[161tb] - based radiopeptides

Abstract

To provide a radionuclide therapy which decays by emission of medium energy β - particles and emits γ - rays suitable for imaging purposes. Furthermore, the combination with appropriate targeting agents provides a coordinately acting radionuclide therapy.SOLUTION: A radiopeptide is provided comprising (a) a radionuclide that is terbium -161, (b) a chelating agent that coordinates terbium -161, and (c) a peptide or peptide analog that is a somatostatin receptor (SSTR) antagonist. The radioactive peptides are suitable for use in the treatment of tumor diseases.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] Neuroendocrine tumors (NENs) are a group of biologically and clinically heterogeneous malignant tumors that arise primarily in the diffuse neuroendocrine system of the gastropancreatic or bronchopulmonary ducts [1,2]. Peptide receptor radionuclide therapy (PRRT) using radiolabeled somatostatin (SST) analogs has been employed for nearly 30 years to treat low- to intermediate-grade NENs that express somatostatin receptors (SSTRs) [1,3,4]. Initially used [ 111 [In]In-octreotide was effective in palliation of symptoms but rarely induced objective radiographic responses in NEN. The short tissue penetration range of Auger electrons (<1 μm) was insufficient for effective cancer treatment, and dose escalation was not possible due to the substantial gamma radiation emitted by indium-111 (Eγ = 171 keV, I = 91%; 245 keV, I = 94%) [5,6]. Yttrium-90 (Eβ 平均 = 934 keV, T 1 / 2 = 2.67d) has been more successful in PRRT applications [7]. However, the high-energy β - Over time, it was recognized that particles (up to 10 mm in tissue range) are undesirable due to their potential for nephrotoxicity [8,9]. - The particle-emitting species lutetium-177 (Eβ 平均 =134keV,T 1 / 2 = 6.65d; tissue range < 2mm

[10] ) has emerged as the preferred radionuclide for PRRT [11,12]. 177 Lu-labeled somatostatin analogs have been shown to be effective in the treatment of NEN with a generally favorable safety profile [8]. The gamma-ray emission of lutetium-177 has been used in gamma scintigraphy and SPECT imaging. Currently, lutetium-177 is used in [ 177 Lu]Lu-DOTATATE (Lutathera™) [13,14] or [ 177 Lu]Lu-DOTATOC

[15] is the most commonly used radiometal for PRRT.

[0002] Recently, α-particle emitting species (LET = 50–230 keV / μm) have been proposed for targeted radionuclide therapy

[16] . In the case of NEN, [ 213 Bi]Bi-DOTATOC / DOTATATE and [ 225 Ac]Ac-DOTATOC / DOTATATE have shown promising results in preclinical studies [17-20]. 213 Bi]Bi-DOTATOC, 90 Y]Y- / [ 177 It was used to treat patients (n=7) with progressive neuroendocrine liver metastases refractory to treatment with [Lu]Lu-DOTATOC

[21] . 225 Ac]Ac-DOTATATE 177 Actinium-225 was investigated in a previous study in 32 patients who had stable or progressive disease after partial remission or stable disease with [Lu]Lu-DOTATATE therapy

[22] . However, there are many uncertainties regarding the application of actinium-225, among which its inability to be used for imaging and, finally, its challenging manufacturing scenario that generally makes it difficult to obtain [23-25]. The complex decay scheme of actinium-225 and the resulting increased risk of unwanted side effects are due to the high cost of rapidly internalizing radioligands (e.g., [Lu]Lu-DOTATATE). 225 This limits its application to other radiopharmaceuticals (e.g., Ac, Ac-based PSMA radioligands) and precludes its use with cell surface-localized radiopharmaceuticals such as somatostatin antagonists.

[0003] The purpose of this study is to 177 As explained for Lu, the medium-energy β - The goal was to identify radionuclide therapies that decay by releasing particles and emit gamma rays suitable for imaging purposes, and to enable synergistic action of the radionuclide therapies in combination with appropriate targeting agents.

[0004] The solution to that object is defined by the subject matter of claim 1. The invention is based on the discovery that the combination of terbium-161 with an antagonistic somatostatin analogue results in enhanced PRRT. Terbium-161 exhibits decay properties for targeted radionuclide therapy similar to those of lutetium-177. It is a medium-energy beta - decays by the emission of particles (E β Average=154keV;T 1 / 2 = 6.9d

[27] ) and emits gamma rays suitable for imaging (Eγ = 48.9 keV, I = 17%, 74.6 keV, I = 10%)

[28] . Advantageously, terbium-161 co-emits a significant number of Auger and conversion electrons (due to its high linear energy transfer (LET) of 4-26 keV / μm) for tumor therapy, particularly when targeting single cancer cells [26, 29]. Terbium-161 has been found to be advantageously combined with antagonistic somatostatin (SST) analogs as targeting agents, especially when the targeting agent is found to preferentially localize to the cell surface rather than be internalized. Such combinations have been shown to benefit from the effects of co-emitted conversion and Auger electrons (high-LET electrons).

[0005] (Details of the invention) According to a first aspect, the present invention solves the object by providing a radioactive peptide comprising (a) a radioisotope / radionuclide that is terbium-161, (b) a chelator of terbium-161, and (c) a peptide or peptide analog that is a somatostatin receptor (sstr) antagonist. Thus, the term "radiopeptide" is understood to mean that the claimed entity comprises a radioisotope and a peptide component. The terms "radionuclide" and "radioisotope" are used interchangeably herein.

[0006] The present invention is based on the identification of terbium-161 as a trivalent radioactive metal isotope (i.e., a radioisotope), which, in combination with other components of the radioactive peptides of the present invention, can be used to produce β( -) particle (tumor) therapy. Its decay properties (T 1 / 2 =6.89d) is 177 The decay properties of terbium-161 are similar to those of Lu. Therefore, the decay properties of terbium-161 are suitable for PRRT. Furthermore, the co-emission of Auger electrons allows for the - / Auger electron combination therapy (~12e - / Decay: Auger / Conversion electron) is possible. Experimental evidence of the present invention is 161 Tb-labeled somatostatin analogs 161 Tb and 177 Although Lu and Lu are both very similar trivalent radioactive lanthanides, 177 It was confirmed that the 111L-labeled somatostatin analog was more effective than the 111L-labeled somatostatin analog.

[0007] 161 Tb production is 160 Gd(n,γ) 161 Gd → 161 This is advantageously done by utilizing Tb nuclear reactions. 160 The Gd target is irradiated for 2-3 weeks at a spallation neutron source, for example at the Paul Scherrer Institute (Switzerland), or for 1 week at a high neutron flux reactor, such as the Laue-Langevin Institute in France or the reactor at Necsa (Pelindaba, South Africa). The product is separated from the Gd target material using ion exchange (resin) chromatography, and the final product can be obtained in a small amount of dilute hydrochloric acid (for example, 1 ml or less). The amount obtained can exceed 10 GBq. The purity of the final product is usually greater than 98% or 99% ( 161 The radionuclide terbium-161 of the radioactive peptide may be specifically provided as non-carrier added terbium-161 (nca terbium-161), which defines that no non-radioactive terbium is present in the final product.

[0008] Terbium-161 is typically provided in a stable coordination state with a chelating agent according to the present invention. A chelating agent is a molecule that binds or coordinates to the central radionuclide atom via the chelating agent atom. The chelating agent is typically a bifunctional molecule that (i) forms a complex with the radionuclide (a) and (ii) links the chelating agent (b) to the peptide component (c) of the radioactive peptide of the present invention. In one embodiment, the chelating agent has an open-chain structure, such as DTPA (diethylenetriaminepentaacetic acid) or a DTPA derivative. In another preferred embodiment, the chelating agent has a cyclic structure. In particular, the chelating agent may be a macrocyclic chelating agent. Furthermore, the chelating agent may be a multidentate chelating agent, such as a tetradentate chelating agent.

[0009] The ring structure of the (macro)cyclic chelating agent may contain a heteroatom, such as O or N, particularly N, for coordinating the radioisotope. In one embodiment, the macrocyclic chelating agent is based on a carbocyclic structure containing 4 to 6, particularly 4, heteroatoms, more particularly 4 N atoms, which serve as atoms for bonding or coordinating to the central radionuclide. Advantageously, the (macro)cyclic base structure of the chelating agent of the radiopeptide of the present invention is derived from cyclen (1,4,7,10-tetraazacyclododecane), which corresponds to a 12-membered tetraaza ring system. The tetraaza ring system may be further modified by substituents, e.g., those that bond or coordinate to the central radionuclide. This may involve, for example, two substituents establishing an octahedral coordination geometry of the complex in addition to the coordination by the four N atoms of the cyclen ring system. In one or more of the four, e.g., all four, the tetraaza ring system of cyclen may be modified by replacing the NH center with an NX group having an X other than H. X may be CHCOH or other substituents having a carboxy functionality. In this regard, the chelator may be DOTAGA(-tetra)(2-[1,4,7,10-tetraazacyclododecane-4,7,10-tris(t-butylacetic acid)]-pentaedionic acid-1t-butyl ester), AAZTA (1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-methylperhydro-1,4-diazepine), DOTA (dodecanetetraacetic acid), or a DOTAGA, AAZTA, or DOTA derivative, e.g., CyAAZTA, DATA, or AAZ3A. DOTA derivatives may exhibit substitution patterns on the cyclen ring system other than the DOTA substitution pattern. Thus, DOTA derivatives may exhibit a different substitution pattern at at least one of the heteroatoms of the ring system than acetic acid (which characterizes DOTA).

[0010] The DOTA derivative employed by the present invention may be selected from the group consisting of: DO3A, DO2A, HP-DO3A, BT-DO3A, PCTA, DO3AP, DO2A2P, DOA3P, DOTP, DOTPMB, DOTAMAE, DOTMA, TCE-DOTA, and DOTAMAP.

[0011] DOTA or one of its derivatives may be a preferred chelating agent for trivalent radiometals, such as terbium-161. The radiopeptide chelating agents of the present invention, particularly DOTA or DOTA derivatives, can be advantageously complexed with terbium-161 by complexation at elevated temperatures (e.g., 70°C to 95°C), application of microwave irradiation, use of solvent mixtures, or a combination thereof to facilitate complex formation. In contrast, complexation at room temperature proceeds rather slowly without the application of more elaborate conditions. Once the radioisotope enters the cavity of DOTA or a DOTA derivative, it remains stable there at room temperature. Therefore, the complex can be stored at room temperature without loss of activity due to dissociation of the terbium-161 radioisotope from the chelating agent.

[0012] More specifically, DOTA is used as a chelating agent for the radioactive peptides of the present invention.

[0013] In one embodiment of the present invention, the radioactive peptide is a conjugate compound characterized by covalently coupling (c) a somatostatin receptor (SSTR) antagonist to (b) a chelating agent. The coupling can involve the N-terminal amino group of the peptidic somatostatin receptor (SSTR) antagonist. The peptidic somatostatin receptor (SSTR) antagonist and the chelating agent can thereby form, for example, an amide bond, particularly by reaction of the N-terminal amino group with a carboxyl functional group of the chelating agent, such as a carboxyl functional group of a substituent of the cyclen ring system. In this regard, for example, the carboxyl functional group of the chelating agent, such as its N-CHCOH group (where N is the nitrogen atom of the cyclen ring system), can react with, for example, the N-terminal amino group of the peptidic component (c) to form a conjugate compound. The following formulas illustrate embodiments of the radioactive peptides of the present invention in which various chelating agents (DOTA or DOTA derivatives) are linked to the peptidic component (c) (not shown in the formula below) of the radioactive peptides of the present invention via an amide bond: [ka] The radioactive peptide of the present invention is further characterized by its component (c). Component (c) is a somatostatin receptor antagonist. Targeting somatostatin receptors (sstr) with sstr antagonists is a valuable therapeutic option, for example, for the treatment of neuroendocrine neoplasms. The sstr antagonist (c) advantageously forms a conjugate compound with a chelator (b), as described above. The sstr antagonist defined for component (c) of the present invention can bind to any sstr subtype. However, more specifically, it preferentially or exclusively binds to SSTR-2 (sst2). Somatostatin receptor 2 is a G protein-coupled receptor (GPCR) typically expressed in neuroendocrine tumors. SSTR2 is the receptor for somatostatin (SST)-14 and (SST)-28, respectively. Overexpression of SSTR2 in tumors can be used to selectively deliver radioactive peptides to tumors. Upon binding to tumor cells, the radioactivity of the radioisotope, or rather the particle emission of the radionuclide (a), can damage and ultimately destroy the tumor cells. Thus, peptide component (c) may be a particularly selective SSTR-2 (sst2) antagonist.

[0014] Therefore, somatostatin receptor (SSTR) antagonists enable the radioactive peptides of the present invention to bind to tumor cells. Therefore, selecting an SSTR antagonist as component (c) advantageously supports the accumulation of the radioactive peptides of the present invention on tumor cells, thereby supporting a much higher accumulation of radioactivity on the cell membrane of tumor cells. This is because the SSTR antagonist recognizes far more binding sites on the SSTR anchored to the cell membrane than the radiolabeled SSTR agonist. This allows the radiolabeled SSTR antagonist to express a much higher radioactivity on tumor cells than its agonistic counterpart.

