JPS62429A - Plasminogen activation factor preparation originated from human uterus - Google Patents

Abstract

PURPOSE:To obtain a drug preparation containing plasminogen activation factor originated from human uterus (uTPA), having remarkably improved stability and keeping the stabilizing effect even after freeze-drying, by adding a polyoxyethylene hardened castor oil derivative to uTPA. CONSTITUTION:A polyoxyethylene hardened castor oil derivative is added to uTPA produced by genetic recombination technique or collected from the tissue of human uterus. The amount of the derivative is preferably 0.001-0.1W/V% based on the uTPA solution. uTPA can be produced e.g. by disintegrating human uterus tissue in the presence of an RNA decomposition enzyme inhibitor, centrifuging the product to separate whole RNA, separating a fraction containing uTPA-specific mRNA from the RNA, preparing a cDNA library using an RNA recovered from the above fraction, constructing a uTPA cDNA therefrom, inserting into a proper expression vector, transforming the obtained expression vector to a proper host cell and culturing the cell.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention relates to a human uterine tissue-derived plasminogen activator preparation, and in particular to human uterine m# plasminogen activator produced by genetic recombination technology. The present invention relates to factor preparations.

(Prior art) In recent years, a tissue plasminogen activator that is structurally and immunologically different from urokinase and has even stronger fibrin affinity has been discovered, and its application as a thrombolytic agent superior to n1 urokinase is expected. ing.

The present inventors attempted to produce, purify, and formulate a human uterine tissue-derived plasminogen activator in large quantities by genetic recombination technology.

(Problems to be solved by the invention) However, the uTPA produced by genetic recombination is unstable and gradually loses its biological activity.In addition, its biological activity is lost when it is frozen and thawed or freeze-dried. The decrease in clinical activity was significant, and it was necessary to implement some kind of stabilization method to provide uTPAl produced by genetic engineering as a pharmaceutical product.

(Means for Solving the Problems) The present inventors have conducted various studies on the stabilization method of uTPA, but surprisingly, adding a polyoxyethylene hydrogenated castor oil derivative to the uTPA solution significantly stabilized the uTPA solution. Does sex improve? By further adding a polyoxyethylene hydrogenated castor 7 oil derivative and freeze-drying, the activity of uTPA during freeze-drying was significantly reduced.
The present invention was completed based on the new knowledge that the plant is settled.

That is, the present invention is a human uterine tissue-derived plasminogen activator preparation containing 2% of human uterine tissue-derived glasminogen activator 2 and a polyoxyethylene hydrogenated castor oil derivative. Contains uTPA taken from human uterine tissue-derived plasmino-miya tissue.

uTPA produced by genetic recombination technology! :Ha, u
UTPA is produced by E. coli, yeast, animal cells, etc. into which a vector containing the TPA gene has been inserted.

The uTPA used in the present invention is produced, for example, as follows:
After disrupting in the presence of an NA-degrading enzyme inhibitor, total RNA1 was isolated by centrifugation.

■ Metsenger RNA was extracted from the obtained human uterine tissue total RNA using oligo dT affinity column chromatography.
(mRNA) was isolated, and the mRNA was further size-fractionated by silosaccharide density gradient sedimentation. A fraction containing uTPA-specific mRNA1 was obtained by the Northern IL method.

■ uTPA-specific mRNA identified as above
RNA (z) is recovered from the fraction containing f, single-stranded cDNA corresponding to the RNA is produced using reverse transcriptase, and double-stranded cDNA is produced from the heavy-chain cDNA using DNA polymerase.
Ai was created.

(2) The obtained double-stranded cDNAi S I was treated with euclease, and then an oligo dc tail was added to the terminal end to prepare cDNAf having an oligo da end.

■ The oligo dc-terminated cD obtained as above.
The NA was inserted into a linear pBR 822 plasmid with an oligo-dG end. Using the obtained vector 1, Escherichia coli was transformed to prepare a cDNA library.

■ From the cDNA library prepared as described above, positive cDNA was isolated using the colony hybridization method.
DNA clones were isolated.

One plasmid DNA was isolated from the isolated clone.
The sequence of A was determined.

■ Multiple Cs obtained as above and whose sequences were determined
DNA? A uTPA cDNA li containing the entire uTPA=r-ding region was constructed by combining cleavage with a DNA side restriction enzyme and ligation with a DNA ligase.

(2) The uTPA cDNA constructed as described above was inserted into an appropriate expression vector by combining cleavage with an appropriate restriction enzyme, modification of the ends, and ligation with a DNA ligase.

