Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/04—X-ray contrast preparations
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
  • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
  • A61K49/0032—Methine dyes, e.g. cyanine dyes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
  • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
  • A61K49/10—Organic compounds
  • A61K49/12—Macromolecular compounds

Definitions

• the present invention relates to a method of in vivo optical imaging, of the margins around tumours, which comprises an optical imaging contrast agent.
• the optical imaging agents comprise conjugates of near-infrared dyes with synthetic polyethyleneglycol (PEG) polymers having a molecular weight in the range 15-45 kDa. Also disclosed are optical imaging contrast agents, pharmaceutical compositions and kits.
• cancerous tissues are, however, often difficult to distinguish from normal tissues, or are too small to be detected (e.g. occult tumors).
• traditional surgical techniques do not ensure that all cancerous tissue has been found or removed and there is a need for agents, which can specifically identify cancer tissue, particularly tumor margins, with a very high resolution and sensitivity.
• Wohrle et al [Makromol. Symp., 5_9, 17-33 (1992)] studied polymer-conjugation to porphyrin photosensitisers as a potential method of improving the uptake in target tissue in vivo for the photodynamic therapy of cancer.
• the polymers studied were rat serum albumin, synthetic polyethers and polyacohols. Wohrle et al concluded that the conjugation of a polymer carrier could improve the tumour uptake.
• US 5,622,685 discloses that polyether-substituted anti-tumour agents comprising a porphyrin, phthalocyanine or naphthalocyanine exhibit improved properties for both in vivo tumour diagnosis and therapy.
• the polyether substituents comprise polyethyleneglycol (PEG) whose terminal hydroxyl group is etherif ⁇ ed or esterified with C I-) 2 alkyl or Ci - 12 acyl groups respectively.
• the alkyl group is most preferably a methyl group.
• US 5,622,685 teaches (column 2) that the total molecular weight of the conjugate is preferably at least 10,000 Da (10 kDa).
• US 6,083,485 and counterparts discloses in vivo near-infrared (NIR) optical imaging methods using cyanine dyes having an octanol-water partition coefficient of 2.0 or less. Also disclosed are conjugates of said dyes with "biological detecting units" of molecular weight up to 30 kDa which bind to specific cell populations, or bind selectively to receptors, or accumulate in tissues or tumours.
• the dyes of US 6,083,485 may also be conjugated to a range of "non-selectively bonding" macromolecules, such as polylysine, dextran, carboxydextran, polyethylene glycol, methoxypolyethylene glycol, polyvinyl alcohol, or a cascade polymer-like structure. The molecular weight of the conjugates is taught to range from 100 Da to over 100,000 Da (0.1 to over 100 kDa). No specific dye-macromolecule conjugates are disclosed.
• US 6,350,431 discloses light imaging contrast agents having a molecular weight in the range 500 to 500,000 Da, comprising a polyalkylene oxide (PAO) of molecular weight 60 to 100,000 Da having at least two chromophores (i.e. dye molecules) linked thereto.
• the polyalkylene oxide (PAO) moiety is taught to have a preferred molecular weight range of 200 to 100,000 Da, more preferably 250 to 50,000 Da, especially preferably 250 to 25,000 Da, most preferably 400 to 15,000 Da.
• the contrast agents of US 6,350,431 may further comprise a targeting vector.
• the Examples of US 6,350,431 employ the following PAO polymers: (i) PEG-diamine 3,400 Da molecular weight: Examples 1, 2, 6, 16, 18 and 25;
• Licha et al [SPIE VoI 3196 p. 98-102 (1998)] disclose contrast agents for in vivo fluorescence imaging which comprise poly(ethyleneglycol) (PEG) polymers based on methoxypolyethyleneglycol (MPEG).
• PEG poly(ethyleneglycol)
• MPEG methoxypolyethyleneglycol
• the conjugates thus have a heptamethine cyanine dye conjugated at one terminus of the PEG polymer and a methyl group at the other terminus:
• Licha was a dye conjugate in which 2 MPEG chains were conjugated to a single cyanine dye (NIR96307, molecular weight ca. 41 kDa):
• n was not determined, but the mean molecular weight of the conjugate was said to be 41 kDa.
• the polymer conjugates of Licha were synthesized from the corresponding MPEG amine, ie. H 2 NCH 2 [CH 2 OCH 2 J n CH 2 OCH 3 .
• Montet et al [Radiology, 242(3), 751-758 (2007)] reported fluorescence molecular tomography (FMT) of angiogenesis using the near-infrared probes AngioSense 680 and AngioSense 750. These were described as high molecular weight (250 kDa) pegylated graft copolymers with an indocyanine-type fluorophore optimized for non- quenching.