[0015] Advantageously, radioactive peptides containing the antagonist component (c) as tumor cell conjugates (peptides) are not significantly internalized by tumor cells due to their low internalization rate, but remain on the surface of the tumor cells. Therefore, less than 30%, less than 25%, less than 20%, or less than 10% of the administered radioactive peptide can be internalized by tumor cells under in vivo or in vitro conditions. Therefore, component (c) may advantageously represent an antagonistic somatostatin analog (or SSTR antagonist) that targets sstr, more specifically sstr2, but is not internalized by the target tumor cells or is internalized only in small amounts (e.g., less than 30% or less than 20%). The retention of most of the radioactive peptide on the cell surface membrane upon binding to the target sstr may act therapeutically synergistically or may be related to the β-antibody catabolism by terbium-161. - / Auger electron emission may further contribute to the therapeutic effect.

[0016] Radioactive peptidic somatostatin receptor antagonists may be peptides or peptide analogs composed of natural or non-naturally occurring amino acids. The radioactive peptides of the present invention may be in the form of the free peptide or a salt, solvate, or tautomer thereof. As used herein, the term "peptide," which defines the peptidic component of a "radiopeptide," refers to any sequence of amino acids, regardless of their length, modification, or nature. Natural or non-naturally occurring amino acids may be D- or L-amino acids, i.e., in the D- or L-configuration. They are linked to each other via amide bonds. While any reactive side chain group of the somatostatin receptor antagonist may be capable of covalently binding to a chelator component to form a conjugate compound, the N-terminal free amino group of the antagonist is particularly suitable for covalent binding to the radioactive peptidic chelator component. Peptidic antagonists typically consist of at least eight natural or non-naturally occurring amino acids. More specifically, the number of amino acids is 8 to 14, 8 to 10, or 8.

[0017] In one embodiment, the antagonist as component (c) of the radioactive peptide of the present invention is provided as a cyclic peptide. The cyclic structure may be introduced by a covalent bond between two side chain moieties. In particular, the ring closure by the two side chain moieties may be achieved by two cysteine ​​residues forming a disulfide bridge with their free thiol groups. When forming a cyclic structure based on a disulfide bridge, the cysteine ​​residues may be positioned within the antagonist peptide sequence so as to be spaced apart by at least three, at least four, or at least five amino acids, more specifically, three, four, or five amino acids. In a specific embodiment, the cysteine ​​residues are spaced apart by four amino acids. The more N-terminal cysteine ​​residue involved in disulfide bridge formation may be located at position 2 or 3 of the antagonist peptide sequence. The more C-terminal cysteine ​​residue involved in disulfide bridge formation may be located at position 7 or 8 of the antagonist peptide sequence. In particular, the two disulfide bridge-forming cysteine ​​residues may be located at positions 2 and 7, respectively, of the peptide sequence of the antagonist.

[0018] The carboxy terminus of the radioactive peptide peptide antagonist of the present invention may be amidated ("-NH2"), for example, as shown in formula (I) below or in embodiments (i) to (xxv) below.

[0019] The non-naturally occurring amino acid of component (c) may specifically be selected from the group consisting of (4-amino-Phe(carbamoyl)), (Aph(Cbm), D-Agl(NMe, benzoyl), Aph(Hor), Aph(CONH-OCH), substituted Phe, and Cpa, all of which may be provided as D- or L-enantiomers. Furthermore, all naturally occurring amino acids may be used as D-enantiomers.

[0020] According to one embodiment, the somatostatin receptor antagonist as component (c) of the radiopeptide of the invention may consist of a sequence according to formula I: X1-cyclo[D-Cys-X3-X4-Lys-Thr-Cys]-D-Tyr-NH2 (Formula (I)) or a salt, solvate or tautomer thereof, wherein X1, X3 and X4 may be selected from naturally or non-naturally occurring D- or L-amino acids.

[0021] More specifically, (i) X1 can be selected from the group consisting of naturally occurring Phe, substituted Phe having one or more substitutions in the phenyl ring system, and Cpa; (ii) X3 can be selected from the group consisting of Aph(Hor), Leu, L-Agl(NMe, benzoyl), D-Agl(NMe, benzoyl), Aph(Cbm), Tyr, Aph(CONH-OCH3), Aph(CONH-OH); and / or (iii) X4 can be selected from the group consisting of D-Trp and D-Aph(Cbm) (D-4-amino-Phe(carbamoyl)). In particular, the peptidic antagonist of formula (I) can contain amino acids as defined above for X1 according to (i), X3 according to (ii), and X4 according to (iii).

[0022] When X1 is a substituted Phe residue, at least one phenyl ring substituent may be located at the ortho (o), meta (m), or para (p) position of the phenyl ring system, more specifically at the para (p) ring position. At least one substituent on the phenyl ring at X1 is typically not bulky, but may represent a sterically inconspicuous substituent such as a halogen, amino, hydroxy, or nitro group. Typically, the phenyl ring system of the substituted Phe residue may be substituted with one or two substituents, more specifically, with one substituent. When the phenyl ring system is substituted with one substituent, the substituent may be located particularly at the para position of the Phe ring system. It may be selected from, for example, a nitro group or a halogen such as Cl.

[0023] More specific embodiments according to formula (I) may be based on the following: (i) X1 may be selected from the group consisting of pNO2-Phe, pCl-Phe, and Cpa; (ii) X3 may be selected from the group consisting of Tyr, Aph(Cbm), and Aph(Hor); and / or (iii) X4 may be selected from D-Trp and D-Aph(Cbm). In particular, peptidic antagonists of formula (I) may contain amino acids as defined above for X1 according to (i), X3 according to (ii), and X4 according to (iii) as defined by this paragraph.

[0024] In more specific embodiments, particularly for formula (I), the radioactive peptides of the present invention may comprise a somatostatin receptor antagonist selected from the group consisting of LM3 ([p-Cl-Phe-cyclo[D-Cys-Tyr-D-Aph(Cbm)-Lys-Thr-Cys]-D-Tyr-NH2]), JR11 (Cpa-cyclo[D-Cys-Aph(Hor)-D-Aph(Cbm)-Lys-Thr-Cys]-D-Tyr-NH2), JR10 (p-NO2-Phe-cyclo[D-Cys-Tyr-D-Aph(Cbm)-Lys-Thr-Cys]-D-Tyr-NH2), and BASS (pNO2-Phe-cyclo[D-Cys-Tyr-D-Trp-Lys-Thr-Cys]-D-Tyr-NH2). The antagonists LM3, JR10, JR11, and BASS all act as sstr-2 selective antagonists.

[0025] According to another embodiment, the antagonist as component (c) of the radioactive peptide of the present invention may be selected from the group consisting of: (i) pNO2-Phe-cyclo[D-Cys-Tyr-D-Trp-Lys-Thr-Cys]-D-Tyr-NH2; (ii) pNO2-Phe-cyclo[D-Cys-Tyr-D-Aph(Cbm)-Lys-Thr-Cys]-D-Tyr-NH2; (iii) H2N-pNO2-Phe-cyclo[D-Cys-Tyr-D-Aph(Cbm)-Lys-Thr-Cys]-2Nal-NH2; (iv) pNO2-Phe-cyclo[D-Cys-Tyr-D-Aph(Cbm)-Lys-Thr-Cys]-2Nal-NH2; (v) pNO2-Phe-cyclo[D-Cys-Aph(Hor)-D-Aph(Cbm)-Lys-Thr-Cys]-2Nal-NH2; (vi) Cpa-cyclo[D-Cys-L-Agl(NMe.benzoyl)-D-Trp-Lys-Thr-Cys]-2Nal-NH2; (vii) Cpa-cyclo[D-Cys-D-Agl(NMe.benzoyl)-D-Trp-Lys-Thr-Cys]-2Nal-NH2; (viii) Cpa-cyclo[D-Cys-Leu-D-Trp-Lys-Thr-Cys]-2Nal-NH2; (ix) Cpa-cyclo[D-Cys-Aph(Cbm)-D-Trp-Lys-Thr-Cys]-2Nal-NH2; (x) Cbm-Cpa-cyclo[D-Cys-Aph(Cbm)-D-Trp-Lys-Thr-Cys]-2Nal-NH2; (xi) [beta]Ala-Cpa-cyclo[D-Cys-Aph(Cbm)-D-Trp-Lys-Thr-Cys]-2Nal-NH2; (xii) Cpa-cyclo[D-Cys-Aph(Cbm)-D-Trp-Lys-Thr-Cys]-2Nal-NH2; (xiii) Cpa-cyclo[D-Cys-Aph(Cbm)-D-Trp-Lys-Thr-Cys]-NH2; (xiv) Cpa-cyclo[D-Cys-Aph(Cbm)-D-Trp-Lys-Thr-Cys]-Cha-NH2; (xv) Cpa-cyclo[D-Cys-Aph(Cbm)-D-Trp-Lys-Thr-Cys]-Aph(Hor)-NH2; (xvi) Cpa-cyclo[D-Cys-Aph(Cbm)-D-Trp-Lys-Thr-Cys]-DAph(Cbm)-NH2; (xvii) Cpa-cyclo[D-Cys-Aph(Cbm)-D-Trp-Lys-Thr-Cys]-Aph(Cbm)-NH2; (xviii) Cpa-cyclo[D-Cys-Aph(Cbm)-D-Trp-Lys-Thr-Cys]-D-Aph(Cbm)-GlyOH; (xix) Cpa-cyclo[D-Cys-Aph(CONH-OCH3)-D-Trp-Lys-Thr-Cys]-2Nal-NH2; (xx) Cpa-cyclo[D-Cys-Aph(CONH-OH)-D-Trp-Lys-Thr-Cys]-2Nal-NH2; (xxi) Cpa-cyclo[D-Cys-Aph(Cbm)-5F-D-Trp-Lys-Thr-Cys]-2Nal-NH2; (xxii) Cpa-cyclo[D-Cys-Aph(Cbm)-5F-Trp-Lys-Thr-Cys]-2Nal-NH2; (xxiii) Cpa-cyclo[D-Cys-Tyr-D-Aph(Cbm)-Lys-Thr-Cys]-2Nal-NH2; (xxiv) Cpa-cyclo[D-Cys-Aph(Hor)-D-Aph(Cbm)-Lys-Thr-Cys]-2Nal-NH2; and (xxv) Cpa-cyclo[D-Cys-Aph(Hor)-D-Aph(Cbm)-Lys-Thr-Cys]-D-Tyr-NH2.

[0026] or a salt, solvate or tautomer thereof.

[0027] The radioactive peptides of the present invention can be selectively accumulated in tumor tissue in tumor patients in vivo, thereby preventing significant accumulation in tissues other than tumor tissue. Preferential accumulation in tumor tissue can be expressed by the ratio of radioactive peptide uptake in tumor cells to the uptake in other tissues, such as the kidney, liver, and blood. The tumor / blood uptake ratio characterizing the radioactive peptides of the present invention may be at least 50.0, or at least 75, or at least 100. The tumor / liver uptake ratio of the radioactive peptide may be at least 10.0 or at least 20. This ratio may be determined as described in the in vivo biodistribution study below (Section III). The tumor / kidney uptake ratio may be at least 2.0. The measurement may be performed at least 2 hours after administration of the radioactive peptide to the subject, typically 2 or 4 hours after administration of the radioactive peptide to the subject.

[0028] According to a specific embodiment of the present invention, the radioactive peptide of the present invention has the following structure: [ka] In this study, the radionuclide terbium-161 is complexed with a DOTA chelator, establishing an octahedral coordination geometry for the complex. The DOTA chelator is covalently conjugated via an amide linker to the N-terminus of the sstr2-selective antagonist LM3, whose carboxy terminus is amidated.

[0029] More generally, the peptide antagonist or peptide antagonist / chelator conjugate, or the radioactive peptide described herein above, may form solvates with water (such as a hydrate or hemihydrate) or common organic solvents. As used herein, the term "tautomer" is used in its broadest sense and includes peptides or peptide / chelator conjugates or radioactive peptides of the present invention that can exist in equilibrium between two isomeric forms. Such compounds may differ in the bond connecting two atoms or groups and the position of these atoms or groups in the compound.

[0030] The radioactive peptides of the present invention can be administered in the form of pharmaceutically or veterinarily acceptable non-toxic salts, such as acid addition salts or metal complexes with, for example, zinc, iron, calcium, barium, magnesium, aluminum, etc. Such non-toxic salts include hydrochloride, hydrobromide, sulfate, phosphate, tannate, oxalate, fumarate, gluconate, alginate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate, etc.

[0031] Another aspect of the present invention refers to a pharmaceutical composition comprising a radioactive peptide described herein and at least one pharmaceutically acceptable excipient. More specifically, the pharmaceutical composition may be a liquid formulation. It may be an aqueous solution, optionally containing a buffer system. Aqueous pharmaceutical compositions of the present invention may contain another water-miscible solvent, such as ethanol. Typically, aqueous solutions should not contain more than 10% by volume of other solvents, such as ethanol. The pharmaceutical composition may have a pH in the range of pH 3 to pH 7, more specifically in the range of pH 3.5 to pH 6 or pH 4 to pH 6.

[0032] The pharmaceutical composition according to the present invention contains 0.001 to 1 mg / ml of the radioactive peptide as defined herein, depending on the subject or disease being treated. Specifically, the concentration of the radioactive peptide may range from 0.01 to 1 or 0.5 mg / ml, or from 0.05 to 0.5 mg / ml.

[0033] The pharmaceutical composition may comprise at least one, such as one, two or three, of the following additives: gentisic acid, ethanol, acetate, NaCl and ascorbic acid / ascorbate.

