CN114404619A

CN114404619A - Aqueous radiopharmaceutical solution, process for its preparation and its use

Info

Publication number: CN114404619A
Application number: CN202210309886.2A
Authority: CN
Inventors: 田佳乐; 杜泽天; 阳国桂
Original assignee: Beijing Cotimes Biotech Co Ltd
Current assignee: Beijing Cotimes Biotech Co Ltd
Priority date: 2022-03-28
Filing date: 2022-03-28
Publication date: 2022-04-29

Abstract

The present application relates to a method for producing an aqueous radiopharmaceutical solution comprising a radionuclide complex and a stabilizer, particularly active ingredient of [ solution ]¹⁷⁷Lu]The radionuclide complex of Lu-EB-PSMA, the stabilizer is preferably one or more of gentisic acid, ethanol and methionine.

Description

Aqueous radiopharmaceutical solution, process for its preparation and its use

Technical Field

The invention relates to a radiopharmaceutical aqueous solution with high chemical stability and high radiochemical stability and a preparation method thereof, in particular to a radiopharmaceutical aqueous solution containing an active ingredient of [ alpha ], [ beta ] -cyclodextrin¹⁷⁷Lu]A medicinal aqueous solution of radionuclide complex of Lu-EB-PSMA and a preparation method thereof.

Background

The prostatic cancer is a common malignant tumor of old men, is the second most common cancer of men worldwide at present, has the first incidence rate of malignant tumor of men who live in Europe and America, and has a trend of increasing incidence rate year by year in China. Most prostate cancer patients in China are in the middle and late stages at the time of treatment, and the disease condition can be controlled through endocrine treatment, but most prostate cancer patients finally develop metastatic castration resistant prostate cancer (mCRPC), namely metastatic prostate cancer of which the disease still progresses after castration level (< 50ng/dL or 1.7 nmol/L) is achieved through Androgen Deprivation Therapy (ADT) testosterone.

PSMA is a type II transmembrane protein highly expressed in prostate cancer and its tumor tissues or neovascular tissues, and is expressed 100 to 1000 times higher in prostate cancer cells than in normal cells, especially at the advanced stages of prostate cancer and metastatic castration-resistant prostate cancer (mCRPC), and some studies have shown that PSMA expression in cancer cells increases with increasing tumor grade. Because PSMA is composed of groups inside and outside cells, the external group can be connected with ligands with different functions, the internal group contains functional factors of endocytosis reaction, and the endocytosis reaction and the intracellular biochemical circulation can be started, thereby increasing the aggregation of the radioactive tracer inside the cells and improving the imaging or curative effect. These characteristics make the PSMA antigen a very promising biological target for imaging and therapy.

Radionuclide-labeled compounds are modern reagents widely used in the field of isotopic labeling of many subjects. A variety of drugs are currently being developed based on PSMA, for example,¹⁷⁷the Lu-labeled PSMA-617 antibody was shown to be an effective agent for prostate cancer. The treatment principle is that¹⁷⁷Lu-labeled drugs can be specifically bound to tumor cell surface antigens or even internalized, and then utilized¹⁷⁷The short-range beta rays of Lu strongly kill tumor cells so as to inhibit the growth of the tumor and even eliminate the tumor.

The generation of diagnostic or therapeutic effects based on the arrival of radionuclides at the tumor site to emit particles or radiation is one of the main applications of radiopharmaceuticals. After the radioactive drug is administrated to a tumor patient, the carrier molecule of the radioactive drug has the property of specifically targeting a certain target spot and is delivered to tumor cells, the diagnosis effect is realized by capturing radioactive signals in vitro to monitor, locate, classify and the like the tumor, or the killing effect is generated on the tumor cells by energy released in the decay process of radioactive nuclide, and the adverse effect of particles or rays on healthy tissues near the tumor is avoided to the maximum extent. However, the radioactive nuclides decay continuously to release high-energy particles or rays, so that the covalent bonds of molecules in the pharmaceutical preparation are broken during the production and storage of the radiopharmaceutical, which is also called radiolysis and also called radiation degradation.

Radiolysis results in an increase in chemical and radiochemical purity of the radiopharmaceutical formulation, i.e., a decrease in the chemical and radiochemical purity of the pharmaceutically active ingredient (API). Radiolytic impurities, particularly radioactive radiolytic impurities, can increase the noise signal of diagnostic radiopharmaceuticals, cause inadequate therapeutic efficacy of therapeutic radiopharmaceuticals, and can cause unnecessary radiation damage to other normal tissues. This also makes the radiolysis problem a significant problem in the radiopharmaceutical development process. How to effectively maintain the stability of the API and reduce the generation of radiolytic impurities becomes a problem to be solved urgently by the technical personnel in the field. A functional linker is disclosed in US2016/0228548a1 to allow binding of a prostate cancer inhibitor to a chelator and chelation of a radionuclide. It is also disclosed in the TW202005669A patent that modification with evans blue increases the half-life of the drug in vivo to prolong its residence time in the blood.

The reference (J. Med. chem. 2021, 64, 4960-4971) reports PSMA targeting molecules¹⁷⁷2 stabilizer schemes in the Lu-PSMA-617 preparation process, 1) sodium ascorbate is added into a reaction phase, and 2) 30 percent ethanol (volume percentage) is added to serve as a stabilizer. The initial radiochemical purity (RCPs) obtained by the two schemes is more than or equal to 95 percent and is very close to 95 percent (the initial chemical purity refers to the radiochemical purity obtained by immediately detecting the prepared radiopharmaceutical after the preparation of the radiopharmaceutical is finished, namely the radiochemical purity at the 0 moment after the preparation). Although about 95% of the initial radiochemical purity meets GMP requirements, the lower initial radiochemical purity necessarily limits the shelf life of the radiopharmaceutical to a short time, which is not conducive to storage and transport of the radiopharmaceutical and greatly hinders the availability of the radiopharmaceutical.

Patent CN112584875A (WO2020/021322) discloses a somatostatin receptor-binding peptide drug comprising gentisic acid and salts thereof and ascorbic acid and salts thereof as stabilizers, but experiments prove that the ascorbic acid and salts thereof in the drug combination are not favorable for radioactive molecules [ 2 ]¹⁷⁷Lu]The stability of Lu-EB-PSMA, on the contrary, the addition of ascorbic acid and its salt aggravates¹⁷⁷Lu]Radiolysis by Lu-EB-PSMA. Ascorbic acid and its salts are therefore not preferred stabilizers for this molecule.

Therefore, it remains a challenge to develop a prescription process that provides a higher initial radiochemical purity and that maintains the stability of the radiopharmaceutical for a longer period of time.

Disclosure of Invention

It is an object of the present application to provide a method for the preparation of an aqueous radiopharmaceutical solution comprising radiationA radionuclide complex, particularly having an active ingredient of [ 2 ]¹⁷⁷Lu]The radionuclide complexes of Lu-EB-PSMA, in particular, the present invention relates to the following:

1. a method for preparing an aqueous radiopharmaceutical solution comprising a radionuclide complex formed from a radionuclide and a molecule targeting PSMA, comprising the steps of:

mixing a solution containing a first stabilizer with a solution containing a radionuclide in a reaction vessel;

after a given time, adding a solution containing the PSMA-targeting molecule into the reaction container, preferably, the given time is 0.1-20 minutes, and further preferably, 3-10 minutes;

the PSMA-targeting molecule reacts with a radionuclide to obtain the radionuclide complex;

adding a solution containing a second stabilizing agent into the reaction vessel after the reaction is carried out for a given time;

recovering the resulting aqueous radiopharmaceutical solution.

2. The method of item 1, wherein the solution containing the radionuclide is added to the reaction vessel after being removed from the raw material bottle, the method further comprising:

and washing the raw material bottle by using a washing liquid, and transferring the washed solution into the reaction container to be mixed with the solution containing the radionuclide.

