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WO2025171887A1 - Complexes radiomarqués et compositions pharmaceutiques les comprenant - Google Patents

Complexes radiomarqués et compositions pharmaceutiques les comprenant

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Publication number
WO2025171887A1
WO2025171887A1 PCT/EP2024/072834 EP2024072834W WO2025171887A1 WO 2025171887 A1 WO2025171887 A1 WO 2025171887A1 EP 2024072834 W EP2024072834 W EP 2024072834W WO 2025171887 A1 WO2025171887 A1 WO 2025171887A1
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WIPO (PCT)
Prior art keywords
moiety
upar
dota
binding
alb
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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PCT/EP2024/072834
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English (en)
Inventor
Roger Schibli
Cristina MÜLLER
Christian VACCARIN
Darja BEYER
Deberle LUISA MARIA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Scherrer Paul Institut
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Scherrer Paul Institut
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Publication of WO2025171887A1 publication Critical patent/WO2025171887A1/fr
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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
    • A61K51/08—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
    • A61K51/0497—Organic compounds conjugates with a carrier being an organic compounds
    • 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
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/121—Solutions, i.e. homogeneous liquid formulation
    • 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 the field of radiolabeled complexes, in particular to novel radiopeptides targeting the urokinase-type plasminogen activator receptor (uPAR), and pharmaceutical compositions including the same.
  • uPAR urokinase-type plasminogen activator receptor
  • Radiopharmaceuticals are drugs, which contain radioactive isotopes (radionuclides). Radiopharmaceuticals can be used to treat various conditions, including cancers, blood disorders and hyperthyroidism.
  • radionuclide therapy of cancer a molecule labeled with a radionuclide is used to deliver a toxic level of radiation to disease sites. Accordingly, the molecule is used to "target" the disease site, e.g. specific cancer cells. Accordingly, the radionuclide complex combines the specificity of cancer cell targeting with the known antitumor effects of ionizing radiation. Thereby, not only the primary tumor site, but also its metastases can be targeted.
  • the choice of the molecule that carries the radiation to the tumor is usually determined by its selectivity and affinity to the tumor's target structures, such as antigens or receptors. Even if a target structure is not selective for a certain kind of cancer, overexpressed target structures are of interest, because they allow the delivery of the radionuclide complex after its systemic administration in high concentration to those (overexpressing) target cells while leaving other cells (with no or minor expression only) essentially unaffected. Radionuclides are usually linked to the targeting molecule through chelating agents. Thereby, strong complexes with the metal ion of the radionuclide can be formed.
  • the radioactive decay of the radionuclides can cause significant damage to cancer cells by releasing high energy electrons, positrons or alpha particles as well as gamma rays at the target site.
  • the human urokinase-type plasminogen activator receptor is a glycosyl- phosphatidyl-inositol (GPI) membrane-anchored protein (Ploug M, Ronne E, Behrendt N, Jensen AL, Blasi F, Dano K. Cellular receptor for urokinase plasminogen activator. Carboxyl- terminal processing and membrane anchoring by glycosyl-phosphatidylinositol. J Biol Chem.
  • uPAR expression has been found in solid tumors including breast, gastric, pancreatic, colorectal, prostate, ovarian, oral and esophageal cancer, where it has been associated with tumorigenesis, metastasis, angiogenesis, tumor proliferation and invasion (Duffy MJ. The urokinase plasminogen activator system: role in malignancy. Curr Pharm Des. 2004;10:39- 49. doi:10.2174/1381612043453559; Alpizar-Alpizar W, Christensen IJ, Santoni-Rugiu E, Skarstein A, Ovrebo K, lllemann M, et al.
  • Urokinase plasminogen activator receptor on invasive cancer cells a prognostic factor in distal gastric adenocarcinoma. Int J Cancer. 2012;131 :E329-36. doi:10.1002/ijc.26417; Christensen A, Kiss K, Lelkaitis G, Juhl K, Persson M, Charabi BW, et al.
  • Urokinase-type plasminogen activator receptor (uPAR), tissue factor (TF) and epidermal growth factor receptor (EGFR) tumor expression patterns and prognostic value in oral cancer. BMC Cancer. 2017;17:572.
  • uPAR is present not only in tumors, but also in a number of tumor- associated cells, including angiogenic endothelial cells and macrophages (Mazar AP et al., 2011, supra).
  • Cleaved uPAR present in the blood plasma was proposed as a biomarker for the diagnosis and therapy of cancer (Rasch MG, Lund IK, Almasi CE, Hoyer-Hansen G. Intact and cleaved uPAR forms: diagnostic and prognostic value in cancer. Front Biosci.
  • uPAR may be a relevant tumor-associated target for diagnostic and therapeutic radiopharmaceuticals.
  • uPAR-targeting agents were identified using phage display and affinity maturation techniques to develop synthetic uPAR-binding peptides (Ploug M, Ostergaard S, Gardsvoll H, Kovalski K, Holst-Hansen C, Holm A, et al. Peptide-derived antagonists of the urokinase receptor. Affinity maturation by combinatorial chemistry, identification of functional epitopes, and inhibitory effect on cancer cell intravasation. Biochem. 2001 ;40:12157-68. doi:10.1021/bi010662g; Ploug M.
  • AE105 was modified with various chelators including NOTA, NODAGA and DOTA, to enable the complexation of a variety of radiometals such as copper-64, gallium-68 and lutetium-177.
  • the resultant radiopeptides were investigated in preclinical studies using various tumor mouse models; for example, [ 64 Cu]Cu-DOTA-AE105 was used successfully to visualize small foci in a model of disseminated prostate cancer in mice (Li ZB, Niu G, Wang H, He L, Yang L, Ploug M, et al. Imaging of urokinase-type plasminogen activator receptor expression using a 54 Cu-labeled linear peptide antagonist by microPET. Clin Cancer Res. 2008;14:4758-66.
  • PSMA prostate-specific membrane antigen
  • uPAR urokinase-type plasminogen activator receptor
  • the word “substantially” does not exclude “completely” e.g., a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.
  • the term “about” in relation to a numerical value x means x + 20%, preferably x + 10%, more preferably x ⁇ 5%, even more preferably x ⁇ 2% and still more preferably x ⁇ 1 %.
  • the present invention provides a radiolabeled complex targeting the urokinase-type plasminogen activator receptor as defined in the appended claims.
  • uPAR urokinase-type plasminogen activator receptor
  • the radiolabeled complex comprises a conjugate comprising a targeting moiety, a chelating moiety and an albumin-binding moiety (linked via a common trifunctional linker), wherein a radionuclide as defined herein is complexed by the chelating moiety.
  • targeting peptide refers to a peptide or polypeptide, which is able to bind (specifically) to the urokinase-type plasminogen activator receptor (uPAR).
  • uPAR urokinase-type plasminogen activator receptor
  • the amino acids of the targeting peptide or polypeptide may be modified, for example by phosphorylation, acetylation, hydroxylation and/or methylation.
  • the binding of the targeting peptide to the urokinase-type plasminogen activator receptor (uPAR) may be reversible or irreversible.
  • the targeting peptide moiety comprises at least 5 amino acids, preferably at least 6 amino acids, more preferably at least 7 amino acids, still more preferably at least 8 amino acids and most preferably at least 9 amino acids.
  • the targeting peptide may e.g. comprise 7 to 20 amino acids, i.e. the targeting peptide may comprise 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 1 7, 18, 19 or 20 amino acids.
  • the targeting peptide comprises 8 to 15 amino acids, in a still more preferred embodiment, the targeting comprises 9 to 13 amino acids.
  • the targeting peptide moiety comprises the amino acid sequence:
  • (Xaa 5 ) is preferably selected from the group consisting of (D-Arg), (Arg), (Tyr), (D-Tyr), and (Gin), and is more preferably (D-Arg);
  • (Xaa 6 ) is preferably selected from the group consisting of (Tyr), ([beta]-cyclohexyl-L-alanine), N-(2,3-dimethoxybenzyl)glycine, and is more preferably (Tyr);
  • (Xaa 7 ) is preferably selected from the group consisting of (Leu) and (D-Phe);
  • (Xaa 8 ) is preferably selected from the group consisting of (Trp), ([beta]-1 -naphthyl-L-alanine), ([beta]-2-naphthyl-L-alanine), (N-(3-indolylethyl)glycine), (N-benzylglycine), (N- (methylnaphthalyl)glycine) and (N-(2,3-dimethoxybenzyl)glycine), and is more preferably (Trp); and
  • the targeting peptide moiety may comprise an amino acid sequence selected from the group consisting of (D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) (SEQ ID NO: 1 ), (Leu)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Gln)-(Tyr)-(Leu)-(Trp)-(Ser) ((SEQ ID NO: 2),
  • the targeting peptide moiety may comprise the amino acid sequence:
  • (Xaa 5 ) is selected from (D-Arg), (D-Lys), (D-Cys), and (D-Ser);
  • (Xaa6 ) is selected from (Tyr) and (Pro);
  • the targeting peptide moiety may comprise an amino acid sequence selected from the group consisting of
  • the targeting peptide moiety comprises the amino acid sequence (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)- (Ser) (SEQ ID NO: 4).
  • This uPAR-binding nonapeptide is also referred to as AE105:
  • the targeting peptide moiety may also comprise a mutated form of the AE105 nonapeptide, such as (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Glu)-(Ser) (SEQ ID NO: 24), for example.
  • a mutated form of the AE105 nonapeptide such as (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Glu)-(Ser) (SEQ ID NO: 24), for example.
  • the targeting peptide moiety may comprise a cyclized variant of AE105, e.g. having the amino acid sequence (Lys)-(Ser)-(Asp)-([beta]-cyclohexyl-L- alanine)-(Phe)-(D-Ser)-(D-Lys)-(Tyr)-(Leu)-(Trp)-(Ser)-(Ser)-(Lys) (SEQ ID NO: 27) referred to as AE147.
  • the targeting peptide moiety of the inventive conjugate/radiolabeled complex preferably comprises or consist of a AE105 nonapeptide, or a derivative or mutated form thereof, or a (cyclized) variant thereof.
  • Radionuclide Various radionuclides (radioisotopes) are known to be useful in the field of radionuclide therapy.
  • the term “radionuclide” refers to isotopes of natural or artificial origin with an unstable neutron to proton ratio that disintegrates with the emission of corpuscular (i.e. positrons (beta plus-radiation); helium nuclei (alpha-radiation) or electrons (beta minus-radiation, Auger and conversion electron radiation) or electromagnetic radiation (gamma-radiation).
  • positrons beta plus-radiation
  • helium nuclei alpha-radiation
  • electrons beta minus-radiation, Auger and conversion electron radiation
  • electromagnetic radiation gamma-radiation
  • Said radionuclide may preferably be useful for cancer imaging or therapy.
  • Non-limiting examples of suitable radionuclides include 99m Tc, 1 l l ln, 67 Ga, 68 Ga, 86 Y, 90 Y, 177 Lu, 161 Tb, 149 Tb, 155 Tb, 152 Tb, 186 Re, 188 Re, 61 Cu, 64 Cu, 67 Cu, 55 Co, 57 Co, 43 Sc, 44 Sc, 47 Sc, 225 Ac, 213 Bi, 212 Bi, 212 Pb, 227 Th, 153 Sm, 166 Ho, 166 Dy, 169 Er, 16S Er, 103 Pd, 109 Pd, 103m Rh, 18 F, 123 l, 124 l, 131 l, and 2l 1 At.
  • the radionuclide of the inventive radiolabeled complex may be any one of the before-mentioned examples.
  • the choice of suitable radionuclides may depend inter alia on the chemical structure and chelating capability of the chelating agent, and the intended application of the resulting (complexed) conjugate (e.g. diagnostic vs. therapeutic).
  • the beta-minus emitters such as 90 Y, l31 l, 161 Tb and 177 Lu may be used for systemic radionuclide therapy.
  • DOTA as chelating agent, may advantageously enable the use of 177 Lu, 161,155, 152, 149 Tb, 43,44,47 Sc, 225 Ac, 213, 212 Bi, or 212 Pb as radionuclides, while NODAGA is more favorably used for chelation of 68,67 Ga, and 61 ' 64,67 Cu.
  • the electron-emitting radionuclide may be selected from 177 Lu and 161 Tb, and alpha-emitting radionuclides may be selected from 149 Tb and 212 Pb whereof the latter decays via a beta minus emission to the alpha-particle emitting 212 Bi.
  • the radionuclide metal ion usually forms a non-covalent bond with functional groups of the chelating moiety, e.g. amines or carboxylic acids.
