CN107865824B - Stable recombinant human endostatin subcutaneous injection composition - Google Patents

Abstract

The invention relates to a recombinant human vascular endothelial inhibin injection, which mainly comprises a stabilizer, a buffer solution and recombinant human vascular endothelial inhibin. The recombinant human endostatin injection has the characteristics of high concentration and good stability, can replace the traditional intravenous drip solution, obviously shortens the administration time, reduces the physiological pain of patients and improves the clinical compliance of the patients.

Description

Stable recombinant human endostatin subcutaneous injection composition

Technical Field

The invention relates to the technical field of a recombinant human vascular endothelial inhibin pharmaceutical composition.

Background

Recombinant human endostatin (rhEndostatin,

the name of Chinese:

) (ZL00107569.1) is prepared by modifying the nucleotide coding sequence of human endostatin to make its N-terminal carry 9 additional amino acid sequences. The produced recombinant human vascular endothelial inhibin consists of 192 amino acids, and the amino acid sequence is as follows: (M) GGSHHHHHHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMTASK.

O 'Reilly teaches that substances (Endostatin ) capable of inhibiting the growth of vascular endothelial cells were isolated from the culture medium of the mouse endothelial cell line EOMAD in 1997, and could significantly inhibit the growth of various primary tumors by inhibiting the generation of tumor blood vessels (O' Relly, M.S., et al.cell.1997,88: 277-285). The recombinant human vascular endostatin is used for inhibiting tumor growth based on the theory of 'starvation tumor therapy', and is clinically combined with an NP chemotherapy scheme for non-small cell lung cancer patients.

The recombinant human endostatin sold in the market at present is a common injection, and in clinical use, a patient needs to inject once a day, a treatment course comprises 14 continuous days, and the next treatment course is continued after a rest for one week. And when the injection is injected every day, the intravenous drip time is at least 3-4 h, and the clinical compliance of patients is poor. Therefore, the recombinant human endostatin is developed into a subcutaneous injection preparation, can replace the traditional intravenous drip solution, obviously shortens the administration time, reduces the physiological pain of patients and improves the clinical compliance of the patients.

The administration route of the recombinant human vascular endothelial inhibin injection produced at home at present is intravenous drip, and the specification is 15mg/3 mL. When a subcutaneous injection is developed, the concentration is increased to 15mg/1mL to 15mg/0.5 mL. However, the increase of the concentration of the recombinant human vascular endothelial inhibin brings about the obvious increase of visible foreign matters or particles, and the recombinant human vascular endothelial inhibin subcutaneous injection is not available in China.

Disclosure of Invention

The invention aims to provide the recombinant human endostatin injection which is suitable for subcutaneous injection, and the administration time is obviously shortened, so that the physiological pain of a patient is reduced, and the clinical compliance of the patient is improved.

The technical problem to be solved by the invention is to provide a recombinant human endostatin subcutaneous injection composition with high concentration and good stability.

After a great deal of experiments, the inventor surprisingly discovers that the combination of the sugar and the surfactant in a buffer system with specific pH can obviously inhibit the formation of visible foreign matters or particles of the recombinant human endostatin at high concentration.

The subcutaneous injection composition of the recombinant human vascular endothelial inhibin comprises a stabilizer, a buffer solution and the recombinant human vascular endothelial inhibin.

The stabilizer provided by the invention can inhibit the formation of visible foreign matters or particles of the recombinant human vascular endothelial inhibin at high concentration. The stabilizer in the composition comprises saccharides and a surfactant.

Wherein the saccharide is preferably one or more of sucrose, lactose, glucose, trehalose, mannitol and sorbitol, and more preferably one or more of trehalose, mannitol and sucrose.

Preferably, the saccharide concentration is 10-100 mg/ml, further selected from 20-80 mg/ml, and preferably 40 mg/ml.

Wherein the surfactant is preferably one or more of polysorbate and poloxamer.

Wherein the polysorbate is preferably one or more of tween 20 and tween 80.

