WO2019117812A1

WO2019117812A1 - Near infra-red molecular probes for use in diagnosis of fibrotic conditions and screening of anti-fibrotic drugs

Info

Publication number: WO2019117812A1
Application number: PCT/SG2018/050608
Authority: WO
Inventors: Kanyi Pu; Chenjie Xu; Qingqing MIAO; Chen Loong David YEO
Original assignee: Nanyang Technological University
Priority date: 2017-12-12
Filing date: 2018-12-12
Publication date: 2019-06-20

Abstract

We disclose a compound of formula I: [X]n-[Y]m-Z where X represents a fluorophore group covalently linked to the rest of the molecule by way of an oxygen atom or an NH group; Y is a self-immolative linking group; Z represents an N-blocked dipeptide Gly-Pro; n is 1 -2; and m is 0-1. The fluorophore X does not fluoresce when attached to the rest of the molecule by way of a covalent bond, but is capable of fluorescence following cleavage of said covalent bond. The compounds of formula I are useful as a fluorescent probe in the diagnosis of fibrotic conditions in vivo, such as keloidal and/or hypertrophic scarring, and may also be used in an in vitro test to screen for compounds useful to treat such fibrotic conditions. Also disclosed herein is the use of RepSox and thiazovivin, whether alone or in combination, to treat fibrotic conditions.

Description

Near Infra-Red Molecular Probes for Use in Diagnosis of Fibrotic Conditions and

Screening of Anti-Fibrotic Drugs

Field of Invention

The current invention relates to molecular probes that can be used in the fields of diagnosis and screening. The current invention also relates to the discovery of anti-fibrotic activity in a number of known compounds, where such activity was not known.

Background

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Abnormal scarring is a result of aberrant wound healing and may develop following any insult to the deep dermis like burn injury, lacerations, abrasions, surgery, piercings and vaccinations. In the developed world alone, a total of 100 million patients develop scars each year as a result of 55 million elective surgeries and 25 million operations after trauma. Therefore, the detection of skin diseases at an early stage is critical to their timely treatment. For example, as keloids, which are a fibrous scar lesions, overgrow their wound boundaries due to over-exuberant healing following skin injury they can cause limited joint mobility, psychological distress and significant pain and itch to those afflicted. Current keloid management strategies such as injection of steroids and/or anti-proliferative drugs, surgical excision and radiotherapy are typically performed following visual assessment in the clinic. Therapy on mature keloids is less effective compared to preventing immature keloids from progressing which risks aggravation or recurrence. The difficulties in treating mature keloid scars necessitates the development of specific and sensitive diagnostic tools for their detection.

In addition to visual assessment, diffuse reflectance spectroscopy has been used to distinguish collagen alignment in keloids from healthy skin morphology. However, because it simply relies on the intrinsic signals from clusters of endogenous biomolecules, this method is insensitive and only limited to imaging mature keloids. Thus, there remains a need for improved and more sensitive diagnostic tools for the in vivo detection of keloid biomarkers. There have been few therapeutic advances over the last few decades in the treatment of keloids and other fibrotic conditions. Used individually, corticosteroid injections and surgical excisions (common first-line options) are ineffective (leading to recurrence), causing pain, psychological distress and inconvenience to patients. Alternative modalities such as radiotherapy, laser therapy and pressure dressing are limited by malignancy, efficacy and adherence concerns. In this light, novel therapeutics with high specificity and efficacy by targeting specific molecular pathways are highly desired.

Drug discovery and development involves the screening of compounds for their specificity and efficacy. There is currently no dedicated screening method to identify drugs for abnormal scarring. Drug screening from a library of compounds can be performed through mass spectrometry, genomic/genetic analysis, cell reporter genes and even computational modeling. However, these methods are limited by the requirement for specialist training (i.e. computational modeling) as well as laborious processes. Thus, there also remains a need for such a dedicated screening test.

Summary of Invention

It has been surprisingly found that specific fluorescent molecular probes can be used to detect fibrotic conditions in vivo and can also be used to identify drug candidates.

Thus, in a first aspect of the invention there is provided a compound of formula I:

[X]_n-[Y]_m-Z I

wherein:

X represents a fluorophore group covalently linked to the rest of the molecule by way of an oxygen atom or an NH group;

Y is a self-immolative linking group;

Z represents a peptide group of formula la:

la where:

the wavy line represents the point of attachment to the rest of the molecule;

R¹ represents Ci-e alkyl or OR²;

R² represents Ci-e alkyl that is unsubstituted or substituted by one or more groups selected from halo, C1 -3 alkyl and aryl;

n is 1 or 2; and

m is 0 or 1 , or pharmaceutically acceptable salts or solvates thereof, provided that:

when m is 0 then n is 1 ; and

when X represents a fluorophore group covalently linked to the rest of the molecule by way of an oxygen atom then m is 1.

In embodiments of the invention, X may be independently selected from the group consisting of:

where the wavy line represents the point of attachment to the rest of the molecule.

In particular embodiments mentioned herein, X may be independently selected from the group consisting of:

5 In embodiments of the invention, Z may be independently selected from the group consisting of:

In embodiments of the invention, when present, Y may be independently selected from the group consisting of:

; and

In particular embodiments mentioned herein, Y may be independently selected from the 5 group consisting of:

In particular embodiments, the compound formula I may be selected from:

In more particular embodiments, the compound of formula I may be selected from:

It will be appreciated that embodiments of the first aspect may be combined in any technically sensible manner.

In a second aspect of the invention, there is provided a method of detecting a fibrotic condition in a subject, the method comprising the steps of providing a compound of formula I, as defined in the first aspect and any technically sensible combination of its embodiments, or a pharmaceutically acceptable salt or solvate thereof to a subject, subjecting a tissue or organ suspected of suffering from a fibrotic condition to irradiation with light and detecting fluorescence from the irradiated tissue or organ, wherein an increase in fluorescence compared to a control indicates the presence of a fibrotic condition. In a third aspect of the invention, there is provided a compound of formula I, as defined in the first aspect and any technically sensible combination of its embodiments, or a pharmaceutically acceptable salt or solvate thereof for use in the detection of a fibrotic condition. In a fourth aspect of the invention, there is provided a use of a compound of formula I, as defined in the first aspect and any technically sensible combination of its embodiments, or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for use in the detection of a fibrotic condition.

In embodiments of the second to fourth aspects of the invention, the fibrotic condition may be keloidal scarring and/or hypertrophic scarring.

In a fifth aspect of the invention, there is provided a cell based method for the identification of compounds suitable to treat a fibrotic condition, the method comprising:

(a) providing cells expressing a fibrotic condition;

(b) contacting the cells with a test compound for a first period of time;

(c) after the first period of time, contacting the cells with a compound of formula I, as defined in the first aspect of the invention, or any technically sensible combination of its embodiments, or a pharmaceutically acceptable salt or solvate thereof for a second period of time;

(d) after the second period of time, irradiating the cells with light and quantitatively measuring the fluorescence, wherein a reduced quantitative fluorescence readout in the presence of a test compound compared to a blank is indicative of antifibrotic activity. In embodiments of this aspect, the fibrotic condition may be keloidal scarring and/or hypertrophic scarring.

In a sixth aspect of the invention, there is provided a method of treating and/or preventing fibrotic scarring in a subject, the method comprising the steps of providing a therapeutically effective amount of RepSox and/or thiazovivin, or a pharmaceutically acceptable salt or solvate thereof to a subject in need thereof.

In a seventh aspect of the invention, there is provided a use of RepSox and/or thiazovivin, or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for use in treating fibrotic scarring.

In an eighth aspect of the invention, there is provided a RepSox and/or thiazovivin or a pharmaceutically acceptable salt or solvate thereof for use in treating fibrotic scarring.

In embodiments of the sixth to eighth aspects of the invention, the fibrotic condition may be keloidal scarring and/or hypertrophic scarring. Drawings

Figure 1. (a) Design and mechanism of FNPs for imaging of FAPa. (b) Synthesis of FNP1 and FNP2. Reagents and conditions: (i) resorcinol, K2CO₃, acetonitrile (ACN), 50 °C, 6 h; (ii) triphosgene, anhydrous dichloromethane (DCM), 25 °C, 0.5 h; (iii) A/,/\/-disuccinimidyl carbonate, A/,/\/-Diisopropylethylamine (DIPEA), anhydrous ACN, 25 °C, overnight (iv) N,N’- dimethylethylenediamine, DIPEA, anhydrous tetrahydrofuran (THF), 25 °C, 8 h; (v) CyOOCI, K2CO₃, anhydrous DCM, 25 °C, 5 h.

Figure 2. UV-Vis absorption spectra (a) and fluorescence (b) of FNP1 or FNP2 (10 mM) in the absence or presence of FAPa (9.0 x 10^-4 U mL·¹) for 25 min at 37 °C in HEPES buffer (50 mM, pH = 7.4) containing BSA (1 mg/ml_) and glycerol (5%). Excitation: 660 nm. Inset: white light (a) and fluorescence (b) images of FNP1 (left panel) or FNP2 (right panel) (10 pM) in the absence or presence of FAPa (9.0 x 10^-4 U mL·¹) for 25 min at 37 °C in HEPES buffer (50 mM, pH = 7.4) containing 1 mg/ml_ BSA and 5% glycerol. The fluorescence images were acquired at 720 nm upon excitation at 640 nm. High performance liquid chromatography (HPLC) traces of the incubation mixture of FNP1 (c) or FNP2 (d) in the absence (upper panel) or presence (middle panel) of FAPa (9.0 x 10^-4 U mL·¹), and HPLC traces of CyOH in water (lower panel). Wavelength: 600 nm. (e) Kinetics studies of FNP1 and FNP2 towards FAPa. (f) Fluorescence intensities of FNP1 (5.5 pM) at 710 nm after incubation with indicated enzymes for 30 min at 37 °C. Excitation: 660 nm.

Figure 3. Time-dependent UV-Vis spectra of FNP1 (a) or FNP2 (c) (10 pM) after incubation with FAPa (9.0 x 10⁴ U mL·¹) at 37 °C in HEPES buffer (50 mM, pH = 7.4) containing 1 mg/mL BSA and 5% glycerol (b) The ratio of the absorption intensity at 682 nm to that at 590 nm (A682/A590) of FNP1 after incubation with different concentrations of FAPa as a function of incubation time (d) HPLC traces of the incubation mixture of FNP2 in the absence (upper) or presence (middle) of FAPa (9.0 x 10^-4 U mL^-1) for 120 min, and HPLC traces of CyOH in water (lower).

Figure 4. Non-linear regression analysis of FNP1 (a) or FNP2 (b) cleavage rate V (nmol min ¹) as a function of FAPa concentration. The error bars represent the standard deviation from three separate measurements.

