WO2016151297A1 - Optical probe for thrombin - Google Patents

Optical probe for thrombin Download PDF

Info

Publication number
WO2016151297A1
WO2016151297A1 PCT/GB2016/050763 GB2016050763W WO2016151297A1 WO 2016151297 A1 WO2016151297 A1 WO 2016151297A1 GB 2016050763 W GB2016050763 W GB 2016050763W WO 2016151297 A1 WO2016151297 A1 WO 2016151297A1
Authority
WO
WIPO (PCT)
Prior art keywords
fluorophore
probe
quencher
enzyme
mmp
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/GB2016/050763
Other languages
French (fr)
Inventor
Mark Bradley
Sunay Vijaykumar Chankeshwara
Alicia MEGIA-FERNANDEZ
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Edinburgh
Original Assignee
University of Edinburgh
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 University of Edinburgh filed Critical University of Edinburgh
Publication of WO2016151297A1 publication Critical patent/WO2016151297A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • 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
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology

Definitions

  • the invention relates to the field of optical probes, more specifically to optical probes for the detection of target species, such as thrombin or two or more target species such as thrombin and a metalloproteinase.
  • Fibroproliferative diseases of the lung and other organs constitute a heavy burden of morbidity and untimely deaths. Fibrosis results in permanent loss of the tissue's ability to function optimally. In the lung, gas exchange ability is impaired by the formation of scar tissue. Often, Idiopathic Pulmonary Fibrosis (IPF) is diagnosed late (on CT scans/lung biopsy) and has a high mortality with a median survival time from diagnosis of 3 years. It is currently impossible to identify patients with Adult Respiratory Distress Syndrome (ARDS) and other inflammatory lung diseases that are developing secondary fibrosis. Moreover there are no effective therapies for fibrosis despite it being a highly active cellular process which should be accessible to intervention. Part of the problem is the time required to establish drug effectiveness in vivo and the poor utility of existing biomarkers. Thus, there is an urgent need to develop diagnostic methodologies that will permit the more effective and rapid determination of both disease activity and efficacy of emerging anti-fibrotic drugs.
  • Idiopathic Pulmonary Fibrosis IPF
  • Optical probes are typically developed for each specific target for effective diagnostic imaging (Biochemistry 2010, 49, 1364-1376).
  • Activatable optical probes can provide functional details of molecular events, and provide advantages such as providing information at the molecular level. Furthermore, they can be used with a small portable system, provide information quickly, are low cost, and can be microdosed ( ⁇ 100 ⁇ g), thereby reducing the risk of side effects.
  • fCFM fibre-Confocal Fluorescence Microscopy
  • Its use in combination with optical probes for specific enzymes can provide valuable information.
  • FRET optical probes have not previously allowed the detection of thrombin in vivo due to the instability of the probes from non-specific cleavage by enzymes other than thrombin typically found in tissue or tissue samples to be tested, such as plasmin, matrix metalloproteinases (MMPs) and factor Xa. Accordingly, it is an object of the present invention to provide improved optical probes that are capable of detecting activated thrombin in the presence of enzymes typically found in tissue.
  • MMPs matrix metalloproteinases
  • optical probes that are capable of detecting specific proteinases, that are preferably stable in vivo.
  • an optical probe comprising a first probe element, and a second probe element; the first probe element and the second probe element connected to a core; the first probe element comprising a first fluorophore connected to a first quencher by a first cleavable linker; the second probe element comprising a second fluorophore connected to a second quencher by a second cleavable
  • the first or second fluorophore of the first or second probe element is separated from the first or second quencher and therefore, the fluorescence of the first or second fluorophore is no longer quenched by the first or second quencher. Accordingly, upon illumination of the target area with an appropriate wavelength of light, the first or second fluorophore of the first or second probe element fluoresces.
  • the first cleavable linker is cleaved by a first cleaving agent
  • the second cleavable linker is cleaved by a second cleaving agent.
  • One or both of the first and second cleaving agents may be an enzyme or a biochemical stimulus.
  • the biochemical stimulus may be selected from a particular biochemical condition, such a pH value or redox potential value, the presence of a particular chemical species or a particular concentration of such a species.
  • suitable biochemical stimuli include a particular pH range or a redox potential range or the presence of a chemical species such as peroxide, oxygen (0 2 ) or superoxide, or a particular concentration range of such a species.
  • the first cleaving agent may be a first enzyme, and the first cleavable linker may correspond to a first enzyme cleavable peptide sequence.
  • the second cleaving agent may be a second enzyme and the second cleavable linker may correspond to a second enzyme cleavable peptide sequence.
  • the first cleaving agent is a first enzyme and the second cleaving agent is a second enzyme.
  • the first cleavable linker is cleaved when a first cleaving enzyme cleaves the first enzyme cleavable peptide sequence
  • the second cleavable linker is cleaved when a second cleaving enzyme cleaves the second enzyme cleavable peptide sequence. Therefore, cleavage of the first enzyme cleavable peptide sequence typically corresponds to cleavage of the first cleavable linker, and cleavage of the second enzyme cleavable peptide sequence corresponds to cleavage of the second cleavable linker. Accordingly, in
  • the term "cleavage of the cleavable linker” refers to cleavage of the enzyme cleavable peptide sequence unless stated otherwise.
  • the first and/or second cleaving enzyme may be produced or expressed by the indigenous cells within the target area.
  • the first and/or second cleaving enzyme may be produced or expressed by additional cells produced by a subject that have migrated to the target area, such as leukocytes, for example neutrophils.
  • the first and/or second cleaving enzyme may be produced or expressed by an infective agent in the target area, such as a bacterial or fungal cell, for example.
  • the first enzyme may be overexpressed and/or activated in a first disease
  • the second enzyme may be overexpressed and/or activated in a second disease.
  • the first disease may be unrelated to the second disease.
  • the first disease may be related to the second disease or the first and second diseases may be the same disease. Therefore, the probe of the invention may provide a fast, effective and reliable way of detecting the first and second disease using a single probe, or may allow two related diseases to be differentiated and positively identified using a single probe or may allow two signals from a single disease to be identified.
  • the first enzyme may be overexpressed and/or activated in a first stage of a disease
  • the second enzyme may be overexpressed and/or activated in a second stage of the disease. Therefore, the probe of the invention may allow a first stage of a disease to be differentiated from a second stage of the disease.
  • the first enzyme may be overexpressed and/or activated in a first disease process
  • the second enzyme may be overexpressed and/or activated in a second disease process. Therefore, the probe of the invention may increase specificity of disease detection.
  • the first enzyme and the second enzyme may both be overexpressed and/or activated in the same one or more diseases.
  • fluorescence of the first fluorophore and/or the second fluorophore is indicative of the presence of the first enzyme and/or the second enzyme.
  • the presence of the first enzyme and/or the second enzyme may be indicative of the presence of specific pathogenic cells or tissue, and/or a specific disease.
  • fluorescence of the first fluorophore and/or the second fluorophore may be indicative of the presence of specific pathological cells or tissue, and/or a specific disease.
  • the probe of the invention may allow the presence of a specific disease to be detected by two independent indicators, thereby minimising false positive results due to the presence of or more of the first or second enzyme only (i.e. only the first enzyme or only the second enzyme) which may be overexpressed and/or activated in diseases in addition to the specific disease.
  • the first and second enzymes may be active in a particular disease stage, such that fluorescence of only one of the fluorophores is observed and can be indicative of the disease stage.
  • the first enzyme may be a matrix metalloproteinase (MMP).
  • MMP matrix metalloproteinase
  • the first enzyme one or more of MMP-2, MMP-9, and/or MMP-13.
  • the first enzyme cleavable peptide sequence is selectively cleavable by MMP-2, MMP-9 and MMP-13.
  • the first enzyme cleavable peptide sequence may comprise one of SEQ ID N0.1 to SEQ ID NO.14 (Table 1 below).
  • the first enzyme cleavable peptide sequence comprises one of SEQ ID N0.1 to SEQ ID NO.14, it allows the probe of the invention to detect MMP, and therefore, fluorescence of the first probe element is indicative of the presence of MMP.
  • MMP is overexpressed and/or activated in diseases such as fibrosis, cirrhosis, cancer, arthritis, particularly osteoarthritis, and atherosclerosis, and therefore, fluorescence of the first probe element may be indicative of fibrosis, cirrhosis, cancer, arthritis or atherosclerosis.
  • MMPs are upregulated in any inflammatory disease and as such these probes may be useful in their diagnosis.
  • the first enzyme cleavable peptide sequence comprises one of SEQ ID NO.5, or SEQ ID NO.7. More preferably, the first enzyme cleavable peptide sequence comprises SEQ ID NO.7.
  • SEQ ID NO.5 More preferably, the first enzyme cleavable peptide sequence comprises SEQ ID NO.7.
  • SEQ ID NO.7 These sequences have surprisingly been found to be selectively cleaved by MMP-2, MMP-9 and MMP-13 over other MMPs and other similar enzymes, such as, plasmin, thrombin and factor Xa. Accordingly, embodiments comprising one or SEQ ID NO.5, or SEQ ID NO.7 allows selective and specific detection of MMP-2, MMP-9 and MMP- 13.
  • the second enzyme may be thrombin.
  • the second enzyme cleavable peptide sequence may comprise one of SEQ ID NO.15 to SEQ ID N0.38 (Table 2).
  • the second enzyme cleavable peptide sequence comprises one of SEQ ID N0.25, SEQ ID N0.31 , or SEQ ID N0.37.
  • the second enzyme cleavable peptide sequence comprises SEQ ID NO.25.
  • Probe elements that comprise one of these sequences have been surprisingly found to be resistant to non-specific cleavage and breakdown whilst still retaining selectivity for cleavage by thrombin. Therefore, probes comprising two probe elements comprising these sequences allow the reliable detection of thrombin in the presence of other enzymes, such as MMP, plasmin and factor Xa.
  • the first enzyme is MMP-2, MMP-9 or MMP-13
  • the second enzyme is thrombin.
  • a probe having two probe elements allows the detection of MMP-2, MMP-9 or MMP-13, and thrombin.
  • MMP-2, MMP-9 or MMP-13, especially MMP-9 and MMP-13, and thrombin are overexpressed and/or activated in fibrosis, and activity of these enzymes is significantly increased in fibrotic tissue. Therefore, the probe of the invention having two probe elements allows the simultaneous detection of MMP-9 and thrombin, for example, using a single probe in a single procedure, to thereby allow the presence of fibrotic tissue and an active fibrotic process to be detected.
  • a probe that allows the detection of MMP and thrombin expression and activity may allow the reliable detection of active fibroproliferation within fibrotic tissue.
  • the probe can minimise false positive detection of fibrotic tissue. For instance due to the selectivity of a cleavable linker for a particular MMP, accurate detection may be achieved even in the presence of other similar enzymes.
  • the presence of a second probe element can allow the selective detection of another enzyme, such as thrombin, which may also be capable of cleaving the cleavable linker of the first probe element, thereby indicating a false positive.
  • a probe that allows the detection of MMP and thrombin expression and activity may allow the clinical stage of the fibro-proliferative disease to be more accurately determined.
  • the probe is advantageous over more invasive techniques, such as a biopsy, because its use does not require tissue to be excised.
  • the probe can be delivered by endoscope, spray, injection, topically, or ingestion and then the delivery site illuminated in situ to detect fluorescence in the target zone.
  • the probe may provide guidance to areas of active disease, improving biopsy sampling.
  • the first or second cleaving enzyme may be proteinase 3 and the enzyme cleavable peptide sequence may comprise V-A-D-C-A-D-Y.
  • the first or second cleaving enzyme may be a caspase and the enzyme cleavable peptide sequence may comprise D-E-V-D. Indeed, the first or second cleaving enzyme may be any enzyme with an appropriate target peptide substrate sequence.
  • the first and or second cleaving agent may be a reactive oxygen species.
  • the reactive oxygen species may be superoxide, or hydrogen peroxide.
  • the first cleavable linker, and/or the second cleavable linker may be a modified boronic acid based linker, such as that described in J. Am. Chem. Soc, 2014, 874, Roger Y. Tsien.
  • the reactive oxygen species may be generated by the same cells or tissues that express the enzyme.
  • the fluorescence of a first fluorophore may be suppressed, or "quenched", by a neighbouring moiety.
  • the neighbouring moiety, or “quencher” may be a second
  • SUBSTITUTE SHEET RULE 26 fluorophore Where the first fluorophore and the neighbouring second fluorophore are of the same type (i.e. they are the same chemical entity with the same excitation and emission spectra), the first and second fluorophores may quench the fluorescence of each other, and "self-quench". For example, carboxy fluorescein, Cy5, and 7-nitrobenz-2-oxa-1 ,3- diazole (NBD) may self-quench.
  • NBD 7-nitrobenz-2-oxa-1 ,3- diazole
  • first fluorophore and the neighbouring second fluorophore are of different types (i.e. they are different chemical entities and have different excitation and emission spectra)
  • energy may be transferred from one to the other fluorophore via Fluorescence (or Forster) Resonance Energy Transfer (FRET).
  • FRET Fluorescence (or Forster) Resonance Energy Transfer
  • they may form a "FRET pair”
  • the second fluorophore may quench the fluorescence of the first fluorophore.
  • the second fluorophore absorbs the fluorescence of the first fluorophore and fluoresces itself at a different wavelength.
  • the second fluorophore may be a "fluorescent quencher".
  • FRET pairs that comprise two fluorophores include (fluorophore/quencher) Cy3/Cy5, and carboxy fluorescein/seminaphthorhodamine carboxylate derivatives.
  • the neighbouring moiety may be a chemical entity that does not fluoresce.
  • the neighbouring moiety may still quench the fluorescence of the first fluorophore, but instead of fluorescing itself, the neighbouring moiety disperses the energy it received from the fluorophore as heat to its surroundings, and is a "dark quencher".
  • FRET pairs that comprise a dark quencher include (fluorophore/quencher) fluorescein/dimethylaminoazobenzenesulfonic acid (DABSYL), carboxy fluorescein/BHQ-1 , carboxy fluorescein/methyl red, NBD/methyl red, carboxy naphthofluorescein/QSY21 , carboxy naphthofluorescein/BHQ-3, seminaphthorhodamine carboxylate derivatives/BHQ-3, seminaphthorhodamine carboxylate derivatives/QSY21 , Cy3/QSY21 , Cy5/QSY21 , and Cy5/BHQ-3.
  • DBSYL fluorescein/dimethylaminoazobenzenesulfonic acid
  • the first quencher and the first fluorophore may quench.
  • a first probe element comprising a first quencher and a first fluorophore that quench is substantially dark and does not produce significant fluorescence before the first enzyme cleavable peptide sequence is cleaved.
  • the first quencher may be the same type of fluorophore as the first fluorophore, and the first quencher and the first fluorophore may self-quench.
  • the second quencher and the second fluorophore may quench.
  • a second probe element comprising a second quencher and a second fluorophore that quench is substantially dark
  • the second quencher may be the same type of fluorophore as the second fluorophore, and the second quencher and the second fluorophore may self-quench.
  • the fluorophore may be selected from fluorescein, or a derivative thereof, seminaphthorhodamine carboxylate or a derivative thereof, a cyanine fluorophore, such as Cy2, Cy3, Cy5, Cy5.5 or Cy7, rhodamine or derivative thereof, a fluorescent protein, such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), or cyan fluorescent protein (CFP), or 7- nitrobenz-2-oxa-1 ,3-diazole (NBD).
  • fluorescein or a derivative thereof, seminaphthorhodamine carboxylate or a derivative thereof
  • a cyanine fluorophore such as Cy2, Cy3, Cy5, Cy5.5 or Cy7, rhodamine or derivative thereof
  • a fluorescent protein such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), or cyan fluorescent protein (CFP), or 7- nitrobenz-2-oxa-1 ,3-diazole (NBD).
  • the first quencher is a different type of fluorophore than that of the first fluorophore, and is a fluorescent quencher. Accordingly, before the first enzyme cleavable peptide sequence is cleaved a first probe element comprising a fluorescent quencher, fluoresces at the wavelength of light emitted by the fluorescent quencher and does not fluoresce at the wavelength of light emitted by the first fluorophore.
  • the second quencher is a different type of fluorophore than that of the second fluorophore, and is a fluorescent quencher.
  • a second probe element comprising a fluorescent quencher fluoresces at the wavelength of light emitted by the fluorescent quencher and does not fluoresce at the wavelength of light emitted by the second fluorophore.
  • the first quencher may be a dark quencher. Accordingly, a first probe element comprising a dark quencher is substantially dark and does not produce fluorescence before the first enzyme cleavable peptide sequence is cleaved.
  • the second quencher may be a dark quencher. Accordingly, a second probe element comprising a dark quencher is substantially dark and does not produce fluorescence before the second enzyme cleavable peptide sequence is cleaved.
  • the first fluorophore may be any fluorophore that may form a FRET pair with a suitable first quencher.
  • the second fluorophore and the second quencher are different chemical entities, the second fluorophore may be any fluorophore that may form a FRET pair with a suitable second quencher. Accordingly, a given fluorophore and quencher are chosen as a pair to ensure that they have appropriate excitation and
  • fluorophore/quencher pairs include Cy3/Cy5, Cy3/QSY21 , Cy5/QSY21 , Cy5/BHQ-3, carboxy fluorescein/tetramethylrhodamine, fluorescein/methyl red, carboxy fluorescein/BHQ-1 , NBD/methyl red, carboxy naphthofluorescein/QSY21 , carboxy naphthofluorescein/BHQ-3, seminaphthorhodamine carboxylate derivatives/BHQ-3, seminaphthorhodamine carboxylate derivatives/QSY21 , cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP), carboxy fluorescein (FAM)/methyl red, etc.
  • FRET pairs may be readily identified by the skilled
  • first fluorophore and first quencher and the second fluorophore and second quencher, should be chosen such that the first fluorophore is not substantially fluorescently quenched by the second quencher, and the second fluorophore is not substantially fluorescently quenched by the first quencher.
  • first fluorophore/first quencher pair may be FAM/methyl red and the second fluorophore/second quencher pair may be Cy5/QSY21 or carboxy naphthofluorescein/QSY21.
  • the first probe element may comprise a plurality of first fluorophores.
  • the second probe element may comprise a plurality of second fluorophores or second quenchers.
  • the first probe element comprises a plurality of first fluorophores each connected to a first quencher by a separate first cleavable linker.
  • the first probe element may comprise a single first quencher that substantially fluorescently quenches each first fluorophore within the plurality of first fluorophores.
  • the first probe element may comprise a plurality of first quenchers, and each first fluorophore within the plurality of first fluorophores may be connected to a first quencher by a first cleavable linker.
  • the second probe element comprises a plurality of second fluorophores each connected to a second quencher by a separate second cleavable linker.
  • the second probe element may comprise a single second quencher that substantially fluorescently quenches each second fluorophore within the plurality of second fluorophores.
  • the second probe element may comprise a plurality of second quenchers, and each second fluorophore within the plurality of second fluorophores may be connected to a second quencher by a second cleavable linker.
  • the probe may comprise a third probe element connected to the core, the third probe element may comprise a third fluorophore connected to a third quencher by a third cleavable linker.
  • the third cleavable linker may comprise a third enzyme cleavable peptide sequence that may be cleaved by a third enzyme.
  • the third enzyme may be associated with the same disease as the first and/or second enzymes.
  • the third enzyme may be associated with a different disease to that associated with the first and/or second enzyme.
  • the probes of the invention are operable to be used to detect a first and a second cleaving agent in a target zone.
  • the target zone may be a portion of tissue within a subject, and the method may be carried out in vivo.
  • the portion of tissue may be a portion of the heart, lung, liver, connective tissue, skin, intestine, or joints of a subject.
  • the probes of the invention may be used in the respiratory system, the circulatory system, the nervous system, the digestive system or the reproductive system.
  • the target area may be a portion of the lung of a subject.
  • the target zone may be a portion of a cell culture, a tissue sample such as a biopsy sample, or a liquid sample such as a bodily fluid sample.
  • the target zone may be a target area.
  • the target zone may be a target volume.
  • the probes of the invention may be used in vivo, ex vivo or in vitro.
  • the probes of the invention may be delivered to a target zone by any means known in the art.
  • the probes of the invention may be delivered by endoscope, spray, injection, topically, or ingestion.
  • the probes may be delivered to the target zone using a bronchoscope.
  • Illumination of a suitable wavelength to excite the first and second fluorophores of the probe may be delivered to the target zone by any conventional means known in the art.
  • the light is delivered by means of an optical fibre or similar.
  • the fluorescence from the probes in the target zone may be collected by an optical fibre or similar.
  • the fluorescence from the probes in the target zone may be collected by the same optical fibre that delivered the illumination light.
  • the collected fluorescence is typically delivered to a recording device, such as a charge-coupled device (CCD) or similar.
  • the fluorescence from the probes in the target zone may be directly collected by a recording device, such as a CCD or similar.
  • a bronchoscope may be used to both deliver the probe to the target zone, to
  • SUBSTITUTE SHEET RULE 26 deliver light to the target zone, and to detect fluorescence from the target zone.
  • individual instruments may be used to deliver the probe to the target zone, to deliver light to the target zone and to detect fluorescence from the target zone.
  • fluorescence may be detected from the tissue of a target area using fiber confocal fluorescence microscopy (fCFM).
  • the subject is a human subject.
  • the subject may be a non-human animal such as equine, ovine, bovine, feline, canine or rodent, for example.
  • the first fluorophore may be connected to the first enzyme cleavable peptide sequence by a spacer.
  • the second fluorophore may be connected to the second enzyme cleavable peptide sequence by a spacer.
  • the first quencher may be connected to the first enzyme cleavable peptide sequence by a spacer.
  • the second quencher may be connected to the second enzyme cleavable peptide sequence by a spacer.
  • the spacer may be a saturated or unsaturated hydrocarbon chain, an ether, a polymer, a polyethylglycol (PEG), a poly glycol, a poly ether or similar.
  • the spacer may be a peptide.
  • the spacer is a peptide
  • the peptide may be 1-10 amino acids in length, 1-20 amino acids in length, or 1- 30 amino acids in length.
  • the spacer may be a mixture of amino acids and a saturated or unsaturated hydrocarbon chain, an ether, a polymer, a PEG, a poly glycol, a poly ether or similar.
  • the spacer may comprise 6-aminohexanoic acid (Ahx), or PEG, or an alternating chain of PEG and amino acids.
  • the linker may increase the solubility of the probe in aqueous media. Therefore, the provision of a probe comprising at least one spacer comprising a polar or hydrophilic group may allow the probe to be more readily soluble in biologically acceptable media for direct application to a target area without the requirement for additional surfactants, for example.
  • the probe may comprise at least one reporter fluorophore that is not substantially fluorescently quenched.
  • the at least one reporter fluorophore fluoresces at a wavelength that is different to the wavelength of light at which the first fluorophore and the second fluorophore fluoresces. Accordingly, the at least one reporter fluorophore fluoresces before and after the first cleavable linker is cleaved, and before and after the second cleavable linker is cleaved. Therefore, the at least one reporter fluorophore fluoresces when illuminated with a suitable wavelength of light. Accordingly, the presence or location of the
  • SUBSTITUTE SHEET RULE 26 probe in a target zone may be monitored with and without the presence of the first cleaving agent and the second cleaving agent.
  • the first probe element may be connected directly to the core.
  • the second probe element may be directly connected to the core.
  • the first probe element may be connected indirectly to the core via a linker.
  • the second probe element may be connected indirectly to the core via a linker.
  • the linker may be a saturated or unsaturated hydrocarbon chain, an ether, a polymer, a polyethylglycol (PEG), a poly glycol, a poly ether or similar.
  • the linker may comprise one or more amino acids.
  • the linker may be selected from the list: [-(lysine)-(PEG 2 )-]i- 2 , [-(PEG-k)-]i -3 , and [-(PEG-k) 0 -2-NH-(CH 2 )3-O-CH2-].
  • the linker may increase the solubility of the probe in aqueous media. Therefore, the provision of a probe comprising at least one linker comprising a polar or hydrophilic group may allow the probe to be more readily soluble in biologically acceptable media for direct application to a target area without the requirement for additional surfactants, for example.
  • the linker comprises at least one D-amino acid. More preferably, the linker comprises at least one D-lysine residue.
  • a linker comprising a D-amino acid, such as a D-lysine residue has surprisingly been found to increase the stability and longevity of the probes in the presence of enzymes such as plasmin, and factor Xa. Accordingly, the provision of at least one D-lysine residue, for example, in the linker may prevent nonspecific cleavage of the linker by enzymes present in vivo.
  • the core we refer to a common moiety that joins the plurality of probe elements to form a single unit. Accordingly, the core could be a single atom, or comprise a functional group, a saturated or unsaturated hydrocarbon chain or a polyglycol (linear, branched, or cyclical), a peptide sequence, a heterocycle, or a polymer.
  • the core is typically chosen to have the correct valency for the number of probe elements that are required to be connected to the core. For example, in embodiments with a first probe element and a second probe element, the core is chosen to have a valency of two or more, such that the first probe element and the second probe element may be connected to the core.
  • the core may comprise a plurality of connectors that may bind to each of the probe elements, thereby connecting each probe element to the core.
  • the first probe element may comprise a solubilising group at the distal end of the first probe element to the core.
  • the second probe element may comprise a solubilising group at the distal end of the second probe element to the core.
  • the or each solubilising group preferably comprises polar or hydrophilic moieties.
  • the or each solubilising group may comprise a polyethylglycol (PEG), a poly glycol, a poly ether or similar, an alcohol, acid, amine, or amide group or similar, such as an amino acid.
  • PEG polyethylglycol
  • a probe according to the present aspect comprising one or more solubilising groups may be more soluble in aqueous media than probes without one or more solubilising groups.
  • the first fluorophore is released from the probe when the first cleavable linker is cleaved, and the second fluorophore may be released from the probe when the second cleavable linker is cleaved.
  • the first quencher may be released from the probe when the first cleavable linker is cleaved, and the second quencher may be released from the probe when the second cleavable linker is cleaved.
  • a method of detecting a first and a second enzyme in a target zone comprising the steps:
  • a probe according to the first aspect comprising a first enzyme cleavable peptide sequence and a second enzyme cleavable peptide sequence;
  • significant fluorescence of the first fluorophore is indicative of the presence of the first enzyme in the target zone
  • significant fluorescence of the second fluorophore is indicative of the presence of the second enzyme in the target zone
  • the term “significant fluorescence” we refer to the fluorescence of a fluorophore that results from sufficient separation of that fluorophore from the quencher of a probe to prevent the quencher quenching the fluorescence of the fluorophore, that is above the background or, where present, autofluorescence in the target area.
  • the autofluorescence of the indigenous cells or tissue within the target area may have a shorter fluorescent lifetime than the fluorophore of the first probe.
  • the autofluorescence of the indigenous cells or tissue within the target area may reduce over time at a faster rate than that of the fluorophore of the probe. Accordingly, fluorescence observed in the target area that decays more slowly over
  • SUBSTITUTE SHEET RULE 26 time may be indicative of the probe, and fluorescence observed in the target area that reduces more quickly over time may be indicative of autofluorescence.
  • the first enzyme is MMP. More preferably, the first enzyme is MMP-2, MMP-9 or MMP-13. In some embodiments, the first enzyme cleavable peptide sequence is selectively cleavable by MMP-2, MMP-9 and MMP-13, and therefore, significant fluorescence of the first fluorophore may be indicative of the presence of MMP-2, MMP-9 and MMP-13 in the target zone.
  • the second enzyme is thrombin.
  • the first enzyme is MMP-2, MMP-9 or MMP-13 and the second enzyme is thrombin, such that significant fluorescence of the first and/or second fluorophores is indicative of a disease in which MMP-2, MMP-9 or MMP-13, and/or thrombin are overexpressed and/or activated.
  • MMP-2, MMP-9, MMP-13 and thrombin are overexpressed and/or activated in fibrotic tissue, and therefore the method of the invention may allow the presence of fibrotic tissue in the target zone to be detected.
  • the first or second cleaving enzyme may be proteinase 3 and the enzyme cleavable peptide sequence may comprise V-A-D-C-A-D-Y.
  • the first or second cleaving enzyme may be a caspase and the enzyme cleavable peptide sequence may comprise D-E-V-D.
  • the at least one fluorophore of the probe may be selected dependent on the location of the target zone. For example, if the target zone is directly observable, such as on the skin or within the lung of a subject, the at least one fluorophore may be selected to fluoresce in the visible region of the spectrum. If the target area is to be observed through the skin or tissue, the at least one fluorophore may be selected to fluoresce in the infrared region of the spectrum, such that the fluorescence of the probe is not significantly absorbed by the skin or tissue and therefore be observable through the skin of tissue. For example, the at least one fluorophore may be selected to fluoresce in the near infrared (fluorescence wavelength of between 600nm to 950nm). Infrared imaging techniques are well known to the skilled person in the art.
  • the target zone may be a portion of tissue within a subject, and the method may be carried out in vivo.
  • the portion of tissue may be a portion of the heart, lung, liver, connective tissue, skin, intestine, or joints of a subject, for example.
  • the target zone may be a portion of the lung of a subject.
  • the method of the invention may be carried out in the circulatory system, the nervous system, the digestive system or the reproductive system.
  • the target zone may be a portion of a cell culture, a tissue sample such as a biopsy sample, or a liquid sample such as a bodily fluid sample.
  • the target zone may be a target area.
  • the target zone may be a target volume.
  • the method of the invention may be carried out in vivo, ex vivo or in vitro.
  • the probes of the invention may be delivered to a target zone by any means known in the art.
  • the probes of the invention may be delivered by endoscope, spray, injection, topically, or ingestion.
  • the probes may be delivered to the target zone using an endoscope, such as a bronchoscope.
  • Illumination of a suitable wavelength to excite fluorophores of the probe may be delivered to the target zone by any conventional means known in the art.
  • the light is delivered by means of an optical fibre or similar.
  • the fluorescence from the probes in the target zone may be collected by an optical fibre or similar.
  • the fluorescence from the probes in the target zone may be collected by the same optical fibre that delivered the illumination light.
  • the collected fluorescence is typically delivered to a recording device, such as a charge-coupled device (CCD) or similar.
  • the fluorescence from the probes in the target zone may be directly collected by a recording device, such as a CCD or similar.
  • fluorescence may be detected from the tissue of a target area using fiber confocal fluorescence microscopy (fCFM).
  • fCFM fiber confocal fluorescence microscopy
  • the method may comprise the steps of (a) illuminating the target zone with an appropriate wavelength of light to excite the first fluorophore, (b) measuring the fluorescence of the first fluorophore, (c) illuminating the target zone with an appropriate wavelength of light to excite the second fluorophore, and then (d) measuring the fluorescence of the second fluorophore.
  • the first cleavable linker of the probe may be cleaved by the first enzyme at a different rate than the second cleavable linker of the probe is cleaved by the second enzyme.
  • the method may allow the fluorescence from the first fluorophore to be determined separately from the fluorescence from the second fluorophore.
  • the target zone may be illuminated by appropriate wavelengths of light appropriate to excite the first and second fluorophores, and the fluorescence from the first and second fluorophores may be determined separately.
  • the fluorescence from the first and second fluorophores may be split before being directed to a recording or measuring device.
  • Fluorescence from the probes may be imaged indirectly.
  • the fluorescence may be converted into acoustic waves by using photoacoustic imaging.
  • Photoacoustic imaging may allow high resolution images of the target area to be generated.
  • the subject is a human subject.
  • the subject may be a non-human animal such as equine, ovine, bovine, or rodent, for example.
  • the invention extends in a third aspect to a kit of parts comprising the probe according to the first aspect in a suitable diluent or buffer.