[0034] In one embodiment, the pharmaceutical composition contains the antioxidant ascorbic acid / ascorbate as a stabilizer. The presence of ascorbic acid / ascorbate, which typically acts as a scavenger, can stabilize the pharmaceutical composition, thereby improving the shelf life of the pharmaceutical composition, while maintaining the pharmaceutical composition suitable for administration to humans and other mammalian subjects. Therefore, the pharmaceutical composition may contain more than about 5 mg of ascorbic acid per milliliter, or more than about 10 mg of ascorbic acid per milliliter, or more than about 20 mg of ascorbic acid per milliliter, or more than about 30 mg of ascorbic acid per milliliter, or more than about 40 mg of ascorbic acid per milliliter, or more than about 50 mg of ascorbic acid per milliliter, or more than about 100 mg of ascorbic acid per milliliter, or more than about 200 mg of ascorbic acid per milliliter. Thus, the pharmaceutical composition may contain 5-100 mg / ml ascorbic acid / ascorbic acid, or 25-500 mg / ml, or 50-200 mg / ml. Alternatively stated, the pharmaceutical composition may contain ascorbic acid / ascorbate in the range of 0.5 mM-0.5M, particularly 1 mM-100 mM or 10 mM-100 mM.

[0035] The pharmaceutical composition may exhibit a radioactivity in the range of 50-800 MBq / ml, or 100-600 MBq / ml, or 200-400 MBq / ml.

[0036] In another aspect, the present invention relates to a method for treating a disease, particularly a tumor disease. The method of treatment may comprise administering a radioactive peptide of the present invention or a composition of the present invention to a subject in need of disease treatment, particularly neoplasm or tumor treatment. The subject may be a mammal, particularly a human.

[0037] The radioactive peptides of the present invention or their non-toxic salts are typically combined with pharmaceutically or veterinarily acceptable carriers / excipients to form pharmaceutical compositions of the present invention, which may be administered to animals, including humans and other mammals, intravenously, subcutaneously, intramuscularly, transdermally, e.g., intranasally, intracerebrospinally, or orally. Specifically, the radioactive peptides of the present invention or pharmaceutical compositions of the present invention may be administered systemically, preferably intravenously, e.g., by infusion. The pharmaceutical composition may be administered intravenously to a subject over an extended period of time, e.g., 10 to 60 minutes, for example, via a prepared catheter or heparin lock line, and may be flushed with an appropriate saline and / or heparin solution. Administration may be via a Luer lock on the catheter or heparin lock line. The administration site depends on the patient's tumor / metastasis profile. A specific administration site may be a vein in the arm.

[0038] The treatment protocol depends on the tumor disease, its severity, and the course of treatment, and is usually monitored by PET / CT or SPECT / CT scans after each treatment cycle. SPECT can preferably serve as a technique for studying the distribution of radioactive peptides in the body. In one embodiment, the treatment protocol can consist of 2 to 10 treatment cycles, for example, 4, 5, 6, or 7 treatment cycles. Therefore, the treatment protocol can foresee two or more treatment cycles every 4 to 10 weeks, for example, every 5 to 8 weeks, or about every 6 weeks. In the event of disease progression, re-treatment is expected after the initial treatment cycle protocol.

[0039] Such pharmaceutical compositions designed for use in the treatment of tumors, e.g., benign or malignant tumors, can be administered in amounts sufficient to treat or control the tumor, including treating its metastases in other tissues. The radioactive peptide should be at least about 90% pure, and preferably at least about 98% pure; however, lower purities may be effective and may be used in mammals other than humans. This purity means that the intended peptide constitutes the weight percent of all homologous peptides and peptide fragments present in the composition. The required dosage will vary depending on the particular condition being treated, the severity of the condition, and the intended duration of treatment.

[0040] More specifically, the diseases to be treated with the radioactive peptides or pharmaceutical compositions of the present invention are neoplasms arising from cells of the endocrine (hormonal) system and (peripheral) nervous system. The abnormal cells of the neoplasm or tumor to be treated typically express sstr2 on their cell surface. They can occur in many different tissues, but most commonly occur in the intestine. Thus, the neoplasm may be a neuroendocrine neoplasm in the gastrointestinal tract, pancreas (e.g., foregut, midgut, hindgut, or pancreas: islet cells) or bronchopulmonary tract. It is understood that the methods of the present invention may include the treatment of neuroendocrine neoplasms and / or neuroendocrine tumor metastases, particularly liver metastases, in a wider variety of tissues. Neuroendocrine neoplasms / tumors may be benign or malignant. Their histological grade is classified as G1, G2, or G3 according to the WHO classification. Differentiated neuroendocrine neoplasms of the gastrointestinal pancreatic system classified as G1, G2, and G3 are defined as neuroendocrine tumors, while poorly differentiated neuroendocrine neoplasms of the gastrointestinal pancreatic system classified as G3 correspond to neuroendocrine carcinomas. The present invention provides methods for treating differentiated or poorly differentiated neuroendocrine tumors or carcinomas of the gastrointestinal pancreatic system, e.g., pancreatic tumors or carcinomas.

[0041] Various other organs may be affected by neuroendocrine neoplasms / tumors and their metastases. Therefore, pulmonary neuroendocrine tumors, e.g., pulmonary or bronchial neuroendocrine carcinomas, including small cell lung cancer, can be treated according to the present invention. Adrenal tumors, particularly adrenal medulla tumors, such as pheochromocytoma, can also be treated according to the present invention. Peripheral nervous system tumors, e.g., paraganglioma, schwannoma, or neuroblastoma, can also be targeted for treatment according to the present invention. Tumors of the thymus, thyroid, or pituitary gland, such as neuroendocrine thyroid tumors, particularly medullary thyroid carcinoma, can also be treated. Breast cancer or genitourinary cancer can also be treated according to the present invention. Prostate cancer, particularly prostate cancer of neuroendocrine origin, can also be treated according to the present invention. The present invention can also be applied to the treatment of meningiomas, renal cell carcinomas, and non-Hodgkin's lymphoma.

[0042] Advantageously, the therapeutic methods described herein may be preceded by an imaging step (e.g., PET / CT scan and / or MRI) that allows for the determination of the uptake of the tracer dose of the radioactive peptide of the present invention by the aberrant cells of the tumor neoplasm, for example by determining the uptake value (SUV). This allows for the selection of tumor patients with high expression of somatostatin receptors, e.g., somatostatin receptor subtype 2 (sstr2), for successful treatment with the approach of the present invention. Alternatively or additionally, tumor cells may be analyzed by molecular biological or in vitro diagnostic methods to determine sstr-positive or sstr2-positive cells, i.e., the level of sstr expression on the tumor cell surface.

[0043] This therapeutic approach is highly effective in treating tumors while minimizing side effects on non-tumor tissues. The radioactive peptide accumulates at a rapid rate at the tumor site in the body. The radiation from the radioactive peptide is essentially absorbed at the tumor site or excreted in the urine, leaving other tissues with no radioisotope (β -) are essentially unaffected by particle radiation. The terbium-161 labeled antagonistic somatostatin analog binds to or enters, and preferably binds to, tumor cells damaged by the radiation of the radioisotope. The tumor cells then undergo apoptosis or necrosis, resulting in a reduction in the tumor burden in the subject as a result of treatment.

[0044] In a specific embodiment, the therapeutic methods of the present invention are applied to patients suffering from a primary endocrine neoplasm / tumor and, potentially, its metastases.

[0045] In another embodiment, the radioactive peptide-based therapy of the present invention can be performed in conjunction with a concomitant treatment. In particular, nephroprotection by concomitant injection of a solution containing an amino acid such as Arg or Lys can be envisioned. Concomitant treatment with such an amino acid solution can be initiated, for example, 3 hours to 0.2 hours, typically about 1 hour to about 1.5 hours, before the end of administration of the radioactive peptide, and can continue for an additional 1 hour to 5 hours after the end of administration of the radioactive peptide. Furthermore, patients may be pretreated with antiemetics and / or corticosteroids before a treatment cycle with the radioactive peptide of the present invention.

[0046] In a specific embodiment, the present invention provides a method for treating a cancer cell comprising: 90 Y-DOTATOC, 177 Lu-DOTATOC, ( 177 Lu)-oxodotreotide (Lutathera®); 177 Lu-DOTATATE and other radiolabeled somatosteine ​​analogs, especially 177 Lu-DOTATOC or ( 177 Lu)-oxodotreotide (Lutathera®); more particularly ( 177 Lu)-oxodotreotide (Lutathera®); 177 It allows for the treatment of neuroendocrine neoplasms / tumors and other SSTR2-positive neoplasms that are stable or refractory to treatment with Lu-DOTATATE. [ka] According to another aspect, the present invention provides a kit or kit of parts.

[0047] The labeling reaction with a radionuclide may need to be performed in a clinical clinic or laboratory prior to treatment. In such cases, the various reaction components may be provided to the user in the form of a "kit" or "kits." Thus, a kit for preparing a pharmaceutical composition according to the present invention may include: (i) a somatostatin receptor (SSTR) antagonist described herein conjugated to a radionuclide chelator; (ii) an inert pharmaceutically acceptable carrier and / or formulation agent with optional adjuvants; (iii) a solution of a salt of the radioactive metal isotope terbium-161, typically in its salt form; and, optionally, (iv) instructions for use with a prescription for reacting the components present in the kit. Components (i), (ii), (iii), and optionally (iv) are typically provided as separate parts of the kit. Alternatively, component (iii) may be provided in a first kit, and components (i) and (ii) may be provided as parts of a separate second kit. Thus, the first and second kits are delivered to the site of use as separate entities, allowing for "on-time" provision of the radionuclide for further formulation and subsequent therapeutic use.

[0048] The somatostatin antagonist (peptide component (c) of the radioactive peptide) can be conjugated by reaction with a chelator (component (b) of the radioactive peptide), as defined herein. The resulting peptide / chelator conjugate provides an advantageous entity for stable complexation of the radioisotope terbium-161 (component (a) of the radioactive peptide) at the site of use in a convenient manner. The peptide / chelator conjugate can be provided in a dried form, e.g., in the form of its salt, or more typically, in a solution, e.g., a buffered or unbuffered aqueous solution, as part of a kit. If provided in a dried form, it may be provided in a lyophilized form, and the lyophilized conjugate must be dissolved in a solution at the site of use. Due to the nature of the injectable solution, sterility is desirable. If the components are in a dry state, the user preferably uses sterile saline, optionally buffered, as a solvent.

[0049] When the radionuclide / isotope is present in a kit containing all parts, or (iii) in a separate kit containing the radionuclide as the only chemical entity, the radionuclide or its salt is typically provided in an acidic aqueous solution, e.g., hydrochloric acid, exhibiting a pH of less than 2 or less than 1. Since the radionuclide is provided as a separate kit or separate part of the kit, it must be prepared so that it can be bound to the peptide / chelator conjugate by a complexation reaction. Advantageously, both a solution containing the peptide / chelator conjugate and a solution containing the radionuclide are combined. The solution containing the peptide / chelator conjugate may advantageously contain a buffer, such as acetate, that buffers the pH in the acidic range, allowing the complexation reaction to occur under appropriate acidic conditions. To prevent precipitation of the radiometal (e.g., by the formation of a hydroxy salt), the complexation reaction should not proceed under alkaline conditions. Advantageously, the complexation reaction is carried out under conditions in which the equilibrium is shifted in favor of complexation, for example by heating a mixed solution of the radionuclide and the peptide / chelator conjugate, for example to a temperature close to the boiling point of the solution.

[0050] Furthermore, the resulting product may be finally reformulated with the addition of a stabilizing additive, whereby the above-described terbium-161 complexed peptide / chelator conjugate may be stabilized with a suitable stabilizer, such as ethanol, ascorbic acid, gentisic acid, or salts of these acids.

[0051] [Brief description of the drawing] Figure 1. Chemical structures of somatostatin (SST) analogs: (a) DOTATOC (a somatostatin receptor (SSTR) agonist) [15,41]; (b) DOTA-LM3 (a SSTR antagonist)

[42] . (Figure 2) 161 Tb- and 177 Representative HPLC chromatograms of Lu-labeled peptides. (a) [ 161 (b) [Tb]Tb-DOTATOC; 177 Lu]Lu-DOTATOC;(c)[ 161 Tb]Tb-DOTA-LM3 ) ;(d)[ 177 Lu]Lu-DOTA-LM3. Traces of unreacted terbium-161 or lutetium-177 appeared with a retention time of 2.5 min