Figure 5. UV-Vis absorption spectra (a), white-light (b, upper panel), fluorescence (b, lower panel) images, and HPLC traces (c) of FNP1 (5.5 pM) in the absence (Blank) or presence of FAPa (9.0 x 10⁴ U mL·¹) in HEPES buffer (50 mM, 1 mg/mL BSA, 5% glycerol, pH = 7.4), talabostat, inhibitor (100 mM) with FAPa (9.0 x 10 ⁴ U mL·¹) in HEPES buffer (50 mM, 1 mg/ml_ BSA, 5% glycerol, pH = 7.4), DPPIV (1.2 x 10³ U mL·¹) in HEPES buffer (25 M, pH 8.0), MMP-1 (3.4 x 10⁴ U mL·¹) in Tris buffer (50 mM, 250 mM NaCI, 10 mM CaCI₂, pH 7.4), MM P-2 (8.4 x 10-⁴ U mL·¹) in Tris buffer (50 mM, 250 mM NaCI, 10 mM CaCI₂, pH 7.4), MMP-13 (5.0 x 10-⁴ U mL·¹) in Tris buffer (50 mM, 250 mM NaCI, 10 mM CaCI₂, pH 7.4), Caspase-3 (1.0 x 10³ U mL·¹) in HEPES buffer (50 mM, 50 mM NaCI, 0.1 % Chaps, 10 mM EDTA, 5% Glycerol, 1 mM DTT, pH 7.4) or tPA (3.8 x 10³ U mL·¹) in Tris buffer (50 mM, pH 8.5) at 37 °C for 30 min. (d) The percentage of FNP1 activation into CyOH after incubation with FAPa (9.0 x 10^-4 U mL·¹) and talabostat at different concentrations at 37 °C for 30 min.

Figure 6. Human skin tissue histology (a) Cross-sectional view of skin sections implanted with KF and NDF cells, their FNP1 signal (purple signal, 710 nm) imaged with epifluorescence microscope. Έrϊ’ - epidermis layer,‘Der’ - dermis layer. Scale bar: 100 pm. (b) FNP1 signal in KF- and NDF-implanted skin tissue with respect to skin depth. *p<0.05.

Figure 7. FNP1 probe in cell culture (a) Fluorescence microscopy of KF cells after treatment with FNP1 (5 mM, purple) for 1 h and stained with nucleus indicator (Hoechst 33342, blue) for 30 min and lysosome indicator (LysoTracker™, white) for 30 min. Scale bar: 20 pm. (b) Fluorescence microscopy of HaCaT, NDF, KF and NDF cells stimulated with TGF-bI (10 ng mL·¹) after treatment with FNP1 (5 pM, purple) for 1 h and stained with nucleus indicator (Hoechst 33342, blue) for 30 min. Scale bar: 100 pm. (c) Quantification of fluorescence intensities of the cells (HaCaT, NDF, KF, NDF+TGF-bI) after incubation with FNP1 in Figure 7b using multiplate reader. The fluorescence intensities of FNP1 were normalized by total cell nuclei signal (NucBlue™). (d) Relative gene expression of FAPa in HaCaT, NDF, KF, NDF+TGF-bI normalized by GAPDH and NDF expression levels using qRT-PCR. The error bars represent the standard deviation from three separate measurements. *:p < 0.05, **:p < 0.01.

Figure 8. Cross-section view of FNP1 distribution in epidermis-removed (left) and epidermis- intact skin (right). Skin sections in Phase contrast, 461 nm (blue), 570 nm (red, green), 710 nm (purple, from left - right) fluorescence emission channels. Scale bar: 100 pm.

Figure 9. (a) Schematic illustration of microneedle-assisted penetration of FNP1 for FAPa imaging in keloid disease models (i) Skin tissue pre-treated with microneedles 10 to generate micro-channels 20 (5 min, 16.7 kPa pressure), (ii) micro-channels 20 facilitate FNP1 30 penetration, (iii) fluorescence imaging (b) Representative fluorescence imaging of unmodified skin, skin implanted with HaCaT, NDF or KF cells after treatment with FNP1 (20 mI_, 250 mM) for 6 h. (c) Quantification of fluorescence intensities of the skins from Figure 9b. The fluorescence intensities derived from FNP1 were normalized by total cell number (@ 570 nm). (d) Representative fluorescence imaging of skin implanted with different amounts of KF cells (1.5^c10⁶, 4.5^c10⁵, 1.5^c10⁵ and 4.5^c10⁴) after treatment with FNP1 (20 mI_, 250 mM) for 6 h. (e) Quantification of fluorescence intensities of the skins in Figure 9d. The fluorescence intensities (@ 710 nm) derived from FNP1 were normalized by background fluorescence. The error bars represent the standard deviation from three separate measurements. *: p< 0.05.

Figure 10. FNP1 performance validation in skin fibroblast cells (a) H&E staining of the abnormal scar tissue: vertical lines denote margins of wound/scar (S), U: unwounded skin, E: epidermis (uppermost skin layer), D: dermis (lower skin layer); (b) FNP1 signals in NDF, KF, HSF and HaCaT with Hoechst 33342 signal as the reference signal; (c) FAP-a gene expression in NDF, KF, HSF and HaCaT with GAPDH as the reference gene; (d) Correlation of FAP-a gene expression with normalized FNP1 signal. **: P<0.01.

Figure 11. FNP1 probe for accessing TQRb modulation in fibroblast cells (a) FNP1 and Hoechst staining of untreated NDF, NDF treated with 2 ng/ml TQRb1 , untreated HSF, HSF treated with 25 mM RepSox, untreated KF, and KF treated with 25mM Repsox; (b) Fluorescence quantification in (a); (c) Fluorescence quantification of FNP1 signal in NDF treated with a range of TQRb1 (0.016, 0.08, 0.4, 2, 10, 50 ng/ml); (d) Correlation of FAP-a gene expression with normalized FNP1 signal. *: P<0.05, **: P<0.01.

Figure 12. PCR quantification of FAPa mRNA expression in NDF following the TQRb1 treatment (0, 0.4, 2, 10, 50 ng/ml). FAPa mRNA expression was normalized by GAPDH mRNA expression.

Figure 13. Drug screening with FNP1 probe: Normalized FNP1 signal and cell viability under different treatment conditions for (a) HSF and (b) KF. The different drugs are grouped to negative controls that have no anti-scarring response (negative), positive controls with known anti-scarring properties (positive), and drugs with unknown effects (drug screening). FNP1 (bar graph) and viability (line graph) signals read from the left and right axis respectively. The treatment groups consist of UT (untreated), TGF (TΰRb1 , 2ng/ml), Rsox (RepSox, 25 mM), Rapa (Rapamycin, 20 nM), Dec (Decorin, 100 nM), Simv (Simvastatin, 20 mM), Perf (Pirfenidone, 50 pg/ml), Thia (Thiazovivin, 500 nM), DMSO (dimethyl sulfoxide, 2.5%), VPA (valproic acid, 500 mM), Tran (Tranylcypromine, 5 mM), PD (PD0325901 , 1 mM), FSK (forskolin, 10 mM), CHIR (CHIR99021 , 10 mM), Vitc (Vitamin C, 10 pg/ml). Figure 14. Scratch assay to study the migration of (a) HSFs and (b) KFs after the treatment with TGF-b, Rsox, and Thia. Cells were pre-labelled with Dil. The vertical lines between the respective untrested (UT) and Rsox-treated panels, respectively, represent the approximate width of the initial scratch (Time = 0).

Figure 15. Effects of newly-identified anti-scarring drugs on the cellular expression of (a) COL1A1 and (b) CTGF in KF and HSF after the treatment of Rsox, Thia, and CHIR. *:p<0.05, **:p<0.01.

Figure 16. PCR analysis of (a) COL1A1 and (b) CTGF expression in KF and HSF treated with drug candidates normalized to values from NDF (normal dermal fibroblasts).

Figure 17. Effects of newly-identified anti-scarring drugs on the expression of a-SMA protein in UT (untreated), Thia (thiazovivin)-treated, Rsox (RepSox)-treated, Simv (simvastatin)- treated HSF and KF cells. Scale bars are: 20 pm.

Figure 18. Immunostaining of a-smooth muscle actin (a-SMA) in untreated and TGF-bI treated normal dermal fibroblasts (NDFs). Scale bars are: 20 pm.

Description

Disclosed herein is the design and syntheses of near-infrared (NIR) fluorescent activatable molecular probes for the specific detection of fibroblasts (e.g. keloid-derived fibroblasts (KF)). These probes can turn on their NIR fluorescence in the presence of fibroblast activation protein-alpha (FAPa), which is overexpressed in activated (e.g. keloid) fibroblasts compared to normal fibroblasts. As a type II transmembrane serine protease, FAPa may play a role in keloids overgrowing their wound borders since it facilitates degradation of extracellular matrix (ECM) components such as gelatin and type I collagen, which correlates with the “invasiveness” of keloids. Said marker may play a role in other fibrotic conditions and so may also be useful in detecting such conditions too. Thus, in a first aspect of the invention there is provided a compound of formula I:

[X]n-[Y]m-Z

wherein: X represents a fluorophore group covalently linked to the rest of the molecule by way of an oxygen atom or an NH group;

Y is a self-immolative linking group;

Z represents a peptide group of formula la:

la

where:

the wavy line represents the point of attachment to the rest of the molecule;

R¹ represents Ci-e alkyl or OR²;

R² represents Ci-e alkyl that is unsubstituted or substituted by one or more groups selected from halo, C_1-3 alkyl and aryl;

n is 1 or 2; and

when m is 0 then n is 1 ; and

The compounds disclosed herein are selective (discriminating keratinocytes and normal fibroblasts from keloid derived fibroblasts) and sensitive (e.g. compounds disclosed herein may only need as few as 20,000 cells to provide a readout). Thus, the compounds disclosed herein show promise as both a diagnostic tool and as a screening tool.

In embodiments herein, the word“comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word“comprising” may be replaced by the phrases“consists of” or“consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word“comprising” and synonyms thereof may be replaced by the phrase“consisting of” or the phrase“consists essentially of’ or synonyms thereof and vice versa. References herein (in any aspect or embodiment of the invention) to compounds of formula I includes references to such compounds per se, to tautomers of such compounds, as well as to pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of such compounds.

Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula I with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of formula I in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.

Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium.

Examples of acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene-2- sulphonic, naphthalene-1 , 5-disulphonic and p-toluenesulphonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)- (1 S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1 , 2-disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL- lactic), lactobionic, maleic, malic (e.g. (-)-L-malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulphonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, tartaric (e.g.(+)-L-tartaric), thiocyanic, undecylenic and valeric acids.

Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.

As mentioned above, also encompassed by formula I are any solvates of the compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.

The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and di hydrates.

For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et al., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

Compounds of formula I, as well as pharmaceutically acceptable salts, solvates and pharmaceutically functional derivatives of such compounds are, for the sake of brevity, hereinafter referred to together as the“compounds of formula I”.

Compounds of formula I may contain double bonds and may thus exist as E ( entgegen ) and Z (zusammen) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.

Compounds of formula I may exist as regioisomers and may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.

Compounds of formula I may contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a‘chiral pool’ method), by reaction of the appropriate starting material with a‘chiral auxiliary’ which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.

The term“halo”, when used herein, includes references to fluoro, chloro, bromo and iodo.

Unless otherwise stated, the term“aryl” when used herein includes Ce-u (such as Ce-io) aryl groups. Such groups may be monocyclic, bicyclic or tricyclic and have between 6 and 14 ring carbon atoms, in which at least one ring is aromatic. The point of attachment of aryl groups may be via any atom of the ring system. However, when aryl groups are bicyclic or tricyclic, they are linked to the rest of the molecule via an aromatic ring. Ce-14 aryl groups include phenyl, naphthyl and the like, such as 1 ,2,3,4-tetrahydronaphthyl, indanyl, indenyl and fluorenyl. Embodiments of the invention that may be mentioned include those in which aryl is phenyl.

