OPTICAL PROBES FOR MATRIX METALLOPROTEINASES

Abstract

An optical probe is presented comprising at least one fluorophore connected to at least one quencher by an enzyme cleavable peptide sequence; the or each fluorophore being substantially fluorescently quenched by the at least one quencher when connected to the enzyme cleavable peptide sequence; the or each fluorophore is separated from the at least one quencher when the enzyme cleavable peptide sequence of the at least one probe element is cleaved; and the enzyme cleavable peptide sequence is selectively cleavable by one or more matrix metalloproteinase (MMP). Methods of use of the optical probe are also presented.

Claims

1. An optical probe comprising at least one fluorophore connected to at least one quencher by an enzyme cleavable peptide sequence; the at least one fluorophore being substantially fluorescently quenched by the at least one quencher when connected to the enzyme cleavable peptide sequence; wherein the at least one fluorophore is separated from the at least one quencher when the enzyme cleavable peptide sequence of at least one probe element is cleaved; wherein the enzyme cleavable peptide sequence comprises an amino acid sequence of SEQ ID NO:7, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14 and is selectively cleavable by one or more matrix metalloproteinase (MMP).

2. The probe according to claim 1, wherein the enzyme cleavable peptide sequence is selectively cleavable by MMP-2 and/or MMP-9 and/or MMP-13.

3. The probe of claim 1, wherein the enzyme cleavable peptide sequence comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:5, SEQ ID NO:7 SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.

4. The probe according to claim 3, wherein the enzyme cleavable peptide sequence comprises the amino acid sequence of SEQ ID NO:7.

5. The probe according to claim 1, wherein the at least one quencher is the same type of fluorophore as the at least one fluorophore, and the at least one quencher and the at least one fluorophore self-quench.

6. The probe of claim 1, wherein the at least one quencher is a different type of fluorophore to the at least one fluorophore, and is a fluorescent quencher.

7. The probe of claim 1, wherein the at least one quencher is a dark quencher.

8. The probe according to any claim 1, wherein the at least one fluorophore is fluorescein or a derivative thereof, a cyanine fluorophore, Cy2, Cy3, Cy5, or Cy7, rhodamine or derivative thereof, a fluorescent protein, green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), or 7-nitrobenz-2-oxa-1,3-diazole (NBD).

9. The probe according to claim 4, wherein the at least one fluorophore and the at least one quencher form a FRET pair and are chosen from the list (fluorophore/quencher): Cy3/Cy5, Cy3/QSY21, Cy5/QSY21, Cy5/BHQ-3, fluorescein/tetramethylrhodamine, fluorescein/methyl red, NBD/methyl red, cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP), and carboxy fluorescein/methyl red.

10. (canceled)

11. The probe according to claim 1, wherein the at least one fluorophore is connected to the enzyme cleavable peptide sequence by a spacer.

12. The probe according to claim 11, wherein the spacer comprises 6-aminohexanoic acid (Ahx), polyethyleneglycol (PEG).

13. The probe according to claim 1, wherein the unit of the at least one fluorophore connected to the enzyme cleavable peptide sequence corresponds to a probe element and wherein the probe comprises a plurality of probe elements; each of the plurality of probe elements comprising at least one fluorophore connected to an enzyme cleavable peptide sequence.

14. (canceled)

15. The probe according to claim 13, wherein the probe comprises a core, and the plurality of probe elements are connected to the core.

16. (canceled)

17. The probe according to claim 15, wherein each probe element within the plurality of probe elements is independently connected indirectly to the core via a linker.

18. The probe according to claim 17, wherein the linker is selected from the group consisting of: [-(lysine)-(PEG.sub.2)-].sub.1-2, [-(PEG-k)-].sub.1-3, and [-(PEG-k).sub.0-2-NH(CH.sub.2).sub.3OCH.sub.2].

19. The probe according to claim 17, wherein the linker comprises a D-amino acid.

20. The probe according to claim 13, wherein the at least one fluorophore of each probe element within the plurality of probe elements self-quenches, such that the at least one fluorophore of a first probe element substantially fluorescently quenches the at least one fluorophore of a second probe element.

21. The probe according to claim 15, wherein the core comprises the at least one quencher.

22. The probe according to claim 13, wherein each of the plurality of probe elements comprise at least one quencher.

23. The probe according to claim 13, wherein one or more of the plurality of probe elements comprises at least two fluorophores.

24. The probe according to claim 13, wherein the probe comprises at least three probe elements.

25. The probe according to claim 1, wherein the probe comprises at least one reporter fluorophore that is not substantially fluorescently quenched.

26. The probe according to claim 25, wherein the at least one reporter fluorophore fluoresces at a wavelength that is different to the wavelength of light at which the at least one fluorophore fluoresces.

27. A method of detecting MMP in a target zone, the method comprising the steps: a. providing a probe according to claim 1; b. applying the probe to the target zone; c. illuminating the target area with an appropriate wavelength of light to excite the at least one fluorophore of the probe; and d. determining the fluorescence intensity of the at least one fluorophore, wherein significant fluorescence of the at least one fluorophore of the probe is indicative of the presence of MMP in the target zone.

28. The method according to claim 27, wherein the target zone is a portion of tissue of an animal.

29. The method according to claim 28, wherein the tissue type is heart, lung, liver, connective tissue, skin, or intestine.

30. The method according to claim 27, wherein the presence of MMP in the target zone is indicative of active fibroproliferation within the target zone.

31. The method according to claim 27, wherein the target zone is a joint of a subject.

32. The method according to claim 31, wherein the presence of MMP in the target zone is indicative of active arthritis in the target zone.

33. The method according to claim 27, wherein the MMP is MMP-2, and/or MMP-9 and/or MMP-13.

34. The method according to claim 33, wherein the MMP is MMP-9 and/or MMP-13.

35. The method according to claim 27, wherein the presence of MMP in the target zone is indicative of cancer within the target zone.

36. A method of assessing a portion of tissue; the method comprising the steps of: a. applying a probe according to claim 1 to the portion of tissue; b. illuminating the portion of tissue 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 probe is indicative of the presence of MMP in the portion of tissue, and the presence of MMP in the portion of tissue is indicative of a disease in which MMP is expressed or overexpressed.

37. The method of claim 36, wherein the disease is active fibroproliferation, cirrhosis, cancer or arthritis.

38. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0124] FIG. 1. Probe fluorescence (ex 485 nm/em 528 nm) was measured for all FRET monomers (10 M) with MMP-9 (30 nM) in the absence or presence of Marimastat (20 M).

[0125] FIG. 2. Probe fluorescence (ex 485 nm/em 528 nm) was measured after 5 min for all FRET monomers (10 M) in the absence and presence of Elastase (30 nM).

[0126] FIG. 3. Probe fluorescence (ex 485 nm/em 528 nm) was measured for FRET monomers (10 M) with MMPs (30 nM); (A) Probe SVC-24: FAM-PEG.sub.2-G-P-K-G-L-K-G-K(MR)-NH.sub.2; (B) Probe SVC-25 (Control): FAM-PEG.sub.2-G-P-K-G-(D)L-K-G-K(MR)-NH.sub.2 (Red dotted line represents background fluorescence for each probe).

[0127] FIG. 4. An illustration of how probes according to an embodiment of the invention comprising three probe elements fluoresce in the presence of MMP-9 and MMP-13.

[0128] FIG. 5. Schematic illustrations of examples of probes according to embodiments of the invention.

[0129] FIG. 6. Probe Background: Background fluorescence signal for MMP probes. Probes at 10 M were incubated at 37 C. in MMP buffer and assessed in a fluorimeter (Synergy H1 Hybrid Reader, BioTek instruments Ltd) at excitation/emission 485/528 nm. Signal was shown as relative fluorescence units after 6 min. The scaffold structure and the number/position of fluorophores and quenchers has an important effect in the quenching efficiency and therefore in the fluorescent background signal.

