IL29465A - Crosslinked kallikrein inactivator and its production - Google Patents

Description

3Ί9Ί »ti0 Tainan †»Rip»VRp aapa CrOselinked kallikreia inactivator and its production s It is knows to link proteins intramol cularly9 without causing any far-=reac-hing modification of their str cure, by the reaction with esters of dicarbox;iiS-idie acids .

It has now been found that di-(imido estera) of succinic acid can be reacted on the kallikrein inactivator obtained from bovine organs „ whereby „ in addition to iaonomeric products p dimerSj, trimers and oligomers are formed by cross-linking. The resultant amidine derivatives are distinguished from the starting material with regard to their pharmacological properties 9 in that their inhibitory effects on different enzymes are modified to a varying degree* Conse e tl ( they have modified pharmacological properties in comparison with the starting material. As can be seen from the following Tcble A9 thoy have5 for example 9 an antitrypsin activity and an antifibrinolytic activity which decrease from the monomer to the oligomer3 whereas the coagulation inhibitory activity increases * Table A Molecular Anti-trypsin Kallikrein Anti-fibri size activity inhibition activity unchanged 7000 KlU/mg 7000 KlU/mg 7000 KlU/ inactivetor 29465/2 The kallikrein inactivator used as starting material is fully known as to its composition and sequence of amino acids [Z. Physiol. Chemie 33 , 230 (1963); Z. Naturforsch. 20b, 462 (1965); J. Biol. Chem. 241, 1568 (1966)]. It contains in its sequence 4 lysine radicals the free £ -amino groups of which [J.A.C.S: 88, 3890 (1966)] are capable of reaction with imido^ .esters. By using esters of suceinimidic acid cross-linking between polypeptides can be obtained .. When this procedure is used for the kallikrein inactivator, compounds are obtained the structural principle of which can be regarded as simplified by. the following formula: ' ' ·. wherein n can amount to between 1 and about 20 or more and m between 0 and 10 or more. The compounds formed in this way wer fractionated according to their molecular size on dextran gel.

During pharmacological examination of the fractions it was seen that particularly those of high molecular weight where n : 4 to 10 or more show pharmacologically valuable properties which are applicable chemotherapeutically. The high molecular compounds with the strongest inhibitory effects in the coagulation forephase are at least first used themselves and not in combination with the low molecular compounds. 29465/2 The reaction of the kallikrein inactivator with the di-(imido esters) of succinic acid is expediently carried out at about pH 10.5 with the addition' of a buffer, at room temperature and with continuous stirring. The reaction solution is subjected to a gel-filtration. The high-molecular components are the first to migrate through the column, whereas the low-molecular components are retained. The fractions are collected according to their molecular size and, for desalting, placed in an exchanger column where any adherent buffer salts are retained. The column filtrates are then freeze-dried. ' v The invention is illustrated by the following non- limitative examples: ' . .

EXAMPLE 1 0.5 g (77 μ mole) of kallikrein inactivator from bovine organs are dissolved in 50 ml of 0.1M sodium phosphate buffer (pH 10.5). 50.5 mg (321 μ mole) of succinic acid di- imidomethyl ester hydrochloride are added to this solution at room temperature and with continuous stirring in the course of one hour. The succinic acid diimidomethyl ester was prepared - 6 - / by the method of S.M. McElvain and J. Schroeder [J. Am. Chem. Soc, 21» Pa e 40 (1949)]. Stirring is continued for one hour and the mixture is then evaporated in a vacuum to a volume of about 20 ml. This solution is purified, if desired, from a small amount of precipitate by centrifuglng and then chromatographed through a column of dextran gel with a diameter of 9 cm and a height of 90 cm. The column has previously been equilibrated with the following buffer which is also used for elution: 0.1M sodium chloride, 0.02M sodium phosphate, 0.001M ethylene-diamine-tetraacetic acid, pH 7.5. The rate of elution should amount to about 60 ml/hour.

The high-molecular reaction products, trimers, dimers and monomers, leave the column in succession.

The individual fractions are concentrated in a vacuum to about 10 ml, desalted through an ion-exchanger column and freeze-dried.

