Compound bone peptide enteric micro-pellets and a preparation method thereof

Abstract

The application discloses a compound bone peptide enteric micro-pellet and a preparation method thereof, and relates to the technical field of biological medicine. The raw materials of the compound bone peptide enteric micro-pellet include compound bone peptide, a filling agent, a disintegrating agent, a binder and a lubricant. The application develops a compound bone peptide oral preparation, namely, the compound bone peptide enteric micro-pellet, and solves the problem of inconvenient administration of injection dosage forms. The compound bone peptide enteric micro-pellet is a novel compound bone peptide oral dosage form, has high bioavailability, can break through the stomach barrier, realizes local drug release in the small intestine, and maintains an effective therapeutic concentration. The application provides a safer and more convenient administration route and method of the compound bone peptide, and has high practicability. The compound bone peptide enteric micro-pellet provided by the application has the advantages of simple preparation method, low cost, no environmental pollution, and convenience in storage and transportation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a compound bone peptide enteric micro-pellet and a preparation method thereof. BACKGROUND

[0002] The compound bone peptide injection is a sterile powder injection prepared by biological extraction and freeze-drying of pig four-limb bones and scolopendra, which has the characteristics of promoting fracture healing, anti-inflammatory analgesia, small toxic and side effects, good patient tolerance and definite curative effect, and is widely used in the treatment of rheumatism, rheumatoid arthritis, hyperosteogeny, fracture and other diseases. Since injection administration is inconvenient, especially for the elderly, there is a problem of long-term medication, therefore, it is necessary to develop a compound bone peptide oral preparation. However, due to the poor oral stability of polypeptide drugs, there are various physiological barriers in the gastrointestinal tract, such as acid barrier, enzyme barrier, mucus barrier and membrane barrier, which result in extremely low bioavailability. The present application aims to develop a compound bone peptide oral preparation suitable for mass production in factories and having high bioavailability. SUMMARY

[0003] The present application aims to provide a compound bone peptide enteric micro-pellet and a preparation method thereof to solve the above-mentioned problems in the prior art. The compound bone peptide enteric micro-pellet has high bioavailability, can break through the gastric barrier, realize local drug release in the small intestine and maintain an effective therapeutic concentration.

[0004] To achieve the above-mentioned purpose, the present application provides the following solutions.

[0005] The present application provides a compound bone peptide enteric micro-pellet, the raw materials of which include compound bone peptide, filler, disintegrating agent, binder and lubricant.

[0006] Further, the disintegrating agent is sodium carboxymethyl starch, and the lubricant is talc.

[0007] Further, the filler is microcrystalline cellulose.

[0008] Further, the binder is water.

[0009] Further, the mass ratio of the compound bone peptide, the filler, the disintegrating agent, the binder and the lubricant is 10:53:2:22.5:0.65.

[0010] The present application also provides a preparation method of the above-mentioned compound bone peptide enteric micro-pellet, which includes the following steps.

[0011] After the compound bone peptide, the filler, the disintegrating agent and the lubricant are uniformly mixed, the binder is added to prepare a soft material, and then the soft material is prepared into a pellet core by using an extrusion-spheronization method.

[0012] The core is sequentially coated with an isolation layer and an enteric layer to obtain the compound bone peptide enteric micro-pellet.

[0013] Further, the isolation layer is coated; and the enteric layer is coated.

[0014] Further, the mass percentage of the isolation layer and the enteric layer relative to the core is 5% and 15%, respectively.

[0015] Further, the extrusion speed of the extrusion-spheronization method is 30 rpm, the spheronization speed is 1100 rpm, and the spheronization time is 5 min.

[0016] The application also provides the use of the compound bone peptide enteric micro-pellet in the preparation of an oral drug for treating osteoporosis.

[0017] The application discloses the following technical effects:

[0018] The application develops a compound bone peptide oral preparation, i.e., a compound bone peptide enteric micro-pellet, and solves the problem of inconvenient administration of an injection dosage form.

