Patent Description:
Neutrophil predominant lung inflammation is a major cause of morbidity and mortality<NUM>. Yet despite decades of investigation, accurate stratification of patients with neutrophil predominant lung injury on intensive care has been hindered by the lack of bedside point of care diagnostics that can reliably and rapidly distinguish acute neutrophilic inflammation<NUM>. The ability to perform bedside diagnostics has the potential to accurately stratify such patients for further neutrophil specific interventions. Excessive neutrophil activity degrades matrix and cellular receptors, activates profibrogenic mediators and contributes to epithelial and endothelial cell damage<NUM>,<NUM>,<NUM>. The involvement of neutrophils in several diseases such as acute lung injury, ischemia-reperfusion injury<NUM>,<NUM>, cystic fibrosis<NUM> and chronic obstructive pulmonary disease<NUM>, makes them important targets for modulation.

In situ in vivo detection of neutrophilic inflammation in human pulmonary inflammation has been reliant upon FDG PET imaging. PET imaging<NUM>, although offering exquisite sensitivity, is cumbersome, expensive and is difficult to implement as a bedside molecular imaging modality. Conversely the advent of confocal endoscopy, such as probe based confocal laser endoscopy has revolutionised the ability to directly visualise the alveolar space in both preclinical and clinical arenas. However, as yet this modality has only been used to image autofluorescent structures within the alveolar space or using non-specific fluorescent dyes<NUM>,<NUM>.

The optical detection of activated neutrophilic activity is feasible with imaging enzymatic activity in whole animals<NUM>. These approaches require substrate specificity
with internally quenched molecular beacons. Often the dequenching may take hours and the substrate may be cleaved by non neutrophil proteases.

Dendrimers are a class of macromolecules possessing a well-defined structure and molecular composition<NUM>.

They are created by the stepwise attachment of monomer units in repeating unit layers, termed generations, which creates branches built upon a central core. These branches terminate in a specific chemical functional group that can be used for further dendrimer growth or modification, or attachment of specific compounds as required.

<CIT> describes the use of dendrimers and polybranched molecules to enhance signals in in vitro fluorescent assay systems<NUM>.

The molecules disclosed in <CIT> comprise cleavage sites which when treated with an appropriate chemical or enzyme lead to cleavage of selective bonds within the molecules and a subsequent change in the fluorescent properties of the molecule, most notably an increase in fluorescence. However, <CIT> only shows the results of in vitro data and there is no suggestion or teaching of how one might use the molecules in an in vivo setting, or indeed if this would in fact be possible.

It is an object of the present invention to obviate and/or mitigate at least one of the aforementioned disadvantages.

It is an object of the present invention to provide means of visualising cells in vivo, such as activated neutrophils within the lung of a subject, using confocal microendoscopy.

In a first aspect there is provided a dye construct for use in a method according to claim <NUM>.

The dye construct is a poly-branched molecule with surface groups linked to a fluorescent dye, as further described herein.

"Detectable increase in fluorescence" is understood to relate to fluorescence which can be detected by confocal microendoscopy techniques. If the dye constructs initially display a low, but detectable level of fluorescence, then a detectable increase can be observed following internalisation by an activated neutrophil cell or cells. Initially the dye constructs for use in the present invention are internally quenched. That is, the dye constructs do not fluoresce or fluoresce poorly due to the fluorescent groups or groups being quenched. However, following internalisation by the cells, dequenching occurs and an increase in fluorescence can be detected.

Typically fluorescence detection is understood to be related to fluorescence intensity, fluorescence lifetime and polarisation may also be detected. Typically the "low or little" amount of fluorescence" is practically not detectable using confocal microendoscopy techniques, or is sufficiently low to permit a clear identification of the "increase" in fluorescence. Typically a suitable increase is understood to be an increase by a factor of <NUM> or more.

The present inventors have observed that combining confocal endoscopic visualisation techniques with the localised administration of a dendrimer dye molecule or poly-branched molecule linked fluorescent dye, it is possible to observe in vivo, by way of fluorescence, specific activated neutrophil cells which internalise a dye construct of the present invention or poly-branched molecule linked fluorescent dye, with a distinct increase in fluorescence. Without wishing to be bound by theory, it is thought that the dye contructs/polybranched-dye molecules of the present invention are internalised or taken up by activated neutrophil cells before internal cellular mechanisms act upon the constructs causing an increase in fluorescence.

The present inventors have observed that through the use of the dye construct/poly-branched molecules described herein, that such molecules are capable of being internalised, by activated neutrophil cells within seconds or a few minutes. However, over time many different cell types may internalise the dye construct/poly-branched molecules described herein and as such, in order to observe the activated neutrophil cells which rapidly internalise the dye construct/poly-branched molecules described herein, the detection of fluorescence should be carried out within a few minutes of administering the dye construct - typically within seconds to minutes, such as <NUM>-<NUM> minutes, typically <NUM>-<NUM> minutes. In this manner, cells which may internalise such constructs over a much longer time period, such as within hours, are not detected and hence the activated neutrophil cells which rapidly internalise the dye constructs are readily discernable, from other cells. This is also advantageous to the patient, as they are subjected to the diagnostic procedure for as short a time as possible. Moreover, if the endoscope were removed, the site of administration of the dye construct may be difficult to relocate.

The confocal endoscope or microendoscope enables real- time in vivo human and animal imaging. The instrument couples a custom built fluorescence slit-scan confocal microscope to a fibre-optic catheter. Further teaching may be found in Thiberville et al<NUM> and <CIT>, to which the skilled reader is directed and the contents of which are hereby incorporated by way of reference.

In a further aspect there is provided a method of imaging neutrophil cells in accordance with claim <NUM>. Activated neutrophils which are characterised by degranulation and protease release may be found at sites of inflammation and may therefore be detected using confocal endoscopy techniques at a variety of locations within the body of a subject such as within in the lung, within the gastrointestinal tract, within the reproductive tract or any other endoscopically accessible orifice.

In a preferred embodiment activated neutrophils are detected in the lung of a subject. Typically the subject may be a subject already hospitalised, such as a patient in intensive care, where early detection of such activated neutrophils would be desirable.

The dye construct or poly-branched molecule linked to fluorescent dye as described herein comprise one or more cleavage sites which are cleavable by appropriate chemical or enzyme means. Preferred molecules comprise three or more, typically six or more branches, such that a significant increase in fluorescence may be observed following dequenching of the fluorescent moieties.

The use of a peptide and/or polyethylene glycol (PEG) portion is intended to improve the solubility of the dendrimer/branched molecules. When present the peptide sequence comprises an enzyme cleavage recognition sequence. or may be random in the sense of not including a recognisable enzyme or chemical cleavage recognition sequence. Without wishing to be bound by theory, when a random peptide sequence is employed, the peptide sequence is not thought to be cleaved by an enzyme present in the cell to be detected. Thus, the increase in fluorescence observed following internalisation of the molecules of the present invention comprising random peptide sequences is not thought to be due to cleavage of the peptide moiety and release of previously quenched fluorescent moieties, in contrast to previous teachings.

When an enzyme cleavable sequence is employed, the peptide sequence is cleaved by an enzyme which may be present outside of the cell and this may result in a low amount of fluorescence being observed. However, a far greater observable increase in fluorescence is observed upon internalisation of the molecules/probes. In this manner the separation of the dequencher moiety from the fluorescent moiety, as well as other cellular mechanisms results in a significant increase in fluorescence being observed. Thus, in an embodiment where a degree of fluorescence may be observed outside of the cell it is to be understood that a detectable increase is observed (i.e. greater than a factor of <NUM> as compared to any fluorescence which is observed outside of the cell) when the construct in internalised. This may in fact be an advantage, as it may allow cells to be generally identified by way of a level of fluorescence being observed outside any cells, but desired cells can more easily be identified once the constructs are internalised and an increase in fluorescence observed.

Dye construct molecules of the present invention include one of the following structures:
<CHM>
or
wherein:.

