CN101272790A - Stable protease composition - Google Patents
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- CN101272790A CN101272790A CNA2005800516550A CN200580051655A CN101272790A CN 101272790 A CN101272790 A CN 101272790A CN A2005800516550 A CNA2005800516550 A CN A2005800516550A CN 200580051655 A CN200580051655 A CN 200580051655A CN 101272790 A CN101272790 A CN 101272790A
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Abstract
A composition is provided, which comprises a serine protease; a reversible inhibitor of said serine protease; and a stabilizing agent M having the formula (I): Also provided are uses of the composition as a medicament, and other uses and methods employing its various properties.
Description
Invention field
The present invention relates to a kind of enzymatic compositions, wherein stablize this enzyme by some additives in compositions of the present invention.More specifically, the present invention relates to a kind of serine stretch protein enzymatic compositions, comprise the reversible inhibitor and the additional stabilizers M of serine protease as giving a definition.
Background technology
Serine protease is an albuminoid hydrolytic enzyme, it is characterized in that having serine and histidine residues on their avtive spot.A lot of well-known enzymes all belong to this type of, for example trypsin, kallikrein, thrombin and fibrinolysin.Have been found that some the practical application in them.Trypsin is used for leather industry.Thrombin is used as hemorrhage to stop wound bleeding.Other two kinds of serine protease-urokinases and tissue plasminogen activator as thrombolytic agent, are used for the treatment of acute myocardial infarction clinically.A lot of such enzymes have been widely used as research tool, for example in protein structure is determined.In addition, these enzymes can use in a lot of diagnostic kits.
For most serine protease, their stable very limited in solution, this mainly be since when being retained in solution in the time they since the character of protease can take place from degrading.In the time must being stored in material in the solution, limited stable be exactly a problem.Present available commercial serine stretch protein enzyme preparation mainly all is frozen soln or freeze-dried powder, and they have conspicuous shortcoming.Dissolved powders or frozen soln is melted up to suitable temperature required extra time is sixty-four dollar question.But these preparations also have other problem.For frozen soln, all need to control temperature (20 ℃) in steps in the institute that makes and be transported to storage.For freeze-dried powder, but the solution that need regenerate has other purity of acceptor level and stable.Simultaneously, this material usually need be in the environment of non-controlled (for example inclement weather or lack the clean water supply) sterile preparation (by these two parts are mixed), need the check powder whether suitably to mix.These all are the major defects of current available product, and they use complex and expensive in addition.
For the thrombin that preferably must use immediately in prevention is hemorrhage, stable problem forces the manufacturer must use the solution of freeze dried thrombin or cryogenic refrigeration.Then, need the regular hour preparation just can use.Two kinds of thrombolytic agent-urokinases and tissue plasminogen activator sell with the form of lyophilized formulations, and they must dissolve before use.Because the thrombolytic treatment that must begin acute myocardial infarction as early as possible after the infraction beginning, any time that these preparations produce postpones all can become problem.
Make a lot of effort and found to stablize the method for various serine proteases.At a good pace from the trypsin of degraded, a kind of simple and efficient stabilizing agent is calcium ion (Sipos T and Merkel J, Biochemistry 9:2766 (1970)) for meeting.It also is to handle for example method of trypsin and fibrinolysin of some enzymes that pH is reduced to below 4, but is not suitable for thrombin, this be because it at pH less than 5 o'clock inactivations irreversibly.Can use reversible protease inhibitor, but and no thoroughfare because when using they itself the time, their understand the effect (seeing below) with deleterious mode interferases.
For stabilizing tissue plasminogen activator (tPA), the general method of using is to add aminoacid-arginine.The tPA material of present clinical use comprises arginine as stabilizing agent.
Simultaneously, done the method that a lot of effort find to stablize thrombin solution.As the example of stablizing additive, advised following material: the carboxylic acid of high concentration, EDTA, each seed amino acid, albumin, polymer be Polyethylene Glycol, polyvinylpyrrolidone and polyvinyl alcohol, glycerol, various inorganic salt, saccharide, gelatin, collagen for example.
Japanese patent application JP2004191367 has described the stable thrombin that comprises test reagent, is used to test the blood coagulation ability.This test reagent comprises thrombin and thrombin inhibitor, also can comprise the chemical compound of one or more stable thrombins, is selected from calcium ion, organic acid, surfactant and protein.
WO 02/100830, WO 02/22575, WO 00/20394, WO 99/11658, WO02/37937 and US 5,409,927 have all described different serpin chemical compounds and they in preparation treatment various diseases situation, the for example application in the thrombosis has wherein pointed out to suppress corresponding serine protease.
People such as Nakamura (J.Chrom.A, 1009, (2003) 133-139) have described the application of fixed protease inhibitor in the affinity chromatography of trypsin-sample protease.
People such as Turner (Biochemistry, 25, (1986) 4929-4935) have described three kinds of right-amidino groups phenylesters, and they irreversibly suppress people's factors IX a.
People such as Tsung Fu Yang (Biomacromolecules, 25, (2004) 1926-1932) have described cation type polymer N, N-diethyl ethylenediamine polyurethane synthetic, this polymer can be used for gene delivery.
A kind of thrombin preparation has been described in US patent application 2001/0033837 (corresponding to EP 1 136 084 A1), comprises non-covalent bonded inhibitor as stabilizing agent.In addition, this inhibitor and other stable additives be sugar or carboxylic acid combination for example, has described these stable additives in the past in patent and other publications.
JP 2000300250 has described the stable of thrombin solution, is included in to add polyvinyl alcohol, gelatin or polyvinylpyrrolidone in the different buffer solution.
In GB 1354761, by a lot of materials, aliphatic alcohol, carboxylic acid, the heterocyclic compound that comprises hydroxyl and fat or aliphatic cyclic amine stabilize proteins enzyme and amylase to some extent for example.
Like this, described the use inhibitor stablize serine protease (for example US2001/0033837 and JP 2004191367, above).The problem of this method is that if do not remove, inhibitor will weaken the effect of enzyme consumingly so before using these goods.If the use effective inhibitors, most enzymatic activity all can be lost.A kind of method preferably is to use the reversible inhibitor of moderate strength.But, even in this case, the enzyme initial activity of considerable part still can be lost, because need to improve the concentration of inhibitor to obtain the good stable effect.
Summary of the invention
Therefore an object of the present invention is to finish a kind of serine stretch protein enzymatic compositions, it is stable in solution, and keeps enzymatic activity to the degree that is enough to actual use said composition.
Another object of the present invention provides a kind of serine stretch protein enzymatic compositions, and it can directly use and need be by the preceding step of cryogenic refrigeration or freeze-dried material preparation.
A further purpose of the present invention is to make the actual use of reversible inhibitor with regard to stablizing purpose of serine protease become possibility by the stable component that other are provided.
Can realize conspicuous these and other purposes herein by the different aspect of the present invention for required protection.