Unless otherwise stated, the term “alkyl” refers to an unbranched or branched, cyclic, saturated or unsaturated (so forming, for example, an alkenyl or alkynyl) hydrocarbyl radical, which may be substituted or unsubstituted (with, for example, one or more halo atoms). Where the term“alkyl” refers to an acyclic group, it is preferably Ci-e alkyl (such as ethyl, propyl, (e.g. n-propyl or isopropyl), butyl (e.g. branched or unbranched butyl), pentyl or, more preferably, methyl). Where the term“alkyl” is a cyclic group (which may be where the group “cycloalkyl” is specified), it is preferably C3-12 cycloalkyl and, more preferably, C5-10 (e.g. C5-7) cycloalkyl. In particular embodiments that may be mentioned herein, the term“alkyl” may be used to refer to unbranched or branched, saturated hydrocarbyl radicals that are substituted or, more particularly, unsubstituted.

When used herein, the term“fluorophore” is intended to refer to a substituent group that does not fluoresce when attached to the rest of the molecule by way of a covalent bond, but which is capable of fluorescence following cleavage of said covalent bond. In other words, the compounds of formula I contain a fluorophore that is initially nonfluorescent because the fluorophore is in a “caged” state due to the covalent bonds reducing the donation of electrons to the fluorophore system. In the presence of FAPa, cleavage of the amide linkage between the peptide portion (Z) leads to a free fluorophore (either directly where no self- immolative linker is present or indirectly following cleavage of the covalent bond attaching the fluorophopre to the self-immolative linker). This cleavage“uncages” the fluorophore and results in increased electron donation into the fluorophore system, thus making the fluorophore capable of fluoresence. Therefore, the compounds of formula I are able to selectively self-destruct to release a fluorescent compound in the presence of FAPa.

While any fluorophore capable of covalent attachment and detachment, where the covalently attached form results in a non-excitable state, is contemplated, the fluorophore X may in particular embodiments be independently selected from the group consisting of:

where the wavy line represents the point of attachment to the rest of the molecule. In yet more particular embodiments, X may be independently selected from the group consisting of:

As noted above, the peptide group Z may have the generic structure of formula la. As will be appreciated, the presence of a peptide group of this generic structure acts as a substrate for FAPa and so is required to be present to induce fluorescence of the fluorophore if there are elevated levels of the FAPa enzyme, which indicates the presence of a fibrotic condition. In more specific embodiments that may be mentioned herein, Z may be independently selected from the group consisting of:

When used herein, the term “self-immolative linker” is a bifunctional or a trifunctional chemical moiety which is capable of covalently linking together two or three spaced chemical moieties into a normally stable tri- or tetrapartite molecule, that can release one of the spaced chemical moieties from the stable molecule by means of enzymatic cleavage and following enzymatic cleavage, can spontaneously cleave from the remainder of the molecule to release the other spaced chemical moiety(ies). An example of this cleavage process is provided in Figure 1a. Any suitable self-immolative linker may be used in the current invention. Examples of suitable self-immolative linkers include, but are not limited to those selected from:

(which self-immolates to produce 4- methylenecyclohexa-2,5-dienimine);

where the wavy line represents the point of attachment to the rest of the molecule. For example, the group Y may be selected from the group of:

; and

where the wavy line represents the point of attachment to the rest of the molecule. As will be noted, these linkers relate to those where n is 1 :

and those in which n is 2:

In embodiments where n is 2, there will be two Z groups. This may help to amplify the fluorescent signal produced (in the case where the same amounts of a compound where m is 2 is used compared to where n is 1), or may allow for less of the compound of formula I to be provided to a subject.

Examples of the compound of formula I (and salts and solvates thereof) include, but are not limited to those listed below. In particular embodiments, the compound formula I (and salts and solvates thereof) may be selected from:

In more particular embodiments, the compound of formula I may be selected from:

Compounds of formula I may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form. Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration.

Compounds of formula I will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy , 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.

Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and/or in accordance with standard and/or accepted pharmaceutical practice.

The amount of compound of formula I in any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the particular patient to be diagnosed, as well as the compound(s) which is/are employed. In any event, the amount of compound of formula I in the formulation may be determined routinely by the skilled person.

For example, a solid oral composition such as a tablet or capsule may contain from 1 to 99 % (w/w) active ingredient; from 0 to 99% (w/w) diluent or filler; from 0 to 20% (w/w) of a disintegrant; from 0 to 5% (w/w) of a lubricant; from 0 to 5% (w/w) of a flow aid; from 0 to 50% (w/w) of a granulating agent or binder; from 0 to 5% (w/w) of an antioxidant; and from 0 to 5% (w/w) of a pigment. A controlled release tablet may in addition contain from 0 to 90 % (w/w) of a release-controlling polymer.

A parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50 % (w/w) active ingredient; and from 50% (w/w) to 99% (w/w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w/w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.

Depending on the disorder to be diagnosed and the patient, as well as the route of administration, compounds of formula I may be administered at varying diagnostically effective doses to a patient in need thereof.

However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a diagnostic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature the condition being diagnosed, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be diagnosed.

Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.01 mg to about 1000 mg per day of a compound of formula I.

In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above- mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

As mentioned above, compounds of formula I may be cleaved in the presence of FAPa and may therefore have utility as diagnostic agents for determining the presence and/or location (either in vivo or in vitro) of a fibrotic condition.

Thus, there is also provided a method of detecting a fibrotic condition in a subject, the method comprising the steps of providing a compound of formula I as defined above, or a pharmaceutically acceptable salt or solvate thereof to a subject, subjecting a tissue or organ suspected of suffering from a fibrotic condition to irradiation with light and detecting fluorescence from the irradiated tissue or organ, wherein an increase in fluorescence compared to a control indicates the presence of a fibrotic condition. There is also provided a compound of formula I, as defined above, or a pharmaceutically acceptable salt or solvate thereof for use in the detection of a fibrotic condition.

There is further provided a use of a compound of formula I, as defined above, or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for use in the detection of a fibrotic condition.

When used herein, the term“fibrotic condition” is intended to cover dermal fibrosis (e.g. hypertrophic scars, keloids, bums, Peyronie's disease, and Dupuytren's contractures) and non-dermal fibrosis (e.g. lung (or pulmonary) fibrosis, liver/hepatic fibrosis, ocular fibrosis, fibrosis of the gut, kidney/renal fibrosis, pancreatic fibrosis, vascular fibrosis, cardiac fibrosis, and myelofibrosis).

Fibrotic conditions of the lung (i.e. , pulmonary fibrosis) include, but are not limited to, idiopathic pulmonary fibrosis (“IFF”); idiopathic pulmonary upper lobe fibrosis (Amitani disease); familial pulmonary fibrosis; pulmonary fibrosis secondary to systemic inflammatory diseases such as, rheumatoid arthritis, scleroderma, lupus, cryptogenic fibrosing alveolitis, chronic obstructive pulmonary disease (“COPD”) or chronic asthma or secondary to radiation exposure; cystic fibrosis; non-specific interstitial pneumonia (“NSIP”); cryptogenic organizing pneumonia (“COP”); progressive massive fibrosis, a complication of coal worker's pneumoconiosis; scleroderma/systemic sclerosis; bronchiolitis obliterans-organizing pneumonia; pulmonary hypertension; pulmonary tuberculosis; silicosis; asbestosis; acute lung injury; and acute respiratory distress (“ARD”; including bacterial pneumonia induced, trauma-induced, and viral pneumonia-induced, ventilator-induced, non-pulmonary sepsis induced).

Fibrotic conditions of the liver (i.e., liver fibrosis) include, but are not limited to, liver cirrhosis due to all etiologies; congenital hepatic fibrosis; obesity; fatty liver; alcohol induced liver fibrosis; non-alcoholic steatohepatitis (NASH); biliary duct injury; primary biliary cirrhosis; infection- or viral-induced liver fibrosis (e.g., chronic hepatitis B and C virus infections); cystic fibrosis; autoimmune hepatitis; necrotizing hepatitis; primary sclerosing cholangitis; hemochromatosis; disorders of the biliary tree; hepatic dysfunction attributable to infections; and fibrosis secondary to radiation exposure.

Fibrotic conditions of the heart and/or pericardium (i.e., heart or pericardial fibrosis, or fibrosis of the associate vasculature) include, but are not limited to, endomyocardial fibrosis; cardiac allograft vasculopathy (“CAV”); myocardial infarction; atrial fibrosis; congestive heart failure; arterioclerosis; atherosclerosis; vascular stenosis; myocarditis; congestive cardiomyopathy; coronary infarcts; varicose veins; coronary artery stenosis and other post- ischemic conditions; and idiopathic retroperitoneal fibrosis.

Fibrotic conditions of the kidney (i.e., kidney fibrosis) include, but are not limited to, glomerulonephritis (including membranoproliferative, diffuse proliferative, rapidly progressive or sclerosing, post-infectious and chronic forms); diabetic glomerulosclerosis; focal segmental glomerulosclerosis; IgA nephropathy; diabetic nephropathy; HIV-associated nephropathy; membrane nephropathy; glomerulonephritis secondary to systemic inflammatory diseases such as lupus, scleroderma and diabetes glomerulonephritis; idiopathic membranoproliferative glomerular nephritis; mesangial proliferative glomerulonephritis; crescentic glomerulonephritis; amyloidosis (which affects the kidney among other tissues); autoimmune nephritis; renal tubuloinsterstitial fibrosis; renal arteriosclerosis; Alport's syndrome; nephrotic syndrome; chronic renal failure; periglomerular fibrosis/atubular glomeruli; combined apical emphysema and basal fibrosis syndrome (emphysema/fibrosis syndrome); glomerular hypertension; nephrogenic fibrosing dermatopathy; polycystic kidney disease; Fabry's disease and renal hypertension.

Fibrotic conditions of the pancreas (i.e., pancreatic fibrosis) include, but are not limited to, stromal remodeling pancreatitis and stromal fibrosis.

Fibrotic conditions of the gastrointestinal tract (i.e., Gl tract fibrosis) include, but are not limited to, Crohn's disease; ulcerative colitis; collagenous colitis; colorectal fibrosis; villous atrophy; crypt hyperplasia; polyp formation; healing gastric ulcer; and microscopic colitis.

Fibrotic conditions of the eye include, but are not limited to, ocular fibrosis, ophthalmic fibrosis, proliferative vitreoretinopathy; vitreoretinopathy of any etiology; fibrosis associated with retinal dysfunction; fibrosis associated with wet or dry macular degeneration; scarring in the cornea and conjunctiva; fibrosis in the corneal endothelium; anterior subcapsular cataract and posterior capsule opacification; anterior segment fibrotic diseases of the eye; fibrosis of the corneal stroma (e.g., associated with corneal opacification); fibrosis of the trabecular network (e.g., associated with glaucoma); posterior segment fibrotic diseases of the eye; fibrovascular scarring (e.g., in retinal or choroidal vasculature of the eye); retinal fibrosis; epiretinal fibrosis; retinal gliosis; subretinal fibrosis (e.g., associated with age related macular degeneration); tractional retinal detachment in association with contraction of the tissue in diabetic retinopathy; congenital orbital fibrosis; corneal subepithelial fibrosis; and Grave's ophthalmopathy.

