FR2657784A1 - CEREBRAL ENDOGENIC FACTOR, PROCESS FOR OBTAINING THERAPEUTIC AND DIAGNOSTIC APPLICATIONS. - Google Patents

Abstract

An endogenous brain factor or a fragment thereof, acting on the serotoninergic functions and having an essentially peptide nature, a molecular weight of approximately 3500 daltons, and a pK of about 5. It is heat stable and soluble in acetone up to 75 %, in primary alcohols up to 90 %, in water and in an acidic medium up to 1M, is 70 % inactive in acetonitrile, reacts negatively with anisaldehyde, and can be obtained by extraction from the cerebral cortex and by chromatographic purification.

Description

 The present invention relates to an endogenous factor of peptide nature or a fragment thereof isolated from the mammalian brain and capable of interacting with the functioning of the cerebral serotonergic system, its process for obtaining and its applications for therapeutic and diagnostic purposes. .

 The serotonergic system has long been demonstrated in the brain; its properties are exercised at the peripheral and central level. At the central level, it is involved in a large number of physiological functions, in particular sleep, thermoregulation, various behaviors of hunger, thirst, fear, aggression, nicteral rhythms, etc. It is also involved in pathological disorders such as migraine, mental retardation in children, senile dementia, depression and impulsivity.

The receptors involved in the functioning of the serotonergic system correspond to 3
Orgrandes classes: 5-HT1, 5-HT2 and 5-HT3.

 Among the high affinity 5-HT1 receptors, it appears that the 5-HT 1b receptors in murids and the 5-HT1d receptors in humans, monkeys, calves, guinea pigs, could play a role as as heterologous presynaptic receptors; in other words, these receptors are located on non-serotonergic neural endings and can modify the functioning of neural endings.

For several years, a set of experimental results and clinical observations strongly suggests the involvement of the serotonin system in phenomena directly involved in depression or in syndromes accompanying it (Coppen, 1974, 1988, Asberg, 1976). Among the various serotonergic sites involved, an important role has been suggested for 5-HT2 and 5-HT1 in particular. During their study, the inventors showed that the heterologous presynaptic receptors of the 5-HT1 type present on the cholinergic terminations of the hippocampus or the cortex are modified by antidepressants. The arguments making it possible to propose the existence of interaction between there are three types of antidepressants and serotonergic sites
1.- Antidepressants have been shown to modify the binding of serotonin to its high affinity 5-HT1 sites.

 2.- These same antidepressants have been shown to modify the adenylcyclastic activity linked to these 5-HT1 sites.

 3.- It has also been shown that antidepressants antagonize the inhibitory effect of serotonin on the neuronal release of acetylcholine.

This effect appears specific, selective for antidepressants since it is not observed with neuroleptics or benzodiazepines.

 The inventors have been led to postulate the existence of an endogenous factor which could play the role of antidepressants by modulating the presynaptic serotonergic activity and have within the framework of their work isolated and purified an endogenous factor capable of modulating the serotonergic activity presynaptic in a similar way to that of antidepressants.

The present invention thus relates to an endogenous cerebral factor or a fragment thereof acting on serotonergic functioning, which is essentially peptide in nature, has a molecular mass of approximately 3500 daltons, a pK of approximately 5, is thermostable , soluble in acetone up to 75%, in primary alcohols up to 90%, in water, in acid medium up to 1M, inactivated in acetonitrile at 70%, has a negative reaction with l anisic aldehyde and can be obtained by
a) extraction from the cerebral cortex,
b) optionally filtration through a gel having a fractionation range of between 10,000 and 100 daltons, and
c) purification by chromatography.

 The endogenous cerebral factor according to the invention is further characterized in that it has, on thin layer chromatography, the eluent being a butanol / acetic acid / H2O: 4/1/1 mixture, an Rf equal to 0.24.

Another aspect of the invention is to provide a process for obtaining the endogenous cerebral factor according to the invention characterized in that
- an extraction is carried out from the cerebral cortex,
- it is optionally filtered through a gel having a fractionation range of between 10,000 and 100 daltons,
- It is purified by chromatography.

 The production of the endogenous cerebral factor according to the invention, its characteristics and its properties will be described below in more detail.

I - Obtaining the endogenous cerebral factor
The purification was undertaken from horse brains and more particularly from the cerebral cortex.

 It can be carried out according to two methods.

