Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
  • C07D213/79—Acids; Esters
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
  • A61K51/0478—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group complexes from non-cyclic ligands, e.g. EDTA, MAG3
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/083—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the peptide being octreotide or a somatostatin-receptor-binding peptide
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies

Definitions

• Some embodiments relate to improved chelators. Some embodiments relate to improved biological targeting constructs incorporating chelators. Some embodiments relate to chelators coupled to a targeting moiety and capable of binding a radioactive isotope to provide targeted in vivo delivery of the radioactive isotope to a desired location within a mammalian subject.
• Radionuclides have potential utility in cancer diagnosis and therapy, particularly if they can be delivered selectively to a target location within the body of a subject. Targeted delivery of radionuclides can be achieved by using constructs that are engineered to both securely retain the radionuclide for in vivo delivery and deliver the radionuclide selectively to a desired location within the body, with a reasonably low level of delivery to non-target regions of the body. [0004] Targeting constructs have been developed that utilize a targeting moiety that targets a desired region of the body (e.g. a tumor-associated antigen) covalently coupled to a chelator to secure radionuclides for such purposes.
• a targeting moiety that targets a desired region of the body (e.g. a tumor-associated antigen) covalently coupled to a chelator to secure radionuclides for such purposes.
• the targeting moiety can be coupled to the chelator via a linker.
• Such targeting constructs may be referred to as radioimmunoconjugates.
• the radioimmunoconjugate is used to chelate a desired radionuclide for in vivo delivery, for example to provide diagnostic imaging, targeted radionuclide therapy using the construct, or both (i.e. as a theranostic construct).
• the chelator can also be used for in vivo delivery of a suitable companion radionuclide for diagnostics.
• [ 155 Tb]Tb 3+ is part of the so-called ‘Terbium theranostic quartet’ which includes the radioisotopes 149 Tb, 152 Tb, 155 Tb, and 161 Tb, and encompasses both PET and SPECT imaging modalities, and all three therapeutic decay types ( ⁇ , ⁇ – , Meitner-Auger electrons). 21–23 Thus, interchange between different Tb 3+ radioisotopes would provide a true theranostic radiopharmaceutical with identical pharmacokinetic and biodistribution properties. [0009] There is a general desire for improved chelators that are useful in the targeted in vivo delivery of radiometals using suitable targeting constructs.
• the functional group is an ester, an amide, an imide, a thioamide, a thioester, or a guanidinium group.
• a radioisotope targeting construct has the structure (II), wherein the chelator is coupled to a biological targeting moiety through one R 1 group or one R 2 group.
• the functional group is an ester, an amide, an imide, a thioamide, a thioester, or a guanidinium group.
• a radioisotope targeting construct has the structure (III), wherein the chelator is coupled to a biological targeting moiety through one R 1 group, one R 2 group, one R 4 group or one R 5 group.
• the chelator can be coupled to a biological targeting moiety to facilitate the targeted in vivo delivery of a radioisotope.
• the radioisotope can be 227 Th, 225 Ac, 155 Tb, 1 77 Lu, 111 In, 132 La, 235 La, 90 Y, 68 Ga, 44 Sc, 203 Pb, 212 Pb, or the like.
• the chelator has the following structure (6): ( 6) .
• Methods of administering an in vivo radioisotope targeting construct comprising the chelator are provided.
• the in vivo radioisotope targeting construct can be administered to a mammalian subject.
• the targeting moiety of the in vivo radioisotope targeting construct can be used to enhance accumulation of the radioisotope at a selected location within the body (e.g. the location of cancerous cells), relative to other locations in the body.
• An imaging procedure can be carried out to evaluate the localization of the in vivo radioisotope targeting construct within the body.
• the in vivo radioisotope targeting construct can be used to cause cell death at the selected location within the body by exposing the cells to radiation from the radioisotope.
• the cells can be cancer cells.
• the mammalian subject can be a human.
• FIG.1 shows the structure of an exemplary in vivo targeting chelate construct.
• FIG.2 shows the chemical structure of H 4 noneunpaX (6) and the 1 H assignment (left side) and 13 C assignment (right side).
• FIG.3 shows the 1 H NMR spectrum of H 4 noneunpaX . 4HCl (6) (400 MHz, D 2 O, 298 K).
• FIG.4 shows 13 C ⁇ 1 H ⁇ NMR spectrum of H 4 noneunpaX .
• FIG.5 shows 1 H – 1 H COSY NMR spectrum of H 4 noneunpaX (6) (300 MHz, D 2 O, 298 K).
• FIG.6 shows 1 H – 13 C HSQC NMR spectrum of H 4 noneunpaX 4HCl (6) (300 MHz, 75 MHz, D 2 O, 298 K).
• FIG.7 shows 1 H – 13 C HMBC NMR spectrum of H 4 noneunpaX 4HCl (6) (300 MHz, 75 MHz, D 2 O, 298 K).
• FIG.9 shows 1 H NMR spectrum of [La(noneunpaX)]- (400 MHz, D 2 O, 298 K, pH 7), and includes the chemical structure of [La(noneunpaX)]- with 1 H assignments indicated.
• FIG.10 shows 1 H – 1 H COSY NMR spectrum of [La(noneunpaX)]- (400 MHz, D 2 O, 298 K, pH 7.0).
• FIG.11 shows 1 H – 1 H COSY NMR spectrum of [La(noneunpaX)]- (400 MHz, D 2 O, 298 K, pH 7.0) (alkyl expansion).
• FIG.12 shows 1 H – 1 H COSY NMR spectrum of [La(noneunpaX)]- (400 MHz, D 2 O, 298 K, pH 7.0) (aromatic expansion).
• FIG.13 shows 1 H NMR spectrum of [Lu(noneunpaX)]- (400 MHz, D 2 O, 298 K, pH 7.0) and includes the chemical structure of [Lu(noneunpaX)]- with 1 H assignments indicated.
• FIG.14 shows 1 H – 1 H COSY NMR spectrum of [Lu(noneunpaX)]- (400 MHz, D 2 O, 298 K, pH 7.0).
• FIG.15 shows 1 H – 1 H COSY NMR spectrum of [Lu(noneunpaX)]- (400 MHz, D 2 O, 298 K, pH 7.0) (alkyl expansion).
• FIG.16 shows 1 H – 1 H COSY NMR spectrum of [Lu(noneunpaX)]- (400 MHz, D 2 O, 298 K, pH 7.0) (aromatic expansion).
• FIG.17 shows 1 H NMR spectrum of [In(noneunpaX)]- (400 MHz, D 2 O, 298 K, pD 4.5) and includes the chemical structure of [In(noneunpaX)]- with 1 H assignments indicated.
• FIG.18 shows 1 H – 1 H COSY NMR spectrum of [In(noneunpaX)]- (400 MHz, D 2 O, 298 K, pD 4.5).
• FIG.20 shows a speciation diagram of H 4 noneunpaX as a function of pH, dotted line indicates pH 7.4.
