Nothing Special   »   [go: up one dir, main page]

WO1998016256A1 - Imagerie par resonance magnetique utilisee detecter un thrombus - Google Patents

Imagerie par resonance magnetique utilisee detecter un thrombus Download PDF

Info

Publication number
WO1998016256A1
WO1998016256A1 PCT/US1997/018412 US9718412W WO9816256A1 WO 1998016256 A1 WO1998016256 A1 WO 1998016256A1 US 9718412 W US9718412 W US 9718412W WO 9816256 A1 WO9816256 A1 WO 9816256A1
Authority
WO
WIPO (PCT)
Prior art keywords
contrast agent
mri contrast
amino acid
group
seq
Prior art date
Application number
PCT/US1997/018412
Other languages
English (en)
Inventor
James O. Tolley
Paul Curtis Mazur
Daniel G. Mullen
Michael D. Pierschbacher
Juerg Tschopp
Original Assignee
The Burnham Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Burnham Institute filed Critical The Burnham Institute
Priority to AU47539/97A priority Critical patent/AU4753997A/en
Publication of WO1998016256A1 publication Critical patent/WO1998016256A1/fr

Links

Classifications

    • 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

Definitions

  • the present invention relates generally to the field of diagnostic imaging and, more specifically, to magnetic resonance imaging for thrombi.
  • Cardiovascular disease is a devastating problem in the United States and worldwide. Diagnosis of thrombus is critical to the detection and management of cardiovascular disease. Major indications for thrombus imaging include coronary artery occlusion, deep vein thrombus, pulmonary emboli, stroke, and other such maladies. The U.S. and European patient populations with a need for diagnostic imaging procedures to be performed related to these maladies number in the tens of millions per year. Several previous methods attempted to provide effective diagnostic imaging of thrombi, but each of these methods has undesirable aspects.
  • Magnetic resonance imaging for diagnostic purposes has previously been used. However, such methods only detected thrombi as ambiguous negative signal areas, which can be misdiagnosed as anatomical irregularities.
  • new software is improving the thrombus-imaging capability of MRI, MRI agents are currently unavailable that can localize a thrombus by rendering a positive image.
  • Another previous method for detecting thrombus was to use radiolabeled small synthetic peptides that specifically home to thrombi. Compared to antibody linked contrast agents, such peptides have shorter clearance times, reduced cross-reactivity, and are more selective for binding to thrombi. Previously, however, such peptides have only been used in conjunction with radioimmunoscintigraphy, which is a technique that uses a ⁇ -camera to detect radioactively labeled agents that are administered to patients.
  • radioimmunoscintigraphy which is a technique that uses a ⁇ -camera to detect radioactively labeled agents that are administered to patients.
  • One study used a peptide labeled with 99ra ⁇ c to detect deep venous thrombi in an animal model and in a preliminary human trial .
  • the present invention satisfies these needs and provides related advantages as well .
  • the present invention provides MRI contrast agents useful for detecting thrombus.
  • the MRI contrast agents comprise a chelator and a chemical compound capable of binding to an integrin.
  • the MRI contrast agent can, though need not, additionally contain a paramagnetic metal.
  • the chelator should be capable of complexing the metal and, if present, does complex the metal. Additionally, the chelator is also coupled to the integrin-binding chemical compound.
  • the integrin-binding compound can be any variety of compounds which bind the RGD site of an integrin and, therefor, have cell attachment activity.
  • the compounds can be linear or cyclic and, generally, will be X-G-D-containing (SEQ ID)
  • the integrin-binding compound has the general structure Y-X 1 -X 2 -G-D-X 3 -X 4 -Z (SEQ ID NO. 1) .
  • Y is an amino-terminal group
  • X x is zero to five amino acids
  • X 2 is a hydrophobic amino acid or a positively charged amino acid
  • X 3 is a hydrophobic amino acid
  • an amino acid that provides an ionic interaction with an integrin receptor or an amino acid that provides a hydrogen bond with an integrin receptor
  • X 4 is zero to five amino acids
  • Z is a carboxy-terminal group.
  • the compounds are cyclized through a bridge between two amino acids, excepting the G and D amino acids in the formation of the bridge.
  • the present invention also provides a composition useful as a medicament comprising an MRI contrast agent and a physiologically acceptable carrier.
  • the present invention further provides a method for detecting thrombus in a subject by administering to a subject an effective amount of an MRI contrast agent, and when the MRI agent homes to a thrombus, detecting the thrombus using magnetic resonance imaging.
  • the present invention provides MRI contrast agents useful for detecting thrombus.
  • the subject MRI contrast agents comprise at least a chelator coupled to and an integrin-binding chemical compound.
  • the agents can further comprise a paramagnetic metal.
  • the chelator is coupled to the integrin-binding chemical compound. It is capable of complexing a paramagnetic metal, and when present, does complex the metal.
  • a paramagnetic metal and when present, does complex the metal.
  • chelator is a molecule with one or more polar groups that act as a ligand for, and can complex with, the metal.
  • the phrase "capable of complexing a paramagnetic metal” means the chelator contains chemical groups which have the ability to complex a paramagnetic metal by noncovalent forces .
  • the phrase chelator complexes the metal means an aggregate of the chelator and metal ion held together by noncovalent forces .
  • Chelators for metals are well known in the art, and include acids with methylene phosphonic acid groups, methylene carbohydroxamic acid groups, carboxyethylidene groups, or carboxymethylene groups.
  • Examples of chelators include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA) , 1,4, 7, 10-tetraazacyclododecane (DOTA) , ethylenediaminetetraacetic acid (EDTA) and 1,4,8,11- tetraazacyclotetradecane-1, 4,8, 11-tetraacetic acid (TETA) .
  • the chelator of the MRI agent is DTPA.
  • the chelator of the MRI contrast agent is coupled to the integrin-binding chemical compound.
  • the two are preferably coupled through the Y or Z substituents or through an amino acid present in X x or X 4 as described in further detail below.
  • the term "coupled” broadly includes any attachment of the chelator to the compound.
  • the attachment can be covalent or noncovalent, although more usually covalent, as with for example, conjugation using DTPA, described in Example III.
  • the coupling may be direct or, more commonly by indirect means of using a coupling or cross-linking reagent.
  • any one of several known intermolecular cross-linking reagents can be used, including N-succinimidyl 3- (2 -pyridyldithio) propionate (SPDP) or N-N' - (1, 3 -phenylene) bismalemide (both highly specific for sulfhydryls, forming irreversible linkages); N-N 1 -ethylene-bis- (iodoacetamide) or other such reagents having 6 and 11 carbon methylene bridges (relatively specific for sulfhydryl groups); 1 , 5-difluoro-2 , 4- dinitrobenzene (forming irreversible linkages with amino and tyrosine groups); p,p' -difluoro-m- m ' dinitrodiphenylsulfone (forming irreversible cross- linkages with amino and phenolic groups) ; dimethyladipimidate (specific for amino groups) ; phenyl- 2-4-dis
  • the chelator and the coupling method will be selected to preserve the paramagneticity and minimize the toxicity of the paramagnetic metal, while preserving the specificity of integrin binding. Such considerations are discussed in Tweedle et al . , Magnetic Resonance Imaging, vol. 1 (Eds. Partain et al . , W.B. Saunders Co., 2nd ed.) pp. 798-806, 1988. INTEGRIN-BINDING COMPOUND
  • the integrin-compound used in the MRI agents of the present invention can be any small, synthetic compound which binds the RGD site of an integrin and, therefore, has cell attachment activity. Such small compounds have rapid clearance times, are sufficiently stable for diagnostic purposes, and have high affinity for integrins, making their application in MRI contrast agents particularly advantageous.
  • the compounds are X-G-D- containing (SEQ ID NO.
  • NO. 23 is meant any molecule which has at least the sequence X-G-D.
  • the chemical compound enables the MRI contrast agent to home to integrins on the surface of activated platelets, thereby enhancing MRI visualization of the thrombus.
  • a critical aspect of the present invention is the ability of compounds of the MRI contrast agent to bind to integrins and do so with sufficient affinity.
  • the structure of the integrin- binding compounds can vary substantially as described above . What is important is that the compound bind the RGD site of an integrin and have sufficient affinity for one or more integrins as to be advantageous in their application to magnetic resonance imaging.
  • a dose to allow a compound to bind with an affinity in the nanomolar range, meaning a range submicromolar, is of the appropriate affinity for MRI.
  • the affinity is 1 to 10 nanomolar.
  • Affinities of integrin-binding compounds can be routinely determined by methods well known in the art and by such methods as described in Example II.
  • the integrin-binding compounds have the general structure Y-
  • Y is an amino-terminal group
  • X x is zero to five amino acids
  • X 2 is a hydrophobic amino acid or a positively charged amino acid
  • X 3 is a hydrophobic amino acid
  • an amino acid that provides an ionic interaction with an integrin receptor or an amino acid that provides a hydrogen bond with an integrin receptor
  • X 4 is zero to five amino acids
  • Z is a carboxy-terminal group.
