allow a person skilled in the art to carry out the examples, and therefore it is not possible to prepare these compositions.

[0005] Matrices based on water-insoluble starch derivatives for controlled drug release are known in the art. They are generally prepared by crosslinking starch derivatives with organic crosslinkers. International Patent Application No. 2019 / 011964(A1) describes maltodextrins crosslinked with dianhydrides, particularly pyromellitic dianhydride, and their use in the administration of biologically active substances such as insulin. The synthesis of these dianhydride-crosslinked maltodextrins is carried out in dimethyl sulfoxide (DMSO) in the presence of triethylamine. The use of organic solvents and harmful reagents such as trimethylamine should generally be avoided.

[0006] There remains a need for new materials that are useful as carriers for active ingredients, particularly pharmaceutically active ingredients, and that do not require, or at least limit, the use of organic solvents and / or hazardous organic reagents. Summary of the Invention

[0007] The inventors have discovered that such materials can be obtained by reticulating certain dextrins, hereinafter defined and classified under the term "dextrins", with a special reticulating agent, sodium trimetaphosphate (STMP), the reaction being carried out in an aqueous medium in the presence of an alkaline agent.

[0008] Thus, in a first aspect, the present invention relates to a method for preparing a water-insoluble cross-linked dextrin-based matrix, the method comprising the following steps: a. providing at least one dextrin or at least one dextrin and at least one cyclodextrin; b. cross-linking the dextrin or dextrin and cyclodextrin with sodium trimetaphosphate (STMP) in an aqueous medium containing an alkaline agent to form a water-insoluble cross-linked dextrin-based matrix; and c. Recovering the mixture of the water-insoluble cross-linked dextrin-based matrix and the aqueous medium.

[0009] In a second aspect, the present invention relates to a matrix based on cross-linked dextrins, in which the dextrins are cross-linked with sodium trimetaphosphate (STMP).

[0010] In further aspects, the present invention relates to various uses of cross-linked dextrin-based matrices, for example, to encapsulate organic compounds in oral delivery systems and as filter media. DETAILED DESCRIPTION OF THE INVENTION

[0011] The method of the present invention for preparing a water-insoluble cross-linked dextrin-based matrix comprises the following steps: a. providing at least one dextrin or at least one dextrin and at least one cyclodextrin; b. cross-linking the dextrin or dextrin and cyclodextrin with sodium trimetaphosphate (STMP) in an aqueous medium containing an alkaline agent to form a water-insoluble cross-linked dextrin-based matrix; and c. Recovering the mixture of the water-insoluble cross-linked dextrin-based matrix and the aqueous medium.

[0012] The crosslinked dextrin-based matrices obtained according to the invention are water-insoluble. In the sense of the present invention, the term "water-insoluble" means that the matrix does not dissolve in water at room temperature, i.e., 18-25°C, at a pH of 7. Preferred crosslinked dextrin-based matrices according to the invention are insoluble in water at room temperature in the pH range of 5-9.

[0013] As used herein, the term "dextrin" includes maltodextrin, glucose syrup with a dextrose equivalent (DE) of 20 to 30, and pyrodextrin. Preferred dextrins within the meaning of the present invention are maltodextrin and pyrodextrin. Maltodextrins are obtained by acid and / or enzymatic hydrolysis of starch and have a DE (or dextrose equivalent) of 20 or less. Pyrodextrins are obtained by dry heating starch under acidic conditions, which generally results in the hydrolysis of the starch and subsequent recombination through α-1,6 bonds. These pyrodextrins are called white or yellow dextrins, or "British gums," depending on the temperature, acidity, and humidity conditions used. The term "dextrin" as used herein does not include cyclodextrins.

[0014] Dextrins suitable for use in the present invention can be prepared from any type of starch. Non-limiting examples of starch sources include, but are not limited to, tuber, cereal, and legume starches. Non-limiting examples of tuber starches include potato and tapioca starches. Examples of cereal starches include, but are not limited to, wheat, maize (also called corn), and barley starches. Examples of legume starches include, but are not limited to, pea, bean, broad bean, horse bean, lentil, alfalfa, lupin, and faba bean starches. Thus, the dextrins used in the present invention are selected from potato, tapioca, wheat, corn, barley, pea, bean, broad bean, horse bean, lentil, alfalfa, lupin, faba bean dextrins and mixtures thereof. Preferably, the dextrins are selected from pea, faba bean and corn dextrins, more preferably from pea and corn dextrins, especially from pea and corn maltodextrins or pyrodextrins.

[0015] In one embodiment, the at least one dextrin is a corn dextrin, particularly a corn pyroextrin.

[0016] In another embodiment, the at least one dextrin used in the process of the present invention is a legume dextrin, preferably derived from legume starch having an amylose content of 25% to 50%, preferably 30% to 40%, particularly 35% to 40%, more preferentially 35% to 38%, where these percentages are expressed as dry weight relative to the dry weight of the starch. The legume dextrin is selected from the group consisting of pea, bean, broad bean, horse bean, lentil, alfalfa, lupin, and faba bean dextrin. Preferably, the dextrin is pea dextrin or faba bean dextrin, more preferably pea dextrin.

