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Blood brain barrier penetrable erythropoietin (EPO) and application thereof

A technology of erythropoietin and blood-brain barrier, applied in erythropoietin, medical preparations containing active ingredients, cardiovascular system diseases, etc., can solve the problem of increased risk of large infarction, increased hematocrit, and increased micro-infarction To achieve the effect of prolonging the effective time window, reducing the volume of cerebral infarction, and strengthening neuroprotection

Inactive Publication Date: 2010-03-17
曹国栋 +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, EPO has a short half-life in the blood, requiring multiple injections to maintain its neuroprotective effects
Large-dose, repeated injections of EPO may increase the hematocrit [8], stimulate platelet production, increase the risk of micro-infarcts, and increase the risk of large infarcts, thus strictly limiting or even preventing the role of EPO in stroke. application in therapy

Method used

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  • Blood brain barrier penetrable erythropoietin (EPO) and application thereof
  • Blood brain barrier penetrable erythropoietin (EPO) and application thereof
  • Blood brain barrier penetrable erythropoietin (EPO) and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: carrier preparation

[0028] 1. Amplification of human EPO cDNA

[0029] Human EPO cDNA encodes 193 amino acids. After the EPO primary protein is synthesized, the signal peptide containing 27 amino acid residues at the amino terminal becomes mature EPO after cleavage. Mature human EPO is thus 166 amino acids. Recent studies have found that the 166th amino acid arginine is also cleaved. Based on the above research results, when we constructed the fusion protein containing the HIV TAT transduction sequence, we added the HIV TAT and the histidine tag used for isolation and purification to the 3' end of the EPO cDNA, and at the same time added the 166th arginine delete. Human EPO cDNA containing the HIVTAT tag was amplified by PCR from a human kidney Marathon-ready cDNA library (Clontech, USA). Primers were designed according to the published human EPO cDNA sequence (gene sequence number: NM000799). The upstream sequence contains EcoRV and KOZAK sequence ...

Embodiment 2

[0032] Embodiment 2: for the establishment of the Chinese hamster ovary (CHO) stable cell line expressing EPO-TAT fusion protein and the preparation and purification of EPO-TAT fusion protein thereof

[0033] 1. Establishment of CHO stable cell lines expressing EPO-TAT fusion protein

[0034] After the vector pEPO-TAT-DHFR was linearized with XhoI enzyme, it was recovered and purified. Using the transfection reagent Lipofectamine 2000 (Invitrogen, USA), the linearized plasmid was introduced into the DHFR-deleted CHO cell line DG44 (gifted by Duke University). After 24 hours of transfection, subculture at 1:12, change to nucleotide-free α-MEM medium (Invitrogen, USA) for culture, and add 5pM methotrene at the same time, select a single clone and transfer it to a 6-well plate after 2 weeks to continue to cultivate. Take 10 μl of culture medium and use Western blot or ELISA to identify EPO-TAT high-expressing cell lines after diluting 1000 times. Take six of the EPO-TAT high-e...

Embodiment 3

[0037] Example 3: Detect whether TAT-mediated EPO can increase EPO passing through the blood-brain barrier

[0038] The purpose of our preparation of TAT-labeled EPO is to improve its penetration of the blood-brain barrier and increase the therapeutic concentration of EPO in the brain. To further prove this problem, we intraperitoneally injected EPO-TAT or wild-type EPO (unfused TAT) to adult rats at a dose of 5000U / kg body weight. After 3 hours of injection, the plasma and cerebrospinal fluid were collected and the EPO content was determined using an ELISA kit (R&D System), the sensitivity of which was 0.8mU / ml. We found no difference in the sensitivity of this kit for measuring EPO-TAT and wild-type EPO. As shown in Figure 3, the content of EPO-TAT in blood was significantly higher than that of wild-type EPO. The monitoring results of cerebrospinal fluid samples showed that the efficiency of EPO-TAT passing through the BBB was significantly higher than that of wild-type EP...

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Abstract

The invention discloses a blood brain barrier penetrable erythropoietin (EPO) which belongs to the field of biological pharmacy. The blood brain barrier penetrable EPO is characterized by being connected with a PTD with a protein transduction function at the carboxyl tail end of a wild type EPO or a derivative of the wild type EPO, and the PTD is selected from the following components: (1) HIV TAT; (2) multi-polyarginine comprising 5 to 10 arginine; (3) an isogeny structure domain of a fruit fly control antenna gene; and (4) herpes simplex virus VP22. The novelty of the invention is in the following two advantages of the EPO-TAT compared with the wild type EPO: first, the EPO-TAT can obviously reduce the needed dosage of the EPO for treating cerebral apoplexy, thereby obviously lighteningthe side effect of the EPO; and second, the EPO-TAT can prolong the treatment time of the cerebral apoplexy, thereby more patients with the cerebral apoplexy are treated.

Description

technical field [0001] The invention relates to an erythropoietin and its application, in particular to a blood-brain barrier-penetrating erythropoietin and its application, belonging to the field of biopharmaceuticals. Background technique [0002] In recent years, erythropoietin (EPO) has become one of the most promising drugs in the neuroprotective treatment of ischemic stroke. In studies of ischemic injury models both in vitro and in vivo, EPO has been shown to have a strong neuroprotective effect. Recently, a clinical trial using EPO in the treatment of acute stroke proved that: EPO can reduce the size of cerebral infarction in stroke patients and promote the recovery of neurological function. Therefore, in the treatment of stroke, especially for patients who miss the treatment time window and are not suitable for treatment with tissue plasminogen activator, EPO shows a potentially encouraging clinical application prospect. However, the clinical application of EPO is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/505A61K38/18A61P9/10A61P25/00A61P11/00A61P9/00A61P43/00
Inventor 曹国栋陈俊罗玉敏吉训明邢娟
Owner 曹国栋
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