*F is carboxyfluorescein (FAM), or rhodamine. Other structures part of the invention are shown in claims <NUM> and <NUM>.

The molecules of the present invention may comprise a peptide linkage, represented in the above structures as (AA)n, where AA means any amino acid and n may be zero or is a positive integer from <NUM>-<NUM> such as <NUM>-<NUM>, or <NUM>-<NUM>. Such peptide sequences may therefore be random sequences, or conform to known sequences contained within peptides or proteins. Sequences which are recognised by the enzyme neutrophil elastase include A-A-P-V, A-A-A-P-V-K, E-E-I-Nle-R-R. Many other peptide sequences are known to the skilled addressee and may be used in probes of the present invention, examples include G-P-K-G-L-K-G (for MMP-<NUM>), V-A-D-C-A-D-Y (for proteinase <NUM>), A-A-P-F, or F-V-T-Gnf-S-W where Gnf= nonproteinogenic <NUM>-guanidine-I-phenylalanine) (for cathepsin G) and D-C-V-D (for Caspase). There is also provided methods of preparing such molecules as described hereinafter.

The molecules of the present invention are initially quenched, that is they display little or no fluorescence in terms of fluorescence which may be detected from the fluorescent moiety following appropriate excitation. However, following internalisation of the molecules by an activated neutrophil cell or cells to be detected, a de-quenching of the molecules occurs and an increase in fluorescent signal, following excitation using light of a suitable wavelength, can be detected<NUM>.

Additionally, the present inventors have observed that certain molecules of the present invention which do not have recognisably cleavable peptide sequences are nevertheless internalised by activated neutrophils and a de-quenching i.e. increase in fluorescence can be observed. Without wishing to be bound by theory, it is thought that the probes may be internalised into acidified vacuolar structures that directly effect internal quenching efficiency.

Thus, in a manner different to that described, for example, by <CIT>, the molecules of the present invention do not necessarily have to possess recognisable enzyme cleavable peptide sequences in order to visualise activated neutrophils.

The present inventors are able through confocal endoscopy to visualise cells in situ in vivo. As such the term "in vivo" is to be understood to relate to cells within the living body and hence is to be distinguished from visualising cells obtained from tissue samples which have been extracted or excised from the body. The present methods may be conducted on or within any organ into which an endomicroscopic catheter may be inserted. This may be, for example, the gut including the large and small intestine; arteries and veins; the respiratory system including the lungs, the brain such as via an intracranial catheter; and the reproductive system including the womb and fallopian tubes.

In a particularly preferred embodiment, the methods of the present invention may be carried out whilst visualising cells in the lung, such as in the alveolar space.

The present inventors have observed that through the use of the dye constructs/poly-branched molecules described herein, that such molecules are capable of being internalised, by activated neutrophils. Such internalisation by activated neutrophils occurs very rapidly, within a few seconds or minutes and as such activated neutrophils may be visualised within <NUM>-<NUM> minutes, typically <NUM>-<NUM> minutes of the molecules of the present invention being administered to the subject at the site of investigation, such as within the lung. As mentioned above, the methods of detection as described herein should typically be conducted within a short period of time, following local administration of the initially quenched molecules, typically within a few minutes of administration, so that only cells, such as activated neutrophils, which internalise or take up the molecules of the present invention rapidly, are detected. Other cell types may also internalise the molecules, but over a much longer period of time. Thus, following the techniques of the present invention, it is possible to rapidly detect activated neutrophils in a mixed population of cells. In a particularly preferred embodiment, it is possible to detect activated neutrophils within the lung, such as in the alveolar space, of a subject.

Moreover, due to the sensitivity and increase in fluorescence following dequenching of the fluorescent moieties, it is possible to detect fluorescence from only microdosed amounts (typically less than <NUM>µg) such as less than <NUM>µg or even <NUM>µg or less of the dye construct which has been administered. This is particularly advantageous in terms of certain possible regulatory issues concerning the use of larger quantities and toxicity concerns when administering any exogenous molecule - although the molecules of the present invention may not in fact be significantly toxic in any case. It is in fact particularly surprising that such low microdose amounts of molecule when administered are capable of eliciting a signal, which is detectable using microendoscopy techniques.

The present invention enables use of a dendrimer dye molecule or poly-branched molecule linked fluorescent dye of the present invention in an amount of less than <NUM>µg, <NUM>µg, or <NUM>µg or less, for visualising cells in vivo using confocal endoscopy.

The dye construct may be provided by a catheter or other suitable administration device comprising a microdose (i.e. less than <NUM>µg, <NUM>µg, or <NUM>µg or less) amount of a dye construct or poly-branched molecule linked fluorescent dye of the present invention, for administration to a subject, such that an activated neutrophil cell or cells is capable of being visualised by confocal endoscopy.

One potential advantage of the present invention is that it may be carried out on subjects who are being given respiratory support in terms of being administered oxygen or air and who as such may have a face mask covering their mouth and/or nose or who are being intubated. Even in such a situation, it is possible to insert a confocal microendoscopy catheter into the lungs through the nasal passage or via the endotracheal tube. In this manner the present invention can truly be carried out at the bedside, without necessarily having to move the subject unduly. Moreover, for such subjects, the ability to detect any activated neutrophils, which are a key marker of an inflammatory response, is of paramount importance and as such the present invention may find particular use in being conducted on such ill patients where moving them to another location may be undesirable and/or problematic.

The present invention will now be further described by way of example and with reference to the following figures which show:.

Commercially available reagents were used without further purification. NMR spectra were recorded using Bruker AC spectrometers operating at <NUM>, <NUM> and <NUM> for <NUM>H. Chemical shifts are reported on the δ scale in ppm and are referenced to residual non-deuterated solvent resonances. Normal phase purifications by column chromatography were carried out on silica gel <NUM> (<NUM>-<NUM> mesh).

Analytical reverse-phase high-performance liquid chromatography (RP-HPLC) was performed on an HP1100 system equipped with a Discovery C18 reverse-phase column (<NUM> x <NUM>, <NUM>) with a flow rate of <NUM>/min and eluting with H<NUM>O/MeOH/HCOOH (<NUM>/<NUM>/<NUM>) to H<NUM>O/MeOH/HCOOH (<NUM>/<NUM>/<NUM>), over <NUM>, holding at <NUM>% MeOH for <NUM>, with detection at <NUM> and <NUM> and by evaporative light scattering. Semi-preparative RP-HPLC was performed on an HP1100 system equipped with a Phenomenex Prodigy C18 reverse-phase column (<NUM> x <NUM>, <NUM>) with a flow rate of <NUM>/min and eluting with <NUM>% HCOOH in H<NUM>O (A) and <NUM>% HCOOH in CH<NUM>CN (B), with a gradient of <NUM> to <NUM>% B over <NUM> and an initial isocratic period of <NUM>. Electrospray ionization mass spectrometry (ESI-MS) analyses were carried out on an Agilent Technologies LC/MSD Series <NUM> quadrupole mass spectrometer (QMS) in an ESI mode. MALDI spectra were acquired on a Voyager-DE™ STR MALDI-TOF MS (Applied Biosystems) with a matrix solution of sinapinic acid (<NUM>/ml) in <NUM>% MeCN in water with <NUM>% TFA.

The monomer <NUM> was synthesised in six steps<NUM> as shown in Scheme <NUM>. Thus, the <NUM>,<NUM> addition of the hydroxy groups of <NUM>,<NUM>,<NUM>-tris(hydroxymethyl)amino-methane onto acrylonitrile, followed by amino protection (Boc), and reduction of the nitrile groups with borane-THF complex gave <NUM>. This was treated with Dde-OH to give the tris-Dde (<NUM>-acetyl-dimedone) protected amine <NUM>. Following removal of the Boc protecting group, the isocyanate <NUM> was prepared following the procedure of Knölker<NUM>.

