Chelate nanoemulsion for MRI

Abstract

The present invention relates to an oil-in-water nanoemulsion composition for MRI, comprising: an aqueous phase, representing 70% to 90% by weight of the composition, advantageously 75% to 85% and more advantageously from 78% to 82% a lipid phase comprising an oil, representing 9.5% to 29.5% by weight of the composition, advantageously 14% to 25% and more advantageously 17% to 21%, a surfactant at the interface between the aqueous and lipid phases, the surfactant comprising at least one amphiphilic paramagnetic metal chelate and optionally an amphiphilic lipid; the total content of surfactant by weight relative to the oil being between 4% and 10% and advantageously between 5% and 8%; the total content of surfactant by weight relative to the composition being between 0.35% and 2.95% and advantageously between 0.5% and 2%; the oil comprising at least 70%, advantageously at least 80%, advantageously at least 95% by weight and especially at least 97% of saturated C6-C18, advantageously C6-C14 and more advantageously C6-C10 fatty acids.

Claims

1. An oil-in-water nanoemulsion composition for MRI comprising nanodroplets, said oil-in-water nanoemulsion composition comprising: an aqueous phase, representing 70% to 90% by weight of the composition, a lipid phase comprising an oil, representing 9.5% to 29.5% by weight of the composition, a surfactant at the interface between the aqueous and lipid phases, the surfactant comprising at least one amphiphilic paramagnetic metal chelate, at least one amphiphilic targeting biovector and an amphiphilic lipid, said surfactant comprising by weight: 50% to 95% of amphiphilic lipid, 5% to 50% of amphiphilic paramagnetic metal chelate, and 0.05% to 5% of amphiphilic targeting biovector; the total content of surfactant by weight relative to the oil being between 4% and 10%; the total content of surfactant by weight relative to the composition being between 0.35% and 2.95%; the oil comprising at least 70% of saturated C6-C18 fatty acids, wherein the amphiphilic paramagnetic metal chelate is a macrocyclic chelate selected from the group consisting of DOTA, DO3A, HPDO3, BTDO3A, PCTA, DOTAM, DOTMA, DOTA-GA, AAZTA, HOPO, multimers thereof and derivatives thereof in which one or more carboxylic groups are in the form of a corresponding salt, ester or amide, or in which one or more carboxylic groups are replaced with a phosphonic and/or phosphinic group, and wherein the amphiphilic targeting biovector is of formula Bio-L-Lipo, in which: Bio is a biological recognition part located on the outer surface of the nanodroplets selected from the group consisting of: peptides, pseudopeptides, peptidomimetics, amino acids, integrin targeting agents, glycoproteins, lectins, biotin, pteroic or aminopteroic derivatives, folic and antifolic acid derivatives, antibodies or antibody fragments, avidin, steroids, oligonucleotides, ribonucleic acid sequences, deoxyribonucleic acid sequences, hormones, proteins, which may be recombinant or muted, mono- or polysaccharides, compounds of benzothiazole, benzofuran, styrylbenzoxazole/thiazole/imidazole/quinoline or styrylpyridine backbone; Lipo is a lipophilic group for inserting Bio into the surfactant; L is a linking group connecting Bio and Lipo, L being: a single bond, squarate, C.sub.1-6 alkylene, PEG, for example CH.sub.2—(CH.sub.2—O—CH.sub.2)k-CH2 with k=1 to 10, (CH.sub.2).sub.3—NH, NH—(CH.sub.2).sub.2—NH, NH—(CH.sub.2).sub.3—NH, (CH.sub.2).sub.n, (CH.sub.2).sub.n—CO—, —(CH.sub.2).sub.nNH—CO— with n=2 to 10, (CH.sub.2CH.sub.2O).sub.q(CH.sub.2).sub.r—CO—, (CH.sub.2CH.sub.2O)q(CH.sub.2).sub.r—NH—CO— with q=1-10 and r=2-10, (CH.sub.2).sub.n—CONH—, (CH.sub.2).sub.n—CONH-PEG, (CH.sub.2).sub.n—NH—HOOC—CH.sub.2—O—(CH.sub.2).sub.2—O—(CH.sub.2).sub.2—O—CH.sub.2—COOH; HOOC—(CH).sub.2—CO.sub.2—(CH.sub.2).sub.2—OCO—(CH.sub.2).sub.2—COOH; HOOC—CH(OH)—CH(OH)—COOH; HOOC—(CH.sub.2).sub.n—COOH; NH.sub.2—(CH.sub.2).sub.n—NH.sub.2, with n=0-20; NH.sub.2—(CH.sub.2).sub.n—CO.sub.2H; NH.sub.2—CH.sub.2— (CH.sub.2—O—CH.sub.2).sub.n—CO.sub.2H with n=1 to 10, or P1-1-P2, which may be identical or different, P1 and P2 being chosen from O, S, NH, nothing, CO.sub.2, NHCO, CONH, NHCONH, NHCSNH, SO.sub.2NH—, NHSO.sub.2—, squarate with 1=alkyl, alkoxyalkyl, polyalkoxyalkyl (PEG), alkyl interrupted with one or more squarates or with one or more aryls, advantageously phenyls, alkenyl, alkynyl, alkyl interrupted with one or more groups chosen from —NH—, —O—, —CO—, —NH(CO)—, —(CO)NH—, —O(CO)—, or —(OC)O—.

