SK419391A3 - Isolating method of plasminogene activators usable as therapeutic and diagnostic agents - Google Patents

Abstract

The isolation procedure utilizes the high affinity of urokinase k fibrin precipitated on a solid adsorption matrix. The method described may be a high affinity form plasminogen activator isolated directly from the urine or of the renal tissue culture medium. The method is economical and provides a high yield of activator that is useful as a thrombolytic agent. So prepared the plasminogen activator has a molecular weight of about 56 000 Daltons, a specific activity of about 40,000 to 50,000 CTA units / mg, apparent single chain structure a high affinity for fibrin.

Description

Method of isolation of plesminogen activators useful 3 ' 3 as therapeutic and diagnostic agents

The invention relates to the preparation of plasminogen activators useful as therapeutic and diagnostic agents.

BACKGROUND OF THE INVENTION Plasminogen activators have been found to be useful in the treatment of blood clots. By introducing such activators into the blood stream of a person in sufficient amount for a time sufficient to cause the blood clot to dissolve.

Previously used for the isolation of plasminogen activators have been characterized by their complexity and cost, and in the case of urokinase, plasminogen activators found in urine, the yield of others is so low that large amounts of urine must be collected and processed to treat one patient. Isolation of urokinase b from other sources, such as from kidney tissue culture medium, or isolation of other plasminogen activators such as streptokinase, also involves many steps that are either expensive, or may be associated with undesirable immunological reactions. In addition, commercially available activators for clinical use (abbokinase and streptokinase) are known to have a low affinity for fibrin inclusions and are therefore not optimally effective or may be associated with undesirable side effects due to the resulting proteolysis.

The work on the prior isolation of urokinase is as follows:

Barlow Q.H .. Mozen M.M., The Isolation & / Whit e W.P. j and Charecterization of Plasminogen éctivatora (Urokinaae) from Human Urine. Biochemistry, vol. 5, p. 2160-2169, 1966, b / Pye EK, Maciag T., Kelly P., Jyznger R., Purification of Urokinase by APfinity Chromium Atography In Thrombosis and Urokinase, edited by S.Palatti and S. Sherry, ecademic Press , New York, New York, San Prancisco 1977.

SUMMARY OF THE INVENTION

The present invention overcomes the above problems and makes it possible to isolate a relatively large amount of plasminogen activator from relatively small amounts of starting material. The invention also provides a novel class of therapeutic and diagnostic agents in the form of plasminogen activator, characterized by a strong affinity for fibrin.

According to the present invention, the plasminogen activator is isolated from urine or tissue culture medium by preparing a solid adsorption matrix (preferably dlathumble clay) having fibrin on its surface, the matrix being exposed to urine-based mother liquors or a fibrin-containing culture. The plasminogen activator molecules in the mother liquors having affinity for fibrin bind to the fibrin molecules, the mother liquor is removed, and the plasminogen activator is separated from the fibrin-containing matrix.

The invention is based on the discovery that fibrin precipitated on a solid adsorption matrix retains considerable affinity for certain plasminogen activators, particularly urokinase species, which are herein designated as having high affinity.

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-3tu. In contrast, when fibrin is covalently attached to activated egarose / i. cyanogen bromide-activated agarose / was not such an affinity for plasminogen activator. ** Since the origin of this difference is not experimentally established, it was now expected that fibrin precipitated on the adsorption matrix without covalent bonds left certain £-amino groups of fibrin lysine residues free. These? -Amino groups of lysine residues are expected to contribute to the newly discovered affinity. Conversely, when fibrin is covalently attached to activated agarose, the ε-amino acids of the lysine residues may be covalently attached to the agarose in such a way that they substantially block affinity. For these reasons, it is clear that the existence of high affinity urokinase has been overlooked.

In preferred embodiments of the isolation method of the invention, many conditions are preferred. The plasminogen activator is exposed to a fibrin-containing matrix by mixing fibrin solids in a bath of liquid in which the plasminogen activator is present. The fibrin-bound activator precipitated on the solid matrix is eluted from the fibrin surface. the fibrinate containing fibrin surface activator containing filtrate is passed through a gel filtration column to separate the high affinity plasminogen activator from the eluents and fibrin contaminants.

Preferably, to obtain a solid matrix with flbrin on the surface, fibrinogen is treated with thrombin in the presence of the matrix by precipitation of fibrin onto the matrix. the surface can be effectively completely occupied by fibrin.

