CN111904932A

CN111904932A - Micelle preparation containing glucocorticoid and preparation method thereof

Info

Publication number: CN111904932A
Application number: CN201910377695.8A
Authority: CN
Inventors: 吴翠栓; 王欣欣; 代文兵; 张强; 程晓波; 马凤中
Original assignee: Beijing Deli Furui Medical Science & Technology Co ltd
Current assignee: Beijing Deli Furui Medical Science & Technology Co ltd
Priority date: 2019-05-08
Filing date: 2019-05-08
Publication date: 2020-11-10
Anticipated expiration: 2039-05-08
Also published as: CN111904932B

Abstract

The invention provides a micelle preparation containing glucocorticoid and a preparation method thereof, the micelle preparation comprises the glucocorticoid, phospholipid, cholate and water, the weight ratio of the glucocorticoid to the sum of the dosages of the phospholipid and cholate is 1: 5-1: 20, and the weight ratio of the phospholipid to the cholate is 1: 0.5-1: 2.5. The micelle preparation containing the glucocorticoid realizes the efficient encapsulation of the glucocorticoid, the encapsulation rate is more than 90 percent, the particle size is uniform, the preparation method is easy to industrialize, and the micelle preparation has the effect of increasing the curative effect.

Description

Micelle preparation containing glucocorticoid and preparation method thereof

Technical Field

The invention belongs to the field of medicinal preparations, and particularly relates to a micelle preparation containing glucocorticoid and a preparation method thereof.

Background

Glucocorticoids, also known as "adrenocortical hormones", are a class of steroid hormones secreted by the adrenal cortex. It has wide pharmacological action, and has effects in regulating biosynthesis and metabolism of sugar, fat, and protein, inhibiting immune response, relieving inflammation, resisting toxicity, and resisting shock. The glucocorticoid is called as 'glucocorticoid' because the activity of regulating carbohydrate metabolism is recognized for the first time, and the glucocorticoid medicine is widely applied in clinic at present.

However, glucocorticoid drugs also show serious side effects in clinical application, especially adverse effects caused by long-term mass application, mainly including hypercortical hyperfunction syndrome (full moon face, buffalo back, hypertension, hirsutism, diabetes, thinning skin), induced or aggravated infection, induced or aggravated ulcer disease, induced hypertension and arteriosclerosis, osteoporosis, muscular atrophy, delayed wound healing, induced psychosis and epilepsy, etc.

In response to the above-mentioned problems with glucocorticoid drugs, the prior art has sought to improve by various approaches. Chinese patent publication No. CN1731981A discloses a liposome preparation entrapping water-soluble glucocorticoid, which achieves the same drug effect as the original drug when the dose is reduced by 10 times, supposing that the potential toxic and side effects caused by the dose can be significantly reduced; PCT patent (WO 94/07466) adopts PEG modified liposome technology to entrap glucocorticoid, and is expected to increase the stability of the preparation in vivo and prolong the circulation time of the liposome, thereby improving the specific release of the glucocorticoid liposome at the inflammation center and increasing the curative effect; chinese patent CN102366411A discloses a dexamethasone palmitate liposome injection with the same effect.

However, the liposome technology for encapsulating glucocorticoid drugs has limited encapsulation effect, the liposome preparation is unstable, the drugs are easy to leak in the storage process, and for the PEG modified liposome, when the drugs are repeatedly administered, antibodies are generated in vivo, and the half-life period is reduced due to the combination of the antibodies and the liposome, and at the same time, serious immune response is generated, which is clinically undesirable;

the mode of administration in the emulsion loaded with the glucocorticoid medicaments is a more common mode in the existing clinical application. Patent CN104706574A discloses a dexamethasone palmitate fat emulsion concentrated solution; but the prescription adds a large amount of Tween80, and the skilled person knows various problems of Tween 80; patent CN1596899A discloses a dexamethasone palmitate freeze-dried emulsion, in the formula of the freeze-dried emulsion, besides oil phase and emulsifier, a large amount of freeze-drying protective agent is added, the freeze-drying process is complex, the reproducibility has certain problems, the industrial production is easy to generate unstable factors, and in addition, when the emulsion is diluted for use, the uniformity of automatic dispersion is poor, and the use effect is affected.

