EP0912893A1

EP0912893A1 - METHODS FOR DIAGNOSING NEURODEGENERATIVE DISEASES $i(IN VITRO), AND KITS THEREFOR

Info

Publication number: EP0912893A1
Application number: EP97934586A
Authority: EP
Inventors: Denis Barritault; Marie-Claude Tournaire; Michel Troupel; Claire Banissi
Original assignee: Valbiofrance Ste
Current assignee: Valbiofrance Ste
Priority date: 1996-07-17
Filing date: 1997-07-17
Publication date: 1999-05-06
Also published as: FR2751412B3; FR2751412A1; AU3773997A; WO1998003869A1; CA2260829A1; JP2000517048A

Abstract

The use of any method for detecting and, where applicable, assaying derivatives that comprise an indole or oxindole ring, to carry out a method for diagnosing human or animal neurodegenerative diseases in vitro, is disclosed. Said in vitro diagnosis methods, as well as kits therefor, are also disclosed.

Description

METHODS OF IN VITRO DIAGNOSIS OF NEURODEGENERATIVE DISEASES AND KITS FOR THE IMPLEMENTATION OF THESE METHODS.

The present invention relates to new methods of in vitro diagnosis of human or animal neurodegenerative diseases, as well as kits (or kits) for the implementation of these methods.

From 1988, a cyclic voltammetry technique made it possible to highlight on the urinary voltammograms obtained, a peak (at 850 mV, visible during the reduction cycle) common to human degenerative dementias

(Alzheimer's type dementia, mixed dementia and Pick's disease) and human spongiform encephalopathies (Creutzfeldt-Jakob disease) and animal (scrapie or scrapie and bovine spongiform encephalopathy). The studies carried out by the inventors in humans have focused on 88 elderly subjects divided into five groups: non-demented controls (n = 38), dementias of the Alzheimer type (n = 30), dementias of vascular origin (n ^** = 13), mixed dementias (π = 5), Creutzfeldt-Jakob (n = 2). The peak is found in the urine of certain demented subjects of the Alzheimer type (12 positive cases, 5 "doubtful" cases, 13 negative cases) and mixed dementia (1 positive case, 2 "doubtful" cases, 2 negative cases) and in two cases of Creutzfeldt-Jakob. The peak has never been demonstrated in the urine from subjects with pure vascular dementia (13 negative cases). On the other hand, it was found in the urine of one of the 38 non-demented subjects (1 positive case, 37 negative cases). The monitoring over time of certain subjects suffering from dementia of the Alzheimer type has made it possible to show that the expression of this peak is subject to strong variations so that this peak is not permanently detectable in the urine of sick subjects . The percentage of cases detected from a single analysis is therefore only 40% (12 cases / 30) for subjects suffering from dementia of the Alzheimer type, (Banissi-Sabourdy C. et al. - Bioelectrochemistry and Bioenergetics, 1992, 28: 127-147).

The studies carried out by the inventors in ruminants concerned 35 sheep (16 suffering from scrapie and 19 healthy) and 110 cows suffering from spongiform encephalopathy (BSE), each of the diagnoses having been subsequently confirmed by anatomopathology. The peak was found in the urine of 87% (14 positive cases / 16) of scrapie sheep and 95% (104 positive cases / 110) of cows with BSE. The woodpecker was never found in the urine of control animals (19 negative cases / 19 healthy sheep, 12 negative cases / 12 healthy cows). (Brugère H. et al. - Bull. Acad. Vét. De France, 1991, 64: 139 - 145; Brugère H. et al. - Commission of the European Communities, Brussels, 14-15 Sept. 1993).

It can be concluded from the following works that the use of cyclic voltammetry makes it possible to detect, from a single analysis, 81% and 85% of the cases of declared spongiform encephalopathy, (i.e. the diagnosis of which can be performed by clinical signs), respectively in sheep and cows. The difference between these two percentages is not significant (p> 0.8). In both cases, these are diseases of similar etiologies, both evolving within comparable time frames, generally of the order of 1 to 6 months.

Studies in humans have shown that a single urine analysis by cyclic voltammetry can detect only 40% of cases of dementia declared Alzheimer type. This percentage is very significantly different (p <0.005) from that observed in ruminants suffering from spongiform encephalopathies. Indeed, we then compare two diseases of different etiologies, evolving in equally different timeframes (a few months for spongiform encephalopathies of ruminants against several years for dementia of the Alzheimer type).

The present invention aims to provide reliable methods of in vitro diagnosis of human or animal neurodegenerative diseases.

The object of the invention is more specifically to provide in vitro diagnostic methods, making it possible to confirm that a man or an animal is or is not suffering from a declared neurodegenerative disease, in particular in the case where the clinical signs observed in this man or this animal may leave a doubt as to the diagnosis of this disease.

Another object of the present invention is to provide in vitro diagnostic methods making it possible to detect that a man or an animal suffers or not from a neurodegenerative disease which is not yet declared, that is to say whose clinical signs are not yet apparent. Another object of the present invention is to provide in vitro diagnostic methods making it possible to distinguish unambiguously within a herd of animals, those of animals suffering from a neurodegenerative disease (in particular of a spongiform encephalopathy) declared or no, of those not affected by such a disease, only the latter then being likely to be sent to a slaughterhouse for obtaining meat intended for food.

