CN105087530A - PEG (polyethylene glycol)-modified LMW-UK (low-molecular-weight urokinase) and preparation method and application thereof - Google Patents

PEG (polyethylene glycol)-modified LMW-UK (low-molecular-weight urokinase) and preparation method and application thereof Download PDF

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CN105087530A
CN105087530A CN201510499115.4A CN201510499115A CN105087530A CN 105087530 A CN105087530 A CN 105087530A CN 201510499115 A CN201510499115 A CN 201510499115A CN 105087530 A CN105087530 A CN 105087530A
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molecular weight
low molecular
weight urokinase
polyethylene glycol
urokinase
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苗丕渠
韩大雄
厉道娟
耿春晨
王筱蒙
池正昌
沈小宁
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YANGZHOU AIDEA BIOTECH CO Ltd
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Abstract

The invention provides PEG (polyethylene glycol)-modified LMW-UK (low-molecular-weight urokinase) and a preparation method and application thereof. According to the preparation method, the PEG is adopted to modify the LMW-UK, and the PEG is connected with the N-terminal of the LMW-UK in a covalent bond manner, so that a PEG-modified product of the LMW-UK is obtained. The modified product is good in homogeneity, simple in separation and purification technologies, high in activity recovery rate and capable of prolonging half-life in vivo remarkably, treating hyperfibrinogenemia effectively and preventing AS (atherosclerosis), thromboembolic complications, diabetic vascular complications, diabetic nephropathy and the like.

Description

Polyethyleneglycol modified low molecular weight urokinase and its preparation method and application
Technical field
The invention belongs to biotechnology and pharmacy field, relate to polyethyleneglycol modified protein medicaments, particularly polyethyleneglycol modified low molecular weight urokinase and its preparation method and application.
Background technology
Urokinase (Urokinase, be abbreviated as UK) be a kind of fibrinolysin activator (PlasminogenActivator) of being closed adult urine release by people's kidney, also double chain urokinase type plasminogen activator (tcu-PA) is claimed, uPA (pro-UK, also Single-chain Urokinase-type Plasminogen Activator is claimed, scu-PA) under the effects such as plasmin, trypsinase and kallikrein, Lys 158-Ile 159between peptide bond rupture after the enzyme having plasminogen activation function that formed.Due to Lys 158-Ile 159between peptide bond rupture after the A chain that formed and B chain still by disulfide linkage Cys 148-Cys 279connect, molecular weight is identical with Pro-UK, therefore is called high molecular weight urokinase (High-Molecular-WeightUrokinase, HMW-UK), and molecular weight is about 54000 dalton.Plasmin continues hydrolysis HWK-UK, at Lys 135-Lys 136fracture loses EGF-like district and Kringle district, and become low molecular weight urokinase (Low-Molecular-WeightUrokinase, LMW-UK), molecular weight is about 33000 dalton.Urokinase is thrombolytic drug important clinically, be widely used in the treatment of the place such as cardiovascular and cerebrovascular and peripheral blood vessel thrombotic diseases, but because low molecular weight urokinase lacks fibrin-specific, easily cause side reaction such as systematicness hemorrhage grade, Clinical practice is restricted, therefore, in pharmacopoeial requirements urokinase bulk drug, HMW-UK ratio need be greater than 90%, needs to remove low molecular weight urokinase in urokinase purge process.
The thrombolytic effect mechanism of high molecular weight urokinase and low molecular weight urokinase is different, they itself are not directly used in clot itself, but first swash intravital Profibrinolysin, become activated plasmin, plasmin degradation fibrin clot, also Fibrinogen, factor V and the platelet cofactor Ⅰ etc. that can degrade in circulation of blood, it is made to resolve into soluble polypeptide, the tissue of the urokinase energy specific combination pathology of high molecular, but low molecular weight urokinase does not have specific recognition function.In the crude product for extraction purification urokinase, the ratio of LMW-UK is up to 30%, and be not fully used in medicine research and development at present, this patent will develop emphatically its fibrinogenolytic function, and the medicine of hyperfibrinogenemia is effectively treated in exploitation.
Fibrinogen (Fibrinogen, Fg) also known as factor I, being synthesized by human liver cell and a kind of glycosylated protein secreted, is the topmost hematoglobin protein promoting platelet aggregation, 80% is present in blood plasma, and normal plasma content is 2 ~ 4g/L.Fasting plasma fibrinogen level >4.0g/L person is mostly called hyperfibrinogenemia (Hyperfibrinogenemia) by current research.Under various pathological state, the exception rising of plasma fibrinogen can increase blood viscosity, promotes platelet aggregation, participate in the combination with inflammatory cell, endotheliocyte and other protein, by changing hemorheology, injured blood vessel endothelium, inducing inflammatory reaction and participating in the pathologic process that the number of ways such as thrombosis participate in body various diseases.Large quantity research shows, plasma fibrinogen level rising is the important risk factor of atherosclerosis (AS), thromboembolic complication and diabetic vascular complications, the generation and development of diabetic nephropathy also play an important role, and strict these Hazard Factor that control are prevention and the important measures delaying vascular complication generation and development.