In summary, it is easy to find that although the existing liposome encapsulation technology, emulsion technology, etc. can increase the curative effect to some extent and reduce the potential side effects of glucocorticoid drugs, there are various problems, and therefore, there is still an urgent need to find other ways.

Disclosure of Invention

In view of the above-mentioned disadvantages or shortcomings of the prior art, it is an object of the present invention to provide a micelle preparation containing glucocorticoid and a method for preparing the same.

To achieve the object of the present invention, the inventors provide the following solutions:

a micelle preparation containing glucocorticoid consists of glucocorticoid, phospholipid, cholate and water, wherein the dosage percentage of the glucocorticoid is 0.05-2% (g/mL), the weight ratio of the glucocorticoid to the sum of the dosages of the phospholipid and the cholate is 1: 5-1: 20, the weight ratio of the phospholipid to the cholate is 1: 0.5-1: 2.5, and the balance is water.

Preferably, the glucocorticoid is used in an amount of 0.05% to 1% (g/mL).

Preferably, the weight ratio of the glucocorticoid to the sum of the dosages of the phospholipid and the cholate is 1: 5-1: 15.

Preferably, the weight ratio of the phospholipid to the bile salt is 1: 1-1: 1.5.

The glucocorticoid is one or more selected from dexamethasone, dexamethasone palmitate, prednisone, prednisolone, cortisone, hydrocortisone, and betamethasone.

The phospholipid is selected from one or more of hydrogenated soybean phospholipid, soybean lecithin, egg yolk lecithin, cephalin, serine phospholipid, and polyene phosphatidylcholine.

The cholate is selected from one or more of taurocholic acid, glycocholic acid, glycodeoxycholic acid, taurodeoxycholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid or pharmaceutically acceptable salts thereof.

The micelle preparation containing the glucocorticoid can also comprise a freeze-drying protective agent, and micelle freeze-drying powder is prepared, wherein the freeze-drying protective agent accounts for 0-30% (g/mL) of the weight-volume ratio of the liquid micelle preparation, and the freeze-drying protective agent is at least one selected from sucrose, lactose, trehalose and mannitol.

In addition, the invention also provides a preparation method of the glucocorticoid micelle preparation, which comprises the following steps:

(1) dissolving glucocorticoid medicine, phospholipid and cholate in absolute alcohol.

(2) And (3) carrying out rotary evaporation on the dissolved ethanol mixed solution on a rotary evaporator until the ethanol solution is completely volatilized, so that a uniform film is formed in the system.

(3) Adding water into the film, and hydrating in water bath under ultrasonic condition to obtain the medicine carrying mixed micelle solution.

The dosage of the glucocorticoid in the glucocorticoid micelle preparation is 0.05-2% (g/mL), in a preferred embodiment, the glucocorticoid is dexamethasone palmitate, the dosage of the dexamethasone palmitate is 0.5% (g/mL), the weight ratio of the sum of the dosages of the phospholipid and cholate is 1: 6.4, the weight ratio of the phospholipid and the cholate is 1: 1, the phospholipid is egg yolk lecithin, the dosage of the phospholipid is 1.6% (g/mL), the cholate is sodium glycocholate, the dosage of the cholate is 1.6% (g/mL), and the balance is water.

In another preferred embodiment, the glucocorticoid is betamethasone in an amount of 1% (g/mL), the weight ratio of the betamethasone to the sum of the amounts of the phospholipid and the cholate is 1: 10, the weight ratio of the phospholipid to the cholate is 1: 1.2, the phospholipid is egg yolk lecithin in an amount of 4.54% (g/mL), the cholate is taurodeoxycholic acid in an amount of 5.46% (g/mL), and the balance is water.

The micelle preparation containing the glucocorticoid, which has the advantages of good encapsulation efficiency, good drug loading capacity, uniform particle size distribution and good stability, can be obtained, and the preparation method of the micelle preparation is simple and easy to industrialize.

