A PET probe conjugated with an SSTR agonist and an inhibitor, and its preparation method and application

Abstract

The present invention relates to the fields of high molecular heterocyclic compounds and nuclear medicine imaging technology, and particularly relates to a PET probe obtained by coupling an SSTR agonist and an inhibitor, and a preparation method and application thereof. The PET molecular probe comprises a targeting molecule obtained by coupling an SSTR agonist and an inhibitor, a radioisotope, and a linker, and the linker connects the targeting molecule and the radioisotope. The heterodimer obtained by coupling the SSTR agonist and the inhibitor can improve the PET detection sensitivity by increasing the binding affinity with the target through a synergistic effect; has higher specificity and can specifically and efficiently target SSTR for in vivo imaging in a variety of tumor microenvironments; not only makes the distribution of the radioligand more uniform, but also can effectively improve the clinical effect of radionuclide therapy after labeling with therapeutic radionuclides such as <supgt;177< / supgt;Lu. Compared with the prior art, the molecular probe has stronger affinity, higher stability and higher target-to-background ratio; meanwhile, the preparation method of the PET molecular probe is simple and stable, and has good market promotion and application prospects.

Description

Technical Field

[0001] The present invention relates to the technical fields of polymer heterocyclic compounds and nuclear medicine imaging technology, and particularly relates to a PET probe in which an SSTR agonist and an inhibitor are coupled, and a preparation method and application thereof. Background Art

[0002] Neuroendocrine neoplasms (NENs) are a relatively rare type of tumors derived from neuroendocrine cells, with high heterogeneity, and are mainly divided into well-differentiated neuroendocrine tumors (NET) and poorly-differentiated neuroendocrine carcinomas (NEC). NENs can occur in various parts of the body, and are most commonly found in the gastrointestinal tract, pancreas, and lungs. Their incidence is showing an increasing trend year by year. Surgical resection of the lesion is currently the most effective method for treating NEN. However, nearly half of the patients have already metastasized at the time of diagnosis and thus lose the opportunity for surgical treatment, mainly because most NENs are non-functional and, due to their slow proliferation rate, most NENs are relatively resistant to chemotherapy drugs. Due to the heterogeneity of NEN (different in pathology, secreted hormones, and biochemical and clinical characteristics), therefore, how to sensitively and accurately identify the lesion and clarify the stage is crucial for the clinical decision-making and treatment formulation of NEN patients.

[0003] The overexpression of somatostatin receptor (SSTR) on the surface of tumor cells is one of the characteristics of NENs. Somatostatin analogs (SSAs) play an anti-tumor role by binding to SSTR, and are the key targets for the diagnosis and treatment of NET. Radioactively labeled somatostatin analogs have been clinically used for imaging or peptide receptor radionuclide therapy, among which gallium-68 ( 68 Ga)-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-SSAs, such as 68 Ga-DOTATATE and 68 Ga-DOTATOC PET have become the gold standard for somatostatin receptor imaging and have been written into clinical guidelines. However, currently, the commonly used somatostatin receptor radioactive drugs in clinics are all SSTR2 agonists. Recently, studies on radioactively labeled SSTR inhibitors have found that: 68Ga-DOTA-JR11, an SSTR inhibitor, has a low background in normal tissues and organs, resulting in a higher tumor-to-organ target ratio. Compared with SSTR agonists, inhibitors have a higher detection efficiency for lesions. This is because SSTR inhibitors can recognize more receptor binding sites on each cell. However, the internalization rate of SSTR inhibitors is very low, only 20% of the cell binding activity. And the detection efficiency of SSTR inhibitors for bone lesions is not as good as that of agonists. Therefore, there is still a need to develop more effective detection and treatment methods for the diagnosis and treatment of neuroendocrine tumors in clinical practice. Summary of the Invention

[0004] To solve the above technical problems, the present invention proposes a PET probe conjugated with an SSTR agonist and an inhibitor, aiming to achieve the complementary advantages of a single SSTR agonist molecular probe and an SSTR inhibitor molecular probe, obtain a PET molecular probe targeting SSTR with stronger affinity, higher stability and higher target-to-background ratio, perform in vivo imaging of the expression level of SSTR, and thus achieve in vivo real-time imaging of tumors related to the expression level of SSTR for imaging diagnosis of tumors and evaluation of the efficacy of immunotherapeutic drugs.

