TW202005669A - The structural molecule of peptide derivative for PSMA-targeted radiotherapy diagnosis and treatment - Google Patents

Abstract

A PSMA-targeted peptide derivative for radiotherapy, which is a novel structural molecule developed for diagnosis or treatment of prostate cancer, and prostate-specific membrane antigen (PSMA) is a protein present on the surface of healthy prostate cells, which is often at a high level of expression on the surface of prostate cancer cells, and the molecular composition of PSMA inhibitor is mainly composed of glutamic acid, urea and lysine, in addition to the linker of the present invention, PSMA inhibitor can be combined with a chelating agent and truncated Evans Blue, which can be labeled with radionuclides Ga-67, Ga-68, In-111, Lu-177, Cu-64 and Y-90, used for image analysis and analysis of human prostate cancer tumor pattern as a new PSMA-targeted peptide receptor radionuclide therapy (PRRT), and which has a longer half-life in vivo and is featured by specific binding of prostate-specific membrane antigens for radiotherapy diagnosis or treatment.

Description

PSMA-targeted radioactive peptide derivatives

This creation is about the end cap structure of a liquid container, especially a peptide derivative with specific binding radioactivity for PSMA and a longer half-life in vivo.

According to the World Health Organization (WHO) International Cancer Research Agency (IARC) GLOBOCAN 2012 statistics on global incidence, death and prevalence data, prostate cancer ranks fourth among common cancers regardless of gender, and ranks second among common cancers among men About 1.1 million men worldwide have been diagnosed with prostate cancer.

The clinical non-invasive diagnosis methods for prostate cancer (PCa) include digital rectal examination, rectal ultrasound, PSA detection, CT, MRI, radionuclide angiography and other imaging examination methods. The above methods have different disadvantages. Here are the points:

The traditional imaging diagnosis of prostate cancer mainly relies on rectal ultrasound, CT, and MRI. Like these diagnostic methods, there is no specificity for prostate cancer. For example, in the clinic, transrectal ultrasound (TURS) guided prostate systemic biopsy for prostate cancer (prostate cancer, PCa) can be performed. However, the commonly used false negative rate of the TURS six-point system puncture method is about 30%, and there are serious complications.

CT examination cannot distinguish between cancerous tissue and benign hyperplastic tissue, so it is not clear whether there is prostate cancer (PCa). MRI soft tissue has a higher resolution and is superior to CT and ultrasound in the diagnosis of prostate lesions, and can effectively distinguish between prostate cancer (PCa) and benign prostatic hyperplasia. However, prostate cancer (PCa) is prone to lymph node metastasis and distal bone metastasis, which reduces the value of MRI in the diagnosis and staging of prostate cancer (PCa). Tc-99m-MDP angiography is comparable to X-ray examination. It can detect bone metastases 3 to 6 months in advance, which helps to judge the accurate clinical stage of prostate cancer (PCa), but its sensitivity is high but its specificity is poor. About 50% of patients with prostate cancer (PCa) bone metastasis die within 30 to 35 months after diagnosis. Therefore, the current traditional imaging and slice methods have been difficult to meet the clinical requirements for early diagnosis and accurate staging of prostate cancer (PCa).

Blood PSA index test; the blood PSA index is used as a tumor biomarker for prostate cancer (PCa) and is currently commonly used as a sensitive indicator for the diagnosis of prostate cancer (PCa). However, when the blood PSA index is 4-10 μg/L, it is difficult to diagnose prostate cancer (PCa). And the blood PSA index can not reflect the characteristics of clinicopathological slices, there is no specificity, and it cannot effectively distinguish local tumors or distant metastases.

The main technical contents of nuclear medicine molecular imaging are as follows:

(A) F-18-NaF angiography; clinical studies indicate that Tc-99m-MDP angiography has a sensitivity of 50.8% and a specificity of 82% for the detection of prostate cancer (PCa) bone metastases. In contrast, F-18-NaF PET/CT imaging has a sensitivity of 93% and a specificity of 54%, and it has a higher spatial resolution, and can perform CT anatomical positioning and three-dimensional imaging, which is expected to become an early discovery. Prostate cancer (PCa) clinical imaging examination method for bone metastasis. Although F-18-NaF can find more metastases, it does not change the clinical treatment plan.

(B) F-18-FDG angiography; F-18-FDG PET/CT can provide effective clinical data for early diagnosis, staging, protocol optimization, and prognostic evaluation of prostate cancer (PCa). But similar to normal cells, prostate cancer (PCa) Glut expression level is low, it is difficult to distinguish benign lesions, and F-18-FDG is mainly excreted through the urinary system, which will interfere with the diagnosis of prostate cancer (PCa), resulting in F-18- FDG has a low detection rate of prostate cancer (PCa) and limited diagnostic value.

