CN107115522A

CN107115522A - A kind of recombinant human vascular endothelial inhibin pharmaceutical composition

Info

Publication number: CN107115522A
Application number: CN201610101173.1A
Authority: CN
Inventors: 赵群; 陈倩洁; 姜桂香
Original assignee: Shandong Simcere Bio Pharmaceutical Co Ltd; Jiangsu Simcere Pharmaceutical Co Ltd
Current assignee: Shandong Simcere Bio Pharmaceutical Co Ltd; Jiangsu Simcere Pharmaceutical Co Ltd
Priority date: 2016-02-24
Filing date: 2016-02-24
Publication date: 2017-09-01

Abstract

The present invention relates to recombinant human vascular endothelial inhibin pharmaceutical composition technical field.The recombinant human vascular endothelial inhibin pharmaceutical composition that a kind of PH is 5.0 6.5 sodium-acetate buffers containing acetic acid is specially provided, said composition, which has, significantly improves stability, the effect of the generation of reduction polymer.

Description

A kind of recombinant human vascular endothelial inhibin pharmaceutical composition

Technical field

The present invention relates to recombinant human vascular endothelial inhibin pharmaceutical composition technical field.

Background technology

In the 1960s, Harvard Medical School of the U.S. doctor Folkman propose " tumour growth dependence angiogenic growth " this It is assumed that i.e. angiogenesis plays an important role in implanted solid tumor growth and transfer process, and " tumour hungry to death was proposed in 1971 Therapy " is theoretical.For vasostimulant generation, tumour cell can raise a series of angiogenesis factors, while can also produce one A little Angiostatins.The opening of blood vessels in tissue generation phenotype will depend on tissue local regional vessel with closing and generate thorn Swash dynamic equilibrium (Folkman, the J.Nat.MED.1 between the factor and inhibiting factor:27-31,1995；Hanahan,D.,et al.Cell.86:353-364,1996).The endogenic Angiostatin of research discovery, such as angiotensins (Angiostatin), Vascellum esoderma inhibin (Endostatin) etc., can suppress growth (O ' Relly, M.S., et of mouse interior tumor al.Cell.88:277-285,1997)。

Vascellum esoderma inhibin (Endostatin) be O ' Relly in 1997 from mouse endothelial cells system EOMAD nutrient solution Isolated is a kind of with material (O ' Relly, M.S., the et al.Cell.88 for suppressing Endothelial Cell Function:299-285,1997). The amino acid sequence analysis material is the degradation fragment of collagen XV III carboxyl terminal, and molecular weight is 20kD or so.

Recombinant human vascular endothelial inhibin (rhEndosratin) has 85% with recombinant murine vascellum esoderma inhibin on amino acid sequence Homology.Entremed companies of the U.S. in 1996 produce recombinant human vascular endothelial inhibin using yeast as expression system. Produced with recombinant DNA technology, using Escherichia coli as expression system, the recombinant human vascular endothelial inhibin that gives expression to it is previous Endostatin compares, and expression is higher, and curative effect is stronger, and does not cause vivo immunization because of additional N-terminal sequence Originality.

But the recombinant human vascular endothelial inhibin obtained using traditional Bacillus coli expression method is difficult to renaturation and is easily formed Precipitation, and it is huge with the production cost spent by pichia pastoris phaff expression system, and two methods fail to solve recombined human The problem of vascellum esoderma inhibin carries out industrial production.Compiled for this researcher by modifying the nucleotides of human Endostatin Code sequence, produces recombinant human vascular endothelial inhibin (rhEndostatin, trade name that N-terminal carries annex amino acid sequence), purification step is enormously simplify, the purity (ZL00107569.1) of product is improved.Produced Recombinant human vascular endothelial inhibinBy 192 Amino acid profiles, its amino acid sequence is：

MGGSHHHHHHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFL SSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPT WPQKSVWHGSDPNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCI ENSFMTASK

In general, the recombinant human vascular endothelial inhibin that clinical research is entered at present uses intravenously administrable, or is subcutaneously injected Administration.By taking the rhEndostatin parenteral solution that existing market is sold as an example, its specification is 15mg/3ml/ branch, adds this product during Clinical practice In 250~500ml physiological saline, at the uniform velocity intravenous drip is instiled 3~4 hours time.This product combine with NP chemotherapy regimens to During medicine, at the 1st~14 day for the treatment of cycle, it is administered once a day, successive administration 14 days, rests one week, be further continued for next control The treatment cycle.2~4 treatment cycles can generally be carried out.

