CN108653234B

CN108653234B - Solid particles loaded with polypeptide protein drugs, double-enteric solid preparation containing particles, and preparation method and application thereof

Info

Publication number: CN108653234B
Application number: CN201710212881.7A
Authority: CN
Inventors: 甘勇; 胡磊; 范未伟; 郭仕艳; 朱春柳; 王瑞
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2017-04-01
Filing date: 2017-04-01
Publication date: 2021-02-26
Anticipated expiration: 2037-04-01
Also published as: CN108653234A

Abstract

The invention relates to solid particles loaded with polypeptide protein drugs, a double enteric solid preparation containing the solid particles, and a preparation method and application thereof. The solid particles comprise polypeptide protein drugs, absorption enhancers and protease inhibitors. The double enteric solid preparation prepared by the invention can resist the degradation of polypeptide protein medicines by gastric acid and gastrointestinal enzymes when being orally taken, has a colon positioning effect and can effectively promote the absorption of the polypeptide protein medicines in intestinal tracts.

Description

Solid particles loaded with polypeptide protein drugs, double-enteric solid preparation containing particles, and preparation method and application thereof

Technical Field

The invention relates to the field of biological pharmacy, in particular to solid particles loaded with polypeptide protein medicines, a double enteric solid preparation containing the solid particles, and a preparation method and application of the double enteric solid preparation.

Background

Polypeptide protein drugs, such as insulin, human growth hormone and calcitonin, are administered by injection. However, the injection administration has many disadvantages, such as short biological half-life of insulin, and 1-2 times of insulin injection per day can cause the side effects of local red swelling, subcutaneous nodules, subcutaneous lipoatrophy and the like of patients, thereby bringing great pain and inconvenience to the patients. Therefore, the research on safe, convenient, cheap and effective oral administration preparations to replace injections greatly facilitates patients.

However, if the polypeptide protein drugs such as insulin are directly orally taken without being processed by a special embedding technology, the drugs are easily degraded by the acid environment in the stomach and the proteolytic enzyme in the gastrointestinal tract, and meanwhile, the drugs have large molecular weight and poor lipid solubility, are difficult to permeate the epithelial cell barrier of the digestive tract, and have the bioavailability of only 0.1-2 percent, so the oral administration of the polypeptide protein drugs faces huge challenges.

In recent years, researches show that the absorption rate of polypeptide protein medicines in the gastrointestinal tract can be effectively improved by adopting a proper medicine carrying system and then adding a certain protease inhibitor and an absorption enhancer. For example, insulin encapsulated in liposome developed by Diatome corporation has a direct targeting function of liver cells, and not only can rapidly reduce blood sugar through a physiological pathway of liver, but also can maintain the concentration of peripheral blood insulin at a normal level. However, the preparation has poor stability, so the clinical application needs a long time. Emisphere corporation developed a series of small molecule compounds as absorption enhancers for polypeptide protein drugs (such as sodium N- (8- [ 2-hydroxybenzoyl ] amino) caprylate (SNAC), WO 2008/028859), which have been used in clinical studies of oral administration of macromolecules by several companies, such as the study on oral Somalutide and oral insulin by Novonide, and the study on oral insulin by Oramed, but the oral tablet also has the disadvantages of large dosage of absorption enhancer, easy dilution in vivo and reduced drug effect. Still others have dissolved insulin in a hydrophobic solvent or oil phase to make an oral preparation, such as WO 95/13795 where insulin is dissolved in an oil phase preparation and the hydrophilic solvent is removed by rotary evaporation, spray drying, or freezing under vacuum conditions of less than 0.1MPa for 48 h. In general, the preparation process is complicated, the production scale is limited, and the cost is increased.

Therefore, designing an oral preparation which has simple and mild preparation method and stable property and can promote the oral absorption of polypeptide protein medicines is always a difficult problem for preparation workers.

Disclosure of Invention

The invention aims to solve the existing problems and provide a double enteric solid preparation loaded with polypeptide protein drugs, which is safe, reliable, easy to accept by patients and can be used for oral administration.

According to one aspect of the present invention, there is provided a solid particulate material loaded with a polypeptide protein drug, comprising:

0.5 to 90 parts by weight, preferably 1 to 50 parts by weight of a polypeptide protein drug;

0.5 to 90 parts by weight, preferably 5 to 50 parts by weight, of an absorption enhancer;

0 to 50 parts by weight, preferably 1 to 30 parts by weight, of a protease inhibitor; and

5 to 90 parts by weight, preferably 10 to 90 parts by weight of an enteric material.