[44] . (Figure 3) Bar graph showing the percentage of intact radioactive peptide examined 1, 4, and 24 hours after preparation and dilution in saline (10 MBq / 250 μL). (a) Intact [ 161 Tb]Tb-DOTATOC and [ 177 ] Lu-Lu-DOTATOC; (b) the ratio of intact [ 161 Tb]Tb-DOTA-LM3 and [ 177 ]Ratio of Lu-Lu-DOTA-LM3. (Figure 4) In vitro tumor cell uptake and internalization of radioactive peptides after incubation of AR42J tumor cells for 0.5, 2, and 4 hours. (a) [ 161 Tb]Tb-DOTATOC and [ 177 (b) [Lu]Lu-DOTATOC; 161 Tb]Tb-DOTATOC and [177 (c) [Lu]Lu-DOTATOC plus excess peptide to block SSTR; 161 Tb]Tb-DOTA-LM3 and [ 177 Lu]Lu-DOTA-LM3;(d)[ 161 Tb]Tb-DOTA-LM3 and [ 177 Lu]Lu-DOTA-LM3 with excess peptide to block SSTR. (Figure 5) Graph showing the uptake and internalization rate of radioactive peptides applied at various molar amounts using AR42J tumor cells. AR42J tumor cells were incubated with each radioactive peptide for 2 hours. (a) [ 177 (b) [Lu]Lu-DOTATOC; 177 Lu]Lu-DOTA-LM3. (Figure 6) Graphs of cellular uptake and localization studies of radioactive peptides. (a) Cellular localization of radiolabeled DOTATOC; (b) Cellular localization of radiolabeled DOTA-LM3. (Figure 7) Graph showing AR42J tumor cell viability assay (MTT). (a) [ 161 Tb]Tb- / [ 177 (b) [Lu] results for cells treated with Lu-DOTATOC; 161 Tb]Tb- / [ 177 Results for cells treated with]Lu-DOTA-LM3. (Figure 8) Graph showing the survival rate of AR42J tumor cells (clone formation assay). (a) [ 161 Tb]Tb-DOTATOC or [ 177 (b) [Lu] results for cells treated with Lu-DOTATOC; 161 Tb]Tb-DOTA-LM3 or [ 177 Results for cells treated with]Lu-DOTA-LM3. (Figure 9) Quantification of γ-H2AX-positive AR42J tumor cells representing DNA DSBs after exposure to radioactive peptides (2.5MBq and 10MBq). (a) γ-H2AX-positive AR42J tumor cells after mock treatment; (b) [ 161 Tb]Tb-DOTATOC or [ 177(c) γ-H2AX-positive AR42J tumor cells after treatment with [Lu]Lu-DOTATOC; 161 Tb]Tb-DOTA-LM3 or [ 177 γ-H2AX-positive AR42J tumor cells after treatment with [Lu]Lu-DOTA-LM3 (2.5 and 10 MBq). The number of positive cells is expressed as a percentage of the number of positive cases in mock-treated AR42J (set at 1.0). (Figure 10A) Shown as maximum intensity projection (MIP), 161 Tb]Tb-DOTATOC (15 MBq, 0.5 nmol / mouse) and [ 177 Dual-isotope SPECT / CT images of AR42J tumor-bearing mice 2, 4, and 24 hours after injection of [Lu]Lu-DOTATOC (15 MBq, 0.5 nmol / mouse). (a / b / c) Scans 2 hours after injection of the radioactive peptide, (d / e / f) Scans 4 hours after injection of the radioactive peptide, and (g / h / i) Scans 24 hours after injection of the radioactive peptide. (a / d / g) Terbium-161 X-ray and gamma-ray reconstructions, (b / e / h) Terbium-161 X-ray and gamma-ray, lutetium-177 gamma-ray reconstructions, and (c / f / i) Lutetium-177 gamma-ray reconstructions. AR42J = SSTR-positive tumor xenograft; Ki = kidney; Bl = bladder. (FIG. 10B) Dual-isotope SPECT / CT images of a blocking study performed in AR42J tumor-bearing mice. Images are from [Lu]Lu-DOTATOC (15 MBq, 0.5 nmol / mouse). 161 Tb]Tb-DOTATOC (15 MBq, 0.5 nmol / mouse) and [ 177 Shown as maximum intensity projections (MIPs) 2 and 4 hours after injection of [Lu]Lu-DOTATOC (15 MBq, 0.5 nmol / mouse) and excess unlabeled DOTATOC (20 nmol / mouse). (A / B / C) Scans 2 hours after injection of the radioactive peptide; (D / E / F) Scans 4 hours after injection of the radioactive peptide. (A / D) Terbium-161 X-ray and gamma reconstruction; (B / E) Terbium-161 X-ray and gamma reconstruction, lutetium-177 gamma reconstruction; (C / F) Lutetium-177 gamma reconstruction. AR42J = SSTR2-positive tumor xenograft; Ki = kidney; Bl = bladder. (Figure 11A) Shown as maximum intensity projection (MIP), 161 Tb]Tb-DOTA-LM3 (15 MBq, 0.5 nmol / mouse) and [ 177 Dual-isotope SPECT / CT images of AR42J tumor-bearing mice 2, 4, and 24 hours after injection of [Lu]Lu-DOTA-LM3 (15 MBq, 0.5 nmol / mouse). (a / b / c) Scans 2 hours after injection of the radioactive peptide, (d / e / f) Scans 4 hours after injection of the radioactive peptide, and (g / h / i) Scans 24 hours after injection of the radioactive peptide. (a / d / g) Terbium-161 X-ray and gamma-ray reconstructions, (b / e / h) Terbium-161 X-ray and gamma-ray, lutetium-177 gamma-ray reconstructions, and (c / f / i) Lutetium-177 gamma-ray reconstructions. AR42J = SSTR-positive tumor xenograft; Ki = kidney; Bl = bladder. (FIG. 11B) Dual-isotope SPECT / CT images of a blocking study performed in AR42J tumor-bearing mice. Images are from [Lu]Lu-DOTA-LM3 (15 MBq, 0.5 nmol / mouse). 161 Tb]Tb-DOTA-LM3 (15 MBq, 0.5 nmol) and [ 177 Shown as maximum intensity projections (MIPs) 2 and 4 hours after injection of [Lu]Lu-DOTA-LM3 (15 MBq, 0.5 nmol) and excess unlabeled DOTA-LM3 (20 nmol / mouse). (A / B / C) Scans 2 hours after injection of the radioactive peptide; (D / E / F) Scans 4 hours after injection of the radioactive peptide. (A / D) Terbium-161 X-ray and gamma reconstructions; (B / E) Terbium-161 X-ray and gamma reconstructions, lutetium-177 gamma reconstructions; (C / F) Lutetium-177 gamma reconstructions. AR42J = SSTR2-positive tumor xenograft; Ki = kidney; Bl = bladder. (Figure 12) Biodistribution data obtained in AR42J tumor-bearing mice. (a / b) Tissue distribution 2 hours after injection of the radioactive peptide; (c / d) Tissue distribution 24 hours after injection of the radioactive peptide. (a / c) [ 161 Tb]Tb-DOTATOC and [ 177 Lu]Data obtained with Lu-DOTATOC (p>0.05);(b / d)[ 161 Tb]Tb-DOTA-LM3 and [177 (a) [Lu]Lu-DOTA-LM3 (p>0.05). (Figure 13) Biodistribution data obtained in AR42J tumor-bearing mice 2 hours after injection of different molar amounts of radioactive peptide. (b) [Lu]Lu-DOTA-LM3 (p>0.05). 161 (b) [Tb]Tb-DOTATOC tissue distribution; 177 (c) [Lu]Lu-DOTATOC tissue distribution; 161 Tb]Tb-DOTA-LM3 tissue distribution; (d) [ 177 Tissue distribution of [Lu]Lu-DOTA-LM3. Results are presented as percentage of injected activity per tissue mass (%IA / g). (Figure 14) Biodistribution data obtained after injecting radioactive peptide (0.2 nmol per mouse) into AR42J tumor-bearing mice. (a) [ 161 (b) [Tb]Tb-DOTATOC; 161 Tissue distribution profile of [Tb]Tb-DOTA-LM3. Results are expressed as percent injected activity per tissue mass (%IA / g). (Figure 15) AR42J tumor-bearing mice 161 Tb and 177 Treatment study with Lu-labeled DOTATOC and DOTA-LM3. (a) Sham-treated mice (group A), [ 161 Tb]Tb-DOTATOC (10 MBq, 0.2 nmol, administered on days 0 and 7) (group B), and [ 177 (b) Sham-treated (Group A), [Lu]Lu-DOTATOC (10 MBq, 0.2 nmol, administered on days 0 and 7) (Group C). 161 Tb]Tb-DOTA-LM3 (10 MBq, 0.2 nmol, administered on days 0 and 7) (group D), [ 177 Tumor growth curves for mice receiving [Lu]Lu-DOTA-LM3 (10 MBq, 0.2 nmol, administered on days 0 and 7) (Group E). Data are shown until the first mouse in each group reached the endpoint. (c) Kaplan-Meier plot for Groups A / B / C; (d) Kaplan-Meier plot for Groups A / D / E. (Figure 16) In AR42J tumor-bearing mice, 161 Tb- and 177Analysis of the therapeutic trial conducted with Lu-SST agonist and antagonist (2 × 10 MBq; 0.2 nmol): (a) Tumor growth delay in the AE group; (b) Tumor doubling time in the AE group. (Figure 17) Relative body weight (RBW) of treated mice. (a) Mice that did not receive treatment (Group A), [ 161 Tb]Tb-DOTATOC (group B) and [ 177 (b) RBW of untreated mice (group A), [ 161 Tb]Tb-DOTA-LM3 (group D) and [ 177 RBW of [Lu]Lu-DOTA-LM3 (Group E). Data are shown until the first mouse in each group reached the endpoint. (Appendix) Two somatostatin receptor (SSTR) targeting peptides, DOTATOC (SSTR agonist) and DOTA-LM3 (SSTR antagonist) 161 Tb or 177 They were labeled with Lu and evaluated in preclinical experiments. Their behavior was studied in preclinical studies. Figure 18 corresponds to a poster published by the inventors in November 2020.

[0052] [Example] The examples of the present disclosure demonstrate the influence of cellular localization on the therapeutic efficacy of terbium-161 versus lutetium-177 in vitro and in vivo. DOTATOC and DOTA-LM3 were used, respectively. 161 Tb labeling and 177 To investigate whether the effects of conversion and Auger electrons emitted from terbium-161 depend on the cellular localization of the radionuclide, 161 Tb radioactive peptide 177 The head-to-head comparison of the therapeutic effects of terbium-161 and lutetium-177 was 161 Tb- and 177 This was possible because Lu- exhibits comparable behavior in terms of cellular uptake in vitro and biodistribution in vivo

[44] .

[0053] The cellular localization of the radioactive peptide was found to affect its therapeutic efficacy, which was dependent on the radionuclide used.

[0054] 161 Tb-labeled peptides 177 Differences in efficacy between Lu-labeled peptides were evident, most notably in the case of DOTA-LM3. 161 Tb]Tb-DOTA-LM3 177 Lu]Lu-DOTA-LM3 was found to be 102-fold more effective in reducing cell viability in vitro.

[0055] Preclinical therapeutic research 161 Tb-labeled somatostatin analogs 177 It was confirmed to be more effective in vivo than Lu-labeled somatostatin analogs. The therapeutic effect of radiolabeled DOTA-LM3 was significantly more pronounced than that of radiolabeled DOTATOC. This is likely due to the higher tumor uptake of radiolabeled DOTA-LM3, as demonstrated by biodistribution data. It is localized in the cell membrane. 161 The Tb-labeled somatostatin receptor antagonist (DOTA-LM3) proved to be more beneficial with terbium-161 than with lutetium-177, suggesting that the effective target of terbium-161 as an Auger electron emitter is the cell membrane.

[0056] I. Production Terbium-161 and lutetium-177 were used to compare the effects of targeting agents based on their cellular localization. For this purpose, two peptides were used: (i) DOTATOC, which is primarily localized in the cytoplasm, and (ii) DOTA-LM3, which is primarily retained at the cell membrane.

[0057] A. Methods for producing radionuclides and peptides Terbium-161, as previously reported [1, 2], 160 Gd(n,γ) 161 Gd → 161Terbium-161 was produced using a Tb nuclear reaction. Target materials were irradiated at Necsa's SAFARI-1 reactor in Pelindaba, South Africa, or at the RHF of the Laue-Langevin Institute in Grenoble, France, or at the Spallation Neutron Source SINQ at Villigen-PSI, Switzerland. Chemical separation of terbium-161 was performed at PSI as previously reported [2]. Terbium-161 was separated by the addition of no carriers (nca) [ 161 Lutetium-177 was obtained from ITM Medical Isotopes GmbH, Germany, as nca[Tb]TbCl3 in 0.04 M HCl. 177 Lu]LuCl3.

[0058] DOTA-[Tyr 3 ]-Octreotide (DOTATOC) was provided by ITM GmbH, Germany (Fig. 1a). DOTA-LM3 was custom synthesized by CSBio (Silicon Valley Menlo Park, California, USA) based on the structure published by Fani et al. (see Fig. 1b) [3, 4].

[0059] Terbium-161 was produced with high yields (average of over 10 GBq, up to 15 GBq / production at the end of isolation) at activity concentrations of 11–21 MBq / µL and radionuclide and radiochemical purities of over 99%

[43] . Radiolabeling of peptides with up to 100 MBq / nmol was possible up to two weeks after chemical isolation, making it possible to plan and execute preclinical studies. The precise product specifications of terbium-161 were comparable to those of nca lutetium-177 reported by Gracheva et al.

[43] .

[0060] B. Preparation of Radioactive Peptides [ 161 Tb]Tb-DOTATOC and [ 161 Tb]Tb-DOTA-LM3, and [ 177 Lu]Lu-DOTATOC and [ 177[Lu]Lu-DOTA-LM3 was prepared with high molar activity to evaluate and compare the radioactive peptides in vitro and in vivo.

[0061] Stock solutions of DOTATOC and DOTA-LM3 were prepared in Milli-Q water to a final concentration of 1 mM. Somatostatin analogs were labeled with terbium-161 or lutetium-177 at a molar activity of up to 100 MBq / nmol using a 1:5 (v / v) mixture of sodium acetate (0.5 M) and hydrochloric acid (0.05 M) at pH 4.5. The reaction mixture was incubated at 95 °C for 10 min, followed by quality control using HPLC. For this purpose, a Merck Hitachi LaChrom HPLC system equipped with a D-7000 interface, an L-7200 autosampler, a radioactivity detector (LB506B; Berthold), and an L-7100 pump connected to a reversed-phase C18 column (Xterra™ MS, C18, 5 μm, 150 × 4.6 mm; Waters) was used. The mobile phase was 0.1% (v / v) TFA (A) and acetonitrile (B) in Milli-Q water. A 15-min linear gradient of solution A (95-20%) and solvent B (5-80%) was used at a flow rate of 1.0 mL / min. Radioactive peptides were diluted with Milli-Q water containing pentasodium diethylenetriaminepentaacetic acid (Na5-DTPA; 50 μM) before injection into the HPLC.