Additional fibrotic disorders or fibrosis resulting from any one of the aforementioned conditions include, but are not limited to, spinal cord injury/fibrosis or central nervous system fibrosis such as fibrosis after a stroke, fibrosis associated with neurodegenerative disorder such as Alzheimer's disease or multiple sclerosis; vascular restenosis; uterine fibrosis; endometriosis; ovarian fibroids; Peyronie's disease; polycystic ovarian syndrome; disease related pulmonary apical fibrosis in ankylosing spondylitis; and fibrosis incident to microbial infections (e.g., bacterial, viral, parasitic, fungal etc.).

In particular embodiments of the invention, the fibrotic condition for diagnosis may be keloidal scarring and/or hypertrophic scarring.

The aspects of the invention described herein (e.g. the above-mentioned compounds, combinations, methods and uses) may have the advantage that, in the diagnosis of the conditions described herein, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, have better selectivity over, be more selective than, be more sensitive than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the diagnosis of those conditions or otherwise.

The compounds of formula I (and their salts and solvates) may also be useful in screening for potential drug candidates. Thus, in a further aspect of the invention, there is provided a cell based method for the identification of compounds suitable to treat a fibrotic condition, the method comprising:

(a) providing cells expressing a fibrotic condition;

(b) contacting the cells with a test compound for a first period of time;

(c) after the first period of time, contacting the cells with a compound of formula I, as defined above, or a pharmaceutically acceptable salt or solvate thereof for a second period of time;

(d) after the second period of time, irradiating the cells with light and quantitatively measuring the fluorescence, wherein a reduced quantitative fluorescence readout in the presence of a test compound compared to a blank is indicative of antifibrotic activity. Any of the fibrotic conditions mentioned herein may benefit from this screening method. However, particular conditions that may benefit include, but are not limited to, keloidal scarring and/or hypertrophic scarring.

Examples of cells expressing a fibrotic condition include activated fibroblasts such as keloid- derived fibroblasts (KF) and fibroblasts derived from hypertrophic scar (HSFs) which, compared to normal fibroblasts, overexpress fibroblast activation protein-alpha (FAPa) and other biomarkers, which may include, but are not limited to, Col1a1 , CTGF, fibronectin, a- smooth muscle actin(SMA), TQRb-GbobrΐqG I etc. Cells which are genetically engineered to overexpress FAPa may also be considered to express a fibrotic condition.

The screening method disclosed herein has been used to identify two compounds that show an anti-fibrotic effect, namely RepSox and thiazovivin. It is noted that the screening method disclosed herein is phenotypic in nature, as RepSox and thiazovivin have different mechanisms of action. Thus the screening test is able to work across a range of different potential mechanisms of action and may be useful in a drug discovery programme for new chemical entities that act as anti-fibrotic agents, or in screening existing drugs that are known to be safe and well-tolerated for anti-fibrotic activity.

Given the anti-fibrotic activity identified herein, there is also disclosed:

(a) a method of treating and/or preventing fibrotic scarring in a subject, the method comprising the steps of providing a therapeutically effective amount of RepSox and/or thiazovivin, or a pharmaceutically acceptable salt or solvate thereof to a subject in need thereof;

(b) use of RepSox and/or thiazovivin, or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for use in treating and/or preventing fibrotic scarring; and

(c) RepSox and/or thiazovivin or a pharmaceutically acceptable salt or solvate thereof for use in treating and/or preventing fibrotic scarring.

To prevent abnormal scarring from a fibrotic condition, RepSox and/or thiazovivin can be applied just after wound closure and prior to the emergence of an abnormal scar lesion in a subject who is susceptible to such scarring. As will be appreciated, if the subject in question also has existing abnormal scarring as the result of a fibrotic condition, then these scars will be treated at the same time as new fibrotic scarring is prevented. If the subject has no history of abnormal scarring, the test methods described herein may be used to establish if the subject is susceptible to such scarring, which can then be prevented accordingly. Finally, if the subject in question has no recent wounds but suffers from fibrotic/abnormal scarring, then the use of RepSox and/or thiazovivin can be used to treat these existing scars.

Any of the fibrotic conditions mentioned herein may benefit from these compounds to treat said conditions. However, particular conditions that may benefit include, but are not limited to, keloidal scarring and/or hypertrophic scarring.

RepSox and/or thiazovivin (and suitable salts and solvates) may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form. Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration.

RepSox and/or thiazovivin (and suitable salts and solvates) will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.

The amount of RepSox and/or thiazovivin (and suitable salts and solvates) in any pharmaceutical formulation(s) used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is/are employed. In any event, the amount of RepSox and/or thiazovivin (and suitable salts and solvates) in the formulation(s) may be determined routinely by the skilled person.

Depending on the disorder, and the patient, to be treated, as well as the route of administration, RepSox and/or thiazovivin (and suitable salts and solvates) may be administered at varying therapeutically effective doses to a patient in need thereof.

However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.

Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.01 mg to about 1000 mg per day of RepSox and/or thiazovivin (and suitable salts and solvates).

As will be appreciated, RepSox and/or thiazovivin act by differing mechanisms of action. Therefore, the compounds may be used individually as monotherapies, or may be combined together to provide a combination therapy. In this circumstance, the combined therapy may be provided as a single formulation (e.g. a single pill) or as separate formulations that may be administered sequentially, simultaneously or concomitantly, as determined by a skilled physician.

The aspects of the invention described herein in relation to RepSox and thiazovivin (e.g. the combinations, methods and uses) may have the advantage that, in the treatment of the conditions described herein, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, have better selectivity over, have a broader range of activity than, be more potent than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the treatment of those conditions or otherwise.

Further aspects and embodiments of the invention will now be described by reference to the following non-limiting examples.

Examples

Materials and Methods

All chemicals and reagents except otherwise mentioned were obtained from Sigma-Aldrich. High glucose (4.5g/L) Dulbecco’s modified eagle medium (DMEM) containing L-glutamine was obtained from Lonza. Fetal bovine serum (FBS), Trypsin-EDTA (0.05%), and penicillin- streptomycin (10 000 U/ml) were purchased from Gibco. Vybrant Dil Cell-Labeling Solution and NucBlue Live ReadyProbes (Hoescht 33342) Reagent were purchased from Thermo- Fisher Scientific. TRIzol reagent was obtained from Invitrogen, while M-MLV reverse transcriptase was from Promega. iQ SYBRGreen Supermix was purchased from Bio-Rad.

Cell cultures

Unless otherwise stated herein, normal dermal fibroblasts (NDFs), fibroblasts derived from human keloid scar tissue (KFs), fibroblasts derived from human hypertrophic scar tissue (HSFs) and immortalized keratinocyte (HaCaT) cells were cultured in high-glucose DMEM with L-glutamine and supplemented with 10% FBS in humidified condition (37 °C, 5% CO2). Culture medium was replaced every 2-3 days and cells were sub-cultured when they reached 95% confluency.

FNP1 application and imaging

Unless otherwise stated herein, cells were stained with FNP1 at 2 mM final concentration in culture medium for 30 min. Excess probes were rinsed off twice with PBS, prior to nuclear staining with Hoescht 33342 for 15min as in manufacturer’s protocol. Fluorescence images of cells were taken with Laser Scanning Microscope LSM800 (Zeiss) or LX71-inverted fluorescence microscope (Olympus) with Retiga-2000R CCD camera. Throughout the experiments, image capture settings were kept constant for FNP channel (500 ms, 8x gain) and Hoechst 33342 channel (50 ms, 3x gain) with 100x magnification for respective fluorescence channels and transmitted light channel. Background fluorescence was removed with ImageJ software.

Statistical analysis

T-test or one-way ANOVA was carried out to calculate the significance P-value. A suitable post-hoc test was chosen using the IBM SPSS Statistics 22 software. For each experiment, values are reported as mean ± standard deviation of at least 3 independent samples.

High Performance Liquid Chromatography (HPLC) purification

The as-synthesised compounds were separated and purified by HPLC using the conditions listed in Tables 1 and 2.

Table 1. HPLC conditions for the purification of FNP1 , FNP2 and their precursors (for Examples 1 and 2).

Time (minute) Flow (ml/min.) H2O % CH₃CN %

0 12Ό 70 30

3 12.0 70 30

35 12.0 10 90

37 12.0 10 90

38 12.0 70 30

40 12.0 70 30 Table 2. HPLC conditions for the enzymatic analysis and purification of the enzymatic mixtures of FNP1 and FNP2 (for Example 3). The HPLC chromatograms are as shown in Figure 2c, d, and Figure 3d.

Time (minute) Flow (ml/min.) H₂O % CH₃CN %

0 3Ό 70 30

3 3.0 70 30

35 3.0 10 90

37 3.0 10 90

38 3.0 70 30

40 3.0 70 30

Preparation 1

Synthesis of CyOH

K₂CO₃ (530 mg, 5.0 mmol) was added to a solution of resorcinol (550 mg, 5.0 mmol) in CH₃CN (50 ml_) at room temperature and stirred for 20 min under N₂ atmosphere. Then a solution of IR-775 chloride (1 ,295 mg, 2.5 mmol) in CH₃CN (20 ml_) was added to the above solution and stirred for 6 h at 50 °C. After reaction, the solvent was evaporated under reduced pressure and the crude product was purified by silica gel column chromatography (CH₂CI₂/CH₃OH = 25: 1), obtaining product CyOH (733 mg, 70%) as a blue-green solid. MS of compound CyOH: calculated for C₂₆H₂₆NO_2, [M⁺]: 384.19; obsvd. ESI-MS: m/z 384.40. ¹ H NMR of compound CyOH (400 MHz, CDCI₃) d (ppm): 8.03 (d, J = 13.2 Hz, 1 H), 7.31 (s, 1 H), 7.29-7.25 (m, 2 H), 7.24 (d, J = 9.2 Hz, 1 H), 7.03 (t, J = 7.6 Hz, 1 H), 6.83 (d, J = 7.6 Hz,

1 H), 6.76 (d, J = 9.2 Hz, 1 H), 6.54 (s, 1 H), 5.57 (d, J = 13.6 Hz, 1 H), 3.34 (s, 3 H), 2.66 (t, J = 6.0 Hz, 2 H), 2.61 (d, J = 6.0 Hz, 2 H), 1.89 (t, J = 5.8 Hz, 2 H), 1.67 (s, 6 H).

Example 1

Synthesis of Cbz-GIv-Pro-OH Peptide Cbz-Gly-Pro-OH (470 mg, 1.54 mmol) was prepared by solid phase peptide synthesis (SPPS) based on the procedure described in Nature Protocols, 2007, 2, 2222- 2227. MS of compound Cbz-Gly-Pro-OH: calculated for C15H19N2O5_, [(M+H)⁺]: 307.13; obsvd. ESI-MS: m/z 307.12. ¹H NMR of compound Cbz-Gly-Pro-OH (d₄-CD₃OD, 400 MHz) d (ppm): 7.29-7.38 (m, 5 H), 5.10 (s, 2 H), 4.4 (m, 1 H), 3.98 (m, 2 H), 3.46-3.63 (m, 2 H), 2.01-2.30 (m, 4 H).

