Method for radiolabeling GRPR antagonist and kit thereof

Abstract

The present invention relates to a method for radiolabeling a GRPR antagonist, such as NeoB, and a kit thereof. In particular, the present invention relates to a method of labeling a gastrin-releasing peptide receptor (GRPR) antagonist with a radioisotope, preferably 68Ga, 67Ga or 64Cu, said method comprising the steps of i. Providing a first vial comprising said GRPR antagonist in dry form, ii. Adding a solution of said radioisotope to said first vial, and iii. Labeling said GRPR antagonist with said radioisotope. The present invention relates to a method for preparing a GRPR antagonist labeled with a radioisotope, the method comprising the steps of i. Providing a solution of said GRPR antagonist and said radioisotope, iii. Mixing the solution obtained in ii. With at least a buffer and incubating for a time sufficient to obtain said GRPR antagonist labeled with said radioisotope, and iv. Optionally adjusting the pH of said solution.

Description

[0001] This application is a divisional application. The application number of the original application is 202080079681.9, the application date is September 16, 2020, the earliest priority date is September 17, 2019, and the name of the invention is "Method for radiolabeling GRPR antagonists and kit thereof". Technical Field

[0002] The present disclosure relates to methods and kits for radiolabeling GRPR antagonists, such as NeoB. Background Art

[0003] Bombesin was originally isolated from the European frog Bombina bombina and was shown to mimic mammalian gastrin-releasing peptide (GRP) and neuromedin B (NMB): Erspamer, V. Discovery, Isolation, and Characterization of Bombesin-like Peptides. Ann NY Acad Sci 547:3-9, 1988; Jensen, RT; Battey, JF; Spindel, ER; Benya, RV International union of pharmacology. LXVIII. Mammalian bombesin receptors: Nomenclature, distribution, pharmacology, signaling, and functions in normal and disease states. [International Union of Pharmacology. LXVIII. Mammalian bombesin receptors: nomenclature, distribution, pharmacology, signaling, and function in normal and disease states] Pharmacol. Rev. [Pharmacological Reviews] 2008, 60, 1-42].

[0004] Gastrin-releasing peptide (GRP) is a bombesin-like peptide growth factor that regulates multiple functions in the gastrointestinal tract and central nervous system, including the release of gastrointestinal hormones, smooth muscle cell contraction, and epithelial cell proliferation. It is a potent mitogen for physiological and tumor tissues and may be involved in growth disorders and carcinogenesis.

[0005] The effects of GRP are primarily mediated through binding to its receptor, the GRP receptor (GRPR), a G protein-coupled receptor originally isolated from a small cell lung cancer cell line. Upregulation of the GRP / GRPR pathway has been reported in several cancers, including breast, prostate, uterine, ovarian, colon, pancreatic, gastric, lung (small cell and non-small cell lung cancer), head and neck squamous cell carcinoma, and various brain tumors and neuromas.

[0006] GRPR is highly overexpressed in prostate cancer, with studies in human prostate cancer cell lines and xenograft models showing high affinity (nM levels) and high tumor uptake (%ID / g), but the relative expression of GRPR in the evolving disease environment from early to late stages has not been fully elucidated [Waters, et al. 2003, Br J Cancer. Jun 2;88(11):1808-1816].

[0007] In patients with colorectal cancer, the presence of GRP and the expression of GRPR have been determined by immunohistochemistry in randomly selected colon cancer samples (including LN and metastatic lesions). More than 80% of the samples aberrantly expressed GRP or GRPR, and more than 60% of the samples expressed both GRP and GRPR, while no expression was observed in the adjacent normal healthy epithelium [Scopinaro F, et al. Cancer Biother Radiopharm [Cancer Biotherapy and Radiopharmaceuticals] 2002, 17 (3): 327-335].

[0008] GRP is physiologically present in pulmonary neuroendocrine cells and plays a role in stimulating lung development and maturation. However, it also appears to be associated with growth disorders and carcinogenesis. GRP stimulation leads to increased release of epidermal growth factor receptor (EGFR) ligands, followed by activation of EGFR and mitogen-activated protein kinase downstream pathways. Using non-small cell lung cancer (NSCLC) cell lines, it has been demonstrated that both EGF and GRP stimulate NSCLC proliferation, while inhibition of EGFR or GRPR can lead to cell death [Shariati F, et al. Nucl Med Commun [Nuclear Medicine Communications] 2014, 35 (6): 620-625].

[0009] In nuclear medicine, peptide receptor agonists have long been the ligand of choice for tracer development and utilization. The rationale for using agonist-based constructs is that the receptor-radioligand complex is internalized, resulting in a high accumulation of radioactivity in the target cell. In the case of radioactive metal-labeled peptides, efficient receptor-mediated endocytosis in response to agonist stimulation provides high in vivo radioactivity uptake in target tissues, which is a key prerequisite for optimizing malignant tumor imaging. However, the paradigm shift occurred when receptor-selective peptide antagonists exhibited better biodistribution (including significantly higher in vivo tumor uptake) compared to highly efficient agonists. Another advantage of GRPR antagonists is safer clinical use (from a current diagnostic perspective, it is more about larger doses for potential therapeutic purposes than tracer doses), because the use of antagonists does not foresee acute biological side effects [Stoykow C, et al. Theragnostics [Therapeutic Diagnostics] 2016, 6 (10): 1641-1650].

[0010] In nonclinical models, [ 68 Ga]-NeoB and [ 177 Lu]-NeoB (also known as [ 68 Ga]-NeoBOMB1 and [ 177 Lu]-NeoBOMB1) shows high affinity for GRPR expressed in breast tumors, prostate tumors, and gastrointestinal stromal tumors (GISTs), as well as low internalization after binding to the specific receptor. The ability of the radiolabeled peptide to target tumors expressing GRPR has been demonstrated in in vivo imaging and biodistribution studies in animal models [Dalm et al. Journal of nuclear medicine 2017, vol. 58(2):293-299; Kaloudi et al. Molecules, 2017 Nov 11;22(11); Paulmichl A et al. Cancer Biother Radiopharm, 2016 Oct;31(8):302-310].

[0011] However, no method has been developed for 68 Ga, 67 Ga or 64 Cu-labeled NeoB to obtain labeled NeoB solution for the purpose of imaging GRPR-positive tumors in human patients. In particular, a rapid, efficient and safe procedure is needed that will provide labeled GRPR antagonists of high radiochemical purity, such as [ 68 Ga]NeoB for intravenous administration in a human subject in need thereof. Summary of the invention

[0012] A first aspect of the present disclosure relates to a method for treating a patient with a radioactive isotope, preferably 68 Ga, 67 Ga or 64 A method for Cu labeling a gastrin releasing peptide receptor (GRPR) antagonist, the method comprising the following steps:

[0013] i. providing a first vial containing the GRPR antagonist in dry form,

[0014] ii. adding the solution of the radioisotope to the first vial, thereby obtaining a solution of the GRPR antagonist and the radioisotope,

[0015] iii. mixing the solution obtained in ii. with at least a buffer and incubating for a time sufficient to obtain said GRPR antagonist labeled with said radioactive isotope, and

[0016] iv. Optionally, adjusting the pH of the solution.

[0017] In a specific embodiment, the radioisotope is 68 Ga and a radiochemical purity of at least 90% as measured in HPLC, and optionally, free 68 The percentage of Ga3+ (in HPLC) is 2% or less, and / or uncomplexed 68 The percentage of Ga3+ species (in ITLC) is 5% or less.

[0018] In other specific embodiments, the radioisotope is 67 Ga and a radiochemical purity of at least 90% as measured in HPLC, and optionally, free 67 The percentage of Ga3+ (in HPLC) is 2% or less, and / or uncomplexed 67 The percentage of Ga3+ species (in ITLC) is 5% or less.

[0019] In other specific embodiments, the radioisotope is 64 Cu and a radiochemical purity of at least 90% as measured in HPLC, and optionally, free 64 The percentage of Cu2+ (in HPLC) is 2% or less, and / or uncomplexed 64 The percentage of Cu2+ species (in ITLC) is 5% or less.

[0020] Preferably, the GRPR antagonist is a NeoB compound having formula (I):

[0021]

[0022] (DOTA-(p-aminobenzylamine-diglycolic acid))-[D-Phe-Gln-Trp-Ala-Val-Gly-His-NH-CH[CH2-CH(CH3)2]2.

[0023] In another aspect, the present disclosure relates to a solution comprising a GRPR antagonist labeled with a radioisotope, which solution is obtainable by or obtained by the method disclosed herein, for use as an injectable solution for in vivo detection of tumors by imaging in a subject in need thereof.

[0024] Another object of the present disclosure is to provide a powder for injection, which comprises the following components in dry form:

[0025] i. A GRPR antagonist having the formula:

[0026] CSP

[0027] in:

[0028] C is a chelating agent capable of chelating the radioactive isotope;

[0029] S is an optional spacer that covalently links C to the N-terminus of P;

[0030] P is a GRPR peptide antagonist, preferably having the following general formula:

[0031] Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Z;

[0032] Xaa1 is absent or selected from the group consisting of the following amino acid residues: Asn, Thr, Phe, 3-(2-thienyl)alanine (Thi), 4-chlorophenylalanine (Cpa), α-naphthylalanine (α-Nal), β-naphthylalanine (β-Nal), 1,2,3,4-tetrahydronorharman-3-carboxylic acid (Tpi), Tyr, 3-iodotyrosine (oI-Tyr), Trp and pentafluorophenylalanine (5-F-Phe) (all L-isomers or D-isomers);

[0033] Xaa2 is Gln, Asn or His;

[0034] Xaa3 is Trp or 1,2,3,4-tetrahydronorharman-3-carboxylic acid (Tpi);

[0035] Xaa4 is Ala, Ser or Val;

[0036] Xaa5 is Val, Ser, or Thr;

[0037] Xaa6 is Gly, sarcosine (Sar), D-Ala, or β-Ala;

[0038] Xaa7 is His or (3-methyl)histidine (3-Me)His;

[0039] Z is selected from -NHOH, -NHNH2, -NH-alkyl, -N(alkyl)2 and -O-alkyl

[0040] or Z is

[0041]

[0042] wherein X is NH (amide) or O (ester) and R1 and R2 are the same or different and are selected from proton, optionally substituted alkyl, optionally substituted alkyl ether, aryl, aryl ether or alkyl-, halogen, hydroxy, hydroxyalkyl, amine, amino, amido or amido-substituted aryl or heteroaryl;

[0043] ii. radiolysis protectants, such as gentisic acid;

[0044] iii. a bulking agent, such as mannitol; and,

[0045] iv. Optionally, a surfactant, such as polyethylene glycol 15 hydroxystearate.

