Peptide Imaging Agents

Field of the Invention

The piesent invention relates to labelled cMet binding peptides suitable foi optical imaging /// \ ιvo The peptides are labelled with an optical reporter group suitable for imaging in the red to near-mfraied region Also disclosed are in vivo imaging methods, especially of use in the diagnosis of colorectal cancer (CRC)

Background to the Invention

WO 2005/030266 discloses that there is a medical need for early diagnosis of colorectal cancer (CRC) WO 2005/030266 discloses optical imaging contrast agents which have affinity for a biological target abnormally expressed in CRC. The biological target is selected from COX-2, beta-catcnin, E-cadheπn, P-cadheπn, various kinases, Hei-2, matrix metalloprotemases (MMPs), cychns, P53, thymidylate synthase, VEGF leceptois, EGF receptors, K-ras, adenomatous polyposis coll protein, cathepsin B, uPAR, cMet, mucins and gastiin receptors Prefeπed such targets (p 7 lines 1 1-12) are said to be- cMet, MMP- 14, COX-2, beta-catenm and Cathepsin B The vectoi of WO 2005/030266 can be' a peptide, peptoid moiety, oligonucleotide, oligosaccharide, hpid-related compound or tiaditional organic diug-like small molecule The reportei moiety is preferably a dye that mteiacts with light m the wavelength region from the ultraviolet to the infrared pait of the electromagnetic spectrum

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 ieceptor (cMet) is implicated in tumoui growth, invasion and metastasis

Knudsen et al have reviewed the role of HGF and cMet in prostate cancer, with possible implications for imaging and therapy [Adv Cancer Res , 9_[, 31-67 (2004)] Labelled anti-met antibodies for diagnosis and therapy aie desciibed in WO 03/057155

WO 2004/078778 discloses polypeptides oi multimeπc peptide constructs which bind

cMet oi a complex comprising cMet and HGF Appioximately 10 different structural classes of peptide are desci ibed WO 2004/078778 discloses that the peptides can be labelled w ith a detectable label for in utro and /// vivo applications, or with a diug for therapeutic applications The detectable label can be an enzyme, fluorescent compound, an optical dye, a paramagnetic metal ion, an ultrasound contiast agent or a iadtonuchde Prefeπed labels of WO 2004/078778 are stated to be radioactive oi paramagnetic, and most preferably comprise a metal which is chelated by a metal chelatoi

The Present Invention

The present invention piovides imaging agents suitable for in vivo optical imaging, which comprise cMet binding cyclic peptides, and a benzopyiyhum dye suitable for imaging the mammalian body in vivo using light of red to near-infrared wavelength 600- 1200 nm

The cMet binding cyclic peptides are related to one of the stiuctural classes of peptide of WO 2004/078778, and have optimal binding affinity for cMet These peptides weie derived from phage display and selected by their affinity for cMet and lack of competition with HGF, as described in WO 2004/078778 The cMet binding peptides of the present invention preferably have at least one of their termini protected by metabolism inhibiting groups (M ¹⁰ ) That is an important consideration for in vivo applications, whei e endogenous enzymes and peptidases would otherwise rapidly metabolise the peptide, with consequent loss of cMet binding affinity, and hence loss of selective targeting m vivo.

The present invention teaches that the best way of using cMet binding peptides foi in vivo imaging of superficial lesions involves the use of an optical reportei, as opposed to othei imaging modalities (eg nuclear, MRI or ultrasound), and also provides prefeπed optical imaging reportei s The ied to near-infrared region (light of wavelength 600-1200 nm) is preferred, since that region has minimal spectral overlap with endogenous tissues and materials, such as haemoglobin, porphyims, melanin, and collagen [Licha, Topics Cuπ .Chem , 222, 1 -29 (2002)] Other important contributors to tissue autofluoiescence are NADH, FAD and elastin

Detailed Description of the Invention

In a fu st aspect, the piesent im ention piovidcs an imaging agent which compi ises a conjugate of Fomiula I

(I) wheie

Z ¹ is attached to the N-termmus of cMBP, and is H 01 M ^IG ,

Z ⁷ is attached to the C-temiinus of cMBP and is OH, OB\ 01 M ¹⁰ , wheie B ^c is a biocompatible cation, cMBP is a cMct binding cyclic peptide of 17 to 30 ammo acids which compiises the ammo acid sequence (SEQ-I) wheiein X ¹ is Asn, His oi Tyi,

X ^" is GIy, Sei , Tm oi Asn, X ⁴ is Ala, Asp, GIu, GIy oi Sei, , X is Asp oi GIu, and Cys ^{a d} aie each cysteine iesidues such that iesidues a and b as well as c and d are cyclised to form two sepaiate disulfide bonds, M ^IG is a metabolism inhibiting gioup which is a biocompatible gioup which inhibits oi suppi esses in vivo metabolism of the peptide,

L is a synthetic hnkei gioup of foimula -(A) ₁₁₁ - wheiein each A is independently -CR,- , -CR=CR- , -C≡C- , -CR ₇ CO ₂ - , -CO ₂ CR ₂ - , -NRCO- , -CONR- , -NR(C=O)NR-, -NR(C=S)NR-, -SO ₂ NR- , -NRSO ₂ - , -CR ₂ OCR,- , -CR ₂ SCR ₇ - , -CR ₇ NRCR ₂ - , a C _{4 8} cycloheteioalkylene gioup, a C _{4 8} cycloalkylene gioup, a C _^ ι ₂ aiylenc gioup, oi a Ci p heteioaiylene gioup, an ammo acid, a sugai oi a monodispeise polyethyleneglycol (PEG) building block,

each R is independently chosen from H, Ci _-4 alkyl, C ₂ ^ alkenyl, C ₂ - ₄ alkynyl,

C|- ₄ alkoxyalkyl or Ci_ ₄ hydroxyalkyl; m is an integer of value 1 to 20; n is an integer of value 0 or 1 ;

Bzp ^M is a benzopyrylium dye of Fomiula II:

where:

Y is a group of Formula Y ^d or Y

(Y ^a (Y ^b )

R' -R ⁴ and R ⁹ -R ¹³ are independently selected from H, -SO ₃ M ¹ , Hal, R ^a or C _{3- I 2} aryl, where each M ¹ is independently H or B ^c , and B ^c is a biocompatible cation;

R ³ is H, C ₁ - ₄ alkyl, Ci _-6 carboxyalkyl, C _{3-I 2} arylsulfonyl, Cl, or R ^""1 together with one of R ⁶ , R ¹⁴ , R ¹³ or R ¹⁶ may optionally form a 5- or 6- membered unsaturated aliphatic, unsaturated heteroaliphatic or aromatic ring;

R ⁶ and R ¹⁶ are independently R ^a groups;

7 R

R and R are independently C ₁ - ₄ alkyl, Ci _-4 sulfoalkyl or Ci _-6 hydroxyalkyl or optionally together with one or both of R ⁹ and/or R ^{! 0} may form a 5- or 6- membered N-containing heterocyclic or heteroaryl ring;

X is -CR ¹⁴ R ¹⁵ -, -O-, -S-, -Se-, -NR ¹⁶ - or -CH-CH- , where R ¹⁴ to R ¹⁶ are independently R ^a groups;

R ^a is Ci_ ₄ alkyl, Ci _-4 sulfoalkyl, Ci_ ₆ carboxyalkyl or Cu ₁ hydroxyalkyl; w is 1 or 2;

J is a biocompatible anion, with the pi oviso that B/p ^M compiises at least one sulfonic acid substituent chosen fiom the R to R ⁶ gioups

^ By the teπn "imaging agent" is meant a compound suitable foi imaging the mammalian body w vn o Piefeiably, the mammal is a human subject The imaging may be invasive (eg mtia-opeiative 01 endoscopy) or non-invasive The piefened imaging method is endoscopy Whilst the conjugate of Formula I is suitable foi /// vivo imaging, it may also have in viti o applications (eg assays quantifying cMet m0 biological samples 01 visualisation of cMet m tissue samples) Piefeiably, the imaging agent is used foi in vivo optical imaging

By the teπn "optical imaging" is meant any method that foπns an image foi detection, staging oi diagnosis of disease, follow up of disease development 01 foi follow up of5 disease tieatment based on intet action with light m the ied to neai-mfiaied iegion (wavelength 600- 1200 nm) Optical imaging fuithci includes all methods ftom dnect visualization without use of any device and involving use of devices such as vaiious scopes, catheteis and optical imaging equipment, eg computei-assisted haidwaie foi tomogiaphic piesentations The modalities and measuiement techniques include, but0 aie not limited to luminescence imaging, endoscopy, fluoiescence endoscopy, optical coheience tomogiaphy, tiansmittance imaging, time lesolved ti ansmittance imaging, confocal imaging, nonhneai micioscopy, photoacoustic imaging, acousto- optical imaging, spectioscopy, ieflectance spectioscopy, mteifeiometiy, coheience mteifeiometiy, diffuse optical tomogiaphy and fluoiescence mediated diffuse optical-> tomogiaphy (continuous wave, time domain and frequency domain systems), and measurement of light scatteimg, absoiption, polanzation, luminescence, fluoiescence lifetime, quantum yield, and quenching Fuithei details of these techniques aie piovided by (Tuan Vo-Dmh (editoi) "Biomedical Photonics Handbook" (2003), CRC Pi ess LCC, Mycek & Pogue (editois) "Handbook of Biomedical Fluoiescence"0 (2003), Maicel Dekkei, Inc , Sphntei & Hoppei "An Intioduction to Biomedical Optics" (2007), CRC Piess LCC