[0130] FIG. 7. Schematic illustrations of Probe A, Probe B and Probe C.

[0131] FIG. 8. Enzyme specificity of the FRET branched probes A, B and C. Data represents the average fold change in fluorescence over background signal provided by probe (10 M) with exogenous enzymes using a multiwell plate fluorimeter (Synergy H1 Hybrid Reader, BioTek instruments Ltd) at excitation/emission 485/528 nm. Recombinant human catalytic domain MMPs -1, -2, -3, -7, -8, -9, -10, -11, -12, -13 were used at 30 nM. Recombinant human Thrombin, Plasmin and Factor Xa were used at 5 U/ml, 30 nM, 0.5 M, respectively.

[0132] FIG. 9. Probe specificity by target inhibition for probes A and probe B. Data represents the fluorescence signal provided by the probe (1 M) after 30 min using a multiwell plate fluorimeter at excitation/emission 485/528 nm. To validate the specificity and ability of molecular probe to detect the target enzyme, probe fluorescence (initiated by cleavage) was measured in the presence of enzyme with/without inhibitor. Inhibitors Marimastat (pan-MMP inhibitor), AZD1236 (MMP-2/9 inhibitor), Inhibitor I (MMP-9 inhibitor) were used at 200 nM.

[0133] FIG. 10. Ex-vivo analysis of molecular probe detection of MMP-9 activity. Sheep zymography (A), Human zymography (B), in the presence or absence of an MMP-9 inhibitor.

[0134] FIG. 11. Ex-vivo analysis of MMP-9 activity using molecular probes in sheep lung. Fluorescence data shown as increase from normal segment (%). Sheep fibrotic lung tissue biopsies were obtained from Ovine Pulmonary Adenocarcinoma-(OPA).

[0135] FIG. 12. Ex-vivo analysis of MMP-9 activity using molecular probes in human lung. Fluorescence data shown as increase from normal segment (%).

[0136] FIG. 13. Haemolytic assay of molecular probes A and B. The haemolytic activity of the molecular probes was evaluated in human red blood cells.

[0137] FIG. 14. An example of a probe according to one embodiment of the invention. The probe comprises one reporter fluorophore that is not substantially quenched plus a FRET pair.

[0138] FIG. 15. Enzyme specificity of the polymerisable probe SVC-180 (SVC-01-180) in comparison with FRET monomer AMF-92.

[0139] FIG. 16. Ex-vivo analysis of detection of MMP-9 activity by the polymerised hydrogel of probe SVC-180 (SVC-01-180).

SPECIFIC DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0140] 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.

[0141] 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.

Enzyme Cleavable Peptide Sequence Optimization as MMP-2/9 Substrate

Generating Molecular Probe Sequences to Optimally Measure the MMP-2/9 Activity:

[0142] The initial activatable probes contain a fluorophore and a quencher bonded to an amino acid sequence that is cleavable by the target enzyme (acting as an enzyme cleavable peptide sequence). In the inactive state, the emission from the fluorophore moiety is absorbed by the quencher by fluorescence resonance energy transfer (FRET), but in the presence of the target enzyme, the amino acid sequence is cleaved, separating the fluorophore from the quencher, and thereby causing a remarkable increase in fluorescence.

[0143] Initial substrate G-P-K-G-L-K-G (SEQ ID NO.1) was selected according to Proteomics studies [Mol. and Cell. Proteomics 2010, 9, 894-911].

[0144] A library of probes (listed in Table 2 below) containing FAM (Carboxyfluorescein) as the fluorophore (donor) and MethylRed (MR) as the quencher (acceptor) and a variety of peptide sequences was synthesized by manual standard solid-phase Fmoc peptide chemistry (described below). The specificity for MMP of the probes was evaluated by measuring the fluorescence of the probes in the presence of MMP-9 and in absence or presence of the MMP suppressor Marimastat, and the results are shown in FIG. 1.

TABLE-US-00002 TABLE2 ProbeStructures Code FAM-PEG.sub.2-G-P-K-G-L-K-G-K(MR)-NH.sub.2 SVC-01-024/ SVC-024b,c FAM-PEG.sub.2-G-P-K-G-DL-K-G-K(MR)-NH.sub.2 SVC-01-025/ SVC-025,b FAM-PEG.sub.2-G-P-K-G-I-K-G-K(MR)-NH.sub.2 SVC-01-026/ SVC-026 FAM-PEG.sub.2-G-P-K-G-Nle-K-G-K(MR)-NH.sub.2 SVC-01-027/ SVC-027 FAM-PEG.sub.2-P-Cha-G-M-F-G-K(MR)-NH.sub.2 SVC-01-015/ SVC-015 FAM-PEG.sub.2-P-F-G-M-K-A-K(MR)-NH.sub.2 SVC-01-016/ SVC-016/ SVC-069 FAM-PEG.sub.2-P-F-G-L-K-A-K(MR)-NH.sub.2 AMF-23 FAM-PEG.sub.2-P-F-G-I-K-A-K(MR)-NH.sub.2 AMF-24 FAM-PEG.sub.2-P-F-G-Nle-K-A-K(MR)-NH.sub.2 AMF-25 FAM-PEG.sub.2-P-F-G-DM-K-A-K(MR)-NH.sub.2 AMF-26 FAM-PEG.sub.2-P-Cha-G-M-W-G-K(MR)-NH.sub.2 SVC-01-017/ SVC-017 FAM-PEG.sub.2-P-Cha-G-M-Y(Me)-G-K(MR)-NH.sub.2 SVC-01-018/ SVC-018 FAM-PEG.sub.2-P-Cha-G-M-Y-G-K(MR)-NH.sub.2 SVC-01-019/ SVC-019 FAM-PEG.sub.2-P-Cha-G-M-K-A-K(MR)-NH.sub.2 SVC-01-020/ SVC-020 FAM-PEG.sub.2-P-Cha-G-M-H-G-K(MR)-NH.sub.2 SVC-01-021/ SVC-021 FAM-PEG.sub.2-P-Cha-G-M-K-G-K(MR)-NH.sub.2 SVC-01-022/ SVC-022

[0145] The selectivity of the probes, together with further probes AMF-14b, SVC-030 (both defined in Table 6 below) and SVC-068 (defined in Table 4 below), to detect MMP over elastase, another proteinase relevant in lung disease, was tested and the results for all probes are shown in FIG. 2.

[0146] Enzyme specificity of the lead probes with exogenous enzymes was tested using a multiwell plate fluorimeter at excitation/emission 485/528 nm (Table 3). Recombinant human catalytic domain MMPs -1, -2, -3, -7, -8, -9, -10, -11, -12, -13 were used at 30 nM. Cleavage site for each sequence is indicated in italics. For probe SVC-24, results are shown in FIG. 3A as SVC-24c. For probe SVC-025, results are shown in FIG. 3B as SVC-25b.

