Optical Imaging Agents

Field of the Invention

The piesent invention ielates to imaging agents suitable foi /// vn o optical imaging, which compiise conjugates of benzopyiyhum dyes with biological taigetmg moieties, such as peptides Also disclosed aie pharmaceutical compositions and kits, as well as m vn o imaging methods

10 Backgtound to the Invention

US 6750346 discloses lasei-compatible neai-mfraied (NIR) maikeis dyes of formulae

A, B oi C

I S whet ci n

n is 1 , 2 or 3,

R to R , 14 aie the same oi diffeient and aic chosen from H, Cl, Bi, an aliphatic

01 mononucleai aiomatic gioup, of up to 12 caibon atoms which may contain as a substituted gioup in addition to C and H, up to 4 oxygen atoms and 0, 1 oi

2 nitiogen atoms oi a sulfur atom oi a sulfui and a nitiogen atom oi iepresent an ammo function, having a nitiogen atom to which thete is bound, H oi at least one substituent having up to 8 caibon atoms, said substituent selected fiom the gioup consisting of C, H and up to two sulfonic acid gioups

The dyes of US 6750346 aie chosen such that piefeiably at least one of R to R 14 contains a solubilising oi ionisable gioup Such gioups aie said to include cyclodextπn, sugai, SCh , PCh ² , CO ₂ oi NR-, ⁺ US 6750346 teaches that the dyes, as well as systems denved fiom them (conjugates) can be used in optical, especially in fluoiescence optical qualitative and quantitative deteimmation methods foi the diagnosis of cell piopeities, in biosensois (pomt-of-caie measuiements), exploiation of the genome and in mimatuiisation technology Typical such applications being in the fields of cytometiy, cell soiling, fluoiescence coiielation spcctioscopy (FCS), ultia-high thioughput scieening (UHTS), multicoloui fluoiescence /// situ hybπdization (FISH) and m mictoaπays (gene and piotein chips)

US 6924372 discloses asymmetπcal polymethine dyes of formula D oi E

(D)

(E) whei e n is 0, 1, 2 oi 3,

R ^! -R ⁹ aie the same oi diffeient and may be H, alkyl-, tei t-alkyl, aryl-, caiboxyatyl-, dicaiboxyaiyl, hetcioaiyl-, cycloalkyl-, heteiocycloalkyl-, alkyloxy-, alkylmeicapto- (with "alkyl" and "cycloalkyl" also including olefin linkage lesidues), aryloxy-, aiylmeicapto-, heteioatyloxy-, heteroaiylmercapto-, hydioxy-, nitio- oi cyano iesidues and R ¹ and R ² , R ² and R ³ , R^ and R ⁴ , R ^{^} and R ⁷ can form one oi moie aliphatic, heteioaliphatic oi aiomatic ling

At least one of the R ^! -R ⁹ substituents of US 6924372 may optionally be a solubihsmg oi ionising substituent (eg SO ₃ , POr , CO ₂ H, OH, NR-, ^" , cyclodextnn 01 sugai), 01 may optionally be a leactive gioup (eg isothiocyanate, hydiazine, active estei, maleimide 01 lodacetamide) permitting covalent linkage of the dye to anothei molecule The dyes of formulae D and E aie said to be useful in diagnosing cell chaiacteiistics 01 biosensois, typically cytometiy and cell sorting

Lisy et al [1 Biomed Optics, 11(6) 064014 (2006)] disclose a method of diagnosis of peiitonitis using neai -mfi ai ed optical imaging and labelled monocytes 01 macrophages The monocytes-macrophages could be labelled in viti o with the dye DY-676 (Dyomics GmbH) Admmistiation of the dye DY-676 itself m an animal model of peiitonitis in vivo led to inci cased fluoiescence in the aiea of peiitonitis The authors concluded that monocyte-maciophage labelling had occuned in vivo

Lisy et al [Invest Radiol , 42(4) 235-241 (2007)] disclose bimodal (MRI and optical) contiast agents which compiise nanopai tides labelled with fluoiescent magnetosomes The fluoiescent magnetosome nanopaiticles weic used to label maciophages by a piocess of phagocytosis The dye used to label the magnetosomes was again DY-676

The Dyomics GmbH website (w w v, d\omics com) includes an image couitesy of I Hilgei (FSU lena) entitled "Visualisation of Aithπtis m a Rat by Accumulation of

DY-676 m loints" No fuithei details aie to m ¹ ven

WO 2007/139815 discloses imaging and theiapeutic methods involving piogemtoi cells Conjugates of the fomuila shown aie disclosed A _B -X

A _B compπses a vitamin oi analog that binds to CD 133 Flkl endothelial piogemtoi cells, X is a quantifiable maikei The quantifiable maikei can be eg a ladioactive piobe oi a fluoiescent piobe Suitable fluoiescent ptobes aie stated to be fluoiescein, lhodamine, Texas Red, phycoeiythπn, Oicgon Green, Alcxa Fluor 488 , Cy3, Cy5, Cy7, and the like Example 30 of WO 2007/139815 discloses a single benzopyiylium dye (DyLight ^{I M} 680) conjugated to folate via a 5-mei peptide hnkei (Asp-Aig-Asp-Asp-Cys)

The Piesent Invention

The piesent invention piovides imaging agents suitable foi /// \ ιvo optical imaging, which compiise a specific class of benzopyiylium dye conjugated to a biological taigetmg moiety (BTM) The piesent invcntois have identified sulfonated benzopyiylium dyes which aie suitable foi /// vivo optical imaging applications as pait of such covalently-bonded BTM conjugates

The benzopyi ylium dyes (Bzp ^M ) of the piesent invention possess a combination of piopeities which make them useful foi in vivo optical imaging applications (i) capability of conjugation to biological taigetmg molecules (BTM),

(ii) watei solubility, (in) absoiption and emission m the led, fai red oi neai mfta-ied poition of the electiomagnetic spectium, (iv) high extinction coefficients, (v) low blood plasma piotem binding,

(V ₁ ) high photostabihty and bnghtness, (vii) high stability of dye and dye-BTM conjugate in blood, (vin) i apid cleai ance fi om the blood /;/ \ n o, (ix) lack of potentially dangeious metabolites (by Meteoi/Deiek analyses)

Detailed Description of the Invention.

In a first aspect, the present invention provides a pharmaceutical composition which comprises an imaging agent suitable for /;/ vivo optical imaging of the mammalian body, together with a biocompatible carrier, said composition being in a form suitable for mammalian administration, wherein said imaging agent comprises a conjugate of Formula I:

[BTM]-(L) _n -Bzp ^M

(I) where:

BTM is a biological targeting moiety; n is an integer of value 0 or 1 ;

L is a synthetic linker group of formula -(A) ₁₁₁ - wherein m is an integer of value 1 to 20, and each A is independently -CR ₂ - , -CR=CR- , -C≡C- ,

-CR ₂ CO ₂ - , -CO ₂ CR ₂ - , -NRCO- , -CONR- , -NR(C=O)NR-, -NR(C=S)NR-,

-SO ₂ NR- , -NRSO ₂ - , -CR ₂ OCR ₂ - , -CR ₂ SCR ₂ - , -CR ₂ NRCR ₂ - , a C ₄ - ₈ cycloheteroalkylene group, a C ₄ _s cycloalkylene group, a C ₅ .12 arylene group, or a C _{3-I 2} heteroarylene group, an ammo acid, a sugar or a monodisperse polyethyleneglycol (PEG) building block; wherein each R is independently chosen from H, C _K4 alkyl, C ₂ - ₄ alkenyl, C ₂ . ₄ alkynyl, Ci _-4 alkoxyalkyl or Ci _-4 hydroxyalkyl;

Bzp ^M is a benzopyrylium dye of Formula II:

where:

Y ¹ is a group of Formula Y ^a or Y ^b

R' -R ⁴ and R ⁹ -R ^π aie independently selected fiom H, -SO ₁ M ¹ , Hal, R ^a 01 Ci ₁₂ atyl, wheie each M ¹ is independently H 01 B ^c , and B ^c is a biocompatible cation, R ^{^} is H, Ci ₄ alkyl, Ci ₆ caiboxyalkyl, C ₃ p aiylsulfonyl, Cl, 01 R ³ togethei with one of R ¹ , R , R ⁵ 01 R ⁶ may optionally form a 5- 01 6- membeicd unsatuiated aliphatic, unsatuiated heteioaliphatic 01 aiomatic ling, R ⁶ and R ¹⁶ aie independently R ¹ gioups,

