CN108117613A - Low molecular weight heparin and heparin are used to prepare the purposes of pulmonary fibrosis - Google Patents

Low molecular weight heparin and heparin are used to prepare the purposes of pulmonary fibrosis Download PDF

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CN108117613A
CN108117613A CN201710374073.0A CN201710374073A CN108117613A CN 108117613 A CN108117613 A CN 108117613A CN 201710374073 A CN201710374073 A CN 201710374073A CN 108117613 A CN108117613 A CN 108117613A
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molecular weight
heparin
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邢新会
闫昳姝
季洋
王怡
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Tsinghua University
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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • C—CHEMISTRY; METALLURGY
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    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
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Abstract

The present invention relates to the purposes that low molecular weight heparin and heparin are used to prepare pulmonary fibrosis.Low molecular weight heparin of the present invention, the scope of its number-average molecular weight (Mn) is 3000~12000Da, the scope of weight average molecular weight (Mw) is 5000~20000Da, it is preferred that the scope of its number-average molecular weight (Mn) is 3500~11000Da, the scope of weight average molecular weight (Mw) is 5500~17000Da, the scope of its further preferred number-average molecular weight (Mn) is 3600~10500Da, the scope of weight average molecular weight (Mw) is 6000~15000Da, still further preferably the scope of its number-average molecular weight (Mn) is 3700~11000Da, the scope of weight average molecular weight (Mw) is 7000~12000Da.

Description

Low molecular weight heparin and heparin are used to prepare the purposes of pulmonary fibrosis
Technical field
The present invention relates to a kind of low molecular weight heparin and its be used to prepare prevention or treat pulmonary fibrosis drug use On the way, prevention is used to prepare the invention further relates to heparin or treat the purposes of the drug of pulmonary fibrosis in addition.
Background technology
Fibrotic disease is after being organized in and being damaged because improper, excessive reparation cause it is collagen-rich Extracellular matrix (ECM) is deposited in tissue and organ, and parenchyma is made to lose original function.Fibrotic disease includes involving The disease of multisystem, such as systemic sclerosis, multifocal fibrosis, chorionitis, the multisystem fibrosis of kidney source property.Also device is included Official's tissue specific disease, such as lung, liver, renal fibrosis disease.
Because organ that different fibrosis lesions is related to and tissue are different, exposed to different environmental factors, and group Into cell type it is different, but fibrotic processes are respectively provided with similar pathologic process：Continuation damage, secretion occur for epithelial cell Excessive cell factor and growth factor.The recruitment and activation of the further mesenchymal precursor of these factors form flesh Fibroblast then secretes substantial amounts of extracellular matrix.Under normal physiological conditions, organize after reparation, fibrosis Matrix will be degraded, and apoptosis will occur for fibroblast or reverse to become non-living cells.But under the conditions of fibrotic disease, Normal cleaning and reversal program are destroyed, and parenchyma is gradually substituted by myofibroblast, loses normal function, leads The generation of fibrotic disease is caused.
In the Western countries, fibrotic disease finally contributes to up to 45% percentage for the death rate, in development China Family still lacks statistics.Although fibrotic disease hair patient it is numerous, be for the Study on Molecular Mechanism being related to it is preliminary, More lack effective therapy.
Idiopathic pulmonary fibrosis (Idiopathic Pulmonary Fibrosis, IPF) is a kind of progressivity, to diffuse Property pulmonary fibrosis, cause impairment of pulmonary function and the Interstitial Lung Disease that is characterized of expiratory dyspnea.During the average survival time of the patient Between only 3.2 years, be a kind of fatal disease that prognosis is worst in chronic non-tumour respiratory disease.Morbidity current IPF Rate is 1.25-27.9/10 ten thousand.There are many sick pathogenic factor, and the more specific cause of disease has sucking dust, gas, virus, thin Bacterium, drug and radioactive damage etc..In China, because process of industrialization is accelerated, air pollution is on the rise, with acute and slow Property pulmonary fibrosis patient dramatically increasing, year IPF death rate average out to 4-10/10 ten thousand from 1999 to 2012 is annual to increase 2-3%.The pathogenesis of IPF is simultaneously indefinite, is caused by chronic inflammation, but due to anti-inflammatory or immunosuppressor Therapeutic effect unobvious in IPF treatments, more focus on the variation of pulmonary epithelial cells, fibroblast, cause at present Fibrocyte activation, pulmonary fibrosis and lung remodeling imbalance.
Develop the active drug for curing IPF not yet both at home and abroad at present, mainly some delay what IPF further deteriorated Drug, such as pirfenidone (Pirfenidone,) and Nintedanib (Nintedanib,).The non-Buddhist nun of pyrrole Ketolysis mechanism is by inhibiting the generation of TGF-β reduction fibroblast and playing anti-inflammatory work by TNF-α and IL-1 β With.Nintedanib is a kind of inhibitor of intracellular multiple tyrosine kinase, including the inhibition work to VEGF, FGF, PDGF With.
The content of the invention
Heparin is a kind of sulphation, polydispersion, linear glycosaminoglycan (Glycosaminoglycans, GAGs), is most One of important anticoagulant.Clinically it is widely used in preventing thrombotic disease.In addition, heparin and its derivative tool There is extensive biological activity, combined including coordinating cell adhesion, regulating cell growth and multiplication, growth course, cell surface Lipoprotein lipase and other oroteins, new blood vessel generation, poisoning intrusion and metastases etc..Have preclinical and clinical grind The symptom for showing unassorted macromolecule heparin to IPF is studied carefully with effect is significantly reduced, and can delay the process of pulmonary fibrosis (Gunther,A.,Lubke,N.,Ermert,M.,Schermuly,R.T.,Weissmann,N.,Breithecker, A.,...&Seeger,W.(2003).Prevention of bleomycin-induced lung fibrosis by aerosolization of heparin or urokinase in rabbits.American Journal of Respiratory and Critical Care Medicine, 168 (11), 1358-1365 and Gunther, A., Lubke,N.,Ermert,M.,Schermuly,R.T.,Weissmann,N.,Breithecker,A.,...&Seeger,W. (2003).Prevention of bleomycin-induced lung fibrosis by aerosolization of heparin or urokinase in rabbits.American Journal of Respiratory and Critical Care Medicine,168(11),1358-1365.)。
But macromolecule heparin molecule common at present is not the optimum structure for treating fibrotic disease in itself.This It is the microheterogeneity because heparin structure, causes have very strong binding ability with a variety of factors, so as to which " dilution " mainly divides Subsequence and the interaction of target spot, and generate side effect.In addition, heparin molecule has in itself, bleeding is more, decrease of platelet Deng more serious side effect.
For obtained through a variety of biodegrading process low molecular weight heparin (Low molecular weight heparins, LMWH the effect of fibrotic disease) is intervened, it is also at present to have been reported that more.But due to different biodegrading process identification heparin Sequence site is different, also completely different to the intervention effect effect of fibrotic disease.For example, using β-degradation mode, i.e. through into The Enoxaparin (Enoxaparin) that salt, esterification, basic hydrolysis technique obtain intervenes pulmonary fibrosis mice caused by Bleomycin, For the no any improved effect of the formation of pulmonary fibrosis (Laxer, U., Lossos, I.S., Gillis, S., Or, R., Christensen,T.G.,Goldstein,R.,&Breuer,R.(1999).THE EFFECT OF ENOXAPARIN ON BLEOMYCIN-INDUCED LUNG INJURY IN MICE.Experimental Lung Research,25(6),531- 541).Therefore, for treating the LMWH classes drug of fibrotic disease, it is necessary to which systematicness screens its activity, and carries out structure effect and close System's research, the active principle for the disease that can just obtain medical treatment so as to improve molecule to various diseases therapeutic choice, reach structure work( The best match state of energy.
The present inventor is directed to the innovation research of heparin industrial technology, utilizes maltose-binding protein (Maltose Binding Protein, MBP) amalgamation and expression technology, realize high activity, solubility expression and the industrialization of serial heparinase Production, serial heparinase is listed in Chinese Pharmacopoeia standard enzyme, and (Heparinase is respectively MBP-HepI, MBP-HepII, MBP- HepIII, can respectively referring to Chinese patent ZL200410038098.6, ZL201010259905.2 and ZL201010259913.7).And it tentatively establishes combination enzyme process and prepares the new process of LMWH (referring to Chinese patent ZL201210328649.7).On this basis, the new low of the present invention has successfully been obtained by depth studying in the present inventor Molecular weight heparin, and find that the low molecular weight heparin of the present invention has the function of to prevent or treat pulmonary fibrosis.
The present invention relates to herein below：
1. a kind of low molecular weight heparin, the scope of number-average molecular weight (Mn) is 3000~12000Da, weight average molecular weight (Mw) scope be 5000~20000Da, preferably the scope of its number-average molecular weight (Mn) be 3500~11000Da, Weight-average molecular The scope of (Mw) is measured as 5500~17000Da, the scope of its further preferred number-average molecular weight (Mn) is 3600~10500Da, The scope of weight average molecular weight (Mw) is 6000~15000Da, and still further preferably the scope of its number-average molecular weight (Mn) is 3700 ~11000Da, the scope of weight average molecular weight (Mw) is 7000~12000Da.
2. according to the low molecular weight heparin described in item 1, wherein, the molecular weight distribution of weight average molecular weight is：Molecular weight is small Below the 30wt% of the low molecular weight heparin whole is accounted in the heparin of 3000Da, preferably in below 25wt%, molecular weight is more than The heparin of 8000Da accounts for more than the 30wt% of the low molecular weight heparin whole, preferably in more than 35wt%, further preferably exists More than 40wt%.
3. the low molecular weight heparin according to item 1 or 2, wherein, the molecular weight distribution of weight average molecular weight is：Molecular weight Below the 30wt% of the low molecular weight heparin whole is accounted in the heparin of 3000~5000Da, preferably in below 25wt%.
4. according to the low molecular weight heparin any one of item 1~3, wherein, the scope of number-average molecular weight (Mn) is 3000~7000Da, the scope of weight average molecular weight (Mw) is 6000~12000Da, and the scope of preferably its number-average molecular weight (Mn) is 3500~6000Da, the scope of weight average molecular weight (Mw) is 6500~11000Da.
5. according to the low molecular weight heparin any one of item 1~3, wherein, the scope of number-average molecular weight (Mn) is 7000~10000Da, the scope of weight average molecular weight (Mw) are the scope of 8000~12000Da, preferably its number-average molecular weight (Mn) For 8000~9800Da, the scope of weight average molecular weight (Mw) is 9000~11000Da.
6. according to the low molecular weight heparin described in item 1, the scope of number-average molecular weight (Mn) is 4000~11000Da, heavy The scope of average molecular weight (Mw) is 7000~12000Da, and it does not have anticoagulating active.
7. according to the low molecular weight heparin described in item 6, the scope of number-average molecular weight (Mn) is 4000~8000Da, and weight is equal The scope of molecular weight (Mw) is 7000~12000Da.