Therefore, one aspect of the present invention provides a kind of stable serine stretch protein enzymatic compositions, comprises a) serine protease; B) reversible inhibitor of described serine protease; And c) have the stabilizing agent M of formula I:
Wherein
N is 0,1 or 2;
X is O, N or CH
2
R
1-R
4Identical or different, be selected from H ,-CH
2-R
6,-CH
2-O-R
6,-CH
2-S-R
6,-CH
2-NH-R
6,-CO-O-R
6,-CO-NH-R
6,-CH
2-NH-CO-R
6,-CH
2-O-CO-R
6,-CH
2-NH-CO-NHR
6,-CH
2-NH-CO-OR
6,-CH
2-NH-CS-NHR
6With-CH
2-O-CO-NHR
6
R
5Be R
1-R
4Or P-Q;
P is selected from-(CH
2)
m-and-(CH
2)
m-Y-(CH
2)
m-, wherein m is that 1-6 and Y are O, NH or S;
Q is selected from H ,-SO
3,-COOH ,-NH
2,-OH and-CONH
2
R
6Be selected from H independently of one another, replace or unsubstituted low alkyl group, replace or unsubstituted cycloalkyl, replace or unsubstituted benzyl, replacement or unsubstituted aryl or list-, two-, or trinucleated hetero-aromatic ring and the nonaromatic heterocycles that have one or more heteroatomic not replacements or replace, the substituent group of the group of described replacement is selected from low alkyl group, halogen, replacement or unsubstituted aryl, replacement or unsubstituted mixing-aromatic, nonaromatic heterocycles, alkoxyl, alkylamino;
Or the acceptable salt of its pharmacy.
The present invention originates from first to the thrombin Study on Stability, wherein is surprisingly found out that, the combination of the reversible inhibitor of this enzyme and stabilizing agent M of the present invention as defined above has intensive Stabilization for the enzyme in the solution.When thrombin inhibitor and stabilizing agent M use separately thrombin is all had Stabilization, but combination is than any all good several times (seeing embodiment 1) in them.Therefore, when enzyme inhibitor and morpholine, MOPS or the relevant chemical compound of low concentration make up, obtained the Stabilization very strong to enzyme.Some are tried compositions is stable, as shown, when 37 ℃ following more than 2 months the time, activity has only reduced less than 30%.Data according to having the document and the inventor to confirm earlier at room temperature are 6 months accordingly, or are 2.5 years under refrigerated storage temperature.The result of initial research can expand to the test that comprises other serine proteases, in these trials, has also observed wonderful Stabilization.
Following institute exemplifies, and does not contain composition b of the present invention) with c) the enzymatic compositions of combination compare, according to compositions display of the present invention the stability of very big improvement.When using method of the present invention, can use the serpin of low concentration, acquisition stable still reached gratifying degree.For example, the concentration of inhibitor can be lower than the concentration of being advised among the previous for example US2001/0033837.When using the inhibitor of low concentration like this, in stable enzymatic solution, kept much bigger enzymatic activity.
Making of should be noted in the discussion above that combination by reversible serpin and stabilizing agent M causes stablized enhancing and should be considered to be the additional inhibitory action that M provides.In fact, as described in the illustrative embodiment A, M lacks the inhibition ability to any serine protease.Do not wish to be bound by theory, the inventor believes that the wonderful Stabilization enhancing that is observed is what to realize by favourable synergism between reversible serpin in the compositions of the present invention and the stabilizing agent M.The invention provides the combination of reversible serpin and stabilizing agent M in stable serine stretch protein enzymatic compositions, and this is combined in the application in the stabilization filament propylhomoserin protease composition.
In one embodiment of the invention, the serine protease in the said composition is selected from the activated form of trypsin, kallikrein, thrombin, fibrinolysin, urokinase, tissue plasminogen activator, the IX factor, the activated form of the X factor and the activated form of the XI factor.In a more particular embodiment, serine protease is a thrombin.In another specific embodiment, serine protease is a fibrinolysin.In another specific embodiment, serine protease is a trypsin.
The reversible inhibitor of serine protease is well known by persons skilled in the art, and the preferred inhibitor that uses is according to employed concrete serine protease and different.Usually, for desired effect, importantly inhibitor intensity is little.In other words, inhibitory action is appropriateness enough, so that enzymatic activity remains on high level effectively.As guidance, have been found that the K of inhibitor when in compositions according to the present invention, using
iFor 0.01mM is suitable to 2mM, preferred range is that 0.04mM is to 0.5mM.
In one embodiment, wherein serine protease is a thrombin, and reversible inhibitor can be selected from N-(2 '-phenoxy group)-4-aminopyridine and derivant thereof, benzenecarboximidamide, N, N-diethyl ethylenediamine, aminobenzene carbonamidine, amidino groups pyridine and tert-butyl group amidine.In another embodiment, wherein serine protease is a thrombin, and reversible inhibitor is selected from N-(2 '-phenoxy group)-4-aminopyridine and derivant thereof, N, N-diethyl ethylenediamine, amidino groups pyridine and tert-butyl group amidine.In a more particular embodiment, wherein serine protease is a thrombin, and reversible inhibitor is N-(2 '-phenoxy group)-4-aminopyridine or derivatives thereof.In another embodiment, wherein serine protease is a fibrinolysin, and reversible inhibitor is selected from N, N-diethyl ethylenediamine, aminobenzene carbonamidine and benzenecarboximidamide.In another embodiment, wherein serine protease is a trypsin, and reversible inhibitor is selected from aminobenzene carbonamidine and benzenecarboximidamide.The combination of these enzymes and inhibitor all is exemplary example, can not be interpreted as restriction.
In one embodiment of the invention, the value of n is 1 or 2 among the formula I.In a more particular embodiment, n is 1 among the formula I.
Compositions according to the present invention comprises the stabilizing agent M with above-mentioned general formula I.In embodiments of the invention, stabilizing agent M is the chemical compound of formula II:
Wherein
R
1-R
4Identical or different, be selected from H ,-CH
2-R
6
R
5Be R
1-R
4Or P-Q;
P is selected from-(CH
2)
m-and-(CH
2)
m-Y-(CH
2)
m-, wherein m is that 1-6 and Y are O, NH or S;
Q be selected from H ,-SO
3,-COOH ,-NH
2,-OH and-CONH
2
R
6Be selected from independently of one another H, replacement or unsubstituted low alkyl group, replacement or unsubstituted cycloalkyl, replacement or unsubstituted benzyl, replacement or unsubstituted aryl or single-, two-or trinucleatedly have one or more heteroatomic hetero-aromatic ring and nonaromatic heterocycles that do not replace or replace, the substituent group of the group of described replacement be selected from low alkyl group, halogen, replacement or unsubstituted aryl, replacement or unsubstituted assorted-aromatic, nonaromatic heterocycles, alkoxyl, alkylamino;
Or the acceptable salt of its pharmacy.
Therefore, in some embodiments, stabilizing agent M is the chemical compound of formula III:
Wherein
R
5Be-CH
2-R
6Or P-Q;
P is selected from-(CH
2)
m-or-(CH
2)
m-Y-(CH
2)
m-, wherein m is that 1-6 and Y are O, NH or S;
Q be selected from H ,-SO
3,-COOH ,-NH
2,-OH and-CONH
2
R
6The substituent group that is selected from the group of replacement or unsubstituted low alkyl group, replacement or unsubstituted cycloalkyl, replacement or unsubstituted benzyl, replacement or unsubstituted aryl, described replacement independently of one another is selected from low alkyl group, halogen, replacement or unsubstituted aryl, replacement or unsubstituted mixing-aromatic, nonaromatic heterocycles, alkoxyl, alkylamino;
Or the acceptable salt of its pharmacy.
In some embodiments of the present invention, stabilizing agent M is selected from morpholine, 3-(N-morpholino) propane sulfonic acid (MOPS), morpholino butyl sulfonic acid, morpholino propyl group carboxylic acid, morpholino ethyl alcohol and morpholino ethylsulfonic acid.Therefore, the example of the chemical compound M that uses in the compositions in this aspect of the invention is morpholine and 3-(N-morpholino) propane sulfonic acid (MOPS).In a specific embodiment of the present invention, stabilizing agent M is a morpholine.
