FR2721030A1 - New leucine contg. peptide(s) that modify serotoninergic transmission - Google Patents

Abstract

Leucine-contg. peptides of formula (I) are new: X-Leu-Y (I). X = H, Ala or Leu-Ser-Ala; Y = OH, 1-10 amino acid peptide residue with C-terminus as amide or opt. substd. hydrocarbyl ester, but not OH if X = H; included are cpds. with CONH replaced by a bond resistant to proteases or having a peptide skeleton that includes 1 or more intercalated gp. that impart enzymatic resistance to peptide bonds. Also new are: (1) in vitro diagnosis of disorders of the serotoninergic system by measuring the level of Leu-Ser-Ala-Leu (II) in a body fluid; (2) mono- or poly-clonal antibodies (Ab) specific for (II) and their fragments; (3) kits contg. Ab for determn. of (II); (4) method for screening antidepressants occupying the same conformational site as (II) in which binding inhibition tests for binding to the 5-HT recapture site and to the 5-HT1B/1D site are used to discriminate between the effects of recapture and of binding to 5-HT1B/1D.

Description

The subject of the present invention is

  compounds with the property of modifying the transmission

  serotonergic sion as well as therapeutic applications

canics of these compounds.

  The cerebral serotonergic system is a cerebral and peripheral neurotransmission system, which implements serotonin or 5 hydroxyxyptamine

  (5-HT) discovered in the years 1949-50.

  At the central level, this system is particu-

  to bind, insofar as it is extremely centralized (all the cellular bodies are located in the posterior region of the brain, the raphe) and where it is projected in practically all the regions of the brain. It is

  characterized by an extremely high number of varicosi-

  tees (or equivalent of neural endings) along the axons; this arrangement multiplies its points of interaction with other cerebral neural systems. In addition, a very large part of these

  spider veins does not have a synaptic profile, that is

  that is to say that the 5-HT released by these "endings" will diffuse and reach the targets (receptors) located at a certain distance; this mechanism requires the use of specific receivers having a

  high affinity for the amine. This structured arrangement

  rale is entirely favorable to a function of control of all the neuronal cells of the brain, and thereby to an essential role in the maintenance of homeostasis

of the central nervous system.

  To exercise this control, the 5-HT system puts

  uses a wide variety of specific receptors.

  These are indeed several families, some of which are well characterized (5-HT ,, 5-HT2, 5-HT3, 5-HT4) and others highlighted more recently are still little known (5-HT5, 5-HT., 5-HT). Some of them are represented by many subtypes; this is particularly the case for the 5-HTJ family, characterized by a nanomolar affinity for 5-HT for these sites, and which comprises the 5-HT1ABDEF subtypes 5-HT1 type receptors. and 5-HTlD are

  located on the serotonergic and non-serotonin terminations

  serotonergic, where they modulate the release of the neurotransmitter contained in these terminations, in particular they can regulate the release of 5-HT itself (autoreceptors) and then play a determining role in the activity of serotoninergic neurotransmission. From a fundamental point of view, the serotonergic system is involved in very many

  physiological functions and in many patholo-

  gies, especially in depressive syndrome.

  Depression is a psychiatric disorder still poorly understood despite the very many studies that have been devoted to it. However, it is now fairly clear that a very large part if not all of the depressions involve the serotonergic system and in particular, it is generally accepted that there is a

  relationship between a serotonergic deficit and suicide.

  In addition, many of the antidepressant drugs interact with the newer, more effective, and less effective serotonergic system.

  are most often of the "serotoniner-" type

  these substances all end up, by

  various mechanisms, to facilitate serotoni-

  nergic (monoamine oxidase inhibitors, reuptake inhibitors, 5-HTlA agonists). But on the one hand, their effectiveness is limited, and on the other hand, they still possess for many of them important side effects, and finally, their time of action is important

(> 2 weeks).

  Recent results from the literature and the inventors indicate that the 5-HT18 / lD1 receptors could play an important role in the syndrome

  depressive and in the mechanism of action of antidepressants-

  sisters. It has indeed been shown that not only do they modulate the release of acetylcholine, but they also modulate that of 5-HT itself, and thereby effectively regulate serotonergic transmission, the importance of which is known in the depressive syndrome. In addition, they are the target of antidepressants which interact in a likely non-competitive manner; in other words, antidepressants would recognize a site distinct from the 5-HTD 'receptors, but closely interactive with

these latter.

  The work of the inventors has made it possible to demonstrate an endogenous cerebral compound recognizing a site carried by the 5HTlB / iD receptors which modulate the

release of 5-HT itself.

  The brain endogenous compound highlighted is a tetrapeptide of sequence:

  Leu - Ser - Ala - Leu (LSAL sequence).

  The inventors also tested the activity of peptides comprising the modified LSAL sequence and showed that a modification (for example an acetylation) of the NH2 terminal end results in a compound no longer having ligand activity on the receptor 5 -HT,

  while modifications to the COOH end allow

  were to get a peptide that still had a

important activity.

  For example, the addition of the sequence Gly-

  Gly-Gly-Tyr at the COOH end makes it possible to obtain a compound having a still significant activity. However, the addition of the same sequence, but whose residue Tyr

  was iodized resulted in a compound no longer active

  ligand activity of the 5-HT receptor, probably due to steric hindrance due to the large size of the iodine atoms which would prevent the binding of the peptide as well

modified on its receiver.

  The inventors have further shown that the peptides of sequence Leu-Ser, Ala-Leu and Ala-Leu-Ser have the property of binding to the 5-HT receptor and exert at high concentrations an activity

  similar to that of the endogenous tetrapeptide.

