ACE1 receptor targeted nuclide polypeptide probe as well as preparation method and application thereof
By developing ACE1 receptor-targeted nuclide polypeptide probes, the problem of lack of effective targeted treatment and diagnosis of TNBC was solved, and efficient imaging and individualized tumor treatment were achieved.
Patent Information
- Application Number
- CN202411781216.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-05
- Publication Date
- 2025-04-11
AI Technical Summary
Existing breast cancer treatment methods lack effective targeted treatments for triple-negative breast cancer (TNBC), and there are difficulties in diagnosis and monitoring.
A probe for targeting ACE1 receptor nuclide polypeptide was developed, which was prepared by amidation reaction and labeled with a variety of radionuclides (such as 68Ga, 111In, 177Lu, etc.) for targeting TNBC tumors.
This probe can significantly improve the imaging effect of TNBC tumors, achieve accurate tumor positioning and efficacy monitoring, and provide the possibility for individualized targeted therapy.
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Figure CN119804850A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of radiopharmaceuticals and nuclear medicine, and more specifically, relates to an ACE1 receptor-targeted radionuclide polypeptide probe, a preparation method of the ACE1 receptor-targeted radionuclide polypeptide probe, and multiple uses of the probe in nuclear medicine imaging and treatment. Background Art
[0002] Angiotensin-converting enzyme 1 (ACE, also known as ACE1) belongs to the type I membrane-anchored dipeptidyl carboxypeptidase family. It controls blood pressure through the renin-angiotensin system (RAS). ACE1 converts angiotensin I into active vasoconstrictor, which indirectly causes vasoconstriction and increases blood pressure. ACE1 is also involved in the degradation of bradykinin and other peptides. It was first discovered in 1956. Researchers obtained an enzyme in a semi-purified form from horse plasma through ammonium sulfate fractionation and isoelectric precipitation. In animal experiments, the enzyme can rapidly convert angiotensin I into angiotensin II, which is also one of the important physiological functions of ACE1. However, it was not until 2003 that the crystal structure of ACE1 was published in Nature. The three-dimensional structural analysis of ACE1 showed that ACE is not very similar to carboxypeptidase A, but is similar to neurolytic enzymes and pyrococcal carboxypeptidase-zinc metallopeptidase. There is evidence that ACE1 and other RAS system components are abnormally expressed in some cancer tissues and various tumor blood vessels, including human glioblastoma, epithelial ovarian cancer, melanoma, gastric cancer, oral squamous cell carcinoma, etc.
[0003] Triple-negative breast cancer (TNBC) is a heterogeneous subtype of breast tumors, accounting for 20% of all invasive breast cancers. Its immunohistochemical characteristics are negative for three receptors: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Characteristics of TNBC include younger age of onset, unique molecular features, larger average tumor size, invasiveness, higher tumor grade, obvious metastatic pattern, and lack of targeted therapy. TNBC is the most challenging breast cancer subtype due to the inconsistency of current treatment methods, poor prognosis, high recurrence rate, and frequent metastasis to the viscera and central nervous system. Current treatments include surgical resection, chemotherapy, and radiotherapy. Researchers have found that ACE1 can be used as a new target for the diagnosis and treatment of TNBC. Nuclear medicine molecular imaging probes have the unique advantages of real-time and non-invasive detection of tumors. Therefore, imaging of ACE1 is expected to perform real-time differential diagnosis, staging, precise localization of lesions, and efficacy monitoring of TNBC lesions. Summary of the invention
[0004] The purpose of the present invention is to provide an ACE1 receptor-targeted nuclide polypeptide probe and its preparation method and application. The probe is a radiolabeled BPP, and the labeled compound has good affinity and selectivity for tumors. The labeling method of the present invention is simple, easy to operate, short in time, high in labeling rate, and stable in labeling. The probe of the present invention is expected to be used for differential diagnosis, staging, precise localization of lesions, and efficacy monitoring of triple-negative breast cancer malignancies.
