WO2001068068A2 - Compounds capable of binding with the cytoskeleton - Google Patents

Abstract

The invention concerns the use of steroids and non-toxic compounds, capable of crossing the blood brain barrier characterised in that they are capable of binding the same site as pregnenolone on proteins constituting or associated with elements of the cytoskeleton and of displacing the MAP-bound pregnenolone, and of influencing the assembling and stabilisation of microtubules. The invention is particularly useful for treating the nervous system, for fighting against ageing of cells containing MAPs, and for treating cancers.

Description

"Compounds capable of binding to constituent proteins or associated with cytoskeleton elements and their applications for the manufacture of medicaments".

The invention relates to compounds capable of binding to constituent proteins or associated with elements of the cytoskeleton.

It aims more specifically their applications for the manufacture of medicaments for the treatment of pathologies involving dysfunctions of these proteins.

The cytoskeleton is a complex network (see bibliographic reference (1) at the end of the description) which includes the primary networks of three classes of cytoskeletal filaments (microtubules, microfilaments, and intermediate filaments) to which is added a microtrabecular network which includes the majority proteins associated with microtubules, microfilaments and intermediate filaments. The MAP protein (family of Microbubules Associated Proteins described below ¹ is a characteristic example of a component of this network: it has ^" a binding domain to microtubules, but also to microfilaments (2,3) and intermediate filaments (4, 5).

associated proteins; dont le rôle est de faciliter l'assemblage de la tubulme, mais auss- l'organisation et les fonctions du reseau icrctubulaire La tubulme présente une grande hétérogénéité. Elle comprend en effet de nombreuses isoformes provenant a la fois de l'expression de plusieurs gènes et de très nombreuses modifications post-traductionnelles (6).The microtubules are hollow cylinders with an external diameter of 25 mm, the wall of which is made up of protoflaments, themselves formed by the stack of globular α and β subunits of tubule (50 Kd). Tubod heterodimeres are periodically associated with proteins called MAP _S

associated proteins; whose role is to facilitate the assembly of the tubule, but also the organization and functions of the icrctubular network The tubule presents a great heterogeneity. It indeed includes numerous isoforms originating from both the expression of several genes and numerous post-translational modifications (6).

MAP _S are divided into two groups, namely, "structural" MAP involved in the assembly of tubulins and MAP _Ξ "motor" which use microtubules as rails to ensure the movement of certain molecules and organelles.

The structural MAP _Ξ targeted in the invention include several families of proteins, namely, the MAPi A and B proteins (350 Kd) encoded by separate genes, the high molecular weight MAP A and B proteins (300 Kd), the proteins MAP. Low molecular weight C and D (around 70 Kd), MAP proteins A and B (180 Kd) present in neurons up to 10 days after birth, MAP proteins ₄ A, B and C (215 to 240 Kd ) ubiquitous, with properties very close to those of MAP., Tau proteins (50-67 Kd or 110-120 Kdj, STOP proteins (145 Kd), very stabilizing molecules with microtubules.

In the neuron, microtubules are involved in many functions (neutral growth, maintenance of complex morphologies such as dendritic arborizations, axonal transport of various molecules). For example, MAP proteins. participate in the dendπtique arborization, while the tau proteins are essential for axonal growth. We also know that the different isotypes of each MAP have a specific location and roles. MAP. B increases his expression from fetal to adult stage. MAP- A is only expressed from a certain stage of development. MAP ₂ C (2), which is mainly expressed in the early stages of development, is still present in the olfactory bulb (7) and in the photoreceptor cells of the retina (8) in adults. MAP ₂ D is localized in the hippocampus from the fetal stage (3).

Microfilaments are polymers made of actin (43 Kd). Very abundant in the cortical region of the axon, they are also found in the central region where they participate in its dynamic architecture.

The intermediate filaments of neurons, polymers formed by a triplet of proteins (68, 150, and 200 Kd), are present in differentiated neurons. Interconnected with microfilaments and microtubules, they help maintain the neuronal architecture, MAP _S serving as a bridge between the three types of cytoskeletal filamentary structures.

After demonstrating a saturable, high affinity binding in the cytosol of the rat brain (fetus and young adult) with steroids, the inventors have found that highly purified preparations of MAP ₂ have the same binding characteristics.