[0046] Typically, the powder for injection contains the following components:

[0047] i. NeoB having the following formula (I), in an amount between 20 and 60 μg, typically 50 μg;

[0048]

[0049] ii. gentisic acid in an amount between 50 and 250 μg, typically 200 μg, and,

[0050] iii. mannitol in an amount between 10 and 30 mg, for example 20 mg, and,

[0051] iv. polyethylene glycol 15 hydroxystearate, in an amount between 250 and 750 μg, for example 500 μg.

[0052] The present disclosure also relates to a kit for implementing the above-mentioned labeling method, the kit comprising

[0053] i. The first vial contains the following components in dry form

[0054] i. NeoB having the following formula (I):

[0055]

[0056] ii. radiolysis protectants, such as gentisic acid,

[0057] iii. Optionally, a bulking agent, such as mannitol, and,

[0058] iv. optionally, a surfactant, such as polyethylene glycol 15 hydroxystearate; and,

[0059] ii. a second vial comprising at least a buffer, preferably in dry form; and,

[0060] iii. Optionally, an accessory kit for eluting radioisotopes produced by the radioisotope generator.

[0061] Another kit disclosed herein comprises a single vial containing the following components in dry form:

[0062] i. NeoB having the following formula (I):

[0063]

[0064] i. Radiolysis protectants, such as gentisic acid,

[0065] ii. Optionally, a bulking agent, such as mannitol,

[0066] iii. Optionally, a surfactant, such as polyethylene glycol 15 hydroxystearate, and,

[0067] iv. at least a buffer, preferably in dry form; and,

[0068] ii. Optionally, an accessory kit for eluting radioisotopes produced by the radioisotope generator.

[0069] For example, the kit may comprise a first or single vial comprising the following components:

[0070] i. NeoB having the following formula (I), in an amount between 20 and 60 μg, typically 50 μg;

[0071]

[0072] ii. gentisic acid, in amounts of 50 and 250 μg, usually 200 μg,

[0073] iii. mannitol in an amount between 10 and 30 mg, for example 20 mg, and,

[0074] iv. Optionally, polyethylene glycol 15 hydroxystearate in an amount between 250 and 750 μg, such as 500 μg. DETAILED DESCRIPTION

[0075] In general, the present disclosure relates to using preferably 68 Ga, 67 Ga or 64 A method for labeling a gastrin releasing peptide receptor (GRPR) antagonist with a radioisotope of Cu, the method comprising the following steps:

[0076] (i) providing a first vial comprising said GRPR antagonist in dry form,

[0077] (ii) adding the solution of the radioisotope to the first vial, thereby obtaining a solution of the GRPR antagonist and the radioisotope,

[0078] (iii) mixing the solution obtained in ii. with at least a buffer and incubating for a time sufficient to obtain said GRPR antagonist labeled with said radioactive isotope, and

[0079] (iv) optionally, adjusting the pH of the solution.

[0080] The radiolabeled GRPR antagonist obtained by the disclosed method is preferably a radiolabeled GRPR antagonist useful as a contrast agent for PET / CT, SPECT or PET / MRI imaging.

[0081] The preferred radiolabeled GRPR antagonists obtained by the disclosed methods are labeled with a radioisotope (preferably 68 Ga, 67 Ga or 64 Cu) labeled NeoB compound, which is suitable for use as a contrast agent for PET / CT, SPECT or PET / MRI imaging. In a preferred embodiment, 67 Ga is used for SPECT imaging, 68 Ga and 64 Cu is used in PET imaging, such as PET / CT or PET / MRI.

[0082] The methods of the present disclosure can advantageously provide excellent radiochemical purity of radiolabeled compounds, for example using 68 The Ga radiolabeled NeoB compound typically has a radiochemical purity of at least 92% as measured by HPLC and, optionally, free 68 The percentage of Ga3+ (in HPLC) is 2% or less, and / or uncomplexed 68 The percentage of Ga3+ species (in ITLC) is 3% or less.

[0083] The determination of radiochemical purity and free68 Determination of Ga3+.

[0084] definition

[0085] The phrases "treatment of" and "treating" include amelioration or cessation of a disease, disorder, or symptom thereof. In particular, with respect to the treatment of tumors, the term "treating" may refer to inhibiting the growth of a tumor or reducing the size of a tumor.

[0086] In line with the International System of Units, "MBq" is the abbreviation for the radioactive unit "megabecquerel".

[0087] As used herein, "PET" stands for Positron Emission Tomography.

[0088] As used herein, "SPECT" stands for single photon emission computed tomography.

[0089] As used herein, "MRI" stands for magnetic resonance imaging.

[0090] As used herein, "CT" stands for computed tomography.

[0091] As used herein, the term "effective amount" or "therapeutically effective amount" of a compound refers to the amount of the compound that will elicit a biological or medical response in a subject (e.g., alleviating symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing disease).

[0092] As used herein, the term "substituted" or "optionally substituted" refers to a group optionally substituted with one or more substituents selected from the group consisting of halogen, -OR', -NR'R", -SR', -SiR'R"R'", -OC(O)R', -C(O)R', -C02R', -C(O)NR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R'", -NR"C(O)OR', -NR-C(NR'R"R'")=NR ””, -NR-C(NR'R")=NR'"-S(O)R', -S(O)2R', -S(O)2NR'R", -NRSO2R', -CN, -NO2, -R', -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy, and fluoro(C1-C4)alkyl, ranging in number from zero to the total number of ring-opening valences on the aromatic ring system; wherein R', R", R'" and R"" can be independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl. When the compounds of the present disclosure contain more than one R group, for example, when more than one of these groups is present, each R group is independently selected to be each R', R", R'" and R"" group.

[0093] As used herein, the term "alkyl" by itself or as part of another substituent refers to a straight or branched chain alkyl functional group having 1 to 12 carbon atoms. Suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl, pentyl and its isomers (e.g., n-pentyl, isopentyl) and hexyl and its isomers (e.g., n-hexyl, isohexyl).

[0094] As used herein, the term "heteroaryl" refers to a polyunsaturated aromatic ring system having a single ring or multiple aromatic rings fused together or linked covalently, containing 5 to 10 atoms, wherein at least one ring is aromatic and at least one ring atom is a heteroatom selected from N, O and S. The nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. Such rings may be fused to an aryl, cycloalkyl or heterocyclyl ring. Non-limiting examples of such heteroaryl groups include furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl, thiatriazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, oxazinyl, dioxazinyl, thiazinyl, triazinyl, indolyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothienyl, isobenzothienyl, indazolyl, benzimidazolyl, benzoxazolyl, purinyl, benzothiadiazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, and quinoxalinyl.

[0095] As used herein, the term "aryl" refers to a polyunsaturated aromatic hydrocarbon radical having a single ring or multiple aromatic rings fused together, containing 6 to 10 ring atoms, at least one of which is aromatic. The aromatic ring may optionally include one to two additional rings (cycloalkyl, heterocyclyl or heteroaryl as defined herein) fused thereto. Suitable aryl groups include phenyl, naphthyl and a benzene ring fused to a heterocyclyl, such as benzopyranyl, benzodioxolyl, benzodioxane, and the like.

[0096] As used herein, the term "halogen" refers to a fluoro (-F), chloro (-Cl), bromo (-Br), or iodo (-I) group.

[0097] As used herein, the term "optionally substituted aliphatic chain" refers to an optionally substituted aliphatic chain having 4 to 36 carbon atoms, preferably 12 to 24 carbon atoms.

[0098] As used herein, the term "chelator" refers to a molecule having a functional group, such as an amine or carboxyl group, suitable for complexing a radioisotope via non-covalent bonding.

[0099] As used herein, the term "radiolysis protectant" refers to a stabilizer that protects an organic molecule from radiolytic degradation, for example, when the gamma rays emitted from a radionuclide cleave the bonds formed between the atoms of the organic molecule and the free radicals, those free radicals are then scavenged by the stabilizer, which prevents the free radicals from undergoing any other chemical reactions that may result in undesirable, potentially ineffective or even toxic molecules. Therefore, these stabilizers are also referred to as "free radical scavengers" or simply "free radical scavengers". Other alternative terms for those stabilizers are "radiostability enhancers", "radiostabilizers" or simply "quenchers".

[0100] As used herein, the term "radiochemical purity" refers to the percentage of the stated radionuclide present in the stated chemical or biological form.Radiochromatography, such as HPLC or instant thin layer chromatography (iTLC), is the most commonly accepted method for determining radiochemical purity in nuclear pharmacy.

[0101] If not otherwise specified herein, "about" means ±20%, preferably ±10%, more preferably ±5%, even more preferably ±2%, even more preferably ±1%. The term "about" is used herein synonymously with "ca."

[0102] Step (i) providing a first vial containing the GRPR antagonist in dry form

[0103] GRPR antagonists

[0104] As used herein, the GRPR antagonist has the formula:

[0105] CSP

[0106] in:

[0107] C is a chelating agent capable of chelating radioactive isotopes;

[0108] S is an optional spacer that covalently links C to the N-terminus of P;

[0109] P is a GRPR peptide antagonist, preferably having the following general formula:

[0110] Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Z;

[0111] Xaa1 is absent or selected from the group consisting of the following amino acid residues: Asn, Thr, Phe, 3-(2-thienyl)alanine (Thi), 4-chlorophenylalanine (Cpa), α-naphthylalanine (α-Nal), β-naphthylalanine (β-Nal), 1,2,3,4-tetrahydronorharman-3-carboxylic acid (Tpi), Tyr, 3-iodotyrosine (oI-Tyr), Trp and pentafluorophenylalanine (5-F-Phe) (all L-isomers or D-isomers);

[0112] Xaa2 is Gln, Asn or His;

[0113] Xaa3 is Trp or 1,2,3,4-tetrahydronorharman-3-carboxylic acid (Tpi);

[0114] Xaa4 is Ala, Ser or Val;

[0115] Xaa5 is Val, Ser, or Thr;

[0116] Xaa6 is Gly, sarcosine (Sar), D-Ala, or β-Ala;

[0117] Xaa7 is His or (3-methyl)histidine (3-Me)His;

[0118] Z is selected from -NHOH, -NHNH2, -NH-alkyl, -N(alkyl)2 and -O-alkyl

[0119] or Z is

[0120]

[0121] wherein X is NH (amide) or O (ester) and R1 and R2 are the same or different and are selected from proton, optionally substituted alkyl, optionally substituted alkyl ether, aryl, aryl ether or alkyl-, halogen, hydroxy, hydroxyalkyl, amine, amino, amido or amido substituted aryl or heteroaryl.