The compositions that shows Stabilization according to the present invention is, wherein serine protease is a thrombin, and reversible inhibitor is N-(2 '-phenoxy group)-4-aminopyridine, and stabilizing agent M is a morpholine.
Another compositions that shows Stabilization according to the present invention is, wherein serine protease is a thrombin, and reversible inhibitor is N-(2 '-phenoxy group)-4-aminopyridine, and stabilizing agent M is 3-(N-morpholino) propane sulfonic acid (MOPS).
Another compositions that shows Stabilization according to the present invention is, wherein serine protease is a thrombin, and reversible inhibitor is the aminobenzene carbonamidine, and stabilizing agent M is a morpholine.
Another compositions that shows Stabilization according to the present invention is, wherein serine protease is a fibrinolysin, and reversible inhibitor is N, the N-diethyl ethylenediamine, and stabilizing agent M is a morpholine.
Another compositions that shows Stabilization according to the present invention is, wherein serine protease is a fibrinolysin, and reversible inhibitor is the aminobenzene carbonamidine, and stabilizing agent M is a morpholine.
For example in the serine stretch protein enzymatic compositions in the wound site administration, existing problem is that said composition flows easily or washes away from employed site in the part.In order to address this problem, can in this enzymatic compositions, add the adhesiveness polymer, so just reached and make said composition skin or wound site more toughness and adhering purpose.As one embodiment of the invention, in compositions of the present invention, add the adhesiveness polymer it is stablely had an extra unexpected and useful effect.Like this, add polymer and just reached dual purpose, both strengthened the viscosity and the adhesiveness of compositions, simultaneously even further help the stable of enzyme.
In some embodiments of the present invention, said composition further comprises viscosity and the adhesiveness polymer that is selected from polysaccharide and gelatin.Like this, this polymer can for example be a polysaccharide, for example is selected from starch, its derivant, cellulose, its derivant and composition thereof.Particularly, the non-limitative example that can be used as the starch of additive in compositions according to the present invention comprises corn starch and potato starch and composition thereof, and useful cellulose derivative non-limitative example is carboxymethyl cellulose and ethylhydroxyethylcellulose and composition thereof.In a specific embodiment, polysaccharide is a carboxymethyl chitosan.In further embodiment of the present invention, the concentration of described polysaccharide is 0.1-5%.But also imagining this polymer is gelatin, for example from the gelatin of cold water fish.In some embodiments of the present invention, the concentration of described gelatin is 0.5-20%.
In one embodiment of the invention, the concentration of described serine protease is 0.001-2mg/ml.In a more particular embodiment, the concentration of described serine protease is 0.01-1mg/ml.
In one embodiment of the invention, wherein serine protease is a thrombin, and the concentration of thrombin is 5-3500 active unit/ml.
In one embodiment of the invention, wherein serine protease is a thrombin, and the concentration of thrombin is 200-1000 active unit/ml.In one embodiment of the invention, wherein serine protease is a thrombin, and the concentration of thrombin is 5-20 active unit/ml.
In one embodiment of the invention, the concentration of the reversible inhibitor of described serine protease is 0.1-10mM.In a more particular embodiment, the concentration of the reversible inhibitor of described serine protease is 0.5-2mM.
In one embodiment of the invention, the concentration of described stabilizing agent M is 0.02-0.5M.In a more particular embodiment, the concentration of described stabilizing agent M is 0.1-0.3M.
According to another aspect, the invention provides of the application of aforesaid compositions as medicine.
It is that the described compositions of thrombin realizes giving application in hemorrhage patient's hemostatic medicine in preparation that another aspect of the present invention is paid close attention to serine protease wherein.Related aspect of the present invention provides a kind of realization to hemorrhage patient's hemostatic method, comprises that wherein serine protease is a thrombin in the said composition to be enough to reduce or stop described hemorrhage amount using compositions of the present invention on hemorrhage site.
Using thrombin compound according to the present invention as aspect the application or method of medicine, the stability of compositions of the present invention provides benefit under its employed condition.Usually, thrombin compound is to use in the situation of emergency treatment, and wherein key is to stop patient's bleeding.In identical situation, it is very difficult using conventional hemostasis thrombin preparation, because their need trouble and time consuming step usually, promptly melts (if freezing) and/or dissolves (if lyophilizing).The present invention can produce the hemorrhage of solution form, and the stable of them makes them for example be easy to store in the long time in the helicopter of ambulance or emergency treatment, uses when the site in accident or the generation of similar problem needs.At this time point, can directly use them, and can be owing to preparation has any delay.
Be used to stop hemorrhage conventional formulation to comprise the thrombin of suitable high concentration, be 200-1000 active unit/ml.When the plastic surgery uses, can see having the danger that cicatrization increases.At present clinically by diluting the thrombin solution that dense thrombin solution prepares low concentration.The preparation that does not also have plug and play.Therefore, one further aspect, the invention provides a kind of stable thrombin compound, it has the thrombin of remarkable low concentration, is 5-20 active unit/ml and its application in the plastic surgery.
Another aspect of the present invention is to utilize fibrinolysin, and urokinase or tPA are as the known properties of thrombolytic agent.Therefore, the invention provides above-mentioned composition and be used for the application of the medicine of thrombolytic treatment in preparation, wherein serine protease is selected from fibrinolysin, urokinase and tissue plasminogen activator.A related aspect provides a kind of method of carrying out thrombolytic treatment in the patient of needs, comprise with the amount that is enough to carry out described treatment and use above-mentioned compositions to the patient, wherein serine protease is selected from fibrinolysin in the said composition, urokinase and tissue plasminogen activator.At these two related aspects, the nonrestrictive example of described thrombolytic treatment is, in order to treat myocardial infarction or in order to treat apoplexy.
As described in Composition Aspects of the present invention, the stable enhancing that is caused by reversible serpin and stabilizing agent M should not be regarded as the additional inhibitory action that M provides.In fact, as described in the illustrative embodiment A, M lacks the inhibition ability to any serine protease.Do not wish to be bound by theory, the inventor believes that the wonderful Stabilization enhancing that is observed is what to realize by favourable synergism between reversible serpin in the compositions of the present invention and the stabilizing agent M.
Therefore, in yet another aspect, the invention provides
A) reversible serpin and
B) the stabilizing agent M of formula I:
Wherein
N is 0,1 or 2;
X is O, N or CH
2
R
1-R
4Identical or different, be selected from H ,-CH
2-R
6,-CH
2-O-R
6,-CH
2-S-R
6,-CH
2-NH-R
6,-CO-O-R
6,-CO-NH-R
6,-CH
2-NH-CO-R
6,-CH
2-O-CO-R
6,-CH
2-NH-CO-NHR
6,-CH
2-NH-CO-OR
6,-CH
2-NH-CS-NHR
6With-CH
2-O-CO-NHR
6
R
5Be R
1-R
4Or P-Q;
P is selected from-(CH
2)
m-and-(CH
2)
m-Y-(CH
2)
m-, wherein m is that 1-6 and Y are O, NH or S;
Q be selected from H ,-SO
3,-COOH ,-NH
2,-OH and-CONH
2
R
6Be selected from independently of one another H, replacement or unsubstituted low alkyl group, replacement or unsubstituted cycloalkyl, replacement or unsubstituted benzyl, replacement or unsubstituted aryl or single-, two-trinucleated substituent group with group of one or more heteroatomic hetero-aromatic rings that do not replace or replace and nonaromatic heterocycles, described replacement be selected from low alkyl group, halogen, replacement or unsubstituted aryl, replacement or unsubstituted assorted-aromatic, nonaromatic heterocycles, alkoxyl, alkylamino;
Be combined in application in the stabilization filament propylhomoserin protease composition, wherein reversible serpin and stabilizing agent M performance synergism are to produce the effect of stablizing serine protease.
