WO2013045662A1 - Peptide margin imaging agents - Google Patents

Abstract

The present invention relates to labelled c-Met binding peptides suitable for optical imaging of tumour margins in vivo. The peptides are labelled with an optical reporter group suitable for imaging in the red to near-infrared region. Also disclosed are in vivo imaging methods, especially of use in the diagnosis of cancer and imaging of tumour margins during surgical resection.

Description

Peptide Margin Imaging Agents.

Field of the Invention.

The present invention relates to labelled c-Met binding peptides suitable for optical imaging of tumour margins in vivo. The peptides are labelled with an optical reporter group suitable for imaging in the red to near-infrared region. Also disclosed are in vivo imaging methods, especially of use in the diagnosis of cancer, and imaging of tumour margins during surgical resection. Background to the Invention.

Despite great advances in scientific knowledge and the development of various therapeutic modalities, surgery remains the most frequently used and single most effective treatment of solid tumours in their early stages. Physically removing the tumour reduces symptoms, reduces the chance of the cancer spreading, decreases the amount of cancer in the body and helps other treatments to be more effective. 60 to 70 percent of cancer patients will have surgery either by itself (40% of all cancers are treated with surgery alone), or in conjunction with other therapies (usually radiation therapy or chemotherapy). Surgery is used to diagnose, stage, treat or manage complications during the course of disease in more than 90% of all cancer patients. Whilst surgery is the oldest and most common form of cancer therapy, it is also the least standardized intervention, and there is a need for new tools to help tracking diseased organs and differentiating normal and cancerous tissues.

Surgeons traditionally depend on sight and touch (inspection and palpation) and any available pre-operative diagnostic imaging information to localize the tumour. Cancerous tissues are, however, often difficult to distinguish from normal tissues, or are too small to be detected (e.g. occult tumours). Thus, traditional surgical techniques do not ensure that all cancerous tissue has been found or removed and there is a need for agents, which can specifically identify cancer tissue, particularly tumour margins, with a very high resolution and sensitivity.

Wohrle et al [Makromol. Symp., 59, 17-33 (1992)] studied polymer-conjugation to porphyrin photosensitisers as a potential method of improving the uptake in target tissue in vivo for the photodynamic therapy of cancer. The polymers studied were rat serum albumin, synthetic polyethers and polyacohols. Wohrle et al concluded that the conjugation of a polymer carrier could improve the tumour uptake.

US 5,622,685 discloses that polyether-substituted anti-tumour agents comprising a porphyrin, phthalocyanine or naphthalocyanine exhibit improved properties for both in vivo tumour diagnosis and therapy. The polyether substituents comprise polyethyleneglycol (PEG) whose terminal hydroxyl group is etherified or esterified with Ci-12 alkyl or C_1-12 acyl groups respectively. US 6,083,485 and counterparts discloses in vivo near-infrared (NIR) optical imaging methods using cyanine dyes having an octanol-water partition coefficient of 2.0 or less. Also disclosed are conjugates of said dyes with "biological detecting units" of molecular weight up to 30 kDa which bind to specific cell populations, or bind selectively to receptors, or accumulate in tissues or tumours. The dyes of US 6,083,485 may also be conjugated to a range of "non-selectively bonding" macromolecules, such as polylysine, dextran, carboxydextran, polyethylene glycol, methoxypolyethylene glycol, polyvinyl alcohol, or a cascade polymer-like structure.

US 6,350,431 (Ny corned Imaging AS) discloses light imaging contrast agents having a molecular weight in the range 500 to 500,000 Da, comprising a polyalkylene oxide (PAO) of molecular weight 60 to 100,000 Da having at least two chromophores (i.e. dye molecules) linked thereto. The contrast agents of US 6,350,431 may further comprise a targeting vector. Yuan et al [Cancer Res., 55, 3752-3756 (1995)] studied the vascular permeability of human tumour cells to dye-labelled macromolecules, and concluded that tumour vessels are in general more leaky and less permselective than normal cells. The tumour cell permeability was reported to vary twofold in the macromolecule molecular weight range 25 kDa to 160 kDa.

Licha et al [SPIE Vol 3196 p. 98-102 (1998)] disclose contrast agents for in vivo fluorescence imaging which comprise poly(ethyleneglycol) (PEG) polymers based on methoxypolyethyleneglycol (MPEG). The conjugates thus have a heptamethine cyanine dye conjugated at one terminus of the PEG polymer and a methyl group at the other terminus. In a related publication [Licha et al, SPIE Vol 3196, p. 103-1 10 (1998)] describe tumour detection in animals using the above MPEG conjugates. In particular, the interest was in the effect of the molecular weight of the PEG conjugate on: (i) their tolerability; (ii) the pharmacokinetic behaviour; and (iii) the contrast between malignant and normal tissue. They observed that increasing molecular weight prolonged the blood circulation time in vivo. They concluded that increased retention in the tumour environment and improved tumour contrast was observed at later times for dye-MPEG conjugates with a molecular weight above 6 kDa. Montet et al [Radiology, 242(3), 751-758 (2007)] reported fluorescence molecular tomography (FMT) of angiogenesis using the near-infrared probes AngioSense 680 and AngioSense 750. These were described as high molecular weight (250 kDa) pegylated graft copolymers with an indocyanine-type fluorophore optimized for non- quenching. The agent contains MPEG attached to a polylysine backbone. Montet et al report that the agent exhibited a prolonged blood half-life (more than 5 hours), with no tumour extravasation up to 30 minutes post-administration, but increasing tumour uptake (and hence imaging brightness) with time thereafter.

Sadd et al [J. Control. Rel., 130, 107-1 14 (2008)] studied the characteristics of 3 different nanocarriers (linear polymer; dendrimers and liposome) on the efficacy of chemotherapy and imaging in vitro and in vivo. The linear polymer studied comprised a targeted PEG polymer of the type:

[LHRH]-[PEG polymer]-Cy5.5

where: LHRH is a synthetic analogue of luteinizing hormone-releasing peptide;

Cy5.5 is a specific cyanine dye.

The PEG polymer used had a molecular weight of about 3 kDa. Figure 4 (p. I l l) of Sadd et al compares the tumour uptake of the above conjugate with the non-targeted analogue, PEG-Cy5.5. Sadd et al concluded that the LHRH targeting polymer conjugate exhibits enhanced accumulation in cancer cells compared to the non- targeted analogue.

WO 2010/106169 discloses a method of in vivo optical imaging, of the margins around tumours, which comprises an optical imaging contrast agent. The optical imaging agents comprise conjugates of near-infrared dyes with synthetic poly ethylenegly col (PEG) polymers having a molecular weight in the range 15-45 kDa.

Hepatocyte growth factor (HGF), also known as scatter factor (SF), is a growth factor which is involved in various physiological processes, such as wound healing and angiogenesis. The HGF interaction with its high affinity receptor (c-Met) is implicated in tumour growth, invasion and metastasis. Knudsen et al have reviewed the role of HGF and c-Met in prostate cancer, with possible implications for imaging and therapy [Adv. Cancer Res., 91, 31-67 (2004)]. Labelled anti-met antibodies for diagnosis and therapy are described in WO 03/057155.

WO 2004/078778 discloses polypeptides or multiiiieric peptide constructs which bind c-Met or a complex comprising c-Met and HGF. Approximately 10 different structural classes of peptide are described. WO 2004/078778 discloses that the peptides can be labelled with a detectable label for in vitro and in vivo applications, or with a drug for therapeutic applications. The detectable label can be: an enzyme, fluorescent compound, an optical dye, a paramagnetic metal ion, an ultrasound contrast agent or a radionuclide. Preferred labels of WO 2004/078778 are stated to be radioactive or paramagnetic, and most preferably comprise a metal which is chelated by a metal chelator.

WO 2008/139207 discloses optical imaging agents which target c-Met in vivo, which comprise a peptide having the amino acid sequence shown labelled with an optical reporter imaging moiety suitable for imaging using light of wavelength 600-1200 nm:

Cys^X^Cys^X^Gly-Pro-Pro-X^Phe-Glu-Cys^Trp-Cys^Tyr-X^X^X⁶; wherein X¹ is Asn, His or Tyr;

X² is Gly, Ser, Thr or Asn;

X³ is Thr or Arg;

X⁴ is Ala, Asp, Glu, Gly or Ser;

X⁵ is Ser or Thr;

X⁶ is Asp or Glu;

and Cys^a"d are each cysteine residues such that residues a and b as well as c and d are cyclised to form two separate disulfide bonds. WO 2008/139207 relates to the discovery whether or not a tumour is present in a given patient.

It is critical that curative surgery does not leave behind any tumour even of microscopic size. Residual and occult tumour tissue, undetectable during primary surgery, might evolve into a recurring cancer. That is why surgeons must ensure that no tumour has been left behind and the "margins" around the excised tumour are negative. Margins, also known as "margins of resection," refer to the distance between a tumour and the edge of the surrounding tissue, which is expected to consist mainly of healthy cells, that is removed along with it. The excised tumour and surrounding tissue are subsequently examined by a pathologist in vitro. They are rolled in special ink so that the margins are clearly visible under a microscope. In clinical practice, the margins around a surgically-excised tumour are described as:

(i) positive margins: cancer cells extend out to the edge of the tissue, where the ink is;

(ii) negative margins: no cancer cells are found in the ink;

(iii) close margins: any situation that falls between positive and negative is considered "close". Knowing how close cancer cells are to the edge of the excised tissue helps in making patient treatment decisions. If the margins are positive, additional surgery is needed. If the margins are close, surgery may or may not be needed or more surgery and the addition of radiotherapy or chemotherapy might be necessary. If the margins are negative, surgery is sufficient. The definition of "negative margins" varies from one hospital to another. In some places, if there is even one normal cell between the ink and the cancer cells, this is considered a negative margin. In other places, the pathologist will require at least two millimeters of tissue without cancer cells between the ink and the tumour before using the category "negative margins". Typically, this analysis is performed after the surgery is complete so the identification of a "negative margin" before the patient has left the operating table would be of great benefit. The Present Invention.

The present invention provides a method of tumour margin imaging, using a contrast agent which targets c-Met. The contrast agent comprises a c-Met binding cyclic peptide of 17 to 30 amino acids conjugated to an optical reporter imaging moiety (IM). The imaging moiety is suitable for imaging the mammalian body in vivo using light of wavelength 500-1000 mil.