[0122] According to an embodiment, Z is selected from one of the following formulae, wherein X is NH or O:

[0123]

[0124] According to an embodiment, P is DPhe-Gln-Trp-Ala-Val-Gly-His-Z; wherein Z is as defined above.

[0125] According to an embodiment, P is DPhe-Gln-Trp-Ala-Val-Gly-His-Z;

[0126] Z is selected from Leu-ψ(CH2N)-Pro-NH2 and NH-CH(CH2-CH(CH3)2)2

[0127] or Z is

[0128]

[0129] wherein X is NH(amide) and R2 is CH(CH2-CH(CH3)2, and R1 and R2 are the same or different (CH2N)-Pro-NH2.

[0130] According to an embodiment, the chelating agent C is obtained by grafting a chelating agent selected from the following list:

[0131]

[0132] In a specific embodiment, C is obtained by grafting a chelating agent selected from the group consisting of:

[0133]

[0134] According to an embodiment, S is selected from the group consisting of:

[0135] a) an aromatic group-containing residue having the formula:

[0136]

[0137] wherein PABA is p-aminobenzoic acid, PABZA is p-aminobenzylamine, PDA is phenylenediamine, and PAMBZA is (aminomethyl)benzylamine;

[0138] b) dicarboxylic acids, ω-aminocarboxylic acids, ω-diaminocarboxylic acids or diamines of the formula:

[0139]

[0140] wherein DIG is diglycolic acid and IDA is iminodiacetic acid;

[0141] c) PEG spacers of various chain lengths, in particular PEG spacers selected from the group consisting of:

[0142]

[0143] d) α-amino acids and β-amino acids, either in single chains or in homologous chains of different chain lengths or in heterologous chains of different chain lengths, in particular:

[0144]

[0145] GRP(1-18), GRP(14-18), GRP(13-18), BBN(1-5) or [Tyr4]BB(1-5); or

[0146] e) A combination of a, b, c and d.

[0147] According to a specific embodiment, the radiolabeled GRPR antagonist is selected from the group consisting of compounds having the following formula:

[0148]

[0149] wherein C and P are as defined above, M is a radioisotope, preferably M is selected from 68 Ga, 67 Ga or 64 Cu.

[0150] According to a preferred embodiment, the GRPR-antagonist is NeoB (also known as NeoBOMB1) having formula (I):

[0151]

[0152] (DOTA-(p-aminobenzylamine-diglycolic acid))-[D-Phe-Gln-Trp-Ala-Val-Gly-His-NH-CH[CH2-CH(CH3)2]2.

[0153] According to an embodiment, the radiolabeled GRPR-antagonist is radiolabeled NeoB2 having the formula (III):

[0154]

[0155] (M-N4(p-aminobenzylamine-diglycolic acid)-[D-Phe-Gln-Trp-Ala-Val-Gly-His-NH-CH[CH2-CH(CH3)2]2;

[0156] Where M is the radionuclide.

[0157] According to another specific embodiment, the GRPR-antagonist is ProBOMB1 having the following formula (II):

[0158]

[0159] (DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ψ(CH2N)-Pro-NH2)

[0160] Synthesis of compounds of formula (I), (II) and (III)

[0161] Compounds of formula (I), (II) and (III) can be synthesized using the method disclosed in the reference "Positron Emission Tomography Imaging of the Gastrin-Releasing Peptide Receptor with a Novel Bombesin Analogue" ACS Omega 2019, 4, 1470-1478.

[0162] The first vial containing the GRPR antagonist

[0163] In certain embodiments, the radiolabeling method uses a single vial kit. In this embodiment, the first vial contains the GRPR antagonist and buffer, both in dry form.

[0164] Alternatively, the radiolabeling method uses a two-vial kit. In this example, the first vial contains the GRPR antagonist and the second vial contains the buffer.

[0165] For example, the GRPR antagonist, typically a NeoB compound, is contained in the first vial in an amount between 20 and 60 μg, typically 50 μg.

[0166] The first vial optionally contains additional excipients, such as radiolysis protectants, bulking agents, and tensioactive agents.

[0167] In a preferred embodiment, gentisic acid may be used as a radiolysis protectant, preferably in an amount between 50 and 250 μg, typically 200 μg.

[0168] In a preferred embodiment, mannitol may be used as a bulking agent, for example in an amount between 10 and 30 mg, typically 20 mg.

[0169] In a preferred embodiment, polyethylene glycol 15 hydroxystearate can be used as a surfactant, for example in an amount between 250 and 750 μg, typically 500 μg. The surfactant advantageously reduces nonspecific adhesion of the NeoB compound to glass or plastic surfaces, thereby optimizing the yield of the labeling process.

[0170] A preferred example of the first vial (vial 1 of a two-vial kit) is given in the Examples.

[0171] The first vial is preferably obtained by freeze drying using methods well known in the art. Thus, the first vial can be provided in a freeze-dried or spray-dried form.

[0172] As used herein, a buffer is a buffer suitable for obtaining a pH between 3.0 and 6.0, preferably 3.0 and 4.0 in the incubation step (iii). A "buffer of pH 3.0 to 6.0, preferably 3.0 to 4.0" may advantageously be a formic acid buffer with sodium hydroxide.

[0173] The buffer may be further contained in the first vial in the embodiment of the kit using a single vial, or may be further contained in a separate second vial in the embodiment of the kit using two vials.

[0174] Step (ii) adding the radioactive isotope solution to the first vial

[0175] Radioisotopes used in radiolabeling methods include those suitable for use as contrast agents in PET and SPECT imaging, including:

[0176] 111 In, 133m In, 99m Tc, 94m Tc, 67 Ga, 66 Ga, 68 Ga, 52 Fe, 72 As, 97 Such as 203 Pb, 62 Cu, 64 Cu, 86 Y. 51 Cr, 52m Mn, 157 Gd, 169 Yb, 172 Tm, 117m Sn, 89 Zr, 43 Sc, 44 Sc.

[0177] According to a preferred embodiment, the radioisotope is 68 Ga, 67 Ga or 64 Cu. In a preferred embodiment, 67 Ga is used for SPECT imaging, 68 Ga and 64 Cu is used in PET imaging, such as PET / CT or PET / MRI.

[0178] The metal ion of such a radioisotope is capable of forming a non-covalent bond with a functional group of the chelating agent (eg, a carboxylic acid of the GRPR antagonist).

[0179] In a specific embodiment, said solution of said radioactive isotope is an eluate obtained from:

[0180] i. Producing radioisotopes from parent non-radioactive elements by means of radioisotope generators,

[0181] ii. separating the radioisotope from the parent non-radioactive element by elution in HCl as elution solvent,

[0182] iii. Recover the eluate,

[0183] Thereby a solution of the radioisotope in HCl is obtained.

[0184] The radioisotope 68 The solution of Ga is the eluate usually obtained from the following steps:

[0185] i. From the parent element through the generator 68 Ge 68 Ga element,

[0186] ii. Optionally, by making the element 68 Ge / 68 Ga passes through a suitable column to generate 68 Ga element and 68 Ge element is separated and eluted in HCl 68 Ga,

[0187] Thereby a solution of the radioisotope in HCl is obtained.

[0188] This kind of 68 Ge / 68 Ga generator produces 68Ga methods are well known in the art, as described, for example, in: Martiniova L, et al. Gallium-68 in Medical Imaging. Curr Radiopharm. 2016;9(3):187-20; Dash A, Chakravarty Radionuclide generators: the prospect of availing PET radiotracers to meet current clinical needs and future research demands. R Am J Nucl Med Mol Imaging. 2019 Feb 15;9(1):30-66.

[0189] Contains radioactive isotopes 68 The solution of Ga may be an eluate preferably obtained from a cyclotron production. Such production is described, for example, in Am J Nucl Med Mol Imaging [American Journal of Nuclear Medicine and Molecular Imaging] 2014; 4(4): 303-310 or BJB Nelson et al. / Nuclear Medicine and Biology [Nuclear Medicine and Biology] 80-81 (2020) 24-31.

[0190] Preferably, 68 Ga can be produced by a cyclotron, more preferably using a proton beam with an energy between 8 and 18 MeV (even more preferably between 11 and 14 MeV). 68 Ga can be delivered using either solid or liquid target systems 68 Zn(p,n) 68 Ga reaction is produced. The target is enriched 68 Zn metal or 68 After irradiation, the target was transferred for further chemical processing, where ion exchange chromatography was used to separate 68 Ga. 68 Ga was eluted in HCl solution.

[0191] Alternatively, the radioisotope is 67 Ga. Produced using zinc (enriched or natural) or copper or germanium targets with protons, deuterons, alpha particles or helium (III) as bombarding particles 67Various methods for producing Ga have been summarized and reported as follows: Helus, F., Maier-Borst, W., 1973. A comparative investigation of methods used to produce 67Ga with acyclotron. In Radiopharmaceuticals and Labelled Compounds, Vol. 1, IAEA, Vienna, pp. 317-324, ML Thakur Gallium-67 and indium-111 radiopharmaceuticals Int. J. Appl. Rad. Isot., 28 (1977), pp. 183-201, and T.,Holtebekk,T.,1993.Production of 67 Ga at Oslo cyclotron 67 Ga]. University of Oslo Report [Oslo University Report] OUP8-3-1, pp.3-5. Bombardment with medium energy protons (up to 64 MeV) nat Ge targets are also suitable for methods of producing 67Ga, as described in: T Horiguchi, H Kumahora, H Inoue, Y Yoshizawa Excitation functions of Ge(p,xnyp)reactions and productionof 68Ge [Excitation functions of Ge(p,xnyp)reactions and production of 68Ge], Int. J. Appl. Radiat. Isot. [International Journal of Applied Radiation and Isotopes], 34 (1983), pp. 1531-1535.

