WO1982003772A1 - New medicine comprised by an aprotinine-plasmine complex and method for the preparation of such new complex - Google Patents

New medicine comprised by an aprotinine-plasmine complex and method for the preparation of such new complex Download PDF

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Publication number
WO1982003772A1
WO1982003772A1 PCT/FR1982/000078 FR8200078W WO8203772A1 WO 1982003772 A1 WO1982003772 A1 WO 1982003772A1 FR 8200078 W FR8200078 W FR 8200078W WO 8203772 A1 WO8203772 A1 WO 8203772A1
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WO
WIPO (PCT)
Prior art keywords
plasmin
aprotinin
complex
plasminogen
activator
Prior art date
Application number
PCT/FR1982/000078
Other languages
English (en)
French (fr)
Inventor
Sa Choay
Original Assignee
Vairel Edmond
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Vairel Edmond filed Critical Vairel Edmond
Publication of WO1982003772A1 publication Critical patent/WO1982003772A1/en
Priority to DK578682A priority Critical patent/DK578682A/da
Priority to IT83361/83A priority patent/IT1195616B/it

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6435—Plasmin (3.4.21.7), i.e. fibrinolysin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin

Definitions

  • the present invention relates to a new product constituted by an aprotinin-plasmin complex and more particularly to aprotinin-lys human plasmin and to the use of this new complex. as a medicament, especially as a thrombolytic.
  • Aprotinin is a competitive natural inhibitor of a number of endopeptidases, extracted from cattle organs such as lungs, pancreas, parotids, in which it is present in cellular microsomes. Its inhibitory activity, in particular with respect to trypsin, kallikrein and plasmin, has enabled its use in therapy, in particular in cataclysmic fibrinolyses due to its antiplasmin activity.
  • the Inventor cf. VAIREL EG, ANTOINE G., Ann. Anesth.
  • aprotinin is very widely used in the treatment of pancreatitis, without causing the development of intravascular coagulation syndromes, although in all cases hypercoagulation is observed accompanied by hyperfibrinolysis.
  • the inventor has shown [VAIREL EG, HUREAU J., Ann. Pharm. Franc., 1973, 31 (6), 409] that the acceleration of coagulation by injection, in dogs, of kaolin or trypsin, is not modified by the administration of aprotinin and that n generally observe no intravascular coagulation phenomenon.
  • epsilon-amino-caproic acid which is a synthetic fibrinolysis inhibitor
  • aprotinin would be, in vivo, only an inhibitor of fibrinogenolysis (fibrinogen being, as we know, the precursor of fibrin, and being transformed into the latter under the action of thrombin), its antifibrinolytic action explained by its action of inhibiting plasmin with which it forms in situ an enzyme-inhibitor complex, considered in the literature to be devoid of activity with respect to both fibrinogen and fibrin.
  • Plasmin-aprotinin complexes have been prepared in the prior art [cf. "The Journal of Biological Chemistry, vol. 250, n ° 10, May 25, 1975, p. 3988-3995, L. SUMMARIA, L. ARZADON, P. BERNABE and K.C. ROBBINS:
  • B. WIMAN was, for example, to study the kinetics of the plasmin inactivation reaction with aprotinin by the formation of a complex present during a first stage of the reaction, in a reversible form and, during a second step, in a modified form, also reversible, from a mixture of plasmin with an excess (60%) of aprotinin relative to plasmin.
  • SUMMARIA and Alia confirmed, in their aforementioned study, that the plasmineaprotinin complexes are inactive as enzymes. None of these publications has considered the possibility of using these complexes in therapy, the clearly aimed aim of their Authors being to try to explain biochemical mechanisms by using the property of aprotinin to inhibit plasmin in situ, using in vitro trained models.
  • the inventor has now demonstrated that the aprotinin-plasmin complex is not inert towards fibrin, under certain conditions, and that, therefore, it can be used as a new fibrinolytic drug. .