Such agents have been previously described (eg. in WO 2008/139207), to detect previously unknown tumour or pre-cancerous lesions within a human patient. The agent of the present invention help to ensure that known tumours are resected completely, based on clean margins being visible in real time during the surgical procedure. When there is a lack of uptake of the agents of the invention in the margin region of the resected patient intra-operatively, that demonstrates lack of c-Met activity, and hence in turn lack of cancerous or pre-cancerous tissues remaining non- excised within the patient. Conversely, if the margin region still shows uptake - then not all the cancerous/pre-cancerous tissue has been excised. This helps guide more effective surgical resection of the tumour. In addition, the excised tumour itself (or a portion thereof) can also be imaged ex vivo to further confirm the situation. The agent of the present invention is essentially fixed at the c-Met site within the tissue, and does not re-distribute during imaging or surgery. That is an advantage over tumour margin agents which localise via other mechanisms (e.g. due to trapping by low diffusion of high molecular weight species), since the margin region can still be imaged after the disturbance to the tissue caused by surgery. c-Met has a relatively uniform distribution within normal background tissue [Prat et a/, Int.J.Cancer, 49, 323-328 (1991)]. Consequently, elevated uptake within a given tissue or organ is a reliable indicator of tumourous tissue. Although a target to background ratio (tumour:background ratio) of 1.3 : 1 was observed in human colorectal cancer patients with the imaging agents of the present invention, that permits good image quality parameters in the fluorescence image in spite of the small concentration of imaging agent. The agent fluorescence is significantly stronger than tissue autofluorescence at the imaging time point, with imaging and detection at longer wavelengths to minimise the impact of tissue autofluorescence. The photobleachiiig half-life in tissue was measured to be over three hours when imaged with an imaging system providing 10 mW/cm² surface irradiance. Hence, imaging can be carried out at multiple time points, because each irradiation with light does not result in loss of fluorescent signal. In addition, blood is hypofluorescent at the imaging time point and any small amounts of blood present do not completely mask the fluorescence image.

The agents of the present invention exhibit a level of fluorescence which can be imaged in real time in vivo. Thus, the dye is robust to photobleachiiig and also 'bright' (fluorescence quantum efficiency and absorption are both relatively high) enabling imaging in real time. Simultaneous colour and fluorescence images can be produced in parallel by separating the fluorescence and white light spectral components.

The agents of the present invention exhibit the ability to view small millimetre sized lesions at up to a few mm depth below the surface being imaged.

Detailed Description of the Invention.

In a first aspect, the present invention provides a method of in vivo optical imaging of the tumour margins of a tumour of an animate subject, during or after surgical resection of said tumour, said method comprising:

(i) providing an optical imaging contrast agent suitable for in vivo imaging, said contrast agent comprising a conjugate of Formula (I):

(I)

(ii) generating an optical image of a region of interest of said subject to which said contrast agent has been administered, said region of interest comprising said tumour; where:

Z¹ is attached to the TV-terminus of cMBP, and is H or M^IG;

Z² is attached to the ( '-terminus of cMBP and is OH, OB^c, or M^IG,

where B^c is a biocompatible cation;

cMBP is a c-Met binding cyclic peptide of 17 to 30 amino acids which comprises the amino acid sequence (SEQ-1):

Cys^X^Cys^X^Gly-Pro-Pro-X^Phe-Glu-Cys^Trp-Cys^Tyr-X^X^X⁶; wherein X¹ is Asn, His or Tyr;

X² is Gly, Ser, Thr or Asn;

X³ is Thr or Arg;

X⁴ is Ala, Asp, Glu, Gly or Ser;

X⁵ is Ser or Thr;

X⁶ is Asp or Glu;

and Cys^a"d are each cysteine residues such that residues a and b as well as c and d are cyclised to form two separate disulfide bonds;

M^IG is a metabolism inhibiting group which is a biocompatible group which inhibits or suppresses in vivo metabolism of the peptide;

L is a synthetic linker group of formula -(A)_m- wherein each A is independently -CR₂- , -CR=CR- , -C≡C- , -CR₂C0₂- , -C0₂CR₂- , -NRCO- , -CONR- , -NR(C=0)NR-, -NR(C=S)NR-, -S0₂NR- , -NRS0₂- , -CR₂OCR₂- ,

-CR₂SCR₂- , -CR₂NRCR₂- , a C_4-8 cycloheteroalkylene group, a C_4-8 cycloalkylene group, a C_5-12 arylene group, or a C_3-12 heteroarylene group, an amino acid, a sugar or a monodisperse polyethyleneglycol (PEG) building block;

each R is independently chosen from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl,

C_1-4 alkoxyalkyl or C_1-4 hydroxyalkyl;

in is an integer of value 1 to 20;

n is an integer of value 0 or 1;

ΓΜ is an optical reporter imaging moiety suitable for imaging the mammalian body in vivo using light of wavelength 500-1000 niii, which is a cyanine dye of Formula III:

(III)

where:

R¹ and R² are independently H or SO₃M¹, and at least one of R¹ and R² is SO₃M¹, where M¹ is H or B^c;

R³ and R⁴ are independently C_1-4 alkyl or C_1-6 carboxyalkyl;

R⁵, R⁶, R⁷ and R⁸ are independently R^a groups;

wherein R^a is C_1-4 alkyl, C_1-6 carboxyalkyl or -(CH^_kSOsM¹, where k is an integer of value 3 or 4;

with the proviso that the cyanine dye has a total of 1 to 4 SO₃M¹ substituents in the R¹, R² and R^a groups.

By the term "imaging agent" or "contrast agent" is meant a compound suitable for imaging the mammalian body in vivo. Preferably, the mammal is a human subject. The imaging may be invasive (eg. intra-operative or endoscopic) or non-invasive.

By the term "optical imaging" is meant any method that forms an image for detection, staging or diagnosis of disease, follow up of disease development or for follow up of disease treatment based on interaction with light in the red to near-infrared region (wavelength 600-1000 11111). Optical imaging further includes all methods from direct visualization without use of any device and involving use of devices such as various scopes, catheters and optical imaging equipment, e.g. computer-assisted hardware for tomographic presentations. The modalities and measurement techniques include, but are not limited to: luminescence imaging; endoscopy; fluorescence endoscopy; optical coherence tomography; transmittance imaging; time resolved transmittance imaging; confocal imaging; nonlinear microscopy; photoacoustic imaging; acousto- optical imaging; spectroscopy; reflectance spectroscopy; interferometry; coherence interferometry; diffuse optical tomography and fluorescence mediated diffuse optical tomography (continuous wave, time domain and frequency domain systems), and measurement of light scattering, absorption, polarization, luminescence, fluorescence lifetime, quantum yield, and quenching. Further details of these techniques are provided by: (Tuan Vo-Dinh (editor): Biomedical Photonics Handbook (2003), CRC Press LCC; Mycek & Pogue (editors): Handbook of Biomedical Fluorescence (2003), Marcel Dekker, Inc.; Splinter & Hopper: An Introduction to Biomedical Optics (2007), CRC Press LCC

By the term "tumour margins" is meant the interstitial space on the periphery of the tumour between the tumour and the normal cells surrounding the bulk of the tumour. Any tumour cells present in the margin may facilitate tumour growth when the tumour is intact, or represent a risk of tumour re-growth following surgical removal of the tumour core mass.

By the term "animate subject" is meant a living mammalian patient, preferably a living human subject.

By the term "during surgical resection" is meant intraoperatively, i.e. in real time during surgery to remove a known or previously diagnosed tumour from within the patient. The terms '"excision" and "resection" have their conventional meaning and refer to cutting out of the tumour by surgery. By the term "after surgical resection" is meant either imaging the patient in vivo after the surgical resection of the tumour, or imaging the excised tumour (or a portion thereof) ex vivo.

By the term "optical reporter imaging moiety" (IM) is meant a fluorescent dye or chromophore which is capable of detection either directly or indirectly in an optical imaging procedure using light of wavelength 500-1000 mil). Preferably, the IM has fluorescent properties.

The term "region of interest" or ROI has its conventional meaning in the field of in vivo medical imaging.

The term "comprising" has its conventional meaning throughout this application and implies that the composition must have the components listed, but that other, unspecified compounds or species may be present in addition. The term 'comprising' includes as a preferred subset "consisting essentially of which means that the composition has the components listed without other compounds or species being present. By the term "c-Met binding cyclic peptide" (cMBP) is meant a peptide which binds to the hepatocyte growth factor (HGF) high affinity receptor, also known as c-Met (c- Met or hepatocyte growth factor receptor). Suitable cMBP peptides of the present invention have an apparent K_D for c-Met of c-Met HGF complex of less than about 20 nM. The cMBP peptides comprise proline residues, and it is known that such residues can exhibit cis trans isomerisation of the backbone amide bond. The cMBP peptides of the present invention include any such isomers.

The Z¹ group substitutes the amine group of the last amino acid residue of the cMBP. Thus, when Z¹ is H, the amino terminus of the cMBP terminates in a free NH₂ group of the last amino acid residue. The Z² group substitutes the carbonyl group of the last amino acid residue. Thus, when Z² is OH, the carboxy terminus of the cMBP terminates in the free C0₂H group of the last amino acid residue, and when Z² is OB^c that terminal carboxy group is ionised as a C0₂B^c group. By the term "metabolism inhibiting group" (M^I&) is meant a biocompatible group which inhibits or suppresses in vivo metabolism of the cMBP peptide at either the amino terminus (Z¹) or carboxy terminus (Z²). Such groups are well known to those skilled in the art and are suitably chosen from, for the peptide amine terminus:

V-acylated groups -NH(C=0)R^G where the acyl group -(C=0)R^G has R^G chosen from: Ci-6 alkyl, C_3-10 aryl groups or comprises a polyethyleneglycol (PEG) building block. Suitable PEG groups are described for the linker group (L), below. Preferred such PEG groups are the biomodifiers of Formula I A or IB. Preferred such amino terminus M^I& groups are acetyl, benzyloxycarbonyl or trifluoroacetyl, most preferably acetyl.