[0017] In this specification, the term "pea" is considered in its broadest sense and particularly includes all wild varieties of "round pea" and all mutant varieties of "round pea" and "wrinkled pea", regardless of the usual purpose of the variety (human food, animal feed, and / or other uses). Such mutant varieties are specifically known as "r mutants", "rb mutants", "rug3 mutants", "rug4 mutants", "rug5 mutants", and "lam mutants", as described in the paper by CL Heydley et al. (HEYDLEY CL (1996) "Developing novel pea starches", Proceedings of the Symposium of the Industrial Biochemistry and Biotechnology Group of the Biochemistry Society, pp. 77-87). Preferred pea varieties are round pea varieties, particularly wild round pea varieties.

[0018] The at least one dextrin used in the present invention is selected from maltodextrins, particularly legume maltodextrins, especially faba bean or pea maltodextrins, more particularly pea maltodextrins. Preferably, the maltodextrin has a weight-average molecular weight selected within the range of 5,000 to 15,000 Daltons (Da), preferably 10,000 to 15,000 Da, more preferably 10,000 to 14,000 Da. The weight-average molecular weight can be determined by steric exclusion chromatography (SEC).

[0019] The use of maltodextrins, in particular legume maltodextrins, in particular faba bean or pea maltodextrins, more particularly pea maltodextrins, is of particular interest as it results in a crosslinked matrix with particularly advantageous properties, in particular with regard to swelling.

[0020] The at least one dextrin used in the present invention may also be selected from pyrodextrins, in particular corn pyrodextrins.

[0021] The at least one dextrin, particularly in the case of pyrodextrin, may be heated prior to the cross-linking step. The resulting paste is preferably cooled to room temperature prior to cross-linking.

[0022] The at least one dextrin can be used alone or in combination with at least one cyclodextrin. As used herein, the term "cyclodextrin" includes all cyclodextrins known in the art, such as natural, unsubstituted cyclodextrins containing 6 to 12 glucose units linked by a covalent bond between carbon 1 and carbon 4, including α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, each containing 6, 7, and 8 glucose units, respectively. Preferred cyclodextrins of the present invention are α-, β-, and γ-cyclodextrin, with natural β-cyclodextrin being most preferred.

[0023] In step b) of the process of the present invention, at least one dextrin, or at least one dextrin and at least one cyclodextrin, is crosslinked with sodium trimetaphosphate (STMP) in an aqueous medium containing an alkaline agent to form a water-insoluble crosslinked dextrin-based matrix.

[0024] Advantageously, step b is carried out in the absence of organic solvents, i.e., the aqueous medium does not contain organic solvents. Those skilled in the art can easily determine the reaction conditions and amounts of reagents to be used.

[0025] The term "alkaline agent" as used herein means a basic ion salt of an alkali metal or alkaline earth metal, such as a hydroxide or carbonate. The alkaline agent is specifically selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, sodium carbonate, or a mixture thereof. The preferred alkaline agent is sodium hydroxide.

[0026] Preferably, the alkaline agent is used in a molar ratio of alkaline agent / STMP greater than 1, preferably 1.5 or greater, more preferably 2.0 or greater, and even more preferably 2.5 or greater. In fact, the inventors have found that when the molar ratio of alkaline agent / STMP, particularly NaOH / STMP, is less than 1 or less than 2, phosphorylation of the dextrin and / or cyclodextrin prevails over reticulation. Preferably, this molar ratio is less than 5.0, more preferably 4.5 or less, even more preferably 4.0 or less, even more preferably 3.5 or less. Even more preferably, it is about 3, for example 3.1.

[0027] Preferably, the alkaline agent is present in an amount such that the pH of the aqueous medium is 8 to 14, preferably 10 to 14, especially about 12, before the addition of the dextrin and sodium trimetaphosphate.

[0028] The crosslinking reaction is carried out at a temperature of 18° C. to 40° C., preferably 18° C. to 30° C. Generally, the crosslinking step is carried out at room temperature, ie, at a temperature of 18 to 25° C.

[0029] The reaction time is related to the temperature at which crosslinking is carried out and can be easily adjusted by those skilled in the art, and is generally 10 minutes to 5 hours, preferably 15 minutes to 4 hours.

[0030] The STMP / dextrin or STMP / (dextrin and cyclodextrin) ratio varies depending on the dextrin or dextrin / cyclodextrin mixture used. The selection of an appropriate ratio is possible with the basic expertise of a person skilled in the art. Preferably, this ratio, expressed as dry weight / dry weight, is 80% or less, preferably 70% or less, preferably 60% or less, preferably 10% to 60%, and even more preferably 15% to 50%. It is preferably greater than 15%, more preferably greater than 20%, more preferably 25% or more, even more preferably greater than 25%, even more preferably 30% or more, even more preferably 35% or more, even more preferably 40% or more, and even more preferably 45% or more. For example, it is about 50%.

[0031] After the water-insoluble cross-linked dextrin-based matrix is ​​formed in step c), a mixture of the matrix and an aqueous medium is recovered in step c). The mixture may then be subjected to step d) to separate the water-insoluble cross-linked dextrin-based matrix from the aqueous medium. Separation can be performed by any suitable method known in the art, such as centrifugal filtration, filtration, or freeze-drying.

[0032] The separated matrix may be dried in step e) Optionally, the separated matrix may be washed with, for example, demineralized water and / or an alcohol, such as ethanol, after separation step d) and before drying.

