WO2010054503A1 - 抗凝血多肽及其应用 - Google Patents
抗凝血多肽及其应用 Download PDFInfo
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- WO2010054503A1 WO2010054503A1 PCT/CN2008/001881 CN2008001881W WO2010054503A1 WO 2010054503 A1 WO2010054503 A1 WO 2010054503A1 CN 2008001881 W CN2008001881 W CN 2008001881W WO 2010054503 A1 WO2010054503 A1 WO 2010054503A1
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- Prior art keywords
- polypeptide
- peptide
- fprp
- qgdfepipedayde
- anticoagulant
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention relates to an anticoagulant polypeptide and an application thereof, and belongs to the field of biomedicine.
- Coagulation is an important physiological defense process of the body, but pathological thrombosis is a serious threat to human health.
- anticoagulant drugs used clinically at home and abroad mainly include unfractionated heparin, low molecular weight heparin and warfarin. These drugs have disadvantages such as thrombocytopenia and bleeding, and require close laboratory monitoring.
- Peptide anticoagulant drugs, such as hirudin, bivalirudin, and orally administrable small peptide drugs (such as ximelagatran) have outstanding advantages such as fast onset and less side effects, and are anticoagulation in the future. The key development direction of blood medicine.
- Thrombin plays a role in thrombus formation: (1) cleavage of fibrinogen to form fibrin; (2) activators, ⁇ 1 and XI, which in turn form more thrombin; (3) stimulate the blood platelets.
- fibrin is cross-linked by the activation factor VDI to stabilize the embolization.
- Thrombin has three binding regions: a fibrin binding region, a substrate catalytic (active) region, and a heparin binding region.
- Thrombin consists of a 36-amino acid A chain and a 259 amino acid B chain. The A and B chains are linked by disulfide bonds. Like other serine proteases, it includes a typical catalytic ternary (Aspl02, Hi s57).
- the S 1 binding region is like a deep pocket, and the bottom Aspl89 can form a hydrogen bond or electrostatic interaction with the amino or sulfhydryl group of the substrate, and the hydrophobic region of the region can bind to the hydrophobic group.
- the hydrophobic region of the region can bind to the hydrophobic group.
- the acidic Glul 92 residue is at the entrance to the pocket and is important for identifying substrates and inhibitors.
- the fibrin binding region and heparin-binding region of thrombin are mainly composed of several arginine and lysine, and can be combined with electronegative amino acids and groups.
- Hirudin is the first direct inhibitor of peptide thrombin, which is composed of 65 amino acid residues. There are many hirudin variants in nature. Among them, the main anticoagulant activities are HV1, HV2 and HV3 (Salzet M). .Curr Pharm Des, 2002, 8(7): 493-503). Hirudin binds to the substrate catalytic region of fibrin and the fibrin binding region, forming a 1:1 complex with thrombin (binding constant Ki up to 20 fM) (Krsttenansky JL, et al.
- Hirudin has the disadvantages of being unable to be taken orally, and being easily degraded in the body, and can be overcome by structural modification. A long half-life can be obtained by using a PEG molecule or a phospholipid modified with a lipophilic compound such as stearic acid or oleic acid. RGD-fused hirudin has anti-platelet aggregation.
- the genetically engineered bifunctional hirudin (RGD-hirudin) for injection has been approved by the State Food and Drug Administration in 2005 for clinical research.
- the clinical indications are anticoagulation and antithrombotic therapy after vascular anastomosis, and can be used for treatment.
- Bivalirudin is a synthetic hirudin derivative containing 20 amino acid residues, the amino terminus of which binds to the substrate catalytic region of thrombin, and its acidic carboxyl terminus binds.
- Thrombin The fibrin binding region (Bourdon P, et, al. FEBS Lett, 1991, 293: 163-166) with a flexible link between the four Gly in the middle.
- Bivalirudin has a wider therapeutic window and safety than hirudin (Hartmann F, et al. Curr Pharm Des. 2008, 14(12): 1 191-6), but the disadvantage is that neither can be taken orally, in vivo Easy to degrade, with a short half-life.
- Small molecule peptide anticoagulants mainly include argatroban, ximelagatran and dabigatran.
- Argatroban is engineered from N- ⁇ -benzenesulfonyl-L-arginine methyl ester and reversibly and selectively binds to the thrombin catalytic site (Preville P, et al. Bioorg Med Chem Lett, 1997, 7(12): 1563-1566).
- some argatroban analogs have been synthesized (Salvagnini C, et al. Eur J Med Chem. 2007, 42(1): 37-53.), such as replacing their strongly basic thiol groups with lipophilic Side chains to increase cell permeability and oral availability.
- Melagatran is an analog of the D-Phe-Pro-Arg tripeptide sequence, in which phenylhydrazine replaces arginine, azetidine-2-carboxylic acid replaces valine, and D-type cyclohexylglycine replaces D-phenylene Amino acid, melagatran can reversibly inhibit the catalytic site of thrombin (Gustafsson D, et. al. J Intern Med. 2003, 254(4): 322-334).
- the carboxyl group of the Melagatran group is converted into an ester group, which is converted into an oral prodrug - ximelga group (Boos CJ, et. al.
- ximelagatran is the first orally available anticoagulant that emerged more than 50 years after the development of heparin. It is less interfered by food and other drugs, its efficacy is predictable, and anticoagulation monitoring is not required. However, due to its hepatotoxicity, in February 2006 Astrazeneca announced the withdrawal of ExantaTM from the market. Dabigatran (BIBR 953 ) contains a trisubstituted benzimidazole core backbone containing a 4-amino phenylalanine analog that binds to the thrombin catalytic site (Bates SM, et.al. Br J Pharmacol.
- the ideal anticoagulant should have the following characteristics: good anticoagulant activity, predictable efficacy, no need for laboratory monitoring, oral administration, fast onset, and low side effects. Current anticoagulants have not met these requirements. Small peptides and small peptides or compounds that can be taken orally will be the focus of future anticoagulant drugs, and small molecule peptide thrombin inhibitors have made great progress, and reducing side effects is the future direction.
- the primary structure of the anticoagulant polypeptide provided by the present invention is as follows - (D) - FPRP-X - QGDFEPI PEDAYDE-NH 2 ;
- the hydrazine is alanine; D is aspartic acid; E is glutamic acid; F is phenylalanine; (D) - F is D-type phenylalanine; G is glycine; I is different Leucine: K is a lysine modified with a fatty acid (such as stearic acid); P is a proline; Q is glutamine; R is arginine; S is serine; W is tryptophan; Y is Tyrosic acid, AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid, and E-NH 2 is carboxyl amidated Glutamate.
- Each of the above amino acids is an L-form amino acid unless otherwise specified.
- the above polypeptide is one of the following 8 polypeptides:
- Peptide 2 (D) -FPRP- AEEA- G-QGDFEPIPEDAYDE- NH 2 ,
- Peptide 3 (D) -FPRP-GKGG-QGDFEPIPEDAYDE-NH2 ,
- Peptide 4 (D) -FPRP-GG G-QGDFEPI PEDAYDE- NH 2 ,
- Peptide 5 (D) -FPRP-GGGK-QGDFEPIPEDAYDE-NH2 ,
- Peptide 7 (D) -FPRP- RGDWP- QGDFEPIPEDAYDE- NH 2 ,
- Peptide 8 (D) -FPRP- KGGG- QGDFEPIPEDAYDE- NH 2 .
- the amino acid sequence of polypeptide 1 is shown in SEQ ID NO: 1 in the sequence listing
- the amino acid sequence of polypeptide 2 is shown in SEQ ID NO: 2 in the sequence listing
- the amino acid sequence of polypeptide 3 is as shown in SEQ ID NO: 3 in the sequence listing
- the amino acid sequence of polypeptide 4 is 90
- the amino acid sequence of the polypeptide 5 is as shown in the sequence 5 in the sequence listing
- the amino acid sequence of the polypeptide 6 is as shown in the sequence 6 in the sequence listing
- the amino acid sequence of the polypeptide 7 is as shown in the sequence 7 in the sequence listing.
- the amino acid sequence of polypeptide 8 is shown in SEQ ID NO:8 in the Sequence Listing.
- polypeptide 1, polypeptide 2, polypeptide 3 and polypeptide 6 are preferably the anticoagulant activity, and polypeptide 3 is the most excellent in anticoagulant activity.
- the polypeptide provided by the present invention can simultaneously bind to the substrate catalytic region and fibrin binding region of thrombin, and has strong anticoagulant activity.
- the amino terminus is a hydrophobic (D)-FPRP sequence that binds to the bottom of the thrombin: catalytic region, and thrombin can slowly cleave the amide bond between RPs after binding (Witting JI, et.al. Biochem J, 1992, 283: 737-743), inactivated.
- the amino acid sequence at the carboxy terminus thereof is completely electronegative and binds to the fibrin binding region of thrombin.
- the 100 column replaces the twelve amino acid residues (NGDFEEIPEEYL) at the carboxy terminus of biruludin (hirulog-1), which has a stronger electronegativity at the carboxy terminus and a stronger affinity for thrombin.
- the carboxy terminus and the amino terminus are linked by a flexible amino acid sequence, and at least one amino acid in the flexible sequence is replaced with a synthetic organic compound.
- lysine with a stearic acid side chain is introduced into the sequence. Acid, in order to slow down the degradation of the polypeptide by thrombin and enhance the anticoagulant activity.
- the polypeptide of the present invention can be produced by solid phase synthesis, liquid phase synthesis or the like.