2. The composition as claimed in claim 1, wherein the amphiphilic targeting biovector represents 0.5% to 1% by weight of the total surfactant.

3. The composition as claimed in claim 1, wherein the surfactant represents 5% to 8% by weight of the oil.

4. The composition as claimed in claim 1, wherein the saturated C6-C18 fatty acids are in the form of saturated fatty acid triglycerides.

5. The composition as claimed in claim 1, wherein the oil comprises saturated fatty acids in the following proportions: C6-C18>70%, or C6-C14>70%, or C8+C10>70%.

6. The composition as claimed in claim 1, wherein the amphiphilic paramagnetic metal chelate is a macrocyclic chelate selected from the group consisting of DOTA, DO3A, HPDO3, BTDO3A and PCTA.

7. The composition as claimed in claim 1, wherein the amphiphilic lipid is a phospholipid.

8. The composition as claimed in claim 1, wherein the surfactant also comprises an amphiphilic stealth agent.

9. The composition as claimed in claim 1, wherein the paramagnetic metal of the amphiphilic paramagnetic metal chelate is selected from the group consisting of: manganese, iron, cobalt, nickel, copper, molybdenum, ruthenium, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium and ytterbium.

10. A process for preparing a composition as claimed in claim 1, comprising the steps of: preparation of a lipid phase comprising optionally a first amphiphilic lipid surfactant an oil comprising at least 70% by weight, of C6-C18 saturated fatty acids an amphiphilic paramagnetic metal chelate an amphiphilic targeting biovector; dispersion of the lipid phase in an aqueous solution so as to form an oil-in-water nanoemulsion; and recovery of the nanoemulsion composition obtained.

11. A method of diagnosing cancerous, inflammatory, neurodegenerative and/or cardiovascular diseases comprising administering a therapeutic amount of the composition as claimed in claim 1 to a patient in need thereof.

12. The composition as claimed in claim 7, wherein the phospholipid is selected from the group consisting of phosphatidylcholine, dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidylserine, phosphatidylinositol and lecithin.

13. The composition as claimed in claim 8, wherein the amphiphilic stealth agent is a PEG derivative, a ganglioside derivative or a polysaccharide.

14. The composition as claimed in claim 9, wherein the paramagnetic metal of the amphiphilic paramagnetic metal chelate is Gd(III), Mn(II), europium or dysprosium.

15. The composition as claimed in claim 1, wherein the amphiphilic targeting biovector is ##STR00038## ##STR00039## ##STR00040## ##STR00041## ##STR00042##

Description

EXAMPLE 1

Synthesis of a Lipophilic DTPA Derivative

(1) ##STR00017##
Step 1:

(2) 6 g of DTPA bis-anhydride are suspended in 240 ml of DMF. The suspension is heated to 50° C. and dissolution takes place. Octadecylamine is added in a single portion. The reaction is maintained at 50° C. overnight. The reaction medium is cooled and then filtered through a sinter funnel. The precipitate is washed once with DMF and then thoroughly with methanol. 13.5 g of yellow-white powder are obtained in a yield (Yld) of 90%. The mass spectrometry analysis is performed by infusion of the sample in ES+.

(3) C.sub.50H.sub.97N.sub.5O.sub.8; m/z (ES+)=896

(4) Step 2:

(5) 13.4 g of ligand (Int3) are suspended in 600 ml of methanol. 6.67 g of GdCl.sub.3.6H.sub.2O are added. Dissolution takes place instantaneously. The pH of the solution is adjusted to 7 with a solution of sodium methoxide in methanol (2.68 g of CH.sub.3ONa in 400 ml of CH.sub.3OH). The solution is refluxed for 45 minutes. The methanol is evaporated off and the residue is taken up in water. The powder is washed thoroughly with water. 15 g of crude product are obtained in a yield of 96%. The product is purified by flash chromatography on silica gel. 15 g are purified with an eluent phase composed of 90/10 methanol/dichloromethane. After purification, 10 g of pure product are obtained (greasy white powder).

(6) C.sub.50H.sub.94GdN.sub.5O.sub.8; m/z (ES−)=1049

EXAMPLE 2

Synthesis of Lipophilic PCTA Derivatives

EXAMPLE 2.1

Dspe-Pcta

(7) ##STR00018##
Step 1:

(8) 576 mg of Int 1 are suspended in 3 mL of DMSO. The activating agents are introduced, 1.1 eq. of EDCI, i.e. 219 mg, and 1.1 eq, of NHS, i.e. 131 mg. After leaving overnight, dissolution takes place (the ester has formed).