  • the compounds are cyclized through a bridge between two amino acids, excepting the G and D amino acids in the formation of the bridge.
  • amino acid is meant in its broadest sense to include naturally occurring proteogenic amino acids, D-amino acids and imino acids, as well as non-naturally occurring or chemically modified amino acids and imino acids and amino acid mimics.
  • proteogenic indicates that the amino acid can be incorporated into a protein in a cell through well known metabolic pathways.
  • a reference herein to an amino acid includes, for example, naturally occurring proteogenic L-amino acids and D-amino acids.
  • the choice of including an L- or a D-amino acid depends, in part, on the desired characteristics of the compound of the MRI contrast agent. For example, the incorporation of one or more D-amino acids can confer increased stability on the MRI contrast agent in vivo and can allow the agent to remain in the body for an extended period of time.
  • amino acid also encompasses chemically modified amino acids such as amino acid analogs, naturally occurring non-proteogenic amino acids such as norleucine and chemically synthesized compounds having properties known in the art to be characteristic of an amino acid.
  • amino acids and derivatives thereof used herein are as follows :
  • a “mimic” or “mimetic” of an amino acid means an amino acid analog or non-amino acid moiety that has the same or similar functional characteristic of a given amino acid.
  • an amino acid mimic of a hydrophobic amino acid is one that is non-polar and retains hydrophobicity, generally by containing an aliphatic functional group.
  • an arginine mimic can be an arginine analog containing a side chain with a positive charge at physiological pH, which is characteristic of the guanidinium side chain of arginine .
  • Amino acid mimics also encompass modifications to the chemical or peptide bonds and backbone. Such modifications can be made to the amino acid, amino acid derivative, non-amino acid moiety or a peptide on either side. What is critical is that such modifications mimic a peptide backbone with substantially the same spatial arrangement and distance that is typical for traditional peptide bonds and backbones.
  • An example of such a modification is reducing a carbonyl of the amide peptide backbone to an amine.
  • Several reagents are available and well known for reducing amides to amines, such as those disclosed in Wann et al . , J. Org. Chem. 46:257 (1981) and Raucher et al . , Tetrahedron Lett .. 21:14061 (1980).
  • An amino acid mimic is, therefor, an organic molecule that retains the similar amino acid pharmacophore groups as is present in the corresponding amino acid and which exhibits substantially the same spatial arrangements between functional groups.
  • Substituting amino acid mimics and non- naturally occurring amino acids for naturally occurring amino acids can enhance the overall activity or properties of an individual compound. These alterations to the compound of the MRI contrast agent can enhance the compound's stability to enzymatic breakdown.
  • Y-X 1 -X 2 -G-D-X 3 -X 4 -Z (SEQ ID NO. 1) will now be described in detail, first with reference to the core structure, X 1 -X 2 -G-O-X 3 -X i (SEQ ID NO. 2), followed by a detailed description of the terminal groups, Y and Z.
  • X- L is zero to five amino acids
  • X 2 is a hydrophobic amino acid or a positively charged amino acid
  • X 3 is a hydrophobic amino acid, an amino acid that provides an ionic interaction with an integrin receptor, or an amino acid that provides a hydrogen bond with an integrin receptor
  • X 4 is zero to five amino acids .
  • hydrophobic amino acid is intended to include naturally-occurring hydrophobic amino acids, including Ala, Val, He, Leu, Phe, Tyr and Trp as well as non-naturally-occurring hydrophobic amino acids, including the D-form of the hydrophobic amino acids, amino acid derivatives and amino acid mimics, which are non-polar.
  • Hydrophobic amino acid derivatives and mimetics useful in the invention can have a range of structural types and hydrophobicities . Examples of such include, but are not limited to, Nle, Nve, Cha, Nap, 2-Nal, Cit, Tyr derivatives, Phe derivatives and Trp derivatives.
  • additional hydrophobic amino acids include Chg, t-BuG, Msa, Npg,
  • Tyr derivatives include Tyr-OMe, Tyr-OEt, O- ⁇ -hexyl-Tyr, O- ⁇ -Bu-Tyr, 3,5-diiodo- Tyr and the like.
  • Phe derivatives include p- chloro-Phe, homoPhe, p-nitro-Phe, Phg, p-iodo-Phe, p- amino-Phe and the like.
  • Tryptophan derivatives are, for example, Trp amino acid analogs with a substituted indole ring, substituted with, for example, one or more halogen atoms including iodo, chloro, fluoro, and bromo atom(s), and/or one or more alkyl groups, such as methyl, ethyl, and the like.
  • the term "positively charged amino acid” refers to those amino acids which occur in nature, including Arg, Lys and His, and the D-form of these naturally-occurring amino acids as well as amino acid derivatives and amino acid mimics, which are positively charged. Examples of additional positively charged amino acids include, but are not limited to, Orn and homoArg.
  • an amino acid that provides an ionic interaction with an integrin receptor means any amino acid which interacts with an integrin receptor by attractive forces, preferably through providing a negative charge, as would be provided by such amino acids as Asp, Glu, ⁇ - (1 (2) H-tetrazol-5-yl) -alanine or analogs or mimics thereof such as the sulfonamide derivatives Msa, Psa, Tfsa or other such negatively charged amino acids or mimics thereof .
  • an amino acid that provides a hydrogen bond with an integrin receptor means any amino acid that provides an attractive interaction between its covalently linked hydrogen atom and a neighboring atom or group of atoms in the integrin receptor such as, for example, Asn, Ser, Thr, or mimics or analogs thereof.
  • X 2 and X 3 are both a hydrophobic amino acid and X 4 is one to five amino acids, at least one of which is a positively charged amino acid directly adjacent to X 3 .
  • the hydrophobic amino acid of either X 2 or X 3 are, independently, Ala, t-BuG, Tic, Val , He, Leu, Phe, Tyr, Trp, a Tyr derivative, a Phe derivative, Nle, Nve, Cha, Nap, 2-Nal or Cit ; and X 4 is at least one positively charged amino acid selected from Arg, Lys, His, Orn or homoArg which amino acid is directly adjacent to X 3 .
  • Arg, Lys, His, Orn or homoArg which amino acid is directly adjacent to X 3 .
  • X 2 is Ala, Val, He, Leu, Phe, Tyr, Trp, Nve, Nle, Cha or
  • X 3 is Phe, a Phe derivative, Tyr, a Tyr derivative or Trp; and X 4 contains at least an Arg amino acid residue directly adjacent to X 3 . Even more preferably, X 2 is
  • Ala, Leu or Nle and X 3 is Tyr-OMe.
  • Preferred compounds falling within these formulae include (Nle) GD (Y-OMe) REK- NH 2 (SEQ ID NO. 3); CNPAGD (Y-OMe) RC-NH, (SEQ ID NO. 4); and CNP (Nle) GD (Y-OMe) RC-NH, (SEQ ID NO. 5) .
  • X 2 and X 3 both being a hydrophobic amino acid and X 4 being one to five amino acids, at least one of which is a positively charged amino acid directly adjacent to X 3
  • the compound encompassed by these formulae have a particular affinity for the ⁇ IIb ⁇ 3 integrin receptor.
  • they have a higher affinity for ⁇ IIb ⁇ 3 at low calcium concentration, which is typical at the site of a clot as compared to « ⁇ i b ⁇ 3 at higher, physiological calcium concentration found elsewhere throughout the blood. Further characterization of these compounds is provided in Example II.
  • X 2 is a positively charged amino acid and X 3 is a hydrophobic amino acid.
  • X 2 is Arg, Lys, His, Orn or homo Arg; and X 3 is Ala, t-BuG, Tic, Val, He, Leu, Phe, Tyr, Trp, a Tyr derivative, a Phe derivative, Nle, Nve, Cha, Nap, 2-Nal or Cit .
  • X 2 is Arg and X 3 is Phe, a Phe derivative, Tyr, a Tyr derivative or Trp.
  • the compound selectively binds to IIb ⁇ 3 and X 3 is a Tyr derivative and, more preferably, Tyr-OMe or Tyr- O- ⁇ -Bu.
  • Preferred compounds falling within these formulae include CNPRGD (Y-OMe) RC-NH-, (SEQ ID NO . 6) ; CNPRGD (Y-OMe) ACR (SEQ ID NO. 7) ; CNPRGD (Y-OMe) PC-NH, (SEQ ID NO. 8); CNPRGD (Y-OMe) ECR (SEQ ID NO. 9); and CNPRGD (Y- Q-n-Bu)RC-NH, (SEQ ID NO. 10) .
  • X 2 is a positively charged amino acid
  • X 3 is either an amino acid that provides an ionic interaction with an integrin receptor or, alternatively, is an amino acid that provides a hydrogen bond with an integrin receptor
  • X 4 is one to five amino acids, at least one of which is either a hydrophobic amino acid or a positively charged amino acid.
  • X 2 is Arg, Lys, His, Orn or homoArg
  • X 3 is
  • X 4 is at least one amino acid selected from Ala, tBuG,
  • Tic Pro, Val, He, Leu, Phe, Tyr, Trp, a Tyr derivative, a Phe derivative, Nle, Nve, Cha, Nap, 2-Nal, Cit, Arg, Lys, His, Orn, or homoArg. More preferably, X 2 is Arg;
  • X 3 is Asp, Asn, Ser or Thr; and X 4 is at least one amino acid selected from tBuG, Phe, Tic, Pro or Arg.
  • integrin-binding compounds of the above formulae corresponding to SEQ ID NO. 1 are selective for one or more integrin receptors, whereas others are not selective and bind multiple integrins.
  • the terms "selective" and “selectively” mean that the integrin-binding compound used in the MRI contrast agent of the present invention preferentially binds to an integrin receptor with greater affinity than it binds to other integrins. It is recognized that a compound of the invention can specifically bind more than one integrin, yet still be considered selective because it does not non-specifically bind any integrin.
  • the binding characteristics and selectively for one or more integrins can readily be determined, as described in Example II and other means well known in the art.
  • the compound binds multiple integrins, but does not selectively bind any integrin.