- solid phase synthesis an amino acid sequence is coupled one by one to the resin to form a polypeptide of the present invention.
- the polypeptide of the present invention can be synthesized using an ABI433 type solid phase synthesizer, and the modification of the polypeptide is carried out manually.
- the amino acid used in the synthesis is protected by Fmoc, and the resin used is Rink resin or Wang resin.
- 1-hydroxybenzotriazole (HoBt) is dissolved in N-methylpyrrolidone (NMP) as an activator.
- DCC Dicyclohexylcarbodiimide
- a protecting group is removed using Piperidine.
- the amino acids all have an L-type chemical structure (except Fmoc-D-Phe- 0H) which are sequentially coupled to Rink or Wang resin.
- the amount of the resin used and the amount of the Fmoc-protected amino acid used were in a molar ratio of 1:5, and the protected amino acids were as follows: Fmoc-D-Phe- 0H, Fmoc-Pro- 0H, Fmoc-Arg (Pbf) - 0H, Fmoc-Gly - 0H, 115 Fmoc-Asn (Trt) -0H> Fmoc- Asp (OtBu) - 0H, Fmoc-Phe-0H, Fmoc-Glu (OtBu) - 0H, Fmoc_Ile_0H, Fmoc-Tyr (tBu) -0H> Fmoc Leu- 0H, Fmoc-Gln (Trt) -OH, Fmoc- Ala-OH, and artificially modified Fmoc- Lys (stearic) - 0H, Fm
- the lysate for the above-mentioned synthesized peptide resin (composition: dithiol threitol (DTT) 0.5 g, water 120 0.5 ml, trifluoroacetic acid (TFA) 8. 8 ml and triisopropylsilyl (TIPS) 0 2ml) cleavage 2. 5 ⁇ 3. 0 hours, filtration, the filtrate is evaporated to the most trifluoroacetic acid by rotary evaporator, precipitated with pre-cooled anhydrous ether, filtered to obtain the initial peptide, with deionized water or diluted The solid was dissolved in aqueous ammonia, filtered, and the filtrate was lyophilized.
- DTT dithiol threitol
- TIPS triisopropylsilyl
- the above lyophilized crude peptide was purified by reverse phase HPLC.
- the purification column was reversed (:, 8 semi-preparative column (Zorbax, 300SB-C18, 9. 4 mm x 25 cm), and the gradient eluent was acetonitrile with different gradients (including 0). 1% TFA) 125 / water (containing 0.1% TFA), the target peak was collected, most of the acetonitrile was removed by rotary evaporation, and lyophilized to obtain a pure peptide.
- the polypeptide of the present invention can be used for the prevention and treatment of post-operative deep vein thrombosis, for anticoagulant therapy in patients with unstable angina pectoris undergoing percutaneous transluminal coronary angioplasty (PTCA), or with platelet glycoprotein Il b/IIIa Inhibitors are used in combination with anticoagulant therapy in patients undergoing percutaneous coronary intervention (PCI), and in patients who replace heparin for heparin-induced thrombocytopenia requiring anticoagulation therapy.
- PTCA percutaneous transluminal coronary angioplasty
- PCI percutaneous coronary intervention
- polypeptide of the present invention has stronger anticoagulant activity than bivalirudin.
- the polypeptide of the present invention can be used for the preparation of a medicament for preventing and/or treating a thrombotic disease.
- Another object of the present invention is to provide a medicament for preventing and/or treating a thrombotic disease.
- the medicament for preventing and/or treating a thrombotic disease provided by the present invention has an active ingredient which is a polypeptide prepared as described above.
- a drug comprising a polypeptide, a truncated derivative, a derivative, and a composition of the present invention may be administered directly to a patient or may be administered to a patient after mixing with a suitable carrier or excipient.
- the carrier comprises: a water-soluble carrier such as polyethylene glycol, polyvinylpyrrolidone or an organic acid; a poorly soluble carrier such as ethyl cellulose or cholesterol stearate; an enteric carrier such as cellulose acetate. Phthalate or carboxymethylcellulose.
- the medicament of the present invention can be formulated into the following dosage forms: tablets, suppositories, solutions, capsules, aerosols, effervescent tablets and drops, and the like.
- Injectable administration including intravenous, subcutaneous or intraluminal injection; mucosal administration, such as nasal administration, in the local effect or transmucosal absorption; systemic administration, such as rectal administration, local It works or absorbs the whole body.
- Figure 1 shows the HPLC analysis of purified peptide 1.
- Figure 2 is a HPLC analysis of the purified polypeptide 2.
- Figure 3 is a HPLC analysis of the purified peptide 3.
- Figure 4 is a HPLC analysis of the purified peptide 6.
- Figure 5 shows the HPLC analysis of purified peptide 7.
- Figure 6 shows the APTT activity of polypeptide 3 in rats.
- Benzotriazole (HoBt, a product of Advanced Chemtech, USA) is dissolved in N-methylpyrrolidone (product of ⁇ , ⁇ company) as an activator, using dicyclohexylcarbodiimide (DCC Acros) as a condensing agent, Piperidine (Pieridine, Shanghai Jill Biochemical) deprotection, according to the operating instructions of the US Applied Biosystems ABI433A solid phase synthesizer, appropriate extension coupling time (60-90min) and deprotection time (20-30 min), synthetic peptide - Resin.
- DCC Acros dicyclohexylcarbodiimide
- the above crude peptide was taken up to 30.0 mg, which was purified by HPLC.
- the column was a reverse phase C 18 semi-preparative column (Zorbax, 300SB-C18, 9. 4mm x 25cm).
- Mobile phase A, acetonitrile (containing 0.1% by volume of TFA); B, water (containing 0.1% by volume of TFA).
- the elution gradient was: l-25 min, 15%-70% by volume A, flow rate 3 ml/min, UV214nm detection, 5 mg per load.
- the target fractions were collected, 175 was evaporated to remove most of the acetonitrile, and lyophilized to give a pure peptide. 19. 2 mg o
- the purity analysis of the polypeptide 1 is shown in Fig. 1.
- A acetonitrile (containing 0.1% by volume of TFA);
- B water (including 0.1% by volume)
- Analytical column Kromasil, C-18 column (Beijing Analytical Instrument Factory), 5 m, ⁇ 4. 6 hidden x250 ⁇ .
- the amino acid sequence of polypeptide 1 was determined as shown in SEQ ID NO:1 in the sequence listing.
- C IS column is a reverse phase semi-preparative column (Zorbax, 300SB-C18, 9. 4mrax25cm).
- Mobile phase A, acetonitrile (containing 0.1% by volume of 205 TFA); B, water (containing 0.1% by volume of TFA).
- the elution gradient was: 1- 25 min, 15% - 70% by volume A, flow rate 3 ml/min, UV 214 nm detection, 5 mg per load.
- the target fractions were collected, most of the acetonitrile was removed by rotary evaporation, and lyophilized to give a pure peptide of 21. 8 mg.
- the purity analysis of polypeptide 2 is shown in Figure 2.
- A acetonitrile (containing 0.1% by volume of TFA);
- B water (containing 0.1% by volume of TFA);
- Analytical column Kromasil, C- 18 column (Beijing Analytical Instrument Factory), 5 ⁇ ⁇ , ⁇ 4. 6 mm x 250 mm. 220
- the amino acid sequence of polypeptide 2 was determined as shown in SEQ ID NO: 2 in the sequence listing.
- the hydroxy group is 0. 5mmol
- the amount of 1-hydroxyl is 0. 5mmol
- the amount of the 1-hydroxyl group is 0. 5mmol
- the amount of 1-hydroxyl Benzotriazole is dissolved in N-methylpyrrolidone (product of ⁇ , ⁇ company) as an activator, dicondensed with dicyclohexylcarbodiimide (DCC, Acros) Mixture, deprotected with piperidine (Pieridine, Shanghai Jill Biochemical), according to the operating instructions of the American Applied Biosystems ABI433A solid phase synthesizer, properly extend the coupling 260 time (60-90min) and deprotection time (20-30 min) , synthetic peptide-resin.
- DCC dicyclohexylcarbodiimide
- the above crude peptide was taken up to 30.0 mg, which was purified by HPLC.
- the column was a reverse phase C 18 semi-preparative column (Zorbax, 300SB-C18, 9. 4mm x 25cm).
- Mobile phase A, acetonitrile (containing 0.1% by volume of TFA); B, water (containing 0.1% by volume of TFA).
- the elution gradient was: 1- 25 min, 15% - 70% 270 vol. A, flow rate 3 ml/min, UV 214 nm detection, 5 mg per load. 5mg ⁇
- the target component was collected, the acetonitrile was removed by rotation and lyophilized to give a pure peptide 16. 5mg.
- the purity analysis of peptide 6 is shown in Figure 4.
- Analytical column Kromasi l, C- 18 column (Beijing Analytical Instrument Factory), 5 ⁇ ⁇ , ⁇ 4 ⁇ 6 mmx250 mm.
- the amino acid sequence of polypeptide 6 was determined as shown in SEQ ID NO:6 in the Sequence Listing.
- 5 ⁇ Triazole (HoBt, a product of Advanced Chemtech, USA) is dissolved in N-methylpyrrolidone (product of ⁇ , ⁇ company) as an activator, condensed with dicyclohexyl carbyl carbodiimide (DC Acros) Mixture, deprotected with piperidine (Piperidine, Shanghai Jill Biochemical), according to the operating instructions of the American Applied Biosystems ABI433A solid phase synthesizer, properly extend the coupling time (60-90min) and deprotection time (20-30min), Synthetic peptide-resin.
