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EP0814849A1 - Substances chimiotactiques marquees magnetiquement comme agents de contraste pour l'imagerie par rmn de tissus vivants cibles - Google Patents

Substances chimiotactiques marquees magnetiquement comme agents de contraste pour l'imagerie par rmn de tissus vivants cibles

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Publication number
EP0814849A1
EP0814849A1 EP96932764A EP96932764A EP0814849A1 EP 0814849 A1 EP0814849 A1 EP 0814849A1 EP 96932764 A EP96932764 A EP 96932764A EP 96932764 A EP96932764 A EP 96932764A EP 0814849 A1 EP0814849 A1 EP 0814849A1
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EP
European Patent Office
Prior art keywords
mmol
peptides
water
solution
compounds
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EP96932764A
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German (de)
English (en)
Inventor
Michael F. Tweedle
Krishan Kumar
Stephen Eaton
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Bracco International BV
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Bracco International BV
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Publication of EP0814849A1 publication Critical patent/EP0814849A1/fr
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    • 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
    • A61K49/08—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
    • A61K49/18—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
    • A61K49/1833—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 a small organic molecule
    • A61K49/1839—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 a small organic molecule the small organic molecule being a lipid, a fatty acid having 8 or more carbon atoms in the main chain, or a phospholipid
    • 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
    • A61K49/1866—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
    • 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
    • A61K49/1896—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes not provided for elsewhere, e.g. cells, viruses, ghosts, red blood cells, virus capsides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins

Definitions

  • the present invention addresses magnetically labeled chemoattractants, e.g. chemotactic peptides, for the detection and investigation of diseased or disordered tissue sites or organs in the body of human and animal patients.
  • disordered sites one wishes to include sites of infection, inflammation, or other trauma.
  • the invention also concerns physiologically acceptable administrable compositions or formulations comprising the labeled chemoattractants, labeled white blood cells, methods for preparing the chemoattractants and the formulations, and methods of using the formulations for detecting, localizing and diagnosing infected or inflammatory sites, or other trauma in the organism.
  • conjugates in which bio olecules targeted to specific sites or receptors in the organism of patients are coupled to ligands acting as signal generator means to provide diagnostically useful information has become widely known in recent years.
  • Said ligands involve elements capable of providing signals detectable and processable into recorded data by suitable medical equipment . Such data can thereafter be displayed and interpreted by medically trained persons into diagnostically pertinent information.
  • Useful signal generator means include, for instance radioactive emitters (radiotracers) , injectable X-ray opacifiers, e.g. iodinated compounds, and magnetic contrast enhancers in magnetic resonance imnaging (MRI) .
  • WO-A-90/14881 there is disclosed the attachment of polyaminocarboxylic chelators to homing proteins by using bridging functional groups such as isocyanato-, isothiocyanato-, bromoacetamido-,diazo-, N-hydroxysuccini- mide esters and inter-molecular or intra-molecular anhydrides .
  • administrable magnetic microparticles for instance magnetite coupled to substances having binding affinity for organic tissues.
  • Said tissue-specific substances include antibodies neurotransmitters, hormones, metabolites enzymes, toxins, and natural or synthetic drugs.
  • the magnetite particles are conveniently coupled to the tissue-specific substances by coating the particles with compounds (for instance biodegradable polymers) carrying reactive functional groups and linking to the tissues- specific substances through said reactive groups.
  • the tissue-specific targeted magnetite microparticles are administerd by injection into the blood stream (or otherwise) , whereby they will be transported to targeted organs or tissues where they operate as contrast enhancers in MRI investigations of said organs.
  • a formulation comprising a leukostimulatory reagent (a lectin) bound to a useful metal species under conditions allowing the reagent to attach to leukocytes, whereby the concentrations of the stimulated leukocytes are subsequently ascertained by metal detection and measuring means.
  • a leukostimulatory reagent a lectin
  • conjugates of polyaza-macrocycles N-substituted by electrophilic groups such as hydroxyalkyl, alkoxyalkyl, carboxyalky1 , phosphate, sulfonate, or phosphonate, and carrying antibodies designed for locating specific tissue types.
  • the acrocycles are provided to complex with metal atoms, suitably radioisotopes of Tc, Re, Co, Cu, Au, Ag, Pb, Bi, In and Ga .
  • the compounds are transported to target sites, and the metals are acting as signal sources to be detected by scintillation or other radiotracer detecting means .
  • the recognition agent involves monoclonal antibodies and peptides capable of selectively interacting with receptors having enhanced expression on activated leukocytes. More particularly, the reference discloses an imaging method utilizing a chemotactic peptide containining an affinity label and a radionuclide label conjugated to leukocytes to in vivo target inflammation sites.
  • the monoclonal antibodies useful in the disclosed method are directed against epitopes on cell surface antigens which are up-regulated upon leukocyte activation.
  • labeling is effected by linking a signal generating moiety containing a radionuclide to the recognition agent.
  • radionuclides are 1:L1 In, 198 Au, 113 Ag, ⁇ Ag, 123 I, 125 I,
  • the reference also provides Examples of chelating compounds to complex the radionuclides and methods to attach the chelatants to the target head of the recognition agent.
