WO2023036351A1 - Fluorescently labelled polymer for tumour visualization, method of its preparation and use thereof - Google Patents

Fluorescently labelled polymer for tumour visualization, method of its preparation and use thereof Download PDF

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WO2023036351A1
WO2023036351A1 PCT/CZ2022/050088 CZ2022050088W WO2023036351A1 WO 2023036351 A1 WO2023036351 A1 WO 2023036351A1 CZ 2022050088 W CZ2022050088 W CZ 2022050088W WO 2023036351 A1 WO2023036351 A1 WO 2023036351A1
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group
groups
polymer
general formula
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Tomas Etrych
Robert POLA
Eliska GROSMANOVA
Michal Pechar
Dominik HORAK
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Ustav Makromolekularni Chemie Av Cr, V.V.I.
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    • A—HUMAN NECESSITIES
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    • 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
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    • 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
    • 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
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    • 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
    • A61K49/0056—Peptides, proteins, polyamino acids
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    • A61K49/00—Preparations for testing in vivo
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    • A61K49/0013—Luminescence
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    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
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    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/58—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing oxygen in addition to the carbonamido oxygen, e.g. N-methylolacrylamide, N-(meth)acryloylmorpholine
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    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/58—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing oxygen in addition to the carbonamido oxygen, e.g. N-methylolacrylamide, N-(meth)acryloylmorpholine
    • C08F220/585—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing oxygen in addition to the carbonamido oxygen, e.g. N-methylolacrylamide, N-(meth)acryloylmorpholine and containing other heteroatoms, e.g. 2-acrylamido-2-methylpropane sulfonic acid [AMPS]
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    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/60—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen
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    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/60—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen
    • C08F220/603—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen and containing oxygen in addition to the carbonamido oxygen and nitrogen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/60—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen
    • C08F220/606—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen and containing other heteroatoms
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    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites

Definitions

  • the present invention relates to a targeting fluorescent polymer with activatable fluorescence composed of a polymeric carrier with side chains containing fluorophores, which is intended to visualise tumour tissue.
  • the use of the polymer is aimed at highlighting the tumour tissue for subsequent surgical removal or non- invasive monitoring of the cancer disease progression.
  • Fluorescence imaging is based on the illumination of tissue with light that excites near-infrared (NIR) or shortwave -infrared (SWIR) fluorophores or contrast agents. Due to the permeability of light in the NIR and SWIR regions through tissues, up to several cm, it is possible to use these fluorophores for visualisation as part of marking tumour foci.
  • NIR near-infrared
  • SWIR shortwave -infrared
  • DDSs are based on water-soluble or amphiphilic polymeric carriers carrying low-molecular-weight active molecules, for example, covalently bound by biodegradable linkers, intended for controlled release and activation of the carried active molecules in desired tissues or cells.
  • biocompatible, non-toxic and non-immunogenic copolymers based on N-(2 -hydroxypropyl) methacrylamide (pHPMA), polyethylene glycol) (PEG), poly(caprolactone) (PCE), poly(lactic acid)/poly(lactide-co-glycolide acid) (PEA/PEGA) and their copolymers as carriers of biologically active molecules and targeting groups.
  • polymeric carriers of bioactive molecules are designed to optimise the pharmacokinetics of active molecules, prolong their blood circulation time, improve localisation in tumours, inflammations and target cells, reduce secondary toxicity and immunogenicity, and solubilise water-insoluble active molecules.
  • the active molecules carried can be released/activated by enzymatic hydrolysis, reduction, or pH-induced hydrolysis by lowering the pH from 7.4 (blood) to 5-6.5 (endosomes/lysosomes; tumour microenvironment; inflammatory environment).
  • targeted systems containing targeting groups for example, antibodies or oligopeptides, or systems having a structure and size enabling ‘passive’ targeting based on the EPR (enhanced permeability and retention) effect using high-molecular- weight (HMW) carriers, for example, star-shaped polymers, nanoparticles or micelles, are studied.
  • HMW high-molecular- weight
  • pHPMA-based polymeric therapeutics and diagnostics have been developed that have increased accumulation in solid tumours.
  • therapeutics carrying anti-tumour drugs such as doxorubicin and paclitaxel, have been found to have anti -cancer activity in various tumour models in mice.
  • Polymeric systems containing both the drug and a bound fluorescent label were not only therapeutically active but were able to monitor tumour development using fluorescence imaging (FI).
  • Linear pHPMA constructs containing NIR dye Dyomics-633 and EGF (epidermal growth factor) targeting oligopeptides GE -7 and GE- 11 were described not long ago.
  • the in vivo biodistribution study showed very promising results in tumour visualisation.
  • the polymeric probe was mainly localised at the tumour border, showing the potential for image-guided surgery.
  • high-molecular-weight delivery systems for fluorescence imaging and tumour surgery have been described, for example, in WO 2020/245447 Al (fluorescent polypeptides). These high molecular weight systems are used for imaging tumour tissue. Although they meet the condition of biocompatibility and can be excreted from the body, none bring a selective increase in fluorescence locally in the tumour tissue.
  • the present invention relates to a targeting fluorescent polymer, which forms a polymeric probe capable of activating a fluorescent signal at the tumour site.
  • the fluorescent polymer according to the present invention enables targeted accumulation in tumour tissue and subsequent activation of a fluorescent signal to visualise tumours.
  • the polymeric system itself contains a polymeric carrier, a fluorescent label linked to the polymeric carrier by a bond that is hydrolytically, enzymatically or reductively degradable in the tumour environment, alternatively further containing a targeting group, which directs the polymer to the tumour or endothelial cells and possibly further containing a fluorescence quencher.
  • the resulting fluorescent polymer makes it possible to amplify the signal and achieve a significant tumour/healthy tissue contrast around the tumour by activating the fluorescence of the transported label only in the tumour tissue. While the fluorescence yield of the fluorescent label drops significantly after binding the fluorophore to the polymeric carrier because of self-quenching of fluorescent molecules localised in the vicinity due to non-radiative energy transfer between the fluorophores, after the release of the label from the polymeric carrier in the tumour tissue, the fluorescence signal restores, leading to a significant increase in the signal versus noise ratio in the tumour environment. This surprising effect of increasing the fluorescence signal contrast between healthy and tumour tissue has not been described before.
  • Copolymers based on A-(2- hydroxypropyl)methacrylamide (HPMA) are suitable precursors for preparing the described fluorescent polymers with activatable fluorescence. Functional groups are introduced along the chain of this copolymer, to which suitable fluorophore derivatives can be attached via biodegradable linkers and targeting groups.
  • the entire system according to the present invention thus enables increased visualisation of tumour foci for subsequent surgical resection navigated by a fluorescent signal.
  • Another use is the application of fluorescent polymers to monitor the progression and regression of cancer during treatment with another therapy. It is preferred to use fluorescent polymers for tumours that are not localised in the depth of the tissue or that require endoscopic techniques, for example, head and neck cancer, breast cancer, colorectal tumours and melanomas.
  • the object of the present invention is a fluorescent polymer suitable for tumour visualisation, which contains a semitelechelic statistical linear copolymer, wherein the semitelechelic statistical linear copolymer is selected from the group comprising poly(A-(2- hydroxypropyljmethacrylamide), polyacrylamide, polymethacrylamide, polyacrylate and polymethacrylate in which from 0.1 to 10 mol% monomer units, preferably from 0.4 to 6 mol% monomer units, more preferably from 0.5 to 4 mol% of monomer units, based on the total number of monomer units, are statistically replaced by monomer units of the general formula (I) fluorophore
  • A is selected from the group consisting of a linear or branched carbon alkylenyl chain having from 1 to 7 carbons ((Cl to C7)alkylenyl); -(CH2) p -(C(O)-NH-(CH2) r ) P -;
  • L is a linker containing a triazole bridge, formed, for example, by the reaction of a propargyl or DBCO group with an azide group; thus, L can have, for example, the following structure: wherein p is an integer in the range of from 1 to 5, and r is selected from 1, 2 and 3; wherein one or more hydrogen atoms in the CH 2 groups of the substituent A
  • fluorophore fluorophore or its amino derivative, NCS ester or A-hydroxysuccinimidyl derivative
  • the fluorophore has molecular weights in the range of from 350 to 1,500 g/mol, excitation wavelengths in the range of from 300 to 850 nm, and emission wavelengths in the range of from 350 to 1,200 nm, and is
  • the molecular weight M n of the fluorescent polymer is in the range of from 6,000 to 100,000 g/mol, preferably from 10,000 to 60,000 g/mol, more preferably from 15,000 to 40,000 g/mol (corresponding to 100 to 280 monomer units), more preferably from 20,000 to 30,000 g/mol (corresponding to 134 to 210 monomer units).
  • the fluorescent polymer according to the present invention enables targeted accumulation in tumour tissue and subsequent activation of the fluorescent signal for the visualisation of tumours.
  • the fluorophore is bound in the monomer unit (I) by a bond that is hydrolytically, enzymatically or reductively degradable in the tumour environment, which enables the amplification of the signal and the achievement of a significant tumour/healthy tissue contrast around the tumour by activating the fluorescence of the transported label only in the tumour tissue.
  • the fluorescence yield of the fluorescent label drops significantly after binding the fluorophore to the polymeric carrier because of self-quenching of fluorescent molecules localised in the vicinity due to non-radiative energy transfer between the fluorophores, after the release of the fluorophore from the polymeric carrier in the tumour tissue, the fluorescence signal restores, leading to a significant increase in the signal versus noise ratio just in the tumour environment. This effect allows the claimed compounds to be used in fluorescence-guided surgery.
  • Natural amino acids shall be understood as being histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, arginine, cysteine, glutamine, glycine, proline, tyrosine, alanine, aspartic acid, asparagine, glutamic acid, serine, selenocysteine.
  • Side chains are chains attached to the alpha-carbon of an amino acid.
  • alkylenyl means a divalent linear or branched carbon chain, for example, -CH 2 -,
  • alkyl means a single-bond linear or branched carbon chain formed from an alkane by the removal of one hydrogen atom.
  • Ma-AP-TT stands for 3-(3-methacrylamidopropanoyl)thiazolidine-2-thione.
  • Ma-AP stands for 3-(3-methacrylamidopropanoyl).
  • Ma-Acap stands for 6-methacrylamidohexanoic acid.
  • Ma-Acap-NH-NH 2 stands forA-(6-methacrylamidohexanoyl)hydrazine
  • Mo-Acap-NH-NH-Boc stands for /V-(tert-butoxycarbonyl)-JV’-(6-methacrylamidohexanoyl)hydrazine.
  • TT stands for thiazolidine-2-thione.
  • DBCO stands for dibenzocyclooctyne.
  • Ma-AP-DBCO stands for 3-(3-methacrylamidopropanoyl)dibenzocyclooctyne.
  • GG stands for dipeptide glycylglycine.
  • GFG stands for glycylphenylalanylglycine tripeptide.
  • OPB 4-(2-oxopropyl)benzenecarboxylic acid.
  • OPB-Cy7 stands for Cy-7 fluorophore modified with OPB acid.
  • Aba stands for aminobenzyl alcohol.
  • the Az-Val-Cit-Aba-DY-676 structure indicates the Dyomics-676 fluorophore bound to the Az-Val-Cit- Aba peptide chain.
  • GFLG-DY-676 stands for the fluorophore DY-676 linked to the tetrapeptide GFLG (SEQ. no. 8), i.e. Gly- Phe-Leu-Gly.
  • COP stands for 5 -cyclohexyl-5 -oxopentanoic acid.
  • COP-DY-676 indicates the COP to which the DY-676 fluorophore is bound.
  • £> stands for a dispersity, calculated as a ratio of weight average by number average molecular weight.
  • fluorophore means an organic molecule with at least one aromatic ringcapable of fluorescence, attached to the substituent B by a covalent bond.
  • a fluorophore is a fluorescent label or a derivative thereof, containing an amino group, a keto group, or an S-S bond, added to the fluorophore structure by reacting the fluorescent label with a selected oxoacid or reagent containing an activated disulphide, for example, by reacting with 3-(2-pyridyldithio)propionate, 5 -cyclohexyl-5 -oxopentanoic acid, 4-(2- oxopropyl)benzenecarboxylic acid and 4-oxo-4-(2-pyridyl)butanoic acid.
  • the groups formed by this reaction are part of the group B defined above.
  • the fluorophore is selected from the group comprising (2E)-2-[(E)-3-(7-azaniumylidene-2-tert-butylchromen-4-yl)prop-2-enylidene]-l-[6-(2,5-dioxopyrrolidin- l-yl)oxy-6-oxohexyl]-3,3-dimethylindole-5-sulphonate (DY-615), sodium 2-[3-[2-tert-butyl-7-[ethyl(3- sulphonatopropyl)azaniumylidene]chromen-4-yl]prop-2-enylidene]-l-(5-carboxypentyl)-3,3- dimethylindole-5 -sulphonate (DY-633), sodium 3-(3-carboxypropyl)-2-[3-(9-ethyl-6,8,8-trimethyl-2- phenylpyran
  • fluorophores are modified with oxoacids using the amide reaction of the amino group of the fluorophore with the activated carboxyl of the oxoacid, the resulting product being an oxo derivative of the fluorophore.
  • the fluorophore is modified with cystamine using an amide bond between the cystamine and the fluorophore.
  • the fluorophore is modified with A-(5-azidopentanoyl-valyl-citrulyl)-4-aminobenzyl (4-nitrophenyl) carbonate or with N-(5- azidopentanoyl -valyl -citrulyl) by reaction with its amino group.
  • the fluorophore can be attached to group B via an oligopeptide linker of 2 to 5 amino acids that can be enzymatically degradable by lysosomal enzymes, a pH-sensitive hydrolytically degradable hydrazone bond or a reductively biodegradable disulphide bond, which enables the controlled release of the fluorophore in the tumour environment.
  • An oligopeptide linker of 2 to 5 amino acids can be, for example, Val-Cit or Val-Cit-Aba.
  • the end groups of the resulting fluorescent polymer contain residues of the polymerisation initiator and transfer agent molecules.
  • the initiator can be, for example, 2,2'-azobis[A-(2-carboxyethyl)-2- methylpropionamidine] (V-70)) and the transfer agent can be, for example, A-(3-azidopropyl)-4- ethylsulphanylcarbothioylsulphanyl-4-methyl-pentanamide (CTA-N3)), or their derivatives formed by reaction with, for example, 2,2'-azobisisobutyronitrile (AIBN).
  • AIBN 2,2'-azobisisobutyronitrile
  • the end groups of the copolymer therefore, contain an azide group, which can subsequently be further modified by reaction with dibenzocyclooctyne - maleimide (DBCO-MI) to another reactive maleimide functional group, by reaction with dibenzocyclooctyne-carboxyl (DBCO-Cbx), which is subsequently modified with thiazolidine-2-thione to the thiazolidine-2-thione reactive functional group, by reaction with dibenzocyclooctyne -N- hydroxysuccinimidyl ester (DBCO-NHS) to the A-hydroxysuccinimidyl ester reactive functional group.
  • DBCO-MI dibenzocyclooctyne - maleimide
  • DBCO-Cbx dibenzocyclooctyne-carboxyl
  • DBCO-NHS dibenzocyclooctyne -N- hydroxysuccinimidyl ester
  • the fluorescent polymer defined above is directed to the tissues of solid tumours due to its high molecular weight and the longer circulation time during which the fluorescent polymer accumulates in solid tumours (due to EPR effect).
  • substituent A of the fluorescent polymer is selected from the group consisting of a linear or branched carbon alkylenyl chain having from 1 to 7 carbons;
  • linker -A-B- is degradable in an acidic environment (for example, in tumour tissue).
  • said combination also leads to a linker structural segment -A-B-, connecting the fluorophore to the polymeric carrier, which has a formula selected from:
  • linker -A-B- is degradable in an acidic environment (for example, in tumour tissue), in the reductive environment of the cytoplasm of tumour cells, and, at the same time, the peptide character of the linker enables its enzymatic degradation.
  • substituent A of the fluorescent polymer is selected from the group consisting of -(CH 2 )p-(C(O)-NH-(CH2)r)p-C(O)-NH-C6H4-CH 2 -O-C(O)-; and -(CH 2 ) p -C(O)-NH-(CH 2 )p-L-(CH 2 )p-(C(O)-NH-(CH 2 ) r )p-C(O)-NH-C6H 4 -CH 2 -O-C(O)-, wherein L is a linker containing a triazole bridge; p is an integer in the range of from 1 to 5, and r is selected from 1, 2 and 3; and substituent B is a bond.
  • Said combination leads to a linker structural segment -A-B-, connecting the fluorophore to the polymeric carrier, which has the formula: -(CH 2 )p-(C(O)-NH-(CH 2 ) r ) p -C(O)-NH-C 6 H4-CH 2 -O-C(O)-; or -(CH 2 ) p -C(O)-NH-(CH 2 )p-L-(CH 2 )p-(C(O)-NH-(CH 2 ) r )p-C(O)-NH-C 6 H4-CH 2 -O-C(O)-.
  • linker -A-B- is enzymatically degradable.
  • the fluorescent polymer further contains targeting groups to enhance the targeting of the fluorescent polymer to tumour tissues. These targeting groups can be attached to the end groups of the linear chain of the fluorescent polymer and/or they can be attached to the side chains.
  • the targeting group for directing the fluorescent polymer to tumour cells of head and neck tumours, breast tumours, melanomas and colorectal tumours or to tumour endothelial cells is selected from the group comprising oligopeptides with the number of amino acids from 3 to 20, and proteins, preferably antibodies, more preferably monoclonal antibodies targeting tumour cells of head and neck tumours, breast tumours, melanomas and colorectal tumours or tumour endothelial cells.
  • oligopeptides withthe number of amino acids from 3 to 20 are selected from the group comprising YESIKVAVS (SEQ. no. 1), SIGYPLP (SEQ. no. 2), RGD (tumour endothelium), CPLHQRPMC (SEQ. no. 3; prostate tumours), NPVVGYIGERPQYRDL (SEQ. no. 4) called GE7 and YHWYGYTPQNVI (SEQ. no. 5) called GE11 (to the EGF receptor), WHYPWFQNWAMA (SEQ. no. 6; head and neck tumour cells), DMPGTVLP (SEQ. no. 7; breast tumours), CNGRC (SEQ. no.
  • the monoclonal antibodies are selected from the group comprising herceptin, erbitux, daratuzumab, trastuzumab.
  • these targeting peptides and/or proteins are attached by conjugation of the amino group or introduced propargyl, DBCO, or SH groups of the targeting peptide/protein with an azide, maleimide, thiazolidine-2-thione, or A-hydroxysuccinimidyl ester end group of the linear chain of the fluorescent polymer.
  • the fluorescent polymer defined above also contains monomer units of the general formula (II)
  • A is as defined above, , wherein L is as defined above; and Z is a targeting group for directing the fluorescent polymer to tumour cells of head and neck tumours, breast tumours, melanomas, and colorectal tumours, or tumour endothelial cells, also defined as above, and is attached to the X group via an amide bond, maleimide, azide, or propargyl.
  • the targeting group is atached to the X group via azide or propargyl.
  • the amount of monomer units of general formula (II) is from at least one monomer unit to 8 mol%, based on the total number of monomer units.
  • the total number of monomer units of the general formula (I) and (II) in the fluorescent polymer is at most 10 mol%, based on the total number of monomer units.
  • the targeting group may optionally be modified prior to a conjugation to the fluorescent polymer to contain at least one group selected from -NH2, -SH, sDBCO (sulphodibenzocyclooctyne group), DBCO (dibenzocyclooctyne group), azide, propargyl, to which the fluorescent polymer is covalently attached via a functional group, preferably selected from A-hydroxysuccinimidyl ester, thiozolidine-2-thione amide, maleinimide, azide, DBCO, sDBCO, or propargyl.
  • a functional group preferably selected from A-hydroxysuccinimidyl ester, thiozolidine-2-thione amide, maleinimide, azide, DBCO, sDBCO, or propargyl.
  • the semitelechelic statistical linear copolymer is poly(/V-(2- hydroxypropyl)methacrylamide) .
  • the fluorescent polymer can further contain from 0 to 9.9 mol% of monomer units of the general formula (III), based on the total number of monomer units,
  • the fluorescent polymer contains from at least one monomer unit of the general formula (III) to 9.9 mol% monomer units of the general formula (III).
  • Said monomer unit of the general formula (III) in the structure of the fluorescent polymer is formed during the synthesis by reactions of unreacted thiazolidine-2-thione groups, (2,3,4,5,6-pentafluorofenyl)oxy groups, (succinimidyl)oxy groups, with an amino alcohol selected from the group comprising NH 2 -(CH 2 ) a - CH 2 (OH); NH 2 -(CH 2 )b-CH(OH)-CH 3 ; NH 2 -(CH 2 )b-CH(OH)-(CH 2 )c-CH 3 ; wherein a is an integer in the range of from 0 to 4, b is an integer in the range of from 0 to 3 and c is an integer in the range of from 1 to 4, preferably with 1 -aminopropan -2-ol, and/or by reaction of unreacted NH 2 groups with acetythiazolidin- 2-thione, and/or by reaction of unreacted disulphide groups
  • the fluorescent polymer is a linear statistical copolymer of HPMA and monomer units of general formula (I), and optionally (II) and/or (III).