  • An example of a compound which binds multiple integrins, but not any one selectively, is (Pen) FARGDS (Tic) C-NH, (SEQ ID NO. 11) .
  • the compounds selectively bind multiple integrins, such G(Pen)RARGDNPCA (SEQ ID NO. 12) ; which selectively bind Ilb ⁇ 3 and 5 ⁇ 1# and (Pen) (Y-OMe) ARGDN (Tic) C-NH, (SEQ ID NO. 13) which selectively binds v ⁇ 3 , v ⁇ 5 , and a 5 ⁇ > 1 .
  • G(Pen)RARGDNPCA SEQ ID NO. 12
  • Ilb ⁇ 3 and 5 ⁇ 1# and (Pen) (Y-OMe) ARGDN (Tic) C-NH
  • SEQ ID NO. 13 which selectively binds v ⁇ 3 , v ⁇ 5 , and a 5 ⁇ > 1 .
  • the invention also encompases compounds which selectively bind to one integrin, such as those described above which are selective for ⁇ IIb ⁇ 3 , as well as others.
  • An example of other such compounds includes one which selectively binds to v ⁇ 3 and has the structure (Mpa) RGDD ( -BuG) CK-NH-, (SEQ ID NO . 14) .
  • compounds preferably having X 3 as a small, aliphatic amino acid containing a hydroxyl or amide group can selectively bind to ⁇ v ⁇ s .
  • X 3 is preferalby Ser, Thr, Asn or Gin and specific examples are those compounds having the structures G(Pen) FRGDSFCA (SEQ ID NO.
  • the substituents X ⁇ and X 4 are, independently, zero to five amino acids. When present and between one and five amino acids, X and X 4 can contain any naturally-occurring or non-naturally- occurring amino acids, including the D-form of the amino acids, amino acid derivatives and amino acid mimics, so long as the desired function and activity of the peptide is maintained.
  • X and/or X are one to five amino acids, wherein at least one amino acid is specified to meet a certain criteria.
  • X 4 must have a positively charged amino acid directly adjacent to X 3 .
  • X 4 can contain either a hydrophobic or a positively charged amino acid also adjacent to X 3 .
  • X ⁇ also preferably contains an amino acid capable of forming a bridge with another amino acid within the chemical compound.
  • the bridging amino acid is Cys, Pen, Mpa, Pmp, Pmc, Pas, Glu, Asp, Lys or Orn.
  • X 4 also preferably contains an amino acid capable of forming a bridge with another amino acid within the chemical compound.
  • the bridging amino acid of X 4 is
  • the length of the integrin-binding compounds can vary, but generally they are small synthetic compounds on the order of about 14 amino acids or shorter. With those compassed encompassed by the formula of SEQ ID NO. 1, the compounds will of course vary in length depending on the substitutions for X ⁇ and X 4 . The compounds must at least comprise X 2 GDX 3 (SEQ ID NO. 18) and, therefore, are at least 4 amino acids, or the equivalent, in length.
  • the cyclic peptides can be as long as 14 amino acid residues. It should, however, be appreciated to those of skill in the art that the addition of one to a few amino acids to increase the peptide length beyond 14 residues could likely yield similar activity and, therefore, would not depart from the spirit and concept of the present invention.
  • the length of the compounds will also depend on whether either or both X and X 4 are present.
  • compounds encompassed within the present invention include those of the formulas Y-X 1 -X 2 - GD-X 3 -Z (SEQ ID NO. 19) and Y-X 2 -GD-X 3 -X 4 -Z (SEQ ID NO.
  • the length of the compounds are preferably less than about 10 amino acids, and more preferably, less than about 7 amino acids in length.
  • the Y substituent in the above formula is an amino-terminal group.
  • amino-terminal group refers to the amino group common to the amino terminus of a peptide (i.e., NH 2 and therfore Y is a hydrogen atom) or an amino group commonly employed to block or protect the amino functionality, for example, from enzymatic degradation.
  • Examples of such amino- terminal groups used to block or protect the amino functionality include COCH 3 , CO-alkyl, an alkyl group, CH 2 Ph, COPh, COOCH 2 Ph, COO-alkyl and CO-alkyl-NH 2 .
  • the abbreviation "Ph” indicates a "phenyl” group (C 6 H 5 ) .
  • alkyl or "alkyl group” mean a C x to C 6 alkyl, including such radicals as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, amyl , hexyl and the like.
  • the Z substituent is a "carboxy-terminal group" which refers to the carboxyl group common to the carboxy terminus of a peptide (i.e., COOH and therfore Z is a hydoxyl group) or a carboxy group commonly employed to block or protect the carboxy functionality, for example, from enzymatic degradation.
  • carboxy- terminal groups used to block or protect the carboxy functionality include NH 2 , NH-NH 2 , O-alkyl, SH, S-alkyl, N-alkyl, NHCH 2 Ph and NH-alkyl-NH 2 .
  • the abbreviation "Ph” and term “alkyl” are the same as defined above with reference to the Y substituent.
  • a compound of the MRI contrast agent can be modified at either or both the amino or carboxyl terminus.
  • modifications should take into account tht there must a terminus or side chain of an amino acid capable of coupling to the chelator as described above.
  • Y is a hydrogen atom
  • the unprotected primary amine at the amino terminus can be used for coupling.
  • amino-terminus can be protected, for example with and acetyl group, and the carboxy terminus can be modified such that a free primary amine is available for coupling to the chelator, as for example when Z is NH 2 , NH-NH 2 , NH-alkyl-NH 2 .
  • side chains of amino acids such as the primary amine of Lys can be used in coupling the chelator to the compound.
  • modifications for the amino terminus (Y) include, COCH 3 ("Ac"), CO-alkyl, an alkyl group, CH 2 Ph, COPh, COOCH 2 Ph, COO-alkyl and C0- alkyl -NH 2 .
  • Modifications for the carboxyl terminus (Z) include, NH 2 , NH-NH 2 , O-alkyl, SH, S-alkyl, N-alkyl, NHCH 2 Ph and NH-alkyl-NH 2 .
  • peptides and peptidic-like compounds can be manipulated, for example, while still attached to a resin to obtain N-terminal modified agents such as an acetylated peptide or can be removed from the resin using hydrogen fluoride or an equivalent cleaving reagent and then modified.
  • N-terminal modified agents such as an acetylated peptide
  • Compounds synthesized containing the C-terminal carboxyl group can be modified after cleavage from the resin, or in some cases, prior to solution phase synthesis. Methods are well known in the art for acetylation, amidation and any of the other above- described modifications.
  • X 2 , X 3 , X , Y and Z can be combined, in any combination, to arrive at yet further embodiments of the present invention than those specific combinations identified above and in the claims.
  • the integrin-binding compounds used in the subject MRI contrast agents can be synthesized by any of the suitable methods well known in the art including methods of chemical synthesis.
  • the linear counterpart can be synthesized using commercially available automated peptide synthesizers such as those manufactured by Applied Biosystems, Inc., Foster City, CA.
  • the compounds can be synthesized using amino acids or amino acid analogs, the active groups of which are protected as necessary using, for example a t- butyldicarbonate (t-Boc) group or a fluorenylmethoxy carbonyl (Fmoc) group.
  • Amino acids and amino acid analogs can be purchased commercially (Sigma Chemical Co.; Advanced Chemtec) or synthesized using methods known in the art.
  • MRI contrast agent can be a cyclic compound resulting from a bridge between two amino acids within the compound.
  • the cyclic bridge within a specifically described compound is identified herein by underlining ( ) .
  • the compound can be cyclized through a bridge between any two of the other amino acids present in a given integrin-binding compound of the MRI contrast agent, provided that the two amino acids are on opposite sides of the G-D sequence. Therefore, with reference to SEQ ID NO.
  • the compounds can be cyclized through (1) X and X 4 , (2) X 2 and X 4 , (3) X 2 and X 3 , and (4) X 3 and X ⁇ .
  • the cyclization is the result of bridging between (1)) X x and X 4 or (2) X 2 and X 4 , particularly when X 3 is a hydrophobic amino acid, an amino acid that provides an ionic interaction with an integrin receptor or when X 3 is an amino acid that provides a hydrogen bond with an integrin receptor.
  • X 3 is a hydrophobic amino acid
  • X 3 is an amino acid that provides a hydrogen bond with an integrin receptor.
  • cyclic refers to a compound having an intramolecular bond between the two amino acids forming a bridge within a given compound.
  • the intramolecular bond includes backbone to backbone, side-chain to backbone, and side-chain to side-chain cyclizations .
  • An example of a backbone to backbone cyclization is a bridge formed between the amino group of the amino terminus and the carboxylic acid of the carboxy terminus.
  • Examples of a side-chain to side-chain cyclization include, for instance, disulfide bridges formed through cysteine residues or other sulfur containing amino acids capable of forming such bridges, or alternatively, a bridge formed between the side chain of a basic amino acid, such as Lys, and the side chain of an acidic amino acid, such as Glu.
  • cyclization can be achieved where the compound contains two sulfur-containing amino acids, or other moieties through a disulfide bond.
  • useful sulfur-containing moieties are Cys, Pen, Mpa, Pmp and Pmc.
  • a bridging structure can be created through a disulfide bond between Pmp and Cys (or similar structures) .
  • residues contain sulfhydryls
  • a disulfide bridge can be formed by oxidizing a dilute aqueous solution of the peptides with K 3 [F e (CN) 6 ] .
  • cyclization can be accomplished through the formation of a peptide bond or alkyl bridge structures using, for example, Pas.
  • an alkyl bond between Pas and an amino acid methylene moiety can be used for forming alkyl bridge structures.
  • the cyclized compounds of the present invention can also be prepared by forming a peptide bond between non-adjacent amino acid residues.
