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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
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  • A61K49/186—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule the organic macromolecular compound being obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. PLGA the organic macromolecular compound being polyethyleneglycol [PEG]
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  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
  • A61K49/085—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier conjugated systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
  • A61K49/10—Organic compounds
  • A61K49/14—Peptides, e.g. proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
  • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
  • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
  • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
  • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
  • A61K49/1851—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule
  • A61K49/1863—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule the organic macromolecular compound being a polysaccharide or derivative thereof, e.g. chitosan, chitin, cellulose, pectin, starch
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
  • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
  • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
  • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
  • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
  • A61K49/1866—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle the nanoparticle having a (super)(para)magnetic core coated or functionalised with a peptide, e.g. protein, polyamino acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
  • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

• the invention provides novel diagnostic compounds for apoptosis-related diseases, their method of preparation and their use in medical imaging.
• Apoptosis refers to the mechanisms that lead to the suicide of eukaryotic cells in response to certain stimuli. This physiological phenomenon ensures the balance between cells that proliferate and those that must be naturally eliminated. In pathological conditions, it can be increased and have devastating functional consequences in some tissues.
• the role of apoptosis has been widely demonstrated in chronic neurodegenerative diseases (Alzheimer's disease, Parkinson's disease), in ischemic diseases (myocardial infarction, cerebral ischemia) and in inflammatory diseases (atherosclerosis, arthritis). Conversely, apoptosis can play a beneficial role when it leads to the death of tumor cells during radiotherapy or chemotherapy treatments.
• apoptosis in imaging is a new diagnostic tool that is very useful in helping clinicians to assess the severity of a disease, predict the risk of occurrence of a serious event, and monitor treatment efficacy through characterization. fine lesions.
• the detection of apoptosis in vivo is difficult because of the fact that the phenomenon is not always expressed, and that it is relatively brief since it takes place over a period of 6 to 24 hours maximum, after which the Cell debris is removed by phagocytic cells.
• the prior art describes several attempts to develop contrast agents intended to target apoptotic cells.
• agents typically comprise a targeting part called a biovector intended for the specific recognition of the biological target associated with the apoptosis mechanism, and a part of the signal capable of being detected by the imaging methods: optical imaging, scintigraphy, magnetic resonance imaging (MRI).
• the signal part is for example a paramagnetic metal chelate such as Gadolinium, linear or macrocyclic, in particular DTPA, DTPA BMA, DTPA BOPTA, D03A, DOTA.
• PS phosphatidylserine
• telomeres proteins have high affinity and specificity for PS. It is in particular annexin V. It is an endogenous protein of 35 kDa of molecular weight, present intra or extra cellular. It was linked to different reporter groups according to the imaging modalities chosen. In particular, it has been conjugated to superparamagnetic iron oxide particles (Schellenberger EA, et al., Mol Imaging, 2002, 1: 102-107). In vitro, this contrast agent made it possible to obtain an MRI image in Jurkat cells made apoptotic after treatment with camptothecin.
• apoptosis usually occurs 24 to 48 hours after the initial accident.
• Anti-apoptotic drugs acting for example on the caspase pathway, would certainly be useful if they could be associated with an imaging method.
• immunosuppressive drugs in cases of organ transplant rejection. There are therefore many indications for a contrast agent capable of effectively detecting apoptotic cells in vivo.
• the applicant has succeeded in obtaining compounds comprising a targeting portion of apoptosis zones, effective not only in vitro but also and especially in vivo MRI.
• Such compounds are indeed difficult to obtain because it is necessary not only to identify an effective biovector in vitro, but to obtain an effective compound in human clinical diagnostic imaging. This is particularly the case for MRI, which is known to be a highly requested technique because it has no radioactivity, but its sensitivity is much lower than that of nuclear medicine.
• the compound obtained must both have sufficient affinity to recognize its target, a high specificity to be a distinctive indicator of the disease state, a stability suitable not to be degraded or modified in vivo; and additionally without the signal part interfering in a way with these different parameters (for example without altering the affinity and stability of the ligand).
• the following correspondence table is used.
• Signal represents a signal entity
• Link present or not, represents a chemical bond
• Peptide represents a peptide comprising a peptide targeting apoptosis, the peptide targeting apoptosis being chosen from the peptides of the following formula and their functional equivalents:
• X1-X2-X3-X4-X5-X6 (1) (SEQ ID NO: 1) wherein X1 and X2 independently of one another are leucine or isoleucine, X3 and X4 are lysine, X5 represents proline and X6 represents phenylalanine, advantageously peptide LIKKPF (SEQ ID No. 11) and its functional equivalents.
• D-A-H-S-X7-S (2) (SEQ ID NO: 2) wherein X7 is phenylalanine or leucine;
• HG-XI 0-LS-X11 (4) (SEQ ID No. 4) wherein X10 is aspartic acid or histidine, and XI is threonine or isoleucine; - VLGERG (5) (SEQ ID No. 5); and the pharmaceutically acceptable salts of these compounds.
• the expression peptide targeting apoptosis and disease associated with apoptosis, it is meant that the applicant's compounds are compounds capable of targeting biological zones (cells, tissues, organs, etc.) undergoing an apoptosis mechanism. This mechanism results in a disease related to apoptosis at an early stage or already advanced.
• This diagnostic information then allows the clinician more appropriate treatment, such as the administration of an anti-apoptosis drug adapted to the biological territory concerned, and possibly any additional diagnostics.
• the invention relates to compounds of formula I and their use for targeting areas
• peptide targeting apoptosis is also referred to as PEPTIDE P in the application.
• Advantageously Peptide represents a PEPTIDE P.