[0019] The application provides a safer and more convenient administration route and method of the compound bone peptide, and has high practicability. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the application, and for those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 Pictures of extruded strips for different administration amounts; wherein A-C are extruded strips for administration proportions of 40wt%, 20wt% and 15wt%, respectively;

[0022] Figure 2 Pictures of cores prepared under different process conditions; wherein A-C are cores prepared under different spheronization speeds of 1000 rpm, 1100 rpm and 1200 rpm; and D-F are cores prepared under different spheronization times of 3 min, 5 min and 7 min;

[0023] Figure 3Figure 1 is a photograph of the core of the compound bone peptide enteric micro pill;

[0024] Figure 4 Figure 2 is a release curve of the compound bone peptide enteric micro pill of each prescription;

[0025] Figure 5 Figure 3 is a photograph of the compound bone peptide enteric micro pill;

[0026] Figure 6 Figure 4 is a hydroxyproline average drug-time curve of the compound bone peptide solution and the compound bone peptide enteric micro pill;

[0027] Figure 7 Figure 5 is a comparison chart of the area under the curve of the average blood drug concentration of the compound bone peptide solution group and the compound bone peptide enteric micro pill group using hydroxyproline as an index;

[0028] Figure 8 Figure 6 is a glutamic acid average drug-time curve of the compound bone peptide solution and the compound bone peptide enteric micro pill;

[0029] Figure 9 Figure 7 is a comparison chart of the area under the curve of the average blood drug concentration of the compound bone peptide solution group and the compound bone peptide enteric micro pill group using glutamic acid as an index;

[0030] Figure 10 Figure 8 is a typical 3D image of the femur Micro-CT scan of each group of rats (n=6);

[0031] Figure 11 Figure 9 is the results of bone tissue morphological parameter analysis (n=6); wherein a is the results of bone trabecular bone volume fraction (BV / TV) analysis; b is the results of bone trabecular number (Tb.N) analysis. DETAILED DESCRIPTION

[0032] The various exemplary embodiments of the present application will now be described in detail, which should not be considered limiting on the present application, but rather as a description of certain aspects, features, and embodiments of the present application.

[0033] It should be understood that the terms used in the present application are merely used to describe particular embodiments and are not intended to limit the present application. In addition, for the numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range within any stated value or stated range, and between any other stated value or stated range, is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0034] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, unless indicated otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application. The description and examples are illustrative of the application and are not limiting.

[0035] Many modifications and variations of this application of the application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative of the application and are not limiting.

[0036] As used herein, the terms "comprise", "comprising", "include", "including", "have" and "having" or the like are used synonymously to mean and disclosed as open-ended language that means "including, but not limited to".

[0037] Compound bone peptide used in the following examples was purchased from Hebei Zhitong Biopharmaceutical Co., Ltd.

[0038] Example 1

[0039] 1. Preparation of the core

[0040] Compound bone peptide, filler, disintegrant (sodium carboxymethyl starch), binder and lubricant (talc) were mixed by extrusion and spherification to prepare the compound bone peptide core. The prescription and process factors affecting the quality of the core were investigated.

[0041] The specific operation was as follows: a certain amount of compound bone peptide powder, filler, disintegrant and lubricant were mixed and passed through a 60-mesh sieve. The binder was slowly added and the soft material was manually prepared. The extrusion was performed three times and the spherification was transferred to the fluidized bed for drying.

[0042] 1.1 Investigation of prescription factors

[0043] The prescription composition of the pellets has a great influence on the shaping of the pellets. Therefore, in the test, the prescription factors of the amount of drug, filler and binder were selected to control the same process parameters. The powder properties were used as the index to investigate the prescription factors affecting the shape of the pellets.

[0044] 1.1.1 Effect of the amount of drug

[0045] The first step in the extrusion-spheronization method is to extrude well-formed strips. In this experiment, microcrystalline cellulose was used as the filler, sodium carboxymethyl starch as the disintegrant, water as the binder, and talc as the lubricant to prepare compound bone peptide pellet cores. Different addition ratios of compound bone peptides were set (40wt%, 20wt%, and 15wt%), and the extruded strips were as follows... Figure 1 As shown.

[0046] The compound bone peptide pellet core formula with a 40wt% addition ratio is as follows: 26g compound bone peptide, 37g microcrystalline cellulose, 2g sodium carboxymethyl starch, 25mL water and 0.65g talc.

[0047] The compound bone peptide pellet core formula with a 20wt% addition ratio is as follows: 13g compound bone peptide, 50g microcrystalline cellulose, 2g sodium carboxymethyl starch, 25mL water and 0.65g talc.

[0048] The compound bone peptide pellet core formula with a 15wt% addition ratio is as follows: 10g compound bone peptide, 53g microcrystalline cellulose, 2g sodium carboxymethyl starch, 25mL water and 0.65g talc.