[0062] [ 161 Tb]Tb-DOTATOC and [ 177 Lu]Lu-DOTATOC, and [ 161 Tb]Tb-DOTA-LM3 and [ 177 Lu]Lu-DOTA-LM3 was obtained with radiochemical purity of >98% up to a molar activity of 100 MBq / nmol (see Figure 2).

[0063] C. Radiolytic Stability of Radioactive Peptides The stability of the radiolabeled somatostatin analogue was tested to ensure completeness in in vitro and in vivo evaluation.

[0064] The radiolytic stability of radiolabeled somatostatin analogs was evaluated over a 24-hour period (n = 2). For this purpose, DOTATOC and DOTA-LM3 were labeled with terbium-161 or lutetium-177 at a molar activity of 50 MBq / nmol. After quality control using HPLC (t = 0, radiochemical purity ≥ 98%, set as 100%), the labeled solution was diluted with saline to a final volume of 250 μL and an activity concentration of 40 MBq / mL. The solution was incubated at room temperature (RT) with or without the addition of l-ascorbic acid (120 μg / 10 MBq in a final volume of 250 μL). Potential degradation of the radioactive peptides was determined after 1, 4, and 24 hours by analyzing the samples using HPLC. Quantitative evaluation of the chromatograms was performed by expressing the integrated peak area of ​​the intact product as a percentage of the sum of the integrated peak areas of the entire chromatogram consisting of released terbium-161 and lutetium-177, as well as unidentified degradation products.

[0065] Regardless of whether the radioactive peptides were labeled with terbium-161 or lutetium-177, all radioactive peptides were stable (more than 90% intact radioactive peptide) for up to 1 hour after preparation (see Figure 3). However, after 4 and 24 hours, the intact fraction decreased to 90% and 60%, respectively, due to radiolysis. In published data [36, 43], the intact fraction 161 Tb-labeled and 177 The percentage of Lu-labeled product was identical, indicating that the possible formation of additional reactive oxygen species (ROS) was due to short-range conversion and that Auger electrons

[45] did not affect the integrity of the radioactive peptide. Stabilization of radioactive peptides for clinical applications is commonly achieved by the addition of ascorbic acid

[46] . 161 Tb and 177 It was also effective in stabilizing the long-term administration of Lu-labeled somatostatin analogs (see Figure 3).

[0066] The addition of l-ascorbic acid was not necessary when the radioactive peptide was used at concentrations below 40 MBq / mL and / or immediately after preparation. Dual-isotope SPECT / CT imaging studies performed at activity concentrations above 40 MBq / mL (>4 MBq in 100 µL per mouse) required the addition of l-ascorbic acid (~300 µg per 30 MBq in 100 µL) to ensure radioactive peptide integrity.

[0067] Measurement of Dn-octanol / PBS partition coefficient (LogD value) The n-octanol / PBS partition coefficient (logD value) was evaluated for each radioactive peptide to assess hydrophilicity and allow comparison of the three analogs.

[0068] The log D values ​​of the radiolabeled analogs (DOTATOC and DOTA-LM3; 30 MBq / nmol) were determined by the shake-flask method as previously described

[47] . An aliquot of radiolabeled peptide (0.5 MBq, 25 μL) was added to a mixture of 1475 μL of PBS, pH 7.4, and 1500 μL of n-octanol. Each tube was vortexed for 60 seconds, centrifuged at 2500 rpm for 6 minutes, and then analyzed by a γ-counter (Perkin The activity concentration of an aliquot of each layer was measured using an Elmer, Wallac Wizard 1480. The log D value was calculated as the logarithm of the ratio of cpm measured in the n-octanol phase to cpm measured in the PBS phase at pH 7.4. Experiments were performed three times with five replicates for each radioactive peptide, and the log D value is expressed as the mean ± standard deviation (SD) of the values ​​obtained in each experiment. Data were analyzed for significance using a two-way analysis of variance with Tukey's multiple comparison test. A p value of <0.05 was considered statistically significant.

[0069] The radioactive peptide exhibited hydrophilic properties as indicated by the low logD values. 161 Tb- and 177 Lu-labeled counterparts showed similar logD values ​​(p>0.05) (Table 1).

[0070] [Table 1]

[0071] II. In vitro testing A.Cell culture In this study, the AR42J tumor cell line, an SSTR-positive rat pancreatic exocrine carcinoma cell line

[48] , was used for in vitro and in vivo tests.

[0072] AR42J tumor cells (ECACC 93100618; Health Protection Agency Culture Collections, Salisbury, UK) were maintained in RPMI 1640 medium supplemented with glutamine, antibiotics, and 20% fetal calf serum as previously described

[44] . Cell culture medium containing glutamine and antibiotics, supplemented with 1% FCS only (referred to herein as "assay medium"), was used for all in vitro assays. Cells were incubated under standard culture conditions at 37°C in a humidified atmosphere of 5% CO2 unless otherwise indicated. Phosphate-buffered saline (PBS), pH 7.2, was used for in vitro experiments unless otherwise indicated. Polystyrene well plates were coated with poly-l-lysine (0.5 mg / mL) for all in vitro experiments to promote cell adhesion and prevent adhesion of the radioactive peptide to the well plate material.

[0073] B. Cellular uptake and internalization: 161 Tb-labeled peptides 177 Comparison of Lu-labeled peptides Each radioactive peptide 161 Tb-labeled and 177 Cellular uptake and internalization studies were performed using Lu-labeled products.

[0074] Cellular uptake and internalization studies were performed using AR42J tumor cells according to a previously reported procedure [5]. 6Cells (2 mL) were seeded onto poly-l-lysine-coated 12-well plates and incubated overnight in supplemented RPMI medium. After washing the cells with PBS (pH 7.4), assay medium (975 μL) and 25 μL of radioactive peptide solution (~15 kBq, ~0.75 pmol / well) were added to each well, resulting in a radioligand concentration of 0.75 nM. The well plates were incubated for 0.5, 2, or 4 hours under standard cell culture conditions. SSTR blocking experiments were performed using 1 μM DOTANOC or 1 μM DOTA-LM3. Total uptake and internalized fractions were determined using a γ-counter (Perkin Elmer, Wallac Wizard 1480). Sample activity was normalized to the average protein concentration (~0.3 mg) in each well using a Micro BCA Protein Assay kit (Pierce, Thermo Scientific). Experiments were performed in triplicate, either twice or three times. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison post-hoc test. A p-value <0.05 was considered statistically significant.

[0075] Uptake and internalization into AR42J tumor cells 161 Tb- and 177 The results were comparable for the Lu-labeled SSTR analogs (p>0.05) (see Figure 4). 161 Tb]Tb- / [ 177 The uptake of [Lu]Lu-DOTATOC was highest after 4 hours of incubation (approximately 15% of the total activity added). Acid-washed cells retained 40-50% of the total uptake, indicating efficient internalization of the radioactive peptide-receptor complex, as previously reported

[49] . 161 Tb]Tb- / [ 177 [Lu]Lu-DOTA-LM3 showed uptake in AR42J cells at all time points investigated, reaching nearly 70% after 4 hours of incubation. However, the internalized fraction of radiolabeled DOTA-LM3 was almost negligible (less than 10% of the total uptake)

[42] . Addition of excess DOTANOC resulted in [ 161 Tb]Tb- / [177 The cellular uptake of [Lu]Lu-DOTATOC was inhibited, confirming SSTR-specific binding of the radioactive peptide. However, DOTANOC was not effective in completely blocking the uptake of radiolabeled DOTA-LM3 (data not shown). This is because SSTR-antagonists have access to more binding sites on the cell membrane than SSTR-agonists (up to 14-fold)

[50] . Blocking was achieved using an excess of unlabeled DOTA-LM3.

[0076] C. Cellular uptake and internalization of increasing molar amounts of peptides The purpose of this in vitro study was to add radioactive peptide to varying amounts of DOTATOC and DOTA-LM3 to determine the amount of peptide that would result in SSTR saturation of AR42J tumor cells.

[0077] The uptake and internalization rate of the peptide in AR42J tumor cells was measured using the same method as above, except that the radioactive peptide was applied using variable molar amounts of peptide. After washing the AR42J tumor cells with PBS (pH 7.4), 0.375–75 pmol of [ 177 Lu]Lu-DOTATOC or [ 177 Cells were incubated with [Lu]Lu-DOTA-LM3 (25 μL, ~15 kBq). After 2 h of incubation at 37 °C, cellular uptake and internalized fractions were determined, and sample activity was normalized to the average protein concentration (~0.3 mg) in each well. Experiments were performed twice in triplicate. In all cases, the uptake and internalization of each radioactive peptide decreased with increasing molar amount of unlabeled peptide (see Figure 5). No saturation of SSTR was observed at peptide amounts of 0.375 pmol and 0.75 pmol, but at peptide amounts of 1.5 pmol and 7.5 pmol [ 177 The uptake and internalization of [Lu]Lu-DOTATOC was reduced by approximately 30%. 177 The uptake and internalization of [Lu]Lu-DOTATOC was reduced by ~90%. 177Lu]Lu-DOTA-LM3 uptake was 177 Similar to [Lu]Lu-DOTATOC, there was a ~30% decrease when 1.5 pmol of the peptide was applied. 177 The uptake of [Lu]Lu-DOTA-LM3 was reduced by ∼80% at a molar amount of 7.5 pmol, whereas under the same conditions, [ 177 Lu]Lu-DOTATOC uptake was reduced by only 30%.

[0078] D. Nuclear localization of radioactive peptides After incubating AR42J tumor cells with each radioactive peptide, cell nuclei were isolated and the percentage of radioactive peptide localized in the nucleus was determined.

[0079] AR42J tumor cells (10 × 10 6) were seeded onto PLL-coated Petri dishes and incubated overnight at 37°C with 15 mL of cell culture medium containing supplements at 5% CO2. The next day, the medium was removed, the tumor cells were washed with PBS (pH 7.4), and 19.5 mL of assay medium was added. 0.5 mL (2.5 MBq, 125 pmol) of radioactive peptide was added, and the tumor cells were incubated at 37°C for 2 hours. The cells were then washed several times with PBS (pH 7.4) to completely remove excess radioactive peptide. Nuclei and cytoplasmic / membrane fractions were then collected using the Nuclei EZ Prep Nuclei Isolation Kit (Sigma-Aldrich, USA) according to the manufacturer's protocol. Ice-cold Nuclei EZ lysis buffer (4 mL) was added to each Petri dish to lyse the tumor cells. The cell suspension was then transferred to an Eppendorf tube and centrifuged at 500 rcf for 5 minutes at 4°C. The supernatant containing the cytoplasmic / membrane fraction was transferred to a tube for activity measurement using a γ-counter. The pellet was suspended in 4 mL of ice-cold Nuclei EZ lysis buffer and further centrifuged at 500 rcf for 5 minutes at 4°C. The remaining supernatant containing the cytoplasmic / membrane fraction was collected for activity measurement, and the pellet was suspended in 200 μL of Nuclei EZ storage buffer and transferred to a tube for activity measurement using a γ-counter. The measured activity in the nuclear and cytoplasmic / membrane fractions was defined as 100% intracellular uptake. Nuclear localization was expressed as a percentage of total intracellular uptake. The collected fractions were stained with 0.4% trypan blue solution and analyzed microscopically to confirm that the nuclei were properly separated from other cellular fragments.

[0080] [ 177 Lu]Lu-DOTATOC and [ 177 The nuclear uptake of [Lu]Lu-DOTA-LM3 was less than 1% and ~2% of the total cellular uptake. These results were combined with the uptake and internalization data (see Figure 5), and the intracellular distribution of the single radioactive peptides was calculated and presented in a pie chart (see Figure 6). 177The internalization rate of [Lu]Lu-DOTA-LM3 was approximately 9% of the total uptake, which was much lower than that of radiolabeled DOTATOC (approximately 81% of the total uptake). The nuclear localization rate was less than 2% (see Figure 6).

[0081] E. Cell Viability Assay Cell viability tests were performed using AR42J tumor cells. 161 Tb- and 177 The potentially different effects of Lu-labeled DOTATOC and DOTA-LM3 were evaluated.

[0082] A total of 7,500 AR42J tumor cells were seeded in 200 μL of cell culture medium in PLL-coated 96-well plates. After overnight incubation to allow cells to adhere, the medium was removed and the cells were incubated with DOTATOC or DOTA-LM3 diluted in assay medium and radiolabeled with terbium-161 or lutetium-177 at a molar activity of 100 MBq / nmol. The applied activity concentration per well ranged from 0.001 MBq / mL to 40 MBq / mL (0.01–400 pmol / mL). After 2 h of incubation at 37°C, cells were washed once with PBS, followed by the addition of fresh cell culture medium with supplements. Tumor cells were grown for 6 days at 37°C without changing the cell culture medium. Cell viability was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described

[51] . After incubating the cells with the MTT reagent for 2 hours, the formed formazan crystals were dissolved in dimethyl sulfoxide. The absorbance at 560 nm was measured using a microplate reader (560 nm, Victor™ X3, Perkin Elmer, Waltham, MA, USA). The absorbance measured for untreated control cells was defined as 100% tumor cell viability. The viability of treated tumor cells (n = 12 per concentration) was expressed as a percentage of the absorbance of control cells. Cell viability was plotted against the applied activity concentration (logarithmically transformed) and fitted to a dose-response curve. Cell viability inhibition was calculated as the activity concentration required to reduce the viability of AR42J tumor cells to 50% of that of untreated control cells (EC 50 ). EC 50 Values ​​were determined in at least four independent experiments.

