DUAL-TARGET IMAGING MOLECULAR PROBE, PREPARATION METHOD THEREFOR, AND APPLICATIONS THEREOF

Abstract

The present invention discloses a targeting polypeptide compound having dual targets, comprising a TATE cyclic peptide structure, an RGD cyclic peptide structure and a NOTA chelating group, wherein the TATE cyclic peptide structure, the RGD cyclic peptide structure and the NOTA chelating group are respectively linked by a PEG segment having a polymerization degree of 1 to 5 or directly linked to a same glutamic acid; the structure of the polypeptide compound can be represented as NOTA-PEGn-Glu{PEGm-TATE}-PEGP-RGD, where m, n and p are an integer from 0 to 5 respectively. The present invention further discloses a TATE-RGD dual-target radioactive molecular probe based on the polypeptide compound. The TATE-RGD dual-target polypeptide drug of the present invention may simultaneously bind to SSTR, integrin v3, has higher receptor binding affinity and uptake, more excellent non-target tissue clearance rate, and better in vivo and in vitro stability.

Claims

1. A targeting polypeptide compound having dual targets, comprising a TATE cyclic peptide structure, an RGD cyclic peptide structure and a NOTA chelating group, wherein the TATE cyclic peptide structure, the RGD cyclic peptide structure and the NOTA chelating group are respectively linked by a PEG segment having a polymerization degree of 1 to 5 or directly linked to a same glutamic acid molecule; the structure of the polypeptide compound can be simplified as NOTA-PEG.sub.n-Glu{PEG.sub.m-TATE}-PEG.sub.P-RGD, where m, n and p are an integer from 0 to 5 respectively.

2. The polypeptide compound of claim 1, wherein the TATE cyclic peptide structure, the NOTA chelating group and the RGD cyclic peptide structure are linked to a same glutamic acid molecule by a PEG segment having a degree of polymerization of 2 to 5 respectively.

3. The polypeptide compound of claim 2, wherein the TATE cyclic peptide structure, the NOTA chelating group and the RGD cyclic peptide structure are linked to the same glutamic acid molecule by a PEG.sub.4 segment respectively.

4. The polypeptide compound of claim 1, wherein the TATE cyclic peptide structure and the RGD cyclic peptide structure are linked to two carboxyl terminals of the same glutamic acid molecule by a PEG4 molecular segment respectively to form a stable amide bond; the NOTA chelating group is linked to the amino terminal of the same glutamic acid molecule by a PEG.sub.4 segment; the polypeptide compound is designated as NOTA-3PEG.sub.4-TATE-RGD, and the specific structure thereof is as shown in the formula (I) below: ##STR00003##

5. A TATE-RGD dual-target radioactive molecular probe, which is a radionuclide-labeled polypeptide complex, wherein the polypeptide complex takes a targeting polypeptide compound having dual targets as a ligand; the targeting polypeptide compound comprises a TATE cyclic peptide structure, an RGD cyclic peptide structure and a NOTA chelating group, wherein the TATE cyclic peptide structure, the RGD cyclic peptide structure and the NOTA chelating group are respectively linked by a PEG segment having a polymerization degree of 1 to 5 or directly linked to a same glutamic acid molecule; the structure of the polypeptide compound can be simplified as NOTA-PEGn-Glu{PEGm-TATE}-PEGP-RGD, where m, n and p are an integer from 0 to 5 respectively.

6. The TATE-RGD dual-target radioactive molecular probe of claim 5, wherein the radionuclide is selected from any one of .sup.68Ga, .sup.64Cu, .sup.18F, .sup.89Zr or .sup.177Lu; preferably from any one of 68Ga, 64Cu Or 18F; and most preferably 68Ga.

7. The TATE-RGD dual-target radioactive molecular probe of claim 5, wherein it is a radionuclide .sup.68Ga-labeled polypeptide complex, the polypeptide complex takes the targeting polypeptide compound as a ligand, and the dual-target radioactive molecular probe is simply represented as .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD.

