CN101219207A - Application of recombined human vascellum esoderma inhibin in medicine preparation - Google Patents
Application of recombined human vascellum esoderma inhibin in medicine preparation Download PDFInfo
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- CN101219207A CN101219207A CNA2008100060632A CN200810006063A CN101219207A CN 101219207 A CN101219207 A CN 101219207A CN A2008100060632 A CNA2008100060632 A CN A2008100060632A CN 200810006063 A CN200810006063 A CN 200810006063A CN 101219207 A CN101219207 A CN 101219207A
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Abstract
The invention relates to the application of a recombinant human blood vessel endothelium inhibin in pharmacy, in particular to the application for preparing medicines for treating endometriosis. The recombinant human blood vessel endothelium inhibin can specifically inhibit neovascular proliferation of ectopic focus and has the advantages of innocuity safety, no drug resistance, significant effect on combined surgery or other pharmaceutical treatments, high curative rate and good application potential.
Description
Technical field
The present invention relates to biological product technical field, be specifically related to the pharmaceutical applications of recombinant human vascular endothelial inhibin, this medicine can be used for the treatment of endometriosis.
Background technology
Normal endometrium is covered in the cavity of uterine body face, as make endometrium in other position growths of health because of certain factor, can become endometriosis.Endometriosis is a kind of invasive common gynecological disease that has, can invade the peritoneum that holds pelvic cavity, ligament, the neighbour-bladder in uterus, the rectum in suspention uterus, and omentum majus, vermiform appendix, small intestinal, incision of abdominal wall, the internal organs such as liver, gallbladder, kidney, lungs, thoracic cavity, limb muscle and skeleton that reach far away, even the interior ectopic focus of the greater vestibular gland of occurring in is arranged, tubal wall can also be involved sometimes.Sickness rate that in recent years should disease has the trend of obvious rising.The endometriosis clinical manifestation is stimulated the menstrual flow, chronic pelvic pain, former or infertility, menoxenia, sexual anhedonia, intestinal function changes and urinary system symptom etc.The infertile incidence rate of endometriosis brings than major injury for women's physical and mental health up to 30%～40%.
Human Endostatin (endostatin) is that the segment molecule amount size of collagen XVIII c-terminus is the enzyme action product of 20kDa, have the vascular endothelial cell proliferation of inhibition, migration, with the activity of angiogenesis in the body, be present the strongest known endogenous vascularization inhibitive factor.
Recombinant human vascular endothelial inhibin is to utilize DNA recombined engineering technology, with protokaryon or carrier for expression of eukaryon, and the protein that the angiogenesis inhibiting activity form is arranged that the human Endostatin gene is obtained through clone, expression and purification.
(patent publication No. is CN1324818A to Chinese invention patent, it open day was calendar year 2001 December 5 days, denomination of invention is " method of producing endostatin ") a kind of improvement is provided, with the method for higher productive rate and simpler purifying process production recombinant human endothelial inhibin.This invention utilizes DNA recombined engineering technology, with escherichia expression system, produce the human Endostatin (wherein Xaa represents neutral amino acid or do not exist) that N-terminal has the additional aminoacid sequence of (Met) GlyGlyXaaHisHisHisHisHis in enormous quantities with simple method and lower cost, not only kept the biologic activity completely of endostatin, and do not produced because of adding immunity in the body due to the additional N terminal sequence.Above-mentioned additional aminoacid sequence has not only improved transcribing and translation initiation efficient of endostatin structural gene, help the purification of recombiant protein, and the structure of additional aminoacid sequence has also increased the stability of recombiant protein.
Hormonotherapy and operation are two kinds of main therapeutic modalities that prior art is used for the treatment of endometriosis.Use the side reaction of hormone more, as hair increase, sound chap, edema, weight increase etc., usually cause drug withdrawal midway, affect the treatment, thereby limited the clinical practice of these medicines.The operative treatment short treating period, especially more suitable to serious symptom person, fibrosis is many, adhesion is tight, medicine is difficult for proving effective case.Even but limited surgical intervention also can cause fertility rate significantly to descend, and nearly 40% patient also needed operation once more in 5 years, and list can not be expected to cure this disease with operation.
