EP1280758A1

EP1280758A1 - Gallic acid derivatives and use for exploring endogenetic coagulation

Info

Publication number: EP1280758A1
Application number: EP02722368A
Authority: EP
Inventors: Jean-Paul Roisin; Stéphane Steurs; Gérard Quentin
Original assignee: Diagnostica Stago SAS
Current assignee: Diagnostica Stago SAS
Priority date: 2001-03-23
Filing date: 2002-03-22
Publication date: 2003-02-05
Also published as: AR034221A1; HUP0302136A2; FR2822462A1; MXPA02011565A; FR2822462B1; HUP0302136A3; WO2002076921A1; JP2004519507A; CN100432046C; PL357449A1; US20030157582A1; JP4563648B2; BR0204494A; US7208266B2; HK1054737A1; RU2002131457A; KR20030005359A; CA2407779A1; CN1460098A

Abstract

The invention concerns contact activators for the endogenetic pathway of coagulation and their use for exploring anomalies of coagulation. The inventive activators are gallic acid derivatives and preferably polyethylene glycol gallates.

Description

GALLIC ACID DERIVATIVES AND THEIR USE IN EXPLORING ENDOGENIC COAGULATION

The present application relates to compounds which activate endogenous coagulation and to their use for its exploration.

Coagulation initiated by the "endogenous route" results in vitro from the exposure of blood to contact with a negatively charged surface. In vivo, it secondarily enhances the growth of the fibrin clot.

The exploration of coagulation initiated by the “endogenous pathway” (hereinafter referred to as “endogenous coagulation”) is used in the context of tests for in vitro detection, coagulation abnormalities or for monitoring of patients treated with substances with anticoagulant activity.

The endogenous coagulation pathway is initiated in vitro by the activation of factor XII (F.XII) in contact with negative charges. In the presence of other enzymatic factors such as factor XI (F.XI), pre-Kalikrein (PK), high molecular weight kininogens (KHPM), factor IX (F. IX) also called anti-hemophilic factor B and a cofactor, anti-hemophilic factor A (F.VIIIc), a series of cascade activations, of the enzymatic type, activates in the simultaneous presence of platelet phospholipids (PF3) and calcium (Ca ++). At the end of this series of activations, the activated factor IX (F.IXa) in turn initiates the cascade activation of a second series of enzymes, comprising factor X

(FX), factor II (F. II) also called prothrombin; this activation takes place in the presence of platelet factor (PF3) of calcium (Ca ++) and a cofactor, factor V (FV). This second cascade of activations results in the formation of thrombin (Fila), which transforms fibrinogen into fibrin, a major constituent of the blood or plasma clot. The simplified diagram of the endogenous (in vitro) pathway of coagulation can be illustrated as follows:

F.XII

Negative charges KHPM - PK F.XI

F.IX

FXIIa

Ca ++

F.X

F. JXIa FVIIIc

PF3

F.IXa F.II

FX,

Ca ++ Fibrinogen

F.V

PF3 F.IIa →

Fibrin

In general, the exploration of coagulation is practiced to look for in a patient, a biological anomaly which can participate in a hemorrhagic syndrome or to follow the consequences of a pathology at the level of coagulation or also to ensure a follow-up of medical treatment likely to directly or indirectly modify coagulation.

To this end, various tests are currently proposed to seek, from the qualitative or quantitative observation of factors intervening in the endogenous coagulation pathway, various anomalies affecting the formation of fibrin resulting from the transformation of fibrinogen by thrombin. .

Different tests are available on the market to explore endogenous coagulation (also called intrinsic coagulation) and in particular we will mention the method of determining the time of partially activated thromboplastin.

(“Activated Partial Thromboplastin Time” or APTT) which can detect on a plasma sample, abnormalities or deficits occurring at the level of factors involved in the endogenous pathway, anomalies resulting in coagulation times outside the limits of normality.

The APTT test is used in particular for the detection of the most frequent constitutional bleeding disorders, namely Willebrand's disease and hemophilia A and B. This test is also frequently used for the control of heparin therapy.

Schematically, the principle of an APTT test consists in adding to the plasma sample tested, an activator of factor XII negatively charged (contact activator) and, a substitute for phospholipids of the platelet membrane as well as calcium ions.

The measured coagulation time corresponds to the period between the addition of the contact activator, the platelet substitute, calcium, and the appearance of a coagulum. This is the time required to transform 1% of the plasma prothrombin into thrombin. The fibrin training which then takes place takes place over a short period of time. Calcium is the trigger for the system.

There are a relatively large number of activators suitable for carrying out APTT tests. These include, for example, solid activators such as silica, glass or kaolin, or liquid activators, such as ellagic acid and its derivatives or sulfatides.

Although reagents based on solid activators remain widely used, these are not without their drawbacks. Thus, the kaolin or silica particles which they contain tend to sediment during the coagulation test. It is therefore necessary to provide continuous stirring, hence a problem of instability of the reagents and difficulties for carrying out tests in automated devices. In addition, activators such as kaolin do not allow optical coagulation measurements to be made.

Liquid activators are an alternative to solid activators.

However, it is known that activators such as ellagic acid are likely to induce variations in test results. In addition, their sensitivity is reduced compared to that of the previous ones. Also, various associations have have been proposed to improve the performance of such reagents. For example, in patent application WO 91/16453, a reagent based on ellagic acid, phenol and a metal ion is recommended, preferably associated with dextran sulfate. Other contact activators in solution have been described in particular in patent EP 0525035 for which the contact activator is a hydroxy-substituted aromatic compound chosen from propyl gallate or tannin, in patent application WO 98/44352 for which the contact activator can in particular be ellagic acid or in American patent US 5,550,028 in which the contact activator is tetrahydroxy-1, 4-quinone.