R ⁷ and R ⁸ aie independently Ci 4 alkyl, C] ₄ sulfoalkyl 01 Ci 6 hydioxyalkyl 01 optionally togethei with one 01 both of R ⁹ and/01 R ¹⁰ may form a 5- 01 6- membeied N-containing heteiocychc ot hcteioaiyl πng,

X is -CR ¹⁴ R ¹ '-, -O-, -S-, -Sc-, -NR ¹⁶ - 01 -CH=CH- , wheie R ^{1 +} to R ¹⁶ aie independently R ^a gioups,

R ^a is Ci ₄ alkyl, Ci ₄ sulfoalkyl, Ci ₍₁ caiboxyalkyl 01 Ci ₆ hydioxyalkyl, W IS 1 Ol 2,

J is a biocompatible anion, with the pi oviso that Bzp ^lVI compπses at least one sulfonic acid substituent chosen fiom the R ¹ to R ¹⁶ gioups

By the term "imaging agent" is meant a compound suitable foi optical imaging of a region of mteiest of the whole (ie intact) mammalian body in vivo Piefeiably, the mammal is a human subject The imaging may be invasive (eg mtia-opeiative 01 endoscopic) 01 non-mvasive The imaging may optionally be used to facilitate biopsy (eg \ ia a biopsy channel m an endoscope mstiument), 01 tumoiu i esection (eg duimg mtia-opeiative pioceduies via tumoui maigin identification)

By the temi "optical imaging" is meant any method that foims an image foi detection, staging 01 diagnosis of disease, follow up of disease development 01 foi follow up of disease tieatment based on intei action with light m the gieen to neai-infiaied legion

(wavelength 500- 1200 nm) Optical imaging fuithei includes all methods fiom diiect visualization without use of any device and involving use of devices such as \ aπous scopes, cathetcis and optical imaging equipment, eg computei -assisted haidwai c foi tomogiaphic piesentations The modalities and measui ement techniques include, but ^ ai e not limited to luminescence imaging, endoscopy, fluoiescence endoscopy, optical coheience tomography, tiansmittance imaging, time lesolved tiansmittance imaging, confocal imaging, nonhneai micioscopy, photoacoustic imaging, acousto- optical imaging, spectioscopy, ieflectance spectioscopy, mteifeiometiy, coheience mteifetometiy, diffuse optical tomogiaphy and fluoiescence mediated diffuse optical

10 tomogiaphy (continuous wave, time domain and ficquency domain systems), and measuiement of light scattetmg, absoiption, polanzation, luminescence, fluoiescence lifetime, quantum yield, and quenching Further details of these techniques aie pi ovided by (Tuan Vo-Dmh (editoi) "Biomedical Photonics Handbook" (2003), CRC Pi ess LCC, Mycek & Pogue (editois) "Handbook of Biomedical Fluoiescence" l "> (2003), Maicel Dekkei, lnc , Sphntei & Hoppei "An Intioduction to Biomedical Optics" (2007), CRC Piess LCC

The gieen to neai-infi aied iegion light is suitably of wavelength 500-1200 nm, piefei ably of wavelength 550- 1000 nm, most piefeiably 600-800 nm The optical 0 imaging method is piefeiably fluoi escence endoscopy The mammalian body of the sixth aspect is piefeiably the human body Piefeπed embodiments of the imaging agent are as desciibed foi the fust aspect (above) In paiticulai, it is piefeπed that the Bzp ^M dye employed is fluoiesccnt S By the term "biocompatible caiπei" is meant a fluid, especially a liquid, m which the imaging agent can be suspended oi dissolved, such that the composition is physiologically toleiable, ie can be admmisteied to the mammalian body without toxicity oi undue discomfoit The biocompatible caπiei is suitably an injectable caπiei liquid such as stenle, pyiogen-fiee watei foi injection, an aqueous solution0 such as salme (which may advantageously be balanced so that the final pioduct foi injection is isotonic), an aqueous solution of one ot moie tomcity-adjustmg substances (eg salts of plasma cations with biocompatible countcπons), sugars (c g glucose oi suet ose), sugai alcohols (eg soibitol oi mannitol), glycols (eg glyceiol), oi othei non- ionic polyol matenals (eg polyethyleneglycols, piopylcne glycols and the like)

Picfeiably the biocompatible caπiei is pyiogen-fi ee watei foi injection 01 isotonic saline

By the term "conjugate" is meant that the BTM, (L) _n gioup and B?p ^M dye aie linked by covalent bonds

Whilst the conjugate of Formula I is suitable foi in vivo imaging, it may also have in viti o applications (eg assays quantifying the BTM m biological samples 01 visualisation of BTM m tissue samples) Piefeiably, the imaging agent is used foi in vivo imaging

By the teim "sulfonic acid substituent" is meant a substituent of formula -SChM ¹ , wheie M ¹ is H oi B\ and B ^L is a biocompatible cation The -SChM ¹ , substituent is covalently bonded to a caibon atom, and the caibon atom may be ai yl (ie sulfoaiyl such as when R ¹ oi R ² is -SO ₁ M ¹ ), oi alkyl (ie a sulfoalkyl gioup) By the teim "biocompatible cation" (B ^c ) is meant a positively chaiged counteπon which forms a salt with an ionised, negatively chaiged gioup (in this case a sulfonate gioup), wheie said positively chaiged countenon 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 Piefened biocompatible cations aie sodium and potassium, most piefeiably sodium

By the teim "biocompatible anion" (J) is meant a negatively chaiged countenon which forms a salt with an ionised, positively chaiged gioup (m this case an mdohnium gioup), wheie said negatively chaiged countenon is also non-toxic and hence suitable foi admimstiation to the mammalian body, especially the human body The countenon (J ) iepiesents an anion which is piesent in a molai equivalent amount, thus balancing the positive chaige on the Bzp ^M dye The anion (J) is suitably smgly- ot multiply-chaiged, as long as a chai ge-balancing amount is piesent The anion is suitably denved ftom an moiganic oi oiganic acid Examples of suitable anions include hahde ions such as chloiide oi biomide, sulfate, nitiate, citiate, acetate, phosphate and boiate A piefened anion is chloiide

By the term "biological taigeting moiety" (BTM) is meant a compound which, aftei admimstiation to the mammalian body in m o, is taken up selectively 01 localises at a paiticulai site of said mammalian body Such sites may foi example be implicated m a paiticulai disease state be indicative of how an oigan 01 metabolic piocess is functioning The biological taigeting moiety piefeiably compiises 3-100 mei peptides, peptide analogues, peptoids oi peptide mimetics which may be lmeai peptides oi cyclic peptides oi combinations theieof, oi enzyme substtates, enzyme antagonists oi enzyme mhibitois, synthetic ieceptoi -binding compounds, oligonucleotides, oi ohgo-DNA oi oligo RNA fiagments

By the term "peptide" is meant a compound compiising two oi moie ammo acids, as defined below, linked by a peptide bond (ic an amide bond linking the amine of one ammo acid to the caiboxyl of anothei) The term "peptide mimetic" oi "mimetic" iefeis to biologically active compounds that mimic the biological activity of a peptide oi a piotein but aie no longei peptidic in chemical natuie, that is, they no longei contain any peptide bonds (that is, amide bonds between amino acids) Heie, the teim peptide mimetic is used m a broadei sense to include molecules that aie no longei completely peptidic in natuie, such as pseudo-peptides, semi-peptides and peptoids The term "peptide analogue" iefeis to peptides compiising one ot moie amino acid analogues, as desciibed below See also "Synthesis of Peptides and Peptidomimetics", M Goodman et al, Houben-Weyl E22c, Thieme

By the teim "ammo acid" is meant an L- oi Z)-amino acid, ammo acid analogue (eg naphthylalanine) oi ammo acid mimetic which may be natuially occunmg oi of puiely synthetic oiigin, and may be optically puie, i e a single enantiomei and hence chnal, oi a mixtuie of enantiomei s Conventional 3-lettei oi single lettei abbreviations foi ammo acids are used heiein Piefeiably the ammo acids of the piesent invention aie optically pure By the teim "ammo acid mimetic" is meant synthetic analogues of natuially occunmg amino acids which aie isosteies, i e have been designed to mimic the steiic and electiomc stiucture of the natuial compound Such isosteies aie well known to those skilled in the ait and include but aie not limited to dcpsipeptides, letio-inveiso peptides, thioamides, cycloalkanes oi 1,5- disubstituted tetiazolcs [see M Goodman, Biopolymeis, 24, 137, (1985)]