Suitable metabolism inhibiting groups for the peptide carboxyl terminus include: carboxamide, fert-butyl ester, benzyl ester, cyclohexyl ester, amino alcohol or a polyethyleneglycol (PEG) building block. A suitable M^I& group for the carboxy terminal amino acid residue of the cMBP peptide is where the terminal amine of the amino acid residue is V-alkylated with a C_1-4 alkyl group, preferably a methyl group. Preferred such M^IG groups are carboxamide or PEG, most preferred such groups are carboxamide. Formula I denotes that the -(L)_n[IM] moiety can be attached at any suitable position of Z¹, Z² or cMBP. For Z¹ or Z², the -(L)_n[IM] moiety may either be attached to the M^IG group when either of Z /Z is a M . When Z¹ is H or Z is OH, attachment of the -(L)_n[IM] moiety at the Z¹ or Z² position gives compounds of formulae [F ]-(L)_n- [cMBP]-Z² or Z¹-[cMBP]-(L)„-[IM] respectively. Inhibition of metabolism of the cMBP at either peptide terminus may also be achieved by attachment of the -(L)_n[IM] moiety in this way, but -(L)_n[IM] is outside the definition of M^IG of the present invention.

The -(L)_n- moiety of Formula I may be attached at any suitable position of the IM. The -(L)n- moiety either takes the place of an existing substituent of the IM, or is covalently attached to the existing substituent of the IM. The -(L)_n- moiety is preferably attached via a carboxyalkyl substituent of the IM.

By the term "biocompatible cation" (B^c) is meant a positively charged counterion which forms a salt with an ionised, negatively charged group, where said positively charged counterion is also non-toxic and hence suitable for administration to the mammalian body, especially the human body. Examples of suitable biocompatible cations include: the alkali metals sodium or potassium; the alkaline earth metals calcium and magnesium; and the ammonium ion. Preferred biocompatible cations are sodium and potassium, most preferably sodium.

By the term "amino acid" is meant an L- or D-amino acid, amino acid analogue (eg. naphthylalanine) or amino acid mimetic which may be naturally occurring or of purely synthetic origin, and may be optically pure, i.e. a single enantiomer and hence chiral, or a mixture of enantiomers. Conventional 3 -letter or single letter abbreviations for amino acids are used herein. Preferably the amino acids of the present invention are optically pure. By the term "amino acid mimetic" is meant synthetic analogues of naturally occurring amino acids which are isosteres, i.e. have been designed to mimic the steric and electronic structure of the natural compound. Such isosteres are well known to those skilled in the art and include but are not limited to depsipeptides, retro-inverso peptides, thioamides, cycloalkanes or 1,5- disubstituted tetrazoles [see M. Goodman, Biopolymers, 24, 137, ( 1985)]. By the term "peptide" is meant a compound comprising two or more amino acids, as defined above, linked by a peptide bond (ie. an amide bond linking the amine of one amino acid to the carboxyl of another). The term "peptide mimetic" or "mimetic" refers to biologically active compounds that mimic the biological activity of a peptide or a protein but are no longer peptidic in chemical nature, that is, they no longer contain any peptide bonds (that is, amide bonds between amino acids). Here, the term peptide mimetic is used in a broader sense to include molecules that are no longer completely peptidic in nature, such as pseudo-peptides, semi-peptides and peptoids.

The linker group (L) of Formula I, when present, suitably has a molecular weight of up to 1500 Daltons, preferably up to 1000 Daltons, more preferably up to 750 Daltons.

It is envisaged that one of the roles of the linker group L of Formula I is to distance the IM from the active site of the cMBP peptide. This is particularly important when the imaging moiety is relatively bulky, so that interaction with the target receptor is not impaired. This can be achieved by a combination of flexibility (e.g. simple alkyl chains), so that the bulky group has the freedom to position itself away from the active site and/or rigidity such as a cycloalkyl or aryl spacer which orientate the IM away from the active site. The nature of the linker group can also be used to modify the biodistribution of the imaging agent. Thus, e.g. the introduction of ether groups in the linker will help to minimise plasma protein binding.

When -(A)_m- comprises a polyethyleneglycol (PEG) building block or a peptide chain of 1 to 10 amino acid residues, the linker group may function to modify the pharmacokinetics and blood clearance rates of the imaging agent in vivo. Such "biomodifier" linker groups may accelerate the clearance of the imaging agent from background tissue, such as muscle or liver, and/or from the blood, thus giving a better diagnostic image due to less background interference. A biomodifier linker group may also be used to favour a particular route of excretion, eg. via the kidneys as opposed to via the liver. By the term "sugar" is meant a mono-, di- or tri- saccharide. Suitable sugars include: glucose, galactose, maltose, mannose, and lactose. Optionally, the sugar may be functionalised to permit facile coupling to amino acids. Thus, eg. a glucosamine derivative of an amino acid can be conjugated to other amino acids via peptide bonds. The glucosamine derivative of asparagine (commercially available from

NovaBiochem) is one example of this: Preferred features.

The molecular weight of the imaging agent is suitably up to 8000 Daltons. Preferably, the molecular weight is in the range 2000 to 6000 Daltons, most preferably 3000 to 4500 Daltons, with 3200 to 4000 Daltons being especially preferred.

Preferred imaging agents of the present invention have both peptide termini protected by M^IG groups, i.e. preferably both Z¹ and Z² are M^IG, which will usually be different. As noted above, either of Z Z² may optionally equate to -(L)_n[IM]. Having both peptide termini protected in this way is important for in vivo imaging applications, since otherwise rapid metabolism would be expected with consequent loss of selective binding affinity for c-Met. When both Z¹ and Z² are M^I&, preferably Z¹ is acetyl and Z² is a primary amide. Most preferably, Z¹ is acetyl and Z² is a primary amide and the -(L)_n[IM] moiety is attached to the epsilon amine side chain of a lysine residue of cMBP.

The cMBP peptide of Formula (I) is preferably functionalised only with Z¹, Z² and -(L)_n[IM]. Thus, the amino acid residues of the cMBP peptide are preferably not functionalised with macromolecular species such as polyethyleneglycol (PEG) polymers.

Preferred cMBP peptides of the present invention have a K_D for binding to c-Met or to a c-Met/HGF complex of less than about 10 nM (based on fluorescence polarisation assay measurements), most preferably in the range 1 to 5 nM, with less than 3nM being the ideal.

The peptide sequence (SEQ-1)

Cys^X^Cys^X^Gly-Pro-Pro-X^Phe-Glu-Cys^Trp-Cys^Tyr-X^X^X⁶

(SEQ-1)

of the cMBP of Formula I is a 17-nier peptide sequence, which is primarily responsible for the selective binding to c-Met. When the cMBP peptide of the present invention comprises more than 17 amino acid residues, the remaining amino acids can be any amino acid apart from cysteine. Additional, unprotected cysteine residues could cause unwanted scrambling of the defined Cys^a-Cys^b and Cys^c-Cys^d disulfide bridges. The additional peptides preferably comprise at least one amino acid residue with a side chain suitable for facile conjugation of the -(L)_n[IM] moiety. Suitable such residues include Asp or Glu residues for conjugation with amine-functionalised -(L)_n[IM] groups, or a Lys residue for conjugation with a carboxy- or active ester- functionalised -(L)_n[IM] group. The amino acid residues for conjugation of -(L)_n[IM] are suitably located away from the 17-mer binding region of the cMBP peptide (SEQ- 1), and are preferably located at the C- or N- terminus. Preferably, the amino acid residue for conjugation is a Lys residue.

Substitution of the tryptophan residue of SEQ-1 was evaluated with the known amino acid substitutes phenylalanine and napthylalanine. Loss of c-Met affinity was, however, found suggesting that the tryptophan residue is important for activity.

It is preferred that the cMBP peptide further comprises an TV-terminal serine residue, giving the 18-mer (SEQ-2):

Ser-Cys^X^Cys^X^Gly-Pro-Pro-X^-Phe-Glu-Cys^Trp-Cys^Tyr-X^X^X⁶.

(SEQ-2)

In addition to SEQ-1, or preferably SEQ-2, the cMBP most preferably further comprises either:

(i) an Asp or Glu residue within 4 amino acid residues of either the C- or N- peptide terminus of the cMBP peptide, and -(L)_nIM is functionalised with an amine group which is conjugated to the carboxyl side chain of said Asp or Glu residue to give an amide bond; (ii) a Lys residue within 4 amino acid residues of either the C- or TV- peptide terminus of the cMBP peptide, and -(L)_nIM is functionalised with a carboxyl group which is conjugated to the epsilon amine side chain of said Lys residue to give an amide bond.

Preferred cMBP peptides comprise the 22-mer amino acid sequence (SEQ-3):

Ala-Gly-Ser-Cys^X^Cys^X^Gly-Pro-Pro-X^Phe-Glu-Cys^Trp-Cys^Tyr- X⁴-X⁵-X⁶-Gly-Thr. (SEQ-3) The cMBP peptides of the present invention preferably have X³ equal to Arg.

The cMBP peptide preferably further comprises in addition to SEQ-1, SEQ-2 or SEQ-3, at either the N- or C- terminus a linker peptide which is chosen from:

-Gly-Gly-Gly-Lys- (SEQ-4), -Gly-Ser-Gly-Lys- (SEQ-5) or

-Gly-Ser-Gly-Ser-Lys- (SEQ-6).

The Lys residue of the linker peptide is a most preferred location for conjugation of the -(L)_n[IM] moiety. Especially preferred cMBP peptides comprise SEQ-3 together with the linker peptide of SEQ-4, having the 26-nier amino acid sequence (SEQ-7):

Ala-Gly-Ser-Cys^a-Tyr-Cys^c-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys^d-Trp-Cys^b- Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys. (SEQ-7)

The cMBP peptides of SEQ-1, SEQ-2, SEQ-3 and SEQ-7 preferably have Z¹ = Z² = M^I&, and most preferably have Z¹ = acetyl and Z² = primary amide.

The -(L)n[IM] moiety is suitably attached to either of the Z or Z groups or an amino acid residue of the cMBP peptide which is different to the c-Met binding sequence of SEQ-1. Preferred amino acid residues and sites of conjugation are as described above. When the -(L)_n[IM] moiety is attached to Z¹ or Z², it may take the place of Z¹ or Z² by conjugation to the N- or C- terminus, and block in vivo metabolism in that way. Preferred dyes of Formula III are chosen such that at least one C_1-6 carboxyalkyl group is present, in order to facilitate conjugation to the cMBP.