  • the chelating moiety has at least two such complexing functional groups to be able to form a chelate complex.
  • chelating moiety refers to polydentate (multiple bonded) ligands capable of forming two or more separate coordinate bonds with (coordinating") a central (metal) ion, in particular the radionuclide metal ion. Specifically, such molecules or molecules sharing one electron pair may also be referred to as resortLewis bases".
  • the central (metal) ion is usually coordinated by two or more electron pairs to the chelating agent.
  • the terms, placedbidentate chelating agent”, frequentlytridentate chelating agent”, and particularlytetradentate chelating agent” are known in the art and refer to chelating agents having two, three, and four electron pairs, respectively, which are readily available for simultaneous donation to a metal ion coordinated by the chelating agent.
  • the electron pairs of a chelating moiety forms coordinate bonds with a single central (metal) ion; however, in certain examples, a chelating moiety may form coordinate bonds with more than one metal ion, with a variety of binding modes being possible.
  • Coordinating and Coordination refer to an interaction in which one multi- electron pair donor coordi natively bonds (is “Coordinated") to, i.e. shares two or more unshared pairs of electrons with, one central (metal) ion.
  • the chelator or chelating moiety is preferably a macrocyclic bifunctional chelator having a metal chelating group at one end and a reactive functional group at the other end, which is capable to bind to other moieties, e.g. peptides.
  • the chelator may be selected such that the chelator forms a square bi-pyramidal complex for complexing the radionuclide. In another embodiment, the chelator does not form a planar or a square planar complex.
  • the chelating moiety is selected from 1 ,4,7,10-tetraazacyclododecane-1 , 4, 7,10- tetraacetic acid (DOTA), 1 ,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7- bis(carboxymethyl)-1 ,4,7-triazonan-1 -yl)pentanedioic acid (NODAGA), N,N'-bis[2- hydroxy-5-(carboxyethyl)-benzyl]ethylenediamine-/V / /V'-diacetic acid (HBED-CC), 2- (4,7,10-tris(carboxymethyl)-1 ,4,7,10-tetraazacyclododecan-1 -yl)-pentanedioic acid
  • the chelating moiety is selected from DOTA (which may be characterized by Formula (1 a)), NOTA (which may be characterized by Formula (1g)), NODAGA (which may be characterized by Formula (1 h)) and DOTAGA (which may be characterized by Formula (1 b)).
  • the chelating moiety is selected from DOTA (1 ,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic aacciidd, and DOTAGA (2-(4,7,10- tris(carboxymethyl)-1 ,4,7,10-tetraazacyclododecan-1 -yl)-pentanedioic acid) or derivatives thereof.
  • the chelating agent is DOTA.
  • DOTA effectively forms complexes with diagnostic (e.g. 68 Ga) and therapeutic (e.g. 161 Tb or 177 lu) radionuclides and thus enables the use of the same conjugate (targeting molecule linked to the chelating agent) for both imaging and therapeutic purposes, i.e. as a theragnostic agent.
  • DOTA derivatives capable of complexing Scandium radionuclides ( 43 Sc, 44 Sc, 47 Sc), including DO3AP, DO3AP PrA , or DO3AP ABn (which may be characterized by Formulae (I d), (1 e) and (If)) may also be preferred and are described in Kerdjoudj et al. Dalton Trans., 2016, 45, 1398-1409.
  • the chelating agent for example DOTA, may be complexed with any appropriate radionuclide (in particular with the radionuclide as described above) as a central (metal) ion. It is within the skill and knowledge of the skilled person in the art to select suitable combinations of conjugates and radionuclides.
  • the chelator may be DOTA and the radionuclide may be 68 Ga.
  • the chelator may be NODAGA and the radionuclide may be 68 Ga.
  • the chelator may be DOTA and the radionuclide may be 44 Sc.
  • the chelator may be DOTA and the radionuclide may be 64 Cu.
  • the chelator may be NODAGA and the radionuclide may be 61 Cu, 64 Cu or 67 Cu. In other embodiments, the chelator may be DOTAM and the radionuclide may be 212 Pb. In a preferred embodiment, the chelator may be DOTA and the radionuclide may be 161 Tb. In another preferred embodiment, the chelator may be DOTAGA and the radionuclide may be 177 Lu. In another preferred embodiment, the chelator may be DOTA and the radionuclide may be , 77 Lu.
  • the inventive radiolabeled complex comprises an albumin-binding moiety which is preferably capable of selectively binding to serum albumin, e.g. to mouse serum albumin or human serum albumin (HSA).
  • serum albumin e.g. to mouse serum albumin or human serum albumin (HSA).
  • HSA human serum albumin
  • selectively binding means that a compound binds with a greater affinity to its intended target than it binds to another, non-target entity.
  • Binding affinity is the strength of the binding interaction between a ligand (e.g. a small organic molecule, protein or nucleic acid) to its target/binding partner.
  • the albumin-binding moiety may be any known or newly developed albumin-binding moiety. Particularly preferred albumin-binding moieties are described herein below.
  • the albumin-binding moiety may preferably bind non-covalently to serum albumin, preferably HSA, typically with a binding affinity of less than about 100 ⁇ m (micromolar), e.g. of about 3 ⁇ m (micromolar) to 50 ⁇ m (micromolar).
  • Human Serum Albumin is the most abundant protein in human blood plasma and constitutes about half of serum protein.
  • Human Serum Albumin or "HSA” as used herein preferably refers to the serum albumin protein encoded by the human ALB gene. More preferably, the term refers to the protein as characterized under UniProt Acc. No. P02768 (entry version 240, last modified May 10, 2017, or functional variants, isoforms, fragments or (post-translational ly or otherwise modified) derivatives thereof.
  • the albumin-binding moiety of the inventive radiolabeled complex preferably extends circulation half-life of the conjugates, and effects compartmentalization of the inventive complexes in the blood and improved delivery to the uPAR-expressing (tumor) target cells or tissues.
  • the albumin- binding moiety is thus envisaged to confer improved pharmacokinetic properties to the inventive complex, preferably without interfering with (reducing or abolishing) the desired function of the chelating moiety and the uPAR binding moiety.
  • the albumin-binding moiety may be any albumin-binding moiety known in the art. Particularly preferred albumin-binding entities are described herein below.
  • the albumin- binding moiety may preferably bind non-covalently to HSA, typically with a binding affinity less than about 100 ⁇ m.
  • typical albumin-binding moieties in accordance with the present invention comprise linear and branched lipophilic groups comprising 1 -40 carbon atoms and a distal acidic group.
  • Suitable albumin-binding entities are inter alia described in US 2010/172844 A1 , WO 2013/024035 A1 and WO 2008/053360 A2, which are incorporated by reference in their entirety herein.
  • R 6 and R 7 are each independently selected from H or branched, unbranched or cyclic C 1-12 hydrocarbyl, and X is selected from O, N, P or S.
  • R 1 and R 2 may be in ortho-, meta or para-position.
  • said cyclic structure is preferably a linear or branched hydrocarbyl chain of 3-12, more preferably 3-10, even more preferably 3-9, 3-8, 3-7, 3-6, 3-5, 3-4 or 4 carbon atoms bonded at two positions to the phenyl ring, i.e. forming two bonds to said phenyl ring, such as to form a ring structure fused to said phenyl ring.
  • said cyclic structure may be selected from (substituted or unsubstituted) adamantyl.
  • said two bonds are preferably situated at the meta (3-) and para (4-) positions, at the ortho (2-) and meta positions or at the ortho and para positions of said phenyl ring.
  • Said cyclic structure is optionally interrupted by up to 2, preferably 1 or no heteroatoms.
  • said cyclic structure may be a C 4 chain fragment (1 ,4-diradical) linked by its 1 - and 4- atoms to said phenyl ring to form a six- membered ring fused to said phenyl ring, preferably at the meta and para positions of said phenyl ring, i.e., preferably forming a meta- and para-fused six-membered ring.
  • R 1 and R 2 may each be independently selected from H, halogen, preferably iodine or bromine, and C 1-6 alkyl, preferably C 1-3 alkyl, even more preferably methyl. More preferably, R 1 is H and R 2 is selected from halogen, preferably iodine or bromine, and C 1-6 alkyl, preferably C1.3 alkyl, even more preferably methyl. Even more preferably, R 1 is H and R 2 is H or is in the para position and selected from iodine, bromine and methyl.
  • Y may be a linear or branched, optionally substituted, C 1 -C 12 hydrocarbyl, more preferably a linear or branched, optionally substituted, C 1 -C 10 hydrocarbyl, even more preferably a linear or branched, optionally substituted, C 1 -C 6 hydrocarbyl, even more preferably a a linear or branched, optionally substituted, C 1 -C 3 hydrocarbyl.
  • Y may be -(CH 2 ) 3 -.
  • X may be O.
  • albumin-binding moiety according to Formula (2) may preferably comprise any one of Formulae (2a)-(21):
  • the albumin-binding moiety comprises a group, which may be characterized by Formula (2c) or (2e).
  • the albumin-binding moiety comprises a group selected from an iodophenyl moiety, and a tolyl moiety.
  • the albumin-binding moiety may comprise an ibuprofen moiety, which is shown in Formula (21).
  • a conjugate according to the present invention, in which ibuprofen is used as an albumin-binding moiety is shown in Formula (9) below.
  • Evans Blue or derivatives thereof e. g. truncated Evans Blue
  • albumin with high affinity
  • Orit Jacobson, Dale O Kiesewetter, Xiaoyuan Chen Albumin-Binding Evans Blue Derivatives for Diagnostic Imaging and Production of Long-Acting Therapeutics, Bioconjug Chem. 2016 Oct 19;27(10):2239-2247. doi: 10.1021/acs.bioconjchem.6b00487. Epub 2016 Oct 6
  • albumin-binding moiety in the inventive conjugates/radiolabeled complexes.
  • the present invention provides novel uPAR-binding conjugates/radiolabeled complexes with improved tumor-targeting properties and favorable pharmacokinetic profiles.
  • the term simply refers to the stability, bioavailability, absorption; biodistribution, biological half-life and/or clearance of a therapeutic or diagnostic agent in a subject.
  • the present invention provides novel conjugates by covalently coupling a human serum albumin (HSA) binding moiety to an uPAR-binding peptide moiety on the one hand and a chelating moiety capable of complexing therapeutic/diagnostic radionuclides on the other hand, via suitable linkers and spacers, respectively.
  • HSA human serum albumin
  • the linker and spacer moieties connecting the binding entities and chelator were found to be crucial for the targeting and pharmacokinetic properties of the resulting conjugates/radiolabeled complexes.
  • the novel conjugates/radiolabeled complexes preferably exhibit superior and specific cellular uptake and internalization characteristics. Introduction of a HSA binding entity thereby advantageously improves biodistribution and, eventually, therapeutic efficacy of the inventive compounds.
  • the (uPAR binding) targeting peptide moiety, the chelating moiety and the albumin-binding moiety are linked via a common trifunctional linker moiety L1.
  • the present invention provides a conjugate represented by the general structure (I): wherein Abm is an albumin-binding moiety, Cm is a chelating moiety, and Tpm is an uPAR- binding targeting peptide moiety.
  • linker moiety L1 constitutes a "branching point" in the inventive conjugate/radiolabeled complex.
  • linking bonds between the targeting peptide moiety and the linker moiety L1 , the chelating moiety and the linker moiety L1 , and the albumin-binding moiety and the linker moiety L1 , respectively, are covalent or non-covalent bonds. Preferably the bonds are covalent.
  • the term "linker” is used herein to specifically refer to the group connecting or linking and thus spanning the distance between the moieties of the inventive conjugate/radiolabeled complex, i.e. the targeting peptide moiety (i.e. uPAR-binding moiety), the chelating moiety and the albumin-binding moiety, respectively.
  • the linker may preferably avoid sterical hindrance between the targeting peptide moiety (i.e. uPAR-binding moiety), the chelating moiety and the albumin-binding moiety and ensure sufficient mobility and flexibility. Further, the linker may preferably be designed so as to confer, support and/or allow sufficient HSA binding, high affinity uPAR binding, and rapid and optionally selective penetration of uPAR positive cells through internalization of the inventive radiolabeled complex.
  • the trifunctional linker moiety L1 comprises an amino acid residue or a derivative thereof.
  • the targeting peptide moiety i.e. uPAR-binding moiety
  • the chelating moiety and the albumin-binding moiety are linked, preferably covalently linked, by an amino acid residue or a derivative thereof, wherein preferably one of the moieties may be bound to the amino group of the amino acid, one moiety may be bound to the carboxy group of the amino acid, and one moiety may be bound to a side-chain functional group, e.g. a further amino (NH2-) group, a further carboxy (COOH-) group, a hydroxyl (OH-) group, or a thio (SH-) group of the amino acid's side chain.
