CN1240364A - Platelet substitutes and conjugation methods suitable for their preparation - Google Patents
Platelet substitutes and conjugation methods suitable for their preparation Download PDFInfo
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- CN1240364A CN1240364A CN97180739A CN97180739A CN1240364A CN 1240364 A CN1240364 A CN 1240364A CN 97180739 A CN97180739 A CN 97180739A CN 97180739 A CN97180739 A CN 97180739A CN 1240364 A CN1240364 A CN 1240364A
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Abstract
Platelet substitutes, comprising fibrinogen, or analogous products useful in therapy, which further comprise an insoluble carrier to which is bound an essentially non-degraded active protein including the sequence RGD. Such conjugates can be made by a conjugation process comprising 0.01 to 2.5% by weight active fibrinogen, and no more than 50% inactive fibrinogen.
Description
Invention field
The present invention relates to platelet substitutes, promptly contain fibrinogenic compositions, and relate to particularly Fibrinogen in conjunction with the conjugation methods on the row specific carrier.
Background of invention
The covalently bound conjugate that contains active medicine and carrier can be used as the means that medicine are transported to for example specific action site.Existing people proposes albumin as this carrier.WO-A-9618388 discloses albumin microsphere granule, its preparation method and as the purposes of carrier.
Macromole and albumen link the many problems of meeting generation to the covalency of human serum albumin's microcapsule (HSA).Because contacting of albumen and surface of microcapsule is bad, therefore can not obtains enough binding sites and carry out crosslinked.And when using cross-linking agent short or the distance of zero mark degree for example when glycolaldehyde or EDC, intramolecular crosslinking is than the difficult control of intermolecular cross-linking.This can make the useful load of microcapsule very low and can make the albumen inactivation.
Agam and Livne propose in a series of paper Blood 55:186-191 (1983), Thromb.Haemostasis 51:145-9 (1984) and 59:504-6 (1988) and Eur.J.Clin.Invest. (1991), be coated on the gathering of the Fibrinogen meeting platelet increasing on the fixed platelet, in suffering from the rat of thrombocytopenia, be surrounded by fibrinogenic erythrocyte the bleeding time is shortened.Fixation comprises the method for using formaldehyde.
People such as Coller have described " blood coagulation erythrocyte " in J.Clin.Invest.89:546-555 (1992), it is a kind of autologous, semiartificial platelet transfusion substitute.For fear of limitation and the defective of using fresh platelet to bring, use the bifunctional cross-linker that erythrocyte is combined on the peptide that contains the RGD cell identification sequence.
(11-16 day June nineteen ninety-five is at Jerusalem in the XV time meeting of thrombosis and hemostasis international association, Israel holds) summary in, Yen etc. have reported " the blood coagulation ball " that potential hemostatic capability is arranged, it is crosslinked HSA microsphere, average diameter is 1.1-1.3 μ m, and wherein human fibrinogen's covalent bond in its surface.In thrombocytopenia rabbit model, the ear bleeding time has shortened.It is reported that this HSA microsphere is to prepare with the method among the US-A-5069936, promptly use the dissolving/desolvation process of glutaraldehyde, use ethanol to precipitate, add the surface that surfactant is modified the crosslinking protein molecule as cross-linking agent.These steps can not be controlled the size of microsphere, may make conjugated protein degraded, and are unsuitable for producing in enormous quantities platelet substitutes.
US-A-5069936 discloses the covalent bond of different biological molecules, but does not comprise Fibrinogen.It proposes with polyacetals as covalent crosslinking agent.Embodiment 12 and 14 comes binding antibody and enzyme (alkali phosphatase) with glutaraldehyde respectively.
WO-A-9639128 (open at December 12 in 1996) also discloses " blood coagulation ball ".But do not provide concrete preparation example.
Fibrinogen is the cohesiveness glycoprotein that contains RGD (S) sequence.It can mediate cancerous cell with other glycoprotein (comprising fibronectin and collagen) and be attached on the subendothelial layer.These glycoproteins and integrin of in cancerous cell, finding such as the GPIIb/IIIa acceptor interaction on fibronectin receptor and the platelet; Referring to Dardik et al, Int.J.Cancer 70:201-7 (1997).
To a undergo surgery subject matter of treatment of cancer is to increase the danger of cancerous cell to the blood circulation diffusion.This is the patient that suffers from carcinoma of prostate one of the reason that the sequela rate increases of performing the operation.People wish to eliminate the transfer of cancerous cell in blood circulation, or suppress its deposition in blood vessel surface.
