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WO2010002976A2 - Constructions fluor-désactivateur contenant un colorant clivable par une enzyme - Google Patents

Constructions fluor-désactivateur contenant un colorant clivable par une enzyme Download PDF

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Publication number
WO2010002976A2
WO2010002976A2 PCT/US2009/049380 US2009049380W WO2010002976A2 WO 2010002976 A2 WO2010002976 A2 WO 2010002976A2 US 2009049380 W US2009049380 W US 2009049380W WO 2010002976 A2 WO2010002976 A2 WO 2010002976A2
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compound
leu
lys
quencher
enzyme
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WO2010002976A3 (fr
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Karen E. Linder
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Bracco Imaging SpA
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Bracco Imaging SpA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/0008—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain
    • C09B23/0025—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain the substituent being bound through an oxygen atom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/0066—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain being part of a carbocyclic ring,(e.g. benzene, naphtalene, cyclohexene, cyclobutenene-quadratic acid)
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
    • C09B23/086—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines more than five >CH- groups
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B56/00—Azo dyes containing other chromophoric systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96486—Metalloendopeptidases (3.4.24)
    • G01N2333/96491—Metalloendopeptidases (3.4.24) with definite EC number
    • G01N2333/96494—Matrix metalloproteases, e. g. 3.4.24.7

Definitions

  • Novel constructs for in vitro assays and for in vivo fluorescence imaging using a construct of the form Fluor-enzyme cleavable substrate-Quencher.
  • the fluor emits fluorescent emissions in the Near Infrared and the quencher is a moiety that quenches the fluorescence of the fluor by contact quenching such as, for example, IR Dye800CW-(en/yme cleavable substrate)-BHQ-3.
  • the enzyme cleavable substrate is cleaved selectively by one or more MMPs and is not substantially cleaved in vivo by enzymes present in normal tissue or blood, such as Neprilysin.
  • Arthritis is a highly debilitating disease that can lake many forms, including osteoarthritis, rheumatoid arthritis, septic arthritis, gout and pseudogout, juvenile idiopathic arthritis, Still's disease and ankylosing spondylitis.
  • the most common form of arthritis, osteoarthritis (degenerative joint disease) is a result of trauma to the joint, infection of the joint, or age.
  • Rheumatoid arthritis and psoriatic arthritis are believed to be autoimmune diseases in which the body attacks itself.
  • Septic arthritis is caused by an infection of the joint.
  • Gouty arthritis and pseudogout are respectively caused by deposition of uric acid or calcium pyrophosphate crystals in the joint, causing inflammation.
  • Diagnostic and monitoring methods can include physical examination, scoring systems to determine the degree of symptoms such as pain and inflammation, laboratory tests, and imaging studies.
  • ANA Antinuclear Antibodies
  • WBC white blood cell count
  • RBC red blood cell count
  • hemoglobin hemoglobin
  • hematocrit several red blood cell indices and a platelet count. Elevated white blood cell counts suggest the possibility of an active infection, but patients taking corticosteroids may have an elevated
  • Low hemoglobin and hematocrit may be indicative of anemia associated with chronic diseases or possible bleeding caused by medications.
  • the platelet count is often high in rheumatoid arthritis patients, while some potent arthritis medications can cause platelets to be low.
  • HLA Tissue Typing Human Leukocyte Antigens are proteins on the surface of cells. Specific HLA proteins are genetic markers for some of the rheumatic diseases, including ankylosing spondylitis and rheumatoid arthritis; however, such markers only indicate the potential for disease. High levels of uric acid in the blood can indicate the potential that crystals have been deposited in the joints and tissues, causing painful gout attacks. For certain types of systemic rheumatic diseases, biopsies of certain organs can provide important diagnostic info ⁇ nation. Also, joint fluid analysis can provide a doctor with many details about the health of a person's joint.
  • Imaging methods that have been used for the detection and staging of arthritis so far include X-ray imaging, MRI and ultrasound imaging. Biopsies are also used, but are highly invasive.
  • X-Ray imaging has the limitation that by the time erosions of the bone can be seen, the disease has already progressed to the point of irreversible damage. X-Ray provides no information about the causative agents for the observed destruction of bone and cartilage.
  • MRI although it is able to detect inflammatory changes such as edema, synovitis, and bone loss, is expensive and time-consuming to carry out.
  • MRl provides no biochemical information regarding the nature of the agent(s) responsible for bone and cartilage destruction.
  • Ultrasound has also been used for the diagnosis and staging of arthritis, but again, provides only morphological information. When diagnosing or treating a disease such as arthritis, it is important to identify morphological changes caused by the disease, but a determination of the biochemical changes that are occurring, especially the presence of the enzymes that ultimately cause tissue damage, would be highly advantageous.
  • MMPs for example MMPs such as MMP-2, MMP-9 and MMP-13
  • RA rheumatoid arthritis
  • Active MMPs cleave selective cut sites in cartilage and bone and can also cause proteolysis of tissue.
  • MMPs have been reported to be responsible for the degradation of bone, tendons and cartilage in arthritis.
