CN100402092C - Gas bearing activator of dry powder in use for diagnosing and/or treating thrombus - Google Patents

Abstract

The present invention relates to a gas-containing dried powder activating agent for diagnosing and/or treating thrombus, and a preparing technology thereof. The activating agent is freeze-dried powder, and is prepared from polypeptide molecules, lipids, surface active agents, stabilizing agents and gas. The active molecule is polypeptide, and can be specifically combined with activated blood platelets with high affinity during thrombosis. In addition, the active molecule has high inhibiting effect on the activating agent, and has high affinity for the target of thrombus and high thrombolysis effect. When the activating agent is used, solvent is added to the activating agent to form medicine liquid by oscillation, and is admitted by intravenous injection. The activating agent can realize the target ultrasonic imaging of thrombus, and has high thrombolysis activity.

Description

Thrombosis diagnosis and/or treatment gas bearing activating agent and preparation technology thereof

Technical field

The present invention relates to a kind of activating agent and preparation technology thereof of peptide molecule guiding, particularly a kind of thrombosis diagnosis and/or treatment gas bearing activating agent and preparation technology thereof.

Background technology

The medicine (acoustic contrast agent) of thrombus target diagnosis and treatment is used to study and the clinical value of showing it as diagnostic tool already, and the history of existing decades, for example be used for cardiovascular system and organize microcirculation perfusion etc.The method of utilizing various contrast agent enhanced diagnosis acoustic pictures is also arranged, comprise solid microparticle suspension, emulsive big nano-particle, bubble, the bubble of other material bag quilt, liquid or its dry powder formulations.Targeted drug (acoustic contrast agent) should have (1) can produce detectable signal with the ultrasound wave radiation interaction; (2) specific target spot (for example pathological particular organization cellularity) is arranged, and have affinity and specificity with target molecule; (3) activity of maintenance medicine.

Acoustic contrast agent is put down in writing by many disclosed data, and the portioned product list marketing is arranged, as the product Sonovue of Bracco company.These acoustic contrasts are made up of the gassiness microvesicle of similar erythrocyte size, have the characteristic of linear scattering and nonlinear scattering down ultrasonic behind the injection vein, can make the abundant organa parenchymatosum of blood flow produce stronger contrast and develop.

The example of the acoustic contrast agent of guiding has: for example EP-A-0727225 has described the acoustic contrast agent of guiding, this report component and surfactant or albumin are carrier related, the cell adhesion molecule part that comprises protein as carrier, peptide, a sugared substrate, recognize that now this method can produce the uncontrollable problem of growth of microvesicle, the potential possibility (for example cardiovascular and cerebrovascular vessel thrombosis) that causes thrombosis is arranged.

The unilamellar liposome that WO-A-9320802 has described tissue specificity part (as antibody, peptide) connection is used to strengthen ultra sonic imaging, for there not being the ultrasonic enhancing contrast agent of gas.Present technique has also been described at fibrin, thrombosis, and the guiding radiography in atherosclerosis zone is used.Publication that some guiding acoustic contrast agents are also arranged is spoken of and can be utilized monoclonal antibody as carrier material, but all do not provide details, WO-A-9300933 for example, WO-A-9401140, WO-A-9428874.

CN 1234742A and CN1302211A described by the film forming surface of shape live give birth to agent material monolayer stable types of gas filled microbubbles, comprise the diagnosis of guiding and/or the component of therapeutic agent, be reported as aqueous suspensions, this component surfactant employing phospholipid and derivant thereof, lipopeptid, can with the functional group in the multiple open source information of receptors bind, wherein also narrated and blood viscosity, hemorrhage, thrombosis, known part and receptor that thrombolytic is relevant have also provided the preparation of a thrombosis guiding lipopeptid and histolytica's activator of plasminogen microvesicle in addition.These report receptors of components and part be for open, its mainly the specific function group of the surfactant by its component carry out in conjunction with to form stable microvesicle.To guide molecule (be target molecule: specificity is attached to the molecule of interior certain position of body or tissue), be exactly the effective molecule that is attached to thrombosis specifically, do not provide detailed important actual detail.

Summary of the invention

The object of the present invention is to provide a kind of long-time polypeptide that guarantees, albumen, stability of drug and activity, and have thrombosis diagnosis and/or the treatment gas bearing activating agent and the preparation technology thereof of high-affinity of the convenience of use.

For achieving the above object, the technical solution used in the present invention is: 1) polypeptide H-Pro-A-B-C-Asp-D-OH's is synthetic: the amino-acid reagent bottle that at first adopts 1mmol, on the automated peptide synthesizer, with the scale of 0.1mmol aminoacid is synthesized polypeptide with the Fmoc resin, before synthetic polypeptide with the pre-activated amino acid of HBTU, synthetic polypeptide was placed TFA solution 2-3 hour, obtain crude product,, use the crude product of preparation HPLC purification gained again with 5ml/ minute flow and 20-40%B gradient; 2) lipid-Pro-A-B-C-Asp-D-OH's is synthetic: earlier described polypeptide is incorporated among the PBS of pre-cooling and stir until dissolving fully, the final concentration that makes polypeptide is 0.1mol/L, room temperature cools, 4 ℃ of following pre-coolings 2 hours, in the polypeptide PBS of pre-cooling solution, be that 1: 99～99: 1 mol ratio adds lipid by lipid: H-Pro-A-B-C-Asp-D-OH, under 4 ℃ of conditions, stirred 4 hours, after treating to become the milky suspension fully, lyophilization below 4 ℃; 3) preparation of DPPG and lipid-Pro-A-B-C-Asp-D-OH gassiness lyophilized powder: lipid-Pro-A-B-C-Asp-D-OH and DPPG are dissolved in the water by 20: 1～1: 20 mol ratio, making the mixed mass percent concentration of lipid-Pro-A-B-C-Asp-D-OH and DPPG is 0.5%, at 40 ℃ of sufficient condition stirring and evenly mixings after about 40～60 minutes, it is 1～20ppm that the adding surfactant makes its final concentration, add stabilizing agent under the room temperature behind the stirring and evenly mixing, the mass percent concentration that makes stabilizing agent is 1-10%, mixing adds lyophilization under the low temperature below 4 ℃ in the lyophilizing bottle after 30 minutes, behind the bone dry,, the gas sealing that adds 0.01-1.0Mpa gets final product.