■ Does the expression vector related to the present invention have the following DNA sequence? include. In other words, in addition to the uTPA cDNA, all or part of the bovine cyst virus DNA sequence, a part of the pBR822 plasmid DNA, the NA sequence necessary for the expression of the uTPA c DNA, and the entire NA sequence necessary for transcription termination, and in some cases, the expression It also contains an NA sequence that is effective for selecting transformants by the vector.

(2) The expression vector obtained as described above is transformed into a suitable host cell, such as mouse cell C127, and the resulting transformed cells are propagated by culture to obtain the desired uTPA.
A volume of medium was grown in i medium.

The polyoxyethylene hydrogenated castor oil derivative used in the present invention is obtained by addition polymerizing ethylene oxide to hydrogenated castor oil, which is obtained by completely adding hydrogen to the bipartite bond sites in the castor oil constituent molecules. It is a type of nonionic surfactant, for example HCO-400, HCO-60 (Nikko Chemicals)
] is preferably used.

There are no particular regulations regarding the amount of polyoxyethylene hydrogenated castor oil derivative added, but it is preferably 0.00 to the uTPA solution.
1w/v%~0. It is appropriate to add it at lW/'v%.

In the present invention, a polyoxyethylene hydrogenated castor oil derivative is added to the uTPA solution, but it may be added at any step of culturing, purification, freezing or lyophilization of uTPA,
Adding it from the time of culture increases the stability of uTPA,
This is particularly advantageous in terms of increasing the yield.

When providing the uTPA preparation of the present invention as a pharmaceutical, tonicity agents, buffering agents, pH adjusters, soothing agents, excipients, etc. may be added according to conventional methods. The present invention will be specifically explained by giving examples, but the present invention is not limited to these in any way.

Example 1 Mouse C- into which a vector containing the uTPA gene was inserted
Using uTPAi obtained by culturing and purifying 127 cells, a uTPA solution (0.1M phosphate buffer pH 2.0) having an activity of 8000 units/11It was obtained. Add this uTPA solution to polyoxyethylene hydrogenated castor oil and HCO-60 manufactured by Tsuko Chemicals) k O, 01w/
The solution was added to the solution at a volume of 1.5 cm, stored in a polypropylene container at 25°C, and sampled over time to measure residual activity. The residual activity rate (%) was calculated from the obtained measured values and shown in Table 1. As a control, a similar experiment was conducted using a uTPA solution containing polyoxyethylene hydrogenated castor oil.

Nari activity measurement beam, C, Riken S, Bio
chim, Biophys.

Measurements were made using the fibrin clot lysis time method according to Acta 580140 (1979). The activity was determined by comparing the dissolution time of fibrin clots using diluted urokinase as a standard.

Example 2 uTPA' solution C″0 with activity of 8000 units/-
．． 1M phosphate buffer (pH 7.0), polio, diethylene hydrogenated castor oil, HCO-600 (manufactured by Tsuko Chemicals)'
k O,0001,0,001,0,old,0.08+0
．． It was added at a concentration of 1 w/v%. This uTPA solution was prepared in a polypropylene test tube, frozen (-80°C),
Melting was repeated (1 time, 3 times). The residual activity was measured and the residual activity rate (%) is shown in Table 2.

Wc2 table Example 8 The 1 uTPA solution containing polyoxyethylene hydrogenated castor oil prepared in Example 2 was lyophilized by a conventional method @The resulting uTPA lyophilized powder was redissolved and the total residual activity was measured to determine the stabilizing effect. compared. The results are shown in Table 8.

As is clear from the Examples in Table 8 and above, the stabilization method of the present invention allows TPA to be produced by genetic manipulation in a solution state or during each operation such as freeze-thaw, freeze-drying, etc.
can be kept stable.

Therefore, polyoxyethylene hydrogenated castor oil derivatives are u
It is possible to culture TPA and use it in the can manufacturing process, and it is harmless, so it is number 1! It can be used in medicine as a uTPA formulation.

Example 4 uTPA produced by genetic manipulation in the same manner as Example 1
solution (containing 20,000 units of uTPA f in 1)
To polyoxyethylene hydrogenated castor oil, add HCO-60 (manufactured by Tsuko Chemicals) and mannitol to a final concentration of 0.01% and 1.01, respectively, and freeze-dry this to obtain a stable uTPA preparation. I was able to do that.

(Effects of the invention) The present invention uses n-uTPA produced by genetic recombination technology.
By adding a polyoxyethylene hydrogenated castor oil derivative to the composition, its stability is significantly improved. Furthermore, even if it is freeze-dried, its stabilizing effect is not lost. Further, even when the uTPA preparation of the present invention is administered to blood vessels, hemolysis does not occur.

Claims (1)

[Claims] A human uterine tissue-derived plasminogen activator preparation comprising a human uterine tissue-derived plasminogen activator and a polyoxyethylene hydrogenated castor oil derivative.