  • an optical probe comprising at least one fluorophore connected to at least one quencher by a cleavable linker; the at least one fluorophore being substantially fluorescently quenched by the at least one quencher when connected to the at least one quencher by the cleavable linker, and the at least one fluorophore is separated from the at least one quencher when the cleavable linker is cleaved; wherein the cleavable linker comprises an enzyme cleavable peptide sequence comprising one of SEQ ID NO.15 to SEQ ID NO.38 and is selectively cleavable by thrombin.
  • the enzyme cleavable peptide sequence comprises one or more D-amino acid residues.
  • the enzyme cleavable peptide sequence comprises one of SEQ ID NO.25, SEQ ID NO.31 , or SEQ ID NO.37. More preferably, the enzyme cleavable peptide sequence comprises SEQ ID NO.25.
  • the inventors have surprisingly found that the inclusion of a D-amino acid at certain positions within the enzyme cleavable peptide sequence results in the enzyme cleavable peptide sequence being more resilient to non-specific cleavage by enzymes such as plasmin, MMP and factor Xa, whilst still being selectively cleavable by thrombin. Accordingly, the probes of the invention allow the presence of thrombin to be reliably and accurately detected in vivo and in tissue samples where proteinases are present.
  • the at least one fluorophore and at least one quencher of the probe of the present aspect may be selected as has been described above in relation to the first aspect, and for the sake of brevity, this text is not repeated here.
  • the at least one fluorophore may be connected to the enzyme cleavable peptide sequence by a spacer.
  • the at least one quencher may be connected to the enzyme cleavable peptide sequence by a spacer.
  • the spacer may be a saturated or unsaturated hydrocarbon chain, an ether, a polymer, a polyethylglycol (PEG), a poly glycol, a poly ether or similar.
  • the spacer may be a peptide. In embodiments where the spacer is a peptide, the peptide may be 1-10 amino acids in length, 1-20 amino acids in length, or 1-30 amino acids in length.
  • the spacer may be a mixture of amino acids and a saturated or unsaturated hydrocarbon chain, an ether, a polymer, a PEG, a poly glycol, a poly ether or similar.
  • the spacer may comprise 6-aminohexanoic acid (Ahx), or PEG, or an alternating chain of PEG and amino acids.
  • the linker may increase the solubility of the probe in aqueous media. Therefore, the provision of a probe comprising at least one spacer comprising a polar or hydrophilic group may allow the probe to be more readily soluble in biologically acceptable media for direct application to a target area without the requirement for additional surfactants, for example.
  • a probe element may comprise at least one fluorophore connected to an enzyme cleavable peptide sequence. Accordingly, the probe element may be depicted as:
  • A a spacer, which may be individually present or absent
  • Seq a peptide sequence comprising the enzyme cleavable peptide sequence
  • the probe comprises a single probe element and a quencher.
  • the probe may comprise a plurality of probe elements, and each of the plurality of probe elements may comprise at least one fluorophore connected to an enzyme cleavable peptide sequence.
  • Optical probes known in the art are often unstable in vivo or in other cases, such as tissue lysates, tissue samples or samples of bodily fluids, due to processes such as non-specific cleavage of the probe by enzymes. Such non-specific cleavage may produce fluorescence of the probe and be mistaken as indicative of the presence of MMP in a target area. Alternatively, non-specific cleavage may break the probe down and prevent the probe from being observed at all.
  • an optical probe comprising a plurality of probe elements in combination with determined sequences improves the stability of the probe in vivo.
  • the inventors suggest that the provision of a plurality of probe elements may shield each probe element from unspecific cleavage, thereby increasing the structural resilience of the probe.
  • a probe comprising a plurality of probe elements provides an increased number of fluorophores per probe, thereby providing an increase in fluorescence per probe. Furthermore, this increase in fluorescence per probe may provide a greater signal-to-noise ratio that may allow lower concentrations of thrombin to be detected, or lower concentrations of the probe to be used.
  • the probe may comprise a plurality of probe elements connected by a core. Each probe element within the plurality of probe elements may be connected to the core directly. Each probe element within the plurality of probe elements may be connected to the core indirectly via a linker.
  • the meaning of the term "core" is described above in relation to the first aspect. The person skilled in the art will appreciate that the linker of the present aspect may be selected as described above in relation to the first aspect and for the sake of brevity, this text is not repeated here.
  • the probe may comprise at least two probe elements.
  • the probe may comprise at least three probe elements. For example, the probe may comprise two or three probe elements. The more probe elements, the greater the enhancement of the fluorescence upon cleavage of the enzyme cleavable peptide sequence.
  • the optical probe of the present aspect may be suitable to be incorporated into an optical probe according to the first aspect of the invention.
  • the optical probe may comprise two probe elements and two or more, for instance three, fluorophores ( Figures 1 B-D).
  • an optical probe comprising a first probe element, and a second probe element; the first probe element and the second probe element connected to a core; the first probe element comprising a first fluorophore, a first cleavable linker and a first quencher, the first fluorophore connected to the first quencher by the first cleavable linker and the core, the first quencher being a second fluorophore and being connected to the core; the second probe element comprising the second fluorophore, a second cleavable linker and a second quencher, the second fluorophore connected to the second quencher by the core and the second cleavable linker; the first fluorophore being substantially fluorescently quenched by the first quencher when connected to the first quencher by the first cleavable linker and core; the second fluorophore being substantially fluorescently quenched by the second quencher when connected to the second quencher by the second
  • the first quencher which is also the second fluorophore, is quencher for the first fluorophore of cleavable linker 1 ( Figure 1 B-D).
  • the second quencher is not a fluorophore ( Figure 1 B).
  • the second quencher may be selected from BHQ-3 (strong absorption from 620 nm to 730 nm), QSY21 (intense absorption about 661 nm maxima but no fluorescence making it useful as an acceptor in fluorescence resonance energy transfer (FRET) applications).
  • the second quencher may be a third fluorophore ( Figures 1 C, D).
  • the second quencher When the second quencher is a third fluorophore, it may or may not be fluorescently quenched by the first quencher. In one embodiment, when the second quencher is a third fluorophore, it is not fluorescently quenched by the first quencher. In this way, the first quencher quenches the first fluorophore until cleavage of the first cleavable linker and the second quencher quenches the second fluorophore until cleavage of the second cleavable linker.
  • the second quencher/third fluorophore would fluoresce under illumination both before and after cleavage of the second cleavable linker, because it is not quenched by a quencher on the probe ( Figure 1 C).
  • the second quencher may be methylblue derivatives, Alexa Fluor® 680 or IRDye 680, all of which are fluorescent and capable of quenching a second fluorophore which is the first quencher, such as a seminapthorhodamine.
  • a second quencher/third fluorophore can be provided which is substantially fluorescently quenched by the first quencher.
  • the first quencher which is also the second fluorophore, is quencher for the first fluorophore of cleavable linker 1 and the third fluorophore of cleavable linker 2 (the second quencher) at the same time.
  • the third fluorophore (the second quencher) is quencher for the second fluorophore of cleavable linker 2.
  • the second quencher/third fluorophore will not fluoresce under illumination before cleavage of the second cleavable linker, but will after cleavage ( Figure 1 D).
  • the first fluorophore and the first quencher may be a FRET pair
  • the second fluorophore (first quencher) and second quencher may be a FRET pair, such that the first quencher and the second quencher are a FRET pair
  • the first quencher (second fluorophore) may be a Seminaphthorhodafluor (SNARF® dyes)
  • At least one fluorophore may be an independent reference fluorophore for the enzyme activity; at least one fluorophore may be a pH sensor and/or at least one fluorophore may be a pC0 2 sensor or Ca 2+ sensor; and at least one fluorophore may display Long-Wavelength Dual-Emission.
  • a Long-Wavelength Dual-Emission fluorophore is one which fluoresces at two sets of wavelengths in response to a single excitation wavelength.
  • the two emission wavelengths respond differently to pH. The higher wavelength emission can increase upon alkalinisation, whilst the lower wavelength emission remains constant or decreases upon alkalinisation.
  • SNARF® dyes Seminaphthorhodafluor (SNARF® dyes) carboxylate derivatives can be used as the fluorophore for Long-Wavelength Dual-Emission, pH sensor, pC0 2 , Ca 2+ sensor and as a fluorophore for reporting thrombin and/or MMP-9 activity, and the longer wavelength of emission is pH dependent.
  • an optical probe is provided in which the first, second and third fluorophores fluoresce at different wavelengths.
  • a method of detecting thrombin activity in a target zone comprising the steps:
  • the term “significant fluorescence” we refer to the fluorescence of a fluorophore that results from sufficient separation of that fluorophore from the quencher of a probe to prevent the quencher quenching the fluorescence of the fluorophore, that is above the background or, where present, autofluorescence in the target area.
  • the autofluorescence of the indigenous cells or tissue within the target area may have a shorter fluorescent lifetime than the fluorophore of the first probe.
  • SUBSTITUTE SHEET RULE 26 within the target area may reduce over time at a faster rate than that of the fluorophore of the probe. Accordingly, fluorescence observed in the target area that decays more slowly over time may be indicative of the probe, and fluorescence observed in the target area that reduces more quickly over time may be indicative of autofluorescence.
  • the method may comprise the step of determining the fluorescence intensity of the target zone over a period of time, determining the rate of decay of fluorescence during the period of time, and determining the fluorescence intensity of that fluorescence with a slower rate of decay, wherein the fluorescence with a slower rate of decay corresponds to the fluorescence of the probe.
  • the probes of the fourth aspect are surprisingly resilient to non-specific cleavage whilst retaining specificity for cleavage by thrombin. Therefore, the method of the present aspect allows the detection of thrombin in the presence of similar cleaving enzymes, such as plasmin, MMP and factor Xa.
  • Thrombin has been identified as playing a critical role in early fibroproliferation, especially in fibroproproliferative ARDS, and during pulmonary fibrosis thrombin activity is significantly increased. Therefore, the provision of a method that allows the reliable detection of thrombin in the presence of other enzymes such as MMP, plasmin and factor Xa, may allow the detection of fibroproliferation and/or pulmonary fibrosis.
  • Thrombin probes can detect the upregulation of the coagulation cascade.
  • Such probes can be used in the diagnosis of diseases associated with the upregulation of the coagulation cascade, such as cancer.
  • probes to detect thrombin can be used in the diagnosis of one or more of thrombosis, atherosclerosis, cancer and coronary heart disease.
  • the at least one fluorophore of the probe may be selected dependent on the location of the target zone. For example, if the target zone is directly observable, such as on the skin or within the lung of a subject, the at least one fluorophore may be selected to fluoresce in the visible region of the spectrum. If the target area is to be observed through the skin or tissue, the at least one fluorophore may be selected to fluoresce in the infrared region of the
  • the at least one fluorophore may be selected to fluoresce in the near infrared (fluorescence wavelength of between 600nm to 950nm). Infrared imaging techniques are well known to the skilled person in the art.
  • the target zone may be a portion of tissue within a subject, and the method may be carried out in vivo.
  • the portion of tissue may be a portion of the heart, lung, liver, connective tissue, skin, intestine, or joints of a subject, for example.
  • the target zone may be a portion of the lung of a subject.
  • the method of the invention may be carried out in the circulatory system, the nervous system, the digestive system or the reproductive system.
  • the target zone may be a portion of a cell culture, a tissue sample such as a biopsy sample, or a liquid sample such as a bodily fluid sample.
  • the target zone may be a target area.
  • the target zone may be a target volume.
  • the method of the invention may be carried out in vivo, ex vivo or in vitro.
  • the probes of the invention may be delivered to a target zone by any means known in the art.
  • the probes of the invention may be delivered by endoscope, spray, injection, topically, or ingestion.
  • the probes may be delivered to the target zone using an endoscope, such as a bronchoscope.
  • Illumination of a suitable wavelength to excite fluorophores of the probe may be delivered to the target zone by any conventional means known in the art.
  • the light is delivered by means of an optical fibre or similar.
  • the fluorescence from the probes in the target zone may be collected by an optical fibre or similar.
  • the fluorescence from the probes in the target zone may be collected by the same optical fibre that delivered the illumination light.
  • the collected fluorescence is typically delivered to a recording device, such as a charge-coupled device (CCD) or similar.
  • the fluorescence from the probes in the target zone may be directly collected by a recording device, such as a CCD or similar.
  • a CCD charge-coupled device
  • SUBSTITUTE SHEET RULE 26 deliver the probe to the target area, to deliver light to the target zone and to detect fluorescence from the target area.
  • fluorescence may be detected from the tissue of a target area using fibered confocal fluorescence microscopy (fCFM).
  • fCFM fibered confocal fluorescence microscopy
  • the fluorophore may also be an optoacoustic fluorophore.
  • Optoacoustic fluorophores generate ultrasound when excited by intense illumination, such as by a laser tuned to the absorption range of the fluorophore. The generated ultrasound can then be detected.
  • Probes comprising optoacoustic fluorophores can be illuminated and detected by multispectral optoacoustic tomography (MSOT). MSOT illuminates the target zone with light pulses at multiple wavelengths and detects the acoustic waves generated by the thermoelastic expansion of the environment surrounding the optoacoustic fluorophore in response to the pulses.
  • MSOT multispectral optoacoustic tomography
  • the subject is a human subject.
  • the subject may be a non-human animal such as equine, ovine, bovine, or rodent, for example.
  • the presence of thrombin in the target area may be indicative of fibrosis within the target zone.
  • the invention extends in a seventh aspect to a kit of parts comprising the probe of the fourth aspect in a suitable diluent or buffer.
  • the probe comprises a first probe element having a solubilising tail, thrombin cleavable sequence, fluorescent dye 1 and quencher Q1 forming a FRET probe element and a second probe element bound to the first probe element by a core, said second probe element having a solubilising tail, MMP-9 cleavable sequence, fluorescent dye 2 and quencher Q2 forming a FRET probe element;
  • the probe comprises a first probe element having a spacer, first fluorophore F1 , first cleavable linker 1 and first quencher Q1 which is a
  • SUBSTITUTE SHEET RULE 26 second fluorophore F2 connected to a core, first fluorophore F1 and first quencher Q1 forming a FRET pair, a second probe element having a spacer, second quencher Q2, second cleavable linker 2 and a second fluorophore F2, second fluorophore F2 and second quencher Q2 forming a FRET pair and the core connected to both first and second probe elements;
  • the probe comprises a first probe element having a spacer, first fluorophore F1 , first cleavable linker 1 and first quencher Q1 which is a second fluorophore F2 connected to a core, first fluorophore F1 and quencher Q1 forming a FRET pair, a second probe element having a spacer, second quencher Q2 which is a third fluorophore F3, second cleavable linker 2 and a second fluorophore F2, second fluorophore F2 and second quencher Q2 forming a FRET pair and the core connected to both first and second probe elements; and
  • the probe comprises a first probe element having a spacer, first fluorophore F1 , first cleavable linker 1 and first quencher Q1 which is a second fluorophore F2 connected to a core, first fluorophore F1 and first quencher Q1 forming a FRET pair, a second probe element having a spacer, second quencher Q2 which is a third fluorophore F3, second cleavable linker 2 and a second fluorophore F2, second fluorophore F2 and second quencher Q2 forming a FRET pair and third fluorophore F3 and first quencher Q1 forming a FRET pair and a core connected to both first and second probe elements.
  • F1/F2/F3 represent fluorophore
  • Q1/Q2/Q3 represent quencher together with cleavable linker 1/2/3, core and spacer.
  • Figure 3 Evaluation of 1 st and 2 nd generation probes. Data shown represent the average fold change in fluorescence over background signal provided by probe (1 ⁇ and 0.1 ⁇ with MMP-9) with exogenous enzymes after 6 min using a multiwell plate fluorimeter at excitation/emission 485/528 nm and 640/670 nm. Recombinant human catalytic domain MMP-9 was used at 30nM. Recombinant human Thrombin and Plasmin were used at 5U/ml and 30nM respectively.
  • Figure 4 Data represents the fluorescence signal provided by probe (1 ⁇ ) after 5 min using a multiwell plate fluorimeter at excitation/emission 485/528nm (left) and 640/670 (right).
  • A Fluorescence signal in presence of enzymes and inhibitors
  • B Average fold change in fluorescence over background signal with exogenous enzymes.
  • Recombinant human catalytic domain MMPs -1 , -2, -3, -7, -8, -9, -10, -1 1 , -12, -13 were used at 30nM.
  • Recombinant human Thrombin, Plasmin and Factor Xa were used at 5U/ml, 30nM and 0.5 ⁇ respectively.
  • Anti-thrombin III inhibitor was used at 3.85 ⁇ and Marimastat at 200 nM.
  • FIG. 6 Ex-vivo assay with human tissue. Data shown represent the fluorescence signal provided by probe AMF-185 (1 ⁇ ) with human fibrotic tissue without (upper chart) and with (lower chart) Marimastat (broad spectrum MMP inhibitor) using a multiwell plate fluorimeter at excitation/emission 485/528 nm and 640/670 nm.
  • FIG. 7 MALDI spectra after assay with human fibrotic lung tissue shows intact probe and additional fragment for Thrombin cleavage (top) and intact probe in presence of Pl-Protease inhibitor (bottom).
  • FIG. 13 MALDI spectra (A) and HPLC trace (B) for compounds AMF-152 (top), AMF-162 (middle) and AMF-185 (bottom).
  • Figure 15 Evaluation of different thrombin specific peptide sequences.
  • Data represents the fluorescence signal provided by 1 st generation probes (1 ⁇ ) using a multiwell plate fluorimeter at excitation/emission 485/528nm after 1 1 min in the presence of Thrombin, human bronchoalveolar lavage fluid (BALF), sheep BALF, human neutrophil lysate, human neutrophil supernatant, human macrophages supernatant and murine macrophages supernatant.
  • BALF human bronchoalveolar lavage fluid
  • BALF human neutrophil lysate
  • human neutrophil supernatant human macrophages supernatant
  • murine macrophages supernatant murine macrophages supernatant.
  • Figure 16 Data represents the fluorescence signal provided by probe SVC-1 13 (1 ⁇ ) using a multiwell plate fluorimeter at excitation/emission 485/528nm after 5 min in presence of Thrombin, plasmin and Factor Xa.
  • Figure 17 Data represents the average fold change in fluorescence over background signal for thrombin probes (1 ⁇ ) with exogenous enzymes Thrombin and Plasmin used at 5U/ml and 0.5 ⁇ respectively, after 5 min using a multiwell plate fluorimeter at excitation/emission 485/528nm.
  • Thrombin probes containing D-amino acid residues showed selectivity for Thrombin over Plasmin (2 nd generation compounds AMF-166, AMF-167 and AMF-168)
  • Figure 18 Data represents the change in fluorescence signal provided by probe AMF-166 (A) and AMF-176 (B) (10 ⁇ ) with exogenous enzymes after 32 min using a multiwell plate fluorimeter at excitation/emission 485/528nm.
  • Recombinant human MMPs -1 , -2, -3, -7, -8, - 9, -10, -11 , -12, -13 were used at 30nM.
  • Recombinant human Thrombin, Plasmin and Factor Xa were used at 5U/ml, 0.5 ⁇ and 30nM respectively.
  • Figure 19 Data represents the fluorescence signal provided by probe AMF-176 (10 ⁇ ) with exogenous enzymes after 5 min using a multiwell plate fluorimeter at excitation/emission 485/528nm.
  • Recombinant human MMP-9 and Plasmin were used at 30nM.
  • Recombinant human Thrombin was used at 5U/ml respectively.
  • Anti-thrombin III inhibitor was used at 3.85 ⁇ and Marimastat at 200 nM.
  • FIG. 20 Ex-vivo assay with human tissue. Data shown represent the fluorescence signal provided by: (A) probe AMF-166 (1 ⁇ ) and (B) AMF-176 (10 ⁇ ) with human fibrotic tissue homogenate at excitation/emission 485/528 nm.
  • Figure 21 Ex-vivo assay with human tissue.
  • A Structure and MALDI spectra provided by probe AMF-166 in the presence of Thrombin and
  • B after treatment with five different human fibrotic tissues showing intact probe (i) and/or the fragment obtained after specific cleavage by Thrombin (ii).
  • the probes of the invention are made up of Thrombin and MMP-9 cleavable linkers, as well as two fluorophores and two quenchers strategically placed ( Figure 2).
  • the probes can be constructed following different strategies, varying in fluorophore/quencher selection and in general bio-orthogonal conjugation strategies', e.g. -metal and metal-free azide-alkyne cycloaddition or inverse electron-demand Diels-Alder reaction.
  • Individual probes containing complementary functional groups have been synthesized by standard Fmoc solid-phase peptide synthesis. With these compounds the final coupling reaction was done by bio-orthogonal conjugation strategies. Specificity of DualProbes 1 st and 2 nd Generation
  • Dualprobes 1 st and 2 nd generation are activated by Thrombin and MMP-9 showing an increase in the fluorescent signal.
  • the enzymatic cleavage of the peptide substrate causes the disruption of the FRET system and the fluorescence for each dye is recovered and measured independently at two different wavelengths (see Figure 3).
  • the main achievement in 3 rd Generation dualprobes is the selectivity for Thrombin and MMP-9 over plasmin.
  • the fluorescence signal for each dye increases after enzymatic activation and can be knocked-down (i.e. reduced) with known inhibitors for each enzyme (Figure 4).
  • Dyes/quenchers act independently without interfering in each other response.
  • MALDI spectra and HPLC trace after enzymatic reaction confirms the right and specific cleavage by exogenous enzymes MMP-9 and Thrombin ( Figures 5-7).
  • the first generation of thrombin probes were synthesized containing:
  • -Different aminoacid sequences (all containing L-aa) were evaluated initially as thrombin substrates. The best sequence was selected based on its activation by thrombin, inhibition with thrombin inhibitors, stability to Factor Xa, human BALF, sheep BALF, human neutrophil lysate, human neutrophil supernatant, human macrophages supernatant and murine macrophages supernatant (Figure 15).
  • SUBSTITUTE SHEET RULE 26 -Different linkers containing hydrophilic or charged groups: ethylenglycol units (PEG), L- Lysine, and alternative ethylenglycol units (PEG) and D-Lysine.
  • the linkers improve the aqueous solubility of the compounds.
  • the presence of unnatural D-amino acid improves the in vivo stability of the compounds making them resistant to proteinases.
  • Additional reactive groups can be added to allow subsequent reaction and formation of multiple probes. These groups do not affect the activity of the probe.
  • Improvements from the first generation are achieved when modifying some key amino acids with their D-unnatural amino acid counterparts. That modification generate probes that are selective to Thrombin over plasmin ( Figure 17). Also fluorophore and quenchers positions can be exchanged without altering the activity of the probes (i.e. AMF-166 vs AMF-176, Figure 18) and additional functional groups are also tolerated (i.e. alkyne group in AMF-176) and do not affect the activation or specificity of the probe. In addition, the activity of the probes was tested in the presence of thrombin inhibitors to show that the increase in fluorescence of the probes was the result of cleavage by thrombin (see Figure 19).
  • 4-Pentynoic acid succinimidyl ester A solution of 4-pentynoic acid (0.5 g, 5.1 mmol) and /V-Hydroxysuccinimide (0.59 g, 1 eq) in EtOAc-Dioxane (1 : 1 , 50 mL) was stirred at 0°C and DCC (1.0g, 1 eq) was added allowing the mixture to reach room temperature (rt) and kept at these conditions for 12 h. The DCU formed was filtrated and the filtrate concentrated under vacuum. EtOAc (100 mL) was added and washed with 5% NaHC0 3 (2x40 mL), water (40 mL) and brine (40 mL).
  • Fmoc-Lys-(/V-4-pentynoyl)-OH Fmoc-Lys-OH HCI (1.67g, 1 eq) was dissolved in DMF (12 mL), DIPEA (0.79 mL, 1.1 eq) was added followed by dropwise addition of a solution of 4- Pentynoic acid succinimidyl ester (0.81 g, 1 eq) in anhDMF (4 mL). The reaction mixture was stirred for 3h. The solvent was removed under vacuum. The oily residue was dissolved in EtOAc (50 mL) and washed with 5% citric acid solution (2x25 mL), water (25 mL) and brine (25 mL).
  • Boc-Lys/V-4-Pentynoyl]-OH Boc-Lys-OH (1.25 g, 5.1 mmol) was dissolved in anhDMF (15 mL), DIPEA (0.97 mL) was added followed by dropwise addition of a solution of 4-Pentynoic acid succinimidyl ester (5.1 mmol) in anhDMF (8 mL). The reaction mixture was stirred at rt for 3h. The solvent was removed under vacuum. To the crude HCI 1 N (30 mL) was added and extracted with EtOAc (3x40 mL). The combined organic phase was dried over anhNa 2 S0 4 and evaporated under vacuum to afford a white solid (1.36g, 82%).
  • MethylRed-Lys-(/V-4-pentynoyl)-OH Boc-Lys[/V-4-Pentynoyl]-OH (1.0 g, 3.0 mmol) was dissolved in 20%TFA in dichloromethane (10 mL) and the resulting mixture stirred for 3h. The solvent was removed under vacuum and co-evaporated with toluene. The crude was dissolved in anhDMF (5 mL). MethylRed-NHS ester [R. C. Brown, Z. Li, A. J. Rutter, X. Mu, O. H. Weeks, K. Smith and I. Weeks, Org. Biomol. Chem. 2009, 7, 386-394.] (1.1 g, 1 eq)
  • FRET peptide sequences for MMP and thrombin were individually synthesized by standard Fmoc solid-phase peptide chemistry. Dyes and quenchers were coupled also by solid phase. General procedures are as follows:
  • Fmoc-Rink linker (4-[(R,S)-a-[1-(9H-Fluoren-9-yl)-methoxy- formamido]-2,4-dimethoxybenzyl-phenoxyacetic acid) (0.54 g, 1.0 eq) was dissolved in DMF (10 mL) and Oxyma (0.14 g, 1.0 eq.) was added and the mixture was stirred for 10 min.
  • Aminoacid coupling A solution of the appropriate D- or L-amino acid (3.0 eq per amine) and Oxyma (3.0 eq) in DMF (0.1 M) was stirred for 10 min. DIC (3.0 eq) was added and stirred for 1 min. The pre-activated mixture was then added to the resin pre-swollen in DCM and the reaction heated at 50°C for 30 min. The solution was drained and washed with DMF (3x10 ml_), DCM (3x10 ml_) and MeOH (3x10 ml_). The completion of the coupling and deprotection reactions was monitored by Kaiser test or Chloranil test when secondary amines are involved. The side chain protecting group used was Boc for arginine, tryptophan and lysine. Fmoc-Lys(Dde)-OH was used as orthogonal reagent to introduce the dyes.
  • Coupling of other carboxylic acids Coupling of ⁇ 2-[2-(Fmoc-amino)ethoxy]ethoxy ⁇ acetic acid (PEG), 5-Carboxyfluorescein (FAM), Fmoc-Lys(N 3 )-OH and MethylRed-Lys-(4- pentynoyl)-OH was done following the same procedure described for Aminoacid coupling.
  • Dde deprotection (a) Dde deprotection in non-containing Fmoc peptides was done following the next procedure: to the resin pre-swollen in DCM was added 2% hydrazine in DMF and stirred at rt (5x10 min). The solution was drained and the resin washed with DMF (3x10 ml_), DCM (3x10 ml_) and MeOH (3x10 ml_). (b) Selective Dde deprotection in Fmoc- protected peptides was done with a solution containing Imidazole (1.35 mmol) and Hydroxylamine hydrochloride (1.80 mmol) in NMP (5 ml_). [Diaz-Mochon, J.
  • Sulfo-Cy5 dye coupling A solution containing sulfo-Cy5 (1 eq) in anh DMF (10 mg/mL) was activated with N,N,N',N'-Bis(tetramethylene)-0-(N-succinimidyl)uronium hexafluorophosphate (HSPyU) (1 eq) and DIPEA (3 eq) at 40°C for 1 h. Once the activation is complete the solution is added to the resin together with DIPEA (3 eq) and shaken at rt overnight. The solution was drained and the resin washed with DMF until colourless wash solution, DCM (3x5 ml_) and MeOH (3x5 ml_).
  • MethylRed-NHS coupling MethylRed-NHS ester (1 eq) coupling in solid phase was done in anhDMF (0.1M) containing DIPEA (3 eq) at rt for 12 h. The solution was drained and the resin washed with DMF until colourless wash solution, DCM (3x5 mL), MeOH (3x5 mL) and finally ether (3x5 mL).
  • Thrombin substrates were built on resin, cleaved and purified following the general procedures previously described.
  • Thrombin substrates were built on resin, cleaved and purified following the general procedures previously described.
  • MMP substrates were built on resin, cleaved and purified following the general procedures previously described.
  • AMF-140 aa aa n -: -GPKGLKG- ;
  • X -CH2-NH-CO-CH2OCH2CH2OCH2CH2- for AM F- 154-03: aa aa n -:-PFGNIeKpA;
  • X -CH2-NH-CO-CH2OCH2CH2OCH2CH2- for AMF-181 : aa aa n -: -PFGNIeK A;
  • the dendrimer scaffold was synthesised by following the prior art reported in WO 2012/136958 A2 (Aslam et al).
  • Multi-valent probe synthesis required the preparation of the monomer (V) which was synthesised in six steps 1 as shown in Scheme 6.
  • Monomer (V) was prepared by the 1 ,4 addition of the hydroxy groups of 1 , 1 , 1 - fr/ ' s(hydroxymethyl)amino-methane onto acrylonitrile, followed by amino group protection (Boc). Reduction of the nitrile groups with Pt0 2 /H 2 gave (III) which was treated with DdeOH to give the tris-Dde protected amine (IV). Following removal of the Boc protecting group, the isocyanate (V) was prepared following the procedure of Knolker. 2
  • MMP substrates were built on resin, cleaved and purified following the general procedures previously described.
  • MethylRed-K ( 4-pentynoyl )-PEG-NleWPRGWR(D)LK(5-FAM)-PEG-(D)K-PEG-(D)K-PEG-(D)K
  • alkyne-peptide fragment (AM F- 146-01 or AMF-176) (50 ⁇ _, 1 mM), azide-peptide fragment (AMF-140, AMF-154-03 or AMF-181) (50 ⁇ _, 1 mM), premixed CuS0 4 and THPTA (40 ⁇ _ CuS0 4 20mM and 80 ⁇ _ THTPA 50 mM), aminoguanidine hydrochloride (250 ⁇ _, 100 mM) and finally sodium ascorbate (250 ⁇ _, 100 mM).
  • Optimized conditions for the click reaction using biomolecules were used: in an eppendorf tube the following aqueous reagents were mixed: alkyne-peptide three-branched fragment (AMF-212) (50 ⁇ _, 1 mM), azide-peptide fragment (AMF-216) (50 ⁇ _, 1mM), premixed CuS0 4 and THTPA (40 ⁇ _ CuS0 4 20mM and 80 ⁇ _ THPTA 50 mM), aminoguanidine hydrochloride (250 ⁇ _, 100 mM) and finally sodium ascorbate (250 ⁇ _, 100 mM).
  • alkyne-peptide three-branched fragment AMF-212
  • AMF-216 azide-peptide fragment
  • premixed CuS0 4 and THTPA 40 ⁇ _ CuS0 4 20mM and 80 ⁇ _ THPTA 50 mM
  • aminoguanidine hydrochloride 250 ⁇ _, 100 mM
  • sodium ascorbate 250 ⁇
  • Tetrazine ligation between fragments containing Tetrazines and fragments containing dienophiles such as Norbornene can be used for the synthesis of dualprobes.
  • thrombin probe with FAM/MR containing Norbornene (AMF1 19-03 m/z calc for C 14 4H212N 33 0 3 4 [M+H] + 2949.476, obs: 2948.724) is shown below:
  • MMP substrate peptide containing Tetrazine (m/z calc for [M+H] + 2223.634, obs: 2224.565) is shown below:
  • Tetrazine ligation in solution phase In an eppendorf tube aqueous solutions of tetrazine- peptide fragment (20 ⁇ _, 1 mM) and norbornene-peptide fragment (20 ⁇ _, 1 mM) were mixed and the reaction was allowed to proceed for 2h. The tetrazine adduct was detected by MALDI. (AMF1 16 m/z calc for C212H361 N58O56 [M+H] + 4618.563, obs: 4617.500).
  • Thrombin probe containing dibenzocyclooctyl (DBCO), a strained alkyne moiety The functionalised peptide was synthesized by either standard Fmoc solid-phase peptide chemistry or solution phase functionalization of fully protected probes by following the general procedures.
  • DBCO-NHS Dibenzocyclooctyne-NHS coupling in solution: To a solution of fully protected peptide fragments in anhDMF (0.1 M) a solution of DBCO-NHS ester (1 eq.) in anhDMF (0.1 M) containing DIPEA (3 eq.) was added and the reaction mixture was kept at rt for 12 h. The reaction mixture was evaporated in vacuo, washed with water and lyophilized to afford the solid compound. To characterise the isolated peptide fragment, it is further treated with 20% TFA in DCM at room temperature.
  • Scheme 11 Structure for DBCO-Thrombin probe: Strain-Promoted Alkyne-Azide Cycloadditions (SPAAC) with protected peptides. Reaction scheme of ligated peptides. SVC-01-134 (m/z calc for C163H228N36O43 [M+H]+ 3276.8, obs: 3276.337).
  • SPAAC reaction in water In an eppendorf tube aqueous solutions of DBCO-peptide (20 ⁇ _, 1 mM) and azide-peptide fragment (20 ⁇ _, 1 mM) or (2-(2-azidoethoxy)ethoxy)acetic acid (1 mg) were mixed and the reaction was allowed to proceed for 2h. The click adduct was detected by MALDI.
  • the enzyme assays were run in a 384-well format on a PCR opaque microplate (Thermo Scientific). All dilutions and reactions were prepared in MMP buffer (50mM Tris, 10mM CaCI 2 , 0.15M NaCI, 0.05% Brij-35, pH 7). Proteolytic activity was determined by calculating the fold change in fluorescence over background signal provided by the corresponding dilution of the probe and/or inhibitors with exogenous enzymes using a-multiwell plate fluorimeter (Synergy H1 Hybrid Reader, BioTek instruments Ltd) at excitation/emission 485/528nm.
  • Recombinant human MMPs (Catalytic domain MMP-1 , -2, -3, -7, -8, -9, -10, -11 , -12, -13 (Enzo Life Sciences) and Full-length MMP-2 , -9, -12 and -13 (Merck/Millipore)) were used at 30nM.
  • Pro- MMP- 13 (R & D Systems) was activated by incubating with 1 mM 4- aminophenylmercuric acetate (APMA) for 2hrs at 37°C.
  • APMA 4- aminophenylmercuric acetate
  • Marimastat Teoris Bioscience
  • AZD1236 AstraZeneca
  • Inhibitor I Sigma-Aldrich
  • SB-3CT Sigma- Aldrich
  • Human fibrotic lung tissue biopsies were obtained from Idiopathic Pulmonary Fibrosis-(IPF) patients at the Royal Infirmary, Edinburgh. Under sterile condition, the tissue was dissected and stored at -70°C for further analysis. Sheep fibrotic lung tissue biopsies were obtained from Ovine Pulmonary Adenocarcinoma-(OPA) animals at the Roslin Institute, Edinburgh. Under sterile condition, the tissue was dissected and stored at -70°C for further analysis. For the preparation of tissue supernatant, frozen tissue was suspended in PBS and homogenised (Bio-Gen PRO200 homogeniser, Pro-Scientific) on ice.
  • Probes 1-6 correspond to probes for MMP
  • probes 7-20 correspon to probes for thrombin
  • probes 21-26 correspond to probes for both MMP and thrombin.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Physics & Mathematics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pathology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Optical probes are presented, the probes comprising a first probe element, and a second probe element connected to a core. The first probe element comprises a first fluorophore connected to a first quencher by a first cleavable linker. The second probe element comprises a second fluorophore connected to a second quencher by a second cleavable linker. The first fluorophore is separated from the first quencher when the first cleavable linker is cleaved, and the second fluorophore is separated from the second quencher when the second cleavable linker is cleaved. Methods of detecting a first and second enzyme using the optical probe are also presented.