  • peptide bond or "peptide linkage” refers to an amide linkage between a carboxyl group of one amino acid and the -amino group of another amino acid.
  • Side-chain to side-chain cyclizations can be performed by using N"- Boc-amino acids together with OFm/Fmoc side-chain protection for Asp and Lys residues as described by Felix et al., Int. J. Peptide Protein Res.. 31:231 (1988). Alternatively, side-chain to backbone cyclizations can be performed using this procedure.
  • Alternative amino acids useful for side-chain to side-chain or side-chain to backbone cyclizations are Glu and Orn.
  • Backbone to backbone cyclizations can be performed in solution on side chain protected peptides.
  • Alternative methods of making cyclized peptides are disclosed, for example, in WO 91/01331 entitled “SMALL CYCLIC PEPTIDE AGGREGATION INHIBITORS,” published 7 February 1991.
  • Bridges can also be formed where the amide bond cyclizes the peptide to form a lactam, as in the case of (Nle) GD (Y-OMe) REK-NH-, (SEQ ID NO. 3) .
  • Lactam bridges can also be used for end- to-end cyclizations with compounds of the formula X 2 -G- D-X 3 (SEQ ID NO. 21), such as for example, R-G-D-K (SEQ ID NO. 22) .
  • Backbone to backbone cyclizations can be performed in solution on side chain protected peptides. Briefly, linear side chain protected peptides are synthesized on O-chlorotrityl resin (Barlos et al . ,
  • the peptide After removal of the acetic acid by rotary evaporation, the peptide is cyclized in solution with the BOP reagent. The solvent is removed by rotary evaporation and the side chain protection then is removed by treatment with trifluoroacetic acid to provide the backbone to backbone cyclic compound.
  • Bromoacetyl-Gly-Arg g-2, 2,5,7,8- pentamethylchroman- ⁇ -sulfonyl
  • -Gly-Asp beta-t-butyl
  • Cys S-triphenylmethyl
  • polymer resin can be prepared using standard solid phase peptide synthesis utilizing Fmoc protecting group chemistry on a p-alkoxybenzyl alcohol resin.
  • the compounds of the used in the subject MRI agents have cell attachment activity.
  • the phrase "cell attachment activity” means that a compound has the functional property of being able to induce a cell to adhere or connect or bind to the compound, thereby forming contact between the compound and the cell .
  • Such activity includes inhibition of platelet aggregation.
  • a paramagnetic metal enhances contrast during MRI.
  • a "paramagnetic metal” is a metal ion with one or more unpaired electrons, which confer a magnetic moment.
  • a variety of paramagnetic metals are known to useful for enhancing contrast during MRI, any one of which can be used with the present invention including, for example, Mn 2+ , Cu 2+ , Fe 2+ , Co 2+ , Ni 2+ , Gd 3+ , Eu 3+ , Dy 3+ , Pr 3+ , Cr 3+ , Co 3+ , Fe 3+ , Ti 3+ , Nd 3+ , Sm 3+ , Y 3+ , Tb 3+ , Ho 3+ , Er 3+ , Pa 4+ and V 4 ".
  • the skilled artisan will readily select a metal according to dose required to detect a thrombus and the toxicity of the metal to a subject.
  • the selection of the metal is discussed Tweedle et al . , supra , pp. 796-797.
  • the desired dose for an individual metal will be proportional to its relaxivity, modified by the biodistribution, pharmacokinetics and metabolism of the metal.
  • the metal of the MRI agent is a trivalent cation, for example Gd 3+ , Eu 3+ , Dy 3+ , Pr 3+ , Cr 3+ , Co 3+ , Fe 3+ , Ti 3+ , Nd 3+ , Sm 3+ , Y 3+ , Tb 3+ , Ho 3+ and Er 3+ .
  • a particularly useful metal to enhance MRI contrast is Gd 3+ , which has a comparatively high relaxivity and low toxicity, combined with the further advantage that it exists in only one biologically accessible oxidation state, thereby reducing undesirable metabolism of the metal.
  • Another particularly useful metal is Cr 3+ , which is relatively inexpensive.
  • the MRI contrast agent comprises a Gd 3+ paramagnetic metal, a DTPA chelator, and a compound having one of the following structures : (Nle) GD (Y-OMe) REK-NH, (SEQ ID NO. 3); CNPAGD (Y-OMe) RC-
  • the MRI contrast agent comprises a Gd 3+ paramagnetic metal, a DTPA chelator, and a compound having one of the following structures: (Mpa) RGDD ( t-
  • BuG CK-NH, (SEQ ID NO. 14); CNPRGD (Y-OMe) RC-NH, (SEQ ID NO. 6) ; CNPRGD (Y-O-n-Butyl) RC-NH, (SEQ ID NO. 10) ; G(Pen)FRGDSFCA (SEQ ID NO. 15); GRGDTFEK-NH, (SEQ ID NO. 16); G(Pen)RARGDNPCA (SEQ ID NO. 12); (Pen) (Y- OMe)ARGDN(Tic)C-NH, (SEQ ID NO. 13); or (Pen) FARGDS (Tic) C- NH 2 (SEQ ID NO. 11) , wherein the Gd 3+ is complexed with DTPA and the compound is conjugated to DTPA.
  • the present invention also provides a composition useful as a medicament comprising an MRI contrast agent and a physiologically acceptable carrier.
  • the MRI contrast agents can be combined with a physiologically acceptable carrier to form a composition useful for the detection of thrombus.
  • Physiologically acceptable carriers are well known in the art and include aqueous solutions such as physiologically buffered saline or other buffers or solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters.
  • a physiologically acceptable carrier can contain acceptable compounds that act, for example, to stabilize the MRI contrast agent, increase the absorption of the agent, or extend the half-life in the circulation.
  • physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.
  • carbohydrates such as glucose, sucrose or dextrans
  • antioxidants such as ascorbic acid or glutathione
  • chelating agents such as ascorbic acid or glutathione
  • the present invention further provides a method of detecting thrombus in a subject.
  • This method comprises administering to the subject an effective amount of a magnetic resonance imaging agent, and when the agent homes to a thrombus, detecting the thrombus using magnetic resonance imaging.
  • thrombus means a clot formed from blood constituents and attached to a vessel within the cardiovascular system.
  • the thrombus can be the result of any variety of pathologies or conditions, such as cardiovascular lesions, atherosclerotic plaques, vascular clots or thromboembolic events, such as myocardial infarction and other organ infarcts .
  • the detection of thrombus can have veterinary uses. Accordingly, by “subject” is meant a human being or other vertebrates that are the object of research, treatment, diagnosis, or experimentation. The subject is preferably a human.
  • the "agent homes" to a thrombus when it proceeds or is drawn to the thrombus location, as for example, by binding to an integrin at the site of thrombi .
  • Magnetic resonance imaging involves detecting differences in the relaxation rates for protons in water molecules of a subject's body.
  • a magnetic field is applied to the protons, which have a magnetic moment, they reach an excited state, but revert to their original state by a process termed "relaxation.
  • MRI allows medical diagnosis by visualizing differences in proton relaxation times in the subject's body.
  • the MRI contrast agent of the present invention enhances the visualization by increasing the rate of relaxation, thereby increasing the contrast between water molecules at a thrombus and water molecules elsewhere in the subject's body.
  • the MRI contrast agent must be present in sufficient amounts to enable detection by an external camera, using magnetic field strengths that are reasonably attainable and compatible with patient safety and instrumental design.
  • the requirements for such agents are well known in the art for those agents that have their effect upon water molecules in the medium, and are disclosed, for example, in Pykett, Scientific
  • the effectiveness of the MRI contrast agent is concentration-dependent, and there is normally an optimum concentration of the paramagnetic metal for maximum efficacy.
  • the optimal concentration will vary with the particular MRI contrast agent used, the locus of imaging, the mode of imaging, and the composition of the carrier. These factors, and their relative importance are known in the art .
  • the method of administering the MRI contrast agent is preferably parenterally, meaning intravenously, intraarterially, intrathecally, interstitially or intracavitarily .
  • parenterally meaning intravenously, intraarterially, intrathecally, interstitially or intracavitarily .
  • intravenous or intraarterial administration is preferred.
  • a subject will receive a dosage of MRI contrast agent sufficient to enhance the MRI signal at the site of a thrombus anywhere from 20 to 500%, the amount being a function of the particular metal, compound and mode of administration.
  • the MRI contrast agent homes to a thrombus, the thrombus is scanned with a conventional MRI camera to visualize the thrombus .
  • This example provides methods of chemically synthesizing compounds that are capable of binding to an integrin.
  • a compound was coupled to a chelator that complexes a paramagnetic metal to form an MRI contrast agent of the present invention.
  • Integrin-binding compounds cyclized through disulfide bridges including CNPAGD (Y-OMe) RC-NH, (SEQ ID NO. 4) and CNP (Nle) GD (Y-OMe) RC-NH, (SEQ ID NO. 5), were synthesized as follows. Compounds were synthesized by the solid-phase method on an automated synthesizer (Applied Biosystems, Inc. Model 431A) (see Stewart and Young, in Solid Phase Peptide Synthesis, 2nd ed. (Pierce Chemical Co., Rockford, IL, 1984). Compounds having a C- terminal amide were synthesized using p- methylbenzhydrylamine (pMBHA) resin.
  • pMBHA p- methylbenzhydrylamine
  • Compounds having a C-terminal acid can be synthesized using, for example, a chloromethylated resin.
  • Compounds having an N-terminal acetyl group can be acetylated using a mixture of acetic anhydride (20 eq) and diisopropylethylamine (20 eq) in N-methylpyrrolidone .
  • N-terminal tertbutyloxycarbonyl (Boc) protection was used for all amino acids.