(9) C.sub.24H.sub.28GdN.sub.5O.sub.10; m/z (ES−)=703

(10) Step 2:

(11) DSPE (1 eq., 711 mg) is dissolved in a minimum amount of pyridine at 90° C. Once dissolved, the solution is poured slowly into the DMSO solution containing the activated ester; the reaction is left for 10 minutes at 90° C. and is then left to react while allowing the temperature to fall overnight. The reaction medium is precipitated from cold water and centrifuged. The pellet is washed with water and then centrifuged again. The pellet is taken up in methanol and then evaporated to dryness. About 400 mg of crude product are obtained, which product is then purified by flash chromatography on silica gel (30 g cartridges).

(12) In a first stage, an eluent phase consisting of 88-12 DCM/MeOH with formic acid is used to remove the residual DSPE, and the expected product is then detached with the 65/25/4/1 DCM/MeOH/water/formic acid quaternary mixture.

(13) C.sub.61H.sub.105GdN.sub.5O.sub.15P; m/z (ES−)=1335

EXAMPLE 2.2

Other Examples of Lipophilic PCTA Chelates

EXAMPLE 19 OF WO 2006/100305

b) Gadolinium complex of 3-[(2-{3,4-dioxo-2-[3-(3,6,9-tris-carboxymethyl-3,6,9,15-tetraaza-bicyclo[9.3.1]pentadeca-1(14),11(15),12-trien-13-yl)propylamino]cyclobut-1-enyl-amino}ethoxy)hydroxyphosphoryloxy]-2-octadecanoyloxypropyl octadecanoate

(14) ##STR00019##

(15) According to the procedure of step a) of Example 18 of WO 2006/100305, starting with 500 mg of the compound prepared in step a) of Example 12 of WO 2006/100305 and 520 mg of DSPE.

(16) m=350 mg

(17) m/z: ES− 1417

EXAMPLE 20 OF WO 2006/100305

a) Gadolinium complex of 2-(3,9-bis-carboxymethyl-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(14),11(15),12-trien-6-yl)-5-hexadecanoylaminopentanoic acid

(18) ##STR00020##

(19) According to the procedure of step a) of Example 6 of WO 2006/100305, starting with the compound obtained in step c) of Example 13 of WO 2006/100305 (300 mg) and 150 mg of palmitic acid chloride. m=230 mg

(20) m/z: ES− 829

EXAMPLE 21 OF WO 2006/100305

a) Gadolinium complex of 3-({2-[5-(3,9-bis-carboxymethyl-3,6,9,15-tetraaza-bicyclo[9.3.1]pentadeca-1(14),11(15),12-trien-6-yl)-5-carboxypentanoyl-amino]ethoxy}hydroxyphosphoryloxy)-2-hexadecanoyloxypropyl hexadecanoate

(21) ##STR00021##

(22) According to the procedure of step a) of Example 8 of WO 2006/100305, starting with 100 mg of the compound prepared in step d) of Example 15 of WO 2006/100305 and 120 mg of DPPE.

(23) m=80 mg

(24) m/z: ES− 1293

EXAMPLE 5 OF WO 2006/100305

a) Gadolinium complex of 3-({2-[4-(3,9-bis-carboxymethyl-3,6,9,15-tetra-azabicyclo[9.3.1]pentadeca-1(14),11(15),12-trien-6-yl)-4-carboxy-butyrylamino]ethoxy}hydroxyphosphoryloxy)-2-octadecanoyloxypropyl octadecanoate

(25) ##STR00022##

(26) 200 mg of the compound obtained in step c) of Example 3 of WO 2006/100305 are dissolved in 10 ml of dimethylformamide. To this solution are added 204 mg of N,N′-dicyclohexylcarbodiimide and 40 mg of N-hydroxysuccinimide The mixture is stirred for 1 hour at room temperature and a solution of 250 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE, Avanti® Polar Lipids, Inc.) in 5 ml of pyridine is added. The reaction medium is stirred for 20 hours at room temperature and then precipitated from 50 ml of ethanol. The product is then purified on silica gel. m=190 mg.

(27) m/z: ES− 1335

EXAMPLE 6 OF WO 2006/100305

a) Gadolinium complex of 2-(3,9-bis-carboxymethyl-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(14),11(15),12-trien-6-yl)-4-(4-octadec-9-enoylaminophenyl)butyric acid

(28) ##STR00023##

(29) 500 mg of the compound obtained in step j) of Example 1 of WO 2006/100305 are dissolved in 30 ml of anhydrous DMSO. 230 mg of triethylamine are added, followed by 400 mg of oleic acid chloride (Aldrich®). The mixture is stirred for 6 hours at room temperature and precipitated from ethanol. The product is then purified on silica gel. m=300 mg.

(30) m/z: ES− 917

EXAMPLE 8 OF WO 2006/100305

a) Gadolinium complex of 2-hexadecanoyloxy-3-(hydroxy{2-[2-(3,6,9-tris-carboxymethyl-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-trien-12-yloxy)acetyl-amino]ethoxy}phosphoryloxy)propyl hexadecanoate

(31) ##STR00024##

EXAMPLE 3

Synthesis of Lipophilic DOTA Derivatives

EXAMPLE 3.1

Dota-Dspe

(32) ##STR00025##

(33) 200 mg of DOTA-Gd carboxylate are dissolved in 1.5 ml of water. 238 mg of 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine (DSPE) dissolved in 70 ml of pyridine at 80° C. are added, along with 85 mg of EDCI and 22 mg of HOBT. The reaction medium is stirred at 40° C. for 24 hours. The pyridine is then evaporated off and the residue is taken up in ethanol and then filtered.

