CN102641506B - Bivalirudin-polyethylene glycol compound - Google Patents
Bivalirudin-polyethylene glycol compound Download PDFInfo
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- CN102641506B CN102641506B CN 201210104327 CN201210104327A CN102641506B CN 102641506 B CN102641506 B CN 102641506B CN 201210104327 CN201210104327 CN 201210104327 CN 201210104327 A CN201210104327 A CN 201210104327A CN 102641506 B CN102641506 B CN 102641506B
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Abstract
The invention relates to a bivalirudin-polyethylene glycol compound and a preparation method thereof, a medicinal composition containing the compound and application. The bivalirudin-polyethylene glycol compound is a new structural compound which is obtained by cysteine substitution for any glycine from the 5th site to the 8th site in a bivalirudin sequence and polyethylene glycol modification, keeps the activity of bivalirudin and prolongs the half-life period of the bivalirudin. The application of the bivalirudin-polyethylene glycol compound in medicaments for treating or preventing thrombus diseases comprises application in medicaments for unstable angina pectoris, peripheral artery intervention therapy, hearth and lung transplantation and thrombus preventing treatment.
Description
[technical field]
The present invention relates to the Pegylation complex of bivalirudin, its preparation method, the pharmaceutical composition that contains them with and application in the medicine for the treatment of or prevention thrombus disease, be included in the application in the medicine in unstable angina pectoris, peripheral arterial interventional therapy, heart-lung transplant operation and the anti-bolt treatment.
[background technology]
The thrombotic disease of the initiations such as atherosclerosis has become the in the world the first cause of death, and its sickness rate is in rising trend in China, and anticoagulation is very important in the treatment of this disease.In at present numerous anticoagulations, the bivalirudin (Angiomax of Medicines company
TM) be a kind of anticoagulant new drug that FDA goes on the market in approval in 2000, its anticoagulant composition is hirudin analog (fragment).This medicine is that thrombin is direct, special, reversible inhibitor, no matter thrombin is in the blood circulation still is combined with thrombosis, specific binding all can occur with its catalytic site and anion binding site (claiming again the substrate recognition site) in it, thus the activity of direct Trombin inhibiting.It has overcome the shortcoming of heparin, Low molecular heparin and hirudin, demonstrates good effect in unstable angina pectoris, peripheral arterial interventional therapy, heart-lung transplant operation and anti-bolt treatment.
But bivalirudin is shorter to the inhibition persistent period in thrombin activity site, about 25 minutes of the half-life in human body.Realize the long-time inhibition to the thrombin activity site, sometimes need repeated multiple times drug administration by injection.And bivalirudin is expensive, and the multiplexing medicine of multiple reversal has increased patient's the cost of seeking medical advice greatly, and therefore, it is very necessary to develop long-acting, stable, economic similar medicine or preparation.
Protein drug is the effect of energy significant prolongation after Polyethylene Glycol (polyethylene glycol, PEG) is modified, and strengthens water solublity and stability, reduces immunogenicity and antigenicity, and the protein drug that existing a plurality of PEG modify goes on the market.The present invention is by introducing cysteine in the bivalirudin structure, to realize that fix a point method and the application of pegylation there is not yet bibliographical information to bivalirudin.
[summary of the invention]
One of purpose of the present invention provides bivalirudin-polyethylene glycol complex.Chemically synthesized polypeptide medicine bivalirudin is the direct inhibitor of thrombin, it has overcome the shortcoming of heparin, Low molecular heparin and hirudin, demonstrates good effect in unstable angina pectoris, peripheral arterial interventional therapy, heart-lung transplant operation and anti-bolt thromboembolism preventing treatment.But the single dose of bivalirudin is 250 milligrams, and price is about 1000 yuan of every grams at present.The PEGization modified outcome by synthetic bivalirudin is wished in this research, can prolong the half-life of bivalirudin, reduces dosage or administration frequency, will bring more wide market for bivalirudin, creates larger economic benefit and social benefit.Bivalirudin derives from the hirudin complex, the polypeptide that is formed by 20 amino acid residues, molecular weight is 2180, and its structural formula is D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Gl u-Ile-Pro-Glu-Glu-Tyr-Leu-OH.Studies show that its N-end Pro-Arg-Pro and C-end Glu-Glu-Ile-Pro-Glu-Glu are its two critical sites of being combined with thrombin, therefore 4 glycine of 5 to 8 have been interconnect function, and selecting 5,6,7 or 8 s' glycine to modify is desirable site.