[0005] To achieve the above object, the present invention first provides a PET probe conjugated with an SSTR agonist and an inhibitor, comprising a targeting molecule, a linking group, a radioisotope and a chelating group. The linking group connects the targeting molecule and the radioactive binding group. The targeting molecule is an SSTR agonist and an SSTR inhibitor linked by lysine. The SSTR agonist is octreotide or an octreotide derivative, and the SSTR inhibitor is selected from somatostatin antagonist peptides or somatostatin antagonist peptide derivatives.

[0006] Preferably, the SSTR agonist is selected from any one of Octreotide and D-Phe1-Tyr3-octreotide, as shown in Formula 1 and Formula 2 below:

[0007]

[0008] Preferably, the SSTR inhibitor is selected from any one of JR11 and SST2-ANT, as shown in Formula 3 and Formula 4:

[0009]

[0010] Preferably, the linking group has a structure shown in any one of Formula 5, Formula 6 or Formula 7 below:

[0011]

[0012] Wherein, in Formula 5, Formula 6 or Formula 7, n is an integer from 1 to 10.

[0013] Preferably, the chelating group is one of DOTA, NOTA, and HYINC; the radioisotope is 68 Ga, 177 Lu, AI 18 F, 225 Ac, 99 any one of mTc.

[0014] Preferably, the PET probe conjugated with the SSTR agonist and inhibitor is prepared from the radioisotope-labeled precursor compound NOTA-OJ-1, and the structural formula of the precursor compound is shown in Formula 8 below:

[0015]

[0016] Preferably, the structural formula of the PET molecular probe is shown in Formula 9 below:

[0017]

[0018] Preferably, x in Formula 9 is a radioisotope 68 Ga.

[0019] Based on a general inventive concept, the present invention also provides a preparation method of a PET probe conjugated with an SSTR agonist and inhibitor, comprising the following steps:

[0020] S1. Mix the probe precursor with an aqueous NaOAc solution evenly and transfer it to a reaction tube;

[0021] S2. Elute the radioisotope into the reaction tube with HCl and react for 10 min under the condition of 90 °C;

[0022] S3. After cooling the solution in the reaction tube in step S2, perform solid-phase extraction through a C18 column, and elute the product into a product bottle equipped with a filter membrane to obtain the PET probe conjugated with the SSTR agonist and inhibitor.

[0023] Preferably, in step S2, the radioisotope is eluted with HCl until the radioactivity of the eluent is 35 mCi.

[0024] Based on a general inventive concept, the present invention also provides an application of a PET probe conjugated with an SSTR agonist and inhibitor in the preparation of a PET for targeting neuroendocrine tumors.

[0025] The imaging principle of the PET probe of the present invention is:

[0026] Traditional PET molecular probes targeting SSTR are mostly agonists or inhibitors of SSTR. There has been no report on coupling these two molecules into heterodimers or multimers. Given the literature reports that SSTR agonists and inhibitors have their respective advantages in terms of affinity, number of binding sites, and internalization when binding to the target SSTR; compared with SSTR agonists, inhibitors have higher detection efficiency for lesions. This is because SSTR inhibitors can recognize more receptor binding sites on each cell. However, the internalization rate of SSTR inhibitors is very low, only 20% of the cell binding activity. And the detection efficiency of SSTR inhibitors for bone lesions is not as good as that of agonists. Therefore, the present invention creatively proposes for the first time to couple and dock SSTR agonists and inhibitors for the construction of molecular probes to achieve the complementary advantages of individual SSTR agonist molecular probes and SSTR inhibitor molecular probes, with the expectation of obtaining a targeted SSTR PET molecular probe with stronger affinity, higher stability, and higher target-to-background ratio.