The first method includes the 3 stages: extraction, filtration and chromatography.

a) Extraction
Horse brains were collected at slaughterhouses, transported on ice and dissected in the laboratory. The cerebral cortex was removed, cut into pieces, the total mass being about 2 kg of tissue. After adding half a volume of distilled water (volume / weight) the mixture was brought to the boil for 5 minutes and then cooled to 4C.

I, the whole was homogenized (Virtis) and the volume made up to 2 l with distilled water and glacial acetic acid (final concentration: 1 M). The homogenate was centrifuged for 20 minutes at 17500 g and the supernatants collected. These were then lyophilized for approximately 18 hours.

The lyophilisates obtained were resuspended in 750 ml of 75% acetone. After 10 minutes the soluble material was separated by filtration on a conical sintered filter of porosity equal to 3. The water and the acetone of the medium were then evaporated at
Rotavapor; the residue thus obtained was resuspended in 100 ml of double-distilled water, and lyophilized.

b) Filtration on ael
The acetone extract previously obtained was resuspended in 30 to 40 ml of buffer and centrifuged at 100,000 g for 30 minutes. The supernatant obtained was filtered through a 0.22 micrometer filter and stored at -80 ° C in 2 ml portions until use. On the day of use, after thawing, the portions were injected directly into a Perkin-Elmer 400 series HPLC device, equipped with a gel permeation column.
TSK-KW 40S (fractionation range corresponding to molecular weights of 10,000 to 100 daltons) with a length of 30 cm and a diameter of 1.6 cm.

 The chromatographic separation conditions were as follows: the mobile phase consisted of a 50 mM ammonium acetate buffer pH7.

The flow rate was 1 ml per minute. The wavelength at which the fractions obtained were analyzed was 280 nm. The fractions collected corresponded to a duration of 2 minutes.

 Fractions Nos. 16 to 20 of each chromatographic separation by gel filtration were collected and pooled (elution time corresponding to approximately 32 to 40 minutes). These fractions were lyophilized (3 successive lyophilizations) and stored at -80 ° C. The active fraction corresponds to an estimated molecular mass close to 4000 daltons.

c) Chromatoqranhies
Normal phase chromatography.

 The various lyophilisates obtained by gel filtration were resuspended in a ternary injection mixture consisting of 70% acetonitrile, 20% methanol and 10% double distilled water.

 Portions of 500 microliters were injected into a column of Lichrosorb-Diol 7 micrometers Merck (250 mm × 10 mm) coupled to a Perkin-Elmer type device. The chromatography conditions were as follows: the mobile phase consisted of the above ternary mixture. The elution gradient was as follows: linear gradient in 20 minutes allowing a ternary mixture of 50% acetonitrile, 40% methanol and 10% double distilled water to be reached. The flow rate was 4 ml / min. The optical observation of the various fractions was carried out at 230 nanometers, the collection of the fractions corresponding to the different separation peaks was carried out manually.

 Various peaks of optical absorption were obtained. The peak corresponding to a retention time of 5.54 + 0.2 minutes was collected, the corresponding fraction reconcentrated by evaporation using a Speedvac coupled to a lyophilizer.

 Thin layer chromatography.

 The fraction thus obtained was resuspended in 100 microliters of double-distilled water, then deposited online on a plate of silica gel 60 F 250 (Merck) 0.2 mm thick on aluminum foil, 1 cm from the bottom edge.

 The solvent used was a butanol / acetic acid / H2O: 4/1/1 mixture. The revelation was carried out by the Reindel-Hope reagent in UV light at 254 nm.

 The separate material corresponded to an Rf of 0.24; it was collected by scraping the plate and then carrying out a sonication in two volumes of double-distilled water; finally, it was centrifuged for 3 minutes, filtered through a 0.22 micrometer filter and finally, lyophilized.

 Normal phase HPLC chromatography.

 The lyophilisate obtained by thin layer chromatography was resuspended in a ternary injection mixture composed of 70% acetonitrile, 20% methanol and 10% water; then centrifuged at 12000 g for 3 minutes and filtered through 0.22 micrometer filters. It was then injected into a normal phase chromatography column identical to that described above. The elution conditions were also identical to those of the normal phase chromatography step described above.

 A peak was observed, the retention time of which was 5.5 ± 0.2 minutes. This was collected, distributed in portions and reconcentrated using a Speedvac connected to a freeze dryer.

 The portions containing the desired product were stored at -80 ° C.

 The purification can also be carried out by a second method comprising an extraction step, and another of reverse phase chromatography.

 a) Extraction.