[0192] Preferably, 67 Ga can be produced by cyclotron. 68 Zn(p,2n) 67 Ga production 67The method of Ga is well known in the art, for example as described in Alirezapour B et al. Iranian Journal of Pharmaceutical Research (2013), 12(2): 355-366. More preferably, the method uses a proton beam with an energy between 10 and 40 MeV. 67 Ga can be 67 Zn(p,n) 67 Ga or 68 Zn(p,2n) 67 Ga reactions are produced using solid or liquid target systems. The target consists of an enriched 67 Zn or 68 After irradiation, the target is transferred for further chemical processing, where ion exchange chromatography is used to separate 67 Ga. Finally, it is evaporated from the HCl aqueous solution to obtain 67 GaCl3 can then be added to the individual vials for the labeling process.

[0193] Alternatively, the radioisotope is obtained from cyclotron production 64 Cu. Such a production method is described, for example, in WO 2013 / 029616.

[0194] generally, 64 Cu can be produced by cyclotron, preferably using a proton beam with an energy between 11 and 18 MeV. 64 Cu can be obtained using solid or liquid target systems. 64 Ni(p,n) 64 The target is produced by 64 After irradiation, the target is transferred for further chemical processing, where ion exchange chromatography is used to separate 64 Cu. Finally, it is evaporated from HCl aqueous solution to obtain 64 CuCl2 can then be added to the first vial for the labeling process.

[0195] Step (iii) mixing the solution obtained in step (ii) with at least a buffer and incubating for a time sufficient to obtain the GRPR antagonist labeled with the radioisotope

[0196] After mixing the first vial containing the GRPR antagonist (e.g., NeoB compound) with the vial containing the radioactive isotope (typically 68 Ga, 67 Ga or 64After mixing a solution of 5% Cu) in a suitable buffer as disclosed above, radiolabeling is initiated.

[0197] In a specific embodiment, the incubation step is performed at a temperature of 80°C to 100°C, preferably 90°C to 100°C, typically about 95°C.

[0198] In particular embodiments, the incubation step is carried out for a period of time comprising 5 to 10 minutes, such as 6 to 8 minutes, typically about 7 minutes.

[0199] At the end of the labeling process, radioactive isotopes (e.g. 68Ga, 67 The radioactive isotope is chelated with a chelating agent with a specific affinity to Ga or 64Cu to chelate the unreacted portion of the isotope. This complex formed by the sequestering agent and the unreacted radioisotope can then be discarded to increase the radiochemical purity after radiolabeling.

[0200] use 68 Preferred embodiment of the method for radiolabeling NeoB with Ga

[0201] This disclosure more specifically relates to 68 A method for Ga labeling a NeoB compound having formula (I),

[0202]

[0203] (DOTA-(p-aminobenzylamine-diglycolic acid))-[D-Phe-Gln-Trp-Ala-Val-Gly-His-NH-CH[CH2-CH(CH3)2]2; the method comprises the following steps:

[0204] i. providing a first vial containing about 50 μg of NeoB and 50 to 250 μg of gentisic acid in dry form, ii. 68 A solution of Ga in HCl was added to the first vial,

[0205] iii. The solution obtained in ii. is mixed with a buffer for adjusting the pH in the range of 3.0 to 4.0 and incubated for a period of time sufficient to obtain 68 The time of Ga-labeled NeoB compound,

[0206] iv. Optionally adjusting the pH of the solution.

[0207] In a specific embodiment of the method, the 68 Said solution of Ga in HCl is the eluate obtained from the following steps:

[0208] i. From the parent element through the generator 68 Ge 68Ga element,

[0209] ii. Optionally, by making the element 68 Ga / 68 Ge passes through the appropriate column to generate 68 Ga element and 68 Ge element is separated and eluted in HCl 68 Ga,

[0210] Thereby a solution of the radioisotope in HCl is obtained.

[0211] Typically, the buffer consisted of 60 mg of formic acid and 56.5 mg of sodium hydroxide.

[0212] Advantageously, in certain embodiments, simple labeling of GRPR antagonists can be performed using commercially available 68 Ge / 68 Ga generator in HCl 68 The Ga eluate is obtained without any treatment of the eluate or any additional purification steps.

[0213] Powder for injection

[0214] The present disclosure also relates to a powder for injection, which comprises the following components in dry form:

[0215] i. A GRPR antagonist as defined above - typically NeoB having formula (I) as defined above;

[0216] ii. radiolysis protectants, such as gentisic acid;

[0217] iii. a bulking agent, such as mannitol; and,

[0218] iv. Optionally, a surfactant, such as polyethylene glycol 15 hydroxystearate.

[0219] The preferred embodiment includes the following components:

[0220] i. NeoB having the following formula (I), in an amount between 20 and 60 μg, typically 50 μg;

[0221]

[0222] ii. gentisic acid in an amount between 50 and 250 μg, typically 200 μg, and,

[0223] iii. mannitol in an amount between 10 and 30 mg, for example 20 mg, and,

[0224] iv. polyethylene glycol 15 hydroxystearate, in an amount between 250 and 750 μg, for example 500 μg.

[0225] Radiolabeling kit of the present disclosure

[0226] The present disclosure also relates to a kit for implementing the above-mentioned labeling method, the kit comprising

[0227] i. The first vial contains the following components in dry form

[0228] i. a GRPR antagonist as defined above,

[0229] ii. radiolysis protectants, such as gentisic acid,

[0230] iii. Optionally, a bulking agent, such as mannitol, and,

[0231] iv. optionally, a surfactant, such as polyethylene glycol 15 hydroxystearate; and,

[0232] ii. a second vial comprising at least a buffer, preferably in dry form; and,

[0233] iii. Optionally, an accessory kit for eluting radioisotopes produced by the radioisotope generator.

[0234] Preferably, the first or single vial comprises the following components:

[0235] i. NeoB having the following formula (I), in an amount between 20 and 60 μg, typically 50 μg;

[0236]

[0237] ii. gentisic acid, in an amount between 50 and 250 μg, usually 200 μg,

[0238] iii. mannitol in an amount between 10 and 30 mg, for example 20 mg, and,

[0239] iv. Optionally, polyethylene glycol 15 hydroxystearate in an amount between 250 and 750 μg, such as 500 μg.

[0240] The second vial or single vial may contain a buffer for maintaining a pH between 3.0 and 4.0. For example, the second vial contains formic acid and sodium hydroxide as a buffer.

[0241] Preferably, all components of the first, second or single vial are in dry form.

[0242] The radioisotope used to label the GRPR antagonist may be provided with the kit as a ready-to-use product, i.e., for mixing and incubation with the first vial and buffer provided with the kit, or alternatively may be used immediately before and shortly before mixing and incubation with the first vial and buffer, particularly in cases where the radioisotope has a relatively short half-life, e.g. 68 Ga, 67 Ga and 64 In the case of Cu, elution from a radioisotope generator.

[0243] Preferably, the components are inserted into a sealed container which may be packaged together with instructions for performing the methods according to the present disclosure.

[0244] The kit can also be used as part of an automated system or remote control mechanism system to automatically perform the elution and / or subsequent mixing and heating of the gallium 69 generator. In this embodiment, the vial containing the GRPR antagonist (the first vial) is directly connected to the elution system and / or the heating system.

[0245] The kit may be particularly suitable for use in the methods disclosed in the next section.

[0246] In a specific embodiment, the GRPR-antagonist is NeoB as defined above.

[0247] Uses of the kit according to the present disclosure

[0248] The kit as defined above may be particularly suitable for use in the labelling method disclosed in the preceding paragraph.

[0249] Advantageously, the invention comprises the use of a radioisotope (e.g. 68 Ga, 67 Ga or 64 The solution of GRPR antagonist (eg, NeoB compound) labeled with Cu can be obtained by the labeling method disclosed in the previous section or by the labeling method disclosed in the previous section.

[0250] Such solutions may be ready-to-use injectable solutions, for example for in vivo detection of tumors by imaging in a subject in need thereof.

[0251] In certain aspects, the subject is a mammal, such as, but not limited to, a rodent, a canine, a feline, or a primate. In preferred aspects, the subject is a human.

[0252] The requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, JB Lippincott Company, Philadelphia, Pennsylvania, eds. Banker and Chalmers, pp. 238-250 (1982), and SHP Handbook on Injectable Drugs, Trissel, 15th ed., pp. 622-630 (2009)).

[0253] Typically, the solution, used as an injectable solution, provides a single dose of 150-250 MBq of [68Ga]-NeoB for administration to a subject in need thereof.

[0254] In a specific embodiment, the subject in need is a subject suffering from cancer, more specifically, a patient suffering from a tumor selected from prostate cancer, breast cancer, small cell lung cancer, colon cancer, gastrointestinal stromal tumor, gastrinoma, glioma, glioblastoma, renal cell carcinoma, gastroenteropancreatic neuroendocrine tumor, esophageal squamous cell tumor, neuroblastoma, head and neck squamous cell carcinoma, and ovarian tumors, endometriomas and pancreatic tumors that may be GRPR-positive.

[0255] Typically, PET / MRI, SPECT or PET / CT imaging can be performed between 1 hour and 4 hours after administration of the radiolabeled GRPR antagonist to the subject, and more preferably between 2 and 3 hours after administration of the radiolabeled GRPR antagonist to the subject.

[0256] Implementation

[0257] The following specific embodiments are disclosed:

[0258] 1. A method preferably comprising: 68 Ga, 67 Ga or 64 A method for labeling a gastrin releasing peptide receptor (GRPR) antagonist with a radioisotope of Cu, the method comprising the following steps:

[0259] i. providing a first vial containing the GRPR antagonist in dry form,

[0260] ii. adding the solution of the radioisotope to the first vial, thereby obtaining a solution of the GRPR antagonist and the radioisotope,

[0261] iii. mixing the solution obtained in ii. with at least a buffer and incubating for a time sufficient to obtain said GRPR antagonist labeled with said radioactive isotope, and

[0262] iv. Optionally, adjusting the pH of the solution.

[0263] 2. The method of embodiment 1, wherein the first vial of step i. is a reaction vial containing the GRPR antagonist and a buffer, preferably both in dry form.

[0264] 3. The method as described in Example 1, wherein step iii comprises mixing the solution obtained in ii. with a reaction solution comprising at least a buffer and incubating for a time sufficient to obtain the GRPR antagonist labeled with the radioactive isotope.

