CN101224297A - Application of recombinant human endostain in preparing medicine - Google Patents
Application of recombinant human endostain in preparing medicine Download PDFInfo
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- CN101224297A CN101224297A CNA2008100060581A CN200810006058A CN101224297A CN 101224297 A CN101224297 A CN 101224297A CN A2008100060581 A CNA2008100060581 A CN A2008100060581A CN 200810006058 A CN200810006058 A CN 200810006058A CN 101224297 A CN101224297 A CN 101224297A
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Abstract
The invention relates to an application of a recombinant human vascular endothelial inhibin in pharmacy, which more particularly discloses an application of the recombinant human vascular endothelial inhibin in the preparation of the medicines for treating tumor. The recombinant human vascular endothelial inhibin can inhibit the metastasis of tumor through the specific inhibition of the multiplication of new blood vessels of tumor tissues. The method is safe and nontoxic and has no drug resistance; the invention has significant curative effect by combining chemotherapeutics, and can improve the effective rate and survival rate of tumor treatment.
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 all kinds of entity tumors.
Background technology
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.
Malignant tumor is the commonly encountered diseases of a class serious harm human life health, and the main means that are used for oncotherapy at present have operation, radiotherapy, chemotherapy and Biotherapeutics.Chemotherapy is a kind of systemic treatment means of routine, and poor selectivity is obtaining the same toxic and side effects that occurs often in various degree of therapeutic effect, and life-time service easily produces drug resistance and causes the treatment failure.
Summary of the invention
Obviously there is the new vessels dependency in growth of tumor, tumor vascular activation, propagation, absorption and the sophisticated process that depends on vascular endothelial cell.The present invention is based on the upward consideration of this mechanism that tumor takes place and develops, if can suppress the propagation of ectopic focus new vessels specifically with some treatment means, make tumor tissues can not get growing necessary a large amount of nutrition and oxygen, stop growing at last or necrosis, therefore can open up a kind of new means for the treatment of tumor.
Tumor traditional chemical therapy toxicity is big in order to overcome, the defective of poor selectivity, 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 tumor, 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 tumor.
Described herein recombinant human vascular endothelial inhibin has kept the biologic activity completely of endos tatin, 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 shown in SEQ ID NO:1, the disclosed SEQ ID of this sequence and Chinese invention patent (patent publication No. is CN1324818A)
NO:2 is identical, 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 tumor 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 tumor.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 tumor medicine can with the medication combined application of chemotherapy of tumors class, as with vinorelbine (NVB, Vinorelbine Navelbine) cisplatin (PDD, Cisplatin or Diamminedichloroplatinum), paclitaxel (Paclitaxe, PTX), Docetaxel (Docetaxel), gemcitabine chemotherapeutics couplings such as (Gemcitabine), also can cooperate the treatment of tumor post-operation.
Tumor described in the present invention is a solid tumor, and there are the propagation of new vessels in the tissue of these solid tumors and periphery thereof.
The invention also discloses a kind of recombinant human vascular endothelial inhibin injection for the treatment of tumor, it is characterized in that it is formulated by recombinant human vascular endothelial inhibin and pharmaceutic adjuvant: contain recombinant human vascular endothelial inhibin 1mg～20mg by every milliliter of injection.The preferred mannitol of described pharmaceutic adjuvant contains 10mg～80mg by every milliliter of injection; Can add the PH regulator in addition, regulating pH value is 4.0～8.5.
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 entity tumor that a variety of causes causes, entity tumor includes but not limited to pulmonary carcinoma, colon cancer, neuroendocrine tumour, osteocarcinoma, hepatocarcinoma, gastric cancer, cancer of pancreas, oral cancer, breast carcinoma, lymphatic cancer, esophageal carcinoma, renal carcinoma, cancer of biliary duct, malignant melanoma, cervical cancer, nasopharyngeal carcinoma, sarcoma or the like, therapeutic dose can be taken the circumstances into consideration plus-minus according to the state of an illness or tumor body size, generally adopts the method for pathological changes local injection to use.