  • W093/17719 and WO94/19024 disclose 99 Tc labeled peptides for diagnostic imaging.
  • the peptides are selected to target sites in-vivo, i.e. to specifically bind to sites of infection and inflammation, or also to thrombotic, atherosclerotic or tumoral sites.
  • the reference particularly discloses a specific peptide and a bisamino-bisthiol radiolabeled moiety covalently linked to the peptide via amide bond.
  • the reference also discloses methods for radiolabeling the targeted peptides by reacting with 99m pertechnetate in the presence of a reducing agent such as dithionite, or Sn 2+ - or Fe 2+ , as well as a solid state method for making the peptide, and a method of using the radiolabeled peptide for diagnostically image various organs .
  • a reducing agent such as dithionite, or Sn 2+ - or Fe 2+
  • EP-A-0 398 143 (A.J. Fischman et al . ; The General
  • X is a protective formyl, acetyl or t-Boc group
  • Y is methionine or norleucine
  • W is a label, e.g. an EDTA or DTPA chelate of a radioactive or paramagnetic isotope covalently attached to the peptide
  • Z is a bond or a linker, e.g. Lys or Nle-Tyr-Lys.
  • the reference also describes the injection of the radioactive labeled chemotactic peptides into experimental rats previously infected in the thighs with strains of E . col i , and the subsequent localization of the infection sites by hourly serial ⁇ -camera images.
  • the present invention proposes novel conjugates of chemotactic peptides with paramagnetic macrocyclic chelates or magnetite particles .
  • novel conjugates have the general formula (F) : N(X) -Y-Leu-Phe-Z-A-W (F)
  • (X) is H or a protective group such as formyl, acetyl, t-Boc, or the like; Y is Nle or Met; Z is a chemical bond, an aminoacid or an oligopeptide, e.g. (N( ⁇ ))Lys, lie,
  • Nle-Tyr-Lys or (Gly) n , n being 1 to 4;
  • A is a linker; and
  • W is a suitably derivatized paramagnetic macrocyclic chelate or magnetic particle.
  • Fig. 1 is a graph showing the results of competition binding experiments using native and Gd-labeled chemotactic peptides.
  • Fig. 2 is a graph showing the variation of binding strength of Gd-labeled peptides in function to the Gd-formyl interatomic distance.
  • Fig. 3 is a graph showing PMNs intensity response in function to Gd-labeled peptide concentration.
  • Fig. 4 is a schematic representation of Gd-labeled molecules/cell association using PMNs and labeled peptides.
  • Fig. 5 is a schematic representation of the uptake of 99m Tc-DTPA and Gd-labeled peptide in infected and non- infected muscle.
  • Fig. 6 (a and b) are graphs showing the rate of reduction of NBT by human PMN stimulated by magnetite- labeled hexapeptide.
  • the general structure of the macrocyclic ligand to form paramagnetic macrocyclic chelates (W) is preferably selected from the derivatives of 1 , 4 , 7 , 10- etraaza- cyclododecane ring compounds of formula I
  • X is an alkyl substituent carrying one or more oxygen containing functions (e.g. OH, CO, COOH) .
  • Compound I has further acetoxy groups affixed to the N atoms and, optionally, other oxygen containing alkyl substituents.
  • Compounds I useful in the present invention are disclosed in K. Kumar et al . J. Liquid. Chromatography 17 (1994) , 3735- 3746.
  • Preferred compounds are those in which X is H (D03A) , acetoxy (DOTA) , -CH 2 -CHOH-CH 3 (HP-D03A, gadoteridol, PROHANCE®) and -CH(CH 3 )COOH (DOTMA) .
  • Other suitable macrocyclic chelates are disclosed in documents WO87/05030 and WO89/01476 incorporated herein by reference.
  • Another class of macrocyclic chelates useful in the present invention is that derived from starburst® dendrimers of the following structure (II) :
  • R's being derivatized chelates of paramagnetic metals, e.g.isothiocyanato-DTPA (ITC-DTPA) , SCN-Ph-NH-CO-CH2-D03A (IPA-D03A) , or having the formula
  • the chelates or chelatant molecules are suitably derivatized to carry groups to effect bonding in the preparation of the magnetically labeled chemoattractants of the invention.
  • the paramagnetic metal ions are conveniently selected from lanthanides (for instance Gd and other lanthanides) and suitable transition elements such as Mn, Co, Ni, Cr, Fe, Cu and the like.
  • the linker A can be selected from chemical sequences having required functions to achieve a conjugation bridge between the peptides and the macrocyclic chelate molecules.
  • Preferred linkers are selected from substituents attached, or to be attached, to the carbon or nitrogen atoms of the chelatants and carrying reactive functions capable of bonding to native or derivatized aminoacid acceptor groups of the chemotactic peptides (e.g. -SH, -OH, -COOH, NH 2 , and the like) .
  • D03A can be successively converted to its p-aminophenyl- and p- iothiocyanatophenyl-acetamido derivatives according to the following scheme:
  • the coupling of the chelate with the peptide is effected by reacting the isothiocyanato function with a terminal amino function of the peptide.
  • the coupling is effected directly on the acetylamino-p-aminophenylderivative 0 of the chelatant in the presence of triethylamine, a carboxylic functions of the peptide (designated here by R) being activated by (benzotriazol-1-yl ) -tris- (dimethylamino) - phosphonium hexafluorophosphate (BOP) .