Preferred individual dyes of Formula III are summarised in Table 1 : Table 1 : chemical structures of individual cyanine dyes.

-(CH₂)₅COOH.

Especially preferred dyes of Formula II are Cy5** and Alexa647, with Cy5** being the ideal.

When a synthetic linker group (L) is present, it preferably comprises terminal functional groups which facilitate conjugation to [IM] and Z^fcMBPJ-Z². When L comprises a peptide chain of 1 to 10 amino acid residues, the amino acid residues are preferably chosen from glycine, lysine, arginine, aspartic acid, glutamic acid or serine. When L comprises a PEG moiety, it preferably comprises units derived from oligomerisation of the monodisperse PEG-like structures of Formulae IA or IB:

(IA)

17-amino-5-oxo-6-aza-3, 9, 12, 15-tetraoxaheptadecanoic acid of Formula IA wherein p is an integer from 1 to 10. Alternatively, a PEG-like structure based on a propionic acid derivative of Formula IB can be used:

(IB)

where p is as defined for Formula IA and q is an integer from 3 to 15.

In Formula IB, p is preferably 1 or 2, and q is preferably 5 to 12.

When the linker group does not comprise PEG or a peptide chain, preferred L groups have a backbone chain of linked atoms which make up the -(A)_m- moiety of 2 to 10 atoms, most preferably 2 to 5 atoms, with 2 or 3 atoms being especially preferred. A minimum linker group backbone chain of 2 atoms confers the advantage that the imaging moiety is well-separated so that any undesirable interaction is minimised.

In Formula I, n is preferably 0 or 1, most preferably 0, i.e. no linker group is present.

Preferred imaging agents of the present invention are of Formula IV:

M^IG-Ala-Gly-Ser-Cys^a-Tyr-Cys^c-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys^d-Trp- Cys^b-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys-M^IG

(L)„[IM]

(IV)

wherein the (L)_n[IM] group is attached to the epsilon amino group of the Lys residue. Preferred imaging agents of Formula IV have M^I& (N-terminal Ala) equal to acetyl and M^IG (C -terminal Lys) equal to primary amide. In Formula IV, n is preferably zero and F is preferably a cyanine dye, most preferably a cyanine dye of Formula II. Especially preferred imaging agents of Formula IV have IM = Cy5** or Alexa647, ideally Cy5**.

In the method of the first aspect, the contrast agent preferably comprises a pharmaceutical composition of the conjugate of Formula (I), together with a biocompatible carrier, in a form suitable for mammalian administration. The "biocompatible carrier" is a fluid, especially a liquid, in which the imaging agent can be suspended or dissolved, such that the composition is physiologically tolerable, ie. can be administered to the mammalian body without toxicity or undue discomfort. The concentration of the imaging agent in the biocompatible carrier can be adjusted to enable ease of administration to match e.g. either an intravenous bolus injection or a slow intravenous infusion. The biocompatible carrier is suitably an injectable carrier liquid such as sterile, pyrogen-free water for injection; an aqueous solution such as saline (which may advantageously be balanced so that the final product for injection is isotonic); an aqueous solution of one or more tonicity-adjusting substances (eg. salts of plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose), sugar alcohols (eg. sorbitol or mannitol), glycols (eg. glycerol), or other non-ionic polyol materials (eg. polyethyleneglycols, propylene glycols and the like). Preferably the biocompatible carrier is pyrogen-free water for injection or isotonic saline. The imaging agents and biocompatible carrier are each supplied in suitable vials or vessels which comprise a sealed container which permits maintenance of sterile integrity and/or radioactive safety, plus optionally an inert headspace gas (eg. nitrogen or argon), whilst permitting addition and withdrawal of solutions by syringe or cannula. A preferred such container is a septum-sealed vial, wherein the gas-tight closure is crimped on with an overseal (typically of aluminium). The closure is suitable for single or multiple puncturing with a hypodermic needle (e.g. a crimped-on septum seal closure) whilst maintaining sterile integrity. Such containers have the additional advantage that the closure can withstand vacuum if desired (eg. to change the headspace gas or degas solutions), and withstand pressure changes such as reductions in pressure without permitting ingress of external atmospheric gases, such as oxygen or water vapour.

Preferred multiple dose containers comprise a single bulk vial (e.g. of 10 to 30 cm³ volume) which contains multiple patient doses, whereby single patient doses can thus be withdrawn into clinical grade syringes at various time intervals during the viable lifetime of the preparation to suit the clinical situation. Pre-filled syringes are designed to contain a single human dose, or "unit dose" and are therefore preferably a disposable or other syringe suitable for clinical use. The pharmaceutical compositions of the present invention preferably have a dosage suitable for a single patient and are provided in a suitable syringe or container, as described above.

The pharmaceutical composition may optionally contain additional excipients such as an antimicrobial preservative, pH-adjusting agent, filler, stabiliser or osmolality adjusting agent. By the term "antimicrobial preservative" is meant an agent which inhibits the growth of potentially harmful micro-organisms such as bacteria, yeasts or moulds. The antimicrobial preservative may also exhibit some bactericidal properties, depending on the dosage employed. The main role of the antimicrobial preservative(s) of the present invention is to inhibit the growth of any such micro-organism in the pharmaceutical composition. The antimicrobial preservative may, however, also optionally be used to inhibit the growth of potentially harmful micro-organisms in one or more components of kits used to prepare said composition prior to administration. Suitable antimicrobial preservative(s) include: the parabens, ie. methyl, ethyl, propyl or butyl paraben or mixtures thereof; benzyl alcohol; phenol; cresol; cetrimide and thiomersal. Preferred antimicrobial preservative(s) are the parabens.

The term "pH-adju sting agent" means a compound or mixture of compounds useful to ensure that the pH of the composition is within acceptable limits (approximately pH 4.0 to 10.5) for human or mammalian administration. Suitable such pH-adjusting agents include pharmaceutically acceptable buffers, such as tricine, phosphate or TRIS [ie. tra(hydroxymethyl)aminomethane], and pharmaceutically acceptable bases such as sodium carbonate, sodium bicarbonate or mixtures thereof. When the composition is employed in kit form, the pH adjusting agent may optionally be provided in a separate vial or container, so that the user of the kit can adjust the pH as part of a multi-step procedure.

By the term "filler" is meant a pharmaceutically acceptable bulking agent which may facilitate material handling during production and lyophilisation. Suitable fillers include inorganic salts such as sodium chloride, and water soluble sugars or sugar alcohols such as sucrose, maltose, mannitol or trehalose.

The pharmaceutical compositions may be prepared under aseptic manufacture (ie. clean room) conditions to give the desired sterile, non-pyrogenic product. It is preferred that the key components, especially the associated reagents plus those parts of the apparatus which come into contact with the imaging agent (eg. vials) are sterile. The components and reagents can be sterilised by methods known in the art, including: sterile filtration, terminal sterilisation using e.g. gamma-irradiation, autoclaving, dry heat or chemical treatment (e.g. with ethylene oxide). It is preferred to sterilise some components in advance, so that the minimum number of manipulations needs to be carried out. As a precaution, however, it is preferred to include at least a sterile filtration step as the final step in the preparation of the pharmaceutical composition. The pharmaceutical composition may optionally be prepared from a kit, wherein said kit comprises the imaging agent of the first aspect in sterile, solid form such that, upon reconstitution with a sterile supply of the biocompatible carrier, dissolution occurs to give the desired pharmaceutical composition. In that instance, the imaging agent, plus other optional excipients as described above, may be provided as a lyophilised powder in a suitable vial or container. The agent is then designed to be reconstituted with the desired biocompatible carrier to the pharmaceutical composition in a sterile, apyrogenic form which is ready for mammalian administration. A preferred sterile, solid form of the imaging agent is a lyophilised solid. The sterile, solid form is preferably supplied in a pharmaceutical grade container, as described for the pharmaceutical composition (above). When the kit is lyophilised, the formulation may optionally comprise a cryoprotectant chosen from a saccharide, preferably mannitol, maltose or tricine.

The method of the first aspect is preferably carried out intraoperatively, to assist in the resection of the tumour from said subject.

In the method of the first aspect, the imaging agent or pharmaceutical composition thereof has preferably been previously administered to said mammalian body. By "previously administered" is meant that the step involving the clinician, wherein the imaging agent is given to the patient e.g. as an intravenous injection or infusion, has already been carried out prior to imaging.

A preferred optical imaging method of the first aspect is Fluorescence Reflectance Imaging (FRI). In FRI, the contrast agent of the present invention is administered to a subject to be diagnosed or treated, and subsequently a tissue surface of the subject is illuminated with an excitation light - usually continuous wave (CW) excitation. The light excites the cyanine dye of the contrast agent. Fluorescence from the contrast agent, which is generated by the excitation light, is detected using a fluorescence detector. The returning light is preferably filtered to separate out the fluorescence component (solely or partially). An image is formed from the fluorescent light. Usually minimal processing is performed (no processor to compute optical parameters such as lifetime, quantum yield etc.) and the image maps the fluorescence intensity. The contrast agent is designed to concentrate in the area of interest, producing higher fluorescence intensity (i.e. positive contrast in a fluorescence intensity image). The image is preferably obtained using a CCD camera or chip, such that real-time imaging is possible.

The wavelength for excitation varies depending on the particular dye used. The apparatus for generating the excitation light may be a conventional excitation light source such as: a laser (e.g., ion laser, dye laser or semiconductor laser); an array of LEDs; halogen light source or xenon light source. Various optical filters may optionally be used to obtain the optimal excitation wavelength.

A preferred FRI method comprises the steps as follows:

(i) a tissue surface of interest within the mammalian body is illuminated with an excitation light;

(ii) fluorescence from the imaging agent, which is generated by excitation of the imaging moiety (IM), is detected using a fluorescence detector;

(iii) the light detected by the fluorescence detector is optionally filtered to separate out the fluorescence component;

(iv) an image of said tissue surface of interest is formed from the fluorescent light of steps (ii) or (iii).

In step (i), the excitation light is preferably continuous wave (CW) in nature. In step (iii), the light detected is preferably filtered.