For better explanation the present invention, its purposes in the medicine of preparation treatment tumor 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, recombinant human vascular endothelial inhibin suppresses the experimentation of melanoma angiogenesis
The B16 malignant melanoma of cryopreservation is organized in the hot water about 43 ℃ intensification 20-30s rapidly, press the centrifugal 10min of 1000rpm then, aseptic cotton-wool after drawing supernatant shreds tumor tissue, adds normal saline and makes tumor cell suspension (cell concentration 1 * 10
7/ ml), in groin injection tumor cell suspension 0.2ml/ only.Observe inoculation position Liquid Absorption, tumor growth process and mice situation, treat tumor growth 10d, adopt lottery to be divided into three groups at random, 20 every group, after the strain of inoculation melanoma cell, gave medicine on the 10th day.Three groups of medication are as follows: (1) recombinant human vascular endothelial inhibin low dose group: the recombinant human vascular endothelial inhibin injection is injected 0.75mg (kgd) down in the mouse tumor perithelium
-1(2) recombinant human vascular endothelial inhibin high dose group: the recombinant human vascular endothelial inhibin injection is injected 1.5mg (kgd) down in the mouse tumor perithelium
-1(3) Endostatin group: the Endostatin injection is injected 1.5mg (kgd) down in the mouse tumor perithelium
-1Endostatin described in the literary composition is a human Endostatin albumen, is the aminoacid sequence by the human Endostatin structural gene coding, does not contain any modification sequence.Blank group, the inoculation back mice feeding of normally drinking water.With the caudal vertebra dislocation method two groups of mices are all put to death behind the administration 10d, separate tumor tissue, formalin fixed, paraffin embedding, make the thick serial section of 4 μ m, the conventional hematoxylin-eosin staining of row, microscopically is observed and comparison of tumor is organized mimicry sample of blood pipe and endothelium-dependent relaxation number of blood vessel.The execution mice is left and taken part fresh specimens glutaraldehyde simultaneously and fixes, and makes electron microscopic section, electron microscope observation tumor microvessel structure.
The result: light microscopic is observed down, can see endothelium-dependent relaxation blood vessel in the tumor tissues, be also shown in the lacuna spline structure that has oncocyte to surround in the tumor tissue, in have the erythrocyte of structural integrity to exist, prompt for VM (vasculogenic mimicry, angiogenesis mimicry).Count endothelium-dependent relaxation blood vessel and VM number respectively, see Table 1.Endothelium-dependent relaxation blood vessel in three groups of mouse tumor tissues of medication and VM number and matched group comparing difference have statistical significance (P＜0.05).
The number of two kinds of blood vessels of table 1 in endostatin group and matched group
| Group | n | The endothelium-dependent relaxation blood vessel | The angiogenesis mimicry |
| Low dose group | 20 | 3.97±1.32 * | 1.87±1.16 * |
| High dose group | 20 | 3.55±1.19 * | 1.65±1.02 * |
| Endostatin organizes matched group | 20 20 | 3.82±1.22 * 6.57±1.48 | 1.77±1.19 * 3.76±1.57 |
Annotate: with blank group ratio, * P＜0.05.
Endotheliocyte in the endothelium-dependent relaxation blood vessel presents the phenomenon of infantilism.
Conclusion: the recombinant human vascular endothelial inhibin treatment can obviously suppress melanoma subcutaneous transplantation tumor new vessels and generate, the mouse tumor vessel density that receives treatment obviously lowers, and tumor tissues occurs large stretch of downright bad, and then tumor vessel density is higher for matched group, cell proliferation is active, and is downright bad rare.Therefore the effect of recombinant human vascular endothelial inhibin with reliable inhibition tumor endothelial dependency blood vessel and VM new life, thus the blocking-up tumor cell obtains nutrition, reaches the purpose that suppresses its growth and transfer.
2, the experiment of recombinant human vascular endothelial inhibin treatment human osteosarcoma nude mice model
40 of Balb/c-nu/nu nude mices, age in 3-4 week, male and female half and half, average weight 16-20g, human osteosarcoma cell's strain 9901 cells of the phase growth of taking the logarithm, preparation concentration of cell suspension 5 * 10
7/ ml.At nude mice oxter inoculated tumour cell 0.2ml.Treated tumor growth 3～4 days, to volume 40mm
3The time random packet be: [dosage is 1.5mg (kgd) to matched group, Endostatin group
-1], [dosage is 0.75mg (kgd) to recombinant human vascular endothelial inhibin low dose therapy group
-1] and recombinant human vascular endothelial inhibin high-dose therapy group [dosage is 1.5mg (kgd)
-1], matched group gives equivalent PBS, successive administration 14 days.Measure Mus weight, tumor major diameter and minor axis therebetween every day, gross tumor volume=major diameter * minor axis
2/ 2.Test and put to death animal on the 15th day, tumor is weighed, and the calculating tumour inhibiting rate (tumor inhibitionrate, TIR), TIR=(matched group gross tumor volume-experimental group gross tumor volume)/matched group gross tumor volume * 100%.Draw the growth of xenografted curve.Quick cutting tumor under aseptic condition, frozen section, row histanoxia and blood vessel labelling.Conventional 10% neutral formalin is liquid-solid fixed, hematoxylin-eosin (HE) dyeing and S-P method immunohistochemical staining.The propagation degree of cell proliferation antigenic label Ki67 immunohistochemical staining tumor cells showed, vascular endothelial cell surface antigen molecule CD31 immunohistochemical staining show the microvessel density of tumor.The TUNEL method detects apoptosis.Preceding 3 hours tail vein injection EF5 of sacrifice of animal (2-[2-nitro-1H-imidazole-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide); Tumor tissues 50 μ m frozen sections are used the EF5 antibody of Cy3-labelling respectively, and the IgG two anti-immunofluorescence dyeings that carry out of CD31 monoclonal antibody and FITC labelling, and laser confocal microscope is observed.