  • Dicyclohexylcarbodiimide and hydroxybenzyltriazole were used in the coupling reactions. The extent of the reactions was monitored by the standard ninhydrin test.
  • the compounds were removed from the resin and deprotected by adding anhydrous HF (10 ml/g of resin-bound compound) containing anisole (1 ml/g) at 0°C for 60 min.
  • the HF was removed by evaporation and the residue was washed with anhydrous ether.
  • the crude compounds were extracted with water or 15% aqueous acetic acid and the aqueous fractions were combined and cyclized as described below.
  • Cyclic compounds that have a lactam bridge such as (Nle) GD (Y-OMe) REK-NH, (SEQ ID NO. 3), can be synthesized as outlined here.
  • the protected compound resin was synthesized using the pMBHA resin.
  • the lactam bridge was formed while the compound was still on the resin using the method described by Felix et al . , Int. J. Pept . Prot . Res . 31:231 (1988) and by Felix et al . , Int. J. Pept. Prot. Res. 32:441 (1988).
  • the crude compounds were purified via preparative RP-HPLC on a C 18 silica gel column (Waters Delta-PakTM, 15 ⁇ m, 300 ⁇ , 47 x 300 mm) eluting with a linear acetonitrile gradient (0-30%) with a constant concentration of trifluoroacetic acid (TFA; 0.1%, v/v) over 30 min at a flow rate of 40 ml/min.
  • the purified compounds were analyzed by analytical RP-HPLC using a C-18 column (VydacTM, 5 ⁇ m, 300 ⁇ , 4.5 x 250 mm) .
  • the purified compounds were recovered by lyophilization of the HPLC fractions, and had a purity of at least 95%.
  • a binary solvent system was used
  • the compound can be adjusted to a neutral pH and potassium ferricyanide added to the TFA compound to minimize polymerization that may result from the presence of the reducing agent .
  • the potassium ferricyanide was removed by ion exchange chromatography and the compounds were lyophilized.
  • the compounds were characterized by fast atom bombardment mass spectroscopy and by amino acid analysis.
  • Amino acid analysis was performed on a Pickering Labs-Trione amino acid analyzer that was equipped with a Spectra-Physics UV detector.
  • Hydrolysis of the compound samples for amino acid analysis was performed on 1 mg samples with 1 ml constant boiling 6N HC1. Samples were degassed, sealed under vacuum and heated for 24 hr. at 110°C.
  • This example provides methods for characterizing the integrin-binding properties of the compounds described in Example I, including such characteristics as selectivity, even at varying calcium concentration, as well as cell attachment activity.
  • A. Selective binding of compounds to one or more inte ⁇ rins :
  • Integrin-binding compounds can bind selectively to one or more integrins.
  • affinity of the compounds for integrins ⁇ Ilb ⁇ 3 , ⁇ v ⁇ 3 , ⁇ v ⁇ s and ⁇ 5 ⁇ x were determined and compared.
  • the binding affinity of a compound for an integrin was assessed by the ability of the compound to displace the binding of an integrin to its integrin-specific ligand, such as fibrinogen, fibronectin or vitronectin.
  • the affinity of a compound to an integrin was determined by using a competitive enzyme-linked immunosorbent assay (ELISA) .
  • ELISA enzyme-linked immunosorbent assay
  • an integrin ligand such as fibrinogen, fibronectin or vitronectin
  • the binding of solubilized integrin to the immobilized ligand was detected with an anti-integrin antibody (see, e.g., Newman et al . , Blood, 65:227-232 (1985)) followed by a labeled secondary conjugate.
  • an anti-integrin antibody see, e.g., Newman et al . , Blood, 65:227-232 (1985)
  • the affinity of the compound for the integrin was determined from the reduced level of binding of solubilized integrin to the ligand.
  • Such ELISA assays were performed as follows.
  • Microtiter plates were coated with 110 ⁇ l/well of human fibronectin (2 ⁇ g/ml in TBS) . The plates were washed three times with TBS-Tween buffer (TBS with 0.05% Tween 20) . Fifty ⁇ l of ⁇ g ⁇ n . in TBS (containing 20 mM octylglucoside, 2 mM NmCl 2 ) was added to each well. Fifty ⁇ l of an integrin-binding compound in the same buffer was added in 10-fold serial dilutions. The plates were incubated for 3 hrs at room temperature, then washed with 200 ⁇ l of TBS-Tween buffer. One hundred ⁇ l of affinity-purified rabbit anti- ⁇ 5 ⁇ ! antibody was added to the wells and the plates were incubated for an additional 2 hrs. The wells were washed twice with TBS- Tween and then with distilled water.
  • TBS-Tween buffer TBS with 0.05% Tween
  • Affinity-purified goat anti-rabbit IgG conjugated to horseradish peroxidase (100 ⁇ l/well) was added and incubated for 16 hrs at room temperature. The following day, the plates were washed twice with TBS- Tween and then with distilled water. One hundred ⁇ l of substrate mixture (10 mg o-phenylenediamine in 25 ml 0.1 M citrate-phosphate buffer, pH 5.0, plus 6 ⁇ l of 30% H 2 0 2 added just before use) was added to the plates and allowed to develop. The development process was stopped by adding 50 ⁇ l of 4N H 2 S0 4 to each well. The optical density at 492 nm compared to 405 nm was recorded and IC 50 values were determined.
  • Microtiter plates were coated with 100 ⁇ l/well of 10 ⁇ g/ml purified fibrinogen and allowed to stand overnight at 4°C. The plates were washed three times with TBS-Tween buffer (0.150 M NaCl , 0.05 M Tris, pH 7.4 at room temperature, 0.05% Tween-20) .
  • integrin-binding compound 50 ⁇ l was added in various dilutions along with 100 mg/ml ⁇ IIb ⁇ 3 purified from human platelets (in 20 mM octyl glucosamine, 1 mM CaCl 2 , 1 mM MgCl 2 , 150 mM NaCl, 50mM Tris, pH 7.4), incubated on a plate shaker at room temperature for 4 hrs. The plates were then washed three times with TBS-Tween and 100 ⁇ l of a polyclonal or monoclonal antibody specific for ⁇ IIb ⁇ 3 (such as 1 ⁇ g/ml AP3) . After 1 hr incubation at room temperature on a plate shaker, the samples were washed three times with TBS-Tween.
  • Microtiter plates were coated with 2 ⁇ g/ml human vitronectin dissolved in PBS (50 ml/well) and stored overnight at 4°C. The plates were washed twice with wash buffer (PBS, 0.05% Tween-20) and blocked by incubating for 1 hr with about 150 ⁇ l/well of assay buffer (1% BSA in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM MgCl 2 , ImM CaCl 2 , ImM MnCl 2 ) .
  • assay buffer 1% BSA in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM MgCl 2 , ImM CaCl 2 , ImM MnCl 2
  • integrin-binding compound prepared in various dilutions in assay buffer
  • 20 ⁇ l/well of integrin-binding compound prepared in various dilutions in assay buffer
  • 20 ⁇ l/well of integrin-binding compound prepared in various dilutions in assay buffer
  • Twenty-five ⁇ l of a 30 ⁇ g/ml solution of purified v ⁇ 3 in assay buffer was added to the well and the reaction was incubated on a plate shaker for 1 hr.
  • a antibody mixture was prepared (1.5 ⁇ g/ml mouse monoclonal anti- ⁇ v ⁇ 3 antibody solution with a 1:6000 dilution of anti-mouse-Fc-HRP antibody conjugate) and incubated for 1 hr.
  • the assay plates were washed 4 times with PBS-Tween, and 50 ⁇ l/well of the antibody mixture was added. After a 1 hr incubation, the plate was then washed 4 times.
  • the color reaction was developed with 50 ⁇ l/well of OPD substrate was added (0.67 mg/ml o-phenylenediamine in 16 mM citric acid, 50 mM NaHP0 4 , pH 5.0, plus 0.0125% H 2 0 2 added just before use). The reaction was stopped with 50 ⁇ l 1M H 2 S0 4 . The optical density at 492 nm compared to 405 nm was recorded and IC 50 values were determined.
  • ELISA assay for v ⁇ 5 was performed by a substantially similar method, except using ⁇ v ⁇ 5 and anti- ⁇ v ⁇ 5 antibodies.
  • Table I compares the relative affinities of various compounds for the indicated integrins in competitive receptor binding assays. It should be noted that in the present example, certain integrin-binding compounds were assayed in the form of closely related analogs. For example, SEQ ID Nos. 6, 10, 13 and 11 were assayed in their acetylated form. Compounds designated SEQ ID Nos. 14 and 16 were assayed in the form of analogs omitting a lysine near the C-terminus. There is an expectation that the results from the compounds of the present invention would be the same and that the results presented are therefore indicative of the selective affinities of such compounds.
  • Affinity was shown in terms of the concentration of each compound required to inhibit 50% of the binding of an integrin to its integrin-specific ligand (IC 50 ) . Values of IC 50 that were less than 50 nM indicate that the compound has a strong binding affinity to the integrin.
  • SEQ ID NO. 15 selectively binds to only one integrin assayed, v ⁇ 5 .
  • SEQ ID NO. 12 selectively binds to more than more integrin, namely ⁇ ⁇ b ⁇ 3 , ⁇ v ⁇ 3 and ⁇ 5 ⁇ x .
  • the affinity of integrin-binding compounds can be cation-dependent.
  • competitive ELISA assays were performed as described in Example II. A. In such assays, the concentration of calcium in different reactions was varied from 0.01 mM to 100 mM Ca 2+ , and the ability of compounds to inhibit the binding of fibrinogen to IIb ⁇ 3 purified was determined. Table II compares the relative affinities of various compounds for IIb ⁇ 3 in the presence of different calcium concentrations. It should be noted that compounds designated SEQ ID Nos.
  • affinities are presented in terms of the fold difference in IC 50 when compared to a base value of the affinity in the presence of 0.01 mM Ca 2 *.
  • Routine assays such as JY cell attachment assays or platelet aggregation assays are useful for assessing cell attachment activity of integrin-binding compounds .
  • JY cell attachment assays such as JY cell attachment assays or platelet aggregation assays are useful for assessing cell attachment activity of integrin-binding compounds .