In the solid particles carrying the polypeptide protein drugs, the average particle size of the solid particles is preferably about 0.1-2000 μm; and/or 90% of the particles of the solid particulate matter have a particle size of not more than 2000 μm, more preferably the average particle size of the solid particulate matter is in the range of about 0.1 to 1000 μm.

In the solid particulate matter loaded with the polypeptide protein drug of the present invention, preferably, the polypeptide protein drug is a polypeptide protein drug having pharmacological activity, such as insulin and its analogs, exenatide, calcitonin, recombinant human parathyroid hormone, erythropoietin, human granulocyte colony stimulating factor, human growth hormone, interleukin, cyclosporine, epidermal growth factor, GLP-1 analogs, interferon, and the like, and more preferably insulin, exenatide, or calcitonin.

In the solid particles loaded with the polypeptide protein drug of the present invention, preferably, the absorption enhancer may be: n- (8- [ 2-hydroxybenzoyl ] amino) caprylic acid and its derivatives (e.g., sodium N- [8- (2-hydroxybenzoyl) amino ] caprylate (SNAC), sodium N- (10- [ 2-hydroxybenzoyl- ] amino) caprate (SNAD)), medium chain fatty acids and their salts (e.g., sodium caprate), bile acids and their derivatives, ethylenediaminetetraacetic acid (EDTA) or its salts, or combinations thereof, more preferably SNAC, SNAD, or sodium caprate.

In the solid particulate matter carrying the polypeptide protein drug of the present invention, preferably, the protease inhibitor may be trypsin inhibitor, cysteine protease inhibitor, threonine protease inhibitor, aspartic protease inhibitor, metalloprotease inhibitor, or the like. More preferably soybean trypsin inhibitor (SBTI), aprotinin or ovomucoid.

In the solid granules loaded with the polypeptide protein drug of the present invention, preferably, the enteric material can be all enteric coating materials without limitation, such as one or more mixed materials of Cellulose Acetate Phthalate (CAP), hypromellose phthalate (HPMCP), polyvinyl acetate phthalate (PVAP), acrylics (such as Eudragit S100, Eudragit L100), shellac, etc., preferably Eudragit S100, Eudragit L100, HPMCP or a combination thereof.

According to another aspect of the present invention, there is provided a method for preparing the solid particulate matter loaded with polypeptide protein drugs, which comprises the following steps:

1) dissolving a polypeptide protein drug, an absorption enhancer and a protease inhibitor in an aqueous solution to form a solution (A), wherein the mass ratio of the polypeptide protein drug to the absorption enhancer is about 100: 1-1: 100, preferably about 1: 10-1: 40, and the mass ratio of the polypeptide protein drug to the protease inhibitor is about 100: 1-1: 100, preferably about 1: 1-1: 10;

2) dissolving an enteric material in a solvent to form a solution (B), wherein the concentration of the enteric material is about 1 mg/mL-200 mg/mL, preferably about 5-100 mg/mL; and

3) adding the solution (A) into the solution (B) at room temperature, wherein the volume ratio of the solution (A) to the solution (B) is about 1: 10-1: 100, preferably about 1: 5-1: 25, forming a uniform solution, and drying to obtain the solid particles.

The aqueous phase solution in step 1) of the above preparation method may be prepared in a pharmaceutically acceptable solvent in the art. The aqueous phase solution is preferably water, phosphate buffer solution, ethanol aqueous solution, acid aqueous solution or alkali aqueous solution; the aqueous alkali solution is preferably an aqueous solution of sodium hydroxide, potassium hydroxide or ammonia; the aqueous acid solution is preferably an aqueous solution of hydrochloric acid, phosphoric acid or acetic acid. Wherein, the pH value and the concentration of the aqueous phase solution can be properly adjusted along with the change of materials.

The solvent in step 2) of the above preparation method may be water, ethanol, tert-butanol, dichloromethane or a combination thereof in any ratio.

In step 3) of the above preparation method, preferably, the drying may be a spray freeze drying method, a spray drying method, a vacuum drying method, or the like.