In vi tro work by the inventors has also demonstrated a specific action of steroids on the polymerization of cytoskeleton structures. However, it is currently considered that the effects of steroids are exerted via soluble intracellular receptors of another protein class. These receptors have been cloned and often classified as "nuclear" because of their mode of action as transcription factors at the genome level.

A series of observations published over the past 20 years indicates that the receptors for steroids also function at the level of the plasma membrane of target cells, the action of neurosteroids at the level of receptors for neurotransmitters being an important example.

By demonstrating a direct interaction of steroids, more specifically pregnenolone (PREG for short), with proteins constituting or associated with elements of the cytoskeleton, the inventors have revealed a new mechanism of action for these compounds, which can be advantageously put take advantage to intervene on all functions involving microtubules, intermediate filaments and microflamaments, including growth, differentiation, plasticity of neurons, prevention and repair of neurodegenerative conditions and lesions, and more generally all cell division phenomena .

In general, this new mechanism of action concerns any non-toxic compound, capable of crossing the blood-brain barrier, and capable of binding the same site as the

PREG on said proteins, or to move PREG, and to exert an effect on the assembly and stabilization of microtubules.

The object of the invention is therefore to provide compounds capable of binding to the proteins constituting or associated with the elements of the cytoskeleton as identified by a screening method, comprising bringing these compounds into contact with said proteins, under conditions allowing the establishment of a bond when the protein, MAP for example, is a receptor for the compound.

The invention particularly relates to the use of such compounds for the manufacture of medicaments for the treatment of pathologies involving these proteins.

The compounds which can be used according to the invention for the manufacture of medicaments are more especially steroid hormones, (this term being used in the description and the claims to cover their conjugates, such as sulphate esters, or fatty acid esters), their precursors, their metabolites, free or conjugated, as well as their analogs, steroid or non-steroid.

As precursor of steroid hormones, mention will be made in particular of pregnenolone or its esters, in particular pregnenolone sulfate (PREGS), for which no intracellular receptor has been identified to date.

Analogs are pharmaceutical agents capable of increasing the formation of microtubules, stabilize under the conditions given in the examples relating to PREG, to compete with PREG for binding to MAPc and to cross the blood-brain barrier while remaining sequestered in the nervous system if possible (9, 10, 11).

They include polycyclic compounds, natural or enantiomeric, optionally in mixtures, their sulfates, or their lipid derivatives.

As new products, the invention relates to addition and / or substitution products produced from said compounds on the one hand and proteins constituting or associated with elements of the cytoskeleton on the other hand.

The invention relates in particular to steroid coupling products - associated proteins - microtubules, - in particular, steroids - MAP-, where appropriate in the form of copolymerized with tubulin, or steroids - tau proteins in the form of copolymerized with tubulin.

The invention also relates to the use of proteins constituting or associated with the elements of the cytoskeleton as receptors for compounds as defined above.

The action exerted by these compounds on the polymerization and stabilization of the structures of the cytoskeleton, as illustrated in the examples, gives them great interest in manufacturing medicaments.

The invention therefore relates to the use of said compounds for manufacturing medicaments capable of binding specifically to constituent proteins or associated with elements of the cytoskeleton with cytoplasmic or nuclear localization for the treatment of pathologies with dysfunction of these proteins.

It relates in particular to the use of said compounds for manufacturing medicaments for the treatment of the nervous system, central nervous system or peripheral nervous system. Such drugs make it possible in particular to stimulate the differentiation / maturation of neurons.

The invention relates in particular to the manufacture of drugs adm istrable in oral form, for the treatment of the central nervous system, or in injectable form, for administration by the general route or m if you.

Other forms of administration fall within the scope of the invention, such as retard solutions or pre-drugs with conjugation, for example, to a fatty acid.

To increase the bioavailability, it is also planned, according to the invention, to use the active ingredients in the form of suitable derivatives, such as fatty acid esters.

In these applications, the medicaments produced in accordance with the invention are advantageously used in combination with growth factors (NGF) and / or cellular regulators, such as mterferon or cytomes. Applications of great interest include the prevention or treatment of conditions such as Alzheimer's disease, dementia of vascular origin, the consequences of trauma and stroke in the nervous system and neurodegenerative diseases.

The invention also relates to the manufacture of medicaments, using said compounds, for combating the aging of nerve cells, or of any other cell type containing MAP ₃ , such as epithelial cells, thyroid cells, or endocrine glands , steroidogens in particular.