8. according to the low molecular weight heparin described in item 6, the scope of number-average molecular weight (Mn) is 9000~11000Da, heavy The scope of average molecular weight (Mw) is 9500~12000Da.
9. according to the low molecular weight heparin any one of item 1~8, wherein, the low molecular weight heparin is to utilize liver Obtained from plain enzyme degradation heparin.
10. according to the low molecular weight heparin any one of item 1~9, wherein, the low molecular weight heparin is to utilize liver Obtained from plain enzyme I or Heparinase I II degradation heparin.
11. according to the low molecular weight heparin described in item 4 or item 7, wherein, the low molecular weight heparin is to utilize Heparinase I Obtained from degradation heparin.
12. according to the low molecular weight heparin described in item 5 or item 8, wherein, the low molecular weight heparin is to utilize heparinase Obtained from III degradation heparin.
13. according to the low molecular weight heparin any one of item 6~8, wherein, the low molecular weight heparin is first to liver Element carries out anti-freezing and handles, then obtained from heparinase degrades heparin.
14. purposes of the heparin in the drug for treating or preventing pulmonary fibrosis is used to prepare.
15. according to the purposes described in item 14, wherein, the heparin is the low molecular weight liver any one of item 1~13 Element.
16. according to the purposes described in item 14, wherein, the heparin be the scope of number-average molecular weight (Mn) be more than 12000Da, the scope of weight average molecular weight (Mw) is the undegradable heparin more than 20000Da.
17. purposes of the heparin in being used to prepare to alleviate the drug of formation of lung's collagen.
18. according to the purposes described in item 17, wherein, the heparin is the low molecular weight liver any one of item 1~13 Element.
19. according to the purposes described in item 18, wherein, the heparin be the scope of number-average molecular weight (Mn) be more than 12000Da, the scope of weight average molecular weight (Mw) is the undegradable heparin more than 20000Da.
20. a kind of method for treating or preventing pulmonary fibrosis, including：To have required mammal or people to Medicine heparin.
21. according to the method described in item 20, wherein, the heparin is the low molecular weight liver any one of item 1~13 Element.
22. according to the method described in item 20, wherein, the heparin be the scope of number-average molecular weight (Mn) be more than 12000Da, the scope of weight average molecular weight (Mw) is the undegradable heparin more than 20000Da.
23. it is a kind of for alleviating the method that lung's collagen is formed, including：To having required mammal or people Heparin is administered.
24. according to the method described in item 23, wherein, the heparin is the low molecular weight liver any one of item 1~13 Element.
25 method according to item 23, wherein, the heparin be number-average molecular weight (Mn) scope be more than 12000Da, the scope of weight average molecular weight (Mw) is the undegradable heparin more than 20000Da.
26. one kind removes anticoagulant heparin, the scope of number-average molecular weight (Mn) is more than 11000Da, weight average molecular weight (Mw) Scope be more than 12000Da, the scope of preferred number average molecular weight (Mn) is more than 12000Da, the model of weight average molecular weight (Mw) It encloses to be more than 13000Da.The scope of preferred number average molecular weight (Mn) is more than 13000Da, and the scope of weight average molecular weight (Mw) is More than 14000Da.The scope of preferred number average molecular weight (Mn) be more than 13500Da, the scope of weight average molecular weight (Mw) be more than 15000Da does not have anticoagulating active.
27. remove anticoagulant heparin according to item 26, be by heparin molecule is carried out N-terminal go sulfate radical and into What row acetylation obtained removes anticoagulant heparin.
28. purposes of the heparin described in 26 or 27 in the drug for treating or preventing pulmonary fibrosis is used to prepare.
29. use of the heparin described in 26 or 27 in being used to prepare to alleviate the drug of formation of lung's collagen On the way.
30. a kind of method for treating or preventing pulmonary fibrosis, including：To have required mammal or people to Anticoagulant heparin is removed described in medicine item 26 or item 27.
31. it is a kind of for alleviating the method that lung's collagen is formed, including：To having required mammal or people Anticoagulant heparin is removed described in administration item 26 or item 27.
Description of the drawings
H＆E dyeing characterization low molecular weight heparin is to the Bleomycin injury of lungs induced and pulmonary fibrosis in Fig. 1 experimental examples 2 Influence.(a) negative control group；(b) I-2 groups；(c) I-11 groups；(d) III-1 groups；(e) III-2 groups；(f) healthy group；(g)I- 16 groups；(h) NS groups；(i) N1 groups；(j) N2 groups；(k) N4 groups；(l) N5 groups；(m) N3 groups；(n) H groups.
Specific embodiment
Hereinafter, embodiments described herein is specifically described.
<The low molecular weight heparin of the present invention>
The present invention relates to the low molecular weight heparin for treating and/or preventing pulmonary fibrosis.In the implementation of the present invention In scheme, it is related to a kind of low molecular weight heparin, the scope of number-average molecular weight (Mn) is 3000~12000Da, weight average molecular weight (Mw) scope be 5000~20000Da, preferably the scope of its number-average molecular weight (Mn) be 3500~11000Da, Weight-average molecular The scope of (Mw) is measured as 5500~17000Da, the scope of its further preferred number-average molecular weight (Mn) is 3600~10500Da, The scope of weight average molecular weight (Mw) is 6000~15000Da, and still further preferably the scope of its number-average molecular weight (Mn) is 3700 ~11000Da, the scope of weight average molecular weight (Mw) is 7000~12000Da.Its number of low molecular weight heparin i.e. of the present invention Average molecular weight (Mn) can be for example, about 3100Da, 3200Da, 3300Da, 3400Da, 3800Da, 3900Da, 4000Da, 4100Da, 4200Da, 4300Da, 4400Da, 4500Da, 4600Da, 4700Da, 4800Da, 4900Da, 5000Da, 5100Da, 5200Da, 5300Da, 5400Da, 5500Da, 5600Da, 5700Da, 5800Da, 5900Da, 6000Da, 6100Da, 6200Da, 6300Da, 6400Da, 6500Da, 6600Da, 6700Da, 6800Da, 6900Da, 7000Da, 7100Da, 7200Da, 7300Da, 7400Da, 7500Da, 7600Da, 7700Da, 7800Da, 7900Da, 8000Da, 8100Da, 8200Da, 8300Da, 8400Da, 8500Da, 8600Da, 8700Da, 8800Da, 8900Da, 9000Da, 9100Da, 9200Da, 9300Da, 9400Da, 9500Da, 9600Da, 9700Da, 9800Da, 9900Da, 10100Da, 10200Da, 10300Da, 10400Da, 10600Da, 10700Da, 10800Da, 10900Da, 11100Da, 11200Da, 11300Da, 11400Da, 11500Da, 11600Da, 11700Da, 11800Da, 11900Da.Its weight average molecular weight (Mw) of low molecular weight heparin of the present invention can be for example, about 5100Da, 5200Da, 5300Da, 5400Da, 5600Da, 5700Da, 5800Da, 5900Da, 6100Da, 6200Da, 6300Da, 6400Da, 6500Da, 6600Da, 6700Da, 6800Da, 6900Da, 7000Da, 7100Da, 7200Da, 7300Da, 7400Da, 7500Da, 7600Da, 7700Da, 7800Da, 7900Da, 8000Da, 8100Da, 8200Da, 8300Da, 8400Da, 8500Da, 8600Da, 8700Da, 8800Da, 8900Da, 9000Da, 9100Da, 9200Da, 9300Da, 9400Da, 9500Da, 9600Da, 9700Da, 9800Da, 9900Da, 10000Da, 10100Da, 10200Da, 10300Da, 10400Da, 10500Da, 10600Da, 10700Da, 10800Da, 10900Da, 11000Da, 11500Da, 12000Da, 12500Da, 13000Da, 13500Da, 14000Da, 14500Da, 15500Da, 16000Da, 16500Da, 17500Da, 18000Da, 18500Da, 19000Da, 19500Da。
In one embodiment of the invention, it is related to a kind of low molecular weight heparin, the molecular weight point of weight average molecular weight Cloth is：Heparin of the molecular weight less than 3000Da accounts for below the 30wt% of the low molecular weight heparin whole, preferably in below 25wt%, It is preferred that in below 24wt%, preferably in below 23wt%.Heparin of the molecular weight more than 8000Da accounts for low molecular weight heparin whole More than 30wt%, preferably in more than 35wt%, preferably in more than 36wt%, further preferably in more than 38wt%, preferably exist More than 40wt%.
In one embodiment of the invention, it is related to a kind of low molecular weight heparin, the molecular weight point of weight average molecular weight Cloth is：Molecular weight accounts for below the 30wt% of the low molecular weight heparin whole in the heparin of 3000~5000Da, preferably in 25wt% Hereinafter, preferably in below 23wt%, preferably in below 22wt%, molecular weight accounts for the low molecular weight in the heparin of 5000~8000Da Below the 30wt% of heparin whole, preferably in below 25wt%, preferably in below 22wt%.
In one embodiment of the invention, it is related to a kind of low molecular weight heparin, the scope of number-average molecular weight (Mn) For 3000~10000Da, the scope of weight average molecular weight (Mw) is the model of 6500~12000Da, preferably its number-average molecular weight (Mn) It encloses for 3500~9500Da, the scope of weight average molecular weight (Mw) is 7000~11000Da, which is to utilize heparin What enzyme III or Heparinase I were degraded.
In one embodiment of the invention, it is related to a kind of low molecular weight heparin, the molecular weight point of weight average molecular weight Cloth is：Molecular weight accounts for more than the 2wt% and below 25wt% of the low molecular weight heparin whole in the heparin of 3000~5000Da, into One step is preferably in more than 3wt% and below 20wt%.It is complete that molecular weight in the heparin of 5000~8000Da accounts for the low molecular weight heparin More than the 5wt% and below 30wt% in portion, further preferably in more than 8wt% and below 25wt%, molecular weight is less than 3000Da Heparin account for below the 30wt% of the low molecular weight heparin whole, preferably below 25wt%, molecular weight is more than the heparin of 8000Da More than the 30wt% and below 90wt% of the low molecular weight heparin whole are accounted for, preferably in more than 35wt% and below 85wt%, on The total amount of four kinds of distributions is stated as 100wt%, which degrades to obtain using Heparinase I or Heparinase I II.
In one embodiment of the invention, it is related to a kind of low molecular weight heparin, the scope of number-average molecular weight (Mn) For 3000~7000Da, the scope of weight average molecular weight (Mw) is the scope of 6000~12000Da, preferably its number-average molecular weight (Mn) For 3500~6000Da, the scope of weight average molecular weight (Mw) is 6500~11000Da, which is to utilize heparinase What I degraded.