(34) C.sub.60H.sub.108GdN.sub.5O.sub.17P; m/z (ES.sup.−)=1358

EXAMPLE 3.2

(35) ##STR00026##
Step 1:

(36) 1.043 g of N-dioctadecylamine and 200 mg of succinic anhydride are dissolved in 10 ml of pyridine. After 2 hours at 50° C., the reaction medium is precipitated from 100 ml of acidified water; filtered and washed with acidified water. After drying under vacuum, 1.12 g of a white powder are obtained.

(37) C.sub.40H.sub.79NO.sub.3; m/z (ES.sup.−)=621

(38) Step 2:

(39) The activated ester of NHS is obtained by reacting 300 mg of the compound obtained in step 1 in 5 ml of dichloromethane with 100 mg of dicyclohexylcarbodiimide and 56 mg of N-hydroxysuccinimide After 30 minutes, the precipitate formed is filtered off. The filtrate is engaged in the following step without concretization.

(40) C.sub.44H.sub.82N.sub.2O.sub.5; m/z (ES.sup.−)=718

(41) Step 3:

(42) The filtrate obtained in step 2 is added dropwise to 360 mg of gadolinium complex dissolved in 3 ml of DMSO and 20 μl of triethylamine. The reaction medium is stirred for 3 hours at room temperature. After evaporating off the dichloromethane, the reaction medium is precipitated from water and then filtered. The precipitate is then purified on normal silica with elution with a dichloromethane/methanol mixture. 30 mg of product are obtained.

(43) C.sub.64H.sub.111GdN.sub.6O.sub.10; m/z (ES.sup.−)=1280.8

EXAMPLE 3.3

(44) ##STR00027##
Step 1:

(45) The reaction is performed under the same conditions as in step 1 of Example 3.2, the N-dioctadecylamine being replaced with 1.495 g of DSPE.

(46) C.sub.45H.sub.86NO.sub.11P; m/z (ES−)=846.6

(47) Step 2:

(48) Identical to step 2 of Example 3.2; starting with the compound obtained in step 1, 41 mg of NHS and 73 mg of DCC.

(49) C.sub.49H.sub.89N.sub.2O.sub.13P; m/z (ES.sup.+)=944

(50) Step 3:

(51) Identical to step 2 of Example 3.2

(52) C.sub.69H.sub.118GdN.sub.6O.sub.18P; m/z (ES.sup.−)=1508

EXAMPLE 3.4

Example 33 OF WO 2010/066815

e) Synthesis of (4,7-bis-carboxymethyl-10-dioctadecylcarbamoylmethyl-1,4,7,10-tetraaza-cyclododec-1-yl)acetic acid

(53) ##STR00028##

(54) 40 mg of the intermediate obtained in d) of WO 2010/066815 (0.037 mmol; 1 eq.) are dissolved in 4 mL of trifluoroacetic acid and 1 mL of CH.sub.2Cl.sub.2 for 5 hours in a 25 mL round-bottomed flask. After evaporating off the mixture of solvents, the yellow solid (32 mg) is precipitated from diethyl ether and then filtered off on a sinter funnel.

(55) C.sub.52H.sub.101N.sub.5O.sub.7; MALDI-TOF positive mode m/z 908.76

(56) The following examples illustrate the synthesis of amphiphilic targeting ligands, with use of certain linkers; a person skilled in the art knows how to adapt the protocols for other linkers, for example C1-C10 alkyl, PEG, C1-C10 alkylene-PEG-C1-C10 alkylene, squarate, alkylene-PEG-alkylene.

EXAMPLE 4

Synthesis of a Lipophilic RGD Peptide (Linear RGD Peptide)

(57) ##STR00029##
Step 1

(58) 100 mg (0.15 mmol) of peptide H-Gly-(D)-Phe-(L)-Val-(L)-Arg-Gly-(L)Asp-NH.sub.2 (H-GfVRGD-NH2) purchased from Bachem are dissolved under argon in 3 ml of DMSO dried over sieves. 23 μl of 3,4-diethoxy-3-cyclobutene-1,2-dione (0.15 mmol; 1 eq.) and 25 μl of triethylamine are added. The reaction medium is left overnight at 40° C., and then precipitated from 40 ml of diethyl ether. After filtration, 98 mg of a white powder are obtained (yield: 84%).