[0049] Depend on Figure 1 It can be seen that the extruded strips did not form properly when the dosage was 40wt% and 20wt%, due to the excellent water solubility and high viscosity of the compound bone peptide. Therefore, the dosage of the core formulation was determined to be 15wt%.

[0050] 1.1.2 Influence of filler type

[0051] Microcrystalline cellulose, starch, and lactose were used as fillers, and the ratio of compound bone peptide to each filler was kept constant. Compound bone peptide enteric-coated microspheres were prepared by extrusion-spheronization method. The extrusion effect and spheronization effect were used as evaluation indicators to investigate the effect of filler type on microsphere formation and quality. The results are shown in Table 1.

[0052] Compound Bone Peptide Pill Core Formula: 10g compound bone peptide, 53g filler, 2g sodium carboxymethyl starch, 25mL water and 0.65g talc.

[0053] Table 1 Results of the investigation on filler types

[0054]

[0055] The experimental results show that the compound bone peptide becomes sticky after meeting water in the granulation process, the soft material viscosity increases by adding starch as a filler, under the same extrusion speed condition, a large amount of water is extruded in the screen plate during the extrusion process, the strip is difficult to form, and the extrusion cannot be smoothly carried out, so the pellets cannot be formed, and even if other ingredients are added, the defects of the starch cannot be compensated. The micro-pellets prepared by using microcrystalline cellulose as the filler are uniform, the particle size distribution range is narrow, and the roundness is good. The extrusion effect of lactose is also good, but the cost is high. Therefore, the filler of the application is microcrystalline cellulose.

[0056] 1.1.3 Influence of the type of binder

[0057] When microcrystalline cellulose is used as the filler to prepare the pellets, a certain amount of binder needs to be added to increase the plasticity of the soft material and the hardness of the pellet core. Water, 80% ethanol and 8% starch slurry are respectively used as the binder to prepare the pellets, and the quality of the extruded strips, the pellet forming property and the roundness of the pellets, i.e. the extrusion effect and the rounding effect, are used as the indexes to investigate the influence of the type of the binder on the quality of the pellets, and the results are shown in Table 2.

[0058] The compound bone peptide pellet core formula is: compound bone peptide 10 g, microcrystalline cellulose 53 g, sodium carboxymethyl starch 2 g, binder 25 mL and talc 0.65 g.

[0059] Table 2 Investigation results of the type of binder

[0060] Adhesive Water 80% ethanol 8% starch paste Extrusion effect Strips Strips, powder Strips Spheronization effect Spherical Powder Short rod Summary Suitable as adhesive Not suitable as adhesive Not suitable as adhesive

[0061] It is found through the investigation that when 80% ethanol is selected as the binder, the viscosity of the prepared soft material is too small, and the soft material is easily broken into powder during the rounding; when 8% starch slurry is selected as the binder, the viscosity of the prepared soft material is too large, and the soft material cannot be rounded into pellets and is formed into short rod shape. The pellets prepared by using the above binders have unsatisfactory effects. The pellets prepared by using water as the binder have good pellet forming property. Therefore, water is selected as the binder in the application.

[0062] 1.2 Investigation of process factors

[0063] The process parameters have a great influence on the pellet forming, the extrusion- rounding method is used to prepare the pellets, the extrusion speed, the rounding speed and the rounding time are selected as the three process factors, and the influence of the process factors on the preparation of the pellets is investigated by investigating the powder properties of the pellets.

[0064] The following process factor investigation experiment uses the compound bone peptide pellet core formula: compound bone peptide 10 g, microcrystalline cellulose 53 g, sodium carboxymethyl starch 2 g, water 25 mL and talc 0.65 g.

[0065] 1.2.1 Influence of extrusion speed

[0066] The fixed rounding time was 5 min, the rounding speed was 1100 rpm, and the extrusion speed was 20 rpm, 30 rpm and 40 rpm respectively to prepare the pellets, and the influence of the extrusion speed on the pellet morphology was investigated, and the results were shown in Table 3.

[0067] Table 3 Influence of extrusion speed

[0068] Extrusion speed (rpm) Extrusion effect Spheronization effect Summary 20 Cannot be extruded / Not suitable 30 Smooth extruded strips Spherical Suitable 40 Rough and loose extruded strips Powder, spherical Not suitable

[0069] It could be known from Table 3 that the extrusion speed played a key role in the preparation of the pellet cores. When the extrusion speed was low, the strip could not be extruded. When the extrusion speed was high, the material was loose, and fine powder was easy to be produced. When the extrusion speed was 30 rpm, the smooth strip could be extruded, and the extrusion rate was high, so the extrusion speed could be set at 30 rpm.