Description

Optical Probe for Thrombin Field of the Invention The invention relates to the field of optical probes, more specifically to optical probes for the detection of target species, such as thrombin or two or more target species such as thrombin and a metalloproteinase.
Background of the Invention
Fibroproliferative diseases of the lung and other organs constitute a heavy burden of morbidity and untimely deaths. Fibrosis results in permanent loss of the tissue's ability to function optimally. In the lung, gas exchange ability is impaired by the formation of scar tissue. Often, Idiopathic Pulmonary Fibrosis (IPF) is diagnosed late (on CT scans/lung biopsy) and has a high mortality with a median survival time from diagnosis of 3 years. It is currently impossible to identify patients with Adult Respiratory Distress Syndrome (ARDS) and other inflammatory lung diseases that are developing secondary fibrosis. Moreover there are no effective therapies for fibrosis despite it being a highly active cellular process which should be accessible to intervention. Part of the problem is the time required to establish drug effectiveness in vivo and the poor utility of existing biomarkers. Thus, there is an urgent need to develop diagnostic methodologies that will permit the more effective and rapid determination of both disease activity and efficacy of emerging anti-fibrotic drugs.
Molecular imaging offers a viable approach to interrogate non-invasively living samples in real time with spatial resolution when combining the technical instrumentation with adequate optical imaging probes. Optical probes are typically developed for each specific target for effective diagnostic imaging (Biochemistry 2010, 49, 1364-1376). Activatable optical probes can provide functional details of molecular events, and provide advantages such as providing information at the molecular level. Furthermore, they can be used with a small portable system, provide information quickly, are low cost, and can be microdosed (< 100 μg), thereby reducing the risk of side effects. fCFM (fibre-Confocal Fluorescence Microscopy) has been used for studying in vivo the alveolar structure of the human lung during bronchoscopy [Eur. Resp. J. 2009, 33, 974-985. Proc. Am. Thorac. Soc. 2009, 4, 444-449]. Its use in combination with optical probes for specific enzymes can provide valuable information.
1
SUBSTITUTE SHEET RULE 26 Many proteinase probes exploit the FRET (Forster Resonance Energy Transfer, also known as Fluorescence Resonance Energy Transfer) phenomenon to detect enzymatic activity [Biotechnol. J. 2014, 9, 266-281 , Chem. Comm. 2008, 4250-4260], where the protease substrate is located between a fluorophore/quencher pair. Alternatively, the "self-quenching" effect in multi-branched systems previously described for the detection of AspN Endoproteinase [Angew. Chem. 2002, 41 , 17, 3233-3236] and more recently Human neutrophil elastase (HNE) [Org. Biomol. Chem. 2013, 1 1 , 4414-4418] has been applied as well for cathepsin S [J. Med. Chem. 2006, 49, 4715-4720]. FRET optical probes have not previously allowed the detection of thrombin in vivo due to the instability of the probes from non-specific cleavage by enzymes other than thrombin typically found in tissue or tissue samples to be tested, such as plasmin, matrix metalloproteinases (MMPs) and factor Xa. Accordingly, it is an object of the present invention to provide improved optical probes that are capable of detecting activated thrombin in the presence of enzymes typically found in tissue.
Several proteinase probes have been investigated over the years, including those based on potent matrix metalloproteinase (MMP) inhibitors and activatable fluorescent probes. To date, design and development of labelled substrates for these enzymes have largely focused on sensing tumour-related activity [review Cancer biotherapy and Radiopharmaceuticals, 21 , 5, 2006, 409-416], as well as for osteoarthritis or atherosclerosis [Chem. BioChem. 2012, 13, 2002-2020; Contrast Media Mol. Imaging 2014, 9, 187-210]. Despite many efforts, the probes known in the art are limited in usefulness in vivo at least, due to poor specificity and in vivo stability.
Accordingly, it is an object of the present invention to provide improved optical probes that are capable of detecting specific proteinases, that are preferably stable in vivo.
Statements of the Invention
According to a first aspect of the invention, there is presented an optical probe comprising a first probe element, and a second probe element; the first probe element and the second probe element connected to a core; the first probe element comprising a first fluorophore connected to a first quencher by a first cleavable linker; the second probe element comprising a second fluorophore connected to a second quencher by a second cleavable
2
SUBSTITUTE SHEET RULE 26 linker; the first fluorophore being substantially fluorescently quenched by the first quencher when connected to the first quencher by the first cleavable linker; the second fluorophore being substantially fluorescently quenched by the second quencher when connected to the second quencher by the second cleavable linker; wherein the first fluorophore is separated from the first quencher when the first cleavable linker is cleaved, and the second fluorophore is separated from the second quencher when the second cleavable linker is cleaved.
As a result of cleavage of the first or second cleavable linker, the first or second fluorophore of the first or second probe element is separated from the first or second quencher and therefore, the fluorescence of the first or second fluorophore is no longer quenched by the first or second quencher. Accordingly, upon illumination of the target area with an appropriate wavelength of light, the first or second fluorophore of the first or second probe element fluoresces. Preferably, the first cleavable linker is cleaved by a first cleaving agent, and the second cleavable linker is cleaved by a second cleaving agent. One or both of the first and second cleaving agents may be an enzyme or a biochemical stimulus. The biochemical stimulus may be selected from a particular biochemical condition, such a pH value or redox potential value, the presence of a particular chemical species or a particular concentration of such a species. Examples of suitable biochemical stimuli include a particular pH range or a redox potential range or the presence of a chemical species such as peroxide, oxygen (02) or superoxide, or a particular concentration range of such a species.
The first cleaving agent may be a first enzyme, and the first cleavable linker may correspond to a first enzyme cleavable peptide sequence. The second cleaving agent may be a second enzyme and the second cleavable linker may correspond to a second enzyme cleavable peptide sequence. Preferably, the first cleaving agent is a first enzyme and the second cleaving agent is a second enzyme. Accordingly, in preferred embodiments of the invention, the first cleavable linker is cleaved when a first cleaving enzyme cleaves the first enzyme cleavable peptide sequence, and the second cleavable linker is cleaved when a second cleaving enzyme cleaves the second enzyme cleavable peptide sequence. Therefore, cleavage of the first enzyme cleavable peptide sequence typically corresponds to cleavage of the first cleavable linker, and cleavage of the second enzyme cleavable peptide sequence corresponds to cleavage of the second cleavable linker. Accordingly, in
3
SUBSTITUTE SHEET RULE 26 embodiments where the first and/or second cleavable linker comprises an enzyme cleavable peptide sequence, the term "cleavage of the cleavable linker" refers to cleavage of the enzyme cleavable peptide sequence unless stated otherwise. The first and/or second cleaving enzyme may be produced or expressed by the indigenous cells within the target area. The first and/or second cleaving enzyme may be produced or expressed by additional cells produced by a subject that have migrated to the target area, such as leukocytes, for example neutrophils. The first and/or second cleaving enzyme may be produced or expressed by an infective agent in the target area, such as a bacterial or fungal cell, for example.
In embodiments where the first and second cleavable linkers are cleaved by a first enzyme and a second enzyme, the first enzyme may be overexpressed and/or activated in a first disease, and the second enzyme may be overexpressed and/or activated in a second disease. The first disease may be unrelated to the second disease. Alternatively, the first disease may be related to the second disease or the first and second diseases may be the same disease. Therefore, the probe of the invention may provide a fast, effective and reliable way of detecting the first and second disease using a single probe, or may allow two related diseases to be differentiated and positively identified using a single probe or may allow two signals from a single disease to be identified.
In an alternative embodiment, the first enzyme may be overexpressed and/or activated in a first stage of a disease, and the second enzyme may be overexpressed and/or activated in a second stage of the disease. Therefore, the probe of the invention may allow a first stage of a disease to be differentiated from a second stage of the disease.
In a further alternative embodiment, the first enzyme may be overexpressed and/or activated in a first disease process, and the second enzyme may be overexpressed and/or activated in a second disease process. Therefore, the probe of the invention may increase specificity of disease detection.
Preferably, the first enzyme and the second enzyme may both be overexpressed and/or activated in the same one or more diseases. Typically, fluorescence of the first fluorophore and/or the second fluorophore is indicative of the presence of the first enzyme and/or the second enzyme. The presence of the first enzyme and/or the second enzyme may be indicative of the presence of specific pathogenic cells or tissue, and/or a specific disease.
SUBSTITUTE SHEET RULE 26 Therefore, fluorescence of the first fluorophore and/or the second fluorophore may be indicative of the presence of specific pathological cells or tissue, and/or a specific disease.
Accordingly, the probe of the invention may allow the presence of a specific disease to be detected by two independent indicators, thereby minimising false positive results due to the presence of or more of the first or second enzyme only (i.e. only the first enzyme or only the second enzyme) which may be overexpressed and/or activated in diseases in addition to the specific disease. In some circumstances, only one or other of the first and second enzymes may be active in a particular disease stage, such that fluorescence of only one of the fluorophores is observed and can be indicative of the disease stage.
The first enzyme may be a matrix metalloproteinase (MMP). Preferably, the first enzyme one or more of MMP-2, MMP-9, and/or MMP-13. In some embodiments, the first enzyme cleavable peptide sequence is selectively cleavable by MMP-2, MMP-9 and MMP-13.
The first enzyme cleavable peptide sequence may comprise one of SEQ ID N0.1 to SEQ ID NO.14 (Table 1 below).
Figure imgf000006_0001
5
SUBSTITUTE SHEET RULE 26 (wherein Nle=norleucine; Cha=3-cyclohexylalanine; βΑ= β-alanine; Y(Me)=4-methoxyphenyl alanine)
Table 1
In embodiments where the first enzyme cleavable peptide sequence comprises one of SEQ ID N0.1 to SEQ ID NO.14, it allows the probe of the invention to detect MMP, and therefore, fluorescence of the first probe element is indicative of the presence of MMP. MMP is overexpressed and/or activated in diseases such as fibrosis, cirrhosis, cancer, arthritis, particularly osteoarthritis, and atherosclerosis, and therefore, fluorescence of the first probe element may be indicative of fibrosis, cirrhosis, cancer, arthritis or atherosclerosis. Indeed, MMPs are upregulated in any inflammatory disease and as such these probes may be useful in their diagnosis. Preferably, the first enzyme cleavable peptide sequence comprises one of SEQ ID NO.5, or SEQ ID NO.7. More preferably, the first enzyme cleavable peptide sequence comprises SEQ ID NO.7. These sequences have surprisingly been found to be selectively cleaved by MMP-2, MMP-9 and MMP-13 over other MMPs and other similar enzymes, such as, plasmin, thrombin and factor Xa. Accordingly, embodiments comprising one or SEQ ID NO.5, or SEQ ID NO.7 allows selective and specific detection of MMP-2, MMP-9 and MMP- 13.
The second enzyme may be thrombin. The second enzyme cleavable peptide sequence may comprise one of SEQ ID NO.15 to SEQ ID N0.38 (Table 2).
Figure imgf000007_0001
6
SUBSTITUTE SHEET RULE 26 25 Nle-W-P-R-G-W-R-(D)L
26 Nle-T-R-G-W-R-(D)L
27 L-W-P-R-G-W-(D)R-L
28 L-T-P-R-G-W-(D)R-L
29 l-W-P-R-G-W-(D)R-L
30 l-T-R-G-W-(D)R-L
31 Nle-W-P-R-G-W-(D)R-L
32 Nle-T-R-G-W-(D)R-L
33 L-W-P-R-G-W-(D)R-(D)L
34 L-T-P-R-G-W-(D)R-(D)L
35 l-W-P-R-G-W-(D)R-(D)L
36 l-T-R-G-W-(D)R-(D)L
37 Nle-W-P-R-G-W-(D)R-(D)L
38 Nle-T-R-G-W-(D)R-(D)L
Table 2
Preferably, the second enzyme cleavable peptide sequence comprises one of SEQ ID N0.25, SEQ ID N0.31 , or SEQ ID N0.37.
More preferably, the second enzyme cleavable peptide sequence comprises SEQ ID NO.25. Probe elements that comprise one of these sequences have been surprisingly found to be resistant to non-specific cleavage and breakdown whilst still retaining selectivity for cleavage by thrombin. Therefore, probes comprising two probe elements comprising these sequences allow the reliable detection of thrombin in the presence of other enzymes, such as MMP, plasmin and factor Xa.
Preferably, the first enzyme is MMP-2, MMP-9 or MMP-13, and the second enzyme is thrombin.
Accordingly, in some preferred embodiments of the invention, a probe having two probe elements allows the detection of MMP-2, MMP-9 or MMP-13, and thrombin. MMP-2, MMP-9 or MMP-13, especially MMP-9 and MMP-13, and thrombin are overexpressed and/or activated in fibrosis, and activity of these enzymes is significantly increased in fibrotic tissue. Therefore, the probe of the invention having two probe elements allows the simultaneous detection of MMP-9 and thrombin, for example, using a single probe in a single procedure, to thereby allow the presence of fibrotic tissue and an active fibrotic process to be detected.
7
SUBSTITUTE SHEET RULE 26 Furthermore, a probe that allows the detection of MMP and thrombin expression and activity may allow the reliable detection of active fibroproliferation within fibrotic tissue. The probe can minimise false positive detection of fibrotic tissue. For instance due to the selectivity of a cleavable linker for a particular MMP, accurate detection may be achieved even in the presence of other similar enzymes. Furthermore, the presence of a second probe element can allow the selective detection of another enzyme, such as thrombin, which may also be capable of cleaving the cleavable linker of the first probe element, thereby indicating a false positive. Furthermore, a probe that allows the detection of MMP and thrombin expression and activity may allow the clinical stage of the fibro-proliferative disease to be more accurately determined.
Additionally, such a probe is advantageous over more invasive techniques, such as a biopsy, because its use does not require tissue to be excised. For instance, the probe can be delivered by endoscope, spray, injection, topically, or ingestion and then the delivery site illuminated in situ to detect fluorescence in the target zone. Further still, where biopsy is still required or advantageous, the probe may provide guidance to areas of active disease, improving biopsy sampling. Alternatively, the first or second cleaving enzyme may be proteinase 3 and the enzyme cleavable peptide sequence may comprise V-A-D-C-A-D-Y. The first or second cleaving enzyme may be cathepsin G and the enzyme cleavable peptide sequence may comprise A- A-P-F, or F-V-T-Gnf-S-W (where Gnf= 4-guanidine-L- phenylalanine). The first or second cleaving enzyme may be a caspase and the enzyme cleavable peptide sequence may comprise D-E-V-D. Indeed, the first or second cleaving enzyme may be any enzyme with an appropriate target peptide substrate sequence.
In alternative embodiments of the invention, the first and or second cleaving agent may be a reactive oxygen species. For example, the reactive oxygen species may be superoxide, or hydrogen peroxide. The first cleavable linker, and/or the second cleavable linker may be a modified boronic acid based linker, such as that described in J. Am. Chem. Soc, 2014, 874, Roger Y. Tsien. In embodiments where the first cleaving agent is a reactive oxygen species, and the second cleaving agent is an enzyme, the reactive oxygen species may be generated by the same cells or tissues that express the enzyme.
Typically, the fluorescence of a first fluorophore may be suppressed, or "quenched", by a neighbouring moiety. The neighbouring moiety, or "quencher", may be a second
SUBSTITUTE SHEET RULE 26 fluorophore. Where the first fluorophore and the neighbouring second fluorophore are of the same type (i.e. they are the same chemical entity with the same excitation and emission spectra), the first and second fluorophores may quench the fluorescence of each other, and "self-quench". For example, carboxy fluorescein, Cy5, and 7-nitrobenz-2-oxa-1 ,3- diazole (NBD) may self-quench.
If the first fluorophore and the neighbouring second fluorophore are of different types (i.e. they are different chemical entities and have different excitation and emission spectra), then if the emission spectra of the first fluorophore and the excitation spectra of the second fluorophore overlap, energy may be transferred from one to the other fluorophore via Fluorescence (or Forster) Resonance Energy Transfer (FRET). In other words, they may form a "FRET pair", and the second fluorophore may quench the fluorescence of the first fluorophore. The second fluorophore absorbs the fluorescence of the first fluorophore and fluoresces itself at a different wavelength. Accordingly, the second fluorophore may be a "fluorescent quencher". For example, FRET pairs that comprise two fluorophores include (fluorophore/quencher) Cy3/Cy5, and carboxy fluorescein/seminaphthorhodamine carboxylate derivatives.
The neighbouring moiety may be a chemical entity that does not fluoresce. The neighbouring moiety may still quench the fluorescence of the first fluorophore, but instead of fluorescing itself, the neighbouring moiety disperses the energy it received from the fluorophore as heat to its surroundings, and is a "dark quencher". For example, FRET pairs that comprise a dark quencher include (fluorophore/quencher) fluorescein/dimethylaminoazobenzenesulfonic acid (DABSYL), carboxy fluorescein/BHQ-1 , carboxy fluorescein/methyl red, NBD/methyl red, carboxy naphthofluorescein/QSY21 , carboxy naphthofluorescein/BHQ-3, seminaphthorhodamine carboxylate derivatives/BHQ-3, seminaphthorhodamine carboxylate derivatives/QSY21 , Cy3/QSY21 , Cy5/QSY21 , and Cy5/BHQ-3. The first quencher and the first fluorophore may quench. A first probe element comprising a first quencher and a first fluorophore that quench is substantially dark and does not produce significant fluorescence before the first enzyme cleavable peptide sequence is cleaved. For instance, the first quencher may be the same type of fluorophore as the first fluorophore, and the first quencher and the first fluorophore may self-quench.
The second quencher and the second fluorophore may quench. A second probe element comprising a second quencher and a second fluorophore that quench is substantially dark
SUBSTITUTE SHEET RULE 26 and does not produce significant fluorescence before the second enzyme cleavable peptide sequence is cleaved. For instance, the second quencher may be the same type of fluorophore as the second fluorophore, and the second quencher and the second fluorophore may self-quench.
In embodiments where the fluorophore and the quencher of a probe element self-quench, (i.e. the fluorophore and the quencher are the same type of fluorophore), the fluorophore may be selected from fluorescein, or a derivative thereof, seminaphthorhodamine carboxylate or a derivative thereof, a cyanine fluorophore, such as Cy2, Cy3, Cy5, Cy5.5 or Cy7, rhodamine or derivative thereof, a fluorescent protein, such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), or cyan fluorescent protein (CFP), or 7- nitrobenz-2-oxa-1 ,3-diazole (NBD).
In another embodiment, the first quencher is a different type of fluorophore than that of the first fluorophore, and is a fluorescent quencher. Accordingly, before the first enzyme cleavable peptide sequence is cleaved a first probe element comprising a fluorescent quencher, fluoresces at the wavelength of light emitted by the fluorescent quencher and does not fluoresce at the wavelength of light emitted by the first fluorophore. Similarly, in some embodiments the second quencher is a different type of fluorophore than that of the second fluorophore, and is a fluorescent quencher. Accordingly, before the second enzyme cleavable peptide sequence is cleaved a second probe element comprising a fluorescent quencher fluoresces at the wavelength of light emitted by the fluorescent quencher and does not fluoresce at the wavelength of light emitted by the second fluorophore. In a further embodiment, the first quencher may be a dark quencher. Accordingly, a first probe element comprising a dark quencher is substantially dark and does not produce fluorescence before the first enzyme cleavable peptide sequence is cleaved. Similarly, in a further embodiment, the second quencher may be a dark quencher. Accordingly, a second probe element comprising a dark quencher is substantially dark and does not produce fluorescence before the second enzyme cleavable peptide sequence is cleaved.
In embodiments where the first fluorophore and the first quencher are different chemical entities, the first fluorophore may be any fluorophore that may form a FRET pair with a suitable first quencher. In embodiments where the second fluorophore and the second quencher are different chemical entities, the second fluorophore may be any fluorophore that may form a FRET pair with a suitable second quencher. Accordingly, a given fluorophore and quencher are chosen as a pair to ensure that they have appropriate excitation and
SUBSTITUTE SHEET RULE 26 emission spectra for the transfer of energy from the fluorophore to the quencher (i.e. they form a FRET pair). For example, typical fluorophore/quencher pairs include Cy3/Cy5, Cy3/QSY21 , Cy5/QSY21 , Cy5/BHQ-3, carboxy fluorescein/tetramethylrhodamine, fluorescein/methyl red, carboxy fluorescein/BHQ-1 , NBD/methyl red, carboxy naphthofluorescein/QSY21 , carboxy naphthofluorescein/BHQ-3, seminaphthorhodamine carboxylate derivatives/BHQ-3, seminaphthorhodamine carboxylate derivatives/QSY21 , cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP), carboxy fluorescein (FAM)/methyl red, etc. Further examples of FRET pairs may be readily identified by the skilled person.
It will be evident to the person skilled in the art that the first fluorophore and first quencher, and the second fluorophore and second quencher, should be chosen such that the first fluorophore is not substantially fluorescently quenched by the second quencher, and the second fluorophore is not substantially fluorescently quenched by the first quencher. For example, the first fluorophore/first quencher pair may be FAM/methyl red and the second fluorophore/second quencher pair may be Cy5/QSY21 or carboxy naphthofluorescein/QSY21.
The first probe element may comprise a plurality of first fluorophores. The second probe element may comprise a plurality of second fluorophores or second quenchers.
In some embodiments of the invention, the first probe element comprises a plurality of first fluorophores each connected to a first quencher by a separate first cleavable linker. The first probe element may comprise a single first quencher that substantially fluorescently quenches each first fluorophore within the plurality of first fluorophores. The first probe element may comprise a plurality of first quenchers, and each first fluorophore within the plurality of first fluorophores may be connected to a first quencher by a first cleavable linker.
In further embodiments of the invention, the second probe element comprises a plurality of second fluorophores each connected to a second quencher by a separate second cleavable linker. The second probe element may comprise a single second quencher that substantially fluorescently quenches each second fluorophore within the plurality of second fluorophores. The second probe element may comprise a plurality of second quenchers, and each second fluorophore within the plurality of second fluorophores may be connected to a second quencher by a second cleavable linker.
11
SUBSTITUTE SHEET RULE 26 The probe may comprise a third probe element connected to the core, the third probe element may comprise a third fluorophore connected to a third quencher by a third cleavable linker. The third cleavable linker may comprise a third enzyme cleavable peptide sequence that may be cleaved by a third enzyme. The third enzyme may be associated with the same disease as the first and/or second enzymes. The third enzyme may be associated with a different disease to that associated with the first and/or second enzyme.
Typically, the probes of the invention are operable to be used to detect a first and a second cleaving agent in a target zone. The target zone may be a portion of tissue within a subject, and the method may be carried out in vivo. The portion of tissue may be a portion of the heart, lung, liver, connective tissue, skin, intestine, or joints of a subject. The probes of the invention may be used in the respiratory system, the circulatory system, the nervous system, the digestive system or the reproductive system. For example, the target area may be a portion of the lung of a subject.
The target zone may be a portion of a cell culture, a tissue sample such as a biopsy sample, or a liquid sample such as a bodily fluid sample.