At reversible serpin of the present invention and combination of stabilizers in the application in stabilization filament propylhomoserin protease composition, the substituent selection of operable concrete component and chemical compound M such as above-mentionedly discuss in Composition Aspects of the present invention.
In yet another aspect, the invention provides a kind of method of stable serine protease, comprise the serine protease and a) reversible inhibitor of described serine protease; And b) the stabilizing agent M of formula I:
Wherein
N is 0,1 or 2;
X is O, N or CH
2
R
1-R
4Identical or different, be selected from H ,-CH
2-R
6,-CH
2-O-R
6,-CH
2-S-R
6,-CH
2-NH-R
6,-CO-O-R
6,-CO-NH-R
6,-CH
2-NH-CO-R
6,-CH
2-O-CO-R
6,-CH
2-NH-CO-NHR
6,-CH
2-NH-CO-OR
6,-CH
2-NH-CS-NHR
6With-CH
2-O-CO-NHR
6
R
5Be R
1-R
4Or P-Q;
P is selected from-(CH
2)
m-and-(CH
2)
m-Y-(CH
2)
m-, wherein m is that 1-6 and Y are O, NH or S;
Q be selected from H ,-SO
3,-COOH ,-NH
2,-OH and-CONH
2
R
6Be selected from independently of one another H, replacement or unsubstituted low alkyl group, replacement or unsubstituted cycloalkyl, replacement or unsubstituted benzyl, replacement or unsubstituted aryl or single-, two-trinucleated substituent group with group of one or more heteroatomic hetero-aromatic rings that do not replace or replace and nonaromatic heterocycles, described replacement be selected from low alkyl group, halogen, replacement or unsubstituted aryl, replacement or unsubstituted assorted-aromatic, nonaromatic heterocycles, alkoxyl, alkylamino;
Or the acceptable salt of its pharmacy mixes.
In the method for this stabilization filament propylhomoserin protease composition of the present invention, the substituent selection of operable concrete component and chemical compound M such as above-mentionedly discuss in Composition Aspects of the present invention.
What further aspect of the present invention related to is the above-mentioned application of compositions in being adsorbed onto the decorating film body, so that this decorating film body can provide described enzymatic activity.Particularly, interestingly in a lot of surgeries are used be, enter and from tremulous pulse, come out especially to have only as far as possible little injury for hemorrhage simultaneously.In order to stop arterial hemorrhage, " tremulous pulse plug " used in previous suggestion, and (this object is also referred to as tremulous pulse closing device, strand access into closed device (when using femoral artery to enter, for example in angiography), the blood vessel hemostasis device and the puncture locking device) form, for example form by collagen or the degradable material preparation of other biological.According to this aspect of the invention, advantageously can use compositions according to the present invention to apply this plug, wherein serine protease is a thrombin.This plug can more promptly seal the opening of tremulous pulse, and wherein the thrombin in the said composition is in order to make plug blood coagulation on every side.Therefore, aspect this, the invention provides a certain amount ofly according to composition adsorbs of the present invention blood vessel hemostasis device thereon, wherein serine protease is a thrombin.Preferably for example collagen, chitosan or other biological learn that polymer makes to this blood vessel hemostasis device with biodegradable solid or semisolid material.
Another aspect of the present invention relates to and newly identifies N, and the N-diethyl ethylenediamine is as serpin.Therefore, aspect this, the invention provides N, the N-diethyl ethylenediamine is as the application of the inhibitor of serine protease, and a kind of method that suppresses serine protease, comprises that the N-diethyl ethylenediamine mixes with its N with amount of suppression.In some embodiments aspect this of the present invention, serine protease is a fibrinolysin.In other embodiments aspect this of the present invention, serine protease is a thrombin.
In order to reach all advantages of different aspect of the present invention, general preferred compositions according to the present invention is the form that is selected from solution and gel.Aspect this, more preferably aqueous solution and aqueous gel.
Definition
Term used herein " low alkyl group " is meant unbranched or side chain, ring-type, and the alkyl of saturated or unsaturated (alkenyl or alkynyl), it can be that replace or unsubstituted.When being ring-type, alkyl is C3-C12 preferably, more preferably C5-C10, most preferably C5-C7.When being non-annularity, alkyl is C1-C10 preferably, more preferably C1-C6, more preferably methyl, ethyl, propyl group (n-pro-pyl, isopropyl), butyl (side chain or unbranched) or amyl group, most preferable.
Term used herein " aryl " is meant aromatic group, phenyl or naphthyl for example, or comprise one or more, be preferably selected from N, the heteroatomic list of O and S-, two-or tricyclic heteroaryl, pyridine radicals for example, pyrrole radicals, quinolyl, furyl, thienyl ， oxadiazole base, thiadiazolyl group, thiazolyl oxazolyl, pyrazolyl, triazolyl, tetrazole radical isoxazolyl, isothiazolyl, imidazole radicals, pyrimidine radicals, indyl, pyrazinyl, indazolyl, pyrimidine radicals, thienyl, pyranose, carbazyl, acridinyl, quinolyl, benzimidazolyl, benzothiazolyl, purine radicals, cinnolines base, pteridyl.
Term used herein " functional group " is meant under unprotected situation: hydroxyl-, mercapto-, amido functional group, carboxylic acid, and under the situation of protection be: lower alkoxy, N-, O-, S-acetyl group, carboxylate.
Term used herein " heteroaryl " be meant comprise one or more preferably from N, the heteroatomic aryl of O and S, for example pyridine radicals, pyrrole radicals, quinolyl, furyl, thienyl ， oxadiazole base, thiadiazolyl group, thiazolyl ， oxazolyl, pyrazolyl, triazolyl, imidazole radicals, pyrimidine radicals, indyl, pyrazinyl or indazolyl.
Term used herein " nonaromatic heterocycles " be meant comprise one or more, be preferably selected from N, the heteroatomic non-aromatic ring shape group of O and S, for example ring is amino, pyrrolidinyl for example, piperidyl, piperazinyl, morpholinyl or cyclic ethers be tetrahydrofuran base for example, monosaccharide.
Term used herein " halogen " is meant fluorine, chlorine, bromine or iodine.
Term used herein " replacement " is meant that related group is by functional group's hydroxyl for example, amino, sulfur (sulfide), silicyl, replacements such as aryl.
The example that is used for the pharmacy acceptable addition salt of compositions of the present invention comprises by mineral acid hydrochloric acid for example, hydrobromic acid, phosphoric acid, Metaphosphoric acid, nitric acid and sulphuric acid and by organic acid tartaric acid for example, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic, gluconic acid, succinic acid and aryl sulfonic acid deutero-those.The acceptable excipient of pharmacy as herein described for example vehicle, adjuvant, carrier or diluent is well known to a person skilled in the art, and is easy to open purchase and arrives.Pharmaceutically acceptable carrier can be to reactive compound be chemical inertness and under service condition with no harmful side-effects or toxic those.Can be from for example Remington:The Science and Practice of Pharmacy, 19
ThEdition, Mack Printing Company, Easton, Pennsylvania (1995) finds pharmaceutical preparation.