  It therefore appears that it is by its NH2 terminal end that the endogenous peptide binds

on the 5-HT receiver.

  It has also been shown in the laboratory that certain antidepressants (whether or not they are amine reuptake inhibitors) are capable of interacting with the function of the 5-HTlB / D receptor involved in the functioning of the serotonergic system.

  cerebral (Harel-Dupas et al., Eur. Neuro-Psychopharma-

  col., 1, 1991, 157-164; Bolanos-Jimenez et al., Eur. J.

Pharmacol., 1993, 242, 1-6).

  This interaction could be due to a

  direct binding of these molecules to the 5- receptor

  HTlB / D. Support for the latter hypothesis is

  killed by the fact that various antidepressants alter the binding of 5-HT to 5-HT1 receptors

  (Fillion and Fillion, Nature, 1981, 292, (5821) 349-351).

  On this assumption, the fact that the interaction

  observed does not appear competitive suggests that if there is a direct interaction of antidepressants

  with the 5-HT: D receptor, this would be done by the

  through a different interaction site stricto-

  sensu of the 5-HT binding site. In other words,

  these antidepressants would interact with the 5-

  HT 1B / D as allosteric modulators of this receptor

  tor. The subject of the invention is therefore a compound of the following peptide sequence: Leu - Ser - Ala - Leu - Y

  in which Y represents OH, NH2, a hydrocarbon residue

  substituted or unsubstituted loxy, or a peptide sequence

  that having from 1 to 10 amino acids, as well as the corresponding compounds in which the peptide bond is replaced by a bond resistant to the enzymatic degradation of the proteases or in which the peptide backbone comprises one or more intercalated groups making the peptide bond resistant to the degradation enzymatic.

  Thus, the peptide linkage group (-CO-

  NH-) is advantageously replaced by a linking group -CO-NR'-, -CR1R2-CR3R4-, -CO-CR1R2-, R1, R2, R3 and R4, identical or different, representing H or a CQ alkyl group -C6, in particular a methyl group, and R '

  representing a C1-C6 alkyl group.

  Among the groups which can be intercalated in the peptide backbone, mention may be made of the following groups -CR1R2-, -NR, - and -O-, R1 and R2 being defined

like before.

  The nature of the amino acids of X is indifferent

  rent, the limit being that when it comes to modified amino acids (not genetically coded), these are not substituted by a bulky group (having a size

  greater than that of the radius of the iodine atom.

  The term “hydrocarbyloxy residue” is understood to mean in particular an alkyloxy, alkenyloxy or alkynyl group, with a straight or branched chain, having from 1 to 10 carbon atoms unsubstituted or substituted by one or more groups chosen from OH, NH2, NO-, Cl, Br , F, CF3, for example the group OCH3, 0C2H5, OCHOH, OCH3, OCHOH-CH20H, OCH2CH2NH2,

0C3H7, etc ...

  The compounds according to the invention include in particular the peptides of the following sequence: Leu - Ser - Ala - Leu Leu - Ser - Ala - Leu - OCH3 Leu - Ser - Ala - Leu - NH2 Leu - Ser - Ala - Leu - Gly - Gly - Gly - Tyr as well as analogous compounds in which the peptide bond is replaced by a bond as defined above, or whose backbone comprises a

  interleaved group as defined above.

  The most active compounds are those of which

  the constituent amino acids are in the L form,

  ie the natural form for peptide structures

defined above.

  The compounds according to the invention are prepared by extraction from lyophilized brain as described below or more advantageously by conventional peptide synthesis, for example by the phase method

  solid from MERRIFIELD when they are of an exclusive nature-

  peptide ment, or alternatively by modification of the amino acid end groups and condensation of the residues thus obtained, the reactive functions other than those involved in the connection between the successive residues having been previously protected using the groups

  protectors well known to those skilled in the art.

  According to an example of preparation of a peptide

  according to the invention, a resin is fixed, by means of

  diary of its carboxylic group, the residue Ser whose

  amine function is protected by a terbutyloxycar- group

  bonyle, then, after deprotecting the amino function by washing the resin with trifluoroacetic acid in dichloromethane, we couple in dimethylformamide the second amino acid residue whose amino function is protected as above, thus fixing each after the others the amino acid residues which will constitute

  the portion of the peptide according to the invention. After deprotec-

  tion, the amino function of the N-terminal residue can be acetylated by the action of an excess of acetic anhydride in

  presence of diisopropylethylamine.

  The side chain of serine can be

  protected for example by a benzyl group.

  After removing the protective group, the peptide according to the invention is released from the solid support, for example with hydrofluoric acid. The crude product is lyophilized and subjected to liquid pressure chromatography at medium pressure, making it possible to obtain

  a practically pure product; this one is then character-

  risé by high pressure liquid chromatography as well as by analysis of its composition

  amino acids and by mass spectrometry.

  The characterized peptides have the property of modifying serotonergic transmission by its interactions with autoreceptors (and also heteroreceptors) and could play a key role in various psychiatric pathologies where the -HT system is notoriously involved (stress, anxiety,

  depression, compulsive obsessions, impaired appetite

  tit, sleep, behavioral problems, aggression,

etc ...).

  A subject of the invention is also a compound capable of fulfilling the role of ligand of the receptor on which the endogenous peptide of sequence Leu-Ser-Ala-Leu (LSAL) is fixed, characterized in that it comprises a group whose structure spatial is substantially identical to that of a peptide of sequence x - Leu - Y 'in which X represents H, Ala or Leu-Ser-Ala, Y' represents Y as defined above or Ser, X and Y

  not simultaneously representing H and OH, respectively-

  is lying. Such a compound can be an agonist or a

ntagonist of the LSAL peptide.

