Methods for radiolabeling psma binding ligands and kits therefor

Abstract

This disclosure involves the use of radioactive isotopes, preferably... 68 Ga, 67Ga or 64 Methods for radiolabeling PSMA-binding ligands with Cu, and their kits.

Description

Technical Field

[0001] This disclosure relates to methods and kits for radiolabeling PSMA binding ligands. Background Technology

[0002] Prostate cancer is one of the most common cancers in the United States and Europe. In particular, metastatic prostate cancer (mCRPC) is associated with poor prognosis and decreased quality of life.

[0003] Recently, a new development stream for the treatment of prostate cancer has been represented by internal radiotherapy based on PSMA ligands, as PSMA is considered a suitable target for imaging and therapy due to its overexpression in both primary carcinomatous lesions and soft tissue / bone metastases. Furthermore, PSMA expression appears to be higher in the most aggressive castration-resistant variant of the disease, representing a patient population with unmet medical needs. (Marchal et al., Histol Histopathol [Histology and Histopathology], July 2004; 19(3):715-8; Mease et al., Curr Top Med Chem [Current Topics in Medicinal Chemistry], 2013, 13(8):951-62).

[0004] Among the many small-molecule ligands targeting PSMA, urea-based low-molecular-weight agents are the most extensively studied. These agents have been shown to be useful in clinical evaluation of prostate cancer and in PRRT therapy (Kiess et al., QJ Nucl Med Mol Imaging, 2015; 59:241-68). Some of these agents use glutamate-urea-lysine (GUL) as a targeting scaffold. A class of molecules has been created using a strategy of attaching a linker between the chelating agent and the GUL moiety. This approach allows urea to reach the binding site while retaining the metal chelating moiety outside the binding site. This strategy has been successful in xenograft PSMA-positive tumors because it exhibits high uptake and retention as well as rapid renal clearance (Banerjee et al., J Med Chem, 2013; 56:6108-21). Further studies have shown that these molecules can be used... 68 Ga labeling was used to detect prostate cancer lesions via PET imaging (Eder et al. Pharmaceuticals 2014, 7, 779-796).

[0005] However, no solution has yet been developed for use. 68 Ga、 67 Ga or 64An optimized method is needed to label PSMA-binding ligands with Cu to obtain labeled PSMA-binding ligand solutions for tumor imaging in human patients with prostate cancer. Specifically, a rapid, efficient, and safe procedure is required that will provide labeled PSMA-binding ligands with high radiochemical purity, such as […]. 68 Ga]PSMA binding ligands were used for intravenous injection in human subjects in need. Summary of the Invention

[0006] A first aspect of this disclosure relates to a method using a radioactive isotope, preferably 68 Ga、 67 Ga or 64 A method for Cu-labeled PSMA binding ligands, the method comprising the following steps:

[0007] i. Provide a first vial containing the PSMA-binding ligand in dry form and an optional extender.

[0008] ii. Add the solution of the radioactive isotope to the first vial to obtain a solution of the PSMA binding ligand and the radioactive isotope.

[0009] iii. The solution obtained in ii. is mixed with at least a buffer and incubated for a period of time sufficient to obtain the PSMA-binding ligand labeled with the radioisotope, and

[0010] iv. Optionally, adjust the pH of the solution.

[0011] In a specific embodiment, the radioactive isotope is 68 Ga and the radiochemical purity measured by HPLC is at least 92%, and optionally, free 68 Ga 3+ The percentage (in HPLC) is 2% or less, and / or uncomplexed. 68 Ga 3+ The percentage of the substance (in ITLC) is 3% or less.

[0012] In other specific embodiments, the radioactive isotope is 67 Ga and the radiochemical purity measured by HPLC is at least 90%, and optionally, free 67 Ga 3+ The percentage (in HPLC) is 2% or less, and / or uncomplexed. 67 Ga 3+ The percentage of the substance (in ITLC) is 5% or less.

[0013] In other specific embodiments, the radioactive isotope is 64Cu and its radiochemical purity, measured by HPLC, is at least 92%, and optionally, free... 64 Cu 2+ The percentage (in HPLC) is 2% or less, and / or uncomplexed. 64 Cu 2+ The percentage of the substance (in ITLC) is 3% or less.

[0014] Preferably, the PSMA-binding ligand is a compound having formula (I):

[0015]

[0016] in:

[0017] Z is either tetrazolium or COOQ, preferably Z is COOQ;

[0018] Q is independently H or a protecting group, preferably Q is H;

[0019] m is an integer selected from the group consisting of 1, 2, 3, 4 and 5, preferably m is 4;

[0020] q is an integer selected from the group consisting of 1, 2, 3, 4, 5 and 6, preferably q is 1;

[0021] R is selected from the following groups: C6-C 10 Aryl and heteroaryl containing 5-10 ring atoms, wherein the aryl and heteroaryl are substituted by X once or more;

[0022] X is -VY;

[0023] V is a bond or a C1-C6 alkylene group, preferably V is a bond;

[0024] Y is a halogen;

[0025] L is a linker selected from the group consisting of: C1-C6 alkylene, C3-C6 cycloalkylene, and C6-C 10The arylene group, wherein the alkylene group, cycloalkylene group, and arylene group are optionally substituted with one or more substituents selected from the following: -OR', =O, =NR', =N-OR', -NR'R”, -SR', -halogen, -SiR'R”R”', -OC(O)R', -C(O)R', -CO2R', -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”')=NR””, -S(O)R', -S(O)2R', -S(O)2NR'R”, -NRSO2R', -CN, and -NO2, wherein the number of substitutions ranges from zero to (2m'+1), where m' is the total number of carbon atoms in such groups. R', R”, R”' and R”” can each independently refer to hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl;

[0026] W chooses freedom-NR 2 -(C=O), -NR 2 -(C=S), -(C=O)-NR 2 - and -(C=S)-NR 2 -

[0027] Preferably, in the group consisting of W, W is -(C=O)-NR 2 -;

[0028] L and W can be the same or different each time they appear;

[0029] R 2 It is H or C1-C4 alkyl, preferably R. 2 It is H;

[0030] n is an integer that can be selected from the groups consisting of 1, 2, and 3;

[0031] Ch is a chelating agent, usually DOTA.

[0032] In another aspect, this disclosure relates to a solution containing a PSMA-binding ligand labeled with a radioisotope that is available or obtainable by the method, which is used as an injectable solution for in vivo detection of tumors, typically PSMA-expressing tumors, by imaging in desired subjects.

[0033] Another object of this disclosure is to provide an injectable powder comprising the following components in a dry form:

[0034] i. PSMA-binding ligands of formula (I):

[0035]

[0036] in:

[0037] Z is either tetrazolium or COOQ, preferably Z is COOQ;

[0038] Q is independently H or a protecting group, preferably Q is H;

[0039] m is an integer selected from the group consisting of 1, 2, 3, 4 and 5, preferably m is 4;

[0040] q is an integer selected from the group consisting of 1, 2, 3, 4, 5 and 6, preferably q is 1;

[0041] R is selected from the following groups: C6-C 10 Aryl and heteroaryl containing 5-10 ring atoms, wherein the aryl and heteroaryl are substituted by X once or more;

[0042] X is -VY;

[0043] V is a bond or a C1-C6 alkylene group, preferably V is a bond;

[0044] Y is a halogen;

[0045] L is a linker selected from the group consisting of: C1-C6 alkylene, C3-C6 cycloalkylene, and C6-C 10 The arylene group, wherein the alkylene group, cycloalkylene group, and arylene group are optionally substituted with one or more substituents selected from the following: -OR', =O, =NR', =N-OR', -NR'R”, -SR', -halogen, -SiR'R”R”', -OC(O)R', -C(O)R', -CO2R', -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”')=NR””, -S(O)R', -S(O)2R', -S(O)2NR'R”, -NRSO2R', -CN, and -NO2, wherein the number of substitutions ranges from zero to 2m', where m' is the total number of carbon atoms in such groups. R', R”, R”' and R”” can each independently refer to hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl;

[0046] W chooses freedom-NR 2 -(C=O), -NR 2 -(C=S), -(C=O)-NR 2 - and -(C=S)-NR 2 -

[0047] Preferably, in the group consisting of W, W is -(C=O)-NR 2 -;

[0048] L and W can be the same or different each time they appear;

[0049] R 2 It is H or C1-C4 alkyl, preferably R. 2 It is H;

[0050] n is an integer that can be selected from the groups consisting of 1, 2, and 3;

[0051] Ch is a chelating agent, usually DOTA; and

[0052] ii. Incremental agents, such as mannitol.

[0053] Typically, the powder for injection contains the following components:

[0054] i. A PSMA-binding ligand having formula (II) in an amount of 10 μg to 100 μg, preferably 15 μg to 60 μg, even more preferably about 30 μg;

[0055] and

[0056] ii. Mannitol, in an amount of 5 mg to 50 mg, preferably 10 mg to 30 mg, or even more preferably about 20 mg.

[0057] This disclosure further relates to a kit for carrying out the method, the kit comprising...

[0058] i. The first vial contains the following components in dry form.

[0059] i. PSMA-binding ligands of formula (II):

[0060] and

[0061] ii. Optionally, a extender, such as mannitol, and,

[0062] ii. A second vial containing at least a buffer, preferably in a dry form; and,

[0063] iii. Optionally, an accessory cassette for eluting radioactive isotopes generated by a radioactive isotope generator or a cyclotron.

[0064] Another kit disclosed in this article contains:

[0065] i. A single vial containing, preferably in a dry form, the following components:

[0066] i. PSMA-binding ligands of formula (II):

[0067] and

[0068] ii. Optionally, an extender, such as mannitol,

[0069] iii. at least one buffer, and,

[0070] ii. Optionally, an accessory cassette for eluting radioactive isotopes generated by a radioactive isotope generator or a cyclotron.

[0071] For example, the kit may contain a first or a single vial, which contains the following components:

[0072] i. A PSMA-binding ligand having formula (II), wherein the amount is from 10 μg to 100 μg, preferably from 15 μg to 60 μg, and even more preferably about 30 μg.

[0073] and

[0074] ii. Mannitol, in an amount of 5 mg to 50 mg, preferably 10 mg to 30 mg, or even more preferably about 20 mg. Detailed Implementation

[0075] Generally speaking, this disclosure relates to a method using a radioactive isotope, preferably 68 Ga、 67 Ga or 64 A method for Cu-labeled PSMA binding ligands, the method comprising the following steps:

[0076] i. Provide a first vial containing the PSMA-binding ligand in dry form and an optional extender.

[0077] ii. Add the solution of the radioactive isotope to the first vial to obtain a solution of the PSMA binding ligand and the radioactive isotope.

[0078] iii. The solution obtained in ii. is mixed with at least a buffer and incubated for a period of time sufficient to obtain the PSMA-binding ligand labeled with the radioisotope, and

[0079] iv. Optionally, adjust the pH of the solution.

[0080] The radiolabeled PSMA-binding ligands obtained by the disclosed method are preferably radiolabeled PSMA-binding ligands used as contrast agents in 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 for PET imaging, such as PET / CT or PET / MRI.

[0081] The preferred radiolabeled PSMA-binding ligands obtained by the disclosed method are PSMA-binding ligands having formula (II):

[0082]

[0083] Its radioisotope (preferably) is suitable for use as a contrast agent in PET / CT, SPECT or PET / MRI imaging. 68 Ga、 67 Ga or 64 (Copper) mark.

[0084] The method disclosed herein can advantageously provide excellent radiochemical purity of radiolabeled compounds, such as PSMA-binding ligands of formula (II) radiolabeled with 68Ga, typically with a radiochemical purity of at least 92% as measured in HPLC, and optionally, the percentage of free 68Ga3+ (in HPLC) is 2% or less, and / or the percentage of uncomplexed 68Ga3+ material (in ITLC) is 3% or less.