At present, for hyperfibrinogenemia patient, clinical treatment mainly contains: 1. apply antithrombotic reagent if acetylsalicylic acid or clopidogrel are to treat hyperfibrinogenemia; 2. fibrinolytic agent, the tPA etc. such as fiber eliminating enzyme is adopted to fall fine preparation to reduce fibrinogen level; 3. adopt treatment by Low molecule heparin, but short-term can only be used for high risk patient; 4. for serious hyperfibrinogenemia, the external plasma lipid strainer treatment of normal employing, JX-DELP external lipid strainer lipid-lowering therapy, effectively reduces triglyceride, fatty liver, high blood viscosity, hyperlipemia etc., and safety is controlled.But these above methods for the treatment of all exist certain defect and limitation, research finds that the medicine such as acetylsalicylic acid or clopidogrel is in the process of the diabetes complicated high microsteping proteinemia for the treatment of, and allergy, infarct and the side reaction such as hemorrhage easily appear in patient; And in the neuronal death models of inducing at some excitotoxins and stroke model, tPA can promote neuronal death, also can increase the risk of hematencephalon; Fiber eliminating enzyme class medicine is owing to carrying out source problem, and immunogenicity is higher, and side effect is larger.Therefore, develop long-acting, efficient and immunogenicity low fine medicine falls, to delaying developing of hyperfibrinogenemia relative disease, reduce morbidity and the mortality risk important in inhibiting of these diseases.
Urokinase, especially low molecular weight urokinase, can plasminogen activation effectively and rapidly, high-caliber Fibrinogen in degraded blood plasma is the potential medicine for the treatment of hyperfibrinogenemia, atherosclerosis (AS), thromboembolic complication and diabetic vascular complications.It can effective plasminogen activation, makes it change the activated plasmin of tool into, and then acts on the Fibrinogen in thrombus, makes it change scleroproein and polypeptide into and thrombolysis.Also can directly act on Fibrinogen α chain, release α chain material, make blood clot be decomposed into polypeptide, peptide material is eliminated in blood.And, urokinase extracts from people's urine, and immunogenicity is low, but low molecular weight urokinase medicine exists transformation period short, the shortcoming such as stability is not high, investigator is trying to explore the effective way improving these medicine effects, wherein the effective method of polyethyleneglycol modified imputed one.
Polyethylene glycol modified protein technology is widely used in biomedicine field, and polyethylene glycol modified protein and other compounds have at least more than 40 to plant so far.The PEG modified product of FDA approved has: Pegylation adenosine deaminase, Pegylation asparaginase, Peg-Intron, PEG-G-CSF etc.
Polyoxyethylene glycol (PEG) is neutral, nontoxic, non-immunogenicity, the high molecular polymer having good biocompatibility, there is the wetting ability of height, there is larger ydrodynamics volume in aqueous, when being coupled to drug molecule surface, its biological distribution behavior and solvability can be changed, produce physical barrier, reduce the enzymolysis of medicine, avoid being eliminated fast in the metabolism of kidney.Therefore, protein and peptide drugs is carried out PEG modification, medicine stability in vivo can be improved, to improve Half-life in vivo.
The PEGization of protein and peptide usually with functional groups such as the amino in its molecule, carboxyl and sulfydryls for avtive spot selects the PEG of respective type to modify.Amido modified is the PEG modifying method commonly used the most, α or ε that early stage PEG modifies mainly for Methionin is amino, and the required condition of this kind of modifier reaction is comparatively gentle, relatively little on medicine stability impact.But its reaction preference is low, degree of crosslinking heterogeneity, by product are many, bond is closed unstable and the interference of PEG self impurity is comparatively serious, make the separation and purification of albumen and qualification very difficult, be unfavorable for suitability for industrialized production, thus gradually substitute by PEG of new generation.Definitely, Typical Representative is mPEG-propionic aldehyde to a new generation's PEG decorating site, finds after deliberation, and aldehyde radical and primary amine, by Schiff, form a stable primary amine, and when pH value is relatively low, the amino and aldehyde radical of the α of N-terminal has very high selectivity.Relative to random modification, pointed decoration can be modified for special groups or single-minded site, is conducive to the activity keeping pharmaceutical grade protein, and product is easy to characterize, and quality product more easily controls, and has become the study hotspot of PEG modifying protein medicine.
Summary of the invention
The object of the invention is the deficiency existed for prior art, the polyethyleneglycol modified low molecular weight urokinase of a kind of tool pharmaceutically long-acting, efficient, reduced immunogenicity is provided.
