Targeted delivery of theranostic drugs

Abstract

The present invention relates to compounds and methods for the targeted delivery of diagnostic / therapeutic radionuclides to cancer tissue. In particular, the present invention relates to the targeted delivery of diagnostic / therapeutic radionuclides to cholecystokinin-2 receptor positive cancers and methods for the diagnosis and treatment of cancer.

Description

[Technical field]

[0001] The present invention relates to compounds and methods for the targeted delivery of diagnostic / therapeutic radionuclides to cancer tissue. In particular, the present invention relates to the targeted delivery of diagnostic / therapeutic radionuclides to cholecystokinin-2 receptor positive cancers and methods for the diagnosis and treatment of cancer. [Background technology]

[0002] The basic principle in the field of theranostic radiopharmaceuticals is to deliver therapeutic ( thera peutic or diagnostic nostic The development of targeted proprietary compounds (such as short peptides) that function as therapeutic (clinical) compounds. This rapidly developing field of personalized medicine has already proven highly successful in treating patients with metastatic malignancies, showing remarkable efficacy and low toxicity (Hofman, MS, et al., Lancet, 2020, 395(10231), 1208-1216; Hofman, MSP, et al., Lancet Oncology, 2018, 19(6), 825-833; Kaltsas, G., et al., Neuroendocrinology 2017, 105(3), 245-254). These remarkable achievements are due in part to the unique ability of clinicians to "see what they treat." The increased sensitivity and specificity of these compounds also allows clinicians to more accurately stage patients before, during, and after treatment to ensure they receive the most appropriate disease management (Velikyan, I. Pharmaceuticals (Basel), 2020, 13, 39). The introduction of peptide-based theranostics in oncology is currently limited to only a few cancers. These include somatostatin-2R-positive neuroendocrine tumors and prostate-specific membrane antigen (PSMA)-positive prostate cancer.

[0003] The G protein-coupled receptor cholecystokinin-2 (CCK-2R), whose natural ligand is the gastrin hormone G17, is an important molecular target for theranostic applications and is overexpressed in many cancers. These cancers include medullary thyroid cancer (MTC), somatostatin-2R-negative neuroendocrine tumors (Gotthardt,M.,et al.,Endocrine-Related Cancer,2006,13(4),1203-1211; Reubi,JC,Schaer,J.-C.,Waser,B.,Cancer Research,1997,57(7),1377-1386; Behe,M.,Behr,TM,Peptide Science 2002,66(6),399-418; Reubi,JC,Maecke,HR,Journal of Nuclear Medicine 2017,58(Supplement 2),10S-16S), and stromal ovarian cancer (incidence 100%; Reubi,JC,Schaer,J.-C.,Waser,B.,Cancer Research,1997,57(7),1377-1386). Research, 1997, 57(7), 1377-1386), and small cell lung cancer (incidence 56%; Roy, J., et al., Oncotarget 2016, 7(12), 14605-14615; Reubi, JC, Macke, HR, Krenning, EP, Society of Nuclear Medicine 2005, 46 Suppl 1, 67s-75s; Copps, J., Murphy, RF, Lovas, S., Protein Pept Lett 2009, 16(12), 1504-1518; Zeng, Q., et al., Frontiers in Endocrinology 2020, 11(112)).

[0004] Due to the well-established association of CCK-2R with cancer, much work has already been done to understand the key features of the G17 peptide in an attempt to establish modified CCK-2R peptides suitable for use as theranostic radiopharmaceuticals in nuclear medicine (Roosenburg, S., et al., Amino Acids, 2010, 41, 1049-1058). Several radiolabeled gastrin-derived peptide analogs have been developed, including DGlu-MG0 derived from human minigastrin, a truncated form MG11 lacking the pentaglutamic acid sequence, and other gastrin and cholecystokinin derivatives (Rottenburger, C., et al., J. Nucl. Med., 2020, 61, 520-526; Erba, PA, et al., Pol. Arch. Intern. Med., 2018, 128, 791-795; Klinger, M., et al., Journal of Nuclear Medicine 2018). CP04 and its closely related analog PP-F11N, which contain an N-terminal hexa-D-glutamic acid sequence, exhibit reduced renal uptake and retention (minimal nephrotoxicity) and enhanced metabolic stability, desirable properties required for theranostic compounds. Both CP04 and PP-F11N have been investigated in recent clinical trials for imaging and targeting CCK-2R in humans (Rottenburger, C., et al., J. Nucl. Med., 2020, 61, 520-526; Erba, PA, et al., Pol. Arch. Intern. Med., 2018, 128, 791-795). Despite the accumulation of both peptides in MTC lesions, they have not been used to treat neuroendocrine tumors. 177 Suboptimal tumor radiation doses were observed compared to [Lu]Lu-DOTA-TATE, indicating that improved agents remain in demand. High tumor uptake and long residence time are paramount for successful delivery of peptide receptor radionuclide therapy (PRRT) and therefore determine the absorbed dose to the tumor.

[0005] von Guggenberg et al. developed the peptide MGS5 by N-methylation of the C-terminal residue of MG11 using unnatural aromatic amino acids. Radiolabeled MGS5 showed improved in vitro and in vivo properties, increased metabolic stability and improved tumor targeting compared to CP04 and PP-F11N (Klinger, M., et al., Journal of Nuclear Medicine 2018; Klinger, M., et al., Journal of Nuclear Medicine 2019, 60(1), 1010-1016; Klinger, M., et al., Theranostics, 2018, 8, 2896-2908; Maximilian, K., Anton Amadeus, H., Guggenberg, Ev, Current Medicinal Chemistry, 2020, 27, 1-21). In recent years, several drugs have emerged from these studies [ 68 Clinical application of [Ga]Ga-DOTA-MG5S has been reported in two patients, demonstrating the concept of PET imaging of CCK-2R in MTC patients (Guggenberg, Ev, et al., Eur. J. Nucl. Med. Mol. Imaging, 2019, 46(S1), S268; Uprimny, C., et al., Eur. J. Nucl. Med. Mol. Imaging, 2020).

[0006] So far, there are only a limited number of studies that elucidate the effect of amino acid modifications on the conformation of radiolabeled G17-based CCK-2R binding ligands (CP04, PP-F11N, and MGS5) and therefore on receptor binding and biological activity (Kolenc Peitl, P., et al., Bioconjugate Chemistry, 2015, 26, 1113-1119). The critical sequence required for peptide-CCK-2R interaction is known to be the C-terminal tetrapeptide Trp-Met-Asp-Phe-NH2, which accounts for only 10% of the total biological potency of G17 (measured by gastric acid secretion in rats). Interestingly, the gradual incorporation of polyglutamic acid residues at the N-terminus leads to conformational changes as evidenced by CD spectroscopy, gradually increasing biological potency by up to 90% (Peggion, E., Foffani, MT, Mammi, S., Structure-Function Relationship of Gastrin Hormones: A Rational Approach to Drug Design. In Bioorganic Chemistry in Healthcare and Technology, Pandit, UK; Alderweireldt, FC, Eds. Springer US: Boston, MA, 1991, 121-133). Peggion et al. concluded that although the N-terminus of G17 likely adopts an α-helical conformation, there must be interactions between the "head" and "tail" of the peptide to explain the increased potency and conformational changes seen upon extension of the pentaglutamyl sequence. Ala 11 -Trp 14 It was suggested that there is a beta turn centered around the residue, likely capping the N-terminal alpha helix and bringing the two ends of the peptide into close proximity. This work highlights the pharmacological significance of a likely "U-shaped" hairpin fold in which the alpha helix N-terminus stabilizes the biologically active conformation of the C-terminus (Copps, J., Murphy, RF, Lovas, S., Protein Pept. Lett., 2009, 16, 1504-1518).

[0007] The three-dimensional structure of receptor ligands is clearly important for binding specificity, affinity, and functionality and should be considered during the design and optimization of targeting peptides intended as theranostic compounds. However, the design of short to medium chain peptides with defined secondary structures is difficult to implement because they are inherently flexible and unlikely to form ordered structures as found in larger proteins and biomolecules. The development of targeting peptides requires detailed mimicry of protein-protein contact points, often requiring the reproduction of not only the primary structure but also the secondary structure of biopolymers. Cyclization and stapling of peptides have been used to restrict peptide conformation through the formation of covalent bonds. However, cyclization and stapling do not result in much secondary structure and are less useful when linear peptides are required for functionality.