[0085] The examples further describe in detail the measurement of radiochemical purity and free radicals in HPLC or ITLC. 68 Ga 3+ The determination method.

[0086] definition

[0087] The terms “PSMA-binding ligand” and “PSMA ligand” are used interchangeably in this disclosure. They refer to molecules capable of interacting with (e.g., binding to) PSMA enzymes.

[0088] The phrase "treatment of" and "treating" encompasses the improvement or cessation of a disease, condition, or its symptoms. Specifically, regarding the treatment of tumors, the term "treatment" can refer to the inhibition of tumor growth or the reduction of tumor size.

[0089] Consistent with the International System of Units (SI), "MBq" is an abbreviation for "megabecquerel," the unit of radioactivity.

[0090] As used in this article, "PET" stands for positron emission tomography.

[0091] As used in this article, "SPECT" stands for Single Photon Emission Computed Tomography.

[0092] As used in this article, "MRI" stands for magnetic resonance imaging.

[0093] As used in this article, "CT" stands for computed tomography.

[0094] As used herein, the term “effective amount” or “therapeutic effective amount” of a compound refers to the amount of a compound that will elicit a biological or medical response in a subject (e.g., improve symptoms, alleviate symptoms, slow or delay disease progression, or prevent disease).

[0095] As used herein, the terms “substituted” or “optionally substituted” refer to a group that is optionally substituted by one or more substituents selected from: halogen, -OR', -NR'R”, -SR', -SiR'R”R”', -OC(O)R', -C(O)R', -CO2R', -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, the number of which ranges 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 disclosed herein contain more than one R group, for example, when more than one of these groups is present, each R group is independently selected as R’, R”, R”’ and R”” groups, respectively.

[0096] As used herein, the term "alkyl" itself, or as part of another substituent, refers to a straight-chain or branched alkyl functional group having 1-12 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).

[0097] As used herein, the term "heteroaryl" refers to a polyunsaturated aromatic ring system having multiple aromatic rings, either monocyclic or fused together or covalently linked, comprising 5-10 atoms, wherein at least one ring is an aromatic ring 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 aryl, cycloalkyl, or heterocyclic rings. Non-limiting examples of such heteroaryl groups include: furanyl, thiopheneyl, pyrroleyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiazolyl, tetrazolyl, oxtriazolyl, thiazolyl, pyridyl, pyrazinyl, pyridazinyl, oxazinyl, dioxazinyl, thiazolyl, triazinyl, indolyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothiopheneyl, isobenzothiopheneyl, inazolyl, benzimidazolyl, benzooxazolyl, purinyl, benzothiazolyl, quinolinyl, isoquinolinyl, cinolinyl, quinazolinyl, and quinoxolinyl.

[0098] As used herein, the term "aryl" refers to a polyunsaturated aromatic hydrocarbon group having a monocyclic or fused aromatic rings containing 6-10 ring atoms, at least one of which is aromatic. The aromatic ring may optionally include one or two additional rings fused to it (such as cycloalkyl, heterocyclic, or heteroaryl as defined herein). Suitable aryl groups include phenyl, naphthyl, and benzene rings fused to heterocyclic groups, such as benzopyranyl, benzo-m-dioxacyclopentenyl, benzodioxacyclohexyl, etc.

[0099] As used herein, the term "halogen" refers to a fluorine (-F), chlorine (-Cl), bromine (-Br), or iodine (-I) group.

[0100] As used herein, the term "dry form" refers to a pharmaceutical composition that has been dried into a powder and has a moisture content of less than about 10% by weight, typically less than about 5% by weight, preferably less than about 3%.

[0101] As used herein, the term "chelating agent" refers to a molecule having functional groups, such as amines or carboxyl groups, suitable for chelating radioisotopes via non-covalent bonds.

[0102] As used herein, the term "antioxidant" refers to a compound that inhibits the oxidation of organic molecules. Antioxidants include gentian acid and ascorbic acid.

[0103] As used herein, the term "radiochemical purity" refers to the percentage of a stated radionuclide present in its stated chemical or biological form. Radiochromatography, such as HPLC or instantaneous thin-layer chromatography (iTLC), is the most widely accepted method for determining radiochemical purity in nuclear pharmacology.

[0104] Step (i) provides a first vial containing the PSMA-binding ligand in a dried form.

[0105] PSMA binding ligand

[0106] Advantageously, the PSMA binding ligand is a molecule comprising a) a urea having two amino acid residues, typically a glutamic acid-urea-lysine (GUL) moiety, and b) a chelating agent that can coordinate with a radioisotope.

[0107] According to one embodiment, the PSMA-binding ligand is a compound having formula (I):

[0108]

[0109] in:

[0110] Z is either tetrazolium or COOQ, preferably Z is COOQ;

[0111] Q is independently H or a protecting group, preferably Q is H;

[0112] m is an integer selected from the group consisting of 1, 2, 3, 4 and 5, preferably m is 4;

[0113] q is an integer selected from the group consisting of 1, 2, 3, 4, 5 and 6, preferably q is 1;

[0114] R is selected from the following groups: C6-C 10 Aryl and heteroaryl containing 5-10 ring atoms, wherein the aryl and heteroaryl are substituted by X once or more;

[0115] X is -VY;

[0116] V is a bond or a C1-C6 alkylene group, preferably V is a bond;

[0117] Y is a halogen;

[0118] L is a linker selected from the group consisting of: C1-C6 alkylene, C3-C6 cycloalkylene, and C6-C 10The arylene group, wherein the alkylene group, cycloalkylene group, and arylene group are optionally substituted with one or more substituents selected from the following: -OR', =O, =NR', =N-OR', -NR'R”, -SR', -halogen, -SiR'R”R”', -OC(O)R', -C(O)R', -CO2R', -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”')=NR””, -S(O)R', -S(O)2R', -S(O)2NR'R”, -NRSO2R', -CN, and -NO2, wherein the number of substitutions ranges from zero to 2m', where m' is the total number of carbon atoms in such groups. R', R”, R”' and R”” can each independently refer to hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl;

[0119] W chooses freedom-NR 2 -(C=O), -NR 2 -(C=S), -(C=O)-NR 2 - and -(C=S)-NR 2 -

[0120] Preferably, in the group consisting of W, W is -(C=O)-NR 2 -;

[0121] L and W can be the same or different each time they appear;

[0122] R 2 It is H or C1-C4 alkyl, preferably R. 2 It is H;

[0123] n is an integer that can be selected from the groups consisting of 1, 2, and 3;

[0124] Ch is a chelating agent, usually DOTA.

[0125] Compounds having formula (I) include stereoisomers having formulas (Ia), (Ib), (Ic), and (Id):

[0126]

[0127] The phrase "wherein each occurrence of L and W may be the same or different" means that when the variable "n" is 2 or 3, one "L" group can be a C1-C6 alkylene group, while another one or more "L" groups can be C3-C6 cycloalkylene or arylene groups, or, in other embodiments, each "L" group can be, for example, a C1-C6 alkylene group. Similarly, for example, when "n" is 2 or 3, a "W" group can be -(C=O)-NR. 2The other one or more "W" groups can be -(C=S)-NR 2 - Or in other embodiments, each "W" can be, for example, -(C=O)-NR 2 -

[0128] According to one embodiment, L is a connector selected from the group consisting of: C1-C6 alkylene, C3-C6 cycloalkylene, and C6-C 10 The arylene group, wherein the alkylene, cycloalkylene, and arylene are optionally substituted by one or more substituents selected from the following: -OR', =O, =NR', -NR'R”, -halogen, -OC(O)R', -C(O)R', -CO2R', -C(O)NR'R”, -OC(O)NR'R”, -NR”C(O)R', -NR'-C(O)NR”R”', -NR”C(O)OR', with the number of substitutions ranging from zero to 2m', where m' is the total number of carbon atoms in such groups. R', R”, R”', and R”” can each independently refer to hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0129] According to one embodiment, L is a linker selected from the group consisting of: a C3-C6 alkylene group optionally substituted with one or more substituents selected from the group consisting of: -OR', =O, =NR', -NR'R”, -halogen, -OC(O)R', -C(O)R', -CO2R', -C(O)NR'R”, -OC(O)NR'R”, -NR”C(O)R', -NR'-C(O)NR”R”', -NR”C(O)OR', with the number of substitutions ranging from zero to 2m', where m' is the total number of carbon atoms in such groups. R', R”, R”' and R”” can each independently refer to hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl.

[0130] According to one embodiment, R is selected from the group consisting of: C6-C substituted with one or more halogens. 10 Aryl groups and pyridines substituted with one or more halogens.

[0131] According to one embodiment, R is selected from the group consisting of:

[0132] and;

[0133] Where p is an integer selected from the group consisting of 1, 2, 3, 4 and 5, preferably p is 1.

[0134] According to a specific embodiment, R is selected from...

[0135] and And more preferably R is

[0136] According to one specific embodiment, X is selected from Br and I.

[0137] Advantageously, R is

[0138] Ch can be selected from the following groups:

[0139]

[0140]

[0141] and According to a specific embodiment, Ch is

[0142]

[0143] According to one embodiment, W is -(C=O)-NR 2 - and Ch is

[0144] According to one embodiment, m is 4, Z is COOQ, and Q is H.

[0145] In a specific embodiment, according to one example, R is And Ch is

[0146] According to a preferred embodiment, the PSMA-binding ligand is a compound having formula (II):

[0147]

[0148] Compounds having formula (II) can be called PSMA-R2.

[0149] According to another embodiment, the PSMA-binding ligand is a compound having formula (III):

[0150]

[0151] Compounds having formula (III) can be called PSMA-Cpd2.

[0152] The first vial containing the PSMA-binding ligand

[0153] In some embodiments, the radiolabeling method uses a kit in a single vial. In this embodiment, the first vial contains the PSMA-binding ligand, a buffer, and an optional expander, all in dry form.

[0154] Alternatively, the radiolabeling method uses a two-vial kit. In this embodiment, the first vial contains the PSMA-binding ligand and an optional incrementing agent, and the second vial contains a buffer.

[0155] For example, the PSMA binding ligand, typically a PSMA binding ligand having formula (II), is contained in the first vial in an amount of 10 μg to 100 μg, preferably 15 μg to 60 μg, or even more preferably about 30 μg.

[0156] In a preferred embodiment, mannitol can be used as an extender, preferably in an amount of 5 mg to 50 mg, more preferably 10 mg to 30 mg, and even more preferably about 20 mg.

[0157] In one specific embodiment, the first dose or single dose does not contain an antioxidant. For example, the first vial or a single vial does not contain gentianic acid.

[0158] The example provides a preferred example of the first vial (vial 1 of the two-vial kit).

[0159] The first vial is preferably obtained by freeze-drying using methods well known in the art. Therefore, the first vial can be provided in the form of freeze-dried or spray-dried vials.

[0160] As used herein, the buffer is a buffer suitable for obtaining a pH of 2.5 to 4.0, preferably 2.8 to 4.0, more preferably 3.0 to 4.0, and even more preferably 3.2 to 3.8 in the incubation step (iii). "A buffer with a pH of 2.5 to 4.0, preferably 2.8 to 4.0, more preferably 3.0 to 4.0, and even more preferably 3.2 to 3.8" can advantageously be a formic acid buffer containing sodium hydroxide.

[0161] In one specific embodiment, the first vial or individual vial does not contain an antioxidant, for example, the first vial or individual vial does not contain gentian acid, and the buffer is a buffer suitable for obtaining a pH of 2.5 to 4.0, preferably 2.8 to 4.0, more preferably 3.0 to 4.0, or even more preferably 3.2 to 3.8 in the incubation step (iii).

[0162] The buffer may be further contained in a first vial in embodiments using a single vial of the kit, or further contained in a separate second vial in embodiments using a two-via-vial kit.

