CN1483473A - New application of recombinant hirudin - Google Patents

Abstract

The invention discloses a new application of recombinant hirudin, specifically the application in the preparation of medicaments for preventing and treating cataract diseases.

Description

New application of recombinant hirudin

technical field

The present invention relates to the new application of recombinant hirudin, especially the application in the field of pharmacy.

Background technique

Hirudin is a protein containing 65-66 amino acids extracted from the salivary glands of medical leeches. It is a single-chain polypeptide with a molecular weight of 7000u. The secondary structure makes this domain stable, and the linear C-terminal sequence is used to bind thrombin. As a specific inhibitor of thrombin, it has strong anticoagulant activity, can effectively prevent and treat disseminated intravascular coagulation, and has a significant effect on diseases such as cardiovascular thrombosis and cerebral thrombosis. Due to the limited source of natural hirudin, bioengineering technology is widely used to prepare recombinant hirudin. The two most widely used cell expression systems are Escherichia coli (E.coli) and Saccharomyces cerevisiae (S.cerevisiae). The preparation method of recombinant hirudin is disclosed in CN1319671A. Clinically, recombinant hirudin is mostly used in the treatment of unstable angina (USA), acute myocardial infarction (AMI), angioplasty, postoperative thrombosis, hemodialysis, extracorporeal circulation, disseminated intravascular coagulation (DIC) etc. (MarkwardtF .Hirudin as an inhibitor of thrombin[J].Methods in Enzymelogy, 1970, 19: 924). Clinical experiments have shown that recombinant hirudin does not cause allergies, immune reactions, and does not cause circulatory dysfunction. But there is no pharmacological report and clinical application of prevention and treatment of cataract diseases.

Contents of the invention

The invention discloses a new application of recombinant hirudin, in particular the application of recombinant hirudin in the preparation of medicines for preventing and treating cataract diseases.

During the occurrence of cataract, there are various biochemical metabolic changes in the lens: such as accelerated lipid peroxidation, increased activity of aldose reductase, decreased activity of superoxide dismutase and glutathione reductase, and decreased ATP concentration. Decrease, some amino acids, vitamins and reducing substances decrease, electrolyte balance disorder and crystal protein polymerization and structural properties change. Therefore, there are many ways to prevent and treat cataracts: such as using antioxidants or antioxidant activators to remove or neutralize harmful damage to the crystals such as ultraviolet rays, ionizing radiation, chemical substances, and metabolites, supplementing vitamins, coenzymes (NADP) , glutathione and ATP, etc., to reduce the changes in biochemical metabolism in the crystal, and to protect the protein from damage caused by the formation of adducts of salty chemical substances by blocking some active groups of the protein. In a pharmacological test, to test whether a drug is beneficial to improve cataract, usually to test whether the drug can increase the activity of SOD and decrease the content of MDA in the lens.

Our experiments found that recombinant hirudin has no irritation to the eyes, can significantly delay the occurrence of cataract, reduce the degree of lens turbidity, reduce the content of MDA, and increase the activity of SOD. It has a significant preventive effect on cataracts.

In order to better exert the curative effect, the recombinant hirudin is prepared into common pharmaceutical preparations, and the preferred preparations are eye drops, eye ointments, and injections. In the preparation of eye drops, in addition to the buffer solution, non-irritating absorption enhancers, thickeners, isotonic agents, and bacteriostatic agents can also be added. The dosage of topical eye drops is generally 0.001-0.01g/day, and it can also exceed the dosage range according to different situations.

The following are some pharmacological tests and data of the present invention, and the recombinant hirudin used is prepared according to CN1319671A method:

1. Multiple eye irritation test Test animals: 4 rabbits, 1.8-2.3Kg. Nanjing Anlimo Technology Co., Ltd., license number: SCXK (Su) 20020-0002 Test method: According to the standards of the Ministry of Health of the People's Republic of China in the "Compilation of Guiding Principles for Preclinical Research", recombinant hirudin eye drops (0.5g /100ml) into the conjunctival sac. Then lightly close the eyelids for about 10 seconds to ensure sufficient contact between the test substance and the local area. The other eye was instilled with solvent into the conjunctival sac as a control.

Observation period and observation indicators: Eye examinations were performed at 1, 24, 48, and 72 hours, and the observation was continued for 7 days. In addition to conjunctiva, cornea, and iris should be observed, other observed damage should also be recorded and reported. For each examination, the ocular response score should be recorded. Test results: Add up the irritant response scores of the cornea, iris and conjunctiva of each animal to get the total score, divide the total score of a group by the number of animals to get the final score, see the table below.

The final score of eyes of each group at each observation time

Test animal group 1h 24h 48h 72h 4d 5d 6d 7d

Eye 0 0 0 0 0 0 0 0 0

The eye of the control side 0 0 0 0 0 0 0 0 0 0 0 0 0 Test conclusion: the recombinant hirudin eye drops did not irritate the cornea, iris and conjunctiva of the eye of the administration side, which was the same as the eye of the control side; the recombinant hirudin eye drops The agent is non-irritating to the eyes.