  • the ⁇ 2antiplasmins contained in the plasma are capable of inhibiting both approximately one third of the plasmin that can be generated in plasma, in respect of which they play the role of natural inhibitors present in circulating blood; therefore, for fibrinolysis to occur, the level of ⁇ 2 antiplasmin has to be lowered to a certain point, in order to allow plasmin to play its role vis-à-vis fibrin, which results in a very high consumption of circulating plasminogen, which necessitates providing an external supply of plasmin or plasminogen.
  • the inventor has been able to establish that when the plasmin is complexed with aprotinin in accordance with the invention, it is inert with respect to ⁇ 2 antiplasmin, which means that the supply of plasmin necessary for fibrinolysis is considerably reduced and the latter is likely to exert its action immediately.
  • the present invention therefore relates to a new medicament constituted by an aprotinin-plasmin complex.
  • the new drug consists of an aprotinin-human plasmin lys complex.
  • aprotinin and plasmin are present in the complex in equimolecular quantities, this 1/1 ratio being expressed in what follows by the abbreviation AP.
  • aprotinin and plasmin are present in the complex in an aprotinin / plasmin ratio of 2/1, this 2/1 ratio being expressed in the following by the abbreviation APA.
  • the subject of the present invention is also a new process for preparing the aprotininplasmin complex, which consists in bringing plasmin or its precursor, the plasminogen associated with an activator, into contact with aprotinin, in appropriate quantities, in a suitable solvent. , the aprotinin-plasmin complex. formed being purified by passage over a Sepharose-lysine column, so as to eliminate the possible excess of aprotinin and, where appropriate, the activator if the latter has been previously added and the complex then being eluted with epsilon acid -amino-caproic at a molar concentration of at least 0.05 M.
  • the plasmin precursor namely plasminogen
  • it is a partially pre-activated plasminogen such than that which has been described in the CHOAY patent n ° 73 45289 filed on December 18, 1973 and published under n ° 2 254 316.
  • the activator associated with plasminogen is urokinase.
  • the mixture of aprotinin and plasmin comprises at least 1400 to 2000 IUU of aprotinin per 1 microkatal of plasmin in the case where it is sought to obtain a complex in which aprotinin and plasmin must be present in equimolecular amounts.
  • the mixture of aprotinin and plasmin comprises at least 3500 to 5000 IUU of aprotinin per 1 microkatal of plasmin, and advantageously 3600 to 4000 IUU of aprotinin for 1 microkatal of plasmin, in the case where it is sought to obtain a complex in which aprotinin and plasmin must be present in a 2/1 ratio.
  • the complexation of aprotinin and plasmin is carried out by incubation of a mixture of aprotinin and plasmin, or of plasminogen associated with a activator, at a temperature between ambient and 40 ° C.
  • the duration of the incubation is sufficient - of the order of approximately one hour - to cause the transformation of the plasminogen plasmin and complexation of the latter with aprotinin, this time can be reduced to a few minutes in the case where plasmin is used.
  • the pH of the mixture is of the order of 7.4 ⁇ 0.4.
  • the complex formed according to the invention is lyophilized.
  • the Applicant has been able to demonstrate that the aprotinin-plasmin complex according to the invention binds to fibrin, a certain amount of aprotinin is then released and entrained in the plasma.
  • the complex thus modified by fibrin then has fibrinolytic properties which are exerted directly on the fibrin support by hydrolyzing it.
  • This modification has been demonstrated, in accordance with the invention, by assaying the inhibitory activity of aprotinin on trypsin, according to the method of the French Pharmacopoeia, on the one hand directly and, on the other hand, in the 5% trichloroacetic supernatant from which the precipitated plasmin is eliminated.
  • the invention relates more particularly to the new medicament according to the invention, as well as the process and the means used for its preparation and its use.
  • AGLME N- ⁇ -acetyl glycyl-L-lysine
  • Affinity chromatography of the incubated mixture is carried out on a lysine-Sepharose column (Pharmacia) 9 mm in diameter and 14 cm long, equilibrated with phosphate buffer.