In these applications for the treatment of nervous system and aging of cells, said compounds can be used in combination with stabilizing effect compounds of the cytoskeleton, such as Taxol ^® and Taxotere ^®.

To the extent that the two types of molecules, compounds like steroids or steroidal or non-steroidal, first, Taxol ^® or Taxotere ^® or equivalent, on the other hand, have a stabilizing effect on microtubules, it is possible to consider a synergistic phenomenon if the two drugs are used simultaneously. In this case, the doses of the two drugs required for good efficacy will be much lower than those required when the drugs are given as monotherapy.

According to another aspect of great interest of the invention, the above drugs can be used for the treatment cancers, in combination with anticancer agents, and allow the effect of the latter to be modulated, in particular in the event of chemo-resistance.

Such associations with agents with microtubular tropism are indeed capable of potentiating the respective effects of the products.

In these different applications, the compounds used are combined with pharmacologically acceptable vehicles suitable for the dosage form chosen. The unit doses are of the order of 100 to 500 mg and the daily doses of the order of 500 mg to 1 g for administration by general route, this dose being reduced for administration in si tu.

Other characteristics and advantages of the invention are given in the description which follows, with reference to FIGS. 1 to 7, which relate, respectively,

- Figure 1, the equilibrium binding of PREG [ ³ H] to fetal brain cytosol,

FIG. 2, by fetal brain cytosol chromatography by FPLC on a Mono Q ion exchange column,

FIG. 3, by chromatography of the Mono Q peaks by FPLC on a column of Superose 12 filtration gel,

FIG. 4, with one immunoblot of the cytosol and of the fractions of one of the peaks eluted from the columns of ion exchange and gel filtration, - Figure 5, the equilibrium binding of PREG [ ^J H] to MAP. purified, complexed or not with purified tubule,

FIG. 6, the assembly kinetics of microtubules, and

- Figure 7, 1 immunocytochemistry of fetal rat brain neurons.

1 - Materials and methods

Animals

Sprague-Dawley rats from the January farm (Le Genest St Isle, France) are used. The rats are subjected to a light-dark alternation of 12 h (lights at 8 h) at 20 ° C and fed to satiety.

Full females (the morning after mating is designated by EO) are housed in the pet store at least 7 days before sacrifice at E18. Adult males 60 to 70 days old are also used.

The calf brains used are free of meninges and large blood vessels. They are stored in an ice-cold isotonic saline solution.

Animals are treated in accordance with the provisions of the Council of the Union Directive

European of November 24, 1986 (86/609 / EEC). materials

- Steroids:

The PREG [7- ^J H] (22.5 Ci / mmol) used is a product marketed by NEN (Boston, MA, EUA).

Progesterone, pregnenolone sulfate, testosterone, corticosterone, tπamc olone acetonide, 17α-OH PREG, 17α-OH progesterone and cholesterol are Sigma-Aldrich products (St Quentin Fallavier, France)

- Enzymes:

The protemase K (Merck, Darmstadt Germany) and Pronase (Roche Molecular Biochemicals, Meylan, France) are dissolved 1 mg / ml in TNM buffer containing lO M Tris, 0.1M NaCl, 3mM MgCl _^ (pH 7.4). Deoxyπbonuclease I and ribonuclease A are Sigma-Aldrich products and are adjusted, respectively, to 100 IU / l and 5 IU / ml. Phospholipase A2 (Roche Molecular Biochemicals) is diluted to 20 IU / ml in TNM.

- Antibodies

The anti-α-tubulm monoclonal antibody N-356 is an Amersham Pharmacia Biotech product (Freiburg, Germany). The monoclonal antibody 152 is an antibody produced by one of the inventors which reacts specifically with a MAP2 of high molecular weight. The antibody antibodies are diluted in PBS containing 3 of BSA.

Methods . Cytosol preparation

Fetal brains are recovered, from which the meninges are removed and subjected to rinsing with a homogenization buffer at 4 ° C. [10 mM Tπs-HCl, pH 8.5 containing 1.5 mM EDTA, ImM dithiothreitol, 10 ° _o glycerol, a mixture of Complete protease inhibitors ^® (Roche Molecular Biochemical), called TEDG buffer]

The brains of adult male rats are recovered after perfusion with PBS containing 2 IU / ml of heparm. The brains are weighed and homogenized with 4 vol. of homogenization buffer, in Potter glass / Teflon ^R homogenizers, and centrifugal at 800 g for 10 mm.