8. A method of preparing the targeting polypeptide compound having dual targets of claim 1, comprising the following steps: a) mixing a protected glutamic acid with polypeptide PEG.sub.n-TATE in a molar ratio of 1-10:1-10, and carrying out an amino condensation reaction to obtain a first product linked by PEG.sub.n segment of TATE polypeptide and the protected glutamic acid, where n is an integer from 0 to 5; b) deprotecting the group Fmoc of the first product obtained in the step a) under the piperidine condition to obtain a second product, simply represented as Glu-PEG.sub.n-TATE, wherein n is an integer from 0 to 5; c) reacting the second product obtained in step b) with NOTA-PEG.sub.m-NHS under DIPEA condition to obtain a third product of Boc-protected glutamic acid that is linked to the NOTA group and the TATE peptide by a PEG.sub.m segment and a PEG.sub.n segment respectively, wherein n and m are integers from 0 to 5 respectively; d) deprotecting the group Fmoc of the third product obtained in the step c) under the TFA conditions to obtain a fourth product of glutamic acid that is linked to the NOTA group and the TATE peptide by a PEG m segment and a PEG.sub.n segment respectively, simply represented as NOTA-PEG.sub.m-Glu(PEG.sub.n-TATE), where n and m are integers from 0 to 5 respectively; e) reacting the fourth product obtained in step d) with the polypeptide PEG.sub.p-RGD under the DIPEA condition, where p is an integer of 0 to 5, finally obtaining a tumor targeting polypeptide compound having dual targets NOTA-PEG.sub.n-Glu{PEG.sub.m-TATE}-PEG.sub.P-RGD.

9. The method of preparing dual- target radioactive molecular probe of claim 7, comprising the following steps: dissolving the NOTA-3PEG.sub.4-TATE-RGD of claim 4 in deionized water; rinsing germanium-gallium (.sup.68Ge/.sup.68Ga) generator into a EP tube with 5 mL of 0.1 mol/L high-purity hydrochloric acid solution, collecting 1 mL of the solution containing the highest content of radioactivity, adding 93 L of 1.25 mol/L sodium acetate to adjust the pH of the mixture to 4-4.5; adding 20 g of the precursor to the mixture and mix well, heat to 100 C. for 10 min; after completion of the reaction, cooling the reaction solution to room temperature, then adding 4 mL of sterile water for injection, filtering the solution to a sterile product bottle through a sterile filter membrane (0.22 m, 13 mm).

10. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 shows .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD HPLC pattern.

[0035] FIG. 2 shows a microPET imaging result of small cell lung cancer H69 model of nude mice. From the left to the right, the imaging results of injection with .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD (Control), .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD+RGD (Block+RGD), .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD+TATE (Block+TATE) and .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD+RGD+TATE (Block+TATE+RGD) from tail veins, respectively.

[0036] FIG. 3 shows a microPET imaging result of non-small cell lung cancer A549 model of nude mice. From the left to the right, the imaging results of injection with .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD (Control), .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD+RGD (Block+RGD),.sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD+TATE(Block+TATE) and .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD+RGD+TATE (Block+TATE+RGD) from tail veins, respectively.

[0037] FIG. 4 shows the radioactive uptake % ID/g of each organ at different time points after injection of .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD in normal mice.

[0038] FIG. 5 shows comparison of imaging results of .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD of the present invention with the existing .sup.68Ga-NOTA-RGD probe in patients with non-small cell lung cancer, wherein the left figure is the imaging result of .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD of the invention, and the right figure is the imaging result of existing .sup.68Ga-NOTA-RGD, and the arrow points to the lesion.

[0039] FIG. 6 shows comparison of imaging results of .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD with the existing .sup.68Ga-NOTA-RGD probe in patients with small cell lung cancer, wherein the left figure is the imaging result of .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD of the invention, and the right figure is the imaging result of existing .sup.68Ga-NOTA-RGD, and the arrow points to the lesion.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] In order to explain the technical solutions of the present invention more clearly, the present invention will be further illustrated and described below with reference to the accompanying drawings and embodiments, but the technical solutions of the present invention are not limited to the specific embodiments and some examples described below. The chemical products and reagents used in the following embodiments herein are existing or commercially available products.

