CN103864959A - Application of heparin oligosaccharide dp12 in atherosclerosis resistance - Google Patents

Application of heparin oligosaccharide dp12 in atherosclerosis resistance Download PDF

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Publication number
CN103864959A
CN103864959A CN201410131880.6A CN201410131880A CN103864959A CN 103864959 A CN103864959 A CN 103864959A CN 201410131880 A CN201410131880 A CN 201410131880A CN 103864959 A CN103864959 A CN 103864959A
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heparin
application
atherosclerosis
heparin oligosaccharide
group
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何书英
钱轩
王文荟
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China Pharmaceutical University
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China Pharmaceutical University
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Abstract

The invention discloses an application of heparin oligosaccharide dp12 in atherosclerosis resistance. According to the application disclosed by the invention, heparin oligosaccharide dp12 is obtained in the laboratory by preliminary study and shown in ZL201010139398.9. In the patent, the atherosclerosis-resistant function of heparin oligosaccharide dp12 is further studied by animal experiments, and the study meets the modern development direction of medicines in China and has important social significance and potential economic benefits in the field of fundamental studies and the field of application studies.

Description

The antiatherogenic purposes of a kind of heparin-12-sugar
Technical field
The present invention relates to biomedicine field, be specifically related to a kind of heparin-12-sugar, it has antiatherogenic effect.
Background technology
Heparin is a kind of by glucosamine, and L-idose aldehyde glycosides, N-Acetyl-D-glucosamine and D-Glucose aldehydic acid is the mucopolysaccharide sulfuric acid ester of composition alternately.Wide clinical application, in anti-freezing antithrombotic, is the choice drug of the thrombotic diseases such as control deep-vein thrombosis formation.But due to its molecular weight large (3000-30000D), biological activity heterogeneity, and have the side effects such as hemorrhage, thrombopenia, abnormalities of sugar/lipid metabolism and osteoporosis, limit to a certain extent its clinical application.
Low molecular heparin is obtained by unfractionated heparin depolymerization, molecular weight 3000-8000D, and its anti thrombotic action is better than heparin, and the feature such as blood coagulation resisting function is lower than heparin, has bioavailability high, and Half-life in vivo is long, and bleeding tendency is little, oral easy absorption.It has been successfully used to clinical at present, has the gesture that replaces heparin.
Along with the raising of people's living standard and the change of diet style, atherosclerosis (AS) has become the common disease of serious harm human health, is the important pathologic basis that cardiovascular and cerebrovascular disease occur.Its generation is relevant with hyperlipemia and the variation of vessel wall composition, often with hypertension, hypercholesterolemia or diabetes etc.Atherosclerosis starts the Childhood and continues progress, conventionally occurs symptom in middle age or person in middle and old age.
Heparin-12-sugar molecular weight is lower than Low molecular heparin, and structure is also more single, and its biological activity requires study.This laboratory obtains a kind of heparin-12-sugar by early-stage Study, see patent ZL201010139398.9, this patent is further studied its study of anti-atherogenic effect by experimentation on animals, this research meets the modern developing direction of China's medicine, has important social effect and potential economic benefit in fundamental research field and Applied research fields.
Summary of the invention
Heparin-12-sugar of the present invention can add water for injection and be prepared into injection liquid or lyophilized injection, or adds pharmaceutically acceptable auxiliary material composition and be prepared into pharmaceutically conventional formulation for clinical, as Heparin Oligosaccharides injection liquid or Heparin Oligosaccharides lyophilized injection etc.
Intracorporeal active experiment proves the antiatherogenic effect excellence of Heparin Oligosaccharides of the present invention.In foley's tube damage new zealand rabbit carotid artery the Atherosclerosis Model with high lipid food induction, high dosage heparin-12-sugar (5mg/kg/d) significantly reduces total plasma cholesterol (TC), low-density lipoprotein (LDL-C) level, and uses and be better than positive drug trapidil at reduction TC, LDL-C.Low-dose heparin ten disaccharides (2.5mg/kg/d) do not reduce the effect of TC, not obvious to the reducing effect of LDL-C.
Part pharmacology test and result below:
One, laboratory animal grouping
Blank group (8), model control group (8), positive controls (8), medicine low dose group (8,2.5mg/kg/d), medicine high dose group (8,5mg/kg/d).
Two, setting up carotid atherosclerosis model and administration raises