- N-methylpyrrolidone product of ⁇ , ⁇ company
- DC Acros dicyclohexyl carbyl carbodiimide
- A acetonitrile (containing 0.1% by volume of TFA);
- B water (containing 0.1% by volume of TFA);
- amino acid sequence of polypeptide 7 was determined as shown in SEQ ID NO: 7 in the Sequence Listing.
- polypeptide 4 For the synthesis and purification methods of polypeptide 4, polypeptide 5 and polypeptide 8, reference is made to the methods for synthesizing and purifying the above polypeptide 1, polypeptide 2, polypeptide 3, polypeptide 6 and polypeptide 7.
- the amino acid sequence of the polypeptide 4 is as shown in the sequence 4 in the sequence listing
- the amino acid sequence of the polypeptide 5 is as shown in the sequence 5 in the sequence listing
- the amino acid sequence 320 of the polypeptide 8 is as shown in the sequence 8 in the sequence listing.
- the buffer solution 0. 05 ⁇ ol / L NaCl 0. 05mol / L Tris-HC1 solution, PH7. 4.
- fibrinogen solution including pig, cow and human fibrinogen solution, porcine 325 fibrinogen was purchased from Bio-rad, human fibrinogen was purchased from China National Institute for the Control of Pharmaceutical and Biological Products, Bovine 'fibrinogen was purchased from sigma, prepared with the above buffer).
- Thrombin solutions with different concentration gradients including pig, cow and human thrombin solutions, pig, cow and human thrombin were purchased from China National Institute for the Control of Pharmaceutical and Biological Products, sigma and China National Institute for the Control of Pharmaceutical and Biological Products): 20NIH/ml (for a titration volume, both IV is 0.1 ATU),
- Each anticoagulant polypeptide solution 5 mg of each of the polypeptides prepared in the above Example 1 was weighed and prepared into a solution having a concentration of 2.5 mmol/L using the above buffer solution. The anticoagulant activity of each polypeptide was further diluted to a suitable concentration during the experiment.
- Bivalirudin (Himlog-1): purchased from Chengdu Kaijie Biomedical Technology Co., Ltd., weighed 5mg, and prepared a solution of 2. 5mmol/L with the above buffer. Dilute to the appropriate concentration during the experiment.
- the anticoagulant activity was measured by thrombin titration (Markwardt F. Methods Enzymol, 1970, 69: 924-932. Chen Huayou et al., Biotechnology, 2002, 12(6): 24-25).
- the anticoagulant activity of polypeptide 3 is exemplified by polypeptide 3 and porcine thrombin at a concentration of 0.1 mM. Specific steps such as
- Example 1 the anticoagulant activity of the polypeptide 3 prepared in the above Example 1 was 101 V, that is, 10.1 ATU, and the experiment was set to three repetitions, and the average value of three repetitions was taken as an experimental result.
- Anticoagulant peptide Anticoagulant activity (ATU/paint ol) Anticoagulant activity (ATU/ ⁇ ol) Anticoagulant activity (ATU/implicit ol)
- the clotting activity is 1-4 times that of the control peptide
- the anticoagulant activity against bovine thrombin is 3-9 times that of the control peptide
- the anticoagulant activity against human thrombin is equal to or nearly 2 times that of the control peptide.
- Buffer 0.01 M Hepes/0.01 M Tris, 0.1 M NaCl, 0.1% PEG 6000, pH 7.4.
- Substrate Chromozym TH (Tos-Gly-Pro-Arg-PNA, available from Roche), formulated to a concentration of 25 ⁇ 33 ⁇ , 40 ⁇ , 50 ⁇ , 100 ⁇ , 125 ⁇ , 200 ⁇ , 330 ⁇ , 500 365 ⁇ , 1000 ⁇ .
- Bovine thrombin (purchased from sigma) Solution: formulated into 5 NIH/mlo
- Concentration of anticoagulant peptide The polypeptide 1 prepared in the above Example 1, the polypeptide '3, the polypeptide 6 and the polypeptide 7 were formulated into 0.05 ⁇ , 0.10 ⁇ , and 0.15 ⁇ , respectively.
- polypeptide prepared in the above Example 1 and the control peptide bivalirudin (hirulog-1) inhibited the inhibition constant Ki value of bovine thrombin, as shown in Table 2.
- Example 4 Activated partial thromboplastin time (APTT) Evaluation of activity of polypeptide 3 in animals Activated partial thrombin time assay kit (Germany TEC0 GmbH product, batch number:
- Coatron Ml coagulometer and disposable coagulation cuvette are made by TEC0 in Germany; 3K15 refrigerated centrifuge is Sigma.
- SD rats male, weighing 300 ⁇ 20 g, were provided by the Experimental Animal Center of the Academy of Military Medical Sciences.
- the SD rats were grouped into groups of 8 rats each with 1.0, 0.6 and 0.3 mol/kg in the tail vein.
- the APTT value of 405 peptides reached the peak plasma concentration was significantly higher than that of biruludine (Hirulog-1), which was more than twice that of Hirulog-1.
- Hirulog-1 biruludine
- the activity of peptide 3 in vivo was long, 12 hours later.
- the APPT value is still 4 seconds higher than the APPT value at Oh, while Hirulog-1 is inactive at 4 hours. 0. 3 ol/kg Peptide 3
- the APTT value was still higher than the APPT value of Oh for 2 seconds, indicating that Peptide 3 has high anticoagulant activity and excellent pharmacokinetic properties.
- SD rats male, weighing 200 ⁇ 20 g, were provided by the Experimental Animal Center of the Academy of Military Medical Sciences.
- the SD rats were divided into normal saline group, Hirulog-l-30min group, Hirulog-1-10min group, polypeptide 3-2h group, and polypeptide 3-4h group, 8 rats in each group.
- the doses of the Hirulog-l-30min group and the Hirulog-10-10rain group were ⁇ ⁇ /kg body weight, and the doses of the peptide 3- 2h group and the polypeptide 3-4h group were 1.
- the above groups of rats were intraperitoneally injected with 3% by mass of sodium pentobarbital solution for anesthesia at a dose of 50 mg/kg body weight.
- the saline or anticoagulant peptide was injected into the tail vein, 30 min after the saline group, 30 min after the Hirulog-1- 30 min group, 10 min after the Hirulog-1-lOmin group, and 2 h after the peptide 3-2 h group, the peptide 3-
- use a scalpel to cut at 2 rounds from the tip of the rat, and put it quickly.
- the interval time refers to the time from the administration to the end of the tail.
- Wi star rats male and female, weighing 300 ⁇ 20 g, were provided by the Experimental Animal Center of the Academy of Military Medical Sciences.
- the rats were randomly divided into groups of 8 rats, respectively, saline group and Hirulog-1 group. And peptide 3 in different dose groups.
- the dose of the Hirulog-1 group was 1.0 mol/kg, and the doses of the peptide 3 group were 1, 0.6, 0.3, and 0. ⁇ /kg o
- Example 1 administered 1 polypeptide prepared in Example 3 above 0.6 or 0. 3 ⁇ ⁇ 1 dose of almost complete inhibition of venous thrombosis, give 0. ⁇ / 5h before ligation
- the prepared polypeptide 3 still has stronger anti-venous thrombogenic activity than Hirulog-1.
- the invention designs and synthesizes 450 kinds of anticoagulant polypeptides according to the structural characteristics and action modes of thrombin and the anticoagulant drug bivalirudin, and the polypeptide of the invention not only can efficiently and specifically inhibit thrombin activity, but also has the same
- the number of amino acids is comparable to that of bivalirudin, and the synthesis is easy and the anticoagulant activity is stronger.
