CN102258771A - Application of recombinant hirudin in preventing and treating diabetic cataract - Google Patents
Application of recombinant hirudin in preventing and treating diabetic cataract Download PDFInfo
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- CN102258771A CN102258771A CN2011101954954A CN201110195495A CN102258771A CN 102258771 A CN102258771 A CN 102258771A CN 2011101954954 A CN2011101954954 A CN 2011101954954A CN 201110195495 A CN201110195495 A CN 201110195495A CN 102258771 A CN102258771 A CN 102258771A
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Abstract
The invention discloses new application of recombinant hirudin, and in particular relates to application of recombinant hirudin in preventing and treating diabetic cataract.
Description
Technical field
The invention belongs to biotech medicine product field or protein and peptide drug world, relate to and adopt the application of hirudin in the control diabetic cataract.
Background technology
The diabetes serious harm mankind's is healthy, and diabetic cataract (diabetic cataract DC) then is a kind of main diabetic complication and blinding oculopathy.Along with living standard improves constantly and the population senescence, global diabetics day by day increases, and diabetic cataract patient number also increases severely thereupon, according to statistics among China's type ii diabetes patient cataract incidence up to 62.37%.
Though cataract can be extractd by operation, posterior lens capsule film muddiness complication such as (after cataract) causes in operation back regular meeting.Even more noteworthy, the diabetics cataract often is not suitable for this operative treatment, because this posterior lens capsule film muddiness complication such as (after cataract) in many corrective surgeries treatment back can be particularly serious.Therefore, the Drug therapy of diabetic cataract receives Medical Technologist's concern always, and especially at the initial stage of cataract morbidity, doctors tend to the choice drug treatment, develop with the relief of symptoms disease controlling.Therefore, seek effective diabetic cataract control medicine and have important application value.
Hirudin is a kind of natural anticoagulant protein and peptide, is made of its molecule such as Tinea Ranae shape 65～66 aminoacid.The hirudin molecule is made up of 2 domains, they respectively with two different surface site effects of thrombin molecular surface.Its N-end structure territory is the core texture of a spherical densification that is formed by amino acid/11-47, and it combines with the avtive spot (active site) of thrombin, suppresses its catalysis activity; Its electronegative carboxyl terminal domain aminoacid (49-65/66) then combines site, the outside (anion binding exosite with the anion of thrombin, or title Fibrinogen recognition site) combination, thereby Trombin inhibiting and fibrinogenic interaction.Between these two domains, exist a finger (finger like structure) from complex, to give prominence to and stretch out, do not contact with thrombin.This finger is that hirudin amino acid residue 27-31 and 36-40 form an antiparallel beta sheet (chain of antiparallel β-sheet), finger tip forms flexible β-corner by amino acid residue 32-35, studies show that this finger does not participate in the inhibitory action of thrombin, belongs to the not function required area.
Hirudin is the most strong thrombin inhibitor of finding at present, and it can suppress tremulous pulse, venothrombotic formation and disseminated inravascular coagulation (DIC) effectively, has high medical value aspect the anti-treatment of cardiovascular and cerebrovascular vessel thrombotic disease.Because hirudin can play inhibitory action (heparin can only act on free thrombin) to free thrombin and the thrombin that is combined on the thrombosis, the thrombocytopenia side effect that does not have heparin allergy to cause simultaneously, so the action effect of hirudin is better than heparin.In addition, hirudin all has huge potential clinical value at aspects such as control unstable angina pectoris, disseminated inravascular coagulation, brain blood coagulation, thrombophlebitis and crown arterial thrombus.
(Chinese invention patent such as Wu Wutong, the patent No.: 03132224.7) pharmacological testing proof lepirudin 023 ludon is to all having remarkable prevention effect with external model in D-galactose and the inductive animal cataract of the sodium selenite body, not experimental results show that lepirudin 023 ludon is to streptozotocin (streptozotocin, STZ) prevention effect of inductive diabetic cataract model but see.
Summary of the invention
The invention discloses a kind of new purposes of lepirudin 023 ludon, especially the application in the control diabetic cataract.
One, experiment material
1. be subjected to the reagent product: lepirudin 023 ludon (III) eye drop, be colourless transparent liquid, its compound method is seen the Chinese invention patent (patent No.: 03132224.7).Be made into 2000U/ml, 1000U/ml and 500U/ml respectively with hirudin eye drop substrate, in 4 ℃ of refrigerator cold-storages.