  • This linear statistical copolymer can be represented by a general formula (IV), wherein the molecular weight M n of the statistical linear copolymer is in the range of from 6,000 to 100,000 g/mol, preferably in the range of from 15,000 to 45,000 g/mol (corresponding to 100 to 300 monomer units), more preferably the molecular weight is in the range of from 20,000 to 40,000 g/mol (corresponding to 134 to 280 monomer units);
  • the remaining monomer units are HPMA monomer units.
  • the end groups of the resulting linear copolymer of the general formula (IV) contain parts of the polymerisation initiator and transfer agent molecules as mentioned above.
  • the general formula (IV) contains from 1 to 30 monomer units of the general formula (I), from 1 to 24 monomer units of the general formula (II) and from 1 to 29 monomer units of the general formula (III), wherein the maximum total number of monomer units of formula (I) + formula (II) + formula (III) being 30.
  • the remaining monomer units are HPMA monomer units.
  • the fluorescent polymer is a semitelechelic statistical linear copolymer that can be prepared by radical polymerisation.
  • the end groups of the resulting linear copolymer are blocked with, for example, 2,2' - azobisisobutyronitrile (AIBN).
  • AIBN 2,2' - azobisisobutyronitrile
  • Another object of the present invention is, therefore, also a method of preparing the fluorescent polymer (fluorescent polymer with activatable fluorescence and possibly with a bound targeting group), which includes the following steps:
  • the targeting structure is selected from the group comprising oligopeptides with the number of amino acids from 3 to 20 and proteins, especially monoclonal antibodies; wherein the targeting structure can be attached to the end of the linear chain and/or to the side chains of the fluorescent polymer (as part of the monomer unit of general formula (II)).
  • Providing monomers of the linear copolymer comprises providing monomers selected from the group comprising A-(2-hydroxypropyl)methacrylamide (HPMA), acrylamide, methacrylamide, acrylate and methacrylate, and monomers of the general formula (V)
  • V which are the precursors for the monomer units of general formulae (I), (II) and (III), wherein A is as defined above; and D is -carbonyl-thiazoline-2-thione group (TT), (4-nitrophenyl)oxy group, (2, 3, 4,5,6- pentafluorophenyljoxy group, (succinimidyl)oxy group, carboxyl, hydrazide, azide, activated disulphide group or NH2 group.
  • the amino group and the hydrazide group may optionally be protected by a protecting group, for example, a tert-butoxycarbonyl (Boc) protecting group.
  • A-(2-hydroxypropyl)methacrylamide (HPMA) and other monomers of the acrylamide, methacrylamide, acrylate, and methacrylate types are commercially available.
  • Substances of the general formula (V) were either obtained from commercially available sources, for example, methacryloyl-6-aminopropyl amine protected on the amine groups by tert-butyloxycarbonyl groups (MA-Pr-NH-Boc), or were prepared according to procedures reported in the literature (Etrych T., et al., A-(2-Hydroxypropyl)methacrylamide-Based Polymer Conjugates with pH-Controlled Activation of Doxorubicin. I. New Synthesis, Physicochemical Characterization and Preliminary Biological Evaluation, J.Appl.Pol.Sci. 109, 3050-3061 (2008), V.
  • Ad (b) Polymerisation of the monomers of the statistical linear copolymer is carried out by controlled radical RAFT (reversible addition-fragmentation chain-transfer) polymerisation of monomers from step (a) with a content of from 0.1 to 10 mol% of monomers of the general formula (V), and at least 90 mol% (90 to 99.9 mol%) of monomer units, selected from the group comprising JV-(2-hydroxypropyl)methacrylamide (HPMA), acrylamide, methacrylamide, acrylate, methacrylate, preferably selected from the group comprising HPMA, acrylamide, methacrylamide, most preferably HPMA.
  • HPMA JV-(2-hydroxypropyl)methacrylamide
  • the reaction typically takes place at a temperature ranging from 30 to 100 °C, preferably from 40 to 80 °C, and a solvent preferably selected from the group comprising dimethylsulphoxide, dimethylacetamide, dimethylformamide, methanol, ethanol, dioxane, tetrahydrofuran, propanol, tert-butanol or their mixture.
  • a solvent preferably selected from the group comprising dimethylsulphoxide, dimethylacetamide, dimethylformamide, methanol, ethanol, dioxane, tetrahydrofuran, propanol, tert-butanol or their mixture.
  • the reaction is initiated by an initiator, preferably selected from the group comprising in particular azoinitiators 2,2'-azobis(2-methylpropionitril) (AIBN), 4,4'-azobis(4-cyanopentanoic acid) (ACVA), 2,2'- azobis(4-methoxy-2,4-dimethylpentannitril) (V70), optionally in the presence of a transfer agent, preferably selected from the group containing 2-cyano-2-propylbenzodithioate, 4-cyano-4- (thiobenzoylthio)pentanoic acid, 2-cyano-2-propyldodecyl-trithiocarbonate, 2-cyano-2- propylethyltrithiocarbonate, 4-cyano-4-[(dodecylsulphanylthio-carbonyl)sulphanyl]pentanoic acid, [4-(3 - azidopropylamino) - 1 -cyano- 1 -methyl
  • the molar mass M n of the linear statistical copolymers thus prepared is in the range of from 4,000 to 100,000 g/mol, preferably from 25,000 to 50,000 g/mol.
  • the resulting semitelechelic linear copolymer contains end reactive groups (for example, azide and TT).
  • the prepared linear copolymer from step (b) can optionally be further subjected to the removal of protecting groups protecting the hydrazide groups of the side chains (for example, Boc groups). Deprotection can be accomplished by established procedures known to those skilled in the art, such as the removal of the Boc group with trifluoroacetic acid or by heating the copolymer in water.
  • the resulting copolymer/product can be stored without the risk of its decomposition.
  • the product of this step is thus a semitelechelic statistical linear copolymer, which contains a linear polymer selected from the group comprising polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate and poly( '-(2 -hydroxyp ropy I (methacryl am ide), in which from 0.1 to 10 mol% of monomer units are statistically replaced by a monomer unit of the general formula (VI)
  • Ad (c) binding the fluorophore to D group of the statistical linear copolymer from step (b) is carried out by conjugation of carbonyl -thiazoline -2 -thione groups, (4-nitrophenyl)oxy groups, (2, 3, 4,5,6- pcntafluorophcnyljoxy groups, (succinimidyl)oxy groups, carboxyl, hydrazide, azide, activated disulphide groups and NH2 groups (while the amino group and the hydrazide group, if protected by a protecting group in the previous step, must be deprotected before this conjugation) of monomer units of the general formula (VI) of the semitelechelic statistical linear copolymer from step b), with a low-molecular-weight fluorophore.
  • the fluorophore is a fluorescent label or a derivative thereof that contains suitable reactive groups (for example, an amine, carboxyl, activated carboxyl, activated disulphide or keto group) and can be used in free form or the form of a salt with an acid, for example, HC1; wherein the low-molecular-weight fluorophore is a fluorophore having a molecular weight in the range of from 350 to 1,500 g/mol.
  • suitable reactive groups for example, an amine, carboxyl, activated carboxyl, activated disulphide or keto group
  • the fluorophore is selected according to excitation and emission wavelengths, with excitation wavelengths ranging from 300 to 850 nm and emission wavelengths ranging from 350 to 1,200 nm; the low- molecular- weight fluorophore molecule is bound to the linear copolymer by an amide, disulphide or hydrazone bond.
  • the product of this step is thus a semitelechelic statistical linear copolymer containing monomer units of general formula (I), (VI), and further containing monomer units selected from the group comprising acrylamide, methacrylamide, acrylate, methacrylate and /V-(2-hydroxypropyl)methacrylamide.
  • Ad (dl) Binding of the targeting structure to the monomer units of the general formula (VI) of the semitelechelic statistical linear copolymer from step (c) - step of conjugation of the targeting structure with introduced amino groups, SH, azide, propargyl sulphodibenzocyclooctyne (sDBCO) or dibenzocyclooctyne (DBCO) groups to the D group defined above.
  • sDBCO propargyl sulphodibenzocyclooctyne
  • DBCO dibenzocyclooctyne
  • the targeting structure is selected from the group comprising oligopeptides with the number of amino acids from 3 to 20, and proteins, preferably antibodies, more preferably monoclonal antibodies, targeting tumour cells of head and neck tumours, breast tumours, melanomas and colorectal tumours or tumour endothelial cells, wherein oligopeptides are attached using a click reaction of the terminal azide with DBZO groups on the polymer; antibodies are linked by a click reaction of the maleimide end group of the semitelechelic fluorescent polymer with the thiol group of the antibody molecule introduced therein by mild reduction.
  • oligopeptides with the number of amino acids from 3 to 20 are selected from the group comprising YESIKVAVS, SIGYPLP, RGD and cyclic RGDfK (tumour endothelium), CPLHQRPMC (prostate tumours), NPWGYIGERPQYRDL called GE7 and YHWYGYTPQNVI called GE11 (to the EGF receptor), WHYPWFQNWAMA (head and neck tumour cells), DMPGTVLP (breast tumours), CNGRC, HEWSYLAPYPWF and SYSMEHFRWGKPV. Oligopeptides and proteins that target tumour tissues in the body are known to those skilled in the art.
  • the monoclonal antibodies are selected from the group comprising herceptin, erbitux, daratuzumab, trastuzumab.
  • sDBCO sulphodibenzocyclooctyne
  • DBCO dibenzocyclooctyne
  • Ad (d2) Binding the targeting structure to the ends of the semitelechelic statistical linear fluorescent polymer from step (c) - the step of conjugation of the targeting structure with amino groups introduced by SH, azide, propargyl, sulphodibenzocyclooctyne (sDBCO) or dibenzocyclooctyne (DBCO) groups to the end group of the linear polymer, which can be maleinimidyl group, TT group, azide, propargyl, DBCO, or sDBCO.
  • the targeting structure is as defined above.
  • Any unreacted D groups can be removed by reaction with amino alcohol selected from the group comprising NH2-(CH 2 ) a -CH 2 (OH); NH 2 -(CH 2 ) b -CH(OH)-CH 3 ; NH 2 -(CH 2 ) b -CH(OH)-(CH 2 ) c -CH 3 ; wherein a is an integer in the range of from 0 to 4, b is an integer in the range of from 0 to 3 and c is an integer in the range of from 1 to 4, preferably with l-aminopropan-2-ol, and/or any unreacted NH 2 groups can be removed by reaction with acetylthiazolidin-2-thione, and/or any unreacted disulphide groups are removed by reaction with 2-hydroxyethanethiol; to form a
  • the product of this step is a fluorescent polymer according to the present invention, i.e. a semitelechelic statistical linear copolymer, containing monomer units of the general formula (I), optionally (II), and optionally (III), defined as above, and further containing monomer units selected from the group comprising acrylamide, methacrylamide, acrylate, methacrylate and /V-(2-hydroxypropyl)methacrylamide.
  • a fluorescent polymer according to the present invention i.e. a semitelechelic statistical linear copolymer, containing monomer units of the general formula (I), optionally (II), and optionally (III), defined as above, and further containing monomer units selected from the group comprising acrylamide, methacrylamide, acrylate, methacrylate and /V-(2-hydroxypropyl)methacrylamide.
  • An object of the present invention is also a star-shaped fluorescent polymer that contains a multivalent carrier to which at least one fluorescent polymer according to the present invention is bound, wherein the multivalent carrier is selected from the group comprising poly(amidoamine)dendrimer of the second or third generation, 2,2-bis(hydroxymethyl)propion dendrimer or dendron of the second to the fourth generation; polyols with the number of hydroxyl groups from 2 to 8, glycerol, pentaerythritol, bis(2- hydroxyethyl)aminotris(hydroxy-methyl)methane, dipentaerythritol .
  • the multivalent carrier is selected from the group comprising poly(amidoamine)dendrimer of the second or third generation, 2,2-bis(hydroxymethyl)propion dendrimer or dendron of the second to the fourth generation; polyols with the number of hydroxyl groups from 2 to 8, glycerol, pentaerythritol, bis(2- hydroxye
  • the star-shaped fluorescent polymer is a copolymer of general formula (VII) that contains a multivalent carrier and at least one fluorescent polymer of general formula (IV), as defined above.
  • the multivalent carrier is a second or third generation poly(amidoamine) (PAMAM) dendrimer (for example, with an ethylenediamine, 1,4-diaminobutane, 1,6-diaminohexane, 1,12-diaminododecane, cystamine core) or 2,2-bis(hydroxymethyl)propion dendrimer or dendron (for example, with a trimethylol propane core) of the second to the fourth generation; polyols with the number of hydroxyl groups from 2 to 8, for example, ethylene glycol, polyethylene glycol, trimethylolpropane (1,1,1- tris(hydroxymethyl)propane), glycerol, pentaerythritol, bis(2- hydroxyethyl)aminotris(hydroxymethyl)methane, dipentaerythritol; wherein A, B, C and Z are as defined above;
  • Y is selected from the group consisting of a primary amino group; primary hydroxy group; alkyne groups with the number of carbons from 3 to 6, for example, propargyl; and a cyclooctyne group, which may optionally be further independently substituted by one or more groups selected from an alkyl group having 1 to 6 carbons and an aryl having 6 carbons, for example, DBCO;
  • W is an amide bond between the primary amino group Y of the multivalent carrier and the carboxyl end group of the polymer of general formula (IV); or an ester bond between the primary hydroxy group Y of the multivalent carrier and the carboxyl end group of the polymer of the general formula (IV) or a triazole linker, formed by the reaction of the azide end group of the polymer of the general formula (IV) and the alkyne or cycloalkyne group Y of the dendrimer or dendron (multivalent carrier).
  • the star-shaped fluorescent copolymer therefore, consists of a central molecule of the carrier (for example PAMAM dendrimer, 2,2-bis(hydroxymethyl)propion dendrimer or dendron or polyol) to the end groups of which at least one chain of the fluorescent polymer is bound, preferably of a linear statistical copolymer of the general formula (IV), as defined above.
  • the carrier for example PAMAM dendrimer, 2,2-bis(hydroxymethyl)propion dendrimer or dendron or polyol
  • End groups of the multivalent carrier are amino groups, hydroxy groups or alkyne groups with the number of carbons from 3 to 6, for example, propargyl, or cyclooctyne groups, which can optionally be further independently substituted by one or more groups selected from an alkyl group with the number of carbons from 1 to 6 and an aryl having 6 carbons, for example, DBCO, and the fluorescent polymer (for example, a linear copolymer of formula (IV)) is attached to them via an amide, ester or triazole linker).
  • the fluorescent polymer for example, a linear copolymer of formula (IV)
  • the star-shaped fluorescent copolymer contains from 2 to 48 linked fluorescent polymer chains, more preferably from 3 to 32 linked fluorescent polymer chains, most preferably from 4 to 24 linked fluorescent polymer chains.
  • the fluorescent polymer is a linear statistical copolymer of the general formula (IV).
  • the molar mass M n of the star-shaped fluorescent copolymer is preferably in the range of from 60,000 to 1,000,000 g/mol, preferably from 70,000 to 400,000 g/mol.
  • the molar mass of each chain of the fluorescent polymer linked to the multivalent carrier is in the range of from 6,000 to 100,000 g/mol, preferably from 40,000 to 70,000 g/mol, while the molar mass of the multivalent carrier itself, which is part of the starshaped fluorescent copolymer, does not exceed 50,000 g/mol.
  • n is an integer in the range of from 1 to 48
  • Y is a primary amino group, a hydroxy group, a C3-C6 alkyne group, or a cyclooctyne group of a poly(amidoamine) or 2,2-bis(hydroxymethyl)propion dendrimer or dendron or polyol;
  • V is an azide or TT end reactive group introduced by a transfer agent to the end of the polymer chain of the fluorescent polymer of general formula (IV) during RAFT polymerisation;
  • W is an amide bond, an ester bond or a triazole linker formed by the reaction of the azide of the polymer of general formula (IV) and the alkyne group or the cycloalkyne group of the multivalent carrier.
  • Scheme 1 Scheme for the preparation of a star-shaped copolymer.
  • Scheme 2 Examples of structures of multivalent carriers for the synthesis of a star-shaped fluorescent copolymer: (i) schematic representation of bis-MPA dendron with DBCO groups; (ii) PAMAM dendrimer with amino groups; (iii) bis-MPA dendrimer with propargyl groups; (iv) polyols with the number of hydroxyls from 4 to 8; and (v) an example of the structure of a star-shaped fluorescent copolymer with the PAMAM dendrimer carrier, in which the chains containing the linear fluorescent polymer are marked schematically with a wavy line.
  • the maximum number of chains containing the linear fluorescent polymer that can be attached to a multivalent carrier is equal to the number of end groups of the multivalent carrier (in this case, 16 end amino groups of the dendrimer).
  • An object of the present invention is also a method of preparing the star-shaped fluorescent polymer defined above, comprising the following steps:
  • a multivalent carrier selected from the group comprising the second or third generation poly(amidoamine) dendrimer, 2,2-bis(hydroxymethyl)propion dendrimer or the second to fourth generation dendron; polyols with the number of hydroxyl groups from 2 to 8, glycerol, pentaerythritol, bis(2- hydroxyethyl)aminotris(hydroxymethyl)methane, dipentaerythritol, porphyrin derivatives; terminated by groups Y selected from primary amino groups, keto groups, azide groups, hydroxy groups, alkyne groups with a number of carbons from 3 to 6, for example, propargyl; and cyclooctyne groups, which may optionally be further independently substituted by one or more groups selected from an alkyl group having 1 to 6 carbon atoms and an aryl having 6 carbon atoms, for example, DBCO.
  • a multivalent carrier selected from the group comprising the second or third generation poly(amidoamine)
  • Said multivalent carriers are commercially available, optionally end DBCO groups can be prepared by reaction of end primary amino groups with DBCO-NHS.
  • step (iii) grafting end reactive functional groups (for example, azide or TT) of the statistical linear copolymer prepared in step (ii) onto the groups Y of the multivalent carrier from step (i), to form a star-shaped polymer, which contains in its arms a statistical linear copolymer selected from the group comprising polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate and poly(A'-(2- hydroxypropyl)methacrylamide) in which from 0. 1 to 10 mol% of monomer units are statistically replaced by a monomer unit of the general formula (VI).
  • a statistical linear copolymer selected from the group comprising polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate and poly(A'-(2- hydroxypropyl)methacrylamide) in which from 0. 1 to 10 mol% of monomer units are statistically replaced by a monomer unit of the general formula (VI).
  • the grafting reaction of polymers onto multivalent carriers takes place in a solvent preferably selected from the group comprising dimethylsulphoxide, dimethylacetamide, dimethylformamide, methanol and ethanol.
  • the molar mass M n of the star-shaped polymers prepared in this way is in the range of from 60,000 to 1,000,000 g/mol, preferably from 100,000 to 400,000 g/mol; (iv) linking of the fluorophore to group D of the statistical linear copolymer from step (iii) by conjugation of carbonyl -thiazoline-2-thione groups, (4-nitrophenyl)oxy groups, (2,3,4,5,6-pentafluorophenyl)oxy groups, (succinimidyl)oxy groups, carboxyl, hydrazide, azide, activated disulphide groups or NH2 groups of monomer units of general formula (VI) with an amine, carboxyl, activated carboxyl, activated disulphide or keto group of the fluorophore
  • the linking of the fluorophore to group D is carried out by conjugation of free hydrazide groups, amino groups, activated disulphide groups or thiazolidine-2-thione (TT) groups of monomer units of the general formula (VI) of the statistical linear copolymer described above with a low-molecular-weight fluorescent label or its derivative (fluorophore) which contains suitable reactive groups (for example, an amine, carboxyl, activated carboxyl or keto group) and can be used in its free form or in the form of a salt with an acid, for example, HC1; wherein the low-molecular-weight fluorophore is a fluorophore having a molecular weight in the range of from 350 to 1,500 g/mol.
  • suitable reactive groups for example, an amine, carboxyl, activated carboxyl or keto group
  • the fluorophore is selected according to excitation and emission wavelengths, with excitation wavelengths ranging from 300 to 850 nm and emission wavelengths ranging from 350 to 1,200 nm; the low-molecular-weight fluorophore molecule is bound to the linear copolymer by an amide, disulphide or hydrazone bond.
  • Linking of the targeting structure is carried out by reacting group D of the monomer unit of general formula (VI) with -NH2, propargyl, sDBCO or DBCO groups present on the targeting structure. The reaction takes place in the order of minutes and with a high yield, and the resulting conjugate retains its biological activity unaffected.