  • a side-chain functional group e.g. a further amino (NH2-) group, a further carboxy (COOH-) group, a hydroxyl (OH-) group, or a thio (SH-) group of the amino
  • the trifunctional linker moiety L1 is an amino acid selected from the group consisting of a lysine residue, an arginine residue, a glutamine residue, an asparagine residue, an aspartic acid residue, glutamic acid residue, serine residue, threonine residue, cysteine residue, or a derivative of said amino acid residues, more preferably selected from a lysine residue, an arginine residue, a glutamine residue, an asparagine residue or a derivative of said amino acid residues, most preferably a lysine residue or a derivative thereof.
  • a derivative of an amino acid residue may e.g. be an amino acid having a shortened or extended hydrocarbon chain.
  • a derivative of an amino acid may also be a substituted amino acid, e.g. a methylated amino acid, such as e.g. methyllysine, methylarginine etc.
  • An example of a conjugate comprising, as a trifunctional linker moiety L1 , a lysine derivative having a shortened side chain (diaminopropanoic acid) will be shown below (Formula (8)).
  • a radiolabeled complex obtained from the above conjugate according to Formula (3) has advantageous properties with respect to an enhanced blood circulation time and an increased uptake into xenografts, in particular when compared to prior art radiopeptide ([ 177 Lu]Lu-DOTA-AE105) lacking the albumin- binding entity.
  • an (additional) linker moiety L2 is located between the trifunctional linker moiety L1 and the albumin-binding moiety providing a greater distance between the albumin-binding moiety to the chelating moiety and the targeting peptide moiety, respectively.
  • the present invention provides a conjugate represented by the following general structure (II): wherein Abm is an albumin-binding moiety, Cm is a chelating moiety, and Tpm is an uPAR- binding targeting peptide moiety, each as defined above, and L1 is a linker moiety as defined above.
  • the linker moiety L2 which provides further distance between the albumin-binding moiety and the chelating and uPAR-binding targeting peptide moieties, respectively, may be selected from any suitable and pharmaceutical acceptable linker moieties known in the art.
  • the linker moiety L2 is selected from the group consisting of amino acids, polyethyleneglycols (PEGs), poly(N/-vinylpyrrolidone), polyacrylamides, polybetaines, poly(2-oxazoline)s, polyesters, polysarcosine, and alkyl groups, more preferably from the group consisting of amino acids, polyethyleneglycols (PEGs), and alkyl groups.
  • the linker moiety L2 may comprise at least one amino acid which is preferably selected from the group consisting of lysine, arginine, glutamine, asparagine, aspartic acid, glutamic acid, serine, tyrosine, threonine, phenylalanine, cysteine, proline, leucine, isoleucine, valine, histidine, alanine, and diaminobutyric acid, or a derivative of said amino acids, or a combination of said amino acid residues.
  • the linker moiety L2 comprises at least one lysine residue or a derivative thereof.
  • the linker moiety L2 comprises a PEG moiety.
  • the PEG based linker may comprise about 1 to 30 PEG units, and preferably comprises 1 to 10 PEG units, more preferably 2 to 7 PEG units, most preferably 3 to 5 PEG units.
  • the trifunctional linker moiety L1 is a lysine residue or a derivative thereof, and the linker moiety L2 is a PEG moiety, preferably a PEG 3 to PEG 5 m oiety, e.g. a PEG 4 moiety.
  • DOTA-uPAR-Alb- 02 An exemplary conjugate according to the present invention (referred to as DOTA-uPAR-Alb- 02), which comprises the nonapeptide AE105 as a (uPAR-binding) targeting peptide moiety, DOTA as a chelating moiety, iodophenyl as an albumin-binding moiety, lysine as a trifunctional linker moiety L1 , and a PEG 4 linker as a linker moiety L2, is represented by formula (4):
  • a radiolabeled complex obtained from the above conjugate according to Formula (4) has particularly advantageous properties with respect to an enhanced blood circulation time and an increased uptake into xenografts.
  • DOTA- uPAR-Alb-1 1 Another exemplary conjugate according to the present invention (referred to as DOTA- uPAR-Alb-1 1 ), which comprises the nonapeptide AE105 as a (uPAR-binding) targeting peptide moiety, DOTA as a chelating moiety, p-tolyl as an albumin-binding moiety, lysine as a trifunctional linker moiety L1 , and a PEG 4 linker as a linker moiety L2, is represented by formula (5):
  • a radiolabeled complex obtained from the above conjugate according to Formula (5) has particularly advantageous properties with respect to an enhanced blood circulation time and an increased uptake into xenografts.
  • DOTA- uPAR-Alb-14 Another exemplary conjugate according to the present invention (referred to as DOTA- uPAR-Alb-14), which comprises the nonapeptide AE105 as a targeting peptide (uPAR- binding) moiety, DOTA as a chelating moiety, p-tolyl as an albumin-binding moiety, diaminopropanoic acid (instead of a lysine residue) as a trifunctional linker moiety L1 , and a PEG 4 linker as a linker moiety L2, is represented by formula (8):
  • a radiolabeled complex obtained from the above conjugate according to Formula (8) has advantageous properties with respect to an enhanced blood circulation time and an increased uptake into xenografts.
  • Another exemplary conjugate according to the present invention which comprises the nonapeptide AE105 as a targeting peptide (uPAR-binding) moiety, DOTA as a chelating moiety, ibuprofen as an albumin-binding moiety, lysine as a trifunctional linker moiety L1 , and a PEG4 linker moiety as a linker moiety L2, is represented by formula (9):
  • DOTAGA- uPAR-Alb-17 Another exemplary conjugate according to the present invention (referred to as DOTAGA- uPAR-Alb-17), which comprises the nonapeptide AE105 as a targeting peptide (uPAR- binding) moiety, DOTAGA as a chelating moiety, tolyl as an albumin-binding moiety, lysine as a trifunctional linker moiety L1 , and a PEG 4 linker moiety as a linker moiety L2, is represented by formula (10):
  • the linker moiety L2 may comprise an alkyl group.
  • the alkyl linker may e.g. comprise about 1 to 30 C atoms.
  • the alkyl linker comprises about 5 to 20 C atoms, more preferably about 7 to 14 C atoms.
  • An exemplary conjugate according to the present invention which comprises the nonapeptide AE105 as a targeting peptide (uPAR-binding) moiety, DOTA as a chelating moiety, tolyl as an albumin-binding moiety, lysine as a trifunctional linker moiety L1 , and a C7 alkyl group as a linker moiety L2, is represented by formula (1 1 ):
  • Another exemplary conjugate according to the present invention which comprises the nonapeptide AE105 as a targeting peptide (uPAR-binding) moiety, DOTA as a chelating moiety, tolyl as an albumin-binding moiety, lysine as a trifunctional linker moiety L1 , and two C7 alkyl groups as a linker moiety L2, is represented by formula (12):
  • Linker moiety L3 In another preferred embodiment of the inventive radiolabeled complex, a linker moiety L3 is located between the trifunctional linker moiety L1 and the chelating moiety thereby extending the distance between the chelating moiety with respect to the uPAR-binding targeting peptide moiety and the albumin-binding moiety, respectively.
  • the present invention provides a conjugate represented by the general structure (III): wherein Abm is an albumin-binding moiety, Cm is a chelating moiety, and Tpm is an uPAR- binding targeting peptide moiety, each as defined above, and L1 is a linker moiety as defined above.
  • the linker moiety L3 which provides further distance between the chelating moiety and the albumin-binding and uPAR-binding targeting peptide moieties, respectively, may be selected from any suitable and pharmaceutical acceptable linker moiety known in the art.
  • the linker moiety L3 is selected from the group consisting of amino acids, polyethyleneglycols (PEGs), poly(AZ-vinylpyrrolidone), polyacrylamides, polybetaines, poly(2-oxazoline)s, polyesters, polysarcosine, and alkyl groups, more preferably from the group consisting of amino acids, polyethyleneglycols (PEGs), and alkyl groups.
  • the linker moiety L3 located between the trifunctional linker moiety Ll and the chelating moiety may comprise a PEG moiety.
  • the PEG based linker may comprise about 1 to 30 PEG units, and preferably comprises 1 to 10 PEG units, more preferably 2 to 7 PEG units, most preferably 3 to 5 PEG units.
  • the linker moiety L3 located between the trifunctional linker moiety L1 and the chelating moiety comprises at least one amino acid which is preferably selected from the group consisting of a lysine residue, an arginine residue, a glutamine residue, an asparagine residue, an aspartic acid residue, a glutamic acid residue, a serine residue, a threonine residue, a cysteine residue, or a derivative of said amino acid residues, or a combination of said amino acid residues, more preferably selected from a lysine residue, an arginine residue, a glutamine residue, an asparagine residue or a derivative thereof, or a combination of said amino acid residues or derivatives thereof.
  • L3 is a lysine residue or a derivative thereof.
  • the trifunctional linker moiety L1 is a lysine residue or a derivative thereof
  • the linker moiety L3 located between the trifunctional linker moiety L1 and the chelating moiety is a further lysine residue or a derivative thereof.
  • a radiolabeled complex obtained from the above conjugate according to Formula (13) has advantageous properties with respect to an enhanced blood circulation time and an increased uptake into xenografts, respectively.
  • a linker moiety L3 is located between the trifunctional linker moiety L1 and the (uPAR binding) peptide moiety thereby extending the distance between the targeting peptide moiety with respect to the chelating moiety and the albumin-binding moiety, respectively.
  • the present invention provides a conjugate represented by the general structure (IV): wherein Abm is an albumin-binding moiety, Cm is a chelating moiety, and Tpm is an uPAR- binding targeting peptide moiety, each as defined above, and L1 is a linker moiety as defined above.
  • the linker moiety L3 which provides further distance between the uPAR-binding targeting peptide moiety and the albumin-binding and chelating moieties, respectively, may be selected from any suitable and pharmaceutical acceptable linker moiety known in the art.
  • the linker moiety L3 is selected from the group consisting of amino acids, polyethyleneglycols (PEGs), poly(Mvinylpyrrolidone), polyacrylamides, polybetaines, poly(2-oxazoline)s, polyesters, polysarcosine, and alkyl groups, more preferably from the group consisting of amino acids, polyethyleneglycols (PEGs), and alkyl groups.
  • the linker moiety L3 located between the trifunctional linker moiety L1 and the (uPAR binding) peptide moiety may comprise at least one amino acid which is preferably selected from the group consisting of a lysine residue, an arginine residue, a glutamine residue, an asparagine residue, an aspartic acid residue, a glutamic acid residue, a serine residue, a threonine residue, a cysteine residue, or a derivative of said amino acid residues, or a combination of said amino acid residues, more preferably selected from a lysine residue, an arginine residue, a glutamine residue, an asparagine residue or a derivative thereof, or a combination of said amino acid residues or derivatives thereof.
  • L3 is a lysine residue or a derivative thereof.
  • the linker moiety L3 located between the trifunctional linker moiety L1 and the (uPAR binding) peptide moiety comprises a PEG moiety.
  • the PEG based linker may comprise about 1 to 30 PEG units, and preferably comprises 1 to 10 PEG units, more preferably 2 to 7 PEG units, most preferably 3 to 5 PEG units.
  • the trifunctional linker moiety L1 is a lysine residue or a derivative thereof
  • the linker moiety L3 located between the trifunctional linker moiety L1 and the (uPAR binding) peptide moiety is a PEG moiety, preferably a PEG 3 to PEGs moiety.
  • the inventive conjugate/radiolabeled complex of the present invention may comprise both, a linker moiety L3 located between the trifunctional linker moiety L1 and the chelating moiety, and a linker moiety L3 located between the trifunctional linker moiety L1 and the (uPAR-binding) targeting peptide moiety, thus providing additional distance between the chelating moiety and the albumin-binding moiety as well as the (uPAR-binding) targeting peptide moiety.
  • the present invention provides a conjugate represented by the general structure (V): wherein Abm is an albumin-binding moiety, Cm is a chelating moiety, and Tpm is an uPAR- binding targeting peptide moiety, each as defined above, and L1 and L3 are as defined above.
