WO2017212298A1 - Agents d'imagerie et leurs procédés d'utilisation - Google Patents

Agents d'imagerie et leurs procédés d'utilisation Download PDF

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Publication number
WO2017212298A1
WO2017212298A1 PCT/GB2017/051701 GB2017051701W WO2017212298A1 WO 2017212298 A1 WO2017212298 A1 WO 2017212298A1 GB 2017051701 W GB2017051701 W GB 2017051701W WO 2017212298 A1 WO2017212298 A1 WO 2017212298A1
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Prior art keywords
xaa
compound
peptide
leu
tyr
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PCT/GB2017/051701
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English (en)
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Christophe Frederic PORTAL
Ian Andrew Wilson
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Edinburgh Molecular Imaging Limited
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Publication of WO2017212298A1 publication Critical patent/WO2017212298A1/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
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/0008—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/0008—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain
    • C09B23/0016—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain the substituent being a halogen atom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
    • C09B23/083—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines five >CH- groups
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/12—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain being branched "branched" means that the substituent on the polymethine chain forms a new conjugated system, e.g. most trinuclear cyanine dyes

Definitions

  • the present invention relates to imaging agents, and methods of use thereof.
  • the present invention relates to molecular imaging agents, pharmaceutical formulations containing them, method of their preparation, and their use in treatment, surgery and/or diagnosis.
  • the molecular imaging agent can be used in imaging techniques such as (but not limited to) near infrared (NIR) imaging, optical (e.g., fluorescence) imaging and positron emission tomography (PET).
  • NIR near infrared
  • PET positron emission tomography
  • the imaging agents can be used to image compounds (such as particular enzymes) that are known or thought to have a role in particular disease processes.
  • One such enzyme family is the transglutaminases, which form extensively cross-linked, generally insoluble protein polymers. These biological polymers are indispensable for an organism to create barriers and stable structures.
  • One enzyme in this family is Factor XIII, and Factor Xllla in particular.
  • Factor XIII is a plasma glycoprotein which is present in blood and certain tissues in a catalytically inactive (or zymogen) form. Factor XIII is transformed into its active form, Factor Xllla, by thrombin in the presence of calcium ions. Factor Xllla is also known as plasma transglutaminase, fibrinoligase or fibrin-stabilising factor.
  • Factor Xllla is a tetrameric transglutaminase and plays a role in several disease processes such as thrombosis, rheumatoid arthritis and
  • Factor Xllla is an important mediator of fibrinolytic resistance: by crosslinking fibrin ⁇ -chains and a-chains and by covalently binding molecules that impede plasmin activity (such as a2-antiplasmin), Factor XI I la increases the stability of thrombi.
  • the final step in the formation of a blood clot is the covalent crosslinking of the fibrin which is formed by the proteolytic cleavage of fibrinogen by thrombin. Fibrin molecules align and Factor Xllla catalyses covalent crosslinking of the NH2 and CONH2 groups of lysyl and glutaminyl residues respectively giving structural rigidity to the blood clot.
  • the crosslinking stabilises the fibrin clot structure and confers resistance to fibrinolysis.
  • the crosslink formation is an important facet of normal blood coagulation and wound healing as well as pathological conditions such as thrombosis.
  • Factor Xllla substrates may allow diagnosis of stroke. It, and other transglutaminases, may also be implicated in atherosclerosis,
  • WO 91/16931 discloses that radiolabeled analogues of Factor XIII (in which the active site has been inactivated by amino acid substitution) are useful as thrombus imaging radiopharmaceuticals.
  • Factor Xllla is present both intracellular ⁇ and at the cell surface of alveolar macrophages and within the extravascular alveolar space, potentially acting as an indicator of inflammation within the distal lung.
  • Factor Xllla is also known to catalyse the incorporation of low molecular weight amines into the ⁇ -glutamine sites of proteins.