The ied to neai-infiaied iegion light is piefeiably of wavelength 650-1000 nm The optical imaging method is picfetably fluoiescence endoscopy

By the term "conjugate" is meant that the cMBP and Bzp ^M dye aie linked by covalent bonds, optionally via the (L) _n gioup

The Z ¹ gioup substitutes the amine gioup of the last amino acid iesidue Thus, when Z ¹ is H, the ammo terminus of the cMBP teimmates m a flee NH ₂ gioup of the last amino acid iesidue The Z ^" gioup substitutes the caibonyl gioup of the last ammo acid iesidue Thus, when Z ^" is OH, the caiboxy teimmus of the cMBP terminates in the fiee CO ₂ H gioup of the last amino acid iesidue, and when Z is OB ⁰ that terminal caiboxy gioup is ionised as a CC> ₂ B ^C caiboxylate gioup

By the teπn "metabolism inhibiting gioup" (M ¹⁰ ) is meant a biocompatible gioup which inhibits 01 suppi esses /// vivo metabolism of the cMBP peptide at eithei the ammo teimmus (Z ¹ ) 01 caiboxy terminus (Z ² ) Such gioups aie well known to those skilled in the art and aie suitably chosen fiom, foi the peptide amine terminus N-acylated gioups -NH(C=O)R ⁰ wheie the acyl gioup (C=O)R ⁰ has R° chosen fiom Ci (, alkyl, C-, ₁₀ aiyl gioups 01 compiises a polyethyleneglycol (PEG) building block Suitable PEG gioups aie desciibed foi the hnkei gioup (L), below Piefened such PEG gioups aie the biomodifieis of Formula IA 01 IB Piefened such ammo terminus M ¹⁰ gioups aie acetyl, benzyloxycaibonyl 01 tπfluoioacetyl, most piefeiably acetyl

Suitable metabolism inhibiting gioups foi the peptide caiboxyl terminus include caiboxamide, te/ r-butyl estei, benzyl estei, cyclohexyl estei, ammo alcohol 01 a polyethyleneglycol (PEG) building block A suitable M ⁱ⁰ gioup foi the caiboxy terminal ammo acid iesidue of the cMBP peptide is wheie the terminal amine of the ammo acid iesidue is N-alkylated with a Ci ₄ alkyl gioup, piefetably a methyl gioup Piefened such M ¹⁰ gioups aie caiboxamide 01 PEG, most piefened such gioups aie caiboxamide

Formula I denotes that the (L) _n [Bzp ^M ] moiety can be attached at eithei Z ¹ , Z ² 01 cMBP Foi Z ¹ 01 Z ² , the (L) _n [Bzp ^M ] moiety may eithei be attached to the M ^1G gioup when eithei of Z'/Z ² is a M ¹⁰ When Z ¹ is H 01 Z ² is OH, attachment of the (L) _n [Bzp ^M ] moiety at the Z ¹ 01 Z ² position gives compounds of formulae [Bzp ^M ]-(L) _n -[cMBP]-Z ²

ot Z'-[cMBP]-(L) _n -[Bzp ^M ] lespcctively Inhibition of metabolism of the cMBP at eithei peptide terminus may also be achieved by attachment of the (L) _n [Bzp ^VI ] moiety in this way, but (L) _n [Bzp ^vl ] is outside the definition of M ^IG of the piesent invention

The -(L) _n - moiety of Foimula I may be attached at any suitable position of the Bzp ^M of Foimula Il The (L) _n - moiety eithei takes the place of an existing substituent (eg one of the R to R , 16 gioups), 01 is covalently attached to the existing substituent of the Bzp ^M The -(L) _n - moiety of Foimula I is piefeiably attached via a caiboxyalkyl substituent of the Bzp ^M

By the term "cMet binding cyclic peptide" (cMBP) is meant a peptide which binds to the hepatocyte giowth factoi (HGF) high affinity leceptoi, also known as cMet (c-Met, Met, Met ieceptoi oi hepatocyte giowth factoi teceptoi) Suitable cMBP peptides of the piesent invention have an appaient K _d foi cMet of cMet/HGF complex of less than about 20 nM The cMBP peptides compiise pioline lesidues, and it is known that such iesidues can exhibit cis/tians isomeiisation of the backbone amide bond The cMBP peptides of the piesent invention include any such isomcis

By the teim "biocompatible cation" (B ^c ) is meant a positively chaiged counteiion which forms a salt with an ionised, negatively chaiged gioup, wheie said positively chaiged counteiion is also non-toxic and hence suitable foi admimstiation to the mammalian body, especially the human body Examples of suitable biocompatible cations include the alkali metals sodium oi potassium, the alkaline eaith metals calcium and magnesium, and the ammonium ion Piefeπed biocompatible cations are sodium and potassium, most piefeiably sodium By the term "biocompatible anion" (J) is meant a negatively chaiged counteπon which forms a salt with an ionised, positively chaiged gioup (m this case an mdohmum gtoup), wheie said negatively chaiged countetion is also non-toxic and hence suitable foi admmisttation to the mammalian body, especially the human body The countei ion (J ) iepiesents an anion which is piesent m a molai equivalent amount, thus balancing the positive chaige on the Bzp ^vl dye The anion (I) is suitably singly- oi multiply-chaiged, as long as a chaige-balancmg amount is piesent The anion is suitably denved fiom an moigamc oi oi game acid Examples of suitable anions include halidc ions such as chloiide oi biomide, sulfate, nitiate, cittate, acetate, phosphate and boiate A pi efened anion is

chloride

By the term "ammo acid" is meant an L- or D- ammo acid, ammo acid analogue (eg naphthylalanine) or ammo 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 ammo acids are used heiem. Prefeiably the amino acids of the present invention are optically pure By the term "ammo acid mimetic" is meant synthetic analogues of naturally occurring ammo acids which are isosteies, i e have been designed to mimic the steric and electronic structuie of the natural compound Such isosteres are well known to those skilled m the art and include but are not limited to depsipeptides, retro-mverso peptides, thioamides, cycloalkanes or 1 ,5- disubstituted tetrazoles [see M Goodman, Biopolymeis, 24, 137, (1985)]

By the term "peptide" is meant a compound comprising two oi more ammo acids, as defined above, linked by a peptide bond (ie an amide bond linking the amine of one ammo 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 oi a protein but are no longer peptidic in chemical nature, that is, they no longer contain any peptide bonds (that is, amide bonds between ammo acids) Here, the term peptide mimetic is used in a broader sense to include molecules that are no longer completely peptidic m nature, such as pseudo-peptides, semi-peptidcs and peptoids

Suitable imaging agents of the invention are those wherein the Bzp ^lVI is of Formula Ha or lib

(Ha)

(lib) wheie X, w and R -R and T aie as defined foi Formula II

When R ³ togethei with one of R ⁶ /R ^l4 -R ¹⁶ forms a 5- oi 6- membeied unsatuiated aliphatic, unsaturated heteioaliphatic oi aiomatic ling, suitable such aiomatic iings include phenyl, fuian, thiazole, pyndyl, pyπole oi pyiazole πngs Suitable unsatuiated nngs compπse at least the C=C to which R ^{^} is attached

When R ⁷ and/oi R ⁸ togethei with one oi both of R ⁹ and/oi R ¹⁰ foim a 5 oi 6- mcmbeied N-containing hctciocyclic ot heteioaiyl ling, suitable such nngs include thiazole, pyndyl, pynole oi pyiazole nngs ot paitially hydiogenated veisions theieof pi efei ably pyndyl oi dihydiopyndyl

By the term "sulfonic acid substituent" is meant a substituent of foimula -SChM , wheie M ¹ is H oi B ^c , and B ^L is a biocompatible cation (as defined above) The -SChM ¹ , substituent is covalently bonded to a caibon atom, and the caibon atom may be aiyl (ie sulfoaiyl such as when R ¹ oi R ² is -SO ₃ M ¹ ), 01 alkyl (ie a sulfoalkyl gioup)

It is envisaged that one of the ioles of the hnkei gtoup -(A) _m - of Formula I is to distance Bzp ^M fiom the binding site of the cMBP peptide, to minimise any steiic impairment of intei action with the binding site This can be achieved by a combination of flexibility (eg simple alkyl chains), so that the bulky gtoup has the fteedom to position itself away ftom the active site and/01 ngidity such as a cycloalkyl 01 aiyl spacer which onentate the Bzp ^M away fiom the binding site The natuie of the lmkei gioup can also be used to modify the biodistnbution of the imaging agent Thus, eg the mtioduction of ethet gioups 111 the hnkei will help to minimise plasma piotein

binding When -(A) ₁₁₁ - compiises a polyethyleneglycol (PEG) building block 01 a peptide chain of 1 to 10 amino acid lesidues, the hnkei gioup may function to modify the pharmacokinetics and blood cleaiance iates of the imaging agent /// vivo Such "biomodifiei" hnkei gioups may acceleiate the cleaiance of the imaging agent fiom backgiound tissue, such as muscle oi hvei, and/oi fiom the blood, thus giving a bettei diagnostic image due to less backgiound mteifeience A biomodifiei hnkei gioup may also be used to favoui a particulai ioute of excietion, eg via the kidneys as opposed to via the hvei