Suitable enzyme substi ates, antagonists or mhibitois include glucose and glucose analogues such as fluorodeoxyglucose, fatty acids, or elastase, Angiotensin II or metallopiotemase inhibitors A preferred non-peptide Angiotensin II antagonist is Losartan Suitable synthetic receptor-bmdmg compounds include estradiol, estrogen, progestin, progesteione and other steroid hormones, hgands foi the dopamine D- I or D-2 receptor, oi dopamine transporter such as tropanes, and hgands for the serotonin receptoi When the receptor-bmdmg compound is folate, the linker gioup preferably does not compiise the 5-mer peptide Asp-Arg-Asp-Asp-Cys Most preferably, the receptor-bmdmg compound is not folate

The benzopyiylmm dye (Bzp ^M ) of Formula II is a fluorescent dye oi chromophorc which is capable of detection either directly or indiiectly in an optical imaging procedure using light of gieen to near-mfrared wavelength (500-1200 nm, prefei ably 550- 1000 nm, moie preferably 600-800 nm) Preferably, the Bzp ^M has fluorescent properties

It is envisaged that one of the roles of the linker gioup -(A) ₁₁₁ - of Formula 1 is to distance the Bzp ^M fiom the binding site of the BTM This is particularly important because the Bzp' is l elatively bulky, so adverse steric interactions are possible This can be achieved by a combination of flexibility (eg simple alkyl chains), so that the Bzp ^M has the freedom to position itself away from the binding site and/oi rigidity such as a cycloalkyl or aryl spacer which orientate the Bzp ¹ away from the binding site The nature of the linkci group can also be used to modify the biodistπbution of the imaging agent Thus, eg the introduction of ethei groups in the linker will help to minimise plasma protein binding When -(A) ₁₁₁ - comprises a polyethyleneglycol (PEG) building block or a peptide chain of 1 to 10 amino acid residues, the linker gioup 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 hvei , and/or from the blood, thus givmg a bettei diagnostic image due to less background interference A biomodifier linker group may also be used to favour a particular ioute of excietion, eg via the kidneys as opposed to via the liver

By the term "sugar" is meant a mono-, di- or tπ- saccharide Suitable sugars include

glucose, galactose, maltose, mannose, and lactose Optionally, the sugai may be functionalised to permit facile coupling to ammo acids Thus, eg a glucosamine deπvative of an ammo acid can be conjugated to othei amino acids via peptide bonds The glucosamine deπvative of aspaiagme (commeicially available fiom NovaBiochem) is one example of this

Formula I denotes that the -(L) _n [Bzp ^M ] moiety can be attached at any suitable position of the BTM Suitable such positions foi the -(L),,[Bzp ^M ] moiety aie chosen to be at positions away fiom that part of the BTM which is iesponsible foi binding to the active site in \ ιvo The [BTM]-(L) _n - moiety of Formula I may be attached at any suitable position of the Bzp ^M of Formula II The [BTM]-(L) _n - moiety eithei takes the place of an existing substituent (eg one of the R ¹ to R ¹⁶ gioups), oi is covalently attached to the existing substituent of the Bzp ^M The [BTM]-(L) _n - moiety is piefeiably attached via a caiboxyalkyl substituent of the Bzp ^M

Suitable imaging agents of the invention aie those wheicin the Bzp ^M is of Formula Ha oi lib

(Ha)

(lib) whete X, w, J and R -R i π aie as defined foi Fomiula II

When R ^{^} togethei with one of RVR -R ⁶ foims a 5- oi 6- membeied unsatuialed aliphatic, unsatuiated heteioaliphatic oi aiomatic πng, suitable such aiomatic imgs include phenyl, fuian, thiazolc, pyπdyl, pyπole oi pyiazole πngs Suitable unsatuiated πngs compiise at least the C=C to which R ^{^} is attached

When R ⁷ and/oi R ^s togethei with one oi both of R ⁹ and/oi R ¹⁰ foim a 5- oi 6- membeied N-contaimng heteiocyclic oi heteioaiyl πng, suitable such πngs include thiazole, pyπdyl, pynole ot pyi azole nngs oi paitially hydiogenated veisions theieof piefeiably pyndyl oi ciihydiopyi idyl

In an altematn e embodiment, the dyes of Foi inula lib may optionally be chosen such that at least one of R ¹ to R ⁴ is F oi (CF->) _t -F, wheie f is an mtegci of value 1 to 4

The phaimaccutical composition is supplied in suitable vials oi vessels which compiise a sealed containei which permits maintenance of stenle mtegπty, plus optionally an meit headspace gas (eg nitiogen oi aigon), whilst permitting addition and withdiawal of solutions by syiinge oi cannula A piefeπed such containei is a septum-scaled vial, wheiein the gas tight closuie is ciimped on with an oveiseal (typically of aluminium) The closuie is suitable foi single oi multiple punctuimg with a hypodeimic needle (e g a ci imped on septum seal closuie) whilst maintaining stenle integiity Such containei s have the additional advantage that the closuie can withstand vacuum if desned (eg to change the headspace gas oi degas solutions), and withstand piessuie changes such as i eductions m piessuie without permitting mgiess of external atmospheiic gases, such as oxygen oi watei vapoui

Ptefeπed multiple dose containeis compπse a single bulk vial (e g of 10 to 30 cm ¹ volume) which contains multiple patient doses, wheieby single patient doses can thus be withdiawn into clinical giade syimges at vaπous time mteivals duimg the viable lifetime of the picpaiation to suit the clinical situation Pie-filled syimges ai e designed to contain a single human dose, oi "unit dose" and aie theiefoie piefeiably a disposable oi othei syimgc suitable foi clinical use The pharmaceutical compositions of the ptesent invention piefeiably have a dosage suitable foi a single patient and aie piovided in a suitable syi mge oi contamei , as desci ibed above

The pharmaceutical composition may optionally contain additional excipients such as an antimiciobial piescivative, pH-adj listing agent, fillei, stabilise! oi osmolality adjusting agent By the teim "antimiciobial piescivative" is meant an agent which inhibits the growth of potentially harmful miα o-oigamsms such as bactena, yeasts oi moulds The antimiciobial pieseivative may also exhibit some bacteiicidal piopeities, depending on the dosage employed The mam iole of the antimiciobial pi eseivative(s) of the piesent invention is to inhibit the giowth of any such micio-otgamsm in the pharmaceutical composition The antimiciobial pieseivative may, howevei, also optionally be used to inhibit the giowth of potentially harmful micio-oiganisms in one oi moie components of kits used to piepaie said composition pπoi to admmistiation Such kits aie desci ibed in the second aspect (below) Suitable antimiciobial pieseivative(s) include the parabens, ie methyl, ethyl, piopyl oi butyl paiaben oi mixtuies theieof, benzyl alcohol, phenol, ciesol, cetiimide and thiomeisal Prefcπed antimiciobial pieseivative(s) aie the paiabens

The term "pH-adjustmg agent" means a compound oi mixtuie of compounds useful to ensuie that the pH of the composition is within acceptable limits (appioximately pH 4 0 to 10 5) foi human ot mammalian admmistiation Suitable such pH-adjustmg agents include pharmaceutically acceptable buffeis, such as tncme, phosphate oi TRIS [ie fm(hydioxymethyl)aminomethane], and pharmaceutically acceptable bases such as sodium caibonate, sodium bicaibonate oi mixtui es theieof When the composition is employed in kit form, the pH adjusting agent may optionally be piovided in a sepai ate vial oi contamei , so that the usei of the kit can adjust the pH as pait of a multi-step pioceduie

By the term "fillet" is meant a pharmaceutically acceptable bulking agent which may facilitate material handling during production and lyophihsation. Suitable fillers include inotganic salts such as sodium chloride, and water soluble sugars or sugar alcohols such as suciose, maltose, mannitol 01 trehalose

The pharmaceutical compositions of the first aspect may be piepared 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) aie sterile The components and reagents can be sterilised by methods known in the art, including sterile filtration, terminal sterilisation using e g gamma-irradiation, autoclavmg, 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 pieferred to include at least a sterile filtration step as the final step in the preparation of the pharmaceutical composition

The pharmaceutical composition of the first aspect is piefcrably prepared from a kit, as described for the second aspect below

Preferred features

The molecular weight of the imaging agent is suitably up to 30,000 Daltons

Preferably, the molecular weight is in the range 1 ,000 to 20,000 Daltons, most prefeiably 2000 to 18,000 Daltons, with 2,500 to 16,000 Daltons being especially preferred.