  • Factor Xllla also catalyses the incorporation of low molecular weight glutamine analogues into lysyl residues.
  • low molecular weight amines or glutamine analogues
  • WO 89/00051 describes a method for targeting fibrin deposits using a labelled compound which is covalently bound to fibrin by Factor Xllla.
  • the fibrin binding compound is stated to be "any peptide that is a substrate for the blood enzyme commonly known as Factor Xllla".
  • Preferred peptides are said to include the tetrapeptide sequence Asn-Gln-Glu-Gln- [SEQ ID NO. 1 ] (or NQEQ in standard amino acid abbreviation notation).
  • NH2-Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Thr-Leu-Leu- Lys-OH [SEQ ID NO. 2] together with a synthetic analogue: NH 2 -Asn-Gln- Glu-Gln-Val-Ser-Pro-Tyr-Thr-Leu-Leu-Lys-OH [SEQ ID NO. 3], (denoted NQEQVSPLTLLK and NQEQVSPYTLLK respectively).
  • the latter was radiolabeled with 125 l and shown to be taken up in thrombin clots in vitro.
  • WO 99/60018 discloses synthetic analogues of lysine and glutamine that are substrates for Factor Xllla and that are labelled with radiometals, in particular 99m Tc, chelated to a chelate that is part of the structure.
  • Factor Xllla molecular imaging agents Whilst several examples of Factor Xllla molecular imaging agents exist, it is believed that none are currently used in NIR, optical and/or PET imaging in a clinical context. Moreover, designing new compounds for use in NIR, optical and/or PET imaging and incorporating known Factor Xllla and transglutaminase substrates can lead to changes in solubility, which can create problematic pharmacokinetics. Also, it has been observed that the synthetic routes to many Factor Xllla molecular imaging agents are complex, and involve solubility problems in aqueous solvents in particular.
  • a further object of the invention is to provide alternative imaging agents that can be used to image one or more transglutaminase, in particular using N IR, optical and/or PET imaging.
  • a still further object of the invention is to provide alternative imaging agents that can be used to image Factor Xllla, in particular using NIR, optical and/or PET imaging.
  • X is selected from the group consisting of: -NRJ, -OR, -OJ, -
  • Y is -(B)p(Z), each R is independently selected from the group consisting of: H, CI, C1-4 alkyl, C1-4 alkenyl, C1-4 alkynyl, C1-4 alkoxyalkyl and C1-4 hydroxyalkyl;
  • R 1 is -(L)nM
  • each n is independently an integer of value 0 to 25;
  • each B is independently chosen from Q or A, and where (B) p is a peptide or a pseudopeptide that is a substrate for one or more transglutaminase;
  • Q is a cyclic peptide
  • each A is independently an amino acid residue or an amino acid analogue residue
  • p is an integer of value 4 to 45;
  • J is R or R 1 ;
  • Z is a protecting group
  • n 0 or 1 ;
  • M is a detectable moiety or is configured to bind a detectable moiety.
  • (B)p may be a peptide or a pseudopeptide that is a substrate for Factor Xllla.
  • Y may comprise one or more peptide fragments selected from the group consisting of: a2-antiplasmin, fibronectin, beta-casein, tetanus, amyloid, trappin, and polyglutamine residues, said peptide fragments comprising at least four amino acid residues.
  • the peptide fragment may be from a2-antiplasmin.
  • the amino acid in the 2-position from the peptide N-terminus may be glutamine.
  • X may be -NRJ, giving the formula:
  • R may be H.
  • n may be an integer of value 0 to 8.
  • n is 4.
  • J may be R.
  • Z may be acetyl (Ac) and m may be 1 .
  • p may be an integer of value 4 to 16, optionally an integer of value 8 to 12. Typically, p is 12.
  • the detectable moiety may be detectable by at least one of PET, optical imaging, NIR imaging, NIR tomography and optical tomography.