Summary of the invention
Endometriosis is child-bearing period women's commonly encountered diseases and frequently-occurring disease, though be optimum, its generation, the development similar with tumor, have tangible new vessels dependency.Studies confirm that, endometriosis generates at intraperitoneal, must provide enough blood confessions by new vessels, the activity that patient's peritoneal fluid medium vessels generates the factor also obviously increases, and finds have newborn blood capillary to center on around the typical Ectopic Endometrium focus with laparoscopic visualization.These evidences are pointed out us, and an important pathological manifestations of endometriosis is not only in the growth of new vessels, also may play an important role in pathogenic process.Consider from this mechanism,, then may bring new breakthrough for the treatment endometriosis if can suppress the propagation of ectopic focus new vessels specifically with some treatment means.
In order to overcome the defective that the side reaction of endometriosis traditional remedies is big, relapse rate is high, the invention provides a kind of pharmaceutical applications of recombinant human vascular endothelial inhibin, specifically be to use the pharmaceutical applications of the medicine of recombinant human vascular endothelial inhibin preparation treatment endometriosis, this medicine has high specificity, nontoxic, the advantage that has no drug resistance.
Technical solution of the present invention relates to the application of recombinant human vascular endothelial inhibin in the medicine of preparation treatment endometriosis.
Described herein recombinant human vascular endothelial inhibin has kept the biologic activity completely of endostatin, can use genetic engineering means to obtain, it is gene constructed in escherichia coli (E.Coli) or Pichia sp. (Pichia pastoris) expression system maybe will to change the human Endostatin of structure by the vascellum esoderma inhibin gene with the people, by fermentation, means such as separation, purification obtain.
Preferred recombinant human vascular endothelial inhibin is to add 0-4 arbitrary amino acid and 2-8 histidine sequence behind the initial amino acid Met of human Endostatin encoding amino acid sequence successively among the present invention.The same Chinese invention patent of its preparation method (patent publication No. is CN1324818A, open day be calendar year 2001 December 5 days, and denomination of invention is " method of production endostatin ").
The initial amino acid Met of above-mentioned recombinant human vascular endothelial inhibin may be cut, and reason is may be deleted in the protein translation post-treatment process in expressive host.
The aminoacid sequence of the preferred recombinant human vascular endothelial inhibin of the present invention is shown in SEQ ID NO:1, this sequence is identical with the disclosed SEQ IDNO:2 of Chinese invention patent (patent publication No. is CN1324818A), and the proteinic preparation method that this sequence constitutes is identical with the preparation embodiment of above-mentioned publication.
The preparation of the medicine of above-mentioned treatment endometriosis can be injection or lyophilized formulations.The described active component of stating medicine can be recombinant human vascular endothelial inhibin or the recombinant human vascular endothelial inhibin microsphere that recombinant human vascular endothelial inhibin, PEG modify.The molecular weight of described PEG is 5000-30000, and PEG links to each other with recombinant human vascular endothelial inhibin by covalent bond (as: succinimido or sulfydryl).Described recombinant human vascular endothelial inhibin microsphere is to be mixed with by host material and recombinant human vascular endothelial inhibin to form, and its host material can be polylactic-co-glycolic acid block copolymer (PLGA), polyglycolic acid (PLA) etc.
Also contain protective agent and pH value regulator in the medicine of above-mentioned treatment endometriosis.Described protective agent is selected from polyhydric alcohol, saccharide, aminoacid, surfactant, human albumin, EDTA and NaCl.The pH value scope of described medicine is 4.0-8.5.
Described in the present invention the treatment endometriosis medicine can with operation or hormone medicine use in conjunction.