The target zone may be a target area. The target zone may be a target volume.
Accordingly, the probes of the invention may be used in vivo, ex vivo or in vitro.
The probes of the invention may be delivered to a target zone by any means known in the art. For example, the probes of the invention may be delivered by endoscope, spray, injection, topically, or ingestion. For example, where the probes are to be delivered to a portion of the lung, the probes may be delivered to the target zone using a bronchoscope.
Illumination of a suitable wavelength to excite the first and second fluorophores of the probe may be delivered to the target zone by any conventional means known in the art. Typically, in embodiments where the probe is to be used within the body of a subject, the light is delivered by means of an optical fibre or similar. The fluorescence from the probes in the target zone may be collected by an optical fibre or similar. The fluorescence from the probes in the target zone may be collected by the same optical fibre that delivered the illumination light. The collected fluorescence is typically delivered to a recording device, such as a charge-coupled device (CCD) or similar. Alternatively, the fluorescence from the probes in the target zone may be directly collected by a recording device, such as a CCD or similar. For example, a bronchoscope may be used to both deliver the probe to the target zone, to
12
SUBSTITUTE SHEET RULE 26 deliver light to the target zone, and to detect fluorescence from the target zone. Alternatively, individual instruments may be used to deliver the probe to the target zone, to deliver light to the target zone and to detect fluorescence from the target zone. For example, fluorescence may be detected from the tissue of a target area using fiber confocal fluorescence microscopy (fCFM).
Preferably, the subject is a human subject. However, the subject may be a non-human animal such as equine, ovine, bovine, feline, canine or rodent, for example.
The first fluorophore may be connected to the first enzyme cleavable peptide sequence by a spacer. The second fluorophore may be connected to the second enzyme cleavable peptide sequence by a spacer. The first quencher may be connected to the first enzyme cleavable peptide sequence by a spacer. The second quencher may be connected to the second enzyme cleavable peptide sequence by a spacer. The spacer may be a saturated or unsaturated hydrocarbon chain, an ether, a polymer, a polyethylglycol (PEG), a poly glycol, a poly ether or similar. The spacer may be a peptide. In embodiments where the spacer is a peptide, the peptide may be 1-10 amino acids in length, 1-20 amino acids in length, or 1- 30 amino acids in length. The spacer may be a mixture of amino acids and a saturated or unsaturated hydrocarbon chain, an ether, a polymer, a PEG, a poly glycol, a poly ether or similar. For example, the spacer may comprise 6-aminohexanoic acid (Ahx), or PEG, or an alternating chain of PEG and amino acids.
In embodiments where the spacer comprises polar or hydrophilic groups, such as PEG, or poly ether for example, the linker may increase the solubility of the probe in aqueous media. Therefore, the provision of a probe comprising at least one spacer comprising a polar or hydrophilic group may allow the probe to be more readily soluble in biologically acceptable media for direct application to a target area without the requirement for additional surfactants, for example.
The probe may comprise at least one reporter fluorophore that is not substantially fluorescently quenched. Preferably, the at least one reporter fluorophore fluoresces at a wavelength that is different to the wavelength of light at which the first fluorophore and the second fluorophore fluoresces. Accordingly, the at least one reporter fluorophore fluoresces before and after the first cleavable linker is cleaved, and before and after the second cleavable linker is cleaved. Therefore, the at least one reporter fluorophore fluoresces when illuminated with a suitable wavelength of light. Accordingly, the presence or location of the
13
SUBSTITUTE SHEET RULE 26 probe in a target zone may be monitored with and without the presence of the first cleaving agent and the second cleaving agent.
The first probe element may be connected directly to the core. The second probe element may be directly connected to the core. Alternatively, the first probe element may be connected indirectly to the core via a linker. The second probe element may be connected indirectly to the core via a linker. The linker may be a saturated or unsaturated hydrocarbon chain, an ether, a polymer, a polyethylglycol (PEG), a poly glycol, a poly ether or similar. The linker may comprise one or more amino acids. For example, the linker may be selected from the list: [-(lysine)-(PEG2)-]i-2, [-(PEG-k)-]i-3, and [-(PEG-k)0-2-NH-(CH2)3-O-CH2-].
In embodiments where the linker comprises polar or hydrophilic groups, such as PEG, or poly ether for example, the linker may increase the solubility of the probe in aqueous media. Therefore, the provision of a probe comprising at least one linker comprising a polar or hydrophilic group may allow the probe to be more readily soluble in biologically acceptable media for direct application to a target area without the requirement for additional surfactants, for example.
Preferably, the linker comprises at least one D-amino acid. More preferably, the linker comprises at least one D-lysine residue. The provision of a linker comprising a D-amino acid, such as a D-lysine residue, has surprisingly been found to increase the stability and longevity of the probes in the presence of enzymes such as plasmin, and factor Xa. Accordingly, the provision of at least one D-lysine residue, for example, in the linker may prevent nonspecific cleavage of the linker by enzymes present in vivo.
By the term "core" we refer to a common moiety that joins the plurality of probe elements to form a single unit. Accordingly, the core could be a single atom, or comprise a functional group, a saturated or unsaturated hydrocarbon chain or a polyglycol (linear, branched, or cyclical), a peptide sequence, a heterocycle, or a polymer. The core is typically chosen to have the correct valency for the number of probe elements that are required to be connected to the core. For example, in embodiments with a first probe element and a second probe element, the core is chosen to have a valency of two or more, such that the first probe element and the second probe element may be connected to the core. The core may comprise a plurality of connectors that may bind to each of the probe elements, thereby connecting each probe element to the core.
14
SUBSTITUTE SHEET RULE 26 The first probe element may comprise a solubilising group at the distal end of the first probe element to the core. The second probe element may comprise a solubilising group at the distal end of the second probe element to the core. The or each solubilising group preferably comprises polar or hydrophilic moieties. For example, the or each solubilising group may comprise a polyethylglycol (PEG), a poly glycol, a poly ether or similar, an alcohol, acid, amine, or amide group or similar, such as an amino acid. A probe according to the present aspect comprising one or more solubilising groups may be more soluble in aqueous media than probes without one or more solubilising groups. In some embodiments, the first fluorophore is released from the probe when the first cleavable linker is cleaved, and the second fluorophore may be released from the probe when the second cleavable linker is cleaved. In alternative embodiments, the first quencher may be released from the probe when the first cleavable linker is cleaved, and the second quencher may be released from the probe when the second cleavable linker is cleaved.
According to a second aspect of the invention, there is provided a method of detecting a first and a second enzyme in a target zone comprising the steps:
a. applying to the target zone a probe according to the first aspect comprising a first enzyme cleavable peptide sequence and a second enzyme cleavable peptide sequence;
b. illuminating the target zone with appropriate wavelengths of light to excite the first and second fluorophores; and
c. determining whether the intensity of fluorescence of the first and second fluorophores has increased within the target area,
wherein significant fluorescence of the first fluorophore is indicative of the presence of the first enzyme in the target zone, and significant fluorescence of the second fluorophore is indicative of the presence of the second enzyme in the target zone.
By the term "significant fluorescence" we refer to the fluorescence of a fluorophore that results from sufficient separation of that fluorophore from the quencher of a probe to prevent the quencher quenching the fluorescence of the fluorophore, that is above the background or, where present, autofluorescence in the target area. The autofluorescence of the indigenous cells or tissue within the target area may have a shorter fluorescent lifetime than the fluorophore of the first probe. The autofluorescence of the indigenous cells or tissue within the target area may reduce over time at a faster rate than that of the fluorophore of the probe. Accordingly, fluorescence observed in the target area that decays more slowly over
15
SUBSTITUTE SHEET RULE 26 time may be indicative of the probe, and fluorescence observed in the target area that reduces more quickly over time may be indicative of autofluorescence.
Preferably, the first enzyme is MMP. More preferably, the first enzyme is MMP-2, MMP-9 or MMP-13. In some embodiments, the first enzyme cleavable peptide sequence is selectively cleavable by MMP-2, MMP-9 and MMP-13, and therefore, significant fluorescence of the first fluorophore may be indicative of the presence of MMP-2, MMP-9 and MMP-13 in the target zone. Preferably, the second enzyme is thrombin.
Preferably, the first enzyme is MMP-2, MMP-9 or MMP-13 and the second enzyme is thrombin, such that significant fluorescence of the first and/or second fluorophores is indicative of a disease in which MMP-2, MMP-9 or MMP-13, and/or thrombin are overexpressed and/or activated.
MMP-2, MMP-9, MMP-13 and thrombin are overexpressed and/or activated in fibrotic tissue, and therefore the method of the invention may allow the presence of fibrotic tissue in the target zone to be detected.
Alternatively, the first or second cleaving enzyme may be proteinase 3 and the enzyme cleavable peptide sequence may comprise V-A-D-C-A-D-Y. The first or second cleaving enzyme may be cathepsin G and the enzyme cleavable peptide sequence may comprise A- A-P-F, or F-V-T-Gnf-S-W (where Gnf= 4-guanidine-L- phenylalanine). The first or second cleaving enzyme may be a caspase and the enzyme cleavable peptide sequence may comprise D-E-V-D.
The at least one fluorophore of the probe may be selected dependent on the location of the target zone. For example, if the target zone is directly observable, such as on the skin or within the lung of a subject, the at least one fluorophore may be selected to fluoresce in the visible region of the spectrum. If the target area is to be observed through the skin or tissue, the at least one fluorophore may be selected to fluoresce in the infrared region of the spectrum, such that the fluorescence of the probe is not significantly absorbed by the skin or tissue and therefore be observable through the skin of tissue. For example, the at least one fluorophore may be selected to fluoresce in the near infrared (fluorescence wavelength of between 600nm to 950nm). Infrared imaging techniques are well known to the skilled person in the art.
16
SUBSTITUTE SHEET RULE 26 The target zone may be a portion of tissue within a subject, and the method may be carried out in vivo. The portion of tissue may be a portion of the heart, lung, liver, connective tissue, skin, intestine, or joints of a subject, for example. For example, the target zone may be a portion of the lung of a subject. In addition, the method of the invention may be carried out in the circulatory system, the nervous system, the digestive system or the reproductive system.
The target zone may be a portion of a cell culture, a tissue sample such as a biopsy sample, or a liquid sample such as a bodily fluid sample.
The target zone may be a target area. The target zone may be a target volume.
Accordingly, the method of the invention may be carried out in vivo, ex vivo or in vitro.
The probes of the invention may be delivered to a target zone by any means known in the art. For example, the probes of the invention may be delivered by endoscope, spray, injection, topically, or ingestion. For example, where the probes are to be delivered to a portion of the lung, the probes may be delivered to the target zone using an endoscope, such as a bronchoscope.
Illumination of a suitable wavelength to excite fluorophores of the probe may be delivered to the target zone by any conventional means known in the art. Typically, in embodiments where the probe is to be used within the body of a subject, the light is delivered by means of an optical fibre or similar. The fluorescence from the probes in the target zone may be collected by an optical fibre or similar. The fluorescence from the probes in the target zone may be collected by the same optical fibre that delivered the illumination light. The collected fluorescence is typically delivered to a recording device, such as a charge-coupled device (CCD) or similar. Alternatively, the fluorescence from the probes in the target zone may be directly collected by a recording device, such as a CCD or similar. For example to both deliver the probe to the target zone, to deliver light to the target area, and to detect fluorescence from the target zone. Alternatively, individual instruments may be used to deliver the probe to the target zone, to deliver light to the target zone and to detect fluorescence from the target zone.
For example, fluorescence may be detected from the tissue of a target area using fiber confocal fluorescence microscopy (fCFM).
17
SUBSTITUTE SHEET RULE 26 The method may comprise the steps of (a) illuminating the target zone with an appropriate wavelength of light to excite the first fluorophore, (b) measuring the fluorescence of the first fluorophore, (c) illuminating the target zone with an appropriate wavelength of light to excite the second fluorophore, and then (d) measuring the fluorescence of the second fluorophore. It may be that the first cleavable linker of the probe may be cleaved by the first enzyme at a different rate than the second cleavable linker of the probe is cleaved by the second enzyme. Accordingly, the method may allow the fluorescence from the first fluorophore to be determined separately from the fluorescence from the second fluorophore. Alternatively, the target zone may be illuminated by appropriate wavelengths of light appropriate to excite the first and second fluorophores, and the fluorescence from the first and second fluorophores may be determined separately. For example, the fluorescence from the first and second fluorophores may be split before being directed to a recording or measuring device.
Fluorescence from the probes may be imaged indirectly. For example, the fluorescence may be converted into acoustic waves by using photoacoustic imaging. Photoacoustic imaging may allow high resolution images of the target area to be generated. Preferably, the subject is a human subject. However, the subject may be a non-human animal such as equine, ovine, bovine, or rodent, for example.
The invention extends in a third aspect to a kit of parts comprising the probe according to the first aspect in a suitable diluent or buffer.
According to a fourth aspect of the invention, there is provided an optical probe comprising at least one fluorophore connected to at least one quencher by a cleavable linker; the at least one fluorophore being substantially fluorescently quenched by the at least one quencher when connected to the at least one quencher by the cleavable linker, and the at least one fluorophore is separated from the at least one quencher when the cleavable linker is cleaved; wherein the cleavable linker comprises an enzyme cleavable peptide sequence comprising one of SEQ ID NO.15 to SEQ ID NO.38 and is selectively cleavable by thrombin.
Preferably, the enzyme cleavable peptide sequence comprises one or more D-amino acid residues. Preferably, the enzyme cleavable peptide sequence comprises one of SEQ ID NO.25, SEQ ID NO.31 , or SEQ ID NO.37. More preferably, the enzyme cleavable peptide sequence comprises SEQ ID NO.25.
18
SUBSTITUTE SHEET RULE 26 Probes known in the art for the detection of thrombin are typically non-specifically cleaved in the presence of enzymes such as plasmin, MMPs and factor Xa, and therefore, such probes are not useful for detecting thrombin in vivo or in tissue samples where these enzymes are present.
The inventors have surprisingly found that the inclusion of a D-amino acid at certain positions within the enzyme cleavable peptide sequence results in the enzyme cleavable peptide sequence being more resilient to non-specific cleavage by enzymes such as plasmin, MMP and factor Xa, whilst still being selectively cleavable by thrombin. Accordingly, the probes of the invention allow the presence of thrombin to be reliably and accurately detected in vivo and in tissue samples where proteinases are present.
The person skilled in the art will appreciate that the at least one fluorophore and at least one quencher of the probe of the present aspect may be selected as has been described above in relation to the first aspect, and for the sake of brevity, this text is not repeated here.
The at least one fluorophore may be connected to the enzyme cleavable peptide sequence by a spacer. The at least one quencher may be connected to the enzyme cleavable peptide sequence by a spacer. The spacer may be a saturated or unsaturated hydrocarbon chain, an ether, a polymer, a polyethylglycol (PEG), a poly glycol, a poly ether or similar. The spacer may be a peptide. In embodiments where the spacer is a peptide, the peptide may be 1-10 amino acids in length, 1-20 amino acids in length, or 1-30 amino acids in length. The spacer may be a mixture of amino acids and a saturated or unsaturated hydrocarbon chain, an ether, a polymer, a PEG, a poly glycol, a poly ether or similar. For example, the spacer may comprise 6-aminohexanoic acid (Ahx), or PEG, or an alternating chain of PEG and amino acids.
In embodiments where the spacer comprises polar or hydrophilic groups, such as PEG, or poly ether for example, the linker may increase the solubility of the probe in aqueous media. Therefore, the provision of a probe comprising at least one spacer comprising a polar or hydrophilic group may allow the probe to be more readily soluble in biologically acceptable media for direct application to a target area without the requirement for additional surfactants, for example.
19
SUBSTITUTE SHEET RULE 26 It is convenient to define the unit of at least one fluorophore connected to an enzyme cleavable peptide sequence as a "probe element". In other words, a probe element may comprise at least one fluorophore connected to an enzyme cleavable peptide sequence. Accordingly, the probe element may be depicted as:
(F-A-Seq)- (1) where F = at least one fluorophore
A = a spacer, which may be individually present or absent
Seq = a peptide sequence comprising the enzyme cleavable peptide sequence
In one embodiment of the invention, the probe comprises a single probe element and a quencher.
The probe may comprise a plurality of probe elements, and each of the plurality of probe elements may comprise at least one fluorophore connected to an enzyme cleavable peptide sequence. Optical probes known in the art are often unstable in vivo or in other cases, such as tissue lysates, tissue samples or samples of bodily fluids, due to processes such as non-specific cleavage of the probe by enzymes. Such non-specific cleavage may produce fluorescence of the probe and be mistaken as indicative of the presence of MMP in a target area. Alternatively, non-specific cleavage may break the probe down and prevent the probe from being observed at all.
Surprisingly, the provision of an optical probe comprising a plurality of probe elements in combination with determined sequences improves the stability of the probe in vivo. Without wishing to be bound by theory, the inventors suggest that the provision of a plurality of probe elements may shield each probe element from unspecific cleavage, thereby increasing the structural resilience of the probe.
In addition, the provision of a probe comprising a plurality of probe elements provides an increased number of fluorophores per probe, thereby providing an increase in fluorescence per probe. Furthermore, this increase in fluorescence per probe may provide a greater signal-to-noise ratio that may allow lower concentrations of thrombin to be detected, or lower concentrations of the probe to be used.
20
SUBSTITUTE SHEET RULE 26 The probe may comprise a plurality of probe elements connected by a core. Each probe element within the plurality of probe elements may be connected to the core directly. Each probe element within the plurality of probe elements may be connected to the core indirectly via a linker. The meaning of the term "core" is described above in relation to the first aspect. The person skilled in the art will appreciate that the linker of the present aspect may be selected as described above in relation to the first aspect and for the sake of brevity, this text is not repeated here. The probe may comprise at least two probe elements. The probe may comprise at least three probe elements. For example, the probe may comprise two or three probe elements. The more probe elements, the greater the enhancement of the fluorescence upon cleavage of the enzyme cleavable peptide sequence. The optical probe of the present aspect may be suitable to be incorporated into an optical probe according to the first aspect of the invention.
The optical probe may comprise two probe elements and two or more, for instance three, fluorophores (Figures 1 B-D).
In a fifth aspect, an optical probe is provided comprising a first probe element, and a second probe element; the first probe element and the second probe element connected to a core; the first probe element comprising a first fluorophore, a first cleavable linker and a first quencher, the first fluorophore connected to the first quencher by the first cleavable linker and the core, the first quencher being a second fluorophore and being connected to the core; the second probe element comprising the second fluorophore, a second cleavable linker and a second quencher, the second fluorophore connected to the second quencher by the core and the second cleavable linker; the first fluorophore being substantially fluorescently quenched by the first quencher when connected to the first quencher by the first cleavable linker and core; the second fluorophore being substantially fluorescently quenched by the second quencher when connected to the second quencher by the second cleavable linker and core; wherein the first fluorophore is separated from the first quencher when the first cleavable linker is cleaved, the second fluorophore is separated from the second quencher when the second cleavable linker is cleaved.
In this way, the first quencher, which is also the second fluorophore, is quencher for the first fluorophore of cleavable linker 1 (Figure 1 B-D).
21
SUBSTITUTE SHEET RULE 26 In one embodiment, the second quencher is not a fluorophore (Figure 1 B). For instance, the second quencher may be selected from BHQ-3 (strong absorption from 620 nm to 730 nm), QSY21 (intense absorption about 661 nm maxima but no fluorescence making it useful as an acceptor in fluorescence resonance energy transfer (FRET) applications).
Alternatively, the second quencher may be a third fluorophore (Figures 1 C, D).
When the second quencher is a third fluorophore, it may or may not be fluorescently quenched by the first quencher. In one embodiment, when the second quencher is a third fluorophore, it is not fluorescently quenched by the first quencher. In this way, the first quencher quenches the first fluorophore until cleavage of the first cleavable linker and the second quencher quenches the second fluorophore until cleavage of the second cleavable linker. The second quencher/third fluorophore would fluoresce under illumination both before and after cleavage of the second cleavable linker, because it is not quenched by a quencher on the probe (Figure 1 C). For instance, the second quencher may be methylblue derivatives, Alexa Fluor® 680 or IRDye 680, all of which are fluorescent and capable of quenching a second fluorophore which is the first quencher, such as a seminapthorhodamine.
In another embodiment, when the second quencher is a third fluorophore and the third fluorophore is separated from the first quencher when the second cleavable linker is cleaved, a second quencher/third fluorophore can be provided which is substantially fluorescently quenched by the first quencher. In this way, the first quencher, which is also the second fluorophore, is quencher for the first fluorophore of cleavable linker 1 and the third fluorophore of cleavable linker 2 (the second quencher) at the same time. Similarly, the third fluorophore (the second quencher) is quencher for the second fluorophore of cleavable linker 2. Thus, the second quencher/third fluorophore will not fluoresce under illumination before cleavage of the second cleavable linker, but will after cleavage (Figure 1 D).
In another embodiment, the first fluorophore and the first quencher may be a FRET pair, and the second fluorophore (first quencher) and second quencher may be a FRET pair, such that the first quencher and the second quencher are a FRET pair. For instance, the first fluorophore may be selected from a carboxy fluorescein, BODIPY, and Rhodamine derivatives (AabS = 460nm-500nm; Aem = 510nm -550nm), and/or the first quencher (second fluorophore) may be a Seminaphthorhodafluor (SNARF® dyes)
