Method for extending in vivo half life of protein medicine by making red cell as carrier

A protein and red blood cell technology, applied in peptide/protein components, drug combinations, pharmaceutical formulations, etc., can solve problems such as re-embolization and bleeding, and achieve the effects of preventing side effects, reducing side effects, and prolonging the action time

Inactive Publication Date: 2005-06-22
SHANGHAI FUCHUN ZHONGNAN BIOTECH
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AI Technical Summary

Problems solved by technology

However, reactions such as bleeding and re-

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0037] Example 1 Preparation of Hirudin and Red Blood Cell Complex

[0038] 1.1 Biotin labeling of red blood cells

[0039] Collect whole blood, add heparin for anticoagulation, centrifuge to collect red blood cells, wash three times with pH 7.4 phosphate buffer (PBS, 125mM NaCl+20mM PB), and suspend in PBS-G (PBS+5mMglucose) at 10% red blood cell volume. Dissolve NHS-biotin at 200mg / ml in 90% DMSO, then add 0.055mg of NHS-biotin per milliliter of red blood cells to mix, incubate at 37°C for 1 hour, and then wash three times with PBS to obtain labeled red blood cells.

[0040] 1.2 Biotin labeling of hirudin

[0041] Use DMF to make 1mg / ml solution of NHS-Biotin, then use 0.1M, pH 9.0 NaHCO 3 Dilute the hirudin to 1mg / ml. Mix NHS-Biotin and hirudin at a weight ratio of about 1:7, react for 4 hours under stirring at room temperature, put them into a dialysis bag, and dialyze 0.05M pH 7.2 PBS overnight at 4°C to remove unreacted NHS-biotin.

[0042] 1.3 Preparation of hirudin-erythro...

Example Embodiment

[0047] Example 2 Preparation of Hirudin and Red Blood Cell Complex

[0048] 2.1 Biotin labeling of red blood cells

[0049] Same as step 1.1 of Example 1.

[0050] 2.2 Streptavidin labeling of hirudin

[0051] Dissolve the frozen hirudin in 0.1M pH 6.8PB buffer containing 1.25% glutaraldehyde, the final concentration is 10mg / ml, and act overnight at room temperature; put it into a dialysis bag, and dialyze 0.05M pH 7.2PBS at 4℃ 12 hours; take out the dialysate, add 15mg streptavidin and 0.5ml 1M pH 9.5Na per ml hirudin solution 2 CO 3 Put it at 4°C for 24 hours; add 50ul 0.2M lysine to terminate the reaction at room temperature for 2 hours; put it into a dialysis bag and dialyze it against 0.05M pH 7.2PBS at 4°C overnight. Centrifugation removes the macromolecular polymer to obtain streptavidin-labeled hirudin.

[0052] 2.3 The formation of hirudin-erythrocyte complex

[0053] Mix the biotinylated red blood cells with streptavidin-labeled hirudin (calculated based on the combinati...

Example Embodiment

[0056] Example 3 In vivo half-life comparison of hirudin-erythrocyte complex and hirudin alone

[0057] Thirty New Zealand rabbits were divided into six groups and injected intravenously with hirudin or hirudin-erythrocyte complex. The dose (calculated based on the protein weight of hirudin) was high (0.4 mg / kg) and medium (0.2 mg / kg) , Low (0.1 mg / kg) three groups, then 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours after injection Blood was taken, and then the concentration of hirudin was measured by sandwich ELISA. The data obtained is used to calculate T1 / 2 with P87 software (α phase is not calculated, only β phase is calculated). The results showed that the half-life of hirudin fused with red blood cells was greatly extended.

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Abstract

The invention provides a method for extending in vivo half life of protein medicine by making red cell as carrier, which can not only extend the half life of protein medicine, but also reduce medicament consumption, prevent medicament from diffusing to outside the blood vessel, thus reduce the side effects of the medicine. The invention also relates to a protein medicament-erythrocyte compound, the protein medicine employs erythrocyte as carrier to be transferred in vivo.

Description

technical field [0001] The invention relates to a method for prolonging the half-life of protein medicine by using red blood cells as carriers. Background technique [0002] In recent years, due to the popularization and application of genetic engineering, the clinical application of protein drugs (including the treatment of cardiovascular diseases) has become more and more extensive. However, due to their physical and chemical properties, most protein drugs are not stable, and their half-life in the body is very short. Many drugs need to be administered every day. At the same time, due to the presence of proteases in the digestive tract, protein drugs are difficult to be administered orally, and continuous infusion or continuous injection will bring mental and physical torture to patients. Therefore, more and more research institutions are concentrating on the formulation research of protein drugs, mainly long-acting drugs and oral and spray ad...

Claims

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Application Information

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IPC IPC(8): A61K38/43A61K38/58A61K47/54A61P9/00
Inventor 倪健杨子义杨武剑
Owner SHANGHAI FUCHUN ZHONGNAN BIOTECH
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