Use of a modified chitosan and nanocomposites comprising the same

Abstract

The present application relates to the use of modified chitosan having structural units shown in the following formula as a carrier of protein polypeptide drugs: wherein R in each structural unit is the same or different, and each is independently hydrogen, acetyl, or a residue obtained by amidation reaction of cholic acid compound with amino in raw chitosan, wherein the weight average molecular weight of the raw chitosan is 1,000-1,000,000, and the degree of deacetylation is 70%-95%, wherein the degree of substitution of cholic acid compound in the modified chitosan is 0.5%-20%. The present application also relates to a nanocomposite comprising the modified chitosan, a preparation method and use thereof. The nanocomposite is mainly used for promoting the oral absorption of protein polypeptide drugs, and has a good effect of improving the biological efficacy of protein polypeptide drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, more particularly, to the use of a modified chitosan, a nanocomposite comprising the modified chitosan, a preparation method and use thereof. The nanocomposite described in the present application is mainly used for promoting the oral absorption of protein polypeptide drugs, and has the effect of improving the biological efficacy of protein polypeptide drugs. TECHNICAL BACKGROUND

[0002] With the development of biotechnology, protein polypeptide drugs with specific pharmacological activities play an increasingly important role in clinical applications. So far, dozens of protein polypeptide drugs have been marketed worldwide. As the most convenient administration method, the research on the oral administration of protein polypeptide drugs has shown its urgency and importance.

[0003] However, due to the degradation of protein polypeptide drugs in the acidic environment of the stomach, the degradation of protein hydrolytic enzymes in the gastrointestinal tract, the large molecular weight of the drugs, the poor lipid solubility, and the difficulty of penetrating biological membranes, the oral administration of protein polypeptide drugs faces great challenges.

[0004] Currently, the research on the oral administration of protein polypeptide drugs mainly includes the following five aspects: enzyme inhibitors, absorption promoters, bioadhesive drug delivery systems, microparticle carrier drug delivery systems, and colon-targeted drug delivery systems.

[0005] Protein polypeptide drug nanocarriers belong to the microparticle carrier drug delivery system. As drug carriers, they have many advantages, such as sustained-release characteristics, prolonged drug action time, reduced drug dosage under the premise of ensuring drug action, reduced drug toxicity and side effects, improved drug stability, and convenient drug storage. Furthermore, due to the ultra-small size and high drug loading capacity of nanocarriers, they can improve local drug concentration, promote the absorption of difficult-to-penetrate membrane protein polypeptide drugs, and thus improve the therapeutic effect.

[0006] Chitosan is a natural high-molecular-weight alkaline polysaccharide with good biocompatibility and biodegradability, and is widely used as a drug carrier material. Chitosan-based nanodrug delivery systems have gradually become one of the popular drug carriers for protein polypeptide drugs due to their high capacity for loading protein polypeptide drugs and their ability to improve the transmembrane absorption of protein polypeptide drugs to some extent. By modifying the structure of chitosan to optimize the properties of nanocarriers and further improve the transmembrane transport capacity to improve the oral absorption of protein polypeptide drugs, it is expected to achieve the oral administration of protein polypeptide drugs. Such carriers have become one of the research hotspots in recent years.

[0007] CN 102614498A discloses an insulin-anion surfactant nanocomposite to improve the stability of insulin in the gastrointestinal tract and promote the penetration of the drug through the biological membrane, but the nanocomposite preparation process involves organic solvents, which has a certain impact on the stability of insulin. CN 102120781A discloses a nanocarrier with chitosan as the matrix, which is modified by arginine and lysine to improve the water solubility of chitosan and promote the absorption effect, but due to the strong hydrophilicity and large molecular weight of protein polypeptide drugs, it is extremely difficult for such drugs to passively diffuse through the small intestinal epithelial cell monolayer, and ordinary nanocarriers cannot improve the oral absorption of protein polypeptide drugs to the ideal level.

[0008] Therefore, it is always a difficult problem for formulators to design a nanocarrier which has a simple and mild preparation method, stable properties, can be stored for a long time, and can effectively improve the membrane permeability of protein polypeptide drugs and the oral absorption of protein polypeptide drugs. SUMMARY

[0009] The inventors of the present application have found through in-depth and meticulous research that the modified chitosan obtained by modifying chitosan with cholic acid compounds (such as deoxycholic acid, which is a substrate of apical sodium-dependent bile acid transporter ASBT, and taurocholic acid) can be used as a carrier for protein polypeptide drugs, which helps to maintain the stability of the loaded protein polypeptide drugs, promotes the transmembrane of protein polypeptide drugs, effectively promotes the absorption thereof, and further improves the biological efficacy of protein polypeptide drugs. The modified chitosan can be self-assembled with protein polypeptide drugs to form a nanocomposite. The nanocomposite can effectively protect protein polypeptide drugs from degradation by proteases in the intestinal tract, thereby improving the stability of the drug in the intestinal tract. In addition, the nanocomposite can be stored for a long time after being dried into a powder. Furthermore, the preparation method is simple and mild.

