Classifications

• • 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
  • A61K51/041—Heterocyclic compounds
  • A61K51/044—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
  • A61K51/0455—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
• • 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
  • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
• • 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
  • A61K51/1027—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 against receptors, cell-surface antigens or cell-surface determinants
  • A61K51/1033—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 against receptors, cell-surface antigens or cell-surface determinants against receptors for cytokines, lymphokines or interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the present invention relates to bifunctional compounds and in particular compounds for use in molecular imaging and therapy.
• the compounds may be conjugated to a targeting group so that the compounds target specific cells or tissues in a subject.
• Molecular imaging may be defined as the three-dimensional mapping of molecular processes, such as gene expression, blood flow, physiological changes (pH, [0 2 ] etc.), immune responses, and cell trafficking, in vivo. It can be used to detect and diagnose disease, select optimal treatments, and to monitor the effects of treatments to obtain an early readout of efficacy.
• a number of distinct technologies can in principle be used for molecular imaging, including positron emission tomography (PET), single photon emission tomography (SPET) , optical (01) and magnetic resonance imaging (MRI). Combinations of these modalities are emerging to provide improved clinical applications, e.g. PET/CT and SPET/CT (“multi-modal imaging”) .
• Radionuclide imaging with PET and SPET has the advantage of extremely high sensitivity and small amounts of administered contrast agents (e.g. picomolar in vivo), which do not perturb the in vivo molecular processes.
• the targeting principles for radionuclide imaging can be applied also in targeted delivery of radionuclide therapy.
• the isotope that is used as a radionuclide in molecular imaging is
• radiotracer that is pharmaceutically acceptable to the subject.
• Many radiotracers have been developed with a range of properties.
• fluorodeoxyglucose 18 F is a labelled glucose derivative that is frequently used in molecular imaging with PET.
• WO 2009/021947 describes tripodal chelators for use as MRI contrast agents. Hydroxypyridinone chelating groups with a hydrophilic R group are described. The hydrophilic group is required to help solubilise the chelator. In addition, the chelator may be coupled to large molecules, such as a dendrimer, in order to increase the relaxivity of the RI contrast agent.
• radiotracers with sensitive functional moieties.
• incorporation of radioisotopes into the radiotracer may involve elevated temperatures that would disrupt protein structure.
• sensitive functional moieties into radiotracers and so it is a need to provide radiotracers that may be prepared using mild conditions. As a result imaging conjugates with improved functionality and improved molecular imaging properties may be produced.
• DOTA 1,4,7, 10-tetraazacyclododecane-N, N ' , ⁇ ' ' , ⁇ ' ' ' - tetraacetic acid
• DOTA has a long radiolabelling time of around 30 minutes (relative to the half-life of 68 Ga ⁇ 68 minutes) .
• chelation of gallium by DOTA derivatives often requires a high labelling temperature of around 95°C.
• the present inventors have found bifunctional molecules that are able to quickly chelate radionuclides at room temperature, whilst retaining adequate or even enhanced stability towards
• the bifunctional molecules have a reactive portion to couple the bifunctional molecule to a functional moiety, such as targeting group which can target, for example, cells, tissues or biological molecules in the body.
• the present invention provides bifunctional molecules for molecular imaging having a tripodal hexadentate tris (hydroxypyridinone) chelating portion to couple to a radionuclide or an imaging label and a reactive functionality to couple to a targeting group for targeting specific cells or tissues in a subject or to a delivery vehicle for delivering a drug, toxin or other such molecule so that the in vivo
• distribution and/or final location of the target group or delivery vehicle may be monitored.
• the present invention provides a bifunctional compound for use in a method of molecular imaging, wherein the bifunctional compound is represented by the formula
• radionuclide and is selected from:
• R, R 2 , R 3 and R 4 are independently hydrogen or an optionally substituted Ci_ 7 alkyl group
• B is a linker group for linking the chelating group to the reactive group, wherein is represented by the formula: wherein each Q is independently selected from a group consisting of -NR S -, -C(0)NR 5 -, -C(0)0-, -NR 5 C(0)NR 5 -, -NR 5 C(S)NR 5 - and -0-, each R 5 is independently hydrogen or an optionally substituted Ci-7 alkyl group, each q and s are independently selected from 0 to 6 and each r is independently selected from 1 to 6;
• A* is a reacted reactive group that is coupled to T, T being a targeting group capable of binding to a target of interest in the subject;
• the bifunctional compound of the first aspect may circulate the biological system to a targeted location. Then, when the radionuclide is introduced into the subject, the radionuclide may quickly pass though the system and chelate with the bifunctional compound. In this way, the radionuclide may be fixed in a location of interest within a short time of being introduced into the biological system so that the efficacy of the radionuclide is maximised .
• s is independently selected from 0 to 6
• each r is independently selected from 1 to 6
• q is selected from 1 to 6.
• the method of molecular imagining is PET or SPET.
• the present invention provides a bifunctional compound for chelating a radionuclide for use in molecular imaging, wherein the compound is represented by the formula:
• R, R 2 , R 3 and R 4 are independently hydrogen
• Ci- 7 alkyl group optionally substituted Ci- 7 alkyl group
• B is a linker group for linking the chelating portion to the reactive group, wherein is represented by the formula:
• each Q is independently selected from a group consisting of -NR 5 - , -C(0)NR 5 -, -C(0)0-, -NR 5 C(0)NR 5 -, -NR 5 C(S)NR 5 - and -0-, wherein each R 5 is independently hydrogen or an optionally substituted Ci_ 7 alkyl group, each q and s are independently selected from 0 to 6 and each r is independently selected from 1 to 6;
• A is a reactive group for coupling to a biological moiety, a targeting group, a protein, a polypeptide or a delivery vehicle.