The invention also discloses a kind of recombinant human vascular endothelial inhibin injection for the treatment of endometriosis, it is characterized in that it is formulated by recombinant human vascular endothelial inhibin and pharmaceutic adjuvant: contain recombinant human vascular endothelial inhibin 1mg～10mg, mannitol 5mg～80mg by every milliliter of injection, it is 4.0～8.5 that pH value is regulated in the pH regulator agent.
Also disclose the preparation technology of recombinant human vascular endothelial inhibin lyophilized formulations in the embodiments of the invention, the lyophilized formulations product stability that this technology makes is good, can long preservation.Lyophilized formulations need be dissolved in the water for injection when using, and use amount takes the circumstances into consideration to use according to lesion degree.
The present invention is applicable to the treatment of the endometriosis that a variety of causes causes, therapeutic dose can be taken the circumstances into consideration plus-minus according to the state of an illness, generally adopts pathological changes local injection or intravenous method to use.
For better explanation the present invention, its purposes in the medicine of preparation treatment endometriosis is described below in conjunction with drug study, zoopery and clinical experiment.Employed recombinant human vascular endothelial inhibin has the aminoacid sequence shown in the SEQ ID NO:1 among experimental example hereinafter and the preparation embodiment.If no special instructions, hereinafter related experiment all can obtain by commercial sources with material.
1, endometriosis chick chorioallantoic membrane model experiment
(1mm * 1mm * 1mm) is inoculated in the relative avascular area on the chick chorioallantoic membrane of 8 days gestational ages with endometriosis patient endometrial tissue on the throne, set up endometriosis chick chorioallantoic membrane model, set up inoculation group, inoculation treatment group (recombinant human vascular endothelial inhibin) and blank group separately, preceding two groups each 20 pieces.Behind the inoculation 24h, with the sterile gelatin sponge is carrier, every of inoculation group gives normal saline 20 μ l/d, inoculation treatment group gives recombinant human vascular endothelial inhibin 5 μ g/20 μ l every every day, blank group is established blank vehicle group and simple blank group in addition, each 10 pieces, do not inoculate endometrial tissue, blank vehicle group is a carrier with the sterile gelatin sponge, give normal saline 20 μ l/d, simple blank group is then promptly given aseptic pad pasting and is sealed after windowing, in order to get rid of the interference of non-processing factors such as after stain, operating technology to experimental result of windowing of embryo egg.Add fixedly 15min of fixative behind the inoculation 72h, cut chick chorioallantoic membrane, be tiled in filter paper and take the photograph sheet.The vessel branch of 5mm scope is counted around the microscopically counting carrier plate.Part chick chorioallantoic membrane 10% formaldehyde fixed, paraffin embedding, section, HE dyeing, om observation.
The result: inoculation group chick chorioallantoic membrane angiogenesis significant reaction strengthens, and number of blood vessel is significantly higher than matched group; Inoculation treatment group number of blood vessel significantly is lower than inoculation group (seeing Table 1); Inoculation treatment group focus is visible obviously downright bad under light microscopic, and recombinant human vascular endothelial inhibin can obviously suppress the endometriosis focus and form.
Table 1 vascellum esoderma inhibin is to the inhibitory action of endometriosis focus angiogenesis
| Grouping | n | Vessel branch is counted |
| The simple blank winding kind treatment group of the blank vehicle group of inoculation group | 20 10 10 20 | 35.2±5.14 * 21.6±3.89 ** 18.6±2.76 19.4±3.65 |
* P＜0.01, inoculation group and blank vehicle group, simple blank group and inoculation treatment group are compared;
* P＞0.05, blank vehicle group and simple blank group are compared.
Conclusion: endometriosis endometrium on the throne can obviously promote the angiogenesis of chick chorioallantoic membrane, recombinant human vascular endothelial inhibin can significantly suppress the vascularization of endometriosis focus, and suppress endometrial growth, illustrate that anti-angiogenic approach is one of available strategy of treatment endometriosis.