The yields and the dissociation constant of the various fractions of the trypsin complexes are stated in the following Table B: Table B Fract.No. Yield Molecular weight K ' mg 7. (gel filtration) approx.

I 37.1 7.1 37-58,000 oligomer 2.5 . lo-9 II 47.7 9.2 21,000 trimer 0.8 . lo-9 III 86.8 16.5 10,000 dimer 0.9 . lo"9 " EXAMPLE 2 4.0 g (0.62 millimole) of kallikrein inactivator are dissolved in 200 ml of 0.1M phosphate buffer, pH 10.5. 404 rag (1.85 millimole) of succinic acid diimidomethyl ester hydrochloride are added to this solution at room temperature and with continuous stirring in the course of one hour. During this time, the pH of the solution is kept at pH 10.5 by the addition of a 2N sodium hydroxide solution. Stirring is continued for one hour, the pH is then adjusted to 7 by means of 2N hydrochloric acid, and a small amount of precipitate is removed by filtering off with suction through a glass frit G 3. The precipitate is rinsed with some water. The combined filtrates are then concentrated in a vacuum to about .80 ml.

The inhibitory capacity of the filtrate for trypsin amounted to 4000 U, i.e. 27% of the inhibitory capacity of the inactivator used.

The concentrated filtrate is chromatographed through a column (9.0 x 90 cm) which is filled with dextran gel Sephadex G 50 (R and equilibrated with 0.05M ammonium acetate, pH 4.7. The monomeric and dimeric components are separated. The high-molecular fractions are combined, freeze-dried and re-chromatographed in an analogous manner through a column with dextran gel (Sephadex G 100) . Those fractions which, according to the gel fractionation, have a molecular weight of more than 30,000, are combined and freeze-dried. The average molecular weight after gel filtration is 100,000. Yield 1.26 g.

The preparation so produced has the following pharmacological properties: Inhibitory capacity for trypsin: (test with benzoyl-arginine-p_-nitroanilide as substrate) 0.65 U/mg corresponding to 1000 KlU/mg, i.e. 14% of the effect of the kallikrein inactivator.

Recalcification time (plasma of the rat): 72,800 KlU/mg (effect 10.4 times stronger than that of kallikrein inactivator) .

Thromboplastin time (bovine plasma): 46,900 KlU/mg (effect 6.7 times stronger than that of kallikrein inactivator).

Thromboplastography (mouse): delay of coagulation at concentrations above 2.10"^ g/ml, reduced thrombus resistance. The course of the reaction is similar to the effect of heparin.

Thrombin time (plasma of the dog) : inhibitory effect about 0.01% of heparin. Kallikrein inactivator is not effective in this test.

Thrombopenia after 200 NIU units thrombin/kg i.v. (mouse): no effect on thrombin- hrombopenia, in contrast to the effect of heparin.

The indications for use are as latent or manifest activation of the coagulation process and as prophylaxis for thrombosis. The dosage is 100 to 300 mg per dose, eventually as long term infusion at 20 to 100 mg per hour. The freeze- dried products are dissolved before use in physiological salt solution or in some other usual physiological electrolyte solutioi and can thereafter be administered by injection.

Claims (1)

11 A process for production of proteins which comprises reacting kallikrein inactivator with a of succinimidic A process according to Claim 1 wherein the reaction is out at a pH of about at room the reaction solution ia subjected to the high and molecular weight filtrate fractions are separated and the fractions are individually A process according to Claim wherein said actions are trimers and A process for the production of an amidine fron the of a of acid with the inactivator of bovine which comprises acting succinimidic acid ester hydrochloride and the kallikrein inactivator buffered to pH with sodium the solution through dextran gel separating the filtrate actions according to differences weight and sise and the fraction She reaction product of a succinimidic acid with kallikrein being the suceindiimidoyl derivative of the and fractions of proteins which are the reaction products of kallikrein inactivator and a of succinimidic acid obtained by the process of Claim The high molecular weight fractions of Claim 6 dissolved insufficientOCRQuality