[0041] Embodiment 1

[0042] A targeting polypeptide compound having dual targets, comprising a TATE cyclic peptide structure, an RGD cyclic peptide structure and a NOTA chelating group, formed by linking the same glutamic acid molecule by the TATE cyclic peptide structure, the NOTA chelating group and the RGD cyclic peptide structure respectively, wherein the TATE cyclic peptide structure and the RGD cyclic peptide structure are linked to the two carboxyl terminals of the glutamic acid molecule through a PEG4 molecular segment respectively to form a stable amide bond; the NOTA chelating group is linked to the amino terminal of the glutamic acid molecule by a PEG4 molecule; the obtained polypeptide compound is designated as NOTA-3PEG.sub.4-TATE-RGD, and the specific structure thereof is as shown in the formula (I) below:

##STR00002##

[0043] A method of preparing the NOTA-3PEG.sub.4-TATE-RGD comprises the following steps:

[0044] 1) Synthesis of Compound Glu-PEG.sub.4-TATE:

[0045] 9.5 mg of Fmoc-Glu(Boc)-OH (Fmoc and Boc protected glutamic acid), 25 L of diisopropylethylamine (DIPEA) and 5 L of diethylphosphonium chloride (DECP) were added to a 20 mL glass vial containing 27.5 mg PEG.sub.4-TATE (a commercially available tumor targeting peptide dissolved in 2.6 mL dimethylformamide (DMF)), after mixed and dissolved, the mixed solution was stirred at room temperature for 2 h, then subjected to LC-MS analysis. Results showed that the Fmoc and Boc protected amino condensation polypeptide product was obtained, recorded as intermediate compound I; then 0.6 mL piperidine was added to the intermediate compound I solution and stirred at room temperature for 1 h, to remove the Fmoc protecting group of intermediate compound I and give the product Glu-PEG.sub.4-TATE. After purification by HPLC and lyophilization, about 17.5 mg of pure compound was obtained, with a yield of 68%.

[0046] 2) Synthesis of Compound NOTA-2PEG.sub.4-TATE:

[0047] 17.5 mg of the compound Glu-PEG4-TATE obtained in step 1) dissolved in 2 mL of dimethyl sulfoxide (DMSO), 20 L DIPEA and 24.0 mg NOTA-PEG4-NHS (1,4,7-triazacyclononane-N,N,N-triacetic acid activated ester, 2 equivalents) were mixed and dissolved. The mixture was stirred at room temperature for 20 min and the reaction process was monitored by HPLC. After the compound Glu-PEG.sub.4-TATE in the above step was consumed, the generation of Boc-protected NOTA-PEG.sub.4-TATE was detected by LC-MS. Then 0.1 mL of trifluoroacetic acid (TFA) was added to remove the protecting group Boc to obtain a target compound formed by linking glutamic acid to the PEG4 segment respectively, simply represented as NOTA-2PEG.sub.4-TATE. The mixture obtained by lyophilization and removal of the solvent DMSO was separated and purified by HPLC, and after lyophilization, about 5.5 mg of target product was obtained, with a yield of 25.6%.