2, endometriosis mouse model experiment
Get endometriosis patient endometrial tissue on the throne, subtract into 1～2mm
3Size, per 15 fritters add 200 μ l PBS and are made into tissue suspension.Choose female severe plyability immunodeficiency (severecombined immtmodefieiency disease, SCID) mice is 50,6～8 ages in week, body weight 20～25g, be divided into 5 groups (seeing Table 2) at random, SPF (specific pathogen free) condition is raised.Mice with etherization after, the sterilization abdominal part is got umbilicus under and to be hit exactly point of puncture, it is subcutaneous with No. 16 syringe needle tissue suspension to be inoculated in mice.Inoculation back the 2nd day is in mice leg muscle injection 0.02ml (1mg/ml) estradiol benzoate, to promote the growth of plantation inner membrance.The inoculation back begins to give medication (seeing Table 2) the 3rd week, injects for 2 weeks continuously.Every 2 days with the length of the subcutaneous focus of vernier caliper measurement and wide, according to formula volume V (mm
3)=0.52 * length * wide
2The calculating foci volume.The last injection was all put to death laboratory animal after 24 hours, and paraformaldehyde was fixed after ectopic focus was weighed.The embedding of focus specimen routine paraffin wax, section, hematoxylin-eosin (HE) dyeing, the variation of om observation body of gland, a matter and epithelium; Adopt SABC to detect microvessel density, microvessel density (micro vascular density, calculating reference literature MVD).
The result: it is basic identical respectively to organize the ectopic focus growing state before the medication, and treatment group focus volume growth is slow after the medication, and medication later stage focus volume has the trend of dwindling.Treatment group focus weight and volume are lower than matched group (P＜0.05), see Table 2.Specimen HE dyeing, visible treatment group body of gland atrophy under the light microscopic, structure is imperfect, and a matter is with necrosis in different degree.
Table 2 vascellum esoderma inhibin is to the influence of mice ectopic focus weight and volume
| Grouping | n | Dosage/μ g.kg -1 | Ectopic focus weight (g) | Ectopic focus volume (mm 3) |
| Dosage treatment group low dose therapy group in the normal saline matched group alarelin matched group high-dose therapy group | 10 10 10 10 10 | - 3 200 100 50 | 0.139±0.016 0.092±0.013 * 0.087±0.015 * 0.098±0.012 * 0.112±0.015 | 219.80±13.42 138.62±12.32 * 130.28±10.27 * 144.18±11.17 * 171.28±13.44 |
Annotate: with normal saline matched group ratio, * P＜0.05
Vascular endothelial cell labelled antigen CD34 immunohistochemical staining (Tjaima W, Van Marck E, Weyler J, et al.Quantification and prognostic relevance ofangiogenic parameters in invasive cervical cancer.Br J Cancer, 1998,78 (2): 170～174.) be positioned vascular endothelial cell, be pale brown color, the formation tube chamber that blood capillary has, what have only is single endotheliocyte or endotheliocyte bunch.Inoculate 4 all ectopic focus MVD (14.76//HPF (high power field)) than (4.23/HPF) significantly increase (P＜0.05) of 1 all MVD.Matched group MVD is 14.76 ± 3.22/HPF, and treatment group MVD is 5.34 ± 1.86/HPF, and difference has statistical significance (P＜0.05).
Conclusion: recombinant human vascular endothelial inhibin has the effect that suppresses growth of endometriosis focus and angiogenesis.
Said medicine test and animal experiment study show: endometriosis endometrium on the throne can obviously promote the angiogenesis of chick chorioallantoic membrane, recombinant human vascular endothelial inhibin can significantly suppress the vascularization of endometriosis focus, and suppressing endometrial growth, focus is visible obviously downright bad under light microscopic; Recombinant human vascular endothelial inhibin can suppress growth of endometrium of mouse dystopy disease focus and angiogenesis, visible treatment group body of gland atrophy under the light microscopic, and structure is imperfect, and a matter is with necrosis in different degree.
Below further set forth the beneficial effect of medicine of the present invention by clinical observation on the therapeutic effect.