  By "sensitively identifiable" spatial structure

  that ", it is understood within the meaning of the present invention that the average position of the atoms forming the group which binds to the receptor differs only by more than 5%, and preferably by more than 2% from the average position of

  atoms forming the corresponding peptide.

  Among the preferred compounds, mention may be made of those comprising a group whose spatial structure is substantially identical to the following di-, tri- and tetrapeptides: Ala-Leu Leu-Ser Ala-Leu-Ser Leu-Ser-Ala-Leu. The compounds according to the invention are developed from the spatial structure (s) of the peptides according to the invention by computer modeling and conventional chemical synthesis from the structure

obtained after modeling.

  Advantageously, these compounds are further developed by relying on the spatial structure of known antidepressant molecules active on this same site.

  The modeling technique allows in particular

  to define and characterize the structure of molecules which do not affect the amine transport sites (unlike most antidepressants currently known) but which affect the only allosteric site

  carried by 5-HTlB / D receptors.

  The compounds of the invention have the pro-

  request to modify serotonergic transmission by its interactions with autoreceptors (and also heteroreceptors) and play a key role in various psychiatric pathologies where the 5-HT system is notoriously involved (stress, anxiety, depression, compulsive obsessions, appetite,

  sleep, behavioral problems, aggression, etc.).

  The subject of the invention is also a therapeutic composition comprising a compound as defined above, capable of crossing the blood-brain barrier.

  To cross the blood-brain barrier

  that, the peptide is preferably administered under the

  form of a pro-drug, especially of the glycosyl type

  phospho-triester, in which the peptide is linked for example by serine to the phosphate group, as described

  in J. MED. CHEM. 92, Vol. 35, p. 30-39.

  Such a pro-drug, as well as in general a precursor molecule capable of releasing in vivo a compound according to the invention, constitutes another

subject of the invention.

  The therapeutic composition is advantageously used in pathologies in which the serotonergic system is involved, in particular in pathologies linked to a deficiency in serotonergic transmission. The composition according to the invention can also be used at peripheral level in o pathologies such as circulation disorders,

  migraine and immune response disorders.

  Thus, when the compound according to the invention is a peptide or a compound mimicking the effect of the LSAL peptide, the composition according to the invention can be used in particular in the treatment of depression, but also in all the indications currently covered by antidepressants, such as obsessive compulsive disorder, generalized anxiety, panic attack, appetite, sleep disorders, impulsivity, sexual disorders, aggression, and more generally those of the compounds facilitating the

serotonergic transmission.

  When the compound according to the invention is a spatial analog of the LSAL peptide antagonizing the effect of the endogenous LSAL peptide, the therapeutic composition can be used in mood and behavioral disorders or vascular and immune disorders involving the action of endogenous peptide on its site

receiver.

  The composition according to the invention is advantageously in injectable or oral form, or in

  any other known pharmaceutical form.

  The effective therapeutic dose is easily determined by those skilled in the art depending on the nature and severity of the pathology to be treated, as well as the weight and age of the patient. The compounds according to the invention are not toxic. They are added to a pharmaceutically acceptable vehicle or an excipient

well known to those skilled in the art.

  The invention also relates to a method for in vitro diagnosis of a condition linked to the serotonergic system in a patient, characterized in that an assay is carried out in a biological fluid of the patient of a peptide of sequence Leu - Ser - Ala - Leu The assay is carried out in particular by an immunological method comprising bringing a biological sample into the presence of an antibody specifically directed against a peptide according to the invention and

  evidence of the antigen-antibody complex thus formed.

  These methods can be based on a radioimmunological method of the RIA, RIPA or IRMA type, or on an immunoenzymatic method, for example of the

ELISA.

  The biological fluid can be blood,

  urine or cerebrospinal fluid.

  The antibodies can be monoclonal or polyclonal and constitute, as well as their fragments

  Fab, Fab ', F (ab') 2 and Fc, another object of the invention.

  The antibodies are obtained by coupling a peptide according to the invention to a peptide or a protein

immunogenic carriers.

  The subject of the invention is also a diagnostic kit for assaying a peptide according to the invention in a body fluid of a patient in whom a pathology linked to a deficiency in serotonergic transmission is suspected, in particular masked depression, comprising at least one antibody as defined above and optionally the reagents for the

dosage work.

  The peptides of the invention have been found

  inactive at the 5-HT reuptake site.

  The existence of an inactive brain endogenous factor at the reuptake site constitutes a standard

  benchmark in terms of activity and thus allows for approxi-

  a process for the selection of antidepressant substances

  sives, consisting in discriminating between the recap-

  ture and interaction effect on the 5-HTlB / lD site.

  This process applies to antidepressants

  known and also allows the development of

new things.

  In practice, the discrimination between the reuptake effect and the effect on the 5-HTB / 5D site can be carried out by carrying out tests of inhibition of binding between on the one hand the activity of increasing amounts of antidepressants and the capture of 5-HT in synaptosomes, 5-HT being radiolabelled and on the other hand

  by carrying out tests of the same type on the receiver 5-

  HTB / 1D by varying the quantity of antidepressants in the presence of a radiolabeled ligand specific for the site of the peptide on the 5HT1B / 1D receptors, and by determining the

  Respective Ki or more generally the CIs0.

  The choice of antidepressants with activity

  selective on the 5-HT1B ID site will be dictated by the substan-

  these exhibiting a high Ki or a low IC50 in the reuptake assay and conversely a low Ki or a high IC50 in the binding test at the recognition site of the peptide of the 5-HT11 / lD receptor. We can

  also and more easily use the radiomar- peptide

  qué (tritium) to identify and characterize the specific site of recognition of the peptide, and thus directly to study the capacities of various substances to move (competition) this peptide by measuring their Ki and by comparing this last to the values of Ki obtained for these same substances to inhibit uptake of -HT (or displace a reuptake inhibitor (3H paroxe-

tine for example).