Optionally, the preparation method of the solid particulate matter loaded with the polypeptide protein drug further comprises the following steps:

1) dissolving polypeptide protein drugs, absorption enhancers and protease inhibitors in an aqueous solution containing a surfactant to form a solution (A); wherein the mass ratio of the polypeptide protein drug to the absorption enhancer is about 100: 1-1: 100, preferably about 1: 10-1: 40; the mass ratio of the polypeptide protein drugs to the protease inhibitor is about 100: 1-1: 100, preferably about 1: 1-1: 10;

2) dissolving an enteric material in a solvent to form a solution (B), wherein the concentration of the enteric material is about 1 mg/mL-200 mg/mL, preferably about 5-100 mg/mL;

3) adding the solution (A) into the solution (B) at room temperature, wherein the volume ratio of the solution (A) to the solution (B) is about 1: 1-1: 100, preferably about 1: 1-1: 50, and forming a W/O water-in-oil emulsion (C);

4) slowly adding the emulsion (C) obtained in the step 3) into a solvent (D) containing a surfactant, wherein the volume ratio of the emulsion (C) to the solvent (D) is about 1: 1-1: 100, preferably about 1: 5-1: 25, stirring at normal temperature, and adding an organic solvent to solidify particles; and

5) drying the particles in the step 4) to obtain the solid particles.

The solution of the polypeptide protein drug in step 1) of the preparation method can be prepared by dissolving the polypeptide protein drug in a pharmaceutically acceptable solvent. The pharmaceutically acceptable solvent is preferably water, phosphate buffer, aqueous ethanol, aqueous acid or aqueous alkali; the aqueous alkali solution is preferably an aqueous solution of sodium hydroxide, potassium hydroxide or ammonia; the aqueous acid solution is preferably an aqueous solution of hydrochloric acid, phosphoric acid or acetic acid.

The solvent in step 2) of the above production method may be t-butanol, dichloromethane, chloroform or the like or a combination thereof with water or ethanol in any ratio, and

the solvent in the surfactant-containing solvent (D) in step 4) may be a non-aqueous solvent phase solvent such as paraffin, cottonseed oil, soybean oil, etc.

In steps 1) and 4) of the above preparation method, preferably, the surfactant may be an anionic surfactant, a cationic surfactant or a nonionic surfactant, and more preferably tween 20, span 60 or span 80.

In step 4) of the above preparation method, preferably, the solvent may be a common organic solvent, and more preferably n-pentane, n-hexane, or n-heptane.

In step 5) of the above preparation method, preferably, the drying may be a spray freeze drying method, a spray drying method, a vacuum drying method, or the like.

According to another aspect of the present invention, there is provided a dual enteric solid formulation of an oral polypeptide protein drug, comprising the solid particulate matter and a colon-specific material as described above.

According to another aspect of the present invention, a method for preparing a dual enteric solid preparation of an oral polypeptide protein drug is provided, which comprises the steps of preparing the solid particles loaded with the polypeptide protein drug, and preparing the prepared solid particles and a colon positioning material into a solid preparation.

In the method for preparing a dual enteric solid preparation of the present invention, preferably, the step of preparing the prepared solid particles and the colon specific material into a solid preparation is selected from any one of the following methods:

a) filling the solid particles, optionally with medicinal additives, into common gelatin capsules, and coating with one or more of colon-specific materials to obtain capsules. Preferably, the medicinal additive in a) is selected from mannitol, starch, lactose, microcrystalline cellulose and other pharmaceutically acceptable additives;

b) and (3) filling the solid particles, optionally together with medicinal additives, into a capsule shell containing the colon positioning material to prepare the capsule. Preferably, the medicinal additive in b) is selected from mannitol, starch, lactose, microcrystalline cellulose and other pharmaceutically acceptable additives;

c) granulating the solid particles, and coating with one or more than two of colon positioning materials to obtain enteric-coated pellets;

d) mixing the solid granules with medicinal additives, tabletting, coating with one or more colon-specific materials, and making into tablet. Preferably, said pharmaceutical additives described in d) are selected from the group of pharmaceutically acceptable additives comprising microcrystalline cellulose, starch, povidone and magnesium stearate; or

e) Mixing the solid particles with colon positioning material, and optionally medicinal additive, tabletting, and making into enteric coated tablet. Preferably, the pharmaceutical additives in e) are selected from the group consisting of microcrystalline cellulose, starch, povidone, and magnesium stearate, among other pharmaceutically acceptable additives.