Result: as shown in table 2, recombinant human vascular endothelial inhibin low dose group and high dose group and matched group have notable difference (P＜0.01), and high dose group is migrated inhibition apparently higher than low dose group to endotheliocyte, show that recombinant human vascular endothelial inhibin can significantly suppress migrating of endotheliocyte, and tangible dose-effect relationship is arranged.
The inhibitory action that table 2 recombinant human vascular endothelial inhibin is migrated the osteosarcoma cell induction of vascular endothelial
| Group | n | Endotheliocyte migration rate (%) |
| Normal control group Endostatin group recombinant human vascular endothelial inhibin low dose group recombinant human vascular endothelial inhibin high dose group | 10 10 10 10 | 67.33±2.08 25.21±1.78 * 30.67±1.53 * 12.67±0.57 * |
Annotate: with blank group ratio, * P＜0.01.
Low dose group and high dose group are respectively 22.92% and 35.83% to inhibition rate of tumor growth, see Table 3.
Table 3 endostatin is to the tumor-inhibiting action of human osteosarcoma cell's transplanted tumor in nude mice
| Group | n | Dosage mg/kg | The body weight of animal (g) | Average tumor heavy (g) | Tumour inhibiting rate % | The P value | |
| Beginning | Finish | ||||||
| Matched group | 10 | - | 17.6 | 21.4 | 2.40±0.11 | - | - |
| The Endostatin group | 10 | 1.5 | 18.3 | 21.5 | 1.78±0.07 | 25.77 | ＜0.01 |
| The recombinant human vascular endothelial inhibin low dose group | 10 | 0.75 | 18.5 | 21.6 | 1.85±0.08 | 22.92 | ＜0.01 |
| The recombinant human vascular endothelial inhibin high dose group | 10 | 1.5 | 18.2 | 21.5 | 1.54±0.08 | 35.83 | ＜0.01 |
Said medicine test and animal experiment study show: recombinant human vascular endothelial inhibin can significantly suppress the tumor growth of human osteosarcoma nude mice model, the mouse tumor vessel density of accepting the recombinant human vascular endothelial inhibin treatment obviously lowers, vascular occlusion, necrosis, tumor tissues occur large stretch of downright bad; Recombinant human vascular endothelial inhibin can significantly suppress the subcutaneous inhibition tumor of B16 melanoma mouse model new vessels and generate, and the mouse tumor vessel density of receiving treatment obviously lowers, and tumor tissues occurs large stretch of downright bad.
Below further set forth the beneficial effect of medicine of the present invention by clinical observation on the therapeutic effect.
Case one
The male, 70 years old.Process PET/CT and pathological examination were diagnosed as the left adenocarcinoma of lung IV phase in February, 2006 (sends out mediastinal lymph nodes, lymphonodi coeliaci, bilateral in two lungs.Adrenal gland, intracranial and multiple bone shift).The multiple chemotherapy regimens of using once based on gemcitabine such as scheme, temozolomide's capsule, capecitabine sheet and semustine capsule are invalid, and the state of an illness continues progress.Use gefitinib in May, 2006 instead, treatment is uncomfortable in chest after January, pain and weakly all alleviate, appetite and body constitution obviously improves, the check breast CT points out that many pieces of enlarged lymph nodes before dwindle in the more preceding remarkable minimizing of the multiple millet appearance focus of two lungs, the mediastinum; Head MRI prompting intracranial metastatic lesion slightly dwindles.Short term effect is evaluated as stable (SD), and quality of life obviously improves.To in January, 2007, symptom repeatedly, PET/CT check to find disease progression.Rise in February, 2007 and use recombinant human vascular endothelial inhibin injection associating NP treatment, 2 all after dates (in March, 2007) check PET/CT shows that pulmonary lesions obviously dwindles, brain lesion disappears, the standardization picked-up value (SUV) of whole body many places bone metastasis descends, the both adrenal glands focus is dwindled, therapeutic evaluation is that part is alleviated (PR), uses for 5 cycles altogether in May, 2007, keeps the PR time to reach for 18 weeks.In JIUYUE, 2007, PET/CT checks disease progression once more, changes modern Chinese medicine preparation and Supporting Therapy into.In November, 2007, the patient dies from pulmonary infection.Total life span reaches 20 months.