  • step (vi) optionally, linking of the targeting group to the ends of the linear chain of the fluorescent star-shaped polymer from step (iv) or (v).
  • Linking of the targeting structure (a peptide structure with amino groups or introduced azide, maleinimidyl, propargyl, sulphodibenzocyclooctyne (sDBCO) or dibenzocyclooctyne (DBCO) groups) is carried out by reacting end group, TT group, azide, propargyl, DBCO or sBCO group or SH group with groups present on the targeting structure. The reaction takes place in the order of minutes and with a high yield, and the resulting conjugate retains its biological activity unaffected.
  • sDBCO sulphodibenzocyclooctyne
  • DBCO dibenzocyclooctyne
  • Any unreacted thiazolidine-2-thione groups, (4-nitrophenyl)oxy groups, (2, 3, 4,5,6- pentafluorophenyl)oxy groups, (succinimidyl)oxy groups can be removed by reaction with amino alcohol selected from the group consisting of NH2-(CH2) a -CH2(OH); NH2-(CH2)b-CH(OH)-CH3; NH2-(CH2)b- CH(OH)-(CH2) C -CH3; wherein a is an integer in the range of from 0 to 4, b is an integer in the range of from 0 to 3 and c is an integer in the range of from 1 to 4; preferably with l-aminopropan-2-ol, and/or the unreacted NH2 groups can be removed by reaction with acetylthiazolidin-2-thione.
  • Activated disulphide groups can be removed by reaction with mercaptoethanol. Carboxyles, hydrazides and azides do not need
  • the present invention further relates to the use of fluorescent probes for the visualisation of tumours for diagnostic purposes and for image-guided surgery.
  • the fluorescent probe means the fluorescent polymer according to the present invention as defined above, and the star-shaped fluorescent polymer according to the present invention as defined above.
  • the fluorescent probe can carry a combination of a fluorophore, the fluorescence of which is activated in the tumour, and a targeting group to increase the specificity of visualisation of the tumour tissue.
  • the statistical linear fluorescent copolymer further contains at least one targeting group for directing the fluorescent probe to tumour tissues, which is selected from the group comprising oligopeptides with the number of amino acids from 3 to 20 and proteins for targeting the fluorescent polymer to tumour cells of head and neck tumours, breast tumours, melanomas and colorectal tumours or to tumour endothelial cells.
  • the targeting group can be attached to the end group of the linear chain of the fluorescent probe or it can be part of the monomer unit (II).
  • An object of the present invention is, therefore, the use of the fluorescent probe according to the present invention in medical diagnostics, whole-body imaging and/or fluorescence-guided surgery, preferably in the diagnosis and monitoring of the success of treatment in cancer diseases, diseases of the hematopoietic system (leukaemia, lymphomas, and hematopoietic failure) and the immune system.
  • Fluorescent probes according to the present invention can be used, for example, in whole-body imaging techniques based on fluorescence detection for the detection of tumour tissue; in fluorescence -guided surgery for marking and imaging fluorescence in target structures of body organs and tissues.
  • fluorescent probes are activatable linear and star-shaped fluorescent polymers according to the present invention, in which the fluorophore is bound to the polymeric carrier by a biologically degradable amide, hydrazone, or disulphide bond, and enable the circulation time of the bound fluorophore in the organism to be significantly extended, which enables the fluorophore to be transported on a high-molecular- weight polymeric carrier into the tumour and released here in its original form.
  • Another advantage of these activatable fluorescent probes according to the invention is the activation of fluorescence, which is given by the release of the fluorophore from the polymeric carrier.
  • Polymeric activatable fluorescent probes according to the invention are further characterised by the fact that the binding of the fluorophore to the polymer chain is relatively stable, only at most 10 percent of the fluorophore is released during 24 h transport in the bloodstream and body fluids, and it is hydrolytically, enzymatically or reductively cleavable in the tumour environment and inside target tumour cells in lysosomes. This means that the fluorophore is transported through the bloodstream in an inactive, low- fluorescent form, and its release and activation of fluorescence mainly occur after entering the tumour tissue or after penetration into the target tumour cells.
  • Activation of the fluorophore only in the target cells leads to a significant increase in fluorescence in the tumour, in the tumour/healthy tissue contrast and thus to a clear visualisation of the tumour tissue.
  • a polymeric carrier prepared preferably on the basis of HPMA copolymers, whose molecular weight and thus the efficiency of accumulation in the tumour tissue can be controlled by changes in the structure of the polymeric carrier (linear polymer, high-molecular-weight biodegradable star-shaped polymer).
  • Polymeric probes can preferably also be targeted actively by means of linked targeting structures, oligopeptides and proteins, which further increases the selectivity of accumulation in tumour tissue and supports an increase in tumour/healthy tissue contrast.
  • the object of the invention is a targeting and activatable fluorescent polymer and a star-shaped fluorescent polymer for enhancing the visualisation of solid tumours. Due to their hydrodynamic size in solution and targeting to receptors on tumour cells, they are significantly accumulated in solid tumours, resulting in a significant enrichment of the tumour tissue with the fluorophore present. In addition, the carried fluorophore will be released in the tumour tissue environment, which results in an increase in the fluorescence signal due to a decrease in fluorescence quenching associated with the binding of the fluorophore to the polymeric system. As a result, there is a very significant increase in the fluorescence contrast between tumour and non-tumour tissue, significantly contributing to the visualisation of tumour tissue during guided tumour surgery. Contrast enhancement significantly advances the boundaries of guided surgery compared to the current state of the art.
  • Fig. 1 Fluorescence intensity of the fluorophore before and after hydrolysis of the pol-PYR-Cy7 conjugate in Example 10.
  • Fig. 2 Rate of release of the fluorophore from conjugates with DY 676 attached to the polymer by pH- sensitive linkers with different structures in Example 11.
  • Fig. 3 Confocal microscopy images - Example 13.
  • Fig. 4 In vivo fluorescence imaging of polymer systems with fluorescence label Dyomic 676; A - polymer system with a label firmly bound by an amide bond to a non-degraded linker; B- polymer system with a label bound via a hydrazone linker formed by OPB-Dy-676 - Example 14
  • the copolymer poly(HPMA-co-Ma-AP-TT) was prepared by controlled solution radical copolymerisation of HPMA (93 mol%, 100 mg) and 3-(3-methacrylamidopropanoyl)thiazolidin-2-thione (Ma-AP-TT) (7 mol%, 14.0 mg) carried out in the presence of the initiator 2,2'-azobis(4-methoxy-2,4- dimethylvaleronitrile) (V70) and transfer agent 4-cyano-4-thiobenzoyl-sulphanylpentanoic acid (CTA).
  • HPMA 93 mol%, 100 mg
  • Mo-AP-TT 3-(3-methacrylamidopropanoyl)thiazolidin-2-thione
  • V70 2,2'-azobis(4-methoxy-2,4- dimethylvaleronitrile)
  • CTA 4-cyano-4-thiobenzoyl-sulphanylpentanoic acid
  • the polymerisation mixture was dissolved in tert-butyl alcohol (751 pL), CTA dissolved in 10 vol% DMA (83 pL), and all transferred to a glass ampoule where the mixture was bubbled with Ar, and the ampoule was sealed. After 24 h at 40 °C, the polymer was isolated by precipitation into acetone/diethylether, the precipitate was then washed with diethyl ether and dried in a vacuum. The end dithiobenzoate groups were removed from the copolymer by reaction with AIBN (10-fold molar excess) in DMA (15% polymer solution) under an argon atmosphere for 3 h at 70 °C in a sealed ampoule.
  • AIBN 10-fold molar excess
  • the polymeric conjugate was isolated by precipitation into acetone. The precipitate was washed with diethyl ether and dried in a vacuum to dryness.
  • the click reaction precursor was prepared by reacting poly(HPMA-co-Ma-AP-TT) (100 mg, 39.3 pmol) with DBCO-NH2 (5.5 mg, 19.9 pmol) in DMA (1 mb) in the presence of DIPEA base (3.4 pL, 19.9 pmol). The progress of the reaction was monitored using HPLC. For some polymers prepared for label binding via amide bond, the polymer poly(HPMA-co-Ma-AP-TT-co-MA-AP-DBCO) was separated, and in the case of polymers for label binding via click chemistry, the remaining TT groups on the polymer were removed by adding l-aminopropan-2-ol (3.0 pl, 39.3 pmol).
  • the polymeric precursor poly(HPMA-co-Ma-AP- DBCO) was precipitated into acetone/diethylether mixture and re-precipitated from MeOH, washed with diethylether and dried.
  • the same polymeric precursors were also used for targeted conjugates with either an enzymatically cleavable fluorophore or control conjugates with a firmly bound fluorophore.
  • poly(HPMA-co-Ma-XX-NH-NH-Boc) precursors were used, resulting in binding of conjugates with fluorescent labels bound via pH-sensitive hydrazone linkers, where the Boc protecting groups were removed in TFA and the poly(HPMA)-co-Ma-XX-NH-NH2) polymer was precipitated after 10 min into diethyl ether, re-precipitated from MeOH and dried.
  • the disulphide polymer was prepared by reacting polyiHPMA-co-Ma-XX-NH-NFE) with succinimidyl 3- (2-pyridyldithio)propionate) (SPDS) to form the polymeric precursor poly(HPMA-co-Ma-XX-NH-NH- PDS).
  • a reactive copolymer p(HPMA- co-Ma-Pentyl-NHNH-Boc)-N3 was prepared using the transfer agent azide-CTA, '-(3 -azidopropyl ethylsulphanylcarbothioylsulphanyl-4-methyl-pentanamide, containing an azide group, in a similar manner to the reactive copolymer in Example 1.
  • the synthesis of the star-shaped copolymer took place in three steps.
  • pentaerythritol was modified using dibenzocyclooctyne -A-hydroxysuccinimidyl ester.
  • a reactive copolymer p(HPMA-co-Ma-Pentyl-NHNH-Boc)-N3 was prepared using the transfer agent azide-CTA, N-(3- azidopropyl)-7-cthylsulphanylcarbothioylsiilphanyl-4-mcthyl-pcntanamidc. containing an azide group, in a similar manner to the reactive copolymer in Example 1.
  • the reactive copolymer p(HPMA- co-Ma-Pentyl-NHNH-Boc)-TT was prepared using the transfer agent TT-CTA, [l-cyano-l-methyl-4-oxo- 4-(2-thioxothiazolidine-3-yl)butyl]benzenecarbodithioate, containing a TT group, in a similar manner to the copolymer in Example 1.
  • the bis-MPA dendrimer containing amino groups was reacted with p(HPMA-co-Ma-Pentyl-NH-NH)-TT in methanol for 2 h.
  • Scheme 5 An example of the structure of a second generation bis-MPA dendron with a linear copolymer linked via an amide bond.
  • the maximum number of linear copolymer chains that can be attached to the dendron is equal to the number of end groups of the dendron (in this case 4 end amino groups).
  • the dipeptide derivative was prepared by solid-phase peptide synthesis on 2-chlorotrityl chloride resin (0.5 g, substitution 1 mmol/g) by reacting 0.2 M solutions of amino acids (Fmoc-Cit-OH, Fmoc-Val-OH, 5- azidopentanoic acid). The product was cleaved from the resin with a 30% solution of HFIP in DCM, yielding 121 mg (0.23 mmol, 46 %) of the azido-peptide derivative.
  • N-(5-azidopentanoyl-valyl-citrullyl)-4-aminobenzyl alcohol Az-Val-Cit-OH (80 mg, 0.2 mmol), 4-aminobenzyl alcohol (26 mg, 0.21 mmol), 1 -hydroxybenzotriazole (35 mg, 0.23 mmol) and DIC (36 pL, 0.23 mmol) were dissolved in 2 mb of DMF: DCM mixture (3:2). The reaction mixture was stirred for 1 h at 0 °C and then allowed to react for 16 h at 25 °C.
  • Az-Val-Cit-Aba-Npc (0.8 mg, 1.2 pmol) and the amino derivative DY -676 (1 mg, 1.2 pmol) were dissolved in DMA (0.5 mL) with DIPEA (0.25 pL, 1.4 pmol). The progress of the reaction was monitored by HPLC. After 24 h, the dye and the linker reacted off and, according to HPLC, there was one peak corresponding to the product Az-Val-Cit-Aba-DY-676.
  • Derivatives with other fluorescent labels were prepared in a similar way.
  • Carboxylic keto acids such as 5 -cyclohexyl-5 -oxopentanoic acid (COP), 4-(2 -oxopropyl) benzenecarboxylic acid (OPB) and 4-oxo-4-(2-pyridyl)butanoic acid (PYR) were activated by reaction with thiazolidine-2-thione. This reaction is shown in Scheme 6.
  • activated acids were then derivatised with an amino derivative of a fluorescent molecule, e.g. Cyanine7 (Cy7) or Dyomics 676 (DY -676).
  • Cyanine7 Cyanine7
  • Dyomics 676 DY -676
  • 1.39 mg (4.65 pmol) of COP acid and 3.35 mg (4.65 pmol, 1.0 mol equiv. to the keto acid) of Cy7 were dissolved in 1.193 mb (256.5 dm 3 -mol -1 ) of solvent (dimethylformamide - methanol, 3/1) and then mixed.
  • a 2.6M solution of sodium hydroxide (1.10 mol equiv. to the keto acid) was added to the solution.
  • the reaction was carried out at room temperature for three hours with constant stirring. The progress of the reaction was monitored using HPEC and TEC (ethyl acetate). The reaction was terminated by the evaporation of the solvent.
  • the yields of these reactions range between 94 and
  • Oligopeptides GE-7, GE-11, CNGRC, cyclic RGDfK, HEWSYLAPYPWF and SYSMEHFRWGKPV were prepared by solid-phase synthesis using a microwave peptide synthesiser by the standard Fmoc method from the C terminus of the peptide using an /V-Fmoc-protected amino acid (2.5 equiv.), DIC (2.5 equiv.) as an activator, and oxyma (2,5 equiv.) as a base in DMF.
  • Example 8 Preparation of polymeric conjugates containing a fluorescent label by polymer analogous reaction
  • Polymeric conjugate poly(HPMA-co-Ma-GFLG-DY-676) with an aminolytically bound dye via the GFLG linker was prepared by reacting the amino derivative of the dye (2 mg) with the polymeric precursor poly( N- (2-hydroxypropyl)methacrylamide-co-A-methacryloylglycyl-leucylphenylalanyl-glycine thiazolidine-2- thione) (poly(HPMA-co-Ma-GLFG-TT) (98 mg) in 1 mb DMA in the presence of DIPEA base (equiv. to dye).
  • the reaction was monitored on HPLC and after binding of all the free dye, the remaining TT groups on the polymer were removed by the addition of I-aminopropan-2-ol (equiv. to TT at the beginning of the reaction).
  • the reaction mixture was purified by gel filtration (Sephadex LH20) and the polymer fraction was concentrated and precipitated into the acetone/diethylether mixture and washed with diethylether. The precipitate was dried to constant weight.
  • the dye content was determined and the molar mass was measured using SEC of the prepared polymeric conjugate.
  • a control polymeric conjugate poly(HPMA-co-Ma-AP-DY-676) with a firmly bound dye without a degradable sequence was also prepared from the polymeric precursor poly(A-(2- hydroxypropyl)methacrylamide -co-methacrylamidopropanoyl)thiazolidine -2 -thione) (poly (HPMA-co - Ma-AP-TT).
  • the contents of fluorescent labels in the polymeric conjugates are shown in Table 2.
  • the polymeric conjugate poly(HPMA-co-Ma-AP-DBCO-Az-ValCit-Aba-DY-676) was prepared by atwo- step synthesis.
  • (poly(HPMA-co-Ma-AP-TT) was converted to poly(HPMA-co-Ma-AP- DBCO) by reaction with DBCO-NH2 in the presence of DIPEA base
  • the product fractions were concentrated by evaporation of methanol under reduced pressure and precipitated into ethyl acetate, centrifuged and dried to constant weight. The yields of these reactions range between 50 and 70 %. Subsequently, the fluorophore content was determined spectrophotometrically and the molar mass was measured using SEC.
  • Peptide-targeted conjugates were prepared by click reaction of azide derivative of peptide GE -7 with the polymeric precursor having DBCO groups, which was prepared from the precursor poly(HPMA-co-Ma- GFLG-TT) converted to poly(HPMA-co-Ma-GFLG-TT-co-Ma-GFLG-DBCO) by reaction with 0.5 - 4 mol% DBCO-NH2 in the presence of DIPEA base (equiv. to DBCO-NH2). After binding the fluorophore, see Example 8, the azide derivative of the peptide was attached by an metal-free click reaction in DMA.
  • Peptide-targeted conjugates with a pH-sensitive bond were prepared as in Example 9, and further DBCO- NHS was attached to the remaining hydrazide groups in the presence of DIPEA base (equiv. to DBCO- NHS) in methanol. The azide derivative of the peptide was then attached to the polymeric conjugate by a click reaction via DBCO.
  • Antibody-targeteded conjugates were prepared from the semitelechelic precursor poly(HPMA-co-Ma- Pentyl-NH-NH-Boc)-TT, terminated with a reactive TT group prepared using the transfer agent TT-CTA as in Example 4.
  • the end TT group was converted to maleimide by reaction with N-(2- aminoethyl)maleimide, which was used, after deprotection of the Boc protecting group and attachment of the fluorophore, for reaction with the reduced antibody rituximab.
  • the antibody was reduced by gentle reduction with dithiothreitol in PBS buffer and after reduction, it was purified from free dithiothreitol on a PD 10 column. It was subsequently mixed with the polymeric conjugate bearing a hydrazone-linked fluorescent label and a maleimide end group.
  • the prepared targeted conjugates see Table 2, were characterised, the dye content was determined and the peptide/antibody content was determined through amino acid analysis.
  • the molar mass could not be measured due to the interaction of the laser used in the GPC detectors with the fluorophore, which made it impossible to determine these characteristics.
  • the activation of the fluorescent signal can be used to increase the fluorescence within the tumour tissue in tumour cells, where after passive or active accumulation, the fluorophore is released either due to the reduced pH of the tumour tissue or due to the activity of lysosomal enzymes after the conjugates enter the tumour cells.
  • This activation of the signal subsequently leads to a significant increase in the contrast of the fluorescent signal in the tumour environment and beyond.
  • the increase in contrast can be used within the framework of navigated tumour surgery, when the improved tumour/non-tumour tissue contrast will allow the surgeon to clearly highlight the tumour mass and, therefore, to perform a precise resection of this tissue.
  • the release of fluorophores was measured by HPLC. Fluorescent polymers with a hydrazone -bound fluorophore were incubated in a phosphate buffer (0.3M, pH 7.4) with a concentration of 0,1 g-dnT 3 at 37 °C. At 0, 14, 1, 2, 4, 6, 8 and 10 hours, the amount of released fluorophore was measured. From Fig. 2 it can be seen that the release rate of the fluorophore is dependent on the pH and the type of linker. Cleavage at physiological pH 7.4 is slower than cleavage at acidic pH, simulating tumour tissue.
  • Example 15 In vivo testing
  • mice HslCpb:NMRI-Foxnl nu bearing DLD-1 colorectal tumour.
  • the mice were examined using a non-invasive imaging system, Fig. 4.

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Abstract

The present invention relates to a fluorescently labelled polymer, to which a fluorescent label is bound in an amount of from 0.1 to 10 mol %, related to the total number of monomer units. The fluorescence of said polymer is activated in tumour tissue, therefore a significant visualization of tumour tissue is provided. It may therefore be used in diagnostic methods or image-guided surgery. Preferably, the fluorescently labelled polymer is targeted towards tumour cells and tumour neovasculature using specific targeting groups. The present invention further relates to methods of preparation of the fluorescently labelled polymer, and its use as a fluorescence probe in diagnostic methods as a contrast agent for tumour visualization, whole body imaging and/or for image-guided surgery.

Description

Fluorescently labelled polymer for tumour visualization, method of its preparation and use thereof
Field of Art
The present invention relates to a targeting fluorescent polymer with activatable fluorescence composed of a polymeric carrier with side chains containing fluorophores, which is intended to visualise tumour tissue. The use of the polymer is aimed at highlighting the tumour tissue for subsequent surgical removal or non- invasive monitoring of the cancer disease progression.
Background Art
Precise and complete resection of the entire tumour without unnecessary removal of adjacent healthy tissue is a prerequisite for a successful outcome of oncological surgery. Unfortunately, the visual distinction between malignant and healthy tissue with the naked eye is often complicated, if not impossible, for the surgeon. However, radical resection with adequate negative margins is one of the most important factors influencing the prognosis of patients with, for example, head and neck squamous cell carcinoma (HNSCC). Usually, the safe margin is defined as greater than 5.0 mm. In the context of oncological endoscopic surgery, accurately delineating tumour boundaries remains a key issue. At the moment, it is possible to partially use non-invasive imaging, such as autofluorescence (AF) of tumour tissues, for imaging tumour margins. However, the AF method is inaccurate and only used for certain types of cancer. Currently, no method works reliably to guide the surgeon during surgery. Fluorescence imaging (FI) is based on the illumination of tissue with light that excites near-infrared (NIR) or shortwave -infrared (SWIR) fluorophores or contrast agents. Due to the permeability of light in the NIR and SWIR regions through tissues, up to several cm, it is possible to use these fluorophores for visualisation as part of marking tumour foci.