  • R carboxylic functions of the peptide
  • the paramagnetic ion can be introduced at an intermediate stage or after the final stage if appropriate protecting groups are used.
  • Chelatants mixed anhydrides R ' -CO-O-CO-R ' obtained from chelatant tetramethylguanidine salts and isobutyl chloroformate) and peptides having lysine functional -NH 2 .
  • the latter are derivatized with materials to become linker A or to effect bonding with linker A.
  • This can be accomplished for example by first coating the particles with a polymeric material provided (or to be provided later) with functional groups capable of bonding with the chemoattractant peptides.
  • the linking techniques are similar to those disclosed herebefore.
  • the magnetic particles can be coated with derivatized polyacrylics, polystyrenes, dextran and other polysaccharides .
  • the latter are silanized with compounds provided with functions capable of binding to proteins, with or without the assistance of additional intermediate sequences.
  • silanizazion can be carried out using reactive trialkoxysilanes such as isocyanatoalkylsilanes , isothiocyanoalkyl si lanes, 3-aminopropyltrimethoxysilane, hydroxypropyltriethoxysilane, 4-chlorobutyltrimethoxysilane and the like. All details about silanization of magnetic particles (including also the preparation of the particles themselves from iron salts solutions) are found for instance in document EP-125 995 (M.S. Chagnon et al . ; Advanced Magnetics) incorporated herein by reference. Once the manetic particles have been silanized they can be coupled to the chemotactic peptides by the same means disclosed above in the case of the paramagnetic chelates .
  • the latter are coated with a phospholipid and an amphiphilic copolymer having lipophilic (POP) and hydrophilic (PEG) segments (see EP-A-607 401 incorporated herein by reference) .
  • the block copolymer is derivatized with functions capable of linking to proteins and peptides such as the chemotactic peptides of the present invention.
  • Derivatised block copolymers such as Pluronic® and Synperonic® are illustrated in WO-A-95 06251 (University of Utah) which discloses derivatized Pluronic® and Tetronic® compounds carrying reactive groups at the end of the PEG blocks.
  • the derivatized copolymers are used to coat by adsorption hydrophobic surfaces (e.g. polystyrene beads and similar carriers), which coated surface then function as substrates for immobilizing proteins and alike substances.
  • hydrophobic surfaces e.g. polystyrene beads and similar carriers
  • the following derivatized amphiphilic copolymers have been syn-thesized
  • gadolinium acetate (602 mg, 1.48 mmol, 1.33 equiv) in water (3.5 mL) was added to a suspension of 10- [N- (4-nitropheny)acetamido] -1,4,7, 10-tetraazacyclodode- cane-1, 4, 7-tri-acetic acid (580 mg, 1.114 mmol) in water (5 mL) at 65°C. Initially, a clear solution was obtained, but after 25 min, a pale yellow solid precipitated. Filtration and washing of the solid with water (2 x 2 mL) gave the gadolinium chelate in 62% yield (470 mg) .
  • Thiophosgene (lOOmL) was suspended in chloroform (0.5 mL) and was mixed with the gadolinium salt of 10- [N- (4- aminophenyl)acetamido] -1,4,7, 10-tetraazacyclododecane-l,4,7- triacetic acid (53.06 mg, 0.0798 mmol) .
  • the biphasic mixture was stirred vigrously for 90 min at room temperature.
  • the aqueous phase was separated from the chloroform layer and the pH was adjusted to 7 with sodium hydroxide.
  • the chloroform layer was washed with water carefully, and the aqueous extracts were combined.
  • N-Formyl-L-norleucyl-L-leucyl-L-phenylalanyl-L-norleu- cyl-L-tyrosyl-L-lysine (79.8 mg, 0.074 mmol) was dissolved in dimethyl formamide/triethyl amine (99:1, 8.0 mL) and the pH of the peptide was adjusted to 9.0.
  • the peptide was mixed with the aqueous solution of the isothiocyanato compound. Some insoluble material was seen upon the progress of the reaction which was dissolved by addition of more base and/or DMF. The reaction mixture was stirred for 18 h.
  • reaction mixture was analyzed by an HPLC method, which confirmed the completion of the reaction.
  • the crude material was purified by a semi-prep HPLC gradient method by using a PLRP-S column. The fractions were collected and the volume of the purified material was reduced at 40°C under the stream of nitrogen. The concentrated solution was lyophilized to a solid material (60 mg overall yield 51.4%) which was analyzed by mass spectral analysis.
  • FAB-MS m/z: 1515 [ (M+H) + with gadolinium pattern. Anal. Calcd.
  • a solution of carrier-added 153 GdCl 3 (504 mCi, 0.126 mmol) was mixed with 10- [N- (4-aminophenyl ) acetamido] - 1, 4,7, 10-tetraazacyclododecane 1, 4,7-triacetic acid triethyl ammonium salt (Example 2a, 76.43 mg, 0.12 mmol) .
  • the reaction mixture was heated and the pH of the solution was raised very slowly to 7.
  • the reaction mixture was analyzed by a reversed phase HPLC/analytical method, which confirmed the formation of the product (by comparison to the compound of example lc) .