  • Levels of MMP- 1 , 3, 9, and 13 have been reported to be strongly stimulated by inflammatory cytokines such as TNF- ⁇ and IL-I, for example.
  • MMP-2 and MMP-9 correlates with bone erosions (but with wide variation). Patients who are responding to therapy are known to show declines in joint total MMPs.
  • MMPs are also known to be upregulated in various types of cancer and in artherosclerosis. The MMPs play an important role in tissue remodeling which occurs in angiogenesis, cirrhosis, metastasis and various inflammatory processes.
  • Some MMPs can be detected in the serum using laboratory assays, but such tests do not provide information about their expression levels in a particular involved joint. At present, MMP levels in joints of patients afflicted by arthritis are most readily delected using biopsy, a highly invasive procedure (vide supra).
  • MMPs exist, they are typically unable to distinguish between the Proenzyme form of the MMP (ProMMPs), which are proieolytically inactive, and active MMPs, which are capable of enzymatic cleavage. MMPs may also be present in tissue or blood as complexes with TIMPs (Tissue inhibitor of matrix metalloproteinase) or as ⁇ -macroglobulin complexes. These latter forms of MMP are also inactive. As these in vitro assays for MMPs cannot distinguish between overexpression of Pro, active and TIMP forms of the MMP, the existing assays only provide information on total MMP levels. Furthermore, only active forms of MMPs cause proteolytic damage.
  • ProMMPs Proenzyme form of the MMP
  • active MMPs which are capable of enzymatic cleavage.
  • MMPs may also be present in tissue or blood as complexes with TIMPs (Tissue inhibitor of matrix metalloproteinase) or as ⁇ -macro
  • MMP expression also correlates with carcinoma survival time. For example, in one study, patients with matrilysin-posilive carcinoma had a significantly shorter overall survival time than did those with malrilysin-negalive carcinoma (H Yamamoto, J Clin Oncol. 2001 Feb 15;19(4): 1 1 18-27). K. Sakata et al (Im J Oncol. 2000; 17(4):673-81 ) found that MMP-2, MTl -MMP, TIMP-2, and MMP-9 and down-regulation of TIMP-I may contribute to the development or enhanced growth capacity of ovarian tumors. Over- expression of MMP-2 has been correlated with poor prognosis in cervical cancer (B Davidson el al, Gynecol Oncol. 1999 Jun;73(3):372-82. Other cancers where MMPs have been implicated include breast, colorectal, endometrial, lung, pancreatic, thyroid and other cancers.
  • MMPs have also been demonstrated in the selling of myocardial ischemia, reperfusion injury, and during the progression to congestive heart failure. MMPs are also believed to be major contributors to the progression of atherosclerotic lesions. They are also suspected of contributing to the progression of chronic obstructive pulmonary disease (COPD) and multiple sclerosis (MS), and are implicated in periodontal disease as well.
  • COPD chronic obstructive pulmonary disease
  • MS multiple sclerosis
  • NlR Near Infrared
  • fluorescent dyes When excited with the proper wavelength of light, fluorescent dyes absorb light. which places the dye in an excited stale. The dye then returns to the ground slate from the excited stale by emitting light (fluorescence) of a different (longer) wavelength than that of the light used to excite the compound. This emission can be detected using fluorescence detectors. Detection methods include reflectance imaging and fluorescence molecular tomography (“FMT”), among others.
  • Green and analogs can be used lo detect arthritis and tumors.
  • the fluor is injected, and the fluorescence emitted in parts of the body (e.g. the tumor or arthritic joint) is detected as a function of time.
  • Several fluorescent agents that contain a targeting group have also been reported. Il has been found in studies with such constructs that the kinetics of clearance of the fluorescent compound from diseased tissue is modified relative to that of normal tissue, so by watching clearance curves, the presence of such diseased tissue can be detected. This is due either to changes in pharmacokinetics of untargeted agents between diseased and normal tissue, or due to specific targeting, for fluorescent constructs that contain a targeting group such as a receptor binding moiety.
  • Fluor-Enzyme Cleavable Substrate-Quencher that are optically silent due to the phenomenon known as FRET, or Fluorescence Resonant Energy Transfer, also known as F ⁇ rsier type energy transfer.
  • FRET Fluorescence Resonant Energy Transfer
  • F ⁇ rsier type energy transfer also known as F ⁇ rsier type energy transfer.
  • the acceptor can be another fluorophore or a non-fluorescent molecule. If the acceptor is a fluorophore, the transferred energy can be emitted as fluorescence. If the acceptor is a non-fluorescent molecule (or quencher), no fluorescence is observed. In such constructs, fluorescence is "quenched" until the linker between the fluor and quencher is broken.
  • DNA probes that use FRET detect many types of reactions involving DNA, RNA, proteins, and inorganic substances (see e.g. Vladimir V. Didenko, DNA Probes Using Fluorescence Resonance Energy Transfer (FRET): Designs and Applications, Biotechniques, 2001 November : 31 (5): 1 106-1 121).