(C) C-11-Choline angiography; compared with F-18-NaF, C-11-Choline prostate cancer (PCa) has obvious advantages in angiography. C-11-Choline can accumulate in tumor cells and be phosphorylated in tumor cells and stay in the cells. Sutinen et al's C-11-CholinePET study showed that although there is also a concentration of contrast agent in the bladder, the concentration is low, and the prostate can be determined by its location and morphology. Picchio et al's retrospective study of 100 patients with prostate cancer (PCa) confirmed that the diagnostic ability of C-11-Choline PET for prostate cancer (PCa) is better than F-18-FDG, and the positive rate of angiography is 47%, while F- 18-NaF is only 27%. N-[F-18] fluoromethyl Choline (F-18-fluoro-methylcholine, F-18-FECH) has a sensitivity of 84.7% and a specificity of 91.1%, showing that C-11/F-18-FECH has an effect on the prostate The sensitivity and specificity of tumor diagnosis are significantly better than Tc-99m-MDP and F-18-NaF. Yamaguchi (Eur J Nucl Med Mol Imaging. 2005) and other scholars compared the sensitivity of C-11-CholinePET and MRI to locate primary prostate cancer (PCa) and found that the sensitivity of C-11-CholinePET is close to 100%, while the MRI is only 60 %. However, C-11-Choline angiography cannot effectively identify the primary tumor of prostate cancer (PCa). F-18-FACBC (anti-1-amino-3-F-18-fluorocyclobutane-1-carboxylic acid) contrast; F-18-FACBC is an L-type amino acid transporter 1 and alanine, serine and cysteine The matrix of the acid transporter is almost not excreted by the kidney, so the pelvis can be clearly visualized and can be used for the detection of prostate cancer (PCa). Schuster et al reported the results of F-18-FACBC PET/CT examination of nearly 100 patients with suspected recurrent prostate cancer (PCa). The positive detection rate of primary prostate cancer (PCa) was 74%, and the positive detection of metastatic tumors The rate is 96%. F-18-FACBC angiography can detect lymph node metastasis that F-18-FDG cannot detect, and the effect is significantly better than CT or PET alone. Schuster et al. reviewed 598 cases of F-18-FACBC multi-center joint study radiography and showed that the probe metabolized in vivo faster, and the radiography was better 30 minutes after injection. At present, F-18-FACBC has undergone Phase III clinical trial (NCT01666808) in the mainland.

(D) 16β-[F-18]fluoro-5-dihydrotestosterone (16β-F-18-fluoro-5-dihydrotestosterone, F-18-DHT) androgen receptor imaging; in prostate tissue, DHT is The main androgen, its concentration is 5 times that of testosterone, and its affinity with androgen receptor is 10 times that of testosterone. F-18-DHT has a high ratio of prostate to soft tissue radioactivity, and is expected to be used in the diagnosis, staging, prognosis, and evaluation of hormone therapy for prostate cancer (PCa). A comparison study of F-18-DHT and F-18-FDG in 7 patients with prostate cancer (PCa) showed that F-18-DHT is metabolized faster in the body, and the uptake of the lesion site increases with time. Of the 59 lesions detected in general, F-18-DHT found 46 of them (detection rate 78%), while F-18-FDG detected 57 lesions (detection rate 97%). The researchers also conducted a comparative study of the clinical diagnostic efficacy of F-18-DHT and F-18-FDG in advanced prostate cancer (PCa) (NCT00588185), using computational methods to analyze androgen receptor expression and F-18-DHT uptake The relationship further confirmed the feasibility of its clinical application in early diagnosis of prostate tumors. In 2014, the research group started to use F-18-DHT as a molecular probe, using PET and MR to simultaneously study the diagnosis and treatment of prostate cancer (PCa), and promote the clinical application of F-18-DHT probe in the diagnosis and treatment of prostate tumor hormones .

(E) New molecular probes targeting prostate specific membrane antigen (PSMA); with the deepening of molecular biology research, PSMA receptors have become the molecular imaging and target of prostate cancer (PCa) The ideal target for treatment. According to the different action sites of drugs [including monoclonal antibodies (abbreviated as monoclonal antibodies), peptides, and small molecules] and PSMA, PSMA targeting antibodies are divided into intracellular domain antibodies (such as 7EI1, PM2J004.5) and extracellular domain antibodies (such as J591, J415, PEQ226.6) etc. In the above diagnosis methods including F-18-FDG, some cases cannot judge prostate cancer, especially in metastatic cases. Current research has found that prostate cancer cell surface highly expresses prostate specific membrane antigen (PSMA), so as long as it targets anti-PSMA, relevant radioactive nuclear medicines can be developed. At present, a variety of antibodies or peptides (J591, PSMA-11, etc.) have been developed and can be diagnosed with the Ga-68 marker. However, clinical progress has moved toward the concept of PRRT (peptide receptor radionuclide therapy), hoping to synchronize diagnosis and treatment.

At present, there are quite a few types developed on the basis of PSMA, which are mainly used for diagnosis. Only the PSMA-617, which is subsequently improved, can be treated with the Lu-177 mark. At present, the drug has entered clinical phase three (expected to be completed in 2020), proving that it can already achieve effective treatment. However, because the half-life of the drug is 10.8 hours, the patient needs about 4 times in the whole course of treatment, which will consume more time and money.