In the past few decades, various polymeric materials have been studied as biocompatible, non-immunogenic and non-toxic biomaterials for medical applications. Biomaterials designed as vectors for transporting biologically active molecules and materials for tissue engineering and diagnostics have been and are currently being studied. Systems for the targeted transport and controlled release of drugs, so-called drug delivery systems (DDS), are intensively researched. In their framework, polymeric drugs are studied as new formulations of ‘classical’ medications to provide an increased therapeutic function for antineoplastic, anti- inflammatory and antimicrobial treatment while eliminating side effects of treatment. Many described DDSs are based on water-soluble or amphiphilic polymeric carriers carrying low-molecular-weight active molecules, for example, covalently bound by biodegradable linkers, intended for controlled release and activation of the carried active molecules in desired tissues or cells. Much attention has been focused on biocompatible, non-toxic and non-immunogenic copolymers based on N-(2 -hydroxypropyl) methacrylamide (pHPMA), polyethylene glycol) (PEG), poly(caprolactone) (PCE), poly(lactic acid)/poly(lactide-co-glycolide acid) (PEA/PEGA) and their copolymers as carriers of biologically active molecules and targeting groups. In general, polymeric carriers of bioactive molecules are designed to optimise the pharmacokinetics of active molecules, prolong their blood circulation time, improve localisation in tumours, inflammations and target cells, reduce secondary toxicity and immunogenicity, and solubilise water-insoluble active molecules. The active molecules carried can be released/activated by enzymatic hydrolysis, reduction, or pH-induced hydrolysis by lowering the pH from 7.4 (blood) to 5-6.5 (endosomes/lysosomes; tumour microenvironment; inflammatory environment). To increase accumulation in the target tissue, for example, tumour tissue, while minimising side effects, targeted systems containing targeting groups, for example, antibodies or oligopeptides, or systems having a structure and size enabling ‘passive’ targeting based on the EPR (enhanced permeability and retention) effect using high-molecular- weight (HMW) carriers, for example, star-shaped polymers, nanoparticles or micelles, are studied. Recently, pHPMA-based polymeric therapeutics and diagnostics have been developed that have increased accumulation in solid tumours. These systems, therapeutics carrying anti-tumour drugs, such as doxorubicin and paclitaxel, have been found to have anti -cancer activity in various tumour models in mice. Polymeric systems containing both the drug and a bound fluorescent label were not only therapeutically active but were able to monitor tumour development using fluorescence imaging (FI). Linear pHPMA constructs containing NIR dye Dyomics-633 and EGF (epidermal growth factor) targeting oligopeptides GE -7 and GE- 11 were described not long ago. The in vivo biodistribution study showed very promising results in tumour visualisation. The polymeric probe was mainly localised at the tumour border, showing the potential for image-guided surgery. Recently, high-molecular-weight delivery systems for fluorescence imaging and tumour surgery have been described, for example, in WO 2020/245447 Al (fluorescent polypeptides). These high molecular weight systems are used for imaging tumour tissue. Although they meet the condition of biocompatibility and can be excreted from the body, none bring a selective increase in fluorescence locally in the tumour tissue.
Disclosure of Invention
The present invention relates to a targeting fluorescent polymer, which forms a polymeric probe capable of activating a fluorescent signal at the tumour site. The fluorescent polymer according to the present invention enables targeted accumulation in tumour tissue and subsequent activation of a fluorescent signal to visualise tumours. The polymeric system itself contains a polymeric carrier, a fluorescent label linked to the polymeric carrier by a bond that is hydrolytically, enzymatically or reductively degradable in the tumour environment, alternatively further containing a targeting group, which directs the polymer to the tumour or endothelial cells and possibly further containing a fluorescence quencher. The resulting fluorescent polymer makes it possible to amplify the signal and achieve a significant tumour/healthy tissue contrast around the tumour by activating the fluorescence of the transported label only in the tumour tissue. While the fluorescence yield of the fluorescent label drops significantly after binding the fluorophore to the polymeric carrier because of self-quenching of fluorescent molecules localised in the vicinity due to non-radiative energy transfer between the fluorophores, after the release of the label from the polymeric carrier in the tumour tissue, the fluorescence signal restores, leading to a significant increase in the signal versus noise ratio in the tumour environment. This surprising effect of increasing the fluorescence signal contrast between healthy and tumour tissue has not been described before. Copolymers based on A-(2- hydroxypropyl)methacrylamide (HPMA) are suitable precursors for preparing the described fluorescent polymers with activatable fluorescence. Functional groups are introduced along the chain of this copolymer, to which suitable fluorophore derivatives can be attached via biodegradable linkers and targeting groups. The entire system according to the present invention thus enables increased visualisation of tumour foci for subsequent surgical resection navigated by a fluorescent signal. Another use is the application of fluorescent polymers to monitor the progression and regression of cancer during treatment with another therapy. It is preferred to use fluorescent polymers for tumours that are not localised in the depth of the tissue or that require endoscopic techniques, for example, head and neck cancer, breast cancer, colorectal tumours and melanomas.
The object of the present invention is a fluorescent polymer suitable for tumour visualisation, which contains a semitelechelic statistical linear copolymer, wherein the semitelechelic statistical linear copolymer is selected from the group comprising poly(A-(2- hydroxypropyljmethacrylamide), polyacrylamide, polymethacrylamide, polyacrylate and polymethacrylate in which from 0.1 to 10 mol% monomer units, preferably from 0.4 to 6 mol% monomer units, more preferably from 0.5 to 4 mol% of monomer units, based on the total number of monomer units, are statistically replaced by monomer units of the general formula (I)
Figure imgf000004_0001
fluorophore
(I), wherein
A is selected from the group consisting of a linear or branched carbon alkylenyl chain having from 1 to 7 carbons ((Cl to C7)alkylenyl); -(CH2)p-(C(O)-NH-(CH2)r)P-;
-(CH2)P-(C(O)-NH-(CH2)r)P-C(O)-; -(CH2)P-(C(O)-NH-(CH2)r)P-C(O)-NH-C6H4-CH2-O-C(O)-; and - (CH2)P-C(O)-NH-(CH2)P-L-(CH2)P-(C(O)-NH-(CH2)r)P-C(O)-NH-C6H4-CH2-O-C(O)-, wherein L is a linker containing a triazole bridge, formed, for example, by the reaction of a propargyl or DBCO group with an azide group; thus, L can have, for example, the following structure:
Figure imgf000005_0001
wherein p is an integer in the range of from 1 to 5, and r is selected from 1, 2 and 3; wherein one or more hydrogen atoms in the CH2 groups of the substituent A may be further substituted with the same or different side chains of a natural amino acid; preferably, these side chains are selected from methyl, isopropyl, isobutyl, -CH(CH3)(CH2CH3), -CH2OH,
-CH(OH)(CH3), -CH2-(C6H4)OH, -(CH2)2-S-CH3, -CH2SH, -(CH2)4-NH2, -CH2COOH,
-CH2C(O)NH2, -(CH2)2COOH, -(CH2)2C(O)NH2, -(CH2)3NH-C(=NH)(NH2),
-(CH2)3NH-C(=O)(NH2), benzyl;
B is selected from the group consisting of a bond,
Figure imgf000005_0002
and wherein the fluorophore (fluorophore or its amino derivative, NCS ester or A-hydroxysuccinimidyl derivative) has molecular weights in the range of from 350 to 1,500 g/mol, excitation wavelengths in the range of from 300 to 850 nm, and emission wavelengths in the range of from 350 to 1,200 nm, and is
I
/C. covalently bound to the =CH-,
Figure imgf000005_0003
or -S-S- group of the substituent B of the monomer unit of the general formula I of the semitelechelic statistical linear copolymer via its primary amino group, or NCS group, A-hydroxysuccinimidyl group, keto group, aldehyde group or a disulphide group, whereby the groups formed after the binding of the fluorophore are subsequently part of the group B; with the proviso that when B is a bond, then A is -(CH2)p-(C(O)-NH-(CH2)r)p-C(O)-; or
-(CH2)p-(C(O)-NH-(CH2)r)p-C(O)-NH-C6H4-CH2-O-C(O)-; or -(CH2)p-C(O)-NH-(CH2)p-L-(CH2)p-(C(O)-NH-(CH2)r)p-C(O)-NH-C6H4-CH2-O-C(O)-; and wherein the molecular weight Mn of the fluorescent polymer is in the range of from 6,000 to 100,000 g/mol, preferably from 10,000 to 60,000 g/mol, more preferably from 15,000 to 40,000 g/mol (corresponding to 100 to 280 monomer units), more preferably from 20,000 to 30,000 g/mol (corresponding to 134 to 210 monomer units).
The fluorophore is thus attached to the side chain of the semitelechelic statistical linear copolymer via a labile hydrazone or disulphide bond or a peptide linker (Val-Cit (-NH-CH(C(CH3)2)-C(=O)-NH- CH((CH2)3-NH-C(=O)-NH2)-C(=O)-), Val-Cit-Aba linker (-NH-CH(C(CH3)2)-C(=O)-NH-CH((CH2)3- NH-C(=O)-NH2)-C(=O)-NH-C6H4-CH2-O-C(=O)-), which is hydrolytically, enzymatically and/or reductively degradable in the tumour environment. The fluorescent polymer according to the present invention enables targeted accumulation in tumour tissue and subsequent activation of the fluorescent signal for the visualisation of tumours. The fluorophore is bound in the monomer unit (I) by a bond that is hydrolytically, enzymatically or reductively degradable in the tumour environment, which enables the amplification of the signal and the achievement of a significant tumour/healthy tissue contrast around the tumour by activating the fluorescence of the transported label only in the tumour tissue. While the fluorescence yield of the fluorescent label drops significantly after binding the fluorophore to the polymeric carrier because of self-quenching of fluorescent molecules localised in the vicinity due to non-radiative energy transfer between the fluorophores, after the release of the fluorophore from the polymeric carrier in the tumour tissue, the fluorescence signal restores, leading to a significant increase in the signal versus noise ratio just in the tumour environment. This effect allows the claimed compounds to be used in fluorescence-guided surgery.
Natural amino acids shall be understood as being histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, arginine, cysteine, glutamine, glycine, proline, tyrosine, alanine, aspartic acid, asparagine, glutamic acid, serine, selenocysteine. Side chains are chains attached to the alpha-carbon of an amino acid.
The term ‘alkylenyl’ means a divalent linear or branched carbon chain, for example, -CH2-,
— (CH2)2 — , -(CH2)5- or -(CH2)2-(CH(CH3))-CH2- formed from an alkane by the removal of two hydrogen atoms located on two different carbon atoms.
The term ‘alkyl’ means a single-bond linear or branched carbon chain formed from an alkane by the removal of one hydrogen atom.
Ma stands for methacrylamide.
Ma-AP-TT stands for 3-(3-methacrylamidopropanoyl)thiazolidine-2-thione.
Ma-AP stands for 3-(3-methacrylamidopropanoyl).
Ma-Acap stands for 6-methacrylamidohexanoic acid.
Ma-Acap-NH-NH2 stands forA-(6-methacrylamidohexanoyl)hydrazineMa-Acap-NH-NH-Boc stands for /V-(tert-butoxycarbonyl)-JV’-(6-methacrylamidohexanoyl)hydrazine.
TT stands for thiazolidine-2-thione.
DBCO stands for dibenzocyclooctyne.
Ma-AP-DBCO stands for 3-(3-methacrylamidopropanoyl)dibenzocyclooctyne.
GG stands for dipeptide glycylglycine.
GFG stands for glycylphenylalanylglycine tripeptide.
OPB stands for 4-(2-oxopropyl)benzenecarboxylic acid.
OPB-Cy7 stands for Cy-7 fluorophore modified with OPB acid. Aba stands for aminobenzyl alcohol.
The Az-Val-Cit-Aba-DY-676 structure indicates the Dyomics-676 fluorophore bound to the Az-Val-Cit- Aba peptide chain.
GFLG-DY-676 stands for the fluorophore DY-676 linked to the tetrapeptide GFLG (SEQ. no. 8), i.e. Gly- Phe-Leu-Gly.
COP stands for 5 -cyclohexyl-5 -oxopentanoic acid.
COP-DY-676 indicates the COP to which the DY-676 fluorophore is bound.
£> stands for a dispersity, calculated as a ratio of weight average by number average molecular weight.
The term ‘fluorophore’ means an organic molecule with at least one aromatic ringcapable of fluorescence, attached to the substituent B by a covalent bond. A fluorophore is a fluorescent label or a derivative thereof, containing an amino group, a keto group, or an S-S bond, added to the fluorophore structure by reacting the fluorescent label with a selected oxoacid or reagent containing an activated disulphide, for example, by reacting with 3-(2-pyridyldithio)propionate, 5 -cyclohexyl-5 -oxopentanoic acid, 4-(2- oxopropyl)benzenecarboxylic acid and 4-oxo-4-(2-pyridyl)butanoic acid. After binding the fluorophore to the substituent B of the general formula (I), the groups formed by this reaction are part of the group B defined above.
In a preferred embodiment, the fluorophore is selected from the group comprising (2E)-2-[(E)-3-(7-azaniumylidene-2-tert-butylchromen-4-yl)prop-2-enylidene]-l-[6-(2,5-dioxopyrrolidin- l-yl)oxy-6-oxohexyl]-3,3-dimethylindole-5-sulphonate (DY-615), sodium 2-[3-[2-tert-butyl-7-[ethyl(3- sulphonatopropyl)azaniumylidene]chromen-4-yl]prop-2-enylidene]-l-(5-carboxypentyl)-3,3- dimethylindole-5 -sulphonate (DY-633), sodium 3-(3-carboxypropyl)-2-[3-(9-ethyl-6,8,8-trimethyl-2- phenylpyrano [3 ,2-g]quinolin-4-ylidene)prop- 1 -enyl] -3 -methyl- 1 -(3 -sulphonatopropyl)indol- 1 -ium-5 - sulphonate (DY-676), sodium 3-(3-carboxypropyl)-2-[3-[8,8-dimethyl-2-phenyl-6-(sulphonatomethyl)-9- (3 -sulphonatopropyl)pyrano [3 ,2-g] quinolin-4-ylidene]prop- 1 -enyl] -3 -methyl- 1 -(3 - sulphonatopropyl)indol-l -ium-5 -sulphonate (DY-678), 2-[5-(2-tert-butyl-7- diethylazaniumylidenechromen-4-yl)penta-2,4-dienylidene] - 1 -(5 -carboxypentyl) -3 ,3 -dimethylindole-5 - sulphonate (DY-730), sodium 2-[5-(2-tert-butyl-9-ethyl-6,8,8-trimethylpyrano[3,2-g]quinolin-4- ylidene)penta- 1 ,3 -dienyl] - 1 -(5 -carboxypentyl) -3 ,3 -dimethylin-dol- 1 -ium-5 -sulphonate (DY -750), sodium 3-(3-carboxypropyl)-2-[5-(9-ethyl-6,8,8-trimethyl-2-phenylpyrano[3,2-g]quinolin-4-ylidene)penta-l,3- dienyl] -3 -methyl- 1 -(3 -sulphonatopropyl)indol-l -ium-5 -sulphonate (DY -776), sodium 2- [5 -[4-tert-butyl-7 - [ethyl(3-sulphonatopropyl) azaniumylidene]chromen-2-yl]penta-2,4-dienylidene]-3-(3-carboxypropyl)-3- methyl-l-(3-sulphonatopropyl)indole-5-sulphonate (DY-782), l-[2-[dimethoxy(phenyl)methyl] phenoxy]- 3,4-diphenylisoquinolin (CY-7), lH-benz[e]indolium, 2-[2-[3-[2-(l,3-dihydro-l,l,3-trimethyl-2H-benz[e] indol-2-ylidene)ethylidene] - 1 -cyclohexen- 1 -yl] ethenyl] -3 -[6-[(2,5 -dioxo- 1 -pyrrolidi-nyl)oxy] -6- oxohexyl]- 1,1 -dimethylisoquinoline (CY-7.5), sodium 4-[(2E)-2-[(2E,4E,6E)-7-[l,l-dimethyl-3-(4- sulphonatobutyl)benzo[e]indol-3-ium-2-yl]hepta-2,4,6-trienylidene]-l,l-dimethyl-benzo[e]indol-3- yl]butane-l -sulphonate (ICG), 2,3,5-trichloro-4-[2-[[6-(2,5-dioxopyrrolidin-l-yl)oxy-6-oxohexyl]amino]- 2-oxoethyl]sulphanyl-6-[6,7,7,19,19,20-hexamethyl-9,17-bis(sulpho-natomethyl)-2-oxa-20-aza-6- azoniapentacyclo [12.8.0.03,12.05, 10.016,21]docosa-l(14),3,5,8, 10,12,15,17,21-nonaen-13-yl]benzoate (Alexa Fluor 610), or their derivatives containing a functional group selected from an amino group, an isothiocyanate group and an A-hydroxysuccinimide group. Optionally, fluorophores are modified with oxoacids using the amide reaction of the amino group of the fluorophore with the activated carboxyl of the oxoacid, the resulting product being an oxo derivative of the fluorophore. Optionally, the fluorophore is modified with cystamine using an amide bond between the cystamine and the fluorophore. Optionally, the fluorophore is modified with A-(5-azidopentanoyl-valyl-citrulyl)-4-aminobenzyl (4-nitrophenyl) carbonate or with N-(5- azidopentanoyl -valyl -citrulyl) by reaction with its amino group.
An example of a fluorophore that can be successfully attached to group B of general formula (I) are commercially available:
DY -782, DY-782-NHS ester, DY -728- carboxylic acid, DY-728-maleimide, DY-728-amine, DY -728- azide, DY-676, DY-676-NHS ester, DY -676- carboxylic acid, DY-676-maleimide, DY-676-amine, Cy7, Cy7-NHS ester, Cy7-amine, Cyl, 5, Cy7,5-NHS ester, Cy7,5-amine, Cy7,5-azide. The fluorophore can be attached to group B via an oligopeptide linker of 2 to 5 amino acids that can be enzymatically degradable by lysosomal enzymes, a pH-sensitive hydrolytically degradable hydrazone bond or a reductively biodegradable disulphide bond, which enables the controlled release of the fluorophore in the tumour environment. An oligopeptide linker of 2 to 5 amino acids can be, for example, Val-Cit or Val-Cit-Aba.
The end groups of the resulting fluorescent polymer contain residues of the polymerisation initiator and transfer agent molecules. The initiator can be, for example, 2,2'-azobis[A-(2-carboxyethyl)-2- methylpropionamidine] (V-70)) and the transfer agent can be, for example, A-(3-azidopropyl)-4- ethylsulphanylcarbothioylsulphanyl-4-methyl-pentanamide (CTA-N3)), or their derivatives formed by reaction with, for example, 2,2'-azobisisobutyronitrile (AIBN). The end groups of the copolymer, therefore, contain an azide group, which can subsequently be further modified by reaction with dibenzocyclooctyne - maleimide (DBCO-MI) to another reactive maleimide functional group, by reaction with dibenzocyclooctyne-carboxyl (DBCO-Cbx), which is subsequently modified with thiazolidine-2-thione to the thiazolidine-2-thione reactive functional group, by reaction with dibenzocyclooctyne -N- hydroxysuccinimidyl ester (DBCO-NHS) to the A-hydroxysuccinimidyl ester reactive functional group.
During biodistribution, the fluorescent polymer defined above is directed to the tissues of solid tumours due to its high molecular weight and the longer circulation time during which the fluorescent polymer accumulates in solid tumours (due to EPR effect). In one embodiment, substituent A of the fluorescent polymer is selected from the group consisting of a linear or branched carbon alkylenyl chain having from 1 to 7 carbons; and
-(CH2)p-(C(O)-NH-(CH2)r)p-, wherein p is an integer in the range of from 1 to 5, and r is selected from 1, 2, and 3; and substituent B is selected from the group consisting
Figure imgf000009_0001
-S-S-. Said combination leads to a linker structural segment -A-B-, connecting the fluorophore to the polymeric carrier, which has a formula selected from:
-(CH2)Z-C(=O)-NH-N=CH-; -(CH2)Z-C(=O)-NH-N=C<; -(CH2)Z-S-S-; wherein z is an integer in the range of from 1 to 7. This type of linker -A-B- is degradable in an acidic environment (for example, in tumour tissue).