The invention summary
According to an aspect of the present invention, a kind of peptide (representing any peptide, polypeptide, albumen or its conjugate) is allowed to insert sept (for example little peptide or fatty acid) between albumen and the microcapsule as the new method that Fibrinogen is attached on the microcapsule.More particularly, the present invention has utilized this fact, and promptly carrier such as HSA contain free sulfhydryl groups, difunctional compound can with its reaction, wherein difunctional compound contain can with the group of wanting bonded active component (medicine) selective reaction.
According to the present invention, can make cross-linking reaction controlled, this is because the linking group of the free sulfhydryl groups on carrier such as the HSA has specificity.Controllable cross-linking reaction is an importance of the present invention, because its active directly related with binding molecule.
According to the needs of using, sept can comprise can be by the peptide of enzymatic lysis, be easy to by the key of acid or alkaline lysis, and has different length.The length of sept can be another importance of the present invention, because it can determine and the binding ability of target receptor that for example Fibrinogen is in conjunction with the binding ability of GPIIb/IIIa.
According to another aspect of the present invention, for example, provide a kind of new pharmaceutically acceptable product that can be used as platelet substitutes by using this new method.That this product contains is bonded with Fibrinogen, do not make the insoluble carrier of fibrinogenic loss of activity basically, the albumin that for example is stabilized.In conjunction with being non-chemical bond, for example combination by Absorption, or chemical bond, the combination of for example using the connector that grows to few 10nm to carry out.
That the present invention provides for the first time is pure, stable, treat acceptable platelet substitutes.Purification can be realized under the condition that does not have chemical cross-linking agent and/or surfactant.These platelet substitutes are suitable for treating thrombocytopenia.
Another characteristics of the present invention are that product of the present invention can be used in conjunction with other active substance because Fibrinogen is used as target reagent.The selection of this class material and site of action (normally wound or other bleeding part) and the character of clear and definite problem relevant.Invention is described
The carrier that the present invention uses preferably prepares by spray drying under the condition that particle diameter and particle size distribution are well controlled.For example, can pass blood capillary in order to make granule, preferred particle diameter is 6 μ m, for example 1-4 μ m to the maximum.
WO-A-9218164, WO-A-9615814 and WO-A-9618388 full disclosure suitable material and preparation method, and stablize the method for microparticle by heating or chemical crosslinking, these patents are incorporated by reference in the present invention.As being explained in the publication of back, described condition does not influence functional group, for example albuminous sulfydryl, so functional group is still keeping the ability with biomolecular reaction.
The microparticle that the present invention uses can have the physics characteristic of describing in above-mentioned two pieces of publications, for example be slick and spheric, and contain air.In order to obtain insoluble, crosslinked microcapsule, can be with spray drying product and chemical cross-linking agent reaction.Yet heating and γ-radiation are preferred, the dry powder product can also be carried out sterilization treatment.
In an embodiment of the invention, can under the condition of connector Fibrinogen (or other RGD peptide) be attached on the carrier not having, for example pass through Absorption.This can be by realizing on the surface of peptide being precipitated the row microsphere that for example by control pH value and other condition, this is conspicuous for those of ordinary skills.Then excessive/unconjugated Fibrinogen is washed off.
In yet another embodiment of the present invention, use conventional difunctionality reagent such as polyacetals that Fibrinogen is attached on the carrier.Glycolaldehyde is preferred.Another example of sept is 4-(iodoacetyl) amino benzoic Acid sulfo group succimide ester (water-insoluble).Its length is about 1nm.
Aspect first in described integrating step, binding peptide preferably comprises Fibrinogen at least of the present invention.Other example has blood coagulation factor VIII, plasma thromboplastin component, other blood factor, coagulation cascade reaction (coagulation cascade) albumen, thrombolytic agent, antibody, α-1-antitrypsin.Unite use by for example Fibrinogen and blood coagulation factor VIII, product of the present invention can be treated hemophilia effectively.In addition, or thing replaces thrombolytic medicine such as urokinase as an alternative, with the ultrasonic blood clot of handling.For this processing, the microcapsule that contains air among the present invention is specially suitable.
The difunctional compound that uses among the present invention is (as Y
1-Y-Y
2) can pass through better simply chemical compound Y
1-Y
3-Y
4With Y
5-Y
6-Y
2Reaction obtains, wherein Y
1Be sulfydryl-specific reaction group, Y
4And Y
5One reacts makes Y
3And Y
6Form spacer groups Y together, Y
2It is the drug reaction group.Therefore, for example at ICH
2Among the COOH, Y
1Be sulfydryl reaction I of group and/or Y
4Be COOH.More specifically, add EDC (can add N-maloyl imines and promote the catalytic amidation process of EDC, reaction generates active succimide ester) and can make the iodoacetic acid activation.Then with the material and the peptide that are activated or contain free amine group (Y
5) and terminal carboxyl group (Y
2) molecule incubation together.Suitable peptide contains just like 3-6 aminoacid such as glycine or alanine.