3. The method according to item 2, characterized in that the rinsing liquid is an aqueous solution, preferably selected from the group consisting of a solution containing a first stabilizer, a solution containing a buffer salt, water or sodium chloride injection; more preferably, the rinsing with the rinsing solution is repeated one or more times.

4. The method according to item 1, wherein the solution containing the radionuclide is a solution containing a radionuclide¹⁷⁷Lu and hydrochloric acid, and in the step of reacting the PSMA-targeting molecule with a radionuclide,¹⁷⁷the specific activity range of Lu is more than or equal to 20 Ci/mg, preferably more than or equal to 60 Ci/mg, and most preferably more than or equal to80 Ci/mg。

5. The method of item 1, wherein the PSMA-targeting molecule comprises a chelating group selected from DOTA, NOTA, PCTA, DTPA, NTA, EDTA, DO3A, or NOC, preferably DOTA.

6. The method of any one of claims 1 to 5, wherein the PSMA-targeting molecule is selected from EB-PSMA, PSMA-617, or PSMA-11.

7. The method of item 6, wherein the radionuclide complex is¹⁷⁷Lu-EB-PSMA。

8. The method according to any one of items 1 to 7, wherein in the step of reacting the PSMA-targeting molecule with the radionuclide, the molar ratio between the PSMA-targeting molecule and the radionuclide is 1.5 to 50, preferably 5 to 20.

9. The method according to item 1, wherein the step of reacting the PSMA-targeting molecule with the radionuclide is performed at a reaction temperature of 50 to 95 ℃, preferably 60 to 80 ℃, and for a reaction time of 5 to 60 minutes, preferably 10 to 30 minutes.

10. The method according to item 1, wherein the first stabilizer is one or more selected from gentisic acid and salts thereof, ascorbic acid and salts thereof, histidine, cysteine and salts thereof, methionine, selenomethionine, thiosulfate, maltose, inositol, benzyl alcohol, trehalose, povidone, nicotinamide, ethanol, curcumin, melatonin, and preferably gentisic acid.

11. The method of item 10, wherein in the step of reacting the PSMA-targeting molecule with a radionuclide, the concentration of the first stabilizer in the reaction phase solution is 0.6-20.0 mg/mL.

12. The method according to item 1, wherein the second stabilizer is one or more selected from gentisic acid and salts thereof, ascorbic acid and salts thereof, histidine, cysteine and salts thereof, methionine, selenomethionine, thiosulfate, maltose, inositol, benzyl alcohol, trehalose, povidone, nicotinamide, ethanol, curcumin, melatonin, preferably gentisic acid, ethanol or methionine.

13. The method of claim 12, wherein the concentration of the second stabilizer is 0-400 mg/mL.

14. The method of item 1, wherein a buffered salt solution is added prior to reacting the PSMA-targeting molecule with the radionuclide, preferably wherein the buffered salt solution is present in the solution comprising the first stabilizing agent.

15. The method according to claim 14, characterized in that the buffered salt solution is selected from an acetate, citrate, phosphate or formate solution, preferably an acetate-sodium acetate buffered salt solution.

16. The method of item 1, wherein the step of adding a solution containing a second stabilizer to the reaction vessel after the reaction for a given time further comprises adding a co-solvent to the reaction vessel.

17. The method according to item 16, wherein the cosolvent is selected from one or two of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, polyoxyethylated castor oil, span, preferably polysorbate 80.

18. The method according to item 1, wherein the step of adding a solution containing a second stabilizer to the reaction vessel after the reaction for a given time further comprises adding a free nuclide chelating agent to the reaction vessel, wherein the chelating agent may be selected from pentetic acid and salts thereof, preferably pentetic acid.

19. The method of any one of claims 1 to 18, further comprising filter sterilizing the aqueous radiopharmaceutical solution through a 0.22 μm filter, preferably after adding a solution comprising a second stabilizer.

20. The method according to any one of claims 1 to 19, further comprising diluting the radioactive aqueous solution, preferably by adding sodium chloride injection after adding a solution containing a second stabilizing agent.

22. An aqueous radiopharmaceutical solution prepared by the method of any one of items 1 to 21.

Effects of the invention

The preparation method of the radiopharmaceutical aqueous solution provided by the application has the following beneficial effects:

in the application, the concentration of gentisic acid in the reaction phase solution is controlled to be 0.6-20.0 mg/mL. Below 0.6 mg/mL, the anti-radiolytic effect of gentisic acid is insufficient, and above 20.0 mg/mL, a high concentration of gentisic acid will slow down the reaction kinetics, disadvantageously prolonging the time required for the reaction. The control range is to minimize the concentration of gentisic acid to avoid adverse effects on reaction kinetics, while ensuring that the solution is stable.

Before the solution containing the molecule of the target PSMA is mixed with the nuclide solution, the nuclide solution is mixed with the solution containing the first stabilizer, and after a given time, the solution containing the molecule of the target PSMA is added, so that the first stabilizer is fully contacted with the nuclide solution to quench a large amount of free radicals caused by radiation decomposition in the nuclide solution, thereby protecting the molecule of the target PSMA added into a reaction system from being attacked by active free radicals and ensuring the initial radiochemical purity of the product. The process can lead the initial radiochemical purity to reach 95.0 to 99.5 percent, and the initial radiochemical purity of the product obtained by the synthesis process of directly mixing the solution containing the molecule of the targeting PSMA with the nuclide solution is about 89 to 93 percent.

The process method in the application can ensure that the labeling rate is at least over 90%, preferably over 95%, and most preferably over 99%, so that the reaction does not generally contain purification steps for removing radioactive impurities, such as preparative liquid phase separation, solid phase extraction separation and the like. If sterility of the radionuclide complex solution is required, the step of recovering the product may further comprise passing the solution through a 0.22 μm sterile filter, and optionally further diluting the solution depending on the amount used.

Detailed Description

The following detailed description of the present application is provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

It should be noted that certain terms are used throughout the description and claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the application, however, the description is made for the purpose of illustrating the general principles of the application and is not intended to limit the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.

The present application relates to an aqueous radiopharmaceutical solution comprising a drug labelled with a radioactive metallic nuclide and a stabilizer.

In one particular embodiment, the radioactive metal species is¹⁷⁷Lu (lutetium-177).

In further embodiments, the radiometal species may also be selected from Tc, Ga, Cu, In, Y, Zr, Rb, Ho, Ac, Pb, Bi, Th, especially^99mTc、⁶⁸Ga、⁶⁴Cu、、⁶⁷Cu、¹¹¹In、⁹⁰Y、⁸⁹Zr、⁸²Rb、¹⁶⁶Ho、²²⁵Ac、²¹²Pb、²¹³Bi、²¹²Bi、²²⁷Th。

In a specific embodiment, the radionuclides-labeled drug is a complex formed by a radionuclide and a molecule that targets PSMA.

In a specific embodiment, the drug labeled with a radioactive metal nuclide is a radioactive metal nuclide¹⁷⁷Lu (lutetium-177) forms a complex with a molecule that targets PSMA.

In a specific embodiment, the PSMA-targeting molecule may be any molecule that targets PSMA, consisting of a targeting group and a chelating group linked by a covalent bond, such that the molecule is capable of forming a stable complex with a radioactive metallic nuclide. In some specific embodiments, the chelating group may be selected from DOTA, NOTA, PCTA, DTPA, NTA, EDTA, DO3A, NOC. In some preferred embodiments, the PSMA-targeting molecule is selected from PSMA-617, EB-PSMA, PSMA-11, each of which has a formula as shown below. In a preferred embodiment, the PSMA-targeting molecule is EB-PSMA.

In a specific embodiment of the present application, the stabilizer in the radiopharmaceutical aqueous solution is a stabilizer against radiolytic degradation, specifically, the stabilizer is one or more selected from gentisic acid and salts thereof, ascorbic acid and salts thereof, histidine, cysteine and salts thereof, methionine, selenomethionine, thiosulfate, maltose, inositol, benzyl alcohol, trehalose, povidone, nicotinamide, ethanol, curcumin, melatonin, and preferably one or more selected from gentisic acid, ethanol, and methionine.