Prior art U.S. Published Bulletin US 2016/0228587 A1 discloses a functional linker that enables a prostate cancer inhibitor to bind to a chelating agent and chelate with a radioactive nucleus to serve as a marker for prostate cancer tracking and diagnosis, but this drug has a short half-life , Multiple treatment sessions are required. The difference between the present invention and the foregoing prior art is to use Evans blue or Evans blue to modify the compound disclosed in US 2016/0228587 A1, so as to increase the compound's function of binding to blood albumin, and effectively reduce the compound's rapid elimination by the kidney after entering the living body. Disadvantages, and reduce the dosage of drugs by more than 75%. In addition, the prior art Chinese published CN 104650217 A patent application discloses the use of Evans blue or Evans blue derivatives to modify Exendin-4 for the treatment of diabetes type II and myocardial infarction, although Evans blue or Iraq is used in the same way as the present invention Vanceline modified the compound, but CN 104650217 A patent application discloses that the modified compound has completely different types and functions. The present invention provides a linking group that can be combined with a chelating agent and truncated Evans blue, the chelating agent can mark the radioactive nuclear species Ga-67, Ga-68, In-111, Lu-177, Cu-64, Y-90, Used for image analysis and evaluation of human prostate cancer tumor model. The PSMA-7165 derivative provided by the present invention has a high degree of binding to the PSMA receptor. It is improved on the basis of the above PSMA-617, and the truncated Evans blue is added by using a linker to increase the half-life in the body to prolong its blood circulation. The duration of stay can reach the goal of completing the course of treatment with a single injection.

The main object of the present invention is to provide a peptide derivative of PSMA targeted radiotherapy and diagnosis, which is based on PSMA-617 and synthesizes a PSMA-7165 diagnostic tracer with radiolabeling function, and uses the PSMA-7165 derivative It has a high degree of binding to the PSMA receptor, and the PSMA receptor will be expressed in prostate cancer (PCa), which can be used as a target diagnostic or therapeutic drug carrier for prostate cancer (PCa), and is improved based on PSMA-617. Evans Blue is added to prolong its stay in the blood and increase the half-life in the body, which can achieve the goal of completing the treatment with a single injection, thereby reducing the burden of patients' time and money.

Another object of the present invention is to provide a peptide derivative of PSMA targeted radioactive diagnosis and treatment. The PSMA-7165 label is convenient and shortens time. The label efficiency does not need to be purified by other columns after labeling, and the label efficiency can reach more than 95%. From the experimental data of tumor animals, it can be seen that it has a high degree of binding to tumors. In the prostate cancer (PCa) animal model (PSMA+), it takes only 2 to 4 hours to see obvious tumor accumulation images, and it still maintains a high accumulation amount until 72 hours.

Another object of the present invention is to provide a peptide derivative for PSMA targeted radiotherapy diagnosis and treatment. The product marked with PSMA-7165 peptide derivative can be administered by injection in the form of radioactive injection, or by combination of frozen crystals, in addition to increase The convenience of delivery and the selectivity of back-end use increase the promotion of the pharmaceutical market.

In order to make the above objects, effects and features of the present invention more specific, the following drawings are as follows:

Please refer to FIG. 1 for the chemical structure of the PSMA-7165 of the present invention; in a feasible embodiment, the chemical synthesis of the PSMA-7165 can be implemented through the contents disclosed in Schemes 1, 2, and 3 below.

Route 1: Compound 1 (Boc glutamic acid) was ice-bathed in dichloromethane for 10 minutes, triphosgene was added to react and stirred at 0°C for 6 hours to obtain intermediate product 2 (isocyanate). Compound 3 (an amine acid derivative) and 2-chlorotrityl resin were reacted in dichloromethane at room temperature for 2 hours to obtain intermediate product 4. Intermediate product 2 and intermediate product 4 were stirred at room temperature for 16 hours for coupling to obtain intermediate product 5. The intermediate product 5, tetrakis(triphenylphosphine) palladium and morpholine were stirred at normal temperature in dichloromethane for 3 hours, and the allyloxy protecting group was removed to obtain intermediate product 6. Fluorinated chloro-3-(2-naphthalene)-L-ionic acid, HBTU, DIPEA and intermediate product 6 were stirred at room temperature for 16 hours to obtain intermediate product 7. The intermediate product 8 was obtained by stirring with tranexamic acid derivative, HBTU, DIPEA and intermediate product 7 at room temperature for 16 hours. Then, N-Succinimidyl-S-acetylthiopropionate (STPA) and sodium hydrogen phosphate were stirred with intermediate product 8 in dimethyl sulfoxide for 10 hours at room temperature to obtain intermediate product 9. The intermediate product 9 is reacted with hydroxylamine to remove the acetyl group, and then trifluoroacetic acid is added to obtain the first semi-finished product (sulfhydryl-modified PSMA-617, PSMA-7165 intermediate).