In view of important function of the recombinant human vascular endothelial inhibin in field of antineoplastic medicaments, more superior for research stability Recombinant human vascular endothelial inhibin pharmaceutical composition is very important.

The content of the invention

It is an object of the invention to provide a kind of recombinant human vascular endothelial inhibin pharmaceutical composition with good stability, the medicine Composition is made up of recombinant human vascular endothelial inhibin, Acetic acid-sodium acetate buffer solution.

The pH value of the composition is 5.0-6.5, preferably 5.0-6.0,5.0-5.5, more preferably 5.5 ± 0.2

Sodium acetate concentration in the composition is preferably 30-180mM, more preferably 30mM, 60.mM, 120mM, 180mM。

The concentration of recombinant human vascular endothelial inhibin in the composition is preferably the preferred 2.0-6.0mg/ml of 2.0-10.0mg/ml, More preferably 4.0mg/ml or 5.0mg/ml.

Recombinant human vascular endothelial inhibin in the composition is rhEndostatin (rhEndostatin, Endostar).It is of the present invention Be gene recombinant protein class medicine, by its physicochemical properties, chemical stability and biological stability just Pacing is determined, and is used as the foundation for formulating prescription.After measured, rhEndostatin basic amino acids account for 16.6% acid amino Acid accounts for 7.8%, and theoretical isoelectric point is 9.3, is surveyed as 9.1, meta-alkalescence.

Described composition can add other pharmaceutically acceptable excipient substances and be further prepared into for parenteral solution, can also entering one Prepared by step turn into freeze-dried powder so that clinic needs.

Composition of the present invention has good stability, particularly, for polymer, can be very good to control it It is used as the generation of impurity.

Embodiment

The screening of the buffer system of embodiment 1

PH value of human body is 7.4, typically requires parenteral solution pH value between 4-9.In addition, to ensure finished product in storage period Inner liquid medicine steady quality, need to determine the rational pH value range of decoction, and the present inventor uses 10mM sodium citrates-lemon respectively Lemon acid buffering system, 30mM sodium acetate buffer systems, are 6.0-6.5,5.0-5.5 with NaOH, HCl regulation pH, RhEndostatin contents are 5.0mg/ml, and embedding is preserved three months for 2-8 DEG C in cillin bottle, the clarity of investigation this product, The indexs such as content, purity, result of the test is shown in Table 1.

Influence of the different buffer systems of table 1 to the rhEndostatin qualities of the pharmaceutical preparations

The detection method of indices is with reference to as follows in table 1：Non SDS-PAGE electrophoresis according to《Middle traditional Chinese medicines Allusion quotation》Carry out；The chromatographic column of HPLC methods is C₁₈Reversed-phase column, using the aqueous solution of 0.1% trifluoroacetic acid as mobile phase A liquid, With the acetonitrile of 0.1% trifluoroacetic acid easily for Mobile phase B liquid, gradient elution 24min (A liquid from 64%-40%, B liquid from 36%-60%), flow velocity is 1.3ml/min, and Detection wavelength is 214nm.

Visible in the range of pH 5.0-6.5 under two kinds of buffer systems according to table 1, rhEndostatin stability is good, but In accelerated test, albumen is precipitated during citric acid-sodium citrate buffer system the tenth day at 37 DEG C, and sodium acetate is then (result of the test is shown in Table 2) is produced without precipitation.