  • JY cells are human-derived B lymphocyte cells that express the ⁇ v ⁇ 3 VnR but do not express the v ⁇ 5 VnR. Integrin-binding compounds can analyzed for their ability to inhibit the attachment of JY cells to vitronectin- coated plates.
  • the JY attachment assay when compared with the receptor-specific enzyme- linked immunosorbent assays (ELISAs) described above, demonstrates the selectivity of RGD peptides for inhibiting binding due to the v ⁇ 3 VnR.
  • JY cells are grown in suspension in RPMI media (Irvine Scientific; Irvine, CA) supplemented with 10% heat inactivated fetal bovine serum (FBS) and 200 mM glutamine and containing 100 IU/ml penicillin and 100 ⁇ g/ml streptomycin.
  • FBS heat inactivated fetal bovine serum
  • a 96 well ELISA plate (Linbro,
  • Titertek is coated with 100 ⁇ l/well of 5 ⁇ g/ml human Vn in sodium carbonate (pH 9.5) and incubated overnight at 4°C. The plate is rinsed 3x with PBS and blocked by adding 100 ⁇ l/well of 2 mg/ml BSA in PBS and incubating for 1 hr. Following blocking, the wells are washed 2x with PBS.
  • the JY cells are rinsed 2x with PBS, then resuspended to 10 x 10 5 cells/ml in serum-free RPMI containing 2 mg/ml BSA, 200 mM glutamine, 100 IU/ml penicillin, 100 ⁇ g/ml streptomycin and 0.5 mM MnCl 2 .
  • 50 ng/ml phorbol myristate acetate (PMA; Sigma) is added to the cell suspension. PMA stimulates the cells and allows them to bind Vn.
  • Integrin-binding compounds are diluted in the serum-free RPMI and 50 ⁇ l of each dilution is added to duplicate wells. Fifty ⁇ l of the JY cell suspension is added to each well and the plate incubated at 37°C for 45 min, 7% C0 2 . Nonadherent cells are gently aspirated from the wells and the wells rinsed lx with PBS. Cells are fixed in 10% formalin in PBS for 10 min, then stained with 0.1% toluidine blue for 1 hr . The plate is rinsed with distilled water and the cells solubilized using 1% SDS . The amount of staining is determined by spectrophotometry at 595 nm and the IC50 calculated as the concentration of an integrin-binding compound that inhibited JY cell binding to Vn by 50%.
  • Platelet aggregation assays also provide an indicia of an integrin-binding compound's cell attachment activity.
  • This example provides platelet aggregation studies for various peptides of the present invention. The studies compare the effect of the peptide on aggregation in both citrated and heparinized plasma. Because citrate is a chelating agent for calcium ions, the citrated plasma has a low calcium concentration as is characteristic of a clot site. In contrast, heparinized plasma has a calcium concentration similar to that of physiological conditions elsewhere in the blood stream.
  • Platelet-rich plasma was prepared by centrifuging heparinized (20 Units lithium porcine mucosa heparin/ml whole blood) or citrated (0.38% sodium citrate) whole blood for 12 min. at 200 relative centrifugal force (RCF) .
  • Platelet-poor plasma is prepared by centrifuging PRP at 750 RCF for 12 min. and collecting the supernatant. Blood cell counts were performed using a Serano Baker automated cell counter. PRP platelet counts were adjusted to 200,000/ ⁇ l by dilution with PPP.
  • Platelet aggregation was determined spectrophotometrically utilizing a four channel aggregometer (Chrono-Log 570) by measuring the increase of light transmission through a stirred suspension of PRP maintained at 37°C. Dilutions of the peptides were added to the suspensions and aggregation induced with 10 ⁇ M adenosine diphosphate (ADP) . Values were expressed as percentage of aggregation in the absence of peptides. This represents the percentage of light transmission standardized to PRP and PPP samples yielding 0% and 100% light transmission respectively.
  • Peptide anti-aggregation potencies were determined from dose-responsive curves for the inhibition of the maximum aggregation responses stimulated by high doses of ADP (10 ⁇ m) .
  • the 50% inhibitory concentration of each peptide (IC 50 ) was determined by regression analysis of these curves.
  • Peptides of the present invention which were tested using the above procedures include CNPAGD (Y-
  • peptides of the present invention have a higher apparent affinity for GP-IIb/HIa at low calcium concentration as compared to GP-IIb/HIa at higher calcium concentration. Peptides of the present invention would inhibit platelet aggregation only at the site of a clot and not throughout the whole blood stream.
  • This example provides a general method for synthesizing the MRI contrast agent of the present invention by first coupling a chelator to an integrin- binding compound described in Examples I and II, and then complexing a paramagnetic metal to the chelator.
  • the resulting MRI contrast agent can be analyzed by spectral and HPLC methods.
  • a chelator was covalently linked to an integrin-binding compound.
  • DTPA diethylenetriaminepentaacetic acid
  • the dianhydride of DTPA can covalently link the DTPA to a primary amine of a compound (see Hnatowich et al . , Science 220: 613-615 (1983).
  • DTPA anhydride which is commercially available from Sigma Chemical Co., St. Louis, MO, was suspended in dry chloroform. An aliquot of the slurry was placed in a glass reaction vial and the chloroform was blown off with nitrogen.
  • a paramagenetic metal was complexed with the chelator.
  • a solution of GdCl 2 (1 M in water) was then added to the DTPA-compound solution in a 2:1 ratio (Gd : compound) .
  • the reacted solution was applied to an Alltech "Maxi-Clean”TM C 18 cartridge with a 1 ml bed volume (preactivated with methanol and washed with water and HBS, pH 6.75) .
  • the column was washed with 5 ml pH 6.75 HBS, then the product was eluted with 50% acetonitrile HBS.
  • DTPA exhibits an absorbance peak at 215 nm.
  • the integrin-binding compound has absorbance peaks at 220 nm and 280 nm. By observing the optical density at 215 nm and 280 nm, the elution positions of DTPA and compound can be differentiated.
  • the resulting product was also analyzed by HPLC on a VydacTM Type 214TP54 C4 protein column. Two hundred ⁇ l samples of a 1:100 dilution of the product were loaded on the column with a flow rate of 1 ml/min using the following gradient: 0 min to 10 min at 5% acetonitrile, 5 min to 50 min in a linear gradient from 10% to 50% acetonitrile, 50 min to 52 min in a linear gradient from 50% to 80% acetonitrile, 52 min to 54 at 80% acetonitrile, 54 min to 56 min in a linear gradient from 80% to 5% acetonitrile, and 56 min to 70 min at 5% acetonitrile. The uncoupled compound eluted at 17.9 minutes and the product eluted at 21 minutes.
  • the stability of the product was monitored by RP-HPLC.
  • the product was tested for stability by mixing with, and incubated in, whole blood, plasma, serum, and water at 4°c, 20°c and 37°C. Samples at various time points were taken over a 36-hour period and diluted and injected on an analytical HPLC with appropriate reverse phase columns as described above. Quantification of the product's stability was performed by comparing the peak area integration of the product sample with standard concentrations of the integrin-binding compound and DTPA. Unmodified integrin-binding compounds have been previously demonstrated to be very stable in whole blood in vi tro and in vivo in comprehensive stability and pharmacokinetic studies. (Nishit et al .. J. Cardiovasc . Pharmacol .. 25:888-897 (1995)).
  • This example provides a method for screening candidate agents for affinity to human blood clots.
  • agents can be, for example, an integrin-binding compound or an MRI imaging agent .
  • Whole blood is drawn from human subjects from the antecubital vein using a 21 gauge butterfly infusion set and syringe. Coagulation of the blood is prevented by gently mixing the blood with one tenth volume of 0.129 M sodium citrate in a sterile polypropylene culture tube. Recalcification, contact with glass, and addition of thrombin are all methods used to induce coagulation and clot formation. Routinely, 100 ⁇ l aliquots of the citrated blood are mixed with 10 ⁇ l of 10 U/ml thrombin in 1.5 ml polypropylene microtubes . The thrombi are allowed to form and contract for 2 hrs at room temperature.
  • Preformed clots are washed with buffer and incubated with the candidate agents in buffer, or alternatively blood or plasma. One hour of incubation of 100 ⁇ l of a candidate agent was sufficient for saturable binding.
  • An alternative method for doing an initial in vi tro screen is to purify the DTPA-compound over the
  • Maxi-CleanTM C 18 cartridge then add the paramagnetic metal, for example 51 Cr on a 1:1 molar ratio. Free metal is then removed from the product by size-exclusion or cation-exchange chromatography. The radioactivity that is incorporated in the clot can be determined with a gamma-counter.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Radiology & Medical Imaging (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Magnetic Resonance Imaging Apparatus (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention a pour objet des agents de contraste pour imagerie par résonance magnétique (IRM), qui sont utilisés pour détecter un thrombus. Les agents de contraste pour IRM comprennent un agent chélatant, qui est capable de former des complexes avec un métal paramagnétique. Ledit agent chélatant est associé à un composé chimique capable de se lier à une intégrine. L'agent de contraste pour IRM peut contenir en outre le métal paramagnétique qui forme des complexes avec ledit agent chélatant. L'invention se rapporte également à des compositions contenant lesdits agents de contraste pour IRM et un support physiologiquement acceptable, et à l'utilisation desdits agents pour détecter la présence d'un thrombus chez un patient.
PCT/US1997/018412 1996-10-16 1997-10-15 Imagerie par resonance magnetique utilisee detecter un thrombus WO1998016256A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU47539/97A AU4753997A (en) 1996-10-16 1997-10-15 Magnetic resonance imaging of thrombi