To a solution of tris(hydroxymethyl)aminomethane (<NUM>, <NUM> mmol) in THF (<NUM>), were added sequentially <NUM>% KOH aqueous solution (<NUM>) and acrylonitrile (<NUM>, <NUM> mmol) and the resulting solution was stirred overnight. The solvent was removed in vacuo and water (<NUM>) was added to the residue. The aqueous layer was extracted with dichloromethane (3x100 ml), and the organic layer was dried with Na<NUM>SO<NUM>. The organic solvent was evaporated in vacuo and the product (<NUM> of an oil, <NUM>%) was used in the next step without further purification; <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (s, <NUM>, NH<NUM>), <NUM> (t, J = <NUM>, <NUM>, OCH<NUM>), <NUM> (s, <NUM>, CH<NUM>O), <NUM> (t, J = <NUM>, <NUM>, CH<NUM>CN); MS (ES) m/z: <NUM> [(M+<NUM>)+, <NUM>], <NUM> [(M+Na)+, <NUM>]. These data are in good agreement with the literature<NUM>.

To a stirred solution of amine (<NUM>) (<NUM>, <NUM> mmol) in THF (<NUM>) was added a solution of di-tert-butyl dicarbonate (<NUM>, <NUM> mmol) in THF (<NUM>) at <NUM> followed by the addition of DIEA (<NUM>, <NUM> mmol). The reaction was allowed to warm to room temperature and was stirred overnight. The THF was evaporated in vacuo and the residue was dissolved in ethyl acetate (<NUM>). The organic layer was washed with 1N KHSO<NUM> (<NUM>), saturated NaHCO<NUM> (<NUM>) and brine (<NUM>), dried over Na<NUM>SO<NUM> and the solvent was evaporated to give the compound (<NUM>) as oil (<NUM>, <NUM>%); <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (s, <NUM>, NH), <NUM> (t, J = <NUM>, <NUM>, OCH<NUM>), <NUM> (s, <NUM>, CH<NUM>O), <NUM> (t, J = <NUM>, <NUM>, CH<NUM>CN), <NUM> (s, <NUM>, CH<NUM>); MS (ES) m/z: <NUM> [(M+Na)+, <NUM>], <NUM> [(M-Boc)+, <NUM>]. Data in good agreement with the literature<NUM>.

To a stirred solution of tris-nitrile (<NUM>) (<NUM>, <NUM> mmol) in dry THF (<NUM>) was added dropwise BH<NUM>·THF complex (<NUM> solution in THF, <NUM> mmol, <NUM>) and the resulting mixture was stirred at <NUM> for <NUM>. Following cooling, <NUM> HCl was added to give an apparent pH between <NUM>-<NUM>. The mixture was neutralized with NaOH (aq <NUM>), and the solvent was removed in vacuo. The crude product was used without purification for the next step.

The crude product (<NUM>) (<NUM>, <NUM> mmol) was dissolved in methanol (<NUM>) and DIPEA (<NUM>, <NUM> mmol) was added. A solution of <NUM>-acetyl-dimedone<NUM> (DdeOH, <NUM>, <NUM> mmol) in dichloromethane (<NUM>) was added and the resulting mixture was stirred overnight. The solvents were removed in vacuo and the residue was purified using column chromatography (eluting with dichloromethane/methanol <NUM>/<NUM>) to afford the product as a colourless oil (<NUM>, <NUM>%); <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (s, <NUM>, CH<NUM>O), <NUM>-<NUM> (m, <NUM>, CH<NUM>), <NUM> (s, <NUM>, CH<NUM>), <NUM> (s, <NUM>, CH<NUM>), <NUM>-<NUM> (m, <NUM>, CH<NUM>), <NUM> (s, <NUM>, CH<NUM>), <NUM> (s, <NUM>, CH<NUM>); MS (ES) m/z: <NUM> [M+, <NUM>]. Data were in good agreement with the literature<NUM>.

The protected amine (<NUM>) (<NUM>, <NUM> mmol) was dissolved in <NUM>% TFA in dichloromethane (<NUM>) and the resulting mixture was stirred for <NUM>. The solvent was removed in vacuo and the residue was dissolved in dichloromethane (<NUM>) and washed with saturated aqueous NaHCO<NUM> solution (<NUM>) and water (<NUM>). The organic layer was dried with Na<NUM>SO<NUM> and the solvents removed in vacuo. The crude product (<NUM>) was used directly in the next step without purification; <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (bs, <NUM>, NH<NUM>), <NUM> (s, <NUM>, CH<NUM>O), <NUM>-<NUM> (m, <NUM>, CH<NUM>), <NUM> (s, <NUM>, CH<NUM>), <NUM> (s, <NUM>, CH<NUM>), <NUM>-<NUM> (m, <NUM>, CH<NUM>), <NUM> (s, <NUM>, CH<NUM>); MS (ES) m/z: <NUM> [M+, <NUM>], <NUM> [(M+<NUM>)+, <NUM>], <NUM> [(M+<NUM>)+, <NUM>]; HPLC tR = <NUM>. Data were in good agreement with the literature<NUM>.

A solution of Boc<NUM>O (<NUM>, <NUM> mmol) in dry DCM (<NUM>) was added dropwise to a mixture of amine <NUM> (<NUM>, <NUM> mmol) and DMAP (<NUM>, <NUM> mmol) in dry DCM (<NUM>) and the reaction mixture was stirred for <NUM>. The solvent was removed in vacuo to give <NUM> (<NUM>, <NUM> %). The isocyanate <NUM> was used immediately. <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM>-<NUM> (m, <NUM>, CH<NUM>), <NUM> (s, <NUM>, CH<NUM>), <NUM> (s, <NUM>, CH<NUM>), <NUM>-<NUM> (m, <NUM>, CH<NUM>), <NUM> (s, <NUM>, CH<NUM>); MS (ES) m/z: <NUM> [M+, <NUM>]; IR (neat) v (cm-<NUM>): <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. Data were in good agreement with the literature<NUM>.

Monomer (<NUM>) used in the preparation of <NUM>-branched dendrimer was synthesised in <NUM> steps using α-resorcylic acid as a starting material (Scheme <NUM>). Esterification of (<NUM>) in methanol provides methyl benzoate (<NUM>), which then undergoes alkylation with <NUM>-(Boc-amino)ethyl bromide in the presence of potassium carbonate in DMF to yield (<NUM>). The latter was then subjected to saponification of the methyl ester by using NaOH/MeOH/dioxane mixture, followed by removal of the Boc protective group using HCl to give amine as the hydrochloride salt (<NUM>). Subsequently, the amine salt was selectively protected with FmocOSu to give the branching monomer (<NUM>), which was utilised in the Fmoc-based solid support synthesis.

A suspension of (<NUM>-bromoethyl)amine (<NUM>, <NUM> mmol) and di-tert-butyl dicarbonate (<NUM>, <NUM> mmol) in dichloromethane (<NUM>) was cooled to <NUM>, and triethylamine (<NUM>, <NUM> mmol) was added dropwise. After stirring for <NUM>, dichloromethane (<NUM>) was added and the solution was washed with <NUM> KHSO<NUM>, water and brine, the mixture was dried (Na<NUM>SO<NUM>) and concentrated in vacuo. Product was isolated as clear yellow oil (<NUM>, <NUM>%); <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (<NUM>, br s, NH), <NUM>-<NUM> (<NUM>, m, BrCH<NUM>, CH<NUM>NH), <NUM> (<NUM>, s, C(CH<NUM>)<NUM>); <NUM>C-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (C=O), <NUM> (C), <NUM> (CH<NUM>), <NUM> (CH<NUM>), <NUM> (CH<NUM>). Data were in good agreement with the literature<NUM>.

