Cyclodecapeptide compounds for use as drugs

Abstract

The invention relates to novel cyclodecapeptide compounds having formula (I) for use as drugs and, more specifically, for use in the diagnosis, prevention and/or treatment of neurodegenerative diseases, such as Wilson's disease and Alzheimer's disease, and for use in the diagnosis, prevention and/or treatment of poisoning with metal ions, such as copper and mercury ions. The invention also relates to pharmaceutical compositions comprising at least one compound having formula (I) as an active principle. ##STR00001##

Claims

1. A pharmaceutical composition comprising: at least one cyclodecapeptide compound corresponding to the following formula ##STR00008## (I): in which: the cysteine amino acids Cys.sub.2 and Cys.sub.7 may or may not be linked by a covalent bond Cys.sub.2-Cys.sub.7 via their sulfur atoms, X.sub.1, X.sub.3, X.sub.4, X.sub.5, X.sub.6, X.sub.8, X.sub.9 and X.sub.10, which are identical or different, are amino acids present in their dextrorotary (D) or levorotary form (L), n.sub.1, n.sub.3, n.sub.6, and n.sub.8, which are identical or different, are equal to 0 or 1, and wherein at least one of n.sub.t, n.sub.3, n.sub.6, and n.sub.8 must be equal to 1, Y.sub.1, Y.sub.3, Y.sub.6, and Y.sub.8, which are identical or different, represent groups C(O)CHNL, C(O)EL or NHEL, in which L is a ligand for hepatic or neuronal cells, and E is a spacer arm selected from polyols and optionally substituted alkyl chains having 1 to 12 carbon atoms, and wherein at least one of the groups herein at least one of the groups Y.sub.1, Y.sub.3, Y.sub.6, and Y.sub.8 represents a group C(O)CHNL, wherein the dipeptide sequences X.sub.4-X.sub.5 and X.sub.9-X.sub.10, which are identical or different, are chosen from the dipeptides (D)Pro-(L)X or (L)Pro-(D)X, in which X and X are amino acids, and at least one pharmaceutically acceptable vehicle, optionally, one of the amino acids X.sub.4, X.sub.5, X.sub.9, and X.sub.10, or one of the groups Y.sub.1, Y.sub.3, Y.sub.6, and Y.sub.8, may be substituted with a group selected from: CO-fluorophore, NH-fluorophore, C(S)NH-fluorophore, SO2-fluorophore, CH-fluorophore and -E-fluorophore, where E is a spacer arm selected from phenyl and triazole.

2. The compound as claimed in claim 1, wherein at least one of the amino acids X.sub.1, X.sub.3, X.sub.6, and X.sub.8 is a lysine.

3. The compound as claimed in claim 1, wherein X and X are chosen from glycine, lysine, glutamate or aspartate.

4. The compound as claimed in claim 1, wherein the ligand L for hepatic or neuronal cells is selected from monosaccharides.

5. The compound as claimed in claim 1, wherein the fluorophore is selected from the group consisting of rhodamine, fluorescein, pyronin, coumarin, benzophenone, anthrone, fluorenone, pyridine, quinoleine, acridine, naphthalene, anthracene, naphthacene, pentacene and xanthene.

6. The compound as claimed in claim 1, wherein one of the amino acids X.sub.4, X.sub.5, X.sub.9, and X.sub.10, or one of the groups Y.sub.1, Y.sub.3, Y.sub.6, and Y.sub.8 is substituted with a group selected from the group consisting of CO-fluorophore, NH-fluorophore, C(S)NH-fluorophore, SO2-fluorophore, CH-fluorophore, and E-fluorophore.

7. The compound as claimed in claim 1, wherein at least one of the amino acids X.sub.1, X.sub.3, X.sub.6, and X.sub.8 is a lysine bearing a group Y.sub.1, Y.sub.3, Y.sub.6 and Y.sub.8.

8. A method for the treatment of Wilson's disease or for the treatment of Alzheimer's disease comprising the step of administering the cyclodecapeptide compound corresponding to the following formula (I) of claim 1 to a patient in need thereof.

9. A method for the diagnosis or treatment of poisoning with metal ions comprising the step of administering the compounds of claim 1 to a patient in need thereof.

10. The method as claimed in claim 9, wherein the patient has been poisoned with copper ions or mercury ions.

11. The method as claimed in claim 9, wherein the metal ion is selected from the group consisting of silver, cadmium, cobalt, copper, mercury, nickel, gold, lead and zinc ions.