• the bifunctional conjugate compounds provide a tripodal
• hexadentate tris (hydroxypyridinone) chelating portion which is able to chelate metallic radionuclides in a very short time (in the order of 5 min or less) and is able to do so at room
• the reactive portion is linked to the chelating portion and allows the bifunctional molecule to be conjugated with a targeting group or other vehicle, such as a polypeptide or other biomolecule, drug or nanoparticle .
• a targeting group or other vehicle such as a polypeptide or other biomolecule, drug or nanoparticle .
• the efficiency of the chelating reaction also allows labelling at very low delivery vehicle concentration. In this way, a significant proportion of the delivery vehicle is radiolabelled with the radionuclide and, as a result, leading to a very high specific activity. In this way, the bifunctional molecule provides an excellent precursor for a radiolabelling conjugate.
• the reactive group A is the protein-reactive
• the protein reactive group may react with proteins or modified proteins or peptides or other vehicles derivatised for the purpose.
• the protein-reactive group is a maleimide group, an aldehyde, an ester, or "click" reagent such as an alkyne, azide, alkene, hydrazine, hydrazine derivative, alkoxyamine, alkoxyamine derivative, aminoxy or thiol group.
• Maleimide, aldehyde and ester groups efficiently react with peptide thiol- or amine-containing residues (cysteine, lysine) and so a conjugate can easily form.
• Other bioorthogonal functional groups can be engineered into peptides and proteins for the purpose of conjugating them with alkyne, azide, alkene, hydrazine, aminoxy or thiol groups.
• R 1 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
• R 2 , R 3 and R 4 are independently H or CH 3 . It is preferred to have relatively small substituents on the
• R 2 , R 3 and R 4 are H.
• one or more methyl substituents on the heterocyclic group may be used to tailor the solubility and/or the hydrophobicity of the molecule. Accordingly one or more of R 2 , R 3 and R 4 may be CH 3 .
• m is 2 or 3.
• the linker group B connects the tripodal chelating portion and the reactive functionality (and the e.g. targeting group or carrier vehicle when conjugated) .
• the linking group may be tailored by varying the groups Q, q, r and/or s in order to vary, for example, the length of the linker.
• the linker group may be arranged in either direction so that either A or the quaternary carbon (attached to each arm having a chelating group) or both may be attached to Q.
• B is arranged so that the bifunctional compound is represented by the formula:
• the ordering of the atoms in the functional group represented by Q is not limited.
• the order of the group from the chelating portion towards the reactive functionality may be -NR 5 - then -C(O)- or vice versa.
• At least one Q is an amide (-C(O)NR 5 -).
• q is 1, r is 2 and/or s is 1. More preferably q is 1, r is 2 and s is 1.
• R 5 is hydrogen.
• B is represented by one of the following formulae:
• r is selected from 1 to 6 and s selected from 0 to 6.
• r is 2 and/or s is 2.
• the bifunctional compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
• the radionuclide is an isotope of technetium, rhenium, copper, cobalt, gallium, yttrium, lutetium or other lanthanide, indium, zirconium, scandium.
• the radionuclide is Tc-99m, Re-186, Re-188, Co-57, In-Ill, Cu-60, Cu-61, Cu-62, Cu-64, Cu-67, Tc-94m, Ga-68, Ga-67, Ga-66, Y-90, Y-86, Sc-44, Sc-47, Fe-52, Sn-117m, Tb-149, Gd-153, Ho-166, Lu- 177, Zr-89, Bi-213 or Co-55 and more preferably the radionuclide is gallium, and most preferably Ga-68 or Ga-67.
• the targeting group is a member of a specific binding pair that is capable of binding to a binding partner at the target of interest. More preferably the targeting group and target of interest are a receptor and ligand, or an antibody and antigen, or metabolic probe e.g. glucose transporter/glucose, hypoxia/hypoxia responsive moiety, or bone
• RNA and antisense RNA RNA and antisense RNA.
• the targeting group is a peptide, protein, antibody, aptamer or small molecule ligand capable of binding to a binding partner at the target of interest. More preferably the targeting group is a polypeptide capable of binding to phosphatidylserine (PS) so that the bifunctional conjugate composition can be employed in apoptosis or cell death imaging studies. More preferably the targeting group includes Annexin V and the C2 domain of a synaptotagmin, TIMP-2, CEA, RGD peptide, somatostatin receptor targeting peptide, bombesin, gastrin or VCAM targeting peptide .
• PS phosphatidylserine
• the target of interest is an in vivo molecular target. More preferably the target of interest is a ligand or receptor expressed on diseased cells or tissue (of which the abundance or ligand occupancy is to be determined) , a cell surface antigen associated with a disease state, tumour markers, such as a cancer specific marker or a tissue specific marker or a marker of a normal process which is up- or down-regulated in a disease state.
• the target of interest is a location, an organ, a tissue type or physiological property in a subject undergoing molecular imaging.
• Reactive group A may be for coupling to a delivery vehicle.
• a delivery vehicle as defined herein is a molecular structure for carrying a functional molecule, such as a small molecule drug or toxin with the purpose of delivering the functional molecule for example in a biological system.
• the functional molecule may be contained within or bonded to a surface of the delivery vehicle.