Use the carboxyl on the EDC activated intermediate conjugate then.Activatory sept is cultivated with albumen, and then the lysine amino on carboxyl on the sept and the albumen side chain has formed a peptide bond.Have only an end to can be incorporated on the albumen on the sept, therefore, cross-linking reaction does not take place this moment.Most of serum albumin do not contain free sulfhydryl groups, but HSA is an exception, has therefore avoided crosslinked in the generation protein molecular.
For example, sept contain can be optionally with the cysteine that is positioned at the HSA microcapsule-34 residue on the terminal iodoacetyl of the N-functional group of free sulfhydryl groups reaction.Sept can also contain can be by 1-ethyl-3 for example, 3-dimethylamino-propyl carbodiimide (EDC) activation and can be connected in free carboxy on the amino of peptide.
Activate that sept can be 6 at pH, carry out in the 0.05M 4-morpholino ethyl sulfonic acid buffer (MES buffer) with EDC.For fear of any fibrinogenic Absorption takes place,, immediately buffer is changed into pH and is 8 0.1M sodium borate buffer liquid in case Fibrinogen reacts with activatory N-iodoacetyl peptide such as Gly-Leu-Phe.Under this higher pH fibrinogenic Absorption can not take place.Fibrinogen and HSA microcapsule any combine all be because, the result that the carbon atom that iodine molecule adjoined on the free sulfhydryl groups of HSA microcapsule and the sept N-end reacts.The interactional optimal pH of sulfydryl-iodine is 8.
For fortifying fibre proteinogen (for example) through the combining of sept and microcapsule, can use Traut reagent (2-imino group Tetramethylene sulfide) to increase the content of microcapsule free sulfhydryl groups.This reagent becomes the epsilon-amino on the lysine into sulfydryl, makes can combine with sept and so increased with the quantity of the bonded free sulfhydryl groups of Fibrinogen.
By with want bonded protein binding before, the aminoacid or the peptide (for example cysteine, reductive glutathion) that will contain sulfydryl are crosslinked on microcapsule, can increase the carrying capacity of microcapsule.Can use chemical cross-linking agent such as imino group Tetramethylene sulfide (imiothiolane) to introduce and dredge base (with increasing its quantity).If be introduced into sulfydryl on other protein surface, also can prepare microcapsule with them, with as utilize its have HSA the substitute of inherent characteristic (being free sulfhydryl groups).
With albumen and sept with the HSA microcapsule incubation that contains free sulfhydryl groups.In WO-A-9615184 and WO-A-9618388, described by spray drying and prepared the method that this functional group does not have the microcapsule of loss.
It is crosslinked with microcapsule that mol ratio between sept and the albumen answers Gao Lieneng that enough groups are attached on the albumen, but also should be low to moderate the degree that does not make the albumen inactivation.Be prepared into sept ICH with this concrete method
2CO (A)
nCOOH, wherein (A)
nRepresent n identical or different amino acid whose residue.According to concrete application, the length of selecting n to come the control interval thing, 10-600nm (1-60 dust) for example, normally 50nm at least.
Above-mentioned preparation process can be carried out in the solvent system that mainly is based on water.The by-product that reaction generates can easily be removed.
Crosslinking technological should controllably be connected in peptide and albumen on the microcapsule, keeps proteic activity preferably, and the length and the division degree of energy control interval thing.
As mentioned above, containing fibrinogenic product among the present invention can work at tumor sites.Therefore, go up cytotoxic agent by for example connecting with the ad hoc approach among the present invention or as the described method of WO-A-9618388, these products can be used for treating tumor.Suitable cytotoxic agent comprises methotrexate, amycin, cisplatin or 5-fluoro-2 '-BrdU.
Be used for directly with cytosis or participate in gathering and the deposition of fibrin in the cell attachment site by product of the present invention is made as carrier, medicine can carry out the target attack to tumor cell.
Product of the present invention can be used for load cytotoxic agent or combined loading cytotoxic agent and targeting agent.Be used for that also the tumor cell that is dispersed in blood circulation is carried out target and attack, this is to carry out special interaction (seek and destroy) or realize by participating in the platelet aggregation process of carrying out on the attachment site by the glycoprotein receptor with cell.In both cases, cytotoxic drug all concentrates on the site of attacking tumor cell.
Or by product of the present invention is wrapped in surface of tumor cells, and blocking-up activates hematoblastic site/mechanism, also can suppress tumor cell in blood circulation or even the gathering on attachment site.Therefore this makes the natural defending system of human body can help to eliminate tumor cell.