  • FRET Fluorescence Resonance Energy Transfer
  • the specific limited distance at which energy transfer is effective forms a basis for utilization of FRET as a "molecular ruler”, measuring 0.5-10-nm distances within biomolecules with high precision (Selection of Fluorophore and Quencher Pairs for Fluorescent Nucleic Acid Hybridisation Probes, Salvatore A. E. Marras, Methods in Molecular Biology: Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols.
  • the degree of FRET quenching is strongly affected by the distance between the donor and the acceptor molecule. Typical effective distances between the donor and acceptor molecules are in the 10 to 100 A range.
  • a special case of static quenching is self-quenching, where the fluorophore and the quencher are identical. [00032] However, for contact quenching to occur, the fluor and quencher should be close to one another, as shown schematically in Fig. 2, and described in Salvatore A. E. Marras, Selection of Fluorophore and Quencher Pairs for Fluorescent Nucleic Acid Hybridization Probes, Methods in Molecular Biology: Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols. Edited by: V V. Didenko. In, for example, probes that can hybridize, bringing fluor and quencher close to one another, contact quenching is observed. However, if the fluor and quencher are separated, contact quenching is not observed, and the compounds fluoresce. This rule has been used to determine nanometer distances in biomolecules.
  • a compound capable of being used in a method to detect active MMPs in the body is needed. It would also be highly useful to have a fluorescent compound that could detect the presence or absence of MMP proteolytic activity and that could be used to determine the efficacy of matrix metalloproteinase inhibitors.
  • a fluorescent compound that could detect the presence or absence of MMP proteolytic activity and that could be used to determine the efficacy of matrix metalloproteinase inhibitors.
  • the present invention relates, inter alia, to constructs (also referred to herein as compounds) which comprise a fluorescent moiety that is linked to a quencher by an enzymatically cleavable substrate where the linkage prevents the compound from emitting fluorescence (also referred to herein as being “optically silent” or “non-fluorescent") until the linkage is cleaved.
  • the linkage can be an enzyme-cleavable substrate. Upon cleavage by an enzyme, the fluorescent moiety and the quencher become separated, allowing the generation of a fluorescent signal if illuminated with light. In embodiments described below the light is in the Near Infrared range.
  • NlR Near Infrared
  • the compounds of embodiments of the present invention have been shown to be cleaved selectively by enzymes such as active MMPs, generating, post cleavage, a fluorescent signal that can be used for imaging.
  • enzymes such as active MMPs
  • These constructs comprise a fluorescent moiety that is linked to a quencher by an enzymalically cleavable substrate; the substrate prevents the compound from emitting fluorescence due to the enforced proximity of the fluor and quencher moieties.
  • the fluorescent moiety and the quencher become separated and migrate away from each other in solution as they are no longer tethered by the substrate.
  • the resulting distance between the fluor and the quencher exceeds the maximum distance allowable for the quenching of the fluor by the quencher.
  • the fluorescence emission due to the fluor is no longer quenched and the emitted light can be detected when the system is illuminated with light in the Near Infrared.
  • the presence and magnitude of a fluorescence signal in an affected tissue such as a joint ofa patient with rheumatoid arthritis, following administration of Fluor-enzyme cleavable substrate-Quencher constructs of the present invention, can provide valuable diagnostic information about the nature and levels of the enzymes present at the imaged site.
  • FIG. I depicts a donor fluorescent dye emission spectrum overlapping the absorption spectrum of an acceptor or quencher.
  • FlG. 2 is a diagram of contact quenching.
  • FIG. 3 is a bar graph showing quenching of Fluor-Enzyme cleavable substrate quencher constructs.
  • FIG. 4 depicts in vitro cleavage curves of IRDyeSOOCW-enzyme cleavable substrate-BHQ3 constructs by MMP-2, 9 and 13.
  • FIG. 5 is a schematic of the synthesis of the SEQ ID NO. 053.
  • the present invention is directed, inter alia, to constructs which comprise a fluorescent moiety that is linked to a quencher by an enzymatically cleavable substrate where the linkage substantially prevents the compound from emitting fluorescence (also referred to herein as being “optically silent” or “non-fluorescent") until the linkage is cleaved.
  • the linkage can be an enzyme-cleavable substrate. Upon cleavage by an enzyme, the fluorescent moiety and the quencher become separated, allowing the generation of a fluorescent signal if illuminated with light in the Near Infrared.
  • Embodiments of the invention are directed to novel fiuor/quencher constructs, particularly those containing the fluor known as IRDyeSOOCW and analogs thereof and the quencher known as Black Hole.Quencher-3 (BHQ-3) and analogs thereof.
  • the fluor and quencher are linked to one another by an enzyme cleavable substrate and are substantially optically silent (non-fluorescent) until such cleavage occurs.
  • the fluor and quencher of the constructs are linked by an enzyme cleavable substrate that can be selectively cleaved by enzymes known as Matrix Metalloproteinases.
  • the enzyme cleavable substrate which links the fluor and the quencher, can be selectively cleaved by one or more MMPs but cannot be substantially cleaved in vivo by the enzymes present in normal tissue or blood, such as the enzyme known as Neprilysin.
  • the enzyme cleavable substrate may be a peptide, a polypeptide, a monomer, a dimer, a multimer, a peptidomimetic, a non-peptide, an antibody fragment, an antibody (humanized or non-humanized), a protein, a hormone, a growth factor, a cytokine or a drug.