Case two
The male, 45 years old.In December, 2004 in the outer court because of the left renal pelvis cancer row peritoneoscope bottom left nephrectomy, left ureter is cut entirely and the part cystectomy, the postoperative pathological replacement: " left renal pelvis transitional cell carcinoma II level, 8cm * 3cm * 4cm; Transitional cell carcinoma of bladder II level, 2cm * 1cm * 1cm; It is special that left ureter and perirenal fat capsule are not seen ".Capable irrigation of bladder chemotherapy of postoperative application table amycin and IFN-α+IL-2 immunization therapy 1.5 years.In June, 2006 is because of bladder body recurrence operation once more, cut-out bladder.The breast flank pain appears in JIUYUE, 2006, and the activity of can not bending over and get up is checked discovery T through CT and MRI
12Multiple metastasis in bone destruction of thoracic vertebra leading edge and the liver.In JIUYUE, 2006 rises uses recombinant human vascular endothelial inhibin injection associating GP scheme chemotherapy, treat 1 all after date osteodynia and alleviates, and physical situation improvement can out-of-bed activity; 2 all after dates (in November, 2006) CT evaluating liver metastatic lesion is PR, T
12Thoracic vertebra bone destruction control; 6 all after dates (in March, 2007) CT check still is among the PR, is using the 7th cycle therapy of this scheme row, and existing life can be taken care of oneself.
Case three
The male, 27 years old.In JIUYUE, 2006 row mediastinal tumor is appeased excision, sees in the art that tumor is positioned at anterior mediastinum, about 15cm * 10cm * 8cm, the capsule reality, in see most caseous necrosises, owe clearly on the border.Postoperative pathology binding immunoassay group consideration yolk sac tumor companion is downright bad, and cancerous tissue involves thymus.Postoperative CT (in October, 2006) shows: the irregular soft tissue focus of anterior mediastinum, and get involved in the pericardium part, and lower-left pulmonary branch is not opened, consolidation, the left side hydrothorax.In row PVB (cisplatin+vincristine+bleomycin) 1 cycle of scheme chemotherapy, check CT shows: the anterior mediastinum focus increases, and the lower-left pulmonary consolidation is more preceding obviously, bottom right lung tuberosity shadow.In November, 2006, the capable PEB of December (cisplatin+etoposide+bleomycin) 2 cycles of scheme chemotherapy, check the CT prompting state of an illness in January, 2007 and make progress rapidly.Begin to use recombinant human vascular endothelial inhibin injection associating DP scheme chemotherapy in January, 2007, uncomfortable in chest behind 2 end cycles, out of breath, pain and feed freely obviously do not alleviate, check breast CT and show in March, 2007: in the mediastinum, pericardium edge and the real sexually transmitted disease (STD) kitchen range of upper left visceral pleura edge capsule is more preceding obviously dwindles right upper lung tuberosity foci disappearance.Clinical evaluation obtains PR, and the patient goes these 3 cycles of scheme chemotherapy at present, keeps PR, and quality of life obviously improves.
Case four
The women, 55 years old.2003 06 month patient looks into CT and shows the leftlobe of liver occupy-place because of losing weight, lobectomy, cholecystectomy outside on 06 29th, 2003 left livers of row, and the art journey is smooth, and postoperative pathology shows Hepatocholangiocarcinoma, chronic cholecystitis.At 2 mmc of outer court's row (mitomycin c) 10mg, 5-fu (5-fluorouracil) 1.0g, epi (epirubicin) 40mg united treatments such as getting involved chemotherapy and kind peaceful, Methyl cantharidinimide Methyleantharidinimide Methylcantharidimide to postoperative on 07 30th, 2003 and on 08 30th, 2003.The check B ultrasonic showed the multiple occupy-place of liver in 2004 06 month, and CT shows multiple low-density shadow in the liver, considers metastatic tumo(u)r.Began to use recombinant human vascular endothelial inhibin injection associating TP scheme chemotherapy in 2004 07 month, the check abdominal CT is shown that liver lesion is more preceding and is obviously dwindled behind 4 end cycles.Evaluation of clinical curative effect obtains PR, and quality of life obviously improves.