Case one
The woman is 28 years old, married.2 years half unpregnancies of living together of marrying, dysmenorrhea, through preceding first quarter moon distending pain of the breast, tenderness, it is single-phase that temperature curve is, prompting: do not ovulate.Inspections such as woman's inspection, B ultrasonic are diagnosed as endometriosis, and right ovary chocolate cyst, card belong to the resistance of the uterus stasis of blood and infertile.Use the recombinant human vascular endothelial inhibin injection, once a day, each 7.5mg recombinant human vascular endothelial inhibin, use 14 days continuously after, dysmenorrhea obviously alleviates, and the distending pain of the breast degree alleviates, and the time shortens to 4 days, menolipsis 36 days, the urine pregnancy test positive is diagnosed as early pregnancy, and the back produces a boy baby.
Case two
The woman is 43 years old, married.Dysmenorrhea, menoxenia, blood is dark red, and is not to the utmost dripping, is diagnosed as endometriosis.Use the recombinant human vascular endothelial inhibin injection, once a day, each 7.5mg recombinant human vascular endothelial inhibin, use 14 days continuously after, palace blood stops, stomachache disappears.Clinical efficacy situation statistics with 297 example endometriosis diseases of recombinant human vascular endothelial inhibin injection for treating is as follows:
Case is selected: endometriosis patient 297 examples, at 18～45 years old age, average 34 years old, the course of disease was the shortest 1 year, the longest 18 years, married 255 examples, married infertile 102 examples, ectopic sites: posterior fornix enclosed mass 121 examples, utero-sacral ligament 15 examples, cardinal ligament 12 examples, a side and bilateral adnexa capsule enclosed mass 18 examples meet the diagnostic criteria of the 3rd endometriosis that academic conference is revised of Chinese Association of the Integration of Traditional Chinese Medicine and Western Medicine department of obstetrics and gynecology Professional Committee.
Intravenous drip this product is cured 96 examples, cure rate 32.3%, used after operation this product wherein, cure rate 83.3%; Produce effects 142 examples, effective 55 examples, effective percentage 98.7%; Invalid 4 examples.Have no side effect or other untoward reaction take place, show this medicine clinical safe in utilization effectively, used after operation medicine particularly, cure rate height.
The invention has the beneficial effects as follows:
1) excavates the therapeutic use of the new indication of recombinant human vascular endothelial inhibin, opened up a new application.
2) propagation of the special inhibition ectopic focus of recombinant human vascular endothelial inhibin of the present invention new vessels, safety non-toxic has no drug resistance, and indicating has good application prospects.
3) effect of recombinant human vascular endothelial inhibin combined surgery of the present invention or other medicines treatment is remarkable, the cure rate height.After operation, use recombinant human vascular endothelial inhibin can make remaining focus atrophy, disappear, effectively control the recurrence of endometriosis.
The specific embodiment
The following examples can make those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.
Embodiment 1
Preparation contains the injection of recombinant human vascular endothelial inhibin, makes its every milliliter to contain vascellum esoderma inhibin 5mg, mannitol 40mg, and adding concentration is 5.5 for 40mM sodium acetate adjusting pH value.Filtration sterilization, sterile filling, every 3ml.
Usage and consumption: one of every day, inject 300～500ml normal saline or glucose injection, intravenous drip 3～4 hours.
Embodiment 2
Recombinant human vascular endothelial inhibin protein solution ultrafiltration dialysis preparation 151.5ml concentration after using acetic acid-sodium acetate buffer system about 30mM pH5.5 ± 0.5 to purification is the recombinant human vascular endothelial inhibin solution of 9.9mg/ml, add 20% mannitol 60ml and 20% sucrose solution 7.5ml, add the acetic acid-sodium-acetate buffer 2.98ml about 1.5M pH5.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the pre-encapsulated injector, in 4 ℃ of preservations.