• the agent contains MPEG attached to a polylysine backbone.
• Montet et al report that the agent exhibited a prolonged blood half-life (more than 5 hours), with no tumour extravasation up to 30 minutes post- administration, but increasing tumour uptake (and hence imaging brightness) with time thereafter.
• the linear polymer studied comprised a targeted PEG polymer of the type: [LHRH]-[PEG polymer]-Cy5.5 where: LHRH is a synthetic analogue of luteinizing hormone -releasing peptide; Cy5.5 is a specific cyanine dye.
• the PEG polymer used had a molecular weight of about 3 kDa.
• Figure 4 (p. 111) of Sadd et al compares the tumour uptake of the above conjugate with the non-targeted analogue, PEG-Cy5.5. Sadd et al concluded that the LHRH targeting polymer conjugate exhibits enhanced accumulation in cancer cells compared to the non- targeted analogue.”
• the present invention provides a method of in vivo optical imaging of the margins around tumours, using an optical imaging contrast agent.
• the optical imaging agents comprise conjugates of near-infrared dyes with synthetic polyethyleneglycol (PEG) polymers having a molecular weight in the range 15-45 kDa. Also disclosed are optical imaging contrast agents, pharmaceutical compositions and kits.
• the present invention provides imaging agents capable of detecting sub-millimetre (down to 0.2-0.3 mm) foci of disease at the section level. The detection of the cancer foci can thus be achieved by the surgeon intraoperatively.
• the agent provides surgical guidance and/or identification of residual disease.
• imaging agents help to standardize surgery, irrespective of the volume of cancer patients operated by a surgeon and/or the experience of the pathologist.
• the agents help improve the efficiency of tumour surgery, maximising "negative margins" (as defined above), whilst minimising unnecessary excision of normal tissue from the patient.
• the present invention provides a method of in vivo optical imaging of the tumour margins of a tumour in an animate subject known to have at least one such tumour, said method comprising:
• an optical imaging contrast agent suitable for in vivo imaging said contrast agent comprising a conjugate of a synthetic polyethyleneglycol polymer of molecular weight 15 to 45 kDa, with one or two groups Opt R ;
• optical imaging any method that forms an image for detection, staging or diagnosis of disease, follow up of disease development or for follow up of disease treatment based on interaction with light in the green to near-infrared region (wavelength 500-1200 nm).
• Optical imaging further includes all methods from direct visualization without use of any device and involving use of devices such as various scopes, catheters and optical imaging equipment, eg. computer-assisted hardware for tomographic presentations.
• the modalities and measurement techniques include, but are not limited to: luminescence imaging; endoscopy; fluorescence endoscopy; optical coherence tomography; transmittance imaging; time resolved transmittance imaging; confocal imaging; nonlinear microscopy; photoacoustic imaging; acousto- optical imaging; spectroscopy; reflectance spectroscopy; mterferometry; coherence interferometry; diffuse optical tomography and fluorescence mediated diffuse optical tomography (continuous wave, time domain and frequency domain systems), and measurement of light scattering, absorption, polarization, luminescence, fluorescence lifetime, quantum yield, and quenching.
• optical imaging contrast agent a compound suitable for optical imaging of a region of interest of the whole (ie. intact) mammalian body in vivo.
• the mammal is a living human subject.
• the imaging may be invasive (eg. intra-operative or endoscopic) or non-invasive.
• the imaging is used to facilitate tumour resection (i.e. during intraoperative procedures) via tumour margin identification.
• tumor margins is meant the interstitial space on the periphery of the tumour between the lumen of the new tumour blood vessels and the tumour and normal cells surrounding the bulk of the tumour, wherein the leakiness of the new tumour blood vessels permits larger macromolecules to extravasate from the blood and get trapped or be temporarily concentrated in that interstitial area. This phenomenon is known as enhanced permeability and retention (EPR).
• EPR enhanced permeability and retention
• cancer cells require additional nutrients to sustain their increased growth rates, and achieve this via angiogenesis.
• Angiogenesis is the process of new blood vessel formation. These new blood vessels also tend to have less structure than established vessels and are sometime termed “leaky” vasculature in that the junctions between the endothelial cells lining these vessels are not as tight and rigid as in established vessels. The angiogenic development of leaky microvasculature is common to all solid tumors [Folkman, Semin.Cancer Biol., 3, 65-71 (1992) and Folkman, Nature Med., I, 27-31 (19
• ananimate subject is meant a living mammalian patient, preferably a living human subject.
• synthetic has its conventional meaning, i.e. man-made as opposed to being isolated from natural sources. Such compounds have the advantage that their manufacture and impurity profile can be fully controlled.