Sulfatides have also been proposed as activating compounds for intrinsic coagulation. However, their level of activity has not always been considered sufficient, and these compounds also have the disadvantage of being relatively expensive compared to the other available activators. There is therefore still a need to have new coagulation reagents or to improve pre-existing reagents in order to achieve products which, while remaining economically attractive, have sufficient stability and sensitivity, induce activation times. short and can be used on analysis machines for carrying out tests in large series.

The invention is concerned with such alternative contact activators capable of participating in the endogenous coagulation pathway and in the production of systems and tests making it possible to explore this endogenous coagulation pathway and to detect abnormalities therein. The invention also relates to the use of these activators in an endogenous coagulation exploration test, of the APTT type.

In a particular embodiment of the invention, the endogenous coagulation activator is associated with compounds which make it possible to modulate the response sensitivity of a reagent used in APTT, with respect to certain coagulation time anomalies encountered in pathologies. The inventors have been interested, in the context of the search for substances capable of activating endogenous coagulation, in compounds having advantageous solubility properties compared to the compounds used until now as activators of coagulation in APTT tests and in particular have sought, from gallic acid, derivatives capable of activating endogenous coagulation.

In a field unrelated to that of the invention, patent application DE 2750560 A1 describes esters obtained by esterification of gallic acid with ethylene glycol or various polyethylene glycols. These esters are used as protective material for glass fibers incorporated into a cement-based composite material.

Still in another field, that of antioxidant compounds which can be used against the deterioration of fats and oils in food, Weetall HH (Applied Biochemistry and Biotechnology vol. 11, 1985) proposes to synthesize esters of gallic acid using particular alcohols or diols. The publication specifies that if esters and diol esters have indeed been synthesized in the presence of tannase from Aspergillus niger, the precise nature of the products remains to be determined.

The reagents in question are contact activators of the endogenous coagulation pathway, capable of having a procoagulant activity, that is to say of activating factor XII into activated factor Xlla, after incubation of a plasma sample, 37 ° C in the presence of PF3 and calcium.

Preferably for carrying out the invention, these compounds are soluble or capable of being made soluble if necessary after modification. The subject of the invention is therefore a compound of formula

R

AT

(I) in which:

- A is chosen from the following formulas:

M ^{q +} representing a cation, q being equal to 1 or 2 and p being such that pxq = 2,

- R represents or

m being equal to 2, 3, 4 or 5 and n being an integer between 1 and

170 or,

AA being a natural or exotic amino acid and r being an integer between 1 and 10.

Among the compounds thus defined, the invention relates, according to a particular embodiment, those which have the advantage of being able to be put in solution and in particular in aqueous solution. Preferably, they are completely soluble.

The side chain constituted by R and the meaning of which has been given above is such that the value of n is preferably equal to or greater than 1 and less than or equal to 170. In particular, n is between 1 and 50, for example between 1 and 30, advantageously between 1 and 20.

In a particularly preferred manner, n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. A first family of preferred compounds according to the invention is defined by the following formula:

in which

R represents or

m being equal to 2, 3, 4 or 5 and n being an integer between 1 and

170 or,

- AAι-AA ₂ - -AA _r

AA being a natural or exotic amino acid and r being an integer between 1 and 10. According to a first preferred embodiment of the invention, in one of the preceding formulas (I) or (II), R represents

m and n meeting the definitions given above.

Advantageously in the context of the invention, the compound of formula (II) is such that:

R represents with n being an integer between 1 and 170 and preferably between 1 and 10.

This preferred form of the compound (II) according to the invention corresponds to the oxidized form of the polyethylene glycol gallates.

This oxidized form is the quinone form resulting from the oxidation in an alkaline medium of one of the -OHs of the aromatic nucleus present in formula (II).

According to another aspect, the compounds of the invention can also be found to constitute a family corresponding to the following formula corresponding to an ionized form of formula (III), associated with mono or bivalent cations defined within the framework of the invention:

in which: R represents

m being equal to 2, 3, 4 or 5 and n being an integer between 1 and 170 and

M ^{q +} is a cation with q equal to 1 or 2 and p being such that pxq = 2.

This second particular family of compounds according to the invention corresponding to formula (III) comprises, in addition to the quinone in position 4, the 2 -OH functions (in positions 3 and 5) in ionized form (sodium alcoholate) formed when the pH middle is greater than pKa. In particular, the invention relates to the oxidized mono- or polyethylene glycol gallate, in its basic form, said compound being associated with cations, in an alkaline medium.

Preferably, it is the basic form of ethylene glycol gallate in which R has the meaning given above with m = 2 and n = 1.

This basic form of oxidized ethylene glycol gallate is soluble in water.

The pH of the composition obtained must be lowered to a physiologically acceptable value for its use. The shift in balance to sodium quinone (according to formula III) at physiological pH can be accelerated and stabilized by the addition of cations giving it immediate coagulant properties.

Among the compounds corresponding to formula (I) identified above, the invention advantageously relates to the compounds corresponding to the following definition:

in which

- A is:

- R is - CH ₂ - CH ₂ - OH.

M ^{q +} being a cation, q being equal to 1 or 2 and p being such that pxq = 2. These compounds are respectively the acid form of oxidized ethylene glycol gallate and the basic or salified form of oxidized ethylene glycol gallate.

The basic salted quinone form is characterized by a yellow color detectable at a wavelength of 355 nm and is particularly advantageous because of its solubility in aqueous medium.

For the production of the compounds according to the invention as defined above and also in the following pages, the cation M ^{q +} is advantageously a metal cation, in particular Mn ²⁺ or Cu ²⁺ or Co ^{2 + -}

It can alternatively consist of a non-metallic cation such as a counterion which is provided during the preparation of the compounds of the invention by heating in an alkaline medium. Such a preparation process is defined in the following pages and the cation M ^{q +} is advantageously Na ⁺ , K ⁺ or NH ⁺ .