• the functional molecule is typically reversibly associated with the delivery vehicle so that the functional molecule may be released at a given location in the biological system.
• Many such delivery vehicles are known in the art.
• the delivery vehicle is a nanoparticle or liposome.
• Functional molecules, such as pharmaceutical drugs may be bound to the surface of
• the bifunctional compounds of the present invention are coupled to the delivery vehicle and a radionuclide is chelated by the chelating group R 1 , the passage of a drug (carried by the delivery vehicle) through a subject may be monitored by imaging of the radionuclide.
• the reactive group may (in addition to a biological moiety, a targeting group, a protein, a polypeptide or a delivery vehicle) be for coupling to a secondary imaging label.
• the secondary imaging label is an optical label, a paramagnetic label, such as a SPIO, or a quantum dot.
• the imaging label is a paramagnetic label which is a MRI contrast agent with a paramagnetic probe.
• the imaging label is an optical label which is an organic molecule or metal complex with fluorescent or luminescent properties.
• the label may, for example in the case of an optical label or paramagnetic label, allow for the bifunctional compound to be used in multi-modal imaging .
• the reactive group A may couple with further biological moieties, targeting groups, proteins, polypeptides or delivery vehicles.
• the bifunctional compound may couple the
• the method of molecular imaging may include introducing the bifunctional compound (or conjugate thereof) into a biological system (e.g. a subject) either before the radionuclide is introduced into the biological system (i.e. pre-targeting) or after the radionuclide has been chelated to the chelating portion of the bifunctional compound (i.e. pre-assembled) .
• the bifunctional compound When the bifunctional compound (or conjugate) is introduced into the biological system before the radionuclide, the bifunctional compound may circulate the biological system to a preferred or targeted location. Then, when the radionuclide is introduced, the radionuclide may quickly pass though the system and chelate with the bifunctional molecule (or conjugate) . In this way, the radionuclide may be fixed in a location of interest within a short time of being introduced into the biological system so that the efficacy of the radionuclide is maximised.
• the bifunctional compound When the radionuclide is chelated to the bifunctional compound (or conjugate) before introduction into the biological system, the bifunctional compound may be used to monitor the passage of the bifunctional compound or the conjugate having a biological moiety, a targeting group, a protein, a polypeptide a carrier vehicle or a secondary imaging label attached to the compound through the biological system.
• the biological moiety, targeting group, protein, polypeptide or delivery vehicle are bound to the bifunctional compound to form a bifunctional compound conjugate before introduction of the bifunctional compound composition into the biological system or subject.
• the therapy may be radionuclide therapy.
• the therapy may be cancer treatment using the radionuclide or a chemotherapeutic agent associated with the bifunctional molecule, for example via the delivery vehicle to which it is attached.
• the bifunctional molecule is associated with or bound to a
• the subject may be a subject in whom the biodistribution of the targeting molecule or delivery vehicle is being evaluated.
• the present invention provides a bifunctional compound conjugate for chelating a radionuclide for use in molecular imaging, wherein the conjugate is represented by the formula :
• R, R 2 , R 3 and R 4 are independently hydrogen
• B is a linker group for linking the chelating portion to the reactive group, wherein is represented by the formula: wherein each Q is independently selected from a group consisting of -NR 5 - , -C(0)NR 5 -, -C(0)0-, -NR 5 C (0) NR 5 -, -NR 5 C ( S ) NR 5 - , -0-, wherein each R 5 is independently hydrogen or an optionally substituted Ci -7 alkyl group, each q and s are independently selected from 0 to 6 and each r is independently selected from 1 to 6; and
• A* is a reacted reactive group for coupling to T, wherein T is a biological moiety, a targeting group, a protein, a polypeptide or a delivery vehicle.
• the present invention provides a kit for use in a method of molecular imaging or therapy comprising: a bifunctional molecule composition having a bifunctional compound as described herein, wherein the compound is linked to a biological moiety, a targeting group, a protein, a polypeptide or a delivery vehicle through reactive group A; and an imaging composition comprising a radionuclide as described herein.
• the present invention provides a kit for use in a method of molecular imaging or therapy comprising: a bifunctional molecule composition having a bifunctional compound as described herein; and a biological moiety, a targeting group, a protein, a polypeptide or a delivery vehicle for coupling to the bifunctional molecule.
• the kit may also include an imaging composition comprising a radionuclide as described herein.
• the present invention provides a method of molecular imaging a target of interest in a biological system, the method comprising:
• composition comprising bifunctional compound as described herein, wherein the compound is linked to a biological moiety, a targeting group, a protein, a polypeptide or a delivery vehicle through reactive group A;
• the invention provides a method of molecular imaging a target of interest or passage of interest in a biological system, the method comprising:
• the radionuclide is a radionuclide capable of delivering radiotherapy to the target of interest.
• the radionuclide is Cu-67, Re-186, Re-188, Y-90, Lu-177, Sc-47 or Bi-213.
• Figure 1 shows the radiolabelling yield of chelating portion CP256 with 67 Ga after 5 minutes at room temperature compared to the radiolabelling yield of DOTA with 67 Ga after 40 minutes at 80°C;
• Figure 2. shows the elution profiles of 6 Ga-CP256 from a PD10 column for radiolabelled solutions at 2.27 ⁇ (A) and 0.227 ⁇ (B) of CP256 after incubation with apotransferrin, compared to 67 Ga-citrate (C) .
• Figure 3 shows retention of 67 Ga-citrate, 67 Ga-CP256, 67 Ga-DOTA and 67 Ga-DTPA to 30 kDa M CO filters, after incubation with apotransferrin .