Embodiment 3:
Recombinant human vascular endothelial inhibin protein solution ultrafiltration dialysis preparation 90.9ml concentration after using acetic acid-sodium acetate buffer system about 30mM pH5.5 ± 0.5 to purification is the recombinant human vascular endothelial inhibin solution of 9.9mg/ml, add 20% mannitol 60ml, add the acetic acid-sodium-acetate buffer 4.20ml about 1.5M pH5.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the cillin bottle, add butyl rubber plug, medicinal liquid places in the freeze drying box, and products temperature drops to-40 ℃, keeps 3-4 hour, evacuation, the dividing plate heating makes products temperature be increased to-20 ℃, kept 8 hours, continue heating elevated temperature to 25 ℃, kept 6 hours, change when little to vacuum, take out after the vacuum tamponade, roll lid.
Embodiment 4:
Recombinant human vascular endothelial inhibin protein solution ultrafiltration dialysis preparation 151.5ml concentration after using acetic acid-sodium acetate buffer system about 30mM pH5.5 ± 0.5 to purification is the recombinant human vascular endothelial inhibin solution of 9.9mg/ml, add 20% mannitol 60ml and 20% sucrose solution 15ml, add the acetic acid-sodium-acetate buffer 2.98ml about 1.5M pH5.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the cillin bottle, add butyl rubber plug, medicinal liquid places in the freeze drying box, and products temperature drops to-40 ℃, keeps 4-5 hour, evacuation, the dividing plate heating makes products temperature be increased to-25 ℃, kept 10 hours, continue heating elevated temperature to 30 ℃, kept 4 hours, change when little to vacuum, take out after the vacuum tamponade, roll lid.
Embodiment 5:
Recombinant human vascular endothelial inhibin protein solution ultrafiltration dialysis preparation 97.8ml concentration after using acetic acid-sodium acetate buffer system about 30mM pH5.5 ± 0.5 to purification is the recombinant human vascular endothelial inhibin solution of 30.61mg/ml, add 20% mannitol 60ml and 20% sucrose solution 60ml, add the acetic acid-sodium-acetate buffer 4.04ml about 1.5M pH5.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the cillin bottle, add butyl rubber plug, medicinal liquid places in the freeze drying box, and products temperature drops to-45 ℃, keeps 6 hours, evacuation, the dividing plate heating makes products temperature be increased to-30 ℃, kept 6 hours, the reheat elevated temperature kept 6 hours to-25 ℃, continue heating elevated temperature to 25 ℃, kept 8 hours, and changed when little to vacuum and take out after the vacuum tamponade, roll lid.
Embodiment 6:
Recombinant human vascular endothelial inhibin protein solution ultrafiltration dialysis preparation 147ml concentration after using acetic acid-sodium acetate buffer system about 30mM pH5.5 ± 0.5 to purification is the recombinant human vascular endothelial inhibin solution of 30.61mg/ml, add 20% mannitol 60ml, 20% sucrose solution 60ml and 20% aqueous trehalose 30ml, add the acetic acid-sodium-acetate buffer 3.06ml about 1.5M pH5.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the cillin bottle, add butyl rubber plug, medicinal liquid places in the freeze drying box, and products temperature drops to-45 ℃, keeps 2-4 hour, evacuation, the dividing plate heating makes products temperature be increased to-30 ℃, kept 12 hours, continue heating elevated temperature to 30 ℃, kept 6 hours, change when little to vacuum, take out after the vacuum tamponade, roll lid.
Embodiment 7:
Recombinant human vascular endothelial inhibin protein solution ultrafiltration dialysis preparation 30ml concentration after using citric acid-sodium citrate buffer system about 10mM pH6.0 ± 0.5 to purification is the recombinant human vascular endothelial inhibin solution of 9.9mg/ml, add 2% glycine 15ml, tween 80 0.15ml, add the citric acid-sodium citrate buffer 2.70ml about 1.0M pH6.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the cillin bottle, add butyl rubber plug, medicinal liquid places in the freeze drying box, and products temperature drops to-40 ℃, keeps 2-4 hour, evacuation, the dividing plate heating makes products temperature be increased to-30 ℃, kept 8 hours, continue heating elevated temperature to 30 ℃, kept 8 hours, change when little to vacuum, take out after the vacuum tamponade, roll lid.