[0265] 4. The method of any one of embodiments 1-3, wherein the solution having the radioactive isotope further comprises a buffer.

[0266] 5. The method of any one of embodiments 1-4, wherein the radioisotope is 68 Ga and a radiochemical purity of at least 92% as measured in HPLC, and optionally, free 68 The percentage of Ga3+ (in HPLC) is 2% or less, and / or uncomplexed 68 The percentage of Ga3+ species (in ITLC) is 3% or less.

[0267] 6. The method of any one of embodiments 1-4, wherein the radioisotope is 67 Ga and a radiochemical purity of at least 92% as measured in HPLC, and optionally, free 67 The percentage of Ga 3+ (in HPLC) is 2% or less, and / or uncomplexed 67 The percentage of Ga 3+ species (in ITLC) is 3% or less.

[0268] 7. The method of any one of embodiments 1-4, wherein the radioisotope is 64 Cu and a radiochemical purity of at least 92% as measured in HPLC, and optionally, free 64 The percentage of Cu2+ (in HPLC) is 2% or less, and / or uncomplexed 64 The percentage of Cu2+ species (in ITLC) is 3% or less.

[0269] 8. The method of any one of embodiments 1-7, wherein the GRPR antagonist is a compound having the formula:

[0270] CSP

[0271] in:

[0272] C is a chelating agent capable of chelating the radioactive isotope;

[0273] S is an optional spacer that covalently links C to the N-terminus of P;

[0274] P is a GRPR peptide antagonist, preferably having the following general formula:

[0275] Xaa1-Xaa2—Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Z;

[0276] Xaa1 is absent or selected from the group consisting of the following amino acid residues: Asn, Thr, Phe, 3-(2-thienyl)alanine (Thi), 4-chlorophenylalanine (Cpa), α-naphthylalanine (α-Nal), β-naphthylalanine (β-Nal), 1,2,3,4-tetrahydronorharman-3-carboxylic acid (Tpi), Tyr, 3-iodotyrosine (oI-Tyr), Trp and pentafluorophenylalanine (5-F-Phe) (all L-isomers or D-isomers);

[0277] Xaa2 is Gln, Asn or His;

[0278] Xaa3 is Trp or 1,2,3,4-tetrahydronorharman-3-carboxylic acid (Tpi);

[0279] Xaa4 is Ala, Ser or Val;

[0280] Xaa5 is Val, Ser, or Thr;

[0281] Xaa6 is Gly, sarcosine (Sar), D-Ala, or β-Ala;

[0282] Xaa7 is His or (3-methyl)histidine (3-Me)His;

[0283] Z is selected from -NHOH, -NHNH2, -NH-alkyl, -N(alkyl)2 and -O-alkyl

[0284] or Z is

[0285]

[0286] wherein X is NH (amide) or O (ester) and R1 and R2 are the same or different and are selected from proton, optionally substituted alkyl, optionally substituted alkyl ether, aryl, aryl ether or alkyl-, halogen, hydroxy, hydroxyalkyl, amine, amino, amido or amido substituted aryl or heteroaryl; and,

[0287] 9. The method of embodiment 8, wherein P is DPhe-Gln-Trp-Ala-Val-Gly-His-NH-CH(CH2-CH(CH3)2)2.

[0288] 10. The method of embodiment 8, wherein the GRPR antagonist is a NeoB compound having formula (I):

[0289]

[0290] DOTA-(p-aminobenzylamine-diglycolic acid))-D-Phe-Gln-Trp-Ala-Val-Gly-His-NH-CH[CH2-CH(CH3)2]2.

[0291] 11. The method of any one of embodiments 1-10, wherein the GRPR antagonist is contained in the first vial in an amount between 20 and 60 μg, typically 50 μg.

[0292] 12. The method of any one of embodiments 1-11, wherein the first vial further comprises gentisic acid as a radiolysis protectant, preferably in an amount between 50 and 250 μg, typically 200 μg.

[0293] 13. The method of any one of embodiments 1-12, wherein the first vial further comprises mannitol as a bulking agent, for example in an amount between 10 and 30 mg, typically 20 mg.

[0294] 14. The method of any one of embodiments 1-13, wherein the first vial further comprises polyethylene glycol 15 hydroxystearate as a surfactant, for example in an amount between 250 and 750 μg, typically 500 μg.

[0295] 15. The method of any one of embodiments 1-14, wherein the buffer is present in an amount suitable to obtain a pH between 3.0 and 4.0 in the incubation step (iii).

[0296] 16. The method of any one of embodiments 1-15, wherein the buffer comprises formic acid and sodium hydroxide.

[0297] 17. The method of any one of embodiments 1-16, wherein the incubation step is performed at a temperature of 80°C to 100°C, preferably 90°C to 100°C, typically about 95°C.

[0298] 18. The method of any one of embodiments 1-17, wherein the incubation step is performed for a period of time comprising 5 to 10 minutes, such as 6 to 8 minutes, typically about 7 minutes.

[0299] 19. The method of any one of embodiments 1-18, wherein the solution of the radioactive isotope is an eluate obtained from:

[0300] i. Producing radioisotopes from parent non-radioactive elements by means of radioisotope generators,

[0301] ii. separating the radioisotope from the parent non-radioactive element by elution in HCl as elution solvent,

[0302] iii. Recover the eluate,

[0303] Thereby a solution of the radioisotope in HCl is obtained.

[0304] 20. A method of 68 A method for Ga labeling a NeoB compound having formula (I),

[0305]

[0306] (DOTA-(p-aminobenzylamine-diglycolic acid))-D-Phe-Gln-Trp-Ala-Val-Gly-His-NH-CH[CH2-CH(CH3)2]2, the method comprising the following steps:

[0307] i. providing a first vial containing about 50 μg of NeoB and 50 to 250 μg of gentisic acid in dry form, ii. 68 A solution of Ga in HCl was added to the first vial,

[0308] iii. The solution obtained in ii. is mixed with a buffer for adjusting the pH in the range of 3.0 to 4.0 and incubated for a period of time sufficient to obtain 68 The time of Ga-labeled NeoB compound,

[0309] iv. Optionally adjusting the pH of the solution.

[0310] 21. The method of embodiment 20, wherein 68 Said solution of Ga in HCl is the eluate obtained from the following steps:

[0311] i. From the parent element through the generator 68 Ge 68 Ga element,

[0312] ii. Optionally, by making the element 68 Ga / 68 Ge passes through the appropriate column to generate 68Ga element and 68 Ge element is separated and eluted in HCl 68 Ga,

[0313] Thereby a solution of the radioisotope in HCl is obtained.

[0314] 22. The method of embodiment 20 or 21, wherein the buffer consists of 60 mg of formic acid and 56.5 mg of sodium hydroxide.

[0315] 23. The method of any one of embodiments 20-22, wherein the incubation step is performed at a temperature of 80°C to 100°C, preferably 90°C to 100°C, typically about 95°C.

[0316] 24. The method of any one of embodiments 20-23, wherein the incubation step is performed for a period of time comprising 5 to 10 minutes, such as 6 to 8 minutes, typically about 7 minutes.

[0317] 25. A solution comprising a GRPR antagonist labeled with a radioisotope, which solution is obtainable by the method of any one of Examples 1-24 or obtained by the method of any one of Examples 1-24, for use as an injectable solution for in vivo detection of tumors by imaging in a subject in need thereof.

[0318] 26. A method comprising 68 A solution of a Ga-labeled NeoB compound obtainable by or obtained by the method of any one of Examples 20-24 is used as an injectable solution for in vivo detection of tumors by imaging in a subject in need thereof.

[0319] 27. A solution for use according to embodiment 25 or embodiment 26, wherein the tumor is selected from tumors expressing GRPR, preferably, the tumor expressing GRPR is selected from GRPR-positive prostate cancer, breast cancer, small cell lung cancer, colon cancer, gastrointestinal stromal tumor, gastrinoma, renal cell carcinoma, gastroenteropancreatic neuroendocrine tumor, esophageal squamous cell tumor, neuroblastoma, head and neck squamous cell carcinoma, and ovarian tumors, endometriomas and pancreatic tumors showing neoplasia-related vasculature.

[0320] 28. A powder for injection, comprising the following components in dry form:

[0321] i. A GRPR antagonist having the formula:

[0322] CSP

[0323] in:

[0324] C is a chelating agent capable of chelating the radioactive isotope;

[0325] S is an optional spacer that covalently links C to the N-terminus of P;

[0326] P is a GRPR peptide antagonist, preferably having the following general formula:

[0327] Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Z;

[0328] Xaa1 is absent or selected from the group consisting of the following amino acid residues: Asn, Thr, Phe, 3-(2-thienyl)alanine (Thi), 4-chlorophenylalanine (Cpa), α-naphthylalanine (α-Nal), β-naphthylalanine (β-Nal), 1,2,3,4-tetrahydronorharman-3-carboxylic acid (Tpi), Tyr, 3-iodotyrosine (oI-Tyr), Trp and pentafluorophenylalanine (5-F-Phe) (all L-isomers or D-isomers);

[0329] Xaa2 is Gln, Asn or His;

[0330] Xaa3 is Trp or 1,2,3,4-tetrahydronorharman-3-carboxylic acid (Tpi);

[0331] Xaa4 is Ala, Ser or Val;

[0332] Xaa5 is Val, Ser, or Thr;

[0333] Xaa6 is Gly, sarcosine (Sar), D-Ala, or β-Ala;

[0334] Xaa7 is His or (3-methyl)histidine (3-Me)His;

[0335] Z is selected from -NHOH, -NHNH2, -NH-alkyl, -N(alkyl)2 and -O-alkyl

[0336] or Z is

[0337]

[0338] wherein X is NH (amide) or O (ester) and R1 and R2 are the same or different and are selected from proton, optionally substituted alkyl, optionally substituted alkyl ether, aryl, aryl ether or alkyl-, halogen, hydroxy, hydroxyalkyl, amine, amino, amido or amido-substituted aryl or heteroaryl;

[0339] ii. radiolysis protectants, such as gentisic acid;

[0340] iii. a bulking agent, such as mannitol; and,

[0341] iv. Optionally, a surfactant, such as polyethylene glycol 15 hydroxystearate.