• FIG.28 shows concentration-dependent radiolabelling studies of H 4 noneunpa, H 4 noneunpaX, and DOTA with: [ 44 Sc]Sc 3+ (1.2 MBq) in NaOAc (0.1 M, pH 4.5) (panel (A)), [ 111 In]In 3+ (1.0 MBq) in NH 4 OAc (0.5 M, pH 5.8) (panel (B)), [ 155 Tb]Tb 3+ (40 kBq) in NH 4 OAc (0.5 M, pH 6.0) (panel (C)), [ 177 Lu]Lu 3+ (150 kBq) in NH 4 OAc (0.5 M, pH 6.0) (panel (D)), [ 213 Bi]Bi 3+ (680 kBq) in MES (1.0 M, pH 5.5), (F) [ 225 Ac]Ac 3+ (40 kBq) in NH 4 OAc (1.0 M, pH 7.3) (panel (E)).
• FIG.29 shows human serum stability studies of H 4 noneunpa and H 4 noneunpaX with: [ 111 In]In 3+ (54 GBq/ ⁇ mol) (panel (A)), [ 155 Tb]Tb 3+ (1.0 GBq/ ⁇ mol) (panel (B)), [ 177 Lu]Lu 3+ (2.0 GBq/ ⁇ mol) (panel (C)), [ 225 Ac]Ac 3+ (134 MBq/ ⁇ mol) (panel (D)).
• FIG.30 shows DFT-optimised structures of [La(noneunpaX)]- in panel (A) (left-hand orientation), and panel (B) front orientation, and [Lu(noneunpaX)]- in panel (c) (left-hand orientation), and panel (D) (front orientation). Selected hydrogens have been omitted for clarity.
• FIG.31 shows concentration – dependent radiolabelling of bifunctional H 4 noneunpaX with: (A) [ 44 Sc]Sc 3+ (400 kBq) in NaOAc (0.1 M, pH 4.5), (B) [ 111 In]In 3+ (1.06 MBq) in NH 4 OAc (0.5 M, pH 5.5), (C) [ 177 Lu]Lu 3+ (350 - 500 kBq) in NH 4 OAc (0.5 M, pH 6.0), (D) [ 133/135 La]La 3+ (400 kBq) in NH 4 OAc (0.2 M, pH 7.0), (E) [ 155 Tb]Tb 3+ (140 kBq) in NH 4 OAc (0.5 M, pH 7.0) and (F) [ 225 Ac]Ac 3+ (40 kBq) in NH 4 OAc (0.5 M, pH 7.2).
• FIG.32 shows a molar activity study of H 4 noneunpaX-Bn-NH 2 with [ 177 Lu]LuCl 3 (20 MBq) in NH 4 OAc (0.5 M, pH 6.0) at RT monitored over 10 minutes.
• Highest molar activity 250 GBq/ ⁇ mol
• ligand-to-metal ratio [L]:[M]; [174:1]
• Human serum stability challenge of [ 177 Lu][Lu(noneunpaX-Bn-NH 2 ]- (5.2 GBq/ ⁇ mol) conducted at 37 °C and monitored over 7 days via radio-iTLC (n 3) (right panel).
• FIG.33 shows radiolabelling studies of bifunctional H 4 noneunpaX and corresponding peptide-conjugates:
• A Concentration-dependent radiolabelling with [ 225 Ac]Ac 3+ (40 kBq) in NH 4 OAc buffer (0.5 M, pH 7) (RT, 10 min.).
• B Human serum stability challenge of [ 225 Ac]Ac 3+ -labelled compounds.
• C Concentration-dependent radiolabelling with [ 155 Tb]Tb 3+ (120 kBq) in NH 4 OAc buffer (0.5 M, pH 6) (RT, 10 min.).
• D Human serum stability challenge of [ 155 Tb]Tb 3+ -labelled bioconjugates.
• FIG.34 shows radio-HPLC traces of [ 155 Tb]Tb 3+ control and [ 155 Tb][Tb 3+ labelled H 4 noneunpaX-Bn-NH 2 and H 4 noneunpaX-Bn-NCS.
• FIG.35 shows radio-HPLC traces for [ 155 Tb][Tb(noneunpaX-Ahx-Tyr 3 -TATE)], Method: A: H 2 O (0.1% TFA), B: MeCN (0.1% TFA); 100% A to 60% B over 15 min., 1 mL/min.
• FIG.36 shows radio-HPLC traces for [ 155 Tb][Tb(noneunpaX-PEG 2 -Tyr 3 -TATE)], Method: A: H 2 O (0.1% TFA), B: MeCN (0.1% TFA); 100% A to 60% B over 15 min., 1 mL/min.
• FIG.37 shows coronal views of maximum intensity projections (MIPs) from quantitative dynamic SPECT/CT scans at 0 – 1 h post administration of [ 155 Tb][Tb(noneunpaX-Ahx-Tyr 3 -TATE)] (top) and [ 155 Tb][Tb(noneunpaX-PEG 2 -Tyr 3 -TATE)] (bottom) in NRG mice bearing AR42J exocrine tumour xenografts (left shoulder).
• MIPs maximum intensity projections
• FIG.38 shows sagittal views of maximum intensity projections (MIPs) from SPECT/CT scans at 1, 2 and 4 h post administration of [ 155 Tb][Tb(noneunpaX-Ahx-Tyr 3 - TATE)] (top) and [ 155 Tb][Tb(noneunpaX-PEG 2 -Tyr 3 -TATE)] (bottom) in NRG mice bearing AR42J pancreatic exocrine tumour xenografts (left shoulder).
• MIPs maximum intensity projections
• FIG.39 shows representative time-activity plots of (A) [ 155 Tb][Tb(noneunpaX-Ahx- Tyr 3 -TATE)] and (B) [ 155 Tb][Tb(noneunpaX-PEG-Tyr 3 -TATE)] in NRG mice bearing AR42J exocrine/pancreatic tumour xenografts.
• Standardised uptake values (SUVs) were extracted for ROIs in relevant organs from calibrated SPECT/CT images.
• FIG.40 shows decay-corrected biodistribution studies of [ 155 Tb][Tb(noneunpaX-Ahx- Tyr 3 -TATE)] at 2 h and 4 h post-injection and [ 155 Tb][Tb(noneunpaX-PEG 2 -Tyr 3 -TATE)] at 4 h post-injection in NRG mice bearing AR42J exocrine/pancreatic tumour xenografts.
• H 4 noneunpaX a novel chelating ligand, H 4 noneunpaX, having an inverted arrangement of functional groups, and have compared the characteristics of this novel ligand with H 4 noneunpa.
• the ligand displays preferential complexation with trivalent Ln 3+ ions, forming single isomeric species in solution, which exhibits high thermodynamic stability and kinetic inertness.
• H 4 noneunpaX having the structure (6) was prepared and comparisons to the symmetric counterpart H 4 noneunpa/H 4 oxyaapa were assessed.
• a bifunctional analogue of H 4 noneunapX was prepared, to firstly demonstrate the synthetic accessibility of bifunctional asymmetric/inverted chelators, and to investigate their performed in vivo.
• prophylaxis includes preventing, minimizing the severity of, or preventing a worsening of a condition.
• the terms treat or treatment include reversing or lessening the severity of a condition.
• the term antibody includes all forms of antibodies including polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, multimeric antibodies, and the like.