[0027] The targeting molecule in the PET probe coupled with SSTR agonist and inhibitor constructed by the present invention is prepared by linking SSTR agonist and SSTR inhibitor through lysine. Among them, SSTR agonist and SSTR inhibitor are synthesized by standard Fmoc chemical methods. The prepared targeting molecule is then coupled with a linker and a chelating group to obtain the precursor compound of the PET probe of the present invention. The precursor compound is then radiolabeled to obtain the PET probe coupled with SSTR agonist and inhibitor of the present invention.

[0028] The present invention creatively proposes for the first time to couple and dock SSTR agonists and inhibitors to form a heterodimer. This structure can bind multiple peptides to target cells, thereby generating a tighter target binding. At the same time, this binding also reduces the possibility of complete dissociation of the ligand. Since one ligand binds to its receptor, the unbound peptides of the multimer remain near the cell surface, making the unbound peptides of the heterodimer or multimer remain near other free target receptors. This increases the possibility of interaction between the unbound peptides of the multimer and other free receptors on the surface of tumor cells, and at the same time increases the possibility of rebinding in the case of ligand dissociation. It further increases the local concentration of receptors near the peptide binding site, thereby increasing the possibility of further binding.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The present invention creatively couples SSTR agonists and inhibitors into heterodimers. The special structure of the heterodimer can increase the binding affinity with the target through synergistic effects to improve the PET detection sensitivity; it has higher specificity and can specifically and efficiently target SSTR for in vivo imaging in a variety of tumor microenvironments.

[0031] 2. The coupling of SSTR agonists and inhibitors into heterodimers can make the distribution of radioactive ligands more uniform, increasing the therapeutic effect of tumor-targeted radionuclide therapy. Due to the relatively large size of heterodimer or multimer constructs, the metabolic stability of peptides as radioactive ligands may be improved, thereby enhancing bioavailability.

[0032] 3. The PET probe provided by the present invention can be better taken up by tumors, and the imaging effect is clearer and more obvious. It is not only conducive to the diagnosis and analysis of tumors, but also can effectively improve 177 the clinical efficacy of radionuclide therapy after labeling with therapeutic radionuclides such as Lu.

[0033] 4. The PET probe provided by the present invention also has the characteristics of high specificity, high sensitivity, non-invasive, non-invasive detection, and real-time whole-body imaging in vivo. The preparation method of this molecular probe is simple and stable, with mild conditions. The purity of the prepared molecular probe is greater than 95%, and its in vitro stability is good, which is conducive to clinical promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0035] Figure 1 It is the synthetic route diagram of the precursor compound NOTA-OJ-1 in Example 1;

[0036] Figure 2 It is the HPLC diagram of the chemical purity of NOTA-OJ-1 in Example 1;

[0037] Figure 3 It is the mass spectrum diagram of NOTA-OJ-1 in Example 1;

[0038] Figure 4 For the experimental example 68 the HPLC purity of the [Ga]-OJ-1 product injection;

[0039] Figure 5 For the MC38 cell uptake in Experimental Example 2 68 [Ga]-OJ-1, 68 [Ga]-DOTATATE, and 68 [Ga]-DOTA-JR11 experimental result diagram;

[0040] Figure 6 For Experimental Example 3 68 [Ga]-OJ-1 and 68 [Ga]-DOTATATE,68 Results of the comparative imaging experiment of the Ga-DOTA-JR11 probe. Among them, Figure 6 A is 68 PET imaging of mice in the Ga-OJ-1 group, Figure 6 B is 68 PET imaging of mice in the Ga-DOTA-TATE group, Figure 6 C is 68 PET imaging of mice in the Ga-DOTA-JR11 group. Specific implementation mode

[0041] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0042] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement made to the methods, steps or conditions of the present invention belongs to the scope of the present invention.

[0043] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the examples are all commercially available.