Synthesis of 1 -1

A/-Ethoxycarbonyl-2-ethoxy-1 ,2-dihydroquinoline (EEDQ, 741 mg, 3.0 mmol) was added to anhydrous CH2CI2 dissolved Cbz-Gly-Pro-OH (470 mg, 1.54 mmol) under N2 atmosphere and the reaction mixture was stirred for 20 min. Then p-aminobenzyl alcohol (369 mg, 3 mmol) dissolved in CH2CI2 was added via a syringe and stirred overnight at room temperature. The pure product 1-1 (525 mg, 83%) was obtained after HPLC purification. MS of compound

1-1 : calculated for C₂₂H₂₅N₃Na0_5, [(M+Na)⁺]: 434.17; obsvd. ESI-MS: m/z 434.17. ¹H NMR of compound 1 -1 (d₄-CD₃OD, 400 MHz) d (ppm): 7.53 (d, J = 12.0 Hz, 2 H), 7.28-7.37 (m, 7 H), 5.10 (s, 2 H), 4.56 (m, 3 H), 4.00 (m, 2 H), 3.61-3.67 (m, 2 H), 1.94-2.26 (m, 4 H).

Synthesis of 3-1

A/,/\/ -Disuccinimidyl carbonate (DSC, 96 mg, 0.375 mmol) was dissolved in anhydrous CH3CN. Then, the mixture of compound 1-1 (60 mg, 0.15 mmol) and DIPEA (76.5 mI_, 0.45 mmol) were added via a syringe. The resulting reaction mixture was stirred overnight at room temperature under N2 atmosphere. After reaction, the solvent was removed under reduced pressure and the product was used in the next step without further purification. Such crude active carbonate mixture was added to the solution of N,N’- dimethylethylenediamine (132 mg, 1.5 mmol) and DIPEA (132 mg, 1.5 mmol) in anhydrous THF. After stirring for 8 h at room temperature under N2 atmosphere, the solvent was removed under reduced pressure and the residue was dissolved in EtOAc. The organic phase was washed with aq. 10% NH4CI solution and deionized water. After extraction, the organic phase was collected, dried over Na2S0₄, filtered and evaporated. The pure product 3-1 (54 mg, 68%) was purified by HPLC. MS of compound 3-1 : calculated for C27H36N5O6_, [(M+H)⁺]: 526.27; obsvd. ESI-MS: m/z 526.22. ¹H NMR of compound 3-1 (d₄-CD₃OD, 400 MHz) d (ppm): 7.53 (d, J = 12.0 Hz, 2 H), 7.20-7.28 (m, 7 H), 5.10 (s, 2 H), 4.48 (m, 3 H), 3.93 (m, 2 H), 3.52-3.63 (m, 4 H), 3.09 (br, 2 H), 2.89 (s, 3 H), 2.62 (s, 2 H), 1.94-2.26 (m, 4 H).

Synthesis of FNP1

Hemicyanine (CyOH, 40 mg, 0.10 mmol; form preparation 1) was added dropwise to the solution of triphosgene (15 mg, 0.05 mmol) in anhydrous CH2CI2 at 0 °C under N2 atmosphere. After reaction at room temperature for 0.5 h, comminuted K₂CC>₃ (138 mg, 1.0 mmol) was added and stirred for another 0.5 h. Then, compound 3-1 (42 mg, 0.08 mmol) was added dropwise and the reaction mixture was stirred for 5 h at room temperature under N2 atmosphere and the pure product FNP1 (46 mg, 62%) was obtained after HPLC purification. MS of compound FNP1 : calculated for C54H59N6O9_, [M⁺]: 935.4338; obsvd. HR- MS: m/z 935.4348. ¹H NMR of compound FNP1 (d₄-CD₃OD, 400 MHz) d (ppm): 8.82 (d, J = 20 Hz, 1 H), 8.64 (m, 1 H), 6.86-7.64 (m, 16 H), 6.58-6.64 (m, 1 H), 5.1 1 (m, 4 H), 4.57 (m, 1 H), 4.03 (m, 2 H), 3.90 (m, 3 H), 3.55-3.75 (m, 6 H), 2.99-3.10 (m, 6 H), 2.70-2.82 (m, 4 H), 1.95-2.10 (m, 6 H), 1.72-1.86 (m, 6 H). Variable-temperature (VT) ¹H NMR of compound FNP1 (d₄-CD₃OD, 400 MHz) d (ppm): 8.52-8.92 (br, 2 H), 6.86-7.64 (m, 16 H), 6.59 (d, J = 16 Hz, 1 H), 5.1 1 (s, 4 H), 4.57 (m, 1 H, covered by broad water peak), 4.03 (s, 2 H), 3.90 (s, 3 H), 3.55-3.75 (m, 6 H), 2.99-3.10 (m, 6 H), 2.70-2.82 (m, 4 H), 1.95-2.10 (m, 6 H), 1.72- 1.86 (br, 6 H).

Example 2

A/-(Fluorenylmethyloxycarbonyloxy)succinimide (Fmoc-OSu, 3.7 g, 1 1 mmol) dissolved in p- dioxane (30 ml_) was added dropwise to the solution (10 ml_) of p-aminobenzyl alcohol (1.23 g, 10 mmol) in p-dioxane (10 ml_). The reaction was stirred for 48 h at room temperature, and then H2O (40 ml_) was poured into the above mixture resulting in the precipitation of product. The product (3.1 g, 90%) was isolated by filtration and washed with H2O (3 x 50 ml_). MS of Fmoc-p-aminobenzyl alcohol: calculated for C₂₂HisNNa0_3, [(M+Na)⁺]: 367.12; obsvd. ESI-MS: m/z 367.41. ¹H NMR of compound Fmoc-p-aminobenzyl alcohol (d₆-DMSO, 300 MHz) d (ppm): 9.66 (s, 1 H), 7.92 (d, J = 9.0 Hz, 2 H), 7.75 (d, J = 6.0 Hz, 2 H), 7.33- 7.46 (m, 6 H), 7.20 (d, J = 6.0 Hz, 2 H), 5.07 (t, 1 H), 4.48 (d, J = 9.0 Hz, 2 H), 4.41 (d, J = 6.0 Hz, 2 H), 4.31 (t, 1 H).

Synthesis of 1-2

Anhydrous pyridine (322 pl_, 4 mmol) was added to the solution of Fmoc-p-aminobenzyl alcohol (690 mg, 2 mmol) in anhydrous THF (10 ml_), then 2-chlorotrityl chloride polystyrene resin (1 g, 1 mmol) was added to the above mixture. The solution was stirred and heated at 60 °C for 12 h under N₂ atmosphere. The resin was isolated from the solution through a sintered glass with sand core filter and was washed with DCM (3 x 10 ml_). Then the glass loaded with resin was added to a mixture solvent of DCM/MeOH/DIPEA (5:0.5:0.17 ml_) and was shaken for 10 min, followed by washing with DCM (2 x 5 ml_), DMF (2 x 5 ml_), DCM (3 x 5 ml_). The Fmoc-p-aminobenzyl alcohol loaded resin was dried under high vacuum overnight and used for the next SPPS. 1-2 (95.7 mg, 0.3 mmol) was prepared by solid phase peptide synthesis (SPPS). MS of compound 1-2: calculated for C16H22N3O4_, [(M+H)⁺]: 320.16; obsvd. ESI-MS: m/z 320.20. ¹H NMR of compound 1-2 (d₄-CD₃OD, 300 MHz) d (ppm): 7.53 (d, J = 9.0 Hz, 2 H), 7.30 (d, J = 9.0 Hz, 2 H), 4.54 (m, 3 H), 4.07 (m, 2 H), 3.59- 3.72 (m, 2 H), 2.04-2.31 (m, 4 H), 2.00 (s, 3 H).

Synthesis of 3-2

A/,/\/-Disuccinimidyl carbonate (DSC, 96 mg, 0.375 mmol) was dissolved in anhydrous CH₃CN. Then, the mixture of compound 1-2 (48 mg, 0.15 mmol) and DIPEA (76.5 mI_, 0.45 mmol) were added via a syringe. The resulting reaction mixture was stirred overnight at room temperature under N₂ atmosphere_. After reaction, the solvent was removed under reduced pressure and the product was used in the next step without further purification. Such crude active carbonate mixture was added to the solution of N,N’- dimethylethylenediamine (132 mg, 1.5 mmol) and DIPEA (132 mg, 1.5 mmol) in anhydrous THF. After stirring for 8 h at room temperature under N₂ atmosphere, the solvent was removed under reduced pressure and the residue was dissolved in EtOAc. The organic phase was washed with aq. 10% NH4CI solution and deionized water. After extraction, the organic phase was collected, dried over Na₂SC>4, filtered and evaporated. The pure product 3-2 (46 mg, 70%) was obtained after HPLC purification. MS of compound 3-2: calculated for C21 H₃2N5O5_, [(M+H)⁺]: 434.24; obsvd. ESI-MS: m/z 434.08. ¹H NMR of compound 3-2 (d₄- CD₃OD, 400 MHz) d (ppm): 7.60 (d, J = 12.0 Hz, 2 H), 7.37 (d, J = 8.0 Hz, 2 H), 5.1 (s, 2 H), 4.57 (m, 1 H), 4.10 (m, 2 H), 3.62-3.73 (m, 4 H), 3.21 (br, 2 H), 2.99 (s, 3 H), 2.74 (s, 3 H), 2.07-2.33 (m, 4 H), 2.02 (s, 3 H).

Synthesis of FNP2

Hemicyanine (CyOH, 40 mg, 0.10 mmol; from Preparation 1) was added dropwise to the solution of triphosgene (15 mg, 0.05 mmol) in anhydrous CH2CI2 at 0 °C under N₂ atmosphere. After reaction at room temperature for 0.5 h, comminuted K₂CC>₃ (138 mg, 1.0 mmol) was added and stirred for another 0.5 h. Then, compound 3-2 (35 mg, 0.08 mmol) was added dropwise and the reaction mixture was stirred for 5 h at room temperature under N2 atmosphere and the pure product FNP2 (46 mg, 62%) was obtained after HPLC purification. MS of compound FNP2: calculated for C48H₅5N₆C>8_, [M⁺]: 843.4076; obsvd. HR- MS: m/z 843.4044. ¹H NMR of compound FNP2 (d₄-CD₃OD, 400 MHz): 8.80 (d, J = 20 Hz, 1 H), 8.64 (m, 1 H), 7.14 -7.59 (m, 10 H), 6.89-6.99 (m, 1 H), 6.56-6.61 (m, 1 H), 5.1 1 (m, 2 H), 4.56 (m, 1 H), 4.04 (m, 2 H), 3.89 (s, 3 H), 3.52-3.73 (m, 6 H), 2.94-3.10 (m, 6 H), 2.69-2.80 (m, 4 H), 1.73-2.04 (m, 15 H). VT ¹H NMR of compound FNP2 (d₄-CD₃OD, 400 MHz): 8.60- 8.94 (br, 2 H), 8.64 (m, 1 H), 7.15 -7.65 (m, 10 H), 7.01 (br, 1 H), 6.61 (d, J = 16 Hz, 1 H), 5.13 (m, 2 H), 4.56 (m, 1 H, covered by broad water peak), 4.06 (m, 2 H), 3.92 (s, 3 H), 3.52- 3.73 (m, 6 H), 2.94-3.18 (m, 6 H), 2.69-2.88 (m, 4 H), 1.73-2.30 (m, 15 H).