In one embodiment of the invention, it is related to a kind of low molecular weight heparin, the molecular weight point of weight average molecular weight Cloth is：Molecular weight accounts for more than the 5wt% and below 25wt% of the low molecular weight heparin whole in the heparin of 3000~5000Da, into One step is preferably in more than 7wt% and below 20wt%.It is complete that molecular weight in the heparin of 5000~8000Da accounts for the low molecular weight heparin More than the 10wt% and below 30wt% in portion, further preferably in more than 13wt% and below 25wt%, molecular weight is less than The heparin of 3000Da accounts for more than the 5wt% and below 30wt% of the low molecular weight heparin whole, preferably more than 6wt% and 25wt% Hereinafter, heparin of the molecular weight more than 8000Da accounts for more than the 30wt% and below 80wt% of the low molecular weight heparin whole, preferably In more than 35wt% and below 75wt%, the total amount of above-mentioned four kinds of distributions is 100wt%, which is to utilize heparin Enzyme I degrades.
In one embodiment of the invention, it is related to a kind of low molecular weight heparin, the scope of number-average molecular weight (Mn) For 7000~10000Da, the scope of weight average molecular weight (Mw) is the model of 8000~12000Da, preferably its number-average molecular weight (Mn) It encloses for 8000~9800Da, the scope of weight average molecular weight (Mw) is 9000~11000Da, which is to utilize heparin Enzyme III degrades.
In one embodiment of the invention, it is related to a kind of low molecular weight heparin, the molecular weight point of weight average molecular weight Cloth is：Molecular weight accounts for below the 10wt% of the low molecular weight heparin whole in the heparin of 3000~5000Da, further preferably exists More than 2wt% and below 8wt%.Molecular weight accounts for more than the low molecular weight heparin whole 5wt% in the heparin of 5000~8000Da And below 25wt%, further preferably in more than 8wt% and below 23wt%, heparin of the molecular weight less than 3000Da accounts for this low point Below the 10wt% of son amount heparin whole, preferably below 5wt%, heparin of the molecular weight more than 8000Da account for the low molecular weight heparin Whole more than 70wt%, preferably in below 90wt%, the total amounts of above-mentioned four kinds of distributions are 100wt%, the low molecular weight heparin It degrades to obtain using Heparinase I II.
In one embodiment of the invention, it is related to a kind of low molecular weight heparin, the scope of number-average molecular weight (Mn) For 4000~11000Da, the scope of weight average molecular weight (Mw) is 7000~12000Da, and it does not have anticoagulating active, this is low Molecular weight heparin is first to carry out anti-freezing to heparin to handle, and is then degraded using Heparinase I II or Heparinase I.
In one embodiment of the invention, it is related to a kind of low molecular weight heparin, the molecular weight point of weight average molecular weight Cloth is：Molecular weight accounts for below the 30wt% of the low molecular weight heparin whole in the heparin of 3000~5000Da, further preferably exists More than 2wt% and below 25wt%.Molecular weight accounts for more than the low molecular weight heparin whole 5wt% in the heparin of 5000~8000Da And below 25wt%, further preferably in more than 8wt% and below 23wt%, heparin of the molecular weight less than 3000Da accounts for this low point Below the 15wt% of son amount heparin whole, preferably below 12wt%, heparin of the molecular weight more than 8000Da account for the low molecular weight liver Plain whole more than 40wt%, preferably in below 90wt%, the total amounts of above-mentioned four kinds of distributions are 100wt%, the low molecular weight liver Element is first to carry out anti-freezing to heparin to handle, and is then degraded using Heparinase I II or Heparinase I.
In one embodiment of the invention, it is related to a kind of low molecular weight heparin, the scope of number-average molecular weight (Mn) For 4000~8000Da, the scope of weight average molecular weight (Mw) is 7000~12000Da, the low molecular weight heparin be first to heparin into Row goes anti-freezing to handle, and is then degraded using Heparinase I, and it does not have anticoagulating active.
In one embodiment of the invention, it is related to a kind of low molecular weight heparin, the molecular weight point of weight average molecular weight Cloth is：Molecular weight accounts for below the 30wt% of the low molecular weight heparin whole in the heparin of 3000~5000Da, further preferably exists More than 4wt% and below 25wt%.Molecular weight accounts for more than the low molecular weight heparin whole 5wt% in the heparin of 5000~8000Da And below 25wt%, further preferably in more than 8wt% and below 23wt%, heparin of the molecular weight less than 3000Da accounts for this low point Below the 15wt% of son amount heparin whole, preferably below 12wt%, heparin of the molecular weight more than 8000Da account for the low molecular weight liver Plain whole more than 40wt%, preferably in below 85wt%, the total amounts of above-mentioned four kinds of distributions are 100wt%, the low molecular weight liver Element is first to carry out anti-freezing to heparin to handle, and is then degraded using Heparinase I, and it does not have anticoagulating active.
In one embodiment of the invention, it is related to a kind of low molecular weight heparin, the scope of number-average molecular weight (Mn) For 9000~11000Da, the scope of weight average molecular weight (Mw) is 9500~12000Da, which is first to heparin It carries out anti-freezing to handle, then be degraded using Heparinase I II, and it does not have anticoagulating active.
In one embodiment of the invention, it is related to a kind of low molecular weight heparin, the molecular weight point of weight average molecular weight Cloth is：Molecular weight accounts for below the 20wt% of the low molecular weight heparin whole in the heparin of 3000~5000Da, further preferably exists More than 1wt% and below 15wt%.Molecular weight accounts for more than the low molecular weight heparin whole 5wt% in the heparin of 5000~8000Da And below 20wt%, further preferably in more than 8wt% and below 18wt%, heparin of the molecular weight less than 3000Da accounts for this low point Below the 10wt% of son amount heparin whole, preferably below 8wt%, heparin of the molecular weight more than 8000Da account for the low molecular weight heparin Whole more than 60wt%, preferably in below 90wt%, the total amounts of above-mentioned four kinds of distributions are 100wt%, the low molecular weight heparin It is first to carry out anti-freezing to heparin to handle, is then degraded using Heparinase I II, and it does not have anticoagulating active.
In the present invention, well known method may be employed in above-mentioned weight average molecular weight, number-average molecular weight and molecular weight distribution Detection, such as according in Wu, Jingjun et al. " Controllable production of low molecular weight heparins by combinations of heparinase I/II/III."Carbohydrate polymers 101 (2014):Recorded method is detected in 484-492, and specific detecting step may refer to described in following embodiments Method.If when the result of other well known detection method detection and the inconsistent testing result of this method, involved by the present invention And low molecular weight heparin weight average molecular weight, number-average molecular weight and molecular weight distribution with the side employed in the embodiment of the present invention Subject to method.
<Low molecular weight heparin is used to inhibit the effect of pulmonary fibrosis>
Low molecular weight heparin of the present invention can be used in treating or preventing pulmonary fibrosis.
The present invention relates to purposes of the low molecular weight heparin in the drug for treating or preventing pulmonary fibrosis is prepared.
Pulmonary fibrosis in the present invention refers to fibroblast proliferation and the aggregation of a large amount of extracellular matrixs and with inflammation The whole latter stage for the major class lung disease that damage, institutional framework destruction are characterized changes, that is, normal alveolar tissue is damaged Cause textural anomaly by abnormal reparation afterwards (scar is formed).Pulmonary fibrosis in the present invention includes idiopathic pulmonary fibrosis, original The pulmonary fibrosis of hair property, secondary pulmonary fibrosis, interstitial lung fibrosis.The present invention relates to the low molecular weight heparin prepare treatment or Prevent the drug of idiopathic pulmonary fibrosis, idiopathic pulmonary fibrosis, secondary pulmonary fibrosis and/or interstitial lung fibrotic disease In purposes.
Low molecular weight heparin of the present invention can effectively inhibit the death rate for the mouse for inducing pulmonary fibrosis, and And carry out sections observation for the lung tissue of the mouse after administration and show, after the low molecular weight heparin of the administration present invention, mouse Substantially complete alveolar structure is presented in lung, and cell monolayer structure is presented in most of alveolar, and cellular morphology is similar with normally organizing, nothing Apparent inflammatory infiltration feature, illustrates that fibrosis also have the different degrees of trend that lightens.
The inventors discovered that in terms of lung tissue change, after administration low molecular weight heparin of the present invention with it is common The effect of macromolecule heparin is basically identical, but is had during the low molecular weight heparin of the administration present invention without macromolecule heparin Some side effects, i.e. bleeding risk increase and easily induce thrombopenia.Several low molecular weights involved in the present invention Although heparin inhibits pulmonary fibrosis effect and the effect of macromolecule effects of heparin pulmonary fibrosis is similar, low molecular weight liver Plain drug metabolism is controllable, and the bleeding risk of patient need not be monitored in clinic.
In addition low molecular weight heparin of the present invention has certain effect to the oedema for inhibiting lung, is effectively reduced The content of hydroxyproline in mouse lung tissue, that is, significantly reduce the conversion ratio of lung's collagen, reduce lung Portion's collagen content, since the deposition of collagen is the most apparent feature of fibrosis lesion, collagen content Reduction shows that pulmonary fibrosis is under control.Currently used Enoxaparin, then without inhibit pulmonary fibrosis effect (referring to Laxer U,Lossos I S,Gillis S,et al.The effect of enoxaparin on bleomycin- induced lung injury in mice[J].Experimental lung research,1999,25(6):531- 541), it is seen that the low molecular weight heparin that the present invention obtains is different from the structure of Enoxaparin, and effect is also entirely different.
Although being not intended to be restricted by theory, the low molecular weight heparin that the present invention obtains is low point obtained by enzymic degradation Sub- heparin, mechanism of degradation are totally different from the Enoxaparin obtained by chemical method, divide according in embodiment for molecular weight The analysis data of cloth can also be it is clearly seen that it be distinguished.
Low molecular weight heparin prepared by enzyme edman degradation Edman of the present invention, can significantly inhibit the formation of injury of lungs, inhibit Inflammation during injury of lungs occurs, and so as to fundamentally prevent and inhibit the formation of pulmonary fibrosis, reduces the heavy of collagen Product.And the pulmonary fibrosis resistant class drug to emerge at present, it is only capable of alleviating the symptom that pulmonary fibrosis is formed, the treatment in IPF treatments Effect is not obvious.
The invention further relates to the heparin derivatives for eliminating anticoagulating active, can be seen that according to the data of following embodiments Either eliminate anticoagulating active heparin derivatives still further degraded to it using above-mentioned enzyme solution after obtain Low molecular weight the death rate removed anticoagulant heparin derivative, effectively inhibit the mouse for inducing pulmonary fibrosis.