40 adaptability of male new zealand rabbit are fed 1 week, are divided into two groups: 8 blank group and 32 operation groups of feeding with high lipid food of feeding with standard rabbit feed.Every of operation group every day give high lipid food 120g, be not enough to common rabbit feed and supplement, Normal group does not apply intervention factor.Operation is organized high fat and is fed after 1 week row carotid artery sacculus damage art simultaneously and strip off blood vessel endothelium.Operation group is divided at random 4 groups by the postoperative number that survives according to rabbit, after 2 weeks, starts administration, and at any time adjust dosage according to the variation of the weight of animals every day, successive administration 6 weeks.Model group is still fed and is added normal rabbit feed with high lipid food, and Normal group is only fed with normal rabbit feed.Every rabbit sub-cage rearing, freely drinks water.Animal is put to death in administration after 6 weeks, draws materials, and analysed for plasma total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) level.All data statistics difference adopts Student's t-test to analyze.P < 0.01 is for there being utmost point significant difference.Detected result is in table 1, table 2.
Table 1 total plasma cholesterol (TC) level
Figure BSA0000102585690000021
Figure BSA0000102585690000022
##with relatively p < 0.01 of blank group, *with relatively p < 0.01 of model group
Table 2 low density lipoprotein cholesterol (LDL-C) level
Figure BSA0000102585690000023
##with relatively p < 0.01 of blank group, *with relatively p < 0.05 of model group, *with relatively p < 0.01 of model group
Three, pathological section HE staining examine
Get operation side arteria carotis communis 0.5cm and be placed in the fixing 24h of 10% formalin, conventional dehydration, sample is with paraffin embedding, serial section, and HE dyes, microscopy.
Under light microscopic, the HE visible blank group arteria carotis communis wall that dyes is divided into inner membrance, middle film and adventitia, demarcate clear, and thickness homogeneous.Elastic membrane is complete, and inner membrance is made up of monolayer endothelial cell, and the main component of middle film is smooth muscle cell, is spindle shape.Inner membrance and middle film are to be wavy interior elastic plate as boundary, and beyond middle film and adventitia, elastic plate is boundary, as Fig. 1.Model group blood vessel shows diffusivity intimal thickening, visible a large amount of foam cells, and tube chamber obvious stenosis, hyperplasia inner membrance is take the vascular smooth muscle of propagation as main, as Fig. 2.Positive drug group and heparin-12-sugar high dose group intimal thickening obviously alleviate, and patch obviously dwindles, and luminal stenosis degree is lighter, as Fig. 3,4.Heparin-12-sugar low dose group is not obvious to intimal thickening restraining effect, as Fig. 5.
Accompanying drawing explanation
Fig. 1 is blank group arteria carotis communis HE dyeing microscopic examination figure.
Fig. 2 is control group arteria carotis communis HE dyeing microscopic examination figure.
Fig. 3 is positive drug group arteria carotis communis HE dyeing microscopic examination figure.
Fig. 4 is heparin-12-sugar high dose group arteria carotis communis HE dyeing microscopic examination figure.
Fig. 5 is heparin-12-sugar low dose group arteria carotis communis HE dyeing microscopic examination figure.
Embodiment
Embodiment mono-
1) high lipid food formula
6% peanut oil+2% cholesterol+92% normal diet
2) foundation of rabbit Carotid Balloon Injury model
Experimental rabbit, in preoperative use 20% urethane 5mL/kg auricular vein injecting anesthetic, is lain on the back and is fixed on rabbit platform, neck cropping preserved skin, 75% alcohol disinfecting, drape.Along the about 3-4cm otch of neck median line longitudinal incision, blunt separation subcutis, muscle also expose tracheae, with stripping needle and carefully free right carotid (CCA), internal carotid artery (ICA) and external carotid artery (ECA) of hook tweezer.With thin silk thread, by the ligation of ECA distal end, CCA proximal part closes with the interim folder of bulldog clamp.Then do " V " type otch with eye scissors at the proximal part of ECA ligation place, size is about 1/3 of vessel wall Zhou Jing.With blood vessel drag hook pull-up arteriotomy place, direct-view retrograde inserts the about 4cm of PTCA foley's tube until CCA.In sacculus, inject air with 8atm (1atm=101.325kpa) with connecting hand propelled pressure pump, it is fully expanded.Maintain pressure, the sacculus of pulling back at a slow speed, to the inside and outside aortic bifurcation of neck place, repetitive operation 3 times is fully to strip off endotheliocyte.Finally withdraw from conduit, then ligation ECA proximal part, unclamp bulldog clamp, recover blood flow.Layer-by-layer suture subcutaneous lipids and skin incision.Above all operations all carries out under aseptic condition.The continuous 3d of postoperative muscle injection penicillin 100,000 U/kg, preventing infection.
3) HE dyeing
(1) the conventional dewaxing of paraffin section: dimethylbenzene (I) 15min → dimethylbenzene (II) (should be completely transparent) 10min; (2) descending gradient alcohol immersion aquation step by step: 100% ethanol (I) 2min → 100% ethanol (II) 2min → 95% ethanol 2min → 80% ethanol 2min → tap water rinses a moment; (3) dyeing: distilled water quarters → Hematorylin liquid of developing a film dyes core 5min → tap water and rinses a moment → 1% acidic alcohol differentiation 30s (carry insert several under) → flowing water and rinse the anti-indigo plant several seconds → flowing water of a moment → weak ammonia liquor aqueous solution flushing 10min → set to 0 in the aqueous solution of .5% Yihong and redye 10min; (4) ascending gradient ethanol dehydration step by step: tap water rinses (differentiation Yihong) → 95% ethanol (I) 2min → 95% ethanol (II) 2min → 100% ethanol (I) 2min → 100% ethanol (II) 2min in a moment; (5) transparent: dimethylbenzene (I) 5min → dimethylbenzene (II) 5min; (6) fixing, mounting: under cover glass resinene fix, mounting.