According to a specific aspect of the invention for high affinity urokinase, the invention includes providing an adsorption solid matrix, preferred diatomaceous earth particles, fibrin precipitation on its surface by treating fibrinogen with thrombin in the presence of matrix, urine or tissue culture medium on a fibrin-containing matrix. of a species that was discovered as high. the fibrin affinity binds to it, unbound material is removed with a buffer solution, the high affinity urokinase is separated from the fibrin surface by an eluting agent comprising arginine, lysine and β-aminocaproic acid in aqueous solution, and then the urokinase is separated from the agent .

In this method, the preferred urine or tissue culture medium is exposed to the fibrin-bearing matrix by mixing the particles with urine or tissue culture media, and the liquid is removed by decanting and filtering followed by repeated washing of the fibrin matrix and bound activator buffer.

The novel product according to the invention is a high affinity plasminogen activator from urine and cultures, obtained as described above.

The improved thrombolytic agent of the invention essentially comprises urokinase, which is highly affinity for fibrin and is available to physicians in a lyophilized form.

It is to provide a specific novel product comprising a plasminogen activator isolated from a biological source such as urine or culture. This new product is characterized by a molecular weight of about 56,000 Daltons, a specific activity of 40,000 to 50,000 CTA units / mg in a fibrin plate assay and the detection of a single chain structure, corresponding to a molecular weight of 56,000 Daltons. such as <··· '' · ''. '-: ··' · ^: / '·' - '. .-.- '. · ^{; ::} ··.! Γ - '·, - ·? .- /

Apparent from a single lane of protein on a 7% polyacrylamide gel in the dodecyl sulfate-gel electrophoresis of the novel product in an unreduced state · The polynogene activator maintains a single strand structure as seen from a single lane on a 7.9-polyecrylamide gel if sodium dodecyl sulphate-plasminogen activator gel electrophoresis, which was reduced with 0.1M di-thiothreltol. Most importantly, the plasminogen activator exhibits a substantial binding affinity for fibrin as evidenced by its high affinity for celite fibrin. The high affinity of plasminogen activator for fibrin is the swelling that makes it, when radiolabelled, a new diagnostic agent for the specific detection of fibrin thrombi by nuclear detection.

The process utilizes the properties of certain plasminogen activators to bind fibrin. This principle is applied in the examples below to isolate plasminogen activator from urine having high affinity for fibrin (High APF Urokinase, HÄK), but is also useful for extracting high affinity plasminogen activators from other sources, such as extracts of kidney tissue culture medium.

The process utilizes a matrix containing fibrinogen absorbing material. In a preferred example, the cellular diatomaceous earth has further proved to be suitable for this purpose. In order to obtain a large specific surface, a powdered form of this material is used. Thereafter, the matrix is mixed with flbrinogen, which is converted to fibrin by the action of thrombin, resulting in the precipitation of fibrin on the matrix. This matrix prevents the formation of a gel that normally forms when fibrinogen is exposed to thrombin. When fibrin-celite is mixed in urine, HÄJK is bound to the affinity matrix. Then the unweighed activator

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removed from fibrin-celite by washing with an eluant. The eluted HAUK is contaminated with only a small amount of fibrin, eluted from the column with an eluant, and is easily separated from the elution material by gel filtration. The final solution containing the activator is then lyophilized.

DETAILED DESCRIPTION OF THE INVENTION

method

The powdered diatomaceous earth / Celite Aialytical filter ald, Pisher Scientific Co./, matrix, was washed with distilled water and precipitated with 2 percent human fibrinogen (Kabi, Stockholm, Sweden) in 25 ml buffer (0.05 M sodium phosphate, pH 7, 4 containing 0.1 M HaCl and 1 m M EDTV. Thrombin / Parke Davis, Bovine topical / 100 µl in 1 ml of buffer is added under constant stirring at room temperature to achieve good mixing to prevent precipitation on the fibrin matrix. After 30 minutes, the fibrin matrix is filtered through a sintered glass funnel and washed thoroughly with the same buffer (1 liter) followed by a buffer containing 0.2 M arginine. The final wash is performed with 0.05 M sodium phosphate buffer, pH 7.4, containing 1mM EDTA and 0.31a NaCl. The washed fibrin matrix (15 ml of settled volume) was added to 1 liter of fresh human urine and the mixture was stirred for 1 hour in cold (4 ° C). After filtration, the fibrin matrix is loaded onto the column, washed with equilibration buffer (8-10 column volumes). The activator is then eluted from fibrin by arginime (0.2 M in the above buffer) in one sharp peak. Alternatively, the elution may be performed with lysine or β-ananocepronic acid. Arginine and any fibrin derivatives contaminating the activator are then gelled. filtration on Sephadex 0-100 (Pharmacia Chemicals, Upsala, Sweden) and then the solution containing the activator is lyophilized.