[0070] 1.2.2 Influence of rounding speed

[0071] The fixed rounding time was 5 min, the extrusion speed was 30 rpm, and the extrusion speed was 1000 rpm, 1100 rpm and 1200 rpm respectively to prepare the pellets, and the influence of the rounding speed on the powder parameters of the pellets was investigated, and the results were shown in Table 4 and A-C in Figure 2 .

[0072] Table 4 Influence of rounding speed

[0073]

[0074]

[0075] It could be known from Table 4 and A-C in Figure 2 that the rounding speed had little influence on the bulk density of the pellet cores, but had great influence on the yield and roundness of the pellet cores. When the speed was too high, fine powder was easy to be produced, and the yield was low. When the speed was too low, the roundness of the pellet cores was not enough. The rounding speed of 1100 rpm had the largest yield of the pellet cores, and had good roundness and flowability, so the rounding speed could be set at 1100 rpm.

[0076] 1.2.3 Influence of rounding time

[0077] The fixed extrusion speed was 30 rpm, the rounding speed was 1100 rpm, and the rounding time was 3 min, 5 min and 7 min respectively to prepare the pellets, and the influence of the rounding speed on the powder parameters of the pellets was investigated, and the results were shown in Table 5 and D-F in Figure 2 .

[0078] Table 5 Influence of rounding time

[0079] Spheronization time (min) Yield of pellet cores (%) Bulk density (g / mL) Sphericity (°) 3 48.25 0.78 38.1 5 51.94 0.71 32.6 7 45.60 0.74 36.8

[0080] It could be known from Table 5 and D-F in Figure 2As shown in the DF (Difference of Dimensions), the rounding time has little effect on the bulk density, but it has a significant impact on the core yield and sphericity. If the rounding time is too short, the rounding process is not completed, resulting in insufficient sphericity of the cores; if the rounding time is too long, the core surface becomes sticky, and the particle size increases. When the rounding time is 5 minutes, the core yield is relatively high, and the cores have good sphericity and flowability. Therefore, the rounding time can be set at 5 minutes.

[0081] The final formulation and process parameters for the compound bone peptide pellet core were determined as follows: 10g compound bone peptide, 53g microcrystalline cellulose, 2g sodium carboxymethyl starch, 0.65g talc, and 25mL water were used to form a soft mass. The mass was extruded three times at 30 rpm, with a spheroidizing speed of 1100 rpm and a spheroidizing time of 5 minutes. The pellet core prepared according to the optimal formulation is shown in the image. Figure 3 .

[0082] 1.2.4 Reproducing the Experiment

[0083] The optimal formulation and process were repeated several times, and the weight of the microspheres was determined by sieving, and the particle size distribution of the microspheres was calculated. Data from several batches of experiments are shown in Table 6.

[0084] Table 6. Particle size distribution of different batches of microspheres

[0085]

[0086] 2. Preparation of Compound Bone Peptide Enteric-Coated Microspheres

[0087] 2.1 Reagent Preparation

[0088] Isolation coating solution: Weigh out Dispersed in ultrapure water and magnetically stirred for 45 min, a 10% (w / w) solution was prepared. Coating solution.

[0089] Enteric coating solution: Weigh out Dispersed in excess water, magnetically stirred for 60 minutes, and passed through an 80-mesh sieve, a 20% (w / w) solution was prepared. Coating solution.

[0090] Simulated gastric juice: Accurately measure 9 mL of hydrochloric acid and dissolve it in 1 L of ultrapure water.

[0091] Simulated intestinal fluid: Accurately weigh 6.8g KH2PO4 and 0.944g NaOH and dissolve them in 1L of ultrapure water, then adjust the pH to 6.8.

[0092] 2.2 Coating

[0093] Prepare 65 g of compound bone peptide core according to the optimal prescription process determined in 1.2.3, sieve, collect the 18-24 mesh range of the core as the subsequent coating raw material. In the coating machine, coat the isolation layer and the enteric layer in turn, and the compound bone peptide enteric micro-pellets are obtained. The coating process parameters are shown in Table 7, and the coating ratio is shown in Table 8.