A among the polypeptide H-Pro-A-B-C-Asp-D-OH of the present invention is selected from alanine, (Ala) or leucine, (Leu) or arginine, (Arg) or lysine, (Lys) or agedoite, (Asn) or methionine, (Met) or aspartic acid, (Asp) or phenylalanine, (Phe) or cysteine, (Cys) or proline, (Pro) or glutamine, (Gln) or serine, (Ser) or glutamic acid, (Glu) or threonine, (Thr) or glycine, (Gly) or tryptophan, (Trp) or histidine, (His) or tyrosine, (Tyr) or isoleucine, (Ile) or valine, (Val); B is selected from alanine (Ala) or leucine (Leu) or arginine (Arg) or lysine (Lys) or agedoite (Asn) or methionine (Met) or aspartic acid (Asp) or phenylalanine (Phe) or cysteine (Cys) or proline (Pro) or glutamine (Gln) or serine (Ser) or glutamic acid (Glu) or threonine (Thr) or glycine (Gly) or tryptophan (Trp) or histidine (His) or tyrosine (Tyr) or isoleucine (Ile) or valine (Val); C is alanine (Ala) or leucine (Leu) or arginine (Arg) or lysine (Lys) or agedoite (Asn) or methionine (Met) or aspartic acid (Asp) or phenylalanine (Phe) or cysteine (Cys) or proline (Pro) or glutamine (Gln) or serine (Ser) or glutamic acid (Glu) or threonine (Thr) or glycine (Gly) or tryptophan (Trp) or histidine (His) or tyrosine (Tyr) or isoleucine (Ile) or valine (Val); D is alanine (Ala) or leucine (Leu) or arginine (Arg) or lysine (Lys) or agedoite (Asn) or methionine (Met) or aspartic acid (Asp) or phenylalanine (Phe) or cysteine (Cys) or proline (Pro) or glutamine (Gln) or serine (Ser) or glutamic acid (Glu) or threonine (Thr) or glycine (Gly) or tryptophan (Trp) or histidine (His) or tyrosine (Tyr) or isoleucine (Ile) or valine (Val); Lipid is distearyl acid PHOSPHATIDYL ETHANOLAMINE, two Palmic acid PHOSPHATIDYL ETHANOLAMINE, phosphatidyl glycerol, cholesterol, sphingosine, lipid sphyngomyelin, ceramide, cerebroside, sulfolipide, ganglioside, glucoside (fat) ceramide, two hydrogen (nerve) sphingols of 4-hydroxyl; L-a-phosphoric acid lecithin, platelet activating factor, phosphatidyl glycerol, PHOSPHATIDYL ETHANOLAMINE, phosphatidylinositols, phosphatidylinositol-4phosphate, phosphatidylinositols-4,5-diphosphate, cuorin, phosphatidic acid, lysophosphatide, PHOSPHATIDYL ETHANOLAMINE-NH2 end group, this NH2 end group comprise caproamide group, laurylamide group, phosphatidyl mercaptamine, succinyl group, glutaryl group, dodecane carboxylic group, biotin group, phosphatidyl ethylene glycol, biotinylated phospholipid medicated cap; Lysophosphatidylcholine, lysophosphatidyl ethanolamine, lysophosphatidyl glycerol, lysophosphoglyceride, lysophosphatidic acid, alkyl phosphate choline, the lipid of oxidation, biotinylated lipid, plasmalogen, polymeric phospholipid, Halogenated Phospholipids, the phospholipid of poly-PEGization, the PEG end has the poly-PEGization phospholipid DSPE-mPEG-Carboxylic Acid of functional group, DSPE-mPEG-Maleimide, DSPE-mPEG-PDP, DSPE-mPEG-Amine, DSPE-mPEG-Biotin or DSPE-mPEG-BTC; Surfactant is Palmic acid, oleic acid or linolenic acid; Stabilizing agent is mannitol, glycerol or Polyethylene Glycol; Gas is nitrogen, oxygen, carbon dioxide, hydrogen, sulfur fluoride, fluoridized ketone, fluoridized ether, sulfur hexafluoride, perfluoropropane, perfluorinated butane or perflenapent.

Every medicine of gas bearing activating agent of making according to preparation method of the present invention contains surfactant, the stabilizing agent of 0.1-5.0mg and the gas of 0.01-1.0Mpa of DPPG, 0.01-0.4mg of lipid-Pro-Ser-Nva-Gly-Asp-Trp-OH, the 25-1mg of 1-25mg.

The present invention is based on the specific binding molecules of thrombosis, the peptide molecule of serial thrombosis specificity high-affinity has been synthesized in screening, and based on the lyophilized powder reporter of this molecule, wherein lyophilized powder also seals the gas that contains certain pressure in certain container.Like this, for example, adopt and mix the enhanced microvesicle of lipid formation elasticity, it strengthens ultrasonic echo performance, and wherein the effect of the bioactive molecule of high-affinity makes aggregate concentration increase the echo reinforcement.Like this, the peptide molecule of application is intensive platelet suppressant drug, and anti-thrombosis function is arranged.Can guarantee long-time polypeptide like this, albumen, stability of drug and activity, and have the convenience of use.

Description of drawings

Fig. 1 is the mass spectrum of sample identification of the present invention, and wherein abscissa is a molecular weight, and vertical coordinate is a percentage ratio intensity;

Fig. 2 is a high-efficient liquid phase chromatogram of the present invention, and wherein abscissa is the time, and vertical coordinate is an intensity;

Fig. 3 is image intensity figure, and wherein abscissa is for before and after the injection, and vertical coordinate is a development intensity.

The specific embodiment

Below in conjunction with accompanying drawing the present invention is described in further detail.