Also can transmit and contain GPIb receptor (with Von willebrands factor interaction) for example or collagen or the product of the receptor of subcutaneous matrix components in other, with by interior subcutaneous substrate is covered the binding site of blocking tumor cell potentially.Product of the present invention should remain on itself and platelet are interacted, and invades blood vessel wall but also should limit any tumor cell that is fixed.
A significant advantage of the present invention is that the activity of Fibrinogen (or other RGD peptide) can keep substantially.Can measure to determine the content of activated fibre proteinogen by the ELISA of fibrinopeptide A (FPA).
In the analysis of FPA, with sample (or standard sample) incubation, the result has generated antigen-antibody complex with the excessive fibrinopeptide A antibody of constant basis.The amount of FPA is inversely proportional in the concentration of remaining excessive antibodies and the sample (or standard sample).
In order to measure the concentration of antibody,, carry out incubation subsequently with waiting culture of branch fraction to transfer in the reactor that scribbles excessive FPA.Wall-bonded the antigen-antibody complex that obtains with formed sandwich complex with the resisting of peroxidase labelling-lgG antibody.Can directly determine the concentration of FPA in the sample with the amount of these complex.
By peroxidase and H
2O
2The enzyme reaction of/o-phenylenediamine (chromogen) and the spectrophotometry that carries out at 492nm subsequently can be measured the amount of the sandwich complex of acquisition.Because the activity and the antigen concentration of bonded enzyme are inversely proportional to, therefore when the concentration of FPA in the sample increased, measured absorbance will reduce.Set up one with reference to curve with the known standard sample of concentration, measurement result is carried out evaluation.
Platelet substitutes of the present invention contains at least 0.01%, preferred at least 0.015%, more preferably at least 0.02%, most preferably at least 0.025% activated fibre proteinogen usually.For fear of gathering, fibrinogenic content can not be too high, for example up to 1,1.5,2 or 2.5%.For the Fibrinogen that is contained, should have at least 50%, preferably at least 70%, more preferably at least 90% to be activated.The content of activated fibre proteinogen can be measured according to fibrinogenic total content, and the available method as ELISA of fibrinogenic total content is measured.Fibrinogenic total content also can be by for example using with radio-labeled
125I measures, and calculates with conventional method.
Fibrinogen can be to be derived from blood, genetically modified or reorganization, its total length or any activated fragment.Its active fragment is disclosed, and is particularly open by above-mentioned people such as Coller.
As therapeutic agent, product of the present invention can mix to come administration with itself or with the suitable carriers known to those of ordinary skills.The amount of product administration mainly is to determine according to the order of severity of wound or other disease that will treat.Dosage generally is 1.5 * 10
9Individual microcapsule/kg body weight.
The following examples are to illustrate of the present invention.
The Fibrinogen that uses among the embodiment is total length, be derived from blood, commercially available, the product that carried out twice inactivation of virus.
The HSA microcapsule that uses among the embodiment prepares by spray drying, and makes it stable by heating subsequently, (as described in WO-A-9615814).Before use, microcapsule is soaked into 1% Tween 80, then with the PFPW thorough washing to remove Tween 80 and excipient.
PFPW=does not contain the purified water of pyrogen.
DTNB=5,5-dithio two (2-nitrobenzoic acid).
Carry out Ellman with DTNB and analyze the content of measuring free sulfhydryl groups.This reagent participates in the sulfydryl exchange process of free sulfydryl in the test proteins, and discharges the material (TNB) that can carry out the UV/VIS spectrophotometry at the 412nm place.Embodiment 1
In the mixed liquor of methanol and distilled water, reaction is 1 hour under iodoacetic acid N-hydroxy-succinamide ester (IAAE) and four alanine (AAAA) room temperature.Contrast with the AAAA in the distilled water, add EDC with 1.2 moles ratios, the time is 5 minutes.Add the Fibrinogen that is suspended in again in the distilled water then.Stirring at room 1 hour adds microcapsule, and mixture at room temperature continued to stir 16 hours.
In order to remove all unreacted Fibrinogen and septs, distilled water wash microcapsule totally 6 times of reaction back are suspended in microcapsule in the distilled water again, and the final microcapsule concentration that obtains is 100mg/ml.
(20mg) compares with microcapsule, and the consumption that calculates Fibrinogen (54mg) is 0.5 molar equivalent.This be considered to be in the microcapsule free sulfhydryl content possible obtain maximum loading.
ELISA result shows: every 100mgHSA is loaded with Fibrinogen 0.5mg.The slide test that carries out with the microcapsule and the 0.15 unit thrombin of 5mg labelling obtains positive findings.Assemble immediately after adding thrombin.