RhEndostatin has heparin affinity regions, under conditions of pH 5.5 with a large amount of positive charges be easy to it is electronegative Ions binding, the pKa of sodium acetate is 4.75, most of negatively charged under conditions of pH 5.5, energy and rhEndostatin Sat linkage is formed to stablize rhEndostatin.Thus the present invention can select the sodium acetate buffer system of pH5.0-6.0.

RhEndostatin stability tests at 2. 37 DEG C of table

Influence experiment of the sodium acetate concentration of embodiment 2 to rhEndostatin

It is determined that from needing to study influence of the concentration to rhEndostatin stability after sodium acetate buffer system, because in theory, Ionic strength has an impact to the stability of protein.Thus 5 kinds of sodium acetate concentrations have been selected to do 45 DEG C of accelerated test studies Relation between rhEndostatin polymers formation speed and concentration.Sodium acetate concentration is followed successively by 10mM, 30mM, 60mM, 120mM, 180mM；RhEndostatin concentration is every 2ml of 4.0mg/ml, is accelerated in 45 DEG C of water-baths Test (result of the test is shown in Table 3), at 1 hour, 2 hours, it is many that non-reduced SDS-PAGE detections are done in end sampling in 4 hours Aggressiveness amount, electrophoresis result is analyzed through the gel Cheng Xiangyi of Kodak 120, is as a result shown, with the increase poly of sodium acetate concentration The formation speed of body is without bigger difference.We from 30mM sodium acetates as final preparation consumption because 10mM Sodium acetate, which is not enough to contend with, is adding pH changes caused by other auxiliary materials, and 30mM is then sufficient for requiring.

Influence experiment of the sodium acetate concentration of table 3. to rhEndostatin

The stability of the different protein concentration preparations of embodiment 3

By being carried out to rhEndostatin contents for 4.0mg/ml preparation after accelerated test investigation, find it in acetic acid It is stable in the system of sodium 30mM, pH 5.5；Then two protein concentrations are have selected around the concentration again：2.0 Mg/ml, 6.0mg/ml do 37 DEG C of accelerated tests to investigate whether adding ingredient contains the stabilization of albumen dependent on albumen Measure, result of the test shows, rhEndostatin preparation stabilities and the 4.0mg/ml rhEndostatin of above two concentration Preparation is consistent.

The comparative test of the different dosage forms of embodiment 4

In the systems of rhEndostatin sodium acetate 30mM pH 5.5, freeze-dried powder is made, 2~8 DEG C preserve 3 months, Indices are determined, and are compared with parenteral solution, it is as a result basically identical.

Above result of the test shows that rhEndostatin has higher stability in the systems of 30mM sodium acetates pH 5.5.

Claims

1. a kind of recombinant human vascular endothelial inhibin pharmaceutical composition, is mainly made up of recombinant human vascular endothelial inhibin, Acetic acid-sodium acetate buffer solution, wherein, the pH value of the composition is 5.0-6.5.

2. the composition according to any one of claim 1, it is characterised in that the pH value of the composition is 5.0-6.0.

3. the composition according to any one of claim 1, it is characterised in that the pH value of the composition is 5.0-5.5.

4. the composition according to any one of claim 1, it is characterised in that the pH value of the composition is 5.5.

5. the composition according to any one of claim 1, it is characterised in that the sodium acetate concentration of the composition is 30-180mM.

6. the composition according to any one of claim 1, it is characterised in that the sodium acetate concentration of the composition is 30mM, 60mM, 120mM, 180mM.

7. the composition according to any one of claim 1, it is characterised in that the concentration of the recombinant human vascular endothelial inhibin is 2.0-10.0mg/ml.

8. the composition according to any one of claim 1, it is characterised in that the concentration of the recombinant human vascular endothelial inhibin is 2.0-6.0mg/ml, preferably 4.0g/ml or 5.0mg/ml.

9. the composition according to any one of claim 1-7, it is characterised in that the recombinant human vascular endothelial inhibin is Endostar.

10. the composition according to any one of claim 1-7, it is characterised in that also further contain other pharmaceutically acceptable auxiliary materials.