In a particular embodiment, the total concentration of the stabilizer in the aqueous pharmaceutical solution is 0.5-400 mg/mL, and may be, for example, 0.5, 100, 150, 200, 250, 300, 350, 400 mg/mL. Preferably, it is 1 to 80mg/mL, and may be, for example, 1, 10, 20, 30, 40, 50, 60, 70 or 80 mg/mL.

In a particular embodiment, the stabilizer is added separately during the complexation reaction that forms the nuclide complex and after the reaction is complete. Wherein, adding during the complexation reaction means that the stabilizer and the radionuclide solution forming the complex and the molecular solution targeting PSMA jointly form a reaction phase solution when the conditions sufficient for the complexation reaction to occur are achieved; the addition after the reaction is finished means that the stabilizer is added after the complex reaction is carried out for a certain time and the complex is formed. Further, the stabilizer added during the complexation reaction is a first stabilizer, and the stabilizer added after the reaction is finished is a second stabilizer. The first stabilizer is typically a small molecule compound with antioxidant properties to reduce radiolysis at high radiation. The primary role of the second stabilizer is to maintain radiochemical purity of the formulation during storage. The first stabilizer and the second stabilizer may be selected from the same stabilizer or different stabilizers.

In a specific embodiment, the first stabilizer is selected from one or more of gentisic acid and salts thereof, ascorbic acid and salts thereof, histidine, cysteine and salts thereof, methionine, selenomethionine, thiosulfate, maltose, inositol, benzyl alcohol, trehalose, povidone, niacinamide, ethanol, curcumin, melatonin, preferably gentisic acid.

In a specific embodiment, the second stabilizer is one or more selected from gentisic acid and salts thereof, ascorbic acid and salts thereof, histidine, cysteine and salts thereof, methionine, selenomethionine, thiosulfate, maltose, inositol, benzyl alcohol, trehalose, povidone, niacinamide, ethanol, curcumin, melatonin, preferably gentisic acid, ethanol or methionine.

In a preferred embodiment, the first and second stabilizers are the same and are each selected from gentisic acid or a salt thereof. Wherein, the concentration of gentisic acid (namely the first stabilizing agent) in the reaction system is in the range of 0.6-20 mg/mL, preferably 2-10 mg/mL, and can be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/mL. Gentisic acid (i.e. the second stabiliser) is added to the preparation after the end of the reaction such that gentisic acid is present in the total concentration of 0.1-10mg/mL, preferably 0.5-5mg/mL, and may be, for example, 0.5, 1.0, 1.5, 2.0, 2.5, 2.8, 3.0, 3.2, 3.5, 3.8, 4.0, 4.5, 5.0 mg/mL throughout the aqueous drug solution.

In other preferred embodiments, the stabilizer is two different stabilizers.

In a specific embodiment, the first stabilizer added to the reaction system during the complexation reaction is gentisic acid or a salt thereof. It is present in the aqueous pharmaceutical solution at a concentration of 0.5-5mg/mL, preferably 0.5-2 mg/mL, and may be, for example, 0.5, 0.8, 1.0, 1.2, 1.5, 1.8, 2.0 mg/mL. The second stabilizer added after the end of the reaction is ethanol, which is present in the aqueous drug solution at a concentration of 0-400 mg/mL, preferably 10-120 mg/mL, and may be, for example, 10, 30, 50, 60, 70, 80, 100, 120 mg/mL. The volume fraction is from 0% to 50%, preferably from 1% to 15%, and may be, for example, 1%, 3%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%.

In a specific embodiment, the first stabilizer added to the reaction system during the complexation reaction is gentisic acid or a salt thereof, which is present in the aqueous pharmaceutical solution at a concentration of 0.5-5mg/mL, preferably 0.5-2 mg/mL, and may be, for example, 0.5, 0.8, 1.0, 1.2, 1.3, 1.4, 1.5, 1.6, 1.8, 2.0 mg/mL. The second stabilizer added after the end of the reaction is L-methionine, which is present in the aqueous pharmaceutical solution in a concentration of 0-50 mg/mL, preferably 1-10mg/mL, and may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/mL.

In other embodiments, the two stabilizers preferably do not contain ascorbic acid and salts thereof.

In a specific embodiment, the aqueous pharmaceutical solution further comprises a buffer. The buffer solution can be added during the complexation reaction to adjust the pH of the reaction phase solution, or can be added again after the reaction is finished to adjust the pH of the preparation solution. The buffer added in the two times may be the same or different. The buffer solution can be selected from acetate system (such as acetic acid-sodium acetate system, sodium acetate system), citrate system (such as citric acid-sodium citrate system), phosphate system (such as sodium dihydrogen phosphate-disodium hydrogen phosphate system), and formate system (such as formic acid-sodium formate system). In a preferred embodiment, the concentration of the buffer salt in the reaction phase solution is 0.01 to 2.0M. In a preferred embodiment, the total buffer salt concentration in the final aqueous pharmaceutical solution is between 0.005 and 0.5M.

In a specific embodiment, the aqueous pharmaceutical solution further comprises a co-solvent, which acts to reduce adsorption of the API to the surfaces of the respective contact materials, particularly glass and plastic surfaces. The cosolvent is one or more selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, polyoxyethylene castor oil and span, preferably polysorbate 80. In a particular embodiment, the concentration of the co-solvent in the aqueous pharmaceutical solution is from 0.01 to 10mg/mL, preferably from 0.05 to 1.0 mg/mL, and may be, for example, 0.05, 0.1, 0.3, 0.5, 0.6, 0.7, 0.8, 1.0 mg/mL.

In a specific embodiment, the aqueous pharmaceutical solution further comprises a chelating agent for a free metal nuclide. The chelating agent is used for complexing unreacted free nuclide ions in the medicine water solution so as to reduce unnecessary irradiation of the free radionuclide ions to healthy tissues in vivo. Therefore, the chelating agent is required to have strong capability of complexing with nuclide ions, and can rapidly react with free nuclide ions under a low concentration condition even after the injection enters a living body and is diluted by plasma, and the complexing reaction needs to be rapid and mild, and can be completely carried out under a room temperature condition. In a particular embodiment, the chelating agent is pentetic acid or a salt thereof, preferably pentetic acid. The concentration of the chelating agent in the aqueous pharmaceutical solution is 0.005 to 0.1mg/mL, and may be, for example, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 mg/mL. Within this range, pentetic acid has sufficient complexing ability for the free nuclide ions, and the complex is also stable for at least 48 hours, more preferably 72 hours, under radiolysis, i.e. free nuclide ions are not released due to radiolysis of the chelating agent.

In a specific embodiment, the aqueous pharmaceutical solution provided herein is capable of providing at least a radiochemical purity of the API of not less than 90% within 48h, more preferably not less than 90% within 72 h, as determined by HPLC, under storage conditions at 32 ℃ and 60% RH.

The present application further relates to a method for preparing an aqueous radiopharmaceutical solution, which, in one embodiment, comprises the steps of:

recovering the resulting aqueous radiopharmaceutical solution.

In one embodiment of the present application, the aqueous radiopharmaceutical solution comprises a radionuclide complex formed from a radionuclide and a molecule that targets PSMA.

In a specific embodiment, the solution containing the radionuclide is taken out of the raw material bottle and then added to the reaction vessel, and after the solution containing the radionuclide is taken out, the raw material bottle is rinsed with a rinsing solution to extract the nuclide solution remaining in the raw material bottle, and the rinsed solution is transferred to the reaction vessel and mixed with the solution containing the radionuclide.

In a particular embodiment, the rinsing solution is an aqueous solution, preferably selected from the group consisting of a solution containing a first stabilizing agent, a solution containing a buffer salt, water or sodium chloride injection.