22
SUBSTITUTE SHEET RULE 26 carboxylate derivatives (AabS = 490-560nm; Aem = 565nm-660nm) and/or the second quencher (third fluorophore) may be selected from methylblue derivatives, Alexa Fluor® 680, IRDye 680 or other fluorescent labels. In one such embodiment, at least one fluorophore may be an independent reference fluorophore for the enzyme activity; at least one fluorophore may be a pH sensor and/or at least one fluorophore may be a pC02 sensor or Ca2+ sensor; and at least one fluorophore may display Long-Wavelength Dual-Emission. A Long-Wavelength Dual-Emission fluorophore is one which fluoresces at two sets of wavelengths in response to a single excitation wavelength. Seminaphthorhodamine can be used as a pH sensor because it emits at two sets of wavelengths (typically at A=580nm and 665nm) in response to a single excitation wavelength (between 488 nm and 530 nm). The two emission wavelengths respond differently to pH. The higher wavelength emission can increase upon alkalinisation, whilst the lower wavelength emission remains constant or decreases upon alkalinisation. Seminaphthorhodafluor (SNARF® dyes) carboxylate derivatives can be used as the fluorophore for Long-Wavelength Dual-Emission, pH sensor, pC02, Ca2+ sensor and as a fluorophore for reporting thrombin and/or MMP-9 activity, and the longer wavelength of emission is pH dependent. In a further such embodiment, an optical probe is provided in which the first, second and third fluorophores fluoresce at different wavelengths.
According to a sixth aspect of the invention, there is provided a method of detecting thrombin activity in a target zone, the method comprising the steps:
a. applying a probe according to the first aspect to the target zone;
b. illuminating the target zone with an appropriate wavelength of light to excite fluorophores of the probe; and
c. determining the fluorescence intensity of probe,
wherein significant fluorescence of the or each fluorophore of the probe is indicative of the presence of thrombin in the target zone.
By the term "significant fluorescence" we refer to the fluorescence of a fluorophore that results from sufficient separation of that fluorophore from the quencher of a probe to prevent the quencher quenching the fluorescence of the fluorophore, that is above the background or, where present, autofluorescence in the target area. The autofluorescence of the indigenous cells or tissue within the target area may have a shorter fluorescent lifetime than the fluorophore of the first probe. The autofluorescence of the indigenous cells or tissue
23
SUBSTITUTE SHEET RULE 26 within the target area may reduce over time at a faster rate than that of the fluorophore of the probe. Accordingly, fluorescence observed in the target area that decays more slowly over time may be indicative of the probe, and fluorescence observed in the target area that reduces more quickly over time may be indicative of autofluorescence.
Therefore, the method may comprise the step of determining the fluorescence intensity of the target zone over a period of time, determining the rate of decay of fluorescence during the period of time, and determining the fluorescence intensity of that fluorescence with a slower rate of decay, wherein the fluorescence with a slower rate of decay corresponds to the fluorescence of the probe.
It has not previously been possible to detect the presence of thrombin, and therefore, associated diseases, using FRET optical probes due to the instability of the probes from non-specific cleavage by enzymes other than thrombin typically found in tissue or tissue samples to be tested. As discussed above in relation to the fourth aspect, the probes of the fourth aspect are surprisingly resilient to non-specific cleavage whilst retaining specificity for cleavage by thrombin. Therefore, the method of the present aspect allows the detection of thrombin in the presence of similar cleaving enzymes, such as plasmin, MMP and factor Xa. Thrombin has been identified as playing a critical role in early fibroproliferation, especially in fibroproproliferative ARDS, and during pulmonary fibrosis thrombin activity is significantly increased. Therefore, the provision of a method that allows the reliable detection of thrombin in the presence of other enzymes such as MMP, plasmin and factor Xa, may allow the detection of fibroproliferation and/or pulmonary fibrosis.
Thrombin probes can detect the upregulation of the coagulation cascade. Thus, such probes can be used in the diagnosis of diseases associated with the upregulation of the coagulation cascade, such as cancer. Thus, such probes to detect thrombin can be used in the diagnosis of one or more of thrombosis, atherosclerosis, cancer and coronary heart disease.
The at least one fluorophore of the probe may be selected dependent on the location of the target zone. For example, if the target zone is directly observable, such as on the skin or within the lung of a subject, the at least one fluorophore may be selected to fluoresce in the visible region of the spectrum. If the target area is to be observed through the skin or tissue, the at least one fluorophore may be selected to fluoresce in the infrared region of the
24
SUBSTITUTE SHEET RULE 26 spectrum, such that the fluorescence of the probe is not significantly absorbed by the skin or tissue and therefore be observable through the skin of tissue. For example, the at least one fluorophore may be selected to fluoresce in the near infrared (fluorescence wavelength of between 600nm to 950nm). Infrared imaging techniques are well known to the skilled person in the art.
The target zone may be a portion of tissue within a subject, and the method may be carried out in vivo. The portion of tissue may be a portion of the heart, lung, liver, connective tissue, skin, intestine, or joints of a subject, for example. For example, the target zone may be a portion of the lung of a subject. In addition, the method of the invention may be carried out in the circulatory system, the nervous system, the digestive system or the reproductive system.
The target zone may be a portion of a cell culture, a tissue sample such as a biopsy sample, or a liquid sample such as a bodily fluid sample.
The target zone may be a target area. The target zone may be a target volume.
Accordingly, the method of the invention may be carried out in vivo, ex vivo or in vitro.
The probes of the invention may be delivered to a target zone by any means known in the art. For example, the probes of the invention may be delivered by endoscope, spray, injection, topically, or ingestion. For example, where the probes are to be delivered to a portion of the lung, the probes may be delivered to the target zone using an endoscope, such as a bronchoscope.
Illumination of a suitable wavelength to excite fluorophores of the probe may be delivered to the target zone by any conventional means known in the art. Typically, in embodiments where the probe is to be used within the body of a subject, the light is delivered by means of an optical fibre or similar. The fluorescence from the probes in the target zone may be collected by an optical fibre or similar. The fluorescence from the probes in the target zone may be collected by the same optical fibre that delivered the illumination light. The collected fluorescence is typically delivered to a recording device, such as a charge-coupled device (CCD) or similar. Alternatively, the fluorescence from the probes in the target zone may be directly collected by a recording device, such as a CCD or similar. For example to both deliver the probe to the target zone, to deliver light to the target area, and to detect fluorescence from the target zone. Alternatively, individual instruments may be used to
25
SUBSTITUTE SHEET RULE 26 deliver the probe to the target area, to deliver light to the target zone and to detect fluorescence from the target area.
For example, fluorescence may be detected from the tissue of a target area using fibered confocal fluorescence microscopy (fCFM).
The fluorophore may also be an optoacoustic fluorophore. Optoacoustic fluorophores generate ultrasound when excited by intense illumination, such as by a laser tuned to the absorption range of the fluorophore. The generated ultrasound can then be detected. Probes comprising optoacoustic fluorophores can be illuminated and detected by multispectral optoacoustic tomography (MSOT). MSOT illuminates the target zone with light pulses at multiple wavelengths and detects the acoustic waves generated by the thermoelastic expansion of the environment surrounding the optoacoustic fluorophore in response to the pulses.
Preferably, the subject is a human subject. However, the subject may be a non-human animal such as equine, ovine, bovine, or rodent, for example.
In embodiments where the target zone is a portion of the lung of a subject, the presence of thrombin in the target area may be indicative of fibrosis within the target zone.
The invention extends in a seventh aspect to a kit of parts comprising the probe of the fourth aspect in a suitable diluent or buffer. Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the accompanying drawings.
Brief Description of the Figures Figure 1. Concept of probes according to embodiments of the invention, A, in which the probe comprises a first probe element having a solubilising tail, thrombin cleavable sequence, fluorescent dye 1 and quencher Q1 forming a FRET probe element and a second probe element bound to the first probe element by a core, said second probe element having a solubilising tail, MMP-9 cleavable sequence, fluorescent dye 2 and quencher Q2 forming a FRET probe element;
another embodiment of the invention, B, in which the probe comprises a first probe element having a spacer, first fluorophore F1 , first cleavable linker 1 and first quencher Q1 which is a
26
SUBSTITUTE SHEET RULE 26 second fluorophore F2 connected to a core, first fluorophore F1 and first quencher Q1 forming a FRET pair, a second probe element having a spacer, second quencher Q2, second cleavable linker 2 and a second fluorophore F2, second fluorophore F2 and second quencher Q2 forming a FRET pair and the core connected to both first and second probe elements;
another embodiment of the invention, C, in which the probe comprises a first probe element having a spacer, first fluorophore F1 , first cleavable linker 1 and first quencher Q1 which is a second fluorophore F2 connected to a core, first fluorophore F1 and quencher Q1 forming a FRET pair, a second probe element having a spacer, second quencher Q2 which is a third fluorophore F3, second cleavable linker 2 and a second fluorophore F2, second fluorophore F2 and second quencher Q2 forming a FRET pair and the core connected to both first and second probe elements; and
another embodiment of the invention, D, in which the probe comprises a first probe element having a spacer, first fluorophore F1 , first cleavable linker 1 and first quencher Q1 which is a second fluorophore F2 connected to a core, first fluorophore F1 and first quencher Q1 forming a FRET pair, a second probe element having a spacer, second quencher Q2 which is a third fluorophore F3, second cleavable linker 2 and a second fluorophore F2, second fluorophore F2 and second quencher Q2 forming a FRET pair and third fluorophore F3 and first quencher Q1 forming a FRET pair and a core connected to both first and second probe elements.
Figure 2. General structures of probes according to embodiments of the invention. F1/F2/F3 represent fluorophore, Q1/Q2/Q3 represent quencher together with cleavable linker 1/2/3, core and spacer.
Figure 3. Evaluation of 1st and 2nd generation probes. Data shown represent the average fold change in fluorescence over background signal provided by probe (1 μΜ and 0.1 μΜ with MMP-9) with exogenous enzymes after 6 min using a multiwell plate fluorimeter at excitation/emission 485/528 nm and 640/670 nm. Recombinant human catalytic domain MMP-9 was used at 30nM. Recombinant human Thrombin and Plasmin were used at 5U/ml and 30nM respectively.
Figure 4. Data represents the fluorescence signal provided by probe (1 μΜ) after 5 min using a multiwell plate fluorimeter at excitation/emission 485/528nm (left) and 640/670 (right). (A) Fluorescence signal in presence of enzymes and inhibitors, (B) Average fold change in fluorescence over background signal with exogenous enzymes. Recombinant human catalytic domain MMPs -1 , -2, -3, -7, -8, -9, -10, -1 1 , -12, -13 were used at 30nM. Recombinant human Thrombin, Plasmin and Factor Xa were used at 5U/ml, 30nM and 0.5μΜ respectively. Anti-thrombin III inhibitor was used at 3.85 μΜ and Marimastat at 200 nM.
27
SUBSTITUTE SHEET RULE 26 Figure 5. MALDI spectra (A) and HPLC trace (B) after enzymatic reaction confirms the right cleavage by exogenous enzymes MMP9 and Thrombin.
Figure 6. Ex-vivo assay with human tissue. Data shown represent the fluorescence signal provided by probe AMF-185 (1 μΜ) with human fibrotic tissue without (upper chart) and with (lower chart) Marimastat (broad spectrum MMP inhibitor) using a multiwell plate fluorimeter at excitation/emission 485/528 nm and 640/670 nm.
Figure 7. MALDI spectra after assay with human fibrotic lung tissue shows intact probe and additional fragment for Thrombin cleavage (top) and intact probe in presence of Pl-Protease inhibitor (bottom).
Figure 8. HPLC trace of probe AMF-185 in PBS solution at room temperature; (left) day 0; (right) after 2 months.
Figure 9. HPLC trace and MALDI spectra for compounds AM F- 146-01 (top) and AMF-176 (bottom).
Figure 10. MALDI spectra for FRET peptide with sulfo-Cy5 and QSY21 exemplified for compound AMF-216.
Figure 11. HPLC trace (A) and MALDI spectra (B) for compounds AMF-140 (top), AMF-154- 03 (middle) and AMF-181 (bottom).
Figure 12. MALDI spectra for FRET branched-peptide with FAM and MR exemplified for compound AMF-216.
Figure 13. MALDI spectra (A) and HPLC trace (B) for compounds AMF-152 (top), AMF-162 (middle) and AMF-185 (bottom).
Figure 14. MALDI spectra for compound AMF-217.
Figure 15. Evaluation of different thrombin specific peptide sequences. Data represents the fluorescence signal provided by 1st generation probes (1 μΜ) using a multiwell plate fluorimeter at excitation/emission 485/528nm after 1 1 min in the presence of Thrombin, human bronchoalveolar lavage fluid (BALF), sheep BALF, human neutrophil lysate, human neutrophil supernatant, human macrophages supernatant and murine macrophages supernatant.
Figure 16. Data represents the fluorescence signal provided by probe SVC-1 13 (1 μΜ) using a multiwell plate fluorimeter at excitation/emission 485/528nm after 5 min in presence of Thrombin, plasmin and Factor Xa.
Figure 17. Data represents the average fold change in fluorescence over background signal for thrombin probes (1 μΜ) with exogenous enzymes Thrombin and Plasmin used at 5U/ml and 0.5μΜ respectively, after 5 min using a multiwell plate fluorimeter at excitation/emission 485/528nm. Thrombin probes containing D-amino acid residues showed selectivity for Thrombin over Plasmin (2nd generation compounds AMF-166, AMF-167 and AMF-168)
28
SUBSTITUTE SHEET RULE 26 compared with the 1 generation analogous AMF-146-01 which contain L-aa in the substrate sequence.
Figure 18. Data represents the change in fluorescence signal provided by probe AMF-166 (A) and AMF-176 (B) (10μΜ) with exogenous enzymes after 32 min using a multiwell plate fluorimeter at excitation/emission 485/528nm. Recombinant human MMPs -1 , -2, -3, -7, -8, - 9, -10, -11 , -12, -13 were used at 30nM. Recombinant human Thrombin, Plasmin and Factor Xa were used at 5U/ml, 0.5μΜ and 30nM respectively.
Figure 19. Data represents the fluorescence signal provided by probe AMF-176 (10μΜ) with exogenous enzymes after 5 min using a multiwell plate fluorimeter at excitation/emission 485/528nm. Recombinant human MMP-9 and Plasmin were used at 30nM. Recombinant human Thrombin was used at 5U/ml respectively. Anti-thrombin III inhibitor was used at 3.85 μΜ and Marimastat at 200 nM.
Figure 20. Ex-vivo assay with human tissue. Data shown represent the fluorescence signal provided by: (A) probe AMF-166 (1 μΜ) and (B) AMF-176 (10μΜ) with human fibrotic tissue homogenate at excitation/emission 485/528 nm.
Figure 21. Ex-vivo assay with human tissue. (A) Structure and MALDI spectra provided by probe AMF-166 in the presence of Thrombin and (B) after treatment with five different human fibrotic tissues showing intact probe (i) and/or the fragment obtained after specific cleavage by Thrombin (ii).
Specific Description of Embodiments of the Invention
While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.
Thrombin and MMP-9 probes
29
SUBSTITUTE SHEET RULE 26 The probes of the invention ("dual-probes") are made up of Thrombin and MMP-9 cleavable linkers, as well as two fluorophores and two quenchers strategically placed (Figure 2). The probes can be constructed following different strategies, varying in fluorophore/quencher selection and in general bio-orthogonal conjugation strategies', e.g. -metal and metal-free azide-alkyne cycloaddition or inverse electron-demand Diels-Alder reaction. Individual probes containing complementary functional groups have been synthesized by standard Fmoc solid-phase peptide synthesis. With these compounds the final coupling reaction was done by bio-orthogonal conjugation strategies. Specificity of DualProbes 1st and 2nd Generation
Dualprobes 1st and 2nd generation are activated by Thrombin and MMP-9 showing an increase in the fluorescent signal. The enzymatic cleavage of the peptide substrate causes the disruption of the FRET system and the fluorescence for each dye is recovered and measured independently at two different wavelengths (see Figure 3).
Specificity of DualProbe 3rd Generation AMF-185
The main achievement in 3rd Generation dualprobes is the selectivity for Thrombin and MMP-9 over plasmin. The fluorescence signal for each dye increases after enzymatic activation and can be knocked-down (i.e. reduced) with known inhibitors for each enzyme (Figure 4). Dyes/quenchers act independently without interfering in each other response. MALDI spectra and HPLC trace after enzymatic reaction confirms the right and specific cleavage by exogenous enzymes MMP-9 and Thrombin (Figures 5-7).
The stability of the Dual Probes was tested over a period of two months at room temperature, and the results are shown in Figure 8.
First Generation Thrombin probes
The first generation of thrombin probes were synthesized containing:
-NBD or FAM as fluorophores; MethylRed or BHQ1 as quenchers.
-Different aminoacid sequences (all containing L-aa) were evaluated initially as thrombin substrates. The best sequence was selected based on its activation by thrombin, inhibition with thrombin inhibitors, stability to Factor Xa, human BALF, sheep BALF, human neutrophil lysate, human neutrophil supernatant, human macrophages supernatant and murine macrophages supernatant (Figure 15).
30
SUBSTITUTE SHEET RULE 26 -Different linkers containing hydrophilic or charged groups: ethylenglycol units (PEG), L- Lysine, and alternative ethylenglycol units (PEG) and D-Lysine. The linkers improve the aqueous solubility of the compounds. The presence of unnatural D-amino acid improves the in vivo stability of the compounds making them resistant to proteinases.
Additional reactive groups (such as azide, alkyne, norbornene) can be added to allow subsequent reaction and formation of multiple probes. These groups do not affect the activity of the probe.
31
SUBSTITUTE SHEET RULE 26 COMPOUND Code
FAM- ■PEG 2 - ■L-T-P-R-G-V-R-L-K(MR)-NH2 SVC-01-001
FAM- ■PEG 2 - ■l-T-P-R-G-V-R-L-K(MR)-NH AMF-06
FAM- ■PEG 2 - ■Nle-T-P-R-G-V-R-L-K(MR)-NH2 AMF-07
NBD- ■PEG 2 - ■L-W-P-R-G-V-R-L-K(MR)-NH2 SVC-01-003
NBD- ■PEG 2 - ■L-T-P-R-G-W-R-L-K(MR)-NH2 SVC-01-004
FAM- ■PEG 2 - ■L-W-P-R-G-V-R-L-K(MR)-NH2 SVC-01-005
FAM- ■PEG 2 - ■L-T-P-R-G-W-R-L-K(MR)-NH2 SVC-01-006
FAM- ■PEG 2 - ■l-T-P-R-G-W-R-L-K(MR)-NH SVC-01-007
FAM- ■PEG 2 - ■Nle-T-P-R-G-W-R-L-K(MR)-NH2 SVC-01-008
FAM- ■PEG 2 - ■L-W-P-R-G-W-R-L-K(MR)-NH2 AMF-15
FAM- ■PEG 2 - ■l-W-P-R-G-W-R-L-K(MR)-NH AMF-16
FAM- ■PEG 2 - ■Nle-W-P-R-G-W-R-L-K(MR)-NH2 AMF-17
FAM- ■PEG 2 - ■Nle-T-P-R-G-W-R-L-K(MR) '-PEG 2 -K- PEG 2 -NH 2 SVC-112
FAM- ■PEG 2 - ■Nle-W-P-R-G-W-R-L-K(MR) '-PEG 2 -K-PEG 2 -NH 2 SVC-113
MR-K(Ac)-PEG2-Nle-T-P-R-G-W-R-L-K(FAM)-(PEG2-k)3-NH2 sVC-115
Norbornene-K(FAM)-PEG2-Nle-T-P-R-G-W-R-L-K(MR)-(PEG2-k)3-NH2 AMF-119-03
N3-PEG2-K(FAM)-PEG2-Nle-T-P-R-G-W-R-L-K(MR)-(PEG2-k)3-NH2 SVC-01 -131 -MR
N3-PEG2-K(FAM)-PEG2-Nle-T-P-R-G-W-R-L-K(BHQ1)-(PEG2-k)3-NH2 SVC-01 -131 -BHQ1
Alkyne-K(FAM)-PEG2-Nle-T-P-R-G-W-R-L-K(MR)-(PEG2-k)3-NH2 AMF-119-02
MR-K(Alkyne)-PEG2-Nle-W-P-R-G-W-R-L-K(FAM)-(PEG2-k)3-NH2 AMF-146-01
Table 3: 1st generation Thrombin FRET probes (*l=lle)
32
SUBSTITUTE SHEET RULE 26
Figure imgf000034_0001
1 = AMF-166, 2 = AMF-167 and (3) = AMF-168
Improvements from the first generation are achieved when modifying some key amino acids with their D-unnatural amino acid counterparts. That modification generate probes that are selective to Thrombin over plasmin (Figure 17). Also fluorophore and quenchers positions can be exchanged without altering the activity of the probes (i.e. AMF-166 vs AMF-176, Figure 18) and additional functional groups are also tolerated (i.e. alkyne group in AMF-176) and do not affect the activation or specificity of the probe. In addition, the activity of the probes was tested in the presence of thrombin inhibitors to show that the increase in fluorescence of the probes was the result of cleavage by thrombin (see Figure 19).
COMPOUND Code
FAM-PEG2-Nle-WPRGWR(D)LK(MR)-(PEG2-k)3-NH2 AMF-166
FAM-PEG2-Nle-WPRGW(D)RLK(MR)-(PEG2-k)3-NH2 AMF-167
FAM-PEG2-Nle-WPRGW(D)R(D)LK(MR)-(PEG2-k)3-NH2 AMF-168
MR-K(Alkyne)-PEG2-Nle-WPRGWR(D)LK(5-FAM)-(PEG2-k)3-NH2 AMF-176
MR-K(Alkyne)-PEG2-Nle-WP(D)RGWR(D)LK(5-FAM)-(PEG2-k)3-NH2 AMF-201
QSY21 -K(N3)-PEG2-Nle-WPRGWR(D)LK(Cy5)-(PEG2-k)3-NH2 AMF-216
Table 4: 2nd generation Thrombin FRET probes
Ex-vivo assays of lead Thrombin probes
The lead probes were evaluated with human lung tissue homogenate from patients with fibroproliferation showing an increase in the signal after activation of the probe (Figure 20). Also MALDI spectra were done after these experiments confirming the activation of the probe due to specific cleavage by Thrombin (Figure 21). Materials and Methods
General
Commercially available reagents were used without further purification. NMR spectra were recorded using Bruker AC spectrometers operating at 500MHz for 1 H. Chemical shifts are reported on the δ scale in ppm and are referenced to residual non-deuterated solvent resonances. Normal phase purifications by column chromatography were carried out on silica gel 60 (230-400 mesh). Analytical reverse-phase high-performance liquid
34
SUBSTITUTE SHEET RULE 26 chromatography (RP-HPLC) was performed on an HP1 100 system equipped with a Discovery C18 reverse-phase column (5 cm x 4.6 mm, 5 μηι) with a flow rate of 1 mL/min and eluting with H20/CH3CN/HCOOH (95/5/0.1) to H20/CH3CN/HCOOH (5/95/0.1), over 6 min, holding at (B) for 2 min (GE10) or over 10 min, holding at (B) for 4 min (GE15), with detection at 254, 500 and 650 nm and by evaporative light scattering. Semi-preparative RP- HPLC was performed on an HP1 100 system equipped with a Zorbax Eclipse XDB-C18 reverse-phase column (250 x 9.4 mm, 5 μηι) with a flow rate of 2.0 mL/min and eluting with 0.1 % HCOOH in H20 (A) and 0.1 % HCOOH in CH3CN (B), with a gradient of 5 to 95% B over 30 min and additional isocratic period of 5 min. Electrospray ionization mass spectrometry (ESI-MS) analyses were carried out on an Agilent Technologies LC/MSD Series 1100 quadrupole mass spectrometer (QMS) in an ESI mode. MALDI spectra were acquired on a Bruker Ultraflextreme MALDI TOF/TOF with a matrix solution of sinapic acid (10 mg/mL) in H20/CH3CN/TFA (50/50/0.1).
35
SUBSTITUTE SHEET RULE 26 Synthesis of Fmoc-Lvs-(/V-4-pentvnoyl)-OH and MethylRed-Lvs-(A/-4-pentvnoyl)-OH
MethylRed-Lys-(A/-4-pentynoyl)-OH
Figure imgf000037_0001
Fmoc-Lys[A/-4-Pentynoyl]-OH
Scheme 1
4-Pentynoic acid succinimidyl ester: A solution of 4-pentynoic acid (0.5 g, 5.1 mmol) and /V-Hydroxysuccinimide (0.59 g, 1 eq) in EtOAc-Dioxane (1 : 1 , 50 mL) was stirred at 0°C and DCC (1.0g, 1 eq) was added allowing the mixture to reach room temperature (rt) and kept at these conditions for 12 h. The DCU formed was filtrated and the filtrate concentrated under vacuum. EtOAc (100 mL) was added and washed with 5% NaHC03 (2x40 mL), water (40 mL) and brine (40 mL). After drying over anhNa2S04 and concentrating it was recrystallized from DCM/Hexane to obtain a white solid that was used in the next step without further purification. 1H-NMR (400 MHz, CDCI3) δ: 2.90 (t, 2H, J 7.0 Hz), 2.86 (s, 4H), 2.64 (td, 2H, J 7.0, 2.7 Hz), 2.07 (t, 1 H, J 2.7 Hz).
Fmoc-Lys-(/V-4-pentynoyl)-OH: Fmoc-Lys-OH HCI (1.67g, 1 eq) was dissolved in DMF (12 mL), DIPEA (0.79 mL, 1.1 eq) was added followed by dropwise addition of a solution of 4- Pentynoic acid succinimidyl ester (0.81 g, 1 eq) in anhDMF (4 mL). The reaction mixture was stirred for 3h. The solvent was removed under vacuum. The oily residue was dissolved in EtOAc (50 mL) and washed with 5% citric acid solution (2x25 mL), water (25 mL) and brine (25 mL). The organic phase was dried over anhNa2S04 and evaporated under vacuum. Recrystallization from DCM/hexane afforded the compound as a white solid (1.66g, 90%). MS (ES)+ m/z 449 [M+H]+, 1H-NMR (400 MHz, CDCI3) δ: 7.78 (d, 2H, J 7.6 Hz), 7.63 (m, 2H), 7.42 (t, 2H, J 7.5 Hz), 7.33 (t, 2H, J 7.6 Hz), 5.92 (m, 1 H), 5.69 (m, 1 H), 4.48-4.34 (m, 3H), 4.24 (t, 1 H, J 7.0 Hz), 3.32 (m, 2H), 2.53 (t, 2H, J 7.0 Hz), 2.40 (t, 2H, J 7.0 Hz), 2.03 (s, 1 H), 1.94 (m, 3H), 1.77 (m, 2H), 1.58 (m, 2H), 1.46 (m, 2H), 1.18 (m, 3H).