The cation concentration can be defined as a function of the coagulation time which it is desired to observe for a normal control sample. Advantageously, for metal cations, this concentration is between 5 and 15 μM, preferably around 10 μM.

According to another particular embodiment, the invention relates to a family of compounds corresponding to formula (I)

in which

A is chosen from the following formulas

- R represents NH-CH ₂ -CH ₂ -NH ₂ with n being an integer between 1 and 170. - "n

In particular in the context of the definition of R, n is between 1 and 50, for example between 1 and 30, advantageously between 1 and 20.

A preferred form of the polyethylene diamine gallate is the quinone form resulting from the oxidation of one of the -OHs of the aromatic ring. It corresponds to the following formula j_

in which n is an integer between 1 and 170; preferably between 1 and 50, for example between 1 and 30, advantageously 1 and 20.

In a particularly preferred manner in the above formulas, n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. Advantageously, it is ethylene diamine gallate (n = 1).

The compounds of the invention can also be in the form associated with mono or bivalent cations in particular with metal cations. These compounds correspond to the following formula:

- in which n is an integer between 1 and 170.

- M ^{q +} is a cation, q is equal to 1 or 2 and p is such that pxq = 2.

In particular, n is between 1 and 50, for example between 1 and 30, advantageously between 1 and 20.

In a particularly preferred manner, n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

According to a third embodiment, the invention relates to a compound characterized in that it corresponds to formula (I)

in which

- A is chosen from the following formulas:

M ^{q +} representing a cation, q being equal to 1 or 2 and p being such that pxq = 2. - R is an amino acid residue or a peptide. Preferably, as indicated in the case of the first two embodiments of the invention, the compounds of the invention are in the quinone form resulting from the oxidation of one of the -OHs of the aromatic ring:

or, in the form associated with mono-, or bivalent cations:

in which M, q and p have the above definitions.

A preferred amino acid for the production of the above compounds is serine where

O

II = - NH-CH-C-OH I

CH ₂ I OH

In this case, the compound of the invention advantageously has the following formula:

M, q and p corresponding to the previous definitions.

The subject of the invention is also a composition, characterized in that it comprises a mixture of several compounds, in particular a mixture of at least two compounds among those identified previously, whether they are compounds belonging to the same family. or to the same group or, on the contrary, to compounds belonging to families or to groups different from those defined above.

In particular, a composition is characterized in that it comprises at least two different compounds of formula:

these compounds being identical or different for the group A and being identical or different for the group R, A and R having the meanings given above. The invention preferably relates to a composition characterized in that it comprises a mixture of compounds (IV) and (V), constituting respectively the acid and basic forms of the oxidized ethylene glycol gallate (GEGox), of the following formulas:

M, q and p having the definitions indicated above. This composition therefore comprises a mixture of the acid and basic forms of oxidized ethylene glycol gallate, corresponding to the products of the keto-enolic equilibrium obtained from ethylene glycol gallate when it is heated in an alkaline medium, or put in the presence of metal cations. The balance between the acid and basic forms of oxidized ethylene glycol gallate can be shifted to one or other of these forms by adjusting the reaction conditions, and in particular by modifying the pH or the concentration of cations. present in the reaction medium.

Advantageously, the composition thus defined is characterized in that it is a solution and preferably an aqueous solution.

The subject of the invention is in particular, within the framework of the preceding definition, a composition in which the basic form corresponding to formula (V) of oxidized ethylene glycol gallate is predominant.

This basic, water-soluble form has entirely advantageous properties for constituting a composition which activates endogenous coagulation, in particular for carrying out an in vitro test for the exploration of endogenous coagulation.

According to a particular embodiment of the invention, the composition corresponding to one of the preceding definitions has a physiologically acceptable alkaline pH. This pH is adjusted after oxidation of the GEG, for example in the presence of buffer molecules: MOPS ('acid - (/ V-Morpholino) propanesulfonic) or TRICINE: (Λ / -Tris (hydroxymethyl) methyl-glycine). Advantageously, the MOPS molecule is chosen.

According to another embodiment of the invention, the composition is characterized in that it comprises a mixture of the different forms of gallate, produced by the keto-enolic balance and corresponding to the formula:

M, q and p corresponding to one of the definitions above.

The presence of one or the other of the forms of the enolate of the gallic acid ester is dependent on the conditions of the reaction and the keto-enolic balance can therefore be shifted to obtain a greater or lesser amount. of one of the forms of gallate produced.

In the context of the invention, there will be a tendency to favor the presence of the basic form of the oxidized gallate, of formula (V).

The preferred compounds and compositions according to the invention can also be defined as resulting from the implementation of the process comprising the following steps:

- Esterification of gallic acid with ethylene glycol and recovery of one of the reaction products consisting of ethylene glycol gallate (GEG) of formula:

- Displacement of the keto-enolic balance by bringing the GEG into contact with an alkaline medium and heating to a temperature between 70 and 90 ° C. The reaction for forming the keto-enolic balance is advantageously carried out for a period of 24 h at 80 ° C. The preparation according to the above process leads to a mixture of the 3 forms of gallate, said mixture being able to be moved towards the oxidized forms.

Preferably, the composition thus obtained is characterized in that the method further comprises the steps of: - stabilization of the keto-enolic balance obtained by adjusting the pH. - recovery of the compounds of formula (IV) and (V) in solution, at alkaline pH.

The oxidized forms of GEG are formed in the presence of sodium hydroxide and in particular, excess sodium hydroxide will be used to shift the keto-enolic balance to the basic form of oxidized ethylene glycol gallate. The heating treatment is carried out at a temperature between 70 and 90 ° C, preferably a temperature of 80 ° C for a period of 24 hours.