In the above method for preparing a double enteric solid preparation, preferably, the colon-specific material may be one or a mixture of two or more of pH-sensitive polymers (such as acrylic resins, e.g., Eudragit S100 and Eudragit L100), natural polysaccharides (such as chitosan, pectin, and calcium pectin), and azo polymers. Preferably Eudragit S100, pectin or calcium pectin.

According to another aspect of the invention there is provided the use of the solid particulate material in the manufacture of a medicament.

According to yet another aspect of the invention, there is provided a pharmaceutical composition comprising the solid particulate or the dual enteric solid formulation.

Advantageous effects

The double-enteric-coated solid preparation carrying the polypeptide protein medicine has the characteristics of stability under an acidic condition and dissolution under the condition that the pH value is more than or equal to 7. The pH value of the upper parts of the digestive tract stomach, the duodenum and the small intestine is less than 6.5, so the double enteric solid preparation can not be dissolved and damaged at the parts, the early release of the preparation is avoided, the polypeptide protein medicines are effectively protected from being degraded by protease in the intestinal tract, and the stability of the medicines in the intestinal tract is improved.

The absorption enhancer and the protease inhibitor are added into the solid particles, so that the transmembrane of the polypeptide protein medicines can be effectively promoted, and the degradation of the polypeptide protein medicines by the protease in intestinal tracts can be inhibited, thereby improving the oral biological curative effect of the polypeptide protein medicines.

The method has the characteristics of simple preparation process, easy realization, low required cost and the like.

Drawings

Fig. 1 shows an SEM image of insulin particles prepared in preparation example 1;

fig. 2 is a graph showing in vitro release results of enteric capsules of insulin granules prepared in preparation example 9;

FIG. 3 is a graph showing the in vivo hypoglycemic effects of enteric-coated capsules of insulin granules prepared in preparation example 9;

FIG. 4 is a graph showing the in vivo absorption-promoting effect of enteric capsules of insulin particles prepared in preparation example 9;

fig. 5 shows an in vivo hypoglycemic picture of exenatide granule enteric-coated capsules prepared in preparation example 10.

Detailed Description

The invention will be further described with reference to specific examples and data. It is to be understood that these examples are intended only to illustrate the present invention and are intended to illustrate the specific formulation, preparation, function and effect of the invention, and not to limit the scope of the invention in any way. In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

In the present invention, the source and trade name of the reagents and equipment used are indicated at the first appearance, and the same reagents used thereafter are the same as those indicated at the first appearance unless otherwise specified, and conventional unlabelled reagents are purchased from national pharmaceutical group chemical agents, ltd. Wherein insulin is purchased from Kyoho gold bridge pharmaceutical Co., Ltd, N- [8- (2-hydroxybenzoyl) amino ] sodium caprylate (SNAC) is purchased from Shanghai Bo medicine Co., Ltd, exenatide and salmon calcitonin are purchased from Aladdin reagent Co., Ltd, aprotinin and trypsin inhibitor are purchased from Aladdin reagent Co., Ltd, sodium cholate is purchased from Guo reagent group Co., Ltd, hypromellose phthalate (HPMCP) is purchased from Yangtze river plant of Xin chemical industry Co., Ltd, Utex (Eudragit S100, L100) is purchased from Yingchun Industrial group, and calcium pectin is purchased from Shanghai Xinjiang biological research institute.

Experimental animals: healthy SD rats 36, male, with a weight of 200- & 220g, were obtained from the animal center of Shanghai drug institute. The test animals were acclimatized at the test site 1-2 weeks before the test day. All animal experiments were approved by the IACUC committee of the shanghai institute of drug research.

Preparation examples

Preparation example 1 preparation of Eudragit S100 coated insulin particles

100mg of SNAC was dissolved in 1mL of 0.01M NaOH, and 10mg of insulin (Ins) was dissolved in 1mL of 0.01M NaOH aqueous solution and then added to the SNAC solution to form a homogeneous solution (A); 500mg of Eudragit S100 was added to 48mL of t-butanol (containing 5mL of water) and appropriately heated to dissolve it sufficiently to form a uniform solution (B); slowly adding A into B, mixing by vortex at 500r/min to form a uniform solution, and preparing Eudragit S100 coated insulin particles by adopting a spray freeze drying technology.