• polyethyleneglycol polymer or "PEG” has its conventional meaning, as described eg. in "The Merck Index", 14 th Edition entry 7568, i.e. a liquid or solid polymer of general formula H(OCH 2 CH 2 ) n OH where n is an integer greater than or equal to 4.
• the polyethyleneglycol polymers of the present invention may be linear or branched (i.e. dendrimeric), but are preferably linear.
• the polyethyleneglycol polymer is suitably polydisperse.
• polymer terminus is meant the functional group(s) which form the end of the polyether chains of the PEG polymer chains - in the above general formula the two hydroxy (-OH) groups.
• conjugate is "meant” a derivative in which the "optical reporter” (Opt R ) is covalently bonded to the polyethyleneglycol polymer.
• biocompatible non-toxic and hence suitable for administration to the mammalian body, especially the human body, without adverse reaction, or pain or discomfort on administration.
• optical reporter i.e. Opt R
• Opt R a fluorescent dye or chromophore which is capable of detection either directly or indirectly in an optical imaging procedure using light of wavelength 600-850 nm. Since the optical reporter must be suitable for imaging the mammalian body in vivo, it must also be biocompatible.
• the Opt R has fluorescent properties, and it preferably comprises a fluorescent, biocompatible dye.
• region of interest or ROI has its conventional meaning in the field of in vivo medical imaging.
• the molecular weight of polyethyleneglycol polymer is preferably 20-43 kDa, more preferably 22-40 kDa, and most preferably 25-38 kDa, with 27-35 kDa being the ideal.
• the polyethyleneglycol polymer is preferably a linear polymer.
• the polyethyleneglycol polymer preferably only has conjugated thereto the Opt R group(s). Thus, the polymer preferably does not have conjugated thereto a biological targeting molecule or other polymer.
• biological targeting moiety is meant a compound which, after administration, is taken up selectively or localises at a particular site of the mammalian body. Such sites may for example be implicated in a particular disease state be indicative of how an organ or metabolic process is functioning.
• a biological targeting moiety typically comprises: 3-100 mer peptides, peptide analogue, peptoids or peptide mimetics which may be linear peptides or cyclic peptides or combinations thereof; or enzyme substrates, enzyme antagonists or enzyme inhibitors; synthetic receptor-binding compounds; oligonucleotides, or oligo- DNA or oligo-RNA fragments.
• the conjugate of the first aspect is preferably of Formula I:
• Y 1 and Y 2 are independently Opt R or a functional group chosen from -OH; -0(CMO alkyl); -NH 2 Or -NH(CO)(C 1-10 alkyl); wherein Opt R is as defined above; with the proviso that at least one of Y 1 and Y 2 is Opt R .
• amino acid is meant an L- or Z)-amino acid, amino acid analogue (eg. naphthylalanine) or amino acid mimetic which may be naturally occurring or of purely synthetic origin, and may be optically pure, i.e. a single enantiomer and hence chiral, or a mixture of enantiomers.
• amino acid analogue eg. naphthylalanine
• amino acid mimetic which may be naturally occurring or of purely synthetic origin, and may be optically pure, i.e. a single enantiomer and hence chiral, or a mixture of enantiomers.
• sugar a mono-, di- or tri- saccharide.
• Suitable sugars include: glucose, galactose, maltose, mannose, and lactose.
• the sugar may be functionalised to permit facile coupling to amino acids.
• a glucosamine e.g. a glucosamine
• derivative of an amino acid can be conjugated to other amino acids via peptide bonds.
• the glucosamine derivative of asparagine (commercially available from NovaBiochem) is one example of this:
• each of Y 1 and Y 2 is Opt R .
• X and X' are preferably chosen to be -NHCO- or -CONH- such that the conjugate is prepared from a diamino-PEG or dicarboxy-PEG polymer.
• PEG polymers thus correspond to H 2 N-[POLYMER]-NH 2 or HOOC-[POLYMER]-COOH respectively, wherein the biocompatible dye of Opt R is conjugated to the polymer at each terminus via an amide bond.
• each of Y 1 and Y 2 is Opt R
• the Opt R groups of Y 1 and Y 2 each comprise the same biocompatible reporter. That has three advantages. Firstly, when the two chromophores of the biocompatible reporters are the same, the contrast agent exhibits an enhanced fluorescent signal for effectively the same molecular weight (because the molecular weight of the reporter is so much less than that of the polymer). Secondly, possible unwanted interference and/or quenching of fluorescence between the signals from two different biocompatible reporters is avoided. Thirdly, symmetric bifunctional-PEGs are easy to synthesise.
• m of the L group is preferably an integer of value 1 to 5, most preferably 1 to 3.