[0048] 3) Synthesis of Compound NOTA-3PEG4-TATE-RGD:

[0049] In a 20 mL glass reaction vial containing 6.5 mg of NOTA-2PEG.sub.4-TATE (dissolved in 1 mL DMSO) obtained in the step 2), 6.0 mg PEG.sub.4-RGD and 10 L DIPEA were added to mix and dissolve. The mixture was stirred at room temperature for 1 h and purified by HPLC. Purification conditions: diluted with 4 mL of water and preparative HPLC (with C18 column) was applied to inject samples in two portions, and purified at a flow rate of 12 mL/min according to the following gradient. The eluent ingredients: Buffer A: 0.1% TFA in H.sub.2O; Buffer B 0.1% TFA in CH.sub.3CN; gradient elution: 0-20 min: 20-50% Buffer B. After lyophilization, about 1.5 mg of product was obtained, with a yield of 13.5% and a purity of more than 97%. The product was identified by LC-MS: [(MHH)/2].sup.++=2930.14, and the calculated (m/z) was 2930.92 (C.sub.131H.sub.193N.sub.27O.sub.43S.sub.3). The target product NOTA-Bn-p-SCN-PEG.sub.4-Glu{PEG.sub.4-Cyclo[Arg-Gly-Asp-(D-Tyr)-Lys]}-PEG.sub.4-(D-Phe)-Cys-Tyr-(D-Trp)-Lys-Thr-Cys-Thr was determined.

[0050] Embodiment 2

[0051] A TATE-RGD dual-target radioactive molecular probe comprising a TATE polypeptide, an RGD polypeptide and a radionuclide .sup.68Ga, wherein the TATE and RGD polypeptides are linked by a PEG.sub.4 molecule to form a TATE-3PEG.sub.4-RGD polypeptide and .sup.68Ga is linked by a NOTA. The TATE-RGD dual-target radioactive molecular probe is .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD.

[0052] The method of preparing .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD dual-target radioactive molecular probe, comprising the following steps:

[0053] (1) .sup.68Ga Rinsing

[0054] 5 mL of 0.1 mol/L HCl was drawn by a 5 mL syringe to elute the germanium-gallium generator slowly, at the same time, the eluent was collected to a 1.5 mL EP tube, 1 mL per tube, a total of 5 tubes. Radioactivity determination was performed for each EP tube, and the most deactivated tube was used for labeling;

[0055] (2) Labeling of .sup.68Ga-NOTA-3PEG4-TATE-RGD

[0056] 1.25 mol/L sodium acetate solution was added to a 1 mL.sup.68Ga eluent, the pH of solution was adjusted to 4.0-4.5, then mixed well, and 20 g of NOTA-3PEG.sub.4-TATE-RGD prepared in the Embodiment 1 was added to mix well, heated to 100 C. and kept for 10 min; after the reaction was completed, the reaction solution was cooled to room temperature, and 4 mL of sterile water for injection was added, and then filtered to a sterile product bottle through a sterile filter membrane (0.22 pm, 13 mm);

[0057] (3) Quality Control

[0058] HPLC conditions: C18 column (4.6 mm250 mm), mobile phase A: E 0.1% trifluoroacetic acid, mobile phase B: water (0.1% trifluoroacetic acid), and flow rate of 1 mL/min: 05 min, mobile phase A 5%;10 min, mobile phase A: 80%; 15 min, mobile phase A: 100%; 18 min, mobile phase A: 100%; 20 min, mobile phase A 5%. The UV detection wavelength was 210 nm and the column temperature was 20 C. Radioactive detection was performed by a dedicated radioactive detector for HPLC. HPLC analysis showed that the retention time of .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD was 11.6 min (FIG. 1), and the radiochemical purity was >99%, without requiring further purification.

[0059] (4) Determination of In Vitro Stability

[0060] After .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD labeling, fetal bovine serum was added and the radiochemical purity was determined by HPLC at 60 and 120 min, respectively. After determination, the radiochemical purity was 98% and 97%, respectively.