• the term antigen binding fragment of an antibody refers to any portion of an antibody that is capable of binding to an antigen and includes by way of example only and without limitation Fab fragments, F(ab’) 2 fragments, Fv fragments, scFv fragments, minibodies, diabodies, and the like. Reference to a specific antibody includes reference to any antibodies that are determined to be biosimilar to that specific antibody by any regulatory authority.
• peptidomimetic means a small protein-like molecule designed to mimic a peptide, and includes without limitation modified peptides, peptidic foldamers, structural mimetics and mechanistic mimetics.
• a chelator composition for radiometals is disclosed. A method of using and making the composition is also disclosed. The composition can be used as a therapeutic and/or diagnostic agent.
• the inventors have now determined that chelators having the general structure (6) can coordinate radioisotopes under mild conditions and produce a complex that is stable under in vivo conditions, making such chelators particularly suitable for example for application in radiotherapeutic, diagnostic and/or theranostic constructs.
• H 4 noneunpaX can be directly coupled to a biological targeting moiety, optionally with a linker interposing the H 4 noneunpaX and the biological targeting moiety, by coupling the biological targeting moiety or linker directly to one of the carboxyl groups of structure (6).
• one or more of the oxygen atoms of the carboxyl group is substituted by a different heterotatom, e.g. N or S.
• the functional group provided on the bifunctional H 4 noneunpaX chelator to couple the chelator to the biological targeting moiety can be a carboxyl, an ester, an amide, an imide, a thioamide, a thioester, a guanidinium, or the like.
• the functional group is a carboxyl, an ester, an amide, an imide, a thioamide, a thioester, a guanidinium, or the like.
• a radioisotope targeting construct has the structure (II), wherein one of the R 1 groups or one of the R 2 groups is a biological targeting moiety.
• each R 5 when both R 4 together form a cyclohexyl moiety, each R 5 is independently H or a functional group. In some embodiments, when both R 5 together form a cyclohexyl moiety, each R 4 is independently H or a functional group. In some embodiments, the functional group is an ester, an amide, an imide, a thioamide, a thioester, or a guanidinium group or the like. In some embodiments, a radioisotope targeting construct has the structure (III), wherein the chelator is coupled to a biological targeting moiety through one R 1 group, one R 2 group, one R 4 group or one R 5 group.
• a radioisotope targeting construct has the structure (III), wherein one of the R 1 groups, one of the R 2 groups, one of the R 4 groups or one of the R 5 groups is a biological targeting moiety. ( III) .
• the chelator has one of the structures shown below: [0073] It has been found by other groups, for example Milenic et al. 58 , that DTPA derivatives comprising a cyclohexyl moiety in the backbone of the chelator show comparable specific activities when incorporated into a chelate-antibody conjugate.
• an in vivo targeting chelate construct 120 has a targeting moiety 122 coupled to a chelator 126.
• a linker 124 interposes targeting moiety 122 and chelator 126.
• targeting moiety 122, linker 124 (if present) and chelator 126 comprise in vivo targeting construct 130.
• chelator 126 is used to chelate a radionuclide 128 that is suitable for in vivo imaging and/or radiotherapy.
• Radionuclide 128 together with in vivo targeting construct 130 provides an in vivo targeting chelate construct 120 suitable for targeted in vivo delivery of the radionuclide 128 payload as assisted by targeting moiety 122.
• Any moiety suitable for directing the targeted delivery of in vivo targeting chelate construct 120 in vivo can be used as targeting moiety 122.
• the targeting moiety 122 of the targeting construct 120 is a hapten, antigen, aptamer, affibody molecule, enzyme, protein, peptide, antibody, antigen-binding fragment of an antibody, peptidomimetic, receptor ligand, steroid, hormone, growth factor, cytokine, molecule that recognizes cell surface receptors (including molecules involved in growth, metabolism or function of cells), lipid, lipophilic group, carbohydrate, or any other molecule or targeting component capable of selectively directing a construct to a specific location within the body.
• the targeting moiety can be produced in any suitable manner, e.g. as a biologic, semisynthetically, or synthetically.
• Examples of targeting moieties that have been developed to deliver radioisotope targeting constructs to desired locations within the body of a mammalian subject in vivo include antibodies targeting specific markers associated with specific types of cancers, peptidomimetics targeting proteins that are highly expressed in cancer cells, and the like.
• Exemplary non-limiting examples of suitable targeting moieties are listed in Table 1 (Lau, J.; Rousseau, E.; Kwon, D.; Lin, K.; Benard, F.; Chen, X., Insight into the Development of PET Radiopharmaceuticals for Oncology. Cancers 2020, 12, 1312, the entirety of which is incorporated by reference herein).
• targeting moieties selectively interact with biological targets, including antigens, proteins, carbohydrates or other molecules present on the surface of cells that are overexpressed in cancer cells relative to normal cells, e.g. tumor-associated antigens.
• biological targets including antigens, proteins, carbohydrates or other molecules present on the surface of cells that are overexpressed in cancer cells relative to normal cells, e.g. tumor-associated antigens.
• suitable targets are listed in Table 1. Suitable targets and/or targeting moieties for radiopharmaceuticals, whether now known or discovered or developed in the future, would be known to a person skilled in the art.
• targeting moiety 122 is an antibody or an antigen-binding fragment of an antibody.
• targeting moiety 122 is a peptidomimetic.
• the targeting moiety 122 is one of the targeting moieties listed in Table 1, with any chelator present in the referenced molecule replaced by H 4 noneunpaX as the chelator. In some embodiments, the targeting moiety 122 interacts selectively with one of the targets listed in Table 1. Table 1. Exemplary targeting moieties and biological targets for targeted radiation therapy.
• linker 124 can be used as linker 124 to couple chelator 126 to targeting moiety 122.
• suitable linkers can include: • a hydrocarbon linker containing between 1 and 10 carbon atoms (C1-C10), including 2, 3, 4, 5, 6, 7, 8 or 9 carbon atoms that is optionally saturated or unsaturated, optionally substituted with one or more heteroatoms or having one or more substituents; the hydrocarbon linker can be linear, cyclic and/or branched, e.g.8- aminooctanoic acid, 6-aminohexanoic acid; • an aromatic linker containing an aromatic moiety such as a benzyl group, e.g.
• aminophenylacetic acid • an amino acid linker having between 1 and 10 amino acid residues, including 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues, any one or more of which may be naturally occurring amino acid residues, D-amino acid residues or other non-naturally occurring residues, examples of which include GlyGly, GluGluGlu, GlySerGlySer; • a cyclized linker, or cyclized ring structure, optionally a cyclized amino acid linker, e.g. aminocyclohexanecarboxylic acid; • a PEG-linker of any suitable length; • cationic linkers, whether formed from amino acid residues or other residues, e.g..
• Pip 4-(2-aminoethyl)-1-carboxymethyl-piperazine (Acp); • anionic linkers, whether formed from amino acid residues or other residues, e.g. AspAsp, GluGlu; • a carbohydrate containing linker; • click chemistry linkers (triazoles); • any other suitable linker; • or combinations or modifications of the foregoing.
• linkers that have been developed in the art for other radiopharmaceutical targeting constructs are known to those skilled in the art.