As detailed description of the present invention, may selecting of the inhibitor that uses in according to the compositions and methods of the invention is " N-(2 '-phenoxy group)-4-aminopyridine and derivant thereof ".This is meant the chemical compound with formula IV:
Wherein
R
1Be selected from H, C1-C6-alkyl, C3-C7-cycloalkyl, phenyl, benzyl acetyl group and benzoyl;
X is selected from oxygen, nitrogen and sulfur;
R
2And R
3Be independently selected from H, halogen, hydroxyl, C1-C6-alkyl, C3-C7-cycloalkyl, C1-C6-alkyl oxygen respectively; With
R
4Be selected from H, C1-C6-alkyl, aryl alkyl and acyl group.
Preferred this inhibitor has formula V:
Wherein
R
1Be selected from C1-C6-alkyl, C3-C7-cycloalkyl, phenyl and benzyl;
R
2And R
3Be independently selected from H, halogen, hydroxyl, C1-C6-alkyl, C3-C7-cycloalkyl and C1-C6-alkyl oxygen respectively.
Embodiment
The following examples are to be used to explain the present invention, and should not be construed as restriction.
Below in the description to the experiment carried out according to the present invention, use to reach time that initial activity 70% spent stable numerical value as enzymatic solution.Select this value to be labeled as " T 70% ", reason is that it is corresponding to the active maximum admissible forfeiture of acceptable in the life-span of commercial product.
In experimentation, use high temperature (37 ℃), and the enzyme of high concentration.This is in order reasonably to obtain stability data in the short period, and need not to wait for several months or several years.After deliberation stability to the dependency degree of temperature, the result provides in embodiment 1.This studies show that, compares with the process (37 ℃) in practical measurement, and at room temperature inactivation process has slowed down about 3 times, has slowed down about 20 times under refrigerated storage temperature.
In addition, inactivation process is a concentration dependent, and deactivation is more rapid under the situation of the enzyme of higher concentration.The concentration of the thrombin that uses in embodiment 1 is that (or 3300 units/ml) promptly are higher than the 0.1-0.3mg/ml concentration of using to 1mg/ml in goods that current commerce can be purchased and/or device.Studies show that of concentration dependent, inactivation process than slow 3-4 under the concentration of using among the embodiment 1 doubly under these concentration.
This is comprehensively provided the factor between the 10-12, it be multiply by T 70% value, to obtain for example comprising the corresponding value of room temperature storage condition of the agent for stanching of thrombin with the commercial product that comprises serine protease.In table 1, the T 70% with compositions of N-(2 '-phenoxy group)-4-aminopyridine and MOPS is 120 days.This and 0.1-0.3mg/ml product surpass 1200 days at ambient temperature, and the value that promptly surpasses 3 years is corresponding.Aspect thrombin stable, this has obviously surpassed any material that had before obtained in solution.
Stablizing of embodiment 1-human thrombin
In order to determine the procoagulant activity of thrombin solution, be determined at add specific human thrombin (from blood plasma, 3300 units/mg, Biovitrum AB, Sweden) setting time of fibrinogen solution (1.3mg/ml) after the different diluent of solution.(Amelungen Germany) measures setting time with Amelungen Kc 1 coagulo meter.In order to study the stable of the thrombin solution that contains different additives, be incubation in 37 ℃ the thermostatic chamber keeping temperature with sample.Take out equal portions liquid at interval with different time, be determined at the thrombin activity that keeps in these equal portions liquid.Can make up the decay of activity curve by the value that is obtained.
At 10mM HEPES, 0.13M NaCl buffer, among the pH 7.4, the decay of activity curve of the thrombin solution of 1mg/ml shows, is about 1.6 days in 37 ℃ of T, 70% value.Corresponding experiment under room temperature (about 21 ℃) shows that T 70% value is 5.4 days, and after (about about 5 ℃) were stored in cold preservation, T 70% value was 36 days.Like this, as desired, have intensive temperature dependency.
The solution that comprises the thrombin of 1mg/ml in the 0.13MNaCl buffer of 10mM HEPES and pH 7.4 that will have specified stable additive places thermostatic chamber, determines their decay of activity curve.Resulting result is as shown in table 1.The data that comprise the corresponding 1mg/ml thrombin solution that does not comprise additive are to be used for comparison.
Obviously, test-compound all has Stabilization, still, as making up with these resulting good result proves, has synergism according to inhibitor of the present invention and the combination of compounds that comprises morpholine.As above shown in the table, add the stable enhancing of 0.20M MOPS separately, the factor is 4.7, adds the reversible thrombin inhibitor aminobenzene carbonamidine of 0.5mM, the stable enhancing, and the factor is 12.5.But this thrombin compound has been stablized in combination of the present invention much betterly, and the factor is 42.5.MOPS of the present invention and N, the N-diethyl ethylenediamine to be combined in the stabilized enzyme aspect also better than independent component.Similarly, very the highland enhancing is stable in the combination of 0.20M MOPS and 1.9mM N-(2 '-phenoxy group)-4-aminopyridine, and the factor is 75, and the stable factor of component enhancing is respectively 4.7 and 22 separately.
The thrombin that uses in this research is the human thrombin from blood plasma.Also after deliberation recombinant human thrombin, it has shown identical characteristic basically.
Stablizing of embodiment 2-thrombin of beef
Studied the stable of thrombin of beef.Experimental arrangement is identical with embodiment 1, but the concentration of employed thrombin of beef (Baxter) is 0.4mg/ml.When storing down for 37 ℃, T 70% value that the thrombin solution in the HEPES buffer shows is 1.3 days.T 70% value that adds the thrombin solution demonstration among 3.0mM N-(2 '-phenoxy group)-4-aminopyridine and the 0.20M MOPS at the HEPES buffer is 54 days.
Resulting result shows it is how much more unstable that thrombin of beef compares the human thrombin goods of being studied, but obtained very good stable effect by combination of the present invention.
The thrombin of embodiment 3-low concentration
Studied the Stabilization of thrombin in the compositions of the thrombin that comprises low concentration.Stabilization according to combination of the present invention has also shown the thrombin that can be used for suitable low concentration.
The HEPES buffer, and the human thrombin of 15.0 active units among the pH 7.4/ml (from blood plasma, 3300 units/mg, Biovitrum AB, T 70% value that solution Sweden) shows is 23 days.T 70% value that HEPES buffer, pH 7.4 add the corresponding solution demonstration among 2.0mM N-(2 '-phenoxy group)-4-aminopyridine and the 0.20M MOPS is 92 days.
Stablizing of embodiment 4-fibrinolysin
Mensuration is according to the Stabilization of the solution of fibrinolysin of the present invention.(Pentapharm, Switzerland), the activity of fibrinolysin is determined in the change of measuring absorption in spectrophotometer at the 405nm place with color development peptide substrates Chromozym TH.To be included in 10mM HEPES and 0.13M NaCl, fibrinolysin (specific activity 3.2 units/mg of 100 μ g/ml among the pH 7.4, Sigma-Aldrich) and the solution of stabilizing agent as shown in table 2 below at 37 ℃ of following incubations, active determine being used for different time samplings.Resulting result is as shown in table 2.
These results have proved, use combination according to the present invention to obtain very strong Stabilization.It is 4 that 0.20M morpholine strengthens the factor of fibrinolysin composition stable, 0.13M N, and the factor of N-diethyl ethylenediamine is 2.7, the factor of 1.3mM aminobenzene carbonamidine is 17.But, morpholine and N, the factor that the combination of N-diethyl ethylenediamine strengthens the fibrinolysin composition stable is 7.3, it is 72 that the combination of morpholine and aminobenzene carbonamidine strengthens the stable factor.