  Another object of the invention also consists in a process for detecting ligands of the site of the 5-HT1 receptor, iD for the peptide LSAL, characterized in that the steps consisting in: - bringing into contact a molecule or a mixture containing different molecules, possibly not identified with a recombinant cell expressing on its surface the 5-HTlB / lD receptor in the presence of a compound

  according to the invention under conditions allowing the inte-

  reaction between the 5-HT1B1 / D receptor and the said molecule (s), in the event that the latter has an affinity for the 5HTlB / lD receptor; - detecting the amount of said compound according to the invention linked to the receptor; - deduce from it the possible fixing of said

(said) molecule (s).

  This process can be used for setting up

  evidence of agonists or antagonists of the LSAL peptide.

  Another subject of the invention relates to a method for demonstrating modulators of the site of the 5-HTlB / lD receptor for the LSAL peptide, characterized in that the steps consisting in: - bringing a molecule or a mixture containing different molecules, possibly unidentified, with a recombinant cell expressing on its surface the 5-HT18 / lD receptor in the presence of a compound according to the invention, under conditions allowing interaction between the 5-HTlB / lDet receptor said compound according to the invention; - detect molecules capable of modulating

  the activity of the compound according to the invention on said receptor

  tor. Ligands or modulators obtained according to a process as described above can be used as active principle of a therapeutic composition useful in the treatment of psychiatric disorders such as depression, circulatory disorders such as migraine, or

  immunologicals linked to 5-HTÎB / lD receptors.

  The compounds according to the invention are also useful as diagnostic tools for labeling the 5-HTlB, / D receptors, and more particularly the site of

  binding of the endogenous peptide to these receptors.

  To this end, it is advantageous to use a diagnostic composition comprising a compound according to the invention radiolabelled in an imaging technique using an emission tomography scanner

of positons.

  The isolation and the

  characterization of the Leu-Ser-Ala-Leu peptide according to the invention

  tion, as well as the binding properties and activi-

  pharmacological tees on the 5-HT1./1 receptors. of the LeuSer-Ala-Leu peptide with reference to the appended figures in which: - Figure 1 represents the interactions of

  bond with [125I] cyanopindolol.

  The binding of [125I] cyanopindolol was measured as described in HOYER D. et al., Eur. J.

Pharmacol. 118, 1-12 (1985).

  - Figure 1A represents the results obtained with cortical membranes of rat brain (50 pg of proteins), incubated for 60 minutes at 37 C

  in a total volume of 100 μl in the presence of [125I] cyano-

0.3 nM pindolol.

  The incubation was carried out in a 5 mM tris-HCl buffer, pH 7.5, containing 154 mM NaCl, 1 μM of

  pargyline, 30 µM isoproterenol and various concentrates

  peptide trations (10- to 10-8 M). Each point corresponds to

  spends an average plus or minus the standard deviation of five

independent determinations.

  - Figure lB represents the results

  obtained with membranes of NIH 3T3 cells trans-

  fected with the 5-HT1 receptor gene (50 μg of proteins), incubated for 60 minutes at 37 ° C. in the presence of 0.3 nM cyanopindolol. The liaison was

measured as in Figure 1A.

  FIG. 1C represents the results obtained from the cortical membranes of the rat brain (50 μg of proteins) incubated for 60 minutes at 37 ° C. in a total volume of 100 μl in the presence of increasing concentrations of [125I] cyanopindolol (0.06 to 0.35 nM) with

  (0) or without (O) the 1 nM peptide. Each point corresponds to

  lays the mean plus or minus the standard deviation of three

independent determinations.

  - Figure 1D represents the curve of

  Scatchard of saturation curves.

  - Figure 2 shows the results of the effects of LSAL on the stimulated adenylylcyclase activity

by forskolin.

  Membranes of NIH 3T3 cells expressing the gene for the 5-HT receptor, of mice were suspended and homogenized with a Dounce homogenizer in a 50 mM tris-HCl buffer pH 7.4 containing MgCl2 to

  3 mM and 0.2 mM EGTA (TME buffer).

  The adenylyl cyclase activities were measured in 50 μl aliquots (30-50 μg of proteins) of homogenates in a final volume of 100 μl containing 0.1 mM, [a32P] ATP (lpCi), 1 mM MgCl2, 50 pM GTP, 10 pM forskolin, 100 mM NaCl, 10 mM theophylline, mM phosphocreatinine, 0.2 mg / ml creatine kinase, 1 mM [3H] cAMP (= 15,000 cpm / test) and the substances to be studied. The membranes were incubated for 20 minutes at 30 C. The reaction was stopped by adding 200 μl of 5 mM ATP, 5 mM cAMP and dodecyl sulfate

  1% sodium dissolved in 50 mM Tris-HCl buffer.

  The [32P] cAMP formed was isolated by the two-column chromatography method described by Salomon et al. (Adv. Cyclic Nucleotide Res., 10, 35-55 (1979).,

  modified by De Vivo and Maayani (J. Pharmacol. Exp.

Therap. 238, 248-253 (1986)).

  - Figure 2A expresses the effect of LSAL as a function of its concentration on the enzymatic activity stimulated by forskolin in the presence of 0.1 pM of 5-HT. - Figure 2B expresses the dose-response curve of the effect of 5-HT on the enzymatic activity stimulated by forskolin in the absence (--- 0), or in the presence

(O ---- O) from LSAL (10-9 M).