10 By the term "sugai" is meant a mono-, di- ot tπ- sacchaiide Suitable sugais include glucose, galactose, maltose, mannose, and lactose Optionally, the sugai may be functionahsed to permit facile coupling to ammo acids Thus, eg a glucosamine denvative of an ammo acid can be conjugated to othei ammo acids via peptide bonds The glucosamine denvative of aspaiagme (commeicially available fiom i s NovaBiochem) is one example of this

Piefened featuies

The moleculai weight of the imaging agent is suitably up to 8000 Daltons Piefeiably, the moleculai weight is in the iange 2800 to 6000 Daltons, most piefeiably 3000 to 0 4500 Daltons, with 3200 to 4000 Daltons being especially piefened

Piefened imaging agents of the piesent invention have both peptide termini piotected by M ^IG gioups, ie piefeiably both Z ¹ and Z ² aie M ^IG , which will usually be diffeient As noted above, eithei of Z'/Z ⁷ may optionally repiesent -(L) _n [Bzp ^M ] Having both <ϊ peptide termini piotected in this way is impoitant foi in vivo imaging applications, since otherwise iapid metabolism would be expected with consequent loss of selective

binding affinity foi cMet When both Z ¹ and Z" aie M ^ICl , piefeiably Z ¹ is acetyl and Z ^? is a pnmaiy amide Most piefeiably, Z is acetyl and Z is a pnmaiy amide and the (L) _n [Bzp ^M ] moiety is attached to the epsilon amine side chain of a lysine iesidue of cMBP

Piefeired cMBP peptides of the picsent invention have a K _c i foi binding to cMet of less than about 10 nM (based on fluorescence polaiisation assay measuiements), most piefeiably less than 5 nM, with less than 3 nM being the ideal

The peptide sequence (SEQ-I)

Cys ^d -X ^l -Cys ^c -X ² -Gly-Pio-Pio-X'-Phe-Glu-Cys ^d -Trp-Cys ^b -Tyi -X ⁴ -X'-X ⁶ of the cMBP of Foimula I is a 17-mei peptide sequence, which is piimanly iesponsible foi the selective binding to cMet When the cMBP peptide of the piesent invention compπscs moie than 17 ammo acid lesidues, the iemammg ammo acids can be any ammo acid apait fiom cysteine Additional, unpiotccted cysteine iesidues could cause unwanted sciambling of the defined Cys ^a -Cys ^b and Cys ^L -Cys ^d disulfide budges The additional peptides piefeiably comprise at least one ammo acid iesidue with a side chain suitable foi facile conjugation of the -(L) _n [Bzp ^M ] moiety Suitable such iesidues include Asp oi GIu i esidues foi conjugation with aminc-functionahsed -(L) _n Bzp ^M gioups, oi a Lys residue foi conjugation with an amine- functionalised

-(L) _n Bzp ^M gioup The ammo acid iesidues foi conjugation of -(L) _n [Bzp ^M ] ate suitably located away fiom the 17-mei binding iegion of the cMBP peptide (SEQ-I ), and aie piefeiably located at the C- oi N- tei minus Piefeiably, the ammo acid iesidue foi conjugation is a Lys i esidue

Substitution of the tiyptophan iesidue of SEQ-I was evaluated with the known amino acid substitutes phenylalanine and napthylalanme Loss of cMet affinity was, howevci, found suggesting that the tiyptophan iesidue is important foi activity

It is piefeπed that the cMBP peptide fuithei compiises a N-termmal senne residue, giving the 18-mei (SEQ-2)

Sei-Cys ^a -X ¹ -Cys ^L -X ² -Gly-Pio-Pio-X'-Phe-Glu-Cys ^d -Tip-Cys ^b -Tyi-X ⁴ -X"-X ⁽¹

In addition to SEQ-I , oi piefei ably SEQ-2, the cMBP most piefeiably fuither

compπses eithei

(i) an Asp or GIu tesidue within 4 amino acid iesidues of eithei the C- 01 N- peptide terminus, and (L) _n Bzp ^λl is functionahsed with an amine gioup which is conjugated to the caiboxyl side chain of said Asp or GIu iesidue to give an amide bond,

(ii) a Lys i esidue withm 4 amino acid iesidues of eithei the C- oi N- peptide terminus, and (L) _n Bzp ^M is functionahsed with a caiboxyl gioup which is conjugated to the epsilon amine side chain of said Lys iesidue to give an amide bond

Picfeπed cMBP peptides compiisc the 22-mei ammo acid sequence (SEQ-3)

Ala-Gly-Sei-Cys ^a -X'-Cys ^c -X ² -Gly-Pio-Pio-X ³ -Phe-Glu-Cys ^d -Tφ-Cys ^b -Tyi-

The cMBP peptides of the piesent invention pi efeiably have X^ equal to Ai g

The cMBP peptide piefeiably fuithei compi ises in addition to SEQ- I , SEQ-2 ot SEQ-3, at eithei the N- oi C- terminus a hnkei peptide which is chosen fiom -Gly-Gly-Gly-Lys- (SEQ-4), -Gly-Sei -Gly-Lys- (SEQ-5) oi -Gly-Sei-Gly-Sei-Lys- (SEQ-6)

The Lys iesidue of the hnkei peptide is a most piefened location foi conjugation of the -(L) _π [Bzp ^M ] moiety Especially pi efen ed cMBP peptides compose SEQ-3 togethei with the hnkei peptide of SEQ-4, having the 26-mei ammo acid sequence (SEQ-7) Ala-Gly-Sei-Cys ^a -Tyi-Cys ^c -Sei-Gly-Pio-Pio-Aig-Phe-Glu-Cys ^d -Tip-Cys ^b -

Tyi -Glu-Tht -Glu-Gly-Thi -Gly-Gly-Gly-Lys

cMBP peptides of SEQ-I , SEQ-2, SEQ-3 and SEQ-7 piefeiably have Z ¹ = Z ² = M ^Kj , and most piefeiably have Z ¹ = acetyl and Z ² = pnmaiy amide

The -(L) _n [Bzp ^vl ] moiety is suitably attached to eithei of the Z ¹ oi Z ¹ gioups oi an ammo acid iesidue of the cMBP peptide which is diffeient to the cMet binding sequence of SEQ- I Pt efeπed ammo acid iesidues and sites of conjugation aie as desciibed above When the -(L) _n [Bzp ^M ] moiety is attached to Z ¹ oi Z ² , it may take

the place of Z 01 Z ^" by conjugation to the N- or C- terminus, and block in vivo metabolism in that way

The [Bzp ^M ]-(L) _n - moiety of Foimula I is prefeiably attached at positions R\ R ⁶ , R ¹⁴ , R" oi R ¹⁶ of the Bzp ^M of Foimula II, moie piefeiably at R ⁶ , R ¹⁴ , R ¹ " 01 R ^{1 6} most piefeiably at R ⁶ , R ¹⁴ 01 R ¹ " In oidei to facilitate the attachment, the lelevant R\ R ⁶ , R ¹⁴ , R ^{1 "1} oi R ¹⁶ substituent is piefeiably Ci ₆ caiboxyalkyl, mote piefeiably C^, caiboxyalkyl

The beiVopyiylmm dye (Bzp ^M ) pi efeiably has at least 2 sulfonic acid substituents, moic pieferably 2 to 6 sulfonic acid substituents, most piefeiably 2 to 4 sulfonic acid substituents Piefeiably, at least one of the sulfonic acid substituents is a Ci ₄ sulfoalkyl gioup Such sulfoalkyl gioups aic piefei ably located at positions R ⁶ , R ⁷ , R ⁸ , R ¹⁴ , R ^{1 "1} or R ¹⁶ , moie piefeiably at R ⁶ , R ⁷ , R ^s , R ¹⁴ 01 R ^] \ most piefeiably at R ⁶ togethci with one 01 both of R ⁷ and R ^s of Foi mula II The sulfoalkyl gioups of Foi mula II, aie pt efeiably of formula -(CFL)κSO M ¹ , wheie M ¹ is H 01 B ^c , k is an integei of value 1 to 4, and B ^c is a biocompatible cation (as defined above) k is piefeiably 3 01 4

In Formula II, w is piefeiably 1 R ^"1 is pi efeiably H 01 Ci ₄ caiboxyalkyl, and is most ptefeiably H X is piefeiably -CR ¹⁴ R ¹ "- 01 -NR ¹⁶ - , and is most piefeiably -CR ¹⁴ R ¹ '-

Piefeited Bzp dyes aie of Foimula III

(III) wheie Y ¹ , R ¹ - R ⁴ , R ⁶ , R ¹⁴ , R ¹ " and I ate as defined foi Formula II Suitable dyes of Formula III aie of Formula Ilia 01 HIb

(Ilia)

(HIb)

Preferred R ¹ -R ⁴ and R ⁶ -R ¹³ groups of Formulae III, Ilia and IHb are as described above for Formulae Ha and lib. In Formulae III, Ilia and IHb, R ¹⁴ and R ¹³ are preferably chosen such that one is an R ^b group and the other is an R ^c group. R ^b is Ci _-2 alkyl, most preferably methyl. R ^c is Ci _-4 alkyl, Ci _-6 carboxyalkyl or C|_ ₄ sulfoalkyl, preferably C _3-6 carboxyalkyl or -(CH ₂ XSChM ¹ where k is chosen to be 3 or 4.