The anticoagulant heparin is gone to derive in the heparin derivatives for eliminating anticoagulating active and low molecular weight of the administration present invention After object, the lung of mouse is presented substantially complete alveolar structure, and most of alveolar is presented cell monolayer structure, cellular morphology with It normally organizes similar, without apparent inflammatory infiltration feature, illustrates that fibrosis also have the different degrees of trend that lightens.In addition, It is administered after this heparan, has certain effect, significantly reduced in mouse lung tissue to the oedema for inhibiting lung The content of hydroxyproline significantly reduces the conversion ratio of lung's collagen, reduce lung's collagen content, due to The deposition of collagen is the most apparent feature of fibrosis lesion, therefore the reduction of collagen content shows that pulmonary fibrosis obtains Control.Experimental example according to the present invention the results show that administration eliminate anticoagulating active heparin derivatives and administration to this The low molecular weight heparin derivative of anticoagulating active is respectively provided with said effect, but does not have big point during the low molecular weight heparin of the administration present invention Side effect possessed by son amount heparin, i.e. bleeding risk increase and easily induce thrombopenia.Involved in the present invention Although several low molecular weight heparins inhibit pulmonary fibrosis effect and the effect of macromolecule effects of heparin pulmonary fibrosis is similar, It is that low molecular weight heparin drug metabolism is controllable, the blood risk of patient need not be detected in clinic.
<The method for removing anticoagulant heparin is produced in the present invention>
In addition to unfractionated heparin, anticoagulant heparin is additionally used in the present invention and produces low molecular weight liver as raw material Element.
Usually heparin, low molecular weight heparin, pentose are all the speed that clotting factor is inactivated by accelerating antithrombin Ⅲ It spends and plays anticoagulation, the main function of such drug is anti-Xa and anti-IIa factor actives.By to the anti-Xa of heparin class drug The active activity with anti-IIa the study found that Anti-Xa activity is insensitive to molecular mass, anti-IIa activity then relies on molecular mass Size.Molecular mass is bigger, and anti-IIa activity is stronger.Heparin depends on heparin-antithrombase-IIa factors to the inactivation of the IIa factors The formation of three compounds, heparin is in combination in antithrombase and factor IIa, will realizing that this connection heparin at least will at this time Containing 18 sugared units, wherein playing " bridge " needs 13 monose, 5 monose are separately needed as identification segment.Each monose Average molecular mass is 300Da, therefore molecular mass has to reach more than 5400Da just with anti-IIa activity.Unfractionated heparin Average molecular mass 15000-19000Da, most molecules are about with anti-IIa activity ratios in more than 5400Da, anti-Xa 1.Low molecular weight heparin average molecular mass is 4000-5000Da, and molecular mass is in the molecule fragment proportion of more than 5400Da It is smaller, its anti-Xa under normal circumstances:Anti- IIa activity about 1.5:1~5:1.
In the art, it is used to remove the anticoagulating active of heparin there are a variety of methods, the present invention does not limit to obtain The method for removing anticoagulant heparin.In a specific embodiment, what is used in the present invention goes the preparation method of anticoagulant heparin It is to remove the sulfate radical of heparin N-terminal and then the method for acetylation is carried out to it (heparin so obtained is hereinafter also referred to as N- Acetylated-heparin), specific preparation method may refer to Lapierre F, Holme K, Lam L, et al.Chemical modifications of heparin that diminish its anticoagulant but preserve its heparanase-inhibitory,angiostatic,anti-tumor and anti-metastatic properties [J].Glycobiology,1996,6(3):The method that 355-366 is reported.
As shown in following embodiments and comparative example, in the present invention, there is the anti-Xa potency of the heparin raw material of anticoagulating active For 187 ± 21IU/mg, anti-IIa potency is 177 ± 6IU/mg.And the method that make use of the removal anticoagulant heparin activity of the present invention The anti-Xa potency of the heparin of anticoagulating active is eliminated as 15 ± 2IU/mg, anti-IIa potency is 12 ± 0.6IU/mg.In addition, as after Shown in continuous embodiment, using the low molecular weight heparin obtained after heparin enzymatic treatment raw material liver element anti-Xa and anti-IIa potency with The raw material heparin not digested is compared to have and reduced to a certain degree, but it is still the low molecular weight heparin with anticoagulating active.
The heparin without anticoagulating active defined in the present invention refers to carry out heparin sufficiently anti-freezing to be gone to handle Heparin, its anti-Xa potency of this heparin and anti-IIa potency is obtained afterwards to degrade to obtain far below raw material heparin and by heparinase Low molecular weight heparin, such as anti-Xa potency be below 20IU/mg, anti-IIa potency be below 20IU/mg.
Remove the anticoagulant heparin that goes that anticoagulant methods handle using above-mentioned, the scope of number-average molecular weight (Mn) be more than 11000Da, the scope of weight average molecular weight (Mw) is more than 12000Da.The scope of preferred number average molecular weight (Mn) be more than 12000Da, the scope of weight average molecular weight (Mw) is more than 13000Da.The scope of preferred number average molecular weight (Mn) be more than 13000Da, the scope of weight average molecular weight (Mw) is more than 14000Da.The scope of preferred number average molecular weight (Mn) be more than 13500Da, the scope of weight average molecular weight (Mw) is more than 15000Da.
Molecular weight accounts for below the 15wt% of the low molecular weight heparin whole in the heparin of 3000~5000Da, further preferably In below 10wt%.Molecular weight accounts for below the 20wt% of the low molecular weight heparin whole in the heparin of 5000~8000Da, into one For step preferably in below 15wt%, heparin of the molecular weight less than 3000Da accounts for below the 10wt% of the low molecular weight heparin whole, excellent Below 8wt% is selected, heparin of the molecular weight more than 8000Da accounts for more than the 80wt% of the low molecular weight heparin whole, preferably exist More than 90wt%, the total amount of above-mentioned four kinds of distributions is 100wt%, which does not have anticoagulating active.
<The production of the low molecular weight heparin of the present invention>
Different types of heparinase is used during low molecular weight heparin of the present invention is produced, these heparin Enzyme can be the heparinase obtained by any method, including Heparinase I, II and III, as long as the activity with heparinase , wherein it is preferable to use Heparinase I, II, III.In the prior art, No. E.C. of Heparinase I be E.C.4.2.2.7, heparin No. E.C. of enzyme III is E.C.4.2.2.8.The heparinase of purchase can also be used, such as purchased from Sigma companies or IBEX companies Heparinase I, II, the III bought.Heparinase can also be by molecular biology method build restructuring Heparinase I, II and The fusion protein that III or Heparinase I, II and III are formed with any fusion partner.A preferred embodiment party according to the present invention Case, Heparinase I, II and III are the fusion protein of Heparinase I, II and III, especially the Heparinase I comprising MBP, II and III Fusion protein.
Heparinase I, Heparinase I I and Heparinase I II can also be the fusion proteins formed with any fusion partner, as long as Activity with Heparinase I, II and III.According to a preferred embodiment herein, Heparinase I, II and III are livers The fusion protein that plain enzyme I, II and III and fusion partner are formed, especially maltose-binding protein (MBP) and Heparinase I, II and The fusion protein of III.The fusion protein of Heparinase I and MBP occasionally are referred to herein as MBP-HepA (referring to Chinese patent ZL200410038098.6, Authorization Notice No. CN1312183C), the fusion protein of Heparinase I I and MBP hereinafter sometimes by Referred to as MBP-HepB (referring to Chinese patent ZL 201010259905.2, Authorization Notice No. CN101942024B), Heparinase I II MBP-HepC occasionally is referred to herein as with the fusion protein of MBP (referring to Chinese patent ZL 201010259913.7, to authorize Notification number CN101942025B).It is preferred that Heparinase I, II and III are respectively provided with Chinese patent ZL201210328649.7, authorize SEQ ID NO in the sequence table of notification number CN103173506B:Sequence described in 1~3.
When producing low molecular weight heparin of the present invention, Heparinase I, II, III or their any combination and substrate The mode that heparin is reacted can be in batches, it is continuous or semi-continuous, those of ordinary skill in the art can be according to production Needs properly select.For the time of reaction, reaction unit, as long as the low molecular weight heparin of target can be obtained , can suitably be determined by those of ordinary skill in the art.
In a specific embodiment, in the method for the low molecular weight heparin of the production present invention, into reactor Substrate heparin solution is added in, is then added in more than one or both of Heparinase I, II or III, is carried out with substrate heparin anti- It should.With the progress of reaction, heparin is gradually degraded, reaction solution is monitored at regular intervals, in due course eventually Only react.Mixed solution by above-mentioned reaction terminating carries out initial filter with cellulose membrane vacuum initial filter device, recycles ultrafiltration dress It puts progress ultrafiltration and obtains time filtrate.So with addition ethyl alcohol after mixing, centrifugation abandons supernatant and collects precipitation, then adds into precipitating Enter acetone washing and carry out evaporated under reduced pressure with Rotary Evaporators and obtain low molecular weight heparin product powder.Those skilled in the art It is appreciated that the above method was merely exemplary, other methods can also be used to obtain the low molecule after degrading by enzyme reaction Measure heparin.
Heparinase I, II and the respective dosages of III, those of ordinary skill in the art may be referred to the active basis of different enzymes It is suitably determined needed for production, preferably the dosage of each enzyme is scope of the Heparinase I in every liter of reaction solution 10IU~500IU, excellent It is selected in the scope of 100IU~250IU.Heparinase I I every liter of reaction solution 10IU~500IU scope, preferably 100IU~ The scope of 200IU.Heparinase I II is in the scope of every liter of reaction solution 10IU~500IU, preferably 25~100IU.Wherein IU is represented： It is 30 DEG C in temperature, under the conditions of pH7.4, the enzyme amount per minute for generating 1 μm of 4,5 unsaturated ends product of ol.
Substrate heparin for producing the low molecular weight heparin of the present invention can be commercially available, can also directly be carried from animal It takes, such as can be extracted from pig intestinal mucosa.Heparin disaccharide unit is mainly the amino Portugal of L- iduronic acids and N- sulphations Grape sugar passes through α (1 → 4) glucosides key connection.
Substrate for producing the low molecular weight heparin of the present invention can be bought from such as Hebei Changshan biochemistry medicine company share The heparin of Co., Ltd, Yintai Dongcheng Biochemical Co., Ltd, Shenzhen City HaiPuRui Pharmaceutical Co., Ltd, Changzhou thousand are red Biochemical pharmacy limited company, Amphastar (Nanjing) Pharmaceutical Co., Ltd. etc..
It can be determined by one skilled in the art using the concentration of substrate heparin in the present invention, do not limited specifically, Preferably 1~100g/L.The substrate heparin used in the present invention is the unassorted heparin of macromolecule, and molecular weight such as may be used To be distributed across 5000~30000, average molecular weight 20000.
Before the production method of the present invention is carried out, substrate heparin can be added in buffer solution, it is suitable to be formulated into Concentration.As long as used buffer solution does not damage the enzyme activity of Heparinase I, II, III or combination thereof.It is specific at one Production method in, using 20mM Tris, 20mM CaCl2, 50mM NaCl, and with 1mM salt acid for adjusting pH be 7 or so, Such as 7.4~7.6 buffer solution.In another specific production method, using 5.0mM CaCl2With 200mM NaCl Deionized water solution in, then with 1M HCl solutions adjust pH to 7.0 buffer solution.
Temperature when reacted with substrate heparin Heparinase I, II, III or combination thereof is not particularly limited, only If the temperature of Heparinase I, II and III inactivations will not be made, for example, it can be set to for 10~45 DEG C, most preferably 30 DEG C.