• LIKKPF SEQ ID No. 11
• LIKKPF peptide SEQ ID No. 11
• X1 is leucine L or another substituted alkyl hydrophobic amino acid (isoleucine, alanine, valine),
• X2 is isoleucine I or another substituted alkyl hydrophobic amino acid (leucine, alanine, valine),
• X3 and X4 are lysine K or another amino acid comprising a basic function (arginine, histidine, ornithine), but preferentially lysine which is very advantageous for the interaction of the peptide with its target X5 is proline P
• X6 is phenylalanine F or another hydrophobic aromatic amino acid (eg, tryptophan, tyrosine, histidine, biphenylalanine, naphthylalanine)
• the functional equivalents are analogous or derivative peptides which have an imaging efficiency similar to or better than that of the peptide of the formula (2) to (5).
• the invention also relates to the compounds of formula (I) functionally equivalent to the compound of formula (I), that is to say having an activity in vitro and in vivo comparable to these compounds, the affinity and the specificity of the peptides equivalent to the peptide of the compound (I) being evaluated by appropriate screening methods, such as that described in the detailed description.
• Peptide comprising at least one peptide targeting apoptosis
• PEPTIDE P a peptide having the peptide sequence of recognition of the biological target of PEPTIDE P, optionally flanked at the N and / or C terminus of a chemical group not interfering on this sequence.
• the peptide targeting apoptosis and its derivatives is designated by "PEPTIDE P”.
• these peptides are excluded when Signal represents a lipid nanoparticle.
• Signal entity is understood to mean a chemical entity that makes it possible to obtain a signal in medical imaging, in particular: a chelate capable of being coupled to a paramagnetic metal or to a radionuclide, a metal nanoparticle, especially a superparamagnetic iron oxide nanoparticle a lipid nanoparticle, advantageously in the form of an emulsion, this nanoparticle carrying at least one chelate capable of being coupled to a paramagnetic metal (in this case, PEPTIDE P is grafted to the emulsion lipid nanoparticle which is it even a carrier of chelates, the peptide is bound to the lipid nanoparticle by, for example, a chemical linking group).
• Signal represents a signal entity chosen from:
• a chelate coupled or not to a paramagnetic metal M or to a radionuclide
• a superparamagnetic nanoparticle coated with an organic layer preferably having an iron oxide core and a marker for optical imaging
• the signal entity comprises at least one chelate Ch (in the form of complexing a metal M).
• the chelate is coupled to the metal M.
• the metal M is a paramagnetic metal ion, or a radionuclide.
• it is a paramagnetic metal.
• the complex formed by the chelate and the metal M is stable under physiological conditions so as to avoid undesired release of the metal M into the body.
• the chelate or the signal entity comprises at least one functional group making it possible to connect the signal entity with the link or directly with the peptide.
• the invention also relates to the compounds of formula (I) in which the chelate is not complexed with the metal.
• Ch is a linear chelate chosen from: EDTA, diethylenetriaminopentaacetic acid DTPA, N- [2- [bis (carboxymethyl) amino] -3- (4-ethoxyphenyl) propyl] -N- [2- [bis (carboxymethyl) amino ] ethyl-L-glycine (EOB-DTPA), mono- or bis-amide derivatives of DTPA, such as N, N-bis [2- [carboxymethyl [(methylcarbamoyl) methyl] amino] ethyl] glycine (DTPA) -BMA), 4-carboxy-5,8,1-tris (carboxymethyl) -1-phenyl-2-oxa-5,8,1,1-triazatridecan-13-oic acid (BOPTA).
• EDTA diethylenetriaminopentaacetic acid
• DTPA N- [2- [bis (carboxymethyl) amino] -3- (4-ethoxyphenyl) propy
• Ch is a macrocyclic chelate selected from 1,4,7,10-tetraazacyclododecan-l, 4,7,10-tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecan-l, 4,7 -triacetic acid (D03A), 10- (2-hydroxypropyl) -1,4,7,10- tetraazacyclododecan-l, 4,7-triacetic acid (HPDO3A) 2-methyl-1,4,7,10-tetraazacyclododecan-l, 4,7,10-tetraacetic acid (MCTA), (alpha, alpha ', alpha ", 1 ', 4,7,10-tetraacetic acid (DOTMA), 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15') -tetramethyl-1,4,7,10-tetraazacyclododecan ), 11, 13-triene-3,6,9-
• Ch may also be a derivative of those compounds in which one or more carboxylic groups are in the form of a salt, ester, corresponding amide; or a corresponding compound in which one or more carboxylic groups are replaced by a phosphonic and / or phosphinic group.
• a chelate chosen from: DOTA gadofluorins, OD3A, HPDO3A, TETA, TRITA, HETA, DOTA-NHS, M4DOTA, M4DO3A, PCTA and their derivatives, advantageously chosen from. DOTA, DTPA, DO3A, HPDO3A, TRITA, TETA, BOPTA, NOTE, PCTA, DOTMA, AAZTA, HOPO and their derivatives.
• the chelate (s) forming the signal entity may satisfy the following formula of WOO 1/60416:
• the compounds DTPA, DOTA, NOTA, DO3A and derivatives may be used. Mention may also be made of the chelates recalled in WO03 / 011115, in particular of the following formulas:
• X a group capable of coordinating a metal cation, preferably O-, OH, NH 2 , OPO 3 -, NHR with R an aliphatic chain, Y a chemical bond.
• X1 to X4 and K1 to C18 of the above chelates representing H or a C 1 -C 20 chain
• R 1, R 2, R 3, R 4, R 5 independently represent -COOH, -P (O) (OH) 2 ; and being selected such that the chelate comprises at least one function capable of being coupled to a PEPTIDE P directly or via the link.