• Hydrophilic or charged linkers such as PEG-linkers or cationic/anionic linkers may be used to increase the overall water solubility of the targeting construct.
• linkers that have been developed in the art for other radiopharmaceutical targeting constructs are described, by way of example only and without limitation, by Bene ⁇ ová et al., Barnaski et al. and Kuo et al.
• a construct such as construct 120 is prepared by carrying out suitable reactions to couple targeting moiety 122 and chelator 126, for example via suitable chemical reaction, to yield an in vivo targeting construct 130, optionally with linker 124 interposing targeting moiety 122 and chelator 126.
• the radionuclide 128 is then added and bound to chelator 126, e.g. at a later time and in a hospital or clinic setting, to form the desired in vivo targeting metal chelate construct 120.
• radionuclide 128 could be first chelated with chelator 126, and then chelator 126 is conjugated with targeting moiety 122 in any suitable manner to yield in vivo targeting chelate construct 120.
• the radionuclide 128 is bound to chelator 126 (including as part of construct 130) under mild temperature conditions, e.g. less than about 65°C, 60°C, 55°C, 50°C, 45°C, 40°C, 35°C or 30°C.
• the mild temperature conditions are between about 10°C and 65°C, including any value or subrange therebetween, e.g.15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C or 60°C.
• the radionuclide 128 is conjugated to chelator 126 or construct 130 at room temperature, i.e.
• the radionuclide 128 or construct 130 is combined with chelator 126 to form a metal chelate under mild pH conditions, e.g. between about 5.0 and about 7.4, including any value or subrange therebetween, e.g.5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0 or 7.2.
• the radionuclide 128 is conjugated to chelator 126 at approximately neutral pH, i.e.
• a pH of approximately 7.0 e.g. between about 6.8 and 7.2 including any value therebetween, e.g.6.9, 7.0 or 7.1.
• the radionuclide 128 is conjugated to chelator 126 at approximately physiological pH, i.e. at approximately pH 7.4, e.g. between about 7.2 and 7.6 including any value therebetween, e.g.7.3, 7.4 or 7.5.
• radionuclide 128 is combined with chelator 126 or construct 130 in aqueous solution.
• the aqueous solution is free or substantially free of alcohol such as ethanol.
• the radionuclide 128 is combined with chelator 126 or construct 130 for an incubation period to allow a chelated metal complex to form.
• the incubation period is between about 5 minutes and about 6 hours, including any period therebetween, e.g.10, 15, 20, 25, 30, 45, 60 or 90 minutes, or 2, 3, 4 or 5 hours.
• the incubation period is between about 5 minutes and about 30 minutes.
• the concentration of chelator 126 or construct 130 that is present when conjugated to radionuclide 128 is between about 10 -4 to 10 -7 M, including any value therebetween, e.g.10 -5 or 10 -6 M.
• in vivo radioisotope targeting chelate construct 120 is present in mammalian serum, optionally in human serum. In some embodiments, in vivo radioisotope targeting chelate construct 120 is stable in mammalian serum, optionally in human serum.
• in vivo radioisotope targeting chelate construct 120 is present in mammalian serum within the body of a mammal, optionally in human serum within the body of the human. In some embodiments, in vivo radioisotope targeting chelate construct 120 is present in mammalian blood, optionally in human blood. In some embodiments, in vivo radioisotope targeting chelate construct 120 is present in mammalian blood within the body of a mammal, optionally in human blood in the body of the human. In some embodiments, in vivo radioisotope targeting chelate construct 120 is present within the body of a mammal, optionally the body of a human.
• in vivo radioisotope targeting chelate construct 120 is present in a mammalian cell, optionally a human cell.
• radionuclide 128 is delivered to a selected location within the body of a mammalian subject by administering to the subject an in vivo radioisotope targeting chelate construct 120 incorporating the radionuclide 128 and a targeting moiety 122 that specifically directs the in vivo radioisotope targeting chelate construct 120, including the bound radionuclide 128, to the selected location within the body of the subject.
• the method includes allowing the targeting moiety 122 to enhance the accumulation of the in vivo radioisotope targeting chelate construct 120 at the selected location within the body relative to other locations in the body to selectively deliver a dose of radiation to the selected location.
• the in vivo radioisotope targeting chelate construct 120 is used to cause cell death at the selected location by delivering a targeted dose of radiation.
• the cells that are killed at the selected location are cancer cells.
• the radiation is alpha radiation; beta or gamma radiation could be used in other embodiments.
• in vivo radioisotope targeting chelate construct 120 is internalized by a cell within the mammalian subject, for example by endocytosis or otherwise.
• in vivo radioisotope targeting chelate construct 120 is present within a mammalian cell.
• the in vivo radioisotope targeting chelate construct 120 is present within a human cell.
• the in vivo radioisotope targeting chelate construct 120 is prepared prior to administration of construct 120 to a subject by combining an in vivo radioisotope targeting construct 130 having a targeting moiety 122, a chelator 126 and optionally a linker 124 with a radionuclide 128 to form the in vivo radioisotope targeting chelate construct 120.
• the combining is carried out at a mild temperature, e.g.
• the combining is carried out at a mild pH, e.g. an approximately neutral pH or an approximately physiological pH.
• the mild pH is a pH of between about 5.0 and about 7.4, including any value therebetween e.g.5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0 or 7.4. In some embodiments, the mild pH is approximately 6.0. In some embodiments, the combining is carried out a physiological pH, e.g. in the range of about. 7.0 to 7.4 including any value therebetween, e.g.7.1, 7.2 or 7.3. In some embodiments, radionuclide 128 is combined with in vivo radioisotope targeting construct 130 in aqueous solution. In some embodiments, the aqueous solution is free or substantially free of alcohols such as ethanol.
• in vivo targeting chelate construct 120 is used in diagnostic applications.
• in vivo targeting chelate construct 120 may be administered to a subject in any suitable manner, and any suitable imaging technology or procedure may be used to evaluate the localization of the targeting chelate construct 120 within the body via targeting moiety 122 by visualizing the location of bound radionuclide 128, e.g. positron emission tomography (PET) imaging or single-photon emission computerized tomography (SPECT) imaging.
• PET positron emission tomography
• SPECT single-photon emission computerized tomography
• Such imaging procedures can be carried out for example to diagnose a subject as having a particular disorder or type of cancer, or to localize regions of the subject’s body affected by the particular disorder or type of cancer.
• localization of targeting chelate construct 120 to a target organ, region or plurality of loci within the body as evaluated by such imaging technology may be indicative that the subject has a particular form of cancer, and/or can be used to evaluate the extent of the cancer and or locations within the body wherein cancerous cells are or may be located, and/or can be used to evaluate the extent of metastasis of the cancer.
• constructs such as targeting chelate construct 120 are used in therapeutic applications, for example to carry out targeted radionuclide therapy.
• targeting chelate construct 120 may be administered to a subject in any suitable manner, and the targeting effect imparted by targeting moiety 122 can be used to deliver the chelated radionuclide 128 to a desired location within the subject’s body.
• radiation from radionuclide 128 is used to kill cells at the desired location.
• the cells that are killed at the desired location are cancer cells.