12. The compound as claimed in claim 4, wherein the monosaccharide is selected from the group consisting of glucose, galactose and N-acetylgalactosamine.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In addition to the preceding arrangements, the invention also comprises other arrangements which will emerge from the additional description which follows, which relates to examples demonstrating the complexation properties, and more particularly the copper Cu(I) complexation properties, in the hepatic cells, of the compounds of the invention, and to the accompanying figures in which:

(2) FIG. 1 represents the UV assay of a compound P according to the invention with Cu(CH.sub.3CN)PF.sub.6, in a phosphate buffer solution at 20 mM, of pH=7.4, at a temperature of 298 K,

(3) FIG. 2 represents the ES-MS mass spectrum of a compound P according to the invention, in the presence of one equivalent of Cu(I),

(4) FIG. 3 shows the variation of the quantity of free copper Cu(I), detected with bathocuproine disulfonate (BCS), and measured by UV absorption of the complex Cu(BCS).sub.2, in a phosphate buffer solution at 20 mM, of pH=7.4, and with [P.sup.3]=[P.sup.1]=[P.sup.2]=50 M, [Cu(I)]=40 M and [BCS]=100 M,

(5) FIG. 4 represents images obtained by fluorescence microscopy (magnification 63) showing the variation of a compound P.sup.3-TRITC according to the invention (0.2 m) in HepG2 cells, after 2 hours, 7 hours and 26 hours of incubation,

(6) FIG. 5 represents images obtained by fluorescence microscopy (magnification 63) showing the variation of a compound P.sup.3-TRITC according to the invention (0.2 m) in Can 10 cells, after 2 hours, 7 hours and 26 hours of incubation,

(7) FIG. 6 represents images obtained by fluorescence microscopy (magnification 63) showing the location of the ATP7B protein in WIF-B9 hepatocytes, under basal conditions (image A) and in the presence of 1 M of copper Cu(I) (image B), and

(8) FIG. 7 represents images obtained by fluorescence microscopy (magnification 63) produced in the presence of 1 M of copper Cu(I), in the absence of a compound P.sup.3 according to the invention (images A) and in the presence of 10 M of a compound P.sup.3 according to the invention (images B).

DETAILED DESCRIPTION

Examples

(9) AMethods of Characterization

(10) 1/High Performance Liquid Chromatography (HPLC)

(11) HPLC chromatography is carried out on a VWR system equipped with RP18 columns (L=250 mm, =4.6 mm and p=5 m, for the analytical column; L=250 mm, =50 mm and p=10 m, for the preparative column).

(12) The flow rates used are 1 mL/min for the analytical column and 75 mL/min for the preparative column, with UV detection at 214 nm.

(13) The elution conditions are the following: solvent A: water/trifluoroacetic acid (TFA) mixture (99.925/0.075), and solvent B: acetonitrile (CH.sub.3CN)/water/trifluoroacetic acid (TFA) mixture (90/10/0.1).
2/UV-Visible Spectroscopy

(14) The UV-visible spectra were recorded on a Varian Cary 50 spectrophotometer.

(15) 3/Mass Spectrometry

(16) The mass spectra were recorded on an LXQ THERMO SCIENTIFIC type spectrometer, equipped with a source of ionization in electrospray mode (ESI).

(17) 4/Fluorescence Microscopy

(18) The fluorescence microscopy images are obtained on an AxioVert 200M (Carl Zeiss) inverted microscope equipped with an NHBO 103 mercury vapor lamp and a HAL 100 W halogen lamp, and with a fluorescence measuring device.

(19) The images are produced with a 63 magnification.

(20) BSynthesis

(21) Synthesis of Cyclodecapeptide Compounds P.sup.1 and P.sup.2 Corresponding to the Formula (Ia) of the Invention (the Amino Acids Cys.sub.2 and Cys.sub.7 not being Linked to Each Other by a Covalent Bond):