  • Peptides, monomers, dimers and multimers may optionally contain one or more unnatural amino acids and or D-amino acids.
  • the enzyme cleavable substrate is a peptide, and in an especially preferred embodiment, the peptide is a substrate for one or more MMP.
  • residues in the enzyme cleavable substrate may optionally be substituted with solubilizing charged or uncharged substituents such as SOj " , or COO ' , or with polymers (e.g PEG, polyglycine, polyproline, polyhydroxymeihylacrylate, polylysinealkyl amines or N-acylated polylysines, polyaspartic acid, polyglutaminc acid or quanidines) to modify its pharmacokinetics.
  • substituents such as SOj " , or COO '
  • polymers e.g PEG, polyglycine, polyproline, polyhydroxymeihylacrylate, polylysinealkyl amines or N-acylated polylysines, polyaspartic acid, polyglutaminc acid or quanidines
  • the fluorescent dye in the fluor-enzyme cleavable substrate-quencher construct is a dye that is capable of emitting fluorescence with an emission maximum wavelength from about 700 to about 900 nm.
  • Suitable dyes include, for example tetrapyrrole, telraazapyrrole, xanthine, phenoxazine, phenolhiazine, and especially polymethine dyes such as cyanine dyes.
  • IR Dye® 800CW IR Dye® 800CW
  • IRDye® 680 IRDye® 700DX
  • Cy5.5 and Cy7 GE Life Sciences
  • Alexa® Fluor 750 Invilrogen
  • HiLylePlusTM750 available from AnaSpec.
  • Cy5.5 has excitation/emission maxima at 675 nm/694 nm, making it a borderline candidate labeling agent for in vivo applications.
  • a recently developed fluorochrome, IRDye 800CW has its excitation/emission maxima at 785 nm/810 nm, precisely centered in the region known to give optimal signal to background for optical imaging in living systems.
  • HiLytePlus750, or IRDye 800CW are preferred, with !RDye800CW being particularly preferred.
  • the fluor is a construct of Formula I:
  • R I , R2, R5, R6, R7, R8, R9, R 10 and R 1 1 are each independently H, Me, substituted or unsubsliluted alkyl, halo, carboxy, amino, sulfonate, R l 2-COOH, R12OR13, R12SR13, or R12COORI3, wherein R12 is a bond or alkylene and R 13 is a substituted or unsubstituted alkyl,
  • R4 is -O-Aryl, NH-Aryl, substituted or unsubstituted Aryl, substituted or unsubstituted C-I-ClO alkyl, halo, S-Aryl or S ⁇ 2 -Aryl, where the aryl ring can, in all cases, be substituted or unsubstituted.
  • it may be a linker (e.g. a substituted or unsubstituted alkyl, substituted or unsubstituted Aryl, PEG, alkyl amine or quanidine) that is either covalently bonded (e.g. via an amine, thiol or acid functionality) to an enzymatically cleavable substrate or contains a reactive moiety suitable for coupling to an en/.yme cleavable substrate.
  • a linker e.g. a substituted or unsubstituted alkyl, substituted or unsubstituted Aryl, PEG, alkyl amine
  • Tl and T2 are each H, or are joined together to form a substituted or unsubstituted 5- or 6-membered ring.
  • X I , X2, X3 and X4 independently represent SOjH, COOH or physiologically acceptable salts thereof, or polyethylene glycol, or one or more ofX 1 -X4 may be a linker (e.g. a substituted or unsubstituted alkyl, substituted or unsubstituled Aryl, PEG, alkyl amine or quanidi ⁇ e) lhai is either covalently bonded (e.g.
  • X l and R3 and/or X2 and R9 can be cycli/.ed together to form a 5- or 6 membered saturated or unsaturated ring that is optionally derivatized with H, substituted or unsubstituted C1 -C6 alkyl, polyethylene glycol, substituted or unsubstituied aryl, halogen, a cyano, carboxy, and/or a sulfo group(s).
  • Y 1 and Y2 are independently C, N (in which case R2 and/or R8 is absent), or O, S,
  • the Linker is a substituted or unsubstituted C l-C lO alkyl chain, a polyethylene glycol derivative, or a substituted or unsubstiluied aryl group.
  • Z is -O-, -S-, -SC-O)-, -SOr -NH.
  • Fluor-Enzyme cleavable substrate-Quencher compounds containing a fluor of the structure shown below:
  • Cal+ is a cation.
  • Cations include, but are not limited to H “ , Na" , K 1 ,
  • the cation is preferably one that is physiologically acceptable.
  • fluorophore known as HyLitePlus75O, a proprietary NIR fluorophore whose structure is unknown. It is commercially available from AnaSpec.
  • fluorophore known as HyLitePlus75O, a proprietary NIR fluorophore whose structure is unknown. It is commercially available from AnaSpec.