Example 3

In vitro analysis of FAPa activation and selectivity study

FNP1 or FNP2 (10 mM; from Examples 1 and 2, respectively) was incubated with FAPa at 37 °C in HEPES buffer (50 mM, pH = 7.4) containing 1 mg mL ¹ BSA and 5% glycerol. The incubation time and the concentration of FAPa were indicated in respective figure caption. For selectivity study, FNP1 (5.5 pM) was treated with FAPa (9.0 x 10⁴ U mL·¹) in HEPES buffer (50 mM, 1 mg mL·¹ BSA, 5% glycerol, pH = 7.4), DPPIV (1.2 x 10 ³ U mL·¹) in HEPES buffer (25 mM, pH 8.0), MMP-1 (3.4 x 10 ⁴ U mL·¹) in Tris buffer (50 mM, 250 mM NaCI, 10 mM CaCI₂, pH 7.4), MMP-2 (8.4 x 10⁴ U mL·¹) in Tris buffer (50 mM, 250 mM NaCI, 10 mM CaCI₂, pH 7.4), MMP-13 (5.0 x 10 ⁴ U mL·¹) in Tris buffer (50 mM, 250 mM NaCI, 10 mM CaCI₂, pH 7.4), Caspase-3 (1.0 x 10³ U mL·¹) in HEPES buffer (50 mM, 50 mM NaCI, 0.1 % Chaps, 10 mM EDTA, 5% Glycerol, 1 mM DTT, pH 7.4) or tPA (3.8 x 10 ³ U mL·¹) in Tris buffer (50 mM, pH 8.5) at 37 °C for 30 min. After incubation, the solutions were recorded on fluorescence spectrofluorometer, UV-Vis spectrophotometer, imaged by I VIS Spectrum imaging system and injected into HPLC for analyses. Fluorescence images were captured with a 0.1 s acquisition time with excitation at 640 ± 10 nm and emission at 720 ± 10 nm using the I VIS Spectrum imaging system. MMP-1 and MMP-2 were activated by 4-aminophenylmercuric acetate (APMA) before usage according to activation protocol. Unit definition: 1 U of enzyme will hydrolyze 1 pmol of the corresponding substrate at optimized condition.

To validate the FAPa-induced cleavage as illustrated in Figure 1a and compare kinetics between FNP1 and FNP2 towards FAPa, absorption, fluorescence spectra and HPLC characterization were used to monitor the change of the probes from“caged” to“uncaged” state in both qualitative and quantitative ways (Figure 2a-e). Both FNP1 and FNP2 had the absorption maximum at 590 nm and were initially non-fluorescent. Upon treatment with FAPa (9.0 x 10⁴ U mL·¹) for 25 min, FNP1 showed obvious decrease in the absorption peak at 590 nm with the emergence of a new peak at 682 nm assigned to the released free CyOH (Figure 2a and Figure 3a). In comparison, FNP2 showed subtle change in its absorption even after incubation with FAPa for 120 min (Figure 2a and Figure 3c). The ratiometric absorption signal A682/A590 (the ratio of the absorption intensity at 682 nm to that at 590 nm) was quantified as a function of incubation time at different concentrations of FAPa (Figure 3b). FNP1 showed increased A682/A590 with increased incubation time and reached its plateau at 25 min, indicating the complete conversion of FNP1 into free CyOH. At this time point, FNP1 showed 45-fold enhancement in the fluorescence intensity at 710 nm, which was only 10-fold for FNP2. (Figure 2b). HPLC and electrospray ionization-mass spectrometry analyses further demonstrated that FNP1 (HPLC retention time, T_R = 24.8 min) was totally converted into free CyOH (TR = 21.9 min) after 25 min incubation with FAPa; in contrast, FNP2 (TR = 17.9 min) had only 14 and 33 % conversion after FAPa treatment for 25 and 120 min, respectively (Figure 2c, d and Figure 3d).

Example 4

Kinetic assay

Various concentrations of FNP1 (7, 14, 28, 56, 84, 112 or 224 mM) or FNP2 (30, 60, 120, 250, 500, 800 or 1000 pM) were incubated with FAPa (9.0 x 10⁴ U mL ¹) at 37 °C for 5 min in HEPES buffer (50 mM, pH = 7.4) containing 1 mg mL^-1 BSA and 5% glycerol. After incubation, the mixture was injected into HPLC for quantification analysis. Then initial reaction velocity (nmol min^-1) was calculated, plotted against the concentration of FNP1 or FNP2, and fitted to a Michaelis-Menten curve. The kinetic parameters were calculated by use of the Michaelis-Menten equation shown below:

V = \ nax*[S] (Km + [S]) where V is initial velocity, and [S] is substrate concentration.

To quantitatively study the probe sensitivity, the enzymatic Michaelis-Menten constants (K_m) of FAPa towards FNP1 and FNP2 were calculated, which were 46 and 185 mM, respectively (Figure 4). This confirmed that the binding affinity of FNP1 to FAPa was 4.0-fold higher as compared with that of FNP2. Additionally, the catalytic rate constants (k_cat) of FAPa towards FNP1 and FNP2 were 0.755 and 0.0793 s^-1 , respectively. Therefore, the catalytic efficiencies (, k_cat/K_m ) of FAPa towards FNP1 was calculated to be 1.64 x 10⁴ ± 1.04 x 10³ M ¹ s^-1, 38.1-fold higher than that of FNP2. Thus, FNP1 was selected for detection of FAPa for the following studies. To determine its specificity, FNP1 activation was tested against FAPa in the presence of its inhibitor Val-boroPro (talabostat) or other enzymes relevant to skin diseases including dipeptidyl peptidase IV (DPPIV), matrix metalloproteinase (MMP)-1 , MMP-2, MMP- 13, caspase-3, and tissue plasminogen activator (tPA). As shown in Figure 2f and Figure 5, the fluorescence intensity of FNP1 barely increased when FAPa was treated with inhibitor talabostat or in the presence of other enzymes, further validating its high selectivity towards FAPa.

Example 5

FAPa detection in KF cells

Cell culture and TGF-31 treatment

Normal dermal fibroblasts (NDF), keloid-derived fibroblasts (KF), and immortalized keratinocyte (HaCaT) cells were cultured in high-glucose DMEM (4.5 g L·¹) containing 4 mM L-glutamine and supplemented with 10% FBS at humidified condition (37 °C, 5% CO2). Culture medium was replaced every 2-3 days. For TGF-bI -treatment experiment, NDF cells were treated with TGF-bI (10 ng mL·¹) in low serum medium (1 % FBS) for 2 days.

Cell imaging

After the seeded cells reached 70-80% confluency, the cells were washed three times using PBS buffer (10 mM, pH 7.4) and then incubated with FAP-1 (5 mM) for 1 h at 37 °C in an atmosphere of 5% CO2 and 95% humidified air. After incubation, the medium was removed and the cells were washed 3 times using PBS buffer (10 mM, pH 7.4). Then the cells were stained with lysotracker (LysoTracker®, Thermo Fisher) for the lysosome and with Hoechst 33342 (NucBlue Live ReadyProbes Reagent, Thermo Fisher) for the nuclei as protocol. Excitation/emission wavelengths were 350 ± 20/460 ± 20 nm for Hoechst 33342, 488 ± 20/525 ± 20 nm for lysosome indicator, and 680 ± 20/710 ± 20 nm for FNP1. Fluorescence imaging

Fluorescence images of cells were acquired using LX71-inverted fluorescence microscope (Olympus) with Retiga-2000R CCD camera (Figure 7b). Throughout all the experiments, capture settings were fixed for FNP1 channel (400ms, 8x gain), Dil channel (100 ms, 3 x gain) and Hoescht33342 channel (40 ms, 3 x gain). ImageJ software was utilized to remove signal background and quantify cellular fluorescence intensity. Confocal imaging was performed on Laser Scanning Microscope LSM800 (Zeiss), with 200 x magnification for respective fluorescence channels and transmitted light channel ESID (Figure 7a). Z-stack images of 2 pm were taken on fixed cells after treatment with 10% neutral-buffered formalin (NBF) for 10 min.

Multiplate-reader measurement

Fluorescence intensities of NDF, KF and HaCaT cells seeded in 48 well-plates and labeled with FNP1 and Hoechst 33342 were analyzed with Synergy™ H4 (BioTek). Readings at 660/710 nm (FNP1) and 358/461 nm (Hoechst) were taken respectively and normalized values were reported.

RT-qPCR analysis

4 x 10⁵ NDF, KF, HaCaT or TGF-bI -treated NDF were resuspended and lysed with TRIzol reagent. Following RNA extraction, cDNA conversion was performed with M-MLV Rnase H(-) Mutant kit. Primer sequences for FAP-a and GAPDH mRNA are listed in Table 3. CT values of both genes were obtained through quantitative PCR steps on CFX ConnectTM PCR System (Biorad). 2-AACT formula was utilized to compare FAP-a expression level between groups (normalized against NDF).

Table 3. Primers used for RT-qPCR analysis (h: human)

Primer Sequences (5’ to 3’)

hFAP-a primer-forward; SEQ ID NO: 1 CGGCCCAGGCATCCCCATTT

hFAP-a primer-reverse; SEQ ID NO: 2 CACTCT GACTGCAGGGACCACC hGAPDH primer-forward; SEQ ID NO: 3 GAAGGT GAAGGTCGGAGT

hGAPDH primer-reverse; SEQ ID NO: 4 GAAG AT GGT GATGGG ATTT C

Histology

For histological evaluation, skin explant samples after FNP1 treatment were fixed in 10% NBF solution for 24 h. Skins were then rinsed thrice with PBS, and treated with 4% sucrose (w/v) for 8 h and 30% sucrose for another 16 h. Thereafter, skins were immersed in optimal cutting temperature (OCT) solution and exposed to liquid nitrogen for cryosectioning. Thin skin slices (15 p ) were stained with Hoechst 33342 according to manufacturer’s protocol and imaged under fluorescence microscope. The results are shown in Figure 6.

Discussion

With its high sensitivity and fast kinetics, FNP1 was then applied to detect KF cells in vitro along with several control skin cells including: HaCaT (epidermis origin) and normal dermal fibroblasts (NDF, dermis origin). After a short incubation period (1 h), strong NIR fluorescence was detected for KF (Figure 7a). Co-staining studies confirm that FNP1 signal was mainly localized in the cytoplasm, including the cell lysosome. In contrast, weak fluorescence signal was observed in other cells including HaCaT and NDF (Figure 7b). Fluorescence quantification further revealed that the NIR fluorescence of FNP1 in KF cells was 19.2 and 2.23-fold higher than in HaCaT and NDF, respectively (Figure 7c). Moreover, NDF cells were stimulated using transforming growth factor (TGF)^1 , which is known to increase FAPa expression levels. As shown in Figures 7b and 7c, the NIR fluorescence of FNP1 in NDF cells was enhanced by 4.15-fold after TGF-bI stimulation, confirming that the higher NIR signal in KF cells was a result of its higher FAPa expression levels relative to normal skin cells (i.e. NDF). To test if the signal intensity was closely correlated with the expression level of FAPa, reverse transcription-quantitative polymerase chain reaction (RT- qPCR) was performed to quantify the gene expression of FAPa for these cells. The expression values of HaCaT, KF, NϋR+TQRb1 were 0.03, 4.32 and 23.2-fold relative to NDF expression, respectively. Such an expression trend obtained from gene expression analysis was consistent with that for the fluorescence signals (Figures 7c and 7d). Thus, these data proved that FNP1 probe could be specifically activated by FAPa, allowing for distinguishing KF cells from other normal skin cells (i.e. NDF, HaCaT).