Drawings

FIG. 1 transmission electron micrograph of glucocorticoid-phospholipid/cholate nanomicelle in example 1.

Figure 2. glucocorticoid-phospholipid/bile salt nanomicelle formulation in test example 3. the swelling curve of the feet was studied in vivo pharmacodynamics.

FIG. 3 in Experimental example 3 the glucocorticoid-phospholipid/cholate nanomicelle formulation was used for in vivo pharmacodynamic study of disease accumulation index.

FIG. 4 shows the in vivo pharmacodynamic study of the weight change curve of glucocorticoid-phospholipid/cholate nanomicelle formulation in Experimental example 3.

FIG. 5 spleen index of glucocorticoid-phospholipid/bile salt nanomicelle formulation in Experimental example 3 was studied in vivo pharmacodynamics.

FIG. 6 is a H & E staining diagram of ankle joint in the in vivo pharmacodynamic study of glucocorticoid-phospholipid/cholate nano-micelle preparation in experimental example 3.

Figure 7. glucocorticoid-phospholipid/bile salt nanomicelle formulation in test example 4. the swelling curve of the feet was studied in vivo pharmacodynamics.

FIG. 8 in Experimental example 4 the glucocorticoid-phospholipid/cholate nanomicelle formulation was studied for disease accumulation index in vivo pharmacodynamics.

The specific implementation mode is as follows:

in order to solve the problems of the encapsulation efficiency of the micelle preparation on the glucocorticoid medicaments and the particle size of the micelle preparation, the inventor of the invention has made a great deal of research, and based on the constitutional elements of the micelle, the inventor screens the proportion of the glucocorticoid medicaments to the micelle materials, the composition of the micelle materials, the preparation method of the micelle preparation and the like. After intensive research, the inventors of the present invention have unexpectedly found that when the micelle preparation has a composition of: the dosage of the glucocorticoid medicaments is between 0.05 percent and 2 percent (g/mL), the micelle material is a mixture of phospholipid and cholate, the weight ratio of the glucocorticoid to the sum of the dosages of the phospholipid and the cholate is 1: 5 to 1: 20, the weight ratio of the phospholipid to the cholate is 1: 0.5 to 1: 2.5, and the balance is water, and the micelle preparation prepared by combining the preparation method has the encapsulation rate of more than 90 percent, the particle size of 50nm to 200nm, and the micelle preparation has good physical and chemical stability.

The present invention will be described in further detail below based on examples and test examples. In the present invention, all the devices and materials are commercially available or commonly used in the industry. The following examples and test examples are only for specifically describing the present invention, and the present invention is not limited thereto.

Example 1

Prescription:

dexamethasone palmitate	500mg
		Egg yolk lecithin	1.6g
Glycocholic acid sodium salt	1.6g
		Sterilized water for injection	Adding to 100mL

The process comprises the following steps:

(1) weighing the dexamethasone palmitate, the egg yolk lecithin and the sodium glycocholate with the prescription amount into a 500mL round-bottom flask, and adding a proper amount of absolute ethyl alcohol to completely dissolve the dexamethasone palmitate, the egg yolk lecithin and the sodium glycocholate.

(2) And (3) carrying out rotary evaporation on the ethanol mixed solution obtained in the step (1) on a rotary evaporator until the ethanol solution is completely volatilized, so that a uniform film is formed in the system.

(3) Adding sterilized water for injection into the film, and slightly shaking and hydrating in a water bath under ultrasonic conditions to obtain the medicine-carrying mixed micelle solution.

The particle size and the encapsulation rate of the prepared dexamethasone palmitate micelle preparation are measured, and the result shows that the average particle size of the micelle preparation is 49.2nm, the encapsulation rate is 94.5%, and a transmission electron microscope image of the preparation is shown in figure 1.