  • the inventive conjugate/radiolabeled complex of the present invention comprises a linker moiety L2 located between the trifunctional linker moiety L1 and the albumin-binding moiety, as well as a linker moiety L3 located between the trifunctional linker moiety L1 and the chelating moiety and/or a linker moiety L3 located between the trifunctional linker moiety L1 and the (uPAR-binding) targeting peptide moiety, thus providing additional distance between the albumin-binding moiety and the chelating moiety as well as the (uPAR-binding) targeting peptide moiety.
  • the inventive conjugate/radiolabeled complex may also comprise a spacer moiety S1 .
  • spacer is used herein to specifically refer to a group connecting and spanning the distance between several moieties of the inventive conjugate/radiolabeled complex, i.e. "spacing" a distinct group apart from the remaining groups/entities of the conjugate/radiolabeled complex.
  • a spacer moiety S1 may be located between the trifunctional linker moiety L1 and the albumin-binding moiety to further enlarge the distance between the albumin-binding moiety and the chelating moiety, and the uPAR-binding targeting peptide moiety, respectively.
  • an albumin-binding entity e.g. an iodophenyl moiety, a tolyl moiety, or an ibuprofen moiety
  • the spacer S1 may be located between linker moieties L1 and L2.
  • the spacer moiety S1 is located between the linker moiety L2 and the albumin-binding moiety.
  • the present invention provides a conjugate represented by the general structure (VII): wherein Abm is an albumin-binding moiety, Cm is a chelating moiety, and Tpm is an uPAR- binding targeting peptide moiety, each as defined above, and L1 and L2 are linker moieties as defined above.
  • a linker moiety L3 may be located between the trifunctional linker moiety L1 and the targeting peptide moiety and/or between the trifunctional linker moiety L1 and the chelating moiety.
  • the present invention also provides a conjugate represented by the general structure (VIII): wherein Abm is an albumin-binding moiety, Cm is a chelating moiety, and Tpm is an uPAR- binding targeting peptide moiety, each as defined above, and L1 , L2 and L3 are linker moieties as defined above.
  • VIII conjugate represented by the general structure (VIII): wherein Abm is an albumin-binding moiety, Cm is a chelating moiety, and Tpm is an uPAR- binding targeting peptide moiety, each as defined above, and L1 , L2 and L3 are linker moieties as defined above.
  • any spacer moiety known in the art can suitably be used as a spacer moiety S1 .
  • the spacer moiety S1 is an aromatic spacer.
  • aromatic spacer moieties S1 which may be used in the inventive conjugates/radiolabeled complexes are amino- and carboxy- substituted aromatic structures, such as 2-, 3-, and 4-aminobenzoic acids, 2-, 3-, and 4- (aminomethyl)benzoic acids, 2-, 3-, and 4-(aminophenyl)acetic acids, 2-, 3-, and 4- (aminomethylphenyl)acetic acid (AMPA), (aminomethyl)pyrrole carboxylic acids, (aminomethyl)thiophene carboxylic acids, (aminomethyl)furan carboxylic acids, (aminomethyl)pyrrole acetic acids, (aminomethyl)thienyl acetic acids, and aminobiphenylcarboxylic acids.
  • Further examples of the spacer moiety S1 are more hydrophilic moi
  • a 4-(aminomethyl)benzoic acid (AMBA) moiety is used as a spacer moiety S1 .
  • An exemplary conjugate according to the present invention (referred to as DOTA-uPAR-Alb- 03), which comprises the nonapeptide AE105 as a (uPAR-binding) targeting peptide moiety, DOTA as a chelating moiety, p-iodophenyl as an albumin-binding moiety, lysine as a trifunctional linker moiety L1 , a PEG 4 linker moiety as a linker moiety L2, and a 4- (aminomethyl)benzoic acid (AMBA) moiety as a spacer moiety S1 is represented by formula (15):
  • a radiolabeled complex obtained from the above conjugate according to Formula (15) has particularly advantageous properties with respect to an enhanced blood circulation time and an increased uptake into xenografts, respectively.
  • the present invention provides a pharmaceutical composition comprising a radiolabeled complex according to the present invention.
  • the uPAR-binding targeting peptide moiety may comprise a peptide as described above, in particular a peptide comprising 7 to 20, preferably 8 to 15, more preferably 9 to 13 amino acids.
  • the uPAR-binding targeting peptide moiety may comprise the amino acid sequence:
  • the chelator may be a macrocyclic chelator or a linear chelator characterized by any one of the above Formulae (1 a) to (I ff).
  • L3 is a linker moiety as defined above, which is preferably selected from the group consisting of amino acids, polyethyleneglycols (PEGs), poly(/V- vinylpyrrolidone), polyacrylamides, polybetaines, poly(2-oxazoline)s, polyesters, polysarcosine, and alkyl groups, and is more preferably selected from a PEG based linker, preferably comprising 1 to 30 PEG units, more preferably comprising 1 to 10 PEG units, still more preferably comprising 1 to 5 PEG units, most preferably comprising 3 to 5 PEG units, and an amino acid residue, preferably a lysine residue, an arginine residue, a glutamine residue, an asparagine residue, or a derivative of said amino acid residues, most preferably a lysine residue or a derivative thereof; and
  • S1 is a spacer moiety, as defined above, preferably comprising an amino- and carboxy-substituted aromatic structure, more preferably selected from 2-, 3-, and 4- aminobenzoic acids, 2-, 3-, and 4-(aminomethyl)benzoic acids, 2-, 3-, and 4- (aminophenyl)acetic acids, 2-, 3-, and 4-(aminomethylphenyl)acetic acid (AMPA), (aminomethyl)pyrrole carboxylic acids, (aminomethyl)thiophene carboxylic acids, (aminomethyl)furan carboxylic acids, (aminomethyl)pyrrole acetic acids, (aminomethyl)thienyl acetic acids, and aminobiphenylcarboxylic acids, most preferably a 4- (aminomethyl)benzoic acid moiety.
  • AMPA aminomethylphenylcarboxylic acids
  • the conjugate may e.g. be represented by any one of the above formulae (3) to (15) which forms a radiolabeled complex with a radionuclide as defined above, for example with 177 Lu or 161 Tb, preferably with 177 Lu, or with 67 Ga or 68 Ga.
  • Stabilizer e.g. be represented by any one of the above formulae (3) to (15) which forms a radiolabeled complex with a radionuclide as defined above, for example with 177 Lu or 161 Tb, preferably with 177 Lu, or with 67 Ga or 68 Ga.
  • the pharmaceutical composition according to the present invention comprises a radiolabeled complex, as described above, and preferably also comprises a stabilizer to provide stability against radiolytic degradation.
  • the stabilizer may be able to scavenge radicals, which may be generated, for example, when the radionuclide emits a gamma ray and the gamma ray cleaves a bond between the atoms of organic molecules, thereby forming radicals. Therefore, the stabilizer can avoid or reduce that radicals undergo other chemical reactions, which might lead to undesired, potentially ineffective or even toxic molecules.
  • the pharmaceutical composition of the present invention may comprise any stabilizer against radiolytic degradation known in the art.
  • Stabilizing agents have been evaluated by Larenkov et al. (Larenkov, A., Mitrofanov, I., Pavlenko, E., Rakhimov, M.: Radiolysis- Associated Decrease in Radiochemical Purity of 177Lu-Radiopharmaceuticals and Comparison of the Effectiveness of Selected Quenchers against this Process. Molecules 2023, 28, 1884. https://doi.org/10.3390/ molecules28041884), which is hereby incorporated by reference.
  • stabilizers include, without being limited thereto, ascorbic acid or a salt thereof (e.g.
  • gentisic acid (2,5-dihydroxybenzoic acid), or a salt thereof, para-amino benzoic acid, Se-methionine, DMSA, cysteine, vanillin, adenine, dobesilic acid, thymine, uracil, nicotinamide, meglumine, and mannitol, or a combination of said stabilizers.
  • the stabilizer comprises ascorbic acid (L-ascorbic acid, vitamin C) and/or a salt thereof (e.g. sodium ascorbate).
  • ascorbic acid L-ascorbic acid, vitamin C
  • a salt thereof e.g. sodium ascorbate
  • the pharmaceutical composition does not comprise further stabilizers in addition to ascorbic acid and/or a salt thereof.
  • ascorbic acid and/or a salt thereof may be the only stabilizers present in the pharmaceutical composition.
  • the stabilizer comprised in the pharmaceutical composition may (exclusively) consist of ascorbic acid and/or a salt thereof.
  • salts of ascorbic acid are known in the art and readily available.
  • salt refers to an ionic assembly of cations and anions, which is composed of related numbers of cations and anions, so that the product (the salt) is electrically neutral (without net charge).
  • the salts are typically formed with the ascorbate anion.
  • Preferred salts of ascorbic acid include the alkali salts of ascorbic acid.
  • alkali salt refers to salts that produce hydroxide ions when dissolved in water.
  • Non-limiting examples of preferred salts of ascorbic acid include sodium, potassium, calcium, magnesium and lithium salts of ascorbic acid; such as sodium ascorbate, sodium ascorbyl phosphate, potassium ascorbate, calcium ascorbate, magnesium ascorbate, magnesium ascorbyl phosphate and lithium ascorbate.
  • the salt of ascorbic acid is a sodium salt of ascorbic acid, in particular sodium ascorbate.
  • the pharmaceutical composition according to the present invention is preferably an aqueous solution, in particular a radiopharmaceutical aqueous solution.
  • an "aqueous solution” is usually a solution of one or more solute(s) in water.
  • the pharmaceutical composition may be for intravenous (i.v.) use/application/administration.
  • the pharmaceutical composition is typically stable, concentrated, and ready-to-use.
  • the pharmaceutical composition may comprise a buffer, e.g. an acetate buffer, a citrate buffer or a phosphate buffer.
  • a buffer e.g. an acetate buffer, a citrate buffer or a phosphate buffer.
  • ascorbic acid and/or a salt thereof not only provides increased stability to the radiolabeled complex, but also may function as a buffer (i) during radiolabeling of the complex and (ii) in the formulation of the pharmaceutical composition (to maintain a suitable pH for parenteral injection). Therefore, additional buffers may not be required.
  • the composition may not contain any of an acetate buffer, a citrate buffer, and a phosphate buffer.
  • the pharmaceutical composition does not contain any additional buffer (in addition to ascorbic acid and/or the salt thereof, which are present as stabilizer(s) and also provide buffering functionality).
  • the only excipients (i.e., components of the pharmaceutical composition, which are not active ingredients, such as the radiolabeled complex) comprised in the pharmaceutical composition may be ascorbic acid and/or a salt thereof, and water (e.g., (sterile) water for injection and/or highly purified water).
  • the pharmaceutical composition may consist (essentially) of
  • a further buffer e.g. as described herein, may be necessary in order not to leave the range of permitted osmolarity.
  • the pharmaceutical composition may comprise both, ascorbic acid as well as a salt thereof (as described above).
  • the pharmaceutical composition may comprise ascorbic acid and sodium ascorbate (and no further stabilizer as described above).
  • the weight ratio of the salt of ascorbic acid to ascorbic acid in the pharmaceutical composition may be between 30 : 1 and 70 : 1 , preferably between 40 : 1 and 60 : 1 , more preferably between 45 : 1 and 55 : 1 , even more preferably between 45 : 1 and 50 : 1. Accordingly the amount (by weight) of the salt of ascorbic acid (in particular of sodium ascorbate) preferably exceeds the amount (by weight) of ascorbic acid considerably, as described above.
  • the concentration of ascorbic acid in the composition is preferably well below the concentration of the salt of ascorbic acid (in particular sodium ascorbate).
  • the concentration of ascorbic acid in the pharmaceutical composition is in the range from 0.5 to 5.0 mg/ml, preferably in the range from 0.7 to 3.0 mg/ml, more preferably in the range from 0.8 to 2.0 mg/ml, even more preferably in the range from 0.9 to 1.5 mg/ml, and still more preferably in the range from 1 .0 to 1.25 mg/ml.
  • the concentration of ascorbic acid in the pharmaceutical composition may be about 1 .1 1 mg/ml.
  • the concentration of the salt of ascorbic acid is in the range from 10 mg/ml to 100 mg/ml, preferably in the range from 20 mg/ml to 90 mg/ml, more preferably in the range from 30 mg/ml to 80 mg/ml, even more preferably in the range from 40 mg/ml to 70 mg/ml, and still more preferably in the range from 50 mg/ml to 60 mg/ml.
  • the concentration of the salt of ascorbic acid, in particular sodium ascorbate in the pharmaceutical composition may be about 51 mg/ml.
  • the pharmaceutical composition is substantially free of ethanol. Higher concentrations of ethanol may be associated with tolerability issues, such that ethanol may be restricted or avoided.