• Figure 4 shows the radioactive elution profile, using a PD10 size exclusion cartridge, of C2Ac and C2Ac-(7) after 15 minutes incubation with Gallium-68 in acetate buffer;
• Figure 6. shows a PET scan of a mouse 90 minutes after
• Figure 8 PD10 size exclusion chromatography profile of ni In after incubation of nl In-acetate and ul In-CP256 in serum, compared to ul In-CP256 in buffer.
• FIG. 11A (left): PET/CT scan (maximum intensity projection) of a mouse from group 3 showing pre-targeting using CP256-SER4: 8 week-old C57B1/6 injected with CP256-SER4 followed by Ga-68 acetate, showing liver/spleen uptake. Image taken at 1.5h post injection.
• Fig. 11B (right): for comparison, mouse injected with Ga-68 acetate only, showing retention in blood pool and accumulation in joints.
• bifunctional compound is for coupling to a targeting molecule
• a wide range of primary targeting groups may be linked to the bifunctional compounds to direct the bifunctional
• the target of interest will comprise a member of a specific binding pair that is capable of specifically binding to the primary targeting group in the system, such that they will be members of a pair of molecules which have particular specificity for each other and which in normal conditions bind to each other in preference to binding to other molecules.
• specific binding pairs are well known in the art and include for example receptors and ligands, enzymes and substrates, antibodies and antigens.
• primary targeting groups may be peptides, proteins or other biological molecules, such as aptamers, or small molecule ligands, that bind to specific in vivo molecular targets.
• Classes of targets of interest include ligands or receptors or transporters expressed on diseased cells or tissue, cell surface antigen associated with disease states, or tumour markers, e.g. cancer specific markers or tissue specific markers.
• One group of primary targeting groups are polypeptides capable of binding to phosphatidylserine (PS) so that the bifunctional molecule conjugates of the present invention can be employed in apoptosis or cell death imaging studies.
• polypeptides include Annexin V and the C2 domain of a synaptotagmin .
• Polypeptides that comprise one or more C2 domains are well known in the art. While some polypeptides have only one C2 domain, others have two or more C2 domains, and the domains are generally described by attaching a letter (in alphabetical order) to the end of the name (e.g., C2A, C2B, and so on).
• C2 domain For a protein that contains only one C2 domain, the domain is simply referred to as C2 domain. While the examples below use the C2A domain of rat synaptotagmin I, other C2 domains that are capable of binding to PS could be employed instead, for example a C2A domain of a synaptotagmin of another species. Further examples of proteins that contain a C2 domain include but are not limited to synaptotagmin 1-13, protein kinase C family members of serine/threonine kinases, phospholipase A2, phospholipase 51, cofactors in the coagulation cascade including factors V and VIII, and members of the copine family. Human synaptotagmins include synaptotagmin 1-7, 12 and 13.
• the present invention can employ an anti-CD33 antibody, or fragment thereof, for imaging cancer cells expressing CD33 such as cells of myelomonocytic lineage and leukaemic cells, see Emberson et al., J. Immunol. Methods. 305 (2 ): 135-51, 2005.
• a further example is the use of a tissue inhibitor of
• TIMPs metalloproteinases
• TIMP-2 metalloproteinases
• CR2 complement receptor 2
• Antibodies capable of binding to the glycoprotein carcinoembryonic antigen (CEA) may also be used as primary targeting groups as members of this family of glycoproteins are expressed on colorectal cancer cells, gastric cancer cells, pancreatic cancer cells, lung cancer cells, medullary thyroid cancer cells and breast cancer cells.
• a further example may exploit the affinity or the peptide sequence arginine-glycine-aspartic acid (RGD) for the ⁇ ⁇ ⁇ 3 integrin expressed highly in the endothelium of tissues
• Atherosclerotic plaque and repairing diseased tissue such as infarcted myocardium by linking an RGD peptide derivative to the bifunctional compound by means of a suitable reactive group A.
• RGD peptide derivative by linking an RGD peptide derivative to the bifunctional compound by means of a suitable reactive group A.
• a further example may exploit the affinity of the peptide octreotide or other related analogue of somatostatin, which may bind to the somatostatin receptor expressed highly at the surface of cancer cells e.g. in carcinoid, medullary thyroid carcinoma and other neuroendocrine tumours, by linking a somatostatin analogue peptide to the bifunctional compound by means of a suitable reactive group A.
• Another example may exploit the affinity of antibodies to cell adhesion molecules.
• One example is the affinity of the
• sialoadhesin is found on the surface of
• macrophages and, for example, in high amounts on macrophages of the spleen, liver, lymph node, bone marrow colon and lungs.
• targeting groups such as bombesin, gastrin, or VCAM targeting peptide.
• the primary targeting group may be linked to the bifunctional compound as a conjugate having a functional group for binding to the bifunctional compound and the reactive group for linking to the primary targeting group.
• a reactive group for site-specific linkage to the primary targeting group may be employed, for example a maleimide group for site-specific linkage to thiol groups in a biomolecule such as a peptide, polypeptide or antibody.
• the maleimide group can then be site-specifically linked to a thiol group, for example of a cysteine residue incorporated site-specifically into the peptide or protein for the purpose.
• the reactive group in the bifunctional chelator would be an aldehyde or ketone group able to react site-specifically with a protein or peptide to which a hydrazine or similarly reactive group has been site-specifically incorporated (e.g. using a hydrazinonicotinic acid derivative) to form a hydrazone or similar link.