Bivalirudin (bivalirudin of the present invention, BVLD)-and polyethylene glycol complex is to contrast the arbitrary glycine that cuts down the 5th to the 8th (holding the C-end by N-) in Lu's fixed sequence to carry out the alternative rear novel compound that carries out obtaining behind the pegylation in cysteine side chain of cysteine, described complex is the chemical compound with formula (I) structure:
[Cys(PEG-M)]
X-BVLD (I)
Wherein
X is the arbitrary integer among the 5-8, and the 5th to the 8th arbitrary glycine in the expression bivalirudin sequence substituted by cysteine; PEG is RO (CH
2CH
2O) n-CH
2CH
2, R=H or CH
3, n=5-1000; Cys is cysteine, and it links to each other with M group covalency by its side chain sulphur atom.Particularly, the structure of this complex comprises [Cys (mPEG
2000-MAL)]
7-BVLD, [Cys (mPEG
5000-MAL)]
7-BVLD and [Cys (mPEG
10000-MAL)]-BVLD.
Another object of the present invention provides the preparation method of bivalirudin polyethylene glycol complex, and described preparation method comprises: with [Cys]
X-BVLD is dissolved in the pure water, regulate pH to 7～8 with sodium bicarbonate, the PEG that adds 2-3 times of molar equivalent, stirring at room reaction 2-3 hour, with reversed-phase high-performance liquid chromatography (RP-HPLC) monitoring reaction process and separate targets product, target product gets bivalirudin-polyethylene glycol complex [Cys (PEG-MAL)] through lyophilization
X-BVLD, wherein PEG is dimaleoyl imino Polyethylene Glycol, vinyl Polyethylene Glycol or iodo acetyl group Polyethylene Glycol.
The present invention substitutes glycine to introduce cysteine at 7, then carries out the polyethyleneglycol modified example that is: adopt first general solid-phase polypeptide synthesis strategy to synthesize 7 bivalirudins by the cysteine substituted glycinic acid, i.e. [Cys]
7-bivalirudin, its aminoacid sequence is
D-Phe-Pro-Arg-Pro-Gly-Gly-Cys-Gly-Asn-Gly-Asp-Phe-Glu-Gl u-Ile-Pro-Glu-Glu-Tyr-Leu-OH is then with [Cys]
7Additive reaction occurs and obtains bivalirudin-polyethylene glycol complex [Cys (mPEG-MAL)] in-bivalirudin and mPEG-MAL
7BVLD.This bivalirudin polyethylene glycol complex is to prepare by the sulfydryl that is incorporated into the cysteine in the bivalirudin sequence and mPEG-MAL reaction, and the process of sulfydryl reaction is as follows in the structure of PEG and complex thereof and mPEG-MAL and the peptide chain:
[Cys]
7The concrete preparation process of-bivalirudin comprises that preparation, the mensuration of substitution degree, coupling amino acid, the peptide resin of amino-acid resin obtain [Cys]
7-bivalirudin crude product, through anti-phase-high performance liquid chromatography (RP-HPLC) purification, lyophilizing obtains [Cys]
7-bivalirudin sterling dry powder.
Among the present invention, first with [Cys] behind the purification
7-bivalirudin is soluble in water, then with saturated sodium bicarbonate solution pH regulator is arrived alkalescence, is between the 7-8 such as pH, and the mean molecule quantity that then adds respectively the 2-3 molar equivalent is 2000,5000,10000 mPEG-MAL, at room temperature reacts 2-3 hour.By RP-HPLC to reaction monitor, target product separates and purity analysis.
Target product [Cys (mPEG-MAL)]
7-BVLD structure obtains conclusive evidence by carrying out amino acid composition analysis, HPLC and mass spectrum (MALDI-TOF-MS) analyses.
Bivalirudin-the polyethylene glycol complex of new construction is through the anticoagulating active evaluation, prothrombin time (PT), thrombin time (TT) and activated partial thromboplastin time (APTT) experimental result show the anticoagulating active that it has kept bivalirudin.Can know the polyethyleneglycol modified of bivalirudin by inference based on the existing knowledge of polyethyleneglycol modified drug research and prolong again its half-life.