[0008] There is therefore a need to develop novel theranostic compounds to expand their transformative role in the management of other malignancies. Summary of the Invention [Means for solving the problem]

[0009] Novel compounds and methods are provided for diagnosing and treating cancer in a subject. Accordingly, in one aspect of the present invention, a compound represented by the following formula (I): [ka] (In the formula, R 1 is the side chain of an amino acid selected from phenylalanine, 3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine, or tyrosine, wherein the phenylalanine residue is selected from the group consisting of one or more of halo, -NH, cyano, C 1-6 Alkyl, haloC 1-6 optionally substituted with alkyl, or acetyl; R 2 is selected from H or C1-C3 alkyl; A is a radionuclide binding ligand; -L- is the amino acid sequence: -D-Glu-D-Glu-D-Ala-D-Glu-D-Glu-D-Glu-. or a pharma- ceutically acceptable salt thereof.

[0010] In one embodiment, the compound of formula (I) has the following formula (Ia): [ka] It is expressed as:

[0011] In another embodiment, the compound of formula (I) has the following formula (Ib): [ka] (In the formula, each R 3 are independently halo, -NH2, cyano, -OH, C 1-6 Alkyl, haloC 1-6 alkyl, or acetyl; n is 0 to 5.

[0012] In a further embodiment, the compound of formula (I) has the following formula (Ic): [ka] It is expressed as:

[0013] In one embodiment, the compounds of the present invention further comprise a radionuclide complexed to said radionuclide binding ligand.

[0014] In another aspect, the present invention provides a method for identifying a CCK-2R positive cancer in a subject, comprising administering to the subject an effective amount of a compound according to the present invention or a pharma- ceutically acceptable salt thereof and detecting the radionuclide.

[0015] In a further aspect, the present invention provides a compound according to the invention, or a pharma- ceutically acceptable salt thereof, for use in a method of identifying CCK-2R positive cancer in a subject, said method comprising administering to said subject an effective amount of the compound or a pharma- ceutically acceptable salt thereof and detecting said radionuclide.

[0016] In another aspect, there is provided a method of treating CCK-2R positive cancer in a subject in need thereof, comprising administering to the subject a compound according to the present invention or a pharma- ceutically acceptable salt thereof.

[0017] In yet another aspect of the present invention, there is provided a pharmaceutical composition comprising a compound according to the present invention or a pharma- ceutically acceptable salt thereof.

[0018] These and other aspects of the present invention will become more apparent to those skilled in the art from a reading of the following detailed description in conjunction with the accompanying examples and claims.

[0019] The invention is herein described, by way of example only, with reference to the following non-limiting drawings. [Brief description of the drawings]

[0020] [Figure 1] Figure 1 is a graphical representation showing the binding curves for IC50 determination of peptides A-F, Comparative Example 1, and CP04 for [177Lu]Lu-DOTA-CP04 in A431-CCK2R cells. Curves are representative of 2-4 biological replicates performed with 3 technical replicates (n=3). [Diagram 2] 2 is a graphical representation showing the binding curves for IC50 determination of compounds 1-4 and Comparative Example 2 for [177Lu]Lu-DOTA-CP04 in A431-CCK2R cells. Curves are representative of 2-4 biological replicates performed with 3 technical replicates (n=3). [Diagram 3]Figure 3 is a graphical representation showing the CD spectra of compounds natGa-1-4 and natGa-Cmp ex-2 in water and 5 mM DPC micelles. Three spectra accumulated at 20°C were averaged and baseline corrected. [Figure 4] Figure 4 is a graphical representation showing the metabolic stability of compounds [68Ga]Ga-1-4 and [68Ga]Ga-Cmp ex-2 incubated in human serum, liver homogenate, and kidney homogenate. Samples were assayed at various time points over a 90 minute period. Results are average values ​​(n=2). [Diagram 5] Figure 5 shows: a) Distribution of compounds [68Ga]Ga-1-4 and [68Ga]Ga-Cmp ex-2 in female BALB / c nu / nu mice (8-10 weeks old). b) Tumor uptake analysis of compounds [68Ga]Ga-1-4 and [68Ga]Ga-Cmp ex-2 at 1 and 2.5 hours post-injection (reported in %ID / g) with co-administration of blocking agent YM022. c) Quantitative analysis of compounds [68Ga]Ga-1-4 and [68Ga]Ga-Cmp ex-2 accumulated in selected organs at 1 hour (reported in %ID / g). d) Quantitative analysis showing tumor-to-organ ratio of compounds [68Ga]Ga-1-4 and [68Ga]Ga-Cmp ex-2 at 1 hour post-injection. T:B is tumor-to-blood ratio and T:Stom is tumor-to-stomach ratio. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Foldamers offer several advantages, such as engineerable secondary structure, controllable orientation of side chain functional groups, and resistance to proteolysis. Foldamers also address the thermodynamic basis of protein-protein interactions (PPIs) by minimizing conformational freedom, thereby minimizing the entropic penalty incurred upon binding and resulting in higher binding affinity (Du, X., et al., International Journal of Molecular Sciences 2016,17(2),144).

[0022] Foldamers have an inherent tendency to form secondary and sometimes tertiary structures through hydrogen bonding, giving rise to a wide range of conformations including all forms of β-turns, hairpins, and helices.

[0023] In one embodiment, the present invention provides a method for the preparation of a compound of formula (I): [ka] (In the formula, R 1 is the side chain of an amino acid selected from phenylalanine, 3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine, or tyrosine, wherein the phenylalanine residue is selected from the group consisting of one or more of halo, -NH, cyano, C 1-6 Alkyl, haloC 1-6 optionally substituted with alkyl, or acetyl; R 2 is selected from H or C1-C3 alkyl; A is a radionuclide binding ligand; -L- is the amino acid sequence: -D-Glu-D-Glu-D-Ala-D-Glu-D-Glu-D-Glu-. or a pharma- ceutically acceptable salt thereof.

[0024] Substitution of amino acid residues at the N-terminus of the C-terminal tetrapeptide Trp-Met-Asp-Phe-NH2 with the turn-inducing residue N-methylglycine was found to improve the binding affinity of the resulting compound. The incorporation of N-methylglycine appears to constrain the compound into a β-hairpin conformation. This structure is further stabilized by interactions between the aromatic and glutamic acid side chains.

[0025] In another embodiment, the present invention provides a compound according to the present invention, comprising the compound of formula (II): [ka] (In the formula, R 1is the side chain of an amino acid selected from phenylalanine, 3-(1-naphthyl)alanine, 3-(2-naphthylalanine), or tyrosine, wherein the phenylalanine residue is selected from the group consisting of one or more of halo, -NH, cyano, C 1-6 Alkyl, haloC 1-6 optionally substituted with alkyl, or acetyl; R 2 is selected from H or C1-C3 alkyl; -L- is the amino acid sequence: -D-Glu-D-Glu-D-Ala-D-Glu-D-Glu-D-Glu-. or a pharma- ceutically acceptable salt thereof.

[0026] In one embodiment, the peptide of formula (II) or a pharma- ceutically acceptable salt thereof has the following formula (IIa): [ka] It is expressed as:

[0027] In another embodiment, the peptide of formula (II) or a pharma- ceutically acceptable salt thereof has the following formula (IIb): [ka] (In the formula, each R 3 are independently halo, -NH2, cyano, -OH, C 1-6 Alkyl, haloC 1-6 alkyl, or acetyl; n is 0 to 5.

[0028] In a further embodiment, the peptide of formula (II) or a pharma- ceutically acceptable salt thereof has the following formula (IIc): [ka] It is expressed as:

[0029] In another embodiment, the present invention provides a peptide of formula (II) selected from those listed in Table 1.

[0030] [Table 1]

[0031] 1. Nal = 3-(1-naphthyl)-L-alanine; 2. Phe(4-F) = 4-fluoro-L-phenylalanine; 3. Phe(3,4-diF) = 3,4-difluoro-L-phenylalanine; 4. Phe(3,4,5-F) 4 = 3,4,5-trifluoro-L-phenylalanine; 5. Phe(4-NH2) = 4-amino-L-phenylalanine; 6. 2-Nal = 3-(2-naphthyl)-L-alanine; 7. Phe(4-CN) = 4-cyano-L-phenylalanine; 8. Phe(4-CF3) = 4-(trifluoromethyl)-L-phenylalanine; 9. Phe(2,3,4,5,6-F) = 2,3,4,5,6-pentafluoro-L-phenylalanine; 10. Phe(3-F) = 3-fluoro-L-phenylalanine; 11. Phe(4-Ac) = 4-(acetyl)-L-phenylalanine.