  • the detectable moiety may be detectable by at least one of PET, fluorescence imaging and fluorescence tomography.
  • the detectable moiety may comprise at least one of a fluorescence emitter and a positron emitter.
  • the detectable moiety may comprise a positron emitter selected from the group consisting of: 18 F, 11 C, 13 N, 15 0, 68 Ga, 89 Zr, and 82 Rb.
  • the detectable moiety may comprise 18 F.
  • the detectable moiety may comprise a fluorescence emitter selected from the group consisting of: cyanine dyes, indolenine based dyes,
  • benzoindolenine based dyes phenoxazines, BODIPY dyes, rhodamines, Si-rhodamines, Alexa dyes, and derivatives thereof.
  • the detectable moiety may comprise a cyanine dye selected from the group consisting of: Cy5, Cy5.5, Cy7, Cy7.5, and derivatives thereof.
  • the detectable moiety may comprise Cy5 or a derivative thereof.
  • the cyanine dye may comprise at least one hydrophilic group.
  • the cyanine dye may comprise at least one sulphonate group.
  • the cyanine dye may comprise at least two sulphonate groups.
  • the compound may comprise the formula: where E is a peptide that is a substrate for at least one
  • transglutaminase comprising the amino acid sequence:
  • Xaa 1 is any amino acid
  • Xaa 2 is Lys, Tyr, Cys, Homolysine, Ornithine, Homocysteine, or Homotyrosine;
  • r is an integer of value 0 to 41 ;
  • Xaa 2 further comprises at least one detectable moiety or comprises at least one moiety configured to bind at least one detectable moiety.
  • the compound may comprise the formula:
  • Xaa 2 may be Lys, Tyr, or Cys. Typically, Xaa 2 is Lys. r may be an integer of value 0 to 12. Optionally, r is an integer of value 4 to 8. Typically, r is 8.
  • Xaa 1 may comprise the amino acid sequence:
  • Xaa 3 is Val, Ala, lie, Leu, Met, Phe, Tyr, or Trp;
  • Xaa 4 is Ser, Thr, Asn, Gin, or Nle;
  • Xaa 5 is Pro or a pseudoproline derivative
  • Xaa 6 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp;
  • Xaa 7 is Thr, Ser, Asn or Glu
  • Xaa 8 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp
  • Xaa 9 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp
  • Xaa 10 is Lys, Tyr, Cys, Homolysine, Ornithine, Homocysteine, or Homotyrosine.
  • Xaa 1 may comprise the amino acid sequence:
  • Xaa 6 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp;
  • Xaa 7 is Thr, Ser, Asn or Glu;
  • Xaa 8 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp
  • Xaa 9 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp
  • Xaa 10 is Lys, Tyr, Cys, Homolysine, Ornithine, Homocysteine, or Homotyrosine.
  • Xaa 1 may comprise the amino acid sequence:
  • Xaa 6 is Leu or Tyr.
  • Xaa 1 may comprise the amino acid sequence:
  • E may be a peptide that is a substrate for Factor XI I la.
  • Xaa 1 is any amino acid
  • Xaa 2 is Lys, Tyr or Cys Homolysine, Ornithine,
  • Homocysteine or Homotyrosine
  • s is an integer of value 0 to 41 ;
  • Xaa 2 further comprises at least one detectable moiety.
  • the peptide may be a substrate for at least one transglutaminase.
  • s may be an integer of value 0 to 12.
  • s is an integer of value 4 to 8.
  • s is 8.
  • Xaa 2 may be Lys, Tyr, or Cys. Typically, Xaa 2 is Lys.
  • Xaa 1 may comprise the amino acid sequence:
  • Xaa 3 is Val, Ala, lie, Leu, Met, Phe, Tyr, or Trp;
  • Xaa 4 is Ser, Thr, Asn, Gin, or Nle;
  • Xaa 5 is Pro or a pseudoproline derivative
  • Xaa 6 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp;
  • Xaa 7 is Thr, Ser, Asn or Glu;
  • Xaa 8 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp
  • Xaa 9 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp
  • Xaa 10 is Lys, Tyr, Cys, Homolysine, Ornithine, Homocysteine, or Homotyrosine.