An alternative imaging method of the fifth aspect uses FDPM (frequency-domain photon migration). This has advantages over continuous-wave (CW) methods where greater depth of detection of the IM within tissue is important [Sevick-Muraca et al, Curr.Opin.Chem.Biol., 6, 642-650 (2002)]. For such frequency/time domain imaging, it is advantageous if the ΓΜ has fluorescent properties which can be modulated depending on the tissue depth of the lesion to be imaged, and the type of instrumentation employed.

The FDPM method is as follows:

(a) exposing light-scattering biological tissue of said mammalian body having a heterogeneous composition to light from a light source with a predetermined time varying intensity to excite the imaging agent, the tissue multiply-scattering the excitation light;

(b) detecting a multiply-scattered light emission from the tissue in response to said exposing;

(c) quantifying a fluorescence characteristic throughout the tissue from the emission by establishing a number of values with a processor, the values each corresponding to a level of the fluorescence characteristic at a different position within the tissue, the level of the fluorescence characteristic varying with heterogeneous composition of the tissue; and

(d) generating an image of the tissue by mapping the heterogeneous composition of the tissue in accordance with the values of step (c). The fluorescence characteristic of step (c) preferably corresponds to uptake of the imaging agent and preferably further comprises mapping a number of quantities corresponding to adsorption and scattering coefficients of the tissue before administration of the imaging agent. The fluorescence characteristic of step (c) preferably corresponds to at least one of fluorescence lifetime, fluorescence quantum efficiency, fluorescence yield and imaging agent uptake. The fluorescence characteristic is preferably independent of the intensity of the emission and independent of imaging agent concentration.

The quantifying of step (c) preferably comprises: (i) establishing an estimate of the values, (ii) determining a calculated emission as a function of the estimate, (iii) comparing the calculated emission to the emission of said detecting to determine an error, (iv) providing a modified estimate of the fluorescence characteristic as a function of the error. The quantifying preferably comprises determining the values from a mathematical relationship modelling multiple light-scattering behaviour of the tissue. The method of the first option preferably further comprises monitoring a metabolic property of the tissue in vivo by detecting variation of said fluorescence characteristic.

Peptides of formula Z^fcMBPJ-Z² of the present invention may be obtained by a method of preparation which comprises:

(i) solid phase peptide synthesis of a linear peptide which has the same peptide sequence as the desired cMBP peptide and in which the Cys^a and Cys^b are unprotected, and the Cys^c and Cys^d residues have thiol-protecting groups;

(ii) treatment of the peptide from step (i) with aqueous base in solution to give a monocyclic peptide with a first disulphide bond linking Cys^a and Cys^b;

(iii) removal of the Cys^c and Cys^d thiol-protecting groups and cyclisation to give a second disulphide bond linking Cys^c and Cys^d, which is the desired bicyclic peptide product Z^fcMBPJ-Z².

By the term "protecting group" is meant a group which inhibits or suppresses undesirable chemical reactions, but which is designed to be sufficiently reactive that it may be cleaved from the functional group in question under mild enough conditions that do not modify the rest of the molecule. After deprotection the desired product is obtained. Amine protecting groups are well known to those skilled in the art and are suitably chosen from: Boc (where Boc is tert-butyloxycarbonyl), Fmoc (where Fnioc is fluorenylmethoxycarbonyl), trifluoroacetyl, allyloxycarbonyl, Dde [i.e. l-(4,4- dimethyl-2,6-dioxocyclohexylidene)ethyl] or Npys (i.e. 3-nitro-2-pyridine sulfenyl). Suitable thiol protecting groups are Trt (Trityl), Acm (acetamidomethyl), t-Bu (tert- butyl), ifert-Butylthio, methoxybenzyl, iiietliylbenzyl or Npys (3-nitro-2-pyridine sulfenyl). The use of further protecting groups are described in Protective Groups in Organic Synthesis, Theorodora W. Greene and Peter G. M. Wuts, (John Wiley & Sons, 1991). Preferred amine protecting groups are Boc and Fmoc, most preferably Boc. Preferred amine protecting groups are Trt and Acm.

Examples 1 and 2 provide further specific details. Further details of solid phase peptide synthesis are described in P. Lloyd-Williams, F. Albericio and E. Girald; Chemical Approaches to the Synthesis of Peptides and Proteins, CRC Press, 1997. The cMBP peptides are best stored under inert atmosphere and kept in a freezer. When used in solution, it is best to avoid pH above 7 since that may cause scrambling of the disulfide bridges.

The imaging agents can be prepared by conjugation of a functionalised cyanine dye to the cMBP. The cyanine dye suitably comprises a group G, where G is a reactive or functional group suitable for attaching to the cMBP peptide. The G group reacts with a complementary group of the cMBP peptide forming a covalent linkage between the cyanine dye fluorophore and the cMBP peptide. G may be a reactive group that may react with a complementary functional group of the peptide, or alternatively may include a functional group that may react with a reactive group of the cMBP peptide. Examples of reactive and functional groups include: active esters; isothiocyanate; maleimide; haloacetamide; acid halide; hydrazide; vinylsulfone; dichlorotriazine; phosphoramidite; hydroxyl; amino; sulfhydryl; carbonyl; carboxylic acid and thiophosphate. Preferably G is an active ester.

By the term "activated ester" or "active ester" is meant an ester derivative of the associated carboxylic acid which is designed to be a better leaving group, and hence permit more facile reaction with nucleophile, such as amines. Examples of suitable active esters are: TV-hydroxysuccinimide (NHS), sulfosuccinimidyl ester, pentafluorophenol, pentafluorothiophenol, /?ara-nitrophenol, hydroxybenzotriazole and PyBOP (ie. beiizotriazol-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate). Preferred active esters are N-hydroxysuccinimide or pentafluorophenol esters, especially N-hydroxysuccinimide esters.

The imaging agents of the first aspect can be obtained using a method of preparation which comprises one of steps (i) to (iv):

(i) reaction of a cMBP peptide of formula Z^fcMBPJ-Z² wherein Z¹ is H and Z² is a M^I& with a compound of formula Y^L)_n-[TM], to give the imaging agent of Formula I wherein [IM] is conjugated at the Z¹ position;

(ii) reaction of a cMBP peptide of formula Z^fcMBPJ-Z² wherein Z¹ = Z² = M^IG and cMBP comprises an Asp or Glu residue within 4 amino acid residues of either the C- or N- cMBP peptide terminus, and all other Asp/Glu residues of the cMBP peptide are protected, with a compound of formula Y²-(L)_n-[IM], to give the imaging agent of Formula I wherein [IM] is conjugated at said Asp or Glu residue of the cMBP peptide;

(iii) reaction of a cMBP peptide of formula Z^fcMBPJ-Z³ wherein Z¹ is M^I& and Z³ is a Z² group or an activated ester and all other Asp/Glu residues of the cMBP peptide are protected, with a compound of formula

Y -(L)_n-[IM], to give the imaging agent of Formula I wherein [IM] is conjugated at the Z² position;

(iv) reaction of a cMBP peptide of formula Z^fcMBPJ-Z² wherein Z¹ = Z² = M^IG and cMBP comprises a Lys within 4 amino acid residues of either the C- or N- cMBP peptide terminus, with a compound of formula Y^L^-flM], to give the imaging agent of Formula I wherein [IM] is conjugated at a Lys residue of the cMBP peptide;

wherein Z¹, cMBP, Z², M^IG, L, n and IM are as defined in the first aspect (above), and

Z³ is a Z² group or an activated ester;

Y¹ is a carboxylic acid, activated ester, isothiocyanate or thiocyanate group; Y² is an amine group. The terms "activated ester" or "active ester" and preferred embodiments thereof are as described above. Y² is preferably a primary or secondary amine group, most preferably a primary amine group.

The compound Z^fcMBPJ-Z² preferably has both Z¹ and Z² equal to M^IG. It is preferred that the cMBP peptide comprises an Asp, Glu or Lys residue to facilitate conjugation to the cMBP peptide. It is especially preferred that the cMBP peptide comprises a Lys residue, as described in step (iv).

The preparation of the Z^fcMBPJ-Z² is described above. The Z^fcMBPJ-Z³ peptide where Z³ is an active ester can be prepared from Z^fcMBPJ-Z², where Z² is OH or a biocompatible cation (B^c), by conventional methods.

Optical reporter cyanine dyes (FM) functionalised suitable for conjugation to peptides are commercially available from GE Healthcare Limited, Atto-Tec, Dyomics, Molecular Probes and others. Most such dyes are available as NHS esters.

Methods of conjugating suitable optical reporter dyes (IM) to amino acids and peptides are described by Licha {vide supra), as well as Flanagan et al [Bioconj .Chem., 8, 751-756 (1997)]; Lin et al, [ibid, 13, 605-610 (2002)] and Zaheer [Mol.Imaging, 1(4), 354-364 (2002)]. Methods of conjugating the linker group (L) to the cMBP peptide use analogous chemistry to that of the dyes alone (see above), and are known in the art.

In a second aspect, the present invention provides a method of detection, staging, diagnosis, monitoring of disease progression, monitoring of treatment, or treatment of a cancer of the mammalian body which comprises the in vivo optical imaging method of the first aspect.

Preferred aspects of the imaging agent and imaging method in the second aspect, are as described in the first aspect (above).

The cancer of the second aspect is preferably cancer of the following types: breast cancer; ovarian cancer; prostate; head and neck cancer; liver; colorectal; stomach; pancreas; thyroid; kidney; urinary bladder; skin or lung.

In a third aspect, the present invention provides the use of the contrast agent of Formula (I) as defined in the first aspect in the method of optical imaging of the first aspect or the method of detection, staging etc of the second aspect.

Preferred aspects of the imaging agent and imaging method in the third aspect, are as described in the first aspect (above).

Description of the Figures.

Figure 1 shows white light images (left) and fluorescence image (right) of two lesions observed in the colon using Compound 3. The contrast afforded by the agent is modest, but the boundaries of the lesions are relatively sharp. The lesions are a few mm in lateral dimensions.

Figure 2 shows cryoslice images according to Example 9. The invention is illustrated by the non-limiting Examples detailed below. Example 1 provides the synthesis of a cMBP peptide of the invention (Compound 1). Example 2 provides the synthesis of cyanine dye Cy5**, a preferred dye of the invention. Example 3 provides the synthesis of an active ester of Cy5**. Example 4 provides the conjugation of cyanine dyes of the invention to peptides (cMBP peptide and control). Compounds 2 to 6 were prepared in this way. Example 5 provides a method of determination of the affinity of the peptides to c-Met in vitro. The results show that the binding is selective, even when an optical reporter imaging moiety (a cyanine dye) is attached. Example 6 provides data on the in vivo testing of Compound 3 in an animal model of cancer.