• the primary targeting group may comprise a suitable polypeptide or protein, or a fragment or domain thereof.
• polypeptides are fragments of a full length protein that have the ability to retain structure independent of the full length protein, typically forming a stable and folded three-dimensional
• Protein domains vary in length from between about 25 amino acids up to 500 amino acids in length, or from 50 amino acids to 250 amino acids, or from 75 amino acids to 150 amino acids.
• polypeptide is an antibody
• this term describes an immunoglobulin whether natural or partly or wholly synthetically produced.
• the term also covers any polypeptide or protein comprising the antigen binding domain of an antibody; antibody fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies. It is possible to take monoclonal and other antibodies and use
• Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the
• CDRs complementarity determining regions
• Antibodies can be modified in a number of ways and the term "antibody molecule" should be construed as covering any specific binding member or substance having an antibody antigen-binding domain with the required specificity. Thus, this term covers antibody fragments and derivatives, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP 0 120 694 A and EP 0 125 023 A. It has been shown that fragments of a whole antibody can perform the function of binding antigens.
• binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S.
• Fv, scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and VL domains (Reiter et al, Nature Biotech, 14: 1239-1245, 1996).
• Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu et al, Cancer Res., 56: 3055-3061, 1996).
• Affibodies may also be used as a primary targeting group.
• Affibodies are small high affinity proteins that may bind specifically to a large number of target proteins or peptides. Affibodies imitate monoclonal antibodies in many respects and are classed as antibody mimetics.
• the selection of affibody should not be limited and many affibodies are known in the art, such as unconjugated anti-TNF- ⁇ affibody.
• Anti-TNF- ⁇ affibody may be produced in E. coli and targets human cytokine tumor necrosis factor a, TNF-a.
• TNF- a is a pleiotropic inflammatory cytokine. The cytokine is produced by several types of cells, but
• TNF-a is an acute phase protein which initiates a cascade of cytokines and increases vascular permeability, thereby recruiting macrophage and neutrophils to a site of infection. Therefore, TNF-a is an important target for inflammation in atherosclerotic lesions and inflammations.
• Each bifunctional compound may be linked to a plurality of primary targeting groups, thereby increasing (a) the affinity of the primary targeting groups for the target of interest as the overall affinity increases as the product of the affinity of each targeting group for the target and/or (b) the rate of binding to the target, i.e. the likelihood of binding ensuing from each encounter between target and primary targeting group in vivo.
• Imaging probeslhe imaging probes that are capable of binding to the bifunctional compounds comprise a radionuclide (bound through the chelating group) .
• the imaging probes (metallic radioisotopes) used in accordance with the present invention have the property of binding to the chelating portion of the bifunctional compounds either in vitro or in vivo, in either the pre-targeted or pre-assembled
• Radionuclides that have an intrinsic binding affinity for chelating portion of the bifunctional compounds.
• 68 Ga 3+ is capable of binding to the hydroxypyridinone groups of the chelating portion and can be used in PET imaging techniques.
• radionuclide probes used in accordance with the present invention may use a range of different radionuclides depending on the application for which the probes are intended.
• radionuclides that may form part of the probes of the present invention include radionuclides of technetium, rhenium, copper, cobalt, gallium, yttrium, lutetium, indium, zirconium, scandium, namely Tc-99m, Ga-67, In-Ill (SPET), Cu-64, Cu-60, Cu-61, Cu-62, Tc-94m, Ga-68, Co-55, Zr-89, Sc-44 (PET), Cu-67, Re-186, Re-188, Y-90, Lu-177, Sc-47 or Bi-213 (radionuclide therapy) .
• the present invention may employ the radionuclides alone or in combinations .
• the radionuclide may be in any form, such as a solvate, hydrate, salt or weak chelate. Solvates, hydrates, salts and weak chelates of radionuclides are known in the art.
• the radionuclide When the radionuclide is to be administered to a subject in a radionuclide composition (for example, when the bifunctional compound chelates the radionuclide in the subject after administration) , the radionuclide should be in a pharmaceutically acceptable form.
• the radionuclide may be a citrate or acetate salt of the metal ion.
• the present invention may also involve the use of secondary imaging labels, such as an optical label or a paramagnetic label (through the reactive group A) that may be linked to or
• optical probes include fluorophores such as fluorescein and luminescent
• PET and MR imaging can provide the advantage of high sensitivity (PET, SPET), quantification of signal (PET) and anatomical resolution (MR) and measurement of the microenvironment (MR contrast enhancement).
• the bifunctional compounds of the present invention may use a range of different chemistries and techniques for linking the biological moiety, targeting group, label, protein or polypeptide to the bifunctional compounds and/or for introducing further groups and properties into the bifunctional compounds of the present invention.
• the reactive group provides a way of coupling the targeting group to the bifunctional compound, for example when the reactive group is a maleimide group (to bind to the thiol side chain of cysteine in the protein/peptide sequence)
• the bifunctional compounds of the present invention may be used for the molecular imaging of diseases, such as cancer,
• the bifunctional compounds may also be employed for radionuclide therapy for treating cancer and arthritis.
• the applications of the bifunctional compounds and radionuclides of the present invention include a wide range of imaging and spectroscopic applications that can employ the radionuclide and/or a further label, for example, in multi-modal imaging studies.
• the bifunctional compounds are particularly useful for in vivo imaging applications such as cell death imaging, for example using bifunctional compounds for the detection of apoptosis. This might be useful in a number of different medical or research applications, for example in the fields of oncology (e.g. in monitoring response to chemotherapy), cardiovascular medicine (e.g.