[0094] Table 7 Coating process parameters

[0095] Process parameters Isolation layer Enteric layer Spheronization speed (rpm) 200 300 Peristalsis speed (rpm) 8 10 Air blowing frequency (Hz) 20 20 Material temperature (°C) 35 29 Air inlet temperature (°C) 60 55 Nozzle diameter (mm) 1 1 Rotary disc diameter (mm) 250 250

[0096] Table 8 Coating prescription

[0097]

[0098] Note: * Indicates the percentage relative to the mass of the compound bone peptide core.

[0099] 2.3 Release rate determination of compound bone peptide enteric micro-pellets

[0100] 2.3.1 Determination of drug loading of micro-pellets

[0101] Take 2 g of compound bone peptide enteric micro-pellets, crush in a mortar, disperse in 10 mL of ultrapure water, magnetically stir for 30 min, take 5 mL of sample, filter with a 0.8 μm filter membrane, discard the first 2 mL, and determine the protein concentration of the filtrate by BCA method. The mass of the micro-pellets is denoted as m (g), the protein concentration of the filtrate is denoted as C (mg / mL), and the drug loading L of the micro-pellets is calculated by the following formula:

[0102] L = 0.2C / m.

[0103] 2.3.2 Determination of release rate of micro-pellets

[0104] According to the 2015 edition of Chinese Pharmacopoeia, select the small cup method 100 rpm as the release condition. Accurately take 0.5 g of compound bone peptide enteric micro-pellets, use 200 mL of simulated gastric fluid as the release medium for the first 2 h, and use 200 mL of simulated intestinal fluid as the release medium for the last 45 min. Take 5 mL of sample at regular intervals, add 5 mL of release medium at the same time, filter with a 0.8 μm filter membrane, discard the first 2 mL of filtrate, and determine the protein concentration of the subsequent filtrate by BCA method. The mass of the micro-pellets is denoted as m, the drug loading of the micro-pellets is denoted as L, the volume of the release medium is denoted as V, and the protein concentration of the subsequent filtrate is denoted as C. The release percentage P of the compound bone peptide is calculated by the following formula:

[0105] P = C*V / m / L.

[0106] The release rate of the compound bone peptide enteric micro-pellets of each prescription is shown in Figure 4 . From Figure 4It can be seen that the uncoated compound bone peptide pellets (R1) release faster in simulated gastric fluid, and are basically completely released within 15 min, without sustained release effect. In the case of the same weight gain of the isolation layer coating (R2 and R3, R5 and R6, R8 and R9), with the increase of the weight gain of the enteric layer coating, the release rate of the compound bone peptide in the simulated gastric fluid is significantly reduced. This effect may be related to the thickness of the coating layer, and the increase of the thickness of the coating layer can significantly increase the diffusion path of the compound bone peptide, thereby effectively reducing the release rate of the compound bone peptide. When the weight gain of the enteric layer coating is 0 (R4 and R7), the release rate of the pellets in the simulated gastric fluid is basically the same as that of the uncoated pellets, indicating that the isolation layer coating material has no sustained release effect. When the weight gain of the enteric layer coating is the same (R2, R5 and R8, R3, R6 and R9), with the increase of the weight gain of the isolation layer coating, the release rate of the compound bone peptide in the simulated gastric fluid is reduced to a certain extent. This effect may be related to the barrier effect of the isolation layer, and the presence of the isolation layer can well hinder the interaction between the compound bone peptide and the enteric layer, ensure the integrity of the enteric layer, and thereby effectively improve the efficiency of the enteric layer.

[0107] According to the 2015 edition of Chinese Pharmacopoeia, the standard for enteric preparation is that the release in simulated gastric fluid is less than 10% in 2 h, and the release in simulated intestinal fluid is more than 70% in 45 min. R6 and R9 can both reach the enteric standard, but considering the economy, the present application selects R6 as the final enteric coating prescription, that is, the weight gain of the isolation layer coating is 5%, and the weight gain of the enteric layer coating is 15%. The prepared compound bone peptide enteric pellets are as shown in Figure 5 .

[0108] 3. Preliminary study on the pharmacokinetics of compound bone peptide enteric pellets in rats in vivo

[0109] The common methods for studying the running rules of gastric retention preparations, small intestine positioning drug release and colon positioning drug delivery preparations in the gastrointestinal tract are as follows: in vivo local drug concentration monitoring method, blood drug concentration method, and y-scintillation scanning method. The first two methods are difficult to comprehensively obtain the in vivo process information such as the time of drug staying in the stomach and transporting in the small intestine, the time of reaching each part of the body, the time and part of starting and completely releasing the drug, and the distribution of the drug in the body. The y-scintillation scanning method (Pharmacoscintigraphy) can show the specific part of the drug in the gastrointestinal tract, and provides an intuitive basis for the transportation of the drug in the gastrointestinal tract. However, for the multi-unit drug delivery system such as pellets, there is no obvious difference between the release of radioactive substances from the preparation, and both are in a diffuse distribution in the body. Therefore, the y-scintillation scanning method cannot accurately reflect the transportation process of the pellets in the body. For the drug which can be positioned in the small intestine to release the drug and achieve the effect of systemic absorption, it is also necessary to use the method of determining the blood drug concentration to evaluate the small intestine positioning drug delivery system.