Examples 2 to 45

The glucocorticoid micelle preparation of the present invention was prepared by the same preparation method as in example 1 according to the formulations shown in tables 1 to 4 below. The freeze-drying protective agent is added into the prescription corresponding to the embodiments 22-27, the corresponding preparation process is that on the basis of the process described in the embodiment 1, the prescription amount of the freeze-drying protective agent is dissolved in sterile water for injection in advance, and then the solution is hydrated and freeze-dried to obtain the corresponding freeze-dried preparation, and the obtained freeze-dried preparation powder cake has good appearance and does not shrink or collapse.

The particle size and encapsulation efficiency of the formulations of examples 1 to 27 were measured, and the results are shown in tables 1 to 4.

TABLE 1

	Unit of	Example 2	Example 3	Example 4	Example 5	Example 6	Example 7
								Dexamethasone palmitate	mg	50	100	200	500	750	1000
Hydrogenated soybean phospholipids	g	0.667
								Soybean lecithin	g		0.428
Egg yolk lecithin	g			0.667
								Polyene phosphatidyl choline	g				1.25
Cephalin	g					2.4
								Serine phospholipids	g						4.5
Glycocholic acid sodium salt	g	0.333	1.072	1.333	1.25	3.6	5.5
								Water (W)	ml	Adding to 100ml	Adding to 100ml	Adding to 100ml	Adding to 100ml	Adding to 100ml	Adding to 100ml
Average particle diameter	nm	90.5	75.8	58.2	70.5	75.4	71.3
								Encapsulation efficiency	％	91.5	92.8	95.0	90.1	92.2	93.3

TABLE 2

	Unit of	Example 10	Example 11	Example 12	Example 13	Example 14	Example 15
								Dexamethasone palmitate	mg	500	500	500	500	1500	2000
Egg yolk lecithin	g	1.67	2.5	2.85	3.75	7.5	11.4
								Taurocholic acid sodium salt	g	0.83
Glycocholic acid sodium salt	g		2.5
								Glycodoxycholic acid sodium salt	g			7.15
Taurodeoxycholic acid sodium salt	g				3.75
								Glycine sodium deoxycholate	g					7.5
Sodium taurochenodeoxycholate	g						28.6
								Water (W)	ml	Adding to 100ml	Adding to 100ml	Adding to 100ml	Adding to 100ml	Adding to 100ml	Adding to 100ml
Average particle diameter	nm	72.5	88.2	69.4	78.3	76.5	93.1
								Encapsulation efficiency	％	90.8	92.5	94.1	95.3	95.5	95.7

TABLE 3

	Unit of	Example 16	Example 17	Example 18	Example 19	Example 20	Example 21
								Dexamethasone	mg		200
Prednisone	mg		500
								Prednisolone	mg			2000
Cortisone	mg				500
								Hydrocortisone	mg					500
Betamethasone	mg						500
								Egg yolk lecithin	g	1.6	2.5	10	3.33	1.67	2.5
Glycocholic acid sodium salt	g	1.6	2.5	10	1.67	3.33	2.5
								Water (W)	ml	Adding to 100ml	Adding to 100ml	Adding to 100ml	Adding to 100ml	Adding to 100ml	Adding to 100ml
Average particle diameter	nm	65.6	70.2	97.3	68.6	65.9	70.4
								Encapsulation efficiency	％	93.6	94.8	90.6	95.3	94.5	94.6

TABLE 4

	Unit of	Example 22	Example 23	Example 24	Example 25	Example 26	Example 27
								Dexamethasone palmitate	mg	500	500	500	500	500	500
Egg yolk lecithin	g	1.6	2.5	3.33	3.75	1.67	2.85
								Glycocholic acid sodium salt	g	1.6	2.5	1.67	3.75	0.83	1.75
Sucrose	g		10			20										10
								Lactose	g						15
Trehalose	g			15	30		15									15
								Mannitol	g					10			5
Water (W)	ml	Adding to 100ml	Adding to 100ml	Adding to 100ml	Adding to 100ml	Adding to 100ml	Adding to 100ml
								Average particle diameter	nm	62.6	84.2	95.3	76.6	88.9	58.4
Encapsulation efficiency	％	95.6	91.8	90.6	95.3	90.5	94.6

From the above examples 1 to 27, it is understood that the glucocorticoid micelle preparation having a particle size of 100nm or less and an entrapment rate of 90% or more can be obtained by the present invention.