Furthermore, said combination also leads to a linker structural segment -A-B-, connecting the fluorophore to the polymeric carrier, which has a formula selected from:
-(CH2)p-(C(O)-NH-(CH2)r)p-C(=O)-NH-N=CH-; -(CH2)p-(C(O)-NH-(CH2)r)p-C(=O)-NH-N=C<; a - (CH2)p-(C(O)-NH-(CH2)r)p-S-S-; where p is an integer in the range of from 1 to 5, and r is selected from 1, 2 and 3. This type of linker -A-B- is degradable in an acidic environment (for example, in tumour tissue), in the reductive environment of the cytoplasm of tumour cells, and, at the same time, the peptide character of the linker enables its enzymatic degradation.
In one embodiment, substituent A of the fluorescent polymer is selected from the group consisting of -(CH2)p-(C(O)-NH-(CH2)r)p-C(O)-NH-C6H4-CH2-O-C(O)-; and -(CH2)p-C(O)-NH-(CH2)p-L-(CH2)p-(C(O)-NH-(CH2)r)p-C(O)-NH-C6H4-CH2-O-C(O)-, wherein L is a linker containing a triazole bridge; p is an integer in the range of from 1 to 5, and r is selected from 1, 2 and 3; and substituent B is a bond. Said combination leads to a linker structural segment -A-B-, connecting the fluorophore to the polymeric carrier, which has the formula: -(CH2)p-(C(O)-NH-(CH2)r)p-C(O)-NH-C6H4-CH2-O-C(O)-; or -(CH2)p-C(O)-NH-(CH2)p-L-(CH2)p-(C(O)-NH-(CH2)r)p-C(O)-NH-C6H4-CH2-O-C(O)-.
This type of linker -A-B- is enzymatically degradable.
In one embodiment, the fluorescent polymer further contains targeting groups to enhance the targeting of the fluorescent polymer to tumour tissues. These targeting groups can be attached to the end groups of the linear chain of the fluorescent polymer and/or they can be attached to the side chains.
The targeting group for directing the fluorescent polymer to tumour cells of head and neck tumours, breast tumours, melanomas and colorectal tumours or to tumour endothelial cells is selected from the group comprising oligopeptides with the number of amino acids from 3 to 20, and proteins, preferably antibodies, more preferably monoclonal antibodies targeting tumour cells of head and neck tumours, breast tumours, melanomas and colorectal tumours or tumour endothelial cells.
In a preferred embodiment, oligopeptides withthe number of amino acids from 3 to 20 are selected from the group comprising YESIKVAVS (SEQ. no. 1), SIGYPLP (SEQ. no. 2), RGD (tumour endothelium), CPLHQRPMC (SEQ. no. 3; prostate tumours), NPVVGYIGERPQYRDL (SEQ. no. 4) called GE7 and YHWYGYTPQNVI (SEQ. no. 5) called GE11 (to the EGF receptor), WHYPWFQNWAMA (SEQ. no. 6; head and neck tumour cells), DMPGTVLP (SEQ. no. 7; breast tumours), CNGRC (SEQ. no. 10), cyclic RGDfK (SEQ. no. 13), HEWSYLAPYPWF (SEQ. no. 11) and SYSMEHFRWGKPV (SEQ. no. 12). Oligopeptides and proteins that target tumour tissues in the body are known to those skilled in the art.
In a preferred embodiment, the monoclonal antibodies are selected from the group comprising herceptin, erbitux, daratuzumab, trastuzumab.
In an embodiment where the targeting groups are attached to the end groups of the linear chain of the fluorescent polymer, these targeting peptides and/or proteins are attached by conjugation of the amino group or introduced propargyl, DBCO, or SH groups of the targeting peptide/protein with an azide, maleimide, thiazolidine-2-thione, or A-hydroxysuccinimidyl ester end group of the linear chain of the fluorescent polymer.
In an embodiment where the targeting groups are attached to the side chains of the fluorescent polymer, the fluorescent polymer defined above also contains monomer units of the general formula (II)
Figure imgf000010_0001
(II), wherein A is as defined above,
Figure imgf000010_0002
, wherein L is as defined above; and Z is a targeting group for directing the fluorescent polymer to tumour cells of head and neck tumours, breast tumours, melanomas, and colorectal tumours, or tumour endothelial cells, also defined as above, and is attached to the X group via an amide bond, maleimide, azide, or propargyl. Preferably the targeting group is atached to the X group via azide or propargyl. These targeting groups are, therefore, covalently bound
I
Figure imgf000011_0001
-CH=CH-, -CH=CH- in the structure of DBCO, N3 or -S-S- group of the substituent X of the monomer unit of the general formula (II) of the semitelechelic statistical linear copolymer via its primary amino group, or an introduced azide, propargyl, DBCO or SH group, wherein the groups formed after the binding of the targeting group are subsequently part of the X group.
In this embodiment, the amount of monomer units of general formula (II) is from at least one monomer unit to 8 mol%, based on the total number of monomer units.
The total number of monomer units of the general formula (I) and (II) in the fluorescent polymer is at most 10 mol%, based on the total number of monomer units.
The targeting group may optionally be modified prior to a conjugation to the fluorescent polymer to contain at least one group selected from -NH2, -SH, sDBCO (sulphodibenzocyclooctyne group), DBCO (dibenzocyclooctyne group), azide, propargyl, to which the fluorescent polymer is covalently attached via a functional group, preferably selected from A-hydroxysuccinimidyl ester, thiozolidine-2-thione amide, maleinimide, azide, DBCO, sDBCO, or propargyl. Modifications of oligopeptides and proteins to introduce -NH2, -SH, sDBCO, DBCO or azide into the structure are known to those skilled in the art.
In a preferred embodiment, the semitelechelic statistical linear copolymer is poly(/V-(2- hydroxypropyl)methacrylamide) .
The fluorescent polymer can further contain from 0 to 9.9 mol% of monomer units of the general formula (III), based on the total number of monomer units,
Figure imgf000011_0002
(III), wherein A is as defined above and C is selected from the group comprising -S-S-(CH2)2-OH, -C(=O)-NH-(CH2)a-CH2(OH); -C(=O)-NH-(CH2)b-CH(OH)-CH3; -C(=O)-NH-(CH2)b-CH(OH)-(CH2)c- CH ,: and -NH-C(=O)-CH3, wherein a is an integer in the range of from 0 to 4, b is an integer in the range of from 0 to 3, and c is an integer in the range of from 1 to 4. In total, there are at most 10 mol% of monomer units of the general formula (I), (II) and (III) in the fluorescent polymer, based on the total number of monomer units. Preferably, substituent C is selected from the group comprising -C(=O)-NH-CH2(OH); -C(=O)-NH- CH(OH)-CH3; -C(=O)-NH-(CH2)2-CH2(OH); -NH-C(=O)-CH3.
Preferably, the fluorescent polymer contains from at least one monomer unit of the general formula (III) to 9.9 mol% monomer units of the general formula (III).
In a preferred embodiment, A is selected from the group comprising (III) ethan-l,2-diyl (-CH2-CH2-); propan- 1,3 -diyl (-CH2-CH2-CH2-); butan- 1,4, -diyl (-CH2)4-); pentan- 1,5 -diyl (-CH2)s-); hexan-l,6-diyl (- CH2)6-); heptan-l,7-diyl (-Ctftft: -CH2-C(=O)-NH-CH2- (derived from the dipeptide Gly-Gly); -CH2- C(=O)-NH-CH(CH2-CH(CH3)2)-C(=O)-NH-CH2- (derived from the tripeptide Gly-Leu-Gly); -CH2- C(=O)-NH-CH(CH2Ph)-C(=O)-NH-CH2- (derived from the tripeptide Gly-Phe-Gly); -CH2-C(=O)-NH- CH(CH2-CH(CH3)2)-C(=O)-NH-CH(CH2Ph)-C(=O)-NH-CH2- (derived from the tetrapeptide Gly-Leu- Phe-Gly); -CH2-C(=O)-NH-CH(CH2Ph)-C(=O)-NH-CH(CH2-CH(CH3)2)-C(=O)-NH-CH(CH2Ph)-C(=O)- NH-CH2- (derived from the pentapeptide Gly-Phe-Leu-Phe-Gly); -(CH2)4-C(=O)-NH-CH(C(CH3)2)- C(=O)-NH-CH((CH2)3-NH-C(=O)-NH2)-C(=O)-NH-C6H4-CH2-O- C(=O)- (derived from N-(5- azidopentanoyl-valyl-citrulyl)-4-aminobenzyl carbonyl), -(CH2)4-C(=O)-NH-CH(C(CH3)2)-C(=O)-NH- CH((CH2)3-NH-C(=O)-NH2)-C(=O)- (derived from A-(5-azidopcntanoyl -valyl -citrulyl).
Said monomer unit of the general formula (III) in the structure of the fluorescent polymer is formed during the synthesis by reactions of unreacted thiazolidine-2-thione groups, (2,3,4,5,6-pentafluorofenyl)oxy groups, (succinimidyl)oxy groups, with an amino alcohol selected from the group comprising NH2-(CH2)a- CH2(OH); NH2-(CH2)b-CH(OH)-CH3; NH2-(CH2)b-CH(OH)-(CH2)c-CH3; wherein a is an integer in the range of from 0 to 4, b is an integer in the range of from 0 to 3 and c is an integer in the range of from 1 to 4, preferably with 1 -aminopropan -2-ol, and/or by reaction of unreacted NH2 groups with acetythiazolidin- 2-thione, and/or by reaction of unreacted disulphide groups with 2 -hydroxyethanethiol; to form a group C selected from the group comprising carboxyl; hydrazide; azide; -S-S-(CH2)2-OH, -C(=O)-NH-(CH2)a- CH2(OH); -C(=O)-NH-(CH2)b-CH(OH)-CH3; -C(=O)-NH-(CH2) -CH(OH)-(CH2)c-CH3; a -NH-C(=O)- CH3, wherein a is an integer in the range of from 0 to 4, b is an integer in the range of from 0 to 3, and c is an integer in the range of from 1 to 4, preferably forming a group -C(=O)-NH-CH2(OH), -C(=O)-NH-CH(OH)-CH3, -C(=O)-NH-(CH2)2-CH2(OH) or -NH-C(=O)-CH3.
Thiazolidine-2-thione groups, (2,3,4,5,6-pentafluorophenyl)oxy groups, (succinimidyl)oxy groups, NH2 groups and disulphide groups were originally intended to react with the fluorophore, but if they did not all react with the fluorophore, they are converted to groups C as defined above. Group C is, therefore, present in the fluorescent polymer as a residue after the removal of reactive groups that did not react with the fluorophore to form a monomer unit of the general formula (I). In one embodiment, the fluorescent polymer is a linear statistical copolymer of HPMA and monomer units of general formula (I), and optionally (II) and/or (III). This linear statistical copolymer can be represented by a general formula (IV), wherein the molecular weight Mn of the statistical linear copolymer is in the range of from 6,000 to 100,000 g/mol, preferably in the range of from 15,000 to 45,000 g/mol (corresponding to 100 to 300 monomer units), more preferably the molecular weight is in the range of from 20,000 to 40,000 g/mol (corresponding to 134 to 280 monomer units);
Figure imgf000013_0001
CH3 \ Xx C fluorophore z
(IV), wherein A, B, C and Z are as defined above, wherein the total number of monomer units in the fluorescent polymer is in the range of from 100 to 300, the content of units of general formula (I) in this statistical linear copolymer is from 0. 1 to 10 mol%, based on the total number of monomer units, which corresponds to 1 to 30 monomer units of the general formula (I); and the content of units of general formula (II) in this statistical linear copolymer is from 0 to 8 mol%, based on the number of monomer units, which corresponds to from 0 to 24 monomer units of general formula (II); and the content of units of general formula (III) in this statistical linear copolymer is from 0 to 9.9 mol%, based on the number of monomer units, which corresponds to from 0 to 29 monomer units of the general formula (III), wherein the total content of monomer units of the general formula (I) and (II) and (III) is at most 10 mol%, based on the total number of monomer units, which corresponds to a maximum total number of 30 units of formula (I) + formula (II) + formula (III).
The remaining monomer units (from about 270 to 298 monomer units) are HPMA monomer units. The end groups of the resulting linear copolymer of the general formula (IV) contain parts of the polymerisation initiator and transfer agent molecules as mentioned above.
In a preferred embodiment, the general formula (IV) contains from 1 to 30 monomer units of the general formula (I), from 1 to 24 monomer units of the general formula (II) and from 1 to 29 monomer units of the general formula (III), wherein the maximum total number of monomer units of formula (I) + formula (II) + formula (III) being 30. The remaining monomer units are HPMA monomer units.
The fluorescent polymer is a semitelechelic statistical linear copolymer that can be prepared by radical polymerisation. The end groups of the resulting linear copolymer are blocked with, for example, 2,2' - azobisisobutyronitrile (AIBN). Another object of the present invention is, therefore, also a method of preparing the fluorescent polymer (fluorescent polymer with activatable fluorescence and possibly with a bound targeting group), which includes the following steps:
(a) providing monomers of the fluorescent polymer,
(b) polymerisation of these monomers,
(c) binding the fluorophore to the statistical linear copolymer,
(d) optionally binding the targeting structure to the fluorescent polymer, wherein the targeting structure is selected from the group comprising oligopeptides with the number of amino acids from 3 to 20 and proteins, especially monoclonal antibodies; wherein the targeting structure can be attached to the end of the linear chain and/or to the side chains of the fluorescent polymer (as part of the monomer unit of general formula (II)).
Ad (a) Providing monomers of the linear copolymer comprises providing monomers selected from the group comprising A-(2-hydroxypropyl)methacrylamide (HPMA), acrylamide, methacrylamide, acrylate and methacrylate, and monomers of the general formula (V)
Figure imgf000014_0001
(V), which are the precursors for the monomer units of general formulae (I), (II) and (III), wherein A is as defined above; and D is -carbonyl-thiazoline-2-thione group (TT), (4-nitrophenyl)oxy group, (2, 3, 4,5,6- pentafluorophenyljoxy group, (succinimidyl)oxy group, carboxyl, hydrazide, azide, activated disulphide group or NH2 group. The amino group and the hydrazide group may optionally be protected by a protecting group, for example, a tert-butoxycarbonyl (Boc) protecting group. Carbonyl means a -C(=O)- group.
A-(2-hydroxypropyl)methacrylamide (HPMA) and other monomers of the acrylamide, methacrylamide, acrylate, and methacrylate types are commercially available. The provision of these monomers, therefore, means their commercial acquisition.
Substances of the general formula (V) were either obtained from commercially available sources, for example, methacryloyl-6-aminopropyl amine protected on the amine groups by tert-butyloxycarbonyl groups (MA-Pr-NH-Boc), or were prepared according to procedures reported in the literature (Etrych T., et al., A-(2-Hydroxypropyl)methacrylamide-Based Polymer Conjugates with pH-Controlled Activation of Doxorubicin. I. New Synthesis, Physicochemical Characterization and Preliminary Biological Evaluation, J.Appl.Pol.Sci. 109, 3050-3061 (2008), V. Subr, et al., Synthesis and properties of new /V-(2- hydroxypropyl)-methacrylamide copolymers containing thiazolidine-2-thione reactive groups, React. Funct. Polym. 66 (12) (2006) 1525-1538). The provision of substances of general formula (V), therefore, means either their commercial acquisition or synthesis.
Ad (b) Polymerisation of the monomers of the statistical linear copolymer is carried out by controlled radical RAFT (reversible addition-fragmentation chain-transfer) polymerisation of monomers from step (a) with a content of from 0.1 to 10 mol% of monomers of the general formula (V), and at least 90 mol% (90 to 99.9 mol%) of monomer units, selected from the group comprising JV-(2-hydroxypropyl)methacrylamide (HPMA), acrylamide, methacrylamide, acrylate, methacrylate, preferably selected from the group comprising HPMA, acrylamide, methacrylamide, most preferably HPMA.
The reaction typically takes place at a temperature ranging from 30 to 100 °C, preferably from 40 to 80 °C, and a solvent preferably selected from the group comprising dimethylsulphoxide, dimethylacetamide, dimethylformamide, methanol, ethanol, dioxane, tetrahydrofuran, propanol, tert-butanol or their mixture.
The reaction is initiated by an initiator, preferably selected from the group comprising in particular azoinitiators 2,2'-azobis(2-methylpropionitril) (AIBN), 4,4'-azobis(4-cyanopentanoic acid) (ACVA), 2,2'- azobis(4-methoxy-2,4-dimethylpentannitril) (V70), optionally in the presence of a transfer agent, preferably selected from the group containing 2-cyano-2-propylbenzodithioate, 4-cyano-4- (thiobenzoylthio)pentanoic acid, 2-cyano-2-propyldodecyl-trithiocarbonate, 2-cyano-2- propylethyltrithiocarbonate, 4-cyano-4-[(dodecylsulphanylthio-carbonyl)sulphanyl]pentanoic acid, [4-(3 - azidopropylamino) - 1 -cyano- 1 -methyl -4 -oxobutyl] benzene -carbodithioate , N-(3 -azidopropyl) -4 -cyano-4- ethylsulphanyl-carbothioylsulphanyl-pentanamide, [l-cyano-l-methyl-4-oxo-4-(2-thioxothiazolidin-3- yl)butyl]benzencarbodithioate and 2-ethylsulphanylcarbothioyl-sulphanyl-2-methyl-5-oxo-5-(2-thioxo- thiazolidin-3-yl)pentan-nitrile. The molar mass Mn of the linear statistical copolymers thus prepared is in the range of from 4,000 to 100,000 g/mol, preferably from 25,000 to 50,000 g/mol. The resulting semitelechelic linear copolymer contains end reactive groups (for example, azide and TT).
The prepared linear copolymer from step (b) can optionally be further subjected to the removal of protecting groups protecting the hydrazide groups of the side chains (for example, Boc groups). Deprotection can be accomplished by established procedures known to those skilled in the art, such as the removal of the Boc group with trifluoroacetic acid or by heating the copolymer in water. The resulting copolymer/product can be stored without the risk of its decomposition.
The product of this step is thus a semitelechelic statistical linear copolymer, which contains a linear polymer selected from the group comprising polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate and poly( '-(2 -hydroxyp ropy I (methacryl am ide), in which from 0.1 to 10 mol% of monomer units are statistically replaced by a monomer unit of the general formula (VI)
Figure imgf000016_0001
(VI), wherein A and D are as defined above.
Ad (c) binding the fluorophore to D group of the statistical linear copolymer from step (b) is carried out by conjugation of carbonyl -thiazoline -2 -thione groups, (4-nitrophenyl)oxy groups, (2, 3, 4,5,6- pcntafluorophcnyljoxy groups, (succinimidyl)oxy groups, carboxyl, hydrazide, azide, activated disulphide groups and NH2 groups (while the amino group and the hydrazide group, if protected by a protecting group in the previous step, must be deprotected before this conjugation) of monomer units of the general formula (VI) of the semitelechelic statistical linear copolymer from step b), with a low-molecular-weight fluorophore. The fluorophore is a fluorescent label or a derivative thereof that contains suitable reactive groups (for example, an amine, carboxyl, activated carboxyl, activated disulphide or keto group) and can be used in free form or the form of a salt with an acid, for example, HC1; wherein the low-molecular-weight fluorophore is a fluorophore having a molecular weight in the range of from 350 to 1,500 g/mol. The fluorophore is selected according to excitation and emission wavelengths, with excitation wavelengths ranging from 300 to 850 nm and emission wavelengths ranging from 350 to 1,200 nm; the low- molecular- weight fluorophore molecule is bound to the linear copolymer by an amide, disulphide or hydrazone bond. The product of this step is thus a semitelechelic statistical linear copolymer containing monomer units of general formula (I), (VI), and further containing monomer units selected from the group comprising acrylamide, methacrylamide, acrylate, methacrylate and /V-(2-hydroxypropyl)methacrylamide.
Ad (dl) Binding of the targeting structure to the monomer units of the general formula (VI) of the semitelechelic statistical linear copolymer from step (c) - step of conjugation of the targeting structure with introduced amino groups, SH, azide, propargyl sulphodibenzocyclooctyne (sDBCO) or dibenzocyclooctyne (DBCO) groups to the D group defined above. The targeting structure is selected from the group comprising oligopeptides with the number of amino acids from 3 to 20, and proteins, preferably antibodies, more preferably monoclonal antibodies, targeting tumour cells of head and neck tumours, breast tumours, melanomas and colorectal tumours or tumour endothelial cells, wherein oligopeptides are attached using a click reaction of the terminal azide with DBZO groups on the polymer; antibodies are linked by a click reaction of the maleimide end group of the semitelechelic fluorescent polymer with the thiol group of the antibody molecule introduced therein by mild reduction. In a preferred embodiment, oligopeptides with the number of amino acids from 3 to 20 are selected from the group comprising YESIKVAVS, SIGYPLP, RGD and cyclic RGDfK (tumour endothelium), CPLHQRPMC (prostate tumours), NPWGYIGERPQYRDL called GE7 and YHWYGYTPQNVI called GE11 (to the EGF receptor), WHYPWFQNWAMA (head and neck tumour cells), DMPGTVLP (breast tumours), CNGRC, HEWSYLAPYPWF and SYSMEHFRWGKPV. Oligopeptides and proteins that target tumour tissues in the body are known to those skilled in the art.