Boc-Lys[/V-4-Pentynoyl]-OH: Boc-Lys-OH (1.25 g, 5.1 mmol) was dissolved in anhDMF (15 mL), DIPEA (0.97 mL) was added followed by dropwise addition of a solution of 4-Pentynoic acid succinimidyl ester (5.1 mmol) in anhDMF (8 mL). The reaction mixture was stirred at rt for 3h. The solvent was removed under vacuum. To the crude HCI 1 N (30 mL) was added and extracted with EtOAc (3x40 mL). The combined organic phase was dried over anhNa2S04 and evaporated under vacuum to afford a white solid (1.36g, 82%). MS (ES)" m/z 325 [M-H]", spectroscopic data identical to those reported previously in the literature. [D. P. Nguyen, H. Lusic, H. Neumann, P. B. Kapadnis, A. Deiters and J. W. Chin, J. Am. Chem. Soc. 2009; 131 , 8720-8721.]
MethylRed-Lys-(/V-4-pentynoyl)-OH: Boc-Lys[/V-4-Pentynoyl]-OH (1.0 g, 3.0 mmol) was dissolved in 20%TFA in dichloromethane (10 mL) and the resulting mixture stirred for 3h. The solvent was removed under vacuum and co-evaporated with toluene. The crude was dissolved in anhDMF (5 mL). MethylRed-NHS ester [R. C. Brown, Z. Li, A. J. Rutter, X. Mu, O. H. Weeks, K. Smith and I. Weeks, Org. Biomol. Chem. 2009, 7, 386-394.] (1.1 g, 1 eq)
SUBSTITUTE SHEET RULE 26 and DIPEA (1.5 mL, 3eq) were added and the mixture stirred overnight at rt. The solvent was removed under vacuum and the crude mixture dissolved in DCM (150 mL). HCI 1 N (100 mL) was added and extracted again with DCM (2x100 mL). The combined organic phase was dried over anhNa2S04, evaporated under vacuum and purified with silica column chromatography (1 : 10 to 1 :3 MeOH/EtOAc) to afford MethylRed-Lys-(/V-4-pentynoyl)-OH as a dark red solid (0.67 g, 47%). m.p. 153-155°C; 1H-NMR (500 MHz, CD3OD) δ: 8.15 (dd, 1 H, J 7.8, 1.4 Hz), 7.98 (d, 2H, J 8.9 Hz), 7.78 (d, 1 H, J 7.9 Hz), 7.57 (td, 1 H, J 7.6, 1.3 Hz), 7.49 (t, 1 H, J 7.6 Hz), 6.86 (d, 2H, J 9.1 Hz), 4.65 (t, 1 H, J 5.2 Hz), 3.12 (s, 6H), 3.08 (t, 2H, J 6.1 Hz), 2.41-2.37 (m, 2H), 2.30-2.26 (m, 2H), 2.24 (t, 1 H, J 2.6 Hz), 2.02 (m, 1 H), 1.87 (m, 1 H), 1.49 (m, 2H), 1.43(m, 2H); 13C-NMR (125 MHz, CD3OD) δ: 179.1 , 173.8, 168.2, 155.0, 152.3, 144.8, 133.0, 131.6, 131.0, 130.2, 127.5, 1 17.4, 1 12.8, 83.5, 70.4, 56.4, 40.4, 40.3, 36.1 , 33.6, 30.1 , 24.3, 15.8; MS (ES)+ m/z 478 [M+H]+; HPLC tR 5.29 min (GE10).
Synthesis of individual FRET peptides
The FRET peptide sequences for MMP and thrombin were individually synthesized by standard Fmoc solid-phase peptide chemistry. Dyes and quenchers were coupled also by solid phase. General procedures are as follows:
Manual peptide synthesis was performed on Aminomethyl-ChemMatrix resin using Rink amide linker.
Coupling of rink amide linker: Fmoc-Rink linker (4-[(R,S)-a-[1-(9H-Fluoren-9-yl)-methoxy- formamido]-2,4-dimethoxybenzyl-phenoxyacetic acid) (0.54 g, 1.0 eq) was dissolved in DMF (10 mL) and Oxyma (0.14 g, 1.0 eq.) was added and the mixture was stirred for 10 min. Diisopropylcarbodiimide (DIC, 155 μί, 1.0 eq.) was then added and the solution stirred for 1 min before adding it to Aminomethyl-ChemMatrix resin (1.0 g, 1.0 mmol/g). The resulting mixture was stirred at 50°C for 45 min and washed with DMF (3x10 mL), DCM (3x10 mL) and MeOH (3x10 mL). Finally the resin was treated with Ac20:Py:DMF (2:3: 15) for 30 min at rt in order to cap any remaining free amino group and it was washed again with DMF (3x10 mL), DCM (3x10 mL) and MeOH (3x10 mL). Resin loading was calculated after that as -0.58 mmol/g.
Fmoc deprotection: In general, to the resin pre-swollen in DCM was added 20% piperidine in DMF and stirred at rt (2x10 min). The solution was drained and the resin washed with DMF (3x10 mL), DCM (3x10 mL) and MeOH (3x10 mL). In the cases were Fmoc
38
SUBSTITUTE SHEET RULE 26 deprotection was done in Cy5 containing peptides, a solution of 2% DBU in DMF (2 x 10 min, rt) was used instead.
Aminoacid coupling: A solution of the appropriate D- or L-amino acid (3.0 eq per amine) and Oxyma (3.0 eq) in DMF (0.1 M) was stirred for 10 min. DIC (3.0 eq) was added and stirred for 1 min. The pre-activated mixture was then added to the resin pre-swollen in DCM and the reaction heated at 50°C for 30 min. The solution was drained and washed with DMF (3x10 ml_), DCM (3x10 ml_) and MeOH (3x10 ml_). The completion of the coupling and deprotection reactions was monitored by Kaiser test or Chloranil test when secondary amines are involved. The side chain protecting group used was Boc for arginine, tryptophan and lysine. Fmoc-Lys(Dde)-OH was used as orthogonal reagent to introduce the dyes.
Coupling of other carboxylic acids: Coupling of {2-[2-(Fmoc-amino)ethoxy]ethoxy}acetic acid (PEG), 5-Carboxyfluorescein (FAM), Fmoc-Lys(N3)-OH and MethylRed-Lys-(4- pentynoyl)-OH was done following the same procedure described for Aminoacid coupling.
Dde deprotection: (a) Dde deprotection in non-containing Fmoc peptides was done following the next procedure: to the resin pre-swollen in DCM was added 2% hydrazine in DMF and stirred at rt (5x10 min). The solution was drained and the resin washed with DMF (3x10 ml_), DCM (3x10 ml_) and MeOH (3x10 ml_). (b) Selective Dde deprotection in Fmoc- protected peptides was done with a solution containing Imidazole (1.35 mmol) and Hydroxylamine hydrochloride (1.80 mmol) in NMP (5 ml_). [Diaz-Mochon, J. J.; Bialy, L; Bradley, M. Org Lett 2004, 6 (7), 1127-1 129]. After complete dissolution 5 volumes of this solution were diluted with 1 volume of CH2CI2 and the resin was treated with the final mixture for 3h at room temperature. The solution was drained and the resin washed with DMF (3x10 ml_), DCM (3x10 ml_) and MeOH (3x10 ml_).
Sulfo-Cy5 dye coupling: A solution containing sulfo-Cy5 (1 eq) in anh DMF (10 mg/mL) was activated with N,N,N',N'-Bis(tetramethylene)-0-(N-succinimidyl)uronium hexafluorophosphate (HSPyU) (1 eq) and DIPEA (3 eq) at 40°C for 1 h. Once the activation is complete the solution is added to the resin together with DIPEA (3 eq) and shaken at rt overnight. The solution was drained and the resin washed with DMF until colourless wash solution, DCM (3x5 ml_) and MeOH (3x5 ml_). QSY21 coupling: N-terminal capping with QSY21-NHS ester (1 eq) was done in anhDMF (0.1 M) containing DIPEA (3 eq) at rt for 12 h. The solution was drained and the resin washed
39
SUBSTITUTE SHEET RULE 26 with DMF until colourless wash solution, DCM (3x5 mL), MeOH (3x5 mL) and finally ether (3x5 mL).
MethylRed-NHS coupling: MethylRed-NHS ester (1 eq) coupling in solid phase was done in anhDMF (0.1M) containing DIPEA (3 eq) at rt for 12 h. The solution was drained and the resin washed with DMF until colourless wash solution, DCM (3x5 mL), MeOH (3x5 mL) and finally ether (3x5 mL).
Cleavage and purification: The resin pre-swollen in DCM was treated with a cleavage cocktail of TFA:triisopropylsilane(TIS):water (95:2.5:2.5) for 3h at room temperature. The reaction solution was drained and the resin washed again with cleavage cocktail. The combined solution was precipitated against cold ether, centrifuged (x3) and purified by RP- HPLC on a Ci8 semi-preparative column. The desired fractions containing the product were collected and lyophilized to afford the products that were characterized by MALDI and analytical HPLC.
Thrombin probes containing alkyne, FAM and MR. Synthesis and Characterization data:
Thrombin substrates were built on resin, cleaved and purified following the general procedures previously described.
40
SUBSTITUTE SHEET RULE 26
Figure imgf000042_0001
41
SUBSTITUTE SHEET RULE 26 :15
MF
Figure imgf000043_0001
i) 20% Piperidine/DMF
ii) Fmoc-aa-OH, Oxyma, DIC, DMF
Fmoc-PEG-NleW(Boc)PR(Boc)GW(Boc)R(Boc)- X-K(Dde)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-Rink
Figure imgf000043_0002
i) NH2OH.HCI, Imidazole, NMP, DCM
ii) 5-FAM, Oxyma, DIC, DMF
Fmoc-PEG-NleW(Boc)PR(Boc)GW(Boc)R(Boc)- X-K(5-FAM)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-Rink
Figure imgf000043_0003
i) 20% Piperidine/DMF
ii) MethylRed-K(4-pentynoyl)-OH, Oxyma, DIC, DMF
MR-K(4-pentynoyl)-PEG-NleW(Boc)PR(Boc)GW(Boc)R(Boc)- X-K(5-FAM)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-Rink —
TFA, TIS, H20 95:2.5:2.5
X : Leu for AMF- 146» MR-K(4-pentynoyl)-PEG-NleWPRGWR- X-K(5-FAM)-PEG-(D)K-PEG-(D)K-PEG-(D)K -NH2
Scheme 2. Synthesis of FRET Thrombin peptides with FAM, MR and alkyne moiety.
MethylRed-K(A/-4-pentvnovn-PEG-aai-aan-K(A/-5-FAM)-PEG-(D)K-PEG-(D)K-PEG-(D)K- NH?
AMF-146-01 : HPLC tR = 4.0 min, MALDI calc. for C148H211 N34 V [M+H]+: 2994.520; found: 2994.846. AMF-176: HPLC tR = 4.2 min, MALDI calc. for C148H211 N34 V [M+H]+: 2994.520; found: 2994.037. (Figure 9)
An alternative thrombin structure with FAM and MR was also synthesised using similar chemistry as described above, following Scheme 3 below:
42
SUBSTITUTE SHEET RULE 26 SYNTHESIS OF TH
(
Figure imgf000044_0001
i) 20% Piperidine/DMF
ii) 5-FAM, Oxyma, DIC, DMF
Figure imgf000044_0002
TFA, TIS, H20 95:2.5:2.5
Compound 1 : X: Arg: Y: D-Lei*
Compound 2: X: D-Arg: Y: lef:
¾¾}ift ¾y^^ 5-FAM-PEG-NleWPRGW-X-Y-K(MR)-PEG-(D)K-PEG-(D)K-PEG-(D)K-NH2
Scheme 3. Synthesis of 2 Generation Thrombin Probes
Thrombin probes containing azide, sulfo-Cy5 & QSY21. Synthesis and Characterization data
Thrombin substrates were built on resin, cleaved and purified following the general procedures previously described.
43
SUBSTITUTE SHEET RULE 26 SYNTHESIS OF THI H,N
(Rink Amide Linker) :15
MF
Figure imgf000045_0001
i) 20% Piperidine/DMF
ii) Fmoc-aa-OH, Oxyma, DIC, DMF
Fmoc-K(N3)PEG-NleW(Boc)PR(Boc)GW(Boc)R(Boc)(D)LK(Dde)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-Rink— i) NH2OH.HCI, Imidazole, NMP, DCM
ii) sulfo-Cy5, HSPyU, DIPEA, anhDMF
Fmoc-K(N3)-PEG-NleW(Boc)PR(Boc)GW(Boc)R(Boc)(D)LK(sulfo-Cy5)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-Rink— M
i) 2% DBU/DMF
ii) QSY21 -NHS, DIPEA, anhDMF
QSY21 -K(N3)-PEG-NleW(Boc)PR(Boc)GW(Boc)R(Boc)(D)LK(sulfo-Cy5)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-Rink— l§ll
TFA, TIS, H20 95:2.5:2.5
QSY21 -K(N3)-PEG-NleWPRGWR(D)LK(sulfo-Cy5)-PEG-(D)K-PEG-(D)K-PEG-(D)K-NH2
Scheme 4. Synthesis of FRET peptide with sulfo-Cy5 and QSY21 exemplified for AMF-216.
44
SUBSTITUTE SHEET RULE 26
Figure imgf000046_0001
MMP probes containing azide, Cy5 & QSY21. Synthetic scheme and Characterization data:
MMP substrates were built on resin, cleaved and purified following the general procedures previously described.
Figure imgf000047_0001
for AMF-140: aa aan-: -GPKGLKG- ; X: -CH2-NH-CO-CH2OCH2CH2OCH2CH2- for AM F- 154-03: aa aan-:-PFGNIeKpA; X: -CH2-NH-CO-CH2OCH2CH2OCH2CH2- for AMF-181 : aa aan-: -PFGNIeK A; X: -CH2-
SYNTHESIS OF MMP RA MEW
FOR DUALPROBE
Figure imgf000048_0001
i) 20% Piperidine/DMF
ii) Fmoc-aa-OH, Oxyma, DIC, DMF
Fmoc-K(N3)-PEG-PFGNIeK(Boc)pAK(Dde)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-Rink i) NH2OH.HCI, Imidazole, NMP, DCM
ii) sulfo-Cy5 or carboxy-NF, HSPyU, DIPEA, anhDMF
Fmoc-K(N3)-PEG-PFGNIeK(Boc)pAK(sulfo-Cy5/carboxy-NF)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-Rin i) 2% DBU/DMF
ii) QSY21 -NHS, DIPEA, anhDMF
QSY21 -K(N3)-PEG-PFGNIeK(Boc)pAK(sulfo-Cy5/carboxy-NF)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-PEG-(D)K(Boc)-Rink
TFA, TIS, H20 95:2.5:2.5
QSY21 -K(N3)-PEG-PFGNIeKpAK(sulfo-Cy5/carboxy-NF)-PEG-(D)K-PEG-(D)K-PEG-(D)K-NH2
Scheme 5. Synthesis of FRET peptides with Cy5 or carboxy-naphtofluorescein (carboxy-NF) and QSY21 exemplified for compound AMF-181 and SVC-02-009.
QSY21-K(X')-PEG-aai-aan-K(sulfo-Cv5)-PEG-(D)K-PEG-(D)K-PEG-(D)K-NH,
AMF-140: HPLC tR = 3.9 min, MALDI calc. for. Ci63H232N33038S3 + [M]+: 3358.021 ; found: 3358.851. AM F- 154-03: HPLC tR = 4.0 min, MALDI calc. for Ci65H228N3i037S3 + [M]+: 3333.998; found: 3333.765. AMF-181 : HPLC tR = 4.5 min, MALDI calc. for Ci59H217N3o034S3+ [M]+: 3188.840; found: 3188.966. (Figure 1 1)
MMP probes containing FAM & MethylRed. Synthetic scheme and Characterization data:
Synthesis of Branched dendrimer scaffold:
The dendrimer scaffold was synthesised by following the prior art reported in WO 2012/136958 A2 (Aslam et al).
47
SUBSTITUTE SHEET RULE 26 Synthesis of monomer (V)
Multi-valent probe synthesis required the preparation of the monomer (V) which was synthesised in six steps1 as shown in Scheme 6. Monomer (V) was prepared by the 1 ,4 addition of the hydroxy groups of 1 , 1 , 1 - fr/'s(hydroxymethyl)amino-methane onto acrylonitrile, followed by amino group protection (Boc). Reduction of the nitrile groups with Pt02/H2 gave (III) which was treated with DdeOH to give the tris-Dde protected amine (IV). Following removal of the Boc protecting group, the isocyanate (V) was prepared following the procedure of Knolker.2
Figure imgf000049_0001
DdeOH, DIPEA
MeOH
Figure imgf000049_0002
IV
V
Scheme 6. Synthesis of monomer (V) used in the preparation of FRET branched-peptide with FAM and MR exemplified for AMF-216 in Scheme 7 below
MMP substrates were built on resin, cleaved and purified following the general procedures previously described.
48
SUBSTITUTE SHEET RULE 26
Figure imgf000050_0001
i) 20% Piperidine/DMF
ii) Fmoc-aa-OH, Oxyma, DIC, DMF
Fmoc-K(Dde)-PEG-(D)K(Boc)-PEG-K( /V-4-pentynoyl)-Rink
Figure imgf000050_0002
i) NHjOH.HCI, Imidazole, NMP, DCM
ii) MethylRed-NHS, DIPEA, anhDMF
Figure imgf000050_0003
Scheme 7. Synthesis of FRET branched-peptide with FAM and MR exemplified for AMF- 216.
Figure imgf000051_0001
Chemical Formula: C238H312N4o06o Molecular Weight: 4693.3340
The above molecular weight was confirmed by MALDI and is shown in Figure 12.
DUALPROBES. Synthesis and Characterization data:
Example 1 : Cu-catalysed azide-alkyne cycloaddition
QSY21 -K( N3)-PEG-PFGNIeK AK(sulfo-Cy5)-PEG-(D)K-PEG-(D)K-PEG-(D)K-NH 2
+
MethylRed-K( 4-pentynoyl )-PEG-NleWPRGWR(D)LK(5-FAM)-PEG-(D)K-PEG-(D)K-PEG-(D)K
CuS04 /Ascorbate
THTPA
Aminoguanidine
H20
QSY2 GNIeK AK(sulfo-Cy5)-PEG-(D)K-PEG-(D)K-PEG-(D)K-NH 2
Figure imgf000052_0001
MethylRed-K-PEG-NleWPRGWR(D)LK(5-FAM)-PEG-(D)K-PEG-(D)K-PEG-(D)K -NH2 Scheme 8. Synthesis of DualProbes exemplified for compound AMF-185
General procedure for the synthesis of DualProbes by Cu-catalysed azide-alkyne cycloaddition: Optimized conditions for the click reaction using biomolecules were used [Hong, V., Presolski, S. I., Ma, C, and Finn, M. G. Angew. Chem. Int. Ed. 2009, 48, 9879.]. In an eppendorf tube the following aqueous reagents were mixed: alkyne-peptide fragment (AM F- 146-01 or AMF-176) (50 μΙ_, 1 mM), azide-peptide fragment (AMF-140, AMF-154-03 or AMF-181) (50 μΙ_, 1 mM), premixed CuS04 and THPTA (40 μΙ_ CuS04 20mM and 80 μΙ_ THTPA 50 mM), aminoguanidine hydrochloride (250 μΙ_, 100 mM) and finally sodium ascorbate (250 μΙ_, 100 mM). The click-chemistry reaction was allowed to proceed at 30°C for 5h, the reaction mixture was lyophilised and purified by HPLC to give the final dual smartprobes, which were characterized by MALDI and analytical HPLC: AMF-152: HPLC tR = 4.21 min, MALDI calc. for C3i i H442N6707iS3 + [M+]: 6351.534; found: 6351.492. AMF-162: HPLC tR = 4.24 min, MALDI calc. for C3i3H438N6507oS3 + [M+]: 6327.51 1 ; found: 6327.699. AMF-185, HPLC tR = 4.15 min, MALDI calc. for C3o7H427N64067S3 + [M+]: 6182.353; found: 6182.343. (see Figure 13).
51
SUBSTITUTE SHEET RULE 26
Figure imgf000053_0001
Branched-DualProbes. Synthesis and Characterization data:
Optimized conditions for the click reaction using biomolecules were used: in an eppendorf tube the following aqueous reagents were mixed: alkyne-peptide three-branched fragment (AMF-212) (50 μΙ_, 1 mM), azide-peptide fragment (AMF-216) (50 μΙ_, 1mM), premixed CuS04 and THTPA (40 μΙ_ CuS04 20mM and 80 μΙ_ THPTA 50 mM), aminoguanidine hydrochloride (250 μΙ_, 100 mM) and finally sodium ascorbate (250 μΙ_, 100 mM). The click- chemistry reaction was allowed to proceed at 30°C for 5h, the reaction mixture was lyophilised and purified by HPLC to give the final branched-dualprobe, which was characterized by MALDI and analytical HPLC: AMF-217: HPLC tR = 4.68 min, MALDI calc. for C4i 9H558N79096S3 + [M+]: 8333.709; found: 8333.424.
Figure imgf000054_0001
Scheme 9. Synthesis of Branched-DualProbes exemplified for compound AMF-217
53
SUBSTITUTE SHEET RULE 26
Figure imgf000055_0001
Chemical Formula: C419H558N79096S3+ Molecular Weight: 8333.7095
The molecular weight of the above was confirmed by MALDI and is shown in Figure 14.
Example 2: Tetrazine ligation
Tetrazine ligation between fragments containing Tetrazines and fragments containing dienophiles such as Norbornene can be used for the synthesis of dualprobes.
Coupling of norbornene and tetrazine moieties was done in solid phase: The peptide was synthesized by standard Fmoc solid-phase peptide chemistry following the general procedures. exo-5-Norbornenecarboxylic acid coupling on solid phase: to the resin previously swollen with DCM a solution of exo-5-Norbornenecarboxylic acid (3.0 eq per amine) and Oxyma (3.0 eq) in DM F (0.1 M) was stirred for 10 min. DIC (3.0 eq) was added and stirred for 1 min. The pre-activated mixture was then added to the resin pre-swollen in DCM and the reaction heated at 50°C for 30 min. The solution was drained and washed with DMF (3x10 ml_), DCM (3x10 ml_) and MeOH (3x10 ml_).
The structure of thrombin probe with FAM/MR containing Norbornene (AMF1 19-03 m/z calc for C144H212N33034 [M+H]+ 2949.476, obs: 2948.724) is shown below:
55
SUBSTITUTE SHEET RULE 26
Figure imgf000057_0001
The structure of MMP substrate peptide containing Tetrazine (m/z calc for
Figure imgf000057_0002
[M+H]+ 2223.634, obs: 2224.565) is shown below:
Figure imgf000057_0003
Chemical Formula: C 0oH168N3o0
Molecular Weight: 2222.62700
Tetrazine ligation in solution phase: In an eppendorf tube aqueous solutions of tetrazine- peptide fragment (20 μΙ_, 1 mM) and norbornene-peptide fragment (20 μΙ_, 1 mM) were mixed and the reaction was allowed to proceed for 2h. The tetrazine adduct was detected by MALDI. (AMF1 16 m/z calc for C212H361 N58O56 [M+H]+ 4618.563, obs: 4617.500).
DUAL SUBS TRA1 E Tetrazine-K(Ac)-PEG-GPKGLKGK(Ac)-PEG-(D)K-PEG-(D)K-PEG-(D)K-NH2 l e trazir lie ation
+
Figure imgf000058_0001
Ac-K-PEG-NleTPRGWRLK(Ac)-PEG-(D)K-PEG-(D)K-PEG-(D)K-NH2
57
SUBSTITUTE SHEET RULE 26
Figure imgf000059_0001
Scheme 10: Tetrazine ligation. Reaction scheme and full structure of dualsubstrate.
Example 3: Strain-Promoted Alkyne-Azide Cycloadditions (SPAAC)
Thrombin probe containing dibenzocyclooctyl (DBCO), a strained alkyne moiety: The functionalised peptide was synthesized by either standard Fmoc solid-phase peptide chemistry or solution phase functionalization of fully protected probes by following the general procedures.
Dibenzocyclooctyne-NHS coupling in solid phase: to the resin previously swollen with DCM a solution of DBCO-NHS ester (1 eq.) in anhDMF (0.1 M) containing DIPEA (3 eq.) was added and the reaction mixture was kept at rt for 12 h. The solution was drained and the resin washed with DMF (3x5 ml_), DCM (3x5 ml_), MeOH (3x5 ml_) and finally ether (3x5 ml_).
Cleavage of fully protected peptide fragments: To the 2-chlorotrityl chloride resin, previously swollen with DCM, a solution of 10% HFIP (hexafluoro-2-propanol) in DCM was added and mixed at room temperature. After 30 min filter off resin and wash with HFIP/DCM (1 :9) and DCM. Remove solvent in vacuum, redissolve in a DCM and evaporate in vacuo to afford solid.
To characterise the isolated peptide fragment, it is further treated with 20% TFA in DCM at room temperature. After 1 hour all volatiles are removed in vacuo and the solid product was washed with diethyl ether and dried (SVC-01 -162;
Figure imgf000060_0001
MW: 2903.34, MALDI- TOF/TOF (m/z): 2905.0 [M+H]+).
Dibenzocyclooctyne-NHS (DBCO-NHS) coupling in solution: To a solution of fully protected peptide fragments in anhDMF (0.1 M) a solution of DBCO-NHS ester (1 eq.) in anhDMF (0.1 M) containing DIPEA (3 eq.) was added and the reaction mixture was kept at rt for 12 h. The reaction mixture was evaporated in vacuo, washed with water and lyophilized to afford the solid compound. To characterise the isolated peptide fragment, it is further treated with 20% TFA in DCM at room temperature. After 1 hour all volatiles are removed in vacuo and the solid product was washed with diethyl ether and dried (SVC-01-163;
Figure imgf000060_0002
MW: 3190.66, MALDI- TOF/TOF (m/z): 3188.5.0 [M]+). Strain-Promoted Alkyne-Azide Cycloadditions (SPAAC) with protected peptide:
DBCO-functionalised fully protected peptide fragment was treated with N3-PEG2-OH in 50%
59
SUBSTITUTE SHEET RULE 26 acetonitrile (0.1M; aqueous) at room temperature. After 2 hours the reaction mixture was lyophilised to afford solid compound. The product was further treated with 20% TFA in DCM at room temperature. After 1 hour all volatiles were removed in vacuo and the solid product was washed with diethyl ether and dried (SVC-01-163Tz; C163H228N36O43; MW: 3379.83, MALDI-TOF/TOF (m/z): 3382.7 [M+H]+).
Figure imgf000061_0001
60
SUBSTITUTE SHEET RULE 26
Figure imgf000062_0001
Scheme 11 : Structure for DBCO-Thrombin probe: Strain-Promoted Alkyne-Azide Cycloadditions (SPAAC) with protected peptides. Reaction scheme of ligated peptides. SVC-01-134 (m/z calc for C163H228N36O43 [M+H]+ 3276.8, obs: 3276.337).
61
SUBSTITUTE SHEET RULE 26
Figure imgf000063_0001
Figure imgf000063_0002
Figure imgf000063_0003
Scheme 12: Structure for DBCO-Thrombin probe AM F-146-02 (m/z calc for C162H220N35O34 [M+H]+ 3201 .753, obs: 3201 .698) and after reactio with azide-PEG (m/z calc for ΟΙΜΗΜΙ ΝΜΟΜ [M+H]+ 3390.924, obs: 3390.860).
SPAAC reaction in water: In an eppendorf tube aqueous solutions of DBCO-peptide (20 μΙ_, 1 mM) and azide-peptide fragment (20 μΙ_, 1 mM) or (2-(2-azidoethoxy)ethoxy)acetic acid (1 mg) were mixed and the reaction was allowed to proceed for 2h. The click adduct was detected by MALDI.
SYNTHESIS OF DUALPROBE QSY21-K(PEG N3)-PEG-GPKGLKGK(sulfo-Cy5)-PEG-(D)K-PEG-(D)K-PEG-(D)K-NH 2 Cu-froo reaction
+
MethylRed-K(DBCO)-PEG-NleWPRGWR(D)LK(5-FAM)-PEG-(D)K-PEG-(D)K-PEG-(D)K -NH2
H20
eK AK(sulfo-Cy5)-PEG-(D)K-PEG-(D)K-PEG-(D)K-NH 2
Figure imgf000064_0001
leWPRGWR(D)LK(5-FAM)-PEG-(D)K-PEG-(D)K-PEG-(D)K -NH2
63
SUBSTITUTE SHEET RULE 26
Figure imgf000065_0001
Scheme 13. Synthetic scheme and full structure of dualprobe AMF-150 synthesized by SPAAC (m/z calc for C325H451 Ν68θ72 obs:6555.453).
Enzyme assay
The enzyme assays were run in a 384-well format on a PCR opaque microplate (Thermo Scientific). All dilutions and reactions were prepared in MMP buffer (50mM Tris, 10mM CaCI2, 0.15M NaCI, 0.05% Brij-35, pH 7). Proteolytic activity was determined by calculating the fold change in fluorescence over background signal provided by the corresponding dilution of the probe and/or inhibitors with exogenous enzymes using a-multiwell plate fluorimeter (Synergy H1 Hybrid Reader, BioTek instruments Ltd) at excitation/emission 485/528nm. Recombinant human MMPs (Catalytic domain MMP-1 , -2, -3, -7, -8, -9, -10, -11 , -12, -13 (Enzo Life Sciences) and Full-length MMP-2 , -9, -12 and -13 (Merck/Millipore)) were used at 30nM. Pro- MMP- 13 (R & D Systems) was activated by incubating with 1 mM 4- aminophenylmercuric acetate (APMA) for 2hrs at 37°C. Human neutrophil elastase (Elastase Product Company, used at 2.5ug/ml), neutrophil lysate (lysed human neutrophils), and recombinant human Thrombin (Sigma-Aldrich, used at 5U/ml), Plasmin (Sigma-Aldrich, used at 30nM) and Factor Xa (Sigma-Aldrich, used at 0.5μΜ) were used to identify the lead molecular probe sequences. For inhibition assays, enzyme and inhibitor were pre-incubated for 1 hr at 37°C before the addition of molecular probe. Inhibitors Marimastat (Toris Bioscience), AZD1236 (AstraZeneca), Inhibitor I (Sigma-Aldrich) and SB-3CT (Sigma- Aldrich) were used at 200nM for in-vitro and 50μΜ for ex-vivo assays, respectively.
Human and Ovine tissue supernatant
Human fibrotic lung tissue biopsies were obtained from Idiopathic Pulmonary Fibrosis-(IPF) patients at the Royal Infirmary, Edinburgh. Under sterile condition, the tissue was dissected and stored at -70°C for further analysis. Sheep fibrotic lung tissue biopsies were obtained from Ovine Pulmonary Adenocarcinoma-(OPA) animals at the Roslin Institute, Edinburgh. Under sterile condition, the tissue was dissected and stored at -70°C for further analysis. For the preparation of tissue supernatant, frozen tissue was suspended in PBS and homogenised (Bio-Gen PRO200 homogeniser, Pro-Scientific) on ice.
65
SUBSTITUTE SHEET RULE 26 Samples were centrifuged at 13000rpm for 15min at 4°C and the debris-free supernatant collected. Total protein concentrations wer determined using at Pierce™ BCA kit (Thermo Scientific). The samples were aliquoted and stored at -20°C or -70°C until further analysis.
COMPOUND Code m/zca|C (Da) MALDI-TOF
m/z (Da)
1 QSY21-K(PEG-N3)-PEG2-G-P-K-G-L-K-G-K(Cy5)-PEG2-k-PEG2-k-PEG2-k-NH2 AMF-140 3358.021[M]+ 3358.851
2 QSY21-K(PEG-N3)-PEG2-P-F-G-Nle-K-pA-K(Cy5)-PEG2-k-PEG2-k-PEG2-k-NH2 AMF-154-03 3333.998[M]+ 3333.765
3 QSY21-K(N3)-PEG2-P-F-G-Nle-K-pA-K(Cy5)-PEG2-k-PEG2-k-PEG2-k-NH2 AMF-181 3188.840[M]+ 3188.966
4 QSY21-K(N3)-PEG2-P-F-G-Nle-K-pA-K(carboxy-NP)-PEG2-k-PEG2-k-PEG2-k- SVC-02-009 3002.5205[M]+ 3002.066
NH2
0\ 5 QSY21-K(N3)-PEG2-P-F-G-(D)Nle-K-pA-K(Cy5)-PEG2-k-PEG2-k-PEG2-k-NH2 AMF-199 3188.840[M]+ 3188.904
6 [FAM-PEG2-P-F-G-Nle-K-pA]3dendr-Lys(M R)-PEG2-k-PEG2-K(Alkyne)-NH2 AMF-212 4694.341[M+H]+ 4694.450
7 Alkyne-K(FAM)-PEG2-Nle-T-P-R-G-W-R-L-K(M R)-(PEG2-k)3-NH2 2909.415[M+H]+ 2909.990
AMF-119-02
8 Norbornene-K(FAM)-PEG2-Nle-T-P-R-G-W-R-L-K(M R)-(PEG2-k)3-NH2 2949.476[M+H]+ 2948.724
AMF-119-03
SVC-01-131- 3000.481[M+H]+ 3000.512
9 N3-K(FAM)-PEG2-Nle-T-P-R-G-W-R-L-K(M R)-(PEG2-k)3-NH2
MR
SVC-01-131- 3234.7[M+H]+ 3235.7
10 N3-K(FAM)-PEG2-Nle-T-P-R-G-W-R-L-K(BHQl)-(PEG2-k)3-NH2
BHQ1
11 M R-K(Alkyne)-PEG2-Nle-W-P-R-G-W-R-L-K(FAM)-(PEG2-k)3-NH2 2994.520[M+H]+ 2994.846
AMF-146-01
Figure imgf000068_0001
o
67
SUBSTITUTE SHEET RULE 26 QSY21-K(PEG2)-PEG2-PFGNIeKPA-K(Cy5)-(PEG2-k)3-NH2
triazole
23 I AMF-162 6327.511 [M]+ 6327.699
MR-K-PEG2-NleWPRGWRLK(FAM)-(PEG2-k)3-NH2
QSY21 -K-PEG2-PFGNIeK A-K(Cy5)-(PEG2-k)3-NH2
24 triazole
I AMF-185 6182.353 [M]+ 6182.343
MR-K-PEG2-NleWPRGWR(DJLK(5-FAM)-(PEG 2-k)3-NH2
QSY21-K-PEG2-PFGNIeK A-K(carboxy napthofluoresceinHPEG 2-k)3-NH2
25 triazole
I SVC-02-034 5995.0355 [M+] 5995.051
MR-K-PEG2-NleWPRGWR(DJLK(5-FAM)-(PEG2-k)3-NH2
[FAM-PEG2-PFGNIeK A]3-K(IVIR)-PEG2-k-PEG2-K-NH2
26 triazole AMF-217 8333.709 [M]+ 8333.424
QSY21 -K-PEG2-NleWPRGWR(DJLK(Cy5)-(PEG2-k)3-NH2
Table 5 shows characterisation data for example probes of the invention. Probes 1-6 correspond to probes for MMP, probes 7-20 correspon to probes for thrombin, and probes 21-26 correspond to probes for both MMP and thrombin.

