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
  • A61K49/00—Preparations for testing in vivo
  • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
  • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
  • A61K49/0032—Methine dyes, e.g. cyanine dyes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
  • A61K49/0052—Small organic molecules
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
  • A61K49/10—Organic compounds
  • A61K49/14—Peptides, e.g. 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
  • A61K51/06—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules
  • A61K51/065—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules conjugates with carriers being macromolecules
• • 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
  • A61K51/082—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the peptide being a RGD-containing peptide
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/64—Cyclic peptides containing only normal peptide links
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

• the invention relates to the field of cyclic peptides as ligands for cellular surface receptors, in particular, as ligands for av 6-integrin. It furthermore relates to conjugates of such peptides with effector moieties that are suitable for use as therapeutic agent, diagnostic agent, targeting moiety and biomolecular research tool.
• the invention specifically relates to the use of derivatives of such peptides with signalling moieties or radionuclides for in-vivo addressing of av 6-integrin.
• Integrins are a class of 24 heterodimeric cellular transmembrane receptors, all comprising one out of 18 a- and 8 b-subunits. They mediate the selective binding of cells to various extracellular matrix proteins, such as vitronectin, fibronectin, collagen, or laminin, and furthermore are involved in signalling pathways.
• 1 anb ⁇ is one of 8 integrin subtypes that recognize the arginine-glycine-aspartate (RGD) peptide sequence.
• anb ⁇ integrin levels in adult tissues are generally low.
• 2 anb ⁇ integrin levels in adult tissues are generally low.
• 3 Expression of anb ⁇ integrin is restricted to epithelial cells. 4 Accordingly, many tumors of epithelial origin (carcinomas) show an enhanced anb ⁇ integrin expression, 5 above all, pancreatic, 6 but also cholangiocellular, 7 gastric, 8,9 breast, 10 ovarian, 11 12 colon, 13 and those of the upper aereodige stive tract.
• avb6-integrin has furthermore been described as a marker for increased invasiveness and malignancy of several carcinomas and thus, poor prognosis. 5,8 11 13 Hence, avb6-integrin has been proposed as a target for in- vivo addressing of carcinoma tissue for the purpose of molecular imaging and targeted therapy. 15 Moreover, avb6-integrin is involved in the epithelial-mesenchymal transition (EMT), e.g., during development of biliary, 16 renal, 17 as well as pulmonary 18 fibrosis, and thus might serve as a fibrosis marker.
• EMT epithelial-mesenchymal transition
• the cyclic nonapeptide cyclo(FRGDLAFp(/VMe)K) 36,37 (herein abbreviated Phe ) was reported to show a high affinity to or ⁇ 6-integrin (0.26 nM), a remarkable selectivity against other integrins (anb3: 632 nM; a5b1: 73 nM; anb5 and aIP>b3: >1 mM), and full stability in human plasma up to 3 h.
• Derivatives of Phe2 were equipped with various chelators for radiometal binding 38,39 and their in-vivo properties evaluated in tumor-bearing mice.
• the present invention solves this problem by providing conjugates comprising specific cyclic nonapeptides targeting or ⁇ 6-integrin.
• These cyclic nonapeptides are characterized by the following amino acid sequences: c y c 1 o ( Y R G D L A Y p ( AM c ) K ) . hereinafter termed Tyn, c y c 1 o ( F R G D L A Y p ( A'M c ) K ) . hereinafter termed FRGD, and c y c 1 o ( Y R G D L A F p ( A ' M c ) K ) . hereinafter termed YRGD.
• Tyr , FRGD and YRGD are structurally related to Phe and they are all encompassed by the general teaching of patent application WO 2017/046416 Al. Flowever, this patent application does not specifically disclose Ty and it also does not disclose any specific conjugates with Ty , FRGD and/or YRGD and/or any tissue specific binding characteristics of conjugates comprising Ty , FRGD and/or YRGD.
• conjugates of Ty , FRGD and/or YRGD in particular those containing more than one Ty , FRGD and/or YRGD moiety, show a high target-specific tissue uptake and retention, accompanied by low unspecific uptake in av 6-integrin negative tissues (particularly, the liver) and a rapid clearance from the blood pool, as compared to, e.g., structurally equivalent derivatives of Phe2.
• conjugates allow for selective and specific addressing of av 6-integrin positive tissues in vivo, in particular, for high-contrast in-vivo imaging of such tissues.
• the invention thus relates to conjugates of Ty , FRGD and/or YRGD wherein an effector moiety is covalently attached to the terminal amino group of the AM c -lysine residue or at least one cyclic nonapeptide selected from Ty , FRGD and YRGD.
• the invention relates in particular to conjugates comprising more than one Ty , FRGD and/or YRGD moiety, which exhibit higher affinities and integrin subtype selectivities than comparable compounds comprising only one such moiety.
• These conjugates can be characterized by the following general formula (I):
• Cp represents a cyclopeptide selected from Ty , FRGD and/or YRGD, n is an integer selected from 1 to 4 and E represents the effector moiety.
• effector moieties can be used, including moieties suitable for diagnostic uses, as well as pharmacologically active moieties for therapeutic uses.
• conjugates with moieties for diagnostic uses include moieties comprising radionuclides (for nuclear imaging or radioguided surgery), fluorophors (for fluorescence imaging or fluorescence-guided surgery), or signalling units for magnetic resonance imaging (MRI).
• the effector moiety may for instance contain a radionuclide (endoradiotherapy) or chemotherapeutic agent (targeted drug delivery).
• Yet another aspect of the present invention pertains to uses of the above-mentioned conjugates in diagnostic methods or therapeutic methods.
• the cyclopeptide Tyn is novel. Building blocks containing one of Tyn, FRGD and YRGD combined with a spacer element adapted for Click chemistry couplings are also novel. Another aspect of the present invention therefore relates to the provision of these compounds.