(59) C.sub.34H.sub.48N.sub.10O.sub.11; m/z=773 (ES.sup.+)

(60) Step 2

(61) 95 mg of the intermediate obtained in step 1 (0.12 mmol; 1 eq.) and 430 mg (0.15 mmol, 1.25 eq.) of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt) are dissolved in 3 mL of DMSO dried over molecular sieves in the presence of 25 μl of triethylamine. The reaction medium is stirred for 48 hours at room temperature, and then precipitated from 40 ml of diethyl ether. After filtration, 400 mg of a white powder are obtained. The product thus obtained is then purified by flash chromatography on a C4 cartridge with a gradient of 10 mM pH6 ammonium formate/methanol. 260 mg of a white powder are obtained (yield: 62%). C.sub.164H.sub.305N.sub.12O.sub.64P; MALDI-TOF: positive mode m/z=3501

(62) Other example:

(63) ##STR00030##

EXAMPLE 5

Synthesis of a Cyclic RGD Lipophilic Peptide

(64) ##STR00031##

(65) Same procedure as for Example 4, starting with 90 mg of cyclic peptide RGDfK purchased from Bachem. C.sub.163H.sub.302N.sub.11O.sub.63P; MALDI-TOF: positive mode m/z=3456

EXAMPLE 6

Synthesis of a Lipophile with an RGD Peptidomimetic; Example of a Naphthyridine Compound

(66) Synthetic Scheme:

(67) ##STR00032## ##STR00033##
Step 1:

(68) 1 g of Int 1 is dissolved in 5 ml of CH.sub.2Cl.sub.2. 5 ml of TFA are added to the medium. The mixture is left for 3 hours at room temperature and then evaporated to dryness. The residue is taken up in 2*40 ml of iso-ether, and an oil is recovered, which is dried by evaporation. m.sub.obt=0.8 g; Yld=90%; C.sub.26H.sub.36N.sub.4O.sub.8S; MALDI-TOF: positive mode m/z=564

(69) Step 2:

(70) TABLE-US-00003 Reagents Amounts Solvents Int 2 M = 0.564 g (0.001 ml) DMFV = 10 ml Int 3 M = 0.235 g (0.00023 ml) HOBT M = 0.131 g DIPEA M = 0.286 g EDCI V = 0.2 ml Int. 4

(71) The acid is dissolved in DMF, HOBT and EDCI are then added and the mixture is left for 1 hour under argon.

(72) Int 2 and DIPEA are added; the mixture is left for 18 hours at room temperature under argon. After evaporation, the oil is taken up in CH.sub.2Cl.sub.2 and washed with dilute Na.sub.2CO.sub.3 solution; after evaporation, an oil is obtained.

(73) m.sub.obt=0.600 g; Yld=77%; C.sub.39H.sub.52N.sub.6O.sub.9S; M/Z=780

(74) Step 3

(75) TABLE-US-00004 Reagents Amounts Solvents Int 4 M = 0.6 g (0.0077 ml) MeOH V = 30 ml Pd/C 10% 1 spatula-full Int. 5

(76) Int 4 is dissolved in methanol, and the solution is placed in a 125 ml autoclave; the catalyst is added and the mixture is left for 3 hours under hydrogen pressure (P=5 bar) at 30° C.

(77) After filtering off the catalyst and evaporating, an oil is obtained, which is washed with 50 ml of iso-ether.

(78) m.sub.obt=0.300 g; Yld=60%; C.sub.31H.sub.46N.sub.6O.sub.7S; HPLC=90%; M/Z=646

(79) Step 4:

(80) TABLE-US-00005 Reagents Amounts Solvents Int 5 M = 0.300 g (0.000442 ml) DMSO V = 10 ml Diethyl squarate M = 0.286 g TEA V = 0.25 ml Int. 6

(81) Int 5 is dissolved in DMSO, followed by addition of diethyl squarate and a few drops of TEA. The mixture is left overnight at room temperature under argon. It is poured into ether: a white paste is obtained.

(82) m.sub.obt=0.330 g; Yld=97%; C.sub.37H.sub.50N.sub.6O.sub.10S; M/Z=770

(83) Step 5:

(84) TABLE-US-00006 Reagents Amounts Solvents Int. 6 M = 0.330 g (0.00043 ml) DMSO V = 10 ml DSPE-PEG.sub.2000-NH.sub.2 M = 1.07 g (0.000385 ml) Saturated Na.sub.2CO.sub.3 M = 0.131 g solution Int. 7

(85) Int 6 and DSPE-PEG2000-NH.sub.2 are dissolved in DMSO, and 3 drops of saturated Na.sub.2CO.sub.3 solution and 2 ml of H.sub.2O are added. The reaction medium is stirred at room temperature for 48 hours and is precipitated from ether. The paste obtained is dissolved in methanol and is then purified on silica, eluting with CH.sub.2Cl.sub.2. After combining and evaporating the correct fractions, crystals are obtained.

(86) COMMENT: The product obtained is in the acid form by cleavage of the methyl ester due to the presence of Na.sub.2CO.sub.3.

(87) m.sub.obt=0.170 g; Yld=17%; C.sub.166H.sub.308N.sub.9O.sub.63PS; M/Z=3500

EXAMPLE 7

(88) ##STR00034##
Step 1:

(89) 150 mg of compound e) of Example 11 of patent application WO 2004/112839 are reacted with 35 μl of diethyl squarate according to the same procedure as in step 4 of Example 6 of this patent.

(90) C.sub.35H.sub.45N.sub.9O.sub.11; m/z (ES.sup.−)=766

(91) Step 2:

(92) The compound obtained in step 1 is reacted with 440 mg of DSPE-PEG2000-NH2 as described in step 5 of Example 6.