To a solution of <NUM>,<NUM>-dihydroxybenzoic acid (<NUM>, <NUM> mmol) in methanol (<NUM>) was added a catalytic amount of sulphuric acid (<NUM>). After stirring at reflux overnight, the mixture was cooled and neutralized with <NUM> NaOH (aq. After concentration, the residue was dissolved in ethyl acetate and washed with water and brine. The organic layer was dried (Na<NUM>SO<NUM>) and concentrated in vacuo. Compound <NUM> was isolated as a white solid (<NUM>, <NUM>%); m. <NUM>-<NUM> (ethyl acetate); <NUM>H-NMR (<NUM>, d6-DMSO) δ: <NUM> (<NUM>, s), <NUM> (<NUM>, d, J <NUM>, CHar), <NUM> (<NUM>, d, J <NUM>, CHar), <NUM> (<NUM>, s, CH<NUM>); <NUM>C-NMR (<NUM>, d6-DMSO) δ: <NUM> (C=O), <NUM> (C x2), <NUM> (C), <NUM> (CH), <NUM> (CH x2), <NUM> (CH<NUM>); MS (ES)- m/z: <NUM> [M-H]-; HPLC tR = <NUM>; IR (neat) v (cm-<NUM>): <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. Data were in good agreement with the literature<NUM>.

A mixture of <NUM>-(boc-amino)ethyl bromide (<NUM>, <NUM> mmol), compound (<NUM>) (<NUM>, <NUM> mmol), potassium carbonate (<NUM>, <NUM> mmol) in anhydrous dimethylformamide (<NUM>) was stirred at <NUM> for <NUM>. The mixture was filtered through Celite® and the filtrate was reduced. The residue was dissolved in ethyl acetate and washed with water and brine, the organic layer was dried (Na<NUM>SO<NUM>) and concentrated in vacuo. Crystallisation (EtOAc/hexane) afforded compound (<NUM>) as a white solid (<NUM>). Remaining mother liquor was reduced in vacuo and the residual oil was purified with silica column chromatography using <NUM>% EtOAc in hexane to give <NUM> of (<NUM>) (total yield <NUM>, <NUM>%); m. <NUM>-<NUM> (EtOAclhexane); <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (<NUM>, d, J <NUM>, CHar), <NUM> (<NUM>, t, J2. <NUM> Hz, CHar), <NUM> (<NUM>, s, NH), <NUM> (<NUM>, t, J <NUM>, CH<NUM>), <NUM> (<NUM>, s, CH<NUM>), <NUM>-<NUM> (<NUM>, m, CH<NUM>), <NUM> (<NUM>, s, C(CH<NUM>)<NUM>); <NUM>C- NMR (<NUM>, CDCl<NUM>) δ: <NUM> (C=O), <NUM> (C x2), <NUM> (C=O), <NUM> (C), <NUM> (CH x2), <NUM> (CH), <NUM> (C), <NUM> (CH<NUM>), <NUM> (CH<NUM>), <NUM> (CH<NUM>), <NUM> (CH<NUM>); MS (ES)+ m/z: <NUM> [M+Na]+; HPLC tR = <NUM>; IR (neat) v (cm-<NUM>): <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. Data were in good agreement with the literature<NUM>.

Compound <NUM> (<NUM>, <NUM> mmol) was dissolved in dioxane/methanol/<NUM> NaOH(aq) (<NUM>:<NUM>:<NUM>, <NUM>) and stirred for <NUM>. The pH of the mixture was adjusted to <NUM> with <NUM> KHSO<NUM> and the mixture was concentrated in vacuo. The residue was dissolved in dichloromethane and water. The organic layer was washed with water and brine, and dried (Na<NUM>SO<NUM>). Compound (<NUM>) was isolated as a white solid (<NUM>, <NUM>%); m. <NUM>-<NUM> (dichloromethane); <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (<NUM>, broad s, CHar), <NUM> (<NUM>, broad s, CHar), <NUM> (<NUM>, s, NH), <NUM>-<NUM> (<NUM>, m, CH<NUM>) , <NUM>-<NUM> (<NUM>, m, CH<NUM>), <NUM> (<NUM>, s, C(CH<NUM>)<NUM>); <NUM>C- NMR (<NUM>, CDCl<NUM>) δ: <NUM> (C=O), <NUM> (C x2), <NUM> (C=O), <NUM> (C=O), <NUM> (C), <NUM> (CH x2), <NUM> (CH), <NUM> (C), <NUM> (CH<NUM>), <NUM> (CH<NUM>), <NUM> (CH<NUM> x2), <NUM> (CH<NUM>), <NUM> (CH<NUM>); MS (ES)+ m/z: <NUM> [M+Na]+; HRMS (ESI)+ m/z: Calculated for C<NUM>H<NUM>N<NUM>O<NUM> [M+H]+ <NUM>, Found <NUM>; HPLC tR = <NUM>; IR (neat) v (cm-<NUM>): <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Benzoic acid (<NUM>) (<NUM>, <NUM> mmol) was dissolved in dichloromethane (<NUM>) and diethyl ether (<NUM>) saturated with hydrochloric acid was added. After stirring for <NUM>, the mixture was concentrated in vacuo. The hydrochloride salt (<NUM>) was obtained as a white solid (<NUM>, quantitative); m. <<NUM> (dichloromethane/ether); <NUM>H-NMR (<NUM>, D<NUM>O) δ: <NUM> (<NUM>, d, J2. <NUM>, CHar), <NUM> (<NUM>, t, J2. <NUM>, CHar), <NUM> (<NUM>, t, J5. <NUM>, CH<NUM>), <NUM> (<NUM>, t, J <NUM>, CH<NUM>); <NUM>C-NMR (<NUM>, D<NUM>O) δ: <NUM> (C=O), <NUM> (C x2), <NUM> (C), <NUM> (CH x2), <NUM> (CH), <NUM> (CH<NUM>), <NUM> (CH<NUM>); MS (ES)+ m/z: <NUM> [M+Na]+; HRMS (ES)+ m/z: Calculated for C<NUM>H<NUM>N<NUM>O<NUM> [M+H]+ <NUM>, Found <NUM>; HPLC tR = <NUM>.

Hydrochloride salt (<NUM>) (<NUM>, <NUM> mmol) was dissolved in acetone:water (<NUM>:<NUM>, <NUM>) containing sodium carbonate (<NUM>, <NUM> mmol). To this solution was added Fmoc-Osu (<NUM>, <NUM> mmol) in acetone (<NUM>) dropwise at room temperature. The solution was stirred at room temperature for <NUM>. The reaction mixture was concentrated and the residue dissolved in water and extracted with ether (<NUM> × <NUM>). The aqueous layer was cooled in an ice bath and acidified with <NUM> HCl to pH3. The white solid (<NUM>) obtained was filtered, washed with water and dried under vacuum (<NUM>, <NUM>%); m. <NUM>-<NUM> (water); <NUM>H-NMR (<NUM>, d6-DMSO) δ: <NUM> (<NUM>, broad s, OH), <NUM> (<NUM>, d, J <NUM>, CHar), <NUM> (<NUM>, d, J <NUM>, CHar), <NUM> (<NUM>, t, J <NUM>, NH), <NUM> (<NUM>, t, J <NUM>, CHar), <NUM> (<NUM>, t, J7. <NUM>, CHar), <NUM> (<NUM>, d, J <NUM>, CHar), <NUM> (<NUM>, broad s, CHar), <NUM> (<NUM>, d, J <NUM>, <NUM>×CH<NUM>), <NUM> (<NUM>, t, J <NUM>, 2xCH), <NUM> (<NUM>, t, J <NUM>, CH<NUM>), <NUM>-<NUM> (<NUM>, m, CH<NUM>); <NUM>C-NMR (<NUM>, d6-DMSO) δ: <NUM> (C=O), <NUM> (C=O x2), <NUM> (C x4), <NUM> (C x4), <NUM>, <NUM>, <NUM> & <NUM> (CH), <NUM> (CH x2), <NUM> (CH), <NUM> (CH<NUM> x2), <NUM> (CH<NUM>×<NUM>), <NUM> (CH<NUM> x2), <NUM> (CH x2); MS (ES)+ m/z: <NUM> [M+Na]+; HRMS (ESI)+ m/z: Calculated for C<NUM>H<NUM>N<NUM>O<NUM> [M+H]+<NUM>, Found <NUM>; HPLC tR = <NUM>; IR (neat) v (cm-<NUM>): <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Resin <NUM> was synthesized using a <NUM>-[(<NUM>,<NUM>-dimethoxyphenyl)-(Fmoc-amino)methyl]phenoxyacetic acid (Rink amide linker) attached to aminomethyl PS resin (<NUM> mmol/g, <NUM>% DVB, <NUM>-<NUM> mesh). Thus, Fmoc-Rink-amide linker (<NUM>, <NUM> mmol) was dissolved in DMF (<NUM>) and HOBt (<NUM>, <NUM> mmol) was added and the mixture was stirred for <NUM>. DIC (<NUM>, <NUM> mmol) was then added and the resulting mixture was stirred for further <NUM>. The solution was added to aminomethyl polystyrene resin (<NUM>, <NUM> mmol/g) and shaken for <NUM>. The resulting resin was washed with DMF (<NUM>×<NUM>), DCM (<NUM>×<NUM>) and MeOH (<NUM>×<NUM>).