The time reacted for Heparinase I, II, III or combination thereof with substrate heparin is not particularly limited, this Field technology personnel can suitably select according to the temperature of the enzyme activity of the heparinase added, the concentration of substrate and reaction, In a specific method, the time of Heparinase I, II, III or combination thereof and substrate reactions can be small with 5 minutes~10 When, or 10 minutes~4 it is small when.
In the production method of low molecular weight heparin of the present invention, at Heparinase I, II, III or combination thereof and bottom During object heparin is reacted, the mode that the solution of reaction is monitored can suitably be selected according to reaction system, In a specific method, using the variation of the absorbance at UV spectrophotometer measuring 231nm, with the progress of reaction Absorbance A at 231nm231It is continuously increased, so as to determine the degree of reaction progress by the increase of absorbance.
During reaction, when measure reaction proceeds to desired degree according to the method described above, it can terminate anti- Should, further to separate to obtain ultra-low molecular weight heparin or low molecular weight heparin.Method wherein for terminating reaction, ability Field technique personnel can select according to the knowledge of its grasp, such as add in the reagent for terminating reaction or improve temperature so that enzyme Inactivation.In a specific embodiment, salt acid for adjusting pH value is added when terminating reaction to stopping 3 minutes after 2.0, then uses 2.0M NaOH pH value is recalled to 7.0.From the viewpoint of never adding other impurity, preferably improving the temperature of reaction system loses enzyme It is living to be reacted so as to terminate.In a specific embodiment, entire reaction system is placed in 100 DEG C of water-baths 10 minutes, So that the reaction terminating of enzyme degradation substrate.
In production method of the present invention, if necessary during the heparinase of two kinds or more species of addition, Ke Yixian A kind of heparin enzyme-to-substrate heparin reaction is added, after reaction carries out a period of time, reaction is terminated, then adds other livers The reaction was continued for plain enzyme, and finally terminates reaction.Two or more heparinase can also be added in simultaneously react wait to react Reaction is terminated when proceeding to desired degree.
The heparin used in the method for the production low molecular weight heparin of the invention described above can be common macromolecular liver Element or remove anticoagulant heparin as described above.
Embodiment
The preparation of 1 low molecular weight heparin 1 of embodiment (hereinafter also referred to as low molecular weight heparin I-2)
5g heparin (is bought from Changshan biochemistry medicine company, ProductName：Heparin sodium, molecular weight distribution 5000~30000, Average molecular weight be 20000) with 100mLTris buffer solutions (20mM Tris, 50mM NaCl, 20mM CaCl2) be configured to it is molten Liquid, disposably added into the 50g/L heparin solutions 100mL of the configuration total enzyme activity be 100IU according to Heparinase I prepared by ZL200410038098.6.It is monitored using the quartz colorimetric utensil and ultraviolet specrophotometer that optical path difference is 1cm Light absorption A231 of the solution at 231nm.When A231 reaches reaction controlling point (A231=20), terminate reaction, end Method is that 5~10min of enzyme-deactivating in reaction solution is then taken out reaction system and is cooled to room temperature in 100 DEG C of boiling water baths, The absolute ethyl alcohol of 6 times of volumes is added into reaction solution, 10min is stirred at room temperature, then at room temperature with 4000r/min's Speed centrifuges 15min, collects precipitation, adds in the deionized water dissolving that quality is 2~3 times of precipitation, using 0.22 μm of membrane filtration, It collects permeate and is placed on -80 DEG C of low temperature refrigerators and be frozen into solid ice cube, be then fed into freeze dryer (condenser temperature is -50 DEG C) It is lyophilized, powder then is ground into mortar or micromill, low molecular weight heparin 1 is obtained and (is also named as：Product I -2).
Then the analysis of molecular weight and molecular weight distribution is carried out for product I -2.Using gel exclusion high-efficient liquid phase color Spectrometry measures the weight average molecular weight (Mw) of low molecular weight heparin, number-average molecular weight (Mn) and breadth coefficient (P).Chromatographic column is TSK- GEL G2000SWXL (TOSOH, Japan), coutroi velocity 0.5mL/min, 35 DEG C of column temperature, sampling volume is 25 μ L.Using WATERS (1525, the U.S.) chromatographic system, UV detector and Composition distribution are sequentially connected in series in chromatographic column Outlet, UV detector wavelength are 234nm.Molecular weight and its distribution determination method may be referred to Wu, Jingjun et al. " Controllable production of low molecular weight heparins by combinations of heparinase I/II/III."Carbohydrate polymers 101(2014):Recorded method in 484-492.
By above method analysis the results show that the number-average molecular weight of product I -2 is 5528Da, weight average molecular weight is 10219Da, wherein molecular weight distribution are that heparin of the weight average molecular weight less than 3K accounts for the 6.189% of -2 total amount of product I, Weight-average molecular The heparin measured as 3K-5K accounts for the 7.757% of -2 total amount of product I, and the heparin of weight average molecular weight 5K-8K accounts for -2 total amount of product I 15.181%, heparin of the weight average molecular weight more than 8K accounts for the 70.873% of -2 total amount of product I.
Further anti-Xa, IIa factor active of the product I -2 of above-mentioned acquisition is detected
The anticoagulating active of heparin needs to measure its acceleration antithrombase (hereinafter referred to as ATIII) inhibition Xa by vitro test The activity of the factor (hereinafter referred to as Anti-Xa factor) and the IIa factors (the hereinafter referred to as anti-IIa factors) determines.It is used in the present invention Anti- Xa and anti-IIa factor actives detection method can refer to European Pharmacopoeia.International unit (the International of anti-Xa and anti-IIa Unit, IU) refer to determine amount heparin or low molecular weight heparin international standard substance contained by activity.The anti-freezing of heparin test sample to be measured Activity carries out comparing calculation by activity corresponding to international standard substance and obtains.
(1) solution is prepared：
Tris-HCl buffer solutions (pH7.4)：Tris 6.08g and NaCl 8.77g are taken, water 500mL is added to be allowed to dissolve, adds ox Haemocyanin 10g adjusts pH value to 7.4 with HCl, is diluted with water to 1000mL.
Tris-EDTA buffer solutions (pH8.4)：Tris 3.03g, NaCl 5.12g and EDTA2Na1.4g are taken, adds water 250mL is allowed to dissolve, and adjusts pH value to 8.4 with HCl, is diluted with water to 500mL.Heparin Standard product and test sample solution：Liver Plain activity criteria's product are purchased from EDQM's (European Directorate for the Quality of Medicines) heparin low-molecular-mass for assay BRP(Biological Reference Preparation) (H0185000,for detection of anti-factor Xa activity and anti-factor IIa activity).Standard items (S) and test sample (T) are diluted to 4 various concentrations respectively with Tris-HCl buffer solutions (pH7.4) Solution, the agent between each dosage is away from than controlling 1:0.7~1:0.6.The concentration should be in the range of linearity of dosage logarithm~reaction Interior, generally every milliliter of 0.025IU~0.2IU when detecting Anti-Xa factor detects anti-IIa because the period of the day from 11 p.m. to 1 a.m is generally every milliliter 0.015IU~0.075IU.
ATIII solution：ATIII is purchased from Chromogenix companies (Sweden).Delayed when detecting Anti-Xa factor with Tris-HCl Fliud flushing (pH7.4) is configured to the solution of 1IU/mL；Anti- IIa is detected because the period of the day from 11 p.m. to 1 a.m is configured to Tris-HCl buffer solutions (pH7.4) The solution of 0.5IU/mL.
Chromophoric substrate solution：With chromophoric substrate S-2765 (N- α-benzyloxycarbonyl during detection Anti-Xa factor
- D-arginyl-L-glycyl-L-arginine-p-nitroaniline-dihydrochlo ride), it is purchased from Chromogenix companies (Sweden).Anti- IIa is detected because of period of the day from 11 p.m. to 1 a.m chromophoric substrate S-2238 (H-D-phenylalanyl-L- Pipecolyl-arginine-p-nitroaniline-dihydrochloride), purchased from Chromogenix companies (Sweden).Two kinds of chromophoric substrates spend ion water making into the solution storage of 0.003M, before use with Tris-EDTA buffer solutions (pH8.4) it is diluted to 0.0005M.
Anti-Xa factor solution：It is prepared with Tris-HCl buffer solutions (pH7.4), debugs concentration, be allowed to replace with 0.9%NaCl In the anti-Xa experiments of generation (super) low molecular weight heparin, the absorbance at 405nm is between 0.6~0.7.
Anti- IIa factor solutions：It is dissolved with Tris-HCl buffer solutions (pH7.4) and is diluted to the solution of 5IU/mL.
Assay method：
1.5mL centrifuge tubes 16 are taken, mark T respectively1, T2, T3, T4And S1, S2, S3, S4.Each concentration is parallel to do two pipes.Often manage The test sample (T) or 50 μ l of standard items (S) dilution and 50 μ l AT III solution, mixing for being separately added into 4 kinds of concentration pay attention to Not have bubble.By S1, S2, S3, S4, T1, T2, T3, T4, T1, T2, T3, T4, S1, S2, S3, S4It is ranked sequentially, 37 DEG C of water-bath balances 1 Often add in the 100 anti-Xa of μ l (or anti-IIa) factor solutions after minute in pipe, 37 DEG C accurate be incubated 1min after add in chromophoric substrate solution 250 μ l, mixing, 37 DEG C of water-baths keep the temperature 4 minutes and immediately 30% 375 μ l of acetum are respectively added to terminate reaction.With the half of 1cm light paths Microcolorimetric ware with Tris-HCl buffer solutions (pH7.4) for blank, measures the absorbance at 405nm.With Tris-HCl buffer solutions (pH7.4) test solution (parallel to do two pipes) is replaced, as blank control pipe, in 16 pipe beginning and end, to divide with method operation Not Ce Ding blank control pipe absorbance.The two absorbance must not have significant difference.Using absorbance as ordinate, standard items are molten Liquid (or test solution) is that log concentration value is that abscissa does linear regression, parallel by the quantitative response in Bioassay-statistical method 4 × 4 method experimental design of line principle calculates potency and experimental error.Average letter limit rate (FL%) is not greater than 15%.