For achieving the above object, the technical scheme that the low molecular weight urokinase that the present invention is polyethyleneglycol modified adopts is:
A polyethyleneglycol modified low molecular weight urokinase, is connected with covalent linkage with low molecular weight urokinase by polyoxyethylene glycol, and the N-end of low molecular weight urokinase connects polyoxyethylene glycol by covalent linkage.
Peg molecule connects low molecular weight urokinase molecule subunit by covalent linkage and forms.
Described polyoxyethylene glycol is mono methoxy polyethylene glycol.
Mono methoxy polyethylene glycol is one or more in mono methoxy polyethylene glycol succinimdyl carbonate, mono methoxy polyethylene glycol succinimide carboxylicesters, mono methoxy polyethylene glycol aldehyde.
Mono methoxy polyethylene glycol molecule is straight chain type or branching type.
Mono methoxy polyethylene glycol is mono methoxy polyethylene glycol aldehyde.
Described molecular weight polyethylene glycol is 5kDa ~ 60kDa.As preferably, described molecular weight polyethylene glycol is 5kDa ~ 40kDa.As preferred again, described molecular weight polyethylene glycol is 20kDa ~ 40kDa.
Described low molecular weight urokinase is the low molecular weight urokinase of natural extract or the low molecular weight urokinase of restructuring that obtained by genetic engineering technique.As preferably, described low molecular weight urokinase be extract from Healthy People urine, purifying obtains.
The present invention also provides a kind of preparation method of polyethyleneglycol modified low molecular weight urokinase, and the method step is simple, is easy to realize, is applicable to suitability for industrialized production.
For achieving the above object, the preparation method of the low molecular weight urokinase that the present invention is polyethyleneglycol modified adopts technical scheme to be:
A preparation method for described polyethyleneglycol modified low molecular weight urokinase, comprises the following steps:
(1) prepare modification reaction damping fluid, ionic strength is 10 ~ 500mmol/L, and pH value is 4 ~ 9;
(2) conditioned response that provides in step (1) of low molecular weight urokinase and activated polyethylene glycol 2 ~ 48 hours, temperature of reaction is 4 ~ 60 DEG C;
(3) modified outcome be obtained by reacting through step (2) carries out separation and purification through cation exchange chromatography, polyethyleneglycol modified low molecular weight urokinase elution peak described in wash-out collection, obtains described polyethyleneglycol modified low molecular weight urokinase after concentrated.
In step (3), (a) positively charged ion chromatography gel is SP-SepharoseFastFlow, CM-SepharoseFastFlow, SP-HighPerformance or Mono-S, preferred SP-SepharoseFastFlow; B () regulates pH value and the conductance of sample solution, by the SP post loading balanced, then rinse with balance liquid; C () continuation washing fluid rinses, collect and rinse peak; D () finally carries out wash-out with elutriant, collect elution peak.
In step (b), SP chromatography column balance liquid is 0.01 ~ 0.2M acetate buffer (pH4.0 ~ 8.0), and sodium chloride concentration is 0 ~ 0.2M sodium-chlor.Preferred 0.1M acetate buffer, sodium chloride concentration is 0M, pH5.5.
In step (b), the pH value regulating sample solution is 4.0 ~ 8.0, conductance 1 ~ 10mS/cm.Optimum condition pH5.5, conductance 5mS/cm.
Step (c) is rinse step, and use pH4.0 ~ 8.0,0.01 ~ 0.2M acetate buffer, containing 0.05M ~ 0.3M sodium-chlor.Preferred 0.1M acetate buffer, containing 0.05M sodium-chlor, pH5.5 rinses.
Step (d) is elution step, and use pH4.0 ~ 8.0,0.01 ~ 0.2M acetate buffer, containing 0 ~ 1.0M sodium-chlor.Preferred 0.1M acetate buffer, adds 0.1M sodium-chlor, and pH5.5 carries out wash-out.
In step (b), collection loading and balance penetrate, mainly containing the free PEG-ALD having neither part nor lot in modification;
In step (c), collect washing fluid, mainly containing the low molecular weight urokinase having neither part nor lot in modification;
In step (d), collect elutriant, main containing the low molecular weight urokinase compound after PEG-ALD modification.
Modification reaction damping fluid is phosphate buffer soln, borate buffer solution or acetate buffer solution.As preferably, modification reaction damping fluid is phosphate buffer soln.
As preferably, the molar ratio that above-mentioned polyoxyethylene glycol and low molecular weight urokinase generation covalent attachment are reacted is 1 ~ 50.As preferred again, the molar ratio that described polyoxyethylene glycol and low molecular weight urokinase generation covalent attachment are reacted is 3 ~ 25.