Test examples

The inventor experimentally examines main factors influencing the particle size and encapsulation efficiency of the preparation in the glucocorticoid micelle preparation.

Restated again: the following experiments are only exemplary experiments among many experiments in the development process of the present invention, and do not cover all the experiments performed by the inventors for the present invention, and the purpose is to illustrate the effect of different components and amounts on the particle size and encapsulation efficiency of the micelle preparation of the present invention by using the following data.

Test example I Effect of the ratio of glucocorticoid to micellar Material on glucocorticoid micellar formulations

The micellar material described in the patent refers to phospholipid and bile salt, and the ratio of the glucocorticoid drug to the micellar material (hereinafter, all referred to as drug-lipid ratio) is a key factor for preparing a qualified and stable micellar preparation, so the inventor examines the drug-lipid ratio under the condition of preliminarily determining that the dosage ratio of the phospholipid and the bile salt is 1: 1 (w/w).

The micelle preparation containing dexamethasone palmitate is prepared according to the formulas 1-8 in the table 5 and the preparation method described in the example 1, and the appearance, stability, average particle size and encapsulation efficiency results of the corresponding preparation are shown in the table 5. The results show that only when the ratio of the drug to the lipid is in the range of 1: 5-1: 20, the dexamethasone palmitate micelle preparation with uniform particle size and good stability can be prepared.

TABLE 5 prescription and investigation results of micelle preparation with different mixture ratios of dexamethasone palmitate and micelle material

Test example II Effect of phospholipid to bile salt ratio on glucocorticoid micelle formulations

The inventor of the invention has conducted a great deal of research and surprisingly found that the ratio (w/w) of phospholipid and cholate in the formula has a significant influence on the stability, particle size and encapsulation efficiency of the micelle preparation, and the inventor determined that the ratio of phospholipid and cholate is 1: 10 and examined the ratio.

The micelle preparation containing dexamethasone palmitate is prepared according to the formulas 9-16 in the table 6 and the preparation method described in the example 1, and the appearance, stability, average particle size and encapsulation efficiency results of the corresponding preparation are shown in the table 6. The results show that the dexamethasone palmitate micelle preparation with uniform particle size and good stability can be prepared only when the ratio of the phospholipid to the cholate is in the range of 1: 0.5-1: 4.

TABLE 6 formulation recipe and investigation results corresponding to different ratio of phospholipid and bile salt

Test example III in vivo pharmacodynamic study of dexamethasone palmitate micelle preparation

SD male rats (120-. Tail vein administration is started on day 9 after the model is made, and the drug-loaded mixed nano-micelle is prepared according to the specific embodiment 1, wherein the administration components are 5 groups, and each group comprises 6 drugs which are respectively: model group, micelle low, medium, high concentration group, and emulsion medium dosage group.

The administration dosages of the dexamethasone palmitate-phospholipid/cholate mixed nano micelle are 0.2mg/kg, 0.4mg/kg and 0.8mg/kg respectively; the dosage of the emulsion group is 0.4 mg/kg. The administration is once every other day.

(1) Measuring foot volume: initial foot volume was recorded before inflammation, measured every two days after inflammation and compared to the initial foot volume to plot the foot swelling curve, the results of which are shown in figure 2. The results of the figure show that each dosage group of the micelle has certain drug effect and shows dose dependence. The area under the curve from day 10 to day 28 was taken as the cumulative disease index, and the results are shown in fig. 3, and the micelle dosage group showed some advantages and significant differences compared with the same dosage emulsion group.

(2) Body weight change curve: rats were weighed every 2 days after the onset of inflammation with a balance. As shown in FIG. 4, the body weight of the rats in the model group was slightly reduced, and no significant weight loss occurred in each administration group.