Example 7 provides a summary of clinical imaging studies with Compound 3 in colorectal cancer patients. The fluorescence pattern was found to be relatively uniform in healthy colon tissue, which greatly aids the visualisation of lesions despite mild imaging contrast. The uniform fluorescence in background healthy tissue also makes it possible to apply automated and standardised image enhancement algorithms without introducing artefacts into the image. Example 8 demonstrates the feasibility of tumour margin detection with Compound 3 of the invention, using a pork meat phantom.

Example 9 provides cryoslice imaging of the agents of the invention in tumour- bearing mice. The images obtained are qualitative in nature, but clearly show a significantly higher accumulation of fluorescence in the tumours of mice injected with the contrast agent over the negative control substance over time, see Figure 2. The images show specific accumulation of the contrast agent in the HT-29 tumours. The contrast agent shows accumulation in the tumour border region and less accumulation in the necrotic core of the tumour (arrows in the contrast agent image). In contrast, the negative control compound shows greater accumulation in the tumour necrotic core (arrows at 60 minutes) than the tumour margin. The data are also consistent with the rapid wash-out of both the contrast agent and the negative control from other organs and support excretion through the kidneys. Signals from the negative control in tumours were confined to the central necrotic core of the tumours, further supporting the specific binding of the contrast agent to the desired target. Example 10 provides evidence from abdomen images of relatively uniform biodistribution of the compounds of the invention. cMBP peptides of the invention labelled with Cy5** exhibited lower background signal compared to such peptide labelled with other dyes such as Cy5 and Cy5.5.

Abbreviations.

Conventional single letter or 3-letter amino acid abbreviations are used.

Acm: Acetamidomethyl

ACN (or MeCN): Acetonitrile

Boc: fert-Buty 1 oxy carb ony 1

DCM: Di chl oromethane

DMF: Dimethylformamide

DMSO: Dimethylsulfoxide

Fnioc: 9-Fluorenylmethoxycarbonyl

HBTU: (9-Beiizotriazol- 1 -yl- V, V, V, V-tetramethyluronium hexafluorophosphate

HPLC: High performance liquid chromatography

HSPyU O-(N-succinimidvl)-NjV,N ,N -tetramethvleneuronium hexafluorophosphate

NHS: N-hydroxy-succinimide

NMM: N-Methylmorpholine

NMP: 1 -Methyl-2-pyrrolidinone

Pbf: 2,2,4,6,7-Pentamethyldihydrobenzofuran- 5-sulfonyl

PBS: Phosphate-buffered saline

tBu: t-butyl

TFA: Trifluoroacetic acid

TIS: Trii sopropyl silane

Trt: Trityl.

Table 2: structures of compounds of the invention. Example 1: Synthesis of Compound 1.

Step (a): synthesis of protected precursor linear peptide.

The precursor linear peptide has the structure:

Ac-Ala-Gly-Ser-Cys-Tyr-Cys(Acm)-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys(Acm)-Trp- Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys-NH₂

The peptidyl resin H-Ala-Gly-Ser(tBu)-Cys(Trt)-Tyr(tBu)-Cys(Acm)-Ser(tBu)-Gly-

Pro-Pro-Arg(Pbf)-Phe-Glu(OtBu)-Cys(Acm)-Trp(Boc)-Cys(Trt)-Tyr(tBu)-

Glu(OtBu)-Thr(v^{Me Me}pro)-Glu(OtBu)-Gly-Thr(tBu)-Gly-Gly-Gly-Lys(Boc)-Polymer was assembled on an Applied Biosystems 433A peptide synthesizer using Fmoc chemistry starting with 0.1 mmol Rink Amide Novagel resin. An excess of 1 nimol pre-activated amino acids (using HBTU) was applied in the coupling steps. Glu-Thr pseudoproline (Novabiocheiii 05-20-1 122) was incorporated in the sequence. The resin was transferred to a nitrogen bubbler apparatus and treated with a solution of acetic anhydride (1 mmol) and NMM (1 mmol) dissolved in DCM (5 mL) for 60 min. The anhydride solution was removed by filtration and the resin washed with DCM and dried under a stream of nitrogen.

The simultaneous removal of the side-chain protecting groups and cleavage of the peptide from the resin was carried out in TFA (10 mL) containing 2.5 % TIS, 2.5 % 4- thiocresol and 2.5 % water for 2 hours and 30 min. The resin was removed by filtration, TFA removed in vacuo and diethyl ether added to the residue. The formed precipitate was washed with diethyl ether and air-dried affording 264 nig of crude peptide. Purification by preparative HPLC (gradient: 20-30 % B over 40 min where A = H₂O/0.1 % TFA and B = ACN/0.1 % TFA, flow rate: 10 niL/min, column: Phenomeiiex Luna 5μ C 18 (2) 250 x 21.20 mm, detection: UV 214 nm, product retention time: 30 min) of the crude peptide afforded 100 mg of pure Compound 1 linear precursor. The pure product was analysed by analytical LTPLC (gradient: 10-40 % B over 10 min where A = H₂O/0.1 % TFA and B = ACN/0.1 % TFA, flow rate: 0.3 niL/min, column: Phenomeiiex Luna 3μ C18 (2) 50 x 2 mm, detection: UV 214 nm, product retention time: 6.54 min). Further product characterisation was carried out using electrospray mass spectrometry (MH₂ ²⁺ calculated: 1464.6, MH₂ ²⁺ found: 1465.1). Step (b): Formation of Monocyclic Cys4-16 disulfide bridge.

Cys4-16; Ac-Ala-Gly-Ser-Cys-Tyr-Cys(Acm)-Ser-Gly-Pro-Pro-Arg-Phe-Glu- Cys(Acm)-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Tlir-Gly-Gly-Gly-Lys-NH_2. The linear precursor from step (a) (100 mg) was dissolved in 5 % DMSO/water (200 mL) and the solution adjusted to pH 6 using ammonia. The reaction mixture was stirred for 5 days. The solution was then adjusted to pH 2 using TFA and most of the solvent removed by evaporation in vacuo. The residue (40 mL) was injected in portions onto a preparative HPLC column for product purification. Purification by preparative HPLC (gradient: 0 % B for 10 min, then 0-40 % B over 40 min where A = H₂O/0.1 % TFA and B = ACN/0.1 % TFA, flow rate: 10 niL/min, column:

Phenomenex Luna 5μ C 18 (2) 250 x 21.20 mm, detection: UV 214 nm, product retention time: 44 min) of the residue afforded 72 mg of pure Compound 1

monocyclic precursor. The pure product (as a mixture of isomers PI to P3) was analysed by analytical HPLC (gradient: 10-40 % B over 10 min where A = H₂O/0.1 % TFA and B = ACN/0.1 % TFA, flow rate: 0.3 mL/niin, column: Phenomenex Luna 3μ C 18 (2) 50 x 2 mm, detection: UV 214 nm, product retention time: 5.37 min (PI); 5.61 min (P2); 6.05 min (P3)). Further product characterisation was carried out using electrospray mass spectrometry (MH₂ ²⁺ calculated: 1463.6, MH₂ ²⁺ found: 1464.1 (PI); 1464.4 (P2); 1464.3 (P3)).

Step (c): Formation of Second Cys6-14 disulfide bridge (Compound 1).

The monocyclic precursor from step (b) (72 mg) was dissolved in 75 % AcOH/water (72 mL) under a blanket of nitrogen. 1 M HC1 (7.2 mL) and 0.05 M I₂ in AcOH (4.8 mL) were added in that order and the mixture stirred for 45 min. 1 M ascorbic acid (1 mL) was added giving a colourless mixture. Most of the solvents were evaporated in vacuo and the residue (18 mL) diluted with water/0.1 % TFA (4 mL) and the product purified using preparative HPLC. Purification by preparative HPLC (gradient: 0 % B for 10 min, then 20-30 % B over 40 min where A = H₂O/0.1 % TFA and B = ACN/0.1 % TFA, flow rate: 10 niL/min, column: Phenomenex Luna 5μ C 18 (2) 250 x 21.20 mm, detection: UV 214 nm, product retention time: 43-53 min) of the residue afforded 52 mg of pure Compound 1. The pure product was analysed by analytical HPLC (gradient: 10-40 % B over 10 min where A = H₂O/0.1 % TFA and B = ACN/0.1 % TFA, flow rate: 0.3 niL/min, column: Pheiiomenex Luna 3μ C 18 (2) 50 x 2 mm, detection: UV 214 nm, product retention time: 6.54 mill). Further product characterisation was carried out using electrospray

2+ 2+

mass spectrometry (MH₂ calculated: 1391.5, MH₂ found: 1392.5).

Example 2: Synthesis of the Cyanine Dye 2-{(lE,3E.,5E)-5-[l-(5-carboxypentvn- 3,3-dimethyl-5-sulfo-l,3-dihydro-2H-indol-2-ylidenelpenta-l,3-dienyl}-3-methyl- l,3-Z>½f4-sulfobutvn-3H-indolium-5-sulfonate (Cv5**).

Cy5 **

(3a) 5-Methyl-6-oxoheptane-l -sulfonic acid.

Ethyl 2-methylacetoacetate (50g) in DMF (25ml) was added to a suspension of sodium hydride (12. Og of 60% NaH in mineral oil) in DMF (100ml), dropwise with ice-bath cooling over 1 hour, (internal temperature 0-4°C). This mixture was allowed to warm to ambient temperature for 45mins with stirring before re-cooling. A solution of 1,4-butanesultone (45g) in DMF (25ml) was then added dropwise over 15 minutes. The final mixture was heated at 60°C for 18hours. The solvent was removed by rotary evaporation and the residue partitioned between water and diethyl ether. The aqueous layer was collected, washed with fresh diethyl ether and rotary evaporated to yield a sticky foam. This intermediate was dissolved in water (100ml) and sodium hydroxide (17.8g) added over 15 minutes with stirring. The mixture was heated at 90°C for 18 hours. The cooled reaction mixture was adjusted to ~pH2 by the addition of concentrated hydrochloric acid (~40ml). The solution was rotary evaporated and dried under vacuum. The yellow solid was washed with ethanol containing 2% hydrochloric acid (3x150ml). The ethanolic solution was filtered, rotary evaporated and dried under vacuum to yield a yellow solid. Yield 70g. (3b) 2,3-Dimethyl-3-(4-sulfobutyl)-3H-indole-5-sulfonic acid, dipotassium salt.