[0044] Example 1

[0045] Preparation of a PET probe conjugated with an SSTR agonist and inhibitor ( 68 Ga-OJ-1)

[0046] 1. First, synthesize Fragment 1 polypeptide

[0047] Synthesized by the standard Fmoc chemistry method:

[0048] 1) Resin preparation: At 20 °C, CTC resin (0.5 mmol, 1.00 eq, Sub 0.5 mmol / g), Fmoc threonyl (1.0 eq) and DIEA (3.0 eq) (3.0 eq) 2 were added to DCM (20 mL) and stirred for 2 hours. Then methanol (0.5 mL) was added to the resin and stirring was continued for 30 min. Then the mixture was filtered to obtain the resin.

[0049] 2) Deprotection: Added to 20% piperidine DMF solution (30.0 mL), and stirring was continued for 30 min. The resin was washed with DMF (30.0 mL * 5), and then the resin was filtered to obtain.

[0050] 3) Coupling: Add a solution of Fmoc-Cyc(Trt)-OH (3.0 mL), HOAt (3.0 eq), and DIC (3.0 mL) in DMF (15.0 mL) to the resin and stir at 20 °C for 60 min. Then wash the resin with DMF (30.0 mL * 5).

[0051] 4) Repeat the above steps 2 - 3 for the coupling steps of amino acids 3 - 9 in Table 1: The required materials and coupling reagents are shown in Table 1:

[0052] Table 1 Materials and Reagents Required for the Synthesis of Fragmengt 1 Polypeptide

[0053] # Material Coupling reagent 1 Fmoc-Threonol(1.0eq) DIEA(3.0eq) 2 Fmoc-Cys(Mmt)-OH(3.0eq) HOAt(3.0eq)and DIC(3.0eq) 3 Fmoc-Thr(tBu)-OH(3.0eq) HOAt(3.0eq)and DIC(3.0eq) 4 Fmoc-Lys(Boc)-OH(3.0eq) HOAt(3.0eq)and DIC(3.0eq) 5 Fmoc-Trp(Boc)-OH(3.0eq) HOAt(3.0eq)and DIC(3.0eq) 6 Fmoc-Phe-OH(3.0eq) HOAt(3.0eq)and DIC(3.0eq) 7 Fmoc-Cys(Mmt)-OH(3.0eq) HOAt(3.0eq)and DIC(3.0eq) 8 Fmoc-Phe-OH(3.0eq) HOAt(3.0eq)and DIC(3.0eq) 9 Succinic Anhydride(3.0eq) DIEA(4.0eq)

[0054] 5) Wash the resin with methanol (100 mL * 3) and dry it under vacuum. Then treat the peptide resin (2.8 g) with 30 mL of cleavage solution (5% TFA / 5% Tis / 95% DCM) for 60 min, repeating 2 times.

[0055] 6) Collect the filtrate, concentrate it under reduced pressure to form a residue. Precipitate the peptide with cold isopropyl ether, centrifuge (3000 rpm, 2 min), wash it 2 times with isopropyl ether, and dry it under vacuum for 2 hours. The content of the crude linear peptide is 0.9 g.

[0056] 7) Add a solution of I2 in methanol (0.1 M) to a solution of the crude peptide (0.9 g) in DMF (180 mL) at 26 °C until the yellow color persists.

[0057] 8) After 2 min, add sodium thiosulfate (0.1 M in water) dropwise until the yellow color disappears. Concentrate the reaction mixture under reduced pressure to obtain a residue.

[0058] 9) Purify by prep-HPLC (A: 0.075% TFA in water, B: ACN) to obtain Fragment 1 (200 mg, 123.82 μmol), as a white solid.

[0059] 2. Synthesis of Polypeptide:

[0060] 1) Resin Preparation: Add Rink amide MBHA resin (0.1 mmol, 1.00 eq, Sub 0.5 mmol / g) to DMF and stir at 20 °C for 2 hours. Then filter the mixture to obtain the resin.

[0061] 2) Deprotection: Add a solution of 20% piperidine in DMF (10.0 mL) to the resin, continue stirring under N2 protection for 30 min. Wash the resin with DMF (10.0 mL * 5), and then filter to obtain the resin.