In a preferred embodiment, the monoclonal antibodies are selected from the group comprising herceptin, erbitux, daratuzumab, trastuzumab.
Peptide and protein structures with introduced amino, azide, sulphodibenzocyclooctyne (sDBCO) or dibenzocyclooctyne (DBCO) groups; groups of the fluorescent polymer react with -NH2, propargyl, sDBCO, or DBCO groups present on the targeting structure in the order of minutes and providing high yield, and the resulting conjugate retains its biological activity unaffected.
Ad (d2) Binding the targeting structure to the ends of the semitelechelic statistical linear fluorescent polymer from step (c) - the step of conjugation of the targeting structure with amino groups introduced by SH, azide, propargyl, sulphodibenzocyclooctyne (sDBCO) or dibenzocyclooctyne (DBCO) groups to the end group of the linear polymer, which can be maleinimidyl group, TT group, azide, propargyl, DBCO, or sDBCO. The targeting structure is as defined above.
Any unreacted D groups (thiazolidine-2-thione groups, (2,3,4,5,6-pentafluorophenyl)oxy groups, (succinimidyl)oxy groups) can be removed by reaction with amino alcohol selected from the group comprising NH2-(CH2)a-CH2(OH); NH2-(CH2)b-CH(OH)-CH3; NH2-(CH2)b-CH(OH)-(CH2)c-CH3; wherein a is an integer in the range of from 0 to 4, b is an integer in the range of from 0 to 3 and c is an integer in the range of from 1 to 4, preferably with l-aminopropan-2-ol, and/or any unreacted NH2 groups can be removed by reaction with acetylthiazolidin-2-thione, and/or any unreacted disulphide groups are removed by reaction with 2-hydroxyethanethiol; to form a group C selected from the group comprising carboxyl; hydrazide; azide; -S-S-(CH2)2-OH, - C(=O)-NH-(CH2)a-CH2(OH); -C(=O)-NH-(CH2)b-CH(OH)-CH3; -C(=O)-NH-(CH2)b-CH(OH)-(CH2)c- CH3; and -NH-C(=O)-CH3, wherein a is an integer in the range of from 0 to 4, b is an integer in the range of from 0 to 3 and c is an integer in the range of from 1 to 4, preferably to form -C(=O)-NH-CH2(OH), - C(=O)-NH-CH(OH)-CH3, -C(=O)-NH-(CH2)2-CH2(OH) or -NH-C(=O)-CH3 group. Carboxyles, hydrazides and azides do not need to be removed.
The product of this step is a fluorescent polymer according to the present invention, i.e. a semitelechelic statistical linear copolymer, containing monomer units of the general formula (I), optionally (II), and optionally (III), defined as above, and further containing monomer units selected from the group comprising acrylamide, methacrylamide, acrylate, methacrylate and /V-(2-hydroxypropyl)methacrylamide.
An object of the present invention is also a star-shaped fluorescent polymer that contains a multivalent carrier to which at least one fluorescent polymer according to the present invention is bound, wherein the multivalent carrier is selected from the group comprising poly(amidoamine)dendrimer of the second or third generation, 2,2-bis(hydroxymethyl)propion dendrimer or dendron of the second to the fourth generation; polyols with the number of hydroxyl groups from 2 to 8, glycerol, pentaerythritol, bis(2- hydroxyethyl)aminotris(hydroxy-methyl)methane, dipentaerythritol .
In one embodiment, the star-shaped fluorescent polymer is a copolymer of general formula (VII) that contains a multivalent carrier and at least one fluorescent polymer of general formula (IV), as defined above.
Figure imgf000018_0001
(VII), wherein the multivalent carrier is a second or third generation poly(amidoamine) (PAMAM) dendrimer (for example, with an ethylenediamine, 1,4-diaminobutane, 1,6-diaminohexane, 1,12-diaminododecane, cystamine core) or 2,2-bis(hydroxymethyl)propion dendrimer or dendron (for example, with a trimethylol propane core) of the second to the fourth generation; polyols with the number of hydroxyl groups from 2 to 8, for example, ethylene glycol, polyethylene glycol, trimethylolpropane (1,1,1- tris(hydroxymethyl)propane), glycerol, pentaerythritol, bis(2- hydroxyethyl)aminotris(hydroxymethyl)methane, dipentaerythritol; wherein A, B, C and Z are as defined above;
Y is selected from the group consisting of a primary amino group; primary hydroxy group; alkyne groups with the number of carbons from 3 to 6, for example, propargyl; and a cyclooctyne group, which may optionally be further independently substituted by one or more groups selected from an alkyl group having 1 to 6 carbons and an aryl having 6 carbons, for example, DBCO;
W is an amide bond between the primary amino group Y of the multivalent carrier and the carboxyl end group of the polymer of general formula (IV); or an ester bond between the primary hydroxy group Y of the multivalent carrier and the carboxyl end group of the polymer of the general formula (IV) or a triazole linker, formed by the reaction of the azide end group of the polymer of the general formula (IV) and the alkyne or cycloalkyne group Y of the dendrimer or dendron (multivalent carrier).
The star-shaped fluorescent copolymer, therefore, consists of a central molecule of the carrier (for example PAMAM dendrimer, 2,2-bis(hydroxymethyl)propion dendrimer or dendron or polyol) to the end groups of which at least one chain of the fluorescent polymer is bound, preferably of a linear statistical copolymer of the general formula (IV), as defined above. End groups of the multivalent carrier are amino groups, hydroxy groups or alkyne groups with the number of carbons from 3 to 6, for example, propargyl, or cyclooctyne groups, which can optionally be further independently substituted by one or more groups selected from an alkyl group with the number of carbons from 1 to 6 and an aryl having 6 carbons, for example, DBCO, and the fluorescent polymer (for example, a linear copolymer of formula (IV)) is attached to them via an amide, ester or triazole linker).
Preferably, the star-shaped fluorescent copolymer contains from 2 to 48 linked fluorescent polymer chains, more preferably from 3 to 32 linked fluorescent polymer chains, most preferably from 4 to 24 linked fluorescent polymer chains. In the most preferred embodiment, the fluorescent polymer is a linear statistical copolymer of the general formula (IV).
The molar mass Mn of the star-shaped fluorescent copolymer is preferably in the range of from 60,000 to 1,000,000 g/mol, preferably from 70,000 to 400,000 g/mol. The molar mass of each chain of the fluorescent polymer linked to the multivalent carrier is in the range of from 6,000 to 100,000 g/mol, preferably from 40,000 to 70,000 g/mol, while the molar mass of the multivalent carrier itself, which is part of the starshaped fluorescent copolymer, does not exceed 50,000 g/mol. This ensures that after the disintegration of the star-shaped fluorescent copolymer into a multivalent carrier and fluorescent polymer in the tumour tissue and after the release of the fluorophore, all the resulting fragments have a molecular weight below the renal filtration limit and are, therefore, easily excreted from the body by renal filtration.
The method of preparing the star-shaped polymer of general formula (VII) is shown in Scheme 1 , wherein n is an integer in the range of from 1 to 48, Y is a primary amino group, a hydroxy group, a C3-C6 alkyne group, or a cyclooctyne group of a poly(amidoamine) or 2,2-bis(hydroxymethyl)propion dendrimer or dendron or polyol;
V is an azide or TT end reactive group introduced by a transfer agent to the end of the polymer chain of the fluorescent polymer of general formula (IV) during RAFT polymerisation;
W is an amide bond, an ester bond or a triazole linker formed by the reaction of the azide of the polymer of general formula (IV) and the alkyne group or the cycloalkyne group of the multivalent carrier.
Figure imgf000020_0001
Scheme 1 : Scheme for the preparation of a star-shaped copolymer.
Examples of schematic structures of multivalent carriers for the preparation of star-shaped fluorescent
5 polymers are shown in Scheme 2 below:
Figure imgf000020_0002
Figure imgf000021_0001
Figure imgf000022_0001
Scheme 2: Examples of structures of multivalent carriers for the synthesis of a star-shaped fluorescent copolymer: (i) schematic representation of bis-MPA dendron with DBCO groups; (ii) PAMAM dendrimer with amino groups; (iii) bis-MPA dendrimer with propargyl groups; (iv) polyols with the number of hydroxyls from 4 to 8; and (v) an example of the structure of a star-shaped fluorescent copolymer with the PAMAM dendrimer carrier, in which the chains containing the linear fluorescent polymer are marked schematically with a wavy line. The maximum number of chains containing the linear fluorescent polymer that can be attached to a multivalent carrier (for example, a dendrimer) is equal to the number of end groups of the multivalent carrier (in this case, 16 end amino groups of the dendrimer).
An object of the present invention is also a method of preparing the star-shaped fluorescent polymer defined above, comprising the following steps:
(i) providing a multivalent carrier selected from the group comprising the second or third generation poly(amidoamine) dendrimer, 2,2-bis(hydroxymethyl)propion dendrimer or the second to fourth generation dendron; polyols with the number of hydroxyl groups from 2 to 8, glycerol, pentaerythritol, bis(2- hydroxyethyl)aminotris(hydroxymethyl)methane, dipentaerythritol, porphyrin derivatives; terminated by groups Y selected from primary amino groups, keto groups, azide groups, hydroxy groups, alkyne groups with a number of carbons from 3 to 6, for example, propargyl; and cyclooctyne groups, which may optionally be further independently substituted by one or more groups selected from an alkyl group having 1 to 6 carbon atoms and an aryl having 6 carbon atoms, for example, DBCO.
Said multivalent carriers are commercially available, optionally end DBCO groups can be prepared by reaction of end primary amino groups with DBCO-NHS. The term “providing a multivalent carrier”, therefore, means the commercial acquisition of a multivalent carrier, possibly followed by the modification of its end groups.
(ii) preparation of a statistical linear copolymer that contains a linear polymer selected from the group consisting of polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate and poly(A-(2- hydroxypropyl)methacrylamide) in which from 0. 1 to 10 mol% of monomer units are statistically replaced by a monomer unit of general formula (VI) that contains end reactive groups (for example azide, DBCO, hydrazide, amino groups and TT); this preparation is described above under steps a) and b) of the preparation of the linear fluorescent polymer;
(iii) grafting end reactive functional groups (for example, azide or TT) of the statistical linear copolymer prepared in step (ii) onto the groups Y of the multivalent carrier from step (i), to form a star-shaped polymer, which contains in its arms a statistical linear copolymer selected from the group comprising polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate and poly(A'-(2- hydroxypropyl)methacrylamide) in which from 0. 1 to 10 mol% of monomer units are statistically replaced by a monomer unit of the general formula (VI). The grafting reaction of polymers onto multivalent carriers takes place in a solvent preferably selected from the group comprising dimethylsulphoxide, dimethylacetamide, dimethylformamide, methanol and ethanol. The molar mass Mn of the star-shaped polymers prepared in this way is in the range of from 60,000 to 1,000,000 g/mol, preferably from 100,000 to 400,000 g/mol; (iv) linking of the fluorophore to group D of the statistical linear copolymer from step (iii) by conjugation of carbonyl -thiazoline-2-thione groups, (4-nitrophenyl)oxy groups, (2,3,4,5,6-pentafluorophenyl)oxy groups, (succinimidyl)oxy groups, carboxyl, hydrazide, azide, activated disulphide groups or NH2 groups of monomer units of general formula (VI) with an amine, carboxyl, activated carboxyl, activated disulphide or keto group of the fluorophore; wherein both the fluorophore and its linking to group D are described above; to form a fluorescent star-shaped copolymer containing monomer units of the general formula (I) and (VI) and further containing monomer units selected from the group comprising acrylamide, methacrylamide, acrylate, methacrylate and /V-(2-hydroxypropyl)methacrylamide.
The linking of the fluorophore to group D is carried out by conjugation of free hydrazide groups, amino groups, activated disulphide groups or thiazolidine-2-thione (TT) groups of monomer units of the general formula (VI) of the statistical linear copolymer described above with a low-molecular-weight fluorescent label or its derivative (fluorophore) which contains suitable reactive groups (for example, an amine, carboxyl, activated carboxyl or keto group) and can be used in its free form or in the form of a salt with an acid, for example, HC1; wherein the low-molecular-weight fluorophore is a fluorophore having a molecular weight in the range of from 350 to 1,500 g/mol. The fluorophore is selected according to excitation and emission wavelengths, with excitation wavelengths ranging from 300 to 850 nm and emission wavelengths ranging from 350 to 1,200 nm; the low-molecular-weight fluorophore molecule is bound to the linear copolymer by an amide, disulphide or hydrazone bond.
(v) optionally, linking of the targeting structure defined above (oligopeptides with the number of amino acids from 3 to 20 or protein, especially monoclonal antibodies), to groups D of the monomer units of the general formula (VI) of the fluorescent star-shaped copolymer from step (iv).
Linking of the targeting structure (a peptide structure with introduced amino groups, azide, sulphodibenzocyclooctyne (sDBCO) or dibenzocyclooctyne (DBCO) groups) is carried out by reacting group D of the monomer unit of general formula (VI) with -NH2, propargyl, sDBCO or DBCO groups present on the targeting structure. The reaction takes place in the order of minutes and with a high yield, and the resulting conjugate retains its biological activity unaffected.
(vi) optionally, linking of the targeting group to the ends of the linear chain of the fluorescent star-shaped polymer from step (iv) or (v).
Linking of the targeting structure (a peptide structure with amino groups or introduced azide, maleinimidyl, propargyl, sulphodibenzocyclooctyne (sDBCO) or dibenzocyclooctyne (DBCO) groups) is carried out by reacting end group, TT group, azide, propargyl, DBCO or sBCO group or SH group with groups present on the targeting structure. The reaction takes place in the order of minutes and with a high yield, and the resulting conjugate retains its biological activity unaffected. (vii) Any unreacted thiazolidine-2-thione groups, (4-nitrophenyl)oxy groups, (2, 3, 4,5,6- pentafluorophenyl)oxy groups, (succinimidyl)oxy groups can be removed by reaction with amino alcohol selected from the group consisting of NH2-(CH2)a-CH2(OH); NH2-(CH2)b-CH(OH)-CH3; NH2-(CH2)b- CH(OH)-(CH2)C-CH3; wherein a is an integer in the range of from 0 to 4, b is an integer in the range of from 0 to 3 and c is an integer in the range of from 1 to 4; preferably with l-aminopropan-2-ol, and/or the unreacted NH2 groups can be removed by reaction with acetylthiazolidin-2-thione. Activated disulphide groups can be removed by reaction with mercaptoethanol. Carboxyles, hydrazides and azides do not need to be removed.
The present invention further relates to the use of fluorescent probes for the visualisation of tumours for diagnostic purposes and for image-guided surgery.
The fluorescent probe means the fluorescent polymer according to the present invention as defined above, and the star-shaped fluorescent polymer according to the present invention as defined above.
In a preferred embodiment, the fluorescent probe can carry a combination of a fluorophore, the fluorescence of which is activated in the tumour, and a targeting group to increase the specificity of visualisation of the tumour tissue. In this embodiment, the statistical linear fluorescent copolymer further contains at least one targeting group for directing the fluorescent probe to tumour tissues, which is selected from the group comprising oligopeptides with the number of amino acids from 3 to 20 and proteins for targeting the fluorescent polymer to tumour cells of head and neck tumours, breast tumours, melanomas and colorectal tumours or to tumour endothelial cells. The targeting group can be attached to the end group of the linear chain of the fluorescent probe or it can be part of the monomer unit (II).
An object of the present invention is, therefore, the use of the fluorescent probe according to the present invention in medical diagnostics, whole-body imaging and/or fluorescence-guided surgery, preferably in the diagnosis and monitoring of the success of treatment in cancer diseases, diseases of the hematopoietic system (leukaemia, lymphomas, and hematopoietic failure) and the immune system. Fluorescent probes according to the present invention can be used, for example, in whole-body imaging techniques based on fluorescence detection for the detection of tumour tissue; in fluorescence -guided surgery for marking and imaging fluorescence in target structures of body organs and tissues.
Thus, fluorescent probes are activatable linear and star-shaped fluorescent polymers according to the present invention, in which the fluorophore is bound to the polymeric carrier by a biologically degradable amide, hydrazone, or disulphide bond, and enable the circulation time of the bound fluorophore in the organism to be significantly extended, which enables the fluorophore to be transported on a high-molecular- weight polymeric carrier into the tumour and released here in its original form. Another advantage of these activatable fluorescent probes according to the invention is the activation of fluorescence, which is given by the release of the fluorophore from the polymeric carrier. Polymeric activatable fluorescent probes according to the invention are further characterised by the fact that the binding of the fluorophore to the polymer chain is relatively stable, only at most 10 percent of the fluorophore is released during 24 h transport in the bloodstream and body fluids, and it is hydrolytically, enzymatically or reductively cleavable in the tumour environment and inside target tumour cells in lysosomes. This means that the fluorophore is transported through the bloodstream in an inactive, low- fluorescent form, and its release and activation of fluorescence mainly occur after entering the tumour tissue or after penetration into the target tumour cells. Activation of the fluorophore only in the target cells leads to a significant increase in fluorescence in the tumour, in the tumour/healthy tissue contrast and thus to a clear visualisation of the tumour tissue. By binding the fluorophore to the polymer chain, there will be a significant increase in the molecular weight of the contrast agent and thus an increase in its circulation time in the bloodstream and increase in its bioavailability. Responsible for the targeted transport to the tumour or tumour cells is a polymeric carrier prepared preferably on the basis of HPMA copolymers, whose molecular weight and thus the efficiency of accumulation in the tumour tissue can be controlled by changes in the structure of the polymeric carrier (linear polymer, high-molecular-weight biodegradable star-shaped polymer). Due to the increased molecular weight of the polymeric carrier, the entire conjugate is accumulated in solid tumours due to the EPR effect. Polymeric probes can preferably also be targeted actively by means of linked targeting structures, oligopeptides and proteins, which further increases the selectivity of accumulation in tumour tissue and supports an increase in tumour/healthy tissue contrast.
Summary
The object of the invention is a targeting and activatable fluorescent polymer and a star-shaped fluorescent polymer for enhancing the visualisation of solid tumours. Due to their hydrodynamic size in solution and targeting to receptors on tumour cells, they are significantly accumulated in solid tumours, resulting in a significant enrichment of the tumour tissue with the fluorophore present. In addition, the carried fluorophore will be released in the tumour tissue environment, which results in an increase in the fluorescence signal due to a decrease in fluorescence quenching associated with the binding of the fluorophore to the polymeric system. As a result, there is a very significant increase in the fluorescence contrast between tumour and non-tumour tissue, significantly contributing to the visualisation of tumour tissue during guided tumour surgery. Contrast enhancement significantly advances the boundaries of guided surgery compared to the current state of the art.
Description of Drawings
Fig. 1: Fluorescence intensity of the fluorophore before and after hydrolysis of the pol-PYR-Cy7 conjugate in Example 10.
Fig. 2: Rate of release of the fluorophore from conjugates with DY 676 attached to the polymer by pH- sensitive linkers with different structures in Example 11. Fig. 3: Confocal microscopy images - Example 13.
Fig. 4: In vivo fluorescence imaging of polymer systems with fluorescence label Dyomic 676; A - polymer system with a label firmly bound by an amide bond to a non-degraded linker; B- polymer system with a label bound via a hydrazone linker formed by OPB-Dy-676 - Example 14
Examples
Examples of the synthesis of intermediates and conjugates according to the invention
Example 1 : Synthesis of polymer precursors
The copolymer poly(HPMA-co-Ma-AP-TT) was prepared by controlled solution radical copolymerisation of HPMA (93 mol%, 100 mg) and 3-(3-methacrylamidopropanoyl)thiazolidin-2-thione (Ma-AP-TT) (7 mol%, 14.0 mg) carried out in the presence of the initiator 2,2'-azobis(4-methoxy-2,4- dimethylvaleronitrile) (V70) and transfer agent 4-cyano-4-thiobenzoyl-sulphanylpentanoic acid (CTA).
The polymerisation mixture was dissolved in tert-butyl alcohol (751 pL), CTA dissolved in 10 vol% DMA (83 pL), and all transferred to a glass ampoule where the mixture was bubbled with Ar, and the ampoule was sealed. After 24 h at 40 °C, the polymer was isolated by precipitation into acetone/diethylether, the precipitate was then washed with diethyl ether and dried in a vacuum. The end dithiobenzoate groups were removed from the copolymer by reaction with AIBN (10-fold molar excess) in DMA (15% polymer solution) under an argon atmosphere for 3 h at 70 °C in a sealed ampoule. The polymeric conjugate was isolated by precipitation into acetone. The precipitate was washed with diethyl ether and dried in a vacuum to dryness. The prepared poly(HPMA-co-Ma-AP-TT) precursor had , = 28,000 g/mol, £> = 1.05, TT content 5.9 mol%.