Another object of the present invention is to provide kits (or kits) for the implementation of these in vitro diagnostic methods. The present invention follows from the discovery made by the inventors, that the aforementioned peak, appearing on the voltammograms measured from the urine of men or animals suffering from declared neurodegenerative diseases, corresponds to the presence in these urines of a marker corresponding to a derivative comprising an indole or oxindole ring, in particular to a derivative corresponding to the general formula (I) indicated below, and more particularly to a derivative of formula (Ib2) corresponding to the oxidized form of the tautomeric form of formula (Ib indicated below.

The invention will be illustrated with the aid of the following figures:

- Figure 1: separation profile of samples by cyclic voltammetry of urine from healthy ewes (Figure la), scrapie ewes (Figure lb), cows with BSE (Figure le), humans healthy elderly people (figure 1d), and humans suffering from senile dementia of the Alzheimer's type (figure le),

- Figures 2 to 4, 8 and 13 (a and b): monitoring by electro-voltammerogram of the extraction and purification of the above-mentioned marker from sheep urine suffering from scrapie,

- Figures 6 and 7: UV spectra of the above-mentioned marker,

FIG. 9: ion chromatography of the abovementioned marker,

- Figure 10: thin layer chromatography on cellulose plate of the above-mentioned marker,

FIG. 11: mass spectrum of the marker of formula (Ib2) mentioned above,

- Figure 12: proton NMR spectrum of the marker of formula (Ib2) above.

The subject of the invention is the use of any method of detection, and if necessary of assaying, of derivatives comprising an indole or oxindole ring, and more particularly of at least one derivative among those corresponding to the general formula (I) next :

in which :

- Rj represents a hydrogen atom, or a sulphate group SO3 ^" , or a group R _a , R _a representing a conjugated group (such as a carbohydrate or carbohydrate group), in particular a glucuronide, a glucose, a deoxyribose, or a fructose,

R2 represents a hydrogen atom or a group R _a as defined above,

- n -, n2, and n3, independently of one another, represent zero or one, said derivatives corresponding to the following three tautomeric forms:

1) that of formula (la) in which:

- the reduced form corresponds to the derivatives of formula (Ial) below:

in which R and R2 have the meanings indicated above,

the oxidized form corresponds to the derivatives of formula (Ia2) below:

in which R \ has the meaning indicated above,

2) that of formula (Ib) in which:

- the reduced form corresponds to the derivatives of formula (Ibl) below

(I b 1)

in which R \ has the meaning indicated above,

- the oxidized form corresponds to the derivative of formula (Ib2) below

3) that of formula (le) in which:

- the reduced form corresponds to the derivatives of formula (here) below

in which R and R2 have the meanings indicated above, - the oxidized form corresponds to the derivatives of formula (Ic2) below

in which R2 has the meaning indicated above,

for the implementation of a method of in vitro diagnosis of human or animal neurodegenerative pathologies, in particular degenerative diseases of the central nervous system, and more particularly human degenerative dementias, or human and animal encephalopathies

It is understood that the dotted lines represented on the above general formula (I) indicate the possible presence of double bonds between the different atoms constituting the ring and / or the heterocycle of the compounds of formula (I)

The invention relates more particularly to the abovementioned use of any detection device, and where appropriate of assay, of at least one derivative of formula (Ib) as defined above, in reduced form (Ib 1), in oxidized form (lb2) The invention more particularly relates to the aforementioned use of any method of detection, and if necessary of assay, of the derivative of formula (Ibl) in which RI = H, or of its oxidized form correspondent of formula (Ib2)

The subject of the invention is also any method of in vitro diagnosis of neurodegenerative diseases as defined above, characterized in that it comprises a step of detection, and if necessary of assay, in a suitable human or animal biological sample at least one derivative comprising an indole or oxindole ring, and more particularly at least one derivative among those corresponding to the general formula (I) mentioned above.

The invention also relates to any method of in vitro diagnosis as defined above, comprising a step of detection, and if necessary of assay, of at least one derivative of formula (Ib) as defined above, under reduced form (Ibl), or in oxidized form (Ib2).

The invention relates more particularly to any in vitro diagnostic method as defined above, comprising a step of detection, and if necessary of assay, of the derivative of formula (Ibl) in which R _j = H, or of its form corresponding oxide of formula (Ib2).

The subject of the invention is more particularly the application of the abovementioned methods to the in vitro diagnosis of the following pathologies Alzheimer type dementia in humans, mixed dementia in humans, Pick's disease in humans, human spongiform encephalopathies, especially sickness

Creutzfeldt-Jakob, animal spongiform encephalopathies, especially scrapie (or scrapie) in sheep and bovine spongiform encephalopathy (E S B)

The invention relates more particularly to the application of the abovementioned methods to the in vitro diagnosis of neurodegenerative diseases defined above, the clinical signs of which are already apparent in humans or animals, these methods making it possible in particular to follow the evolution of the sickness

Advantageously, the diagnostic methods of the invention allow the screening of these neurodegenerative diseases in apparently carriers healthy, in which the characteristic clinical signs of these diseases are not yet apparent

The biological sample used in the context of the implementation of the diagnostic methods of the invention is preferably urine, or blood, or other biological fluids, such as cerebrospinal fluid (CSF)