• Ch is DTPA or DOTA or their derivatives.
• the relaxivity ri of these chelates is typically of the order of 4 to 20 s "1 mMol " 1 Gd "1 at a field of 0.5 to 1.5 T. It is recalled that the Longitudinal relaxivity of a paramagnetic contrast product gives the measure of its magnetic efficiency and allows its influence on the recorded signal to be appreciated. In medical imaging MRI, the contrast products modify the relaxation time of the protons, and the increase the relaxivity obtained makes it possible to obtain a higher signal.
• the coupling of chelates with biovectors in particular peptides is described in the prior art, and generally involves a chemical bond (Link) as described in WO01 / 60416.
• the structure and the chemical nature of the chemical bond are defined to allow the chemical coupling between the peptidic part of PEPTIDE P and the chelate (s) used, and so as to obtain an affinity of the PEPTIDE P part for its target and a specificity of the recognition suitable for use.
• a large number of bonds may be used, as long as they are capable of interacting with at least one biovector functional group and at least one chelate functional group.
• Link represents: a) a group of formula Q1-I-Q2, in which Q1 and Q2, which are identical or different, represent O, S, NH, CO 2 , -NHCO, CONH, NHCONH, NHCSNH, SO 2 NH- or NHSO 2 -, and 1 represents an alkyl group (preferably C 1 -C 1 0), alkoxyalkyl
• alkenyl preferably C 2 -C O
• alkynyl preferably C 2 -C O
• polyalkoxyalkylène alkyl interrupted by one or more squarates, with one or more aryls, advantageously phenyl, or with one or more groups selected from -NH-, -O-, -CO-, -NH (CO) -, - (CO) NH-, - O (CO) -, or - (OC) O- ;
• Signal represents a marker for optical imaging (fluorescent molecule used in optical imaging).
• markers for optical imaging include those of US2006 / 0018830 and in particular those cited on pages 11 and 12 paragraph IB, with precise imaging modalities described in column 33 ([0259]) in paragraph 6 (techniques and chromophores and fluorophores described in detail).
• Signal represents quantum dots (inorganic fluorophores comprising nanocrystals).
• Signal represents a superparamagnetic nanoparticle coated with an organic layer, advantageously commonly referred to as SPIO or USPIO ("ultra small particles of iron oxide”).
• the nanoparticle comprises a core of oxide or iron hydroxide, in particular magnetite (Fe3 ⁇ / i), maghemite ( ⁇ -Fe 2 ⁇ 3).
• a nanoparticle covered with a bisphosphonate coating, advantageously gew-bisphosphonate, described in WO2004058275 is used, the particle and the coupling method between the peptide and the nanoparticle being detailed in the examples of the present application.
• the magnetic nanoparticles used are acidic nanoparticles based on an iron compound, and covered by a layer comprising one or more identical or different gembisphosphonate compounds, the covering layer of the nanoparticle having the following formula (C):
• the link L2 represents an organic group linking the function T to the gem-bisphosphonate function -CH (PO 3 H 2 ) 2 ;
• T represents a chemical function coupled to the PEPTIDE P or the link of the present application.
• T-L2 represents the bond of the compound of formula (I).
• the composition is in the form of a stable aqueous solution of nanoparticles.
• the complexation rate (of the layer with the peptide) of the compound (C) on the particles is greater than 50%, advantageously 70%, and preferably greater than 80, 90, 95%. It is particularly preferred that the acid magnetic particles (p) are complexed on at least 90% of their protonated sites with compounds of formula (C).
• a part of the functions T of the layer is coupled to a PEPTIDE P, and part of the functions T is coupled to a hydrophilic compound, in particular a compound carrying hydroxyl groups and in particular a hydrophilic aminoalcoholic compound designated AAG1AA28 described in WO2004058275 ( Example 8) or a PEG group.
• the magnetic particles (p) have a hydrodynamic diameter of between 5 and 300 nm, preferably between 5 and 60 nm, more preferably between 5 and 30 nm.
• the linker L2 makes it possible to connect and / or space the gew-bisphosphonate function and the reactive entity T capable of ensuring the covalent grafting of the PEPTIDE P (the biovector) on the nanoparticle, via or without the Link. .
• the link L2 represents a divalent group.
• the link L 2 is chosen from: an aliphatic group; alicyclic; alicyclic alicyclic; aromatic; aliphatic aromatic, said aliphatic, alicyclic and aromatic groups being optionally substituted by a methyl, hydroxy, methoxy, acetoxy, amido, or a halogen atom, advantageously a chlorine, iodine or bromine atom; a group -IrNHCO-I 2 where Ii and I 2 , identical or different, represent an aliphatic group; alicyclic; aromatic; aliphatic alicyclic or aliphatic aromatic alicyclic group, said groups possibly being optionally substituted with a methyl, hydroxy, methoxy, acetoxy, amido group or a chlorine, iodine or bromine atom.
• L 2 represents a substituted or unsubstituted aliphatic group, and more preferably a - (CH 2 ) p - group, where p is an integer of 1 to 5, or preferably - (CH 2 ) n -NHCO- (CH 2 ) m - where n and m represent an integer from 0 to 5.
• T represents a group -COOH or -NH 2 and L 2 a substituted or unsubstituted aliphatic group, advantageously a group - (CH 2 ) P -, where p is an integer of 1 to 5.
• the most preferred layer is following formula (Cl)
• Signal represents a lipid nanoparticle comprising at least one chelate.
• the lipid nanoparticles may be in the form of a nanoparticulate emulsion, containing or not perfluorocarbons, such as those described in the documents WO 03/062198, US 5,958,371, US 5,080,885 and US 6,403,056.