A further object of the invention provides institute's bivalirudin-polyethylene glycol complex, the pharmaceutical composition that contains them with and application in the medicine for the treatment of or prevention thrombus disease, and be used for the treatment of purposes in the medicine in unstable angina pectoris, peripheral arterial interventional therapy, heart-lung transplant operation and the anti-bolt treatment.Pharmaceutical composition of the present invention contains at least a bivalirudin-polyethylene glycol complex and pharmaceutic adjuvant.
The present invention adopts simple and easy to do method, and the anticoagulating active that can keep bivalirudin for preparing new construction prolongs again the bivalirudin polyethylene glycol complex of its anticoagulant time.
[description of drawings]
Accompanying drawing 1 is [Cys]
7The HPLC of-BVLD analyzes collection of illustrative plates.
Accompanying drawing 2 is [Cys]
7The ESI-MS of-BVLD analyzes collection of illustrative plates.
Accompanying drawing 3 is [Cys (mPEG
2000-MAL)]
7The HPLC of-BVLD analyzes collection of illustrative plates.
Accompanying drawing 4 is [Cys (mPEG
2000-MAL)]
7The MALDI-TOF-MS of-BVLD analyzes collection of illustrative plates.
Accompanying drawing 5 is [Cys (mPEG
5000-MAL)]
7The HPLC of-BVLD analyzes collection of illustrative plates.
Accompanying drawing 6 is that the MALDI-TOF-MS of [Cys (mPEG5000-MAL)] 7-BVLD analyzes collection of illustrative plates.
Accompanying drawing 7 is that the HPLC of [Cys (mPEG10000-MAL)] 7-BVLD analyzes collection of illustrative plates.
Accompanying drawing 8 is [Cys (mPEG
10000-MAL)]
7The MALDI-TOF-MS of-BVLD analyzes collection of illustrative plates.
The specific embodiment
The used aminoacid configuration of the present invention is the L-type except specified otherwise, the relevant abbreviation of use has following implication:
TBu: the tert-butyl group
2-CTC Resin:2-chlorine trityl chloride resin
DCM: dichloromethane
DIC:N, the N-DIC
DMF:N, dinethylformamide
EDT:1, the 2-dithioglycol
The Fmoc:9-fluorenylmethyloxycarbonyl
The HOBt:1-hydroxy benzo triazole
The NMP:N-methyl pyrrolidone
Pbf:2,2,4,6,7-pentamethyl Dihydrobenzofuranes-5-sulfonyl
TFA: trifluoroacetic acid
Ts-Cl: paratoluensulfonyl chloride
Fmoc-Arg (Pbf)-OH:N-fluorenes methoxy carbonyl acyl group-2,2,4,6,7-pentamethyl Dihydrobenzofuranes-5-sulphonyl-L-arginine
Fmoc-Asn (Trt)-OH:N-fluorenylmethoxycarb-nyl-nyl O-tert-butyl-L-Aspartic acid
Fmoc-Asp (OtBu)-OH:N-fluorenylmethyloxycarbonyl-trityl-altheine
Fmoc-Cys (Trt)-OH:N-fluorenylmethyloxycarbonyl-S-trityl-Cys
Fmoc-Glu (OtBu)-OH:N-fluorenylmethoxycarb-nyl-nyl O-tert-butyl-Pidolidone
Fmoc-Gly-OH:N-fluorenylmethyloxycarbonyl glycine
Fmoc-Ile-OH:N-fluorenylmethyloxycarbonyl-ILE
Fmoc-Leu-OH:N-fluorenylmethyloxycarbonyl-L-Leu
Fmoc-D-Phe-OH:N-fluorenylmethyloxycarbonyl-D-phenylalanine
Fmoc-Phe-OH:N-fluorenylmethyloxycarbonyl-L-Phe
Fmoc-Pro-OH:N-fluorenylmethyloxycarbonyl-L-PROLINE
Fmoc-Tyr (tBu)-OH:N-fluorenylmethoxycarb-nyl-nyl O-tert-butyl-TYR
MALDI-TOF-MS: matrix assisted laser desorption ionization time-of-flight mass spectrometry
PEG-MAL: dimaleoyl imino Polyethylene Glycol
Among the present invention, the reagent that the Kaiser detection method mainly adopts is 1,2,3-indantrione monohydrate, and reaction is purple or blue showing has free amino to exist on the resin, shows that coupling is incomplete.