As preferably, the modification reaction pH of cushioning fluid that above-mentioned polyoxyethylene glycol and low molecular weight urokinase generation covalent attachment are reacted is 5 ~ 7.
As preferably, the temperature that described polyoxyethylene glycol and low molecular weight urokinase generation covalent attachment are reacted is 4 ~ 37 DEG C.
As preferably, the reaction times that described polyoxyethylene glycol and low molecular weight urokinase generation covalent attachment are reacted is 4 ~ 24 hours.
As preferably, above-mentioned polyoxyethylene glycol and low molecular weight urokinase generation covalent attachment are reacted and are adopted NaCNBH 3as catalyzer.
As preferably, above-mentioned positively charged ion chromatography gel is SP-SepharoseFastFlow, CM-SepharoseFastFlow, SP-HighPerformance or Mono-S.
Present invention also offers the application of polyethyleneglycol modified low molecular weight urokinase.The polyethyleneglycol modified low molecular weight urokinase that the present invention obtains has the feature of the prolonged action preparations such as long-acting, efficient and reduced immunogenicity, for the prolonged action preparation of disease such as preparation treatment hyperfibrinogenemia and prevention of arterial atherosis (AS), thromboembolic complication, diabetic vascular complications, diabetic nephropathy etc. is laid a good foundation.
A purposes for polyethyleneglycol modified low molecular weight urokinase, for the preparation for the treatment of hyperfibrinogenemia and prevention of arterial is atherosis, the medicine of thromboembolic complication, diabetic vascular complications, diabetic nephropathy.
Present invention also offers a kind of medicine for the treatment of hyperfibrinogenemia.
Treat a medicine for hyperfibrinogenemia, it contains polyethyleneglycol modified low molecular weight urokinase as effective constituent.
Present invention also offers that a kind of prevention of arterial is atherosis, the medicine of thromboembolic complication.
Prevention of arterial is atherosis, a medicine for thromboembolic complication, and it contains polyethyleneglycol modified low molecular weight urokinase as effective constituent.
Present invention also offers the medicine of a kind of prevent diabetes vascular complication, diabetic nephropathy.
A medicine for prevent diabetes vascular complication, diabetic nephropathy, it contains polyethyleneglycol modified low molecular weight urokinase as effective constituent.
The present invention compared with prior art, has the following advantages:
The present invention adopts the modifying method high to the N-terminal selective of low molecular weight urokinase, namely the polyoxyethylene glycol activated is connected by the mode of covalent linkage with the N-end of low molecular weight urokinase, that homogeneity and the stability of product is greatly enhanced, easy purifying, the activity of purified product can be retained preferably.Product after modification significantly improves stability, extend Half-life in vivo and alleviate side effect, add action specificity.Polyethyleneglycol modified low molecular weight urokinase can be used in disease such as treatment hyperfibrinogenemia and prevention of arterial atherosis (AS), thromboembolic complication, diabetic vascular complications, diabetic nephropathy etc.Polyethyleneglycol modified low molecular weight urokinase can effectively treat hyperfibrinogenemia and effect of the disease such as prevention of arterial atherosis (AS), thromboembolic complication, diabetic vascular complications, diabetic nephropathy as the medicine of effective constituent.The preparation method of polyethyleneglycol modified low molecular weight urokinase, the method step is simple, is easy to realize, and is applicable to suitability for industrialized production.
Accompanying drawing explanation
Fig. 1 adopts SP-SepharoseFastFlow chromatography column to carry out the SDS-PAGE electrophorogram after purifying to the LMW-UK after mPEG-ALD-20K modification.
Fig. 2 is the reverse-phase chromatography figure that before purifying, mPEG-ALD-20K modifies LMW-UK sample.
Fig. 3 is the reverse-phase chromatography figure that after purifying, mPEG-ALD-20K modifies LMW-UK sample.
Fig. 4 is the MALDI-TOFMS detected result figure of mPEG-ALD-20K and LMW-UK reaction mixture.
Fig. 5 is the Plasma Concentration variation diagram in time after mPEG-ALD-20K coupling protein and the administration of LMW-UK former albumen rat vein.
Embodiment
Below in conjunction with the drawings and specific embodiments, illustrate the present invention further, these embodiments should be understood only be not used in for illustration of the present invention and limit the scope of the invention, after having read the present invention, the amendment of those skilled in the art to the various equivalent form of value of the present invention has all fallen within the application's claims limited range.
As Fig. 1, swimming lane 1: for mPEG-ALD-20K is to the sample after LMW-UK modification; Swimming lane 4: be 0.1M acetate+0.1MNaCl, ALD-20K-LMW-UK pure protein prepared by pH5.5 buffer solution elution; Swimming lane 7: albumen Marker.