Under the situation that does not have IAAE, AAAA or EDC to exist, microcapsule and the Fibrinogen of also using same amount have carried out controlled trial.The ELISA result of this sample shows: be loaded with Fibrinogen 0.06mg among every 100mg HSA.This is to take place under the situation of not using cross-linking agent.This sample also provides male slide test result, but just occurs gathering after 12 seconds until add thrombin.Embodiment 2
Repeat the step among the embodiment 1, but, then use reversed-phase HPLC in order to optimize the reaction of IAAE and AAAA.For this reason, be necessary research needed AAAA amount when all IAAE are transformed into product.Unwanted side reaction during all unreacted IAAE may participate in and further synthesize.Therefore, the AAAA of reaction amount is increased to 4 molar equivalents from 1, and keeps the consumption of IAAE constant.Embodiment 3
Repeat the step of embodiment 1, but in order to study the needed sept amount of specified rate Fibrinogen, the ratio of using IAAE and AAAA is 1: 4, and uses excessive EDC with respect to 1.2 molar equivalents of AAAA.The molal quantity of using the IAAE that exists calculates used sept consumption, because IAAE is the active constituent and the limiting factor (s) of preparation sept.Like this, IAAE-AAAA-EDC and fibrinogenic ratio were increased to 1: 5 from 1: 1.
ELISA result shows: when the ratio of Fibrinogen and IAAE-AAAA-EDC is 1: 2, can obtain the Fibrinogen of peak load renewablely.Calculate and be loaded with 0.06 gram Fibrinogen among every 100mgHSA, sample was assembled in 5 seconds when using slide test.Embodiment 4
In further optimization experiment, repeat embodiment 1 but reduce fibrinogenic amount.Changing the ratio of IAAE-AAAA-(EDC)-Fibrinogen and microcapsule, is respectively 0.5,0.3,0.1,0.05 and equivalent.
ELISA result shows: except that 1: 0.01 ratio, all samples has acceptable Fibrinogen useful load.The excursion of sample is among every 100mg HSA the 0.1-0.2mg Fibrinogen to be arranged, and experiment shows: when reducing Fibrinogen to 0.05 molar equivalent only, must be listed as the loading value up to 0.5 molar equivalent.Embodiment 5
Because embodiment 4 resulting results are very unified, have therefore carried out two experiments.Table 1 shows: the consumption that the research Fibrinogen loads in first experiment, and used material proportion is as follows:
IAAE∶AAAA(1∶4)
IAAE-AAAA-EDC: Fibrinogen (2: 1)
IAAE-AAAA-(EDC)-Fibrinogen: microcapsule (0.3: 1)
Table 2 is summarized needed amount in second experiment, and this is tested each material consumption and tests with first, but has done following change:
IAAE-AAAA-(EDC)-Fibrinogen: microcapsule (0.05: 1)
Table 1
| Reagent | Consumption | The nanomole number |
| ????IAAE | ????52μg | ????182 |
| ????AAAA | ????210μg | ????695 |
| ????EDC | ????160μg | ????834 |
| Fibrinogen | ????31mg | ????91 |
| Microcapsule | ????20mg | ????303 |
Table 2
| Reagent | Consumption | The nanomole number |
| ????IAAE | ????8.6μg | ????30.4 |
| ????AAAA | ????35μg | ????115.8 |
| ????EDC | ????26.6μg | ????139 |
| Fibrinogen | ????5.2mg | ????15.2 |
| Microcapsule | ????20mg | ????303 |
In these two experiments, in the buffer of the 0.1M of pH8 sodium phosphate and 0.15M sodium chloride, reaction is 16 hours under microcapsule and the sept room temperature.Use PFPW thorough washing microcapsule then, microcapsule is suspended again obtain the final microcapsule that concentration is 100mg/ml.
Ratio is that the ELISA result that 0.3 and 0.05 sample obtains shows: in every 100mgHSA, they are loaded with 0.38 and the 0.42mg Fibrinogen respectively.The slide test result of two kinds of samples all provides positive findings, and assembles after about two minutes at the adding thrombin.Embodiment 6
Preparation 1mg/ml four alanine.Take by weighing 3mg four alanine (Sigma) and join in the little glass bottle of nut of 7ml, dissolve in 3ml PAPW, the vortex vibration mixes.
Preparation 3mg/ml iodoacetic acid N-hydroxyl succinimido ester (IAAE).Take by weighing 3mgIAAE (Sigma) and join in the little glass bottle of 7ml nut, dissolve in 1ml methanol, the vortex vibration mixes.