The present invention is described in further detail below in conjunction with concrete example, but the invention is not restricted to following examples.
Embodiment 1[Cys (mPEG
2000-MAL)]
7The preparation of-BVLD
Weighing m PEG
2000-OH 20g (10mmol) places reaction bulb, adds dichloromethane 50mL, adds triethylamine 7.5mL (50mmol) and paratoluensulfonyl chloride 9.5g (Ts-Cl, 50mmol) behind the dissolution of solid again, the stirring at room reaction.After the TLC detection reaction was complete, the rotary evaporation desolventizing added the 50mL absolute ether and is settled out solid, adds water with dissolution of solid again, separatory, minute water-yielding stratum is with absolute ether 50mL washing 2 times, water with dichloromethane 75mL extraction 2 times, is collected organic facies, dried overnight again.Revolve and boil off solvent, add the absolute ether sedimentation and go out solid.Obtain mPEG after the drying
2000-OTs 14.2g, yield 71%.
MPEG-OTs 14g (7mmol) is dissolved in 30mL DMF, the potassium phthalimide 3.88g (21mmol) of adding, under the nitrogen protection, 120 ℃ of reaction 4h.The vacuum rotary steam desolventizing adds dehydrated alcohol, adds hydrazine hydrate 4.5mL, reflux 4h.The vacuum rotary steam desolventizing adds dichloromethane 100mL, and the elimination insoluble matter is washed three times anhydrous Na with saturated nacl aqueous solution 50mL
2SO
4Dry 12h, the elimination desiccant, the vacuum rotary steam desolventizing, the ether sedimentation, sucking filtration gets mPEG after the drying
2000-NH
210.8g, yield 77%.
With mPEG
2000-NH
210g (5mmol) is dissolved among the 20mL DMF, adds maleic anhydride 4g, 80 ℃ of stirring reaction 30min.The pressure reducing and steaming solvent, the ether sedimentation, filter collection solid gets 9.5g after the drying.Solid is dissolved in the 30mL acetic anhydride, adds anhydrous sodium acetate 4.9g, 100 ℃ of stirring reaction 45min.Solid in the filtering reactant liquor adds the water of 10 times of volumes in the reactant liquor, then uses the dichloromethane extraction water three times, organic facies after saturated nacl aqueous solution is washed three times, anhydrous Na
2S0
4Dry 12h.Filter, the vacuum rotary steam desolventizing, the ether sedimentation, centrifugal, sucking filtration, the dry mPEG that gets
2000-MAL 6.8g, yield 68%.
Take by weighing 2-CTC Resin 3.8g (5mmol, 1.3mmol/g) to reactor, add an amount of DMF washing after, take out solution, add the about 30min of an amount of DCM swelling.Take by weighing the Fmoc-Leu-OH of 2.9g in beaker, add an amount of DMF dissolving, add the DIPEA (1.4mL) of 1/3 overall accumulated amount in above-mentioned Freamine Ⅲ, stir ice-water bath 10～15min.After the resin swelling finishes, take out DCM, the protected amino acid feed liquid that dissolving is good is poured in the reactor, behind reaction 10～15min, adds the DIPEA (2.85mL) of 2/3 cumulative volume, room temperature reaction 3～4h.Reaction is taken out reactant liquor after finishing, and adds an amount of DMF washing 3 times, and the ratio that adds volume is the mixed solution sealing unreacted radical of 17: 2: 1 DCM, methanol, DIPEA, sealing twice, at every turn about 20min.After sealing finished, the DMF that adds an amount of volume washed 3 times, uses the absolute methanol wash shrinkage 2 times again, each about 10 minutes.Vacuum drying obtains about Fmoc-Leu-CTC resin 4.6g, and recording substitution degree is 0.52mmol/g.