The BTM may be of synthetic or natuial oiigm, but is prefeiably synthetic The term "synthetic" has its conventional meaning, ie man-made as opposed to being isolated from natural sources eg from the mammalian body Such compounds have the advantage that their manufacture and impurity profile can be fully controlled. Monoclonal antibodies and fragments thereof of natural origin are therefore outside the scope of the term 'synthetic' as used herein

The BTM is preferably chosen from a 3-100 mer peptide, enzyme substrate, enzyme

antagonist 01 enzyme mhibitoi BTM is most piefeiably a 3- 100 mei peptide or peptide analogue When the BTM is a peptide, it is piefeiably a 4-30 mei peptide, and most pi efeiably a 5 to 28-mei peptide

5 The [BTM]-(L) _n - moiety of Formula I is piefeiably attached at positions R\ R ⁶ , R ¹⁴ , R" oi R ¹⁶ of the Bzp ^M of Formula II, moie piefeiably at R ⁶ , R ¹⁴ , R ¹ ' oi R ¹⁶ most piefeiably at R ⁶ , R ¹⁴ oi R ¹ " In oidei to facilitate the attachment the lelevant R\ R ⁶ , R ¹⁴ , R ¹ " oi R ¹⁶ substituent is piefeiably Ci ₆ caiboxyalkyl, moie piefeiably C _{3 6} caiboxyalkyl, with the caiboxy gioup used as an active estet

10

The benzopyi yhum dye (Bzp ) piefei ably has at least 2 sulfonic acid substituents, moie piefei ably 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 aie piefeiably located at positions R ⁶ , R ⁷ , R ⁸ ,

I ^ R ¹⁴ , R" 01 R ¹⁶ , moie piefeiably at R ⁶ , R ⁷ , R ⁸ , R ¹⁴ 01 R* \ most piefeiably at R ⁶ togcthei with one 01 both of R and R ^s of Foimula II The sulfoalkyl gioups of Formula II, aie piefeiably of formula -(CHiXSOiM ¹ , wheic M ¹ is H 01 B\ k is an mtegei of \ alue 1 to 4, and B ^c is a biocompatible cation (as defined above) k is pieieiably 3 01 4 0

In Formula II, w is piefeiably 1 R ^{^} is piefeiably H 01 C| ₄ caiboxyalkyl, and is most piefeiably H X is piefeiably -CR ¹⁴ R ¹ "- 01 NR ¹⁶ - , and is most piefeiably -CR ¹⁴ R"-

Piefeπed Bzp ¹ dyes aie of Formula III

(HI) w hcie Y ¹ , R ¹ - R ⁴ , R ⁶ , R ¹⁴ , R ¹ " and J aie as defined foi Formula II Suitable dyes of Foimula III aie of Formula IHa 01 TIIb

(Ilia)

(IHb)

Piefeπed R' -R ⁴ and R ⁶ -R ⁿ gioups of Foimulae III, HIa and IHb die as desciibed above foi foimulae Ha and lib In Foimulae III, Ilia and HIb, R ¹⁴ and R ^{1 ^} aie piefeiably chosen such that one is an R ^b gioup and the othei is an R ^L gioup R ^b is Ci -i alkyl, most piefeiably methyl R ^c is Ci ₄ alkyl, Ci ₆ caiboxyalkyl 01 Ci ₄ sulfoalkyl, piefeiably C^ ₆ caiboxyalkyl 01 "(CH ₂ ) _L SO ₁ M ¹ wheie k is chosen to be 3 01 4

Piefeiably the dyes of Foimula III have a Ci ₆ caiboxyalkyl substituent to peraiit facile covalent attachment to the BTM

In Foimula II 01 III, when R ⁷ and/or R ^s togethet with one 01 both of R ⁹ and/01 R ¹⁰ form a 5- 01 6- membeied N-contammg heteiocyclic 01 heteioaiyl πng, piefeπed such lings aie pyndyl or dihydiopyiidyl A piefeπed such Y ¹ gioup wheiein an R ^s gioup has been cyclised with R is of Foimula Y ^c

(T)

A piefeπed such Y gioup wheiein both R and R gioup been cyclised is of Foimula Y ^d

(Y ^ϋ )

R ⁷ , R ⁹ and R ¹¹ -R ¹ aie as defined abo\ e, each X ¹ is independently H oi C| ₄ alkyl

In Formula Y\ it is piefened that each X ' IsCH ₁ , R ^l) = R"=H, R ^P isH, R ^p is CH, oi -C(CH,),, moie pi efei ably -C(CH ₃ ) i

In Formula Y ^c , it is piefeπed that R ⁹ = H, R ^p isH, R ^p ispicfeiablyCH oi -C(CH^, moie piefetably -C(CHi

It is piefened that the -NR R gioup of Foimula III is eithei

(i) in open chain foim, ic the R 1 / /TR-. S gioups aie not cyclised with one oi both of R 19 /R 1 » Piefeπed such R and R gioups aie independently

I S

chosen from Ci 4 alkyl or Ci 4 sulfoalkyl, most preferably ethyl or Cv ₄ sulfoalkyl,

(11) cycliscd to give a cyclic Y ¹ substituent of Formula Y ^L or Y ⁽ⁱ , more preferably of Formula Y ^c .

The open chain form (1) is most preferred.

Especially preferred dyes of Formula III are of Formula IHc, IHd or IHe-

(IIIc)

(IHd)

(IIIc)

where:

M ¹ and J are as defined above;

R ^{1 7} and R ¹⁸ are independently chosen from Ci _-4 alkyl or Ci _-4 sulfoalkyl;

R ¹⁹ is H or Ci _-4 alkyl;

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

R ²¹ is C]_ ₄ sulfoalkyl or Ci _-6 carboxyalkyl;

R ²² is C|_ ₄ alkyl, Ci _-4 sulfoalkyl or Ci _-6 carboxyalkyl;

X ² , X ³ and X ⁴ are independently H or Ci _-4 alkyl.

The dyes of Formulae IHd, HIe and IHf are preferably chosen such that one or more of R ²⁰ -R ²² is C _M sulfoalkyl.

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

DY-631

DY-633

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

DY-652 Piefened specific dyes ate DY-631 and DY-652, with DY-652 being most piefened

When the BTM is a peptide, piefened such peptides include somatostatin, octieotide and analogues, peptides which bind to the ST leccptoi, w heie ST iefeis to the heat-stable toxin pioduced by E coli and othei micio-oiganisms,

10 - lammin fiagments eg YIGSR, PDSGR, IKVAV, LRE and

KCQAGTFALRGDPQG,

N-formyl peptides foi taigetmg sites of leucocyte accumulation, Platelet factoi 4 (PF4) and fiagments theieof, RGD (Aig-Gly-Asp)-contammg peptides, which may eg taiget l -i angiogenesis [R Pasquahni et al , Nat Biotechnol 1997 Jun, 15(6) 542-6],

[E Ruoslahti, Kidney Int 1997 May,51 (5) 1413-7] peptide fiagments of α->-antiplasmm, fibionectm oi beta-casem, fibimogen oi thiombospondm The ammo acid sequences of α->-antiplasmm, fibionectm, beta-casem, fibimogen and thiombospondm can be found in 0 the following i efeiences α ₂ -antiplasmin piecuisoi [M Tone e/ a/ ,

J Biochem, 102, 1033, (1987)], beta-casem [L Hansson et al Gene, L39, 193, (1994)], fibionectm [A Gutman et al, FEBS Lett , 207, 145, ( 1996)] , thiombospondm- 1 piecuisoi [V Dixit et al, Pi oc Natl Acad Sci , USA, 83, 5449, (1986)], R F Doohttle, Ann Re\ Biochem , 53, 195, (1984), *1 - peptides which aic substiates oi mhibitoi s of angiotensin, such as angiotensin II Asp-Aig-Val-Tyt-Ile-His-Pio-Phe (E C Toigensen et al, J

Med Chem , 1979, VoI 22, 9, 1038- 1044)

[Sai , He] Angiotensin II Sai-Ai g-Val-Tyi-Ile-His-Pio-Ile (R K Tuikei et al , Science, 1972, 177, 1203 )