[0163] Step (ii) adds the solution of the radioactive isotope to the first vial.

[0164] Radioactive isotopes used in radiolabeling methods include those suitable for use as contrast agents in PET and SPECT imaging, including:

[0165] 111 In、 133m In、 99m Tc, 94m Tc, 67 Ga、 66 Ga、 68 Ga、 52 Fe、 72 As、 97 Ru、 203 Pb, 62 Cu、 64 Cu、 86 Y、 51 Cr 52m Mn, 157 Gd, 169 Yb、 172 Tm、 177m Sn、 89 Zr、 43 Sc、 44 Sc、 55 Co.

[0166] According to a preferred embodiment, the radioactive isotope 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 for PET imaging, such as PET / CT or PET / MRI.

[0167] The metal ions of this radioactive isotope can form non-covalent bonds with the functional groups of chelating agents (such as carboxylic acids of PSMA-binding ligands).

[0168] In a specific embodiment, the solution containing the radioactive isotope is an eluent obtained from the following steps:

[0169] i. Generating radioactive isotopes from non-radioactive parent elements using a radioactive isotope generator.

[0170] ii. Separating the radioactive isotope from the parent non-radioactive element by elution in HCl as the elution solvent, and

[0171] iii. Recover the eluent.

[0172] Thus, a solution of the radioactive isotope in HCl is obtained.

[0173] In a specific embodiment, the solution containing the radioactive isotope is a radioactive isotope containing metal ions (e.g.,68 Ga 3+ , 67 Ga 3+ or 64 Cu 2+ An aqueous solution containing the radioactive isotope. The solution may contain HCl. 68 GaCl3, 67 GaCl3 or 64 Aqueous solution of CuCl2.

[0174] The containing radioactive isotopes 68 The Ga solution is typically obtained from the following steps:

[0175] i. From the parent element via the generator 68 Ge production 68 Ga element, and

[0176] ii. Optionally, by making the element 68 Ge / 68 Ga passing through a suitable pillar will generate 68 Ga element and 68 Ge element was separated and eluted in HCl. 68 Ga

[0177] Thus, a solution of the radioactive isotope in HCl is obtained.

[0178] from 68 Ge / 68 Ga generator produces 68 This method of Ga is well known in the art and is described, for example, in: Martinova L. et al. Gallium-68 in Medical Imaging. CurrRadiopharm. 2016; 9(3):187-20; Dash A, Chakravarty Radionuclidegenerators: the prospect of availing PET radiotracers to meet current clinical needs and future research demands. R Am J Nucl Med Mol Imaging. 15 Feb 2019; 9(1):30-66.

[0179] Contains radioactive isotopes 68The solution of Ga can be an eluent commonly obtained from cyclotron production. Such production is described, for example, in Am J Nucl Med Mol Imaging 2014; 4(4):303-310 or BJB Nelson et al. / Nuclear Medicine and Biology 80-81(2020)24-31.

[0180] generally, 68 Ga can be produced by a cyclotron, preferably using a proton beam with energies in the range of 8 MeV to 18 MeV, and more preferably in the range of 11 MeV to 14 MeV. 68 Ga can be used with solid or liquid target systems. 68 Zn(p,n) 68 Ga is produced through a reaction. The target is enriched with... 68 Zn metal or 68 The Zn liquid solution composition. After irradiation, the target is transferred for further chemical processing, in which ion exchange chromatography is used for separation. 68 Ga. 68 Ga was eluted in HCl solution.

[0181] Alternatively, the radioactive isotope is 67 Ga. Protons, deuterium nuclei, alpha particles, or helium(III) are produced using zinc (enriched or natural) or copper or germanium targets as bombardment particles. 67 Various 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 67 Ga with a cyclotron. 67 [Comparative Study of Ga Methods] This article is published in: Radiopharmaceuticals and Labelled Compounds, Vol. 1, IAEA, Vienna, Vol. 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 67Ga at Oslo cyclotron. [Oslo cyclotron produces...] 67 [Ga] University of Oslo Report, OUP8-3-1, pp. 3-5. Bombardment with intermediate-energy protons (up to 64 MeV). nat Ge targets are also a suitable method for producing 67Ga, as described below: T Horiguchi, H Kumahora, H Inoue, Y Yoshizawa Excitation functions of Ge(p,xnyp) reactions and production of 68Ge, Int. J. Appl. Radiat. Isot., 34 (1983), pp. 1531-1535.

[0182] Preferably, 67 Ga can be produced by cyclotrons. 68 Zn(p,2n) 67 Ga production 67 The Ga method is well known in the art, for example, as described in the Iranian Journal of Pharmaceutical Research by Alirezapour B et al. (2013), 12(2):355-366. More preferably, the method uses a proton beam with energies in the range of 10 MeV to 40 MeV. 67 Ga can be accessed through 67 Zn(p,n) 67 Ga or 68 Zn(p,2n) 67 Ga is produced using solid or liquid target systems. The target is composed of enriched... 67 Zn or 68 Zn is composed of metal or liquid solution. After irradiation, the target is transferred for further chemical processing, including separation using ion exchange chromatography. 67 Ga was finally obtained by evaporation from an aqueous HCl solution. 67 GaCl3 can then be added to the individual vials for labeling.

[0183] Alternatively, the radioactive isotope is obtained from cyclotron production. 64 Cu. This production method is described, for example, in WO2013 / 029616.

[0184] generally, 64Cu can be produced by a cyclotron, preferably using a proton beam with energies between 11 and 18 MeV. 64 Cu can be used with solid or liquid target systems. 64 Ni(p,n) 64 Produced by the reaction of Cu. The target is made from... 64 The target consists of Ni metal or a 64Ni liquid solution. After irradiation, the target is transferred for further chemical processing, in which ion-exchange chromatography is used for separation. 64 Cu. Produced from the final evaporation of aqueous HCl. 64 CuCl2 can then be added to the first vial for labeling.

[0185] Step (iii) involves mixing the solution obtained in step (ii) with at least a buffer and incubating for a period of time sufficient to obtain the PSMA-binding ligand labeled with the radioisotope. Step (iii) is preferably carried out at a sufficiently high temperature, for example at least 50°C, preferably between 50°C and 100°C.

[0186] In a first vial containing a PSMA-binding ligand (e.g., a PSMA-binding ligand of formula (II)) and a radioactive isotope (typically in a suitable buffer as described above) is mixed. 68 Ga、 67 Ga or 64 After the solution of Cu (as described above) is prepared, radioactive labeling begins.

[0187] In a specific embodiment, the incubation step is performed at a temperature of 50°C to 100°C. In a specific embodiment, the incubation step lasts for a period of 2 to 25 minutes.

[0188] In a specific embodiment, the incubation step is carried out at a temperature of 80°C to 100°C, preferably 90°C to 100°C, and typically about 95°C.

[0189] In other specific embodiments, the incubation step is carried out at a temperature of 50°C to 90°C, preferably 60°C to 80°C, and typically about 70°C.

[0190] In a specific embodiment, the incubation step is carried out for 2 to 20 minutes, preferably 5 to 10 minutes, more preferably 6 to 8 minutes, and even more preferably about 7 minutes.

[0191] In other specific embodiments, the incubation step is performed for 5 to 25 minutes, preferably 10 to 20 minutes, more preferably 12 to 18 minutes, and even more preferably for a period of about 15 minutes.

[0192] At the end of the labeling process, add the radioactive isotope (e.g.) 68 Ga、 67 Ga or64 Cu) chelating agents with specific affinities chelate the unreacted portions of the co-isotope. This complex, formed by the sequestering agent and the unreacted radioisotope, can then be discarded to increase the radiochemical purity after radiolabeling.

[0193] use 68 Preferred embodiment of the method for radiolabeling PSMA-binding ligands having formula (II) with Ga

[0194] This disclosure relates more specifically to the use of 68 Method for Ga to label PSMA-binding ligands having formula (II)

[0195]

[0196] The method includes the following steps:

[0197] i. Provide a first vial containing approximately 30 μg of a PSMA-binding ligand of formula (II) in dry form.

[0198] ii. will 68 A solution of Ga in HCl was added to the first vial.

[0199] iii. Mix the solution obtained in ii with a reaction solution containing a buffer to adjust the pH to the range of 3.2 to 3.8, and incubate at a sufficiently high temperature for a sufficient period of time to obtain the solution. 68 Ga-labeled PSMA-binding ligands, and

[0200] iv. Optionally adjust the pH of the solution.

[0201] In a specific embodiment of the method, the 68 The solution of Ga in HCl is the eluent obtained from the following steps:

[0202] i. From the parent element via the generator 68 Ge production 68 Ga element, and

[0203] ii. Optionally, by making the element 68 Ga / 68 Ge will be generated when it passes through a suitable pillar. 68 Ga element and 68 Ge element was separated and eluted in HCl. 68 Ga

[0204] Thus, a solution of the radioactive isotope in HCl is obtained.

[0205] Typically, the buffer consists of 60 mg of formic acid and 56.5 mg of sodium hydroxide.

[0206] In one specific embodiment, the powder for injection does not contain antioxidants. For example, the powder for injection does not contain gentianic acid.

[0207] Advantageously, in certain embodiments, simple labeling of PSMA-binding ligands can be achieved using commercially available [products / methods]. 68 Ge / 68 Ga generator in HCl 68 Ga is obtained from the eluent without any treatment of the eluent or any additional purification steps.

[0208] Powder for Injection

[0209] This disclosure also relates to an injectable powder comprising the following components in a dry form:

[0210] i. PSMA-binding ligands of formula (I):

[0211]

[0212] in:

[0213] Z is either tetrazolium or COOQ, preferably Z is COOQ;

[0214] Q is independently H or a protecting group, preferably Q is H;

[0215] m is an integer selected from the group consisting of 1, 2, 3, 4 and 5, preferably m is 4;

[0216] q is an integer selected from the group consisting of 1, 2, 3, 4, 5 and 6, preferably q is 1;

[0217] R is selected from the following groups: C6-C 10 Aryl and heteroaryl containing 5-10 ring atoms, wherein the aryl and heteroaryl are substituted by X once or more;

[0218] X is -VY;

[0219] V is a bond or a C1-C6 alkylene group, preferably V is a bond;

[0220] Y is a halogen;

[0221] L is a linker selected from the group consisting of: C1-C6 alkylene, C3-C6 cycloalkylene, and C6-C 10The arylene group, wherein the alkylene group, cycloalkylene group, and arylene group are optionally substituted with one or more substituents selected from the following: -OR', =O, =NR', =N-OR', -NR'R”, -SR', -halogen, -SiR'R”R”', -OC(O)R', -C(O)R', -CO2R', -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”')=NR””, -S(O)R', -S(O)2R', -S(O)2NR'R”, -NRSO2R', -CN, and -NO2, wherein the number of substitutions ranges from zero to 2m', where m' is the total number of carbon atoms in such groups. R', R”, R”' and R”” can each independently refer to hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl;

[0222] W chooses freedom-NR 2 -(C=O), -NR 2 -(C=S), -(C=O)-NR 2 - and -(C=S)-NR 2 -The group consisting of W, preferably -(C=O)-NR 2 -;

[0223] L and W can be the same or different each time they appear;

[0224] R 2 It is H or C1-C4 alkyl, preferably R. 2 It is H;

[0225] n is an integer that can be selected from the groups consisting of 1, 2, and 3;

[0226] Ch is a chelating agent, usually DOTA; and

[0227] ii. Incremental agents, such as mannitol.

[0228] The preferred embodiment includes the following components:

[0229] i. A PSMA-binding ligand having formula (II) in an amount of 10 μg to 100 μg, preferably 15 μg to 60 μg, even more preferably about 30 μg;

[0230] and

[0231] ii. Mannitol, in an amount of 5 mg to 50 mg, preferably 10 mg to 30 mg, or even more preferably about 20 mg.