To the resin (pre-swollen in DCM) was added <NUM>% piperidine in DMF (<NUM>) and the reaction mixture was shaken for <NUM>. The solution was then drained and the resin was washed with DMF (<NUM>×<NUM>), DCM (<NUM>×<NUM>) and MeOH (<NUM>×<NUM>). This procedure was repeated twice.

To resin <NUM> (<NUM>, <NUM> mmol), pre-swollen in DCM (<NUM>), was added a solution of isocyanate monomer <NUM> (<NUM>, <NUM> mmol), DIPEA (<NUM>, <NUM> mmol) and DMAP (<NUM>, <NUM> mmol) in a mixture of DCM/DMF (<NUM>:<NUM>, <NUM>) and the mixture was shaken overnight and the reaction was monitored by a quantitative ninhydrin test. The solution was drained and the resin was washed with DMF (<NUM>×<NUM>), DCM (<NUM>×<NUM>), MeOH (<NUM>×<NUM>) and finally by ether (<NUM>×<NUM>).

To the resin (<NUM>, <NUM> mmol), pre-swollen in DCM (<NUM>), was added <NUM>% hydrazine in DMF (<NUM>) and the reaction mixture was shaken for <NUM>. The solution was then drained and the resin (<NUM>) was washed with DMF (<NUM>×<NUM>), DCM (<NUM>×<NUM>) and MeOH (<NUM>×<NUM>).

A solution of the monomer (<NUM>) (<NUM>, <NUM> mmol, <NUM> eq) and oxyma (<NUM>, <NUM> mmol, <NUM> eq) in DMF (<NUM>, <NUM>) was stirred for <NUM>. DIC (<NUM>µL, <NUM> mmol, <NUM> eq) was then added and the resulting solution was stirred for further <NUM>. The solution was then added to resin (<NUM>) (<NUM>, <NUM> mmol, <NUM> eq), pre-swollen in DCM (<NUM>), and the reaction mixture was shaken for <NUM>. The solution was drained and the resin (<NUM>) was washed with DMF (<NUM>×<NUM>), DCM (<NUM>×<NUM>) and MeOH (<NUM>×<NUM>).

A solution of <NUM>(<NUM>)-carboxyfluorescein (<NUM> eq) and oxyma (<NUM> eq) in DMF (<NUM>µl) was stirred for <NUM>. DIC (<NUM> eq) was then added and the resulting solution was stirred for further <NUM>. This solution was added to the appropriate resin (<NUM> eq), pre-swollen in DCM, and the reaction mixture was shaken for <NUM>. The solution was drained and the resin washed with DMF (×<NUM>), DCM (×<NUM>) and MeOH (×<NUM>). The coupling reactions were monitored by a quantitative ninhydrin test<NUM>. Before cleavage, the resin was washed with <NUM>% piperidine to remove any fluorescein phenol esters<NUM>.

A solution of <NUM>(<NUM>)-tetraethylrhodamine (<NUM> eq) and oxyma (<NUM> eq) in DMF (<NUM>µl) was stirred for <NUM>. DIC (<NUM> eq) was then added and the resulting solution was stirred for further <NUM>. This solution was added to the appropriate resin (<NUM> eq), pre-swollen in DCM, and the reaction mixture was shaken for <NUM>. The solution was drained and the resin washed with DMF (×<NUM>), DCM (×<NUM>) and MeOH (×<NUM>). The coupling reactions were monitored by a quantitative ninhydrin test.

A solution of the appropriate D- or L-Fmoc-amino acid (<NUM> eq per amine) and HOBt or Oxyma (<NUM> eq per amine) in DMF (<NUM>) was stirred for <NUM>. DIC (<NUM> eq per amine) was then added and the resulting solution was stirred for further <NUM>. The solution was then added to the appropriate resin <NUM>/<NUM> (<NUM> eq), pre-swollen in DCM (<NUM>), and the reaction mixture was shaken for <NUM>-<NUM>. The solution was drained and the resin was washed with DMF (<NUM>×<NUM>), DCM (<NUM>×<NUM>) and MeOH (<NUM>×<NUM>). The coupling reactions were monitored by a quantitative ninhydrin test<NUM>.

A solution of the {<NUM>-[<NUM>-(Fmoc-amino)ethoxy]ethoxy}acetic acid (<NUM> eq per amine) and Oxyma (<NUM> eq per amine) in DMF (<NUM>) was stirred for <NUM>. DIC (<NUM> eq per amine) was then added and the resulting solution was stirred for further <NUM>. The solution was then added to the appropriate resin <NUM>/<NUM> (<NUM> eq), pre-swollen in DCM (<NUM>), and the reaction mixture was shaken for <NUM>. The solution was drained and the resin was washed with DMF (<NUM>×<NUM>), DCM (<NUM>×<NUM>) and MeOH (<NUM>×<NUM>). The coupling reactions were monitored by a quantitative ninhydrin test<NUM>.

The appropriate resin (<NUM>), pre-swollen in DCM, and treated with a cleavage cocktail of TFA/DCM/TIS (<NUM>/<NUM>/<NUM>, <NUM>µl) for <NUM>. The solution was drained and the resin was washed with the cleavage cocktail and the solution was removed in vacuo. The crude material was dissolved in a minimum amount of cleavage cocktail (<NUM>µl) and added to ice-cold ether (<NUM>). The precipitated solid was collected by centrifugation and the solvent removed by decantation and the precipitate was washed with cold ether (3x5 ml). The precipitate was then purified by reverse phase preparative HPLC and the required fractions were pooled and lyophilized to afford IQR<NUM> - IQR<NUM>; Reporter IQR<NUM>: HPLC: tR = <NUM>, purity><NUM>% by ELSD; MALDI: C<NUM>H6<NUM>N<NUM>O<NUM>: [M+] calcd: <NUM>, [M+<NUM>]+ found: <NUM>; Reporter IQR<NUM>: HPLC tR = <NUM>, purity><NUM>% by ELSD; Reporter IQR<NUM>: HPLC tR = <NUM> purity><NUM>% by ELSD; Reporter IQR<NUM>: HPLC tR = <NUM>; HRMS (ESI)+ m/z: Calcd for C<NUM>H<NUM>N<NUM>O<NUM> ([M+<NUM>]/<NUM>)+ <NUM>, Found <NUM>; IR (neat) v (cm-<NUM>): <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>; Reporter IQR<NUM>: HPLC tR = <NUM> purity><NUM>% by ELSD; Reporter IQR<NUM>: HPLC tR = <NUM> purity><NUM>% by ELSD; Reporter IQR<NUM>: HPLC tR = <NUM>; MALDI: [M+H]+ <NUM>; HRMS (ESI)+ m/z: Calcd for C<NUM>H<NUM>N<NUM>O<NUM> ([M+<NUM>]/<NUM>)+ <NUM>, Found <NUM>; IR (neat) v (cm-<NUM>): <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Human peripheral blood leukocytes from healthy volunteers were prepared as previously described<NUM>. Briefly, citrated blood was centrifuged at room temperature for <NUM> at <NUM>, and platelet-rich plasma was removed. Autologous serum was prepared by recalcification of platelet-rich plasma by addition of CaCl<NUM> to a final concentration of <NUM>. Leukocytes were separated from erythrocytes by dextran sedimentation using <NUM>% dextran T500 (Pharmacia, Milton Keynes, UK), and the leukocyte-rich upper layer was then fractionated using isotonic Percoll (Pharmacia). Neutrophils and mononuclear leukocytes (PBMC) were harvested from the <NUM>%/<NUM>% and <NUM>/<NUM>% interfaces, respectively. In some experiments, neutrophils were labelled with DiD (Invitrogen, molecular probes) (<NUM>) in D-PBS (w/o Ca/Mg) for <NUM> at room temperature.