  • the APA complex is eluted by the phosphate buffer supplemented with 0.05 M of ⁇ -araino-caproic acade (cf. arrow 3). It appears in a narrow peak (2), representing in this test, 6 ml of eluate.
  • the new complex according to the invention has the following physicochemical characteristics:. its molecular weight after filtration on "ULTROGEL" is around 90,000 for the complex in which aprotinin and plasmin are present in a 2/1 ratio, and it is around 84,000 for the complex wherein aprotinin and plasmin are present in equimolecular amounts; . the association constant of the AP complex calculated by
  • Chem., 1969, 350, 1531, is equal to 3 ⁇ 10 8 .
  • the complex can be partly dissociated in the presence of fibrin, as described below and acquires, in particular with regard to the APA complex, due to its partial dissociation, fibrinolytic properties.
  • the biological characteristics of the complex according to the invention are as follows:. it is inactive with respect to fibrinogen, in a plasma medium,. it is inactive vis-à-vis an excess of plasmin. Determination of aprotinin released from the APA complex
  • the aprotinin released from the complex is assayed, by proceeding as described below, in order to determine the amount of aprotinin fixed by microkatal of plasmin, in the complex according to the invention.
  • the aprotinin-plasmin complex according to the invention is formed from: 25 microkatals of human Lys-plasminogen whose activity has been previously measured,. urokinase,
  • the aprotinin-lys human plasmin complex obtained is eluted with ⁇ -amino-caprolic acid, then dialyzed to eliminate the latter.
  • the complex obtained is dissolved in physiological saline.
  • To this solution is added concentrated trichloroacetic acid in sufficient quantity to obtain a final concentration of 5% trichloroacetic acid in the complex solution. A precipitate containing the uninhibited plasmin is formed, the oil is eliminated by centrifugation.
  • the supernatant, which contains aprotinin, is collected; the pH is adjusted to 8, then the aprotinin present in the supernatant is assayed, by inhibition of the action of trypsin on BAEE, at 253 nm, according to the method of the French Pharmacopoeia.
  • the assay shows that 100,000 PIUs of aprotinin have been released from the complex, ie approximately 4,000 PIUs per microkatal of plasmin
  • the assay of aprotinin in the AP complex can be carried out in the same way. Identification and differentiation of the complex in legacy aprotinin and plasmin appeared in a 2/1 ratio (APA) and of the complex in leg uel they are present in a 1/1 ratio (AP)
  • Plasmin is suspended in a buffer close to neutral; a large excess of aprotinin is added to this solution; after incubation at laboratory temperature (approximately 20 ° C) for 30 minutes, the solution is passed through a column of lysine-Sepharose.
  • the 2/1 complex is formed which is retained on the column, while the excess aprotinin is in the effluent.
  • the entire fibrin is then mechanically dispersed in 50 ml of buffer.
  • This suspension is used to form a column 15 cm long and 0.9 cm in diameter.
  • This column is continuously supplied by a pump, the effluent is analyzed by a UV reader before being distributed by a fraction collector (LKB material).
  • the balance of the column is obtained by passing the Tris buffer solution at a flow rate of 10 ml / hour for 2 hours.
  • the buffer is added with 0.05 M of ⁇ -amino-caproic acid, the dissolution of fibrin does not continue and the complex, as further flow, is read from fibrin; it appears in a fairly broad peak.
  • the APA complex whose association constant is very strong, is partially dissociated in the presence of fibrin. It is as if the aprotininplasmin complex transformed as described below, forms a transient complex with fibrin. 3 °) Behavior of the aprotininr-plasmin APA complex in human plasma medium
  • the fibrin column being produced as above, 20 ml of human citrate plasma diluted 1/2 with Tris buffer were used to balance the column at a flow rate of 12.5 ml / hour.
  • the APA complex according to the invention is made to pass comprising 10 m i c r o k a t a l s of plasmin and 40,000 uIP of aprotinin (1 microkatal for