[0005] In order to achieve the above object, the present invention provides an ACE1 receptor targeting nuclide polypeptide probe, which is BPP or BFC-BPP labeled with a radionuclide, wherein the BFC is a bifunctional chelating agent for radionuclide labeling; the BPP has a structure shown in Formula I:
[0006]
[0007] In the ACE1 receptor targeting nuclide polypeptide probe of the present invention, the bifunctional chelator can be any conventional bifunctional chelator used for radionuclide labeling, preferably DOTA, NOTA, NODGA 、 NODA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, Df, DFO, TACN, NO2A / NOTAM, CB-DO2A, Cyclen, NOTA-AA, DO3A or DO3AP.
[0008] In the ACE1 receptor targeting radionuclide polypeptide probe of the present invention, the radionuclide can be either a diagnostic radionuclide or a therapeutic radionuclide. The diagnostic radionuclide is preferably 68 Ga, 18 F. 64 Cu, 124 I. 111 In and 89 At least one of Zr; the therapeutic radionuclide is preferably 90 Y. 177 Lu, 225 Ac, 124 / 125 I and 213 At least one of Bi.
[0009] According to a specific embodiment of the present invention, the probe is 68 Ga, 111 In, 177 Lu-labeled DOTA-BPP, or 18 F. 64 Cu-labeled NOTA-BPP / NODGA-BPP, or 124 / 125I-labeled BPP. Specifically preferably, the probe is 68 Ga-DOTA-BPP, 111 In-DOTA-BPP, 177 Lu-DOTA-BPP, 64 Cu-NOTA-BPP, or 124 / 125 I-BPP.
[0010] The second aspect of the present invention provides a method for preparing the above-mentioned ACE1 receptor targeting nuclide polypeptide probe, comprising the following steps:
[0011] The labeled precursor BFC-BPP or BPP, the radionuclide eluent and the buffer solution are mixed to carry out radionuclide labeling to obtain BPP or BFC-BPP labeled with radionuclide. When the labeling rate is insufficient, separation and purification can be optionally carried out, for example, separation and purification using Sep-pak C18 Column. The radiochemical purity of the purified probe can be greater than 95%, preferably greater than 98%.
[0012] The labeling precursor BFC-BPP is prepared by amidation reaction of BFC and BPP.
[0013] The precursor BPP in the present invention can be synthesized or commercially available. According to a specific embodiment of the present invention, the precursor BPP is synthesized on a CS Bio CS336 instrument using a Fmoc solid phase extraction method for polypeptides.
[0014] The radionuclide eluent (eluent) can be prepared by conventional methods in the art, for example, 68 The eluent of Ga is the eluent obtained by eluting the germanium-gallium generator with HCL, and the present invention has no particular limitation on this.
[0015] According to the present invention, preferably, the buffer is NaAc buffer or PBS buffer.
[0016] Depending on the type of radionuclide to be labeled, the labeling of the radionuclide includes the following steps:
[0017] (a) 68 Ga labeling of peptides:
[0018] To the labeled precursor DOTA-BPP, 0.8-1.2 M NaAc buffer and 7.4-740 MBq 68 The GaCl3 eluent was reacted at 95-100°C for 15-25 min. The product obtained by the reaction was optionally separated and purified using a Sep-pak C18 Column to obtain 68 Ga-DOTA-BPP; preferably obtained 68The radiochemical purity of Ga-DOTA-BPP is greater than 95%;
[0019] (b) 111 In labeling of peptides:
[0020] Add 0.8-1.2M NaAc buffer and 20-300MBq of DOTA-BPP to the labeled precursor. 111 InCl3 eluent, react at 80-90°C for 15-25 min, and the product obtained by the reaction is optionally separated and purified using Sep-pak C18 Column to obtain 111 In-DOTA-BPP; preferably obtained 111 The radiochemical purity of In-DOTA-BPP is greater than 98%;
[0021] (c) 177 Lu labeling of peptides:
[0022] Labeled precursor DOTA-BPP is added to 0.8-1.2M NaAc buffer, 0.04-0.06M HCl, 35-400MBq 177 LuCl3 eluent, react at 95-100℃ for 15-25min, and the reaction product is optionally separated and purified using Sep-pak C18 Column to obtain 177 Lu-DOTA-BPP; preferably obtained 177 The radiochemical purity of Lu-DOTA-BPP is greater than 95%;
[0023] (d) 64 Cu labeling of peptides:
[0024] Labeled precursor DOTA-BPP is added to 0.08-0.12M NaAc buffer, 150-200MBq 64 The eluent is CuCl2, and the reaction is carried out at 95-100°C for 15-25 min. The product obtained by the reaction is optionally separated and purified using a Sep-pak C18 Column to obtain 64 Cu-DOTA-BPP; preferably obtained 64 The radiochemical purity of Cu-DOTA-BPP is greater than 95%;
[0025] (e)Al 18 F labeling of polypeptides:
[0026] Add 0.4-0.6M KHP buffer, 1-2mM AlCl3, 150-200MBq Na 18The eluent F was reacted at 105-115°C for 15-25 min. The product obtained by the reaction was optionally separated and purified using a Sep-pak C18 Column to obtain Al 18 F-NOTA-BPP; Al 18 The radiochemical purity of F-NOTA-BPP was greater than 95%.