PBMC-derived macrophages were generated from mononuclear leukocytes as follows (<NPL>. Regulation of macrophage phagocytosis of apoptotic cells by cAMP). Mononuclear leukocytes were allowed to adhere to poly-d-lysine coated coverslips for <NUM> hour before washing to remove non-adherent cells. Adherent cells were cultured for <NUM>-<NUM> days in vitro (DIV) in IMDM containing <NUM>% autologous serum.

A549 cells (ATCC CCL-<NUM>) were maintained at <NUM>-<NUM> % confluence in culture medium which consisted of Dulbecco's modified Eagle's medium (<NUM>. l-<NUM> glucose) supplemented with <NUM> % (v/v) heat inactivated fetal calf serum (FCS), L-glutamine (<NUM>), streptomycin (<NUM>µg. ml-<NUM>) and penicillin (<NUM> units. ml-<NUM>) in a thermostatted (<NUM>) and humidified atmosphere of <NUM> % CO<NUM>/ <NUM> % air. Suspensions of exponentially growing cells (<NUM>×<NUM><NUM> cells), detached following trypsin/EDTA exposure were then seeded onto coverslips or <NUM> well microplates and grown to confluence over <NUM>-<NUM>.

Bronchoalveolar lavage (BAL) was obtained from patients with Idiopathic Pulmonary Fibrosis (IPF). Written informed consent was obtained from all subjects. The study was approved by the Lothian Research Ethics Committee. BAL was performed as part of ongoing clinical research studies. <NUM> of saline was instilled into the right middle lobe and lavaged in <NUM> aliquots. Samples were kept on ice and <NUM>µl aliquots pipetted onto coverslips followed by immediate staining as detailed and live confocal imaging in a POC mini (perfusion open and closed) (PeCon GmbH) cultivation chamber.

A laser-scanning confocal imaging system (LSM510; Carl Zeiss, Jena, Germany), incorporating an upright Axioskop FS2 microscope (<NUM>× objective) was used for image acquisition and processing. Exposure to <NUM> light was limited to <NUM>-<NUM>% of the maximum laser power in order to minimize photobleaching and toxicity. In all cases, images were obtained without Kalman averaging and typically with a pixel dwell time of <NUM> with a pinhole diameter corresponding to <NUM> Airy unit. In multiple-labelling experiments pinhole diameters were adjusted to give optical Z-sections of equivalent depths, corresponding to <NUM> Airy unit for the longest excitation wavelength. All live time-lapse imaging of was performed in IMDM. Coverslips were transferred to a thermostatted environmental stage (POC Mini) maintained at <NUM><NUM>. Drugs were added by bath addition. In experiments where fluorescein was examined alone, the fluorophore was excited with a dedicated <NUM> line, and emitted light reflected from a NFT545 filter and passed through an LP505 filter.

In multiple labelling experiments involving membrane (DiD, Invitrogen), or dextran-<NUM>, and IQR probes, images were obtained simultaneously. DiD or dextran-<NUM> was excited with a dedicated <NUM> line, and emitted light detected with meta detector (<NUM>-<NUM>), whereas Fluorescein was excited with a dedicated <NUM> line, and emitted light reflected from a NFT545 filter and passed through an LP505 filter.

In multiple labelling experiments using IQR probes in combination with syto-<NUM> (Invitrogen, <NUM>, <NUM> minutes, <NUM>), images were acquired sequentially. Fluorescein was excited with a dedicated <NUM> line and emitted light reflected from a NFT545 filter and passed through an LP505 filter whereas syto-<NUM> were excited with a dedicated <NUM> line, and emitted light detected with meta detector (<NUM>-<NUM>).

Neutrophils and mononuclear cells (<NUM>×<NUM><NUM> cells total), or PBMC-derived macrophages, were seeded onto glass coverslips pre-coated with <NUM>µg/ml fibronectin (Sigma). Cells were allowed to adhere and <NUM> (final concentration) IQR1. <NUM> added to wells prior to transfer to POC mini and placement in environmental chamber. Live imaging commenced at <NUM> intervals. Baseline images were acquired for <NUM>-<NUM> prior to addition of A21387 (<NUM>, Sigma) and live time-lapse confocal images acquired for further <NUM>. Where sivelestat or dynasore was included, cells were pretreated for <NUM> minutes prior to imaging.

A549 cells grown to confluence (><NUM>%) in <NUM> well plates on coverslips were used and cultured as described above. Coverslips were transferred to POC mini and freshly isolated neutrophils added (<NUM>-<NUM> ×<NUM><NUM>). The coculture was allowed to settle for <NUM> prior to addition of PAF (platelet activating factor) (<NUM>, Sigma) for <NUM> then fMLP (formyl-met-leu-phe)(<NUM>, Sigma) (<NUM> calcium). For these experiments IQR was present throughout at <NUM>. Z-stack images for 3D reconstruction were captured every <NUM> overnight for <NUM>.

Images were acquired at the correct Nyquist sampling rate. Scanning area was reduced to minimum to allow quicker scanning times per z-section. Images were deconvolved using Hyugens Essential (<NUM> iterations maximum).

To induce lipopolysaccharide (LPS) lung inflammation, methods as detailed previously were used<NUM>. Briefly, LPS (<NUM> ug/mouse E. coli LPS) was instilled by direct intubation to induce a neutrophilic alveolitis. <NUM> hours later, BAL was obtained after euthanasia of mice with <NUM> ul aliquots of ice cold PBS into the exposed and intubated murine trachea on three occasions.

IQR4 (<NUM>) was incubated with of human neutrophil elastase (HNE; <NUM>. 3ug/ml) in reaction buffer (<NUM> Hepes buffer, pH <NUM>, <NUM> NaCl, <NUM>% Igepal CA-<NUM> (v/v)) with or without of sivelestat (<NUM>). The timecourse of fluorescence dequenching was followed for <NUM> with a fluorescence microplate reader (excitation <NUM>/<NUM>, emission <NUM>/<NUM>). A fluorescence increase is observed only in presence of HNE, and this is inhibited by the presence of sivelestat.

For experiments using neutrophil lysate, IQR4 (<NUM>) is incubated with freeze-thawed neutrophil lysate (<NUM>×<NUM><NUM> cells/ml) in IMDM with or without sivelestat (<NUM>). The timecourse of fluorescence dequenching was followed for <NUM> with a fluorescence microplate reader (excitation <NUM>/<NUM>, emission <NUM>/<NUM>).

Human neutrophils were isolated as described above and suspended in PBS with or without activating agents. A fibreoptic confocal system (<NUM> Cellvizio) was used to acquire images in eppendorfs of cells both in the presence of free FAM and IQRs and after washing cells of free FAM and IQR in media. Imaging was performed for <NUM> and representative still frames converted to bmp format.

Commercially available sheep were purchased. Sheep were sedated, intubated and ventilated. Cells: neutrophils, activated neutrophils, and activated monocytes were instilled (<NUM> million in <NUM>) into disparate ovine subsegments via direct visualisation (under bronchoscopy) to the <NUM>rd order bronchi and then a microcatheter was instilled into the working channel and the cells were visualised. As a control, <NUM> mls of IQR <NUM> was also insilled into a subsegment. Following this the microcatheter was replaced and <NUM> of <NUM> concentration of IQR2. <NUM> was instilled <NUM> later into the designated subsegments. Five minutes later, alveoscopy was performed by passing a alveoflex (fibreoptic bundle) down the working channel and live imaging commenced upon distal alveoscopy. Images were captured at <NUM> frames per second and representative frames converted to jpeg format.