[0032] References to the "side chain" of an amino acid have their standard meaning in the art. Examples of amino acid side chains are shown below.

[0033] [ka]

[0034] As used herein, non-natural amino acids include compounds that have both amino and carboxyl functionalities, derivatives thereof, or derivatives of natural amino acids. These amino acids form part of a peptide chain by linking through their amino and carboxyl groups. Alternatively, these derivatives can be linked to other natural or non-natural amino acids to form non-peptide bonds.

[0035] It will be appreciated that in addition to the negatively charged side chains shown above, many side chains can be protonated and therefore positively charged, such as the side chains of lysine, and the present invention contemplates within its scope these protonated side chains.

[0036] It will be understood that the compounds of the present invention can exist in one or more stereoisomeric forms (e.g., diastereomers). The present invention includes within its scope all of these stereoisomeric forms in isolated (e.g., enantiomeric separation) or in combination (including racemic and diastereomeric mixtures). The present invention contemplates the use of both L- and D-forms of amino acids, including the use of each amino acid independently selected from the L- and D-forms, for example, if a compound contains two Glu residues, each Glu residue can have the same or opposite absolute stereochemistry.

[0037] The compounds of the invention may further comprise a radionuclide complexed or covalently bound to a radionuclide binding ligand. As described above, the compounds of the invention are useful as theranostic compounds in that the same peptides can be covalently bound to a radionuclide binding ligand suitable for binding a radionuclide useful for the diagnosis of cancer and / or a radionuclide binding ligand suitable for binding a radionuclide useful for the treatment of cancer. In one embodiment, it is envisaged that the radionuclide binding ligand suitable for binding a radionuclide useful for the treatment of cancer is the same as the radionuclide binding ligand suitable for binding a radionuclide useful for the diagnosis of cancer. In a further embodiment, the radionuclide binding ligand suitable for binding a radionuclide for the treatment of cancer is different from the radionuclide binding ligand suitable for binding a radionuclide for the diagnosis of cancer.

[0038] Reference to a "radionuclide-binding ligand" will be understood to mean a ligand or chelator that binds strongly to a radionuclide (radioisotope). The radionuclide-binding ligand is covalently attached to a compound such that, when a compound of the invention is administered to a subject, the compound can deliver the radionuclide to a target site with no or minimal loss, effectively providing a site-specific radioactive source in vivo for imaging or therapy. Examples of suitable radionuclide-binding ligands include DOTA (1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid), DOTA-NHS-ester, p-SCN-Bn-DOTA (C-DOTA), DOTAGA, DOTAGA-anhydride, CB-DO2A (4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane), TCMC (1,4,7,10- Tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), p-SCN-Bn-TCMC, 3p-C-DEPA (2-[(carboxymethyl)-[5-(4-nitrophenyl-1-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]pentan-2-yl)-amino]acetic acid), 3p-C-DEPA-NCS, p-NH2-Bn-Oxo -DO3A, TETA (1,4,8,11-tetraazacyclotetradecane 1,4,8,11-tetraacetic acid), BAT, p-NH2-Bn-TE3A, C-TETA, CB-TE2A (4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]-hexadecane), CB-TE1A1P, CB-TE2P, MM-TE2A, DM-TE2A, TE2A, Diamsar, SarAr (1- N-(4-aminobenzyl)-3,6,10,13,16,19-hexazabicyclo[6.6.6]-eicosane-1,8-diamine), AmBaSar, BaBaSar, NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), p-SCN-Bn-NOTA, NODASA, NODAGA, NETA ({4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl}-acetic acid), NETA-monoamide, C-NE3TA-NCS, C-NETA-NCS, 3p-C-NETA, TACN-TM (N,N',N''-tris(2-mercaptoethyl)-1,4,7-triazacyclononane), DTPA (diethylenetriaminepentaacetic acid), p-SCN-Bn-1B-DTPA, p-SCN-Bn-1B4M-DTPA, CHX-A''-DTPA (2-(p-isothiocyanatobenzyl)-cyclohexyldiethylenetriaminepentaacetic acid), p-SCN-Bn-CHX-A''-DTPA PA, TRAP (1,4,7-triazacyclononane 1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid]), AAZTA (1,4-bis(hydroxycarbonylmethyl)-6-[bis(hydroxylcarbonylmethyl)]amino-6-methylperhydro-1,4-diazepine), NOPO, H2dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane), H4octapa (N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid), H2 azapa (N,N'-[1-benzyl-1,2,3-triazol-4-yl]methyl-N,N'-[6-(carboxy)pyridin-2-yl]-1,2-diaminoethane), H5decapa (N,N''-[[6-(carboxy)pyridin-2-yl]methyl]-diethylenetriamine-N,N',N''-triacetic acid), p-SCN-Bn-H4octapa, HBED (N,N'-bis(2-hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid), HBED-CC, (HBED-CC)TFP, SHBED (N,N'-bis (2-hydroxy-5-sulfobenzyl)-ethylenediamine-N,N'-diacetic acid), BPCA, CP256 (4-acetylamino-4-[2-[(3-hydroxy1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-carbamoyl]-ethyl]-heptanedioic acid bis[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-amide]), PCTA (3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-3,6,9-triacetic acid), p-SCN-Bn-PCTA, DFO, p-SCN-Bn-DFO, H6phospha (N,N'-(methylene phosphonate)-N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane), HEHA (1,4,7,10,13,16-hexaazacyclohexadecane-N,N',N'',N'''',N'''''-hexaacetic acid), p-SCN-Bn-HEHA, PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N'''',N''''-pentaacetic acid), p-SCN-Bn-PEPA, Crown, MACROPA (4-amino-6-[[16-[(6-carboxypyridin-2-yl)methyl]- Examples of suitable radioactive ligands include, but are not limited to, 1,4,10,13-tetraoxa-7,16-diazacyclooctadec-7-yl]methyl]pyridine-2-carboxylic acid), MACROPA-NCS, pypa, py4pa (6,6'-(((azanediylbis(ethane-2,1-diyl))bis(carboxymethyl)azanediyl))bis(methylene))dipicolinic acid), noneunpa (6,6'-(((oxybis(ethane-2,1-diyl))bis(carboxymethyl)azanediyl))bis(methylene))dipicolinic acid), DOTAM (2,2',2'',2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetamide), and derivatives thereof. In one embodiment, the radionuclide binding ligand is DOTA.

[0039] Alternatively, the radionuclide-binding ligand may be a peptide or small molecule organic moiety to which a radionuclide has been covalently attached. For example, the radionuclide-binding ligand may be a short peptide or organic moiety to which a radionuclide such as fluorine-18, iodine-124, or iodine-131 has been covalently attached at an appropriate position. It will be understood that the radionuclide-binding ligand should be selected so as not to compete with the compound for the binding site of the CCK-2 receptor.

[0040] In one embodiment, the present invention provides a method for identifying a CCK-2R positive cancer in a subject, comprising administering to the subject an effective amount of a compound according to the present invention, or a pharma- ceutically acceptable salt thereof, and detecting a radionuclide.

[0041] In another embodiment, the present invention provides a compound according to the present invention, or a pharma- ceutically acceptable salt thereof, for use in a method for identifying CCK-2R positive cancer in a subject, the method comprising administering an effective amount of the compound to the subject and detecting the radionuclide.

[0042] The compounds of the present invention localize to cancers that overexpress the CCK-2 receptor, such as medullary thyroid carcinoma (MTC), somatostatin-2R negative neuroendocrine tumors, stromal ovarian cancer (100% incidence), and small cell lung cancer (56% incidence).

[0043] After administering the compound, for example by intravenous injection, the subject is placed in the scanner. As the injected radionuclide decays, it emits a positron, which annihilates with an electron to produce a pair of gamma rays or photons traveling in opposite directions. Typically, the emitted photons are detected when they reach the scintillator material of the scanning device, producing a burst of light that is detected by a photomultiplier tube. Suitable scanning methods include computed tomography (CT), positron emission tomography (PET), and combined PET-CT procedures.