• the Opt R preferably comprises a biocompatible dye capable of detection either directly or indirectly in an optical imaging procedure using light of wavelength 610-
• the biocompatible dye of Opt R preferably has fluorescent properties.
• Particular examples of such dyes include: indocyanine green, the cyanine dyes Cy5, Cy5.5, Cy7, and
• Alexa Fluor 633 Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750.
• the biocompatible dye is preferably a cyanine dye or benzopyrylium dye, most preferably a cyanine dye.
• Preferred cyanine dyes which are fluorophores are of Formula II:
• each X' is independently selected from: -C(CHs) 2 , -S-, -O- or
• Q' is independently selected from the group consisting of: H, SO 3 M 1 , NH 2 , COOM ,
• M 1 is H or B c ; where B c is a biocompatible cation;
• z is an integer of value 2 or 3;
• n is an integer from 1 to 5;
• G is a reactive or functional group suitable for attaching to the PEG polymer.
• biocompatible cation By the term “biocompatible cation” (B c ) is meant a positively charged counterion which forms a salt with an ionised, negatively charged group, where said positively charged counterion is also non-toxic and hence suitable for administration to the mammalian body, especially the human body.
• suitable biocompatible cations include: the alkali metals sodium or potassium; the alkaline earth metals calcium and magnesium; and the ammonium ion.
• Preferred biocompatible cations are sodium and potassium, most preferably sodium.
• the G group reacts with a complementary group of the PEG polymer forming a covalent linkage between the cyanine dye fluorophore and the polymer.
• the location of the G groups in Formula II is such that the PEG can suitably be conjugated at positions, Q', X' or Y'.
• G may be a reactive group that may react with a complementary functional group of the PEG, or alternatively may include a functional group that may react with a reactive group of the PEG.
• reactive and functional groups include: active esters; isothiocyanate; maleirmde; haloacetamide; acid halide; hydrazide; vinylsulfone; dichlorotriazine; phosphoramidite; hydroxyl; amino; sulfydryl; carbonyl; carboxylic acid and thiophosphate.
• G is an active ester.
• activated ester or “active ester” is meant an ester derivative of the associated carboxylic acid which is designed to be a better leaving group, and hence permit more facile reaction with nucleophile, such as amines.
• suitable active esters are: N-hydroxysuccinimide (NHS), sulfo-succinimidyl ester, pentafluorophenol, pentafluorothiophenol, /? ⁇ ra-nitrophenol, hydroxybenzotriazole and PyBOP (i.e. benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate).
• Preferred active esters are N-hydroxysuccinimide or pentafluorophenol esters, especially N-hydroxysuccinimide esters.
• Preferred cyanine dyes based on Formula II are as defined in Formula Ha:
• Y 3 and Y 4 are independently -O-, -S-, -NR 5 - or -CR 6 R 7 - and are chosen such that at least one of Y 3 and Y 4 is -CR 6 R 7 -;
• R 1 and R 2 are independently H, -SO 3 M 1 or R a ;
• R 3 to R 5 are independently C 1 - 5 alkyl, Ci -6 carboxyalkyl or R a ;
• R 6 is H or Ci -3 alkyl, R 7 is R a or Ci _ 6 carboxyalkyl;
• R a is independently Ci -4 sulfoalkyl; where M 1 and z are as defined in Formula II; with the proviso that the cyanine dye of Formula Ha comprises at least one R a group and a total of 1 to 6 sulfonic acid substituents from the R 1 , R 2 and R a groups.
• sulfonic acid substituent is meant a substituent of formula -SO 3 M 1 , where M 1 is as defined above.
• the -SO 3 M 1 substituent is covalently bonded to a carbon atom, and the carbon atom may be aryl (such as the R 1 or R 2 groups), or alkyl (i.e. an R a group).
• the R a groups are preferably of formula -(CH 2 XSO 3 M 1 , where M 1 is as defined above, and k is an integer of value
• Particularly preferred cyanine dyes are of Formula lib:
• R and R .10 are independently H or SO 3 M , and at least one of R and R 10 is SO 3 M 1 ;
• R 11 and R 12 are independently Cj -4 alkyl or C 1-6 carboxyalkyl;
• R 13 , R 14 , R 15 and R 16 are independently R b groups; wherein R b is Ci -4 alkyl, d_ 6 carboxyalkyl Or -(CH 2 ) C1 SO 3 M 1 , where q is an integer of value 3 or 4; where M 1 is as defined for Formulae II and Ha; with the proviso that the cyanine dye has a total of 1 to 4 SO 3 M 1 substituents in the R 9 , R 10 and R b groups.
• Preferred cyanine dyes of Formula lib are chosen to comprise at least one Cj -6 carboxyalkyl group, or activated ester thereof, in order to facilitate conjugation to the PEG polymer.