Preparation example 2 preparation of protease inhibitor-containing insulin granules coated with Eudragit S100

100mg of SNAC was dissolved in 1mL of 0.01M NaOH, and 10mg of Ins and 10mg of aprotinin were dissolved in 1mL of 0.01M NaOH aqueous solution and then added to the SNAC solution to form a homogeneous solution (A); 500mg of Eudragit S100 was added to 23mL of t-butanol (containing 2mL of water) and appropriately heated to dissolve it sufficiently to form a uniform solution (B); slowly adding A into B, mixing by vortex at 500r/min to form a uniform solution, and preparing Eudragit S100 coated insulin particles by adopting a spray freeze drying technology.

Preparation example 3 preparation of protease inhibitor-containing insulin granules coated with Eudragit S100

200mg of SNAC was dissolved in 1mL of 0.01M NaOH, and 10mg of Ins and 10mg of aprotinin were dissolved in 1mL of 0.01M NaOH aqueous solution and then added to the SNAC solution to form a homogeneous solution (A); 400mg of Eudragit S100 was added to 23mL of t-butanol (containing 2mL of water) and properly heated to be sufficiently dissolved to form a uniform solution (B); slowly adding A into B, mixing by vortex at 500r/min to form a uniform solution, and preparing Eudragit S100 coated insulin particles by adopting a spray freeze drying technology.

Preparation example 4 preparation of HPMCP-coated insulin particles containing protease inhibitors

200mg of SNAC was dissolved in 1mL of 0.01M NaOH, and 10mg of Ins and 10mg of aprotinin were dissolved in 1mL of 0.01M NaOH aqueous solution and then added to the SNAC solution to form a homogeneous solution (A); adding 400mg HPMCP into 48mL of water, and heating properly to fully dissolve the HPMCP to form a uniform solution (B); slowly adding A into B, mixing with vortex at 500r/min to obtain uniform solution, and spray drying to obtain HPMCP coated insulin granule.

Preparation example 5 preparation of Eudragit S100 Encapsulated Exenatide granules containing protease inhibitor

Dissolving 200mg of SNAC in 1mL of 0.01M NaOH, and adding 10mg of exenatide and 100mg of aprotinin in 4mL of 0.01M NaOH aqueous solution to the SNAC solution to form a homogeneous solution (A); 400mg of Eudragit S100 was added to 25mL of t-butanol (containing 2mL of water) and properly heated to be sufficiently dissolved to form a uniform solution (B); slowly adding A into B, mixing by vortex at 500r/min to form a uniform solution, and preparing Eudragit S100-coated exenatide particles by adopting a spray freeze drying technology.

Preparation example 6 preparation of Eudragit S100 Encapsulated Exenatide granules containing protease inhibitor

400mg of SNAC was dissolved in 1mL of 0.01M NaOH, and 10mg of exenatide and 100mg of aprotinin were dissolved in 4mL of 0.01M NaOH aqueous solution and then added to the SNAC solution to form a uniform solution (A); 400mg of Eudragit S100 was added to 25mL of t-butanol (containing 2mL of water) and properly heated to be sufficiently dissolved to form a uniform solution (B); slowly adding A into B, mixing by vortex at 500r/min to form a uniform solution, and preparing Eudragit S100-coated exenatide particles by adopting a spray freeze drying technology.

Preparation example 7 emulsification method for preparing Eudragit S100 coated protease inhibitor-containing insulin particles

40mg of SNAC was dissolved in 0.5mL of 0.01M aqueous NaOH solution, and 2mg of Ins and 2mg of aprotinin were dissolved in 0.5mL of 0.01M aqueous NaOH solution (containing 1% Tween 20) and then added to the SNAC solution to form a uniform solution (A); adding 80mg of Eudragit S100 to 4mL of ethanol/dichloromethane (ethanol: dichloromethane ═ 1:4), which was sufficiently dissolved to form a homogeneous solution (B); slowly adding the A into the B, forming W/O emulsion by vortex mixing at 500r/min, and then slowly dripping the (C) into 50mL of liquid paraffin (containing 1% span 80) to form W/O/O microspheres; and adding normal hexane to solidify the microspheres, continuing stirring for 1.5h, washing for 3 times by the normal hexane, and drying the microspheres in a vacuum drying oven for 24h to obtain the Eudragit S100-coated insulin particles.