  • the amount of ethanol in the pharmaceutical composition is no more than 5%, preferably no more than 2%, more preferably no more than 1 % in the final pharmaceutical composition (to be injected/infused). Even more preferably, the solution is free of ethanol.
  • the pharmaceutical composition may comprise a sequestering agent, such as diethylentriaminepentaacetic acid (DTPA) or a salt thereof.
  • DTPA diethylentriaminepentaacetic acid
  • the term "sequestering agent” refers to an agent suitable to complex/chelate traces of unreacted radionuclide metal ions, which then can be excreted quickly via kidneys (Eur J Nucl Med Mol Imaging 2003 Feb;30(2):312-5. doi: 10.1007/s00259-002-1054-4. Epub 2002 Nov 29).
  • the pharmaceutical composition according to the present invention can provide a stability of > 93% over at least 24 h, in particular when stored at room temperature (RT).
  • RT room temperature
  • the use of the specific stabilizer(s) as described herein ensures high radiolytic stability, in particular at least 93%, 94%, 95%, 96%, 97%, 98% or 99% intact radiopeptides, even after 24 hours, with respect to the value obtained immediately after labeling.
  • radiochemical purity may be determined by HPLC as known in the art; for example utilizing reversed phase chromatography (e.g., column: Acclaim 120, C18, 3 gm, 3 x 150 mm), e.g. at gradient conditions, with UV and radio-chemical detection.
  • the pharmaceutical composition according to the present invention may be provided as single-dose product, e.g. in a vial containing a single dose of the radiolabeled complex.
  • the vial may contain about 10 to 25 ml of the pharmaceutical composition, preferably 15 to 20 ml of the pharmaceutical composition, more preferably 16 to 19 ml of the pharmaceutical composition, and even more preferably about 18 ml of the pharmaceutical composition.
  • a single dose may allow delivery of 7.5 GBq + 10% of radioactivity at injection time.
  • each of the one or more the stabilizer(s) present in the (final) pharmaceutical composition is/are already present during complex formation (radiolabeling).
  • the expression “present during complex formation” is intended to refer to such agents/compounds, which are present in the reaction mixture (also referred to as “radiolabeling composition”) for the complex formation (radiolabeling).
  • the radionuclide solution is added to the solution containing the conjugate comprising the chelating moiety linked to the targeting molecule and the albumin-binding entity (or vice versa).
  • any agent/compound present during complex formation may be contained in either the radionuclide solution, in the solution containing the conjugate comprising the chelating moiety linked to the targeting molecule and the albumin-binding entity, or in a separate solution to be added.
  • elevated temperatures may be applied to the radiolabeling composition (including the agents/compounds comprised therein) for a defined time window to facilitate the complex formation (radiolabeling).
  • each of the one or more the stabilizer(s) present in the (final) pharmaceutical composition is/are already present during complex formation (radiolabeling).
  • the concentrations and/or weight ratios of the stabilizer(s) in the radiolabeling composition (reaction mix) during complex formation (radiolabeling) are preferably distinct from the concentrations and/or weight ratios of the stabilizer(s) in the (final) pharmaceutical composition.
  • one or more of the stabilizers present during complex formation (radiolabeling) may be additionally added after the complex formation (radiolabeling).
  • the expression “after the complex formation (radiolabeling)” refers to the time when the complex forming (radiolabeling) reaction is completed.
  • “after the complex formation (radiolabeling)” may refer to a time when the radiolabeling composition (radiolabeling reaction mixture) is no longer exposed to an elevated temperature (for example, when ambient temperature is reached again, e.g. by cooling down the radiolabeling composition).
  • “after the complex formation (radiolabeling)” may refer to the formulation of the (final) pharmaceutical composition, e.g. by dilution of the radiolabeling mix with water.
  • ascorbic acid and/or a salt thereof is/are present during complex formation (radiolabeling). It is also preferred that ascorbic acid and/or a salt thereof is/are added after complex formation (radiolabeling). More preferably, ascorbic acid and/or sodium ascorbate is/are present during complex formation (radiolabeling) and ascorbic acid and/or sodium ascorbate is/are added after complex formation (radiolabeling).
  • ascorbic acid and a salt thereof, in particular sodium ascorbate are present during complex formation (i.e., in the radiolabeling composition) at a weight ratio (sodium ascorbate : ascorbic acid) of about 2 : 1 to 6 : 1 , preferably about 3 : 1 to 5 : 1 , more preferably about 3.5 : 1 to 4.5 : 1 , even more preferably about 3.75 : 1 to 4.25 : 1 , still more preferably about 4 : 1 .
  • ascorbic acid is present during complex formation (i.e., in the radiolabeling composition) at a concentration of 1 - 50 mg/ml, preferably 5 - 40 mg/ml, more preferably 7 - 30 mg/ml, even more preferably 10 - 20 mg/ml, still more preferably 10 - 15 mg/ml, such as about 13.3 mg/ml.
  • the salt of ascorbic acid in particular sodium ascorbate, (but preferably not ascorbic acid) is added after complex formation (during formulation of the pharmaceutical composition).
  • ascorbic acid and a salt thereof, in particular sodium ascorbate are present during complex formation (radiolabeling); and the salt of ascorbic acid, in particular sodium ascorbate, (but preferably not ascorbic acid) is added after complex formation (during formulation of the pharmaceutical composition).
  • the radionuclide in particular 177 Lu, is present in the pharmaceutical composition at a concentration providing volumetric radioactivity of from 0.25 to 0.6 GBq/ml, preferably 0.3 to 0.55 GBq/ml, more preferably 0.35 to 0.5 GBq/ml.
  • Effective doses of the inventive conjugates may be determined by routine experiments, e.g. by using animal models. Such models include, without implying any limitation, rabbit, sheep, mouse, rat, dog, pig and non-human primate models.
  • Therapeutic efficacy and toxicity of inventive conjugates or radiolabeled complexes can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • the data obtained from the cell culture assays and animal studies can be used in determining a dose range for use in humans.
  • the dose of said conjugates lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • therapeutically or diagnostically effective doses of the inventive conjugates may range from about 0.001 mg to 10 mg, preferably from about 0.01 mg to 5 mg, more preferably from about 0.1 mg to 2 mg per dosage unit or from about 0.01 nmol to 1 mmol per dosage unit, in particular from 1 nmol to 1 mmol per dosage unit, preferably from 1 micromol to 1 mmol per dosage unit. It is also envisaged that therapeutically or diagnostically effective doses of the inventive conjugates (compounds) may range (per kg body weight) from about 0.01 mg/kg to 10 g/kg, preferably from about 0.05 mg/kg to 5 g/kg, more preferably from about 0.1 mg/kg to 2.5 g/kg.
  • the pharmaceutical composition is administered parenterally, in particular via intravenous or intratumoral injection, and is accordingly formulated in liquid or lyophilized form for parenteral administration.
  • Parenteral formulations may be stored in vials, IV bags, ampoules, cartridges, or prefilled syringes and can be administered as injections, inhalants, or aerosols, with injections being preferred.
  • the pharmaceutical composition may comprise a pharmaceutically acceptable excipient, diluent or carrier.
  • pharmaceutically acceptable refers to a compound or agent that is compatible with the components of the pharmaceutical composition, in particular the active (anti-cancer) compounds, and does not interfere with and/or substantially reduce its therapeutic activities.
  • Pharmaceutically acceptable carriers preferably have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a subject to be treated.
  • the sodium, calcium and, optionally, potassium salts may occur in the form of their halogenides, e.g. chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc.
  • examples of sodium salts include e.g. NaCI, Nal, NaBr, Na 2 CO 3 , NaHCO 3 , Na 2 SO 4
  • examples of the optional potassium salts include e.g. KCI, KI, KBr, K 2 CO 3 , KHCO3, K 2 SO 4
  • examples of calcium salts include e.g. CaCI 2 , Cal 2 , CaBr 2 , CaCO 3 , CaSO 4 , Ca(OH) 2 .
  • organic anions of the aforementioned cations may be contained in the buffer.
  • the pharmaceutical composition may be provided in lyophilized form. Lyophilized pharmaceutical compositions are preferably reconstituted in a suitable buffer, advantageously based on an aqueous carrier, prior to administration. The pharmaceutical compositions are also provided for use in the preparation of a medicament for the treatment of cancer.
  • the present invention also provides the use of the pharmaceutical composition as described above in medicine.
  • the pharmaceutical composition as described above may be preferably used in the treatment or in the (in vitro) diagnosis of cancer (e.g., by using an isolated sample, for example a blood sample or tumor tissue).
  • the present invention also provides a method for treating cancer or initiating, enhancing or prolonging an anti-tumor-response in a subject in need thereof comprising administering to the subject the pharmaceutical composition as described above.
  • the pharmaceutical composition usually comprises an effective amount of the radiolabeled complex.
  • an effective amount means an amount of the agent(s) that is sufficient to allow for diagnosis and/or significantly induce a positive modification of the disease to be treated.
  • an “effective amount” may be small enough to avoid serious side-effects, that is to say to permit a sensible relationship between advantage and risk.
  • Anguereffective amount may vary depending on the particular condition to be diagnosed or treated and also with the age and physical condition of the patient to be treated, the severity of the condition, the duration of the treatment, the nature of the accompanying therapy, of the particular pharmaceutically acceptable excipient or carrier used, and similar factors.
  • an "effective amount" may be readily determined in a specific situation by the physician.
  • effective doses may be determined by routine experiments, e.g. by using animal models. Such models include, without implying any limitation, rabbit, sheep, mouse, rat, dog, pig and non-human primate models.
  • Therapeutic efficacy and toxicity of radiolabeled complexes can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD5O/ED5O.
  • the data obtained from the cell culture assays and animal studies can be used in determining a dose range for use in humans.
  • the dose of said conjugates lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • diagnosis refers to act of identifying a disease from its signs and symptoms and/or as in the present case the analysis of biological markers (such as genes or proteins) indicative of the disease.
  • the term “treatment” or “treating” of a disease includes preventing or protecting against the disease (that is, causing the clinical symptoms not to develop); inhibiting the disease (i.e., arresting or suppressing the development of clinical symptoms; and/or relieving the disease (i.e., causing the regression of clinical symptoms).
  • the term “prophylaxis” will be understood to constitute a type of “treatment” that encompasses both "preventing” and “suppressing.”
  • the term “treatment” thus includes “prophylaxis”.
  • the term “treatment” includes prophylactic treatment (before onset of the disease) as well as therapeutic treatment (after onset of the disease).
  • compositions as described herein are typically administered parenterally. Administration may preferably be accomplished systemically, for instance by intravenous (i.v.), subcutaneous, intramuscular or intradermal injection. Alternatively, administration may be accomplished locally, for instance by intra-tumoral injection.
  • the pharmaceutical compositions as described above may be administered to a subject in need thereof several times a day, daily, every other day, weekly, or monthly.
  • compositions of the invention in particular pharmaceutical compositions comprising a radiolabeled complex comprising a uPAR-binding targeting peptide moiety linked to an albumin-binding moiety and a chelating moiety complexing a radionuclide, may be used in the treatment or diagnosis of any cancer expressing an urokinase-type plasminogen activator receptor (uPAR).
  • uPAR urokinase-type plasminogen activator receptor
  • the presence of uPAR-expressing cells or tissues may be indicative of a solid tumor, neuroendocrine tumor and/or a hematologic malignancy.
  • the cancer is preferably selected from breast, brain (e.g. glioblastoma), gastric, pancreatic, colorectal, prostate, ovarian, oral and esophageal cancer.
  • Hematologic malignancies may be selected from multiple myeloma and acute leukemias.
  • compositions of the invention in particular pharmaceutical compositions comprising a radiolabeled complex comprising an uPAR-binding targeting peptide moiety linked to an albumin-binding moiety and a chelating moiety complexing a radionuclide, may be used in the treatment or diagnosis of any cancer or hematologic malignancy expressing an urokinase-type plasminogen activator receptor (uPAR).
  • uPAR urokinase-type plasminogen activator receptor
  • radiographic imaging may be accomplished using any means and methods known in the art.
  • radiographic imaging may involve positron emission tomography (PET) or single-photon emission computed tomography (SPECT).
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • Figure 1 represents a schematic overview of the design of AE105-based uPAR targeting radiopeptides using distinct albumin-binding and linker/spacer entities.
  • Figure 2 shows for Example 6 albumin-binding curves of the radiopeptides synthesized in Example 1 using mouse (panels A and B) and human (panels C and D) blood plasma dilutions.