[0062] 3) Coupling: Add a solution of Fmoc-Tyr(tBu)-OH (3.0 eq), HOAt (3.0 eq), and DIC (3.0 eq) in DMF (5.0 mL) to the resin and stir for 60 min. Then wash the resin with DMF (10.0 mL * 5).

[0063] 4) Repeat the above steps 2 - 3 for the coupling steps of amino acids 2 - 12 in Table 2.

[0064] Table 2 Materials required for the synthesis of NOTA - OJ - 1

[0065]

[0066]

[0067] 3. Synthesis 68 Ga - OJ - 1

[0068] In this example, the chelating group NOTA, the targeting molecule, and the linker PEG3 are combined into a precursor compound (NOTA - OJ - 1) as shown in Formula 8 below:

[0069]

[0070] The synthetic route of NOTA - OJ - 1 is as Figure 1 shown.

[0071] The structure of the linker PEG3 is as shown in Formula 5 below:

[0072]

[0073] Among them, n = 3 in Formula 5;

[0074] The radioisotope is 68 Ga.

[0075] The HPLC chromatogram of the chemical purity of the precursor compound NOTA - OJ - 1 is as Figure 2 shown; the mass spectrum is as Figure 3 shown.

[0076] Radioactive labeling method of the precursor compound NOTA - OJ - 1:

[0077] 1. Add 1 mL of 0.25 M aqueous NaOAc to an EP tube containing 40 μg of the precursor compound NOTA - OJ - 1 and mix well;

[0078] 2. Transfer the NOTA - OJ - 1 precursor solution to a 10 mL reaction tube;

[0079] 3. Use 4 mL of 0.05 M HCl to 68Ga was eluted into the reaction tube with a radioactivity of 35 mCi;

[0080] 4. React in the reaction tube at 90 °C for 10 min;

[0081] 5. After cooling, the system was subjected to solid-phase extraction through a C18 column;

[0082] 6. The radioactive product 68 Ga-OJ-1 passed through a sterile membrane to obtain an injectable product solution.

[0083] Experimental Example 1

[0084] Detection 68 HPLC purity of Ga-OJ-1 product injection

[0085] HPLC purity analysis: Mobile phase A was distilled water containing 0.1% TFA, mobile phase B was acetonitrile containing 0.1% TFA, and the chromatographic column was ZORBAX SB-C18. The elution method was gradient elution (0 - 2 min: 5% acetonitrile; 2 - 14 min: 90% acetonitrile), 68 The HPLC results of the radiochemical purity of Ga-OJ-1 are as Figure 4 shown, and it can be seen from Figure 4 that the peak emergence time of the product 68 Ga-OJ-1 was about 10.5 min, and the purity was greater than 95%.

[0086] Experimental Example 2

[0087] Investigate 68 Cell uptake of Ga-OJ-1

[0088] MC38 transfected SSTR2 cells were evenly spread in a 24-well plate, with 2×10 5 cells per well. Cultured in an incubator for 24 hours to confirm whether monolayer adherent cells were formed. The next day, the culture medium in each well was discarded. Every four wells were grouped, and a solution containing the radiopharmaceutical 68 Ga-OJ-1 was added to each well and incubated in a cell culture incubator. At the corresponding times (30, 60, 120 min) respectively, the culture medium was discarded, and after digestion, its radioactivity was counted.

[0089] Comparative Example 1

[0090] Cell uptake of SSTR agonist or inhibitor

[0091] The method was the same as that in Experimental Example 2, and the probe 68 Ga-OJ-1 was replaced with 68 Ga-DOTA-TATE or 68 Ga-DOTA-JR11.

[0092] The results are as follows Figure 5 shown: At 30, 60, and 120 min, the uptake rates of the MC38 cells for 68 the Ga-OJ-1 probe were significantly higher than those for 68 Ga-DOTA-TATE or 68 Ga-DOTA-JR11, and the uptake rate increased from 2.2 ID% / g to 2.6 ID% / g within 30 - 60 min, and there was no obvious downward trend at 120 min, indicating that the PET probe of the SSTR agonist-coupled inhibitor prepared in the present invention optimized the low internalization rate of the SSTR agonist or inhibitor.