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US73204396A 1996-10-16 1996-10-16
US08/732,043 1996-10-16

Publications (1)

Publication Number Publication Date
WO1998016256A1 true WO1998016256A1 (fr) 1998-04-23

Family

ID=24941964

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/018412 WO1998016256A1 (fr) 1996-10-16 1997-10-15 Imagerie par resonance magnetique utilisee detecter un thrombus

Country Status (2)

Country Link
AU (1) AU4753997A (fr)
WO (1) WO1998016256A1 (fr)

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19744004C1 (de) * 1997-09-26 1999-07-22 Schering Ag Lipophile Metall-Komplexe für Nekrose und Infarkt-Imaging
WO1999058162A2 (fr) * 1998-03-31 1999-11-18 Du Pont Pharmaceuticals Company Produits pharmaceutiques pour l'imagerie de troubles angiogenes
WO2000035488A2 (fr) * 1998-12-18 2000-06-22 Du Pont Pharmaceuticals Company Medicaments antagonistes du recepteur de la vitronectine
WO2000035492A2 (fr) * 1998-12-18 2000-06-22 Du Pont Pharmaceuticals Company Medicaments antagonistes du recepteur de la vitronectine
WO2002022011A1 (fr) 2000-09-15 2002-03-21 Institut für Diagnostikforschung GmbH an der Freien Universität Berlin Procede de representation par images et de diagnostic de thrombus par tomographie a spin nucleaire faisant appel a des agents de contraste particulaires
WO2002069799A1 (fr) * 2001-03-07 2002-09-12 The University Of Queensland Methode de prediction d'evolution d'accident cerebro-vasculaire a l'aide de l'imagerie par resonance magnetique
US6511649B1 (en) 1998-12-18 2003-01-28 Thomas D. Harris Vitronectin receptor antagonist pharmaceuticals
US6524553B2 (en) 1998-03-31 2003-02-25 Bristol-Myers Squibb Pharma Company Quinolone vitronectin receptor antagonist pharmaceuticals
US6537520B1 (en) 1998-03-31 2003-03-25 Bristol-Myers Squibb Pharma Company Pharmaceuticals for the imaging of angiogenic disorders
US6548663B1 (en) 1998-03-31 2003-04-15 Bristol-Myers Squibb Pharma Company Benzodiazepine vitronectin receptor antagonist pharmaceuticals
US6558649B1 (en) 1998-12-18 2003-05-06 Bristol-Myers Squibb Pharma Company Vitronectin receptor antagonist pharmaceuticals
US6569402B1 (en) 1998-12-18 2003-05-27 Bristol-Myers Squibb Pharma Company Vitronectin receptor antagonist pharmaceuticals
WO2004006965A2 (fr) * 2002-07-10 2004-01-22 Schering Aktiengesellschaft Utilisation de complexes metalliques contenant du perfluoroalkyle en tant qu'agents de contraste en imagerie par resonance magnetique pour la representation de thrombus intravasculaires
US6794518B1 (en) 1998-12-18 2004-09-21 Bristol-Myers Squibb Pharma Company Vitronectin receptor antagonist pharmaceuticals
EP1720521A2 (fr) * 2004-02-10 2006-11-15 Barnes-Jewish Hospital Efficacite et securite ameliorees d'agents particulaires diriges a l'aide de systemes leurres
US7344704B2 (en) 2002-07-10 2008-03-18 Schering Ag Use of perfluoroalkyl-containing metal complexes as contrast media in MR-imaging for visualization of intravascular thrombi

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4879237A (en) * 1985-05-24 1989-11-07 La Jolla Cancer Research Foundation Use of peptides in control of cell attachment and detachment
US4988496A (en) * 1988-05-31 1991-01-29 Neorx Corporation Metal radionuclide chelating compounds for improved chelation kinetics
US5659041A (en) * 1993-07-19 1997-08-19 Resolution Pharmaceuticals, Inc. Hydrazino-type radionuclide chelators having an N3 S configuration

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4879237A (en) * 1985-05-24 1989-11-07 La Jolla Cancer Research Foundation Use of peptides in control of cell attachment and detachment
US4988496A (en) * 1988-05-31 1991-01-29 Neorx Corporation Metal radionuclide chelating compounds for improved chelation kinetics
US5659041A (en) * 1993-07-19 1997-08-19 Resolution Pharmaceuticals, Inc. Hydrazino-type radionuclide chelators having an N3 S configuration