  • Other fluors suitable for use in embodiments of the present invention include the various fluors that emit in the NIR region that have been developed in recent years, including derivatives of the various cyanine compounds reported in EP 1 480 683 B 1 (Kawakami et al, Near Infrared Fluorescent Contrast Agent and Method for Fluorescence Imaging), Indocyanine Green (as described by Li, X. et al, SPIE 2005, SPlE Vol. 2389 p.
  • Quenchers The quenchers on the Fluor-Enzyme cleavable substrate-
  • Quencher constructs are electron deficient compounds that comprise diazo-aromatic or nitro- aromatic rings.
  • Quenchers useful in embodiments of the present invention include Deep Dark Quenchers, Dabcyl, Eclipse, Iowa Black FQ and RQ, Blackberry Quencher 650, QSY- 7, QSY-21, and Black Hole Quenchers 0, 1 , 2, and 3. It is to be appreciated that other quenchers known to those skilled in the art can also be used.
  • the quencher shown below (Black Hole Quencher 3) is particularly preferred.
  • An enzyme cleavable substrate is a key component of the Fluor-Enzyme cleavable substrate-Quencher constructs of the invention.
  • This substrate may be branched or unbranched, and may be a substrate for any proteolytic enzyme, although compounds comprising a sequence thai is selectively cleaved by one or more active MMP(s) are preferred.
  • Quencher constructs of the invention showed that the constructs are cleaved by active MMPs, such as MMP-I , -2, -3, -7, -8, -9, - 10, and - 13, generating significant NIR fluorescence.
  • active MMPs such as MMP-I , -2, -3, -7, -8, -9, - 10, and - 13, generating significant NIR fluorescence.
  • MMPs human matrix metalloproteinases
  • the enzyme cleavable substrates need not be substrates for active MMPs.
  • IR Dye800CW-enzyme cleavable subsirate-BHQ-3 constructs can be constructed, having sensitivity to and selectivity for the wide range of proteolytic enzymes that exist in nature.
  • proteolytic enzymes are known, including those that are described in the Handbook of Proteolytic Enzymes, 2nd Edition, VoI 1 and 2, A, J. Barrett el al.
  • the enzyme-cleavable constructs, properly designed could serve as selective substrates for a large variety of enzymes including, for example,
  • Aspartic peptidases such as the Pepsins, Cathepsins, Presenilins, Renin and the like
  • Cysteine Peptidases such as the Calhepsins, Ubiquitin-specific proteases, Caspases, Dipeptidyl peptidase and the like,
  • Metallopeptidases such as aminopeptidases, Angiotensin-converting enzyme, Neprilysin, Endolhelin-converling enzyme, Matrix Metalloproleases including Collagenase-1 , -3, and -4, Gelatinase A and B, Stromelysin 1 , 2 and 3, Matrilysin, Membrane-type matrix melalloproteinases such as Membrane-type matrix metalloproteinase 1-6, the ADAM melalloproteinases, ADAMSTS metal loproteases, TNF- ⁇ converting enzyme, the Carboxypeptidases, aminopeptidases such as Leucyl and Methionyl aminopeptidases,
  • Lipases such as Phospholipase A 1 ,A2, B, C and D,
  • Serine and Threonine peptidases such as trypsin, chymotrypsin, leukocyte elastase, kallikreins. Complement factors. Plasminogen activators, Plasmin, Hyaluronon-binding protease, Oligopeplidases, Serine carboxypeptidase D and the like.
  • proteases are examples and are not intended to be limiting.
  • Preferred Fluor-Enzyme cleavable substrate-Quencher constructs are those that are selectively cleaved by proteolytic enzymes that are overexpressed in a disease state. In one embodiment these substrates are not substantially cleaved by enzymes present in normal tissues.
  • the Fluor-Enzyme cleavable substrate-Quencher constructs may be provided as freeze-dried solids that are reconstituted with a physiologically acceptable solution prior to administration, or may be provided in a physiologically acceptable aqueous or nonaqueous solution, in the presence of such buffers, stabilizers, and solubilizers as are necessary to prepare a stable solution of said constructs.
  • the agents may also be formulated as micelles, liposomes and the like. Prior to administration, the solid or solution may stored frozen or at room temperature, depending on the stability requirements of the compound.
  • the part of the body to be studied is positioned under the detector, and a slow or fast bolus of the compounds of the invention of sufficient quantity to provide a diagnostic image is administered by intravenous, intraperitoneal, subcutaneous or intramuscular injection.
  • the agent may alternatively be administered to the surface of an organ or disease site, e.g. the lumen of an artery, esophagus, colon etc. Scanning can be initiated within 1 -2 minutes or may be delayed, depending upon the pharmacokinetics of the test compound.
  • Light of suitable wavelength(s) is used to illuminate the subject.
  • the florescence that is emitted is detected in target and non-target organs, using (e.g.) a fluorescence detector or an endoscopic or fiber optic probe that is sensitive to fluorescent emissions.
  • the relative amount of fluorescence in the tissues of interest can be determined using regions of interest, or using time-activity curves, using methods known to those skilled in the art. Either reflectance or tomographic images may be obtained.
  • the agent may be administered repeatedly over time, to determine the changes in the fluorescent images that may have occurred. Said images may be reflective of rises or falls in proteolytic enzyme levels due to changes in disease status, an increase or decrease in inflammation etc. The differences in the images over lime can be used to detect response to drug therapy.