[0027] The probe prepared by the above method can be tested by the following quality control method: the radioactivity purity of the probe is determined by HPLC, and the HPLC analysis conditions are: the chromatographic analysis column is a C-18 column, 4.6×250 mm, the mobile phase A is a 0.1% by mass trifluoroacetic acid TFA aqueous solution, the mobile phase B is a 0.1% by mass trifluoroacetic acid TFA acetonitrile solution, the flow rate is 1 mL / min; the gradient elution condition is 0-10 min, the mobile phase B increases from 20% to 65%, and the detection wavelength is 280 nm. The radioactivity detection uses a HPLC-specific radioactivity detector.
[0028] The ACE1 receptor-targeted nuclide polypeptide probe of the present invention can be used to prepare tumor diagnostic reagents and / or tumor therapeutic drugs; the tumor is, for example, a triple-negative breast cancer malignant tumor. The probe has good targeting to the tumor, can improve the effect of tumor imaging, provides a visualization tool for tumor differential diagnosis, tumor staging, precise lesion positioning and efficacy monitoring, and can realize tumor radio-targeted therapy, and is an integrated diagnosis and treatment imaging agent.
[0029] The present invention innovatively modifies BPP with BFC, and then realizes labeling of multiple radionuclides to obtain corresponding probes. These probes are expected to be used for the diagnosis of malignant tumors with high expression of ACE1 receptors, and are expected to provide early warning for patient screening, efficacy monitoring, drug resistance and / or recurrence and metastasis during treatment, and realize individualized treatment of targeted drugs in the treatment of malignant tumors.
[0030] The probe of the present invention belongs to a labeled compound of a polypeptide, has a small molecular weight, low immunogenicity, good tissue penetration ability, high affinity and selectivity for ACE1, and has good application prospects. The probe preparation process of the present invention is simple, easy to operate, short in time, high in labeling rate, and stable in labeling, which is convenient for further application in clinical, scientific research and drug development.
[0031] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.
[0033] Figure 1Aand Figure 1B They are HPLC and mass spectrometry quality control charts of BPP respectively.
[0034] Figure 2A and Figure 2B They are HPLC and mass spectrometry quality control charts of DOTA-BPP respectively.
[0035] Figure 3 Radio-HPLC determination is shown 68 Ga-DOTA-BPP radioactivity purity results.
[0036] Figure 4 Shown is a normal mouse injected 68 Results of radioactivity uptake in various organs at different time points after Ga-DOTA-BPP.
[0037] Figure 5 Shown is a normal mouse injected 68 Blood pharmacokinetics profile after Ga-DOTA-BPP.
[0038] Figure 6 Shown is a normal mouse injected 68 Micro-PET / CT analysis after Ga-DOTA-BPP.
[0039] Figure 7 shows the injection of MDA-MB-231 tumor-bearing mice 68 Micro-PET / CT dynamic imaging after Ga-DOTA-BPP.