As shown in Figures 2 and 3, Fig. 2 is mPEG-ALD-20K modified protein before purifying, is the mixture of the LMW-UK of LMW-UK and the mPEG-ALD-20K modification of unmodified, can finds out that modification rate is higher than 50%.Fig. 3 is mPEG-ALD-20K modified protein after purifying, and anti-phase detected result display modified protein is single absorption peak, and mono-modified product purity is more than 95%.
As Fig. 4, the MALDI-TOFMS detected result figure of mPEG-ALD-20K and LMW-UK reaction mixture, detected result finds, the LMW-UK containing unmodified in mixture and the ALD-20K-LMW-UK albumen after modifying, the molecular weight of modified protein is about 53kDa, conforms to expection.
As Fig. 5, detected by the pharmacokinetics of Isotope tracer labelling method to LMW-UK and ALD-20K-LMW-UK.Experimental result shows, and compared with the LMW-UK of unmodified, the Plasma Concentration of ALD-20K-LMW-UK reduces comparatively slow, and the transformation period rises appreciably, and AUC significantly increases, and plasma clearance speed obviously declines.
1, polyethylene glycol aldehyde is to the modification of low molecular weight urokinase:
(1) with the phosphate buffer soln of 10 ~ 100mM, pH5.0 ~ 7.0, high purity and low molecular weight urokinase (LMW-UK) lyophilized powder is mixed with the solution of 1 ~ 30mg/ml, the phosphate buffer soln of preferred 50mM, pH6.5 dissolves; Preferred LMW-UK reaction density is 1 ~ 10mg/ml.
(2) LMW-UK:PEG-aldehyde in molar ratio: reductive agent=1:3 ~ 25:30 ~ 250 are reacted 4 ~ 24 hours under the condition of 4 ~ 37 DEG C; Preferred reaction ratio is LMW-UK:PEG: the preferred PEG-propionic aldehyde (PEG-ALD) of reductive agent=1:20:200, PEG-aldehyde, molecular weight 20kDa; Preferred temperature of reaction is 25 DEG C, and the reaction times is preferably 12 hours, and preferred reductive agent is NaCNBH 3.
The separation and purification of 2, polyethyleneglycol modified low molecular weight urokinase:
A () selects ion exchange chromatography, preferred SP-SepharoseFastFlow;
B () regulates pH value and the conductance of sample solution, by the SP post loading balanced, then rinse with balance liquid;
C () continuation washing fluid rinses, collect and rinse peak;
D () finally carries out wash-out with elutriant, collect elution peak.
In step (b), SP chromatography column balance liquid is 0.01 ~ 0.2M acetate buffer (pH4.0 ~ 8.0), and sodium chloride concentration is 0 ~ 0.2M sodium-chlor, preferred 0.1M acetate buffer, and sodium chloride concentration is 0M, pH5.5.
In step (b), the pH value regulating sample solution is 4.0 ~ 8.0, conductance 1 ~ 10mS/cm; Optimum condition pH5.5, conductance 5mS/cm.
Step (c) is rinse step, and use pH4.0 ~ 8.0,0.01 ~ 0.2M acetate buffer, containing 0.05M ~ 0.3M sodium-chlor, preferred 0.1M acetate buffer, containing 0.05M sodium-chlor, pH5.5 rinses.
Step (d) is elution step, and use pH4.0 ~ 8.0,0.01 ~ 0.2M acetate buffer, containing 0 ~ 1.0M sodium-chlor, preferred 0.1M acetate buffer, adds 0.1M sodium-chlor, and pH5.5 carries out wash-out.
In step (b), collection loading and balance penetrate, mainly containing the free PEG-ALD having neither part nor lot in modification.
In step (c), collect washing fluid, mainly containing the low molecular weight urokinase having neither part nor lot in modification.
In step (d), collect elutriant, main containing the low molecular weight urokinase compound after PEG-ALD modification.
embodiment 1:mono methoxy polyethylene glycol propionic aldehyde (mPEG-ALD) is to the selection of the end modified condition of low molecular weight urokinase (LMW-UK) N-
The selection of pH value in reaction: respectively taking 0.5mgLMW-UK dry powder, to be dissolved in 0.5mlpH value be respectively in the 50mMPB buffered soln of 5.0,5.5,6.0,6.5,7.0, respectively add mPEG-ALD-20K solid 12.12mg again, modification mol ratio is made to be 1:20, dissolve, mixing, termination reaction after room temperature reaction spends the night, SDS-PAGE compares modification rate, determines optimum modification pH value.
Result shows: under these modification conditions, all can obtain polyethyleneglycol modified LMW-UK, when wherein pH value is 6.5, modification rate is the highest.