[0342] 29. The powder for injection as described in Example 28, wherein the GRPR antagonist is a NeoB compound having the following formula (I):

[0343]

[0344] 30. The powder for injection as described in Example 29, wherein the amount of the NeoB compound contained is between 20 and 60 μg, usually 50 μg.

[0345] 31. The powder for injection as described in any one of Examples 28-30, wherein the amount of gentisic acid contained therein is between 50 and 250 μg, typically 200 μg.

[0346] 32. The powder for injection as described in any one of embodiments 28-31, wherein the bulking agent is mannitol, and the amount thereof is between 10 and 30 mg, for example 20 mg.

[0347] 33. The powder for injection as described in any one of Examples 28-32, wherein the surfactant is polyethylene glycol 15 hydroxystearate, and the amount thereof is between 250 and 750 μg, for example, 500 μg.

[0348] 34. The powder for injection according to any one of embodiments 28-33, comprising the following components:

[0349] - NeoB having the following formula (I), in an amount between 20 and 60 μg, typically 50 μg;

[0350]

[0351] - gentisic acid in an amount between 50 and 250 μg, typically 200 μg, and,

[0352] - mannitol in an amount between 10 and 30 mg, for example 20 mg, and,

[0353] - Polyethylene glycol 15 hydroxystearate in an amount between 250 and 750 μg, for example 500 μg.

[0354] 35. A kit for implementing the method described in Example 20, comprising

[0355] i. The first vial contains the following components in dry form

[0356] -NeoB having the following formula (I):

[0357]

[0358] - radiolysis protectants, such as gentisic acid,

[0359] -optionally, a bulking agent, such as mannitol, and,

[0360] - optionally, a surfactant, such as polyethylene glycol 15 hydroxystearate; and,

[0361] ii. a second vial comprising at least a buffer, preferably in dry form; and,

[0362] iii. Optionally, an accessory kit for eluting radioisotopes produced by the radioisotope generator.

[0363] 36. A kit for implementing the method described in Example 20, comprising

[0364] i. A single vial containing the following components in dry form

[0365] -NeoB having the following formula (I):

[0366]

[0367] - radiolysis protectants, such as gentisic acid,

[0368] -optionally, a bulking agent, such as mannitol,

[0369] - optionally, a surfactant, such as polyethylene glycol 15 hydroxystearate, and,

[0370] - at least a buffer, preferably in dry form; and,

[0371] ii. Optionally, an accessory kit for eluting radioisotopes produced by the radioisotope generator.

[0372] 37. The kit of embodiment 35 or 36, wherein the amount of the NeoB compound contained is between 20 and 60 μg, typically 50 μg.

[0373] 38. The kit of any one of embodiments 35-37, wherein the amount of gentisic acid contained is between 50 and 250 μg, typically 200 μg.

[0374] 39. A kit as described in any of embodiments 35-38, wherein the bulking agent is mannitol and its amount is between 10 and 30 mg, such as 20 mg.

[0375] 40. The kit of any one of embodiments 35-39, wherein the surfactant is polyethylene glycol 15 hydroxystearate in an amount between 250 and 750 μg, such as 500 μg.

[0376] 41. The kit of any one of embodiments 35-40, wherein the first or single vial comprises the following components:

[0377] - NeoB having the following formula (I), in an amount between 20 and 60 μg, typically 50 μg;

[0378]

[0379] - gentisic acid, in an amount between 50 and 250 μg, typically 200 μg,

[0380] - mannitol in an amount between 10 and 30 mg, for example 20 mg, and,

[0381] - Optionally, polyethylene glycol 15 hydroxystearate, in an amount between 250 and 750 μg, such as 500 μg.

[0382] 42. The kit of any one of embodiments 35-41, wherein the second vial or single vial comprises a buffer for maintaining a pH between 3.0 and 4.0.

[0383] 43. The kit of any one of embodiments 35-42, wherein the second vial comprises formic acid and sodium hydroxide as buffering agents.

[0384] 44. The kit of any one of embodiments 35-43, wherein all components of the first, second or single vial are in dry form.

[0385] Hereinafter, the present disclosure will be described in more detail and specifically with reference to Examples, but these Examples are not intended to limit the present invention.

[0386] Radiochemical purity determined by ITLC

[0387] Preparation of mobile phase solution:

[0388] Second Ammonium chloride 5M: Accurately weigh 3.85 g (3.84615 ÷ 3.85385 g) of ammonium acetate into a 10 mL volumetric flask and dissolve it in 10 mL of MilliQ water.

[0389] Ammonium acetate / MeOH: Using a measuring cylinder, add 1 mL of ammonium acetate solution 5 M, 2 mL of MilliQ water and 7 mL of methanol. Transfer the eluate to a TLC chamber.

[0390] ITLC-SA preparation: Cut a 115 mm piece of ITLC-SA from each bottle, draw a line 20 mm from the bottom (where a drop of 5 uL sample is placed) and draw a line 100 mm from the bottom (where the chromatographic display must be abandoned).

[0391] 68 Ga-NeoB: reference factor 0.6-0.9

[0392] 68 Ga uncomplexed substances: reference factor = 0.0 ÷ 0.1

[0393] ( 68 The Ga-uncomplexed substance is 68 Ga colloidal substances and free 68 Ga. )

[0394] Radiochemical purity determined by HPLC

[0395] Table 1 Chromatographic conditions

[0396]

[0397] Example 1: Development of a method for radiolabeling NeoB with 68gallium using a two-vial kit

[0398] 1.2- Description and composition of the vial kit

[0399] Applicants have developed a sterile 2-vial kit consisting of:

[0400] ● Vial 1: Mix NeoB (50 μg, powder for injection) with 68 Ge / 68 Gallium chloride-68 ( 68 GaCl3) solution in HCl for reconstitution;

[0401] ● Vial 2: Reaction buffer.

[0402] Add vial 2 to the reconstituted vial 1.

[0403] An accessory cartridge is used to reduce the amount of germanium-68 (68Ge) ions that may be present in the generator eluate.

[0404] This kit must be used with 68 Ge / 68 Ga generator provides 68 A solution of Ga in HCl is used in combination to obtain 68 Ga-NeoB Injectable Solution, a radiolabeled imaging product, can be injected directly into the patient.

[0405] For injection 68 The volume of Ga-NeoB solution (corresponding to the radioactive dose to be administered) is calculated according to the estimated injection time, based on the current activity provided by the generator and the physical decay of the radionuclide (half-life = 68 min). It is a single-dose product.

[0406] Vial 1 is a powder for injection containing 50 μg NeoB as the active ingredient in a 10 mL glass vial.

[0407] The composition of vial 1 is provided in Table 2.

[0408] Table 2 Composition of powder for injection in vial 1

[0409]

[0410] The composition of Vial 2 is provided in Table 3.

[0411] Table 3

[0412] Components Composition (per bottle) Function Formic acid 60mg Buffer Sodium hydroxide 56.5mg Buffer Water for injection Appropriate amount to 1mL Solvents

[0413] 2. Drug development

[0414] As described above, Vial 1 (NeoB, 50 μg, powder for injection) is part of a radiopharmaceutical kit that also contains reaction buffer (Vial 2) and an accessory kit.

[0415] This kit must be used with 68 Ge / 68 Ga generator provides 68 A solution of Ga in HCl is used in combination to obtain 68 Ga-NeoB Injectable Solution, a radiolabeled imaging product, can be injected directly into the patient.

[0416] 2.1 Components of the drug product

[0417] The drug product contains NeoB as the active ingredient and gentisic acid, mannitol and Kolliphor HS 15 as excipients.

[0418] 2.1.1 API

[0419] The active substance is the NeoB peptide, a 7-mer amino acid sequence covalently bound to a chelating agent (DOTA) via a PABZA-DIG linker, as shown in the following formula (I):

[0420]

[0421] 2.1.2 Excipients

[0422] Excipients selected for the components of vial 1 are added to maintain the stability of the active substance in the final formulation to ensure the safety and efficacy of the drug product and to obtain 68 The desired radiochemical purity of the Ga-NeoB solution. The selected excipients produce a drug product with the desired pharmacotechnical properties.

[0423] A non-pharmacopoeial excipient, gentisic acid, was added to the drug product composition with a specific function related to the purity and stability of the radiolabeled imaging product obtained after reconstitution.

[0424] A brief description of each excipient is as follows:

[0425] Mannitol

[0426] Mannitol is used as a bulking agent. Since peptide drugs are very potent, very small amounts are required in the drug product. In the absence of bulking agents, product processing becomes unsuitable from a technical point of view. Bulking agents allow drug processing and production of presentable lyophilized products.

[0427] Gentisic acid

[0428] Gentisic acid is a non-pharmacopoeial excipient used as an antioxidant in drug product formulations.

[0429] ●Kolliphor HS15 (Polyethylene glycol 15 hydroxystearate)

[0430] Kolliphor HS15 is a water-soluble, nonionic solubilizer for parenteral formulations. As a solubilizer, it is particularly suitable for parenteral and oral dosage forms.

[0431] Due to the non-specific binding of peptides used as active ingredients in the NeoB radiopharmaceutical kit, Kolliphor HS 15 was used as a tensioactive agent for peptides that have a tendency to adhere to glass and plastic surfaces. 68 There is no risk of interference during Ga labeling.

[0432] 2.2 Pharmaceutical products

[0433] 2.2.1 Formulation Development

[0434] Formulation development has been conducted with the goal of identifying products that would allow for direct reconstitution based use from commercially available 68 Ge / 68 The eluate from the Ga generator simply constitutes the reaction mixture for labeling the DOTA peptide without any treatment of the eluate or any additional purification steps.

[0435] The goal was to develop a bombesin-like peptide antagonist (NeoB) for use as a radiotracer for the detection of GRPR-positive tumors.

[0436] Vial 1 contains the peptide as the active ingredient, a lyophilized powder, which is used in the radiolabeling procedure. 68 Ga was radiolabeled.

[0437] Initial efforts to develop a suitable formulation for NeoB (Vial 1) involved testing of bulk solutions prior to sterilization and lyophilization processes for bench-scale preparation.

[0438] Development efforts focus on selecting appropriate excipients related to the peptide properties to obtain a final product that will be used to 68 Ga radiolabeled NeoB products have the following target radiochemical purities:

[0439] ● 68 Ga-NeoB (HPLC) → > 92%

[0440] ● Free 68 Ga 3 + (HPLC) → ≤ 2%

[0441] ● Uncomplexed 68 Ga 3 + Substance (ITLC) → ≤ 3%

[0442] The components selected for the final formulation were as follows:

[0443] Table 4 Selected components for vial 1 composition (powder for injection)

[0444]

[0445] The development work including the relevant conducted studies is described, starting from the selection of the amount of active ingredient and appropriate excipients.