[0040] Figure 8 Shown is the injection of 4T1 tumor-bearing mice 68 Micro-PET / CT dynamic imaging after Ga-DOTA-BPP. DETAILED DESCRIPTION
[0041] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.
[0042] Example 1
[0043] This example is used to illustrate the preparation of the precursor DOTA-BPP.
[0044] BPP was synthesized on a CS Bio CS336 instrument using the Fmoc solid phase extraction peptide method. Weigh 50 mg of DOTA / NOTA and dissolve it in 5 mL of water, and dissolve 9.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in 1 mL of water, and mix the two. Adjust the pH to 5 with 0.1 mol / L NaOH and react for 10 minutes. Move the reaction bottle to an ice bath and continue the reaction for 30 minutes. Weigh 25 mg of BPP and dissolve it in 3 mL of water, add it to the above reaction solution, and then adjust the pH of the reaction solution to 8.5 with 0.1 mol / L NaOH and react overnight. After the reaction is completed, dialyze and freeze-dry to obtain solid DOTA-BPP with the structural formula shown in Formula II. After bottling and sealing, store at -20°C. The HPLC and mass spectrometry quality controls of BPP and DOTA-BPP are as follows: Figure 1A , 1B and Figure 2A , 2B shown.
[0045]
[0046] Example 2
[0047] This example is used to illustrate 68 Labeling of Ga-DOTA-BPP.
[0048] (1) 68 Elution of Ga: Use a syringe to take 4 mL of 0.05 mol / L HCl to elute the germanium-gallium generator at a rate of 1-2 mL / min. Discard the first 1 mL of HCl, collect the remaining 3 mL of eluent, and record the radioactivity.
[0049] (2) Take the precursor DOTA-BPP prepared in Example 1, dissolve it in DMF, and prepare a 2 mg / mL solution. Take 4 μL of the prepared sample in an ampoule bottle, add 3 mL of 92.5 MBq of freshly washed in step (1) 68 Ga, then add 65 μL / mL 1.0 M sodium acetate solution to adjust the pH to 3.5-4.5, gently shake to mix, and place in an incubator at 95°C for 20 min.
[0050] (3) The system was taken out and measured with a 2 mL syringe, and then added to an activated Sep-pak C-18 column. The impurities were washed with 3.0 mL of pure water and discarded. Using a 0.2 μm microporous filter, 1.0 mL of 80% ethanol solution was collected in a sterile vacuum bottle to obtain the product - radioactive 68 Ga-DOTA-BPP.
[0051] HPLC analysis conditions: the chromatographic analysis column is a C-18 column (4.6×250 mm), the injection volume is 10 μL, the mobile phase A is pure water containing 0.1% trifluoroacetic acid TFA by mass, and B is acetonitrile containing 0.1% trifluoroacetic acid TFA by mass, and the flow rate is 1 mL / min; the gradient elution condition is 0-10 min, B increases from 20% to 65%, the detection wavelength is 280 nm, and the radioactivity detection adopts a HPLC-specific radioactivity detector. 68 The radioactivity of Ga-DOTA-BPP was determined by Radio-HPLC. Figure 3 As shown, the purity is over 99%.
[0052] Example 3
[0053] This example is used to illustrate 68 Biodistribution of Ga-DOTA-BPP in normal mice.
[0054] 0.2 mL (1.85 MBq) was injected into the tail vein of mice 68 Ga-DOTA-BPP (prepared in Example 2) was used to kill mice (3 mice in each group) at 5, 30, 60 and 120 min, respectively, and the blood, brain, heart, liver, spleen, lung, kidney, stomach, large intestine, small intestine and muscle were collected and weighed. The radioactivity count was measured with a γ counter, and the uptake of each organ and tissue (%ID / g) was calculated. The experimental results are expressed as SD. 68 The radioactivity uptake of each organ at different time points after Ga-DOTA-BPP was shown in Figure 4 ,Depend on Figure 4 The results show that 68 Ga-DOTA-BPP is mainly metabolized by the kidney.