(93) C.sub.165H.sub.302N.sub.11O.sub.64P; m/z (ES.sup.−)=3493

EXAMPLE 8

(94) ##STR00035##

(95) 0.517 g of compound e) of Example 11 of patent application WO 2004/112839 are reacted with 500 mg of the compound obtained from step 2 of Example 3.2 in 5 ml of DMSO, 93 mg of NHS and 166 mg of DCC. After 24 hours at room temperature, the reaction medium is precipitated from 50 ml of water and filtered. After drying under vacuum, 515 mg of a yellow powder are obtained.

(96) C.sub.69H.sub.118N.sub.10O.sub.10; m/z (ES.sup.−)=1246

EXAMPLE 9

(97) Step 1:

(98) 200 mg of cyclic peptide 8-amino-3,6-dioxaoctanoylcyclo-Cys-Met-Lys(TFA)-Thr-Asp-Thr-Arg-Leu-Cys-COOH synthesized by Polypeptide are reacted in 1 ml of DMSO and 0.23 μl of diethyl squarate according to the same procedure as in step 4 of Example 6 of this patent.

(99) C.sub.55H.sub.87F.sub.3N.sub.14O.sub.21S.sub.3; m/z (ES.sup.−)=1432

(100) Step 2:

(101) The compound obtained in step 1 is reacted with 362 mg of DSPE-PEG2000-NH.sub.2 as described in step 5 of Example 6.

(102) C.sub.185H.sub.344F.sub.3N.sub.16O.sub.74PS.sub.3; m/z (ES.sup.−)=4160

(103) Step 3:

(104) The compound obtained in the preceding step is dissolved in 0.2 M piperidine in methanol for 3 hours at 0° C.

(105) C.sub.183H.sub.345N.sub.16O.sub.73PS.sub.3; m/z (ES.sup.−)=4064

EXAMPLE 10

(106) ##STR00036##
Step 1:

(107) 200 mg of cyclic peptide 8-amino-3,6-dioxaoctanoylcyclo-Cys-Pro-Ser-Ile-Tyr-Pro-Leu-Leu-Cys-NH.sub.2 synthesized with Polypeptide are reacted in 1 ml of DMSO and 0.23 μl of diethyl squarate according to the same procedure as in step 4 of Example 6 of this patent.

(108) C.sub.58H.sub.87N.sub.11O.sub.17S.sub.2; m/z (ES.sup.−)=1273

(109) Step 2:

(110) The compound obtained in step 1 is reacted with 350 mg of DSPE-PEG2000-NH.sub.2 as described in step 5 of Example 6.

(111) C.sub.188H.sub.344N.sub.13O.sub.70PS.sub.2; m/z (ES.sup.−)=4000

EXAMPLE 11

(112) ##STR00037##
Step 1:

(113) 100 mg of {4-[6-(2-aminoethoxy)benzoxazol-2-yl]phenyl}dimethylamine are reacted with 50 μl of diethyl squarate according to the same procedure as in step 4 of Example 6 of this patent.

(114) m.sub.obt=43 mg; Yld=34%; C.sub.23H.sub.23N.sub.3O.sub.5; m/z (ES.sup.−)=421

(115) Step 2:

(116) The compound obtained in step 1 is reacted with 256 mg of DSPE-PEG2000-NH.sub.2 as described in step 5 of Example 6.

(117) m.sub.obt=182 mg; Yld=63%; C.sub.153H.sub.280N.sub.5O.sub.58P; m/z (ES.sup.−)=3148

EXAMPLE 12

(118) Synthesis of an emulsion containing the compound of Example 1 and PEG-2000 10 g of Miglyol®, 420 mg of Lipoid S75 (Lipoid GmbH), 30 mg of DSPE-PEG-2000 (Lipoid) and 150 mg of the compound of Example 1 are dissolved in a chloroform/methanol mixture (90/10). This represents a content of 6% by mass of total surfactants relative to the mass of oil used.

(119) This mixture is treated on a rotary evaporator in order to remove the solvents. A perfectly homogeneous oily phase is obtained. 40 ml of water containing 2.5 m/m % of glycerol are added and then pre-emulsified using an Ultra-Turrax homogenizer. The pre-emulsion is then finished in a microfluidizer (Microfluidics M-110-S) by recycling for 3 to 4 minutes, which corresponds to about 25 passes in the cell.

(120) The mass content of total surfactants relative to the final solution is 1.18% (0.6/50.6)

(121) The pH is monitored and then adjusted to ˜7. The emulsion is filtered through a 0.45μ membrane. Gentallin is added at a rate of 7 μl/100 ml in order to ensure conservation.

(122) The hydrodynamic diameter (Zetasizer from Malvern) of the emulsion obtained is 190 nm.

EXAMPLE 13

Synthesis of an Emulsion Containing the Compound of Example 2

(123) 3 g of Miglyol® and 180 mg of the compound of Example 2 are dissolved in a chloroform/methanol mixture (90/10). This represents a content of 6% by mass of total surfactants relative to the mass of oil used. This mixture is treated on a rotary evaporator in order to remove the solvents. A perfectly homogeneous oily phase is obtained. 27 ml of water containing 2.5 m/m % of glycerol are added and then pre-emulsified using an Ultra-Turrax homogenizer. The pre-emulsion is then finished in a microfluidizer (Microfluidics M-110-S) by recycling for 3 to 4 minutes, which corresponds to about 25 passes in the cell.