70 μ l, four alanine are pipetted in the little glass bottle of 7ml nut, 5.7 μ l IAAE are being moved into wherein, the vortex vibration mixes.The mol ratio of IAAE and four alanine is 1: 4.
With glucose preparation 2.3g microcapsule.Mixture is made up of about 800mg protein and 1600mg glucose.Preparation concentration is the protein of 50mg/ml in the Tween 80 (Sigma) of 1% (v/v).
The vibration of foregoing thing vortex mixes, and leaves standstill under the room temperature about 30 minutes, and microcapsule is sunk.Get 400 μ l aliquot and join among the Eppendorf, the vortex vibration mixes.In BeckmannGS-15, make this sample with centrifugal 2 minutes of the speed of 3000rpm (relative centrifugal force(RCF) RCF=1502 radian per second) under the room temperature.PFPW with 1ml washs 3 times then.The shot-like particle room temperature preservation that obtains during to needs till.
Again be mixed with a bottle human fibrinogen in 20ml 0.1M normal saline/0.025M sodium phosphate buffer (pH7.2), producing fibrinogenic theoretical concentration is 60mg/ml.The PEG solution of buffered 35% (w/v) of adding 4ml is made the fibrinogen solution with respect to 5.8% (w/v) of PEG1000.Only mix the solution that obtains, on ice cube, cooled off 15-20 minute then by reversing.The centrifugal precipitate that obtains (4000rpm, 4 minutes, BeckmanGS15).Remove supernatant and measure its volume.
Add 7% (w/v) PEG/0.1M normal saline/0.025M sodium phosphate buffer (pH7.2) washing shot-like particle.Use this shot-like particle of the former washing of initial fibrillin of half volume.This shot-like particle is suspended in the buffer again, and mixes with the cuvette agitator.The solution that obtains centrifugal 4 minutes with the speed of 4000rpm.From the shot-like particle of washing, remove supernatant and measurement volumes.In 20ml 0.025M sodium phosphate buffer/0.1M normal saline (pH7.2), reformulate shot-like particle.Measure the fibrinogen solution volume of purification.Use BCA and analyze definite total protein concentration.Read uv absorption at the 280nm place and calculate fibrinogenic concentration.Because the extinction coefficient of fibrinogen solution at wavelength 280nm place of known 1% (w/v) are 15.5, therefore can calculate fibrinogenic concentration.(referring to Haemostasis and Thrombosis the 1st volume, the 3rd edition, 492 pages, R.F.Doolittle).
In PFPW, prepare fresh 1mg/ml EDC.Take by weighing 3mg EDC (Sigma) and join in the little glass bottle of 7ml nut, dissolve in 3ml PFPW again.The vortex vibration mixes.From blender, remove and contain the little glass bottle of nut of reacting space thing.Getting 53 μ l EDC joins in the reactant mixture.The mol ratio of EDC and four alanine is 1: 1.2.The vortex vibration mixed about 5 minutes.
Add the 10.3mg Fibrinogen, in 258 μ l with 40mg/ml.The mol ratio of Fibrinogen and HSA microcapsule is 0.1: 1.IAAE and fibrinogenic mol ratio are 2: 1.Reverse the little glass bottle of this nut and mixing about 60 minutes.
The microcapsule that 20mg was washed is suspended in the buffer again, and promptly pH is in 0.1M sodium hydrogen phosphate/0.15M sodium chloride buffer of 8.Hydrochloric acid with 12N is adjusted pH value.In microcapsule, add 613 μ l buffer.Again it is joined in the 387 μ l reactant mixtures.Obtaining final volume like this is 1ml.Stir under the room temperature and spend the night.This reacted about 16 hours.
From agitator disk, remove the little glass bottle of nut.With centrifugal 1 minute of the speed of 4500rpm (RCF=3379 radian per second).Remove supernatant and carry out elisa assay.
Washing pellet is totally 6 times in 1ml PFPW.Preserving cleaning mixture simultaneously makes ELISA determine weight balancing.Shot-like particle is suspended among the 200 μ l PFPW again.Be ELISA (100 μ l) and (the 100 μ l) sampling of slide test mensuration.
Outturn sample is preserved down at 4 ℃.Embodiment 7
Prepare the microcapsule of 1 bottle allotment, it contains have an appointment 1g protein and 2g mannitol.Postradiation bottle stores down at 4 ℃.The protein of preparation 50mg/ml concentration in 1% (v/v) Tween 80.The vortex vibration mixes this content, leaves standstill under the room temperature about 30 minutes, is settled out capsulae vacuus.