Embodiment 8:
Recombinant human vascular endothelial inhibin protein solution ultrafiltration dialysis preparation 73.5ml concentration after using sodium dihydrogen phosphate-sodium hydrogen phosphate buffer system about 10mM pH7.0 ± 0.5 to purification is the recombinant human vascular endothelial inhibin solution of 30.61mg/ml, add 2%EDTA 30ml, 20% mannitol solution 30ml and 20% sucrose solution 7.5ml, add the sodium dihydrogen phosphate-sodium hydrogen phosphate buffer 2.26ml about 1.5M pH7.0 ± 0.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the cillin bottle, add butyl rubber plug, medicinal liquid places in the freeze drying box, and products temperature drops to-45 ℃, keeps 8 hours, evacuation, the dividing plate heating makes products temperature be increased to-30 ℃, kept 12 hours, continue heating elevated temperature to 25 ℃, kept 10 hours, change when little to vacuum, take out after the vacuum tamponade, roll lid.
Embodiment 9:
Recombinant human vascular endothelial inhibin protein solution ultrafiltration dialysis preparation 15ml concentration after using acetic acid-sodium acetate buffer system about 30mM pH5.5 ± 0.5 to purification is the recombinant human vascular endothelial inhibin solution of 9.9mg/ml, add 10% glutamic acid 15ml and 10% threonine 15ml, add the acetic acid-sodium-acetate buffer 5.7ml about 1.5M pH5.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the cillin bottle, add butyl rubber plug, medicinal liquid places in the freeze drying box, and products temperature drops to-40 ℃, keeps 4-5 hour, evacuation, the dividing plate heating makes products temperature be increased to-25 ℃, kept 10 hours, continue heating elevated temperature to 30 ℃, kept 4 hours, change when little to vacuum, take out after the vacuum tamponade, roll lid.
Embodiment 10:
Recombinant human vascular endothelial inhibin protein solution ultrafiltration dialysis preparation 151.5ml concentration after using acetic acid-sodium acetate buffer system about 30mM pH5.5 ± 0.5 to purification is the recombinant human vascular endothelial inhibin solution of 9.9mg/ml, add 20% human albumin's solution 30ml, add the acetic acid-sodium-acetate buffer 2.98ml about 1.5M pH5.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the cillin bottle, add butyl rubber plug, medicinal liquid places in the freeze drying box, and products temperature drops to-40 ℃, keeps 4-5 hour, evacuation, the dividing plate heating is increased to products temperature--and 15 ℃, kept 10 hours, continue heating elevated temperature to 20 ℃, kept 10 hours, change when little to vacuum, take out after the vacuum tamponade, roll lid.
Embodiment 11:
Take by weighing recombinant human vascular endothelial inhibin microsphere (PLGA enclose) 30g, interior enclose recombinant human vascular endothelial inhibin 3g, add 20% mannitol 60ml, 20% sucrose solution 15ml adds the acetic acid-sodium-acetate buffer 6.0ml about 1.5MpH5.5, adds injection water to 300ml, be sub-packed in the cillin bottle, add butyl rubber plug, medicinal liquid places in the freeze drying box, and products temperature drops to-40 ℃, kept 4-5 hour, evacuation, the dividing plate heating makes products temperature be increased to-25 ℃, kept 10 hours, continue heating elevated temperature to 30 ℃, kept 4 hours, change when little to vacuum, take out after the vacuum tamponade, roll lid.
Sequence table
＜110〉Shandong Xiansheng Maidejin Biological Pharmaceutical Co., Ltd.
