CN108117613B - Low molecular weight heparin and application of heparin in preparing medicine for treating pulmonary fibrosis - Google Patents

Low molecular weight heparin and application of heparin in preparing medicine for treating pulmonary fibrosis Download PDF

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CN108117613B
CN108117613B CN201710374073.0A CN201710374073A CN108117613B CN 108117613 B CN108117613 B CN 108117613B CN 201710374073 A CN201710374073 A CN 201710374073A CN 108117613 B CN108117613 B CN 108117613B
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邢新会
闫昳姝
季洋
王怡
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Abstract

The invention relates to low molecular weight heparin and the use of heparin for the preparation of pulmonary fibrosis. The low molecular weight heparin has a number average molecular weight (Mn) in the range of 3000 to 12000Da, a weight average molecular weight (Mw) in the range of 5000 to 20000Da, preferably a number average molecular weight (Mn) in the range of 3500 to 11000Da, preferably a weight average molecular weight (Mw) in the range of 5500 to 17000Da, more preferably a number average molecular weight (Mn) in the range of 3600 to 10500Da, more preferably a weight average molecular weight (Mw) in the range of 6000 to 15000Da, even more preferably a number average molecular weight (Mn) in the range of 3700 to 11000Da, and more preferably a weight average molecular weight (Mw) in the range of 7000 to 12000 Da.

Description

Low molecular weight heparin and application of heparin in preparing medicine for treating pulmonary fibrosis
Technical Field
The invention relates to low molecular weight heparin and application thereof in preparing a medicament for preventing or treating pulmonary fibrosis.
Background
Fibrotic diseases are diseases in which parenchymal cells lose their original functions after tissue damage due to deposition of collagen-rich extracellular matrix (ECM) in tissues and organs caused by abnormal, excessive repair. Fibrotic diseases include diseases involving multiple systems, such as systemic sclerosis, multifocal fibrosis, scleroderma, nephrogenic multiple system fibrosis. Also included are organ and tissue specific diseases, such as pulmonary, hepatic, renal fibrosis.
Because different fibrotic lesions involve different organs and tissues, are exposed to different environmental factors, and have different cell types, the fibrotic processes all have similar pathological processes: epithelial cells undergo sustained damage and secrete excessive amounts of cytokines and growth factors. These factors further stimulate recruitment and activation of mesenchymal cell precursors, forming myofibroblasts, which subsequently secrete large amounts of extracellular matrix. Under normal physiological conditions, after the tissue is repaired, the fibrotic matrix will be degraded and the fibroblasts will undergo apoptosis or reversion to inactive cells. However, under the condition of fibrotic diseases, the normal clearance and reversion procedures are destroyed, and parenchymal cells are gradually replaced by myofibroblasts, so that the normal functions are lost, and the fibrotic diseases are caused.
In the western world, fibrotic disease ultimately contributes to mortality as high as 45% in percentage, and there is no statistical data available in developing countries. Although the onset of fibrotic diseases is numerous, the study of the molecular mechanisms involved is preliminary, and an effective therapeutic method is further lacking.
Idiopathic Pulmonary Fibrosis (IPF) is a progressive, Pulmonary interstitial disease characterized by diffuse Pulmonary Fibrosis leading to impairment of lung function and dyspnea. The average survival time of the patient is only 3.2 years, and the patient is a fatal disease with the worst prognosis in chronic non-tumor respiratory diseases. The current incidence of IPF is 1.25-27.9/10 ten thousand. The disease is caused by many factors, and the more clear causes include inhalation dust, gas, virus, bacteria, drugs, radiation injury, and the like. In China, because the industrialization process is accelerated and the air pollution is getting worse, the number of patients suffering from acute and chronic pulmonary fibrosis is increasing remarkably, the IPF death rate is 4-10/10 ten thousand on average from 1999 to 2012, and the IPF death rate is increased by 2-3% every year. The pathogenesis of IPF is not clear and is traditionally considered to be caused by chronic inflammation, but because the therapeutic effect of anti-inflammatory or immunosuppressive agents on IPF treatment is not obvious, more attention is paid to changes in lung epithelial cells and fibroblasts, which leads to fibroblast activation, pulmonary fibrosis and lung tissue remodeling disorder.
At present, no effective medicine for curing IPF is developed at home and abroad, and mainly some medicines for delaying further deterioration of IPF, such as Pirfenidone (Pirfenidone,
Figure GDA0002125140510000021
) And Nintedanib (Nintedanib,
Figure GDA0002125140510000022
) Pirfenidone mechanism of action reduces fibroblast production by inhibiting TGF- β and acts as an anti-inflammatory agent by TNF- α and IL-1 β nintedanib is an inhibitor of intracellular multiple tyrosine kinases including inhibition of VEGF, FGF, PDGF.
Disclosure of Invention
Heparin is a class of sulfated, polydisperse, linear Glycosaminoglycans (GAGs) that are one of the most important anticoagulant drugs. Is widely applied to preventing and treating thromboembolic diseases clinically. In addition, heparin and its derivatives have a wide range of biological activities, including coordinating cell adhesion, regulating cell growth and proliferation, developmental processes, cell surface binding to lipoprotein lipase and other proteins, neoangiogenesis, viral invasion and tumor metastasis, among others. Preclinical and clinical studies have shown that unfractionated large molecular weight heparin has a significant reducing effect on symptoms of IPF, delaying the progression of pulmonary fibrosis (Gunther, a., lubbe, n., erm, m., schermuli, r.t., Weissmann, n., breitecker, a., & seger, W. (2003), prediction of pulmonary-induced fibrosis of pulmonary tissue, 168, (1358) -and Gunther of pulmonary tissue, a., lubbe, n., mer, m., schermuli, r.t., prosthesis, n., brewag, n., brewag, m., schermuir, r.t., brewag, n., schermuli, r.1368-and cement, a., milometer, p.s., r.t., brewag, n., schermuli, p.s., p.1368, and g. 1365, cement, p.c., p. trailer, p.c., p. 1, and g. reflector, p.c., p. heir.c., p. 1, p. reflector, p.
However, the large molecular weight heparin molecules that are currently common are not themselves the optimal structure for the treatment of fibrotic diseases. This is because the micro-heterogeneity of heparin structure leads to strong binding capacity with various factors, thus "diluting" the interaction of the main molecular sequences with the target and causing side effects. In addition, heparin molecules themselves have relatively serious side effects such as increased bleeding and thrombocytopenia.
For example, β -degradation mode, i.e., Enoxaparin (Enoxaparin) obtained by salification, esterification and alkaline hydrolysis processes, is adopted to intervene IN mice with pulmonary fibrosis caused by Bleomycin, and has no improving effect on pulmonary fibrosis formation (Laxer, u., Lossos, i.e., Gillis, s., Or R., christentn, t.g., Goldstein, R., & Breuer, R. (1999), THE EFFECT OF enxaparinton-b-r.e., r.g., R. b. c., R. g., R. b. c., R. b.
The inventor of the present invention is dedicated to the innovative research of heparin industry technology, and utilizes the Maltose Binding Protein (MBP) fusion expression technology to realize the high activity, soluble expression and industrial production of a series of heparinases, wherein the series of heparinases are listed as Chinese pharmacopoeia standard enzymes (Heparinase, MBP-HepI, MBP-HepII and MBP-HepIII respectively, which can be seen in Chinese patents ZL200410038098.6, ZL201010259905.2 and ZL 201010259913.7). And a novel process for preparing LMWH by using the combined enzyme method is initially established (see Chinese patent ZL 201210328649.7). On the basis, the inventors have conducted intensive studies to successfully obtain the novel low molecular weight heparin of the present invention, and found that the low molecular weight heparin of the present invention has a function of preventing or treating pulmonary fibrosis.
The present invention relates to the following:
1. a low molecular weight heparin having a number average molecular weight (Mn) in the range of 3000 to 12000Da, a weight average molecular weight (Mw) in the range of 5000 to 20000Da, preferably a number average molecular weight (Mn) in the range of 3500 to 11000Da, a weight average molecular weight (Mw) in the range of 5500 to 17000Da, more preferably a number average molecular weight (Mn) in the range of 3600 to 10500Da, and a weight average molecular weight (Mw) in the range of 6000 to 15000Da, even more preferably a number average molecular weight (Mn) in the range of 3700 to 11000Da, and a weight average molecular weight (Mw) in the range of 7000 to 12000 Da.
2. The low molecular weight heparin according to item 1, wherein the molecular weight distribution of the weight average molecular weight thereof is: heparin with a molecular weight of less than 3000Da accounts for less than 30 wt%, preferably less than 25 wt%, and heparin with a molecular weight of greater than 8000Da accounts for more than 30 wt%, preferably more than 35 wt%, more preferably more than 40 wt% of the total low molecular weight heparin.
3. The low molecular weight heparin according to item 1 or 2, wherein the molecular weight distribution of the weight average molecular weight thereof is: the heparin with the molecular weight of 3000-5000 Da accounts for less than 30 wt% of the total low molecular weight heparin, and preferably less than 25 wt%.
4. The low molecular weight heparin according to any one of claims 1 to 3, wherein the number average molecular weight (Mn) is in the range of 3000 to 7000Da, the weight average molecular weight (Mw) is in the range of 6000 to 12000Da, preferably the number average molecular weight (Mn) is in the range of 3500 to 6000Da, and the weight average molecular weight (Mw) is in the range of 6500 to 11000 Da.
5. The low molecular weight heparin according to any one of items 1 to 3, wherein the number average molecular weight (Mn) thereof is in the range of 7000 to 10000Da, and the weight average molecular weight (Mw) thereof is in the range of 8000 to 12000Da, preferably the number average molecular weight (Mn) thereof is in the range of 8000 to 9800Da, and the weight average molecular weight (Mw) thereof is in the range of 9000 to 11000 Da.
6. The low molecular weight heparin according to item 1, which has a number average molecular weight (Mn) ranging from 4000 to 11000Da and a weight average molecular weight (Mw) ranging from 7000 to 12000Da, and which has no anticoagulant activity.
7. The low molecular weight heparin according to item 6, wherein the number average molecular weight (Mn) is in the range of 4000 to 8000Da and the weight average molecular weight (Mw) is in the range of 7000 to 12000 Da.
8. The low molecular weight heparin according to item 6, wherein the number average molecular weight (Mn) is in the range of 9000 to 11000Da and the weight average molecular weight (Mw) is in the range of 9500 to 12000 Da.
9. The low molecular weight heparin according to any one of claims 1 to 8, wherein the low molecular weight heparin is obtained by degrading heparin with heparinase.
10. The low molecular weight heparin according to any one of claims 1 to 9, wherein the low molecular weight heparin is obtained by degrading heparin with heparinase I or heparinase III.