[0033] In a second aspect, the present invention relates to a water-insoluble crosslinked dextrin-based matrix, in which at least one dextrin or at least one dextrin and at least one cyclodextrin are crosslinked with sodium trimetaphosphate. The at least one dextrin and at least one cyclodextrin are used in the above-described method for preparing a crosslinked dextrin-based matrix. The dextrin-based matrix of the present invention can be obtained according to this method. Therefore, the dextrin-based matrix of the present invention is preferably free of any organic solvent. The expression "free of organic solvent" in the sense of the present invention means that the matrix does not contain even traces of organic solvents resulting from the preparation method using one or more organic solvents.

[0034] The crosslinked dextrin-based matrix of the present disclosure may contain crosslinking components other than dextrin and cyclodextrin, as long as they do not interfere with the desired properties of the dextrin-based matrix. However, the crosslinked dextrin-based matrix of the present disclosure preferably contains no more than 30% by dry weight of crosslinking components other than dextrin and cyclodextrin, preferably no more than 20%, more preferably no more than 10%, more preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0%. The crosslinked dextrin-based matrix, which can be obtained according to the above-described method for preparing a crosslinked dextrin-based matrix, preferably comprises at least one dextrin crosslinked with sodium trimetaphosphate or at least one dextrin and at least one cyclodextrin. In other words, the dextrin-based matrix of the present disclosure preferably does not contain crosslinking components other than dextrin and cyclodextrin.

[0035] The cross-linked dextrin-based matrices of the present invention are water-insoluble. In the sense of the present invention, the term "water-insoluble" means that the matrix does not dissolve in water at room temperature, i.e., 18-25°C, at a pH of 7. Preferred cross-linked dextrin-based matrices of the present invention are insoluble in water at room temperature in the pH range of 5-9.

[0036] However, the cross-linked dextrin-based matrices of the present invention swell in water. The swelling capacity of the matrix can be characterized by the swelling index (SI), which is defined as follows: SI%=(Ws-Wd) / Wd * 100 where Wd = dry weight of matrix and Ws = weight of swollen matrix. To determine SI%, 1 g of dry matrix is ​​dispersed in 100 mL of demineralized water and allowed to swell for 24 hours. After 24 hours of contact, the mixture of dispersed matrix in water is centrifuged to separate the supernatant (water) from the bottom layer (swollen matrix or gel). The swollen matrix is ​​then weighed.

[0037] The swelling index of the crosslinked dextrin-based matrix of the present invention is preferably at least 200%, more preferably at least 500%, even more preferably at least 600%, more preferably at least 700%, more preferably at least 800%, more preferably at least 900%, even more preferably at least 1000%, even more preferably more than 1000%, even more preferably at least 1100%, even more preferably at least 1200%, even more preferably at least 1300%, even more preferably at least 1400%, even more preferably at least 1500%, even more preferably at least 1600%. It is generally at most 4000%, even at most 3500%, even at most 3000%, even at most 2500%, even at most 2000%.

[0038] Advantageously, the water-insoluble crosslinked matrices of the present invention have a negative zeta potential. Preferably, the zeta potential is between -10 mV and -50 mV, more preferably between -20 mV and -30 mV. The zeta potential can be determined by electrophoretic mobility as described in the Examples section.

[0039] The water-insoluble crosslinked matrix of the present invention can be in the form of particles. The average diameter of the matrix particles is, for example, 100 nm to 1000 nm, specifically 150 nm to 500 nm, and more specifically 200 nm to 300 nm. The matrix may be milled to obtain the appropriate particle size. Preferably, the matrix has a polydispersity index of 0.10 to 0.50, preferably 0.15 to 0.45, and more preferably 0.20 to 0.40. The average diameter and polydispersity index can be determined by laser light scattering, as described in the Examples section.

[0040] In one embodiment, the at least one dextrin cross-linked with STMP is a corn dextrin, particularly a corn pyrodextrin.

[0041] In another embodiment, the at least one dextrin cross-linked with STMP is a legume dextrin, preferably derived from legume starch having an amylose content of 25% to 50%, preferably 30% to 40%, particularly 35% to 40%, and more preferentially 35% to 38%, where these percentages are expressed as dry weight relative to the dry weight of the starch. The legume dextrin is selected from the group consisting of pea, bean, broad bean, horse bean, lentil, alfalfa, lupin, and faba bean dextrin. Preferably, the dextrin is pea dextrin or faba bean dextrin, more preferably pea dextrin.

[0042] In this specification, the term "pea" is considered in its broadest sense and particularly includes all wild varieties of "round pea" and all mutant varieties of "round pea" and "wrinkled pea", regardless of the usual purpose of the variety (human food, animal feed, and / or other uses). Such mutant varieties are specifically known as "r mutants", "rb mutants", "rug3 mutants", "rug4 mutants", "rug5 mutants", and "lam mutants", as described in the paper by CL Heydley et al. (HEYDLEY CL (1996) "Developing novel pea starches", Proceedings of the Symposium of the Industrial Biochemistry and Biotechnology Group of the Biochemistry Society, pp. 77-87). Preferred pea varieties are round pea varieties, particularly wild round pea varieties.