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The results

Approximately 12.00-30.00 CTA HAOK units are obtained from 1 liter of fresh human urine. · The HÄJK molecular weight was determined to be approximately 56,000 Balton gel filtration on Sephedex G-200 superfine ✓ P. Aidrews visa technique, ethods Biochem. AIALA. 8, 1-553 (1970). The HÄJK material converts plasminogen to plasmin as demonstrated by a chromogenic synthetic substrate, and induces rapid lysis of fibrin on plasminogen-rich fibrin plates. * demonstrates specific activity of 40000-50000 GTA units / mg in a fibrin plate study / technique, see P. Brokman, Fibrinolysis: A Standardized Fibrin Plate Method and the Fibrinolytic Assay of Plasminogen, published by Scheltim and Holkem, Aasterodam, pp.1-124 / 1967 // ·

Comparison of HAUK with commercial urokinase / Abbott /

Eferly found two fundamental differences and one of the same property that are summarized in the board longer.

High affinity urokinase table HJSJK urokinase / podie / Abbott / vynélezu /

molecular weight 36000 56000 / Dalton / fibrin binding <5% 100% / fibrin-celite column / binding to sepharose <1% * 1%

In addition, more efficient fibrin lyses were found under physiological conditions for HADK compared to commercial urokinase.

Commercial urolinase (Abbott) mainly contains a low molecular weight form. The high-molecular-weight urokinase activity found in commercial urokinase (Abbott) appears to be a two-chain structure, as can be seen from the two protein bands (see K.Weber and M. Os bon) , The proteins, eds. Weurath, H. and Hill, L.L. (Academic Press, New York), Vol. 1, pp. 179-223 (1975). This high molecular weight form of urokinase has a low affinity for fibrin, while low molecular weight urokinase has none. Summary of commercial low molecular weight urokinase mixtures are known to have a very low affinity for fibrin as described above.

HAUK has a molecular weight of about 96,000 Dal tonds, which is comparable to a commercial high molecular weight urokinase. However, HAUK appears to be a single chain structure as seen from a single protein band after reduction with 0.1M dithiothreitol sodium sodium dodecyl sulfate-gel electrophoresis to 7, 5% polyacrylamide gel (ibid Weber et al.).

The binding of HADK, a type of urokinase of the invention of high molecular weight and one chain to fibrin celite under the conditions used for its adsorption, indicates a high affinity for fibrin.

It is believed that HAJK, which was first isolated, maintains the native molecular form, a step-wise, rapid method of isolation allowing the single-phase structure to be heated.

on the contrary, with a relatively slow, multistage approach, and

according to the prior art it is believed that it leads to the molecule's overgrowth and to the double-stranded structure and low affinity for fibrin.

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9The presented procedure has many advantages over the existing ones:

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1. fast, one-step instead of a long, multi-step insulation process. The long process usually results in undesirable contamination, autodegradation, or urokinase degradation by other proteolytic enzymes,

2. the method separates the high affinity activator of plasminogen from low affinity and first determines that fresh urine contains a high affinity urokinase (40-80% of the total urokinase depending on the subject)

The method provides a high yield (about 60%) of HÄJK in urine and there is no low affinity urokinase. In contrast to the commercially available urokinase (Abbokinase, Abbott), HÄJK is essentially a low affinity plasminogen activator.

application

I. Generally, since the method described is economical and rapid, it should provide a relatively inexpensive and abundant supply of human plasminogen activator characterized by high fibrin activity for both scientific research and clinical purposes.

II. therapy

Two commercially available plasminogen activators - Streptokinase (Hoechst and Kabi) and Urokinese (Abbott), which have been adopted by the PDA for thrombolytic treatment of pulmonary embolism, are low affinity activators. As a result, a large amount of activator (100,000 - 200,000 CTA / h) must be infused to achieve lysis of the precipitate.

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Since plasmin is an enzyme with a broad specificity, the effect of infusion of these activators is an undesirable state of proteolysis that degrades some plasma proteins (fibrinogen, factors V and VIII).