According to a preferred embodiment of the invention, the above-mentioned diagnostic methods comprise a step, physical or biological separation of at least one derivative of formula (I) to be detected, in particular in its oxidized form, and more particularly the derivative of the formula (Ib2), of the other constituents of the biological sample, and if appropriate, of direct identification of the drift, in particular by HPLC, gas chromatography, or capillary electrophoresis,

- if necessary, a step of detecting the possible presence of said drift in this biological sample, in particular using one (or more) reactιf (s) brand (s)

A diagnostic method as described above by physical separation and direct identification of said drift, particularly preferred is that carried out by implementing an HPLC method coupled with fluorescence detection. Such a method can be carried out in particular according to the method described by Vohcer et al in the context of the detection of abnormal forms of 5-hydroxytryptophan and serotonin in CSF (Vohcer et al, Arch Neurol, 1985, vol 42, 1158-1161)

Another detection method which can be coupled with HPLC is that of electrochemistry (a Coulometπc detection device such as an ESA 5100 makes it possible to carry out detection and quantitative measurement) Advantageously, the use of standard molecules produced in synthesis and corresponding to the formula to be detected, will allow the measurements to be calibrated and the conditions for elution of the molecules described in the formulas la, Ib, and the, to be identified, in particular in the oxidized form tautomer Ib2. The detection of this latter form will require a first oxidation cycle, without effect on this molecule, followed by a reduction making it possible to detect a signal corresponding to that described at 850 V in the cyclic electrovoltamperometry technique (see Figure 1) The marker will then advantageously be detected during a second oxidation cycle

Another measurement method consists in separating the molecules corresponding to the general formula on gas chromatography (depending on whether or not there is an OH group, sililation will be necessary). The samples will then be measured by mass spectrometry. It should be noted that the shape oxidized according to the formula Ib2 is detectable without prior sililation steps and that its mass is 147 or 148 (with two hydrogens or one hydrogen less than in the reduced form).

Another detection method is mass spectrometry arranged in series (HPLC, Mass, Mass). According to another particularly advantageous embodiment of the invention, the above-mentioned diagnostic methods comprise a step of biological separation of the derivatives of formulas (I) which may be present in the biological sample, from the other constituents of this sample biological, in particular with the aid of antibodies directed against these derivatives of formula (I), in particular with the aid of antibodies directed against the derivative of formula (Ib2).

As such, the invention more particularly relates to any in vitro diagnostic method as described above, and comprising the following steps:

- bringing the biological sample into the presence of antibodies, directed against at least one derivative of formula (I) mentioned above, said antibodies being fixed on a solid support, which leads to the production of complexes between said antibodies and said derivatives likely to be present in the biological sample,

- rinsing the solid support with an appropriate solution,

addition to the solid support of a labeled reagent capable of specifically recognizing the above-mentioned complexes, in particular labeled anti-immunoglobulins, recognizing said complexes,

- rinsing of the solid support,

- Detection of the possible presence of the labeled reagent on the solid support. By way of illustration, the diagnostic methods of the invention are advantageously carried out according to the ELISA technique (Enzyme Linked

Immunosorbent Assay) in which the anti-immunoglobulins used to reveal the complexes formed between the antibodies fixed on the solid support and the derivatives to be detected, are labeled by coupling to an enzyme itself capable of reacting specifically with a determined substrate. Such a method can for example be carried out according to the method described in the article by Yie et al. - Clinical

Chemistry, 1993, vol. 39, 2322-2325.

A subject of the invention is also the polyclonal or monoclonal antibodies directed against at least one derivative of formula (I) mentioned above, and more particularly against the derivative of formula (Ib2). A more particular subject of the invention is polyclonal antibodies directed against one or more, or even all, of the derivatives of formula (I) mentioned above. Such polyclonal antibodies can be obtained by immunization of an animal with one or more, or even all of the derivatives of formula (I), followed by the recovery of the antibodies produced by the immune system of said animal.

The invention also relates to the monoclonal antibodies directed against derivatives of the above-mentioned formula (I), and as produced by any hybridoma capable of being formed, by conventional methods, from the spleen cells of an animal, in particular of mice. or rat, immunized against one of the derivatives of formula (I) on the one hand, and cells of an appropriate myeloma on the other hand, and to be selected by its capacity to produce monoclonal antibodies recognizing the derivative of formula (I) above, initially used for the immunization of animals.

As regards the derivatives of formula (I) mentioned above, used for obtaining the antibodies described above, these can be obtained either in purified form from an appropriate biological sample, in particular from blood, d urine or cerebrospinal fluid, taken from a man or animal suffering from a neurodegenerative disease, for example according to the purification process described below.

These derivatives of formula (I) in reduced form, and more particularly those of formulas (lai), (Ibl) or (Here) above, in which Ri and / or R2 represent H, can also be obtained by chemical synthesis, in particular according to the method described in the article by Yamaguchi et al. - Wakayama Med.

Rept. , 1971, vol. 15, 127-134, or according to the method described in the article by Beckett A. H. et al. - Tetrahedron, 1968, vol. 24, 6093-6109.