(22) ##STR00005##

(23) The protected linear precursors HArg(Pbf)-Cys(Trt)-Ser(tBu)-Pro-Gly-Ser(tBu)Cys(Trt)-Trp(Boc)Pro-Gly-OH and H-Trp(Boc)-Cys(Trt)-Glu(tBu)-Pro-Gly-Glu(tBu)-Cys(Trt)-Asp(tBu)-Pro-GlyOH were prepared by solid phase peptide synthesis on a 2-chlorotrityl chloride resin (substitution 0.5 mmol/g, 500 mg) by Fmoc chemistry (peptide synthesis on a solid support using 9-fluorenylmethoxycarbonyl as protecting group, R. Sheppard, J. Peptide Sci., 2003, 9: 545-552). The couplings are carried out by mixing the N--Fmoc-protected amino acids (2 equivalents), with benzotriazol-1-yl-oxytripyrrolidinophosphonium (PyBOP) (2 equivalents) and N,N-diisopropylethylamine (DIEA) (6 equivalents), for 30 minutes. After each coupling, the resin is treated with a DMF/pyridine/Ac.sub.2O mixture (v/v/v=7/2/1) in order to acetylate the unreacted amino groups (25 minutes). The deprotection of the Fmoc groups is carried out by treatment with a DMF/piperidine mixture (v/v=4/1, 35 minutes). The yield obtained for each peptide is monitored by UV-visible spectrometry (.sup.300 nm=7800 L.Math.mol.sup.1.Math.cm.sup.1 for the piperidine-dibenzofulvene adduct). The peptide is then detached from the resin by treatment with 15 mL of a mixture of dichloromethane (CH.sub.2Cl.sub.2) and trifluoroacetic acid (TFA) (v/v=99/1) (23 minutes). The cleavage is carried out rapidly, and the solution is introduced into 15 mL of a methanol/pyridine solution (v/v=8/2). After concentration, the residue is precipitated several times in ice-cold diethyl ether in order to obtain a white powder. The linear precursor is then reacted in CH.sub.2Cl.sub.2 (0.5 mM) with PyBOP (3 equivalents) and DIEA (4 equivalents). The formation of the cyclic peptide is monitored by HPLC analysis, and the reaction is stopped after 10 minutes. The dichloromethane (CH.sub.2Cl.sub.2) is then evaporated. The oily residue is precipitated with a CH.sub.2Cl.sub.2/Et.sub.2O mixture in order to obtain a cyclic peptide in the form of a powder. The chains are then deprotected by treatment with a solution of 1.4 g of dithiothreitol (DTT) in a TFA/TIS (triisopropylsilane)/H.sub.2O mixture (v/v/v=95/2.5/2.5) (peptide concentration=10 mM). After stirring for 2 hours, the solution is evaporated under reduced pressure in order to give a yellow oil which is precipitated several times with ice-cold diethyl ether. The solid residue obtained is then dissolved in a water/acetonitrile mixture, and then passed over a polytetrafluoroethylene (PTFE) filter whose pore diameter is 0.45 m, and then purified by reversed phase HPLC chromatography (gradient from 5 to 45% of B over 30 minutes), in order to give a compound P in the form of a white powder (52 mg, 19% yield), or a compound P.sup.2 in the form of a white powder (144 mg, 53% yield).

(24) Compound P.sup.1:

(25) HPLC analysis, purity: 96%, t.sub.R=15.7 min (gradient 5 to 60% of B over 30 minutes).

(26) MS: calculated for C.sub.43H.sub.62N.sub.14O.sub.12S.sub.2, [M+H.sup.+].sup.+=1031.41, exp=[M+H.sup.+].sup.+=1031.45.

(27) Compound P.sup.2:

(28) HPLC analysis, purity: 98%, t.sub.R=23.6 min (gradient 5 to 45% of B over 30 minutes).

(29) MS: calculated for C.sub.45H.sub.59N.sub.11O.sub.16S.sub.2, [M+H.sup.+].sup.+=1074.36, exp=[M+H.sup.+].sup.+=1074.65.

(30) Synthesis of Cyclodecapeptide Compounds P.sup.3 and P.sup.3-TRITC Corresponding to the Formula (I) of the Invention (the Amino Acids Cys.sub.2 and Cys.sub.7 being Linked by a Covalent Bond):

(31) ##STR00006##

(32) The cyclodecapeptide compounds P.sup.3 and P.sup.3-TRITC corresponding to the formula (I) of the invention are synthesized according to scheme 1 below. The oxyamine blocks Fmoc-Lys[BocSer(tBu)]-OH and O--D-galactopyranosyl (GaINAcONH.sub.2, compound 6) are synthesized as described in the literature (Renaudet et al., Org. Biomol. Chem., 2006, 4: 2628-2636; S. Fouillard et al., J. ORG. Chem., 2008, 73: 983-991).