[0110] Compound bone peptide enteric micro-pellets is a new oral dosage form, which is expected to release drugs locally in the small intestine to avoid being destroyed in the stomach and maintain effective therapeutic concentration. In order to verify the rationality of the prescription composition of compound bone peptide enteric micro-pellets and the stability of the preparation process, the relevant in vivo drug release is determined.

[0111] The indicators for evaluating drug absorption include half-life (t 1 / 2 ), AUC, bioavailability (Bioavailability), etc. Bioavailability is an important indicator for measuring the effect of drug absorption, and plays a crucial role in drug efficacy and drug dosage adjustment. Bioavailability is expressed in two ways: absolute bioavailability and relative bioavailability. Absolute bioavailability refers to the ratio between extravascular administration and intravenous injection bioavailability. Relative bioavailability refers to the comparison between different drug dosage forms or different administration routes. This experiment studies the blood drug concentration of compound bone peptide after direct oral administration of compound bone peptide solution and compound bone peptide enteric micro-pellets, calculates the relative bioavailability of oral compound bone peptide, and evaluates the absorption and distribution of the drug.

[0112] 3.1 Relative bioavailability of hydroxyproline

[0113] Hydroxyproline is a very special amino acid that exists specifically in collagen. In addition, hydroxyproline has a high content and specificity in it, is stable in the body, and is not easily decomposed. Collagen is an important protein that constitutes bone tissue, and bone polypeptide drugs often exert their effects by binding to collagen in bone tissue. Therefore, hydroxyproline was selected as an indicator in this experiment to evaluate the bioavailability of compound bone peptide drugs through quantitative detection.

[0114] 3.1.1 Experimental method

[0115] 6 healthy SD rats were randomly divided into 2 groups, namely compound bone peptide solution group and compound bone peptide enteric micro-pellets group. Before the experiment, the rats were fed normally and free to drink water. The dosage was calculated according to the body weight of 126 mg / kg.

[0116] 3.1.2 Administration method

[0117] Compound bone peptide solution group: according to the calculation of 30 mg / mL bone polypeptide content, the rats were weighed before administration, and the administration amount was calculated. The required drug solution was prepared. An appropriate amount of bone peptide for injection was taken, and physiological saline was added before use to prepare a certain concentration of clear solution to ensure uniform mixing, and was administered by gavage.

[0118] Compound bone peptide enteric micro-pellets group: according to the actual drug loading of the micro-pellets, a certain amount of compound bone peptide enteric micro-pellets was weighed for administration, and 5 mL of warm water was used for gavage.

[0119] 3.1.3 Sample collection and processing

[0120] Oral gavage administration: each group of male rats were given compound bone peptide solution and compound bone peptide enteric microspheres (2.2 part of R6 prescription) by gavage, after fasting, their body weight was measured, the actual amount of administration was calculated, and it was ensured that the amount of administration did not cause the rat's trachea to be inaccurate. About 0.5 mL of blood was taken from the eye fundus venous plexus before administration and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 14 h after administration, and was left to stand at room temperature for 2 h, and was high-speed centrifuged. After centrifugation, obvious stratification was observed, and the separated serum was stored at -80°C in the refrigerator for use.

[0121] 100 μL of the above treated serum sample was placed in a centrifuge tube, 200 μL of hydrolysis solution was accurately added, mixed and shaken, and was hydrolyzed in boiling water. The pH value of the solution was adjusted to 6.0-6.8 until the solution turned yellow-green, and was made up to 2 mL with distilled water, and was vortexed. Activated carbon was added for adsorption, mixed and centrifuged, and the supernatant was detected for OD value.

[0122] The content of hydroxyproline in the serum was detected according to the kit instructions. The absorbance of hydroxyproline in the serum sample treated at different time points was measured by an enzyme marker, and the corresponding blood drug concentration was calculated according to the formula, and the blood drug concentration-time curve was analyzed by using DAS software, and the corresponding pharmacokinetic parameters were obtained, and the pharmacokinetic curve was fitted, and the results were expressed as mean ± SD. AUC was calculated by the linear trapezoidal method recommended by FDA.