The oxidized form of these derivatives in the aforementioned reduced form can be obtained by oxidation of the reduced form in an electric field not exceeding a few volts. The reduced derivative is introduced into a solution which may consist of a mixture of water and one or more organic solvents (for example an alcohol, an amide or a nitrile) and made conductive of the electric current by adding a support electrolyte such as a mineral acid (HC1, H2SO4 ...) or a salt of an alkali metal. The electrochemical cell is, in a conventional manner and well known to those skilled in the art, consisting of two glass containers separated by an ion exchange membrane or any other device making it possible to avoid the diffusion of the reduced or oxidized derivative towards the negative electrode (cathode). The electro-oxidation of the reduced derivative is brought about at an anode which may consist of solid carbon or of fiber or of an unassailable metal, for example platinum. Electrolysis is carried out by imposing, using an original potentiostat

Radiometer Tacussel or other, with respect to a reference electrode, for example with Calomel, a potential such as to cause the oxidation of the reduced derivative. The sulfoconjugation of the derivatives of formulas (la). (Ib) or (le) above, in which R \ represents H, can be obtained using an enzymatic reaction using a sulfotransferase type M (EC 2.8.2.1) purified from rat liver or human platelets according to R. Sekura, 5 M. Duf el, W. Jakoby (Methods in Enzymology, Jakoby W, ed., Académie Press,

New York, 1981, volume 77, pages 197 to 206) and the sulfoconjugation protocol is that described by H. Soliman, P. Boudou, JM Vilette and J. Fiet (J. Steroid Biochem. Molec.Biol., 1993, vol 46, Pages 631 to 634) for the sulfoconjugation of 5-Pregnen-3-ol-20-one (PREG). The abovementioned derivatives are thus obtained in l () in which R \ represents SO3 ^" . 'Acting in the preparation of the derivatives of formulas (la), (Ib) or (le) above, in which R \ and / or R2 represent a group R _a , R _a being as defined above, this can be carried out according to a protocol derived from those described in the articles by Yamaguchi et al., And Beckett AH et al. 15 above, or in the work methods in Enzymology, Jakoby W, ed., Académie Press, New York, 1981, volume 77.

The derivatives of formula (I) used for obtaining the antibodies described above, can advantageously be coupled to carrier molecules usually used for haptens, according to techniques known to those skilled in the art, in particular in position a or c on formula (I), in order to increase the immunogenic properties of said derivatives, and thus to increase the production of antibodies.

The subject of the invention is also any method of in vitro diagnosis of neurodegenerative diseases as defined above, characterized in that the detection, or even the assay, of the derivative (s) of formula (I) is carried out without prior separation of said derivative and of the other constituents of the biological sample, in particular by carrying out a colorimetric reaction between said derivative and a molecule or group of molecules reacting specifically with said derivative by causing the appearance of a specific coloration of this reaction , this coloration being either that of the products resulting from the reaction between said derivative and the aforementioned molecule or group of molecules, or that of a substrate transformed by reaction with one of the products of the aforementioned reaction.

The subject of the invention is also the use of polymers synthesized by methods of combinatorial chemistry (polypeptides,

35 polyoligosaccharides for example), and screened according to affinity criteria with the compounds corresponding to formula I, thus allowing a specific identification of these compounds and to select the most affine polymers (these methods, techniques, aptameres or Selex method, being described in "Methods in enzymology", Volume 267, edited by John Abelson, Académie press)

Another assay method for establishing a diagnostic kit is advantageously developed from molds produced by impression or "impπnting" techniques (K Mosbach and O Ramstrom, Biotechnology, 14, 163-169, 1996)

The subject of the invention is also any diagnostic method as described above, including a prior step of treatment of the biological sample, in particular by acid hydrolysis, for example using hydrochloric acid (pHl) during 2 hours at 80 ° C., in order to desulfate those of the derivatives of formula (I) in which Rj represents SO ^{^} likely to be present in the biological sample, to obtain that derivatives of formula (I) in which R] repiescnte H in said sample, this step being followed by a step of detecting, or even assaying, the derivatives of formula (I) in which R \ represents

H, such as the derivative of formula (Ibl), in particular according to the techniques described above

The subject of the invention is also the kits (or kits) for implementing one of the diagnostic methods of the invention Advantageously the kits of the invention comprise

- Antibodies as described above, directed against one or less of the derivatives of formula (I), in particular antibodies directed against the derivative of formula (Ib2), these antibodies being advantageously fixed on a solid support, in particular in wells of titration plates, - if necessary a medium conducive to the establishment of an immunological reaction between said antibodies, and the derivative (s) of formula (I) capable of being present in the biological sample analyzed, a solution for rinsing the solid support, reagents suitable for the detection of complexes possibly formed between said antibodies and said derivatives of formula (I), in particular anti-immunoglobulins labeled, for example in a radioactive manner, enzymatic or by fluorescence

The invention will be further illustrated with the aid of the following detailed description of the purification and the characterization of a potential marker for degenerative diseases of the central nervous system.

As we have seen previously, this marker excreted in the urine is common to human degenerative dementias (dementia of the Alzheimer type, mixed dementia and Pick disease) and human spongiform encephalopathies (Creutzfeldt-Jakob disease) and animal (scrapie and bovine spongiform encephalopathy).

The presence of this marker is demonstrated directly on human or animal urine samples by a cyclic voltammetry technique on a graphite powder electrode (Figures la to le).

The purification of the marker was carried out by usual methods such as: extraction, dialysis, chromatography on an ion exchange column, etc.

The characterization of the marker in its reduced form was carried out by standard analytical techniques: U .V spectroscopy. , ion chromatography, mass spectrometry, 1 H NMR.