In a specific embodiment, the first stabilizer is one or more selected from gentisic acid and salts thereof, ascorbic acid and salts thereof, histidine, cysteine and salts thereof, methionine, selenomethionine, thiosulfate, maltose, inositol, benzyl alcohol, trehalose, povidone, nicotinamide, ethanol, curcumin, melatonin, preferably gentisic acid.

In a preferred embodiment, the irrigation solution is selected from water for injection or sodium chloride injection.

In a preferred embodiment, the rinsing is repeated one or more times with the rinsing liquid.

In a specific embodiment of the present application, the solution containing a radionuclide is a solution containing¹⁷⁷Lu and hydrochloric acid, in particular, in a step of complexing with a molecule targeting PSMA¹⁷⁷The specific activity of Lu is not less than 20 Ci/mg, preferably not less than 60 Ci/mg, and most preferably not less than 80 Ci/mg. Of too low specific activity¹⁷⁷Lu affects radiolabeling efficiency.

In a specific embodiment of the present application, a solution containing a first stabilizer is mixed with a solution containing a radionuclide in a reaction vessel, and after a given time, a solution containing the PSMA-targeting molecule is added to the reaction vessel. The given time can enable the first stabilizer to be in sufficient contact with the nuclide solution, and quench a large number of free radicals brought by radiolysis existing in the nuclide solution, so that the target PSMA molecules added into a reaction system subsequently are protected from being attacked by active free radicals, and initial radiochemical purity of a final product is improved.

In a preferred embodiment, the predetermined time is 0.1 to 20 minutes, more preferably 3 to 10 minutes, and may be, for example, 3, 4, 5, 6, 7, 8, 9, or 10 minutes.

In a specific embodiment of the present application, the PSMA-targeting molecule comprises a chelating group and a PSMA-targeting group, wherein the chelating group can be selected from DOTA, NOTA, PCTA, DTPA, NTA, EDTA, DO3A or NOC, preferably DOTA. The chelating group is connected with the targeting group through a covalent bond.

In a specific embodiment, the PSMA-targeting molecule can be any PSMA-targeting molecule, including PSMA-617, EB-PSMA, PSMA-11. In a preferred embodiment, the PSMA-targeting molecule is EB-PSMA.

In a specific embodiment, the solution containing the PSMA-targeting molecule is added to a reaction phase solution and reacted with a radionuclide to obtain the radionuclide complex.

In a specific embodiment, the PSMA-targeting molecule (label precursor) solution is selected from an aqueous compound solution with a concentration of 0.05-5.0 mg/mL, and is prepared by dissolving a lyophilized powder of the label precursor in sterile water for injection.

In a preferred embodiment, the radionuclide complex is¹⁷⁷Lu-EB-PSMA。

In a particular embodiment, the first stabilizer present during the reaction of the PSMA-targeting molecule with the radionuclide to give the radionuclide complex is gentisic acid, which is present in the reaction phase in a concentration of 0.6-20.0 mg/mL, preferably 2-10 mg/mL, most preferably 3.0-5.0 mg/mL, and may be, for example, 3.0, 3.2, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0 mg/mL. When the concentration of the gentisic acid in the reaction phase system exceeds the control range, the reaction rate can be greatly slowed down, and the whole synthesis process is not facilitated; when the gentisic acid concentration is lower than the control concentration, radiation degradation impurities increase due to insufficient concentration of the stabilizer.

In a specific embodiment, the PSMA-targeting molecule and the radionuclide are reacted to form a reaction phase solution, and the molar ratio between the PSMA-targeting molecule and the radionuclide is 1.5 to 50, preferably 5 to 20, and may be, for example, 5, 8, 10, 12, 15, 18, 20. The molar ratio refers to the ratio of the molar amount of PSMA-targeted molecules (label precursors) to the radionuclide in the reaction system. In the reaction phase solution, the increase of the molar ratio is beneficial to the complete reaction of the radionuclide, so that the labeling rate is increased, but the unlabeled labeled precursor competes with the API in the organism. However, the too low molar ratio results in the lack of a carrier for the API, and the content of the API in the organism is easily lost by the combination of other non-specific targets, so that the expected therapeutic or diagnostic effect cannot be achieved.

In the embodiments of the present application, the concentration of the reaction phase in the reaction phase solution may also be controlled. Theoretically, the higher the concentration of the reaction phase, the faster the labeling reaction rate, but at the same time, the stronger the radiolysis effect caused by the radionuclide, so the reaction phase concentration cannot be too high, while too low a concentration of the reaction phase increases the reaction volume, limiting the mass production of nuclide complexes. For the preparation method of the present application, the concentration of the PSMA-targeting molecule in the reaction phase solution is in the range of 0.01-1.0 mg/mL, preferably 0.05-0.5 mg/mL, and may be, for example, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5 mg/mL.

In an embodiment of the present application, the step of performing a complex reaction between the PSMA-targeting molecule and the radionuclide is performed by controlling the reaction temperature and time to achieve a reaction labeling rate of >90%, a chemical purity of >90%, and an radiochemical purity of > 90%. In a particular embodiment, the reaction temperature is between 50 and 95 ℃, preferably between 60 and 80 ℃, and may be, for example, 60, 62, 65, 68, 70, 72, 75, 78, 80 ℃; the reaction time is 5 to 60 minutes, and may be, for example, 5, 10, 12, 15, 18, 20, 25, 30, 40, 50, or 60 minutes, preferably 10 to 30 minutes, and most preferably 10 to 20 minutes.

In a specific embodiment, the second stabilizer is added after the PSMA-targeting molecule and the radionuclide react for the above reaction time to form a complex. Specifically, the second stabilizer is one or more selected from gentisic acid and salts thereof, ascorbic acid and salts thereof, histidine, cysteine and salts thereof, methionine, selenomethionine, thiosulfate, maltose, inositol, benzyl alcohol, trehalose, povidone, nicotinamide, ethanol, curcumin and melatonin, and is preferably gentisic acid, ethanol or methionine.

In a specific embodiment, the concentration of the second stabilizer in the post-reaction system is 0 to 400mg/mL, and may be, for example, 0, 50, 100, 150, 200, 250, 300, 350, 400 mg/mL.

In embodiments of the present application, the method of preparing further comprises adding a buffered salt solution prior to reacting the PSMA-targeting molecule with the radionuclide, preferably, the buffered salt solution is present in the solution containing the first stabilizing agent.

In a particular embodiment, the buffered salt solution is selected from an acetate, citrate, phosphate or formate solution, preferably an acetate-sodium acetate buffered salt solution.

The pH of the reaction system can be adjusted by adding the buffer salt solution, and the pH of the reaction phase system can be controlled to be in the range of 3.5 to 6.0, for example, 3.5, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.5, 6, preferably 3.5 to 5. In a particular embodiment, the pH of the final formulation solution is controlled to be 4-6, for example, 4, 4.2, 4.5, 4.8, 5, 5.2, 5.5, 5.8, 6.

In a specific embodiment, the step of adding a solution containing a second stabilizer to the reaction vessel after the reaction for a given time further comprises adding a co-solvent to the reaction vessel.

In a specific embodiment, the cosolvent is one or more selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, polyoxyethylated castor oil, and span, preferably polysorbate 80. In a particular embodiment, the cosolvent is added to give a concentration of 0.01-10 mg/mL, preferably 0.05-1.0 mg/mL, and may be, for example, 0.05, 0.1, 0.3, 0.5, 0.6, 0.7, 0.8, 1.0 mg/mL in the aqueous pharmaceutical solution.

In a particular embodiment, the step of adding a solution containing a second stabilizer to the reaction vessel after the reaction for a given time further comprises adding a free nuclide chelating agent to the reaction vessel, said chelating agent being selected from pentetic acid and salts thereof, preferably pentetic acid. In a preferred embodiment, the chelating agent is added to give a concentration of 0.005-0.1 mg/mL, for example 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1mg/mL in the aqueous pharmaceutical solution.