• the lipid nanoparticles can be suspended in an aqueous or hydrophilic medium. These nanoparticles have a diameter of the order of 10 nm to 500 nm, especially 20 to 250 nm.
• the emulsion nanoparticles may comprise or be coupled with a large number of chelates, for example 10,000 to 100,000 chelates per nanoparticle.
• the emulsions comprise sufficient compounds of formula (I) and thus peptide to allow recognition of the apoptosis zone. Mention may be made, as nanoparticles, of liposomes, unilamellar or multilamellar, micelles, microgels, oil droplets, lipoproteins, such as HDL, LDL, IDL, VLDL, chylomicrons, fluorocarbon nanoparticles, nanobulles, or the like of which the surface is lipophilic.
• the chelate is lipophilic and attached to the membrane of the nanoparticle.
• the bond of the compound of formula (I) is sufficiently lipophilic to couple the peptide to the membrane of the lipid nanoparticle, the PEPTIDE P being sufficiently expressed on the outer part of the nanoparticle for the specific recognition of the apoptotic target.
• the Link is for example a group lipophilic such as a C10-C20 alkylene chain, this chain being inserted into the lipid layer of the nanoparticle and thus enabling the peptide to be attached to the nanoparticle.
• the chelate carries a long lipophilic chain (phospholipid for example) which is inserted into the membrane of the lipid nanoparticle (liposome, micelle, nanoemulsion).
• the PEPTIDE P advantageously carries a lipophilic chain (the Link) which is inserted into the membrane of the lipid nanoparticle.
• the lipid nanoparticle includes perfluorocarbons as described in US Pat. No.
• the liquid emulsion containing nanoparticles comprising a perfluorocarbon with a relatively high boiling point (for example between 50 and 90 ° C.) surrounded by a coating.
• a coating composed of a lipid and / or a surfactant.
• the surfactant is capable of coupling directly to a targeting biovector or to include an intermediate compound covalently bound to the biovector, optionally using a chemical linker.
• Phosphatidylcholine dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine and phosphatidylethanolamine are commonly used as membrane forming phospholipids of the nanoparticles.
• compositions are described, for example, in US Pat. No. 5,989,520 and US Pat. No. 5,958,371, as recalled in document US 20040248856, which notably mentions perfluorocarbon compounds: perfluorodecalin, perfluorooctane, perfluorodichlorooctane, perfluoro-n-octyl bromide, perfluoroheptane and the like.
• suitable methods and lipid compositions recalled in US Pat. No. 6,010,682 will be used to prepare contrast agents according to the invention, in particular as regards the detailed description of the lipid composition and the preparation of liposomes. , micelles, emulsions.
• the emulsions are heterogeneous lipid mixtures obtained suitably by mechanical stirring and / or addition of emulsifying agents.
• the lipophilically rendered chelates are mechanically mixed with organic solvents such as chloroform. After evaporation of the solvent, the lipids are resuspended in an aqueous medium such as PBS to obtain an emulsion which then typically undergoes sonication and microfluidization.
• the emulsions obtained can be lyophilized with the use, if appropriate, of anti-agglutination agents.
• To formulate the desired paramagnetic contrast agent emulsion typically 1 to 75% by weight of lipophilic chelate compound is used relative to the total ingredients of the emulsion.
• the contrast agent-forming composition is preferably administered intravascularly, depending on the patient under examination, for example 0.1 mg to 1 g of lipophilic chelate compound and 1 to 50 micromoles of paramagnetic metal ion per kg. of patient.
• the lipid compositions obtained are optionally formulated using additives recalled in US 6 010 682, in particular for administration by intravenous injection. These include dextrose, sodium chloride, antimicrobials.
• compositions according to the invention it is possible to obtain an increase in relaxivity by ion.
• the following characteristics are typically obtained, which can vary according to the precise compositions of the emulsions and their method of preparation: polydispersity index: 0.2 to 0.3
• [Gd 3+ ] 2 to 10 mM, preferably 3 to 7 mM concentration in particles: 50 to 100 nM- 1 (InM 1 S 1 Gd- 1 ): 5 to 40, preferably 10 to 40
• the invention also relates to those compounds of formula (I) wherein the Peptide contains a modified peptide sequence, but without altering the affinity and specificity for the target and the effectiveness of the compound in vivo.
• PEPTIDE P is for example modified using appropriate methodologies described in the prior art for example in US2005100963 (column 20-21 paragraphs [529] to [541] in the case of peptides targeting KDR receptors), in order to select compounds of formula (I) effective:
• hydrophobic amino acids leucine X1 and isoleucine X2 the use of another linear or branched C1-10 aliphatic side chain (alkyl, alkenyl, alkynyl) can be tested. It will thus be possible to test the substitution of the amino acids X1, X2 with other alkyl-substituted hydrophobic amino acids (alanine, leucine, isoleucine, valine, norleucine, S-2-aminobutyric acid).
• proline other hydrophobic amino acids, in particular derivatives with a secondary amine, are also used.
• X3 and X4 lysine
• other dibasic amino acids arginine, histidine, ornithine
• derivatives of lysine or of these other amino acids in particular the alkyl, alkenyl and aryl derivatives; such as N-epsilon-isopropyl-lysine derivatives.
• the derivatives mentioned in paragraph [533] of US2005100963 may be tested;
• the peptides are also studied with: A replaced by leucine, isoleucine, Valine and H derivatives replaced by lysine or arginine and S derivatives replaced by threonine and L derivatives replaced by isoleucine, valine and derivatives.