• An especially preferred such dye of Formula lib is Cy7:
• benzopyrylium dye has its conventional meaning. Suitable benzopyrylium dyes of the present invention are denoted Bzp M and are of Formula III:
• R > 2 ⁇ 5 i ⁇ s H, oxyalkyl, C 3-I2 arylsulfonyl, Cl, or R 2 ⁇ 5 C M alkyl, C] -6 carb together with one of R ,26 , negligence R34 ,ticianR35 or R , 36 may optionally form a 5- or 6- membered unsaturated aliphatic, unsaturated heteroaliphatic or aromatic ring;
• R 26 and R 36 are independently R g groups
• R 27 and R 28 are independently Ci -4 alkyl, Ci -4 sulfoalkyl or Ci - 6 hydroxyalkyl or for Y a may optionally together with one or both of R 29 and/or R 30 may form a 5- or 6- membered N-containing heterocyclic or heteroaryl ring, or for Y b may optionally together with one or both of R 30 and/or R 30 may form a 5- or 6- membered N- containing heterocyclic or heteroaryl ring;
• R g is Ci -4 alkyl, Cj -4 sulfoalkyl, Ci -6 carboxyalkyl or Ci -6 hydroxyalkyl; w is 1 or 2;
• J is a biocompatible anion; where M 1 is as defined for Formula II; with the proviso that Bzp M comprises at least one sulfonic acid substituent chosen from the R 21 to R 36 groups.
• biocompatible anion a negatively charged counterion which forms a salt with an ionised, positively charged group (in this case an indolinium group), where said negatively charged counterion is also non-toxic and hence suitable for administration to the mammalian body, especially the human body.
• the counterion (J " ) represents an anion which is present in a molar equivalent amount, thus balancing the positive charge on the Bzp M dye.
• the anion (J) is suitably singly- or multiply-charged, as long as a charge-balancing amount is present.
• the anion is suitably derived from an inorganic or organic acid. Examples of suitable anions include: halide ions such as chloride or bromide; sulfate; nitrate; citrate; acetate; phosphate and borate. A preferred such anion is chloride.
• Suitable contrast agents of the invention are those wherein the Bzp M is of Formula Ilia or HIb:
• R 25 together with one of R 26 /R 34 -R 36 forms a 5- or 6- membered unsaturated aliphatic, unsaturated heteroaliphatic or aromatic ring
• suitable such aromatic rings include: phenyl, furan, thiazole, pyridyl, pyrrole or pyrazole rings.
• R 2 , R .3 j 0 ⁇ or R j l form a 5- or 6- membered N-containing heterocyclic or heteroaryl ring
• suitable such rings include: thiazole, pyridyl, pyrrole or pyrazole rings or partially hydrogenated versions thereof, preferably pyridyl or dihydropyridyl.
• the PEG polymer is preferably attached at positions R 25 , R 26 , R 34 , R 35 or R 36 of the
• R 25 , R 26 , R 34 , R 35 or R 36 substituent preferably comprises Ci -6 carboxyalkyl, more preferably C 3-6 carboxyalkyl.
• the benzopyrylium dye (Bzp M ) preferably has at least 2 sulfonic acid substituents, more preferably 2 to 6 sulfonic acid substituents, most preferably 2 to 4 sulfonic acid substituents.
• at least one of the sulfonic acid substituents is a Ci -4 sulfoalkyl group.
• Such sulfoalkyl groups are preferably located at positions R 26 , R 27 ,
• R , 26 together with one or both of R ,27 and R 28 of Formula III.
• the sulfoalkyl groups of Formula III are preferably of formula -(CH 2 XSO 3 M 1 , where M 1 is H or B c , k is an integer of value 1 to 4, and B c is a biocompatible cation (as defined above), k is preferably 3 or 4.
• w is preferably 2.
• R 25 is preferably H or C 1-4 carboxyalkyl, and is most preferably H.
• X is preferably -CR 34 R 35 - or -NR 36 - , and is most preferably - CR R 35 -.
• the contrast agent preferably comprises a pharmaceutical composition of the conjugate, together with a biocompatible carrier.
• a pharmaceutical composition of the conjugate preferably comprises a pharmaceutical composition of the conjugate, together with a biocompatible carrier.
• Such pharmaceutical compositions are as described in the third aspect (below).
• a preferred optical imaging method of the sixth aspect is Fluorescence Reflectance Imaging (FRI).
• FRI Fluorescence Reflectance Imaging
• the contrast agent of the present invention is administered to a subject to be diagnosed, and subsequently a tissue surface of the subject is illuminated with an excitation light
• the light excites the Opt R of the contrast agent. Fluorescence from the contrast agent, which is generated by the excitation light, is detected using a fluorescence detector. The returning light is preferably filtered to separate out the fluorescence component (solely or partially). An image is formed from the fluorescent light. Usually minimal processing is performed (no processor to compute optical parameters such as lifetime, quantum yield etc.) and the image maps the fluorescence intensity.
