DE2128257A1 - Procedure for the isolation of anti coagulate ttel - Google Patents

Description

Patent attorneys
Dr. Ing.Walter Abitt
_Dr. Dieter F. Morf
Dr. Har · "- *. Brauns

8München86, Pienzeneue «ti.28

June 7, 1971 2694

Abbott Laboratories,
Bbrth Chicago, Illinois (V.St.A,)

Method of Isolating Anticoagulants

The invention is concerned with a new single stage Process for the purification and isolation of the thrombin-like fraction of the Malayan snake venom Color viper (pit viper). The simple procedure used Affinity chromatography for fractionation of the native snake venom using special buffered feed and feed solutions. The chromatography column uses an agmatine-coupled one Agarose pack,.

For several years it has been known that snake venom some scar vipers (pit vipers) e.g. Ancistrodon Rhodostoma, which contains a component that is used as an anti " coagulant is effective. Recently, it has been found that this component is actually a coagulant for blood. Forms in its effect the thrombin-like material with which the invention is concerned, uncrosslinked fibrin polymer, which is easily removed by the reticulo-endothelial system and / or the fibrinolytic system of the body, and thus depletes or depletes the fibrinogen of the blood. It thus creates an anticoagulant effect.

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Unfortunately, the previously known cleaning process leaves a lot to be desired. The best known method at the moment is a two-step chromatographic process that_. two columns with different packings and two differently buffered solutions with different requirements in terms of extraction solvents in the two stages. The most used The substrate for the first stage of this chromatographic method is triethylaminoethylcellulose, which unfortunately does not provide reliable and easily reducible results. Worst of all, it's like that Purified snake venom solution still has the entire hemorrhage factor, which is the most dangerous component of the viper snake venom is. Required in every pail further purification of the snake venom solution fractionated in this way, for example by a similar chromatographic method Method using a differently buffered solution of the effective fraction from the first partition and a different column substrate.

It is therefore an object of the invention to provide a simplified process for the production of pure thrombin-like Material from the snake venom of the Malay pit viper; another task consists in a reproducible process for the isolation of the thrombin-like fraction of pit viper snake venom and further the invention provides a one-step isolation method for pure thrombin-like efficacy from scarred viper snake poison in purified form.

These and other objects are achieved by using a clear, dilute aqueous solution of native scarred viper snake venom, which to a pH of about

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pH 7.5 "to 8.1 is buffered to one with agmatine coupled agarose beads packed column is brought, the column with a to a pH of 8.1 + 0.1 buffered linear gradient sodium chloride solution and the fractions containing the thrombin-like active material are pooled. the above reference to agmatine coupled agarose is intended to describe "agarose beads" to which 4-aminobutylguanidine (Agmatine) is covalently bound. Agmatine is a competing inhibitor of the thrombinähnlicheη effective Material of scar viper venom and prevents that the active material is eluted from the column until virtually all other protein materials are eluted are, with a suitably buffered linear gradient sodium chloride solution having a molarity from 0.01 to 1.0 is used.

The term "native" used herein to describe snake venom in the present specification is intended to be define the snake venom that has not previously been treated by other chromatographic or chemical methods has been. Consequently, it contains other protein materials previously referred to as Fraction I - VIII, including the hemorrhage factor and fractions V and VII, i.e. those that have similar physico-chemical Have properties such as the coagulation factor which this isolation method is concerned with. The unwanted ones Fractions and components are obtained from the native snake venom by the method according to the invention completely eliminated and no prior ion exchange treatment is required. On the other hand, be on it pointed out that the expression defined above

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"Native" is not to be understood as meaning that the snake venom actually precedes the one-stage according to the invention Separation must be unaffected; it can be centrifuged - to remove sediments, lyophilized, in order to improve the solubility and of course must be solved in order to meet the requirements that it is used as a clear, dilute aqueous solution. If necessary, the snake venom solution can be pretreated with ammonium sulfate to prevent certain undesirable effects Remove proteins from it. However, as shown below, such treatment is not necessary.