[0043] In another embodiment, the at least one dextrin cross-linked with STMP is derived from a starch having an amylose content of 25% to 50%, preferably 30% to 40%, particularly 35% to 40%, and more preferably 35% to 38%, where these percentages are expressed as dry weight relative to the dry weight of the starch. These dextrins are preferably legume dextrins. These dextrins are selected from the group consisting of pea, bean, broad bean, horse bean, lentil, alfalfa, lupin, and faba bean dextrins. Preferably, the dextrin is pea dextrin or faba bean dextrin, more preferably pea dextrin.

[0044] A water-insoluble cross-linked matrix in which at least one dextrin is derived from a starch having an amylose content as defined above has particularly advantageous properties, especially with regard to swelling.

[0045] The at least one crosslinked dextrin in the matrix of the present invention is selected from maltodextrins, particularly legume maltodextrins, specifically faba bean or pea maltodextrins, more specifically pea maltodextrins. Preferably, the maltodextrin has a weight-average molecular weight selected within the range of 5,000 to 15,000 Daltons (Da), preferably 10,000 to 15,000 Da, more preferably 10,000 to 14,000 Da. The weight-average molecular weight can be determined by steric exclusion chromatography (SEC).

[0046] The water-insoluble crosslinked matrix, in which at least one dextrin is a maltodextrin, in particular a legume maltodextrin, in particular a faba bean or pea maltodextrin, more in particular a pea maltodextrin, has particularly advantageous properties, in particular with regard to swelling.

[0047] The at least one cross-linked dextrin in the matrix of the present invention may also be chosen from pyrodextrins, in particular corn pyrodextrins.

[0048] The at least one dextrin, particularly in the case of pyrodextrin, may be heated prior to the cross-linking step. The resulting paste is preferably cooled to room temperature prior to cross-linking.

[0049] At least one dextrin is crosslinked with STMP, either alone or together with at least one cyclodextrin. The term "cyclodextrin" as used herein includes all cyclodextrins known in the art, such as natural, unsubstituted cyclodextrins containing 6 to 12 glucose units linked by a covalent bond between carbon 1 and carbon 4, including α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, each containing 6, 7, and 8 glucose units, respectively. Preferred cyclodextrins of the present invention are α-, β-, and γ-cyclodextrin, with natural β-cyclodextrin being most preferred.

[0050] The water-insoluble crosslinked dextrin-based matrix of the present invention can be loaded with active ingredients. Therefore, a third aspect of the present invention relates to the use of the water-insoluble crosslinked dextrin-based matrix of the present invention as a carrier for organic compounds. The matrix of the present invention can actually load various types of organic compounds, including cationic compounds, nonionic compounds, and complex compounds such as polypeptides. These organic compounds are particularly selected from active ingredients. Within the meaning of the present invention, the term "pharmaceutical active ingredient" includes both low-molecular-weight active ingredients and high-molecular-weight active ingredients. High-molecular-weight active ingredients include, but are not limited to, proteins such as insulin, antibodies, and nucleotides. The active ingredient may be, for example, a pharmaceutical active ingredient, a bioactive ingredient, or a food active ingredient. Preferably, the active ingredient of the present disclosure is a high-molecular-weight active ingredient. More preferably, the active ingredient of the present disclosure is a protein, even more preferably insulin.

[0051] The water-insoluble cross-linked dextrin-based matrix of the present invention is particularly useful for the sustained release of active ingredients in the human or animal body by oral administration. Thus, in a fourth aspect, the present invention relates to an oral delivery system comprising the water-insoluble cross-linked dextrin-based matrix of the present invention and an active ingredient, wherein the active ingredient is supported in the matrix. In other words, the water-insoluble cross-linked dextrin-based matrix of the present invention is used as a carrier for the active ingredient. Suitable active ingredients are those described above.

[0052] Due to their ability to retain compounds, the water-insoluble crosslinked matrices of the present invention are also useful for capturing pollutants in water or air. In particular, they can be used to retain cationic organic pollutants or metal cations. Cationic organic pollutants include, for example, cationic small molecule active ingredients and cationic dyes. The crosslinked dextrin-based matrices of the present invention can be used, for example, as filter media for filtering air or water.

[0053] The present invention will be better understood with reference to the following illustrative and non-limiting examples and drawings. [Brief explanation of the drawings]

[0054] [Figure 1] 1 shows insulin release over time from an insulin-loaded matrix of the present invention at pH=1.2. [Figure 2] 1 shows insulin release over time from an insulin-loaded matrix of the present invention at pH=6.8. [Example]

[0055] The following starting materials were used in the synthesis of the crosslinked matrix:

[0056] KLEPTOSE Linecaps® 17 (Roquette Freres): Pea maltodextrin.

[0057] Stabilys® A025 (Roquette Freres): Corn pyrodextrin.

[0058] Stabilys® A053 (Roquette Freres): Corn pyrodextrin.

[0059] Sodium trimetaphosphate (STMP, Na3P3O9, CAS No. 7785-84-4): Sigma-Aldrich, purity 95%.

[0060] Example 1: Synthesis of a maltodextrin-based matrix of the present invention cross-linked with 60% STMP A glass reactor equipped with a mechanical stirrer was charged with 105.2 g of Linecaps 17 (residual moisture 4.9 wt. %, 100 g dry matter).