4-Hydrazinobenzenesulfonic acid (40g), 5-methyl-6-oxoheptane-l -sulfonic acid (from 3a; 60g) and acetic acid (500ml) were mixed and heated under reflux for 6hrs. The solvent was filtered, rotary evaporated and dried under vacuum. The solid was dissolved in methanol (lL). To this was added 2M methanolic potassium hydroxide (300ml). The mixture was stirred for 3 hours and then the volume of solvent reduced by 50% using rotary evaporation. The resulting precipitate was filtered, washed with methanol and dried under vacuum. Yield 60g. MS (LCMS) : MH⁺ 362. Acc. Mass: Found, 362.0729. MH⁺ =

requires m/z 362.0732 (-0.8ppm).

(3 c) 2,3-Dimethyl-l,3- ? (4-sulfobutyl)-3H-indolium-5-sulfonate, dipotassium salt.

2,3-Dimethyl-3-(4-sulfobutyl)-3H-indole-5-sulfonic acid (from 3b; 60g) was heated with 1,4 butane sultone (180g) and tetramethylene sulfone (146ml) at 140°C for 16 hours. The resulting red solid was washed with diethyl ether, ground into a powder and dried under vacuum. Yield 60g.

(3d) Cy5**, as TFA salt.

l-(5'-Carboxypentyl)-2,3,3-triiiietliyl-indolenium bromide-5-sulfonic acid, K⁺ salt (2.7g), malonaldehyde Z>/s(phenylimine) monohydrochloride (960mg), acetic anhydride (36ml) and acetic acid (18ml) were heated at 120°C for 1 hour to give a dark brown-red solution. The reaction mixture was cooled to ambient temperature. 2,3-Dimethyl-l,3-Z⁾ (4-sulfobutyl)-3H-indolium-5-sulfonate (from 3c; 8. lg) and potassium acetate (4.5g) were added to the mixture, which was stirred for 18 hours at ambient temperature. The resulting blue solution was precipitated using ethyl acetate and dried under vacuum. The crude dye was purified by liquid chromatography (RPCis. Water + 0.1% TFA/ MeCN + 0.1%TFA gradient). Fractions containing the principal dye peak were collected, pooled and evaporated under vacuum to give the title dye, 2g. UV/Vis (Water+0.1%TFA): 650nm. MS (MALDI-TOF): MH+ 887.1. IVIH = C3₈H₅oN₂Oi4S4 requires m/z 887.1. Example 3: Synthesis of 2-[(lE,3E,5E)-5-(l-{6-[(2,5-dioxopyrrolidin-l-yl)oxyl-6- oxohexyl}-3,3-dimethyl-5-sulfo-l,3-dihvdro-2H-indol-2-ylidene)penta-l,3- dienyll-3-methyl-l,3-bis(4-sulfobutyl)-3H-indolium-5-sulfonate,

diisopropylethylamine salt (NHS Ester of Cv5**).

Cy5** (Example 2; lOmg) was dissolved in anhydrous DMSO (3ml); to this were added HSPyU (20mg) and Ν,Ν^' -diisopropylethylamine (80μ1). The resulting solution was mixed for 3 hours, whereupon TLC (RPC 18. Water/MeCN) revealed complete reaction. The dye was isolated by precipitation in ethyl acetate/di ethyl ether, filtered, washed with ethyl acetate and dried under vacuum. UV/Vis (Water) 650nm. MS (MALDI-TOF) MH+ 983.5. MH⁺ = C42H53N3O16S4 requires 111/z 984.16.

Example 4: Conjugation of Dyes, Synthesis of Compounds 3 to 5.

Cys4-16, 6-14; Ac-Ala-Gly-Ser-Cys-Tyr-Cys-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys- Trp-Cy s-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Ly s(Cy 5)-NH₂ (Compound 3 ).

Compound 1 (10 mg), NMM (4 μί) and Cy5 NHS ester (5.7 mg; GE Healthcare PA15104) were dissolved in NMP (1 mL) and the reaction mixture stirred for 7 firs. The reaction mixture was then diluted with 5 % ACN/water (8 mL) and the product purified using preparative HPLC.

Purification by preparative HPLC (gradient: 5-50 % B over 40 min where A = H₂O/O. I % HCOOH and B = ACN/0.1 % HCOOH, flow rate: 10 mL/min, column: Phenomenex Luna 5μ C 18 (2) 250 x 21.20 mm, detection: UV 214 nm, product retention time: 35.5 min) of the crude peptide afforded 8.1 mg of pure Compound 3. The pure product was analysed by analytical HPLC (gradient: 5-50 % B over 10 min where A = H₂O/0.1 % HCOOH and B = ACN/0.1 % HCOOH, flow rate: 0.3 mL/min, column: Phenomenex Luna 3μ C 18 (2) 50 x 2 mm, detection: UV 214 11111, product retention time: 8.15 min). Further product characterisation was carried out using electrospray mass spectrometry (MH₂ ²⁺ calculated: 1710.6, MH₂ ²⁺ found: 171 1.0).

Other dye-peptide conjugates (Compounds 3 and 4) were prepared by analogous methods. Alexa647was purchased from Molecular Probes (A20106):

Compound 3 (MH₂ ²⁺ calculated: 1825.7, MH₂ ²⁺ found: 1825.9),

Compound 4 (MH₂ ²⁺ calculated: 181 1.7, MH₂ ²⁺ found: 1812.0).

Example 5: In Vitro Fluorescence polarisation assay.

Fluorescence polarisation was used to measure Kd. Monochromatic light passes through a horizontal polarizing filter and excites fluorescent molecules in the sample. Molecules oriented in the vertically polarized plane adsorb light, become excited, and subsequently emit light. The emitted light is measured in both the horizontal, I_→ and vertical, I_†, polarisation planes. The anisotropy value (γ), is the ratio between the light intensities defined via the following the equation:

The fluorescence anisotropy measurements were performed in 384-well microplates in a volume of 40 ^iL in binding buffer (PBS, 0.01%Tween-20, pH 7.5) using a Tecan Safire fluorescence polarisation plate reader (Tecan, US) at ex646/eni678 nm. The concentration of Dye-labelled peptide was held constant (5nM) and the concentrations of the target (human and murine versions) were varied from 0-150 iiM. Binding mixtures were equilibrated in the microplate for 10 niin at 30°C. The observed change in anisotropy was fitted to the equation:

where γ_ο1κ is the observed anisotropy, Yf_ree is the anisotropy of the free peptide, ybound is the anisotropy of the bound peptide, Kd is the dissociation constant, cT is the total target concentration, and P is the total Dye-labelled peptide concentration. The equation assumes that the synthetic peptide and the receptor form a reversible complex in solution with 1 : 1 stoichiometry. Data fitting was performed via nonlinear regression using SigmaPlot software (version 10) to obtain the Kd value. Compound 2 was tested for binding towards human and mouse c-Met (Fc chimera). The results showed a K_d value of 3+/- 0.5nM for binding towards human c-Met, and no binding towards murine c-Met was observed. Using the same method, Compounds 3 and 4 were found to have a K_d value of 2 +/- 0.5nM and 1.5 +/- O. lnM for binding towards human c-Met, respectively.

Example 6: In Vivo testing of Compound 3.

(a) Animal Model.

54 Female BALB c/A nude (Bom) mice were used in the study. The use of the animals was approved by the local ethics committee. BALB c/A nude is an inbred immunocompromised mouse strain with a high take rate for human tumours as compared to other nude mice strains. The mice were 4 weeks old upon arrival and with a body weight of approx. 20 grams at the start of the study. The animals were housed in individually ventilated cages (IVC, Scanbur BK) with LTEPA filtered air. The animals had ad libitum access to "Rat and Mouse nr. 3 Breeding" diet (Scanbur BK) and tap water acidified by addition of HCl to a molar concentration of 1 niM (pH 3.0).

The colon cancer cell HCT-15 is derived from human colon carcinomas and is reported to express c-Met according to Zeng et al [Clin. Exp. Metastasis, 21, 409-417. (2004)]. The cell line was proven to be tumourigenic when inoculated subcutaneously into nude mice [Flatmark et al, Eur. J. Cancer 40, 1593-1598 (2004)].

HCT-15 cells were grown and prepared for subcutaneous inoculation in RPMI (Sigma Cat # R0883) with 10% serum and penicillin/streptomycin. Stocks were made at passage number four (P4) and frozen down for storage in liquid nitrogen at 3xl0⁷ cells/vial in the culture media containing 5% DMSO. On the day of the transplantation, the cells were thawed quickly in a 37°C water bath (approx. 2 niin), washed and resuspended in PBS/2% serum (centrifugation at 1200 rpm for 10 min). Thorough mixing of cells in the vials was ensured every time cells were aspirated into the dosing syringe. Volumes of 0.1 ml of cell suspension were injected s.c. at the shoulder and at the back using a fine bore needle (25 G) while the animals were under light gas anaesthesia. The animals were then returned to their cages and the tumours were allowed to grow for 13-17 days. The animals were allowed an acclimatisation period of at least 5 days before the inoculation procedure.

(b) procedure.

All test substances were reconstituted with PBS from freeze-dried powder. A small stack of white printer paper was imaged to obtain a flat field image which was used to correct for illumination inhomogeneities. The test substances were injected intravenously in the lateral tail vein during physical fixation. The injection volume was 0.1ml, which corresponds to a dose of lnniol test substance per animal. After injection the animals were returned to their cages. The animals were sacrificed immediately before imaging by cervical dislocation. The optimal imaging time point for each test substance was estimated based on comparison of wash out rates in the skin and in muscle tissue in a limited number of animals (n=l-6). The imaging time point for Compounds 3 and 4 was 120 minutes post injection. For each animal the subcutaneously grown tumours were excised post mortem. A thin slice, approximately 1.6mm thick and 3-4mm in diameter, was cut off the edge of one of the tumours. The tumour slice was then imaged against an area of normal colon from the same animal.