Route 2: Compound 11 (o-dimethylbenzidine) and di-tert-butyl dicarbonate were reacted in dichloromethane at room temperature for 5 hours. Using di-tert-butyl dicarbonate as the limiting reagent, intermediate product 12 (Boc- Benzylamine), and then reacted with sodium nitrite and hydrochloric acid to form diazonium salt intermediate product 13, dissolved in water with 1-amino-8-naphthol-2,4-disulfonic acid sodium and sodium bicarbonate , Slowly add intermediate product 13 and react in the solution for 12 hours to obtain intermediate product 14, then add trifluoroacetic acid as intermediate product 14 to remove the boc protecting group, add Boc-Lys-Fmoc and HATU at room temperature and stir for 6 hours to obtain intermediate product 15. The intermediate product 15 was added to piperidine and stirred at room temperature for 4 hours to obtain intermediate product 16. After pre-stirring NOTA, HATU and DIPEA in DMF for 15 minutes, intermediate product 16 was added and reacted at room temperature until overnight to obtain intermediate product 17. The intermediate product 17 was added to trifluoroacetic acid and DMF solvent and stirred at room temperature for 2 hours to obtain a second semi-finished product (DOTA-EB-Lys, PSMA-7165 intermediate).

其中n為阿拉伯數字1，m為阿拉伯數字1，p為阿拉伯數字1，R1為金屬螯合基DOTA，R2為化學結構萘分子（naphthalene），即為PSMA-7165。Route 3: Stir the second semi-finished product (DOTA-EB-Lys) and the dioxolyl ester derivative at room temperature to obtain the intermediate product 19, and finally mix the intermediate product 19 and the first semi-finished product (PSMA617-SH) in PBS buffer solution and DMF Stir at room temperature for 2 hours to obtain PSMA-7165. Its chemical structural formula (as shown in Figures 1 and 19) is as follows:

Among them, n is Arabic numeral 1, m is Arabic numeral 1, p is Arabic numeral 1, R1 is metal chelate group DOTA, R2 is chemical structure naphthalene molecule (naphthalene), namely PSMA-7165.

Please refer to Figures 2 to 4b, it can be seen that the preparation method of the Ga-68 radioactive marker of the PSMA-7165 of the present invention includes: "Preparation of quantitative PSMA-7165" S11 step, "Add reaction buffer" S12 step, "Add Ga -68 shot source" S13 step, "constant temperature reaction" S14 step, "quality control" S15 step, "finished product output" S16 step, etc.; wherein the "preparation of quantitative PSMA-7165" S11 step is made by PSMA-7165 Prepare 20mg/mL in DMSO, divide into 10μg in microcentrifuge tubes, and store at -20℃. When marking, take out a 1.5mL microcentrifuge tube containing 10µg PSMA-7165.

"Add reaction buffer" step S12, in step S11, add a 1.5 mL microcentrifuge tube containing 10 µg PSMA-7165 to 1, 1.5 or 3 M sodium acetate buffer solution, the pH value is 6.0, 6.0 or 7.0, and let the final The pH values of the reaction solution were 4.0, 5.0 and 6.0, respectively.

"Add Ga-68 source" step S13 is to oscillate the mixture of step S12 for 1~2min through ultrasonic wave, then add Ga-68 source (the initial activity is about 1.3mCi), and mix thoroughly and finally The reaction volume is 165.5 µL.

Step S14 of "Constant temperature reaction" is to put it in a 95°C precision thermostat for 15, 20 or 30 minutes, and shake at 500 rpm during heating.

"Quality control" step S15, after completely cooling, take an appropriate amount of sample for radio-ITLC analysis, the development solution for analysis is 0.1M citric acid, in this analysis system, the origin is the standard On the radioactive Ga-68-PSMA-7165, the mobile end of the solution is unreacted Ga-68.

"Product output" step S16, under different conditions, with 1.5M sodium acetate buffer (pH 5.0) after 15 minutes of marking, the marking efficiency can reach 95%, and the reaction time is increased to 20 or 30 minutes, the marking efficiency can be Up to 100%, the logo data is shown in Figures 3 to 4b.

Please refer to Figs. 5-7, it can be seen that the preparation method of the Ga-67 radioactive marker of the PSMA-7165 of the present invention includes: "Preparation of quantitative PSMA-7165" S21 step, "Add reaction buffer" S22 step, "Add Ga -67 ray source" S23 step, "constant temperature reaction" S24 step, "quality control" S25 step, "finished product output" S26 step, etc.; wherein the "preparation of quantitative PSMA-7165" step S21 is made by PSMA-7165 Prepare 20mg/mL with DMSO, divide it into microcentrifuge tubes at 20µg, and store at -20℃. When marking, take out a 1.5mL microcentrifuge tube containing 20µg PSMA-7165.

"Add reaction buffer" step S22, add 0.4 M sodium acetate buffer solution, the pH value is 6.0.

"Add Ga-67 source" step S23, after the shock of the mixture of step S22 is oscillated by ultrasound for 1~2min, then add Ga-67 source (the initial activity is about ~3mCi), complete mixing and then finally The reaction volume is 150µL.

Step S24 of "Constant temperature reaction" is to put it in a 95°C precise constant temperature controller for 15, 20 or 30 minutes, and shake at 500 rpm during heating.

"Quality Control" step S25, after completely cooling, take a proper amount of sample for radio-ITLC analysis, the development solution for analysis is 0.1M citric acid, in this analysis system, the origin is the standard On the radioactive Ga-67-PSMA-7165, the mobile end of the solution is unreacted Ga-67.