Take by weighing Fmoc-Leu-CTC Resin 3.5g (1.82mmol, 0.52mmol/g) in reactor, add the about 30min of an amount of DCM swelling, take out.Adding 20% an amount of piperidines/DMF solution deprotection 2 times, be 10min for the first time, be 20min for the second time, needs an amount of DMF of adding to wash between twice deprotection.Wash 5 times with an amount of DMF again after having taken off protection, wash 1min at every turn.The Kaiser method detects, and resin is navy blue, shows that Fmoc removes.Take by weighing Fmoc-Tyr (tBu)-OH 1.1g, HOBt 0.6g puts in the beaker, adds an amount of DMF dissolving, beaker is put into the about 5min of ice bath ice bath, add again DIC 1.3mL in solution, add in the reactor room temperature reaction 2～3h behind ice bath activation 10～15min.Behind the reaction 2h, the Kaiser method detects, if observe resin and solution all show light yellow, shows that then reaction is complete, if behind the reaction 3h, the Kaiser method detects resin and still is blueness, then need repeat coupling.According to [Cys]
7The sequence of-bivalirudin is held N end from C, repeats above operating procedure, successively complete last the aminoacid Fmoc-D-Phe-OH of coupling and remove the Fmoc blocking group of N-end after obtain full guard [Cys]
7-BVLD-peptide resin: D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Cys (Trt)-Gly-Asn (Trt)-Gly-Asp (OtBu)-Phe-Glu (OtBu)-Glu (OtBu)-Ile-Pro-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Leu-CTC resin.16g will be got after the peptide resin drying.Peptide resin is joined in advance (TFA/ thioanisole/EDT/ methyl phenyl ethers anisole=90/5/3/2) in the freezing 150mL lysate, and then about 2.5 hours of room temperature cracking filters and sedimentation in freezing absolute ether.Centrifugal, washing, the about 14h of vacuum drying obtains [Cys]
7The thick peptide 3.2g of-BVLD.The thick peptide of above-mentioned gained gets after the lyophilizing [Cys] through the HPLC purification
7-BVLD1.4g, purity 96%, total recovery 34%.
Take by weighing [Cys]
7-bivalirudin 220mg is dissolved in the pure water, regulates pH to 7～8 with sodium bicarbonate, adds mPEG
2000-MAL 600mg, room temperature reaction 2 hours is used the RP-HPLC separated product, gets [Cys (mPEG after the lyophilizing
2000-MAL)]
7-BVLD 294mg, purity is greater than 95%, yield 70%.
[Cys (mPEG
2000-MAL)]
7-BVLD analyzes through MALDI-TOF-MS, finds to have near 4232 a series of peaks, adjacent two peak molecular weight to differ about 44, has the typical structure feature of Polyethylene Glycol.The composition of amino acid analysis of acid hydrolysis (6N aqueous hydrochloric acid solution, 110 ℃, 22 hours) conforms to theoretical value: Asp, 1.99 (2); Glu, 4.11 (4); Pro, 3.09 (3); Arg, 0.96 (1); Gly, 4.21 (4); Tyr, 1.07 (1); Phe, 1.92 (2); Ile, 0.99 (1); Leu, 0.98 (1).
Embodiment 2[Cys (mPEG
5000-MAL)]
7The preparation of-BVLD
Weighing m PEG
5000-OH 25g (5mmol) places reaction bulb, adds dichloromethane 100mL, adds triethylamine 7.5mL (5mmol) and paratoluensulfonyl chloride 9.5g (Ts-Cl, 50mmol) behind the dissolution of solid again, the stirring at room reaction.After the TLC detection reaction was complete, the rotary evaporation desolventizing added the 150mL absolute ether and is settled out solid, adds water with dissolution of solid again, separatory, minute water-yielding stratum is with absolute ether 100mL washing 2 times, water with dichloromethane 150mL extraction 2 times, is collected organic facies, dried overnight again.Revolve the steaming desolventizing, add the absolute ether sedimentation and go out solid, filter, obtain mPEG after the drying
5000-OTs 21.6g, yield 86%.
With mPEG
5000-OTs 20g (4mmol) is dissolved in 30mL DMF, the potassium phthalimide 2.2g (12mmol) of adding, and under the nitrogen protection, 120 ℃ of reaction 4h.The vacuum rotary steam desolventizing adds dehydrated alcohol, adds hydrazine hydrate 2.0mL, reflux 4h.The vacuum rotary steam desolventizing adds dichloromethane 100mL, and the elimination insoluble matter is washed anhydrous Na three times with saturated NaCl solution 50mL
2SO
4Dry 12h, the elimination desiccant, the vacuum rotary steam desolventizing, the ether sedimentation, sucking filtration, drying gets mPEG
5000-NH
217.5g, yield 82%.