The supernatant is then centrifuged at 100,000 g for 1 h at 4 ° C. The samples are used immediately for testing or are stored in liquid nitrogen until needed.

Protein concentrations are determined using the Bio-Rad kit (Bio-Rad, Hercules, Canada) with BSA as a reference standard.

. Bond constants at equilibrium

Just before the binding test, each sample is purified with a suspension of dextran-carbon and samples of 2 ml of supernatant (purified cytosol) are added to a series of tubes containing PREG [ ^J H] at a rate of 1 to 500 nM, with or without unmarked steroid in excess of 1000 times.

The tubes are incubated at 45 ° C for 30 mm. The separation of the bound steroid and the free steroid was carried out on a mini-column of Sephadex LH-20 (Amersham Pharmacia Biotech).

The counting of the radioactivity in the flasks was carried out in a liquid scintillation spectrometer with correction of the attenuation (Tπ-carb 2100 TR, Packard).

. Nature of the pregnenolone binding component

1 ml of brain cytosol is incubated with 0.2 ml of an enzyme solution at 37 ° C for 30 mm. The binding activity of the samples (0.2 ml) having undergone the enzyme treatment or of the control samples diluted with the buffer alone are studied.

. Competition tests

In the competition tests between pregnenolone and different steroids for the studied bond, 0.2 ml of cytosol is incubated with 100 nM of PREG [° H] and an excess of 1, 10, 100 and 1000 times in non-radioactive steroid . . Anion exchange chromatography

The FPLC system (Amersham Pharmacia Biotech) was equipped with an anion exchange column (5ml Econo-Pac High Q,

The column was equilibrated with a TEDG buffer containing 0.03% CHAPS and 100 nM PREG (TEDGCP buffer) at 4 ° C.

The cytosol (20 ml, 60 mg of protein) is applied and eluted at a speed of 1 ml / mm with the TEDGCP buffer for 20 mm.

2 ml fractions are collected. A linear gradient of NaCl is then applied in TEDGCP to reach a concentration of 1M NaCl after 30 mm., Followed by the isocratic application of 1M NaCl in TEDGCP buffer.

The appropriate fractions are combined; the salts are removed by passing through Hi Trap® mini-desalting columns (Amersham Pharmacia Biotech).

- Gel filtration

The Superose 12 column (1 x 30 cm, Amersham Pnarmacia Biotech) is balanced with TEDGCP buffer containing 0.15 M NaCl, at 4 ° C.

The flow rate is adjusted to 0.25 ml / mm. and 0.4 ml fractions are collected. - Control of protein purification

At each purification step, the fractions collected are incubated with PREG [ ^J H] 100 nM in the absence or with a 100-fold excess of non-radioactive PREG. The saturable bond is then determined.

- Western blots

Raw cytosol and partially purified samples

(10 μg of protein) are separated by 10% SDS-PAGE, then transferred to nitrocellulose membranes. Membranes were blocked by incubation in 5% skim milk containing 0.1% Tween 20, added to a Tris ^® buffer (10 mM Tris, 50 mM NaCl, 2 mM EDTA, pH 8.8) for 1 h, at room temperature. All washes are performed in Tris buffer containing 0.1% Tween 20 ^® (TNT buffer). The membranes are then incubated with anti-tubulin antibodies (1: 2000), or anti-MAP2 monoclonal antibodies (1: 1000), at 4 ° C., for approximately 14 h.

Incubation with a secondary anti-mouse IgG antibody [fragment F (ab ') ₂ ], coupled to peroxidase, 1/10000, (Pierce, Rockford, 111., EUA) is carried out in a TNT buffer containing 5% milk, for 45 min., at room temperature. The signal is detected by an ECL chemiluminescence system (Amersham Pharmacia Biotech), with a Kodak Y-Omat film, following the manufacturer's instructions. - Preparation of microtubule proteins

Calf brains are washed 2 times in L buffer (0.1 M MES, 1 M EGTA, 0.1 mM EDTA, 1 mM MgCl, 1 mM dithiothreitol and 1 mM PMSF, pH 6.4).

The microtubules are prepared from calf brain or adult or fetal rat brain according to an assembly / disassembly process, as a function of temperature, m vi tro (12).

The calf tubule is purified according to the protocol of Weingarten et al (13), but the step using phosphocellulose is replaced by chromatography on a cation exchange column (Fractogel Merck) (14). The microtubules and the tube are prepared in buffer L, supplemented with 1 mM GTP (buffer A).