  • the citrated blood (1/20 of sodium citrate at 9%) is centrifuged to obtain a plasma poor in platelets
  • the citrate plasma is recalcified and 0.5 ml is immediately introduced into the central tube with a porous bottom, while at the same time time, the same level is obtained in the external tube, with a buffer solution pH 7.4, 0.15 mg.
  • the tubes are maintained at 37 ° C.
  • washing is obtained by adding buffer at 37 ° C. to the external tube; the difference in level obtained means that the tampon penetrates through the clot from bottom to top, and performs its washing.
  • the washing liquid after passing through the clot is removed by siphoning. This washing can be stopped after 3 hours.
  • the tubes are maintained at 37 ° C.
  • a clot formed from normal blood has not undergone any modification; a clot formed from blood containing the aprotinin-plasmin complex according to the invention has undergone partial or total dissolution.
  • Observation of the plasma clot lysis of an animal which has previously received an injection of the aprotinin-plasmin APA complex means that the aprotinin-plasmin APA complex is still present in the blood at the time of collection. This shows that the complex is capable of continuing its action for a prolonged period, which is not observed with the fibrinolytics currently used in clinical practice (streptokinase, urokinase or plasmin).
  • the inventor practiced this test by drawing blood from the central artery of the ear before the administration of the aprotinin-plasmin APA complex, then 1 min after, 30 minutes after, 2 hours, 4 hours and 5 hours 30 minutes after. injection into the marginal ear vein.
  • the results observed after injection of 25 microkatals of lys-plasmin inhibited by 2 mg of pure aprotinin, are recorded in the table below.
  • the "+" sign indicates that the clot has lysed.
  • the sign "O" that there has been no lysis. It is noted that, in 5 animals out of 6, the complex is still present at the 2nd hour and that in 3 animals out of 4, it is still present during the 5th hour.
  • downstream ligature is then moved in the direction of the upstream ligature, so that the downstream ligature is upstream of the puncture hole, then the upstream ligature is lifted very slightly to allow a little penetration blood in the bag formed by the two ligatures.
  • the blood introduced coagulates under the effect of the thrombin previously introduced into the bag.
  • the two ligatures are left in place for 1/4 hour after which the upstream ligature is completely removed.
  • the thrombin contained in the clot then diffuses and spreads in non-bare areas.
  • plasmin solution or complex Into hemolysis tubes placed in a water bath at 37 ° C., 0.1 ml of plasmin solution or complex is introduced at different concentrations, then 0.1 ml of thrombin solution at 25 NIH / ml.
  • - Figure 3 shows the evolution curve of said lysis surface in the case where plasmin is deposited in the three respective forms above, in solutions at 15 ⁇ katals / ml
  • - Figure 4 shows the curve evolution of the lysis surface in the case where plasmin is deposited in the three respective forms above in solutions at 30 ⁇ katals / ml.
  • the new aprotinin-plasmin complex in accordance with the invention is particularly indicated for all fibrinolytic treatments and in particular for the treatment of embolism, thrombosis, capillary micro clots, disseminated intravascular coagulation (DIC) frequent in nephrology, etc.
  • stability of the complex according to the invention could advantageously be used to obtain local therapeutic fibrinolysis, in particular in arterial thrombosis, because its activity is prolonged for several hours, instead of only lasting a few minutes as is the case for currently known fibrinolytics.
  • the complex is advantageously. to be administered from 50 to 300 microkatals of plac ⁇ r: i :: G: complexed with aprotinin per 24 hours.
  • the complex according to the invention is advantageously administered in combination with an acceptable pharmaceutical vehicle, such as the isotonic chlorinated solution, for example, parenterally, preferably intravenously.
  • an acceptable pharmaceutical vehicle such as the isotonic chlorinated solution