Claims (3)

1. Heparin Oligosaccharides ten dimers of a structural formula (I):
Figure FSA0000102585680000011
2. a pharmaceutical composition, wherein contains Heparin Oligosaccharides ten dimers and the pharmaceutically acceptable carrier of claim 1.
3. Heparin Oligosaccharides ten dimers of claim 1 are for the preparation of the purposes of the medicine for the treatment of atheromatosis.
CN201410131880.6A 2014-04-03 2014-04-03 Application of heparin oligosaccharide dp12 in atherosclerosis resistance Pending CN103864959A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4788307A (en) * 1986-04-30 1988-11-29 Choay S.A. Oligosaccharidic fractions devoid or practically devoid of antithrombotic activity
CN1852926A (en) * 2003-07-24 2006-10-25 安万特医药股份有限公司 Oligosaccharide mixtures derived from heparin, preparation thereof and pharmaceutical compositions containing them
CN101824100A (en) * 2010-04-02 2010-09-08 中国药科大学 Heparin-12-sugar, preparation method thereof and application thereof in resisting vascular smooth muscle cell proliferation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4788307A (en) * 1986-04-30 1988-11-29 Choay S.A. Oligosaccharidic fractions devoid or practically devoid of antithrombotic activity
CN1852926A (en) * 2003-07-24 2006-10-25 安万特医药股份有限公司 Oligosaccharide mixtures derived from heparin, preparation thereof and pharmaceutical compositions containing them
CN101824100A (en) * 2010-04-02 2010-09-08 中国药科大学 Heparin-12-sugar, preparation method thereof and application thereof in resisting vascular smooth muscle cell proliferation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
李巍等: "肝素十二糖对血管平滑肌细胞增殖作用的研究", 《中国临床药理学与治疗学》 *

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