(c) Imaging.

Imaging was performed through a clinical laparoscope adapted to use a light source to excite the reporter and a filtering system to extract the fluorescence component. A 635nm laser was used for excitation of the reporter molecule. A Hamamatsu ORCA ERG CCD camera was used as the detector. The camera was operated in 2x2 binning mode with 0 gain. Standard exposure time for colon imaging was 10s. System calibration measurements indicate that the 10s exposure time with the animal imaging system corresponds to 40ms exposure with a clinically relevant light source, field of view, and distance to the tissue surface. The intensity distribution in the image was corrected for illumination inhomogeneities through system calibration data. A target to background ratio was computed from regions of interest placed over the tumour, and normal colon background. The images were visually scored using the standard scoring system employed for receiver operating characteristic (ROC) analysis.

(d) Results.

Compound 3 had a tumour to normal ratio of 1.46: 1 and a readily identifiable tumour image, with an area under the curve of 0.97 for the ROC analysis. Example 7: In Vivo Imaging with Compound 3 in Human Patients.

Imaging studies with Compound 3 were carried out in colorectal cancer patients. The dose of Compound 3 was given as a bolus intravenous injection. A target to background ratio of 1.3 : 1 was observed in the human patients. The contrast was limited due to the degree of c-Met expression, but the fluorescence pattern was found to be relatively uniform in healthy colon tissue. Thus, uniform distribution was consistently observed in 20 healthy volunteers and 15 patients. Relatively sharp boundaries were observed for most colorectal cancer lesions - see Figure 1. Due to the relatively 'sharp' border to the lesion, and the uniform fluorescence signal in healthy colon, the boundary of the lesion was clearly visible.

Example 8: Phantom Imaging Studies.

Fluorescent reference standards were prepared which comprised small mm sized plugs of epoxy material, loaded with an absorber and scattering particles to match the optical properties of tissue in the wavelength region of Compound 3. They also include a controlled quantity of quantum dots to provide a known and calibrated fluorescence signal. A small plug of this material of 2mm diameter and 2mm depth, matched to the fluorescence intensity produced by lesions in the colon, was used as the test target. Test imaging was carried out with the plug inserted into the pork meat in vitro. Surface detection was possible, but when the plug was buried 5mm below the surface, the presence of the plug was no longer discernable in the fluorescence image. The plug was just discernable in the fluorescence image at 2-3 mm below the surface.

To simulate a tumour margin remnant, a shaving was taken from a cylinder of fluorescence reference standard material and placed on the surface of the pork meat phantom. This shaving has less than hum thickness. The shaving was clearly visible in fluorescence when placed on the surface, indicating the possibility of visualising small volumes of tumour tissue remaining during surgical resection with Compound 3. Example 9: Cryoslice Imaging Studies.

HT-29 tumour bearing mice were frozen down in liquid nitrogen at different time points (5, 30, 60, 120 and 240 min) after injection of 1 nniol of contrast agent (Compound 3) or a negative control peptide (scrambled peptide sequence lacking c- Met binding, labelled with Cy5), and subsequently sliced (75 micron thick slices) and scanned using a fluorescence scanner. Non-injected animals were included as controls. Non-injected control animals showed in particular fluorescent signals from the large bowel content (not in colon wall, from chlorophyll in the animal diet), and some autofluorescence from the liver.

Representative images are shown in Figure 2. Images are displayed on a linear 'negative' grey scale, i.e. white indicates low fluorescence signal and black indicates high fluorescence signal.

Example 10: Abdomen Imaging Studies.

For the 54 animals used in Example 6, fluorescence images were acquired of the open abdomen. These images were processed in standard format and reviewed. The purpose of the review was to gain any additional information from the images, specifically to assess common image features in background tissues and evidence of image and/or biodistribution artefacts. The signal from background tissue was found to be relatively uniform. Skin appeared to be brighter than other tissues and areas assumed to be associated with connective tissue/fascia. Higher skin and connective tissue signal is also indicated in the cryoslice data of Example 9. Cy5** labelled peptide (Compound 3) showed relatively lower skin and connective tissue/fascia binding than Cy5 and Cy5.5 labelled compounds.

Claims

CLAIMS.

1. A method of in vivo optical imaging of the tumour margins of a tumour of an animate subject, during or after surgical resection of said tumour, said method comprising:

(i) providing an optical imaging contrast agent suitable for in vivo imaging, said contrast agent comprising a conjugate of Formula (I):

(I)

(ii) generating an optical image of a region of interest of said subject to which said contrast agent has been administered, said region of interest comprising said tumour;

where:

Z¹ is attached to the N-terminus of cMBP, and is H or M^IG;

Z² is attached to the C -terminus of cMBP and is OH, OB^c, or M^IG,

where B^c is a biocompatible cation;

cMBP is a c-Met binding cyclic peptide of 17 to 30 amino acids which comprises the amino acid sequence (SEQ-1):

Cys^X^Cys^X^Gly-Pro-Pro-X^Phe-Glu-Cys^Trp-Cys^Tyr-X^X^X⁶; wherein X¹ is Asn, His or Tyr;

X² is Gly, Ser, Thr or Asn;

X³ is Thr or Arg;

X⁴ is Ala, Asp, Glu, Gly or Ser;

X⁵ is Ser or Thr;

X⁶ is Asp or Glu;

and Cys^a"d are each cysteine residues such that residues a and b as well as c and d are cyclised to form two separate disulfide bonds; M is a metabolism inhibiting group which is a biocompatible group which inhibits or suppresses in vivo metabolism of the peptide;

L is a synthetic linker group of formula -(A)_m- wherein each A is independently -CR₂- , -CR=CR- , -C≡C- , -CR₂C0₂- , -C0₂CR₂- , -NRCO- , -CONR- , -NR(C=0)NR-, -NR(C=S)NR-, -S0₂NR- , -NRS0₂- , -CR₂OCR₂- , -CR₂SCR₂- , -CR₂NRCR₂- , a C4-8 cycloheteroalkylene group, a C_4-8 cycloalkylene group, a C_5-12 arylene group, or a C_3-12 heteroarylene group, an amino acid, a sugar or a monodisperse polyethyleneglycol (PEG) building block;

each R is independently chosen from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl,

C_1-4 alkoxyalkyl or C_1-4 hydroxyalkyl;

111 is an integer of value 1 to 20;

11 is an integer of value 0 or 1;

ΓΜ is an optical reporter imaging moiety suitable for imaging the mammalian body in vivo using light of wavelength 500-1000 mil, which is a cyanine dye of Formula III:

(III)

where:

R¹ and R² are independently H or SO₃M¹, and at least one of R¹ and R² is SO₃M¹, where M¹ is H or B^c;

R³ and R⁴ are independently C_1-4 alkyl or C_1-6 carboxyalkyl;

R , R°, R' and R are independently R^a groups;

wherein R^a is C_1-4 alkyl, C_1-6 carboxyalkyl or -(CH^_kSOsM¹, where k is an integer of value 3 or 4;

with the proviso that the cyanine dye has a total of 1 to 4 SO₃M¹ substituents in the R¹, R² and R^a groups.

2. The method of Claim 1, where in addition to SEQ-1, the cMBP further comprises an Asp or Glu residue within 4 amino acid residues of either the C- or TV- cMBP peptide terminus, and -(L)_nIM is functionalised with an amine group which is conjugated to the carboxyl side chain of said Asp or Glu residue to give an amide bond.

3. The method of Claim 1 or Claim 2, where in addition to SEQ-1, the cMBP comprises a Lys residue within 4 amino acid residues of either the C- or TV- cMBP peptide terminus, and -(L)_nIM is functionalised with a carboxyl group which is conjugated to the epsilon amine side chain of said Lys residue to give an amide bond.

4. The method of any one of Claims 1 to 3, wherein cMBP comprises the amino acid sequence of either SEQ-2 or SEQ-3 :

Ser-Cys^X^Cys^-X^Gly-Pro-Pro-X^Phe-Glu-Cys^Trp-Cys^Tyr-X^X^X⁶. (SEQ-2);

Ala-Gly-Ser-Cys^X^Cys^X^Gly-Pro-Pro-X^Phe-Glu-Cys^Trp-Cys^Tyr- X⁴-X⁵-X⁶-Gly-Thr (SEQ-3).

5. The method of any one of Claims 1 to 4, wherein X³ is Arg.

6. The method of any one of Claims 1 to 5, wherein in addition to SEQ-1, SEQ-2 or SEQ-3, cMBP further comprises at either the TV- or C- terminus a linker peptide which is chosen from -Gly-Gly-Gly-Lys- (SEQ-4), -Gly-Ser-Gly-Lys- (SEQ-5) or - Gly-Ser-Gly-Ser-Lys- (SEQ-6).

7. The method of Claim 6, where cMBP has the amino acid sequence (SEQ-7):

Ala-Gly-Ser-Cys^a-Tyr-Cys^c-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys^d-Trp-Cys^b- Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys.

8. The method of any one of Claims 1 to 7, where both Z¹ and Z² are independently M^IG.

9. The method of Claim 8, where Z¹ is acetyl and Z² is a primary amide.

10. The method of any one of Claims 1 to 9, where n is 0.

1 1. The method of any one of Claims 1 to 10, where cMBP is as defined in Claim 7, Z¹ and Z² are as defined in Claim 9.

12. The method of any one of Claims 1 to 1 1, where the contrast agent comprises a pharmaceutical composition of the conjugate of Formula (I) as defined in any one of Claims 1 to 1 1, together with a biocompatible carrier, in a form suitable for mammalian administration.

13. The method of any one of Claims 1 to 12, where the optical imaging is carried out intraoperatively, to assist in the resection of said tumour from said subject.

14. The method of any one of Claims 1 to 12, where the optical imaging is carried out ex vivo on the resected tumour, or a portion thereof.

15. A method of detection, staging, diagnosis, monitoring of disease progression, monitoring of treatment, or treatment of a cancer of the mammalian body which comprises the in vivo optical imaging method of any one of claims 1 to 14.

16. The method of claim 15, where the cancer is breast cancer, ovarian cancer, prostate cancer or head and neck cancer.

17. Use of the contrast agent of Formula (I) as defined in any one of claims 1 to 1 1 in the method of any one of claims 1 to 16.