The marker in its reduced form is identified by comparison with molecules synthesized as being a sulfated derivative of oxindole.

1) Purification of the urinary marker and characterization.

Having found that ruminants with spongiform encephalopathies excrete the marker at relatively higher concentrations than human subjects with Alzheimer's disease, we chose to use the urine of sheep with scrapie. However, this protocol applies to urine whatever its origin.

After acidification to pH = 1 of the urine with aqueous HC1, a voltammetric tracing is carried out in order to verify the presence in sufficient quantity of the marker.

1. 1.) Treatment with ethyl acetate

After centrifugation, the urine is subjected to several extractions with ethyl acetate in order to eliminate the maximum of urine compounds which are not loaded at this pH (catecholamines, p-cresol, etc.), the compound corresponding to the marker n 'being apparently not soluble in most conventional organic solvents (ether, chloroform, dichloromethane, ethyl acetate, isopropanol, ...). Thus to a volume of urine (20 ml) is added at room temperature 20 ml of ethyl acetate and the mixture is stirred vigorously in a separating funnel and centrifuged at 10,000 rpm for 10 minutes in order to separate the phases. The ethyl acetate phase is eliminated and the same experiment is repeated 4 times. After extraction of the neutral compounds, a new electrochemical control is carried out on an aliquot of 1 ml of the aqueous phase (FIG. 2).

1 .2.) Cross dialysis Since the marker is not soluble in most organic solvents, even at pH ^* = 1, the inventors deduced therefrom that it was a very hydrophilic product, probably charged at this pH. The inventors therefore attempted to extract it from the urine by cross-dialysis experiments carried out using ionic membranes. They found that the marker does not cross the cationic membranes but that it is on the contrary found in the dialysates when the experiment is carried out using an anionic membrane. It is therefore a negatively charged species, even in very acidic environments. They further observed that the dialysate is in the form of a practically colorless and clear liquid, which is obviously not the case with the initial urines, so that all the subsequent experiments were systematically carried out after dialysis at through an anionic membrane.

Dialysis is carried out from the aqueous phase obtained after extraction of the neutral compounds with ethyl acetate. The aqueous phase is evaporated under vacuum using a rotary evaporator at a temperature below 40 ° C after the fold has been brought to 5-6 by addition of sodium hydroxide in order to avoid degradation of the marker by acid hydrolysis. . The residue is then taken up in HC1 10 ^~ -> N and then subjected to a cross dialysis which is carried out using a conditioned anion exchange membrane (brand RAI model R n ^c 1030) in a glass dialysis cell specially made. The concentration compartment contains 1 M NaCl at pH = 3, these conditions having been chosen in order to optimize the transmembrane exchanges. Dialysis is carried out for 24 to 48 hours with magnetic stirring in a cold room at 4 ° C. An aliquot of the concentration compartment is taken in order to check by voltammetry the presence of the marker. At the same time, a UV spectrum of the mixture is produced.

(figure 3).

1.3.) Purification on an ion exchange column

The dialysate is evaporated to dryness under vacuum, taken up in absolute ethanol, then filtered in order to remove the salts. After evaporation of the ethanol, the residue is dissolved in a minimum volume of 10 ^" 3 N HCl before being injected onto an open column of anion exchange poly ethylene imine cellulose (PEI). The various anionic constituents are separated in a cold room with an NaCl gradient. Partially purified fractions are then obtained which show the characteristics of the marker in electrochemistry. We used a column of Poly Ethylene

Imine 15 cm high and 1 cm in diameter. The sample containing the marker is applied to the column with a flow rate of 1 ml / minute and the elution obtained and the position of the marker are presented in Figures 4 and 5. The elution volume is 400 ml of NaCl and the 1 ml fractions are collected and subjected to electro-voltammetry (Co = 4.5 x 10-2 M - Cl = 9.5 x 10-2 M).

1 .4. ) UV spectroscopy

The UV spectra of the fractions containing the marker have a maximum absorption such that λmax = 249-250 nm (Figures 6 and 7). The shoulder at 280 nm shows the existence of a C = O bond.

1 .5. ) Detection of sulfate ions

The marker being negatively charged at pH = 1, the inventors assumed that it was a compound excreted in the urine in form condensed with the sulfate ion (derivative "sulfo-conjugate"). In order to test this hypothesis, a small quantity of the purified product was subjected to acid hydrolysis at 80 ° C for 2 hours (this hydrolysis can be obtained by adding HCl so that the pH is

1). Likewise, the UV spectrum corresponding to the hydrolysis sample differs from that obtained before hydrolysis (Figure 7). We can therefore deduce that the marker is sensitive to acid hydrolysis. A voltammogram of the sample after hydrolysis was performed and was found to be different from that of the same sample before hydrolysis, (Figure 8). The sulphate, phosphate, nitrates and chlorides ions were measured in the samples before and after hydrolysis by ion chromatography on an anionic DIONEX ION / PAC column AS4A-SC Analytical column (isocratic separation - detection by conductivity - eluent: 1.8 mM Na2Cθ 1.7 mM NaHCO3 conductimetric The analysis revealed the presence of free sulphates in an amount four times greater in the hydrolysis sample than in the sample before hydrolysis taken as reference. This result confirms the hypothesis according to which the marker would be a derivative eliminated in the urine in sulfo-conjugated form (Figure 9).