• the present invention is particularly relevant to nuclear medicine imaging techniques, such as Single Photon Emission Tomography (SPET) , an imaging technique that detects gamma rays emitted from a radionuclide to produce a three dimensional image of the distribution of the radionuclide in a sample or subject, and Positron Emission Tomography (PET), an imaging technique that provides three-dimensional images by detecting pairs of gamma rays emitted indirectly by a positron-emitting radionuclide introduced into a sample or subject.
• SPET Single Photon Emission Tomography
• PET Positron Emission Tomography
• SPET studies can be carried out using In-Ill and PET studies using Ga ⁇ 68.
• the skilled person will be aware of other suitable SPET and PET radionuclides that can be employed in the present invention.
• the present invention may be employed for positron emission tomography (PET), single photon emission tomography (SPET), optical (01) and/or magnetic resonance imaging (MRI) by appropriate selection of imaging probe, or for
• radionuclide therapy by selection of the appropriate radionuclide (e.g. Y-90, Lu-177) .
• the bifunctional compounds of the present invention may be used in methods of multi-modal imaging, that is where information or images are derived from two different techniques, either by the detection of the imaging probe capable of detection using two different techniques or by providing a second label at the site in the biological system where the bifunctional compounds become localised, most conveniently by linking or associating the second label with the bifunctional compounds as explained in detail above.
• Multi-modal studies will be co-registered and may entail simultaneous imaging with two modalities or may need to take place in two steps, but generally employ the same sample so that spatial information obtained using the two techniques can be compared. Examples of multi-modal imaging include PET/CT, SPET/CT, PET/MR and S PET/MR
• nitro group of 61 was then reduced to an amine to form 62 by hydrogenation with a T-l Raney Nickel catalyst, 5 which was specially prepared from a nickel/aluminium alloy.
• a T-l Raney Nickel catalyst 5 which was specially prepared from a nickel/aluminium alloy.
• Acetylation of 62 with acetyl chloride and purification by column chromatography gave 63 in 72% yield.
• the tert-butyl groups were then removed with formic acid and the product, compound 64 was recrystallised from ethanol/petroleum spirit with a yield of 93%.
• Ga-CP256 and Fe-CP256 complexes Formation of the Ga-CP256 and Fe-CP256 complexes is evident from the mass spectral data shown in Table 1 and Table 2. The protonated form of each complex was detected and the isotopic distribution from the mass spectrum matches the calculated values recorded in table 1.
• the Ga-CP256 complex has two molecular species for (M + H) + , at m/z 806 and m/z 808, due to gallium having two stable isotopes with atomic masses of 69 and 71. No evidence of Ga-CP256 complexes with Ga:ligand ratios other than 1:1 was found.
• the mass spectral data for Fe-CP256 complex match the predicted isotopic distributions.
• HPLC analyses were carried out with FC3600 detector and ⁇ detector probe connected in series.
• the HPLC system was an Agilent 1200 series with quadruple pump, degasser, UV detector and manual syringe injector.
• the HPLC column used for all analyses was an Agilent Eclipse XDB-C18 (5 ⁇ , .6 mm X 150 mm) with a guard column.
• the analytical software used for radio-TLC and radio- HPLC was Laura (Lablogic) . All general reagents and consumables were purchased from Sigma-Aldrich or from Fisher Scientific.
• a 1 mg/ml stock solution of CP256 was prepared in PBS buffer in a 1 ml plastic microcentrifuge tube.
• Radiolabelling solutions were prepared by adding a 10 ⁇ aliquot of the stock solution to 90 ⁇ of labelling buffer in a plastic microcentrifuge tube to give a total ligand concentration of 0.1 mg/ml in 100 ⁇ .
• This solution was radiolabelled by adding 10 ⁇ 67 Ga-citrate . Radiolabelling conditions are shown in table 4.
• Silica TLC plates and ITLC-SG strips were spotted with 1 ⁇ of radiolabelled complex at the origin, which was marked at 5 mm in pencil. Spots were allowed to dry on silica gel TLC plates but not for ITLC-SG strips.
• the plates were placed in a developing chamber (100 ml wide necked sample bottle) filled with a 3 mm depth of the mobile phase. Stationary phases and mobile phases are listed in table 8.
• the TLC plate was developed until the solvent front had reached a distance of up to 65 mm.
• the plates were removed from the developing chamber and dried in an oven set at 80°C. Radiochemical yields were determined using either Laura software or the instant imager software.
• TLC and ITLC analyses were carried out on a Mini-Scan TLC scanner with FC3600 detector and ⁇ detector probe.
• the analytical software used was Laura (Lablogic) . Radioactive samples were counted for 10 seconds (window 101-110) on a 1282 Compugamma Gamma Counter (LKB Wallac) using Ultroterm software.
• the dose calibrator used for measuring radioactivity in samples was a CRC- 25R (Capintec.) Electronic autoradiography was performed on a Packard Instant Imager with Imager version 2.05 software.
• the gas mixture was 1% isobutane, 2.5% carbon dioxide and 96.5% argon (Air products . ) Radiolabelling procedure for radiochemical yield determination by TLC analysis
• concentrations of each ligand in the labelling mixture were 1 mM, 100 ⁇ , 10 ⁇ , ⁇ , 100 nM and 10 nM.