[0232] In one specific embodiment, the powder for injection does not contain antioxidants. For example, the powder for injection does not contain gentianic acid.

[0233] The radiolabeled reagent kit disclosed herein

[0234] This disclosure also relates to a kit for implementing the above-described labeling method, the kit comprising...

[0235] i. The first vial contains the following components in dry form.

[0236] i. PSMA-binding ligands of formula (II):

[0237] and

[0238] ii. Optionally, a extender, such as mannitol, and,

[0239] ii. A second vial containing at least a buffer, preferably in a dry form; and,

[0240] iii. Optionally, an accessory cassette for eluting radioactive isotopes generated by a radioactive isotope generator or a cyclotron.

[0241] Preferably, the first or single vial contains the following components:

[0242] i. A PSMA-binding ligand having formula (II) in an amount of 10 μg to 100 μg, preferably 15 μg to 60 μg, even more preferably about 30 μg;

[0243] and

[0244] ii. Mannitol, in an amount of 5 mg to 50 mg, preferably 10 mg to 30 mg, or even more preferably about 20 mg.

[0245] The second vial or individual vial may contain a buffer for maintaining the pH at 2.5 to 4.0, preferably 2.8 to 4.0, more preferably 3.0 to 4.0, and even more preferably 3.2 to 3.8. For example, the second vial contains formic acid and sodium hydroxide as a buffer. The buffer may be in dry or solution form. According to one embodiment, the buffer consists of an aqueous solution of formic acid and sodium hydroxide, wherein the formic acid is present at a concentration of about 60 mg / mL and the sodium hydroxide is present at a concentration of about 56.5 mg / mL.

[0246] Preferably, all components of the first, second, or individual vials are in dry form.

[0247] The radioisotope used to label the PSMA-binding ligand can be provided as a ready-to-use product with the kit, i.e., for mixing and incubation with the first vial and buffer provided with the kit, or alternatively, can be eluted from the radioisotope generator or cyclotron shortly before or shortly before mixing and incubation with the first vial and buffer, especially where the radioisotope has a relatively short half-life, for example... 68 Ga、 67 Ga and 64 Cu. Radioactive isotopes used for labeling, such as... 68 Ga、 67 Ga or 64 Cu can also be produced by a cyclotron.

[0248] Preferably, the components are inserted into a sealed container that can be packaged together with the instructions for performing the method according to this disclosure.

[0249] The kit can also be used as part of an automated system or remote control mechanism that automatically performs elution and / or subsequent mixing and heating of the gallium-68 generator. In this embodiment, the vial containing the PSMA-binding ligand (the first vial) is directly connected to the elution system and / or heating system.

[0250] This kit is particularly suitable for the methods disclosed in the next section.

[0251] In one specific embodiment, the kit does not contain antioxidants. For example, the kit does not contain gentianic acid.

[0252] In one specific embodiment, the kit does not contain antioxidants, for example, the kit does not contain gentianic acid, and the second vial or individual vial contains a buffer for maintaining a pH of 2.5 to 4.0, preferably 2.8 to 4.0, more preferably 3.0 to 4.0, and even more preferably 3.2 to 3.8.

[0253] In a specific embodiment, the PSMA binding ligand is a PSMA binding ligand having formula (II) as defined above.

[0254] According to the intended use of the reagent kit disclosed herein

[0255] The kits defined above are particularly suitable for the labeling methods disclosed in the preceding sections.

[0256] Advantageously, it includes radioactive isotopes (e.g. 68 Ga、 67 Ga or 64 Solutions of Cu-labeled PSMA-binding ligands (e.g., PSMA-binding ligands having formula (II)) can be obtained or acquired by the labeling methods disclosed in the preceding sections.

[0257] Such solutions can be ready-to-use injectable solutions, for example, for detecting tumors in vivo by imaging in subjects who need them.

[0258] In some respects, the subjects are mammals, such as, but not limited to, rodents, dogs, cats, or primates. In a preferred respect, the subjects are humans.

[0259] The requirements for effective drug carriers in injectable compositions are well known to those skilled in the art (see, for example, Pharmaceutics and Pharmacy Practice, JB Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pp. 238-250 (1982), and SHP Handbook on Injectable Drugs, Trissel, 15th edition, pp. 622-630 (2009)).

[0260] Typically, the solution used as an injectable solution provides a single dose of 100-350 MBq, preferably 150-250 MBq, having formula (II) [ 68 Ga]-PSMA binding ligands are used for administration to subjects in need.

[0261] In a specific embodiment, the subject in need is a subject suffering from cancer with tumors or cells expressing PSMA. The tumors or cells expressing PSMA may be selected from the group consisting of: prostate tumors or cells, metastatic prostate tumors or cells, lung tumors or cells, kidney tumors or cells, glioblastoma, pancreatic tumors or cells, bladder tumors or cells, sarcoma, melanoma, breast tumors or cells, colon tumors or cells, germ cells, pheochromocytoma, esophageal tumors or cells, gastric tumors or cells, and combinations thereof. In some other embodiments, the tumors or cells expressing PSMA are prostate tumors or cells.

[0262] Typically, PET / MRI, SPECT, or PET / CT imaging can be obtained 20 to 120 minutes, preferably 50 to 100 minutes, and more preferably 2 to 3 hours after intravenous administration of the radiolabeled PSMA-binding ligand to the subject.

[0263] Synthesis of compounds having formulas (I), (II) and (III)

[0264] Compounds having formulas (I), (II) and (III) can be synthesized using the methods disclosed in WO2017 / 165473.

[0265] Specifically, compounds having formula (II) can be synthesized as disclosed in scheme 1. The modified p-bromobenzyl group of Glu-Lys urea 2 can be prepared by reductive alkylation of Glu-Lys urea 1 with p-bromobenzaldehyde in methanol in the presence of sodium cyanoborohydride. This procedure has been described in the literature (Tykvart et al. (2015) Journal of Medicinal Chemistry 58, 4357-63). Then, Boc-6-aminohexanoic acid can be coupled to the same ε-Lysamine of 2 to produce compound 3, for example, using a base (such as N,N-diisopropylethylamine) and a coupling agent (such as N,N,N′,N′-tetramethyl-O-(N-succinimide)urea tetrafluoroborate or 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate). Compound 3 can then be deprotected, for example, using an acid such as trifluoroacetic acid, to produce compound 4. Finally, conjugation with a commercially available DOTA-NHS ester can be performed to produce compound (II).

[0266] Scheme 1: Synthesis of compounds having formula (II)

[0267]

[0268] Implementation

[0269] The following specific embodiments are disclosed:

[0270] 1. A method using a radioactive isotope, preferably 68 Ga、 67 Ga or 64 A method for Cu-labeled PSMA binding ligands, the method comprising the following steps:

[0271] i. Provide a first vial containing the PSMA-binding ligand in dry form and an optional extender.

[0272] ii. Add the solution of the radioactive isotope to the first vial to obtain a solution of the PSMA binding ligand and the radioactive isotope.

[0273] iii. The solution obtained in ii. is mixed with at least a buffer and incubated for a period of time sufficient to obtain the PSMA-binding ligand labeled with the radioisotope, and

[0274] iv. Optionally, adjust the pH of the solution.

[0275] 2. The method as described in Example 1, wherein the first vial in step i is a vial containing, preferably entirely in dry form, the PSMA binding ligand, a buffer, and optionally a extender.

[0276] 3. The method as described in Example 1, wherein step iii comprises mixing the solution obtained in ii. with a reaction solution containing at least a buffer and incubating it at a sufficiently high temperature for a sufficiently long time to obtain the PSMA binding ligand labeled with the radioisotope.

[0277] 4. The method as described in any one of Examples 1-3, wherein the solution containing the radioactive isotope further comprises HCl.

[0278] 5. The method as described in any one of Examples 1-4, wherein the radioactive isotope is 68 Ga and the radiochemical purity measured by HPLC is at least 92%, and optionally, free 68 Ga 3+ The percentage (in HPLC) is 2% or less, and / or uncomplexed. 68 Ga 3+ The percentage of the substance (in ITLC) is 3% or less.

[0279] 6. The method as described in any one of Examples 1-4, wherein the radioactive isotope is 64Cu and its radiochemical purity, as measured by HPLC, is at least 92%, and optionally, free... 64 Cu 2+ The percentage (in HPLC) is 2% or less, and / or uncomplexed. 64 Cu 2+ The percentage of the substance (in ITLC) is 3% or less.

[0280] 7. The method as described in any one of Examples 1-4, wherein the radioactive isotope is 67 Ga and the radiochemical purity measured by HPLC is at least 92%, and optionally, free 67 Ga 3+ The percentage (in HPLC) is 2% or less, and / or uncomplexed. 67 Ga 3+ The percentage of the substance (in ITLC) is 3% or less.

[0281] 8. The method as described in any one of Examples 1-7, wherein the PSMA-binding ligand is a compound having formula (I):

[0282]

[0283] in:

[0284] Z is either tetrazolium or COOQ, preferably Z is COOQ;

[0285] Q is independently H or a protecting group, preferably Q is H;

[0286] m is an integer selected from the group consisting of 1, 2, 3, 4 and 5, preferably m is 4;

[0287] q is an integer selected from the group consisting of 1, 2, 3, 4, 5 and 6, preferably q is 1;

[0288] R is selected from the following groups: C6-C 10 Aryl and heteroaryl containing 5-10 ring atoms, wherein the aryl and heteroaryl are substituted by X once or more;

[0289] X is -VY;

[0290] V is a bond or a C1-C6 alkylene group, preferably V is a bond;

[0291] Y is a halogen;

[0292] L is a linker selected from the group consisting of: C1-C6 alkylene, C3-C6 cycloalkylene, and C6-C 10 The arylene group, wherein the alkylene group, cycloalkylene group, and arylene group are optionally substituted with one or more substituents selected from the following: -OR', =O, =NR', =N-OR', -NR'R”, -SR', -halogen, -SiR'R”R”', -OC(O)R', -C(O)R', -CO2R', -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”')=NR””, -S(O)R', -S(O)2R', -S(O)2NR'R”, -NRSO2R', -CN, and -NO2, wherein the number of substitutions ranges from zero to 2m', where m' is the total number of carbon atoms in such groups. R', R”, R”' and R”” can each independently refer to hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl;

[0293] W chooses freedom-NR 2 -(C=O), -NR 2 -(C=S), -(C=O)-NR 2 - and -(C=S)-NR 2 -The group consisting of W, preferably -(C=O)-NR 2 -;

[0294] L and W can be the same or different each time they appear;

[0295] R 2 It is H or C1-C4 alkyl, preferably R. 2 It is H;

[0296] n is an integer that can be selected from the groups consisting of 1, 2, and 3;

[0297] Ch is a chelating agent, usually DOTA.

[0298] 9. The method as described in Example 8, wherein the PSMA-binding ligand is a compound having formula (II):

[0299]

[0300] 10. The method as described in any one of Examples 1-9, wherein the PSMA binding ligand is contained in the first vial in an amount of 10 μg to 100 μg, preferably 15 μg to 60 μg, or even more preferably about 30 μg.

[0301] 11. The method as described in any one of Examples 1-10, wherein the first vial further comprises mannitol as an extender, preferably 5 mg to 50 mg, preferably 10 mg to 30 mg, and even more preferably about 20 mg.

[0302] 12. The method as described in any one of Examples 1-11, wherein the buffer in incubation step (iii) is present in an amount suitable for obtaining a pH of 2.5 to 4.0, preferably 2.8 to 4.0, more preferably 3.0 to 4.0, and even more preferably 3.2 to 3.8.

[0303] 13. The method of any one of Examples 1-12, wherein the buffer comprises formic acid and sodium hydroxide as buffers.