[0123] Hydroxyproline (μg / mL) = (A 测定 -A 空白 ) / (A 标准 -A 空白 ) × C 标准 × V 水解液 / V 样 .

[0124] 3.1.4 In vivo pharmacokinetic study of hydroxyproline

[0125] According to the calculated concentration of hydroxyproline in the plasma, the pharmacokinetic curve was drawn as shown in Figure 6 The measured data were processed by DAS Ver2.0 pharmacokinetic software, and the pharmacokinetic parameters of compound bone peptide solution and compound bone peptide enteric microspheres were calculated, and the results are shown in Table 9.

[0126] Table 9 Pharmacokinetic parameters of hydroxyproline in plasma after administration (n = 3)

[0127] Parameter Unit Compound bone peptide solution Compound bone peptide enteric micro-pellets AUC (0-t) ]] μg / L·h 14.88±0.68 61.15±8.84 [CAT max ]]> H 1.50±0.00 5.00±0.00 C max ]]> μg / L 7.61±0.93 10.45±0.17 t 1 / 2 ]]> H 0.94±0.31 4.36±3.91 MRT (0-t) ]]> H 1.68±0.09 6.08±0.53

[0128] 3.1.5 Summary

[0129] The experiment was carried out by developing the pharmacokinetics of compound bone peptide to calculate the bioavailability and evaluate the absorption effect of the drug. The experiment showed that the AUC of oral administration of compound bone peptide solution was 14.88±0.68 μg / L·h, and the AUC of oral administration of compound bone peptide enteric-coated pellets was 61.154±8.84 μg / L·h. The maximum blood drug concentration was reached at 5h after administration, and the residence time in the body was longer than that of the injection administration group, and hydroxyproline was metabolized completely in about 14h, which had a long in vivo cycle and was the basis for exerting drug efficacy. Through Figure 7 It can be seen that the AUC of compound bone peptide enteric-coated pellets is significantly higher than that of oral compound bone peptide solution, which is 4.1 times that of compound bone peptide solution (P<0.05). Therefore, this part of the experiment will provide a basis for the future application of compound bone peptide as an oral preparation in clinical practice, and has very important significance for the study of the composition and mechanism of action of the drug.

[0130] 3.2 Relative bioavailability study of glutamic acid

[0131] Glutamic acid is a non-essential amino acid in the body, which plays a key role in regulating signal transmission between nerve cells and the balance of excitability and inhibition of neurons. At the same time, it participates in the transamination of amino acids by converting to alpha-ketoglutarate, thereby maintaining the balance of amino acid metabolism, and participating in cell immunity and energy supply of immune cells, regulating immunity. Glutamic acid is one of the main amino acids in compound bone peptide drugs and plays an important role, so glutamic acid is selected to measure the absorption of oral compound bone peptide.

[0132] 3.2.1 Experimental method

[0133] The same as 3.1.1.

[0134] 3.2.2 Dosing method

[0135] The same as 3.1.2.

[0136] 3.2.3 Sample processing and dosing method

[0137] Oral gavage dosing is the same as 3.1.3.

[0138] 3.2.4 In vivo pharmacokinetics of glutamic acid

[0139] According to the calculated concentration of glutamic acid in the plasma, the pharmacokinetic curve was drawn as shown in Figure 8 The measured data was processed by DAS Ver2.0 pharmacokinetic software to calculate the pharmacokinetic parameters of compound bone peptide solution and self-made compound bone peptide enteric-coated pellets, and the results are shown in Table 10.

[0140] Table 10 Pharmacokinetic parameters of glutamic acid in plasma after administration (n=3)

[0141] Parameter Unit Compound bone peptide solution Compound bone peptide enteric micro-pellets AUC (0-t) ]]> μg / L·h 451±90.82 987.52±49.30 [TECHNICAL FIELD] max ]] H 1.33±0.68 5.00±0.00 [C max ]]> μg / L 195.67±68.72 229.03±52.80 <![CDATA[t 1 / 2 ]]> H 1.83±0.69 1.51±1.21 MRT (0-t) ]]> H 2.58±0.11 4.78±0.04