For the 1g preparation, branch things such as 20ml after mixing, are joined in the 50mlBeckmann centrifuge tube in the vortex vibration.Under the room temperature with the speed centrifugal 3 minutes (relative centrifugal force(RCF) RCF=4648 radian per second) on Beckman Avanti J-25 of 5000rpm.PFPW with 20ml washs 2 times then, simultaneously at room temperature with centrifugal 2 minutes of the speed of 3300rpm (RCF=2025 radian per second).
Use 20ml 10mM phosphate buffer (pH=6.0) washing microcapsule at last, under the room temperature centrifugal 2 minutes with the speed of 3300rpm.This shot-like particle is suspended in the 10mM phosphate buffer (pH6.0) of 20ml again, transfers to then in the 70ml Sterilin container that has magnetic stirrer.
The theoretical fibrinogen concentration of regenerated fiber proteinogen generation is 40mg/ml in WFI.Stirred suspension 20 minutes gently in cylinder mixer.The excess fibre proteinogen is sharply freezing and storage (20 ℃) in low temperature nalgene bottle.
For the 1g preparation, under agitation Fibrinogen (0.25ml, concentration 40mg/ml) is joined in the microcapsule.The mol ratio of Fibrinogen and HSA microcapsule is 0.002: 1.Mixture stir about 4 hours.
From agitator disk, remove container, content is transferred in the Beckmann pipe of 50ml.Under the room temperature with centrifugal 2 minutes of the speed of 3300rpm (RCF=2025 radian per second).Abandoning supernatant.The washing shot-like particle is 3 times in the WFI of 20ml.Discard cleaning mixture.Using WFI makes this shot-like particle be suspended into 10ml again.Provide 500 μ l samples to be used for ELISA, slide test mensuration and Ku Erte granulometry.
When finishing the Ku Erte granulometry, adding former mannitol (153mg/ml) and former phosphate buffer (250mM) preparation sample, reach to wait and ooze mannitol (51mg/ml), 25mM phosphate buffer (pH7.0 ± 0.2) and microcapsule number be 1,500 million/milliliter.Embodiment 8
HSA microcapsule (50mg, 0.757 the μ mole) be suspended in again in the 0.1M sodium borate buffer liquid (832 μ l) of pH8.05, add the 2-imino group Tetramethylene sulfide (520 μ g, 3.78 μ mol) in the 0.1M sodium borate buffer liquid (168 μ l) that is dissolved in pH8.05 again.Reaction stirred is 1 hour under the room temperature, uses distilled water wash microcapsule (5 * 5ml) then.At last with the washing of the 0.1M sodium borate buffer liquid of pH8.05, and be suspended in (2ml) in the same buffer again.Contrast
Fibrinogen (20mg, 0.058 μ mol) is suspended in the MES buffer (1.2ml) of pH6.03,0.05M again, stirs 2 hours under the room temperature.In pH8.05 (2ml), 0.1M sodium borate buffer liquid (2ml), add HSA microcapsule (50mg, 0.757 μ mol).At room temperature continued stirring reaction 2 hours.Embodiment
N-iodacetyl Gly-Leu-Phe (3mg, 5.95 μ mol) is suspended among the MES (1.2ml) of pH6.03,0.05M again, adds EDC (2mg, 10.4 μ mol), under the room temperature stirring reaction 5-7 minute.Add Fibrinogen (20mg, 0.058 μ mol), stirred the mixture under the room temperature 2 hours.Add the pH8.05, the 0.1M sodium borate buffer liquid (2ml) that contain HSA microcapsule (50mg, 0.757 μ mol), at room temperature continued stirring reaction 2 hours.
From each contrast and experiment reaction, remove sample and be used for free thiol mensuration.(2 * 5ml) remaining contrasts of washing and experiment material, the concentration that reaches the HSA microcapsule that suspends again then is 100mg/ml with distilled water.
In this embodiment, the content of free thiol increases in the HSA microcapsule, and the SH that every nanomole HSA contains is increased to 1.73 nanomoles from 0.211 nanomole.Do not see that control sample (not having sept) activity has any influence.Yet modified microcapsule and during with the reaction of the Fibrinogen of sept co-cultivation has obtained active sample by slide test.The slide test activity is increased to 5-15 second, and the HSA microcapsule has more high-load free thiol.Fortune shows: the content that increases free HSA microcapsule free thiol causes the activity of final products to increase, and this is therefore to reach such Fibrinogen owing to increase the combination of sept.Embodiment 9
EDC (69.8:1,0.5mg/ml aqueous solution) joins N-iodoacetyl tetraglycine (125.5:1,0.5mg/ml aqueous solution), stirs this mixture 5 minutes under the room temperature.Add Fibrinogen (186:1,55.5mg/ml solution), reaction was at room temperature stirred 1 hour.
Be loaded with the microcapsule of amycin, in every mole of HSA, contain 0.56 mole of medicine (100mg), be suspended in again in the 1.619ml water, it is joined in the activatory fibrinogen solution.Stirred the mixture under the room temperature 16 hours.
With the speed of 3500rpm centrifugal 2 hours, abandoning supernatant was collected microcapsule.Microcapsule wash with water (4 * 5ml), be suspended in again in the lml water, obtain the final about 100mg/ml of HSA concentration.
The activity of working sample finds that fibrinogenic adding does not cause the loss of any amycin in the microcapsule.And the microcapsule of this dual loading shows the activity of thrombin, and this shows: although the existence of amycin, it is active that bonded Fibrinogen also keeps.Use ELISA and measure the fibrinogen level that exists.These results show the Fibrinogen of reasonable loading.
Claims (26)
1. the pharmaceutically acceptable product that contains insoluble carrier, wherein insoluble carrier are combined with and contain RGD sequence and undegradable substantially activated protein.
2. according to the product of claim 1, wherein in conjunction with being non-chemical bond, for example combination by Absorption.
3. according to the product of claim 1, wherein in conjunction with being not have under the situation of surfactant, the covalent bond of being undertaken by chemical connector.
4. according to the product of claim 1, wherein combination is the covalent bond of being undertaken by the chemical connector that grows to few 10nm.
5. according to the product of aforementioned each claim, wherein carrier comprises crosslinked protein particle.
6. according to the product of claim 15, wherein microparticle albumen is albumin.
7. according to the product of aforementioned each claim, wherein RGD albumen is Fibrinogen.
8. according to the product of claim 7, wherein contain the activated fibre proteinogen of 0.01-2.5% weight and be no more than 50% nonactive Fibrinogen.
9. the method for preparation formula X-S-Y-Z covalent conjunct agent, wherein X-SH is the carrier that free sulfhydryl groups is arranged, and Y is a sept, and Z is an active component, and reaction may further comprise the steps:
With active component and formula Y
1-Y-Y
2Difunctionality agent reaction, wherein Y
1Be can with the group of thin based selective reaction, Y
2Be can be with active component but can not with the group of sulfydryl reaction: and
With the Y that obtains
1-Y-Z and carrier reaction.
10. according to the method for claim 9, Y wherein
1Be I.
11. according to the method for claim 9 or 10, wherein Y
2Be COOH.
12. according to the method for claim 10, wherein the difunctionality agent is by sept and the iodoacetic acid reaction that can be activated are obtained.
13. according to each method of claim 9-12, wherein sept comprises fatty acid and peptide chain.
14. according to each method of claim 9-13, wherein active component has NH
2Base.
15. according to each method of claim 9-14, the wherein long 10-600nm of sept.
16. according to each method of claim 9-15, wherein carrier is that form with microparticle exists.
17. according to each method of claim 9-15, wherein active component is the albumen that includes the RGDS sequence.
18. according to the method for claim 17, wherein RGD albumen is Fibrinogen.
19. according to each method of claim 9-18, wherein carrier is an albumin.
20. according to the method for claim 19, wherein carrier is the human serum albumin.
21. by its sulfydryl and the bonded albumin of active component, use therein sept is to the youthful and the elderly 50nm.
22. according to the albumin of claim 21, wherein it is to use the method according to claim 18 or 20 to prepare.
23. according to the albumin of claim 21 or 22, wherein it is that form with microparticle exists.
24. according to each albumin of claim 20-22, wherein active component is a Fibrinogen, as platelet substitutes.
25. according to the albumin of claim 24, wherein Fibrinogen such as claim 8 definition.
26. according to each product of claim 1-8 and 21-25, wherein also contain bonded cytotoxic agent, for example methotrexate, amycin, cisplatin or 5-fluoro-2 '-BrdU.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97180739A CN1240364A (en) | 1996-10-21 | 1997-10-17 | Platelet substitutes and conjugation methods suitable for their preparation |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9621886.2 | 1996-10-21 | ||
| GB9702652.0 | 1997-02-10 | ||
| CN97180739A CN1240364A (en) | 1996-10-21 | 1997-10-17 | Platelet substitutes and conjugation methods suitable for their preparation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1240364A true CN1240364A (en) | 2000-01-05 |
Family
ID=5177843
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN97180739A Pending CN1240364A (en) | 1996-10-21 | 1997-10-17 | Platelet substitutes and conjugation methods suitable for their preparation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1240364A (en) |
-
1997
- 1997-10-17 CN CN97180739A patent/CN1240364A/en active Pending
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