  • the reaction mixture was reduced to approximately 3 mL and was transferred to a vial.
  • the gadolinium-153 salt of 10- [N- (4-aminophenyl) - acetamido] -1,4,7, 10-tetraazacyclododecane-l, 4, 7-triacetic acid (Example 2b) was added to a mixture of thiophosgene (150 L) and chloroform (2.0 mL) .
  • the biphasic reaction mixture was stirred vigrously for 40 min at room temperature.
  • the aqueous phase was separated from the chloroform layer and the pH was adjusted to 7 with sodium hydroxide.
  • the chloroform layer was washed with additional water.
  • N-formyl-L-norleucyl-L-leucyl-L-phenylalanyl-L-norleu- cyl-L-tyrosyl ) -L-lysine 64.81 mg, 0.0795 mmol
  • dimethyl formamide/triethylamine 99:1, 10.0 mL
  • the peptide was mixed with the above isothiocyanate, and the reaction mixture was stirred for 18 h. At the end of this period the reaction mixture was analyzed by an HPLC method, which confirmed the completion of the reaction.
  • the crude material was purified by a semi-prep HPLC gradient method by using a PLRP-S column. The fractions were collected and the volume of the purified material was reduced at 40°C under the stream of nitrogen to near dryness . The recovery of the purified product was 30%.
  • N- Formyl-L-methionyl-L-leucyl-L-phenylalanyl-L-lysine (41.48 mg, 0.05 mmol) at pH 10.1 was added to the aqueous solution of the isothicyanato compound.
  • the pH was adjusted to 10.1 with sodium hydroxide, and the reaction mixture was stirred overnight at ambient temperature.
  • the progress of the reaction was checked by an HPLC method.
  • the product was purified by a semi-prep HPLC method. All fractions containing the product were collected and concentrated to a minimum volume. The sample was lyophilized to a solid material (30 mg yield 55%) .
  • the solid material was assayed for its purity by an HPLC/analytical method, which indicated 97% purity.
  • GdCl 3 solution (0.36 M, 0.6 mL, 0.216 mmol)
  • 153 GdCl 3 500 mCi, 0.73 mCi/mL, 0.329 mCi/mg Gd 2 0 3 , 0.0083 mmol
  • 10-[N-(4- aminophenyl) acetamido] -1,4,7, 10-tetraazacyclododecane-l, 4,7 -triacetic acid triethyl ammonium salt (133 mg, 0.2 mmol) .
  • the chelate was converted to 10- [N- (4 Isothiocyanato- phenyl) acetamido] -1,4,7, 10-tetraazacyclodo-decane-l,4,7- triacetic acid, monogadolinium-153 salt as outlined in Example 2c.
  • Example 5b A solution of LiOH (110 mg, 2.62 mmol) in dioxane- water (5 mL, 4:1) was added to the product in Example 5b (0.70 g, 0.75 mmol) and stirred at room temperature for 4 h.
  • the reaction mixture was diluted to -10 mL with water and pH of the solution adjusted to -4.00 with KHS0 4 solution.
  • the solution was evaporated under vacuum to afford a foamy solid.
  • the soild was then subjected to preparative reversed-phase HPLC purification using water-acetonitrile (0.1% TFA) as eluant to separate diastereoisomers resulting from the D- and L-forms of phenylalanine.
  • GdCl 3 (3.6 mL of a 9.945 mM solution, 0.0358 mmol) was mixed with 10- [ ( (N-formyl-L-methionyl-L-leucyl-L-phenyl- alanyl) -N- (4-aminophenyl)acetamido] -1,4,7, 10-tetraazacyclo- dodecane-1, 4,7-triacetic acid (40 mg, 0.0358 mmol) in water.
  • the reaction mixture was heated at 60°C.
  • the pH of the reaction mixture was raised slowly to 7, while the reaction mixture was heated. When the pH of the reaction mixture reached 7, the sample was allowed to cool, and the product was purified by semi-preparative HPLC method.
  • reaction mixture was then poured into water, pH of the solution was then adjusted to -4.00 using KHS0 4 solution and extracted with CH 2 C1 2 (3 x 100 mL) .
  • the combined organic layer was then washed with water, dried and evaporated on a rotary evaporator to provide a foamy solid of the title compound.
  • the solid was loaded onto a silical gel column as dichloromethane solution and the column was eluted with CH 2 C1 2 -CH 3 0H (98:2) mixture. Fractions containing the compound were collected and evaporated under vacuum to afford the product as a light yellow colored solid (1.68 g, 80%) ; Mp. 135-139 °C .
  • the reaction mixture was then treated with water (10 mL) and evaporated on a rotary evaporator to remove the solvent to afford a thick paste.
  • the paste was then loaded onto a silica gel column as dichloromethane solution and the column was eluted with CH 2 C1 2 -CH 3 0H (95:5) mixture. The fractions with compound were collected and evaporated to provide the product as an off white solid in 88.8% (2.80 g) yield.
