CN100593420C - Endostatin conjugate and its preparation method - Google Patents
Endostatin conjugate and its preparation method Download PDFInfo
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- CN100593420C CN100593420C CN200610043873A CN200610043873A CN100593420C CN 100593420 C CN100593420 C CN 100593420C CN 200610043873 A CN200610043873 A CN 200610043873A CN 200610043873 A CN200610043873 A CN 200610043873A CN 100593420 C CN100593420 C CN 100593420C
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Abstract
The disclosed endotheliochalone compound is prepared as: the free amino with modified rate as 1.22~54.13% to combine 1-8 heparins and salt, and other amino with modified rate as 23.78~76.92% to combine 1-8 carbowaxs and derivative. Compared with natural product, this invention has low antigenicity, long half-life, and high stability, and fit to application in clinic.
Description
Technical field
The present invention relates to a kind of Angiostatin conjugate and preparation method thereof, relate in particular to a kind of endostatin conjugate and preparation method thereof, belong to biomedicine field.
Background technology
(Endostatin ES), is called endostatin again to Endostatin, is that a kind of endogenous new vessels of finding in recent years generates inhibitive factor, thereby it is to block the new type antineoplastic medicine that its nutrition supply is a mechanism of action to suppress tumor neogenetic blood vessels formation.Because Endostatin is that vascular endothelial cell is had specific action, can not cause tangible influence to normal in-house blood vessel, therefore, compare with traditional Therapeutic Method (as chemotherapy, radiotherapy etc.) and have as not developing immunity to drugs, do not have many advantages such as cytotoxicity.Yet its I clinical trial phase result of study shows that the effective dose that Endostatin is applied to human body is 300mg/m every day
2, this dosage is very large for a kind of genetically engineered drug, so big dosage not only can produce many side effect to human body, and the patient also is difficult to bear huge drug cost, is applied to clinical biggest obstacle thereby become Endostatin.In addition, shortcomings such as half-life weak point, poor stability also all are an impediment to its application clinically in the Endostatin body.
If can by certain means increase Endostatin stability, prolong in its body the half-life and will improve the therapeutic effect of Endostatin greatly to reduce dosage or to prolong the administration cycle, reduce drug cost, promote its application clinically.By retrieval, utilize heparin and its esters or Polyethylene Glycol and derivant thereof to modify Endostatin and prepare endostatin conjugate,, realize that the research and the application that improve the Endostatin curative effect yet there are no report to reduce dosage or to prolong the administration cycle.
Summary of the invention
At the deficiencies in the prior art, the objective of the invention is to overcome that above-mentioned existing Endostatin exists to the disadvantageous shortcoming of clinical practice, a kind of new ideal endostatin conjugate and preparation method thereof is proposed, to solve existing problem in the existing Endostatin clinical practice.
Endostatin conjugate of the present invention, be made of the free amino group heparin-binding on the Endostatin molecule and its esters or Polyethylene Glycol and derivant thereof, it is characterized in that: the modification rate of the free amino group on the Endostatin molecule of described heparin-binding and its esters is 1.22%～54.13%; Described modification rate in conjunction with the free amino group on the Endostatin molecule of Polyethylene Glycol and derivant thereof is 23.78%～76.92%; And an Endostatin molecule can be in conjunction with 1～8 heparin and its esters molecule or Polyethylene Glycol and derivative molecular thereof.
In the above-mentioned endostatin conjugate: the molecular weight of described Endostatin molecule is 15000～30000 dalton.
In the above-mentioned endostatin conjugate: the molecular weight of described heparin and its esters is 500～50000 dalton.
Wherein: the molecular weight of described heparin and its esters is preferably 1500～30000 dalton.
Wherein: described heparin and its esters preferably refer to one of heparin sodium, calciparine, low molecular sodium heparin and low molecular heparin calcium.
In the above-mentioned endostatin conjugate: the molecular weight of described Polyethylene Glycol and derivant thereof is 400～20000 dalton.
Wherein: the molecular weight of described Polyethylene Glycol and derivant thereof is preferably 800～10000 dalton.
Wherein: the derivant of described Polyethylene Glycol is preferably mono methoxy polyethylene glycol.