[0061] 20% by weight of NaOH (20 g, 0.5 moles) based on the dry weight of starch was added with 10% NaOH solution (200 g) under stirring.

[0062] The reaction was left under stirring at room temperature (about 20-25° C.) for 3.5 hours.

[0063] Sodium trimetaphosphate (60 g, 0.196 mol) was added under stirring in an amount of 60% by weight based on the dry weight of starch. The reaction mixture was allowed to stand for 1.5 hours.

[0064] After a few minutes the mixture was observed to gel and stirring was stopped.

[0065] The crude material was then collected.

[0066] The solid was crushed and dispersed in a sufficient amount of water to give a stirred suspension. The crude product was neutralized by adding HCl until the residual pH was 6.5.

[0067] The mixture was centrifuged at 4700 rpm for 15 minutes using a VMR Mega Star 1.6 centrifuge. The supernatant was removed and the resulting matrix was washed with demineralized water. After stirring for 15 minutes, the mixture was centrifuged at 4700 rpm for 15 minutes using the same centrifuge as before. The supernatant was removed and the resulting gel was washed two more times with demineralized water.

[0068] The final matrix was collected and precipitated in ethanol with stirring. The resulting white powder was filtered and dried under vacuum. The product was recovered in 59% yield (based on the dry weight of the recovered product / initial amount of dried Linecaps + STMP added to the reaction mixture).

[0069] Example 2: Synthesis of a maltodextrin-based matrix of the present invention cross-linked with 50% STMP A glass reactor equipped with a mechanical stirrer was charged with 105.2 g of Linecaps 17 (residual moisture 4.9%, 100 g of dry matter).

[0070] 20% by weight of NaOH (20 g, 0.5 moles) based on the dry weight of starch was added with 10% NaOH solution (200 g) under stirring.

[0071] The reaction was left under stirring at room temperature (about 20-25° C.) for 3.5 hours.

[0072] Sodium trimetaphosphate (50 g, 0.163 mol) was added under stirring in an amount of 50% by weight based on the dry weight of starch. The reaction mixture was allowed to stand for 1.5 hours.

[0073] After a few minutes the mixture was observed to gel and stirring was stopped.

[0074] The crude material was then collected.

[0075] The solid was crushed and dispersed in a sufficient amount of water to give a stirred suspension. The crude product was neutralized by adding HCl until the residual pH was 6.5.

[0076] The reaction mixture was centrifuged at 4700 rpm for 15 minutes using a VMR Mega Star 1.6 centrifuge. The supernatant was removed and the resulting gel was washed with demineralized water. After stirring for 15 minutes, the mixture was centrifuged at 4700 rpm for 15 minutes using the same centrifuge as before. The supernatant was removed and the resulting matrix was washed two more times with demineralized water.

[0077] The final matrix was collected and precipitated in ethanol with stirring. The resulting white powder was filtered and dried under vacuum. The product was recovered in 54% yield (based on the dry weight of the recovered product / the initial amount of dry Linecaps + STMP added to the reaction mixture).

[0078] Example 3: Synthesis of a maltodextrin-based matrix of the present invention cross-linked with 40% STMP A glass reactor equipped with a mechanical stirrer was charged with 525.8 g of Linecaps 17 (residual moisture 4.9%, 500 g dry matter).

[0079] 16% by weight of NaOH (80 g, 2 moles) based on the dry weight of starch was added with 10% NaOH solution (800 g) under stirring.

[0080] The reaction was left under stirring at room temperature (about 20-25° C.) for 3.5 hours.

[0081] Sodium trimetaphosphate (200 g, 0.653 mol) was added under stirring at 40% by weight based on the dry weight of starch. The reaction mixture was left for 1.5 hours.

[0082] After a few minutes the mixture was observed to gel and stirring was stopped.

[0083] The crude material was then collected.

[0084] The solid was crushed and dispersed in a sufficient amount of water to give a stirred suspension. The crude product was neutralized by adding HCl until the residual pH was 6.5.

[0085] The reaction mixture was centrifuged at 4700 rpm for 15 minutes using a VMR Mega Star 1.6 centrifuge. The supernatant was removed and the resulting gel was washed with demineralized water. After stirring for 15 minutes, the mixture was centrifuged at 4700 rpm for 15 minutes using the same centrifuge as before. The supernatant was removed and the resulting matrix was washed two more times with demineralized water.

[0086] The final matrix was collected and precipitated in ethanol with stirring. The resulting white powder was filtered and dried under vacuum. The product was recovered in 59% yield (based on the dry weight of the recovered product / initial amount of dried Linecaps + STMP added to the reaction mixture).

[0087] Example 4: Synthesis of a maltodextrin-based matrix of the present invention cross-linked with 25% STMP A glass reactor equipped with a mechanical stirrer was charged with 525.8 g of Linecaps 17 (residual moisture 4.9%, 500 g dry matter).

[0088] 10% by weight of NaOH (50 g, 1.25 moles) based on the dry weight of starch was added with 10% NaOH solution (500 g) under stirring.

[0089] 50 g of demineralized water was added to the reaction mixture to ensure good stirring.

[0090] The reaction was left under stirring at room temperature (about 20-25° C.) for 3.5 hours.

[0091] Sodium trimetaphosphate (125 g, 0.408 mol) was added under stirring at 25% by weight based on the dry weight of starch. The reaction mixture was allowed to stand for 1.5 hours.

[0092] After a few minutes the mixture was observed to gel and stirring was stopped.

[0093] The crude material was then collected.

[0094] The solid was crushed and dispersed in a sufficient amount of water to give a stirred suspension. The crude product was neutralized by adding HCl until the residual pH was 6.5.

[0095] The reaction mixture was centrifuged at 4700 rpm for 15 minutes using a VMR Mega Star 1.6 centrifuge. The supernatant was removed and the resulting gel was washed with demineralized water. After stirring for 15 minutes, the mixture was centrifuged at 4700 rpm for 15 minutes using the same centrifuge as before. The supernatant was removed and the resulting matrix was washed two more times with demineralized water.

[0096] The final matrix was collected and precipitated in ethanol with stirring. The resulting white powder was filtered and dried under vacuum. The product was recovered in 58% yield (based on the dry weight of the recovered product / initial amount of dried Linecaps + STMP added to the reaction mixture).

[0097] Example 5: Synthesis of a maltodextrin-based matrix of the present invention cross-linked with 20% STMP A glass reactor equipped with a mechanical stirrer was charged with 525.8 g of Linecaps 17 (residual moisture 4.9%, 500 g dry matter).

[0098] 8% by weight of NaOH (40 g, 1 mole) based on the dry weight of starch was added under stirring with 10% NaOH solution (400 g).

[0099] 140 g of demineralized water was added to the reaction mixture to ensure good stirring.

[0100] The reaction was left under stirring at room temperature (about 20-25° C.) for 3.5 hours.

[0101] Sodium trimetaphosphate (100 g, 0.327 mol) was added under stirring at 20% by weight based on the dry weight of starch. The reaction mixture was left for 1.5 hours.

[0102] After a few minutes the mixture was observed to gel and stirring was stopped.

[0103] The crude material was then collected.

[0104] The solid was crushed and dispersed in a sufficient amount of water to give a stirred suspension. The crude product was neutralized by adding HCl until the residual pH was 6.5.

[0105] The reaction mixture was centrifuged at 4700 rpm for 15 minutes using a VMR Mega Star 1.6 centrifuge. The supernatant was removed and the resulting gel was washed with demineralized water. After stirring for 15 minutes, the mixture was centrifuged at 4700 rpm for 15 minutes using the same centrifuge as before. The supernatant was removed and the resulting matrix was washed two more times with demineralized water.

[0106] The final matrix was collected and precipitated in ethanol with stirring. The resulting white powder was filtered and dried under vacuum. The product was recovered in a 57% yield (based on the dry weight of the recovered product / the initial amount of dry Linecaps + STMP introduced into the reaction mixture).

[0107] Example 6: Synthesis of a pyrodextrin-based matrix of the present invention cross-linked with 60% STMP Example 6a: A glass reactor equipped with a mechanical stirrer was charged with a slurry of 20% by weight of dry substance consisting of 100 g of dry Stabilys® A053 (amount calculated after determining the residual moisture, 100 g of dry substance) and 400 g of demineralized water. The preparation was heated to 95°C, and the slurry became a paste (yellow). After heating at 95°C for 30 minutes, the reaction mixture was cooled to 25°C before adding sodium hydroxide solution.

[0108] 20% by weight of NaOH (20 g, 0.5 moles) based on the dry weight of starch was added under stirring with 10% NaOH solution (200 g).

[0109] The reaction was left under stirring at room temperature (about 20-25° C.) for 3.5 hours.

[0110] Sodium trimetaphosphate (60 g, 0.196 mol) was added under stirring in an amount of 60% by weight based on the dry weight of starch. The reaction mixture was allowed to stand for 1.5 hours.

[0111] After a few minutes the mixture was observed to gel and stirring was stopped.

[0112] The crude material was then collected.

[0113] The solid was crushed and dispersed in a sufficient amount of water to give a stirred suspension. The crude product was neutralized by adding HCl until the residual pH was 6.5.

[0114] The reaction mixture was centrifuged at 4700 rpm for 15 minutes using a VWM Mega Star 1.6 centrifuge. The supernatant was removed and the resulting matrix was washed with demineralized water. After stirring for 15 minutes, the mixture was centrifuged at 4700 rpm for 15 minutes using the same centrifuge as before. The supernatant was removed and the resulting matrix was washed two more times with demineralized water.

[0115] The final matrix was collected and precipitated in ethanol with stirring. The resulting white powder was filtered and dried under vacuum. The product was recovered in a yield of 58% (based on the dry weight of the recovered product / initial amount of dry Stabilys® A053 + STMP added to the reaction mixture).

[0116] Example 6b: Example 3a was repeated, replacing the 20 wt.-% dry matter slurry of Stabilys® A053 with a 15 wt.-% dry matter slurry of Stabilys® A025 (75 g Stabilys® A025 in 425 g demineralized water). The product was recovered in a yield of 68% (based on the dry weight of the recovered product / initial amount of dry Stabilys® A025+STMP added to the reaction mixture).

[0117] Example 7: Solubility of the matrix of the present invention The solubility of the matrices of Examples 1-3 in water was determined according to the following protocol.

[0118] 250 mg of each matrix was placed in a vial. 5 mL of deionized water was added to each vial. All samples were periodically stirred and observed.

[0119] The swelling and dissolution of each sample were observed for up to 72 hours. To confirm the solubility, the viscosity and transparency of the supernatant (water) were carefully observed under a magnifying glass. Swelling and dissolution of the sample could be clearly distinguished by this visual evaluation.

[0120] For each matrix, tests were performed at pH 7, pH 5 (addition of HCl) and pH 9 (addition of NaOH).

[0121] All samples were insoluble under the test conditions, however, they exhibited significant swelling.

[0122] Example 8: Swelling capacity of the matrix of the present invention The swelling index (SI) of the matrices of Examples 2-4 was measured according to the following protocol.

[0123] 1 g (dry weight) of product was dispersed in 100 mL of demineralized water in a graduated cylinder and allowed to swell for 24 hours. After 24 hours of contact, the gel-water mixture was centrifuged to separate the supernatant and the bottom layer (swollen gel). The swollen gel was weighed to determine the amount of water absorbed.

[0124] SI was calculated as above.

[0125] The results are shown in Table 1 below. [Table 1]

[0126] Example 9: pH value, mean diameter and polydispersity of matrices of the invention The pH values ​​of the matrices of Examples 2, 3, 4 and 5 were determined using a pH meter (Orion Model 420A).

[0127] The mean 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, New York, USA), and the zeta potential was determined by electrophoretic mobility using the same instrument.

[0128] Each analysis was performed on a matrix suspension prepared as follows. 1. Prepare a suspension of the coarse powder in distilled water at a concentration of 10 mg / mL by stirring at room temperature. 2. Disperse the suspension using a high shear homogenizer (Ultraturrax®, IKA, Königswinter, Germany) at 24000 rpm for 10 minutes. 3. High pressure homogenization using an EmulsiFlex C5 device (Avastin, USA) at 500 bar back pressure for 90 minutes for further size reduction. 4. The homogenized nanosuspension is purified by dialysis (Spectrapore, cellulose membrane, cut-off 12000 Da) to remove any potentially present synthetic residues. 5. Store the nanosuspension at 4°C.

[0129] The results are shown in Table 2 below. [Table 2]

[0130] Example 10: Methylene Blue Loading Capacity of the Matrix of the Present Invention Methylene blue was used as a model for organic cationic compounds to demonstrate the ability of the matrix of the present invention to retain organic cationic compounds.

[0131] 2 g (dry weight) of cross-linked matrix was added to 10 -5The gel was dispersed in 100 mL of an aqueous methylene blue solution and allowed to swell for 24 hours. After 24 hours of contact, the mixture of gel dispersed in the aqueous methylene blue solution was centrifuged to separate the supernatant and the lower layer (blue swollen gel).

[0132] The concentration of methylene blue remaining in the supernatant was determined using UV-visible spectroscopy.

[0133] The methylene blue absorption capacity (%) was calculated as the ratio of the amount of methylene blue retained by the matrix to the amount of methylene blue initially added × 100. The amount of methylene blue retained by the matrix corresponds to the difference between the amount of methylene blue initially added and the amount of methylene blue present in the supernatant.

[0134] The results are shown in Table 2 below. [Table 3]

[0135] Example 11: Loading of insulin in a matrix of the invention Insulin from bovine pancreas powder was used to prepare a 2 mg / mL solution in distilled water adjusted to pH 2.3 with phosphoric acid. The insulin solution was added to a preformed aqueous nanosuspension of crosslinked matrix (according to the protocol described in Example 9) at a weight ratio of 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 precipitate, which was collected and lyophilized.

[0136] Lyophilized insulin-loaded matrices were prepared from the matrices of Examples 1 and 2 according to this procedure.

[0137] Insulin-carrying capacity The loading capacity was determined from the lyophilized insulin-loaded samples according to the following protocol.

[0138] 2-3 mg of lyophilized insulin-loaded crosslinked matrix was dispersed in 5 mL of distilled water. Insulin was released from the delivery system by sonication (15 min, 100 W) and centrifugation. The supernatant was then analyzed for quantitative insulin determination.

[0139] Quantitative measurement of insulin was performed using high-performance liquid chromatography (HPLC) (PerkinElmer 250B, Waltham, MA) equipped with a spectrophotometer detector (Flexar UV / Vis LC, PerkinElmer, Waltham, MA). An analytical column, C18 (250 mm x 4.6 mm, ODS Ultrasphere 5 μm; 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 μm nylon membrane, and degassed by ultrasound before use. UV detection was fixed at 214 nm, and the flow rate was set at 1 mL / min. The insulin concentration was calculated using the external standard method based on a standard calibration curve. For this purpose, 1 mg of insulin was weighed into a 10 mL flask and dissolved in distilled water adjusted to pH 2.3 with phosphoric acid to obtain the mother liquor. This solution was then diluted with the mobile phase to prepare a series of standard solutions, which were then injected into the HPLC system. A linear standard curve was obtained over the concentration range of 0, 5 to 25 μg / mL with a regression coefficient of 0.999.

[0140] The insulin-loading capacity (%) of the delivery system was calculated as follows: [weight of insulin / weight of lyophilized cross-linked matrix]×100.

[0141] The results are shown in Table 3 below. [Table 4]

[0142] Example 12: Insulin release In vitro drug release kinetics In vitro drug release experiments were performed in a multicompartment rotating disk (a diffusion cell system containing a donor chamber separated by a membrane from a donor compartment) consisting of several donor cells on one side separated from receiver cells on the other side by a cellulose membrane (Spectrapore, cutoff 50 kDa). The lyophilized insulin-loaded crosslinked matrix prepared in Example 11 from the matrix in Example 2 was placed in the donor cell (1 mL). The receiver cells were filled with phosphate-buffered saline (PBS) solutions of pH 1.2 and pH 6.8, respectively. The in vitro release test was carried out for 24 hours, during which the receiver phase was withdrawn at regular intervals and replaced with an equal volume of fresh 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 insulin concentration in the withdrawn samples was subsequently detected by HPLC.

[0143] The results are shown in Figures 1 and 2. Figure 1 shows only the results up to 3 hours, since there was no release after 3 hours.

[0144] The crosslinked matrices of the present invention do not release insulin at stomach pH (FIG. 1, pH 1.2), but can release insulin at intestinal pH (FIG. 2, pH 6.8). In other words, the matrices of the present invention do not release insulin at pHs where insulin is hydrolyzed due to the high acidity of the stomach, but can release insulin in the intestine where insulin absorption is desired. FIG. 2 also shows that the matrices of the present invention continue to release insulin over several hours.

[0145] Furthermore, the crosslinked matrices of the present invention advantageously allow for sustained release of insulin, meaning that insulin may be bioavailable over an extended period of time and are less likely to cause a spike in blood insulin after ingestion of the crosslinked matrices of the present invention. Thus, the crosslinked matrices of the present invention reduce the risk of harmful hypoglycemia caused by a spike in blood insulin.

[0146] In vivo experiments The lyophilized insulin-loaded crosslinked matrix prepared in Example 11 from the matrix of Example 2 was administered to rats via oral gavage into their stomachs. The administered dose of insulin was 2.10 mg / kg. Blood samples were taken at various time points.

[0147] Insulin was extracted from plasma samples obtained from the collected blood samples according to the following protocol. 100 μl of plasma was added to each sample, along with 100 μl of PBS (pH 7.4), 50 μl of acetonitrile, 20 μl of ethylparaben, and 3 mL of dichloromethane / n-hexane (1:1 v / v). The mixture was vortexed for 2 minutes and then centrifuged at 5,000 rpm for 10 minutes. The supernatant was transferred to a test tube. 300 μl of 0.05 N HCl was then added, and the mixture was vortexed for 2 minutes under a nitrogen stream. After complete evaporation of the organic phase under a nitrogen stream, the remaining supernatant was centrifuged at 15,000 rpm for 10 minutes. A clear supernatant was obtained. The supernatant samples were stored in a -18°C freezer and analyzed by HPLC and ELISA.

[0148] HPLC analysis: HPLC analysis was performed on a PerkinElmer 250B HPLC system, and peak integration was performed using Chromera software. The HPLC experimental conditions were as follows: Loop: 20 μl Flow rate: 1ml / min Pressure: 180 bar * Column: Agilent TC-C18(2) 5 mer μm (4.6 mm x 150 mm, USA). λ:214nm Apparatus: PerkinElmer 250B, Waltham, Massachusetts Eluent: a mixture of 42 volumes of mobile phase A (a solution of 28.4 g of anhydrous sodium sulfate in 1000 mL of water, pH = 2.3 using phosphoric acid) and 58 volumes of mobile phase B (a mixture of 550 mL of mobile phase A and 450 mL of acetonitrile).

[0149] The results are shown in Table 4. [Table 5]

[0150] ELISA assay ELISA assays (Sigma-Aldrich ELISA kit) were performed on samples taken at 15, 60, and 360 minutes. They are listed in the table below along with the average absorbance readings at 450 nm measured on a Perkin-Elmer instrument and the corresponding concentrations (µIU / mL). The results are shown in Table 5. [Table 6]

[0151] * The linear regression equation was calculated using a standard curve of insulin prepared according to the protocol provided in the "Certificate of Analysis" of the ELISA kit provided by Sigma-Aldrich. y=0.0061x+0.0819, and the correlation coefficient is 0.954.

[0152] **Samples were diluted 1:4.

[0153] The results of the HPLC analysis and ELISA assays show that the administered insulin is found in the blood, thus confirming that the matrix of the present invention is useful for oral administration of insulin.

Claims

1. 1. A method for preparing a water-insoluble cross-linked dextrin-based matrix, comprising: the cross-linked dextrin-based matrix consists of at least one dextrin or at least one dextrin and at least one cyclodextrin cross-linked with sodium trimetaphosphate; a. providing at least one dextrin or at least one dextrin and at least one cyclodextrin; b. cross-linking the dextrin or dextrin and cyclodextrin with sodium trimetaphosphate (STMP) in an aqueous medium containing an alkaline agent to form a matrix based on the water-insoluble cross-linked dextrin; c) recovering the mixture of the water-insoluble cross-linked dextrin-based matrix and the aqueous medium; The method of preparation, wherein the alkaline agent is used in a molar ratio of alkaline agent / STMP greater than 1.

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

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

4. The method according to any one of claims 1 to 3, wherein the crosslinking is carried out in the absence of organic solvents.

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