Preferably the dyes of Formula III have a Ci _-6 carboxyalkyl substituent to permit facile covalent attachment to the cMBP.

In Formula II or III, when R and/or R together with one or both of R and/or R 10 form a 5- or 6- membered N-containmg heterocyclic or heteroaryl ring, preferred such rings are pyridyl or dihydropyridyl. A preferred such Y ¹ group wherein an R ⁸ group has been cyclised with R ¹⁰ is of Formula Y ^c :

(Y ^L ) A piefeπed such Y ¹ gioup wheiein both R ⁷ and R ^s gioup have been cyclised is of

Formula Y ^d

(Y ^d ) wheie

R ⁷ , R ⁹ and R ^{1 1} -R ¹ ' ate as defined above, each E ¹ is independently H 01 Ci ₄ alkyl In Formula Y\ it is ptefeπed that each X ¹ is OHU, R ⁹ = R ^π = H, R ^P is H, R ¹² is CH, 01 -C(CH ₃ K moie piefei ably - C(CHOi

In Foimula Y ^c , it is piefeπed that

R" = H,

R ¹² Is H,

R ^" is piefeiably CH , ot -C(CHOs, moie piefeiably -C(CHOi

It is piefcπed that the -NR ⁷ R ^S gioup of Formula III is eithei

(1) in open chain foim, ie the R ⁷ /R ^s gioups aie not cyclised with one 01 both of R ⁹ /R ¹⁰ Piefeπed such R ⁷ and R ⁸ gioups aie independently chosen fiom Ci ₄ alkyl 01 Ci ₄ sulfoalkyl, most pi efeiably ethyl 01 C-, ₄

sit lfo alky 1, (u) cyclised to give a cyclic Y ¹ substituent of Formula Y ^c or Y ^c , moie pieferably of Formula Y ^1" The open chain form (i) is most pieferred

Especially preferred dyes of Formula III are of Formula HIc, IIId oi IHe

(HIc)

10 (IIId)

(HIe) where'

M ¹ is as defined above, i s R ¹⁷ and R ¹⁸ arc independently chosen from C| 4 alkyl or Ci 4 sulfoalkyl,

R ¹⁹ is H 01 Ci ₄ alkyl,

R ²⁰ is Ci-4 alkyl, Ci _-4 sulfoalkyl or Ci _-6 carboxyalkyl;

R ^" is Ci „ ₄ sulfoalkyl or C] _-6 carboxyalkyl;

R- ^" is Ci- ₄ alkyl, C ₁ - ₄ sulfoalkyl or Ci _-6 carboxyalkyl;

E ² , E' and E ⁴ are independently H or Ci _-4 alkyl.

The dyes of Formulae IHd, IHe and IHf are preferably chosen such that one or more of R ¹⁹ -R ^" is CM sulfoalkyl.

Preferred specific dyes of Formula Hid are DY-631 and DY-633:

DY-631

DY-633

A preferred specific dye of Formula Hie is DY-652:

DY-652 Preferred specific dyes are DY-631 and DY-652, with DY-652 being most preferred.

When a synthetic linker group (L) is present, it preferably comprises terminal functional groups which facilitate conjugation to [Bzp ^λl J and Z'-[cMBPl-Z ² . When L comprises a peptide chain of 1 to 10 amino acid residues, the amino acid residues are preferably chosen from gly cine. l) sine, 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 Biol or Bio2:

(Biol)

17-amino-5-oxo-6-aza-3, 9, 12, 15-tetraoxaheptadecanoic acid of Formula Biol

wherein p is an integer from 1 to 10. Alternatively, a PEG-like structure based on a propionic acid derivative of Formula Bio2 can be used:

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

In Formula Bio2, p is prefeiably 1 or 2, and q is piefeiably 5 to 12

When the linker group does not comprise PEG oi a peptide chain, preferred L gioups have a backbone chain of linked atoms which make up the -(A) ₁₁₁ - moiety of 2 to 10 > atoms, most preferably 2 to 5 atoms, with 2 oi 3 atoms being especially prefeπed A minimum lmkei group backbone chain of 2 atoms confeis the advantage that the imaging moiety is well-sepaiated so that any undesiiable interaction is minimised

In Formula I, n is preferably 0 or 1 , most piefeiably 0, i e no linker group is present0

Prefeπed imaging agents of the present invention aie of Formula IV

M ^1G -λla-Gly-Sei-Cys ^a -Tyr-Cys ^c -Sei -GIy-Pi o-Pro-Arg-Phe-Glu-Cys ^d -Trp- Cys ^b -Tyr-Glu-Thi-Glu-Gly-Thr-Gly-Gly-Gly-Lys-M ^{1( l}

(L) _n [Bzp ^lVI ] 5 (IV)

wherein the (L) _n [Bzp ^M ] gioup is attached to the epsilon ammo group of the Lys iesidue Preferred imaging agents of Formula IV ha\ e M ^IG (N-termmal Ala) equal to acetyl and M ' (C-termuial Lys) equal to primary amide In Formula IV, n is preferably zero and Bzp ^M is prefeiably of Formula IUa or IHb, moie preferably of Formula IHc, IHd or HIe, most preferably the specific dyes DY-631 , DY-633 or DY- 652. In Formula IV, the most preferred specific dye is DY-652

Peptides of formula Z'-[cMBP]-Z ² of the present invention may be obtained by a5 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 aie unprotected, and the Cys ^c and Cys ^d i esidues have thiol-piotecting gioups, (ii) treatment of the peptide from step (i) with aqueous base in solution to give a monocyclic peptide with a first disulfide bond linking Cys ^a and Cys\

(in) removal of the Cys ^1' and Cys ^c thiol-piotectmg groups and cyclisation to give a second disulfide bond linking Cys ^c and Cys ^d , which is the desired bicychc peptide pioduct Z'-[cMBP]-Z ²

By the teim "piotecting gioup" is meant a gioup which inhibits 01 suppi esses undesiiable chemical leactions, but which is designed to be sufficiently leactive that it may be cleaved fiom the functional gioup in question undei mild enough conditions that do not modify the iest of the molecule Aftei depiotection the desned pioduct is s obtained Amine piotecting gioups aie well known to those skilled m the ait and aie suitably chosen fiom Boc (whei e Boc is teit-butyloxycaibonyl), Fmoc (wheie Fmoc is fluoienylmethoxycaibonyl), tiifluoioacetyl, allyloxycaibonyl, Dde [i e l-(4,4- dimethyl-2,6-dioxocyclohexyhdene)ethyl] 01 Npys (i e 3-mtio-2-pyiidine sulfenyl) Suitable thiol piotecting gioups aie Tit (Tπtyl), Acm (acetamidomethyl), /-Bu (teit-

10 butyl), /e/ Z-Butylthio, methoxybenzyl, methylbenzyl oi Npys (3-nitio-2-pyπdine sulfenyl) The use of fuithet piotecting gioups aie desciibed m 'Piotective Gioups in Oiganic Synthesis', Theoiodoia W Gieene and Petei G M Wuts, (John Wiley & Sons, 1991 ) Piefened amine piotecting gioups aie Boc and Fmoc, most piefeiably Boc Piefened amine protecting gioups aie Tit and Acm i s

Examples 1 and 2 piovide furthei specific details Fuithei details of solid phase peptide synthesis aie desci ibed m P Lloyd-Williams, F Albeiicio and E Gn aid, Chemical Appi oaches to the S] n thesis of Peptides and Pi oteins, CRC Pi ess, 1997 The cMBP peptides aie best stoted undei ineit atmospheie and kept in a fieezei 0 When used in solution, it is best to avoid pH above 7 since that πsks sciamblmg of the disulfide budges

The imaging agents can be piepaied as desci ibed in the thud aspect (below) 5

In a second aspect, the piesent invention piovides a pharmaceutical composition which composes the imaging agent of the fust aspect togethei with a biocompatible cai πei , in a foim suitable foi mammalian admimstiation 0 The "biocompatible caiπei" is a fluid, especially a liquid, in which the imaging agent can be suspended oi dissolved, such that the composition is physiologically tolei able, ie can be administeied to the mammalian body without toxicity oi undue discomfoit The biocompatible caπ iei is suitably an injectable caπ iei liquid such as steπle, pytogen-fiee watei foi injection, an aqueous solution such as saline (which may

advantageously be balanced so that the final pioduct foi injection is isotonic), an aqueous solution of one 01 moie tonicity-adjustmg substances (eg salts of plasma cations w ith biocompatible counteiions), sugais (e g glucose 01 suctose), sugai alcohols (eg soibitol 01 mannitol), glycols (eg glyceiol), 01 othei non-ionic polyol mateuals (eg polyethyleneglycols, piopylene glycols and the like) Piefeiably the biocompatible caπiei is pyrogen-ftee watei foi injection oi isotonic saline