[0010] An object of the present application is to provide the use of chitosan modified with cholic acid compounds as a carrier for protein polypeptide drugs.

[0011] Another object of the present application is to provide a nanocomposite, a preparation method and use thereof.

[0012] One aspect of the present application relates to the use of a modified chitosan having a structural unit shown in the following formula 1 as a carrier for protein polypeptide drugs,

[0013]

[0014] wherein R in each structural unit is the same or different, and each is independently hydrogen, acetyl, or a residue obtained by amidation reaction of a cholic acid compound with an amino group in the raw material chitosan,

[0015] The raw chitosan has a weight average molecular weight of about 1,000 to about 1,000,000, preferably about 10,000 to about 500,000, and particularly about 50,000 to about 300,000, and a degree of deacetylation of about 70% to about 95%, such as about 75%, about 80%, about 85%, about 90%, about 95%, and the like.

[0016] The raw chitosan has a dynamic viscosity of 20 mpa·s to 300 mpa·s at 20°C, which is tested according to the viscosity determination method of chitosan for pharmaceutical use in the 2015 edition of the Chinese Pharmacopoeia: 1.0 g of the raw chitosan is precisely weighed, 100 ml of 1% glacial acetic acid is added, and stirring is performed to completely dissolve the raw chitosan, and a Brookfield LVDV-III Ultra type rotary viscometer is used for determination.

[0017] The degree of substitution of the cholic acid compound in the modified chitosan is about 0.5% to about 20%, and preferably about 1% to about 10%.

[0018] The degree of deacetylation represents the average percentage of the number of structural units (i.e., the number of deacetylated glycosyl groups) removed from the acetyl group in the raw chitosan molecule to the total number of structural units (i.e., the total number of glycosyl groups) in the raw chitosan molecule. The degree of substitution of the cholic acid compound represents the average percentage of the number of structural units (i.e., the total number of glycosyl groups) substituted by the cholic acid compound in the modified chitosan molecule to the total number of structural units (i.e., the total number of glycosyl groups).

[0019] The cholic acid compound is preferably selected from the compounds represented by the following formula 2 or salts thereof:

[0020]

[0021] wherein R1 is selected from -OH, carboxymethylamino, and sulfonic acid ethylamino; R2 is -OH; R3 is -H or -OH; and R4 is -H or -OH.

[0022] Preferably, the cholic acid compound is selected from cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid, isoursodeoxycholic acid, Lago deoxycholic acid, glycocholic acid, taurocholic acid, and salts thereof. The structures of the cholic acid derivatives are as follows:

[0023]

[0024] wherein the names of the cholic acid compounds and the corresponding R1, R2, R3, and R4 groups are as follows:

[0025]

[0026] Most preferably, the cholic acid compound is selected from cholic acid, deoxycholic acid, taurocholic acid, and salts thereof.

[0027] The salt can be an alkali metal salt, an alkaline earth metal salt, or an ammonium salt, and is preferably a potassium salt, a sodium salt, and an ammonium salt.

[0028] In the case where the cholic acid compound is selected from cholic acid, deoxycholic acid, taurocholic acid, and salts thereof, the residue obtained by subjecting the amino group in the raw chitosan to an amidation reaction with the cholic acid compound is a residue represented by Formula 3, Formula 4, or Formula 5 below:

[0029]

[0030]

[0031] wherein * indicates a position bound to the chitosan skeleton.

[0032] The modified chitosan can be prepared by subjecting the raw chitosan to an amidation reaction with a cholic acid compound. The amidation reaction can be performed in the presence of a catalyst. The catalyst can be a catalyst conventionally used in the art for promoting the amidation reaction of a carboxylic acid or a sulfonic acid with an amino group. For example, the catalyst can be selected from the group consisting of l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, 2-(7-oxazolyl)benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and O-benzotriazolyl-tetramethyluronium hexafluorophosphate, and the like. The time for the amidation reaction is not particularly limited, as long as a desired chitosan modified with a cholic acid compound can be obtained, and can be, for example, 1 hour or more, 2 hours or more, 5 hours or more, or 10 hours or more, and 40 hours or less, 30 hours or less, 25 hours or less, and the like. The temperature for the amidation reaction is not particularly limited, as long as the reaction can be performed, and can be performed, for example, at room temperature.

[0033] In the amidation reaction, the molar ratio of the catalyst to the raw chitosan can be selected to be about 2: 1 to about 1:2, and the mass ratio of the cholic acid compound to the raw chitosan can be about 1:10 to about 1:2. In the amidation reaction, the pH of the reaction mixture is not particularly required, as long as the amidation reaction can be performed, and preferably, the pH of the reaction mixture is in the range of about 4 to about 7, preferably about 4.5 to about 6.5.