  • FIG 4 shows for Example 9 SPECT/CT images shown as maximum intensity projections (MIPs) of HEK-uPAR xenograft-bearing mice acquired 1 h after injection of the radiopeptide [ 177 Lu]Lu-DOTA-AE105 or 177 Lu-DOTA-uPAR- ALB radiopeptides (25 MBq; 0.5 nmol per mouse).
  • MIPs maximum intensity projections
  • Figure 5 shows for Example 9 SPECT/CT images shown as maximum intensity projections (MIPs) of HEK-uPAR xenograft-bearing mice acquired 4 h after injection of the radiopeptide [ 177 Lu]Lu-DOTA-AE105 or 177 Lu-DOTA-uPAR- ALB radiopeptides (25 MBq; 0.5 nmol per mouse).
  • MIPs maximum intensity projections
  • Figure 6 shows for Example 9 SPECT/CT images shown as maximum intensity projections (MIPs) of HEK-uPAR xenogaft-bearing mice acquired 24 h after injection of the radiopeptide [ 177 Lu]Lu-DOTA-AE105 or 177 Lu-DOTA-uPAR- ALB radiopeptides (25 MBq; 0.5 nmol per mouse).
  • MIPs maximum intensity projections
  • Figure 7 shows for Example 10 decay-corrected blood retention of the radiopeptides in HEK-uPAR xenograft-bearing mice.
  • Figure 8 shows for Example 10 decay-corrected uptake of the radiopeptides in HEK- uPAR xenografts of mice.
  • Figure 9 shows for Example 10 decay-corrected uptake of the radiopeptides in the kidneys of HEK-uPAR-xenografted mice.
  • Figure 10 shows for Example 10 decay-corrected uptake of the radiopeptides in the liver of HEK-uPAR-xenografted mice.
  • Figure 11 shows for Example 10 xenograft-to-blood ratios determined based on biodistribution data obtained at 4 h, 24 h and 48 h injection of respective radiopeptides.
  • Figure 14 represents a schematic overview of the design of 2 nd generation AE105-based uPAR targeting peptides using variable chelators and linker entities.
  • PEG polyethylene glycol
  • Lys lysine
  • DAP diaminopropanoic acid
  • Figure 15 shows for Example 16 albumin-binding curves of the radiopeptides synthesized in Example 1 1 using human (panels A and B) and mouse (panels C and D) blood plasma dilutions.
  • Figure 16 shows for Example 17 cell uptake (panels A and D), internalization (panels B and E) and blocking studies (panels C and F) of distinct uPAR-targeting radiopeptides performed in HEK-uPAR cells after 2 h and 4 h incubation at 37 °C.
  • the data are represented as average ⁇ SD of three independent experiments.
  • Figure 17 shows for Example 19 SPECT/CT images shown as maximum intensity projections (MIPs) of HEK-uPAR xenogaft-bearing mice acquired 1 h after injection of the radiopeptide [ 177 Lu]Lu-DOTA-AE105 or 177 Lu-DOTA-uPAR- ALB radiopeptides (25 MBq; 0.5 nmol per mouse).
  • MIPs maximum intensity projections
  • Figure 18 shows for Example 19 SPECT/CT images shown as maximum intensity projections (MIPs) of HEK-uPAR xenogaft-bearing mice acquired 4 h after injection of the radiopeptide [ 177 Lu]Lu-DOTA-AE105 or 177 Lu-DOTA-uPAR- ALB radiopeptides (25 MBq; 0.5 nmol per mouse).
  • MIPs maximum intensity projections
  • Figure 19 shows for Example 19 SPECT/CT images shown as maximum intensity projections (MIPs) of HEK-uPAR xenogaft-bearing mice acquired 24 h after injection of the radiopeptide [ 177 Lu]Lu-DOTA-AE105 or 177 lu-DOTA-uPAR- ALB radiopeptides (25 MBq; 0.5 nmol per mouse).
  • MIPs maximum intensity projections
  • Figure 20 shows for Example 19 SPECT/CT images shown as maximum intensity projections (MIPs) of HEK-uPAR xenogaft-bearing mice acquired 48 h after injection of the radiopeptide [ 177 Lu]Lu-DOTA-AE105 or 177 Lu-DOTA-uPAR- ALB radiopeptides (25 MBq; 0.5 nmol per mouse).
  • MIPs maximum intensity projections
  • Figure 22 shows for Example 20 decay-corrected uptake of the radiopeptides in HEK- uPAR xenografts of mice.
  • Figure 23 shows for Example 20 decay-corrected uptake of the radiopeptides in the kidneys of HEK-uPAR-xenografted mice.
  • Figure 24 shows for Example 20 decay-corrected uptake of the radiopeptides in the liver of HEK-uPAR-xenografted mice
  • Figure 25 shows for Example 20 decay-corrected xenograft-to-blood ratios determined based on biodistribution data obtained at 4 h, 24 h and 48 h injection of respective radiopeptides.
  • Figure 26 shows for Example 20 xenograft-to-kidney ratios determined based on biodistribution data obtained at 4 h, 24 h and 48 h after injection of respective radiopeptides.
  • Figure 27 shows for Example 20 xenograft-to-liver ratios determined based on biodistribution data obtained at 4 h, 24 h and 48 h after injection of respective radiopeptides.
  • Figure 28 shows for Example 21 cell uptake and internalization of the radiopeptides in HEK-uPAR cells after 2-h and 4-h incubation period at 37 °C.
  • A Uptake and internalization of [ 67 Ga]Ga-DOTA-uPAR-ALB-1 1 ;
  • B Uptake and internalization of [ 67 Ga]Ga-DOTA-uPAR-ALB-18.
  • C The radiopeptides in the presence of excess AE105 to block uPAR. The data are represented as average ⁇ SD of two independent experiments.
  • Figure 29 shows for Example 21 SPECT/CT images shown as maximum intensity projections (MIPs) of HEK-uPAR xenograft-bearing mice acquired 1 h and 4 h after injection of 67 Ga-labeled uPAR-targeting radiopeptides (10 MBq; 0.5 nmol per mouse).
  • MIPs maximum intensity projections
  • Dde-Lys(Fmoc)-OH was conjugated to the terminal a-amino functionality of RI-AE105 following the procedure reported above.
  • the Fmoc group present on the sidechain of the newly inserted lysine residue was removed and the resulting primary ⁇ -amino group was subsequently coupled with 4-(p-iodophenyl)butanoic acid.
  • the Dde protecting group present at the ⁇ -amino group of the lysine residue was removed using a solution of 2% ( v/v hydrazine hydrate in DMF two times for 30 minutes.
  • DOTA-uPAR-ALB-04 was synthesized in analogy to the procedure described for DOTA-uPAR-ALB-01 , but an additional lysine residue was included as a spacer before the conjugation of DOTA-tris( t Bu)ester.
  • Dde-Lys(Fmoc)-OH (0.4 mmol, 4.00 equiv) was conjugated to the resin-immobilized AE105 peptide chain (RI-AE105; 0.1 mmol, 1.00 equiv).
  • RI-AE105 0.1 mmol, 1.00 equiv
  • the ⁇ -amino group of the lysine residue was reacted with a Fmoc-Namido-PEC4-acid entity followed by conjugation of 4-(p-tolyl)butanoic acid.
  • the Dde protecting group of the lysine residue was cleaved using a mixture of 2% hydrazine in DMF two times for 30 minutes before conjugation of the DOTA-tris(‘Bu)ester.
  • the chemical purity of the final products was determined by LC-MS analysis using an Acquity SQD2 LC-MS (Waters, Milford, MA, USA) system equipped with a reversed-phase C18 column (Acquity UPLC BEH, 1.7 ⁇ m, 2.1 x 50 mm, Waters, Milford, MA, USA).
  • the DOTA-AE105 and DOTA-uPAR-ALB peptides were labeled with lutetium-177 followed by investigation of the radiopeptides' radiolytic stability.
  • DOTA-AE105 and the DOTA-uPAR-ALB peptides were prepared in Milli-Q water at a concentration of 1 mM.
  • DOTA-uPAR-ALB-02 and DOTA- uPAR-ALB-03 dimethyl sulfoxide (DMSO) was added to facilitate the dissolution (28% and 36%, respectively).
  • DMSO dimethyl sulfoxide
  • Quality control (QC) of the radiolabeled peptides was performed by HPLC (Merck Hitachi HPLC system, Darmstadt, Germany, equipped with a radiodetector LB 508, Berthold Technologies) using a C-18 reversed-phase column (XterraTM MS C-18, 5 pm, 15 cm x 4.6 cm, Waters, Milford, MA, U.S.) and a linear gradient of Milli-Q water containing 0.1 % TFA (95-20%) and ACN (5- 80%) over 15 min at a flow rate of 1 .0 mL/min.
  • HPLC Merck Hitachi HPLC system, Darmstadt, Germany, equipped with a radiodetector LB 508, Berthold Technologies
  • C-18 reversed-phase column XterraTM MS C-18, 5 pm, 15 cm x 4.6 cm, Waters, Milford, MA, U.S.
  • ACN 5- 80%
  • the stability of [ 177 Lu]Lu-DOTA-AE105 and the ,77 Lu-DOTA-uPAR-ALB radiopeptides was assessed in vitro.
  • the radiopeptide solutions were diluted with 0.9% NaCI to obtain an activity concentration of 150 MBq/300 pL in the absence and presence of L-ascorbic acid (3 mg in 20 pL) together with NaOAc (0.5 M, 30 pL) to compensate for the acidic pH. Aliquots of the solutions (without and with L-ascorbic acid) were sampled after an incubation period of 1 h, 4 h and 24 h at room temperature (RT) followed by analysis using HPLC.
  • the integrated peak area of the intact radioligand peak was expressed as the percentage of the sum of the integrated peak areas of each peak present in the chromatogram.
  • L-ascorbic acid (3 mg, 20 pL) as a scavenger after radiolabeling of the peptides.
  • the acidic pH value was compensated by the addition of NaOAc (0.5 M, 30 pL).
  • the radiopeptides were added to mouse blood plasma (Lot: 32321 , Rockland Inc.) or in human blood plasma ( founded Blutspende SRK Aargau-Solothurn, Switzerland) at a concentration of 10 MBq/200 pL and incubated at 37 °C for up to 24 h. Control samples were prepared by dilution of the radiopeptides in 0.9% NaCl to obtain the same activity concentration.
  • TLC thin layer chromatography
  • [ 177 Lu]Lu -DOTA-uPAR-ALB-01 , [ 177 Lu]Lu - DOTA-uPAR-ALB-02, [ 177 Lu]Lu -DOTA-uPAR-ALB-03, [ 177 Lu]Lu -DOTA-uPAR-ALB-05, [ 177 Lu]Lu -DOTA-uPAR-ALB-1 1 were stable (>95% intact radiopeptide) in murine and human blood plasma for up to 24 h.
  • [ 177 Lu]Lu-DOTA-uPAR-ALB-04 showed >76% intact radiopeptide after 24 h when incubated in human blood plasma, and >84% intact radiopeptide after 24 h when incubated in mouse blood plasma.
  • the distribution coefficients (logD values) of [ 177 Lu] Lu-DOT A-AE105 and the 177 Lu-DOTA- uPAR-ALB peptides were determined in a mixture of /7-octanol and phosphate-buffered saline (PBS) to estimate their hydrophilic/lipophilic properties.
  • [ 177 Lu]Lu-DOTA-AE105 and the 177 Lu-DOTA-uPAR-ALB peptides were diluted in PBS pH 7.4 to obtain an activity concentration of 10 MBq/500 pL.
  • the diluted radiopeptides (-0.5 MBq, 25 pL, 0.01 nmol) were added to a mixture of PBS pH 7.4 (1475 pL) and /7-octanol (1500 pL).
  • the vials were vortexed vigorously for 1 min followed by centrifugation (6 min, 560 ref) for phase separation. Aliquots were taken from each phase and measured in a y-counter ((PerkinElmer, Wallac Wizard 1480)).
  • the distribution coefficients were calculated as the logarithmic value of the ratio of counts per minute (cpm) measured in the /7-octanol phase relative to the cpm measured in the PBS phase. The results were listed as average ⁇ SD of the data obtained from 3 independent experiments, each performed with five replicates.
  • [ 177 Lu]Lu-DOTA-uPAR-ALB-03 (logD value: 0.51 ⁇ 0.13) showed the lowest logD value due to the lipophilic AMBA linker integrated into the chemical structure.
  • the radiopeptides comprising a />iodophenyl-based albumin-binding entity were more lipophilic (logD values: 0.24-0.78) than [ 177 Lu
  • the binding of the , 77 Lu-DOTA-uPAR-ALB peptides to serum albumin in mouse and human blood plasma was determined and compared.