The imaging agents and biocompatible camel aie each supplied in suitable vials oi vessels which compnse a sealed containei which permits maintenance of stenle mtegiity, plus optionally an ineit headspace gas (eg nitiogen oi aigon), whilst peimitting addition and withdiawal of solutions by syπnge oi cannula A piefeπed such contdinei is a septum-sealed vial, wheiein the gas-tight closuie is cπmped on with an oveiseal (typically of aluminium) The closuie is suitable foi single oi multiple punctuimg with a hypodermic needle (e g a ctimpcd-on septum seal closuie) whilst maintaining stenle mtegiity Such containei s have the additional advantage that the closuie can withstand vacuum if desued (eg to change the headspace gas oi degas solutions), and withstand piessuie changes such as i eductions m piessuie without peimitting ingiess of external atmospheiic gases, such as oxygen oi watei vapour

Piefened multiple dose containei s compnse a single bulk vial (e g of 10 to 30 cm' volume) which contains multiple patient doses, wheieby single patient doses can thus be withdiawn into clinical giade syiinges at vaiious time inteivals dining the viable lifetime of the piepaiation to suit the clinical situation Pie-filled syimges aie designed to contain a single human dose, oi "unit dose" and aie theicfoie piefeiably a disposable oi othei synnge suitable foi clinical use The pharmaceutical compositions of the piesent invention piefeiably have a dosage suitable foi a single patient and aie piovided m a suitable synnge oi containei, as descnbed above

The pharmaceutical composition may optionally contain additional cxcipients such as an antimiciobial pieseivative, pH-adj listing agent, fillei, stabilise! oi osmolality adjusting agent By the teim "antimiciobial pieseivative" is meant an agent which inhibits the giowth of potentially harmful micio-oigamsms such as bactena, yeasts oi moulds The antimiciobial pteseivatn e may also exhibit some bactencidal pioperties, depending on the dosage employed The mam iole of the antimiciobial pieservative(s)

of the picsent invention is to inhibit the giowth of any such micio-oiganism in the phaimaceutical composition The antimiciobial pieseivative may, however, also optionally be used to inhibit the giowth of potentially harmful micio-oiganisms m one oi moie components of kits used to piepaie said composition pnoi to admmistiation Suitable antimiciobial pieseivative(s) include the paiabens, ie methyl, ethyl, piopyl oi butyl paiaben 01 mixtuies theieof, benzyl alcohol, phenol, ciesol, cetiimide and tluomeisal Ptcfeπed antimiciobial pieseivative(s) aie the paiabens

The term "pH-adjustmg agent" means a compound 01 mixtme of compounds useful to ensuie that the pH of the composition is within acceptable limits (appioximately pH

4 0 to 10 5) foi human oi mammalian admmistiation Suitable such pH-adjusting agents include pharmaceutically acceptable buffei s, such as tiicme, phosphate oi TRIS

[ie /m(hydioxymethyl)ammomethane], and phaimaceutically acceptable bases such as sodium caibonate, sodium bicaibonate oi mixtuies theieof When the composition is employed in kit form, the pH adjusting agent may optionally be piovided in a sepaiate vial oi containet, so that the usei of the kit can adjust the pH as pait of a multi-step pioceduie

By the term "fillei" is meant a pharmaceutically acceptable bulking agent which may facilitate mateπal handling dm ing pioduction and lyophilisation Suitable filleis include moiganic salts such as sodium chlonde, and watei soluble sugais oi sugai alcohols such as suciose, maltose, manmtol oi tiehalose

The pharmaceutical compositions of the second aspect may be piepaied undei aseptic manufactuie (ie clean loom) conditions to give the desned stenle, non-pyiogenic pioduct It is piefened that the key components, especially the associated ieagents plus those paits of the appaiatus which come into contact with the imaging agent (eg vials) aie stenle The components and ieagents can be steπlised by methods known m the ait, including stenle filtiation, terminal stei ihsation using e g gamma-irradiation, autoclavmg, dty heat oi chemical treatment (e g with ethylene oxide) It is piefeπed to steiihse some components m advance, so that the minimum numbei of manipulations needs to be earned out As a piccaution, howevei, it is piefened to include at least a stenle filtiation step as the final step m the pi epaiation of the

phaimaceutical composition

The phaimaceutical composition of the second aspect may optionally be piepaied fiom a kit, as desciibed foi the fouith aspect below

In a thud aspect, the piesent invention piovides a method of piepaiation of the imaging agent of the fust aspect, which composes one of steps (i) to (iv)

(i) ieaction of a peptide of formula Z ¹ ^cMBP]-Z ⁰ wheiein Z ¹ is H and Z ¹ is a M ^{I( l} with a compound of formula j '-(L) _n -[Bzp ^M ], to give the imaging agent of

Foimula I wheiem Bzp ^v( is conjugated at the Z ¹ position, (n) ieaction of a peptide of formula Z'-[cMBP]-Z ⁷ wheiem Z ¹ = Z ⁷ = M ^1G and cMBP compiises an Asp or GIu residue withm 4 ammo acid iesidues of eithei the C- oi N- cMBP peptide terminus, and all othei Asp/Glu iesidues of the cMBP peptides ate piotected, with a compound of formula

I -(L) _n -[Bzp' ], to give the imaging agent of Formula I whei ein Bzp is conjugated at said Asp oi GIu iesidue of the cMBP peptide, (in) ieaction of a peptide of formula Z'-fcMBPJ-Z ¹ wheiem Z ¹ is M ^l(l and Z ¹ is a 77 gioup oi an activated estei and all othei Asp/Glu iesidues of the cMBP peptides are piotected, with a compound of formula

T ⁷ -(L) _n -[B7p ^M ], to give the imaging agent of Formula I wheiem Bzp ^M is conjugated at the Z ^~ position,

(iv) ieaction of a peptide of formula Z'-fcMBPJ-Z ² wheiein Z ¹ = Z ^" " = M ¹⁰ and cMBP compiises a Lys within 4 ammo acid iesidues of eithei the C oi N- cMBP peptide terminus, with a compound of foimula T ¹ (L) _n -[Bzp ^M ], to give the imaging agent of Formula I wheiem B/p ^M is conjugated at a Lys iesidue of the cMBP peptide, wheiem Z ¹ , cMBP, M ^IG , L, n and B/p ^M aie as defined in the fust aspect, Z is a Z ^" gioup ot an activated estei ,

T ¹ is a caiboxyhc acid, activated estei, isothiocyanate oi thiocyanate gioup, I ^" IS an amine gioup

By the tetm "activated estei" oi "active estei" is meant an estei denvative of the

carboxylic acid which is designed to be a better leaving gioup, and hence permit moie facile ieaction with nucleophilc, such as amines Examples of suitable active esteis are N-hydioxysuccmimide (NHS), pentafluoiophenol, pentafluorothiophenol, para- nitiophenol and hydroxybenzotπazole Preferred active esters are N- hydioxysuccinimide or pentafluorophenol esteis

J ^" is pieferably a pnmaiy or secondaiy amine group, most preferably a pπmaiy amine group

The compound Z'-[cMBP]-Z ² preferably has both Z ¹ and Z ² equal to M ^IG Preferred cMBP peptides and Z'/Z ² groups aie as dcscπbed in the first aspect In particular, it is preferred that the cMBP peptide compiises an Asp, GIu or Lys residue to facilitate conjugation as described fot the pieferred cMBP peptides of the fust aspect It is especially prefeπed that the cMBP peptide comprises a Lys residue, as described in step (iv)

The pieparation of the Z' -[cMBP]-Z ² is described m the fust embodiment (above) The Z'-[cMBP]-Z^ peptide wheie Z' is an active ester can be prepared from Z'-[cMBP]-Z ² , where Z ² is OH oi a biocompatible cation (B ^c ), by conventional methods

Foi conjugation with the cMBP, the Bzp ¹ suitably compiises a reactive oi functional group (G) gioup which reacts with a complementary group of the cMBP peptide forming a covalent linkage between the dye and the cMBP peptide G may be a reactive group (Q ^a ) 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, malcimide, haloacetamide, acid halide, hydrazide, vmylsulfone, dichlorotπazme, phosphoiamidite, hydro xyl, ammo, sulfydryl, carbonyl, carboxylic acid and thiophosphate Preferably G is a reactive gioup (Q ^a ) Q ^a is prefeiably an active ester

Benzopyrylium dyes (Bzp ^M ) functionalised suitable for conjugation to cMBP aie

commeicially available fiom Dyomics (Dyomics GmbH, Wmzeilaei Stt 2A, D- 07745 Tena, Germany, w ww dyomics com), wheie the leactive gioup (Q ^a ) is NHS estei, maleimide, ammo 01 caiboxyhc acid Piecuisois suitable foi the synthesis of benzopyiyhum dyes can also be piepaied as descπbed m US 5405976 Methods of i conjugating optical tepoitci dyes, to ammo acids and peptides aie descπbed by Licha [Topics Cuπ Chem , 222, 1-29 (2002), Adv Diug Dehv Rev , 57, 1087-1 108 (2005)], as well as Flanagan et al [Bioconj Chem , 8, 751-756 (1997)], Lm et al, [ibid, J_3, 605-610 (2002)] and Zaheei [MoI Imaging, 1(4), 354-364 (2002)] Methods of conjugating the hnkei gioup (L) to the cMBP peptide use analogous chemistiy to that0 of the dyes alone (see above), and aie known in the art