[0304] 14. The method as described in any one of Examples 1-13, wherein the incubation step is performed at a temperature of 50°C to 100°C.

[0305] 15. The method as described in any one of Examples 1-14, wherein the incubation step is performed for a period of 2 to 25 minutes.

[0306] 16. The method as described in any one of Examples 1-15, wherein the incubation step is performed at a temperature of 80°C to 100°C, preferably 90°C to 100°C, or even more preferably about 95°C.

[0307] 17. The method as described in any one of Examples 1-16, wherein the incubation step is performed for a period of 2 to 20 minutes, preferably 5 to 10 minutes, more preferably 6 to 8 minutes, and even more preferably about 7 minutes.

[0308] 18. The method as described in any one of Examples 1-15, wherein the incubation step is performed at a temperature of 50°C to 90°C, preferably 60°C to 80°C, typically about 70°C.

[0309] 19. The method as described in any one of Examples 1-15 or 18, wherein the incubation step is performed for a period of 5 to 25 minutes, preferably 10 to 20 minutes, more preferably 12 to 18 minutes, and even more preferably about 15 minutes.

[0310] 20. The method of any one of Examples 1-19, wherein the solution of the radioactive isotope is the eluent obtained from the following steps:

[0311] i. Generating radioactive isotopes from non-radioactive parent elements using a radioactive isotope generator.

[0312] ii. Separating the radioactive isotope from the parent non-radioactive element by elution.

[0313] iii. Recover the eluent.

[0314] Thus, a solution of the radioactive isotope is obtained.

[0315] 21. The method as described in any one of Examples 1-19, wherein the solution of the radioactive isotope is the eluent obtained from the following steps:

[0316] i. Producing radioactive isotopes from non-radioactive or radioactive elements using a cyclotron.

[0317] ii. Separating the radioactive isotope from the non-radioactive or radioactive element by elution.

[0318] iii. Recover the eluent.

[0319] Thus, a solution of the radioactive isotope is obtained.

[0320] 22. The method as described in any one of Examples 1-21, wherein the first vial or individual vial does not contain an antioxidant, for example, the first vial or individual vial does not contain gentianic acid, and the buffer is a buffer suitable for obtaining a pH of 2.5 to 4.0, preferably 2.8 to 4.0, more preferably 3.0 to 4.0, or even more preferably 3.2 to 3.8 in the incubation step (iii).

[0321] 23. The method as described in any one of Examples 1-22, wherein the first vial or individual vial does not contain gentianic acid.

[0322] 24. A method for using 68Ga method for binding ligands to PSMA with formula (II)

[0323]

[0324] The method includes the following steps:

[0325] i. Provide a first vial containing approximately 30 μg of a PSMA-binding ligand of formula (II) in dry form.

[0326] ii. will 68 A solution of Ga in HCl was added to the first vial.

[0327] iii. Mix the solution obtained in ii with a reaction solution containing a buffer to adjust the pH to the range of 3.2 to 3.8, and incubate at a sufficiently high temperature for a sufficient period of time to obtain the solution. 68 Ga-labeled PSMA binding ligands

[0328] iv. Optionally adjust the pH of the solution.

[0329] 25. The method as described in Example 24, wherein... 68 The solution of Ga in HCl is the eluent obtained from the following steps:

[0330] i. From the parent element via the generator 68 Ge production 68 Ga element,

[0331] ii. By making elements 68 Ga / 68 Ge passes through a suitable column and is eluted in HCl. 68 Ga will generate 68 Ga element and 68 Ge element separation

[0332] Thus, a solution of the radioactive isotope in HCl is obtained.

[0333] 26. The method as described in Example 24, wherein... 68 The solution of Ga in HCl is the eluent obtained from the following steps:

[0334] i. From elements such as 68 Zn is produced via a cyclotron. 68 Ga element,

[0335] ii. By making elements 68 Ga / starting element is passed through a suitable column and eluted in HCl. 68 Ga will generate 68The Ga element is separated from the starting element.

[0336] Thus, a solution of the radioactive isotope in HCl is obtained.

[0337] 27. A method for using 67 Method for Ga to label PSMA-binding ligands having formula (II)

[0338]

[0339] The process includes the following steps:

[0340] i. Provide a first vial containing approximately 30 μg of a PSMA-binding ligand of formula (II) in dry form.

[0341] ii. will 67 A solution of Ga in HCl was added to the first vial.

[0342] iii. Mix the solution obtained in ii with a reaction solution containing a buffer to adjust the pH to the range of 3.2 to 3.8, and incubate at a sufficiently high temperature for a sufficient period of time to obtain the solution. 67 Ga-labeled PSMA binding ligands

[0343] The pH of the solution may be adjusted optionally.

[0344] 28. A method for using 64 Method for labeling PSMA-binding ligands with formula (II) using Cu

[0345]

[0346] The process includes the following steps:

[0347] i. Provide a first vial containing approximately 30 μg of a PSMA-binding ligand of formula (II) in dry form.

[0348] ii. will 64 A solution of Cu in HCl was added to the first vial.

[0349] iii. Mix the solution obtained in ii with a reaction solution containing a buffer to adjust the pH to the range of 3.2 to 3.8, and incubate at a sufficiently high temperature for a sufficient period of time to obtain the solution. 64 Cu-labeled PSMA binding ligands

[0350] iv. Optionally adjust the pH of the solution.

[0351] 29. The method as described in Examples 24-28, wherein the first vial or individual vial does not contain an antioxidant, for example, the first vial or individual vial does not contain gentianic acid, and the buffer is a buffer suitable for obtaining a pH of 2.5 to 4.0, preferably 2.8 to 4.0, more preferably 3.0 to 4.0, or even more preferably 3.2 to 3.8 in the incubation step (iii).

[0352] 30. The method of any one of Examples 24-29, wherein the buffer consists of 60 mg formic acid and 56.5 mg sodium hydroxide.

[0353] 31. The method of any one of Examples 24-29, wherein the buffer is composed of an aqueous solution of formic acid and sodium hydroxide, wherein the formic acid is present at a concentration of about 60 mg / mL and the sodium hydroxide is present at a concentration of about 56.5 mg / mL.

[0354] 32. The method as described in any one of Examples 24-31, wherein the incubation step is performed at a temperature of 50°C to 100°C.

[0355] 33. The method as described in any one of Examples 24-32, wherein the incubation step is performed for a period of 2 to 25 minutes.

[0356] 34. The method as described in any one of Examples 24-27 and 29-33, 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.

[0357] 35. The method as described in any one of Examples 24-27 or 29-34, wherein the incubation step is performed for a period of 2 to 20 minutes, preferably 5 to 10 minutes, preferably 6 to 8 minutes, and even more preferably about 7 minutes.

[0358] 36. The method as described in any one of Examples 28-33, wherein the incubation step is performed at a temperature of 50°C to 90°C, preferably 60°C to 80°C, typically about 70°C.

[0359] 37. The method as described in any one of Examples 28-33 or 36, wherein the incubation step is performed for a period of 5 to 25 minutes, preferably 10 to 20 minutes, more preferably 12 to 18 minutes, and even more preferably about 15 minutes.

[0360] 38. The method of any one of Examples 24-37, wherein the first vial or individual vial does not contain an antioxidant, such as gentianic acid.

[0361] 39. A solution comprising a PSMA-binding ligand labeled with a radioisotope, which can be obtained or acquired by any of the methods described in any of Examples 1-23, said solution being used as an injectable solution for in vivo detection of tumors, typically PSMA-expressing tumors, by imaging in a subject in need.

[0362] 40. The solution as described in Example 39, wherein the radioactive isotope is selected from the group consisting of: 111 In、 133m In、 99m Tc, 94m Tc, 67 Ga、 66 Ga、 68 Ga、 52 Fe、 72 As、 97 Ru、 203 Pb, 62 Cu、 64 Cu、 86 Y、 51 Cr 52m Mn, 157 Gd, 169 Yb、 172 Tm、 177m Sn、 89 Zr、 43 Sc、 44 Sc、 55 Co.

[0363] 41. A method that can be obtained or acquired by any one of Examples 24-38, comprising... 68 Ga、 67 Ga or 64 A Cu-labeled solution of a PSMA-binding ligand of formula (II), said solution being used as an injectable solution for in vivo detection of tumors, typically PSMA-expressing tumors, by imaging in subjects in need.

[0364] 42. A powder for injection, comprising the following components in dry form:

[0365] i. PSMA-binding ligands of formula (I):

[0366]

[0367] in:

[0368] Z is either tetrazolium or COOQ, preferably Z is COOQ;

[0369] Q is independently H or a protecting group, preferably Q is H;

[0370] m is an integer selected from the group consisting of 1, 2, 3, 4 and 5, preferably m is 4;

[0371] q is an integer selected from the group consisting of 1, 2, 3, 4, 5 and 6, preferably q is 1;

[0372] R is selected from the following groups: C6-C 10 Aryl and heteroaryl containing 5-10 ring atoms, wherein the aryl and heteroaryl are substituted by X once or more;

[0373] X is -VY;

[0374] V is a bond or a C1-C6 alkylene group, preferably V is a bond;

[0375] Y is a halogen;

[0376] L is a linker selected from the group consisting of: C1-C6 alkylene, C3-C6 cycloalkylene, and C6-C 10 The arylene group, wherein the alkylene group, cycloalkylene group, and arylene group are optionally substituted with one or more substituents selected from the following: -OR', =O, =NR', =N-OR', -NR'R”, -SR', -halogen, -SiR'R”R”', -OC(O)R', -C(O)R', -CO2R', -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”')=NR””, -S(O)R', -S(O)2R', -S(O)2NR'R”, -NRSO2R', -CN, and -NO2, wherein the number of substitutions ranges from zero to 2m', where m' is the total number of carbon atoms in such groups. R', R”, R”' and R”” can each independently refer to hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl;

[0377] W chooses freedom-NR 2 -(C=O), -NR 2 -(C=S), -(C=O)-NR 2 - and -(C=S)-NR 2 -The group consisting of W, preferably -(C=O)-NR 2 -;

[0378] L and W can be the same or different each time they appear;

[0379] R 2 It is H or C1-C4 alkyl, preferably R. 2 It is H;

[0380] n is an integer that can be selected from the groups consisting of 1, 2, and 3;

[0381] Ch is a chelating agent, usually DOTA; and

[0382] ii. Incremental agents, such as mannitol.

[0383] 43. The powder for injection as described in Example 42, wherein the PSMA binding ligand has formula (II):

[0384]

[0385]

[0386] 44. The powder for injection as described in Examples 42 or 43, wherein the PSMA binding ligand is contained in an amount of 10 μg to 100 μg, preferably 15 μg to 60 μg, or even more preferably about 30 μg.

[0387] 45. The powder for injection as described in any one of Examples 42-44, wherein the extender is mannitol in an amount of 5 mg to 50 mg, preferably 10 mg to 30 mg, and even more preferably about 20 mg.

[0388] 46. ​​The powder for injection as described in any one of Examples 42-45, comprising the following components:

[0389] i. A PSMA-binding ligand having formula (II) in an amount of 10 μg to 100 μg, preferably 15 μg to 60 μg, even more preferably about 30 μg;

[0390] and

[0391] ii. Mannitol, in an amount of 5 mg to 50 mg, preferably 10 mg to 30 mg, or even more preferably about 20 mg.

[0392] 47. The powder for injection as described in any one of Examples 42-46, wherein the powder does not contain an antioxidant, for example, the powder does not contain gentianic acid.

[0393] 48. A kit for carrying out the method as described in any one of Examples 24-28, the kit comprising...

[0394] i. The first vial contains the following components in dry form.

[0395] i. PSMA-binding ligands of formula (II):

[0396] and

[0397] ii. Optionally, a extender, such as mannitol, and,

[0398] ii. A second vial containing at least a buffer, preferably in a dry form; and,

[0399] iii. Optionally, an accessory cassette for eluting radioactive isotopes generated by a radioactive isotope generator or a cyclotron.