  • compositions of this invention may be administered for imaging by more than one modality.
  • the compositions may be used for imaging by optical imaging alone, or may be used for photoacoustic imaging.
  • NMP N- Methylpyrrolidinone
  • DMF N,N-dimethylformamide
  • Piperidine Sequencing grade, redistilled 99+%) and trifluoroacelic acid (specirophotometric grade or sequencing grade) were purchased from Sigma-Aldrich Corporation (Milwaukee, WI) or from the Fluka Chemical Division of Sigma- Aldrich Corporation.
  • N, N'-Diisopropylcarbodiimide DIC
  • DIEA N,N- diisopropylethylamine
  • TIS lriisopropylsilane
  • Fmoc-proiecled amino acids O-(benzotriazol-l-yl)-N,N,N',N'- tetiamethyluronium hexafluorophosphate (HBTU) and N-hydroxybenzotriazole (HOBt) were purchased from Novabiochem (San Diego, CA).
  • BHQ3-OSu was purchased from Biosearch Technologies, lnc (Novato, CA) and IRDye 800CW--NHS esier was purchased from LI-COR
  • HyLitePlusTM 750 NHS ester was obtained from AnaSpec, lnc
  • Detection of compounds was accomplished using UV either at 220 and 254 nm (deuterium lamp) or at 701 and 790 nm (tungsten lamp).
  • Preparative HPLC was conducted on a Shimad/u LC-8A dual pump gradient system equipped with a SPD-10AV UV detector. Generally the solution containing the crude peptide was loaded onto a reversed phase Cl 8 column, depending on the compound characteristics, using a third pump attached to the preparative Shimadzu LC-8A dual pump gradient system. After the compound was applied to the preparative HPLC column solvents present in the reaction mixture, such as DMF or DMSO were eluted from the column at low organic phase composition; then the desired product was eluted using a gradient elution of the stronger eluanl into the weaker eluant.
  • solvents present in the reaction mixture such as DMF or DMSO
  • Mass Spectrometer using API-ES in -/+ ion mode The molecular weight of the target peptides exceeds 2000, thus the mass spectra usually exhibited doubly or triply negatively charged ion mass values rather than the molecular ion. Doubly or triply charged ion mass values of the desired peptide were generally employed for selection of fractions for collection and combination to obtain the pure peptide during HPLC purification. After careful collection of fractions by comparing MS results and HPLC purities and freeze-drying process, a small amount of the isolated fluffy solid was dissolved in water-acetonitrile (1 : 1) (0.25 mg/mL) and this solution was analyzed by HPLC and MS for final purity determination of the purified peptide.
  • SPPS Solid Phase Peptide Synthesis
  • HBTU-HOBt-DIEA reagent in NMP was activated with HBTU (1.0 mmol, 4 eq.), HOBt (1.0 mmol, 4 eq.) and DIEA (2.5 mmol, 10 eq.) using NMP as the solvent in an activation chamber, and transferred to the reaction vessel containing resin (0.25 mmol). After agitating the reaction mixture for 63 min, the resin was washed thoroughly with NMP.
  • Arg(Pmc)-NovaSyn TGT (0.2 mmol/g) resin was used.
  • Step 1 Preparation of peptide sequences containing Black Hole Quencher (BHQ3) [00089]
  • BHQ3-OSu 5 mg, 0.0063 tnmol
  • DIEA 10 mg, 0.077 mmol
  • the reaction mixture was diluted to 10 mL with anhydrous DMF, piperidine (0.2 mL) was added and the mixture was stirred for 30 min.
  • the reaction mixture was diluted with water to 50 mL and loaded onto the preparative HPLC column (Waters, XTerra® Prep MS Cl 8, 1 O ⁇ , 12 ⁇ A, 19 x 300 mm) which had been pre-equilibrated with 5% acetonilrile in water (0.1% TFA).
  • the flow of the equilibrating eluent from the preparative HPLC system was stopped.
  • Step 2 Preparation of peptide sequences containing IRDye 800CVV and BHQ3 [00092)
  • a solution of the peptide-BHQ3 conjugate seqO35 (3 mg, 0.0017 mmol) in anhydrous DMF (0.3 mL) was added FRDye800CW-NHS ester (3 nig, 0.0026 mmol) followed by DFEA ( 10 mg, 0.077 mmol) and the solution was stirred for 8 h at ambient temperature (flask was wrapped with aluminum foil to avoid light).
  • protecting groups such as Boc or Pmc group(s) on the peptide, if any, were removed as follows.
  • reaction mixture was evaporated to remove the volatiles on a rotary evaporator, treated with a cleavage cocktail of TFA:water:phenol (0.5 mL, 95:3:2, v/v/w) and stirred for 2 h at ambient temperature. After deprotection was completed (as determined using HPLC and MS), the reaction mixture was diluted with water to 20 mL and loaded onto the preparative HPLC column (Waters, XTerra® Prep MS C 18, lO ⁇ , 120A, 19 x 300 mm) which had been pre-equilibrated with 5% acetonitrile in water (0.1% TFA).