Angiotensin I Asp-Aig-Val-Tyi-lle-His-Pio-Phe-His-Leu

When the BTM is a peptide, one oi both teimini of the peptide, piefeiably both, have conjugated theieto a metabolism inhibiting gioup (M ¹⁰ ) Having both peptide termini pi otected in this way is impoitant for in vivo imaging applications, since otherwise i apid metabolism would be expected with consequent loss of selective binding affinity

10 fot the BTM peptide By the tet m "metabolism inhibiting group" (M ^IG ) is meant a biocompatible gioup which inhibits oi suppi esses enzyme, especially peptidase such as caiboxypeptidase, metabolism of the BTM peptide at eithei the ammo terminus oi caiboxy teiminus Such gioups ate paiticulaily impoitant foi /// vivo applications, and aie well known to those skilled in the art and ai e suitably chosen fiom, foi the peptide

I ^s ! amine tei minus

N-acylated gi oups -NH(C=O)R ⁰ wheie the acyl gioup -(C=O)R ⁰ has R° chosen fi om C] _& alkyl, C , io aiyl gioups oi composes a polyethyleneglycol (PEG) building block Suitable PEG gioups ai e desci ibed foi the hnkei gioup (L), below Piefened such PEG gi oups ai e the biomodifiei s of Formulae Bio l oi Bio2 (below) Pi efen ed 0 such amino terminus M ^IG gtoups ai e acetyl, benzyloxycatbonyl oi tπfluoi oacetyl, most pi efei ably acetyl

Suitable metabolism inhibiting gioups fot the peptide caiboxyl terminus include caiboxamide, tei /-butyl estei, benzyl ester, cyclohexyl estei , ammo alcohol oi a 1 polyethyleneglycol (PEG) building block A suitable M ¹⁰ gioup foi the caiboxy terminal ammo acid iesidue of the BTM peptide is wheie the teimmal amine of the ammo acid iesidue is N-alkylated with a Ci ₄ alkyl gioup, pi efei ably a methyl gi oup Pi efei red such M ^IG gioups aie caiboxamide 01 PEG, most prefeπed such gioups aie caiboxamide 0

When eithei ot both peptide termini aie protected with an M ¹⁰ gioup, the -(L) _n [Bzp ^M ] moiety may optionally be attached to the M ¹⁰ gi oup Pi efei ably, at least one peptide terminus has no M ^{l( l} gi oup, so that attachment of the -(L) _n [Bzp ^M ] moiety at that position gives compounds of Formulae IVa 01 IVb l espectively

??

[Bzp ^M ]-(L) _n -[BTM]-Z ^{^} (IVa),

Z'-[BTM]-(L) _n -[Bzp ^M ] (IVb), whet c Z ¹ is attached to the N-temiinus of the BTM peptide, and is H 01 M ^1G ,

Z ² is attached to the C-terminus of the BTM peptide and is OH, OB ^C , ot M ¹⁰ , wheie B ^c is a biocompatible cation (as defined above)

In Formula IVa and IVb, Z ¹ and z ^" " aie piefeiably both independently M ^IC| Piefeπed such M ^lfl gioups foi Z ¹ and Z ² aie as desciibed above for the peptide termini Whilst inhibition of metabolism of the BTM peptide at eithei peptide terminus may also be achieved by attachment of the -(L) _n [Bzp ^vl ] moiety in this way, -(L) _n [Bzp ^M ] itself is outside the definition of M ^Kj of the piesent invention

The BTM peptide may optionally compπse at least one additional ammo acid iesidue which possesses a side chain suitable foi facile conjugation of the Bzp ^NI , and forms pait of the A iesidues of the lmkei gioup (L) Suitable such ammo acid iesidues include Asp oi GIu iesidues foi conjugation with amme-functionahsed Bzp ^M dyes, oi a Lys iesidue foi conjugation with a caiboxy- oi active estei- functionalised Bzp ^M dye

The additional amino acid ιesidue(s) foi conjugation of Bzp ¹ aie suitably located away fiom the binding tegion of the BTM peptide, and aie piefeiably located at eithei the C- oi N- tei minus Piefeiably, the amino acid iesidue foi conjugation is a Lys iesidue

When a synthetic linkei gioup (L) is piesent, it piefeiably composes teiminal functional gioups which facilitate conjugation to [BTM] and Bzp ^M Suitable such gioups (Q' ¹ ) aie desciibed below When L compiises a peptide chain of 1 to 10 ammo acid iesidues, the amino acid iesidues aie piefeiably chosen fiom glycine, lysine, aigmme, aspaitic acid, glutamic acid oi senne When L compiises a PEG moiety, it pieleiably compiises units deπved fiom ohgomeiisation of the monoαispeise PEG- likc stiuctuies of Foimulae Biol oi Bio2

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

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

(Bio2) where p is as defined for Formula Biol and q is an integer from 3 to 15 In Formula Bio2, p is piefcrably 1 or 2, and q is prefeiably 5 to 12

When the linker group does not comprise PEG or a peptide chain, prefericd L groups have a backbone chain of linked atoms which make up the -(A) _n -,- moiety of 2 to 10 atoms, most preferably 2 to 5 atoms, with 2 or 3 atoms being especially piefeπed A minimum hnkei gioup backbone chain of 2 atoms confers the advantage that the Bzp ^M is well-sepaiated so that any undesirable interaction is minimised

BTM peptides which are not commercially available can be synthesised by solid phase peptide synthesis as described m P Lloyd- Williams, F Albeπcio and E Girald, Chemical Approaches to the Sy nthesis of Peptides and Pi otems, CRC Press, 1997.

The imaging agents can be prepared as follows

In order to facilitate conjugation of the Bzp ^M to the BTM, the Bzp ^vι suitably has attached thereto a reactiv e functional group (Q' ¹ ) The Q ^a group is designed to react with a complementary functional gioup of the BTM, thus forming a covalent linkage

between the Bzp ^M and the BTM The complementaiy functional gioup of the BTM may be an mtimsic pait of the BTM, oi may be intioduced by use of dem atisation with a bifunctional gioup as is known in the art Table 1 shows examples of leactive gioups and then complementaiy counterparts

Table 1 Reactive Gioups and Complementaiy Gioups Reactive Theiewith

By the term "activated estei" oi "active estei" is meant an estei denvative of the caiboxyhc acid which is designed to be a bettei leaving gtoup, and hence permit moie facile ieaction with nucleophile, such as amines Examples of suitable active esteis ate N-hydioxysuccmimide (NHS), pentafluoiophenol, pentafluoiothiophenol, pai a- nitiophenol and hydioxybcnzotπazole Piefened active esteis aie N- hydioxysuccimmide or pentafluoiophenol estets

Examples of functional gioups piesent in BTM such as piotems, peptides, nucleic acids caibohydiates and the like, include hydioxy, ammo, sulfydiyl, caibonyl (including aldehyde and ketone) and thiophosphate Suitable Q' ¹ gioups may be selected fiom caiboxyl, activated esteis, isothiocyanate, maleimide, haloacetamide, hydiazide, vmylsulfone, dichloiotiiazine and phosphoiamidite Piefeiably, Q ¹ is an activated estei of a caiboxyhc acid, an isothiocyanate, a maleimide oi a haloacetamide

When the complementaiy gioup is an amine oi hydioxyl, Q ^a is piefeiably an activated estei, with piefened such esteis as desciibed above A piefened such substituent on

the Bzp is the activated estei of a 5-caibo\ypentyl gioup When the complementai y gioup is a thiol, Q ^a is piefeiably a maleimide 01 iodoacetamide gioup

Geneial methods foi conjugation of dyes to biological molecules aie desciibed by s Licha et al [Topics Cuπ Chem , 222, 1 -29 (2002), Adv Di ug Dehv Rev , 57, 1087- 1 108 (2005)] Peptide, piotem and oligonucleotide substiates foi use m the invention may be labelled at a terminal position, oi alternatively at one oi moie internal positions Foi leviews and examples of piotem labelling using fluoiescent dye labelling leagcnts, see "Non-Radioactive Labelling, a Piactical Intioduction", Garman,0 A J Academic Piess,1997, "Bioconjugalion - Piotem Coupling Techniques foi the Biomedical Sciences", Aslam, M and Dent, A , Macmillan Refetence Ltd, (1998) Piotocols aie available to obtain site specific labelling in a synthesised peptide, foi example, see Hermanson, G T , "Bioconμigate Techniques", Academic Pi ess ( 1996) -) Piefeiably, the method of piepai ation of the imaging agent compi iscs eithci