• targeting construct 120 is used to perform targeted radionuclide therapy.
• targeting construct 120 is used to perform targeted alpha therapy.
• a pharmaceutical composition is provided, the pharmaceutical composition comprising a construct such as targeting construct 120 and a pharmaceutically acceptable carrier.
• the pharmaceutical composition may include any suitable excipient, vehicle, buffer, diluent, binder, thickener, lubricant, preservative or the like, and may be provided in any desired state, e.g. as a liquid, suspension, emulsion, paste, or the like.
• the pharmaceutical composition can be administered in any suitable manner, e.g. orally, intravenously, intramuscularly, subcutaneously, intraperitoneally, intratumorally, by inhalation, or the like.
• a method of prophylaxis and/or treatment of a subject having or believed to have cancer is provided.
• the method comprises administering an in vivo targeting chelate construct 120 or a pharmaceutical composition comprising such a targeting chelate construct 120 to the subject. In some embodiments, the method comprises administering a therapeutically and/or prophylactically effective amount of the targeting chelate construct 120 to the subject.
• the subject is a mammal. In some embodiments, the subject is a human. In alternative embodiments, the subject is livestock or a pet, e.g. a horse, cow, sheep, goat, cat, dog, rabbit, or the like. In some embodiments, the subject is a monkey.
• the metals that can be used as metal 128 include actinides, lanthanides, rare earth metals, or main group metals.
• the lanthanide is La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb or Lu.
• the lanthanide is Gd, Lu, Pr, Nd, Ho, Er or Yb. In some embodiments, the lanthanide is a radiolanthanide. In some embodiments, the actinide is Ac, Th, Pa, U, Np, Pu, Am, Cm, Bk, Cf, Es, Fm, Md, No or Lr. In some embodiments, the actinide is Ac, Th or U. In some embodiments, the actinide is a radioactinide. In some embodiments, the rare earth metal is Sc, Y, La, Ce, Pr, Nd, Pm, Sm, Gd, Tb, Dy, Ho, Er, Tm, Yb or Lu.
• the metal is a trivalent lanthanide ion.
• the metal is a radioisotope.
• the radioisotope is any desired radioisotope, e.g.
• the metal is actinium (Ac), lutetium (Lu), bismuth (Bi), gallium (Ga), indium (In), terbium (Tb), thorium (Th), or Caesium (Cs).
• the metal is actinium (III) (Ac 3+ ), lutetium (III) (Lu 3+ ), bismuth (III) (Bi 3+ ), gallium (III) (Ga 3+ ), indium (III) (In 3+ ), terbium (Tb 3+ ), thorium (III) (Th 3+ ), or Cesium (I) (Cs 1+ ) .
• the metal is 225 Ac, 177 Lu, 213 Bi, 232 Th, 230 Th, 228 Th, 68 Ga, 161 Tb, 155 Tb, 152 Tb, 149 Tb, 111 In, or 137 Cs. In some embodiments, the metal is 227 Th, 225 Ac, 155 Tb, 177 Lu, 111 In, 132 La, 235 La, 90 Y, 68 Ga, 44 Sc, 203 Pb, or 212 Pb. [0097] In some embodiments, H 4 noneunpaX is bound to a metal ion to form a coordination complex. In some embodiments, the coordination complex is referred to as a metal chelate.
• the metal chelate or H 4 noneunpaX as the chelating ligand is associated with one or more cations as counter ions, for example Na + , K + , Ca 2+ or the like.
• the metal chelate or the chelating ligand is fully protonated.
• the metal chelate or the chelating ligand is in its free acid form.
• the metal chelate or the chelating ligand is in a partially protonated state.
• the coordination complex is present in mammalian serum, optionally human serum. In some embodiments, the coordination complex is stable in mammalian serum, optionally human serum.
• the coordination complex is present in mammalian serum within the body of the mammal, optionally present in human serum within the body of the human.
• the coordination complex is present in blood, optionally human blood.
• the coordination complex is stable in mammalian blood, optionally human blood.
• the coordination complex is present in mammalian blood within the body of the mammal, optionally present in human blood within the body of the human.
• the coordination complex is present within the body of a mammal, optionally present within the body of a human.
• the coordination complex is present within a cell of a mammalian subject, optionally present within a cell of a human subject. Examples [0099] Further embodiments are described with reference to the following examples, which are intended to be illustrative and not limiting in nature.
• Example 1.0 - Synthesis and Characterisation of H 4 noneunpaX [0100] The nonadentate chelating ligand, H 4 noneunpa, has been previously reported 8,13 and was synthesised following established procedures. 8 Preparation of H 4 noneunpaX was carried out in accordance with the linear synthetic route outlined in Scheme 1.
• Scheme 1. Synthesis of H 4 noneunpaX . 4HCl . 5H 2 O from commercially available 2-(2- aminoethoxy)ethan-1-ol, over 5 steps with a cumulative yield of 33%.
• Methyl (6-bromomethyl) picolinate (515 mg, 2.824 mmol) was added to a solution of di-tert-butyl 2,2'-((2-(2-aminoethoxy)ethyl)azanediyl) diacetate (4) (372 mg, 1.12 mmol) in dry MeCN (15 mL) under argon.
• Diisoproylethylamine (585 ⁇ L, 434 mg, 3.36 mmol) was added and the resulting pale-yellow solution stirred for 1 h at ambient temperature. The reaction mixture was subsequently heated to 50 °C and stirred overnight.
• the NMR spectra for the Lu 3+ complex reveal a single, asymmetric isomer, wherein chemically distinct resonances for each picolinic acid donor arm are seen in the aromatic region.
• one picolinate donor arm H l , H m , H n
• the second picolinate arm H h , H i , H j
• exhibits a significant downfield shift indicating closer coordination to the Lu 3+ metal centre and a change in ligand conformation at this position.
• the Lu 3+ complex shows further splitting for all eight protons in the backbone, with large differences in chemical shift for mutually coupled protons (e.g. Hc and H c’ ) which suggests a rigid complexation environment and close coordination to the metal centre.
• Hc and H c mutually coupled protons
• the metal complexes of H 4 noneunpaX share similar spectral characteristics to those reported for H 4 noneunpa; whereby fully saturated metal coordination spheres were achieved with La 3+ and Lu 3+ ions, as indicated by diastereotopic splitting of the pendent donor arm resonances and backbone ethylene bridges.
• H 4 noneunpaX The larger difference in basicity between the terminal amines of H 4 noneunpaX, in comparison to H 4 noneunpa/Oxyaapa, may be advantageous for metal ion coordination, whereby the less basic dipyridyl nitrogen favours complexation at a lower pH, while the more basic iminodiacetate nitrogen will act as a stronger donor group to metal ions.
• affinity of H 4 noneunpaX towards metal ions of medicinal importance e.g.
• a KML defined as [ML]/([M][L]); KMHL defined as [MHL]/([ML][H]); KM(OH)L defined as [M(OH)L]/([ML][OH]) (charges omitted for clarity).