With mPEG
5000-NH
215g (3mmol) is dissolved among the 30mL DMF, adds maleic anhydride 1.25g, 80 ℃ of stirring reaction 30min.The pressure reducing and steaming solvent, the ether sedimentation, filter collection solid gets 13.8g after the drying.Solid is dissolved in the 30mL acetic anhydride, adds anhydrous sodium acetate 5g, 100 ℃ of stirring reaction 45min.Solid in the filtering reactant liquor adds the water of 10 times of volumes in the reactant liquor, then uses the dichloromethane extraction water three times, organic facies after saturated nacl aqueous solution is washed three times, anhydrous Na
2SO
4Dry 12h.Filter, the vacuum rotary steam desolventizing, the ether sedimentation, centrifugal, sucking filtration, the dry mPEG that gets
5000-MAL 12.6g, yield 80%.
Take by weighing [Cys]
7-bivalirudin 220mg is dissolved in the pure water, regulates pH to 7～8 with sodium bicarbonate, adds mPEG
5000-MAL 1.25g, room temperature reaction 3 hours is used the RP-HPLC separated product, gets [Cys (PEG after the lyophilizing
5000-MAL)]
7-BVLD 587mg, purity is greater than 95%, yield 81%.
[Cys (mPEG
5000-MAL)]
7-BVLD analyzes through MALDI-TOF-MS, and a series of peaks are arranged near 7322, and it is about 44 that adjacent two peak molecular weight differ, and has the typical structure feature of Polyethylene Glycol.The composition of amino acid analysis of acid hydrolysis (6N aqueous hydrochloric acid solution, 110 ℃, 22 hours) conforms to theoretical value: Asp, 2.00 (2); Glu, 4.31 (4); Pro, 3.18 (3); Arg, 1.03 (1); Gly, 4.13 (4); Tyr, 1.02 (1); Phe, 1.82 (2); Ile, 1.06 (1); Leu, 1.03 (1).
Embodiment 3[Cys (mPEG
10000-MAL)]
7The preparation of-BVLD
Weighing m PEG
10000-OH 25g (2.5mmol) places reaction bulb, adds dichloromethane 100mL, adds triethylamine 3.75mL (2.5mmol) and paratoluensulfonyl chloride 4.75g (Ts-Cl, 25mmol) behind the dissolution of solid again, the stirring at room reaction.After the TLC detection reaction was complete, the rotary evaporation desolventizing added the 150mL absolute ether and is settled out solid, adds water with dissolution of solid again, separatory, minute water-yielding stratum is with absolute ether 100mL washing 2 times, water with dichloromethane 150mL extraction 2 times, is collected organic facies, dried overnight again.Revolve and boil off solvent, add the absolute ether sedimentation and go out solid, filter, obtain mPEG after the drying
10000-OTs 24g, yield 92%.
With mPEG
10000-OTs 20g (2mmol) is dissolved in 30mL DMF, the potassium phthalimide 1.1g (6mmol) of adding, and under the nitrogen protection, 120 ℃ of reaction 4h.The vacuum rotary steam desolventizing adds dehydrated alcohol, adds hydrazine hydrate 1.0mL, reflux 4h.The vacuum rotary steam desolventizing adds dichloromethane 100mL, and the elimination insoluble matter is washed anhydrous Na three times with saturated NaCl solution 50mL
2SO
4Dry 12h, the elimination desiccant, the vacuum rotary steam desolventizing, the ether sedimentation, sucking filtration, drying gets mPEG
10000-NH
217.2g, yield 86%.