In one embodiment, the methods of the present application further comprise filter sterilizing the aqueous radiopharmaceutical solution, and in one embodiment, filter sterilizing the aqueous radiopharmaceutical solution through a 0.22 μm filter.

In a specific embodiment, the preparation method of the present application further comprises diluting the radioactive aqueous solution, preferably, adding sodium chloride injection for dilution for recovery. The final formulation has a specification of 5-20 mCi/mL, preferably 8-15 mCi/mL, for example 8, 9, 10, 11, 12, 13, 14, 15 mCi/mL, considering that too high a radioactive concentration causes radiolysis problems and too low a radioactive concentration poses dosing challenges.

In a preferred embodiment, the filter sterilization and dilution are performed after the addition of the solution containing the second stabilizer. The sequence of the filter sterilization and the dilution steps is not limited in the application, and the filter sterilization can be carried out firstly and then the dilution is carried out, or the dilution is carried out firstly, and then the filter sterilization is carried out by a filter membrane, and then the recovery is carried out.

In a specific embodiment, the present application provides a process for the preparation of¹⁷⁷Method for preparing Lu-EB-PSMA radioactive medicine aqueous solution:

a. will contain 300mCi¹⁷⁷Transferring the nuclide solution of Lu and hydrochloric acid from the raw material bottle to a reaction bottle;

b. 0.3mL of a rinsing solution containing 2.7M acetic acid-sodium acetate buffer salt and 100mg/mL gentisic acid was added to the raw material bottle to rinse the residual in the raw material bottle¹⁷⁷Lu solution;

c. transferring the mixed solution in the washed raw material bottle into a reaction bottle;

d. adding 2mL of water for injection into the raw material bottle for flushing the raw material bottle;

e. transferring the mixed solution in the washed raw material bottle into a reaction bottle;

f. standing the reaction bottle containing the solution at room temperature for 8 minutes;

g. adding a solution containing 1mg of a labeled precursor EB-PSMA into a reaction bottle;

h. heating the reaction bottle to 65 ℃ and reacting for 20 minutes;

i. after the reaction is finished, cooling the reaction bottle, and adding 3mL of a mixed solution containing 1.05mg/mL pentetic acid, 300mg/mL ethanol and 3.5mg/mL polysorbate 80 into the reaction bottle;

j. filtering the obtained solution through a 0.22 mu m filter membrane for sterilization;

k. the resulting solution was diluted with 23mL of sodium chloride injection;

recovering the product obtained.

Examples

The experimental methods used in the following examples are all conventional methods, unless otherwise specified.

The chemical precursor EB-PSMA used in the following examples was synthesized according to the literature method (Bioconjugate chem. 2018, 29, 3213-3221).

Gentisic acid used in the following examples was purchased from Douguery scientific development Co., Ltd, and pentetic acid was purchased from Jiangxi alpha high-tech pharmaceutical Co., Ltd.

Other materials, reagents, etc., are commercially available without specific reference.

Example 1:¹⁷⁷preparation of Lu-EB-PSMA medicine water solution

Mixing 10 μ L EB-PSMA solution and 10mCi¹⁷⁷LuCl₃Mixing the solution with 10 μ L acetic acid-sodium acetate buffer salt solution (containing 100mg/mL gentisic acid) and 100 μ L sterile water for injection to obtain reaction phase solution, heating the reaction phase solution to allow sufficient reaction, cooling to room temperature, adding 35 μ L adjuvant solution (containing 100mg/mL gentisic acid, 3.0mg/mL pentetic acid, 10mg/mL tween-80) and normal saline, and making the total volume of the preparation to be 1 mL.

Example 2:¹⁷⁷preparation of Lu-EB-PSMA medicine water solution

Mixing 10 μ L EB-PSMA solution and 10mCi¹⁷⁷LuCl₃Mixing the solution with 10 μ L acetic acid-sodium acetate buffer salt solution (containing 100mg/mL gentisic acid) and 100 μ L sterile water for injection to obtain reaction phase solution, heating the reaction phase solution to allow sufficient reaction, cooling to room temperature, adding 100 μ L adjuvant solution (containing 80mg/mL gentisic acid, 1.05mg/mL pentetic acid, and 3.5mg/mL tween 80) and normal saline, and making the total volume of the preparation to be 1 mL.

Example 3:¹⁷⁷preparation of Lu-EB-PSMA medicine water solution

Mixing 10 μ L EB-PSMA solution and 10mCi¹⁷⁷LuCl₃Mixing the solution with 10 μ L acetic acid-sodium acetate buffer salt solution (containing 100mg/mL gentisic acid) and 100 μ L sterile water for injection to obtain reaction phase solution, heating the reaction phase solution to allow sufficient reaction, cooling to room temperature, adding 100 μ L adjuvant solution (containing 5mg/mL gentisic acid, 1.05mg/mL pentetic acid, and 3.5mg/mL tween-80) and normal saline, and making the total volume of the preparation to be 1 mL.

Example 4:¹⁷⁷preparation of Lu-EB-PSMA medicine water solution

Mixing 10 μ L EB-PSMA solution and 10mCi¹⁷⁷LuCl₃Mixing the solution with 10 μ L acetic acid-sodium acetate buffer salt solution (containing 100mg/mL gentisic acid) and 100 μ L sterile water for injection to obtain reaction phase solution, heating the reaction phase solution to allow sufficient reaction, cooling to room temperature, adding 100 μ L adjuvant solution (containing 300mg/mL ethanol, 1.05mg/mL pentetic acid, 3.5mg/mL tween-80) and normal saline, and making the total volume of the preparation to be 1 mL.

Example 5:¹⁷⁷preparation of Lu-EB-PSMA medicine water solution

Mixing 10 μ L EB-PSMA solution and 10mCi¹⁷⁷LuCl₃Mixing the solution with 10 μ L acetic acid-sodium acetate buffer salt solution (containing 100mg/mL gentisic acid) and 100 μ L sterile water for injection to obtain reaction phase solution, heating the reaction phase solution to allow sufficient reaction, cooling to room temperature, adding 200 μ L adjuvant solution (containing 400mg/mL ethanol, 0.525mg/mL pentetic acid, 1.75mg/mL tween-80) and normal saline, and making the total volume of the preparation to be 1 mL.

Example 6:¹⁷⁷Lu-EB-PSMpreparation of aqueous drug solution

Mixing 10 μ L EB-PSMA solution and 10mCi¹⁷⁷LuCl₃Mixing the solution with 10 μ L acetic acid-sodium acetate buffer salt solution (containing 100mg/mL gentisic acid) and 100 μ L sterile water for injection to obtain reaction phase solution, heating the reaction phase solution to allow sufficient reaction, cooling to room temperature, adding 200 μ L adjuvant solution (containing 0.525mg/mL pentetic acid, 1.75mg/mL tween 80), 400mg ethanol and normal saline to make the total volume of the preparation be 1 mL.

Example 7:¹⁷⁷preparation of Lu-EB-PSMA medicine water solution

Mixing 10 μ L EB-PSMA solution and 10mCi¹⁷⁷LuCl₃Mixing the solution with 10 μ L acetic acid-sodium acetate buffer salt solution (containing 100mg/mL gentisic acid) and 100 μ L sterile water for injection to obtain reaction phase solution, heating the reaction phase solution to allow sufficient reaction, cooling to room temperature, adding 100 μ L adjuvant solution (containing 10mg/mL methionine, 1.05mg/mL pentetic acid, 3.5mg/mL tween-80) and normal saline, and making the total volume of the preparation to be 1 mL.