Wherein the poloxamer is preferably one or more of poloxamer 105, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 401, poloxamer 403 and poloxamer 407, and more preferably one or two of poloxamer 188 and poloxamer 407.

Preferably, the dosage concentration of the surfactant is 0.1-2 mg/ml, and preferably 0.2-2 mg/ml.

The concentrations mentioned above are calculated on the basis of the volume of the injection solution.

Wherein the buffer solution is preferably acetate buffer solution or histidine buffer solution, preferably acetic acid-sodium acetate buffer solution or histidine-histidine hydrochloride buffer solution.

Wherein the pH of the buffer system is preferably 5.0-6.5, and preferably 5.0-6.0.

The concentration of the buffer system is preferably 20 mM-120 mM, and preferably 20 mM-60 mM.

The recombinant human vascular endothelial inhibin of the invention is protein, fragment or modified form thereof which can inhibit endothelial cell proliferation. The recombinant human endostatin can be liquid, freeze-dried or spray-dried solid. Preferably, the recombinant human endostatin is rhEndostatin (Endotatin), and the concentration of the recombinant human endostatin is 15 mg/mL-30 mg/mL.

The invention has the advantages that:

the composition adopts the combination of the saccharides and the surfactant as the stabilizer, can obviously inhibit the formation of visible foreign matters or particles of the recombinant human vascular endothelial inhibin at high concentration in a buffer system with specific pH, and improves the stability;

compared with the traditional intravenous drip, the subcutaneous injection of the invention obviously shortens the administration time, reduces the physiological pain of patients and improves the clinical compliance of the patients.

Detailed Description

The present invention is described in more detail by the following examples, but the scope of the present invention is not limited thereto.

EXAMPLE 1 screening of buffer systems

(1) Prescription preparation

The pH value of the human body is 7.4, and the pH value of the injection is generally required to be 4-9. In addition, in order to ensure that the quality of the finished product liquid medicine is stable in the storage period, and a reasonable pH value range of the liquid medicine needs to be determined, the inventor prepares solutions according to the table 1 respectively, and encapsulates the solutions in a penicillin bottle for storage, wherein in the buffer system, an acetate buffer solution is an acetic acid-sodium acetate buffer solution, a citrate buffer solution is a citric acid-sodium citrate buffer solution, a histidine buffer solution is a histidine-histidine hydrochloride buffer solution, and a phosphate buffer solution is a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution.

TABLE 1 prescription List

(2) Accelerated test

The above formulations were checked for appearance and purity by accelerated testing for 2 weeks at 25 deg.C, 2 weeks at 40 deg.C, and 4 weeks at 40 deg.C, respectively.

For purity check, the purity of rhEndostatin is determined by HPLC, and the chromatographic column of HPLC method is C₁₈And (3) performing gradient elution on the reversed-phase column by using a 0.1% trifluoroacetic acid aqueous solution as a mobile phase A liquid and using 0.1% trifluoroacetic acid acetonitrile as a mobile phase B liquid for 24min (the A liquid is 64-40% and the B liquid is 36-60%), wherein the flow rate is 1.3ml/min, and the detection wavelength is 214 nm.

Appearance results are shown in table 2:

TABLE 2 appearance observation results

The purity check results are shown in table 3:

TABLE 3HPLC purity results

Conditions of standing	Prescription 1	Prescription 2	Prescription 3	Prescription 4	Prescription 5	Prescription 6	Prescription 7	Prescription 8
									Day 0	98.60	98.74	98.57	98.67	98.63	98.57	98.55	98.56
2 weeks at 25 ℃	98.65	98.69	98.45	98.54	98.60	98.59	98.50	98.44
									2 weeks at 40 ℃	97.64	97.87	ND*	ND*	97.74	97.98	ND*	ND*
4 weeks at 40 ℃	92.78	93.91	ND*	ND*	92.84	ND*	ND*	ND*

ND: indicating that no measurement was performed.