Example 6

FAPa detection in KF cells-containing skin tissues and Microneedle-assisted FAPa detection in KF-implanted skin tissues

Ex vivo skin disease model

Preparation of skin disease models was contracted to Cell Research Corporation Pte Ltd (Singapore). Human skin tissue explants (> 1.2 ^c 1.2 cm²) cultured in cell culture medium were modified by injecting blank gel or gel-containing HaCaT, NDF, KF cells (100 pL Geltrex + 100 pL medium, 200 pL total volume) at a cell density of 7.5 x 10⁶ cells mL ¹. This resulted in 1.5 x 10⁶ cells per skin tissue explant. For subsequent cell number limiting dilution studies, designated densities of 3 x 10⁵, 1 x 10⁶, 3 x 10⁶ or 1 x 10⁷ cells mL^-1 were used. This is equivalent to 1.5 x 10⁶, 4.5 x 10^s, 1.5 x 10⁵ and 4.5 x 10⁴ keloid cells respectively injected into a piece of skin tissue. These modified skin explants were given 24 h incubation period prior to probe application. FNP1 (20 pl_, 250 mM) is thoroughly mixed with Aquaphor® (1 :1 weight ratio) and topically applied on modified skin, following application of poly(methyl methacrylate) (PMMA) microneedle for 5 min at -16.7 kPa (18-fold lower than breaking strength of healthy skin). 6 h later, excess probe was wiped off and the skin samples were imaged with a 0.1 s acquisition time with excitation at 640 ± 10 nm and emission at 720 ± 10 nm using I VIS Spectrum imaging system (PerkinElmer, Inc.). Cell quantification was enabled by Dil-labelled cells with excitation at 535 ± 10 nm and emission at 580 ± 10 nm. Using dual fluorescence channel imaging allows normalization of FNP1 signal (Em: 720 nm) with total cell signal (Em: 580 nm) for accurate signal quantification. FNP1 signal (Em: 720 nm) was subtracted by background values before dividing by the cell signal (Em: 580 nm). Control skin samples (Skin) were set at a nominal value of 1.0 with HaCaT, NDF and KF signal values further divided relative to it. Fluorescence images were analyzed by ROI analysis of equal areas using the Living Image 4.0 Software.

Discussion

The ability of FNP1 to detect KF cells was subsequently evaluated in live, metabolically- active human skin tissue models containing diseased KF cells as a proof-of-concept. To detect the implanted KF cells, FNP1 was mixed with Aquaphor® ointment to form an emulsion to help it cross the uppermost skin epidermal barrier to interact with dermis residing KF cells for topical application. Initial trials using skin stripped of the epidermis (uppermost skin layer), showed that the probe readily diffused throughout the skin dermis, and detected KF cells within tissue at the depth of 1.4 mm at least. This confirmed that FNP1 was suitable for imaging keloid scars found < 2 mm from the skin surface (Figure 8). However, when whole-skin models with intact epidermis barrier was used, the probe signal was mainly observed on the skin surface with negligible signal in the skin dermis (Figure 8). These data showed that FNP1 was likely to be trapped in the uppermost skin layer, failing to cross the epidermis.

To facilitate transdermal penetration of the hydrophilic FNP1 probe 30, microneedles 10 were employed to create microchannels 20 (Figure 9ai). Microneedle device (500 pm in height per needle) is sufficient to insert into skin at the early stages of scar formation, allowing FNP1 probe emulsion 30 to traverse beyond the epidermis barrier layer 40. The KF cells 60 were located at the dermis layer 50. The microneedles were weighted down to deform skin at 18-fold pressure magnitude below that required to break skin (i.e. 300 kPa). After 5-minutes, the microneedles 10 were removed and the FAPa probe 30 was topically applied to the skin surface (Figure 9aii) and incubated for 6 hrs before I VIS (in vivo imaging system) imaging was carried out subjecting the sample with an excitation light 70 and detecting the emission 80 detected with a camera 90 (Figure 9aiii). As shown in Figures 9b and 9c, the NIR fluorescence of FNP1 from KF-implanted skin was 14.5, 6.5, and 2.4-fold higher than unmodified skin, HaCaT-implanted and NDF-implanted skin, respectively. This demonstrates how microneedle-assisted topical application of FNP1 allows it to cross the skin epidermis barrier, before selectively detecting dermis-residing KF cells 60.

To evaluate the sensitivity of FNP1 for detecting KF cells in metabolically-active skin models, skins implanted with different amounts of KF cells (1.5 x 10⁶, 4.5 x 10⁵, 1.5 x 10⁵ and 4.5 x 10⁴ cells) were tested. As shown in Figure 9d, the NIR fluorescence signal of FNP1 was still detectable with cell numbers as low as 4.5 x 10⁴ cells. An exponential relationship was observed between the NIR signal magnitude and the number of KF cells (Figure 9e). Its theoretical relationship suggests that FNP1 could potentially detect a minimum of 20,000 KF cells by assuming a minimum observable signal of ~0.1. This is ~50-fold smaller than the estimated number of cells (-1 ,000,000) within a mature keloid scar of 1 cm radius (assuming a spherical shape and cell density of 2.3 x10⁵ cells/mL). Therefore, the FNP1 probe is a highly specific, easy-to-use diagnostic strategy suitable to provide early indications of abnormal scarring before progression into mature keloid scars.

Example 7

FNP1 performance validation in cells

FNPTs sensitivity was further evaluated by applying it to normal dermal fibroblasts (NDF), keloid fibroblasts (KF), hypertrophic scar fibroblasts (HSF) and keratinocytes (HaCaT, non expressing cells), by the procedures set out above in the materials and methods section. This was compared to conventional gene expression analysis, PCR.

PCR analysis

1 x 10⁵ NDFs, KFs, HSFs or HaCaT (treated or untreated with molecular compounds) were suspended and lysed with TRIzol reagent. Following RNA isolation, cDNA conversion was done using M-MLV RNase H(-) Mutant kit. Primer sequences for genes evaluated (FAP-a, COL1a1 , CTGF and GAPDH) are listed in Table 4. CT values of these genes were obtained through quantitative PCR with CFX Connect™ PCR System (Biorad). 2^_AACT formula was utilized to compare gene expression level between different treatment groups (normalized against NDF). Table 4. Primers used for PCR analysis (h: human)

Discussion

Fibroblasts derived from abnormal (keloid, hypertrophic) scar lesions (Figure 10a,‘S’ region) express higher FAPa than normal dermal fibroblasts (NDFs) derived from undiseased skin. Hypertrophic, keloid scar-derived fibroblasts (HSF, KF respectively) and keratinocytes (cells derived from the epidermis layer, HaCaT, Figure 10a, Έ’ region) were compared with NDF.

The FNP1 signal was obtained at excitation: 680 nm, emission: 710 nm using a fluorescence multi-plate reader. NDF, KF, HSF and HaCaT were evaluated. Being of skin epidermal lineage, HaCaT cells, devoid of FAPa, express a weak FNP1 signal (Figure 10b). NDF produced a fluorescence signal 2.4-fold higher by comparison. However, the FNP1 signal was 5.76-fold and 4.32-fold higher in keloids and hypertrophic scar fibroblasts respectively (compared to HaCaT). When considering NDF as the baseline, FNP1 signals from KF and HSF were >1.7-fold and >1.4-fold higher respectively than that from NDF (Figure 10b). FNP1 signal in HaCaT was 0.41 folds of that in NDF. PCR analysis of FAPa expression trended in a similar way to FNP1 analysis. FAPa in KF, HSF, HaCaT was 7.3-fold, 8-fold and 0.6-folds of that in NDF respectively (Figure 10c). The indirect quantification of FAPa using FNP1 method was comparable to the direct quantification of FAPa expression with PCR (R², coefficient of determination was 0.8712, Figure 10d). Thus, the FNP1 probe can discriminate between skin cells with differential FAPa expression and this confirms the reliability of FNP1 assay to quantify cellular FAPa.

Example 8

Initial analysis of (TGF)-fis role in promoting FAPa release and identification of suitable inhibitor through use of FNP1 Probe There are some links between fibrosis, transforming growth factor (TQR)-b signalling and FAPa ( Journal of Molecular and Cellular Cardiology 87 , 194-203 (2015)). This suggests that modulating TQRb signalling activity within normal (NDF) and diseased (KF, HSF) fibroblasts brings about changes in FAPa expression levels. To examine FNP1 probe sensitivity, NDF, HSF and KF were treated with different stimulants and inhibitors of TQRb activity. In HSF and KF, cells that overexpress FAPa, the TGF-b receptor inhibitor, RepSox was applied. On the other hand, NDF with minimal FAPa expression was treated with TQRb1 growth factor to assess its stimulatory effects on FAPa (Figure 11a and b). Here, RepSox treatment led to a 50% reduction in FNP1 signal for both HSF and KF (Figure 11a and b).

Compared to untreated NDFs, TQRb1 treatment (2 ng/ml, 48 h) led to FNP1 signal increase using the parameters of excitation/emission of 680 nm/710 nm (Figure 11a). HSFs and KFs that express FAPa strongly express a comparatively higher FNP1 signal than NDF (Figure 11a). Following RepSox treatment (25 mM, 48 h), representative fluorescence images showed a decrease in FNP1 fluorescence signal. This demonstrated the sensitivity of the FNP1 probe in tracking FAPa expression induced by TQRb stimulants and inhibitors (TQRb1 and RepSox respectively).

To further assess FNP1 sensitivity, NDFs (with low endogenous FAPa expression and TQRb activity) were titrated with TQRb1 using a range of concentrations (0.016 - 50 ng/ml, Figure 11 c). Normalized FNP1 probe signal was significantly higher in 10 and 50 ng/ml TQRb1 compared to untreated NDF. This was found to be 3.07 and 2.89-fold higher respectively. The smallest quantity of TQRb1 (0.016 ng/ml) resulted in a 1.57-fold increase in FNP1 signal demonstrating FNP1 sensor sensitivity (Figure 11 c). In general, an ascending trend in FNP1 signal with respect to increasing TQRb1 concentration was observed. Even 0.016 ng/ml - 3,125-fold lower than the maximum TQRb1 concentration showed an increase in FNP1 signal.

Correspondingly, a 9.2-fold increase in FAPa gene expression was confirmed using PCR analysis (Figure 12).