TABLE-US-00003 TABLE3 Inflammatorymediators Neutro- Neutro- Probe Probe MMP-9 MMP-2 MMP-12 MMP-13 phil phil code sequence (39.0kDa) (20.3kDa) (20.3kDa) (20.4kDa) lysate lysate Thrombin SVC-24/ -G-P-K-G-L-K-G- 3.50 1.98 2.34 9.30 1.93 1.30 1.31 SVC24b SVC-26 -G-P-K-G-Ile-K-G- 4.22 2.10 1.54 9.33 13.49 11.97 1.37 SVC-27 -G-P-K-G-Nle-K-G- 4.53 2.70 1.46 11.75 3.06 1.97 1.48 SVC-69/ SVC-16 -P-F-G-M-K-A- 5.21 3.11 1.88 13.57 1.07 1.06 0.99 AMF-23 -P-F-G-L-K-A- 2.50 1.87 3.03 6.41 0.99 0.10 0.89 AMF-24 -P-F-G-Ile-K-A- 2.29 1.59 1.65 5.05 3.29 2.25 0.96 AMF-25 -P-F-G-N/e-K-A- 2.02 1.88 1.53 5.27 0.96 0.99 0.96 SVC-15 -P-Cha-G-M-F-G- 5.89 3.27 2.45 11.75 2.02 1.07 0.94 SVC-17 -P-Cha-G-M-W-G- 5.07 2.59 2.59 6.5 0.84 1.04 1.05 -P-Cha-G-M- SVC-18 Y(Me)-G- 4.94 2.54 2.15 10.04 0.75 1.00 0.97 SVC-19 -P-Cha-G-M-Y-G- 6.90 4.07 2.87 11.26 1.89 1.08 1.04 SVC-20 -P-Cha-G-M-K-A- 9.61 5.22 3.21 15.9 1.40 1.19 G- 1.00 SVC-21 -P-Cha-G-M-H-G- 7.53 4.01 3.05 12.93 1.27 1.08 0.97 SVC-22 -P-Cha-G-M-K-G- 8.21 4.24 2.61 14.88 1.95 1.44 1.02

[0147] An increase in fluorescence was observed with MMP-9 and site specific cleavage, as revealed by MALDI, was confirmed for all the sequences. The other MMPs tested shared the same cleavage site. Control probes (containing the sequences -G-P-K-G-(D)L-K-G- and -P-F-G-(D)M-K-A-) with D-amino acids in the cleavage site were tested and no increase in fluorescence was observed.

[0148] Specific inhibition of fluorescence signal using Marimastat was successful for all of the probes. In order to choose the best probes for our application, additional parameters were characterized and compared. The fluorescence increase in presence of other related enzymes (Thrombin, Elastase), macrophages, and bronchoalveolar lung lavage fluid (BALF) was also measured and used as a selection criteria. Despite the positive results for probes containing Methionine in the cleavage site, these probes were avoided due to potential stability problems caused by sulphide oxidation. The best probes were also evaluated in an ex vivo assay with human tissue homogenate confirming only specific cleavage and inhibition with Marimastat for -G-P-K-G-L-K-G-K(MR)-(SEQ ID NO.15) and -P-F-G-Nle-K-A-K(MR)-(SEQ ID NO.16). In each case, the enzyme cleavable peptide sequence that is specifically cleaved by MMP is shown in bold. All assays were carried out using FRET monomers (i.e. they comprise one probe element) to overcome ineffectiveness of the final MMP probes due to poor specificity and in vivo stability, one of the main difficulties found when applying a probe in vivo.

[0149] Control probes are listed in Table 4 below containing FAM (Carboxyfluorescein) as the fluorophore (donor), MethylRed (MR) as the quencher (acceptor) and a variety of peptide sequences. The control probes were synthesized by manual standard solid-phase Fmoc peptide chemistry (described below). SVC-068 is an oxidised version of SVC-01-16, which contains methionine in the cleavable sequence. Oxidation of SVC-01-16 introduces a SO group. It is apparent from a FIG. 2 that the oxidised cleavable sequence was not recognised by MMP.

TABLE-US-00004 TABLE4 ProbeStructures Code FAM-PEG.sub.2-P-F-G-M(S= O)-K-A-K(MR)-NH.sub.2 SVC-01-68/ SVC-068 FAM-PEG.sub.2-G-p-k-G-L-k-G-K(MR)-NH.sub.2 AMF-73 FAM-PEG.sub.2-G-p-k-G-I-k-G-K(MR)-NH.sub.2 AMF-74 FAM-PEG.sub.2-G-G-G-G-G-K(MR)-NH.sub.2 ETA-12

[0150] Synthesising aqueous soluble molecular probes: The two selected sequences A and B were implemented in new probes flanked by ethylenglycol units and Lys residues to increase the solubility. Good results were achieved with alternate D-Lys and PEG units.

[0151] A number of structural modifications were carried out to improve the solubility of the probes, and to determine the optimal fluorophores and quenchers to be used.

Solubility:

[0152] Different linker/spacer composition consisting of hydrophilic/hydrophobic or charged groups: ethylenglycol units (PEG), L-Lysine, and alternative ethylenglycol units (PEG) and D-Lysine were generated. The linkers improve the aqueous solubility of the compounds. The presence of unnatural D-aminoacid improves the in vivo stability of the compounds making them resistant to proteolysis. Furthermore, this allowed greater understanding of the effect of linkers on structure and function of the probes.

Expansion of Fluorophore and FRET System:

[0153] Various fluorophores, including FAM, Cy5 and carboxy naphthofluorescein (NF), and quenchers (MR, BHQ-1/3 and QSY-21) labelled on optimised sequences to expand the scope of probe, not limited to, near infrared range (i.e. probes that emit radiation having a wavelength of 0.75-1.4 m) with the aim of producing a higher signal-to-noise ratio, which may enable lower levels of MMP detection at lower effective probe concentrations and to rule out the non-specific activation by introducing additional label which is aimed to be as a reference dye (for example, see FIG. 13). Example structures are shown in Table 5 below, where -k- represents -(D)-Lysine-.

[0154] Probe SVC-01-180 in Table 5 below is a polymerisable probe containing an ethylenically unsaturated monomer moiety. This can be polymerised in the presence of water to form a hydrogel and is discussed in more detail below.

TABLE-US-00005 TABLE5 ProbeStructures Code FAM-PEG.sub.2-G-P-K-G-L-K-G-K(MR)-K-K-PEG.sub.2-PEG.sub.2-NH.sub.2 AMF-91 FAM-PEG.sub.2-P-F-G-Nle-K-A-K(MR)-K-K-PEG.sub.2-PEG.sub.2-NH.sub.2 AMF-92 FAM-PEG.sub.2-G-P-K-G-(D)L-K-G-K(MR)-K-K-PEG.sub.2-PEG.sub.2-NH.sub.2 ODHS-1 FAM-PEG.sub.2-G-P-K-G-L-K-G-K(MR)-PEG.sub.2-PEG.sub.2-NH.sub.2 AMF-96 FAM-PEG.sub.2-P-F-G-Nle-K-A-K(MR)-PEG.sub.2-PEG.sub.2-NH.sub.2 AMF-97 MR-K(Ac)-PEG.sub.2-GPKGLKG-K(FAM)-PEG.sub.2-k-PEG.sub.2-k-PEG.sub.2-k-NH.sub.2 AMF-111 QSY21-K(PEG-N.sub.3)-PEG.sub.2-GPKGLKG-K(Cy5)-PEG.sub.2-k-PEG.sub.2-k-PEG.sub.2-k-NH.sub.2 AMF-140 FAM-PEG.sub.2-GPkGLkGK(MR)-PEG.sub.2-k-PEG.sub.2-k-NH.sub.2 SVC-01-181DD FAM-PEG.sub.2-GPkGLKGK(MR)-PEG.sub.2-k-PEG.sub.2-k-NH.sub.2 SVC-01-181DL MR-PEG.sub.2-P-F-G-Nle-K-A-K(FAM)-PEG.sub.2-k-PEG.sub.2-k-PEG.sub.2-k-NH.sub.2 AMF-154-01 QSY21-PEG.sub.2-P-F-G-Nle-K-A-K(Cy5)-PEG.sub.2-k-PEG.sub.2-k-PEG.sub.2-k-NH.sub.2 AMF-154-02 QSY21-PEG.sub.2-P-F-G-Nle-K-A-K(naphthofluorescein)-(PEG.sub.2-k)3-NH.sub.2 SVC-02-008 QSY21-K(PEG-N.sub.3)-PEG.sub.2-P-F-G-Nle-K-A-K(Cy5)-PEG.sub.2-k-PEG.sub.2-k-PEG.sub.2-k-NH.sub.2 AMF-154-03 QSY21-K(N.sub.3)-PEG.sub.2-P-F-G-Nle-K-A-K(Cy5)-PEG.sub.2-k-PEG.sub.2-k-PEG.sub.2-k-NH.sub.2 AMF-181 QSY21-K(N.sub.3)-PEG.sub.2-P-F-G-(D)Nle-K-A-K(Cy5)-PEG.sub.2-k-PEG.sub.2-k-PEG.sub.2-k-NH.sub.2 AMF-199 FAM-PEG.sub.2-K(tzCy5)-PEG.sub.2-P-F-G-Nle-K-A-K(MR)-[PEG.sub.2-k].sub.3-NH.sub.2 AMF-210 Cy5-K-(DBCO)-PEG.sub.2-G-P-K-G-L-K-G-K(BHQ-3)-PEG.sub.2-k-PEG.sub.2-k-PEG.sub.2-k-NH.sub.2 SVC-01-161 MR-K(Acryloyl)-PEG.sub.2-G-P-K-G-L-K-G-K(FAM)-PEG.sub.2-k-PEG.sub.2-k-NH.sub.2 SVC-01-180 SVC-180