The selection of modifier molecular weight: each 0.5ml of LMW-UK getting the 1mg/ml that 4 parts of pH6.5 dissolve, add mPEG-ALD-10K, mPEG-ALD-20K, mPEG-ALD-30K, mPEG-ALD-40K solid 6.06mg, 12.12mg, 18.18mg, 24.24mg more respectively, modification mol ratio is made to be 1:20, dissolve, mixing, termination reaction after room temperature reaction spends the night, SDS-PAGE compares modification rate, determines the optimum weight of modifier.
Result shows: the PEG-ALD of four kinds of different molecular weights all can modify LMW-UK, and wherein the modification rate of PEG-ALD-20K is the highest.
The selection of temperature of reaction: the LMW-UK0.5ml getting the 1mg/ml that three parts of pH6.5PB dissolve, add mPEG-ALD-20K solid 12.12mg more respectively, dissolve, mixing, then be placed in respectively 4 DEG C, 25 DEG C, 37 DEG C reactions spend the night after termination reaction, SDS-PAGE compares modification rate, determines best modification temperature.
Result shows: can obtain polyethyleneglycol modified LMW-UK at these tem-peratures, and wherein the modification rate of 25 DEG C is the highest.
LMW-UK reacts the selection of mol ratio with mPEG-ALD-20K: respectively get the LMW-UK(1mg/ml that 0.5ml is dissolved in pH6.5 phosphate buffered saline buffer) 5 parts, add mPEG-ALD-20K solid 1.82mg, 3.03mg, 6.06mg, 12.12mg, 30.30mg(to be respectively equivalent to mPEG-ALD-20K and to be 3:1,5:1,10:1,20:1,50:1 with the mol ratio of LMW-UK), dissolve, mixing, reacts termination reaction after spending the night.Relatively modification rate, determines modification condition.
Result shows: can obtain polyethyleneglycol modified LMW-UK under these modification ratios, when mPEG-ALD-20K with the mol ratio of LMW-UK at 20:1 time modification rate reached the highest, the amount modification rate increasing mPEG-ALD-20K further is not also significantly increased.
embodiment 2:prepare mono methoxy polyethylene glycol propionic aldehyde-20K(mPEG-ALD-20K) LMW-UK that modifies
LMW-UK lyophilized powder is dissolved to prepare the solution becoming 1 ~ 30mg/ml with the phosphate buffered saline buffer of 50mMpH6.5, use PEG-ALD-10K respectively, PEG-ALD-20K, PEG-ALD-30K, PEG-ALD-40K(is purchased from Jiankai Science and Technology Co., Ltd., Beijing), according to mol ratio LMW-UK:PEG: reductive agent=1:20:200 reacts, after 25 DEG C of reaction 12h, add 1M glycine termination reaction.Prepare 4 kinds of modified outcome ALD-10K-LMW-UK(conjugates 1), ALD-20K-LMW-UK(conjugate 2), ALD-30K-LMW-UK(conjugate 3) and, ALD-40K-LMW-UK(conjugate 4).By C18 analytical column and SDS-PAGE electrophoresis detection, the modification rate of conjugate 1, conjugate 2, conjugate 3, conjugate 4 is respectively 55%, 52%, 45%, 30%.ALD-20K-LMW-UK(conjugate 2) SDS-PAGE electrophoresis detection the results are shown in accompanying drawing 1.
embodiment 3:mono methoxy polyethylene glycol propionic aldehyde-20K(mPEG-ALD-20K) separation and purification of LMW-UK of modifying
(1) chromatography removes unreacted PEG-ALD
Chromatography conditions: SP weak cation exchange post is (purchased from GE company, SPSephroseFastFlow), the acetate buffer (pH5.5) of A liquid: 100mM, B liquid: the acetate buffer (pH5.5) of the 100mM containing 1M sodium-chlor, flow velocity 3ml/min, determined wavelength is 280nm.
Balance: A liquid rinses consistent with A liquid with conductance to effluent liquid pH value.
Loading: the acetic acid of above-mentioned modification reaction thing 0.5M is adjusted to pH5.5, is bonded to SP weak cation exchange post.
Rinse: A liquid+5%B liquid rinses 8 column volumes, collect washing fluid.
(2) chromatography purification ALD-20K-LMW-UK conjugate
Chromatographic condition: after the A liquid+5%B liquid flushing of above-mentioned steps, rinse with the acetate buffer (pH5.5) of the 100mM containing 0.1M sodium-chlor, collect washing fluid, flow velocity is 3ml/min, and determined wavelength is 280nm.