  - Figure 3 represents the effect of the peptide on the release of serotonin in synaptosomes of the hippocampus of rat brain. The results are expressed as percentages of the release of [3H] 5-HT (P2 / Pl ratio) in the absence of peptide. Each point is the mean plus or minus the standard deviation of triplicate determinations obtained using 3-6 distinct hippocampal homogenates.

  The method used is the method

  previously cited (BOLANOS-JIMENEZ B. et al., Eur. J. Pharmacol. 242, 1-6 (1993)) with the following modifications: [3H] acetylcholine was substituted with 60 nM [3H] 5-HT and 0 0.01% ascorbic acid was added to the

incubation medium.

  1. Isolation and purification of the peptide Leu-

  Ser-Ala-Leu Using as a discrimination criterion the ability of a potential factor to interact with the recognition of 5-HT by the 5-HT1, l1 receptor of rat brain synaptosome membranes, as described in FR- 2,657,784, an active compound was purified from extracts of rat, horse and bovine brains. The purification methodology used gel permeation separations, normal phases

  or reversed or ion exchanges.

  It is summarized in the table below: Steps in the purification of the endogenous peptide

  STEPS OF THE CONDITIONS OF THE FRACTION

  CHROMATOGRAPHY ACTIVE CHROMATOGRAPHY

  (Elution time __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ or retention retention) SIZE EXCLUSION Isocratic elution with a buffer (TSK HW 40S, Merck, of ammonium acetate TE = 80 min 600 x 26 mm) (CH3COONH4) 50 mM (pH 5) Flow rate: 2 ml.min- 'Linear elution gradient from A to B for 12 minutes: REVERSE PHASE A: 50 mM CH3 COONH4 (pH 5); (C ,, Ultrabase. Shandon, B: 50 mM CH3 COONH, (pH 5) / CH3CN, 88/12 TR = 20 min. 250 x 10 mm) Gradient in stages for 5 min. with C. C: 50 mM CH3 COONH, (pH 5) / CH3CN, 50/50 Flow rate: 4 ml.min 'SIZE EXCLUSION Isocratic elution with (Sephadex G, 10 mM CH3 COONH, (pH 5) TE = 240 min Pharmacia, Flow rate: 0.3 ml.min '450 x 16 mm) Linear elution gradient from A to B for 15 minutes INVERSE PHASE AT 50 mM CH3 COONH. (pH 5) / CH3CN, 85/15 (C ,,. Ultrabase, Shandon, B: 50 mM CH3 COONH4 (pH 5) / CH3CN, 75/25 TR = 5.75 min. 250 x 10 mm) Gradient in stages for 5 min. with C. C: 50 mM CH, COONH, (pH 5) / CHCN. 50/50 Flow speed: 4 ml. min- 'REVERSE PHASE Linear elution gradient from A to B (C, ". Ultrabase, Shandon, for 35 minutes 250 x 10 mm) A: 50 mM CH3 COONH, (pH 5); TR = 28 min. B: 50 mM CH3 COONH, (pH 5) / CH3CN, 70/30 Flow rate: 4 ml.min- 'PHASE Linear elution gradient from A to B PSEUDO-INVERSE for 30 minutes (Coal column, A: 50 mM CH3 COONH, (pH 5), TR = 12.71 min Hypercarb., B: 50 mM CH3 COONH, (pH 5) / CH3CN, 70/30 Shandon, 100 x 3 mm) Flow rate: 1 mlmin 'INVERSE PHASE Isocratic elution with 0.05% (C,., Ultrabase, Shandon, TFA / CHCN, 83/17 T = 5.14 min. X 4 mm) Flow rate: 1 ml.min-' Characterization of the fractions obtained was carried out essentially on the basis of analysis by

  NMR, amino acid analysis and amino acid sequencing

  cides. The purified compound consisted of Leu-Ser-Ala-Leu.

  The same fraction was also obtained from horse and bovine brain, which means

  that this peptide is conserved in mammals.

  2. Interactions of the purified peptide with the 5-HT1B / 1D receptors The synthetic peptide LSAL was tested for its capacity to "mimic" the effects of the purified fraction obtained from brain extracts. LSAL inhibits the binding of [3H] -5-HT to 5-HT1 receptors with an IC50 close to 10-1M and a maximum inhibitory effect of 30%, occurring at a concentration of 10-9M. The 5-HT1o bond, which under experimental conditions

  implemented, mainly represents sites 5-

  HT1 B / D, is inhibited up to a maximum value of 75%, as shown in Table 2 below with an identical IC50. This last observation indicates that the 5-HT 1 B / D receptors are particularly sensitive to the peptide. This is why tests have been carried out on homogenates of rat brain tissue in which the 5-HTib receptors were specifically labeled with [125I] cyanopindolol with an IC50 of 10-1M and a maximum effect of 60% inhibition. , as shown in Figure

1A.

* In addition, the activity of this peptide on the 5-HTD receptors has been demonstrated in the guinea pig and on the human brain using [3 H] 5-HT. The IC50 value was again 10-1m. These results suggest

  strongly that in vitro 5-HT 1 B / D receptors are

kill the target of the LSAL peptide.

  The activity of the peptide was also evaluated on the binding to 5-HT1 receptors. in NIH 3T3 cells transfected with the 5-HT receptor gene,

the mouse.

  The peptide inhibitory effect was very similar to that observed in tissue homogenates

cerebral (Figure 1B).

  The maximum effect corresponded to 70% inhibition

  tion of the specific binding of [125I] cyanopindolol with a CId0 value of 10- "M. This result clearly demonstrates that LSAL interacts directly with

5-HT1B receptors.

  To further study the type of interaction of the tetrapeptide with the 5-HT1 B / D receptors, saturation curves for the radiolabelled ligand were established in the presence of the peptide. The binding results obtained

  suggest that the inhibitory activity of the corresponding peptide

  lies in a non-competitive interaction (Figures lC, 1D).

  This result was observed with [3H] 5-HT, as well as with ['25I] cyanopindolol to label the 5-HT1B receptors and is in agreement with the previous hypothesis of the existence of a specific site for the distinct but closely related peptide at the site of

5-HT binding.

  The pharmacological specificity of the partially purified fractions was studied during a subsequent purification using rat brain homogenates. The tests were performed using synthetic LSAL to determine if the

  peptide interacted or not with other receptors 5-

  HT, or those of other neuro-transmitters.

  The 5-HT 1A receptors were not affected at a concentration of the peptide which strongly antagonized the binding to 5-HT 1, B- '

  Similarly, the radio link

  ligands to the 5-HT, E 5-HT1, F 5-HT2 and 5-HT3 receptors were not affected (Table 2), which indicates that among the serotonergic receptors studied, only the 5-HT class. é, D was sensitive to the peptide. The binding of other specific radio-ligands to receptors of various neurotransmitters has been examined: adrenergic, dopaminergic, muscarinic, histaminergic, opioid and benzodiazepine receptors a and P. The binding to these receptors was not significantly reduced by LSAL at concentrations which had a maximum inhibitory effect for binding to the -HT 1B / D receptors (Table 2).

Table 2

  Inhibitory effect of the peptide on different radioligand bonds in rat brain tissue

INHIBITOR EFFECT

L! GANDS RECEIVERS IN%

WITNESS

SEROTONINERGIC

  5-HTlA [3H] 8-OH-DPAT (3 nM) 0 -HT, B ['251lcyanopindolol (0.3 nM) + 30 pM isoproterenol 70 + 2 + 0.1 nM 8-OH DPAT -HT, NONA [3H] 5-HT (30 nM) + 0.1 pM 8-OH-DPAT 75 + 3 5-HT, EFC [3H] 5-HT (30 nM) + 20 nM 5-CT 0 + 10 -HT2A [3H] ketanserine (5 nM) 10 + 5 -HT2A [3H] DOB (5 nM) 2 + 8 -HT3 [3H] BRL 43694 (3 nM O + _ 10

ADRENERGIC

  a, [3H] prazos: rne (2 nM) 5 + 3 f3 [3H] dihyarca! preno! ol <3 nM) 10 15 + 5-HT 10-r M

DOPAMINERGICS

  D2 [3H] spiperone (2 nM) + 10 pM 5-HT 2 3

HISTAMINERGIC

  Hi [3H] mepyramine (5 nM) 10 10 MUSCARINICS [3H] quinuclidinyl benzylate (3 nM) 0 2 OPIACEATES [3H] naloxone (2 nM) 10 10 BENZODIAZEPINES [3H] flunitrazepam (3 nM) 2 2 Legends from Table 2 : Rat brain brain membranes (200 pg) were incubated for 30 minutes at 250C with different specific radio-ligands (see Table) in

  presence or absence of 1 nM of the peptide.

  The different binding conditions used for serotonergic receptors are those described above (PALACIOS J.M. et al., Methods in Neurosciences 12, 238-262 (1993)). For the other bonds, the incubation medium consisted of 50 mM Tris-HCl pH 7.4 containing 120 mM NaCl and 50 mM KCl ([3H] spiroperidol, [3H] quinuclidinyl benzylate,

  [3H] naloxone, or 4 mM CaCl2 and 4 mM MgCl2 ([3H] flu-

  nitrazepam), or 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2 and 1 mM MgSO4 ([3H] prazosin), or 90 mM NaCl

([3H] dihydroalprenolol).

  Each point corresponds to an average plus or

  minus the standard deviation of five independent determinations.

  The identified tetrapeptide did not show

  other effect on the uptake of 5-HT in preparations

  synaptosomal tions. It also did not show any effect on the reuptake of other neurotransmitters (or their

  precursors), for example choline, GABA, histami-

  ne, dopamine and norepinephrine. this result indicates that the peptide is different from endogenous compounds previously suspected of interacting with transport

amines.

  3) Interaction of LSAL with the activity

  of the 5-HT1 receptors. (transducer system-

  tion) The 5-HT1 B / D receptors are known to be mainly coupled to the Gi protein, by inhibiting the activity of adenylyl cyclase. The interaction of LSAL was examined on the enzymatic activity correlated with

  stimulation of the 5-HT1 receptor expressed in cells

NIH 3T3.

  The peptide had no effect on basal cAMP production. However, it significantly increased the inhibitory effect of 5-HT on the adenylyl cyclase activity stimulated by forskolin (Figure 2). This effect was dependent on the concentration

  with a bell-shaped dose-response curve. The acti-

  speed of the peptide on the homogenates of substantia nigra

  was almost similar. These results indicate

  that LSAL interacts with the activity of the

5-HTIB receptor.

  4) Effects of the peptide on the functional activity

  cellular concentration of 5-HT 1B / D receptors The activity of the peptide on the release of [3H] 5-HT from hippocampal synaptosomes,

  previously charged rat brain was examined.

  The release of [3H] 5-HT elicited by K 'decreased dosedependently in the presence of LSAL with an apparent IC50 close to 10-1lM. The maximum effect, observed at a concentration of 1-10M, corresponds to a 20 to 25% inhibition of the induced release and

  decreases at higher concentrations (Figure 3).

  These results demonstrate that LSAL interacts with cellular function correlated with the activity of 5-HT1 B / D receptors and regulates the inhibitory activity of

5-HT.