[0142] 3.2.5 Summary

[0143] The experiment determines the bioavailability of glutamic acid in the compound bone peptide medicine, evaluates the absorption effect of the medicine, and verifies the bioavailability of hydroxyproline, so that the result is more real and reliable. Figure 9 As shown in the formula, the AUC of the compound bone peptide solution after oral administration is 451±90.82 μg / L·h, the maximum drug concentration of the compound bone peptide solution is 195.67 μg / L, and the metabolism is fast. The AUC of the compound bone peptide enteric-coated pellets after oral administration is 987.52±49.30 μg / L·h, which is 2.2 times of the compound bone peptide solution, and the maximum blood drug concentration is 229.03 μg / L at 5 h, and the concentration of glutamic acid in the body is reduced to 0 at 12 h after administration. The absorption effect is good, so it can be inferred that the compound bone peptide enteric-coated pellets can play a better curative effect, and provide a reference for its application in clinic.

[0144] 4 Pharmacodynamic study

[0145] The present application establishes a rat model of osteoporosis caused by retinoic acid, observes the therapeutic effect of the compound bone peptide enteric-coated pellets on osteoporosis, and aims to provide experimental basis for further clinical application.

[0146] 4.1 Animal grouping and model preparation

[0147] Female SD rats were adaptively fed for 1 w, and then randomly divided into 4 groups: normal control group (Control), oral administration of compound bone peptide enteric-coated pellets low-dose group (Compound Bone Peptide enteric coated pellet low dose, CEP-L), oral administration of compound bone peptide enteric-coated pellets high-dose group (Compound Bone Peptide enteric coated pellet high dose, CEP-H) and model group (Model), 6 rats in each group. Except the normal control group, the rest of the rats were given retinoic acid 100 mg / kg by gavage every day, and the administration period was 14 d. After the model was successful, the low-dose and high-dose groups of compound bone peptide enteric-coated pellets were given compound bone peptide enteric-coated pellets (15 mg / kg, 90 mg / kg) respectively, and the normal control group and the model group were given distilled water, and the continuous administration was 14 d.

[0148] 4.2 Sample collection

[0149] After the last gavage administration, the rats were fasted overnight without water, anesthetized to death, and the complete left and right femur and tibia were taken, the residual connective tissue and muscle were fully removed, wrapped with gauze dipped in normal saline, and stored in a-80℃ refrigerator for standby.

[0150] 4.3 Bone histomorphometric parameters detection

[0151] The femur was taken and soaked in 4% paraformaldehyde for 24 h, and then detected by small animal Micro-CT. The bone analysis parameters included: bone trabecular bone volume fraction (BV / TV) and bone trabecular number (Tb.N).

[0152] 4.4 Experimental results

[0153] The bone histomorphology of rats in each group was seen Figure 10 The bone histomorphometric parameter analysis results were seen Figure 11 It can be seen that the BV / TV and Tb.Th of the Model group were reduced, the separation degree of bone trabecula was increased. The porosity of bone trabecula was increased, the continuity was reduced, and the bone trabecular coverage area was also significantly reduced. The BV / TV and Tb.N of rats in each administration group were increased, the number of bone trabecula was increased, and the improvement of bone mass was more significant. The results showed that the compound bone peptide enteric-coated pellets had improvement effects on bone mass and bone microstructure, and the difference had statistical significance.

[0154] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A compound bone peptide enteric-coated microcapsules, characterized in that, The raw materials include compound bone peptides, fillers, disintegrants, binders, and lubricants; The disintegrant is sodium carboxymethyl starch, and the lubricant is talc. The filler is microcrystalline cellulose; The adhesive is water; The mass ratio of the compound bone peptide, the filler, the disintegrant, the binder, and the lubricant is 10:53:2:22.5:0.

65. The preparation method of the compound bone peptide enteric-coated microcapsules includes the following steps: After the compound bone peptide, the filler, the disintegrant and the lubricant are mixed evenly, the binder is added to produce a soft material, and then the soft material is prepared into a pellet core by extrusion-spheronization method. The pellet core is coated with an isolation layer and an enteric coating layer in sequence to obtain the compound bone peptide enteric-coated microgranules; The insulating layer coating is Coating; the enteric coating is Coating; The isolation layer coating and the enteric coating have a mass percentage of 5% and 15% respectively relative to the pellet core; The extrusion-rounding method has an extrusion speed of 30 rpm, a rounding speed of 1100 rpm, and a rounding time of 5 min.

2. The use of the compound bone peptide enteric-coated microspheres as described in claim 1 in the preparation of an oral medicament for treating osteoporosis.

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