  • the albumin-binding properties of 177 Lu-DOTA-uPAR-ALB peptides in mouse blood plasma (Rockland Immunochemicals, Inc., USA) and human blood plasma ( founded Blutspende SRK Aargau -So loth urn, Switzerland) were determined using an ultrafiltration method and compared to that of [ 177 Lu]Lu-DOA-AE105, which does not comprise a designated albumin- binder.
  • the 177 Lu-DOTA-uPAR-ALB peptides (50 MBq/nmol, ⁇ 300 kBq, 0.006 nmol in 15 pL) were added to samples of mouse and human blood plasma (150 ⁇ L), followed by incubation of the samples at 37 °C for 30 min. Ice-cold PBS (150 ⁇ L, pH 7.4) was added before loading the blood plasma sample on Amicon centrifugal filters (cut-off of 10 kDa; Merck Millipore) followed by centrifugation (14'000 ref, 30 min, 4 °C) to allow the separation of the plasma protein-bound from the plasma-unbound (free) fractions of each sample.
  • the inserts of the filter devices were inverted and centrifuged at 200 ref for 3 min to recover the protein-bound radiopeptide.
  • the activity of the protein-bound fraction was measured using a y-counter (PerkinElmer, Wallac Wizard 1480).
  • the activity in the filtrate and filter unit was measured in a y-counter (PerkinElmer, Wallac Wizard 1480) and the counts were combined assuming that the fraction retained in the filter membrane was not bound to proteins.
  • the protein-bound fraction was expressed as percentage of the whole activity (i.e. plasma protein-bound activity, activity measured in the filtrate and activity measured in the filter (set as 100%)).
  • the relative affinity of the radiopeptides to albumin in mouse and human blood plasma was determined to compare the properties of the candidates.
  • mice serum albumin (MSA) and human serum albumin (HSA) were determined using an ultrafiltration method.
  • the amount of mouse serum albumin (MSA) and human serum albumin (HSA) in mouse and human blood plasma was defined as 550 ⁇ M and 800 ⁇ M, respectively, based on measurements using a dry chemistry analyzer (DRI-CHEM 4000i, FUJIFILM, Japan).
  • HEK cells transfected with human uPAR herein referred to as HEK-uPAR cells
  • HEK-uPAR cells were obtained from Innoprot, Innovative Technologies in Biological Systems S.L. (Bizkaia, Spain).
  • the cells were cultured in DMEM medium supplemented with 10% fetal calf serum, L- glutamine, 1 % non-essential amino acids and antibiotics.
  • Hygromycin B 50 pg/ml was added to maintain uPAR expression.
  • the cells were subcultured twice a week using trypsin/EDTA and cultured under standard cell culture conditions using 5% CO 2 at 37 °C in a humidified atmosphere.
  • HEK-uPAR cells were seeded in polylysine-coated 12-well plates (1 x 10 6 cells in 2 ml per well) using DMEM cell culture medium with supplements. The cells were incubated at 37 °C and 5% CO 2 overnight to allow adhesion and growth. After removal of the supernatant, the HEK-uPAR cells were rinsed with PBS before adding DMEM without supplements (975 pl per well). The radiopeptides (50 MBq/nmol) were added to each well in a volume of 25 pl (0.75 pmol, 38 kBq). In some wells, the HEK-uPAR cells were coincubated with an excess of AE105 (5 pM) to block uPAR.
  • AE105 5 pM
  • the protein concentration of each sample was determined using a Micro BCA Protein Assay kit (Pierce, Thermo Scientific) to standardize the measured activity to the average content of protein in a single well.
  • the HEK-uPAR cell uptake of [ 177 Lu]Lu-DOTA-AE105 was higher (44 ⁇ 4% and 46 ⁇ 5% after 2 h and 4 h, respectively) than for the albumin-binding radiopeptides, which showed uptake in the range of 21-33% and 18-34% after 2 h and 4 h, respectively.
  • the 177 Lu-DOTA-uPAR-ALB peptides showed an internalized fraction of 16- 24% and 12- 26% after 2 h and 4 h, respectively, whereas the internalized fraction of [ 177 Lu]Lu-DOTA-AE105 was 11 + 1 % and 17 + 3% after 2 h and 4 h, respectively.
  • the uPAR-binding affinity of the radiopeptides was investigated using HEK-uPAR cells.
  • HEK-uPAR cells were seeded in polylysine-coated 48-well plates (0.25 x 10 6 in 0.5 mL per well) using DMEM culture medium with supplements. The cells were incubated at 37 °C and 5% CO 2 overnight to allow adhesion and growth. After removing the supernatant, the well plates with HEK-uPAR cells were placed on ice to avoid internalization of the peptides. Cells were rinsed with PBS followed by the addition of DMEM culture medium without supplements (450 pL/well) in the presence or absence of AE105 (40 ⁇ m) used as a blocking agent.
  • the K o values of [ 177 Lu]Lu-DOTA- uPAR-ALB-01 , [ 177 Lu]Lu-DOTA-uPAR-ALB-02 and [ 177 Lu]Lu-DOTA-uPAR-ALB-1 1 were in the range of 30-40 nM.
  • mice All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. In particular, all animal experiments were carried out according to the guidelines of the Swiss Regulations for Animal Welfare. The preclinical studies have been ethically approved by the Cantonal Committee of Animal Experimentation and permitted by the responsible cantonal authorities (License N° 75721 ).
  • Five-week-old female CD1 nude (Crl:CD1 -Foxn nu ) mice were obtained from Charles River Laboratories (Sulzfeld, Germany) and fed with standard rodent chow ad libitum. The mice were subcutaneously inoculated with HEK-uPAR cells (7 x 10 6 cells in 100 pL PBS) on the right shoulder.
  • the images were acquired using Nucline Software (version 10.2, MEdiso Ltd., Budapest, Hungary).
  • the real-time CT reconstruction used a cone-beam filtered beckprojection.
  • the reconstruction of SPECT data was performed with HiSPECT software (version 1.4.3049, Scivis GmbH, Gottingen, Germany) using y- energies of 56.1 keV ( ⁇ 10%), 112.9 keV ( ⁇ 10%) and 208.4 keV ( ⁇ 10%) for lutetium- 177.
  • the images were prepared using VivoQuant post-processing software (version 3.5, inviCRO Imaging Services and Software, Boston, U.S.).
  • [ 177 Lu]Lu-DOTA-AE105 showed rapid clearance from the blood mainly via kidneys, which showed the highest accumulation at 1 h after injection, while only moderate uptake of the radiopeptide was seen in the xenograft. After 4 h, [ 177 Lu]Lu- DOTA-AE105 was almost entirely cleared from the blood circulation and the signal in the xenograft and kidneys was comparable, but low.
  • the ,77 Lu-DOTA-uPAR-ALB peptides showed considerable retention in the blood and heart early after injection, which can be ascribed to their albumin-binding properties.
  • accumulation of activity in the xenograft was rather low, whereas [ 177 Lu]Lu-DOTA-uPAR- ALB-02 and [ 177 Lu]Lu-DC)TA-uPAR-ALB-05 visualized the xenograft better at this early timepoint.
  • albumin-binding affinity of the radiopeptides had a substantial impact on their tissue distribution profiles, not only with regard to the blood residence time, but also with regard to the renal excretion and, most importantly, accumulation in the xenograft. While high albumin-binding properties, as was the case for [ 177 Lu]Lu-DOTA-uPAR-ALB-03, led to lower accumulation in the xenograft at early time points (1 h, 4h p.i.) and increased accumulation at a later time point (24h p.i.), moderate albumin-binding properties, as is the case for [ 177 Lu]Lu-DOTA-uPAR-ALB-1 1 , results in less retention of activity in the blood but faster accumulation in the xenograft.
  • radiopeptides [ 177 Lu]Lu-DOTA-uPAR-ALB-02, [ 177 Lu]Lu-DOTA-uPAR-ALB-03 and [ 177 Lu]Lu-DOTA-uPAR-ALB-1 1 which show distinct tissue distribution patterns in imaging experiments using SPECT/CT, were used in order to quantify the uptake in xenografts and normal tissue. Biodistribution studies were performed in HEK-uPAR xenografted nude mice.
  • mice were injected with uPAR-targeting radiopeptides (5 MBq, 0.5 nmol, 100 pL) in 0.9% NaCI containing 0.05% BSA and sacrificed at 4 h, 24 h or 48 h after injection of the radiopeptides. Selected tissues and organs were collected, weighed and measured using a y- counter (PerkinElmer, Wallac Wizard 1480). The results were listed as a percentage of the injected activity per gram of tissue mass (% lA/g) using counts of a standard (defined volume of the original injection solution) measured at the same time to enable the calculation of decay-corrected values.
  • Biodstribution data were obtained at variable timepoints after injection of [ 177 Lu]Lu-DOTA- AE105 (Table 9), [ 177 Lu]Lu-DOTA-uPAR-ALB-02 (Table 10), [ 177 Lu]Lu-DOTA-uPAR-ALB-03 (Table 1 1 ) and [ 177 Lu]Lu-DOTA-uPAR-ALB-1 1 (Table 12).
  • the most relevant differences among the radiopeptides were observed regarding their residence time in the blood ( Figure 7), their accumulation in HEK-uPAR xenografts (Figure 8), the kidneys ( Figure 9) and the liver (Table 10).
  • Table 11 Biodistribution data and tumor-to-background ratios obtained in HEK-uPAR- bearing mice at various time points after injection of [ 177 Lu]Lu-DOTA-uPAR-ALB-03.
  • [ 177 Lu]Lu-DOTA-uPAR-ALB-1 1 showed increased retention in the blood as compared to [ 177 Lu]Lu-DOTA-AE105 (Table 12), however, due to the reduced albumin-binding affinity, it was less pronounced than in the cases of [ 177 Lu]Lu-DOTA-uPAR-ALB-02 and [ 177 Lu]Lu- DOTA-uPAR-ALB-03.
  • the xenograft-to-kidney ratios of accumulated activity were lowest for [ 177 Lu]Lu-DOTA-AE105 ( ⁇ 1 ). The highest ratio was obtained for [ 177 Lu]Lu-DOTA-uPAR- ALB-1 1 (3.9-4.9), followed by [ 177 Lu]Lu-DOTA-uPAR-ALB-02 (2.0-3.3) and [ 177 Lu]Lu- DOTA-uPAR-ALB-03 (1.0-2.0). The ratios were higher for all albumin-binding radiopeptides at later timepoints, which can mainly be ascribed to renal clearance of activity over time.
  • the radiolabeling of the peptides was performed by the addition of lutetium-177 (no-carrier-added 177 LuCl3 in 0.04 M HCI; ITM Medical Isotopes GmbH, Germany) to a 1 :5 (v/v) mixture of sodium acetate (0.5 M) and HCI (0.05 M) at pH 4.5 followed by addition of the respective peptide (stock solution of 1 mM, i.e. 1 nmol corresp. 1 pl) to obtain molar activities up to 50 MBq/nmol. The reaction mixture was incubated for 10 min at 95 °C.
  • the 177 Lu-labeled uPAR-targeting peptides were investigated with regard to their stability after incubation in blood plasma.
  • L-ascorbic acid (3 mg, 20 pL) as a scavenger after radiolabeling of the peptides.
  • the acidic pH value was compensated by the addition of NaOAc (0.5 M, 30 pL).
  • the radiopeptides were added to mouse blood plasma (Lot: 32321 , Rockland Inc.) or to human blood plasma ( founded Blutspende SRK Aargau-Solothurn, Switzerland) at a concentration of 10 MBq/200 pL and incubated at 37 °C for up to 24 h. Control samples were prepared by dilution of the radiopeptides in 0.9% NaCI to obtain the same activity concentration.
  • TLC thin layer chromatography
  • the 177 Lu- uPAR-ALB peptides were stable (>95% intact radiopeptide) in murine and human blood plasma for up to 24 h. The only exception was [ 177 Lu]Lu-DOTAGA-uPAR-ALB-17, which showed degradation products when incubated in murine blood plasma (>63% intact radiopeptide after 24 h).
  • the distribution coefficients (logD values) of the 177 Lu-labeled uPAR-targeting peptides were determined in a mixture of /7-octanol and phosphate-buffered saline (PBS) to estimate their hydroph i I ic/lipophi lie properties.
  • [ 177 Lu]Lu-DOTA-AE105 and 177 Lu-uPAR-ALB peptides were diluted in PBS pH 7.4 to obtain an activity concentration of 10 MBq/500 pL.