• Figure 1 Exemplary positron emission tomography (PET) scans (maximum intensity projections) of the same H2009 tumor-bearing SCID mouse, 75 min after injection of Ga-68-TRAP(Phe 2 ) 3 (left) and Ga-68-C-7 (right). The time between both scans was 24 h.
• PET positron emission tomography
• Figure 3 Biokinetics of Ga-68-TRAP(Phe 2 ) 3 (left) and Ga-68-C-7 (right), obtained from a region-of- interest based evaluation of 90-min dynamic PET scans.
• Ga-68-TRAP(Phe 2 ) 3 a) Ga-68-TRAP(Phe 2 ) 3 ; b) Ga-68-TRAP(Phe 2 ) 3 , blockade; c) Ga-68-C-l l; d) Ga-68-C-l l, blockade; e) Ga-68-C-9; f) Ga-68-C-9, blockade; g) Ga-68-C-8; h) Ga-68-C-8, blockade; i) Ga-68-C-10; k) Ga-68-C-10, blockade; 1) Ga-68-C-7; m) Ga-68-C-7, blockade.
• Figure 5 Exemplary positron emission tomography (PET) scans (maximum intensity projections) of the same H2009 tumor-bearing SCID mouse, 75 min after injection of Ga-68-TRAP(Phe 2 ) 3 , Ga-68-C- 9, Ga-68-C-8, and Ga-68-C-7 (from left to right). The time between the scans was 24 h. %IA/mL means percent of injected activity per mL tissue.
• PET positron emission tomography
• FIG. 6 Biokinetics of Ga-68-TRAP(Phe 2 ) 3 , Ga-68-C-9, Ga-68-C-8, and Ga-68-C-7 (from left to right), obtained from a region-of-interest based evaluation of 90-min dynamic PET scans.
• %IA/mL means percent of injected activity per mL tissue.
• the term “derived from” indicates that an atomic group contained in the conjugate has the same structure as the compound from which it is derived, the only difference being the replacement of a hydrogen atom by a covalent bond for binding the atomic group to the remainder of the conjugate.
• heavy atom is used herein to characterize any atom other than hydrogen, deuterium or any other isotope thereof.
• a bivalent atomic group there must be at least one heavy atom with at least two free valences. If a heavy atom with more than two free valences is present, the remaining valences may be saturated by hydrogen or other heavy atoms.
• amino acids are L-stereoisomers. Unless specified otherwise, amino acid moieties are linked to each other via peptide bonds. Unless specified otherwise, standard one-letter or three-letter code for amino acids applies. Unless specified otherwise, lower case letters indicate that the amino is in the D- configuration while upper case letters indicate that the amino acid is in the L-configuration.
• Me refers to a methyl group.
• N-Me-amino acid refers to a group, wherein the a-amino group carries a methyl group.
• references to the “compound of the invention”, “conjugate of the invention” or the like are to be understood as references not only to the compound, conjugate, etc. of the present invention as described hereinbelow and/or as specified in the appended claims, but also as references to the pharmaceutically acceptable salts, esters, solvates, and polymorphs thereof.
• substitution or “substituted with” includes the implicit proviso that such substitution is in accordance with the permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
• substituted is meant to include all permissible substituents of organic compounds.
• the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
• the permissible substituents can be one or more.
• Substituents may be selected from alkyl, preferably Ci- 6 -alkyl, alkenyl, preferably C2-6- alkenyl, alkynyl, preferably C2-e-alkynyl, alkoxy, preferably Ci- 6 -alkoxy, acyl, preferably C2-e-acyl, amino (including simple amino, mono and di-Ci- 6 -alkylamino, mono and di-Ce- M -arylamino, and C1-6- alkyl-Ce- [ 4 -aryl amino).
• conjugates and other compounds of the present invention are “pharmaceutically acceptable” which means that the respective compounds are suitable for use with humans and/or animals without causing adverse effects (such as irritation or toxicity), commensurate with a reasonable benefit/risk ratio.
• Root temperature can be any temperature from 20°C to 25°C and preferably it is 22°C.
• Ga-68-TRAP(Phe 2 ) 3 refers to a compound previously described as “Ga-68-TRAP(AvB6) 3 " by Maltsev et al. 38
• chelating group refers to a group that is capable to forming two or more, preferably three, four, five, six, seven or eight, coordinative bonds to a metal ion.
• Tyr 2 c y c 1 o ( Y R G D L A Y p ( AM c ) K ) .
• each Cp represents a cyclopeptide independently selected from Tyr 2 , FRGD and/or YRGD
• ii is an integer selected from 1 to 4, preferably from 2 to 4 and more preferably 3 or 4
• E represents an effector moiety.
• n may be an integer selected from 2 to 100, preferably 10 to 30.
• Suitable polymeric scaffolds include polyethyleminines, polysaccarides, polyamides, polypeptides, poly(amidoamine) (PAMAM) dendrimers, poly(propylene imine) (PPI) dendrimers, polyether- copolyester (PEPE) dendrimers, polyether dendrimers, polyester dendrimers, and polyaryl ether dendrimers.
• the one or more cyclopeptides are each covalently bonded to the effector moiety via the terminal amino group in the sidechain of the NMe-Lys residue.
• the conjugate of formula (I) contains 2, 3 or 4 cyclopeptide moieties. Most preferably, the conjugate of formula (I) contains 3 or 4 cyclopeptide moieties.
• conjugates of the present invention containing two or more cyclopeptide moieties, these multiple cyclopeptide moieties may be the same or different from each other. All of the following specific conjugates are encompassed by the scope of the present invention:
• E(FRGD)i(YRGD)i E( FRG D) 2 (YRG D)i, E(FRGD)I(YRGD) 2 , E(FRGD) 2 (YRGD) 2 , E( FRG D)i(YRG D) 3 ,
• cyclopeptides different from to Tyr 2 , FRGD and YRGD covalently attaching cyclopeptides different from to Tyr 2 , FRGD and YRGD to the effector moiety.