• FIG. 2 shows the elution profiles of 67 Ga-CP256 from a PD10 column for radiolabelled solutions at 100 ⁇ (A) and 10 ⁇ (B) of CP256. After dilution and incubation in apo-transferrin, the concentrations of CP256 in these solutions were 227 ⁇ and 2.27 nM respectively. After 4 hours incubation in apo-transferrin, the profile is unchanged from that of the incubation in the reference buffer, indicating no binding of 67 Ga to transferrin or loss of 67 Ga from the ligand, for either of the 2.27 ⁇ or 227 nM solutions.
• spectrophotometry of apo-transferrin was performed on a Cary UV spectrophometer with Cary WinUV software. The wavelength was set at 280nm.
• Table 8 Details of mobile phases and stationary phases used for TLC and ITLC quality control of 6 Ga-CP256, 67 Ga-DOTA and 67 Ga-DTPA .
• a reference solution of 67 Ga-citrate was produced by incubating 20 ⁇ 67 Ga-citrate in 200 ⁇ PBS. This solution was incubated at 37°C in a metal heating block and samples were removed for analysis in the same manner as for the serum incubation.
• TRIS-HCl buffer pH 7.5
• Sodium bicarbonate was added to a concentration of 25 mM.
• a 2.5 mg/ml (32 ⁇ ) solution of apo-transferrin was prepared in this buffer.
• Apo-transferrin solution 200 ⁇ in a plastic microcentrifuge tube was incubated at 37°C in a metal heating block. To this was added 20 ⁇ of each pH-neutralised tracer. 20 ⁇ of each tracer was also incubated in a reference buffer (containing no apo- transferrin) of 50 mM TRIS-HCl containing 25 mM sodium
• the 50 mM TRIS-HCl solution (200 ⁇ , not containing apo- transferrin) was incubated in a heating block at 37°C.
• the appropriate pH adjusted tracer (20 ⁇ ) was added to the
• radioactive 500 ⁇ of the 2.5 mg/ml solution was loaded onto a PDlO column, eluted with PBS buffer and 0.5 ml fractions were collected. The samples were loaded into a 1 ml quartz cuvette and diluted to 0.9 ml with an extra 0.4 ml of PBS. Absorbance was determined at 280 nm with PBS as a reference standard.
• the analysis was done in triplicate after 4 hours for the apo-transferrin incubations. The analysis was done 6 times (6 tubes) for the incubations in the 50 mM TRIS-HCl reference buffer. Average values were taken as described above.
• N-hydroxysuccinimide (NHS; 2.3 g, 20 mmol)
• the residue was loaded on a solid phase extraction column by dissolving in 1ml 0.1% formic acid solution.
• the column was firstly washed with 0.1% formic acid (10 mL) to remove triethylamine hydrochloride salt, followed by elution of methanol (10 mL) .
• the fraction of the methanol solution was concentrated and dried by vacuum oven to yield a white powder (80%).
• C2A is a small protein that recognises phosphatidylserine displayed on cells undergoing apoptotis. It has been engineered with a cysteine residue to facilitate site-specific labelling with thiol-reactive agents (see Tavare R, Torres Martin de
• the average molecular weight of C2Ac is 14997 and so the C2Ac-(7) conjugate was expected to have an average molecular weight of 15918.44.
• the found average molecular weight was 15970.85, which corresponds to the C2Ac-(7) conjugate with an additional iron atom and without 3 hydrogen atoms.
• the mixture was purified using a PD-10 column pre- equilibrated with PBS that had been stored over Chelex and analysed by LC-MS.
• Example 7 Labelling of C2Ac protein conjugate with 6a Sa and functional data on labelled protein
• the C2Ac-(7) conjugate was radiolabelled with 68 Ga according to the method described herein.
• Figure 4 shows a high degree of chelation after only 15 minutes of incubation with 68 Ga in acetate buffer.
• the Ga-labelled conjugate showed specific calcium dependent binding to phosphatidylserine (PS) in a red blood cell binding assay (see Fig. 5) Experimental Details
• Unchelated 68 Ga in buffer was prepared as follows. The generator was eluted with in 2ml 0.1M HC1 to give 25 MBq 6B Ga eluate. 1.5ml of cone. HCl (36%) was added to the elution to give a final concentration of 4M HCl to form gallium tetrachloride anion, and the solution passed through an anion exchange column (SAX SPEC, Chromafix 30-PS-HCO3, 45mg, Macherey-Nagel, Germany) , trapping all activity.
• SAX SPEC anion exchange column
• the column was then washed with 250 ⁇ 5M NaCl and the activity then eluted with 300 ⁇ distilled water and the eluate (pH ⁇ 1, -70 MBq) buffered by adding 5 ⁇ of 1M NaOH and 30 ⁇ of 1M ammonium acetate buffer, pH 6, raising pH to 7. 100 ⁇ of PBS, pH 6 was then added.
• Reactions were prepared with 1 nM 68 Ga labelled C2AcH and calcium; RBC were then added, and the reaction (1 ml) was incubated for 8 min at RT . The cells were then centrifuged (3 min at 7500 RCF) , the
• radiolabelled protein bound at saturating calcium concentrations.
• Curve fitting was performed using a nonlinear curve fit by a routine based on the Levenberg-Marquardt algorithm using
• Image reconstruction OSEM with SSRB 2D LOR, energy window: 400- 600 keV, filter: Ramlak cutoff 1, number of iterations/subsets: 8/6. Animals were killed at 90 min and explanted organs counted in a gamma counter to determine biodistribution .
