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PolyHb-rPA complex, preparation method and application of PolyHb-rPA complex

A complex, hemoglobin technology, applied in the field of biomedicine, can solve the problems of restricting the widespread use of r-PA, improper administration of bleeding, and high price of t-PA, achieving the possibility of increasing the dosage of drugs, reducing the risk of bleeding, low immunogenicity

Active Publication Date: 2013-04-17
山东圣奥生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the current price of t-PA is relatively high, and because of its short half-life in vivo (11-19 minutes, recombinant t-PA is about 5 minutes), in order to maintain the effective concentration after the first intravenous injection, it must be injected again at an interval of 30 minutes. Improper administration will cause the risk of bleeding, which limits the widespread use of r-PA in clinical practice

Method used

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  • PolyHb-rPA complex, preparation method and application of PolyHb-rPA complex
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  • PolyHb-rPA complex, preparation method and application of PolyHb-rPA complex

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: Preparation of PolyHb-rPA complex

[0019] 1. Preparation of stroma-free hemoglobin (Hb)

[0020] Stroma-free hemoglobin is prepared by lysing bovine red blood cells with low osmosis, extracting with toluene, purifying by high-speed centrifugation, and ion-exchange chromatography. The final solution contained 0.1-0.15 g / mL hemoglobin. In order to reduce the formation of methemoglobin, the operation process was carried out at 4°C under a nitrogen atmosphere, and the pH value of the hemoglobin solution was 7.4.

[0021] (1) Separation of bovine red blood cells

[0022] Take fresh bovine blood and put it into several aseptically treated centrifuge tubes, 4000 x g Centrifuge for 10 minutes. After taking out, suck out the upper layer of plasma and middle layer of white blood cells, and mix the lower layer of red blood cells with 0.9% normal saline, 4000 x g Centrifuge for 10 minutes. The supernatant was discarded, and the red blood cells in the lower la...

Embodiment 2

[0030] Example 2: Detection of activity of reteplase (r-PA) and determination of half-life in rats

[0031] 1. Activity detection of reteplase (r-PA)

[0032] Weigh 125 mg of agarose, add 23 mL of normal saline solution, boil to dissolve, and equilibrate in a 60°C water bath; add 5 mL of thrombin (100 u / mL), 100 μL of plasminogen (0.5 mg / mL), add as you go Shake well; add 1 mL of human fibrinogen (5 mg / mL), and shake constantly; pour the plate (diameter 8 cm) immediately after cloudiness, place horizontally and fully solidify, and place at 4°C for at least 30 min before use. The reference substance was diluted with physiological saline to different dilutions. At the same time, the vehicle was used as a blank control. Punch holes (diameter 2 mm) in the formed fibrin plate, spot 10 mL per hole, spot 2 holes for each sample to be tested and standard substance, and place them horizontally in a humid box at 37°C for 24 h. Measure the diameter of the dissolved ring twice vertical...

Embodiment 3

[0037] Example 3: The rapid thrombolytic effect of PolyHb-rPA complex on the formation of thrombus induced in vivo

[0038]1. Protection of PolyHb-rPA collagen-adrenaline-induced thrombosis in mice

[0039] Clean-grade Kunming mice, male, weighing 18-20g (Experimental Animal Center of Shandong University), fully soak collagen in saline, homogenate to a concentration of 1.0 mg / mL, then add epinephrine 40 μg / mL and mix well , which is the inducer; the test drug inducer was formulated into solutions of different concentrations and equal volumes, and injected into the tail vein of mice, and the administration volume was 0.1 mL / 10g (body weight). Immediately after injection, the number of mice that died within 5 minutes and the number of mice that recovered from hemiplegia within 15 minutes were observed. Calculate the protection rate of the drug against cerebral thrombosis in mice, and perform statistical analysis with the exact probability method. The results are shown in the t...

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Abstract

The invention provides a PolyHb-rPA complex, a preparation method and an application of the PolyHb-rPA complex. Reteplase (r-PA) is cross-linked with high-purity bovine hemoglobin (Hb) by glutaraldehyde to form the PolyHb-rPA complex, so that the stability and the half life of the r-PA in vivo after intravenous injection are effectively improved. The volume of the cross-linked complex PolyHb-rPA is hundreds of times smaller than that of red blood cells in the blood, the hemoglobin in the PolyHb-rPA can form capillaries to supply oxygen to embolized ischemic tissues, and the capillaries are narrow at an early stage. After the r-PA is cross-linked with the hemoglobin, the half life is prolonged, the activity is reduced, and the effect is more stable and durable, so that the risk of bleeding is reduced.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a PolyHb-rPA complex and its preparation method and application. The PolyHb-rPA complex is generated by cross-linking bovine hemoglobin and reteplase with glutaraldehyde to prepare a therapeutically significant method of thrombolytic drugs. Background technique [0002] Reteplase (r-PA) is the drug of choice for the treatment of acute myocardial infarction (AMI), r-PA is a part of human tissue plasminogen activator (t-PA), Compared with other thrombolytic drugs, r-PA has the advantages of significant curative effect, rapid onset, convenient use (intravenous injection), and less adverse reactions. However, the current price of t-PA is relatively high, and because of its short half-life in vivo (11-19 minutes, recombinant t-PA is about 5 minutes), in order to maintain the effective concentration after the first intravenous injection, it must be injected again at an interval of 30 minute...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/96A61K47/48A61K38/49A61P7/02
Inventor 谷劲松王革
Owner 山东圣奥生物科技有限公司
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