• one embodiment relates to compounds as described above, but wherein one, two or three of the cyclopeptide moieties Tyr 2 , FRGD and/or YRGD is/are replaced by the cyclopeptide moiety Phe 2 mentioned in the introduction, wherein Phe 2 is linked to the remainder of the conjugate in the same way as the other cyclopeptide moieties, i.e.
• n is the number of cyclopeptide moieties, the number of Phe 2 moieties is no more than n-1 while at least one cyclopeptide moiety is selected from Tyr 2 , FRGD and YRGD.
• no further cyclopeptides are present.
• the effector moiety is an atomic group having from 10 to 1000 heavy atoms, preferably from 20 to 200 heavy atoms and more preferably from 30 to 150 heavy atoms. It is further characterized by the following characteristics:
• the effector may in some embodiments be characterized by the following general formulae (II) and
• Aa'(Cg)k(S) n (II') wherein Aa stands for an active atom or active atomic group that is capable of being bonded via chelation, Aa’ stands for an active atom or active atomic group that is capable of being bonded via covalent bonding, Cg stands for a chelating group, k is 0 or 1, S stands for an atomic group acting as a spacer and n is as defined above with respect to formula (I) with the proviso that n does not exceed the number of free valences of the chelating group and with the proviso that n is 1 if k is 0, i.e. that a single spacer is directly bonded to the active atom or active atom group if no chelating group is present.
• formula (II) with formula (I) yields the following formula (la):
• an active atom or active atomic group Aa’ is covalently bonded to the chelating group or to the spacer.
• the conjugate of this embodiment is characterized by the following formula (la’):
• Aa'(Cg) k (SCp) n (la') wherein Aa’ is an active atom or active atomic group capable of forming covalent bonds, Cg, S, Cp and n have the same meanings as defined above with respect to formulae (I) and (II); k is 0 or 1; if k is 1, Aa’ is covalently bonded to Cg; if k is 0, Aa’ is covalently bonded to S. In this case, n is 1, i.e. there is only a single spacer which forms covalent bonds to Aa’ and Cp.
• Aa(Cg)(SCp) n' SAa') (lb) wherein Aa, Cg, S and Cp have the same meanings as in formula (la) above, and wherein Aa’ is an active atom or active atomic group different from Aa insofar as it is covalently bonded to the spacer and not via a chelating group, n’ is 1, 2 or 3 with the proviso that n’+l is the number of free valences of the chelating group or less.
• different linkers may be connected via a non-chelating central moiety.
• the active atom or active atomic group is covalently bonded to another part of the molecule, which can be either the central moiety, a spacer or a cyclopeptide.
• the conjugate of this embodiment is characterized by the following formulae (Ic), (Id), (Ie) and (If):
• the formula (Ic) corresponds to the above formula (la’), but wherein the chelating group is replaced by a central moiety Cm.
• S, Cp and n have the same meanings as defined above with respect to formulae (I), (la), and (II); k is 0 or 1.
• Aa’ is an active atom or active atomic group that is capable of forming covalent bonds. In formula (Ic), it is bonded via a covalent bond to Cm if k is 1 and it is bonded to S if k is 0. In the latter case, n must be 1, i.e. there is only a single spacer binding both Aa’ and Cp.
• the central moiety Cm can be any atom or atomic group having at least n+1 valences to accommodate n spacer-cyclopeptide moieties and 1 active atom or active atomic group.
• Cm preferably has 1 to 30 atoms selected from C, N, O, S and P. The remaining valences are saturated by hydrogen.
• Preferred Cm groups are aromatic groups such as phenyl, naphthyl, or derived from larger condensed aromatic groups containing 3 or 4 6-membered rings such as anthracen, phenantren, benzpyrene, etc.; non aromatic cyclic groups including C5-7 carbocycles such as cyclopentane, cyclohexane, cycloheptane, condensed groups containing 2, 3 or 4 rings, each consisting of 5 to 7 ring members such as fully or partially hydrogenated forms of naphthalene, anthracen, phenantren, benzpyrene, etc., bi- or tricyclic groups having 7 to 10 carbon atoms such as norbomene or adamantane.
• central moieties may be heterocyclic groups containing 1, 2, 3 or 4 condensed rings each having a ring size independently selected from 5, 6 or 7 ring members. These groups may be aromatic, partially or fully saturated. Alternatively, the central moiety may be a single atom selected from C, N and P.
• Formula (Id) characterizes conjugates, wherein the cyclopeptide moieties and the active atom or active atomic group Aa’ are all linked to the central moiety via spacers. That is, the active atom or active atomic group Aa’ is covalently bonded to one of the spacers.
• the meanings of Cm, Aa’, S, Cp and n are the same as explained above with respect to formula (I), (II), (la) and (Ic).
• the spacer carrying the active atom or active atomic group Aa’ may additionally carry a cyclopeptide Cp; hence, m may be 0 or 1.
• the active atom or active atomic group Aa’ and its point of attachment must be selected such that detrimental interactions with the cyclopeptide are avoided or at least minimized, e.g. by attaching the two moieties to different atoms of the spacer, which are at least 5 covalent bonds apart from each other.
• the number of spacers carrying the active atom or group Aa’ is characterized by o and it can be any integer from 1 to n.
• the number of active atoms Aa’ bonded to an individual spacer is characterized by p and it can be 1 or 2.
• Formula (Ie) is characterized by the active atom or active atomic group Aa’ being bonded to the cyclopeptide Cp.
• the meanings of Cm, Aa’, S, Cp and n are the same as explained above with respect to formula (I), (II), (la) and (Ic).
• the variable o indicates the number of cyclopeptides Cp carrying an active atom or group Aa’. This can be any integer from 1 to n.