(3) Spleen index: the rats were sacrificed on day 28, spleens were isolated after dissection, weighed, and the ratio of them to the body weight of the rats was used as a spleen index. The results are shown in FIG. 5. The spleen enlargement is serious in the model group, the spleen index is increased, the spleen index of each administration group is reduced, the high dose group is close to a normal value, and the micelle and the emulsion in the same dose have significant advantages.

(4) Observation of ankle pathology: the ankle joints of rats are taken, the epidermis and redundant tissues are removed, paraffin sections are prepared, H & E staining is carried out, and the joint damage and recovery conditions are observed. As a result, as shown in FIG. 6, the model group showed narrowing of the joint space, formation of pannus, and severe destruction of articular cartilage and bone. The improvement of each administration group is obvious.

Test example four, in vivo pharmacodynamic study of dexamethasone palmitate micelle preparation

SD male rats (120-. Starting articular cavity administration by 100 microliters at 9 days after molding, preparing the drug-loaded mixed nano-micelle according to the specific example 1, wherein the administration components are 4 groups, and each group comprises 6 drugs: model group, micelle low, medium, high concentration group.

The single administration dosage of each group of dexamethasone palmitate-phospholipid/cholate mixed nano micelle is as follows: 0.05mg, 0.1mg, 0.2 mg. Given every second day.

The foot volume was measured every two days after administration and compared with the initial values to evaluate the efficacy.

The results are shown in FIG. 7. The swelling degree of each dose group is obviously reduced.

The area under the curve from day 10 to day 28 was taken as the cumulative disease index and the results are shown in fig. 8, where the micelle low, medium and high dose groups all had significant differences compared to the model group.

Claims

1. A micelle preparation containing glucocorticoid is characterized by comprising glucocorticoid, phospholipid, cholate and water, wherein the dosage percentage of the glucocorticoid is 0.05-2% (g/mL), the weight ratio of the glucocorticoid to the sum of the dosages of the phospholipid and cholate is 1: 5-1: 20, the weight ratio of the phospholipid to the cholate is 1: 0.5-1: 2.5, and the balance is water.

2. The micelle formulation comprising a glucocorticoid according to claim 1, wherein the glucocorticoid is present in an amount of between 0.05% and 1% (g/mL).

3. The micelle formulation comprising a glucocorticoid according to claim 1, wherein the weight ratio of the glucocorticoid to the sum of the amounts of phospholipid and cholate is 1: 5 to 1: 15.

4. The micelle formulation comprising a glucocorticoid according to claim 1, wherein the weight ratio of the phospholipid to the bile salt is 1: 1 to 1: 1.5.

5. The micelle formulation comprising a glucocorticoid according to claim 1, wherein the glucocorticoid is selected from one or more of dexamethasone, dexamethasone palmitate, prednisone, prednisolone, cortisone, hydrocortisone, betamethasone.

6. The micelle formulation comprising a glucocorticoid according to claim 1, characterized in that the phospholipids are selected from one or more of the group consisting of hydrogenated soy phospholipids, soy lecithin, egg lecithin, cephalin, serine phospholipids, polyene phosphatidylcholines.

7. The micelle formulation comprising a glucocorticoid according to claim 1, wherein the cholate is selected from one or more of taurocholic acid, glycocholic acid, glycodeoxycholic acid, taurodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, or a pharmaceutically acceptable salt thereof.

8. The micelle preparation containing the glucocorticoid according to any one of claims 1 to 7, which further comprises a lyoprotectant, wherein the lyoprotectant is prepared as micelle freeze-dried powder, and the lyoprotectant accounts for 0 to 30% (g/mL) of the weight-volume ratio of the liquid micelle preparation.

9. The micelle formulation comprising a glucocorticoid according to claim 8, wherein the lyoprotectant is at least one selected from the group consisting of sucrose, lactose, trehalose, and mannitol.

10. The preparation method of the glucocorticoid micelle preparation is characterized by comprising the following steps:

11. The micelle preparation according to any one of claims 1 to 7, comprising a glucocorticoid, for use in intravenous and/or intra-articular injection for the treatment of rheumatoid arthritis.