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PCT/FR1982/000078 1981-04-30 1982-04-29 New medicine comprised by an aprotinine-plasmine complex and method for the preparation of such new complex WO1982003772A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
DK578682A DK578682A (da) 1981-04-30 1982-12-29 Nyt laegemiddel bestaaende af et aprotinin-plasmin-kompleks og fremgangsmaade til fremstilling af det nye kompleks
IT83361/83A IT1195616B (it) 1982-04-29 1983-04-05 Medicamento costituito da un complesso aprotina-plasmina e procedimento di preparazione di questo complesso

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR8108681810430 1981-04-30
FR8108681A FR2504921A1 (fr) 1981-04-30 1981-04-30 Medicament nouveau constitue par un complexe aprotinine-plasmine et procede de preparation de ce nouveau complexe

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WO1982003772A1 true WO1982003772A1 (en) 1982-11-11

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EP (1) EP0077796A1 (enrdf_load_stackoverflow)
JP (1) JPS58500610A (enrdf_load_stackoverflow)
FR (1) FR2504921A1 (enrdf_load_stackoverflow)
WO (1) WO1982003772A1 (enrdf_load_stackoverflow)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0065256A3 (en) * 1981-05-14 1984-01-18 Behringwerke Aktiengesellschaft Process for the preparation of plasminogen, and plasminogen so prepared
US7202066B2 (en) * 2002-01-29 2007-04-10 Carrington Laboratories, Inc. Combination of a growth factor and a protease enzyme
CN117568445A (zh) * 2024-01-18 2024-02-20 西南交通大学 一种tat、pic复合质控品的制备方法及其应用

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0768274B2 (ja) * 1987-03-27 1995-07-26 株式会社ミドリ十字 プラスミノゲンの製造方法
GB8721951D0 (en) * 1987-09-18 1987-10-28 Thrombosis Research Trust Organic compounds
JP2764264B2 (ja) * 1987-10-01 1998-06-11 株式会社ミドリ十字 線溶活性増強剤
DE19903693A1 (de) * 1998-04-24 1999-10-28 Centeon Pharma Gmbh Protease zur Aktivierung des Gerinnungsfaktors VII
DE10023923A1 (de) 1999-06-10 2000-12-14 Aventis Behring Gmbh Verfahren zur Bestimmung der Aktivität der Faktor VII-aktivierenden Protease aus Proteinlösungen

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2711164A1 (de) * 1976-03-18 1977-09-29 Leuven Research & Dev V Z W Lo Antiplasmin und ein antiserum dagegen

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2711164A1 (de) * 1976-03-18 1977-09-29 Leuven Research & Dev V Z W Lo Antiplasmin und ein antiserum dagegen

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Volume 94, No. 23, 08 June 1981, (Columbus, Ohio, US), E. SUENSON et al. "A Novel Method for the Preparation of Plasmin Enabling the Study of Glu-Plasmin and its Role in Fibrinolysis", see page 227, Abstract 187521b, Protides Biol. Fluids, 1980, 28th, 383-6 (Eng.), see the whole Abstract *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0065256A3 (en) * 1981-05-14 1984-01-18 Behringwerke Aktiengesellschaft Process for the preparation of plasminogen, and plasminogen so prepared
US7202066B2 (en) * 2002-01-29 2007-04-10 Carrington Laboratories, Inc. Combination of a growth factor and a protease enzyme
CN117568445A (zh) * 2024-01-18 2024-02-20 西南交通大学 一种tat、pic复合质控品的制备方法及其应用
CN117568445B (zh) * 2024-01-18 2024-04-09 西南交通大学 一种tat、pic复合质控品的制备方法及其应用

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EP0077796A1 (fr) 1983-05-04
FR2504921B1 (enrdf_load_stackoverflow) 1985-05-17
JPS58500610A (ja) 1983-04-21
FR2504921A1 (fr) 1982-11-05

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