The preparation method of endostatin conjugate of the present invention, form by following steps:
(1) be that 1%～10% heparin and its esters and mass percent concentration are that one of 1%～13% sodium metaperiodate, Bromine cyanide., cyanuric chloride carry out priming reaction with mass percent concentration; Perhaps, one of Polyethylene Glycol and derivant thereof and Bromine cyanide., cyanuric chloride are carried out priming reaction;
Wherein: when selecting for use heparin and its esters and sodium metaperiodate to carry out priming reaction, can make heparin and its esters and sodium metaperiodate at pH 4.0～7.0, under 0～4 ℃ of the temperature, continuous stirring reaction 5～20 hours, and be 2%～10% sodium sulfite solution titration with mass percent concentration to color by achromaticity and clarification → muddy shape aubergine → finally be achromaticity and clarification, get activatory heparin and saline solution thereof, stand-by;
When selecting for use Polyethylene Glycol and derivant thereof and cyanuric chloride to carry out priming reaction, can make Polyethylene Glycol and derivant thereof and cyanuric chloride be (5～7) with weight ratio: 1 ratio joins in the anhydrous benzene jointly, room temperature stirred 10～16 hours down, filtered, and was settled out product with absolute ether, reuse anhydrous benzene dissolution precipitation, so repeated multiple times detects no cyanuric chloride until UV scanning and only absorbs conventional vacuum drying, get Powdered activated polyglycol of white solid and derivant thereof, stand-by;
(2) Endostatin behind the purification is dissolved with carbonate buffer solution, add above-mentioned activatory heparin and saline solution thereof, under pH 8～10,0～4 ℃ of condition of temperature, slowly stir and carry out modification reaction, react after 10～24 hours, add glycine and stop modification reaction, get heparin and its esters-endostatin conjugate;
Wherein, above-mentioned Endostatin and activatory heparin and saline solution thereof in mg than mL, by (14～16): 1 quantitative response;
Perhaps, with the Endostatin behind the Polyethylene Glycol after the above-mentioned activation and derivant and the purification, (40 ± 2) in molar ratio: 1 ratio is dissolved in the sodium tetraborate buffer, under pH 8～10,0～4 ℃ of condition of temperature, slowly stir and carry out modification reaction, react after 10～24 hours, add glycine and stop modification reaction, get Polyethylene Glycol and derivant-endostatin conjugate thereof;
Wherein, above-mentioned Endostatin carries out mole calculating with endostatin protein content;
(3) adopt gel chromatography respectively above-mentioned heparin and its esters-endostatin conjugate or Polyethylene Glycol and derivant-endostatin conjugate thereof to be carried out separation and purification, through lyophilization, make the pure product of heparin and its esters-endostatin conjugate or Polyethylene Glycol and derivant-endostatin conjugate thereof again.
In the preparation method of above-mentioned endostatin conjugate: the concentration of described carbonate buffer solution is 0.1～0.3mol/L, and preferred concentration is the Na of 0.3mol/L
2CO
3-NaHCO
3Buffer, the concentration of described sodium tetraborate buffer are 1～10mmol/L, and preferred concentration is the sodium tetraborate buffer of 10mmol/L.
In the preparation method of above-mentioned endostatin conjugate: described gel chromatography preferably adopts Sephadex G-50 or Sephadex G-75 post that heparin and its esters-endostatin conjugate or Polyethylene Glycol and derivant-endostatin conjugate thereof are carried out separation and purification.
The Low molecular heparin that the present invention makes and its esters-endostatin conjugate are compared with natural Endostatin with Polyethylene Glycol and derivant-endostatin conjugate thereof, not only kept the effect of the original inhibition vascular endothelial cell proliferation of Endostatin, but also have that antigenicity is low, long half time, stable high characteristic, can more give full play to it and suppress tumor growth and other blood vessel formation against function, thereby further promote Endostatin application clinically.
Prove through activity research, Endostatin after the modification has the obvious suppression tumor growth, suppresses vascular endothelial cell proliferation, suppresses the effect of chick chorioallantoic membrane blood vessel and the inductive rabbit corneal blood capillary proliferation of alkali burn, and suppresses the Endostatin that effect obviously is better than unmodified.
Low molecular heparin of the present invention and its esters-endostatin conjugate and Polyethylene Glycol and derivant-endostatin conjugate thereof have overcome the excessive problem of consumption that Endostatin exists to a certain extent in the clinical practice of treatment tumor, and, also can be used for treating diabetic retina blood capillary proliferation disease.