Step S26 of "Finished Product Output" is to use 0.4M sodium acetate buffer solution (pH 6.0) after marking for 15 minutes to achieve 100% marking efficiency. The marking data is shown in Figures 6 to 7.

Please refer to Figures 8 to 10, it can be seen that the preparation method of the PSMA-7165 In-111 radioactive marker of the present invention includes: "Preparation of quantitative PSMA-7165" S31 step, "Add reaction buffer" S32 step, "Add In -111 shot source" S33 step, "constant temperature reaction" S34 step, "quality control" S35 step, "finished product output" S36 step, etc.; wherein the "preparation of quantitative PSMA-7165" step S31, by PSMA-7165 Prepare 20mg/mL with DMSO, divide it into microcentrifuge tubes at 20µg, and store at -20℃. When marking, take out a 1.5mL microcentrifuge tube containing 20µg PSMA-7165.

"Add reaction buffer" S32 step is to add 1M sodium acetate buffer solution, the pH value is 6.0.

"Adding In-111 source" step S33 is to oscillate the mixture of step S32 through ultrasound for 1~2min, and then add In-111 source (the initial activity is about ~5.7mCi), after complete mixing The final reaction volume is 300 µL.

Step S34 of "Constant temperature reaction" is to put it in a 95°C precise constant temperature controller for 5, 10, 15 or 20 minutes, and shake at 500 rpm during the heating process.

"Quality Control" step S35, after completely cooling, take an appropriate amount of sample for radio-ITLC analysis. The developing solution for analysis is 0.1M citric acid and 0.1M sodium citrate in a ratio of 2 to 8 by volume Configuration, in this analysis system, the origin is marked with radioactive In-111-PSMA-7165, and the mobile end of the solution is unreacted In-111.

Step S36 of "finished product output" is to use 1M sodium acetate buffer solution (pH 6.0) after 10 minutes of marking to achieve a marking efficiency of 97%. The marking data is shown in Figures 9-10.

Please refer to FIG. 11, it can be seen that in the in vitro cell binding activity test of In-111-PSMA-7165, the present invention combines LNCaP cells expressing PSMA and PC3 cells expressing PSMA with ⁷ ×10 ⁵ cells and different peptides. The concentration of In-111-PSMA-7165 was incubated at 37°C for 45 minutes, and then washed with iced PBS buffer to remove the In-111-PSMA-7165 that was not bound to the cells. After centrifugation, the precipitated cells were collected. -Counter readings of radiometer readings. LNCaP cell lines that exhibit PSMA at different peptide concentrations of 2.5nM, 25nM or 250nM all have higher readings (CPM).

Please refer to FIG. 12, and it can be known that the PSMA-7165 logo In-111 of the present invention is distributed in 48 hours after the LNCaP (PSMA+) and PC3 (PSMA-) prostate tumor mouse angiography test. LNCaP human prostate cancer cells expressing PSMA were inoculated into the right forelimb of SCID mice at 4× ^{10 6} or PC-3 human prostate cancer cells not expressing PSMA were inoculated into the hind limbs of BALB/c nude mice at 2×10 ⁶ . After the animals were inoculated for about 3 weeks, PSMA-7165 was labeled with the radioisotope In-111, and then the radioactive thin layer analysis method was used to confirm that the drug's labeling efficiency was 100%. The marked product was adjusted to approximately 500µCi/100µL per injection with injection water, and In-111-PSMA-7165 was given via tail vein injection for nanoSPECT/CT imaging after 1, 4, 24, and 48 hours.

The NanoSPECT/CT contrast and image semi-quantitative steps include: (1) Injecting In-111-PSMA-7165 drug with known activity into LNCaP tumor mice via tail vein injection. (2) Prepare a fixed activity prosthesis as a reference substance and fix it to the bed of NanoSPECT/CT instrument together with tumor mice. During the imaging process, tumor mice use the animal drug Aining (Attane, Baoling Fujinsheng) Technology company) to perform in vivo anesthesia. (3) NanoSPECT/CT imaging is performed at different time points according to the test design. During the process, Nucline (V.2.00) software developed by Mediso is used with SPECT: 60 sec/frame, Energy Window 245±10% keV&171±10% keV Perform imaging and use HiSPECT (V.1.43049) image reconstruction software developed by Scivis to reconstruct the image. (4) The reconstructed image uses the InVivoScope (V.1.44) image analysis software developed by Mediso to perform the preliminary inspection of SPECT and CT images and the production of image files. (5) Use PMod (V.3.4) image analysis software developed by PMOD Technologies to perform semi-quantitative image analysis: use the SPECT count value obtained from the reference substance with known activity at each time point to derive the SPECT count value and actual activity The linear ratio formula between degrees, and then use the SPECT count value obtained by circled each target organ to obtain the activity/volume of each target organ by interpolating or extrapolating the linear formula. Assuming 1 g per 1 mL of injection, and 1 g per 1 cm3 of the circled area in the mouse, due to the known activity volume of the administered injection and the weight of the experimental rat, it can be deduced that each gram of drug in each organ Biological distribution ratio (%ID/g).