Proteins tau and MAP. are purified according to the protocol of Fellous et al (12) by denaturation of microtubules by heat and gel filtration column, Sephacryl ^® 300 being used instead of Ultrogel ACA 34. tau proteins are then further purified according to Lmdwall and Cole method (15).

- Measurement of the assembly of microtubules

This assembly is able by controlling the turbidity increases with a spectrophotometer Uvicon ^® (Kontron Instruments, Montigny-le-Bretonneux, France). - Culture of neurons

Primary cultures of fetal neurons E17 are carried out according to El-Etr et al (16). Briefly, this method consists in spreading the dissociated cells (200,000 cells / cm-) on glass coverslips coated with poly-1-ornιthme and cultured in a completely defined medium in 5 ° αe CO; at 37 ° C. The culture conditions used make it possible to obtain a practically pure neuronal population.

24 h after sowing, 1 micromolar pregnenolone (final ethanol concentration 0.01%) or the solvent is added and the cultures are continued for 2 days. The cells are fixed in 4% paraformaldehyde, then frozen at -80 ° C. In good time, they are rehydrated by passages of 5 mm in ethanol at concentrations of 100, 90 and 70%. After 2 washes in PBS for 5 mm, the cells were incubated in PBS containing 3% BSA and 0.1% Triton X-100 ^® at room temperature for 1 h.

After 4 washes in PBS, the cells are incubated at 37 ° C. for 1 h, then at 4 ° C. for approximately 14 h, in the presence of either anti-α-tubulm monoclonal antibody diluted to 1/2000 or d monoclonal antibody antι-MAP2 diluted to 1/1000 or anti-double cortin antiserum diluted to 1/300.

After 3 washes in PBS, the secondary antibody is added at room temperature, for 45 mm. It acts either of rabbit IgG [fragment F (ab ') _] anti-mouse Ig conjugated with biot e, diluted to 1/350 (Roche Molecular Biochemicals) or biotmyle anti-rabbit IgG, diluted to 1/300 (Boehrmger).

After 3 washes, the peroxidase-streptavidme amplification complex (Dako, Denmark) is added for 30 mm at room temperature.

The cells are washed and the AEC substrate (amino-ethyl-carbazole, Sigma-Aldrich, Saint-Quentin Fallavier, France) is deposited on the cells for 15 mm. The reaction is stopped by adding distilled water. The cells are counter-stained with hematoxylm and mounted on Glycergel.

- Electron microscopy

After polymerization of the microtubules induced by MAP ₂ in the absence or in the presence of the test substance, an aliquot of the experimental sample is deposited on a hydrophilic grid (300 mesh) coated with carbon. The grids are treated with a 1% aqueous solution of uranyl acetate. The observations are made with a Philips CM12 electron microscope.

- Statistical analyzes

The equilibrium binding data are analyzed with the Mac Ligand program, adapted from Munson and Rodbard (17). The curves are plotted with the kaleidograph TM 3.0 software (Abelbeck software, Ritme Informatique, Paris). 2 - Results

Pregnenolone binding sites in the rat brain.

The cytosol samples (0.2 ml, 2.5 mg protein / ml), previously deteroidized by adsorption on carbon / dextran, are incubated with increasing concentrations of PREG [ ³ H] in a TEDG buffer at pH 8 , 5, at 40 ° C, for 30 mm.

The bound steroid is separated by gel filtration on Sephadex LH20 ^® at 0-4 ° C. The binding constants are determined with the Mac Ligand program.

FIG. 1 represents a Scatchard diagram of the specific bond.

A Kd constant of 31.0 ± 0.8 nM and a maximum concentration of binding sites (Bmax) of 1.2 ± 0.1 pmol / mg of protein are observed (mean ± standard error, n = 3).

To study the effects of hydrolytic enzymes on the binding of PREG [ ^J H], samples of 1 ml of cytosol were used, incubated with 0.2 ml of an enzyme solution, at 37 ° C, for 30 mm . The results obtained, expressed in ° ₀ of the control incubation (cytosol diluted in buffer) are reported in Table 1 (mean ± standard deviation (n-3)). TABLE 1

Enzyme Concentration / ml% of control

Protemase K 0.17 mg 42.2 ± 7.5

Pronase 0.17 mg 40.5 ± 5.7

Deoxyπbonuclease 1 17 IU 99.4 ± 12.7

Ribonuclease A 0.8 IU 82.4 ± 9.2

Phospholipase A2 33 IU 85.0 ± 7.3

It can be seen that the PREG [ ³ H] binding sites are destroyed at about 60% by protemase K and Pronase ^κ . On the contrary, no significant effect is observed with DNAase, RNAase and phospholipase A2.