(33) ##STR00007##
Synthesis of Compound 4:

(34) The linear precursor 1 bearing protecting groups is prepared by solid phase peptide synthesis on a 2-chlorotrityl chloride resin (substitution 0.4 mmol/g, 0.507 g, 0.202 mmol) by Fmoc chemistry. The resin is swollen with dichloromethane (CH.sub.2Cl.sub.2) (10 mL, 110 min) and DMF (10 mL, 110 min). The couplings are carried out by mixing the N--Fmoc-protected amino acids or Fmoc-Lys[BocSer(tBu)]-OH (2.5 equivalents, 0.5 mmol), with benzotriazol-1-yl-oxytripyrrolidinophosphonium (PyBOP) (2.5 equivalents, 0.5 mmol) and N,N-diisopropylethylamine (DIEA) (pH8-9) in DMF (10 mL), for 30 minutes. After washing with DMF (10 mL, 41 min) and CH.sub.2Cl.sub.2 (10 mL, 21 min), the deprotection of the N--Fmoc groups is carried out by treatment with a DMF/piperidine mixture (v/v=4/1, 10 mL, 310 min). After the last wash with DMF (10 mL, 61 min), the end of the deprotection reaction is checked by UV-visible spectrometry (.sup.300 nm=7800 L.Math.mol-1.Math.cm-1 for the piperidine-dibenzofulvene adduct). After the last coupling reaction, the functionalized resin 1 is obtained (0.13 mmol, 64% yield).

(35) The functionalized resin 1 (0.13 mmol) is then swollen with dichloromethane (CH.sub.2Cl.sub.2) (10 mL, 110 min) and DMF (10 mL, 110 min). Iodine (0.660 g, 2.60 mmol) and DMF (10 mL) are added. The reaction mixture is stirred at room temperature for 1.5 hours. After filtration, the resin is washed with DMF (10 mL, 65 min), a DMF/water mixture (v/v=1/1) (10 mL, 25 min), DMF (10 mL, 15 min) and CH.sub.2Cl.sub.2 (10 mL, 35 min).

(36) The peptide is then detached from the resin by treatment with a CH.sub.2Cl.sub.2/TFA mixture (v/v=99/1, 10 mL, 102 min). The filtrate is then recovered, and N,N-diisopropylethylamine (DIEA) (1 mL) is added in order to avoid deprotection during the evaporation step. After concentration, the residue is precipitated in diethyl ether. The linear precursor is then reacted in DMF (0.5 mM) with PyBOP (0.074 g, 0.14 mmol) and DIEA (0.08 mL, 0.39 mmol), for 2 hours. The DMF is evaporated under reduced pressure. The oily residue is precipitated with a CH.sub.2Cl.sub.2/Et.sub.2O mixture, in order to give a cyclic peptide 3 in the form of a powder. The chains are then deprotected by treatment with a TFA/H.sub.2O mixture (v/v=90/10, 20 mL). After stirring for 2 hours, the solution is evaporated in order to give a yellow oil which is precipitated with diethyl ether, in order to give a deprotected peptide 4 in the form of a white powder (0.097 g, 0.067 mmol, 33% yield).

(37) HPLC analysis, purity: 83%, t.sub.R=6.13 min (linear gradient A/B:95/5 to 60/40, over 15 minutes).

(38) MS: calculated for C.sub.60H.sub.105N.sub.19O.sub.18S.sub.2, [M+H.sup.+].sup.+=1444.74, exp: [M+H.sup.+].sup.+=1444.58, [M+2H.sup.+].sup.+=722.92, [M+3H.sup.+].sup.3+=482.33.

(39) Synthesis of Compound 5:

(40) Sodium periodate (0.380 g, 1.77 mmol) is added to a solution of compound 4 (0.064 g, 0.044 mmol) in water (8 mL). After 15 minutes, the reaction mixture is injected into an RP-HPLC column (t.sub.R=14 min, linear gradient A/B:95/5 to 60/40, over 15 minutes), in order to give a compound 5 in the form of a white powder (0.009 g, 0.0068 mmol, 15% yield) after freeze-drying.

(41) MS: calculated for C.sub.56H.sub.85N.sub.15O.sub.18S.sub.2, [M+H.sup.+].sup.+=1320.57, exp=[M+H.sup.+].sup.+=1320.5.