[0034] In one example, the amidation reaction is carried out as follows: after the raw chitosan is completely dissolved in about 1% (v / v) aqueous acetic acid solution, a cholic acid compound and a dimethyl sulfoxide solution of a catalyst 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are added, the pH is adjusted to about 4.5 to about 6.5, and after the reaction is carried out for about 16 to about 24 hours, the modified chitosan is obtained, the pH is adjusted to about 8.0 to about 10.0 with an aqueous base solution, the precipitate is separated by centrifugation, and the modified chitosan solid is obtained after drying. The molar ratio of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to the raw chitosan is preferably about 2:1 to about 1:2, and the mass ratio of the cholic acid compound to the raw chitosan is preferably about 1:10 to about 1:2.

[0035] The raw chitosan has a structural unit shown in the following Formula 6:

[0036]

[0037] wherein R in each structural unit is the same or different and each is independently hydrogen or an acetyl group;

[0038] The raw chitosan has a weight average molecular weight (Mw) of about 1,000 to about 1,000,000, preferably about 10,000 to about 500,000, and particularly about 50,000 to about 300,000, and a degree of deacetylation of about 70% to about 95%, such as about 75%, about 80%, about 85%, about 90%, about 95%, and the like.

[0039] The raw chitosan has a dynamic viscosity of 20 mpa·s to 300 mpa·s at 20°C, and the dynamic viscosity is tested according to the viscosity determination method for chitosan as a pharmaceutical excipient in the 2015 edition of the Chinese Pharmacopoeia: 1.0 g of the raw chitosan is accurately weighed, 100 ml of 1% glacial acetic acid is added, and stirring is performed to completely dissolve the chitosan, and the Brookfield LVDV-III Ultra type rotational viscometer is used for determination.

[0040] According to another aspect of the present application, a nanocomposite is provided, comprising, in weight percentage:

[0041] about 0.5% to about 90%, preferably about 5% to about 80%, of the modified chitosan as described above;

[0042] about 0.5% to about 90%, preferably about 5% to about 90%, more preferably about 20% to about 50%, of the protein polypeptide drug;

[0043] about 0% to about 25%, preferably about 0.5% to about 15%, of the anionic crosslinking agent; and

[0044] about 0% to about 25%, preferably about 0% to about 20% of a backbone polymer.

[0045] The average particle size of the nanocomposite is preferably no more than 800 nm, more preferably about 100 to about 500 nm. Preferably, 90% of the particles of the nanocomposite have a particle size of no more than 2 μm, preferably in the range of about 200 to about 1,000 nm. Preferably, the Zeta potential of the nanocomposite is no more than +65 mV, preferably about +15 mV to about +45 mV.

[0046] The protein polypeptide drug can be any pharmacologically active protein polypeptide drug, preferably selected from the group consisting of calcitonin, insulin, recombinant human parathyroid hormone, epidermal growth factor, GLP-1 (human glucagon-like peptide-1) analogs, and interferon, etc.

[0047] The anionic crosslinking agent can be any anionic compound capable of crosslinking the modified chitosan and the protein polypeptide drug, for example, it can be a water-soluble compound that is negatively charged after dissolution. The addition of the anionic crosslinking agent to the nanocomposite of the present application can further improve the stability of the nanocomposite in a physiological environment. Preferably, the anionic crosslinking agent can be selected from the group consisting of polyglutamic acid and sodium polyphosphate.

[0048] The backbone polymer can be any high molecular material capable of encapsulating and compressing the nanocomposite, for example, it can be a water-soluble high molecular material. The addition of the backbone polymer to the nanocomposite of the present application can further improve the stability of the nanocomposite. Preferably, the backbone polymer is selected from the group consisting of polyacrylamide, poly(beta-amino ester), and polyvinyl alcohol.

[0049] In addition to the above components, the nanocomposite of the present application can further include excipients commonly used in pharmaceutical agents such as Eudragit, polylactic acid-glycolic acid copolymer (PLGA), and polylactic acid (PLA), etc. according to actual needs.

[0050] In another aspect, the present application provides a method for preparing the nanocomposite, the preparation mechanism of which is that when the pH of the system is less than the isoelectric point of the modified chitosan, the modified chitosan is positively charged, and when the pH of the system is greater than the isoelectric point of the protein polypeptide drug, the protein polypeptide drug is negatively charged, the modified chitosan and the protein polypeptide drug can form a nanocomposite through electrostatic adsorption, thereby preparing a functional nanocomposite. In the case of additional addition of an anionic crosslinking agent and / or a backbone polymer, the nanocomposite can be further stabilized.

[0051] The preparation method according to the present application can be carried out as follows: uniformly mixing a solution of a protein polypeptide drug with a solution of the modified chitosan described above, adjusting the pH to about 4.0 to about 7.0, and then optionally adding an anionic crosslinking agent and an aqueous solution of a backbone polymer to self-assemble into a nanocomposite.

[0052] The solution of the protein polypeptide drug can be prepared by dissolving the protein polypeptide drug in a pharmaceutically acceptable solvent. The solution of the modified chitosan can be prepared by dissolving the modified chitosan in an aqueous acetic acid solution, such as an aqueous solution of about 1% acetic acid.