• V replaced by leucine or isoleucine
• L replaced by isoleucine
• arginine replaced by valine
• E derivatives replaced by aspartic acid R replaced by lysine
• the invention relates to contrast products for MRI comprising a compound of formula (I) as previously described, the paramagnetic metal ion M of which is of atomic number 21-29, 42-44 or 58, 70, preferably gadolinium.
• Paramagnetic metals M include lanthanides of atomic number 58-70 and transition metals of atomic number 21-29, 42 or 44, for example scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, molybdenum , ruthenium, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, and ytterbium.
• the elements Gd (III), Mn (II), europium and dysprosium are particularly preferred, advantageously M is chosen from Gd, Mn, Fe, Dy and Tm.
• the invention relates to contrast agents for imaging.
• X-ray or CT comprising a compound (I) as described above whose heavy metal ion M is atomic number 21-31, 39-50, 56-80, 82, 83 or 90.
• the invention according to another aspect of the radiopharmaceutical products, comprising a compound of formula (I) as described above whose chelate is coupled with a radionuclide or a radiohalogen known to those skilled in the art, typically gadolinium, technetium, chromium gallium, indium, ytterbium, rhenium, lanthanium, yttrium, dysprosium, copper or the like.
• the radionuclides include the radioactive forms of the elements Sm, Ho, Y, Pm, Gd, La, Lu, Yb, Sc, Pr, Tc, Re, Ru, Rh, Pd, Pt, Cu, Au, Ga, In, Sn, Cr, Pb, especially 99 Tc, 117 Sn,
• the invention relates to a diagnostic method and a radiopharmaceutical treatment method using a compound of formula (I) as described above.
• the present invention furthermore relates to a composition comprising at least one compound of general formula (I) as described above and a pharmaceutically acceptable excipient, advantageously intended for parenteral administration. It further relates to a process for preparing such a composition comprising the addition of a compound of general formula (I) as defined above to an injectable medium comprising the pharmaceutically acceptable excipient.
• the invention relates to the use of a composition according to the present invention for the diagnosis of a pathology associated with apoptosis.
• the diagnostic and radiopharmaceutical compositions according to the invention can be used as described in US 2002/0090342, US 2002/0098149, WO 02/055111 for anticancer indications.
• the invention further relates to the compounds of general formula (I) as defined above for their use as diagnostic agent for diseases associated with apoptosis.
• the invention also relates to the use of the compounds described above for the preparation of a diagnostic or radiopharmaceutical composition intended for the diagnosis and / or treatment of diseases associated with apoptosis, advantageously chosen from chronic neurodegenerative diseases, ischemic diseases and inflammatory pathologies.
• a peptide targeting apoptosis of formulas (1) to (5) as defined above for the preparation of a diagnostic composition for diseases associated with apoptosis.
• the compounds of the Applicant's general formula (I) will be used as a diagnostic agent, or as a therapeutic treatment agent at apoptosis zones, or as a diagnostic and diagnostic agent. therapeutic treatment, or diagnostic follow-up of therapeutic efficacy. If appropriate, the compound will be co-administered simultaneously or in a delayed manner with other diagnostic and / or therapeutic agents targeting apoptosis.
• the invention also relates to an imaging method comprising the synthesis of a compound comprising a paramagnetic metal according to the invention, capable of targeting a pathological zone, its administration to a patient, MRI imaging.
• the invention also relates to an imaging method comprising the synthesis of a radiopharmaceutical compound according to the invention, capable of targeting a pathological zone, its administration to a patient, SPECT or planar gamma scintigraphy imaging, or emission tomography. of positrons.
• intravenous administration by injection usually in saline is typically at a metal ion dose of 0.001 to 1.5 mmol / kg body weight, for example 1 to 500 ⁇ mol Gd / kg.
• intravenous administration by injection usually in saline is typically at a dose of 1 to 100 mCi per 70 kg body weight, preferably 5 to 50 mCi, with diagnostic imaging for example 180 minutes after injection for 99 Tc.
• concentration of the heavy atom is typically from 0.1 M to 5 M, with concentrations by intravenous administration of the order of 0.5 to 1.5 mmol / kg.
• the invention relates to the use of a compound of formula (I) as described above for the preparation of a composition intended for optical imaging.
• compositions for medical imaging are described in the prior art, for example in WO 0226776 and US 2006/0018830 column 36 ([0282]) paragraph 8 (assays and additives).
• Pharmaceutically physiologically acceptable vehicles for forming diagnostic compositions (contrast products) comprising the compounds described above are known from the prior art. For example, salts (sodium, calcium, meglumine), pH control agents (acetic acid, citric acid, fumaric acid, antioxidants) will be used.
• the invention also relates to a process for preparing compounds comprising coupling a peptide with at least one chelate.
• the Applicant has also studied the possibilities of combining a PEPTIDE P coupled to several chelates in the compound of formula (I).
• the applicant has also studied compounds of formula (I) having an assembly between one or more peptides of the compound of formula (I) targeting apoptosis, and one or more chelates; and so that access to the target is not impeded despite the presence of the chelate (s).
• the chelate is spaced from the PEPTIDES P by the link of sufficient size and a chemical structure such that the recognition of the peptide or peptides by their target is not impaired.
• biovector associations peptides or possible other biovectors
• a central biovector peptide connected to several identical or different chelates
• a central chelate connected to several identical or different peptides a first set [chelate-carrying peptide (s)] coupled via a hydrophobic or hydrophilic link to a second set [peptide carrying chelates (s)]
• a first set [chelate-carrying peptide (s)] coupled via a hydrophobic or hydrophilic link to a second set [peptide carrying chelates (s)]
• the method for constructing multimeric compounds described in US2005 / 0100963 (WO2006 / 031885, line 25 to page 69, line 30) will be used in the case of peptides targeting KDR receptors (for example using the method 13).