An ex vivo ovine lung was ventilated and perfused with human blood (<NUM>% haematocrit). Following this, a bronchoscope was used to instill 500mcg of E. coli LPS into the upper right segment. PBS as control was instilled into the upper left segment. <NUM> hours later, IQR1. <NUM> was instilled (10mcg) into each segment and probe based confocal laser endomicroscopy was performed with immediate imaging.

All amino acids, Aminomethyl Polystyrene Resin (<NUM>. 23mmol/g, <NUM>-<NUM> mesh,<NUM>% DVB) and Rink Amide Linker were purchased from GL Biochem (Shangai) Ltd and NovaBiochem. <NUM>(<NUM>)-carboxyfluorescein was from NovaBiochem and Oxyma from Apollo Scientific.

Fmoc-Lys(Dde)-OH is prepared in <NUM> steps.

Synthesis of Dde-OH: Dimedone (<NUM>, <NUM> mmol, <NUM> eq), <NUM>-(Dimethylamino)pyridine (DMAP, <NUM>, <NUM> mmol, <NUM> eq) and <NUM>-(<NUM>-Dimethylaminopropyl)-<NUM>-ethylcarbodiimide hydrochloride (EDCI. HCI, <NUM>, <NUM> mmol, <NUM> eq) were dissolved in DMF (<NUM>). Acetic acid (<NUM>, <NUM> mmol, <NUM> eq) was added and the reaction was stirred overnight. The DMF was removed in vacuo, the residue was dissolved in EtOAc (<NUM>) and washed with <NUM> HCl (<NUM> × <NUM>) and water (<NUM> × <NUM>). The organic phase was dried over MgSO<NUM>, filtered, concentrated and dried in vacuo to give a yellowish solid (<NUM>, <NUM>%), which was used without further purification. (Ref: <NPL>).

Synthesis of Fmoc-Lys(Dde)-OH: Fmoc-Lys-OH. HCI (<NUM>, <NUM> mmol) was dissolved in H<NUM>O, N, N-diisopropylethylamine (DIPEA <NUM> eq, <NUM>, <NUM> mmol) was added and the resulting solid was collected by filtration and dried in a vacuum oven overnight. To a stirred suspension of Fmoc-Lys-OH (<NUM>, <NUM> mmol, <NUM> eq) in ethanol (<NUM>), Dde-OH (<NUM>, <NUM> mmol, <NUM> eq) and TFA (<NUM>µL, <NUM> mmol, <NUM> eq) were added. The reaction was refluxed for <NUM> hours. After the reaction mixture was cooled to room temperature, the solvent was removed in vacuo and the residue was dissolved in EtOAc (<NUM>), washed with <NUM> KHSO<NUM> (<NUM> × <NUM>) and <NUM> HCl (<NUM> × <NUM>). The organic phase was dried over MgSO<NUM>, filtered, and evaporated in vacuo. Fmoc-Lys(Dde)-OH was isolated by flash column chromatography (elute with <NUM>% acetic acid / ethyl acetate) and crystallised from ethyl acetate/hexane as an off white solid (<NUM>, <NUM>%).

Peptide coupling based on Fmoc deprotection strategy using solid support.

Fmoc-Rink Amide-PS resin was prepared using a <NUM>-[(<NUM>,<NUM>-dimethoxyphenyl)-(Fmoc-amino)methyl]phenoxyacetic acid (Rink amide linker) attached to aminomethyl PS resin (<NUM> mmol/g, <NUM>% DVB, <NUM>-<NUM> mesh). Thus Fmoc-Rink-amide linker (3eq) was dissolved in DMF (<NUM>) and Oxyma (3eq) was added and the mixture was stirred for <NUM>. DIC (3eq) was then added and the resulting mixture was stirred for further <NUM>. The solution was added to aminomethyl polystyrene resin (1eq) and shaken for <NUM> hours. The resulting resin was washed with DMF (<NUM>×<NUM>), DCM (<NUM>×<NUM>) and MeOH (<NUM>×<NUM>).

After the tri-branched monomer was attached using DIPEA/DMAP at r. overnight (Tet. <NUM>, <NUM>, <NUM>). <NUM>% hydrazine in DMF solution was used (x2) for <NUM> to deprotect the Dde-group.

All Fmoc deprotections are carried out with <NUM>% piperidine in DMF for <NUM>. The solution was then drained and the resin was washed with DMF (<NUM>×<NUM>), DCM (<NUM>×<NUM>) and MeOH (<NUM>×<NUM>). This procedure was repeated twice. The deprotection was monitored by the Kaiser test (primary amines) and the chloranil test for secondary amines.

Solid phase couplings: A solution of the appropriate Fmoc-amino acid or or Methyl red (MR) or <NUM>(<NUM>)-carboxyfluorescein (FAM) (10eq) and Oxyma (10eq) in DMF (<NUM>) was stirred for <NUM>. DIC (10eq) was then added and the resulting solution was stirred for further <NUM>. The solution was then added to the resin (1eq), pre-swollen in DCM (<NUM>), and the reaction mixture was shaken for <NUM> hours. The solution was drained and the resin washed DMF (<NUM>×<NUM>), DCM (<NUM>×<NUM>) and MeOH (<NUM>×<NUM>). The coupling reactions were monitored by the Kaiser and chloranil tests.

After the FAM coupling the resin was washed with <NUM>% piperidine to remove any fluorescein phenol esters.

The probe was then released from the resin using a cocktail of TFA/TIS/DCM (<NUM>/<NUM>/<NUM>) for <NUM>. To the filtrate, cold ether was added to precipitate the product.

Initial purification was done by ether precipitation. Cold ether was added and collected by centrifugation. Washing was repeated with cold ether <NUM> times.

Purification of the probe was performed on a RP-HPLC (HP1100) system equipped with a Discovery C18 reverse-phase column (<NUM> × <NUM>, <NUM>) with a flow rate <NUM>/min and eluting with <NUM>% HCOOH in H<NUM>O (A) and <NUM>% HCOOH in CH<NUM>CN (B), with a gradient of <NUM> to <NUM>% B over <NUM> and an initial isocratic period of <NUM> (tr = <NUM>).

Analysis of the probe was performed on a RP-HPLC (HP1260) system equipped with a Discovery C18 reverse-phase column (<NUM> × <NUM>, <NUM>) with a flow rate <NUM>/min and eluting with <NUM>% HCOOH in H<NUM>O (A) and <NUM>% HCOOH in CH<NUM>CN (B), with a gradient of <NUM> to <NUM>% B over <NUM> and an initial isocratic period of <NUM> (tr = <NUM>).

Initially we exposed quiescent and activated human neutrophils to a series of fluorophores and demonstrated the activation dependent uptake of fluorophores in human neutrophils. Only activated neutrophils or permeabilised neutrophils bound dye in a punctuate manner. Neutrophils were imaged continuously in the presence of the fluorophores (<FIG>). The active uptake of the fluorophores was inhibited by the presence of a dynamin inbitory agent (<FIG>) showing that this process was dynamin dependent. Importantly, monocytes and lymphocytes did not take up the dyes.

Having demonstrated high levels of endocytosis in neutrophils and cells such as macrophages. We applied IQR1 to macrophages and demonstrated no dequenching or fluorescent amplification of IQR1. This demonstrates that a closely related cell to neutrophils shows absolutely no dequenching despite having a high endocytic rate (<FIG>).