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Description
抗凝血多肽及其应用 技术领域
本发明涉及抗凝血多肽及其应用, 属于生物医药领域。
背景技术
凝血是机体的重要生理防御过程, 但病理性血栓严重危害人类健康。 目前国 内外临床使用的抗凝血药物主要有普通肝素、 低分子量肝素和华法林等, 这些药 物存在导致血小板减少和出血等副作用或需要密切的实验室监测等缺点。 而多肽 类抗凝血药物, 如水蛭素、 比伐卢定以及可口服的小分子肽类药物 (如希美加群 等) 等, 具有起效快、 副作用较小等突出优点, 是今后抗凝血药的重点发展方 向。
凝血酶在血栓形成过程中起以下作用: (1 ) 裂解纤维蛋白原形成纤维蛋白; ( 2 ) 激活因子 、 \1及 XI, 这些因子反过来形成更多的凝血酶; (3 ) 刺激血 小板。 此外, 通过激活因子 VDI使纤维蛋白交联, 稳定栓塞。 凝血酶具有三个结合 区: 纤维蛋白结合区、 底物催化 (活性) 区及肝素结合区。 凝血酶由含 36 个氨 基酸的 A链及 259个氨基酸的 B链组成, A链与 B链由二硫键连接, 像其他丝氨 酸蛋白酶一样, 它包括一个典型的催化三元体 (Aspl02、 Hi s57及 Ser l95 ) , 其 S 1结合区象一个深的口袋, 底部的 Aspl89能与底物的氨基或胍基形成氢键或静 电相互作用, 该区的疏水区可结合疏水基团。 与其他胰蛋白酶不同的是还有第二 个疏水区 (S2 ) , 该区含有疏水的 YPPW环。 此外, 酸性的 Glul 92残基处于口袋 的入口处, 对识别底物及抑制剂十分重要。 凝血酶的纤维蛋白结合区及肝素结合' 区主要由数个精氨酸及赖氨酸组成, 可与电负性氨基酸及基团结合。
水蛭素是最早发现的多肽类凝血酶直接抑制剂, 由 65 个氨基酸残基组成, 自然界中有多种水蛭素变异体, 其中, 抗凝活性较 的主要有 HV1、 HV2 及 HV3 等 (Salzet M.Curr Pharm Des,2002,8(7):493-503 ) 。 水蛭素可结合凝血酶的底物 催化区和纤维蛋白结合区, 与凝血酶形成 1: 1 的复合物 (结合常数 Ki 高达 20 fM ) ( Krsttenansky J L,et al.FEBS Lett, 1990, 269: 425-429 ) , 阻止凝血酶将纤维 蛋白原降解成纤维蛋白单体, 从而抑制纤维蛋白单体的凝结 (Greinacher A,et al. Thromb Haemost. 2008, 99(5):819-29)。 水蛭素具有不能口服、 体内易降解等缺 点, 可通过结构修饰来克服。 如用 PEG分子或硬脂酸、 油酸等亲脂化合物修饰的 水蛭素可获得较长的半衰期。 RGD 融合水蛭素则具有抗血小板聚集功能。 注射用 的基因工程双功能水蛭素 (RGD-hirudin ) 已于 2005年获国家食品药品监督管理 局批准进入临床研究, 临床适应症为血管吻合术后的抗凝、 防栓治疗, 并可用于 治疗心绞痛、 深静脉血栓等疾病。
由水蛭素衍生出的小分子肽很多, 有的只结合凝血酶催化区, 有的只结合凝 血酶的纤维蛋白结合区, 活性不理想, 有的活性虽然较髙, 但合成较难。 比伐卢 定 (Bivalirudin , hirulog-1 ) 是人工合成的水蛭素衍生物, 含有 20 个氨基酸残 基, 其氨基端能结合凝血酶的底物催化区, 而其呈酸性的羧基端则能结合凝血酶
的纤维蛋白结合区 (Bourdon P,et,al. FEBS Lett, 1991, 293: 163-166 ) , 中间由 4 个 Gly 组成一个柔性的连接部分。 比伐卢定比水蛭素有更宽的治疗窗及安全性 (Hartmann F, et al. Curr Pharm Des. 2008, 14(12): 1 191-6), 但缺点是两者都不能 口服, 体内易降解, 半衰期较短。
小分子肽类抗凝剂主要有阿加曲班、 希美加群和达比加群等。 阿加曲班由 N - α -苯磺酰基 -L-精氨酸甲酯改造而成, 能可逆、 选择性地结合凝血酶催化位点 (Preville P, et al. Bioorg Med Chem Lett, 1997, 7(12): 1563-1566 ) 。 近年来, 一 些阿加曲班类似物被合成 (Salvagnini C,et al. Eur J Med Chem. 2007, 42(1): 37- 53. ) , 如将其强碱性的胍基替换成亲脂性的侧链, 以提高其细胞通透性及口服 利用度。 美拉加群是 D- Phe-Pro- Arg 三肽序列的类似物, 其中苯脒基代替精氨 酸, azetidine-2-羧酸代替脯氨酸, D-型环己基甘氨酸代替 D-苯丙氨酸, 美拉加 群能可逆地抑制凝血酶的催化位点 (Gustafsson D,et.al. J Intern Med. 2003, 254(4): 322-334 ) 。 将美拉加群的羧基变成酯基, 脒基加胺肟基, 可转化成口服的前药- 希美加群 (Boos C J,et.al. Eur J Intern Med, 2005, 16(4):267-278), 希美加群是继肝 素发展 50多年后出现的第一个可口服的抗凝血药, 它受食物和其他药物的干扰 小, 药效可以预测, 不需要抗凝血监测, 但由于其肝毒性, 2006 年 2 月 Astrazeneca 公司宣布将希美加群 (ExantaTM ) 从市场上撤回。 达比加群 (dabigatran, BIBR 953 ) 包含三取代的苯并咪唑核心骨架, 含有一个 4-氨基的 苯丙氨酸类似物, 可结合凝血酶催化位点 (Bates S M,et.al. Br J Pharmacol. 2005 ,144(8): 1017- 1028 ) , 达比加群酯化后转变成可口服的 BIBR 1048 (dabigatran exexilate) , 目前已进入 III期临床试验 (Sanford M,et al. n gs, 2008, 68(12): 1699-709)。
理想的抗凝血药物应该具有如下特性: 抗凝活性良好、 药效可以预测、 不需 要实验室监测、 可口服、 起效快和副作用小等。 目前的抗凝药还未能满足这些要 求。 小肽及可口服的小分子肽类或化合物将是今后抗凝血药物的重点研究方向, 而小分子肽类凝血酶抑制剂已经取得了很大进展, 减少副作用是今后的努力方 向。
发明公开
本发明的目的是提供抗凝血多肽。
本发明所提供的抗凝血多肽的一级结构通式如下- (D) -FPRP-X -QGDFEPI PEDAYDE-NH2;
其中: 选自 G、 AEEA、 K和 R中的任意一个, 选自 G、 K和 R中的任意一 个或 缺失, X3选自 G、 K和 D中的任意一个或 ¾缺失, Χ4选自 G、 K、 W和 D中 的任意一个, X5选自 P和 S中的任意一个或 X5缺失;
其中, 所述 Α为丙氨酸; D为天冬氨酸; E为谷氨酸; F为苯丙氨酸; (D) - F 为 D型苯丙氨酸; G为甘氨酸; I 为异亮氨酸: K为修饰有脂肪酸 (如硬脂酸) 的赖氨酸; P为脯氨酸; Q为谷氨酰胺; R为精氨酸; S为丝氨酸; W为色氨酸; Y为酪氧酸, AEEA为 2- (2- (2-氨基乙氧基)乙氧基)乙酸, E- NH2为羧基端酰胺化
的谷氨酸。
上述各氨基酸如未特别指明, 均为 L型氨基酸。
上述多肽为如下 8个多肽之一:
80 多肽 1 : (D) - FPRP- GGGG- QGDFEPIPEDAYDE- NH2,
多肽 2: (D) -FPRP- AEEA- G-QGDFEPIPEDAYDE- NH2,
多肽 3: (D) -FPRP-GKGG-QGDFEPIPEDAYDE-NH2 ,
多肽 4: (D) -FPRP-GG G-QGDFEPI PEDAYDE- NH2,
多肽 5: (D) -FPRP-GGGK-QGDFEPIPEDAYDE-NH2 ,
85 多肽 6: (D) - FPRP- GRGDS- QGDFEP I PEDAYDE- NH2,
多肽 7: (D) -FPRP- RGDWP- QGDFEPIPEDAYDE- NH2,
多肽 8: (D) -FPRP- KGGG- QGDFEPIPEDAYDE- NH2。
多肽 1的氨基酸序列如序列表中序列 1所示, 多肽 2的氨基酸序列如序列表 中序列 2所示, 多肽 3的氨基酸序列如序列表中序列 3所示, 多肽 4的氨基酸序 90 列如序列表中序列 4所示, 多肽 5的氨基酸序列如序列表中序列 5所示, 多肽 6 的氨基酸序列如序列表中序列 6所示, 多肽 7的氨基酸序列如序列表中序列 7所 示, 多肽 8的氨基酸序列如序列表中序列 8所示。
上述多肽中, 抗凝活性较好的是多肽 1、 多肽 2、 多肽 3和多肽 6, 抗凝活 性最好的是多肽 3。
95 本发明所提供的多肽能同时结合凝血酶的底物催化区和纤维蛋白结合区, 具 有很强的抗凝血活性。 其氨基端为疏水性的(D) - FPRP 序列, 能结合凝血酶的底: 物催化区, 而凝血酶在与其结合后能缓慢切割 R-P 之间的酰胺键 (Witting J I, et.al. Biochem J, 1992,283:737-743 ) , 使其失活。 其羧基端的氨基酸序列整体呈 电负性, 能结合凝血酶的纤维蛋白结合区, 本发明用水蛭素变种 III的羧基端序
100 列 (QGDFEPIPEDAYDE ) 替换比伐卢定 (hirulog-1 ) 的羧基端的十二个氨基酸残基 ( NGDFEEIPEEYL ) , 替换后的羧基端具有更强的电负性, 与凝血酶的亲和力更 强。 而羧基端与氨基端之间则用柔性的氨基酸序列连接起来, 用人工合成的有机 化合物替换了柔性序列中的至少一个氨基酸, 另外, 在序列中引入了带有硬脂酸 侧链的赖氨酸, 以期减缓凝血酶对多肽的降解, 增强抗凝活性。