• the variable p characterizes the number of active atoms Aa’ bonded to an individual cyclopeptide and it can be 1 or 2.
• Formula (If) characterizes conjugates of the present invention, which do not contain any central moiety and/or spacer. Instead, the active atom or active group Aa’ is directly bonded to the cyclopeptide.
• an iodine atom or radioisotope is attached to a 3-position of one or both of the tyrosine residues present in Ty , FRGD or YRGD, so that the resulting cyclopeptides are cyclo(3-I-YRGDLAYp(AMe)K); cyclo(3-I-YRGDLA3-I- Yp(AMe)K); cyclo(YRGDLA3-I-Yp(AMe)K); cyclo(3-I-YRGDLAFp(AMe)K); cyclo(FRGDLA3-I- Yp(AMe)K); wherein 3-I-Y represents a Tyr residue that carries an iodine atom in the 3-position of the 3-position of the
• the compounds of formula (If) may have a dual character: as long as the binding of Aa’ does not lead to significant deterioration of the affinity to av 6-integrin, i.e. binding affinity of the Aa’ -carrying cyclopeptide being 5 nM or lower when determined in accordance with the methods described in references 36 and 37, they may serve as conjugates of the present invention. In addition, they may also be incorporated into larger conjugates, e.g. of formula (le), and thus serve as a building block of the invention.
• a compound of formula (la) or (la’) may carry one or more cyclopeptides which themselves carry one or more active atoms or active groups Aa’.
• the present invention also relates to conjugates of formula (la) or (la’), wherein one or more of the cyclopeptides carries one or two iodine atoms or radioisotopes bonded to the 3 -position of tyrosine residues.
• the active atom or active atomic group Aa, Aa’ may include the following:
• (b-1) a non-radioactive isotope or a radioisotope of a metal ion selected from La 3+ , Ce 3+ ,
• a metal ion selected from the group consisting of Ga 3+ , Gd 3+ , Cu 2+ , Sc 3+ , Y 3+ , and Lu 3+ and mixtures thereof.
• the radioisotope may specifically be selected from 43 Sc, 44 Sc, 46 Sc, 47 Sc, 55 Co, 99m Tc, 2 03 Pb, 212 Pb, 66 Ga, 67 Ga, 68 Ga, 72 As, n Tn, 113m In, 114m In, 97 Ru, 62 Zn, 61 Cu, 62 Cu, 64 Cu, 52 Fe, 5 2m Mn, 51 Cr, 186 Re, 188 Re, 77 As, 86 Y, 90 Y, 67 Cu, 169 Er, 117m Sn, 121 Sn, 127 Te, 142 Pr, 143 Pr, 198 Au, 1 99 Au, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 109 Pd, 165 Dy, 149 Pm, 151 Pm, 153 Sm, 157 Gd, 166 Ho, 172 Tm, 169 Yb, 1 75 Yb, 177 Lu, 105 Rh, in Ag, 88 Zr, 89 Zr,
• non-metal radioisotope which is selected from n C, 13 N, 15 0, 18 F, 123 I, 124 1, 125 I, or 131 I, preferably 18 F or 123 I.
• said non-metal radioisotope may also be an active atom Aa’ that is present anywhere else within the molecule, where it may replace any other covalently bonded atom that already exists as part of the remaining molecule and which has an appropriate number of bonding partners.
• (b-3) a chromophore of a fluorescence or non-fluorescent dye and preferably moieties derived from ThermoFisher’s commercially available Cy® series such as CY® 3, 5, 5.5, 7, 7.5 and the AlexaFluor® series such as AlexaFluor® 350, 405, 488, 532, 546, 555, 568, 594, 647, 680, and 750 as well as Fluorescein, Pyren, Rhodamin, BODIPY dyes and their analogues;
• (b-4) a contrast agent for magnetic resonance imaging (MRI), preferably Gd, Fe, Mn and most preferably Gd in the form of Gd(III) in the form of a chelate complex;
• MRI magnetic resonance imaging
• (b-5) an atom or atomic group suitable for imaging by X-ray based technology, preferably iodine or atomic groups containing iodine.
• (b-6) an atom or atomic group derived from a therapeutic agent.
• Said atom or atomic group may have therapeutic activity as such or after cleavage of cyclopeptide -containing moieties to thereby release the underlying therapeutic agent.
• said therapeutic agent is a therapeutic agent suitable for the treatment of cancer or fibrosis.
• the therapeutic agent is preferably selected from alkylating agents, anti-metabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors and other anti-tumor drugs. More specifically, the following can be mentioned: platinum based compounds, antibiotics with anti-cancer activity, anthracyclines, anthracenediones, alkylating agents, antimetabolites, Antimitotic agents, taxanes, taxoids, microtubule inhibitors, Vinca alkaloids, folate antagonists, topoisomerase inhibitors, antiestrogens, antiandrogens, aromatase inhibitors, GnRh analogs, inhibitors of 5a-reductase, bisphosphonates, a metabolic inhibitor, preferably a mTOR inhibitor; an epigenetic inhibitor, preferably a DNMT inhibitor; an anthracycline antibiotic; a camptotheca; an anthracycline; histone deacetylase (HDAC) inhibitors, proteasome inhibitor
• HDAC histone
• Said anticancer drug is preferably selected from the group consisting of 5-fluorouracil, cisplatin, irinotecan hydrochloride, epirubicin, paclitaxel, docetaxel, camptothecin, doxorubicin, rapamycin, 5-azacytidine, doxorubicin irinotecan, topotecan (type 1 topoisomerase inhibitors), amsacrin, etoposide, etoposide phosphate and teniposide (topoisomerase-type 2 inhibitors); UFT, capecitabine, CPT-II, oxaliplatin, cyclophosphamide, methotrexate, navelbine, epirubicin, mitoxantrone, raloxifen, mitomycin, carboplatinum, gemcitabine, etoposide and topotecan.