11. The low molecular weight heparin according to item 4 or item 7, wherein the low molecular weight heparin is obtained by degrading heparin using heparinase I.
12. The low molecular weight heparin according to item 5 or 8, wherein the low molecular weight heparin is obtained by degrading heparin using heparinase III.
13. The low molecular weight heparin according to any one of claims 6 to 8, wherein the low molecular weight heparin is obtained by anticoagulation treatment of heparin and degradation of heparin by heparinase.
14. Use of heparin for the manufacture of a medicament for the treatment or prevention of pulmonary fibrosis.
15. The use according to claim 14, wherein the heparin is a low molecular weight heparin according to any one of items 1 to 13.
16. Use according to item 14, wherein the heparin is undegraded heparin with a number average molecular weight (Mn) ranging over 12000Da and a weight average molecular weight (Mw) ranging over 20000 Da.
17. Use of heparin for the manufacture of a medicament for alleviating the formation of collagen in the lung.
18. The use of item 17, wherein the heparin is the low molecular weight heparin of any one of items 1-13.
19. Use according to claim 18, wherein the heparin is undegraded heparin having a number average molecular weight (Mn) in the range of more than 12000Da and a weight average molecular weight (Mw) in the range of more than 20000 Da.
20. A method for treating or preventing pulmonary fibrosis, comprising: heparin is administered to a mammal or human in need thereof.
21. The method of item 20, wherein the heparin is the low molecular weight heparin of any one of items 1-13.
22. The method of item 20, wherein the heparin is undegraded heparin having a number average molecular weight (Mn) in the range of greater than 12000Da and a weight average molecular weight (Mw) in the range of greater than 20000 Da.
23. A method for mitigating pulmonary collagen formation, comprising: heparin is administered to a mammal or human in need thereof.
24. The method of item 23, wherein the heparin is the low molecular weight heparin of any one of items 1-13.
The method of item 23, wherein the heparin is undegraded heparin having a number average molecular weight (Mn) in the range of greater than 12000Da and a weight average molecular weight (Mw) in the range of greater than 20000 Da.
26. A anticoagulated heparin, which has a number average molecular weight (Mn) in the range of more than 11000Da and a weight average molecular weight (Mw) in the range of more than 12000Da, preferably has a number average molecular weight (Mn) in the range of more than 12000Da and a weight average molecular weight (Mw) in the range of more than 13000 Da. Preferably, the number average molecular weight (Mn) is in the range of greater than 13000Da and the weight average molecular weight (Mw) is in the range of greater than 14000 Da. Preferably the number average molecular weight (Mn) is in the range of more than 13500Da and the weight average molecular weight (Mw) is in the range of more than 15000Da, which has no anticoagulant activity.
27. The anticoagulated heparin according to item 26, which is a anticoagulated heparin obtained by N-terminal sulfation and acetylation of a heparin molecule.
28. Use of a heparin according to item 26 or 27 for the preparation of a medicament for the treatment or prevention of pulmonary fibrosis.
29. Use of the heparin of item 26 or 27 for the preparation of a medicament for alleviating the formation of collagen in the lung.
30. A method for treating or preventing pulmonary fibrosis, comprising: administering to a mammal or human in need thereof a de-anticoagulated heparin according to item 26 or item 27.
31. A method for mitigating pulmonary collagen formation, comprising: administering to a mammal or human in need thereof a de-anticoagulated heparin according to item 26 or item 27.
Drawings
Figure 1H & E staining in experimental example 2 characterizes the effect of low molecular weight heparin on Bleomycin-induced lung injury and pulmonary fibrosis. (a) A negative control group; (b) group I-2; (c) group I-11; (d) group III-1; (e) group III-2; (f) a healthy group; (g) group I-16; (h) NS group; (i) group N1; (j) group N2; (k) group N4; (l) Group N5; (m) group N3; and (n) group H.
Detailed Description
Hereinafter, embodiments of the present invention will be specifically described.
< Low molecular weight heparin of the present invention >
The present invention relates to low molecular weight heparins having the function of treating and/or preventing pulmonary fibrosis. In one embodiment of the invention, the low molecular weight heparin has a number average molecular weight (Mn) in the range of 3000 to 12000Da, a weight average molecular weight (Mw) in the range of 5000 to 20000Da, preferably a number average molecular weight (Mn) in the range of 3500 to 11000Da, a weight average molecular weight (Mw) in the range of 5500 to 17000Da, more preferably a number average molecular weight (Mn) in the range of 3600 to 10500Da, and a weight average molecular weight (Mw) in the range of 6000 to 15000Da, more preferably a number average molecular weight (Mn) in the range of 3700 to 11000Da, and a weight average molecular weight (Mw) in the range of 7000 to 12000 Da. That is, the low molecular weight heparins to which the invention relates may have a number average molecular weight (Mn) of, 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, 6000, 6100Da, 6200Da, 6300Da, 6400Da, 6500Da, 6600Da, 6700Da, 6800Da, 6900Da, 7100Da, 7200Da, 7300Da, 7400Da, 7500Da, 7600, 7700, 7800Da, 8000Da, 8100, 8200Da, 9100Da, 8500Da, 8600, 7900Da, 7900, 9200, 7900Da, 20400, 7900, 20400, 9200, 20400, 4300, 7900, 4300Da, 10800, 9800, 4300, 7900, 10800, 4300, 7900, 4300, 11400Da, 11500Da, 11600Da, 11700Da, 11800Da and 11900 Da. The low molecular weight heparins of the invention may have a weight average molecular weight (Mw) of, 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, 8200, 8300Da, 8400Da, 8500Da, 88000 Da, 8600Da, 8700Da, 890 Da, 9000Da, 9100Da, 9200Da, 9300Da, 9400, 9500Da, 9600, 9700Da, 9800Da, 9900Da, 10000Da, 10100Da, 10300Da, 19500Da, 13000Da, 15500Da, 15500Da, 12500 Da.
In one embodiment the invention relates to a low molecular weight heparin having a weight average molecular weight with a molecular weight distribution of: heparin with a molecular weight of less than 3000Da comprises less than 30 wt%, preferably less than 25 wt%, preferably less than 24 wt%, preferably less than 23 wt% of the total low molecular weight heparin. The heparin with molecular weight more than 8000Da accounts for more than 30 wt%, preferably more than 35 wt%, preferably more than 36 wt%, more preferably more than 38 wt%, preferably more than 40 wt% of the total low molecular weight heparin.
In one embodiment the invention relates to a low molecular weight heparin having a weight average molecular weight with a molecular weight distribution of: the heparin with the molecular weight of 3000-5000 Da accounts for less than 30 wt%, preferably less than 25 wt%, preferably less than 23 wt%, preferably less than 22 wt% of the total low molecular weight heparin, and the heparin with the molecular weight of 5000-8000 Da accounts for less than 30 wt%, preferably less than 25 wt%, preferably less than 22 wt% of the total low molecular weight heparin.
In one embodiment of the invention, the low molecular weight heparin is obtained by degrading heparinase III or heparinase I, wherein the number average molecular weight (Mn) of the low molecular weight heparin is 3000-10000 Da, the weight average molecular weight (Mw) of the low molecular weight heparin is 6500-12000 Da, preferably the number average molecular weight (Mn) of the low molecular weight heparin is 3500-9500 Da, and the weight average molecular weight (Mw) of the low molecular weight heparin is 7000-11000 Da.
In one embodiment the invention relates to a low molecular weight heparin having a weight average molecular weight with a molecular weight distribution of: the heparin having a molecular weight of 3000-5000 Da accounts for 2 wt% or more and 25 wt% or less, and more preferably 3 wt% or more and 20 wt% or less of the total low molecular weight heparin. Heparin with a molecular weight of 5000-8000 Da accounts for more than 5 wt% and less than 30 wt% of the total low molecular weight heparin, more preferably more than 8 wt% and less than 25 wt%, heparin with a molecular weight of less than 3000Da accounts for less than 30 wt% and less than 25 wt% of the total low molecular weight heparin, heparin with a molecular weight of more than 8000Da accounts for more than 30 wt% and less than 90 wt% and preferably more than 35 wt% and less than 85 wt% of the total low molecular weight heparin, the total amount of the four distributions is 100 wt%, and the low molecular weight heparin is obtained by degrading heparinase I or heparinase III.
In one embodiment of the invention, the low molecular weight heparin is obtained by degrading heparin I, wherein the number average molecular weight (Mn) of the low molecular weight heparin is 3000-7000 Da, the weight average molecular weight (Mw) of the low molecular weight heparin is 6000-12000 Da, preferably the number average molecular weight (Mn) of the low molecular weight heparin is 3500-6000 Da, and the weight average molecular weight (Mw) of the low molecular weight heparin is 6500-11000 Da.
In one embodiment the invention relates to a low molecular weight heparin having a weight average molecular weight with a molecular weight distribution of: the heparin having a molecular weight of 3000-5000 Da accounts for 5 wt% or more and 25 wt% or less, and more preferably 7 wt% or more and 20 wt% or less of the total low molecular weight heparin. Heparin with a molecular weight of 5000-8000 Da accounts for more than 10 wt% and less than 30 wt% of the total low molecular weight heparin, preferably more than 13 wt% and less than 25 wt%, heparin with a molecular weight of less than 3000Da accounts for more than 5 wt% and less than 30 wt% of the total low molecular weight heparin, preferably more than 6 wt% and less than 25 wt%, heparin with a molecular weight of more than 8000Da accounts for more than 30 wt% and less than 80 wt% of the total low molecular weight heparin, preferably more than 35 wt% and less than 75 wt%, the total amount of the four distributions is 100 wt%, and the low molecular weight heparin is degraded by heparinase I.
In one embodiment of the invention, the low molecular weight heparin has a number average molecular weight (Mn) in the range of 7000 to 10000Da and a weight average molecular weight (Mw) in the range of 8000 to 12000Da, preferably a number average molecular weight (Mn) in the range of 8000 to 9800Da and a weight average molecular weight (Mw) in the range of 9000 to 11000Da, and is obtained by degrading the low molecular weight heparin by heparinase III.
In one embodiment the invention relates to a low molecular weight heparin having a weight average molecular weight with a molecular weight distribution of: the heparin having a molecular weight of 3000-5000 Da accounts for 10 wt% or less, more preferably 2 wt% or more and 8 wt% or less of the total low molecular weight heparin. Heparin with a molecular weight of 5000-8000 Da accounts for more than 5 wt% and less than 25 wt% of the total low molecular weight heparin, more preferably more than 8 wt% and less than 23 wt%, heparin with a molecular weight of less than 3000Da accounts for less than 10 wt% and preferably less than 5 wt% of the total low molecular weight heparin, heparin with a molecular weight of more than 8000Da accounts for more than 70 wt% and preferably less than 90 wt% of the total low molecular weight heparin, the total amount of the four distributions is 100 wt%, and the low molecular weight heparin is obtained by degrading with heparinase III.