(42) Synthesis of Compound P.sup.3:

(43) O--D-galactopyranosyl oxyamine (compound 6) is added (0.045 g, 0.192 mmol) to a solution of compound 5 (0.025 g, 0.019 mmol), in an AcOH/H.sub.2O mixture (4 mL, v/v=1/9).

(44) The reaction mixture is stirred at room temperature for 1 hour. The mixture is then injected into an RP-HPLC column (t.sub.R=14-16 min, linear gradient A/B:95/5 to 60/40, over 15 minutes), in order to give a compound P.sup.3 in the form of a white powder (0.021 g, 0.0096 mmol, 50% yield) after freeze-drying.

(45) HPLC analysis, purity: 95%, t.sub.R=7.0 min (linear gradient A/B:95/5 to 60/40, over 15 minutes).

(46) MS: calculated for C.sub.88H.sub.141N.sub.23O.sub.38S.sub.2, [M+H.sup.+].sup.+=2192.92, exp=[M+H.sup.+].sup.+=2193.5.

(47) Synthesis of Compound P.sup.3-TRITC:

(48) A marker, TetraMethylRhodaminelsoThioCyanate (TRITC) is added (0.003 g, 0.0067 mmol) with a few drops of DIEA (pH8-9) to a solution of a compound P.sup.3 (0.012 g, 0.0055 mmol) in DMF (2 mL). The reaction mixture is then stirred at room temperature for 2 hours, and then injected into an RP-HPLC column (t.sub.R=19 min, linear gradient A/B:95/5 to 60/40, over 15 minutes) in order to give a compound P.sup.3-TRITC in the form of a white powder (0.0012 g, 0.00046 mmol, 8% yield) after freeze-drying.

(49) MS: calculated for C.sub.113H.sub.163N.sub.26O.sub.41S.sub.3.sup.+, [M].sup.+=2637.1, exp=[M].sup.+=2636.6, [M.sup.++H.sup.+].sup.2+=1318.9.

(50) CCharacterization of the Copper Cu(I) Complexes Formed with the Peptides P.sup.1 and P.sup.2 Procedure

(51) Since the thiol functional groups SH of the cysteine amino acids are subject to oxidation in the air, all their solutions were prepared in a glove box under an argon atmosphere. Solutions of ligands were then prepared, before each experiment, by using water deoxygenated and purified by a Millipore Milli-Q system containing 20 mM of a phosphate buffer solution (pH=7.4) and acetonitrile (v/v:9/1).

(52) The final concentration of the solution was determined by measuring the concentration of the free thiol functional groups, following the Ellman procedure described in the literature (P. W. Riddles et al., Methods Enzymol., 1983, 91, pp. 49-60). This method uses 5,5-dithiobis-2-nitrobenzoic acid (DNTB) as indicator, each free thiol group present in the ligand leading to 1 equivalent of TNB.sup.2 (.sup.412 nm (TNB.sup.2)=14 150 M.sup.1.cm.sup.1, .sup.412 nm being the molar extinction coefficient of TNB.sup.2 at 412 nm). The concentrations of the solutions of ligands are between 30 and 100 M.

(53) The copper Cu(I) solutions were prepared by dissolving an appropriate quantity of Cu(CH.sub.3CN).sub.4 PF.sub.6 in deoxygenated acetonitrile. The final concentration is determined by adding an excess of sodium bathocuproine disulfonate (Na.sub.2BCS) and by measuring the absorbance of Cu(BCS).sub.2.sup.3 (.sub.max=483 nm, =13 300 M.sup.1.cm.sup.1).

(54) For the measurements of affinity constants, the complex is prepared by adding to the ligand solution a solution of acetonitrile (CH.sub.3CN) containing 0.8-0.9 equivalent of copper Cu(I), in a phosphate buffer solution at 20 mM (pH=7.4) and acetonitrile (CH.sub.3CN) (v/v:9/1).

(55) The formation of the complex is then carried out by stirring the mixture for 10 minutes, under argon. Aliquots of a bathocuproine disulfonate (BCS) solution in the same buffer solution are then added to the ligand-copper complex. The UV-visible spectra are recorded, and the stability of the absorbance is checked before the addition of the other aliquots.

(56) 1UV-Visible Spectroscopy

(57) The formation of the Cu(I) complexes was monitored by UV-visible spectroscopy.

(58) FIG. 1 gives an example of a UV assay of the compound P.sup.1 with Cu(CH.sub.3CN)PF.sub.6 (Cu(I)) in phosphate buffer at 20 mM, at a pH of 7.4 and at 298 K.