[0083] A reduction in the viability of AR42J cells to less than 10% compared to the viability of untreated control cells (set as 100%) was achieved at variable activity concentrations depending on the somatostatin analogue and radionuclide employed, respectively. In all cases, 161 Tb-labeled somatostatin analogs 177It was observed to be more potent than its Lu-labeled counterpart (see Figure 7). 161 Tb]Tb-DOTATOC 177 It was five times more potent than [Lu]Lu-DOTATOC. 161 Tb]Tb-DOTA-LM3 177 It was 102-fold more potent than [Lu]Lu-DOTA-LM3. 161 EC of Tb-labeled somatostatin analogues 50 Comparing [ 161 Tb]Tb-DOTA-LM3 161 It showed 157-fold higher potency than [Tb]Tb-DOTATOC (Table 2). 177 The situation is different in Lu-DOTA-LM3. 177 It was 7.7-fold more potent than [Lu]Lu-DOTATOC (Table 2).

[0084] [Table 2]

[0085] F. Cell viability (clone formation assay) 161 Tb- and 177 The ability of single AR42J tumor cells exposed to Lu-radiolabelled DOTATOC or DOTA-LM3 to grow into colonies was determined by performing a clonogenic assay [8].

[0086] To allow for proper colony formation, 300 μL of Matrigel (Growth Factor Reduced Basement Membrane Matrix, Corning Inc, New York, USA; 2 mg / mL) diluted in additive-free RPMI cell culture medium was added to each well of a 6-well plate coated with PPL. AR42J tumor cells were seeded on the solidified Matrigel at a density of 2000 cells per well in 2 mL of cell culture medium (with supplements) and incubated overnight. The next day, the medium was removed, and the cells were 161 Tb- and 177The cells were incubated with Lu-radiolabelled somatostatin analogs (30 MBq / nmol) at activity concentrations ranging from 0.01 MBq / mL to 0.5 MBq / mL (0.3-15 pmol / mL) for 2 hours at 37°C and 5% CO2. The same procedure was performed on untreated control cells. After incubation, the supernatant was discarded, and the tumor cells were washed with PBS before adding fresh cell culture medium. After 2 weeks of incubation at 37°C and 5% CO2, the medium was removed and the wells were washed once with PBS. Colonies were stained with crystal violet solution (0.5% crystal violet, 6% glutaraldehyde in water, 800 μL). The number of colonies ≥0.1 mm was determined visually by selecting five 0.5 cm × 0.5 cm squares under a microscope. Plating efficiency (PE) and survival rate (SF) were calculated according to the following formulas: PE = ([number of colonies formed (untreated)] / [number of cells plated]) * 100; SF = ([number of colonies formed after treatment] / [number of cells plated * PE]) * 100

[52] .

[0087] SF following exposure to various radioactive concentrations of radioligand was measured in at least three independent experiments, with triplicates per experiment. Data were analyzed by two-way ANOVA with Sidak's multiple comparison post-hoc test. A p-value <0.05 was considered statistically significant.

[0088] In the colony formation assay, 161 Tb-labeled peptides 177 It was confirmed that the Lu-labeled peptide had a greater effect on reducing cell viability than the untreated control cells (see Figure 8). 161 The viability of cells treated with [Tb]Tb-DOTATOC was less than 3%. 177 To achieve a similar effect with [Lu]Lu-DOTATOC, a 10-fold higher active concentration (5 MBq / mL) had to be applied (n=1). 161 Tb]Tb-DOTA-LM3 reduced cell viability to less than 5% at an active concentration of 0.1 MBq / mL, whereas [ 177 Lu]Lu-DOTA-LM3 was applied at a 10-fold higher concentration (1 MBq / mL) and reduced viability to only about 15%.

[0089] G. Assessment of DNA Damage [ 161 Tb]Tb-DOTATOC and [ 177 Lu]Lu-DOTATOC, or [ 161 DOTA-LM3 and [Tb]Tb 177 Experiments were performed using AR42J tumor cells treated with [Lu]Lu-DOTA-LM3 to determine the number of double-strand breaks (DSBs) induced in each case.

[0090] The number of DNA DSBs was assessed by immunostaining of γ-H2AX in AR42J cells treated with 2.5 MBq / mL or 10 MBq / mL of each radioactive peptide. Cells were seeded (5 × 10 6Cells were grown overnight (cells / Petri dish). The following day, the medium was removed, and the cells were treated with radioactive peptide diluted in assay medium for 2 hours. The supernatant was removed, and AR42J tumor cells were washed with PBS before adding fresh medium. After 24 hours of incubation, the cells were washed with PBS, scraped with 1.5 mL of PBS, and centrifuged in a 1.5 mL Eppendorf tube. The cell pellet was fixed in 1 mL of 4% neutral buffered formalin, incubated at room temperature for 24 hours, and then replaced with PBS. After paraffin embedding, 4 μm-thick sections were prepared. Briefly, cells were deparaffinized for 20 minutes at 98°C using a solution containing EDTA (pH 9), followed by antigen retrieval by incubation with REAL Antibody Diluent (Agilent) for 30 minutes at room temperature and hydrogen peroxide (Agilent) for 10 minutes at room temperature. Sections were incubated with primary antibody (cell signaling rabbit monoclonal antibody (Ser139); dilution 1:200) at RT for 1 h. The Envision horseradish peroxidase rabbit (Agilent Technologies, Inc.) detection system was used with DAB substrate buffer (Agilent). Immunostained sections were scanned with a digital slide scanner (NanoZoomer-XR C12000; Hamamatsu, Japan), and the total number of positive and negative cells was quantified using the pathology image analysis software VIS (Visiopharm Integrator System, Version 208 2019.02.2.6239, Visiopharm, Hoersholm, Denmark). First, a decision forest classification method was used to outline the tissue cell pellet as a region of interest (ROI). Then, cell nuclei within each ROI were detected using a cell classification method and classified as positive (brown) or negative (blue). Separation of nuclear types was performed by training the software with predefined options: "standard positive nuclei" and "standard negative nuclei." Results were expressed as total positive and total negative cell counts. Data were analyzed by one-way ANOVA with Dunnett's multiple comparison post-hoc test. A p-value <0.05 was considered statistically significant.

[0091] At high activity concentrations (10 MBq / mL), all 161 The number of γ-H2AX-positive foci increased in Tb-treated samples compared with controls, but did not reach significant levels (p<0.05). 177 The Lu-labeled somatostatin analog produced only a small increase in γ-H2AX-positive foci, which was only evident at highly active concentrations (p>0.05). The results are shown in Figure 9.

[0092] III. In vivo testing In vivo studies were performed to evaluate the tissue distribution of the radioactive peptide and to optimize the molar dose of the injected peptide. Furthermore, the therapeutic efficacy and early side effects of the radioactive peptide were evaluated.

[0093] All applicable international, national, and / or institutional guidelines regarding animal care and use were followed. In particular, all animal experiments were conducted in accordance with the guidelines of the Swiss Animal Welfare Regulations. Preclinical studies were ethically approved by the Cantonal Animal Experimentation Committee and permitted by the competent cantonal authorities (permit numbers 75721 and 79692). Five-week-old female athymic nude mice (CD-1 Foxn-1 / nu) were obtained from Charles River Laboratories (Sulzfeld, Germany). Mice were inoculated with AR42J tumor cells (5 × 10 in 100 μL PBS). 6 SPECT / CT imaging, biodistribution, and therapeutic studies were performed. SPECT / CT and biodistribution studies were performed 10-14 days after tumor cell inoculation, when tumor size reached approximately 250 mm. 3 It was carried out when it reached

[0094] A. Dual-Isotope SPECT / CT Imaging Study The purpose is to 161 Tb- and 177 The primary objectives were to demonstrate that the Lu-labeled somatostatin analogs have the same in vivo distribution and that tumor uptake is SSTR-specific. A second objective was to compare the tissue distribution of the three somatostatin analogs.

[0095] Dual-isotope SPECT / CT scans were performed using a dedicated small-animal SPECT / CT scanner (NanoSPECT / CT, Mediso Medical Imaging Systems, Budapest, Hungary; see Supplementary Material) as previously described

[44] . Scans were acquired using Nucline software (version 1.02, Mediso Ltd., Budapest, Hungary). Simultaneous acquisition of counts originating from terbium-161 and lutetium-177 was achieved by selecting different energy windows for the two radionuclides. For terbium-161, two energy windows were selected: 47.7 keV ± 10%, which allows detection of X-rays and gamma rays (46.0 keV, 48.9 keV, and 52.0 keV), and 74.6 keV ± 10%, which allows detection of gamma rays at 74.6 keV. For lutetium-177, the windows were set to 208.4 ± 10% and 112.9 keV ± 10% to detect γ-rays at 208.4 keV and 112.9 keV, respectively. SPECT data were iteratively reconstructed using HiSPECT software (version 1.4.3049, Scivis GmbH, Gottingen, Germany). CT scans were reconstructed in real time using backprojection with a cone-beam filter. Fusion datasets of SPECT and CT scans were analyzed using VivoQuant post-processing software (version 3.5, Invicro Imaging Services and Software, Boston, MA, USA). A Gaussian post-reconstruction filter (FWHM = 1.0 mm) was applied. Mice were treated with PBS (pH 7.4) containing 0.05% BSA and ascorbic acid (∼300 μg / 30 MBq) [ 161 Tb]Tb-DOTATOC (~15 MBq, 0.5 nmol / mouse) and [ 177 a mixture with [Lu]Lu-DOTATOC (~15 MBq, 0.5 nmol / mouse), or [ 161 Tb]Tb-DOTA-LM3 and [ 177A mixture of [Lu]Lu-DOTA-LM3 was injected intravenously. Blocking studies were performed under the same experimental conditions, except that an excess (20 nmol / mouse) of unlabeled DOTATOC or DOTA-LM3 was added to the injection solution. SPECT / CT scans were acquired 2, 4, and 24 hours after radioactive peptide injection using a dual-isotope SPECT acquisition protocol with a frame time of 60 seconds and a scan time of 45 minutes. During in vivo scans, mice were anesthetized by inhalation of a mixture of isoflurane and oxygen. SPECT / CT images of AR42J tumor-bearing mice were simultaneously injected [ 161 Tb]Tb-DOTATOC and [ 177 The same observation was made with [Lu]Lu-DOTATOC in vivo (see Figure 10). 161 Tb]Tb-DOTA-LM3 and [ 177 This was also done for [Lu]Lu-DOTA-LM3 (see Figure 11)

[44] .

[0096] SPECT / CT images showed accumulation of activity in AR42J xenografts, and the amount of accumulation was [ 161 Tb]Tb-DOTA-LM3 and [ 177 Lu]Lu-DOTA-LM3 is better, [ 161 Tb]Tb-DOTATOC and [ 177 The activity of [Lu]Lu-DOTATOC was higher at all time points investigated. Activity was efficiently eliminated over time through the kidney, with almost complete excretion after 24 hours. Due to the favorable uptake of radiolabeled DOTA-LM3 in tumor tissue, the tumor-to-kidney ratio was higher compared to that obtained after injection of radiolabeled DOTATOC

[44] .

[0097] Co-injection of excess unlabeled peptides inhibited radioactive peptide accumulation in AR42J tumors, demonstrating SSTR-specific uptake, as shown in Figures 10B and 11B.

[0098] B. Biodistribution Studies: 161 Tb-labeled peptides177 Comparison of Lu-labeled peptides Biodistribution studies include: 161 Quantitatively assess the tissue distribution of Tb-labeled peptides. 177 This was done to compare with the Lu-labeled peptide.

[0099] Diluted in PBS (pH 7.4) containing 0.05% BSA [ 161 Tb]Tb-DOTATOC or [ 177 Biodistribution studies were performed after intravenous injection of [Lu]Lu-DOTATOC (5 MBq; 1 nmol in 100 μL per mouse) into mice. 161 Tb]Tb-DOTA-LM3 or [ 177 [Lu]Lu-DOTA-LM3 was also used under the same conditions. Mice were sacrificed 2 and 24 hours post-injection (pi). Selected tissues and organs were collected, weighed, and the accumulated activity was counted using a γ-counter. Decay-corrected data were expressed as the percentage of injected activity per gram of tissue mass (%IA / g). Data were analyzed for significance using two-way ANOVA with Tukey's multiple comparison post-hoc test. A p-value <0.05 was considered statistically significant.

[0100] 161 Biodistribution of Tb-labeled DOTATOC 177 The biodistribution was comparable to that of Lu-labeled DOTATOC, as indicated by similar activity accumulation in both tissues and organs examined after 2 and 24 hours, respectively (p>0.05). 161 Tb labeling and 177 Comparable tissue distribution of the Lu label was observed (see Figure 12, Tables A1 / A2). These results suggest that terbium-161 and lutetium-177 161 Tb- and 177 As previously demonstrated with Lu-labeled folate conjugates and PSMA ligands [34, 36], the exchangeability of radioactive peptides was confirmed without altering their pharmacokinetic properties.

[0101] [Table 3] [Table 4]

[0102] C. Biodistribution Study: Evaluation of the Optimal Injection Molar Dose The purpose is to 161 Tb- and 177 The purpose of this study was to evaluate the effect of peptide injection volume on the biodistribution of Lu-labeled DOTATOC and DOTA-LM3.

[0103] Radiolabeled DOTATOC or DOTA-LM3 (3 MBq, 0.04 nmol / mouse; 5 MBq, 0.2 nmol / mouse; or 5 MBq, 1.0 nmol / mouse) in 100 μL of PBS (pH 7.4) containing 0.05% BSA was injected intravenously into mice (n = 3 per group). Mice were sacrificed 2 h later, and selected tissues and organs were collected, weighed, and counted for accumulated activity using a γ-counter (Perkin Elmer). Decay-corrected data were expressed as % IA / g. Data were analyzed for significance using two-way ANOVA with Tukey's multiple comparison post-hoc test. A p-value <0.05 was considered statistically significant.