The pH of the solution is an alkaline pH, and the action of the soda can be stopped by the addition of a buffer which contributes to stabilizing the keto-enolic balance obtained. In a particularly preferred manner, the pH of the composition according to the invention is between 7.6 and 7.8.

The added buffer can for example be a MOPS or TRIS buffer.

The composition contains the compounds according to the invention in a concentration of between 100 and 1000 μM.

The compounds and compositions thus defined in the context of the invention are capable of activating endogenous coagulation on contact with a plasma sample, in other words are capable of activating factor XII into factor Xlla, after having was brought into contact and incubated with a plasma sample in the presence of PF3 and calcium, at a temperature of approximately 37 ° C.

The invention also relates to a reagent system which can be used for the in vitro examination of endogenous coagulation on a blood sample, which system comprises, among its reagents, a compound or composition as defined above.

A preferred reagent according to the invention comprises a salt of gallic acid

(gallate) in activated form, corresponding to the formula:

M, q and p corresponding to the above definitions.

R represents or

with m equal to 2, 3, 4 or 5 and n being an integer between 1 and 170 or,

- AA-t-AAr -AA _r AA being a natural or exotic amino acid and r being an integer between 1 and 10.

In a particularly preferred manner in the above formulas, n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

The determination of the concentrations of the compounds according to the invention takes account of the fact that in the APTT system, the expected coagulation times for plasmas originating from healthy subjects are generally between 28 and 40 seconds. In this context, the concentration of the compounds according to the invention and of the other constituents used for the coagulation test can be defined from the coagulation time sought for samples of healthy plasmas. In particular, the concentration of the compounds according to the invention can vary from 100 to 1000 μM. The optimization of the concentrations of the main constituents of the reagent aims to obtain, on a sample library of plasmas from at least 30 healthy subjects, clotting times included in this interval.

In addition, the optimization of the biochemical environmental parameters of the active principles of the APTT reagent makes it possible, by lengthening the times, to specifically adjust the detection sensitivity of certain pathologies covered by an APTT test. Thus, for example, it an extension of time compared to the healthy subject control will be sought, of at least 20% in the cases of Lupus Anticoagulant syndromes. For plasmas from subjects treated with unfractionated heparin-type anticoagulants, time extensions comprised in ratios of 1.5 to 3.0 times the control time will be sought. For plasmas of hemophiliacs type A and B, the greatest possible sensitivity is required and we will seek to obtain significant time extensions.

Preferably, the reagent thus formed comprises activated ethylene glycol gallate. The activity results from the keto-enolic balance obtained and from its stabilization at a physiologically acceptable pH.

In a particular embodiment of the invention, the concentration of gallate, in particular, of ethylene glycol gallate in the composition or the reagent system described, is advantageously between 100 and 1000 μM, preferably 100 μM.

The subject of the invention is also any compound or any composition among those defined above, for constituting a reagent for exploring endogenous coagulation, in particular for carrying out an APTT test, associated with a constituent having a modulation activity on the response sensitivity of the compound or composition of the invention.

The modulation of the sensitivity can consist of an attenuation of the lengthening of the coagulation time observed on samples of plasmas from patients affected by a pathology of the Lupus Anticoagulant type or from patients under anticoagulant treatment for example based on unfractionated heparin. Alternatively, this sensitivity modulation can consist in lowering the coagulation time measured on samples of non-pathological plasmas.

One such modulator of the sensitivity of a reagent prepared with the compounds or compositions of the invention is a polyamino acid, preferably partially substituted, the substitution affecting the amino groups, which are substituted by succinyl groups (- CO-CH ₂ -CH ₂ -COOH) or glutaryl (-QC (CH ₂ ) ₃ CO). The substitution must not affect the spatial conformation and / or the stability of the starting constituent, under conditions such that the substituted polyamino acid could no longer exert its sensitivity modulating effect. For these reasons, a fully substituted polyamino acid would be excluded a priori.

According to a particular embodiment of the invention, the modulator constituent is a substituted polylysine (either D- or L-polylysine or a mixture of these), in particular succinylated and for example it is L-polylysine substituted , advantageously succinylated L-polylysine. The inventors notably used the succinylated L-polylysine available in the catalog of the SIGMA company (2001-2002). Glutarylated L-Polylysine can also be used.

Preferably, succinylated L-polylysine is used, in a molecular weight range ranging from 20,000 to 300,000 Da, preferably from 50,000 to 300,000 Da.

The concentration, in the reagent prepared to carry out the APTT test, of the modulating component, and in particular of succinylated L-Polylysine is in the range of 10 to 100 μg / ml, preferably from 50 to 100 μg / ml. The substituted polyamino acid, in particular succinylated polylysine, can be added at any time during the preparation of the reagent intended for use in an endogenous coagulation exploration test, of the APTT type.

The reagent system according to the invention comprises, in addition to the contact activator of the endogenous coagulation pathway, a substitute for the phospholipids of the platelet membrane. This substitute can be cephalin: suspension of phospholipids extracted from mammalian brains. The invention also relates to a reagent system which further comprises, in a form separate from the other reagents, Ca ⁺ ions for triggering the activation of factors involved in the endogenous pathway.

The reagents of the invention and, where appropriate, the other constituents used in the implementation of an APTT test can be combined in the form of a kit.

The use of the compounds, compositions or reagents according to the invention for the exploration of coagulation and more particularly of endogenous coagulation, can be implemented for measuring the time of a blood sample and in particular a plasma sample. partially activated thromboplastin (APTT). In this regard, use is advantageously made of the use of platelet-poor plasma.