The anti-Xa potency of product I -2 is detected as 74 ± 10IU/mg according to the above method, anti-IIa potency is 91 ± 10IU/ mg。
The preparation of 2 low molecular weight heparin 2 of embodiment (hereinafter also referred to as low molecular weight heparin I-11)
Total enzyme activity is disposably added in the 50g/L heparin solutions 100mL configured to method similarly to Example 1 as 100IU According to ZL200410038098.6 prepare Heparinase I.Use the quartz colorimetric utensil and uv-spectrophotometric that optical path difference is 1cm Light absorption A231 of the meter monitoring solution at 231nm.When A231 reaches reaction controlling point (A231=46.3), tie reaction Beam, the method for end are that it is cold to then take out reaction system to 5~10min of enzyme-deactivating in reaction solution in 100 DEG C of boiling water baths But to room temperature, the absolute ethyl alcohol of 6 times of volumes is added into reaction solution, 10min is stirred at room temperature, then at room temperature with The speed centrifugation 15min of 4000r/min, collects precipitation, adds in the deionized water dissolving that quality is 2~3 times of precipitation, use 0.22 μm membrane filtration, collect permeate and be placed on -80 DEG C of low temperature refrigerators and be frozen into solid ice cube, be then fed into freeze dryer (cold-trap Temperature is -50 DEG C) it is lyophilized, powder then is ground into mortar or micromill, low molecular weight heparin 2 is obtained and (also names For：Product I -11).
Then the analysis of molecular weight and molecular weight distribution is carried out for product I -11 according to the method for embodiment 1.Pass through The above method analysis the results show that the number-average molecular weight of product I -11 be 3768Da, weight average molecular weight 7160Da, wherein dividing Son amount is distributed as heparin of the weight average molecular weight less than 3K and accounts for the 22.273% of -11 total amount of product I, and weight average molecular weight is 3K-5K's Heparin accounts for the 15.842% of -11 total amount of product I, and the heparin of weight average molecular weight 5K-8K accounts for the 21.674% of -11 total amount of product I, weight Heparin of the average molecular weight more than 8K accounts for the 40.211% of -11 total amount of product I.
Then detect the anti-Xa potency of product I -11 according to the method for embodiment 1 and anti-IIa potency, moderate resistance Xa potency are 86 ± 10IU/mg, anti-IIa potency are 43 ± 10IU/mg.
The preparation of 3 low molecular weight heparin 3 of embodiment (hereinafter also referred to as low molecular weight heparin III-1)
Total enzyme activity is disposably added in the 50g/L heparin solutions 100mL configured to method similarly to Example 1 as 100IU According to ZL 201010259913.7 prepare Heparinase I II.Use the quartz colorimetric utensil and ultraviolet spectrometry that optical path difference is 1cm Photometer monitors light absorption A231 of the solution at 231nm.When A231 reaches reaction controlling point (A231=7.77), make reaction Terminate, the method for end is to then take out reaction system to 5~10min of enzyme-deactivating in reaction solution in 100 DEG C of boiling water baths Be cooled to room temperature, into reaction solution add 6 times of volumes absolute ethyl alcohol, 10min is stirred at room temperature, then at room temperature with The speed centrifugation 15min of 4000r/min, collects precipitation, adds in the deionized water dissolving that quality is 2~3 times of precipitation, use 0.22 μm membrane filtration, collect permeate and be placed on -80 DEG C of low temperature refrigerators and be frozen into solid ice cube, be then fed into freeze dryer (cold-trap Temperature is -50 DEG C) it is lyophilized, powder then is ground into mortar or micromill, low molecular weight heparin 3 is obtained and (also names For：Product I II-1).
Then the analysis of molecular weight and molecular weight distribution is carried out for product I II-1 according to the method for embodiment 1.It is logical Cross the above method analysis the results show that the number-average molecular weight of product I II-1 be 9476Da, weight average molecular weight 10622Da, Middle-molecular-weihydroxyethyl is distributed as heparin of the weight average molecular weight less than 3K and accounts for the 1.022% of product I II-1 total amounts, weight average molecular weight 3K- The heparin of 5K accounts for the 3.252% of product I II-1 total amounts, and the heparin of weight average molecular weight 5K-8K accounts for product I II-1 total amounts 10.810%, heparin of the weight average molecular weight more than 8K accounts for the 84.916% of product I II-1 total amounts.
Then the anti-Xa potency of product I II-1 and anti-IIa potency, moderate resistance Xa potency are detected according to the method for embodiment 1 For 158 ± 10IU/mg, anti-IIa potency is 155 ± 10IU/mg.
The preparation of 4 low molecular weight heparin 4 of embodiment (hereinafter also referred to as low molecular weight heparin III-2)
Total enzyme activity is disposably added in the 50g/L heparin solutions 100mL configured to method similarly to Example 1 as 100IU According to ZL 201010259913.7 prepare Heparinase I II.Use the quartz colorimetric utensil and ultraviolet spectrometry that optical path difference is 1cm Photometer monitors light absorption A231 of the solution at 231nm.When A231 reaches reaction controlling point (A231=18.56), make anti- It should terminate, the method for end is to then take out reactant to 5~10min of enzyme-deactivating in reaction solution in 100 DEG C of boiling water baths System is cooled to room temperature, and the absolute ethyl alcohol of 6 times of volumes is added into reaction solution, 10min is stirred at room temperature, then at room temperature 15min is centrifuged with the speed of 4000r/min, collects precipitation, the deionized water dissolving that quality is 2~3 times of precipitation is added in, uses 0.22 μm of membrane filtration collects permeate and is placed on -80 DEG C of low temperature refrigerators and is frozen into solid ice cube, is then fed into freeze dryer (condenser temperature be -50 DEG C) is lyophilized, is then ground into powder with mortar or micromill, obtain low molecular weight heparin 4 ( It is named as：Product I II-2).
Then the analysis of molecular weight and molecular weight distribution is carried out for product I II-2 according to the method for embodiment 1.It is logical Cross the above method analysis the results show that the number-average molecular weight of product I II-2 be 8659Da, weight average molecular weight 10151Da, Middle-molecular-weihydroxyethyl is distributed as heparin of the weight average molecular weight less than 3K and accounts for the 1.848% of product I II-2 total amounts, weight average molecular weight 3K- The heparin of 5K accounts for the 4.963% of product I II-2 total amounts, and the heparin of weight average molecular weight 5K-8K accounts for product I II-2 total amounts 13.611%, heparin of the weight average molecular weight more than 8K accounts for the 79.578% of product I II-2 total amounts.
Then the anti-Xa potency of product I II-2 and anti-IIa potency, moderate resistance Xa potency are detected according to the method for embodiment 1 For 132 ± 10IU/mg, anti-IIa potency is 138 ± 10IU/mg.
The preparation of embodiment 5N- acetylated-heparins derivative (NS)
5g heparin (is bought from Changshan biochemistry medicine company, ProductName：Heparin sodium, molecular weight distribution 5000~30000, 20000) average molecular weight is by styrene cation exchange resin short column (HL-1200, the wide limited public affairs of sharp biotechnology in Shanghai Department), at 4 DEG C, it is monitored with the flow velocity of 2.2mL/12min and to the variation of ultraviolet region light absorption, further, efflux uses Pure pyridine is neutralized, and the heparin pyridiniujm of white powder is obtained through further freezing.
Heparin pyridiniujm obtained above is dissolved in the 95% DMSO aqueous solutions of 300mL, 50 DEG C, heating 5h are adopted PH to 8.0 is adjusted with NaOH (1M), the heparin derivatives that N-terminal removes sulfate radical are obtained after freeze-dried.By sample obtained in the previous step Product are dissolved into the NaHCO of saturation3In solution, 200 μ L aceticanhydrides are added at 4 DEG C, and pH to 7.5-8.4 is adjusted with NaOH solution. It dialyses desalination in the bag filter that reaction solution is 1kDa in molecular cut off, lyophilized to obtain product, hereinafter also referred to NS heparin is (specific Preparation method may refer to：Lapierre F,Holme K,Lam L,et al.Chemical modifications of heparin that diminish its anticoagulant but preserve its heparanase- inhibitory,angiostatic,anti-tumor and anti-metastatic properties[J] .Glycobiology,1996,6(3):355-366.)。
Then molecular weight analysis is carried out for product NS.By the analysis of 1 method of embodiment the results show that the number of product NS Average molecular weight is 14071Da, and weight average molecular weight 16148Da, wherein molecular weight distribution are the heparin that weight average molecular weight is less than 3K The 1.1% of product NS total amounts is accounted for, weight average molecular weight is that the heparin of 3K-5K accounts for the 1.28% of product NS total amounts, weight average molecular weight 5K- The heparin of 8K accounts for product NS
The 5.09% of total amount, heparin of the weight average molecular weight more than 8K account for the 92.53% of product NS total amounts.
Then the anti-Xa potency of product NS and anti-IIa potency are detected according to the method for embodiment 1, moderate resistance Xa potency is 15 ± 2IU/mg, anti-IIa potency are 12 ± 2IU/mg.
The preparation of experimental example 6N- acetylation low molecular weight heparin derivatives (N1)
According to method similarly to Example 1, NS heparin solutions (the NS heparin concentrations of 5 preparation of configuration 100mL above-described embodiments For 50g/L, 20mM Tris, 50mM NaCl, 20mM CaCl2, pH 7.6), to the 50g/LNS heparin solutions 100mL of the configuration In disposably add total enzyme activity be 100IU according to ZL200410038098.6 prepare Heparinase I.The use of optical path difference is 1cm Light absorption A231 at 231nm of quartz colorimetric utensil and ultraviolet specrophotometer monitoring solution.When A231 reaches reaction controlling During point (A231=18.9), terminate reaction, the method for end is to the enzyme-deactivating 5 in reaction solution in 100 DEG C of boiling water baths ~10min then takes out reaction system and is cooled to room temperature, and the absolute ethyl alcohol of 6 times of volumes is added into reaction solution, at room temperature 10min is stirred, 15min is then centrifuged with the speed of 4000r/min at room temperature, collects precipitation, it is precipitation 2~3 to add in quality Times deionized water dissolving, using 0.22 μm of membrane filtration, collect permeate and be placed on -80 DEG C of low temperature refrigerators be frozen into it is solid Ice cube is then fed into freeze dryer (condenser temperature is -50 DEG C) and freezes, is then ground into powder with mortar or micromill, N- acetylation low molecular weight heparins derivative 1 is obtained (to be also named as：Product N1).
Then the analysis of molecular weight and molecular weight distribution is carried out for product N1 according to the method for embodiment 1.By upper State that method is analyzed the results show that the number-average molecular weight of product N1 is 7534Da, wherein weight average molecular weight 11511Da, molecule Amount is distributed as heparin of the weight average molecular weight less than 3K and accounts for the 1.446% of product N1 total amounts, and weight average molecular weight is that the heparin of 3K-5K accounts for The 6.291% of product N1 total amounts, the heparin of weight average molecular weight 5K-8K account for the 10.635% of product N1 total amounts, and weight average molecular weight is big The 81.608% of product N1 total amounts is accounted in the heparin of 8K.
The preparation of experimental example 7N- acetylation low molecular weight heparin derivatives (N2)
Method is configured to 100mL NS heparin solutions similarly to Example 6, to the 50g/LNS heparin solutions 100mL of configuration In disposably add total enzyme activity be 100IU according to ZL200410038098.6 prepare Heparinase I.The use of optical path difference is 1cm Light absorption A231 at 231nm of quartz colorimetric utensil and ultraviolet specrophotometer monitoring solution.When A231 reaches reaction controlling During point (A231=40), terminate reaction, the method for end be in 100 DEG C of boiling water baths to the enzyme-deactivating 5 in reaction solution~ 10min then takes out reaction system and is cooled to room temperature, and the absolute ethyl alcohol of 6 times of volumes is added into reaction solution, is stirred at room temperature 10min is mixed, 15min is then centrifuged with the speed of 4000r/min at room temperature, collects precipitation, it is 2~3 times of precipitation to add in quality Deionized water dissolving, using 0.22 μm of membrane filtration, collect permeate and be placed on -80 DEG C of low temperature refrigerators and be frozen into solid ice Block is then fed into freeze dryer (condenser temperature is -50 DEG C) and freezes, is then ground into powder with mortar or micromill, obtains It (is also named as to N- acetylation low molecular weight heparins derivative 2：Product N2).
Then the analysis of molecular weight and molecular weight distribution is carried out for product N2 according to the method for embodiment 1.By upper State method analysis the results show that the number-average molecular weight of product N2 be 4022Da, weight average molecular weight 7160Da, middle-molecular-weihydroxyethyl It is distributed as heparin of the weight average molecular weight less than 3K and accounts for the 11.803% of product N2 total amounts, weight average molecular weight is that the heparin of 3K-5K accounts for The 21.342% of product N2 total amounts, the heparin of weight average molecular weight 5K-8K account for the 20.776% of product N2 total amounts, and weight average molecular weight is big The 46.079% of product N2 total amounts is accounted in the heparin of 8K.
The preparation of experimental example 8N- acetylation low molecular weight heparin derivatives (N4)
The 100mL NS heparin solutions of method configuration similarly to Example 6, to the 50g/LNS heparin solutions 100mL of configuration In disposably add total enzyme activity be 100IU according to ZL 201010259913.7 prepare Heparinase I II.It is using optical path difference Light absorption A231 of quartz colorimetric utensil and ultraviolet specrophotometer the monitoring solution of 1cm at 231nm.When A231 reaches reaction control During point (A231=4.92) processed, terminate reaction, the method for end is to the enzyme-deactivating in reaction solution in 100 DEG C of boiling water baths 5~10min then takes out reaction system and is cooled to room temperature, and the absolute ethyl alcohol of 6 times of volumes is added into reaction solution, in room temperature Then lower stirring 10min centrifuges 15min with the speed of 4000r/min at room temperature, collect precipitation, add in quality be precipitation 2~ 3 times of deionized water dissolving, using 0.22 μm of membrane filtration, collect permeate and be placed on -80 DEG C of low temperature refrigerators be frozen into it is solid Ice cube, it is lyophilized to be then fed into freeze dryer (condenser temperature be -50 DEG C), is then crushed into powder with mortar or micromill End obtains N- acetylation low molecular weight heparins derivative 4 and (is also named as：Product N4).
Then the analysis of molecular weight and molecular weight distribution is carried out for product N4 according to the method for embodiment 1.By upper State that method is analyzed the results show that the number-average molecular weight of product N4 is 10001Da, wherein weight average molecular weight 11134Da, molecule Amount is distributed as heparin of the weight average molecular weight less than 3K and accounts for the 1.29% of product N4 total amounts, and weight average molecular weight is that the heparin of 3K-5K accounts for The 2.909% of product N4 total amounts, the heparin of weight average molecular weight 5K-8K account for the 9.943% of product N4 total amounts, and weight average molecular weight is more than The heparin of 8K accounts for the 85.858% of product N4 total amounts.
The preparation of experimental example 9N- acetylation low molecular weight heparin derivatives (N5)
The 100mL NS heparin solutions of method configuration similarly to Example 6, to the 50g/LNS heparin solutions 100mL of configuration In disposably add total enzyme activity be 100IU according to ZL201010259913.7 prepare Heparinase I II.It is using optical path difference Light absorption A231 of quartz colorimetric utensil and ultraviolet specrophotometer the monitoring solution of 1cm at 231nm.When A231 reaches reaction control During point (A231=14.20) processed, terminate reaction, the method for end is to go out in 100 DEG C of boiling water baths to the enzyme in reaction solution 5~10min living, then takes out reaction system and is cooled to room temperature, and the absolute ethyl alcohol of 6 times of volumes is added into reaction solution, in room The lower stirring 10min of temperature, then centrifuges 15min with the speed of 4000r/min at room temperature, collects precipitation, it is precipitation 2 to add in quality ~3 times of deionized water dissolving using 0.22 μm of membrane filtration, collects permeate and is placed on -80 DEG C of low temperature refrigerators and be frozen into heavily fortified point Real ice cube is then fed into freeze dryer (condenser temperature is -50 DEG C) and freezes, is then crushed into powder with mortar or micromill End obtains N- acetylation low molecular weight heparins derivative 5 and (is also named as：Product N5).
Then the analysis of molecular weight and molecular weight distribution is carried out for product N5 according to the method for embodiment 1.By upper State that method is analyzed the results show that the number-average molecular weight of product N5 is 9900Da, wherein weight average molecular weight 10392Da, molecule Amount is distributed as heparin of the weight average molecular weight less than 3K and accounts for the 0.37% of product N5 total amounts, and weight average molecular weight is that the heparin of 3K-5K accounts for The 5.059% of product N5 total amounts, the heparin of weight average molecular weight 5K-8K account for the 12.106% of product N5 total amounts, and weight average molecular weight is big The 82.465% of product N5 total amounts is accounted in the heparin of 8K.
Comparative example 1
From the biochemical purchase unfraction heparin of Hebei Changshan, the number-average molecular weight Mn of the heparin (UFH, also referred to as H groups) is 14000, weight average molecular weight 24899.
Then detect the anti-Xa potency of product UFH according to the method for embodiment 1 and anti-IIa potency, moderate resistance Xa potency are 187 ± 21IU/mg, anti-IIa potency are 177 ± 6IU/mg.
Comparative example 2
Yi Nuo heparin is bought from Hebei Changshan biochemistry medicine company, the number-average molecular weight of the heparin is 4372, and weight average molecular weight is 5679.Molecular weight analysis is carried out to Yi Nuo heparin using method described in the present invention.
By above method analysis the results show that the molecular weight distribution of Yi Nuo heparin is liver of the weight average molecular weight less than 3K Element accounts for the 20.71% of Yi Nuo heparin total amounts, and weight average molecular weight is that the heparin of 3K-5K accounts for the 55.42% of Yi Nuo heparin total amounts, and weight is equal The heparin of molecular weight 5K-8K accounts for the 13.16% of Yi Nuo heparin total amounts, and heparin of the weight average molecular weight more than 8K accounts for Yi Nuo heparin total amounts 10.71%.
The preparation of 3 low molecular weight heparin 5 (product I -16) of comparative example
Total enzyme activity is disposably added in the 50g/L heparin solutions 100mL configured to method similarly to Example 1 as 100IU According to ZL200410038098.6 prepare Heparinase I.Use the quartz colorimetric utensil and uv-spectrophotometric that optical path difference is 1cm Light absorption A231 of the meter monitoring solution at 231nm.When A231 reaches reaction controlling point (A231=98.6), tie reaction Beam, the method for end are that it is cold to then take out reaction system to 5~10min of enzyme-deactivating in reaction solution in 100 DEG C of boiling water baths But to room temperature, the absolute ethyl alcohol of 6 times of volumes is added into reaction solution, 10min is stirred at room temperature, then at room temperature with The speed centrifugation 15min of 4000r/min, collects precipitation, adds in the deionized water dissolving that quality is 2~3 times of precipitation, use 0.22 μm membrane filtration, collect permeate and be placed on -80 DEG C of low temperature refrigerators and be frozen into solid ice cube, be then fed into freeze dryer (cold-trap Temperature is -50 DEG C) it is lyophilized, powder then is ground into mortar or micromill, low molecular weight heparin 5 is obtained and (also names For：Product I -16).
Then the analysis of molecular weight and molecular weight distribution is carried out for product I -16 according to the method for embodiment 1.Pass through The above method analysis the results show that the number-average molecular weight of product I -16 be 2523Da, weight average molecular weight 4341Da, wherein dividing Son amount is distributed as heparin of the weight average molecular weight less than 3K and accounts for the 34.707% of -16 total amount of product I, and weight average molecular weight is 3K-5K's Heparin accounts for the 34.637% of -16 total amount of product I, and the heparin of weight average molecular weight 5K-8K accounts for the 17.908% of -16 total amount of product I, weight Heparin of the average molecular weight more than 8K accounts for the 12.748% of -16 total amount of product I.
The preparation of comparative example 4N- acetylation low molecular weight heparin derivatives (N3)
The 100mL NS heparin solutions of method configuration similarly to Example 6, to the 50g/LNS heparin solutions 100mL of configuration In disposably add total enzyme activity be 100IU according to ZL200410038098.6 prepare Heparinase I.The use of optical path difference is 1cm Light absorption A231 at 231nm of quartz colorimetric utensil and ultraviolet specrophotometer monitoring solution.When A231 reaches reaction controlling During point (A231=92), terminate reaction, the method for end be in 100 DEG C of boiling water baths to the enzyme-deactivating 5 in reaction solution~ 10min then takes out reaction system and is cooled to room temperature, and the absolute ethyl alcohol of 6 times of volumes is added into reaction solution, is stirred at room temperature 10min is mixed, 15min is then centrifuged with the speed of 4000r/min at room temperature, collects precipitation, it is 2~3 times of precipitation to add in quality Deionized water dissolving, using 0.22 μm of filtering, collect permeate and be placed on -80 DEG C of low temperature refrigerators and be frozen into solid ice Block is then fed into freeze dryer (condenser temperature is -50 DEG C) and freezes, is then ground into powder with mortar or micromill, obtains It (is also named as to N- acetylation low molecular weight heparins derivative 3：Product N3)..
Then the analysis of molecular weight and molecular weight distribution is carried out for product N3 according to the method for embodiment 1.By upper State method analysis the results show that the number-average molecular weight of product N3 be 2033Da, weight average molecular weight 4549Da, middle-molecular-weihydroxyethyl It is distributed as heparin of the weight average molecular weight less than 3K and accounts for the 35.481% of product N3 total amounts, weight average molecular weight is that the heparin of 3K-5K accounts for The 32.363% of product N3 total amounts, the heparin of weight average molecular weight 5K-8K account for the 17.69% of product N3 total amounts, and weight average molecular weight is big The 14.466% of product N3 total amounts is accounted in the heparin of 8K.
The molecular weight and molecular weight distribution of each heparin product in 1 embodiment of table and comparative example summarize
1 pulmonary fibrosis Animal Model of experimental example and pulmonary fibrosis pharmacodynamic evaluation
After 1%Averdin (Sigma companies) anesthesia is fixed, ethyl alcohol disappears C57BL/6 mouse (20-25g, 8-12 week old) Malicious skin of neck does the notch of an a length of left and right, blunt separation subcutaneous tissue, exposure tracheae, with 2.8mg/kg agent in the middle part of neck It measures in intratracheal injection Bleomycin (Dalian U.S. logical sequence Technology Co., Ltd.), uprightly at the uniform velocity rotates mouse immediately after injection, Drug is made to be uniformly distributed in intrapulmonary, sutures skin of neck.Carried out it is treated on the day of start to be administered, and will work as heaven-made For the 1st day, dosage regimen was as described below.
Every other day to above-mentioned processed mouse spraying embodiment 1 to 9 and the heparin of comparative example 1 and 3-4 groups to carry out Administration.In addition to administration group, negative control group is set, which is according to the small of method similar to the above processing Mouse every other day gives same amount of physiological saline as negative control；Healthy group (also referred to as positive controls) is set, to just Normal mouse every other day gives same dose of physiological saline and establishes model, and any drug is not given during modeling, every group small Mouse 5.Spray delivery gives the various embodiments described above of 2mg/mL concentration and the drug 5mL of comparative example group, and dosage rate is every other day It is administered once, and the whole mouse of sudden death in the 21st day after administration, lung is taken, and according to Cahill, Emer F. et al. " Hepatocyte Growth Factor Is Required for Mesenchymal Stromal Cell Protection Against Bleomycin-Induced Pulmonary Fibrosis."Stem Cells Translational Medicine(2016):Method described in sctm-2015 calculates the survival rate of mouse.
According to the mouse survival rate that the above method calculates, the results are shown in Table 2, it can be seen that compared with negative control group, Administration embodiment 1-9 groups, the survival rate of mouse of 1 group of comparative example greatly improve.And the mouse of 3,4 groups of comparative example is administered Survival rate does not improve significantly then.
It can be seen from the results above that each group low molecular weight heparin involved in the embodiment of the present invention is for inhibiting The death of the pulmonary fibrosis mice of Bleomycin inductions has certain effect, and unfractionated heparin can also inhibit such mouse in addition Death.
The influence of different heparin and derivative to the Bleomycin mouse pulmonary fibrosis survival rates induced in 2 embodiment 1 of table
Group Mouse survival rate (%) Group Mouse survival rate (%)
Blank control group 100 NS groups 100
Negative control group 12.5 N1 groups 100
I-2 groups 100 N2 groups 87.5
I-11 groups 75 N4 groups 87.5
III-1 groups 100 N5 groups 75
III-2 groups 100 N3 groups 0
I-16 groups 25 H groups 87.5
2 pulmonary fibrosis Animal Model of experimental example and pulmonary fibrosis pharmacodynamic evaluation
It is similar such as the step of experimental example 1, the dosage of Bleomycin is simply changed to 2.5mg/kg.C57BL/6 mouse (20- 25g, 8-12 week old) after 1%Averdin (Sigma companies) anesthesia is fixed, ethanol disinfection skin of neck does one in the middle part of neck The notch of a length of left and right, pure property separate subcutaneous tissue, expose tracheae, and 2.5mg/kg dosage is (big in intratracheal injection Bleomycin Lian Meilun biologies), it is upright immediately after injection to hook speed rotation mouse, drug is made to be uniformly distributed in intrapulmonary, sutures skin of neck.Into Having gone the treated same day starts to be administered, and using the same day as the 1st day, dosage regimen is as described below.
Every other day to above-mentioned processed mouse spraying embodiment 1 to 9 and comparative example 1, the heparin administration of 3-4 groups.It removes Outside administration group, negative control group is set, which is according to the mouse of method similar to the above processing, Mei Geyi It gives same amount of physiological saline as negative control；Positive controls are set, normal mouse are every other day given similary The physiological saline of dosage establishes model, and any drug is not given during modeling, otherwise referred to as healthy group below the group, every group Mouse 5.The drug 5mL of the various embodiments described above and comparative example group mouse 2mg/mL concentration is given in spraying, and dosage rate is every other day It is administered once, and the whole mouse of sudden death in the 15th day after administration, take lung tissue.
It takes and according to Cahill, Emer F. et al. " Hepatocyte Growth Factor Is Required for Mesenchymal Stromal Cell Protection Against Bleomycin-Induced Pulmonary Fibrosis."Stem Cells Translational Medicine(2016):It is identical in method described in sctm-2015 Method obtain pathologic section coloration result, as shown in Figure 1.
After Bleomycin is handled 15 days, the visible apparent fibrosis of lung tissue changes (referring to (a) in Fig. 1 for display Figure), illustrate the mouse that pulmonary fibrosis is also obtained in experimental example 2.Normal group mouse lung tissue surface is smooth, have compared with Good elasticity, appearance pinkiness (referring to (f) figure in Fig. 1), negative control group lung tissue are presented typical cellular shape character, are in Fibrillatable pathological feature.Compared to negative control group, embodiment 1-9, the substantially complete alveolar structure of 1 group of presentation of comparative example, greatly Cell monolayer structure is presented in part alveolar, and cellular morphology is similar with normally organizing, and without apparent inflammatory infiltration feature, illustrates fiber Change degree also has the different degrees of trend that lightens.And comparative example 3-4 groups then present stronger fibrosis trend.
Then, the mouse lung tissue for accurately weighing each group of 30~100mg of weight in wet base is put into test tube, accurately adds hydrolyzate 1ml, mixing.95 DEG C or boiling water bath hydrolyze 20 minutes (during hydrolysis 10 minutes mixing once make hydrolysis more abundant) after capping.So PH value is adjusted afterwards to 6.0~6.8 or so.Add distilled water to 10ml, mixing；The diluted hydrolyzates of 3~4ml is taken to add proper amount of active carbon (about 20~30mg or so, clear, colorless after supernatant of being subject to centrifuges), mixing, 3500 revs/min centrifuge 10 minutes, carefully take Then clear 1ml builds up hydroxyproline determination kit come hydroxyl dried meat ammonia in determination sample using purchase as detection sample from Nanjing The content of acid, and the content to characterize collagen contained in lung.
The results are shown in Table 3.It can be seen that H groups (1 group of comparative example), the embodiment 1-9 groups of administration unfractionated heparin are effective Ground reduces the content of hydroxyproline, that is, significantly reduces the content of lung's collagen, shows that heparin and embodiment is administered In obtained low molecular weight heparin can alleviate the formation of collagen, it was demonstrated that fibrosis mitigate, and comparative example 3-4 groups are then Fibrosis do not have significant change.
The influence of hydroxyproline content, knot in the mouse lung tissue that the different heparin of table 3 and derivative induce Bleomycin Fruit is expressed as (result ± SD)
The deposition for generally believing collagen is the most apparent feature of fibrosis lesion.Collagen -1 (ColA-1) is most There is characteristic collagen index.Therefore, the deposition of collagen can be predicted by measuring ColA-1 on transcriptional level Amount, predicts the development of fibrosis lesion.According to method (Yang D, Atkins G J, Turner the A G, et of the reports such as Yang al.Differential effects of 1,25-dihydroxyvitamin D on mineralisation and differentiation in two different types of osteoblast-like cultures[J].The Journal of steroid biochemistry and molecular biology,2013,136:166-170.) to reality The mRNA for testing the collagen of the lung of each group mouse obtained in example 2 is quantitative determined, and the results are shown in Table 4, find to Medicine embodiment 1-9 groups, 1 group of conversion ratio that can be effectively reduced collagen of comparative example, so as to inhibit the hair of pulmonary fibrosis It is raw.
The influence of collagen mRNA, knot in the mouse lung tissue that the different heparin of table 4 and derivative induce Bleomycin Fruit is expressed as (result ± SD)
Group Collagen mRNA Group Collagen mRNA
Negative control group 20.04±6.30 NS groups 10.75±1.77
I-2 groups 10.20±1.34 N1 groups 10.17±3.45
I-11 groups 12.27±2.57 N2 groups 7.24±2.34
III-1 groups 10.25±4.17 N4 groups 10.87±1.56
III-2 groups 7.72±1.63 N5 groups 7.36±5.67
I-16 groups 25.16±4.37 N3 groups 26.47±1.07
H groups 10.77±3.22
According to the results show of above-mentioned experimental example 1 and experimental example 2 although unfractionated heparin, i.e., do not degraded using enzyme or The heparin for removing anticoagulating active and degrading also has the effect for inhibiting pulmonary fibrosis, but generally believes that macromolecule heparin has Apparent side effect for example, increasing the risk of bleeding, easily induces thrombopenia.And low molecular weight of the present invention Heparin or the low molecular weight heparin gone after anti-freezing then not only there is inhibition pulmonary fibrosis to be formed, and improve the effect of the survival rate of mouse Outside fruit, drug metabolism is controllable, it is contemplated that need not detect the bleeding risk of patient in clinic.

Claims (10)

1. a kind of low molecular weight heparin, the scope of number-average molecular weight (Mn) is 3000~12000Da, weight average molecular weight (Mw) Scope be 5000~20000Da, preferably the scope of its number-average molecular weight (Mn) be 3500~11000Da, weight average molecular weight (Mw) Scope for 5500~17000Da, the scope of its further preferred number-average molecular weight (Mn) is 3600~10500Da, is divided equally again The scope of son amount (Mw) for 6000~15000Da, still further preferably the scope of its number-average molecular weight (Mn) for 3700~ 11000Da, the scope of weight average molecular weight (Mw) is 7000~12000Da.
2. low molecular weight heparin according to claim 1, wherein, the molecular weight distribution of weight average molecular weight is：Molecular weight Heparin less than 3000Da accounts for below the 30wt% of the low molecular weight heparin whole, and preferably in below 25wt%, molecular weight is more than The heparin of 8000Da accounts for more than the 30wt% of the low molecular weight heparin whole, preferably in more than 35wt%, further preferably exists More than 40wt%.
3. low molecular weight heparin according to claim 1 or 2, wherein, the molecular weight distribution of weight average molecular weight is：Molecule The heparin measured in 3000~5000Da accounts for below the 30wt% of the low molecular weight heparin whole, preferably in below 25wt%.
4. low molecular weight heparin according to claim 1, the scope of number-average molecular weight (Mn) is 4000~11000Da, The scope of weight average molecular weight (Mw) is 7000~12000Da, and it does not have anticoagulating active.
5. low molecular weight heparin according to any one of claims 1 to 4, wherein, the low molecular weight heparin is to utilize Obtained from heparinase degradation heparin.
6. according to low molecular weight heparin according to any one of claims 1 to 5, wherein, the low molecular weight heparin is to utilize Obtained from Heparinase I or Heparinase I II degradation heparin.
7. low molecular weight heparin according to claim 4, wherein, the low molecular weight heparin is first heparin resist Solidifying processing, then obtained from heparinase degrades heparin.
8. purposes of the heparin in the drug for treating or preventing pulmonary fibrosis is used to prepare.
9. purposes of the heparin in being used to prepare to alleviate the drug of formation of lung's collagen.
10. purposes according to claim 8 or claim 9, wherein, the heparin is according to any one of claims 1 to 7 low Molecular weight heparin.
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