[0104] Consistent with previous studies

[53] , the amount of peptide injected significantly affected the accumulation activity in various organs and tissues (see Figure 13, Tables A3 / A4).

[0105] [Table 5] [Table 6]

[0106] [ 161 Tb]Tb- / [ 177Tumor uptake of [Lu]Lu-DOTATOC was similar following administration of 0.04 nmol and 0.2 nmol per mouse (~17% IA / g and ~15% IA / g, respectively, p>0.05) but significantly higher than that following injection of 1.0 nmol (~8% IA / g, p<0.05). Gastric uptake was higher following injection of 0.04 nmol compared with injection of 0.2 nmol or 1.0 nmol (p<0.05). Pancreas, adrenal, and lung uptake showed similar trends, but the differences between settings were not significant (p>0.05). Kidney (~10%) and liver (~0.2% IA / g) uptake did not differ regardless of the setting applied. 161 Tb]Tb / [ 177 [Lu]Lu-DOTA-LM3 showed similarly high tumor uptake (~34% and ~38% IA / g, p>0.05) when 0.040 nmol and 0.20 nmol of peptide were injected per mouse. Uptake was lower at 1.0 nmol (~18% IA / g, p<0.05). Uptake in the pancreas, adrenal gland, lung, and stomach was significantly higher when 0.040 nmol of peptide was injected than when 0.2 nmol was injected, with the lowest uptake occurring when 1.0 nmol of peptide was injected (p<0.05). Liver uptake was slightly elevated at the lowest molar dose injected, but not significantly (p>0.05). 161 Tb]Tb / [ 177 Renal uptake of [Lu]Lu-DOTA-LM3 was in the range of 10% IA / g, regardless of the injected dose of peptide. The results are shown in Figure 13.

[0107] As a result of these findings, tumor-to-organ ratios varied depending on the amount of radioactive peptide injected (Tables 3 and 4). For DOTATOC and DOTA-LM3, tumor-to-kidney (tu-to-ki) and tumor-to-liver (tu-to-li) ratios were both favorable when low molar amounts of peptide (0.04 nmol or 0.2 nmol) were injected. Tumor-to-panc (tu-to-panc), tumor-to-adrenal (tu-to-adr), tumor-to-lung (tu-to-lung), and tumor-to-stomach (tu-to-sto) ratios were favorable when 0.2 nmol or 1.0 nmol was injected. 161 Tb]Tb / [ 177 However, these ratios were higher after injection of [Lu]Lu-DOTATOC. 161 Tb- and 177 The tumor uptake after injection of 1.0 nmol of Lu-labeled DOTA-LM3 peptide was better than that after injection of 0.2 nmol of peptide per mouse. The lowest ratio was obtained when 0.04 nmol of peptide was injected per mouse. Because injection of 0.2 nmol of peptide per mouse resulted in the highest tumor uptake and a favorable tumor-to-background ratio in most cases, this molar amount of peptide was used for further in vivo studies.

[0108] [Table 7] [Table 8]

[0109] D. Time-Dependent Biodistribution Studies Time-dependent biodistribution studies were performed to assess the total uptake of the radioactive peptide in tumor and healthy organs and tissues.

[0110] Radiolabeled DOTATOC or DOTA-LM3 (5 MBq, 0.2 nmol / mouse) was injected intravenously into mice (n = 3 per group) in 100 μL of PBS (pH 7.4) containing 0.05% BSA. Mice were sacrificed at 0.5, 2, 4, 24, and 48 h. Selected tissues and organs were collected, weighed, and counted for accumulated activity using a γ-counter (Perkin Elmer). Decay-corrected data were expressed as % IA / g. Data were analyzed for significance using two-way ANOVA with Tukey's multiple comparison post-hoc test. A p-value <0.05 was considered statistically significant.

[0111] [ 161 [Tb]Tb-DOTATOC reached its highest tumor uptake (15±2%) already at 0.5 h postinjection. Activity was retained in tumor tissue for up to 4 h but then disappeared over several hours, reaching 6.4±0.5% IA / g and 3.7±0.7% IA / g at 24 and 48 h postinjection. Significant activity accumulation was observed in the lung, stomach, and pancreas, but was efficiently eliminated, dropping to <1% IA / g at 4 h, ~4–5% IA / g, and 2–3% at 24 and 48 h postinjection (Fig. 14a; see Table A5).

[0112] [Table 9]

[0113] These results are based on the fact that tissue distribution does not change whether the somatostatin analog is labeled with terbium-161 or lutetium-177. 177 It can also be estimated for the Lu-labeled counterpart

[44] . 161 Tumor uptake of [Tb]Tb-DOTA-LM3 was also rapid, reaching 35±7% IA / g 4 hours after injection. After 48 hours, activity in the tumor (21±4% IA / g) remained high. In other organs, such as the lung, stomach, and pancreas, activity was retained for the first 4 hours, but then efficiently disappeared. [Tb]Tb-DOTA-LM3 in the tumor, pancreas, and stomach 161The accumulated activity of Tb]Tb-DOTA-LM3 was [ 161 Tb]Tb-DOTATOC was significantly higher (p<0.05), but renal uptake was 161 This was similar to that observed with [Tb]Tb-DOTATOC (p>0.05) (Fig. 14b; see Table A6).

[0114] [Table 10]

[0115] E. Preclinical therapeutic studies using AR42J tumor-bearing mice The objective of this preclinical study was to evaluate the therapeutic efficacy and potential early side effects of DOTATOC and DOTA-LM3 peptides radiolabeled with terbium-161 or lutetium-177.

[0116] The treatment trial included AR42J tumors with a mean volume of 99 ± 16 mm 3 At the time of reaching 100 mg / kg, mice were randomly assigned to 5 groups (n = 6) and started. On days 0 and 7 of the study, mice were given either vehicle only (Group A: PBS containing 0.05% BSA; sham treatment), 161 Tb]Tb-DOTATOC (group B: 10MBq, 0.2nmol), [ 177 Lu]Lu-DOTATOC (group C: 10MBq, 0.2nmol), [ 161 Tb]Tb-DOTA-LM3 (group D: 10 MBq, 0.2 nmol), and [ 177 Lu]Lu-DOTA-LM3 (Group E: 10 MBq, 0.2 nmol) was intravenously injected (Table 5).

[0117] Relative body weight (RBW) and relative tumor volume (RTV) were defined based on the values ​​at the start of treatment as previously described

[54] . RBW was calculated as [BW x / BW0], where BW xwhere BW is the body weight in grams on a given day x, and BW is the body weight in grams on day 0. Tumor dimensions were determined by measuring the longest axis of the tumor (L) and its perpendicular axis (W) with a digital caliper. Tumor volume (TV) was calculated using the formula [TV = 0.5 × (L × W)]. 2 Relative tumor volume (RTV) was calculated according to the formula [TV / TV x0 ] where TV x is the tumor volume (mm ) on a given day x 3 ), and TV0 is the tumor volume on day 0 (mm 3 )

[0118] Endpoint criteria were defined according to a scoring system. Mice with a score of 3 or higher were euthanized. The following criteria were evaluated every two days, and each criterion was given a score of 0–3: (i) appearance (general condition, skin color, etc.), (ii) behavior (activity, sociability, crouching, etc.), (iii) body weight (stable, >5% but <10%, >10% but <15%, or ≥15% decrease compared to initial weight), and (iv) tumor size (800 mm). 3 Less than 800mm 3 Over 900mm 3 Less than 900mm 3 More than 1000mm 3 Less than 1000mm 3 (v) tumor ulceration. A score of 3 or greater is indicated by, for example: (i) wrinkled, translucent skin; (ii) squatted and / or lethargic mice; (iii) weight loss of 15% or more of the initial body weight; (iv) tumor mass exceeding 1000 mm 3 Tumor volume greater than or equal to 800 mm 3 or greater than 10% combined tumor size and weight loss of 10% or greater, and / or (vi) tumor ulceration.

[0119] The efficacy of treatment was evaluated by comparing the RTV measured every two days for mice in each group using two-way analysis of variance with Sidak's multiple comparison post-hoc test. The mean tumor growth delay, defined herein as the time until tumors stopped growing or decreased in size, was determined for each group of mice. For the subsequent stage, when tumors began to regrow, the tumor volume doubling time was calculated based on the fitted exponential tumor growth curve. The mean ± SD tumor growth delay and tumor volume doubling time in a single mouse were compared between groups using one-way analysis of variance and Tukey's multiple comparison post-hoc test, respectively. Mouse survival times were displayed as Kaplan-Meier curves and analyzed using the log-rank test (Mantel-Cox).

[0120] [Table 11]

[0121] Placebo-treated mice in group A showed exponential tumor growth, reaching the endpoint within the first 14 days in all cases. Treated mice in group BE showed delayed tumor growth, resulting in a significantly longer median survival compared to 9 days in the control group (Figure 15, Table 6). 161 Tb]Tb-DOTATOC-treated mice (group B) showed 177 Tumor growth was slightly slower in mice treated with [Lu]Lu-DOTATOC (group C) compared with mice treated with [Lu]Lu-DOTATOC (group C). Twelve days after the start of treatment, the RTVs of these two groups of mice were significantly different (2.0 ± 0.7 vs. 4.0 ± 3.3, p < 0.05). Tumor growth delay and doubling time were higher in group B (9.0 ± 5.5 days and 3.4 ± 3.6 days, respectively) compared with group C (6.0 ± 4.4 days and 3.4 ± 0.8 days, respectively, p > 0.05) (Figure 16). Therefore, mice in group B were euthanized later (days 20–26) than in group C (days 12–22). However, median survival times (21 days vs. 19.5 days) were comparable between groups (p > 0.05).

[0122] [ 161The tumor growth delay in mice treated with [Tb]Tb-DOTA-LM3 was 44 ± 5 ​​days, whereas [ 177 In mice treated with [Lu]Lu-DOTA-LM3, the tumor growth rate was only 35 ± 7 days (p < 0.05). After that, tumors began to grow exponentially in 5 of 6 mice in both groups, but [ 161 The doubling time of mice treated with [Tb]Tb-DOTA-LM3 was 177 The tumor growth in mice treated with [Lu]Lu-DOTA-LM3 was significantly longer than that in mice treated with [Lu]Lu-DOTA-LM3 (7.4±4.6 days vs. 3.8±1.1 days, p>0.05) (Figure 16). 161 All mice treated with [Tb]Tb-DOTA-LM3 survived to the end of the study, but 177 Only 3 of 6 mice were treated with [Lu]Lu-DOTA-LM3.

[0123] [Table 12]

[0124] F. Early Adverse Reaction Assessment [ 161 Tb]Tb-DOTATOC, [ 177 Lu]Lu-DOTATOC,[ 161 Tb]Tb-DOTA-LM3, or [ 177 The aim was to evaluate signs of early side effects in mice administered 2 × 10 MBq of [Lu]Lu-DOTA-LM3.

[0125] Early adverse events were assessed based on the RBW of each mouse, which was monitored every two days. Upon reaching the endpoint or at the end of the study (49 days), relevant organs and tissues were collected, weighed, and related to the brain mass and body weight of each mouse. This allowed for comparison of organ-to-brain and organ-to-body mass ratios. Additionally, plasma parameters were measured from blood collected immediately after euthanasia.

[0126] RBW, organ mass, and mass ratios at endpoint: RBW was monitored every 2 days. Mice were euthanized when they reached predefined endpoint criteria or at the end of the study on day 49. RBW, organ mass, organ mass ratios (kidney to brain, liver to brain, spleen to brain), and organ mass to body weight ratios (kidney to body weight, liver to body weight, spleen to body weight) were analyzed for significance using one-way ANOVA with Tukey's multiple comparison post-hoc test. A p value of <0.05 was considered statistically significant.

[0127] Plasma chemistry: Blood was collected from the retrobulbar vein immediately before euthanasia of mice that had reached the endpoint. Creatinine (CRE), blood urea nitrogen (BUN), alkaline phosphatase (ALP), total bilirubin (TBIL), and albumin (ALB) levels were measured in plasma after centrifugation using a dry chemistry analyzer (DRI-CHEM 4000i, Fujifilm, Japan). Mean plasma parameters for each group were analyzed for significance using a one-way ANOVA test with Tukey's multiple comparison post-hoc test. A p value of <0.05 was considered statistically significant.

[0128] No obvious early side effects were observed in the treated mice. Relative body weight increased over the study period, indicating the health of the mice (see Figure 17). On day 6, when the first mice in the control group reached the endpoint (p>0.05), no difference in body weight was observed between the control and treated mice (Table 7).

[0129] There were no significant differences (p>0.05) between treated mice (groups B to E) and untreated control mice (group A) in organ mass, organ-to-brain ratio, or organ-to-body weight ratio at the time of euthanasia (Tables 8 and 9).

[0130] Plasma chemistry tests showed no differences between treated and untreated control mice. BUN values ​​were within acceptable limits in all mice, but were significantly higher than those in control mice (6.2 ± 0.7 mmol / L). 161 Tb]Tb-DOTA-LM3 (9.9 ± 1.5 mmol / L; p < 0.05) and [ 177Lu]Lu-DOTA-LM3 (8.2±1.9 mmol / L; p>0.05) in mice treated with LM3 (Table 10).