Modified protein after purifying detects purity by SDS-PAGE and RP-HPLC, and SDS-PAGE electrophoresis detection the results are shown in accompanying drawing 1.
embodiment 4:mono methoxy polyethylene glycol propionic aldehyde-20K(mPEG-ALD-20K) purity detecting of LMW-UK of modifying
Efficient liquid phase chromatographic analysis is carried out to the ALD-20K-LMW-UK sterling of collecting after purifying in the reaction mixture of mPEG-ALD-20K and LMW-UK in embodiment 2, embodiment 3.Chromatographic condition is as follows: chromatographic column is AgilentZDRBAX300SB-C18 post (4.6 × 250mm, 5 μm), with containing 0.1%(v/v) the TFA aqueous solution (A moving phase) and containing 0.1%(v/v) acetonitrile (B moving phase) of TFA carries out gradient elution.0 ~ 40min, 20% ~ 50%B, enter next sample again with after back balance 10min.Flow velocity is 0.5ml/min, and determined wavelength is 280nm, and sample size is 40 μ l, and column temperature is room temperature.As shown in Figure 2, result shows that modifying ALD-20K-LWM-UK ratio mono-modified in rear mixture is greater than 50% to detected result; Modified protein after purifying is single absorption peak, and mono-modified product purity is more than 95%.
embodiment 5:mono methoxy polyethylene glycol propionic aldehyde-20K(mPEG-ALD-20K) molecular weight identification of LMW-UK of modifying
5% acetonitrile is dissolved in, 0.22 μm of membrane filtration by after concentrated for the reaction mixture of mPEG-ALD-20K and LMW-UK in embodiment 2, desalination.The saturated solution (solvent is 0.1% formic acid solution) of extracting sample solution and isopyknic alpha-cyano-4-hydroxycinnamic acid mixes, point sample, measures after sample dries up.4700 types are substance assistant laser desorpted ionized-time-of-flight mass spectrometry (MALDI-TOFMS, AB, the U.S.) take linear positive ion operating mode, with the nitrogen-atoms laser excitation sample of 337nm, each sample at least excites 60 times, detect the generation of positive charge ion, measure the molecular weight of ALD-20K-LMW-UK, detected result is shown in shown in accompanying drawing 3.
embodiment 6:mono methoxy polyethylene glycol propionic aldehyde-20K(mPEG-ALD-20K) Bioactivity of LMW-UK modified measures
The enzyme active valency adopting bubble method mPEG-ALD-20K to be modified to the LMW-UK of front and back measures.The mensuration that the preparation of reagent solution and trial-product are tired is undertaken by " Chinese Pharmacopoeia " version in 2010 two urokinase Determination methods, and result is as shown in the formula calculating:
Urokinase is tired=(trial-product units every milliliter units × extension rate)/sampling amount
Detected result shows, and the enzyme active valency of the LMW-UK after mPEG-ALD-20K modifies is compared with the LMW-UK enzyme active valency of unmodified, and obviously do not reduce, activity preservation rate is more than 80%.
embodiment 7:mono methoxy polyethylene glycol propionic aldehyde-20K(mPEG-ALD-20K) pharmacokinetics of LMW-UK of modifying detects
The present embodiment adopts 125i Isotope tracer labelling method is studied Plasma Concentration in the body of the LMW-UK before and after mPEG-ALD-20K modification.Wherein, in order to reduce the absorption of rat thyroid to labeled drug, experiment precontract 8h abdominal injection 1%KI(traditional Chinese medicines group) Tiroidina of the saturated rat of solution 1ml.Rat is divided into LMW-LUK, ALD-20K-LMW-LUK group at random, often organizes 8, male and female half and half.
Concrete operation method is as follows:
(1) radioiodine replaces the hydroxyl in protein molecule on amino-acid residue phenyl ring, obtains having radioactive tracer effect 125the above-mentioned albumen of I.
(2) be numbered according to coding rule with it rat with picric acid, weigh.After fixing with rat fixator, by tail vein injection administration.Rat is decontroled immediately after administration, freely movable, freely drink water and feeding.
(3) get blood respectively at 0.1h, 0.2h, 0.5h, 2h, 4h, 8h, 16h, 24h, 36h, 60h, 72h after administration from eye socket and be about 0.2ml.And add EDTA anti-freezing.
(4) 5000rpm × 3min centrifugation rat plasma, measure plasma radioactivity activity by gamma counter, Measuring Time is 1min.
(5) reclaim blood plasma after activity measurement, HPLC is separated undegradable Proteinogenic medicine.Drug-time curve as shown in Figure 5.
(6) experimental result shows, the pharmacokinetic model of said medicine belongs to two compartment models.MPEG-ALD-20K coupling protein is compared with LMW-LUK, and the transformation period obviously extends, and area under the drug-time curve (AUC) has had and significantly improves.
The present invention adopts mPEG-aldehyde to carry out N-end pointed decoration to low molecular weight urokinase, modified outcome is homogeneous, quality easily controls, Stability Analysis of Structures, activity retain higher, and effectively can extend the transformation period of albumen, keep or strengthen its thrombolytic efficacy, improve its specificity, reduce hemorrhage untoward reaction of Denging, be more suitable for Clinical practice.Can be used for the exploitation of the depot drug product for the treatment of the disease such as hyperfibrinogenemia and prevention of arterial atherosis (AS), thromboembolic complication, diabetic vascular complications, diabetic nephropathy.