  • Xaa 1 may comprise the amino acid sequence:
  • Xaa 6 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp
  • Xaa 7 is Thr, Ser, Asn or Glu
  • Xaa 8 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp;
  • Xaa 9 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp;
  • Xaa 10 is Lys, Tyr, Cys, Homolysine, Ornithine, Homocysteine, or Homotyrosine.
  • Xaa 1 may comprise the amino acid sequence:
  • Xaa 6 is Leu or Tyr.
  • Xaa 1 may comprise the amino acid sequence:
  • the peptide may be a substrate for Factor XI I la.
  • a preparation for human or animal administration comprising the compound of the first aspect or the peptide of the second aspect.
  • a kit comprising the compound of the first aspect or the peptide of the second aspect useful in the preparation of a composition for human or animal administration.
  • the compound of the first aspect or the peptide of the second aspect for use as a medicament.
  • the compound of the first aspect or the peptide of the second aspect for use in diagnosis, surgery, treatment or therapy monitoring.
  • the compound of the first aspect or the peptide of the second aspect for use as an imaging agent.
  • the compound of the first aspect or the peptide of the second aspect for use in the diagnosis, surgery, treatment or monitoring therapy of rheumatoid arthritis, atherosclerosis, fibrosis or cancer.
  • the compound of the first aspect or the peptide of the second aspect for use in the diagnosis, surgery or treatment of sites of thrombosis, embolism or inflammation.
  • the compound of the first aspect or the peptide of the second aspect for use in detecting tissue transglutaminse.
  • a method of diagnosis, surgery, treatment or therapy monitoring using the compound of the first aspect or the peptide of the second aspect there is provided a method of imaging using the compound of the first aspect or the peptide of the second aspect.
  • a method of diagnosing, treating or monitoring therapy of rheumatoid arthritis, atherosclerosis, fibrosis or cancer using the compound of the first aspect or the peptide of the second aspect.
  • a method of diagnosing, treating or monitoring therapy of sites of thrombosis, embolism or inflammation using the compound of the first aspect or the peptide of the second aspect.
  • a method of detecting tissue transglutaminse using the compound of the first aspect or the peptide of the second aspect.
  • the use of the compound of the first aspect or the peptide of the second aspect in diagnosis, surgery, treatment or therapy monitoring.
  • the use of the compound of the first aspect or the peptide of the second aspect as an imaging agent.
  • the use of the compound of the first aspect or the peptide of the second aspect in the diagnosis, surgery, treatment or therapy monitoring of rheumatoid arthritis, atherosclerosis, fibrosis or cancer.
  • the use of the compound of the first aspect or the peptide of the second aspect in the diagnosis, treatment or therapy monitoring of sites of thrombosis, embolism or inflammation.
  • the use of the compound of the first aspect or the peptide of the second aspect in the detection of tissue transglutaminse.
  • the invention also includes kits for the preparation of the above
  • cyclic peptide is meant a sequence of 5 to 15 amino acids in which the two terminal amino acids (or two other, non-terminal amino acids) are bonded together by a covalent bond which may be a peptide or disulphide bond or a synthetic non-peptide bond such as a thioether, phosphodiester, disiloxane or urethane bond.
  • amino acid is meant an L-or D-amino acid, amino acid analogue or amino acid mimetic which may be naturally occurring or of purely synthetic origin, and may be optically pure, i.e., a single enantiomer and hence chiral, or a mixture of enantiomers.
  • amino acids of the present invention are optically pure.
  • amino acid mimetic synthetic analogues of naturally occurring amino acids which are isosteres, i.e. have been designed to mimic the steric and electronic structure of the natural compound.