[0155] Variants consisting of D-amino acids at the recognition site sequence with or without blocking PEG units were also synthesised and assessed. The D-amino acid variants as well as the variants with PEG or acetyl blocking groups at the ends of the peptide sequence exhibited reduced activation by MMP in vitro.

[0156] A number of further structural modifications were carried out to improve the signal-to-noise ratio and to improve the structural stability of the probes in vivo, and the structures tested are shown in Table 6 below:

TABLE-US-00006 TABLE6 ProbeStructures Code FAM-Ahx-G-P-K-G-L-K-G-3probeelements TWB-140 FAM-Ahx-G-P-K-G-L-K-G-K(MR)-3probeelements AMF-14/AMF-14b FAM-PEG.sub.2-P-F-G-M-K-A-K(MR)-NH-3probeelements SVC-01-030/ SVC-030 FAM-PEG.sub.2-G-P-K-G-L-K-G-K(MR)-PEG.sub.2-k-PEG.sub.2-k-3probeelements AMF-106 FAM-PEG.sub.2-PFGNleKA-PEG.sub.2-k-PEG.sub.2-k-3probeelements SVC-01-188 FAM-PEG.sub.2-PFGNleKA-K(MR)-PEG.sub.2-k-PEG.sub.2-k-3probeelements SVC-01-186/194 FAM-K(FAM)-PEG.sub.2-PFGNleKA-PEG.sub.2-k-PEG.sub.2-k-3probeelements SVC-01-189 FAM-PEG.sub.2-PFGNleKA-K[K(MR).sub.2]-PEG.sub.2-k-PEG.sub.2-k-3probeelements SVC-01-187/195 FAM-K(FAM)-PEG.sub.2-PFGNleKA-K(FAM)-PEG.sub.2-k-PEG.sub.2-k-3probeelements SVC-01-196 Ac-PEG.sub.2-PFGNleKA-K(FAM)-PEG.sub.2-k-PEG.sub.2-k-3probeelements SVC-01-197 Ac-PEG.sub.2-PFGNleKA-K[K(FAM).sub.2]-PEG.sub.2-k-PEG.sub.2-k-3probeelements SVC-01-198 FAM-K(FAM)-PEG.sub.2-PFGNleKA-K(MR)-PEG.sub.2-k-PEG.sub.2-k-3probeelements SVC-02-026 FAM-K(FAM)-PEG.sub.2-k-PEG.sub.2-k-PFGNleKA-PEG.sub.2-3probeelements AMF-164 FAM-PEG.sub.2-k-PEG.sub.2-k-K(FAM)-PFGNleKA-PEG.sub.2-3probeelements AMF-165 NBD-PEG.sub.2-PFGNleKA-K(MR)-PEG.sub.2-k-PEG.sub.2-k-3probeelements SVC-02-030 [FAM-PEG.sub.2-PFGNleKA]3probeelements-Lys(MR)-PEG.sub.2-k-PEG.sub.2- AMF-212 K(Alkyne)-NH.sub.2

Branched Probe:

[0157] Increase the signal-to-noise ratio by examining different probe scaffold designs, change of fluorophore positions, spacer length and location between the fluorophore-quencher or increasing the fluorophore (FAM) or quencher (MethylRed, BHQ-1/3, or QSY-21) number per probe element. FIG. 4 shows how a probe comprising three probe elements, or branches fluoresces in the presence of MMP-9 or MMP-13. FIG. 5 shows schematic illustrations of a number of different probe structures, including a linear probe (FRET monomer), branched probes comprising three probe elements (3Dendrimer, 6Dendrimer and 9Dendrimer, where the number indicates the number of fluorophores in total in the probe) and branched probes comprising three probe elements comprising fluorophores and quenchers (FRET dendrimer).

[0158] SEQ ID NO.7 was implemented in multibranch probes (i.e. probes comprising a plurality of probe elements) to take advantage of the amplification nature of the assay. These probes were designed as self-quenched or FRET quenched systems. These probes consist of the MMP substrate with hydrophilic spacers and N-terminal fluorophores that are released after enzymatic cleavage (self-quenched), and probes with additional Methyl Red (FRET quenched). With all these compounds, the effect caused by spacer location, different number and position of the fluorophores (3, 6 or 9) and the absence or presence of Methyl Red moieties (3 or 6) was evaluated.

[0159] Comparison of the background signal showed very different results for the different architectures. As can be seen from FIG. 7, only the schematically illustrated 3Dendrimer showed any significant background fluorescence in the absence of MMP, indicating that the fluorophores of the majority of the probe structures were successfully quenched. While a substantial decrease in background signal was observed from self-quenched probes when going from 3 to 6 or 9 units of FAM, the maximum quenching efficiency was achieved with FRET Dendrimer probe (SVC-01-186).

[0160] The different compounds were also evaluated according to the fold change in fluorescent signal in presence of MMP-9 and MMP-13, and the results are shown in Table 7. From this comparison, the FRET dendrimers comprising SEQ ID NO.1 and SEQ ID NO.7 (AMF-106 and SVC-186) together with the 9Dendrimer comprising SEQ ID NO.7 (SVC-196) were chosen for further evaluation. These dendrimers are shown in FIG. 7, probe A (AMF-106), probe B (SVC-186) and probe C (SVC-196).

TABLE-US-00007 TABLE7 Probe Probe Probe MMP-9 MMP-13 code sequence scaffold (83kDa) (52kDa) AMF-111 (-G-P-K-G-L-K-G-) FRETmonomer 3.66 10.57 TWB140 3Dendrimer 1.06 1.47 AMF-106 FRETdendrimer 11.20 34.52 AMF-92 (-P-F-G-Nle-K-A-) FRETmonomer 4.75 19.30 SVC-188 3Dendrimer 1.41 1.80 SVC-186 FRETdendrimer 6.49 26.65 SVC-189 6Dendrimer 2.92 7.11 SVC-196 9Dendrimer 3.33 11.80 SVC-195 FRET9Dendrimer 4.84 9.00

[0161] We have assessed and validated the efficacy of FRET or self-quenched probes by including up to three fluorophores or two quenchers for each probe element and have also assessed this with a -amino acid variant designed to enhance stability and specificity.