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOPOLYMERS (PEPTIDE SCIENCE), 1995, Vol. 37, No. 2, CRAIG et al., "Concept and Progress in the Development of RGD-Containing Peptide Pharmaceuticals", pages 157-175. *
CHENG et al., "Design & Synthesis of Novel Cyclic RGD Peptides as Highly Potent & Selective GpIIb/IIIa Antagonists", In: PEPTIDES: CHEMISTRY, STRUCTURE AND BIOLOGY, PROCEEDINGS OF THE THIRTEENTH AMERICAN PEPTIDE SYMPOSIUM, Edited by R.S., June 1993, pages 384-386. *
JOURNAL OF MEDICINAL CHEMISTRY, 07 January 1994, Vol. 37, No. 1, CHENG et al., "Design and Synthesis of Novel Cyclic RGD-Containing Peptides as Highly Potent and Selective Integrin AlphaIIbBeta3 Antagonist", pages 1-8. *

Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19744004C1 (de) * 1997-09-26 1999-07-22 Schering Ag Lipophile Metall-Komplexe für Nekrose und Infarkt-Imaging
US6524553B2 (en) 1998-03-31 2003-02-25 Bristol-Myers Squibb Pharma Company Quinolone vitronectin receptor antagonist pharmaceuticals
WO1999058162A2 (fr) * 1998-03-31 1999-11-18 Du Pont Pharmaceuticals Company Produits pharmaceutiques pour l'imagerie de troubles angiogenes
WO1999058162A3 (fr) * 1998-03-31 2000-04-06 Du Pont Pharm Co Produits pharmaceutiques pour l'imagerie de troubles angiogenes
US7052673B2 (en) 1998-03-31 2006-05-30 Bristol-Myers Squibb Pharma Company Pharmaceuticals for the imaging of angiogenic disorders
US6548663B1 (en) 1998-03-31 2003-04-15 Bristol-Myers Squibb Pharma Company Benzodiazepine vitronectin receptor antagonist pharmaceuticals
US6537520B1 (en) 1998-03-31 2003-03-25 Bristol-Myers Squibb Pharma Company Pharmaceuticals for the imaging of angiogenic disorders
US6322770B1 (en) 1998-03-31 2001-11-27 Dupont Pharmaceuticals Company Indazole vitronectin receptor antagonist pharmaceuticals
US6794518B1 (en) 1998-12-18 2004-09-21 Bristol-Myers Squibb Pharma Company Vitronectin receptor antagonist pharmaceuticals
US6689337B2 (en) 1998-12-18 2004-02-10 Bristol-Myers Squibb Pharma Company Vitronectin receptor antagonist pharmaceuticals
US6511649B1 (en) 1998-12-18 2003-01-28 Thomas D. Harris Vitronectin receptor antagonist pharmaceuticals
US6511648B2 (en) 1998-12-18 2003-01-28 Bristol-Myers Squibb Pharma Company Vitronectin receptor antagonist pharmaceuticals
US7332149B1 (en) 1998-12-18 2008-02-19 Bristol-Myers Squibb Pharma Company Vitronectin receptor antagonist pharmaceuticals
WO2000035492A3 (fr) * 1998-12-18 2001-01-18 Du Pont Pharm Co Medicaments antagonistes du recepteur de la vitronectine
WO2000035488A3 (fr) * 1998-12-18 2000-11-09 Du Pont Pharm Co Medicaments antagonistes du recepteur de la vitronectine
US6558649B1 (en) 1998-12-18 2003-05-06 Bristol-Myers Squibb Pharma Company Vitronectin receptor antagonist pharmaceuticals
US6569402B1 (en) 1998-12-18 2003-05-27 Bristol-Myers Squibb Pharma Company Vitronectin receptor antagonist pharmaceuticals
US7321045B2 (en) 1998-12-18 2008-01-22 Bristol-Myers Squibb Pharma Company Vitronectin receptor antagonist pharmaceuticals
AU766822B2 (en) * 1998-12-18 2003-10-23 Du Pont Pharmaceuticals Company Vitronectin receptor antagonist pharmaceuticals
US7090828B2 (en) 1998-12-18 2006-08-15 Bristol-Myers Squibb Pharma Company Vitronectin receptor antagonist pharmaceuticals
US6683163B2 (en) 1998-12-18 2004-01-27 Bristol-Myers Squibb Pharma Company Vitronectin receptor antagonist pharmaceuticals
WO2000035488A2 (fr) * 1998-12-18 2000-06-22 Du Pont Pharmaceuticals Company Medicaments antagonistes du recepteur de la vitronectine
US6743412B2 (en) 1998-12-18 2004-06-01 Bristol-Myers Squibb Pharma Company Vitronectin receptor antagonist pharmaceuticals
US7018611B2 (en) 1998-12-18 2006-03-28 Bristol-Myers Squibb Pharma Company Vitronectin receptor antagonist pharmaceuticals
WO2000035492A2 (fr) * 1998-12-18 2000-06-22 Du Pont Pharmaceuticals Company Medicaments antagonistes du recepteur de la vitronectine
US6818201B2 (en) 1998-12-18 2004-11-16 Bristol-Myers Squibb Pharma Company Vitronectin receptor antagonist pharmaceuticals
EP1317208A1 (fr) * 2000-09-15 2003-06-11 INSTITUT FÜR DIAGNOSTIKFORSCHUNG GmbH AN DER FREIEN UNIVERSITÄT BERLIN Procede de representation par images et de diagnostic de thrombus par tomographie a spin nucleaire faisant appel a des agents de contraste particulaires
WO2002022011A1 (fr) 2000-09-15 2002-03-21 Institut für Diagnostikforschung GmbH an der Freien Universität Berlin Procede de representation par images et de diagnostic de thrombus par tomographie a spin nucleaire faisant appel a des agents de contraste particulaires
AU2002234436B2 (en) * 2001-03-07 2005-08-18 The University Of Queensland Method of predicting stroke evolution utilising MRI
WO2002069799A1 (fr) * 2001-03-07 2002-09-12 The University Of Queensland Methode de prediction d'evolution d'accident cerebro-vasculaire a l'aide de l'imagerie par resonance magnetique
WO2004006965A3 (fr) * 2002-07-10 2004-07-15 Schering Ag Utilisation de complexes metalliques contenant du perfluoroalkyle en tant qu'agents de contraste en imagerie par resonance magnetique pour la representation de thrombus intravasculaires
WO2004006965A2 (fr) * 2002-07-10 2004-01-22 Schering Aktiengesellschaft Utilisation de complexes metalliques contenant du perfluoroalkyle en tant qu'agents de contraste en imagerie par resonance magnetique pour la representation de thrombus intravasculaires
US7344704B2 (en) 2002-07-10 2008-03-18 Schering Ag Use of perfluoroalkyl-containing metal complexes as contrast media in MR-imaging for visualization of intravascular thrombi
EP1720521A2 (fr) * 2004-02-10 2006-11-15 Barnes-Jewish Hospital Efficacite et securite ameliorees d'agents particulaires diriges a l'aide de systemes leurres
EP1720521A4 (fr) * 2004-02-10 2008-11-05 Barnes Jewish Hospital Efficacite et securite ameliorees d'agents particulaires diriges a l'aide de systemes leurres

Also Published As

Publication number Publication date
AU4753997A (en) 1998-05-11

Similar Documents

Publication Publication Date Title
US6217846B1 (en) Synthetic peptides for use in thrombus detection
US5759515A (en) Polyvalent peptide pharmaceutical applications
EP0629133B1 (fr) Applications pharmaceutiques a base d'ions metalliques et peptides
AU684069B2 (en) Alpha-ketoamide derivatives as inhibitors of thrombosis
US5886142A (en) Radiolabeled thrombus imaging agents
US6027711A (en) Structurally determined metallo-constructs and applications
WO1998016256A1 (fr) Imagerie par resonance magnetique utilisee detecter un thrombus
EP0677060A1 (fr) Inhibiteurs peptidiques de l'adhesion cellulaire
US20060292075A1 (en) Labelled glutamine and lysine analogues
PL178252B1 (pl) Nowe cykliczne pochodne aminokwasowe i nowe znakowane cykliczne pochodne aminokwasowe
WO1996040781A1 (fr) INHIBITEURS PEPTIDIQUES CYCLIQUES D'ADHERENCE CELLULAIRE INDUITE PAR INTEGRINE β1 ET β¿2?
US20030224998A1 (en) Radiolabeled peptides for the diagnosis and treatment of breast and prostate tumors and metastases of such tumors
KR100322161B1 (ko) 고리형헥사펩티드소마토스타틴유사체
US6194386B1 (en) Labelled peptide compounds
Bovy et al. Structure-activity relationships for the carboxy-terminus-truncated analogs of angiotensin II, a new class of angiotensin II antagonists
JPH09509653A (ja) キレート基を有するセリンプロテアーゼ阻害剤
EP1001977B1 (fr) Peptides radiomarques permettant de diagnostiquer et de traiter des tumeurs du sein et de la prostate et des metastases de ces tumeurs
US5858972A (en) Antithrombotic agents and methods of use
EP0885017A2 (fr) Procede de detection et de localisation de tumeurs malignes chez l'homme a l'aide de ligands de recepteurs de cck-b marques
MXPA00011242A (en) Labelled glutamine and lysine analogues

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase