WO2016024104A1

WO2016024104A1 - Method for detecting inflammatory cells

Info

Publication number: WO2016024104A1
Application number: PCT/GB2015/052318
Authority: WO
Inventors: Dawn A SIM; Marcus FRUTTIGER; Adnan TUFAIL
Original assignee: UCL Business Ltd
Current assignee: UCL Business Ltd
Priority date: 2014-08-11
Filing date: 2015-08-11
Publication date: 2016-02-18
Anticipated expiration: 2017-02-11
Also published as: GB201414181D0

Abstract

The present invention provides a method for detecting inflammatory cells that have been recruited to a site of local inflammation in a subject, which comprises the systemic administration of indocyanine green (ICG) dye to the subject, wherein the presence of inflammatory cells which have taken up the ICG is determined.

Description

METHOD FOR DETECTING INFLAMMATORY CELLS

FIELD OF THE INVENTION The present invention relates to methods for detecting inflammatory cells and to methods for detecting and monitoring inflammation and inflammatory disease in a subject.

BACKGROUND TO THE INVENTION

Inflammation plays a key role in many diseases, including sight-threatening diseases of the eye. There is emerging evidence that ocular inflammation occurs not only in the form of uveitic syndromes, but that its cellular participants also act as drivers for pathological angiogenesis, the two main causes of blindness in Western industrialised countries, namely, age-related macular degeneration (AMD) and diabetic retinopathy.

Current methods for monitoring inflammation include direct visualisation, for example by endoscopy or PET scan and indirect measurements, for example via biomarker assays such as C-reactive protein assays.

Alternative techniques include Optical Coherence Tomography (OCT), which is a technique for obtaining sub-surface images of translucent or opaque materials at a resolution equivalent to a low-power microscope. The technique is limited to imaging 1 to 2 mm below the surface in biological tissue, because at greater depths the proportion of light that escapes without scattering is too small to be detected. It is not possible to determine the phenotype of cells imaged.

The current gold standard for cellular imaging in humans is the application of in vitro radionuclide labelling of leukocytes from the patient's own blood. Although this allows direct visualization of cell migration patterns, current imaging techniques do not allow sufficient resolution for tracking of single cells. In addition, these cells are labelled outside the circulation - i.e. peripheral venous blood is drawn from a patient, and its leukocytes isolated and tagged before being injected intravenously. These techniques are therefore accompanied by the risks and requirements associated with the ex vivo labelling of cells (i.e. infection and cost).

Thus there is a need for a method of detecting inflammatory cells which is not associated with the above disadvantages. SUMMARY OF ASPECTS OF THE INVENTION

The present inventors have determined that indocyanine Green (ICG) dye is taken up by circulating inflammatory cells when administered systemically to a subject and that cells which have taken up ICG can be detected at sites of local inflammation.

Thus, in one aspect the present invention provides a method for detecting inflammatory cells that have been recruited to a site of local inflammation in a subject, which comprises the systemic administration of indocyanine green (ICG) dye to the subject, wherein the presence of inflammatory cells which have taken up the ICG is determined.

In another aspect, the present invention provides a method for detecting inflammatory cells that have been recruited to a site of local inflammation in a subject, which comprises the steps;

i) administering indocyanine green (ICG) dye to the subject; and ii) determining the presence of cells which have taken up the ICG;

wherein the ICG is administered to circulating inflammatory cells as a slow-release formulation and wherein cells which have taken up the ICG are determined to be inflammatory cells.

In another aspect, the present invention provides a method for detecting inflammatory cells that have been recruited to a local site of inflammation in a subject, which comprises the steps;

wherein the ICG is administered in a manner that facilitates long-term systemic release, and wherein cells which have taken up the ICG are determined to be inflammatory cells. In yet another aspect, the present invention provides a method for detecting inflammatory cells that have been recruited to a local site of inflammation in a subject, which comprises the steps;

wherein the ICG is released in a manner which enables its uptake by circulating inflammatory cells, and wherein cells which have taken up the ICG are determined to be inflammatory cells. In one embodiment, the ICG may be administered as depot formulation.

In one embodiment, the ICG may be administered by intramuscular, intraperitoneal, subcutaneous, oral or suppository administration.

In one embodiment, the inflammatory cells may be monocytes, macrophages or lymphocytes. Therefore, in a preferred embodiment, the inflammatory cells may be circulating monocytes. In particularly preferred embodiment, the inflammatory cells may be CD1 1 b+ monocytes.

In another embodiment, the inflammatory cells may be detected in the eye of the subject. Thus, in a preferred embodiment, the inflammatory cells may be detected in the retina and/or the choroid.

In one embodiment, the subject has or is at risk of a disease associated with ocular inflammation. Therefore, in a preferred embodiment the disease may be selected from the group consisting of uvetis, age-related macular degeneration and diabetic retinopathy.

In one embodiment, the presence of inflammatory cells may be determined using a scanning laser ophthalmoscope.

In yet another embodiment, the presence of inflammatory cells may be determined between 2 and 14 days after the administration of ICG.

In one embodiment, the subject may be a human subject, and the ICG may be administered at a dose of about 50 mg. In another embodiment, the ICG may be administered on multiple occasions and the presence of inflammatory cells may be determined between ICG administrations. In a preferred embodiment, the multiple ICG administrations may be performed at an interval of 5 to 7 days. In one aspect, the present invention provides a method for diagnosing an inflammatory disease in a subject which comprises detecting inflammatory cells according to any one of the methods disclosed herein; wherein the detection of inflammatory cells indicates an inflammatory disease.

In another aspect, the present invention provides a method for monitoring the progression of an inflammatory disease in a subject, which comprises detecting inflammatory ceils according to any one of the methods disclosed herein on multiple occasions; wherein an increase in the levels of inflammatory cells detected indicates a worsening of the inflammatory disease and a decrease in the levels of inflammatory cells detected indicates an improvement in the inflammatory disease.

In yet another aspect, the present invention provides a method for determining the efficacy of an anti-inflammatory agent which comprises the steps of:

a) administering the agent to the subject; and

b) detecting inflammatory cells by the method according to the present invention;

wherein steps a) and b) may be performed in any order.

In one embodiment, the agent may be administered after administration of ICG and before determining the presence of cells which have taken up the ICG.

In another embodiment, the method may comprise:

c) performing step b);

d) performing step a); and

e) repeating step b);

wherein a decrease in the level of inflammatory cells detected in step e) compared to step c) indicates efficacy of the anti-inflammatory therapy.

In one aspect, the present invention provides an ICG depot formulation which is suitable for use in the methods disclosed herein.

In another aspect, the present invention provides a composition comprising ICG and a pharmaceutically acceptable excipient which is suitable for depot administration.

In yet another aspect, the present invention provides ICG for use in detecting inflammatory ceils. In one embodiment, the presence of inflammatory cells is determined by a method disclosed herein. In a further aspect, the present invention provides a method for diagnosing an inflammatory disease, which comprises the steps of:

i) administering indocyanine green (ICG) dye to the subject in a manner that facilitates long-term systemic release of the ICG;

ii) in vivo imaging of a site of local inflammation;

iii) performing image analysis to detect and quantify ICG-positive inflammatory cells;

iv) comparing the number of ICG-positive inflammatory cells to a control or reference level; and

v) making a diagnosis of an inflammatory disease;

wherein the ICG is released in a manner which enables its uptake by circulating inflammatory cells, and wherein cells which have taken up the ICG are determined to be inflammatory cells. In one embodiment, the inflammatory disease may be associated with the recruitment of circulating inflammatory cells.

In another embodiment the control or reference level may be derived from a cohort predetermined not to have an inflammatory disease, and the subject may be diagnosed as having an inflammatory disease if the number of ICG-positive cells is greater than the control or reference level. Thus, in a preferred embodiment the control or reference level is derived from a cohort predetermined not to have an inflammatory disease, and the subject is diagnosed as having an inflammatory disease if the number of ICG-positive cells is at least 1.5, 2, 3, 5, 10, 100, 1000, 5000 or 10000-fold greater than the control or reference level.

In an alternate embodiment, the control or reference level may be derived from a cohort predetermined to have an inflammatory disease, and the subject may be diagnosed as having an inflammatory disease if the number of ICG-positive cells is within 1 , 2, 5, 10 or 20% of the control or reference level.

In one embodiment, the control or reference level may be stored in a database.

In one embodiment, the ICG may be administered by intramuscular, intraperitoneal, subcutaneous, oral or suppository administration. In one embodiment, the inflammatory cells may be monocytes, macrophages or lymphocytes. Thus, in a preferred embodiment the inflammatory cells may be circulating monocytes. In a particularly preferred embodiment the inflammatory cells may be CD1 1 b+ monocytes.

In one embodiment the site of local inflammation may be the eye, or a tissue thereof. Therefore, in a preferred embodiment, the tissue of the eye may be the retina and/or choroid. In one embodiment, the inflammatory disease may be selected from the group consisting of uvetis, age-related macular degeneration, diabetic retinopathy, infection (e.g. leptospirosis, lyme disease, syphilis, tuberculosis), ankylosing spondylitis, enthesitis, inflammatory bowel disease, multiple sclerosis, psoriatic arthritis, reactive arthritis, sarcoidosis and systemic lupus erythematosus.

In one embodiment, step (ii) may be performed with a scanning laser ophthalmoscope. In another embodiment, step (ii) may be performed between 2 and 14 days after step (i) In one embodiment, the subject may be a human subject and the ICG may be administered at a dose of about 50 mg

In one embodiment, steps (i) to (iv) may be performed on multiple occasions. In a preferred embodiment multiple ICG administrations are performed at an interval of 5 to 7 days.

In a final aspect, the present invention provides a method for diagnosing a disease associated with ocular inflammation in a subject, which comprises the steps of:

ii) in vivo imaging of the retina and/or choroid of the subject;

iv) comparing the number of ICG-positive inflammatory cells detected in the retina and/or choroid to a control or reference level; and

v) making a diagnosis of a disease associated with ocular inflammation; wherein the ICG is released in a manner which enables its uptake by circulating inflammatory cells, and wherein cells which have taken up the ICG are determined to be inflammatory cells. In one embodiment, the control or reference level may be derived from a cohort predetermined not to have a disease associated with ocular inflammation, and the subject may be diagnosed as having a disease associated with ocular inflammation if the number of ICG-positive cells detected in the retina and/or choroid is greater than the control or reference level. Thus in a preferred embodiment, the control or reference level may be derived from a cohort predetermined not to have a disease associated with ocular inflammation, and the subject may be diagnosed as having a disease associated with ocular inflammation if the number of ICG-positive cells detected in the retina and/or choroid is at least 1 .5, 2, 3, 10, 100, 1000, 5000 or 10000-fold greater than the control or reference level.

In an alternate embodiment, the control or reference level may be derived from a cohort predetermined to have a disease associated with ocular inflammation, and the subject may be diagnosed as having a disease associated with ocular inflammation if the number of ICG-positive cells detected in the retina and/or choroid is within 1 , 2, 5, 10 or 20% of the control or reference level.

DESCRIPTION OF THE FIGURES

Figure 1 - In vitro labelling of peripheral blood mononuclear cells (PB C) and splenocytes in human and mouse

(A) Blood smear of human blood incubated in a concentration of 6.25 pg/mL ICG for 30 minutes at room temperature. A proportion of cells stained with ICG were visualized on the near-infrared channel at 10X magnification and (B) 2 OX magnification. (C, D) An example of detecting ICG-stained human PBMCs by flow cytometry reveals labelling in around 5% of cells (mean 6.5%, SD +/- 1.3%, N=4). (E, F) In mouse PBMCs a smaller proportion of ICG-labelled cells were detected (mean 1 %, SD +/- 0.3%, N=4), whereas mouse splenocytes labelled more readily with ICG (mean 10.3%, SD +/- 0.7%, N=4) (G, H). Figure 2 - In vivo labelling of infiltrating inflammatory cells in two murine models of ocular inflammation (A) Inflammatory infiltration of the deep retina/choroid of an endotoxin-induced uveitis (EIU) model imaged using scanning laser ophthalmoscope with a near-infrared filter (790 nm diode excitation laser and 800 nm barrier filter). ICG-labelled cells were visualized as white dots throughout the 55 degree field of view after an intraperitoneal (ip) injection of ICG (5 days prior to imaging), and induction of systemic inflammation with an ip injection of lipopolysaccharide (2 days prior to imaging). (B) An image of the deep retina/choroid of the same mouse was taken using a blue-light filter (488 nm solid state excitation laser and 500 nm barrier filter). No white dots are present demonstrating that white dots imaged in (A) are not a consequence of autofluorescence but of ICG-labelled cells. (C) Control mouse (no induction of inflammation) that received ip ICG (3 days prior to imaging). Imaging with a near- infrared filter showed only a few sporadic ICG-labelled cells suggesting a low level circulation of inflammatory cells into the retina. (D) An image of the deep retina/choroid of the same mouse was taken using a blue-light filter showing that white dots were not visible. (E) Inflammation of the retina vein (vasculitis) is visualised using a near-infrared filter in an experimental autoimmune uveitis (EAU) model. ICG- labelled cells were visualized as white dots clustering around a segment retina vasculitis. EAU models received an injection of ip ICG 7 days prior to imaging, and an intraocular injection of uveitogenic proteins 3 days prior to imaging. (F) An infrared- reflectance image (820 diode excitation laser, no barrier filter) of the same mouse was taken, which demonstrates the segment of retinal vein affected by vasculitis with increased reflectance (higher white intensity) of the vein itself, and surrounding tissues. Of note, no white dots were observed in this image, indicating that again, the white dots observed in (C) are not resultant of autofluorescence but of ICG-labelled cells.

Figure 3 - In vivo labelling of infiltrating inflammatory cells in a laser-induced choroidal neovascularisation (CNV) murine model

The scanning laser ophthalmoscope with a near-infrared filter (790 nm diode excitation laser and 800 nm barrier filter) was used for purpose of imaging the retina and choroid. (A) A deep retinal/choroidal image of a control animal that did not receive an intraperitoneal (ip) injection of ICG. No fluorescence was detected using the near-infrared filter. (B) A deep retinal/choroidal image of an animal which received ip ICG and laser-induction of CNV sequentially showing fluorescence in the retina vessels and laser lesions. No obvious leakage of ICG could be seen in the surrounding retinal tissues. (C) A deep retinal/choroidal image of an animal which received ip ICG (10 days prior to imaging) and laser-induction of CNV (7 days prior to imaging) showing an accumulation of ICG-labelled cells in and around the laser lesions. (C) Magnified image of laser lesion and surrounding cells. (D) A deep retinal/choroidal image of the same animal from (C) 3 days later (13 days after ip ICG) showing that the ICG signal has started to fade. (D') Magnified image of laser lesion showing the waning of ICG-signal from surrounding cells. (E) A deep retinal/choroidal image of an animal which received ip ICG (10 days prior to imaging) with six laser-induced CNV lesions (7 days prior to imaging) showing accumulation of ICG-labelled cells in and around all six laser lesions. (F, G) Corresponding fluorescein angiography images of the superficial retina and deep retina/choroid taken 10 minutes after injection with 100 μΙ_ of fluorescein dye, and imaged with a blue-light filter (488 nm solid state excitation laser and 500 nm barrier filter). (G) Deep retinal/choroidal images show that only two of six laser lesions subsequently developed choroidal neovascularisation, and none of these one-week old lesions showed obvious fluorescein dye leakage into the surrounding tissues suggesting that inflammatory cellular infiltration occurred independently of vascular leakage.

Figure 4 - In vivo monitoring of inflammation

(A) ICG was injection intraperitoneally at day 0 before laser-CNV induction at day 3 in C57B6 mice. Imaging with the scanning laser ophthalmoscope using a near-infrared filter (790 nm diode excitation laser and 800 nm barrier filter) was performed on day 5, 7 and 10. (C-E) Increasing cellular infiltration in and surrounding the laser-CNV lesion was observed over time. (B, C'-E') Mean intensity values and standard deviations quantified over a given area and showing an increase in inflammation over time.

Figure 5 - Quantification of ICG-labelled cells

(A) Autofluorescence image acquired using blue-light filter (488 nm solid state excitation laser and 500 nm barrier filter) showing laser-induced CNV lesions. (B) Fluorescein angiogram showing CNV formation and fluorescein leakage into surrounding tissues. (C) ICG-labelled cells in and around CNV lesions acquired with a near-infrared filter (790 nm diode excitation laser and 800 nm barrier filter). (D) An example of thresholding individual CNV lesions from (C). Simple pixel counting for quantification of ICG-labelled cellular infiltration was not possible due to the narrow range of intensity values of cells overlying the CNV. (E) Protocol for intraperitoneal ICG injection, laser-induction of CNV, and imaging with the scanning laser ophthalmoscope. (F) An inverted image of (C) showing that individual cells (black) can be identified qualitatively overlying CNV lesions. (G) A method for quantifying ICG-labelled cellular infiltration over individual CNV lesions (a-e) by the use of histograms.

Figure 6 - ICG dosing for the purpose of cellular labelling

(A) The experimental protocol is shown diagrammatically with intraperitoneal (ip) ICG administered at day 0, laser-CNV induction at day 3, and subsequent imaging at day 10. (B) Table showing ICG doses in a stepwise reduction in mice from 0.5 mg to 0.0125 mg in the left column, and the equivalent human dose in the right column. (C, D) A deep retinal/choroidal image of an animal that received 0.5 mg and 0.25 mg of ip ICG, respectively. Although ICG-labelled cells can be detected, they were fainter compared to the higher dose of 1 mg used in previous experiments (not shown). (E- H) A deep retinal/choroidal image of an animal that received 0.125 mg, 0.05 mg, 0.025 mg, 0.0125 mg of ip ICG respectively. In these further dose reductions, CNV lesions were faintly fluorescent but individual cells could no longer be detected.

Figure 7 - Route of administration of ICG for the purpose of cellular labelling

(A-C) 1 mg of ICG was administered via the intraperitoneal, subcutaneous, and intravenous (tail vein) route at day 0 before laser-CNV induction at day 3 and imaging at day 13 respectively. (D,E) 1 mg of ICG was given by daily oral gavage and the equivalent added to the drinking water for 7 days respectively, before laser-CNV induction, and subsequent imaging 10 days later. The intraperitoneal route of administration (A) was the most effective for cellular labelling. Faint labelling could be detected with the subcutaneous route (B) but not with the intravenous (C), or oral routes of administration (D,E).

Figure 8 - Detection of in vivo ICG-labelled cells by flow cytometry

24 hours after an intraperitoneal injection of indocyanine green (ICG) dye, PBMCs and splenocytes were isolated from C57B6 mice in order to assess the proportion of cells labelled by ICG. (A) 1 .01 % of circulating mouse PBMCs were stained with ICG. This was statistically significant from control animals (p=0.001). (B) A higher proportion of splenocytes (3.48%) were stained with ICG. This was also statistically significant from control animals (p=0.002).

Figure 9 - Characterization of in vivo ICG-labelled cells in a laser-induced choroidal neovascularisation (CNV) murine model

(A) An infrared-reflectance image (820 diode excitation laser, no barrier filter) demonstrating the presence of laser-induced CNV lesions in the deep retina/choroid. (B) A deep retinal/choroidal image taken with a near-infrared filter (790 nm diode excitation laser and 800 nm barrier filter) showing ICG-labelled cells surrounding two laser-induced CNV lesions. (C) A magnified view of ICG-labelled cell surrounding the CNV lesion from the top lesion in (B). (D) A deep retinal/choroidal image of the same mouse in (B) 15 minutes and (E) 30 minutes after a tail vein injection of CD1 1 b-FITC. (F) A magnified view of CD-1 1 b-FTIC-labelled cells surrounding the CNV lesion from figure (E) showing co-labelled in most but not all ICG-labelled cells.

Figure 10 - Distribution of ICG within the abdominal and thorasic cavity after intraperitoneal administration

Dissection of mice was performed 7 days after intraperitoneal administration of 1 mg of indocyanine green (ICG). (A, B) In the thorasic cavity we observed ICG staining of lymphatic tissue along the thorasic wall and (C) mediastinal lymph nodes. (D-F) In the abdominal cavity, strong ICG staining of the greater omentum was observed.

Figure 11 - ICG labels bone marrow derived macrophages (BMD ) by internalization of the dye, but does not appear to cause activation

(A) When analysed by flow cytometry, in vitro labelled BMDMs (dark grey filled peak) appear more strongly labelled than CD4+ lymphocytes (light grey filled peak); unlabelled cells are represented by the dashed black peak (open). (B) Brightfield microscopy of BMDMs cultured for two hours with 0.2 mg/ml ICG identifies regions of visible ICG dye apparently within cells. Applying LPS, (C) 5 ng/ml LPS only and (D) LPS+ICG, did not lead to a marked difference in ICG uptake. (E) There is no evidence of classical activation, as measured by the production of IL-6 from supernatants taken across different time points. Data were combined from two separate experiments (means + SD shown). (F) BMDMs were incubated with 0.2 mg/ml ICG. Following several washes, PBS was incubated with the cells for 5 minutes, then tested by spectrophotometry and compared to supernatants from the same cells post-lysis with 2% Triton. Increasing time of ICG incubation showed increased absorption, consistent with the release of progressive internalized ICG.

Figure 12 - Assessment of cytotoxicity following exposure to ICG

No significant differences in cell death were noted in individual leukocyte populations exposed to ICG in both in vitro and in vivo settings, using flow cytometric analysis. (A) Murine splenocytes were either unlabelled (light grey bars) or labelled with 1.25 pg/mL ICG for 30 minutes (dark grey bars) in an in vitro setting. (B) Splenocytes were analysed 10 days following an intraperitoneal injection of phosphate buffered 760 saline (light grey bars) or ICG (dark grey bars). (C, D) Choroidal neovascularization was induced in control (light grey bars) and ICG-labelled (dark grey bars) 3 days following intraperitoneal administration. Cell death in individual leucocyte populations was determined 7 days following laser induction of choroidal neovascularization.

DETAILED DESCRIPTION

The present invention relates to a method for detecting inflammatory cells which involves administering ICG to a subject.

The term 'detect', as used herein, is synonymous with terms such as identify, observe and visualise. Thus the method of the present invention determines the presence of inflammatory cells based on the uptake of ICG by those cells.

INDOCYANINE GREEN (ICG)

ICG is a fluorescent dye which is used in medicine as an indicator substance (e.g. for photometric hepatic function diagnostics and fluorescence angiography) in cardiac, circulatory, hepatic and ophthalmic conditions.

ICG is also known as sodium 4-[2-[(1 E,3E,5E,7Z)-7-[1,1-dimethyl-3-(4- sulfonatobutyl)benzo[e]indol-2-ylidene]hepta-1 ,3,5-trienyl]-1 ,1-dimethylbenzo[e]indol- 3-ium-3-yl]butane-1 -sulfonate and its structure, in the form of a sodium salt, is provided herein as Structure (i).

Structure (i)

ICG sodium salt is normally available in powder form and can be dissolved in various solvents; 5% (<5% depending on batch) sodium iodide is usually added to ensure better solubility. The sterile lyophilisate of a water-ICG solution is approved in many European countries and the United States under the names ICG-Pulsion and IC- Green as a diagnostic for intravenous use. It is administered intravenously and, depending on liver performance, is eliminated from the body with a half-life of approximately 3-4 minutes.

The absorption and fluorescence spectrum of ICG is in the near infrared region, however, both spectra depend largely on the solvent used and the concentration. ICG absorbs mainly between 600 nm and 900 nm, with a peak spectral absorption at about 800 nm, and emits fluorescence between 750 nm and 950 nm. The large overlapping of the absorption and fluorescence spectra leads to a marked reabsorption of the fluorescence by ICG itself. The fluorescence spectrum is very wide. Its maximum values are -810 nm in water and -830 nm in blood. For medical applications based on absorption, the maximum absorption at -800 nm (in blood plasma at low concentrations) is important. In combination with fluorescence detection, lasers with a wavelength of around 780 nm are used. At this wavelength, it is still possible to detect the fluorescence of ICG by filtering out scattered light from the excitation beam.

Because of its plasma protein binding properties, ICG stays for up to 20-30 minutes in the vessels (intravasally). Thus when the eye is examined, it is retained in tissues with a higher blood flow, such as the choroid and the blood vessels of the retina. In ophthalmology, ICG angiography is therefore used for angiography of the ocular fundus. ICG is also used for non-invasive monitoring of perfusion in a number of tissues and organs (e.g. liver or splanchnic perfusion).

The present invention is based in part on the inventors surprising determination that ICG, when administered in a manner which facilitates long-term systemic release, is taken up by circulating inflammatory cells (i.e. leukocytes). The cells which have taken up ICG can be detected by a variety of known methods which are suitable for detecting fluorescent emission by ICG. This enables inflammation (i.e. the recruitment of systemic inflammatory cells to local sites) to be monitored. The method of the present invention may be performed using any dye which can be taken up by inflammatory cells following systemic administration to a subject and can subsequently enable the inflammatory cells which have taken up the dye to be detected at sites of local inflammation. Such dyes include, but are not limited to, ICG and Evans blue.

DETECTING INFLAMMATORY CELLS

The present invention comprises the step of determining the presence of cells which have taken up ICG. For example, the method of the present invention may involve determining the presence of cells which have taken up ICG at sites of localised inflammation.

In the method of the present invention, cells which have taken up ICG are determined to be inflammatory cells.

The term "inflammatory cell" is used herein to refer to an immune cell which may be recruited to local sites of inflammation. For example the inflammatory cell may be a leukocyte. The inflammatory cell may be a circulating leukocyte which can be recruited to sites of local inflammation. An illustrative list of cells which may take up ICG includes, but is not limited to, CD4⁺ cells, CD8⁺ cells, CD20⁺ cells and CD45⁺/CD1 1 b⁺ cells.

The inflammatory cell may be a monocyte, macrophage or lymphocyte. The inflammatory cell may be a CD457CD1 1 b⁺ monocytes.

The inflammatory cell may be circulating monocyte.

The term "cell which has taken up ICG" is synonymous with terms such as "ICG- positive cell" and "ICG-labelled cell" and refers to a cell which comprises ICG. An ICG-labelled cell is detectable by any method which is suitable for detecting ICG- derived fluorescence. The cell may uptake ICG via an active process. For example, the cell may uptake ICG via phagocytosis.

Cells which are positive for ICG may be detected using any method or device which is suitable for detecting ICG-derived fluorescence. As such, ICG-labelled cells may be detected using an endoscopic device comprising near-infrared filters capable of detecting ICG fluorescence. The endoscopy device used depends on the location of the area of potential inflammation which is to be investigated. For example the fields of gastroenterology, rheumatology and respiratory medicine involve the use of endoscopes, arthroscopes and bronchoscopes respectively. The method of the present invention may also be used in dermatology and dentistry. ICG fluorescence may be imaged using devices such as: photodynamic Eye (PDE) (Hamamatsu): SPY (Novadaq); FDPM imager (Texas); IC-view (Pulsion Medical); or FLARE (Israel Beth Deaconess Hospital). The method of the present invention may be used to determine the presence of inflammatory cells in the eye of a subject. Herein, ICG-positive cells may be detected using a scanning laser ophthalmoscope (SLO) with a near infrared channel.

SLOW RELEASE

ICG formulations presently used in medicine are for intravenous use. Depending on liver performance, ICG from these formulations is generally eliminated from the body with a half-life of approximately 3-4 minutes. In contrast, the method of the present invention may involve the administration of ICG as a slow-release formulation. The ICG may be released over a period of up to 1 week. For example the formulation may release ICG for up to 7, 5 or 4 days or for up to 72, 48, 36, 24 or 12 hours. In particular, in the method of the present invention the ICG is released in a manner which enables its uptake by circulating inflammatory cells, as described herein.

DEPOT FORMULATION

The method of the present invention may involve administering ICG as a depot formulation.

The term "depot formulation" refers to a formulation which releases the active agent (i.e. herein ICG) in a consistent manner over a long period of time. The depot formulation used in the present invention may release ICG over a period of up to 1 week. For example the formulation may release ICG for up to 7, 5 or 4 days or for up to 72, 48, 36, 24 or 12 hours.

The depot formulation for use in the present invention may be suitable for intramuscular, intraperitoneal, subcutaneous, oral or suppository administration. In a further aspect the present invention also provides an ICG depot formulation. The ICG depot formulation is suitable for use in a method according to the first aspect of the invention. Where the depot formulation is suitable for human use, the depot formulation may comprise more than 10 mg ICG. For example, the depot formulation may comprise 10-100 mg, 20-80, 30-70, 40-60 or about 50 mg ICG.

SUBJECT

The subject may be a human or animal subject. The subject may be a mammalian subject.

The subject may have an inflammatory disease, as described herein. 'Having an inflammatory disease' refers to a subject having at least one symptom associated with the condition.

The subject may be at risk of an inflammatory disease, as described herein. 'At risk of an inflammatory disease' refers to a subject who has not yet contracted an inflammatory disease and/or who is not showing any symptoms of the disease. The subject may have a predisposition for, or be thought to be at risk of developing, an inflammatory disease.

OCULAR INFLAMMATION

The method of the present invention may be used to detect inflammatory cells in the eye of a subject having, or at risk of, a disease associated with ocular inflammation.

The disease may be any disease which is associated with ocular inflammation. For example the disease may be uvetis, age-related macular degeneration or diabetic retinopathy.

The disease may be a systemic disease which is associated with ocular inflammation. For example the disease may be, but is not limited to, infection (e.g. leptospirosis, lyme disease, syphilis, tuberculosis), ankylosing spondylitis, enthesitis, inflammatory bowel disease, multiple sclerosis, psoriatic arthritis, reactive arthritis, sarcoidosis and systemic lupus erythematosus.

UVEITIS

'Uveitis' is used herein to refer to inflammation of the eye.

Uveitis is classified anatomically into anterior, intermediate, posterior, and panuveitic forms— based on the part of the eye primarily affected. Anterior uveitis, also known as iridocyclitis and iritis, is the inflammation of the iris and anterior chamber. Anywhere from two-thirds to 90% of uveitis cases are anterior in location. This condition can occur as a single episode and subside with proper treatment or may take on a recurrent or chronic nature. Symptoms include redness of the eye, blurred vision, photophobia, floaters (dark spots which float in the visual field) and headaches.

Intermediate uveitis, also known as pars planitis, consists of vitritis— which is inflammation of cells in the vitreous cavity, sometimes with 'snowbanking', or deposition of inflammatory material on the pars plana. Symptoms include floaters and blurred vision.

Posterior uveitis or chorioretinitis is the inflammation of the retina and choroid. Symptoms include floaters, photophobia and blurred vision.

Pan-uveitis is the inflammation of all the layers of the uvea.

AGE-RELATED MACULAR DEGENERATION Age-related macular degeneration (AMD) is the leading cause of severe visual loss in persons over 65 years old. It is associated with neovascularisation originating from the choroidal vasculature and extending into the subretinal space. Choroidal neovascularisation causes severe visual loss in AMD patients because it occurs in the macula, the area of retina responsible for central vision. The stimuli that lead to choroidal neovascularisation are not understood.

The clinical progression of AMD is characterised in stages according to changes in the macula. The hallmark of early AMD is drusen, which are accumulations of extracellular debris underneath the retina and appear as yellow spots in the retina on clinical exam and on fundus photographs. Depending on the size and quantity of the drusen and the simultaneous presence of other AMD features such as pigmentation changes of the retina, patients with early AMD may progress to advanced AMD. When patients are diagnosed with advanced AMD, which has two types that include geographic atrophy AMD and neovascular AMD, they typically have had noticeable vision loss.

Drusens are categorised by sizes as small (<63 pm), medium (63-124 pm) and large (>124 pm). They are also considered as hard or soft depending on the appearance of their margins on opthalmological examination. While hard drusens have clearly defined margins, soft ones have less defined and fluid margins. The Age-related Eye Disease Study (AREDS) fundus photographic severity scale is one of the main classification systems used for this condition. "Dry" or nonexudative AMD is characterised by discrete regional loss of the retinal pigment epithelium. Wet AMD results from the abnormal growth of blood vessels from the choriocapillaris (choroidal neovascularisation) through Bruch's membrane. The fragility of the blood vessels and inflammatory processes lead to subretinal haemorrhages and fibrovascular scarring. This process can occur de novo or as a progression of dry AMD. Although wet AMD is only responsible for 15% of the total AMD, it is responsible for more than 80% of AMD-related severe visual loss and blindness.

DIABETIC RETINOPATHY

Ocular manifestations of diabetes affect up to 80 percent of all patients who have had diabetes for 10 years or more, and 10 percent of diabetic patients will suffer from vision loss related to macular oedema. Diabetic macular oedema (DME) is caused by leaking macular capillaries and is the most common cause of visual loss in both proliferative and non-proliferative diabetic retinopathy.

In the first stage of diabetic retinopathy, which is called non-proliferative diabetic retinopathy (NPDR), macular oedema may occur in which blood vessels leak contents into the macular region. The symptoms of macular oedema are blurring, darkening or distortion of vision.

In the second stage of diabetic retinopathy, abnormal neovascularisation occurs at the back of the eye as a part of proliferative diabetic retinopathy (PDR). These new vessels are fragile due to their formation in a hypoxic environment and can easily burst and bleed, leading to blurred vision and possible retinal damage. Fibrovascular proliferation can cause tractional retinal detachment and the new blood vessels can grow into the angle of the anterior chamber of the eye and cause neovascular glaucoma. Diabetic retinopathy is the leading cause of blindness in adults of working age. In persons with diabetes mellitus, retinal capillary occlusions develop, creating areas of ischemic retina. Retinal ischemia serves as a stimulus for neovascular proliferations that originate from pre-existing retinal venules at the optic disk or elsewhere in the retina posterior to the equator. Severe visual loss in proliferative diabetic retinopathy (PDR) results from vitreous hemorrhage and tractional retinal detachment. Again, laser treatment (panretinal photocoagulation to ischemic retina) may arrest the progression of neovascular proliferations in this disease but only if delivered in a timely and sufficiently intense manner. Some diabetic patients, either from lack of ophthalmic care or despite adequate laser treatment, go on to sustain severe visual loss secondary to PDR. Vitrectomy surgery can reduce, but not eliminate, severe visual loss in this disease.

RETINA/CHOROID

The method of the present invention may be used to detect inflammatory cells in the retina and/or choroid of the subject.

The term "retina" is used herein according to its normal meaning to refer to the light- sensitive layer of tissue which lines the inner surface of the eye. The retina is a layered structure with several layers of neurons interconnected by synapses. Neurons that are sensitive to light are termed 'photoreceptors'. Neural signals from photoreceptors undergo processing by other neurons of the retina. The output takes the form of action potentials in retinal ganglion cells whose axons form the optic nerve. Several important features of visual perception can be traced to the retinal encoding and processing of light.

The term "choroid" is used herein according to its normal meaning to refer to the vascular layer of the eye between the retina and the sclera. The choroid is generally divided into four layers: (i) Haller's layer; (ii) Sattler's layer; (iii) choriocapillaris and (iv) Bruch's membrane and functions to provide oxygen and nutrients to the outer layer of the eye.

SCANNING LASER OPHTHALMOSCOPE

A scanning laser ophthalmoscope (SLO) is a confocal laser scanning microscope which is used for diagnostic imaging of the retina or cornea of the eye. Such devices and their use are well known in the art - see for example EP 1969996. The SLO is equipped to detect ICG. Examples of suitable devices include the SECTRALIS models HRA or HRA+OCT. REGIMES

The time period between the administration of ICG and determining the presence of ICG-positive cells may be vary according to different embodiments of the present invention. For example the presence of ICG-positive cells may be determined up to two weeks after the administration of ICG. The presence of ICG-positive cells may be determined up to one week after the administration of ICG. The presence of ICG- positive cells may be determined up to 72 or 48 hours after administration of the ICG.

ICG may be administered at a dose of about 50 mg for a human subject. For example, ICG may be administered at a dose of between 10-100 mg, 20-80, 30-70, 40-60 or about 50 mg ICG.

ICG may be administered to the subject on multiple occasions. The presence of ICG- positive cells may be determined between ICG administrations.

The multiple ICG administrations may be performed at an interval of up to 14 days. For example ICG administrations may be performed at an interval of up to 10, 7 or 5 days. ICG administrations may be performed at an interval of 5 to 7 days.

Where ICG is administered on multiple occasions, each dose may comprise 10-100 mg of ICG, or the total of doses in combination may comprise 10-100 mg of ICG. DIAGNOSING INFLAMMATORY DISEASE

In one aspect the present invention provides a method for diagnosing an inflammatory disease, which comprises detecting inflammatory cells by the method of the first aspect of the invention; wherein the detection of inflammatory cells indicates an inflammatory disease. The inflammatory disease may be any disease which is associated with the recruitment of circulating inflammatory cells to local sites of inflammation.

For example the inflammatory disease may be associated with appendicitis, arthritis, bronchitis, colitis, conjunctivitis, cystitis, dermatitis, encephalitis, gastritis, hepatitis, mastitis, meningitis, uveitis or poliomyelitits. The inflammatory disease may be associated with ocular inflammation. For example the disease may be uveitis, age-related macular degeneration or diabetic retinopathy. The disease may also be a systemic disease which is associated with ocular inflammation, as described herein. The detection of inflammatory cells which indicates an inflammatory disease may be determined by comparison to a control.

Reference to a "control" broadly includes data that the skilled person would use to facilitate the accurate interpretation of technical data. As such "control level" is interchangable with "reference level". In an illustrative example, the level or levels of ICG-positive cells are compared to the respective level or levels of ICG-positive cells in one or more cohorts (populations/groups) of control subjects selected from a cohort wherein the subjects have been diagnosed with a condition which is associated with inflammation at a particular site and a cohort wherein the subjects have been predetermined not to have a condition which is associated with inflammation at a particular site.

Where the control is derived from a cohort which has been predetermined not to have a condition which is associated with inflammation at a particular site, numbers/levels of ICG-positive cells which are or are at least 1.5, 2, 3, 5, 10, 100, 1000, 5000 or 10000-fold greater than the control level may indicate an inflammatory disease. Where the control is derived from a cohort in which the subjects have been diagnosed with a condition which is associated with inflammation at a particular site, numbers/levels of ICG-positive cells which are within 1 , 2, 5, 10 or 20% of the control level may indicate an inflammatory disease.

The control or reference levels for the detection of a given level/number of ICG- positive cells at a particular site may be stored in a database and used in order to interpret the results of the method as performed on the subject.

MONITORING THE PROGRESSION OF INFLAMMATORY DISEASE

In one aspect the present invention relates to a method for monitoring the progression of an inflammatory disease in a subject which comprises detecting inflammatory cells according to the method of the first aspect of the invention on multiple occasions, wherein an increase in the levels of inflammatory cells detected indicates a worsening of the inflammatory disease and a decrease in the levels of inflammatory cells detected indicates an improvement of the inflammatory disease. An increase or decrease in the level of inflammatory cells may indicate a 2, 3, 5, 10, 20, 100 or 1000-fold difference in the number of inflammatory cells detected at the same or equivalent site in the same subject at an earlier time point.

"Multiple occasions" indicates that the method of the first aspect of the present invention may be performed on at least one, two, three, four, five, ten or twenty occasions.

Herein, the control value may be the level/number of ICG-positive cells detected at the same, or equivalent, site in the same subject at an earlier time point. Thus, a temporal change in the level of the level/number of ICG-positive cells can be used to identify a progression of the inflammatory disease.

DETERMINING THE EFFICACY OF AN ANTI-INFLAMMATORY THERAPY

In a further aspect the present invention relates to a method for determining the efficacy of an anti-inflammatory agent.

An 'anti-inflammatory agent' refers to an entity which may be useful for reducing levels of inflammation in a subject. The method involves administering an agent to be assessed to a subject and detecting inflammatory cells at a local site of inflammation according to the method of the first aspect of the present invention.

As used herein "determining the efficacy of an anti-inflammatory agent" refers to determining if administration of the agent causes a decrease in the number of inflammatory cells detected at a site of inflammation.

In particular, a decrease in the number of inflammatory cells detected at a site of inflammation following administration of the agent is indicative that the agent is efficacious as an anti-inflammatory therapeutic.

As used herein "a decrease in the number of inflammatory cells" may refer to a reduction of 10, 20, 30, 40, 50, 70, 90 or 95% of number of inflammatory cells that were present before the agent was administered. "A decrease in the number of inflammatory cells" may indicate that essentially no cells that have taken up ICG can be detected following administration of the antiinflammatory agent. The method comprises three steps:

(a) administering an agent to be assessed to the subject;

(b) administering ICG to the subject; and

(c) determining the presence of cells which have taken up the ICG. Steps (a) and (b) may be performed in either order. Thus the method may be performed in the order (a) - (b) - (c) or (b) - (a) - (c).

Steps (b) and (c) are the same steps as performed in the method according to the first aspect of the invention. These steps may be performed according to any description as provided herein.

The method for determining the efficacy of an anti-inflammatory agent may therefore be described as comprising the steps of:

(a') administering the agent to be assessed to the subject; and

(b') detecting inflammatory cells by the method according to the first aspect of the invention;

wherein steps (a') and (b') may be performed in any order.

Step (b') may be performed both before and after (a'). Thus the method may be performed as (b') - (a') - (b').

Where (b') is performed both before and after (a'), a decrease in the number of inflammatory cells detected in the second iteration of (b') indicates that the antiinflammatory therapeutic is efficacious.

The detection of inflammatory cells may be performed up to two weeks after the administration of the anti-inflammatory agent. The presence of ICG-positive cells may be determined up to one week after the administration of the anti-inflammatory agent. The presence of ICG-positive cells may be determined up to 72 or 48 hours after administration of the anti-inflammatory agent.

Step (a') may represent an ongoing treatment course with an anti-inflammatory agent to be assessed. Herein, (b') may be performed on multiple occasions during the treatment course. For example (b') may be performed one, two, three, four, five, ten or twenty times during the treatment course. A decrease in the number of inflammatory cells detected in subsequent performances of (b') indicates that the antiinflammatory agent is efficacious.

COMPOSITION

In another aspect the present invention provides a composition comprising ICG and a pharmaceutically acceptable excipient which is suitable for depot administration.

The ICG may be administered with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as (or in addition to) the carrier, excipient or diluent, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), and other carrier agents.

USE

In a further aspect the present invention also provides the use of ICG for detecting inflammatory cells.

The present invention also provides ICG for use in detecting inflammatory cells in a subject.

The subject may have, or be at risk of, an inflammatory disease as described herein. The inflammatory disease may be a disease associated with ocular inflammation. The use may involve detecting inflammatory cells according to a method as provided by any aspect of the present invention.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

EXAMPLES

Example 1 - In vitro labelling of peripheral blood mononuclear cells (PBMCs) and splenocytes with ICG Whole blood was incubated with ICG for 30 minutes at room temperature and visual inspection by fluorescent microscopy of a blood smear from human blood revealed a small population of fluorescent cells in the NIR channel (Fig. 1A, B). PBMCs isolated from human and mouse blood, and mouse splenocytes were incubated with ICG (30 minutes at room temperature) and then ICG uptake and cell identity was analysed by flow cytometry. In human PBMCs, around 2-5% of all cells were stained with ICG. Of these, 50.5% co-stained with CD45 and CD1 1 b and >99% of the CD45+ CD11 b+ population were stained with ICG (Fig. 1 C, D). In mouse PBMCs <1 % of all cells were stained with ICG (Fig. 1 E, F), compared to 7-10% of mouse splenocytes (Fig. 1 G, H). Again, in these cells >90% of the CD45+ CD1 1 b+ population were stained with ICG.

Although the proportion of ICG-stained cells increased when incubated in higher temperatures and concentrations of ICG, there was an increase in non-specific staining (not shown). A 30 minute incubation of ICG at a concentration of 6.25 pg/mL at room temperature achieved reproducible staining of cells. Furthermore, the minimum dose in which cells could be stained reproducibly was 5 μg/mL, and the minimum dose in which ICG-stained could be detected was 1.56 pg/mL

In vitro, the specificity of ICG-binding to PBMCs was dependent upon concentration, ambient temperature, and period of incubation.

Example 2 - In vivo labelling of inflammatory cells with ICG

1 mg of ICG was administered by intraperitoneal (ip) injection to C57BL6 mice 3 days before they were used in three disease models that are known to trigger invasion of inflammatory cells into the retina. The first model was an endotoxin-induced uveitis (EIU) model based on systemic delivery of lipopolysaccharide (LPS). Two days after LPS administration (and 5 days after ip ICG) ICG labelled cells were identified in the retina by in vivo imaging using fluorescence scanning laser ophthalmoscopy (Fig. 2A), no signal was seen in the fluorescein channel (Fig. 2B). Control animals that were only injected with ICG but not with LPS showed only a few sporadic ICG positive cells (Fig. 2C, D). This suggests a low level invasion of peripheral cells into normal retina, which is dramatically increased after LPS stimulation. The second model was an experimental autoimmune uveoretinitis model based on inducing inflammation in the posterior segment of the eye by injecting uveitogenic proteins S-Ag (a 48-kD protein from the light signal transduction pathway), and IRBP (a 140-kD protein which transports vitamin A in the retinal outer segment) into the vitreous chamber. Unlike the EIU model which produces a diffused, short-lived inflammation in the deep retina and choroid, the prominent features of the EAU model include inflammation around blood vessels (vasculitis) and more sustained inflammation, lasting up to 6 weeks. ICG labelled cells could be visualized in the retina, choroid, and particularly in areas of active vasculitis seven days after immunisation of these animals (Fig. 2E, F).

The third model was the laser-induced choroidal neovascularisation (CNV) model, which is a widely used model to study pathological angiogenesis in the retina. The vascular response in this model is accompanied by an accumulation of inflammatory cells in the laser lesion from the periphery and from within the retina. Since laser injury can induce autofluorescence initially animals were imaged without ICG. In the NIR channel, no fluorescence was detected in the lesion (Fig. 3A). Administration (ip) of ICG immediately after laser was assessed. Although ICG could be visualised the retinal vasculature and laser lesions, overt leakage of ICG into the retina was not apparent (Fig. 3B).

To ensure that there was sufficient time for ICG-labelling of cells in the circulation, and the clearance of ICG from the circulation, ICG was injected 3 days before laser- induction of CNV, which is sufficient for minimizing any ICG in the circulation that may directly leaking into the retina from the blood stream and labelling resident retinal macrophages. In keeping with this, ICG could no longer be detected in the circulation by NIR imaging at this stage (not shown).

7 days after laser (10 days after ICG) a marked accumulation of clearly labelled cells was observed in and around the laser lesions (Fig. 3C, C). After a further 3 days (10 days after laser) the signal started to wane (Fig. 3D, D'). The invasion of ICG positive cells was independent of the angiogenic response. This is illustrated by an example shown in Fig. 3E-G. Here 6 laser lesions were applied. Although all of them accumulated ICG positive cells (Fig. 3E), only 2 lesions showed signs of neovascularisation (Fig. 3G) based on fluorescein angiography (Fig. 3F, G).

The cellular infiltration around the laser-induced CNV lesion was monitored over time. ICG was administered (ip) 3 days prior to laser, and animals were imaged subsequently 2, 5, and 8 days after laser CNV induction (Fig. 4). The number of ICG- labelled cells in and surrounding the CNV lesion could be observed qualitatively to increase over time (Fig. 4C-E). Example 3 - Quantification of cellular infiltration by ICG labelling

A simple pixel count was performed by thresholding individual CNV lesions (Fig. 5). However, due to the narrow range of intensity values within each lesion, it was not possible to visualize individual cells overlying CNV lesion. However, assessing the mean intensity values for a given pixel area using histograms enabled the quantification of cellular infiltration over CNV lesions (Fig. 5G). Using this method, ICG-labelled cellular infiltration can be quantified and monitored over time (Fig. 4B, C'-E'). Example 4 - Assessment of ICG administration routes

Because ICG is routinely used in clinical practice, the dosage and delivery routes normally used in humans (5 mg iv bolus injection) was compared with the mouse protocol described above (1 mg ip bolus injection).

The dosage of ICG was reduced in a stepwise fashion, from 1 mg to 0.0125 mg, which roughly equates to the human dose in ophthalmic use. Invading cells could still be readily detected after administration of 0.5 mg ICG ip but with 0.25 mg they were fainter. With further reduced amounts of ICG (0.125 mg, 0.05 mg, 0.025 mg and 0.0125 mg), the lesions were faintly fluorescent but individual cells could no longer be detected (Fig. 6). Out of the different delivery routes tested, ip was the most efficient at labelling cells (Fig. 7A). A subcutaneous depot (1 mg) produced weaker labelling, but individual cells could still be detected (Fig 7B), whereas iv (1 mg) delivery only very faint staining (Fig. 7C). Oral administration (by gavage or in the drinking water) did not lead to any labelling (Fig. 7D, E).

Example 5 - Characterizing in vivo ICG-labelled cells PBMCs and dissociated spleen were analysed by flow cytometry 24 hours after ICG administration (1 mg ip). In control animals isolated PBMCs did not contain any ICG, whereas PBMCs from ICG injected animals contained a small population (0.3 to 1 %) of ICG labelled cells (Fig. 8A). Of these, 22.8% also stained for CD45+ CD1 1 b+. In the CD45+ CD1 1 b+ population 7.9% were stained with ICG. The spleen, which is known for its role as a reservoir for circulating monocytes, also contained a larger population of ICG-labelled cells (2-5%), compared to circulating PBMCs (Fig. 8B). 2.4% of the CD45+ CD11 b+ splenocytes were stained with ICG.

These findings further confirm the in vitro finding that ICG is taken up by CD45+, CD1 1 b+ monocytes/macrophages. However, it also highlights that ICG may stain other inflammatory cells. A proportion of CD4, CD8, and CD20 which were included in the exclusion channel (R-Phycoerythrin) were also stained with ICG. Furthermore, dissection of the mice 7 days after ICG ip revealed "green" staining of lymphatic tissue in the thorasic cavity, mediastinal lymph nodes, thymus and the greater omentum in the abdominal cavity (Fig. 10). This suggests that inflammatory cells in the circulation, most likely both monocytes and lymphocytes that continuously circulate between the bloodstream and lymphoid organs can be labelled with ICG.

To identify the ICG positive cells invading the retina in the laser-induced CNV model an in vivo staining protocol was used. After ICG imaging (Fig. 9A-C) a fluorescently labelled antibody against CD11 b was injected. Over the course of 30 minutes this labelled a population of cells that spatially matched the ICG labelled cells (Fig. 9D-F). The Cd1 1 b signal was weaker than the ICG signal and there was not a perfect overlapping. Nevertheless, all Cd1 1 b positive cells were also ICG positive. The same approach was also taken with an anti CD45 antibody with the same outcome (not shown). This further confirms the identity of most of the ICG labelled cells in the retina as invading monocytes/macrophages.

MATERIALS AND METHODS In vitro labelling of peripheral blood mononuclear cells and splenocytes with ICG

Mouse blood was drawn via cardiac puncture with a 0.5 M EDTA-coated 23G needle, before either red cell lysis or Ficoll-gradient separation using Histopaque-1077 (Sigma Aldrich, UK) according to manufacturer's guidelines. Cells were stained with ICG, then incubated with Fc-block (BD Biosciences, UK) before primary antibody staining at manufacturer's recommended concentrations at 4°C for 20 minutes. All antibodies were from BD Biosciences.

Human PBMCs were isolated from 20 mL whole blood using Ficoll-gradient.

PBMCs were isolated from murine whole blood using Percoll-gradient separation, and splenocytes mechanically dissociated before being incubated in 6.25 g/mL ICG for 30 minutes at room temperature. PBMCs and splenocytes were washed and co- stained with CD45, CD11 b, CD3 fluorescent antibodies (Miltenyi Biotech, Bisley, UK). Flow cytometric analysis of ICG labeled cells

ICG stained PBMCs, whole blood and splenocytes were analysed using a BD Bioscience LSRII flow cytometer as no commercial machine was available with a near infra-red laser for ideal excitation of ICG. Sub-optimal excitation by the 633 nm red laser nonetheless still resulted in a reliable signal using a 780/60 bandpass filter. A minimum of 10,000 events was collected for each sample and fluorescence-minus- one controls were used to determine the placement of gates. Data was processed using FlowJo v10.1 (TreeStar, Ashton, Oregon, USA)

Animals

All animals were handled in accordance with the UK Animals (Scientific Procedures) Act 1986. Female C57BL/6J mice (Harlan, UK) at seven to eight-weeks of age were used. For in vivo procedures, the mice were anesthetized with an ip injection of medetomindine hydrochloride (1 mg/kg body weight; Domitor; Pfizer Animal Health, New York, NU), and ketamine (60 mg/kg body weight) in water. Pupillary dilation was achieved with 1 drop of 1 % Tropicamide (Bausch and Lomb, Surrey, UK).

Induction of Endotoxin Induced Uveitis

Female C57BL/6J mice (Harlan, UK) at eight-weeks of age received a single ip injection of 0.2 mg of lipopolysaccharide (LPS) from Escherichia coli (Sigma-Aldrich, St Louis, MO, USA) in phosphate-buffered saline (PBS). 1 mg ip bolus injection of ICG was given 24 hours prior to LPS treatment. Imaging was performed 48 hours later, at a previously determined time point where there is peak infiltration by myeloid cells. Induction of Experimental Autoimmune Uveoretinitis

Female C57BL/6J mice (Harlan, UK) at seven-weeks of age received 500 μg of human RBP-1-20 peptide subcutaneously, emulsified in complete Freund's adjuvant (Sigma Aldrich, UK) supplemented with 1 .5 mg/mL M. tuberculosis H37RA (Difco laboratories, BD, Oxford, UK). 1.5 pg of Pertussis toxin was simultaneously administered into the peritoneal space (Tocris Bioscience, Bristol, UK). Imaging was performed 26 days later at the time point determined to be disease peak in our facility using this protocol.

Induction of laser choroidal neovascularisation

In female C57BL/6J mice (Harlan, UK) at seven to eight-weeks of age, laser CNV was induced using a slit-lamp-mounted diode laser system (wavelength 680 nm; Keeler, Windsor, UK). Laser settings used: 200 mW power, 100 ms duration, and 100 μηη spot diameter. Laser CNV lesions were applied at a distance of 3 disc diameters from the optic nerve avoiding large blood vessels.

In vivo imaging

Ocular imaging was performed using a scanning laser ophthalmoscope (Spectralis™ HRA, Heidelberg Engineering, Heidelberg, Germany). A 55° field of view lens was used and a mean of 100 consecutive frames was taken for each image. In order to visualize ICG-labelled cells, the near infrared channel (ICG-channel, 790 nm diode excitation laser and 800 nm long-pass filter) was used. Cell labelling was achieved with various doses (1 mg to 0.0125 mg) (Fig. 6) of 5 mg ICG (Pulsion Medical Systems AG) dissolved in 5 mL of water and administered 3 days prior to laser induction of CNV, ip injection of LPS, imaging of the EAU model. The different routes of administration are summarized in Fig. 7. To assist the identification of laser CNV lesions, infrared-reflectance imaging was performed with a 820 diode excitation laser and no barrier filter. For autofluorescence imaging and fluorescein angiography the fluorescein channel (FA-channel, 488 nm solid state excitation laser and 500 nm long-pass filter) was used. Fluorescein angiography was performed 1 week after laser CNV induction with an ip injection of 0.2 mL fluorescein sodium (2%). Images were acquired at 90 seconds and 7 minutes after injection.

Quantification of ICG positive cells in the retina

Images were exported from the Heidelberg eye explorer version 1.7.1.0. and processed in Adobe Photoshop CS5 (Adobe Systems Incorporated, San Jose, USA) and analysed with ImageJ. Details of image processing and quantification can be found in the results section and Fig. 5.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled the art are intended to be within the scope of the following claims.

Various preferred features and embodiments of the present invention are now described with reference to the following numbered paragraphs. 1. A method for detecting inflammatory cells in a subject which comprises the steps;

wherein the ICG is administered as a slow-release formulation and wherein cells which have taken up the ICG are determined to be inflammatory cells.

2. A method according to paragraph 1 wherein the ICG is administered as a depot formulation. 3. A method according to paragraph 1 or 2 wherein the ICG is administered by intramuscular, intraperitoneal, subcutaneous, oral or suppository administration.

4. A method according to any preceding paragraph wherein the inflammatory cells are monocytes, macrophages or lymphocytes.

5. A method according to any preceding paragraph wherein the inflammatory cells are CD11b+ monocytes.

6. A method according to any preceding paragraph wherein the inflammatory cells are circulating monocytes.

7. A method according to any preceding paragraph wherein the inflammatory cells are detected in the eye of the subject. 8. A method according to paragraph 7 wherein the inflammatory cells are detected in the retina and/or the choroid.

9. A method according to paragraph 7 or paragraph 8 wherein the subject has or is at risk of a disease associated with ocular inflammation.

10. A method according to paragraph 9 wherein the disease is selected from the group consisting of uvetis, age-related macular degeneration and diabetic

retinopathy. 1 1. A method according to any one of paragraphs 7 to 10 wherein the presence of inflammatory cells is determined using a scanning laser opthalmolscope.

12. A method according to any preceding paragraph wherein the presence of inflammatory cells is determined between 2 and 14 days after the administration of

ICG.

13. A method according to any preceding paragraph, wherein the subject is a human subject, and ICG is administered at a dose of about 50 mg.

14. A method according to any preceding paragraph wherein ICG is administered on multiple occasions and the presence of inflammatory cells is determined between ICG administrations. 15. A method according to paragraph 14 wherein the multiple ICG administrations are performed at an interval of 5 to 7 days.

16. A method for diagnosing an inflammatory disease in a subject which comprises detecting inflammatory cells according to the method of any preceding paragraph; wherein the detection of inflammatory cells indicates an inflammatory disease.

17. A method for monitoring the progression of an inflammatory disease in a subject which comprises detecting inflammatory cells according to the method of any one of paragraphs 1 to 15 on multiple occasions; wherein an increase in the levels of inflammatory cells detected indicates a worsening of the inflammatory disease and a decrease in the levels of inflammatory cells detected indicates an improvement in the inflammatory disease. 18. A method for determining the efficacy of an anti-inflammatory agent which comprises the steps of:

a) administering the agent to the subject; and

b) detecting inflammatory cells by the method according to any one of paragraphs 1 to 15;

wherein steps a) and b) may be performed in any order. 19. A method according to paragraph 18, wherein the agent is administered after administration of ICG and before determining the presence of cells which have taken up the ICG. 20. The method according to paragraph 18 which comprises;

c) performing step b);

d) performing step a); and

e) repeating step b);

21. An ICG depot formulation which is suitable for use in the method according to any one of paragraphs 1 to 15. 22. A composition comprising ICG and a pharmaceutically acceptable excipient which is suitable for depot administration.

23. ICG for use in detecting inflammatory cells. 24. The use according to paragraph 23, wherein the presence of inflammatory cells is determined by the method as defined in any one of paragraphs 1 to 20.

Claims

1. A method for detecting inflammatory cells that have been recruited to a site of local inflammation in a subject, which comprises the systemic administration of indocyanine green (ICG) dye to the subject, wherein the presence of inflammatory cells which have taken up the ICG is determined.

2. A method for detecting inflammatory cells that have been recruited to a site of local inflammation in a subject, which comprises the steps;

i) administering indocyanine green (ICG) dye to the subject; and

ii) determining the presence of cells which have taken up the ICG;

wherein the ICG is administered to circulating inflammatory cells as a slow-release formulation and wherein cells which have taken up the ICG are determined to be

inflammatory cells.

3. A method for detecting inflammatory cells that have been recruited to a local site of inflammation in a subject, which comprises the steps;

i) administering indocyanine green (ICG) dye to the subject; and

ii) determining the presence of cells which have taken up the ICG;

wherein the ICG is administered in a manner that facilitates long-term systemic release, and wherein cells which have taken up the ICG are determined to be inflammatory cells.

4. A method for detecting inflammatory cells that have been recruited to a local site of inflammation in a subject, which comprises the steps;

i) administering indocyanine green (ICG) dye to the subject; and

ii) determining the presence of cells which have taken up the ICG;

wherein the ICG is released in a manner which enables its uptake by circulating

inflammatory cells, and wherein cells which have taken up the ICG are determined to be inflammatory cells.

5. A method according to any preceding claim, wherein the ICG is administered as a depot formulation.

6. The method according to any preceding claim, wherein the ICG is administered by intramuscular, intraperitoneal, subcutaneous, oral or suppository administration.

7. The method according to any preceding claim, wherein the inflammatory cells are monocytes, macrophages or lymphocytes.

8. The method according to any preceding claim, wherein the inflammatory cells are circulating monocytes.

9. The method according to any preceding claim, wherein the inflammatory cells are CD11 b+ monocytes.

10. The method according to any preceding claim, wherein the inflammatory cells are detected in the eye of the subject.

11. The method according to claim 10, wherein the inflammatory cells are detected in the retina and/or the choroid.

12. The method according to claim 10 or claim 11 , wherein the subject has or is at risk of a disease associated with ocular inflammation.

13. The method according to claim 12, wherein the disease is selected from the group consisting of uvetis, age-related macular degeneration and diabetic retinopathy.

14. The method according to any one of claims 10-13, wherein the presence of inflammatory cells is determined using a scanning laser ophthalmoscope.

15. The method according to any preceding claim, wherein the presence of inflammatory cells is determined between 2 and 14 days after the administration of ICG.

16. The method according to any preceding claim, wherein the subject is a human subject, and the ICG is administered at a dose of about 50 mg.

17. The method according to any preceding claim, wherein ICG is administered on multiple occasions and the presence of inflammatory cells is determined between ICG administrations.

18. The method according to claim 17, wherein the multiple ICG administrations are performed at an interval of 5 to 7 days.

19. A method for diagnosing an inflammatory disease in a subject which comprises detecting inflammatory cells according to the method of any preceding claim; wherein the detection of inflammatory cells indicates an inflammatory disease.

20. A method for monitoring the progression of an inflammatory disease in a subject, which comprises detecting inflammatory cells according to the method of any one of claims 1 to 18 on multiple occasions; wherein an increase in the levels of inflammatory cells detected indicates a worsening of the inflammatory disease and a decrease in the levels of inflammatory cells detected indicates an improvement in the inflammatory disease.

21. A method for determining the efficacy of an anti-inflammatory agent which comprises the steps of:

a) administering the agent to the subject; and

b) detecting inflammatory cells by the method according to any one of claims 1 to 18;

wherein steps a) and b) may be performed in any order.

22. The method according to claim 21, wherein the agent is administered after administration of ICG and before determining the presence of cells which have taken up the ICG.

23. The method according to claim 21 , which comprises:

c) performing step b);

d) performing step a); and

e) repeating step b);

24. An ICG depot formulation which is suitable for use in the method according to any one of claims 1 to 18.

25. A composition comprising ICG and a pharmaceutically acceptable excipient which is suitable for depot administration.

26. ICG for use in detecting inflammatory cells.

27. The use according to claim 26, wherein the presence of inflammatory cells is determined by the method as defined in any one of claims 1 to 23.

28. A method for diagnosing an inflammatory disease, which comprises the steps of: i) administering indocyanine green (ICG) dye to the subject in a manner that facilitates long-term systemic release of the ICG;

ii) in vivo imaging of a site of local inflammation;

v) making a diagnosis of an inflammatory disease;

wherein the ICG is released in a manner which enables its uptake by circulating inflammatory cells, and wherein cells which have taken up the ICG are determined to be inflammatory cells.

29. The method according to claim 28, wherein the inflammatory disease is associated with the recruitment of circulating inflammatory cells.

30. The method according to claim 28 or claim 29, wherein the control or reference level is derived from a cohort predetermined not to have an inflammatory disease, and wherein the subject is diagnosed as having an inflammatory disease if the number of ICG-positive cells is greater than the control or reference level.

31. The method according to any one of claims 28-30, wherein the control or reference level is derived from a cohort predetermined not to have an inflammatory disease, and wherein the subject is diagnosed as having an inflammatory disease if the number of ICG- positive cells is at least 1.5, 2, 3, 5, 10, 100, 1000, 5000, or 10000-fold greater than the control or reference level.

32. The method according to claim 28 or claim 29, wherein the control or reference level is derived from a cohort predetermined to have an inflammatory disease, and wherein the subject is diagnosed as having an inflammatory disease if the number of ICG-positive cells is within 1 , 2, 5, 10 or 20% of the control or reference level.

33. The method according to any one of claims 28-32, wherein the control or reference level is stored in a database.

34. The method according to any one of claims 28-33, wherein the ICG is administered by intramuscular, intraperitoneal, subcutaneous, oral or suppository administration.

35. The method according to any one of claims 28-34, wherein the circulating inflammatory cells are monocytes, macrophages or lymphocytes.

36. The method according to any one of claims 28-35, wherein the circulating inflammatory cells are CD11 b+ monocytes.

37. The method according to any one of claims 28-36, wherein the site of local inflammation is the eye, or a tissue thereof.

38. The method according to claim 37, wherein the tissue of the eye is the retina and/or the choroid.

39. The method according to claim 37 or claim 38, wherein the inflammatory disease is selected from the group consisting of uvetis, age-related macular degeneration, diabetic retinopathy, infection (e.g. leptospirosis, Iyme disease, syphilis, tuberculosis), ankylosing spondylitis, enthesitis, inflammatory bowel disease, multiple sclerosis, psoriatic arthritis, reactive arthritis, sarcoidosis and systemic lupus erythematosus.

40. The method according to any one of claims 37-39, wherein step (ii) is performed with a scanning laser ophthalmoscope.

41. The method according to any one of claims 28-40, wherein step (ii) is performed between 2 and 14 days after step (i).

42. The method according to any one of claims 28-41 , wherein the subject is a human subject and the ICG is administered at a dose of about 50 mg.

43. The method according to any one of claims 28-42, wherein steps (i) to (iv) are performed on multiple occasions.

44. The method according to claim 43, wherein multiple ICG administrations are performed at an interval of 5 to 7 days.

45. A method for diagnosing a disease associated with ocular inflammation in a subject, which comprises the steps of:

ii) in vivo imaging of the retina and/or choroid of the subject;

v) making a diagnosis of a disease associated with ocular inflammation;

46. The method according to claim 45, wherein the control or reference level is derived from a cohort predetermined not to have a disease associated with ocular inflammation, and wherein the subject is diagnosed as having a disease associated with ocular inflammation if the number of ICG-positive cells detected in the retina and/or choroid is greater than the control or reference level.

47. The method according to claim 45 or claim 46, wherein the control or reference level is derived from a cohort predetermined not to have a disease associated with ocular inflammation, and wherein the subject is diagnosed as having a disease associated with ocular inflammation if the number of ICG-positive cells detected in the retina and/or choroid is at least 1.5, 2, 3, 10, 100, 1000, 5000, or 10000-fold greater than the control or reference level.

48. The method according to claim 45, wherein the control or reference level is derived from a cohort predetermined to have a disease associated with ocular inflammation, and wherein the subject is diagnosed as having a disease associated with ocular inflammation if the number of ICG-positive cells detected in the retina and/or choroid is within 1 , 2, 5, 10 or 20% of the control or reference level.