  • the diluted radiopeptides (-0.5 MBq, 25 pL, 0.01 nmol) were added to a mixture of PBS pH 7.4 (1475 pL) and n- octanol (1500 ⁇ L).
  • the vials were vortexed vigorously for 1 min followed by centrifugation (6 min, 560 ref) for phase separation. Aliquots were taken from each phase and measured in a y-counter ((PerkinElmer, Wallac Wizard 1480)).
  • the distribution coefficients were calculated as the logarithmic value of the ratio of counts per minute (cpm) measured in the n-octanol phase relative to the cpm measured in the PBS phase. The results were listed as average ⁇ SD of the data obtained from 3 independent experiments, each performed with five replicates.
  • the binding of the 177 Lu-labeled uPAR-targeting peptides to serum albumin in mouse and human blood plasma was determined and compared.
  • the albumin-binding properties of the 177 Lu-uPAR-ALB peptides in mouse blood plasma (Rockland Immunochemicals, Inc., USA) and human blood plasma ( founded Blutspende SRK Aargau-Solothurn, Switzerland) were determined using an ultrafiltration method and compared to that of [ 177 Lu]Lu-DOTA-uPAR-ALB-1 1 .
  • Lu-DOTA-AE1 05 and the albumin-binding, uPAR-targeting radiopeptides of the 2 nd generation were added to samples of mouse and human blood plasma (1 50 pL), followed by incubation of the samples at 37 °C for 30 min.
  • Ice-cold PBS (1 50 pL, pH 7.4) was added before loading the blood plasma samples on Amicon centrifugal filters (cut- off of 10 kDa; Merck Millipore) followed by centrifugation (14'000 ref, 30 min, 4 °C) to allow the separation of the plasma protein-bound from the unbound (free) fractions of each sample.
  • the inserts of the filter devices were inverted and centrifuged at 200 ref for 3 min to recover the protein-bound radiopeptide.
  • the activity of the protein-bound fraction was measured using a y-counter (PerkinElmer, Wallac Wizard 1480).
  • the activity in the filtrate and filter unit was measured in a y-counter (PerkinElmer, Wallac Wizard 1480) and the counts were combined assuming that the fraction retained in the filter membrane was not bound to proteins.
  • the protein-bound fraction was expressed as percentage of the whole activity (i.e. plasma protein-bound activity, activity measured in the filtrate and activity measured in the filter (set as 1 00%)). These experiments were performed 3 times for each radiopeptide.
  • the second generation 177 Lu-uPAR-ALB peptides showed a fraction of >79% and >84% bound to albumin in mouse and human blood plasma, respectively, which was considerably higher than the protein-associated fraction of [ 177 Lu]Lu-DOTA- AE105 measured in the same conditions.
  • the relative affinity of the radiopeptides to albumin in mouse and human blood plasma was determined to compare the properties of the candidates.
  • the relative albumin-binding affinities of 177 Lu-uPAR-ALB peptides in mouse blood plasma (Rockland Immunochemicals, Inc., USA) and human blood plasma ( founded Blutspende SRK Aargau-Solothurn, Switzerland) were determined using an ultrafiltration method and the data compared to that of [ 177 Lu]Lu-DOTA-AE105.
  • the amount of mouse serum albumin (MSA) and human serum albumin (HSA) in mouse and human blood plasma was defined as 550 ⁇ .M and 800 ⁇ m, respectively, based on measurements using a dry chemistry analyzer (DRI-CHEM 4000i, FUJIFILM, Japan).
  • radiopeptide 50 MBq/nmol, -300 kBq, 15 ⁇ L, 0.006 nmol
  • a defined volume 150 ⁇ L
  • mouse and human blood plasma 50 MBq/nmol, -300 kBq, 15 ⁇ L, 0.006 nmol
  • the albumin-bound fraction was determined using the ultrafiltration device as described above. The data were analyzed using a semi-logarithmic plot assuming a maximum binding of 100%.
  • HEK cells transfected with human uPAR herein referred to as HEK-uPAR cells
  • HEK-uPAR cells were obtained from Innoprot, Innovative Technologies in Biological Systems S.L. (Bizkaia, Spain).
  • the cells were cultured in DMEM cell culture medium supplemented with 10% fetal calf serum, L-glutamine, 1 % non-essential amino acids and antibiotics.
  • Hygromycin B 50 pg/mL was added to maintain uPAR expression.
  • the cells were subcultured twice a week using trypsin/EDTA and cultured under standard cell culture conditions using 5% CO 2 at 37 °C in a humidified atmosphere.
  • HEK-uPAR cells were seeded in polylysine-coated 12-well plates (1 x 10 6 cells in 2 mL per well) using DMEM cell culture medium with supplements. The cells were incubated at 37 °C and 5% CO 2 overnight to allow adhesion and growth. After removal of the supernatant, the HEK-uPAR cells were rinsed with PBS before adding DMEM without supplements (975 pL per well). The radiopeptides (50 MBq/nmol) were added to each well in a volume of 25 pL (0.75 pmol, 38 kBq). In some wells, the HEK-uPAR cells were coincubated with an excess of AE105 (5 ⁇ m) to block uPAR.
  • an acidic stripping buffer pH 2.8, 50 mM glycine, 100 mM NaCI
  • the cells were lysed using NaOH solution (1 M, aq., 1 mL) and the lysates were transferred to RIA tubes for counting the activity in a y-counter (PerkinElmer, Wallac Wizard 1480).
  • the protein concentration of each sample was determined using a Micro BCA Protein Assay kit (Pierce, Thermo Scientific) to standardize the measured activity to the average content of protein in a single well.
  • kidney accumulation of [ 177 Lu]Lu-DOTA-uPAR-ALB-18 was similar to that [ 177 Lu]Lu-DOTA-uPAR- ALB-11 , however, it was somewhat higher for [ 177 Lu]Lu-DOTA-uPAR-ALB-14, [ 177 Lu]Lu- DOTA-uPAR-ALB-15 and [ 177 Lu]Lu-DOTAGA-uPAR-ALB-17 at all investigated imaging timepoints.
  • the liver uptake of the radiopeptides of the 2 nd generation was low ( ⁇ 2% lA/g) at all investigated timepoints although clearly higher than for [ 177 Lu] Lu-DOT A- AE105 (Table 9).
  • the lowest liver accumulation was seen after injection of [ 177 Lu]Lu-DOTA-uPAR-ALB-18 (1 .2 + 0.1 % lA/g at 4 h p.i., Table 21 ), whereas [ 177 Lu]Lu-DOTA-uPAR-ALB-14 (2.0 ⁇ 0.2% lA/g at 4 h p.i., Table 20) revealed a slightly higher accumulation compared to [ 177 Lu]Lu-DOTA-uPAR-ALB-1 1 (1.8 ⁇ 0.2% lA/g, at 4 h p.i., respectively, Table 12).
  • the 2 nd generation radiopeptides showed lower xenograft-to-blood ratios compared to [ , 77 Lu]Lu-DOTA-uPAR-ALB-1 1 (Table 12), except [ 177 Lu]Lu-DOTA- uPAR-ALB-18, which showed a slightly higher ratio at 4 h p.i. (4.1 + 0.7).
  • [ 177 Lu] Lu-DOT A- AE105 showed particularly high xenograft-to-blood ratios at early timepoints after injection due to its rapid blood clearance (Table 9).
  • the xenograft-to-kidney ratios of accumulated activity of the radiopeptides of the 2 nd generation ranged from 1.7 to 3.8 and were, therewith, lower than those of [ 177 Lu]Lu-DOTA-uPAR-ALB-1 1 (3.9-4.9 between 4 h and 48 h, Table 12).
  • the xenograft-to-kidney ratios of [ 177 Lu]Lu-DOTA-uPAR-ALB-18 were similar but still lower than that for [ 177 Lu]Lu-DOTA-uPAR-ALB-1 1 , in particular at 24 h and 48 h p.i. (3.5 + 0.4 and 3.8 ⁇ 0.7, respectively).
  • the xenograft-to-liver ratios were high for all radiopeptides due to their low accumulation in the liver.
  • the highest ratios were obtained for [ 177 Lu]Lu-DOTA- uPAR-ALB-18 (1 1 + 1 after 4 h, 20 + 3 after 24 h and 18 ⁇ 3 after 48 h), similar to those obtained for [ 177 Lu]Lu-DOTA-uPAR-ALB-11 (9-24 between 4 h and 48 h p.i.).
  • DOTA-uPAR-ALB-11 and DOTA-uPAR-ALB-18 were labeled with gallium-67 and investigated in vitro and in vivo.
  • diagnostic scans performed to investigate tumor accumulation of a specific tumor-targeting agent are commonly performed with 68 Ga- labeled analogues to the 177 Lu-based therapeutics.
  • gallium-67 was used in this study as a surrogate radioisotope for gallium-68 due to the more convenient half-life of 3.26 d vs. only 68 min.
  • the uPAR-targeting peptides DOTA-uPAR-ALB-1 1 and DOTA-uPAR-ALB-18 were labeled with gallium-67 (no-carrier-added [ 67 Ga]GaCl 3 in ⁇ 0.1 M HCI, Curium Netherlands B.V., the Netherlands, via b.e. imaging GmbH (Switzerland); purified at PSI) at molar activities of up to 50 MBq/nmol. Quality control was performed using the same HPLC system as reported for the 177 Lu-labeled counterparts. The radiolytic stability was tested at an activity concentration of (10 MBq/ 100 pL) in 0.9% NaCI.
  • the radiolabeling with gallium-67 was achieved with a radiochemical purity of >98% at a molar activity of 50 MBq/nmol.
  • the radiopeptides were stable (>95%) over 24 h at the concentration that was used to inject animals for SPECT imaging.
  • SPECT/CT experiments were performed with mice, approximately 3-4 weeks after HEK- uPAR cell inoculation. The mice were scanned 1 h and 4 h after injection of the respective radiopeptide (10 MBq, 0.5 nmol, 100 pl, diluted in 0.9% NaCl containing 0.05% BSA). The studies were performed as described for the 177 Lu-labeled counterparts using the same small-animal SPECT/CT scanner (NanoSPECT/CTTM, Mediso Medical Imaging Systems, Budapest, Hungary).
  • the reconstruction of SPECT data was performed with HiSPECT software (version 1.4.3049, Scivis GmbH, Gottingen, Germany) using y-energies of 93.20 keV ( ⁇ 10%), 184.60 keV ( ⁇ 10%) and 300.00 keV (+ 10%) for gallium-67.

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Abstract

La présente invention concerne de nouveaux composés ayant un temps de circulation sanguine prolongé qui sont utiles en tant que produits radiopharmaceutiques, agents d'imagerie et pour le traitement ou le diagnostic du cancer. Dans un mode de réalisation, la présente invention concerne un complexe radiomarqué ciblant le récepteur activateur du plasminogène de type urokinase (uPAR) comprenant (a) une fraction peptidique de ciblage liant le récepteur activateur du plasminogène de type urokinase (uPAR), (b) une fraction chélatante, (c) un radionucléide, et (d) une fraction de liaison à l'albumine, la fraction peptidique de ciblage, la fraction chélatante et la fraction de liaison à l'albumine étant liées par l'intermédiaire d'une fraction de liaison trifonctionnelle commune (L1). Dans un mode de réalisation préféré, la fraction peptidique de ciblage comprend la séquence d'acides aminés (Asp)-([bêta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), la fraction chélatante est le DOTA et la fraction de liaison à l'albumine est une fraction iodophényle ou tolyle.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008053360A2 (fr) 2006-11-03 2008-05-08 Philochem Ag Molécules de liaison à l'albumine et leurs utilisations
WO2013024035A1 (fr) 2011-08-17 2013-02-21 Merck & Cie Conjugués avec des folates d'entités de liaison de l'albumine
WO2013167130A1 (fr) * 2012-05-08 2013-11-14 Rigshospitalet Peptide marqué par 177-lu pour le ciblage de upar spécifique d'un site

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008053360A2 (fr) 2006-11-03 2008-05-08 Philochem Ag Molécules de liaison à l'albumine et leurs utilisations
US20100172844A1 (en) 2006-11-03 2010-07-08 Dario Neri Albumin binding molecules and uses thereof
WO2013024035A1 (fr) 2011-08-17 2013-02-21 Merck & Cie Conjugués avec des folates d'entités de liaison de l'albumine
WO2013167130A1 (fr) * 2012-05-08 2013-11-14 Rigshospitalet Peptide marqué par 177-lu pour le ciblage de upar spécifique d'un site

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