  5) In vivo effects of LSAL Tests have been carried out in vivo in

  mouse, to examine the behavioral effects of LSAL.

  Insofar as neuro-pharmacological studies

  previous logic had shown the involvement of the 5 HT HT IB receptors in stress and anxiety disorders, the effects of the peptide were examined in

two animal models of anxiety.

  Open field test The activity of LSAL was determined in the open field test on C57 / Black-6 mice. At doses of 50 pg ICV per animal, the exploratory activity was significantly reduced compared to control animals which received the excipient in the same

conditions (table 3).

TABLE 3

  Effects of intracerebroventricular injections of LSAL in mice

BEHAVIOUR

OPEN FIELD

  Substance Number Number of Number of Times injected with square animals restorations of immobilization (sec.) Crossed ___ Control 12 115 + 9 23 + 3 12 + 5 (NaCI)

LSAL 12 88 + 9 * 15 + 3 * 47+ 8 *

pg

Raised Cross Labyrinth

  Substance Number Open arms Open arms Total Arm injected with animals INPUTS (%) TIME (%) INPUTS Control 18 61 9 66 + 8 10 + 2 (NaCI) LSAL 2 pg 8 68 + 4 55 4 24 4 ** pg 8 71 t 6 58 + 6 15 3 pg 10 28 t 9 * 22 7 ** 3 + 1 Legends of Table 3 Open field: Male C57 / BL / 6 mice (20-25 g) were used. The open field consisted of a white plastic box (35 x 35 x 20 cm) divided into 25 squares

  equal. LSAL dissolved in 5 ml of 0.9% saline solution

  line in a volume of 5 μl was injected icv into the mice, which were placed at an angle to the box. After two minutes, the number of squares crossed, the U-turns

  performed and downtime were measured.

  The results are expressed as the mean plus or minus the standard deviation. Difference with the witness:

  * p <0.05, ** p <0.005 (Student's Test).

  Raised cross maze Male BALB / C mice weighing 20 & 24 g

have been used.

  The raised cross labyrinth was made of plexiglass with open arms and two closed arms of the same size (12.5 x 5 cm) with walls 15 cm high, these arms starting from a central platform of x 5 cm, placed 40 cm above the ground. LSAL dissolved in 5 ml of 0.9% saline in a volume of 5 μl was injected icv into the mice. Three hours later, the animal was placed on the central platform of the labyrinth. Each entry into an arm and the time spent in each arm was recorded. The duration of the test was 5 minutes. The

  labyrinth was cleaned after each test.

  The LSAL was tested under conditions of low light intensity (BENJAMIN D. et al., Life Sci.

47, 195-203 (1990)).

  The results were expressed as the mean plus or minus the standard deviation of the percentage of entries in the open arms (% of entries in the open arms), percentage of time spent in the open arms (% time in the open arm) and total number of entries in the arms. Difference with the witness:

  * p <0.05, ** p <0.01 (Student's Test).

  The recovery trend was significant.

  tively decreased (60 to 65%) and acts of grooming were abolished. Similar results were observed in BALB / C mice. These results indicate that LSAL is potentially anxiety-provoking at high concentration, because it induces a behavior of "immobilization" distinct from

the sedative effect.

  Test of the "raised cross labyrinth" This experimental test is used to

  determine the degree of anxiety and consists of a labyrin-

  the raised with two arms, one protected by walls and

the other unprotected.

  The animal is supposed to be anxious when it avoids

  entry and reduces the time spent in open arms.

  Administration of tetrapeptide at low concentrations (2 pg ICV) increased the total number of entries in the arms of the apparatus, which corresponded to increased locomotor activity, while at 10 and 15 pg, it reduced significantly the

  time spent by the animal in open arms (Ta-

3).

  This result suggests that under the experimental conditions used, the peptide has effects

  on behavior. The increase in locomotive activity

  trice observed at 2 pg can correspond to a state of disinhibition allowing a more intense exploration or to a state of low anxiety (nervousness or restlessness restless). At 10 and 15 pg, the significant decrease

  of the time spent in open arms is probably

frankly to an anxiety-provoking effect.

  These behavioral modifications observed after in vivo administration of LSAL strongly show that the molecular and cellular modifications induced by the peptide actually lead to

  changes in the function of the central nervous system.

  In fact, LSAL seems capable of modifying the state of anxiety after administration in vivo. LSAL appears as an endogenous regulator, capable of finely modulating the control exerted by 5-HT on neurotransmission to

  central nervous system level. He probably plays-

  play a key role in physiological functions (sleep, thermoregulation, learning and memory,

  behavior, pain, ...), as well as in mechanisms

  my known pathophysiology (stress, anxiety, depression, aggression, eating disorders, etc.) or

  strongly suspected to involve the serotoni-

  energetic. It should also play an important role in pathologies linked to blood pressure and migraine, because 5-HT1 (5-HTl-like (probably 5-HTlD)) receptors have been located in the vessels of the human brain. and in other muscles

smooth.

  It undoubtedly also regulates the immune response insofar as the 5-HT1 t / D receptors are undoubtedly present in immunocompetent tissues, and insofar as the transmission of 5-HT is known to

  interact with the immune response.