Preparation example 8 preparation of Eudragit L100 coated Salmon calcitonin particles

Dissolve 300mg sodium cholate in 5mL H₂O, 10mg of salmon calcitonin and 100mg of trypsin inhibitor are dissolved in 5mL of 0.01M NaOH aqueous solution and then added to the sodium cholate solution to form a uniform solution (A); 400mg of Eudragit L100 was added to 50mL of t-butanol (containing 10mL of water) and appropriately heated to dissolve it sufficiently to form a uniform solution (B); a was slowly added to B and after vortex mixing at 500r/min to form a homogeneous solution Eudragit L100 coated salmon calcitonin particles were prepared using spray freeze drying technique.

Preparation example 9 preparation of enteric capsule filled with insulin granules

The insulin granules prepared in preparation examples 2 and 3 were filled with Pccaps size 9 preclinical capsules (Capsugel, usa), which were then placed in a coating pan and coated with a 15% Eudragit S100 ethanol solution. The average weight gain was 15%.

Preparation example 10 preparation of enteric capsule filled with Exenatide particles

Exenatide granules prepared in preparation examples 5 and 6 were filled with Pccaps size 9 preclinical capsules (Capsugel, usa), and the capsules were subsequently placed in a coating pan and coated with a 10% pectin solution. The average weight gain was 10%.

Preparation example 11 preparation of enteric coated tablet containing insulin particles

The insulin granules prepared in preparation example 2 were mixed with starch, microcrystalline cellulose and magnesium stearate and compressed into tablets using a tablet press, and then the tablets were put into a coating pan and coated with a 15% Eudragit S100 ethanol solution. The average weight gain was 15%.

Preparation example 12 preparation of enteric coated tablet containing insulin particles

The insulin granules prepared in preparation example 2 were mixed with calcium pectin, starch, microcrystalline cellulose and magnesium stearate, and then sieved through a 40-mesh sieve, and compressed into tablets with a tablet press.

Table 1 below lists the respective pharmaceutical ingredients and their contents in the solid granules prepared in preparation examples 1 to 8, and the preparation methods and final dosage forms of the dual enteric solid formulations prepared in preparation examples 9 to 12.

TABLE 1

Experimental examples

Experimental example 1

Insulin particle morphology and particle size

The Eudragit S100 coated insulin particles prepared in preparation example 1 were subjected to ion sputtering coating and observed by a scanning electron microscope SEM (Phenom XL, Netherlands), and the insulin particles were found to be spherical, and the specific results are shown in fig. 1. Wherein 90% of the solid particles have a particle size of no more than 500 μm and an average particle size of about 10 μm.

Experimental example 2

Insulin particle encapsulation efficiency and drug loading rate determination

1. The instrument comprises the following steps: agilent 1200 high performance liquid chromatograph (Agilent corporation, usa); high speed centrifuges (Allegra 64R, Beckman Coulter, USA). Reagent: insulin particles (prepared according to the methods described herein); acetonitrile (chromatically pure, sigma, usa); the water is Milli-Q ultrapure water, and other reagents such as phosphoric acid and the like are analytical reagents.

2. Chromatographic conditions are as follows: a chromatographic column: grace Vydac 218TP C18 column (250 mm. times.4.6 mm, 5 μm, Grace, USA); mobile phase: 0.1M disodium hydrogen phosphate buffer (pH 3.0 adjusted with phosphoric acid) acetonitrile (72:28, v/v); flow rate: 1 mL/min; column temperature: 40 ℃; detection wavelength: 214 nm.

3. The determination method comprises the following steps: 10mg of insulin particles prepared in preparative example 1 according to the present invention were dissolved in 1mL of 95% ethanol (containing 100. mu.L of 0.01M NaOH) to dissolve the microspheres and release Ins, acidified with mobile phase and tested for encapsulated insulin content by HPLC.

And (3) measuring results: the encapsulation efficiency of the insulin particles prepared in preparation example 1 was about 90.66%, and the drug loading was about 14.68%.

Experimental example 3

The insulin solid particles prepared in preparative example 2 and the enteric capsule prepared in preparative example 9 were put into dialysis bags and respectively put into 500mL of buffer solution with a pH value of 1.2, the buffer solution was stirred at 100rpm under a condition of 37 ℃, samples were taken at time points of 30min, 60min, 90min and 120min within 2h, and the content of insulin in the buffer solution was determined by the HPLC method described in experimental example 2. After 2h, the dialysis bags were taken out and placed in a buffer solution of pH 4.5 to detect the amount of insulin released at different time points. The same method was used to measure the release of insulin in the buffers pH6.8 and 7.4, respectively.