An advantageous embodiment of the present invention is based on other discovery, promptly with the bonded guide molecule of pathological tissues thrombosis specificity---synthetic polypeptide, a glycoprotein receptor in the time of can be with platelet activation combines the effective site combination of GPIIIa/IIb, stop hematoblastic further activation and gathering, this character makes it become effective development thrombolytics.This character is to utilize receptor, part combination, the peptide molecule antagonism activation of part realize.These targeted drugs can effectively be attached to the thrombosis surface that is forming by synthetic peptide molecule, and the thrombosis at the ultrasonic development enhancing of thrombosis or the position that can not develop originally develops.It is can be at the thrombotic enhancing ultrasonic echo that provides in early days that another one is utilized a favourable aspect of this guide molecule, after the thrombosis (being the old thrombosis), platelet activation diminuendo or disappearance, guide molecule do not provide enhanced echo-signal in conjunction with diminuendo.The present invention is particularly suitable for the early stage targeting level diagnosis of thrombosis and the discriminating of new and old thrombosis.

The gas of any bio-compatibility all can be present in the reporter, and is sealed in the container.Gas used in the present invention is included under 37 ℃ of normal person's body temperature in fact or is entirely any material of gas, comprises their mixture.For example, these gases comprise, air; Nitrogen; Oxygen; Carbon dioxide; Hydrogen; Noble gas; The sulfur fluoride; Seienium hexafluoride; Halogenated silanes; Low-molecular-weight Hydrocarbon; Alkane (as first, second, third, fourth, pentane); Cycloalkanes (as cyclopropane, Tetramethylene., Pentamethylene .); Alkene (as ethylene, propylene, allene or butylene); Alkynes (as acetylene, propine); Ether; Ketone; Ester; The low-molecular-weight halogenated hydrocarbon; At least one halogen atom in the halo gas is replaced by fluorine.The preferred sulfur hexafluoride of the present invention.

Lipid in the reporter of the present invention be in principle any in liquid phase film-formable natural or synthetic lipid, lipoid.Effectively the example of phospholipid comprises cephalin and lecithin and synthetic, semisynthetic, as distearyl acid PHOSPHATIDYL ETHANOLAMINE, two Palmic acid PHOSPHATIDYL ETHANOLAMINE, phosphatidyl glycerol;

Natural phospholipid comprises toxicide lipid A; Cholesterol; Sphingolipid, as sphingosine, lipid sphyngomyelin, ceramide, cerebroside, sulfolipide class, ganglioside, glucoside (fat) ceramide, phytosphingosine and derivant thereof (as two hydrogen (nerve) sphingols of 4-hydroxyl); Glycerol is the lipid of basic structure, as choline (as L-a-phosphoric acid lecithin, platelet activating factor), as phosphatidyl glycerol, as ethanolamines (as PHOSPHATIDYL ETHANOLAMINE), as the inositol class (as phosphatidylinositols, phosphatidylinositol-4phosphate, phosphatidylinositols-4, the 5-diphosphate), cuorin, phosphatidic acid, lysophosphatide.

Synthetic lipoids comprises:

1, with biological relevant lipid derivate,

(1) as the derivant (replaced as the fatty acid by different carbon chain lengths above the asymmetric carboxyl groups, symmetric fatty acid by identical different carbon chain lengths is replaced) of PHOSPHATIDYL ETHANOLAMINE; As the listed derivant that contains the PHOSPHATIDYL ETHANOLAMINE of asymmetric carbochain of following table

The derivant that contains the PHOSPHATIDYL ETHANOLAMINE of symmetrical carbochain

The carbon number derivant

6: 0 Dicaproyl two caproyls

8: 0 Dioctanoyl two decoyls

Dicapryl two was sad in 10: 0

12: 0 Dilauroyl 22 (alkane) acyls

14: 0 Dimyristoyl two myristoyls

16: 0 Dipalmitoyl two palmityls

16: 0[(CH3) 4] Diphytan oyl two phytane acyls

16: 1 Dipalmitoleoyl two palmitoleoyls (base)

17: 0 Diheptadecanoyl two-heptadecanoyls

18: 0 Distearoyl distearyls

18: 1 (delta9-cis) Dioleoyl two oleoyls

18: 1 (delta9-trans) Dielaidoyl two anti-oleoyls

18: 2 Dilinoeoyl two inferior oleoyls

18: 3 Dilinolenoyl two Caulis et Folium Lini oleoyls

20: 4 Diarachidon oyl two Semen arachidis hypogaeae tetraene acyls

Docosa-hexaenoyl

22∶6

Two or two ten two-Hexaethyl

(2) derivant of GranulestinLecithin

The derivant that contains the GranulestinLecithin of asymmetric fatty acid chain

Carbon number	The 1-acyl group	The 2-acyl group
			14∶0-16∶0	The Myristoyl myristoyl	Palmitoyl
14∶0-18∶0	Myristoyl	Stearoyl 18 (alkane) acyl
			16∶0-14∶0	The Palmitoyl palmityl	The Myristoyl myristoyl
16∶0-18∶0	The Palmitoyl palmityl	Stearoyl 18 (alkane) acyl
			16∶0-18∶1	The Palmitoyl palmityl	The Oleoyl oleoyl
16∶0-18∶2	The Palmitoyl palmityl	The inferior oleoyl of Linoleoyl
			16∶0-20∶4	The Palmitoyl palmityl	Arachidonoyl arachidonic acyl
16∶0-22∶6	The Palmitoyl palmityl	Docosahexaenoyl 22-Hexaethyl
			18∶0-14∶0	Stearoyl 18 (alkane) acyl	The Myristoyl myristoyl
18∶0-16∶0	Stearoyl 18 (alkane) acyl	The Palmitoyl palmityl
			18∶0-18∶1	Stearoyl 18 (alkane) acyl	The Oleoyl oleoyl
18∶0-18∶2	Stearoyl 18 (alkane) acyl	The inferior oleoyl of Linoleoyl
			18∶0-20∶4	Stearoyl 18 (alkane) acyl	Arachidonoyl arachidonic acyl
18∶0-22∶6	Stearoyl 18 (alkane) acyl	Docosahexaenoyl 22-Hexaethyl
			18∶1-14∶0	The Oleoyl oleoyl	The Myristoyl myristoyl
18∶1-16∶0	The Oleoyl oleoyl	The Palmitoyl palmityl
			18∶1-18∶0	The Oleoyl oleoyl	Stearoyl 18 (alkane) acyl

The derivant that contains the GranulestinLecithin of symmetrical fatty acid chain

Carbon number	Derivant
		3∶0	The Propionoyl propionyl
4∶0	The Butanoyl butyryl
		5∶0	The Pentanoyl valeryl
6∶0	The Caproyl caproyl
		7∶0	The Heptanoyl oenanthyl
8∶0	Capryloyl decoyl (base)
		9∶0	The Nonanoyl nonanoyl
10∶0	The Capryl decoyl
		11∶0	Un decanoyl caprinoyl
12∶0	Lauroyl 12 (alkane) acyl
		13∶0	Tridecanoyl 13 (alkane) acyl
14∶0	The Myristoyl myristoyl
		15∶0	The Pentadecanoyl pentadecanoyl
16∶0	The Palmitoyl palmityl
		16∶0[(CH 3) 4]	Phytanoyl phytane acyl
17∶0	The Heptadecanoyl heptadecanoyl
		18∶0	Stearoyl 18 (alkane) acyl
19∶0	Nonadecanoyl ten

	Nine (alkane) acyl
		20∶0	Arachidoyl sweet (alkane) acyl
21∶0	Heniecosanoyl 21 (alkane) acyl
		22∶0	Behen oyl 22 (alkane) acyl
23∶0	Trucisan oyl 23 (alkane) acyl
		24∶0	Lignocer oyl 24 (alkane) acyl

Carbon number	Derivant
		14∶1	9-cis-tetradecenoic 9-is suitable-tetradecene
14∶1	9-trans-tetradecenoic 9-is anti--tetradecene
		16∶1	9-cis-hexadecenoic 9-is suitable-hexadecene
16∶1	9-trans-hexadecenoic 9-is anti--hexadecene
		18∶1	6-cis-octadecenoic 6-is suitable-vaccenic acid
18∶1	9-cis-octadecenoic 9-is suitable-vaccenic acid
		18∶1	9-trans-octadecenoic 9-is anti--vaccenic acid
18∶2	9-cis-12-cis-octadecadienoic 9-is suitable-suitable-18 carbon diene of 12-
		18∶3	9-cis-12-cis-15-cis-octadecatrienoic 9-is suitable-and 12-is suitable-and 15-is along 18 carbon triolefins
20∶1	11-cis-eicosenoic 11-is suitable-eicosylene
		20∶4	5，8，11，14(all-cis)

	Eicosatetraenoic 5,8,11, and 14-is all-cis-eicosylene
		22∶1	Suitable-two dodecylenes of 13-cis-docosenoic 13-
22∶6	4,7,10,13,16,19 (all-cis) docosahexaenoic 4,7,10,13,16,19-is all-cis-two dodecahexaenes
		24∶1	15-cis-tetracosenoic 15-is suitable-tetracosene

(3) derivant of the pure and mild diphosphoinositide of phosphatidyl-4

1，2-Dioctanoyl-sn-Glycero-3-[Phosphoinositol-4，5-Bisphosphate]

1,2-two decoyls-glycerol-3-[phosphoinositide-4,5-diphosphate (ester)]

1，2-Dioctanoyl-sn-Glycero-3-[Phosphoinositol-3，5-Bisphosphate]

1,2-two decoyls-glycerol-3-[phosphoinositide-3,5-diphosphate (ester)]

1，2-Dioctanoyl-sn-Glycero-3-[Phosphoinositol-3，4-Bisphosphate]

1,2-two decoyls-glycerol-3-[phosphoinositide-3,4-diphosphate (ester)]

1，2-Dioctanoyl-sn-Glycero-3-[Phosphoinositol-3，4，5-Trisphosphate]

1,2-two decoyls-glycerol-3-[phosphoinositide-3,4,5-triphosphate (ester)]

L-α-Phosphatidylinositol-4，5-bisphosphate(brain)

L-α-phosphatidylinositols-4,5-diphosphate (ester) (brain)

L-α-Phosphatidylinositol-4-phosphate(brain)

L-α-phosphatidylinositol-4phosphate salt (ester) (brain)

L-α-Phosphatidylinositol(liver)

L-α-phosphatidylinositols (liver)

L-α-Phosphatidylinositol(soy)

L-α-phosphatidylinositols (Semen sojae atricolor)

(4) derivant of phosphatidic acid

The derivant that contains the phosphatidic acid of asymmetric fatty acid chain

Carbon number	The 1-acyl group	The 2-acyl group
			16∶0-18∶1	The Palmitoyl palmityl	The Oleoyl oleoyl
16∶0-18∶2	The Palmitoyl palmityl	The inferior oleoyl of Linoleoyl
			16∶0-20∶4	The Palmitoyl palmityl	Arachidonoyl arachidonic acyl
16∶0-22∶6	The Palmitoyl palmityl	Docosahexaenoyl
			18∶0-18∶1	The Stearoyl stearoyl	The Oleoyl oleoyl
18∶0-18∶2	The Stearoyl stearoyl	The inferior oleoyl of Linoleoyl
			18∶0-20∶4	The Stearoyl stearoyl	Arachidonoyl arachidonic acyl
18∶0-22∶6	The Stearoyl stearoyl	Docosahexaenoyl 22-Hexaethyl acyl

The derivant that contains the phosphatidic acid of symmetrical fatty acid chain

Carbochain	Derivant
			6∶0	Dicaproyl two caproyls
8∶0	Dicapryloyl two decoyls
		10∶0	Dicapryl two is sad
12∶0	Dilauroyl 22 (alkane) acyl
		14∶0	Dimyristoyl two myristoyls
16∶0	Dipalmitoyl two palmityls
		16∶0[(CH 3) 4]	Diphytanoyl two phytane acyls
17∶0	Diheptadecanoyl two-heptadecanoyl
		18∶0	The Distearoyl distearyl
18∶1	Dioleoyl two oleoyls
		18∶2	Dilinoleoyl two inferior oleoyls
20∶4	Diarachidonoyl two Semen arachidis hypogaeae tetraene acyls
		22∶6	Didocosahexaenoyl two-22-Hexaethyl acyl

(5) derivant of phosphatidyl glycerol

The derivant that contains the phosphatidyl glycerol of asymmetric fatty acid chain

The derivant that contains the phosphatidyl glycerol of symmetrical fatty acid chain

The carbon number derivant

Dicaproyl two in 6: 0

Caproyl

8∶0 Dioctanoyl

Two decoyls

Dicapryl two in 10: 0

Sad

12: 0 Dilauroyl 22 (alkane) acyls

Dimyristoyl two in 14: 0

Myristoyl

16: 0 Di palmitoyl two

Palmityl

16: 0[(CH3) 4] Di phytanoyl two

The phytane acyl

Dipalmitoleoyl two in 16: 1

Palmitoleoyl (base)

17: 0 Diheptadecanoyl two-17

The alkane acyl

Distearoyl two in 18: 0

Stearoyl

18: 1 (delta 9-cis) Dioleoyl two

Oleoyl

18: 1 (delta 9-trans) Dielaidoyl two anti-oleoyls

18: 2 Dilinoeoyl two inferior oleoyls

18: 3 Dilinolenoyl two oleum linis

Acyl

20: 4 Diarachidon oyl two

The arachidonic acyl

22∶6 Docosa-hexaenoyl

Two-22-Hexaethyl

(6) cuorin derivant:

Carbochain	Derivant
			14∶0	Tetramyristoyl 4-myristoyl
14∶0	Tetramyristoyl 4-myristoyl
		18∶1	Tetra oleoyl 4-oleoyl

(7) derivant of diglyceride:

Derivant
	1-Oleoyl-2-Acetoyl-sn-Glycerol 1-oleoyl-2-propionyl-sn-glycerol
1,2-Dioctanoyl-sn-Glycerol 1,2-two decoyls-sn-glycerol
	1,2-Di decanoyl-sn-Glycerol 1,2-two caprinoyls-sn-glycerol
1,2-Di lauroyl-sn-Glycerol 1,2-silicon in February acyl-sn-glycerol
	1,2-Di myristoyl-sn-Glycerol 1,2-two myristoyls-sn-glycerol
1,2-Di palmitoyl-sn-Glycerol 1,2-two palmityls-sn-glycerol
	1,2-Dioleoyl-sn-Glycerol 1,2-two oleoyls-sn-glycerol
1-Palmitoyl-2-Oleoyl-sn-Glycerol 1-palmityl-2-oleoyl-sn-glycerol
	The inferior oleoyl of 1-Stearoyl-2-Linoleoyl-sn-Glycerol 1-stearoyl-2--sn-glycerol
1-Stearoyl-2-Arachidon oyl-sn-Glycerol 1-stearoyl-2-arachidonic acyl-sn-glycerol

Derivant
	1, and 2-Dioctanoyl Ethylene Glycol (8: 0EG) 1,2-two decoyl ethylene glycol (8: 0EG)
1, and 2-Dicapryl Ethylene Glycol (10: 0EG) 1, the ethylene glycol (10: 0EG) that 2-two is sad
	1, and 2-Dilauroyl Ethylene Glycol (12: 0EG) 1,2-silicon in February acyl ethylene glycol (12: 0EG)
1, and 2-Di myristoyl Ethylene Glycol (14: 0EG) 1,2-two myristoyl ethylene glycol (14: 0EG)
	1, and 2-Di palmitoyl Ethylene Glycol (16: 0EG) 1,2-two palmityl ethylene glycol (16: 0EG)
1, and 2-Di oleoyl Ethylene Glycol (18: 1EG) 1,2-two oleoyl ethylene glycol (18: 0EG)

2. head contains the derivant of modification group

Have the derivant of the functional group that is connected with albumen, peptide, medicine, as shown below

(1) PHOSPHATIDYL ETHANOLAMINE Phosphatidylethanolamine ,-NH ₂End group Phosphatidylethanolamine

(2), N-Caproylamine-PE, the caproamide group

N-Caproylamine-PE

(3), N-Dodecanylamine-PE laurylamide group

N-Dodecanylamine-PE

4, Phosphatidyl thioethanol phosphatidyl mercaptamine

Phosphatidylthioethanol

5、N-MCC-PE

N-MCC-PE

6、N-MPB-PE

N-MPB-PE

7、N-PDP-PE

N-PDP-PE

8, N-Succiny-PE succinyl group

N-Succinyl-PE

9, N-Glutaryl-PE glutaryl group

N-Glutaryl-PE

10, N-Dodecanyl-PE dodecane carboxylic group

N-Dodecanyl-PE

11, N-Biotinyl-PE biotin group

N-Biotinyl-PE

12, Phosphatidyl Ethylene Glycol phosphatidyl ethylene glycol

Phosphatidyl Ethylene Glycol

13, the biotinylated phospholipid medicated cap of N-Biotinyl Cap-PE

N-Biotinyl Cap-PE

3. the derivant that contains two above function headgroups in addition: as alkyl phosphoric acid salt derivative, methyl-derivatives.The PH sensitive lipid is as DG-succinate (succinate).

The preferred functional group of the present invention is azimidobenzene carbonic ester (BTC), as figure below:

The derivant of fatty acid modifying

Dissolved phosphorus lipid, as lysophosphatidylcholine, lysophosphatidyl ethanolamine, lysophosphatidyl glycerol, lysophosphoglyceride, lysophosphatidic acid; Alkyl phosphate choline; The lipid of oxidation; Biotinylated lipid; Plasmalogen; Polymeric phospholipid (as diacetylene phospholipid); Halogenated Phospholipids; Fluoridize phospholipid; Isotopic labeling phospholipid (as deuterium-labeled); The phospholipid of poly-PEGization; The PEG end has the poly-PEGization phospholipid of functional group, as (DSPE-mPEG-CarboxylicAcid, DSPE-mPEG-Maleimide, DSPE-mPEG-PDP, DSPE-mPEG-Amine, DSPE-mPEG-Biotin, DSPE-mPEG-BTC).The preferred DSPE-mPEG-BTC of the present invention.In addition, preferred two kinds of the present invention, two or more lipids are mixed in proportion the main component that forms dry powder doses, and preferably another lipid is DPPG.

Said surfactant can be any type of film formation material in principle in the reporter of the present invention.For example, anion surfactant (as metal carboxylate, sulfuric ester salt, Sulfonates, phosphates); Cationic surfactant (as amine salt class, quaternary, amino acid pattern, betaine type); Nonionic surfactant (as polyethylene glycol type, the polyhydric alcohol type); Gluconic acid sodium salt; Zinc stearate; Fatty acid (as oleic acid, linoleic acid, Palmic acid).The preferred Palmic acid of the present invention.

The said stabilizing agent of reporter of the present invention (protective agent) can be the protective agent of any lyophilized injectable powder in principle.Alcohols (as aliphatic alcohol, butanols, glycerol) for example; Polyol (as glycerol); Saccharide (as sucrose, mannitol, glucosan); Polyethylene Glycol (as cetomacrogol 1000-10000).Preferred Polyethylene Glycol.

The dry powder formulations of reporter of the present invention quite is easy to reconstruct in water, add solvent (for example sterile distilled water), and (preferred 5-20ml annotates vial) vibrated about 30 seconds with hands in airtight container.The size of the microvesicle that produces is uniformly, has reached goodish controlled size and Billy's range of distribution.The advantage of lyophilization thing is that the medicine of albumen, polypeptide class can be preserved the considerable time activity, and experiment of the present invention is verified under 4 ℃ of conditions, and pharmaceutically active can be stablized preservation more than 18 months.The microvesicle diffusate that reconstruct produces in the water can be saved to minority individual hour in airtight container.

The invention provides the reporter of the thrombus target location imaging of polypeptide guiding, another effectively embodiment carry the thrombolytic curative drug by this report thing.Though for example proposing acoustic contrast agent and drug regimen among CN 1234742A, CN1302211A and the WO-A-9507072, these products lack and the specific carrier of site tool, or existing carrier (part), but carrier (part) is openly.Part of the present invention (polypeptide) is proprietary by the present invention, and the glycoprotein receptor on the thrombosis is had high affinity and specificity.Can be carried in the inside of microvesicle according to the thromboembolism treatment medicine that the present invention utilized, or attached to or be incorporated on the film of microvesicle, the part medicine can finally be dissolved in the solvent in dry powder doses.Like this, medicine can be by active group covalent bond mentioned in this article or ionic bond combination, or the mixing with reporter of physical property.

The representative example of the medicine that the present invention is useful comprises tissue inhibiting such as monoclonal antibody and fragment thereof; Platelet suppressant drug such as thrombin receptor inhibitor, GP IIb/IIIa acceptor inhibitor (as abciximab), adp receptor inhibitor, prostaglandin, aspirin, phosphodiesterase inhibitor; Tissue factor, heparin, hirudin; T-PA, urokinase, fibrinolysin, streptokinase, rt-activator of plasminogen, r-stqphylokinase, anistreplase.

According to product of the present invention, can be used for extracorporeal diagnostic system, as the external thrombus diagnostic kit.Utilize effective target molecule and those known thrombosis diagnostic techniquess, method in external the present invention, utilize fluorescent labeling technology, nanometer labelling technique etc. to realize the external thrombus diagnosis.

Lipid (derivant) according to this utilization has linking group, and functional group then links to each other with linking group, and pattern is: lipid-linking group-functional group, the preferred lipid-mPEG of the present invention (Polyethylene Glycol)-functional group.MPEG is the more a kind of polymer of research during present biotechnology and bio-pharmaceutical are used, PEG has fine solubility in water, each PEG and two or three water molecules and the height aquation, can stop PEG trim surface by other polymer or protein adsorption or covering, PEG is harmless to protein and cell activity in addition, covalently bound PEG is non-immunogenic and nonantigenic, PEG modifies very little to chemical property, the activity influence of decorating molecule, and the PEG junctional complex can make the picked-up of reticuloendothelial system in the body reduce.The used PEG of the present invention selects suitable molecular weight for use, for example between 200 dalton and 20 kilodaltons.Preferred 3400 dalton.

Because guide molecule of the present invention and composition thereof can allow other contrast agent assembly such as Baryan, optical contrast agent, radiocontrast medium, magnetic resonance contrast agent are connected on the reporter, and therefore diagnosis of the present invention and/or medicine also comprise above-mentioned situation.

The preparation of embodiment 1:DSPE DPPG gassiness lyophilized powder and microvesicle are rebuild

DSPE:DPPG is dissolved in the water by 1: 5 (mol ratio), at 40 ℃ of about 40-60 of sufficient condition mixing minutes.After sediments microscope inspection does not have big granule, add the Palmic acid surfactant of 8ppm, mixing is 15 minutes under the room temperature; The back adds PEG4000 as stabilizing agent, mixing 30 minutes; The back adds lyophilization under the low temperature in the lyophilizing bottle, behind the bone dry, adds 1 atmospheric sulfur hexafluoride gas sealing.Again add the about 10 times of volumes of solvent (distilled water), vibration forms the suspension that contains microvesicle, and Particle Size Analyzer is analyzed the microvesicle characteristic.As follows:

Concentration 2-5 * 10 ⁸/ ml

·D ₅₀ 4.0μm

90% threshold value, 8 μ m

SF ₆Bag is by volume 8 μ l/ml

·pH： 6.0to6.5

Stability after reconstitution: 6hours

Embodiment 2: comprise DPPG and DSPE-PEG ₃₄₀₀-BTC-Pro-Ser-Nva-Gly-Asp-Trp-OH gassiness lyophilized powder and biological assessment

This embodiment is the guidance type microvesicle, and this microvesicle contains the peptide molecule of the claim of the present invention of linear structure.

1) polypeptide H-Pro-Ser-Nva-Gly-Asp-Trp-OH's is synthetic

At first adopt the amino-acid reagent bottle of 1mmol, on the automated peptide synthesizer, with the scale of 0.1mmol aminoacid is synthesized polypeptide with the Fmoc resin, before synthetic polypeptide, activate all aminoacid in advance with HBTU, synthetic polypeptide was placed TFA solution 2-3 hour, can from resin, remove peptide and Side chain protective group simultaneously, obtain the 100mg crude product,, get 89mg with the crude product of preparation HPLC purification gained again with 5ml/ minute flow and 20-40%B gradient; After the lyophilization, will obtain pure product and carry out analytical type HPLC analysis and mass spectrography evaluation, as Fig. 1, expection molecular weight 656.6, actual measurement 656.57, referring to Fig. 2, its purity is 98.2%.

2) DSPE-PEG ₃₄₀₀-BTC-Pro-Ser-Nva-Gly-Asp-Trp-OH's is synthetic

Earlier polypeptide is dissolved among the PBS of pre-cooling and stir until dissolving fully, making the final concentration of polypeptide is 0.1mol/L, and room temperature cools, and 4 ℃ of following pre-coolings 2 hours, presses DSPE-PEG in the polypeptide PBS of pre-cooling solution ₃₄₀₀-BTC:H-Pro-Ser-Nva-Gly-Asp-Trp-OH mol ratio is that 5: 1 mol ratio adds lipid, stirs 4 hours under 4 ℃ of conditions, and after treating to become the milky suspension fully, the structure of product is confirmed to form in lyophilization by mass spectrography and magnetic resonance;

3) DPPG and DSPE-PEG ₃₄₀₀The preparation of-BTC-Pro-Ser-Nva-Gly-Asp-Trp-OH gassiness lyophilized powder

With DSPE-PEG ₃₄₀₀-BTC-Pro-Ser-Nva-Gly-Asp-Trp-OH and DPPG are dissolved in the water by the mol ratio of 1:5, make DSPE-PEG ₃₄₀₀The mixed mass percent concentration of-BTC-Pro-Ser-Nva-Gly-Asp-Trp-OH and DPPG is 0.5%, after 40 ℃ of about 40-60 of sufficient condition stirring and evenly mixing minutes, the Palmic acid surfactant that adds 8ppm, add 1mgPEG4000 under the room temperature behind the stirring and evenly mixing as stabilizing agent, mixing adds lyophilization under the low temperature in the lyophilizing bottle after 30 minutes, behind the bone dry, the sulfur hexafluoride gas sealing that adds 0.01-1.0Mpa, again add the about 10 times of volumes of solvent (distilled water), vibration forms the suspension that contains microvesicle, and Particle Size Analyzer is analyzed the microvesicle characteristic.As follows:

Concentration 2-5 * 10 ⁸/ ml

·D ₅₀ 4.0μm

90% threshold value, 8 μ m

SF ₆Bag is by volume 8 μ l/ml

·pH ： 6.0to6.5

Stability after reconstitution: 6hours

In vitro study with the types of gas filled microbubbles after the dry powder doses reconstruct that contains DSPE, DPPG of guide molecule: thrombosis is in conjunction with experimental study

Get human peripheric venous blood, venous blood is applied on the slide, dry under the room temperature.Get c) formed microvesicle suspension, suspension dropwise drips in slide, and suspension covers the thrombosis that forms on the slide fully.Hatched 10 minutes for 37 ℃, PBS flushing three times, microscopically is observed microvesicle in conjunction with the thrombosis situation.

Canis familiaris L. thrombus in vivo imaging experiment

Mongrel with about 20 kilograms of pentobarbital anesthesia.Separate and expose the both sides iliac vein, artificial hemopoietic bolt in the iliac vein of both sides.Get c) formed microvesicle suspension after veins of upper extremity injection, adopted doppler imaging to observe clot strength in 0,1,3,5,10,15,25 minutes in injection respectively and change.And compare with microvesicle after the dry powder doses reconstruct of embodiment 1, referring to Fig. 3, adopt image intensity of the present invention to reach 1200.

Embodiment 3: comprise DPPG and DSPE-PEG ₃₄₀₀The gassiness lyophilized powder of the former monoclonal antibody of-BTC-antifibrin

Utilize identical method preparation described in the embodiment 2 to contain the lyophilized powder of the former monoclonal antibody of antifibrin.In addition, the former monoclonal antibody of antifibrin can physical property or is dispersed in the dry powder doses by ionic bond.

Embodiment 4: comprise DPPG and DSPE-PEG ₃₄₀₀The gassiness lyophilized powder of-BTC-antifibrin monoclonal antibody

Utilize identical method preparation described in the embodiment 2 to contain the lyophilized powder of antifibrin monoclonal antibody.In addition, the antifibrin monoclonal antibody can physical property or is dispersed in the dry powder doses by ionic bond.

Embodiment 5: comprise DPPG and DSPE-PEG ₃₄₀₀The gassiness lyophilized powder of the anti-GP IIb/IIIa of-BTC-monoclonal antibody

Utilize identical method preparation described in the embodiment 2 to contain the lyophilized powder of the monoclonal antibody of anti-GP IIb/IIIa receptor.In addition, the monoclonal antibody of anti-GP IIb/IIIa receptor can physical property or is dispersed in the dry powder doses by ionic bond.

Embodiment 6: comprise DPPG and DSPE-PEG ₃₄₀₀The gassiness lyophilized powder of-BTC-Pro-Ser-Nva-Gly-Asp-Trp-OH and t-PA functionalization

The lipopeptid DSPE-PEG of A, thrombosis affinity ₃₄₀₀-BTC-Pro-Ser-Nva-Gly-Asp-Trp-OH's is synthetic

Utilize embodiment 21) with 22) described in the identical synthetic affine lipopeptid of thrombosis of method preparation.

The modification of B, the t-PA that has

1mg t-PA is dissolved in the 0.2ml ammonium carbonate buffer (a).0.4mg sulfur-SMPB is dissolved in (b) among the 0.5mlDMSO.A liquid and b liquid chamber relaxing the bowels with purgatives of warm nature mixing oscillating reactions 1 hour.The chromatographic column purifies and separates is collected, mass spectrography, and synthetic is identified in magnetic resonance, lyophilization becomes powdered.

C, phosphatidyl mercaptamine-sulfur-SMPB-t-PA's is synthetic

3: 1 in molar ratio ratios of phosphatidyl mercaptamine and B are dissolved among the PBS and vibrated preparation HPLC separation and purification, mass spectrography purification Identification product 1-2 hour under the room temperature.

D, comprise DPPG and DSPE-PEG ₃₄₀₀The preparation of the gassiness lyophilized powder of-BTC-Pro-Ser-Nva-Gly-Asp-Trp-OH and t-PA functionalization

Example 23 is executed in utilization) described in the preparation of identical method contain the gassiness lyophilized powder of t-PA functionalization.T-PA can physical property or is dispersed in the dry powder doses by ionic bond.

Embodiment 7: comprise DPPG and DSPE-PEG ₃₄₀₀The gassiness lyophilized powder of-BTC-Pro-Ser-Nva-Gly-Asp-Trp-OH and urokinase functionalization

Utilize identical method preparation described in the embodiment 6 to contain the urokinase functionalization, or ionic bond, or the dispersive gassiness freeze dried powder of physical property.

Embodiment 8: comprise DPPG and DSPE-PEG ₃₄₀₀The gassiness lyophilized powder of-BTC-Pro-Ser-Nva-Gly-Asp-Trp-OH and streptokinase functionalization

Utilize identical method preparation described in the embodiment 6 to contain the streptokinase functionalization, or ionic bond, or the dispersive gassiness freeze dried powder of physical property.

Embodiment 9: comprise DPPG and DSPE-PEG ₃₄₀₀The gassiness lyophilized powder of-BTC-thrombin

Utilize the identical method described in the embodiment 2, peptide molecule is replaced synthetic comprise DPPG and DSPE-PEG by thrombin ₃₄₀₀The gassiness lyophilized powder of-BTC-thrombin, separation and purification authentication method are also with the method for embodiment 2.

Claims (5)

1. the preparation technology of usefulness gas bearing activating agent is diagnosed and/or treated to a thrombosis, it is characterized in that:

1) polypeptide H-Pro-Ser-Nva-Gly-Asp-Trp-OH's is synthetic

At first adopt the amino-acid reagent bottle of 1mmol, on the automated peptide synthesizer, with the scale of 0.1mmol aminoacid is synthesized polypeptide with the Fmoc resin, before synthetic polypeptide with the pre-activated amino acid of HBTU, synthetic polypeptide was placed TFA solution 2-3 hour, obtain crude product, with 5ml/ minute flow and 20-40%B gradient, use the crude product of preparation HPLC purification gained again;

2) DSPE-PEG ₃₄₀₀-BTC-Pro-Ser-Nva-Gly-Asp-Trp-OH's is synthetic

Earlier described polypeptide is incorporated among the PBS of pre-cooling and stir until dissolving fully, making the final concentration of polypeptide is 0.1mol/L, and room temperature cools, and 4 ℃ of following pre-coolings 2 hours, presses DSPE-PEG in the polypeptide PBS of pre-cooling solution ₃₄₀₀-BTC:H-Pro-Ser-Nva-Gly-Asp-Trp-OH is that 1: 99～99: 1 mol ratio adds lipid, under 4 ℃ of conditions, stirred 4 hours, and after treating to become the milky suspension fully, lyophilization below 4 ℃;

3) DPPG and DSPE-PEG ₃₄₀₀The preparation of-BTC-Pro-Ser-Nva-Gly-Asp-Trp-OH gassiness lyophilized powder

With DSPE-PEG ₃₄₀₀-BTC-Pro-Ser-Nva-Gly-Asp-Trp-OH and DPPG are dissolved in the water by 20: 1～1: 20 mol ratio, make DSPE-PEG ₃₄₀₀The mixed mass percent concentration of-BTC-Pro-Ser-Nva-Gly-Asp-Trp-OH and DPPG is 0.5%; at 40 ℃ of sufficient condition stirring and evenly mixings after 40～60 minutes; it is 1～20ppm that the adding film formation material makes its final concentration; the protective agent that adds lyophilized injectable powder under the room temperature behind the stirring and evenly mixing; the protectant mass percent concentration that makes lyophilized injectable powder is 1-10%; mixing adds lyophilization under the low temperature below 4 ℃ in the lyophilizing bottle after 30 minutes; behind the bone dry, the gas sealing that adds 0.01-1.0Mpa gets final product.

2. thrombosis diagnosis according to claim 1 and/or the treatment preparation technology of gas bearing activating agent, it is characterized in that: said film formation material is Palmic acid, oleic acid or linolenic acid.

3. thrombosis diagnosis according to claim 1 and/or the treatment preparation technology of gas bearing activating agent, it is characterized in that: the protective agent of said lyophilized injectable powder is mannitol, glycerol or Polyethylene Glycol.

4. thrombosis diagnosis according to claim 1 and/or the treatment preparation technology of gas bearing activating agent, it is characterized in that: said gas is nitrogen, oxygen, carbon dioxide, hydrogen, fluoridized ketone, fluoridized ether, sulfur hexafluoride, perfluoropropane, perfluorinated butane or perflenapent.

5. the activating agent that makes with the preparation technology of gas bearing activating agent according to the diagnosis of the described thrombosis of claim 1 and/or treatment, it is characterized in that: every gas bearing activating agent contains the DSPE-PEG of 1-25mg ₃₄₀₀The protective agent of the lyophilized injectable powder of the DPPG of-BTC-Pro-Ser-Nva-Gly-Asp-Trp-OH, 25-1mg, the film formation material of 0.01-0.4mg, 0.1-5.0mg and the gas of 0.01-1.0Mpa.

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