[0053] In one example, the preparation method according to the present application can be carried out as follows:

[0054] The protein polypeptide drug is dissolved in a pharmaceutically acceptable solvent, the modified chitosan is dissolved in an aqueous solution of about 1% acetic acid, and then the two are mixed, the pH is adjusted to about 4.0 to about 7.0 with an aqueous acid or base solution, and after vortex mixing, an anionic crosslinking agent and an aqueous solution of a backbone polymer are added, and after stirring, a functional nanocomposite with an average particle size of no more than 800 nm and 90% of the particle size of no more than 2 μm is self-assembled.

[0055] Preferably, the pharmaceutically acceptable solvent is water, a phosphate buffer, ethanol, a dilute hydrochloric acid solution, or a dilute aqueous sodium hydroxide solution; the base is preferably sodium hydroxide, potassium hydroxide, or aqueous ammonia; and the acid is preferably hydrochloric acid, phosphoric acid, or acetic acid.

[0056] Preferably, the ratio (mass ratio) of the modified chitosan to the protein polypeptide drug is about 1:10 to about 10:1.

[0057] In still another aspect, the present application provides the use of the nanocomposite described above for the preparation of a medicament.

[0058] In still another aspect, the present application provides a pharmaceutical composition comprising the nanocomposite according to the present application described above. In addition, the pharmaceutical composition can also include, as needed, pharmaceutically acceptable adjuvants, such as dry protection agents, coloring agents, coating agents, sweetening agents, flavoring agents, preservatives, lubricants, binders, disintegrating agents, solvents, fillers, and flow aids, etc.

[0059] The pharmaceutical composition can be a solid preparation, such as a tablet, a capsule, a granule, a pill, a paste, and a powder, etc., or a liquid preparation, such as a solution, a tincture, a syrup, a suspension, and a sol, etc.

[0060] In still another aspect, the present application provides a method for preparing a solid preparation comprising the nanocomposite of the present application, comprising the following steps:

[0061] a) powdering the nanocomposite of the present application;

[0062] b) preparing a solid preparation by mixing the powder obtained in step a) with a pharmaceutical additive;

[0063] c) optionally coating the solid preparation obtained in step b) to obtain a coated solid preparation.

[0064] In step a), the method for powdering is not limited, for example, wet granulation or dry granulation, or the nanocomposite of the present application can be powdered by a drying technique. The drying technique can be freeze-drying or spray-drying.

[0065] In step b), the solid dosage form is preferably selected from the group consisting of capsules, tablets and granules; and the pharmaceutical additive is preferably selected from the group consisting of microcrystalline cellulose, starch, povidone and magnesium stearate.

[0066] In step c), the coating can be an enteric coating. The enteric coating can use all enteric coating materials without limitation, and the enteric coating material is preferably selected from the group consisting of Eudragit L100, Eudragit S100, Eudragit L100-55, HPMCP and cellulose acetate phthalate.

[0067] In the present specification, "about" means that the numerical value can vary within an error range acceptable to those skilled in the art, for example, within ±20%, ±15%, ±10%, or ±5%.

[0068] Advantages

[0069] The modified chitosan of the present application can be combined with ASBT and negatively charged proteins on the cell membrane to promote the transmembrane of protein polypeptide drugs, effectively promote their absorption, and thus improve the oral bioavailability of protein polypeptide drugs.

[0070] In addition, the modified chitosan of the present application can effectively self-assemble with protein polypeptide drugs to form nanocomposites, which have uniform particle size distribution, high encapsulation efficiency and drug loading capacity. The nanocomposites can effectively protect protein polypeptide drugs from protease degradation in the intestine and improve the stability of the drugs in the intestine.

[0071] The solid dosage form loaded with functional nanocomposites of the present application can be stored for a long time while maintaining the stability and physiological activity of protein polypeptide drugs during storage. BRIEF DESCRIPTION OF DRAWINGS

[0072] Figure 1 H-NMR spectra of chitosan and the modified chitosan prepared in Preparation Example 1

[0073] Figure 2 Transmission electron microscope (Tecnai G2 Spirit, FEI, USA) image of the modified chitosan / insulin nanocomplex prepared in Preparation Example 4;

[0074] Figure 3 Particle size distribution graph (a) and Zeta potential graph (b) of the modified chitosan / insulin nanocomplex prepared in Preparation Example 4;

[0075] Figure 4 Line graph showing the in-vitro release result of the enteric capsule loaded with the modified chitosan / insulin nanocomplex prepared in Preparation Example 7;

[0076] Figure 5 Line graph showing the in-vivo hypoglycemic effect of the enteric capsule loaded with the modified chitosan / insulin nanocomplex prepared in Preparation Example 7;

[0077] Figure 6 Line graph showing the in-vivo absorption promoting effect of the enteric capsule loaded with the modified chitosan / insulin nanocomplex prepared in Preparation Example 7. DETAILED DESCRIPTION

[0078] The present application will be further described in detail below with specific examples and with reference to data. It should be understood that these examples are only intended to illustrate the present application, and are intended to illustrate the specific formula composition, preparation method, and functions and effects thereof, but not to limit the scope of the present application in any way. In the following examples, various processes and methods not described in detail are conventional methods known in the art.