Clinical observation
Through histopathology and/or cytolgical examination, being all the IV phase of making a definite diagnosis according to " solid tumor diagnostic criteria " controls the malignant solid tumor patient again, comprise nonsmall-cell lung cancer, small cell lung cancer, the esophageal carcinoma, colorectal cancer, renal carcinoma, ovarian cancer, cancer of biliary duct, malignant melanoma, cervical cancer, nasopharyngeal carcinoma, gastric cancer totally 452 examples, previously all accept one or more scheme combined chemotherapy, but disease progression occurred.Have the measurable objective focus of CT or MRI, routine blood test, hepatic and renal function and cardiac function are normal substantially.All case all adopts recombinant human vascular endothelial inhibin injection combined chemotherapy Drug therapy, the chemotherapeutics that gives for this case previously untapped or with the chemotherapeutics of the no crossing drug resistant of treatment previously, scheme 131 examples of wherein uniting NP, associating DP110 example, associating TP123 example, associating GP88 example.The toxicity that G3/4 (Grade3-4) level occurs only has 57 examples inferior, and safety is higher.Have 182 examples (accounting for 40.3%) quality of life (QOL) to improve, have 205 examples (accounting for 45.4%) QOL stable, quality of life improves obviously.Can estimate curative effect totally 390 examples, treat four all after dates and obtain alleviation (CR) 25 examples fully, the PR111 example, the SD162 example, effective percentage is 34.8%, disease control rate is 76.4%.
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) the special inhibition entity tumor of recombinant human vascular endothelial inhibin of the present invention is organized the propagation of new vessels, and safety non-toxic has no drug resistance, and indicating has good application prospects.
3) recombinant human vascular endothelial inhibin combined chemotherapy Drug therapy tumor of the present invention by improving treatment effective percentage and patient's survival rate with the chemotherapeutics synergism, suppresses neoplasm metastasis, does not increase toxic and side effects, can improve patients ' life quality.
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
The aseptic filtration of μ m microporous filter membrane is sub-packed in the cillin bottle, adds 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 makes products temperature be increased to-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>MP071324
<160>1
<170>PatentIn version 3.3
<210>1
<211>192
<212>PRT
<213>Artificial
<220>
＜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 Hi s 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 35 140
Glu Thr Trp Arg Thr Glu Ala Pro Ser Ala Thr Gly Gln Ala Ser Ser
145 150 55 160
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 (13)
1. the application of recombinant human vascular endothelial inhibin in the medicine of preparation treatment tumor.
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 tumor 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 tumor medicine can with the tumor chemotherapeutic drug use in conjunction.
13. the described application of claim 1 is characterized in that described tumor is a solid tumor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100060581A CN101224297A (en) | 2008-02-01 | 2008-02-01 | Application of recombinant human endostain in preparing medicine |
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2008100060581A CN101224297A (en) | 2008-02-01 | 2008-02-01 | Application of recombinant human endostain in preparing medicine |
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| CN101224297A true CN101224297A (en) | 2008-07-23 |
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| CNA2008100060581A Pending CN101224297A (en) | 2008-02-01 | 2008-02-01 | Application of recombinant human endostain in preparing medicine |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009033406A1 (en) * | 2007-09-05 | 2009-03-19 | Jiangsu Simcere Pharmaceutical R & D Co., Ltd. | Modified recombinant human endostatin and its application |
| CN105709209A (en) * | 2016-01-17 | 2016-06-29 | 新乡医学院第一附属医院 | Medicine for treating tumor and preparation method thereof |
| 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 CNA2008100060581A patent/CN101224297A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009033406A1 (en) * | 2007-09-05 | 2009-03-19 | Jiangsu Simcere Pharmaceutical R & D Co., Ltd. | Modified recombinant human endostatin and its application |
| CN105709209A (en) * | 2016-01-17 | 2016-06-29 | 新乡医学院第一附属医院 | Medicine for treating tumor and preparation method thereof |
| 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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Application publication date: 20080723 |