  • Trifluoroacetic acid (5 mL) was added to a solution of TrimethyllO- [ ( (N-t-Boc-isoleucinyl) -N- (4-aminophenyl)aceta ⁇ mido] -1,4, 7, lO-tetraazacyclododecane-1, 4, 7-triacetate (2.00 g, 2.64 mmol) in CH 2 C1 2 (10 mL) and stirred at room temperature for 30 min. The reaction mixture on evaporation provided a thick paste which on trituration with dry ether gave the product as an off white solid in near quantitative yield.
  • GdCl 3 (3.5 mL of 9.945 mM, 0.0348 mmol) was mixed with 10- [ ( (N-for-L-methionyl-L-leucyl-L-phenylalanyl-L-isoleucin- yl) -N- (4-aminophenyl) acetamido] -1,4,7, 10-tetraazacyclodode- cane-1,4, 7-triacetic acid (40 mg, 0.0319 mmol) .
  • the reaction mixture was heated at 40°C.
  • the pH of the reaction mixture was raised slowly to 7, while the reaction mixture was heated.
  • the product was isolated by semi-preparative HPLC.
  • the gadolinium complex was prepared by the method outlined in example 6C . Yield 75%.
  • GdCl 3 (0.93 mL of a 49.7 mM solution, 0.0462 mmol) was mixed with 10- [ ( (N-formyl-L-methionyl-L-leucyl-L-phenyl- alanyl-glycyl-glycyl-glycyl-glycyl-glycyl) -N- (4-aminophenyl) acetamido] -1,4,7, 10-tetraazacyclododecane-l, 4, 7-triacetic acid as triethylammonium salt (64.82 mg, 0.04665 mmol) in water. The reaction mixture was heated at 50°C.
  • GdCl 3 (0.65 mL of a 49.7 mM solution, 0.0323 mmol) was mixed with 10- [ ( (N-L-methionyl-L-leucyl-L-phenylalanyl- glycyl-glycyl-glycyl-glycyl-glycyl) -N- (4-aminophenyl) -acetamido] - 1, 4, 7 , 10-tetraazacyclododecane-l, 4, 7-triacetic acid (43.3 mg, 0.0319 mmol) in water.
  • the reaction mixture was heated at 50°C for several hours.
  • the pH of the reaction mixture was raised slowly to 7.5, while the reaction mixture was heated.
  • GdCl 3 (0.74 mL of a 49.7 mM solution, 0.0367 mmol) was mixed with 10- [( (t-Boc-methionyl-L-leucyl-L-phenylalanyl- glycyl-glycyl-glycyl-glycyl-glycyl) -N- (4-aminophenyl) -acetamido] - 1, 4, 7, 10-tetraazacyclododecane-l, 4, 7-triacetic acid (53.10 mg, 0.0367 mmol) in 3.0 mL water.
  • the reaction mixture was heated at 50°C.
  • the pH of the reaction mixture was raised slowly to 7, while the reaction mixture was heated.
  • Tri-t-butyl-1,4,7, 10-tetraazacyclododecane-l,4,7-tri- acetate hydrochloride (3.3 g, 5.5 mmol) and K 2 C0 3 (3 g) were added in dimethylacetamide (25 ml) .
  • p-n-chloroacetyl- phenylacetic acid (1.1 g, 5 mmol) in dimethylacetamide (10 ml) was added and the reaction mixture was stirred at 45° C for 29 h. The reaction mixture was filtered and evaporated to dryness. The residue was purified by preparative HPLC to obtain the product (1.64 g, yield 46.5 %) .
  • Tri-t-butyl 10- [ (4-carboxy ethylpheny1) acetamido] -1, 4, 7 , 10-tetraazacyclododecane-l, 4, 7-triacetate (105 mg, 60% purity) and carbonyldiimidazole (16.2 mg, 0.1 mmol) were mixed in DMF 0.5 ml and stirred for 10 min under nitrogen atmosphere.
  • N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys (61.8 mg, 0.075 mmol) mixed with Et 3 N (1:1 ratio) in DMF (1 ml) was added. The reaction mixture was then stirred at room temperature for 22 h under nitrogen atmosphere. The reaction mixture was evaporated to dryness.
  • PMNs rabbit polymorphonuclear leukocytes isolated from peritoneal exudate. Isolated PMNs were diluted to 1 x 10 cells/mL in assay buffer, which was varied initially in an effort to identify conditions that optimized total binding.
  • the assay buffer contained 15 mM Hepes, pH 7.3, 148 mM NaCl, 0.15 mM CaCl 2 , and 0.1% BSA (fatty acid free) . Because of the low solubility of native and Gd- (IPA-D03A) -fnLLFnLYK, these two peptides were solubilized in buffer containing 1.5% BSA.
  • the radioligand standard fML 3 HF was diluted in the standardized assay buffer and 25 L was placed into polypropylene tubes containing test chemotactic peptide (25 mL) at dilutions of 10 " M to 10 " M, depending on published dissociation constants of the various test peptides. Tubes containing the peptide mixture were placed in a 15°C water bath and allowed to equilibrate. To each tube, 100 mL of PMNs (1 x 10 ) were added to initiate the reaction. After 45 min at 15°C, the reaction was terminated with the addition of 3 mL ice-cold assay buffer. Cells were collected by vacuum filtration over GF/C filters. Filters were washed 2x with 3 mL each of assay buffer and were then placed in Eco-Scint for liquid scintillation counting. Specific binding (%) was calculated as:
  • FIG. 1 shows the results for five native chemotatic peptides - fMLF, fnLLF, fMLFK, fnLLFnLYKDDD and fnLLFnLYK (open symbols) - and five Gd-labeled derivatives (closed symbols) .As shown in Fig l.both Gd- labeled and unlabeled chemotactic peptides compete with fML 3 HF.