＜120〉application of recombinant human vascular endothelial inhibin in pharmacy
<130>MP071322
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<170>PatentIn version 3.3
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<211>192
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<213>Artificial
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＜223〉recombinant human vascular endothelial inhibin
<400>1
Met Gly Gly Ser His His His His His His Ser His Arg Asp Phe Gln
1 5 10 15
Pro Val Leu His Leu Val Ala Leu Asn Ser Pro Leu Ser Gly Gly Met
20 25 30
Arg Gly Ile Arg Gly Ala Asp Phe Gln Cys Phe Gln Gln Ala Arg Ala
35 40 45
Val Gly Leu Ala Gly Thr Phe Arg Ala Phe Leu Ser Ser Arg Leu Gln
50 55 60
Asp Leu Tyr Ser Ile Val Arg Arg Ala Asp Arg Ala Ala Val Pro Ile
65 70 75 80
Val Asn Leu Lys Asp Glu Leu Leu Phe Pro Ser Trp Glu Ala Leu Phe
85 90 95
Ser Gly Ser Glu Gly Pro Leu Lys Pro Gly Ala Arg Ile Phe Ser Phe
100 105 110
Asp Gly Lys Asp Val Leu Arg His Pro Thr Trp Pro Gln Lys Ser Val
115 120 125
Trp His Gly Ser Asp Pro Asn Gly Arg Arg Leu Thr Glu Ser Tyr Cys
130 135 140
Glu Thr Trp Arg Thr Glu Ala Pro Ser Ala Thr Gly Gln Ala Ser Ser
145 150 155 160
Leu Leu Gly Gly Arg Leu Leu Gly Gln Ser Ala Ala Ser Cys His His
165 170 175
Ala Tyr Ile Val Leu Cys Ile Glu Asn Ser Phe Met Thr Ala Ser Lys
180 185 190
Claims (12)
1. the application of recombinant human vascular endothelial inhibin in the medicine of preparation treatment endometriosis.
2. the described application of claim 1 is characterized in that described recombinant human vascular endothelial inhibin is to add 0-4 arbitrary amino acid and 2-8 histidine sequence behind the initial amino acid Met of human Endostatin encoding amino acid sequence successively.
3. the described application of claim 2 is characterized in that the initial amino acid Met of described recombinant human vascular endothelial inhibin can be cut.
4. the described application of claim 2, the aminoacid sequence that it is characterized in that described recombinant human vascular endothelial inhibin is shown in SEQ ID NO:1.
5. each described application of claim 1-4, the preparation that it is characterized in that the medicine of described treatment endometriosis is injection or lyophilized formulations.
6. the described application of claim 5, the active component that it is characterized in that described medicine are recombinant human vascular endothelial inhibin or the recombinant human vascular endothelial inhibin microspheres that recombinant human vascular endothelial inhibin, PEG modify.
7. the described application of claim 6, the molecular weight that it is characterized in that described PEG is 5000-30000, PEG links to each other with recombinant human vascular endothelial inhibin by covalent bond.
8. the described application of claim 6 is characterized in that described recombinant human vascular endothelial inhibin microsphere is to be mixed with by host material and recombinant human vascular endothelial inhibin to form.
9. the described application of claim 5 is characterized in that also containing in the described medicine protective agent and pH value regulator.
10. the described application of claim 9 is characterized in that described protective agent is selected from polyhydric alcohol, saccharide, aminoacid, surfactant, human albumin, EDTA and NaCl.
11. the described application of claim 9 is characterized in that the pH value scope of described medicine is 4.0-8.5.
12. the described application of claim 1, it is characterized in that described treatment endometriosis medicine can with operation or hormone medicine use in conjunction.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107115522A (en) * | 2016-02-24 | 2017-09-01 | 山东先声生物制药有限公司 | A kind of recombinant human vascular endothelial inhibin pharmaceutical composition |
-
2008
- 2008-02-01 CN CNA2008100060632A patent/CN101219207A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107115522A (en) * | 2016-02-24 | 2017-09-01 | 山东先声生物制药有限公司 | A kind of recombinant human vascular endothelial inhibin pharmaceutical composition |
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