• the therapeutic agent is preferably selected from therapeutic drugs suitable for the treatment of fibrosis.
• therapeutic drugs are disclosed, for instance, in “Cystic Fibrosis in the 21st Century” by Andrew Bush (Ed.), S. Karger, 2006; “Liver Fibrosis: New Insights for the Healthcare Professional: 2013 Edition” by Q. Ahton Acton, ScholarlyEditions, 2013; “Idiopathic Pulmonary Fibrosis: A Comprehensive Clinical Guide” by Keith C. Meyer, Steven D.
• the therapeutic agent may be any agent having experimentally established or suspected activity in the treatment of such infections, irrespective whether they are already used in clinical practice or are still in development stage.
• Agents currently used for or in development for the treatment of Covid-19 infections are for instance antiviral agents, including for example the anti-ebola agent remdesivir or the anti influenza agent favilavir, kinase inhibitors such as ATR-002, anti-inflammatory agents including glucocorticoids, antagonists of IL-1 or IL-6 such as anakinra and tocilizumab, respectively, anti-infective agents such as ivermectin or agents for the treatment of other lung conditions like fibrosis.
• antiviral agents including for example the anti-ebola agent remdesivir or the anti influenza agent favilavir, kinase inhibitors such as ATR-002, anti-inflammatory agents including glucocorticoids, antagonists of IL-1 or IL-6 such as anakinra and tocilizuma
• the active atom or group of atoms can be bonded to the cyclopeptide (or cyclopeptides) via atomic groups acting as a spacer.
• the atomic group acting as a spacer is typically a linear chain of 2 to 20 and preferably 3 to 10 atoms selected from C, N, O, P and S, preferably alkylene groups, which optionally carry one or more substituents, the remaining valences being saturated by hydrogen.
• This linear chain may be interrupted by one or more cyclic structures preferably such having 5 ring atoms, and more preferably a triazole ring.
• Bonding to the amino group of the side chain of M(Me)K is typically accomplished by means of an amide bond. Bonding of the spacer to the active atom or atomic group Aa’ in formula (la’), (lb), (lc) or (Id) can also be accomplished by means of an amide bond but a direct covalent bond is also possible.
• additional divalent functional groups may be present, as shown by the following formulae (Illb) to (Illf):
• one or more of the spacers may carry one or more independently selected substituents.
• substituents are not particularly restricted.
• the substituent is itself a moiety containing a spacer and a cyclopeptide, preferably a spacer S and a cyclopeptide Cp as described herein. It is even possible that the spacer part of said substituents is further substituted to form a dendrimeric structure, which may have up to 3 generations of substituents attached to the 0 generation spacers depicted in formulae (la) to (Ie).
• the spacer may be cleavable under physiological conditions.
• Such cleavable spacers are not particularly limited and may be selected from the spacers described in WO 2009/117531 A, WO 2015/123679 A, Younes et al. N. Engl. J. Med. 2010;363:1812-1821; Dorywalska et al. Mol. Cancer Ther. 2016; 15(5):958— 970, Jain et al., Pharm. Res. 2015;32(11):3526- 3540, and references cited therein.
• the active atom is a metal ion
• bonding is typically accomplished via a chelating group, for example, as described in Chem. Soc. Rev. 2011;40:3019-3049.
• Binding of the metal ion by the chelating group preferably occurs via complex bonds (Lewis acid/base interactions) effected by the N and O atoms of the chelating group.
• the chelating group is however not particularly limited as long as it is capable of forming a chelate complex with the metal ion of interest, which is preferably stable under physiological conditions for a time period that is sufficiently long for carrying out the intended diagnostic method.
• Preferred chelators or chelator-containing functional groups are those mentioned in Chem. Soc. Rev.
• TRAP its tetravalent homologue DOTPI, DOTAZA, and analogues and derivatives of these chelating groups.
• Typical structures of these chelating groups are represented by formulae (IV a) to (IVd) below:
• the conjugates of the invention may be synthesized using standard materials and methods known in the art. If the conjugate is a chelate, the formation of the chelate is typically performed as the last step. That is, a suitable procedure includes one or more steps for forming a precursor, as described below, followed by reaction of the precursor with the atom, atomic group or ion to be chelated. Said final reaction is typically conducted under usual conditions for reactions of this kind which are known to the skilled artisan. In a preferred setting, the reaction is conducted at ambient temperature (room temperature, e.g. 20-25°C). Also preferred, the reaction is conducted at temperatures ranging from ambient temperature (room temperature) to 37 °C.
• Said ion may be provided in the form of a salt, wherein the salt-forming counter-ion may be selected from the group consisting of sulfates, fluorides, chlorides, bromides, nitrates, phosphates, carbonates, hydrogencarbonates, sulfonates, acetates, and mixtures thereof.
• the ion is provided in the form of a solution.
• the precursor is preferably prepared using a modular approach based on Click chemistry to link the chelating group (or central moiety) to the cyclopeptide moiety/moieties.
• the spacer/spacers is/are formed in situ during said coupling reaction.
• the starting materials contain themselves precursors of the spacers with functional groups suitable for Click chemistry couplings at their termini.
• Cyclopeptides carrying precursors of spacers at their (ZVMe)K residue may be obtained by reacting the respective precursor, which carries a carboxyl group at the cyclopeptide -binding terminus and which may be activated using for instance HATU, HOBt and DIPEA, is reacted with the respective cyclopeptide under standard amide coupling conditions for instance as described in Maltsev OV, et al, Angew. Chem. Int. Ed. 2016;55:1535-1539 and/or WO 2017/046416 Al.
• the cyclopeptide can be synthesized by applying suitably adapted materials and procedures described in the literature, for instance in Maltsev OV, et al., Angew. Chem. Int. Ed. 2016;55:1535-1539 and/or WO 2017/046416 Al.