The click reaction precursor was prepared by reacting poly(HPMA-co-Ma-AP-TT) (100 mg, 39.3 pmol) with DBCO-NH2 (5.5 mg, 19.9 pmol) in DMA (1 mb) in the presence of DIPEA base (3.4 pL, 19.9 pmol). The progress of the reaction was monitored using HPLC. For some polymers prepared for label binding via amide bond, the polymer poly(HPMA-co-Ma-AP-TT-co-MA-AP-DBCO) was separated, and in the case of polymers for label binding via click chemistry, the remaining TT groups on the polymer were removed by adding l-aminopropan-2-ol (3.0 pl, 39.3 pmol). The polymeric precursor poly(HPMA-co-Ma-AP- DBCO) was precipitated into acetone/diethylether mixture and re-precipitated from MeOH, washed with diethylether and dried. The same polymeric precursors were also used for targeted conjugates with either an enzymatically cleavable fluorophore or control conjugates with a firmly bound fluorophore.
Polymeric precursors poly(HPMA-co-Ma-X-TT), where X = GG, GLG, GFG, GLFG (SEQ. no. 14), GFLFG (SEQ. no. 9), and poly(HPMA-co-Ma-XX-NH-NH-Boc), where XX= -CH2-CH2-(C=O)- (ethyl); -CH2-CH2-CH2-(C=O)- (propyl); -CH2)4-(C=O)- (butyl); -CH2)5-(C=O)- (pentyl); -CH2)6-(C=O)- (hexyl); -CH2)7-(C=O)- (heptyl); GG, GLG, GFG, GLFG, GFLFG, were prepared analogously according to this procedure. T1
For pH-sensitive binding of fluorophores, poly(HPMA-co-Ma-XX-NH-NH-Boc) precursors were used, resulting in binding of conjugates with fluorescent labels bound via pH-sensitive hydrazone linkers, where the Boc protecting groups were removed in TFA and the poly(HPMA)-co-Ma-XX-NH-NH2) polymer was precipitated after 10 min into diethyl ether, re-precipitated from MeOH and dried. For the synthesis of targeting conjugates with a pH-sensitive hydrazone bond, part of the hydrazide groups were reacted with DBCO-NHS, and for the subsequent click reaction with the peptide, poly(HPMA-co-Ma-XX-NH-NH2-co- Ma-XX-NH-NH-DBCO) was used.
The disulphide polymer was prepared by reacting polyiHPMA-co-Ma-XX-NH-NFE) with succinimidyl 3- (2-pyridyldithio)propionate) (SPDS) to form the polymeric precursor poly(HPMA-co-Ma-XX-NH-NH- PDS).
The characteristics of the prepared polymer precursors are shown in Table 1.
Table 1
Figure imgf000028_0001
Example 2: Synthesis of a star-shaped polymer with a triazole linker
The synthesis of the star-shaped copolymer took place in two steps. First, a reactive copolymer p(HPMA- co-Ma-Pentyl-NHNH-Boc)-N3 was prepared using the transfer agent azide-CTA, '-(3 -azidopropyl ethylsulphanylcarbothioylsulphanyl-4-methyl-pentanamide, containing an azide group, in a similar manner to the reactive copolymer in Example 1. Next, the Boc groups were removed by boiling in water and subsequently the polymer was reacted with a bisMPA dendrimer containing end DBCO or propargyl groups with p(HPMA-co-Ma-Pentyl-NHNH2)-N3 in methanol for 2 h. The resulting star-shaped polymeric conjugate was precipitated into acetone and dried to constant weight. Characterisation of the resulting starshaped polymeric conjugate: Mv =250,000 g/mol, £> = 1.20, content of hydrazide groups = 8 mol%.
By changing the polymer/dendrimer core ratio and by changing the dendrimer generation, it is possible to control the of polymer systems over a wide range. Similarly, it is possible to use a derivatised bis-MPA dendron or PAMAM dendrimer, to which DBCO or propargyl groups are introduced, for the preparation of star-shaped conjugates. Star-shaped copolymers with polymers containing other linkers were prepared in a similar way.
Figure imgf000029_0001
Scheme 3: Synthesis and structure of star-shaped copolymers
Example 3 : Synthesis of a star-shaped conjugate with an ester linker
The synthesis of the star-shaped copolymer took place in three steps. In the first step, pentaerythritol was modified using dibenzocyclooctyne -A-hydroxysuccinimidyl ester. Furthermore, a reactive copolymer p(HPMA-co-Ma-Pentyl-NHNH-Boc)-N3 was prepared using the transfer agent azide-CTA, N-(3- azidopropyl)-7-cthylsulphanylcarbothioylsiilphanyl-4-mcthyl-pcntanamidc. containing an azide group, in a similar manner to the reactive copolymer in Example 1. Next, the Boc groups were removed by boiling in water, and then the polymer was reacted with pentaerythritol containing end DBCO groups with p(HPMA-co-Ma-Pentyl-NHNH2)-N3 in methanol for 2 h. The resulting star-shaped polymeric conjugate was precipitated into acetone and dried to constant weight. Characterisation of the resulting star -shaped polymeric conjugate: Mv = 180,000 g/mol, £> = 1.25, content of hydrazide groups = 8 mol%. Star-shaped copolymers with polymers containing other polyols were prepared in a similar manner.
Figure imgf000030_0001
Scheme 4 An example of the structure of a star-shaped copolymer based on pentaerythritol and a linear copolymer
Example 4: Synthesis of a star-shaped conjugate with an amide linker
The synthesis of this star-shaped conjugate took place in two steps. First, the reactive copolymer p(HPMA- co-Ma-Pentyl-NHNH-Boc)-TT was prepared using the transfer agent TT-CTA, [l-cyano-l-methyl-4-oxo- 4-(2-thioxothiazolidine-3-yl)butyl]benzenecarbodithioate, containing a TT group, in a similar manner to the copolymer in Example 1. In the second step, the bis-MPA dendrimer containing amino groups was reacted with p(HPMA-co-Ma-Pentyl-NH-NH)-TT in methanol for 2 h. The resulting star-shaped polymeric conjugate was precipitated into acetone and dried to constant weight. Characterisation of the resulting starshaped polymeric conjugate: A w = 220,000 g/mol, £> = 1.18, content of hydrazide groups = 8 mol%. By changing the polymer/dendrimer core ratio and by changing the dendrimer generation, it is possible to control the M,. of polymer systems over a wide range. Similarly, it is possible to use bis-MPA dendron or PAMAM dendrimer with amino groups for the preparation of star-shaped conjugates.
Figure imgf000031_0001
Scheme 5 : An example of the structure of a second generation bis-MPA dendron with a linear copolymer linked via an amide bond. The maximum number of linear copolymer chains that can be attached to the dendron is equal to the number of end groups of the dendron (in this case 4 end amino groups).
Example 5 : Preparation of fluorophore derivatives
Preparation of Az-Val-Cit-Aba-DY-676 and Az-Val-Cit-DY-676 derivatives
5-azidopentanoyl-valyl-citrulin (Az-Val-Cit-OH)
The dipeptide derivative was prepared by solid-phase peptide synthesis on 2-chlorotrityl chloride resin (0.5 g, substitution 1 mmol/g) by reacting 0.2 M solutions of amino acids (Fmoc-Cit-OH, Fmoc-Val-OH, 5- azidopentanoic acid). The product was cleaved from the resin with a 30% solution of HFIP in DCM, yielding 121 mg (0.23 mmol, 46 %) of the azido-peptide derivative.
ESI MS (calculated 399.5, measured M+Na 422.5). According to HPLC, 1 peak is visible at tr= 1.39 min. N-(5-azidopentanoyl-valyl-citrullyl)-DY-676 (Az-Val-Cit-DY-676)
Figure imgf000032_0001
Az-Val-Cit-OH (0.5 mg, 1.2 pmol) and the amino derivative of DY-676 (1 mg, 1.2 pmol) were dissolved in DMA (0.5 mb) with DIC (0.3 pL, 1.8 pmol) and HOBt (0.3 mg, 1.8 pmol). The progress of the reaction was monitored by HPLC. After 24 h, the dye and the linker reacted off and, according to HPLC, there was one peak corresponding to the product Az-Val-Cit-DY-676.
N-(5-azidopentanoyl-valyl-citrullyl)-4-aminobenzyl alcohol (Az-Val-Cit-Aba)
Figure imgf000032_0002
Az-Val-Cit-OH (80 mg, 0.2 mmol), 4-aminobenzyl alcohol (26 mg, 0.21 mmol), 1 -hydroxybenzotriazole (35 mg, 0.23 mmol) and DIC (36 pL, 0.23 mmol) were dissolved in 2 mb of DMF: DCM mixture (3:2). The reaction mixture was stirred for 1 h at 0 °C and then allowed to react for 16 h at 25 °C. The solvent was concentrated under reduced pressure and the product was isolated by precipitation into diethylether, filtered off and purified on preparative HPLC (Chromolith C18, water/acetonitrile linear gradient 0-100%). The reaction yield was 53 mg (0.11 mmol, 55 %) of white powder. ESI MS (calculated 504.6, measured M+H 505.6). One peak is visible on HPLC at tr= 1.74 min. N-(5-azidopentanoyl-valyl-citrullyl)-4-aminobenzyl(4-nitrophenyl)carbonate (Az-Val-Cit-Aba-Npc)
Figure imgf000033_0001
Az-Val-Cit-Aba (50 mg, 0.1 mmol) was dissolved in DMA (0.15 mL), 4-nitrophenyl chloroformate (60 mg, 0.3 mmol) was dissolved in DCM (0.5 mL). The solutions were mixed and pyridine (0.15 mL) was added. The progress of the reaction was monitored by HPLC, the product was isolated on preparative HPLC
(Chromolith C18, water/acetonitrile linear gradient 0-100%), and the reaction yield was 50 mg (75 mmol, 75 %) of white powder. ESI MS (calculated 669.7, measured M+H 670.7). On HPLC, there is one peak at tr= 2.55 min. N-[N’-(5-azidopentanoyl-valyl-citrullyl)-4-aminobenzyloxycarbonyl] DY-676
Figure imgf000033_0002
Az-Val-Cit-Aba-Npc (0.8 mg, 1.2 pmol) and the amino derivative DY -676 (1 mg, 1.2 pmol) were dissolved in DMA (0.5 mL) with DIPEA (0.25 pL, 1.4 pmol). The progress of the reaction was monitored by HPLC. After 24 h, the dye and the linker reacted off and, according to HPLC, there was one peak corresponding to the product Az-Val-Cit-Aba-DY-676.
Derivatives with other fluorescent labels (Dyomics-782, Dyomics-633, Dyomics-781, Dyomics-776, Dyomics-777, Dyomics-778, Dyomics 780, Cyanine-7, Cyanine-7.5, Cyanine-5, Cyanine-5.5, indocyanine green) were prepared in a similar way.
Example 6: Derivatisation of keto acids
Carboxylic keto acids such as 5 -cyclohexyl-5 -oxopentanoic acid (COP), 4-(2 -oxopropyl) benzenecarboxylic acid (OPB) and 4-oxo-4-(2-pyridyl)butanoic acid (PYR) were activated by reaction with thiazolidine-2-thione. This reaction is shown in Scheme 6.
Figure imgf000034_0001
Scheme 6
100 mg (0.56 mmol) of OPB acid and 132 mg (0.67 mmol, 1.2 mol equiv. to the keto acid) of N,N'- dicyclohexylcarbodiimide DCC were dissolved in 0.78 m of tetrahydrofuran (THF) (2,45 dm'-mol ’). Furthermore, 39.4 mg (0.59 mmol, 1.05 mol equiv. to the keto acid) of thiazolidine-2-thione TT and 2 mg (catalytic amount) of 4-(dimethylamino)pyridine (DMAP) were were dissolved in 0.30 m of THF (1.12 dm3-mol-1). Subsequently, the solutions were cooled to -18 °C and mixed after twenty minutes. With constant stirring, this temperature was maintained for one hour, and the temperature was 4 °C for the next twenty hours. Subsequently, the reaction mixture was filtered off to remove N, A'-dicyclohexylurea. The filtrate was evaporated under reduced pressure and dissolved in ethyl acetate. This procedure was repeated once more and the product was purified by double crystallisation in a mixture of ethyl acetate: dichloromethane (1: 1). The yields of these reactions range between 30 and 50 %.
The same procedure was performed with COP and PYR.
Thus, activated acids were then derivatised with an amino derivative of a fluorescent molecule, e.g. Cyanine7 (Cy7) or Dyomics 676 (DY -676). 1.39 mg (4.65 pmol) of COP acid and 3.35 mg (4.65 pmol, 1.0 mol equiv. to the keto acid) of Cy7 were dissolved in 1.193 mb (256.5 dm3-mol-1) of solvent (dimethylformamide - methanol, 3/1) and then mixed. A 2.6M solution of sodium hydroxide (1.10 mol equiv. to the keto acid) was added to the solution. The reaction was carried out at room temperature for three hours with constant stirring. The progress of the reaction was monitored using HPEC and TEC (ethyl acetate). The reaction was terminated by the evaporation of the solvent. The yields of these reactions range between 94 and 99 %.
The same procedure was performed with OPB and PYR. Example 7 : Preparation of peptide azido derivatives
Oligopeptides GE-7, GE-11, CNGRC, cyclic RGDfK, HEWSYLAPYPWF and SYSMEHFRWGKPV were prepared by solid-phase synthesis using a microwave peptide synthesiser by the standard Fmoc method from the C terminus of the peptide using an /V-Fmoc-protected amino acid (2.5 equiv.), DIC (2.5 equiv.) as an activator, and oxyma (2,5 equiv.) as a base in DMF.
After binding of the last A-Fmoc-amino acid and removal of the Fmoc group, 5 -azidopentanoic acid (2.5 equiv.), PyBOP (2.5 equiv.) as an activator, and DIPEA (5 equiv.) as a base were attached.
Example 8: Preparation of polymeric conjugates containing a fluorescent label by polymer analogous reaction
Polymeric conjugate poly(HPMA-co-Ma-GFLG-DY-676) with an aminolytically bound dye via the GFLG linker was prepared by reacting the amino derivative of the dye (2 mg) with the polymeric precursor poly( N- (2-hydroxypropyl)methacrylamide-co-A-methacryloylglycyl-leucylphenylalanyl-glycine thiazolidine-2- thione) (poly(HPMA-co-Ma-GLFG-TT) (98 mg) in 1 mb DMA in the presence of DIPEA base (equiv. to dye). The reaction was monitored on HPLC and after binding of all the free dye, the remaining TT groups on the polymer were removed by the addition of I-aminopropan-2-ol (equiv. to TT at the beginning of the reaction). The reaction mixture was purified by gel filtration (Sephadex LH20) and the polymer fraction was concentrated and precipitated into the acetone/diethylether mixture and washed with diethylether. The precipitate was dried to constant weight. The dye content was determined and the molar mass was measured using SEC of the prepared polymeric conjugate.
A control polymeric conjugate poly(HPMA-co-Ma-AP-DY-676) with a firmly bound dye without a degradable sequence was also prepared from the polymeric precursor poly(A-(2- hydroxypropyl)methacrylamide -co-methacrylamidopropanoyl)thiazolidine -2 -thione) (poly (HPMA-co - Ma-AP-TT). The contents of fluorescent labels in the polymeric conjugates are shown in Table 2.
The polymeric conjugate poly(HPMA-co-Ma-AP-DBCO-Az-ValCit-Aba-DY-676) was prepared by atwo- step synthesis. In the first step, (poly(HPMA-co-Ma-AP-TT) was converted to poly(HPMA-co-Ma-AP- DBCO) by reaction with DBCO-NH2 in the presence of DIPEA base
(equiv. to DBCO-NH2). In the second step, a solution of the modified dye Az-Val-Cit-Aba-DY-676 was added to the precursor poly(HPMA-co-Ma-AP-DBCO) (48.6 mg) dissolved in 300 pL DMA to form a triazole ring. After the disappearance of the free dye peak according to HPLC, the reaction mixture was precipitated into a mixture of acetone/diethylether 2: 1. The precipitate was washed with diethylether and dried. The precipitate was dissolved in MeOH and purified by gel filtration (Sephadex LH20). The polymeric fraction was concentrated and precipitated into diethylether. The dye content was determined and the molar mass was measured using SEC of the prepared polymeric conjugate. Example 9: Preparation of conjugates with a pH-sensitive bond
The polymeric conjugate with a pH-sensitive bond poly(HPMA-co-Ma-amidoheptoyl-NH-NH=COP-DY- 676) was prepared by the reaction of the keto acid-modified dye COP-Dy-676 (2 mg) and the polymeric precursor poly(HPMA-co -Ma-amidopentoyl-NH-NEE) (98 mg) in 1 m of MeOH with 40 pL of acetic acid and stirred at room temperature for 48 h. The course of the reaction was monitored by HPLC and TLC (CHCE/MeOH/acetic acid - 8/2/1). Then the reaction products were purified by gel filtration chromatography using Sephadex LH-20 gel, with methanol used as the mobile phase. The product fractions were concentrated by evaporation of methanol under reduced pressure and precipitated into ethyl acetate, centrifuged and dried to constant weight. The yields of these reactions range between 50 and 70 %. Subsequently, the fluorophore content was determined spectrophotometrically and the molar mass was measured using SEC.
Example 10: Preparation of targeted conjugates
Peptide-targeted conjugates were prepared by click reaction of azide derivative of peptide GE -7 with the polymeric precursor having DBCO groups, which was prepared from the precursor poly(HPMA-co-Ma- GFLG-TT) converted to poly(HPMA-co-Ma-GFLG-TT-co-Ma-GFLG-DBCO) by reaction with 0.5 - 4 mol% DBCO-NH2 in the presence of DIPEA base (equiv. to DBCO-NH2). After binding the fluorophore, see Example 8, the azide derivative of the peptide was attached by an metal-free click reaction in DMA. Other targeted conjugates of azide derivatives of peptides GE-11, CNGRC, cyclic RGDfK, HEWSYLAPYPWF and SYSMEHFRWGKPV were prepared in the same way. A control targeted conjugate with a firmly bound dye was prepared from poly(HPMA-co-Ma-AP-TT) as described above.
Peptide-targeted conjugates with a pH-sensitive bond were prepared as in Example 9, and further DBCO- NHS was attached to the remaining hydrazide groups in the presence of DIPEA base (equiv. to DBCO- NHS) in methanol. The azide derivative of the peptide was then attached to the polymeric conjugate by a click reaction via DBCO.
Antibody-targeteded conjugates were prepared from the semitelechelic precursor poly(HPMA-co-Ma- Pentyl-NH-NH-Boc)-TT, terminated with a reactive TT group prepared using the transfer agent TT-CTA as in Example 4. The end TT group was converted to maleimide by reaction with N-(2- aminoethyl)maleimide, which was used, after deprotection of the Boc protecting group and attachment of the fluorophore, for reaction with the reduced antibody rituximab.
The antibody was reduced by gentle reduction with dithiothreitol in PBS buffer and after reduction, it was purified from free dithiothreitol on a PD 10 column. It was subsequently mixed with the polymeric conjugate bearing a hydrazone-linked fluorescent label and a maleimide end group.
The prepared targeted conjugates, see Table 2, were characterised, the dye content was determined and the peptide/antibody content was determined through amino acid analysis. The molar mass could not be measured due to the interaction of the laser used in the GPC detectors with the fluorophore, which made it impossible to determine these characteristics.
Example 11 : Fluorescence quenching on polymer
Fluorescence quenching was experimentally performed by measuring the fluorescence intensity of the fluorescent polymer poly(HPMA-co-Ma-Acap-NH-NH=COP-DY-676) in a phosphate buffer (0.3M, pH 7.4) with a fluorescent polymer concentration of 0.1 g-dm ' and the fluorescence intensity of this solution after total hydrolysis. This was achieved by adding concentrated acetic acid (! of the original volume) to the original solution. This dilution was compensated for by multiplying this intensity by 1 ,25x. It was found that after binding there is a significant decrease in the fluorescence of the bound fluorophores, and the fluorescence is restored again after release from the polymer, see Fig. 1. Similarly, an experiment was carried out with polymeric conjugates with hydrazone -bound OPB and PYR derivatives of the fluorescent label DY-676 and also with identical derivatives of other fluorescent labels, e.g. DY-782, Cy-7, DY -767, and then with the fluorescent label DY -676 or CY -7 bound via enzymatically cleavable linkers GLG, GFG, GLFG, GFLFG, GFLG, Val-Cit and Val-Cit-Aba, where a significant quenching of the fluorescence signal after binding to the polymer and subsequent activation of fluorescence after incubation with the model lysosomal enzyme cathepsin B was also observed.
The activation of the fluorescent signal can be used to increase the fluorescence within the tumour tissue in tumour cells, where after passive or active accumulation, the fluorophore is released either due to the reduced pH of the tumour tissue or due to the activity of lysosomal enzymes after the conjugates enter the tumour cells. This activation of the signal subsequently leads to a significant increase in the contrast of the fluorescent signal in the tumour environment and beyond. The increase in contrast can be used within the framework of navigated tumour surgery, when the improved tumour/non-tumour tissue contrast will allow the surgeon to clearly highlight the tumour mass and, therefore, to perform a precise resection of this tissue.