[0079] In the present application, the source and trade name of the reagents and equipment used are indicated at the first occurrence, and the same reagents used thereafter are the same as the first indicated content unless otherwise specified, and the conventional unmarked reagents are purchased from the National Pharmaceutical Group Chemical Reagent Co., Ltd. Among them, the raw material chitosan is a pharmaceutical excipient special chitosan purchased from Zhejiang Jinshell Pharmaceutical Co., Ltd., sodium deoxycholate and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are purchased from Sigma-Aldrich Company, insulin is purchased from Xuzhou Wanbang Jinqiao Pharmaceutical Co., Ltd., γ-polyglutamic acid is purchased from Shanghai Jiukuan Chemical Co., Ltd., Eudragit L100 is purchased from Wincreate Industrial Group, and recombinant human epidermal growth factor is purchased from Dalian Melun Biotechnology Co., Ltd.

[0080] Experimental animals: 24 healthy SD rats, male, body weight 200-220 g, from Shanghai Institute of Materia Medica Experimental Animal Center. The test animals were acclimated in the test site for 1-2 weeks before the test day. All animal experiments were approved by the IACUC committee of Shanghai Institute of Materia Medica.

[0081] The dynamic viscosity was determined by precisely weighing 1.0 g of the product to be tested, adding 1% glacial acetic acid 100 ml, stirring to completely dissolve, and using a Brookfield LVDV-III Ultra type rotary viscometer.

[0082] Preparation Example

[0083] Preparation of modified chitosan

[0084] Take the raw material chitosan (Mw is 100,000, dynamic viscosity is 65 mpa·s, Zhejiang Jinshell, degree of deacetylation is 90%) 3 g is added to 1% (v / v) acetic acid solution at room temperature, and deoxycholic acid sodium (900 mg) and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) (528 mg) are added in turn, and the pH is adjusted to 5.5 with sodium hydroxide solution. After 24 hours of reaction, the pH is adjusted to 9.0 with sodium hydroxide solution, the precipitate is separated by centrifugation, dialysis bag (MWCO 50,000) dialysis, freeze-drying, and yellow flocculent material 2.5 g is obtained after drying, which is modified chitosan.

[0085] The prepared modified chitosan and the raw material chitosan were dissolved in deuterated acetic acid respectively and subjected to hydrogen nuclear magnetic resonance analysis (Varian-MERCURY Plus-300, USA).

[0086] Comparison Figure 1 The nuclear magnetic resonance spectrum of the raw material chitosan (CS) and the deoxycholic acid coupled chitosan (i.e. modified chitosan DCS) can be seen that the characteristic peak of deoxycholic acid appears at 0-1.5 ppm in the deoxycholic acid coupled chitosan. It can be seen that deoxycholic acid has been successfully grafted onto the chitosan skeleton.

[0087] Deoxycholic acid substitution degree determination method: the percentage content of C, H and N elements of chitosan and deoxycholic acid coupled chitosan was determined by Vario EL III type automatic element analyzer, and the deoxycholic acid substitution degree was calculated according to the following formula.

[0088] The deoxycholic acid substitution degree is the average number of deoxycholic acid contained in each chitosan structural unit, and the calculation method is:

[0089]

[0090] The deoxycholic acid substitution degree is about 1.3%.

[0091] Preparation Example 2 Preparation of modified chitosan

[0092] To the raw chitosan (Mw 200,000, dynamic viscosity 140 mpa-s, Zhejiang Jinshell, degree of deacetylation 85%) 3 g was added 1% (v / v) acetic acid aqueous solution and stirred at room temperature, and then deoxycholic acid sodium (1800 mg) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (1056 mg) were added in turn, and the pH was adjusted to 5.5 with sodium hydroxide aqueous solution. After 24 hours of reaction, the pH was adjusted to 9.0 with sodium hydroxide aqueous solution, the precipitate was separated by centrifugation, dialyzed in a dialysis bag (MWCO 100,000), and freeze-dried. After drying, 2.5 g of yellow flocculent material was obtained, which was the modified chitosan.

[0093] The degree of substitution of deoxycholic acid was about 3.2% as measured by an elemental analyzer.

[0094] Preparation of modified chitosan of Preparation Example 3

[0095] To the raw chitosan (Mw 100,000, dynamic viscosity 65 mpa-s, Zhejiang Jinshell, degree of deacetylation 90%) 3 g was added 1% (v / v) acetic acid aqueous solution and stirred at room temperature, and then sodium taurocholate (900 mg) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (528 mg) were added in turn, and the pH was adjusted to 5.5 with sodium hydroxide aqueous solution. After 24 hours of reaction, the pH was adjusted to 9.0 with sodium hydroxide aqueous solution, the precipitate was separated by centrifugation, dialyzed in a dialysis bag (MWCO 50,000), and freeze-dried. After drying, 2.4 g of yellow flocculent material was obtained, which was the modified chitosan.

[0096] The degree of substitution of sodium taurocholate was about 1.5% as measured by an elemental analyzer.