• H 4 noneunpaX shows a high thermodynamic preference for complexation with trivalent Ln 3+ ions, as indicated by the high stability constants (log K ML ) and pM values, Table 4. Notably, these values are directly comparable to those previously determined for H 4 noneunpa/ Oxyaapa, 13 with H 4 noneunpaX exhibiting marginally higher stability constants across the Ln 3+ series, implying a small thermodynamic benefit to this inverted structural arrangement (which may be a result of a degree of preorganisation of the binding cavity of the ligand).
• pK a 6.17(3)
• the formation of hydroxo species is observed at higher pH, which without being bound by theory is accounted for by considering the mismatch between the size of these small metal ions and the ligand binding cavity.
• the electrode was calibrated daily in hydrogen ion concentration by direct titration of HCl with freshly prepared NaOH solution, and the results were analysed with the Gran procedure 55 to determine the standard potential (E°) and the ionic product of water (pKw), at 298 K, with 0.16 M NaCl as a supporting electrolyte. Solutions under study were titrated with carbonate- free NaOH solution ( ⁇ 0.16 M) which was standardized against freshly recrystallized potassium hydrogen phthalate.
• Speciation diagrams were generated with the calculated protonation constants and stability constants using Hyss software.
• a complex containing a metal ion, M, proton, H and ligand, L has the general formula M p H q L r .
• the stoichiometric index p may also be 0 in the case of protonation equilibria, and negative values of q refers to proton removal from coordinated water, equivalent to hydroxide ion addition during formation of the complex.
• the overall equilibrium constant for the formation of the complexes M p H q L r from its components is designated as log ⁇ .
• Stepwise equilibrium constants log K correspond to the difference in log units between the overall constants of sequentially protonated (or hydroxide) species.
• Radiolabelling studies with [ 44 Sc]Sc 3+ , [ 111 In]In 3+ , [ 177 Lu]Lu 3+ , [ 155 Tb]Tb 3+ , [ 213 Bi]Bi 3+ and [ 225 Ac]Ac 3+ have been performed to investigate the variation in metal ion affinity with changes in ionic radii across a broad size range and coordination number.
• TAT targeted alpha therapy
• Suitable companion radionuclides for [ 225 Ac]Ac 3+ with comparable coordination characteristics and imageable decay properties are required to perform accurate staging of disease progression and assessment of patient suitability for treatments.
• Concentration-dependent radiolabelling studies have shown H 4 noneunpaX to be a highly versatile chelator, exhibiting high affinity for all metal ions tested, with the exception of [ 213 Bi]Bi 3+ , FIG.28.
• H 4 noneunpaX showed comparable coordination characteristics to H 4 noneunpa; each chelate was successfully radiolabelled at high molar activities with [ 111 In]In 3+ (54 GBq/ ⁇ mol), [ 155 Tb]Tb 3+ (1.0 GBq/ ⁇ mol), [ 177 Lu]Lu 3+ (2.0 GBq/ ⁇ mol) and [ 225 Ac]Ac 3+ (134 MBq/ ⁇ mol).
• Concentration-dependent radiolabelling studies of H 4 noneunpa and H 4 noneunpaX with [ 44 Sc]Sc 3+ showed poor radiometal ion compatibility; whereby low RCYs were achieved at high ligand concentration.
• Bi 3+ is also known to exhibit a stereochemically active 6s 2 lone pair in some of its coordination complexes, which can have significant impacts on the favoured conformational geometries of the chelating ligand and their effective denticities.
• N-alkylation of sulfonamide (7) with methyl (6-bromomethyl)picolinate under basic conditions was achieved through mild heating of the reaction mixture overnight, to generate the singly alkylated product, cleanly in high yields. Removal of the Nosyl-protecting group was performed upon treatment of compound (8) with thiophenol to give the corresponding secondary amine (9). An excess of thiophenol (3 equiv.) was found to be necessary to achieve complete deprotection of compound (8) and thus avoid tedious purification of the desired product.
• Scheme 3 Synthetic scheme of alkyne-functionalised picolinate electrophile (S1).
• Scheme 4. Synthetic scheme for the preparation of clickable bifunctional handle (S2).
• Triethylamine (2.00 mL, 1.45 g, 14.3 mmol, 2 equiv.) was added to the reaction mixture, followed by slow addition of 2- nitrobenzenesulphonyl chloride (1.59 g, 7.16 mmol).
• the resulting pale-yellow solution was stirred at 0 °C for 1 h, then allowed to warm to RT and stirred for a further 5 h.
• the reaction mixture was diluted with CH 2 Cl 2 (40 mL) and extracted with de- ionised H 2 O (2 x 50 mL) and brine (50 mL).
• reaction mixture was cooled to room temperature, and the inorganic salts removed via centrifuge.
• the separated salts were washed with CH 2 Cl 2 (3 x 10 mL) and the combined organic phase evaporated in vacuo.
• the resulting residue was re-dissolved in CH 2 Cl 2 (25 mL) and washed with de-ionised H 2 O (2 x 25 mL) and brine (25 mL).
• the volatiles were removed in vacuo and the crude product purified via silica gel chromatography (Combiflash automated purification system; A: CH 2 Cl 2 , B: MeOH; 100% A to 5% B).
• the title product was attained as a pale-yellow oil (249 mg, 88%).
• the resulting aqueous phase was diluted with de-ionised H 2 O (10 mL) and extracted with CH 2 Cl 2 (3 x 15 mL). The combined organic phase was washed with de-ionised H 2 O (10 mL) and brine (10 mL). The volatiles were removed in vacuo and the crude material purified via silica gel chromatography (Combiflash automated purification system; A: CH 2 Cl 2 , B: MeOH; 100% A to 10% B) to attain the title compound as an white aerated solid (211 mg, 76%).
• reaction mixture was stirred for 30 minutes at 0 °C, then allowed to warm to RT and stirred for a further 3.5 h. Upon completion, the reaction solution was cooled to 0 °C and quenched with de-ionised H 2 O (25 mL). The volatiles were removed in vacuo and the resulting aqueous phase extracted with CH 2 Cl 2 (3 x 30 mL). The combined organic phase was dried over Na 2 SO 4 , filtered and evaporated in vacuo to give an off-white solid.
• tert-butyl (4-(2-hydroxyethyl)phenyl)carbamate (S6) Di-tert-butyl dicarbonate (3.50 g, 16.0 mmol, 1.1 equiv.) was added to a solution of 2-(4-aminophenyl)ethanol (2.01 g, 14.6 mmol) and triethylamine (2.0 mL, 1.46 g, 14.6 mmol) in dry THF (50 mL). The reaction mixture was stirred overnight at RT, then heated to 50 °C for a further 5 h.
• Methanesulphonyl chloride (825 mL, 1.22 g, 10.6 mmol, 1.1 equiv.) was added dropwise to a solution of tert-butyl (4-(2- hydroxyethyl)phenyl)carbamate (2.29 g, 9.68 mmol) and DIPEA (1.85 mL, 1.37 g, 10.6 mmol, 1.1 equiv.) in dry EtOAc (20 mL) at 0 °C. The solution was stirred for 10 min over ice, then warmed to RT and stirred for a further 1 h.
• the moderate improvement in RCC at low ligand concentration (10 -5 M) may be attributed to the bifunctional appendage imposing a degree of preorganisation in the binding cavity, thereby favouring metal complexation under these conditions.