The specific embodiment
The key of Endostatin trim preparation is the activation of dressing agent and the control of the condition of modification.The activatory method of heparin and Polyethylene Glycol and derivant thereof has multiple, as sodium metaperiodate activation method, cyanogen bromide activation, cyanuric chloride activation method etc.At this only for sodium metaperiodate, cyanogen bromide-activated heparin and cyanuric chloride activated polyethylene glycol and derivant thereof and the further example of preparation heparin-endostatin conjugate and Polyethylene Glycol and derivant-endostatin conjugate thereof.
Embodiment 1: the preparation of Polyethylene Glycol-endostatin conjugate
(1) activation of Polyethylene Glycol
Polyethylene Glycol (molecular weight is 6000 dalton) 18g, natrium carbonicum calcinatum 2.2g, cyanuric chloride 2.75g are joined among the anhydrous benzene 75mL, room temperature stirs down and spends the night, filter, be settled out product, the dissolving of reuse anhydrous benzene with absolute ether, repeated multiple times like this, detect no cyanuric chloride until UV scanning and only absorb, vacuum drying gets the white solid powder, be activated polyglycol, place 4 ℃ of refrigerator sealings to preserve.
(2) activated polyglycol is to the modification of Endostatin
Polyethylene Glycol and Endostatin (in endostatin protein content) after the activation were dissolved in the sodium tetraborate buffer of pH9.0,10mmol/L in 40: 1 in molar ratio, 4 ℃ of slow stirring reactions, react and add a certain amount of glycine after 15 hours and stop modification reaction, Polyethylene Glycol-endostatin conjugate.
(3) purification of Polyethylene Glycol-endostatin conjugate
Modification reaction thing (Polyethylene Glycol-endostatin conjugate) is carried out separation and purification with Sephadex G-50 post to it, and lyophilization more promptly makes the pure product of Polyethylene Glycol-endostatin conjugate.
Embodiment 2: the preparation of mono methoxy polyethylene glycol-endostatin conjugate
(1) activation of mono methoxy polyethylene glycol
Mono methoxy polyethylene glycol (molecular weight is 5000 dalton) 50g, cyanuric chloride 5.5g, anhydrous natrium carbonicum calcinatum 10g, molecular sieve 5A 5g are dissolved among the anhydrous benzene 400mL, room temperature stirs down and spends the night, float such as centrifugal removal sodium carbonate, molecular sieve 5A, be settled out product with absolute ether, reuse anhydrous benzene 400mL dissolving.So repeated multiple times does not have cyanuric chloride until UV scanning and absorbs.Vacuum drying gets the white solid powder, is activatory mono methoxy polyethylene glycol, places 4 ℃ of refrigerators sealings to preserve.
(2) activatory mono methoxy polyethylene glycol is to the modification of Endostatin
Mono methoxy polyethylene glycol and Endostatin (in endostatin protein content) after the activation were dissolved in the sodium tetraborate buffer of pH 9.0,10mmol/L in 40: 1 in molar ratio, 4 ℃ of slow stirring reactions, react and add a certain amount of glycine after 15 hours and stop modification reaction, mono methoxy polyethylene glycol-endostatin conjugate.
(3) purification of mono methoxy polyethylene glycol-Endostatin
Modification reaction thing (mono methoxy polyethylene glycol-endostatin conjugate) is carried out separation and purification with Sephadex G-50 post to it, and lyophilization more promptly makes the pure product of mono methoxy polyethylene glycol-endostatin conjugate.
Embodiment 3: the preparation of heparin-endostatin conjugate
(1) activation of heparin (sodium metaperiodate activation method)
Take by weighing heparin (mean molecule quantity is 15000 dalton) 0.3g and be dissolved among the distilled water 4.5mL, drip 12% sodium periodate solution 0.5mL under the gentle agitation.The pH value that record solution this moment is about 5.40, is about 5.0 with the pH of the hydrochloric acid accent solution of 0.1mol/L, 4 ℃ of slow stir-activatings in dark place 20 hours.
The solution of priming reaction 20h is taken out from the dark place, slowly stir the priming reaction of Dropwise 5 % sodium sulfite solution termination Low molecular heparin down, solution colour is changed to achromaticity and clarification → muddy shape aubergine → achromaticity and clarification in the cessation reaction process, and the pH that regulates this solution with the sodium carbonate liquor of 2mol/L is 9.0 ± 0.2 then.
(2) activatory heparin is to the modification of Endostatin
Take by weighing the Endostatin 90mg behind the purification, carbonate buffer solution 1mL dissolving with pH 9.5,0.3mol/L, the activatory heparin solution 6mL that adds pH9.0 ± 0.2,4 ℃ of slow stirring reactions, react after 18 hours, add a certain amount of glycine and stop modification reaction, get heparin-endostatin conjugate.