  • MMP assay Fluor/quencher-containing peptides were tested in an in vitro assay to determine their relative rates of cleavage by specific enzymes. The assay procedure described here was used. [00098] Materials: MMPs were obtained from AnaSpec and stored in 50 niM Tris-
  • MMP assay buffer (Cat No. 60907-500) was obtained from AnaSpec and stored at 4 0 C until use.
  • Mouse Neprilysin was obtained from R&D Systems.
  • APMA Aminomethylphenyl mercuric acetate, Cat No.A9563-5G was purchased from Sigma.
  • the 96-well plates used were NUNC MaxiSorb black plate (NUNC Cat No. 4371 1 1 ). The assay was performed on a Near- IRDye fluorescence plate reader (BioTek-Synergy2).
  • Peptide stock solutions 500 ⁇ M were prepared in DMSO and stored at -20 0 C; an aliquot was diluted to 10 ⁇ M using Assay Buffer prior to assay.
  • APMA solutions 10 mM were prepared in water.
  • MMPs supplied at a concentration of 1 ⁇ g in 100 ⁇ L
  • APMA 1 mM APMA just prior to assay.
  • the incubation time required to activate the various MMPs are given below.
  • MMP- 13 (supplied as 1 ⁇ g in 100 ⁇ L) was diluted to 450 ⁇ L in AnaSpec MMP assay buffer. It was mixed with APMA (10 mM, 50 ⁇ L) and incubated at 37 0 C for 45 min. Ii was then cooled in ice (4 0 C) and diluted to 1.25 mL in assay buffer to give a final enzyme concentration of 0.8 ⁇ g/mL. This solution was stored in ice until use.
  • MMP- 13 cleaves the substrate faster than MMP-9, which in turn was faster than MMP-2. (Seq ID 039>Seq ID 37>Seq ID 040). Using such an assay, the relative sensitivity of such compounds to various enzymes can be determined.
  • Neprilysin is a protease that is known to cleave a variety of peptides, especially in the brush borders of the kidney.
  • compounds that are not substantially cleaved in vivo by enzymes found in normal tissue or blood, such as for example, Neprilysin are especially preferred.
  • a compound comprising: a compound of the general formula:
  • F is a fluorescent dye
  • E is an enzyme-cleavable substrate
  • a compound comprising: a compound of the general formula:
  • F is a compound selected from lhe group consisting of Formula I, Formula II and Formula III, wherein Formula 1 comprises:
  • R I , R2, R5, R6, R7, R8, R9, R 10 and R 1 1 are each independently H, Me, substituted or unsubstituted alky I, halo, carboxy, amino, sulfonate, R I 2-COOH, R 12OR13, R 12SR 13, or R12COOR I 3, wherein R12 is a bond or alkylene and R 13 is substituted or unsubstituted alky 1 R4 is -O-aryl, NH-aryl, a substiluied or unsubstituied aryl, a subslituted or unsubstiluted C-I -ClO alkyl, halo, S-aryl or SO2-aryl, where the aryl ring can, in all cases, be substituted or unsubstiluted, or a linker that is covalently bonded (e.g. via an amine, thiol or acid functionality) to an enzymatically cleavable substrate or a linker that
  • Tl and T2 are each H, or are joined together to form a substituted or unsubstiluted 5- or 6-membered ring.
  • X 1 , X2, X3 and X4 independently represent SO3H, COOH or physiologically acceptable salts thereof, or polyethylene glycol, or one or more of X 1-X4 may be a linker (e.g. a substituted or unsubstituied alkyl, substituted or u ⁇ substituted Aryl, PEG, alkyl amine or quanidine) that is either covalently bonded (e.g.
  • a linker e.g. a substituted or unsubstituied alkyl, substituted or u ⁇ substituted Aryl, PEG, alkyl amine or quanidine
  • X 1 and R3 and/or X2 and R9 can be cyclized together to form a 5- or 6 membered saturated or unsaturated ring that is optionally derivatized with H, subslituted or unsubstituted C 1 -C6 alkyl, polyethylene glycol, substituted or unsubstituted aryl, halogen, cyano, carboxy, and/or sulfo group(s).
  • Y 1 and Y2 are independently C, N (in which case R2 and/or R8 is absent), or O, S, Se, sulfide, sulfone or sulfoxide (in which case both
  • Rl and R2 and/or R7 and R8 are absent), and the Linker is a substituted or unsubstituted Cl -C lO alkyl chain, a polyethylene glycol derivative, or a substituted or unsubslituted aryl group;
  • Formula II comprises:
  • Z is -O-, -S-, -S(-O)-, -SO 2 - -NH, HN-NH- or N-Alkyl, or absent and wherein A1-A5 are each independently H, alkyl, halo, carboxy, alkylcarboxy, amino, alkylamino, amino alkyl, or SCVCat+, wherein Cat' is a H * or a cation, and the remaining residues are defined as described above for Fo ⁇ nula I; and wherein Formula III comprises:
  • Cat+ is a cation selected from the group consisting of H * , Na * , K * , NH/, meglumine, a protonated organic base or a physiologically acceptable cation,
  • E is an en/yme-cleavable substrate
  • Q is wherein the compound fluoresces under near infrared light after cleavage of the enzyme-cleavable substrate.