  • the peptides fall into two groups with the native peptide fMLF having the highest binding affinity of the main group and with a minor group of low binding affinity consisting of two gadolinium peptides, Gd-fMLF and Gd-fnLLF.
  • the binding affinities obtained with the 'Ligand' program are summarized in Table I.
  • Table 1 also shows that chelation produced a much greater reduction in the binding affinity of the tripeptides than in the tetrapeptides .
  • Gd- (IPA-D03A) - fMLFK has a binding affinity that is of the same order of magnitude as fMLF.
  • Fig. 2 where the log 1Q of the association constant (K a ) is plotted against the Gd - formyl interatomic distance, determined from computer-generated molecular models (Hyperchem) .
  • the 'native' peptides are also shown on this graph, with their position on the x-axis determined by the Gd-formyl interatomic distance of their Gd-APA-D03A derivatives.
  • Logio ⁇ a as a function of Gd-formyl interatomic distance is shown in Fig. 2.
  • This graph shows that binding strength (of the Gd-peptides to PMNs) increases as the distance between the gadolinium chelate and formyl group (based on extended linear arrangement) of the chemotactic peptide also increases.
  • the native peptides show a less distinct trend of binding vs length.
  • the binding of Gd(IPA-D03A) -f-MLFK is greater than its native peptide.
  • Example 15 A. Real-Time Video Analysis of PMN Chemotaxis in Response to fMLF and G (IPA-D03A) -f-MLFK Ideally, a Gd-chelated chemotactic peptide should serve as an antagonist for the receptor, since avoiding activation would conserve PMNs labeled in the blood for extravassation and chemokinesis to the site of infection.
  • the data confirm that the native peptide fMLF is an agonist and that the Gd-labeled derivatives Gd- fnLLFnLYK and Gd-fMLFK are also agonists.
  • concentration at which PMNs maximally orient in the Zigmond chamber corresponds to the approximate K d of the receptors on the PMNs for the particular peptide
  • the Zigmond chamber results also confirm the Kds of the native and Gd-labeled peptides as determined from competitive binding studies.
  • _ 9 receptors have Kd values of -10 M, three orders of magnitude lower than found with mouse PMNs .
  • Example 16 A. Tn Vitro MRI Experiments with Gd(IPA-D03A) fnLLFnlKY. Gd(IPA-D03A) -fMLFK. and ProHance®
  • Test tube MRI experiments used rabbit peritoneal PMNs rather than rat peripheral blood PMNs, (to avoid inclusion of platelets and other types of leukocytes) .
  • ProHance was included as a non-protein Gd-control and a negative control which contained no PMNs in order to detect images produced by gelled/precipitated Gd-peptide was also included.
  • a careful analysis of the distribution of material between the cells and the supernatant was also performed.
  • the quantity of radiolabeled and unlabeled Gd-peptide was calculated to achieve the desired concentrations in 1 mL final volume containing the peptide, cells, and buffer. Binding experiment may be carried out at room temperature for 30 min on a rocker plate. Cells were collected by centrifugation at 500 xg for 2 min. The test tubes were placed into the magnet and the pellet was imaged. The supernatant liquid was removed and cells were washed twice with buffer and the pellet imaged again. The total Gd- peptide concentration in the pellet and washes was determined by the addition of tracer quantities of Gd- peptide. The specific activity in cpm/mmole was known for each experimental sample.
  • Gd-fnLLFnLYK (cold), [400 ⁇ M] in 1.5% BSA (fatty acid free) /assay buffer.
  • the signal intensity of the pellet as a function of the pellet concentration of gadolinium complex appears to reach a maximum of about 4 (Fig. 3) .
  • the two peptides, Gd(IPA-D03A) -fnLLFnLYK and Gd (IPA-D03 ) -fMLFY are similar and have up to 4 times the signal for the same concentration of ProHance Th s s presumably a function of the different relaxivities of these two classes of compounds.
  • the highest signal intensity with ProHance was 1.6 at an initial concentration of 1050 mM (this signal intensity was achieved with 1 mM Gd- fnLLFnLYK) .
  • Gd- ( IPA-D03A) -fnLLFnLYK and PMNs should provide insight into the mechanism of uptake and allow us to assess if uptake is receptor mediated or if nonspecific interactions lead to nonspecific associations with the PMN surface.
  • the experiment (given below) is in agreement that Gd(IPA-D03A) -fnLLFnLYK in rabbit PMNs internalized as compared to Gd(IPA-D03A) -f-MLFK Association (molecules/cell) of Gd(IPA-D03A) -fnLLFnLYK and Gd(IPA-D03A) - f-MLFK with rabbit peritoneal PMNs is shown in Fig.4.
  • the left rectus muscle of nine male Sprague-Dawley rats (250-350 g) were inoculated with human E. Coli (10 9 ,) 20 hours prior to the study.