1.6.) Purification by thin layer chromatography (TLC)

The fractions containing the marker are evaporated to dryness and taken up in a small volume of HCl 10 ^~ 3 N before being deposited on cellulose plates for thin layer chromatography (Merck supplier). The migration solvent is an 8: 2: 2 butanol / acetic acid / water mixture. After migration, the different separated products are identified under UV light and detached with the adsorbent layer. The cellulose-bound products are taken up in H2O and the cellulose is removed by centrifugation. An electrochemical control is then carried out in order to locate the marker. The purification by CCM is renewed in order to to obtain a sufficient quantity of product to carry out analyzes by NMR and by mass spectroscopy (FIG. 10).

1 .7.) Analysis by mass spectrometry and 1 H NMR

Analysis by mass spectrometry (Figure 1 1) (electronic impact and chemical ionization) of the marker in its reduced form purified by TLC, indicates that it is a compound having an equal molecular mass (or fragment) to 149. This value, as well as those observed for various fragmentations of the molecule may suggest that it is a compound of the hydroxy-oxindole type, the position of the hydroxyl radical not being resolved. We do not highlight the sulfate group, which is not surprising since the analysis by mass spectrometry under the same conditions of a control compound, MHPG-sulfate, gives as molecular peak that of MHPG (having lost the sulfate group).

The 1 H NMR spectrum (FIG. 12) obtained is compatible with the existence of an indole or oxindole nucleus which, taking into account the UV detection of the C = O group, gives as the only possibility that of an oxindole nucleus. Furthermore, the presence of an OH group is also noted in position 5. These analyzes lead us to the sulfated (diagram (a)) and desulfated (diagram (b)) structures to represent this marker.

diagram (a)

diagram (b) 2) Examples of dosage of the Marker.

Several methods can be used to measure the marker in the urine. The cyclic electro-amperometry described above is one of these methods which makes it possible to carry out a quantitative assay. The marker can be dosed in its natural sulfated form or after desulfation. Desulfation can be obtained as before by acid hydrolysis.

In order to be able to quantify this marker, it is necessary to carry out a calibration using a reference product. We therefore synthesized the oxindole form (scheme b) according to the protocol described by A. H Beckett,

R. Daisley and J. Walker (Tetrahedron, 1968, volume 24, pages 6093 to 6109). The sulfoconjugation of the oxindole in position 5 was obtained using an enzymatic reaction using sulfotransferase type M (EC 2.8.2.1) purified from rat liver or human platelets according to R. Sekura, M. Duffel, W. Jakoby (Methods in Enzymology, Jakoby W, ed., Académie Press, New York, 1981, volume 77, pages 197 to 206) and the sulfoconjugation protocol is that described by

H. Soliman, P. Boudou, J.M. Vilette and J. Fiet (J. Steroid Biochem. Molec.Biol.,

1993, vol 46, Pages 631 to 634) for the sulfation of 5-Pregnen-3-ol-20-one

(PREG). This protocol makes it possible, by replacing PREG with oxindole, to obtain a sulfation radiolabelled with sulfur if necessary.

The two forms (diagrams a and b) obtained by enzymatic synthesis and sulfation, give a voltammerogram superimposable on those obtained from the natural compounds of the two forms (Figures 12 and 13).

Several chromatography techniques can be used for the assay, either of the natural form or of the desulfated form.

Without being exhaustive, techniques for high performance chromatography (HPLC) in reverse phase (column C18 for example), or ion exchanger (like the one described above for the purification of the marker), or by electrophoresis capillary, can be used. Detection of the marker can be based on UV absorption, fluorescence, electrochemistry, mass spectrometry etc. , and the quantification by integration of the surfaces of the corresponding peaks and their comparison with that of two peaks obtained with known doses of the marker.

Another way of carrying out the assay is the implementation of a technique which calls for recognition by antibodies. Obtaining poly or monoclonal antibodies and the assay is carried out according to conventional protocols for those skilled in the art and described for example for the assay of melatonin by Shang-Mian Yie,

Erika Johansson and Gregory Brown in Clin.Chem. 1993, volume 39 pages 2322 at 2325). The assays are carried out for example with the measurement of a radioactivity or an enzymatic or colorimetric reaction etc.

The protocol routinely used for this assay was that of HPLC on C18, according to the publication by Mills M. IL, Finlay D.C., Haddad P.R. in J. Chromatography, 1991, vol. 164, 441-445, and Van Haarel A. and Pavel S., J.

Chromatography, 1988, vol. 429, 59-94.

For urine samples, we take 1 ml to which 9 ml of an Ethanol / Acetone solution (50/50) are added. The mixture is centrifuged at low speed

(l OOORPM) 10 minutes and a sample of 100 μl of the supernatant is lyophilized and redissolved in 100 μl of ImM HCl. This volume is injected into the column of

HPLC.

For the blood test, the protocol is as follows: 1 volume of plasma

(200μl) is mixed with 3 volumes of the solution (50/50) Ethanol / Acetone. The mixture is stored at 4 ° C for 30 minutes and then centrifuged at 1000 rpm for 10 minutes at 4 ° C. A 100 μl sample of the supernatant is lyophilized and redissolved in 100 μl of ImM HCl. This volume is injected onto the HPLC column.

For dosing in the cerebrospinal fluid, the 100 μl sample is directly injected on HPLC.

It may be desirable to first treat the samples with an acid solution to reach the pH of 1.