The quantification of FAPa using the FNP1 method was comparable to the PCR quantification (R² was 0.8527, Figure 11 d). Thus, FNP1 is highly sensitive, affirming it can detect changes in FAPa induced by changes in TQRb activity. The FNP1 probe may further identify anti-scarring drugs caused by mechanisms besides TQRb.

Example 9 Anti-scarring drug screening with FNP1

Screening of anti-fibrotic compounds

1.5 x 10⁴ HSF and KF cells were seeded in each well of a 96 well-plate. Upon reaching 85% confluency, cell medium was replaced with DMEM supplemented with 1% FBS. 12 hours later, the candidate compounds (Table 5) were added to wells. Following 48 hr incubation period, the cells were rinsed twice with PBS prior to staining with the probe.

Table 5. Drug compounds used in this study.

Microplate-reader measurement and subsequent analysis

Cellular fluorescence intensity following compound treatment and labelling with FNP1 and Hoescht33342 dye was quantified with Synergy™ H4 microplate reader (BioTek). Fluorescence measurement was performed at wavelength excitation/emission of 684/710 nm (±10nm) and 358/461 nm (±10 nm) for FNP1 and Hoechst channel respectively. Hoechst values were normalized against untreated control to obtain cell viability and proliferation. Finally, FNP1 intensities were normalized with cell density in reported figures. The therapeutic index of distinct molecular compounds was calculated based on equation 1.

Equation

where FNP_xand FNPo represent the FNP intensities of the cells from treatment‘X’ and for the untreated cells, respectively.

Briefly, normalized FNP intensity of compound-treated sample was deduced from the FNP intensity of untreated control (FNPo), before multiplying with its corresponding viability. Subsequently, the calculated therapeutic index for the respective treatments were tabulated as shown in Table 6 below.

Discussion

Untreated and TQRb1 -treated HSFs (Figure 13a) and KFs (Figure 13b) acted as the negative controls. TQRb1 pre-treatment was used to saturate receptors, ensuring the highest FAPa expression as well as FNP1 signal magnitude. Molecules with known anti scarring or anti-fibrotic properties were applied to establish a baseline FNP1 response for positive candidates. These compounds included Pirfenidone (Perf), Rapamycin (Rapa), Decorin (Dec), Simvastatin (Simv). They reduced the cellular FNP1 signal by 58.9%, 52.3%, 69.2%, and 73.1 % of the level in untreated HSFs respectively (Figure 13a). These positive candidates had a similar effect on KFs leading to reduction of FNP1 signal by 71.0%, 58.9%, 51.5% and 54.9% respectively (Figure 13b). Similar to the above experiments, the FNP1 signal was normalized by the nuclei staining.

Next, nine further molecules were screened. These molecules (Table 5) are widely used for stem cell and cell reprogramming cultures to either promote or inhibit particular pathways. Amongst the nine candidates, RepSox (Rsox) and Thiazovivin (Thia) successfully decreased the FNP1 signal in both HSF (Figure 13a) and KF (Figure 13b). The FNP1 signal decreased to 35.2% and 40.2% for HSFs treated with Rsox and Thia. In KF, the signal decreased to 58.0% and 63.9%. The viability of HSFs and KFs post-treatment were further studied (Figures 13a and 13b). Both Rsox and Thia did not show any influence. Interestingly, the positive drug (Simv) led to a decrease in cell viability. The responses of HSF and KF to these molecules are summarized in Table 6 which lists the thereapeutic index of the cells for the respective treatments. Each compound was given a ‘therapeutic index’ value (see‘methods’for calculation) with untreated cells assigned a value of “0”,“1” for maximal suppression of FAP-a activity, and“-1” for maximal enhancement of FAP-a activity. The relative change in FNP1 signal was then normalized by multiplying their respective cell viability value. Comparison of FNP1 signal showed that Rsox- and Thia- treated cells clustered near anti-scarring positive controls. Most of the other candidates did not generate FNP1 signal that is indicative of anti-scarring properties. Relative FNP1 signal (compared to untreated cells) showed that HSF and KF responded differently to drug treatment.

Table 6. Therapeutic index for HSF and KF cells following their respective treatments.

Example 10

Biological examination of identified anti-scarring drug candidates

Scratch assay Following 48 hr treatment and removal of molecular compounds, nearly-confluent NDFs, KFs, HSFs were stained with Dil dye according to manufacturer’s protocol (2 mM concentration for 1 hr) before thoroughly rinsed with PBS. Scratch gap was induced at the centre of each well with 10OmI pipette tips. Then, floating cells were rinsed with PBS and cells were left to migrate and proliferate in complete culture medium. Gap width was then monitored daily through fluorescence imaging for the subsequent 48 hrs. a-SMA immuno-staining

Upon reaching 85-90% confluency, NDFs, KFs or HSFs (treated or untreated with molecular compounds) were rinsed with PBS and fixed with 4% paraformaldehyde in PBS for 10 minutes on ice. Fixed cells were then stained with monoclonal Anti-Actin, a-Smooth Muscle- Cy3 clone 1A4 overnight at 4 °C, with final concentration of 6 pg/ml. Following thorough rinsing with chilled PBS, Hoescht 33342 staining was performed before confocal microscopy imaging. Immunostaining and imaging were performed in triplicate and repeated.

Discussion

The identified Rsox and Thia suppressed FAP expression. Their effects were further evaluated using the following assays: wound healing model (scratch assay), gene expression analysis and immunofluorescence imaging of known abnormal scar biomarkers.

Cell migration, a critical attribute of wound closure/healing, precedes wound resolution and influences scar formation. Here the influence of identified drugs to cell migration in the wound healing model was studied through the scratch assay (Figures 14a and 14b). The gap width (separation) was measured at 0, 24 and 48 hrs following the scratch respectively. Rsox and Thia were observed to suppress cell migration, reducing gap width after 48 hrs particularly for KSF (Figures 14a and 14b).

Thereafter, the drug candidates (Rsox, Thia) were validated by expression of biomarkers known to be well-expressed in abnormal scars and compared to a negative candidate (CHIR). COL1A1 (Figure 15a) is responsible for excess scar tissue generation and CTGF (Figure 15b) is an activation target of TΰRb signaling activity. Keloid and hypertrophic scars express abnormally high COL1A1 and CTGF levels. COL1A1 expressions in KSFs and HSFs were 5.8-fold and 5.1-fold higher than that in NDFs respectively (Figure 16a). Treatment with Rsox and Thia led to significant decreases in COL1A1 expression levels (85% and 87%) in HSF and KSF cells respectively (Figure 15a). On the other hand, treatment with CHIR identified as a negative candidate (Figure 13) did not significantly change the COL1A1 expression levels. Similarly, CTGF was overexpressed for both KSF and HSF (Figure 16b). Rsox and Thia treatment also inhibited the cellular expression of CTGF by 86% and 91% respectively in KF. In HSF, the reduction was 88% and 74% for Rsox and Thia treatment respectively (Figure 15b). And CHIR did not significantly change CTGF expression either.

Rsox and Thia were further assessed using immuno-staining of a-SMA (Figure 17). Both untreated cells displayed typical a-SMA morphology with bundles of well-spread inter cellular actin fibers. Thia and Rsox treatment suppressed the expression of a-SMA. Similarly, the anti-scarring positive control, Simv suppressed a-SMA expression. To further validate that the suppression of anti-scarring activity was responsible for the loss of a-SMA expression, undiseased NDFs (bearing minimal a-SMA expression) were treated with TGF- b1 (2 ng/ml). This resulted in the generation of significant quantities of a-SMA stress fibres (Figure 18). This firmly demonstrates that a-SMA expression was determined by scar promoting activity such as TGF-b.

Molecular biology validation was critical to confirm anti-scarring drug activity. Interestingly, the 2 positive candidates (Thia and Rsox) act via different signalling pathways. Thia acts on Rho-associated coiled-coil containing protein kinase (ROCK), which plays a critical role in single-cell survival of pluripotent stem cells, whereas Rsox selectively inhibits TGF-b type 1 receptor. This suggests that abnormal scarring can be modulated via separate and distinct signalling pathways which may reduce drug resistance brought upon by over-reliance on any single pathway. Thus, anti-scar therapeutic and prophylactic development may involve a combination of efficacious drugs through synergy to improve abnormal scar cell elimination/suppression.

Claims

1. A compound of formula I:

[X]n-[Y]m-Z I

wherein:

Y is a self-immolative linking group;

Z represents a peptide group of formula la:

where:

the wavy line represents the point of attachment to the rest of the molecule;

R¹ represents Ci-e alkyl or OR²;

n is 1 or 2; and

when m is 0 then n is 1 ; and

2. The compound according to Claim 1 , wherein X is independently selected from the group consisting of:

3. The compound according to Claim 2, wherein X is independently selected from the group consisting of:

4. The compound according to any one of the preceding claims, wherein Z is independently selected from the group consisting of:

5. The compound according to any one of the preceding claims, wherein, when present, Y is independently selected from the group consisting of:

6. The compound according to Claim 5, wherein Y is independently selected from the group consisting of:

7. The compound according to any one of the preceding claims, wherein the compound is selected from:

8. The compound according to Claim 7, wherein the compound is selected from:

9. A method of detecting a fibrotic condition in a subject, the method comprising the steps of providing a compound of formula I, as defined in any one of Claims 1 to 8, or a pharmaceutically acceptable salt or solvate thereof to a subject, subjecting a tissue or organ suspected of suffering from a fibrotic condition to irradiation with light and detecting fluorescence from the irradiated tissue or organ, wherein an increase in fluorescence compared to a control indicates the presence of a fibrotic condition.

10. A compound of formula I, as defined in any one of Claims 1 to 8, or a pharmaceutically acceptable salt or solvate thereof for use in the detection of a fibrotic condition.

11. Use of a compound of formula I, as defined in any one of Claims 1 to 8, or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for use in the detection of a fibrotic condition.

12. The method, compound for use and use of Claims 9, 10 and 11 , respectively, wherein the fibrotic condition is keloidal scarring and/or hypertrophic scarring.

13. A cell based method for the identification of compounds suitable to treat a fibrotic condition, the method comprising:

(a) providing cells expressing a fibrotic condition;

(b) contacting the cells with a test compound for a first period of time;

(c) after the first period of time, contacting the cells with a compound of formula I, as defined in any one of Claims 1 to 8, or a pharmaceutically acceptable salt or solvate thereof for a second period of time;

(d) after the second period of time, irradiating the cells with light and quantitatively measuring the fluorescence, wherein a reduced quantitative fluorescence readout in the presence of a test compound compared to a blank is indicative of antifibrotic activity.

14. The method according to Claim 13, wherein the fibrotic condition is keloidal scarring and/or hypertrophic scarring.

15. A method of treating and/or preventing fibrotic scarring in a subject, the method comprising the steps of providing a therapeutically effective amount of RepSox and/or thiazovivin, or a pharmaceutically acceptable salt or solvate thereof to a subject in need thereof.

16. Use of RepSox and/or thiazovivin, or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for use in treating and/or preventing fibrotic scarring.

17. RepSox and/or thiazovivin or a pharmaceutically acceptable salt or solvate thereof for use in treating and/or preventing fibrotic scarring.

18. The method, compound for use and use of Claims 15, 16 and 17, respectively, wherein the fibrotic condition is keloidal scarring and/or hypertrophic scarring.