[0446] 2.2.1.1 Selection of peptide amount

[0447] Use from 1850MBq 68 Ge / 68 The eluate from the Ga generator and the formate buffer were tested with increasing amounts of NeoB peptide (from 15 μg to 100 μg) during labeling in order to identify 68 Minimum peptide amount required for more than 98% incorporation of Ga in HPLC and more than 97% incorporation in ITLC. Based on the results summarized in Table 5, 25 μg is the minimum peptide amount that provides reproducibility (by good radiochemical purity).

[0448] Table 5 - Effect of NeoB amount on labeling efficiency

[0449]

[0450] In parallel, different peptide doses were also tested in in vivo biodistribution experiments. In brief, using a mouse prostate cancer model, two different peptide mass doses were compared: 10 pmol vs. 200 pmol; in these experiments, the total amount of radioactivity injected remained unchanged (1 MBq). When a higher peptide mass dose (200 pmol) was used, injection of radiolabeled NeoB resulted in increased accumulation in the tumor. At the same time, the uptake of non-target organs (such as the pancreas) decreased significantly with increasing peptide mass dose (200 pmol). Therefore, based on these preclinical evaluations, higher peptide mass doses were demonstrated to be preferred because they are associated with reduced uptake in non-target organs (especially the pancreas in this case).

[0451] The final amount of peptide contained in vial 1 was chosen to be 50 μg based on the radiolabeling tests performed (as described in Table 5) and in vivo biodistribution experiments indicating that higher peptide mass doses ensure better performance and safety of the compound.

[0452] Formulation development work also focused on the selection of surfactants, antioxidants and bulking agents. Radiolabeling procedures have also been thoroughly evaluated.

[0453] 2.2.1.2 Selection of key excipients

[0454] ●Choice of surfactant

[0455] During the tests performed to determine the formulation of vial 1 (NeoB 50 μg, powder for injection), peptides seemed to have a particular tendency to stick to glass and plastic surfaces. This phenomenon is called non-specific binding (NSB). Peptides generally exhibit greater NSB problems than small molecules, and especially uncharged peptides can adhere strongly to plastics. The reasons can be different: physical / chemical properties, van der Waals interactions, ionic interactions. Therefore, the addition of excipients known to reduce NSB, including surface tension active agents and solubilizers, was evaluated.

[0456] Organic solvents can improve solubility and prevent adsorption. For example, ethanol can be used in radiopharmaceutical injections to improve the solubility of highly lipophilic tracers or to reduce adsorption to vials, membrane filters, and syringes. In the case of NeoB powders for injection, ethanol is not an option because it is incompatible with the freeze-drying process.

[0457] Human serum albumin (HSA) is also used in many protein formulations as a stabilizer to prevent surface adsorption, but this excipient is not suitable due to its thermal instability.

[0458] Another possible approach to reduce nonspecific binding of peptides is to use surfactants (e.g., polysorbate 20, polysorbate 80, Pluronic F-68, sorbitan trioleate). Special attention has been paid to nonionic surfactants because ionic surfactants may interfere with 68 Ga marker.

[0459] Nonionic surfactants such as Kolliphor HS15, Kolliphor K188, Tween 20, Tween 80, polyvinylpyrrolidone K10 are commercially available as solubilizing excipients in oral and injectable formulations.

[0460] Peptide adhesion tests were performed with different surface tension active agents to evaluate the suitability of the most suitable agent for use in the composition of NeoB powder for injection (vial 1) (see results in Table 6 below).

[0461] Hydroxypropyl beta-cyclodextrin was also evaluated in the formulation alone or in combination with a surfactant. As described below, the presence of hydroxypropyl beta-cyclodextrin had only a limited positive effect on peptide adhesion. In addition, as demonstrated in subsequent testing (see also Section 2.2.1.3, Radiolabeling Procedure), the presence of hydroxypropyl beta-cyclodextrin and a surfactant alone does not improve the radiochemical purity of the final product if compared to a formulation containing only a surfactant. For this reason, hydroxypropyl beta-cyclodextrin was not included in the final formulation.

[0462] Table 6 - Selection of Surface Tension Active Agents - Peptide Adhesion Test

[0463]

[0464]

[0465] The best results in terms of peptide adhesion were obtained using Kolliphor HS 15 and Tween 20. These two excipients were further investigated to determine the final amounts in the kit. The results obtained were good in terms of radiochemical purity and peptide adhesion.

[0466] Table 7 - Comparison between Tween 20 and Kolliphor HS 15 surface tension agents

[0467]

[0468] The final choice was Kolliphor HS 15, since polysorbate (Tween 20) can undergo auto-oxidation, cleavage at the ethylene oxide subunit, and hydrolysis of the fatty acid ester bond caused by oxygen, metal ions, peroxides, or elevated temperature.

[0469] The lowest peptide adhesion was obtained when 0.5 mg Kolliphor HS 15 was used, which was the amount of Kolliphor HS 15 chosen in the final composition of the drug product.

[0470] ● Choice of antioxidant

[0471] The presence of free radical scavengers with antioxidant properties could protect NeoB from radiolysis.

[0472] We considered gentisic acid and ascorbic acid for development studies as antioxidants for use in radiopharmaceutical formulations. Testing was performed to identify the lowest amount of antioxidant that could exert the desired protective function without interfering with the radiolabel.

[0473] Radiolabeling has been tested, varying the amount of antioxidant and keeping other parameters constant mainly to identify the most suitable antioxidant and not interfere with 68 As shown in the table below, gentisic acid was identified as the best antioxidant because it did not interfere with 68 The incorporation of Ga in HPLC was over 98%. The amount of gentisic acid selected was 200 μg.

[0474] Table 8 - Selection of Antioxidants

[0475]

[0476]

[0477] Note: Out-of-specification results are Underlined bold text express

[0478] ● Selection of extender

[0479] The formulation is finalized by adding bulking agents required for the freeze-drying process of the product.

[0480] Among the bulking agents commonly recommended for peptide lyophilization, drug manufacturers have tested inositol and mannitol.

[0481] Table 9 - Selection of Extender Tests

[0482]

[0483]

[0484] Mannitol was chosen because it is the most commonly used in lyophilizates and it is known that cakes produced using it during the lyophilization process have good properties in terms of appearance, stability and moisture. In addition, mannitol is described in the literature as a good scavenger of OH radicals.

[0485] 2.2.1.3 Radiolabeling Procedure

[0486] Based on the 2-vial design, a three-step labeling procedure was developed as follows:

[0487] 1. In a heating block, use 68 Ge / 68 Ga generator provides 68 The lyophilized formulation (Vial 1) was reconstituted directly with a solution of Ga in HCl (verify that the temperature has reached 95°C before starting elution).

[0488] 2. Add the required volume of reaction buffer (vial 2)

[0489] 3. Heat at 95°C for at least 7 minutes (no more than 10 minutes)

[0490] at this time, 68 The Ga-NeoB solution is ready for application.

[0491] During labeling procedure development, different time and temperature conditions have been tested.

[0492] The temperature dependence of labeling efficiency has been studied to identify 68 The short half-life of Ga provides good incorporation values ​​within a compatible time frame.

[0493] Known 68 The incorporation of Ga into the DOTA chelating moiety requires heating to complete.

[0494] The labeling conditions for the first test were: labeling at 80°C, 85°C and 95°C with different reaction times (3, 5 and 7 minutes). These tests were carried out using the following product formulations:

[0495] ●Peptide (50 μg),

[0496] Mannitol (20 mg),

[0497] Gentisic acid (0.2 mg),

[0498] Kolliphor HS15 (0.5 mg),

[0499] ●Hydroxypropyl beta-cyclodextrin (3 mg).

[0500] The formulation tested in these initial tests included the solubilizing agent hydroxypropyl beta-cyclodextrin. However, later in the development process, similar tests were performed using the same formulation except without hydroxypropyl beta-cyclodextrin, and good radiochemical and chemical purity was obtained. In addition, peptide adhesion was also shown to be unaffected by the absence of hydroxypropyl beta-cyclodextrin, so hydroxypropyl beta-cyclodextrin was not included in the final formulation. At 80°C and 85°C, radiometric analysis showed sufficient incorporation within 7 minutes.

[0501] At 95°C, incorporation was complete after only 7 minutes.

[0502] Based on these observations, 95 °C for 7 min appeared to be the most conservative labeling condition, which guaranteed incorporation of more than 98% without significant fragmentation even with temperature fluctuations within ±15 °C.

[0503] Table 10 - Marking at different temperatures and times

[0504]

[0505] Furthermore, to increase the robustness of the labeling procedure, the addition of the reaction buffer (vial 2) at room temperature (RT) was evaluated (and only after the addition of the reaction buffer, the labeling reaction was performed at 95°C). The results shown in Table 11 confirm that good radiochemical purity was also obtained under these conditions.

[0506] Table 11 - Addition of elution and buffer at room temperature

[0507]

[0508] 2.2.1.4 Final selected formulation (vial 1)

[0509] Based on all the above development studies, the final composition of NeoB 50 μg powder for injection (vial 1) is as follows:

[0510] Table 12 - Final composition of powder for injection in vial 1

[0511]

[0512] The final formulation was tested against the radiolabeled product to confirm the results obtained during development.

[0513] Table 13 - Radiolabeling tests performed using final formulations

[0514]

[0515] As shown in Table 13, good radiochemical purity results (>92%) were obtained by both ITLC and HPLC after three independent radiolabeling tests on the final formulation. It is also important to note that free gallium (by HPLC) was always below 2%. Finally, the adhesion of the peptide to glass was also tested during these radiolabeling tests, confirming that the presence of Kolliphor HS15 was necessary to maintain peptide adhesion at acceptable levels.

[0516] 2.2.1.5 Quality Standards Assessment

[0517] In order to properly define the quality specification, a set of preliminary experiments were conducted, which are summarized as follows.