Having demonstrated the neutrophil specific staining by these dyes, we wished to optimise the profiles of the optical detection of neutrophil activation to permit high signal-to-noise ratios. In that regard, we and (more recently) others have shown that multivalent fluorescent peptide dendrimers, display the phenomenon of internal fluorescence quenching<NUM>,<NUM>. Dendrimers possess unique molecular architectures and dimensions compared to traditional linear polymers, are monodisperse, easy to synthesize and their sizes can be accurately controlled<NUM>. Additionally their biocompatibility, low toxicity and important ability to access the intracellular compartment<NUM>,<NUM>, supports their use as scaffolds for permitting fluorophore quenching and hence as potential 'smart' visualisable sensors of neutrophil activation as their quenching may inherently permit high signal to noise discrimination in the inflammatory milieu. Using carboxyfluorescein (FAM) or rhodamine (TAMRA) as exemplar fluorophores, we hypothesised that placing dyes spatially orientated as dendrimeric internally quenched reporters (IQRs) would generate neutrophil activation probes with high signal to noise ratios. Conceptually, the delivery of these probes to the inflammatory milieu would permit sufficient pericellular quenching to readily visualise individual cells which had undergone activation dependent accumulation of dyes. Hence alongside monomeric fluorophore, we synthesised dendrimers with three and six branches, called respectively IQR1 and IQR2 (<FIG>). As the FAM dendrimer displayed a brighter signal in vitro and as the fibreoptic confocal device used in this study was aligned to this wavelength, the FAM dendrimer was chosen to develop the other compounds instead of rhodamine. Initial studies showed that these structures permitted the detection of neutrophil uptake/internalisation (conformed by colocalisation with fluorescently conjugated dextran) (<FIG>). Again these structures permitted the cell specific detection of uptake/internalisation (<FIG>).

For the ultimate delivery of these optical reporters to the human lung, they would require enhanced solubility compared to the probes described above. Hence the enhanced solubility of these IQRs was achieved by incorporating a small series of peptides within the dendrimer backbone (IQR1. <NUM> and IQR2. <NUM>) or pegylating the probes (IQR1. <NUM> and IQR2. <NUM>) (<FIG>). As above, these were initially evaluated in vitro for selectivity against other cells commonly obtained from the inflammatory milieu; monocytes, macrophages and lymphocytes and epithelial cells. Again, we confirmed the cellular specificity of the probes using live confocal imaging (<FIG>). This was extended to freshly isolated cells from the BAL of patients with IPF. BAL analysis revealed a mixed inflammatory infiltrate, but again with only neutrophils activating the dendrimeric probe scaffold both before and after stimulation (<FIG>). Co-cultures of neutrophils and epithelial cells demonstrated that no probe activation occurred on epithelial cells despite <NUM> of exposure of epithelial cells to IQRs (<FIG>) Additionally and surprisingly, this activation dependent labelling was species specific (<FIG>).

Importantly, using an alternative structural strategy to produce an IQR, FRET probes did not permit direct visualisation of cell uptake, but this remains unexplained. (see <FIG>). Thus, these dendrimeric IQRs provide an optimised tool to directly assess neutrophil activation status in freshly isolated biological samples providing enhanced signal to noise ratios.

Experiments were then performed to ascertain dynamic activation in cells with the soluble dendrimeric reporters using live time-lapse confocal microscopy as performed using the monomeric dyes. Freshly isolated human neutrophils were continuously imaged before and immediately after stimulation (<FIG>) and demonstrated a striking, rapid increase in cell-associated fluorescence (<FIG>). To determine the precise cellular localization of fluorescence, live high resolution multi-stack images were acquired after stimulation of neutrophils. These showed prominent perimembranous activity alongside intracellular activation (<FIG>). This colocalized with fluorescent dextran confirming uptake by pinocytosis and again was dynamin dependent.

We compared the non-dendrimeric FAM, with the branched dendrimers of the present invention. As the dendrimer branches increased in number, the quenching increased as expected leading to an increase in the signal to noise ratio obtainable.

We performed experiments utilising latruniculin (actin cytoskeleton inhibitor) that showed increased amplification of fluorescence. These indicated that the IQR neutrophil specific signal may be affected by actin cytosleton rearrangements during the process of cell activation. We assume that the dequencing is due to the combination of endocytosis and degranulation. As such we tested key components of the neutrophil granule against IQR. No differences were seen with myeloperoxidase inhibitors, reactive oxygen species. The conclusion drawn was that the probes of the present invention requires both degranulation and also endocytosis.

Fundamental to applying the direct delivery of these probes for human clinical use, was the requirement to demonstrate no toxicity and in particular no pulmonary toxicity when delivered directly in to the lung. No cellular toxicity was observed in any of the cells we used (<FIG>). Additionally no pulmonary inflammatory response ensued upon direct intratracheal administration of milligrams of probe/kg to mice (<FIG>).

To develop a methodology for detecting activated neutrophils deep within lungs and in particular the alveolar space of humans in the future, it was imperative that we utilise a size relevant model. In that regard the ovine lung provided a potential model<NUM> system to assess the spatiotemporal visualization of activated human neutrophils. We used a strategy employing fibred confocal microendoscopy. This permits cellular resolution at the alveolar level in both humans and animals. Initial characterisation was performed with the Cellvizio fibres in eppendorfs. This clearly demonstrated superior quenching of the <NUM> branch, IQR <NUM>, with excellent signal to noise ratios (<FIG>). In particular that single free dyes that have previously been used to image pinocytosis would not permit such visualisation with fibreoptic confocal (<FIG>). Subsequently, human neutrophils were delivered by microcatheter into a defined subsegment of the ovine lung. Subsequently a minute quantity of IQR was instilled in the same segment (<NUM>µg). Confocal microendocscopy with Cellvzio <NUM> was performed in control segments (pre delivery of activated monocytes, quiescent neutrophils and probe alone) and were compared with imaging in segments that had received activated neutrophils (<FIG>). Only segments that had received activated neutrophils, clearly demonstrated fluorescent cells (<FIG>).

A further experiment (<FIG>) was performed to image recruited activated human neutrophils. In this experiment, an ex vivo ovine lung was ventilated and perfused with human blood. Following this, a subsegment of the lung received lipopolysaccharide to induce the recruitment of human neutrophils. 10µgγ of IQR1. <NUM> was delivered into a control segment and the LPS instilled segment. Only the LPS segment showed signal enhancement imaged using probe based confocal microendoscopy. This experiment conclusively demsonstarted that recruited activated neutrophils can be detected by IQR.

The method and timing of probe delivery are crucial to this approach. Only minute quantities are required (<100µg) and the timing of detection is within minutes. To image differential neutrophil pinocytosis, it is imperative that imaging is conducted immediately as delaying imaging may lead to pinocytic uptake by other cells within a few hours. The directed delivery of minute quantities foregoes any toxicity issues and direct instillation into the distal lung is ideally suited to confocal microendoscopy to permit molecular resolution. Proof of concept in vivo is demonstrated.

In summary, directly visualisable reporters of neutrophil activity provide a potential diagnostic tool. Especially in the setting of neutrophil dominant conditions such as acute lung injury. Developing bedside methodologies could aid in stratifying those patients who have chest x-ray infiltrates due to non inflammatory causes versus those with either sterile or infective lung injury. Confocal laser microendoscopy coupled with the delivery of small concentrations of molecular probes permits the visualisation deep within the lung and provides the detection platform for the delivery and imaging of such probes in humans.

Claim 1:
A dye construct for use in a method of diagnosing inflammation by imaging activated neutrophil cells in vivo using confocal endoscopy, the dye construct having any one of structures (I) to (V):
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
wherein:
AA of is any amino acid and n is from <NUM> to <NUM>;
(AA)n of (II) is a peptide sequence which is recognised by the enzyme neutrophil elastase;
Q* is a dark quencher;
R<NUM> is:
<CHM>
*F is carboxyfluorescein (FAM), or rhodamine,
wherein the dye construct, prior to activated neutrophil internalisation, displays substantially no detectable or only a low amount of fluorescence, but upon activated neutrophil internalisation displays an increase in fluorescence by a factor of <NUM> or more as compared to any fluorescence which is observed outside of the activated neutrophil using confocal endoscopy.