The results are shown in fig. 2, where the solid insulin particles release insulin in a buffer environment at pH 6.8. In order to prevent the insulin from being released in advance, the insulin solid particles are filled into a common capsule for secondary coating. The results show that the Eudragit S100 enteric-coated capsule does not release insulin in the acidic environment with pH 1.4 and 4.5 and the weak acidic environment with pH6.8, and can successfully release insulin in the environment with pH 7.4, and release about 90% of insulin within 1 h. The results show that the enteric capsule of the insulin granule can protect the insulin preparation from resisting the acid environment in the stomach, and the insulin preparation is not released in the stomach and begins to release the insulin after entering the colon.

Experimental example 4

After weighing 12 Sprgue-dawley rats, STZ (streptozotocin) was weighed at a dose of 65mg/kg, dissolved in 0.1M citric acid buffer (pH 4.5), and immediately injected intraperitoneally into the rats. After one week of feeding, rats with blood glucose values above 16.67mM were tested for subsequent experiments. The experimental animals were randomly divided into 3 groups and fasted for 12h before administration without water deprivation. The first group is an oral insulin control group (30IU/kg) (control group), and Eudragit S100 enteric-coated capsules containing a physical mixture of insulin, absorption enhancer and aprotinin are administered; the second group was the group of Eudragit S100 enteric-coated capsules prepared in preparation example 9 (30IU/kg, SNAC: Ins ═ 10: 1); the third group was the group of Eudragit S100 enteric-coated capsules prepared in preparation example 9 (30IU/kg, SNAC: Ins ═ 20: 1); respectively taking blood from tail veins at 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h and 12h, measuring the blood glucose level by a glucometer, and counting the blood glucose reduction effect of insulin.

The results are shown in fig. 3, and the Eudragit S100 coated capsules (dual enteric capsules) loaded with insulin granules exhibited a significant hypoglycemic effect, reducing blood glucose by about 40% within 12h, compared to the control group not prepared as enteric granules. And the hypoglycemic effect can last for 10 hours.

Experimental example 5

After weighing 12 Sprgue-dawley rats, STZ (streptozotocin) was weighed at a dose of 65mg/kg, dissolved in 0.1M citric acid buffer (pH 4.5), and immediately injected intraperitoneally into the rats. After one week of feeding, rats with blood glucose values higher than 16.67mM/L were used for subsequent experiments. The experimental animals were randomly divided into 4 groups and fasted for 12h before administration without water deprivation. The first group is subcutaneous insulin solution (5 IU/kg); the second group was the group of Eudragit S100 enteric-coated capsules prepared in preparation example 9 (30IU/kg, SNAC: Ins ═ 10: 1); the third group was the group of Eudragit S100 enteric-coated capsules prepared in preparation example 9 (30IU/kg, SNAC: Ins ═ 20: 1). Blood is taken from orbital venous plexus at 0h, 1h, 2h, 3h, 4h, 5h, 6h, 8h, 10h and 12h respectively, the concentration of insulin in serum is detected by using an insulin ELISA kit, and a pharmacokinetic curve is drawn.

The results are shown in fig. 4, and show that Eudragit S100 coated dual enteric capsules loaded with insulin particles significantly increased serum insulin concentrations at SNAC: Ins: 20:1, significantly increased serum insulin concentrations were detectable at 4h, and peaked at 5 h. The bioavailability was about 7% relative to the subcutaneous insulin solution group.

Experimental example 6

12 normal Sprgue-dawley rats were weighed and randomized into 4 groups, fasted for 12h before administration, without water deprivation. The first group was a blank control group, no drug was administered; the second group was a group of exenatide saline for subcutaneous injection (10 ug/kg); the third group was a group of orally-administered Eudragit S100 enteric-coated capsules of exenatide granules prepared in preparation example 10 (30IU/kg, SNAC: exenatide ═ 20: 1); the fourth group was a group of orally-administered Eudragit S100 enteric-coated capsules of exenatide granules prepared in preparation example 10 (30IU/kg, SNAC: exenatide ═ 40: 1). Each group of rats was gavaged with 1g/kg of glucose solution at 0h to simulate the postprandial state, and 10. mu.g/kg of the glucose solution was administered to the rats in the injection group. The oral administration is performed 2h before the intragastric administration of glucose. The rats of each group take blood from tail veins at 0h, 0.5h, 1h, 1.5h, 2h, 3h and 4h respectively, and blood glucose level is measured by a glucometer to count the blood glucose reduction effect of each group.