[0044] Diagnostic radionuclides within the scope of the present invention include, but are not limited to, carbon-11, nitrogen-13, oxygen-15, fluorine-18, scandium-44, copper-64, gallium-67, gallium-68, yttrium-86, zirconium-89, technetium-99m, indium-111, iodine-124, iodine-125, and terbium-152.

[0045] In one embodiment, the compounds of the present invention can be administered in tumor ablation therapy applications to detect the extent of damage caused to affected tissue. The compounds localize to CCK-2R positive tumors, indicating tumor size and location to physicians, thus allowing for continuous monitoring to track tumor size and indicate the effectiveness of medical treatment methods. Being able to monitor the effectiveness of ongoing therapeutic treatments can avoid subjects receiving ineffective medical treatments, thus aiding in the development of personalized treatments for patients. This is particularly valuable in areas where a wide range of potential treatments are available, such as cancer treatment, where many chemotherapy and radiation therapies are available. By using the compounds to continuously monitor tumor size, the effectiveness of a particular treatment can be assessed earlier, thus avoiding subjects from being exposed to ineffective treatments for long periods of time. The compounds can indicate the ineffectiveness of medical treatments, allowing physicians to modify or change the course of medical treatment. Such diagnostic tools allow for time-saving measures and improved overall patient outcomes.

[0046] In a further embodiment, the present invention provides a method of treating CCK-2R positive cancer in a subject in need thereof, comprising administering to the subject an effective amount of a compound according to the present invention or a pharma- ceutically acceptable salt thereof.

[0047] In another embodiment, the present invention provides a compound of the present invention, or a pharma- ceutically acceptable salt thereof, for use in the treatment of a CCK-2R positive cancer in a subject in need thereof.

[0048] In yet another embodiment, the present invention provides the use of a compound according to the present invention, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for diagnosing and / or treating CCK-2R positive cancer in a subject in need thereof.

[0049] Typically, radionuclides suitable for cancer treatment are beta-emitting radionuclides.After the compound is administered to CCK-2R positive cancer and localized, the radionuclides complexed to the compound of the present invention directly interact with tumor cells.Cancer treatment radionuclides within the scope of the present invention include, but are not limited to, copper-67, yttrium-90, iodine-131, terbium-149, terbium-161, lutetium-177, rhenium-186, rhenium-188, bismuth-212, bismuth-213, astatine-211, lead-212, and actinium-225.

[0050] As used herein, the term "subject" refers to an animal, such as a bird or a mammal. Particular animals include a rat, a mouse, a dog, a cat, a cow, a sheep, a horse, a pig, or a primate. A subject may be a human, also referred to as a patient. A subject may also be a rodent, such as a mouse or a rat.

[0051] In general, techniques for preparing the compounds of the invention are well known in the art, see, for example, below. a) Alewood, P.; Alewood, D.; Miranda, L.; Love, S.; Meutermans, W.; Wilson, D. Meth. Enzymol., 1997, 289, 14-28; b) Merrifield, RB, J. Am. Chem. Soc., 1964, 85, 2149; c) Bodanzsky, “Principles of Peptide Synthesis”, 2nd Ed., Springer-Verlag (1993); and d) Houghten, Proc. Natl. Acad. Sci. USA, 1985, 82, 5131.

[0052] The compounds of the present invention can be synthesized using known solid-phase or liquid-phase techniques, for example, N-terminus or C-terminus can be bound to a solid support (typically a resin) followed by stepwise synthesis of a linear peptide. Protecting group chemistry for the protection of amino acid residues (including side chains) is well known in the art and can be found, for example, in Theodora W. Greene and Peter GMWuts, Protecting Groups in Organic Synthesis (Third Edition, John Wiley & Sons, Inc, 1999), the entire contents of which are incorporated herein by reference.

[0053] Briefly, compounds of the present invention were synthesized according to general Scheme 1 below.

[0054] [ka]

[0055] N-terminus-free linear peptides A-I were synthesized using a general Fmoc-based solid-phase peptide (SPPS) synthesis protocol using a microwave-assisted automated CEM Liberty peptide synthesis module. N-terminus-free linear peptides A-I were globally deprotected, cleaved from the resin, and purified by HPLC before being coupled to a radionuclide-binding ligand (illustrated as DOTA) in solution. The compounds were then coordinated to radionuclides or natural gallium for binding assays in sodium ascorbate buffer solution (pH 4.5) to give the coordinated compounds in quantitative yields.

[0056] If the compounds of the invention require purification, chromatographic techniques such as reverse phase high performance liquid chromatography (HPLC) can be used. The compounds can be characterized by mass spectrometry and / or other appropriate methods.

[0057] When a compound contains one or more functional groups that can be protonated or deprotonated (e.g., at physiological pH), the compound can be prepared and / or isolated as a pharma- ceutically acceptable salt. It will be understood that a compound can be zwitterionic at a given pH. As used herein, the phrase "pharma-ceutically acceptable salt" refers to a salt of a given compound that is suitable for administration as a medicament. Such salts can be formed, for example, by reaction of an acid or base with an amine or carboxylic acid group, respectively. In the case of zwitterionic compounds, the salt can be an internal salt where the compound contains suitable proton donating and accepting functional groups.

[0058] Pharmaceutically acceptable acid addition salts can be prepared from inorganic and organic acids. Examples of inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Examples of organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

[0059] Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. The corresponding counterions derived from inorganic bases include sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Organic bases include primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), and cyclic amines, including isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamine, theobromine, purine, piperazine, piperidine, and N-ethylpiperidine.

[0060] Acid / base addition salts tend to be more soluble in aqueous solvents than the corresponding free acid / base forms.

[0061] The compounds of the present invention may be in crystalline form or solvated (e.g., hydrated), and both forms are intended to be within the scope of the present invention. The term "solvate" refers to a complex of variable stoichiometry formed by a solute (in the present invention, the peptide of the present invention) and a solvent. Such a solvent should not interfere with the biological activity of the solute. Solvents include, for example, water, ethanol, or acetic acid. Solvation methods are generally known within the art.

[0062] The present invention also provides pharmaceutical compositions comprising a compound according to the invention in association with at least one pharma- ceutically acceptable carrier or diluent.

[0063] As those skilled in the art will readily understand, the nature of the administration route and the pharma- ceutical acceptable carrier will depend on the condition to be treated and the nature of the mammal. Radiolabeled compounds are generally administered to subjects intravenously, enterally, or parenterally as therapeutic and / or diagnostic agents. The selection of a specific carrier or delivery system and administration route will be easily determined by those skilled in the art. In preparing formulations containing the compounds of the present invention, care should be taken to ensure that the activity of the compound is not destroyed during the process and that the compound can reach its site of action without being destroyed. Similarly, the administration route selected should be such that the compound reaches its site of action.

[0064] Those skilled in the art can easily determine suitable formulations for the compounds of the present invention using conventional techniques. Identification of preferred pH ranges and suitable excipients, such as antioxidants, is routine in the art. Buffer systems are routinely used to provide pH values ​​in the desired range, including carboxylic acid buffers, such as acetate, citrate, lactate and succinate buffers. A variety of antioxidants are available for such formulations, including phenolic compounds such as BHT and vitamin E, reducing agents such as methionine and sulfite, and metal chelators such as EDTA.

[0065] The compounds of the present invention are envisioned to be prepared in parenteral administration forms, including those suitable for intravenous, intrathecal, intracerebral or epidural delivery.The pharmaceutical forms suitable for injection use include sterile injectable solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions.They should be stable under the conditions of manufacture and storage, and can be protected against reduction or oxidation, and against the contaminating action of microorganisms such as bacteria and fungi.

[0066] The solvent or dispersion medium for the injectable solution or dispersion may include any of the conventional solvents or carrier systems for the active compounds, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved, if necessary, by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it is preferable to include agents for adjusting the osmotic pressure, for example, sugars or sodium chloride. It is preferable that the injectable preparation is isotonic with blood. Prolonged absorption of the injectable composition can be achieved by using agents that delay absorption in the composition (e.g., aluminum monostearate and gelatin). Pharmaceutical forms suitable for injectable use can be delivered by any suitable route, including intravenous, intramuscular, intracerebral, intrathecal, and epidural injection or infusion.