In one embodiment of the invention, the low molecular weight heparin has the number average molecular weight (Mn) ranging from 4000 to 11000Da and the weight average molecular weight (Mw) ranging from 7000 to 12000Da and has no anticoagulation activity, and is obtained by anticoagulating heparin and then degrading the heparin by heparinase III or heparinase I.
In one embodiment the invention relates to a low molecular weight heparin having a weight average molecular weight with a molecular weight distribution of: the heparin having a molecular weight of 3000-5000 Da accounts for less than 30 wt%, more preferably 2 wt% to 25 wt% of the total low molecular weight heparin. The heparin with the molecular weight of 5000-8000 Da accounts for more than 5 wt% and less than 25 wt% of the total low molecular weight heparin, more preferably more than 8 wt% and less than 23 wt%, the heparin with the molecular weight of less than 3000Da accounts for less than 15 wt% and preferably less than 12 wt% of the total low molecular weight heparin, the heparin with the molecular weight of more than 8000Da accounts for more than 40 wt% and preferably less than 90 wt% of the total low molecular weight heparin, the total amount of the four distributions is 100 wt%, and the low molecular weight heparin is obtained by performing anticoagulation treatment on the heparin and then degrading the heparin by heparinase III or heparinase I.
In one embodiment of the invention, the low molecular weight heparin has the number average molecular weight (Mn) ranging from 4000 to 8000Da and the weight average molecular weight (Mw) ranging from 7000 to 12000Da, is obtained by performing anticoagulation treatment on heparin and then degrading the heparin by heparinase I, and has no anticoagulation activity.
In one embodiment the invention relates to a low molecular weight heparin having a weight average molecular weight with a molecular weight distribution of: the heparin having a molecular weight of 3000 to 5000Da accounts for not more than 30 wt%, more preferably not less than 4 wt% and not more than 25 wt% of the total low molecular weight heparin. The heparin with the molecular weight of 5000-8000 Da accounts for more than 5 wt% and less than 25 wt% of the total low molecular weight heparin, more preferably more than 8 wt% and less than 23 wt%, the heparin with the molecular weight of less than 3000Da accounts for less than 15 wt% and preferably less than 12 wt% of the total low molecular weight heparin, the heparin with the molecular weight of more than 8000Da accounts for more than 40 wt% and preferably less than 85 wt% of the total low molecular weight heparin, the total amount of the four distributions is 100 wt%, the low molecular weight heparin is obtained by performing anticoagulation treatment on the heparin and then degrading the heparin by heparinase I, and the low molecular weight heparin does not have anticoagulation activity.
In one embodiment of the invention, the low molecular weight heparin has a number average molecular weight (Mn) in the range of 9000-11000 Da and a weight average molecular weight (Mw) in the range of 9500-12000 Da, is obtained by performing anticoagulation treatment on heparin and then degrading the heparin by heparinase III, and has no anticoagulation activity.
In one embodiment the invention relates to a low molecular weight heparin having a weight average molecular weight with a molecular weight distribution of: the heparin having a molecular weight of 3000 to 5000Da accounts for 20 wt% or less, more preferably 1 wt% or more and 15 wt% or less of the total low molecular weight heparin. The heparin with the molecular weight of 5000-8000 Da accounts for more than 5 wt% and less than 20 wt% of the total low molecular weight heparin, more preferably more than 8 wt% and less than 18 wt%, the heparin with the molecular weight of less than 3000Da accounts for less than 10 wt% and preferably less than 8 wt% of the total low molecular weight heparin, the heparin with the molecular weight of more than 8000Da accounts for more than 60 wt% and preferably less than 90 wt% of the total low molecular weight heparin, the total amount of the four distributions is 100 wt%, the low molecular weight heparin is obtained by performing anticoagulation treatment on the heparin and then degrading the heparin by heparinase III, and the low molecular weight heparin does not have anticoagulation activity.
In the present invention, the weight average molecular weight, number average molecular weight, and molecular weight distribution can be measured by a known method, for example, by the method described in Wu, Jingjun et al, "control layer chromatography of low molecular weight polymers of Carbohydrate polymers 101(2014):484-492, and the specific measurement procedure can be referred to the method described in the following examples. If the results of other known detection methods are inconsistent with the results of the method, the weight average molecular weight, number average molecular weight, and molecular weight distribution of the low molecular weight heparin according to the present invention are based on the method used in the examples of the present invention.
< Effect of Low molecular weight heparin on inhibition of pulmonary fibrosis >
The low molecular heparin provided by the invention can be used for treating or preventing pulmonary fibrosis.
The invention relates to application of low molecular heparin in preparing a medicament for treating or preventing pulmonary fibrosis.
Pulmonary fibrosis in the present invention refers to the terminal change of a large group of lung diseases characterized by fibroblast proliferation and accumulation of a large amount of extracellular matrix with inflammatory injury and destruction of tissue structure, that is, structural abnormality (scar formation) caused by abnormal repair after normal alveolar tissue is damaged. The pulmonary fibrosis in the invention includes idiopathic pulmonary fibrosis, primary pulmonary fibrosis, secondary pulmonary fibrosis and pulmonary interstitial fibrosis. The invention relates to application of the low molecular heparin in preparing a medicament for treating or preventing idiopathic pulmonary fibrosis, primary pulmonary fibrosis, secondary pulmonary fibrosis and/or pulmonary interstitial fibrosis diseases.
The low molecular weight heparin can effectively inhibit the death rate of mice inducing pulmonary fibrosis, and section observation of lung tissues of the mice after administration shows that after the low molecular weight heparin is administered, the lungs of the mice present a basically complete alveolar structure, most alveoli present a monolayer cell structure, the cell morphology is similar to that of a normal group, no obvious inflammatory infiltration characteristic exists, and the fibrosis degree has the tendency of becoming lighter in different degrees.
The present inventors have found that the low molecular weight heparin according to the present invention has substantially the same effect as the normal high molecular weight heparin in terms of the alteration of lung tissue after administration, but administration of the low molecular weight heparin according to the present invention does not have the side effect of the high molecular weight heparin, i.e., an increase in bleeding risk, and is likely to induce thrombocytopenia. Although the effect of the low molecular weight heparin for inhibiting pulmonary fibrosis is similar to that of the high molecular weight heparin for inhibiting pulmonary fibrosis, the metabolism of the low molecular weight heparin is controllable, and the bleeding risk of a patient does not need to be monitored clinically.
In addition, the low molecular weight heparin has a certain effect on inhibiting pulmonary edema, effectively reduces the content of hydroxyproline in mouse pulmonary tissues, namely effectively reduces the conversion rate of collagen in the lung and reduces the content of collagen in the lung. Currently, enoxaparin has no effect of inhibiting pulmonary fibrosis (see LaxerU, Lossos I S, Gillis S, et al. the effect of enoxaparin on bleomycin-induced lung in cancer [ J ]. Experimental lung research,1999,25(6): 531-541). The low molecular heparin obtained by the present invention has a structure different from enoxaparin, and the efficacy is completely different.
Although not intending to be bound by theory, the low molecular heparin obtained by the present invention is a low molecular heparin obtained by enzymatic degradation, the degradation mechanism is completely different from enoxaparin obtained by chemical method, and the difference can be clearly seen from the analysis data of the molecular weight distribution in the examples.
The low molecular weight heparin prepared by the enzyme degradation method can obviously inhibit the formation of lung injury and the generation of inflammation in the process of lung injury, thereby fundamentally preventing and inhibiting the formation of pulmonary fibrosis and reducing the deposition of collagen. However, the existing anti-pulmonary fibrosis drugs can only relieve the symptoms of pulmonary fibrosis, and the treatment effect on IPF is not obvious.
The present invention also relates to heparin derivatives from which anticoagulant activity has been removed, and it can be seen from the data of the following examples that either heparin derivatives from which anticoagulant activity has been removed or low-molecular-weight anticoagulated heparin derivatives obtained by further degrading them by the above-mentioned enzymatic hydrolysis method are effective in suppressing the mortality of mice induced with pulmonary fibrosis.
After the heparin derivative with the anticoagulant activity removed and the low-molecular-weight anticoagulant heparin derivative are administrated, the lung of a mouse presents a basically complete alveolar structure, most alveoli present a monolayer cell structure, the cell morphology is similar to that of a normal group, no obvious inflammatory infiltration characteristic exists, and the fibrosis degree also has the tendency of becoming lighter in different degrees. In addition, after the heparin is administrated, the heparin has a certain effect on inhibiting pulmonary edema, and effectively reduces the content of hydroxyproline in the lung tissue of a mouse, namely effectively reduces the conversion rate of collagen in the lung and reduces the content of collagen in the lung. The results of the experimental examples according to the present invention show that administration of a heparin derivative with anticoagulation activity removed and administration of a low-molecular-weight heparin derivative with anticoagulation activity have the above-described effects, but administration of the low-molecular-weight heparin according to the present invention does not have the side effects of high-molecular-weight heparin, i.e., an increased bleeding risk, and is likely to induce thrombocytopenia. Although the effect of the low molecular weight heparin for inhibiting pulmonary fibrosis is similar to that of the high molecular weight heparin for inhibiting pulmonary fibrosis, the metabolism of the low molecular weight heparin is controllable, and the blood risk of a patient does not need to be monitored clinically.
< method for producing anticoagulated heparin according to the invention >
Besides normal heparin, anticoagulated heparin is also used as a raw material to produce low molecular weight heparin in the invention.
Generally speaking, heparin, low molecular weight heparin, pentose, and others, act as anticoagulants by increasing the rate at which antithrombin iii inactivates the clotting factor, and the primary effects of these drugs are anti-Xa and anti-IIa activity. Through the research on the anti-Xa activity and anti-IIa activity of heparin drugs, it was found that the anti-Xa activity is insensitive to molecular mass and the anti-IIa activity depends on the size of molecular mass. The greater the molecular mass, the greater the anti-IIa activity. The inactivation of factor IIa by heparin relies on the formation of a heparin-antithrombin-factor IIa triple complex, where heparin binds both antithrombin and factor IIa, to achieve this linkage heparin contains at least 18 saccharide units, of which 13 monosaccharides are required for "bridging" and 5 monosaccharides are required as recognition fragments. The average molecular mass per monosaccharide is 300Da, so that the molecular mass must be above 5400Da to have IIa-resisting activity. The average molecular mass of the common heparin is 15000-19000Da, most molecules are above 5400Da, and the ratio of the anti-Xa activity to the anti-IIa activity is about 1. The average molecular mass of the low molecular heparin is 4000-5000Da, the proportion of the molecular fragments with the molecular mass of more than 5400Da is small, and the anti-Xa, anti-IIa activity is about 1.5: 1-5: 1 under the general condition.