The aqueous solution of the native snake venom can between 0.5 and about 5% by weight of the poison in the accordingly buffered pH range included. The preferred buffer for the solution is a non-toxic salt of tri- (hydroxymethyl) aminomethane, although other non-toxic buffers can be used instead. The preference for a non-toxic buffer is based on the fact that the same buffer is also in the eluent solvent is used and partly remains in the isolated protein fraction, which is the thrombin-like Contains effectiveness. This eluate is usually so pure that it can be used directly for medicinal purposes although it usually requires dilution, because a conveniently injectable solution is usually between 50 and 200 units (defined by Owren in Acta Medica Scandinavia, Suppl. 194 (1947)) of the thrombin-like Contains effectiveness per ml, while the eluate according to the invention often has more than 500 units / ml.

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According to a general embodiment of the invention, the buffered “dilute aqueous solution of the natural snake venom is applied to the agarose beads which have been pretreated with agmatine. The column is then filled with an aqueous sodium chloride linear gradient solution which is adjusted to a pH of 8, 1 + 0.1 is buffered with tri- (hydroxymethyl) aminomethanehydrοchloride or other non-toxic pharmaceutically acceptable buffer, the eluate is collected in small fractions and the fractions contain the thrombin-like material which is combined about 80 $ of the thrombin-like effect. "- · ness of the native snake venom In a preferred embodiment, the fractions are collected with cooling, d" h _# at a temperature below 10 ⁰ C yon,

The agmatine-bound agarose beads are made from a commercially available agarose in bead form prepared in a known manner. The pearls are then placed in a column, where they pack as affinity chromatograms works; they are extremely effective in retaining active snake venom protein while they the other proteins with much lower affinity hold back. During the elution with the linear gradient described above, these other processes are bonds eluted before the effective fraction appears, resulting in an extremely pure isolated thrombin- " like acting material ready for use as an injectable, with the exception of the Dilution to the prescribed concentration. Suitable diluents are water, saline or a dilute tri- (hydroxymethyl) aminomethane hydrochloride solution. The latter material can also be added

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if the pooled eluate obtained has a pH unsuitable for intravenous injections for which the preferred pH range is close to 7 or slightly higher.

The following examples serve to illustrate the invention Method without limiting the invention in any way.

example 1

Agmatine was made with Sepharose 4B (a pearl processed Agarose material available from Pharmacia, Ltd. Schv / eden is sold) according to the Cuatrecasas et al. in Proc. Catl. Acad, Sei, U.S. 61., 636 (1968) with the exception that the pH value during The agarose was held at 11.0 for 15 minutes during CNBr activation and the joining or coupling reaction in tri- (hydroxymethyl) aminomethane hydrochloride pH 8.5 using 0.149 g agmatine sulfate per ml deposited volume of agarose took place * The nitrogen content of the agmatine-agarose material prepared in this way was $ 3.1 + $ 0.1.

A solution of 1.0 g of lyophilized natural snake venom from the Malay scarred viper in 100 ml 0.005 molar tri- (hydroxymethyl) aminomethane hydrochloride of pH 7.5 was placed on a chromatographic column 1.9 cm in diameter with the above Agmatine agarose beads was packed to a height of 13 cm. The column was then at room temperature at a flow rate of 60 to 80 ml / h with a

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Linear gradient sodium chloride solution between 500 ml from a molarity of 0.01 and 500 ml from a molari « tat of 0.5 eluted, with both solutions on one pH 8.1 were buffered with tri- (hydroxymethyl) aminomethane hydrochloride. The eluate was in Fractions of 5 to 7 ml are collected in a refrigerated automatic fraction collector. The first 400 ml of eluate were almost completely inactive; the subsequent fractions, however, were highly active and contained next to the desired coagulant component only inert materials. It was found that the combined, the fractions containing the active material were slightly more than 80% of the initially present thrombin-like Effectiveness included, and $ 90 of gained Effectiveness had a specific effectiveness of 1200 (specific effectiveness = ^^

If the Sepharose used in the above procedure by Bi-GeI A-15m (one of Bio-Rad Laboratories, Richmond, CaI. marketed agarose) was replaced, the pooled fractions obtained showed the same effectiveness and the degree of purity as above. The same result was also obtained when the upper gradient concentration 1 molar sodium chloride.