As part of this measurement, the usual conditions for carrying out this test are implemented, in particular with regard to the preparation of the samples. Thus, the sample of the plasma taken can initially have been treated for example with citrate so as to complex the Ca ²⁺ ions contained in the sample so that the sample can no longer coagulate. The sample is then incubated with a compound or composition according to the invention as a coagulation activator and phospholipid substitutes. Ca ²⁺ ions are then added in an amount equivalent to that which has been complexed, to trigger coagulation.

The activity of the compounds or compositions according to the invention requires that their pH has a physiologically acceptable value.

The compounds, compositions or reagents according to the invention can also be used to determine the coagulation time for whole blood or the plasma coagulation time.

The invention therefore allows the use of a compound or a composition as described above, for the preparation of a reagent system for examining the coagulation factors involved in the endogenous pathway. coagulation. The factors concerned are mainly factors XII, XI, IX, VIII. In this context, the invention relates for example to the detection of anomalies linked to von Willebrand disease, to hemophilia A and B or to the detection of anticoagulants. These anomalies lead to a measured APTT time outside of normal values, these usually being between 20 and 40 seconds.

The invention therefore also relates to a method for measuring the time of partially activated thromboplastin (APTT) comprising the steps of:

- bringing a platelet-poor plasma sample into contact with compounds or compositions according to the invention, in the presence of phospholipid substitutes for the platelet membrane, under conditions allowing activation of the endogenous coagulation pathway,

- addition of Ca ²⁺ ions in an amount equivalent to the amount of these complexed ions in the plasma sample.

- measurement of the time necessary for the transformation of fibrinogen into fibrin. In particular, within the framework of the implementation of the above method, the plasma sample consists of a plasma poor in platelets and a coagulation activator is incubated with the plasma for a period of approximately 2 to 5 minutes, at 37 ° C. The membrane phospholipid substitutes are either added at the start of the reaction, or mixed with the activator, or added individually during the reaction.

Then the calcium ions are added, in the form for example of calcium chloride with a concentration of between 20 mM and 25 mM. The time required for fibrin formation is then measured.

The time measured indicates, when it is outside the time interval corresponding to a normal coagulation situation (that is to say between 20 and 40 seconds), that one of the parameters of the endogenous coagulation pathway is at an abnormal level, including absent, or that there are anticoagulant compounds in the plasma tested.

When the plasma studied comprises a factor from the endogenous coagulation pathway at an abnormal rate, the invention then allows a quantitative analysis of the factor in question, using a system depressed with this factor, and in which all the other parameters are also introduced in excess. In this system, after adding the plasma to be studied, only the factor to be measured is in a limiting quantity.

The invention also relates to a process for the preparation of a compound of the following formula:

in which

A represents one or more of the compounds chosen from the following formulas:

M ^{q +} representing a cation, q being equal to 1 or 2 and p being such that pxq = 2.

R represents o,

m being equal to 2, 3, 4 or 5 and n being an integer between 1 and

170 or,

- AA-ι-AA ₂ - -AA _r AA being a natural or exotic amino acid and r being an integer between 1 and 10. said process comprising the steps of:

- esterification of gallic acid, in the presence of a compound having at least one alcohol function or amidation of gallic acid in the presence of a compound having at least one amino function,

- setting. contact of the gallic acid derivative obtained, with OH- ions to form a compound of formula

addition of a metal cation M ^{q +} (with q equal to 1 or 2), under conditions allowing the formation of a complex of formula:

stabilization of the balance obtained, between the compounds (II) and (III) at a physiologically acceptable pH, by the addition of a buffer such as MOPS or TRICINE

- recovery of the solution obtained.

This process is preferably such that the esterification is carried out in the presence of ethylene glycol to form the ethylene glycol gallate, thereby making it possible to recover, at the end of the reactions implemented, the products of equilibrium keto-enolic including the acid and basic forms of oxidized ethylene glycol gallate and, where appropriate, a residual amount of ethylene glycol gallate.

The definitions of preferred families or groups of compounds or compositions according to the invention are also concerned with the definition of the process.

According to a particular embodiment of the invention, one may wish to recover mainly the basic form of formula (V) of oxidized ethylene glycol gallate and to do this, it may be advantageous to shift the balance towards this basic form. Another method has been developed by the inventors for preparing a compound of formula A according to the invention having activating properties of endogenous coagulation. This process includes the steps of:

- esterification of gallic acid,

heating the gallic acid ester obtained at a temperature between 70 and 90 ° C, preferably at 80 ° C for a period between

16h and 24h preferably 24h at alkaline pH, in a buffer solution,

- recovery of the different forms of the gallic acid ester present in solution.

Other properties and advantages of the invention appear on reading the examples which follow which constitute preferred embodiments of the subject of the invention.

EXAMPLE 1

In this example, the coagulation time is measured with APTT reagents manufactured according to the invention using various cations. Different types of plasmas are tested, normal on the one hand, and representative of pathologies covered by an APTT test on the other:

- CK - Perst = reference APTT reagent whose contact activator is kaolin,

- pool N = mixture in equivalent quantities of plasmas from healthy subjects, - CCN = Coag. Normal Control: Plasma quality control normal level,

- CCP = Coag. Patho control: Plasma quality control pathological level,

- Heparinized = plasmas from patients receiving anticoagulant treatment based on unfractionated heparin in the form of sodium or calcium salts,

- Lupus = plasma from patients with Lupus Anticoagulant syndrome,

- Def. F City = plasma of a patient with a f City deficiency (Hemophiliac A).

The measurements are carried out on a hemostasis machine (STA - Diagnostica Stago).

EXAMPLE 2:

In the following example, the coagulation times of different plasmas are determined from APTT reagents obtained from amides or esters of gallic acid.

- Normal plasmas = pool in equivalent quantities of plasmas of normal subjects,

- CCN = Coag. Control Normal: quality control normal level, - CCP = Coag. Control Patho: pathological level quality control, - Heparinized = plasmas of patients receiving an anticoagulant treatment based on unfractionated heparin in the form of sodium or calcium salts.