[0162] We have assessed a series of alternative quenchers, MethylRed, BHQ-1/3, or QSY-21, with the aim of producing a higher signal-to-noise ratio, which may enable lower levels of MMP detection at lower effective probe concentrations.

Improve the Specificity and Resistance to Proteolytic and Plasmin Degradation.

[0163] We have assessed a number of compounds including variants incorporating D-amino acids and -amino acids at selected positions identified by MALDI analysis of the parent SVC-01-024 and TWB-140 compound as sites susceptible to plasmin degradation. Also, to reduce degradation without altering the amino acid constituents we have synthesised variants including PEG units at the amino and/or carboxy termini in order to block degradation from the ends of the peptide sequence.

[0164] Several inhibitors with IC.sub.50s in the range 3-5 nM were tested in combination with the probes A and B. The fluorescent intensity was significantly reduced after incubation with Marimastat. The knock-down when the enzyme is previously incubated with known MMP inhibitors demonstrates that selective proteolytic activity by MMP is responsible for probe activation (FIG. 9).

[0165] An appropriate model to evaluate the utility of our probes was established by measuring the expression of MMP-9 in the samples of sheep and human lung tissue homogenates using gelatin zymography (FIG. 10). This model is discussed in more detail in the Molecular probe response in an ex-vivo assay section below.

[0166] All compounds have undergone biological assessment. For stability we have assessed each compound in the presence of 0.9% NaCl (Saline) or pooled lavage fluid from patients with acute lung injury and analysed by matrix-assisted laser desorption/ionization (MALDI) or fourier transform mass spectrometry (FTMS). In vitro activation was assessed on benchtop confocal in the presence of compound with lung tissue samples. For the ex vivo and in vivo ovine lung (FIG. 11) and human lung tissue experiments (FIG. 12) each compound was assessed in a control lung segment (instilled with 2 ml PBS) or a fibrotic/cancer segment, the compound was administered to the segment of interest and this was imaged by probe based fCFM.

[0167] Assessment of the different MMP probe variants has illustrated the different mechanistic factors affecting the function of the probe in the lung environment, and specifically clarified the reasons why, out of all the MMP probe variants, only SVC-01-186 (probe B) is able to activate in the presence of MMP-2/9/13 in the lung and is stable in plasmin environment.

[0168] The alternative scaffolds were inferior to the design of SVC-01-186, SVC-01-196, and AMF-106 for this application. A probe variant (SVC-01-198) with 2 fluorophores near the C-terminus exhibited a much lower intensity upon activation by MMP and the presence of an additional quencher (SVC-01-187) did not further improved signal-to-noise ratio.

In Vitro Biolociical Studies:

[0169] Specificity of lead molecular probes

[0170] Three selected probes (A: AMF-106, B: SVC-186 B, and C: SVC-196) were evaluated in vitro in a new experiment including other members of the MMP family (MMPs -1, -2, -3, -7, -8, -9, -10, -11, -12, -13) as well as Thrombin, Plasmin and Factor Xa, and the results are shown in FIG. 8. Probe A (AMF-106) and B (SVC-01-186) showed better selectivity for MMP-2/9 compared with probe C that provided also lower signal-to-noise (fold change above the background) and poor selectivity. While probe A is susceptible to plasmin, probe B is plasmin resistant. The plasmin cleavage site was identified and attempts to make a resistant version replacing with D-aa in these positions were unsuccessful.

[0171] Enzyme kinetics of the molecular probe specificity to human MMP-2, MMP-9, MMP-13 and plasmin. The kinetic constants K.sub.M and V.sub.max were calculated for probe A and B (Table 8 below).

TABLE-US-00008 TABLE 8 Probe .sup.A Probe .sup.B V.sub.t=0 V.sub.t=0 V.sub.max K.sub.m [10 M] V.sub.max K.sub.m [10 M] MMP-2 5.66 0.06 5.62 45.01 0.41 39.10 MMP-9 27.10 0.53 27.75 43.97 0.59 39.61 MMP-13 11.65 0.22 11.40 20.56 0.44 17.97 Plasmin 14.43 0.36 13.93 1.46 0.19 1.05

Validation of the Probe:

[0172] Several inhibitors with IC.sub.50s in the range 3-5 nM were tested in combination with the probes A and B and the results shown in FIG. 9. AZD1236 and also hydroxamate-based MMP inhibitors such as MMP-9-inhibitor I or Marimastat were used. Marimastat is a potent broad-spectrum inhibitor of all the major MMPs and one of the most advanced MMP inhibitors in terms of preclinical and clinical development, with IC.sub.50 values of 3 and 6 nM for MMP-9 and MMP-2 [Pharmacol. Ther. Vol. 75, No. 1, pp. 69-75, 1997]. This group of inhibitors contain a hydroxamate group that binds the zinc atom in the active site of the MMP enzyme.

[0173] The fluorescent intensity was significantly reduced after incubation with Marimastat. The knock-down when the enzyme is previously incubated with known MMP inhibitors demonstrates that selective proteolytic activity by MMP is responsible for probe activation.

Molecular Probe Response in an Ex-Vivo Assay

[0174] The expression of MMP-9 was analysed in several samples of sheep and human lung tissue homogenates using gelatin zymography (FIG. 10) in the presence or absence of inhibitor. After confirming the presence of variable amounts of MMP-9 in the different samples as previously predicted this homogenised lung tissue was considered an appropriate model to evaluate the utility of our probes.

[0175] Furthermore, the haemolytic activity in human blood and preliminary toxicity in animals was assessed for molecular probes, which confirmed the safety and non-toxicity of the compounds (FIG. 13).

Materials and Methods

General

[0176] Commercially available reagents were used without further purification. NMR spectra were recorded using Bruker AC spectrometers operating at 500 MHz for 1 hour. Chemical shifts are reported on the 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 chromatography (RP-HPLC) was performed on an HP1100 system equipped with a Discovery C18 reverse-phase column (5 cm4.6 mm, 5 m) with a flow rate of 1 mL/min and eluting with H.sub.2O/CH.sub.3CN/HCOOH (95/5/0.1) to H.sub.2O/CH.sub.3CN/HCOOH (5/95/0.1), over 6 min, holding at 95% ACN for 2 min, with detection at 254, 495 nm and by evaporative light scattering. Semi-preparative RP-HPLC was performed on an HP1100 system equipped with a Zorbax Eclipse XDB-C18 reverse-phase column (2509.4 mm, 5 m) with a flow rate 2.0 mL/min and eluting with 0.1% HCOOH in H.sub.2O (A) and 0.1% HCOOH in CH.sub.3CN (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 H.sub.2O/CH.sub.3CN/TFA (50/50/0.1).

Methods of Synthesis of Probes

Synthesis of Linear MMP Probes (FAM-PEG.SUB.2.-G-P-K-G-L-K-G-K(MethylRed)-NH.SUB.2. and FAM-PEG.SUB.2.-P-F-G-Nle-K-A-K(MR)-N H.SUB.2.)

[0177] ##STR00001##

Compound 1

[0178] [FAM-PEG.sub.2-G-P-K-G-L-K-G-K(MethylRed)-NH.sub.2 (SVC-01-24)] fragment was synthesised on solid-phase employing Fmoc-strategy, with standard amino acid coupling cycles with DIC and oxyma in peptide grade DMF at 0.1 M reagent concentration. Fmoc deprotection steps were done in 20% piperidine in DMF (25 min). Between each step, the resin was washed with DMF, DCM and MeOH.