  • isosteres are well known to those skilled in the art and include but are not limited to depsipeptides, retro-inverso peptides, thioamides, cycloalkanes or 1 ,5-disubstituted tetrazoles [M. Goodman, Biopolymers, 24, 137, (1985)].
  • protecting group is meant a biocompatible group which inhibits or suppresses in vivo metabolism of the peptide or amino acid at the amino or carboxyl terminus.
  • groups are well known to those skilled in the art and are suitably chosen from, for the N (or amine) terminus: acetyl (Ac), Boc (where Boc is tert-butyloxycarbonyl), Fmoc (where Fmoc is fluorenyimethoxycarbonyl), benzyloxycarbonyl,
  • a “detectable moiety” is a moiety that emits a signal or is suitable for diagnostic imaging of the human body and may be a radioisotope for radiopharmaceutical imaging or therapy, a paramagnetic metal or species for MRI contrast imaging, a radiopaque group or metal for X-ray contrast imaging, a gas microbubble ultrasound contrast agent, or a suitable dye for detection by external or internal light imaging.
  • a detectable moiety may be a moiety that is detectable by PET (e.g., a positron emitter), or a moiety that is detectable by NIR or optical imaging (e.g., a fluorescence emitter).
  • the peptide fragments comprise at least 4 and optionally 4 to 20 amino acid residues, typically 4 to 15 amino acid residues.
  • amino acid sequences of a2-antiplasmin, fibronectin, beta-casein, fibrinogen and thrombospondin can be found in the following references: c(2-antiplasmin precursor [M. Tone et al., J. Biochem, 102, 1033, (1987)]; beta-casein [L. Hansson et al, Gene, 139, 193, (1994)]; fibronectin [A.
  • amino acid sequence can be taken from the N-terminus of:
  • Fluorine may be 18 F.
  • Fluorine may be 18 F.
  • n may be 2, but can be any integer between 2 and 8.
  • Fluorine may be 18 F.
  • n may be 2, but can be any integer between 2 and 8.
  • Figure 1 is a plot of the RFU obtained using compound 1 in a platelet-rich plasma clot test using a Badimon chamber
  • Figure 2 shows the imaging of ex-vivo formed human thrombus using Compound 1 ;
  • Figure 3 is the image of the binding of Compound 1 to in vivo murine model of arterial thrombosis
  • Figure 4 is the imaging of ex vivo binding experiment of 18 F- Compound 2 to thrombus
  • Figure 5 is the imaging of ex vivo binding experiment of 18 F- Compound 2 to thrombus using microPET/CT;
  • Figure 6 is the in vivo microPET/CT (maximum intensity projection) imaging of mouse with active thrombus;
  • Figure 7 is a plot showing data obtained
  • Figure 8 shows the biodistribution of 18 F-Compound 2 (30 seconds, 5 minutes, 10 minutes, 30 minutes, 60 minutes and 120 minutes;
  • Figure 9 shows the specific accumulation of 18 F-Compound 2 in mice with active thrombus in contrast to the contralateral vein and the blockade animal;
  • Figure 10 is a plot of the standard uptake values obtained for mice with active thrombus and blockade animals (contralateral thrombus values also showed).
  • Figure 1 1 is a plot of the standard uptake values obtained for mice with active thrombus and blockade animals (contralateral thrombus values also showed), expressed in g/ml_.
  • WO 99/60018 describes 99m Tc-compounds that are radiotracers developed as an in vivo diagnostic markers of thromboembolic disease.
  • the 12 amino acid peptide (Asn-Gln-Glu-Gln-Val-Ser-Pro-Tyr-Thr-Leu-Leu-Lys [SEQ ID NO. 3]), is based on the N-terminal sequence of human 02- antiplasmin.
  • the N-terminal sequence of a2-antiplasmin has been demonstrated to be a Factor XI I la substrate.