• examples: "preparation of homodimers and heterodimers” but using PEPTIDE P (for example, the peptides of the compounds of FIGS. 44 to 47 of US2005100963 will be replaced by PEPTIDES P).
• the compound may thus advantageously comprise a peptide coupled to several chelates, or a chelate coupled to several identical or different peptides.
• the invention also relates to mixed compounds comprising in addition to PEPTIDE P at least one other biovector targeting apoptosis, the biovector being either another peptide or another biovector but nonpeptide.
• the specificity of the product refers to its specific affinity for at least one marker of apoptosis or any associated pathological disorder, the specificity of the binding typically expressed by Kd and Ka constants, the Kd value for the target markers being less than 10 ⁇ M, preferably less than 1 ⁇ M.
• cation salts of inorganic bases include sodium, sodium, calcium, magnesium, etc.
• organic base cations include organic base cations (ethanolamine, diethanolamine, morpholine, glucamine, N-methylglucamine, N, N). -dimethylglucamine ...), anions of inorganic acids (in particular chlorides, bromides, iodides, sulphates, etc.), organic acid anions (acetate, succinate, citrate, fumarate, maleate, oxalate, trifluoroacetate). .), amino acid ions (taurine, glycine, lysine, arginine, ornithine, aspartic acid, glutamic acid ).
• vascular disease apoptosis at atheromatous plaque areas
• alteration of liver tissue apoptosis of the liver
• alteration of nerve tissue nerve tissue causing brain diseases including Parkinson's disease in particular.
• FIG. 1 represents the competition test with annexin V
• FIG. 2 represents the measurement of the affinity of peptide LIKKPF (SEQ ID No. 11)
• FIG. 3 represents the measurement of the affinity of peptide LIKKPF (SEQ ID No. 11) coupled to a USPIO
• Figure 4 illustrates apoptotic cell assays performed with a USPIO-
• FIG. 5 illustrates the affinity of a DTP A-LIKKPF product with apoptotic cells
• FIG. 6 illustrates the dynamic tracking of the mouse apoptotic liver MRI signal with a DTPA-LIKKPF product
• FIG. 7 shows MRI images on ApoE mice obtained with a product DTPA-LIKKPF
• FIG. 8 represents the corresponding quantitative enhancement.
• M molar concentration (mol / l).
• M / z mass on charge determined by mass spectrometry.
• ES + positive mode electrospray.
• ES " electrospray negative mode
• kD molecular weight unit (kiloDalton)
• TLC thin layer chromatography
• Z ave hydrodynamic diameter measured by PCS
• the iron is assayed by atomic absorption spectroscopy (VARIAN AA10 Spectrophotometer) after mineralization with concentrated HCl and dilution against a standard range of ferric ions (0, 5, 10, 15 and 20 ppm).
• Particle size Particle size:
• T1 and T2 relaxation times were determined by standard procedures on a Minispec 120 (Broker) apparatus at 20 MHz (0.47T) and 37 ° C.
• the longitudinal relaxation time T1 is measured using an inversion recovery sequence and the T2 transverse relaxation time is measured by a CPMG technique.
• R1 or R2 as a function of the concentration is linear, and the slope represents the relaxivity ri (Rl / C) or r2 (R2 / C) expressed in (1 / second) x (1 / mmol / L) ie s " '.mM " ').
• nanoparticles were prepared according to the methods described in the patent WO2004 / 058 275 (US 2004/253181) Examples 8 and 9 for the preparation of the colloidal solutions of magnetic particles and Examples 10 to 12 for the complexation of the magnetic particles by a coating gem -bisphosphonate of Example 1 of WO2004 / 058,275.
• solution B The previous solution (solution B) is stirred at room temperature and the pH is adjusted to
• amino PEG-NH 2 750 (O- (2-aminoethyl) -O'-methyl polyethylene glycol 750 was obtained from FLUKA®).
• AminoPEG 350 is prepared according to the following reaction sequence according to procedures described in the literature.
• aminoPEGs are available from suppliers:
• the peptide (350 mg, 0.323 mmol) is dissolved in 2 ml of water and the polycarboxylated compound (NCS-Bz-DTPA 420 mg, 0.646 mmol, 2 equivalents) dissolved in water is added dropwise.
• the pH of the reaction mixture is adjusted to 10 with NaOH solution and the solution is stirred for 48h.
• the pH is readjusted to 7, and the solution is percolated on a column of silica RPCl 8.
• the fractions containing the condensation product are concentrated and the ligand is complexed with one equivalent of GdCl 3 -OH 2 O (0.235 mmol) for 6 hours.
• Step 1 5- (1,3-Dioxo-1,3-dihydro-isomol-2-yl) -2- (IAJ, 1-Oterraaza-cyclododec-1-yl) -pentanoic acid benzyl ester
• Step 2 5- (1,3-Dioxo-1,3-dihydro-isoindol-2-yl) -2- (4,7,10-tris-ethoxycarbonylmethyl) -1,4,7-benzyl ester 1 Otetraaza-cyclododec-1-yl) pentanoic
• Step 3 5-Amino-2- (4,7,10-tris-carboxymethyl-1,4,7,1-tetraazacyclododec-1-yl) -pentanoic acid
• Step 4 Gadolinium complex of 5-Amino-2- (4,7,10-tris-carboxymethyl-1, 4,7, 1-Otetraaza-cyclododec-1-yP) -pentanoic acid
• step 3 7.2 g of the compound obtained in step 3 (16 mmol) are dissolved in 70 ml of water and the pH is adjusted to 5.5 by addition of 6 N hydrochloric acid. 2.9 g of Gd are added. 2 ⁇ 3 (8 mmol), and the reaction medium is heated to 80 ° C. The pH of the solution increases steadily, and should be maintained between 5.2 and 5.7 by dropwise addition of 6 N hydrochloric acid. After two hours, the pH stabilizes at 5.7. The slight cloudiness is filtered through Whatman® filter and the filtrate is concentrated. Obtain 11.1 g of product in the form of white flakes (100% corrected yield).