To study the specificity of PREG [ ³ H] binding, various steroids were used.

The relative competition indices were determined as ratios of the competitor's concentrations (in denominator) versus PREG leading to a 50% decrease in the specific binding of PREG [ ¹ H] x 100 (mean ± standard deviation, n = 3 ). The results are given in Table 2.

TABLE 2

^" ~~~ ^™ ~~ Stéî dê7 ^" RCR 50

Pregnenolone 100

Pregnenolone sulfate 78.8 ± 7.0

Progesterone 72.1 ± 17.7

Δ5- Pregnene-3β, 20α-diol 68.4 ± 15.3

3β-hydroxy-5α-pregnane 20-one 18.4 ± 4.0

Testosterone 5.6 ± 2.8

Dehydroepiandrosterone 4.3 ± 2.6

Estrone 4.4

Corticosterone 2.8

Androstenediol 1.7

Estradiol 1.3

Triamcinolone acetonide 1

17-hydroxy-pregnenolone 0.85

17α-hydroxy-progesterone 0.79

DHEA sulfate 0.045

Cholesterol 0.02

It is found that the best competitors for the binding of PREG [ ^J H] are pregnenolone sulfate, progesterone, and Δ5-pregnene-3β, 20α-diol (20α-dihydro-pregnenolone).

Another steroid, with a structure similar to PREG, namely 3β-hydroxy-5α-pregnane-20-one (5α-dihydro-PREG) also appears to be an effective competitor, although 5 times less active than PREG. Among the other steroids tested, DHEA is a weak competitor and DHEA sulfate has an almost negligible effect.

The preferred structures are C-21 steroids with a ketone group or an alcohol group in positions 3 and 20 and a ring A or B msature conferring a more planar structure on the skeleton of the steroid.

Other tests carried out with adult rat brains give similar results, showing that the brain contains a PREG binding component, with binding constants similar to those of the fetal brain (Kd = 50.7 nM, Bmax = 0.75 pmol / mg protein).

Purification of the cytosol binding protein. Mono Q column chromatography

The pregnenolone binding sites are eluted using an exchange column Mono Q ^® ions. 60 mg of cytosolic proteins in a TEDGCP buffer are loaded on the column and eluted with a NaCl gradient. 2 ml fractions are collected. FIG. 2 shows the saturable bond of PREG [ ³ H] PREG (dpm x 10 ⁻⁴ ) (-o-) and the protein concentration (mg / ml) (0) in each fraction.

The fractions of peak A (fractιonsl7 at 19: 4.5 mg of protein) and those of peak B (fractions 23 to 25: 1.1 mg of protein) are combined separately. The salts of the 2 fractions are removed and the fractions are concentrated and subjected to a gel filtration. . Chromatography on Superose 12 ^®

Peaks A and B filters on a Superose 12 ^® column (eluting with TEDGCP buffer containing 0.15 M NaCl).

0.4 ml fractions are collected. The specific binding of PREG [ ^J H] is measured on each fraction of the calibrated column with nuclear weight markers. The results obtained in FIG. 3 are reported, which gives, for each fraction, the saturable bond of PREG [ ^J H] (-o-) in dpm x 10 ^-4 and the protein concentration in mg / ml

(Δ). We note the presence of 2 peaks of 40-60 kDA

(fraction A) and more than 440 kDa (fraction B).

The presence of a very high molecular weight component suggests that the cytosol binding protein could be a very high molecular protein associated with microtubules, which are major constituents of the neuronal cytoplasm.

It was necessary to verify the presence in fraction A of components of the microtubular system. To this end, the PREG binding peaks were subjected to an SDS-PAGE, followed by an immunoblot. FIG. 4 reports the immunoblot of the cytosol and of the peak A fractions eluted from the ion exchange columns and ironed on a gel filtration column. Lane 1 corresponds to the raw cytosol; lane 2 at fraction A Mono Q ^® ; lane 3 at fraction A subjected to a new chromotography on Superose 12 ^® . 10 μg of protein are deposited on tracks 1 and 2. The amount of protein in track 3 is below the detection threshold.