  • Detection was carried out at 21 Onm, 220 nm, 254nm, 280nm, 300nm and 647nm when relevant. Fluorescence detection was also performed (excitation: 647nm, emission 670nm) when relevant. Full DAD spectra were also recorded (190-500nm and 190-800 nm when relevant) for all runs.
  • Method 2 (23 min, eluents A and B, column A, 0.8 mL/min): 5 % B during 2.5 min, 5 % to 90 % B over 17 min, then 90% B for 1 .5 min, then 90 % to 5 % B over 0.5 min, then 5 % B for 1.5 min.
  • Method 3 (26 min, eluents A and B, column B, 20 mL/min): 5 % B during 2.5 min, 5 % to 85 % B over 20 min, then 85% B for 0.5 min, then 85 % to 5 % B over 1 min, then 5 % B for 2 min. Fraction collection based on 645nm channel.
  • Mmt deprotection Protecting group was removed by treating the resin with TFA in CH2CI2 (1 :99 v/v) for 15 min. The deprotection step was repeated 3 times.
  • the final compound was purified by semi preparative HPLC using Method 3 (2 injections). The final compound was obtained in good yield (23.9 mg, 1 1 %).
  • Resin Rink amide chemmatrix, 0.5 mmol/g.
  • Peptide couplings Fmoc-AA-OH (3 equiv.) and HBTU (2.9 equiv.) were dissolved in 2 ml_ DMF. Sonication was used if needed to dissolve AA. DIPEA (6 equiv.) was added to the mixture.
  • the aminooxyacetic acid functionality on one of the Lysine side chains was labelled with p-F benzaldehyde following the protocol below: To the oxyamine peptide (5 mg, 2.65 pmol) dissolved in H2O with 0.1 % TFA (500 u L) was added 4-fluorobenzaldehyde (0.57 uL, 2 equiv.). The reaction mixture was heated up to 70°C for 45 min.
  • n is 2, but can be any integer between 2 and 8.
  • the peptide substrate of Compounds 4 and 5 can be synthesised using the same synthetic route as for outlined for Compound 1 and 3.
  • the introduction of the fluorinated PET emitting moiety is carried out by click chemistry as follows:
  • a further agent designed to bind AI 18 F (i.e., configured to bind a detectable moiety), was prepared as follows:
  • FXIII resin (EMIR-002-TB1 -12, 20 mg, 4.22 mmol) was placed in a 1 mL solid phase extractor. Hydroxylamine hydrochloride (26 mg, 90 equiv.) and imidazole (19 mg, 67.5 equiv.) were dissolved in NMP (105 uL) and the solution diluted with CH2CI2 (20 ⁇ _) and poured onto resin. The reaction was placed on an orbital shaker for 3 hours and subsequently washed with DMF (1 ml_, 5 x 30 seconds) and CH2CI2 (1 ml_, 5 x 30 seconds). A positive ninhydrin test was performed.
  • DIPEA (10 ⁇ _, 13 equiv.) in DMF (150 ⁇ _) was added to NOTA-NHS (4.18 mg, 1 .5 equiv.) dissolved in dry DMF (150 ⁇ _) and was subsequently added to the resin in dry DMF (150 ⁇ _).
  • the resin was placed on an orbital shaker for 1 hour and then washed with DMF (1 ml_, 5 x 30 seconds) and CH2CI2 (1 ml_, 5 x 30 seconds).
  • a negative ninhydrin test was performed. The product was cleaved off with TFA/TIS/H2O
  • cyanine dyes used with the optical and NIR imaging agents described herein can be, but are not limited to, derivatives of the following general structure referred to as Cy5:
  • Each R 2 is typically independently selected from the group consisting of: H, CI, Ci-4 alkyl, Ci- 4 alkenyl, Ci -4 alkynyl, Ci -4 alkoxyalkyl and Ci -4 hydroxyalkyl.
  • R 2 may also be the attachment point for a peptide or a substrate for a transglutaminase, or for a moiety that links Cy5 to a peptide or a substrate for a transglutaminase.