• Conjugates of the invention include both the conjugates as shown below as well as the corresponding conjugates obtainable by incorporating a non-radioactive metal ion or a radionuclide such as 68 Ga into the structures shown below.
• the present invention further relates to building blocks that can be used for obtaining the conjugates of the present invention.
• a first type of building block of the present invention is the group of compounds corresponding to the chelate complexes described above, but without the coordinated atom (such as Ga-68). These building blocks of the present invention may be characterized by the following formula (Ila):
• Cg(SCp) connector (Ila) wherein Cg stands for a chelating group, S stands for an atomic group acting as a spacer, each Cp is a cyclopeptide independently selected from Tyn, YRGD and FRGD and n is an integer of from 1 to 4. All of the further information provided above for the corresponding coordinated complexes applies in an analogous fashion to the building blocks of formula (Ila).
• the present invention further relates to building blocks, which are modified cyclopeptides that can be used at earlier stages of the synthetic procedure for synthesizing the conjugates and above-mentioned building blocks of the present invention by the convenient Click chemistry.
• Such building blocks comprise a cyclopeptide moiety selected from Ty , YRGD and FRGD, a functional group that may participate in a Click reaction (e.g. as specified, for instance, in the Wikipedia entry “Click chemistry” in the version of January 24, 2020) and an atomic group linking the cyclopeptide to the functional group via the terminal amino group of the sidechain of the A'Mc-K residue.
• Cp-L-Fg (V) wherein Cp represents the cyclopeptide selected from Ty , YRGD and FRGD, L represents the linking group and Fg represents the functional group for carrying out the Click reaction.
• the functional group is preferably an azide group, an alkyne group including especially a terminal ethyne group, a dibenzylcyclooctyne group, a trans-cyclooctene group, a tetrazine group, a dibenzocyclooctyne group or a bicyclo[6.1.0]nonyne group.
• the linking group typically includes a carbonyl group forming an amide bond with the amino group of the sidechain of the A'Mc-K residue. It further includes a group of 1 to 15 atoms selected from C, N, O, forming a linear chain between the amide bond and the functional group, which is optionally substituted by one or more substituents, the remaining valences of the chain-forming atoms being saturated by hydrogen atoms.
• said group is an alkylene group with 1 to 15, more preferably 2 to 6, methylene groups.
• BB-1 illustrates this concept for a building block of the invention wherein the Tyr2 cyclopeptide is linked to an azide functional group via a C4-alkylene group.
• the Ty cyclopeptide itself is novel and represents another building block of the present invention for obtaining the conjugates of the invention described hereinabove and hereinbelow. The same is true for the iodine-modified cyclopeptides Ty3 ⁇ 4 FRGD and YRGD.
• further building blocks of the invention are cyc1o(3-I-YRGDLAYp(AMc)K); cyc1o(3-I-YRGDLA3-I-Yp(AMc)K); cyclo( YRGDL A3 -I-Yp(AMe)K) ; cyclo(3 -I-YRGDL AFp(AMe)K); cyclo(FRGDLA3 -I- Yp(/VMe)K); wherein 3-I-Y represents a Tyr residue that carries an iodine atom in the 3-position of the phenyl ring, wherein said iodine atom can be any non-radioactive isotope or radioisotope of iodine.
• cyclopeptides of the invention can be synthesized using standard peptide methodology such as solid phase peptide synthesis using Fmoc as a protective group.
• standard peptide methodology such as solid phase peptide synthesis using Fmoc as a protective group.
• Fmoc Fmoc
• Cyclization of the peptide can be effected using standard techniques. For instance, cyclization can be accomplished on the solid support or in solution using FlBTU/FlOBt/DIEA, PyBop/DIEA or PyClock/DIEA reagents. The available cyclization methods are described for instance in WO 2017/046416 A, J. Chatteijee, B. Laufer, FI. Kessler, Nat. Protoc.2012, 7, 432-444 and references cited therein.
• the conjugate may be prepared by analogous use of methods described in the literature. 38,43,44,45
• the conjugates of the present invention are useful for any disease that is associated with an increased expression of avP6-integrin.
• the presence of avP6-integrin in tissue can be determined by immunohistochemistry (IHC). Applying this analytical technique to healthy adult tissue does not give rise to any avP6-integrin signal.
• tissue giving rise to a detectable IHC signal for avP6-integrin is regarded as tissue with increase expression of avP6-integrin.
• tissue exhibiting increased expression of avP6-integrin is tissue deviating from healthy adult tissue, be it due to a disease such as cancer, fibrosis or Covid-19, or due to a condition like an earlier wound resulting in scar tissue formation. Any of these diseases and conditions may be identified using the conjugates of the present invention. Such diseases are described in the literature. 41,42
• NSCLC non-small-cell lung cancer
• pancreatic cancer cholangiocellular cancer
• gastric cancer breast cancer
• breast cancer head-and-neck squamous cell
• basal cell colon cancer
• ovarian cancer ovarian cancer
• PDAC pancreatic ductal adenocarcimoma
• lung adenocarcinoma mammary carcinoma, colon adenocarcinoma, pancreatic adenocarcinoma (PDAC), head and neck squamous cell carcinoma such as oral squamous cell carcinoma, laryngeal squamous cell carcinoma, oropharyngeal squamous cell carcinoma, nasopharyngeal squamous cell carcinoma, hypopharyngeal squamous cell carcinoma.
• PDAC pancreatic adenocarcinoma
• fibrosis Using IHC, anb ⁇ expression in fibrotic tissue was also confirmed (Munger CS, et al., Cell 1999;96:319). Further diseases therefore include fibrosis and especially biliary, renal, endomyocardial fibrosis, Crohn’s disease, arthofibrosis as well as pulmonary fibrosis. Of particular interest is idiopathic pulmonary fibrosis (IPF).