It can be seen that the proteins which react immunologically with the anti-MAP2 and anti-tubule antibodies (α and β) have been enriched in these partially purified preparations, and this in a proportion compatible with the increase in specific binding activity. The low molecular weight of polypeptides recognized by anti-MAP antibodies _. suggests that breaks in the 300 (or 70) Kd MAP protein (s) occurred during purification of the cytosolic protein.

Binding of PREG [ ³ H] to microtubules of purified rat brain.

The binding of PREG [ ^J H] to microtubules prepared by polymerization / depolymerization cycles was determined.

The fetal microtubules have a Kd constant for PREG [ ^J H] of 42.9 nM and a Bmax of 3.7 pmol / mg of protein, which represents a 3-fold enrichment compared to the fetal brain cytosol.

The specificity of the ligand appears unchanged [RCR:

PREG (100)> PREGS (78.8)> PROG (72.1) »Testosterone

(5,6)> DHEA (4,3)> estradιol (1,3)].

Adult microtubules have a Kd constant for PREG [ ^J H] of 54.8 nM and a Bmax of 3.4 pmol / mg of protein, which which represents an enrichment of 4.5 times compared to the cytosol of adult brain.

Binding activities of pregnenolone to MAP _. , purified tubule and tau protein.

Purified calf brain tubing (90 μg / ml), MAP2 (45 μg / ml) or tau protein (25 μg / ml) is incubated with PREG [ ^J H] 100nM without or with a 1000 times excess of non-radioactive PREG.

It is found that neither the tubule, nor the tau protein, nor the rabbit IgG binds PREG [° H] in a saturable manner.

On the contrary, the MAP fraction. shows specific binding sites which increase when co-incubated with tubule.

The equilibrium binding constants of MAP _. purified calves are as follows: Kd = 39.2 nM, Bmax = 16 pmol/mg of MAPo whereas those of the MAP _2- tubulme copolymers are: Kd = 44.0 nM, Bmax = 135 pmol / mg of MAP ₂ .

It therefore appears that the binding of MAP ₂ to the tubule does not modify the affinity of PREG, but on the contrary increases the concentration of the binding sites by more than 8 times.

In FIG. 5, the values B / U are reported as a function of B in nM, (-o-) representing the equilibrium bond of

MAP; purified calf brain and (-o-), the equilibrium binding of MAP. purified calf brain associated with purified calf brain tubing (for experimental details, see Figure 1).

The molar concentration of the binding sites is 14 nM and that of MAP _. , based on an average MW of 200,000, approximately 200 nM.

It is therefore found that less than 10% of the MAP ₂ molecules can bind to PREG [ ^J H].

The replacement of buffer A with TEDG buffer does not affect the binding constants of pregnenolone at equilibrium.

The specificity of binding of steroids to MAP2-tubule copolymers is very close to that of fetal microtubules [CPR: PREG (100)> PREGS (91.5)> PROG (81.3) »estradiol (3.2)> Testosterone and DHEA (2,3)].

Effects of steroids on the assembly kinetics of microtubules

The microtubules are reconstituted m vi tro with purified tubule (1 mg / l) and MAP2 (0.05 mg / ml) in buffer A.

The increase in absorbance is measured at 345 nm, at 37 ° C and recorded every 30 sec. for 15 min.

The results are given in FIG. 6. The assembly of microtubules is measured either in the absence of steroids (- -), or in the presence of 500 nM of steroid: PREG (- -), progesterone (-X-), estradiol (-V-), or in presence of both PREG and progesterone (-o-) or PREG and estradiol (-Δ-), each at a concentration of 500 nM.

The increase in absorbance at 345 nM shows that PREG induces a large increase in both the speed and the extent of tubular assembly. Estradiol, which does not bind to MAP., Is inactive. However, progesterone, which shows an affinity for MAP of the same order as that of PREG, is also without effect. Unlike estradiol, it counteracts the stimulating effect of PREG on the assembly of microtubules. PREG sulfate has effects similar to those of progesterone.

Analysis by electron microscopy

Microtubules polymerized in the presence of PREG were analyzed to verify their appearance.