  • R 3 can be a hydrophilic group such as, for example, sulphonate.
  • Cy5 has the following structure:
  • cyanine dyes that can be used include Cy5.5, Cy7 and Cy7.5 and derivatives of such dyes, general structures for which known in the art.
  • a platelet-rich plasma (PRP) clot test was carried our using a Badimon chamber (ex vivo model of thrombosis that mimics flow conditions within the coronary circulation of man). These ex vivo experiments carried out on human thrombus using compound 1 are shown in graph form in Figure 1 .
  • Table 4 Binding of EMI07678 to ex vivo human thrombi: specificity shown by inhibition of FXIIIa (with iodoacetamide, IAA) and Cy5 control Figure 2 illustrates the same ex vivo experiments carried out on ex vivo formed human thrombus using Compound 1 and that show specific binding of compound 1 to ex vivo human thrombus.
  • Compound 1 (referred to on the image as EM07678) conjugated with Cy5 fluorescent probe readily binds to thrombus (B and C) and this is inhibited by Factor Xllla inhibition (D) . No non-specific binding to thrombus by Cy5 (E) or auto fluorescence (F) was observed and accounted to determine specific binding. Quantification of binding demonstrates discrimination by 2 to 3 orders of magnitude (see Table 1 ). in vivo experiments were carried out as follows. Compound 1 (80 g/kg i.v.) was injected 5 min after application of FeCl3 (10%) to induce thrombosis in the mouse left femoral artery.
  • thrombus can be imaged against background tissues.
  • the positive image of thrombi indicates that clinical imaging of thrombi and areas of activated transglutaminase (e.g., Factor Xllla) is possible.
  • activated transglutaminase e.g., Factor Xllla
  • Positive accumulation and imaging also gives an indication of the stability of Compound 1 .
  • Figure 4 shows on the left side the total binding (incubation with just the radiotracer) and on the right side non-specific binding (incubation with radiotracer and inhibitor, iodoacetamide).
  • Figure 5 shows PET and CT data for the total binding (incubation with just the radiotracer) and on the right side non-specific binding (incubation with radiotracer and inhibitor, iodoacetamide). Total and nonspecific binding are shown side to side.
  • PET Positron Emission Tomography
  • DIPEA diisopropylethylamine
  • TIS triisopropylsilane
  • Npys 3-nitro-2-pyridine sulfenyl
  • AoA aminooxyacetic acid
  • MALDI matrix assisted laser desorption ionisation
  • DAD diode array detector
  • NOTA-NHS 2,2'-(7-(2-((2,5-dioxopyrrolidin-1 -yl)oxy)-2-oxoethyl)-1 ,4,7- triazonane-1 ,4-diyl)diacetic acid
  • Asn-Gln-Glu-Gln Tetrapeptide seguence that is a substrate for the blood enzyme commonly known as Factor Xllla.
  • Xaa 1 is any amino acid
  • Xaa 2 is Lys, Tyr, Cys, Homolysine, Orn, Homocysteine, or Homotyrosine;
  • r is an integer of value 0 to 41 .
  • Peptide seguence or synthetic analogue peptide seguence, that is a substrate for the blood enzyme commonly known as Factor Xllla.
  • SEQ ID NO. 5 SEQ ID NO. 5
  • Xaa 3 is Val, Ala, lie, Leu, Met, Phe, Tyr, or Trp;
  • Xaa 4 is Ser, Thr, Asn, Gin, or Nle;
  • Xaa 5 is Pro or a pseudoproline derivative
  • Xaa 6 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp;
  • Xaa 7 is Thr, Ser, Asn or Glu
  • Xaa 8 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp;
  • Xaa 9 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp;
  • Xaa 10 is Lys, Tyr, Cys, Homolysine, Orn, Homocysteine, or Homotyrosine.