In the art, there are various methods for removing the anticoagulant activity of heparin, and the present invention is not limited to the method for obtaining anticoagulated heparin. In a specific embodiment, the anticoagulant heparin used in the present invention is prepared by removing the sulfate from the N-terminus of heparin and then acetylating the resulting heparin (hereinafter referred to as N-acetylated heparin), as described in Lapierre F, Holme K, Lam L, et al chemical modifications of heparin with respect to the amount of heparin at the site of diagnostic reagents present in the platelet-inhibitor, angiostatic, anti-tumor and anti-viral properties [ J ] Glycobiology,1996,6(3): 355-.
As shown in the following examples and comparative examples, in the present invention, the anti-Xa titer of the heparin material having anticoagulation activity was 187. + -.21 IU/mg, and the anti-IIa titer was 177. + -.6 IU/mg. The heparin from which the anticoagulant activity is removed by the method for removing the anticoagulant activity of heparin according to the present invention has an anti-Xa titer of 15 + -2 IU/mg and an anti-IIa titer of 12 + -0.6 IU/mg. Furthermore, as shown in the subsequent examples, the anti-Xa and anti-IIa potency of the low molecular weight heparin obtained after treatment of the raw material heparin with heparinase is reduced to some extent compared to the non-enzymolyzed raw material heparin, but it is still a low molecular weight heparin with anticoagulant activity.
Heparin having no anticoagulant activity as defined in the present invention means heparin obtained after sufficient anticoagulation treatment of heparin, and the anti-Xa potency and anti-IIa potency of this heparin are much lower than those of low molecular weight heparin obtained by degradation of raw material heparin and heparinase, for example, the anti-Xa potency is 20IU/mg or less and the anti-IIa potency is 20IU/mg or less.
The anticoagulated heparin obtained by the anticoagulation treatment has a number average molecular weight (Mn) in a range of more than 11000Da and a weight average molecular weight (Mw) in a range of more than 12000 Da. Preferably, the number average molecular weight (Mn) is in the range of greater than 12000Da and the weight average molecular weight (Mw) is in the range of greater than 13000 Da. Preferably, the number average molecular weight (Mn) is in the range of greater than 13000Da and the weight average molecular weight (Mw) is in the range of greater than 14000 Da. Preferably, the number average molecular weight (Mn) is in the range of greater than 13500Da and the weight average molecular weight (Mw) is in the range of greater than 15000 Da.
The heparin having a molecular weight of 3000 to 5000Da accounts for 15 wt% or less, and more preferably 10 wt% or less of the total low molecular weight heparin. The heparin with the molecular weight of 5000-8000 Da accounts for less than 20 wt% of the total low molecular weight heparin, more preferably less than 15 wt%, the heparin with the molecular weight of less than 3000Da accounts for less than 10 wt%, preferably less than 8 wt%, the heparin with the molecular weight of more than 8000Da accounts for more than 80 wt%, preferably more than 90 wt%, the total amount of the four distributions is 100 wt%, and the heparin has no anticoagulant activity.
< production of Low molecular weight heparin according to the invention >
In the production of the invention relates to the low molecular weight heparin process using different kinds of heparinase, these heparinase can be any method to obtain heparinase, including heparinase I, II and III, as long as it has heparinase activity, preferably using heparinase I, II, III. In the prior art, the E.C. number of heparinase I is E.C.4.2.2.7, and the E.C. number of heparinase III is E.C. 4.2.2.8. Commercially available heparinases, such as heparinases I, II, III available from Sigma or IBEX may also be used. Heparinase may also be a fusion protein of recombinant heparinase I, II and III or heparinase I, II and III with any fusion partner constructed by molecular biological methods. According to a preferred embodiment of the invention, heparinases I, II and III are fusion proteins of heparinases I, II and III, in particular of heparinases I, II and III comprising MBP.
Heparinase I, heparinase II and heparinase III may also be fusion proteins with any fusion partner as long as they have the activity of heparinase I, II and III. According to a preferred embodiment herein, heparinases I, II and III are fusion proteins of heparinases I, II and III and a fusion partner, in particular of Maltose Binding Protein (MBP) and heparinases I, II and III. The fusion protein of heparinase I and MBP is sometimes referred to as MBP-HepA hereinafter (see Chinese patent ZL200410038098.6, grant No. CN1312183C), the fusion protein of heparinase II and MBP is sometimes referred to as MBP-HepB hereinafter (see Chinese patent ZL201010259905.2, grant No. CN101942024B), and the fusion protein of heparinase III and MBP is sometimes referred to as MBP-HepC hereinafter (see Chinese patent ZL201010259913.7, grant No. CN 101942025B). Preferably, heparinase I, II and heparinase III respectively have sequences which are shown in SEQ ID NO. 1-3 in a sequence table of Chinese patent ZL201210328649.7 and grant publication No. CN 103173506B.
In the production of the invention related to low molecular weight heparin, heparin enzyme I, II, III or their arbitrary combination and substrate heparin reaction mode can be batch, continuous or semi-continuous, the technicians in this field can be according to the production needs to be appropriate selection. The reaction time and reaction apparatus may be appropriately determined by those skilled in the art as long as the target low molecular weight heparin can be obtained.
In a specific embodiment, in the method of producing low molecular weight heparin of the present invention, the substrate heparin solution is added to the reactor, and then one or more of heparinase I, II or III is added to react with the substrate heparin. As the reaction proceeds, heparin is gradually degraded, and the reaction solution is monitored at regular intervals and terminated at appropriate times. And (3) carrying out primary filtration on the mixed solution after the reaction is ended by using a cellulose membrane vacuum primary filtration device, and carrying out ultrafiltration by using an ultrafiltration device to obtain secondary filtrate. And then adding ethanol, uniformly mixing, centrifuging, removing supernatant, collecting precipitate, adding acetone into the precipitate, washing, and performing reduced pressure evaporation by using a rotary evaporator to obtain low-molecular-weight heparin product powder. It will be appreciated by those skilled in the art that the above method is merely exemplary and that other methods may be used to obtain low molecular weight heparin that is degraded by enzymatic reactions.
The amount of each of the heparinases I, II and III is suitably determined by a person skilled in the art according to the production requirements with reference to the activity of the different enzymes, preferably the amount of each enzyme is in the range of 10IU to 500IU, preferably 100IU to 250IU per liter of reaction solution for heparinases I. The heparinase II is in the range of 10 IU-500 IU per liter of reaction liquid, preferably in the range of 100 IU-200 IU. The heparinase III is in the range of 10 IU-500 IU per liter of reaction liquid, and preferably 25-100 IU per liter of reaction liquid. Wherein IU represents: an amount of enzyme which gives 1. mu. mol of 4,5 unsaturated end product per minute at a temperature of 30 ℃ and a pH of 7.4.
The heparin substrate used for the production of the low molecular weight heparins of the invention is either commercially available or can be extracted directly from animals, for example from porcine small intestinal mucosa the heparin disaccharide units are mainly L-iduronic acid and N-sulphated glucosamine linked by α (1 → 4) glycosidic bonds.
Substrates for producing the low molecular weight heparins of the invention can be purchased from, for example, heparin from Hebei Heshan Biochemical pharmaceutical Co., Ltd, tobacco terrace Dongcheng Biochemical Co., Ltd, Shenzhen Shanpu Rui pharmaceutical Co., Ltd, Hezhou Qianhong Biochemical pharmaceutical Co., Ltd, Meiyaxing (Nanjing) pharmaceutical Co., Ltd, and the like.
The concentration of heparin substrate used in the present invention is not particularly limited, and is preferably 1 to 100g/L, which can be determined by those skilled in the art. The substrate heparin used in the present invention is unfractionated heparin with a large molecular weight, and the molecular weight thereof may be, for example, 5000 to 30000, and the average molecular weight thereof may be 20000.
Before carrying out the production method of the present invention, the substrate heparin may be added to a buffer solution to be formulated to an appropriate concentration. The buffer used may be any buffer as long as it does not impair the enzymatic activity of heparinase I, heparinase II, heparinase III or a combination thereof. In a specific production method, 20mM Tris, 20mM CaCl are used250mM NaCl, and 1mM hydrochloric acid to adjust the pH to about 7, for example, a buffer solution of 7.4 to 7.6. In another specific production method, 5.0mM CaCl is used2And 200mM NaCl in deionized water, followed by a buffer solution adjusted to pH 7.0 with 1M HCl solution.
The temperature at which heparinase I, II, III or a combination thereof is reacted with the substrate heparin is not particularly limited as long as it is a temperature at which heparinase I, II and III are not inactivated, and may be set to, for example, 10 to 45 ℃ and most preferably 30 ℃.
The time for the reaction of heparinase I, II, III or a combination thereof with the substrate heparin is not particularly limited, and may be appropriately selected by those skilled in the art according to the enzyme activity of the added heparinase, the concentration of the substrate and the reaction temperature, and in a specific method, the time for the reaction of heparinase I, II, III or a combination thereof with the substrate may be 5 minutes to 10 hours, or may be 10 minutes to 4 hours.
In the method for producing low molecular weight heparin according to the present invention, the method of monitoring the reaction solution during the reaction of heparinase I, heparinase II, heparinase III or a combination thereof with the substrate heparin may be appropriately selected depending on the reaction system, and in a specific method, the change of absorbance at 231nm is detected by an ultraviolet spectrophotometer, and the absorbance A at 231nm as the reaction proceeds231And is increased to determine the extent of reaction progress by the increase in absorbance.
During the reaction, when the determination of reaction to the desired degree, can terminate the reaction, to further separation to obtain ultra low molecular weight heparin or low molecular weight heparin. As for the method of terminating the reaction, a person skilled in the art can select, for example, a method of adding a reagent for terminating the reaction or a method of raising the temperature to inactivate the enzyme, according to his/her knowledge. In one embodiment, hydrochloric acid is added to adjust the pH to 2.0 upon termination of the reaction, and the reaction is allowed to stand for 3 minutes, after which the pH is adjusted back to 7.0 with 2.0M NaOH. From the viewpoint of not adding other impurities, it is preferable to stop the reaction by inactivating the enzyme by raising the temperature of the reaction system. In a specific embodiment, the entire reaction system is placed in a water bath at 100 ℃ for 10 minutes, thereby terminating the reaction of enzymatically degrading the substrate.
In the production method of the invention, if two or more kinds of heparinases are required to be added, one heparinase can be added to react with the substrate heparin firstly, the reaction is stopped after the reaction is carried out for a period of time, then other heparinases are added to continue the reaction, and finally the reaction is stopped. Two or more heparinases may also be added simultaneously to carry out the reaction and the reaction may be terminated when the reaction has progressed to a desired extent.
The heparin used in the method for producing low molecular weight heparin according to the present invention may be a conventional macromolecular heparin, or may be anticoagulated heparin as described above.
Examples
Example 1 preparation of Low molecular weight heparin 1 (hereinafter, also referred to as Low molecular weight heparin I-2)
5g heparin (purchased from dichroa febrifuga biochemical industry, product name: heparin sodium, molecular weight distribution of 5000-30000, average molecular weight of 20000) and 100 mM Tris buffer (20mM Tris, 50mM NaCl, 20mM CaCl) are mixed2) Prepared into solution, and 100mL of the prepared 50g/L heparin solution is added with heparinase I prepared according to ZL200410038098.6 with total enzyme activity of 100IU at one time. The light absorption of the solution at 231nm, A231, was monitored using a quartz cuvette with an optical path difference of 1cm and an ultraviolet spectrophotometer. And when the A231 reaches a reaction control point (A231 is 20), finishing the reaction by inactivating the enzyme in the reaction solution in a boiling water bath at 100 ℃ for 5-10 min, taking out the reaction system, cooling to room temperature, adding 6 times of volume of absolute ethyl alcohol into the reaction solution, stirring at room temperature for 10min, centrifuging at 4000r/min at room temperature for 15min, collecting the precipitate, adding deionized water with the mass 2-3 times of that of the precipitate to dissolve, filtering by using a 0.22 mu m membrane, collecting the permeate, freezing into solid ice blocks in a low-temperature refrigerator at-80 ℃, freeze-drying in a freeze dryer (the cold trap temperature is-50 ℃) and crushing into powder by using a mortar or a small-sized crusher to obtain the low-molecular-weight heparin 1 (also named as product I-2).
The molecular weight, and therefore the molecular weight distribution, was then analyzed for product I-2. The weight average molecular weight (Mw), number average molecular weight (Mn) and distribution coefficient (P) of low molecular weight heparin were determined by gel exclusion high performance liquid chromatography. The column was TSK-GEL G2000SWXL (TOSOH, Japan), the flow rate was controlled to 0.5mL/min, the column temperature was 35 ℃ and the injection volume was 25. mu.L. A WATERS (1525, USA) chromatographic system is adopted, an ultraviolet detector and a differential detector are connected in series in sequence at the outlet of a chromatographic column, and the wavelength of the ultraviolet detector is 234 nm. The molecular weight and the molecular weight distribution can be measured by the method described in Wu, Jingjun et al, "control able product of low molecular weight by molecules of peptides I/II/III," Carbohydrate polymers 101(2014): 484-492.
The results of the analysis by the above method show that product I-2 has a number average molecular weight of 5528Da and a weight average molecular weight of 10219Da, wherein the molecular weight distribution is that heparin with a weight average molecular weight of less than 3K accounts for 6.189% of the total amount of product I-2, heparin with a weight average molecular weight of 3K-5K accounts for 7.757% of the total amount of product I-2, heparin with a weight average molecular weight of 5K-8K accounts for 15.181% of the total amount of product I-2, and heparin with a weight average molecular weight of more than 8K accounts for 70.873% of the total amount of product I-2.
Further detecting the anti-Xa and IIa factor activity of the product I-2 obtained above
The anticoagulant activity of heparin is determined by measuring its activity of accelerating the inhibition of factor Xa (hereinafter, referred to as anti-factor Xa) and factor IIa (hereinafter, referred to as anti-factor IIa) by antithrombin (hereinafter, referred to as ATIII) in an in vitro assay. The method for detecting the activity of Xa-and IIa-resistant factors used in the present invention can be referred to the European pharmacopoeia. The International Units (IU) for anti-Xa and anti-IIa refer to the activity contained in a defined amount of an international standard for heparin or low molecular weight heparin. The anticoagulant activity of the heparin test sample to be tested is obtained by comparing and calculating the corresponding activity of the heparin test sample with that of the international standard.
(1) Solution preparation:
Tris-HCl buffer (pH 7.4): tris 6.08g and NaCl 8.77g are taken, water 500mL is added for dissolution, bovine serum albumin 10g is added, the pH value is adjusted to 7.4 by HCl, and water is added for dilution to 1000 mL.
Tris-EDTA buffer (pH8.4): 3.03g of Tris, 5.12g of NaCl and 1.4g of EDTA-2 Na1 are taken, 250mL of water is added for dissolution, the pH value is adjusted to 8.4 by HCl, and the solution is diluted to 500mL by water. Heparin standard and test sample solutions: heparin activity standards were purchased from EDQM (European director for the Quality of medicines) as part of the low-molecular-mass for assay BRP (Biological Reference preference) (H0185000 for detection of anti-factor Xa activity and anti-factor IIa activity). And (3) respectively diluting the standard substance (S) and the test substance (T) into 4 solutions with different concentrations by using Tris-HCl buffer solution (pH7.4), wherein the dose-to-dose ratio is controlled to be 1: 0.7-1: 0.6. The concentration should be in the log dose to linear range of response, typically 0.025IU to 0.2IU per ml for anti-Xa factors and 0.015IU to 0.075IU per ml for anti-IIa factors.
ATIII solution: ATIII was purchased from Chromogenix corporation (Sweden). When detecting the Xa resisting factor, Tris-HCl buffer solution (pH7.4) is prepared into solution with the concentration of 1 IU/mL; when detecting anti-IIa factor, Tris-HCl buffer (pH7.4) was used to prepare a solution of 0.5 IU/mL.
Chromogenic substrate solution for detecting anti-Xa factor, chromogenic substrate S-2765(N- α -benzyloxycarbonyl-D-arylyl-L-glycosyl-L-arylenine-p-nitroaniline-dihydrazide), available from Chromogenix corporation (Sweden), for detecting anti-IIa factor, chromogenic substrate S-2238 (H-D-phenylallyl-L-piperidinoyl-arylenine-p-nitroaniline-dihydrazide), available from Chromogenix corporation (Sweden), both substrates were stored in a 0.003M solution in deionized water and diluted to 0.0005M in Tris-EDTA buffer (pH8.4) immediately before use.
Anti-factor Xa solution: Tris-HCl buffer (pH7.4) was used to adjust the concentration so that the absorbance at 405nm was between 0.6 and 0.7 in an anti-Xa experiment using 0.9% NaCl instead of (ultra) low molecular weight heparin.
Anti-factor IIa solution: the solution was dissolved and diluted to 5IU/mL with Tris-HCl buffer (pH 7.4).
The determination method comprises the following steps:
taking 16 centrifuge tubes of 1.5mL, and respectively marking T1，T2，T3，T4And S1，S2，S3，S4. Two tubes were made in parallel for each concentration. 50. mu.l of 4 concentrations of the test (T) or standard (S) dilutions and 50. mu.l of AT III solution were added to each tube and mixed well, taking care of the absence of air bubbles. According to S1，S2，S3，S4，T1，T2，T3，T4，T1，T2，T3，T4，S1，S2，S3，S4The sequence was sequenced, after equilibrating in a 37 ℃ water bath for 1 minute, 100. mu.l of the anti-Xa (or anti-IIa) factor solution was added to each tube, after incubation for 1 minute at 37 ℃ exactly 250. mu.l of the chromogenic substrate solution was added, mixed, and quenched by addition of 375. mu.l of 30% acetic acid solution each immediately after incubation in a 37 ℃ water bath for 4 minutes. Absorbance at 405nm was measured using a 1cm optical path semimicroscale cuvette with a Tris-HCl buffer (pH7.4) as a blank. The same procedure was performed with Tris-HCl buffer (pH7.4) instead of the sample solution (two tubes in parallel) as a blank control tube, and at the beginning and end of 16 tubes, the tubes were divided into twoThe absorbance of the blank control tube was measured. The absorbance of the two should not be significantly different. Taking the absorbance as the ordinate, taking the concentration logarithm value of the standard solution (or the test solution) as the abscissa, performing linear regression, and calculating the titer and the experimental error according to the experimental design of the 4 × 4 method based on the quantity response parallel line principle in the biological assay statistical method. The average signal-to-limit ratio (FL%) should not be greater than 15%.
The anti-Xa potency of product I-2 was tested as described above to be 74 + -10 IU/mg and the anti-IIa potency was tested to be 91 + -10 IU/mg.
Example 2 preparation of Low molecular weight heparin 2 (hereinafter, also referred to as Low molecular weight heparin I-11)
Heparinase I prepared according to ZL200410038098.6 having a total enzyme activity of 100IU was added to 100mL of a 50g/L heparin solution prepared in the same manner as in example 1 in a lump. The light absorption of the solution at 231nm, A231, was monitored using a quartz cuvette with an optical path difference of 1cm and an ultraviolet spectrophotometer. When the A231 reaches a reaction control point (A231 is 46.3), the reaction is ended by inactivating the enzyme in the reaction solution in a boiling water bath at 100 ℃ for 5-10 min, taking out the reaction system, cooling to room temperature, adding 6 times of volume of absolute ethyl alcohol into the reaction solution, stirring at room temperature for 10min, centrifuging at 4000r/min at room temperature for 15min, collecting the precipitate, adding deionized water with the mass 2-3 times of that of the precipitate to dissolve, filtering with a 0.22 mu m membrane, collecting the permeate, freezing in a low-temperature refrigerator at-80 ℃ to form solid ice blocks, freeze-drying in a freeze dryer (the cold trap temperature is-50 ℃) and then crushing into powder by using a mortar or a small-size crusher to obtain the low-molecular-weight heparin 2 (also named as product I-11).
The analysis of the molecular weight, and of the molecular weight distribution, was then carried out on product I-11 according to the method of example 1. The results of the analysis by the above method show that product I-11 has a number average molecular weight of 3768Da and a weight average molecular weight of 7160Da, wherein the molecular weight distribution is that heparin with a weight average molecular weight of less than 3K accounts for 22.273% of the total weight of product I-11, heparin with a weight average molecular weight of 3K-5K accounts for 15.842% of the total weight of product I-11, heparin with a weight average molecular weight of 5K-8K accounts for 21.674% of the total weight of product I-11, and heparin with a weight average molecular weight of more than 8K accounts for 40.211% of the total weight of product I-11.
The products I-11 were then tested for anti-Xa and anti-IIa titers according to the method of example 1, with the anti-Xa titer being 86. + -. 10IU/mg and the anti-IIa titer being 43. + -.10 IU/mg.
Example 3 preparation of Low molecular weight heparin 3 (hereinafter, also referred to as Low molecular weight heparin III-1)
Heparinase III prepared according to ZL201010259913.7 having a total enzyme activity of 100IU was added to 100mL of a 50g/L heparin solution prepared in the same manner as in example 1 in a lump. The light absorption of the solution at 231nm, A231, was monitored using a quartz cuvette with an optical path difference of 1cm and an ultraviolet spectrophotometer. When the A231 reaches a reaction control point (A231 is 7.77), the reaction is ended by inactivating the enzyme in the reaction solution in a boiling water bath at 100 ℃ for 5-10 min, taking out the reaction system, cooling to room temperature, adding 6 times of volume of absolute ethyl alcohol into the reaction solution, stirring at room temperature for 10min, centrifuging at 4000r/min at room temperature for 15min, collecting the precipitate, adding deionized water with the mass 2-3 times of that of the precipitate to dissolve, filtering with a 0.22 mu m membrane, collecting the permeate, freezing in a low-temperature refrigerator at-80 ℃ to form solid ice blocks, freeze-drying in a freeze dryer (the cold trap temperature is-50 ℃) and then crushing into powder by using a mortar or a small-sized crusher to obtain the low-molecular-weight heparin 3 (also named as product III-1).
The analysis of the molecular weight, and of the molecular weight distribution, was then carried out on the product III-1 according to the method of example 1. The results of the analysis by the above method show that the product III-1 has a number average molecular weight of 9476Da and a weight average molecular weight of 10622Da, wherein the molecular weight distribution is that heparin with a weight average molecular weight of less than 3K accounts for 1.022% of the total amount of the product III-1, heparin with a weight average molecular weight of 3K-5K accounts for 3.252% of the total amount of the product III-1, heparin with a weight average molecular weight of 5K-8K accounts for 10.810% of the total amount of the product III-1, and heparin with a weight average molecular weight of more than 8K accounts for 84.916% of the total amount of the.
Then, the anti-Xa potency and anti-IIa potency of product III-1 were determined according to the method described in example 1, wherein the anti-Xa potency was 158. + -. 10IU/mg and the anti-IIa potency was 155. + -. 10 IU/mg.
Example 4 preparation of Low molecular weight heparin 4 (hereinafter, also referred to as Low molecular weight heparin III-2)
Heparinase III prepared according to ZL201010259913.7 having a total enzyme activity of 100IU was added to 100mL of a 50g/L heparin solution prepared in the same manner as in example 1 in a lump. The light absorption of the solution at 231nm, A231, was monitored using a quartz cuvette with an optical path difference of 1cm and an ultraviolet spectrophotometer. When the A231 reaches a reaction control point (A231 is 18.56), the reaction is ended by inactivating the enzyme in the reaction solution in a boiling water bath at 100 ℃ for 5-10 min, taking out the reaction system, cooling to room temperature, adding 6 times of volume of absolute ethyl alcohol into the reaction solution, stirring at room temperature for 10min, centrifuging at 4000r/min at room temperature for 15min, collecting the precipitate, adding deionized water with the mass 2-3 times of that of the precipitate to dissolve, filtering with a 0.22 mu m membrane, collecting the permeate, freezing in a low-temperature refrigerator at-80 ℃ to form solid ice blocks, freeze-drying in a freeze dryer (the cold trap temperature is-50 ℃) and then crushing into powder by using a mortar or a small-size crusher to obtain the low-molecular-weight heparin 4 (also named as product III-2).
The analysis of the molecular weight, and of the molecular weight distribution, was then carried out on the product III-2 according to the method of example 1. The results of the analysis by the above method show that the product III-2 has a number average molecular weight of 8659Da and a weight average molecular weight of 10151Da, wherein the molecular weight distribution is that heparin with a weight average molecular weight of less than 3K accounts for 1.848% of the total amount of the product III-2, heparin with a weight average molecular weight of 3K-5K accounts for 4.963% of the total amount of the product III-2, heparin with a weight average molecular weight of 5K-8K accounts for 13.611% of the total amount of the product III-2, and heparin with a weight average molecular weight of more than 8K accounts for 79.578% of the total amount of the.
The product III-2 was then tested for anti-Xa and anti-IIa titers according to example 1, with the anti-Xa titer being 132 + -10 IU/mg and the anti-IIa titer being 138 + -10 IU/mg.
EXAMPLE 5 preparation of N-acetylated heparin derivative (NS)
5g of heparin (purchased from Heishan Biochemical pharmaceutical industry, product name: heparin sodium, molecular weight distribution of which is 5000-30000, average molecular weight of which is 20000) was passed through a styrene cation exchange resin short column (HL-1200, Shanghai Guanghai Biotech Co., Ltd.), at 4 ℃ and at a flow rate of 2.2mL/12min and changes in light absorption in the ultraviolet region were monitored, further, the effluent was neutralized with pure pyridine, and further lyophilized to obtain heparin pyridinium in the form of white powder.
Dissolving the obtained heparin pyridinium in 300mL of 95% DMSO aqueous solution, heating at 50 ℃ for 5h, adjusting the pH to 8.0 by NaOH (1M), and freeze-drying to obtain the heparin derivative with the sulfate radical removed at the N end. Dissolving the sample obtained in the previous step into saturated NaHCO3To the solution, 200. mu.L of acetic anhydride was added at 4 ℃ and the pH was adjusted to 7.5-8.4 with NaOH solution. Dialyzing the reaction solution in a dialysis bag with the molecular weight cut-off of 1kDa to remove salts, and freeze-drying to obtain a product, hereinafter also referred to as NS heparin (see: Lapierre F, Holme K, Lam L, et al. chemical modifications of heparin sodium salts antisense heparin-inhibitor, angiostatic, anti-molecular and anti-viral properties [ J-heparin [ ]].Glycobiology,1996,6(3):355-366.)。
Molecular weight analysis was then performed on the product NS. The results of the analysis by the method of example 1 show that the product NS has a number average molecular weight of 14071Da and a weight average molecular weight of 16148Da, wherein the molecular weight distribution is such that heparin with a weight average molecular weight of less than 3K makes up 1.1% of the total amount of the product NS, heparin with a weight average molecular weight of 3K to 5K makes up 1.28% of the total amount of the product NS, heparin with a weight average molecular weight of 5K to 8K makes up 5.09% of the total amount of the product NS, and heparin with a weight average molecular weight of more than 8K makes up 92.53% of the total.
The anti-Xa and anti-IIa titers of the product NS were then determined according to the method of example 1, with the anti-Xa titer being 15. + -.2 IU/mg and the anti-IIa titer being 12. + -.2 IU/mg.
EXPERIMENTAL EXAMPLE 6 preparation of N-acetylated Low molecular weight heparin derivative (N1)
In the same manner as in example 1, 100mL of the NS heparin solution prepared in example 5 (NS heparin concentration: 50g/L, 20mM Tris, 50mM NaCl, 20mM CaCl2, pH 7.6) was prepared, and heparinase I prepared in accordance with ZL200410038098.6 having a total enzyme activity of 100IU was added to 100mL of the prepared 50g/LNS heparin solution in one portion. The light absorption of the solution at 231nm, A231, was monitored using a quartz cuvette with an optical path difference of 1cm and an ultraviolet spectrophotometer. When the A231 reaches the reaction control point (A231 is 18.9), the reaction is ended by inactivating the enzyme in the reaction solution in a boiling water bath at 100 ℃ for 5-10 min, taking out the reaction system, cooling to room temperature, adding 6 times of volume of absolute ethyl alcohol into the reaction solution, stirring at room temperature for 10min, centrifuging at 4000r/min at room temperature for 15min, collecting the precipitate, adding deionized water with the mass 2-3 times of that of the precipitate to dissolve, filtering with a 0.22 mu m membrane, collecting the permeate, freezing in a low-temperature refrigerator at-80 ℃ to form solid ice blocks, freeze-drying in a freeze-dryer (the cold trap temperature is-50 ℃) and then crushing into powder by a mortar or a small-size crusher to obtain the N-acetylated low-molecular-weight heparin derivative 1 (also named as product N1).
The analysis of the molecular weight, and of the molecular weight distribution, was then carried out on the product N1 according to the method of example 1. The results of the analysis by the above method show that the product N1 has a number average molecular weight of 7534Da and a weight average molecular weight of 11511Da, wherein the molecular weight distribution is that heparin with a weight average molecular weight of less than 3K accounts for 1.446% of the total amount of the product N1, heparin with a weight average molecular weight of 3K-5K accounts for 6.291% of the total amount of the product N1, heparin with a weight average molecular weight of 5K-8K accounts for 10.635% of the total amount of the product N1, and heparin with a weight average molecular weight of more than 8K accounts for 81.608% of the total amount of the.
EXAMPLE 7 preparation of N-acetylated Low molecular weight heparin derivative (N2)
100mL of an LNS heparin solution was prepared in the same manner as in example 6, and heparinase I prepared according to ZL200410038098.6 having a total enzyme activity of 100IU was added at once to 100mL of the prepared 50g/LNS heparin solution. The light absorption of the solution at 231nm, A231, was monitored using a quartz cuvette with an optical path difference of 1cm and an ultraviolet spectrophotometer. When the A231 reaches a reaction control point (A231 is 40), the reaction is ended by inactivating the enzyme in the reaction solution in a boiling water bath at 100 ℃ for 5-10 min, taking out the reaction system, cooling to room temperature, adding 6 times of volume of absolute ethyl alcohol into the reaction solution, stirring at room temperature for 10min, centrifuging at 4000r/min at room temperature for 15min, collecting the precipitate, adding deionized water with the mass 2-3 times of that of the precipitate to dissolve, filtering with a 0.22 mu m membrane, collecting the permeate, freezing in a low-temperature refrigerator at-80 ℃ to form solid ice blocks, freeze-drying in a freeze dryer (the cold trap temperature is-50 ℃) and then crushing into powder by using a mortar or a small-sized crusher to obtain the N-acetylated low-molecular-weight heparin derivative 2 (also named as product N2).
The analysis of the molecular weight, and of the molecular weight distribution, was then carried out on the product N2 according to the method of example 1. The results of the analysis by the above method show that product N2 has a number average molecular weight of 4022Da and a weight average molecular weight of 7160Da, wherein the molecular weight distribution is that heparin with a weight average molecular weight of less than 3K accounts for 11.803% of the total amount of product N2, heparin with a weight average molecular weight of 3K-5K accounts for 21.342% of the total amount of product N2, heparin with a weight average molecular weight of 5K-8K accounts for 20.776% of the total amount of product N2, and heparin with a weight average molecular weight of more than 8K accounts for 46.079% of the total amount of product N2.
EXPERIMENTAL EXAMPLE 8 preparation of N-acetylated Low molecular weight heparin derivative (N4)
100mL of NS heparin solution prepared in the same manner as in example 6 was added at a time with heparinase III prepared according to ZL201010259913.7 having a total enzyme activity of 100IU to 100mL of 50g/LNS heparin solution prepared. The light absorption of the solution at 231nm, A231, was monitored using a quartz cuvette with an optical path difference of 1cm and an ultraviolet spectrophotometer. When the A231 reaches the reaction control point (A231 is 4.92), the reaction is ended by inactivating the enzyme in the reaction solution in a boiling water bath at 100 ℃ for 5-10 min, taking out the reaction system, cooling to room temperature, adding 6 times of volume of absolute ethyl alcohol into the reaction solution, stirring at room temperature for 10min, centrifuging at 4000r/min at room temperature for 15min, collecting the precipitate, adding deionized water with the mass 2-3 times of that of the precipitate to dissolve, filtering with a 0.22 mu m membrane, collecting the permeate, freezing in a low-temperature refrigerator at-80 ℃ to form solid ice blocks, freeze-drying in a freeze-dryer (the cold trap temperature is-50 ℃) and then crushing into powder by a mortar or a small-size crusher to obtain the N-acetylated low-molecular-weight heparin derivative 4 (also named as product N4).
The analysis of the molecular weight, and of the molecular weight distribution, was then carried out on the product N4 according to the method of example 1. The results of the analysis by the above method show that the product N4 has a number average molecular weight of 10001Da and a weight average molecular weight of 11134Da, wherein the molecular weight distribution is that heparin with a weight average molecular weight of less than 3K accounts for 1.29% of the total amount of the product N4, heparin with a weight average molecular weight of 3K-5K accounts for 2.909% of the total amount of the product N4, heparin with a weight average molecular weight of 5K-8K accounts for 9.943% of the total amount of the product N4, and heparin with a weight average molecular weight of more than 8K accounts for 85.858% of the total amount of the.
EXPERIMENTAL EXAMPLE 9 preparation of N-acetylated Low molecular weight heparin derivative (N5)
100mL of NS heparin solution prepared in the same manner as in example 6 was added at a time with heparinase III prepared according to ZL201010259913.7 having a total enzyme activity of 100IU to 100mL of 50g/LNS heparin solution prepared. The light absorption of the solution at 231nm, A231, was monitored using a quartz cuvette with an optical path difference of 1cm and an ultraviolet spectrophotometer. When the A231 reaches the reaction control point (A231 is 14.20), the reaction is ended by inactivating the enzyme in the reaction solution in a boiling water bath at 100 ℃ for 5-10 min, taking out the reaction system, cooling to room temperature, adding 6 times of volume of absolute ethyl alcohol into the reaction solution, stirring at room temperature for 10min, centrifuging at 4000r/min at room temperature for 15min, collecting the precipitate, adding deionized water with the mass 2-3 times of that of the precipitate to dissolve, filtering with a 0.22 mu m membrane, collecting the permeate, freezing in a low-temperature refrigerator at-80 ℃ to form solid ice blocks, freeze-drying in a freeze-dryer (the cold trap temperature is-50 ℃) and then crushing into powder by a mortar or a small-size crusher to obtain the N-acetylated low-molecular-weight heparin derivative 5 (also named as product N5).
The analysis of the molecular weight, and of the molecular weight distribution, was then carried out on the product N5 according to the method of example 1. The results of the analysis by the above method show that the product N5 has a number average molecular weight of 9900Da and a weight average molecular weight of 10392Da, wherein the molecular weight distribution is that heparin with a weight average molecular weight of less than 3K accounts for 0.37% of the total amount of the product N5, heparin with a weight average molecular weight of 3K-5K accounts for 5.059% of the total amount of the product N5, heparin with a weight average molecular weight of 5K-8K accounts for 12.106% of the total amount of the product N5, and heparin with a weight average molecular weight of more than 8K accounts for 82.465% of the total amount of the.
Comparative example 1
Unfractionated heparin was biochemically purchased from Hebei Heshan, and the heparin (UFH, also known as group H) had a number average molecular weight Mn of 14000 and a weight average molecular weight of 24899.
The product UFH was then tested for anti-Xa and anti-IIa titers, as described in example 1, wherein the anti-Xa titer was 187. + -.21 IU/mg and the anti-IIa titer was 177. + -.6 IU/mg.
Comparative example 2
Enoxaparin was purchased from Hebei Heshan Biochemical industries and has a number average molecular weight of 4372 and a weight average molecular weight of 5679. Molecular weight analysis of enoxaparin was performed using the method described in the present invention.
The results of the analysis by the above method show that the molecular weight distribution of the enoxaparin is such that heparin with a weight average molecular weight of less than 3K represents 20.71% of the total weight of the enoxaparin, heparin with a weight average molecular weight of 3K to 5K represents 55.42% of the total weight of the enoxaparin, heparin with a weight average molecular weight of 5K to 8K represents 13.16% of the total weight of the enoxaparin, and heparin with a weight average molecular weight of more than 8K represents 10.71% of the total weight of the enoxaparin.
Comparative example 3 preparation of Low molecular weight heparin 5 (product I-16)
Heparinase I prepared according to ZL200410038098.6 having a total enzyme activity of 100IU was added to 100mL of a 50g/L heparin solution prepared in the same manner as in example 1 in a lump. The light absorption of the solution at 231nm, A231, was monitored using a quartz cuvette with an optical path difference of 1cm and an ultraviolet spectrophotometer. When the A231 reaches a reaction control point (A231 is 98.6), the reaction is ended by inactivating the enzyme in the reaction solution in a boiling water bath at 100 ℃ for 5-10 min, taking out the reaction system, cooling to room temperature, adding 6 times of volume of absolute ethyl alcohol into the reaction solution, stirring at room temperature for 10min, centrifuging at 4000r/min at room temperature for 15min, collecting the precipitate, adding deionized water with the mass 2-3 times of that of the precipitate to dissolve, filtering with a 0.22 mu m membrane, collecting the permeate, freezing in a low-temperature refrigerator at-80 ℃ to form solid ice blocks, freeze-drying in a freeze dryer (the cold trap temperature is-50 ℃) and then crushing into powder by using a mortar or a small-size crusher to obtain the low-molecular-weight heparin 5 (also named as product I-16).
The analysis of the molecular weight, and of the molecular weight distribution, was then carried out on product I-16 according to the method of example 1. The results of the analysis by the above method show that the product I-16 has a number average molecular weight of 2523Da and a weight average molecular weight of 4341Da, wherein the molecular weight distribution is that heparin with a weight average molecular weight of less than 3K accounts for 34.707% of the total amount of the product I-16, heparin with a weight average molecular weight of 3K-5K accounts for 34.637% of the total amount of the product I-16, heparin with a weight average molecular weight of 5K-8K accounts for 17.908% of the total amount of the product I-16, and heparin with a weight average molecular weight of more than 8K accounts for 12.748% of the total amount of the.
Comparative example 4 preparation of N-acetylated Low molecular weight heparin derivative (N3)
100mL of NS heparin solution prepared in the same manner as in example 6 was added at a time with heparinase I prepared according to ZL200410038098.6 having a total enzyme activity of 100IU to 100mL of 50g/LNS heparin solution prepared. The light absorption of the solution at 231nm, A231, was monitored using a quartz cuvette with an optical path difference of 1cm and an ultraviolet spectrophotometer. When the A231 reaches a reaction control point (A231 is 92), the reaction is ended by inactivating the enzyme in the reaction solution in a boiling water bath at 100 ℃ for 5-10 min, taking out the reaction system, cooling to room temperature, adding 6 times of volume of absolute ethyl alcohol into the reaction solution, stirring at room temperature for 10min, centrifuging at 4000r/min at room temperature for 15min, collecting the precipitate, adding deionized water with the mass 2-3 times of that of the precipitate to dissolve, filtering with 0.22 mu m, collecting the permeate, freezing in a low-temperature refrigerator at-80 ℃ to form solid ice blocks, freeze-drying in a freeze dryer (the cold trap temperature is-50 ℃) and then crushing into powder by using a mortar or a small-sized crusher to obtain the N-acetylated low-molecular-weight heparin derivative 3 (also named as product N3). .
The analysis of the molecular weight, and of the molecular weight distribution, was then carried out on the product N3 according to the method of example 1. The results of the analysis by the above method show that product N3 has a number average molecular weight of 2033Da and a weight average molecular weight of 4549Da, wherein the molecular weight distribution is that heparin with a weight average molecular weight of less than 3K accounts for 35.481% of the total amount of product N3, heparin with a weight average molecular weight of 3K-5K accounts for 32.363% of the total amount of product N3, heparin with a weight average molecular weight of 5K-8K accounts for 17.69% of the total amount of product N3, and heparin with a weight average molecular weight of more than 8K accounts for 14.466% of the total amount of product N3.
TABLE 1 summary of molecular weights and molecular weight distributions of each heparin product in examples and comparative examples
Figure GDA0002125140510000251
Figure GDA0002125140510000261
Experimental example 1 pulmonary fibrosis animal model establishment and pulmonary fibrosis pharmacodynamic evaluation
C57BL/6 mice (20-25g, 8-12 weeks old) were anesthetized and fixed with 1% Averdin (Sigma), neck skin was disinfected with ethanol, a left and right incision was made in the middle of the neck, subcutaneous tissue was bluntly isolated, the trachea was exposed, Bleomycin (Dalian Meiren Biotechnology Co., Ltd.) was injected into the trachea at a dose of 2.8mg/kg, the mice were immediately rotated upright at a constant speed after injection to distribute the drug uniformly in the lungs, and the neck skin was sutured. Administration was started on the day after the above treatment, and the day was regarded as day 1, and the administration schedule was as follows.
The heparin of the groups of examples 1 to 9 and comparative examples 1 and 3 to 4 was sprayed every other day to the above-treated mice for administration. A negative control group, which was mice treated in the same manner as described above, was provided except for the administration group, and the same amount of physiological saline was administered every other day as a negative control; healthy groups (also called positive control groups) were set, normal mice were modeled by administering the same dose of physiological saline every other day, and 5 mice per group were not administered any drug during the modeling. 5mL of the drug of each of the above examples and comparative examples was administered at a concentration of 2mg/mL by spray administration, once every other day, and all mice were quenched on day 21 after administration, lungs were taken, and the survival rate of the mice was calculated according to the method described in Cahill, Emer F. et al, "Hepatocyte Growth Factor Is Required for sensory bacterial Cell monitoring and Pulmonary fibrosis," Stem Cells transformation medicine (2016): sctm-2015.
The results of the mouse survival rates calculated according to the above methods are shown in table 2, and it can be seen that the survival rates of the mice administered in the groups of examples 1 to 9 and the group of comparative example 1 were greatly improved as compared with the negative control group. The survival rate of mice in groups of comparative examples 3 and 4 was not significantly improved.
From the above results, it can be seen that the low molecular weight heparins of the groups involved in the examples of the present invention have a certain effect on inhibiting the death of mice with pulmonary fibrosis induced by Bleomycin, and in addition, the common heparin can also inhibit the death of such mice.