 The extraction was carried out as indicated above, except that after the extraction with 75% acetone, an additional extraction is carried out with 250 ml of 90% isopropanol. The solvent is then evaporated and the residue obtained resuspended in 100 ml of double-distilled water and lyophylized.

 b) Reverse phase chromatography.

Reverse column chromatography
C4.

The lyophilisate obtained in the previous step was suspended in 30 ml of 50 mM ammonium acetate pH5 and injected onto a Nucleosil 300 - 5C4 column (250 mm x 10 mm). . -1
The flow rate was set at 3 ml min.

 A first phase (phase 0) composed of 100% 50 mM ammonium acetate pH5, was passed over the column for 1 minute, then a second phase (phase 1) of 50 mM ammonium acetate pH5 and acetonitrile for 10 minutes so as to produce a linear gradient allowing a concentration of acetonitrile from 0% to 10% to be obtained in 10 minutes; finally a third phase (phase) 2 consisting of 50% 50 mM ammonium acetate pH5 and 50% acetonitrile was passed for 5 minutes on the column. The chromatogram obtained is shown in FIG. 1. The fraction of retention time between 4 min. 30 and 6 min. 30 containing the activity was collected.

Reverse column chromatography
C18.

 The fraction obtained previously was resuspended in 30 ml of 50 mM ammonium acetate, pH5 and injected into a Lichrosorb Merck RP18 column (250 x 10 mm).

-1
The flow rate was set at 4 ml. min.

The phases were as follows
- phase 0: 1 min., 100 of 50 mM ammonium acetate pH5,
- phase 1: 10 min., linear gradient of acetonitrile from O to 3%, 50 mM ammonium acetate pH5,
- phase 2: 10 min., linear gradient, from acetonitrile from 3 to 50%, 50 mM ammonium acetate pH5.

 The chromatogram obtained is shown in fig. 2. The active fraction is that corresponding to the retention peak equal to 3.44 + 0.1.

II - Characteristics of the endogenous cerebral factor
a) Physicochemical characteristics.

 The physicochemical characteristics of the endogenous cerebral factor are essentially those of a peptide, whose molecular weight is approximately 3500 daltons.

 Fig. 3 illustrates the amino acid composition. As it appears on this figure the main amino acids found constantly in the analyzes are: aspartate, threonine, serine, glycine, leucine, alanine, valine, phenylalanine and a corresponding amino acid either glutamate, or glutamine.

 b) Biological and cellular activity of the endogenous cerebral factor.

 A - Interaction of this factor with high affinity 5-HT1 serotonin binding.

 The same test was used as that which made it possible to follow the existence of this endogenous factor throughout the purification process.

 This test consists in observing the binding of 3H75HT (5HT = 5-hydroxytryptamine or serotonin) on its specific 5-HT1 binding sites and in observing the interaction capacities of the endogenous factor with it.

The test was carried out using membrane preparations obtained from horse brains. The brains were dissected and the cortical tissue placed in centrifuge tubes where they were homogenized using an apparatus
Ultra-turrax (22000 rpm) for 30 seconds in 5 volumes of Tris-HCl buffer pH = 7.4 (50 mM) containing 10 5 M of phenylmethylsulfonyl fluoride (PMSF) and 5 units / l of Aprotinin. The homogenate was then incubated for 10 minutes at 37 ° C. to dissociate the endogenous serotonin from its recognition sites. The tissue preparation was then diluted 20 times with the dreary buffer at 22 C and centrifuged for 10 minutes at 20,000 g (Kontron Centrikon H401 B) at 4-C.

The pellet obtained was resuspended in 50 ml per gram of fresh tissue of Tris-HCl buffer pH 7.4, 50 mM containing 5.10 b M pargyline and 0.1% ascorbic acid.

0.5 ml portions of the cortical tissue homogenate at 1 mg / ml were incubated for 30 minutes at 22-C in a final volume of 1 ml of buffer
Tris-HCl (50 mM, pH 7.4, containing 5.10 6 M pargyline and 0.1 ascorbic acid) in the presence of; 3H25-HT at a concentration of 5 nM (corresponding to
Kd of 5-HT1 receptors). The incubates were then filtered under vacuum on a Whatman type GF / B glass fiber filter and then washed twice with a total volume of 10 ml of Tris-HCl buffer at 4 C r pH 7.4.

The device used was a Harvester Brandell (New
York), with a capacity of 24 tubes filtered simultaneously under a depression of 15 mm of mercury (2.103 Pa).