[0061] The above TATE-RGD dual-target probe .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD was further prepared into a colorless transparent injection for imaging test or imaging diagnosis. The specific experiments and effects were as follows:

[0062] {circumflex over (1)} MicroPET Imaging of Small Cell Lung Cancer H69 Tumor-Bearing Mice

[0063] The tumor-bearing mice inoculated with H69 tumor subcutaneously in the right forelimb were injected with .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD injection in Embodiment 2 by tail veins at 100-200 Ci for 60 min, and then 10 min static image was collected by a Siemens Inveon micro PET (FIG. 2, wherein the arrow indicated the tumor site). The results showed that, when the probe of Embodiment 2 was injected alone, there was significant radioactivity uptake at the tumor site, the tumor was clearly visible, and the tumor uptake value was 9.782.77; while the co-injection of the unlabeled precursor RGD, TATE and RGD+TATE was effective in reducing tumor uptake to 8.231.08, 1.410.73, and 1.050.13, respectively; when concurrently injected with unlabeled precursors RGD and TATE, the radioactivity uptake of tumor was nearly reduced to the background. Small cell H69 tumors were predominantly expressed by SSTR, and the concentration of the dual-target molecular probe of the present invention in tumors was mainly inhibited by unlabeled TATE.

[0064] {circumflex over (2)} MicroPET Imaging of Non-Small Cell Lung Cancer A549 Tumor-Bearing Mice

[0065] The tumor-bearing mice inoculated with A549 tumors in the right forelimb were injected with .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD of injection of embodiment 2 by tail veins at 100-200 Ci for 60 min, and then 10 min static image was collected by a Siemens Inveon micro PET (FIG. 3, wherein the arrow indicated the tumor site). The results showed that, when the probe of Embodiment 2 was injected alone, there was significant radioactivity uptake at the tumor site, the tumor was clearly visible, and the tumor uptake value was 6.460.59; while the co-injection of the unlabeled precursor RGD, TATE and RGD+TATE was effective in reducing tumor uptake to 1.750.53, 3.800.48 and 1.350.26, respectively; when concurrently injected with unlabeled precursors RGD and TATE, the radioactivity uptake of tumor was nearly reduced to the background. Non-small cell A549 tumors were predominantly expressed by integrin receptor, and the concentration of the dual-target molecular probe of the present invention in tumors was mainly inhibited by unlabeled RGD.

[0066] {circumflex over (4)} Biodistribution of .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD in Healthy Mice

[0067] The distribution of healthy Balb/c was shown in FIG. 4, and .sup.68Ga-NOTA-3PEG4-TATE-RGD of Embodiment 2 was cleared from the blood, heart and liver faster; the kidney was more radioactive, indicating that the molecular probe was mainly excreted through the kidneys. There was small amount of uptake in the heart, liver and lungs, which decreased quickly over time. The imaging agent had a small amount of distribution in the stomach, intestines, spleen, and pancreas, and little distribution in the brain tissues, indicating that it could not pass through the blood-brain barrier.

[0068] {circumflex over (5)} Imaging of .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD in Patients with Small Cell Lung Cancer

[0069] The .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD PET/CT images of clinically diagnosed small cell lung cancer patients were shown in FIG. 5. .sup.68Ga-NOTA-3PEG4-TATE-RGD 3.5 mCi was injected intravenously, 30 min later, the images of truck were collected by Siemens Biograph64 PET/CT, 3 min/bed, and a total of 5 beds were collected. The lesions were clearly displayed and the highest standard uptake value of tumor (SUVmax) was 18.2 (FIG. 5 left). However, the existing single-target imaging agent only showed an increase in the .sup.68Ga-NOTA-RGD uptake of the tumor region, and the SUVmax value was 4.7 (FIG. 5 right).

[0070] {circumflex over (6)} Imaging of .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD in Patients with Non-Small Cell Lung Cancer

[0071] The images of clinically diagnosed non-small cell lung cancer patients were shown in FIG. 6. .sup.68Ga-NOTA-3PEG.sub.4-TATE-RGD 3.0 mCi was injected intravenously, 30 min later, the images were collected by Siemens Biograph64 PET/CT, 3 min/bed, and a total of 5 beds were collected. The lesions were clearly displayed and the highest standard uptake value of tumor (SUVmax) was 3.4 (FIG. 6 left). However, the existing single-target imaging agent showed low tumor uptake of .sup.68Ga-NOTA-RGD, and the SUVmax value was 2.8 (FIG. 6 right).