• the contrast agent is designed to concentrate in the disease area, producing higher fluorescence intensity. Thus the diseased area produces positive contrast in a fluorescence intensity image.
• the image is preferably obtained using a CCD camera or chip, such that real-time imaging is possible.
• the wavelength for excitation varies depending on the particular dye used.
• the apparatus for generating the excitation light may be a conventional excitation light source such as: a laser (e.g., ion laser, dye laser or semiconductor laser); an array of LEDs; halogen light source or xenon light source.
• Various optical filters may optionally be used to obtain the optimal excitation wavelength.
• a preferred FRI method comprises the steps of:
• tissue surface comprising the region of interest within the animate subject is illuminated with an excitation light
• the light detected by the fluorescence detector is optionally filtered to separate out the fluorescence component
• an image of said tissue surface is formed from the fluorescent light of steps (ii) or (iii).
• the excitation light of step (i) is preferably continuous wave (CW) in nature.
• the optical imaging preferably comprises FDPM (frequency- domain photon migration).
• FDPM frequency- domain photon migration
• CW continuous-wave
• the Opt R has fluorescent properties which can be modulated depending on the tissue depth of the lesion to be imaged, and the type of instrumentation employed.
• a preferred FDPM method comprises the steps of: (a) exposing light-scattering biologic tissue having a heterogeneous composition, said tissue forming a region of interest of said animate subject, to light from a light source with a pre-determined time varying intensity to excite the contrast agent, the tissue multiply-scattering the excitation light;
• step (d) generating an image of the tissue by mapping the heterogeneous composition of the tissue in accordance with the values of step (c).
• the fluorescence characteristic of step (c) preferably corresponds to uptake of the contrast agent and preferably further comprises mapping a number of quantities corresponding to adsorption and scattering coefficients of the tissue before administration of said contrast agent.
• the fluorescence characteristic of step (c) preferably corresponds to at least one of fluorescence lifetime, fluorescence quantum efficiency, fluorescence yield and contrast agent uptake.
• the fluorescence characteristic is preferably independent of the intensity of the emission and independent of contrast agent concentration.
• the quantifying of step (c) preferably comprises, (i) establishing an estimate of the values, (ii) determining a calculated emission as a function of the estimate, (iii) comparing the calculated emission to the emission of said detecting to determine an error, (iv) providing a modified estimate of the fluorescence characteristic as a function of the error.
• the quantifying preferably comprises determining the values from a mathematical relationship modelling multiple light-scattering behaviour of the tissue.
• the method of the first option preferably further comprises monitoring a metabolic property of the tissue in vivo by detecting variation of said fluorescence characteristic.
• the contrast agents of the first aspect can be prepared as follows:
• the dye of the Opt R suitably has attached thereto a reactive functional group (Q a ).
• the Q a group is designed to react with a complementary functional group of the polymer, thus forming a covalent linkage between the dye and the polymer.
• Suitable Q a groups may be selected from: carboxyl; activated esters; isothiocyanate; maleimide; haloacetamide; hydrazide; vinylsulfone, dichlorotriazine and phosphoramidite.
• Q a is: an activated ester of a carboxylic acid; an isothiocyanate; a maleimide; or a haloacetamide.
• Q a is an activated ester. Preferred aspects of such activated esters are as described above.
• a preferred starting material is a diamino-PEG.
• the PEG-diamine is preferably of greater than 90% purity, more preferably of over 95% purity, most preferably of over 99% purity.
• the synthesis described by Elbert provides PEG- diamines of the required purity.
• Example 1 provides further details.
• Cyanine dyes functionalised suitable for conjugation to peptides are commercially available from GE Healthcare Limited, Atto-Tec, Dyomics, Molecular Probes and others. Most such dyes are available as NHS esters. Methods of conjugating the linker group (L) to the polymer employ analogous chemistry to that of the dyes alone (see above), and are known in the art. Benzopyrylium dyes are commercially available from Dyomics GmbH, Winzerlaer Str. 2A, D-07745 Jena, Germany; (www.dyomics.com).
• the present invention provides a contrast agent suitable for in vivo optical imaging of the mammalian body which comprises the conjugate as defined in the first aspect.
• a contrast agent suitable for in vivo optical imaging of the mammalian body which comprises the conjugate as defined in the first aspect.
• Preferred embodiments of the conjugate in the contrast agent are as described in the first aspect.