In a fourth aspect, the piesent invention piovides a kit foi the piepaiation of the phaimaceutical composition of the second aspect, which compπses the imaging agent5 of the fiist aspect in stenle, solid form such that, upon ieconstitution with a stenle supply of the biocompatible caπiei of the second aspect, dissolution occuis to give the desned pharmaceutical composition

In that instance, the imaging agent, plus othei optional excipients as desciibed above,0 may be piovided as a lyophilised powdei in a suitable vial or contamei The agent is then designed to be ieconstituted with the desned biocompatible camel to the phaimaceutical composition m a stenle, apyiogemc fomi which is ieady foi mammalian admimstiation A piefeπed stenle, solid fomi of the imaging agent is a lyophilised solid The stenle, solid fomi is piefeiably supplied m a phaimaceutical^ giade contamei, as desciibed foi the pharmaceutical composition (above) When the kit is lyophilised, the foimulation may optionally compπse a cryoptotectant chosen fiom a sacchaπde, piefeiably niannitol, maltose oi tiicme

0 In a fifth aspect, the piesent invention piovides a method of //; vivo optical imaging of the mammalian body which compπses use of eithei the imaging agent of the fust aspect ot the pharmaceutical composition of the second aspect to obtain images of sites of cMet ovei-expiession oi localisation ;// vivo

The term "optical imaging" has the same meaning as foi the fust aspect (abo\ c) The mammalian body of the fifth aspect is piefetably the human body Piefeπed embodiments of the imaging agent aie as desciibed foi the fust aspect (above)

In the method of the fifth aspect, the imaging agent oi pharmaceutical composition has piefei ably been pieviously admimsteied to said mammalian body By "picviously administeied" is meant that the step involving the clinician, wheiein the imaging agent is given to the patient eg as an mtiavenous injection, has aheady been earned out pπoi to imaging This embodiment includes the use of the imaging agent of the fust embodiment foi the manufactuie of a diagnostic agent foi optical imaging in \ n o of disease states of the mammalian body wheie cMet is implicated

A piefeπed optical imaging method of the fifth aspect is Fluoiescence Reflectance Imaging (FRI) In FRI, the imaging agent of the piesent invention is admimsteied to a subject to be diagnosed, and subsequently a tissue sui face of the subject is illuminated w ith an excitation light - usually continuous w a\ e (CVV) excitation The light excites the dye (Bzp ¹ ) Fluoiescence fiom the imaging agent, which is geneiated by the excitation light, is detected using a fluoiescence detectoi The ietuming light is piefeiably filteicd to sepaiate out the fluoiescence component (solely oi paitiallv) An image is formed fiom the fluoiescent light Usually minimal piocessmg is pei formed (no piocessoi to compute optical paiameteis such as lifetime, quantum yield etc ) and the image maps the fluoiescence intensity The imaging agent is designed to concentiate in the disease ai ea, pioducmg highei fluoiescence intensity Thus the disease area pioduces positive contiast m a fluoiescence intensity image The image is piefeiably obtained using a CCD cameia oi chip, such that leal-time imaging is possible

The wavelength foi excitation vanes depending on the type of dye used The appaiatus foi genei ating the excitation light may be a conventional excitation light souice such as a lasei (e g , ion lasei, dye lasei oi semiconductoi lasei ), halogen light souice oi xenon light souice Vai ious optical filteis may optionally be used to obtain the optimal excitation wa\ elength

A piefened FRI method compiises the steps as follows

(l) a tissue sui face of inteiest within the mammalian body is illuminated with an excitation light,

(n ) fluoiescence fiom the imaging agent, which is gcneiated by excitation of the dye (Bzp ^M ), is detected using a fluoiescence detectoi, 5 (in) the light detected by the fluoiescence detectoi is optionally filteied to sepaiate out the fluoiescence component,

(iv) an image of said tissue suiface of mteiest is formed fiom the fluorescent light of steps (n) 01 (in)

In step (i), the excitation light is piefeiably continuous wave (CW) m natuie In step 10 (in), the light detected is piefeiably filteied An especially piefei icd FRI method is fluoiescence endoscopy

An alternative imaging method of the fifth aspect uses FDPM (fiequency-domam photon migiation) This has advantages ovei continuous-w ave (CW) methods wheic

I ¹ ) gieatei depth of detection of the imaging agent within tissue is impoitant [Sevick- Muiaca et al, Cuπ Opin Chem Biol , 6, 642 650 (2002)] Foi such fiequency/time domain imaging, it is advantageous if the Bzp ^M has fluoiescent piopeities which can be modulated depending on the tissue depth of the lesion to be imaged, and the type of insti umentation employed 0

The FDPM method is as follows

(a) exposing light-scatteimg biological tissue of said mammalian body having a heteiogeneous composition to light fiom a light souice with a pie- determmed time vaiying intensity to excite the imaging agent, the tissue5 multiply-scatteimg the excitation light,

(b) detecting a multiply- scatteied light emission fiom the tissue in iesponse to said exposing,

(c) quantifying a fluoiescence chaiacteiistic throughout the tissue fiom the emission by establishing a numbei of values with a piocessoi, the values each0 conespondmg to a level of the fluoiescence chaiacteiistic at a diffeient position within the tissue, the level of the fluoiescence chaiacteiistic vaiying with heteiogeneous composition of the tissue, and

(d) genei atmg an image of the tissue by mapping the heteiogeneous composition of the tissue in accoi dance with the values of step (c)

The fluoiescence characteristic of step (c) piefeiably coπ esponds to uptake of the imaging agent and piefeiably fuithei compiises mapping a numbei of quantities coπesponding to adsotption and scatteiing coefficients of the tissue befote administi ation of the imaging agent The fluoiescence chai acteπstic of step (c) 5 piefeiably conesponds to at least one of fluoiescence lifetime, fluoiescence quantum efficiency, fluoiescence yield and imaging agent uptake The fluoiescence chaiactenstic is piefeiably independent of the intensity of the emission and independent of imaging agent concentiation

The quantifying of step (c) piefeiably compiises (i) establishing an estimate of the

10 values, (n) deteiminmg a calculated emission as a function of the estimate, (in) compaimg the calculated emission to the emission of said detecting to determine an en oi, (iv) piovidmg a modified estimate of the fluoiescence chaiactenstic as a function of the eπoi The quantifying piefeiably compiises determining the values fiom a mathematical ielationship modelling multiple hght-scatteimg behavioui of the

I ¹ } tissue The method of the fust option piefeiably fuither compiises monitoiing a metabolic piopeity of the tissue ;;; M\ O by detecting vanation of said fluoiescence chaiactenstic

The optical imaging of the fifth aspect is piefeiably used to help facilitate the 0 management of coloiectal cancel (CRC) By the term "management of CRC" is meant use m the detection, staging, diagnosis, momtoimg of disease piogiession oi the monitoiing of tieatment Fuithei details of suitable optical imaging methods have been leviewed by Sevick-Muiaca et al [Cuπ Opin Chem Biol , 6, 642-650 (2002)] 5

In a sixth aspect, the piesent invention piovides a method of detection, staging, diagnosis, momtoimg of disease progression or m the momtoimg of tieatment of coloiectal cancel (CRC) of the mammalian body which compiises the in vivo optical imaging method of the fifth aspect 0

The invention is illustiated by the non-limiting Examples detailed below Example 1 piovides the synthesis of a biological taigetmg peptide (Peptide 1 ), which binds to cMct Example 2 piovides methods of conjugating Bzp ^M dyes of the invention to

peptides, m paiticulai Peptide 1 Example 3 piovides data demonstiating that the peptide conjugates of Peptide 1 of the invention tetam affinity foi cMet, i e that the conjugated dye does not inteifeie with the biological binding and selectivity Appiopnate low binding to human seium albumin and high stability m plasma was demonstiated Example 4 shows that the peptide conjugates of the invention exhibit useful turnout backgiound iatios in an animal model of coloiectal cancel Example 5 descπbes the use of piedictive softwaie foi the dyes of the invention, and demonstiates that the dyes of the invention lack potentially dangeious metabolites /// vivo Example 6 descπbes the toxicity testing of Compound 6, showing that the anticipated clinical dose was well tolerated and without any diug substance i elated adveise effects

Table 1 Stiuctuies of Benzopyiylmm dyes of the Exam les

wheie R ^d is -(C H,) SO ₃ H, R ^c is -(CII,),CO->H and R ¹ is -(CH^COH DY-752 has the same nngs and substituent pattern as DY-652, but has a pentamcthme linkage (i e w = 2 and R ^{^} =H) in place of the tπmethine linkage of DY-652

Abbieviations

Conventional 3-letter and single lettei ammo acid abbieviations aie used

Acm Acctamidomethyl

ACN Acetoniti ile

Boc fø/ t-Butyloxycaibonyl

DMF N,N -Dimethylfomiamide

DMSO Dimethylsulfoxide

λθ

Fmoc 9-Fluoienylmethoxycaibonyl

HCl Hydiochloiic acid

HPLC High peiformance liquid chiomatogiaphy

HSPyU O-(N-succinimidyl)-N,N N ,N tetiamethyleneuiomum he\afluoiophosphate lie Isoleucine

LC-MS Liquid chiomatogiaphy mass spectioscopy

NHS TV-hydioxy-succinimide

NMM TV-Methylmoφholme

NMP l-Methyl-2-pyrtohdmone

Pbf 2,2,4,6, 7-Pentamethyldihydiobenzofuran-5-sulfonyl

PBS Phosphate-buffeied saline

TFA Tiifluoioacetic acid

Tit Tiityl

TSTU O-(N-Succinimidyl)-N,N,N',N -tetiamethyluionium tetiafluoioboiate

Example 1 : Synthesis of Peptide 1.