In the presence of calf brain MAP ₂ , the calf brain tubule assembles into microtubules of normal appearance, that is to say characterized by a remarkably constant conformation and a uniform diameter. The microtubule structure remains completely normal when MAP. induces tubing polymerization in the presence of 500 nM PREG. Effects of pregnenolone on neuronal cultures

The fetal rat brain neurons are studied by immunocytochemistry after 3 days of culture. The results are given in FIG. 7 where A, C, E correspond to a control culture for 3 days, B, D, F, to the addition of pregnenolone 1 μM the last 2 days of culture. In A and B, a monoclonal anti-tubulin antibody is added, in C and D, a polyclonal anti-doublecortin antibody and in E and F, a monoclonal anti-MAP ₂ antibody.

Staining with anti-α tubulin or anti-doublecortin monoclonal antibodies does not show any difference between the control cultures and the cultures exposed to pregnenolone 1 μM for 2 days. On the contrary, exposure to pregnenolone increases the immunolabelling of MAP ₂ in cell bodies and induces its extension to nearby neurites (see arrows).

Study of the competition capacity of various compounds with pregnenolone

The results obtained with 11 steroid derivatives are reported in the table below.

pregnenolone

Competition Index

Cytosol MT MAP-2c + Compounds with High Competitive Capacity (Rat) (Beef) Tubulin

PREG Tosylate 107 121 129

3β-0H- 5 , 14 Prégnadiène-20-one 102 95

3β-0H- 5, 14 Pregnadiene-20-one 102 95

Competition Index

Compounds Cytosol MT MAP-2c + to ~ —G. ran — Competitive Capacity (Rat) (Beef) „, Tub„ uline

Pregna-5-ene-3β, 20α-diol 100 (Beef)

acétoxy3β-OH-Pregna-5-ene-20-one-21- ⁸ 0 107

acetoxy

(Sigma) (Piglet!

c≈o S? PREG-acetate 79

CH,

Competition Index

Cytosol MT MAP-2c + Large Capacity Compounds (Rat) (Beef) Tubulin

competitor

PREG-16α-methyl 80

97

PREG-16α-cyclohexylamine 59 81 (Piglet)

Pregna-16α-17α-methylene 62 59

The results show that the steroids tested bind to the same site as PREG on the proteins constituting or associated with the elements of the cytoskeleton.

J _" , J - _*

BIBLIOGRAPHICAL REFERENCE

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Claims

1 / Non-toxic compounds, capable of crossing the blood-brain barrier, characterized in that they are capable of binding the same site as pregnenolone on the proteins constituting or associated with the elements of the cytoskeleton and of displacing the pregnenolone linked to MAP, thus than influencing the assembly and stabilization of microtubules. 2 / Adducts produced from the compounds according to claim 1 or from steroids on the one hand, and proteins constituting or associated with the elements of the cytoskeleton on the other hand. 3 / Adducts according to claim 2, characterized in that they are steroids - proteins associated microtubules, in particular steroids - MAP ₂ , where appropriate in the form of copolymerized with tubulin, or steroids _. .- tau proteins in form copolymerized with tubulin. 4 / Use of proteins constituting or associated with the elements of the cytoskeleton as receptors for steroids or for compounds according to claim 1. 5 / Use of steroids or compounds according to claim 1, for the manufacture of medicaments for the treatment of pathologies involving the proteins constituting or associated with the elements of the cytoskeleton. 6 / Use according to claim 5, characterized in that the steroid is pregnenolone or its sulfate. 7 / Use according to claim 5 or 6, for manufacturing medicaments for the treatment of the nervous system, central nervous system or peripheral nervous system. 8 / Use according to claim 7, characterized in that the drugs are in oral form for the treatment of the nervous system, or in injectable form, for administration by the general route or m if you, or in the form of solutions delay or pre-conjugation drugs, for example, has a fatty acid. 9 / Use according to claim 7 or 8 for manufacturing drugs for the prevention or treatment of conditions such as Alzhei er's disease, dementias of vascular origin, the consequences of trauma and vascular accidents at the level of nervous system and neurodegenerative diseases. 10 / Use according to claim 5 or 6, for manufacturing drugs to fight against the aging of nerve cells, or any other cell type containing MAP _Ξ , such as epithelial cells, thyroid cells, or endocrine glands. 11 / Use according to any one of claims 7 to 10 in combination with compounds having a cytoskeleton stabilizing effect, such as Taxol ^® . 12 / Use according to claim 5 or 6, for manufacturing drugs for the treatment of cancers, in combination with anticancer agents.