WO1991011463A1 - Endogenous brain factor, method for obtaining it and therapeutic and diagnostic uses thereof - 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

Cerebral endogenous factor, process for obtaining it and therapeutic and diagnostic applications.

The present invention relates to an endogenous factor of peptide nature or a fragment thereof which is 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 been demonstrated for a long time in the brain, its properties are exerted 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 main classes: S-HT ₁ , 5-HT ₂ and 5-HT ₃ .

Among the high affinity 5-HT ₁ receptors, it appears that the 5-HT _1b receptors in murids and the 5-HT _1d receptors in humans, monkeys, calves, guinea pigs, could play a role. 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 use of the serotonergic 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-HT ₂ and 5-HT ₁ in particular. During their study, the inventors showed that the heterologous presynaptic receptors of the 5-HT _{1 type} present on the cholinergic terminations of the hippocampus or the cortex are modified by antidepressants. The arguments for proposing the existence of interaction between antidepressants and serotonergic sites are of three types:

1.- Antidepressants have been shown to modify the binding of serotonin to its high affinity 5-HT ₁ sites.

2.- It has been shown that these same antidepressants modify the adenylcyclatic activity linked to these 5-HT ₁ 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 serotoninergic activity presynaptic similarly to that of antidepressants.

The subject of the present invention is therefore an endogenous cerebral factor or fragment thereof, acting on the serotonergic functioning, which presents in chromatography after purification two peaks carrying the biological activity of molecular mass of approximately 3000 and approximately 1500 daltons, has a pK of approximately 5, is thermostable, soluble in acetone up to 75%, in isopropanol up to 90%, in water, in an acid medium up to 1M, insoluble in acetonitrile at concentrations above 70% and in primary alcohols at concentrations above 90% , inactivated in acetonitrile, exhibits activity not affected by trypsin, pronase or protease V8, exhibits a negative reaction with anisaldehyde and can be obtained by:

 a) extraction from the brain, and b) purification by chromatography,

The endogenous cerebral factor according to the invention is further characterized in that it exhibits in thin layer chromatography, the eluent being a butanol / acetic acid / H ₂ O: 4/1/1 mixture, an Rf equal to 0, 24.

 Another aspect of the invention is to provide a method for obtaining the endogenous cerebral factor according to the invention, characterized in that:

 - an extraction is carried out from the cerebral cortex,

 optionally filtering on a gel having a fractionation domain 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 of the endogenous cerebral factor

 The purification was undertaken from horse brains and more particularly from the cerebral cortex.

 It comprises two stages: extraction and purification by chromatography.

 Preferably the endogenous factor according to the invention is obtained by extraction from the cerebral cortex.

I - 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 4 ° C. The whole was homogenized (Virtis) and the volume made up to 2 1 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 on a Rotavapor; the residue thus obtained was resuspended in 100 ml of double-distilled water, and lyophilized.

II - Chromatographies

a) Reverse phase chromatography on a NUCLEOSIL ₅ μm C ₄ column. The isopropanolic extract obtained previously was resuspended in double-distilled water.

500 μl portions were injected into a NUCLEOSIL 5 μm C ₄ column (250 mm × 10 mm) coupled to a Perkin-Elmer type device.

 The chromatography conditions were as follows: the mobile phase consisted of a linear gradient of 0 to 12% acetonitrile in 10 min. The flow rate was 5 ml / min. The observation was made at 220 nm. The fraction collected carrying biological activity corresponded to a peak retention time of 3.16 minutes.

 b) Reverse phase chromatography on a Cl8 ULTRABASE column.

 The fraction obtained previously was resuspended in 500 μl of double-distilled water and then 500 μl were injected onto a C18 ULTRABASE column (250 mm x 10 mm).

 Elutionr was an isocratic elution of double distilled water. The flow rate was 4 ml / min. The observation was made at 220 nm. The collected fraction carrying the biological activity corresponded to a peak retention time 2.82 min.

 c) Reverse phase chromatography on a C18 ULTRABASE column.

 The fraction collected in the previous step was redissolved in a triethylamine acetate buffer 10, mM pH7.

 500 μl portions were injected into a C18 ULTRABASE column (250 mm x 10 mm).

The elution was an isocratic elution of 10 mM triethylamine acetate buffer pH7. The flow rate was 4 ml / min. The observation was made at 220 nm. The fraction collected carrying the biological activity that corresponded to a time peak, retention 2, -84 min.

 d) Reverse phase chromatography on RP18 LICHROSORB column.