2. Experiment of prevention and treatment of cataract in rats Experimental animals: 60 28-day-old SD rats, weighing 53.5-68 g, half male and half male. Provided by the Experimental Animal Center of China Pharmaceutical University, certificate number: SCXK (Su) 2001-001. Test method: 60 28-day-old SD rats, half male and half male, were randomly divided into 6 groups, 10 rats in each group: normal control group (normal saline), model control group (only modeling without administration), positive control group (Bai Nei Ting Pir: 0.4mg/kg), intraconjunctival sac infusion of high, medium and low dose groups (recombinant hirudin 1.68mg/kg, 0.84mg/kg, 0.42mg/kg). The administration volume of each group was 0.25ml/50g. Except for the normal control group, the other groups were intraperitoneally injected with 0.08% D-galactose solution, equivalent to 20mg/kg, once a day, for 14 consecutive days, with a volume of 25ml/kg, and drank 10% D-galactose solution. lactose solution. On the fourth day after galactose cataract was induced, high-dose (1.68mg/kg), medium-dose (0.84mg/kg) and low-dose (0.42mg/kg) recombinant hirudin (5 times/day) were administered to both eyes respectively. From the 3rd day, the turbidity of the rat lens was observed with an ophthalmoscope, and the changes of the lens were recorded on the 8th and 14th day. After the experiment, the rat lens was taken to measure the MDA content and SOD activity in the lens.

The results of lens observation were statistically analyzed by Ridit method; in the results of MDA and SOD measurement, each group was compared with the cataract model group by t-test between groups.

test results:

1. The effect of recombinant hirudin on the lens changes of rats induced by D-galactose. See Table 1 and Table 2 for the results.

Table 1. Effect of recombinant hirudin on lens turbidity in cataract rats (Day 8) (n=10)

Group Dose (mg/kg) / + + ++ + +++ Total

Negative group ---- 10 0 0 0 ^** 10

Model group ---- 0 0 2 8 10

Pir 0.4 8 2 0 0 ^** 10

High dose rH 1.68 7 3 0 0 ^** 10

Medium dose rH 0.84 8 2 0 0 ^** 10

Low dose rH 0.42 8 1 1 0 ^** 10

Total 41 8 3 8 60/: No turbidity +: Slight turbidity ++: Moderate turbidity +++: Severe turbidity ^** means u＞2.58, compared with the model group

      Table 2. Effects of recombinant hirudin on lens turbidity in cataract rats (Day 14) (n=10)

Group Dose (mg/kg) / + + ++ ++ +++ Total

Negative group ---- 10 0 0 0 ^** 10

Model group ---- 0 0 1 9 10

Pir 0.4 9 1 0 0 ^** 10

High dose rH 1.68 8 2 0 0 ^** 10

Medium dose rH 0.84 7 3 0 0 ^** 10

Low dose rH 0.42 8 1 1 0 ^** 10

Total 42 7 2 9 60/: No turbidity +: Slight turbidity ++: Moderate turbidity +++: Severe turbidity ^** means u＞2.58, compared with the model group

The results showed that: the lens turbidity of each administration group was significantly different from that of the model group. 2. Comparison of SOD activity in the lens of rats in each group: the results are shown in Table 3

    Table 3 The effect of recombinant hirudin on the activity of SOD in the lens of cataract rats ( x±S) (n=10)

Group Dose (mg/kg) SOD (NU/mgprot)

Negative group ---- 137.2±13.7 ^**

Model Group ---- - 37.5±19.0

pir 0.4 133.5±31.3 ^**

High dose rH 1.68 130.4±24.9 ^**

Medium dose rH 0.84 123.6±18.8 ^**

Low dose rH 0.42 122.2±19.2 ^{** **} P＜0.01, compared with model group

The results showed that both the recombinant hirudin high-dose group and the positive drug Beinetin could significantly increase the SOD activity, which was significantly different from the model control group (p<0.01), and was positively correlated with the dose.

3. Comparison of MDA content in lens of rats in each group: see Table 4

    Table 4 Effects of recombinant hirudin on MDA content in the lens of cataract rats ( x±S) (n=10)

Group Dose (mg/kg) MDA (nmol/mgprot)

Negative group ---- 6.34±1.19 ^**

Model Group ---- - 24.29±4.72

pir 0.4 14.81±2.91 ^**

High dose rH 1.68 15.14±3.32 ^**

Medium dose rH 0.84 16.19±3.27 ^**

Low dose rH 0.42 16.87±4.24 ^**

^** P＜0.01, compared with model group

The results showed that the high, medium and low dose groups of recombinant hirudin and the positive drug Beinetin could significantly reduce the content of MDA, which was significantly different from the model control group (p<0.01), and there was a positive correlation with the dose. Experimental conclusion: Recombinant hirudin can significantly delay the occurrence of cataract, reduce the degree of lens turbidity, reduce the content of MDA, and increase the activity of SOD. Recombinant hirudin has a significant preventive effect on galactose-induced cataract in rats.

The specific embodiment of recombinant hirudin eye drops formula and preparation method thereof:

Recombinant Hirudin 0.25g

Boric acid 1.116g

Borax 0.191g

Sodium chloride 0.22g

EDTA 0.5g

Chitosan 0.5g

Ethyl p-hydroxybenzoate 0.03g

Add distilled water to 100ml

Boric acid, borax and water are added to prepare a buffer solution with a pH of 7.4, and the buffer solution is used to dissolve recombinant hirudin, EDTA, chitosan, sodium chloride, and hexyl p-hydroxybenzoate, mix well, filter, sterilize and subpackage to obtain The recombinant hirudin eye drops of the present invention.

Claims (1)

1. The application of lepirudin 023 ludon in the medicine of preparation prevention and treatment cataract disease.