[0067] Sterile injectable solutions are prepared by incorporating the aqueous liquids of the invention in the required amount in an appropriate solvent with various other ingredients (such as those listed above), as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above.

[0068] Pharmaceutically acceptable vehicles and / or diluents include any and all solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as it is incompatible with the active ingredient, use of any conventional media or agent in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0069] It is particularly advantageous to formulate the compositions in unit dosage forms for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to physically discrete units suitable as unitary dosages for the mammalian subject to be diagnosed, each unit containing a predetermined amount of nanoparticles calculated to obtain the desired diagnosis together with the necessary pharma-ceutically acceptable vehicle. The specifications for the novel unit dosage forms of the invention are determined by and directly depend on (a) the unique properties of the nanoparticles and the specific diagnosis to be achieved, and (b) the limitations inherent in the technology of compounding nanoparticles for the invention in living subjects with pathological conditions that compromise physical health.

[0070] As mentioned above, the main active ingredient can be formulated with a suitable pharma- ceutically acceptable vehicle in a unit dosage form so that it can be conveniently and effectively administered in an effective amount. A unit dosage form can contain, for example, an amount of nanoparticles ranging from 0.25 μg to about 2000 mg. Expressed as a percentage, the active compound may be present in the range of about 0.25 μg to about 2000 mg per mL of carrier. In the case of a composition containing a supplementary active ingredient, the dosage is determined with reference to the usual dosage and method of administration of the ingredient.

[0071] Throughout this specification and the claims that follow, unless the context clearly indicates otherwise, the term "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers or steps but not the exclusion of other integers or groups of integers.

[0072] Reference herein to any prior publication (or information derived therefrom) or to any known matter is not, and should not be considered, as an admission or acknowledgement, or in any way an indication that the prior publication (or information derived therefrom) or known matter is part of the common general knowledge in the field to which this specification pertains.

[0073] The invention will now be described with reference to the following non-limiting examples.

[0074] Example 1. Examination of D-alanine insertion in a hexaglutamic acid sequence For example, we synthesized a series of peptides in which one of the glutamic acid residues in the hexaglutamic acid sequence of CP04 was replaced with a D-glutamic acid residue. Furthermore, we replaced the Gly-Trp-Met-Asp-Phe-NH2 sequence of CP04 with NMe-Gly-Trp-NMe-Nle-Asp-Nal. The alanine-substituted peptides then bound to the CCK-2 receptor (CCK-2R) and [ 177 The ability of the β-Lu complex to disrupt the binding of [Lu]Lu-DOTA-CP04 was evaluated.

[0075] 1.1. Peptide synthesis Peptides were assembled using standard Fmoc-based solid-phase peptide synthesis (SPPS) procedures on an automated CEM Liberty Blue microwave peptide synthesizer (John Morris Group, Victoria, Australia) using Rink amide resin (0.8 mmol / g). Peptides were assembled on a 0.1 mmol reaction scale. Fmoc deprotection was performed in two steps as follows: the peptide-resin was treated with 20% piperidine / DMF (v / v; 5 mL) containing oxyma (0.1 M) for 30 s (40 W, 40 °C) under microwave irradiation. This was followed by filtration and a second treatment with the same deprotection cocktail under microwave irradiation (45 W, 75 °C; 3 min). The peptide-resin was then rinsed with DMF (3 × 4 mL). Coupling of all standard Fmoc-amino acids was achieved by adding Fmoc-amino acid (5 equiv, 0.5 mmol), DIC (5 equiv), and oxima (10 equiv) in DMF (4 mL) to the Nα-deprotected peptide-resin and stirring the mixture under microwave irradiation for 3 min (30 W, 90 °C). After sequence assembly, the peptide-resin was manually rinsed with dichloromethane (DCM) (3 × 5 mL) before the cleavage step. Global deprotection of the peptide and cleavage from the solid support were performed for 2 h using a TFA / TIPS / HO:DODT:1,3-dimethoxybenzene (85:5:5:2.5:2.5 (% v / v / v / v / v); 8 mL) cocktail. The cleavage mixture was then filtered, the TFA solution evaporated under a stream of nitrogen, and the crude product was isolated by trituration and subsequent washing with diethyl ether (3 × 30 mL).

[0076] Peptides were purified using an Aeris 5 μm PEPTIDE XB-C18 column (10 μm, 100 Å, 250 × 21.2 mm) eluted with a gradient of MeCN:0.1% (v / v) TFA at 8 mL / min.

[0077] 1.2. For in vitro ligand binding assays 177 Radiosynthesis of [Lu]Lu-DOTA-CP04 [ka]

[0078] [ 177 [Lu]Lu-DOTA-CP04 was prepared similarly with minor modifications to a reported method (Ocak, M. et al., Eur J Nucl Med Mol Imaging 2011, 38(8), 1426-35). Briefly, DOTA-CP04 (30 μg, 14.6 nmol) was dissolved in sodium acetate (0.5 M, 100 μL (0.3 μg / μL peptide solution)) and reconstituted in 0.4 M ammonium acetate / 0.24 M 2,5-dihydroxybenzoic acid (200 μL, pH 4.5) containing ethanol (50 μL), L-methionine (50 μL of a 10 mg / mL solution in milliQ water), and sodium ascorbate (50 μL, 0.05 M in milliQ water). To this mixture, 500–1000 MBq of non-carrier added [ 177 [Lu]LuCl3 was added and the resulting solution was heated at 80 °C for 20 min. A portion of this material containing approximately 200 MBq was HPLC purified using a Proteo90Å (250 × 4.6 mm) eluted with a gradient of MeCN (25% MeCN for the first minute, increasing to 65% over 80 min) containing 0.1% TFA and 2% EtOH (v / v) at 1 mL / min. The product eluting at 35 min was manually collected in a vial containing water:ethanol (9:1 v / v, 10 mL) and then captured on a Strata-X SPE cartridge. The product was then eluted from the cartridge using ethanol (1 mL). A portion of this purified peptide was then diluted in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% fetal bovine serum to obtain a final concentration of 4 MBq / mL suitable for ligand binding assays.

[0079] 1.3. Ligand binding assay CCK-2 receptor binding experiments were performed using A431 (human epidermoid carcinoma) cells stably transfected to overexpress the human full-length CCK-2 receptor (A431-CCK-2R). As a negative control, A431 cells stably transfected with an empty vector were analyzed simultaneously (A431-EV). A431 cells were maintained in DMEM (Gibco, Australia) medium supplemented with 10% fetal bovine serum and 250 μg / mL G418 as previously described (Aloj L, et al., J Nucl Med., 2004, Mar; 45(3), 485-94).

[0080] The affinity of the peptides for CCK-2R was determined by the following formula: 177 The peptides were assessed using a competitive binding assay against [Lu]Lu-DOTA-CP04. Briefly, cells were plated at a density of 650,000 cells per well of a 6-well plate 48 hours before the experiment. On the day of the experiment, peptides were diluted in DMEM supplemented with 1% fetal bovine serum to give at least seven different concentrations (0–400 nM), with each dilution containing approximately 50,000 cpm of [Lu]Lu-DOTA-CP04. 177 [Lu]Lu-DOTA-CP04 was added. Cells were washed twice in ice-cold binding buffer (DMEM supplemented with 1% fetal bovine serum) and resuspended in 1.5 mL of the previously prepared compound mixture before incubation at 37°C for 1 h. After incubation, the medium was removed and kept for analysis (unbound portion) and the cells were washed twice with ice-cold binding buffer, which was pooled with the unbound portion. Finally, 1 M NaOH was added to the cells and after 5 min of incubation, the lysate was collected (bound portion). A small amount of the lysate was set aside and protein concentration was measured using the Pierce® BCA Protein Assay Kit (Thermo Fisher, Australia). All samples were analyzed using a Perkin Elmer 2480 Wizard2 TMThe results were analyzed using a gamma counter (PerkinElmer, MA, USA) and normalized according to protein concentration. Half-maximal inhibitory concentrations (IC) were calculated using nonlinear regression using GraphPad Prism Software (GraphPad Software, CA, USA). 50 ) was measured.