The filters were then dried under an infrared lamp for 1 hour and placed in scintillation vials containing 5 ml of OCS (Trademark by
Amersham) (New England Nuclear, Boston, US); the radioactivity contained in these filters was measured by counting for 4 minutes in a liquid scintillation spectrophotometer (Kontron SL 2000). The specific binding of E H25HT was measured by the difference between the radioactivity retained in the absence and in the presence of non-radioactive 5-HT at the concentration of 10 mM.

 For the measurement of the effect of the endogenous factor on the 5-HT bond, the increasing amounts of the fraction corresponding to the endogenous factor were added to the L3H75-HT incubation medium in the presence of the membrane preparations; the effect of the addition of the endogenous factor on the specific binding of i? HJS-HT was determined according to the same method as that described in the preceding paragraph.

 The results obtained appear in fig. 4 which shows that the fractions corresponding to the endogenous cerebral factor have the property of altering the binding of pH15-HT on its specific 5-HT1 sites.

B - Interaction of the endogenous factor with the cellular functional effect of 5-HT on the release of acetYlcholine 3
The release of acetylcholine has been studied using a technique derived from the technique of superfused synaptosomes; the synaptosomes prepared from brain tissue (hippocampus or cortex) of guinea pigs were incubated at 37 ° C. in the presence of 20 mM pH7choline for 10 minutes in Hepes buffered medium. This material was then washed in 25 volumes of 376 Hepes buffer , centrifuged for 4 minutes at 12,000 g; the pellet thus obtained was resuspended in 30 volumes of Hepes buffer at 37 "C., 500 microliters portions of the synaptosomal preparation were distributed in series of tubes containing or not calcium and / or potassium. The release of acetylcholine was measured after centrifugation of the incubates and determination of the radioactivity in the supernatants obtained. The effect of serotonin on the evoked release of acetylcholine was determined in the presence of the amine or of a serotonergic analog agonist, the trifluorophenylmethylpiperazine (TFMPP) effect corresponded to an inhibition of 48 + 5% of the evoked release of acetylcholine.

 Under the conditions described above, it has been shown that antidepressants are capable of antagonizing the inhibitory effect of serotonin on the evoked release of acetylcholine; similarly, the endogenous factor at concentrations which are capable of altering the binding of serotonin is also capable of antagonizing the inhibitory effect of serotonin on the evoked release of acetylcholine, as shown in fig. 5.

 The biological properties of the endogenous cerebral factor show that it is capable of interacting with the functioning of the serotonergic system which modulates the functioning of other serotonergic or non-serotonergic neurons. In addition, this factor mimics the effect of antidepressants at this level. These observations lead to two essential conclusions 1) first of all this endogenous factor is capable of altering the cerebral serotonergic functioning; consequently it can modify certain physiological effects of serotonin on the cerebral level and thereby intervene in behavior, thermal regulation, sleep, learning, memory, etc.

and also interact on serotonergic functioning at peripheral levels, in particular with regard to immune properties.

2) a second conclusion is that the interactions of this factor with the serotonergic system are implicated in the pathology of depression or at least in that of the mechanisms of action of antidepressants suggesting that this natural factor could regulate the modulating effects of serotonin on the functioning of other brain neurons and that this mechanism would be important in the pathology of depression.

 Consequently, the subject of the invention is also a pharmaceutical composition comprising, as active principle, the endogenous cerebral factor according to the invention or an active fragment thereof.

 This pharmaceutical composition is intended for treating pathologies linked to the serotonergic system at the cerebral and peripheral level.

 At the peripheral level, the action of - the composition according to the invention is a modulation of the immunological system by modification of the effects of stress.

 At the brain level, it acts by correcting the effects of serotonin, in particular at the level of behavior, thermal regulation, sleep, etc.

 It intervenes in particular by its antidepressant action, its action on impulsivity or inhibition and its action on anxiety. It can also be used for its activity in Alzheimer's disease.

 The pharmaceutical compositions according to the invention can be administered orally or parenterally. Examples include tablets, capsules, suppositories, injectable solutions or suspensions and delay forms.

 In these compositions, the active principle is generally mixed with one or more acceptable pharmaceutical excipients well known to those skilled in the art.

 The amount of active ingredient administered obviously depends on the patient being treated, the route of administration and the severity of the disease.

 In addition, insofar as the endogenous cerebral factor plays a key role in the pathology of depression, a dosage in peripheral fluids can represent an interesting tool for diagnosing this pathology.

 The invention therefore also relates to monoclonal or polyclonal antibodies directed against the endogenous cerebral factor.

 The present invention also relates to hybridomas producing monoclonal antibodies directed against the endogenous cerebral factor according to the invention.