[0093] Experimental Example 3

[0094] Detection 68 In vivo imaging of the Ga-OJ-1 probe

[0095] 1. Purchase 30 nude mice, construct a subcutaneous MC38 transfected with SSTR2 mouse metastatic tumor model, and use it as a model mouse when the tumor size is about 0.8 cm.

[0096] 2. Molecular probe comparative imaging at the animal level: Randomly select 10 model mice as the 68 Ga-OJ-1 group. After intravenous injection of the 68 Ga-OJ- molecular probe (0.2 - 0.3 mCi) into the tail veins of the model mice, perform PET scanning 45 min later. The results are as Figure 2 shown, and the tumor location of the mice can be clearly observed, indicating that the probe has good targeting to the tumor, 68 and the tumor uptake of the Ga-OJ-1 group is as high as 12.33 ID% / g.

[0097] Comparative Example 2

[0098] In vivo imaging of the SSTR agonist or inhibitor probe

[0099] Randomly divide the other 20 model mice into 2 groups, 68 the Ga-DOTA-TATE and 68 Ga-DOTA-JR11 groups, with 10 mice in each group. The operation method is the same as that in Experimental Example 3, only changing the molecular probe to 68 Ga-DOTA-TATE or 68 Ga-DOTA-JR11 probe.

[0100] The imaging results are as Figure 6 shown. The tumor locations of the mice can be clearly observed in all three groups, indicating that the probe has good targeting to the tumor. Among them, 68 the imaging effect of Ga-OJ-1 (tumor uptake 12.33 ID% / g) is better than that of 68Ga-DOTATATE (tumor uptake 6.8 ID% / g) and 68 Ga-DOTA-JR11 (tumor uptake 7.33 ID% / g) group. It is shown that the imaging effect of the PET probe conjugated with SSTR agonist and inhibitor is better than that of SSTR agonist or inhibitor.

Claims

1. A PET probe conjugated with an SSTR agonist and an inhibitor, characterized in that, It includes a targeting molecule, a linking group, a radioisotope and a chelating group. The linking group connects the targeting molecule and the radioactive chelating group. The targeting molecule is an SSTR agonist and an SSTR inhibitor linked by lysine; The structural formula of the PET molecular probe is shown as Formula 9 below: ； Formula 9; In Formula 9, X is a radioisotope.

2. The PET probe conjugated with an SSTR agonist and an inhibitor according to claim 1, wherein The chelating group can also be one of DOTA or HYINC.

3. The PET probe conjugated with an SSTR agonist and an inhibitor according to claim 1, wherein The radioactive isotope is 68 Ga,[[]] 177 Lu, AI 18 F,[[]] 225 Ac,[[]] 99 any one of mTc.

4. A conjugate of an SSTR agonist and an inhibitor as described in any one of claims 1 to 3 Preparation method of a PET probe, characterized in that, comprises the following steps: S1. Mix the probe precursor and the aqueous NaOAc solution evenly and transfer them to a reaction tube; S2. Wash the radioisotope into the reaction tube with HCl and react for 10 min under the condition of 90 °C; S3. After cooling the solution in the reaction tube in step S2, perform solid-phase extraction through a C18 column, and elute the product into a product bottle equipped with a filter membrane to obtain a PET probe conjugated with an SSTR agonist and an inhibitor.

5. The preparation method according to claim 4, characterized in that, In step S2, the HCl elution washes the radioisotope until the radioactivity of the eluate is 35 mCi.

6. Use of a PET probe conjugated with an SSTR agonist and an inhibitor as described in any one of claims 1 to 3 or a PET probe conjugated with an SSTR agonist and an inhibitor prepared by the preparation method as described in any one of claims 4 to 5 in the preparation of a PET imaging agent for targeting neuroendocrine tumors.

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