[0179] The resin was synthesized using a 4-[(2,4-dimethoxyphenyl)-(Fmoc-amino)methyl]phenoxyacetic acid (Rink amide linker) attached to aminomethyl PS resin (1.6 mmol/g, 1% DVB, 100-200 mesh). Thus, Fmoc-Rink-amide linker (2.6 g, 4.8 mmol) was dissolved in DMF (16 ml) and HOBt (0.7 g, 4.8 mmol) was added and the mixture was stirred for 10 min. DIC (0.7 ml, 4.8 mmol) was then added and the resulting mixture was stirred for further 5 min. The solution was added to aminomethyl polystyrene resin (1 g, 1.6 mmol/g) and shaken for 2 h. The resulting resin was washed with DMF (310 ml), DCM (31 0 ml) and MeOH (31 0 ml). After washing and Fmoc deprotection, the Fmoc-PEG.sub.2-G-P-K-G-L-K-G-K(Dde) sequence was synthesised as described above using, Fmoc-Lys(Dde)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Pro-OH and Fmoc-PEG.sub.2-OH.

FAM and MethylRed Dye Coupling:

[0180] After Fmoc deprotection, 5-carboxyfluorescein (FAM) was coupled to the N-terminus as described above. After the sequence was completed, Dde protecting group was removed with 2% hydrazine in DMF (510 min) followed by the coupling of Methyl Red to the Lys side chain as described above. The coupling reactions were monitored by a ninhydrin test (E. Kaiser, R. L. Colescott, C. D. Bossinger and P. I. Cook, Analytical Biochemistry, 1970, 34, 595-598).

Sulfo-Cy5 Dye Coupling:

[0181] A solution containing sulfo-Cy5 (1 eq.) in anh (anhydrous) DMF (10 mg/ml) was activated with N,N,N,N-Bis(tetramethylene)-O(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 room temperature (rt) overnight. The solution was drained and the resin washed with DMF until colourless wash solution, DCM (35 ml) and MeOH (35 ml).

QSY21 Coupling:

[0182] N-terminal capping with QSY21-NHS ester (1 eq.) 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 (35 ml), MeOH (35 ml) and finally ether (35 ml).

[0183] Before cleavage, the resin was washed with 20% piperidine to remove any fluorescein phenol esters. After washing, the fragment was cleaved off the resin with TFA-TIS-H.sub.2O (95:2.5:2.5) (90 min) and precipitated with cold ether to give SVC-01-24 (MALDI-ToF m/z: 1538.1988, >95% HPLC purity, t.sub.R=6.357 min).

[0184] By following the above procedure, Compound 2 [FAM-PEG.sub.2-P-F-G-Nle-K-A-K(MR)-NH.sub.2 (AMF-025)] was synthesised using Fmoc-Lys(Dde)-OH, Fmoc--Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Nle-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, and Fmoc-PEG.sub.2-OH followed by coupling of FAM and MethylRed. After washing, the fragment was cleaved off the resin with TFA-TIS-H.sub.2O (95:2.5:2.5) (90 min) and precipitated with cold ether to give Compound 2 (MALDI-ToF m/z: 1514.6800; >95% HPLC purity, t.sub.R=6.330 min).

Synthesis of Multi-Fluorophore Linear Probe Scaffold

[0185] ##STR00002##

##STR00003##

[0186] Manual peptide synthesis was performed on Aminomethyl-ChemMatrix resin using Rink amide linker.

Coupling of Rink Amide Linker:

[0187] 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 L, 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 (310 mL), DCM (310 mL) and MeOH (310 mL). Finally the resin was treated with Ac.sub.2O: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 (310 mL), DCM (310 mL) and MeOH (310 mL). Resin loading was calculated after that as 0.58 mmol/g.

Fmoc Deprotection:

[0188] In general, to the resin pre-swollen in DCM was added 20% piperidine in DMF and stirred at rt (210 min). The solution was drained and the resin washed with DMF (310 mL), DCM (310 mL) and MeOH (310 mL). In the cases were Fmoc deprotection was done in Cy5 containing peptides, a solution of 2% DBU in DMF (210 min, rt) was used instead.

Aminoacid Coupling:

[0189] A solution of the appropriate D- or L-amino acid (3.0 eq per amine) and Oxyma (3.0 eq) in DMF (0.1M) 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 (310 mL), DCM (310 mL) and MeOH (310 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:

[0190] Coupling of {2-[2-(Fmoc-amino)ethoxy]ethoxy}acetic acid (PEG), 5-Carboxyfluorescein (FAM) and Fmoc-Lys(N.sub.3)OH was done following the same procedure described for Aminoacid coupling.

Dde Deprotection:

[0191] To the resin pre-swollen in DCM was added 2% hydrazine in DMF and stirred at rt (510 min). The solution was drained and the resin washed with DMF (310 mL), DCM (310 mL) and MeOH (310 mL).

MethylRed-NHS Coupling:

[0192] MethylRed-NHS ester (1 eq) coupling in solid phase was done in anhDMF (0.1M) containing DI PEA (3 eq) at rt for 12 h. The solution was drained and the resin washed with DMF until colourless wash solution, DCM (35 mL), MeOH (35 mL) and finally ether (35 mL).

Cleavage and Purification:

[0193] The resin pre-swollen in DCM was treated with a cleavage cocktail of TFA:triisopropylsilane(TIS):water (95:2.5:2.5) for 3 h 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 (3) and purified by RP-HPLC on a C18 semi-preparative column. The desired fraction containing the product were collected and lyophilized to afford compound AMF-209 that was characterized by MALDI and analytical HPLC: t.sub.R=4.174 min, MALDI calc. for C.sub.127H.sub.187N.sub.28O.sub.33 [M+H].sup.+: 2634.056; Found: 2634.031.

Synthesis of sulfo-Cy5-alkyne

[0194] Sulfo-Cy5-Carboxylic acid (75 mg, 0.11 mmol) was dissolved in anhDMF (8 mL). HSPyU (46 mg, 0.11 mmol) and DIPEA (58 L, 0.33 mmol) were added and the mixture stirred at 40 C. for 1 h. Propargylamine (30 mg, 0.55 mmol) was added together with DIPEA (58 L, 0.33 mmol) and the reaction was stirred overnight. The solvent was removed under vacuum. Purification by column chromatography (10:1 ACN-H.sub.2O) afforded the compound sulfo-Cy5-alkyne as a dark blue solid (55 mg, 70%). .sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 9.28 (t, J=5.5 Hz, NH), 9.23 (d, J=2.3 Hz, 1H), 8.47 (s, 1H), 8.44 (s, 1H), 8.38 (dd, J=8.1, 2.3 Hz, 1H), 7.84 (s, 2H), 7.64 (m, 3H), 7.32 (d, J=8.3 Hz, 2H), 5.84 (m, 2H), 4.15 (dd, J=5.5, 2.5 Hz, 2H), 3.34 (s, 6H), 3.20 (t, J=2.5 Hz, 1H), 1.78 (s, 12H); .sup.13C-NMR (125 MHz, DMSO-d.sub.6) : 174.2, 164.2, 157.3, 152.2, 149.0, 145.6, 142.4, 140.4, 135.9, 127.6, 125.8, 125.2, 119.7, 110.2, 100.7, 80.9, 73.0, 49.0, 31.0, 28.4, 26.7; MS (ES).sup.+ m/z 701 [M].sup.+; HPLC t.sub.R 3.921 min (GE10).