(124) The mass content of total surfactants relative to the final solution is 0.6% (0.18/30.18). The pH is monitored and then adjusted to =7. The emulsion is filtered through a 0.45μ membrane. Gentallin is added at a rate of 7 μl/100 ml in order to ensure conservation.

(125) The hydrodynamic diameter (Zetasizer from Malvern) of the emulsion obtained is 160 nm.

EXAMPLE 14

Synthesis of an Emulsion Containing the Compound of Example 3 and PEG-5000 (Emulsion Containing 20% Oil)

(126) 10 g of Miglyol®, 420 mg of Lipoid S75 (Lipoid GmbH), 30 mg of DSPE-PEG-5000 (Lipoid) and 150 mg of the compound of Example 3 are dissolved in a chloroform/methanol mixture (90/10). This represents a content of 6% by mass of total surfactants relative to the mass of oil used.

(127) This mixture is treated on a rotary evaporator in order to remove the solvents. A perfectly homogeneous oily phase is obtained.

(128) 40 ml of water containing 2.5 m/m % of glycerol are added and then pre-emulsified using an Ultra-Turrax homogenizer.

(129) The pre-emulsion is then finished in a microfluidizer (Microfluidics M-110-S) by recycling for 3 to 4 min, which corresponds to about 25 passes in the cell.

(130) The mass content of total surfactants relative to the final solution is 1.2% (0.6/50.6)

(131) The pH is monitored and then adjusted to ˜7. The emulsion is filtered through a 0.45μ membrane. Gentallin is added at a rate of 7 μl/100 ml in order to ensure conservation. The hydrodynamic diameter (Zetasizer from Malvern) of the emulsion obtained is 210 nm.

EXAMPLE 15

Synthesis of an RGD Vectorized Emulsion Containing DSPE-PEG-2000 and the Compound of Example 1 (Emulsion Containing 20% Oil)

(132) 10 g of Miglyol®, 400 mg of egg phosphatidylcholine (EPC, Lipoid GmbH), 110 mg of DSPE-PEG-2000 (Lipoid), 210 mg of the compound of Example 1 and 60 mg of the compound of Example 4 are dissolved in a chloroform/methanol mixture (90/10). This represents a content of 7.8% by mass of total surfactants relative to the mass of oil used.

(133) This mixture is treated on a rotary evaporator in order to remove the solvents. A perfectly homogeneous oily phase (lipid phase consisting of oil) is obtained.

(134) 40 ml of water containing 2.5% m/m % of glycerol are added and then pre-emulsified using an Ultra-Turrax homogenizer.

(135) The pre-emulsion is then finished with a microfluidizer (Microfluidics M-110-S) by recycling for 3 to 4 min, which corresponds to about 25 passes in the cell.

(136) The mass content of total surfactants relative to the final solution is 1.54%.

(137) The pH is monitored and then adjusted to ˜7. The emulsion is filtered through a 0.45μ membrane. Gentallin is added at a rate of 7 μl/100 ml in order to ensure conservation.

(138) The hydrodynamic diameter (Zetasizer from Malvern) of the emulsion obtained is 168 nm.

(139) Monitoring of the diameter of the emulsion is performed by dynamic light scattering (Zetasizer from Malvern) for one year with conservation of the emulsion at 4° C.

(140) The hydrodynamic diameter at 1 year is 170 nm.

EXAMPLE 16

Synthesis of an RGD Vectorized Emulsion Containing the Compound of Example 1, DSPE-PEG-2000 and Rhodamine (Emulsion Containing 20% Oil)

(141) 10 g of Miglyol®, 330 mg of Lipoid S75 (Lipoid GmbH), 30 mg of DSPE-PEG-2000 (Lipoid) and 150 mg of the compound of Example 1, 90 mg of the compound of Example 4 and 2 mg of DSPE-rhodamine are dissolved in a chloroform/methanol mixture (90/10). This represents a content of 6% by mass of total surfactants relative to the mass of oil used.

(142) This mixture is treated on a rotary evaporator in order to remove the solvents. A perfectly homogeneous oily phase is obtained.

(143) 40 ml of water containing 2.5 m/m % of glycerol are added and then pre-emulsified using an Ultra-Turrax homogenizer.

(144) The pre-emulsion is then finished with a microfluidizer (Microfluidics M-110-S) by recycling for 3 to 4 min, which corresponds to about 25 passes in the cell.

(145) The mass content of total surfactants relative to the final solution is 1.2%.

(146) The pH is monitored and then adjusted to ˜7. The emulsion is filtered through a 0.45μ membrane. Gentallin is added at a rate of 7 μl/100 ml in order to ensure conservation.

(147) The hydrodynamic diameter (Zetasizer from Malvern) of the emulsion obtained is 206 nm.