[0400] 49. A kit for carrying out the method as described in any one of Examples 24-28, the kit comprising...

[0401] i. A single vial containing, preferably in a dry form, the following components:

[0402] i. PSMA-binding ligands of formula (II):

[0403] and

[0404] ii. Optionally, an extender, such as mannitol,

[0405] iii. at least one buffer, and,

[0406] ii. Optionally, an accessory cassette for eluting radioactive isotopes generated by a radioactive isotope generator or a cyclotron.

[0407] 50. The kit as described in Example 48 or 49, wherein the PSMA binding ligand is contained in an amount of 10 μg to 100 μg, preferably 15 μg to 60 μg, or even more preferably about 30 μg.

[0408] 51. The kit as described in any one of Examples 48-50, wherein the extender is mannitol in an amount of 5 mg to 50 mg, preferably 10 mg to 30 mg, and even more preferably about 20 mg.

[0409] 52. The kit as described in any one of Examples 48-51, wherein the first vial or individual vial comprises the following components:

[0410] i. A PSMA-binding ligand having formula (II) in an amount of 10 μg to 100 μg, preferably 15 μg to 60 μg, even more preferably about 30 μg;

[0411] and

[0412] ii. Mannitol, in an amount of 5 mg to 50 mg, preferably 10 mg to 30 mg, or even more preferably about 20 mg.

[0413] 53. The kit as described in any one of Examples 48-52, wherein the second vial or single vial contains a buffer for maintaining the pH at 2.5 to 4.0, preferably at 2.8 to 4.0, more preferably at 3.0 to 4.0, and even more preferably at 3.2 to 3.8.

[0414] 54. The kit as described in any one of Examples 48-53, wherein the kit does not contain an antioxidant, for example, the kit does not contain gentianic acid, and the second vial or individual vial contains a buffer for maintaining a pH of 2.5 to 4.0, preferably 2.8 to 4.0, more preferably 3.0 to 4.0, and even more preferably 3.2 to 3.8.

[0415] 55. The kit as described in any one of Examples 48-54, wherein the first vial or individual vial does not contain gentianic acid.

[0416] 56. The kit as described in any one of Examples 48-55, wherein the second vial or single vial contains formic acid and sodium hydroxide as buffers.

[0417] 57. The kit as described in any one of Examples 48-56, wherein all components of the first vial, second vial, or individual vial are in dry form.

[0418] Example

[0419] The present disclosure will be described in more detail and with specific reference to examples below, but these examples are not intended to limit the invention.

[0420] Radiochemical purity: non-complexed 68 Gallium-based materials (ITLC)

[0421] Preparation of the mobile phase solution:

[0422] Second Ammonium sulfate 5M: Accurately weigh 3.85 g of ammonium acetate into a 10 mL graduated flask and dissolve it in 10 mL of MilliQ water.

[0423] Ammonium acetate / MeOH Using a graduated cylinder, add 1 mL of 5M ammonium acetate solution, 4 mL of MilliQ water, and 5 mL of methanol. Transfer the eluent to a TLC chamber.

[0424] ITLC-SG preparation: Cut a 115mm ITLC-SG from each vial, draw a line 10mm from the bottom (place a 5µL sample drop here), and draw a line 105mm from the bottom (the chromatographic development must be discarded here).

[0425] 68 Ga-PSMA-R2: Reference coefficient 0.7-1.0

[0426] 68 Uncomplexed Ga: Reference factor = 0.0 ÷ 0.1

[0427] ( 68 Ga-uncomplexed substances refer to 68 Ga colloidal substances and free Ga 68 Ga.)

[0428] According to HPLC 68 Radiochemical purity and identification of GaPSMA-R2

[0429] Chromatographic conditions

[0430]

[0431] Example 1: Development using two vials of reagent kit 68 Methods for radiolabeling gallium with PSMA-R2

[0432] 1.2 Description and composition of the vial reagent kit

[0433] The applicant has developed a sterile 2-vial reagent kit, which consists of the following:

[0434] • Vial 1: PSMA-R2, 30μg, powder for injection, from... 68 Ge / 68 Gallium chloride-68 eluted by Ga generator (Ga generator) 68 Reconstruction of GaCl3 solution in HCl;

[0435] • Vial 2: Reaction buffer. Add vial 2 to the reconstructed vial 1.

[0436] The kit and from 68 Ge / 68 Ga generator elution 68 Ga solutions in dilute HCl are used in combination to prepare 68 Ga-PSMA-R2 is a radiolabeled imaging product for intravenous injection.

[0437] Injection 68 The volume of the Ga-PSMA-R2 solution (corresponding to the radioactive dose to be applied) is calculated based on the estimated injection time, the current activity provided by the generator, and the physical decay of the radionuclide (half-life = 68 min).

[0438] Vial 1 is an injectable powder containing 30 μg of PSMA-R2 as the active ingredient, packaged in a 10 mL ultra-inert Type I Plus glass vial.

[0439] The composition of vial 1 is provided in Table 1.

[0440] Table 1 - Composition of Powder for Injection in Vial 1

[0441]

[0442] *Current version

[0443] **Water for injection is eliminated during the freeze-drying process.**

[0444] The composition of vial 2 is provided in Table 2.

[0445] Table 2 - Composition of Powder for Vial 2 Injection

[0446]

[0447]

[0448] 2. Small bottle of powder (small bottle 1)

[0449] As described above, vial 1 (PSMA-R2, 30 μg, powder for injection) is part of a radiopharmaceutical kit, which also includes reaction buffer (via 2) and an accessory kit.

[0450] The kit must be compatible with 68 Ge / 68 Ga generator provides 68 Ga solutions in HCl are used in combination to obtain injectable formulations. 68 Ga-PSMA-R2 solution is a radiolabeled imaging product that can be injected directly into the patient.

[0451] 2.1 Components of the pharmaceutical product

[0452] This drug product contains PSMA-R2 as the active ingredient and mannitol as an excipient.

[0453] 2.1.1 Active pharmaceutical ingredient

[0454] The active substance is the PSMA-R2 peptide, a 7-polyamino acid sequence covalently bound to a chelating agent (DOTA) via a C6 (6-aminocaproic acid) linker. It is a compound having formula (II).

[0455] The sequence of PSMA-R2 is: HO-Glu-CO-Lys(Ne-4-bromobenzyl-Ne′-Ahx-DOTA)-OH, and the molecular formula is: C41H63BrN8O15.

[0456] 2.1.2 Excipients

[0457] Excipients selected for the composition of vial 1 are added to maintain the stability of the active ingredient in the final formulation, ensuring the safety and efficacy of the drug product and achieving [the desired results]. 68 The required radiochemical purity of the Ga-PSMA-R2 solution during reconstruction. The selected excipients produce a pharmaceutical product with the desired pharmaceutical properties.

[0458] A brief description of each excipient is as follows:

[0459] Mannitol

[0460] Mannitol is used as a expander. Because peptide drugs are highly effective, very small amounts are required in the drug product. Without a expander, product processing becomes technically unsuitable. The expander allows for the processing and production of a readily available lyophilized product.

[0461] 2.2 Pharmaceutical Products

[0462] 2.2.1 Formulation Development

[0463] Product development has been carried out, with the aim of being based on commercially available... 68 Ge / 68 The eluent from the Ga generator can be directly reconstructed without any treatment or additional purification steps to identify reaction mixture compositions that allow for simple labeling of DOTA molecules.

[0464] The goal of this project is to develop the PSMA-R2 small molecule as a radiotracer for detecting prostate tumors.

[0465] Vial 1 is a lyophilized powder containing peptides as the active ingredient, used in radiolabeling procedures. 68 Ga was radioactively labeled.

[0466] The initial efforts to develop a suitable formulation for PSMA-R2 (vial 1) involved testing in liquid form.

[0467] Pharmaceutical manufacturers are focusing their development efforts on selecting appropriate excipients related to the properties of PSMA-R2 in order to obtain finished products that meet the specifications typically required for radiopharmaceutical formulations.

[0468] · 68 Ga-PSMA-R2 (HPLC): >92%

[0469] ·free 68Ga3+ (HPLC): <2%

[0470] Non-complexing 68 Ga 3+ Substance (ITLC): <3%

[0471] The development work, including related studies, is described, starting with the selection of the amount of active ingredient and appropriate excipients.

[0472] 2.2.1.1 Selection of PSMA-R2 quantity

[0473] Using Galliapharm, E&Z 68 Ge / 68 A Ga generator (1850 MBq) was used to test incremental PSMA-R2, with the aim of identifying radiochemical purity ≥92% and free... 68 The minimum amount required for Ga < 2% (determined by HPLC analysis).

[0474] The following HPLC analyses are summarized in Table 3, indicating that the labeling using 5 μg PSMA-R2 did not meet specifications (free). 68 Ga% <2%). Labeling with 10 μg indicates free Ga% <2%. 68 The Ga% value was very close to the specification limit. When the amount of PSMA-R2 exceeded 15 μg, the results improved significantly.

[0475] For those with current specific activity 68 Biodistribution studies of the GaPSMA-R2 molecule have shown a favorable biodistribution profile in tumor models (primarily uptake in tumors and kidneys, with relatively low uptake in other organs). In vivo biodistribution data do not indicate a particular need to increase the cold peptide in formulations.

[0476] Therefore, based on all these considerations, 30 μg was chosen as the final amount because it meets our requirements for radiolabeling, stability, and biodistribution.

[0477] Table 3 - Effect of PSMA-R2 Quantity on RCP%

[0478]

[0479] *Results do not meet specifications

[0480] Our development research also focuses on selecting potential antioxidants and extenders. The radiolabeling procedure has also been thoroughly evaluated.

[0481] 2.2.1.2 Selection of Key Excipients

[0482] • Selection of antioxidants

[0483] The presence of free radical scavengers; thanks to their antioxidant properties, they can protect PSMA-R2 from radiation decomposition.

[0484] Attention was focused on gentianic acid. Tests were conducted to identify the minimum amount of antioxidant that could exert the desired protective function without interfering with the labeling process.

[0485] The labeling has been tested, where the amount of antioxidant was varied while other parameters remained constant, primarily to ensure that identification was not hindered. 68 The concentration of Ga incorporated into the DOTA molecule.

[0486] Using activity in the range of 2030 mCi 68 The molecule was labeled with Ga (using an E&Z generator). Labeling was performed at 95°C for 7 minutes using gallium buffer (pH 3.2–3.8). Experiments were conducted by testing different amounts of gentianic acid and peptides (15 μg and 30 μg) as radiolytic scavengers. In all tests, 20 mg of mannitol was added to form a cake.

[0487] The following table summarizes the labeling conditions and the results obtained.

[0488] As shown in Table 4, in the absence of gentianic acid... 68 The radiochemical purity of GaPSMA-R2 remained consistently above 92% for up to 4 hours. Furthermore, when gentianic acid was not used in the formulation, the free... 68 Ga remained below 2%. These preliminary results indicate that the molecule exhibits good stability against radiodegradation.

[0489] The following tests performed by increasing the amount of gentianic acid did not appear to show any improvement in molecular stability. The maximum amount of gentianic acid that gave good radiochemical results was 2 mg, while results using 5 mg of gentianic acid did not meet specifications. This may be due to an interaction between the DOTA chelating agent and gentianic acid (which exhibits a chelating functional group (carboxyl group) suitable for complexing with metal ions). 68 Partial competition for Ga complexation. The effect of gentic acid only becomes noticeable at high doses (5 mg) because it is a much weaker chelating agent than the DOTA molecule.

[0490] Therefore, based on these experimental results, it can be concluded that antioxidants are not required in pharmaceutical product compositions.