##STR00004##

Labelling with sulfo-Cy5-alkyne:

[0195] Click reaction between sulfo-Cy5-alkyne (AMF-208) and azide-FRET peptide (AMF-209) was done in solution phase: in an eppendorf tube the following aqueous reagents were mixed: azide-peptide (AMF-209) (50 L, 10 mM), sulfo-Cy5-alkyne (AMF-208) (50 L, 30 mM), premixed CuSO.sub.4 and THPTA (40 L CuSO.sub.4 20 mM and 80 L THPTA 50 mM), aminoguanidine hydrochloride (250 L, 100 mM) and finally sodium ascorbate (250 L, 100 mM). The click-chemistry reaction was allowed to proceed at 30 C. for 5 h, the reaction mixture was lyophilised and purified by HPLC to give the final FRETplus MMP-probe AMF-210, which was characterized by MALDI and analytical HPLC: AMF-210: HPLC t.sub.R=3.921 min, MALDI calc. for C.sub.163H.sub.223N.sub.32O.sub.40S.sub.2[M].sup.+: 3334.880; Found: 3334.398.

Synthesis of Branched Dendrimer Scaffold:

[0196] The dendrimer scaffold was synthesised by following the prior art reported in WO 2012/136958 A2 (Aslam et al).

Synthesis of Monomer (V)

[0197] Multi-valent probe synthesis required the preparation of the monomer (V) which was synthesised in six steps (M. Ternon, J. J. Diaz-Mochon, A. Belsom, M. Bradley, Tetrahedron, 2004, 60, 8721) as shown in Scheme 3. Monomer (V) was prepared by the 1,4 addition of the hydroxy groups of 1,1,1-tris(hydroxymethyl)amino-methane onto acrylonitrile, followed by amino group protection (Boc). Reduction of the nitrile groups with PtO.sub.2/H.sub.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 Knlker (H. J. Knlker, T. Braxmeier, G. Schlechtingen, Angew. Chem. Int. Ed., 1995, 34, 2497).

Fmoc-Rink Amide ChemMatrix Resin (VI):

[0198] Peptide synthesis was performed on Aminomethyl-ChemMatrix resin using 4-[(2,4-Dimethoxyphenyl)-(Fmoc-amino)methyl]phenoxyacetic acid (Rink amide linker) by following the procedure. Thus, Fmoc-Rink-amide (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 L, 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 (310 mL), DCM (310 mL) and MeOH (310 mL). Finally the resin was treated with Ac.sub.2O: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 (310 mL), DCM (310 mL) and MeOH (310 mL). Resin loading was calculated after that as 0.58 mmol/g. The coupling reaction was monitored by ninhydrin test as discussed above.

[0199] The probes were synthesised on a ChemMatrix resin derivatized with an Fmoc-Rink Amide type linker (Scheme 3). The linker (VI) was loaded with monomer (V) to give the tri-branched scaffold (VII). The appropriate Fmoc-spacers (Pegylation), Fmoc-solubilizers (D-amino acids) and specific sequences were coupled sequentially followed by the attachment of FAM. Following the removal of the Dde groups (2% hydrazine in DMF) the MethylRed NHS ester (MR-NHS) was coupled and the resin was wash with 20% piperdine in DMF before the probe was cleaved from the resin using TFA/TIS/DCM (90/5/5).

##STR00005##

Fmoc Rink-Amide Linker

[0200] Peptide synthesis was performed on Aminomethyl-ChemMatrix resin using Rink amide linker by following the procedure. Fmoc-Rink-amide (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 L, 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 (310 mL), DCM (310 mL) and MeOH (310 mL). Finally the resin was treated with Ac.sub.2O: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 (310 mL), DCM (310 mL) and MeOH (310 mL). Resin loading was calculated after that as -0.58 mmol/g.

General Procedure for the Fmoc Deprotection

[0201] To the resin (pre-swollen in DCM) was added 20% piperidine in DMF (5 mL) and the reaction mixture was shaken for 10 min. The solution was drained and the resin was washed with DMF (310 mL), DCM (310 mL) and MeOH (310 mL). This procedure was repeated twice. The coupling reaction was monitored by the ninhydrin test as described above.

Isocyanate Coupling to Give (VII)

[0202] To resin (0.30 mmol), pre-swollen in DCM (10 mL), was added a solution of isocyanate (6) (920 g, 0.93 mmol), DIPEA (0.2 mL, 0.93 mmol) and DMAP (22 mg, 0.17 mmol) in a mixture of DCM/DMF (1:1, 5 mL) and the mixture was shaken overnight and the reaction monitored by ninhydrin test. The solution was drained and the resin was washed with DMF (320 mL), DCM (320 mL) and MeOH (320 mL) and ether (320 mL). (320 mL). The coupling reaction was monitored by the ninhydrin test as described above.

Pegylation ({2-[2-(Fmoc-amino)ethoxy]ethoxy}acetic acid or PEG.sub.2-OH)

[0203] A solution of PEG.sub.2-OH (3.0 eq per amine, 0.1M) and Oxyma (3.0 eq, 0.1M) in DMF was stirred for 10 min. DIC (3.0 eq, 0.1M) 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 (310 mL), DCM (310 mL) and MeOH (310 mL).

Peptide Synthesis

Peptide Sequence: -P-F-G-Nle-K-A-

[0204] A solution of the appropriate Fmoc-amino acid (3.0 mmol, 10 eq) [Fmoc-Lys(Dde)-OH, Fmoc--Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Nle-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Pro-OH] and Oxyma (3.0 mmol, 10 eq) was added and the mixture was stirred for 5-10 min. DIC (3.0 mmol, 10 eq) was then added and the resulting mixture was stirred for a further 2 min. The solution was added to pre-swollen resin in DCM and the reaction mixture was mixed for 0.5 h at 60 C. The solution was drained and the resin washed DMF (320 mL), DCM (320 mL) and MeOH (320 mL). The coupling reactions were monitored by ninhydrin test as discussed above.

5-carboxyfluorescein (FAM) Labelling

[0205] A solution of FAM (3-10 eq) and oxyma (3-10 eq) in DMF (0.1 M) was stirred for 10-15 min followed by DIC (3-10 eq) and the resulting solution was stirred for further 1-2 min. This solution was added to the appropriate resin (1 eq), pre-swollen in DCM, and the reaction mixture was stirred at room temperature or 60 C. for 0.5-1 h. The solution was drained and the resin washed with DMF (3), DCM (3) and MeOH (3). The coupling reactions were monitored by ninhydrin test. Before cleavage, the resin was washed with 20% piperidine to remove any fluorescein phenol esters.

Dde Deprotection

[0206] Selective Dde deprotection was done with a solution containing Imidazole (1.35 mmol) and Hydroxylamine hydrochloride (1.80 mmol) in NMP (5 mL). [Diaz-Mochn, J. J.; Bialy, L.; Bradley, M. Org. Lett. 2004, 6 (7), 1127-1129]. After complete dissolution 5 volumes of this solution were diluted with 1 volume of CH.sub.2Cl.sub.2 and the resin was treated with the final mixture for 3 h at room temperature. The solution was drained and the resin washed with DMF (310 mL), DCM (310 mL) and MeOH (310 mL).

MethylRed (MR) NHS Ester Labelling

[0207] A solution of MR-NHS ester (3-10 eq) and DIPEA (3-10 eq) in DMF (0.1 M) was added to the appropriate resin (1 eq), pre-swollen in DCM, and the reaction mixture was stirred at room temperature or 60 C. for 0.5-1 h. The solution was drained and the resin washed with DMF (3), DCM (3) and MeOH (3). The coupling reactions were monitored by ninhydrin test as described above.

7-Nitrobenzofurazan (NBD) Coupling

[0208] To a solution of NBD-PEG-NHS (3.0 mmol, 10 eq) in DMF (3 mL) was added DI PEA (3.0 mmol, 10 eq). The resulting solution was added to resin (1 eq), pre-swollen in DCM, and the reaction mixture was shaken for 0.5 h. The solution was drained and the resin washed with DMF (3), DCM (3) and MeOH (3). The coupling reaction was monitored by ninhydrin test as described above.