• IPF idiopathic pulmonary fibrosis
• radiolabeled compounds of the present invention are therefore suitable for in-vivo imaging of post-COVID-19 syndrome in patients.
• avP6-integrin is an activator of transforming growth factor beta (TGF-beta)
• TGF-beta transforming growth factor beta
• diseases could be diagnosed by determining the avP6-integrin expression status of cells in the affected tissues.
• Increased anb6 expression can be exploited for in-vivo targeting using radiolabeled compounds as those of the present invention.
• Conjugates of the present invention are suitable for use as diagnostic agent.
• the conjugates of the present invention are advantageously used, wherein the effector moiety contains an active atom or active atomic group suitable for the imaging method/diagnostic method of interest, as described above.
• a suitable active atom or active atomic group is selected.
• the chosen imaging/diagnostic method also determines the dosage, form and timing of the administration of the conjugate of the present invention.
• the conjugates of the present invention are suitable for virtually any analytical/diagnostic method that involves the use of diagnostic agents.
• the conjugates of the present invention are particularly suitable for imaging methods such as gamma scintigraphy, fluorescence-based imaging, positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnet resonance tomography (MRT), optical imaging or magnetic resonance imaging (MRI), X-ray based CT imaging, scintigraphy, Cherenkov imaging, ultrasonography, thermography and combinations thereof.
• the conjugates of the present invention may be used by applying techniques described in the literature. 33,34,35,38
• the present invention thus provides methods for imaging patients, such as cancer patients, fibrosis patients or patients affected by Covid-19 infection, including post-COVID-19 syndrome, which comprise administration of the conjugate of the present invention to the patient, followed by subjecting the patient to an imaging method selected from gamma scintigraphy, fluorescence-based imaging, positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnet resonance tomography (MRT), optical imaging or magnetic resonance imaging (MRI), X-ray based CT imaging, scintigraphy, Cherenkov imaging, ultrasonography, thermography and combinations thereof, wherein the active atom or active atomic group is suitable for the selected imaging method and wherein the selected imaging method detects a signal resulting from the active atom or active atomic group.
• an imaging method selected from gamma scintigraphy, fluorescence-based imaging, positron emission to
• conjugates of the present invention having an effector moiety with an active atom or active atomic group derived from a drug can be used in the treatment of the diseases associated with upregulation of av 6-integrin, e.g. as listed above.
• the conjugates of the present invention may be administered to the patient for instance by intravenous, transmucosal, transdermal, intranasal administration. Suitable dosages may be in the range of 0.1 to 1000 mg/day, preferably 0.1 to 10 mg/day.
• the conjugates of the present invention may be administered once daily, twice a day, three times a day, etc. for any period of time, wherein multiple periods of time may be interrupted by one or more periods of time where the compounds of the present invention are not administered.
• the conjugates of the present invention may also be used as a component in combination therapy. They may be combined with one or more other therapeutic agents effective in the treatment of cancer such as the therapeutic agents listed above and/or below. Such combination therapy may be carried out by simultaneously or sequentially administering the two or more therapeutic agents.
• conjugates of the present invention particularly those incorporating radionuclides emitting alpha or beta radiation, such as for example, 47 Sc, 67 Cu, 177 Lu, 90 Y, 213 Bi, 225 Ac, 161 Tb, 149 Tb, or 131 I, for targeted radiotherapy.
• the conjugates of the present invention may also be used for diagnosis or treatment of fibrosis.
• the conjugates of the present invention may be used for such purposes by any suitable administration form including intravenous, intra-arterial, transmucosal, pulmonary, and intranasal administration. Dosages and administration schemes can be the same as specified above for the treatment of cancer. Combination therapy is also possible, wherein the one or more other therapeutic agents is selected from other therapeutic agents suitable for the treatment of fibrosis, for instance as cited above by cross-reference to the review article by Gharaee-Kermani et al. which is incorporated herein by reference.
• the conjugates of the present invention as well as the one or more other therapeutic agents can be administered simultaneously or sequentially.
• the conjugates of the present invention may also be used for diagnosis or treatment of Covid-19 infections, including post-COVID-19 syndrome.
• the conjugates of the present invention may be used for such purposes by any suitable administration form including intravenous, intra-arterial, transmucosal, pulmonary, and intranasal administration. Dosages and administration schemes can be the same as specified above for the treatment of cancer. Combination therapy is also possible, wherein the one or more other therapeutic agents is selected from other therapeutic agents suitable for the treatment of Covid-19 infections, for instance immune therapy, dexamethasone or remdesivir.
• the conjugates of the present invention as well as the one or more other therapeutic agents can be administered simultaneously or sequentially.
• the present invention thus provides methods for treating patients suffering from diseases associated with increased anb ⁇ integrin expression, and especially cancer, fibrosis or Covid-19 infections, which include administration of a conjugate of the present invention to the patient, wherein the active atom or active atomic group is derived from a therapeutic agent that is selected to be suitable for treating the respective disease, e.g. as specified under Item (b-6) in the Effector Moiety section above.
• the conjugates of the present invention may also be used for drug targeting as well as in biomolecular research. These uses may be carried out as described in the respective sections of WO 2017/046416 A.
• This approach is of particular interest for the treatment of cancer and especially carcinoma with chemotherapeutics as it may accomplish a “homing” in such av 6-expressing tissues.
• the conjugates of the present invention may be formulated as pharmaceutical compositions. This can be done using conventional means and methods for peptide-based medicaments. Suitable literature is recited for instance in the section on pharmaceutical compositions of WO 2017/046416 A. These disclosures are incorporated herein by reference.
• fPharmaceutical compositions of the present invention may also comprise the nanoparticles mentioned in the preceding section. According to a preferred embodiment, such nanoparticles comprise not only the conjugate of the present invention and the nanoparticle itself, but additionally also a therapeutic agent, preferably chemotherapeutic, within the nanoparticle.