 The fraction obtained previously was resuspended in 200 ml of 0.1% trifluoroacetic acid, pH2 and then injected onto a RP18 LICHROSORB column (250 mm x 10 mm). The elution was an isocratic elution of 0.1% trifluoroacetic acetate, pH 2. The flow rate was 4 ml / min. The observation was made at 220 nm. The collected fraction carrying the biological activity corresponded to a peak retention time of 3.79 min.

 e) Reverse phase chromatography on RP18 LICHROSORB column.

 The fraction harvested previously was resuspended in 50 μl of 90% triethylamine acetate buffer, 10 mM pH7 / 10% acetonitrile, then injected into a RP18 LICHROSORB column (250 mm x 4 mm). The elution was an isocratic elution of 10 mM triethylamine acetate buffer, pH 7, 90% / 10% acetonitrile. The flow rate was 0.8 ml / min. The observation was made at 220 nm. The collected fraction carrying biological activity corresponded to a peak retention time of 0.5 min.

 f) Permeation chromatography on a TSK HW 40S column (Merck).

The fraction obtained previously was resuspended in 400 μl of 50 mM ammonium acetate buffer pH5 and injected on a TSK HW 40S column (250 mm x 1.6 cm). The elution was an isocratic elution of 50 mM ammonium acetate buffer pH5. The flow rate was 0.5 ml / min. The observation was made at 220 nm. The fraction collected carrying the activity biological corresponded to two peaks of respective retention times at 74.64 min and 84.48 min. Both peaks carried biological activity.

 g) Reverse phase chromatography on a NUCLEOSIL column 5 μm C18.

 The fraction collected previously was resuspended in 50 μl of 10 mM triethylamine acetate pH 7, and injected onto a NUCLEOSIL column 5 μm C18 (150 mm x 4 mm): The elution was an isocratic elution of acetate buffer triethylamine mM pH7. The flow rate was 0.6 ml / min. The observation was made at 220 nM. The collected fraction carrying the biological activity corresponded to a retention time peak 2.89 min carrying the biological activity and a peak of homogeneous appearance which was not repurified.

 h) Reverse phase chromatography on RP18 LICHROSORB column (Merck).

 The fraction collected previously carrying the biological activity was resuspended in 50 ml of a 10 mM triethylamine acetate buffer pH7, and injected onto a RP18 LICHROSORB column (250 mm x 7.5 mm). The elution was an isocratic elution of 10 mM triethylamine acetate buffer pH7. The flow rate was 2 ml / min. The observation was made at 220 nM. The collected fraction carrying the biological activity corresponding to a peak of retention time 4.22 min.

II - Characteristics of the endogenous cerebral factor

 a) Physiochemical characteristics.

The endogenous cerebral factor appears after purification in the form of two peaks carrying the biological activity, of molecular masses of approximately 3000 and approximately 1500 daltons. In addition, electrophoresis followed by staining with silver nitrate makes it possible to visualize two protein bands with molecular masses of less than 10,000 daltons. The marker used consisted of a kit of low molecular weight markers for elecrophesis sold by Sigma.

 These results indicate that the endogenous cerebral factor comprises one or two components, the existence of two peaks of activity being able to be interpreted either as an indicator of the presence of two different components or of a component and a subunit of that or a component and an aggregation thereof.

 The two endogenous factor fractions according to the invention are also:

 - thermostable

 - insoluble in acetonitrile at concentrations above 70%, in primary alcohols above 90%

 - soluble in water, in acetone 75%, in isopropanol at 90%

 - their activities are not affected by trypsin, pronase or protease V8

 - a loss of activity seems to occur with organic solvents (such as acetonitrile).

 Fig. 1 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. 1) Effect of brain endogenous factor on central 5-HT ₁ receptors.

A - Interaction of this factor with the high affinity 5-HT ₁ serotonergic bond.