The results are shown in fig. 5, and Eudragit S100 coated capsules (double enteric capsules) loaded with exenatide granules have similar effect of controlling blood glucose as the injection group, wherein the SNAC group of exenatide 40:1 has the best effect of controlling blood glucose.

Claims

1. a method for preparing solid particles loaded with polypeptide protein drugs, comprising the steps:

1) Dissolve the polypeptide protein drug, absorption enhancer, and protease inhibitor in an aqueous solution to form a solution (A), wherein the mass ratio of the polypeptide protein drug to the absorption enhancer is 1:10 to 1:40, and the polypeptide protein The mass ratio of drug-like and protease inhibitors is 1:1~1:10;

2) Dissolving the enteric-coated material in a solvent to form a solution (B), the concentration of the enteric-coated material is 1 mg/mL to 200 mg/mL; and

3) Add (A) solution to (B) solution at room temperature, the volume ratio of (A) solution to (B) solution is 1:10~1:100 to form a homogeneous solution, and then dry to prepare the solid particulates,

Wherein, the solid particles loaded with polypeptide and protein drugs include:

0.5 parts by weight to 90 parts by weight of polypeptide protein drugs;

0.5 to 90 parts by weight of an absorption accelerator;

1 part by weight to 50 parts by weight of protease inhibitor; and

5 parts by weight to 90 parts by weight of enteric material, and

Wherein, the average particle size of the solid particles is 0.1 to 2000 μm,

Wherein, the polypeptide protein drugs are insulin, exenatide, calcitonin, recombinant human parathyroid hormone, erythropoietin, human granulocyte colony stimulating factor, human growth hormone, interleukin, cyclosporine hormones, epidermal growth factor, GLP-1 analogs, or interferon;

The absorption enhancer is N-(8-[2-hydroxybenzoyl]amino) octanoic acid and derivatives thereof, medium chain fatty acids and salts thereof, bile acids and derivatives thereof, ethylenediaminetetraacetic acid or its salts , or a combination thereof;

The protease inhibitor is trypsin inhibitor, cysteine protease inhibitor, threonine protease inhibitor, aspartic protease inhibitor or metalloproteinase inhibitor; and

Described enteric material is selected from cellulose acetate phthalate, hypromellose phthalate, polyvinyl alcohol acetate phthalate, Eudragit S100, Eudragit L100 and shellac one or variety.

2. The method of claim 1, wherein,

The aqueous phase solution in step 1) is water, phosphate buffer, aqueous ethanol, aqueous acid or aqueous alkali; and/or

The solvent in step 2) is water, ethanol, tert-butanol, dichloromethane or any combination thereof.

3. The method according to claim 2, wherein the aqueous solution of the alkali in step 1) is an aqueous solution of sodium hydroxide, potassium hydroxide or ammonia, and the aqueous solution of the acid is an aqueous solution of hydrochloric acid, phosphoric acid or acetic acid.

4. A polypeptide protein drug-loaded solid particle prepared by the method according to any one of claims 1 to 3.

5. A double enteric-coated solid preparation of oral polypeptide protein drugs, which comprises the solid particles as claimed in claim 4 and a colon localization material.

6. The double enteric solid preparation according to claim 5, wherein,

The colon positioning material is one or a mixture of two or more selected from pH-sensitive polymers, natural polysaccharides and azo polymers.

7. a preparation method of double enteric solid preparation as claimed in claim 5, it comprises following any method:

a) after the solid particulate matter of claim 4, and optional pharmaceutical additives, are poured into ordinary gelatin capsules, and one or two or more mixtures in the colon positioning material are used for coating to prepare capsules;

b) filling the solid particulate matter of claim 4, and optional pharmaceutical additives, into a capsule shell containing colon positioning material to prepare a capsule;

c) adopt one or more mixtures in colon positioning material to coat after granulating the solid particulate matter according to claim 4, and prepare enteric-coated pellets;

d) mixing the solid particulate matter of claim 4 with optional pharmaceutical additives, and after tableting, coating with one or more mixtures of colon locating materials to prepare tablets; or

e) The solid particulate matter of claim 4, the colon positioning material, and the optional pharmaceutical additives are mixed and compressed into an enteric-coated tablet.

8. A use of the solid particulate matter as claimed in claim 4 in the preparation of medicine.