 Polyclonal and monoclonal sera can be prepared according to a conventional technique. For this purpose, peptide fragments thereof can be coupled to immunogenic agents, such as KLH (Keyhole Lympet Hemocyanin), ovalbumin, bovine serum albumin, etc., by a coupling agent such as glutaraldehyde. Cerebral endogenous factor can also be injected directly.

 The immunizations can be carried out in a conventional manner, for example in rabbits and in mice, by injecting the animal for example 100 micrograms of the coupling product in the presence of Freund's adjuvant 3 to 4 times at 3 weeks apart. It is thus possible to obtain polyclonal sera from rabbits.

 Hybridomas and monoclonal antibodies can be obtained by conventional methods.

 The present invention also relates to a method for assaying or detecting endogenous cerebral factor according to the invention in tissues and biological fluids.

 For this purpose, one can use in particular an RIA type method using a peptide labeled with a radioisotope, and the competition between this peptide and the endogenous factor to be assayed (Niswender G.D. et al; Pork.

Soc. Exp. Biol. 128, 807, 1968). One can also use an ELISA type method using for example a peptide on a support and the competition between it. peptide and the endogenous cerebral factor to be assayed with respect to the antibodies prepared against it. The antibodies retained by the peptide attached to the support are detected by antibodies directed against the first and linked to an enzyme.

Claims (15)

 1. Cerebral endogenous factor or fragment thereof, acting on serotonergic functioning, which is essentially peptide in nature, has a molecular mass of approximately 3500 daltons, a pK of approximately 5, is thermostable, soluble in acetone up to 75%, in primary alcohols up to 90%, in water, in an acid medium up to 1M, inactivated in 70% acetonitrile has a negative reaction with anisaldehyde and can be obtained by  a) extraction from the cerebral cortex,  b) optionally gel filtration having a fractionation range of between 10,000 and 100 daltons, and  c) purification by chromatography.  2. Cerebral endogenous factor according to claim 1, characterized in that it exhibits in thin layer chromatography, the eluent being a butanol / acetic acid / H2O mixture: 4/1/1, an Rf equal to 0.24.  3. Method for obtaining a cerebral endogenous factor according to claim 1 or 2, characterized in that  a) an extraction is carried out from the cerebral cortex,  b) optionally, filtering on a gel having a fractionation domain of between 10,000 and 100 daltons,  c) purifying by chromatography.  4. Method according to claim 3, characterized in that step a) comprises an extraction with acetic acid followed by an extraction with acetone.  5. Method according to claim 4, characterized in that step a) comprises extraction with glacial acetic acid, centrifugation at 17,500 g for 20 minutes, lyophilization, resuspension in water and extraction with acetone.  6. Method according to claim 3, characterized in that in step b) the filtration is carried out on an HPLC column with 50 mM ammonium acetate as eluent.  7. Method according to claim 3, characterized in that step c) comprises a normal phase HPLC chromatography followed by a thin layer chromatography and a normal phase chromatography.  8. Method according to claim 6, characterized in that the normal phase HPLC chromatographies are carried out with a mobile phase consisting of a mixture composed of 70% acetonitrile, 20% methanol and 10% double distilled water and the chromatography in a thin layer is carried out on silica gel with a butanol / acetic acid / H2O mixture as eluent: 4/1/1.  9. Method according to claim 3, characterized in that an extraction is carried out with acetic acid, an extraction with acetone and an extraction with isopropanol, and a chromatograph is carried out Reverse phase HPLC on a Nucleosil 300 5C4 column followed by reverse phase HPLC chromatography on a Lichrosorb Merck RP18 column, the mobile phase consisting of a gradient of acetonitrile in ammonium acetate.  10. Pharmaceutical composition comprising, as active ingredient, the endogenous cerebral factor according to claim 1 or an active fragment thereof.  11. Pharmaceutical composition according to claim 10, intended to combat pathologies linked to the serotonergic system at the cerebral and peripheral level.  12. Pharmaceutical composition according to claim 11 for the treatment of depression, impulsivity, inhibition, anxiety or Alzheimer's disease.  13. Monoclonal and polyclonal antibodies directed against the endogenous cerebral factor according to claim 1 or 2 or a fragment thereof.  14. Hybridomas, characterized in that they produce monoclonal antibodies according to claim 13.  15. Method for assaying or detecting endogenous cerebral factor according to claim 1 or 2, in tissues and biological fluids, comprising the use of monoclonal and polyclonal antibodies directed against cerebral endogenous factor or an epitope thereof.