Claims
An optical probe comprising a first probe element, and a second probe element; the first probe element and the second probe element connected to a core; the first probe element comprising a first fluorophore connected to a first quencher by a first cleavable linker; the second probe element comprising a second fluorophore connected to a second quencher by a second cleavable linker; the first fluorophore being substantially fluorescently quenched by the first quencher when connected to the first quencher by the first cleavable linker; the second fluorophore being substantially fluorescently quenched by the second quencher when connected to the second quencher by the second cleavable linker; wherein the first fluorophore is separated from the first quencher when the first cleavable linker is cleaved, and the second fluorophore is separated from the second quencher when the second cleavable linker is cleaved.
A probe according to claim 1 , wherein the first cleavable linker is cleaved by a first cleaving agent, and the second cleavable linker is cleaved by a second cleaving agent.
A probe according to either claim 1 or claim 2, wherein the first cleaving agent is a first enzyme, and the first cleavable linker corresponds to a first enzyme cleavable peptide sequence.
A probe according to any preceding claim, wherein the second cleaving agent is a second enzyme and the second cleavable linker corresponds to a second enzyme cleavable peptide sequence.
A probe according to any preceding claim, wherein the first cleaving agent is a first enzyme and the second cleaving agent is a second enzyme.
A probe according to claim 5, wherein the first enzyme and the second enzyme are both overexpressed and/or activated in the same one or more diseases.
A probe according to any one of claim 3 to claim 6, wherein the first enzyme is a matrix metalloproteinase (MMP).
A probe according to claim 7, wherein the MMP is MMP-2, MMP-9, or MMP-13. A probe according to claim 7 or claim 8, wherein the first enzyme cleavable peptide sequence comprises one of SEQ ID N0.1 , SEQ ID NO.5 or SEQ ID NO.7.
A probe according to claim 9, wherein the first enzyme cleavable peptide sequence comprises SEQ ID NO. 1 or SEQ ID NO.7.
A probe according to any one of claim 4 to claim 10, wherein the second enzyme is thrombin.
A probe according to claim 11 , wherein the second enzyme cleavable peptide sequence comprises one of SEQ ID N0.25 to SEQ ID NO.31 or SEQ ID NO.37.
A probe according to claim 12, wherein the second enzyme cleavable peptide sequence comprises SEQ ID NO.25.
A probe according to either claim 1 or claim 2, wherein the first cleaving agent is a reactive oxygen species.
A probe according to any one of claim 1 to claim 3, or 13, wherein the second cleaving agent is a reactive oxygen species.
A probe according to any preceding claim, wherein the first fluorophore is not substantially fluorescently quenched by the second quencher, and the second fluorophore is not substantially quenched by the first quencher.
A probe according to any preceding claim, wherein the first fluorophore and the first quencher form a FRET pair.
A probe according to claim 17, wherein the first fluorophore/first quencher are selected from Cy3/Cy5, Cy3/QSY21 , Cy5/QSY21 , Cy5/BHQ-3, carboxy- Naphtofluorescein/QSY21 , carboxy-Naphtofluorescein/BHQ-3, seminaphthorhodamine carboxylate derivatives/BHQ-3, seminaphthorhodamine carboxylate derivatives/QSY21 , fluorescein/tetramethylrhodamine, fluorescein/methyl red, NBD/methyl red, cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP), and carboxy fluorescein/methyl red.
19. A probe according to any preceding claim, wherein the second fluorophore and the second quencher form a FRET pair.
20. A probe according to claim 19, wherein the second fluorophore/second quencher are chosen from Cy3/Cy5, Cy3/QSY21 , Cy5/QSY21 , Cy5/BHQ-3, carboxy- Naphtofluorescein/QSY21 , carboxy-Naphtofluorescein/BHQ-3, seminaphthorhodamine carboxylate derivatives/BHQ-3, seminaphthorhodamine carboxylate derivatives/QSY21 , fluorescein/tetramethylrhodamine, fluorescein/methyl red, NBD/methyl red, cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP), and carboxy fluorescein/methyl red.
21. A probe according to any preceding claim, wherein the first probe element comprises a plurality of first fluorophores.
22. A probe according to any preceding claim, wherein the second probe element comprises a plurality of second fluorophores.
23. A probe according to any preceding claim, comprising a plurality of first probe elements connected to the core.
24. A probe according to any preceding claim, comprising a plurality of second probe elements connected to the core.
25. A probe according to claim 23 or claim 24, wherein the probe comprises a plurality of first probe elements connected to the core and a plurality of second probe elements connected to the core.
26. A probe according to any preceding claim, wherein the probe comprises a third probe element connected to the core, the third probe element comprising a third fluorophore connected to a third quencher by a third cleavable linker.
27. A method of detecting a first and a second enzyme in a target zone comprising the steps:
a. applying a probe according to any preceding claim to the target zone; b. illuminating the target zone with appropriate wavelengths of light to excite the first and second fluorophores; and c. determining whether the intensity of fluorescence of the first and second fluorophores has increased within the target area,
wherein significant fluorescence of the first fluorophore is indicative of the presence of the first enzyme in the target zone, and significant fluorescence of the second fluorophore is indicative of the presence of the second enzyme in the target zone.
28. The method according to claim 27, wherein the first enzyme is MMP.
29. The method according to claim 28, wherein the MMP is one or more of MMP-2, MMP- 9 and MMP-13.
30. The method according to any one of claim 27 to claim 29, wherein the second enzyme is thrombin.
31. The method according to any one of claim 28 to claim 30, wherein the first enzyme is MMP-2, MMP-9 or MMP-13 and the second enzyme is thrombin, such that significant fluorescence of the first and/or second fluorophores is indicative of a disease in which MMP-2, MMP-9 or MMP-13, and/or thrombin are overexpressed and/or activated.
32. The method of claim 31 , wherein the disease is fibrosis, arthritis, or cancer.
33. A kit of parts comprising the probe according to any one of claim 1 to claim 26 in a suitable diluent or buffer.
34. An optical probe comprising at least one fluorophore connected to at least one quencher by a cleavable linker; the at least one fluorophore being substantially fluorescently quenched by the at least one quencher when connected to the at least one quencher by the cleavable linker, and the at least one fluorophore is separated from the at least one quencher when the cleavable linker is cleaved; wherein the cleavable linker comprises an enzyme cleavable peptide sequence that is selectively cleavable by thrombin comprising one of SEQ ID NO.15 to SEQ ID NO.38.
35. The probe according to claim 34, wherein the enzyme cleavable peptide sequence comprises SEQ ID N0.25, SEQ ID N0.31 or SEQ ID N0.37.
36. The probe according to claim 34 or claim 35, wherein the enzyme cleavable peptide sequence comprises one or more D-amino acid residues. A method of detecting thrombin activity in a target zone, the method comprising the steps:
a. applying a probe according to any one of claim 34 to claim 36 to the target zone;
b. illuminating the target zone with an appropriate wavelength of light to excite fluorophores of the probe; and
c. determining the fluorescence intensity of probe,
wherein significant fluorescence of the or each fluorophore of the probe is indicative of the presence of thrombin in the target zone.
A kit of parts comprising the probe of any one of claim 34 to claim 36 in a suitable diluent or buffer.
An optical probe comprising a first probe element, and a second probe element; the first probe element and the second probe element connected to a core; the first probe element comprising a first fluorophore, a first cleavable linker and a first quencher, the first fluorophore connected to the first quencher by the first cleavable linker and the core, the first quencher being a second fluorophore and being connected to the core; the second probe element comprising the second fluorophore, a second cleavable linker and a second quencher, the second fluorophore connected to the second quencher by the core and the second cleavable linker; the first fluorophore being substantially fluorescently quenched by the first quencher when connected to the first quencher by the first cleavable linker and core; the second fluorophore being substantially fluorescently quenched by the second quencher when connected to the second quencher by the second cleavable linker and core; wherein the first fluorophore is separated from the first quencher when the first cleavable linker is cleaved, the second fluorophore is separated from the second quencher when the second cleavable linker is cleaved.
The probe according to claim 39 wherein the second quencher is not a fluorophore.
The probe according to claim 39 wherein the second quencher is a third fluorophore.
The probe according to claim 41 wherein when the second quencher is a third fluorophore, it is not be fluorescently quenched by the first quencher. The probe according to claim 41 wherein when the second quencher is a third fluorophore, the third fluorophore is separated from the first quencher when the second cleavable linker is cleaved, and the second quencher/third fluorophore is substantially fluorescently quenched by the first quencher.
PCT/GB2016/050763 2015-03-20 2016-03-18 Optical probe for thrombin Ceased WO2016151297A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB1504775.6A GB201504775D0 (en) 2015-03-20 2015-03-20 Optical probe for thrombin
GB1504775.6 2015-03-20