Embodiment 5-is tryptic stable
Mensuration is stable according to tryptic solution of the present invention.Determine the activity of fibrinolysin as substrate and the change that in spectrophotometer, measure to absorb at the 247nm place with TAME (TAME).To be included in 10mM HEPES and 0.13M NaCl, the trypsin TPCK-treated of 100 μ g/ml among the pH 7.4, Sigma-Aldrich) and the solution of stabilizing agent as shown in table 3 below at 37 ℃ of following incubations, active determine being used for different time samplings.Resulting result is as shown in table 3.
Equally, the Stabilization according to compositions of the present invention is maximum.Therefore, single factor with 0.5M morpholine enhancing fibrinolysin composition stable is 13, and single is 72 with the stable factor of 1mM benzenecarboximidamide enhancing.On the other hand, the factor of combination enhanced stability of the present invention is 147.
Embodiment 6-contains the Stabilization of CMC thrombin
Comprise the thrombin solution of 1.0 to 2.0% carboxymethyl celluloses (CMC) and the adhering test of application on human skin and show, add CMC and greatly strengthened viscosity and adhesiveness.But astoundingly, we also find, the stability of these thrombin solutions strengthens further, will comprise the 0.5mM aminobenzene carbonamidine of 2.0%CMC, 0.20M MOPS, 10mM HEPES, 0.13M the 1mg/ml human thrombin of NaCl (from blood plasma, 3300 units/mg, BiovitrumAB, Sweden) solution is determined the decay of activity curve at 37 ℃ of following incubations.Resulting T 70% value is 175 days.
Embodiment 7-is with the Stabilization of other adhesiveness polymer
4 kinds of other polymer have also been studied: ethylhydroxyethylcellulose (EHEC), potato starch, corn starch and cold water fish gelatin.These four kinds of polymer have all strengthened the adhesiveness and the viscosity of thrombin solution.The compatibility and the stability of these polymer and thrombin solution have further been studied, comprise 0.20M MOPS, 0.5mM aminobenzene carbonamidine, 10mM HEPES, the 0.13M NaCl that will comprise various polymer, the 1mg/ml human thrombin is (from blood plasma among the pH 7.4,3300 units/mg, Biovitrum AB, Sweden) solution is at 37 ℃ of following incubations.The concentration of employed polymer is: EHEC, 0.6%; Two kinds of different starch, 4.0%; And gelatin, 12.8%.EHEC and thrombin are compatible fully, and have obtained T 70% value identical with the corresponding solution that does not contain EHEC, promptly about 65 days.T 70% value that comprises the solution of starch is 22 and 26 days.The stable of thrombin is very good in gelatin, and T 70% value is more than 90 days, and this shows that the cold water fish gelatin has extra Stabilization.
The hemorrhage experiment of embodiment 8-
In a series of tests, test compositions of the present invention with rabbit and stop hemorrhage ability.Selected model is at the liver upper cut, and this is a model that often uses.Open the abdominal part of rabbit, expose liver.Produce the long standard otch of 3mm on the liver surface, and the solution that tried of 0.10ml is applied to this wound with syringe.Measure the hemostatic time.Carry out 10-12 experiment with every kind of solution.Calculate the meansigma methods in bleeding time behind the highest and minimum in removing each campaign.For comparing, in this research, also comprise hemorrhage Tisseel (Baxter) commonly used, it is a kind of Fibrin Glue.Basically use Tisseel according to the recommendation of manufacturer.With double syringe the solution of 0.2ml is applied to each wound with mixing chamber.Resulting result provides in following table 4.
Find out apparently that from these results stable thrombin solution according to the present invention is the most effective in the hemostasis finishing fast of bleeding patients, it with commonly used medicament quite or better.
The compatibility of embodiment 9-and how empty material
To comprise the 0.4mg/ml human thrombin (from blood plasma, 3300 units/mg, Biovitrum AB, Sweden) 10mM HEPES, 0.14M NaCl, 0.5mM aminobenzene carbonamidine, 0.20M MOPS, the solution absorbs of pH 7.4 is to a slice polyurethane cream (by with Hartmann Scandicare AB, Anderstorp, Sweden Ligasone sells).The amount of the solution that uses is enough to saturated this urethane film.This sheet is transferred in the test tube, sealed then to avoid evaporating.This test tube is kept under 37 ℃, by the light sample of getting solution with different time at interval of pressing on urethane film.T 70% value of decay of activity curve display is 74 days, and the stable accordingly enhanced factor is 46.
The absorption of embodiment 10-enzyme on solid phase
Measure in the stable solution thrombin to the absorption on surface.With the chitosan of about 3 * 3mm (at least 85% is deacetylated, solid tablets Sigma-Aldrich) 400 units/ml human thrombin (from blood plasma, 3300 units/mg; Biovitrum AB; Sweden) 10mM HEPES, 0.13M NaCl, incubation is 10 minutes in pH 7.4 solution.This solution has following additive: 1) do not have 2) the 0.10M morpholine, 2mM N-(2 '-phenoxy group)-4-aminopyridine, 3) the 0.10M morpholine, 2mM N-(2 '-phenoxy group)-4-aminopyridine, 0.5% carboxymethyl cellulose.Take out these small pieces then, and dry on filter paper.In order to obtain the measured value of thrombin coagulation activity, small pieces are placed test tube, and add the 1.3mg/ml fibrinogen solution of 0.4ml.In order to improve the detection to solidifying, this test tube also comprises a little steel ball.Small pieces initial setting time that obtains in different incubation mixture changed between 1 to 4 minute.In the Eppendorf pipe at 37 ℃ of following incubations after 7 days, at solution 1) in the setting time of small pieces of incubation greatly prolong.This value is between 24 to 27 minutes.On the contrary, at solution 2) and 3) in the setting time of small pieces of incubation in 1 to 2.5 minute scope, promptly identical with initial value.Obviously, with solution 2) and 3) strong Stabilization obtained to thrombin activity.In order to measure styptic activity in the body, in according to embodiment 8 described animal models, will be at solution 3) in the chitosan small pieces of incubation be applied on the wound of liver of rabbit.Hemostatic is 27 seconds (based on 6 tests) average time.
Illustrative embodiment A-morpholine is not a thrombin inhibitor
Having estimated morpholine is the probability of thrombin inhibitor.Usually measure the Fibrinogen coagulation activity of thrombin by setting test, wherein pass through the time of machinery or Optical devices detection fibers proteinogen solution solidifies.Setting test in this experimental arrangement is to carry out in the 0.01MHEPES of pH 7.4,0.13M NaCl buffer, and this experiment is carried out according to standard method (EU pharmacopeia).Use comprise 89 units/ml human thrombin (from blood plasma, 3300 units/mg, Biovitrum AB, Sweden) solution is measured 1/5,1/10 and 1/16 diluent.The dense morpholine solution that is adjusted to pH 7.4 by adding prepares the morpholine solution of variable concentrations in the HEPES buffer.When morpholine was dissolved in the water, pH was increased to 9-10, added HCl like this to obtain pH 7.4.So also strengthened the ionic strength of solution.Table I has shown resulting result.The setting time that observes is converted to the concentration of thrombin with standard curve.
From these results apparently, the concentration of morpholine is brought up to certain level and can prolong setting time.But, when strengthening ionic strength, observed identical phenomenon with NaCl, shown identical characteristic.Like this, in all probabilities, ionic strength strengthens the prolongation that has caused effect.Further, known ion intensity is brought up to 0.22M from 0.15M and is caused fibrinous polymerization to change (B.
, Thrombosis Research, vol.83, (1996), p.1-75, particularly p.18).This is in fact corresponding to the scope of studying in these experimentalists and technicians, and wherein the initial concentration of NaCl is 0.13M, brings up to 0.18M then, and brings up to 0.33M always.This is also corresponding to the platform level that is observed.In a word, morpholine itself is not a kind of inhibitor of thrombin.
Claims (62)
1. stable serine stretch protein enzymatic compositions comprises a) serine protease; B) reversible inhibitor of described serine protease; And c) have the stabilizing agent M of formula I:
Wherein
N is 0,1 or 2;
X is O, N or CH
2
R
1-R
4Identical or different, be selected from H ,-CH
2-R
6,-CH
2-O-R
6,-CH
2-S-R
6,-CH
2-NH-R
6,-CO-O-R
6,-CO-NH-R
6,-CH
2-NH-CO-R
6,-CH
2-O-CO-R
6,-CH
2-NH-CO-NHR
6,-CH
2-NH-CO-OR
6,-CH
2-NH-CS-NHR
6With-CH
2-O-CO-NHR
6
R
5Be R
1-R
4Or P-Q;
P is selected from-(CH
2)
m-and-(CH
2)
m-Y-(CH
2)
m-, wherein m is that 1-6 and Y are O, NH or S;
Q be selected from H ,-SO
3,-COOH ,-NH
2,-OH and-CONH
2
R
6Be selected from independently of one another H, replacement or unsubstituted low alkyl group, replacement or unsubstituted cycloalkyl, replacement or unsubstituted benzyl, replacement or unsubstituted aryl or single-, two-or trinucleatedly have one or more heteroatomic hetero-aromatic ring and nonaromatic heterocycles that do not replace or replace, the substituent group of the group of described replacement be selected from low alkyl group, halogen, replacement or unsubstituted aryl, replacement or unsubstituted assorted-aromatic, nonaromatic heterocycles, alkoxyl, alkylamino;
Or the acceptable salt of its pharmacy.
2. according to the compositions of claim 1, wherein said serine protease is selected from the activated form of trypsin, kallikrein, thrombin, fibrinolysin, urokinase, tissue plasminogen activator, the IX factor, the activated form of the X factor and the activated form of the XI factor.
3. according to the compositions of aforementioned arbitrary claim, the K that wherein said reversible inhibitor shows
iValue is 0.01 to 2mM, and for example 0.04mM is to 0.5mM.
4. according to the compositions of aforementioned arbitrary claim, wherein said serine protease is a thrombin.
5. according to the compositions of claim 4, wherein this reversible inhibitor is selected from N-(2 '-phenoxy group)-4-aminopyridine and derivant thereof, benzenecarboximidamide, N, N-diethyl ethylenediamine, aminobenzene carbonamidine, amidino groups pyridine and tert-butyl group amidine.
6. according to the compositions of claim 4, wherein this reversible inhibitor is selected from N-(2 '-phenoxy group)-4-aminopyridine and derivant thereof, N, N-diethyl ethylenediamine, amidino groups pyridine and tert-butyl group amidine.
7. according to the compositions of claim 4, wherein this reversible inhibitor is N-(2 '-phenoxy group)-4-aminopyridine or derivatives thereof.
8. according to each compositions of claim 1-3, wherein this serine protease is a fibrinolysin.
9. compositions according to Claim 8, wherein this reversible inhibitor is selected from N, N-diethyl ethylenediamine, aminobenzene carbonamidine and benzenecarboximidamide.
10. the compositions arbitrary according to claim 1-3, wherein this serine protease is a trypsin.
11. according to the compositions of claim 10, wherein this reversible inhibitor is selected from aminobenzene carbonamidine and benzenecarboximidamide.
12. according to the compositions of aforementioned arbitrary claim, n is 1 or 2 among its Chinese style I.
13. according to the compositions of aforementioned arbitrary claim, n is 1 among its Chinese style I.
14. according to the compositions of aforementioned arbitrary claim, wherein stabilizing agent M is the chemical compound of formula II:
Wherein
R
1-R
4Identical or different, be selected from H ,-CH
2-R
6
R
5Be R
1-R
4Or P-Q;
P is selected from-(CH
2)
m-and-(CH
2)
m-Y-(CH
2)
m-, wherein m is that 1-6 and Y are O, NH or S;
Q be selected from H ,-SO
3,-COOH ,-NH
2,-OH and-CONH
2
R
6Be selected from independently of one another H, replacement or unsubstituted low alkyl group, replacement or unsubstituted cycloalkyl, replacement or unsubstituted benzyl, replacement or unsubstituted aryl or single-, two-or trinucleatedly have one or more heteroatomic hetero-aromatic ring and nonaromatic heterocycles that do not replace or replace, the substituent group of the group of described replacement be selected from low alkyl group, halogen, replacement or unsubstituted aryl, replacement or unsubstituted assorted-aromatic, nonaromatic heterocycles, alkoxyl, alkylamino;
Or the acceptable salt of its pharmacy.
15. according to the compositions of claim 14, wherein stabilizing agent M is the chemical compound of formula III:
Wherein
R
5Be-CH
2-R
6Or P-Q;
P is selected from-(CH
2)
m-or-(CH
2)
m-Y-(CH
2)
m-, wherein m is that 1-6 and Y are O, NH or S;
Q be selected from H ,-SO
3,-COOH ,-NH
2,-OH and-CONH
2
R
6The substituent group that is selected from the group of replacement or unsubstituted low alkyl group, replacement or unsubstituted cycloalkyl, replacement or unsubstituted benzyl, replacement or unsubstituted aryl, described replacement independently of one another is selected from low alkyl group, halogen, replacement or unsubstituted aryl, replacement or unsubstituted mixing-aromatic, nonaromatic heterocycles, alkoxyl, alkylamino;
Or the acceptable salt of its pharmacy.
16. according to the compositions of claim 15, wherein stabilizing agent M is selected from morpholine, 3-(N-morpholino) propane sulfonic acid (MOPS), morpholino butyl sulfonic acid, morpholino propyl group carboxylic acid, morpholino ethyl alcohol and morpholino ethylsulfonic acid.
17. according to the compositions of claim 16, wherein stabilizing agent M is selected from morpholine and 3-(N-morpholino) propane sulfonic acid.
18. according to the compositions of claim 17, wherein stabilizing agent M is a morpholine.
19. according to the compositions of aforementioned arbitrary claim, wherein serine protease is a thrombin, reversible inhibitor is N-(2 '-phenoxy group)-4-aminopyridine, and stabilizing agent M is a morpholine.
20. according to the compositions of aforementioned arbitrary claim, wherein serine protease is a thrombin, reversible inhibitor is N-(2 '-phenoxy group)-4-aminopyridine, and stabilizing agent M is 3-(N-morpholino) propane sulfonic acid.
21. according to the compositions of aforementioned arbitrary claim, further comprise viscosity and adhesiveness polymer, be selected from polysaccharide and gelatin.
22. according to the compositions of claim 21, wherein said viscosity and adhesiveness polymer are polysaccharide.
23. according to the compositions of claim 22, wherein said polysaccharide is selected from starch, its derivant, cellulose, its derivant and composition thereof.