Example 8:¹⁷⁷preparation of Lu-EB-PSMA medicine water solution

Mixing 10 μ L EB-PSMA solution and 10mCi¹⁷⁷LuCl₃Mixing the solution with 10 μ L acetic acid-sodium acetate buffer salt solution (containing 100mg/mL gentisic acid) and 100 μ L sterile water for injection to obtain reaction phase solution, heating the reaction phase solution to allow sufficient reaction, cooling to room temperature, adding 500 μ L adjuvant solution (containing 20mg/mL methionine, 0.21mg/mL pentetic acid, 0.7mg/mL tween-80) and normal saline, and making the total volume of the preparation to be 1 mL.

Comparative example 1

Mixing 10 μ L EB-PSMA solution and 10mCi¹⁷⁷LuCl₃Mixing the solution with 10 μ L acetic acid-sodium acetate buffer salt solution (containing 100mg/mL gentisic acid) and 100 μ L sterile water for injection to obtain reaction phase solution, heating the reaction phase solution to allow sufficient reaction, cooling to room temperature, adding 100 μ L adjuvant solution (containing 30mg/mL ascorbic acid, 1.05mg/mL pentetic acid, 3.5mg/mL Tween 80) and normal saline to make the total volume of the preparation be 1mL。

Comparative example 2

Mixing 10 μ L EB-PSMA solution and 10mCi¹⁷⁷LuCl₃Mixing the solution with 10 μ L acetic acid-sodium acetate buffer salt solution (containing 10mg/mL gentisic acid) and 100 μ L sterile water for injection to obtain reaction phase solution, heating the reaction phase solution to allow sufficient reaction, cooling to room temperature, adding 100 μ L adjuvant solution (containing 1mg/mL gentisic acid, 1.05mg/mL pentetic acid, and 3.5mg/mL tween-80) and normal saline, and making the total volume of the preparation to be 1 mL.

Comparative example 3

Mixing 10 μ L EB-PSMA solution and 10mCi¹⁷⁷LuCl₃Mixing the solution with 10 μ L acetic acid-sodium acetate buffer salt solution (containing 10mg/mL gentisic acid) and 100 μ L sterile water for injection to obtain reaction phase solution, heating the reaction phase solution to allow sufficient reaction, cooling to room temperature, adding 35 μ L adjuvant solution (containing 3.0mg/mL pentetic acid, 10mg/mL tween-80) and normal saline, and making the total volume of the preparation be 1 mL.

Experimental example 1¹⁷⁷Lu-EB-PSMA medicament aqueous solution stability evaluation experiment

Chemical purification detection experiment at 1.25 DEG C

The aqueous pharmaceutical solution preparations obtained in examples 1 to 8 and comparative examples 1 to 3 were stored at 25 ℃ and 60% RH for 48 hours and then tested for API radiochemical purity by HPLC, and the results are shown in Table 1.

TABLE 1

Stabilizer 1 (trans) Adding in phase)

Preparation of stabilizer 1 In (b) content

Stabiliser 2 (reaction) Post-addition)

Content of stabilizer 2 in the preparation

At 25 ℃ after 48h Purifying by heating

At 32 ℃ after 48h Purifying by heating

Example 1

Gentisic acid

1.0mg/mL

Gentisic acid

3.5mg/mL (i.e., the total concentration of gentisic acid in the formulation) Degree of 4.5 mg/mL)

95.7%

95.3%

Example 2

Gentisic acid

1.0mg/mL

Gentisic acid

8mg/mL (i.e., the total concentration of gentisic acid in the formulation) Is 9.0 mg/mL)

95.8%

95.5%

Example 3

Gentisic acid

1.0mg/mL

Gentisic acid

0.5mg/mL (i.e., the total concentration of gentisic acid in the formulation) Degree of 1.5 mg/mL)

94.4%

93.1%

Example 4

Gentisic acid

1.0mg/mL

Ethanol

30mg/mL

94.6%

93.8%

Example 5

Gentisic acid

1.0mg/mL

Ethanol

80mg/mL

94.1%

94.0%

Example 6

Gentisic acid

1.0 mg/mL

Ethanol

400 mg/mL

95.1%

94.5%

Example 7

Gentisic acid

1.0mg/mL

Methionine

1mg/mL

95.0%

93.0%

Example 8

Gentisic acid

1.0mg/mL

Methionine

10mg/mL

95.0%

93.6%

Comparative example 1

Gentisic acid

1.0mg/mL

Ascorbic acid

3mg/mL

84.4%

81.1%

Comparative example 2

Gentisic acid

0.1mg/mL

Gentisic acid

0.1mg/mL

88.3%

86.4%

Comparative example 3

Gentisic acid

0.1mg/mL

Is free of

/

85.6%

82.2%

Chemical purification detection experiment at 2.32 DEG C

The aqueous pharmaceutical solution preparations obtained in examples 1 to 8 and comparative examples 1 to 3 were stored at 32 ℃ and 60% RH for 48 hours and then tested for API radiochemical purity by HPLC, and the results are shown in Table 1.

And (3) analyzing an experimental result: in the presence of a stabilizerIn the sample of gentisic acid, the total concentration of gentisic acid of 1.5mg/mL already provided a better stabilization of the radiochemical purity of the formulation within 48h, compared to comparative example 3, in which the stabilizer was 0.1mg/mL gentisic acid, and the total concentration of gentisic acid was further increased to 4.5mg/mL, the radiochemical purity of the same formulation stored under the same conditions was further increased by about 1% -2%. On this basis, the increase in radiochemical purity brought by the continued increase in the total concentration of gentisic acid is not obvious, i.e. the stabilizing effect of gentisic acid reaches the plateau region. Similar plateaus occur in other stabilizer combinations, with relatively limited increase in radiochemical purity brought about by increasing stabilizer concentration at ethanol concentrations above 80mg/mL or methionine concentrations above 1 mg/mL. Another stabilizer is gentisic acid + ascorbic acid in pairs¹⁷⁷The Lu-EB-PSMA stabilization was significantly weaker than gentisic acid + gentisic acid, gentisic acid + ethanol and gentisic acid + methionine combinations, indicating that ascorbic acid is not the preferred stabilizer.

Experimental example 2 control of reaction temperature and time

An aqueous precursor solution containing 100. mu.g EB-PSMA and 50 mCi were added to the reactor¹⁷⁷LuCl₃The solution was adjusted to pH 5 using a buffer solution containing 5mg gentisic acid, water for injection was added thereto so that the volume of the reaction phase was 1mL, and the reaction system was heated at a certain temperature for a certain time. From the viewpoint of labeling rate (labeling rate = radioactivity chelated to precursor molecule ÷ total radioactivity), the labeling rate of the reaction can reach 90% when the reaction temperature is 50 to 95 ℃ and the reaction time is 5 to 60 minutes, preferably the reaction temperature is 60 to 80 ℃ and the reaction time is 10 to 30 minutes, and at this time the labeling rate can reach 99% or more. From the viewpoint of radiochemical purity, when the reaction temperature is 50 to 95 ℃ and the reaction time is 5 to 60 minutes, the initial radiochemical purity of the resulting labeled compound is obtained>95%, preferably at a temperature of 60-80 ℃ and a time of 10-30 minutes, at which the initial radiochemical purity of the resulting labelled compound is obtained>99%。

Experimental example 3 control of pH value

An aqueous precursor solution containing 100. mu.g EB-PSMA and 5 was added to the reactor0 mCi ¹⁷⁷LuCl₃The solution was adjusted to a certain pH value using different buffer solutions (5 mg gentisic acid was contained in the buffer solution), water for injection was added thereto to make the volume of the reaction phase 1mL, and the reaction system was heated at 65 ℃ for 20 minutes. When the pH value of the reaction phase solution is 3.5-6.0, the reaction labeling rate is high>99%。