(i) ieaction of an amine functional gioup of a BTM with a compound of foimula l' -(L) _n -|Bzp ^M ], oi

(n) ieaction of a caiboxyhc acid oi activated ester functional gioup of a BTM with a compound of formula ,T-(L) _n -[B7p ^M ], 0 (in) ieaction of a thiol gioup of a BTM with a compound of formula wheiein BTM, M ^IG , L, n and Bzp ^M aie as defined above, and

J is a caiboxyhc acid, activated ester, isothiocyanate oi thiocyanate gioup,

J is an amine gioup, 5 J ' is a maleimide gioup

J ^" is piefeiably a pπmai y oi secondaiy amine gioup, most piefeiably a pnmaiy amine gioup In step (in), the thiol gioup of the BTM is piefeiably fiom a cysteine i csiduc

In steps (i) to (in), the BTM may optionally have othei functional gioups which could potentially ieact with the Bzp ^M denvative, piotected with suitable piotectmg gioups so that chemical ieaction occuis selectn ely at the desned site only By the term "piotectmg gioup" is meant a gioup which inhibits oi suppiesses undesnable

chemical icactions, but which is designed to be sufficiently teactπ e that it may be cleaved fiom the functional gioup m question undei mild enough conditions that do not modify the iest of the molecule Aftei depiotection the desned pioduct is obtained Amine piotectmg gioups aie well known to those skilled in the ait and aie s suitably chosen fiom Boc (whcie Boc is teit-butyloxycaibonyl), Fmoc (wheie Fmoc is fluoienylmethoxycaibonyl), tiifluoioacetyl, allyloxycaibonyl, Dde [i e l -(4,4- dimethyl 2,6-dioxocyclohexylidene)ethyl] 01 Npys (i e 3 nitio-2-pyπdine sulfenyl) Suitable thiol piotectmg gioups aie Tit (Ti ityl), Acm (acetamidomethyl), t-Bu (tert- butyl), /e/ f-Butylthio, methoxybenzyl, methylbenzyl oi Npys (3-mtio-2-pyiidme0 sulfenyl) The use of fuithei piotectmg gioups aie desciibed in 'Piotective Gioups in Oi game Synthesis', Theodoia W Gieene and Petei G M Wuts, (Tohn Wiley & Sons, 1991 ) Piefened amine piotectmg gioups aie Boc and Fmoc, most piefeiably Boc Piefened amine piotectmg gioups aie Tit and Acm :> Beii7opyiyhum dyes (Bzp ¹ ') functionalised suitable foi conjugation to BTM ate commeicially available fiom Dyomics (Dyomics GmbH, Wmzeilaet Sti 2A, D- 07745 lena, Geimany, w ww dyomics com), wheie the leactive functional gioup (Q ^a ) is NHS estei, maleimide, ammo oi caiboxyhc acid Piecuisois suitable foi the synthesis of benzopyiyhum dyes can also be piepaied as desciibed in US 54059760 Methods of conjugating optical ieportei dyes, to ammo acids and peptides aie desciibed by Licha (vide supi a), as well as Flanagan et al [Bioconj Chem , 8, 751-756 (1997)], Lin et al, [ibid, L3, 605-610 (2002)] and Zaheei [MoI Imaging, 1(4), 354-364 (2002)] Methods of conjugating the lmket gioup (L) to the BTM employ analogous chemistiy to that of the dyes alone (see above), and aie known m the ait 5

In a second aspect, the present invention piovides a kit foi the piepaiation of the pharmaceutical composition of the fust aspect, wheiein said kit compiises the conjugate of Formula I in stenle, solid form such that, upon ieconstitution with a stenle supply of the biocompatible caniei, dissolution occuis to give the desned pharmaceutical composition The "conjugate" and "biocompatible earner", togethci with piefened embodiments theieof aie as desciibed in the fust aspect

Foi the kit, the conjugate, plus othei optional excipients as desciibed above, may be

piouded as a lyophilised powdei in a suitable vial 01 containei The powdei is then designed to be ieconstituted with the desited biocompatible camel to give the pharmaceutical composition in a steπle, apyiogenic foiτn which is ieady foi mammalian admmisti ation

A ptefeπed stenle, solid form of the conjugate is a lyophilised solid The stenle, solid form is piefeiably supplied in a pharmaceutical giade containei, as described foi the phaimaceutical composition (above) When the kit is lyophilised, the formulation may optionally comprise a ciyopiotectant chosen from a saccharide, piefeiably mannrtol, 10 maltose oi tiicme

In a thud aspect, the present invention piovides a conjugate of Formula I l ϊ (I) where L and n are as defined foi the fust aspect, and Bzp ^M is of Formula II as defined above, BTM' is a BTM as defined in the fust aspect, which is synthetic and is chosen fi om

(r) a 3-100 mei peptide, 0 (π) an enzyme substiate, enzyme antagonist oi en/yme mhibitoi ,

(in) a receptor -bindrng compound, (iv) an oligonucleotide, (v) an ohgo-DNA oi oligo-RNA fragment 5 The term 'synthetic' has the definition given above Piefeπed embodiments of the Bzp of Formula II in the conjugate aie as desciibed in the first aspect above Picfei ied aspects of BTM' ot (r)-(v) are as descnbed m the fust aspect foi those types of BTM BTM' is preferably a 3-100 met peptide 0 The conjugates of the thud aspect are useful m the prepaiation of the imaging agent pharmaceutical compositions of the invention The conjugates can be piepaied as described in the fust aspect

In a foLiith aspect, the piesent invention piovides a method of in \ n o optical imaging of the mammalian body which compiises use of the phaimaceuUcal composition of the fust aspect to obtain images of sites of BTM localisation in m o

The term "optical imaging" is as defined in the fust aspect (above)

In the method of the fouith aspect, the imaging agent pharmaceutical composition has piefeiably been pieviously admimsteied to said mammalian body By "pieviously admmisteied" 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 piwi to imaging This embodiment includes the use of the conjugate as defined in the fust aspect in the manufactuie of a diagnostic agent foi optical imaging in vivo of disease states of the mammalian body whei e the BTM is implicated

A piefeπed optical imaging method of the fouith aspect is Fluoiescence Reflectance Imaging (FRI) In FRI, the imaging agent of the pi esent invention is admmisteied to a subject to be diagnosed, and subsequently a tissue suiface of the subject is illuminated with an excitation light - usually continuous wave (CW) excitation The light excites the Bzp ^M dye of the imaging agent Fluoiescence fiom the imaging agent, which is geneiated by the excitation light, is detected using a fluoiescence detectoi The ietuming light is piefeiably filteied to sepaiate out the fluoiescence component (solely oi partially) 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 concenti ate m the disease ai ea, pioducmg highei fluoiescence intensity Thus the disease aiea pioduces positive contiast in a fluoiescence intensity image The image is piefeiably obtained using a CCD camei a oi chip, such that leal-tirae imaging is possible

The wavelength foi excitation vanes depending on the paiticulat Bzp ^M dye used, but is typically in the iange 500 - 1200nm foi dyes of the piesent invention The appaiatus foi geneiating the excitation light may be a conventional excitation light souicc such as a lasei (e g , ion lasei, dye lasei oi semiconductoi lasei), halogen light soiπce oi xenon light souice Vanous optical filteis may optionally be used to obtain

the optimal excitation wavelength A piefened FRI method composes the steps as follows

(i) a tissue suiface of mteiest withm the mammalian body is illuminated with an excitation light, (ii) fluoiescence fiom the imaging agent, which is generated by excitation of the Bzp ^M , is detected using a fluoiescence detectoi ,

(in) the light detected by the fluoiescence detectoi is optionally filteied to separate out the fluoiescence component,

(iv) an image of said tissue suiface of mteiest is formed fiom the fluoiescent light of steps (ii) oi (in)

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

An alternative imaging method of the sixth aspect uses FDPM (fiequency-domain photon migiation) This has advantages ovei contmuous-w ave (CW) methods wheie gieatei depth of detection of the dye within tissue is impoitant [Sevick-Muiaca et al, Cuπ Opm 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 mstiumentation employed

The FDPM method is as follows

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

(b) detecting a multiply-scatteied light emission fiom the tissue in iesponse to said exposing, (c) quantifying a fluoiescence chaiacteπstic thioughout the tissue fiom the emission by establishing a numbei of values with a piocessoi, the values each coπesponding to a level of the fluoiescence chaiacteπstic at a diffeient position within the tissue, the level of the fluoi escence chaiacteπstic varying with heteiogeneous composition of the tissue, and