With mPEG
10000-NH
215g (1.5mmol) is dissolved among the 30mL DMF, adds maleic anhydride 0.625g, 80 ℃ of stirring reaction 30min.The pressure reducing and steaming solvent, the ether sedimentation, filter collection solid gets 14.5g after the drying.Solid is dissolved in the 30mL acetic anhydride, adds anhydrous sodium acetate 2.5g, 100 ℃ of stirring reaction 45min.Solid in the filtering reactant liquor adds the water of 10 times of volumes in the reactant liquor, then uses the dichloromethane extraction water three times, organic facies after saturated NaCl solution is washed three times, anhydrous Na
2SO
4Dry 12h.Filter, the vacuum rotary steam desolventizing, the ether sedimentation, centrifugal, sucking filtration, the dry mPEG that gets
10000-MAL 13.6g, yield 90%.
Take by weighing [Cys]
7-BVLD 110mg is dissolved in the pure water, regulates pH to 7～8 with sodium bicarbonate, adds mPEG
10000-MAL 1.0g, room temperature reaction 3 hours is used the RP-HPLC separated product, gets [Cys (PEG after the lyophilizing
10000-MAL)]
7-BVLD 1065mg, purity is greater than 95%, yield 89%.
[Cys (mPEG
10000-MAL)]
7-BVLD analyzes through MALDI-TOF-MS, and a series of peaks are arranged near 11973, and it is about 44 that adjacent two peak molecular weight differ, and has the typical structure feature of Polyethylene Glycol.The composition of amino acid analysis of acid hydrolysis (6N aqueous hydrochloric acid solution, 110 ℃, 22 hours) conforms to theoretical value: Asp, 1.95 (2); Glu, 4.06 (4); Pro, 2.92 (3); Arg, 0.96 (1); Gly, 4.21 (4); Tyr, 1.06 (1); Phe, 1.92 (2); Ile, 1.01 (1); Leu, 0.97 (1).
The anticoagulating active evaluation of embodiment 4 bivalirudins-polyethylene glycol complex
4.1 the mensuration of prothrombin time (PT)
With PT reagent and examined blood plasma, put pre-temperature 5min in 37 ℃ of water-baths.Get 1 in test tube, add and examined blood plasma 0.1mL, pre-temperature 30s in 37 ℃ of water-baths adds pre-warm PT reagent 0.2mL again, and mixing starts manual time-keeping immediately.When test tube to the liquid flow that constantly tilts slowly is tending towards stopping, the record required time.Repeat 3 times, average, make simultaneously normal control.
With PT reagent and examined blood plasma, put pre-temperature 5min in 37 ℃ of water-baths.Get 1 in test tube, add and to be examined blood plasma 0.1mL, and add a certain amount of bivalirudin or its trim makes its final concentration as shown in the table, pre-temperature 30s in 37 ℃ of water-baths adds the PT reagent 0.2mL of pre-temperature again, and mixing starts manual time-keeping immediately.When test tube to the liquid flow that constantly tilts slowly is tending towards stopping, the record required time.Repeat 3 times, average.
The PT experimental result shows that bivalirudin-polyethylene glycol complex all has good anticoagulant effect, wherein [Cys (mPEG
5000-MAL)]
7-BVLD and [Cys (mPEG
10000-MAL)]
7-BVLD anticoagulant effect is better than bivalirudin, and difference has statistical significance (table 1) more than 3s.
PT experimental result (the normal control meansigma methods: 27.4s) of table 1 bivalirudin-polyethylene glycol complex
4.2 thrombin time of blood plasma (TT) is measured
Get and examined blood plasma 0.1mL in test tube, add a certain amount of bivalirudin or its trim, put in 37 ℃ of water-baths, pre-temperature 5min adds the thrombin reagent of 25U/mL again, starts simultaneously stopwatch record PCT, repeat 3 times, average, as normal control.
Get and examined blood plasma 0.1mL in test tube, add a certain amount of bivalirudin or its trim and make its final concentration as shown in the table, put in 37 ℃ of water-baths, pre-temperature 5min adds 0.1mL standardization " thrombin " reagent again, starts simultaneously stopwatch record PCT, repeat 3 times, average.
The TT experimental result shows that bivalirudin-polyethylene glycol complex maintains good anticoagulant effect, wherein [Cys (mPEG
2000-MAL)]
7-BVLD and [Cys (mPEG
5000-MAL)]
7-BVLD anticoagulant effect is better than bivalirudin, and difference has statistical significance (table 2) more than 3s.