[0081] Apparent binding affinity (IC 50 ) (Table 2; Figure 1) compared to Comparative Example 1 and CP04, showing the effect of D-Ala substitutions for each glutamic acid residue in the key hexameric peptide sequence. 177 When tested against [Lu]Lu-DOTA-CP04, the affinity for the CCK-2 receptor was reduced by more than three-fold (from 0.76 nM to 2.33 nM). This loss of affinity very clearly demonstrates the importance of the linker and radiolabeled conjugate in improving the affinity of the peptide for the receptor of interest. Similarly, Comparative Example 1, which contains a hexaglutamic acid sequence, was previously nat Although the affinity for the CCK-2 receptor was estimated to be 0.05 nM in the Ga-DOTA-peptide form, there was a 5-fold decrease in affinity for the receptor. Nevertheless, Comparative Example 1 established an important baseline for the D-Ala analog, with an IC 50 was 0.32 nM.

[0082] [Table 2]

[0083] Peptide A, which had a terminal D-alanine substitution at position 1, had a significant loss of binding affinity to the target receptor. Substitution of the small D-alanine residue at position 1 resulted in a greater than 10-fold loss of activity, clearly demonstrating the important role of the terminal glutamic acid in improving the binding affinity of the peptide sequence to the CCK-2 receptor. Similarly, peptide E, which had a D-alanine substitution at position 5, had a comparable loss of activity towards the receptor. Previous work on this scaffold has shown that the peptide preferentially folds into a β-hairpin structure through intramolecular interactions of the backbone and residues, and this secondary structure is important for its superior activity. The D-alanine substitution may suggest that the D-glutamic acid residues at positions 1 and 5 are important for the formation of this secondary structure, and thus the loss of these critical residues results in loss of activity.

[0084] The biological activity of peptides B, D, and F, which contained repeated D-alanine substitutions at positions 2, 4, and 6, was evaluated and found to be well tolerated while maintaining moderate affinity for the CCK-2 receptor. The resulting activities indicated an approximately 5-fold loss of binding affinity for the target receptor in all three peptides. This result provided insight into other possible substitutions that could be made without severely compromising ligand-receptor affinity, opening up many opportunities for future modifications.

[0085] Peptide C, which has a D-alanine substitution at position 3, yielded the most active ligand among the peptide libraries evaluated. IC 50With a β-hairpin affinity of 0.18 nM, peptide C showed a more than 10-fold improvement over the existing CP04 activity. Moreover, it exceeded the already excellent affinity of the comparison peptide by almost 2-fold. Thus, not only is position 3 well tolerated in modifications, but the substitution of a neutral hydrophobic moiety such as D-alanine also significantly improves ligand-receptor binding. Given the three-dimensional β-hairpin structure of this scaffold, it is highly likely that the polar and bulky D-glutamic acid at this position would be repelled by the corresponding residue in the binding site of the CCK-2 receptor, whereas the small, neutral and hydrophobic D-alanine residue is much more suitable for the receptor's binding pocket.

[0086] Further studies were performed to evaluate the binding affinity of peptides based on Cmp ex-1 in which the 3-(1-naphthyl)alanine residue was replaced with various substituted phenylalanine residues or 3-(2-naphthyl)-L-alanine (Table 3). Most of the resulting peptides showed affinities comparable to Cmp ex-1 or CP04. Notably, replacement of 3-(1-naphthyl)alanine with 3,4,5-trifluoro-phenylalanine increased the affinity over the control.

[0087] [Table 3]

[0088] Finally, studies were performed to evaluate the binding affinity of peptides based on peptide C, in which the 3-(1-naphthyl)alanine residue was replaced with various substituted phenylalanine residues, tyrosine, or 3-(2-naphthyl)-L-alanine, with a D-alanine substitution at position 3 (Table 4). Most of the resulting peptides showed affinities comparable to peptide C.

[0089] [Table 4]

[0090] Example 2. Examination of C-terminal modification of peptide C Following the D-alanine studies, we synthesized a series of compounds in which the NMe-Gly-Trp-NMe-Nle-Asp-Nal sequence of peptide C was modified. We then conjugated the radionuclide binding ligand DOTA to the peptides, nat Chelated with gallium.

[0091] 2.1. Synthesis and purification of DOTA-conjugated compounds Synthesis and purification of peptides were performed as described above. Conjugation of free DOTA with peptides was performed in solution similarly to previously reported procedures with minor modifications (Schottelius, M.; Schwaiger, M.; Wester, H.-J., Tetrahedron Letters, 2003, 44(11), 2393-2396). Briefly, DOTA (1.5 equivalents relative to peptide) was preactivated with N-hydroxysuccinimide (NHS) (1.5 equivalents relative to NHS) using EDCI (1.5 equivalents relative to NHS) and DIPEA (2 equivalents relative to NHS) in anhydrous DMSO (typically 500 μL). The reaction mixture was sonicated for 30 min at 50 °C until all starting materials were completely dissolved. The peptide was then added to a minimum amount of anhydrous DMSO (typically 200-500 μL). The resulting mixture was left shaking for an additional 30 min at room temperature and the progress of the reaction was monitored by MS analysis. Once the reaction was complete, as shown by MS analysis that the m / z peak of the starting peptide had disappeared, distilled water was added to form a 20:80 mixture of DMSO:water, and the crude peptide was purified using reverse-phase HPLC. The HPLC conditions for purification of DOTA-compounds were identical to those reported above for the purification of each compound.

[0092] [Table 5]

[0093] 2.2. nat Synthesis and purification of Gallium-chelating DOTA-compounds DOTA-compounds natGallium chelation was performed according to published procedures (Maurin, M., et al., Nucl Med Rev Cent East Eur., 2015, 18(2), 51-5). Briefly, compounds (2–8 μmol) were suspended in freshly prepared ascorbic acid buffer (3–5 mL of 50 mg / mL) neutralized to pH 4.5 with concentrated NaOH solution. Gallium nitrate (4 equivalents relative to DOTA-peptide) was then added and the resulting mixture was heated at 95 °C for 15 min. The resulting mixture was cooled to room temperature, centrifuged to remove precipitates, and purified using reversed-phase HPLC employing the same chromatographic conditions for the purification of each N-terminal free peptide.

[0094] 2.3. Receptor binding assay Using the methods described above, compounds were found to bind to the CCK-2 receptor (CCK-2R) and 177 The ability to disrupt the binding of [Lu]Lu-DOTA-CP04 was evaluated and compared to Comparative Example 2 (Figure 2; Table 5).

[0095] [Table 6]

[0096] Comparative peptide 2 (Cmp Ex-2), previously evaluated in several preliminary studies, showed favorable biostability, selectivity for the target receptor, and highest tumor uptake in biodistribution mouse studies when evaluated alongside other potential ligand scaffolds. However, this peptide unfortunately also showed the highest renal uptake of the compounds tested. This is due in large part to the highly acidic hexameric terminal peptide chain, which is known to promote renal uptake. Cmp Ex-2 serves as an important template to explore the impact of D-alanine substitutions that reduce the overall acidity of the peptide and how this structural change impacts the binding affinity of the peptide to its receptor of interest. Compounds 1-4 were designed with modifications to key C-terminal recognition sequence residues.

[0097] In improving the binding affinity of linear peptides to receptors, nat The importance of the Ga)-DOTA binding peptides has been previously confirmed. Similarly, hexa-D-glutamate substituted variants of compounds 1, 3 and 4 have been previously investigated and shown to have sub-nanomolar affinity for the CCK-2 receptor. The high aromaticity of the C-terminal position further enhances receptor binding. Further constraining the peptides by introducing N-methylation at key residues stabilizes the peptides but shows a slight decrease in affinity. Ligands were assayed to confirm the effect of D-alanine substitution at position 3 of these peptides and showed that they tolerated the substitution very well with minimal loss of binding affinity for all compounds tested.

