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Staphylokinase intramuscular injection and method for preparing the same

A technology of staphylokinase and injection, which is applied in the field of intramuscular injection of thrombolytic drugs and its preparation, and can solve problems such as hindering the repeated use of Sak

Inactive Publication Date: 2011-04-20
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Neutralizing antibodies appeared in most patients two weeks after administration, and the antibody level gradually increased, which could last for several months, thus hindering the repeated use of Sak (Declerck PJ et al.Thromb Haemost.71.129-133(1994))

Method used

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  • Staphylokinase intramuscular injection and method for preparing the same
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  • Staphylokinase intramuscular injection and method for preparing the same

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preparation example Construction

[0087] A kind of preparation method of preferred staphylokinase intramuscular injection comprises the steps: appropriate amount of staphylokinase and phosphate are dissolved in distilled water, make the staphylokinase intramuscular injection containing the phosphate of 0.5-40mg / ml staphylokinase and 3-15mM , adjust the pH to a suitable range (such as 6.5-7.5), then divide into suitable containers (such as 1ml, 2ml, 5ml or 10ml ampoules, etc.), and freeze-dry.

[0088] The active ingredient staphylokinase that can be used in the staphylokinase intramuscular injection of the present invention can be natural staphylokinase or recombinant staphylokinase, preferably recombinant staphylokinase; Described recombinant staphylokinase includes various mutants produced by gene mutation, and PEGylated Various derivatives of staphylokinase. Especially preferred are low antigenicity staphylokinase mutants.

[0089] A preferred method for preparing staphylokinase intramuscular injection com...

Embodiment 1

[0100] Preparation of Recombinant Staphylokinase with Low Antigenicity

[0101] The results of bioinformatics analysis of staphylokinase showed that Sak contains three non-overlapping antigenic determinants, and the specific positions of two antigenic determinants have been elucidated. In this example, the deletion of the staphylokinase gene leads to changes in its spatial structure, thereby affecting the antigenicity of the glucosylase, and obtaining a low-antigenic staphylokinase.

[0102] Now take the deletion of 21 amino acids at the N-terminal of natural staphylokinase as an example.

[0103] Construction of recombinant staphylokinase gene

[0104] Primers were designed as follows:

[0105] P15’-CAC GAA TTC ATG GGC CCG TAT TTG ATG GTA-3’

[0106] P25’-CAC GGA TCC TTA TTT CTT TTC ATA AAC AAC CTT-3’

[0107] Obtain the mutant gene by PCR, and the reaction steps of PCR are:

[0108] After adding DNA template, buffer, dNTP, primers P1 and P2, make up 50 microliters with ...

Embodiment 2-3

[0122] Preparation of Recombinant Staphylokinase with Low Antigenicity

[0123] A method similar to Example 1 was used to obtain low antigenic staphylokinase that lacked 20 and 22 amino acids at the N-terminal of natural staphylokinase, and the mutant genes were respectively Δ20-Sak (SEQ ID NO: 1) and Δ22-Sak (SEQ ID NO :3); its amino acid sequences are respectively SEQ ID NO: 4 (116 amino acids, molecular weight about 13200 Daltons) and SEQ ID NO: 6 (114 amino acids, molecular weight about 13000 Daltons).

[0124] The determination of low antigenicity was carried out separately, and the results are shown in figure 2 and image 3 .

[0125] The results showed that the staphylokinase obtained from the mutant strain Δ20 and the mutant strain Δ22 had the same low antigenicity as the staphylokinase obtained from the mutant strain Δ21.

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Abstract

The present invention discloses a staphylokinase intramuscular injection and a preparation method thereof; the staphylokinase intramuscular injection contains: (a) 0.5 to 40mg / ml of staphylokinase; (b) 3 to15mM of phosphate. The present invention has easy usage and can be used for treatment and prevention. The present invention also discloses a low-antigen recombinant staphylokinase and the preparation method and usage.

Description

technical field [0001] The invention relates to the field of pharmaceutical preparations, in particular to an intramuscular injection of a thrombolytic drug and a preparation method thereof. Background technique [0002] The drugs currently used for thrombolysis mainly include urokinase (uk), streptokinase (sk) and tissue plasminogen activator (t-PA). It is used in the treatment of thrombolysis by intravenous infusion or bolus injection, or a combination of bolus injection and infusion. There are also methods such as direct injection around the embolus of blood vessels, so that the blood drug concentration reaches a peak in a short time. It can play a role in time for the treatment of acute thrombosis such as myocardial infarction, so as to save valuable time and obtain therapeutic effect. However, it is not suitable for the treatment of chronic arteriovenous thrombosis due to the inconvenience of long-term infusion. [0003] The new thrombolytic drug Staphylokinase (Sak)...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K9/08A61P7/02A61K38/43
Inventor 王家驯李武军
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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