• the present invention provides a pharmaceutical composition which comprises the conjugate as defined in the first aspect, together with a biocompatible carrier.
• Preferred embodiments of the conjugate in the pharmaceutical composition are as described in the first aspect.
• the “biocompatible carrier” is a fluid, especially a liquid, in which the imaging agent can be suspended or dissolved, such that the composition is physiologically tolerable, ie. can be administered to the mammalian body without toxicity or undue discomfort.
• the biocompatible carrier is suitably an injectable carrier liquid such as sterile, pyrogen- free water for injection; an aqueous solution such as saline (which may advantageously be balanced so that the final product for injection is isotonic); an aqueous solution of one or more tonicity-adjusting substances (eg. salts of plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose), sugar alcohols (eg. sorbitol or mannitol), glycols (eg.
• glycerol or other non-ionic polyol materials (eg. polyethyleneglycols, propylene glycols and the like).
• a macromolecular polyol it is suitably of molecular weight up no more than 10 kDa, preferably below 5 kDa - since higher molecular weight species might compete with the contrast agent of the present invention.
• the biocompatible carrier is pyrogen-free water for injection or isotonic saline.
• the contrast agent and biocompatible carrier are each supplied in suitable vials or vessels which comprise a sealed container which permits maintenance of sterile integrity and/or radioactive safety, plus optionally an inert headspace gas (eg. nitrogen or argon), whilst permitting addition and withdrawal of solutions by syringe or cannula.
• a sealed container which permits maintenance of sterile integrity and/or radioactive safety, plus optionally an inert headspace gas (eg. nitrogen or argon), whilst permitting addition and withdrawal of solutions by syringe or cannula.
• a preferred such container is a septum-sealed vial, wherein the gas-tight closure is crimped on with an overseal (typically of aluminium).
• the closure is suitable for single or multiple puncturing with a hypodermic needle (e.g. a crimped-on septum seal closure) whilst maintaining sterile integrity.
• Such containers have the additional advantage that the closure can withstand vacuum if desired (eg. to
• Preferred multiple dose containers comprise a single bulk vial (e.g. of 10 to 30 cm 3 volume) which contains multiple patient doses, whereby single patient doses can thus be withdrawn into clinical grade syringes at various time intervals during the viable lifetime of the preparation to suit the clinical situation.
• Pre-filled syringes are designed to contain a single human dose, or "unit dose” and are therefore preferably a disposable or other syringe suitable for clinical use.
• the pharmaceutical compositions of the present invention preferably have a dosage suitable for a single patient and are provided in a suitable syringe or container, as described above.
• the pharmaceutical composition may optionally contain additional excipients such as an antimicrobial preservative, pH-adjusting agent, filler, stabiliser or osmolality adjusting agent.
• an antimicrobial preservative is meant an agent which inhibits the growth of potentially harmful micro-organisms such as bacteria, yeasts or moulds.
• the antimicrobial preservative may also exhibit some bactericidal properties, depending on the dosage employed.
• the main role of the antimicrobial preservative(s) of the present invention is to inhibit the growth of any such micro-organism in the pharmaceutical composition.
• the antimicrobial preservative may, however, also optionally be used to inhibit the growth of potentially harmful micro-organisms in one or more components of kits used to prepare said composition prior to administration.
• Suitable antimicrobial preservative(s) include: the parabens, ie. methyl, ethyl, propyl or butyl paraben or mixtures thereof; benzyl alcohol; phenol; cresol; cetrimide and thiomersal.
• Preferred antimicrobial preservative(s) are the parabens.
• pH-adjusting agent means a compound or mixture of compounds useful to ensure that the pH of the composition is within acceptable limits (approximately pH 4.0 to 10.5) for human or mammalian administration. Suitable such pH-adjusting agents include pharmaceutically acceptable buffers, such as tricine, phosphate or TRIS [ie. tr ⁇ (hydroxymethyl)aminomethane], and pharmaceutically acceptable bases such as sodium carbonate, sodium bicarbonate or mixtures thereof.
• buffers such as tricine, phosphate or TRIS [ie. tr ⁇ (hydroxymethyl)aminomethane]
• pharmaceutically acceptable bases such as sodium carbonate, sodium bicarbonate or mixtures thereof.
• the pH adjusting agent may optionally be provided in a separate vial or container, so that the user of the kit can adjust the pH as part of a multi-step procedure.
• filler is meant a pharmaceutically acceptable bulking agent which may facilitate material handling during production and lyophilisation.
• suitable fillers include inorganic salts such as sodium chloride, and water soluble sugars or sugar alcohols such as sucrose, maltose, mannitol or trehalose.