Claims (24)

  1. Compounds of the following peptide sequence: Leu - Ser - Ala - Leu - Y   in which Y represents OH, NH2, a hydrocarbon residue   substituted or unsubstituted loxy, or a peptide sequence   that having from 1 to 10 amino acids, as well as the corresponding compounds in which the peptide bond is replaced by a bond resistant to the enzymatic degradation of the proteases or in which the peptide backbone comprises one or more intercalated groups making the peptide bond resistant to the degradation enzymatic.   2. Compounds according to claim 1, in which the peptide linking group -CO-NH- is   replaced by a group chosen from -CO- NR ', -CR1R2-   CR3R4-, -CO-CR1R2-, or in which the backbone peptidi-   that has one or more intercalated groups chosen from the groups -CR R2-, -NRJ-, -O-, Rl, R2, R3 and R4, identical or different, representing H or a CQ-C6 alkyl group and R 'representing a C1-C6 alkyl group. 3. Compounds according to claim 1, comprising a sequence chosen from: Leu - Ser - Ala - Leu Leu - Ser - Ala - Leu - OCH3 Leu - Ser - Ala - Leu - NH2 Leu - Ser - Ala - Leu - Gly - Gly - Gly - Tyr as well as the corresponding compounds in which the   peptide bond is replaced by a strong bond   aunt to the enzymatic degradation of proteases, or in which the peptide backbone contains one or more   intercalated groups making the peptide bond resistant   aunt to enzymatic degradation.   4. Compounds according to one of claims 1   to 3, characterized in that they are in the form L. 5. Compound capable of fulfilling the role of ligand of the receptor on which the endogenous peptide of sequence Leu-Ser-Ala-Leu (LSAL) is fixed, characterized in that it comprises a group whose spatial structure is substantially identical to that a peptide of sequence X - Leu - Y 'in which X represents H, Ala or Leu-Ser-Ala,   Y 'represents Ser or Y as defined in the claim   tion 1, X and Y 'do not simultaneously represent H and OH, respectively.   6. Compound according to claim 5, character-   in that they comprise a group whose spatial structure is substantially identical to the following peptides: Ala-Leu, Leu-Ser, Ala-Leu-Ser, and Leu-Ser-Ala-Leu. 7. Compound according to claim 5 or claim 6, characterized in that they comprise a group in which the average position of the atoms forming the group which binds to the receptor differs only by more than 5% from the average position of the atoms forming the corresponding peptide.   8. A compound according to claim 5 or claim 6, characterized in that they comprise a group in which the average position of the atoms forming the group which binds to the receptor differs only by more than 2% from the average position of atoms forming the corresponding peptide.   9. Process for producing a compound according to   one of claims 5 to 8, characterized in that it   it is a computer modeling from (s) the   (the) spatial structure (s) of the corresponding peptides   dants and classical chemical synthesis from the   structure obtained after modeling.   10. Method according to claim 9, characterized in that the modeling is carried out at   from the spatial structure of antide-   active pressers at the endogenous peptide binding site. 11. Therapeutic composition, characterized in that it comprises at least one compound according to one   any of claims 1 to 8.   12. Method for in vitro diagnosis of a condition linked to the serotonergic system in a patient, characterized in that an assay is carried out in a biological fluid of the patient of a peptide of sequence Leu Ser - Ala - Leu 13. Method according to claim 12, characterized in that the dosage is carried out using   an antibody specific for said peptide.   14. Polyclonal or monoclonal antibody directed specifically against a peptide as defined in   claim 12, as well as the fragments of said anti-   body, in particular the Fab, Fab ', F (ab') 2 or Fc fragments.   15. A diagnostic kit for implementing the method according to claim 12 or claim 13, characterized in that it comprises an antibody or an antibody fragment as defined in claim 14.   16. Method for screening anti-substances   pressives occupying the same conformational site as the peptide Leu-SerAla-Leu, characterized in that discrimination is carried out by inhibition tests between on the one hand their binding to the 5-HT reuptake site and d between their link on the site - HT1B / lD - 17. The method of claim 16,   characterized in that the inhibitory constant is compared   tion Ki, substances to be tested by inhibition tests   competitive in the presence of a peptide according to claim 1 to the inhibition constant Ki2 of these same substances, to inhibit the reuptake of 5-HT   or move a reuptake inhibitor.   18. Diagnostic composition comprising a   compound according to the radiolabelled invention.   19. A method of visualizing the peptide sites of 5HTlB / lD receptors, characterized in that a composition according to claim 18 is used and that the 5-HTlB / lD receptors are visualized using   of a positron emission tomography scanner.   20. Method for detecting ligands of the   5-HTlB /, D receptor site for the LSAL peptide, characteristic   laughed at in that the steps consisting in: - bringing a molecule or a mixture containing different molecules, possibly unidentified, into contact with a recombinant cell expressing on its surface the 5-HTIB / lD receptor in the presence of a compound   according to any one of claims 1 to 4 in   conditions allowing the interaction between the -HT1B / lD receptor and said molecule (s), in the case where   this would have an affinity for the receptor 5-   HTlB / 1D; - detecting the amount of said compound according to the invention linked to the receptor; - deduce from it the possible fixing of said (said) molecule (s).   21. The method of claim 20 for the demonstration of agonists or antagonists of the peptide endogenous LSAL.   22. Method for highlighting modula-   teurs of the site of the receptor 5-HTlB / lD for the peptide LSAL characterized in that one carries out the stages consisting at:   - contacting a molecule or mixture containing different molecules, possibly unidentified, with a recombinant cell expressing on its surface the 5-HTlB / lD receptor in the presence of a   compound according to one of claims 1 to 4, in   conditions for interaction between the receiver   -HTlB / lD and said compound according to one of claims 1 to 4;   - detect molecules capable of modulating   the activity of the compound according to one of claims 1 to 4 on said receiver.   23. Ligand or modulator of the -HTlBulD receptor site for the LSAL peptide, identified according to the method of claim 20 or 22.   24. Therapeutic composition comprising as active ingredient a ligand or modulator according to the claim 23.