Experimental example 4 control of reaction phase concentration

An aqueous precursor solution containing 100. mu.g EB-PSMA and 50 mCi were added to the reactor¹⁷⁷LuCl₃A solution, the pH of which was adjusted to 5 using a buffer solution containing 5mg gentisic acid, and the concentration of EB-PSMA in the reaction phase was controlled by adding different volumes of water for injection thereto, and the reaction system was heated at 65 ℃ for 20 minutes. When the concentration of EB-PSMA in the reaction phase is 0.02-0.7mg/mL, the labeling rate is all>99%。

Experimental example 5 control of the feed ratio

Adding an aqueous EB-PSMA precursor solution and 50 mCi to a reactor¹⁷⁷LuCl₃A solution, the pH of which was adjusted to 5 using a buffer solution containing 5mg gentisic acid, to which water for injection was added to make the volume of the reaction phase 1mL, and the reaction system was heated at 65 ℃ for 20 minutes. When EB-PSMA is reacted with¹⁷⁷LuCl₃When the feeding molar ratio of (A) is 1.5-50, the marking rate is>95%, preferred molar ratios of charge (EB-PSMA:¹⁷⁷LuCl₃) At 5-20, the marking rate>99%。

Experimental example 6 control of the amount of prescription of pentetic acid

Adding pentetic acid aqueous solution and 50 mCi into a reactor¹⁷⁷LuCl₃Adjusting pH value of the solution to 5 by using buffer solution containing 5mg gentisic acid, adding water for injection to make volume of reaction phase be 30mL, reacting the reaction system at room temperature for different time periods, and detecting free hydrochloric acid not chelated by pentetic acid after reaction¹⁷⁷LuCl₃. When pentetic acid is added in an amount of 0.005-0.5mg, the free form which is not chelated by pentetic acid is not present¹⁷⁷LuCl₃In a ratio of<1% and the chelation reaction can be completed within 5 minutes. After standing at room temperature for 96 hoursFree chelated by pentetic acid¹⁷⁷LuCl₃In a ratio of<1%。

Experimental example 7 control of quenching time

(1) Preparation of aqueous drug solutions without quenching time

10 uL EB-PSMA solution and 10mCi are added into the reactor in sequence¹⁷⁷LuCl₃Mixing the solution with 10 μ L acetic acid-sodium acetate buffer salt solution (containing 100mg/mL gentisic acid) and 100 μ L sterile water for injection to obtain reaction phase solution, heating the reaction phase solution to allow sufficient reaction, cooling to room temperature, adding 35 μ L adjuvant solution (containing 100mg/mL gentisic acid, 3.0mg/mL pentetic acid, 10mg/mL tween-80) and normal saline, and making the total volume of the preparation to be 1 mL. The initial marking rate at this point was 95%.

(2) Preparation of an aqueous solution of the drug under 5 minute quenching conditions

Adding 100 μ L sterile water for injection, 10 μ L acetic acid-sodium acetate buffer solution (containing 100mg/mL gentisic acid), and 10mCi into reactor in sequence¹⁷⁷LuCl₃The solution was mixed well and allowed to stand at room temperature for 5 minutes. Standing, adding 10 μ L EB-PSMA solution, mixing to obtain reaction phase solution, heating the reaction phase solution to allow full reaction, cooling to room temperature, adding 35 μ L adjuvant solution (containing 100mg/mL gentisic acid, 3.0mg/mL pentetic acid, 10mg/mL tween-80) and normal saline to make total volume of the preparation be 1 mL. The initial labeling rate at this time was 99%.

(3) Preparation of an aqueous solution of the drug under 15 minute quenching conditions

Adding 100 μ L sterile water for injection, 10 μ L acetic acid-sodium acetate buffer solution (containing 100mg/mL gentisic acid), and 10mCi into reactor in sequence¹⁷⁷LuCl₃The solution was mixed well and allowed to stand at room temperature for 10 minutes. Standing, adding 10 μ L EB-PSMA solution, mixing to obtain reaction phase solution, heating the reaction phase solution to allow full reaction, cooling to room temperature, adding 35 μ L adjuvant solution (containing 100mg/mL gentisic acid, 3.0mg/mL pentetic acid, 10mg/mL tween-80) and normal saline to make total volume of the preparation be 1 mL. The initial labeling rate at this time was 99%.

And (3) analyzing an experimental result: before adding a labeled precursor solution (a targeting molecule connected with a chelating group), standing a mixed solution containing a nuclide solution, a buffer salt and a first stabilizer for a short time (quenching time), and adding a precursor compound for reaction, so that the initial radiochemical purity of the API can be obviously improved. The first stabilizer can be sufficiently contacted with the nuclide solution after the quenching time, so that a large amount of free radicals generated due to high radioactivity in the solution are quenched by the first stabilizer, and the damage of the free radicals to the labeled precursor molecules (the targeting molecules connected with the chelating groups) during the subsequent addition of the labeled precursor is reduced. The standing time is controlled to be 0.1-20 minutes, preferably 3-10 minutes.

Experimental example 8 control of the amount of Co-solvent prescribed

(1) Automatic synthesis of aqueous solution of medicine without cosolvent

In the synthesis hot chamber, an automatic synthesizer, a matched card sleeve and a medicine box are used for carrying out the following automatic synthesis steps, and 300mCi is added into a reaction bottle¹⁷⁷LuCl₃The solution was mixed with 0.3mL of acetic acid-sodium acetate buffer salt solution (containing 100mg/mL gentisic acid) and 3mL of sterile water for injection, and after standing at room temperature for 10 minutes, 0.3mL of EB-PSMA solution was added to the above mixed solution, followed by mixing to obtain a reaction phase solution. The reaction phase solution was heated to react sufficiently, and after cooling to room temperature, 1.05mL of an adjuvant solution (containing 100mg/mL gentisic acid and 3.0mg/mL pentetic acid) and physiological saline were added thereto so that the total volume of the resulting aqueous drug solution was 30 mL. And (3) conveying the obtained drug aqueous solution from the synthesis hot chamber to a subpackaging hot chamber through a PEEK conveying pipe with the length of 1.5 meters for subpackaging and packaging. The final aqueous drug solution had a total activity of 207mCi and a yield of 69%.

(2) Automated synthesis of aqueous drug solutions with a formulation comprising 0.01mg/mL polysorbate 80 as co-solvent

The procedure was the same as in Experimental example 8 (1) except that the adjuvant solution added after the reaction contained 100mg/mL gentisic acid, 3.0mg/mL pentetic acid, and 0.29mg/mL polysorbate 80. The final aqueous drug solution had a total activity of 262mCi and a yield of 87%.

(3) Automated synthesis of aqueous drug solutions with a formulation comprising 1mg/mL polysorbate 80 as co-solvent

The procedure was the same as in Experimental example 8 (1) except that the adjuvant solution added after the reaction contained 100mg/mL gentisic acid, 3.0mg/mL pentetic acid, and 28.6mg/mL polysorbate 80. The final aqueous drug solution had a total activity of 296mCi and a yield of 99%.

(4) Automated synthesis of aqueous drug solutions with a formulation comprising 10mg/mL polysorbate 80 as co-solvent

The procedure was the same as in Experimental example 8 (1) except that the adjuvant solution added after the reaction contained 100mg/mL gentisic acid, 3.0mg/mL pentetic acid, and 285.7mg/mL polysorbate 80. The final aqueous drug solution had a total activity of 298mCi and a yield of 99%.

And (3) analyzing an experimental result: API molecule¹⁷⁷Lu]Lu-EB-PSMA is very easy to be absorbed by a cutting sleeve made of plastic materials, the inner wall of a transmission pipeline and the like due to strong lipophilicity, so that a large amount of activity loss is caused, the yield of automatic synthesis is reduced, and in a prescription without adding a cosolvent, the activity loss rate is up to 31%. The addition of the cosolvent polysorbate 80 can significantly reduce the adsorption loss of the API. The yield can be improved to 87% by adding 0.01mg/mL of polysorbate 80 in the prescription, and the yield of the automatic synthesis is stabilized to about 99% when the prescription concentration of the polysorbate 80 is continuously improved to 1mg/mL or even 10 mg/mL.