[0491] Table 4 - Gentian Acid Content: (for...) 68 Effect of GaPSMA-R2 stability

[0492]

[0493]

[0494] *Results do not meet specifications.

[0495] • Selection of extender

[0496] The formulation is finally completed by adding a extender for the freeze-drying process.

[0497] Mannitol was chosen from commonly recommended expanders for peptide freeze-drying because it produces a cake-like product during the freeze-drying process and exhibits good properties in terms of appearance, stability, and moisture content.

[0498] Radiolabeling tests were performed on two different formulations using an E&Z generator (active 30mCi-1110 MBq) by varying the amount of mannitol instead of using gentic acid as described in the table below. Neither formulation negatively affected the radiolabeling results, but better results were recorded on the formulation containing 20 mg of mannitol. The chosen amount of mannitol was 20 mg. Furthermore, mannitol is described in the literature as a good scavenger of OH radicals.

[0499] Table 5 - Different Incremental Doses

[0500]

[0501] 2.3 Effect of pH on radiochemical purity

[0502] The purpose of these tests was to evaluate the effect of labeled pH on the radiochemical purity of different formulations. pH plays a crucial role not only in coordination chemistry but also in the stability of peptides and small molecules in liquid formulations. Regarding... 68 The chemical properties of Ga and pH changes significantly affect its labeling behavior.

[0503] Due to the aqueous chemistry of gallium-68, a low pH must be maintained to prevent the formation of... 68 Ga oxides and hydroxides.

[0504] On the other hand, the pH must be high enough to allow a sufficient number of chelating agent donors to be functionally deprotonated.

[0505] For these 68 The pH specification defined for Ga-labeled products is 3.2-3.8. This pH range covers the results obtained by using our labeling method with DOTA chelating agents and 68 Complexation-compatible values ​​for GaCl3.

[0506] Based on these considerations, by varying the volume of gallium buffer (vial 2), three different formulations were labeled using an E&Z generator at different pH values ​​(3.0, 3.2, 3.8, 4.0).

[0507] The following formulations have been selected based on the optimal radiochemical purity obtained with the lowest amount of antioxidant (see Table 4).

[0508] -Formulation 1: 30μg PSMA-R2, 20mg mannitol;

[0509] -Formulation 2: 30μg PSMA-R2, 6.0μg gentic acid, 20mg mannitol;

[0510] -Formulation 3: 30μg PSMA-R2, 100μg gentic acid, 20mg mannitol;

[0511] As shown in the table below, all radiolabeling results for formulation 1 with a final pH of 2.90-3.35 were within specifications.

[0512] Table 6 - Formulations labeled at lower pH 1

[0513]

[0514] Table 7 shows the results of preparation 1 at pH > 3.80: all results are in compliance with specifications.

[0515] Table 7 - Formulations labeled at higher pH 1

[0516]

[0517] As shown in Table 8, all radiolabeling tests performed on formulation 2 with a final pH < 3.40 showed results within specifications.

[0518] Table 8 - Formulation 2 labeled at lower pH

[0519]

[0520] Table 9 shows the results of preparation 2 at pH > 3.80: all results are in compliance with specifications.

[0521] Table 9 - Formulations labeled at higher pH 2

[0522]

[0523]

[0524] Table 10 shows the results for formulation 3 at pH < 3.2: all results were non-compliant (RCP% < 92%).

[0525] Table 10 - Formulations labeled at lower pH 3

[0526]

[0527] *Results do not meet specifications

[0528] Table 11 shows the results of preparation 3 at pH > 3.80: all results are in compliance with specifications.

[0529] Table 11 - Formulations labeled at higher pH 3

[0530]

[0531] In summary, the collected results indicate that only when the final labeled pH is low (between 3.0 and 3.2), in formulation 3 (100 μg gentianic acid) 68 The radiochemical purity of GaPSMA-R2 was significantly reduced. When the same formulation was tested at a final pH near the upper limit (approximately pH 3.8), the results were within specifications.

[0532] Tests on formulation 1 (without gentialic acid) and formulation 2 (6 μg gentialic acid) showed that the results were within specifications, whether at lower pH (3.0-3.2) or higher pH (3.8-4.0).

[0533] Based on these considerations, it can be assumed that the negative impact of gentianic acid only exists when the final pH of the labeling is low (approximately 3.2). HPLC results indicate that these specific conditions lead to an increase in radioactive impurities in the radiolabeled product.

[0534] For these reasons, we tested formulations containing varying amounts of gentic acid, consistently maintaining the final labeled pH near the lower limit (pH 3.2). Our aim was to gain a clearer understanding of whether gentic acid has any effect on... 68 The radiochemical purity of GaPSMA-R2 is adversely affected.

[0535] The radiolabeling results collected in Table 12 indicate that 200 μg of gentianic acid in the formulation negatively impacts the RCP% of the product when the final labeled pH is approximately 3.2. Results obtained using 100 μg were slightly better than the specification, while further reductions to below 12 μg yielded significant improvements. Based on all these results, it can be concluded that the presence of gentianic acid negatively affects the radiochemical purity of the radiolabeled solution, promoting the appearance of potential impurities (other radioactive substances) in low-pH solutions.

[0536] Table 12 - Results obtained at pH 3.0–3.2 68 GaPSMA-R2 labeling results

[0537]

[0538]

[0539] *Results do not meet specifications

[0540] 2.4 Radiolabeling Procedure

[0541] Based on the 2-vial reagent kit design, the following 3-step labeling procedure was developed:

[0542] 1. Use 68 Ge / 68 The Ga E&Z generator provides [the solution] in HCl. 68 Ga solution directly reconstructs powder vials.

[0543] 2. Add the necessary volume of reaction buffer.

[0544] 3. Heat at 95°C for at least 7 minutes (but not more than 10 minutes).

[0545] at this time 68 The Ga-PSMA-R2 solution is ready for application.

[0546] During the development of the marker program, different time and temperature conditions have been tested.

[0547] The dependence of labeling efficiency on temperature has been studied to determine its relationship with... 68 Ga’s short half-life (68 minutes) provides good doping values ​​within a compatible time range.

[0548] Known 68 The incorporation of Ga into the DOTA chelate requires heating to complete.

[0549] The test began with elution from the generator, followed by the addition of reaction buffer at room temperature and then heating at 95°C. The results are summarized in the table below.

[0550] Table 13 - Elution and buffer addition during RT

[0551]

[0552] Furthermore, marking at 70℃, 80℃, 90℃, and 95℃ was tested at different reaction times (3, 5, and 7 minutes) and 100℃. A 7-minute reaction at 70℃ was also tested. 68 Ga-radioactive markings 68 Ga doping was insufficient. Increasing the temperature to 80°C promoted doping to over 94%. At 95°C, doping was almost complete after 5 minutes. Based on these observations, 95°C for 7 minutes is the most conservative labeling condition, ensuring over 95% doping without significant fragmentation even with temperature fluctuations within ±5°C.

[0553] Table 14 - Marking at different temperatures and times

[0554]

[0555] *Results do not meet specifications

[0556] Tests were also conducted to evaluate the addition. 68 The permissible delay between the Ga eluent and the added buffer solution still provides a compliant radiolabeled imaging product.

[0557] The reconstitution process was tested by increasing the number of minutes of waiting after reconstitution of the lyophilized formulation and before adding buffer. Radiochemical purity was determined by HPLC.

[0558] The results showed that a buffer addition delay of up to 15 minutes did not affect the success of labeling.

[0559] Table 15 - Effect of buffer addition delay test on stability / purity

[0560]

[0561]

[0562] Also tested in 68 Ge / 68 The possibility of adding gallium buffer to vial 1 before eluting with the Ga generator. Following this procedure, the labeling results obtained by HPLC analysis are reported in Table 19.

[0563] Table 16 - Buffer Added Before Elution: Radiochemical Purity Assessment

[0564]

[0565] *Results do not meet specifications

[0566] Radiolabeling tests performed with gallium buffer prior to the elution step yielded results that did not meet specifications, so this option was discarded.

[0567] use 64 Radiolabeling procedure for Cu

[0568] In addition to targeting 68 Ga, based on a 2-vial reagent kit design 64 The Cu marking program has also been developed as follows:

[0569] 1. Using the power generated by a cyclotron 64 Cu provides in HCl 64 Cu solution directly reconstitutes powder vials.

[0570] 2. Add the necessary volume of reaction buffer.

[0571] 3. Heat at 70°C for at least 15 minutes.

[0572] During the development of the marker program, different time and temperature conditions have been tested.

[0573] The dependence of labeling efficiency on temperature has been investigated to determine a value that provides good incorporation and good stability for up to 24 hours without causing product degradation.

[0574] The tests began with incorporation at room temperature. Table 17 reports the results achieved using the PSMA R2 peptide and the PSMA R2 kit during RT incubation.

[0575] Table 17 - Marking in RT

[0576]

[0577] Labeling conditions at 40°C, 70°C, and 95°C were tested using the different reaction times and pH values ​​reported in Table 18.

[0578] Table 18 - Labeled at different temperatures, times, and pH levels

[0579] *Results are affected by the analysis problem. **Reaction incomplete*** Results exceed specifications.

[0580] In RT 64 The radiolabeling of Cu indicates that when the pH is below 4... 64 Insufficient Cu doping. Increasing the temperature to 70°C promoted the above doping.

[0581]

[0582] 94%. Good incorporation was achieved at 95°C for 7 minutes. Based on these observations, 70°C for 15 minutes is the most conservative labeling condition, ensuring over 94% incorporation for up to 24 hours without significant fragmentation.

[0583] Furthermore, the last three results obtained using 70°C for 15 minutes as a heating step indicate that good results can be obtained using different radioactive concentrations ranging from 100 MBq / mL to 200 MBq / mL with a final volume of 3 to 8 mL.

[0584] These results indicate that, considering the high charge... 68 Ge / 68 The Ga generator can elute approximately 200 MBq / mL, which can simulate the use of 68 To obtain the same range of Ga radiolabel concentration 64 CuPSMA-R2.

[0585] 2.5 Final formulation and detailed composition

[0586] Based on all the development of the above formulations, the final selected formulation for vial 1 is as follows:

[0587] Table 19 - Final Preparations

[0588]

[0589] *Current version

[0590] **Water for injection is eliminated during the freeze-drying process.**

[0591] The following are radiolabeled preparations:

[0592] Table 20 - Final Radiolabeled Preparations

[0593] Components 5 mL of HCl 0.1 N PSMA-R2 content 30μg <![CDATA[ 68 Ga-PSMA-R2 content]]> ≤0.0161μg Total radioactivity ≤1110MBq Specific activity (GBq / total peptides) ≤36.5 GBq / μmol radioactivity concentration ≤202 MBq / mL volume ≤5.5mL Excipient content mg / vial Mannitol 20 Formic acid 30 Sodium hydroxide 28.25 hydrochloric acid 18.22 Water for Injection Add ≤5.5ml

[0594] As demonstrated during product development, even without gentic acid, 68 The radiochemical purity of Ga-PSMA-R2 remained above 92% for up to 4 hours (Table 4). This behavior indicates that the molecule has intrinsic stability against radiodegradation.

[0595] Adding gentianic acid to formulations appears to have an effect on... 68 The GaPSMA-R2 product does not improve stability, therefore this excipient is not included in the final formulation.

[0596] 2.6 Specification Evaluation

[0597] The final formulation has been tested to confirm the results obtained during the development process.

[0598] Table 21 - Labeling results obtained using final preparations

[0599]

[0600] Liquid formulations are prepared with the goal of achieving very high radiochemical purity values. This approach ensures a broad range of possibilities for evolution from R&D liquid formulations to GMP-compliant lyophilized products while maintaining sufficient quality.

[0601] Free ions were monitored using HPLC during the development process. 68 The Ga content of the selected formulation consistently remained within the target limit. Based on this, and considering the non-complexing nature of the ITLC assessment... 68 Ga substances include colloidal and free forms. 68 Ga, therefore, there is no need to continuously monitor the latter parameter.