(d) geneiating an image of the tissue by mapping the heteiogeneous composition of the tissue m accordance with the values of step (c)

The fluoiescence chaiacteπstic of step (c) preferably conesponds to uptake of the imaging agent and preferably furthei compiises mapping a number of quantities coπesponding 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, fluoiescence yield and imaging agent uptake The fluorescence characteiistic 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, (π) determining a calculated emission as a function of the estimate, (in) comparing the calculated emission to the emission of said detecting to determine an en oi, (iv) providing a modified estimate of the fluoiescence characteristic as a function of the error The quantifying piefeiably compiises determining the \ alues from a mathematical relationship modelling multiple hght-scatteimg behaviour of the tissue The method of the first option piefeiably furthei compiises monitoring a metabolic property of the tissue in vivo by detecting variation of said fluoiescence chaiacteiistic

The optical imaging of the fourth aspect is preferably used to help facilitate the management of a disease state of the mammalian body. By the temi "management" is meant use in the detection, staging, diagnosis, monitoiing of disease progression or the monitoring of treatment The disease state is suitably one in which the BTM of the imaging agent is implicated Imaging applications preferably include camera-based sui face imaging, endoscopy and surgical guidance Further details of suitable optical imaging methods have been reviewed by Sevick-Muraca et al [Cun Opm Chem Biol , 6, 642-650 (2002)].

In a fifth aspect, the present invention provides a method of detection, staging, diagnosis, monitoring of disease piogression or monitoring of tr eatment of a disease

state of the mammalian body which compiises the in \ ιvo optical imaging method of the fouith aspect

The invention is illustiated by the non-hmitmg Examples detailed below Example 1 piovides the synthesis of a biological taigeting peptide (Peptide 1), which binds to cMet Example 2 piovides methods of conjugating Bzp ^M dyes of the invention to peptides, m particulai Peptide 1 Example 3 piovides data demonstiatmg that the peptide conjugates of Peptide 1 of the invention ietam affinity foi cMet, i e that the conjugated dye does not mteifeie with the biological binding and selectivity Appiopnate low binding to human seium albumin and high stability in plasma weie demonsti ated Example 4 shows that the peptide conjugates of the invention exhibit useful tumoui backgiound iatios in an animal model of coloiectal cancel Example 5 desciibes the use of piedictive softwaie foi the dyes of the invention, and demonstiates that the dyes of the invention lack potentially dangeious metabolites in vivo Example 6 desciibes the toxicity testing of Compound 6, showing that the anticipated clinical dose was well toleiated and without any diug substance ielated adveise effects

Table 2 Stiuctuies of Benzopyiylium dyes of the Examples

wheie R ^d is -(CII-.) SO H R ^e is -(CH.) CO.H and R ¹ is -(CH.)XO.H

DY-752 has the same imgs and substituent pattern as DY-652, but has a pentamethine linkage (i e w = 2 and R ^{^} =H) m place of the tiimethme linkage of DY-652

Abbreviations

Conventional 3-lettei and single lettei ammo acid abbieuations aie used

Acm Acetamidomethyl

ACN Acetomtπle

Boc tei t-Butyloxycaibonyl

DMF N,N -Dimethylfoimamide

DMSO Dimethylsulfoxide

Fmoc 9-Fluoienylmethoxycaibonyl

HCl Hydiochloiic acid

10 HPLC High peiformance liquid chiomatogiaphy

HSPyU O-(N-succinimidyl)-N,N,N N -tetiamcthyleneuionium hexafluoiophosphatc

He Isoleucine

LC-MS Liquid chiomatogiaphy mass spectioscopy

NHS TV-hydioxy-succinimide

I D NMM yV-Methylmoipholme

NMP l-Methyl-2-pyπohdmone

Pbf 2,2,4,6, 7-Pcntamethyldihydrobenzofui an 5 sulfonyl

PBS Phosphate-buff eied salme

TFA Tiifluoioacetic acid 0 Tit TiUyI

TSTU O-(N-Succimmidyl)-N,N,N ,N -tetiamethyluromum tetiafluoioboiate

Example 1: Synthesis of Peptide 1. i 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-Trp-Cys-Tyi -GIu-

ThI-GIu-GIy-TIn-GIy-GIy-GIy-LyS-NH ₂ ("Peptide 1")

0 Peptide 1

Step (a) Synthesis of piotected lmeai piecuisoi of Peptide 1 The piecuisoi lmeai peptide has the sequence

Ac-Ala-Gly-Sei -Cys-Tyi -Cys(Acm)-Sei -GIy-Pi o-Pi o-Ai g-Phe-Glu-Cys( Acm)-Ti p- CyS-TyI-GIu-ThI-GIu-GIy-ThI-GIy-GIy-GIy-LyS-NH ₂

The peptidyl resin H-Ala-Gly-Sei(tBu)-Cys(Tit)-Tyi(tBu)-Cys(Acm)-Sei(tBu)-Gly- P10-P10-A1 g(Pbf)-Phe-Glu(OtBu)-Cys(Acm)-Tip(Boc)-Cys(Tit)-Tyi(tBu)- GIu(OtBu)-ThI (ψ ^{Me ML} pi o)-Glu(OtBu)-Gly-Thi (tBu)-Gly-Gly-Gly-Lys(Boc)-Polymei was assembled on an Applied Biosystcms 413 A peptide synthesizei using Fmoc chemistiy starting with O 1 mmol Rink Amide Novagel tesin An excess of 1 mmol pie-activated ammo acids (using HBTU) was applied in the coupling steps Glu-Thi pseudopiolme (Novabiochem 05-20-1 122) was incorpoiated in the sequence The iesm was tiansfeπed to a mtiogen bubblei appaiatus and tieated with a solution of acetic anhydπde (1 mmol) and NMM (1 mmol) dissolved in DCM (5 mL) foi 60 mm The anhydπde solution was lemoved by filtiation and the iesin washed with DCM and dned undei a stieam of mtiogen

The simultaneous lemoval of the side-chain piotcctmg gioups and cleavage of the peptide fiom the iesm was earned out in TFA (10 mL) containing 2 5 % TIS, 2 5 % 4- thioctesol and 2 5 % watei foi 2 houis and 30 mm The iesm was lemoved by filtiation, TFA lemoved in vacuo and diethyl cthei added to the iesidue The foπned piecipitate was washed with diethyl ethet and an-diied affoiding 264 mg of ciude peptide

Puiification by piepaiative HPLC (giadient 20-30 % B ovci 40 mm wheie A = H?O/0 1 % TFA and B = ACN/0 1 % TFA, flow iate 10 niL/min, column Phenomenex Luna 5μ C18 (2) 250 x 21 20 mm, detection UV 214 nm, pioduct ictention time 30 mm) of the etude peptide affoided 100 mg of puie Peptide 1 lmeai piecuisor The puie pioduct was analysed by analytical HPLC (giadient 10-40 % B ovei 10 mm wheie A = H ₂ O/0 1 % TFA and B = ACN/0 1 % TFA, flow iate 0 3 mL/min, column Phenomenex Luna 3μ Cl 8 (2) 50 x 2 mm, detection UV 214 nm, pioduct tetention time 6 54 mm) Fuithei pioduct chaiacteiisation was earned out using electiospiay mass spectiometiy (MHi ^"+ calculated 1464 6, MH ₂ ^{" *"} found 1465 1)

M

Step (b) Formation of Cys4-16 disulfide budge

Cys4-16, Ac-Ala-Gly-Sei-Cys-Tyi-Cys(Acm)-Sei-Gly-Pio-Pio-λig-Phe-Glu - CyS(ACm)-TiP-CyS-TyI-GIu-ThI-GIu-GIy-ThI-GIy-GIy-GIy-LyS-NHi The lineai piecuisoi fiom step (a) (100 mg) was dissolved in 5 % DMSO/vvatei (200 niL) 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 residue (40 mL) was injected in poitions onto a piepaiative HPLC column foi ptoduct puiification

Puiification by piepaiative HPLC (gtadient 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 mL/mm, column Phenomenex Luna 5μ C18 (2) 250 x 21 20 mm, detection UV 214 nm, pioduct ietention time 44 mm) of the iesidue affoided 72 mg of puie Peptide 1 monocyclic pi ecui soi