Please refer to Figures 13 to 15b, it can be seen that the preparation method of the Ga-67 radioactive marker of DOTA-EB of the present invention includes: "Preparation of quantitative DOTA-EB" S41 step, "Add reaction buffer" S42 step, "Add Ga -67 ray source" S43 step, "constant temperature reaction" S44 step, "quality control" S45 step, "finished product output" S46 step, etc.; wherein the "preparation of quantitative DOTA-EB" step S41, by DOTA-EB Prepare 20mg/mL with DMSO, divide it into microcentrifuge tubes at 20µg, and store at -20℃. At the time of marking, take out another 20μg DOTA-EB 1.5mL microcentrifuge tube.

"Add reaction buffer" S42 step is to add 1, 1.5, 3M sodium acetate solution reaction buffer solution, the pH value of which is 6.0, 6.0 or 7.0, so that the final reaction solution pH value is 4.0, 5.0 and 6.0.

"Adding Ga-67 source" step S43 is to oscillate the mixture of step S42 through ultrasonic for 1~2min, and then add Ga-67 source (initial activity is about 3mCi), complete mixing and final reaction The volume is 150µL.

Step S44 of "Constant temperature reaction" is to put it in a 95°C precise constant temperature controller for 15, 20, or 30 minutes, and shake at 500 rpm while heating.

"Quality Control" step S45, after completely cooling, take an appropriate amount of sample for radio-ITLC analysis, the development solution for analysis is 10% methanol (Methanol, MeOH). In this analysis system, the original The point is unreacted Ga-67, and the mobile end of the solution is Ga-67-DOTA-EB marked with radioactivity.

"Product output" step S46, under different conditions, with 1M sodium acetate solution (pH 4.0) or 1.5M sodium acetate solution (pH 5.0) after 15 minutes mark, the mark efficiency can reach more than 95%, if the reaction time Increasing to 20 or 30 minutes, the marking efficiency is still above 95%, and the marking data is shown in Figures 14 to 15b.

Please refer to Figures 16 to 18, it can be seen that the preparation method of the DOTA-EB In-111 radioactive marker of the present invention includes: "Preparation of quantitative DOTA-EB" S51 step, "Add reaction buffer" S52 step, "Add In -111 shot source" S53 step, "constant temperature reaction" S54 step, "quality control" S55 step, "finished product output" S56 step, etc.; wherein the "preparation of quantitative DOTA-EB" step S51, by DOTA-EB Prepare 20mg/mL with DMSO, divide it into microcentrifuge tubes at 20µg, and store at -20℃. At the time of marking, take out another 20μg DOTA-EB 1.5mL microcentrifuge tube. .

"Add reaction buffer" step S52 is to add 1, 2 or 3 M sodium acetate solution, the pH value of which is 6.0, 6.0 or 7.0 respectively, so that the pH value of the final reaction solution is 6.0, 6.0 and 7.0 respectively.

"Adding In-111 source" step S53 is to oscillate the mixture of step S52 for 1~2min through ultrasonic wave, then add In-111 source (the initial activity is about 1 or 5mCi), and mix thoroughly The final reaction volume is 300 µL.

Step S54 of "Constant temperature reaction" is to place it in a 95°C precise constant temperature controller for 10 minutes, and shake at 500 rpm during heating.

"Quality Control" step S55, after completely cooling, take an appropriate amount of sample for radio-ITLC analysis, and the developing solution for analysis is 0.1M citric acid and 0.1M sodium citrate in a ratio of 2 to 8 by volume Configuration, in this analysis system, In-111-DOTA-EB is located at the origin, and unreacted In-111 is located at the developing liquid end.

Step S56 of "finished product output" is under different conditions. After 10 minutes of marking, the efficiency of the marking can reach more than 90%. The marking data is shown in Figures 17 to 18.

Based on the above, the peptide derivative of the PSMA targeted radioactive diagnosis and treatment of the present invention can indeed achieve the effect of prolonging the half-life in vivo and specifically binding radioactivity for PSMA, which is actually a novel and progressive invention. Invention patent; but the above description is only for the description of the preferred embodiments of the present invention. Any changes, modifications, changes or equivalent replacements that extend according to the technical means and scope of the present invention should also fall into the present invention. Within the scope of the patent application.