2. positive control medicine: Bernetine Sodium eye drop (catalin), (lot number: 101015), specification: every contains Bernetine Sodium 0.8mg to Wuhan Yuanda Pharmaceutical Group Co., Ltd.'s product, in 4 ℃ of refrigerator cold-storages.
3. laboratory animal: male SPF level SD rat, body weight 130 ± 20g amounts to 120, purchases in west, Shanghai pul-Bi Kai laboratory animal company limited (quality certification number: SCXK (Shanghai) 2008-0016).The experimental mouse pellet is raised, and freely drinks water.
4. other reagent: STZ (U.S. Sigma company provides, China CAS NO.18883-66-4 Pcode:1000830091.); Citric acid (Shanghai chemical reagents corporation of Chinese Medicine group, lot number: 20000801); Sodium citrate (Shanghai reagent four factories, lot number: 080330); Tropicamide and Phenylephrine eye drop (Shenyang Xing Qi Pharmaceutical Co., Ltd, lot number: 110102); Total superoxide dismutase (T-SOD) testing cassete, malonaldehyde (MDA) testing cassete, Coomassie brilliant blue protein determination kit are purchased and are built up bio-engineering research institute in Nanjing.
5. main experimental apparatus: YZ5E type slit lamp microscope (Suzhou six or six vision limited companies)
Two, part pharmacology test data of the present invention.
1.STZ preparation: get 0.21g citric acid adding 10ml distilled water and be made into citric acid solution, get 0.29g sodium citrate adding 10ml distilled water and be made into sodium citrate solution.Get the 100ml buffer solution that above-mentioned citric acid solution 1.1ml and sodium citrate solution 0.86ml are mixed with PH=4.5.Get buffer solution 70ml, add STZ1.4g and fully dissolve, be mixed with 2%STZ solution [1].
2. the therapeutical effect of diabetic cataract model preparation and lepirudin 023 ludon (III) eye drop
Rat fasting 24h can't help water, is divided into 2 groups of normal control group and model group.The above-mentioned buffer solution of rats in normal control group lumbar injection (65mg/kg), model group rats by intraperitoneal injection 2%STZ (65mg/kg, 0.325ml/100g) solution.Injection finishes, normal raising rat.Behind the 72h, rat fasting blood-glucose＞11mmolL
-1Be diabetes model success standard, after 2 weeks of modeling rat used Tropicamide and Phenylephrine eye drop mydriasis, with the muddy degree of slit lamp microscope observation rat lens, record result
[1]According to lenticular muddy degree, the modeling rat is divided into 5 groups at random, make different muddy degree rats can be evenly distributed to model group, positive drug group, high dose group, middle dosage group, low dose group; Other establishes one group is the normal control group, and rat is divided into 6 groups, every group of 16 rats.High dose group, middle dosage group, low dose group gives 2000U/ml respectively, 1000U/ml, 500U/ml lepirudin 023 ludon III eye drip, the positive drug group gives the catalin eye drip, is every day 3 times, each 1.After 4 weeks of administration, use Tropicamide and Phenylephrine eye drop mydriasis, with the muddy degree of slit lamp microscope routine observation rat lens, observe back record result.The lenticular opacity degree is given different score values respectively by table 1 standard, organizes a t check
[2,3]
The muddy degree standards of grading of table 1. rat lens
After 2 weeks of modeling, model group, lepirudin 023 ludon III senior middle school low dose group, positive drug group lenticular opacity degree is compared with the normal control group, and utmost point significant difference (p＜0.01) is arranged.Normal control group crystalline lens is transparent, and other are respectively organized crystalline lens and obvious cavity all occurs, shows that rat diabetes cataract model prepares successfully (see Table 2 and Fig. 1).
The muddy degree of 2 week of table 2. modeling back rat lens (
N=16)
Annotate:
*P＜0.05,
*P＜0.01vs normal control group
After 4 weeks of administration, lepirudin 023 ludon III senior middle school low dose group is compared with model group lenticular opacity degree, utmost point significant difference (p＜0.01) is arranged, senior middle school's low dose group is compared with the positive drug group, there is utmost point significant difference (p＜0.01) to show that each dosage group can significantly alleviate lenticular opacity, and is better than the curative effect (seeing Table 3) of positive drug.