• Radiolabelling studies of H 4 noneunpaX-Bn-NH 2 with [ 177 Lu]Lu 3+ also showed comparable results to the unmodified chelator; achieving quantitative RCYs over a wide concentration range (10 -3 to 10 -6 M) at RT within 10 minutes.
• Further screening of the radiolabelling properties of H 4 noneunpaX-Bn-NH 2 with [ 111 In]In 3+ , [ 177 Lu]Lu 3+ , [ 133/135 La]La 3+ , [ 155 Tb]Tb 3+ and [ 225 Ac]Ac 3+ showed comparable results to the unmodified chelator, with quantitative RCCs being achieved at ligand concentrations of 10 -6 M with each respective radiometal ion.
• the amount of radioactivity used for assessment of RCCs with each radionuclide corresponds to similar molar equivalents of radiometal ions thereby allowing more direct comparison of the concentration-dependence with different radionuclides.
• an evaluation of the maximum molar activity of [ 177 Lu][Lu(noneunpaX-Bn- NH 2 )]- was conducted to determine minimal amount of chelate required to achieve quantitative RCYs using 20 MBq of [ 177 Lu]Lu 3+ , FIG.32 left panel.
• Quantitative radiolabelling of [ 177 Lu]Lu 3+ (20 MBq) was achieved using 80 pmol of H 4 noneunpa-Bn-NH 2 , corresponding to a molar activity of 250 GBq/ ⁇ mol and a ligand-to-metal ratio of 174:1.
• Human serum stability studies of [ 177 Lu][Lu(noneunpaX-Bn-NH 2 )]- showed no transchelation of bound radioactivity over 7 days, thereby confirming that modification of the pendent donor arm does not impact the overall stability of the metal complex, FIG.32 right panel.
• N ⁇ -Fmoc-Ahx-CO 2 H or N ⁇ -Fmoc-PEG-CO 2 H were incorporated as covalent linkers to give the Fmoc-protected linear-peptides (14) and (15) respectively.
• Cyclisation of the linear peptides was achieved by treatment with iodine in DMF, which sequentially removes the acetamido methyl (Acm) protecting groups and mediates the formation of the disulfide bridge between Cys 2 and Cys 7 .
• H 4 noneunpaX-Bn-NCS Conjugation of H 4 noneunpaX-Bn-NCS to the free N-terminus of (18) and (19) was achieved under mild, basic conditions in solution, and final cleavage of N(Boc)-Lys 5 protecting group gave the corresponding chelate-peptide bioconjugates (20) and (21).
• H 4 noneunpaX-Ahx-Tyr 3 -TATE (20) and H 4 noneunpaX-PEG 2 -Tyr 3 -TATE (21) were purified via RP-HPLC and mass spectrometry analysis (ESI/MALDI) performed to confirm isolation of the intended products.
• the resulting mixture was stirred O/N at RT, then treated with 20%TFA in CH 2 Cl 2 (1 mL) and stirred for a further 2 h. After completion, the volatiles were evaporated under a stream of N2 gas, and the resulting residue diluted with H 2 O (0.1% TFA).
• H 4 noneunpaX-Ahx-Tyr 3 - TATE (20) as a white solid.
• ESI-MS H 2 O/MeCN (1:1)
• 1911.9 [M+H] + .
• Synthesis of H 4 noneunpaX-PEG 2 -Tyr 3 -TATE [0163] H 4 noneunpaX-Bn-NCS (2.3 mg, 3.07 ⁇ mol) was added to a solution of H 2 N-PEG 2 - Boc(Lys 6 )-Tyr 3 -TATE (4.0 mg, 3.07 ⁇ mol) in dry DMF (500 ⁇ L). The solution was stirred for 10 min.
• Each of the chelate-bioconjugates attained quantitative RCCs at a concentration of 10 -5 M within 10 min at ambient temperature.
• the decrease in RCC at lower concentrations in comparison to the free bifunctional chelator is typical for peptide-based bioconjugates, which is attributed to the steric influence of the targeting vector on the metal binding cavity, in addition to the lower solubility of the constructs in aqueous solution and thus availability for metal ion coordination.
• Assessment of the serum stability for each of the radiolabelled bioconjugates showed excellent stability over the course of the study, with no significant changes in radiochemical purity from the initial time-points. [0165]
• concentration-dependent radiolabelling studies the following general protocol was applied for radiolabelling with different radiometal ions.
• Reactions with DOTA were carried out at elevated temperatures (85-90 °C) and monitored over 30-60 minutes.
• Each [ 155 Tb]Tb 3+ -labelled radiotracer was successfully prepared at high molar activity ( ⁇ 23.6 MBq/nmol) with a high radiochemical purity (>98%) as confirmed by iTLC and radio- HPLC (FIGs.35-37).
• mice were administered with 13.5 MBq (0.572 nmol) of each radiotracer, while biodistribution studies were performed using 0.80 MBq (0.033 nmol) per subject.
• the [ 155 Tb]Tb 3+ -labelled radiotracers exhibit rapid clearance from blood circulation over the first 1 h after administration, with uptake in the tumour beginning within the first 5 minutes p.i. Both tracers followed the typical pharmacokinetic profile observed for hydrophilic octreotate-based bioconjugates, whereby fast clearance and accumulation in the kidneys and bladder is seen.
• both tracers exhibited very similar overall distribution over the course of the study, with [ 155 Tb][Tb(noneunpaX-PEG 2 -Tyr 3 -TATE)] showing a marginally faster accumulation in the AR42J tumour xenografts, which is accompanied by faster clearance over time compared to [ 155 Tb][Tb(noneunpaX-Ahx-Tyr 3 - TATE)].
• This observation is clearly in line with the expected trend, accounting for the difference lipophilicity between these bioconjugates.
• good contrast in the tumour region is seen after 45 min p.i. with maximum uptake peaking at 2 h p.i.
• the tumours appear non-homogeneous, with regions of necrotic tissue and no radiotracer uptake; hence, the %ID/g in the tumours is likely lower than achievable.
• the primary objective of these studies was to evaluate the in vivo suitability of H 4 noneunpaX as a new bifunctional chelating ligand.
• the imaging and biodistribution studies show good initial results for this chelator, with no evidence of degradation in vivo or release of bound activity, which would be seen as uptake in the bone over time.
• both Tyr 3 -TATE analogues show improved pharmacokinetics with lower non-target tissue accumulation compared to [ 177 Lu][Lu(DOTATATE)] and would be of interest for further development and evaluation in vivo.
• [0176] [ 155 Tb][Tb(nonenunpaX-Ahx-Tyr 3 -TATE)] and [ 155 Tb][Tb(nonenunpaX-PEG 2 -Tyr 3 -TATE)] were prepared with high molar activities (23.6 MBq/nmol and 22.5 MBq/nmol, respectively), suitable for in vivo SPECT/CT imaging and biodistribution studies.
• mice were administered with either [ 155 Tb][Tb(noneunpaX-Ahx-Tyr 3 - TATE (10.2 MBq, 23.6 MBq/nmol) or [ 155 Tb][Tb(noneunpaX-PEG 2 -Tyr 3 -TATE (13.5 MBq, 22.5 MBq/nmol) in PBS (100 ⁇ L) via lateral tail vein.