1‧‧‧ Compound (Boc glutamic acid) 2, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17, 19‧‧‧‧ Intermediate 3‧‧‧ Compound Amino acid derivatives) 11‧‧‧ Compound (o-dimethylbenzidine) S11, S21, S31‧‧‧ Prepare quantitative PSMA-7165S12, S22, S32, S42, S52‧‧‧Add to reaction buffer S13‧‧ ‧Join Ga-68 sources S14, S24, S34, S44, S54‧‧‧ Constant temperature reaction S15, S25, S35, S45, S55‧‧‧Quality control S16, S26, S36, S46, S56 finished product output S23, S43‧‧‧ Added Ga-67 source S33, S53‧‧‧ Added In-111 source S41, S51‧‧‧ Prepare quantitative DOTA-EB

Figure 1 is an explanation of the chemical structure and function (including molecular weight) of PSMA-7165 of the present invention. Figure 2 is a flow chart of the logo of the Ga-68-PSMA-7165 of the present invention. Figure 3 is the Ga-68-PSMA-7165 marker efficiency and marker efficiency analysis table of the present invention. Figures 4a and 4b are the Radio-ITLC analysis results of the Ga-68-PSMA-7165 of the present invention. Figure 5 is a flow chart of the Ga-67-PSMA-7165 logo of the present invention. Figure 6 is the Ga-67-PSMA-7165 logo efficiency and logo efficiency analysis table of the present invention. Figure 7 is the result of Radio-ITLC analysis of Ga-67-PSMA-7165 of the present invention. Figure 8 is a flow chart of the logo of the present invention In-111-PSMA-7165. Figure 9 is the In-111-PSMA-7165 logo efficiency and logo efficiency analysis table of the present invention. Figure 10 is the result of Radio-ITLC analysis of In-111-PSMA-7165 of the present invention. Figure 11 is a test of the binding of In-111-PSMA-7165 of the present invention to LNCaP (PSMA+) or PC3 (PSMA-) prostate cancer cells. FIG. 12 is a NanoSPECT/CT contrast image of In-111-PSMA-7165 of the present invention in an animal model of LNCaP human prostate cancer tumor. Figure 13 is the flow chart of the Ga-67-DOTA-EB logo. Figure 14 is the Ga-67-DOTA-EB logo efficiency and logo efficiency analysis table. Figures 15a and 15b are the results of Radio-ITLC analysis of Ga-67-DOTA-EB. Figure 16 is a flow chart of the In-111-DOTA-EB logo. Figure 17 is the In-111-DOTA-EB logo efficiency and logo efficiency analysis table. Figure 18 is the result of Radio-ITLC analysis of In-111-DOTA-EB. Figure 19 is the chemical structure of PSMA-7165 of the present invention.

Claims (20)

A peptide derivative of target radioactive diagnosis and treatment made according to the 20th method of the patent scope, which includes a nuclear species part (R1), truncated Evans blue, a new functional connector, an old functional connector, and The pharmacologically active structure, which interacts with radioactive isotopes to form radioactive peptides along with diagnostic and therapeutic drugs, its chemical structure is as follows:

Among them, n stands for Arabic numeral 1: m stands for Arabic numeral 1: p stands for Arabic numeral 1: R1 stands for metal chelate group DOTA: R2 stands for chemical structure naphthalene. The peptide derivative of target radioactive diagnosis and treatment as described in item 1 of the scope of patent application, wherein the part with nucleus species includes metal chelate compounds that can be used to mark radioisotopes, such as DOTA, NOTA or DTPA. The peptide derivative of target radioactive diagnosis and treatment as described in item 1 of the patent application scope, wherein the new functional connector has a parameter n which is an integer of 1 to 12. The peptide derivative of the target radioactive diagnosis and treatment as described in item 1 of the scope of the patent application, in which the old functional connector has a parameter R2 naphthalene. The peptide derivative of target radioactive diagnosis and treatment as described in item 1 of the patent scope, wherein the radioisotope is ⁶⁸ Ga, ⁶⁷ Ga, ¹¹¹ In, ¹⁷⁷ Lu, ⁶⁴ C or ⁹⁰ Y, which is suitable for diagnostic or therapeutic radioactivity isotope. The peptide derivative of the target radioactive diagnosis and treatment as described in item 1 of the patent scope, in which the target diagnostic peptide derivative PSMA-7165 is combined with the labeled radioactive isotope Ga-68, can be used for positron diagnosis. The peptide derivative of target radiotherapy for diagnosis and treatment as described in item 1 of the scope of the patent application, in which the target peptide derivative PSMA-7165 combined with the labeled radioisotope Ga-67 can be used for single-photon diagnosis. The peptide derivative of target radiotherapy for diagnosis and treatment as described in item 1 of the patent scope, in which the target peptide derivative PSMA-7165 is combined with the labeled radioisotope In-111, which can be used for single-photon diagnosis or treatment and animal biology Body distribution test. For example, the peptide derivative of target radiotherapy for diagnosis and treatment as described in item 1 of the patent scope, in which the target diagnostic peptide derivative PSMA-7165 is combined with the Lu-177 logo, can be used in animal experiments and in vivo radiotherapy. The peptide derivative of the target radioactive diagnosis and treatment as described in item 1 of the patent application scope, wherein the target diagnostic and therapeutic peptide derivative PSMA-7165 and sodium acetate, mannitol, gentisic acid or ascorbic acid can be formulated into radioactive frozen crystals preparation. The peptide derivative of target radioactive diagnosis and treatment as described in item 10 of the patent application scope, wherein the non-radioactive cryocrystal preparation can be reacted with Ga-68, Ga-67, In-111 or Lu-177 radioisotope to produce radioactive diagnostic therapy drug. The peptide derivative of target radioactive diagnosis and treatment as described in item 10 of the patent application scope, wherein the non-radioactive cryocrystal preparation is a radiopharmaceutical label kit. The peptide derivative of the target radioactive diagnosis and treatment as described in item 12 of the patent application scope, wherein the radioactive label kit is a kit component including an automated process. The peptide derivative of the target radioactive diagnosis and treatment as described in item 12 of the patent application scope, wherein the non-radioactive cryocrystal preparation is a process device that combines with the Ga-68 purification process to automatically mark the Ga-68 radioactive injection. The peptide derivative of target radioactive diagnosis and treatment as described in item 6 of the patent application scope, wherein the Ga-68-labeled PSMA-7165 can be used for positron diagnosis of poorly differentiated, metastatic, and hormone therapy-ineffective prostate cancer. The peptide derivative of target radiotherapy for diagnosis and treatment as described in item 7 of the patent scope, wherein the PSMA-7165 marked with Ga-67 or In-111 can be used for prostate cancer with poor differentiation, metastasis and hormone therapy failure Single photon diagnosis. The peptide derivative of target radiotherapy for diagnosis and treatment as described in Item 8 of the patent scope, wherein the PSMA-7165 marked with Ga-67 or In-111 can be used for prostate cancer with poor differentiation, metastasis and hormone therapy failure Single photon diagnosis. Peptide derivative of target radioactive diagnosis and treatment as described in item 8 of the scope of patent application, wherein the PSMA-7165 high activity drug marked with In-111 can be used for prostate cancer with low differentiation, metastasis and hormone therapy failure In vivo radiation therapy. The peptide derivative of the target radioactive diagnosis and treatment as described in item 9 of the patent application scope, wherein the PSMA-7165 labeled with Lu-177 can be used for the in vivo radiotherapy of poorly differentiated, metastatic, and hormone-free prostate cancer. A method for preparing peptide derivatives for target radiotherapy and diagnosis, including the first, second and third processes (Scheme 1, 2, 3), wherein the first process includes the steps: 　Place compound 1 (Boc glutamic acid) In a dichloromethane ice bath for 10 minutes, add triphosgene reaction and stir at 0°C for 6 hours to obtain intermediate product 2 (isocyanate); 　 Combine compound 3 (isoamine derivative) with 2-chlorotrityl resin in dichloromethane In methyl chloride, react at room temperature for 2 hours to obtain intermediate product 4; 　Intermediate product 2 and intermediate product 4 are stirred at room temperature for 16 hours for coupling to obtain intermediate product 5; 　Intermediate product 5, tetrakis(triphenylphosphine) palladium and Morpholine was stirred in dichloromethane at room temperature for 3 hours to remove the allyloxy protecting group to obtain intermediate product 6; 　Fluorinated chloro-3-(2-naphthalene)-L-isoamine acid, HBTU, DIPEA and intermediate product 6, Stir at room temperature for 16 hours to obtain intermediate product 7; 　Tranexamic acid derivative, HBTU, DIPEA and intermediate product 7 were stirred at room temperature for 16 hours to obtain intermediate product 8; 　N-Succinimidyl-S-acetylthiopropionate (STPA) and hydrogen phosphate Sodium is stirred with intermediate product 8 in dimethyl sulfoxide for 10 hours at room temperature to obtain intermediate product 9; 　 After the intermediate product 9 is reacted with hydroxylamine to remove the acetyl group, trifluoroacetic acid is added to obtain the first semi-finished product (sulfhydryl-modified PSMA-617 , PSMA-7165 intermediate); wherein the second process includes the steps: 　 Reaction of compound 11 (o-dimethylbenzidine) with di-tert-butyl dicarbonate in dichloromethane at room temperature for 5 hours, using di-tert-butyl dicarbonate As a limited amount of reagents, intermediate product 12 (Boc-benzylamine) can be obtained, which is then reacted with sodium nitrite and hydrochloric acid to form diazonium salt intermediate product 13. 　The 1-amino-8-naphthol-2,4 -Dissolve sodium disulfonate and sodium bicarbonate in water, slowly add intermediate product 13 to react in the solution for 12 hours to obtain intermediate product 14, then add trifluoroacetic acid as intermediate product 14 to remove the boc protecting group, add Boc-Lys -Fmoc and HATU, stirred at room temperature for 6 hours to obtain intermediate product 15, which was added to piperidine at room temperature and stirred for 4 hours to obtain intermediate product 16; 　 After pre-stirring NOTA, HATU, DIPEA in DMF for 15 minutes, add Intermediate product 16 was reacted at room temperature until overnight to obtain intermediate product 17; 　Add intermediate product 17 to trifluoroacetic acid and DMF solvent and stir at room temperature for 2 hours to obtain a second semi-finished product (DOTA-EB-Lys, PSMA-7165 intermediate); 　 of which 3. The process includes the steps: 　 Stir the second semi-finished product (DOTA-EB-Lys) and the dioxolyl ester derivative at room temperature to obtain intermediate product 19, and finally buffer the intermediate product 19 and the first semi-finished product (PSMA617-SH) in PBS Solution and DMF at room temperature After stirring for 2 hours, the final product PSMA-7165 was obtained.

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