 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 [ ³ H] 5HT (5HT = 5-hydroxytryptamine or serotonin) on its specific 5-HT ₁ binding sites and in observing the capacities for interaction 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 Ultra-turrax device (22,000 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 endogenous serotonin from its recognition sites. The tissue preparation was then diluted 20 times with the same 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 ^-6 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 Tris-HCl buffer (50 mM, pH 7.4, containing pargyline 5.10 ^-6 M and 0.1% ascorbic acid) in the presence of [ ³ H] 5-HT at a concentration of 5 nM (corresponding to the Kd of 5-HT ₁ 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, pH 7.4. The apparatus used was a Harvester Brandell (New York), with a capacity of 24 tubes filtered simultaneously under a depression of 15 mm of mercury (2.10 ³ 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 Amershairt) (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 [ ³ H] 5HT 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 incubation medium of [ ³ H] 5-HT in the presence of the membrane preparations; the effect of the addition of the endogenous factor on the specific binding of [ ³ H] 5-HT was determined according to the same method as that described in the preceding paragraph.

The results obtained appear in fig. 2 which shows that the fractions corresponding to the endogenous cerebral factor have the property of altering the binding of [ ³ H] 5-HT on its specific 5-HT ₁ sites. On a less purified fraction containing the 2 active peaks, an IC50 of 300 μl is observed on the total 5-HT ₁ bond.

A comparable IC50 is obtained on the 5-HT _1A subclass if the tritiated 8-ODPAT is used as a ligand in place of [ ³ H] -5HT.

 A blank extract produced without brain material has no biological activity.

 B - Specificity.

The endogenous factor was tested at a concentration corresponding to IC50 on the binding of tritiated 5-HT to its total 5-HT ₁ receptors.

For this purpose 400 μl of the different extracts or fractions (columns C ₄ , C ₁₈ and TSK) were incubated at equilibrium (30 min / 22 ° C) in the presence of 500 μl

(1 mg / ml) homogenate of Rat Cortex and 100 μl of tritiated 5-HT (5 nM). Nonspecific binding was determined in the presence of an excess of non-radioactive 5-HT at 10 μM. The free and bound radioactivities were separated by filtration on Whatman GF / B filters). After these had dried, the radioactivity retained on the filters (linked to the membranes) was measured by counting in liquid scintillation.

 In certain experiments, the activity of the endogenous factor (s) was also sought on other types of receptors (see results).

As shown in fig. 4, the endogenous factor does not modify the bonds of ketanserin (5-HT ₂ receptors), QNB (muscarinic receptors), naloxone (opiate receptors), spiroperidol (dopaminergic receptors), DHA (β adrenergic receptors) , FNZP (benzodiazepine receptors), prazonin (α-adrenergic receptors). On the other hand, it inhibits the binding of mepyramine (histaminergic H ₁ receptors).

 C - Interaction of the endogenous factor with the cellular functional effect of 5-HT on the release of acetylcholine

The release of [ ³ H] acetylcholine was 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 [ ³ H] 20 mM choline for 10 minutes in Hepes buffered medium. This material was then washed in 25 volumes of Hepes buffer at 37 ° C, centrifuged for 4 minutes at 12,000 g; the pellet thus obtained was resuspended in 30 volumes of Hepes buffer at 37 ° C., portions of 500 microliters of the synaptosomal preparation were distributed in series of tubes containing or not containing 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 a serotoninergic agonist analog, trifluorophenylmethylpiperazine (TFMPP), the effect of which corresponded to an inhibition of 48 ± 5% of the release. mentioned 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, 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. 3. 2) Effect on the reσaptύre of 5-HT.

100 μl of the different extracts or fractions (columns C ₁₁ , C ₁₈ or TSK) at different dilutions were incubated in the presence of 100 μl of 20 nM tritiated 5-HT and of a preparation of non-lysed synaptbtosomes of rat cortex. The buffer used was Krebs Henseleit pH7 supplemented with glucose and oxygenated. Incubation was carried out at 37 ° C for 10 minutes. The radioactivity captured was separated from the free radioactivity by filtration on a Whatman GF / B filter. After drying, the radioactivity captured was measured by liquid scintillation counting.

 The effect on the recapture of 5-HT is illustrated in fig. 5.

 The endogenous factor inhibits the reuptake of tritiated serotonin by nerve endings.

 The IC50 is approximately 15 μl, which is approximately 20 times lower than that observed on the link.

 This action, however, appears less specific on the binding of 5-HT since the active fractions of the endogenous cerebral factor affect in the same way the reuptake of noradrenaline, dopamine, Gaba and the capture of choline.