  • the rats were anaesthetized with sodium pentobarbital , and 5 mCi (3.8 x 10 moles) of Gd- 153 (for-Nle-Leu-Phe-Nle-Tyr- (N(g) )Lys-APA-D03A) was co-
  • D03A) -fnLLFnLYK showed that the %ID increased with time up to 0.16% at 6 h post injection. This is about 4 times the amount of Gd(IPA-D03A) -fnLLFnLYK found in the normal leg and 2 times the amount of m Tc-DTPA in the same infected leg. Uptake of 99 Tc-DTPA and Gd-fnLLFnLYK in infected and noninfected muscle is shown in Fig.5.
  • the relaxivity measurements were carried out by measuring T ls of several solution of different concentrations of Gd (IPA-D03A) -f-nLI,FnLYK at 20 MHz and at 37°C.
  • the experiments were carried out in different media, e.g., bis-tris buffer, human plasma, rat plasma, and rat whole blood. In all cases, the pH of the medium was adjusted to 7.4 with bis-tris buffer and ionic strength at 0.1.
  • rat plasma experiment two types of experiments were carried out. In the first experiment, the plasma was first separated from whole blood, samples of Gd (IPA-D03A) - fnLLFnLYK with different concentration were prepared, and T x s were measured.
  • Measurements by means of a particle counter apparatus indicated that, depending on the run, the average particle size was in the range 50 - 100 nm ( ⁇ 20-40%) .
  • Example 26 Preparat ion of the S-acetyl thioacetate of the hexapeptide f -Nle-Leu-Phe-Nle-Tyr-Lys .
  • the hexapeptide ( 6 ⁇ ol ) was dissolved in DMSO to make a concentration of 10 mg/ml and 1 equiv . of N- hydroxysuccinimidyl-S-thioacetate ( SATA) in the form of a 14 mg/ml DMSO so lut ion was admixed ; therea f t er , N- ethylmorpholine was added to bring the appsarent pH to 8
  • the hexapeptide S-acetylthioacetate ( 2 mg ) in DMSO solution was added to a HEPES solution of the SBPA- maleininimide derivatized iron oxide particles (2.5 mg Fe) prepared as disclosed in Example 25.
  • the HEPES buffer used also contained EDTA (2.5 mM) and hydroxylamine (50 mM) ; the hydroxylamine acts as a deacetylation reagent.
  • the mixture was kept at room temperature for 2 hrs, then the iron oxide particles were isolated by centrigugation, resuspended in buffer and extensively dialyzed for purification.
  • Example 28 Testing of the magnetite-chemoattractant peptide conjugate affinity with granulocytes .
  • Tissue injury and inflammation by bacterial infection induce granulocyte (PMN) and mononuclear phagocyte formation in response to the chemotactic peptides produced on the infection site.
  • Granulocytes are known to produce O 2 " " radicals which can be assayed by the reduction of the dye Nitro Blue Tetrazolium (NBT) absorbing at 540 nm.
  • Neutrophils were isolated from fresh buffy coats and cultured according to Magn.Res.Imaging 13 (1995), 393-400.
  • the rate of NBT reduction was measured directly in the cells cultured in microplates .
  • the assay media comprised 150 ⁇ l of NBT solution (2 mg/ml) ; 100 ⁇ l of the conjugate solution (this including various concentration samples) ; to which were added 50 ⁇ l of PMN (2.5-10 5 cells) lodoacetamide (an inhibitor of oxidative burst) was added to the medium in the photometer reference well (to 10 mM final concentration) , the cells having been preincubated for 10 min at 37°C in 10 mM iodoacetamide.
  • the details of the assay are disclosed in "Methods in Enzymology" 132 (1995) , 417.
  • Example 29 Conjugation of Gd(III) complexes of linear polyaminocarboxylates (e.g. DTPA and DTPA.BMA) with chemotactic peptides.
  • linear polyaminocarboxylates e.g. DTPA and DTPA.BMA
  • the isothicyanate DTPA derivative, (ITC-DTPA, Structure I) is synthesized according to M.W. Brechbiel et al. in Inorg. Chem. 25 (1986), 2772 or Bioconj . Chem. 2 (1991), 187.
  • the Gd(III) complex of ITC-DTPA is prepared in situ by mixing equimolecular quantities of the ligand and GdC13 in a suitable solvent ad raising the pH of the mixture slowly.
  • the conjugation of the chelate and the chemotactic peptide (see the previous Examples), and its final purification, is achieved according to the method given in the next Example for the conjugation of Gd(IPA-D03A) .
  • ITC- DTPA is made by usual means from the corresponding aniline (Structure II)
  • the amino group of the aniline derivative is protected, for instance with BOC; then (2) the pentaacetic acid is converted to its cyclic bisanhydride according to W.C. Eckelman et al . in J.Phar .Sci .64 (1975), 704.
  • the bisanhydride is then reacted (3) with an alkylamine R H 2 (in which R is C ⁇ _ 6 Alk) according to US-A-5, 087, 439 (S.C. Quay) incorporated herein by reference to provide a bisalkylamide the protecting group is then removed to give the compound of Structure III .