[0055] Example 4
[0056] This example is used to illustrate 68 Drug metabolism of Ga-DOTA-BPP in mice.
[0057] 0.2 mL (1.85 MBq) was injected into the tail vein of mice (n=5) 68 Ga-DOTA-BPP (prepared in Example 2) was injected and blood was collected at the corresponding time points and weighed. After weighing, the radioactivity count was measured using a γ counter, and the blood uptake (%ID / g) at each time point was calculated. The experimental results were expressed as SD. The pharmacokinetic curve was fitted using Prism software, and the distribution phase and excretion phase half-life of the probe in the blood were obtained. Injection 68 The blood radioactivity uptake at different time points after Ga-DOTA-BPP was Figure 5 ,Depend on Figure 5 The results show that 68Ga-DOTA-BPP is metabolized rapidly.
[0058] Example 5
[0059] This example is used to illustrate the injection 68 PET / CT imaging of normal mice with Ga-DOTA-BPP.
[0060] 200 μL (7.4 MBq) was injected into the mouse tail vein 68 Ga-DOTA-BPP (prepared in Example 2). Imaging was performed 30 min, 60 min, and 120 min after injection, and the region of interest (ROI) of the whole body decay-corrected coronal image obtained by Micro-PET scanning was delineated. The results are shown in Figure 6 As shown, mouse kidneys had a higher uptake, while other normal organs had a lower uptake.
[0061] Example 6
[0062] This example is used to illustrate the injection 68 Micro-PET / CT imaging of MDA-MB-231 tumor-bearing mice with Ga-DOTA-BPP.
[0063] 0.4 mL (30 MBq) was injected into the tail vein of MDA-MB-231 tumor-bearing mice 68 Ga-DOTA-BPP (prepared in Example 2) was subjected to 30-minute dynamic scanning and image reconstruction. The ROI of the whole-body decay-corrected coronal image obtained by Micro-PET scanning was delineated. The results are as follows: Figure 7 As shown, the tumor site of the mouse is clearly visible in the figure.
[0064] Example 7
[0065] This example is used to illustrate the injection 68 Micro-PET / CT imaging of 4T1 tumor-bearing mice with Ga-DOTA-BPP.
[0066] 0.4 mL (30 MBq) was injected into the tail vein of 4T1 tumor-bearing mice 68 Ga-DOTA-BPP (prepared in Example 2) was subjected to 30-minute dynamic scanning and image reconstruction. The ROI of the whole-body decay-corrected coronal image obtained by Micro-PET scanning was delineated. The results are as follows: Figure 8 As shown, the tumor site of the mouse is clearly visible in the figure.
[0067] It can be seen from the above studies on two breast cancer cell lines that the ACE1 receptor-targeted radionuclide polypeptide probe of the present invention has the specificity of targeting breast cancer, has a good tumor imaging effect, and has a fast metabolic rate. It is expected to be used for the differential diagnosis, staging, precise positioning of lesions and efficacy monitoring of triple-negative breast cancer malignancies.
[0068] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.
Claims
1. An ACE1 receptor targeting radionuclide polypeptide probe, characterized in that: The probe is BPP or BFC-BPP labeled with a radionuclide, wherein the BFC is a bifunctional chelating agent for radionuclide labeling; the BPP has a structure shown in Formula I:
2. The ACE1 receptor targeting nuclide polypeptide probe according to claim 1, wherein: The bifunctional chelating agent is DOTA, NOTA, NODGA 、 NODA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, Df, DFO, TACN, NO2A / NOTAM, CB-DO2A, Cyclen, NOTA-AA, DO3A or DO3AP.
3. The ACE1 receptor targeting nuclide polypeptide probe according to claim 1, wherein: The radionuclide is a diagnostic radionuclide, and the diagnostic radionuclide is preferably 68 Ga, 18 F. 64 Cu, 124 I. 111 In and 89 At least one of Zr; or The radionuclide is a therapeutic radionuclide, and the therapeutic radionuclide is preferably 90 Y. 177 Lu, 225 Ac, 124 / 125 I and 213 At least one of Bi.