24. according to the compositions of claim 23, wherein said polysaccharide is selected from carboxymethyl cellulose, ethylhydroxyethylcellulose and composition thereof.
25. according to the compositions of claim 22, wherein said polysaccharide is a carboxymethyl chitosan.
26. the compositions arbitrary according to claim 22-25, the concentration of wherein said polysaccharide is 0.1-5%.
27. according to the compositions of claim 21, wherein said viscosity and adhesiveness polymer are gelatin.
28. according to the compositions of claim 27, wherein said gelatin is the cold water fish gelatin.
29. the compositions arbitrary according to claim 27-28, the concentration of wherein said gelatin is 0.5-20%.
30. according to the compositions of aforementioned arbitrary claim, the concentration of wherein said serine protease is 0.001-2mg/ml.
31. according to the compositions of aforementioned arbitrary claim, wherein said serine protease is a thrombin, the concentration of thrombin is 5-3500 active unit/ml.
32. according to the compositions of aforementioned arbitrary claim, wherein said serine protease is a thrombin, the concentration of thrombin is 200-1000 active unit/ml.
33. according to the compositions of aforementioned arbitrary claim, wherein said serine protease is a thrombin, the concentration of thrombin is 5-20 active unit/ml.
34. according to the compositions of aforementioned arbitrary claim, the concentration of the reversible inhibitor of wherein said serine protease is 0.1-10mM.
35. according to the compositions of aforementioned arbitrary claim, the concentration of wherein said stabilizing agent is 0.02-0.5M.
36. according to the compositions of aforementioned arbitrary claim, it is for being selected from solution and gel, for example the form of aqueous solution or aqueous gel.
37. according to the application of the compositions of aforementioned arbitrary claim as medicine.
38. realize to give application in hemorrhage patient's hemostatic medicine according to each compositions of claim 1-36 in preparation, wherein serine protease is a thrombin.
39. realize to give hemorrhage patient's hemostatic method, comprise that wherein serine protease described in the said composition is a thrombin to be enough to reduce or to stop described hemorrhage amount to use each the compositions according to claim 1-36 on hemorrhage site.
40. the application of compositions in the plastic surgery according to claim 33.
41. be used for the application of the medicine of thrombolytic treatment according to each compositions of claim 1-36 in preparation, wherein said serine protease is selected from fibrinolysin, urokinase and tissue plasminogen activator.
42. in the patient of needs, carry out the method for thrombolytic treatment, comprise with the amount that is enough to carry out described treatment and use each the compositions according to claim 1-36 to the patient, wherein serine protease described in the said composition is selected from fibrinolysin, urokinase and tissue plasminogen activator.
43. in reversible serpin and the claim 1 defined stabilizing agent M be combined in application in the stabilization filament propylhomoserin protease composition, wherein said reversible serpin and described stabilizing agent M synergism are to produce the serine protease stablizing effect.
44. according to the application of claim 43, wherein this serine protease be such as claim 2,4,8,10,30,31,32 and 33 in each definition.
45. according to the application of claim 43-44, wherein this reversible inhibitor be such as claim 3,5,6,7,9,11 and 34 in each definition.
46. the application arbitrary according to claim 43-45, wherein stabilizing agent M be such as claim 12-18 and 35 in each definition.
47. according to each application of claim 43-46, wherein said combination further comprises viscosity and the adhesiveness polymer that is selected from polysaccharide and gelatin.
48. according to the application of claim 47, wherein this viscosity and adhesiveness polymer be such as claim 22-29 in each definition.
49. according to each application of claim 43-48, wherein said reversible serpin is that N-(2 '-phenoxy group)-4-aminopyridine and described stabilizing agent M are 3-(N-morpholino) propane sulfonic acid.
50. stabilization filament propylhomoserin protease composition method comprises the serine protease and a) reversible inhibitor of described serine protease; And b) the acceptable salt of the stabilizing agent M of formula I or its pharmacy mixes:
Wherein
N is 0,1 or 2;
X is O, N or CH
2
R
1-R
4Identical or different, be selected from H ,-CH
2-R
6,-CH
2-O-R
6,-CH
2-S-R
6,-CH
2-NH-R
6,-CO-O-R
6,-CO-NH-R
6,-CH
2-NH-CO-R
6,-CH
2-O-CO-R
6,-CH
2-NH-CO-NHR
6,-CH
2-NH-CO-OR
6,-CH
2-NH-CS-NHR
6With-CH
2-O-CO-NHR
6
R
5Be R
1-R
4Or P-Q;
P is selected from-(CH
2)
m-and-(CH
2)
m-Y-(CH
2)
m-, wherein m is that 1-6 and Y are O, NH or S;
Q be selected from H ,-SO
3,-COOH ,-NH
2,-OH and-CONH
2
R
6Be selected from independently of one another H, replacement or unsubstituted low alkyl group, replacement or unsubstituted cycloalkyl, replacement or unsubstituted benzyl, replacement or unsubstituted aryl or single-, two or trinucleatedly have one or more heteroatomic hetero-aromatic ring and nonaromatic heterocycles that do not replace or replace;
The substituent group of the group of described replacement is selected from low alkyl group, halogen, replacement or unsubstituted aryl, replacement or unsubstituted mixing-aromatic, nonaromatic heterocycles, alkoxyl, alkylamino.
51. according to the method for claim 50, wherein this serine protease be as claim 2,4,8,10,30,31,32 and 33 each define.
52. according to the method for claim 50-51, wherein this reversible inhibitor be as claim 3,5,6,7,9,11 and 34 each define.
53. according to each method of claim 50-52, wherein stabilizing agent M be as claim 12-18 and 35 each define.
54., further comprise with serine protease and the viscosity and the adhesiveness polymer mixed that are selected from polysaccharide and gelatin according to each method of claim 50-53.
55. according to the method for claim 54, wherein this viscosity and adhesiveness polymer are that each defines as claim 22-29.
56. according to each method of claim 50-55, wherein said reversible serpin is that N-(2 '-phenoxy group)-4-aminopyridine and described stabilizing agent M are 3-(N-morpholino) propane sulfonic acid.
57. according to each composition adsorbs blood vessel hemostasis device thereon of claim 1-36, wherein serine protease described in the compositions is a thrombin.
58. the blood vessel hemostasis device according to claim 57 comprises biodegradable material, for example is selected from chitosan and collagen.
59.N the N-diethyl ethylenediamine is as the application of serpin.
60. according to the application of claim 59, wherein this serine protease is selected from thrombin and fibrinolysin.
61. suppress the method for serine protease, comprise to the N that wherein sneaks into amount of suppression the N-diethyl ethylenediamine.
62. according to the method for claim 61, wherein this serine protease is selected from thrombin and fibrinolysin.
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| CN106390186A (en) * | 2011-04-27 | 2017-02-15 | 比奥马普公司 | Hemostatic compositions |
| US11052172B2 (en) | 2016-08-12 | 2021-07-06 | Biom'up France SAS | Hemostatic flowable |
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| CN106390186A (en) * | 2011-04-27 | 2017-02-15 | 比奥马普公司 | Hemostatic compositions |
| US10342856B2 (en) | 2011-04-27 | 2019-07-09 | Biom'up | Hemostatic compostions |
| CN106390186B (en) * | 2011-04-27 | 2020-07-03 | 比奥马普公司 | Hemostatic compositions |
| US11052172B2 (en) | 2016-08-12 | 2021-07-06 | Biom'up France SAS | Hemostatic flowable |
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