[0131] [Table 13] [Table 14] [Table 15] [Table 16]

[0132] (References) All documents cited herein are incorporated by reference. 1.Cives M, Strosberg J. Radionuclide therapy for neuroendocrine tumors. Curr Oncol Rep. 2017;19:9. doi:10.1007 / s11912-017-0567-8. 2. Yao JC, Hassan M, Phan A, Dagohoy C, Leary C, Mares JE, et al. One hundred years after "carcinoid": epidemiology of and prognostic factors for neuroendocrine tumors in 35,825 cases in the United States. J Clin Oncol. 2008;26:3063-72. doi: 10.1200 / JCO.2007.15.4377. 3.Krenning EP, Kooij PP, Bakker WH, Breeman WA, Postema PT, Kwekkeboom DJ, et al. Radiotherapy with a radiolabeled somatostatin analogue, [111In-DTPA-D-Phe1]-octreotide. A case history. Ann N Y Acad Sci. 1994;733:496-506. doi:10.1111 / j.1749-6632.1994.tb17300.x. 4.Kwekkeboom DJ, Krenning EP. Peptide receptor radionuclide therapy in the treatment of neuroendocrine tumors. 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Terbium-161 for PSMA-targeted radionuclide therapy of prostate cancer. Eur J Nucl Med Mol Imaging. 2019;46:1919-30. doi:10.1007 / s00259-019-04345-0. 39.Fani M, Andre JP, Maecke HR. 68Ga-PET: a powerful generator-based alternative to cyclotron-based PET radiopharmaceuticals. Contrast Media Mol Imaging. 2008;3:67-77. doi:10.1002 / cmmi.232. 41.Wang LF, Lin L, Wang MJ, Li Y. The therapeutic efficacy of 177Lu-DOTATATE / DOTATOC in advanced neuroendocrine tumors: a meta-analysis. Medicine (Baltimore). 2020;99:e19304. doi:10.1097 / MD.0000000000019304. 42.Fani M, Del Pozzo L, Abiraj K, Mansi R, Tamma ML, Cescato R, et al. PET of somatostatin receptor-positive tumors using 64Cu- and 68Ga-somatostatin antagonists: the chelate makes the difference. J Nucl Med. Rev. 2011;52:1110–8 doi:10.2967 / jnumed.111.087999. 43.Gracheva N, Muller C, Talip Z, Heinitz S, Koster U, Zeevaart JR, et al. 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[0133] (appendix) JPEG2026027389000020.jpg197169

[0134] [1] (a) a radionuclide, which is terbium-161; (b) a chelating agent for terbium-161, and (c) Radioactive peptides, including peptides or peptide analogs that are somatostatin receptor (SSTR) antagonists. [2] 2. The radioactive peptide of embodiment 1, wherein the somatostatin receptor (SSTR) antagonist is covalently attached to (b). [3] 3. The radioactive peptide according to embodiment 1 or 2, wherein the chelating agent is a cyclic chelating agent, in particular a macrocyclic chelating agent. [4] Aspect 4. The radioactive peptide according to any one of Aspects 1 to 3, wherein the chelating agent is a tetradentate chelating agent. [5] Aspect 5. The radioactive peptide of any one of aspects 1 to 4, wherein the chelating agent comprises four nitrogen atoms. [6] Aspect 6. The radioactive peptide of any one of aspects 1 to 5, wherein the chelating agent is a 12-membered tetraaza ring system. [7] 7. The radioactive peptide of any one of aspects 1 to 6, wherein the chelating agent comprises at least one substituent containing at least one carboxy functional group. [8] Aspect 8. The radioactive peptide of any one of aspects 1 to 7, wherein the chelating agent is DOTA or a DOTA derivative. [9] 9. The radioactive peptide of embodiment 8, wherein the DOTA derivative is selected from the group consisting of: [ka]

[10] 10. The radioactive peptide of embodiment 9, wherein the chelating agent is DOTA (dodecanetetraacetic acid).

[11] 11. The radioactive peptide of any one of aspects 1 to 10, wherein the somatostatin receptor antagonist is covalently attached to the chelator via an amide bond.

[12] 12. The radioactive peptide of any one of aspects 1 to 11, wherein the somatostatin receptor antagonist binds to SSTR-2 (sst2) or is an SSTR-2 (sst2) selective antagonist.

[13] 13. The radioactive peptide of any one of aspects 1 to 12, wherein less than 20% of the administered somatostatin receptor antagonist is internalized by cells.

[14] 14. The radioactive peptide of any one of aspects 1 to 13, wherein the somatostatin receptor antagonist is a cyclic peptide.

[15] 15. The radioactive peptide of any one of aspects 1 to 14, wherein the somatostatin receptor antagonist comprises two cysteine ​​residues, preferably forming a disulfide bridge.

[16] 16. The radioactive peptide of embodiment 15, wherein said somatostatin receptor antagonist comprises cysteine ​​residues at positions 2 and 7 of the peptide.

[17] 17. The radioactive peptide according to any one of Aspects 1 to 16, wherein the somatostatin receptor antagonist consists of 8 to 14 amino acids, preferably 8 to 10 amino acids, and more preferably 8 amino acids.

[18] 18. The radioactive peptide of any one of aspects 1 to 17, wherein the somatostatin receptor antagonist is of formula I: X1-cyclo[D-Cys-X3-X4-Lys-Thr-Cys]-D-Tyr-NH2, wherein X1, X3 and X4 may be selected from naturally or non-naturally occurring D- or L-amino acids.

[19] (i) X1 is selected from the group consisting of naturally occurring Phe, substituted Phe having one or more substitutions on the phenyl ring system, and Cpa; (ii) X3 is selected from the group consisting of Aph(Hor), Leu, L-Agl(NMe, benzoyl), D-Agl(NMe, benzoyl), Aph(Cbm), Tyr, Aph(CONH-OCH3), Tyr, Aph(CONH-OH); and / or (iii) X4 is selected from the group consisting of D-Trp and 19. The radioactive peptide of claim 18, wherein X1 is preferably selected from the group consisting of pNOs-Phe, pCl-Phe and Cpa, X3 is preferably selected from the group consisting of Tyr, Aph(Cbm) and Aph(Hor), and / or X4 is preferably selected from the group consisting of D-Trp and D-Aph(Cbm).

[20] 20. The radioactive peptide of claim 18 or 19, wherein the somatostatin receptor antagonist is selected from the group consisting of LM3 ([p-Cl-Phe-cyclo[D-Cys-Tyr-D-Aph(Cbm)-Lys-Thr-Cys]D-Tyr-NH2]), JR11 (Cpa-cyclo[D-Cys-Aph(Hor)-D-Aph(Cbm)-Lys-Thr-Cys]-D-Tyr-NH2), and BASS (pNO2-Phe-cyclo[D-Cys-Tyr-D-Trp-Lys-Thr-Cys]-D-Tyr-NH2). [twenty one] 21. The radioactive peptide of any one of aspects 1 to 20, wherein the terbium-161 is produced by neutron irradiation of gadolinium-160. [twenty two] 22. The radioactive peptide of any one of aspects 1 to 21, wherein the terbium-161 is non-carrier-added terbium-161 (nca terbium-161). [twenty three] 23. The radioactive peptide of any one of aspects 1 to 22, wherein the antagonist is preferentially taken up by tumors compared to other tissues. [twenty four] 24. The radioactive peptide according to embodiment 23, wherein the ratio of radioactive peptide uptake in tumor cells to blood is at least 50.0, the ratio of radioactive peptide uptake in tumor cells to liver cells is at least 10.0, and / or the ratio of radioactive peptide uptake in tumor cells to kidney cells is at least 2.0, preferably measured 2 hours after administration. [twenty five] 25. The radioactive peptide according to any one of aspects 1 to 24, wherein the radioactive peptide has the structure of the following formula: [ka]

[26] A pharmaceutical composition comprising a radioactive peptide according to any one of aspects 1 to 25 and at least one pharmaceutically acceptable excipient, preferably water.

[27] 27. The pharmaceutical composition according to aspect 26, wherein the composition comprises 0.001 to 1 mg / ml, or 0.01 to 1 mg / ml, or 0.05 to 0.5 mg / ml of the radioactive peptide.

[28] 28. The pharmaceutical composition of aspect 26 or 27, wherein the composition comprises at least one member of the group consisting of gentisic acid, ethanol, acetate, NaCl, and ascorbate / ascorbic acid.

[29] 29. The pharmaceutical composition of embodiment 28, wherein the composition comprises an ascorbate salt.

[30] 30. The pharmaceutical composition according to aspect 29, wherein the composition contains 0.5 mM to 0.5 M, in particular 1 mM to 100 mM, or 10 mM to 100 mM, of ascorbate.

[31] Aspect 31. The pharmaceutical composition according to any one of aspects 26 to 30, wherein the pH value of the composition is pH3.5 to pH6 or pH4 to pH6.

[32] A method for treating a disease or a tumor disease, the method comprising administering a radioactive peptide according to any one of aspects 1 to 25, or a composition according to any one of aspects 24 to 31, to a subject in need of such treatment.

[33] 33. The method of embodiment 32, wherein said subject is suffering from a neuroendocrine neoplasm and / or metastasis thereof, particularly liver metastasis.

[34] 34. The method of embodiment 32 or 33, wherein the neoplasm is a gastropancreatic, bronchopulmonary, thyroid, thymus, or pituitary gland neuroendocrine neoplasm.

[35] 34. The method of embodiment 32 or 33, wherein the neuroendocrine neoplasm is selected from the group consisting of gastroenteropancreatic neuroendocrine neoplasm, pulmonary neuroendocrine tumor, pulmonary neuroendocrine carcinoma, particularly small cell lung cancer thymic neuroendocrine tumor, paraganglioma, pheochromocytoma, e.g., malignant pheochromocytoma, meningioma, medullary thyroid carcinoma, thyroid carcinoma, breast carcinoma, renal cell carcinoma, prostate carcinoma, and non-Hodgkin's lymphoma.

[36] 36. The method of embodiment 35, wherein the neuroendocrine neoplasm is a pancreatic tumor.

[37] The method of any one of aspects 32 to 36, wherein the neuroendocrine neoplasm is grade 1, grade 2, or grade 3.

[38] The neuroendocrine neoplasm is 177 38. The method of any one of aspects 32-37, wherein the patient is stable or refractory to treatment with Lu)-oxodotreotide (Lutathera®) or other radiolabeled somatostatin analogues.

[39] Aspect 39. The method according to any one of aspects 32 to 38, wherein the radioactive peptide according to any one of aspects 1 to 25, or the composition according to any one of aspects 26 to 31, is administered systemically, preferably intravenously. [Brief explanation of the drawings]

[0135] [Figure 1] FIG. 1 shows the chemical structures of somatostatin (SST) analogs. [Figure 2] 1 is a graph of a representative HPLC chromatogram of 161Tb- and 177Lu-labeled peptides. [Figure 3] 1 is a bar graph showing the percentage of intact radioactive peptide examined 1 hour, 4 hours, and 24 hours after preparation and dilution in saline (10 MBq / 250 μL). [Figure 4] Figure 1 shows the in vitro tumor cell uptake and internalization of radioactive peptides after incubation of AR42J tumor cells for 0.5, 2, and 4 hours. [Figure 5] 1 is a graph showing the uptake and internalization rate of radioactive peptides applied at varying molar amounts of peptide using AR42J tumor cells. [Figure 6] 1 is a graph of intracellular uptake and localization studies of radioactive peptides. [Figure 7] 1 is a graph depicting AR42J tumor cell viability assay (MTT). [Figure 8] 1 is a graph showing the viability of AR42J tumor cells (clone formation assay). [Figure 9] Quantification of γ-H2AX-positive AR42J tumor cells representing DNA DSBs after exposure to radiopeptides (2.5 MBq and 10 MBq). [Figure 10-1] Dual-isotope SPECT / CT images of AR42J tumor-bearing mice 2, 4, and 24 hours after injection of [161Tb]Tb-DOTATOC (15 MBq, 0.5 nmol / mouse) and [177Lu]Lu-DOTATOC (15 MBq, 0.5 nmol / mouse), shown as maximum intensity projections (MIPs). [Figure 10-2] Dual-isotope SPECT / CT images of a blocking study performed in AR42J tumor-bearing mice. [Figure 11-1]Dual-isotope SPECT / CT images of AR42J tumor-bearing mice 2, 4, and 24 hours after injection of [161Tb]Tb-DOTA-LM3 (15 MBq, 0.5 nmol / mouse) and [177Lu]Lu-DOTA-LM3 (15 MBq, 0.5 nmol / mouse), shown as maximum intensity projections (MIPs). [Figure 11-2] Dual-isotope SPECT / CT images of a blocking study performed in AR42J tumor-bearing mice. [Figure 12] Biodistribution data obtained in AR42J tumor-bearing mice. [Figure 13] Biodistribution data obtained in AR42J tumor-bearing mice 2 hours after injection of different molar amounts of radioactive peptide. [Figure 14] Biodistribution data obtained after injection of radioactive peptide (0.2 nmol per mouse) into AR42J tumor-bearing mice. [Figure 15] This was a therapeutic study in which 161Tb- and 177Lu-labeled DOTATOC and DOTA-LM3 were administered to AR42J tumor-bearing mice. [Figure 16] Analysis of therapeutic trials performed with 161Tb- and 177Lu-SST agonists and antagonists (2 × 10 MBq; 0.2 nmol) in AR42J tumor-bearing mice. [Figure 17] Relative body weight (RBW) of treated mice.

Claims

[Claim 1] (a) a radionuclide which is terbium-161; (b) a chelating agent for terbium-161, and (c) Radioactive peptides, including peptides or peptide analogs that are somatostatin receptor (SSTR) antagonists.

Images