As can be seen from tables 2 and 3, in the formula of the mannitol-containing recombinant human endostatin (rhEndostatin), each buffer system with pH of 5.0-6.5 has better stability at room temperature of 25 ℃. When the buffer solution is acetate buffer solution, the highest stability is shown in the accelerated test process, and the appearance of the test sample is not obviously changed after the test sample is stored for 4 weeks at 40 ℃; however, when the buffer solution is histidine salt, the stability of the buffer solution is different according to different pH values; when the buffer solution is citrate buffer solution and phosphate, the flaky protein is separated out when visible foreign matters exist at 40 ℃ for 2-4 weeks, and the system stability is not high.

Example 2 Effect of stabilizer sugars and surfactant

(1) Prescription preparation

An acetate buffer system and a histidine buffer system are selected to study the influence of saccharides and surfactants on the stability of recombinant human endostatin (rhEndostatin), and the inventor prepares solutions according to the table 4 respectively and encapsulates the solutions in penicillin bottles for storage. In the buffer system, an acetate buffer solution is an acetic acid-sodium acetate buffer solution, a histidine salt buffer solution is a histidine-histidine hydrochloride buffer solution, and a phosphate buffer solution is a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution.

TABLE 4 prescription List

(2) Accelerated test

The above formulations were examined for appearance and purity by accelerated testing, respectively, at 25 ℃ for 2 months, 25 ℃ for 6 months, 40 ℃ for 2 weeks, and 40 ℃ for 4 weeks.

For purity check, the purity of rhEndostatin is determined by HPLC, and the chromatographic column of HPLC method is C₁₈The reversed phase column, using 0.1% trifluoroacetic acid water solution as mobile phase A liquid, using 0.1% trifluoroacetic acid acetonitrile as mobile phase B liquid, gradient eluting for 24min (liquid A from 64% -40%, liquid B from 36% -60%), flow rate of 1.3ml/min, detection wavelength of 214nm。

The appearance observation results are shown in table 5:

TABLE 5 appearance observation results

The purity check results are shown in table 6:

TABLE 6HPLC purity results

The particle size was measured by DLS method (dynamic light scattering method) using a malvern Zetasizer nano Z laser detector, and the results are shown in table 7:

TABLE 7DLS results (mean particle size, nm)

Conditions of standing	Prescription 9	Prescription 10	Prescription 11	Prescription 12	Prescription 13
						Day 0	3.73	3.83	4.87	4.62	6.27
2 weeks at 40 ℃	3.66	3.66	5.04	4.59	10.52
						4 weeks at 40 ℃	3.84	4.02	6.51	4.25	31.96

As can be seen from tables 5, 6 and 7, the acetate buffer system and the histidine buffer system are adopted, the saccharides such as sucrose, mannitol and trehalose are added into the prescription, and the surfactant such as Tween or poloxamer with a certain concentration is added, so that the recombinant human endostatin can be obviously inhibited from forming foreign matters or particles at high concentration (15 mg/ml-30 mg/ml) in the buffer system with specific pH, and has high stability.

Claims (3)

1. The recombinant human vascular endothelial inhibin injection is characterized by comprising the following components:

a) a stabilizer: the stabilizer consists of sugar and a surfactant, wherein the sugar is sucrose, and the surfactant is poloxamer 407; the concentration of the sucrose is 80mg/ml, and the concentration of the surfactant is 0.1-0.5 mg/ml;

b) a buffer solution; the buffer solution is 20mM histidine salt buffer solution, and the pH value is 6.0;

c) recombinant human endostatin: the concentration of the recombinant human endostatin is 15-30 mg/ml.

2. The recombinant human vascular endothelial inhibin injection is characterized by comprising the following components:

a) a stabilizer: the stabilizer consists of saccharide and surfactant, wherein the saccharide is mannitol, and the surfactant is Tween 20; the concentration of the mannitol is 60mg/ml, and the concentration of the surfactant is 2 mg/ml;

b) a buffer solution; the buffer solution is 20mM histidine salt buffer solution, and the pH value is 6.0;

c) recombinant human endostatin: the concentration of the recombinant human endostatin is 15-30 mg/ml.

3. The injection according to claim 1 or 2, wherein the recombinant human endostatin is rhEndostatin.