[0602] In the process of transforming internally derived results into GMP products awaiting local reconfiguration, an ITLC of <5% for non-complexed 68Ga materials is considered sufficient. This specification ensures at least 95% radioisotope incorporation, which meets the general requirements for kit-based radiopharmaceuticals.

[0603] HPLC analysis prior to reagent release 68 Ga-PSMA-R2 radiochemical purity ensures that, when the reconstruction instructions are correctly applied, the reconstructed... 68 Ga-labeled peptides account for more than 90.0% of the total radioactivity.

[0604] In summary, based on the results of formulation development and the general requirements for radiopharmaceutical preparations, the following radiochemical specifications have been established for the release of lyophilized GMP products at the production site:

[0605] · 68 Ga-PSMA-R2 (HPLC): ≥90.0%

[0606] Non-complexing 68 Ga 3+ Intense Total Colored Carbohydrate (ITLC): <5.0%

[0607] 3. Reaction buffer vials (via vial 2)

[0608] 3.1 Formulation Development

[0609] 3.1.1 General

[0610] The formulation development of reaction buffers aims to define formulations that allow for the use of... 68 Ge / 68 The Ga generator provides direct reconstruction of the eluent, labeling DOTA molecules with high and reproducible complexation yields.

[0611] This direct procedure makes the labeling process easy and does not rely on automated synthesis modules that are very expensive and available only in a limited number of rabies pharmacies.

[0612] The proposed reconstitution procedure does not require additional purification steps and provides radiolabeled imaging products that meet predetermined quality standards.

[0613] This method addresses a widely recognized unmet need in the field of nuclear medicine.

[0614] As is well known, a major challenge in developing kits for radiopharmaceutical formulations is related to successful labeling procedures. 68 In the case of Ga isotopes, the procedure is subject to the following limitations:

[0615] 1. It is difficult to maintain a constant and suitable pH value.

[0616] 2. Competition among metallic impurities during the complexation process.

[0617] 3. Product and Key Indicators: Stability of Radiochemical Purity

[0618] These three aspects make it possible for people from 68 Ge / 68 The direct application of the washing solution from the Ga generator in the labeling process is currently impractical.

[0619] Therefore, the first focus of formulation development is to study buffer solutions that can reliably maintain the desired pH value after the total eluent provided by the recovery generator.

[0620] pH value at 68 Ga plays a crucial role in the labeling process because its changes significantly affect the labeling behavior:

[0621] Due to the aqueous chemistry of gallium-68, a low pH must be maintained to prevent the formation of... 68 Ga oxides and hydroxides.

[0622] On the other hand, the pH must be high enough to allow a sufficient number of chelating agent donors to be functionally deprotonated.

[0623] Of the different available buffers, the first one tested is known and used for... 68 Ga is labeled, for example, with HEPES (sulfonic acid derivatives) or acetate buffer.

[0624] 3.1.2 Buffer Selection

[0625] HEPES

[0626] HEPES cannot even tolerate small changes in the volume of HCl solution, therefore, it is almost impossible to apply it to applications designed for use with HCl solutions. 68 Ge / 68 A kit for directly reconstructing the eluent from a Ga generator (whose volume cannot be strictly constant during normal use).

[0627] Furthermore, HEPES should not be left in the injectable solution at such a high concentration, as this would result in final purification after labeling, which is incompatible with the kit method.

[0628] Table 22: pH values ​​obtained after mixing HEPES buffer (500 μL of Na-HEPES 300 mg / mL) with 0.1 N hydrochloric acid

[0629]

[0630] Acetate buffer

[0631] Acetate buffer also because of 68 It is widely known for its use in Ga labeling and provided fairly stable pH values ​​in initial tests. However, it yielded inconsistent results.

[0632] It is worth noting that all successful labeling with HEPES and acetate documented in the literature came from labeling procedures based on a pretreatment step of the eluent and a final purification of the radiolabeled product.

[0633] Alternative buffer solutions

[0634] Subsequently, alternative buffers compatible with injection use were sought, focusing on buffers with a pKa in the range of 3.2–4.2, thereby ensuring compatibility with the intended use. 68 The effective buffering capacity of Ga-labeled substances at their optimal pH values. Table 23 lists common organic acids and their pKa.

[0635] Table 23: pKa of common organic acids

[0636] buffer solution pKa (20℃) Citric acid 3.14 Formic acid 3.75 lactic acid 3.86 Succinic acid 4.22

[0637] Citric acid was excluded because it forms a stable complex with gallium. Well-known SPECT products... 67 The presence of Ga-citric acid also confirms this.

[0638] Lactic acid also demonstrated in preliminary tests that it hindered the binding of DOTA chelators with... 68 Ga complexation provides over 97% of the free... 68 Ga.

[0639] 5 ml of HCl 0.1 68 Ga solution was used to test for succinic acid in the labeling process, but even after establishing a reliable pH of around 3.4, it never provided a satisfactory final result. 68 Ga-labeled DOTA-peptide, free in HPLC 68 The Ga content is always higher than 8%.

[0640] Finally, due to its pKa, formic acid was found to have excellent buffering capacity and is located in a suitable position. 68 The pH center is Ga-complexed. Furthermore, this buffer is considered compatible with the intended pharmaceutical application because formic acid is classified as a Class 3 (solvent with low toxicity potential) residual solvent in the pharmacopoeia and should not be removed from the final injectable solution at the end of labeling if it remains below the permissible daily exposure (PDE).

[0641] Based on the Henderson-Hasselbalch equation describing the behavior of the buffer system, the amounts of formic acid and basic counterparts required to achieve a final pH of approximately 3.5 were calculated, taking into account the contribution of HCl from the generator.

[0642] Sodium hydroxide was chosen as the basic counterpart because it is a strong base, capable of neutralizing strong HCl acid and producing the conjugate base of formic acid required to establish a buffer pair. An amount of 30 mg formic acid and 28.25 mg sodium hydroxide is sufficient to maintain the pH at around 3.5. Furthermore, this amount of formic acid is far below the PDE value of 50 mg for Class 3 solvents.

[0643] Formate buffers at the above concentrations have been shown to maintain the pH in the range of 3.2–3.8 over a considerable volume of HCl, not just after adding standard volumes of eluent (5 mL HCl 0.1N and 4 mL HCl 0.05N), thus mimicking the most common commercially available solutions. 68 Ge / 68 (The characteristics of the eluent from the Ga generator). This ensures optimal labeling conditions, even with reduced eluent recovery from the generator, which is likely to occur in practice where a strictly constant volume cannot be guaranteed.

[0644] Since the kit-based method lacks a pre-concentrator eluent volume, the formic acid concentration in the formulation was optimized to maintain the low-volume buffer required for labeling, thus avoiding further dilution of the reaction mixture. This is an advantage because labeling at nanomolar peptide concentrations requires a smaller reaction volume to maximize labeling yield.

[0645] Tables 24 and 25 summarize the pH values ​​measured after mixing appropriate volumes of formate buffer with variable volumes of 0.1N and 0.05N HCl.

[0646] Table 24: pH values ​​obtained after mixing formate buffer (500 μL of formic acid 60 mg / mL, sodium hydroxide 56.5 mg / mL) with 5 mL of 0.1 N HCl

[0647]

[0648] Table 25: pH values ​​obtained after mixing formate buffer (200 μL of formic acid 60 mg / mL and sodium hydroxide 56.5 mg / mL) with 4 mL of 0.05 N HCl

[0649] Volume of HCl: 0.05 N (mL) Test 1 pH Test 2 pH Test 3 pH Average pH 2.6 3.75 3.73 3.76 3.75 2.8 3.61 3.59 3.63 3.61 3.0 3.52 3.56 3.54 3.54 3.2 3.45 3.50 3.51 3.49 3.4 3.38 3.41 3.43 3.41 3.6 3.31 3.33 3.36 3.33 3.8 3.24 3.25 3.28 3.26 4.0 3.16 3.17 3.19 3.17

[0650] The competence of the formate buffer was then confirmed by the successful labeling, proving that the DOTA component does not exist. 68The interference of Ga chelation. Overall, the formic acid / formate buffer at the above concentrations demonstrates:

[0651] - It can offset the acidity of the total eluent from the generator without reducing or concentrating the eluent volume, making kit-type direct labeling procedures feasible;

[0652] - It maintains a stable pH value even when there are significant changes in the HCl eluent, making it particularly suitable for routine applications where a strictly constant elution volume cannot be expected;

[0653] -Does not negatively interfere with DOTA chelating agents and 68 The complexation of Ga. All these observations have led to interest in formic acid for further development.

[0654] 3.2 Final formulation and detailed composition

[0655] Based on all the development of the above formulations, the final selected formulation for vial 2 is as follows:

[0656] Table 26 - Final Preparations

[0657]

Claims

1. A method for binding ligands to PSMA of formula (II) with radiolabeled isotopes. (II) The radioactive isotope mentioned is 68 Ga、 67 Ga or 64 Cu, the method includes the following steps: i. Provide a first vial containing the PSMA-binding ligand in dry form and the extender mannitol. ii. Add the solution of the radioactive isotope to the first vial to obtain a solution of the PSMA binding ligand and the radioactive isotope. iii. The solution obtained in ii. is mixed with a buffer containing formic acid and sodium hydroxide and incubated for a period of time sufficient to obtain the PSMA-binding ligand labeled with the radioisotope, and iv. Adjust the pH of the solution.

2. A method that can be obtained or acquired by the method of claim 1, comprising... 68 Ga、 67 Ga or 64 The use of a Cu-labeled solution of the PSMA-binding ligand of formula (II) for the preparation of an injectable solution for in vivo detection of tumors by imaging in subjects in need.

3. The use as described in claim 2, wherein the tumor is a tumor expressing PSMA.

4. A kit for carrying out the method of claim 1, the kit comprising... i. The first vial contains the following components in dry form: i. PSMA-binding ligands of formula (II): (II), and ii. mannitol as a extender, and ii. A second vial containing a buffer in dry form, said buffer comprising formic acid and sodium hydroxide; and iii. An accessory box for eluting radioactive isotopes generated by a radioactive isotope generator or cyclotron.

5. The kit of claim 4, wherein the first vial comprises the following components: i. The amount of the PSMA-binding ligand of formula (II) described herein is from 10 μg to 100 μg; and ii. Mannitol, in amounts of 5 mg to 50 mg.

6. The kit of claim 5, wherein the amount of the PSMA binding ligand of formula (II) is from 15 μg to 60 μg.

7. The kit of claim 5, wherein the amount of the PSMA binding ligand of formula (II) is 30 μg.

8. The kit of claim 5, wherein the amount of mannitol is 10 mg to 30 mg.

9. The kit of claim 5, wherein the amount of mannitol is 20 mg.

10. The kit according to any one of claims 4-9, wherein the second vial contains formic acid and sodium hydroxide as buffers for maintaining pH between 2.5 and 4.

0.

11. The kit of claim 10, wherein the second vial contains formic acid and sodium hydroxide as buffers for maintaining pH between 2.8 and 4.

0.

12. The kit of claim 10, wherein the second vial contains formic acid and sodium hydroxide as buffers for maintaining pH at 3.0 to 4.

0.

13. The kit of claim 10, wherein the second vial contains formic acid and sodium hydroxide as buffers for maintaining pH at 3.2 to 3.

8.

14. The kit according to any one of claims 4-9 and 11-13, wherein the kit does not contain antioxidants.

15. The kit of claim 10, wherein the kit does not contain antioxidants.

16. The kit according to any one of claims 4-9 and 11-13, wherein the kit does not contain the antioxidant gentianic acid.

17. The kit of claim 10, wherein the kit does not contain the antioxidant gentianic acid.

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