LEGEND OF FIGURES

Figure 1 Separation profile of urine samples from:

- the: healthy sheep

- Ib: sheep with scrapie

- le: cows with spongiform encephalopathy

- ld: healthy elderly humans - le: humans suffering from senile dementia of the Alzheimer type.

A 1 ml sample of urine acidified to pH1 with hydrochloric acid is subjected to an oxidation-reduction by passing an electric current with cyclic scanning.

The redox potentials are noted on the abscissa (in mVolts) measured with respect to the normal hydrogen electrode. On the ordinate, the intensity of the current is measured in milliamps.

The cyclic voltammetry is a system with three electrodes, one in platinum, one with hydrogen and one working with graphite powder. We can note the characteristic peak of the marker at 850 mv indicated by the arrow.

Figure 2 Electrovoltamperogram of sheep urine with scrapie after treatment of urine with a volume of ethyl acetate, centrifugation and recovery of the aqueous phase.

We can note the characteristic peak of the marker at 850 mv indicated by the arrow.

Figure 3

Electrovoltamperogram of the urine sample after extraction with ethyl acetate and membrane dialysis.

Figure 4

Electrovoltamperogram of the urine sample after purification by an anionic column (polyethylene imine) corresponding to fraction No. 70 of elution of this column by an NaCl gradient (0 and 1 M).

Figure 5

Electrovoltamperogram of the urine sample after purification by an anionic column (polyethylene imine) corresponding to fraction No. 95 of elution of this column by a NaCl gradient (0 and 1 M).

Figure 6

UV spectrum of the fractions recovered and combined comprised between fraction No. 70 and 95 of the PEI column. On the abscissa, the wavelength (nm), on the ordinate, the optical density.

The main peak at 249 nm (arrow) is that at 286, 1 nm corresponding to a group C = 0.

We note on all these voltammograms the presence of a reduced peak (lower part of the graph, noted by an arrow) at 850 m volts, clearly distinct in Figures lb, le, le, corresponding to spongiform pathologies and

Alzheimer's. Figure 7

Idem in Figure 6, sample 88 had been hydrolyzed by HCl about 2 hours at 80 ° C. It can be noted that the peak at 249 nm is now at 254.6 nm.

Figure 8

Voltammogram of the product (fraction 88 of the PEI column). Note the peak at 850 mV in oxidation (upper part of the graph) on the second scan.

Figure 9

Ion chromatography of the urine marker on an anionic Dionex column after acid hydrolysis. The abscissa represents a measure of conductivity. The retention times are shown on the ordinate. A sulphate peak appears at the sulphate calibration retention time and a chloride peak linked to the use of HCl for the hydrolysis of the label.

Figure 10

Purification of the marker by thin layer chromatography on cellulose plate.

The samples corresponding to the fractionations enriched in markers after chromatography on a PEI column are separated by thin layer chromatography on cellulose.

The area indicated by an arrow corresponds to the marker. This area is removed from the plate and the marker eluted on cellulose gel in water, centrifuged and subjected to voltammetric analyzes.

Figure 11

Mass spectrum of the marker in its reduced form. The mass spectrum was measured by two techniques. The electronic impact

(Figure l ia) and chemical ionization with ammonia (Figure 11b).

A main peak appears at a mass of 149 in FIG. 11a corresponding to the molecular mass of the marker, the minor peaks corresponding to contaminants or degradation products. The spectrum after chemical ionization with ammonia shows a peak with a mass of 150, corresponding to the mass of the marker increased by that of hydrogen and a second peak at 167 corresponding to the mass of the marker increased by 18 (that of NH4). Figure 12

Proton NMR spectrum

On the abscissa the ppm which show the presence of the NH group beyond 10 ppm, the area from 7.55 to 6.95 has been enlarged and is represented in the lower part of the figure. This makes it possible to see that the protons are in position 7, 6, 4 and that position 5 is occupied by the hydroxyl This interpretation results from the integration of the peaks Ha, Hb, Hc

Figures 13 a and 13 b

Electrovoltamperogram of the synthesized product of the oxindole form The peak can be noted at 850 mV (and at 960 mV in oxidation at the second dotted scan)

Electrovoltampérogramme of the product synthesizes oxindole and sulfated by enzymatic way

We can note the peak at 850 mV in reduction at the first sweep and the peak at 1100 mV in oxidation, characteristic of sulfates

The oxidation peak at 960 mV on the second scan (dotted line) which was also observed in Figures 2, 3, 4 and 5

Claims

I. Use of a process for the detection or determination of derivatives comprising an indole or oxindole ring, and more particularly of at least one derivative among those corresponding to the following general formula (I):

in which :

R2 represents a hydrogen atom or a group R _a as defined above,

- n-, n2, and n3, independently of one another, represent zero or one, said derivatives corresponding to the following three tautomeric forms:

1) that of formula (la) in which:

- the reduced form corresponds to the derivatives of formula (Ial) below:

in which Rj and R2 have the meanings indicated above, the oxidized form corresponds to the derivatives of formula (Ia2) below

in which R \ has the meaning indicated above,

2) that of formula (Ib) in which:

- the reduced form corresponds to the derivatives of formula (Ibl) below

in which Rj has the meaning indicated above,

- the oxidized form corresponds to the derivative of formula (Ib2) below

3) that of formula (le) in which:

- the reduced form corresponds to the derivatives of formula (here) below

in which Rj and R2 have the meanings indicated above, - the oxidized form corresponds to the derivatives of formula (Ic2) below

in which R2 has the meaning indicated above, for the implementation of a method of in vitro diagnosis of human or animal neurodegenerative pathologies, in particular degenerative diseases of the central nervous system, and more particularly human degenerative dementias, or human and animal encephalopathies.