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 i ate 0 3 mL/mm, column Phenomenex Luna 3μ Cl 8 (2) 50 x 2 mm, detection UV 214 nm, pioduct 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 ₂ ²⁺ calculated 1463 6, MH ₇ ² ' found 1464 1 (Pl), 1464 4 (P2), 1464 3 (P3))

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

The monocyclic precuisoi fiom step (b) (72 mg) was dissolved in 75 % AcOH/watei (72 mL) undei a blanket of mtiogen 1 M HCl (7 2 mL) and 0 05 M I ₂ in AcOH (4 8 mL) weie added in that oidei and the mixtuie stilted foi 45 mm 1 M ascoibic acid (1 mL) was added giving a colourless 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 foi 10 mm, then 20-30 % B ovei 40 mm wheie A = PI 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 ietention time 43-53 mm) of the iesidue affoided 52 mg of puie Peptide 1 The puie pioduct was analysed by analytical HPLC

3 i

(giadient 10-40 % B ovei 10 mm wheie A = H ₇ O/0 1 % TFA and B = ACN/0 1 % TFA, flow iatc 0 3 mL/min, column Phenomenex Luna 3μ C18 (2) 50 x 2 mm, detection UV 214 nm, product ietention time 6 54 mm) Fuithei pioduct chaiacteπsation was earned out using electiospiay mass spectiometiy (MH: calculated 1391 5, MH, ^2" found 1392 5)

Example 2: S>nthesis of Peptide Conjugates of Benzopyrylium Dyes.

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

Puiification and chaiacteiisation

Puiification by piepaiative HPLC (giadient 20-40 % B ovei 40 mm wheie A = H,O/0 1 % TFA and B = ACN/0 1 % TFA, flow iate 10 mL/min, column Phenomenex Luna 5μ C 18 (2) 250 x 21 2 mm, detection UV 214 nra) of the ciiide peptide affotded puie [Peptide 1]-Bzp conjugate The pine pioduct was analysed by analytical HPLC (giadient 10-40 % B ovei 5 mm wheie A = H-.O/0 1 % TFA and B = ACN/0 1 % TFA, flow iate 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 in Table 3

16

Table 3 Peptide-dye conjugates of Peptide 1

Example 3: In Vitro Fluorescence polarisation assay.

Fluoicscence polaπsation assay was used to examine the affinity binding ol the imaging agent towaids the cMet taiget as well as the binding piopeities telated to plasma pioteins The piinciple of the fluoiescence polaiisation method can bnefly be desciibed as follows Monochiomatic light passes thiough a honzontal polaiizing filtei and excites fluotescent molecules m the sample Only those molecules that oπented piopeily in the vertically polanzed plane adsoib light, become excited, and subsequently emit light The emitted light is mcasuied m both honzontal and vertical planes The amsotiopy value (A), is the iatio between the light intensities following the equation

A = Intensity with hoi izontul polanzei - Intensity \\ ith vei tical polai izei

Intensity λ\ ιth honzontal poku izei + 2 ^* Intensity with vet tical polar izei

The fluoiescence amsotiopy measuiements weie peiformed in 384 well micioplates in a volume of 10 μL in binding buffei (PBS, 0 01%Tween-20, pH 7 5) using a Tecan

Safne fluoiescence polaiisation plate ieadei (Tecan , US) at Ex 635/Em 678 nm The concenti ation of dye-labelled peptide was held constant (5nM) and the concentiation of the human c Met/ Fc chimeia (R&D Systems) was vaned ftom 0-250 iiM Binding mixtuies weie equihbiated m the micioplate foi 10 mm at 30 ⁰ C The obsen ed change in amsotiopy was fitted to the equation

/M - /';>« -K λII,' : - J _j > )

where rafo is the observed anisotropy. rjree is the anisotropy of the free peptide. rhoimd 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 Kj value (one-site binding).

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

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 w ith 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 ⁰ C.

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 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 8 weeks old upon anival and with a body weight of appiox 20 giams at the start of the study The animals were housed in individually ventilated cages (IVC, Scanbui BK) with HEPA 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 molai concentration of 1 niM (pH 3 0)

The colon cancer cell HT-29 is derived from human colon carcinomas and is reported to expiess c-Met according to Zeng et al [Clin Exp Metastasis, 2J_, 409-417 (2004)] The cell line was proven to be tumoπgenic when inoculated subcutaneously into nude mice [Flatmark et al, Eur J Cancer 40,1593-1598 (2004)]

HT-29 cells were grown in McCoy's 5a medium (Sigma # M8403) supplemented with 10% fetal bovine serum and penicillin/streptomycin Stocks were made at passage number four (P4) and frozen down for storage m liquid nitrogen at 10 ⁷ cells/vial in the respective culture media containing 5% DMSO. On the day of the transplantation, the cells were thawed quickly in 37 ⁰ C water bath (approx 2 nun), washed and resuspended m PBS/2% seium (centπfugation at 1200 rpm for 10 mm) Thorough mixing of cells in the vials was ensured every time the cells were aspirated into the dosing syringe A volume of 0 1 ml of cell suspension was injected s c at the shoulder and at the back using a fine bore needle (25 G) The animals were then returned to their cages and the tumours were allowed to grow foi 13-17 days The animals were allowed an acclimatisation period of at least 5 days before the inoculation piocedure

[b) procedure

All test substances were reconstituted with PBS fiom freeze-dned powder A small stack of white printer paper was imaged to obtain a flat field image which was used to coπect foi illumination inhomogeneities

For immobilisation during the optical imaging procedure, the animals were anaesthetized in a coaxial open mask to light surgical level anaesthesia with

19

Isofluiane (typically 1 3-2%), using oxygen as the camel gas A small piece of skin (3-5mm) was lemoved ovei paits of the tumoui and adjacent muscle using a suigical foiceps and fine scissois while the animal was anaesthetized This was done to measuie the signal fiom 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)

The iespnation and body tempeiatuie of the animal was monitoied with a BioVet system (m2m Imaging Corp, NJ, USA) using a pneumatic sensoi underneath the animal and a iectal tempeiatui e piobe The BioVet system also supplied external heating using a heating mat set to 40 ⁰ C to sustain normal body tempeiatuie foi the duiation of the imaging piocedui e (2 houis) A Venflon cathetei was placed m the tail vein foi contiast agent admmistiation 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 pπoi to injection and then eveiy 30 seconds foi 2 houis

(c) Imaging

Imaging was pei formed thiough a clinical lapaioscope adapted to use a light souice to excite the iepoiter and a filteπng system to extiact the fluoiescence component A 635nm lasei was used foi excitation of the iepoitei molecule A Hamamatsu ORCA ERG CCD camera was used as the detectoi The camei a was opeiated in 2x2 binning mode with 0 gam Standaid exposuie time foi colon imaging was 4s The intensity distiibution m the image was collected foi illumination inhomogeneities thiough system calibiation data A taiget to backgiound iatio was computed fiom legions of mteiest placed ovei the exposed tumoui and normal muscle backgiound

(d) Results

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

Table 5 turnout 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 fot piedicting toxicity of new chemical entities based on known stiuctuie-dcpendent toxicity Similatly, Meteoi pi edicts likely metabolites of novel chemicals Both tools aie based on published and unpublished (but veiificd) data foi chemical compounds The chemical stiuctuie of dye DY-652 was input No potentially dangeious metabolites /// vn o weie picdicted

Example 6: Toxicity Testing of Compound 6.

A limited acute dose toxicity study was conducted to investigate the toleiancc of Compound 6 at 100 times the pieclinical imaging dose (50 nmol/kg body weight)

The compound was injected mtiavcnously in male iats, and the animals weie saciificed at 1 , 14, 21 and 28 days post injection (p i ) At neciopsy, the majoi oigans weie inspected foi gioss pathology, and the kidneys weie taken into neutial buffcied foi malm foi subsequent histomorphological evaluation A weak blue colouiation of the skin and a modeiate blue colouiation of the mine weie obseived immediately after injection, which disappeaied within 1 day p i At neciopsy, the kidneys weie diffusely gteen on day 1 p i Light micioscopy showed no Compound 6-t elated findings in the kidneys The othei minoi changes seen weie incidental and common in young adult laboiatoiy tats Stiong fluoiescence staining of blood vessels in 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 degeneration, necrosis or inflammation was noted in any of the treated animals, suggesting that the nephrotoxicity of the compound is low It was concluded that a single intravenous administration of Compound 6 to male rats at 100 times the anticipated clinical dose was well tolerated and without any drug substance related adverse effects.