- Lupus = plasma from patients with Lupus Anticoagulants syndrome

- F VIII def. = plasma from patients with F city deficiency (hemophilia A)

From this table, it therefore appears that the results closest to those obtained with the reference reagent, CK-Perst, are those obtained with ethylene glycol gallate.

EXAMPLE 3

After heating for 24 hours at 80 ° C, the ethylene glycol gallate in solution

500 μM, buffered to pH 7.00, becomes active in an APTT coagulation test.

The table below shows the activation kinetics of the molecule. The stimulating activity of the ethylene glycol gallate solution is compared to that of a reference reagent: CK-PREST, APTT reagent whose activator is kaolin. The device used to measure coagulation times is a STA Diagnostica Stago hemostasis machine (serial number 307)

In parallel with the appearance of coagulating activity, a yellow coloration measurable at 355 nm appears.

The plasmas tested are quality control plasmas:

CCN = Coag Normal Control. CCP = Coag Pathological Control lot n ° 980901. APTT reagent produced = Ethylene glycol gallate at 500 μM oxidized at 80 ° C + Tricin 50 mM, pH 7.00 + Cephalin (substitute for platelet phospholipids).

EXAMPLE 4:

Various buffer molecules, chosen from those usually used in biological systems, make it possible to obtain significant procoagulant activities within a reasonable time of heating (<4 days).

10 those of the reference reagent CK-PREST. These are the following molecules:

TRICIN: / V-Tris (hydroxymethyl) methyl-glycine

HEPES: Λ / -2-Hydroxyethylpiperazine- / V-2-ethanesulfonic acid - MOPS: acid- (V-Morpholino) propanesulfonic

- TRIS: [rr / s- (hydroxymethyl) aminomethane]

The Tricine buffer and MOPS are preferably used, because with TRIS, a precipitate appears during the activation of the ethylene glycol gallate, and with the HEPES buffer, it is necessary to conduct the heating for a longer period. In the following table, the performances obtained are recorded on 2 control plasmas (normal and pathological), 1 pool of plasmas from healthy donors and 4 plasmas representative of pathologies detectable in an APTT-type coagulation system;

* Pool N = pool of several normal plasmas

* CCN = Coag. Control Normal: normal level quality control

* CCP = Coag. Pathological control: quality control at pathological level,

* LA: plasma of patients with Lupus Anticoagulant syndrome * Hep: Plasma of patients receiving anticoagulant treatment based on unfractionated heparin in the form of calcium salt. Two plasmas, with 2 heparinemia levels are tested: 0.09 and 0.34 International units per ml.

* Def. City = hemophiliac plasma A at 40% activity in City factor.

The coagulation times obtained are expressed in seconds.

The response sensitivity obtained with each of the reagents manufactured with the 4 buffers is objectified by the calculation of a ratio (R) carried out by dividing the coagulation time obtained on the patient's plasma, by that obtained with the normal pool, or the coagulation time of the pathological control (CCP) by that of the normal control (CCN).

Patient clotting time

K " ^~

Pool N coagulation time

EXAMPLE 5:

The use of an APTT reagent can be conceived according to 2 purposes:

- global exploration of the endogenous pathway of blood coagulation, with a normal-abnormal response;

10 - analytical and quantitative exploration of the coagulation factors involved in the endogenous coagulation pathway: F XII-XI-IX-Vlllc.

- It is possible to assay in a patient plasma, any coagulating factor of the endogenous path, using a system specifically screened for this factor. All the other parameters being entered in excess, only the

15 factor to be dosed is then in limiting quantity. Its activity becomes quantifiable after comparison with a calibration curve beforehand established under the same conditions. Depending on the size of the deficit, the coagulation time is more or less extended (see example for the City factor).

Sensitivity of the reagent APTT-ethylene glycol gallate, to FVHlc.com deficit compared to the reference CK-Perst

20 40 60 80 100 120 140

F City (%) EXAMPLE 6

The coagulating activity of an APTT reagent requires the joint action of two active ingredients:

- a molecule activating the contact phase of the endogenous pathway. In this case, it is ethylene glycol gallate activated by heating to 80 ° C.

- a substitute for phospholipids in the platelet membrane, cephalin, a preparation obtained from chloroform extraction from rabbit brains.

- The optimal activity of the APTT reagent thus formed depends, however, on the concentrations of ethylene glycol and cephalin gailate.

- The following tables summarize the results obtained on plasmas representative of different pathologies as well as on controls.

CK Perst lot n ° 981201 34.2 47.6 38.3 57.1 121.5 38.9

The choice can be made for concentrations between 100 and 1000 μM. Our preference is at a concentration of 100 μM. Determination of the optimal dilution of cephalin

CK Perst lot n ° 981201 33.0 38.6 84.8 49.5

EXAMPLE 7 Choice of Manganese Chloride Concentration

In this example, the coagulation time is measured with APTT reagents produced according to the invention, and for which the concentration of chloride of

Manganese is variable.

The plasmas tested are Quality Control plasmas. Two levels are used: a normal level and a pathological level. The commercial reagent CK Perst is tested in parallel and constitutes the reference.

Preferably, the 10 μM concentration is used.

EXAMPLE 8:

It may be advantageous to be able to modulate the response sensitivity of an APTT reagent with respect to certain coagulation time anomalies encountered in various pathophysiological situations. This modulation aims to obtain a reagent with specific characteristics in terms of response sensitivity, reproducible from one manufacturing batch to another.