TFA Cleavage and Purification of Reporter Compound 4 (SVC-01-188)

[0209] The resin, pre-swollen in DCM, was treated with a cleavage cocktail of TFA/TIS/DCM (90/5/5, 0.5 mL) for 1.5 h. The solution was drained and the resin was washed with the cleavage cocktail and added to ice-cold ether (7.5 mL). The precipitated solid (22 mg) was collected by centrifugation and the solvent removed by decantation and the precipitate was washed with cold ether (35 mL). The precipitate was then purified by preparative reverse phase HPLC and the desired fractions were pooled and lyophilized to afford products that were characterized by MALDI and analytical HPLC.

##STR00006## ##STR00007## ##STR00008## ##STR00009## ##STR00010## ##STR00011##

MALDI-TOF:

[0210] Probe was added to saline or pooled BALF from patients with fibrosis and incubated for 30 minutes. A ZipTip (C-18, 0.2 L) with 5 L MeCN (with 0.1% TFA as an additive) followed by 20 L of H.sub.2O was washed. The ZipTip was loaded with the sample, washed and eluted into 5 L of 80% aq. MeCN (with 0.1% TFA as an additive). The sample was analysed by MALDI TOF/TOF (Bruker Ultraflextreme mass spectrometer).

Compound 10:

[0211] Polymerisable probe SVC-01-180 (SVC-180). The specificity of the polymerisable probe SVC-180 (SVC-01-180) was tested with MMP-9 and MMP-13 and compared with FRET monomer AMF-92. The results are shown in FIG. 15.

[0212] By following the procedure for the synthesis of Compound 1, FAM was introduced on the probe by selective Dde group deprotection, followed by coupling of last Fmoc-Lys(Dde)-OH to finish the probe. MethylRed was introduced on N-terminus and finally the polymerisable acryloyl moiety was coupled to complete the synthesis of probe on solid support. After washing the resin with 20% piperidine in DMF followed by DCM, the fragment was cleaved off the resin with TFA-TIS-H.sub.2O (95:2.5:2.5) (90 min) and precipitated with cold ether to give Compound 10 (MALDI-ToF m/z: 2265.7; >95% HPLC purity, t.sub.R=4.178 min).

Acryloyl Coupling:

[0213] N-terminal capping with acryloyl-NHS ester (1 eq.) was done in anhDMF (0.1M) containing DI PEA (3 eq.) at rt for 12 h. The solution was drained and the resin washed with DMF (35 ml), DCM (35 ml), MeOH (35 ml) and finally ether (35 ml).

Compound 11:

[0214] The polymerisable probe compound 10 (SVC-01-180) was polymerised in water to form a hydrogel. The hydrogel obtainable by the polymerisation of monomer compound 10 can be represented by the polymeric structure:

[0215] The polymerisation can be carried out in the presence of a sugar to increase the porosity of the resulting hydrogel. Increasing the porosity improves the access of the enzyme to the polymerised probe.

Acrylamide Polymerisation:

[0216] A) Hydrogel monomer solution was prepared by mixing acrylamide (1.20 g) PEGDA 700 (100 L), TMED (10 L) in distilled water (3.20 mL) and degassed by purging with N.sub.2 for 10 min;
B) Probe monomer solution was prepared by mixing polymerisabable probe (25 mg) in distilled water (1 mL) and degassed by purging with N.sub.2 for 10 min;
C) The monomer solution (500 L) and ammonium persulfate or potassium persulfate (10% in water, 60 L), and distilled water (250 L) were mixed with or without sugar/salt (50 L, 50% in distilled water) and polymerisable probe solution (50 L) for 1 min. The resulting solution (100 L/well) was transferred to a 96 well plate and allowed to polymerise at 40 C. After 12 h, the excess of monomer solution was washed away with distilled water until the washings are clear (5100 L/well). The hydrogels were allowed to dry at 40 C. for 12 under vacuum protecting from direct light.

[0217] A control hydrogel was prepared without probe solution by following the above procedure.

[0218] The polymerised probe hydrogel was tested for cleavage by MMP-9 and the results are shown in FIG. 16. Since the probe is prepared already immobilised as hydrogel in the 96 well plate, the MMP-9 enzyme (39 and 83 kDa, 5-30 nm in buffer) was added to the wells and the fluorescence from cleaved fragment released in buffer was measured from the control and probe containing hydrogel wells using a plate reader.

Animals

[0219] Adult (25-35 g) specific pathogen-free BALB/c and CD1 wild type mice were used. All studies were done under UK Home Office license 60/4434.

Enzyme Assay

[0220] 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 (50 mM Tris, 10 mM CaCl.sub.2, 0.15M NaCl, 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 485/528 nm. 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 30 nM. Pro-MMP-13 (R & D Systems) was activated by incubating with 1 mM 4-aminophenylmercuric acetate (APMA) for 2 hrs at 37 C. Human neutrophil elastase (Elastase Product Company, used at 2.5 ug/ml), neutrophil lysate (lysed human neutrophils), and recombinant human Thrombin (Sigma-Aldrich, used at 5 U/ml), Plasmin (Sigma-Aldrich, used at 30 nM) and Factor Xa (Sigma-Aldrich, used at 0.5 M) 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 200 nM for in-vitro and 50 M for ex-vivo assays, respectively.

Human and Ovine Tissue Supernatant

[0221] 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 an Ovine Pulmonary Adenocarcinoma-(OPA) animal 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. Samples were centrifuged at 13000 rpm for 15 min at 4 C. and the debris-free supernatant collected. Total protein concentrations were determined using at Pierce BCA kit (Thermo Scientific). The samples were aliquoted and stored at 20 C. or 70 C. until further analysis.

Gelatin Zymography

[0222] To assess the MMP activity in human and ovine tissue, gelatin zymography (Novex, Life technologies) was performed. Total protein concentrations of tissue supernatants were standardised (1 g/l, Human and 1.5 g/l Ovine) and 20 l of supernatant mixed with 20 l of 2 Tris-Glycine SDS sample buffer without boiling. 15 l of mixed samples were then electrophoresed for 90 min, 150V at 4 C. on 10% Tris-Glycine gel containing 0.1% gelatin. After electrophoresis, the gel was washed with deionised water at room temperature to remove SDS. The gel was suspended in 1 Renaturing Buffer for 90 min at 4 C. before being developed (1 Developing buffer) overnight at 37 C. in Developing Buffer with or without inhibitor (50 M Marimastat, pan-MMP inhibitor). The gel was washed, fixed and stained with the Colloidal Blue Staining Kit (Novex, Life technologies) for 3 hrs at room temperature, and destained with deionised water at room temperature. Areas of protease activity appear as a clear band against a dark background.

Haemolytic Toxicity Assay

[0223] The haemolytic activities of the molecular probes were evaluated in human red blood cells using a previously reported method (Lequin et al., 2006; Lequin, O. et al. Dermaseptin S., Biochemistry 45, 468-480 (2006)). Fresh human red blood cells were incubated with molecular probe (10 M final concentration) at 37 C. for 45 min. After centrifugation (350 g for 10 min), the supernatant absorbance was measured at 350 nm. In addition, control samples for 0 and 100% haemolysis were incubated with 0.9% (w/v) NaCl (negative control) or Triton-X-100 (positive control), respectively. Percent haemolysis was calculated according to the following eq: Haemolysis (%)=(Probe/Positive control)100. Each measurement was taken in duplicate. The HC.sub.50 was defined as the mean probe concentration that produced 50% haemolysis.

Statistical Analysis

[0224] The statistical values are expressed as mean standard error of the mean (SEM). The statistical analyses were performed using Microsoft Excel, and the datasets were tested using Student's t-test. Indications of significance correspond to p<0.05 (*) and p<0.01 (**).