SDS-polyacrylamide gel electrophoresis, different modifying response time free amino group number by the different modifying response time in modification changes mensuration, different modifying response time Endostatin activity change is measured and monitored its modification reaction.
(3) purification of heparin-endostatin conjugate
Modification reaction thing (heparin-endostatin conjugate) is carried out separation and purification with Sephadex G-50 post to it, and lyophilization more promptly makes the pure product of heparin-endostatin conjugate.
Embodiment 4: the preparation of Low molecular heparin-endostatin conjugate
(1) activation of Low molecular heparin (cyanogen bromide activation)
Take by weighing Low molecular heparin (mean molecule quantity is 5000 dalton) 4g, be dissolved among the carbonate buffer solution 20mL of pH9.0,0.1mol/L of pre-cooling, add and be dissolved with among the cold same buffer 20mL of Bromine cyanide. 1.60g, ice bath stirring reaction 10min, add the 4mol/L sodium hydroxide between the reaction period and keep pH10.4～10.8, will precipitate sucking filtration among the reactant liquor impouring cold acetone 100mL rapidly, use the cold acetone thorough washing, drain.Collecting precipitation adds above-mentioned buffer 7～8mL dissolving in the 25mL small beaker, and the pH that regulates this solution with the sodium carbonate liquor of 2mol/L is 9.0 ± 0.2 then.
(2) activatory Low molecular heparin is to the modification of Endostatin
Take by weighing the Endostatin 90mg behind the purification, carbonate buffer solution 1mL dissolving with pH9.5,0.3mol/L, the activatory Low molecular heparin solution 6mL that adds pH 9.0 ± 0.2,4 ℃ of slow stirring reactions, react after 17 hours, add a certain amount of glycine and stop modification reaction, sub-heparin-endostatin conjugate makes low score.
SDS-polyacrylamide gel electrophoresis, different modifying response time free amino group number by the different modifying response time in modification changes mensuration, different modifying response time Endostatin activity change is measured and monitored its modification reaction.
(3) purification of Low molecular heparin-endostatin conjugate
Modification reaction thing (Low molecular heparin-endostatin conjugate) is carried out separation and purification with Sephadex G-75 post to it, and lyophilization more promptly makes the pure product of Low molecular heparin-endostatin conjugate.
Claims (10)
1. endostatin conjugate, be made of the free amino group heparin-binding on the Endostatin molecule and its esters or Polyethylene Glycol and derivant thereof, it is characterized in that: the modification rate of the free amino group on the Endostatin molecule of described heparin-binding and its esters is 1.22%～54.13%; Described modification rate in conjunction with the free amino group on the Endostatin molecule of Polyethylene Glycol and derivant thereof is 23.78%～76.92%; And an Endostatin molecule can be in conjunction with 1～8 heparin and its esters molecule or Polyethylene Glycol and derivative molecular thereof.
2. endostatin conjugate as claimed in claim 1 is characterized in that: the molecular weight of described Endostatin molecule is 15000～30000 dalton.
3. endostatin conjugate as claimed in claim 1 is characterized in that: the molecular weight of described heparin and its esters is 500～50000 dalton.
4. endostatin conjugate as claimed in claim 3 is characterized in that: the molecular weight of described heparin and its esters is 1500～30000 dalton.
5. as claim 3 or 4 described endostatin conjugates, it is characterized in that: described heparin and its esters are meant one of heparin sodium, calciparine, low molecular sodium heparin and low molecular heparin calcium.
6. endostatin conjugate as claimed in claim 1 is characterized in that: the molecular weight of described Polyethylene Glycol and derivant thereof is 400～20000 dalton.
7. endostatin conjugate as claimed in claim 6 is characterized in that: the molecular weight of described Polyethylene Glycol and derivant thereof is 800～10000 dalton.
8. as claim 6 or 7 described endostatin conjugates, it is characterized in that: described polyethyleneglycol derivative is a mono methoxy polyethylene glycol.