• Figure 7 shows size exclusion radiochromatography (PD10 column) of chelation of 68 Ga by CP256 in serum and control experiments to show Ga-chloride incubated in serum, preformed Ga-CP256-(7) complex incubated with serum and 69 Ga incubated in buffer.
• PD10 column size exclusion radiochromatography
• l u In-chloride formed a complex with CP256 (589 ⁇ ) in about 90% yield after 5 min, as determined by ITLC-SG (HSA) .
• the complex showed ⁇ 2% release of In-111 or binding to serum proteins when incubated in human serum for 2 h, as determined by PD10 size exclusion chromatography (Fig. 8).
• Example 11 in vivo imaging of antibody-CP256 conjugate via the ⁇ retargeting mode with 67 Ga.
• a monoclonal IgG antibody, SER4 against a sialoadhesin antigen expressed by pro-inflammatory macrophages was conjugated with CP256-malemide (7) by first reducing antibody disulfide bonds with 2-mercaptoethanol, then treating the thiol groups so formed with CP256-maleimide (7) to give CP256-SER4.
• mice of SER4 labelled with 99m Tc The typical biodistribution in mice of SER4 labelled with 99m Tc (unpublished data) observed previously reflects rapid clearance from circulation with uptake predominantly in spleen and to some extent also liver.
• CP256-SER4 was labelled with 6 Ga to give 6 Ga-CP256-SER4.
• Groups of wild type mice were injected with either directly labelled 67 Ga-CP256-SER4 (group 1), or SER4 (unconjugated) followed 5 min later by 67 Ga citrate (group 2) or CP256-SER4 followed by 67 Ga citrate .
• biodistribution of radioactivity determined. The results are shown in Fig. 9. For each organ, biodistribution data for Group 1 are shown in left hand column, biodistribution data for Group 2 are shown in the central column, and
• biodistribution data for Group 3 are shown in the right hand column .
• Group 1 showed strong targeting of 57 Ga-CP256-SER4 to spleen (36% ID/g) , whereas group 2 showed very low spleen uptake of 67 Ga citrate (4% ID/g) .
• group 3 showed that pre- injection of CP256-SER4 was able to increase spleen uptake of 6 Ga citrate from 4% ID/g to 10% ID/g, suggesting that the 67 Ga followed the location of antibody conjugate to a significant extent .
• Group 1 also showed high activity of 67 Ga-CP256-SER4 in
• the antibody was then washed by centrif gation at 3000 rpm for 3 x 15 min in a 50,000 mwco Vivaspin2 ultracentrifugation tube.
• the antibody was resuspended between centrifugation steps and the volume adjusted to 2 ml by addition of 0.1M metal-free phosphate buffer, pH 7.
• the concentrated and washed antibody was collected in approximately 300 pL 0.1 M phosphate buffer.
• 2-mercaptoethanol 5 pL was diluted to 20 pL in 0.1 M phosphate buffer, pH 7. 3.8 pL (1000 fold excess) of the diluted 2-mercaptoethanol was added to the antibody and the reduction allowed to proceed for 30 min at room temperature .
• the volume was adjusted to 1 ml by addition of 0.1 M phosphate buffer and the removed by applying the reduced antibody to a disposable size exclusion column (PD10) .
• PD10 disposable size exclusion column
• Fractions containing the reduced antibody were combined and CP256-maleimide (0.77 mg dissolved in 8 pL DMSO, a 40 fold excess) was added immediately.
• the conjugation reaction was heated at 37°C for 30 min.
• the conjugated antibody was then washed using a 50,000 mwco Vivaspin2 ultracentrifugation tube as before but using 0.1 M ammonium acetate buffer, pH6 to remove the excess ligand.
• the concentrated CP256-antibody was collected in 0.1 M ammonium acetate buffer, pH6.
• 67 Gallium radiolabelling was performed by addition of 45 pL 67 Ga citrate (0.6-0.75 MBq) to 90 pL (90 pg) CP256-SER4.
• Radiolabelling was allowed to proceed for 10 min before dilution to 350 pL with phosphate buffered saline. Injections containing 25 pg of labelled antibody were drawn up. Analysis of the radiolabelled antibody was performed by size exclusion HPLC using 0.1 M phosphate buffer containing 2 mM EDTA as the mobile phase to ensure good radiolabelling had been achieved prior to
• Example 12 in vivo Imaging of ant ⁇ hody-CP256 conjugate via the pretargeting mode with 68 Ga.
• SER4 conjugated with CP256-malemide (7) as described in Example 11, was labelled with 68 Ga as described above in example 7, to give 68 Ga-CP256-SER4.
• group 3 showed that pre-inj ection of CP256-SER4 was able to increase spleen uptake of 68 Ga acetate from 2-5% ID/g to 17-40% ID/g, suggesting that the 68 Ga followed the location of antibody conjugate to a significant extent.
• FIG. 11A A NanoPET/CT image of a mouse from group 3 is shown in Fig. 11A.
• the mouse was image using the pre-targeting method of the invention using CP256-SER4: 8 week-old C57B1/6 injected with
• FIG. 11 B shows retention in blood pool and accumulation in joints.
• the antibody was conjugated to CP256 as described in Example 11.
• 68 Ga was eluted from a 68 Ge/ 68 Ga generator and concentrated as described in Example 7 to give 68 Ga acetate.
• 150 pL (105 pg) CP256-SER4 was added 150 pL 58 Ga acetate (16 MBq) .
• the radiolabelling reaction was allowed to proceed for 5 min at room temperature. Injections containing 35 pg of the
• radiolabelled antibody was carried out as described in Example 11.

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