Experimental example 9 [ alpha ], [ alpha ]¹⁷⁷Lu]Stability Studies of labeled other complexes

[¹⁷⁷Lu]Labeling of Lu-PSMA-617

Mixing 10 μ L of PSMA-617 solution and 10mCi¹⁷⁷LuCl₃Mixing the solution with 10 μ L acetic acid-sodium acetate buffer salt solution (containing 100mg/mL gentisic acid) and 100 μ L sterile water for injection to obtain reaction phase solution, heating the reaction phase solution to allow sufficient reaction, cooling to room temperature, adding 35 μ L adjuvant solution (containing 100mg/mL gentisic acid, 3.0mg/mL pentetic acid, 10mg/mL tween-80) and normal saline, and making the total volume of the preparation to be 1 mL. The resulting aqueous drug solution was stored at 25 ℃ and 60% RH for 48h and then its radiochemical purity was determined to be 95.1% by HPLC.

Experimental example 10 stability study of EB-PSMA Complex labeled with other radionuclides

（1） [⁶⁸Ga]Labeling of Ga-EB-PSMA

Leaching a commercial germanium-gallium generator with 0.1M hydrochloric acid to obtain⁶⁸Ga hydrochloric acid solution, taking⁶⁸Ga hydrochloric acid solution 10mCi is mixed with 10 muL EB-PSMA solution, 10 muL sodium acetate buffer solution (containing 100mg/mL gentisic acid) and 100 muL sterile water for injection to form reaction phase solution, the reaction phase solution is heated to fully react, 35 muL adjuvant solution (containing 100mg/mL gentisic acid, 3.0mg/mL pentetic acid and 10mg/mL tween-80) and normal saline are added after cooling to room temperature, and the total volume of the preparation is 1 mL. After the resulting aqueous drug solution was stored at 25 ℃ and 60% RH for 5h, its radiochemical purity was determined to be 93.3% by HPLC.

（2） [⁹⁰Y]Labeling of Y-EB-PSMA

Mixing 10 μ L EB-PSMA solution and 10mCi⁹⁰Mixing the solution Y with 10 μ L acetic acid-sodium acetate buffer salt solution (containing 100mg/mL gentisic acid) and 100 μ L sterile water for injection to obtain reaction phase solution, heating the reaction phase solution to allow sufficient reaction, cooling to room temperature, adding 35 μ L adjuvant solution (containing 100mg/mL gentisic acid, 3.0mg/mL pentetic acid, 10mg/mL tween-80) and normal saline, and making the total volume of the preparation to be 1 mL. The obtained aqueous solution of the drug was stored at 25 ℃ and 60% RH for 48 hours, and then its radiochemical purity was 90.9%.

（3） [²¹²Pb]Labeling of Pb-EB-PSMA

10 uL EB-PSMA solution, 10 uCi²¹²Mixing the Pb solution with 10 μ L sodium acetate buffer solution (containing 100mg/mL gentisic acid) and 100 μ L sterile water for injection to obtain reaction phase solution, heating the reaction phase solution to allow sufficient reaction, cooling to room temperature, adding 35 μ L adjuvant solution (containing 100mg/mL gentisic acid, 3.0mg/mL pentetic acid, 10mg/mL tween-80) and normal saline, and making the total volume of the preparation to be 1 mL. The radiochemical purity of the API at 0 after the marking is completed is 85.0%, and the radiochemical purity of the obtained drug aqueous solution is 78.3% after the drug aqueous solution is stored for 24 hours at the temperature of 25 ℃ under the condition of 60% RH.

（4） [⁶⁴Cu]Labeling of Cu-EB-PSMA

Mixing 10 μ L EB-PSMA solution and 4mCi⁶⁴Mixing the Cu solution with 10 μ L acetic acid-sodium acetate buffer salt solution (containing 100mg/mL gentisic acid) and 100 μ L sterile water for injection to obtain reaction phase solution, heating the reaction phase solution to allow sufficient reaction, cooling to room temperature, adding 35 μ L adjuvant solution (containing 100mg/mL gentisic acid, 3.0mg/mL pentetic acid, 10mg/mL tween-80) and normal saline, and making the total volume of the preparation to be 1 mL. The obtained aqueous solution of the drug was stored at 25 ℃ and 60% RH for 48 hours, and then its radiochemical purity was 92.1%.

And (3) analyzing an experimental result: the mechanism of degradation of radiochemical purity and chemical purity of API by radiolysis is divided into direct action, in which API molecules are directly ionized or excited by charged particles and the molecular structure is destroyed thereby, and indirect action, in which particles interact with other chemical substances (e.g. water molecules) around API molecules to generate a large number of active radicals, which destroy the API molecular structure. Among the two effects, the chain reaction of free radicals and secondary free radicals are the main factors causing radiolysis. The addition of the stabilizer can effectively quench a large amount of active free radicals in the solution, and plays a role in protecting API molecules. The above experimental examples show that although different radiopharmaceuticals have different physicochemical properties, the stabilizers mentioned in the present application have relatively wide applicability to the quenching effect of free radicals generated by different nuclides and different radiation types, and have a better stabilizing effect on the radiopharmaceuticals exemplified in the experimental examples, which effect is reflected to different degrees in different aqueous solutions of the radiopharmaceuticals.

Although the present disclosure has been described with reference to particular embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present disclosure, and the scope of the present disclosure should be limited only by the terms of the appended claims.

Claims

recovering the resulting aqueous radiopharmaceutical solution.

2. The method of claim 1, wherein the solution containing the radionuclide is added to the reaction vessel after being removed from the raw material bottle, the method further comprising:

3. The method according to claim 2, wherein the rinsing liquid is an aqueous solution, preferably selected from the group consisting of a solution containing a first stabilizer, a solution containing a buffer salt, water or sodium chloride injection.

4. The method of claim 1, wherein the solution containing the radionuclide is a solution containing a radionuclide¹⁷⁷Lu and hydrochloric acid, and in the step of reacting the PSMA-targeting molecule with a radionuclide,¹⁷⁷the specific activity range of Lu is more than or equal to 20 Ci/mg.

5. The method of claim 1, wherein the PSMA-targeting molecule comprises a chelating group selected from DOTA, NOTA, PCTA, DTPA, NTA, EDTA, DO3A, or NOC.

6. The method of claim 5, wherein the radionuclide complex is¹⁷⁷Lu-EB-PSMA。

7. The method according to any one of claims 1 to 6, wherein in the step of reacting the PSMA-targeting molecule with a radionuclide, the molar ratio between the PSMA-targeting molecule and the radionuclide is 1.5 to 50.

8. The method of claim 1, wherein the step of reacting the PSMA-targeting molecule with a radionuclide is performed at a temperature of 50-95 ℃ for a time of 5-60 minutes.

9. The method of claim 8, wherein in the step of reacting the PSMA-targeting molecule with a radionuclide, the concentration of the first stabilizer in the reaction phase solution is 0.6-20.0 mg/mL.

10. The method of claim 9, wherein the concentration of the second stabilizer is 0-400 mg/mL.

11. The method of claim 1, wherein a buffered saline solution is added prior to reacting the PSMA-targeting molecule with the radionuclide.

12. The method of claim 1, wherein the step of adding a solution comprising a second stabilizer to the reaction vessel after the reaction for a given time further comprises adding a co-solvent to the reaction vessel.

13. The method of any one of claims 1 to 12, further comprising filter sterilizing the aqueous radiopharmaceutical solution through a 0.22 μm filter.

14. The method of any one of claims 1 to 13, further comprising diluting the radioactive aqueous solution.

15. The method of claim 13 or 14, further comprising filter sterilizing or diluting the radioactive aqueous solution after adding the second stabilizing agent.

16. An aqueous radiopharmaceutical solution prepared by the process of any one of claims 1 to 15.