Publications (1)

Publication Number Publication Date
WO2016151297A1 true WO2016151297A1 (en) 2016-09-29

Family

ID=53052162

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2016/050763 Ceased WO2016151297A1 (en) 2015-03-20 2016-03-18 Optical probe for thrombin

Country Status (2)

Country Link
GB (1) GB201504775D0 (en)
WO (1) WO2016151297A1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108379598A (en) * 2018-02-07 2018-08-10 中国人民解放军总医院 Fibrin ferment intelligent response fluorescence/multi-modal molecular probes of MRI and its preparation method and application
WO2020081454A1 (en) * 2018-10-15 2020-04-23 The Board Of Trustees Of The Leland Stanford Junior University Multi-enzyme-activated imaging probes and related compositions and methods
WO2023076640A3 (en) * 2021-10-29 2023-06-08 Glympse Bio, Inc. Ex vivo protease activation and detection
WO2024039724A3 (en) * 2022-08-16 2024-04-04 Massachusetts Institute Of Technology Rna-triggered protein cleavage and applications by the crispr cas7-11-csx29 complex
US12305217B2 (en) 2020-09-11 2025-05-20 Sunbird Bio, Inc. Ex vivo protease activity detection for disease detection/diagnostic, staging, monitoring and treatment

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012136958A2 (en) * 2011-04-08 2012-10-11 The University Court Of The University Of Edinburgh Optical imaging probes
US20130078188A1 (en) * 2003-10-31 2013-03-28 The Regents Of The University Of California Activatable cell penetrating peptides with quenched fluorophores

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130078188A1 (en) * 2003-10-31 2013-03-28 The Regents Of The University Of California Activatable cell penetrating peptides with quenched fluorophores
WO2012136958A2 (en) * 2011-04-08 2012-10-11 The University Court Of The University Of Edinburgh Optical imaging probes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ROY WEINSTAIN ET AL: "In Vivo Targeting of Hydrogen Peroxide by Activatable Cell-Penetrating Peptides", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 136, no. 3, 22 January 2014 (2014-01-22), US, pages 874 - 877, XP055274513, ISSN: 0002-7863, DOI: 10.1021/ja411547j *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108379598A (en) * 2018-02-07 2018-08-10 中国人民解放军总医院 Fibrin ferment intelligent response fluorescence/multi-modal molecular probes of MRI and its preparation method and application
WO2020081454A1 (en) * 2018-10-15 2020-04-23 The Board Of Trustees Of The Leland Stanford Junior University Multi-enzyme-activated imaging probes and related compositions and methods
US12305217B2 (en) 2020-09-11 2025-05-20 Sunbird Bio, Inc. Ex vivo protease activity detection for disease detection/diagnostic, staging, monitoring and treatment
US12492422B2 (en) 2020-09-11 2025-12-09 Sunbird Bio, Inc. Ex vivo protease activity detection for disease detection/diagnostic, staging, monitoring and treatment
WO2023076640A3 (en) * 2021-10-29 2023-06-08 Glympse Bio, Inc. Ex vivo protease activation and detection
WO2024039724A3 (en) * 2022-08-16 2024-04-04 Massachusetts Institute Of Technology Rna-triggered protein cleavage and applications by the crispr cas7-11-csx29 complex

Also Published As

Publication number Publication date
GB201504775D0 (en) 2015-05-06

Similar Documents

Publication Publication Date Title
US10507247B2 (en) Optical probes for matrix metalloproteinases
Kele et al. Dual labeling of biomolecules by using click chemistry: a sequential approach
Chevalier et al. Bioconjugatable azo‐based dark‐quencher dyes: synthesis and application to protease‐activatable far‐red fluorescent probes
JP6204147B2 (en) Fluorescent probe for pancreatic juice detection with high sensitivity and pancreatic juice detection method
US8968700B2 (en) Imaging of protease activity in live cells using activity based probes
US20140140930A1 (en) Non-peptidic quenched fluorescent imaging probes
JP2010535167A (en) Caspase imaging probe
AU2008221036B2 (en) Imaging probes
JP2009500448A (en) Optical imaging contrast agent
JP7149614B2 (en) Protease-activated contrast agents for in vivo imaging
JP2018511556A (en) Protease-activated contrast agent for in vivo imaging
US10383956B2 (en) Fluorescent probe for detecting dipeptidyl peptidase IV
Marshall et al. Expanding the fluorescence toolkit: molecular design, synthesis and biological application of unnatural amino acids
Leriche et al. A FRET-based probe with a chemically deactivatable quencher
CN101652149B (en) Imaging probes
Renault et al. Fluorogenic Behaviour of the Hetero‐Diels–Alder Ligation of 5‐Alkoxyoxazoles with Maleimides and their Applications
US8859223B1 (en) Compositions and methods for imaging beta-secretase activity in living cells and organisms
US20220341922A1 (en) Fluorescent probe for use in detection of brain tumor
WO2016133913A1 (en) Fluorescently labeled molecules containing modified tryptophan
Evans Synthesis of optical probes for the visualization of human neutrophil elastase
Hadzima Synthesis of inhibitors and substrates for the inhibition, visualization and targeting of proteases
HK1141243B (en) Imaging probes

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16712410

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16712410

Country of ref document: EP

Kind code of ref document: A1