(59) The thiolate.fwdarw.Cu(I) charge transfer band appears clearly around 260 nm. This band increases up to 1 equivalent for the two peptides P.sup.1 and P.sup.2. The Cu(I) complexes obtained therefore have an overall stoichiometry of 1:1 (Cu:L) for these ligands (L) comprising two cysteines.

(60) 2Mass Spectrometry

(61) The stoichiometry for the complex is also demonstrated by mass spectrometry in electrospray ionization mode, the mass spectra having been recorded with an LXQ THERMO SCIENTIFIC type spectrometer. The Cu(P.sup.1) complex is clearly detected on the spectra for the compound P in the presence of 1 equivalent of Cu(I) (cf. FIG. 2).

(62) 3Affinity Constants

(63) For the measurements of affinity constants, the complex is prepared by adding to the ligand solution a solution of acetonitrile (CH.sub.3CN) containing 0.8-0.9 equivalent of copper Cu(I), in a phosphate buffer solution at 20 mM (pH=7.4) and acetonitrile (CH.sub.3CN) (v/v:9/1).

(64) The affinity of the compounds of the invention for Cu(I) is an important matter since it makes it possible to quantify the capacity of the compounds of the invention to complex this ion. The affinity constants were measured using a known competitor having a high affinity for Cu(I), bathocuproine disulfonate (BCS), which forms complexes with copper Cu(I) of known stability according to the reaction below (Z. Xiao et al., J. Am. Chem. Soc., 2004, 126: 3081-3090; P. Rousselot-Paillet et al., Inorg. Chem., 2006, 45: 5510-5520):

(65) $\mathrm{Cu}\left(I\right)+2\mathrm{BCS}={\mathrm{Cu}\left(\mathrm{BSC}\right)}_2$$K=\frac{\left[{\mathrm{Cu}\left(\mathrm{BCS}\right)}_2\right]}{{\left[\mathrm{Cu}\right]\left[\mathrm{BCS}\right]}^2}={10}^{19.8}\mathrm{at}298K$

(66) These competition experiments made it possible to quantify the affinity of the compounds P.sup.1 and P.sup.2 of the invention for copper Cu(I): the apparent copper Cu(I) complexation constants in a phosphate buffer solution at 20 mM of pH 7.4, at a temperature of 298 K, as defined below, are given in table II.

(67) $K_{\mathrm{app}}=\frac{{\left[\mathrm{Cu}\right]}_{\mathrm{complexed}}}{{{\left[\mathrm{Cu}\right]}_{\mathrm{free}}\left[L\right]}_{\mathrm{free}}}$

(68) TABLE-US-00002 TABLE II P.sup.C Compound P.sup.1 Compound P.sup.2 logK.sub.app 16.5 16.7 15.5

(69) It appears clearly that the peptides of the invention in which the thiol functional groups of the cysteine amino acids are free have high affinities for Cu(I). Moreover, the affinities of P.sup.1 and P.sup.2 are comparable to those obtained with the cyclopeptide P.sup.c (reference) modeling the Atx1 yeast copper chaperone loop (P. Rousselot-Paillet et al., Inorg. Chem., 2006, 45: 5510-5520). These results demonstrate the capacity of the compounds of the invention to complex copper Cu(I) in excess in an intracellular medium.

(70) D/ Characterization of the Complexes Between the Peptides and P.sup.2 and Other Metal Ions

(71) Procedure:

(72) The procedures are those described in the article Rousselot-Paillet et al., Inorg. Chem., 2006, 4: 2628-2636.

(73) The complexation of mercury Hg(II) by the compounds P.sup.1 and P.sup.2 is very effective (high affinity constants), and may therefore be of interest for the detoxification of this toxic metal. The complexation of zinc Zn(II) was also studied because this nontoxic metal ion is present in vivo, in the hepatic cells targeted. The compounds have a much lower affinity for zinc Zn(II) than for copper Cu(I) and mercury Hg(II). This selectivity is crucial because it makes it possible to detoxify the target metal (copper Cu(I) or mercury Hg(II)) without complexing zinc. This parameter is expressed by the selectivity Sel. M/M between two metals M and M. Table III below assembles the apparent constants obtained at pH=7.4, with the cyclodecapeptide compounds of the invention, and their selectivity for the ions targeted relative to zinc Zn(II).