• Solvent B CH 3 CN; UV detection at 201 nm; Tr: 10 min.
• Step 5 5- (2-Ethoxy-3,4-dioxo-cyclobut-1-enylamino) -2- (4,7,10-tris-carboxymethyl-1, 4,7, 1) gadolinium complex Otetraaza-cyclododec-1-yl) pentanoic
• Step 1 Coupling Peptide Nos. 1, 2, 3, 4, 5, 6 or the Squarate Derivative
• the compound obtained in Step 5 of Example 3 (100 mg, 1.35 ⁇ 10 -4 mol) is dissolved in 15 ml of aqueous Na 2 CO 3 solution pH 9.4.
• the protected peptide (1.6 ⁇ 10 -4 mol) is introduced while maintaining the pH at 9.4 by addition of Na 2 CO 3. If the peptide is not soluble in water, a few drops of DMF are added until After 48 hours of reaction at room temperature, the medium is precipitated in an ethanol / ethyl ether mixture and the precipitate is filtered and dried.
• step 1 The compound obtained in step 1 above is dissolved in a mixture of 10 cm 3 of TFA / TIS / H 2 O in the proportions 90/5/5. The medium is stirred for 5 hours at ambient temperature and the solvent is then evaporated off under reduced pressure. The residue is taken up in ethyl ether and the precipitate is filtered and dried. The product is then purified by HPLC preparative on a Symmetry® column with an eluent consisting of water / TFA PH 3 /
• Example 3 210 mg of the compound obtained in step 5 of Example 3 are dissolved in 20 ml of water and the pH is adjusted to 9 with a solution of Na 2 CO 3 . 350 mg of protected peptide (peptide No. 7) are added and the pH is adjusted to 9.2. The medium is stirred at ambient temperature and then the solution is dialyzed on a 1 kD cut-off membrane for 48 h and then chromatographed on an RP-18 column (MeOH / water (50/50) mixture).
• Peptides are synthesized on a solid phase using 2.5 equivalents of each Fmoc protected amino acid at each coupling cycle. Activation of the carboxylic acids is carried out with HATU, N-methylmorpholine in DMF. The Fmoc groups are cut by piperidine treatment (20% in DMF). After introduction of the last amino acid of the peptide sequence and cleavage of the Fmoc protecting group, the N-terminal amine of the peptide is acylated by the compound prepared in step 1 (2.5 equivalents) dissolved in CH 2 Cl 2 in the presence of HoBt and N-methylmorpholine.
• the peptide is then released from the resin and the protective groups of the side functions are cleaved by the action of a trifluoroacetic acid / thioanisole mixture (95/5) for 30 minutes at 0 ° C. and then for 2 hours at room temperature.
• the resin is removed and the solvent is evaporated under reduced pressure.
• the lipopeptide is precipitated in ethyl ether.
• the products are purified by preparative HPLC on a Vydac ODS® column, eluting with a water / acetonitrile / TFA mixture.
• Example IL1 Peptide LIKKPF (SEQ ID No. 11)
• the affinity of peptide LIKKPF (SEQ ID No. 11) is measured on immobilized PS (ELISA plate).
• the peptide is incubated at increasing concentrations of 5.10 14 M to 4.2 ⁇ 10 7 M.
• Annexin V-biotin is then added to the concentration of 5.10 10 M which corresponds to its K ⁇ j value for PS. Incubation at 37 ° C is continued for 1.5 hours.
• Bound annexin V is detected by addition of a solution of streptavidin-HRP and then of an ABTS substrate solution containing 0.05% H 2 O 2 .
• the DO 4 05 is measured for calculation of the IC 50 value of the peptide.
• USPIO-LIKKPF The affinity of USPIO-LIKKPF is measured on immobilized PS (120 ⁇ g / mL). After saturation of nonspecific sites, the USPIO-LIKKPF is incubated at increasing concentrations (122 nM - 4 mM iron) for 2 hours at 37 ° C. After washing the wells, the PS-bound molecules are digested in a volume of 5 N HCl. The samples are digested in a volume of 5N HCl, 48h at 37 ° C, and the iron assay is made by Prussian Blue ( D0 6 3o).
• In vitro MRI is performed on apoptotic cells (or controls) preincubated with USPIO-LIKKPF or nonfunctional USPIO (3 or 4 mM iron) for 2 hours at 37 ° C.
• the apoptosis of Jurkat cells is induced by treatment with 2 ⁇ M camptothecin for 24 hours.
• Example 113 Contrast product Gd-DTPA-LIKKPF
• the peptides LIKKPF (SEQ ID No. 11) and FKIPKL ("scramble" peptide - mixed sequence) are grafted separately on a Gd-DTPA chelate.
• Peptides protected by TFA on two lys are attached to the 8-amino-3,6-dioxooctanoyl linker.
• the protected peptide is coupled to DTPA-isothiocyanate (NCS-Bz-DTPA), then the Lys are deprotected in a basic medium.