Claims (10)

1. a polyethyleneglycol modified low molecular weight urokinase, is characterized in that: be connected with covalent linkage with low molecular weight urokinase by polyoxyethylene glycol, and the N-end of low molecular weight urokinase connects polyoxyethylene glycol by covalent linkage.
2. polyethyleneglycol modified low molecular weight urokinase according to claim 1, is characterized in that: peg molecule connects low molecular weight urokinase molecule subunit by covalent linkage and forms.
3. polyethyleneglycol modified low molecular weight urokinase according to claim 1 and 2, is characterized in that: described polyoxyethylene glycol is mono methoxy polyethylene glycol.
4. polyethyleneglycol modified low molecular weight urokinase according to claim 3, is characterized in that: mono methoxy polyethylene glycol be selected from mono methoxy polyethylene glycol succinimdyl carbonate, mono methoxy polyethylene glycol succinimide carboxylicesters, mono methoxy polyethylene glycol aldehyde one or more.
5. polyethyleneglycol modified low molecular weight urokinase according to claim 4, is characterized in that: mono methoxy polyethylene glycol is mono methoxy polyethylene glycol aldehyde.
6. polyethyleneglycol modified low molecular weight urokinase according to claim 1 and 2, is characterized in that: described molecular weight polyethylene glycol is 5kDa ~ 60kDa.
7. polyethyleneglycol modified low molecular weight urokinase according to claim 1 and 2, is characterized in that: described low molecular weight urokinase is the low molecular weight urokinase of natural extract or the low molecular weight urokinase of restructuring that obtained by genetic engineering technique.
8., according to the preparation method of the polyethyleneglycol modified low molecular weight urokinase of claim 1 to 7 described in any one, it is characterized in that comprising the following steps:
Preparation modification reaction damping fluid, ionic strength is 10 ~ 500mmol/L, and pH value is 4 ~ 9;
The conditioned response that low molecular weight urokinase and activated polyethylene glycol provide in step (1) 2 ~ 48 hours, temperature of reaction is 4 ~ 60 DEG C;
The modified outcome be obtained by reacting through step (2) carries out separation and purification through cation exchange chromatography, and the polyethyleneglycol modified low molecular weight urokinase elution peak described in wash-out collection, obtains described polyethyleneglycol modified low molecular weight urokinase after concentrated.
9. the preparation method of polyethyleneglycol modified low molecular weight urokinase according to claim 8, is characterized in that: modification reaction damping fluid is phosphate buffer soln, borate buffer solution or acetate buffer solution.
10. according to the purposes of the polyethyleneglycol modified low molecular weight urokinase of claim 1 to 7 described in any one, it is characterized in that: for the preparation for the treatment of hyperfibrinogenemia with prevention of arterial is atherosis, the medicine of thromboembolic complication, diabetic vascular complications, diabetic nephropathy.
CN201510499115.4A 2015-08-14 2015-08-14 PEG (polyethylene glycol)-modified LMW-UK (low-molecular-weight urokinase) and preparation method and application thereof Pending CN105087530A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108226363A (en) * 2017-12-04 2018-06-29 赵欣雨 It is a kind of using HPLC detect urokinase molecular components than method
WO2022095973A1 (en) * 2020-11-05 2022-05-12 杭州远大生物制药有限公司 Urate oxidase preparation and use thereof
CN114569707A (en) * 2020-11-30 2022-06-03 南京理工大学 Application of adenosine deaminase or modified substance thereof in preparation of diabetes treatment drug

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
N.SAKURAGAWA等: "Studies on the effect of PEG-modified urokinase on coagulation-fibrinolysis using beagles", 《THROMBOSIS RESEARCH》 *
叶建新等: "聚乙二醇修饰对尿激酶原性质的影响", 《军事医学》 *
孙天霄等: "高分子量和低分子量尿激酶的分离纯化及动力学性质研究", 《生物化学杂志》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108226363A (en) * 2017-12-04 2018-06-29 赵欣雨 It is a kind of using HPLC detect urokinase molecular components than method
WO2022095973A1 (en) * 2020-11-05 2022-05-12 杭州远大生物制药有限公司 Urate oxidase preparation and use thereof
CN114569707A (en) * 2020-11-30 2022-06-03 南京理工大学 Application of adenosine deaminase or modified substance thereof in preparation of diabetes treatment drug
CN114569707B (en) * 2020-11-30 2024-05-07 南京理工大学 Application of adenosine deaminase or modification thereof in preparation of diabetes therapeutic drugs

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Application publication date: 20151125