The pooled fractions containing the thrombin-like material were identified with a previously identified one pure sample of the isolated thrombin-like material from scar viper venom obtained by previously used methods was compared: It was found that the two samples in the polyacrylamide gel electrophoresis method using 7.0 gels at pH 8.9

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were identical to those of Ornstein in Annals of N.Y. Acad. of Science, Vol. 121, p. 321 (1964) is, with identical electrophoretic mobilities obtained by gel filtration on Sephadex G-100 result, which indicates the same molecular weight, and based on criteria of enzymatic activity, namely the enzyme specificity and effectiveness including in vivo difibrogenation. The coagulant effectiveness test according to the thrombin coagulation .bzw. Clumping time method shows no difference between the two samples, and the hemorrhage factor was completely absent in both samples. Both materials coagulated in purified plasma fibrinogen.

Example 2

A solution of 50 mg of lyophilized snake venom from Bothrops atrox in 5 ml of the 0.01 molar buffer measured example 1 at pH 8.1 + 0.1 was centrifuged for clarification and applied to a 1x15 cm agmatine-agarose column. The column was at room temperature at 20 to 30 ml / h with 5 column bed volumes of the above buffer at pH 8.1 +0.1 and with a linear sodium chloride gradient between 150 ml of the above buffer and 150 ml of the same buffer made 0.3 molar with sodium chloride was eluted. 5 ml fractions were as in Example 1 collected «

The separated fractions were tested for their thrombin-like activity. By application clear activity peaks were found in their effectiveness against the number of fractions. Although the materials of all three tips thrombin-like effective

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possessed, only the material of the third tip resulted in a purified fibrinogen Qerinsel, and it is therefore that. obtained in Example 1 Material most similar.

Example 5

By repeating the procedure of Example 2 with the snake venom from Echis carinatus, peaks were obtained two thrombin-effectiveness _. Both materials showed efficacies similar to that described in Example 1.

Example 4

By repeating the above procedure with a more dilute sample of the snake venom Grotalus adamanteus was able to test the thrombin-like activity of this snake venom from other protein fractions with appeared associated with snake venom, separated and preserved in a highly purified form.

It turns out that the method according to the invention is the process for isolating the active coagulating component made from snake venom considerably simplified. In particular, it should be noted that only a simple Affinity chromatogram is required in order to obtain a pure material that has so far only passed through a two step process to produce a material with identical potency, molecular size, anticoagulant potency and which also agree with regard to other physical and chemical identification, was available. It is particularly noteworthy

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and surprisingly that a simple affinity chromatogram the only requirement is to get a purified to obtain highly effective directly usable thrombin-like material from previously untreated snake venom.

In view of the surprising effectiveness of the invention Cleaning and isolation process and the fact that the solution obtained is directly for their intended medical purpose can be used with the exception of adjusting the concentration, it follows that that the buffer and the salt concentration of the snake venom solution should be made with pyrogen-free materials and non-toxic salts as buffers. For this reason, physiologically acceptable salts are used as buffers in such amounts that a physiologically result in a suitable concentration, such as an isotonic solution.

Optionally, a preservative can be added to the snake venom solution at the beginning, or such a component can subsequently be added to the purified isolated coagulant fraction if the solution is to be stored or to extend its useful life. Numerous preservatives are suitable for this purpose, for example benzyl alcohol, methyl and / or propyl p-hydroxybenzoate, chlorobutanol, and the like. Usually 0.05 to 1 percent of such a preservative is sufficient for all normal storage requirements.

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Claims (5)

H Claims * Procedure for the isolation of the thrombin-like acting Component of a pit viper snake venom, characterized in that there is essentially a clear, dilute aqueous solution of native snake venom that. to a pH of 7.5 to 8.1 is buffered, on a column packed with agmatine-coupled agarose beads, the Column with a linear gradient sodium chloride solution, which is buffered to a pH of 8.1 + 0.1, elutes and the active thrombin-like Material containing fractions together. 2. The method according to claim 1, characterized in that a native snake venom solution is used, which contains between 0.5 and 5 wt .- $ snake venom. 3 »The method according to claim 1, characterized in that a sodium chloride solution is used, the linear gradient of which begins with a molarity of 0.01 and ends with a molarity of 1.0. 4. The method according to claim 1, characterized in that .that a sodium chloride solution is used, the linear gradient of which begins with a molarity of 0.01 and starts with ends with a molarity of 0.5. 5. The method according to claim 1, characterized in that both the snake venom solution and the Eluting liquid with a non-toxic tri * (hydroxymethyl) aminomethane salt are buffered. «11 - 109851 / -1848 6 # Method according to claim 5 »characterized in that that the hydrochloride salt is used as the non-toxic salt. «12 - 109851/184