  • NC No Cleavage
  • LC Little cleavage
  • PC Partial cleavage

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Abstract

L’invention concerne de nouveaux composés qui contiennent un fluor et un désactivateur reliés par un substrat clivable par une enzyme. Les constructions contiennent des fonctions de fluor/désactivateur qui sont optiquement silencieuses jusqu’à leur clivage par des enzymes, selon un mode de réalisation préféré par des métalloprotéases de la matrice (MMP). Après le clivage, les constructions deviennent fluorescentes. Les fonctions fluorescentes résultantes fournissent des émissions pouvant être imagées qui peuvent être utilisées pour l’évaluation in vitro de l’activité enzymatique ou pour la détection in vivo d’une maladie. Selon un mode de réalisation préféré, le fluor est détectable dans le proche infrarouge (proche IR). Contrairement aux procédés d’imagerie qui fournissent des informations sur la morphologie, ces constructions fournissent des informations biochimiques sur la maladie.
PCT/US2009/049380 2008-07-03 2009-07-01 Constructions fluor-désactivateur contenant un colorant clivable par une enzyme Ceased WO2010002976A2 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
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WO2012054784A1 (fr) 2010-10-20 2012-04-26 Li-Cor, Inc. Imagerie de fluorescence à colorants à base de cyanine substituée
CN103242822A (zh) * 2013-04-26 2013-08-14 华东理工大学 用于检测n-乙酰转移酶2的荧光探针
US10646591B2 (en) 2011-09-09 2020-05-12 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Imaging agents for imaging protease activity and uses thereof
WO2021198228A1 (fr) * 2020-03-30 2021-10-07 Original G B.V. Peptide de diagnostic destiné à être utilisé dans un procédé de diagnostic d'une infection virale, kit et système
CN113683602A (zh) * 2021-09-08 2021-11-23 中国人民解放军陆军军医大学 一种用于缺氧肿瘤多模态治疗的七甲川花菁小分子和制备方法及应用
CN114878725A (zh) * 2021-06-22 2022-08-09 长春大学 一种食品中二氧化硫的检测方法
WO2022197186A1 (fr) * 2021-03-19 2022-09-22 Original G B.V. Procédé de diagnostic in vitro d'une infection
EP3983334A4 (fr) * 2019-02-01 2023-08-23 Glympse Bio, Inc. Détection de remodelage architectural dans une matrice extracellulaire de cellules, et dans le micro-environnement tissulaire
US12305217B2 (en) 2020-09-11 2025-05-20 Sunbird Bio, Inc. Ex vivo protease activity detection for disease detection/diagnostic, staging, monitoring and treatment

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US8227621B2 (en) * 2005-06-30 2012-07-24 Li-Cor, Inc. Cyanine dyes and methods of use
US20080085893A1 (en) * 2006-10-06 2008-04-10 Shyh-Ming Yang Matrix metalloprotease inhibitors

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012054784A1 (fr) 2010-10-20 2012-04-26 Li-Cor, Inc. Imagerie de fluorescence à colorants à base de cyanine substituée
US10646591B2 (en) 2011-09-09 2020-05-12 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Imaging agents for imaging protease activity and uses thereof
CN103242822A (zh) * 2013-04-26 2013-08-14 华东理工大学 用于检测n-乙酰转移酶2的荧光探针
EP3983334A4 (fr) * 2019-02-01 2023-08-23 Glympse Bio, Inc. Détection de remodelage architectural dans une matrice extracellulaire de cellules, et dans le micro-environnement tissulaire
WO2021198228A1 (fr) * 2020-03-30 2021-10-07 Original G B.V. Peptide de diagnostic destiné à être utilisé dans un procédé de diagnostic d'une infection virale, kit et système
US12492422B2 (en) 2020-09-11 2025-12-09 Sunbird Bio, Inc. Ex vivo protease activity detection for disease detection/diagnostic, staging, monitoring and treatment
US12305217B2 (en) 2020-09-11 2025-05-20 Sunbird Bio, Inc. Ex vivo protease activity detection for disease detection/diagnostic, staging, monitoring and treatment
WO2022197186A1 (fr) * 2021-03-19 2022-09-22 Original G B.V. Procédé de diagnostic in vitro d'une infection
KR20230149829A (ko) * 2021-03-19 2023-10-27 오리지날 헤 베.페. 감염의 체외 진단을 위한 방법
KR102725628B1 (ko) 2021-03-19 2024-11-04 오리지날 헤 베.페. 감염의 체외 진단을 위한 방법
CN114878725B (zh) * 2021-06-22 2023-08-11 长春大学 一种食品中二氧化硫的检测方法
CN114878725A (zh) * 2021-06-22 2022-08-09 长春大学 一种食品中二氧化硫的检测方法
CN113683602A (zh) * 2021-09-08 2021-11-23 中国人民解放军陆军军医大学 一种用于缺氧肿瘤多模态治疗的七甲川花菁小分子和制备方法及应用

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