[0097] Preparation of modified chitosan / insulin nanocomposite of Preparation Example 4

[0098] To 20 mg of the modified chitosan prepared in Preparation Example 1 was added 10 mL of 1% (v / v) acetic acid aqueous solution, and 1 mg of γ-polyglutamic acid (γ-PGA) was weighed and dissolved in 5 mL of water, and 20 mg of insulin was weighed and dissolved in 10 mL of 0.01 M NaOH. 10 mL each of the chitosan solution and the insulin solution, and 5 mL of the γ-PGA (γ-polyglutamic acid) solution were mixed, and the pH was adjusted to 6.5 with NaOH aqueous solution, and stirred at room temperature for 10 min. A transparent solution having a light blue opalescence was obtained, which was the modified chitosan / insulin nanocomposite.

[0099] Preparation of modified chitosan / recombinant human epidermal growth factor nanocomposite of Preparation Example 5

[0100] Take the modified chitosan prepared in the preparation of example 2 2mg, dissolved in 1 mL 1% (v / v) acetic acid aqueous solution, take γ-polyglutamic acid (γ-PGA) 1mg, dissolved in 10 mL water, take recombinant human epidermal growth factor 2mg, dissolved in 1 mL water. Take 1 mL of modified chitosan solution and recombinant human epidermal growth factor solution, 0.5 mL of γ-PGA solution, mix, adjust pH to 5.5 with NaOH aqueous solution, stir at room temperature for 10 min to obtain modified chitosan / recombinant human epidermal growth factor nanocomposite.

[0101] Preparation of modified chitosan / insulin nanocomposite of preparation example 6

[0102] Take the modified chitosan prepared in the preparation of example 3 10mg, dissolved in 5 mL 1% (v / v) acetic acid aqueous solution, take γ-polyglutamic acid (γ-PGA) 1mg, dissolved in 5 mL water, take insulin 10mg, dissolved in 5 mL of 0.01M NaOH, take poly(β-amino ester) 1mg, dissolved in 1 mL water. Take 10 mL of chitosan solution and insulin solution, 5 mL of γ-PGA (γ-polyglutamic acid) solution, 1 mL of poly(β-amino ester) solution, mix, adjust pH to 6.0 with NaOH aqueous solution, stir at room temperature for 10 min to obtain a transparent solution with light blue opalescence as modified chitosan / insulin nanocomposite.

[0103] Preparation of enteric capsule loaded with modified chitosan / insulin nanocomposite of preparation example 7

[0104] Take 10 mL of modified chitosan / insulin nanocomposite prepared in preparation example 4 and mix with an equal volume of 2.5% trehalose aqueous solution, freeze-dry to obtain white powder. Take the freeze-dried powder and fill into No. 9 Pccaps preclinical capsules (Capsugel, USA), then put the capsules into the coating pot and coat with 15% Eudragit L100 ethanol solution.

[0105] Preparation of enteric tablet of modified chitosan / insulin nanocomposite of preparation example 8

[0106] Take 10 mL of modified chitosan / insulin nanocomposite prepared in preparation example 4 and mix with an equal volume of 2.5% trehalose aqueous solution, freeze-dry to obtain white powder. Take the freeze-dried powder and mix with starch, microcrystalline cellulose and magnesium stearate, then press into tablets with a tablet press, and then put the tablets into the coating pot and coat with 15% Eudragit L100 ethanol solution.

[0107] Experimental example

[0108] Experimental example 1

[0109] Observation of morphology and particle size of modified chitosan / insulin nanocomplexes

[0110] The modified chitosan / insulin nanocomplexes 20 μL solution (DNC) prepared in Preparation Example 4 was observed by transmission electron microscopy (Tecnai G2 Spirit, FEI, USA) after being negatively stained with uranium acetate. It was found that the nanocomplexes were in a spherical shape, with a size of about 200 nm. For specific results, please refer to Figure 2 .

[0111] Determination of particle size and potential of modified chitosan / insulin nanocomplexes

[0112] The modified chitosan / insulin nanocomplexes 100 μL solution prepared in Preparation Example 4 was measured by Zetasizer ZS particle size instrument (Malvern Instruments, Malvern, UK). The average particle size of the nanocomplexes was 231.4 nm, and the Zeta potential was +21.8 mV. For specific particle size distribution chart, please refer to Figure 3 a, the analysis results are shown in Table 1, and for specific Zeta potential chart, please refer to Figure 3 b, the analysis results are shown in Table 2.

[0113] Table 1

[0114]

[0115] Table 2

[0116]

[0117] Determination of drug encapsulation efficiency and drug loading of modified chitosan / insulin nanocomplexes

[0118] 1. Instruments: Agilent 1200 high performance liquid chromatograph (Agilent, USA); high speed centrifuge (Allegra64R, Beckman Coulter, USA). Reagents: modified chitosan / insulin nanocomplexes (prepared according to the method described in the present application); acetonitrile (chromatographic pure, sigma, USA); water is Milli-Q ultrapure water, and other reagents such as phosphoric acid are all analytical pure.

[0119] 2. Chromatographic conditions: chromatographic column: Grace Vydac 218TP C18 column (250 mm x 4.6 mm, 5 μm, USA Grace Company); mobile phase: 0.1 M sodium phosphate buffer (phosphoric acid to adjust pH = 3.0): acetonitrile (72:28, v / v); flow rate: 1 mL / min; column temperature: 40℃; detection wavelength: 214 nm.

[0120] 3. Assay method: 1.5 mL of the modified chitosan / insulin nanocomposite solution prepared in Preparation Example 4 was centrifuged at 16,000 g for 30 min, and the supernatant was taken. The concentration of free drug in the supernatant was determined by high performance liquid chromatography. The encapsulation efficiency of the nanocomposite solution was calculated by comparing the amount of free drug determined with the total amount of insulin added.

[0121] The results of the assay showed that the encapsulation efficiency of the drug in the modified chitosan / insulin nanocomposite prepared in Preparation Example 4 was about 73.5%, and the drug loading was about 33.0%.

[0122] Experimental Example 2

[0123] The enteric capsule prepared in Preparation Example 7 was placed in 10 mL of phosphate buffer having pH values of 2.5 and 6.6, respectively, and the buffer was shaken at 100 rpm at 37°C. At 0, 30, 60, 90 and 120 min, 200 μL of the buffer was taken and the insulin content in the buffer was determined by the HPLC method described in Experimental Example 1.

[0124] The results are shown in Table 2. Figure 4 The results showed that the enteric capsule containing the modified chitosan / insulin nanocomposite of the present application could protect the insulin preparation from the acidic environment in the stomach, and did not release the nanocomposite in the stomach, but released the nanocomposite after entering the small intestine, and was completely released in 2 hours.

[0125] Experimental Example 3

[0126] Twelve Sprgue-dawley rats were weighed, and STZ (streptozotocin) was weighed at a dose of 65 mg / kg, dissolved in 0.1 M citric acid buffer (pH = 4.5), and immediately injected intraperitoneally into the rats. After one week of feeding, the blood glucose was measured, and rats having blood glucose values higher than 16.67 mM were used for the subsequent experiment. The experimental animals were randomly divided into 4 groups, and were fasted for 12 h before administration, but were not deprived of water. The first group was an oral insulin control group (30 IU / kg) (abbreviated as S group), and was given an enteric capsule containing insulin and trehalose powder; the second group was a subcutaneous injection of insulin solution group (5 IU / kg) (abbreviated as S-SC group); the third group was an enteric capsule containing a common chitosan / insulin nanocomposite (30 IU / kg) (abbreviated as NC group); and the fourth group was an enteric capsule containing the modified chitosan / insulin nanocomposite prepared in Preparation Example 7 (30 IU / kg) (abbreviated as DNC group). At 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 h, blood was taken from the tail vein, and the blood glucose level was measured using a blood glucose meter, and the hypoglycemic effect of insulin was calculated.

[0127] The results are shown in Table 3. Figure 5The results show that the enteric capsule loaded with the modified chitosan / insulin nanocomposite can produce a significant hypoglycemic effect. The blood glucose starts to decrease 1 h after oral administration, and decreases to 50% 4 h later, and the hypoglycemic effect can last for 10 h.

[0128] Experimental Example 4

[0129] Twelve Sprgue-dawley rats were weighed, and STZ (streptozotocin) was weighed at a dose of 65 mg / kg, dissolved in 0.1 M citric acid buffer (pH = 4.5), and immediately injected intraperitoneally into the rats. After one week of feeding, the blood glucose was measured, and the rats with blood glucose values higher than 16.67 mM were used for subsequent experiments. The experimental animals were randomly divided into four groups, and were fasted for 12 h before administration without water. The first group was an oral insulin control group (30 IU / kg) (S group), and was given enteric capsules loaded with insulin and trehalose powder; the second group was a subcutaneous injection of insulin solution group (5 IU / kg) (S-SC group); the third group was an enteric capsule loaded with ordinary chitosan / insulin nanocomposite (30 IU / kg) (NC group); and the fourth group was an enteric capsule loaded with modified chitosan / insulin nanocomposite prepared in Preparation Example 7 (30 IU / kg) (DNC group). Blood was taken from the orbital venous plexus at 0, 1, 2, 4, 6, 8, 10 and 12 h, respectively, and the concentration of insulin in the plasma was detected using an insulin ELISA kit, and a pharmacokinetic curve was drawn.

[0130] The results are shown in Figure 6 The results show that the enteric capsule loaded with the modified chitosan / insulin nanocomposite of the present application can significantly improve the oral absorption of insulin. The blood concentration significantly increases 2 h after oral administration, reaches a peak value at 4 h, and the insulin concentration is higher than that of the enteric capsule loaded with ordinary chitosan / insulin nanocomposite in 2-8 h.

Claims

1. The use of a modified chitosan having the structural unit shown in Formula 1 for preparing an oral carrier for protein and polypeptide drugs. in, The R groups in each structural unit may be the same or different, and each is independently a residue obtained by amidation of a bile acid compound with an amino group in the raw material chitosan. The raw material chitosan has a weight-average molecular weight of 50,000–300,000 g / mol and a degree of deacetylation of 80%–95%. The degree of substitution of bile acid compounds in the modified chitosan is 1% to 10%. Among them, protein and polypeptide drugs are selected from insulin and human glucagon-like peptide-1 analogs; The bile acid compounds are selected from the compounds shown in Formula 2 or their salts: R1 is selected from -OH, carboxymethylamino and sulfonate ethylamino; R2 is -OH; R3 is -H or -OH; R4 is -H or -OH. The modified chitosan and the protein / peptide drug self-assemble to form a nanocomposite, which is prepared as follows: the solution of the protein / peptide drug and the solution of the modified chitosan are uniformly mixed, the pH is adjusted to 4.0-7.0, and the nanocomposite is self-assembled.

2. The use according to claim 1, wherein, The cholic acid compounds are selected from cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid, isursodeoxycholic acid, lago deoxycholic acid, glycocholic acid, taurocholic acid and their salts.

3. The use according to claim 1, wherein, The cholic acid compounds are selected from deoxycholic acid, taurocholic acid, cholic acid and their salts.

4. An oral pharmaceutical composition comprising a nanocomposite and pharmaceutically acceptable excipients, said nanocomposite comprising, by weight percentage: 5% to 80% of the modified chitosan as described in any one of claims 1-3; 5% to 50% of protein and polypeptide drugs; 0.5%–25% anionic crosslinking agent; and 0%–25% of backbone polymers, in, The protein-peptide drugs are selected from insulin and human glucagon-like peptide-1 analogs. The nanocomposite is prepared as follows: a solution of protein-peptide drug is uniformly mixed with a solution of modified chitosan, the pH is adjusted to 4.0-7.0, and then an aqueous solution of anionic crosslinking agent and an aqueous solution of non-essential backbone polymer are added to form a nanocomposite through self-assembly.

5. The oral pharmaceutical composition according to claim 4, wherein, By weight percentage, it comprises 20% to 50% protein and polypeptide drugs.

6. The oral pharmaceutical composition according to claim 4, wherein, It contains 0.5% to 15% anionic crosslinking agent by weight percentage.

7. The oral pharmaceutical composition according to claim 4, wherein, It comprises 0% to 20% of backbone polymers by weight percentage.

8. The oral pharmaceutical composition according to claim 4, wherein, The average particle size of the nanocomposite does not exceed 800 nm.

9. The oral pharmaceutical composition according to claim 8, wherein, The average particle size of the nanocomposite is 100–500 nm.

10. The oral pharmaceutical composition according to claim 4, wherein, 90% of the particles in the nanocomposite have a particle size of no more than 2 μm.

11. The oral pharmaceutical composition according to claim 10, wherein, 90% of the particles in the nanocomposite have a particle size in the range of 200–1,000 nm.

12. The oral pharmaceutical composition according to claim 4, wherein, The zeta potential of the nanocomposite does not exceed +65mV.

13. The oral pharmaceutical composition according to claim 12, wherein, The zeta potential of the nanocomposite is +15mV to +45mV.

14. The oral pharmaceutical composition according to claim 4, wherein, The anionic crosslinking agent is a water-soluble compound that carries a negative charge after dissolution.

15. The oral pharmaceutical composition according to claim 14, wherein, The anionic crosslinking agent is selected from polyglutamic acid and sodium polyphosphate.

16. The oral pharmaceutical composition according to claim 4, wherein, The skeleton polymer is a water-soluble polymer material.

17. The oral pharmaceutical composition according to claim 16, wherein, The backbone polymer is selected from polyacrylamide, poly(β-amino ester) and polyvinyl alcohol.

18. The oral pharmaceutical composition according to any one of claims 4-17, wherein, The nanocomposite was prepared as follows: a solution of protein-peptide drugs was uniformly mixed with a solution of modified chitosan, the pH was adjusted to 4.0-7.0, and then an aqueous solution of anionic crosslinking agent and an aqueous solution of non-essential backbone polymer were added to form a nanocomposite through self-assembly.

19. The oral pharmaceutical composition according to claim 4, wherein, The pharmaceutical composition is a solid dosage form, which is selected from tablets, capsules, granules, and pills.

20. A method for preparing the oral pharmaceutical composition according to any one of claims 4-19, comprising the following steps: a) The nanocomposite is made into a powder; b) The powder obtained in step a) is mixed with pharmaceutical additives to prepare a solid dosage form; c) It is not necessary to coat the solid dosage form obtained in step b) to obtain a coated solid dosage form.

21. The method according to claim 20, wherein, In step a), the nanocomposite is made into powder by drying technology.

22. The method according to claim 20, wherein, In step b), the pharmaceutical additive is selected from microcrystalline cellulose, starch, povidone and magnesium stearate, and the solid dosage form is selected from capsules, tablets and granules.

23. The method of claim 20, wherein, In step c), the coating is an enteric coating.

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