A 26-mei bicyclic peptide having 2 Cys-Cys bonds (Cys4-16 and 6-14) having the following sequence was used

Ac-Ala-Gly-Sei-Cys-Tyi-Cys-Sei -GIy-Pi o-Pio-Aig-Phe-Glu-Cys-Tφ-Cys-Tyi -GIu-

Thi-Glu-Gly-Thi -GIy-GIy-GIy-LyS-NH ₂ ("Peptide 1 ")

Peptide 1

Step (a) Synthesis of piotected linear pi ecuisoi of Peptide 1

The piecuisoi hneai peptide has the sequence

Ac- Ala-Gly-Sei-Cys-Tyi-Cys(Acm)-Sei -GIy-Pi o-Pio- Ai g-Phe-Glu-Cys( Acm)-Tφ-

CyS-TyI-GIu-ThI-GIu-GIy-ThI-GIy-GIy-GIy-LyS-NH ₂

The peptidyl iesm H-Ala-Gly-Sei(tBu)-Cys(Tit)-Tyi(tBu)-Cys(Acm)-Sei(tBu)-Gly- Pio-Pio-Ai g(Pbf)-Phe-Glu(OtBu)-Cys(Acm)-Tip(Boc)-Cys(Tit)-Tyi(tBu)-

^Me pio)-Glu(OtBu)-Gly-Thi(tBii)-Gly-Gly-Gly-Lys(Boc)-Polymci was assembled on an Applied Biosystems 433 A peptide synthesize! using Fmoc chemistiy stalling with 0 1 mmol Rink Amide Novagel iesin An excess of 1 mraol pie-acti\ ated ammo acids (using HBTU) was applied in the coupling steps Glu-Thi pseudopiolme (Novabiochem 05-20-1 122) was mcoipoiated in the sequence The iesin was tiansfeπed to a nittogen bubblei appaiatus and tieated with a solution of acetic anhydnde (1 mmol) and NMM (1 mmol) dissolved m DCM (5 mL) foi 60 mm The anhydnde solution was lemoved by filtiation and the iesm washed with DCM and dned undei a stream of nitiogen

The simultaneous temoval of the side-chain piotectmg gioups and cleavage of the peptide fiom the iesin was earned out m TFA (10 mL) containing 2 5 % TIS, 2 5 % A- thiociesol and 2 5 % watei foi 2 houis and 30 mm The iesin was lemoved by filtiation, TFA lemoved /// vacuo and diethyl ethei added to the iesidue The foimed piecipitate was washed with diethyl ethei and aii-dπed affoidmg 264 mg of ciude peptide

Puπfication by piepaiative HPLC (giadient 20-30 % B ovei 40 mm wheie A = H _^ O/0 1 % TFA and B = ACN/0 1 % TFA, flow iate 10 niL/mni, column Phenomenex Luna 5μ C18 (2) 250 x 21 20 mm, detection UV 214 nm, pioduct ietention time 30 mm) of the ciude peptide affoided 100 mg of puie Peptide 1 lineai piecuisoi The puie pioduct was analysed by analytical HPLC (giadient 10-40 % B ovei 10 mm wheie A = H ₂ CVO 1 % TFA and B = ACN/0 1 % TFA, flow iate 0 3 mL/min, column Phenomenex Luna 3μ C18 (2) 50 x 2 mm, detection UV 214 nm, pioduct ietention time 6 54 mm) Fuithei pioduct chaiacteiisation was earned out uussiinngg eeklectiospiay mass spectiometiy (MHT ⁺ calculated 1464 6, MH ₂ ²⁺ found 1465 1 )

Step (b) Foimation of Cys4-16 disulfide budge Cys4-16, Ac-Ala-Gly-Sei-Cys-Tyi-Cys(Acm)-Ser-Gly-Pio-Pio-Aig-Phe-Glu- CyS(ACm)-Ti-P-CyS-TyI-GIu-ThI-GIu-GIy-ThI-GIy-GIy-GIy-LyS-NH ₂

The lmeai piecuisoi fiom step (a) (100 mg) was dissolved in 5 % DMSO/watei (200 mL) and the solution adjusted to pH 6 using ammonia The ieaction mixtuie was stiiied foi 5 days The solution was then adjusted to pH 2 using TFA and most of the

solvent lemoved by evapoiation in vacuo The tesidue (40 niL) was injected in poitions onto a ptepaiative HPLC column foi pioduct puiification

Puiification by piepaiative HPLC (giadient 0 % B foi 10 mm, then 0-40 % B ovei 40 mm wheie A = H ₂ O/0 1 % TFA and B = ACN/0 1 % TFA, flow iate 10 niL/niin, column Phenomenex Luna 5μ Cl 8 (2) 250 x 21 20 mm, detection UV 214 nm, pioduct ietention time 44 mm) of the tesidue affoided 72 mg of puie Peptide 1 monocyclic piecuisoi The puie pioduct (as a mixtuie of isomeis Pl to P3) was analysed by analytical HPLC (giadient 10-40 % B ovei 10 mm wheie A = H?O/0 1 % TFA and B = ACN/0 1 % TFA, flow iate 0 3 mL/min, column Phenomenex Luna 3μ C18 (2) 50 x 2 mm, detection UV 214 nm, ptoduct ietention time 5 37 mm (Pl), 5 61 mm (P2), 6 05 mm (P3)) Fuithei pioduct chaiacteiisation was earned out using electiospiay mass spectiometiy (MH? ^" calculated 1463 6, MH? ^{" 1} found 1464 1 (Pl ), 1464 4 (P2), 1464 3 (P3)

Step (c) Fomiation of Cys6-14 disulfide budge (Peptide 1)

The monocyclic piecuisoi fiom step (b) (72 mg) was dissolved in 75 % AcOH/watei (72 HiL) undei a blanket of nitiogen 1 M HCl (7 2 mL) and 0 05 M I? m AcOH (4 8 ITiL) weie added in that oidei and the mixtuie stiπcd foi 45 mm 1 M ascoibic acid (1 mL) was added giving a colouiless mixtuie Most of the solvents weie evapoiated in vacuo and the iesidue (18 mL) diluted with watei/0 1 % TFA (4 mL) and the pioduct puiified using piepaiative HPLC

Puiification by piepaiative HPLC (giadient 0 % B for 10 mm, then 20-30 % B ovei 40 mm wheie A = H ₂ CVO 1 % TFA and B = ACN/0 1 % TFA, flow iate 10 mL/mm, column Phenomenex Luna 5μ C18 (2) 250 x 21 20 mm, detection UV 214 nm, pioduct retention time 43-53 min) of the iesidue affoided 52 mg of puie Peptide 1 The pure product was analysed by analytical HPLC (giadient 10-40 % B ovei 10 mm wheie A = H?O/0 1 % TFA and B = ACN/0 1 % TFA, flow iate 0 3 mL/mm, column Phenomenex Luna 3μ Cl 8 (2) 50 x 2 mm, detection UV 214 nm, pioduct ietention time 6 54 mm) Fuithei pioduct chatacteπsation was caiπed out using electiospiay mass spectiometiy (MH ₂ ^{2 H} calculated 1391 5, MH ₂ ²⁺ found 1392 5)

Example 2: Synthesis of Peptide Conjugates of Benzopyry Hum Dλ es.

Gcneial coni ligation method

To a solution of Peptide 1 (fiom Example 1 , 4 mg, 1 4 μmol) in DMF (0 5 niL) was added a solution of Bzp ^M NHS estei (1 mg, 1 μmol) and sym -collidme (8 μL, 60 μmol) m DMF (0 5 mL) The ieaction mixtuie was heated (miciowave assisted) at 60 0C foi 1 hi, then at RT overnight The ieaction mixtuie was then diluted with 20 % ACN/watei/0 1 % TFA (7 mL) and the pioduct puiified using piepaiative HPLC

Puπfication and chaiacteiisation

10 Puiification by piepaiative HPLC (giadient 20-40 % B ovei 40 mm wheie A = H ₂ O/0 1 % TFA and B = ACN/0 1 % TFA, flow iate 10 mL/min, column Phenomenex Luna 5μ C18 (2) 250 x 21 2 mm, detection UV 214 nm) of the ciude peptide affoided pine [Peptide l]-Bzp ^M conjugate The puic pioduct was analysed by analytical HPLC (giadient 10-40 % B ovei 5 mm wheie A = H ₇ CVO 1 % TFA and B is = ACN/0 1 % TFA, flow iatc 0 6 mL/min, column Phenomenex Luna 3μ Cl 8 (2) 20 x 2 mm, detection UV 214 nm) Fuithei pioduct chaiacteiisation was earned out using electiospiay mass spectiometiy The compounds piepaied aie given m Table 3

Table 3 Peptide-dyc conjugates of Peptide 1

20

Example 3: In Vitro Fluorescence polarisation assay.