(74) TABLE-US-00003 TABLE III logK.sup.app P.sup.C Compound P.sup.1 Compound P.sup.2 Cu(I) 16.5 16.7 15.5 Zn(II) 6.8 6.6 5.9 Hg(II) >18.6 >18.7 >17.5 Sel. Cu/Zn 9.7 10.1 9.6 Sel. Hg/Zn >11.8 >12.1 11.6

(75) The compounds P.sup.1 and P.sup.2 of the invention exhibit good affinities and good selectivities relative to zinc Zn(II) present in the cells, which makes them very promising for the selective complexation of copper having an oxidation state +I, which oxidation state is favored in the intracellular medium and which may therefore be targeted in Wilson type diseases. These compounds are also candidates for the selective complexation of mercury Hg(II) during poisoning by this metal.

(76) E/ Characterization of the Copper Cu(I) Complexes Formed with the Peptide P.sup.3

(77) BCS was used to determine the concentration of copper Cu(I) not complexed by the compound P.sup.3. The results are represented in FIG. 3. When the compound P.sup.3 is alone, it does not complex copper (100% of Cu(I) is complexed by BCS), since the thiol functional groups of the compound P.sup.3 are masked by the disulfide bridges (SS) of the molecule. On the other hand, in the presence of a reducing agent (GSH 1 mM as in the cells), capable of reducing the SS bridges in order to regenerate the free thiol functional groups, the compound P.sup.3 evolves into an effective complexing agent for copper (the two cysteines Cys.sub.2 and Cys.sub.7 then being free as in the compounds P or P.sup.2), since the quantity of copper detected by BCS drops to 40%. This percentage corresponds to an intermediate apparent stability constant between the peptides P.sup.1 and P.sup.2: log K.sup.app (P.sup.3 reduced)=16.

(78) These results show that the compound P.sup.3 complexes copper Cu(I) in a reducing medium, with an affinity similar to the compounds P.sup.1 and P.sup.2, the compounds of the invention, in which the cysteine amino acids Cys.sub.2 and Cys.sub.7 are linked by a Cys.sub.2-Cys.sub.t covalent bond, becoming metal-chelating agents only in the target cells, and therefore causing no side effects linked to the undesirable complexation of metals at other sites in the body.

(79) F/ Biological Results on Hepatic Cells

(80) 1Entry of the Compound P.sup.3-TRITC into Hepatic Cells

(81) Procedure:

(82) The cells (10.sup.5-10.sup.6/mL) are deposited on cover glass at the bottom of culture wells and immersed in the appropriate culture medium. After a variable incubation time in the presence of the compound P.sup.3-TRITC, each cover glass is washed, fixed with a 10% formaldehyde solution (Sigma) and mounted on an observation slide in the presence of mounting fluid (Sigma). Each slide is then observed under a fluorescence microscope in order to locate the TRITC marker in the cells. About thirty fields are observed on each cover glass in order to obtain a significant statistical result. The experiment is repeated on cells from various batches.

(83) Results:

(84) The entry of the compound P.sup.3-TRITC (0.2 M) into HepG2 type hepatic cells, WIF-B9 (C. Decaens et al., 1996, J. Cell Sci., 109 (Pt 6): 1623-1635) and Can10 (X. Peng et al., 2006, Cell Tissue Res., 323: 233-243) was studied by fluorescence microscopy by monitoring the emission in the red of the TRITC marker (cf. FIG. 4). Kinetics were established at two concentrations (0.2 and 2 M) in order to evaluate the entry time of the molecule into these different cells.

(85) The hepatocytes of the HepG2 line incorporate the compound P.sup.3-TRITC from 2 hours of incubation. Over time, the cells become enriched with compound P.sup.3-TRITC.

(86) The study was continued with hepatocytes of the Can10 (cf. FIG. 5) and WIF-B9 lines, which have the characteristic feature of forming bile canaliculi after about ten days of culture. This characteristic makes it possible to monitor the presence of the compound P.sup.3-TRITC in a polarized cell, which is closer to the physiology than the HepG2 line which does not become polarized. Compared with the situation of a hepatocyte in the liver, the culture medium represents the blood plasma and the bile canaliculus represents the natural site of excretion of copper.

(87) The Can10 cells incorporate the compound P.sup.3-TRITC from 2 hours of incubation. After 24 hours of incubation, some canaliculi are fluorescent, which demonstrates that the compound P.sup.3-TRITC has crossed the cells. After 48 hours, all the canaliculi are fluorescent. The results obtained with the cells of the WIF-B9 line are similar.