 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, in particular with regard to the action on the reuptake of 5-HT. These observations lead to two essential conclusions

1) first of all this endogenous factor is capable of altering the cerebral serotonergic functioning; in Consequently, it can modify certain physiological effects of serotonin at 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 by 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. She can besides being used for its activity in Alzheimer's disease.

 The pharmaceutical compositions according to the invention can be administered orally or parenterally. As examples, mention may be made of tablets, capsules, suppositories, solutions or suspensions for injection and retard 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 momoclonal 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. The endogenous brain 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 into 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, it is possible in particular to use 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 GD et al; Porc. 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 this peptide and the endogenous cerebral factor to be assayed vis-à-vis 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

 1. Cerebral endogenous factor or fragment thereof, acting on the serotonergic functioning, which presents in chromatography after purification two peaks carrying the biological activity of molecular mass of approximately 3000 and approximately 1500 daltons, has a pK of approximately 5 , is thermostable, soluble in acetone up to 75%, in isopropanol up to 90%, in water, in an acid medium up to 1M, insoluble in acetonitrile at concentrations above 70% and in primary alcohols at concentrations greater than 90%, inactivated in acetonitrile, exhibits activity not affected by trypsin, pronase or protease V8, exhibits a negative reaction with anisaldehyde and can be obtained by:  a) extraction from the brain, and  b) purification by chromatography. 2. Cerebral endogenous factor according to claim 1, characterized in that it is obtained by extraction from the cerebral cortex. 3. Cerebral endogenous factor according to claim 1, characterized in that it has, on thin layer chromatography, the eluent being a butanol / acetic acid / H ₂ O mixture: 4/1/1, an Rf equal to 0, 24.  4. Method for obtaining an endogenous cerebral factor according to any one of claims 1 to 3, characterized in that:  a) an extraction is carried out from the cerebral cortex, and  b) purifying by chromatography. 5. Method according to claim 4, characterized in that step a) comprises an extraction with 1 acetic acid followed by an extraction with acetone and isopropanol extraction.  6. Method according to claim 5, characterized in that step a) comprises an extraction with 1M acetic acid, centrifugation at 17,500 g for 20 minutes, lyophilization, resuspension in water and extraction with acetone.  7. Method according to claim 4, characterized in that step b) comprises five reverse phase chromatography steps, a permeation chromatography step followed by two reverse phase chromatography steps.  8. Method according to claim 7, characterized in that the purification step comprises: a) reverse phase chromatography on a NUCLEOSIL 5 μm C ₄ column, with a mobile phase consisting of a gradient of 0 to 12% acetonitrile, in bidistilled H ₂ O, b) reverse phase chromatography on a C18 ULTRABASE column with isocratic elution with bidistilled H ₂ O,  c) reverse phase chromatography on a C18 ULTRABASE column with isocratic elution with a 10 mM triethylamine acetate buffer pH7,  d) reverse phase chromatography on a RP18 LICHROSORB column with isocratic elution with a 0.1% pH2 trifluoroacetic buffer,  e) reverse phase chromatography on a RP18 LICHROSORB column with isocratic elution with a buffer of 10 mM triethylamine acetate pH7 and 10% acetonitrile,  f) permeation chromatography on a TSK HW 4OS column with isocratic elution with a 50 mM ammonium acetate buffer pH5, g) reverse phase chromatography on a NUCLEOSIL 5 μm Cl8 column with isocratic elution with a 10 mM triethylamine acetate buffer, pH7, and  h) reverse phase chromatography on a LICHROSORB RP18 column with isocratic elution with a 10 mM triethyalmine acetate buffer pH7.  9. Pharmaceutical composition comprising, as active principle, the endogenous cerebral factor according to claim 1 or an active fragment thereof.  10. Pharmaceutical composition according to claim 9, intended to combat pathologies linked to the serotonergic system at the cerebral and peripheral level.  11. Pharmaceutical composition according to claim 10 for the treatment of depression, impulsivity, inhibition, anxiety or Alzheimer's disease.  12. Monoclonal and polyclonal antibodies directed against the endogenous cerebral factor according to any one of claims 1 to 3 or a fragment thereof.  13. Hybridomas, characterized in that they produce monoclonal antibodies according to claim 12.  14. Method for assaying or detecting endogenous cerebral factor according to any one of claims 1 to 3, in tissues and biological fluids, comprising the use of monoclonal and polyclonal antibodies directed against cerebral endogenous factor or an epitope of this one.