EXAMPLE 17

Synthesis of an RGD Vectorized Emulsion Containing the Compound of Example 1, DSPE-PEG-2000 and Rhodamine

(148) 10 g of Miglyol®, 330 mg of egg phosphatidylcholine (EPC, Lipoid GmbH), 30 mg of DSPE-PEG-2000 (Lipoid), 150 mg of the compound of Example 1, 90 mg of the compound of Example 5 and 2 mg of DSPE-rhodamine are dissolved in a chloroform/methanol mixture (90/10). This represents a content of 6% by mass of total surfactants relative to the mass of oil used.

(149) This mixture is treated on a rotary evaporator in order to remove the solvents. A perfectly homogeneous oily phase is obtained.

(150) 40 ml of water containing 2.5 m/m % of glycerol are added and then pre-emulsified using an Ultra-Turrax homogenizer.

(151) The pre-emulsion is then finished with a microfluidizer (Microfluidics M-110-S) by recycling for 3 to 4 min, which corresponds to about 25 passes in the cell.

(152) The mass content of total surfactants relative to the final solution is 1.2%.

(153) The pH is monitored and then adjusted to ˜7. The emulsion is filtered through a 0.45μ membrane. Gentallin is added at a rate of 7 μl/100 ml in order to ensure conservation.

(154) The hydrodynamic diameter (Zetasizer from Malvern) of the emulsion obtained is 206 nm.

EXAMPLE 18

Relaxivity Measurements

(155) The relaxivity measurements are performed on Minispec relaxometers at 20 and 60 MHz.

(156) The stock solution is diluted over 6 range points in Milli-Q water in order to be able to study the linearity of the relaxation rates as a function of the concentration. The concentration range is from 0.1 to 2.5 mM of Gd.

(157) The relaxivity measurement is performed at 37° C. The Gd assay is performed by ICP-AES on all the range points.

(158) TABLE-US-00007 20 MHz 60 MHz Emulsion r1(mM.sup.−1s.sup.−1) r2(mM.sup.−1s.sup.−1) r1(mM.sup.−1s.sup.−1) r2(mM.sup.−1s.sup.−1) Example 12 16 19 14 20 Example 13 39 43 29 52 Example 14 18 22 16 20 Example 15 23 25 24 35 Example 16 27 28 24 34

EXAMPLE 19

Toxicity Tests on the Emulsion of Example 15

(159) In Vivo Test:

(160) On “Swiss” mice weighing about 25 g: manual conscious caudal IV injection at 2 mL/min in isovolume (200 μl/animal, i.e. 6.67 ml/kg).

(161) At 24 hours: anesthesia with isoflurane, collection of a sublingual blood sample for hematology analysis on an MS4 automated analyzer followed by exsanguination with syringe+heparinized needles.

(162) Symptomatology: no lethality or deleterious clinical signs observed at the test dose and period.

(163) Hematology: normal hematological analysis.

(164) Weight change of the mice after injection: no significant weight variation.

(165) In vitro test: MTT test on L929 at 24 hours.

(166) The CEL50 is higher than the test dose, which is 3 mM of Gd.

EXAMPLE 20

Measurement of IC50

(167) The IC50 measurement of the emulsions is performed on HUVEC cells overexpressing ∞vβ3 by measurement of competition with echistatin .sup.125I.

(168) The HUVEC suspension is distributed in a conical-based 96-well plate, at a rate of 2×10.sup.5 cells in 50 μL of binding buffer. Fifty μL of solutions of increasing concentration of echistatin or of RGD products are added per well. The positive control is made by adding binding buffer without competitor. All the concentration points are produced in duplicate. The plate is incubated for 2 hours at room temperature with agitation. Fifty μL of the echistatin-.sup.125I-SIB solution at 3 nM are then distributed in each well and the plate is again incubated for 2 hours at room temperature with agitation. The reaction mixtures are transferred into vials containing 200 μL of a density cushion composed of paraffin and dibutyl phthalate (10/90). The microtubes are then centrifuged at 12 000 rpm for 3 minutes. The tubes are finally frozen in liquid nitrogen and then sectioned in order to count the cell pellet and the supernatant in a gamma counter. A competition curve is then plotted, where the relative binding of the echistatin.sup.125I-SIB is determined by the following equation:

(169) $\mathrm{Relative}\mathrm{binding}\mathrm{of}\mathrm{echistatin}-I^{125}-\mathrm{SIB}=\frac{\begin{array}{c}\mathrm{Radioactivity}\mathrm{bound}\mathrm{in}\mathrm{the}\\ \mathrm{presence}\mathrm{of}\mathrm{competitor}\left(\mathrm{cpm}\right)\end{array}}{\mathrm{Radioactivity}\mathrm{of}\mathrm{the}\mathrm{control}\mathrm{sample}\left(\mathrm{cpm}\right)}⨯100$

(170) The data are analyzed using the GraphPad Prism® 5.0 software which determines the IC.sub.50 values for each product from the competition curve.

(171) TABLE-US-00008 Compound IC50 (nM of targeting ligand) IC50 (nM of emulsion) Example 4   300 Example 5    1 Example 6    0.4 Example 15   4500 2 Example 16 13 500 2.1