[0098] Example 3. Radiosynthesis of Gallium-68 Labeled DOTA-Compounds

[0099] [ka]

[0100] 3.1. Radiolabeling of DOTA-compounds DOTA-Compounds 1-4 and Comparative Example 2 were radiolabeled according to published procedures (Haskali, MB et al., EJNMMI Radiopharmacy and Chemistry, 2019, 4(1), 23). Briefly, a mixture of the corresponding DOTA-peptide precursor (30 μg), ethanol (200 μL), 0.05 M sodium ascorbate (200 μL), 0.05 M 2,5-dihydroxybenzoic acid sodium salt (200 μL), and 10 mg / mL methionine (100 μL) in 0.5 M sodium acetate solution (800 μL) was freshly prepared prior to radiosynthesis. The reaction mixture was then transferred into the reactor of a MultiSyn radiochemistry module. Then, 0.05 M HCl (4 mL) was used to radiolabel the ITG. 68 Ge / 68Gallium-68 was delivered to the reaction vessel by performing an elution of the Ga generator. The reaction mixture was heated at 90°C for 480 seconds (pH of the reaction mixture was 4.5). The reaction mixture was then diluted with water (5 mL) and the gallium-68 labeled peptide was captured on a Strata-X SPE cartridge. The captured product was rinsed with water (5 mL), eluted with ethanol (~0.5 mL), and diluted with saline (9 mL) to obtain the compound [ 68 Ga]Ga-1~4 and [ 68 Ga]Ga-Cmp ex-2 was obtained in high radiochemical purity.

[0101] 3.2. Quality control of Gallium-68 labeled DOTA-compounds For each radiolabeled compound, the final formulation appearance, pH, radionuclide identity (half-life study), purity, and radiochemical identity were evaluated (Table 6). Radiochemical identity and purity were determined for compounds [ 68 Ga]Ga-1~4 and [ 68 The radiochemical identity of each gallium-68 labeled compound was assessed by radio-HPLC analysis of [Ga]Ga-Cmp ex-2. The radiochemical identity was assessed by radio-HPLC analysis of each gallium-68 labeled compound and its respective non-radioactive reference standard ( nat The radiochemical purity was determined by integrating all observed radioactive peaks and comparing their relative area percentages.

[0102] [Table 7]

[0103] Example 4. LogD 7.4 Measurement and serum protein binding assay Radiolabeled compound [ 68 Ga]Ga-1~4 and [ 68 Ga]Ga-Cmp ex-2 partition coefficient (LogD 7.4 ) is the aqueous phase (phosphate buffer pH 7.4The partition coefficients (LogD) were determined by measuring the distribution of radioactivity in the organic phase (n-octanol) and the organic phase (n-octanol). To determine the partition coefficients (LogD), Ga-68 radiolabeled compounds were diluted to 20 pmol / mL in PBS (pH 7.4) and an equal volume of n-octanol was added. The mixture was vortexed vigorously for 10 cycles of 1 min at room temperature and then centrifuged for 6 min to separate the two phases. Equal volumes from each phase were dispensed into individual counter tubes and counted using a Perkin Elmer 2480 Wizard2 TM The analysis was performed for 1 min using a gamma counter (PerkinElmer, Massachusetts, USA). The partition coefficient was calculated using the following formula:

number

[0104] Ga-68 labeled compound [ 68 Ga]Ga-1~4 and [ 68 Ga]Ga-Cmp ex-2 is LogD 7.4 The LogD values ​​were low, ranging from -4.1 to -2.5, indicating high hydrophilicity (Table 7). 7.4 This is consistent with other peptide-based theranostics that have been successful in clinical studies, resulting in rapid renal excretion and low background uptake. Furthermore, metabolic stability and bioavailability are regulated in part by serum protein binding, reduced glomerular filtration and enzymatic degradation, and increased retention in tumor degradation (Smith, D., Di, L. & Kerns, E., Nat Rev Drug Discov. 2010, 9, 929-939). Compound [ 68 Ga]Ga-1-4 showed moderate to high plasma protein binding (59-84%).

[0105] [Table 8]

[0106] Example 5. Circular dichroism Circular dichroism (CD) measurements were acquired on a Chirascan-plus spectropolarimeter (Applied photophysics, UK). Where applicable, samples were prepared from diluted NMR samples. CD experiments were performed in triplicate from 195 nm to 260 nm with a 1 nm step size, 1 nm bandwidth, 1 s time per point, and a 1 mm quartz cell (Starna, UK). Signals were recorded in millidegrees, set to zero at 260 nm, and normalized to obtain units of mean residue ellipticity (MRE) according to the following formula:

number

[0107] Peptides A-F, compounds 1-4, and comparative examples 1 and 2 were analyzed by CD spectroscopy to examine changes in secondary structure (Figure 3). Peptides C and D showed a minimum at about 228 nm in water, indicating an alpha-helical structure (determined in DPC micelles). The overall alignment of comparative example 1 and peptides A and B in water indicates a random coil structure.

[0108] The CD spectra of the remaining peptides and compounds showed two maxima at 200 nm and 230 nm, and a minimum at approximately 220-227 nm.

[0109] Example 6. In vitro metabolic assay Drug detoxification, drug metabolism, and metabolic stability are important determinants of the efficacy of theranostic candidates in the clinical setting. To assess metabolic stability, radiolabeled compounds [ 68 Ga]Ga-1 to 4 and Comparative Example 2 ([ 68[Ga]Ga-Cmp ex-2) was validated by incubating in either human serum, mouse liver or kidney homogenates, and HEPES buffer as a negative control. Duplicate samples (750–900 pmol / mL) were incubated at 37 °C with pooled human serum, 15% mouse kidney homogenate, 30% mouse liver homogenate, or 20 mM HEPES (pH 7.3, control). Human blood was collected and serum was prepared using SSTII Advance Vacutainer tubes according to the manufacturer's instructions (Becton Dickinson, NJ, USA). Mouse kidney and liver homogenates were prepared by washing the tissue in ice-cold HEPES buffer (pH 7.3) and placing it into pre-cooled tubes containing HEPES buffer and 2.38 mm metal beads (Mo-Bio Laboratories, Hilden, Germany). Tissues were homogenized using a PowerLyzer24 homogenizer (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Ga-68 radiolabeled peptides were incubated with the various lysates at 37°C and samples were collected after 15, 30, 60 and 90 min. Reactions were terminated by adding an equal volume of acetonitrile and centrifugation at 15,000 rpm for 2 min. Supernatants were diluted in water and then analyzed by HPLC using a Kinetex C18 XB column (5 μm 4.6 × 150 mm) eluted at 1.5 mL / min with a gradient of 20-60% MeCN in water with 0.1% formic acid over 7 min.

[0110] The experiments showed that the tested compounds remained intact when incubated with human serum at 37° C. for 90 min (FIG. 4a). 68 Ga]Ga-1 and [ 68 Both Ga]Ga-3 underwent significant metabolism in liver and kidney homogenates during the course of the experiment (Figures 4b and 4c). 68 [Ga]Ga-3 was particularly susceptible to degradation, with approximately 35% of the peptides intact in liver microsomes and only 20% of the peptides intact in kidney homogenates after 90 min. 68Ga]Ga-1 showed a more stable metabolic profile, with 85% of the peptides intact and 75% of the parent peptide intact when incubated with liver microsomes at 90 min. 68 Ga]Ga-2, [ 68 Ga]Ga-4, and [ 68 Ga]Ga Cmp ex-2 was extremely stable in liver and kidney homogenates, with no observable metabolites detected at 90 min (Figure 4).

[0111] The metabolically labile nature of the terminal Phe residue present in compound 3 has been previously shown by Corlett et al. (Corlett, A. et al., J. Med. Chem 2021, 64, 4841-4856). The results presented herein remain consistent with those previously examined, with compound 3 showing significant degradation at 90 minutes. Further analysis of the metabolites confirmed the cleavage site to be at position 13, the terminal Phe residue. Enzymatic cleavage at the Phe residue can be prevented by replacing the native Phe moiety with a non-natural N-methylated Phe, as shown in compound 2. The incorporation of N-methylated residues is a known strategy to increase protease resistance. This was again shown in compound 1 and its N-methylated variant at position 13, compound 4. Although not as susceptible to metabolic degradation as compound 3, N-methylation of the terminal naphthylalanine residue prevents enzymatic degradation of the peptide, leaving 100% of the parent peptide intact.