• Animal subjects were maintained at constant body temperature using a blanket on a heated bed, maintained under continuous stream of 1.5-2% isoflurane, and the respiration rate monitored throughout the duration of each scan.
• SPECT images were reconstructed using pixel-based ordered-subset expectation maximization (POSEM) reconstruction algorithm using a voxel size of 0.4 mm 3 , 16 subsets with 6 iterations (96 MLEM equivalent).
• SPECT images were decay corrected and attenuation factors applied based on CT acquisitions at each time-point.
• 53 A calibration factor relating (counts/voxel) to radioactivity concentration was previously determined by measurement of a known source of 155 Tb.
• Spherical volumes of interest (VOIs) (3 mm diameter) were drawn using AMIDE (v.1.0.4) software to determine the pharmacokinetic profile of the tracer in target organs of interest.
• SUVs radioactivity concentration
• MBq body weight
• FWHM 3 mm
• image rendering was carried out post-reconstruction for data visualisation purposes only.
• mice were allowed to roam freely in their cages and sacrificed at 2 h or 4 h post-injection by CO 2 asphyxiation under 2% isoflurane anesthesia. Cardiac puncture was performed immediately after sacrifice to recover blood, and organs of interest were harvested, rinsed with PBS and blotted dry. Each organ was weighed, and the radioactivity measured using a calibrated gamma counter (Packard Cobra II Auto-gamma counter, Perkin Elmer, Waltham, MA, USA) with a 1 min acquisition time per sample.
• a calibrated gamma counter Packard Cobra II Auto-gamma counter, Perkin Elmer, Waltham, MA, USA
• Flash column chromatography was performed using Siliaflash F60 silica gel (60 ⁇ , 40 ⁇ 63 ⁇ m particle size, 230 ⁇ 400 mesh) from Silicycle Inc. Automated column chromatography was performed using a Teledyne Isco (Lincoln, NE) CombiFlash Rf automated purification system equipped with RediSep Rf Gold HP prepacked reusable silica and neutral alumina column cartridges. Low-resolution mass spectrometry (LR-MS) was performed using a Waters 2965 ZQ spectrometer with an electrospray/chemical ionization (ESI/CI) source. High-resolution mass spectrometry (HR- MS) was performed using a Waters Micromass LCT TOF instrument.
• LR-MS Low-resolution mass spectrometry
• ESI/CI electrospray/chemical ionization
• HR- MS High-resolution mass spectrometry
• Elemental analyses were carried out using a Thermoflash 2000 elemental analyzer. 1 H and 13 C ⁇ 1 H ⁇ NMR spectra were recorded using Bruker AV300 and AV400 spectrometers; all spectra are reported on the delta scale referenced to residual solvent peaks.
• Analytical and semipreparative high-performance liquid chromatography was carried out using a Waters 600 system equipped with a Waters 2487 dual wavelength absorbance detector monitoring at 254 and 210 nm and a Phenomenex Synergi 250 mm ⁇ 21.2 mm 4 ⁇ m hydro- RP80 ⁇ column (10 mL/min).
• HPLC solvent system 1 (A: H 2 O (0.1%TFA), B: MeCN (0.1%TFA), HPLC solvent system 2: (A: H 2 O (0.01%TFA), B: MeCN).
• Radioactivity was quantified using a calibrated high-purity germanium (HPGe) detector (Mirion Technologies (Canberra)Inc.) with Genie 2000 software. All work with radionuclides at TRIUMF was undertaken in shielded fume hoods to minimize dose to experimenters (and special precautions were used to prevent contamination). Peptides were prepared using an AAPPTec Focus Xi semi-automated solid phase peptide synthesiser. SPECT/CT studies were performed using a multimodal VECTor/CT system (MILabs, Netherlands) in combination with an extra ultra high sensitivity (XUHS) 2-mm pinhole collimator. Image analysis was performed using AMIDE (v.1.0.4) software.
• HPGe high-purity germanium
• H 4 noneunpaX A new nonadentate chelating ligand, H 4 noneunpaX, was synthesised to investigate the influence of donor group arrangement on metal binding characteristics within the ‘NON backbone’. Characterisation of the metal complexation of H 4 noneunpaX through NMR spectroscopy, mass spectrometry, radiolabelling, solution thermodynamic stability studies and DFT calculations, showed excellent compatibility with a wide range of large trivalent metal cations, with a particular preference for hard lanthanide ions.
• H 4 noneunpaX The mild conditions required for radiolabelling of H 4 noneunpaX are particularly well suited for combination with thermally sensitive antibody-based targeting vectors and thus it can be soundly predicted that there is a high likelihood that H 4 noneunpaX will have good utility as a chelator for the targeted in vivo delivery of radiometals.
• the objectives of this work were to investigate the influence of donor group substitution about a common backbone and study the impact of inverted group placement on metal ion affinity. H 4 noneunpaX was developed to this affect and studied in direct comparison to H 4 noneunpa.
• H 4 noneunpaX may be particularly well suited to applications in targeted alpha therapy (TAT) involving [ 225 Ac]Ac 3+ in combination with appropriate imaging radionuclides for dosimetry evaluations ([ 135 La]La 3+ , [ 155 Tb]Tb 3+ , [ 111 In]In 3+ , [ 44 Sc]Sc 3+ etc).
• TAT targeted alpha therapy
• imaging radionuclides for dosimetry evaluations [ 135 La]La 3+ , [ 155 Tb]Tb 3+ , [ 111 In]In 3+ , [ 44 Sc]Sc 3+ etc.
• H 4 octapa Synthesis, Solution Equilibria and Complexes with Useful Radiopharmaceutical Metal Ions. Dalt. Trans.2017, 42, 14647–14658. https://doi.org/10.1039/c7dt02343j. (13) Hu, A.; Keresztes, I.; MacMillan, S. N.; Yang, Y.; Ding, E.; Zipfel, W. R.; Distasio, R. A.; Babich, J. W.; Wilson, J. J. Oxyaapa: A Picolinate-Based Ligand with Five Oxygen Donors That Strongly Chelates Lanthanides. Inorg. Chem.2020, 59, 5116–5132.
• Lutathera® The First FDA-and EMA-Approved Radiopharmaceutical for Peptide Receptor Radionuclide Therapy. Pharmaceuticals. 2019. https://doi.org/10.3390/ph12030114. (16) Müller, C.; Van Der Meulen, N. P.; Bene ⁇ ová, M.; Schibli, R. Therapeutic Radiometals beyond 177Lu and 90Y: Production and Application of Promising ⁇ - Particle, ⁇ --Particle, and Auger Electron Emitters. Journal of Nuclear Medicine.2017, pp 91S-96S. https://doi.org/10.2967/jnumed.116.186825. (17) Jain, A. K.; Raut, R.; Tuli, J.

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  • 2022-05-06 AU AU2022271362A patent/AU2022271362A1/en active Pending
  • 2022-05-06 WO PCT/CA2022/050712 patent/WO2022232943A1/en not_active Ceased
• 2023
  • 2023-10-26 ZA ZA2023/10013A patent/ZA202310013B/en unknown

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