  • Xaa 6 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp;
  • Xaa 7 is Thr, Ser, Asn or Glu
  • Xaa 8 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp;
  • Xaa 9 is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp;
  • Xaa 10 is Lys, Tyr, Cys, Homolysine, Orn, Homocysteine, or Homotyrosine.
  • Peptide sequence or synthetic analogue peptide sequence, that is a substrate for the blood enzyme commonly known as Factor Xllla.
  • Xaa 6 is Leu or Tyr.
  • Peptide sequence or synthetic analogue peptide sequence, that is a substrate for the blood enzyme commonly known as Factor Xllla.
  • Synthetic analogue peptide sequence of the a2-antiplasmin enzyme SEQ ID NO. 10
  • Synthetic analogue peptide sequence of the a2-antiplasmin enzyme SEQ ID NO. 12

Abstract

La présente invention concerne des agents d'imagerie et des procédés d'utilisation dans l'imagerie du Facteur XIIIa. La présente invention concerne également l'utilisation d'agents d'imagerie et des procédés de détection de la transglutaminase et/ou de l'activité du facteur XIIIa. Les agents d'imagerie comportent une entité peptide ou pseudopeptide qui est un substrat pour une ou plusieurs transglutaminases, et une entité détectable qui est détectable en imagerie NIR, optique et/ou TEP. Les agents d'imagerie peuvent être utilisés dans l'imagerie, le diagnostic, la chirurgie, le traitement ou la surveillance thérapeutique. Par exemple, les agents d'imagerie peuvent être utilisés dans le diagnostic, la chirurgie, le traitement ou la surveillance thérapeutique de la polyarthrite rhumatoïde, de l'athérosclérose, de la fibrose ou du cancer. Les agents d'imagerie peuvent également être utilisés dans le diagnostic, la chirurgie ou le traitement de sites de thrombose, d'embolie ou d'inflammation.
PCT/GB2017/051701 2016-06-10 2017-06-09 Agents d'imagerie et leurs procédés d'utilisation WO2017212298A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113229213A (zh) * 2021-05-14 2021-08-10 福州大学 通过近红外荧光探针标记血栓实现肺栓塞造模及无创定量检测的方法
WO2023164956A1 (fr) * 2022-03-04 2023-09-07 浙江省肿瘤医院 Sonde fluorescente à région ii proche infrarouge conjuguée à un anticorps, procédé de construction et utilisation

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999060018A1 (fr) * 1998-05-15 1999-11-25 Nycomed Amersham Plc Analogues de glutamine et de lysine marques
US20030143158A1 (en) * 2000-12-23 2003-07-31 Wescott Charles R. Fibrin binding moieties useful as imaging agents
WO2004037297A1 (fr) * 2002-10-25 2004-05-06 Ge Healthcare Limited Conjugues de complexes tc et de fragments de ciblage et leur utilisation dans le diagnostic par irm

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999060018A1 (fr) * 1998-05-15 1999-11-25 Nycomed Amersham Plc Analogues de glutamine et de lysine marques
US20030143158A1 (en) * 2000-12-23 2003-07-31 Wescott Charles R. Fibrin binding moieties useful as imaging agents
WO2004037297A1 (fr) * 2002-10-25 2004-05-06 Ge Healthcare Limited Conjugues de complexes tc et de fragments de ciblage et leur utilisation dans le diagnostic par irm

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113229213A (zh) * 2021-05-14 2021-08-10 福州大学 通过近红外荧光探针标记血栓实现肺栓塞造模及无创定量检测的方法
CN113229213B (zh) * 2021-05-14 2022-06-14 福州大学 通过近红外荧光探针标记血栓实现肺栓塞造模及无创定量检测的方法
WO2023164956A1 (fr) * 2022-03-04 2023-09-07 浙江省肿瘤医院 Sonde fluorescente à région ii proche infrarouge conjuguée à un anticorps, procédé de construction et utilisation

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