105 本发明的多肽可以通过固相合成、 液相合成等方法进行制备。 例如, 采用固 相合成, 在树脂上逐个偶联氨基酸序列, 以形成本发明的多肽。
本发明的多肽可以用 ABI433型固相合成仪合成, 多肽的修饰手工完成。 该 合成使用的氨基酸以 Fmoc保护, 使用的树脂为 Rink树脂或 Wang树脂。 合成时 将 1_羟基苯并*** (HoBt ) 溶解在 N-甲基吡咯烷酮 (NMP) 中作为活化剂, 使用
110 二环己基碳二亚胺 (DCC ) 作为缩合剂, 使用哌啶 (Piperidine) 除去保护基。 氨 基酸均具有 L-型化学结构 (Fmoc-D-Phe- 0H除外) , 它们被依次偶联在 Rink或 Wang树脂上。
树脂的使用量与 Fmoc保护氨基酸的使用量按 1 : 5的摩尔比进行, 保护氨基 酸如下: Fmoc- D-Phe- 0H、 Fmoc-Pro- 0H、 Fmoc-Arg ( Pbf ) - 0H、 Fmoc-Gly- 0H、
115 Fmoc-Asn (Trt) -0H> Fmoc- Asp (OtBu) - 0H、 Fmoc-Phe-0H、 Fmoc- Glu (OtBu) - 0H、 Fmoc_Ile_0H、 Fmoc-Tyr (tBu) -0H> Fmoc Leu- 0H、 Fmoc-Gln (Trt) -OH, Fmoc- Ala-OH, 而人工改造而成的 Fmoc- Lys (stearic) - 0H、 Fmoc- AEEA- 0H、 Fmoc- PABA以及 Fmoc-PAMBA为本实验室合成, 氨基酸的偶联按照仪器操作规程进行。
上述合成后的肽树脂用裂解液 (组成: 二巯基苏糖醇 (DTT ) 0. 5g , 水 120 0. 5ml , 三氟乙酸 (TFA) 8. 8ml 和三异丙基硅垸 (TIPS ) 0. 2ml ) 裂解 2. 5〜3. 0 小时, 过滤, 滤液用旋转蒸发仪蒸去大部分三氟乙酸后, 用预冷的无水***进行 沉淀, 过滤得到初肽, 用去离子水或稀氨水溶解固体, 过滤, 滤液冻干。
上述冻干粗肽用反相 HPLC进行纯化, 纯化柱为反相(:,8半制备柱 (Zorbax, 300SB- C18, 9. 4 mmx25cm) , 梯度洗脱液为含有不同梯度的乙腈 (含 0. 1%TFA) 125 /水 (含 0. 1%TFA) , 收集目标峰, 旋转蒸发除去大部分乙腈, 冻干得到纯肽。
本发明的多肽可以用于手术后深静脉血栓的预防及治疗, 用于行经皮冠状动 脉腔内成形术 (PTCA) 的不稳定型心绞痛患者的抗凝治疗, 或者与血小板糖蛋白 Il b/IIIa 抑制剂联合用于经皮冠脉介入 (PCI ) 治疗的患者的抗凝治疗, 以及替 代肝素用于需要抗凝治疗的肝素引起的血小板减少症的患者。
130 实验表明, 本发明的多肽与比伐卢定相比, 其抗凝血活性更强。
本发明的多肽可以用于制备预防和 /或治疗血栓性疾病的药物。
本发明的另一个目的是提供一种预防和 /或治疗血栓性疾病的药物。 本发明所提供的预防和 /或治疗血栓性疾病的药物, 其活性成份是上述制备 的多肽。
135 具体应用时, 可以将含有本发明的多肽、 多肽的截断物、 衍生物及组合物的 药物直接给予病人, 或者与适宜的载体或者赋型剂混合后给予病人。 所述载体包 括: 水溶性载体, 如聚乙二醇、 聚乙烯吡咯垸酮或有机酸等; 难溶性载体, 如乙 基纤维素或胆固醇硬脂酸酯等; 肠溶性载体, 如醋酸纤维素酞酸酯或羧甲乙纤维 素等。
140 本发明的药物可以制成如下的剂型: 片剂、 栓剂、 溶液、 胶囊、 气雾剂、 泡 腾片和滴剂等。 采用注射给药, 包括静脉注射、 皮下注射或腔内注射等; 粘膜给 药, 如鼻腔给药, 在局部起效或经粘膜吸收全身发挥作用; 腔道给药, 如经直肠 给药, 局部起效或者经吸收全身发挥作用。 上述给药途径中, 优选的是静脉注射 给药。
145 附图说明
图 1为多肽 1纯化后的 HPLC分析图谱。
图 2为多肽 2纯化后的 HPLC分析图谱。
图 3为多肽 3纯化后的 HPLC分析图谱。
图 4为多肽 6纯化后的 HPLC分析图谱。
150 图 5为多肽 7纯化后的 HPLC分析图谱。
图 6为多肽 3在大鼠体内的 APTT活性。
实施发明的最佳方式
下面结合具体实施例对本发明作进一步说明, 并非对本发明的限定, 依照本 领域公知的现有技术, 本发明的实施方式并不限于此, 因此凡依照本发明公开内
155 容所作出的本领域的等同替换, 均属于本发明的保护范围。
实施例 1、 多肽的合成和纯化
一、 多肽 1的合成和纯化
称取 0. 17g Rink树脂 (0. lramol,取代率 0. 6g/ramol,美国 Advanced Chemtech公司产品), Fmoc保护氨基酸 (美国 Advanced Chemtech公司产 160 品) 的用量为 0. 5mmol , 将 1-羟基苯并*** (HoBt,美国 Advanced Chemtech公 司产品) 溶解在 N-甲基吡咯烷酮 (ΝΜΡ, ΡΕ公司产品) 中作为活化剂, 以二环己 基碳二亚胺 (DCC Acros公司) 为缩合剂, 以哌啶 (Piperidine,上海吉尔生化)脱 保护, 按美国应用***生物的 ABI433A型固相合成仪的操作说明, 适当延长偶合 时间 (60-90min) 及脱保护时间 (20- 30min) ,合成肽-树脂。
165 取上述肽 -树脂 0. 5g, 放入 10ml裂解液 (组成: 0. 5g二巯基苏糖醇
(DTT) , 0. 5ml水, 8. 8ml三氟乙酸 (TFA) , 0. 2ml三异丙基硅垸) 中, 裂解 3. 0小时, 用 G3玻璃砂芯漏斗过滤, 用旋转蒸发仪蒸去大部分滤液至残余液体 约 2ml后, 用 100ml预冷的无水***进行沉淀, 静置 1小时, G4玻璃砂芯漏斗 过滤得到初肽, 用 1%体积百分含量的稀氨水溶解初肽固体, 滤液冻干得粗肽, 170 纯度约为 60%。
取上述粗肽 39. 0mg, 用 HPLC纯化。 色谱柱为反相 C18半制备柱 (Zorbax, 300SB-C18, 9. 4mmx25cm) 。 流动相: A, 乙腈 (含 0. 1%体积百分含量 TFA) ; B, 水 (含 0. 1%体积百分含量 TFA) 。 洗脱梯度为: l-25min, 15%-70% 体积百分含量 A, 流速 3ml/min, UV214nm检测, 每次上样 5mg。 收集目标组分, 175 旋转蒸发除去大部分乙腈, 冻干得到纯肽 19. 2mg o 多肽 1的纯度分析见图 1。
多肽 1纯化后的 HPLC分析图谱- 流速: lml/rain,
检测波长: 214nm,
柱温: 25°C,
180 梯度: 洗脱梯度为
0- lmin, (0-15%) A
1- 25rain, ( 15%- 70%) A
. 25- 27min, (70%- 90%) A
A, 乙腈 (含 0. 1%体积百分含量 TFA) ; B, 水 (含 0. 1%体积百分含
185 量 TFA) ;
上样量: 50 g,
分析柱: Kromasil , C-18柱 (北京分析仪器厂) , 5 m, Φ4. 6 隱 x250匪。 经测定, 多肽 1的氨基酸序列如序列表中序列 1所示。
二、 多肽 2的合成和纯化
190 称取 0. 17gRink树脂 (0. lmmol,取代率 0. 6g/mmol,美国 Advanced
Chemtech公司产品) ,各 Fmoc保护氨基酸 (美国 Advanced Chemtech公司产 品) 的用量为 0. 5瞧 ol , 将 1-羟基苯并*** (HoBt,美国 Advanced Chemtech公 司产品) 溶解在 N-甲基吡咯垸酮 (ΝΜΡ, ΡΕ公司产品) 中作为活化剂, 以二环己 基碳二亚胺 (DCC Acros公司) 为缩合剂, 以哌啶(Piperidine,上海吉尔生化)脱
195 保护, 按美国应用***生物的 ABI433A型固相合成仪的操作说明, 适当延长偶合 时间 (60- 90min) 及脱保护时间 (20- 30min) ,合成肽-树脂。
取上述肽-树脂 0. 5g , 放入 10ml 裂解液 (组成: 0. 5g 二巯基苏糖醇 ( DTT ) , 0. 5ml 水, 8. 8ml 三氟乙酸 (TFA ) , 0. 2ml-三异丙基硅垸) 中, 裂解 3. 0 小时, 用 G3 玻璃砂芯漏斗过滤, 用旋转蒸发仪蒸去大部分滤液至残余液体
200 约 2ml后, 用 100ml预冷的无水***进行沉淀, 静置 1 小时, G4玻璃砂芯漏斗 过滤得到初肽, 用 1%体积百分含量的稀氨水溶解初肽固体, 滤液冻干得粗肽, 纯度约为 65%。
取上述粗肽 39. Omg, 用 HPLC 纯化。 色谱柱为反相 CIS半制备柱 (Zorbax, 300SB-C18, 9. 4mrax25cm) 。 流动相: A, 乙腈 (含 0. 1%体积百分含量 205 TFA) ; B, 水 (含 0. 1%体积百分含量 TFA ) 。 洗脱梯度为: 1- 25min, 15%- 70% 体积百分含量 A, 流速 3ml/min, UV214nm检测, 每次上样 5mg。 收集目标组分, 旋转蒸发除去大部分乙腈, 冻干得到纯肽 21. 8mg。 多肽 2的纯度分析见图 2。
多肽 2纯化后的 HPLC分析图谱:
流速: lml/min,
210 检测波长: 214nm,
柱温: 25°C,.
梯度: 洗脱梯度为
0- lmin, (0-15%) A
1- 25min, ( 15%-70%) A
215 25-27min, ( 70%-90%) A
A, 乙腈 (含 0. 1%体积百分含量 TFA) ; B, 水 (含 0. 1%体积百分含 量 TFA) ;
上样量: 50 y g,
分析柱: Kromasil , C- 18柱 (北京分析仪器厂) , 5 μ ιη, Φ4. 6 mmx250mm。 220 经测定, 多肽 2的氨基酸序列如序列表中序列 2所示。
三、 多肽 3的合成和纯化
称取 0. 17g Rink树脂 (0. lmmol,取代率 0. 6g/mmol,美国 Advanced
Chemtech公司产品) ,各 Fmoc保护氨基酸 (美国 Advanced Chemtech公司产 品) 的用量为 0. 5mmol , 将 1-羟基苯并*** (HoBt,美国 Advanced Chemtech公 225 司产品) 溶解在 N-甲基吡咯垸酮 (ΝΜΡ, ΡΕ公司产品) 中作为活化剂, 以二环己 基碳二亚胺 (DCC Acros公司) 为縮合剂, 以哌啶(Piperidine,上海吉尔生化)脱 保护, 按美国应用***生物的 ABI433A型固相合成仪的操作说明, 适当延长偶合 时间 (60- 90min) 及脱保护时间 (20- 30min) ,合成肽-树脂。
取上述肽 -树脂 0. 5g, 放入 10ml裂解液 (组成: 0. 5g二巯基苏糖醇
230 (DTT) 0. 5ml水, 8. 8ml三氟乙酸 (TFA) , 0. 2ml三异丙基硅垸) 中, 裂解 3. 0小时, 用 G3玻璃砂芯漏斗过滤, 用旋转蒸发仪蒸去大部分滤液至残余液体 约 2ml后, 用 100ml预冷的无水***进行沉¾, 静置 1小时, G4玻璃砂芯漏斗 过滤得到初肽, 用 1%体积百分含量的稀氨水溶解初肽固体, 滤液冻干得粗肽, 纯度约为 55%
235 取上述粗肽 38. 0mg, 用 HPLC纯化。 色谱柱为反相 C18半制备柱
(Zorbax, 300SB-C18, 9. 4mmx25cm) 。 流动相: A, 乙腈 (含 0. 1%体积百分含量 TFA) B, 水 (含 0. 1%体积百分含量 TFA) 。 洗脱梯度为: l-25min 15%_70% 体积百分含量 A, 流速 3ml/min UV214nni检测, 每次上样 5mg。 收集目标组分, 旋转蒸发除去大部分乙腈, 冻干得到纯肽 14. 3mg。 多肽 3的纯度分析见图 3 240 多肽 3纯化后的 HPLC分析图谱:
流速: lml/min,
检测波长 ,· 214nm
柱温: 25°C
梯度: 洗脱梯度为
245 0-lmin, (0-45%) A
1- 25min (45%-80%) A
25- 27min 80%- 90%) A
A, 乙腈 (含 0. 1%体积百分含量 TFA) B, 水 (含 0. 1%体积百分含 量 TFA)
250 上样量: 50 g
分析柱: Kroraasil , C- 18柱 (北京分析仪器厂) , 5 u rn, Φ4. 6 250mm 经测定, 多肽 3的氨基酸序列如序列表中序列 3所示。
四、 多肽 6的合成和纯化
称取 0. 17g Rink树脂 (0. lmmol,取代率 0. 6g/mmol,美国 Advanced 255 Chemtech公司产品) ,各 Fmoc保护氨基酸 (美国 Advanced Chemtech公司产 品) 的用量为 0. 5mmol, 将 1_羟基苯并*** (HoBt,美国 Advanced Chemtech公 司产品) 溶解在 N-甲基吡咯垸酮 (ΝΜΡ, ΡΕ公司产品) 中作为活化剂, 以二环己 基碳二亚胺 (DCC, Acros公司) 为缩合剂, 以哌啶(Piperidine,上海吉尔生化)脱 保护, 按美国应用***生物的 ABI433A型固相合成仪的操作说明, 适当延长偶合 260 时间 (60-90min) 及脱保护时间 (20- 30min) ,合成肽-树脂。
取上述肽 -树脂 0. 5g, 放入 10ml裂解液 (组成: 0. 5g二巯基苏糖醇
(DTT) 0. 5ml水, 8. 8ml三氟乙酸 (TFA) , 0. 2ml三异丙基硅烷) 中, 裂解 3. 0小时, 用 G3玻璃砂芯漏斗过滤, 用旋转蒸发仪蒸去大部分滤液至残余液体 约 2ml后, 用 100ml预冷的无水***进行沉淀, 静置 1小时, G4玻璃砂芯漏斗 265 过滤得到初肽, 用 1%体积百分含量的稀氨水溶解初肽固体, 滤液冻干得粗肽, 纯度约为 65%
取上述粗肽 35. 0mg, 用 HPLC纯化。 色谱柱为反相 C18半制备柱 (Zorbax, 300SB-C18, 9. 4mmx25cm) 。 流动相: A, 乙腈 (含 0. 1%体积百分含量 TFA) ; B, 水 (含 0. 1%体积百分含量 TFA) 。 洗脱梯度为: 1- 25min, 15%- 70% 270 体积百分含量 A, 流速 3ml/min, UV214nm检测, 每次上样 5mg。 收集目标组分, 旋转蒸发除去大部分乙腈, 冻干得到纯肽 16. 5mg。 多肽 6的纯度分析见图 4。
多肽 6纯化后的 HPLC分析图谱:
流速: lml/min,
检测波长: 214nm,
275 柱温: 25°C,
梯度: 洗脱梯度为
0- lmin, (0-15%) A
1- 25min, ( 15%- 70%) A
25-27min, ( 70%-90%) A
280 A, 乙腈 (含 0. 1%体积百分含量 TFA) ; B, 水 (含 0. 1%体积百分含 量 TFA) ;
上样量: 50 μ g,
分析柱: Kromasi l , C- 18柱 (北京分析仪器厂) , 5 μ πι, Φ4· 6 mmx250mm。 经测定, 多肽 6的氨基酸序列如序列表中序列 6所示。
285 五、 多肽 7的合成和纯化
称取 0. 17g Rink树脂 (0. lramol,取代率 0. 6g/ramol,美国 Advanced Chemtech公司产品) ,各 Fmoc保护氨基酸 (美国 Advanced Chemtech公司产 品) 的用量为 0. 5mmol, 将 1-羟基苯并*** (HoBt,美国 Advanced Chemtech公 司产品) 溶解在 N-甲基吡咯垸酮 (ΝΜΡ, ΡΕ公司产品) 中作为活化剂, 以二环己 290 基碳二亚胺 (DC Acros公司) 为缩合剂, 以哌啶 (Piperidine,上海吉尔生化)脱 保护, 按美国应用***生物的 ABI433A型固相合成仪的操作说明, 适当延长偶合 时间 (60-90min) 及脱保护时间 (20-30min) ,合成肽-树脂。
取上述肽 -树脂 0. 5g, 放入 10ml裂解液 (组成: 0. 5g二巯基苏糖醇
(DTT) , 0. 5ml水, 8. 8ml三氟乙酸 (TFA) , 0. 2ml三异丙基硅烷) 中, 裂解 295 3. 0小时, 用 G3玻璃砂芯漏斗过滤, 用旋转蒸发仪蒸去大部分滤液至残余液体 约 2ml后, 用 100ml预冷的无水***进行沉淀, 静置 1小时, G4玻璃砂芯漏斗 过滤得到初肽, 用 1%体积百分含量的稀氨水溶解初肽固体, 滤液冻干得粗肽, 纯度约为 60%。
取上述粗肽 40. 0mg, 用 HPLC纯化。 色谱柱为反相 C,8半制备柱
300 (Zorbax, 300SB-C18, 9. 4mmx25cm) 。 流动相: A, 乙腈 (含 0. 1%体积百分含量 TFA) ; B, 水 (含 0. 1%体积百分含量 TFA) 。 洗脱梯度为: 1- 25min, 15%- 70% 体积百分含量 A, 流速 3ml/min, UV214nm检测, 每次上样 5mg。 收集目标组分, 旋转蒸发除去大部分乙腈, 冻干得到纯肽 18. 7mg。 多肽 7的纯度分析见图 5。
多肽 7纯化后的 HPLC分析图谱:
305 流速: lml/min,
检测波长: 214nm,
柱温: 25°C,
梯度: 洗脱梯度为
0-lmin, (0-15%) A
310 l-25min, ( 15%- 70%) A
25-27min, ( 70%- 90%) A
A, 乙腈 (含 0. 1%体积百分含量 TFA) ; B, 水 (含 0. 1%体积百分含 量 TFA) ;
上样量: 50 μ g,
315 分析柱: Kromasil , C- 18柱 (北京分析仪器广) , 5 μ ηι, Φ4. 6 mmx250mm。
经测定, 多肽 7的氨基酸序列如序列表中序列 7所示。
多肽 4、 多肽 5和多肽 8的合成与纯化方法参照上述多肽 1、 多肽 2、 多肽 3、 多肽 6和多肽 7的合成与纯化方法。 其中, 多肽 4的氨基酸序列如序列表中 序列 4所示, 多肽 5的氨基酸序列如序列表中序列 5所示, 多肽 8的氨基酸序列 320 如序列表中序列 8所示。
实施例 2、 抗凝血活性的测定
本实施例中涉及的各种溶液及其配制方法如下:
缓冲液: 含 0. 05匪 ol/L NaCl的 0. 05mol/L的 Tris- HC1溶液, PH7. 4。
0. 5%质量百分含量纤维蛋白原溶液 (包括猪、 牛及人的纤维蛋白原溶液, 猪 325 纤维蛋白原购自 Bio- rad公司, 人纤维蛋白原购自中国药品生物制品检定所, 牛 ·' 纤维蛋白原购自 sigma公司, 用上述缓冲液配制) 。
不同浓度梯度的凝血酶溶液 (包括猪、 牛及人的凝血酶溶液, 猪、 牛及人 的凝血酶分别购自中国药品生物制品检定所、 sigma公司及中国药品生物制品检 定所) : 浓度分别为 20NIH/ml (为 1个滴定体积, 既 IV为 0. 1ATU) 、
330 40NIH/ml (2V) 、 100 NIH/ml (5V)、 200NIH/ml (10V) 、 400NIH/ml (20V) 、
800NIH/ml (40V)及 1000NIH/ml (50V) 。
各抗凝血多肽溶液: 分别称取上述实施例 1制备的多肽各 5mg, 用上述缓冲 液分别配制成浓度为 2. 5mmol/L的溶液。 实验过程中根据各多肽的抗凝血活性再 稀释为合适的浓度。
335 比伐卢定 (Himlog-1 ) : 购自成都凯捷生物医药科技有限公司, 称取 5mg, 用上述缓冲液配制成 2. 5mmol/L的溶液。 实验过程中再稀释为合适的浓度。
抗凝血活性的测定采用凝血酶滴定法 (Markwardt F. Methods Enzymol, 1970,69:924-932.陈华友等, 生物技术, 2002, 12(6):24-25 ) 。 下面以浓度为 0. ImM的多肽 3和猪凝血酶为例, 说明多肽 3的抗凝血活性。 具体操作步骤如
340 下:
取 5支小试管, 分别加入 200 μ ί 0. 5%质量百分含量纤维蛋白原溶液及 50 上述实施例 1制备的浓度为 0. ImM的多肽 3溶液, 37°C温育 5min, 取其一支
试管, 滴加 5V的猪凝血酶溶液 5 PL, 迅速摇匀, lmin内未凝固, 再滴加 20V的 猪凝血酶溶液 5 L, 迅速摇匀, lmin内仍未凝固, 再滴加 20V的猪凝血酶溶液
345 5μί, 迅速摇匀, lmin内仍未凝固, 直至共加入 5次 20V的猪凝血酶溶液时, 出现凝固, 说明上述实施例 1制备的多肽 3的抗凝血活性在 85V- 105V之间; 另 取一支小试管, 加入 40V的猪凝血酶溶液 5μί, 迅速摇匀, lmin内未凝固, 再 加入 40V的猪凝血酶溶液 5μί, 迅速摇匀, lmin内未凝固, 再加入 20V的猪凝 血酶溶液 5 L, 迅速摇匀, lmin内未凝固, 再加入 IV的猪凝血酶溶液 5 uL,
350 迅速摇匀, lmin内出现凝固, 说明上述实施例 1制备的多肽 3的抗凝血活性为 101V, 即 10.1ATU, 实验设三次重复, 取三次重复的平均值作为实验结果。
上述实施例 1制备的其它多肽、 比伐卢定 (Hirulog-1) 及各多肽与牛凝血 酶、 人凝血酶的抗凝活性的测定方法于此相同。 上述实施例 1制备的多肽及比伐 卢定 (Hirulog-1) 的抗凝血活性的测定结果如表 1所示。
355 表 1 部分多肽的抗凝活性实验结果
抗凝肽 抗凝活性 (ATU/画 ol) 抗凝活性 (ATU/誦 ol) 抗凝活性 (ATU/隱 ol)
(猪凝血酶) (牛凝血酶) (人凝血酶) hirulog-1 5.20 105 2.08X106 1.24X107 多肽 1 6.60X105 1.76X107 . 2.24X107 多肽 2 6.40X105 6.16X106 2.08 107 多肽 3 2.02X106 1.28X107 1.24 107 多肽 5 1.62X106 8.80X106 1.08 107 多肽 6 1.82X106 1.84X107 1.84 107 多肽 7 4.40X105 2.32X106 1.24X107 多肽 8 2.20X105 0.88X106 0.48X10' 结果表明, 上述实施例 1制备的多肽 1、 多肽 2、 多肽 3及多肽 6的抗凝活 性明显高于对照肽比伐卢定 (hirulog-1) , 对猪凝血酶的抗凝血活性为对照肽的 1-4倍, 对牛凝血酶的抗凝血活性为对照肽的 3-9倍, 对人凝血酶的抗凝血活性 与对照肽相等或近 2倍。
360 实施例 3、 抑制常数 (Ki) 的测定
本实施例中涉及的各种溶液及其配制方法如下:
缓冲液: 0.01M Hepes/0.01M Tris, 0.1M NaCl, 0.1% PEG6000, PH7.4。 底物: Chromozym TH(Tos-Gly- Pro-Arg- PNA, 购自罗氏公司), 分别配制成 浓度为 25 Μ 33 Μ, 40μΜ、 50μΜ、 100 μΜ、 125 μΜ, 200 μΜ、 330 Μ, 500 365 μΜ、 1000 μΜ。
牛凝血酶 (购自 sigma公司) 溶液: 配制成 5 NIH/mlo
抗凝肽的浓度: 上述实施例 1制备的多肽 1、 多肽' 3、 多肽 6和多肽 7分别 配制成 0.05 μΜ、 0.10 μΜ、 0.15 μΜ。
采用生色底物法 (Cappiello M,et.al. Biochemical. Biochemical Pharmacology, 370 1996,52:1141-1146. Maraganore J M,et.al. Biochemistry, 1990,29:7095 -7101), 底
物 Chromozym TH(Tos-Gly- Pro-Arg- PNA)能被凝血酶切割产生对硝基苯胺, 而 后者在 405mn波长有很强的吸收, 所以可以通过监测对硝基苯胺的生成来确定抗 凝肽对凝血酶的抑制活性。 下面以多肽 3为例, 进行具体的说明。 具体实验过程 如下:
375 将 0.05ml牛凝血酶溶液和 0.05ml抗凝肽 3溶液加入到 0.35ml缓冲液
(0.01M Hepes/0.01M Tris, 0.1M NaCl, 0.1% PEG6000, PH7.4) 中, 混匀, 于 2min内加入 Chromozym TH溶液, 使其在混合液中的终浓度分别为 2.5 μ M、 3.3 μΜ、 4.0μΜ、 5·0μΜ、 10·0μΜ、 12.5μΜ、 20.0 μ Μ、 33·0μΜ、 50.0μΜ、 100. Ou ) , 室温下反应 10min, BEC MAN DU640分光光度计检测其于 405mn波 380 长的吸光度变化。 Km和 Vmax通过双倒数作图法确定, Ki值通过竞争性和非竞争 性抑制方程计算。 实验设三次重复, 取三次重复的平均值作为实验结果。
上述实施例 1制备的多肽以及对照肽比伐卢定 (hirulog- 1) 的抑制牛凝血 酶的抑制常数 Ki值, 见表 2。
表 2 部分多肽的 Ki值
抗凝肽 Ki (nM)
hirulog- 1 21.0
多肽 1 10.4
多肽 3 15.2
多肽 6 15.5
多肽 7 15.0
385 结果表明, 上述实施例 1制备的多肽 1、 多肽 3、,多肽 6和多肽 7的 Ki值均 小于比伐卢定 (hirulog-Ι) 的 Ki值, 说明这些多肽的抗凝活性均比比伐卢定 ( hirulog- 1 ) 强。
实施例 4、 活化部分凝血活酶时间 (APTT) 评价多肽 3在动物体内的活性 活化部分凝血酶时间测定试剂盒 (德国 TEC0 GmbH公司产品, 批号:
390 R067015) ; 凝血酶时间测定试剂盒 (德国 TEC0 GmbH公司产品, 批号:
P01351) ; Coatron Ml凝血仪和一次性凝血测定比色杯均为德国 TEC0公司产 品; 3K15冷冻离心机为 Sigma公司产品。
SD大鼠, 雄性, 体重 300±20g, 由军事医学科学院实验动物中心提供。 将上 述 SD大鼠进行分组, 每组 8只, 分别尾静脉注射给予 1.0、 0.6和 0.3 mol/kg
395 上述实施例 1制备的多肽 3或 1. Ομιιιοΐ/kg比伐卢定 (Hirulog-Ι) 。 注射比伐 卢定 (Hirulog- 1) 的大鼠于注射后 0min、 20min、 40min、 60min、 90min、 120min、 180min及 240min, 或注射多肽 3的大鼠于注射后 0h、 lh、 2h、 3h、 4h、 5h、 8h及 12h分别于眼球后静脉丛取血 0.5nil, 将各组血液分别和质量百分 含量为 3.2%的枸櫞酸钠以 =9: 1的体积比混合。 室温下 2000g离心 15min, 分离
400 出血浆, 立即置于 -20'C冰箱密封保存, 分离、 冻存血浆应在取血后 lh内完成。
取上述各组血浆, 分别测定 APTT值。 实验设三次重复, 取三次重复的平均值作 为实验结果。
多肽 3在大鼠体内的 APTT活性实验结果如图 6所示。 图 6中, Hirulog-S 表示上述实施例 1 制备的多肽 3, Hirulog-1 表示比伐卢定。 结果表明, 相同剂
405 量的多肽 3达到血药浓度峰值时的 APTT值显著高于比伐卢定 (Hirulog-1 ) , 为 Hirulog-1 的 2倍以上; 多肽 3在体内活性的持续时间很长, 12小时后, APPT 值仍比 Oh 时的 APPT 值高出 4 秒, 而 Hirulog-1 在 4 小时时已无活性。 0. 3議 ol/kg 多肽 3在 8小时时 APTT值仍高于 Oh的 APPT值 2秒, 表明多肽 3具 有很高的抗凝活性及优良的药代性质。
410 实施例 5、 出血时间 (BT ) 评价多肽 3的安全性
SD大鼠, 雄性, 体重 200±20g, 由军事医学科学院实验动物中心提供。 将上 述 SD大鼠分为生理盐水组、 Hirulog- l-30min组、 Hirulog- l-10min组, 多肽 3 - 2h组, 多肽 3-4h组, 每组 8只。 Hirulog- l-30min组和 Hirulog- l-10rain组 的给药剂量为 Ι . Ο μ πιοΙ/kg体重, 多肽 3- 2h组和多肽 3-4h组的给药剂量为 1、
415 0. 3和 0. Ι μ πιοΐ/kg体重。
将上述各组大鼠分别腹腔注射 3%质量百分含量戊巴比妥钠溶液进行麻醉, 剂量为 50mg/kg体重。 待大鼠麻醉后尾静脉注射生理盐水或抗凝肽, 生理盐水组 30min后, Hirulog- 1- 30min组 30min后, Hirulog- 1- lOmin组 lOmin后, 多肽 3-2h组 2h后, 多肽 3-4h组 4h后, 用手术刀在距大鼠尾尖 2圆处切断, 迅速放
420 入装有生理盐水的试管中, 记录出血开始至出血停止 (不出血 30s ) 的时间。 实 验设三次重复。 上述实施例 1制备的多肽 3与 Hirulog-1在给药不同时间后测得 的大鼠断尾 BT值结果如表 3所示。
表 3 多肽 3对 SD大鼠 BT的影响 (平均值土标准差) 组别 齐 U量 ( μπιοΐ/kg) 间隔时间 * (min ) 出血时间 (S ) 生理盐水组 一 30 685. 6 ± 108. 35
Hirulog-l-30rain组 1. 0 30 1418. 57 ± 193. 12
Hirulog-1-l Omi n组 1. 0 10 1644. 00 ± 351. 30 多肽 3-2h组 1. 0 120 >3000
多肽 3-4h组 0. 3 240 2258. 00 ± 996. 48 多肽 3-4h组 0. 1 240 1894. 3 ± 200. 2 表 3中, 间隔时间指给药后到断尾之间的时间。
425 结果表明, 虽然多肽 3在高剂量时 BT值高于 Hirulog-1, 但因 Hirulog - 1 半衰期短(25min) , 出血时间也相应短。 本实施例还测定了 0. Ι μπιοΐ/kg剂量的多 肽 3对大鼠的 BT, 结果表明, 该剂量下, 多肽 3注射后的出血时间已经与 Hirulog- l-10mi n组相当, 说明在低剂量下, 多肽 3的预计出血时间倾向于与 Hirulog-1相当。
430 实施例 6、 抗凝肽抗静脉血栓形成实验
Wi star 大鼠, 雌雄各半, 体重 300±20g, 由军事医学科学院实验动物中心 提供。 将上述大鼠随机进行分组, 每组 8只, 分别为生理盐水组、 Hirulog- 1 组
和多肽 3不同剂量组。 Hirulog- 1 组的剂量为 l. O mol/kg, 多肽 3组的剂量分 别为 1、 0. 6、 0. 3和 0. Ιμπιοΐ/kg o
435 将上述各组大鼠按 50mg/kg 体重腹腔注射 3%质量百分含量戊巴比妥钠, 麻 醉后取仰卧位固定, 分离下腔静脉, 于左肾静脉分支下缘近端结扎下腔静脉, 关 闭腹腔。 结扎前 20min 分别于尾静脉注射生理盐水、 Hirulog- 1 或 Ι. ΟμπιοΙ/kg 剂量的上述实施例 1 制备的多肽 3, 结扎前 5h分别于尾静脉注射 1、 0. 6、 0. 3 或 0. Ιμπιοΐ/kg剂量的上述实施例 1制备的多肽 3 (为了进一步验证多肽 3的长
440 效性, 设置了给药后 5h才结扎的血栓模型) 。 结扎 4h后打开腹腔, 于结扎远端 2cm处剪开血管, 取出血栓, 用滤纸吸干血栓表面的血液后称重。
不同剂量的多肽 3 与比伐卢定 (Hirulog- 1 ) 的抗静脉血栓形成活性结果见 表 4。
表 4 多肽 3在大鼠体内抗静脉血栓形成活性
组别 剂量(μιηοΐ/kg) 间隔时间' (min ) 血栓湿重 (mg) 生理盐水组 0 20 14. 9±7. 9
Hirulog- 1组 1 20 3. 8±2. 8 多肽 3组 1 20 1. 3±1. 8
多肽 3-5h组 1 300 0
多肽 3-5h组 0. 6 300 0
多肽 3- 5h组 0. 3 300 0
多肽 3-5h组 0. 1 300 1. 7±0. 2
445 结果表明, 在结扎 5h前给予 1、 0. 6或 0. 3μπιοΐΑβ剂量的上述实施例 1制 备的多肽 3几乎能完全抑制静脉血栓的形成, 给予 0. Ιμπιοΐ/kg剂量的上述实施 例 1制备的多肽 3仍比 Hirulog- 1具有更强的抗静脉血栓形成活性。
工业应用
本发明根据凝血酶和抗凝药物比伐卢定的结构特点及作用方式, 设计合成了 450 了一类抗凝血多肽, 本发明的多肽不仅能高效、 特异抑制凝血酶活性, 而且其所 具有的氨基酸个数与比伐卢定相当, 合成容易, 抗凝活性更强。
Claims
1、 抗凝血多肽, 其一级结构通式如下:
(D) - FPRP- X'-Xs- X3- X4- X5- QGDFEPIPEDAYDE- NH2;
其中: X,选自 G、 AEEA、 K和 R中的任意一个, X2选自 G、 K和 R中的任意一 个或 X2缺失, 选自 G、 K和 D中的任意一个或 X3缺失, Χ4选自 G、 K、 W和 D中 的任意一个, X5选自 P和 S中的任意一个或 X5缺失;
上述多肽中, 所述 Α 为丙氨酸, D 为天冬氨酸, E 为谷氨酸, F 为苯丙氨 酸,. (D) - F为 D型苯丙氨酸, G为甘氨酸, I为异亮氨酸, K为修饰有脂肪酸的赖 氨酸, P为脯氨酸, Q为谷氨酰胺, R为精氨酸, S为丝氨酸, W为色氨酸, Y为 酪氨酸, AEEA为 2- (2- (2-氨基乙氧基)乙氧基)乙酸, E- NH2为羧基端酰胺化的谷 氨酸。
2、 根据权利要求 1所述的多肽, 其特征在于: 所述多肽为如下 8个多肽之 —:
多肽 1 : (D) - FPRP- GGGG- QGDFEPIPEDAYDE- NH2,
多肽 2: (D) -FPRP-AEEA-G-QGDFEPIPEDAYDE-NH2,
多肽 3: (D) -FPRP-GKGG-QGDFEPIPEDAYDE-NH2,
多肽 4: (D) -FPRP-GGKG- QGDFEPIPEDAYDE- NH2,
多肽 5: (D) -FPRP-GGGK- QGDFEPIPEDAYDE- NH2,
多肽 6: (D) -FPRP-GRGDS-QGDFEP I PEDAYDE-NH2 ,
多肽 7: (D) -FPRP-RGDWP- QGDFEPIPEDAYDE- NH2,
多肽 8: (D) - FPRP- KGGG- QGDFEPIPEDAYDE- NH2。
3、 根据权利要求 1或 2所述的多肽, 其特征在于: 所述多肽 3、 多肽 4、 多 肽 5和多肽 8的赖氨酸修饰有硬脂酸。
4、 权利要求 1-3中任一所述的多肽在制备预防和 /或治疗血栓性疾病的药物 中的应用。
5、 一种预防和 /或治疗血栓性疾病的药物, 其活性成份是权利要求 1-3中任 一所述的多肽。
6、 根据权利要求 5所述的药物, 其特征在于: 所述药物中包括载体, 所述 载体为水溶性载体、 难溶性载体或肠溶性载体。
7、 根据权利要求 6所述的药物, 其特征在于- 所述水溶性载体为聚乙二 醇、 聚乙烯吡咯烷酮或有机酸。
8、 根据权利要求 6所述的药物, 其特征在于: 所述难溶性载体为乙基纤维 素或胆固醇硬脂酸酯。
9、 根据权利要求 6所述的药物, 其特征在于: 所述肠溶性载体为醋酸纤维 素酞酸酯或羧甲乙纤维素。
10、 根据权利要求 5-9中任一所述的药物, 其特征在于: 所述药物为片剂、 栓剂、 溶液、 胶囊、 气雾剂、 泡腾片或滴剂。
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CN114773446A (zh) * | 2022-06-16 | 2022-07-22 | 中国农业科学院农业质量标准与检测技术研究所 | 一种蜂毒肽及其分离纯化方法 |
CN114773446B (zh) * | 2022-06-16 | 2022-11-04 | 中国农业科学院农业质量标准与检测技术研究所 | 一种蜂毒肽及其分离纯化方法 |
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