• the pharmaceutical compositions may be prepared under aseptic manufacture (ie. clean room) conditions to give the desired sterile, non-pyrogenic product. It is preferred that the key components, especially the associated reagents plus those parts of the apparatus which come into contact with the imaging agent (eg. vials) are sterile.
• the components and reagents can be sterilised by methods known in the art, including: sterile filtration, terminal sterilisation using e.g. gamma-irradiation, autoclaving, dry heat or chemical treatment (e.g. with ethylene oxide). It is preferred to sterihse some components in advance, so that the minimum number of manipulations needs to be carried out. As a precaution, however, it is preferred to include at least a sterile filtration step as the final step in the preparation of the pharmaceutical composition.
• the pharmaceutical composition is preferably prepared from a kit, as described for the fourth aspect below.
• the present invention provides a kit for the preparation of the pharmaceutical composition of the second aspect, which comprises the contrast agent of the first aspect in sterile, solid form such that, upon reconstitution with a sterile supply of a biocompatible carrier (as described in the third aspect), dissolution occurs to give the desired pharmaceutical composition.
• the contrast agent may be provided as a lyophilised powder in a suitable vial or container.
• the agent is then designed to be reconstituted with the desired biocompatible carrier to give the pharmaceutical composition in a sterile, apyrogenic form which is ready for mammalian administration.
• a preferred sterile, solid form of the contrast agent is a lyophilised solid.
• the sterile, solid form is preferably supplied in a pharmaceutical grade container, as described for the pharmaceutical composition (above).
• the formulation may optionally comprise a cryoprotectant chosen from a saccharide, preferably mannitol, maltose or tricine.
• Example 1 provides the synthesis of a PEG- ⁇ w(dye) conjugate of the invention.
• Example 2 provides the synthesis of other PEG-dye conjugates of the invention.
• Example 3 provides the biological screening model used. The results are shown in Figure 1. Earlier time points were regarded as being somewhat more important, since that is more pertinent to the clinical situation.
• the PEG3.4k conjugate has markedly inferior MSR values at all times.
• the PEG30k conjugate was superior, exhibiting good MSR values at all time points.
• the PEG20k and PEG43k conjugates exhibited good MSR values at earlier times, but were inferior at 24 hours.
• Example 4 examined the effect of changing the dye from the Cy5 range (excitation: 650 nm, emission: 670 nm) to Cy7 (excitation: 743 nm, emission; 767 nm).
• the MSR score either increased or was similar.
• MALDI Matrix assisted laser desorption ionization
• MSR margin to surrounding skin ratio
• PBS Phosphate-buffered saline.
• TFA Trifluoroacetic acid
• Example 1 Synthesis of a Bis-Cyl PEG-31k Conjugate (Compound 1).
• Diamino-PEG was purchased from supplier LaysanBio. It was synthesised from the corresponding PEG-diol (Sigma/Aldrich), using the method of Elbert et al [Biomacromolecules, 2, p 430-441 (2001)].
• the diamine-PEG had an average mass of -31 kDa by GFC and -35 kDa by MALDI. Amine substitution was ca. 100% with no other impurities detectable by proton NMR, in particular no CH 2 -OMs or CH 2 -OH protons observable.
• the fluorescent dye, Cy7-NHS was obtained from GE Healthcare. It had an active ester content of 81.3%.
• the conjugate was prepared as follows:
• PEGs functionalised with a single dye molecule were synthesised in an analogous manner to Example 1, using the appropriate PEG-monoamine with the dye active ester ( ⁇ 1.2-1.5 equivalents).
• the PEG 43 kDa conjugate was prepared by reaction of mono-amino PEG20K with a bifunctional dye (Cy5-bis NHS ester) in a molar ratio of 3.33:1.
• a bifunctional dye Cy5-bis NHS ester
• PEG20K 100 mg was co-evaporated with anhydrous DMF (3x) and redissolved in anhydrous DMF (5ml).
• N-methylmorpholine 4 ⁇ l
• Cy5-bis NHS 0.3 equiv. in 146 ⁇ l of DMSO
• the mixture was stirred in the dark overnight and then purified by HPLC. The pure fraction was concentrated using Amicon 5K MWCO filter.
• Example 3 Screening Model.
• the 13762 Mat B III (a rat mammary gland adenocarcinoma, ATCC # CRL- 1666) cell line was cultured in DMEM (Gibco #10564-011) with 10% FBS and 1% Pen/Strep. Cells were incubated at 37°C in a mixture of air:CO 2 (95%:5%). After cells reached more than 80% confluency, cells were collected, counted, and concentrated to 10 x 10 6 cells/mL of culture media for injection.
• Figure 1 shows the effect of PEGs of differing molecular weights.
• the number of each PEG represents the molecular weight of the agent (i.e. PEG30K has a MW of 30 kDa).

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