[0518] pH marker

[0519] because 68 GaCl3 has a specific chemical behavior and the labeling pH is one of the key parameters to obtain good results in terms of radiolabeled DOTA peptide yield. To determine the pH range in which labeling provides good results, the pH range was kept between 3.0 and 4.0 and the 68 Gallium labeled NeoB formulation. Labeling has been tested varying the volume of reaction buffer added and keeping other parameters constant. As shown in Tables 14 and 15, pH variations in the range of 3.0-4.0 did not affect the success of labeling. The radiolabeled product obtained met the radiochemical purity specifications.

[0520] Table 14 - Testing at lower pH using final formulation

[0521]

[0522] Table 15 - Tests performed at higher pH using final formulations

[0523]

[0524] Gentisic acid and volume activity

[0525] When using 68 Ge / 68 Ga generators were available at the time. 68 The test was performed to evaluate the effect of gentisic acid as a radiation scavenger when labeling with the highest volume activity of GaCl3. In order to obtain the highest possible volume activity, a fractional elution was performed; only the fraction with the highest activity was used for labeling.

[0526] The protective effect was verified by monitoring the peptide fragmentation over time in the presence of different amounts of gentisic acid (0.20 mg and 0.35 mg). The results (see Table 16) confirmed an almost identical positive effect in both tests. Therefore, the lowest amount of gentisic acid (200 μg) sufficient to achieve a good level of radiolytic protection was selected.

[0527] Table 16 - Radiolabeling Tests at Maximum Volume Activity (Step Elution)

[0528]

[0529] In addition, to test whether lower amounts of gentisic acid could still act as an antioxidant in the final formulation, initial tests were performed using 0.1 mg of gentisic acid. The results of the radiolabeling tests performed under these conditions are shown in Table 17 and confirm that good radiochemical purity can be obtained even in the presence of lower amounts of gentisic acid. Nevertheless, in order to ensure good radiochemical purity at higher generator activity, the amount of gentisic acid in the final formulation has been conservatively kept at 200 μg.

[0530] Table 17 - Using higher concentrations 68 Radiolabeling with Ga and lower concentrations of gentisic acid

[0531]

[0532] Zoom in batches - 68 Test results of Ga radiolabeled products

[0533] Table 1 Table 18 summarizes two radiolabeling tests performed using scale-up batch NeoB vial 1. The results show that the radiolabeled drug product obtained using scale-up batch vial 1 68 Ga-NeoB met the radiochemical purity specifications up to 4 hours after the completion of the radiolabeling reaction.

[0534] Table 18 - Radiolabelling tests performed using scaled-up batch (CT005 16001)

[0535]

[0536] References

[0537] 1.Sah BR,Burger IA,Schibli R,Friebe M,Dinkelborg L,Graham K,BorkowskiS,Bacher-Stier C,Valencia R,Srinivasan A et al:Dosimetry and First ClinicalEvaluation ofthe New 18F-Radiolabeled Bombesin Analogue BAY 864367 inPatients with ProstateCancer.J Nucl Med 2015,56(3):372-378.

[0538] 2.Kahkonen E,Jambor I,Kemppainen J,Lehtio K,Gronroos TJ,Kuisma A,Luoto P,Sipila HJ,Tolvanen T,Alanen K et al:In vivo imaging of prostatecancer using[68Ga]-labeled bombesin analog BAY86-7548.Clin Cancer Res 2013,19(19):5434-5443.

[0539] 3.Maina T,Bergsma H,Kulkarni HR,Mueller D,Charalambidis D,KrenningEP,NockBA,de Jong M,Baum RP:Preclinical and first clinical experience withthe gastrin-releasingpeptide receptor-antagonist[(68)Ga]SB3 and PET / CT.Eur JNucl Med Mol Imaging 2016,43(5):964-973.

[0540] 4.Dimitrakopoulou-Strauss A,Hohenberger P,Haberkorn U,Macke HR,Eisenhut M,Strauss LG:68Ga-labeled bombesin studies in patients withgastrointestinal stromal tumors:comparison with 18F-FDG.J Nucl Med 2007,48(8):1245-1250.

[0541] 5.Velikyan I,Xu H,Nair M,Hall H:Robust labeling and comparativepreclinicalcharacterization of DOTA-TOC and DOTA-TATE.Nucl Med Biol 2012,39(5):628-639.

Claims

1. A method preferably comprising: 68 Ga, 67 Ga or 64 A method for labeling a gastrin releasing peptide receptor (GRPR) antagonist with a radioactive isotope of Cu, the method comprising the following steps: i. providing a first vial containing said GRPR antagonist in dry form, ii. adding the solution of the radioisotope to the first vial, thereby obtaining a solution of the GRPR antagonist and the radioisotope, iii. mixing the solution obtained in ii. with at least a buffer and incubating for a time sufficient to obtain said GRPR antagonist labeled with said radioactive isotope, and iv. Optionally, adjusting the pH of the solution.

2. The method of claim 1, wherein the first vial of step i. is a reaction vial comprising the GRPR antagonist and a buffer, preferably both in dry form.

3. The method of claim 1, wherein step iii comprises mixing the solution obtained in ii. with a reaction solution comprising at least a buffer and incubating for a time sufficient to obtain the GRPR antagonist labeled with the radioactive isotope.

4. The method of any one of claims 1-3, wherein the GRPR antagonist is a compound having formula (I): DOTA-(p-aminobenzylamine-diethylene glycol Acid))-D-Phe-Gln-Trp-Ala-Val-Gly-His-NH-CH[CH2-CH(CH3)2]2.

5. The method of any one of claims 1-4, wherein the GRPR antagonist is contained in the first vial in an amount between 20 and 60 μg, typically 50 μg.

6. The method of any one of claims 1 to 5, wherein the first vial further comprises gentisic acid as a radiolysis protectant, preferably in an amount between 50 and 250 μg, typically 200 μg.

7. The method of any one of claims 1-6, wherein the first vial further comprises mannitol as a bulking agent, for example in an amount between 10 and 30 mg, typically 20 mg.

8. The method of any one of claims 1-7, wherein the first vial further comprises polyethylene glycol 15 hydroxystearate as a surfactant, for example in an amount between 250 and 750 μg, typically 500 μg.

9. A solution comprising a GRPR antagonist labeled with a radioisotope, said solution being obtainable by the method of any one of claims 1 to 8 or obtained by the method of any one of claims 1 to 8, for use as an injectable solution for in vivo detection of tumors by imaging in a subject in need thereof.

10. A method comprising 68 A Ga-labeled solution of a compound of formula (I) as defined in claim 4, obtainable by the method of any one of claims 4 to 8 or obtained by the method of any one of claims 4 to 8, for use as an injectable solution for in vivo detection of tumors by imaging in a subject in need thereof.

11. A powder for injection, comprising the following components in dry form: i. A GRPR antagonist having the formula: CSP in: C is a chelating agent capable of chelating the radioactive isotope; S is an optional spacer that covalently links C to the N-terminus of P; P is a GRPR peptide antagonist, preferably having the following general formula: Xaa1-Xaa2—Xaa3—Xaa4—Xaa5—Xaa6—Xaa7—Z; Xaa1 is absent or selected from the group consisting of the following amino acid residues: Asn, Thr, Phe, 3-(2-thienyl)alanine (Thi), 4-chlorophenylalanine (Cpa), α-naphthylalanine (α-Nal), β-naphthylalanine (β-Nal), 1,2,3,4-tetrahydronorharman-3-carboxylic acid (Tpi), Tyr, 3-iodotyrosine (oI-Tyr), Trp and pentafluorophenylalanine (5-F-Phe) (all L-isomers or D-isomers); Xaa2 is Gln, Asn or His; Xaa3 is Trp or 1,2,3,4-tetrahydronorharman-3-carboxylic acid (Tpi); Xaa4 is Ala, Ser or Val; Xaa5 is Val, Ser, or Thr; Xaa6 is Gly, sarcosine (Sar), D-Ala, or β-Ala; Xaa7 is His or (3-methyl)histidine (3-Me)His; Z is selected from -NHOH, -NHNH2, -NH-alkyl, -N(alkyl)2 and -O-alkyl or Z is wherein X is NH (amide) or O (ester) and R1 and R2 are the same or different and are selected from proton, optionally substituted alkyl, optionally substituted alkyl ether, aryl, aryl ether or alkyl-, halogen, hydroxy, hydroxyalkyl, amine, amino, amido or amido-substituted aryl or heteroaryl; ii. radiolysis protectants, such as gentisic acid; iii. bulking agents, such as mannitol; and, iv. Optionally, a surfactant, such as polyethylene glycol 15 hydroxystearate.

12. The powder for injection according to claim 11, wherein the GRPR antagonist is a compound having formula (I):

13. The powder for injection according to any one of claims 11 to 12, comprising the following components: - the following formula (I), in an amount between 20 and 60 μg, usually 50 μg; - gentisic acid in an amount between 50 and 250 μg, typically 200 μg, and, - mannitol in an amount between 10 and 30 mg, for example 20 mg, and, - Polyethylene glycol 15 hydroxystearate in an amount between 250 and 750 μg, for example 500 μg.

14. A kit for implementing the method according to claim 4, comprising i. The first vial contains the following components in dry form - A compound having the following formula (I): - radiolysis protectants, such as gentisic acid, -optionally, a bulking agent, such as mannitol, and, - optionally, a surfactant, such as polyethylene glycol 15 hydroxystearate; and, ii. a second vial comprising at least a buffer, preferably in dry form; and, iii. Optionally, an accessory kit for eluting radioisotopes produced by the radioisotope generator.

15. A kit for implementing the method according to claim 4, comprising i. A single vial containing the following components in dry form - A compound having the following formula (I): - radiolysis protectants, such as gentisic acid, -optionally, a bulking agent, such as mannitol, - optionally, a surfactant, such as polyethylene glycol 15 hydroxystearate, and, - at least a buffer, preferably in dry form; and, ii. Optionally, an accessory kit for eluting radioisotopes produced by the radioisotope generator.

16. The kit of any one of claims 14-15, wherein the first or single vial comprises the following components: - the following compound of formula (I) in an amount between 20 and 60 μg, usually 50 μg; - gentisic acid in amounts of 50 and 250 μg, typically 200 μg, - mannitol in an amount between 10 and 30 mg, for example 20 mg, and, - Optionally, polyethylene glycol 15 hydroxystearate, in an amount between 250 and 750 μg, such as 500 μg.

17. The kit of any one of claims 14-16, wherein all components of the first, second or single vial are in dry form.

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