The inventors have observed that by adding succinylated L-Polylysine to the reagent APTT based on ethylene glycol gallate, it was possible to attenuate the coagulation time prolongations observable on plasmas originating from patients with Lupus Anticoagulants syndrome or of patients on anticoagulant therapy based on unfractionated heparin. Similarly, it has been observed that the use of succinylated L-Polylysine makes it possible to reduce the coagulation time measured on plasmas originating from a normal population. These observations can be obtained by the use of a polyamino acid more or less substituted at the level of the amino groups by succinyl or glytaryl groups in order to define the level of sensitivity of the reagent. One of the preferences of the invention consists in using partially succinylated L-Polylysine. The following tables summarize the results obtained on the various control plasmas used in the context of the examples. • CCN: Coag Control Normal

CCP: Pathological Coag Control

Pool N: Pool of several normal plasmas

LA: Plasma from patients with Lupus Anticoagulant Syndrome

Heparin: Plasmas from patients receiving unfractionated heparin-based anticoagulation therapy

• Def. VIII C: Hemophiliac A plasma with 40% activity in F. VIII C.

For the use of succinylated L-polylysine in the context of the invention, the molecular weight of succinylated L-polylysine is preferably in the range 50,000 - 300,000 Da and the concentration chosen preferably for this range is 50 μg / ml of reagent.

Claims

1. Compound of the following formula

in which

A is chosen from the following formulas

M ⁺ representing a cation and q can be equal to 1 or 2 and p is such that pxq = 2 or

m being equal to 2, 3, 4 or 5 and n being an integer between 1 and 170 or,

- AA ₁ -AA ₂ - -AA _r

AA being a natural or exotic amino acid and r being an integer between 1 and 10, it being understood that the compound is not

when m is 2, 3 or 4.

2. Compound according to claim 1, of the following formula

in which R represents

n being an integer between 1 and 170.

3. Compound according to claim 1, of the following formula

(III) in which

R represents with n being an integer between 1 and 17

- M ⁺ is a cation, q is equal to 1 or 2 and p is such that pxq = 2.

4. Compound according to claim 2 or claim 3 corresponding to one of the formulas (II) or (III) in which R represents

n being an integer between 1 and 10.

5. A compound according to claim 4 in which n is equal to 1.

6. Compound according to any one of claims 1 to 5 of the following formula:

in which -A is

M ⁺ being a cation, q being equal to 1 or 2 and p being such that pxq = 2, -Rest-CH ₂ -CH ₂ -OH.

7. Compound according to claim 1, of the following formula:

in which: -A is

M ⁺ being a cation, q being equal to 1 or 2 and p being such that pxq = 2, - R is a residue of natural or exotic amino acid or a peptide, preferably serine.

8. Composition characterized in that it comprises a mixture of at least two different compounds of formula:

the two compounds in question being identical or different for the group A and being identical or different for the group R, said groups A and R being such that: - A is chosen from the following formulas:

M ⁺ representing a cation, q being equal to 1 or 2 and p being such that pxq = 2;

R represents, or

m being equal to 2, 3, 4 or 5 and n being an integer between 1 and 170 preferably between 1 and 50, 1 and 30 or 1 and 20 or,

9. Composition according to claim 8, characterized in that it comprises a mixture of compounds (IV) and (V), constituting respectively the acid and basic forms of the oxidized ethylene glycol gallate (GEGox), of the following formulas:

in which M ⁺ is a cation, q is equal to 1 or 2 and p is such that pxq = 2.

10. Composition according to any one of claims 8 or 9, characterized in that it comprises the different forms of Ethylene Glycol Gallate produced by the keto-enolic balance and corresponding to the formula

in which

A is chosen from the following formulas

M ⁺ representing a cation, q being equal to 1 or 2 and p being such that pxq = 2,

- R is - CH ₂ - CH ₂ - OH.

11. Compound capable of being obtained by implementing the process comprising the following steps: esterification of gallic acid with ethylene glycol and recovery of one of the reaction products consisting of ethylene glycol gallate ( GEG) of formula:

- displacement of the keto-enolic balance by bringing the GEG into contact with an alkaline medium, heating to a temperature between 70 and 90 ° C, for a period of 24 hours.

12. Compound according to claim 11, characterized in that the method further comprises the steps of:

- stabilization of the keto-enolic balance obtained by adjusting the pH,

- recovery of the compounds of formula (IV) and (V) in solution, at alkaline pH.

13. Compound likely to be obtained by implementing the following steps:

- esterification of gallic acid, heating the gallic acid ester obtained at a temperature between 70 and 90 ° C, for a period of between 16 and 24 hours at alkaline pH, in a buffer solution,

- recovery of the different forms of the gallic acid ester present in solution.

14. Compound according to any one of claims 1 to 7, 11 to 13, characterized .in that it (it) is in the form of an aqueous solution.

15. Composition characterized in that it comprises a mixture of compounds capable of being obtained according to a process according to any one of claims 1 to 13.

16. Composition according to any one of claims 8 to 10, characterized in that it mainly comprises basic GEGox of formula:

M ⁺ being a cation, q being equal to 1 or 2 and p being such that pxq = 2.

17. Composition according to any one of claims 8 to 10 or 16, characterized in that it contains, in the form of a stabilized balance, the compounds of formulas (IV) and (V) and in that it has physiologically acceptable pH.

18. Composition according to any one of claims 8 to 10 or 16, or 17, characterized in that it contains a mixture of the compounds of formulas (IV) and (V) and in that its pH is between 7, 6 and 7.8.

19. Composition according to any one of claims 8 to 10 or 16 to 18, capable of activating endogenous coagulation on contact with a sample of plasma, further comprising a buffer chosen from MOPS or TRICINE buffers.

20. Composition according to any one of claims 8 to 10 or 16 to

19, characterized in that the compounds it contains are in sufficient concentration to activate endogenous coagulation on contact with a plasma sample.