In contrast, the HÄJK discovered and successfully isolated according to the present invention, due to its affinity for fibrin, will provide more specific fibrinolysis at lower activator concentrations.

III. diagnosis

The fibrin-HAUK binding properties can also be utilized in the detection of intravascular clots or thrombi. By engaging a suitable radioactive isotope / e.g. ¹ ^^ I or techneeia / the molecule and then by intravenous injection of radiolabeled HAUK can édentifikovat intravascular fibrin by nuclear pqgmocí analysis. For example, labeled HÄDK can be used to specifically detect pulmonary embolism / pulmonary imaging / pulmonary examination. ^And this diagnosis is difficult to do with conventional methods because these are not specific.

Previous attempts to use radiolabeled plasminogen activators for radionuclear investigations have been relatively unsuccessful since the plasminogen activators used do not have the essential property of high affinity for fibrin. References and and such prior work include Millar W.T. eSmith J.F., Localization of Deepvenous Thrombosis ^ sing Technitium 99 m - labeled Jrokinase / preliminary communication /, Lancet vol.2, pp.695-696, 1974, / b / Kempi V ·,

Van DerLinden W., and van Scheele C., Diagnosis of Deep Vein Thrombosis with 99 mTc-sterptokinase, Clinical Comparison with Phlebography, Medical Medical Journal, Vol.2, pp.748-749, 1974.

Claims (10)

PATENT N O * R O K oči urine, A method for isolating a plasminogen activator from or a culture medium, comprising providing an adsorption matrix having fibrin plated on its surface, treating urine-based mother liquors or culture medium and comprising a fibrin-containing plasminogen activator with a high affinity for fibrin whereby the plasminogen activator molecules having high affinity bind to the fibrin molecules, removing the residual mother liquor and separating the plasminogen activator from the fibrin. 2. The method of claim 1, wherein the plasminogen activator is eluted from the fibrin surface by an eluting agent comprising an agent selected from the group consisting of arginine, lysine and < -amino-caproic acid. Method according to claim 1, characterized in that the matrix used is diatomaceous earth. 4. The method of claim 1, wherein, to provide said fibrin matrix on its surface, fibrinogen is treated with thrombin in the presence of said matrix in a manner causing fibrin to precipitate onto said matrix without gel formation. . 5. The method of claim 4, wherein the adsorption surface of said matrix is treated with fibrinogen in an amount sufficient to effectively coat said adsorption surface, then / thrombin is introduced to convert said adsorbed fluorine to fibrin. '/ Wherein said adsorption surface is effectively occupied by adeorbated fibrin. 6. A method for isolating interest, comprising preparing a solid fibrin matrix by treating fibrinogen with thrombin in the presence of said matrix, such that the fibrin is in the adsorbed state of the substrate, the substrate is treated with urine or sulphurizing medium, taking a species; the urokinesis having fibrin affinity to fibrin, removing unbound material by washing with a buffer solution, separating the fibrin urokinase first by eluting the fibrin-surface urokinase with an eluting agent from the group consisting of arginine, lysine, -amino-caproic acid, and then said urokinase separates from this agent. 7. A method according to claim 1 or 6, wherein said matrix comprises particles wherein the liquid is exposed to a fibrin-bearing matrix by mixing said particles with said flue gas. 8. A method according to claim 1, wherein said liquid is removed by decantation and filtration followed by washing with buffer. A plasminogen activator enzyme concentrate isolated from a biological source by any of the methods of claims 1 to 6 and comprising urokinesis / human / molecular weight of about 56,000 Daltons, having a high affinity for binding to fibrin on an adsorption matrix and having an apparent single chain molecular structure. A urokinase plasminogen activator isolated from urine or culture containing essentially urokinase (human), characterized in that it (a) has a molecular weight of about 56,000 Daltons as was? / - / / .;. · / 'Y í', à. -T 33 · ··; <··; < -m '• l' · '?' j · - ·. as determined by gel filtration, (b) appears as a single protein band, corresponding to a molecular weight of about 56000 Dal, tinted on a 7.5% polyacrylamide gel in sodium dodecylsulfate-gel electrophoresis with unrecognized urokinase, (0) retaining this property if said electrophoresis is performed on urokinase, which was reduced with 0.1M dithiothreitol, thereby demonstrating its single-stranded structure and / or / exhibiting high binding affinity to fibrin precipitated on the diatomic clay.