  • the following steps are involved: (1) The reaction of p-nitrobenzoyl chloride with the chemotactic peptide; (2) the catalytic hydrogenation of the nitro group to the corresponding aniline, and (3) reaction of the latter with thiophosgene to give the isocyanato derivative.
  • the activated peptide is then reacted in the presence of a base with an amino-terminated dendrimer (see Magnetic Resonance in Medicine 31 (1994) to achieve reaction (thiourea bonds) on a few dendrimer amino-functions .
  • the remaining dendrimer end sites can be conjugated with the activated Gd-chelated moieties, e.g. Gd(IPA-D03A) to provide a polytargeted- polychelated dendrimer, i.e an example whith multiple signal generators attached to peptides that can bind to receptors.
  • Dextran coated magnetite particles are disclosed in US-A-5,219, 554 (E.V. Groman et al . ) .
  • the derivative l-[(4- isothiocyanatophenethyl)amino]dihydrodextran (Structure IV) is synthesized according to J.S. Mann et al. in Bioconj .Chem. 3 (1992) , 154 and this activated dextran can be bound to a chemotactic peptide with desired length and distribution of aminoacids using the conjugation procedure oulined in previous Examples.
  • the peptide derivatized dextran can be used to coat magnetite particles, thus providing magnetite particles targeted toward sites of production of receptors.

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Abstract

L'invention concerne des substances chimiotactiques marquées magnétiquement, par exemple, des peptides chimiotactiques, pour effectuer des investigations et des diagnostics sur des sites ou des organes malades ou atteints chez des humains ou chez des animaux. Les peptides chimiotactiques ont la formule générale N(X)-Y-Leu-Phe-Z-A-W. Dans cette formule, (X) est H ou un groupe protecteur tel que le formyle, acétyle, t-Boc et similaire; Y est Nle ou Met; Z est une liaison chimique, un acide aminé ou un oligopeptide, tel que (N(η))Lys, Ile, Asp, Nle-Tyr-Lys ou (Gly)n, n étant compris entre 1 et 4; A est un groupe de liaison; et W est un chélate macrocyclique paramagnétique modifié chimiquement de manière appropriée ou une particule magnétique. L'invention concerne également des compositions administrables et acceptables sur le plan physiologique, contenant les substances chimiotactiques marquées, des procédés pour préparer ces substances, des formulations les contenant et des procédés d'utilisation de ces formulations pour détecter, localiser et diagnostiquer, dans l'organisme, des sites infectés ou atteints d'une inflammation, ou d'autres traumatismes.
EP96932764A 1995-10-19 1996-10-18 Substances chimiotactiques marquees magnetiquement comme agents de contraste pour l'imagerie par rmn de tissus vivants cibles Withdrawn EP0814849A1 (fr)

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JP2002500242A (ja) * 1997-12-24 2002-01-08 ヘンケル・コマンディットゲゼルシャフト・アウフ・アクチエン 過酸素化合物の漂白効果を強化するためのデンドリマー配位子を有する遷移金属錯体の使用
US6872536B1 (en) * 1998-11-10 2005-03-29 Bracco Imaging S.P.A. Chemotactic peptide antagonists for imaging sites of inflammation
JP4511075B2 (ja) * 2001-04-23 2010-07-28 レンゴー株式会社 含硫ウレタン系デンドリマー及びその製造方法
DE60318466T2 (de) 2002-09-27 2008-09-18 Nihon Medi-Physics Co., Ltd. An leukozyten bindende verbindung und diese verbindung im markierten zustand als wirkstoff enthaltende medizinische zusammensetzung
US8175678B2 (en) * 2005-09-13 2012-05-08 Koninklijke Philips Electronics N.V. Multiple contrast agent injection for imaging
KR101502180B1 (ko) * 2006-08-30 2015-03-12 지이 헬스케어 에이에스 동적 핵 분극화 방법 및 이러한 방법에서 사용되는 화합물 및 조성물
US8414926B1 (en) 2006-09-12 2013-04-09 University Of South Florida Nanoparticles with covalently bound surfactant for drug delivery
US10086095B2 (en) 2012-10-09 2018-10-02 Case Western Reserve University Rod-shaped plant virus nanoparticles as imaging agent platforms
US11193111B2 (en) 2013-01-17 2021-12-07 Case Western Reserve University Viral nanoparticle multimers
WO2015123654A1 (fr) 2014-02-17 2015-08-20 The Cleveland Clinic Foundation Nanoparticules amine-passivées pour le traitement et l'imagerie du cancer
US11433123B2 (en) 2016-07-21 2022-09-06 Case Western Reserve University Sustained release cowpea mosaic virus or virus-like particle therapeutic constructs for the treatment of cancer
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US4554088A (en) * 1983-05-12 1985-11-19 Advanced Magnetics Inc. Magnetic particles for use in separations
US4827945A (en) * 1986-07-03 1989-05-09 Advanced Magnetics, Incorporated Biologically degradable superparamagnetic materials for use in clinical applications
NZ314680A (en) * 1989-05-09 2000-08-25 Ortho Pharma Corp Treating localised infection or inflammation with a therapeutically conjugated chemotactic peptide
DE3919873A1 (de) * 1989-06-19 1990-12-20 Behringwerke Ag Magnetische protein-konjugate, verfahren zu ihrer herstellung und ihre verwendung
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