21. Compound according to any one of claims 1 to 7, characterized in that the cation is a metal cation.

22. Compound according to claim 21, characterized in that the metal cation is chosen from Mn ²⁺ 'Cu ²⁺ or Co ^{2 + -}

23. Composition according to any one of claims 8 to 10 or 15 to

20, characterized in that the cation is a metal cation.

24. Composition according to claim 23, characterized in that the metal cation is chosen from Mn ²⁺ , Cu ²⁺ or Co ²⁺ .

25. A reagent system usable for the in vitro examination of endogenous coagulation, comprising a compound according to any one of claims 1 to 7 or 11 to 14 or 21, 22 or a compound corresponding to the formula

in which m is equal to 2, 3 or 4, or a composition according to any one of claims 8 to 10 or 15 to 20 or 23, 24 under conditions such that it (it) constitutes an activator of endogenous coagulation , said system further comprising a substitute for phospholipids of the platelet membrane.

26. Reagent system according to claim 25, characterized in that the endogenous coagulation activator comprises GEG in activated form corresponding to the formula:

in which - M ⁺ is a cation, q is equal to 1 or 2 and p is such that pxq = 2,

R represents

m being equal to 2, 3, 4 or 5 and n being an integer between 1 and 170 or,

- AAι-AA ₂ - -AA _r

27. Composition according to any one of claims 8 to 10 or 15 to 20 or 23, 24 or reagents according to one of claims 25 or 26, characterized in that its GEG concentration is between 100 and 1000 μM.

28. Compound according to any one of claims 1 to 7, or a compound corresponding to the formula

in which m is equal to 2, 3 or 4, or composition according to any one of Claims 8 to 24 or 27 or reagent according to any one of Claims 25 to 26, associated with a constituent capable of modulating its sensitivity, said constituent being a substituted polyamino acid.

29. Compound, composition or reagent according to claim 28, combined with succinylated polylysine.

30. A compound, composition or reagent according to claim 29, associated with the succinylated polylysine having a molecular weight in the range of 20,000 to 300,000 Da.

31. Compound, composition or reagent according to claim 28, combined with glutarylated polylysine.

32. Use of a compound according to any one of claims 1 to 7 or of a compound corresponding to the formula

in which m is equal to 2.3 or 4, or of a composition according to any one of Claims 8 to 10 or 15 to 20 or 23, 24 or of a reagent according to any one of Claims 25 to 27 or of a composition according to one of claims 28 to 30, for the preparation of a test for the in vitro examination of endogenous coagulation.

33. Use of a compound according to any one of claims 1 to 7 or of a compound corresponding to the formula

in which m is equal to 2.3 or 4, or of a composition according to any one of Claims 8 to 10 or 15 to 20 or 23, 24 or of a reagent system according to any one of Claims 25 or 26 or a composition according to one of claims 28 to 30 for the exploration of coagulation by the endogenous route.

34. Use according to claim 33 in which the time of partially activated thromboplastin (APTT) is determined.

35. Use according to claim 32, in which the coagulation time of the activated whole blood is determined.

36. Use according to claim 32 in which the coagulation time of the activated plasma is determined.

37. Use of a compound according to any one of claims 1 to 7 or a compound corresponding to the formula

in which m is equal to 2.3 or 4, or of a composition according to any one of Claims 8 to 10 or 15 to 20 or 23, 24 or of a reagent according to either of claims 25 or 26 or of a composition according to one of claims 28 to 30, for the examination of the coagulation factors involved in the endogenous coagulation pathway.

38. Process for the preparation of a compound of the following formula:

in which

A is chosen from the following formulas

M ⁺ representing a cation, q being equal to 1 or 2 and p being such that pxq = 2 and

R represents

n being an integer between 1 and 170, said method comprising the steps of:

- reaction of gallic acid, in the presence of a compound having an alcohol function, contacting the gallic acid ester obtained, in an alkaline medium, to form a compound of formula

stabilization of the balance obtained, between the compounds (II) and (III) at a physiologically acceptable pH, by the addition of a buffer,

- recovery of the solution obtained.

39. The method of claim 38 characterized in that the esterification is carried out in the presence of ethylene glycol to form ethylene glycol gallate (GEG), and in that a mixture of the forms of GEG involved in keto-enolic balance.

40. Method according to claim 38, characterized in that the solublè form of the GEG corresponding to the formula is recovered

[pM ^{q +} ]

(V)

M ⁺ being a cation, q being equal to 1 or 2 and p being such that pxq = 2.

41. Process for the preparation of a compound of formula

R

AT

(I) in which

A is chosen from the following formulas:

M ⁺ representing a cation, q being equal to 1 or 2 and p being such that pxq

= 2,

R represents -fO-CH2-CH ₂ -) - OH with n being an integer between 1 and 170 n comprising the steps of:

- esterification of gallic acid,

heating the gallic acid ester obtained at a temperature between 70 and 90 ° C, for a period of between 16 hours and 24 hours at alkaline pH, in a buffer solution,

- recovery of the different forms of the gallic acid ester present in solution.

42. Method for measuring the time of partially activated thromboplastin (APTT) comprising the steps of: bringing a sample of platelet-poor plasma into contact with a compound according to any one of claims 1 to 7 or 11 to 14 or 21, 22 or a compound corresponding to the formula

in which m is equal to 2.3 or 4, or a composition according to any one of Claims 8 to 10 or 15 to 20 or 23, 24 or a reagent system according to any one of Claims 25 or 26, or of a composition according to one of claims 28 to 30 in the presence of substitutes for phospholipids of the platelet membrane, under conditions allowing the activation of the endogenous coagulation pathway, - the addition of Ca ²⁺ ions in amount between 20 and 25 mM, - the measurement of the time necessary for the transformation of fibrinogen into fibrin.