4 week of table 3. administration back lepirudin 023 ludon III is to the influence of the muddy degree of rat lens
Annotate:
*P＜0.05,
*P＜0.01vs model group;
#P＜0.05,
##P＜0.01vs positive drug group
The transparent clarification of the crystalline lens of normal control group; The crystalline lens peripheral cortical of model group and nuclear are muddy, have reached the cataractous stage of the degree of depth; Low dose group crystalline lens peripheral cortical muddiness, comparing model group has certain curative effect, similar to positive drug; Middle dosage group crystalline lens has medium cavity, and high dose group has tiny cavity, and it is evident in efficacy to compare model group, and is better than the positive drug group.(see figure 2)
After giving lepirudin 023 ludon III4 week, eyeball is got in rat execution, isolated crystalline lens.Detect rat lens SOD activity, MDA content with superoxide dismutase (SOD) test kit, malonaldehyde (MDA) test kit.With model group relatively, high dose group, middle dosage group and low dose group be extremely remarkable increased SOD activity (p＜0.01) all; High dose group, middle dosage group can extremely significantly reduce MDA content (p＜0.01), and low dose group can significantly reduce MDA content (p＜0.05), show that lepirudin 023 ludon has therapeutical effect preferably to diabetic cataract.Compare with positive controls, high dose group energy increased SOD activity (p＜0.05) shows that the effect of high dose lepirudin 023 ludon is better than catalin (seeing Table 4).
Table 4. lepirudin 023 ludon (III) is to the influence of rat lens SOD activity, MDA content
Annotate: * p＜0.05, compare with model group * * p＜0.01; ##p＜0.05, compare with positive group ##p＜0.01
Three, experiment conclusion: rat diabetes cataract experimental result shows, give (1000U/ml) low (500U/ml) dosage eye drip in the lepirudin 023 ludon height (2000U/ml), back all around slit lamp observation lenticular opacity degree is also measured the SOD activity and MDA content, the result shows, lepirudin 023 ludon can obviously alleviate the lenticular opacity degree, the development of diabetes-alleviating cataract symptom, increase the SOD activity, reduce MDA content, significantly improve the rat diabetes cataract order of severity, effect is better than positive drug (catalin).
Rat cataract crystalline lens after two weeks of Fig. 1 modeling: 1 is the normal control group, and 2 is model group, 3 positive medicine groups, and 4 is high dose group, and 5 is middle dosage group, and 6 is low dose group
Rat cataract crystalline lens after 4 weeks of Fig. 2 administration: 1 is the normal control group, and 2 is model group, 3 positive medicine groups, and 4 is high dose group, and 5 is middle dosage group, and 6 is low dose group
List of references
[1] Ding Zhenghua, Yan Hong, Guo Yong, breathe out gentle and quiet, Wang Jianwei. streptozotocin causes the pathogenesis [J] of rat diabetes cataract. The Fourth Military Medical University's journal (J Fourth Mil Med Univ), 2006,27 (13): 1208-1210.
[2] Bian Xiaoyun, Zhang Yachao, Song Xiujun. taurine is to the experimentation [J] of diabetic cataract control. ophthalmology research, 2000,18 (4): 302-304.
[3] cold non-, Guan Linan, Liu Ping, higher-dimension is strange, Li Zhijian, Ge Hongyan, Zhang Jiaying. and the salvianolic acid B eye drip is to the influence of rat diabetes cataract. Harbin Medical University's journal, 2009,43 (3): 249-251.
Claims (1)
1. the application of lepirudin 023 ludon in prevention and treatment diabetic cataract.
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Cited By (2)
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| US8685462B1 (en) | 2012-09-17 | 2014-04-01 | Biopep Solutions, Inc. | Whole, leech saliva product and applications thereof |
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2011
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| US8685462B1 (en) | 2012-09-17 | 2014-04-01 | Biopep Solutions, Inc. | Whole, leech saliva product and applications thereof |
| US8790711B2 (en) * | 2012-09-17 | 2014-07-29 | Biopep Solutions, Inc. | Treating diabetes with a whole, leech saliva extract |
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| US9597361B2 (en) | 2012-09-17 | 2017-03-21 | Biopep Solutions, Inc. | Treating a bacterial skin infection with a whole, leech saliva extract |
| US10064897B2 (en) | 2012-09-17 | 2018-09-04 | Biopep Solutions, Inc. | Treating a bacterial skin infection with a whole, leech saliva extract |
| CN103071186A (en) * | 2012-12-19 | 2013-05-01 | 华南理工大学 | Surface-modified artificial lens with coating layer carrying drug and preparation method thereof |
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Application publication date: 20111130 |