(88) These results demonstrate that the compound P.sup.3-TRITC is capable of entering into various types of hepatic cells within only a few hours.

(89) 2Complexation of Copper Cu(I) in Hepatic Cells

(90) Procedure:

(91) The cells (10.sup.5-10.sup.6/mL) are deposited on cover glass at the bottom of culture wells and immersed in the appropriate culture medium. After an incubation of 1 to 5 hours in the presence of 1 M of copper Cu(I), and optionally in the presence of 10 M of compound P.sup.3, each cover glass is washed, and then fixed and the cells permeabilized with a pure methanol solution at 20 C. for 4 minutes. After washing, the cover glass are exposed to a medium containing the primary anti-ATP7B antibody (Hernandez et al., Gastoenterology, 2008, 134, 1215-1223) and, in the case of a double labeling, to a medium containing an anti-ZO-1 antibody (ZO-1 being a protein consisting of tight junctions joining the hepatocytes and delimiting the apical membrane, ZO-1 being a marker for the canaliculi). The cover glass are then exposed to a medium containing a secondary antibody, Alexa Fluor 546 goat anti-rat IgG (H+ L) (Invitrogen), fluorescent in the green for the protein ATP7B and in the red for the ZO-1 antibody, and then mounted on an observation slide in the presence of mounting fluid (Sigma), this secondary antibody making it possible to visualize the ZO-1 protein and to signal its position in the cells. Each slide is then observed under a fluorescence microscope in order to locate the ATP7B protein, and optionally ZO-1, in the cells. About thirty fields are observed on each cover glass in order to obtain a significant statistical result. The experiment is repeated on cells obtained from various batches.

(92) Results:

(93) It was shown that the position of the membrane ATP7B protein (Wilson protein) depended on the concentration of intracellular copper Cu(I) in the hepatocytes, such as for example WIF-B9. This protein can therefore be used as indicator for the increase in the intracellular concentration of copper Cu(I). For that, the position of the ATP7B protein in the cell is identified by labeling with a primary anti-Wilson antibody, itself detected by a secondary antibody that is fluorescent in the green. Under basal conditions, the ATP7B protein is located in the region of the Golgi apparatus, whereas in an excess of copper Cu(I), it moves toward the apical membrane, that is to say toward the membrane which surrounds the canaliculi, in order to excrete the excess copper Cu(I) (Y. Guo et al., Am. J. Physiol. Gastrointest. Liver. Physiol., 2005, 289: G904-G916).

(94) The location of the ATP7B protein in the WIF-B9 cells is represented in FIG. 6. Under basal conditions (image A), the ATP7B protein is close to the Golgi apparatus, between the bile canaliculus (Cb) and the nucleus (N). After 2 hours of incubation in the presence of 1 M of copper Cu(I), the ATP7B protein gets closer to the apical membrane, until it surrounds the canaliculi.

(95) The experiments carried out on the WIF-B9 cells also demonstrated that it was possible to see the movement of the ATP7B protein between the basal conditions (copper0.01 M) and an excess of copper Cu(I) (1 M). This movement can therefore be used as a probe for the intracellular concentration of copper Cu(I).

(96) To test the capacity of the compound P.sup.3 to reduce the intracellular concentration of copper Cu(I), the cells incubated in an excess of copper Cu(I) are exposed to the compound P.sup.3 for at least 2 hours. The incubation with the compound P.sup.3 inhibits the movement of the ATP7B protein toward the apical membrane, which demonstrates the absence of an increase in the intracellular concentration of copper Cu(I). The compound P.sup.3 is therefore found to be a chelating agent for copper Cu(I) in cellulo.

(97) The position of the ATP7B protein in the presence of 1 M of copper Cu(I), in the absence (images A) or in the presence (images B) of 10 M of compound P.sup.3 is also represented in FIG. 7 (on the left: image by phase contrast (Nomarski); in the center: fluorescence of the ATP7B protein (visualized by means of the Alexa Fluor 488 antibody); on the right: fluorescence of ZO-1 (protein of the tight junctions or zonula occludens which is visualized by means of the Alexa Fluor 546 antibody; the tight junctions are located at the apex of the hepatocytes and contain numerous proteins, including ZO-1, and they ensure the association of the cells with each other, and therefore the tightness between the inner space of the caniculi and the intercellular medium).