[0112] Example 7. Small Animal PET Imaging and Biodistribution Studies Compound [ 68 Ga]Ga-1~4 and [ 68 The diagnostic efficacy of [Ga]Ga-Cmp ex-2 was evaluated using static small animal PET imaging and by quantifying biodistribution studies. Female BALB / c nu / nu mice (8–10 weeks old) were administered 3 × 10 6 A431CCK2R cells were inoculated subcutaneously into the right flank. Mice were weighed and tumors were measured twice weekly using electronic calipers to determine tumor volume (mm 3) was calculated as length × width × height × π / 6. Mice were assigned to imaging and / or biodistribution groups (tumor volume: 50–500 mm 3 Each Ga-68 labeled tracer (4–5 MBq, 100 μL, 13.7–14.2 pmol) was mixed with vehicle solution (75 μL, DMSO:Tween80:water = 2:2:6 (v / v / v)) and then administered intravenously via tail vein injection to six mice. Three mice were euthanized for biodistribution 1 h after injection, and selected tissues were excised, weighed, and counted using a Capintec (Captus 4000e) gamma counter. Three mice were anesthetized using 1.5% isoflurane and imaged 1 and 2 h later on a G8 small animal PET / CT scanner (Perkin Elmer / Sofie Biosciences). A 10-min static PET scan was acquired, followed by a CT scan immediately after. PET images were acquired using the G8 acquisition engine software and reconstructed using a 3D maximum likelihood-expectation maximization (ML-EM) algorithm. PET images were analyzed using VivoQuant software (version 3.0, invicro Imaging Services and Software) to quantify the maximum standardized uptake value (SUVmax) in the region of interest. After imaging the 2-h time point, mice were excised for biodistribution analysis as described above.

[0113] To block CCK2R binding, we used N-[(3R)-2,3-dihydro-1-[2-(2-methylphenyl)-2-oxoethyl]-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl]-N'-(3-methylphenyl)-urea (YM022; Sigma Aldrich) solution. YM022 (75 μL of 1 mg / mL solution consisting of DMSO:Tween80:water = 2:2:6 (v / v / v)) was added to each Ga-68 labeled tracer (4-5 MBq, 100 μL, 13.7-14.2 pmol), and this mixture (total 175 μL) was administered intravenously by tail vein injection to three mice, and organs were removed for biodistribution analysis at 1 h after injection as described above. All animal experiments were approved by the Peter MacCallum Cancer Centre Animal Experimentation Ethics Committee and were performed in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes, 8th edition (2013).

[0114] Compound [ 68 Imaging and biodistribution properties of [Ga]Ga-1-4 68 Ga]Ga-Cmp ex-2 (Fig. 5). 68 Compound [Ga]Ga-Cmp ex-2 showed the best tumor uptake. However, to the detriment of this compound, it also showed the highest kidney uptake. 68 Ga]Ga-1~4 is [ 68 The renal uptake was approximately half or less than half that of the compound [Ga]Ga-Cmp ex-2. 68 Ga]Ga-4 had the best tumor-to-kidney ratio. All compounds tested had low biodistribution in blood, lung, heart, liver, and spleen.

Claims

1. The following formula (I): 【Chemistry 1】 (In the formula, R １ is the side chain of an amino acid selected from phenylalanine, 3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine, or tyrosine, the phenylalanine residue containing one or more halo, -NH ２ , Cyano, C. １－６ Alkyl, haloC １－６ optionally substituted with alkyl, or acetyl; R ２ is H or C １ -C ３ alkyl; A is a radionuclide binding ligand; -L- is the amino acid sequence: -D-Glu-D-Glu-D-Ala-D-Glu-D-Glu-D-Glu-. or a pharma- ceutically acceptable salt thereof.

2. The following formula (Ia): 【Chemistry 2】 (In the formula, R ２ is as defined in claim 1. The compound according to claim 1, wherein the compound is represented by the formula:

3. The following formula (Ib): 【Chemistry 3】 (In the formula, R ２ is as defined in claim 1; Each R ３ are independently halo, -NH ２ , cyano, -OH, C １－６ Alkyl, haloC １－６ alkyl, or acetyl; n is 0 to 5. The compound according to claim 1, wherein the compound is represented by the formula:

4. 4. The compound of claim 3, or a pharma- ceutically acceptable salt thereof, wherein n is 0.

5. R ３ The compound according to claim 3, wherein n is F and n is 1 to 3, or a pharma- ceutically acceptable salt thereof.

6. 6. The compound of claim 5, or a pharma- ceutically acceptable salt thereof, wherein n is 3.

7. 6. The compound of claim 5, or a pharma- ceutically acceptable salt thereof, wherein n is 1.

8. R ３ Ha-NH ２ and n is 1; or a pharma- ceutically acceptable salt thereof.

9. The following formula (Ic): 【Chemistry 4】 (In the formula, R ２ is as defined in claim 1. The compound according to claim 1, wherein the compound is represented by the formula:

10. R ２ The compound according to any one of claims 1 to 9, or a pharma- ceutically acceptable salt thereof, wherein is methyl.

11. A is DOTA, DOTA-NHS-ester, p-SCN-Bn-DOTA, DOTAGA, DOTAGA-anhydride, CB-DO2A, TCMC, p-SCN-Bn-TCMC, 3p-C-DEPA, 3p-C-DEPA-NCS, p-NH ２ -Bn-Oxo-DO3A, TETA, BAT, p-NH ２ -Bn-TE3A, C-TETA, CB-TE2A, CB-TE1A1P, CB-TE2P, MM-TE2A, DM-TE2A, TE2A, Diamsa r, SarAr, AmBaSar, BaBaSar, NOTA, p-SCN-Bn-NOTA, NODASA, NODAGA, NETA, NETA-Mono Amide, C-NE3TA-NCS, C-NETA-NCS, 3p-C-NETA, TACN-TM, DTPA, p-SCN-Bn-1B-DTPA, p- SCN-Bn-1B4M-DTPA, CHX-A''-DTPA, p-SCN-Bn-CHX-A''-DTPA, TRAP, AAZTA, NOPO, H ２ Dedpa, H ４ octapa, H ２ azapa, H ５ decapa, p-SCN-Bn-H ４ octapa, HBED, HBED-CC, (HBED-CC) TFP, SHBED, BPCA, CP256, PCTA, p-SCN-Bn-PCTA, DFO, H ６ 11. The compound according to any one of claims 1 to 10, which is a radionuclide binding ligand selected from phospha, p-SCN-Bn-DFO, HEHA, p-SCN-Bn-HEHA, PEPA, p-SCN-Bn-PEPA, Crown, MACROPA, MACROPA-NCS, pypa, py4pa, noneunpa, DOTAM, and derivatives thereof, or a pharma- ceutically acceptable salt thereof.

12. 12. The compound according to any one of claims 1 to 11, or a pharma- ceutically acceptable salt thereof, wherein A is DOTA.

13. 13. The compound of any one of claims 1 to 12, or a pharma- ceutically acceptable salt thereof, further comprising a radionuclide complexed to said radionuclide binding ligand.

14. 14. A pharmaceutical composition comprising a compound of claim 13 or a pharma- ceutically acceptable salt thereof.

15. 14. A method for identifying a CCK-2R positive cancer in a subject, comprising administering to the subject an effective amount of a compound of claim 13 or a pharma- ceutically acceptable salt thereof and detecting the radionuclide.

16. 16. The method of claim 15, wherein the radionuclide is detected by positron emission tomography (PET).

17. 17. The method of claim 15 or 16, wherein the radionuclide is selected from carbon-11, nitrogen-13, oxygen-15, fluorine-18, scandium-44, copper-64, gallium-67, gallium-68, yttrium-86, zirconium-89, technetium-99m, indium-111, iodine-124, iodine-125, and terbium-152.

18. 14. A method of treating a CCK-2R positive cancer in a subject in need thereof, comprising administering to the subject an effective amount of the compound of claim 13 or a pharma- ceutically acceptable salt thereof.

19. 19. The method of claim 18, wherein the CCK-2R positive cancer is selected from medullary thyroid cancer, somatostatin-2R negative neuroendocrine tumors, stromal ovarian cancer, or small cell lung cancer.

20. 20. The method of claim 18 or 19, wherein the radionuclide is selected from copper-67, yttrium-90, iodine-131, terbium-149, terbium-161, lutetium-177, rhenium-186, rhenium-188, bismuth-212, bismuth-213, astatine-211, lead-212, and actinium-225.

21. 14. The compound or a pharma- ceutically acceptable salt thereof according to claim 13 for use in a method for identifying a CCK-2R positive cancer in a subject, said method comprising administering to said subject an effective amount of said compound and detecting said radionuclide.

22. 14. The compound of claim 13, or a pharma- ceutically acceptable salt thereof, for use in the treatment of a CCK-2R positive cancer in a subject in need thereof.

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