2. Method for in vitro diagnosis of human or animal neurodegenerative pathologies, in particular degenerative diseases of the central nervous system, and more particularly human degenerative dementias, or human and animal encephalopathies, characterized in that it comprises a detection step, or even an assay, in an appropriate human or animal biological sample, of the presence of at least one derivative comprising an indole or oxindole nucleus, in particular of at least one compound among those corresponding to the following general formula (I):

in which :

- R \ represents a hydrogen atom, or a sulphate group SO3 ^" , or a group R _a , R _a representing a conjugated group (such as a carbohydrate or carbohydrate group), in particular a glucuronide, a glucose, a deoxyribose, or a fructose, R2 represents a hydrogen atom or a group R _a as defined above,

- n i, n2, and n3, independently of one another, represent zero or one, said derivatives corresponding to the following three tautomeric forms

1) that of formula (la) in which

- the reduced form corresponds to the derivatives of formula (Ial) below

I 5 in which R] and R2 have the meanings indicated above, - the oxidized form corresponds to the derivatives of formula (Ia2) below

25 in which R \ has the meaning indicated above,

2) that of formula (Ib) in which ^*

- the reduced form corresponds to the derivatives of formula (Ibl) below

in which Ri has the meaning indicated above, the oxidized form corresponds to the derivative of formula (Ib2) below

3) that of formula (le) in which:

- the reduced form corresponds to the derivatives of formula (here) below

in which Ri and R2 have the meanings indicated above, - the oxidized form corresponds to the derivatives of formula (Ic2) below

in which R2 has the meaning indicated above,

3. Method of diagnosis according to claim 2, characterized in that the pathologies liable to be detected are the following: dementia of the Alzheimer type in humans, mixed dementia in humans, Pick's disease in humans, encephalopathies human spongiforms, including Creutzfeldt-Jakob disease, animal spongiform encephalopathies, especially scrapie and bovine spongiform encephalopathy (BSE)

4. A diagnostic method according to claim 2 or claim 3, characterized in that the biological sample is urine, blood, or cerebrospinal fluid (CSF).

5. Diagnostic method according to one of claims 2 to 4, characterized in that it comprises:

a step of separation, physical or biological, of at least one derivative of formula (I) to be detected, from the other constituents of the biological sample, and where appropriate, of direct identification of the derivative, in particular by HPLC,

- where appropriate, a step of detecting the possible presence of said derivative in this biological sample, in particular using one (or more) labeled reagent (s).

6. Method of diagnosis according to one of claims 2 to 5 characterized in that it comprises the following stages:

- bringing the biological sample into the presence of antibodies, directed against at least one derivative of formula (I) above, said antibodies being fixed on a solid support, which leads to obtaining complexes between said antibodies and said derivatives likely to be present in the biological sample,

- rinsing the solid support with an appropriate solution,

- rinsing of the solid support,

- Detection of the possible presence of the labeled reagent on the solid support.

7. polyclonal or monoclonal antibodies capable of being used in the context of the implementation of an in vitro diagnostic method according to one of claims 2 to 6, said antibodies being directed against at least one compound among those corresponding to the following general formula (I):

in which :

- Rj represents a hydrogen atom, or a sulphate group SO3 ^" , or a group R _a . R _a representing a conjugated group (such as a carbohydrate or carbohydrate group), in particular a glucuronide, a glucose, a deoxyribose, or a fructose,

R2 represents a hydrogen atom or a group R _a as defined above,

- ι, n2, and 113, independently of one another, represent zero or one, said derivatives corresponding to the following three tautomeric forms.

) that of formula (la) in which:

- the reduced form corresponds to the derivatives of formula (Ial) below

in which R \ and R2 have the meanings indicated above, - the oxidized form corresponds to the derivatives of formula (Ia2) below

in which R has the meaning indicated above,

2) that of formula (Ib) in which:

- the reduced form corresponds to the derivatives of formula (Ibl) below

(I b 1)

in which Rj has the meaning indicated above,

- the oxidized form corresponds to the derivative of formula (Ib2) below

3) that of formula (le) in which:

- the reduced form corresponds to the derivatives of formula (here) below

in which R \ and R2 have the meanings indicated above, - the oxidized form corresponds to the derivatives of formula (Ic2) below

in which R2 has the meaning indicated above.

8. Kit (or kit) for implementing a diagnostic method according to one of claims 2 to 6, characterized in that it comprises: - antibodies as defined in claim 7, directed against one or less of the derivatives of formula (I), these antibodies being advantageously fixed on a solid support, in particular in wells of titration plates,

- if applicable:

. a medium conducive to the establishment of an immunological reaction between said antibodies, and the derivative (s) of formula (I) capable of being present in the biological sample analyzed,

. a solution for rinsing the solid support,

. reagents suitable for the detection of complexes possibly formed between said antibodies and said derivatives of formula (1), in particular labeled anti-immunoglobulins, for example radioactive, enzymatic or fluorescent.