Example 12: Release of fluorophores from polymer systems
The release of fluorophores was measured by HPLC. Fluorescent polymers with a hydrazone -bound fluorophore were incubated in a phosphate buffer (0.3M, pH 7.4) with a concentration of 0,1 g-dnT3 at 37 °C. At 0, 14, 1, 2, 4, 6, 8 and 10 hours, the amount of released fluorophore was measured. From Fig. 2 it can be seen that the release rate of the fluorophore is dependent on the pH and the type of linker. Cleavage at physiological pH 7.4 is slower than cleavage at acidic pH, simulating tumour tissue. The significant increase in the fluorescence intensity caused by the release of the fluorophore from the polymer will therefore only occur in the tumour tissue, where the contrast between the tumour and healthy tissue will increase. Fluorescent polymers with the fluorescent label bound via enzymatically degradable linkers were incubated in phosphate buffer (KH2PO4, NaOH, pH = 6.0, 0.001 M EDTA and 0.01 M glutathione) at a concentration of 0.1 g-dnY3 at 37 °C in the presence of cathepsin B (2x l0-7 mold 1). At 0, 1, 2, 8 and 24 hours, the amount of released fluorophore was measured. After cleavage with cathepsin B, the release of the fluorophore and a significant increase in fluorescence were detected.
Example 13: Physical characterisation of conjugates The fluorophore content was determined spectrophotometrically and the fluorophore content in the conjugates was determined: pol-COP-Cy7 (a = 91,000 dm3-mor1-cm"1) 0.4 wt%, pol-OPB-Cy7 (a = 83,000 dm3 -mol-1 -cm-1) 1.8 wt% and pol-PYR-Cy7 (a = 60,000 dm3-mor1-cm"1) 0.4 wt%.
Table 2
Figure imgf000038_0001
Figure imgf000039_0001
Example 14: In vitro testing
Cells with the fluorescent polymer poly(HPMA-co-Ma-Acap-NH-NH=OPB-Cy7) at a concentration of 5 mg-d r3 were incubated at 37 °C for 6 and 24 h. Then, fluorescence was measured using a confocal microscope. From Fig. 3, it is noticeable that at 24 h there was a significant increase in fluorescence due to the cleavage of the fluorophore, as the polymeric conjugate had enough time to enterthe cells. On the contrary, at 6 h, it is clear that no hydrolysis or enzymatic cleavage of the linkers, and therefore no release, has yet occurred.
Example 15: In vivo testing
An in vivo experiment was performed on athymic nude mice (HslCpb:NMRI-Foxnlnu) bearing DLD-1 colorectal tumour. Mice were injected with the fluorescent polymers poly(HPMA-co-Ma-AP-DY-676) or poly(HPMA-co-Ma-Acap-NH-NH=OPB-DY676), which had a fluorescent label attached either by an amide bond to a non-degradable linker in the side chain of the polymeric carrier or by a pH -sensitive linker (hydrazone bond). After 24 h, the mice were examined using a non-invasive imaging system, Fig. 4. It was found that while in mice with a fixed label the fluorescence is spread very homogeneously within the body with a slightly increased intensity at the tumour site (Fig. 4A), in mice with administered pH -sensitive fluorescent polymer (Fig. 4B) the fluorescence is mainly localised in the tumour tissue and the contrast with respect to the healthy tissue is significantly higher than in the stable system.

Claims

39 CLAIMS
1. Fluorescently labelled polymer for tumour visualization, which contains a semitelechelic statistical linear copolymer, selected from the group comprising poly(/V-(2-hydroxypropyl)methacrylamide), polyacrylamide, polymethacrylamide, polyacrylate and polymethacrylate, wherein from 0. 1 to 10 mol% of monomer units, based on the total number of monomer units, are statistically replaced with monomer units of general formula (I)
Figure imgf000040_0001
fluorophore
(I), wherein
A is selected from the group consisting of a linear or branched carbon alkylenyl chain with the number of carbon atoms in the range of from 1 to 7; -(CH2)p-(C(O)-NH-(CH2)r)p-;
-(CH2)p-(C(O)-NH-(CH2)r)p-C(O)-;
-(CH2)P-(C(O)-NH-(CH2)r)P-C(O)-NH-C6H4-CH2-O-C(=O)-; and -(CH2)P-C(O)-NH-(CH2)P-L-(CH2)P-(C(O)-NH-(CH2)r)P-C(O)-NH-C6H4-CH2-O-C(O)-, wherein L is a linker containing a triazole bridge; wherein p is an integer from 1 to 5, and r is selected from 1, 2 and 3; wherein one or more hydrogen atoms in CH2 groups of the substituent A may further be substituted with one or more identical or different natural amino acid side chains;
B is selected from the group consisting of a bond,
Figure imgf000040_0002
and wherein the fluorophore has a molecular weight in the range of from 350 to 1 500 g/mol, an excitation wavelength in the range of from 300 to 850 nm and an emission wavelength in the range of from 350 to I
1,200 nm, and is covalently bound to =CH-,
Figure imgf000040_0003
or -S-S- group of the substituent B of the monomer unit of general formula (I) of the semitelechelic statistical linear copolymer via its primary amine group or NCS group, A-hydroxysuccinimid group, keto group or disulfide group, wherein the groups formed following the fluorophore binding are subsequently part of the group B; 40 and wherein the molecular weight Mn of the fluorescently labelled polymer is in the range of from 6,000 to 100,000 g/mol; with the proviso that if B is a bond, then A is -(CH2)p-(C(O)-NH-(CH2)r)P-C(O)-; or
-(CH2)P-(C(O)-NH-(CH2)r)P-C(O)-NH-C6H4-CH2-O-C(O)-; or
-(CH2)P-C(O)-NH-(CH2)P-L-(CH2)P-(C(O)-NH-(CH2)r)P-C(O)-NH-C6H4-CH2-O-C(O)-.
2. Fluorescently labelled polymer according to claim 1, wherein the substituent A is selected from the group consisting of linear or branched carbon alkylenyl chain with the number of carbon atoms in the range of from 1 to 7; and -(CH2)p-(C(O)-NH-(CH2)r)P-; wherein p is an integer from 1 to 5, and r is selected from 1, 2 and 3; and the substituent B is selected from the group consisting
Figure imgf000041_0001
-S-S-.
3. Fluorescently labelled polymer according to claim 1, wherein the substituent A is -(CH2)P-(C(O)-NH-(CH2)r)P-C(O)-NH-C6H4-CH2-O-C(O)-; or -(CH2)P-C(O)-NH-(CH2)P-L-(CH2)P-(C(O)-NH-(CH2)r)P-C(O)-NH-C6H4-CH2-O-C(O)-, wherein L is a linker containing a triazole bridge; p is an integer from 1 to 5, and r is selected from 1, 2 and 3; and the substituent B is a bond.
4. Fluorescently labelled polymer according to claim 1, 2 or 3, which further contains at least one targeting group for targeting the fluorescently labelled polymer to tumour tissues, said targeting group being selected from the group comprising oligopeptides having from 3 to 20 amino acids; and proteins for targeting the fluorescently labelled polymer towards tumour cells of head and neck carcinomas, breast cancer, melanomas and colorectal cancer or towards cells of tumour endothelium.
5. Fluorescently labelled polymer according to claim 4, wherein the targeting group is bound to an end group of the linear chain of the fluorescently labelled polymer.
6. Fluorescently labelled polymer according to claim 4 or 5, which further comprises monomer units of the general formula (II)
Figure imgf000042_0001
(ii), wherein A is as defined in claim 1 ;
Figure imgf000042_0002
wherein L is as defined in claim 1 ;
Z is a targeting group for targeting the fluorescently labelled polymer to tumour tissues, covalently bound I
Figure imgf000042_0003
-CH=CH-, -CH=CH- in DBCO structure, N3 or -S-S- group of the substituent X of the monomer unit of general formula (II) of the semitelechelic statistical linear copolymer via an amide bond, maleinimide, azide, or propargyl, wherein the groups formed following the binding of the targeting group are subsequently part of the group X; wherein the content of monomer units of the general formula (II) in the fluorescent polymer is in the range of from at least one monomer unit to 8 mol%, based on the total number of monomer units of the fluorescently labelled polymer; wherein the total content of the monomer units of general formulae (I) and (II) in the fluorescent polymer is at most 10 mol%, based on the total number of monomer units of the fluorescently labelled polymer.
7. Fluorescently labelled polymer according to any one of the preceding claims, wherein the semitelechelic statistical linear copolymer is poly(A-(2-hydroxypropyl)methacrylamide.
8. Fluorescently labelled polymer according to any one of the preceding claims, wherein A is selected from the group comprising ethan- 1,2 -diyl; propan-1, 3-diyl; butan-l,4,-diyl; pentan- 1,5 -diyl; hexan-1,6- diyl; heptan-l,7-diyl; -CH2-C(=O)-NH-CH2-; -CH2-C(=O)-NH-CH(CH2-CH(CH3)2)-C(=O)-NH-CH2-; - CH2-C(=O)-NH-CH(CH2Ph)-C(=O)-NH-CH2-; -CH2-C(=O)-NH-CH(CH2-CH(CH3)2)-C(=O)-NH-
CH(CH2Ph)-C(=O)-NH-CH2-; -CH2-C(=O)-NH-CH(CH2Ph)-C(=O)-NH-CH(CH2-CH(CH3)2)-C(=O)- NH-CH(CH2Ph)-C(=O)-NH-CH2-; -(CH2)4-C(=O)-NH-CH(C(CH3)2)-C(=O)-NH-CH((CH2)3-NH-C(=O)- NH2)-C(=O)-NH-C6H4-CH2-O-C(=O)-; and -(CH2)4-C(=O)-NH-CH(C(CH3)2)-C(=O)-NH-CH((CH2)3-NH-C(=O)-NH2)-C(=O)-.
9. Fluorescently labelled polymer according to any one of the preceding claims, which further comprises at least one monomer unit of a general formula (III)
Figure imgf000043_0001
(III), wherein
A is as defined in claim 1 ;
C is selected from the group comprising -C(=O)-NH-(CH2)a-CH2(OH); -C(=O)-NH-(CH2)b-CH(OH)-CH3; -C(=O)-NH-(CH2)b-CH(OH)-(CH2)c-CH3; and -NH-C(=O)-CH3, wherein a is an integer from 0 to 4, b is an integer from 0 to 3 and c is an integer from 1 to 4; or -S-S-(CH2)2-OH; wherein the content of monomer units of the general formula (III) in the fluorescent polymer is in the range of from at least one monomer unit to 9.9 mol%, based on the total number of monomer units of the fluorescently labelled polymer; wherein the total content of the monomer units of general formulae (I), (II) and (III) in the fluorescent polymer is at most 10 mol. %, based on the total number of monomer units of the fluorescently labelled polymer.
10. Fluorescently labelled polymer according to any one of the preceding claims, which is a linear statistical copolymer of the general formula (IV)
Figure imgf000043_0002
(IV), wherein A, B, C, X and Z are as defined in claims 1, 6 and 9; wherein the fluorophore is as defined in claim 1 ; wherein the total number of monomer units in the fluorescently labelled polymer is in the range of from 100 to 300; and wherein the number of monomer units of the general formula (I) is in the range of from 1 to 30; the number of monomer units of the general formula (II) is in the range of from 0 to 24, and the number of monomer units of the general formula (III) is in the range of from 0 to 29; with the proviso that the total number of the monomer units of general formulae (I), (II), and (III) is at most 30.
11. A method of preparation of the fluorescently labelled polymer according to any one of the claims 1 to 10, characterized in that it contains the following steps:
0) providing monomers selected from the group comprising A-(2-hydroxypropyl)mcthacrylamidc. acrylamide, methacrylamide, acrylate, methacrylate; and providing a monomer of the general formula (V)
Figure imgf000044_0001
(V); wherein A is as defined in claim 1 ;
D is selected from the group comprising carbonyl-thiazolin-2-thione group, (4-nitrophenyl)oxy group, (2,3,4,5,6-pentafluorophenyl)oxy group, (succinimidyl)oxy group, carboxylic group, hydrazide group, azide group, disulfide group and NH2 group, wherein the amino group and the hydrazide group may optionally be protected with a protecting group; i) RAFT polymerization of from 90 to 99.9 mol% of monomers selected from the group comprising A-(2- hydroxypropyl)methacrylamide, acrylamide, methacrylamide, acrylate, methacrylate; and of from 0. 1 to 10 mol% of the monomer of the general formula (V); resulting in a statistical linear copolymer, which contains a linear polymer, selected from the group comprising poly(A-(2-hydroxypropyl)methacrylamide), polyacrylamide, polymethacrylamide, polyacrylate and polymethacrylate, wherein from 0.1 to 10 mol% of monomer units are statistically replaced with monomer units of the general formula (VI)
Figure imgf000044_0002
(VI); wherein A is as defined in claim 1 and D is as defined in step 0); ii) optionally, removing of the protecting groups, if any are present, from the group D of the monomer unit
(VI); 44 iii) binding of the fluorophore to the group D of the statistical linear copolymer using conjugation of carbonyl-thiazolin-2-thione groups, (4-nitrophenyl)oxy groups, (2,3,4,5,6-pentafluorophenyl)oxy groups, (succinimidyl)oxy groups, carboxylic, hydrazide, azide, activated disulfide groups or NH2 groups of monomer units of the general formula (VI) with an amine, carboxylic group, activated carboxylic group, activated disulfide group or keto group of the fluorophore; wherein the fluorophore is as defined in claim 1; iv) optionally, binding of the targeting group to the group D of the monomer of the general formula (VI), wherein the targeting group is as defined in claim 4; v) optionally, binding of the targeting group to at least one end of the linear chain of the fluorescently labelled polymer, wherein the targeting group is as defined in claim 4; vi) removal of the eventually non-reacted thiazolidin-2-thione groups, (4-nitrophenyl)oxy groups, (2,3,4,5,6-pentafluorophenyl)oxy groups, (succinimidyl)oxy groups by their reaction with aminoalcohol, selected from the group comprising NH2-(CH2)a-CH2(OH); NH2-(CH2)b-CH(OH)-CH3; NH2-(CH2)b- CH(OH)-(CH2)C-CH3; wherein a is an integer in the range of from 0 to 4, b is an integer in the range of from 0 to 3 and c is an integer in the range of from 1 to 4, preferably the aminoalcohol is 1-aminopropan- 2-ol, and/or removal of non-reacted NH2 groups by their reaction with acetythiazolidin-2-thione; optionally removal of activated disulfide groups by their reaction with mercaptoethanol; resulting in the formation of group C as defined in claim 9; and thus resulting in the formation of the fluorescently labelled polymer according to any one of the preceding claims 1 to 10.
12. A star-shaped fluorescence polymer, which comprises a multivalent carrier, to which at least one fluorescently labelled polymer according to any one of the preceding claims 1 to 10 is bound, wherein the multivalent carrier is selected from the group comprising poly(amidoamine) dendrimer of the second or third generation, 2,2-bis(hydroxymethyl)propionic dendrimer or dendron of the second, third or fourth generation; polyols containing from 2 to 8 hydroxyl groups; glycerol, pentaerythritol, bis(2- hydroxyethyl)aminotris(hydroxymethyl)methane, dipentaerythritol, porphirine derivatives.
13. The star-shaped fluorescence polymer according to claim 12, which has the general formula (VII),
Figure imgf000045_0001
(VII), 45 wherein A, B, C, Z, X and fluorophore are as defined in claims 1, 6, and 9;
Y is selected from the group comprising primary amino group; primary hydroxyl group; (C3 to C6)alkynyl group; cyklooctynyl group, which may optionally be further independently substituted with one or more groups selected from (Cl to C6)alkyl group and (C6)aryl group; DBCO group;
W is an amide bond between the primary amine group Y of the multivalent carrier and an end carboxylic group of the polymer of the general formula (IV); or an ester bond between the primary hydroxyl group Y of the multivalent carrier and an end carboxylic group of the polymer of the general formula (IV); or a triazole linker, formed by a reaction of an azide end group of the polymer of the general formula (IV) and alkynyl or cycloalkynyl group Y of the multivalent carrier.
14. The star-shaped fluorescence polymer according to claim 12 or 13, which has its molar mass Mn in the range of from 60 000 to 1 000 000 g/mol, wherein the molar mass of the multivalent carrier is at most 50 000 g/mol.
15. A method of preparation of the star-shaped fluorescence polymer according to any one of the claims 12 to 14, characterized in that it contains the following steps:
0) providing of the multivalent carrier, selected from the group comprising poly(amidoamine) dendrimer of the second or third generation, 2,2-bis(hydroxymethyl)propionic dendrimer or dendron of the second, third or fourth generation; polyols containing from 2 to 8 hydroxyl groups; glycerol, pentaerythritol, bis(2- hydroxyethyl)aminotris(hydroxymethyl)methane, dipentaerythritol, porphirine derivatives; and comprising
Y end groups, selected from the group comprising primary amino group; primary hydroxyl group; (C3 to C6)alkynyl group; cyklooctynyl group, which may optionally be further independently substituted with one or more groups selected from (Cl to C6)alkyl group and (C6)aryl group; DBCO group; keto groups, and azide groups; i) preparation according to claim 11 of the statistical linear copolymer, which contains a linear polymer, selected from the group comprising poly(/V-(2-hydroxypropyl)methacrylamide), polyacrylamide, polymethacrylamide, polyacrylate and polymethacrylate, wherein from 0.1 to 10 mol% of monomer units is statistically replaced with monomer units of the general formula (VI); and which contains reactive end groups, preferably azide groups, DBCO, hydrazide groups, amine groups or TT groups; ii) grafting of the reactive end groups of the statistical linear copolymer from step i) to Y groups of the multivalent carrier from step 0), resulting in the formation of the star-shaped polymer, which bears on its pendant arms a statistical linear polymer, selected from the group comprising poly(A'-(2-hydroxypropyl)mcthacrylamidc). polyacrylamide, polymethacrylamide, polyacrylate, and polymethacrylate, and wherein from 0.1 to 10 mol% of monomer units are statistically replaced with monomer units of the general formula (VI); iii) optionally, removing of the protecting groups, if any are present, from the group D of the monomer unit of the general formula (VI); 46 iv) binding of the fluorophore to the group D of the star-shaped polymer from step ii) or iii) using conjugation of carbonyl-thiazolin-2-thione groups, (4-nitrophenyl)oxy groups, (2, 3, 4,5,6- pcntafluorophcnyljoxy groups, (succinimidyl)oxy groups, carboxylic, hydrazide, azide, activated disulfide groups or NH2 groups of monomer units of the general formula (VI) with an amine, carboxylic group, activated carboxylic group, activated disulfide group or keto group of the fluorophore; wherein the fluorophore is as defined in claim 1 ; resulting in the formation of the star-shaped fluorescence polymer, containing monomer units of the general formulae (I) and (VI) and further containing monomer units, selected from the group comprising /V-(2- hydroxypropyl)methacrylamide, acrylamide, methacrylamide, acrylate and methacrylate; v) optionally, binding of the targeting group to the group D of the monomer of the general formula (VI), wherein the targeting group is as defined in claim 4; vi) optionally, binding of the targeting group to at least one end of the linear chain of the fluorescently labelled polymer, wherein the targeting group is as defined in claim 4; vii) removal of the eventually non-reacted thiazolidin-2-thione groups, (4-nitrophenyl)oxy groups, (2,3,4,5,6-pentafluorophenyl)oxy groups, (succinimidyl)oxy groups by their reaction with aminoalcohol, selected from the group comprising NH2-(CH2)a-CH2(OH); NH2-(CH2)b-CH(OH)-CH3; NH2-(CH2)b- CH(OH)-(CH2)C-CH3; wherein a is an integer in the range of from 0 to 4, b is an integer in the range of from 0 to 3 and c is an integer in the range of from 1 to 4, preferably the aminoalcohol is 1 -aminopropan- 2-ol, and/or removal of non-reacted NH2 groups by their reaction with acetythiazolidin-2-thione; optionally removal of activated disulfide groups by their reaction with mercaptoethanol; resulting in the formation of group C as defined in claim 9; and thus resulting in the formation of the star-shaped fluorescence polymer according to any one of the claims 12 to 14.
16. A fluorescence probe for use in diagnostics as a contrast agent for tumour visualization, whole body imaging and/or for image-guided surgery, preferably in fluorescence imaging techniques, more preferably selected from the group comprising non-invasive imaging, image-guided surgery, microscopy, fluorescence flow cytometry, and in diagnostics of tumour, inflammatory and bacterial diseases, wherein the fluorescence probe is selected from the group comprising the fluorescently labelled polymer according to any one of the claims 1 to 10, and the star-shaped fluorescence polymer according to any one of the claims 12 to 14.
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