• apoptotic Jurkat cells (2.10 6 cells / ml) are incubated in the presence of the Gd-DTPA-LIKKPF product at concentrations of 400, 200 and 100 ⁇ M in Gd. The cells are incubated for 2 hours at room temperature. After rinsing and centrifugation, the cell pellet is mineralized under acidic conditions and the amounts of Gd are measured by ICP-MS (inductively coupled plasma - mass spectrometry). The targeting efficiency of the Gd-DTPA-LIKKPF product by MRI was also evaluated on an apoptotic liver model in mice.
• Apoptosis is induced by an iv injection (caudal vein) of anti-Fas monoclonal antibody in anesthetized Balb / c mice (Blankenberg et al., 1998, 1999, Rodriguez et al., 1996).
• SI signal intensity
• the SI value is also measured in a tube filled with a 2% aqueous solution of gelatin enriched with 50 ⁇ M Gd-DTPA, used as a reference product, in a region outside the image of the mouse which corresponds to the background brait.
• the MRI protocol is as follows: (1) pre-contrast MRI starts Ihl5 after injection of anti-Fas; (2) the contrast product is injected into the femoral vein at a dose of 60 ⁇ mol Gd / kg approximately 1:45 after injection of anti-Fas; (3) several post-contrast acquisitions are made during 1h30.
• a competition protocol is performed by injection of 100 ⁇ mol peptide / kg 10 minutes before the injection of the contrast product Gd-DTPA-LIKKPF.
• the targeting efficacy of the Gd-DTPA-LIKKPF product was also evaluated on another model of apoptosis: the apoE ⁇ mouse, model of atherosclerosis.
• Female C57B1 ApoE 7 " mice, approximately 15 months old, are subjected to a high cholesterol diet for 3 months prior to MRI studies, and for MRI acquisition the animals are anesthetized with pentobarbital.
• the contrast agent Gd-DTPA- LIKKPF is injected iv at a dose of 100 ⁇ mol Gd / kg All images are acquired at the level of the abdominal aorta, especially the near-renal region which is known for the development of atheromatous plaque due to the presence of the arterial branches
• the parameters of the RARE sequence (Rapid Acquisition with Relaxation Enhancement) are adjusted using a reference tube filled with a 1 mM solution of Gd-DTPA.
• SI values are measured in different regions of interest (abdominal artery wall or whole liver) using OSIRIS image analysis software. The regions are first drawn on the postcontrast images, then duplicated on the pre-contrast images. The SI value is measured on all sections of images where the arterial wall and the liver are visible. Finally, the SI values obtained for serial aortic sections over a length of 3.2 - 8 mm are averaged for each animal.
• VLGERG SEQ ID No. 5
• LIKKPF SEQ ID No. 11
• D-A-H-S-X7-S SEQ ID No. 2
• X7 can be F or L
• HG-X10-LS-XI1 (SEQ ID No. 4) and its functional equivalents, where X 10 is advantageously chosen from D or H, and X 11 is chosen from T or I.
• the affinity (in order of magnitude) of these peptides for PS is between 10 6 and 10 9 M.
• the highly specific peptide LIKKPF (SEQ ID No. 11) has been particularly well characterized. The specificity of interaction of the peptide with the PS is confirmed using the competition assay with Annexin V. The value IC 5 O Annexin V is equal to 1,08.10 "9 M ( Figure 1).
• Example 11.5 LIKKPF Peptide (SEQ ID NO 11) immobilized on PS vitro test is used to calculate a value IC 5 O ( Figure 2).
• Example II.6 USPIO-PEG-PEPTIDE P contrast medium with PEPTIDE P being LIKKPF (SEQ ID No. 11) (also referred to as USPIO-LIKKPF)
• the USPIO-LIKKPF interaction test with the immobilized PS makes it possible to measure a K * d value of 7.85.10 8 M.
• the interaction specificity of the USPIO-LIKKPF with the PS is checked with an annexin V competition test.
• the IC 5 O value is equal to 4.2 ⁇ 10 7 M. (FIG. 3)
• the grafting of the LIKKPF peptide (SEQ ID No. 11) on the Gd-DTPA contrast product makes it possible to preserve the targeting of the apoptotic cells. Indeed, the ICP-MS assay of apoptotic cells incubated with Gd-DTPA-LIKKPF proves that their concentration-dependent interaction is greater than the capture of the product by the control cells (FIG. 5). After iv injection of 60 ⁇ mol Gd / kg of product, the dynamic MRI signal monitoring of the mouse apoptotic liver (FIG. 6) shows an enhancement with Gd-DTPA-LIKKPF greater than that measured with Gd-DTPA-FKIPKL (scramble ) or non-functionalized Gd-DTPA.
• Example IL 8 Imaging of Apoptotic Vascular Tissue
• Gd-DTPA-LIKKPF at a dose of 100 ⁇ mol Gd / kg, can dynamically detect a higher signal enhancement than that measured with the non-grafted product (Gd-DTPA) at the level of the abdominal aorta (FIGS. 8 - RARE sequence, 27 minutes after contrast injection - MRI of serial sections of the abdominal aorta over a length of 3.2 mm).
• Gd-DTPA non-grafted product
• Example II.9 Apoptotic Nervous Tissue Imaging in a Model of Parkinson's Disease, USPIO-LIKKPF Product Injection
• Immunohistochemistry and MRI are very revealing. Sick and healthy mice were studied on the 4th, 5th, 6th day of treatment, and one and three weeks after the end of MPTP treatment. Imaging shows an evolution of the signal during the treatment, the targeting being more marked on the 5th day and then decreasing after. Sections made with these non-vectorized USPIOs (without peptide) show no marked targeting.
• the MRI location of the diseased areas corresponds to the zones containing dopaminergic neurons (immunohistochemistry: detection of dopaminergic neurons by measurement of tyrosine hydroxylase in the basal ganglia).

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