TABLE 2 Effect of different heparins and derivatives on survival of Bleomycin-induced pulmonary fibrosis in mice in example 1
Group of Survival Rate (%) of mouse Group of Survival Rate (%) of mouse
Blank control group 100 NS group 100
Negative control group 12.5 N1 group 100
Group I-2 100 N2 group 87.5
Group I-11 75 N4 group 87.5
Group III-1 100 N5 group 75
Group III-2 100 N3 group 0
Group I-16 25 Group H 87.5
Experimental example 2 pulmonary fibrosis animal model establishment and pulmonary fibrosis pharmacodynamic evaluation
The procedure was as in Experimental example 1 except that the dose of Bleomycin was changed to 2.5 mg/kg. C57BL/6 mice (20-25g, 8-12 weeks old) were anesthetized and fixed with 1% Averdin (Sigma Co.), the neck skin was disinfected with ethanol, a left and right incision was made in the middle of the neck, subcutaneous tissue was isolated purely, the trachea was exposed, 2.5mg/kg dose of Bleomycin (Dalian Melam Bion) was injected into the trachea, the mice were immediately rotated upright at hook speed after injection to distribute the drug evenly in the lungs, and the neck skin was sutured. Administration was started on the day after the above treatment, and the day was regarded as day 1, and the administration schedule was as follows.
Heparin administration of the groups of examples 1 to 9 and comparative examples 1, 3 to 4 was sprayed every other day to the above-treated mice. A negative control group, which was mice treated in the same manner as described above, was provided except for the administration group, and the same amount of physiological saline was administered every other day as a negative control; a positive control group is set, normal mice are given the same dose of physiological saline every other day to establish a model, no drug is given in the molding process, the group is also called a healthy group in some cases, and 5 mice in each group are provided. 5mL of the drug at a concentration of 2mg/mL was administered by spraying to the mice of each of the above examples and comparative examples, once every other day, and all the mice were suddenly killed on the 15 th day after the administration, and lung tissues were taken.
The staining results of pathological sections of lung tissue were obtained in the same manner as described in "tissue growth factors requiring for structural Cell Protection approach in" Stem Cells transformation Medicine (2016): sctm-2015, according to Cahill, EmerF, et al, and as shown in FIG. 1.
It was shown that a significant fibrosis change was observed in lung tissue after 15 days of the Bleomycin treatment (see (a) diagram in fig. 1), indicating that mice with pulmonary fibrosis were also obtained in experimental example 2. The lung tissue of the normal control group mouse has smooth surface, better elasticity and pink appearance (see (f) diagram in fig. 1), and the lung tissue of the negative control group has typical honeycomb-shaped character and is characterized by fibrosis pathology. Compared with a negative control group, the groups of examples 1 to 9 and the group of comparative example 1 show a basically complete alveolar structure, most alveoli show a monolayer cell structure, the cell morphology is similar to that of a normal group, no obvious inflammatory infiltrative characteristic exists, and the fibrosis degree also has a trend of becoming lighter in different degrees. While the groups of comparative examples 3-4 showed a strong tendency to fiberize.
And then accurately weighing the lung tissues of the mice of each group with the wet weight of 30-100 mg, putting the lung tissues into a test tube, accurately adding 1ml of hydrolysis liquid, and uniformly mixing. After covering, hydrolyzing at 95 ℃ or boiling water bath for 20 minutes (after hydrolyzing for 10 minutes, uniformly mixing once to ensure that the hydrolysis is more complete). Then adjusting the pH value to about 6.0-6.8. Adding distilled water to 10ml, and mixing; adding an appropriate amount of activated carbon (about 20-30 mg, taking clear and colorless supernatant after centrifugation of the supernatant) into 3-4 ml of diluted hydrolysate, uniformly mixing, centrifuging at 3500 rpm/min for 10min, taking 1ml of supernatant carefully as a detection sample, determining the content of hydroxyproline in the sample by adopting a hydroxyproline determination kit built from Nanjing, and representing the content of collagen contained in the lung.
The results are shown in Table 3. It can be seen that the group H (comparative example 1) and the groups of examples 1 to 9, which were administered with the normal heparin, effectively reduced the content of hydroxyproline, i.e., effectively reduced the content of collagen in the lung, indicating that both the administration of heparin and the low molecular weight heparin obtained in the examples can alleviate the formation of collagen, demonstrating that the degree of fibrosis is reduced, while the groups of comparative examples 3 to 4 did not significantly change.
TABLE 3 Effect of different heparins and derivatives on the content of hydroxyproline in the lung tissue of Bleomycin-induced mice, the results are expressed as (results. + -. SD)
Figure GDA0002125140510000281
It is generally accepted that collagen deposition is the most prominent feature of fibrotic lesions. Collagen-1 (ColA-1) is the most characteristic collagen index. Therefore, by measuring ColA-1 at the transcription level, the amount of collagen deposition can be predicted, and the progression of fibrotic lesions can be predicted. mRNA of collagen in the lung of each group of mice obtained in Experimental example 2 was quantitatively determined according to the method reported by Yang et al (Yang D, Atkins G J, Turner A G, actual. differential effects of 1, 25-dihydrovitamin D on minor administration and differentiation in two differential types of osteoplast-like cultures [ J ]. the journal of stereo biological and molecular biology,2013,136: 166-170). As a result, as shown in Table 4, it was found that administration of groups of examples 1-9, group of comparative example 1 can effectively reduce the conversion rate of collagen, thereby inhibiting the occurrence of pulmonary fibrosis.
TABLE 4 Effect of different heparins and derivatives on the collagen mRNA in the lung tissue of Bleomycin-induced mice, the results are expressed as (results. + -. SD)
Group of Collagen mRNA Group of Collagen mRNA
Negative control group 20.04±6.30 NS group 10.75±1.77
Group I-2 10.20±1.34 N1 group 10.17±3.45
Group I-11 12.27±2.57 N2 group 7.24±2.34
Group III-1 10.25±4.17 N4 group 10.87±1.56
Group III-2 7.72±1.63 N5 group 7.36±5.67
Group I-16 25.16±4.37 N3 group 26.47±1.07
Group H 10.77±3.22
From the results of the above experimental examples 1 and 2, it was revealed that although normal heparin, i.e., heparin which is not degraded by an enzyme or degraded with anticoagulation activity, has the effect of inhibiting pulmonary fibrosis, it is generally considered that heparin with a large molecular weight has significant side effects, for example, an increased risk of bleeding, and is likely to induce thrombocytopenia. The low molecular weight heparin or the anticoagulated low molecular weight heparin not only has the effects of inhibiting pulmonary fibrosis formation and improving the survival rate of mice, but also has controllable drug metabolism, and is expected to avoid the need of detecting the bleeding risk of patients clinically.

Claims (14)

1. A low molecular weight heparin having a number average molecular weight (Mn) in the range of 3000 to 12000Da and a weight average molecular weight (Mw) in the range of 7000 to 17000Da, wherein heparin having a molecular weight of less than 3000Da accounts for less than 25 wt% of the total low molecular weight heparin, and heparin having a molecular weight of more than 8000Da accounts for 40 wt% or more of the total low molecular weight heparin, and 90 wt% or less, and heparin having a molecular weight of 3000 to 5000Da accounts for 22 wt% or less, and 3 wt% or more of the total low molecular weight heparin.
2. The low molecular weight heparin according to claim 1, wherein the number average molecular weight (Mn) is in the range of 3700 to 11000Da and the weight average molecular weight (Mw) is in the range of 7000 to 12000 Da.
3. Low molecular weight heparin according to any one of claims 1 to 2, wherein the number average molecular weight (Mn) is in the range of 7000 to 10000Da and the weight average molecular weight (Mw) is in the range of 8000 to 12000 Da.
4. Low molecular weight heparin according to any one of claims 1 to 2, wherein the number average molecular weight (Mn) is preferably within the range of 8000 to 9800Da and the weight average molecular weight (Mw) is preferably within the range of 9000 to 11000 Da.
5. The low molecular weight heparin according to claim 1, wherein the number average molecular weight (Mn) is 4000 to 11000Da, the weight average molecular weight (Mw) is 7000 to 12000Da, the anti-Xa potency of the heparin is 20IU/mg or less, and the anti-IIa potency of the heparin is 20IU/mg or less.
6. Low molecular weight heparin according to claim 5, wherein the number average molecular weight (Mn) is in the range of 4000 to 8000Da and the weight average molecular weight (Mw) is in the range of 7000 to 12000 Da.
7. Low molecular weight heparin according to claim 5, wherein the number average molecular weight (Mn) is in the range of 9000 to 11000Da and the weight average molecular weight (Mw) is in the range of 9500 to 12000 Da.
8. The low molecular weight heparin according to any one of claims 1 to 2, wherein the low molecular weight heparin is obtained by degrading heparin using heparinase.
9. The low molecular weight heparin according to any one of claims 1 to 2, wherein the low molecular weight heparin is obtained by degrading heparin using heparinase I or heparinase III.
10. The low molecular weight heparin according to claim 6, wherein the low molecular weight heparin is obtained by degrading heparin using heparinase I.
11. Low molecular weight heparin according to claim 3 or 7, wherein the low molecular weight heparin is obtained by degradation of heparin using heparinase III.
12. The low molecular weight heparin according to any one of claims 5 to 7, wherein the low molecular weight heparin is obtained by anticoagulation treatment of heparin and degradation of heparin by heparinase.
13. Use of heparin for the preparation of a medicament for the treatment or prevention of pulmonary fibrosis, wherein said heparin is a low molecular weight heparin as claimed in any one of claims 1 to 12.
14. Use of heparin for the preparation of a medicament for alleviating the formation of collagen in the lung, wherein said heparin is a low molecular weight heparin as claimed in any one of claims 1 to 12.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102399306A (en) * 2010-09-09 2012-04-04 上海喜恩医药科技发展有限公司 Preparation method of heparin-derived polysaccharide mixture
CN103173506A (en) * 2011-10-09 2013-06-26 清华大学 Method for controlling production of low-molecular-weight heparin
CN103540630A (en) * 2013-10-22 2014-01-29 常山生化药业（江苏）有限公司 Preparation method of low molecular weight heparin

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102399306A (en) * 2010-09-09 2012-04-04 上海喜恩医药科技发展有限公司 Preparation method of heparin-derived polysaccharide mixture
CN103173506A (en) * 2011-10-09 2013-06-26 清华大学 Method for controlling production of low-molecular-weight heparin
CN103540630A (en) * 2013-10-22 2014-01-29 常山生化药业（江苏）有限公司 Preparation method of low molecular weight heparin

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Chemical modifications of heparin that diminish its anticoagulant but preserve its heparanase-inhibitory, angiostatic, anti-tumor and anti-metastatic properties;Lapierre. F;《GLYCOBIOLOGY》;19960630;第6卷(第3期);第355-366页 *
低分子肝素对大鼠百草枯中毒致肺纤维化的作用研究;徐玉辉;《毒理学杂志》;20151031;第29卷(第5期);第331-333页 *

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