4. The ACE1 receptor targeting nuclide polypeptide probe according to claim 1, wherein: The probe is 68 Ga, 111 In, 177 Lu-labeled DOTA-BPP, or 18 F. 64 Cu-labeled NOTA-BPP / NODGA-BPP, or 124 / 125 I-labeled BPP.
5. The ACE1 receptor targeting nuclide polypeptide probe according to claim 4, wherein: The probe is 68 Ga-DOTA-BPP, 111 In-DOTA-BPP, 177 Lu-DOTA-BPP, 64 Cu-NOTA-BPP,Al 18 F-NOTA-BPP or 124 / 125 I-BPP.
6. The method for preparing the ACE1 receptor targeting nuclide polypeptide probe according to any one of claims 1 to 5, comprising the following steps: Mixing the labeled precursor BFC-BPP or BPP, the radionuclide eluent and the buffer, labeling the radionuclide to obtain BPP or BFC-BPP labeled with the radionuclide, and optionally separating and purifying it; The labeling precursor BFC-BPP is prepared by amidation reaction of BFC and BPP.
7. The preparation method according to claim 6, wherein: The buffer is NaAc buffer or PBS buffer; The labeling of the radionuclide comprises the following steps: (a) 68 Ga labeling of peptides: To the labeled precursor DOTA-BPP, 0.8-1.2 M NaAc buffer and 7.4-740 MBq 68 The GaCl3 eluent was reacted at 95-100°C for 15-25 min. The product obtained by the reaction was optionally separated and purified using a Sep-pak C18 Column to obtain 68 Ga-DOTA-BPP; preferably obtained 68 The radiochemical purity of Ga-DOTA-BPP is greater than 95%; (b) 111 In labeling of peptides: Add 0.8-1.2M NaAc buffer, 0.04-0.06M HCl, 20-300MBq of 111 InCl3 eluent, react at 80-90°C for 15-25 min, and the reaction product is optionally separated and purified using Sep-pak C18 Column to obtain 111 In-DOTA-BPP; preferably obtained 111 The radiochemical purity of In-DOTA-BPP is greater than 98%; (c) 177 Lu labeling of peptides: Labeled precursor DOTA-BPP is added to 0.8-1.2M NaAc buffer, 0.04-0.06M HCl, 35-400MBq 177 LuCl3 eluent, react at 95-100℃ for 15-25min, and the reaction product is optionally separated and purified using Sep-pak C18 Column to obtain 177 Lu-DOTA-BPP; preferably obtained 177 The radiochemical purity of Lu-DOTA-BPP is greater than 95%; (d) 64 Cu labeling of peptides: Labeled precursor DOTA-BPP is added to 0.08-0.12M NaAc buffer, 150-200MBq 64 The eluent is CuCl2, and the reaction is carried out at 95-100°C for 15-25 min. The product obtained by the reaction is optionally separated and purified using a Sep-pak C18 Column to obtain 64 Cu-DOTA-BPP; preferably obtained 64 The radiochemical purity of Cu-DOTA-BPP is greater than 95%; (e)Al 18 F labeling of polypeptides: Add 0.4-0.6M KHP buffer, 1-2mM AlCl3, 150-200MBq Na 18 The eluent F was reacted at 105-115°C for 15-25 min. The product obtained by the reaction was optionally separated and purified using a Sep-pak C18 Column to obtain Al 18 F-NOTA-BPP; Al 18 The radiochemical purity of F-NOTA-BPP was greater than 95%.
8. Use of the ACE1 receptor targeting nuclide polypeptide probe according to any one of claims 1 to 5 in the preparation of an imaging agent targeting ACE1.
9. Use of the ACE1 receptor targeting nuclide polypeptide probe according to any one of claims 1 to 5 in the preparation of tumor diagnostic reagents and / or tumor therapeutic drugs.
10. The use according to claim 9, wherein: The tumor diagnosis includes tumor staging, lesion localization, and efficacy monitoring; the tumor is triple-negative breast cancer.