9. the preparation method of the described endostatin conjugate of claim 1, form by following steps:
(1) be that 1%～10% heparin and its esters and mass percent concentration are that one of 1%～13% sodium metaperiodate, Bromine cyanide., cyanuric chloride carry out priming reaction with mass percent concentration; Perhaps, one of Polyethylene Glycol and derivant thereof and Bromine cyanide., cyanuric chloride are carried out priming reaction;
Wherein: when selecting for use heparin and its esters and sodium metaperiodate to carry out priming reaction, can make heparin and its esters and sodium metaperiodate at pH 4.0～7.0, under 0～4 ℃ of the temperature, continuous stirring reaction 5～20 hours, and be 2%～10% sodium sulfite solution titration with mass percent concentration to color by achromaticity and clarification → muddy shape aubergine → finally be achromaticity and clarification, get activatory heparin and saline solution thereof, stand-by;
When selecting for use Polyethylene Glycol and derivant thereof and cyanuric chloride to carry out priming reaction, can make Polyethylene Glycol and derivant thereof and cyanuric chloride be (5～7) with weight ratio: 1 ratio joins in the anhydrous benzene jointly, room temperature stirred 10～16 hours down, filtered, and was settled out product with absolute ether, reuse anhydrous benzene dissolution precipitation, so repeated multiple times detects no cyanuric chloride until UV scanning and only absorbs conventional vacuum drying, get Powdered activated polyglycol of white solid and derivant thereof, stand-by;
(2) Endostatin behind the purification is dissolved with carbonate buffer solution, add above-mentioned activatory heparin and saline solution thereof, under pH8～10,0～4 ℃ of condition of temperature, slowly stir and carry out modification reaction, react after 10～24 hours, add glycine and stop modification reaction, get heparin and its esters-endostatin conjugate;
Wherein, above-mentioned Endostatin and activatory heparin and saline solution thereof in mg than mL, by (14～16): 1 quantitative response;
Perhaps, with the Endostatin behind the Polyethylene Glycol after the above-mentioned activation and derivant and the purification, (40 ± 2) in molar ratio: 1 ratio is dissolved in the sodium tetraborate buffer, under pH 8～10,0～4 ℃ of condition of temperature, slowly stir and carry out modification reaction, react after 10～24 hours, add glycine and stop modification reaction, get Polyethylene Glycol and derivant-endostatin conjugate thereof;
Wherein, above-mentioned Endostatin carries out mole calculating with endostatin protein content;
(3) adopt gel chromatography respectively above-mentioned heparin and its esters-endostatin conjugate or Polyethylene Glycol and derivant-endostatin conjugate thereof to be carried out separation and purification, through lyophilization, make the pure product of heparin and its esters-endostatin conjugate or Polyethylene Glycol and derivant-endostatin conjugate thereof again.
10. as the preparation method of endostatin conjugate as described in the claim 9, it is characterized in that: the concentration of described carbonate buffer solution is 0.1～0.3mol/L; The concentration of described sodium tetraborate buffer is 1～10mmol/L; Described gel chromatography adopts Sephadex G-50 or Sephadex G-75 post that heparin and its esters-endostatin conjugate or Polyethylene Glycol and derivant-endostatin conjugate thereof are carried out separation and purification.
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| CN101143894A (en) * | 2007-06-22 | 2008-03-19 | 中国药科大学 | Highly effective polypeptide for inhibiting angiogenesis, physical chemistry modifying method and application thereof |
| CN101381413A (en) * | 2007-09-05 | 2009-03-11 | 江苏先声药物研究有限公司 | Modified recombinant human endostatin and use thereof |
| CN101265298B (en) * | 2008-04-30 | 2011-11-09 | 中国药科大学 | Endothelium chalone mutant containing non-natural amino acid and derivatives thereof |
| CN102850443B (en) * | 2011-12-27 | 2014-04-16 | 中国药科大学 | Integrin blocker polypeptides and application thereof |
| US9879052B2 (en) | 2011-12-27 | 2018-01-30 | Hanmei Xu | Integrin-blocking polypeptides and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1266064A (en) * | 1999-03-03 | 2000-09-13 | 华西医科大学 | Vascalogenesis inhibition factor-endothelial inhibitor and preparation process thereof |
| CN1401785A (en) * | 2002-09-26 | 2003-03-12 | 山东大学 | Human recombinant secretor type endostatin protein, preparing process and use thereof |
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| CN1266064A (en) * | 1999-03-03 | 2000-09-13 | 华西医科大学 | Vascalogenesis inhibition factor-endothelial inhibitor and preparation process thereof |
| CN1401785A (en) * | 2002-09-26 | 2003-03-12 | 山东大学 | Human recombinant secretor type endostatin protein, preparing process and use thereof |
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