Fluoiescence polansation assay was used to examine the affinity binding of the imaging agent towaids the cMet taiget as well as the binding piopeities ielated to 2 ¹ ) plasma pioteins The pimciple of the fluoiescence polansation method can biiefly be desciibed as follows

Monochiomatic light passes thiough a honzontal polanzmg filtei and excites fluoiescent molecules m the sample Only those molecules that onented piopeily in

the vertically polarized plane adsorb light, become excited, and subsequently emit light. The emitted light is measured in both horizontal and vertical planes. The anisotrop) value (A), is the ratio between the light intensities following the equation

A = Intensity with horizontal polarizer - Intensity with vertical polarizer Intensity with horizontal polarizer + 2* Intensity with vertical polarizer

The fluorescence anisotropy measurements were performed in 384-well microplates in a volume of 10 μL in binding buffer (PBS, 0.01%Tween-20. pll 7.5) using a Tecan Safire fluorescence polarisation plate reader (Tecan , US) at Ex 635/Em 678 nm. The concentration of dye-labelled peptide was held constant (5nM) and the concentration of the human c-Met/ Fc chimera (R&D Systems) was varied from 0-250 nM. Binding mixtures were equilibrated in the microplate for 10 min at 30 ⁰ C. The observed change in anisotropy was fit to the equation

[K- i- cMct T P) ^~ v'(A ^{' >} ífλ/rt + P)I - 4 ^• ckkh p ^'

where robs is the observed anisotropy, rfree is the anisotrop} of the free peptide, rbound is the anisotropy of the bound peptide, K _d is the dissociation constant, cMet is the total c-Met 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 done via nonlinear regression using SigmaPlot software to obtain the K _c i value (one-site binding).

Compounds 1 to 6 were tested for binding towards human c-Met (Fc chimera). The results showed a Kd of nM for the binding of all compounds tested to human c-Met (see Table 4).

The change of the polarization value was used to assess the binding of the Compound to human serum albumin as a low change of polarisation value is associated to low binding being appropriate for in-vivo use. The plasma protein binding (PPB) was confirmed with Biacore measurements. The stability of the imaging agent in plasma was confirmed by measuring the amount of the Compound left after incubation in mouse plasma for 2 hours at 37°C.

^λ ,5

Table 4 ;// vitio piopeities of Compounds 1-6

Example 4: /// Vivo testing of Compounds 2 to 6.

(a) Animal Model Female BALB c/A nude (Bom) mice weie used in the study The use of the animals was appiovcd by the local ethics committee BALB c/A nude is an inbied immunocompiomised mouse stiam with a high take tate foi human tumouis as compaied to othei nude mice stiams The mice weie 8 weeks old upon aiiival and with a body weight of appiox 20 giams at the stait of the study The animals weie housed in individually ventilated cages (IVC, Scanbui BK) with HEPA filteied an The animals had ad libitum access to "Rat and Mouse ni 3 Bleeding" diet (Scanbui BK) and tap watei acidified by addition of HCl to a molai concentiation of 1 niM (pH 3 0)

The colon cancel cell HT-29 is denved fiom human colon caicinomas and is repotted to expiess c-Met accoiding to Zeng et al [CIm Exp Metastasis, 21_, 409-417 (2004)] The cell line was pi oven to be tumoπgenic when inoculated subcutaneously into nude mice [Flatmaik et al, Em J Cancel 40,1593-1598 (2004)]

HT-29 cells weie giown in McCoy's 5a medium (Sigma # M8403) supplemented with 10% fetal bovine seium and penicillm/stieptomycm Stocks weie made at passage numbei foui (P4) and fiozen down foi stoiage m liquid mtiogen at 10 ⁷ cells/vial m the lespective cultuie media containing 5% DMSO On the day of the tiansplantation, the cells were thawed quickly in 37 ⁰ C watei bath (appiox 2 mm),

washed and iesuspended in PBS/2% seium (centπfugation at 1200 rpm foi 10 mm) Thoiough mixing of cells in the vials was ensuied eveiy time the cells weie aspπated into the dosing syiinge A volume of 0 1 ml of cell suspension was injected s c at the shouldei and at the back using a fine boie needle (25 G) The animals w eie then s ietumcd to then cages and the tumouis weie allowed to giow foi 13-17 days The animals weie allowed an acclimatisation penod of at least 5 days befoie the inoculation pioceduie

(b) pioceduie 10 All test substances weie teconstituted with PBS fiom fieezc-dπed powdei A small stack of white pπntei papei was imaged to obtain a flat field image which was used to collect foi illumination inhomogeneities Foi immobilisation dining the optical imaging pioceduie, the animals weie anaesthetized in a coaxial open mask to light suigical level anaesthesia with Isofluiane (typically 1 3-2%), using oxygen as the

I ¹ S caπiei gas A small piece of skin (3-5mm) was lemovcd ovei parts of the tumoui and adjacent muscle using a suigical foiccps and fine scissois while the animal was anaesthetized This was done to measuie the signal from tumoui and muscle without inteifeience fiom the ovei lying skin tissue The wound was coveied by applying a liquid, non-fluoiescent bandage spiay (3M, MN, USA) 0 The iespnation and body tempeiatuie of the animal was momtoied with a BioVet system (m2m Imaging Coip, NJ, USA) using a pneumatic sensoi underneath the animal and a iectal tempeiatuie piobe The BioVet system also supplied external heating using a heating mat set to 40 ⁰ C to sustain normal body tempeiatuie for the dutation of the imaging pioceduie (2 houis) A Vcnflon cathetei was placed in the tail 5 vein foi contiast agent admimstiation Each animal was given one contiast agent injection The injected volume was 0 ImI of test compound followed immediately by a 0 2ml saline flush Fluoiescence images weie acquned just pnoi to injection and then eveiy 30 seconds for 2 houis 0 (c) Imaging

Imaging was peifoimed thiough a clinical lapaioscope adapted to use a light souice to excite the ieportei and a filteiing system to extiact the fluoiescence component A 635nm lasei was used foi excitation of the iepoitet molecule A Hamamatsu ORCA ERG CCD cameia was used as the detectoi The cameia was opeiated in 2x2 binning

mode with 0 gam Standaid exposuie time foi colon imaging was 4s The intensity distiibution in the image was collected foi illumination inhomogeneities thiough system calibiation data A taiget to backgiound tatio was computed fiom legions of mteiest placed ovei the exposed tumoui and nomial muscle backgiound

(d) Results

The test Compounds had the following aveiage tumoui muscle tatios (Table 5)

Table 5 tumoui muscle iatios of Compounds 2 to 6

Example 5: Metabolism and Toxicity Prediction.

The softwaie tools Deiek and Meteoi weie obtained fiom Lhasa Ltd (22-23 Blenheim Ten ace, Leeds LS2 9HD, UK) Deiek is used foi piedicting toxicity of new chemical entities based on known stiuctuie-dependent toxicity Similaily, Meteot piedicts likely metabolites of novel chemicals Both tools aie based on published and unpublished (but venfied) data foi chemical compounds The chemical stnictuie of dye DY-652 was input No potentially dangeious metabolites w vivo wcie piedicted

Example 6: Toxicity Testing of Compound 6.

A limited acute dose toxicity study was conducted to investigate the toleiance of

Compound 6 at 100 times the pieclinical imaging dose (50 nmol/kg body weight) The compound was injected mtiavenously in male tats, and the animals weie saciificed at 1 , 14, 21 and 28 days post injection (p i ) At neciopsy, the majot oigans weie inspected foi gioss pathology, and the kidneys were taken into neutial buffeied foi malm foi subsequent histomorphological evaluation A weak blue colouiation of the skm and a model ate blue colouiation of the mine weie obseived immediately aftei

injection, which disappeaicd within 1 day p i At neciopsy, the kidneys weic diffusely gieen on day 1 p i Light micioscopy showed no Compound 6 i elated findings m the kidneys The othei minoi changes seen weie incidental and common m young adult laboiatoiy iats Stiong fluoiesccnce staining of blood vessels m the kidney was obseived on day 1 p i The staining was i educed by day 14 p i and was not discernible fiom contiol on day 21 p i

No evidence of degeneiation, neciosis oi inflammation was noted m any of the tieated animals, suggesting that the nephiotoxicity of the compound is low It was concluded that a single intiavenous admmistiation of Compound 6 to male iats at 100 times the anticipated clinical dose was well toleiated and without any diug substance i elated adveise effects