TT experimental result (the normal control meansigma methods: 18.0s) of table 2 bivalirudin-polyethylene glycol complex
4.2 blood plasma activated partial thromboplastin time (APTT) is measured
Get APTT reactant liquor 0.1mL and blood plasma 0.1mL, and add 5 μ L normal saline, put pre-temperature 3min in 37 ℃ of water-baths.Add the APTT catalyst 0.1mL of pre-temperature, mixing starts manual time-keeping immediately again.Constantly tilt test tube when having flocculent deposit to generate, the record required time.Repeat 2 times, average, make simultaneously normal control.
Get APTT reactant liquor 0.1mL and examined blood plasma 0.1mL in plastic centrifuge tube, and bivalirudin and trim to its final concentration thereof of adding 5 μ L be shown in the following table, put pre-temperature 3min in 37 ℃ of water-baths.Add the APTT catalyst 0.1mL of pre-temperature, mixing starts manual time-keeping immediately again.Constantly tilt test tube when having flocculent deposit to generate, the record required time.Repeat 2 times, average.
APTT experimental result (the normal control meansigma methods: 33.6s) of table 3 bivalirudin and trim thereof
All kept at bivalirudin-polyethylene glycol complex on the basis of anticoagulating active, can know the polyethyleneglycol modified of bivalirudin by inference based on the existing knowledge of polyethyleneglycol modified drug research can prolong its half-life.
Claims (5)
1. bivalirudin (bivalirudin, BVLD)-and polyethylene glycol complex, it is characterized in that described complex cuts down in Lu's fixed sequence by N-for contrast to hold C-to hold arbitrary glycine of the 5th to the 8th to carry out carrying out the chemical compound that obtains behind the pegylation in cysteine side chain after cysteine substitutes;
Described complex is the chemical compound with formula I structure:
[Cys（PEG-M）]
X-BVLD (Ⅰ)
Wherein
X is the arbitrary integer among the 5-8, and the 5th to the 8th arbitrary glycine in the expression bivalirudin sequence substituted by cysteine; PEG is RO (CH
2CH
2O) n-CH
2CH
2, R=H or CH
3, n=5-1000; Cys is cysteine, and it links to each other with M group covalency by its side chain sulphur atom.
2. described bivalirudin-polyethylene glycol complex according to claim 1, wherein the structure of this complex is [Cys (mPEG
2000-MAL)]
7-BVLD, [Cys (mPEG
5000-MAL)]
7-BVLD and [Cys (mPEG
10000-MAL)]
7-BVLD.
3. a pharmaceutical composition is characterized in that containing at least a bivalirudin-polyethylene glycol complex and pharmaceutic adjuvant among the claim 1-2.
4. the described bivalirudin-polyethylene glycol complex of each claim is characterized in that for the preparation of the application in the medicine for the treatment of or prevention thrombus disease described medicine is used for the treatment of unstable angina pectoris, peripheral arterial interventional therapy, heart-lung transplant operation and anti-bolt treatment according to claim 1-2.
5. the preparation method of bivalirudin-polyethylene glycol complex according to claim 1 is characterized in that comprising
With [Cys]
X-BVLD is dissolved in the pure water, regulate pH to 7～8 with sodium bicarbonate, the PEG that adds 2-3 times of molar equivalent, stirring at room reaction 2-3 hour, with reversed-phase high-performance liquid chromatography (RP-HPLC) monitoring reaction process and separate targets product, target product gets bivalirudin-polyethylene glycol complex [Cys (PEG-MAL)] through lyophilization
X-BVLD, PEG wherein are dimaleoyl imino Polyethylene Glycol, ethylene sulfuryl Polyethylene Glycol or iodo-acetamide base Polyethylene Glycol.
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| CN1532207A (en) * | 2003-03-21 | 2004-09-29 | 中国人民解放军军事医学科学院毒物药 | Polyethylene glycol derivatives of thymosin alphal |
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Non-Patent Citations (2)
| Title |
|---|
| John Eikelboom et al..The Evolving Role of Direct Thrombin Inhibitors in Acute Coronary Syndromes.《Journal of the American College of Cardiology》.2003,第41卷(第4期),p70S-78S. |
| The Evolving Role of Direct Thrombin Inhibitors in Acute Coronary Syndromes;John Eikelboom et al.;《Journal of the American College of Cardiology》;20030219;第41卷(第4期);第71S页右栏第1、2段 * |
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