• CuAAC copper-catalyzed azide-alkyne cycloaddition
• Dde l-(4,4-dimethyl-2,6-dioxocyclohex-l- ylidene)-3ethyl
• DIAD diisopropyl azodicarboxylate
• DIPEA A'.A'-di i sopropy 1 am i n c .
• DMF dimethylformamide
• DPPA diphenyl phosphoryl azide
• Fmoc 9-fluorenylmethoxycarbonyl
• HATU N,N,N A r -lclramclhyl uroni um -hcxanuorophosphalc.
• HFIP 1,1, 1,3,3, 3-hexafluoro-2- propanol
• HOBt 1-hydroxybenzo triazole hydrate
• NMP A'-m ethyl -2-pyrrol i done .
• NOTA 1,4,7- triazacyclononane-l,4,7-triacetic acid
• Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
• PBS phosphate -buffered saline
• TFA trifluoroacetic acid
• THF tetrahydrofuran
• TIPS triisopropyl silane
• TRAP l,4,7-triazacyclononane-l,4,7- tris[methylene(2-carboxyethylphosphinic acid)]
• TRAP(azide)i 38 and TRAP(azidc)A' were synthesized as described previously.
• Semi-preparative reversed-phase HPFC was performed using a Waters system: Waters 2545 (Binary Gradient Module), Waters SFO (System Fluidics Organizer), Waters 2996 (Photodiode Array Detector) and Waters 2767 (Sample Manager). Separations were performed using a Dr.
• Maisch C18-column Reprosil 100 C18, 5 pm, 150x30 mm (Column 1) with a flow rate of 40 mL/min of water (0.1%v/v trifluoroacetic acid and acetonitrile (0.1% v/v trifluoroacetic acid) or a YMC C18-column: YMC-Pack ODS-A, 5 pm, 250x20 mm (Column 2) with a flow rate of 16 mL/min of water (0.1%v/v trifluoroacetic acid) and acetonitrile (0.1% v/v trifluoroacetic acid).
• Analytical F1ESI-F1PLC-MS (heated electrospray ionization mass spectrometry) was performed on a LCQ Fleet (Thermo Scientific) with a connected UltiMate 3000 UF1PLC focused (Dionex) on C18-columns: SI: Flypersil Gold aQ 175 A, 3 pm, 150x2.1 mm (for 8 or 20 minutes measurements); S2: Accucore C18, 80 A, 2.6 pm, 50x2.1 mm (for 5 minute measurements) (Thermo Scientific).
• On-Resin Fmoc-Deprotection The Fmoc-protected peptidyl-resin was treated with a 20% piperidine in DMF (v/v) for 10 min and again for 5 min. The resin was washed with DMF (5 x).
• Radiometal incorporation and radiochemical purity of labeled compounds was determined by radio- TL on ITLC silica impregnated chromatography paper ( Agilent , Santa Clara, USA; eluents: 0.1 M trisodium citrate or a 1:1 (v/v) mixture of 1 M ammonium acetate and methanol), analyzed using a scan-RAM radio-TLC detector by LabLogic systems Inc. (Brandon, USA). 68 Ga-labelling was performed using a fully-automated on-site system ( Gall El ul by Scintomics, Lindach, Germany) as described previously.
• H2009 human lung adenocarcinoma cells (CRL-5911; American Type Culture Collection) were cultivated as recommended by the distributor.
• CB17 SCID mice CultrexBME, type 3 PathClear; Trevigen, GENTAUR GmbH. Mice were used for biodistribution or PET studies when tumors had grown to a diameter of 10-12 mm (4-6 weeks after inoculation).
• mice were anaesthesized with isoflurane for intravenous administration of the radiolabeled compounds.
• the administered activity per mouse ranged between 10 and 15 MBq (100-200 pmol, depending on variations in timing of production and administration).
• PET imaging was performed on a Siemens Inveon small-animal PET system, either dynamic under isoflurane anaesthesia for 90 min, or as single frames 75 min p.i. with an acquisition time of 20 min.
• Data were reconstructed using Siemens Inveon Research Workspace software, employing a three-dimensional ordered subset expectation maximum (OSEM3D) algorithm without scatter and attenuation correction.
• OEM3D three-dimensional ordered subset expectation maximum
• ROIs regions of interest
• 3-6 MBq (between 70-140 pmol) of the radiolabeled compound was injected into the tail vein.
• the mice were sacrificed 90 min after injection, a blood sample was taken and the organs of interest were dissected. Quantification of the activity in weighed tissue samples was done using a 2480 WIZARD 2 automatic g-counter ( PerkinElmer , Waltham, USA). Injected dose per gram tissue (%ID/g) was calculated from the organ weights and counted activities.
• Novel peptidic compounds and conjugates were synthesized and characterized as described above.
• Ga-68-C-7 shows markedly improved biokinetics and imaging properties in comparison to the corresponding state-of-the-art compound, Ga-68-TRAP(Phe 2 ) 3 , 38 substantiating that Ty is advantageously used in av 6-integrin targeted compounds for in-vivo applications.
• Ga-68-C-7 shows the best tumor-to- liver and particularly tumor-to-pancreas ratios, suggesting that it should be most suitable for imaging or ⁇ 6-integrin positive lesions in those organs, such as metastases or primary tumors of the pancreatic adenocarcinoma type.
• Figure 5 corroborates that the peptides FRGD and YRGD, which are featured in Ga-68-C-8 and Ga- 68-C-9, respectively, are also suitable for synthesis of targeted radiolabeled molecules with significantly lower liver uptake than Ga-68-TRAP(Phe 2 ) 3 . Accordingly, Figure 6 shows that the blood clearance of Ga-68-C-8 and Ga-68-C-9 is much faster than that of Ga-68-TRAP(Phe 2 ) 3 , and resembles that of Ga-68-C-10.

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